CN115304657B - Preparation method and application of estradiol phosphate and salts thereof - Google Patents

Abstract

The invention belongs to the technical field of pharmacy, and particularly relates to a preparation method and application of estradiol phosphate and salts thereof. The preparation method takes estradiol as a raw material, phosphorus oxychloride as a phosphate reagent, and the phosphate reaction is carried out in an organic solvent in the presence of organic alkali, and the estradiol phosphate is obtained through hydrolysis reaction. The invention takes estradiol as an initial raw material, reacts with phosphorus oxychloride under alkaline condition to generate estradiol phosphoryl chloride, and then hydrolyzes to generate estradiol phosphate. The estradiol phosphate can be subjected to neutralization reaction with inorganic alkali to prepare the estradiol phosphate salt. The solubility of the estradiol phosphate in water is measured to be 0.88mg/mL, and compared with the solubility of the original medicine in water is less than 0.01mg/mL, the solubility is improved by more than 80 times.

Description

Preparation method and application of estradiol phosphate and salts thereof

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to a preparation method and application of estradiol phosphate and salts thereof.

Background

Estradiol (structural formula 1) is also known as Estradiol and dihydroxyestrone, and is chemically named estra-1, 3,5 (10) -triene-3, 17-beta-diol, and is an estrogenic drug. Can be used for treating functional uterine bleeding, primary amenorrhea, climacteric syndrome and prostate cancer. Estradiol can promote and regulate normal growth of female sexual organs and secondary sexual characteristics, promote maturation and growth and localization of mammary ducts.

Estradiol phosphate is a phosphorylated prodrug (formula 2). The design method is to utilize the free hydroxyl in the medicine molecule to carry out the phosphorylation to obtain the phosphorylation of the parent medicine molecule and the derivative of the salt thereof, so as to improve the water solubility and the pharmacokinetics property of the parent medicine and improve the medicine property. The phosphorylated prodrugs only increase the water solubility of the parent compound, improve its pharmacokinetic properties, do not alter the pharmacological properties of the parent compound, do not introduce toxic pharmacophores, and are endogenous in the sense that phosphate building blocks (e.g., phosphorylated proteins and adenosines, etc.) and enzymes hydrolyse such building blocks are widely present in the organism. Therefore, the disodium salt of the estradiol phosphate can overcome the defect of too low water solubility of the estradiol and can simultaneously maintain the physiological activity of the estradiol.

Spivey et al disclose a process for the preparation of estradiol phosphates (Synlett, 2015,26 (7): 985-990): the corresponding phosphate is obtained by the phosphorylation reaction of estradiol with o-XPCl under the catalysis of pyridine nitroxide, followed by hydrogenolysis (H ₂, pd/C) or deprotection with acids (e.g. HBr or AcOH). The phosphorylating reagent used is difficult to synthesize and is not commercially available, but the pyridine-N-oxide has a high boiling point to remove, which adds difficulty to the post-treatment. Meanwhile, the route also needs hydrogenolysis to remove the protecting group, which increases the reaction steps and the synthesis cost.

Disclosure of Invention

The invention aims to solve the technical problems of providing a preparation method and application of estradiol phosphate and salts thereof with low cost, simple process and high yield aiming at the prior art. The invention takes estradiol as an initial raw material, reacts with phosphorus oxychloride under alkaline condition to generate estradiol phosphoryl chloride, and then hydrolyzes to generate estradiol phosphate. The estradiol phosphate can be subjected to neutralization reaction with inorganic alkali to prepare the estradiol phosphate salt. The solubility of the estradiol phosphate in water is measured to be 0.88mg/mL, and compared with the solubility of the original medicine in water is less than 0.01mg/mL, the solubility is improved by more than 80 times.

The technical scheme provided by the invention is as follows:

A method for preparing estradiol phosphate, comprising the following steps: using estradiol as a raw material, using phosphorus oxychloride as a phosphate reagent, carrying out phosphate esterification reaction in an organic solvent in the presence of organic alkali, and carrying out hydrolysis reaction to obtain estradiol phosphate, wherein the reaction formula is as follows:

in the technical scheme, phosphorus oxychloride has high selectivity to phenolic hydroxyl groups in estradiol, and the reaction condition is mild.

Specifically, the preparation method of the estradiol phosphate comprises the following steps:

1) Dissolving estradiol in an organic solvent, adding organic base as an acid binding agent, slowly dropwise adding phosphorus oxychloride, and reacting to obtain an estradiol phosphoryl chloride intermediate;

2) And adding water into the estradiol phosphoryl chloride intermediate to hydrolyze, then adding inorganic alkali to adjust the reaction liquid to be alkaline, adding ethyl acetate to extract, separating the liquid to obtain a water phase, and adding acid into the water phase to acidify to obtain the estradiol phosphate.

