WO2022057223A1 - 苯并噻唑杂萜类化合物及其衍生物以及制备方法与应用 - Google Patents

苯并噻唑杂萜类化合物及其衍生物以及制备方法与应用 Download PDF

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WO2022057223A1
WO2022057223A1 PCT/CN2021/081434 CN2021081434W WO2022057223A1 WO 2022057223 A1 WO2022057223 A1 WO 2022057223A1 CN 2021081434 W CN2021081434 W CN 2021081434W WO 2022057223 A1 WO2022057223 A1 WO 2022057223A1
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compound
preparation
benzothiazole
crude
extract
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杨献文
谢春兰
鄢庆祥
邹正彪
何志辉
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自然资源部第三海洋研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles 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 in position 2
    • 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
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C—CHEMISTRY; METALLURGY
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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
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/14—Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

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  • the invention relates to the technical field of pharmaceutical compounds, in particular to a benzothiazole heteroterpenoid compound and a derivative thereof, as well as a preparation method and application thereof.
  • Nuclear receptors are a class of ligand-dependent transcriptional regulators that are distributed in the cytoplasm and nucleus of cells and play an important role in maintaining the body's homeostasis.
  • Retinol X receptor (RXR) is an important member of the nuclear receptor family, and is considered to be the most promising core member. It regulates the proliferation of various cancer cells such as lung cancer, breast cancer, liver cancer and prostate cancer. and play an important role in apoptosis.
  • RXR ⁇ contains an N-terminal region, a DNA binding domain and a ligand binding domain (LBD).
  • RXR ⁇ -LBD has a ligand-binding pocket (LBP) for the binding of small molecules to ligands, which can recognize specific hormonal and non-hormonal ligands.
  • LBP ligand-binding pocket
  • RXR ligands can regulate RXR-related signaling pathways through activation or antagonism, and all of them can play a role in metabolic diseases or cancer to varying degrees. Therefore, the discovery of RXR ⁇ ligands is a research hotspot.
  • RXR ⁇ -LBD ligands such as all-trans retinoic acid, 9-cis sulfonic acid, targeting redine, CD3254, K8003, K8008: hydrophobic group, polar region, and central polymer The ethylenic linker structure.
  • the object of the present invention is to provide a kind of benzothiazole heteroterpenoids and derivatives thereof and preparation method and reference, this benzothiazole heteroterpenoids and derivatives thereof are isolated from the fermentation product of Penicillium allii-sativi, which It has significant anti-tumor activity and can be used for the preparation and research and development of anti-cancer drugs.
  • a first aspect of the present invention provides a benzothiazole heteroterpenoid and its derivatives, which are compounds represented by formulas I to III or salts thereof:
  • a second aspect of the present invention provides a method for preparing the above-mentioned benzothiazole heteroterpenoids and derivatives thereof, comprising the following steps:
  • the Penicillium allii-sativi is fermented and cultured to obtain a fermented product; the Penicillium allii-sativi is preserved in the China Marine Microorganism Collection of China (Marine Culture Collection of China, MCCC), and the deposit number is MCCC 3A00580 ;
  • step S2 Extract the fermented product obtained in step S1, and the obtained extract is separated and purified to obtain the benzothiazole heteroterpenoid compound and its derivatives.
  • step S2 includes:
  • step S21 extract the fermented product obtained in step S1 and extract the fermented product with ethyl acetate, take the organic extract for chromatography, and use petroleum ether, dichloromethane and methanol to elute respectively; concentrate the dichloromethane layer to obtain a crude extract thing;
  • step S22 separating the crude extract obtained in step S21 by normal phase silica gel column chromatography, and performing gradient elution with petroleum ether-ethyl acetate system to obtain 8 crude fractions in sequence: Fr.1 ⁇ Fr.8;
  • step S23 The crude fraction Fr.5 obtained in step S22 is firstly separated by ODS column chromatography, and then gradient elution is performed with a water-methanol system to obtain 11 crude fractions in sequence: Fr.5.1 ⁇ Fr.5.11;
  • step S24 separate the crude fraction Fr.5.9 obtained in step S23 with a Sephadex column and a semi-preparative liquid chromatography column to obtain the compound of formula I;
  • step S25 firstly separate the crude fraction Fr.6 obtained in step S22 by Sephadex column chromatography to obtain 3-stage crude fractions: Fr.6.1 ⁇ Fr.6.3;
  • step S26 the crude fraction Fr.6.2 obtained in step S25 is separated by ODS column chromatography and semi-preparative liquid chromatography to obtain the compound of formula II and compound of formula III.
