WO2021008549A1 - 一种新化合物及药物组合物 - Google Patents

一种新化合物及药物组合物 Download PDF

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WO2021008549A1
WO2021008549A1 PCT/CN2020/102060 CN2020102060W WO2021008549A1 WO 2021008549 A1 WO2021008549 A1 WO 2021008549A1 CN 2020102060 W CN2020102060 W CN 2020102060W WO 2021008549 A1 WO2021008549 A1 WO 2021008549A1
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purification
separation
pharmaceutical composition
climycin
new compound
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French (fr)
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姜恩鸿
梁鑫淼
刘艳芳
邱桂侠
姜勋东
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沈阳福洋医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to a new compound and a pharmaceutical composition.
  • Kelimycin also known as Bitespiramycin and Shengjimycin, was developed by the Institute of Biotechnology of the Chinese Academy of Medical Sciences in cooperation with the applicant.
  • the 4" isovaleryl transferase group (4"-o-acyl-transferase) was cloned into the spiramycin producing bacteria, and the spiramycin 4"-OH was acylated and the isovaleryl side chain was added at the 4" position
  • Calinomycin is composed of a variety of spiramycin derivatives.
  • the structural formula is shown in formula (2).
  • the total content of the main active ingredient isovalerylspiramycin (I+II+III) is not less than 60%, 4"
  • the total content of acylated spiramycin is not less than 80%, and it is an acceptable pharmaceutical composition in pharmacy.
  • the central structure is a 16-membered lactone ring, and one molecule of folofamine and one molecule of mycotamine It is connected with a molecule of mycylose. Its main components are isovalerylspiramycin I, II, and III.
  • the difference in structure from spiramycin is that the group attached to the 4" position of mycylmycose is isovaleryl instead of Hydroxy.
  • the chemical structure of climycin contains more than ten components.
  • the composition standard of the finished product of climycin is isovalerylspiramycin III ⁇ 30%, the total proportion of isovalerylspiramycin I, II, and III ⁇ 60%, and the proportion of total acylated spiramycin ⁇ 80%. Unacylated spiramycin ⁇ 5%.
  • Clarithromycin is a white non-crystalline powder, slightly hygroscopic, with a specific rotation of about -80.8°, and a maximum ultraviolet absorption wavelength of 231 ⁇ 232nm. It has a weak fluorescent chromophore, and it is exposed to concentrated sulfuric acid or hydrochloric acid. Violet reaction produces strong purple fluorescence, with maximum absorbance at 231 ⁇ 232nm.
  • the drug has good lipophilicity, strong tissue penetration ability, fast oral absorption, long maintenance time in the body, and continuous post-antibiotic effect. According to the relationship between pharmacodynamics and chemical conformation, after macrolide antibiotics are acylated at the 4" position, their lipophilicity and in vivo activity are improved, in vivo antibacterial activity and clinical therapeutic effects are significantly improved, and the stability of antibiotics in vivo As the carbon chain of the 4" hydroxy ester increases, it increases, that is, isovalerylspiramycin>butyrylspiramycin>propionylspiramycin>acetylspiramycin.
  • composition of clarithromycin is complicated, and the extract obtained from the fermentation broth by solvent extraction method contains too much acetyl and propionylspiramycin, so that the final product obtained cannot meet the component index requirements. Therefore, establishing a reasonable and effective extraction, separation and purification process based on the solvent extraction method, removing impurities, and optimizing the composition ratio is an effective method to improve the quality of climacin products.
  • the technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a new compound and pharmaceutical composition.
  • the present invention adopts the following technical solutions:
  • the present invention also provides the method for separation and purification of the new compound, wherein the method for separation and purification is: firstly analyze the climycin sample to determine the target peak, and then purify, preferably three times, to obtain the New compounds.
  • the retention time of the target peak is 15.015 min, and the RRT is 0.50.
  • the preferred elution conditions are: 0 min, 30% acetonitrile, 70% ammonium acetate; 65 min, 65% acetonitrile , Ammonium acetate 35%; Flow rate is 0.2mL/min; Injection volume is 1 ⁇ L; Wavelength is 232nm.
  • the first purification uses a reversed-phase chromatography column
  • the mobile phase is acetone and ammonium acetate, preferably 58% acetone/10mM ammonium acetate; more preferably, the flow rate is 80mL/min, the injection volume is 30mL, and the sample load is 0.6% , The wavelength is 232nm.
  • the climycin sample is obtained by inoculating, culturing, fermentation and extracting the climycin producing bacteria.
  • the colimycin-producing bacterium is a species capable of producing climycin, including but not limited to cloning the carbomycin-producing bacterium 4" isovaleryltransferase gene to the spiramycin-producing bacterium to make A cloned strain of spiramycin-producing bacteria containing the 4" isovaleryltransferase gene obtained by the expression; or further inactivation of the Lrp gene in the cloned strain of spiramycin-producing bacteria with 4" isovaleryltransferase gene.
  • the spiramycin-producing bacterial strain containing 4" isovaleryltransferase gene may be WSJ195; the Lrp gene in the spiramycin-producing bacterial clone containing 4" isovaleryltransferase gene is inactivated.
  • the colimycin-producing bacteria can be Streptomyces spiramyceticus, which was delivered to the General Microbiology Center of the China Microorganism Collection Management Committee on July 4, 2018. Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing , Deposit number CGMCC No.16055.
  • control tank pressure is 0.04 ⁇ 0.01MPa
  • control ventilation rate is 1:(1 ⁇ 0.1)v/v/min;
  • the extraction first neutralize with water until the pH is acidic, and then bubbling in oxygen for oxidation, and post-processing to obtain the climycin sample.
  • the climycin sample provided by the present invention can be prepared by the following methods, including culture fermentation and extraction, wherein the culture fermentation includes:
  • the strain is colimycin-producing bacteria, and a single colony is selected from the preservation slope of the climycin-producing bacteria including but not limited to WSJ195 or Streptomyces spiramyceticus for subculture, and then selected The colonies with concave folds on the surface were subjected to secondary passage activation culture.
  • the colonies with the concave folds in the middle of the surface are picked for the second subculture activation and then inoculated to the first-level seed medium.
  • the bacteria are cultured to the folds with depressions in the middle of the colony surface at the first passage, they are directly inoculated to the first-level seed medium for culture.
  • This application has undergone multiple subculture and fermentation experiments and found that the bacterial layer formed by a single colony with a concave fold on the surface is relatively smooth or a colony with protrusions, and the viability of the bacterial cells is higher, and the later fermentation effect is better. High potency.
