Preparation method of ionizable cationic lipid LNP5

Abstract

The invention discloses a preparation method of ionizable cationic lipid LNP5 in the technical field of medicines, which comprises the following steps: carrying out nucleophilic substitution reaction on 8-bromooctanoic acid heptadecane-9-yl ester and 8-amino octanoic acid nonyl ester under alkaline conditions to obtain a crude product of 8-[[8-(nonyloxy)-8-oxo-octyl] amino] octanoic acid-1-octyl nonyl ester, and carrying out acid salification refining to obtain the ionizable cationic lipid LNP5. The heptadecane-9-yl8-((2-ethoxyl) (8-(nonyloxy)-8-oxooctyl) amino) octanoate is prepared by reacting heptadecane-9-yl8-((2-ethoxyl) (8-(nonyloxy)-8-oxooctyl) amino) octanoate with 2-haloethanol acetate in a methanol solvent. The method has the advantages of high efficiency, simplicity, easiness in factory operation, good product appearance, high purity, economical and easily available raw materials and good industrialization prospect.

Description

Technical Field

[0001] This invention relates to a method for preparing an ionizable cationic lipid LNP5, belonging to the field of pharmaceutical technology. Background Technology

[0002] The chemical structure of the cationic lipid LNP5, namely heptadecano-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate (CAS: 2089251-33-0), is as follows:

[0003] The cationic lipid LNP5 is used in mRNA-4157, a personalized mRNA tumor vaccine developed by Moderna. It has been granted Breakthrough Therapy Designation by the FDA and Priority Medicines (PRIME) status by the European Medicines Agency. It is indicated for melanoma and non-small cell lung cancer and is expected to become the world's first marketed mRNA tumor vaccine.

[0004] The existing literature mainly describes the following two synthesis techniques: 1. One method uses heptadecano-9-yl-8-bromooctanoate as the starting material, which is first reacted with ethanolamine via a bimolecular nucleophilic substitution amination reaction to obtain 8-(2-hydroxyethyl)aminooctanoate-1-octylnonyl ester, and then subjected to an aminoalkylation reaction with 8-bromooctanoate nonyl ester, followed by chromatographic purification to obtain the target product.

[0005]

[0006] Because both O and N of aminoethanol are reactive, they easily generate O,N-alkylated products, resulting in poor purity of intermediate 3. Further alkylation under alkaline conditions requires column chromatography to obtain qualified product 1. This route attaches the head and long straight chain in the SN2 nucleophilic substitution reaction (aminoalkylation), and the yield after the two-step reaction is only 18.0%.

[0007] 2. Chinese patent CN114874104A discloses a "method for preparing cationic liposome SM-102 and its analogues", which uses 8-bromooctanoic acid nonyl ester as raw material, first reacts with ethanolamine to generate ethanolamine-octanoic acid nonyl ester, and then reacts with 8-bromooctanoic acid-9-heptadecyl ester to prepare compound 1.

[0008]

[0009] Although the patent discloses an improved preparation method, it does not fundamentally solve the defects in the process. It suffers from technical problems such as low synthesis efficiency, very high cost, and difficulty in purification. The product has large impurities and cannot meet the requirements of API raw materials.

[0010] In summary, the market prices of raw materials 2 and 4 are too high, making large-scale production and supply difficult. Therefore, we need to research and develop our own preparation process. Current production processes suffer from numerous side reactions and require multiple purifications of the product. It is necessary to improve the process, reduce the harshness of the reaction conditions, and thus use an efficient and simple process to prepare high-purity cationic lipid LNP5. Summary of the Invention

[0011] To overcome the above-mentioned technical problems, the present invention provides a method for preparing ionizable cationic lipid LNP5, which has the advantages of high efficiency and simplicity, is easy to operate in factories, produces products with good appearance and high purity, and uses readily available and economical raw materials, thus having good prospects for industrialization.

[0012] Therefore, the technical solution of the present invention is as follows: a method for preparing ionizable cationic lipid LNP5, the reaction formula of which is as follows: ; ; The preparation method includes the following steps: S1: Preparation of compound (4) Under nitrogen protection, heptadecanoyl 8-bromooctanoate (2), nonyl 8-aminooctanoate (3), and solvent were added to a reaction vessel, and diisopropylethylamine was added dropwise. After the addition was completed, the temperature was raised to 50-60℃ and the reaction was carried out for 3 hours. The sample was taken for HPLC control, and the mass content of heptadecanoyl 8-bromooctanoate (2) in the raw materials was controlled to be ≤1.0%. After the reaction was completed, the reaction product was crude 8-[[8-(nonoxy)-8-oxo-octyl]amino]octanoate-1-octylnonyl ester (4). The solvent was methanol, ethanol, or acetonitrile. Add ethyl acetate and water to the reaction system and let it stand to separate the layers; add acid to the ethyl acetate layer, stir, and filter to obtain a solid; then add the solid to ethyl acetate and water, adjust the pH value to 7-8 with sodium carbonate solution, let it stand to separate the layers to obtain an organic layer; remove ethyl acetate under reduced pressure to obtain a pale yellow oily liquid, which is the pure product of 8-[[8-(nonoxy)-8-oxo-octyl]amino]octanoic acid-1-octylnonyl ester (4); S2: Preparation of compound (1) Under nitrogen protection, compound (4) and methanol were added into the reaction vessel, alkali was added, and the mixture was stirred and heated to 40-50℃. 2-Haloethanol acetate was added dropwise. After the dropwise addition was completed, the reaction was maintained at 40-50℃ for 12 hours. Samples were taken for HPLC control. The mass content of compound (4) in the raw material was controlled to be ≤0.1%. After the reaction was completed, crude heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate (1) was obtained. Methanol was removed under reduced pressure, and then ethyl acetate and water were added to the reaction system and allowed to stand for separation. The ethyl acetate layer was dried with anhydrous sodium sulfate, and the filtrate obtained by filtering to remove sodium sulfate was concentrated under reduced pressure to obtain a pale yellow oily substance, which is the pure product of heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate (1).

