Preparation and purification method of 1, 2-dioleoyloxy-3-(dimethylamino) propane

Abstract

The invention discloses a preparation method and a purification method of 1, 2-dioleoyloxy-3-(dimethylamino) propane. Specifically disclosed is a preparation method of a compound 3, which comprises the following step: in an organic solvent, under the action of EDCI and alkali, a compound 1 and a compound 2 are subjected to a condensation reaction as shown in the specification to prepare the compound 3. The preparation method and the purification method are simple and convenient to operate, high in yield and suitable for industrial production. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a preparation and purification method of 1,2-dioleoyloxy-3-(dimethylamino)propane. Background Art

[0002] Cationic lipids are one of the earliest and most widely used non-viral gene vectors. Cationic lipids can interact with DNA or RNA to form stable lipid complexes, effectively avoiding nuclease degradation and increasing blood circulation time. In 1987, Felgner et al.

[36] synthesized the cationic lipid DOTMA containing a glycerol backbone structure. Two years later, DOTMA-mediated gene transfection was first achieved in mice. Since then, many cationic lipids have been synthesized and carried nucleic acids into the cell nucleus, showing high transfection efficiency both in vivo and in vitro. After more than 40 years of development, many highly efficient cationic lipids have become commercial transfection reagents, some of which have been used in clinical trials for gene therapy of cancer and other familial genetic diseases. In order to improve the transfection efficiency of cationic lipids, researchers have continuously chemically modified the structure of cationic lipids, including peptide-modified lipids, polymer-modified lipids, dendritic-modified lipids and functional molecule-modified lipids. After structural modification, the delivery efficiency of lipids to nucleic acids is further improved, the immune response is reduced, and the targeting of cells and tissues is increased.

[0003] At present, the cationic lipid DOTAP has the disadvantages of high toxicity and low encapsulation efficiency. Therefore, DODAP is now being developed as an alternative, and it has already entered Phase III clinical trials. At the same time, DODAP can also be further made into derivative compounds, and there are currently multiple derivatives in clinical or cell studies. Some of these compounds have been clinically studied, and some are in cell studies. The next work can start from the structure-activity relationship of cationic liposomes to design derivatives of DODAP, and design compounds with better toxicity and better encapsulation and transfection rates than DOTAP.

[0004]

[0005] It has been reported in the literature that 3-dimethylamino-1,2-propanediol is used as the raw material, first esterified to form a diacyl compound, and then quaternized to obtain DOTAP. Among them, there are two reaction conditions for esterification to form a diacyl compound. One is the esterification reaction with oleic acid chloride as the substrate, and the synthesis of this reaction route is relatively mature. However, there are still many problems: the yield is not high, about 70% overall, and the reagents in the preparation process of acyl chloride are either dangerous or pollute the environment and corrode the equipment. At a time when environmental protection is becoming increasingly stringent, the process production of this route is greatly limited. Therefore, this method is only used for small tests below the gram level in the laboratory.

[0006] Another condition is a condensation reaction represented by DCC / DMAP. This route uses the classic Steglich reaction with an overall yield of more than 85%. The intermediate is easy to purify by column and can be further made into derivative compounds. Therefore, this route is the most studied, with high stability and more suitable for laboratory production. After Ulrich Massing et al. crystallized DOTAP with dimethyl sulfate, the rapid preparation of DOTAP became possible. However, the disadvantage of this preparation method is that the purification of the compound (DODAP) requires repeated column purification, which is not suitable for industrial large-scale production, limiting its further application in industry. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the problem of complicated post-processing operation of DODAP in the prior art, and to provide a preparation and purification method of 1,2-dioleoyloxy-3-(dimethylamino)propane (Compound 3). The preparation method and purification method are simple to operate, have high yield, and are suitable for industrial production.

[0008] The present invention provides a method for preparing a compound 3, which comprises the following steps: in an organic solvent, under the action of EDCI and a base, subjecting a compound 1 and a compound 2 to a condensation reaction as shown below to prepare a compound 3;

[0009]

[0010] In the condensation reaction, the organic solvent may be a conventional solvent for such condensation reactions in the art, preferably a halogenated hydrocarbon solvent, such as dichloromethane.

