Preparation method of cationic lipid compound

Abstract

The invention belongs to the technical field of organic synthesis, and provides a preparation method of a cationic lipid compound, which comprises the following steps: (1) reacting 1, 5-dihalopentane, 2-tert-butyl glycolate and alkali to obtain a product G; (2) hydrolyzing the product G under an acidic condition to obtain a product H; (3) carrying out esterification reaction on the product H, the raw material C, 4-dimethylaminopyridine and a condensing agent in dichloromethane to obtain a product J; and (4) carrying out amination reaction on the product J, the raw material L and alkali in acetonitrile to obtain the cationic lipid compound. Compared with the prior art, the method has the relatively remarkable advantages that when the first part of halogenated long-chain alkyl ester H is synthesized by the method, 1, 5-dihalopentane and 2-tert-butyl glycolate are used as raw materials, the halogenated long-chain alkyl ester J is synthesized by a simple method, the product is obtained at a higher yield in synthesis, and the method is suitable for kilogram-level production. The route synthesis only comprises four steps of chemical reactions, and is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis, and particularly relates to a preparation method of a cationic lipid compound. BACKGROUND

[0002] Nucleic acid-based drugs, such as messenger RNA (mRNA), antisense oligonucleotides, small interfering RNA (SiRNA), plasmids, etc., have broad application prospects, and how to safely and effectively deliver them to target organs and target cells in the body is a difficult problem restricting the development of this technology.

[0003] At present, nucleic acid drug delivery systems can be divided into two categories: viral vector systems and non-viral systems, and the lipid nanoparticle-mediated nucleic acid drug delivery is a main method of non-viral delivery systems.

[0004] In gene therapy and vaccine applications, lipid nanoparticles have been proven to be excellent carriers of nucleic acids for treating various diseases. Lipid nanoparticles formed by cationic lipids and other auxiliary lipids, such as cholesterol, phospholipids and PEGylated lipids, encapsulate nucleic acids, protect nucleic acids from degradation and promote cellular uptake, and reduce immune responses. In addition, cell delivery of bioactive ingredients by lipid nanoparticles has other advantages, such as good tropism, small side effects, good stability and high transfection efficiency, etc.

[0005] Based on the rapid development of the therapeutic field of nucleic acid molecules, there is a greater demand for the delivery of nucleic acid drugs, and therefore it is necessary to develop efficient and safe nucleic acid delivery vectors.

[0006] In view of the synthesis method of the compound of formula (I) in the patent WO2023125738, the reaction has 9 steps of chemical reaction, and the last step uses heavy metal palladium catalytic hydrogenation to remove benzyl group, which has great challenges to production requirements and product quality control. In view of this structure compound, a simple synthesis method is developed. SUMMARY

[0007] The present application aims to overcome the problem of complex synthesis process in the prior art, and provides a preparation method of a cationic lipid compound.

[0008] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a preparation method of a cationic lipid compound, comprising the following steps: (1) reacting 1,5-dihalogenopentane, tert-butyl 2-hydroxyacetate and a base to obtain product G; (2) hydrolyzing product G under acidic conditions to obtain product H; (3) esterifying product H, raw material C, 4-dimethylaminopyridine and a condensing agent in dichloromethane to obtain product J; (4) carrying out an amination reaction of the product J, the raw material L and a base in acetonitrile to obtain the cationic lipid compound.

[0009] Preferably, the structure of the 1,5-dihalogen pentane in step (1) is ; wherein X is chlorine or bromine; The molar ratio of the 1,5-dihalogen pentane to the tert-butyl 2-hydroxyacetate in step (1) is 1-20:1. The molar ratio of the tert-butyl 2-hydroxyacetate to the base in step (1) is 1:1-3. The base in step (1) is one or more of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium hydride, potassium tert-butoxide and sodium tert-butoxide.

[0010] Preferably, the temperature of the reaction in step (1) is -5-30℃ and the time is 3-10h.

[0011] Preferably, the structure of the product G in step (1) is ; wherein X is chlorine or bromine; The acid solution used in the acid condition in step (2) is one or more of hydrogen bromide solution, hydrochloric acid, sulfuric acid solution and trifluoroacetic acid. The concentration of the hydrogen bromide solution is 35-48%; the concentration of the hydrochloric acid is 30-37%; the concentration of the sulfuric acid solution is 25-35%. The molar ratio of the product G to the solute in the acid solution in step (2) is 1:1-20.

[0012] Preferably, the temperature of the hydrolysis in step (2) is 20-50℃ and the time is 2-16h.

[0013] Preferably, the structure of the product H in step (2) is ; wherein X is chlorine or bromine; The structure of the raw material C in step (3) is ; The condensing agent in step (3) is one or more of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N,N'-carbonyldiimidazole. The molar ratio of the product H, the raw material C, 4-dimethylaminopyridine and the condensing agent in step (3) is 1-1.5:1:0.001-0.05:1-2.

[0014] Preferably, the temperature of the esterification reaction in step (3) is 20-30℃ and the time is 5-20h.