  • the normal phase silica gel column chromatography elution solvent used in step S22 is petroleum ether-ethyl acetate system, and the ratios are 100:1, 50:1, 30:1, 10:1, 5:1, 3:1, 1:0.
  • the mobile phase used in the ODS column in step S23 is methanol-water, gradient elution (40% ⁇ 100%, 15 ⁇ 310 mm, 20 ml/min).
  • step S24 the mobile phase used in the Sephadex column is methanol, and the mobile phase used in the semi-preparative liquid chromatography column is acetonitrile-water, gradient elution (ACN-H 2 O, 60% ⁇ 100%, 10 ⁇ 250mm, 5ml/min).
  • the mobile phase used in the Sephadex column in step S25 is methanol.
  • the mobile phase used in the ODS column is methanol-water, gradient elution (40% ⁇ 100%, 15 ⁇ 310 mm, 20ml/min); the mobile phase used in the semi-preparative liquid chromatography column is acetonitrile-water, gradient Elution (ACN- H2O , 40% ⁇ 100%, 10 x 250 mm, 5 ml/min).
  • the fermentation culture conditions of the Penicillium allii-sativi include: connecting the mycelium to a culture solution containing PDB, and culturing to obtain a seed solution; inoculating the seed solution into a fermentation medium , static culture at 28° C. for 30 days; the fermentation medium includes 80 g of oats and 120 ml of seawater with a salinity of 3%.
  • step S1 the mycelium is prepared by the following steps: culturing Penicillium allii-sativi on a PDA plate at 28° C. for 3-4 days to obtain the mycelium.
  • the third aspect of the present invention provides the use of the above-mentioned benzothiazole heteroterpenoids and derivatives thereof and derivatives thereof or salts thereof in the preparation of the following products: 1) tumor cell proliferation inhibitor; 2) prevention and/or Drugs for the treatment of tumor diseases.
  • the tumor cells include, but are not limited to, cervical cancer cells, liver cancer cells, breast cancer cells, and prostate cancer cells.
  • the tumor diseases include, but are not limited to, cervical cancer, liver cancer, breast cancer and prostate cancer.
  • the present invention has the following advantages:
  • the present invention provides three new compounds meroterpenthiazole A (formula I), 4-(5-hydroxy-7-methylbenzo[d]thiazol-4-yl)-2-methylbutanoic acid (formula II), 4-(5- hydroxy-4-methylbenzo[d]thiazol-7-yl)-2-methylbutanoic acid (formula III), which is isolated from the fermentation broth of Penicillium allii-sativi, wherein the compound meroterpenthiazole A is composed of sesquiterpene and benzo
  • the new skeleton compound composed of thiazole ring is a kind of secondary metabolites containing sulfur containing heteroterpenes with novel structure, and the structure containing thiazole ring is very rare in nature.
  • the present invention discovers for the first time a new skeleton compound composed of a sesquiterpene and a benzothiazole ring, which is of great significance for the discovery and research of new RXR ⁇ targets; the method for separating compounds I to III from fermentation broth of the present invention is environmentally friendly, It has the advantages of simple steps and high product purity; the present invention proves that the compounds I to III are combined with the anti-tumor target RXR ⁇ to inhibit the transcription thereof through the RXR ⁇ double reporter gene experiment, the surface ion resonance technology, the molecular docking technology and the cytotoxic activity experiment. activity to achieve anti-cancer effect. Therefore, the three novel compounds provided by the present invention have good application prospects in the preparation of anticancer drugs.
  • Figure 1 shows the results of the transcriptional activity of compounds I to III of the present invention on RXR ⁇ .
  • Figure 2 shows the binding results of Compound I of the present invention and RXR ⁇ -LBD.