Specifically, in step 1, the molar ratio of the reaction raw materials is: estradiol is an acid binding agent, and phosphorus oxychloride=1:1-10:1-10.

Specifically, in the step 1), the organic solvent is any one or a mixture of a plurality of solvents selected from tetrahydrofuran, acetonitrile, chloroform, dichloromethane and 1, 2-dichloroethane.

Specifically, in the step 1), the acid binding agent is triethylamine, pyridine, N-diisopropylethylamine or 4-dimethylaminopyridine or tetrabutylammonium bromide.

Specifically, in the step 2), the inorganic base is sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate or lithium hydroxide.

Specifically, in the step 2), the acid is hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid or hydroiodic acid.

The invention also provides a preparation method of the estradiol phosphate, which comprises the following steps:

1) The estradiol phosphate is prepared by adopting the preparation method of the estradiol phosphate;

2) And (3) carrying out neutralization reaction on the estradiol phosphate and inorganic base to obtain the estradiol phosphate salt.

The invention also provides application of the estradiol phosphate or the estradiol phosphate salt in preparing soluble medicaments or injection.

Compared with the prior art, the invention has the following advantages and effects:

1) The invention has mild reaction condition and simple operation.

2) The adopted reaction raw materials are low in price, the industrial cost is reduced, and the product yield is high.

3) The invention adopts environment-friendly solvent, and does not need to use solvents with high price and high toxicity.

Drawings

FIG. 1 is a standard curve of estradiol phosphate concentration versus absorbance.

Detailed Description

The principles and features of the present invention are described below with examples only to illustrate the present invention and not to limit the scope of the present invention.

Example 1:

Estradiol (0.20 g,0.73 mmol), pyridine (0.06 g,0.73 mmol) and dichloromethane (5 mL) are sequentially added into a 25mL flask, the reaction flask is placed in an ice-water bath for continuous stirring for 20min after stirring for 10min, phosphorus oxychloride (0.11 g,0.73 mmol) is slowly added dropwise into a solution of dichloromethane (10 mL), after 1h dropwise addition, stirring is continued in the ice-water bath for 2h, the temperature is naturally raised to room temperature for continuous stirring for 2h, TLC shows that the raw materials are reacted, 1mL of water is added, and stirring is continued for 2h at room temperature. 10% sodium hydroxide solution is added into the reaction solution, the pH of the reaction solution is regulated to 11, ethyl acetate is added for extraction, water phase is obtained by separating, 10% hydrochloric acid is added into the water phase for acidification until the pH=2, solid is separated out, filtration and drying are carried out, 0.22g of white estradiol phosphate solid is obtained, and ¹H NMR、³¹ P NMR and HR-MS data of the products in the examples 2-8 are consistent with the data of the example 1 in yield 86％.¹H NMR(400MHz,Methanol-d₄)δ8.44(d,J＝8.9Hz,1H),7.93–7.83(m,2H),3.49(s,2H),3.22(d,J＝3.4Hz,1H),3.19(s,1H),3.03(dd,J＝13.9,7.0Hz,3H),2.84–2.45(m,9H),1.36(s,3H);³¹P NMR(162MHz,DMSO-d₆)δ1.32;HR-MS(ESI)calcd for C₁₈H₂₅O₅P[M-H]^-m/z 351.1440,found 351.1360..

Example 2:

Estradiol (0.20 g,0.72 mmol), pyridine (0.12 g,1.44 mmol) and acetonitrile (5 mL) are sequentially added into a 25mL flask, the reaction flask is placed in an ice-water bath for continuous stirring for 20min after stirring for 10min, phosphorus oxychloride (0.22 g,1.46 mmol) is slowly added dropwise into a solution of acetonitrile (10 mL), after 1h dropwise addition, stirring is continued in the ice-water bath for 2h, the temperature is naturally raised to room temperature for continuous stirring for 2h, TLC shows that the raw materials are reacted, 1mL of water is added, and stirring is continued for 2h at room temperature. Adding 10% potassium hydroxide solution into the reaction solution, adjusting the pH of the reaction solution to 11, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% sulfuric acid into the water phase for acidification to pH=2, separating out solid, filtering, and drying to obtain 0.23g white estradiol phosphate solid, wherein the yield is 90%.

