Preparation method of cationic lipid compound

Abstract

The invention belongs to the field of chemistry, and particularly relates to a preparation method of a cationic lipid compound, which comprises the following steps: S1, reacting N, N-bis (3-aminopropyl) methylamine with 1, 2-epoxydecane to obtain an epoxy ring-opening product A1-EP10-O18A-1; s2, in the presence of an acid-binding agent, taking linolenol as an initial raw material, and reacting with acryloyl chloride to obtain linolenic acid ester Lin-AA; s3, the epoxy ring-opening product A1-EP10-O18A-1 and linolenic acid ester are subjected to an addition reaction, and a target product is obtained. The cationic liposome prepared by the method is suitable for being used as a medicinal liposome auxiliary material. Meanwhile, a better choice is provided for the development of an LNP delivery system, and the method has important significance for promoting the industrial development of vaccines. The synthesis process of the cationic liposome, provided by the invention, has the advantages of easily available route raw materials, high reaction selectivity and easiness in product separation, and is beneficial to industrial production.

Description

Technical Field

[0001] The invention belongs to the field of chemistry, and in particular relates to a method for preparing a cationic lipid compound. Background Art

[0002] Lipid nanoparticles have attracted much attention in the pharmaceutical industry as very promising carriers of various therapeutic agents. Liposomes are the earliest type of LNPs that can transport hydrophobic or hydrophilic molecules, including small molecules, proteins and nucleic acids. They are a very versatile nanocarrier platform. In fact, liposomes are also the earliest type of nanodrug delivery platform that has successfully transformed from concept to clinical application. Many liposomal drug preparations have been approved for marketing and successfully used in medical practice. The new generation of LNPs, including solid lipid nanoparticles, nanostructured lipid carriers and cationic lipid-nucleic acid complexes, show more complex internal structures and better physical stability. LNPs can control the targeted location and time of drug delivery in the body and can provide treatment options for a variety of diseases.

[0003] Nucleic acids have a variety of roles in medicine, including gene therapy agents and RNA therapeutics. However, nucleic acids are negatively charged and hydrophilic, which hinders their passive diffusion across the plasma membrane. Therefore, nucleic acids require delivery vehicles to protect them from degradation and deliver them to target cells for effective uptake. Cationic LNPs, which contain a stable complex of synthetic cationic lipids and anionic nucleic acids, are the most widely used non-viral delivery system for nucleic acid drugs.

[0004] The advantage of cationic liposomes as gene carriers is that they can form stable complexes that protect nucleic acids from degradation by nucleases and improve transfection efficiency. Once these complexes fuse with the cell membrane, they release the encapsulated nucleic acids into the cytoplasm, which then enter the nucleus through the endocytic mechanism to achieve gene expression. Cationic liposomes have achieved remarkable results in clinical trials. For example, tumor suppressors for cancer treatment, gene replacement therapy for genetic diseases, and gene immunotherapy for vaccine development. With the continuous development of technology, cationic liposomes, as a safe and effective gene delivery system, are expected to play a greater role in future gene therapy. Summary of the invention

[0005] The purpose of the present invention is to provide a process for preparing cationic liposomes with easily available raw materials, high reaction selectivity and easy product separation. The process can produce high-quality cationic liposomes, which is conducive to industrial production.

[0006] The first aspect of the present invention discloses a process for preparing cationic liposomes, comprising the following steps: S1. The epoxy ring-opening product A1-EP10-O18A-1 is obtained by reacting N,N-bis(3-aminopropyl)methylamine with 1,2-epoxydecane; the reaction formula is: ; S2. In the presence of an acid-binding agent, linolenic acid alcohol is used as a starting material and reacted with acryloyl chloride to obtain linolenic acid ester Lin-AA; the reaction formula is: ; S3. The epoxy ring-opening product A1-EP10-O18A-1 is then added with linolenic acrylate to obtain the target product; the reaction formula is: .

[0007] Preferably, in step S1, the feed ratio of N,N-bis(3-aminopropyl)methylamine to 1,2-epoxydecane should be 5:1-10:1, with the best yield and less by-products (multiple substitution products). Considering the cost and post-processing, the equivalent ratio is preferably 5:1.

[0008] Preferably, in step S1, the reaction temperature should be between 60 and 90° C., preferably 80° C.; the reaction time is 5 to 24 hours, preferably 16 hours.

[0009] Preferably, in step S1, the reaction is carried out in an organic protic solvent, selected as anhydrous ethanol; the mass volume ratio of the substrate to the solvent is 1:1-100 (mg / ml), preferably 1:2 (mg / ml).

[0010] Preferably, in step S1, the reaction progress is preferably tracked and controlled by gas chromatography or liquid chromatography, and the specific reaction temperature and reaction time are determined according to the detection results.

