WO2019227969A1 - 一类安莎全碳环聚酮类抗生素及其在制备抗菌药物或抗肿瘤药物中的应用 - Google Patents

一类安莎全碳环聚酮类抗生素及其在制备抗菌药物或抗肿瘤药物中的应用 Download PDF

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WO2019227969A1
WO2019227969A1 PCT/CN2019/075053 CN2019075053W WO2019227969A1 WO 2019227969 A1 WO2019227969 A1 WO 2019227969A1 CN 2019075053 W CN2019075053 W CN 2019075053W WO 2019227969 A1 WO2019227969 A1 WO 2019227969A1
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kendomycin
verrucosispora
scsio
human
extract
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French (fr)
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鞠建华
孙长利
张善文
桂春
张华�
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中国科学院南海海洋研究所
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    • 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
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D495/18—Bridged systems
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention belongs to the technical field of microorganisms, and particularly relates to a marine wart spore (Verrucosispora sp.) SCSIO07399 and three kinds of new kendomycin compounds kendomycin BD extracted and isolated from the strain.
  • the invention also relates to the preparation of the three new compounds by the strain Methods and applications.
  • Kendomycin is a polyketone compound of the ansamycin antibiotic family.
  • the mother nucleus is characterized by a macrolide mixed with a quinone methyl chromophore. It was originally isolated from Streptomyces AL-71389 by Takeda Pharmaceutical Company as an antagonist of the endothelin receptor in 1996. [Bruck KB, Rychnovsky SD. Formal synthesis of (-)-kendomycin featuring aprins-cyclization method to construct the macrocycle [J] .J Am Chem Soc, 2008.130 (39): 13177-13181]. In 1998, Su et al.
  • Bode and Zeeck determined their absolute configuration through the Mosher reaction [Bode, H, B, Zeeck, A. Structure, and biosynthesis of Kendomycin, a carbocyclic, ansa-compound, from Streptomyces [J]. 58 (3): 323-328].
  • kendomycin antibiotics have good biological activity, not only a wide range of antibacterial spectrum, but also inhibit methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus, and also have a significant number of human tumor cells. Toxic activity, and is expected to develop into endocortin receptor antagonists and anti-osteoporosis agents [Magauer T, Martin HJ, Mulzer J. Ring-closing metathesis and photo-fries reaction for the construction of the ansamycin antibioticskendomycin: development a protecting group free oxidation endgame [J] .Chemistry, 2010,16 (2): 507-519].
  • One of the objectives of the present invention is to provide a marine wart spore (Verrucosispora sp.) SCSIO 07399 capable of producing kendomycin BD, which was deposited at the Guangdong Provincial Center for Microbial Strains (GDMCC) on October 22, 2018, address : 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Provincial Institute of Microbiology, Deposit No .: GDMCC No. 60466.
  • Another object of the present invention is to provide an ansa full carbocyclic polyketide antibiotic kendomycin B-D as shown in formulae (I), (II) and (III), or a pharmaceutically acceptable salt thereof:
  • formula (I) is kendomycin B
  • formula (II) is kendomycin C
  • formula (III) is kendomycin D.
  • the third object of the present invention is to provide the application of marine wart spore (Verrucosispora sp.) SCSIO 07399 in the preparation of kendomycin B-D.
  • the fourth object of the present invention is to provide a method for preparing kendomycins B-D.
  • the kendomycin B-D is prepared from a fermented culture of Verrucosispora sp. SCSIO 07399.
  • kendomycin B-D is prepared from a fermented culture of Verrucosispora sp. SCSIO07399, and specifically includes the following steps:
  • the fraction Fr.B5 is passed through a Sephex LH-20 glucan gel chromatography column, and the volume ratio of CHCl 3 / MeOH is 1: 1. The phases were eluted, and the fractions were collected and purified to obtain kendomycin D.
  • Fr.B7 was passed through a Sephex LH-20 glucan gel column and eluted with CHCl 3 / MeOH volume ratio 1: 1 as the mobile phase. The fractions were collected. After further purification, kendomycin B and kendomycin C were obtained.
  • Verrucosispora sp. SCSIO 07399 is inserted into a seed medium, and a seed culture liquid is obtained by fermentation.
  • the seed culture liquid is connected to a fermentation medium, and a fermentation culture is obtained by fermentation.
  • the formula of the seed medium For: 10g of soluble starch, 4g of yeast extract, 2g of bacteriological peptone and 30g of sea salt per liter, the balance is water; the formula of the fermentation medium is: 20g of soluble starch, 10g of glucose, 10g of maltose, 5g of corn flour per liter , 10g of malt extract powder, 2g of CaCO 3 and 30g of sea salt, the balance is water.
  • a sixth object of the present invention is to provide an antibacterial or antineoplastic drug, which contains an effective amount of the kendomycin B, kendomycin C or kendomycin D, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the antibacterial drug is a drug against Bacillus thuringiensis, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, or methicillin-resistant Staphylococcus aureus;
  • the antitumor drug is anti-human gastric cancer, human lung cancer , Human cervical cancer, human liver cancer, human breast adenocarcinoma, or human colorectal cancer.