  • Penicillium allii-sativi preserved in China Marine Microbial Culture Collection and Management Center, preservation number MCCC 3A00580 was cultured on a PDA plate at 25° C. for 3 days; then fresh mycelium was connected to a PDB containing 400ml of PDB After 24h, inoculate 10ml of seed liquid into 1L Erlenmeyer flasks (100 flasks), each flask contains 80g of oats and 120ml of seawater with a salinity of 3%, and statically culture at 28°C for 30 days;
  • step (2) The fermentation product obtained in step (1) was extracted three times with ethyl acetate, and the organic solvent was evaporated under reduced pressure to obtain organic extracts (200g); these extracts were passed through a normal phase column chromatography column, and petroleum ether was used respectively. , eluted with dichloromethane and methanol; the dichloromethane layer was concentrated to obtain a crude extract (63.0 g);
  • step (3) The crude extract obtained in step (2) is separated by normal phase silica gel column chromatography, and gradient elution is performed with petroleum ether-ethyl acetate system to obtain 8 crude fractions (Fr.1 ⁇ Fr.8);
  • the compound of formula I is a white powder. According to its main ion peak in high-resolution mass spectrometry, its molecular formula is C 26 H 34 N 2 O 4 S. 1 H, 13 C NMR data (Table 1) and DEPT and HMBC spectra showed 26 carbon signals, including 4 methyl, 8 methylene, 3 methine and 11 quaternary carbons, after detailed two-dimensional The data confirmed the planar structure of the compound, and finally determined the relative and absolute configuration of the compound of formula I by NOE spectrum and ECD and 13 C NMR calculations, and named meroterpenthiazole A.
  • the compound of formula II has the formula C13H15NO3S,1H,13C NMR data (Table 1) show 13 carbons including 2 methyl, 2 methylene, 3 methine and 6 quarter carbon. These signals are similar to some of the data for compounds of formula 1 . Through detailed 1D and 2D NMR analysis, the compound of formula II was determined to be: 4-(5-hydroxy-7-methylbenzo[d]isothiazol-4-yl)-2-methylbutanoic acid.
  • the compound of formula III has the formula C13H15NO3S . Its1H , 13C NMR data, with the exception of C-4, C-7 and C-12, were very similar to formula II (Table 1). Through detailed spectral analysis, its structure was determined to be: 4-(5-hydroxy-4-methylbenzo[d]thiazol-7-yl)-2-methylbutanoic acid.
  • Example 2 Detection of RXR ⁇ dual reporter gene activity: to detect whether the compound can affect the transcriptional activity of RXR ⁇ , and to preliminarily explore whether the compound may have the effect of binding to RXR ⁇ .
  • This example adopts the dual-luciferase reporter gene (Dual-luciferase reporter, DLR) detection composed of firefly luciferase (Firefly luciferase, FL) reporter gene and Renilla luciferase (Rellina luciferase, RL) reporter gene to detect system.
  • the RL reporter gene served as an internal control to normalize the FL reporter gene measurements.
  • the receptor RXR ⁇ and a reporter gene containing an RXR ⁇ response element were introduced by transfection, thereby simply mimicking the transcriptional activation process of the receptor in vivo.
  • Negative control group equal volume of culture medium, 0.1% DMSO, containing cells, without adding compounds of formulas I to III
  • Positive control group equal volume of culture medium, RXR ⁇ agonist 9-cis-retinoic acid and antagonist UVI3003 were added respectively;
  • Human embryonic kidney cells (293T) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in a 37°C incubator containing 5% CO 2 . Cells were in logarithmic growth phase.
  • the sample to be tested, 9-cis-retinoic acid (9-cis-RA), and UVI3003 were prepared with DMSO to prepare a stock solution, and the culture medium containing 10% FBS was diluted to the required concentration before use.
  • Human embryonic kidney cells (293T) were seeded at 1 ⁇ 10 4 /well in a 96-well culture plate, and cultured at 37°C for 24 hours; the cells with 80-90% confluency were replaced, and the target plasmid was transfected into the cells with liposomes; Drugs (5 ⁇ M, 10 ⁇ M, 20 ⁇ M) were added 24 h after the cells were transfected, and the drugs continued to act for 12 h; then, the cells were washed with PBS, and 40 ⁇ L of cell lysis buffer PLB (passive lysis buffer) was added.
  • PLB passive lysis buffer
  • Example 3 Biacore experiment: evaluating whether compounds I to III directly bind to RXR ⁇ -LBD protein.
