WO2020056974A1 - 一种泊沙康唑磷酸酯单胆碱盐及其制备方法和用途 - Google Patents

一种泊沙康唑磷酸酯单胆碱盐及其制备方法和用途 Download PDF

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WO2020056974A1
WO2020056974A1 PCT/CN2018/123343 CN2018123343W WO2020056974A1 WO 2020056974 A1 WO2020056974 A1 WO 2020056974A1 CN 2018123343 W CN2018123343 W CN 2018123343W WO 2020056974 A1 WO2020056974 A1 WO 2020056974A1
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formula
compound represented
posaconazole
preparation
compound
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PCT/CN2018/123343
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English (en)
French (fr)
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杨成
陆华龙
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陕西合成药业股份有限公司
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Priority to JP2020570616A priority Critical patent/JP7026264B2/ja
Priority to AU2018441838A priority patent/AU2018441838B2/en
Priority to ES18933805T priority patent/ES2923683T3/es
Priority to US17/048,745 priority patent/US20210147455A1/en
Priority to DK18933805.6T priority patent/DK3770165T3/da
Priority to EP18933805.6A priority patent/EP3770165B1/en
Publication of WO2020056974A1 publication Critical patent/WO2020056974A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics

Definitions

  • the invention relates to a posaconazole phosphate monocholine salt, a preparation method and uses thereof.
  • Fungal infections are common and frequently-occurring diseases in the clinic. Infection can be divided into two types: superficial fungal infection and deep fungal infection. Superficial fungal infections are caused by ringworms invading skin, hair, fingernails, and other body surface parts, with a high incidence and less harmfulness. Deep fungal infections are caused by fungi such as Candida, Aspergillus, and Cryptococcus invading internal organs and deep tissues, which are highly harmful.
  • Posaconazole is a derivative of itraconazole. Its oral suspension was first marketed in Germany in 2005 and approved by the FDA in 2006. It is clinically effective against systemic fungi caused by Aspergillus and Candida. Infections and oropharyngeal candidiasis infections have good curative effects. Currently, they have been approved in more than 70 countries and regions around the world, and have been marketed in more than 40 countries and regions such as the United States and the European Union. However, the degree of absorption of oral suspensions is extremely susceptible to factors such as food and gastrointestinal function, which cause large differences in pharmacokinetic parameters between individuals, large fluctuations in plasma concentration values, and low bioavailability.
  • posaconazole is a weakly alkaline and poorly water-soluble drug, which is not easy to develop into an injection form.
  • Some immunosuppressed patients undergoing chemotherapy or organ transplantation have problems such as nausea and vomiting and gastrointestinal discomfort, which makes oral administration difficult and requires injection.
  • SBE- ⁇ -CD sulfobutyl ether- ⁇ -cyclodextrin
  • the target indications for posaconazole injection are bone marrow transplantation, chemotherapy, etc.
  • a considerable part of this group of patients have renal impairment, especially those with moderate or severe renal insufficiency, the glomerular filtration efficiency is low, SBE- ⁇ -CD in A large amount of accumulation in the body poses a high safety risk.
  • the use of the excipient sulfobutyl ether- ⁇ -cyclodextrin greatly limits the clinical application of the drug.
  • the instructions for posaconazole injection specifically state that the drug is not suitable for patients with moderate to severe renal impairment. Therefore, improving the deficiencies in the prior art, increasing the medication safety and drug applicability of patients with renal injury, has important clinical value.
  • the present invention provides a compound of formula (I) having antifungal infection, a method for preparing the compound, a pharmaceutical composition containing the compound, and an antifungal compound prepared by the compound. Use in medicine for infections.
  • the present invention provides a compound represented by formula (I) (Posaconazole phosphate monocholine salt)
  • n is an integer of 0 to 12, preferably an integer of 0 to 8, and more preferably an integer of 0 to 6.
  • n may be 0, 1, 2, 3, 4, 5, or 6.
  • the present invention provides a method for preparing the compound represented by the above formula (I), which method includes:
  • step (b) hydrolyzing the compound represented by formula C formed in step (a) with a solvent B to form a compound represented by formula D:
  • step (c) reacting the compound represented by formula D obtained in step (b) with choline hydroxide in solvent C to prepare the compound represented by formula (I);
  • the inert gas is selected from one or more of nitrogen, helium, and argon, and is preferably nitrogen or argon.
  • the organic solvent A is selected from the group consisting of aromatic hydrocarbons, halogenated hydrocarbons, nitriles, ketones, ethers, triethylamine, diethylamine, and pyridine. Or one or more of 1,1-methylimidazole, N, N-diisopropylethylamine and esters, preferably ethyl acetate, acetonitrile, tetrahydrofuran, dichloromethane, toluene, acetone, triethylamine , 1-methylimidazole, pyridine or chloroform.
  • the solvent B is selected from one or more of water, an alkaline aqueous solution and an organic solvent aqueous solution.
  • the alkaline aqueous solution is preferably an aqueous sodium hydroxide solution, ammonia, potassium hydroxide or sodium carbonate
  • the organic solvent aqueous solution is preferably a methanol aqueous solution, an ethanol aqueous solution, an isopropanol aqueous solution, or an acetone aqueous solution.
  • the solvent C is selected from one of water, aromatic hydrocarbons, halogenated hydrocarbons, nitriles, ketones, ethers, alcohols, and esters.
  • the reaction temperature is -10 ° C to 50 ° C, and preferably 0 to 35 ° C.
  • the hydrolysis temperature is -20 ° C to 30 ° C, preferably -5 to 10 ° C.
  • the reaction temperature is -10 ° C to 80 ° C, preferably 10 to 40 ° C.
  • the molar ratio between the compound represented by the formula A and the compound represented by the formula B is 1: 1.0 to 20.0, preferably 1: 2.25 ⁇ 10.0.
  • the molar ratio between the compound represented by the formula D and the choline hydroxide is 1: 0.5-2, and preferably 1: 1.05.
  • the present invention provides the use of a compound represented by the above formula (I) for preparing a medicament for antifungal infection; preferably, the fungal infection is an infection caused by Candida or Cryptococcus.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound represented by the formula (I) and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is a tablet, suppository, dispersible tablet, enteric tablet, chewable tablet, orally disintegrating tablet, capsule, dragee, granule, dry powder, oral solution, injection needle, injection Freeze-dried powder injection or large infusion.
  • the pharmaceutically acceptable excipient is selected from one or more of the following: a pH adjuster, a diluent, a solubilizer, a disintegrant, a suspending agent, a lubricant, a binder, a filler, a correction agent Flavors, sweeteners, antioxidants, surfactants, preservatives, encapsulants and pigments.
  • the dosage and method of use of the compound of the present invention depends on many factors, including the age, weight, sex, health status, nutritional status, intensity of the compound's activity, duration of use, metabolic rate, severity of the condition, and subjective judgment of the treating physician. .
  • the preferred dosage is between 2 and 1200 mg / kg; the optimal dosage for 24 hours is 0.2 to 300 mg per kg, and multiple administrations can also be used.
  • the present invention has at least the following beneficial effects:
  • the compound represented by formula (I) of the present application that is, the monocholine salt compound has higher solubility, and has better stability under influencing factors (high temperature, high humidity, light) and long-term storage conditions, which is significantly higher than Other posaconazole phosphate monosalts and posaconazole phosphate disalts.
  • the monocholine salt compound represented by formula (I) of the present application when administered into the body, it functions as a "prodrug" and is converted into a biologically active parent posa in the presence of alkaline phosphatase. Conazole.
  • the monocholine salt compound represented by formula (I) of the present application has low hygroscopicity and thus has unexpectedly improved physical stability, which makes it easier to handle during the preparation process, while maintaining proper solubility, making the former
  • the drug is suitable for oral, topical and parenteral administration.
  • Sample concentration 1mg / ml dilution medium is 50% acetonitrile
  • the reaction solution was added dropwise to 150 mL of pure water at 0 ° C, and the hydrolysis temperature was controlled at 0 to 5 ° C. After stirring for 16 hours, the organic phase and the aqueous phase were transferred to a separation funnel for extraction, and the solid phase was dissolved and extracted with MeOH (150 mL). The organic phases were combined, and then about 25 g of silica gel of 200 to 300 mesh was poured into it, and the solvent was evaporated to dryness on a rotary evaporator. The evaporated silica gel was poured into a column (diameter 4.5 cm) of 25 cm silica gel. The target product began to elute from the column. The collected solvent was removed by rotary evaporation to obtain a yellow solid.
  • Sample concentration 1mg / ml dilution medium is 50% acetonitrile
  • reaction solution was added dropwise to 800 mL of an aqueous sodium hydroxide solution at 0 ° C, and the hydrolysis temperature was controlled at 0 to 5 ° C. After completion of the hydrolysis, the pH was adjusted to 3 to 4 with 10% hydrochloric acid. Filter, wash the cake with acetone, and blow dry at 25-40 ° C. 8.3 g of off-white solid was obtained. Related substances: 0.42% single impurity and 1.02% total impurity.
  • Example 2 The same batch of posaconazole as in Example 1 was used to repeat the steps 1 and 2 of Example 2 for testing, except that the drying method of Step 2 was changed to 35 ⁇ 5 ° C. air drying.
  • the title compound was obtained as a white solid. (Relevant substances: 0.13%; moisture: 9.06%; content 99.87%).
  • the reaction solution was added dropwise to 150 mL of pure water at 10 ° C., and the hydrolysis temperature was controlled at 10 ° C., and stirred for 16 h.
  • the organic phase and the aqueous phase were transferred to a separating funnel for extraction, and the solid phase was dissolved in the extracted organic with MeOH (150 mL).
  • the reaction solution was added dropwise to 150 mL of pure water at 0 ° C, the hydrolysis temperature was controlled at -5 to 0 ° C, and stirred for 16 hours.
