CN111689895B - 两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用 - Google Patents
两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用 Download PDFInfo
- Publication number
- CN111689895B CN111689895B CN202010442011.0A CN202010442011A CN111689895B CN 111689895 B CN111689895 B CN 111689895B CN 202010442011 A CN202010442011 A CN 202010442011A CN 111689895 B CN111689895 B CN 111689895B
- Authority
- CN
- China
- Prior art keywords
- piericidin
- compound
- dichloromethane
- renal cancer
- compounds
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 46
- 206010038389 Renal cancer Diseases 0.000 title claims abstract description 31
- 208000008839 Kidney Neoplasms Diseases 0.000 title claims abstract description 29
- 201000010982 kidney cancer Diseases 0.000 title claims abstract description 29
- 239000003560 cancer drug Substances 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims description 10
- 238000006317 isomerization reaction Methods 0.000 title abstract description 17
- BBLGCDSLCDDALX-LKGBESRRSA-N piericidin A Chemical compound COC=1NC(C\C=C(/C)C\C=C\C(\C)=C\[C@@H](C)[C@@H](O)C(\C)=C\C)=C(C)C(=O)C=1OC BBLGCDSLCDDALX-LKGBESRRSA-N 0.000 claims abstract description 108
- 229930182661 Piericidin Natural products 0.000 claims abstract description 102
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 241000187180 Streptomyces sp. Species 0.000 claims abstract description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 63
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000003208 petroleum Substances 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 3
- 238000002953 preparative HPLC Methods 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 241000187747 Streptomyces Species 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 claims 2
- 201000011510 cancer Diseases 0.000 abstract description 16
- 206010028980 Neoplasm Diseases 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 12
- 230000005764 inhibitory process Effects 0.000 abstract description 11
- 230000022131 cell cycle Effects 0.000 abstract description 5
- 150000002611 lead compounds Chemical class 0.000 abstract description 5
- 239000002246 antineoplastic agent Substances 0.000 abstract description 4
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 4
- 230000001419 dependent effect Effects 0.000 abstract description 3
