CN113354679A

CN113354679A - Preparation process of distearoyl phosphatidyl glycerol sodium

Info

Publication number: CN113354679A
Application number: CN202110754813.XA
Authority: CN
Inventors: 计炜; 徐佩; 胡峻
Original assignee: Nanjing Hengyuan Technology Development Co ltd
Current assignee: Nanjing Hengyuan Technology Development Co ltd
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-09-07

Abstract

The invention relates to a preparation process of distearoyl phosphatidyl glycerol sodium (DSPGNa). The glycerol end is firstly connected to a phosphorus-oxygen bond and then connected with the intermediate 7, so that the utilization rate of the expensive intermediate 7 is improved as much as possible.

Description

Preparation process of distearoyl phosphatidyl glycerol sodium

Technical Field

The invention relates to the field of synthesis, and in particular relates to a preparation process of distearoyl phosphatidyl glycerol sodium.

Background

Bangham teaches that liposomes (liposomes) were first discovered in 1965, and the liposome technology is also referred to as "biolistic" technology or fourth generation targeted drug delivery technology. With the popularization of liposome technology, liposome as a novel drug carrier has the advantages of low toxicity, long in-vivo circulation time, targeting property, slow release and the like, and is increasingly paid more attention to the aspect of drug directional delivery.

Composition of the liposomes: lipid (phospholipid) and additive.

1. Phospholipids: including natural phospholipids and synthetic phospholipids. The structure of phospholipids is characterized by a hydrophilic group consisting of one phosphate group and one quaternary ammonium salt group, and a lipophilic group consisting of two longer hydrocarbon groups. The natural phospholipid is mainly lecithin (phosphatidylcholine, PC), is derived from egg yolk and soybean, and is neutral. The synthetic phospholipid mainly comprises DPPC (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine), DSPG (distearoylphosphatidylglycerol) and the like, which all belong to hydrogenated phospholipids, have the characteristics of stable property, strong oxidation resistance, stable finished product and the like, and are preferred auxiliary materials abroad.

2. Cholesterol: cholesterol and phospholipids are the basic substances that together constitute cell membranes and liposomes. Cholesterol has the effect of regulating membrane fluidity and may be referred to as a liposomal "fluidity buffer".

Natural phospholipids are the major components of biological membranes and are present in almost all cells of the organism. Phospholipids can be classified into glycerophospholipids and sphingophospholipids according to their chemical structure. The glycerophospholipid consists of three parts, namely a glycerol skeleton, fatty acid and a hydrophilic head group containing hydroxyl. Sphingomyelin differs from glycerophospholipids only in the substitution of glycerol with sphingosine. The natural phospholipid is generally separated and extracted from soybean or egg yolk, and phospholipid compounds are obtained by processing, enzyme modification or semi-synthesis, but the phospholipid obtained by the method is a mixture of various phospholipids or homologues of the phospholipids, is not single or high-purity phospholipid, and has certain application limitation.

Distearoyl phosphatidyl glycerol sodium (DSPGNa) is one of important auxiliary materials for preparing liposome, is different from natural phospholipid DSPGNa, belongs to synthetic phospholipid, and has determined chemical components. Due to the unique structure and physicochemical properties, the amifostine liposome plays an extremely important role in the development of some key preparations, wherein the most representative is the liposome drug amifostine (amphotericin B liposome for injection), and the amifostine liposome is suitable for patients infected by deep fungi such as candida, aspergillus, cryptococcus and the like. Amphotericin B is a drug for deep fungal infections, but it is highly toxic to the kidneys. Amphotericin liposome B solves this problem to a large extent, and can be used for patients who cannot use an effective dose of amphotericin B due to renal injury or drug toxicity, or who have been ineffectively treated with amphotericin B. The phospholipid material used by the liposome drug ampirome comprises: DSPG (distearoyl phosphatidyl glycerol), HSPC (hydrogenated soy lecithin), CHO (high purity cholesterol). In view of the more and more extensive application of DSPGNa in liposome research and large market demand, the development of the method for synthesizing distearoyl phosphatidyl glycerol sodium DSPGNa which is suitable for industrial production prospect has important significance.