  • the organic and aqueous phases were transferred to a separation funnel for extraction, and the solid phase was dissolved in MeOH (150 mL) and The extracted organic phases were combined, and then about 25 g of silica gel of 200 to 300 mesh was poured into it.
  • the rotary evaporator was used to evaporate the solvent and mixed with the sample.
  • the evaporated silica gel was poured into a column (diameter 4.5 cm) containing 25 cm silica gel.
  • the target product began to elute from the column, and the collected solvent was removed by rotary evaporation to obtain a yellow solid.
  • choline hydroxide (0.31g, 1.28mmol, 50%) into a beaker containing 5mL of pure water, stir well, and then pour the compound (Formula D) (1g, 1.28mmol) into the beaker, 35 ° C Stir for 1 h to dissolve the solid, filter, pour the filtrate into a beaker containing 50 mL of methanol, stir at room temperature for 12 h, suction filter to obtain a white solid, rinse with 5 mL of methanol, and dry in vacuo to give 0.25 g of a white solid.
  • Related substances single impurity 0.02%, total impurity 0.10%.
  • the reaction solution was added dropwise to 150 mL of pure water at 0 ° C, the hydrolysis temperature was controlled at -5 to 0 ° C, and stirred for 16 hours.
  • the organic and aqueous phases were transferred to a separation funnel for extraction, and the solid phase was dissolved in MeOH (150 mL) and The extracted organic phases were combined, and then about 25 g of silica gel of 200 to 300 mesh was poured into it.
  • the rotary evaporator was used to evaporate the solvent and mixed with the sample.
  • the evaporated silica gel was poured into a column (diameter 4.5 cm) containing 25 cm silica gel.
  • the target product began to elute from the column, and the collected solvent was removed by rotary evaporation to obtain a yellow solid.
  • choline hydroxide (0.31g, 1.28mmol, 50%) into a beaker containing 5mL of pure water, stir well, and then pour the compound (Formula D) (1g, 1.28mmol) into the beaker, 35 ° C Stir for 1h to dissolve the solid, filter, pour the filtrate into a beaker containing 50mL of isopropanol, stir at room temperature for 12h, suction filter to obtain a white solid, rinse with 5mL of isopropanol, and dry in vacuo to obtain a white solid 0.21g.
  • Related substances single impurity 0.22%, total impurity 0.53%.
  • Preparation method take posaconazole phosphate monocholine salt and crush it through an 80 mesh sieve; weigh the prescribed amount of starch and the prescribed amount of posaconazole phosphate monocholine salt, microcrystalline cellulose, and mix well.
  • the material is made into a soft material with a 4% povidone K30 solution, granulated with a 20-mesh sieve, and dried at 40 to 60 ° C. until the moisture in the particles is about 5%.
  • Preparation method take posaconazole phosphate monocholine salt pentahydrate and crush it through an 80 mesh sieve; weigh the prescribed amount of starch and the prescribed amount of posaconazole phosphate monocholine salt pentahydrate, microcrystalline fiber Vegetarian, mix well.
  • the material is made into a soft material with a 4% povidone K30 solution, granulated with a 20-mesh sieve, and dried at 40 to 60 ° C. until the moisture in the particles is about 5%.
  • Preparation method add batch volume of water for injection, weigh the prescribed amount of posaconazole phosphate monocholine salt pentahydrate and mannitol, stir to fully dissolve, adjust the pH to 5-10 with hydrochloric acid, 0.22 ⁇ m micropore Filter membrane filtration, filling; freeze-drying, capping, packaging.
  • Preparation method Add batch volume of water for injection, weigh the prescribed amount of posaconazole phosphate monocholine salt, glucose, stir to fully dissolve, adjust the pH to 5-10 with hydrochloric acid, and filter with 0.22 ⁇ m microporous membrane. Filling; freeze-drying, capping, packaging.
  • Example 11 Preparation of posaconazole phosphate monocholine for injection
  • Preparation method adding batch volume of water for injection, weighing the prescribed amount of posaconazole phosphate monocholine salt and stirring to fully dissolve it, filtering through a 0.22 ⁇ m microporous membrane, filling; freeze drying, capping, and packaging.
  • Example 2 The same batch of posaconazole as in Example 1 was used to repeat steps 1 and 2 of Example 1 for testing, except that the molar ratio of the amount of choline hydroxide charged in step 2 to the amount of compound (Formula D) was replaced. It's 2: 1. The title compound was obtained as a white solid.
  • Example 2 The same batch of posaconazole as in Example 1 was used to repeat steps 1 and 2 of Example 3 for testing, except that the molar ratio of the amount of choline hydroxide charged in step 2 to the amount of compound (Formula D) was replaced. It's 2: 1. The title compound was obtained as a white solid.
  • Example 2 The same batch of posaconazole as in Example 1 was used to repeat steps 1 and 2 of Example 1 for testing, except that the choline hydroxide in step 2 was converted to potassium hydroxide, and potassium hydroxide and the compound (formula D) The molar ratio is 1: 1. The title compound was obtained as a white solid.
  • Example 2 Use the same batch of posaconazole as in Example 1 to repeat steps 1 and 2 of Example 1 for testing, except that the choline hydroxide in step 2 is converted to meglumine, and meglumine and the compound (formula D) The molar ratio is 1: 1. The title compound was obtained as a white solid.
  • Example 2 Use the same batch of posaconazole as in Example 1 to repeat steps 1 and 2 of Example 1 for testing, except that the choline hydroxide in step 2 is converted to arginine, and arginine and the compound (formula D) The molar ratio is 1: 1. The title compound was obtained as a white solid.
  • Example 2 Use the same batch of posaconazole as in Example 1 to repeat steps 1 and 2 of Example 3 for testing, except that the choline hydroxide in step 2 is converted to potassium hydroxide, and potassium hydroxide and the compound (formula D) The molar ratio is 1: 1. The title compound was obtained as a white solid.
  • Example 2 The same batch of posaconazole as in Example 1 was used to repeat the steps 1 and 2 of Example 3 for testing, except that the choline hydroxide in step 2 was converted into sodium hydroxide, and the sodium hydroxide and the compound (formula D) The molar ratio is 1: 1. The title compound was obtained as a white solid.
  • Example 2 Use the same batch of posaconazole as in Example 1 to repeat steps 1 and 2 of Example 1 for testing, except that the choline hydroxide in step 2 is converted to sodium hydroxide, and the sodium hydroxide and the compound (formula D) The molar ratio is 1: 1. The title compound was obtained as a white solid.
  • Example 2 The same batch of posaconazole as in Example 1 was used to repeat the steps 1 and 2 of Example 3 for testing, except that the choline hydroxide in step 2 was converted into sodium hydroxide, and the sodium hydroxide and the compound (formula D) The molar ratio is 2: 1. The title compound was obtained as a white solid.
  • Example 2 Use the same batch of posaconazole as in Example 1 to repeat steps 1 and 2 of Example 1 for testing, except that the choline hydroxide in step 2 is converted to sodium hydroxide, and the sodium hydroxide and the compound (formula D) The molar ratio is 2: 1. The title compound was obtained as a white solid.
  • Step A An oven-dried 1L round-bottomed flask equipped with a mechanical stirrer, a nitrogen inlet adapter, a pressure-equilibrium addition funnel and a temperature probe equipped with a rubber septum, and sodium hydride (2.89g, 0.069mol, 60%) ) And THF (50 mL).
  • Posaconazole a compound of formula A
  • dissolved in 30 mL of THF the same batch of posaconazole as in Example 1 was selected
  • 17.94 g, 0.023 mol was added dropwise at room temperature over 20 minutes to The stirred suspension.
  • reaction mixture was poured into ice water (100 mL).
  • the aqueous phase was separated and extracted with ethyl acetate (3 ⁇ 50 mL).
  • the combined organic extracts were washed with 10% sodium thiosulfite (50 mL), water (50 mL), brine (50 mL), dried over magnesium sulfate and concentrated under reduced pressure.
  • a light yellow oil was obtained (22.8 g, during which the reaction degree of the product was followed by HPLC to over 97% as the end point of the reaction).
  • the crude product (formula IV) is used as is in step B.
  • Step B The round bottom flask is equipped with a magnetic stirrer, a cooling bath, a pH probe, and a nitrogen inlet and outlet, and the product of the above Step A (Formula IV) (7.5 g) dissolved in CH 2 Cl 2 (23 mL) is added thereto and Cool to 0 ° C. To this stirred solution was slowly added trifluoroacetic acid (8.8 mL) and stirred for 3 hours to complete the reaction. HPLC was used to determine whether the reaction was complete. The reaction mixture was poured into a cooled solution of 2N NaOH (64 mL). The reaction mixture was extracted with tert-butyl acetate (2 x 65 mL) to remove all organic impurities.
  • step B may be performed in a continuous process, and details thereof may be determined by those skilled in the art.
  • Step C The product V obtained above was dissolved in methanol (75 mL). L-Lysine (1.8 g) was added to the free acid solution and the pH was maintained at 4.2 to 5.5, and the mixture was heated at 60 ° C for 4.5 hours. The hot reaction was filtered through a layer of diatomaceous earth. The filtrate was concentrated to approximately 5 mL, mixed with ethanol (100 mL) and heated to 65 ° C to crystallize the solvate of the monolysine salt. This solvate was collected on a Buchner funnel and dried under vacuum to give 3.0 g of the title solvate compound as a crystalline solid.
  • the compounds prepared by the present invention have higher solubility, which are higher than the compounds obtained in Comparative Examples 1-11 and the solubility of posaconazole in water, methanol, and isopropanol.
  • the solubility of the compound of the present invention is better than that of the compound obtained in Comparative Examples 1-11 and posaconazole. Therefore, the compound of the present invention has fast onset of action, high bioavailability, and improved formulation stability . Therefore, the compound prepared by the present invention is of great significance for improving its bioavailability and curative effect.