Example 3:

Estradiol (0.20 g,0.73 mmol), triethylamine (0.74 g,7.34 mmol) and acetonitrile (5 mL) are sequentially added into a 25mL flask, the flask is placed into an ice-water bath for continuous stirring for 20min after stirring for 10min, phosphorus oxychloride (0.22 g,1.46 mmol) is slowly added dropwise into a solution of acetonitrile (5 mL), after 1h dropwise addition, stirring is continued in the ice-water bath for 2h, the temperature is naturally raised to room temperature for continuous stirring for 2h, TLC shows that the raw materials are reacted, 1mL of water is added, and stirring is continued at room temperature for 2h. Adding 10% sodium carbonate solution into the reaction solution, adjusting the pH of the reaction solution to 8, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% nitric acid into the water phase for acidification until the pH=2, separating out solid, filtering, and drying to obtain 0.17g white estradiol phosphate solid, wherein the yield is 65%.

Example 4:

Estradiol (0.20 g,0.73 mmol), N-diisopropylethylamine (0.19 g,1.46 mmol) and tetrahydrofuran (5 mL) were added sequentially to a 25mL flask, the flask was placed in an ice-water bath after stirring for 10min, stirring was continued for 20min, phosphorus oxychloride (0.22 g,1.46 mmol) was slowly added dropwise to a solution of tetrahydrofuran (5 mL), after 1h dropwise addition was completed, stirring was continued in the ice-water bath for 2h, natural warming was continued to room temperature for stirring for 2h, TLC indicated that the starting material had reacted, 1mL of water was added, stirring was continued at room temperature for 2h. Adding 10% sodium bicarbonate solution into the reaction solution, adjusting the pH of the reaction solution to 8, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% hydrobromic acid into the water phase for acidification until the pH=2, separating out solid, filtering, and drying to obtain 0.18g of white estradiol phosphate solid, wherein the yield is 73%.

Example 5:

Estradiol (0.20 g,0.73 mmol), 4-dimethylaminopyridine (0.18 g,1.46 mmol) and chloroform (5 mL) were sequentially added to a 25mL flask, the flask was placed in an ice-water bath after stirring for 10min, stirring was continued for 20min, phosphorus oxychloride (0.22 g,1.46 mmol) was slowly added dropwise to a solution of chloroform (5 mL), after 1h dropwise addition was completed, stirring was continued in the ice-water bath for 2h, then naturally warmed to room temperature and stirred for 2h, TLC showed that the starting material had reacted, 1mL of water was added, and stirring was continued at room temperature for 2h. Adding 10% potassium carbonate solution into the reaction solution, adjusting the pH of the reaction solution to 8, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% hydroiodic acid into the water phase for acidification until the pH=2, separating out solid, filtering, and drying to obtain 0.19g white estradiol phosphate solid, wherein the yield is 76%.

Example 6:

estradiol (0.20 g,0.73 mmol), N-diisopropylethylamine (0.095 g,0.73 mmol), 4-dimethylaminopyridine (0.09 g,0.73 mmol) and 1, 2-dichloromethane (5 mL) were added sequentially to a 25mL flask, the flask was placed in an ice-water bath for stirring for 10min, stirring was continued for 20min, phosphorus oxychloride (0.22 g,1.46 mmol) was slowly added dropwise to a solution of 1, 2-dichloromethane (5 mL), after 1h dropwise addition was completed, stirring was continued in the ice-water bath for 2h, natural temperature was raised to room temperature for continuing stirring for 2h, TLC showed that the starting material had reacted, 1mL of water was added, and stirring was continued for 2h at room temperature. Adding 10% potassium bicarbonate solution into the reaction solution, adjusting the pH of the reaction solution to 8, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% hydrochloric acid into the water phase for acidification until the pH=2, separating out solid, filtering, and drying to obtain 0.15g white estradiol phosphate solid, wherein the yield is 15%.

Example 7:

Estradiol (0.20 g,0.73 mmol), pyridine (0.12 g,1.46 mmol) and acetonitrile (5 mL) are sequentially added into a 25mL flask, the reaction flask is placed into an ice-water bath for continuous stirring for 20min after stirring for 10min, phosphorus oxychloride (0.22 g,1.46 mmol) is slowly added dropwise into a solution of acetonitrile (5 mL), after 1h dropwise addition, stirring is continued in the ice-water bath for 2h, the temperature is naturally raised to room temperature for continuous stirring for 2h, TLC shows that the raw materials are reacted, 1mL of water is added, and stirring is continued for 2h at room temperature. Adding 10% potassium hydroxide solution into the reaction solution, adjusting the pH of the reaction solution to 11, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% hydrochloric acid into the water phase for acidification to pH=2, separating out solid, filtering, and drying to obtain 0.19g white estradiol phosphate solid with a yield of 74%.