The preparation of DSPGNa was first mentioned in the Journal of Biological Chemistry (1958),232,895-901 (By Baer, Erich; Buchnea, Ddyro). With phosphorus oxychloride (POCl)₃) D-alpha, beta-diol and D-acetonylglycerol are used as main raw materials, equimolar D-alpha, beta-diol and pyridine are kept at 0 ℃ in ice bath, and are dripped into equimolar phosphorus oxychloride solution, and after dripping, the temperature is raised to room temperature, and stirring is carried out for 1.0 hr. Cooling to 10 deg.C, adding dropwise equimolar D-acetone glycerol and pyridine, heating to room temperature after dropwise addition, stirring for 2.5hr, adding dropwise equimolar water, treating with 2N glacial sulfuric acid solution, saturated sodium carbonate solution and water, and concentrating under reduced pressure to obtain crude product. Dispersing the crude product in acetone solution, treating at-85 deg.C for 2.0hr, and centrifuging to obtain dioleoyl-L-alpha-glycerophosphoryl-L-glycerol.

Adding the dioleoyl-L-alpha-glyceryl phosphoryl-L-glycerol into platinum oxide as a catalyst and ethanol as a solvent, hydrogenating at normal pressure to obtain a crude product, and refining with acetone to obtain the distearoyl-L-alpha-glyceryl phosphoryl-L-glycerol.

This route seems to be simple, but we are trying to directly react the intermediate 7[ (2S) -3-hydroxy-1, 2-propanediyl distearate by a process similar to the one described above]When the intermediate 4 (acetonide condensed glycerol) is coupled in a stepwise manner, the reaction activity of phosphorus oxychloride is high, the selectivity of the reaction with hydroxyl is poor, even if the molar ratio is controlled to be 1:1, phosphorus oxychloride can be coupled with a plurality of alcohols at the same time, so that the product is a mixture of a plurality of phospholipids, and the purity of the crude intermediate 9 is only 40-50%, and the crude intermediate is mixed with a plurality of phospholipid components through mass spectrometric detection and analysis. Among them, the largest impurity (55-65% in total), MS (EI +): m/e-933.5, consistent with the structure of compound 10 below, should be a monostearyldiglycerol impurity; there is also one major impurity (20-30% of total impurities), MS (EI +): m/e-1493.7, corresponding to the structure of compound 11 below, should be a distearoyl monoglycerol impurity;there is one non-major impurity (5-10% of total impurities), MS (EI +): m/e-1311.83, consistent with the structure of compound 12 below, should be a distearoyl impurity. The intermediate 5 is difficult to purify, and can only be subjected to the next reaction after being separated and purified by column chromatography at present. Aiming at the side reaction caused by overhigh activity of phosphorus oxychloride, measures of reducing the reaction temperature, controlling the dropping speed, increasing the reaction system and the like are adopted successively, and the reaction is hopefully controlled at a phosphodiester stage, but the failure is ended. Thus passing through the POCl₃The stepwise substitution of chlorine in the molecule to synthesize diesters of phosphoric acid, although straightforward, is not an effective method.

In addition, we have found that side reactions also occur when the propylidene protecting group is deprotected under acidic conditions, and we refer to the hydrolysis conditions in S.Pedatella et al/Carbohydrate Research 343(2008) 31-38: hydrolysis reaction was carried out with 90% TFA at room temperature for 3.0hr, and it was found that 15-20% of the product was produced by cleavage of one side of the stearic acid chain in the molecular structure, and Lysophospholipid (LSPG) as a by-product 13 was produced, while stearic acid as a by-product 14 was detected. We tried to perform the depropylidene reaction with slightly weaker glacial acetic acid, but heating to above 80 ℃ was required to allow the reaction to proceed, and side reactions were still unavoidable.

The synthetic route is as follows:

the impurity structure contained in intermediate 9 is as follows:

the structure of the acid degradation impurity of the product 1 is as follows:

2015, Shigeki Sano et alUsing Still-Gennari reagent [ O, O' bis (2,2, 2-trifluoroethyl) phosphoacetic acid methyl ester]Ester-type glycerophosphatidic acid (PA), Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC) are efficiently constructed through Horner-Wadsworth-Emmons (HWE) reaction. The HWE reaction is an improvement over the Wittig reaction, which uses a stable phosphonate carbanion, instead of a phosphoylide, to react with aldehydes and ketones to form olefins, the products being predominantly E-type olefins, and the dihydrocarbyl phosphates produced in this reaction are usually disposed of as by-products, but are the major target products in the synthesis of phospholipids. According to this document, we attempted to synthesize DSPGNa using Still-Gennari reagent, and we found the trifluoroacetyl group (CF) in Still-Gennari reagent₃CH₂O-) is a relatively suitable leaving group, has better reaction selectivity with alcohol, can realize gradual coupling with different alcohols, has simple operation, can obtain an intermediate 17 with the purity of 85-90 percent, can be directly used for the next reaction, but still avoids acidic degradation impurities when the propylidene group is removed. The trifluoroethanol group used in the Still-Gennari reagent broadens new ideas for the preparation of phosphodiesters.