  • the purified nuclear receptor RXR ⁇ -LBD protein was first coupled to Biacore's CM5 chip. Then the compounds to be tested were diluted with PBS to prepare solutions of different concentrations and then injected. When the sample to be tested flows over the chip surface, the binding between biomolecules causes an increase in the surface mass of the biosensor, resulting in a change in the refractive index. By monitoring the angle change of the SPR, the kinetic binding and dissociation constants of the analyte can be automatically obtained, affinity and specificity.
  • the Biacore T200 detector can detect the interaction between the target protein and the sample to be tested in real time, and the binding strength is expressed in units of RU (response units, RU) (the change in the concentration of the binding substance on the chip surface 1pg/mm2 is defined as 1RU).
  • RU response units
  • Compound I can bind to RXR ⁇ protein, and its binding mode is rapid binding and rapid dissociation, and its binding constant is 12.3 ⁇ M.
  • Example 1 four tumor cell lines were selected: cervical cancer cells (Hela), liver cancer cells (HepG2), breast cancer cells (MDA-MB231) and prostate cancer cells (LNCap).
  • the cytotoxicity of the compounds I to III prepared in Example 1 was detected by detecting the inhibition rate of the compound samples on these tumor cells.
  • Negative control group equal volume of culture medium, 0.1% DMSO, containing cells, without adding compounds I to III;
  • Blank control group equal volume of culture medium, no cells, and no addition of compounds I to III;
  • the cells were resuspended in the medium and pipetted into a single cell suspension, and then seeded into a 96-well plate with 2000-5000 cells per well, with a volume of 200 ⁇ l per well;
  • the microplate reader measures the light absorption value at 570nm, and calculates the inhibition rate.