[0015] Preferably, the structure of product J in step (3) is as follows: ; Where X is chlorine or bromine; The structure of raw material L in step (4) is as follows: ; The base mentioned in step (4) is one or more of N,N-diisopropylethylamine, triethylamine, pyridine and potassium carbonate; In step (4), the molar ratio of product J, raw material L, and alkali is 1~1.5:1:3~10; The amination reaction in step (4) is carried out at a temperature of 70-80°C for 5-24 hours.

[0016] This invention provides a method for preparing cationic lipid compounds, comprising the following steps: (1) reacting 1,5-dihalopentane, tert-butyl 2-hydroxyacetate, and a base to obtain product G; (2) hydrolyzing product G under acidic conditions to obtain product H; esterifying product H, raw material C, 4-dimethylaminopyridine, and a condensing agent in dichloromethane to obtain product J; (4) amination of product J, raw material L, and a base in acetonitrile to obtain the cationic lipid compound. The significant advantage of this invention is that when synthesizing halo-long-chain alkyl ester H using this method, 1,5-dihalopentane and tert-butyl 2-hydroxyacetate are used as raw materials, resulting in a simple method for synthesizing product J. The synthesis yields the product at a high rate and is suitable for kilogram-scale production. In this process, column chromatography is not required; the target product can be obtained directly by distillation or vacuum distillation, offering significant advantages in production route and synthesis cost. Product J and raw material L are reacted directly under alkaline conditions and purified by column chromatography to obtain a pure product with a total molar yield of 36%. This synthetic route involves only four chemical reactions with no special reactions. Only the final product step requires column purification, making it suitable for industrial production. Compared to the reported nine-step reaction, the synthetic route is significantly shortened, and the molar yield is only 5%. It avoids the use of heavy metal catalysis, demonstrating the clear advantages of this invention. Attached Figure Description

[0017] Figure 1 This is an HPLC-CAD detection image of the cationic lipid compounds in Example 1; Figure 2 This is the mass spectrum of the cationic lipid compound in Example 1. Detailed Implementation

[0018] This invention provides a method for preparing cationic lipid compounds, comprising the following steps: (1) 1,5-Dihalopentane, tert-butyl 2-hydroxyacetate and base were reacted to give product G; (2) Product G is hydrolyzed under acidic conditions to obtain product H; (3) The product H, raw material C, 4-dimethylaminopyridine and condensing agent are esterified in dichloromethane to obtain product J; (4) The product J, the raw material L and the base are subjected to an amination reaction in acetonitrile to obtain the cationic lipid compound.

[0019] In this invention, the structure of 1,5-dihalopentane in step (1) is as follows: ; Where X is chlorine or bromine.

[0020] In this invention, the molar ratio of 1,5-dihalopentane and tert-butyl 2-hydroxyacetate in step (1) is preferably 1 to 20:1, more preferably 5 to 15:1, and even more preferably 8 to 12:1.

[0021] In this invention, the molar ratio of tert-butyl 2-hydroxyacetate to base in step (1) is preferably 1:1 to 3, more preferably 1:1.5 to 2.5, and even more preferably 1:1.8 to 2.2.

[0022] In this invention, the alkali in step (1) is one or more of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium hydride, potassium tert-butoxide, and sodium tert-butoxide.

[0023] In this invention, the temperature of the reaction in step (1) is preferably -5~30℃, more preferably 0~25℃, and even more preferably 0~10℃; the time is preferably 3~10h, more preferably 4~9h, and even more preferably 5~8h.

[0024] In this invention, after the reaction in step (1) is completed, acetic acid is added to quench the reaction, water is added to quench the reaction, the mixture is stirred to separate the layers, the organic phase is washed with water, allowed to stand to separate the layers, and the organic phase is concentrated under reduced pressure. The temperature of the reduced pressure concentration is preferably 90~100℃, more preferably 92~98℃, and even more preferably 94~96℃. The main fraction is removed by reduced pressure distillation to obtain product G.

[0025] In this invention, the reaction in step (1) is as follows: .

[0026] In this invention, the structure of product G in step (1) is as follows: ; Where X is chlorine or bromine.

[0027] In this invention, the acidic solution used in step (2) is one or more of hydrogen bromide solution, hydrochloric acid, sulfuric acid solution and trifluoroacetic acid.

[0028] In this invention, the concentration of the hydrogen bromide solution is preferably 35-48%, more preferably 38-45%, and even more preferably 40-43%; the concentration of the hydrochloric acid is preferably 30-37%, more preferably 31-36%, and even more preferably 32-35%; and the concentration of the sulfuric acid solution is preferably 25-35%, more preferably 26-34%, and even more preferably 28-32%.

[0029] In this invention, the molar ratio of product G to solute in acidic solution in step (2) is preferably 1:1 to 20, more preferably 1:2 to 10, and even more preferably 1:3 to 4.

[0030] In this invention, the hydrolysis temperature in step (2) is preferably 20~50℃, more preferably 20~35℃, and even more preferably 20~30℃; the time is preferably 2~16h, more preferably 3~8h, and even more preferably 3~4h; the hydrolysis is carried out under stirring conditions.