[0011] Preferably, in step S1, the post-treatment operation is: salt washing and extraction, drying and filtration, concentration, and drying to obtain the intermediate A1-EP10-O18A-1.

[0012] Preferably, in step S2, the molar ratio of linolenic alcohol to acryloyl chloride should be between 1:1.05 and 1.5, preferably an equivalent ratio of 1:1.1.

[0013] Preferably, in step S2, the reaction temperature should be between -5 and 10°C, preferably 0 to 5°C; the reaction time is 1 to 5 hours, preferably 2 hours.

[0014] Preferably, in step S2, the reaction occurs under the action of an acid binding agent, and the acid binding agent is selected from an organic base or an inorganic base. Preferably, the organic base is selected from but not limited to one or more of triethylamine, diethylisopropylamine, pyridine, DMAP, and DBU, and the inorganic base is selected from one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate, preferably triethylamine.

[0015] Preferably, in step S2, the equivalent of the acid binding agent should be between 1.05 and 2, preferably 1.1 equivalents.

[0016] Preferably, in step S2, the reaction is carried out in an organic inert aprotic solvent, and the solvent is preferably selected from one or more of dry dichloromethane, tetrahydrofuran, DMF, DMSO, acetone, and dioxane, preferably anhydrous dichloromethane; the mass volume ratio of the substrate to the solvent is 1:1~50 (mg / ml), preferably 1:1 (mg / ml).

[0017] Preferably, in step S2, the post-treatment operation is: acid washing and extraction, filtration, drying and concentration, column chromatography purification, and drying to obtain the intermediate Lin-AA.

[0018] Preferably, in step S3, the molar ratio of A1-EP10-O18A-1 to Lin-AA should be between 1:3.3 and 3.9, preferably 1:3.5.

[0019] Preferably, in step S3, the reaction is carried out without solvent or in an organic solvent, and the organic solvent is selected from one or more of dry acetonitrile, ethanol, isopropanol, tetrahydrofuran, toluene, and dioxane, preferably anhydrous ethanol; the mass volume ratio of A1-EP10-O18A-1 to the solvent is 1:0~20 (mg / ml), preferably 1:1 (mg / ml).

[0020] Preferably, in step S3, the reaction temperature should be between 60 and 90° C., preferably 80° C.; and the reaction time should be between 16 and 48 hours, preferably 16 hours.

[0021] Preferably, in step S3, 0.05 equivalent of BHT should be added as a reaction stabilizer (the reaction stabilizer can prevent polymerization from occurring during the reaction).

[0022] Preferably, in step S3, a suitable amount of catalyst may be added to catalyze the reaction, and the catalyst equivalent should be between 0 and 0.05, preferably 0 equivalent; the catalyst is preferably acetic acid or boric acid.

[0023] Preferably, the step S3 further comprises a step of refining the crude product by column chromatography separation and refining technology. In the column chromatography separation and refining process, the eluent used is preferably a mixture of dichloromethane-methanol, and the volume ratio is preferably 100:1 to 20:1. The product is collected by TLC monitoring (using DCM-MeOH (10:1) as a developing agent and phosphomolybdic acid as a color developer), and the collected product eluents are combined.

[0024] Preferably, in step S3, the post-treatment operation is: concentration, column chromatography purification, concentration through a membrane, and drying to obtain the target product.

[0025] The second aspect of the present invention discloses cationic liposomes produced by the above process.

[0026] Compared with the prior art, the present invention has the following beneficial effects: the present invention develops a new process for preparing cationic liposomes, and the prepared cationic liposomes are suitable as pharmaceutical liposome excipients. At the same time, it provides a better choice for the development of LNP delivery systems, which is of great significance for promoting the industrial development of vaccines.

[0027] The present invention also provides a synthesis process for the cationic liposome, which has easy-to-obtain raw materials, high reaction selectivity, and easy product separation, and is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The schematic diagram is a reaction route for the synthesis of the cationic lipid compound of the present invention.

[0029] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the epoxy ring-opening product A1-EP10-O18A-1 in Example 2.

[0030] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of linolenic acrylate Lin-AA in Example 2.

[0031] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the cationic lipid synthesized product in Example 2.

[0032] Figure 5 This is the carbon NMR spectrum of the cationic lipid synthesized product in Example 2.

[0033] Figure 6 This is the Fourier transform infrared absorption spectrum of the cationic lipid product synthesized in Example 2.

[0034] Figure 7 This is the mass spectrum of the cationic lipids synthesized in Example 2. DETAILED DESCRIPTION

[0035] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0036] Example 1 Process Optimization of Intermediate A1-EP10-O18A-1 (1) Effect of solvent selection on the yield of A1-EP10-O18A-1 When the solvent for the epoxy ring-opening reaction is ethanol, the reaction yield is the best, and other aprotic solvents are basically unresponsive.

