WO2014005408A1 - 抗生素Tetrathiazomycin A及其制备方法和在制备抗肿瘤药物中的应用 - Google Patents
抗生素Tetrathiazomycin A及其制备方法和在制备抗肿瘤药物中的应用 Download PDFInfo
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- WO2014005408A1 WO2014005408A1 PCT/CN2012/087016 CN2012087016W WO2014005408A1 WO 2014005408 A1 WO2014005408 A1 WO 2014005408A1 CN 2012087016 W CN2012087016 W CN 2012087016W WO 2014005408 A1 WO2014005408 A1 WO 2014005408A1
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- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 229940041181 antineoplastic drug Drugs 0.000 title claims abstract description 16
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- 238000000855 fermentation Methods 0.000 claims abstract description 29
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- 150000001875 compounds Chemical class 0.000 claims abstract description 17
- 241001387776 Marinactinospora thermotolerans Species 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
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- 239000003560 cancer drug Substances 0.000 claims description 6
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- 239000011780 sodium chloride Substances 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 241001446247 uncultured actinomycete Species 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 4
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- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
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- 108010080698 Peptones Proteins 0.000 claims description 3
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- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- -1 lactam compound Chemical class 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
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- 201000005202 lung cancer Diseases 0.000 claims description 3
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 208000032612 Glial tumor Diseases 0.000 description 3
- 206010018338 Glioma Diseases 0.000 description 3
- 206010023774 Large cell lung cancer Diseases 0.000 description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 3
- 229960004316 cisplatin Drugs 0.000 description 3
- 201000009546 lung large cell carcinoma Diseases 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical group [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
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- 241000456624 Actinobacteria bacterium Species 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
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- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical group C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/426—1,3-Thiazoles
-
- 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
- C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
- C07D513/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains four or more hetero rings
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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
- C12N1/205—Bacterial isolates
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- 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
- C12P17/185—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system
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- 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
- C12R2001/04—Actinomyces
Definitions
- the invention belongs to the field of industrial microorganisms, and specifically relates to a novel antibiotic Tetrathiazomycin A and a preparation method thereof and application thereof in preparing an antitumor drug. Background technique:
- Malignant tumors are one of the major diseases that seriously endanger human life and quality of life. They have become the main cause of death among Chinese residents, accounting for more than 20% of deaths. Since the second half of the 20th century, the world's malignant tumors and deaths have shown an upward trend, especially after the 1970s, malignant tumors increased at an average annual rate of 3% to 5%. The World Health Organization predicts that by 2020, there will be 20 million new cases of malignant tumors, including 12 million deaths, and the vast majority will occur in developing countries. In the three major therapies for malignant tumors, drug treatment plays an important role. Most of the synthetic chemical drugs are based on natural antitumor active ingredients. According to reports, from 1984 to 1995, natural products accounted for more than 60% of listed anti-tumor drugs.
- a first object of the present invention is to provide a novel tetrathiazyl-containing lactam compound Tetrathiazomycin A or a salt thereof having antitumor activity.
- the tetrathiazole-containing lactam compound Tetrathiazomycin A or a salt thereof of the present invention has a structure represented by the formula (I):
- a second object of the present invention is to provide a process for the preparation of the compound Tetrathiazomycin A, characterized in that the compound Tetrathiazomycin A is isolated and prepared from a fermentation culture of actinomycete Marinactinospora thermotolerans SCSIO 00652.
- Tetrathiazomycin A is isolated from the fermentation culture of actinomycete Marinactinospora thermotolerans SCSIO 00652 by the following method, the specific steps are as follows:
- Extract A is subjected to normal phase silica gel column chromatography and eluted with a gradient of 100: 0-1:1 with chloroform/methanol as eluent.
- the volume ratio of chloroform/methanol is 92: 8 ⁇ 1: 1
- the fermentation culture of Man'wact ww/wra thermotolerans SCSIO 00652 was prepared by adding the activated Marinactinospora thermotolerans SCSIO 00652 to the seed medium at 28 ° C, 200 rpm.
- the seed solution was prepared in 4 days, and the seed liquid was connected to the fermentation medium at a 10% inoculation amount, and cultured at 28 ° C, 200 rpm for 7 days, to prepare a fermentation culture, the seed culture medium and the fermentation culture.
- the formula of the base is: per liter of medium: starch 10 g, (NH 4 ) 2 S0 4 2g, K 2 HP0 4 lg, MgS0 4 -7H 2 0 lg, NaCl lg, peptone lg, yeast extract 0.5g , Trace element solution 0.1 mL, 30 g of crude sea salt, the balance is water, pH 7.4.