[0013] Furthermore, in step S1, the solvent is acetonitrile.

[0014] Furthermore, in step S1, the acid used to form the salt is hydrochloric acid, acetic acid, benzoic acid, propionic acid, or oxalic acid, preferably oxalic acid.

[0015] Further, the base used in step S2 is sodium carbonate, sodium bicarbonate, potassium carbonate, triethylamine, diisopropylethylamine, DBU, tetramethylguanidine, or pyridine. Tetramethylguanidine is preferred.

[0016] Further, in step S2, the 2-haloethanol acetate is 2-chloroethanol acetate, 2-bromoethanol acetate, or 2-iodoethanol acetate. Preferably, it is 2-bromoethanol acetate.

[0017] This invention uses heptadecano-9-yl 8-bromooctanoate (2) and nonyl 8-aminooctanoate (3) under alkaline conditions to carry out a nucleophilic substitution reaction to obtain crude 8-[[8-(nonoxy)-8-oxo-octyl]amino]octanoate-1-octylnonyl ester (4), which is then purified by acid salt formation to obtain compound (4); then, it is reacted with 2-haloethanol acetate in methanol solvent to prepare crude heptadecano-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate (1), which is then purified to obtain compound (1). It overcomes the problem in the prior art that both O and N of aminoethanol are reactive and easily generate O, N-alkylated products; it changes the connection order of the lipid head bromoethanol acetate and the hydrophobic long straight chain (3) in the structure of the target product (1), adopting the method of attaching the side chain first and then attaching the head bromoethanol acetate; high purity compound (1) can be obtained by reacting haloethanol acetate and compound (4) in alcohol solvents. The reaction mechanism is that the acetate of compound (1) and methanol solvent can rapidly generate compound (1) in situ under alkaline conditions. This reduces the difficulty of preparing compound (1) and forms the preparation method of this invention, which has technological foresight and can promote the technological progress of domestic and foreign counterparts. Compared with the prior art, the beneficial effects of this invention are: the preparation method of this invention has the advantages of high efficiency and simplicity, is easy to operate in factories, produces products with good appearance and high purity, and uses readily available and economical raw materials, thus having good industrialization prospects. Attached Figure Description

[0018] Figure 1 This is an HPLC chromatogram of the purity of compound (1) in Example 1. Detailed Implementation Example 1

[0019] A method for preparing an ionizable cationic lipid LNP5, the reaction formula is as follows:

[0020] The specific preparation steps are as follows: S1: Preparation of compound (4) Under nitrogen protection, 46.1 g of compound (2), 57.5 g of compound (3) and 300 g of acetonitrile were added to the reaction vessel, and 15.0 g of diisopropylethylamine was added dropwise over approximately 2 hours. After the addition was completed, the temperature was raised to 50-60℃ and the reaction was carried out for 3 hours. A sample was taken for HPLC control, and the mass content of compound (2) in the raw materials was controlled to be ≤1.0%. The reaction was then terminated. 1000 ml of ethyl acetate and 200 ml of water were added to the reaction system and allowed to stand for separation. 10 g of anhydrous oxalic acid was added to the ethyl acetate layer, and the mixture was stirred for 5 hours. The solid was then filtered. The solid was then added to 500 ml of ethyl acetate and 100 g of water. The pH was adjusted to 7-8 using sodium carbonate, and the mixture was allowed to stand for separation to obtain the organic layer. The aqueous layer was discarded. The ethyl acetate was removed under reduced pressure to obtain 59.9 g of pale yellow oily liquid (4), with a yield of 90.0% and a purity of 99.2% as determined by HPLC.

