WO2021228007A1 - 一种头孢哌酮镁化合物、其制备方法及应用 - Google Patents

一种头孢哌酮镁化合物、其制备方法及应用 Download PDF

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WO2021228007A1
WO2021228007A1 PCT/CN2021/092553 CN2021092553W WO2021228007A1 WO 2021228007 A1 WO2021228007 A1 WO 2021228007A1 CN 2021092553 W CN2021092553 W CN 2021092553W WO 2021228007 A1 WO2021228007 A1 WO 2021228007A1
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cefoperazone
magnesium
magnesium compound
preparation
suspension
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PCT/CN2021/092553
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French (fr)
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刘翠哲
刘金霞
张树峰
毛晓霞
金鹏
白红红
杨禄坤
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承德医学院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D501/14—Compounds having a nitrogen atom directly attached in position 7
    • C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
    • C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
    • C07D501/24—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids with hydrocarbon radicals, substituted by hetero atoms or hetero rings, attached in position 3
    • C07D501/36—Methylene radicals, substituted by sulfur atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D501/02—Preparation
    • C07D501/04—Preparation from compounds already containing the ring or condensed ring systems, e.g. by dehydrogenation of the ring, by introduction, elimination or modification of substituents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D501/02—Preparation
    • C07D501/12—Separation; Purification

Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to a cefoperazone magnesium compound, a preparation method and an application thereof.
  • the third-generation semi-synthetic cephalosporin broad-spectrum antibiotic of cefoperazone has the characteristics of strong antibacterial activity, broad antibacterial spectrum, high blood concentration in the body, and wide distribution. It has a certain degree of broad-spectrum ⁇ -lactamase produced by negative bacilli It is used clinically for the treatment of various infectious diseases caused by sensitive bacteria, such as the treatment of respiratory system, genitourinary system, biliary tract, gastrointestinal tract, chest and abdominal cavity, skin and soft tissue infections, and against influenza bacillus and meningococcus Intracerebral infection caused by it also has a good effect.
  • Cefoperazone sodium is commonly used clinically, with a molecular formula of C 25 H 26 N 9 NaO 8 S 2 and a molecular weight of 667.
  • sodium salt is not suitable for patients with hypernatremia.
  • the present invention provides a cefoperazone magnesium compound with enhanced antibacterial effect and magnesium supplementation effect, and a preparation method and application thereof.
  • the invention can play a synergistic effect by combining cefoperazone with magnesium.
  • Cefoperazone can inhibit the cell wall synthesis of pathogenic bacteria and has a strong effect on gram-negative bacteria. It can significantly inhibit the synthesis of bacterial cell wall after combining with magnesium. The effect is significantly enhanced. Improve the stability of cefoperazone drugs, and at the same time provide a choice for patients who are clinically unsuitable for sodium.
  • a cefoperazone magnesium compound the structural formula is as follows:
  • the molecular formula is: Mg(C 25 H 26 N 9 O 8 S 2 ) 2 ;
  • the molecular weight is: 1288.
  • the preparation method of the cefoperazone magnesium compound includes the following steps:
  • the magnesium compound is crushed into fine powder, sieved, added to purified water, and mixed to obtain a magnesium compound solution or suspension;
  • the filtrate obtained in step (3) is dried and purified to obtain the cefoperazone magnesium compound.
  • step (1) the aperture of the sieved sieve is 60-120 mesh.
  • the molar ratio of the cefoperazone acid in the cefoperazone acid suspension to the magnesium element in the magnesium compound solution or suspension is 2:1.
  • the magnesium compound is one or a mixture of magnesium hydroxide, magnesium bicarbonate, and magnesium acetate.
  • step (3) the temperature for carrying out the reaction is 15-30°C.
  • step (3) the decoloring agent is also added while the stirring reaction is performed.
  • the decolorizing agent is activated carbon.
  • step (4) the drying is spray drying or freeze drying.
  • the spray drying is to use spray drying equipment to disperse the filtrate of step C into particles, and contact the particles with hot air to instantly remove the water, and obtain a cefoperazone magnesium compound with a water content of 2.0% or less by weight .
  • the spray drying equipment used in the present invention is a product currently on the market, such as a product sold under the trade name QZR-5 by Linzhou Dryer Factory Company, Xishan City, Jiangsu province.
  • the freeze-drying is to use freeze-drying equipment to freeze the filtrate of step C to below 0°C, and heat under high vacuum to directly sublime the water from the solid state into water vapor, remove the water, and obtain a water content of 2.0% by weight
  • the freeze-drying equipment used in the present invention is a product currently on the market, such as a product sold under the trade name LGJ-22D by Beijing Sihuan Scientific Instrument Factory Co., Ltd.
  • step (4) the purification is carried out by recrystallization, octadecylsilane reverse phase column, and preparative liquid chromatography, so that the content of the cefoperazone magnesium compound in the product is more than 95% by weight.
  • ODS Octadecylsilane reverse phase column
  • C18 column is a commonly used reverse phase chromatography column, also called C18 column; because it is a long-chain alkyl bonded phase, it has a higher carbon content and better hydrophobicity. It has stronger adaptability to various types of biological macromolecules, so it is the most widely used in biochemical analysis.