[0011] In the condensation reaction, the amount of the organic solvent used can be the conventional amount used in the art for such condensation reactions. The mass volume ratio of the organic solvent to the compound 1 is preferably 0.03-0.05 g / mL.

[0012] In the condensation reaction, the molar ratio of the EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to the compound 1 is preferably 3.5:1.

[0013] In the condensation reaction, the base may be an organic base, preferably DMAP (4-dimethylaminopyridine).

[0014] In the condensation reaction, the molar ratio of the base to the compound 1 is preferably 1:1.

[0015] In the condensation reaction, the molar ratio of compound 2 to compound 1 is preferably 2.2:1.

[0016] The reaction temperature of the condensation reaction is preferably room temperature, such as 25°C.

[0017] The reaction time of the condensation reaction can be monitored by conventional means in the art (such as TLC, HPLC or LC-MS), and is preferably overnight.

[0018] In the condensation reaction, the reaction reagents are the organic solvent, EDCI, base, compound 1 and compound 2 as described above.

[0019] In one embodiment of the present invention, the condensation reaction comprises the following steps: mixing the compound 1 and the organic solvent, adding the EDCI and the base, and then adding the compound 2 to react.

[0020] In a certain embodiment of the present invention, the condensation reaction comprises the following steps: mixing the compound 1 and the organic solvent, adding the EDCI and the base, and then adding the oleic acid to react; the organic solvent is dichloromethane; the mass volume ratio of the organic solvent to the compound 1 is 0.03 g / mL; the molar ratio of the EDCI to the compound 1 is 3.5:1; the base is DMAP; the molar ratio of the base to the compound 1 is 1:1; the molar ratio of the compound 2 to the compound 1 is 2.2:1; the reaction temperature of the condensation reaction is room temperature.

[0021] In one embodiment of the present invention, the condensation reaction further comprises the following post-treatment step: after the reaction is completed, the reaction solution is sequentially washed with 2.5-6.02mol / L sodium chloride solution (e.g., 2.7mol / L sodium chloride solution), 1.2-6.02mol / L sodium chloride solution (e.g., 1.35mol / L sodium chloride solution) and water; the organic phase is dried, and after vacuum distillation, petroleum ether and acetonitrile are added for extraction, and the liquid is separated, and the petroleum ether layer is retained and spin-dried to obtain a crude compound 3. The volume ratio of petroleum ether to acetonitrile is preferably (0.1-10):1, more preferably (1-3.7):1, for example, 1.7:1, 1.9:1, 2:1 or 2.9:1.

[0022] In one embodiment of the present invention, the condensation reaction may further include a purification operation, and the purification operation includes the following steps:

[0023] (1) in an organic solvent, reacting the crude compound 3 with oxalic acid to obtain the oxalate of the compound 3; the organic solvent is an alkane solvent;

[0024] (2) In an organic solvent, in the presence of a base, neutralizing the oxalate of the compound 3 to obtain the compound 3.

[0025] In the salt-forming reaction, the alkane solvent is preferably petroleum ether or n-heptane.

[0026] In the salt-forming reaction, the mass volume ratio of the crude compound 3 to the alkane solvent is preferably 0.16-0.33 g / mL, such as 0.26 g / mL.

[0027] In the salt-forming reaction, the molar ratio of the oxalic acid to the crude compound 3 is preferably (1.1-1.2):1.

[0028] The reaction temperature of the salt-forming reaction is preferably room temperature, more preferably 25°C.

[0029] In the salt-forming reaction, the reaction time of the salt-forming reaction can be monitored by conventional means in the art (such as TLC, HPLC or LC-MS), and the reaction time of the salt-forming reaction is preferably 30-120 min.

[0030] In the neutralization reaction, the organic solvent may be a solvent capable of dissolving the oxalate of compound 3, preferably a halogenated hydrocarbon solvent, such as dichloromethane.

[0031] In the neutralization reaction, the mass volume ratio of the oxalate of the compound 3 to the organic solvent is preferably 0.1-0.2 g / mL, such as 0.12-0.13 g / mL.

[0032] In the neutralization reaction, the base is preferably an inorganic base, more preferably an alkali metal bicarbonate, such as sodium bicarbonate. The amount of the base used can be the conventional amount used in the neutralization reaction in the art.

[0033] The present invention provides a post-treatment method for the compound 3, which comprises the following steps: washing a mixture containing the compound 3 and a condensing agent with a 2.5-6.02 mol / L sodium chloride solution and a 1.2-6.02 mol / L sodium chloride solution in sequence; distilling the organic phase under reduced pressure, adding petroleum ether and acetonitrile for extraction, and spinning the petroleum ether layer to obtain a crude compound 3.

[0034] In a certain embodiment of the present invention, the post-treatment method of the compound 3 may also include the following steps: washing the mixture containing the compound 3 and the condensing agent with 2.7 mol / L sodium chloride solution, 1.35 mol / L sodium chloride solution and water in sequence; drying the organic phase, adding petroleum ether and acetonitrile for extraction after reduced pressure distillation, separating the liquids, and spin-drying the petroleum ether layer to obtain a crude compound 3. The condensing agent is preferably EDCI, DCC (N, N'-dicyclohexylcarbodiimide) or DIC (N, N'-diisopropylcarbodiimide), more preferably EDCI. The volume ratio of petroleum ether to acetonitrile is preferably (0.1-10):1, more preferably (1-3.7):1, for example 1.7:1, 1.9:1, 2:1 or 2.9:1.

[0035] In one embodiment of the present invention, the mixture containing the compound 3 and a condensing agent further comprises a condensation product of oleic acid and monooleic acid. In the mixture containing the compound 3 and a condensing agent, the content of the compound 3 is 70-95%, for example 75%, 80%, 85% or 90%.

[0036] The present invention provides a method for purifying the crude compound 3, which comprises the following steps: (1) subjecting the crude compound 3 to a salt-forming reaction with oxalic acid in an organic solvent to obtain an oxalate of the compound 3; the organic solvent is an alkane solvent;

[0037] (2) neutralizing the oxalate of the compound 3 in an organic solvent in the presence of a base to obtain the compound 3;

[0038] The conditions of the salt-forming reaction and the neutralization reaction are as described above.

[0039] In one embodiment of the present invention, the crude compound 3 can be prepared by the aforementioned method for preparing compound 3. The purity of the crude compound 3 can be 85-95%, preferably 90%.

[0040] In one embodiment of the present invention, the crude compound 3 is a condensation product of compound 3, oleic acid and monooleic acid. mixture.

[0041] The present invention provides the following compounds:

[0042]

[0043] In the present invention, the room temperature refers to 20-30°C.

[0044] In the present invention, overnight refers to 10-12 hours.

[0045] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0046] The reagents and raw materials used in the present invention are commercially available.

[0047] The positive progress of the present invention is that by optimizing the condensation reaction conditions, salt formation, extraction and other process parameters, the present invention provides a 1,2-dioleoyloxy-3-(dimethylamino)propane synthesis route suitable for industrial production. The preparation method and purification method are simple to operate, high in yield, more environmentally friendly, and suitable for industrial production. DETAILED DESCRIPTION

[0048] 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 without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0049] Example 1

[0050]

[0051] 3-Dimethylamino-1,2-propanediol (compound 1, 5 g, 41.5 mmol) was dissolved in dichloromethane (150 mL), EDCI (28.2 g, 146.8 mmol) and DMAP (5.2 g, 41.5 mmol) were added, and stirred for 30 minutes until the reaction solution became colorless and transparent. Oily acid (compound 2, 26.5 g, 92.3 mmol) was added dropwise within 30 minutes, and the reaction was stirred at room temperature overnight. After the reaction, 2.7 mol / L sodium chloride solution (200 mL), 1.35 mol / L sodium chloride solution (200 mL) and water (200 mL) were used to wash 3 times to remove EDCI, and the lower organic phase was dried with anhydrous sodium sulfate, and 200 mL of petroleum ether was added after reduced pressure distillation. The petroleum ether layer was extracted with 70 mL of acetonitrile 3 times, 200 mL of acetonitrile 1 time, and 100 mL of acetonitrile 2 times. After separation, the petroleum ether layer was retained. The acetonitrile layer was divided into two batches, each extracted once with 100mL and 50mL petroleum ether, the acetonitrile layer was discarded, and the petroleum ether layer was retained. The two petroleum ether layers were mixed and extracted with 50mL of acetonitrile for 6 times, and the petroleum ether layer was retained and dried. Compound 3 was obtained as a brown oil with a yield of more than 95% (containing a small amount of oleic acid and solvent) and a purity of 90%. 100mL of petroleum ether was added, followed by oxalic acid (4.2g, 46.2mmol), stirred for 30min, then filtered, and the bottle wall was washed with 50mL of petroleum ether. After the filter cake was dried, the oxalate of compound 3 was obtained as a white solid. The structural confirmation data of the oxalate of compound 3 are as follows: 1 H-NMR (600MHz, CDCl3) δ5.50–5.43(m,1H),5.34(m,4H),4.37(m,1H),4.07(m,1H),3.43(m,1H),3.22(m 1H),2.87(s,6H),2.40–2.29(m,4H),2.01(m,8H),1.62–1.58(m,4H),1.28(m,40H),0.88(t,J=6.9Hz,6H). 13 C NMR (151MHz, CDCl3) δ172.97,130.05,129.66,65.70,62.89,54.78,33.99,31.90,30.41,27.30,24.62,22.68.[M+H] +m / z:662.61,found:649.59.

[0052] The oxalate of compound 3 was dissolved in 200 mL of dichloromethane, washed three times with 200 mL of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 16.3 g of 1,2-dioleoyloxy-3-(dimethylamino)propane as a light yellow oil with a yield of 60% and a purity of 97%. The structural confirmation data of compound 3 are as follows: 1 H-NMR (400MHz, CDCl3): δ5.35-5.30(m,4H),5.18(m,1H),4.35(m,1H),4.07(m,1H),2.42(m,2H),2.29 (m,4H),2.24(s,6H),1.99(m,8H),1.65-1.54(m,4H),1.31-1.22(m,40H),0.90-0.80(t,J=6.9Hz,6H). 13 C-NMR (100MHz, CDCl3): δ173.31,129.99,69.24,63.92,59.40,46.06,29.77,29.52,29.32,29.20,24.91,22.68,14.10.[M+H] + m / z:647.59,found:648.59.

[0053] The condensing agent EDCI in Example 1 was replaced by other condensing agents (DCC / DIC / EDCI / acyl chloride), and other reaction conditions and post-treatment were completely repeated in Example 1, but the product yield was poor.

[0054] The reaction conditions of Example 1 were repeated, but after the condensation reaction, the 2.7 mol / L sodium chloride solution and the 1.35 mol / L sodium chloride solution were replaced with ammonium chloride solution, ammonium bicarbonate, sodium bicarbonate, ethanol or ammonium sulfate solution for post-treatment, and the post-treatment operation of Example 1 was repeated to obtain a brown oil, and the yield and purity were also poor.

[0055] After the oxalic acid in Example 1 was replaced by concentrated hydrochloric acid to form a salt, only a white waxy solid was obtained; after the oxalic acid in Example 1 was replaced by concentrated sulfuric acid, acetic acid, methanesulfonic acid or p-toluenesulfonic acid to form a salt, no solid was generated, only a white oil was obtained, and compound 3 could not be further purified.

Claims

1. A method for preparing compound 3, characterized in that: The method comprises the following steps: in an organic solvent, under the action of EDCI and a base, subjecting compound 1 and compound 2 to a condensation reaction as shown below to prepare compound 3; 2. The method for preparing compound 3 according to claim 1, characterized in that: The condensation reaction satisfies one or more of the following conditions: (1) The organic solvent is a halogenated hydrocarbon solvent, such as dichloromethane; (2) The mass volume ratio of the organic solvent to the compound 1 is 0.03-0.05 g / mL; (3) the molar ratio of EDCI to compound 1 is 3.5:1; (4) The base is an organic base, preferably DMAP; (5) The molar ratio of the base to the compound 1 is 1:1; (6) The molar ratio of compound 2 to compound 1 is 2.2:1; (7) The reaction temperature of the condensation reaction is room temperature, for example 25°C.

3. The method for preparing compound 3 according to claim 1, characterized in that: The condensation reaction satisfies one or more of the following conditions: (1) In the condensation reaction, the reaction reagents are the organic solvent, EDCI, the base, the compound 1 and the compound 2; (2) The condensation reaction comprises the following steps: mixing the compound 1 and the organic solvent, adding the EDCI and the base, and then adding the compound 2 to react; (3) The condensation reaction includes the following post-treatment steps: after the reaction is completed, the reaction solution is washed with 2.5-6.02 mol / L sodium chloride solution, 1.2-6.02 mol / L sodium chloride solution and water in sequence; the organic phase is dried, distilled under reduced pressure, and then petroleum ether and acetonitrile are added for extraction, and the liquids are separated, and the petroleum ether layer is retained and spin-dried to obtain a crude compound 3.

4. The method for preparing compound 3 according to claim 3, characterized in that: The reaction solution was washed with 2.7 mol / L sodium chloride solution, 1.35 mol / L sodium chloride solution and water in sequence; And / or, the volume ratio of petroleum ether to acetonitrile is (0.1-10):1, preferably (1-3.7):1, such as 1.7:1, 1.9:1, 2:1 or 2.9:

1.

5. The method for preparing compound 3 according to claim 1, characterized in that: The condensation reaction may further include a purification operation, which includes the following steps: (1) In an organic solvent, reacting the crude compound 3 as claimed in claim 3 with oxalic acid to form a salt to obtain the oxalate of the compound 3; the organic solvent is an alkane solvent; (2) In an organic solvent, in the presence of a base, neutralizing the oxalate of the compound 3 to obtain the compound 3.

6. The method for preparing compound 3 according to claim 5, characterized in that: The purification operation satisfies one or more of the following conditions: (1) In the salt-forming reaction, the alkane solvent is petroleum ether or n-heptane; (2) In the salt-forming reaction, the mass volume ratio of the crude compound 3 to the alkane solvent is 0.16-0.33 g / mL, for example 0.26 g / mL; (3) In the salt-forming reaction, the molar ratio of the oxalic acid to the crude compound 3 is (1.1-1.2):1; (4) The reaction temperature of the salt-forming reaction is room temperature, preferably 25°C; (5) In the neutralization reaction, the organic solvent is a halogenated hydrocarbon solvent, such as dichloromethane; (6) In the neutralization reaction, the mass volume ratio of the oxalate of the compound 3 to the organic solvent is 0.1-0.2 g / mL, for example 0.12-0.13 g / mL; (7) In the neutralization reaction, the base is an inorganic base, preferably an alkali metal bicarbonate, such as sodium bicarbonate.

7. A post-treatment method for compound 3, characterized in that: The method comprises the following steps: washing a mixture containing compound 3 and a condensing agent with a 2.5-6.02 mol / L sodium chloride solution and a 1.2-6.02 mol / L sodium chloride solution in sequence; distilling the organic phase under reduced pressure, adding petroleum ether and acetonitrile for extraction, and drying the petroleum ether layer to obtain a crude compound 3; 8. The post-treatment method of compound 3 according to claim 7, characterized in that: The post-treatment method of compound 3 satisfies one or more of the following conditions: (1) The post-treatment method of the compound 3 comprises the following steps: washing the mixture containing the compound 3 and the condensing agent with 2.7 mol / L sodium chloride solution, 1.35 mol / L sodium chloride solution and water in sequence; drying the organic phase, distilling under reduced pressure, adding petroleum ether and acetonitrile for extraction, separating the liquids, and spinning the petroleum ether layer to obtain the crude compound 3; (2) The condensing agent is EDCI, DCC or DIC; (3) The volume ratio of petroleum ether to acetonitrile is as described in claim 4; (4) The mixture containing the compound 3 and the condensation agent also includes a condensation product of oleic acid and monooleic acid; (5) In the mixture containing the compound 3 and a condensing agent, the content of the compound 3 is 70-95%.

9. A method for purifying a crude compound 3, characterized in that: The method comprises the following steps: (1) in an organic solvent, subjecting the crude compound 3 as claimed in claim 7 to a salt-forming reaction with oxalic acid to obtain an oxalate of the compound 3; the organic solvent is an alkane solvent; (2) neutralizing the oxalate of the compound 3 in an organic solvent in the presence of a base to obtain the compound 3; The conditions of the salt-forming reaction and the neutralization reaction are as described in claim 5 or 6; Preferably, the crude compound 3 is prepared by the preparation method of compound 3 as described in claims 1-4; the purity of the crude compound 3 can be 85-95%, preferably 90%; the crude compound 3 can be a mixture of compound 3, oleic acid and monooleic acid condensation product.

10. A compound as shown below:

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