  • the present invention provides a strain of marine wart spore (Verrucosispora sp.) SCSIO 07399 capable of producing kendomycin antibiotics.
  • a new compound kendomycin BD can be prepared by using this bacterium. These three compounds have significant antibacterial and antitumor activities and have broad Application prospects.
  • the Verrucosispora sp. SCSIO07399 of the present invention was deposited on October 22, 2018 at the Guangdong Provincial Center for Microbial Strains (GDMCC), Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, deposit number: GDMCC No. 60466.
  • Fig. 1 is a morphological diagram of Verrucosispora sp. SCSIO 07399 on M-ISP4 medium;
  • Figure 2 is a phylogenetic tree of Verrucosispora sp. SCSIO 07399, where 07399 represents Verrucosis pora sp. SCSIO 07399;
  • Figure 3 is an HPLC chart of the production of kendomycin B and kendomycin C by SCSIO 07399 from Verrucosispora sp .;
  • Example 1 Isolation and identification of Verrucosispora sp. SCSIO 07399
  • the Verrucosispora sp. SCSIO 07399 of the present invention is isolated from a sediment sample in the South China Sea.
  • the colonies are small, usually orange-yellow, slender mycelium, branched, and not broken. Basal hyphae were formed, and occasionally sparse slightly white aerial hyphae (as shown in Figure 1).
  • Extract the genomic DNA of the isolated strain PCR-amplify its 16SrDNA sequence (the nucleotide sequence is shown in SEQ ID NO.1) by conventional methods, and perform sequencing analysis to construct a phylogenetic tree based on the 16SrDNA sequence (such as Figure 2) shows that the sequence similarity between strain SCSIO 07399 and Verrucosispora sp. R8-37 16SrDNA is 99%, indicating that strain SCSIO 07399 belongs to Verrucosispora sp.
  • the identified strain SCSIO 07399 belongs to a species of the genus Warthospora, named Verrucosispora sp. SCSIO 07399, which was deposited in the Guangdong Provincial Center for Microbial Strains on October 22, 2018. (GDMCC), Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Provincial Institute of Microbiology, Deposit No .: GDMCC No. 60466.
  • seed medium Each liter of seed medium contains 10g of soluble starch, 4g of yeast extract, 2g of bacteriological peptone, and 30g of sea salt, and the balance is water. The above components are mixed uniformly and the pH is adjusted to 7.0. Prepare 1L seed medium, and then aliquot it into 20 250mL Erlenmeyer flasks, each 50mL, and sterilize at 121 ° C for 30min.
  • Each liter of fermentation medium contains 20g of soluble starch, 10g of glucose, 10g of maltose, 5g of corn flour, 10g of malt extract powder, 2g of CaCO 3 and 30g of sea salt. The balance is water. Mix well and adjust pH to 7.0. Prepare 1L fermentation medium, then aliquot it into 5 1L Erlenmeyer flasks and sterilize at 121 ° C for 30min for later use.
  • the fermented culture of Verrucosispora sp. SCSIO 07399 was centrifuged (3800 ⁇ 4000r / min, 10 ⁇ 15min) to separate the fermentation supernatant from the mycelium; the fermentation supernatant was treated with methyl ethyl ketone, etc. Extraction was performed three times in volume. The methyl ethyl ketone phase was condensed by rotary evaporation to obtain extract A. The mycelium was immersed and extracted with acetone (2L), and the acetone extract was obtained by ultrasonic treatment. Cream B.
  • Extract A and extract B are similar. Extract A and extract B are combined, and subjected to normal phase silica gel column chromatography, using chloroform-methanol as the mobile phase. Different volume ratios (100: 0, 99 : 1, 98: 2, 96: 4, 94: 6, 92: 8, 9: 1, 8: 2, 5: 5, 0: 100), followed by gradient elution, and eluted under a gradient of 100% chloroform.
  • the fraction was recorded as Fr.A1
  • the fraction eluted under a gradient of chloroform-methanol volume ratio of 99: 1 was recorded as Fr.A2
  • the fraction eluted under a gradient of chloroform-methanol volume ratio of 98: 2 was recorded as Fr.A3.
  • Fr.A4 The fraction eluted under a gradient of chloroform-methanol volume ratio of 96: 4 is recorded as Fr.A4, and the fraction eluted under a gradient of chloroform-methanol volume ratio of 94: 6 is recorded as Fr.A5.
  • the fraction eluted under a gradient of 92: 8 was recorded as Fr.A6, the fraction eluted under a gradient of 9: 1 was recorded as Fr.A7, and the volume ratio of chloroform-methanol was determined under a gradient of 8: 2.
  • Compound 1 is a yellow powder with molecular formula C 28 H 40 O 6 , HR-ESI-MS m / z 471.2757 ([MH] - ); [ ⁇ ] 20 D -59 (c 0.21, MeOH); UV (MeOH) ⁇ max (log ⁇ ) 202 (4.276), 307 (3.681) nm; IR (film): v max : 3325, 2937, 2358, 2341, 1602, 1456, 1359, 1195, 1095, 1022 cm -1 .