Example 8:

Estradiol (0.20 g,0.73 mmol), pyridine (0.12 g,1.46 mmol) and acetonitrile (5 mL) are sequentially added into a 25mL flask, the reaction flask is placed into an ice-water bath for continuous stirring for 20min after stirring for 10min, phosphorus oxychloride (1.12 g,7.34 mmol) is slowly added dropwise into a solution of acetonitrile (5 mL), after 1h dropwise addition, stirring is continued in the ice-water bath for 2h, the temperature is naturally raised to room temperature for continuous stirring for 2h, TLC shows that the raw materials are reacted, 1mL of water is added, and stirring is continued for 2h at room temperature. Adding 10% lithium hydroxide solution into the reaction solution, adjusting the pH of the reaction solution to 11, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% hydrochloric acid into the water phase for acidification until the pH=2, separating out solid, filtering, and drying to obtain 0.13g white estradiol phosphate solid with the yield of 50%.

Example 9: solubility determination of estradiol phosphate

1) Determination of detection wavelength: and taking a certain amount of estradiol phosphate reference substance, preparing estradiol phosphate solutions with different concentrations, and scanning within 200-500 nm. The results show that the estradiol phosphate solutions with different concentrations have maximum absorption at 278nm, so 278nm wavelength is chosen as the experimental determination wavelength.

2) Drawing a standard curve: 20.4mg of estradiol phosphate solid is weighed into a 100mL volumetric flask, dissolved by adding methanol and diluted to a scale, and used as a stock solution. Shaking, sequentially weighing stock solutions 1, 2, 4, 6, 8 and 10mL, respectively placing in 100mL volumetric flasks, and adding purified water to dilute to scale. Solutions were prepared at concentrations of 2.04, 4.08, 8.16, 12.24, 16.32 and 20.4. Mu.g/mL, respectively, and the absorbance was measured at 278nm, and the absorbance corresponding to estradiol phosphate at different concentrations is shown in Table 1. The mass concentration (c) was linearly regressed with absorbance (a) to determine a standard curve, as shown in fig. 1, with regression equations a= 3.708c-0.0152 and r ² =0.971.

(3) Solubility test of estradiol phosphate: adding excessive estradiol phosphate into 6mL of purified water, carrying out ultrasonic treatment for 10min, stirring at 25 ℃ for 24h, taking supernatant, filtering with a 0.45 mu m microporous filter membrane, taking 2mL of filtrate, placing into a 100 volumetric flask, adding purified water to dilute to a scale, measuring the absorbance at 278nm, calculating to obtain saturated solution with concentration of 0.88mg/mL according to a standard curve method, and improving the solubility of the estradiol phosphate in water by more than 80 times compared with the solubility of the original medicine in water of less than 0.01 mg/mL.

Table 1 absorbance corresponding to different concentrations of estradiol phosphate

Example 10: examples of preparation of estradiol phosphate

Estradiol phosphate (1.00 g,2.84 mmol) was dissolved in 10mL ethanol, 1mL aqueous solution of sodium hydroxide (0.23 g,5.68 mol) was added, stirred at 25 ℃ for 2 hours, cooled to 0 ℃ and left to stand to precipitate solid, namely disodium estradiol phosphate, filtered and dried to give 0.93g of white solid with a yield of 82%. The solubility test method of the disodium estradiol phosphate is the same as that of the estradiol phosphate, and the solubility of the estradiol phosphate in water is improved by more than 100 times compared with that of the original medicine estradiol.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (2)

1. The preparation method of the estradiol phosphate is characterized by comprising the following steps: sequentially adding 0.72mmol of estradiol, 1.44mmol of pyridine and 5mL of acetonitrile into a 25mL flask, stirring for 10min, placing the reaction bottle into an ice water bath, continuously stirring for 20min, slowly dropwise adding 1.46mmol of phosphorus oxychloride into a 10mL solution of acetonitrile, after 1h dropwise adding, continuously stirring for 2h in the ice water bath, naturally heating to room temperature, continuously stirring for 2h, TLC (thin layer chromatography) shows that the raw materials are reacted, adding 1mL of water, continuously stirring for 2h at room temperature, adding 10% of potassium hydroxide solution into the reaction solution, adjusting the pH of the reaction solution to 11, adding ethyl acetate for extraction, separating to obtain a water phase, adding 10% of sulfuric acid into the water phase, acidifying to pH=2, precipitating solid, filtering and drying to obtain white estradiol phosphate solid.

2. A method for preparing estradiol phosphate, comprising the steps of:

1) The estradiol phosphate prepared by the preparation method of claim 1;

2) And (3) carrying out neutralization reaction on the estradiol phosphate and inorganic base to obtain the estradiol phosphate salt.