- 238000011156 evaluation Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 230000006882 induction of apoptosis Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 38
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108010087230 Sincalide Proteins 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 5
- 238000010609 cell counting kit-8 assay Methods 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 5
- 235000002639 sodium chloride Nutrition 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 229930014626 natural product Natural products 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 101710128742 Cytochrome b6-f complex iron-sulfur subunit 2 Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102100020870 La-related protein 6 Human genes 0.000 description 2
- 108050008265 La-related protein 6 Proteins 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 description 2
- 240000002044 Rhizophora apiculata Species 0.000 description 2
- VORIUEAZEKLUSJ-UHFFFAOYSA-M [(6-chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylidene]-dimethylazanium;trifluoroborane;fluoride Chemical compound [F-].FB(F)F.C1=C(Cl)C=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 VORIUEAZEKLUSJ-UHFFFAOYSA-M 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 201000010174 renal carcinoma Diseases 0.000 description 2
- 238000013341 scale-up Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000284466 Antarctothoa delta Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 230000035519 G0 Phase Effects 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 230000004668 G2/M phase Effects 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- KYIKRXIYLAGAKQ-UHFFFAOYSA-N abcn Chemical compound C1CCCCC1(C#N)N=NC1(C#N)CCCCC1 KYIKRXIYLAGAKQ-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 230000006203 ethylation Effects 0.000 description 1
- 238000006200 ethylation reaction Methods 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UBQKCCHYAOITMY-UHFFFAOYSA-N pyridin-2-ol Chemical compound OC1=CC=CC=N1 UBQKCCHYAOITMY-UHFFFAOYSA-N 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/62—Oxygen or sulfur atoms
- C07D213/69—Two or more oxygen atoms
-
- 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
- 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