  • H-12 and C-14 can be observed in the HMBC spectrum; H-13 and C-14, C-15, C-24; H-15 and C-13, C-14, C-24; H-16 Correlation with C-14 and H-24 with C-13, C-14, C-15, it is inferred that the above two fragments are connected through C-14.
  • 8 typical aromatic carbons in compound 1 are shown: ⁇ C 173.9 (C-1), 96.7 (C-2), 183.9 (C-3), 148.2 (C-4) 113.0 (C-4a), 120.6 (C-19), 143.5 (C-20) and 131.4 (C-20a) are a polysubstituted pyranone ring structure.
  • Compound 3 is a yellow powder with molecular formula C 33 H 47 NO 9 S, HR-ESI-MS m / z 471.2757 ([MH] - ); [ ⁇ ] 20 D -240 (c 0.34, MeOH); UV (MeOH) ⁇ max (log ⁇ ) 202 (4.328), 244 (3.591), 382 (3.453) nm; IR (film): v max : 3363, 2949, 2362, 2330, 1602, 1373, 1010 cm -1 . By nuclear magnetic spectrum assignment, it was determined that compound 3 and 1 contained the same 22-membered polysubstituted pyranolactone ring as the mother core structure.
  • H-27 ( ⁇ H 3.52, ⁇ H 3.87) and C-20 ( ⁇ C 151.5) and C-28 ( ⁇ C 55.1H-28 ( ⁇ H 4.60) and C-27 ( ⁇ C 35.8) C-29 ( ⁇ C 173.9) and C-30 ( ⁇ C 172.7), H-31 ( ⁇ H 1.93) and C-30 ( ⁇ C 172.7) are remotely correlated, and combine 1 H- 1 H
  • the coupling of H-27 ( ⁇ C 35.8) and H-28 ( ⁇ H 4.60) in COSY infers the existence of acetylacetylcysteine.
  • the filter paper sheet method was used to test the antibacterial activity of the compound, and it was found that compounds 1-3 had certain antibacterial activity against most Gram-positive bacteria.
  • Bacillus thuringiensis BT01 Bacillus subtilis BS01, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 292292, Staphyureococcus 745a And methicillin-resistant Staphylococcus aureus shhs-A1
  • 96-well plate method was used to determine the minimum inhibitory concentration, kanamycin, ampicillin and vancomycin were used as positive controls.
  • the minimum inhibitory concentration (MIC) of compound 1 against Staphylococcus aureus ATCC 29213, Staphylococcus aureus 745524, Enterococcus faecalis ATCC 29212, and Bacillus thuringiensis BT01 were 0.5, 1.0, 2.0, 1.0 ⁇ g / mL, respectively, which were better than those of card.
  • the minimum inhibitory concentration (MIC) of Bacillus BT01 was 0.5, 1.0, 1.0, 0.5, 0.5 ⁇ g / mL, which was better than the positive controls of kanamycin, ampicillin, and vancomycin.
  • Compounds 1-3 were applied to human gastric cancer cell line MGC803 (human gastric cancer cell line), human lung cancer cell line A549 (human lung cancer cell line) and human cervical cancer cell line Hela (human cervical cancer cell line) by standard MTT method.