- a third object of the invention is to provide the use of Man'wact ww/wra thermotolerans SCSIO 00652 in the preparation of the compound Tetrathiazomycin A.
- the present inventors have found through experiments that the Tetrathiazomycin A of the present invention is a glioma cell line (SF-268), a breast cancer cell line (MCF-7), a human large cell lung cancer cell line (NCI-H460) and a human liver cancer cell (HepG). -2) having good cytotoxic activity, which IC 5Q respectively 38 ⁇ 10- 2 ⁇ , 43 10 " 2 ⁇ 47 ⁇ 10- 2 ⁇ and 52 ⁇ 10- 2 ⁇ , may be applied to the preparation of anti-tumor drugs.
- a fourth object of the present invention is to provide a use of Tetrathiazomycin A for the preparation of an antitumor drug.
- the antitumor drug is preferably an anti-glioma drug, an anti-breast cancer drug, an anti-lung cancer drug or an anti-liver cancer drug.
- the present invention isolates a new anti-tumor compound Tetrathiazomycin A from a fermentation culture of Actinobacterium Mar ⁇ act ww/wra thermotolerans SCSIO 00652, which opens up a new way for the preparation of the compound Tetrathiazomycin A, and the present invention Compound Tetrathiazomycin
- A has good anti-tumor activity and can be used in the preparation of anti-tumor drugs, providing a lead compound for the development of anti-tumor drugs.
- the actinomycete Man'wact ww/wra thermotolerans SCSIO 00652 of the present invention is a known strain, and is disclosed in the non-patent literature: the establishment of the genetic operating system of the Marinactinospora thermotolerans SCSIO 00652. Li Jun, Zhu Qinghua, Zhang Yun, Ma Junying, Tian Xinpeng, Li Wenjun, Zhang Changsheng, Qi Jianhua. Chinese Antibiotic Journal. 2012 (2) 105 ⁇ 111. The Applicant warrants that the Marinactinospora thermotolerans SCSIO 00652 strain will be available to the public within 20 years from the date of filing. BRIEF DESCRIPTION OF THE DRAWINGS:
- Figure 1 is an electron microscopy image of Marinactinospora thermotolerans. SCSIO 00652, wherein Figures 1A and 1B are SEM scans at different multiples;
- Figure 2 is an X-single diffraction pattern of Tetrathiazomycin A. detailed description:
- Medium A seed medium: per liter of medium contains: starch 10 g, (NH 4 ) 2 S0 4 2g, K 2 HP0 4 lg, MgS0 4 '7H 2 0 lg, NaCl lg, peptone lg, yeast extract 0.5 g, trace element solution 0.1 mL, crude sea salt 30 g, balance is water, pH 7.4. Sterilized at 121 ° C for 30 min;
- Fermentation medium The formula is the same as the seed medium. Sterilized at 121 ° C for 30 min.
- SCSIO 00652 (the SEM scan shown in Figure 1) is connected to 6L seed medium, 28 ° C, 200 rpm, cultured for 4 d to prepare seed solution, 6L seeds The solution was transferred to a 60 L fermentation medium, and cultured at 28 ° C, 200 rpm, and shaken for 7 days to prepare a fermentation culture.
- the fermentation culture was first centrifuged (4000 rmin- 1 , lOmin), the fermentation broth and the mycelium were separated, the fermentation broth was extracted with an equal volume of ethyl acetate, and the ethyl acetate layer was concentrated by distillation to obtain the extract A. (20.1g
- the extract A was subjected to normal phase silica gel column chromatography (silica gel 100 to 200 mesh), and eluted with a gradient of 100: 0-1:1 with chloroform/methanol as an eluent to collect a chloroform/methanol volume ratio of 92: 8 ⁇ 1: 1 Gradient eluted fraction Fr.5-8, and then subjected to silica gel column chromatography, using ethyl acetate/methanol as eluent, gradient elution from a volume ratio of 94: 6-8:2.
- the compound 1 (Tetrathiazomycin A) prepared from the fermentation culture of Mar ⁇ actww/wra thermotolerans.
- SCSIO 00652 of the present invention was identified by structural analysis, and the results were as follows:
- the inhibitory rate and IC 5Q value of Tetrathiazomycin A against glioma cell line SF-268, breast cancer cell line MCF-7, human large cell lung cancer cell line NCI-H460 and human hepatoma cell line HepG-2 were determined.