[0037] Table 1 Effect of solvent on reaction N,N-Bis(3-aminopropyl)methylamine (eq) 1,2-Epoxydecane (eq) Solvents Crude yield (%) 5 1 Ethanol 96.3 5 1 Tetrahydrofuran TLC showed no reaction 5 1 Ethyl acetate TLC showed no reaction 5 1 Acetonitrile TLC showed no reaction (2) Effect of the equivalent ratio of N,N-bis(3-aminopropyl)methylamine to 1,2-epoxydecane on the reaction When the feed ratio of N,N-bis(3-aminopropyl)methylamine to 1,2-epoxydecane is 5:1~10:1, the yield is the best and there are fewer by-products (multiple substitution products). Considering the cost and post-processing, we currently adopt an equivalent ratio of 5:1 for later scale-up.

[0038] Table 2 Effect of the equivalent ratio of N,N-bis(3-aminopropyl)methylamine to 1,2-epoxydecane on the reaction N,N-Bis(3-aminopropyl)methylamine (eq) 1,2-Epoxydecane (eq) TLC Results Crude yield (%) 1 1 Large proportion of di-substitution 41 3 1 Less disubstituted 52 5 1 Less disubstituted 96.3 10 1 Minimum di-substitution 95.2 (3) Effect of reaction solvent on the yield of A1-EP10-O18A Through the screening of reaction solvents, when the reaction solvent is ethanol, the yield of A1-EP10-O18A is the highest. Most of the products of other solvents only stay in the double addition product and the single addition product, resulting in a lower final yield.

[0039] Table 3 Effects of different solvents on the reaction A1-EP10-018A-1 (eq) Linolenic acrylate (eq) Solvents Crude yield (%) 1 3.5 Ethanol 48% 1 3.5 Isopropyl alcohol 30% 1 3.5 Tetrahydrofuran TLC shows -10% 1 3.5 Acetonitrile 13% 1 3.5 Toluene 42% (4) Effect of reaction concentration on the yield of A1-EP10-O18A By screening the reaction concentration, when the reaction concentration of ethanol: A1-EP10-O18A-1 is 1:1, the yield of A1-EP10-O18A is the highest. The neat reaction will produce many small impurities that are difficult to separate, resulting in a low yield; and as the proportion of ethanol solvent increases, alcoholysis becomes more obvious, resulting in a decrease in yield.

[0040] Table 4 Effect of reaction concentration on the yield of A1-EP10-O18A A1-EP10-O18A-1 (eq) Linolenic acrylate (eq) Ethanol: A1-EP10-O18A-1 (mL:mg) Yield (%) 1 3.5 neat 52 1 3.5 1:1 61 1 3.5 5:1 47.8 1 3.5 10:1 54 1 3.5 20:1 33.4 (5) Effect of reaction time on the yield of A1-EP10-O18A Through the screening of reaction time, when the reaction time is 16h, the yield of A1-EP10-O18A is the highest. If the reaction time is too short, the addition is not complete, resulting in low yield; if the reaction time is too long, the alcoholysis byproduct increases.

[0041] Table 5 Effect of reaction time on the yield of A1-EP10-O18A A1-EP10-O18A-1 (eq) Lin-AA (eq) Ethanol: A1-EP10-O18A-1 (mL:mg) Reaction time Yield (%) 1 3.5 1:1 6 48.3 1 3.5 1:1 10 56.5 1 3.5 1:1 16 66.7 1 3.5 1:1 24 57.6 (6) Effect of additives on the yield of A1-EP10-O18A Through screening of additives, it was found that acetic acid had the best reaction effect among the additives currently added, but the yield was not significantly improved compared to that without additives. Therefore, the reaction without additives will be used in the subsequent scale-up.

[0042] Table 6 Effect of additives on the yield of A1-EP10-018A A1-EP10-O18A-1 (eq) Lin-AA (eq) Ethanol: A1-EP10-O18A-1 (mL:mg) additive Yield (%) 1 3.5 neat 0.05eqAcOH 57.2 1 3.5 1:1 0.05eq AcOH 60.4 1 3.5 1:1 1eq TEA 57.7 1 3.5 1:1 <![CDATA[1eq K2CO3]]> 37 In summary, through small-scale test, pilot test and scale-up verification of process conditions, the production process conditions of A1-EP10-O18A are confirmed as follows: S1: Intermediate Lin-AA: Linolenic alcohol (1.0 eq), acryloyl chloride (1.1 eq) and triethylamine (1.1 eq) were reacted in DCM at 0±5°C for 2-4 h, extracted with acid, filtered, dried and concentrated, purified by column chromatography, and dried to obtain intermediate Lin-AA.

[0043] S2: Intermediate A1-EP10-O18A-1: 1,2-Epoxydecane (1.0 eq) and N,N-bis(3-aminopropyl)methylamine (5.0 eq) were reacted in ethanol at 80±5°C for 16 to 24 h, extracted with salt, dried and filtered, concentrated, and dried to obtain intermediate A1-EP10-O18A-1.