  • Purification using an ODS column was performed using an ODS column sold by YMC under the trade name GEL C18AAG12S50 under the condition of 5%-50% acetonitrile-methanol-water as the eluent.
  • the instrument used for preparative liquid chromatography purification was a preparative liquid sold under the trade name CTO-10A by Shimadzu Company, the preparative column was C18, and the liquid phase conditions were acetonitrile-methanol-water (5:50:45).
  • cefoperazone magnesium compound is determined according to high performance liquid chromatography, the instrument used is Agilent 1260, the chromatographic column is ODS C18, the liquid phase conditions are 0.1% phosphoric acid water-acetonitrile (70:30), and the flow rate is 1 mL/min.
  • the detection wavelength is 230nm.
  • Mg 2+ One magnesium ion (Mg 2+ ) reacts with two carboxyl groups on cefoperazone to produce cefoperazone magnesium, the molecular formula is Mg(C 25 H 26 N 9 O 8 S 2 ) 2 , and the molecular weight is 1288.
  • Magnesium is an essential element that participates in the normal life activities and metabolic processes of organisms. Magnesium affects various biological functions of cells, affects potassium ion and calcium ion transport, regulates signal transmission, participates in energy metabolism, protein and nucleic acid synthesis; activates and inhibits catalytic enzymes, and regulates cell cycle, cell proliferation and cell differentiation; magnesium It is also involved in maintaining the stability of the genome, and is also related to the body's oxidative stress and tumorigenesis. In a long-term study, the inventor of the present application found that the combination of magnesium and cefoperazone has a synergistic effect on the treatment of some inflammations.
  • the cefoperazone magnesium compound of the present invention can play a synergistic effect by combining cefoperazone with magnesium.
  • Cefoperazone can inhibit the synthesis of the cell wall of pathogenic bacteria and has a strong effect on gram-negative bacteria. , Combined with magnesium, it can significantly inhibit the synthesis of bacterial cell wall, and the use effect is significantly enhanced.
  • the invention uses the reaction of cefoperazone acid and magnesium ions to generate cefoperazone magnesium compound, improves the stability of the cefoperazone drug, simplifies the preparation production process, and provides a choice for patients who are clinically unsuitable for sodium salt.
  • the cefoperazone magnesium compound described in the present application is a magnesium-containing antibiotic, which has both antibacterial therapeutic effects and magnesium supplementation effects, is beneficial to some inflammations, and has a synergistic effect.
  • cefoperazone magnesium of the present invention has simple operation, no organic solvents, no generation of toxic and harmful substances, high product purity, and convenient industrial production.
  • This embodiment provides a method for preparing a cefoperazone magnesium compound, the molecular formula of the cefoperazone magnesium compound is: Mg(C 25 H 26 N 9 O 8 S 2 ) 2 , the molecular weight is 1288, and the structural formula is as follows:
  • the preparation method of the cefoperazone magnesium compound includes the following steps:
  • step (2) Add the magnesium bicarbonate solution described in step (2) to the cefoperazone acid suspension described in step (1), stir at 25°C for a full reaction, then add 0.1% activated carbon for stirring and decolorization, and filter to obtain the filtrate ;
  • the filtrate obtained in step (3) is first freeze-dried under 50 Pa pressure conditions to obtain a preliminary product with a water content of less than 2%, and then ethanol-water (1:9-9:1) is used at 25°C. The solvent is recrystallized and purified to obtain the cefoperazone magnesium compound product.
  • the content of the cefoperazone magnesium compound in the product is more than 95% by weight.
  • the instrument used for HPLC is Agilent 1260, the chromatographic column is ODS C18, the liquid phase conditions are 0.1% phosphoric acid water-acetonitrile (70:30), the flow rate is 1 mL/min, and the detection wavelength is 230 nm.
  • This embodiment provides a method for preparing the cefoperazone magnesium compound, which includes the following steps:
  • the filtrate obtained in step (3) was first spray-dried under a pressure of 100 Pa to obtain the initial product with a water content of less than 2%, and then purified by an octadecylsilane reverse phase column to obtain the cefoperazone Ketone magnesium compound products. After testing, the content of the cefoperazone magnesium compound in the product is more than 95% by weight.
  • This embodiment provides a method for preparing the cefoperazone magnesium compound, which includes the following steps:
  • step (2) The magnesium hydroxide suspension described in step (2) was added to the cefoperazone acid suspension described in step (1), stirred at 30°C to fully react, and then activated carbon was added for stirring and decolorization, and the filtrate was obtained by filtration ;
  • the filtrate obtained in step (3) is first spray-dried under a pressure of 70 Pa to obtain an initial product with a water content of less than 2%, and then purified by preparative liquid chromatography to obtain the cefoperazone magnesium compound product . After testing, the content of the cefoperazone magnesium compound in the product is more than 95% by weight.
