CN109575040B - 一种具有抗菌活性的化合物及其制备方法 - Google Patents
一种具有抗菌活性的化合物及其制备方法 Download PDFInfo
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- CN109575040B CN109575040B CN201910042207.8A CN201910042207A CN109575040B CN 109575040 B CN109575040 B CN 109575040B CN 201910042207 A CN201910042207 A CN 201910042207A CN 109575040 B CN109575040 B CN 109575040B
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- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/04—Ortho-condensed systems
- C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
- C07D491/048—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being five-membered
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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
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- 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
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
技术领域
本发明涉及一种具有抗菌活性的化合物及其制备方法,属于生物医药领域。
背景技术
近年来,从陆生放线菌中分离得到的先导化合物数量锐减,人们把目光投向了生境特殊的广阔海洋。海洋环境具有高盐度、高压、低温、低营养等特点,在这种特殊环境里生存的海洋放线菌与陆生放线菌显著不同,具有独特的代谢途径,可以产生许多结构新颖和活性多样的天然产物,为临床用药研发提供了宝贵的化学资源。仅2014年,就有1378个新化合物从海洋微生物中被发现,海洋天然产物是新药发现的热点研究领域。
海绵是海洋放线菌的主要来源。海绵是滤食性的多细胞动物,靠过滤海水中微生物和有机颗粒生存,海绵具有丰富的微生物,可占海绵生物量的35%,其中放线菌是海绵微生物的主要类群,据统计,在已获得10400条的海洋放线菌16S rDNA,海绵来源的占21%,且含有许多稀有和新的种属。海绵放线菌也是海洋先导化合物的重要来源,具有抗细菌、真菌、病原虫、肿瘤和病毒等多种活性,如具有抑制白色念珠菌活性的urauchimycins A和B(Imamura等,1993)。近几年,不断有新颖结构的化合物从海绵来源的放线菌被发现。Dharmaraj从印度海岸的海绵中分离出的 94株共生链霉菌均显示出抗细菌和真菌的能力(Dharmaraj等,2009)。Sheila等从4 种地中海海绵共附生链霉菌中分离出3种活性物质,分别是缬氨霉素(valinomycin),星形孢菌素(staurosporine)和丁烯酸内酯(butenolide),这是首次从海绵链霉菌中分离出缬氨霉素的报道(Pimentelelardo等,2010)。2011年Thomas从一株分离自美国佛罗里达群岛海绵的疣孢菌Verrucosispora sp.的代谢产物中得到五个新的环二聚硫环缩肽,对A549肺腺癌细胞具有显著的细胞毒活性(Wyche等,2011)。 Abdelmohsen等从Dysidea tupha的共附生链霉菌RV15分离到4个新环脂肽化合物cyclodysidins A-D(Abdelmohsen等,2012)。