  • Test method 1. Take a dry glass weighing bottle with a stopper (outer diameter: 50mm, height: 15mm), and place it in a suitable constant temperature desiccator at 25 °C ⁇ 1 °C (a saturated ammonium chloride solution is placed in the lower part) the day before the test. Inside, accurately weigh (m 1 ). 2. Take an appropriate amount of the test product and spread it in the above weighing bottle. The thickness of the test product is about 1mm and the weight is accurately measured (m 2 ). 3. Open the weighing bottle and place it with the bottle cap under the above constant temperature and humidity conditions for 24 hours. 4. Close the lid of the weighing bottle and accurately weigh it (m 3 ).
  • Deliquescence Absorb sufficient water to form a liquid.
  • the moisture gain is not less than 15%.
  • Hygroscopicity The moisture gain is less than 15% but not less than 2%
  • the moisture gain is less than 2% but not less than 0.2%.
  • the moisture gain is less than 0.2%.
  • the stability test of the posaconazole phosphate monocholine salt of the present invention and the compound of the comparative example was performed at 25 ° C ⁇ 2 ° C, 65% RH ⁇ 5% RH conditions, and in January and March , June, September, December, and 24 samples were taken to determine properties, related substances, and content. The results are shown in the table below.
  • the posaconazole phosphate monocholine salt prepared by the present invention has undergone accelerated test for 6 months, and no significant change has been observed in various indicators compared with that of 0 month, and the compounds of Comparative Examples 1-11 have undergone accelerated test 6 Compared with the month of January, various inspection indexes were significantly increased, indicating that the stability of the posaconazole phosphate monocholine salt of the present invention is better than that of the compounds of Comparative Examples 1-11.
  • the compounds prepared by the present invention have no significant change when compared with 0 days in the various indicators examined under the conditions of high temperature, high humidity and light for 10 days, indicating that the properties of the compounds of the present invention are relatively stable.
  • Blood cell counting plate paraffin microtome
  • SPX-250B biochemical incubator ultra-clean bench
  • micro-sampler pressure steam sterilizer
  • optical microscope electronic analytical balance
  • Estradiol benzoate injection polyethylene glycol, sandcastle glucose agar solid medium.
  • KM mice weighing 18-22 g, female, were provided by Jiangsu Experimental Animal Center.
  • Candida albicans was purchased from the American Type Culture Collection and the strain number was ATCC10231.
  • mice were randomly divided into groups after weighing: posaconazole group, test compound group and vehicle group, each group of 20 animals.
  • posaconazole group was insoluble in vehicle (normal saline), so the posaconazole group was Commercially available posaconazole injection (Merzaton / Schering-Plough, 3PAR80701, hereinafter the same), solubilized with sulfobutyl ether- ⁇ -cyclodextrin.
  • mice in each group were subcutaneously injected with 0.5ml of estradiol benzoate (2mg / ml) for 6 consecutive days to enter the estrus period, and the injection was continued every 2 days until the end of the experiment. After 6 days, each mouse was injected 20ul of white candida liquid with a concentration of 3.5x10 6 CFU / ml in the vagina, which caused a vaginal infection model. From the first day after infection, the animals in each group were given the corresponding drug 20 mg / kg (calculated as posaconazole) in the tail vein. The administration volume was 0.1 ml / 0 g once a day for 5 consecutive days.
  • the model group was given an equal volume of solvent ( Saline).
  • Saline solvent
  • the concentration of bacterial solution was inoculated on a sandcastle glucose agar solid medium containing 0.5% (W / V) chloramphenicol, and the fungal load of Candida albicans on the vagina was observed.
  • Candida albicans vaginitis intravenous administration: vaginal fungal load in mice of each group
  • Example 6 Experiment of oral administration of compound of the present invention against Candida albicans vaginitis
  • Blood cell counting plate paraffin microtome
  • SPX-250B biochemical incubator ultra-clean bench
  • micro-sampler pressure steam sterilizer
  • optical microscope electronic analytical balance
  • Estradiol benzoate injection polyethylene glycol, sandcastle glucose agar solid medium.
  • KM mice weighing 18-22 g, female, were provided by Jiangsu Experimental Animal Center.
  • Candida albicans was purchased from the American Type Culture Collection and the strain number was ATCC10231.
  • mice After weighing, the mice were randomly divided into groups: posaconazole (CMC-Na) group, test compound group and vehicle group, 20 mice in each group.
  • posaconazole is insoluble in vehicle (normal saline), so in the test, the posaconazole group was a commercially available posaconazole injection (Merzaton / Schering-Plough, 3PAR80701, the same below), and sulfobutyl ether- ⁇ - The cyclodextrin was solubilized, the other tested medicinal saline was dissolved, and the ultrasound was used for administration after clarification.
  • mice in each group were subcutaneously injected with 0.5ml of estradiol benzoate (2mg / ml) for 6 consecutive days to enter the estrus period, and the injection was continued every 2 days until the end of the experiment. After 6 days, each mouse was injected 20ul of white candida liquid with a concentration of 3.5x10 6 CFU / ml in the vagina, which caused a vaginal infection model. From the first day after infection, animals in each group were orally administered with the corresponding drug 20 mg / kg (calculated based on posaconazole) in a volume of 0.1 ml / 10 g once a day for 15 consecutive days. The model group was given an equal volume of solvent ( Saline).
  • mice On the 3rd, 5th, 7th, 11th, and 15th days of infection in each group of mice, wipe the mouse's vagina with a sterile cotton swab, soak the cotton swab in 0.9 ml of main saline, and dilute the bacterial solution in 10-fold increments. A series of concentrations were then taken, and each of 100 ul of each concentration of bacterial solution was taken and inoculated on a sandcastle dextrose agar solid medium containing 0.50 / (W / V) chloramphenicol to observe the fungal load of Candida albicans on the vagina.
  • Candida albicans vaginitis vaginal fungal load of mice in each group
  • Example 7 Experiment of intravenous administration of compounds of the present invention against systemic fungal infection in water rats
  • Multiskan MK3 enzyme-labeled detector water-proof electric heating constant temperature incubator, zo-F160 full temperature shaking incubator, MJX intelligent mold incubator, SW-CT-IF type ultra-purification workbench, UV spectrophotometer.
  • ICR mice weighing 18-22 g, male, were provided by Hubei province Experimental Animal Center.
  • Candida albicans was purchased from the American Type Culture Collection and the strain number was ATCC10231.
  • Candida albicans from the SDA (Safari agar, hereinafter) stored at 4 ° C with an inoculation circle, inoculate it into 1ml YPD (Yeast Extract Peptone Dextrose Medium) culture solution, and shake at 30 ° C, 200rpm Cultivate and activate for 16 hours, so that the fungus is in the late exponential growth stage.
  • YPD Yeast Extract Peptone Dextrose Medium
  • mice were randomly divided into groups of 10 mice, each of which was a posaconazole group, a test compound group, and a vehicle group.
  • the posaconazole group was commercially available because the posaconazole was insoluble in the solvent (normal saline).
  • Posaconazole injection solubilized with sulfobutyl ether- ⁇ cyclodextrin.
  • 20 mg / kg (calculated as posaconazole) was administered to the tail vein of each administration group, and the administration volume was 0.1 ml / 10 g.
  • the model group was given 0.9% sodium chloride.
  • the solution was 0.1 ml / 10 g once a day for 5 consecutive days. Observe the death of the mice and record the survival time. Observed for 7 days. All dead mice were treated with ethanol fire.
  • Example 8 Effect of oral administration of compounds of the present invention on systemic fungal infection in mice
  • Multiskan MK3 enzyme-labeled detector water-proof electric heating constant temperature incubator, zo-F160 full temperature shaking incubator, MJX intelligent mold incubator, SW-CT-IF type ultra-purification workbench, UV spectrophotometer.
  • ICR mice weighing 18-22 g, male, were provided by the Experimental Animal Center of Jiangsu Province.
  • Candida albicans was purchased from the American Type Culture Collection and the strain number was ATCC10231.
  • mice Pick the Candida albicans monoclonal on the SDA plate, inoculate it into Iml YPD medium, incubate at 35 °C, 200rpm for 16h to the end of exponential growth period, inoculate 1% in fresh medium and incubate for 6h, and centrifuge at 1000x g for 5min. Wash with physiological saline three times until the supernatant is colorless, count with a hemocytometer, adjust the cell concentration to 5 * 10 6 cells / ml, and inject 0.1ml / 10g in the tail vein to cause systemic fungal infection in mice. The mice were randomly divided into groups of 10 mice, each of which was a posaconazole group, a test compound group, and a vehicle group.
  • the posaconazole group was a commercially available posaconazole injection, that is, sulfobutyl ether- ⁇ was used.
  • the cyclodextrin was solubilized, the other tested medicinal saline was dissolved, and the ultrasound was used for administration after clarification.
  • the administration group was administered orally with 20 mg / kg (calculated based on posaconazole), the administration volume was 0.1 ml / log, and the model group was given 0.9% / chlorinated
  • the sodium solution was 0.1 ml / log once a day for 5 consecutive days. Observe the death of the mice and record the survival time. Observed for 7 days. All dead mice were treated with ethanol fire.
  • the survival rate of the compounds of the present invention is significantly higher than that of the vehicle group: the survival rate of the listed compounds on day 7 is better than that of the posaconazole group, and the bioavailability is higher.
  • mice Male mice, 6 to 8 weeks old, weighing 190 to 215 grams, and were purchased from Beijing Weili Tonghua Experimental Animal Technology Co., Ltd. Based on the weight of the mice, they were randomly divided into 5 groups of 3 animals each. The doses and routes of administration for mice in each group are shown in the table below.
  • mice were fasted for 16 hours before the pharmacokinetic test.
