CN103265550B - Alkaloid compounds and preparation method thereof and the application in the anti-marine biofouling coating of preparation and antitumor drug - Google Patents
Alkaloid compounds and preparation method thereof and the application in the anti-marine biofouling coating of preparation and antitumor drug Download PDFInfo
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Abstract
Description
技术领域: Technical field:
本发明属于生物技术领域,具体涉及一类生物碱类化合物及其制备方法和在制备抗海洋生物污损涂料与抗肿瘤药物中的应用。 The invention belongs to the field of biological technology, and in particular relates to a class of alkaloid compounds, a preparation method thereof and an application in preparation of anti-marine biofouling coatings and anti-tumor drugs.
背景技术: Background technique:
真菌能产生包括生物碱类、肽类、聚酮类、甾体和萜类等在内的多种结构类型的次生代谢产物,其中很多具有抗肿瘤、抗细菌、抗真菌、抗病毒、抗虫等生物活性。生物碱是天然产物中一类含氮的碱性有机化合物,大多数具有复杂的环状结构,是天然药物中重要的有效成分之一。目前已经有不少生物碱用于临床的实例,如喜树中的喜树碱与长春花中的长春新碱用于抗肿瘤等。此外,海洋生物污损已成为人们充分利用海洋资源所要迫切解决的首要问题之一。随着TBT等有毒油漆在全球被禁用,开发高效、无毒、环境友好的海洋新型防污剂需求紧迫,这方面的研究突有重要的生态意义和经济意义。海洋天然产物防污剂属于无毒(或低毒)防污剂,易降解,且不危害海洋生物的生命,有利于保持生态平衡,因此,研制开发海洋天然产物防污剂已成为获得高效无毒防污剂的重要途径之一。 Fungi can produce secondary metabolites of various structural types including alkaloids, peptides, polyketides, steroids and terpenes, many of which have anti-tumor, anti-bacterial, anti-fungal, anti-viral, anti- Biological activity such as insects. Alkaloids are a class of nitrogen-containing basic organic compounds in natural products, most of which have complex ring structures, and are one of the important active ingredients in natural medicines. At present, there have been many clinical examples of alkaloids, such as camptothecin in camptotheca and vincristine in vinca for anti-tumor. In addition, marine biofouling has become one of the most urgent problems that people need to solve to make full use of marine resources. With the global prohibition of toxic paints such as TBT, there is an urgent need to develop new marine antifouling agents that are efficient, non-toxic, and environmentally friendly. Research in this area has important ecological and economic significance. Marine natural product antifouling agents are non-toxic (or low-toxicity) antifouling agents, easy to degrade, and do not endanger the life of marine organisms, and are conducive to maintaining ecological balance. Therefore, the research and development of marine natural product antifouling agents has become an efficient and non-toxic One of the important ways to poison antifouling agent.
发明内容: Invention content:
本发明的第一个目的是提供一类具有抗肿瘤和抗污损活性的生物碱类化合物。 The first object of the present invention is to provide a class of alkaloid compounds with antitumor and antifouling activities.
本发明通过多种柱层析及一维、二维核磁共振波谱,从真菌Aspergillus westerdijkiae DFFSCS013的发酵液中分离得到一类新的生物碱类化合物,这种生物碱类化合物具有抑制多种癌细胞生长的细胞毒活性,可用于制备抗肿瘤药物;且还有抗海洋生物污损附着活性,可用于制备抗海洋生物污损涂料,从而实现了本发明的目的。 The present invention separates and obtains a new class of alkaloid compounds from the fermentation broth of the fungus Aspergillus westerdijkiae DFFSCS013 through various column chromatography and one-dimensional and two-dimensional nuclear magnetic resonance spectra. This alkaloid compound has the ability to inhibit various cancer cells The growth cytotoxic activity can be used to prepare antitumor drugs; and the anti-marine biofouling adhesion activity can be used to prepare anti-marine biofouling coatings, thereby achieving the purpose of the present invention.
本发明的生物碱类化合物,其结构如式(Ⅰ)所示: The alkaloid compound of the present invention has a structure as shown in formula (I):
式(Ⅰ) Formula (I)
化合物1:R1=H,R2=OH,R3=Cl;化合物2:R1=OH,R2=H,R3=H。 Compound 1: R 1 =H, R 2 =OH, R 3 =Cl; Compound 2: R 1 =OH, R 2 =H, R 3 =H.
本发明的第二个目的是提供如式(Ⅰ)所示的生物碱类化合物的制备方法。 The second object of the present invention is to provide the preparation method of the alkaloid compound represented by formula (I).