  • step (1) and step (2) When culturing in step (1) and step (2), the tank pressure is controlled to 0.04 ⁇ 0.01MPa, and the ventilation rate is 1:(1 ⁇ 0.1)v/v/min.
  • step (3) control the tank pressure to 0.04 ⁇ 0.01Mpa; within the fermentation time of 0-15h, control the aeration rate to 1:(0.3 ⁇ 0.03)v/v/min, and gradually adjust to 1: after 15h: (1.0 ⁇ 0.1)v/v/min.
  • the fermentation of climycin samples requires a suitable amount of dissolved oxygen. When it is too high, the bacteria will grow faster and the product yield will decrease. When it is too low, it will affect the production of the bacteria and then the yield.
  • the dissolved oxygen is controlled to be suitable for the needs of the growth of the bacteria and the production of metabolites at each stage, which can greatly increase the titer of the product.
  • the extraction is as follows: the fermentation broth after culturing and fermentation is treated with aluminum sulfate to obtain the filtrate, the pH is adjusted to 8.5-9.0, and the extraction is carried out with butyl acetate.
  • the butyl acetate extract is carried out with salt-free and 1% NaH 2 PO 4 respectively. Wash, then extract with pH 2.0-2.5 water to obtain an aqueous phase extract, adjust the pH to 4.5-5.5, volatilize to remove residual butyl acetate to obtain a water extract, filter, adjust the filtrate to pH 8.5-9.0 to obtain a precipitate, use Purified water rinses the precipitate to obtain a wet product and dry it.
  • CN101921302A discloses a process for purifying climycin, in which the pH value of the aqueous extract is adjusted to neutral before the aqueous extract obtained in step (4) is crystallized.
  • the neutral adjustment because the clarithromycin will crystallize in the alkaline condition in the water phase, and it will destroy the drug under the condition of overacid and overbase, and the drug will be degraded. Once it is too neutral, it will affect ; And when adjusting the pH value, it is adjusted with NaOH solution.
  • there will be agglomeration This is because the residual butyl ester in the water extract meets alkali. As the pH value increases, the alkali The increase in the dosage will cause the agglomeration to be more prominent, which will cause difficulties in the subsequent blowing of butyl ester, prolong the blowing time, easily lose materials, and affect the yield.
  • the pH value from water extraction from 2.0-2.5 to pH 4.5-5.5 can be adjusted immediately to avoid the influence of the drug over acid conditions for a long time, and is relatively stable under other pH ranges; at the same time, the pH value from water extraction in the present invention is 2.0-2.5 2.5 is adjusted to pH 4.5-5.5 immediately, reducing the amount of alkali and avoiding agglomeration.
  • the clidamycin sample prepared by the present invention contains the novel formula (I) of the present invention.
  • Compound, further experiments have found that the climycin sample containing the new compound represented by formula (I) has better antibacterial effect.
  • the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition contains the new compound of the present invention, preferably, the pharmaceutical composition also contains isovalerylspiramycin I and isovalerylspiramycin At least one of Iovalerylspiramycin III or Isovalerylspiramycin III; more preferably, the mass content of the new compound in the pharmaceutical composition is less than 5%.
  • Isovalerylspiramycin III, II and I are the main active ingredients of climycin.
  • the present invention found that when a small amount of the new compound is contained in the climycin composition, the stability of isovalerylspiramycin III, II and I under acidic conditions can be improved, so that isovalerylspiramycin III, II And I crystallize under acidic conditions, and the content is high; and it was surprisingly found that when a small amount of the new compound is contained in the climycin composition of the present invention, they have a synergistic effect with each other, and the antibacterial effect has achieved a better effect.
  • the pharmaceutical composition can be a combination of the prior art isovalerylspiramycin III, II, I and the new compound of formula (I) of the present invention.
  • the pharmaceutical composition of the present invention can be obtained by the above-mentioned inoculation, culture, fermentation, and extraction methods of the present invention, that is, the pharmaceutical composition of the present invention is the above-mentioned inoculation, culture, fermentation, and And the extracted climycin sample.
  • the advantages of the present invention are that the method is simple to operate, and a new compound is obtained by separating and purifying cleritromycin through a new separation and purification method.
  • Figure 1 is an analysis spectrum of the target peak determined by sample analysis in Example 1 of the present invention.
  • Example 2 is an analysis spectrum diagram of the target peak determined by sample analysis in Example 1 of the present invention
  • Example 3 is a spectrum diagram of the separation method in the first-dimensional purification preparation process in Example 1 of the present invention.
  • Example 4 is a spectrum diagram of fraction analysis during the first-dimensional purification preparation process in Example 1 of the present invention.
  • Example 5 is a spectrum diagram of the separation method in the second-dimensional purification preparation process in Example 1 of the present invention.
  • Example 6 is a spectrum diagram of fraction analysis during the second-dimensional purification preparation process in Example 1 of the present invention.
  • Fig. 7 is a spectrum diagram of fraction analysis during the second-dimensional purification preparation process in Example 1 of the present invention.
  • Example 8 is a spectrum diagram of the separation and purification method in the third-dimensional purification preparation process in Example 1 of the present invention.
  • FIG. 9 is an analysis spectrum diagram of compound purity analysis during the third-dimensional purification preparation process in Example 1 of the present invention.
  • Figure 10 is a high-resolution mass spectrum of a new compound prepared in Example 1 of the present invention.
  • Figure 11 is an NMR characterization spectrum of the new compound prepared in Example 1 of the present invention.
  • inoculate into the first-level seed medium for cultivation control the tank pressure at 0.04MPa, control the tank temperature at 28°C, and control the ventilation rate at 1:(1 ⁇ 0.1)v/v/min, culture 48h, get the first-level seed liquid;
  • the new compound represented by formula (1) can be obtained by separation and purification from the climycin sample
  • the peak with retention time of 15.015min is the target peak of the new compound, RRT is 0.50, and the chromatographic purity is 0.52%.
  • Sample Take 600g of CA1512031 batch of climycin product, add acetone to dissolve, and then add ammonium acetate water to dilute and dissolve, the concentration is 67mg/mL.
  • Chromatographic column RZA10-C18YE-0.05 (10 ⁇ m, 50 ⁇ 255mm, 2017112104)
  • Mobile phase A: acetonitrile, B: triethylamine acetate (pH8.07); configuration of triethylamine acetate (pH8.07): 10L water, add 100mL triethylamine, then add 40mL acetic acid, and adjust the pH to 8.07;
  • Injection volume 21mL, sample loading volume 0.7%;
  • Fraction post-treatment Fraction post-treatment: Desalination by SPE, concentration under reduced pressure at 50°C to obtain 81 mg of the target fraction, which is recorded as CA1512031-7-6.