  • a single dose of the compound was then administered intravenously (1 mL / kg; 1 mg / kg) as shown in Table 2.
  • Jugular vein puncture was used to periodically collect 200 ⁇ L of blood after administration, and for the intravenously administered animal group, 0, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after administration Hours of blood were collected; urine was collected at 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours.
  • the blood sample was collected in a sample tube with EDTA, the blood sample was immediately centrifuged at 4000 rpm for 5 minutes at 4 ° C, and then the plasma was transferred to another sample tube and stored at -20 degrees Celsius.
  • the concentration of posaconazole formed by the conversion of the test compound in the blood and urine samples obtained at each time point is detected, and the pharmacokinetic test is performed on the sample.
  • the methods and instruments used are as follows:
  • Pillar Phenomenex Luna5 ⁇ C18
  • Quantitative method internal standard method
  • the compound E in the comparative example is difficult to hydrolyze into active ingredients by enzymes in the body, but can be rapidly metabolized and enriched in the urine through the circulatory system in the body; greatly reducing the bioavailability of such compounds.
  • the compound of the present invention is not enriched in urine, which is beneficial to its medical application.

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Abstract

本发明涉及一种泊沙康唑磷酸酯的单胆碱盐及其制备方法和用途，本发明所述的泊沙康唑磷酸酯的单胆碱盐具有下式(I)的结构：(I) 其中： n为0至12的整数，优选为0至8的整数，更优选为0至6的整数。

Description

一种泊沙康唑磷酸酯单胆碱盐及其制备方法和用途 技术领域
本发明涉及一种泊沙康唑磷酸酯单胆碱盐及其制备方法和用途。
背景技术
真菌感染是临床上的常见病、多发病，感染可分为浅部真菌感染和深部真菌感染两类。浅部真菌感染是由癣菌侵犯皮肤、毛发、指(趾)甲等体表部位造成的，发病率高，危害性较小。深部真菌感染是由念珠菌、曲霉菌和隐球菌等真菌侵犯内脏器官及深部组织造成的，危害性大。
近年来，随着免疫抑制患者的不断增加，深部真菌感染的发病率明显增加，真菌感染，尤其是深部真菌感染日益引起人们的广泛关注。但目前临床应用的抗真菌药物而言，存在副作用大、易产生耐药性等问题。临床上现有的抗真菌药物按其结构可分为有机酸类、多烯类、氮唑类、烯丙胺类等，其中氮唑类抗真菌药物是一类发展较快的全合成抗真菌化合物，目前己成为临床上治疗深部和浅部真菌感染的主要用药，自上个世纪中叶第一个唑类化合物抗真菌作用被报道以后，第一代三唑类药物氟康唑、伊曲康唑，第二代三唑类药物伏立康唑逐渐出现在抗真菌治疗领域中。
泊沙康唑(posaconazole)是伊曲康唑的衍生物，其口服混悬剂于2005隼首次在德国上市，2006年被FDA批准上市，在临床上对曲霉菌、念珠菌导致的系统性真菌感染以及口咽念珠菌病感染有较好的疗效，目前已在全球70多个国家、地区获批，并在美国、欧盟等40多个国家、地区上市。但口服混悬剂的吸收程度极易受到食物、胃肠功能等因素影响，导致个体间药代动力学参数差异较大，血药浓度值波动范围大，生物利用度较低等问题。且泊沙康唑是一种弱碱性、水溶性差的药物，不易于开发成注射剂型。而接受化疗或器官移植的一些免疫抑制患者存在恶心呕吐以及胃肠道不适等问题，导致口服给药困难，需要采用注射给药。
为了解决泊沙康唑由于溶解度差不易于开发成注射制剂的问题，默沙东公司专利申请CN201180031488.9公开了取代β-环糊精增溶的泊沙康唑静脉输注液制剂，通过采用取代β-环糊精对泊沙康唑增溶，制备注射制剂。目前该注射剂已经在美国获准上市。该注射剂虽然解决了泊沙康唑不溶于水的缺陷，实现了对口服给药不便患者的用药，但由于添加了大量磺丁基醚-β-环糊精(SBE-β-CD)进行增溶，存在潜在的安全风险，且临床前毒理学研究显示，磺丁基醚-β-环糊精导致尿道上皮细胞空泡形成以及激活肝脏和肺内臣噬细胞。临床研究显示，磺丁基醚-β-环糊精(SBE-β-CD)需要通过肾脏代谢，大大增加了肾脏负担，而泊沙康唑注射剂的目标适应症患者为接受骨髓移植、化疗等的免疫抑制、真菌感染风险高的患者，该类患者中相当一部分人群存在肾功能损伤，特别是中度或重度肾功能不全的患者，肾小球滤过效率较低，SBE-β-CD在体内的大量蓄积，存在较高的安全风险。辅料磺丁基醚-β-环糊精的使用，大大限制了该药物的临床应用范围。泊沙康唑注射剂说明书中特别指出，该药物不适用于中、重度的肾损伤患者。因此，改善现有技术中存在的不足，增加肾损伤患者的用药安全及药物适用性，具有重要的临床价值。
发明内容
为了克服现有技术的不足，本发明提供一种具有抗真菌感染的式(I)所示的化合物、所述化合物的制备方法、包含所述化合物的药物组合物以及所述化合物在制备抗真菌感染的药物中的用途。
本发明的上述目的是通过以下技术方案来实现的。
一方面，本发明提供一种式(I)所示的化合物(泊沙康唑磷酸酯单胆碱盐)
Figure PCTCN2018123343-appb-000001
其中：
n为0至12的整数，优选为0至8的整数，更优选为0至6的整数，例如n可以为0、1、2、3、4、5或6。
另一方面，本发明提供上述式(I)所示的化合物的制备方法，所述制备方法包括：
(a)将式A所示的化合物与式B所示的化合物在在惰性气体存在下、无溶剂或有机溶剂A中反应，形成式C所示的化合物，
Figure PCTCN2018123343-appb-000002
Figure PCTCN2018123343-appb-000003
(b)将步骤(a)形成的式C所示的化合物用溶剂B进行水解，以形成式D所示的化合物：
Figure PCTCN2018123343-appb-000004
(c)将步骤(b)得到的式D所示的化合物与氢氧化胆碱在溶剂C中反应以制备所述的式(I)所示的化合物；
优选地，在上述制备方法中，在步骤(a)中，所述惰性气体选自氮气、氦气和氩气的一种或多种，优选为氮气或氩气。
优选地，在上述制备方法中，在步骤(a)中，所述有机溶剂A选自芳香烃类、卤代烃类、腈类、酮类、醚类、三乙胺、二乙胺、吡啶、1-甲基咪唑、N,N-二异丙基乙基胺和酯类中的一种或多种，优选为乙酸乙酯、乙腈、四氢呋喃、二氯甲烷、甲苯、丙酮、三乙胺、1-甲基咪唑、吡啶或氯仿。
优选地，在上述制备方法中，在步骤(b)中，所述溶剂B选自水、碱性水溶液和有机溶剂水溶液中的一种或多种。所述碱性水溶液优选为氢氧化钠水溶液、氨水、氢氧化钾水溶液或碳酸钠水溶液，所述有机溶剂水溶液优选为甲醇水溶液、乙醇水溶液、异丙醇水溶液或丙酮水溶液。
优选地，在上述制备方法中，在步骤(c)中，所述溶剂C选自水、芳香烃类、卤代烃类、腈类、酮类、醚类、醇类、酯类中的一种或多种；优选选自水、乙腈、甲醇、乙醇、丙酮、异丙醇、四氢呋喃、乙酸乙酯、二氯甲烷、甲苯和丁酮中的一种或多种。
优选地，在上述制备方法中，在步骤(a)中，所述反应温度为-10℃～50℃，优选为0～35℃。
优选地，在上述制备方法中，在步骤(b)中，所述水解温度为-20℃～30℃，优选为-5～10℃。
优选地，在上述制备方法中，在步骤(c)中，所述反应温度为-10℃～80℃，优选为10～40℃。
优选地，在上述制备方法中，在步骤(a)中，所述式A所示的化合物与所述式B所示的化合物之间的摩尔比为1:1.0～20.0，优选为1:2.25～10.0。