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用。本发明从链霉菌Streptomyces sp.HBERC‑58855发酵培养物中分离制备得到具有抗肾癌活性的粉蝶霉素piericidin L和piericidin M。通过对多株癌细胞的抑制活性实验评价,发现化合物piericidin L和piericidin M对人肾癌OS‑RC‑2细胞具有明显的选择性抑制活性,IC50分别为2.2和4.5μM,并以浓度依赖型方式使肾癌OS‑RC‑2细胞周期G2/M期受阻,并诱导细胞凋亡;因此支链异构化粉蝶霉素类化合物piericidin L或piericidin M可以做为抗肾癌药物开发的先导化合物。本发明为研制微生物来源新型抗癌药物提供了新的先导化合物。
Description
技术领域
本发明属于天然产物领域,具体涉及两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用。
背景技术
恶性肿瘤已经成为我国居民首要死因,中国恶性肿瘤死亡率近20年来不断呈现上升趋势,处于世界恶性肿瘤死亡率的前列。据我国***统计,中国每年新增肿瘤病人约200万人,目前全国肿瘤患者总数约为450万人。药物治疗在恶性肿瘤的疗法中占有相当重要的地位,发展速度最快。对于恶性肿瘤的治疗,天然产物及其衍生物药物发挥着重要作用。据报道,1981年到2008年间,天然产物来源的抗肿瘤药物占上市抗肿瘤药物的60%以上,而且,新型天然产物及其衍生物作为新的抗肿瘤药物所占数量还在不断增加。肾癌现位居世界恶性肿瘤第16位,其发病率占恶性肿瘤的3%-5%,发病率上升幅度居于首位,另外肾癌在我国泌尿系肿瘤相关死亡中居于首位。研究发现,肾癌患者中30%发生转移,另有30%在10年内有发生转移的危险,并且转移性肾癌对放化疗及***性治疗具有较高的耐受性。
粉蝶霉素(piericidin)类化合物是微生物来源的α-吡啶酮类抗生素,不仅有杀虫、抗菌等活性,而且对部分肿瘤细胞也有抑制活性,支链异构化对粉蝶霉素的活性变化影响较大,从支链异构化的粉蝶霉素类化合物中有望挖掘具有抗肿瘤潜力的先导化合物。
发明内容
本发明的第一个目的是提供两个支链异构化粉蝶霉素类化合物piericidin L和piericidin M。
式(I)所示的任一粉蝶霉素类化合物或其药用盐,
本发明人通过对一株红树林底泥来源的链霉菌Streptomyces sp.HBERC-58855(保藏编号为CCTCC NO:M 2017186)进行摇床放大发酵和提取纯化,获得两个未见报道的结构新颖的粉蝶霉素类化合物piericidin L和piericidin M。化合物piericidin L和piericidin M的结构,相对于其他同类化合物,新颖性在于支链片段发生了异构化,piericidin L具有少见的C-12酮基和Δ11,14共轭片段,piericidin M具有同类化合物少见的C-7乙基化。
通过对这两个支链异构化粉蝶霉素类化合物进行多种癌细胞的体外活性评价,发现piericidin L和piericidin M具有选择性地抑制人肾癌细胞OS-RC-2的活性,半数抑制率IC50分别为2.2和4.5μM。piericidin L和piericidin M以浓度依赖型方式使人肾癌细胞OS-RC-2细胞周期G2/M期受阻,并诱导细胞凋亡。因此,支链异构化粉蝶霉素类化合物piericidin L或piericidin M可以做为抗肾癌药物开发的先导化合物。
因此本发明的第二个目的是提供所述的化合物piericidin L和/或piericidinM,或其药用盐在制备抗肾癌药物中的应用。
本发明的第三个目的是提供一种抗肾癌药物,其特征在于,包括有效量的作为活性成份的权利要求1所述的化合物piericidin L和/或piericidin M,或其药用盐,和药学上可以接受的载体。
本发明的第四个目的是提供所述的化合物piericidin L和/或piericidin M的制备方法,是从链霉菌Streptomyces sp.HBERC-58855的发酵培养物中分离制备得到piericidin L和/或piericidin M。
优选,所述的制备方法,包括以下步骤:
将链霉菌Streptomyces sp.HBERC-58855的发酵培养物用乙酸乙酯萃取,萃取物浓缩后得到提取物,提取物经硅胶柱层析,顺序用石油醚、石油醚:二氯甲烷1:1v/v、二氯甲烷、二氯甲烷:甲醇100:1v/v、二氯甲烷:甲醇100:2v/v、二氯甲烷:甲醇100:3v/v、二氯甲烷:甲醇100:4v/v、二氯甲烷:甲醇100:5v/v、甲醇洗脱,收集石油醚:二氯甲烷1:1v/v洗脱的馏分S2,S2经制备HPLC纯化得到化合物piericidin L和piericidin M。
本发明的第五个目的是提供链霉菌Streptomyces sp.HBERC-58855在制备所述的化合物piericidin L和/或piericidin M中的应用。
本发明的两个支链异构化粉蝶霉素类化合物piericidin L和piericidin M是未经报道的新化合物。本发明揭示piericidin L和piericidin M对肾癌细胞OS-RC-2具有明显的选择性抑制活性,这类粉蝶霉素可以用于制备特异性的抗肾癌药物。因此本发明为开发新的抗肾癌药物提供了备选化合物,对开发中国海洋微生物药物资源具有重要的意义。
本发明涉及的链霉菌Streptomyces sp.HBERC-58855,保藏于中国典型培养物保藏中心,保藏地址为中国武汉武汉大学,保藏日期为2017年4月20日,保藏编号为CCTCC NO:M2017186(该保藏信息公开于专利申请CN201710344174.3中)。