  • Compound collection shaded area.
  • Compound post-treatment Fraction post-treatment: Desalination by SPE, concentration under reduced pressure at 50°C, adding appropriate amount of pure water and freeze-drying to obtain 7.3 mg of the target compound, which is designated as CA1512031-7-6-4.
  • the chromatographic purity of the new compound is 95.02%.
  • inoculate into the first-level seed medium for cultivation control the tank pressure at 0.04 ⁇ 0.01MPa, control the tank temperature at 28 ⁇ 1°C, and control the ventilation rate at 1:(1 ⁇ 0.1)v/v /min, cultivate for 49h to obtain the first-level seed liquid;
  • the new compound represented by formula (1) can be obtained by separation and purification from the climycin sample
  • inoculate into the first-level seed medium for cultivation control the tank pressure at 0.04 ⁇ 0.01MPa, control the tank temperature at 28 ⁇ 1°C, and control the ventilation rate at 1:(1 ⁇ 0.1)v/v /min, cultivate for 45h to obtain first-level seed liquid;
  • the new compound represented by formula (1) can be obtained by separation and purification from the climycin sample
  • inoculate to the first-level seed medium for culture control the tank pressure at 0.04 ⁇ 0.01MPa, control the tank temperature at 28°C, control the ventilation rate at 1:1.1v/v/min, and cultivate for 48h.
  • the new compound represented by formula (1) can be obtained by separation and purification from the climycin sample
  • a pharmaceutical composition comprising a compound of formula (1) and isovalerylspiramycin I, wherein the content of the compound of formula (1) is 1%.
  • a pharmaceutical composition comprising a compound of formula (1) and isovalerylspiramycin II, wherein the content of the compound of formula (1) is 2%.
  • a pharmaceutical composition comprising a compound of formula (1) and isovalerylspiramycin III, wherein the content of the compound of formula (1) is 0.5%.
  • a pharmaceutical composition comprising a compound of formula (1), isovalerylspiramycin I and isovalerylspiramycin II, wherein the compound of formula (1), isovalerylspiramycin I and isovalerylspiramycin
  • the content of II is 0.1%, 44.9% and 55.0% respectively.
  • inoculate to the first-level seed medium for culture control the tank pressure at 0.04 ⁇ 0.01MPa, control the tank temperature at 28 ⁇ 1°C, and control the ventilation rate at 1:0.6v/v/min. 48h, get the first-level seed liquid;
  • the extraction method for extracting the finished product of climaromycin from the fermentation broth is the same as in Example 1, and 7.8 g of the finished product of climamycin is obtained.
  • Test Example 1 In vitro antibacterial test data
  • Standard agar double dilution method was used to determine the minimum inhibitory concentration of each antibacterial drug against various pathogenic bacteria, and the pH value of the medium was 7.0.

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Abstract

属于药物化学领域，具体地说，涉及一种新化合物及药物组合物。所述的新化合物的结构式如式（1）所示，方法操作简单，通过一种新的分离纯化方法从可利霉素中分离纯化得到了一种新化合物。并惊喜地发现当可利霉素组合物中含有少量的新化合物时，产生了协同作用，抗菌作用达到了更好的效果。

Description

一种新化合物及药物组合物 技术领域