  • Six human cancer cell lines including the human hepatocellular carcinoma cell line HepG2, the human breast adenocarcinoma cell line MCF-7, and the colorectal carcinoma cell line RKO Human liver cell line L02 (normal human human hepatic cell line) and human umbilical vein endothelial cell line Huvec-12 (normal human human vein endothelial cell line) were tested for cytotoxic activity, and cisplatin was used as a positive control.

Abstract

安莎全碳环聚酮类抗生素及其在制备抗菌药物或抗肿瘤药物中的应用。海洋疣孢菌(Verrucosispora sp.)SCSIO 07399于2018年10月22日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，保藏编号为：GDMCC No.60466。海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物能够制备新化合物kendomycin B-D，如式(I)-(III)所示，这三种化合物具有显著的抗菌和抗肿瘤活性。

Description

一类安莎全碳环聚酮类抗生素及其在制备抗菌药物或抗肿瘤药物中的应用 技术领域：
本发明属于微生物技术领域，具体涉及一种海洋疣孢菌(Verrucosispora sp.)SCSIO07399及从该菌株中提取分离的三种kendomycin类新化合物kendomycin B-D，本发明还涉及该菌株制备该三种新化合物的方法和应用。
背景技术：
Kendomycin属于ansamycin抗生素家族的聚酮类化合物，母核特征为杂合了醌甲基发色团的大环内酯。最初于1996年由Takeda制药公司作为内皮素受体的拮抗剂从链霉菌Streptomyces AL-71389中分离获得[Bahnck KB,Rychnovsky SD.Formal synthesis of(-)-kendomycin featuring a Prins-cyclization to construct the macrocycle[J].J Am Chem Soc,2008.130(39):13177-13181]。1998年，Su等从链霉菌Streptomyces NRRL-21370中再次分离得到，活性测试结果表明具有抗骨质疏松作用[Xu S,Arimoto H.Strategies for construction of the all-carbon macrocyclic skeleton of the ansamycin antibiotic-kendomycin[J].J Antibioti(Tokyo),2016,47(31):203]。2000年，Bode和Zeeck通过Mosher反应对其绝对构型进行了确定[Bode H B,Zeeck A.Structure and biosynthesis of kendomycin,a carbocyclic ansa-compound from Streptomyces[J].J chem soc perkin Trans,2000,58(3):323-328]。Kendomycin类抗生素的生物合成途径最初由Bode和Zeek用同位素标记法进行了阐述[Bode H B,Zeeck A.Biosynthesis of kendomycin:origin of the oxygen atoms and further investigations[J].J chem soc perkin Trans,2000,16(16):2665-2670]。后Wenzel等克隆了其生物合成基因簇，生物合成分析结果表明该类化合物其母核的形成由混合I型和III型PKS 聚酮合酶负责，这在ansamycin类抗生素中是首次报道[Wenzel SC,Bode HB,Kochems I,et al.A Type I/Type III polyketide synthase hybrid biosynthetic pathway for the structurally unique ansa compound kendomycin[J].Chembiochem,2008,9(16):2711-2721]。据报道，kendomycin类抗生素具有良好的生物活性，不仅抗菌谱广，对耐甲氧西林金黄色葡萄球菌及耐万古霉素金黄色葡萄球菌有抑制作用，同时对人类多种肿瘤细胞也有显著的细胞毒活性，并有望开发成为内皮质素受体拮抗剂及抗骨质疏松剂[Magauer T,Martin HJ,Mulzer J.Ring-closing metathesis and photo-fries reaction for the construction of the ansamycin antibiotic kendomycin:development of a protecting group free oxidative endgame[J].Chemistry,2010,16(2):507-519]。
发明内容：
本发明的目的之一是提供一种能够产生kendomycin B-D的海洋疣孢菌(Verrucosispora sp.)SCSIO 07399，该菌于2018年10月22日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，保藏编号为：GDMCC No.60466。
本发明的目的之二是提供如式(I)、(II)和(III)所示的安莎全碳环聚酮类抗生素kendomycin B-D，或其药用盐：
Figure PCTCN2019075053-appb-000001
其中，式(I)为kendomycin B，式(II)为kendomycin C，式(III)为kendomycin D。
本发明的目的之三是提供海洋疣孢菌(Verrucosispora sp.)SCSIO 07399在制备kendomycin B-D中的应用。