  • cefoperazone sodium in the prior art and the cefoperazone magnesium prepared by the present invention are automatically scanned by the instrument under the ultraviolet absorption spectrum at 200-400nm.
  • the UV absorption spectrum was measured by the comparative method of reference substance.
  • Preparation of liquid culture medium accurately weigh 2g beef extract, 3g yeast, 5g sodium chloride, 700ml distilled water and mix, adjust the pH of the liquid culture medium to about 7.0, autoclave in a steam sterilizer for 30 minutes, and adjust the temperature to Take it out after sterilization at 120°C and put them into 6 conical flasks.
  • the volume of the liquid culture medium in each conical flask is 100ml, which is sealed and ready for use.
  • Preparation of the drug solution accurately weigh cefoperazone sodium and cefoperazone magnesium according to the number of equimolar ⁇ -lactam rings, add distilled water to make the volume to 10ml, and dilute to make the concentration of cefoperazone sodium and cefoperazone magnesium solution respectively 32 ⁇ g/ml, 31.26 ⁇ g/ml, filter with 0.22 ⁇ m filter membrane, take additional filtrate and set aside.
  • Preparation of bacterial solution Pick an inoculation loop amount from the second-generation bacteria of Escherichia coli and Staphylococcus albicans from the nutrient agar slope, and inoculate them into two conical flasks with liquid culture medium, and shake gently After the conical flask is placed in a gas bath constant temperature shaker, the temperature is adjusted to 37°C, and after 24-48 hours of incubation, it is observed that the liquid becomes turbid, that is, the bacteria resurrection is successful.
  • Turbidimetric experiment Before the experiment, autoclave the required experimental supplies at 120°C for 30 minutes, and sterilize the laboratory with a UV lamp for 30 minutes. Under aseptic operating conditions, 28 test tubes were taken and divided into four groups, and each test tube was added with liquid medium to dilute the cefoperazone sodium and cefoperazone magnesium liquids. Add 50 ⁇ l of Staphylococcus albicans solution to 14 of them, and add 50 ⁇ l of Escherichia coli solution to the remaining 14 and place them in a constant temperature incubator for 18-24 hours to observe the turbidity.
  • cefoperazone magnesium is at 31.26, 7.82, 3.90, 1.95, 0.98, 0.48 ⁇ g/ml in terms of its inhibitory effect on Staphylococcus albicans
  • the antibacterial effect is stronger than the same molar number of ⁇ -lactam rings corresponding to the concentration of cefoperazone sodium at 32, 8, 4, 2, 1, 0.5 ⁇ g/ml; in the inhibitory effect on E.
  • cefoperazone magnesium At the concentrations of 1.95, 0.98, and 0.48 ⁇ g/ml, the antibacterial effect is stronger than the same molar number of ⁇ -lactam rings corresponding to the concentration of cefoperazone sodium at 2,1, and 0.5 ⁇ g/ml.
  • cefoperazone magnesium salt The inhibitory effect on Staphylococcus albicans is stronger, stronger than that of cefoperazone sodium.
  • the rats were adaptively reared for one week, their body temperature was monitored every morning and evening for 3 days.
  • the rats with body temperature fluctuation less than 0.5°C were selected into the experiment and randomly divided into 6 groups: blank group, model group, cefoperazone sodium low-dose group, cefoperazone sodium high-dose group, cefoperazone magnesium low-dose group, and cefoperazone magnesium high-dose group Groups of 10 animals per group.
  • the rat is anesthetized, open the mouth of the rat to fully expose the throat, and slowly insert the tracheal tube with a syringe into the trachea through the mouth, push in 0.6ml of bacterial liquid, and push the same volume of normal saline in the blank group.
  • the rat was erected, and the rat was kept upright for about 20s to ensure that the inoculated bacteria liquid entered the lungs due to gravity.
  • the low dose is 180mg/kg
  • the high dose 360mg/kg in view of the different molecular weights of magnesium and sodium, the low and high doses here are both calculated as cefoperazone
  • twice a day continuous
  • the blank group and the model group were given an equal volume of normal saline.
  • the lungs were fixed with 10% formaldehyde solution, routinely embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE). The pathological changes of liver tissues were observed under light microscope.
  • the administration group and the model group began to experience shortness of breath, reduced food intake, and poor response to the surrounding environment.
  • the animals had a significant decrease in eating and mobility, and their hair was straight and curled in the corner of the cage.
  • the cefoperazone sodium (low and high dose) groups were all faster breathing than the cefoperazone magnesium (low and high dose) groups, and the food intake was significantly less than the cefoperazone magnesium group.
  • the appetite and activity of the animals in the administration group increased.
  • the rats in the cefoperazone sodium group were still breathing faster than the cefoperazone magnesium group.
  • the rats in the model group could hear obvious breathing wheezing, weight loss, and obvious skinny.
  • the appetite, activity level, body weight, and body temperature of rats in the blank group did not change significantly compared with those before vaccination.