The synthetic route is as follows:

disclosure of Invention

The invention aims to provide a preparation process of distearoyl phosphatidyl glycerol sodium (DSPGNa) with simple synthetic route, low production cost, high yield and high purity aiming at the technical problems.

The technical scheme of the invention is as follows:

a preparation process of distearoyl phosphatidyl glycerol sodium (DSPGNa) comprises the following reaction equation:

the reaction equation for the preparation of intermediate 19(R ═ TFE) [5- (benzyloxy) -2- (2,2, 2-trifluoroethoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ]:

the method comprises the following specific steps:

1. preparation of intermediate 19(R ═ TFE): phosphorus oxychloride of the intermediate 22 and a compound 23 (2-benzyloxy-1, 3-propylene glycol) are subjected to a disubstituted reaction in the presence of an organic alkali reagent, and are subjected to a monosubstitution reaction with trifluoroethanol in the presence of the organic alkali reagent to obtain an intermediate 19, and the intermediate 19 is subjected to reduced pressure distillation to obtain a pure product, wherein the organic alkali can be DBU (1, 8-diazabicycloundecene-7-ene), DIEA (N, N-diisopropylethylamine), TEA (triethylamine) and the like; preparation of intermediate 19(R ═ p-NP): carrying out disubstituted reaction on the intermediate 22 phosphorus oxychloride and the compound 23 (2-benzyloxy-1, 3-propylene glycol) in the presence of an organic base reagent, carrying out monosubstitution on the phosphorus oxychloride and 4-nitro-phenol (p-NP) in the presence of the organic base reagent to obtain an intermediate 19, and carrying out reduced pressure distillation to obtain a pure product, wherein the organic base can be DBU (1, 8-diazabicycloundecen-7-ene), DIEA (N, N-diisopropylethylamine), TEA (triethylamine) and the like;

2. preparation of intermediate 20: intermediate 7[ (2S) -3-hydroxy-1, 2-propanediyl distearate ] with intermediate 19[5- (benzyloxy) -2- (2,2, 2-trifluoroethoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ], a molar ratio of 1:1 feeding, and carrying out condensation reaction in the presence of an organic base reagent to obtain an intermediate 20(R) -3- { [5- (benzoyloxy) -2-oxo-1, 3, 2-dioxaphosphin-2-yl ] oxy } propane-1, 2-diethyl distearate, wherein the organic base reagent can be DBU, DIEA, TEA and the like;

3. preparation of intermediate 21: intermediate 20 is analogous to boron trifluoride, indium trichloride (InCl)₃) Tin tetrachloride (SnCl)₄) Zinc chloride (ZnCl)₂) Ring opening is carried out under the catalysis of Lewis acid to obtain an intermediate 21(2R) -3- { [2- (benzoyloxy) -3-hydroxypropoxy]-hydroxy-phosphoryloxy } propane-1, 2-diethyldistearate;

4. preparation of compound 1: and removing benzyl (Bzl) from the intermediate 21 under the action of trimethyl iodosilane (TMSI), and sodium methoxide to form sodium salt to obtain the target compound 1.

The invention has the beneficial effects that: according to the structure of the compound, the compound is split into butt joint of two fragments by taking a middle phosphorus-oxygen bond as a center. The glycerol end is firstly connected to a phosphorus-oxygen bond and then connected with the intermediate 7, so that the utilization rate of the expensive intermediate 7 is improved as much as possible.

In addition, the glycerol terminal is connected in a disubstituted mode, so that only one active leaving terminal is exposed in the whole intermediate 19, and the connection of the intermediate 7 is purer. The ring opening of the intermediate 20 is catalyzed by a lewis acid, a possible mechanism of which is: boron trifluoride diethyl etherate is used as a Lewis acid center, so that charges on O deviate, the polarity of a P-O bond is increased, and in this case, a nucleophilic reagent attacks a phosphorus-oxygen bond with small steric hindrance to generate a substitution reaction to generate a compound 21. The ring-opening condition is mild, and the stearoyl phosphoryl bond on the other side is hardly influenced. Finally, the benzyl protecting group can be removed by hydrogenation or by using iodotrimethylsilane, which we prefer from a safety point of view.