据海洋天然产物数据库MarinLit的统计,2013年,从海洋放线菌中共发现411个新天然产物,其中22%是海绵放线菌产生的(Abdelmohsen UR,Bayer K,Hentschel U.Diversity,abundance and natural productsof marine sponge-associated actinomycetes.Natural product reports.2014;31(3):381-99.)。 Cheng等从地中海海绵Agelas oroides的共附生链霉菌SBT345中分离到新颖氯代喹诺酮化合物ageloline A,具有抗沙眼衣原体的活性(Cheng C,Othman EM,Reimer A,Grüne M,Kozjak-Pavlovic V,Stopper H,Hentschel U,Abdelmohsen UR.Ageloline A,new antioxidant and antichlamydial quinolone from the marine sponge-derivedbacterium Streptomyces sp.SBT345.Tetrahedron letters.2016Jun 22;57(25):2786-9.)。刘睿等从羽毛山海绵来源的海绵共附生放线菌糖多孢菌Saccharopolyspora sp.novSP2210发现具有诱导肿瘤细胞坏死的活性(刘睿,方玉春,段琳,杜林,朱天骄,刘红兵,顾谦群, and朱伟明."海绵共附生放线菌Saccharopolyspora sp.nov中抗肿瘤活性成分的研究."PhD diss.,2006.)。赵文英等从海绵共附生放线菌SH6004菌株中发现抗肿瘤活性成分(赵文英,顾谦群,朱伟明.海绵共附生放线菌SH6004菌株中抗肿瘤活性成分的研究.中國海洋大學學報(自然科學版).2008Jan 1;38(1):23-6.)。彭杰等研究了海绵共附生放线菌Streptomyces sp.A01059抗稻瘟病活性(彭杰,吴晓鹏,张开山, 黄惠琴,孙前光,鲍时翔.海绵共附生放线菌Streptomyces sp.A01059抗稻瘟病活性物质的分离研究.中國農學通報.2009May 5;25(9):51-4.)。甄心等从南海丰肉结海绵的相关链霉菌LS-298菌株的发酵产物中同时分离到棘霉素和替达霉素B,不仅具有较强的抗菌活性,亦具有很强的体外抗肿瘤活性(甄心,巩婷,and朱平."丰肉结海绵相关放线菌LS-298活性产物."菌物研究2(2013):148-148.)。张艳凤等从中国南海一株海绵中分离到一株稀有放线菌皮生球属菌株Dermacoccus sp.X4对金黄色葡萄球菌具有较好抑菌活性,并分离吲哚酸酯类化合物(张艳凤,许勇,陈雷,等.海绵内/共生稀有放线菌Dermacoccus sp.X4次级代谢产物分离纯化及结构解析[J].生物工程学报,2016,32(5):599-609.)。因此,海绵放线菌的次级代谢产物,不仅结构新颖,也是抗菌活性新化合物的主要来源。
细菌性病害是人类健康的主要威胁,而且,由于抗菌药物的滥用,耐药性细菌越来越多,寻找安全有效的抗菌药物是人类长期的目标。基于以上背景,开展从海洋放线菌中寻找抗菌药物的发明研究。
发明内容
本发明的目的在于提供一种具有抗菌活性的化合物及其制备方法及其应用。
一种具有抗菌活性的化合物,所述化合物为 (6E,8Z)-11-(1-(2-hydroxy-3,4-dimethyl-8-oxo-6-oxa-3-azatricyclo[3.3.0.02,4]octan-7-yl)et hyl)-8,10-dimethyloxacycloundeca-6,8-diene-2,4,5-trione,分子式为C22H27NO7,基本结构如下:
本发明所涉及的海洋链霉菌Streptomyces sp.FJNU027,该菌株已于2018年9月 7日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2018601;保藏的培养物名称及注明的鉴别特征为海洋链霉菌Streptomyces sp.FJNU027,保藏日为2018年9 月7日,保藏地址为中国武汉武汉大学。
本发明所述结构新颖的化合物制备方法包括:
1)海洋放线菌液体发酵:将海洋放线菌Streptomyces sp.FJNU027(该菌株已于2018年9月7日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M2018601) 斜面菌种活化后,进行液体深层发酵,培养基配方按重量比为:KNO3 0.5-3.0g,K2HPO4 0.1-3g,MgSO40.1-3g,FeSO4 0-0.1g,可溶性淀粉0-50g,葡萄糖0-50g,海水1.0L, 于0.1Mpa和121℃灭菌30min。采用恒温摇床或各式发酵罐进行液体发酵,温度设置23-30℃,发酵时间5-30天;
2)发酵产物处理:液体发酵结束后,除去菌体的发酵液用乙酸乙酯萃取,萃取液经脱水、浓缩,得到有机粗提物浸膏;
3)所述的化合物的分离:将2)所述的有机粗提物浸膏用甲醇溶解,进行反相硅胶柱层析,用甲醇或丙酮与水体积比为30-80:100的不同梯度甲醇或丙酮水溶液为洗脱剂,将含有目标组分的洗脱液浓缩得到亚组分。用甲醇将亚组分溶解,进行凝胶柱层析,用丙酮或甲醇溶液洗脱,分管收集,将含有所述的新化合物组分的洗脱液合并,接着进行正相硅胶柱层析,以氯仿装柱,干法上样,用含有甲醇体积比为1-10%的氯仿溶剂洗脱,得到含有所述的新化合物。
本发明的显著优点在于:
本发明制备的化合物对枯草芽孢杆菌有较好的抑制作用,可以利用菌株Streptomyces sp.FJNU027发酵制得,培养基原料主要为无机盐,来源广泛,制备方法简单,容易实现工业化生产。
附图说明
图1本发明所述化合物的化学结构。
图2本发明所述化合物抗枯草芽孢杆菌Bacilus subtilus的MIC测定。
具体实施方式
以下实施例将对本发明作进一步说明。
实施例1
将海洋放线菌(Streptomyces sp.FJNU027)斜面菌种接种于装有100mL改良高氏1号培养基(配方:每升海水中含KNO3 1.0g,K2HPO4 0.5g,MgSO4 0.5g,FeSO4 0.01g,可溶性淀粉15g,葡萄糖15g)的三角瓶中活化,活化条件为转速230r/min,培养温度28℃,培养时间15天,再进行大批量的液体发酵65L,采用上述的改良高氏1号培养基,pH自然,0.1Mpa,121℃灭菌25min,置于28℃,230r/min恒温摇床中培养。发酵20天后,用离心法将菌丝体与发酵液分离。发酵液用等体积的乙酸乙酯萃取,所得萃取物用无水硫酸钠脱水,再用旋转蒸发仪于40℃条件下真空浓缩,得到有机粗提物浸膏5g。
将上一步骤中得到的有机粗提物浸膏(5g),用甲醇溶解,进行反相硅胶(170 g)柱层析,用70%(vol)甲醇水洗脱2L,流速为15mL/min,每管收集280mL,用旋转蒸发仪真空浓缩后,分别取样进行薄层层析分析合并(展开剂为按体积比计,氯仿:甲醇=10:1,显色剂为碘、10%硫酸乙醇或碘化铋钾),得到含有目标化合物的组分(502.4mg)。
从上一步骤得到的组分(502.4mg),用适量甲醇溶解,进行凝胶(120g,SephadexLH-20)柱层析,甲醇洗脱,流速约为12s/drop,每管收集4mL,根据薄层层析检测合并得到含有目标化合物的组分(255.7mg)。
取上一步骤得到的组分19.5mg进行正相硅胶层析(2g硅胶),氯仿装柱,干法上样,流动相为氯仿:甲醇(60:1),洗脱体积为300mL,薄层层析检测合并得到目标化合物(12.4mg)。
将上一步骤所得的化合物,进行核磁共振波谱(1H-NMR、13C-NMR、HSQC、 HMBC、1H-1H COSY和NOESY)、高分辨质谱(HREI-MS)、旋光([α])、紫外光谱(UV)、红外光谱(IR)测定,并确定所述化合物结构(图1)。
所述的化合物,白色,可溶于甲醇,UV-vis(MeOH),λ/nm:335;IR(KBr)νmax:3372,2975,2362,1724,1595,1475cm-1;HR-ESI-Q-TOF MS:m/z418.1856([M+H]+,calculated for C22H28NO7,418.1866)and m/z 440.1683 ([M+Na]+,calculated for C22H27NO7Na,440.1685)。结合1H和13C NMR谱数据(表1) 确定化合物的分子式为C22H27NO7。