  • Results Compounds I-III had no obvious cytotoxicity (IC 50 >50 ⁇ M) against cervical cancer cells (Hela), liver cancer cells (HepG2), breast cancer cells (MDA-MB231) and prostate cancer cells (LNCap).

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Abstract

一种深海真菌来源的苯并噻唑杂萜类化合物及其衍生物、制备方法以及制备抗肿瘤药物的应用，其为式I～式III所示的化合物或其盐。该化合物分离自深海青霉菌Penicillium allii-sativi的发酵产物，其通过与抗肿瘤靶点RXRα结合来抑制RXRα的转录活性从而达到显著的抗肿瘤效果，可用于抗癌药物的制备和研发，具有良好的应用前景。

Description

苯并噻唑杂萜类化合物及其衍生物以及制备方法与应用 技术领域
本发明涉及药物化合物技术领域，具体涉及一种苯并噻唑杂萜类化合物及其衍生物以及制备方法与应用。
背景技术
肿瘤是当今严重威胁人类健康的疾病之一。核受体是一类配体依赖性的转录调节因子，分布于细胞中胞浆及细胞核内，在维持机体的稳态中发挥重要作用。视黄醇X受体(RXR)是核受体家族中重要的一份子，则被认为是最具有开发前景的核心成员，在调控肺癌、乳腺癌、肝癌和前列腺癌等多种癌细胞的增殖和凋亡中发挥重要作用。RXRα包含一个N-末端区域、一个DNA结合域和一个配体结合域(LBD)。RXRα-LBD具有一个配体结合袋(LBP)用于小分子与配体的结合，它能够识别特定的激素和非激素配体。RXR配体能通过激活或拮抗的方式调节RXR相关信号通路，且都能在不同程度上起到对代谢性疾病或癌症的作用，因此RXRα配体的发现是研究热点。在先前报道的RXRα-LBD配体(如全反式维甲酸、9-顺式磺酸、靶向雷丁、CD3254、K8003、K8008)中有三个基本区域：疏水基、极性区和中心聚烯键连接体结构。
虽然目前已经报道了许多RXRα的天然配体，但它们的选择性较差，毒性较高。海洋微生物由于生存在特殊的海洋环境中能够产生结构新颖的次级代谢产物，且来源于海洋的这些次级代谢产物生物碱类、萜类、黄酮类化合物等可表现出强的抗肿瘤活性。因此，从天然产物中发现高效低毒的RXRα小分子调节剂是开发基于RXRα靶点的抗肿瘤药物的有效策略。
发明内容
本发明的目的在于提供一种苯并噻唑杂萜类化合物及其衍生物以及制备方法和引用，该苯并噻唑杂萜类化合物及其衍生物分离自青霉菌Penicillium allii-sativi的发酵产物，其具有显著的抗肿瘤活性，可用于抗癌药物的制备和研发。
为此，本发明的第一方面提供一种苯并噻唑杂萜类化合物及其衍生物，其为式I～III所示的化合物或其盐：
Figure PCTCN2021081434-appb-000001
本发明的第二方面，提供上述苯并噻唑杂萜类化合物及其衍生物的制备方法，包括以下步骤：
S1、将青霉菌Penicillium allii-sativi进行发酵培养，得到发酵物；所述青霉菌Penicillium allii-sativi保藏于中国海洋微生物菌种保藏管理中心(Marine Culture Collection of China，MCCC)，保藏编号为MCCC 3A00580；
S2、将步骤S1得到的发酵物进行萃取，所得萃取物经分离纯化，得到所述苯并噻唑杂萜类化合物及其衍生物。
优选的，步骤S2包括：
S21、将步骤S1得到的发酵物进行萃取发酵物用乙酸乙酯萃取，取有机萃取物进行层析，分别使用石油醚，二氯甲烷和甲醇进行洗脱；浓缩二氯甲烷层，得到粗提物；
S22、将步骤S21得到的粗提物用正相硅胶柱色谱进行分离，用石油醚-乙酸乙酯体系进行梯度洗脱，依次得到8个粗馏分：Fr.1～Fr.8；
S23、将步骤S22得到的粗馏分Fr.5先使用ODS柱色谱进行分离，使用水-甲醇体系进行梯度洗脱依次得到11段粗馏分：Fr.5.1～Fr.5.11；
S24、将步骤S23得到的粗馏分Fr.5.9用葡聚糖凝胶柱和半制备液相色谱柱分离后得到式I化合物；
S25、将步骤S22得到的粗馏分Fr.6先使用葡聚糖凝胶柱色谱进行分离，得到3段粗馏分：Fr.6.1～Fr.6.3；
S26、将步骤S25得到的粗馏分Fr.6.2用ODS柱色谱和和半制备液相色谱柱分离后得到式II化合物和式III化合物。
进一步的，步骤S22中所用正相硅胶柱色谱洗脱溶剂为石油醚-乙酸乙酯体系， 比例为100:1，50:1，30:1，10:1，5:1，3:1，1:0。
进一步的，步骤S23中ODS柱所用流动相为甲醇-水，梯度洗脱(40％→100％，15×310mm，20ml/min)。