优选地，在上述制备方法中，在步骤(c)中，所述式D所示的化合物与所述的氢氧化胆碱之间的摩尔比为1:0.5～2，优选为1:1.05。
又一方面，本发明提供上述式(I)所示的化合物在制备抗真菌感染的药物中的用途；优选地，所述真菌感染 是由念珠菌属或隐球菌属引起的感染。
再一方面，本发明还提供一种药物组合物，该药物组合物包含上述式(I)所示的化合物以及药学上可接受的辅料。
优选地，所述药物组合物为片剂、栓剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小针、注射用冻干粉针或大输液。
优选地，所述药学上可接受的辅料选自以下中的一种或多种：pH调节剂、稀释剂、增溶剂、崩解剂、悬浮剂、润滑剂、粘合剂、填充剂、矫味剂、甜味剂、抗氧化剂、表面活性剂、防腐剂、包裹剂和色素。
本发明化合物的使用剂量和使用方法取决于诸多因素，包括患者的年龄、体重、性别、健康状况、营养状况、化合物的活性强度、使用时间、代谢速率、病症的严重程度以及诊治医生的主观判断。优选的使用剂量介于2～1200mg/kg；最好24小时的给药量为每公斤0.2～300mg，也可采用多次给药方式。
与现有技术相比较，本发明至少具有以下有益效果如下：
首先，本申请式(I)所示的化合物，即单胆碱盐化合物的溶解度较高，并且在影响因素(高温、高湿、光照)及长期放置的条件下稳定性较好，明显高于其他泊沙康唑磷酸酯单盐和泊沙康唑磷酸酯双盐。
其次，当本申请式(I)所示的单胆碱盐化合物被给药到体内时，其作为“前药”发挥作用，在碱性磷酸酶的存在下转化为具备生物活性的母体泊沙康唑。本申请式(I)所示的单胆碱盐化合物具有低的吸湿性从而具备出乎意料的改进的物理稳定性，这使其在制备过程中更易处理，同时保持合适的溶解度，使得该前药适于口服、局部和胃肠外给药。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
实施例1：泊沙康唑磷酸酯单胆碱盐的制备
Figure PCTCN2018123343-appb-000005
步骤1
Figure PCTCN2018123343-appb-000006
称取泊沙康唑(10g，14.28mmol)置于干燥的250mL的三颈烧瓶中，氮气保护下，加入二氯甲烷(100mL)，搅拌溶解，室温下(25℃)加入三乙胺(5mL)室温(25℃)搅拌反应30min，缓慢加入三氯氧磷(3mL，32.13mmol)，约1min加完，反应6h，反应结束。以过程中HPLC判断反应是否完成。
色谱条件：
流动相：6.8g/L磷酸二氢钾用磷酸调pH为3.0：乙腈＝60:40
检测波长：220nm流速：1.0ml/min柱温：25℃
样品浓度：1mg/ml稀释介质是50％乙腈
将反应液滴加到0℃的150mL纯水中，控制水解温度0～5℃，搅拌16h，将有机相和水相移至分液漏斗中萃取，将固相用MeOH(150mL)溶解与萃取的有机相合并，然后向其中倒入200～300目的硅胶约25g，旋转蒸发仪，蒸干溶剂拌样，将蒸干的硅胶倒入至装有25cm的硅胶的柱子(直径4.5cm)里，目标产品开始从柱子上洗脱下来，旋转蒸发除去收集的溶剂，得到黄色固体，加入150mL水和50mL二氯甲烷，萃取，有机相用无水Na 2SO 4干燥，然后用旋转蒸发仪，蒸干得淡黄色固体6.3g。有关物质：单杂0.42％，总杂1.02％。
步骤2：
Figure PCTCN2018123343-appb-000007
称取氢氧化胆碱(0.31g，1.28mmol，50％)置于盛有5mL纯水的烧杯中，搅拌均匀，然后化合物(式D)(1g，1.28mmol)倒入至烧杯中，30℃搅拌1h，使固体溶解，过滤，滤液倒入至盛有50mL的乙醇的烧杯中，室温搅拌12h，抽滤可得白色固体，再用5mL乙醇淋洗，真空干燥得白色的固体0.76g。(有关物质：0.13％；水分：0.3％；含量99.87％)。
实施例2：泊沙康唑磷酸酯单胆碱盐的制备
Figure PCTCN2018123343-appb-000008
步骤1
Figure PCTCN2018123343-appb-000009
取三氯氧磷40ml置于干燥的250mL的三颈烧瓶中，氮气保护下，降温至-5至5℃，缓慢加入泊沙康唑(10g，14.28mmol)，加完后保温搅拌12小时，反应结束。以过程中HPLC判断反应是否完成。
色谱条件：
流动相：6.8g/L磷酸二氢钾用磷酸调pH为3.0：乙腈＝60:40
检测波长：220nm流速：1.0ml/min柱温：25℃
样品浓度：1mg/ml稀释介质是50％乙腈
将反应液滴加到0℃的800mL氢氧化钠水溶液中，控制水解温度0～5℃，水解完成后，用10％盐酸调节pH至3～4。过滤，滤饼用丙酮洗涤，25～40℃鼓风干燥。得类白色固体8.3g。有关物质：单杂0.42％，总杂1.02％。
步骤2：
Figure PCTCN2018123343-appb-000010
称取氢氧化胆碱(0.31g，1.28mmol，50％)置于盛有3mL纯水的烧杯中，搅拌均匀，然后化合物(式D)(1g，1.28mmol)倒入至烧杯中，35℃搅拌1h，使固体溶解，过滤，滤液倒入至盛有50mL的乙醇的烧杯中，5℃搅拌12h，抽滤可得白色固体，再用5mL乙醇淋洗，真空干燥得白色的固体0.76g。(有关物质：0.13％；水分：0.3％；含量99.87％)。
实施例3：泊沙康唑磷酸酯单胆碱盐五水合物的制备
Figure PCTCN2018123343-appb-000011
选用与实施例1为同一批的泊沙康唑重复实施例2的步骤1、2进行试验，只是将步骤2的干燥方式改为35±5℃鼓风干燥。得到呈白色固体的标题化合物。(有关物质：0.13％；水分：9.06％；含量99.87％)。
实施例4：泊沙康唑磷酸酯单胆碱盐的制备
Figure PCTCN2018123343-appb-000012
步骤1
Figure PCTCN2018123343-appb-000013
称取泊沙康唑(10g，14.28mmol)置于干燥的250mL的三颈烧瓶中，氮气保护下，加入四氢呋喃(20mL)，搅拌溶解，50℃加入三乙胺(5mL)50℃搅拌反应30min，缓慢加入三氯氧磷(1.3mL，14.28mmol)，约1min加完，反应6h，HPLC监测反应，结果反应约20％，继续反应24小时，反应约81％，反应36小时反应基本完全。其中HPLC色谱条件同实施例1。
将反应液滴加到10℃的150mL纯水中，控制水解温度10℃，搅拌16h，将有机相和水相移至分液漏斗中萃取，将固相用MeOH(150mL)溶解与萃取的有机相合并，然后向其中倒入200～300目的硅胶约25g，旋转蒸发仪，蒸干溶剂拌样，将蒸干的硅胶倒入至装有25cm的硅胶的柱子(直径4.5cm)里，目标产品开始从柱子上洗脱下来，旋转蒸发除去收集的溶剂，得到黄色固体，加入150mL水和50mL二氯甲烷，萃取，有机相用无水Na 2SO 4干燥，然后用旋转蒸发仪，蒸干得淡黄色固体5.2g。有关物质：单杂2.5％，总杂4.90％。
步骤2：
Figure PCTCN2018123343-appb-000014
称取氢氧化胆碱(0.31g，1.28mmol，50％)置于盛有5mL纯水的烧杯中，搅拌均匀，然后化合物(式D)(1g，1.28mmol)倒入至烧杯中，60℃搅拌1h，使固体溶解，过滤，滤液倒入至盛有50mL的丙酮的烧杯中，室温搅拌12h，抽滤可得白色固体，再用5mL丙酮淋洗，真空干燥得白色的固体0.45g。有关物质：单杂1.23％，总杂2.54％。
实施例5：泊沙康唑磷酸酯单胆碱盐的制备
Figure PCTCN2018123343-appb-000015
步骤1
Figure PCTCN2018123343-appb-000016
称取泊沙康唑(10g，14.28mmol)置于干燥的250mL的三颈烧瓶中，氮气保护下，加入乙腈(20mL)，搅拌溶解，30℃加入三乙胺(5mL)30℃搅拌反应30min，缓慢加入三氯氧磷(13mL，142.8mmol)，约1min加完，反应6h，HPLC监测反应，反应基本完全。其中HPLC色谱条件同实施例1。
将反应液滴加到0℃的150mL纯水中，控制水解温度-5～0℃，搅拌16h，将有机相和水相移至分液漏斗中萃取，将固相用MeOH(150mL)溶解与萃取的有机相合并，然后向其中倒入200～300目的硅胶约25g，旋转蒸发仪，蒸干溶剂拌样，将蒸干的硅胶倒入至装有25cm的硅胶的柱子(直径4.5cm)里，目标产品开始从柱子上洗脱下来，旋转蒸发除去收集的溶剂，得到黄色固体，加入150mL水和50mL二氯甲烷，萃取，有机相用无水Na 2SO 4干燥，然后用旋转蒸发仪，蒸干得淡黄色固体7.3g。有关物质：单杂0.26％，总杂0.68％
步骤2：
Figure PCTCN2018123343-appb-000017
称取氢氧化胆碱(0.31g，1.28mmol，50％)置于盛有5mL纯水的烧杯中，搅拌均匀，然后化合物(式D)(1g，1.28mmol)倒入至烧杯中，35℃搅拌1h，使固体溶解，过滤，滤液倒入至盛有50mL的甲醇的烧杯中，室温搅拌12h，抽滤可得白色固体，再用5mL甲醇淋洗，真空干燥得白色的固体0.25g。有关物质：单杂0.02％，总杂0.10％。
实施例6：泊沙康唑磷酸酯单胆碱盐的制备
Figure PCTCN2018123343-appb-000018
步骤1
Figure PCTCN2018123343-appb-000019