本发明属于药物化学领域，具体地说，涉及一种新化合物及药物组合物。
背景技术
可利霉素(Kelimycin)，又称必特螺旋霉素(Bitespiramycin)、生技霉素(Shengjimycin)是由中国医科院生物技术研究所与本申请人合作，通过转基因技术将碳霉素产生菌的4”异戊酰基转移酶基团(4”-o-acyl-transferase)克隆至螺旋霉素产生菌中，定向酰化螺旋霉素4”-OH，在4”位加入异戊酰基侧链所形成的以4”位异戊酰基螺旋霉素为主要组分的新型抗生素。
可利霉素是由多种螺旋霉素衍生物组成，结构式如式(2)所示，主要活性成分异戊酰螺旋霉素(Ⅰ+Ⅱ+Ⅲ)总含量不低于60％，4”酰化螺旋霉素的总含量不低于80％，于药学上是一种可接受的药物组合物。中心结构为16元内酯环，与一分子福洛胺糖、一分子碳霉胺糖和一分子碳霉糖连接而成，其主要成分异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ与螺旋霉素结构不同之处在于碳霉糖4”位上连接的基团为异戊酰基而不是羟基。可利霉素的化学结构，如下所示，共包含十余种组分。目前可利霉素成品组成标准为异戊酰螺旋霉素Ⅲ≥30％，异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ的比例总和≥60％，总酰化螺旋霉素的比例≥80％，未酰化的螺旋霉素≤5％。
Figure PCTCN2020102060-appb-000001
可利霉素属于16元大环内酯类抗生素，具有活性基团羧基、烷氧基、环氧基、酮基和醛基以及一对共轭的C＝C，分子量约为884～982。由于具有相似的化学结构，可利霉素与大环内酯类抗生素具有很多共性：易溶于酯类、丙酮、氯仿、醇类等大多数有机溶剂，微溶于石油醚，难溶于水；分子结构中含有两个二甲胺基而呈弱碱性，易溶于酸性水溶液；具有溶解 度随温度的升高而降低的“负溶解度”性质。由于可利霉素主要组分异戊酰螺旋霉素4”位碳链较长，亲水性差，其水中溶解度比螺旋霉素及4”-乙酰螺旋霉素小。
可利霉素是一种白色非结晶粉末，略有引湿性，比旋度约为-80.8°，紫外最大吸收波长为231～232nm，本身带有弱荧光发色基团，遇浓硫酸或盐酸呈紫色反应，产生强紫色荧光，在231～232nm处有最大吸光值。
该药具有亲脂性好，组织渗透能力强，口服吸收快，体内维持时间长，有持续的抗生素后效应。根据药效与化学构象的关系，大环内酯类抗生素4”位酰化后，其亲脂性和体内活性提高，体内抗菌活性与临床治疗效果均得到了显著提升，并且抗生素在体内的稳定性随着4”羟基酯的碳链增长而增强，即异戊酰螺旋霉素＞丁酰螺旋霉素＞丙酰螺旋霉素＞乙酰螺旋霉素。
初步体内外药效学试验表明，该药不仅对多数G +菌有较好抗菌活性，对部分G -菌也有一定作用，各项技术指标明显优于阿奇霉素、红霉素、乙酰螺旋霉素、麦迪霉素，尤其对肺炎支原体的抗菌活性最强，对红霉素耐药菌、淋球菌、肺炎球菌、金葡菌、绿脓假单胞菌、流感杆菌、流感嗜血杆菌、脆弱拟杆菌、军团菌、多行杆菌和产气荚膜梭菌也有一定抗菌活性，对临床耐红霉素的金葡球菌仅有极少交叉耐药性。可利霉素将主要用于治疗革兰氏阳性菌感染性疾病，尤其是上呼吸道感染，并可能用于泌尿系统感染等。
可利霉素成分复杂，用溶媒萃取法从发酵液中提取获得的萃取液由于含有过多乙酰、丙酰螺旋霉素而使最终获得的成品不能达到组分指标要求。因此，在溶媒萃取法的基础上建立合理有效的提取、分离纯化工艺，去除杂质，优化组分配比，是提高可利霉素产品质量的有效方法。
本发明人在对可利霉素进行提取、分离纯化工艺时，通过调整工艺惊喜地得到了一种新化合物，从而完成了本发明。
发明内容
本发明要解决的技术问题在于克服现有技术的不足，提供一种新化合物及药物组合物。
为解决上述技术问题，本发明采用如下技术方案：
一种新化合物，其中，所述的新化合物的结构式如式(1)所示：
Figure PCTCN2020102060-appb-000002
本发明还提供所述的新化合物的分离纯化方法，其中，所述的分离纯化方法为：首先对可利霉素样品进行分析确定目标峰，然后再进行纯化，优选三次纯化，得到所述的新化合物。
进一步的，目标峰的保留时间为15.015min，RRT0.50。
进一步的，所述的三次纯化均为反相色谱分离纯化。
进一步的，对可利霉素样品进行分析确定目标峰采用反相色谱柱，流动相为乙腈和乙酸铵；优选洗脱条件为：0min，乙腈30％，乙酸铵70％；65min，乙腈65％，乙酸铵35％；流速为0.2mL/min；进样体积为1μL；波长为232nm。
进一步的，第一次纯化采用反相色谱柱，流动相为丙酮和乙酸铵，优选58％丙酮/10mM乙酸铵；更优选流速为80mL/min，进样体积为30mL，载样量为0.6％，波长为232nm。
进一步的，第二次纯化采用反相色谱柱，流动相为乙腈和乙酸三乙胺；优选洗脱条件为乙腈：乙酸三乙胺＝56：44；流速：70mL/min；进样体积：21mL，载样量0.7％；波长：232nm；量程：10。
进一步的，第三次纯化采用反相色谱柱，以乙腈：乙酸三乙胺＝62：38等度洗脱；优选流速为10mL/min，进样体积为1mL，波长为232nm。
进一步的，所述的可利霉素样品为将可利霉素产生菌进行接种、培养发酵和提取得到的。
进一步的，所述的可利霉素产生菌为能产生可利霉素的菌种，包括但不限于将碳霉素产生菌4〃异戊酰基转移酶基因克隆至螺旋霉素产生菌，使之表达获得的含4〃异戊酰基转移酶基因的螺旋霉素产生菌克隆菌株；或者进一步将4〃异戊酰基转移酶基因的螺旋霉素产生菌克隆菌株中的Lrp基因失活得到的。
本发明中，含4〃异戊酰基转移酶基因的螺旋霉素产生菌克隆菌株可以为WSJ195；将含4〃异戊酰基转移酶基因的螺旋霉素产生菌克隆菌株中的Lrp基因失活得到的可利霉素产生菌可以为螺旋链霉菌Streptomyces spiramyceticus，于2018年7月4日送交中国微生物菌种保藏管理委员会普通微生物中心保藏，地址：北京市朝阳区北辰西路1号院3号，保藏编号CGMCC No.16055。
进一步的，将可利霉素进行接种时，挑取表面具有凹陷的褶皱的菌落进行培养；
优选的，所述的培养发酵中，控制罐压为0.04±0.01MPa，控制通气量为1：(1±0.1)v/v/min；
优选的，所述的提取中，先用水中和至pH为酸性，然后通入氧气进行氧化，经后处理得到所述可利霉素样品。
作为一种可供选择的实施方案，本发明所提供的可利霉素样品可采用如下方法制备得到，包括培养发酵和提取，其中培养发酵包括：
(1)将菌种进行活化后，接种至一级种子培养基中进行培养，得到一级种子液；
(2)将一级种子液接种至二级种子培养基中进行培养，得到二级种子液；
(3)将二级种子液接种至发酵罐中，控制发酵条件进行培养，得到发酵液。
其中，步骤(1)中，菌种为可利霉素产生菌，从可利霉素产生菌菌种包括但不限于WSJ195 或Streptomyces spiramyceticus的保藏斜面上挑取单菌落进行传代培养，然后挑取表面具有凹陷的褶皱的菌落进行二次传代活化培养。
优选的，第一次代培养一段时间后，挑取表面中部具有凹陷的褶皱的菌落进行二次传代活化培养，然后接种至一级种子培养基。或者，第一次传代时将菌培养至菌落表面中部具有凹陷的褶皱时，直接接种至一级种子培养基进行培养。