The lewis acid catalyzed ring opening mechanism is as follows (taking boron trifluoride diethyl ether as an example):

phosphorus oxychloride is a common chemical raw material, and is cheap and easy to obtain. We engineered it into the structural form of intermediate 19 by means of group modification. The disubstituted structure in the intermediate 19 brings many convenient conditions for the following reaction, so that side reactions caused by too high activity are avoided; in addition, in the whole reaction process, no acid and alkali are involved, the reaction is mild, side reactions are few, and the obtained crude product is simply refined to reach the pharmaceutical adjuvant standard; the preparation process of the product is also relatively simple, all conventional, and only in the preparation of intermediate 19, the final product needs to be purified by reduced pressure distillation.

Detailed Description

Example 1: preparation of sodium distearoyl phosphatidyl glycerol (DSPGNa)

1.1 preparation of intermediate 20

Materials:

the operation is as follows: weighing 710.00 g of intermediate 16.00mmol, 195.22 g of intermediate 16mmol and 100ml of toluene, uniformly mixing, cooling in an ice water bath, controlling the temperature of a reaction solution to be 0-10 ℃, slowly dripping a solution prepared from 4.87g of DBU and 10ml of toluene into the reaction solution, controlling the dripping time to be 10-15min, heating the temperature of the reaction solution to be 20-30 ℃ and stirring for reaction for 2.0-3.0hr, filtering to remove precipitates after the reaction is finished, obtaining an organic phase, fully washing the organic phase with 1% hydrochloric acid solution and saturated sodium chloride solution, drying the obtained organic phase with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 12.83g of white solid, namely the product intermediate 20, wherein the yield is as follows: 94.21 percent. And (3) product purity: 95.42 percent

HPLC conditions:

octadecylsilane chemically bonded silica is used as filler (4.6mm × 250mm, 5 μm) or chromatographic column with equivalent performance; gradient elution was performed according to the following table using water-glacial acetic acid (100:0.1) as mobile phase a and methanol as mobile phase B; the flow rate was 1.0ml per minute; the detector is a CAD detector, and the atomization temperature is 35 ℃; the column temperature was 30 ℃ and the amount of sample was 10. mu.l.

The gradiometer is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	50	50
10	50	50
			40	15	85
45	15	85
			46	50	50
55	50	50

Test solution: weighing about 10mg of the product, accurately weighing, placing in a 20ml measuring flask, adding methanol for dissolving, diluting to scale, and shaking uniformly to obtain a test solution.

The determination method comprises the following steps: the percentage of the main peak area should not be less than 95% as calculated by area normalization.

1.2 preparation of intermediate 21

The operation is as follows:

weighing 2010.00 g of intermediate 11.76mmol, adding 100ml of toluene, mixing uniformly, cooling in ice water bath, controlling the temperature of reaction liquid at 0-10 ℃, slowly dripping a solution prepared from 0.18g of boron trifluoride diethyl etherate solution (47%) and 10ml of toluene into the reaction liquid, controlling the dripping time to be 5-10min, heating the temperature of the reaction liquid to 20-30 ℃, stirring and reacting for 7.0-8.0hr, and after the reaction is finished, using 1% NaHCO for an organic phase₃The solution and a saturated sodium chloride solution were sufficiently washed, and the obtained organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to dryness to obtain 9.21g of a white solid as a product intermediate 21 in yield: 90.19 percent. And (3) product purity: 91.08 percent

HPLC conditions:

The gradiometer is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	50	50
10	50	50
			40	15	85
45	5	95
			46	50	50
55	50	50

The determination method comprises the following steps: the percentage of the main peak area should not be less than 90% as calculated by area normalization.