进一步的，步骤S24中葡聚糖凝胶柱所用流动相为甲醇，半制备液相色谱柱所用流动相为乙腈-水，梯度洗脱(ACN-H 2O，60％→100％，10×250mm，5ml/min)。
进一步的，步骤S25中葡聚糖凝胶柱所用流动相为甲醇。
进一步的，步骤S26中ODS柱所用流动相为甲醇-水，梯度洗脱(40％→100％，15×310mm，20ml/min)；半制备液相色谱柱所用流动相为乙腈-水，梯度洗脱(ACN-H 2O，40％→100％，10×250mm，5ml/min)。
优选的，步骤S1中，所述青霉菌Penicillium allii-sativi的发酵培养条件包括：将菌丝体接到含有PDB的培养液中，经培养获得种子液；将所述种子液接种到发酵培养基中，于28℃静态培养30天；所述发酵培养基包括燕麦80g和盐度3％的海水120ml。
进一步，步骤S1中，所述菌丝体通过以下步骤制备得到：将青霉菌Penicillium allii-sativi在PDA平板上于28℃培养3～4天，获得所述菌丝体。
本发明的第三方面，提供上述苯并噻唑杂萜类化合物及其衍生物化合物及其衍生物或其盐在制备以下产品中的应用：1)肿瘤细胞增殖抑制剂；2)预防和/或治疗肿瘤疾病的药物。
优选的，所述肿瘤细胞包括但不限于宫颈癌细胞、肝癌细胞乳腺癌细胞、前列腺癌细胞。
优选的，所述肿瘤疾病包括包括但不限于宫颈癌、肝癌、乳腺癌和前列腺癌。
本发明的有益效果：
与现有技术相比，本发明具有以下优点：
本发明提供了三个新的化合物meroterpenthiazole A(式I)，4-(5-hydroxy-7-methylbenzo[d]thiazol-4-yl)-2-methylbutanoic acid(式II)，4-(5-hydroxy-4-methylbenzo[d]thiazol-7-yl)-2-methylbutanoic acid(式III)，其分离自深海青霉菌Penicillium allii-sativi的发酵液，其中化合物meroterpenthiazole A是由倍半萜与苯并噻唑环组成的新骨架化合物，是一类结构新颖的含硫杂萜次生代谢产物，而在自然界中，含噻唑环的结构是非常罕见的。本发明首次发现了倍半萜与苯并噻唑环组成的新骨架化合物，这对于发现和研究新的RXRα靶点具有重要意义；本发明从 发酵液中分离化合物I～III的方法，具有环保、步骤简单、产品纯度高等优点；本发明通过RXRα双报告基因实验、表面离子共振技术、分子对接技术以及细胞毒活性实验证明了该化合物I～III是通过与抗肿瘤靶点RXRα结合来抑制其转录活性来达到抗癌效果。因此，本发明提供的三个新化合物在制备抗癌药物方面具有良好的应用前景。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。
图1为本发明的化合物I～III对RXRα的转录活性结果。
图2为本发明的化合物I与RXRα-LBD的结合结果。
实施例1.苯并噻唑杂萜类化合物的制备。
(1)将青霉菌Penicillium allii-sativi(保藏于中国海洋微生物菌种保藏管理中心，保藏编号MCCC 3A00580)在PDA平板上于25℃培养3天；然后将新鲜的菌丝体接到含有400ml PDB的培养液中；24h后，将10ml种子液接种到1L的三角烧瓶中(100瓶)，每瓶中有80g燕麦和120ml盐度3％的海水，在28℃下静态培养30天；
(2)将步骤(1)得到的发酵物用乙酸乙酯萃取三次，减压蒸发掉有机溶剂，得到有机萃取物(200g)；将这些萃取物过正相柱层析柱，分别使用石油醚，二氯甲烷和甲醇进行洗脱；浓缩二氯甲烷层，得到粗提物(63.0g)；
(3)将步骤(2)得到的粗提物使用正相硅胶柱色谱进行分离，用石油醚-乙酸乙酯体系进行梯度洗脱，得到8个粗馏分(Fr.1～Fr.8)；
(4)将步骤(3)中的粗馏分Fr.5(3.5g)先使用ODS柱色谱进行分离，使用水-甲醇体系进行梯度洗脱得到11段粗馏分(Fr.5.1-Fr.5.11)。Fr.5.9(89.1mg)使用葡聚糖凝胶柱(纯甲醇)和半制备液相色谱柱(乙腈-水，60％→100％)分离后得到式I化合物(6.2mg)。Fr.6(7.0g)使用葡聚糖凝胶色谱柱(纯甲醇)，ODS色谱柱(甲醇-水，40％→100％)和半制备液相色谱柱(乙腈-水，40％→100％)分离后得到式II化合物(4.8mg)和式III化合物(3.7mg)。
(5)将上述步骤中得到的式I、式II、式III化合物分别用1D、2D NMR图谱以及高分辨质谱分析确定化合物的平面结构，然后利用ECD计算等确定其绝对构型，详述如下：