本发明的目的之四是提供kendomycins B-D的制备方法，所述的kendomycin B-D是从海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物中制备得到的。
按照本发明，优选所述的kendomycin B-D是从海洋疣孢菌(Verrucosispora sp.)SCSIO07399的发酵培养物中制备得到的，具体包括以下步骤：
(a)制备海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物，将该发酵培养物的发酵上清液和菌丝体分开，发酵上清液用丁酮萃取，丁酮相经浓缩后得到浸膏A，菌丝体用丙酮浸泡萃取，丙酮浸提液经浓缩后得到浸膏B；
(b)将浸膏A和浸膏B合并，采用硅胶柱层析，以氯仿-甲醇作为洗脱剂，从体积比100:0、99:1、98:2、96:4、94:6、92:8、9:1、8:2、5:5、0:100依次进行梯度洗脱，收集氯仿-甲醇体积比为92:8、9:1、8:2和5:5洗脱的馏分Fr.A6～A9，将馏分Fr.A6～A9合并后经反相中压液相色谱，以流动相CH 3CN/H 2O 10％～100％v/v线性梯度洗脱120min，流速10mL/min，每馏分150mL，顺序得到8个馏分Fr.B1～B8，将馏分Fr.B5经Sephex LH-20 葡聚糖凝胶色谱柱，以CHCl 3/MeOH体积比1:1作为流动相洗脱，收集馏分再经纯化后得到kendomycin D，将馏分Fr.B7经Sephex LH-20葡聚糖凝胶色谱柱，以CHCl 3/MeOH体积比1:1作为流动相洗脱，收集馏分再经纯化后得到kendomycin B和kendomycin C。
所述的制备海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物是通过以下方法制备：
将海洋疣孢菌(Verrucosispora sp.)SCSIO 07399接入种子培养基中，发酵得种子培养液，将种子培养液接入到发酵培养基，发酵得到发酵培养物；所述的种子培养基的配方为：每升含有可溶性淀粉10g、酵母提取物4g、细菌学蛋白胨2g和海盐30g，余量为水；发酵培养基的配方为：每升含有可溶性淀粉20g、葡萄糖10g、麦芽糖10g、玉米粉5g、麦芽提取粉10g、CaCO 3 2g和海盐30g，余量为水。
本发明的第五个目的是提供了kendomycin B、kendomycin C或kendomycin D，或其药用盐在制备抗菌药物或抗肿瘤药物中的应用。
本发明的第六个目的是提供一种抗菌药物或抗肿瘤药物，含有有效量的所述的kendomycin B、kendomycin C或kendomycin D，或其药用盐作为活性成分。
优选，所述的抗菌药物为抗苏云金芽孢杆菌、枯草芽孢杆菌、粪肠球菌、金黄色葡萄球菌或耐甲氧西林金黄色葡萄球菌的药物；所述的抗肿瘤药物为抗人胃癌、人肺癌、人***、人肝癌、人乳腺腺癌或人结直肠癌的药物。
本发明提供了一株能够产生kendomycin类抗生素的海洋疣孢菌(Verrucosispora sp.)SCSIO 07399，利用该菌可以制备新化合物kendomycin B-D，这三个化合物具有显著的抗菌和抗肿瘤活性，具有广阔的应用前景。
本发明的海洋疣孢菌(Verrucosispora sp.)SCSIO 07399于2018年10月22日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，保藏编号为：GDMCC No.60466。
附图说明：
图1是海洋疣孢菌(Verrucosispora sp.)SCSIO 07399在M-ISP4培养基上的形态图；
图2是海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的***进化树，其中07399代表海洋疣孢菌(Verrucosispora sp.)SCSIO 07399；
图3是海洋疣孢菌(Verrucosispora sp.)SCSIO 07399发酵生产kendomycin B和kendomycin C的HPLC图谱；
图4是海洋疣孢菌(Verrucosispora sp.)SCSIO 07399发酵生产kendomycin D的HPLC图谱。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的分离与鉴定
本发明的海洋疣孢菌(Verrucosispora sp.)SCSIO 07399是从我国南海海域的沉积物样品中分离得到的。
该菌株的分类学特征是：
1、形态学特征：
菌落小，通常橙黄色，菌丝体纤细，有分支，不断裂。形成基生菌丝，偶见稀疏微白色气生菌丝(如图1所示)。
2、分子生物学分离特征：
提取上述分离的菌株的基因组DNA，通过常规方法PCR扩增其16S rDNA序列(其核苷酸序列如SEQ ID NO.1所示)，并测序分析，构建基于16S rDNA序列的***进化树(如图2所示)，显示菌株SCSIO 07399与Verrucosispora sp.R8-37 16S rDNA的序列相似性为99％，表明菌株SCSIO 07399属于疣孢菌(Verrucosispora sp.)。
综上所述，鉴定菌株SCSIO 07399属于疣孢菌属的一个种，命名为海洋疣孢菌(Verrucosispora sp.)SCSIO 07399，该菌于2018年10月22日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，保藏编号为：GDMCC No.60466。
实施例2：kendomycin B-D的分离鉴定
1、制备海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物：
(1)种子培养基和发酵培养基的配制：
a)种子培养基的配制：每升种子培养基中含有可溶性淀粉10g、酵母提取物4g、细菌学蛋白胨2g和海盐30g，余量为水，将上述组份混合均匀，调节pH至7.0。配制1L种子培养基，然后平均分装于20个250mL锥形瓶中，每瓶50mL，121℃灭菌30min备用。