[0031] In this invention, after hydrolysis in step (2), the organic phase is extracted twice with toluene, the organic phases are combined, the organic phase is washed with 20% sodium chloride solution, and the organic phase is concentrated under reduced pressure to remove toluene. The temperature of the reduced pressure concentration is preferably 50~70℃, more preferably 55~65℃, and more preferably 58~62℃. The product H is obtained by reduced pressure distillation.

[0032] In this invention, the reaction in step (2) is as follows: .

[0033] In this invention, the structure of product H in step (2) is as follows: ; Where X is chlorine or bromine.

[0034] In this invention, the structure of raw material C in step (3) is as follows: .

[0035] In this invention, the condensing agent in step (3) is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N,N'-carbonyldiimidazole.

[0036] In this invention, the preferred ratio of product H, raw material C, 4-dimethylaminopyridine and condensing agent in step (3) is 1~1.5:1:0.001~0.05:1~2, more preferably 1~1.3:1:0.01~0.04:1~1.5, and even more preferably 1~1.1:1:0.02~0.03:1.2~1.3.

[0037] In this invention, the temperature of the esterification reaction in step (3) is preferably 20~30℃, more preferably 22~28℃, and even more preferably 24~26℃; the time is preferably 5~20h, more preferably 10~15h, and even more preferably 11~14h.

[0038] In this invention, after the reaction in step (3) is completed, a saturated sodium bicarbonate solution is added for extraction and separation. The organic phase is then extracted again with a saturated sodium bicarbonate solution and separated. The organic phase is washed with a 20% sodium chloride solution and concentrated under reduced pressure to remove dichloromethane to obtain product J. The temperature of the reduced pressure concentration is preferably 40~60℃, more preferably 45~55℃, and even more preferably 48~52℃.

[0039] In this invention, the reaction in step (3) is as follows: .

[0040] In this invention, the structure of product J in step (3) is as follows: ; Where X is chlorine or bromine.

[0041] In this invention, the structure of raw material L in step (4) is as follows: .

[0042] In this invention, raw material L can be purchased directly or synthesized in a laboratory; this invention provides a method for synthesizing raw material L, comprising the following steps: (a) The raw materials D, E, dichloromethane, 4-dimethylaminopyridine and condensing agent are subjected to esterification reaction to obtain product K; (b) React product K, raw material F and ethanol to obtain raw material L.

[0043] In this invention, the structure of raw material D in step (a) is as follows: .

[0044] In this invention, the structure of raw material E in step (a) is as follows: .

[0045] In this invention, the condensing agent in step (a) is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N'-carbonyldiimidazole.

[0046] In this invention, the molar ratio of raw material D, raw material E, 4-dimethylaminopyridine and condensing agent in step (a) is preferably 1:1~2:0.001~0.2:1~2, more preferably 1:1~1.5:0.01~0.15:1~1.5, and even more preferably 1:1.1~1.2:0.1~0.12:1.2~1.3.

[0047] In this invention, the esterification reaction time in step (a) is preferably 5 to 20 hours, more preferably 10 to 15 hours, and even more preferably 12 to 13 hours. The esterification reaction is carried out under stirring conditions.

[0048] In this invention, after the reaction in step (a) is completed, a saturated sodium bicarbonate solution is added for extraction, and the layers are separated. The organic phase is then extracted again with a saturated sodium bicarbonate solution, and the layers are separated. The organic phase is washed with a 20% sodium chloride solution, and the organic phase is concentrated under reduced pressure to remove dichloromethane. The temperature for the reduced pressure concentration is preferably 40~60℃, more preferably 45~55℃, and even more preferably 48~52℃, to obtain product K.

[0049] In this invention, the reaction in step (a) is as follows: .

[0050] In this invention, the structure of product K in step (a) is as follows: .

[0051] In this invention, the structure of raw material F in step (b) is as follows: .

[0052] In this invention, the molar ratio of product K to raw material F in step (5) is preferably 1:10~50, more preferably 1:20~40, and even more preferably 1:28~32.

[0053] In this invention, the reaction temperature in step (b) is preferably 40~70°C, more preferably 40~55°C, and even more preferably 44~46°C; the reaction time is preferably 2~5h, more preferably 2.5~4.5h, and even more preferably 3~4h.

[0054] In this invention, after the reaction in step (b) is completed, the ethanol is concentrated, ethyl acetate is added to dissolve the organic phase, the organic phase is extracted once with 20% sodium chloride solution, and the phase is allowed to stand and separate. The organic phase is then extracted once more with 20% sodium chloride solution, and the phase is allowed to stand and separate. The organic phase is concentrated under reduced pressure to remove ethyl acetate. The temperature for reduced pressure concentration is preferably 40~60℃, more preferably 45~55℃, and even more preferably 48~52℃, to obtain crude product L. Heptane is added to crude product L and stirred to dissolve at 30℃. After dissolution, the temperature is slowly lowered to crystallize. The crystallization temperature is preferably -10~-30℃, more preferably -15~-25℃, and even more preferably -18~-22℃. The product is then filtered and dried to obtain raw material L.