- the test method is the internationally accepted SRB method: According to the cell growth rate, the tumor cells in the logarithmic growth phase are inoculated into a 96-well plate at 180 ⁇ 7 well, and adherent for 24 hours for re-dosing (RPMI of different concentrations of Compound 1) 1640 solution) 20 ⁇ 7 wells. Set 3 holes for each concentration. The corresponding RPMI-1640 vehicle control and cell-free withering holes were set. Tumor cells are at 37. C.
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Abstract
本发明提供抗生素Tetrathiazomycin A及其制备方法和在制备抗肿瘤药物中的应用。Tetrathiazomycin A其结构式如式（I）所示。本发明从放线菌属Marinactinospora thermotolerans SCSIO 00652的发酵培养物中分离得到新的具有抗肿瘤活性的化合物Tetrathiazomycin A，为化合物Tetrathiazomycin A的制备开辟了新的途径，并且本发明的化合物Tetrathiazomycin A具有较好的抗肿瘤活性，能应用制备抗肿瘤药物中，为抗肿瘤药物的开发提供了先导化合物。
Description
抗生素 Tetrathiazomycin A及其制备方法和在制备抗肿瘤药物中的应用 技术领域：
本发明属于工业微生物领域， 具体涉及新的抗生素 Tetrathiazomycin A及其 制备方法和在制备抗肿瘤药物中的应用。 背景技术：
恶性肿瘤是目前严重危害人类生命和生活质量的主要疾病之一，现已成为我 国居民的主要死因， 占死亡原因 20 %以上。 20世纪下半叶以来， 世界恶性肿瘤 与死亡均呈上升趋势， 尤其是 70年代以后， 恶性肿瘤以年均 3 %〜5 %的速度递 增。 世界卫生组织预测， 到 2020年， 将有 2000万新发恶性肿瘤病例， 其中死亡 人数达 1200万， 且绝大部分将发生在发展中国家。 在恶性肿瘤的三大疗法中， 药物治疗占用重要的地位。在合成化学类药物中多数都是以天然抗肿瘤活性成分 为先导化合物的。据报道， 1984年到 1995年， 天然产物占上市抗肿瘤药物的 60 %以上， 1995- 1999年， 3 个天然产物衍生物作为新的抗肿瘤药物上市， 由此可 见， 天然产物是结构新颖和作用独特的抗肿瘤药物的重要来源。 目前发现的多肽 类化合物具有广泛的生物学活性，据报道， 含有噻唑环和恶唑环的环肽具有很强 的细胞毒活性， 例如 Urukthapelstatin A对人体肺癌细胞的 IC5Q值惊人的达到了 12nM o 发明内容：
本发明的第一个目的是提供一个新的具有抗肿瘤活性的含四噻唑的内酰胺 化合物 Tetrathiazomycin A或其盐。
本发明的含四噻唑的内酰胺化合物 Tetrathiazomycin A或其盐， 其结构如式 ( I ) 所示：
( D o
本发明的第二个目的是提供化合物 Tetrathiazomycin A的制备方法， 其特征 在于， 所述的化合物 Tetrathiazomycin A 是从放线菌 Marinactinospora thermotolerans SCSIO 00652的发酵培养物中制备分离得到的。
优选通过以下方法从放线菌 Marinactinospora thermotolerans SCSIO 00652 的发酵培养物中制备分离得到 Tetrathiazomycin A， 具体步骤如下：
a)制备放线菌 Man 7aC^0¾90ra thermotolerans SCSIO 00652的发酵培养物， 将该发酵培养物的发酵液和菌丝体分离开，发酵液用乙酸乙酯萃取， 乙酸乙酯相 经浓縮后得到浸膏 A;
b )浸膏 A经正相硅胶柱层析， 以氯仿 /甲醇作为洗脱剂，从体积比 100: 0-1： 1进行梯度洗脱， 收集氯仿 /甲醇体积比 92: 8〜1： 1梯度洗脱下来的馏分 Fr.5-8， 再经硅胶柱层析， 用乙酸乙酯 /甲醇作为洗脱剂， 从体积比 94: 6〜8： 2进行梯度 洗脱，收集体积比 9: 1〜8： 2洗脱的组分 Fr.5-8-1 ,再经 ODS反相中压液相色谱， 以体积分数从 10%〜100%的甲醇 /水梯度洗脱， 收集体积分数 80〜90%的甲醇 /水 梯度洗脱下来的馏分， 最后经高效液相色谱纯化后得到 Tetrathiazomycin A。