称取泊沙康唑(10g，14.28mmol)置于干燥的250mL的三颈烧瓶中，氮气保护下，加入氯仿(70mL)，搅拌溶解，30℃加入三乙胺(5mL)30℃搅拌反应30min，缓慢加入三氯氧磷(6mL，64.36mmol)，约1min加完，反应6h，HPLC监测反应，反应基本完全。其中HPLC色谱条件同实施例1。
将反应液滴加到0℃的150mL纯水中，控制水解温度-5～0℃，搅拌16h，将有机相和水相移至分液漏斗中萃取，将固相用MeOH(150mL)溶解与萃取的有机相合并，然后向其中倒入200～300目的硅胶约25g，旋转蒸发仪，蒸干溶剂拌样，将蒸干的硅胶倒入至装有25cm的硅胶的柱子(直径4.5cm)里，目标产品开始从柱子上洗脱下来，旋转蒸发除去收集的溶剂，得到黄色固体，加入150mL水和50mL二氯甲烷，萃取，有机相用无水Na 2SO 4干燥，然后用旋转蒸发仪，蒸干得淡黄色固体7.0g。有关物质：单杂0.33％，总杂0.61％。
步骤2：
Figure PCTCN2018123343-appb-000020
称取氢氧化胆碱(0.31g，1.28mmol，50％)置于盛有5mL纯水的烧杯中，搅拌均匀，然后化合物(式D)(1g，1.28mmol)倒入至烧杯中，35℃搅拌1h，使固体溶解，过滤，滤液倒入至盛有50mL的异丙醇的烧杯中，室温搅拌12h，抽滤可得白色固体，再用5mL异丙醇淋洗，真空干燥得白色的固体0.21g。有关物质：单杂0.22％，总杂0.53％。
实施例7：片剂的制备
处方：
Figure PCTCN2018123343-appb-000021
制法：取泊沙康唑磷酸酯单胆碱盐粉碎过80目筛；称取处方量的淀粉和处方量的泊沙康唑磷酸酯单胆碱盐、微晶纤维素，混匀。用4％聚维酮K30溶液将物料制软材，用20目筛制粒，于40～60℃干燥至颗粒中的水分为5％左右。过20目筛整粒，加入处方量的硬脂酸镁，终混，测中间体含量，定片重；压片。
实施例8：片剂的制备
处方：
Figure PCTCN2018123343-appb-000022
制法：取泊沙康唑磷酸酯单胆碱盐五水合物粉碎过80目筛；称取处方量的淀粉和处方量的泊沙康唑磷酸酯单 胆碱盐五水合物、微晶纤维素，混匀。用4％聚维酮K30溶液将物料制软材，用20目筛制粒，于40～60℃干燥至颗粒中的水分为5％左右。过20目筛整粒，加入处方量的硬脂酸镁，终混，测中间体含量，定片重；压片。
实施例9：注射用泊沙康唑磷酸酯单胆碱的制备
处方：
Figure PCTCN2018123343-appb-000023
制法：加入批量体积注射用水，称取处方量的泊沙康唑磷酸酯单胆碱盐五水合物、甘露醇，搅拌使充分溶解后，用盐酸调节pH至5～10，0.22μm微孔滤膜过滤，灌装；冷冻干燥，轧盖，包装。
实施例10：注射用泊沙康唑磷酸酯单胆碱的制备
处方：
Figure PCTCN2018123343-appb-000024
制法：加入批量体积注射用水，称取处方量的泊沙康唑磷酸酯单胆碱盐、葡萄糖，搅拌使充分溶解后，用盐酸调节pH至5～10，0.22μm微孔滤膜过滤，灌装；冷冻干燥，轧盖，包装。
实施例11：注射用泊沙康唑磷酸酯单胆碱的制备
处方：
Figure PCTCN2018123343-appb-000025
制法：加入批量体积注射用水，称取处方量的泊沙康唑磷酸酯单胆碱盐搅拌使充分溶解后，0.22μm微孔滤膜过滤，灌装；冷冻干燥，轧盖，包装。
对比实施例1：泊沙康唑磷酸酯双胆碱盐的制备
选用与实施例1为同一批的泊沙康唑重复实施例l的步骤1、2进行试验，只是将步骤2的氢氧化胆碱的投料量与化合物(式D)的投料量的摩尔比替换为2:1。得到呈白色固体的标题化合物。
对比实施例2：泊沙康唑磷酸酯双胆碱盐五水合物的制备
选用与实施例1为同一批的泊沙康唑重复实施例3的步骤1、2进行试验，只是将步骤2的氢氧化胆碱的投料量与化合物(式D)的投料量的摩尔比替换为2:1。得到呈白色固体的标题化合物。
对比实施例3：泊沙康唑磷酸酯单钾盐的制备
选用与实施例1为同一批的泊沙康唑重复实施例l的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为氢氧化钾，且氢氧化钾与化合物(式D)摩尔比为1:1。得到呈白色固体的标题化合物。
对比实施例4：泊沙康唑磷酸酯单葡甲胺盐的制备
选用与实施例1为同一批的泊沙康唑重复实施例l的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为葡甲胺，且葡甲胺与化合物(式D)摩尔比为1:1。得到呈白色固体的标题化合物。
对比实施例5：泊沙康唑磷酸酯单精氨酸盐的制备
选用与实施例1为同一批的泊沙康唑重复实施例l的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为精氨酸，且精氨酸与化合物(式D)摩尔比为1:1。得到呈白色固体的标题化合物。
对比实施例6：泊沙康唑磷酸酯单钾盐三水合物的制备
选用与实施例1为同一批的泊沙康唑重复实施例3的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为氢氧化钾，且氢氧化钾与化合物(式D)摩尔比为1:1，。得到呈白色固体的标题化合物。
对比实施例7：泊沙康唑磷酸酯单钠盐三水合物的制备
选用与实施例1为同一批的泊沙康唑重复实施例3的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为氢氧化钠，且氢氧化钠与化合物(式D)摩尔比为1:1。得到呈白色固体的标题化合物。
对比实施例8：泊沙康唑磷酸酯单钠盐的制备
选用与实施例1为同一批的泊沙康唑重复实施例1的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为氢氧化钠，且氢氧化钠与化合物(式D)摩尔比为1:1。得到呈白色固体的标题化合物。
对比实施例9：泊沙康唑磷酸酯二钠盐五水合物的制备
选用与实施例1为同一批的泊沙康唑重复实施例3的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为氢氧化钠，且氢氧化钠与化合物(式D)摩尔比为2:1。得到呈白色固体的标题化合物。
对比实施例10：泊沙康唑磷酸酯二钠盐的制备
选用与实施例1为同一批的泊沙康唑重复实施例1的步骤1、2进行试验，只是将步骤2的氢氧化胆碱变换为氢氧化钠，且氢氧化钠与化合物(式D)摩尔比为2:1。得到呈白色固体的标题化合物。
对比实施例11：式E所示的化合物的制备
Figure PCTCN2018123343-appb-000026
步骤A：烘箱干燥的1L圆底烧瓶装有机械搅拌器、氮气入口转接器、装备橡胶隔片的压力平衡添加漏斗和温 度探针，向其中加入氢化钠(2.89g，0.069mol，60％)和THF(50mL)。将溶于30mL THF中的泊沙康唑(式A所示的化合物)(选用与实施例1为同一批的泊沙康唑)(17.94g，0.023mol)在室温下用20分钟滴加到该搅拌后的悬浮液中。搅拌45分钟后，将溶于30mLTHF中的碘(2.99g，0.0115mol)溶液用10分钟滴加加入，随后用15分钟滴加加入二叔丁基氯甲基磷酸酯(式III)(13.29g，0.035mol，～68％纯度)。反应混合物在约41℃下搅拌4小时使反应完成。以过程中HPLC判断反应是否完成。
Figure PCTCN2018123343-appb-000027
将该反应混合物倒入冰水(100mL)中。分离水相并以乙酸乙酯萃取(3×50mL)，混合的有机萃取物以10％的硫代亚硫酸钠(50mL)、水(50mL)、盐水(50mL)洗涤，硫酸镁干燥并减压浓缩，得到浅黄色油状物(22.8g，过程中用HPLC跟踪产物反应程度至97％以上作为反应结束点)。粗产物(式IV)在步骤B中按原态使用。
步骤B：圆底烧瓶装有磁性搅拌器、冷却浴、pH探针和氮气进出口，向其中加入溶于CH 2Cl 2(23mL)的上述步骤A的产物(式IV)(7.5g)并冷却到0℃。向该搅拌的溶液中缓慢加入三氟乙酸(8.8mL)并搅拌3小时至完成反应。以过程中HPLC判断反应是否完成。将该反应混合物倒入2N NaOH(64mL)的冷却溶液中。反应混合物以乙酸叔丁酯萃取(2×65mL)去除所有的有机杂质。含有双钠盐产物的水层以活性炭(10g)处理并以硅藻土层过滤。清滤液以1N HC1酸化到pH2.5。游离酸产物被萃取到乙酸乙酯(2×50mL)中。混合的有机层以水洗涤，MgSO4干燥，过滤，滤液减压浓缩得到3.39g粗产物V。可选地，在本发明的一个优选方面，步骤B可以连续过程进行，其细节可由本领域普通技术人员来确定。
Figure PCTCN2018123343-appb-000028
步骤C：将上面得到的产物V溶于甲醇(75mL)中。将L-赖氨酸(1.8g)加入该游离酸溶液中并维持pH在4.2到5.5，混合物在60℃加热4.5小时。热反应物以硅藻土层过滤。滤液浓缩到大约5mL，与乙醇(100mL)混合并加热到65℃以结晶出单赖氨酸盐的溶剂化物。该溶剂化物收集到布氏漏斗上并真空干燥，得到3.0g呈结晶固体的标题溶剂化物化合物。
Figure PCTCN2018123343-appb-000029
实验例1：溶解性研究
分别测试本发明得到的化合物、对比实施例2-12得到的化合物与泊沙康唑在水、异丙醇、甲醇中的溶解度。试验结果见下表所示。
测试化合物的溶解度数据
化合物 水 甲醇 异丙醇
实施例1 82mg/ml 35mg/ml 1mg/ml