本申请经过多次传代培养、发酵试验发现，此种表面带有凹陷的褶皱的单菌落形成的菌层相对于表面光滑或者具有突起的菌落，菌体的活力高，后期发酵效果更好，产物效价高。
步骤(1)、步骤(2)中培养时，控制罐压为0.04±0.01MPa，控制通气量为1：(1±0.1)v/v/min。
步骤(3)中发酵培养时，控制罐压为0.04±0.01Mpa；在发酵时间0-15h内，控制通气量为1：(0.3±0.03)v/v/min，15h后逐步调至1：(1.0±0.1)v/v/min。
可利霉素样品的发酵需要合适的溶氧量，过高时，菌体生长快，产物产量反而下降，太低时影响菌体生产，进而影响产量。本申请通过控制种子培养和发酵过程中的罐压和通气量，将溶氧控制在适合菌体生长及产生代谢产物各阶段的需要，可以大大提高产物的效价。
所述的提取为：将培养发酵后的发酵液用硫酸铝处理得滤液，调pH至8.5-9.0，用乙酸丁酯提取，乙酸丁酯提取液分别用无盐水及1％NaH 2PO 4进行洗涤，再用pH2.0-2.5水提取，得水相提取液，调pH至4.5-5.5，挥发除去残余乙酸丁酯得水提取液，过滤，滤液调pH8.5-9.0，得沉淀，用纯化水对沉淀物进行淋洗，得湿品，干燥。
CN101921302A公开了一种可利霉素的纯化工艺，该工艺中在对步骤(4)得到的水相提取液进行结晶前将水相提取液的pH值调节至中性。而对于中性调节不便控制，因为可利霉素在水相中碱性条件产生析晶，在过酸过碱的条件下对药物都有破坏使药物产生降解，一旦过于中性就会产生影响；并且在调节pH值时，是使用NaOH溶液调节，在调节过程中，会有结团现象，这是因为水提液中残留丁酯遇碱造成的，随着pH值的增大，碱的用量增多，结团现象更突出，对后步吹出丁酯操作造成困难，延长了吹酯时间，容易损失物料，影响收率。
本发明中从水提pH2.0-2.5立刻调至pH4.5-5.5可以避免药物长时间过酸的条件影响，相对其他pH值范围下稳定；同时，本发明中从水提pH2.0-2.5立刻调至pH4.5-5.5，减少了碱的用量，避免了结团现象的发生。
通过进一步采用本发明所述的分离纯化方法对本发明所制得的可利霉素样品进行分离纯化，发现本发明所制得的可利霉素样品中含有本发明式(Ⅰ)所示的新化合物，进一步通过试验发现含有式(Ⅰ)所示的新化合物的可利霉素样品具有更好的抗菌效果。
本发明还提供一种药物组合物，其中，所述的药物组合物含有本发明所述的新化合物，优选，所述的药物组合物还含有异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅲ中的至少一种；更优选，所述的药物组合物中新化合物的质量含量小于5％。
异戊酰螺旋霉素Ⅲ、Ⅱ和Ⅰ为可利霉素的主要活性成分。本发明发现，当可利霉素组合物中含有少量的新化合物时，能够提高异戊酰螺旋霉素Ⅲ、Ⅱ和Ⅰ在酸性条件下的稳定性，使异戊酰螺旋霉素Ⅲ、Ⅱ和Ⅰ在酸性条件下结晶，含量高；并惊喜地发现当采用本发明的可 利霉素组合物中含有少量的新化合物时，相互具有协同作用，抑菌作用达到了更好的效果。
本发明中，所述的药物组合物可为现有技术的异戊酰螺旋霉素Ⅲ、Ⅱ、Ⅰ和本发明所述的式(Ⅰ)新化合物进行组合而成。
作为一种可供选择的实施方案，本发明所述的药物组合物可采用本发明上述接种、培养发酵和提取方法得到，即本发明所述的药物组合物即为本发明上述接种、培养发酵和提取得到的可利霉素样品。
本发明的优点在于，该方法操作简单，通过一种新的分离纯化方法从可利霉素中分离纯化得到了一种新化合物。
下面对本发明的具体实施方式作进一步详细的描述。
附图说明
图1为本发明实施例1中样品分析确定目标峰的分析谱图；
图2为本发明实施例1中样品分析确定目标峰的分析谱图；
图3为本发明实施例1中第一维纯化制备过程中分离方法的谱图；
图4为本发明实施例1中第一维纯化制备过程中馏分分析的谱图；
图5为本发明实施例1中第二维纯化制备过程中分离方法的谱图；
图6为本发明实施例1中第二维纯化制备过程中馏分分析的谱图；
图7为本发明实施例1中第二维纯化制备过程中馏分分析的谱图；
图8为本发明实施例1中第三维纯化制备过程中分离纯化方法的谱图；
图9为本发明实施例1中第三维纯化制备过程中化合物纯度分析的分析谱图；
图10为本发明实施例1所制备的新化合物的高分辨质谱图；
图11为本发明实施例1所制备的新化合物的NMR表征谱图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对实施例中的技术方案进行清楚、完整地描述，以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
一、制备可利霉素样品
培养发酵：
(1)从WSJ195菌种的保藏斜面上挑取单菌落进行传代培养，然后挑取表面中部具有凹陷的褶皱的菌落进行二次传代活化培养；
按0.1％的接种量，接种至一级种子培养基中进行培养，控制罐压为0.04MPa，控制罐温为28℃，控制通气量为1：(1±0.1)v/v/min，培养48h，得到一级种子液；
(2)将一级种子液按20％的接种量接种至二级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：(1±0.1)v/v/min，培养24h，得到二级种子液；
(3)将二级种子液按14％的接种量接种至发酵罐中，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，连续进行搅拌；发酵开始到15h内，1：(0.3±0.03)v/v/min，15h后逐步调至1：(1.0±0.1)v/v/min，发酵110h，得到可利霉素发酵液。
提取：
(1)向25L可利霉素发酵液(效价796u/ml)中加入150g聚合氯化铝，板框过滤，获得37.5L滤液；
(2)向滤液中加入乙酸丁酯1.46L，搅拌的同时加入6mol/LNaOH溶液调节pH至8.5，继续搅拌30min后静置，加NaCl后分层获得1.13L酯相提取液；
(3)依次用质量浓度为1％的NaH 2PO 4溶液、0.5％的NaH 2PO 4溶液和0.3％的NaH 2PO 4溶液对丁酯提取液进行洗涤。向洗后体积为1.07L的丁酯提取液中加入纯水310mL，搅拌的同时加入3mol/L盐酸溶液调节pH至2.1，继续搅拌30min后静置、分层，获得325ml水相提取液；
(4)加入2mol/LNaOH溶液调节水相提取液的pH至4.5，向获得的300ml水相提取液中加入2mol/LNaOH溶液调节pH至8.5，静置、抽滤、干燥，获得可利霉素成品8.6g。
二、从可利霉素样品中分离纯化得到式(1)所示的新化合物
按照上述方法连续制备多批可利霉素样品，取CA1512031批次可利霉素样品进行分离纯化得到新化合物。其中分离纯化方法如下：
1、样品分析确定目标峰
仪器：华谱S1反相
样品：CA1512031批次可利霉素产品，乙腈-乙酸铵溶解，浓度20mg/mL；
色谱柱：XBridge C18(3.5μm，2.1×150mm，186003023)
流动相：A：乙腈，C：20mM乙酸铵(pH6.82)
洗脱条件见表1：
表1
Figure PCTCN2020102060-appb-000003
流速：0.2mL/min
进样体积：1μL
波长：232nm
分析谱图见图1和图2所示，谱图中各峰保留时间等如表2所示：
表2