[0055] In this invention, the reaction process in step (b) is as follows: .

[0056] In this invention, the base mentioned in step (4) is one or more of N,N-diisopropylethylamine, triethylamine, pyridine and potassium carbonate.

[0057] In this invention, the molar ratio of product J, raw material L and alkali in step (4) is preferably 1~1.5:1:3~10, more preferably 1~1.3:1:4~8, and even more preferably 1~1.1:1:4~6.

[0058] In this invention, the temperature of the amination reaction in step (4) is preferably 70~80℃, more preferably 72~78℃, and even more preferably 74~76℃; the time is preferably 5~24h, more preferably 10~20h, and even more preferably 14~16h; the reaction is carried out under stirring conditions.

[0059] In this invention, after the reaction in step (4) is completed, ethyl acetate is added, water is stirred and extracted, and the mixture is allowed to stand and separate into layers. The organic phase is then extracted once with 20% sodium chloride solution, allowed to stand and separate into layers, and the organic phase is concentrated under reduced pressure to remove ethyl acetate. The temperature for reduced pressure concentration is preferably 40~60℃, more preferably 45~55℃, and even more preferably 48~52℃. A crude cationic lipid compound is obtained as a pale yellow liquid. The crude product is purified by column chromatography, eluted with a dichloromethane and methanol system, and the concentrated product is the cationic lipid compound.

[0060] In this invention, the reaction in step (4) is as follows: .

[0061] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] Step (1): Add 1,5-dibromopentane (1300g, 8.0eq) and tert-butyl 2-hydroxyacetate (100g, 1.0eq) to a three-necked flask, cool to -5℃, add 60% sodium hydride (45g, 1.5eq) in batches, and after the addition is complete, heat to 25℃ and react for 3h. After the reaction is complete, add acetic acid to quench the reaction, add 500ml of water to quench the reaction, stir to separate the layers, add 500ml of water to the organic phase and wash it again, let it stand to separate the layers, concentrate the organic phase under reduced pressure at 95℃, and distill under reduced pressure to obtain G, 185g of liquid, yield 87%.

[0064] Step (2): Add bromine G (150g, 1.0eq) and 40% hydrogen bromide solution (324g, 3.0eq) to a three-necked flask, stir at 25℃ for 5h, and after the reaction is complete, extract twice with 500ml toluene, combine the organic phases, wash the organic phase once with 300ml 20% sodium chloride solution, concentrate the organic phase under reduced pressure at 60℃ to remove toluene, and distill under reduced pressure to obtain bromine H, 108g of liquid, yield 90%.

[0065] Step (3): Add 1000 ml of dichloromethane, raw material C (100 g, 1.0 eq), bromo H (95 g, 1.1 eq), and 4-dimethylaminopyridine (1.0 g, 0.02 eq) to a three-necked flask. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (104 g, 1.3 eq) in batches. After adding all the ingredients, stir at 25°C for 5 h. Add 500 ml of saturated sodium bicarbonate solution for extraction and separate the layers. Add 600 ml of saturated sodium bicarbonate solution to the organic phase for extraction and separate the layers again. Add 300 ml of 20% sodium chloride solution to the organic phase for washing once. Concentrate the organic phase under reduced pressure at 50°C to remove dichloromethane, and obtain 151 g of bromo J liquid, with a yield of 83%.

[0066] Step (a): Add 1000 ml of dichloromethane, D (110 g, 1.0 eq), E (137 g, 1.1 eq), and 4-dimethylaminopyridine (4.7 g, 0.1 eq) to a three-necked flask. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (157 g, 1.3 eq) in batches. Stir for 5 h after the addition is complete. Extract with 600 ml of saturated sodium bicarbonate solution. After separation, extract the organic phase again with 600 ml of saturated sodium bicarbonate solution. After separation, wash the organic phase once with 300 ml of 20% sodium chloride solution. Concentrate the organic phase under reduced pressure at 50 °C to remove dichloromethane, and obtain 205 g of K yellow liquid, yield 92%.

[0067] Step (b): Add 1000 ml of ethanol, F (1050 g, 30 eq), and K (200 g, 1.0 eq) to a three-necked flask. After adding and stirring evenly, heat to 45 °C and react for 4 h. After the reaction is complete, concentrate to remove ethanol, add 1000 ml of ethyl acetate to dissolve, extract the organic phase once with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, extract the organic phase once more with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, and obtain 181 g of crude pale yellow liquid L. Add 1000 ml of n-heptane to crude L and stir to dissolve at 30 °C. After dissolving, slowly cool to -20 °C to crystallize, filter, and dry to obtain 146 g of white solid L, with a yield of 77% and a purity of 98%.

