Preparation method of dipalmitoyl phosphatidylcholine and intermediate of dipalmitoyl phosphatidylcholine

Abstract

The invention discloses a preparation method of dipalmitoyl phosphatidylcholine and an intermediate of the dipalmitoyl phosphatidylcholine. Specifically disclosed is a preparation method of a compound 4, which comprises the following step: in a solvent, in the presence of ammonia water, carrying out a deprotection reaction as shown in the specification on a compound 3 to obtain the compound 4. The preparation method is simple to operate, good in intermediate stability and low in cost. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a preparation method of dipalmitoylphosphatidylcholine and an intermediate thereof. Background Art

[0002] The most commonly reported phosphating agent is a pentavalent phosphating agent, among which phosphorus oxychloride is the most commonly used phosphating agent. Kevin et al. (Kevin, Burgess, Kwok, et al. Synthesis of a valuable cyclopropylchiron for preparations of 2,3-methanoamino acids [J]. Journal of Organic Chemistry, 1993, 58 (14): 3767-3768) used phosphorus oxychloride 5 as a raw material for coupling to obtain phosphatidylcholine, but phosphorus oxychloride is too active, and the reaction of choline replacing the second chlorine to obtain a phosphodiester is difficult to avoid, and the efficiency is not high. Therefore, a phosphating agent in which one chlorine atom is replaced by an alkyl or aryl group is usually used in the synthesis of phospholipid compounds.

[0003] Lindberg et al. (J Lindberg, J Ekeroth, P Konradsson. Efficient synthesis of phospholipids from glycidyl phosphates [J]. Journal of Organic Chemistry, 2002, 67 (1): 194-199.) successfully prepared diphosphatidylglycerol using phenoxyphosphoryl dichloride 6 as a phosphating agent. The advantage of this phosphating agent is that it can effectively avoid the formation of phosphodiester during the reaction, and the deprotection conditions are mild, making it a good phosphating agent. The disadvantage is that the deprotection of phenoxy requires hydrogen pressure, which makes the reaction complicated. In addition, after cyclophosphoxyl chloride 7 was coupled with a glycerol derivative, it was opened under the action of triethylamine to obtain phosphatidylcholine (Wanibuchi K, Takezawa M, Hosoda K, et al. Antibacterial effect of indene on Helicobacter pylori correlates with specific interaction between its compound and dimyristoyl-phosphatidylethanolamine[J]. Chemistry and Physics of Lipids, 2020, 227: 104871.).

[0004] Trivalent phosphorus is usually used in the synthesis of nucleoside molecules. It is necessary to control the reaction conditions to replace the leaving group in sequence and finally oxidize it to a phosphate ester. There are two main types of phosphitylation reagents that are widely used: 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite 8 and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite 9.

[0005] Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the single preparation method of dipalmitoylphosphatidylcholine in the prior art, and to provide a preparation method of dipalmitoylphosphatidylcholine and its intermediate. The preparation method is simple to operate, the intermediate has good stability and low cost.

[0007] The present invention provides a method for preparing compound 4, which comprises the following steps: in a solvent, in the presence of aqueous ammonia, subjecting compound 3 to a deprotection reaction as shown below to obtain compound 4;

[0008]

[0009] In the deprotection reaction, the solvent may be a conventional solvent for such deprotection reactions in the art, preferably a halogenated hydrocarbon solvent, such as dichloromethane.

[0010] In the deprotection reaction, the molar ratio of the aqueous ammonia to the compound 3 is preferably (4-200):1, for example (10-173):1.

[0011] The reaction temperature of the deprotection reaction is preferably room temperature.

[0012] The reaction time of the deprotection reaction can be monitored by conventional means in the art (such as TLC, HPLC or LC-MS), and is preferably 4-12 h, and more preferably 8 h.

[0013] In the deprotection reaction, the reaction reagents are the solvent, ammonia water and compound 3 as described above.