[0021] S2: Preparation of compound (1) Under nitrogen protection, 66.6 g (4) and 200 g methanol were added to the reaction vessel, and 12.9 g tetramethylguanidine (TMG) was added. The mixture was stirred and heated to 40-50 °C. 17.0 g 2-bromoethanol acetate was added dropwise over 2-3 hours. After the addition was completed, the reaction was maintained at 40-50 °C for 12 hours. A sample was taken for HPLC control. The mass content of compound (4) in the raw materials was controlled to be ≤0.1%. The reaction was then terminated. Methanol was removed under reduced pressure. Then, 500 ml ethyl acetate and 100 ml water were added to the reaction system and allowed to stand for separation. The ethyl acetate layer was dried with 20 g anhydrous sodium sulfate. The filtrate obtained by filtration to remove sodium sulfate was concentrated under reduced pressure to obtain 67.5 g of pale yellow oil, which was compound (1). The yield was 95.0%, and the purity was 99.8% as determined by HPLC.

[0022] With all other substances remaining unchanged, the solvent in step S1 was changed to methanol or ethanol, and compared with Example 1, the data listed in Table 1 below were obtained:

[0023] With all other substances remaining unchanged, the acid used in step S1 was changed to hydrochloric acid, acetic acid, benzoic acid, or propionic acid, and compared with Example 1. The data listed in Table 2 below are obtained:

[0024] Selection of alkali in step S2: With other substances remaining constant, the bases used for comparison were sodium carbonate, sodium bicarbonate, potassium carbonate, triethylamine, DBU, tetramethylguanidine, and pyridine, and compared with Example 1. The data listed in Table 3 below are obtained:

[0025] The selection of 2-haloethanol acetate in step S2: With all other substances remaining constant, a comparison was made between 2-chloroethanol acetate, 2-iodoethanol acetate, and Example 1, yielding the data listed in Table 4 below:

[0026] The table above shows that using 2-bromoethanol acetate results in higher yields and product purity.

[0027] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for preparing an ionizable cationic lipid LNP5, characterized in that, The reaction formula is as follows: ； ； The preparation method includes the following steps: S1: Preparation of compound (4) Under nitrogen protection, heptadecanoyl 8-bromooctanoate (2), nonyl 8-aminooctanoate (3), and solvent were added to a reaction vessel, and diisopropylethylamine was added dropwise. After the addition was completed, the temperature was raised to 50-60℃ and the reaction was carried out for 3 hours. The sample was taken for HPLC control, and the mass content of heptadecanoyl 8-bromooctanoate (2) in the raw materials was controlled to be ≤1.0%. After the reaction was completed, the reaction product was crude 8-[[8-(nonoxy)-8-oxo-octyl]amino]octanoate-1-octylnonyl ester (4). The solvent was methanol, ethanol, or acetonitrile. Add ethyl acetate and water to the reaction system and let it stand to separate the layers; add acid to the ethyl acetate layer, stir, and filter to obtain a solid; then add the solid to ethyl acetate and water, adjust the pH value to 7-8 with sodium carbonate solution, let it stand to separate the layers to obtain an organic layer; remove ethyl acetate under reduced pressure to obtain a pale yellow oily liquid, which is the pure product of 8-[[8-(nonoxy)-8-oxo-octyl]amino]octanoic acid-1-octylnon ester (4); S2: Preparation of compound (1) Under nitrogen protection, compound (4) and methanol were added into the reaction vessel, alkali was added, and the mixture was stirred and heated to 40-50℃. 2-Haloethanol acetate was added dropwise. After the dropwise addition was completed, the reaction was maintained at 40-50℃ for 12 hours. Samples were taken for HPLC control. The mass content of compound (4) in the raw material was controlled to be ≤0.1%. After the reaction was completed, crude heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate (1) was obtained. Methanol was removed under reduced pressure, and then ethyl acetate and water were added to the reaction system and allowed to stand for separation. The ethyl acetate layer was dried with anhydrous sodium sulfate, and the filtrate obtained by filtering to remove sodium sulfate was concentrated under reduced pressure to obtain a pale yellow oily substance, which is the pure product of heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate (1).

2. The method for preparing an ionizable cationic lipid LNP5 according to claim 1, characterized in that, In step S1, the solvent is acetonitrile.

3. The method for preparing an ionizable cationic lipid LNP5 according to claim 1, characterized in that, In step S1, the acid used to form the salt is benzoic acid or oxalic acid.

4. The method for preparing an ionizable cationic lipid LNP5 according to claim 3, characterized in that, The acid in question is oxalic acid.

5. The method for preparing an ionizable cationic lipid LNP5 according to claim 1, characterized in that, The base used in step S2 is sodium carbonate, sodium bicarbonate, potassium carbonate, triethylamine, diisopropylethylamine, DBU, tetramethylguanidine, or pyridine.

6. The method for preparing an ionizable cationic lipid LNP5 according to claim 5, characterized in that, The base is tetramethylguanidine.

7. The method for preparing an ionizable cationic lipid LNP5 according to claim 1, characterized in that, In step S2, the 2-haloethanol acetate is 2-chloroethanol acetate, 2-bromoethanol acetate, or 2-iodoethanol acetate.

8. The method for preparing an ionizable cationic lipid LNP5 according to claim 7, characterized in that, In step S2, the 2-haloethanol acetate is 2-bromoethanol acetate.

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