  保留时间 面积 ％面积   保留时间 面积 ％面积
1 2.083 79277 0.05 24 21.610 733092 0.42
2 2.792 58473 0.03 25 22.289 515851 0.29
3 3.233 13763 0.01 26 23.158 13099978 7.46
4 3.450 18094 0.01 27 24.869 644264 0.37
5 4.087 6769 0.00 28 25.605 6883398 3.92
6 4.564 942267 0.54 29 26.509 1272914 0.72
7 5.402 88502 0.05 30 27.871 624839 0.36
8 6.446 1112937 0.63 31 29.029 217207 0.12
9 6.927 955391 0.54 32 30.225 6586068 3.75
10 8.547 8341 0.00 33 32.253 13794629 7.85
11 9.137 174878 0.10 34 34.684 40600666 23.11
12 10.678 98589 0.06 35 37.676 3115180 1.77
13 11.288 891889 0.51 36 39.735 448069 0.26
14 12.375 21594 0.01 37 40.744 935236 0.53
15 13.072 183787 0.10 38 41.877 64245553 36.56
16 13.710 223006 0.13 39 44.609 535008 0.30
17 14.186 521934 0.30 40 46.229 1117067 0.64
18 15.015 921738 0.52 41 49.247 199629 0.11
19 15.815 4730786 2.69 42 50.349 458669 0.26
20 17.426 4864603 2.77 43 51.603 1089481 0.62
21 18.350 320490 0.18 44 52.705 268782 0.15
22 19.118 386065 0.22 45 54.342 786091 0.45
23 20.218 931505 0.53        
根据质谱分析结果，保留时间在15.015min的峰为新化合物目标峰，RRT0.50，色谱纯度为0.52％。
2、第一维纯化制备
2.1分离方法
样品：取600g CA1512031批次可利霉素产品，加入丙酮溶解，再加入乙酸铵水稀释溶解，浓度67mg/mL。
色谱柱：RZA10-C18YE-0.05(10μm，50×255mm，2017112104)
流动相：58％丙酮/10mM乙酸铵
流速：80mL/min
进样体积：30mL，载样量0.6％
波长：232nm
谱图如图3：
馏分收集：图3中阴影部分
重复制备302针，合并目标馏分。
馏分后处理：经SPE脱盐，50℃减压浓缩得目标馏分5.6克，记为CA1512031-7。
2.2馏分分析
分析方法同前，谱图见图4，谱图中各峰保留时间等见表3。
表3
保留时间 面积 ％面积
16.593 23162 8.56
3、第二维纯化制备
3.1分离方法
样品：取CA1512031-7 5.6克，乙腈-乙酸铵溶解，浓度100mg/mL；
仪器：创新通恒6#；
色谱柱：10-C18TDE(10μm，50×200mm，D20140403混，9#)；
流动相：A：乙腈，B：乙酸三乙胺(pH8.07)；乙酸三乙胺(pH8.07)配置：10L水，加100mL三乙胺，再加入40mL乙酸，pH调节至8.07；
洗脱条件：A：B＝56：44；
流速：70mL/min；
进样体积：21mL，载样量0.7％；
波长：232nm；
量程：10；
制备谱图见图5：
馏分收集：阴影部分
馏分后处理：馏分后处理：经SPE脱盐，50℃减压浓缩得目标馏分81mg，记为CA1512031-7-6。
3.2馏分分析
分析方法同前，谱图见图6和图7，谱图中各峰保留时间见表4。
表4、第1针-F3
  保留时间 面积 ％面积
1 7.699 4153 0.31
2 7.924 3649 0.27
3 9.564 1713 0.13
4 11.773 5062 0.38
5 12.535 9040 0.67
6 13.540 2246 0.17
7 14.000 1521 0.11
8 15.800 21080 1.56
9 16.860 68883 5.11
10 17.489 27402 2.03
11 18.102 566995 42.03
12 18.919 491076 36.40
13 19.740 13918 1.03
14 20.687 98886 7.33
15 22.723 2380 0.18
16 23.725 2872 0.21
17 26.817 23695 1.76
4、第三维纯化制备
4.1分离纯化方法
样品：取CA1512031-7-6 81mg，加乙腈溶解，部分样品未溶解，离心得澄清溶液；
仪器：HBZB-2
色谱柱：C18TDE(5μm，20×250mm，R2014092503)
等度洗脱：乙腈：50mM乙酸三乙胺(pH7.6)＝62：38
流速：10mL/min
进样体积：1mL
波长：232nm
谱图见图8。
化合物收集：阴影部分。化合物后处理：馏分后处理：经SPE脱盐，50℃减压浓缩，加入适量纯水冷冻干燥得目标化合物7.3mg，记为CA1512031-7-6-4。
4.2化合物纯度分析
样品：取新化合物CA1512031-7-6-4适量，加100μL乙腈溶解后分析；
仪器：S6
色谱柱：XBridge C18(3.5μm，2.1×150mm)
流动相：A：乙腈，C：20mM乙酸铵(pH6.82)
洗脱条件见表5：
表5
时间(min) ％A ％C
0 45 55
80 75 25
流速：0.2mL/min
进样体积：5μL
波长：232nm
分析谱图见图9，各峰保留时间等见表6：
表6
  保留时间 面积 ％面积
1 5.006 99743 0.11
2 13.217 552572 0.62
3 13.718 990755 1.11
4 20.767 51138 0.06
5 22.733 578757 0.65
6 24.213 56458 0.06
7 24.707 96422 0.11
8 27.202 84509432 95.02
9 28.500 1046959 1.18
10 35.379 317971 0.36
11 36.782 46971 0.05
新化合物色谱纯度为95.02％。
5、新化合物结构表征
5.1高分辨质谱
质谱图见图10，结构式为式(1)所示：
Figure PCTCN2020102060-appb-000004
化学式：C 51H 88O 16N 2
质谱数据：m/z 985.6037
理论值：m/z 985.6107
误差：1.69ppm
5.2 NMR表征