1.3 preparation of product 1

Materials:

the operation is as follows:

weighing 2110.00 g of intermediate 11.50mmol, adding 100ml of toluene, uniformly mixing, cooling in a ice salt bath, controlling the temperature of a reaction solution to be-10-0 ℃, slowly dripping a solution prepared from 4.60g of iodotrimethylsilane solution and 10ml of toluene into the reaction solution, controlling dripping to be finished within 10-20min, heating the temperature of the reaction solution to be 10-20 ℃, stirring for reaction for 0.5-1.0hr, after the reaction is finished, adding 5ml of methanol solution containing 0.62g of sodium methoxide, separating out white solid, filtering, drying to obtain 8.16g of white solid, wherein the yield is 1: 88.54 percent. And (3) product purity: 97.76 percent

HPLC conditions:

using C8(4.6 mm. times.250 mm, 5 μm) or equivalent performance columns; taking 10mmol/L ammonium acetate buffer solution (adjusting pH to 4.0 with glacial acetic acid) as mobile phase A, taking methanol as mobile phase B, and performing gradient elution according to the following table; the flow rate was 1.0ml per minute; the detector is a CAD detector, and the atomization temperature is 35 ℃; the column temperature was 30 ℃ and the amount of sample was 10. mu.l.

The gradiometer is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	40	60
10	40	60
			40	10	90
45	10	90
			46	40	60
55	40	60

19(R ═ TFE) [5- (benzyloxy) -2- (2,2, 2-trifluoroethoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ] prepared by the method of example 3.

Example 2: preparation of sodium distearoyl phosphatidyl glycerol (DSPGNa)

2.1 preparation of intermediate 20

Materials:

the operation is as follows:

weighing 710.00 g of intermediate 16.00mmol, 195.84 g of intermediate 16mmol and 100ml of dichloromethane, uniformly mixing, cooling in ice water bath, controlling the temperature of reaction liquid at 0-10 ℃, slowly dripping a solution prepared from 3.24g of triethylamine and 10ml of dichloromethane into the reaction liquid, controlling the dripping time to be 10-15min, heating the temperature of the reaction liquid to 20-30 ℃ and stirring for reaction for 2.0-3.0hr, after the reaction is finished, filtering to remove precipitates to obtain an organic phase, fully washing the organic phase with 1% hydrochloric acid solution and saturated sodium chloride solution, drying the obtained organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to obtain 12.83g of white solid, namely the product intermediate 20, wherein the yield is as follows: 94.21 percent. And (3) product purity: 96.32 percent

HPLC conditions:

The gradiometer is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	40	60
10	40	60
			45	5	95
50	5	95
			51	40	60
60	40	60

2.2 preparation of intermediate 21

Materials:

the operation is as follows:

weighing 2010.00 g of intermediate 11.75mmol, adding 100ml of Tetrahydrofuran (THF), mixing uniformly, cooling in ice water bath, controlling the temperature of the reaction solution at 0-10 ℃, slowly dripping a solution prepared from 0.33g of stannic chloride and 10ml of THF into the reaction solution, controlling the dripping time to be 5-10min, heating the temperature of the reaction solution to 20-30 ℃, stirring for reaction for 7.0-8.0hr, and after the reaction is finished, using 1% NaHCO for an organic phase₃The solution and a saturated sodium chloride solution were sufficiently washed, and the obtained organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to dryness to obtain 9.08g of a white solid as a product intermediate 21 in yield: 88.92 percent.

And (3) product purity: 92.07 percent of

HPLC conditions:

The gradiometer is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	30	70
10	30	70
			45	10	90
50	10	90
			51	30	70
60	30	70

2.3 preparation of product 1

Materials:

the operation is as follows:

weighing 2110.00 g of intermediate 11.50mmol, adding 100ml of toluene, uniformly mixing, cooling in a ice salt bath, controlling the temperature of a reaction solution to be-10-0 ℃, slowly dripping a solution prepared from 4.60g of iodotrimethylsilane solution and 10ml of toluene into the reaction solution, controlling dripping to be finished within 10-20min, heating the temperature of the reaction solution to be 10-20 ℃, stirring for reaction for 0.5-1.0hr, after the reaction is finished, adding 5ml of methanol solution containing 0.62g of sodium methoxide, separating out white solid, filtering, drying to obtain 7.95g of white solid, wherein the yield is 1: 86.26 percent. And (3) product purity: 98.13 percent

HPLC conditions: same as example 1 (1.3)

19(R ═ p-NP) [5- (benzyloxy) -2- (4-nitro-phenoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ] was prepared as in example 4.