b)发酵培养基的配制：每升发酵培养基中含有可溶性淀粉20g、葡萄糖10g、麦芽糖10g、玉米粉5g、麦芽提取粉10g、CaCO 3 2g和海盐30g，余量为水，将上述组份混合均匀，调节pH至7.0。配制1L发酵培养基，然后平均分装于5个1L锥形瓶中，121℃灭菌30min备用。
(2)种子的培养：
将活化的海洋疣孢菌(Verrucosispora sp.)SCSIO 07399(保藏编号为：GDMCC No.60466)接入到装有50mL种子培养基的250mL锥形瓶中，于28℃、200rpm的摇床上培养24h，得到种子培养液。
(3)规模发酵培养：
将上述锥形瓶中的50mL种子培养液转接到装有200mL发酵培养基的1L锥形瓶中，于28℃、200r/min的摇床上培养7天，获得海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物。
2、海洋疣孢菌(Verrucosispora sp.)SCSIO 07399所产kendomycin B-D的分离
(1)发酵培养物的萃取
将海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物进行离心分离(3800～4000r/min，10～15min)使发酵上清液与菌丝体分开；发酵上清液使用丁酮进行等体积萃取3次，丁酮相经旋蒸冷凝浓缩之后得到浸膏A；菌丝体使用丙酮(2L)浸泡萃取，超声处理得到丙酮浸提液，丙酮浸提液经旋蒸冷凝浓缩后得到浸膏B。
(2)kendomycin B-D的提取
经HPLC分析，浸膏A和浸膏B的成分类似，将浸膏A和浸膏B合并，经正相硅胶柱层析，用氯仿-甲醇作为流动相，不同体积比(100:0、99:1、98:2、96:4、94:6、92:8、9:1、8:2、5:5、0:100)依次进行梯度洗脱，100％氯仿梯度下洗脱下来的馏分记为Fr.A1，氯仿-甲醇体积比为99:1梯度下洗脱下来的馏分记为Fr.A2，氯仿-甲醇体积比为98:2梯度下洗脱下来的馏分记为Fr.A3，氯仿-甲醇体积比为96:4梯度下洗脱下来的馏分记为Fr.A4，氯 仿-甲醇体积比为94:6梯度下洗脱下来的馏分记为Fr.A5，氯仿-甲醇体积比为92:8梯度下洗脱下来的馏分记为Fr.A6，氯仿-甲醇体积比为9:1梯度下洗脱下来的馏分记为Fr.A7，氯仿-甲醇体积比为8:2梯度下洗脱下来的馏分记为Fr.A8，氯仿-甲醇体积比为5:5梯度下洗脱下来的馏分记为Fr.A9，氯仿-甲醇体积比为0:100梯度下洗脱下来的馏分记为Fr.A10。对上述馏分Fr.A1～A10进行高效液相分析，发现Fr.A6、Fr.A7、Fr.A8和Fr.A9馏分中含有kendomycin B-D(1-3)，进行合并。合并后的馏分经反相中压液相色谱仪(CH 3CN/H 2O10％～100％v/v线性梯度洗脱120min，流速10mL/min)，每馏分150mL，顺序得到8个馏分Fr.B1～B8。经HPLC检测，发现Fr.B5中含化合物3，Fr.B7中含化合物1和化合物2。将馏分Fr.B5和Fr.B7分别经Sephex LH-20葡聚糖凝胶色谱柱纯化(流动相CHCl 3/MeOH1/1v/v等度洗脱)，得Fr.C1～C15及Fr.D1～D15。经HPLC检测，将Fr.C5～C9(含有kendomycin D)合并后通过反相ODS(YMC-Pack ODS-A column，250×20mm,5μm)半制备高效液相色谱分离纯化(流动相CH 3CN/H 2O 55％～100％v/v线性梯度洗脱30min，流速2.5mL/min)(如图4所示)，得到化合物3(6.2mg，保留时间16.2min)，即为kendomycin D。将Fr.D5～D12(含有kendomycin B和kendomycin C)合并后通过反相ODS(YMC-Pack ODS-A column，250×20mm,5μm)半制备高效液相色谱分离纯化(流动相CH 3CN/H 2O70％～100％v/v线性梯度洗脱30min，流速2.5mL/min)(如图3所示)，得到化合物1(5.0mg；保留时间26.0min)和化合物2(4.0mg；保留时间24.5min)，化合物1即为kendomycin B，化合物2即为kendomycin C。
通过结构分析，对本发明的从海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物中制备的化合物1-3鉴定结果如下：
化合物1为黄色粉末，分子式C 28H 40O 6，HR-ESI-MS m/z 471.2757([M-H] -)；[α] 20 D-59(c 0.21,MeOH)；UV(MeOH)λ max(logε)202(4.276),307(3.681)nm；IR(film):v max:3325,2937,2358,2341,1602,1456,1359,1195,1095,1022cm -1。在 1H NMR谱高场显示6个甲基信号δ H 0.73(3H,d,J＝6.8Hz，Me-26)，δ H 0.89(3H,d,J＝6.6Hz，Me-23)，δ H 0.91(3H,d,J＝6.6Hz，Me-21)，δ H 0.95(3H,d,J＝7.0Hz，Me-22)，δ H 0.95(3H,d,J＝6.5Hz，Me-25)和δ H 1.61(3H,m,Me-24)。结合一维 13C NMR，DEPT NMR和二维HSQC数据，显示共有28个碳信号，其中含6个甲基，4个亚甲基，12个次甲基，6个季碳根据COSY和HMBC谱图分析，可以推断化合物1中C-5/C-6(C-21)/C-7/C-8(C-22)/C-9/C-10/C-11/C-12(C-23)/C-13和C-15/C-16(C-25)/C-17/C-18(C-26)两个片段的存在。在HMBC谱中可以观察到H-12与C-14；H-13与C-14,C-15,C-24；H-15与C-13,C-14,C-24；H-16与C-14和H-24与C-13,C-14,C-15的相关，推断出上述两片段通过C-14相连。结合一维 13C NMR及HMBC相关，显示化合物1中的8个典型的芳香碳δ C 173.9(C-1),96.7(C-2),183.9(C-3),148.2(C-4),113.0(C-4a),120.6(C-19),143.5(C-20)和131.4(C-20a)为一个多取代吡喃内醌环的结构。同时，通过H-5与C-4,C-4a,C-20a；H-6与C-4a；H-17与C-19；H-18与C-19,C-20；H-26与C-19的HMBC相关，确定化合物1的平面结构。