所述的 a) 步骤的制备 Man'wact ww/wra thermotolerans SCSIO 00652的发酵 培养物是通过以下方法制备的：将活化的 Marinactinospora thermotolerans SCSIO 00652接入种子培养基中， 28 °C， 200 rpm, 培养 4 d制得种子液， 将种子液按 10%的接种量接到发酵培养基中， 28 °C， 200 rpm, 振荡培养 7d， 而制得发酵培 养物， 所述的种子培养基和发酵培养基的配方都为每升培养基中含有： 淀粉 10 g， (NH4)2S04 2g, K2HP04 lg， MgS04-7H20 lg， NaCl lg， 蛋白胨 lg， 酵母提 取粉 0.5g， trace element solution 0.1 mL, 粗海盐 30 g， 余量为水， pH 7.4。
本发明的第三个目的是提供 Man'wact ww/wra thermotolerans SCSIO 00652 在制备化合物 Tetrathiazomycin A中的应用。
本发明通过实验发现， 本发明的 Tetrathiazomycin A对神经胶质瘤细胞株 ( SF-268 ) , 乳腺癌细胞株 （MCF-7 ) , 人大细胞肺癌细胞株 （NCI-H460 ) 和人 肝癌细胞 （HepG-2 ) 具有较好的细胞毒活性， 其 IC5Q分别为 38χ 10—2μΜ、 43 10"2μΜ 47χ 10—2μΜ和 52χ 10—2μΜ， 可应用于制备抗肿瘤药物。
因此， 本发明的第四个目的是提供 Tetrathiazomycin A在制备抗肿瘤药物中 的应用。
所述的抗肿瘤药物优选为抗神经胶质瘤药物、抗乳腺癌药物、抗肺癌药物或 抗肝癌药物。
本发明从放线菌属 Mar^act ww/wra thermotolerans SCSIO 00652的发酵培 养物中分离得到新的具有抗肿瘤活性的化合物 Tetrathiazomycin A， 为化合物 Tetrathiazomycin A的制备开辟了新的途径，并且本发明的化合物 Tetrathiazomycin
A具有较好的抗肿瘤活性， 能应用制备抗肿瘤药物中， 为抗肿瘤药物的开发提供 了先导化合物。
本发明的放线菌属 Man'wact ww/wra thermotolerans SCSIO 00652是已知菌 种，公开于非专利文献：探侮放线葡 Marinactinospora thermotolerans SCSIO 00652 遗传操作系统的建立. 李军、 朱清华、 张云、 马俊英、 田新朋、 李文均、 张长生、 鞠建华. 中国抗生素杂志. 2012 ( 2 ) 105〜111。 本申请人保证自申请日起 20年 内向公众提供该 Marinactinospora thermotolerans SCSIO 00652菌株。 附图说明：
图 1是 Marinactinospora thermotolerans. SCSIO 00652的电镜扫描图， 其中图 1A 和图 1B是不同倍数下的电镜扫描图；
图 2是 Tetrathiazomycin A的 X-单晶衍射图。 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 :
Tetrathiazomycin A的分离和制备
1、 培养基
A、种子培养基：每升培养基中含有：淀粉 10 g， (NH4)2S04 2g， K2HP04lg， MgS04'7H20 lg, NaCl lg, 蛋白胨 lg， 酵母提取粉 0.5g， trace element solution 0.1 mL, 粗海盐 30g， 余量为水， pH7.4。 121°C， 灭菌 30 min;
B、 发酵培养基： 配方同种子培养基。 121°C， 灭菌 30min。
2、 发酵
将活化的放线菌属 Marinactinospom thermotolerans . SCSIO 00652 (其电镜扫 描图如图 1所示）接入 6L种子培养基中， 28°C， 200 rpm,培养 4 d制得种子液， 将 6L的种子液接入到 60L发酵培养基中， 28 °C， 200 rpm, 振荡培养 7d， 而制 得发酵培养物。
3、 萃取
发酵培养物先进行离心分离（4000 rmin-1, lOmin), 将发酵液和菌丝体分离 开， 发酵液经等体积的乙酸乙酯萃取， 乙酸乙酯层经蒸馏浓縮后得到浸膏 A (20.1g
4、 化合物 TetrathiazomycinA (1) 的提取分离和鉴定