  • the number of white blood cells in the model group increased significantly (P ⁇ 0.01); compared with the model group, the number of white blood cells in the rats in each administration group decreased significantly (P ⁇ 0.01), indicating that both cefoperazone sodium and magnesium have effects on rat pneumonia Treatment effect, and compared with the low-dose cefoperazone sodium group, the white blood cell count of the low-dose cefoperazone magnesium group decreased more significantly (P ⁇ 0.05).
  • the blank group there was a significant difference in the high-dose cefoperazone sodium group (P ⁇ 0.01), and there was no significant difference in the high-dose cefoperazone magnesium group.
  • the percentage of lymphocytes in the model group decreased significantly (P ⁇ 0.01); compared with the model group, the number of lymphocytes in each administration group increased significantly (P ⁇ 0.01, P ⁇ 0.05). It shows that both cefoperazone sodium and cefoperazone magnesium have therapeutic effects on pneumonia in rats, and compared with the low-dose cefoperazone sodium group, the percentage of lymphocytes in the low-dose cefoperazone magnesium group increased significantly (P ⁇ 0.05).

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Abstract

本发明涉及一种头孢哌酮镁化合物、其制备方法及应用，通过采用头孢哌酮与镁结合，能起到协同增效的作用，头孢哌酮能抑制病原菌细胞壁合成，对革兰阴性菌的作用强，联合镁元素后显著抑制细菌细胞壁的合成，使用效果显著增强。本发明通过利用头孢哌酮酸与镁离子反应生成头孢哌酮镁化合物，提高头孢哌酮药物的稳定性，同时为临床上不适合钠盐的患者提供一种选择。本发明所述头孢哌酮镁化合物为含镁抗生素，同时具有抗菌治疗效果和补镁的效果，并对一些炎症有益处，起到协同增效的作用。所述头孢哌酮镁的制备方法，操作简单，不使用有机溶剂，无有毒有害物质生成，产品纯度高，便于工业化生产。

Description

一种头孢哌酮镁化合物、其制备方法及应用 技术领域
本发明属于医药技术领域，具体涉及一种头孢哌酮镁化合物、其制备方法及应用。
背景技术
头孢哌酮第三代半合成头孢菌素类广谱抗生素，具有抗菌活性强、抗菌谱广、体内血药浓度高、分布广等特点，对阴性杆菌产生的广谱β-内酰胺酶有一定的稳定性，临床用于敏感菌引起的各种感染性疾病的治疗，如呼吸系统、生殖泌尿系统、胆道、胃肠道、胸腹腔、皮肤软组织感染的治疗，并对流感杆菌、脑膜炎球菌引起的脑内感染也有较好的疗效。
目前，尽管该药单独使用耐药严重，由于头孢哌酮对铜绿假单胞菌有很好的疗效，该药与酶抑制剂联用的复方制剂仍然被认为具有非常大的临床应用价值。
市场上现有头孢哌酮和舒巴坦钠及三唑巴坦钠的不同配比复方制剂。临床上常用的是头孢哌酮钠，分子式为C 25H 26N 9NaO 8S 2，分子量为667。然而临床应用时，对于高血钠患者来说，钠盐并不适合。
发明内容