查阅文献，化合物1的NMR数据与文献[Magauer T,Martin HJ,Mulzer J.Ring-closing metathesis and photo-fries reaction for the construction of the ansamycin antibiotic kendomycin:development of a protecting group free oxidative endgame[J].Chemistry,2010,16(2):507-519]报道的kendomycin A的NMR数据比对，鉴定化合物1为kendomycin类抗生素新化合物， 命名为kendomycin B，其结构如式(I)所示，碳谱和氢谱数据如表1所示。
化合物2为黄色粉末，分子式C 29H 42O 6S，HR-ESI-MS m/z 517.2633([M-H] -)；[α] 20 D-547(c 0.22,MeOH)；UV(MeOH)λ max(logε)202(4.265),240(3.490),380(3.600)nm；IR(film):v max:3298,2937,2358,2333,1597,1421,1008cm -1。通过 1H， 13C和DEPT NMR谱图的对比，化合物2为1的同系物，其差异在于2比1多一个-SCH 3结构。最终根据HMBC谱中H-27与C-20的相关，确定该-SCH 3与C-20相连，命名为kendomycin C，其结构如式(II)所示，碳谱和氢谱数据如表1所示。
化合物3为黄色粉末，分子式C 33H 47NO 9S，HR-ESI-MS m/z 471.2757([M-H] -)；[α] 20 D-240(c 0.34,MeOH)；UV(MeOH)λ max(logε)202(4.328),244(3.591),382(3.453)nm；IR(film):v max:3363,2949,2362,2330,1602,1373,1010cm -1。通过核磁谱图归属，确定化合物3与1含有相同的22元多取代吡喃内酯环的母核结构。其主要差异在于， 1H NMR谱中，在高场区多了1个甲基信号δ H 1.93(3H，s，Me-31)，低场区多了δ H 3.52(1H，dd，J＝5.0，13.6Hz)，δ H 3.87(1H，dd，J＝7.8，13.6Hz)和δ H 4.60(1H，dd，J＝5.0，7.8Hz)3个信号； 13C NMR中多了5个C信号，其中含1个甲基信号δ C 22.7，1个亚甲基信号δ C 35.8，1个次甲基信号，1个羰基碳δ C 172.6及1个羧基碳δ C 173.9。在HMBC谱图中可观察到H-27(δ H 3.52，δ H 3.87)与C-20(δ C 151.5)及C-28(δ C 55.1H-28(δ H 4.60)与C-27(δ C 35.8)C-29(δ C 173.9)及C-30(δ C 172.7)，H-31(δ H 1.93)与C-30(δ C 172.7)的远程相关，并结合 1H- 1H COSY中H-27(δ C 35.8)与H-28(δ H 4.60)的耦合，推断出氮乙酰半胱氨酸的存在。氮乙酰半胱氨酸的连接位置通过HMBC谱图中H-27(δ H 3.52，δ H 3.87)与C-20(δ C 151.5)的相关确定。氮乙酰半胱氨酸的绝对构型通过化学方法确定，由L和D型氮 乙酰半胱氨酸与底物1在中性条件下反应，通过HPLC及LC-MS分析，最终确定化合物3中C-20连接的为L型氮乙酰半胱氨酸。因此，最终确定化合物3的结构，并命名为kendomycin D，其结构如式(III)所示，碳谱和氢谱数据如表1所示。
Figure PCTCN2019075053-appb-000002
表1 kendomycin B-D的核磁碳谱和氢谱数据
Figure PCTCN2019075053-appb-000003
Figure PCTCN2019075053-appb-000004
a Measured in CD 3OD.
由滤纸片法对化合物进行抑菌活性测试，发现化合物1-3对大多革兰氏阳性菌均有一定的抑菌活性。继续选取苏云金芽孢杆菌(Bacillus thuringiensis)BT01，枯草芽孢杆菌(Bacillus subtilis)BS01，粪肠球菌(Enterococcus faecalis)ATCC 29212，金黄色葡萄球菌(Staphylococcus aureus)ATCC 29213，金黄色葡萄球菌(Staphylococcus aureus)745524及耐甲 氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus)shhs-A1，96孔板法测最小抑菌浓度，卡那霉素、氨苄霉素和万古霉素作为阳性对照。化合物1对金黄色葡萄球菌ATCC 29213、金黄色葡萄球菌745524、粪肠球菌ATCC 29212和苏云金芽孢杆菌BT01的最小抑菌浓度(MIC)分别为0.5、1.0、2.0、1.0μg/mL，优于卡那霉素、氨苄霉素和万古霉素的阳性对照，化合物2对金黄色葡萄球菌ATCC 29213、金黄色葡萄球菌745524、耐甲氧西林金黄色葡萄球菌shhs-A1、粪肠球菌ATCC 29212和苏云金芽孢杆菌BT01的最小抑菌浓度(MIC)分别为0.5、1.0、1.0、0.5、0.5μg/mL，优于卡那霉素、氨苄霉素和万古霉素的阳性对照，化合物3对金黄色葡萄球菌ATCC 29213、金黄色葡萄球菌745524和苏云金芽孢杆菌BT01的最小抑菌浓度(MIC)分别为1.0、2.0、2.0μg/mL，优于卡那霉素、氨苄霉素和万古霉素的阳性对照。测试结果如表2所示。
表2 化合物1-3的MIC值(μg/mL)
Figure PCTCN2019075053-appb-000005
a Vancomycin,kanamycin and ampicilin served as positive controls.The tests were performed in triplicate.