[0044] S3: Product A1-EP10-O18A: A1-EP10-O18A-1 (1.0 eq), linolenic acrylate (3.5 eq), reacted in ethanol at 80±5℃ for 16 ~ 24h, concentrated, purified by column chromatography, concentrated and filtered, and dried to obtain A1-EP10-O18A.

[0045] Example 2 (1) Epoxy ring-opening reaction The reactor was protected by nitrogen, 968 liters of anhydrous ethanol was added, stirring was started, the stirring speed was set to 20 ± 5 rpm, the explosion-proof high and low temperature integrated machine was turned on, the temperature was set to 25 ± 5 ℃, and 484 g (3.1 mol, 1.0 eq.) of 1,2-epoxydecane and 2252 g (15.49 mol, 5.0 eq.) of N,N-bis(3-aminopropyl)methylamine were added in sequence under slow stirring. The stirring speed was set to 100 ± 5 rpm, the temperature of the explosion-proof high and low temperature integrated machine was set to 80 ℃, and the reaction was stirred for 16 hours. About 5 mL of the reaction solution was taken with a sampler, diluted with 10 mL of dichloromethane, and monitored by TLC, with PE-EA (20:1) as the developing agent and phosphomolybdic acid as the color developer. If 1,2-epoxydecane was consumed, continue to the next step; otherwise, the reaction time was extended for 2 hours before sampling and detection until 1,2-epoxydecane was consumed. The reaction temperature was cooled to 25 ± 5 °C, and dichloromethane was added. A 10% sodium chloride solution was added (preparation method: sodium chloride was added to purified water and stirred to dissolve), and the mixture was stirred for 10 ± 5 min at a stirring speed of 100 ± 5 rpm. Isopropanol was added and the mixture was allowed to stand for 15 ± 5 min until layers were separated. The dichloromethane phase was collected in a clean stainless steel barrel, and the aqueous phase was discarded only after it was confirmed that there was no abnormal leakage (using DCM-MeOH (5:1) as the developing agent and phosphomolybdic acid as the color developer, and there should be basically no product spots on the TLC spot plate). The dichloromethane phase was transferred back to the reactor, and the prepared 10% sodium chloride solution (preparation method: add sodium chloride to purified water and stir to dissolve), stirred for 10 ± 5min, stirring speed 100 ± 5rpm, add isopropanol, and stand for 15 ± 5min until stratification; the dichloromethane phase was collected in a clean stainless steel barrel, and the aqueous phase was discarded after confirming that there was no abnormal material leakage (using DCM-MeOH (5:1) as the developing agent and phosphomolybdic acid as the color developer, and the TLC spot plate basically had no product spots.) The dichloromethane phase was transferred back to the reactor, and the prepared 10% sodium chloride solution (preparation method: add sodium chloride to purified water and stir to dissolve), stirred for 10 ± 5min, stirring speed 100 ± 5 rpm, add isopropanol, and stand for 15 ± 5min until stratification; the dichloromethane phase was collected in a clean stainless steel barrel, and the aqueous phase was discarded after confirming that there was no abnormal material leakage (using DCM-MeOH (5:1 ) as the developing solvent, phosphomolybdic acid as the color developer, and the TLC spot plate is basically free of product spots.). The dichloromethane phase is monitored by TLC (using DCM-MeOH (5:1) as the developing solvent and phosphomolybdic acid as the color developer). Whether N,N-bis(3-aminopropyl)methylamine is washed away. Anhydrous sodium sulfate is added to the dichloromethane phase and stirred for 15 ± 5 min at a stirring speed of 100 ± 5 rpm. The filter is filtered through a sand core filter, the filter cake is washed with dichloromethane, and the washing liquid and the filtrate are combined and concentrated.The dichloromethane phase was added to the concentration equipment, the water bath temperature was set to 40 ± 5 ℃, the explosion-proof low-temperature coolant circulation pump was set to -10 ± 5 ℃, and the explosion-proof low-temperature coolant circulation pump was set to -10 ± 5 ℃. The condensate was reduced (vacuum degree <-0.06 MPa) until almost no condensate dripped. After the concentration was completed, the condensate was discharged, the oil pump was changed, and the drying was continued at 25 ± 5 ℃ for more than 4 h (vacuum degree <-0.08 MPa) to obtain 915 grams of A1-EP10-O18A-1 crude light yellow oily product. The yield was 97.98% calculated based on 1,2-epoxydecane. The purity and solvent residue of the sample were tested, and the HPLC-CAD purity was ≥ 70.0%, and the solvent residue was ≤ 5000 ppm for dichloromethane, ≤ 5000 ppm for ethanol, and ≤ 5000 ppm for isopropanol. After passing the test, the product was packaged as required, filled with nitrogen, and stored at -20 ± 5 °C for the next step of feeding reaction.