[0014] In one embodiment of the present invention, the deprotection reaction further comprises the following post-treatment: the reaction solution is washed with an aqueous ammonium chloride solution, extracted with an organic solvent (such as dichloromethane), concentrated under reduced pressure, and the crude product is subjected to column chromatography.

[0015] The preparation method of the compound 4 may further include the preparation method of the compound 3. The preparation method of the compound 3 may include the following steps: in a solvent, in the presence of an oxidant, subjecting the compound 2a to an oxidation reaction as shown below to obtain the compound 3;

[0016]

[0017] In the oxidation reaction, the solvent may be a conventional solvent for such oxidation reactions in the art, preferably a halogenated hydrocarbon solvent, such as dichloromethane.

[0018] In the oxidation reaction, the oxidant may be t BuOOH (tert-butyl hydroperoxide), m-CPBA (meta-chloroperbenzoic acid) or hydrogen peroxide, preferably hydrogen peroxide.

[0019] In the oxidation reaction, the molar ratio of the oxidant to the compound 2a is preferably (0.5-40): 1, for example (2-33): 1. The mass fraction of hydrogen peroxide in the hydrogen peroxide is preferably 30%.

[0020] The reaction temperature of the oxidation reaction is preferably room temperature.

[0021] The reaction time of the oxidation reaction can be monitored by conventional means in the art (such as TLC, HPLC or LC-MS), and is preferably 10-60 min, more preferably 20 min.

[0022] In the oxidation reaction, the reaction reagents are the solvent, hydrogen peroxide and compound 3 as described above.

[0023] In one embodiment of the present invention, the oxidation reaction further comprises the following post-treatment: the reaction solution is washed with a saturated sodium chloride solution, extracted with an organic solvent (such as dichloromethane), and the organic phase is concentrated.

[0024] The preparation method of the compound 4 may further include the following steps: in a solvent, in the presence of tetrazole, subjecting the compound 2 and choline to a substitution reaction as shown below to obtain the compound 2a;

[0025]

[0026] In the preparation method of the compound 2a, in the substitution reaction, the solvent may be a conventional solvent for such substitution reactions in the art, preferably a halogenated hydrocarbon solvent, such as dichloromethane.

[0027] In the preparation method of the compound 2a, in the substitution reaction, the amount of the solvent used can be the conventional amount used in the art for such substitution reactions. The volume mass ratio of the solvent to the compound 2 is preferably 5-15 mL / g, such as 9-10 mL / g.

[0028] In the preparation method of the compound 2a, in the substitution reaction, the molar ratio of the tetrazole to the compound 2 is preferably (2-4):1, for example 2:1.

[0029] In the preparation method of the compound 2a, in the substitution reaction, the molar ratio of the choline to the compound 2 is preferably (1-4):1, more preferably (1-2):1.

[0030] In the preparation method of the compound 2a, the substitution reaction is preferably carried out under gas protection. The gas does not participate in the reaction and can be nitrogen, helium or argon.

[0031] In the method for preparing compound 2a, the reaction temperature of the substitution reaction is preferably room temperature, such as 20°C.

[0032] In the preparation method of the compound 2a, the reaction time of the substitution reaction can be monitored by conventional means in the art (such as TLC, HPLC or LC-MS), and is preferably 8-24 h, and more preferably 18 h.

[0033] In the preparation method of the compound 2a, in the substitution reaction, the reaction reagents are the solvent, tetrazole, choline and compound 2 as described above.

[0034] In a certain embodiment of the present invention, the method for preparing the compound 2a further comprises the following steps: mixing the compound 2, tetrazole and the solvent, and then adding the choline.

[0035] In one embodiment of the present invention, in the preparation method of the compound 2a, the substitution reaction further comprises the following post-treatment: the reaction solution is washed with a saturated sodium bicarbonate solution, an aqueous ammonium chloride solution, and a saturated sodium chloride solution, and the organic phase is concentrated.