取5mg新化合物进行核磁表征，见图11。结果如下： 1H NMR谱中可以观察到4个双键上的质子信号δ H 6.44(dd,J＝15.2,10.5Hz,1H)，6.05(dd,J＝15.0,10.5Hz,1H)，5.70(dd,J＝15.2,9.6Hz,1H)，5.59(ddd,J＝15.0,11.1,4.0Hz,1H)，16个连氧质子信号δ H 5.15(brd,J＝10.9Hz,1H)，5.11(m,1H),5.07(d,J＝3.8Hz,1H)，4.63(d,J＝10.2Hz,1H)，4.46(m,1H)，4.43(m,2H)，4.15(dd,J＝9.6,4.2Hz,1H)，3.72(m,1H)，3.55(m,3H)，3.42(dq,J＝9.1,6.0Hz,1H)，3.33(m,1H),3.27(m,1H)，3.25(brd,J＝8.9Hz,1H)，一组连氧甲基质子信号δ H 3.55(s,3H)，4组连氮甲基质子信号δ H 2.51(s,3H)，2.51(s,3H)，2.22(s,3H)，2.22(s,3H)，9组甲基质子信号δ H 1.26(d,J＝6.2Hz,3H),1.25(d,J＝6.2Hz,3H)，1.23(d,J＝6.2Hz,3H)，1.19(t,J＝7.5Hz,3H)，1.14(d,J＝6.2Hz,3H)，1.12(s,3H)，1.00(d,J＝6.7Hz,3H)，0.99(d,J＝6.6Hz,3H)，0.99(d,J＝6.6Hz,3H)。以上 1H NMR谱特征与化合物可利霉素Ⅲ进行比较，发现二者结构十分相似，不同之处在于化合物CA1512031-7-6-4的C-18位醛基质子信号消失(可利霉素Ⅲδ H9.66)，新出现了2个连氧质子信号δ H 3.55(m,2H)。结合分子式C 51H 88N 2O 16可以推断，18位醛基被还原为羟基，新出现的质子信号为连氧亚甲基。
结果表明：以600g CA1512031可利霉素产品为原料，经多维纯化制备后，获得7.3mg目标新化合物(CA1512031-7-6-4)，色谱纯度＞95％，进而取用5mg新化合物，通过高分辨质谱和核磁表征等波谱分析技术，鉴定其化学结构，最终剩余2mg单体化合物。注：原料剩余量约20克。
实施例2
一、制备可利霉素样品
培养发酵：
(1)从WSJ195菌种的保藏斜面上挑取单菌落进行传代培养，然后挑取表面中部具有凹 陷的褶皱的菌落进行二次传代活化培养；
按0.1％的接种量，接种至一级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：(1±0.1)v/v/min，培养49h，得到一级种子液；
(2)将一级种子液按20％的接种量接种至二级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：(1±0.1)v/v/min，培养25h，得到二级种子液；
(3)将二级种子液按14％的接种量接种至发酵罐中，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，连续进行搅拌；发酵开始到15h内，1：(0.3±0.03)v/v/min，15h后逐步调至1：(1.0±0.1)v/v/min，发酵112h，得到可利霉素发酵液。
提取：
(1)向25L可利霉素发酵液(效价796u/ml)中加入150g聚合氯化铝，板框过滤，获得35L滤液；
(2)向滤液中加入乙酸丁酯1.46L，搅拌的同时加入6mol/LNaOH溶液调节pH至9.0，继续搅拌30min后静置，加NaCl后分层获得1.13L酯相提取液；
(3)依次用质量浓度为1％的NaH 2PO 4溶液、0.5％的NaH 2PO 4溶液和0.3％的NaH 2PO4溶液对丁酯提取液进行洗涤。向洗后体积为1.07L的丁酯提取液中加入纯水310mL，搅拌的同时加入3mol/L盐酸溶液调节pH至2.5，继续搅拌40min后静置、分层，获得325ml水相提取液；
(4)加入2mol/LNaOH溶液调节水相提取液的pH至5.5，向获得的300ml水相提取液中加入2mol/LNaOH溶液调节pH至8.5，静置、抽滤、干燥，获得可利霉素成品8.4g。
二、从可利霉素样品中分离纯化得到式(1)所示的新化合物
同实施例1。
实施例3
一、制备可利霉素样品
培养发酵：
(1)从WSJ195菌种的保藏斜面上挑取单菌落进行传代培养，然后挑取表面中部具有凹陷的褶皱的菌落进行二次传代活化培养；
按0.1％的接种量，接种至一级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：(1±0.1)v/v/min，培养45h，得到一级种子液；
(2)将一级种子液按20％的接种量接种至二级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：(1±0.1)v/v/min，培养24h，得到二级种子液；
(3)将二级种子液按14％的接种量接种至发酵罐中，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，连续进行搅拌；发酵开始到15h内，1：(0.3±0.03)v/v/min，15h后逐步调至1：(1.0±0.1)v/v/min，发酵110h，得到可利霉素发酵液。
提取：
(1)向25L可利霉素发酵液(效价796u/ml)中加入150g聚合氯化铝，板框过滤，获得37.5L滤液；
(2)向滤液中加入乙酸丁酯1.46L，搅拌的同时加入6mol/LNaOH溶液调节pH至8.8，继续搅拌30min后静置，加NaCl后分层获得1.13L酯相提取液；
(3)依次用质量浓度为1％的NaH 2PO 4溶液、0.5％的NaH 2PO 4溶液和0.3％的NaH 2PO4溶液对丁酯提取液进行洗涤。向洗后体积为1.07L的丁酯提取液中加入纯水310mL，搅拌的同时加入3mol/L盐酸溶液调节pH至2.4，继续搅拌30min后静置、分层，获得325ml水相提取液；
(4)加入2mol/LNaOH溶液调节水相提取液的pH至4.5，向获得的300ml水相提取液中加入2mol/LNaOH溶液调节pH至8.8，静置、抽滤、干燥，获得可利霉素成品8.8g。
二、从可利霉素样品中分离纯化得到式(1)所示的新化合物
同实施例1。
实施例4
一、制备可利霉素样品
培养发酵：
(1)从螺旋链霉菌Streptomyces spiramyceticus菌种的保藏斜面上挑取单菌落进行传代培养，然后挑取表面中部具有凹陷的褶皱的菌落进行二次传代活化培养；螺旋链霉菌Streptomyces spiramyceticus，于2018年7月4日送交中国微生物菌种保藏管理委员会普通微生物中心保藏，地址：北京市朝阳区北辰西路1号院3号，保藏编号CGMCC No.16055。
按0.3％的接种量，接种至一级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28℃，控制通气量为1：1.1v/v/min，培养48h，得到一级种子液；
(2)将一级种子液按20％的接种量接种至二级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：(1±0.1)v/v/min，培养24h，得到二级种子液；
(3)将二级种子液按14％的接种量接种至发酵罐中，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，连续进行搅拌；发酵开始到15h内，1：3.2v/v/min，15h后逐步调至1：1.1v/v/min，发酵110h，得到可利霉素发酵液。