[0046] The synthesized product was subjected to NMR analysis, and its characterization data are as follows: 1H NMR (400 MHz, Chloroform-d) δ 3.60 (dddd, J = 9.8, 7.2, 4.4, 2.9Hz, 1H), 2.79 – 2.57(m, 5H), 2.47 – 2.34 (m, 5H), 2.20 (s, 3H), 1.64 (dp, J =18.7, 6.9 Hz, 4H), 1.49 – 1.18 (m, 14H), 0.93 – 0.81 (m, 3H).

[0047] (2) Acylation reaction The reactor was protected by nitrogen, 3900 liters of dichloromethane were added, stirring was started, the stirring speed was set to 20 ± 5 rpm, the explosion-proof high and low temperature integrated machine was turned on, the temperature was set to 25 ± 5 ℃, 3900 g of linolenic alcohol (14.64 mol, 1.0 eq.) and 1630 g of triethylamine (16.10 mol, 1.1 eq.) were added in sequence under slow stirring, and the inner wall of the reactor and the stirring paddle were rinsed with dichloromethane. The stirring speed was set to 100 ± 5 rpm, the temperature of the explosion-proof high and low temperature integrated machine was set to 0 ± 5 ℃, and then 1460 g of acryloyl chloride (16.10 mol, 1.1 eq.) was prepared into a solution with dichloromethane. When the temperature dropped below 5 ℃, it was added dropwise to the reactor through a dropping funnel. The dropping time was 2 ± 0.5 h, and the reaction was stirred for 2 ~4 h. Take about 5 mL of the reaction solution with a sampler and monitor it by TLC (with PE-EA (10:1) as the developing agent and phosphomolybdic acid as the color developer). If the linolenic alcohol is completely consumed, proceed to the next step; otherwise, extend the reaction time for 1 h before sampling and testing until the linolenic alcohol is completely consumed. Slowly add the prepared 0.2 M hydrochloric acid / 10% sodium chloride solution to the reactor (preparation method: add 12 N hydrochloric acid and sodium chloride to purified water, stir to dissolve), stir for 10 ± 5 min, stirring speed 100 ± 5 rpm, stand for 15 ± 5 min until stratification, emulsification will occur during this process, add isopropanol to break the emulsion; collect the dichloromethane phase into a clean stainless steel barrel, and discard the aqueous phase after confirming that there is no abnormal material leakage (with PE-EA (10:1) as the developing agent and phosphomolybdic acid as the color developer, the TLC spot plate basically has no product spots). The dichloromethane phase was transferred back to the reactor, and the prepared 0.2 M hydrochloric acid / 10% sodium chloride solution (preparation method: add 12 N hydrochloric acid and sodium chloride to purified water, stir to dissolve), stirred for 10 ± 5 min, stirring speed 100 ± 5 rpm, and allowed to stand for 15 ± 5 min until stratification. Emulsification will occur during this process, and isopropanol was added to break the emulsion; the dichloromethane phase was collected in a clean stainless steel barrel, and the aqueous phase was discarded after confirming that there was no abnormal material leakage (using PE-EA (10:1) as the developing agent and phosphomolybdic acid as the color developer, and the TLC spot plate basically had no product spots). The dichloromethane phase was monitored by TLC (using PE-EA (10:1) as the developing agent and phosphomolybdic acid as the color developer) to see if triethylamine was completely removed. Add anhydrous magnesium sulfate to the dichloromethane phase, stir for 15 ± 5 min, stir at 100 ± 5 rpm, filter with a sand core filter, wash the filter cake with dichloromethane, and combine the washing liquid and the filtrate for concentration. Add the dichloromethane phase to the concentration equipment, set the heating temperature to 40 ± 5 ℃, set the temperature of the explosion-proof low-temperature coolant circulation pump to -10 ± 5 ℃, and reduce the steam (vacuum degree <-0.06 MPa) until almost no condensate drips.Add n-hexane and silica gel to the glass column, homogenize and press out the excess n-hexane in the glass column with an air pump, add the crude product obtained by concentration, add anhydrous sodium sulfate after loading. Silica gel column chromatography (with HEX-EA (100:1 ~ 30:1) as eluent), collect the product by TLC monitoring (with PE-EA (10:1) as the developing solvent and phosphomolybdic acid as the color developer), and combine the collected product eluents. The heating temperature is set to 40 ± 5 ℃, and the explosion-proof low-temperature coolant circulation pump is set to -10 ± 5 ℃. The collected product eluent is concentrated (vacuum degree <-0.06 MPa) until almost no condensate drips. After the concentration is completed, the condensate is discharged, the oil pump is replaced, and the drying is continued at 25 ± 5 ℃ for more than 4 hours (vacuum degree <-0.08 MPa) to obtain 3050 grams of refined colorless oily product of Lin-AA. The yield calculated based on linolenic alcohol is 65.02%. The purity and solvent residue of the sample were tested. The HPLC-CAD purity was ≥ 90.0%. The solvent residue met the requirements of dichloromethane ≤ 5000 ppm, ethyl acetate ≤ 5000 ppm, isopropanol ≤ 5000 ppm, and n-hexane ≤ 5000 ppm. After passing the test, the product was packaged as required, filled with nitrogen, and stored at -20 ± 5 ℃ for the next step of feeding reaction.

[0048] The synthesized product was subjected to NMR analysis, and its characterization data are as follows: 1H NMR (400 MHz, Chloroform-d) δ 6.40 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.4, 10.4 Hz, 1H), 5.81 (dd, J =10.4, 1.5 Hz, 1H), 5.49 – 5.26(m, 4H), 4.15 (t, J = 6.7 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H), 2.05 (q, J = 6.8Hz, 4H), 1.72 – 1.62 (m, 2H), 1.41 – 1.24 (m, 17H), 0.89 (t, J = 6.8 Hz, 3H.

[0049] (3) Addition reaction The reactor was protected by nitrogen, and 806 liters of anhydrous ethanol were added, followed by 806 grams of A1-EP10-O18A-1 (2.67 mol, 1.0 eq.) and 3000 grams of Lin-AA (9.36 mol, 3.5 eq.). Stirring was started, and the stirring speed was set to 100±5 rpm. The explosion-proof high and low temperature integrated machine was turned on, and the temperature was set to 80±5℃. The reaction was stirred for 16 to 24 hours. About 5 mL of the reaction solution was taken with a sampler, diluted with 10 mL of dichloromethane, and monitored by TLC (using DCM-MeOH (10:1) as the developing agent and phosphomolybdic acid as the color developing agent). If A1-EP10-O18A-1 was consumed, the next step was continued; otherwise, the reaction time was extended for 2 hours before sampling and detection until A1-EP10-O18A-1 was consumed. The reaction solution was cooled to 25 ± 5 ℃, the water bath temperature was set to 45 ± 5 ℃, the explosion-proof low-temperature coolant circulation pump was set to -10 ± 5 ℃, and the evaporation was reduced (vacuum degree <-0.06 MPa) until almost no condensate dripped. Dichloromethane and silica gel were added to the glass column, and the excess dichloromethane in the glass column was pressed out with an air pump after homogenization, and the crude product obtained by concentration was added. After loading, anhydrous sodium sulfate was added to level it. Purification was performed on a silica gel column (using DCM-MeOH (100:1 ~ 20:1) as the eluent), and the product was collected by TLC monitoring (using DCM-MeOH (10:1) as the developing solvent and phosphomolybdic acid as the color developer), and the collected product eluents were combined. The water bath temperature was set at 40 ± 5 ℃, and the explosion-proof low-temperature coolant circulation pump was set at -10 ± 5 ℃. The collected product eluate was concentrated (vacuum degree <-0.06 MPa) until almost no condensate dripped. After the concentration was completed, dichloromethane was added to dissolve, and the microorganisms were removed by passing through a microporous filter membrane (0.22μm). The filtrate was concentrated at 25 ± 5 ℃ (vacuum degree <-0.06 MPa) until almost no condensate dripped, and n-heptane was added to continue concentrating (vacuum degree <-0.06 MPa) until almost no condensate dripped. After the concentration was completed, the condensate was discharged, the oil pump was changed, and the product was dried at 25 ± 5 ℃ for more than 4 h (vacuum degree <-0.08 MPa) to obtain 962 grams of refined light yellow oily product. The yield calculated based on A1-EP10-O18A-1 was 28.49%. Sampling was conducted for purity and solvent residue testing. The HPLC-CAD purity was ≥ 90.0%. The solvent residue met the requirements of n-hexane ≤ 290 ppm, ethyl acetate ≤ 5000 ppm, ethanol ≤ 5000 ppm, dichloromethane ≤ 600 ppm, n-heptane ≤ 5000 ppm, methanol ≤ 3000 ppm, and isopropanol ≤ 5000 ppm. After passing the test, the product was packaged as required.

[0050] This reaction is a convergent reaction route. If the route with the lowest yield is selected, the total yield is: 65.02%*28.49% = 18.53% If the route with the highest yield is selected, the total yield is: 97.98%*28.49% = 27.91% The synthesized product was subjected to hydrogen spectrum analysis, and its characterization data are as follows: From the 1H-NMR spectrum, we can see that the multiple peaks at the chemical shift δ of 0.93-0.84 (m, 12H) are the H peaks on -CH3(a), and the multiple peaks at the chemical shift δ of 1.38-1.23 (m, 60H) are the H peaks on -CH 2- The H peaks on CH2-CH3, -CH-CH2CH2- and -CH2-CH2CH2-(b), the multiplet with chemical shift δ at 1.51-1.40 (m, 2H) is the H peak of -CH(OH)-CH2-(p), the multiplet with chemical shift δ at 1.67-1.55 (m, 12H) is the H peak of -COOCH2-CH2CH2-(f) and -N-CH2-CH2-CH2-N-(k), the quartet with chemical shift δ at 2.05 (q, J = 6.8 Hz, 12H) is the H peak of -CH2-CH2CH=CH-(c), the multiplet with chemical shift δ at 2.16 (s, 3H) is the H peak of N-CH3(m), the multiplet with chemical shift δ at 2.94-2.21 (m, The multiple peaks of chemical shift δ at 3.58 (m, 1H) are H peaks on -N-CH2-CH(OH)-CH2-(o), the multiple peaks of chemical shift δ at 4.10-4.01 (m, 6H) are H peaks on -COOCH2-(g), the multiple peaks of chemical shift δ at 5.43-5.28 (m, 12H) are H peaks on -CH=CH-(e), and the spectrum is consistent with the product structure. The results are as follows Figure 2-4 shown.

[0051] The synthesized product was subjected to carbon spectrum analysis, and its characterization data are as follows: 13C NMR (101 MHz, CDCl3) δ 172.72, 172.67, 130.18, 130.04, 128.00,127.89, 77.32, 77.00, 76.68, 67.83, 64.70, 64.53, 60.95, 55.65, 55.36, 51.86,51.82,49.73, 49.20, 41.93, 34.74, 32.61, 32.58, 31.88, 31.50, 29.88, 29.63,29.58, 29.43, 29.33, 29.28, 29.24, 28.63, 28.60, 27.20, 27.18, 25.91, 25.81,25.61,24.80, 22.66, 22.55, 14.09, 14.05. The results are as follows Figure 5 shown.

[0052] The synthesized product was subjected to FTIR analysis, and its characterization data are as follows: The infrared spectrum showed that at 3458 cm -1 The peak at 3010 cm is the -OH stretching vibration peak. -1 The peak at 2929 cm is the stretching vibration peak of olefin -C=CH- -1 and 2856 cm -1 The peak at 1738 cm is the symmetric stretching vibration peak of -CH2 and -CH3. -1 The peak at 1462 cm is the stretching vibration peak of the ester carbonyl -OC=O. -1 The peak at 1395 m is the -CH2- bending vibration peak. -1 、1378 cm -1 、1349 cm -1 The peak at 1250 cm is the in-plane bending vibration peak of -CH3. -1 The peak at 1177 cm is the CN stretching vibration peak. -1 The peak at 1051 cm is the ester-OC=O stretching vibration absorption peak. -1 The peak at 723 cm is the -CO stretching vibration peak. -1 The peak at is the absorption peak of CH out-of-plane bending vibration. The spectrum is consistent with the structure. Figure 6 shown.

[0053] The synthesized product was subjected to mass spectrometry analysis, and its characterization data are as follows: MS (m / z): [M+H]+= 1262.83. Figure 7 shown.

[0054] In summary, the present invention provides a synthesis process of a cationic lipid compound, which has a short overall synthesis route, low industrial production cost, high yield, and the product has good safety and is suitable as a pharmaceutical liposome excipient.

[0055] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A process for preparing a cationic lipid compound, characterized in that: The following steps are involved: S1. Reaction of N,N-bis(3-aminopropyl)methylamine with 1,2-epoxydecane to obtain epoxy ring-opening product A1-EP10-O18A-1; the reaction formula is: ； S2. In the presence of an acid-binding agent, linolenic acid alcohol is used as a starting material and reacted with acryloyl chloride to obtain linolenic acid ester Lin-AA; the reaction formula is: ； S3, adding the epoxy ring-opening product A1-EP10-O18A-1 to linolenic acrylate Lin-AA to obtain the target product; the reaction formula is: 。 2. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S1, the ratio of N,N-bis(3-aminopropyl)methylamine to 1,2-epoxydecane is 5:1 to 10:

1.

3. The process for preparing the cationic lipid compound according to claim 2, characterized in that: In step S1, the ratio of N,N-bis(3-aminopropyl)methylamine to 1,2-epoxydecane is 5:

1.

4. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S1, the reaction temperature is between 60 and 90° C., and the reaction time is between 5 and 24 hours.

5. The process for preparing the cationic lipid compound according to claim 4, characterized in that: In step S1, the reaction temperature is 80° C. and the reaction time is 16 h.

6. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S1, the reaction is carried out in an organic protic solvent, and the organic protic solvent includes anhydrous ethanol.

7. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S1, the mass volume ratio of the substrate to the solvent is 1:1-100.

8. The process for preparing the cationic lipid compound according to claim 7, characterized in that: In step S1, the mass volume ratio of the substrate to the solvent is 1:

2.

9. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S1, the reaction progress is tracked and controlled by gas chromatography or liquid chromatography.

10. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S1, after the reaction is completed, the product is subjected to salt washing extraction, drying filtration, concentration and drying to obtain the epoxy ring-opening product A1-EP10-O18A-1.

11. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S2, the molar ratio of linolenic alcohol to acryloyl chloride is 1:1.05-1.

5.

12. The process for preparing the cationic lipid compound according to claim 11, characterized in that: In step S2, the molar ratio of linolenic alcohol to acryloyl chloride is 1:1.

1.

13. The process for preparing the cationic lipid compound according to claim 1, characterized in that: In step S2, the reaction temperature is -5~10°C and the reaction time is 1~5h.

14. The process for preparing a cationic lipid compound according to claim 13, characterized in that: In step S2, the reaction temperature is 0-5°C and the reaction time is 2h.

15. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S2, the acid binding agent is selected from an organic base or an inorganic base.

16. The process for preparing a cationic lipid compound according to claim 15, characterized in that: In step S2, the organic base includes one or more of triethylamine, diethylisopropylamine, pyridine, DMAP, and DBU, and the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate.

17. The process for preparing a cationic lipid compound according to claim 15, characterized in that: In step S2, the acid binding agent is preferably triethylamine.

18. The process for preparing a cationic lipid compound according to any one of claims 15 to 17, characterized in that: In step S2, the acid binding agent equivalent is between 1.05 and 2.

19. The process for preparing a cationic lipid compound according to claim 18, characterized in that: The acid binding agent is 1.1 equivalents.

20. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S2, the reaction is carried out in an organic inert aprotic solvent; The organic inert aprotic solvent is selected from one or more of dry dichloromethane, tetrahydrofuran, DMF, DMSO, acetone, and dioxane.

21. The process for preparing a cationic lipid compound according to claim 20, characterized in that: The organic inert aprotic solvent is selected as anhydrous dichloromethane.

22. The process for preparing a cationic lipid compound according to claim 20, characterized in that: The mass volume ratio of the substrate to the organic inert aprotic solvent is 1:1-50.

23. The process for preparing a cationic lipid compound according to claim 22, characterized in that: The mass volume ratio of the substrate to the organic inert aprotic solvent is 1:

1.

24. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S2, after the reaction is completed, the product is acid-washed, extracted, filtered, dried, concentrated, purified by column chromatography and dried to obtain linolenic acrylate Lin-AA.

25. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S3, the molar ratio of A1-EP10-O18A-1 to Lin-AA is between 1:3.3 and 3.

9.

26. The process for preparing a cationic lipid compound according to claim 25, characterized in that: In step S3, the molar ratio of A1-EP10-O18A-1 to Lin-AA is 1:3.

5.

27. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S3, the reaction is carried out in a solvent-free reaction or in an organic solvent.

28. The process for preparing a cationic lipid compound according to claim 27, characterized in that: The organic solvent is selected from one or more of dry acetonitrile, ethanol, isopropanol, tetrahydrofuran, toluene, and dioxane.

29. The process for preparing a cationic lipid compound according to claim 27, characterized in that: The organic solvent is anhydrous ethanol.

30. The process for preparing a cationic lipid compound according to any one of claims 27 to 29, characterized in that: The mass volume ratio of A1-EP10-O18A-1 to the organic solvent is 1:0~20.

31. The process for preparing the cationic lipid compound according to claim 30, characterized in that: The mass volume ratio of A1-EP10-O18A-1 to the organic solvent is 1:

1.

32. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S3, the reaction temperature is between 60 and 90° C., and the reaction time is between 16 and 48 hours.

33. The process for preparing the cationic lipid compound according to claim 32, characterized in that: In step S3, the reaction temperature is selected as 80° C. and the reaction time is selected as 16 h.

34. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S3, 0.05 equivalent of BHT is added as a reaction stabilizer.

35. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S3, a catalyst is added to catalyze the reaction, and the catalyst equivalent is between 0 and 0.05; the catalyst is selected from acetic acid or boric acid.

36. The process for preparing a cationic lipid compound according to claim 1, characterized in that: Step S3 includes the step of refining the crude product by column chromatography separation and refining technology; in the column chromatography separation and refining process, the eluent used is preferably a mixture of dichloromethane and methanol, and the volume ratio is preferably 100:1~20:1; the product is collected by TLC monitoring, DCM-MeOH (10:1) is used as the developing agent, and phosphomolybdic acid is used as the color developer in the TLC monitoring; and the collected product eluents are combined.

37. The process for preparing a cationic lipid compound according to claim 1, characterized in that: In step S3, after the reaction is completed, the product is concentrated, purified by column chromatography, concentrated through a membrane and dried to obtain the target product.

38. A cationic lipid compound prepared by the preparation process according to any one of claims 1-37.

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