为了解决现有技术存在的以上问题，本发明提供了一种抗菌效果增强同时具有补镁功效的头孢哌酮镁化合物、其制备方法及应用。本发明通过采用头孢哌酮与镁结合，能起到协同增效的作用，头孢哌酮能抑制病原菌细胞壁合成，对革兰阴性菌的作用强，联合镁元素后显著抑制细菌细胞壁的合成，使用效果显著增强。提高头孢哌酮药物的稳定性，同时为临床上不适合钠盐的患者提供一种选择。
本发明所采用的技术方案为：
一种头孢哌酮镁化合物，结构式如下：
Figure PCTCN2021092553-appb-000001
分子式为：Mg(C 25H 26N 9O 8S 2) 2；
分子量为：1288。
所述的头孢哌酮镁化合物的制备方法，包括以下步骤：
(1)制备头孢哌酮酸混悬液：
将头孢哌酮酸过筛，加入纯化水中，混匀，得到头孢哌酮酸混悬液；
(2)制备镁化合物溶液或混悬液：
将镁化合物粉碎成细粉，过筛，加入纯化水中，混匀，得到镁化合物溶液或混悬液；
(3)反应：
将步骤(2)所述的镁化合物溶液或混悬液加入到步骤(1)所述头孢哌酮酸混悬液中，搅拌条件下使充分反应，过滤得到滤液；
(4)干燥：
将步骤(3)得到的滤液进行干燥、纯化，即得所述的头孢哌酮镁化合物。
步骤(1)中，所述过筛的筛子孔径为60-120目。
所述头孢哌酮酸混悬液中的头孢哌酮酸与所述镁化合物溶液或混悬液中的镁元素的摩尔比为2:1。
步骤(2)中，所述镁化合物为氢氧化镁、碳酸氢镁、乙酸镁中的一种或几种的混合物。
步骤(3)中，进行所述反应的温度为15-30℃。
步骤(3)中，进行所述搅拌反应的同时还加入脱色剂。
所述脱色剂为活性炭。
步骤(4)中，所述干燥为喷雾干燥或冷冻干燥。
所述的喷雾干燥是采用喷雾干燥设备将步骤C的滤液分散成微粒，并且让其微粒与热空气接触，在瞬间除去其水分，得到水含量为以重量计2.0％以下的头孢哌酮镁化合物。本发明使用的喷雾干燥设备是目前市场上销售的产品，例如由江苏锡山市林州干燥机厂公司以商品名QZR-5型销售的产品。
所述的冷冻干燥是采用冷冻干燥设备将步骤C的滤液冷冻至0℃以下，并在高真空下通过加热使水分直接从固态升华为水汽，除去其水分，得到水含量为以重量计2.0％以下的头孢哌酮镁化合物。本发明使用的冷冻干燥设备是目前市场上销售的产品，例如由北京四环科学仪器厂有限公司以商品名LGJ-22D型销售的产品。
步骤(4)中，所述纯化采用重结晶、十八烷基硅烷反相柱、制备液相色谱法进行纯化，以达到产品中头孢哌酮镁化合物的含量为以重量计95％以上。
重结晶纯化是在乙醇-水(1:9-9:1)溶剂中在25℃条件下进行的。十八烷基硅烷反相柱(ODS)是一种常用的反相色谱柱，也叫C18柱；由于它是长链烷基键合相，有较高的碳含量和更好的疏水性，对各种类型的生物大分子有更强的适应能力，因此在生物化学分析工作中应用的最为广泛。使用ODS柱纯化是在使用由YMC公司以商品名GEL C18AAG12S50销售的ODS柱在5％-50％乙腈-甲醇-水作为洗脱液条件下进行的。
使用制备液相色谱法纯化所用仪器为岛津公司以商品名CTO-10A销售的制备液相，制备色谱柱为C18，液相条件为乙腈-甲醇-水(5:50:45)。
头孢哌酮镁化合物含量是根据高效液相色谱法测定的，所用仪器为Agilent 1260，色谱柱为ODS C18，液相条件为0.1％磷酸水-乙腈(70:30)，流速为1mL/min，检测波长230nm。
一个镁离子(Mg 2+)与两个头孢哌酮上的羧基发生反应，生成头孢哌酮镁，分子式为Mg(C 25H 26N 9O 8S 2) 2，分子量为1288。
所述的头孢哌酮镁化合物在制备抗菌药物中的应用。
镁是一种参与生物体正常生命活动及新陈代谢过程的必不可少的元素。镁影响细胞的多种生物功能，影响钾离子和钙离子转运，调控信号传递，参与能量代谢、蛋白质和核酸合成；催化酶的激活和抑制及对细胞周期、细胞增殖及细胞分化的调控；镁还参与维持基因组的稳定性，并且还与机体氧化应激和肿瘤发生有关。本申请发明人在长期研究中发现，通过将镁元素与头孢哌酮合用，对一些炎症的治疗起到协同增效的作用。
本发明的有益效果为：
(1)本发明所述的头孢哌酮镁化合物，通过采用头孢哌酮与镁结合，能够起到协同增效的作用，头孢哌酮能抑制病原菌细胞壁的合成，对革兰阴性菌的作用强，联合镁元素后可显著抑制细菌细胞壁的合成，使用效果显著增强。本发明通过利用头孢哌酮酸与镁离子反应生成头孢哌酮镁化合物，提高头孢哌酮药物的稳定性，简化制剂生产工艺，同时为临床上不适合钠盐的患者提供一种选择。本申请所述头孢哌酮镁化合物为含镁抗生素，同时具有抗菌治疗效果和补镁的效果，并对一些炎症有益处，起到协同增效的作用。