提取：
(1)向25L可利霉素发酵液(效价796u/ml)中加入150g聚合氯化铝，板框过滤，获得37.5L滤液；
(2)向滤液中加入乙酸丁酯1.46L，搅拌的同时加入6mol/LNaOH溶液调节pH至8.1，继续搅拌30min后静置，加NaCl后分层获得1.13L酯相提取液；
(3)依次用质量浓度为1％的NaH 2PO 4溶液、0.5％的NaH 2PO 4溶液和0.3％的NaH 2PO 4溶液对丁酯提取液进行洗涤。配制0.7～1.0％磷酸二氢钠溶液，用草酸调节使溶液的pH值2.0 ～2.5，备用；将上述配制好的磷酸二氢钠溶液与洗涤后的酯相混合，再加入草酸调节pH，充分搅拌使得混合溶液的pH值2.5，继续搅拌30min后静置、分层，获得325ml水相提取液；
(4)加入2mol/LNaOH溶液调节水相提取液的pH至4.5，向获得的300ml水相提取液中加入2mol/LNaOH溶液调节pH至8.9，静置、抽滤、干燥，获得可利霉素成品9.8g。
二、从可利霉素样品中分离纯化得到式(1)所示的新化合物
同实施例1。
实施例5
一种药物组合物，包括式(1)化合物和异戊酰螺旋霉素Ⅰ，其中式(1)化合物的含量为1％。
实施例6
一种药物组合物，包括式(1)化合物和异戊酰螺旋霉素Ⅱ，其中式(1)化合物的含量为2％。
实施例7
一种药物组合物，包括式(1)化合物和异戊酰螺旋霉素Ⅲ，其中式(1)化合物的含量为0.5％。
实施例8
一种药物组合物，包括式(1)化合物、异戊酰螺旋霉素Ⅰ和异戊酰螺旋霉素Ⅱ，其中式(1)化合物、异戊酰螺旋霉素Ⅰ和异戊酰螺旋霉素Ⅱ的含量分别为0.1％、44.9％和55.0％。
对比例1
(1)从WSJ195菌种的保藏斜面上挑取单菌落进行传代培养，然后挑取表面中部具有凹陷的褶皱的菌落进行二次传代活化培养；
按0.1％的接种量，接种至一级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：0.6v/v/min，培养48h，得到一级种子液；
(2)将一级种子液按20％的接种量接种至二级种子培养基中进行培养，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，控制通气量为1：0.6v/v/min，培养24h，得到二级种子液；
(3)将二级种子液按14％的接种量接种至发酵罐中，控制罐压为0.04±0.01MPa，控制罐温为28±1℃，连续进行搅拌；发酵开始到15h内，1：0.2v/v/min，15h后逐步调至1：0.6v/v/min，发酵110h，得到可利霉素发酵液。
从发酵液中提取可利霉素成品的提取方法与实施例1相同，获得可利霉素成品7.8g。
试验例1、体外抗菌试验数据
1、研究目的
评价可利霉素1(对比例1制得)及可利霉素2(实施例1制得)对我国近2年临床耳鼻咽喉感染及社区获得性肺炎主要分离菌种肺炎链球菌、化脓性链球菌、金黄色葡萄球菌的体外抗菌活性。
2、试验药品
可利霉素1、可利霉素2、红霉素、阿奇霉素
3、试验菌株
3.1标准菌株：金黄色葡萄球菌ATCC29213，肺炎链球菌ATCC49619，流感嗜血杆菌ATCC49247。
3.2临床分离革兰阳性菌105株：
肺炎链球菌Streptococcus pneumoniae(42株)
红霉素敏感肺炎链球菌(19株)
红霉素耐药肺炎链球菌(23株)
化脓性链球菌Streptococcus pyogenes(29株)
红霉素敏感化脓性链球菌(16株)
红霉素不敏感化脓性链球菌(13株)
金黄色葡萄球菌Staphylococcus aureus(34株)
红霉素敏感金黄色葡萄球菌(20株)
红霉素耐药金黄色葡萄球菌(14株)
每株细菌在试验前都经过平板转活分纯，以新鲜菌体用于试验。每次实验均用标准菌株作为敏感实验质控菌。
4、培养基与孵育条件
葡萄球菌在MH培养基，35℃孵育16-20h；链球菌在血培养基(MH培养基中加入5％脱纤维羊血制成)，35℃5％CO 2环境(CO 2培养箱)中孵育20-24h。
5、最低抑菌浓度(MIC)测定
采用标准琼脂二倍稀释法测定各抗菌药物对各种致病菌的最低抑菌浓度，培养基pH值为7.0。
表7、可利霉素1、可利霉素2及对照药对临床分离致病菌MIC结果(mg/L)
Figure PCTCN2020102060-appb-000005
Figure PCTCN2020102060-appb-000006
以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本发明的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许变动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。

Claims (10)

  1. 一种新化合物，其特征在于，所述的新化合物的结构式如式(1)所示：
    Figure PCTCN2020102060-appb-100001
  2. 一种权利要求1所述的新化合物的分离纯化方法，其特征在于，所述的分离纯化方法为：首先对可利霉素样品进行分析、确定目标峰，然后再进行纯化，优选三次纯化，得到所述的新化合物。
  3. 根据权利要求2所述的分离纯化方法，其特征在于，目标峰的保留时间为15.015min，RRT0.50。
  4. 根据权利要求2所述的分离纯化方法，其特征在于，所述的第一次纯化、第二次纯化和第三次纯化均为反相色谱分离纯化。
  5. 根据权利要求4所述的分离纯化方法，其特征在于，对可利霉素样品进行分析、确定目标峰的过程采用反相色谱柱，流动相为乙腈和乙酸铵。
  6. 根据权利要求2-5任意一项所述的分离纯化方法，其特征在于，第一次纯化采用反相色谱柱，流动相为丙酮和乙酸铵；
    第二次纯化采用反相色谱柱，流动相为乙腈和乙酸三乙胺；
    第三次纯化采用反相色谱柱，以乙腈：乙酸三乙胺＝62：38等度洗脱。
  7. 根据权利要求2-6任意一项所述的分离纯化方法，其特征在于，所述的可利霉素样品为将可利霉素产生菌进行培养发酵和提取得到的。
  8. 根据权利要求7所述的分离纯化方法，其特征在于，所述的可利霉素产生菌为将碳霉 素产生菌4〃异戊酰基转移酶基因克隆至螺旋霉素产生菌，使之表达获得的含4〃异戊酰基转移酶基因的螺旋霉素产生菌克隆菌株；或者为进一步将4〃异戊酰基转移酶基因的螺旋霉素产生菌克隆菌株中的Lrp基因失活得到的。
  9. 一种药物组合物，其特征在于，所述的药物组合物含有权利要求1所述的新化合物。
  10. 根据权利要求9所述的药物组合物，其特征在于，所述的药物组合物还含有异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅲ中的至少一种；更优选，所述的药物组合物中新化合物的质量含量小于5％。
PCT/CN2020/102060 2019-07-18 2020-07-15 一种新化合物及药物组合物 WO2021008549A1 (zh)

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