[0036] The preparation method of the compound 4 may further include a preparation method of the compound 2. The preparation method of the compound 2 comprises the following steps: in a solvent, in the presence of tetrazole, subjecting the compound 1 and 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite to a substitution reaction as shown below to obtain the compound 2;

[0037]

[0038] In the method for preparing compound 2, the solvent may be a conventional solvent for such deprotection reactions in the art, preferably a halogenated hydrocarbon solvent, such as dichloromethane.

[0039] In the preparation method of the compound 2, in the substitution reaction, the amount of the solvent used can be the conventional amount used in the art for such substitution reactions. The volume mass ratio of the solvent to the compound 1 is preferably 10-30 mL / g.

[0040] In the preparation method of the compound 2, in the substitution reaction, the molar ratio of the tetrazole to the compound 1 is preferably (2-4):1, more preferably 2:1.

[0041] In the preparation method of the compound 2, in the substitution reaction, the molar ratio of the 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite to the compound 1 is preferably (1-2):1.

[0042] In the preparation method of the compound 2, the substitution reaction is preferably carried out under gas protection. The gas does not participate in the reaction and can be nitrogen, helium or argon.

[0043] In the method for preparing compound 2, the reaction temperature of the substitution reaction is preferably room temperature, such as 20°C.

[0044] In the preparation method of the compound 2, the reaction time of the substitution reaction can be monitored by conventional means in the art (such as TLC, HPLC or LC-MS), and is preferably 2 to 8 hours, and more preferably 4 hours.

[0045] In the preparation method of the compound 2, in the substitution reaction, the reaction reagents are the solvent, tetrazole, 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite and compound 1 as described above.

[0046] In one embodiment of the present invention, the method for preparing compound 2 further comprises the following steps: mixing the 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite, tetrazole and the solvent, and then adding compound 1.

[0047] In one embodiment of the present invention, in the preparation method of compound 2, the substitution reaction further includes the following post-treatment: the reaction solution is washed with a saturated sodium bicarbonate solution, an aqueous ammonium chloride solution, a saturated sodium chloride solution, dried (for example, with anhydrous sodium sulfate), concentrated under reduced pressure, and then subjected to column chromatography.

[0048] The present invention provides a method for preparing a compound 2, comprising the following steps: in a solvent, in the presence of tetrazole, subjecting a compound 1 and 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite to a substitution reaction as shown below, to obtain the compound 2;

[0049]

[0050] In the preparation method of the compound 2, the conditions and operation of the substitution reaction are as described above.

[0051] The present invention provides a method for preparing compound 2a, comprising the following steps: in a solvent, in the presence of tetrazole, subjecting compound 2 and choline to a substitution reaction as shown below to obtain compound 2a;

[0052]

[0053] In the preparation method of the compound 2a, the conditions and operation of the substitution reaction are as described above.

[0054] The present invention provides a method for preparing compound 3, which comprises the following steps: subjecting compound 2a to an oxidation reaction as shown below in a solvent in the presence of an oxidant to obtain compound 3;

[0055]

[0056] The conditions and operation of the oxidation reaction are as described above.

[0057] The present invention provides a method for preparing compound 4, which comprises the following steps: in a solvent, in the presence of aqueous ammonia, subjecting compound 3 to a deprotection reaction as shown below to obtain compound 4;

[0058]

[0059] The conditions and operation of the deprotection reaction are as described above.

[0060] The present invention provides the following compounds:

[0061]

[0062] In the present invention, the room temperature refers to 20-30°C.

[0063] 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.

[0064] The reagents and raw materials used in the present invention are commercially available.

[0065] The positive progressive effect of the present invention is that the present invention provides a method for preparing dipalmitoylphosphatidylcholine, the preparation method is simple to operate, the intermediate has good stability and low cost. DETAILED DESCRIPTION

[0066] 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.