附图说明
图1是化合物piericidin L和piericidin M的重要COSY、HMBC和NOESY相关信息。
图2是化合物piericidin L和piericidin M对OS-RC-2肾癌细胞的细胞周期作用。
图3是化合物piericidin L和piericidin M对OS-RC-2肾癌细胞的细胞凋亡作用。
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:支链异构化粉蝶霉素类化合物piericidin L和piericidin M的制备
1、Streptomyces sp.HBERC-58855的固体培养
链霉菌Streptomyces sp.HBERC-58855(保藏编号为CCTCC NO:M 2017186)是从红树林底泥分离得到,该菌种保存于ISP-2培养基斜面,ISP-2培养基组成为:每升含有酵母提取粉4g,葡萄糖4g,麦芽提取粉10g,粗海盐30g,琼脂粉20g,余量为水,pH 7.2-7.4;制备方法为将上述各组分混合,121℃灭菌,分装备用。
2、Streptomyces sp.HBERC-58855的放大发酵
取少量Streptomyces sp.HBERC-58855斜面菌种进行种子发酵培养,种子发酵培养基:每升含有甘露醇20g,大豆蛋白胨10g,豆油2.5g,磷酸氢二钾0.35g,余量为去离子水,pH7.0。用500毫升三角瓶装100毫升种子发酵培养基,接种培养条件为:28℃,120转/min。培养96h后,进行扩大培养,培养体积扩大到3L,培养基和培养条件不变。培养48h后,进行罐体发酵,发酵体积为30L,罐体发酵培养基:每升含有葡萄糖10g,可溶性淀粉10g,棉籽粉25g,酵母提取物3g,碳酸钙5g,氯化钠2g,余量为去离子水,pH7.0;28℃,培养120h后,收取发酵液,用乙酸乙酯萃取,萃取物经浓缩后得到Streptomyces sp.HBERC-58855菌株发酵物的干燥提取物。
3、提取分离
Streptomyces sp.HBERC-58855菌株发酵物的干燥提取物用少量甲醇溶解后硅胶拌样,进行中压硅胶柱层析(200-300目),用石油醚、石油醚:二氯甲烷1:1v/v、二氯甲烷、二氯甲烷:甲醇100:1v/v、二氯甲烷:甲醇100:2v/v、二氯甲烷:甲醇100:3v/v、二氯甲烷:甲醇100:4v/v、二氯甲烷:甲醇100:5v/v、甲醇顺序洗脱得到9个洗脱部位S1-S9。S2部位(石油醚:二氯甲烷1:1v/v洗脱馏分)经制备HPLC(Hitachi-L2130液相色谱仪,Hitachi L-2455DAD检测器,Phenomenex ODS色谱柱,250mm×10.0mm i.d.,5μm,流动相:乙腈:水45:55v/v洗脱;流速1.5ml/min;柱温40℃)进行反复分离纯化,得到纯化的化合物piericidinL(Rt 14.7min,乙腈:水45:55v/v,1.5mg)和piericidin M(Rt 15.1min,乙腈:水45:55v/v,1.1mg)。
实施例2:支链异构化粉蝶霉素类化合物piericidin L和piericidin M的结构鉴定
对化合物piericidin L和piericidin M进行结构分析测试,得到以下理化性质数据:
Piericidin L:淡黄色油状物,3.1°(c 0.10,MeOH);IR(ATR)νmax 3377,2920,1670,1585,1411,1190,1124,1045,972,721,667,600cm-1;1H和13C NMR数据见表1;(+)-HR-ESIMS m/z430.2583,[M+H]+(calcd for C25H36NO4430.2588)。
Piericidin M:淡黄色油状物,2.4°(c 0.080,MeOH);IR(ATR)νmax3381,2920,1471,1456,1412,1190,1124,1026,966,825,775,669cm-1;1H和13C NMR数据见表1;(+)-HR-ESIMS m/z430.2955,[M+H]+(calcd for C24H36NO4498.2952)。
表1化合物piericidinL和piericidinM的1H(700MHz)和13C NMR(175HMz)数据
ainMeOD,700MHz;b inDMSO,700MHz;
化合物piericidin L和piericidin M的2D NMR相关信息如图1。
化合物piericidin L和piericidin M具有式(I)所示的粉蝶霉素类化合物结构,
式(I);其中,
实施例3:支链异构化粉蝶霉素类化合物piericidin L和piericidin M对肾癌细胞的抑制活性
6株癌细胞株订购于中科院上海细胞资源中心:HL-60细胞株(Cat#TCHu 23);K-562细胞株(Cat#TCHu191);MOLT-4细胞株(Cat#TCHu224);786-O细胞株(Cat#TCHu186),ACHN细胞株(Cat#TCHu199);OS-RC-2细胞株(Cat#TCHu40)。癌细胞抑制活性实验采用CCK-8检测法。收集对数生长期细胞,计数,用完全培养基重新悬浮细胞,调整细胞浓度至合适浓度(依照细胞密度优化试验结果确定),接种96孔板,每孔加100μl细胞悬液。细胞在37℃,100%相对湿度,5%CO2培养箱中孵育24小时。用培养基将待测化合物稀释至合适的作用浓度,按25μl/孔加入细胞。化合物piericidin L和piericidin M的作用终浓度从50μM开始,4倍梯度稀释,9个浓度点。细胞置于37℃,100%相对湿度,5%CO2培养箱中孵育72小时。吸弃培养基,加入含10%CCK-8的新鲜完全培养基置于37℃培养箱中孵育2-4小时。轻轻震荡后在SpectraMax M5 Microplate Reader上测定450nm波长处的吸光度,以650nm处吸光度作为参比,计算抑制率。