(2)本发明所述头孢哌酮镁的制备方法，操作简单，不使用有机溶剂，无有毒有害物质生成，产品纯度高，便于工业化生产。
具体实施方式
实施例1
本实施例提供一种头孢哌酮镁化合物的制备方法，所述头孢哌酮镁化合物 的分子式为：Mg(C 25H 26N 9O 8S 2) 2，分子量为1288，结构式如下：
Figure PCTCN2021092553-appb-000002
所述的头孢哌酮镁化合物的制备方法，包括以下步骤：
(1)制备头孢哌酮酸混悬液：
将0.2mol头孢哌酮酸过100目筛，加入100mL纯化水中，混匀，得到头孢哌酮酸混悬液；
(2)制备碳酸氢镁溶液：
将0.1mol碳酸氢镁粉碎成细粉，过60目筛，加入30mL纯化水中，混匀，得到碳酸氢镁溶液；
(3)反应：
将步骤(2)所述的碳酸氢镁溶液加入到步骤(1)所述头孢哌酮酸混悬液中，在25℃下搅拌使充分反应，再加入0.1％活性炭进行搅拌脱色，过滤得到滤液；
(4)干燥：
将步骤(3)得到的滤液先在50帕压力条件下进行冷冻干燥，得到水含量低于2％的初品，之后在25℃条件下、采用乙醇-水(1:9-9:1)溶剂进行重结晶纯化，即得所述的头孢哌酮镁化合物产品。
经高效液相色谱法测定，产品中头孢哌酮镁化合物的含量为以重量计95％ 以上。高效液相色谱法所用仪器为Agilent 1260，色谱柱为ODS C18，液相条件为0.1％磷酸水-乙腈(70∶30)，流速为1mL/min，检测波长230nm。
实施例2
本实施例提供一种所述头孢哌酮镁化合物的制备方法，包括以下步骤：
(1)制备头孢哌酮酸混悬液：
将0.2mol头孢哌酮酸过120目筛，加入200mL纯化水中，混匀，得到头孢哌酮酸混悬液；
(2)制备乙酸镁溶液：
将0.1mol乙酸镁粉碎成细粉，过120目筛，加入40mL纯化水中，混匀，得到乙酸镁溶液；
(3)反应：
将步骤(2)所述的乙酸镁溶液加入到步骤(1)所述头孢哌酮酸混悬液中，在15℃下搅拌使充分反应，再加入活性炭进行搅拌脱色，过滤得到滤液；
(4)干燥：
将步骤(3)得到的滤液先在100帕压力条件下进行喷雾干燥，得到水含量低于2％的初品，之后采用十八烷基硅烷反相柱进行纯化，即得所述的头孢哌酮镁化合物产品。经检测，产品中头孢哌酮镁化合物的含量为以重量计95％以上。
实施例3
本实施例提供一种所述头孢哌酮镁化合物的制备方法，包括以下步骤：
(1)制备头孢哌酮酸混悬液：
将0.2mol头孢哌酮酸过80目筛，加入150mL纯化水中，混匀，得到头孢哌酮酸混悬液；
(2)制备氢氧化镁混悬液：
将0.1mol氢氧化镁粉碎成细粉，过120目筛，加入50mL纯化水中，混匀，得到氢氧化镁混悬液；
(3)反应：
将步骤(2)所述的氢氧化镁混悬液加入到步骤(1)所述头孢哌酮酸混悬液中，在30℃下搅拌使充分反应，再加入活性炭进行搅拌脱色，过滤得到滤液；
(4)干燥：
将步骤(3)得到的滤液先在70帕压力条件下进行喷雾干燥，得到水含量低于2％的初品，之后制备液相色谱法进行纯化，即得所述的头孢哌酮镁化合物产品。经检测，产品中头孢哌酮镁化合物的含量为以重量计95％以上。
实验例
1、采用紫外吸收光谱(紫外吸收光谱分析设备Agilent 8453)对实施例1制备的头孢哌酮镁化合物进行结构分析。
将现有技术中的头孢哌酮钠与本发明制得的头孢哌酮镁在200-400nm下仪器自动扫描紫外吸收光谱。采用对照品比较法测定紫外吸收光谱。
将2mg头孢哌酮钠与2mg本发明实施例1制备的头孢哌酮镁分别溶于10ml甲醇中，然后测定最大吸收波长，其结果列于表1中。
表1-头孢哌酮与头孢哌酮镁的最大吸收波长
Figure PCTCN2021092553-appb-000003
从表1可以看出，头孢哌酮钠与头孢哌酮镁化合物的紫外最大吸收波长(λ max)相同，它们都在228、265nm处有最大吸收；这些紫外吸收光谱结果表明，镁离子的结合对头孢哌酮的离域共振结构影响不大。
2、头孢哌酮钠与头孢哌酮镁化合物在水中溶解度的比较
采用常规溶解度测定方法，在温度25℃水中，测定了头孢哌酮钠与头孢哌酮镁的溶解度，其结果列于表2中。
表2-头孢哌酮钠与头孢哌酮镁化合物在水中的溶解度
样品 溶解度(g/mL)
头孢哌酮钠 2.07
头孢哌酮镁 4.46
从表2可以看出，头孢哌酮镁的水溶性较好，是头孢哌酮钠的2.15倍。
3、稳定性
分别精密称取头孢哌酮钠、头孢哌酮镁适量，各2份，置于容量瓶中，分别加0.9％氯化钠，5％葡萄糖注射液定容至刻度，配制浓度为4mg/ml的样品液，将各样品分别放置于20℃的恒温水溶中，分别于1、2、4、8h取样测定，计算各待测溶液的药物浓度，取平均值。结果列于表3中。
表3-头孢哌酮钠与头孢哌酮镁的稳定性结果
Figure PCTCN2021092553-appb-000004
由表3可见，本发明所述头孢哌酮镁在不同注射液中的稳定性，含量变化比头孢哌酮钠变化更小，稳定性更好。
4、体外抑菌试验
根据徐淑云编写的《药理实验方法学》(第三版)，第63章中抗菌药物实验 法，进行体外抑菌实验。
液体培养基的制备：精密称取牛肉膏2g、酵母3g、氯化钠5g、蒸馏水700ml混合，调节液体培养基的pH值为7.0左右，在蒸汽灭菌锅里高压灭菌30min，温度调至120℃，灭菌完毕后取出，分别放入6个锥形瓶中，每一个锥形瓶中液体培养基的体积为100ml，密封好，备用。
药液的制备：按照等摩尔的β-内酰胺环数精密称取头孢哌酮钠和头孢哌酮镁，加蒸馏水定容至10ml，稀释后使头孢哌酮钠和头孢哌酮镁药液浓度分别为32μg/ml、31.26μg/ml，采用0.22μm滤膜过滤，取续滤液，备用。
菌液的制备：将大肠埃希菌、白葡萄球菌第二代细菌从营养琼脂斜面上分别挑取一接种环的量，接种到2个带有液体培养基的锥形瓶中，轻轻摇晃锥形瓶后，放置在气浴恒温振荡器中，温度调节至37℃，培养24-48h后，观察液体变浑浊，即细菌复活成功。
比浊法实验：实验前，将所需实验用品120℃下高压灭菌30分钟，用紫外灯消毒实验室30min。在无菌操作条件下，取28支试管，分为四组，每支试管加入液体培养基，倍比稀释头孢哌酮钠和头孢哌酮镁药液。其中14支加入50μl白葡萄球菌液，其余14支加入50μl大肠杆菌液，再次放入恒温培养箱中培养18-24h，观察浊度。
抑菌效果评价：通过紫外分光光度计测量OD600值，3次重复实验均有抑菌效果，视为抑菌作用有效。
结果见表4、表5。
表4-头孢哌酮钠及头孢哌酮镁对白葡萄球菌的抑菌效果
Figure PCTCN2021092553-appb-000005
Figure PCTCN2021092553-appb-000006
表5-头孢哌酮钠及头孢哌酮镁对大肠杆菌的抑菌效果
Figure PCTCN2021092553-appb-000007
从表4和表5可以看出，根据测量OD600值，确定头孢哌酮钠和头孢哌酮镁对2种耐药性细菌均有一定的抑制作用，随着药物浓度的升高，抑菌效果越明显。通过对比相同摩尔的β-内酰胺环数的头孢哌酮钠和头孢哌酮镁的OD值，在对白葡萄球菌的抑制作用上，头孢哌酮镁在31.26，7.82，3.90，1.95，0.98，0.48μg/ml浓度时，抑菌效果分别强于相同摩尔的β-内酰胺环数对应头孢哌酮钠在32、8、4、2、1、0.5μg/ml浓度；在对大肠杆菌的抑制作用上，头孢哌酮镁在1.95、0.98、0.48μg/ml浓度时，抑菌效果分别强于相同摩尔的β-内酰胺环数对应头孢哌酮钠在2、1、0.5μg/ml浓度，综上所述，头孢哌酮镁盐对白葡萄球菌的抑制作用较强，强于头孢哌酮钠。
5、体内抗菌实验
(1)检测方法
将真空冻干保存的肺炎链球菌标准株用液体培养基溶解后，加入到含有10％小牛血清的营养肉汤中，7.5％二氧化碳，37℃培养24h。将复苏后的细菌传代至血琼脂平板培养基上，7.5％二氧化碳，37℃培养24h。使用前，在无菌超净台上用取菌环将细菌挑出，用无菌生理盐水稀释至适当浓度并检测600nm吸光度(A600nm)值为2.489。
大鼠适应性饲养一周后，每天早晚监测体温，连续监测3d。选择体温浮动小于0.5℃的大鼠纳入实验，随机分为6组，分别为空白组、模型组、头孢哌酮 钠低剂量组、头孢哌酮钠高剂量组、头孢哌酮镁低剂量组、头孢哌酮镁高剂量组，每组10只。大鼠麻醉后，撑开大鼠口腔，充分暴露咽喉部，用连接注射器的气管导管经口缓慢插入气管，推入0.6ml菌液，空白组推入相同体积的生理盐水。接种后立即竖立大鼠，使大鼠保持直立位约20s，以保证接种菌液因重力作用而入肺。
尾静脉注射给药，给药剂量低为180mg/kg，高剂量360mg/kg(鉴于镁、钠分子量不同，这里的低剂量和高剂量均是以头孢哌酮计)，一日两次，连续三天，空白组和模型组给予等体积生理盐水。
(2)观察内容及检测指标
观察大鼠生理病理状况是否主动摄食摄水；是否呆滞、竖毛、嗜睡；鼻腔及眼眶有无分泌物；有无气促，有无喘鸣音；尿色、量及粪便变化等。
末次给药12h后，腹腔注射4％水合氯醛10mg/kg麻醉，腹主动脉取血1.5mL于加有EDTA抗凝真空采血管中，混匀，血液分析仪测定全血WBC计数及分类。
取双肺用10％甲醛溶液固定，常规石蜡包埋，切片，苏木素-伊红(HE)染色，于光镜下观察肝组织病理学变化。