化合物1-3通过标准MTT法用对人胃癌细胞系MGC803(human gastric carcinoma cell line)、人肺癌细胞系A549(human lung cancer cell line)、人***细胞系Hela(human cervival carcinoma cell line)、人肝癌细胞系HepG2(human hepatocellular carcinoma cell line)、人乳腺 腺癌细胞系MCF-7(human breast adenocarcinoma cell line)和人结直肠癌细胞系RKO(human colorectal carcinoma cell line)六种癌细胞系及人类肝细胞系L02(normal human hepatic cell line)和人脐静脉内皮细胞系Huvec-12(normal human umbilical vein endothelial cell line)两种非恶性细胞系进行细胞毒活性测试，以顺铂作为阳性对照。化合物1-3均具有显著的抗肿瘤活性。测试结果如表3所示。据Janssen等的报道可知，化合物1和2对A549的细胞毒活性是显著优于kendomycin A的[Janssen CO,Lim S,Lo EP,et al.Interaction of kendomycin and semi-synthetic analogues with the anti-apoptotic protein Bcl-xl[J].Bioorg Med Chem Lett,2008,18(21):5771-5773]。
表3 化合物1-3的IC 50值(μM)
Figure PCTCN2019075053-appb-000006
a Data are expressed as mean of at least three independent experiments.Cis-platinum was used as positive control
Figure PCTCN2019075053-appb-000007
Figure PCTCN2019075053-appb-000008

Claims (10)

  1. 海洋疣孢菌(Verrucosispora sp.)SCSIO 07399，其保藏编号为：GDMCC No.60466。
  2. 如式(I)、(II)和(III)所示的任一安莎全碳环聚酮类抗生素或其药用盐：
    Figure PCTCN2019075053-appb-100001
    其中，式(I)为kendomycin B，式(II)为kendomycin C，式(III)为kendomycin D。
  3. 权利要求1所述的海洋疣孢菌(Verrucosispora sp.)SCSIO 07399在制备权利要求2中所述的kendomycin B、C或D中的应用。
  4. 一种权利要求2所述的kendomycin B-D的制备方法，其特征在于，所述的kendomycin B-D是从权利要求1所述的海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物中制备得到的。
  5. 根据权利要求4所述的kendomycin B-D的制备方法，其特征在于，具体包括以下步骤：
    (a)制备海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物，将该发酵培养物的发酵上清液和菌丝体分开，发酵上清液用丁酮萃取，丁酮相经浓缩后得到浸膏A，菌丝体用丙酮浸泡提取，丙酮浸提液经浓缩后得到浸膏B；
    (b)将浸膏A和浸膏B合并，采用硅胶柱层析，以氯仿-甲醇作为洗脱剂，从体积比100:0、99:1、98:2、96:4、94:6、92:8、9:1、8:2、5:5、0:100依次进行梯度洗脱，收集氯 仿-甲醇体积比为92:8、9:1、8:2和5:5洗脱的馏分Fr.A6～A9，将馏分Fr.A6～A9合并后经反相中压液相色谱，以流动相CH 3CN/H 2O 10％～100％v/v线性梯度洗脱120min，流速10mL/min，每馏分150mL，顺序得到8个馏分Fr.B1～B8，将馏分Fr.B5经Sephex LH-20葡聚糖凝胶色谱柱，以CHCl 3/MeOH体积比1:1作为流动相洗脱，收集馏分再经纯化后得到kendomycin D，将馏分Fr.B7经Sephex LH-20葡聚糖凝胶色谱柱，以CHCl 3/MeOH体积比1:1作为流动相洗脱，收集馏分再经纯化后得到kendomycin B和kendomycin C。
  6. 根据权利要求5所述的制备方法，其特征在于，所述的制备海洋疣孢菌(Verrucosispora sp.)SCSIO 07399的发酵培养物是通过以下方法制备：
    将海洋疣孢菌(Verrucosispora sp.)SCSIO 07399接入到种子培养基中，发酵得种子培养液，将种子培养液接入到发酵培养基，发酵得到发酵培养物；所述的种子培养基的配方为：每升含有可溶性淀粉10g、酵母提取物4g、细菌学蛋白胨2g和海盐30g，余量为水；发酵培养基的配方为：每升含有可溶性淀粉20g、葡萄糖10g、麦芽糖10g、玉米粉5g、麦芽提取粉10g、CaCO 3 2g和海盐30g，余量为水。
  7. 权利要求2所述的kendomycin B、kendomycin C或kendomycin D，或其药用盐在制备抗菌药物或抗肿瘤药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述的抗菌药物为抗苏云金芽孢杆菌、枯草芽孢杆菌、粪肠球菌、金黄色葡萄球菌或耐甲氧西林金黄色葡萄球菌的药物；所述的抗肿瘤药物为抗人胃癌、人肺癌、人***、人肝癌、人乳腺腺癌或人结直肠癌的药物。
  9. 一种抗菌药物或抗肿瘤药物，其特征在于，含有有效量的权利要求2所述的kendomycin B、kendomycin C或kendomycin D，或其药用盐作为活性成分。
  10. 根据权利要求9所述的抗菌药物或抗肿瘤药物，其特征在于，所述的抗菌药物为抗苏云金芽孢杆菌、枯草芽孢杆菌、粪肠球菌、金黄色葡萄球菌或耐甲氧西林金黄色葡萄球菌的药物；所述的抗肿瘤药物为抗人胃癌、人肺癌、人***、人肝癌、人乳腺腺癌或人结直肠癌的药物。
PCT/CN2019/075053 2019-01-28 2019-02-14 一类安莎全碳环聚酮类抗生素及其在制备抗菌药物或抗肿瘤药物中的应用 WO2019227969A1 (zh)

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