按下式计算化合物对癌细胞生长的抑制率:
癌细胞生长抑制率%=[(Ac-As)/(Ac-Ab)]×100%
As:样品的吸光度OA(细胞+CCK-8+待测化合物)
Ac:阴性对照的吸光度OA(细胞+CCK-8+DMSO)
Ab:阳性对照的吸光度OA(培养基+CCK-8+DMSO)
运用软件Graphpad Prism 5并采用计算公式log(inhibitor)vs.normalizedresponse进行IC50曲线拟合并计算出IC50值。
支链异构化粉蝶霉素类化合物piericidin L和piericidin M对6株癌细胞株的增殖抑制作用见表2:
表2化合物piericidinL和piericidinM对6株癌细胞的增殖抑制作用(IC50,μM)
piericidinL | piericidinM | |
HL-60 | 12.3 | 9.8 |
K562 | >50 | >50 |
MOLT-4 | >50 | >50 |
ACHN | >50 | >50 |
786-O | >50 | >50 |
OS-RC-2 | 2.2 | 4.5 |
通过对6株癌细胞的增殖抑制活性评价发现,支链异构化粉蝶霉素类化合物piericidin L和piericidin M对人肾癌细胞OS-RC-2具有选择性的抑制活性,半数抑制率IC50分别为2.2和4.5μM。
为了进一步研究piericidin L和piericidin M抑制人肾癌细胞OS-RC-2的活性,取对数生长期的OS-RC-2细胞,调整细胞浓度为2×106/ml,接种于50mL培养瓶内,分三批,每批两个化合物各分4组,1组为空白对照,2、3、4组分别加入piericidin L或piericidinM,使其终浓度为1.25、2.5和5.0μM。细胞贴壁后,更换培养液并加药,培养至48h光镜观察照相。收集的细胞用4℃预冷的PBS洗涤,离心1000r/min,2×5min。缓冲液重悬细胞后,取100μL细胞悬液于流式管内,加入5μLAnnexin-V和10μLPI溶液,室温避光15min后,流式细胞仪进行检测。
细胞周期分析发现,piericidin L或piericidin M处理后,OS-RC-2细胞处于G0/G1期细胞百分含量减少、S期细胞含量基本不变、G2/M期细胞含量明显增多(图2)。细胞凋亡分析发现,piericidin L(1.25、2.5和5.0μM)处理导致的细胞凋亡率分别为6.83%、9.00%和13.9%;piericidin M(1.25、2.5和5.0μM)处理后,细胞凋亡率分别为5.10%、10.2%和11.1%(图3)。
综上,实验显示,在piericidin L或piericidin M具有选择性地抑制OS-RC-2肾癌细胞的活性中,并以浓度依赖型方式使肾癌OS-RC-2细胞周期G2/M期受阻,并诱导细胞凋亡。因此,支链异构化粉蝶霉素类化合物piericidin L或piericidin M可以做为抗肾癌药物开发的先导化合物。
Claims (6)
2.权利要求1所述的化合物piericidin L和/或piericidin M、或其药用盐在制备抗肾癌药物中的应用。
3.一种抗肾癌药物,其特征在于,包括有效量的作为活性成份的权利要求1所述的化合物piericidin L和/或piericidin M、或其药用盐,和药学上可以接受的载体。
4.一种权利要求1所述的化合物piericidin L和/或piericidin M的制备方法,其特征在于,是从链霉菌Streptomyces sp.HBERC-58855的发酵培养物中分离制备得到piericidin L和/或piericidin M。
5.根据权利要求4所述的制备方法,其特征在于,包括以下步骤:
将链霉菌Streptomyces sp.HBERC-58855的发酵培养物用乙酸乙酯萃取,萃取物浓缩后得到提取物,提取物经硅胶柱层析,顺序用石油醚、石油醚:二氯甲烷1:1v/v、二氯甲烷、二氯甲烷:甲醇100:1v/v、二氯甲烷:甲醇100:2v/v、二氯甲烷:甲醇100:3v/v、二氯甲烷:甲醇100:4v/v、二氯甲烷:甲醇100:5v/v、甲醇洗脱,收集石油醚:二氯甲烷1:1v/v洗脱的馏分S2,S2经制备HPLC纯化得到化合物piericidin L和piericidin M。
6.链霉菌Streptomyces sp.HBERC-58855在制备权利要求1所述的化合物piericidinL和/或piericidin M中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010442011.0A CN111689895B (zh) | 2020-05-22 | 2020-05-22 | 两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010442011.0A CN111689895B (zh) | 2020-05-22 | 2020-05-22 | 两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111689895A CN111689895A (zh) | 2020-09-22 |