式I化合物为白色粉末。根据其在高分辨质谱中的主离子峰确定其分子式为C 26H 34N 2O 4S。 1H、 13C NMR数据(表1)以及DEPT和HMBC图谱显示26个碳信号，包括4个甲基，8个亚甲基，3个次甲基和11个季碳，经过详细的二维数据确定了化合物的平面结构，最后利用NOE图谱和ECD和 13C NMR计算确定了式I化合物的相对及绝对构型，并命名为meroterpenthiazole A。
表1.式I～III化合物的 1H和 13C NMR数据
Figure PCTCN2021081434-appb-000002
a DMSO； b CD 3OD
式II化合物的分子式为C 13H 15NO 3S， 1H、 13C NMR数据(表1)显示有13个碳，包括2个甲基，2个亚甲基，3个次甲基和6个季碳。这些信号与式1化合物中的部分数据相似。通过详细的 1D、2D NMR解析，确定式II化合物为：4-(5-hydroxy-7-methylbenzo[d]isothiazol-4-yl)-2-methylbutanoic acid。
式III化合物的分子式为C 13H 15NO 3S。其 1H、 13C NMR数据，除了C-4、C-7和C-12之外，其他与式II非常相似(表1)。通过详细的波谱解析，确定其结构为：4-(5-hydroxy-4-methylbenzo[d]thiazol-7-yl)-2-methylbutanoic acid。
实施例2.RXRα双报告基因活性检测：检测化合物是否会影响RXRα的转录活性，并初步探讨化合物是否可能存在与RXRα结合的作用。
本实施例采用的是萤火虫荧光素酶(Firefly luciferase,FL)报告基因和海肾荧光素酶(Rellina luciferase,RL)报告基因组成的双萤光素酶报告基因(Dual-luciferase reporter,DLR)检测系统。RL报告基因作为内对照，使FL报告基因的测量结果正态化。在缺少内源性RXRα及其下游信号传导必要组成的细胞中，通过转染的方法引入受体RXRα以及含有RXRα应答元件的报告基因，从而简单模拟体内受体的转录活化过程。
本实施例设置以下3组：
阴性对照组：等量培养液，0.1％DMSO，含细胞，不加入式I～III化合物
阳性对照组：等量培养液，分别加入RXRα激动剂9-顺视黄酸，拮抗剂UVI3003；
实验组：向细胞培养液分别加入式I～III化合物；
具体步骤包括：
(1)细胞的培养和供试品的配制
人胚肾细胞(293T)用含10％胎牛血清(fetal bovine serum，FBS)的DMEM培养基，在含5％CO 2的37℃孵箱中培养，按细胞的生长规律定期传代，实验所用细胞均处于对数生长期。将待测样品、9-顺视黄酸(9-cis-RA)、UVI3003用DMSO配成储存液，临用前用含10％FBS的培养液稀释成所需的浓度。
(2)活性的测定
将人胚肾细胞(293T)以1×10 4/孔接种于96孔培养板，37℃培养24h；80-90％融合度的细胞换液，用脂质体转染目的质粒于细胞中；细胞转染后24h加药(5μM、10μM、20μM)，药物持续作用12h；然后，细胞用PBS洗涤，加入40μL的细胞 裂解液PLB(passive lysis buffer)，在摇床上中速振摇20min后，分出取20μl裂解液转移到96孔遮光板中，每孔加入50μL的荧光素酶检测试剂II(LARII)后立即测量萤火虫荧光素酶的活性，随后加入50μL的Stop&GloTM试剂，以淬灭萤火虫荧光素酶反应，同时激活海肾荧光素酶反应，并立即测量海肾荧光素酶的活性。
结果如图1，当与RXRα的激动剂9-cis-RA联合使用的情况下，化合物I能够抑制9-cis-RA对RXRα的转录作用，并表现出明显的浓度依赖性。
实施例3.Biacore实验：评价化合物I～III与RXRα-LBD蛋白有无直接结合。
先将纯化的核受体RXRα-LBD蛋白与Biacore的CM5芯片偶联。然后将待测化合物用PBS稀释配制成不同浓度的溶液后进样。当待测的样品流过芯片表面时，生物分子间的结合引起生物传感器表面质量的增加，导致折射率的变化，通过监测SPR的角度变化，可自动获得分析物的动力学结合和解离常数、亲和力及特异性等。BiacoreT200检测器能实时检测靶蛋白和待测样品之间的相互作用，以RU(response unites,RU)为单位表示结合强弱(芯片表面结合物质浓度改变1pg/mm2定义为1RU)。我们采用Biacore技术对化合物I～III进行筛选，并通过浓度梯度实验计算出小分子化合物与RXRa-LBD的结合常数K D。结果显示见图2，化合物I能够与RXRα蛋白结合，其结合模式为快速结合快速解离，其结合常数为12.3μM。
实施例4.化合物I～III的细胞毒活性检测。
本实施例选择四株肿瘤细胞：宫颈癌细胞(Hela)、肝癌细胞(HepG2)、乳腺癌细胞(MDA-MB231)和前列腺癌细胞(LNCap)。通过检测化合物样品对这些肿瘤细胞的抑制率，从而检测实施例1制备得到的化合物I～III的细胞毒性。
本实施例设置以下3组：
阴性对照组：等量培养液，0.1％DMSO，含细胞，不加入化合物I～III；
空白对照组：等量培养液，不含细胞，且不加入化合物I～III；
实验组：向细胞培养液分别加入化合物I～III；
具体步骤包括：
(1)细胞经常规消化后，于培养基中重悬并吹打成单细胞悬液，然后以每孔2000～5000个细胞接种到96孔板，每孔体积200μl；
(2)37℃，5％CO 2的培养箱中培养24h，然后分别加入不同浓度的化合物处理 细胞；
(3)继续培养48h后，每孔加10μl的5mg/ml MTT(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide)，37℃避光反应3h；小心吸弃上清液后，每孔加入150μl DMSO，振荡10min，使结晶物充分融解；
(4)酶标仪测定570nm光吸收值，计算抑制率，
结果化合物I～III对宫颈癌细胞(Hela)、肝癌细胞(HepG2)、乳腺癌细胞(MDA-MB231)和前列腺癌细胞(LNCap)均无明显的细胞毒性(IC 50>50μM)。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (8)

  1. 一种苯并噻唑杂萜类化合物及其衍生物，其特征在于，其为式(I～III)所示的化合物或其盐：
    Figure PCTCN2021081434-appb-100001
  2. 如权利要求1所述的苯并噻唑杂萜类化合物及其衍生物的制备方法，其特征在于，包括以下步骤：
    S1、将青霉菌Penicillium allii-sativi发酵培养，得到发酵物；所述青霉菌Penicillium allii-sativi保藏于中国海洋微生物菌种保藏管理中心，保藏编号为MCCC 3A00580；
    S2、将步骤S1得到的发酵物用乙酸乙酯进行萃取，所得萃取物经分离纯化，得到所述苯并噻唑杂萜类化合物及其衍生物。
  3. 根据权利要求2所述的制备方法，其特征在于，所述步骤S2包括：
    S21、将步骤S1得到的发酵物进行萃取，发酵物用乙酸乙酯萃取，取有机萃取物进行层析，分别使用石油醚，二氯甲烷和甲醇进行洗脱；浓缩二氯甲烷层，得到粗提物；
    S22、将步骤S21得到的粗提物用正相硅胶柱色谱进行分离，依次得到8个粗馏分：Fr.1～Fr.8；
    S23、将步骤S22得到的粗馏分Fr.5先使用ODS柱色谱进行分离，依次得到11段粗馏分：Fr.5.1～Fr.5-11；
    S24、将步骤S23得到的粗馏分Fr.5.9用葡聚糖凝胶柱和半制备液相色谱柱分离后得到式I化合物；
    S25、将步骤S22得到的粗馏分Fr.6先使用葡聚糖凝胶柱色谱进行分离，得到3段粗馏分：Fr.6.1～Fr.6.3；
    S26、将步骤S25得到的粗馏分Fr.6.2用ODS柱色谱和和半制备液相色谱柱分离后得到式II和式III化合物。
  4. 根据权利要求2所述的制备方法，其特征在于，步骤S1中，所述青霉菌Penicillium allii-sativi的发酵培养条件包括：将菌丝体接到含有PDB的培养液中，经培养获得种子液；将所述种子液接种到发酵培养基中，于28℃静态培养30天；所述发酵培养基包括燕麦80g和盐度3％的海水120ml。
  5. 根据权利要求4所述的制备方法，其特征在于，步骤S1中，所述菌丝体通过以下步骤制备得到：将青霉菌Penicillium allii-sativi在PDA平板上于28℃培养3～4天，获得所述菌丝体。
  6. 如权利要求1所述的苯并噻唑杂萜类化合物及其衍生物在制备以下产品中的应用：1)肿瘤细胞抑制剂；2)预防和/或治疗肿瘤疾病的药物。
  7. 根据权利要求6所述的应用，其特征在于，所述肿瘤细胞包括宫颈癌细胞、肝癌细胞、乳腺癌细胞和前列腺癌细胞。
  8. 根据权利要求6所述的应用，其特征在于，所述肿瘤疾病包括宫颈癌、肝癌乳腺癌和前列腺癌。
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CN112047901A (zh) * 2020-09-21 2020-12-08 自然资源部第三海洋研究所 苯并噻唑杂萜类化合物及其衍生物以及制备方法与应用

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