本发明的如式(Ⅰ)所示的生物碱类化合物的制备方法,其特征在于,包括以下步骤: The preparation method of the alkaloid compound represented by formula (I) of the present invention is characterized in that it comprises the following steps:
(1)制备真菌Aspergillus westerdijkiae DFFSCS013的发酵液; (1) Prepare the fermentation broth of the fungus Aspergillus westerdijkiae DFFSCS013;
(2)将步骤(1)得到的发酵液用乙酸乙酯、二氯甲烷或氯仿溶剂萃取,浓缩得到乙酸乙酯提取物、二氯甲烷提取物或氯仿提取物; (2) Extract the fermentation broth obtained in step (1) with ethyl acetate, dichloromethane or chloroform solvent, and concentrate to obtain ethyl acetate extract, dichloromethane extract or chloroform extract;
(3)将步骤(2)所述的乙酸乙酯提取物、二氯甲烷提取物或氯仿提取物经过常压硅胶柱层析,以氯仿-甲醇、氯仿-丙酮、氯仿-乙酸乙酯、石油醚-丙酮或石油醚-乙酸乙酯溶剂系统为洗脱剂,从体积比100:0到0:100进行梯度洗脱,用薄层层析追踪合并组分,将在薄层层析上能用体积比95:5至90:10范围内的氯仿-甲醇溶剂系统展开的组分,经过凝胶柱层析得到粗品,经纯化,得到如式(Ⅰ)所示的生物碱类化合物1和2。 (3) The ethyl acetate extract, dichloromethane extract or chloroform extract described in step (2) is subjected to atmospheric pressure silica gel column chromatography, and the chloroform-methanol, chloroform-acetone, chloroform-ethyl acetate, petroleum Ether-acetone or sherwood oil-ethyl acetate solvent system is eluent, carries out gradient elution from volume ratio 100:0 to 0:100, uses thin-layer chromatography to track and merge components, will be able to be obtained on thin-layer chromatography The components developed with the chloroform-methanol solvent system in the range of volume ratio 95:5 to 90:10 are subjected to gel column chromatography to obtain the crude product, and after purification, the alkaloid compound 1 and 2.
步骤(1)中所述的真菌Aspergillus westerdijkiae DFFSCS013的发酵液可以将真菌 Aspergillus westerdijkiae DFFSCS013接种到曲霉属真菌适用的培养基中,在通常的发酵条件下制得。优选的制备方法是将真菌Aspergillus westerdijkiae DFFSCS013接种于PDA琼脂培养基中,26℃培养3天,得到培养有菌种的平板,然后将平板中菌种接种入PDB液体培养基中,于转速200rpm摇床、温度26℃培养2天,得到种子液,再将种子液接种于大米固体培养基中,于温度26℃静置培养26天,得到发酵液。上述PDA琼脂培养基的成分为土豆200g、葡萄糖20g、海盐30g、琼脂20g及1L水;PDB液体培养基的成分为土豆200g、葡萄糖20g、海盐30g及1L水;大米固体培养基的成分为大米400g、酵母浸膏2g、葡萄糖2g、海盐18g及水600mL。 The fermentation broth of the fungus Aspergillus westerdijkiae DFFSCS013 described in step (1) can be prepared by inoculating the fungus Aspergillus westerdijkiae DFFSCS013 into a suitable medium for Aspergillus fungi under normal fermentation conditions. The preferred preparation method is to inoculate the fungus Aspergillus westerdijkiae DFFSCS013 in the PDA agar medium, and cultivate it at 26°C for 3 days to obtain a flat plate with the cultured bacteria, then inoculate the bacteria in the plate into the PDB liquid medium, shake at a speed of 200rpm The bed was cultivated at a temperature of 26° C. for 2 days to obtain a seed liquid, and then the seed liquid was inoculated into a rice solid medium, and cultured at a temperature of 26° C. for 26 days to obtain a fermented liquid. The composition of above-mentioned PDA agar medium is potato 200g, glucose 20g, sea salt 30g, agar 20g and 1L water; The composition of PDB liquid medium is potato 200g, glucose 20g, sea salt 30g and 1L water; The composition of rice solid medium is rice 400g, yeast extract 2g, glucose 2g, sea salt 18g and water 600mL.
步骤(2)所述的萃取最好用乙酸乙酯,所述的浓缩可以采用常规的方法如减压浓缩。 The extraction described in step (2) is best to use ethyl acetate, and the described concentration can adopt conventional methods such as concentration under reduced pressure.
步骤(3)所述的纯化可以采用色谱柱分离或重结晶。 The purification described in step (3) can adopt chromatographic column separation or recrystallization.
通过抗海洋大型污损生物草苔虫幼虫和藤壶幼虫附着活性测试发现,本发明的式(Ⅰ)中化合物1和化合物2抑制草苔虫幼虫附着的EC50值分别为13.51μg/mL和28.12μg/mL,且抑制藤壶幼虫附着的EC50值小于25μg/mL,在所测得最高浓度200μg/mL时对幼虫没显示毒性,具有良好的抗海洋生物污损活性,能用于制备抗海洋生物污损涂料,如将其单独或组合渗入或扩散于成膜天然树脂,聚乙烯乙酸乙酯共聚物以及其它可水解,可溶或不溶性树脂等聚合物中,制成抗污损涂料,抗污损涂料能释放出足够量的有效成分至表面达到防污作用,并且这些化合物是天然活性成分,而且均不易溶于水,极易溶于氯仿、乙酸乙酯等低极性有机溶剂,具有良好的亲油性,因此可单独或组合应用于制备海洋防污剂中,具有良好的应用前景。 Through the anti-adhesion activity test of marine large-scale fouling organisms bryozoan larvae and barnacle larvae, it was found that the EC 50 values of compound 1 and compound 2 in the formula (I) of the present invention for inhibiting the attachment of bryozoan larvae were 13.51 μg/mL and 28.12μg/mL, and the EC 50 value of inhibiting the attachment of barnacle larvae is less than 25μg/mL, and it has no toxicity to larvae at the highest concentration of 200μg/mL, and has good anti-marine biofouling activity, which can be used in the preparation of Anti-marine biofouling coatings, such as infiltrating or diffusing in film-forming natural resins, polyethylene ethyl acetate copolymers and other polymers such as hydrolyzable, soluble or insoluble resins alone or in combination, to make anti-fouling coatings , anti-fouling coatings can release a sufficient amount of active ingredients to the surface to achieve anti-fouling effect, and these compounds are natural active ingredients, and are not easily soluble in water, but easily soluble in low-polarity organic solvents such as chloroform and ethyl acetate , has good lipophilicity, so it can be used alone or in combination in the preparation of marine antifouling agents, and has a good application prospect.
通过体外抗肿瘤活性筛选实验,结果显示:本发明的式(Ⅰ)所示的化合物1和2抑制 人白血病细胞株K562生长的IC50值分别为44.2和52.8μM,且有一定的抑制恶性黑色素瘤细胞株A375、肺癌细胞株A549、宫颈癌细胞株HeLa、乳腺癌细胞株MCF-7、喉癌细胞株Hep-2、肝癌细胞株HepG2、肠癌细胞株SW-620生长的作用。 Through in vitro anti-tumor activity screening experiments, the results showed that the compounds 1 and 2 represented by formula (I) of the present invention inhibited the growth of human leukemia cell line K562 with IC50 values of 44.2 and 52.8 μM, respectively, and had a certain inhibitory effect on malignant melanin The effect on the growth of tumor cell line A375, lung cancer cell line A549, cervical cancer cell line HeLa, breast cancer cell line MCF-7, laryngeal cancer cell line Hep-2, liver cancer cell line HepG2, and intestinal cancer cell line SW-620.
因此,本发明的第三个目的是提供式(Ⅰ)所示的生物碱类化合物或其盐在制备抗海洋生物污损涂料和抗肿瘤药物中的应用。 Therefore, the third object of the present invention is to provide the application of the alkaloid compound represented by formula (I) or its salt in the preparation of anti-marine biofouling coatings and anti-tumor drugs.
所述的抗肿瘤药物优选为抗人体白血病药物。 The anti-tumor drug is preferably an anti-human leukemia drug.
本发明从真菌Aspergillus westerdijkiae DFFSCS013(CCTCC M2013078)发酵液中制备分离得到了一类生物碱类化合物—化合物1和化合物2。化合物1和化合物2具有抗海洋大型污损生物草苔虫和藤壶幼虫附着活性,且能抑制白血病细胞株K562等多种肿瘤细胞株的生长,因此在制备抗海洋生物污损涂料与抗肿瘤药物方面具有良好的应用前景。 The present invention prepares and separates a class of alkaloid compounds—compound 1 and compound 2—from the fermentation broth of the fungus Aspergillus westerdijkiae DFFSCS013 (CCTCC M2013078). Compound 1 and Compound 2 have anti-adhesion activity of marine large-scale fouling organisms Bryophora and barnacle larvae, and can inhibit the growth of various tumor cell lines such as leukemia cell line K562, so they are useful in the preparation of anti-marine biofouling coatings and anti-tumor It has a good application prospect in medicine.
本发明的真菌Aspergillus westerdijkiae DFFSCS013于2013年3月12日保藏于中国典型培养物保藏中心(CCTCC),地址:中国,武汉,武汉大学,保藏号CCTCC M2013078。 The fungus Aspergillus westerdijkiae DFFSCS013 of the present invention was deposited in China Center for Type Culture Collection (CCTCC) on March 12, 2013, address: China, Wuhan, Wuhan University, preservation number CCTCC M2013078.
附图说明:Description of drawings:
图1是化合物1和2的NOESY相关信号,其中1代表化合物1,2代表化合物2。 Figure 1 is the NOESY correlation signals of compounds 1 and 2, where 1 represents compound 1 and 2 represents compound 2.
具体实施方式: Detailed ways:
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。 The following examples are to further illustrate the present invention, rather than limit the present invention.
实施例1:生物碱类化合物1和2的制备 Embodiment 1: Preparation of alkaloid compounds 1 and 2
PDA琼脂培养基是这样配制的:将200克土豆,20克葡萄糖,30克海盐,20克琼脂混合,加入1升水充分溶解,121℃高压蒸汽灭菌25分钟。 The PDA agar medium is prepared as follows: mix 200 grams of potatoes, 20 grams of glucose, 30 grams of sea salt, and 20 grams of agar, add 1 liter of water to fully dissolve, and sterilize under high pressure steam at 121 ° C for 25 minutes.
PDB液体培养基是这样配制的:将200克土豆,20克葡萄糖,30克海盐混合,加入1 升水充分溶解后,再分装于500mL的三角烧瓶中,每瓶约150mL,在121℃高压蒸汽灭菌25分钟。 The PDB liquid medium is prepared as follows: mix 200 grams of potatoes, 20 grams of glucose, and 30 grams of sea salt, add 1 liter of water to fully dissolve, and then divide them into 500 mL Erlenmeyer flasks, each bottle is about 150 mL. Sterilize for 25 minutes.
大米固体培养基是这样配制的:将400克大米、2克酵母浸膏、2克葡萄糖、18克海盐混合,加入600毫升水充分溶解后,装入5000mL的三角烧瓶中,在121℃高压蒸汽灭菌25分钟。 The rice solid medium is prepared as follows: mix 400 grams of rice, 2 grams of yeast extract, 2 grams of glucose, and 18 grams of sea salt, add 600 milliliters of water to fully dissolve it, put it into a 5000-mL Erlenmeyer flask, and heat it under high-pressure steam at 121 ° C. Sterilize for 25 minutes.
用无菌竹签将真菌Aspergillus westerdijkiae DFFSCS013菌种接种于PDA琼脂培养基上,26℃培养3天,得到培养有菌种的平板,然后用无菌竹签从平板中挑约3微升菌种接种入含150mL上述装有PDB液体培养基的三角烧瓶中,于摇床(转速200rpm)温度26℃培养2天,得到种子液,再在上述每瓶大米固体培养基的三角瓶中接种50mL种子液,于温度26℃静置培养26天后,得到发酵液。 Use a sterile bamboo stick to inoculate the fungus Aspergillus westerdijkiae DFFSCS013 on the PDA agar medium, and culture it at 26°C for 3 days to obtain a plate with the cultured bacteria, and then use a sterile bamboo stick to pick about 3 microliters of the bacteria from the plate Inoculate into 150mL of the Erlenmeyer flask containing the above-mentioned PDB liquid medium, culture on a shaker (rotating speed 200rpm) at 26°C for 2 days to obtain the seed liquid, and then inoculate 50mL of seeds in each of the above-mentioned Erlenmeyer flasks of rice solid medium After static culture at 26°C for 26 days, the fermentation broth was obtained.
将经大米固体培养基(共2kg大米)培养得到的发酵液用丙酮-水(体积比80:20)提取3遍,然后将此提取液减压浓缩至无丙酮残留,将浓缩后的水相用等体积乙酸乙酯萃取3遍,减压浓缩得到乙酸乙酯提取物30g。乙酸乙酯提取物用正相硅胶(200-300目)干法拌样后,装入玻璃层析柱(含200-300目的正相硅胶约500g),进行常温柱层析,以氯仿-甲醇作为洗脱剂,从体积比100:0到0:100进行梯度洗脱,根据薄层层析(GF254硅胶板)情况合并各个馏分,回收洗脱溶剂,蒸干馏分用甲醇转移。将在薄层层析上能用体积比95:5至90:10范围内的氯仿-甲醇溶剂系统展开的组分经凝胶柱层析(直径18mm,柱长1600mm,凝胶为sephedex LH-20,流动相为体积比1:1的氯仿-甲醇)分离,收集馏分,析出粗品,粗品采用高效液相半制备(检测波长为254nm,流速为3mL/min,色谱柱为Phenomenex Gemini 5μ C18110A column(250×10mm,5μm),流动相为体积比为65:35的甲醇-水)分别在出峰时间为25 和33分钟时分离纯化得到化合物1和2。 Extract the fermented liquid obtained by culturing the rice solid medium (a total of 2kg of rice) with acetone-water (volume ratio 80:20) for 3 times, then concentrate the extract under reduced pressure until no acetone remains, and the concentrated aqueous phase Extracted 3 times with equal volume of ethyl acetate, concentrated under reduced pressure to obtain 30 g of ethyl acetate extract. After the ethyl acetate extract was mixed with normal phase silica gel (200-300 mesh) by dry method, it was loaded into a glass chromatography column (containing about 500 g of normal phase silica gel of 200-300 mesh), and subjected to normal temperature column chromatography. As the eluent, gradient elution was performed from volume ratio 100:0 to 0:100, and the fractions were combined according to thin layer chromatography (GF 254 silica gel plate), the elution solvent was recovered, and the fractions were evaporated to dryness and transferred with methanol. The components that can be developed with the chloroform-methanol solvent system in the range of volume ratio 95:5 to 90:10 on thin layer chromatography are subjected to gel column chromatography (diameter 18mm, column length 1600mm, gel is sephedex LH- 20. The mobile phase is chloroform-methanol with a volume ratio of 1:1) to separate, collect the fractions, and separate out the crude product. The crude product is semi-preparative with high performance liquid phase (the detection wavelength is 254nm, the flow rate is 3mL/min, and the chromatographic column is Phenomenex Gemini 5μ C18110A column (250×10mm, 5μm), the mobile phase is methanol-water with a volume ratio of 65:35), and compounds 1 and 2 were obtained by separation and purification at the peak eluting time of 25 and 33 minutes, respectively.
其中化合物1的结构解析如下: The structural analysis of compound 1 is as follows:
化合物1为白色固体,其高分辨质谱(HRESIMS)在m/z498.1778[M+H]+处给出准分子离子峰,且同位素峰m/z500.1762[M+H+2]+与准分子离子峰的丰度比为1:3,表明分子中含有1个氯原子,结合NMR光谱数据(Table1),得知化合物1的分子式为C26H28ClN3O5。1HNMR谱显示分子中存在4个甲基[δH0.70(3H,s)、0.78(3H,s)、1.42(6H,s)],4个亚甲基[δH1.77(1H,dd,J=9.5,12.5Hz)、1.84(2H,overlapped)、1.96(1H,dd,J=10.0,12.5Hz)、2.03(1H,m)、2.54(1H,overlapped)、3.41(2H,t,J=6.0Hz)],1个高场次甲基[δH3.76(1H,t,J=9.5Hz)],1个氧化的次甲基[δH5.19(1H,d,J=7.5Hz)],3个烯氢[δH5.84(1H,d,J=10.0Hz)、6.54(1H,d,J=10.0Hz)、6.91(1H,s)],1个羟基氢[δH5.65(1H,d,J=8.0Hz)]以及2个氨基氢[δH8.01(1H,s)、10.81(1H,s)]。13C NMR和DEPT谱(Table2)表明分子中存在26个碳,包括4个甲基(δC18.9、22.5、27.5、27.5),4个亚甲基(δC24.2、28.8、29.5、43.2),1个高场次甲基(δC54.9),1个氧化的次甲基(δC72.9),5个季碳(δC43.1、65.3、68.4、69.0、77.1),8个烯碳(δC105.3、112.0、116.2、122.1、125.2、130.8、138.1、147.7)以及3个羰基(δC168.6、172.5、177.4)。上述数据表明化合物1与已知化合物sclerotiamide具有类似结构[Whyte,A.C.;Gloer,J.B.;Wicklow,D.T.;Dowd,P.F.J Nat Prod.1996,59,1093-1095.],与sclerotiamide相比,化合物1的1H NMR谱中缺少了一个芳香质子信号,其13C NMR和DEPT谱中缺少了一个芳香次甲基信号但却增加了1个芳香季碳信号,进一步对比其分子式的差异,可以推断出化合物1与sclerotiamide的平面结构中唯一的区别是sclerotiamide的1个芳香质子被氯原子取代。由于在氢谱中H-4(δH6.91,s)为单峰,并且在HMBC谱(Figure1)中H-4与C-3/C-5/C-6/C-8 相关,由此推测氯原子的取代位置为C-5。由此,化合物1的平面结构鉴定如式(Ⅰ)所示,其中所有的碳、氢信号还都通过HSQC、HMBC和1H-1H COSY光谱进行了归属。 Compound 1 is a white solid, and its high-resolution mass spectrum (HRESIMS) gives a quasi-molecular ion peak at m/z498.1778[M+H] + , and the isotopic peak m/z500.1762[M+H+2] + is consistent with The abundance ratio of the quasi-molecular ion peak is 1:3, indicating that the molecule contains a chlorine atom. Combined with the NMR spectral data (Table 1), it is known that the molecular formula of compound 1 is C 26 H 28 ClN 3 O 5 . 1 H NMR spectrum shows that there are 4 methyl groups in the molecule [δ H 0.70(3H,s), 0.78(3H,s), 1.42(6H,s)], 4 methylene groups [δ H 1.77(1H,dd, J=9.5,12.5Hz), 1.84(2H,overlapped), 1.96(1H,dd,J=10.0,12.5Hz), 2.03(1H,m), 2.54(1H,overlapped), 3.41(2H,t,J =6.0Hz)], 1 high-field methine [δ H 3.76(1H,t,J=9.5Hz)], 1 oxidized methine [δ H 5.19(1H,d,J=7.5Hz)] , 3 alkene hydrogens [δ H 5.84 (1H, d, J=10.0Hz), 6.54 (1H, d, J = 10.0Hz), 6.91 (1H, s)], 1 hydroxyl hydrogen [δ H 5.65 (1H ,d,J=8.0Hz)] and two amino hydrogens [δ H 8.01(1H,s), 10.81(1H,s)]. 13 C NMR and DEPT spectra (Table 2) indicated that there were 26 carbons in the molecule, including 4 methyl groups (δ C 18.9, 22.5, 27.5, 27.5), and 4 methylene groups (δ C 24.2, 28.8, 29.5, 43.2) , 1 high-field methine (δ C 54.9), 1 oxidized methine (δ C 72.9), 5 quaternary carbons (δ C 43.1, 65.3, 68.4, 69.0, 77.1), 8 alkene carbons (δ C 105.3, 112.0, 116.2, 122.1, 125.2, 130.8, 138.1, 147.7) and three carbonyl groups (δ C 168.6, 172.5, 177.4). The above data show that compound 1 has a similar structure with known compound sclerotiamide [Whyte, AC; Gloer, JB; Wicklow, DT; Dowd, PFJ Nat Prod.1996,59,1093-1095.], compared with sclerotiamide, compound 1 An aromatic proton signal is missing in the 1 H NMR spectrum, and an aromatic methine signal is missing in the 13 C NMR and DEPT spectra, but an aromatic quaternary carbon signal is added. Further comparing the difference in its molecular formula, it can be deduced that the compound The only difference in the planar structure of 1 and sclerotiamide is that 1 aromatic proton of sclerotiamide is replaced by a chlorine atom. Since H-4 (δ H 6.91, s) is a single peak in the hydrogen spectrum, and H-4 is related to C-3/C-5/C-6/C-8 in the HMBC spectrum (Figure 1), thus It is speculated that the substitution position of the chlorine atom is C-5. Thus, the planar structure of compound 1 was identified as shown in formula (I), in which all carbon and hydrogen signals were also assigned by HSQC, HMBC and 1 H- 1 H COZY spectra.
化合物1的立体构型通过NOESY谱(图1)进行了鉴定,H-4、H-10和CH3-24之间的NOE相关信号表明H-10和CH3-24为β构型,且H-10和CH3-24位于环戊烷环的表面并朝向H-4,由此可推断C-3的相对构型如式(Ⅰ)所示;同时,H-19与NH-21/CH3-23具有NOE相关,表明H-19和CH3-23为α构型。由此可知,化合物1的相对构型与sclerotiamide完全相同,又因为sclerotiamide的绝对构型已通过与结构类似物paraherquamide对比的方法鉴定,因此化合物1的绝对构型也同样鉴定为(3R,10S,11R,17S,19S)。化合物1的具体结构如式(Ⅰ)所示,其中R1=H,R2=OH,R3=C1。 The stereo configuration of compound 1 was identified by NOESY spectrum (Figure 1), and the NOE correlation signals between H-4, H-10 and CH 3 -24 indicated that H-10 and CH 3 -24 were in the β configuration, and H-10 and CH 3 -24 are located on the surface of the cyclopentane ring and face H-4, so it can be deduced that the relative configuration of C-3 is shown in formula (I); meanwhile, H-19 and NH-21/ CH 3 -23 has a NOE correlation, indicating that H-19 and CH 3 -23 are in the alpha configuration. It can be seen that the relative configuration of compound 1 is exactly the same as that of sclerotiamide, and because the absolute configuration of sclerotiamide has been identified by the method of comparison with the structural analog paraherquamide, the absolute configuration of compound 1 is also identified as (3R, 10S, 11R, 17S, 19S). The specific structure of compound 1 is shown in formula (I), wherein R 1 =H, R 2 =OH, R 3 =C1.
表1.化合物1和2的NMR数据(500and125MHz,respectively,in DMSO-d6,δppm) Table 1. NMR data of compounds 1 and 2 (500and125MH z , respectively, in DMSO-d6, δppm)
asignals overlapping a signal overlapping
化合物2为白色固体,其高分辨质谱(HRESIMS)在m/z464.2166[M+H]+处给出准分子离子峰,结合NMR光谱数据(Table1),得知化合物2具有与sclerotiamide相同的分子式C26H29N3O5。化合物2的1H和13C NMR谱数据与sclerotiamide十分相近,除了10-CH(δC77.5,δH4.62)和C-3(δC65.7)在化学位移上存在明显差异。进一步对化合物2的1H NMR,13C NMR,HSQC和HMBC数据进行分析,发现化合物2与sclerotiamide具有相同的平面结构,但其立体构型存在差异[1.Whyte,A.C.;Gloer,J.B.;Wicklow,D.T.;Dowd,P.F.J Nat Prod.1996,59,1093-1095.]。化合物2的相对构型通过NOESY光谱(图1)进行了鉴定,10-OH与H-4/H-5/CH3-24的NOE相关信号表明OH-10和CH3-24为β构型,而H-10,H-19,NH-21和CH3-23之间的NOE相关信号表明H-10、H-19和CH3-23为α构型。上述推断表明化合物2为sclerotiamide的差向异构体,二者C-10处的相对构型正好相反,但其余手型碳的构型完全一致,由于sclerotiamide的绝对构型已确定,因此,化合物2的绝对构型鉴定为(3R,10R,11R,17S,19S)。化合物2的具体结构如式(Ⅰ)所示,其中R1=OH,R2=H,R3=H。 Compound 2 is a white solid, and its high-resolution mass spectrum (HRESIMS) gives a quasi-molecular ion peak at m/z464.2166[M+H] + . Combined with NMR spectral data (Table1), it is known that compound 2 has the same Molecular formula C 26 H 29 N 3 O 5 . The 1 H and 13 C NMR spectra of compound 2 are very similar to those of sclerotiamide, except that there are significant differences in chemical shifts between 10-CH (δ C 77.5, δ H 4.62) and C-3 (δ C 65.7). Further analysis of the 1 H NMR, 13 C NMR, HSQC and HMBC data of compound 2 revealed that compound 2 has the same planar structure as sclerotiamide, but there are differences in their three-dimensional configuration [1. Whyte, AC; Gloer, JB; Wicklow , DT; Dowd, PFJ Nat Prod. 1996, 59, 1093-1095.]. The relative configuration of compound 2 was identified by NOESY spectrum (Figure 1), and the NOE correlation signals of 10-OH and H-4/H-5/CH 3 -24 indicated that OH-10 and CH 3 -24 were in the β configuration , while the NOE correlation signals between H-10, H-19, NH-21 and CH 3 -23 indicated that H-10, H-19 and CH 3 -23 were in the α configuration. The above deduction shows that compound 2 is an epimer of sclerotiamide, and the relative configuration at C-10 of the two is just opposite, but the configuration of the remaining chiral carbons is completely consistent. Since the absolute configuration of sclerotiamide has been determined, the compound The absolute configuration of 2 was identified as (3R, 10R, 11R, 17S, 19S). The specific structure of compound 2 is shown in formula (I), wherein R 1 =OH, R 2 =H, R 3 =H.
式(Ⅰ)。 Formula (Ⅰ). the
实施例2:式(I)所示的生物碱类化合物抗草苔虫幼虫附着活性测试 Example 2: Anti-Adhesive Activity Test of Bryonyx Larvae of Alkaloid Compounds Represented by Formula (I)
活性测试模型采用目前实验室最常用的海洋污损生物模型之一多室草苔虫幼虫(Bugula neritina)的附着抑制试验。将采集到的成熟的多室草苔虫放置到用0.22μm滤膜过滤的海水中,用光照刺激,收集成熟多室草苔虫经光照刺激产生的幼虫。将幼虫放置到盛有用0.22μm滤膜过滤的海水的表面皿中。用草苔虫经光照产生的幼虫做活性试验。采用24孔聚苯乙烯板测定化合物的抗幼虫附着活性。将式(I)化合物(化合物1或化合物2)分别溶于DMSO,用无菌过滤海水稀释成不同的浓度溶液(60–140μg/mL)作为测试液。每个孔中加入1mL测试液和20±2个成熟的草苔虫幼虫,每个浓度设5个重复样,无菌过滤海水做空白对照。将24孔细胞培养板置于28℃培养箱中放置3小时。在显微镜下统计附着的幼虫数,用spss11.0软件进行统计分析,计算EC50值。 The activity test model adopts the attachment inhibition test of Bugula neritina, one of the most commonly used marine fouling biological models in the laboratory. The collected mature B. multiloculum was placed in seawater filtered with a 0.22 μm filter membrane, stimulated with light, and the larvae produced by the mature B. multiloculum stimulated by light were collected. Larvae were placed in a watch glass containing seawater filtered through a 0.22 μm filter membrane. The activity test was done with the larvae produced by Bryosa larvae by light. Anti-larval settling activity of compounds was determined using 24-well polystyrene plates. The compound of formula (I) (compound 1 or compound 2) was dissolved in DMSO respectively, and diluted with sterile filtered seawater to obtain solutions of different concentrations (60–140 μg/mL) as the test solution. Add 1mL test solution and 20±2 mature bryozoa larvae to each well, set 5 replicate samples for each concentration, and sterile filtered seawater as blank control. Place the 24-well cell culture plate in a 28°C incubator for 3 hours. The number of attached larvae was counted under the microscope, statistical analysis was performed with spss11.0 software, and the EC 50 value was calculated.
实验结果显示式(I)中化合物1和2抑制草苔虫幼虫附着的EC50值分别为13.52μg/mL和28.12μg/mL,由此说明式(I)所示的生物碱类化合物具有抗草苔虫幼虫附着活性,能用于制备抗海洋生物污损涂料中。 The experimental results showed that the EC 50 values of compounds 1 and 2 in the formula (I) for inhibiting the larvae of B. The bryozoan larvae adhesion activity can be used in the preparation of anti-marine biofouling coatings.
实施例3:式(I)所示的生物碱类化合物抗藤壶幼虫附着活性测试 Example 3: Anti-barnacle larva attachment activity test of alkaloid compounds represented by formula (I)
活性测试模型是采用目前实验室最常用的海洋污损生物模型之一纹藤壶虫金星幼虫(Barnacles larvae)的附着抑制试验。藤壶成虫以纤细角毛藻(Chaetoceros gracilis Schutt)为食物,在0.22μm过滤海水中将藤壶的无节幼体培养到金星幼体阶段,每毫升2个幼虫,培养温度28℃。用产生的金星阶段幼体进行活性试验。采用24孔聚苯乙烯板测定化合物的抗幼虫附着活性。将式(1)化合物(化合物1或化合物2)分别溶于DMSO,用无菌过滤海水稀释成不同的浓度溶液(60–140μg/mL)作为测试液。每个孔中加入1mL测试液和20±2个成熟的藤壶幼虫,每个浓度设5个重复样,无菌过滤海水做空白对照。将24孔细胞培养板置于30℃培养箱中放置24小时。在显微镜下统计附着的幼虫数。 The activity test model is the attachment inhibition test of Barnacles larvae, one of the most commonly used marine fouling biological models in laboratories. Adult barnacles feed on Chaetoceros gracilis Schutt, and culture barnacle nauplii to Venus larva stage in 0.22 μm filtered seawater with 2 larvae per milliliter at 28°C. Activity tests were carried out with the resulting Venus stage larvae. Anti-larval settling activity of compounds was determined using 24-well polystyrene plates. The compound of formula (1) (compound 1 or compound 2) was dissolved in DMSO respectively, and diluted with sterile filtered seawater into different concentration solutions (60–140 μg/mL) as the test solution. Add 1mL test solution and 20±2 mature barnacle larvae to each well, set 5 replicate samples for each concentration, and sterile filtered seawater as blank control. Place the 24-well cell culture plate in a 30°C incubator for 24 hours. The number of attached larvae was counted under a microscope.