[0067] Example 1

[0068]

[0069] Synthesis of intermediate 2:

[0070] 2-(Cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite (0.21 g, 0.70 mmol) and tetrazole (0.50 g, 0.70 mmol) were dissolved in anhydrous dichloromethane (1 mL) and activated for 0.5 h under nitrogen protection. Dipalmitoylglycerol (1, 0.20 g, 0.35 mmol) was dissolved in anhydrous dichloromethane (5 mL) and added thereto. The mixture was stirred at 20°C for 4 h until the reaction was complete. The reaction solution was washed three times with saturated aqueous sodium bicarbonate solution (10 mL×3), once with aqueous ammonium chloride solution (10 mL), and once with saturated aqueous sodium chloride solution (10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate / triethylamine=9:1:0.1) to obtain compound 2, 123.8 mg (colorless oil, yield: 46%, purity 98%). 1 H NMR (400MHz, CDCl3) δ5.25-4.99(m,2H),4.36-4.26(m,2H),4.15(dd,J=11.5,7.3,5.4Hz,1H),4.07-4.00(m,2H),3.96(q,J= 4.7,2.1Hz,4H),2.67-2.57(m,2H),2.32(dt,J=10.1,7.5Hz,4H),1.61(q,J=7.1Hz,4H),1.26(s,56H),0.88(t,J=6.9Hz,8H). 13 C NMR (150MHz, CDCl3) δ173.98,68.38,65.04,58.03,34.12,31.94,29.70,29.66,29.62,29.61,29.47,29.37,29.26,29.14,24.90,22.70,14.13.

[0071] Synthesis of compound 4:

[0072] Compound 2 (0.115 g, 0.15 mmol) and tetrazole (0.021 g, 0.30 mmol) were dissolved in anhydrous dichloromethane (1 mL) and activated for 0.5 h under nitrogen protection. Choline (0.18 g, 0.15 mL) was added to the activated reaction system and stirred at 20 °C for 18 h until the reaction was complete. The reaction solution was washed three times with saturated sodium bicarbonate aqueous solution (5 mL), once with ammonium chloride aqueous solution (5 mL), and once with saturated sodium chloride aqueous solution (5 mL). The organic phase was concentrated under reduced pressure and 30% H2O2 (0.5mL) was reacted for 20min, the reaction solution was washed three times with saturated sodium chloride solution (5mL×3), extracted with dichloromethane solution, the organic phase was concentrated, 1mL of ammonia water was added and stirred at room temperature for 8h, washed three times with ammonium chloride aqueous solution (5mL×3), extracted with dichloromethane solution, the organic phase was concentrated under reduced pressure, and the crude product was subjected to column chromatography (dichloromethane: triethylamine 10:0.1) to obtain 23.34mg of dipalmitoylphosphatidylcholine (white solid powder, yield 21%, purity 96%). 1 H NMR(400MHz, CDCl3) δ5.20(dd,J=6.0,3.1Hz,1H),4.39(dd,J=12.1,2.9Hz,1H),4.31(s,2H),4.13(dd,J=12.0,7.3Hz,1H),4.02–3.86 (m,2H),3.90–3.76(m,2H),3.37(s,9H),2.28(dt,J=11.3,7.7Hz,4H),1.58(q,J=6.9,5.2Hz,4H),1.25(s,48H),0.88(t,J=7.0Hz,6H). 13 C NMR (150MHz, CDCl3) δ173.59,173.25,77.24,77.23,77.03,76.82,70.50,70.45,63.00,59.36,54.34,34.34 ,34.16,31.95,29.76,29.70,29.64,29.62,29.44,29.40,29.25,29.21,25.00,24.92,22.71,14.12.ESI-MS m / z:734.5[M+H] + .

Claims

1. A method for preparing compound 4, characterized in that: The method comprises the following steps: in a solvent, in the presence of aqueous ammonia, subjecting compound 3 to a deprotection reaction as shown below to obtain compound 4; 2. The method for preparing compound 4 according to claim 1, characterized in that: The deprotection reaction satisfies one or more of the following conditions: (1) The solvent is a halogenated hydrocarbon solvent, such as dichloromethane; (2) The molar ratio of the aqueous ammonia to the compound 3 is (4-200):1, for example 173:1; (3) The reaction temperature of the deprotection reaction is room temperature; (4) The deprotection reaction includes the following post-treatment: the reaction solution is washed with an aqueous ammonium chloride solution, extracted with an organic solvent, concentrated under reduced pressure, and the crude product is subjected to column chromatography.