分析1H和13C NMR以及DEPT谱,表明所述的化合物含有5个甲基,1个亚甲基,9个次甲基(其中3个连氧,3个为烯碳),7个季碳(其中4个为碳基,1 个为烯碳)。HMBC实验中观察到甲基H320与C10/C11/C12,甲基H321与 C8/C9/C10,甲基H322与C6/C7/C8,亚甲基H22与C1/C3,H9与C7/C8/C21,H10 与C8/C9/C20/C21,以及H5和H6与C4的碳氢远程相关,和从1H-1HCOSY实验中观察到的H5与H6,H8与H9,H9与H10,H10与H11,H11与H12之间的氢氢相关,以及C1(δC 178.6)、C3(δC 193.5)、C4(δC 181.9)、C5(δC 124.4)、C6(δC 143.9)、 C7(δC 134.2)和C8(δC 138.7)的化学位移值,可以推测出化合物的一个结构片段1a (C1-12和C20-22)(图1)。同样根据从HMBC实验中观察到甲基H318与C15/C16/C17,H319与C15/C16,H11与C13,H12与C13/C15/C17,和H17与 C11/C13/C16/C18的碳氢远程相关,以及根据C15(δC 96.1)、C16(δC 56.7)和C17(δC 59.9)的化学位移值可以推测出化合物的另一个结构片段1b(C12-19)(图1)。综合以上数据,可以推测出所述化合物的基本结构(图1)。
表1所述化合物的NMR波谱数据(DMSO-d6,500M)
实施例2
采用96孔板法进行所述化合物的MIC抑菌试验。无菌操作下,在96孔板的每孔中加入180μL细菌培养基(配方:牛肉膏10g,蛋白胨10g,氯化钠5g,水1 L,pH 7.2-7.5,121℃高压灭菌30min),10μL枯草杆菌菌液(含菌数约2.0×106),和10μL含有不同浓度所述化合物的样品,所述的化合物的终浓度分别2.5、2、1.5、 1.0、0.5、0.25和0μg/mL,设3个平行,同时设添加10μL甲醇为溶剂对照,设青霉素为阳性对照(终浓度为50μg/mL)。另设不接菌的液体培养基为空白对照。置37 ℃恒温箱内培养24h,肉眼观察枯草杆菌生长情况。经观察,所述化合物为0.5μg/mL 时,枯草芽孢杆菌完全不生长(图2),表明所述化合物具有抗菌活性,最低抑制浓度为1.2μM。
图2中1-7列添加不同浓度的所述化合物,终浓度分别为2.5、2、1.5、1.0、 0.5、0.25和0μg/mL。第8列添加氨苄青霉素为阳性对照,终浓度为50μg/mL。第 9列添加不含样品的10μL甲醇为溶剂对照。第10列为空白对照,添加不接种的培养基。第1-5、8和10列的培养基清澈透明,表示无菌生长。第6列培养基中有部分生长。第7和9列的培养基浑浊,枯草芽孢杆菌正常生长。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (5)
2.一种如权利要求1所述的化合物的制备方法,其特征在于:通过发酵培养海洋链霉菌Streptomyces sp. FJNU027,获取发酵物,然后从发酵物中分离纯化得到所述化合物。
3.根据权利要求2所述的化合物的制备方法,其特征在于:具体步骤为:
1) 海洋放线菌液体发酵:将海洋链霉菌Streptomyces sp. FJNU027斜面菌种活化后,进行液体深层发酵,采用恒温摇床或各式发酵罐进行液体发酵,温度设置23-30 ℃,发酵时间5-30天;
2) 发酵产物处理:液体发酵结束后,除去菌体的发酵液用乙酸乙酯萃取,萃取液经脱水、浓缩,得到有机粗提物浸膏;
3) 所述的化合物的分离:将2)所述的有机粗提物浸膏用甲醇溶解,进行反相硅胶柱层析,用甲醇或丙酮与水体积比为30-80:100的不同梯度甲醇或丙酮水溶液为洗脱剂,将含有目标组分的洗脱液浓缩得到亚组分;用甲醇将亚组分溶解,进行凝胶柱层析,用丙酮或甲醇溶液洗脱,分管收集,将含有所述的化合物组分的洗脱液合并,接着进行正相硅胶柱层析,以氯仿装柱,干法上样,用含有甲醇体积比为1-10%的氯仿溶剂洗脱,得到含有所述化合物。
4.根据权利要求3所述的化合物的制备方法,其特征在于:步骤(1)深层发酵的培养基配方按重量比为:KNO3 0.5-3.0 g, K2HPO4 0.1-3 g, MgSO4 0.1-3 g, FeSO4 0-0.1 g, 可溶性淀粉 0-50 g, 葡萄糖0-50 g, 海水1.0 L;将配置好的培养基于0.1 Mpa和121 ℃灭菌30 min。
5.如权利要求1所述的化合物在制备抗菌药物中的用途。
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