实验结果显示,式(I)中化合物1和2在初始浓度25μg/mL时抑制藤壶幼虫附着的抑制率分别为70%和64%,其EC50值应小于25μg/mL(由于样品量有限没进一步测试),在所测得最高浓度200μg/mL时对幼虫没显示毒性,由此说明式(I)所示的生物碱类化合物具有抗藤壶幼虫附着活性,能用于制备抗海洋生物污损涂料中。 The experimental results showed that compounds 1 and 2 in formula (I) inhibited the attachment of barnacle larvae by 70% and 64% at an initial concentration of 25 μg/mL, respectively, and their EC 50 values should be less than 25 μg/mL (due to the limited amount of samples No further test), there was no toxicity to larvae at the highest concentration measured at 200 μg/mL, thus indicating that the alkaloid compound represented by formula (I) has anti-barnacle larva attachment activity and can be used to prepare anti-marine organisms In fouling paint.
实施例4:式(I)所示的生物碱类化合物体外抗肿瘤活性测试 Example 4: In vitro anti-tumor activity test of alkaloid compounds represented by formula (I)
分别收集对数生长期的白血病细胞株K562、恶性黑色素瘤细胞株A375、肺癌细胞株A549、宫颈癌细胞株HeLa、乳腺癌细胞株MCF-7、喉癌细胞株Hep-2、肝癌细胞株HepG2和肠癌细胞株SW-620,用10%血清1640培养基使其悬浮,再接种于96孔培养板,每孔细胞数为5000个/80μL,置于5%CO2培养箱37℃培养。用10%血清1640培养基将式(I)中化合物1和2稀释成浓度为3.125μg/mL,6.25μg/mL,12.5μg/mL,25μg/mL,50μg/mL,100μg/mL,200μg/mL的实验溶液。次日于不同的实验组的培养板中分别加入浓度为3.125μg/mL, 6.25μg/mL,12.5μg/mL,25μg/mL,50μg/mL,100μg/mL,200μg/mL的实验溶液20μL,并使每孔中的终浓度达到测试(实验组浓度采用对倍稀释)。另外阴性对照组中加入等量的10%血清1640培养基。48h后,吸弃实验组和对照组中的培养液,每孔加入MTT20μL(2.5mg/mL),继续培养4h,再每孔加入DMSO100μL终止反应,37℃放置20min,用酶标仪检测各孔在570nm处的吸光度A值,计算细胞生长抑制率。细胞生长率=(实验组OD÷对照组OD)×100%。 Leukemia cell line K562, malignant melanoma cell line A375, lung cancer cell line A549, cervical cancer cell line HeLa, breast cancer cell line MCF-7, laryngeal cancer cell line Hep-2, and liver cancer cell line HepG2 were collected in logarithmic growth phase. and intestinal cancer cell line SW-620 were suspended in 10% serum 1640 medium, then inoculated in 96-well culture plate, the number of cells per well was 5000/80 μL, and cultured in a 5% CO 2 incubator at 37°C. Compounds 1 and 2 in formula (I) were diluted with 10% serum 1640 medium to a concentration of 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL mL of the experimental solution. On the next day, 20 μL of experimental solutions with concentrations of 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL were added to the culture plates of different experimental groups. And make the final concentration in each hole reach the test (experimental group concentration adopts double dilution). In addition, the same amount of 10% serum 1640 medium was added to the negative control group. After 48 hours, discard the culture medium in the experimental group and the control group, add 20 μL (2.5 mg/mL) of MTT to each well, continue to incubate for 4 hours, then add 100 μL of DMSO to each well to stop the reaction, place at 37°C for 20 minutes, and detect each well with a microplate reader The absorbance A value at 570nm was used to calculate the cell growth inhibition rate. Cell growth rate=(experimental group OD÷control group OD)×100%.
实验结果显示,本发明的式(Ⅰ)所示的化合物1和2抑制人白血病细胞株K562生长的IC50值分别为44.2和52.8μM,且有一定的抑制恶性黑色素瘤细胞株A375、肺癌细胞株A549、宫颈癌细胞株HeLa、乳腺癌细胞株MCF-7、喉癌细胞株Hep-2、肝癌细胞株HepG2、肠癌细胞株SW-620生长的作用,IC50值为80~100μM。因此本发明的式(I)所示的生物碱类化合物能用于制备抗肿瘤药物。 Experimental results show that compounds 1 and 2 represented by the formula (I) of the present invention inhibit the growth of human leukemia cell line K562 with IC50 values of 44.2 and 52.8 μM, respectively, and have certain inhibitory effects on malignant melanoma cell line A375, lung cancer cell line The effect on the growth of strain A549, cervical cancer cell line HeLa, breast cancer cell line MCF-7, laryngeal cancer cell line Hep-2, liver cancer cell line HepG2, and intestinal cancer cell line SW-620, with IC 50 values of 80-100 μM. Therefore, the alkaloid compound represented by the formula (I) of the present invention can be used to prepare antitumor drugs.
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