实施例3 84mg/ml 36mg/ml 1mg/ml
对比实施例1 76mg/ml 15mg/ml 0.7mg/ml
对比实施例2 78mg/ml 19mg/ml 0.9mg/ml
对比实施例3 34mg/ml 14mg/ml 0.6mg/ml
对比实施例4 63mg/ml 10mg/ml 0.4mg/ml
对比实施例5 71mg/ml 11mg/ml 0.7mg/ml
对比实施例6 35mg/ml 15mg/ml 1mg/ml
对比实施例7 67mg/ml 8mg/ml 0.3mg/ml
对比实施例8 68mg/ml 9mg/ml 0.4mg/ml
对比实施例9 72mg/ml 10mg/ml 0.8mg/ml
对比实施例10 72mg/ml 10mg/ml 0.8mg/ml
对比实施例11 70mg/ml 14mg/ml 1mg/ml
泊沙康唑 0.05mg/ml 0.06mg/ml 0.1mg/ml
从实验结果看：本发明制备得到的化合物溶解度较高，均高于对比实施例1-11得到的化合物和泊沙康唑在水、 甲醇和异丙醇中的溶解度。同样的载药量，本发明化合物溶解度比对比实施例1-11得到的化合物和泊沙康唑的溶解度好，因此，本发明的化合物起效快、生物利用度高，制剂稳定性也得到了提高。所以本发明制备得到的化合物对提高其生物利用度和疗效具有重要意义。
实验例2：引湿性的研究
参照中国药典2015年版四部通则9103药物引湿性试验指导原则，考察本申请的化合物及对比实施例的化合物的引湿性。
试验方法：1、取干燥的具塞玻璃称量瓶(外径为50mm，高为15mm),于试验前一天置于适宜的25℃±1℃恒温干燥器(下部放置氯化铵饱和溶液)内，精密称定重量(m 1)。2、取供试品适量，平铺于上述称量瓶中，供试品厚度约为1mm，精密称定重量(m 2)。3、将称量瓶敞口，并与瓶盖同置于上述恒温恒湿条件下24小时。4、盖好称量瓶盖子，精密称定重量(m 3)。
Figure PCTCN2018123343-appb-000030
引湿性特征描述与引湿性增重的界定：
潮解：吸收足量水分形成液体。
极具引湿性：引湿增重不小于15％。
有引湿性：引湿增重小于15％但不小于2％。
略有引湿性：引湿增重小于2％但不小于0.2％。
无或几乎无引湿性：引湿增重小于0.2％。
本品经24小时后引湿性试验结果见下表。
测试化合物的引湿性试验结果
化合物 吸湿增重 结论
实施例1 0.12％ 无引湿性
实施例3 0.15％ 无引湿性
对比实施例1 8.76％ 有引湿性
对比实施例2 11.81％ 有引湿性
对比实施例3 1.56％ 略有引湿性
对比实施例4 0.88％ 略有引湿性
对比实施例5 1.02％ 略有引湿性
对比实施例6 1.95％ 略有引湿性
对比实施例7 1.86％ 略有引湿性
对比实施例8 1.92％ 略有引湿性
对比实施例9 12.01％ 有引湿性
对比实施例10 13.91％ 有引湿性
对比实施例11 0.76％ 略有引湿性
实验例3：稳定性研究
一、25℃±2℃、65％R.H±5％R.H条件下的稳定性实验
本实验例对本发明的泊沙康唑磷酸酯单胆碱盐与对比实施例的化合物在25℃±2℃，65％R.H±5％R.H条件进行的稳定性实验，并在0月、3月、6月、9月、12月、24月取样测定性状、有关物质、含量，结果详见下表。
测试化合物的稳定性实验结果
Figure PCTCN2018123343-appb-000031
从实验结果可以看出，泊沙康唑磷酸酯单胆碱盐的稳定性结果较对比实施例1-11化合物稳定性好，有利于工业化生产的储存。
二、40℃±2℃，75％R.H±5R.H条件下的稳定性实验
泊沙康唑磷酸酯单胆碱盐与对比实施例化合物在温度40℃±2℃，75％R.H±5R.H进行的稳定性实验，分别在0月、1月、2月、3月、6月取样测定性状、有关物质、含量，结果详见下表。
Figure PCTCN2018123343-appb-000032
结论：本发明制备得到的泊沙康唑磷酸酯单胆碱盐经加速试验6个月，各项考察指标与0月比较均未见明显变化，而对比实施例1-11化合物经加速试验6个月，各项考察指标与0月比较，有关物质明显增加；说明本发明的泊沙康唑磷酸酯单胆碱盐的稳定性优于对比实施例1-11化合物。
实验例4：影响因素试验
取本实验例发明的泊沙康唑磷酸酯单胆碱盐，放置在高温40℃、光照4500LX、高湿92.5％条件下10天，于0天、5天、10天取样检测性状、有关物质、含量，结果详见下表。
影响因素试验结果
Figure PCTCN2018123343-appb-000033
结论：本发明制备得到的化合物在高温、高湿、光照条件下放置10天各项考察指标与0天比较均未见明显变化，说明本发明化合物性质较稳定。
实验例5:本发明化合物静脉给药对抗白色念珠菌阴道炎实验
1.实验材料
1.1实验仪器
血细胞计数板、石蜡切片机、SPX-250B生化培养箱、超净化工作台、微量加样器、压力蒸汽灭菌器、光学显微镜、电子分析天平。
1.2实验试剂
苯甲酸雌二醇注射液，聚乙二醇，沙堡葡萄糖琼脂固体培养基。
1.3实验动物
KM小鼠，体重18-22g，雌性，由江苏省实验动物中心提供。
1.4实验菌株
标准菌株白色念珠菌购于美国菌种保藏中心，菌株编号为ATCC10231。
2.实验方法
上述小鼠称重后随机分组：泊沙康唑组、受试化合物组和溶媒组，每组20只，其中由于泊沙康唑不溶于溶媒(生理盐水)，因此试验中泊沙康唑组为市售泊沙康唑注射液(默沙东/先灵葆雅，3PAR80701，下同)，即使用磺丁基醚-β-环糊精增溶。感染白色念珠菌前，各组动物连续6天皮下注射给予0.5ml苯甲酸雌二醇(2mg/ml)，使其进入发情期，以后每2天注射1次持续至实验完毕。6天后，每只小鼠阴道注入20ul浓度为3.5x10 6CFU/ml的白色念珠菌液，造成阴道感染模型。感染后第一天起，各组动物尾静脉给以相应药物20mg/kg(以泊沙康唑计算)，给药体积0.1ml/0g，每天一次，连续5天，模型组给予等体积溶剂(生理盐水)。小鼠在感染后的第3、5日，用无菌棉签擦拭小鼠阴道，把棉签浸泡于0.9ml主理盐水中，按10倍递增将该菌液稀释成系列浓度，然后各取100ul各浓度菌液，接种于含0.5％(W/V)氯霉素的沙堡葡萄糖琼脂固体培养基上，观察白色念珠菌在阴道上的真菌载荷量。
3.实验结果
白色念珠菌阴道炎(静脉给药)：各组小鼠阴道真菌载荷量
Figure PCTCN2018123343-appb-000034
注：数据以20只小鼠的CFU值的对数的均值±标准差表示。
从实验结果可以看出，静脉给药5天后，本发明的化合物组小鼠真菌载荷量较溶媒组显著降低，与泊沙康唑组一致，取得了明显的疗效，且避免了使用β-环糊精类辅料进行增溶带来的安全性风险。
实施例6:本发明化合物灌胃给药对抗白色念珠菌阴道炎实验
l、实验材料
1.1实验仪器
血细胞计数板、石蜡切片机、SPX-250B生化培养箱、超净化工作台、微量加样器、压力蒸汽灭菌器、光学显微镜、电子分析天平。
1.2实验试剂
苯甲酸雌二醇注射液、聚乙二醇、沙堡葡萄糖琼脂固体培养基。
1.3实验动物
KM小鼠，体重18-22g，雌性，由江苏省实验动物中心提供。
1.4实验菌株
标准菌株白色念珠菌购于美国菌种保藏中心，菌株编号为ATCC10231。
2.实验方法
上述小鼠称重后随机分组：泊沙康唑(CMC-Na)组、受试化合物组和溶媒组，每组20只。其中由于泊沙康唑不溶于溶媒(生理盐水)，因此试验中泊沙康唑组为市售泊沙康唑注射液(默沙东/先灵葆雅，3PAR80701，下同)，即使用磺丁基醚-β-环糊精增溶，其他受试药用生理盐水溶解，超声至澄清后用于给药。感染白色念珠菌前，各组动物连续6天皮下注射给予0.5ml苯甲酸雌二醇(2mg/ml)，使其进入发情期，以后每2天注射1次持续至实验完毕。6天后，每只小鼠阴道注入20ul浓度为3.5x10 6CFU/ml的白色念珠菌液，造成阴道感染模型。感染后第一天起，各组动物灌胃给药相应药物20mg/kg(以泊沙康唑计算)，给药体积0.1ml/10g，每天一次，连续15天，模型组给予等体积溶剂(生理盐水)。各组小鼠在感染后的第3、5、7、11及15日，用无菌棉签擦拭小鼠阴道，把棉签浸泡于0.9ml主理盐水中，按10倍递增将该菌液稀释成系列浓度，然后各取l00ul各浓度菌液，接种于含0.50/(W/V)氯霉素的沙堡葡萄糖琼脂固体培养基上，观察白色念珠菌在阴道上的真菌载荷量。
3.实验结果
白色念珠菌阴道炎(灌胃)：各组小鼠阴道真菌载荷量
Figure PCTCN2018123343-appb-000035
从实验结果可以看出，用药15天后，采用生理盐水溶解的本发明的化合物组小鼠真菌载荷量较溶媒组显著降低，与泊沙康唑组一致，取得了明显的疗效。
实施例7:本发明化合物静脉给药对抗水鼠系统性真菌感染作用实验
1.实验材料
1.1实验仪器
Multiskan MK3型酶标检测仪、隔水式电热恒温培养箱、zo-F160全温振荡培养箱、MJX型智能霉菌培养箱、SW-CT-IF型超净化工作台、紫外分光光度计。
1.2实验试剂
二甲基亚砜，沙堡葡萄糖琼脂固体培养基(SDA)。
1.3实验动物
ICR小鼠，体重18-22g，雄性，由湖北省实验动物中心提供。
1.4实验菌株
标准菌株白色念珠菌购于美国菌种保藏中心，菌株编号为ATCC10231。
2.实验方法