采用SPSS 19.0统计软件处理实验数据，计量数据以x±s表示。组间均数比较采用单因素方差分析，以P＜0.05表示有统计学意义。
(3)检测结果
(3.1)一般状态
动物接种后第1天，给药组和模型组开始出现呼吸急促、进食减少、对周围环境反应差。第2天动物进食、活动度明显下降，毛发直竖，卷曲于饲养笼角落。头孢哌酮钠(低、高剂量)组均较头孢哌酮镁(低、高剂量)组呼吸急促，且进食量明显少于头孢哌酮镁组。第3天，给药组动物食欲、活动度上升，头孢哌酮钠组大鼠较头孢哌酮镁组呼吸依旧急促，模型组大鼠可闻及明显呼吸 喘鸣声，体重减轻，出现明显骨感。空白组大鼠食欲、活动度、体质量、体温较接种前无明显变化。
(3.2)血常规
(3.2.1)对白细胞数的影响
与空白组比较，模型组大鼠白细胞数显著上升(P＜0.01)；与模型组比较，各给药组大鼠白细胞数显著降低(P＜0.01)，说明头孢哌酮钠、镁均对大鼠肺炎有治疗作用，并且与头孢哌酮钠低剂量组比较，头孢哌酮镁低剂量组的白细胞数降低更为显著(P＜0.05)。与空白组比较，头孢哌酮钠高剂量组差异显著(P＜0.01)，头孢哌酮镁高剂量组无明显差异。
(3.2.2)对中性粒细胞百分比的影响
与对照组比较，模型组大鼠中性粒细胞百分比显著上升(P＜0.01)；与模型组比较，各给药组大鼠中性粒细胞数显著降低(P＜0.01，P＜0.05)，说明头孢哌酮钠、头孢噻肟镁均对大鼠肺炎有治疗作用，并且与头孢哌酮钠低剂量组比较，头孢哌酮镁低剂量组的中性粒细胞百分比降低更为显著(P＜0.05)。与空白组比较，头孢哌酮钠高剂量组差异显著(P＜0.01)，头孢哌酮镁高剂量组无明显差异。
(3.2.3)对淋巴细胞百分比的影响
与对照组比较，模型组大鼠淋巴细胞百分比显著下降(P＜0.01)；与模型组比较，各给药组大鼠淋巴细胞数显著升高(P＜0.01，P＜0.05)。说明头孢哌酮钠、头孢哌酮镁均对大鼠肺炎有治疗作用，并且与头孢哌酮钠低剂量组比较，头孢哌酮镁低剂量组的淋巴细胞百分比升高更为显著(P＜0.05)。
结果见表6所示。
表6-头孢哌酮哌酮镁体内药效实验结果
Figure PCTCN2021092553-appb-000008
Figure PCTCN2021092553-appb-000009
注：与模型组比较， *p＜0.05， **p＜0.01；与空白组比较， #p＜0.05， ##p＜0.01；与头孢哌酮钠低剂量组比较， △P＜0.05， △△P＜0.01。

Claims (10)

  1. 一种头孢哌酮镁化合物，其特征在于，结构式如下：
    Figure PCTCN2021092553-appb-100001
    分子式为：Mg(C 25H 26N 9O 8S 2) 2；
    分子量为：1288。
  2. 权利要求1所述的头孢哌酮镁化合物的制备方法，其特征在于，包括以下步骤：
    (1)制备头孢哌酮酸混悬液：
    将头孢哌酮酸过筛，加入纯化水中，混匀，得到头孢哌酮酸混悬液；
    (2)制备镁化合物溶液或混悬液：
    将镁化合物粉碎成细粉，过筛，加入纯化水中，混匀，得到镁化合物溶液或混悬液；
    (3)反应：
    将步骤(2)所述的镁化合物溶液或混悬液加入到步骤(1)所述头孢哌酮酸混悬液中，搅拌条件下使充分反应，过滤得到滤液；
    (4)干燥：
    将步骤(3)得到的滤液进行干燥、纯化，即得所述的头孢哌酮镁化合物。
  3. 根据权利要求2所述的头孢哌酮镁化合物的制备方法，其特征在于，步骤(1)中，所述过筛的筛子孔径为60-120目。
  4. 根据权利要求2所述的头孢哌酮镁化合物的制备方法，其特征在于，所述头孢哌酮酸混悬液中的头孢哌酮酸与所述镁化合物溶液或混悬液中的镁元素的摩尔比为2:1。
  5. 根据权利要求2所述的头孢哌酮镁化合物的制备方法，其特征在于，步骤(2)中，所述镁化合物为氢氧化镁、碳酸氢镁、乙酸镁中的一种或几种的混合物。
  6. 根据权利要求2所述的头孢哌酮镁化合物的制备方法，其特征在于，步骤(3)中，进行所述反应的温度为15-30℃；
  7. 根据权利要求2所述的头孢哌酮镁化合物的制备方法，其特征在于，步骤(3)中，进行所述搅拌反应后还加入脱色剂。
  8. 根据权利要求7所述的头孢哌酮镁化合物的制备方法，其特征在于，所述脱色剂为活性炭。
  9. 根据权利要求2所述的头孢哌酮镁化合物的制备方法，其特征在于，步骤(4)中，所述干燥为喷雾干燥或冷冻干燥。
  10. 权利要求1所述的头孢哌酮镁化合物在制备抗菌药物中的应用。
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