浸膏 A经正相硅胶柱层析（硅胶 100〜200目）， 以氯仿 /甲醇作为洗脱剂， 从 体积比 100: 0-1： 1进行梯度洗脱， 收集氯仿 /甲醇体积比 92: 8〜1： 1梯度洗脱 下来的馏分 Fr.5-8， 再经硅胶柱层析， 用乙酸乙酯 /甲醇作为洗脱剂， 从体积比 94： 6-8： 2进行梯度洗脱， 收集体积比 9: 1-8： 2洗脱的组分 Fr.5-8-l， 再经 ODS反相中压液相色谱（S-50 μηι, 12 nm; 100 20 mm, YMC)，流速为 15 ml/min, 以体积分数从 10%〜100%的甲醇 /水梯度洗脱 60min， 收集体积分数 80〜90%的甲 醇 /水梯度洗脱下来的馏分， 最后经高效液相色谱 （ODS-A, 250 10 mm, 5 μηι; YMC), 流速为 2.5 ml/min， 以体积分数从 70%〜100%的乙腈 /水梯度洗脱 30min， 在保留时间为 22min得到化合物 1 (TetrathiazomycinA) (16mg)。
通过结构分析，对本发明的从 Mar^actww/wra thermotolerans. SCSIO 00652 的发酵培养物中制备得到的化合物 1 (TetrathiazomycinA) 进行鉴定， 其鉴定结 果如下：
白色固体，其核磁数据归属如表 1所示， 700° (c = 0.2, MeOH 1HNMR (500 MHz, CD3OD) 及 13CNMR(125 MHz，CD3OD)， 见表 1。 熔点： 115〜116°C。 HR-ESI-MS m/z 666.1382 ([M+H]+, 理论值 666.1444). 其 X-单晶衍射图如图 2
所示。
表 1 : 化合物 1 (Tetrathiazomycin A) 的核磁数据 H-NMR数据于 500MHz测定， 括号 中为偶合常数 (Hz)， 13C-NMR数据于 125MHz测定， 溶剂为氘代甲醇） position ， multi. (</ in Hz) HMBC lU-lU COSY
1 171.3, qC
2 6.99, d (8.1) C-l H-3
3 5.50, m 47.1, CH C-5 H-2, 4
4 1.61, d (7.1) 23.0, CH3 C-3, 5 H-3
5 174.3, qC
7 7.74, s 118.2, CH C-5, 8, 10
8 147.2, qC
10 161.1, qC
12 7.72, s 118.3, CH C-10, C-l 3, C-15
13 148.1, qC
15 161.0, qC
17 7.66, s 122.3, CH C-l 5, C-l 8, C-20
18 148.9, qC
20 161.7, qC
22 3.70, d (10.8) 36.3, CH2 C-20, 23, 25 H-23
23 4.81, t (10.5) 78.8, CH C-20, 25 H-22
25 170.3, qC
26 8.19, d (10.2) C-25 H-27
27 5.12, td (3.8, 10.8) 56.4, CH C-25, 28 H-26, 28
28 2.98 , dd (11.4, 13.5); 40.3, CH2 C-27, 29, 30, 34 H-27
3.53, dd (3.8, 13.6)
29 137.1, qC
30/34 7.34, d (7.4) 129.4, CH C-28, 32
31/33 7.27, t (7.4) 128.5, CH C-29, 33
32 7.20, t (7.4) 126.9, CH C-30, 34
35 172.0, qC
36 8.48, d (9.6) C-35 H-37
37 4.70, dd (3.7, 9.6) 58.0, CH C-1, 35, 38, 39, 40 H-36, 38
38 2.12, m 36.6, CH C-41 H-37, 39, 40
39 0.79, dd (7.1, 15.5) 16.0, CH3 C-37, 38, 40 H-38
40 1.02, m; 1.34, m 24.8, CH2 H-38, 41
41 0.49, t (7.4) 11.8, CH3 C-38, 40 H-40 综上所述， 鉴定化合物 1的结构式如式（ I )所示， 命名为 Tetrathiazomycin
(1)。
实施例 2: TetrathiazomycinA的抗肿瘤活性测定
测试 TetrathiazomycinA对神经胶质瘤细胞株 SF-268,乳腺癌细胞株 MCF-7, 人大细胞肺癌细胞株 NCI-H460和人肝癌细胞 HepG-2的抑制率和 IC5Q值测定。