实验前，用接种圈从4℃保存的SDA(沙氏琼脂，下同)培养基上挑取白色念珠菌少量，接种至Iml YPD(Yeast Extract Peptone Dextrose Medium)培养液，于30℃，200rpm振荡培养，活化16h，使真菌处于指数生长期后期。用血细胞计数板计数，以RPMI1640(RoswellPark Memorial Institute1640，下同)培养液调整菌液浓度至1＊10 3～5＊10 3CFU/ml。挑取SDA乎板上的白念珠菌单克隆，接种至Iml  YPD(Yeast Extract Peptone Dextrose Medium，下同)培养基中，35℃，200rpm培养16h至指数生长期后期，以1％接种到新鲜培养基中培养6h，1000x g离心5min，用生理盐水洗涤三次至上清无色，以血球计数板计数，调整细胞浓度至5＊10 6个/ml，尾静脉注射0.1ml/lOg造成小鼠系统性真菌感染。小鼠随机分组，每组10只，分别为泊沙康唑组、受试化合物组和溶媒组，其中由于泊沙康唑不溶于溶媒(生理盐水)，因此试验中泊沙康唑组为市售泊沙康唑注射液，即使用磺丁基醚-β环糊精增溶。在小鼠系统性真菌感染模型建立2h后，各给药组分别尾静脉给药20mg/kg(以泊沙康唑计算)，给药体积0.1ml/l0g，模型组给以0.9％氯化钠溶液0.1ml/l0g，每天一次，连续给药5天。观察小鼠死亡情况，记录存活时间。共观察7天。死亡小鼠全部用乙醇火烧处理。
3.实验结果
系统性真菌感染(静脉给药)：给药后各组小鼠存活率(％)
Figure PCTCN2018123343-appb-000036
从实验数据可以看出，本发明的化合物组小鼠存活率明显高于溶媒组；所列化合物第7天小鼠存活率与泊沙康唑组相同，取得了较好的效果。
实施例8:本发明化合物灌胃给药对抗小鼠系统性真菌感染作用实验
1.实验材料
1.1实验仪器
Multiskan MK3型酶标检测仪、隔水式电热恒温培养箱、zo-F160全温振荡培养箱、MJX型智能霉菌培养箱、SW-CT-IF型超净化工作台、紫外分光光度计。
1.2实验试剂
二甲基亚砜，沙堡葡萄糖琼脂固体培养基(SDA)。
1.3实验动物
ICR小鼠，体重18-22g，雄性，由江苏省实验动物中心提供。
1.4实验菌株
标准菌株白色念珠菌购于美国菌种保藏中心，菌株编号为ATCC10231。
2.实验方法
实验前，用接种圈从4℃保存的SDA培养基上挑取白色念珠菌少量，接种至1mIYPD培养液，于30℃，200rpm振荡培养，活化16h，使真菌处于指数生长期后期。用血细胞计数板计数，以RPMI1640培养液调整菌液浓度至1＊10 3～5＊10 3CFU/ml。挑取SDA平板上的白念珠菌单克隆，接种至Iml YPD培养基中，35℃，200rpm墙养16h至指数生长期后期，以l％接种到新鲜培养基中培养6h，1000x g离心5min，用生理盐水洗涤三次至上清无色，以血球计数板计数，调整细胞浓度至5＊10 6个/ml，尾 静脉注射0.1ml/10g造成小鼠系统性真菌感染。小鼠随机分组，每组10只，分别为泊沙康唑组、受试化合物组和溶媒组，试验中泊沙康唑组为市售泊沙康唑注射液，即使用磺丁基醚-β环糊精增溶，其他受试药用生理盐水溶解，超声至澄清后用于给药。在小鼠系统性真菌感染模型建立2h后，各给药组分别灌胃给药20mg/kg(以泊沙康唑计算)，给药体积0.1ml/log，模型组给以0.9％/氯化钠溶液0.1ml/log，每天一次，连续给药5天。观察小鼠死亡情况，记录存活时间。共观察7天。死亡小鼠全部用乙醇火烧处理。
3.实验结果
系统性真菌感染(灌胃)：给药后各组小鼠存活率(％)
Figure PCTCN2018123343-appb-000037
以实验结果可以看出，本发明的化合物小鼠存活率明显高于溶媒组：所列化合物第7天小鼠存活率优于泊沙康唑组，生物利用度较高。
实施例9：体内药代动力学试验
试验方法：实验动物为雄性小鼠，6至8周龄，体重190至215克，购自北京维利通华实验动物技术有限公司。基于小鼠体重随机分成5组，每组3只动物。各组小鼠的给药剂量和途径见下表。
组 药物 药物剂量(mg/Kg) 途径 数量
组1 实施例1化合物 1 静脉 3
组2 对比实施例的11(化合物E) 1 静脉 3
在药代动力学试验前，将小鼠禁食16小时。然后按照表2中所示经静脉(1mL/kg；1mg/kg)给药单个剂量的化合物。采取颈静脉穿刺的方式在给药后定时收集血液200μL，其中对于经静脉给药的动物组，在给药后0、15分钟、30分钟、1小时、2小时、4小时、8小时和24小时收集血液；于2小时、4小时、8小时、12小时、24小时收集尿液。将血样收集于具有EDTA的样品管中，立即在4℃下以4000rpm离心血样5分钟，然后将血浆转移到另一个样品管中，储存于-20摄氏度下。
检测各时间点取得的血样和尿样中由测试化合物转化形成的泊沙康唑的浓度，由此对样品进行药代动力学检验，采用的方法和仪器如下：
HPLC：Shimadzu
MS：AB API4000Q
柱子：Phenomenex Luna5μC18
流动相：100％乙腈(3mM乙酸铵)和100％水(3mM乙酸铵)
定量方法：内标法
体内药代动力学试验结果如下：
对比实施例11的化合物E的试验结果
Figure PCTCN2018123343-appb-000038
本发明的实施例1的化合物的试验结果：
Figure PCTCN2018123343-appb-000039
由表中数据可知，对比实施例中化合物E在体内通过酶较难水解成活性成分，但在体内能迅速通过循环系统代谢富集在尿液中；极大地降低该类化合物的生物利用度。而本发明的化合物在尿液中无富集，有利于其在医学上的应用。

Claims (7)

  1. 一种式(I)所示的化合物(泊沙康唑磷酸酯的单胆碱盐)
    Figure PCTCN2018123343-appb-100001
    其中：
    n为0至12的整数，优选为0至8的整数，更优选为0至6的整数。
  2. 一种权利要求1所述的式(I)所示的化合物的制备方法，所述制备方法包括：
    (a)将式A所示的化合物与式B所示的化合物在惰性气体存在下、无溶剂或有机溶剂A中反应，形成式C所示的化合物，
    Figure PCTCN2018123343-appb-100002
    Figure PCTCN2018123343-appb-100003
    (b)将步骤(a)形成的式C所示的化合物用溶剂B水解，以形成式D所示的化合物：
    Figure PCTCN2018123343-appb-100004
    (c)将步骤(b)得到的式D所示的化合物与氢氧化胆碱在溶剂C中反应以制备所述的式(I)所示的化合物。
  3. 根据权利要求2所述的制备方法，其特征在于，在步骤(a)中，所述惰性气体选自氮气、氦气和氩气的一种或多种，优选为氮气或氩气；
    优选地，在步骤(a)中，所述有机溶剂A选自芳香烃类、卤代烃类、腈类、酮类、醚类、三乙胺、二乙胺、吡啶、1-甲基咪唑、N,N-二异丙基乙基胺和酯类中的一种或多种，优选为乙酸乙酯、乙腈、四氢呋喃、二氯甲烷、甲苯、丙酮、三乙胺、1-甲基咪唑、吡啶或氯仿。
    优选地，在步骤(b)中，所述溶剂B选自水、碱性水溶液和有机溶剂水溶液中的一种或多种；所述碱性水溶液优选为氢氧化钠水溶液、氨水、氢氧化钾水溶液或碳酸钠水溶液；所述有机溶剂水溶液优选为甲醇水溶液、乙醇水溶液、异丙醇水溶液或丙酮水溶液；
    优选地，在步骤(c)中，所述溶剂C选自水、芳香烃类、卤代烃类、腈类、酮类、醚类、醇类、酯类中的一种或多种；优选选自水、乙腈、甲醇、乙醇、丙酮、异丙醇、四氢呋喃、乙酸乙酯、二氯甲烷、甲苯和丁酮中的一种或多种。
  4. 根据权利要求2或3所述的制备方法，其特征在于，在步骤(a)中，所述反应温度为-10℃～50℃，优选为0～35℃；
    优选地，在上述制备方法中，在步骤(b)中，所述水解温度为-20℃～30℃，优选为-5～10℃；
    优选地，在上述制备方法中，在步骤(c)中，所述反应温度为-10℃～80℃，优选为10～40℃。
  5. 根据权利要求2至4中任一项所述的制备方法，其特征在于，在步骤(a)中，所述式A所示的化合物与所述式B所示的化合物之间的摩尔比为1:1.0～20.0，优选为1:2.25～10.0；
    优选地，在上述制备方法中，在步骤(c)中，所述式D所示的化合物与所述氢氧化胆碱之间的摩尔比为1:0.5～2，优选为1:1.05。
  6. 权利要求1中所述的化合物在制备抗真菌感染的药物中的用途；优选地，所述真菌感染是由念珠菌属或隐球菌属引起的感染。
  7. 一种药物组合物，该药物组合物包含权利要求1中所述的化合物以及药学上可接受的辅料；
    优选地，所述药物组合物为片剂、栓剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、小容量注射液、注射用冻干粉针或大输液；
    优选地，所述药学上可接受的辅料选自以下中的一种或多种：pH调节剂、稀释剂、崩解剂、悬浮剂、润滑剂、粘合剂、填充剂、矫味剂、甜味剂、抗氧化剂、防腐剂、包裹剂和色素。
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AU2018441838A1 (en) 2020-11-12
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EP3770165A4 (en) 2021-04-28
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