[0068] Step (4): Add 300 ml of acetonitrile, L (50 g, 1.0 eq), bromo J (77.4 g, 1.1 eq), and N,N-diisopropylethylamine (97.8 g, 5 eq) to a three-necked flask. After adding and stirring evenly, heat to 75 °C and react for 16 h. After the reaction is complete, add 500 ml of ethyl acetate and 500 ml of water, stir and extract, and allow to stand for separation. Extract the organic phase once with 200 ml of 20% sodium chloride solution, allow to stand for separation, and concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, obtaining 115 g of crude (I) pale yellow liquid. Purify (I) by column chromatography, elute with dichloromethane and methanol system, concentrate the product, and obtain 62 g of pale yellow liquid, with a yield of 57% and a purity of 99%.

[0069] The HPLC-CAD detection chromatogram of the cationic lipid compounds prepared in this embodiment is shown below. Figure 1 As shown, from Figure 1 It can be seen that the prepared sample has a purity of 99% and the product quality is good.

[0070] The mass spectrum of the cationic lipid compound prepared in this embodiment is as follows: Figure 2 As shown, the sample was confirmed to be the target product, and its structure was correct.

[0071] Example 2

[0072] Step (1): Add 1,5-dibromopentane (1300g, 8.0eq) and tert-butyl 2-hydroxyacetate (100g, 1.0eq) to a three-necked flask, cool to -5℃, add potassium tert-butoxide (127g, 1.5eq) in batches, raise the temperature to 25℃ and react for 5h. After the reaction is complete, add acetic acid to quench the reaction, add 500ml of water to quench the reaction, stir to separate the layers, add 500ml of water to the organic phase and wash it again, let it stand to separate the layers, concentrate the organic phase under reduced pressure at 95℃, and distill under reduced pressure to obtain G, 180g of liquid, yield 85%.

[0073] Step (2): Add bromine G (150g, 1.0eq) and 36% hydrochloric acid (162g, 3.0eq) to a three-necked flask, stir at 25℃ for 3h, and when the reaction is complete, extract twice with 500ml toluene, combine the organic phases, wash the organic phase once with 300ml 20% sodium chloride solution, concentrate the organic phase under reduced pressure at 60℃ to remove toluene, and distill under reduced pressure to obtain bromine H, 102g of liquid, yield 85%.

[0074] Step (3): Add 1000 ml of dichloromethane, raw material C (100 g, 1.0 eq), bromo H (95 g, 1.1 eq), and 4-dimethylaminopyridine (1.0 g, 0.02 eq) to a three-necked flask. After stirring evenly, add EDC (98 g, 1.3 eq) in batches. After adding all the ingredients, stir at 25°C for 12 h. Add 600 ml of saturated sodium bicarbonate solution for extraction and separate the layers. Add 600 ml of saturated sodium bicarbonate solution to the organic phase for extraction and separate the layers again. Add 300 ml of 20% sodium chloride solution to the organic phase for washing once. Concentrate the organic phase under reduced pressure at 50°C to remove dichloromethane, and obtain 118 g of bromo J liquid, with a yield of 78%.

[0075] Step (a): Add 1000 ml of dichloromethane, D (110 g, 1.0 eq), E (137 g, 1.1 eq), and 4-dimethylaminopyridine (4.7 g, 0.1 eq) to a three-necked flask. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (157 g, 1.3 eq) in batches. Stir for 5 h after the addition is complete. Extract with 600 ml of saturated sodium bicarbonate solution. After separation, extract the organic phase again with 600 ml of saturated sodium bicarbonate solution. After separation, wash the organic phase once with 300 ml of 20% sodium chloride solution. Concentrate the organic phase under reduced pressure at 50 °C to remove dichloromethane, and obtain 205 g of K yellow liquid, yield 92%.

[0076] Step (b): Add 1000 ml of ethanol, F (1050 g, 30 eq), and K (200 g, 1.0 eq) to a three-necked flask. After adding and stirring evenly, heat to 45 °C and react for 4 h. After the reaction is complete, concentrate to remove ethanol, add 1000 ml of ethyl acetate to dissolve, extract the organic phase once with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, extract the organic phase once more with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, and obtain 181 g of crude pale yellow liquid L. Add 1000 ml of n-heptane to crude L and stir to dissolve at 30 °C. After dissolving, slowly cool to -20 °C to crystallize, filter, and dry to obtain 146 g of white solid L, with a yield of 77% and a purity of 98%.

[0077] Step (4): Add 300ml acetonitrile, L (50g, 1.0eq), brominated J (77.4g, 1.1eq), powdered potassium carbonate (168g, 8eq), and potassium iodide (2.5g, 0.1eq) to a 1000ml three-necked flask. After adding and stirring evenly, heat to 75℃ and react for 13h. After the reaction is complete, filter and wash the filter cake with 500ml ethyl acetate. Add 500ml water to the filtrate and stir to extract. Let stand and separate the layers. Extract the organic phase once with 200ml 20% sodium chloride solution. Let stand and separate the layers. Concentrate the organic phase under reduced pressure at 50℃ to remove ethyl acetate, and obtain 115g of crude (I) pale yellow liquid. Purify (I) by column chromatography, elute with dichloromethane and methanol system, concentrate the product, and obtain 65g of pale yellow liquid with a yield of 60% and a purity of 99%.

[0078] Example 3

[0079] Step (1): Add 1,5-dibromopentane (1300g, 8.0eq) and tert-butyl 2-hydroxyacetate (100g, 1.0eq) to a three-necked flask, cool to -5℃, add sodium tert-butoxide (109g, 1.5eq) in portions, raise the temperature to 25℃ after the addition is complete, react for 6h, add acetic acid to quench, add 500ml of water to quench, stir to separate the layers, add 500ml of water to wash the organic phase again, let stand to separate the layers, concentrate the organic phase under reduced pressure at 95℃, and distill under reduced pressure to obtain G, 195g of liquid, yield 92%.

[0080] Step (2): Add bromo G (150g, 1.0eq) and trifluoroacetic acid (310g, 6.0eq) to a three-necked flask, stir at 25°C for 5h, and after the reaction is complete, concentrate under reduced pressure at 60°C to remove trifluoroacetic acid, and obtain 120g of bromo H liquid, with a yield of 99%.

[0081] Step (3): Add 1000 ml of dichloromethane, raw material C (100 g, 1.0 eq), bromo H (96 g, 1.1 eq), and 4-dimethylaminopyridine (2.5 g, 0.05 eq) to a three-necked flask. After stirring evenly, add CDI (85 g, 1.3 eq) in batches. After adding all the ingredients, stir at 25°C for 5 h. Add 600 ml of saturated sodium bicarbonate solution for extraction and separate the layers. Add 600 ml of saturated sodium bicarbonate solution to the organic phase for extraction and separate the layers again. Add 300 ml of 20% sodium chloride solution to the organic phase for washing once. Concentrate the organic phase under reduced pressure at 50°C to remove dichloromethane, and obtain 101 g of bromo J liquid, with a yield of 81%.

[0082] Step (a): Add 1000 ml of dichloromethane, D (110 g, 10 eq), E (137 g, 1.1 eq), and 4-dimethylaminopyridine (4.7 g, 0.1 eq) to a three-necked flask. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (157 g, 1.3 eq) in batches. Stir for 5 h after the addition is complete. Extract with 600 ml of saturated sodium bicarbonate solution. After separation, extract the organic phase again with 600 ml of saturated sodium bicarbonate solution. After separation, wash the organic phase once with 300 ml of 20% sodium chloride solution. Concentrate the organic phase under reduced pressure at 50 °C to remove dichloromethane, and obtain 205 g of K yellow liquid, with a yield of 92%.

[0083] Step (b): Add 1000 ml of ethanol, F (1050 g, 30 eq), and K (200 g) to a three-necked flask. After adding and stirring evenly, heat to 45 °C and react for 4 h. After the reaction is complete, concentrate to remove ethanol, add 1000 ml of ethyl acetate to dissolve, extract the organic phase once with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, extract the organic phase once more with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, and obtain 181 g of crude pale yellow liquid L. Add 1000 ml of n-heptane to crude L and stir to dissolve at 30 °C. After dissolving, slowly cool to -20 °C to crystallize, filter, and dry to obtain 146 g of white solid L, yield 77%, purity 98%.

[0084] Step (4): Add 300 ml of acetonitrile, L (50 g, 1.0 eq), bromo J (77.4 g, 1.1 eq), and triethylamine (76.6 g, 5 eq) to a three-necked flask. After adding and stirring evenly, heat to 75 °C and react for 15 h. After the reaction is complete, add 500 ml of ethyl acetate and 500 ml of water, stir and extract, and allow to stand for separation. Extract the organic phase once with 200 ml of 20% sodium chloride solution, allow to stand for separation, and concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, obtaining 115 g of crude (I) pale yellow liquid. Purify (I) by column chromatography, elute with dichloromethane and methanol system, concentrate the product, and obtain 57 g of pale yellow liquid, with a yield of 53% and a purity of 99%.

[0085] Example 4

[0086] Step (1): Add 1-bromo-5-chloropentane (1120g, 8.0eq) and tert-butyl 2-hydroxyacetate (100g, 1.0eq) to a three-necked flask, cool to -5℃, add potassium tert-butoxide (1.5eq) in batches, and after the addition is complete, heat to 25℃ and react for 4h. When the reaction is complete, add acetic acid to quench the reaction, add 500ml of water to quench the reaction, stir to separate the layers, add 500ml of water to the organic phase and wash it again, let it stand to separate the layers, concentrate the organic phase under reduced pressure at 95℃, and distill under reduced pressure to obtain G, 175g of liquid, yield 82%.

[0087] Step (2): Add 140 g of chlorinated G (1.0 eq) and 361 g of 40% hydrogen bromide solution (3.0 eq) to a three-necked flask, stir at 25 °C for 5 h, and after the reaction is complete, extract twice with 500 ml of toluene, combine the organic phases, wash the organic phase once with 300 ml of 20% sodium chloride solution, concentrate the organic phase under reduced pressure at 60 °C to remove toluene, and distill under reduced pressure to obtain 108 g of chlorinated H liquid, yield 90%.

[0088] Step (3): Add 1000 ml of dichloromethane, C (100 g, 1.0 eq), chloro H (93 g, 1.1 eq), and 4-dimethylaminopyridine (1.0 g, 0.02 eq) to a three-necked flask. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (125 g, 1.3 eq) in batches. After the addition is complete, stir at 25°C for 5 h. Add 600 ml of saturated sodium bicarbonate solution for extraction and separate the layers. Add 600 ml of saturated sodium bicarbonate solution to the organic phase for extraction and separate the layers again. Add 300 ml of 20% sodium chloride solution to the organic phase for washing once. Concentrate the organic phase under reduced pressure at 50°C to remove dichloromethane, and obtain 162 g of chloro J liquid, with a yield of 83%.

[0089] Step (a): Add 1000 ml of dichloromethane, D (110 g, 1.0 eq), E (137 g, 1.1 eq), and 4-dimethylaminopyridine (4.7 g, 0.1 eq) to a three-necked flask. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (157 g, 1.3 eq) in batches. Stir for 5 h after the addition is complete. Extract with 600 ml of saturated sodium bicarbonate solution. After separation, extract the organic phase again with 600 ml of saturated sodium bicarbonate solution. After separation, wash the organic phase once with 300 ml of 20% sodium chloride solution. Concentrate the organic phase under reduced pressure at 50 °C to remove dichloromethane, and obtain 205 g of K yellow liquid, yield 92%.

[0090] Step (b): Add 1000 ml of ethanol, F (1050 g, 30 eq), and K (200 g, 1.0 eq) to a three-necked flask. After adding and stirring evenly, heat to 45 °C and react for 4 h. After the reaction is complete, concentrate to remove ethanol, add 1000 ml of ethyl acetate to dissolve, extract the organic phase once with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, extract the organic phase once more with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, and obtain 181 g of crude pale yellow liquid L. Add 1000 ml of n-heptane to crude L and stir to dissolve at 30 °C. After dissolving, slowly cool to -20 °C to crystallize, filter, and dry to obtain 146 g of white solid L, with a yield of 77% and a purity of 98%.

[0091] Step (4): Add 300 ml of acetonitrile, L (50 g, 1.0 eq), chloro J (74.4 g, 1.1 eq), and N,N-diisopropylethylamine (97.8 g, 5 eq) to a three-necked flask. After adding and stirring evenly, heat to 75 °C and react for 14 h. After the reaction is complete, add 500 ml of ethyl acetate and 500 ml of water, stir and extract, and allow to stand for separation. Extract the organic phase once with 200 ml of 20% sodium chloride solution, allow to stand for separation, and concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, obtaining 115 g of crude (I) pale yellow liquid. Purify (I) by column chromatography, elute with dichloromethane and methanol system, concentrate the product, and obtain 66 g of pale yellow liquid, with a yield of 61% and a purity of 99%.

[0092] Example 5

[0093] Step (1): Add 1-bromo-5-chloropentane (1120g, 8.0eq) and tert-butyl 2-hydroxyacetate (100g, 1.0eq) to a three-necked flask, cool to -5℃, add cesium carbonate (219g, 1.5eq) in portions, and after the addition is complete, heat to 25℃ and react for 5h. After the reaction is complete, add acetic acid to quench the reaction, add 500ml of water to quench the reaction, stir to separate the layers, add 500ml of water to the organic phase and wash it again, let it stand to separate the layers, concentrate the organic phase under reduced pressure at 95℃, and distill under reduced pressure to obtain G, 160g of liquid, yield 75%.

[0094] Step (2): Add 150 g of chlorinated G (1.0 eq) and 175 g of 36% hydrochloric acid (3.0 eq) to a three-necked flask, stir at 25 °C for 5 h, and after the reaction is complete, extract twice with 500 ml of toluene, combine the organic phases, wash the organic phase once with 300 ml of 20% sodium chloride solution, concentrate the organic phase under reduced pressure at 60 °C to remove toluene, and distill under reduced pressure to obtain chlorinated H, 108 g of liquid, yield 90%.

[0095] Step (3): Add 1000 ml of dichloromethane, C (100 g, 1.0 eq), chloro H (93 g, 1.1 eq), and 4-dimethylaminopyridine (1.0 g, 0.02 eq) to a three-necked flask. After stirring evenly, add EDC (98 g, 1.3 eq) in batches. After adding all the EDC, stir at 25°C for 5 h. Add 600 ml of saturated sodium bicarbonate solution for extraction and separate the layers. Add 600 ml of saturated sodium bicarbonate solution to the organic phase for extraction and separate the layers again. Add 300 ml of 20% sodium chloride solution to the organic phase and wash it once. Concentrate the organic phase under reduced pressure at 50°C to remove dichloromethane, and obtain 163 g of chloro J yellow liquid, with a yield of 84%.

[0096] Step (a): Add 1000 ml of dichloromethane, D (110 g, 1.0 eq), E (137 g, 1.1 eq), and 4-dimethylaminopyridine (4.7 g, 0.1 eq) to a three-necked flask. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (157 g, 1.3 eq) in batches. Stir for 5 h after the addition is complete. Extract with 600 ml of saturated sodium bicarbonate solution. After separation, extract the organic phase again with 600 ml of saturated sodium bicarbonate solution. After separation, wash the organic phase once with 300 ml of 20% sodium chloride solution. Concentrate the organic phase under reduced pressure at 50 °C to remove dichloromethane, and obtain 205 g of K yellow liquid, yield 92%.

[0097] Step (b): Add 1000 ml of ethanol, F (1050 g, 30 eq), and K (200 g, 1.0 eq) to a three-necked flask. After adding and stirring evenly, heat to 45 °C and react for 4 h. After the reaction is complete, concentrate to remove ethanol, add 1000 ml of ethyl acetate to dissolve, extract the organic phase once with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, extract the organic phase once more with 1000 ml of 20% sodium chloride solution, allow to stand and separate the layers, concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate, and obtain 181 g of crude pale yellow liquid L. Add 1000 ml of n-heptane to crude L and stir to dissolve at 30 °C. After dissolving, slowly cool to -20 °C to crystallize, filter, and dry to obtain 146 g of white solid L, with a yield of 77% and a purity of 98%.

[0098] Step (4): Add 300 ml of acetonitrile, L (50 g, 1.0 eq), chlorinated J (74.4 g, 1.1 eq), powdered potassium carbonate (168 g, 8 eq), and potassium iodide (2.5 g, 0.1 eq) to a three-necked flask. After adding and stirring evenly, heat to 75 °C and react for 15 h. After the reaction is complete, filter and wash the filter cake with 500 ml of ethyl acetate. Add 500 ml of water to the filtrate and stir to extract. Let stand and separate the layers. Extract the organic phase once with 200 ml of 20% sodium chloride solution. Let stand and separate the layers. Concentrate the organic phase under reduced pressure at 50 °C to remove ethyl acetate and obtain 115 g of crude (I) pale yellow liquid. Purify (I) by column chromatography, elute with dichloromethane and methanol system, concentrate the product and obtain 54 g of pale yellow liquid with a yield of 50% and a purity of 99%.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a cationic lipid compound, characterized in that, Includes the following steps: (1) 1,5-Dihalopentane, tert-butyl 2-hydroxyacetate and base were reacted to give product G; (2) Product G is hydrolyzed under acidic conditions to obtain product H; (3) The product H, raw material C, 4-dimethylaminopyridine and condensing agent are esterified in dichloromethane to obtain product J; (4) The product J, the raw material L and the base are subjected to an amination reaction in acetonitrile to obtain the cationic lipid compound.

2. The method for preparing the cationic lipid compound as described in claim 1, characterized in that, The structure of 1,5-dihalopentane in step (1) is as follows: ; Where X is chlorine or bromine; In step (1), the molar ratio of 1,5-dihalopentane to tert-butyl 2-hydroxyacetate is 1~20:1; In step (1), the molar ratio of tert-butyl 2-hydroxyacetate to the base is 1:1~3; In step (1), the alkali is one or more of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium hydride, potassium tert-butoxide, and sodium tert-butoxide.

3. The method for preparing the cationic lipid compound as described in claim 2, characterized in that, The reaction in step (1) is carried out at a temperature of -5 to 30°C for 3 to 10 hours.

4. The method for preparing the cationic lipid compound as described in claim 3, characterized in that, The structure of product G in step (1) is as follows: ; Where X is chlorine or bromine; In step (2), the acidic solution used for the acidic conditions is one or more of hydrogen bromide solution, hydrochloric acid, sulfuric acid solution and trifluoroacetic acid; The concentration of hydrogen bromide solution is 35-48%; the concentration of hydrochloric acid is 30-37%; and the concentration of sulfuric acid solution is 25-35%. In step (2), the molar ratio of product G to solute in acidic solution is 1:1~20.

5. The method for preparing the cationic lipid compound as described in claim 4, characterized in that, The hydrolysis temperature in step (2) is 20~50℃ and the time is 2~16h.

6. The method for preparing the cationic lipid compound as described in claim 5, characterized in that, The structure of product H in step (2) is as follows: ; Where X is chlorine or bromine; The structure of raw material C in step (3) is as follows: ; In step (3), the condensing agent is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N,N'-carbonyldiimidazole. In step (3), the molar ratio of product H, raw material C, 4-dimethylaminopyridine and condensing agent is 1~1.5:1:0.001~0.05:1~2.

7. The method for preparing the cationic lipid compound as described in claim 6, characterized in that, The esterification reaction in step (3) is carried out at a temperature of 20~30℃ for 5~20h.

8. The method for preparing the cationic lipid compound as described in claim 7, characterized in that, The structure of product J in step (3) is as follows: ; Where X is chlorine or bromine; The structure of raw material L in step (4) is as follows: ; The base mentioned in step (4) is one or more of N,N-diisopropylethylamine, triethylamine, pyridine and potassium carbonate; In step (4), the molar ratio of product J, raw material L, and alkali is 1~1.5:1:3~10; The amination reaction in step (4) is carried out at a temperature of 70-80°C for 5-24 hours.

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