Example 3: preparation of 19(R ═ TFE) [5- (benzyloxy) -2- (2,2, 2-trifluoroethoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ]

The operation is as follows:

weighing 2311.88 g of intermediate 65.22mmol, DBU29.98g of 195.66mmol and 200ml of dichloromethane, uniformly mixing, cooling in ice-water bath, controlling the temperature of the reaction solution to be 0-5 ℃, slowly dripping a solution prepared from 2210.00 g of phosphorus oxychloride intermediate 65.22mmol and 20ml of dichloromethane into the reaction solution, controlling the dripping time to be 20-30min, heating the temperature of the reaction solution to be 20-30 ℃, stirring and reacting for 0.5-1.0hr, cooling in ice-water bath after the reaction is finished, controlling the temperature of the reaction solution to be 0-5 ℃, slowly dripping a solution prepared from 6.52g of trifluoroethanol 65.22mmol and 10ml of dichloromethane into the reaction solution, controlling the dripping time to be 10-20min, heating the reaction solution to be 20-30 ℃, stirring and reacting for 0.5-1.0hr, filtering to remove precipitates to obtain a dichloromethane solution, distilling under reduced pressure to remove an organic phase, distilling at 140 ℃/5mmHg, the fractions were collected to give 15.5g of a colorless oily liquid as product intermediate 19, yield: 72.86 percent. And (3) product purity: 98.74 percent

HPLC conditions:

The gradiometer is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	95	5
10	95	5
			45	80	20
50	80	20
			51	95	5
60	95	5

Test solution: taking about 40mg of the product, accurately weighing, placing in a 20ml measuring flask, adding methanol for dissolving, diluting to scale, and shaking up to obtain a test solution.

Example 4: preparation of 19(R ═ p-NP) [5- (benzyloxy) -2- (4-nitro-phenoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ]

Materials:

the operation is as follows:

weighing 2311.88 g of intermediate 65.22mmol, DBU29.98g of 195.66mmol and 200ml of dichloromethane, uniformly mixing, cooling in ice-water bath, controlling the temperature of reaction liquid to be 0-5 ℃, slowly dripping solution prepared by 2210.00 g of phosphorus oxychloride intermediate 65.22mmol and 20ml of dichloromethane into the reaction liquid, controlling the dripping time to be 20-30min, heating the temperature of the reaction liquid to be 20-30 ℃, stirring and reacting for 0.5-1.0hr, cooling in ice-water bath after the reaction is finished, controlling the temperature of the reaction liquid to be 0-5 ℃, slowly dripping solution prepared by 9.07g of 65.22mmol of 4-nitro-phenol and 10ml of dichloromethane into the reaction liquid, controlling the dripping time to be 10-20min, heating the temperature of the reaction liquid to be 20-30 ℃, stirring and reacting for 0.5-1.0hr, filtering to remove precipitate, obtaining dichloromethane solution, distilling under reduced pressure to remove an organic phase, 155 ℃ and 160 ℃/5mmHg, collecting the fraction to obtain 17.8g of colorless oily liquid as the product intermediate 19, with the yield: 74.72 percent. And (3) product purity: 99.04% HPLC conditions:

The gradiometer is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	90	10
10	90	10
			35	30	70
40	30	70
			41	90	10
50	90	10

Test solution: taking about 20mg of the product, accurately weighing, placing in a 20ml measuring flask, adding methanol for dissolving, diluting to scale, and shaking up to obtain a test solution.

The determination method comprises the following steps: the percentage of the main peak area should not be less than 97% as calculated by area normalization.

Comparative example 1:

according to the DSPG synthetic method in CN103864840A, the concrete steps are as follows:

cooling (R) -1, 2-Glycerol distearate-Glycerol-3-phosphatidic acid 5.0g (7.09mmol) and 500ml of a chloroform-pyridine mixed solution (chloroform: pyridine ═ 2:1) to 0-5 ℃, and adding dropwise 2, 2-dimethyl-4-methanol0.94g (7.11mmol) of-1, 3-dioxolane was stirred at the same temperature for 5.0hr, and the temperature was raised to 30 ℃ and stirring was continued for 20 hr. The reaction was stopped, the pH was adjusted to 2 with 1N hydrochloric acid solution, washed 3 times with 150ml of saturated sodium chloride solution, and the organic layer was concentrated to dryness under reduced pressure. Adding 20ml of 20% HAc solution into the concentrate, stirring at 60 deg.C for 2.0hr, and adding saturated NaHCO₃The solution was adjusted to pH 7, extracted with chloroform 250ml x 3, washed 3 times with 100ml saturated sodium chloride solution and the organic layer concentrated to dryness under reduced pressure to give the crude product. And (3) carrying out column chromatography purification on 4.5g of the crude product by using 450g of silica gel (100 meshes and 200 meshes), developing agent chloroform, methanol and water at a ratio of 30:6:1 to obtain a product DSPG2.76g, wherein the yield is as follows: 49.95%, product purity: 97.82 percent