采用国际通用的肿瘤细胞株， 即： 神经胶质瘤细胞株 （SF-268), 乳腺癌细 胞株 （MCF-7), 人大细胞肺癌细胞株 （NCI-H460) 和人肝癌细胞 （HepG-2)。 试验方法为国际通用的 SRB法： 根据细胞生长速度， 将处于对数生长期的肿瘤 细胞以 180 μΙ7孔接种于 96孔板， 贴壁生长 24小时再加药 （不同浓度的化合物 1的 RPMI-1640溶液） 20 μΙ7孔。每个浓度设 3复孔。 并设相应的 RPMI-1640溶 媒对照及无细胞凋零孔。 肿瘤细胞在 37 。C、 5%的 C02条件下培养 72小时， 倾 去培养液， 每孔加入 50 μΐ,的 50% 冷 TCA固体细胞， 然后采用 0.4%的 SRB染 色 30分钟， 用 1%的乙酸洗涤 5次， 空气干燥。 最后加入 200μΙ7孔的 Tris溶液， 酶标仪 570nm波长测定 OD值。 以顺铂作为阳性对照。 实验结果见表 2:
表 2: TetrathiazomycinA对肿瘤细胞株的抑制作用 （IC5。， μΜ)
SF-268 MCF-7 NCI-H460 HepG-2
TetrathiazomycinA 38x10— 2 43x10— 2 47x10— 2 52x10— 2
顺铂 Cisplatin" 20x10— 3 47x10— 4 27 10^ 14x10— 3
Positive control
Claims
1、一个含四噻唑的内酰胺化合物 Tetrathiazomycin A或其盐，其结构式如（I) 所示：
( I)
2、一种权利要求 1所述的化合物 Tetrathiazomycin A的制备方法， 其特征在 于，所述的化合物 Tetrathiazomycin A是从方夂线菌 Marinactinospora thermotolerans SCSIO 00652的发酵培养物中制备分离得到的。
3、如权利要求 2所述的化合物 Tetrathiazomycin A的制备方法，其特征在于， 具体步骤如下：
a)制备放线菌 Marinactinospom thermotolerans SCSIO 00652的发酵培养物， 将该发酵培养物的发酵液和菌丝体分离开，发酵液用乙酸乙酯萃取， 乙酸乙酯相 经浓縮后得到浸膏 A;
b )浸膏 A经正相硅胶柱层析， 以氯仿 /甲醇作为洗脱剂，从体积比 100: 0-1： 1进行梯度洗脱， 收集氯仿 /甲醇体积比 92: 8-1： 1梯度洗脱下来的馏分 Fr.5-8， 再经硅胶柱层析， 用乙酸乙酯 /甲醇作为洗脱剂， 从体积比 94: 6-8： 2进行梯度 洗脱，收集体积比 9: 1-8： 2洗脱的组分 Fr.5-8-l，再经 ODS反相中压液相色谱， 以体积分数从 10%~100%的甲醇 /水梯度洗脱， 收集体积分数 80~90%的甲醇 /水 梯度洗脱下来的馏分， 最后经高效液相色谱纯化后得到 Tetrathiazomycin A。
4、 如权利要求 3所述的制备方法， 其特征在于， 所述的 a) 步骤的放线菌
Marinactinospora thermotolerans SCSIO 00652的发酵培养物是通过下述方法制备 的：
将活化的 Marinactinospora thermotolerans SCSIO 00652接入种子培养基中， 28 °C， 200 rpm, 培养 4 d制得种子液， 将种子液按 10%的接种量接到发酵培养 基中， 28 °C， 200 rpm, 振荡培养 7d， 而制得发酵培养物， 所述的种子培养基和
发酵培养基的配方都为每升培养基中含有淀粉 10 g， (NH4)2S042g, K2HP04 lg， MgS04'7H20 lg, NaCl lg, 蛋白胨 lg， 酵母提取粉 0.5g， trace element solution 0.1 mL, 粗海盐 30 g， 余量为水， pH 7.4。
5、权利要求 1所述的化合物 Tetrathiazomycin A或其盐在制备抗肿瘤药物中 的应用。
6、 如权利要求 5所述的应用， 其特征在于， 所述的抗肿瘤药物为抗神经胶 质瘤药物、 抗乳腺癌药物、 抗肺癌药物或抗肝癌药物。
7、 Marinactinospora thermotolerans SCSIO 00652在制备权利要求 1所述的 化合物 Tetrathiazomycin A中的应用。
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| CHUNG-MAO PAN ET AL.: "Progress toward the synthesis of Urukthapelstatin A and two analogues", TETRAHEDRON LETTERS, vol. 53, 29 May 2012 (2012-05-29), pages 4065 - 4069, XP028400870, DOI: doi:10.1016/j.tetlet.2012.05.105 * |
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