3. The method for preparing compound 4 according to claim 1, characterized in that: The preparation method of the compound 3 comprises the following steps: in a solvent, in the presence of an oxidant, subjecting the compound 2a to an oxidation reaction as shown below to obtain the compound 3; 4. The method for preparing compound 4 according to claim 3, characterized in that: The oxidation reaction satisfies one or more of the following conditions: (1) The solvent is a halogenated hydrocarbon solvent, such as dichloromethane; (2) The oxidant is t BuOOH, m-CPBA or hydrogen peroxide; (3) The molar ratio of the oxidant to the compound 2a is (0.5-40):1, for example (2-33):1; (4) The reaction temperature of the oxidation reaction is room temperature; (5) The oxidation reaction includes the following post-treatment: the reaction solution is washed with a saturated sodium chloride solution, extracted with an organic solvent, and the organic phase is concentrated.

5. The method for preparing compound 4 according to claim 3, characterized in that: The preparation method of the compound 2a comprises the following steps: in a solvent, in the presence of tetrazole, subjecting the compound 2 and choline to a substitution reaction as shown below to obtain the compound 2a; 6. The method for preparing compound 4 according to claim 5, characterized in that: In the preparation method of the compound 2a, the substitution reaction satisfies one or more of the following conditions: (1) The solvent is a halogenated hydrocarbon solvent, such as dichloromethane; (2) The volume mass ratio of the solvent to the compound 2 is 5-15 mL / g, for example 9-10 mL / g; (3) the molar ratio of the tetrazole to the compound 2 is (2-4):1; (4) The molar ratio of choline to compound 2 is (1-4):1, preferably (1-2):1; (5) The substitution reaction is carried out under gas protection; the gas may be nitrogen, helium or argon; (6) The reaction temperature of the substitution reaction is room temperature, for example, 20°C; (7) The preparation method of the compound 2a comprises the following steps: mixing the compound 2, tetrazole and the solvent, and then adding choline; (8) The substitution reaction includes the following post-treatment: the reaction solution is washed with a saturated sodium bicarbonate solution, an ammonium chloride aqueous solution, and a saturated sodium chloride solution, and the organic phase is concentrated.

7. The method for preparing compound 4 according to claim 5, characterized in that: The preparation method of the compound 2 comprises the following steps: in a solvent, in the presence of tetrazole, subjecting the compound 1 and 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite to a substitution reaction as shown below to obtain the compound 2; 8. The method for preparing compound 4 according to claim 7, characterized in that: In the preparation method of the compound 2, the substitution reaction satisfies one or more of the following conditions: (1) The solvent is a halogenated hydrocarbon solvent, such as dichloromethane; (2) The volume mass ratio of the solvent to the compound 1 is 10-30 mL / g; (3) The molar ratio of the tetrazole to the compound 1 is (2-4):1, preferably 2:1; (4) the molar ratio of the 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite to the compound 1 is (1-2):1; (5) The substitution reaction is carried out under gas protection; the gas may be nitrogen, helium or argon; (6) The reaction temperature of the substitution reaction is room temperature, for example, 20°C; (7) The preparation method of the compound 2 comprises the following steps: mixing the 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite, tetrazole and the solvent, and then adding the compound 1; (8) The substitution reaction includes the following post-treatment: the reaction solution is washed with a saturated sodium bicarbonate solution, an ammonium chloride aqueous solution, a saturated sodium chloride solution, dried, concentrated under reduced pressure, and subjected to column chromatography.

9. A method for preparing compound 2, characterized in that: The method comprises the following steps: in a solvent, in the presence of tetrazole, subjecting compound 1 and 2-(cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite to a substitution reaction as shown below to obtain compound 2; In the method for preparing compound 2, the conditions and operation of the substitution reaction are as described in claim 7 or 8.

10. A compound as shown below,

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