HPLC conditions: same as example 1 (1.3)

Comparing the results of the comparative example 1 and the experimental examples, the product yield in the examples is higher, and complicated column chromatography purification is avoided, so that the production is more convenient.

Claims

1. A preparation process of distearoyl phosphatidyl glycerol sodium (DSPGNa) is characterized in that the reaction equation is as follows:

2. the process for preparing sodium distearoylphosphatidylglycerol (dspgsna) according to claim 1, wherein the reaction equation for preparing intermediate 19(R ═ TFE) [5- (benzyloxy) -2- (2,2, 2-trifluoroethoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ]:

the method comprises the following specific steps:

preparation of intermediate 19(R ═ TFE): carrying out disubstituted reaction on the intermediate 22 phosphorus oxychloride and the compound 23 (2-benzyloxy-1, 3-propylene glycol) in the presence of an organic base reagent, carrying out monosubstitution on the phosphorus oxychloride and trifluoroethanol in the presence of the organic base reagent to obtain an intermediate 19(R ═ TFE)5- (benzyloxy) -2- (2,2, 2-trifluoroethoxy) -2-oxo-1, 3, 2-dioxaphosphorinane ], and carrying out reduced pressure distillation to obtain a pure product; preparation of intermediate 19(R ═ p-NP): the intermediate 22 phosphorus oxychloride and the compound 23 (2-benzyloxy-1, 3-propylene glycol) are subjected to disubstituted reaction in the presence of an organic alkali reagent, and are subjected to monosubstitution reaction with 4-nitro-phenol (p-NP) in the presence of an organic alkali reagent to obtain an intermediate 19(R ═ p-NP)5- (benzyloxy) -2- (4-nitro-phenoxy) -2-oxo-1, 3, 2-dioxaphosphorinane, and the intermediate is subjected to reduced pressure distillation to obtain a pure product.

3. Process for the preparation of sodium distearoylphosphatidylglycerol (DSPGNa) according to claim 1, wherein the intermediate 20(R) -3- { [5- (benzoyloxy) -2-oxo-1, 3, 2-dioxaphosphin-2-yl ] oxy } propane-1, 2-diethyldistearate is prepared: intermediate 7[ (2S) -3-hydroxy-1, 2-propanediyl distearate ] with intermediate 195- (benzyloxy) -2- (2,2, 2-trifluoroethoxy) -2-oxo-1, 3, 2-dioxaphosphorinane or 5- (benzyloxy) -2- (4-nitro-phenoxy) -2-oxo-1, 3, 2-dioxaphosphorinane in a molar ratio of 1:1, and carrying out condensation reaction in the presence of an organic base reagent to obtain an intermediate 20.

4. The process for the preparation of sodium distearoylphosphatidylglycerol (dspgcna) according to claim 1, wherein the intermediate 21: intermediate 20 is analogous to boron trifluoride, indium trichloride (InCl)₃) Tin tetrachloride (SnCl)₄) Zinc chloride (ZnCl)₂) Ring opening under the catalysis of Lewis acid to obtain an intermediate 21.

5. The process for the preparation of distearoylphosphatidylglycerol sodium (dspgcna) according to claim 1, wherein compound 1: and removing benzyl (Bzl) from the intermediate 21(2R) -3- { [2- (benzoyloxy) -3-hydroxypropoxy ] -hydroxy-phosphoryloxy } propane-1, 2-diethyl distearate under the action of trimethyl iodosilane (TMSI), and obtaining sodium methoxide to obtain the target compound 1.

6. The process for preparing distearoyl phosphatidyl glycerol sodium (DSPGNa) according to claim 2 or 3, wherein the organic base is one or more of DBU (1, 8-diazabicycloundecen-7-ene), DIEA (N, N-diisopropylethylamine) and TEA (triethylamine).