CN111689895B true CN111689895B (zh) | 2021-08-24 |
Family
ID=72477221
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010442011.0A Active CN111689895B (zh) | 2020-05-22 | 2020-05-22 | 两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111689895B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113402453B (zh) * | 2021-06-21 | 2022-08-05 | 中国科学院南海海洋研究所 | 一种吡啶酮粉蝶霉素及其制备方法和在制备抗癌药物中的应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106562963A (zh) * | 2016-10-31 | 2017-04-19 | 中国科学院南海海洋研究所 | 粉蝶霉素类化合物Piericidin A在制备抗肾癌药物中的应用 |
CN107475146B (zh) * | 2017-05-16 | 2020-05-12 | 中国科学院南海海洋研究所 | 一种链霉菌及其代谢产物粉蝶霉素类化合物在抗肾癌中的应用 |
WO2020056425A1 (en) * | 2018-09-14 | 2020-03-19 | University Of Mississippi | Anticancer formulation |
CN109384823B (zh) * | 2018-11-12 | 2020-11-06 | 中国科学院南海海洋研究所 | 两个粉蝶霉素葡萄糖苷及其在抗肾癌药物中的应用 |
-
2020
- 2020-05-22 CN CN202010442011.0A patent/CN111689895B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN111689895A (zh) | 2020-09-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107298671A (zh) | 源于草酸青霉的黑麦酮酸h及制备抗人结肠癌药物的应用 | |
CN107475146B (zh) | 一种链霉菌及其代谢产物粉蝶霉素类化合物在抗肾癌中的应用 | |
CN107353274A (zh) | 源于草酸青霉的黑麦酮酸i及制备抗人食管癌药物的应用 | |
CN111689895B (zh) | 两个支链异构化粉蝶霉素类化合物及其在制备抗肾癌药物中的应用 | |
CN103304635A (zh) | 一种环肽类化合物抗肿瘤的应用及其制备方法 | |
CN102516368A (zh) | 环七肽类化合物及其在制备抗肿瘤药物中的应用 | |
CN112300243B (zh) | 一种环肽化合物及其制备方法和应用 | |
CN103724290B (zh) | 一种环肽化合物clavatustide A及其产生菌、制备方法和应用 | |
CN112939865A (zh) | 一种大环内酰胺化合物FW05328-d及其高效发酵方法 | |
CN105061446B (zh) | 源于桔青霉的penicitrinine A及在制备抗鼻咽癌药物上的应用 | |
CN109384823B (zh) | 两个粉蝶霉素葡萄糖苷及其在抗肾癌药物中的应用 | |
CN110218200A (zh) | 一种红树内生真菌中环缩肽化合物及其制备方法与应用 | |
CN102631664A (zh) | 3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸的应用 | |
CN113402453B (zh) | 一种吡啶酮粉蝶霉素及其制备方法和在制备抗癌药物中的应用 | |
US7939081B2 (en) | Method for producing cercosporamide | |
CN103641791B (zh) | 一种环肽化合物clavatustide B及其制备方法和应用 | |
CN102531906B (zh) | 天然化合物p21在抑制肿瘤细胞生殖生长中的应用 | |
CN108660169A (zh) | 一种发酵制备棘孢菌素类抗生素的方法 | |
CN109384710B (zh) | 三个粉蝶霉素类天然产物及其在制备抗肾癌药物中的应用 | |
CN102051394A (zh) | 一种硫代二酮哌嗪类化合物制备方法及其应用 | |
CN105112322A (zh) | 灰霉醌a和b及其制备方法和医药用途 | |
CN103570652A (zh) | 一种环氧苯并蒽醌类化合物及其制备方法和应用 | |
CN115227691B (zh) | 源于细曲霉的内酯类化合物在抗人乳腺癌药物中的应用 | |
CN116199695B (zh) | 一类桔霉素衍生物及其制备方法和应用 | |
CN103242348A (zh) | 吲哚啉二酮哌嗪类螺环化合物及其制备方法和用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |