WO2018153330A1 - 双黄酮-铜配合物及其制备方法和应用 - Google Patents

双黄酮-铜配合物及其制备方法和应用 Download PDF

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WO2018153330A1
WO2018153330A1 PCT/CN2018/076736 CN2018076736W WO2018153330A1 WO 2018153330 A1 WO2018153330 A1 WO 2018153330A1 CN 2018076736 W CN2018076736 W CN 2018076736W WO 2018153330 A1 WO2018153330 A1 WO 2018153330A1
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ame
copper complex
biflavonoid
copper
complex
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徐莉
杨世龙
曹福亮
卢雯
唐颖
周梦怡
薛华玉
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南京林业大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F1/00—Compounds containing elements of Groups 1 or 11 of the Periodic Table
    • C07F1/005—Compounds containing elements of Groups 1 or 11 of the Periodic Table without C-Metal linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the invention belongs to the field of organic synthesis and medicine, and particularly relates to a biflavonoid-copper complex and a preparation method and application thereof.
  • the biflavonoid compound is a chemical component unique to gymnosperm, such as ginkgo, cypress, etc., and has biological activities such as anti-oxidation, anti-inflammatory, anti-viral, anti-tumor and the like.
  • Amentoflavone (Ame) is a relatively common type of biflavonoids, and its structural formula is as follows:
  • flavonoids of Spruce sinensis inhibit the growth of tumor cells by inhibiting the activity of NF-kappaB, and inhibiting the growth of cancer cells [Chen JH, Chen WL, Liu YCAmentoflavone induces anti-angiogenic and anti- Metastatic effects through suppression of NF-kappa B activation in MCF-7 cells [J]. Anticancer Research, 2015, 35(12): 6685-6693.].
  • Lee et al. found that flavonoids can inhibit the expression of metalloproteinases caused by ultraviolet radiation, thereby exerting anti-oxidation and anti-radiation effects [Lee CW, Na Y., Park N., etal.
  • Amentoflavone inhibits UVB-induced Matrix metalloproteinase-1expression through the modulation of AP-1mponents in normal human fibroblasts [J]. Applied Biochemistry and Biotechnology, 2012, 166: 1137-1147.].
  • Zhang et al. found that flavonoids and ginkgo flavonoids have certain antioxidant activity and strong ability to remove DPPH free radicals [Zhang YP, Shi SY, Wang YX, etal. Target-guided isolation and purification of antioxidants from Selaginella sinensis By offline coupling of DPPH-HPLC and HSCCC experiments [J]. Journal of Chromatography B, 2011, 879: 191-196.]. Li et al.
  • flavonoids have an antioxidant activity, which can effectively scavenge free radicals such as OH - ⁇ , O 2 - , DPPH ⁇ , ABTS + ⁇ and protect DNA from oxidative damage caused by OH - ⁇ Li XC, Wang L., Han WJ, et al. Amentoflavone protects against hydroxyl radical-induced DNA damage via antioxidant mechanism [J].
  • Turkish Journal of Biochemistry-Turk Biyokimya Dergisi, 2014, 39(1): 30-36. Turkish Journal of Biochemistry-Turk Biyokimya Dergisi, 2014, 39(1): 30-36.].
  • quercetin rare earth complexes have stronger ability to scavenge O 2 - ⁇ than quercetin.
  • Quercetin rare earth complex can inhibit a variety of tumors, and anti-tumor activity is stronger than quercetin.
  • the complex has a strong inhibitory effect on bladder tumor cells, but quercetin has no such effect [Zhou J., Wang LF, Wang JY, etal. Synthesis, characterization, antioxidative and antitumor activities of solid quercetin rare earth (III) complexes [J]. Journal of Inorganic Biochemistry, 2001, 83: 41-48.].
  • OBJECTS OF THE INVENTION In view of the deficiencies in the prior art, it is an object of the present invention to provide a flavonoid-copper complex of spikes that meets the needs of anti-tumor and antioxidant active drugs. Another object of the present invention is to provide a process for the preparation of the above-mentioned biflavonoid-copper complex. Still another object of the invention is to provide the use of a biflavonoid-copper complex.
  • the biflavonoid-copper complex has the following structural formula:
  • X is NO 3 - or Cl - .
  • a method for preparing the bisflavonoid-copper complex dissolving a copper salt with an alcohol and adding it to an alcohol solution of a biflavonoid, controlling the pH to 5-7, heating and stirring, reacting for 2-5 hours, and forming a precipitate, which will The precipitate was filtered, washed with alcohol and water, and then recrystallized from dimethyl sulfoxide as a solvent and dried to give a bis-flavonoid-copper complex.
  • the copper salt used is an alcohol-soluble copper salt such as copper nitrate or copper chloride.
  • the solvent used includes ethanol, methanol, and various concentrations of methanol, an aqueous ethanol solution, and the like.
  • the pH is adjusted with an alkali alcohol solution, and the base used includes common bases such as sodium hydroxide, potassium hydroxide, aqueous ammonia, sodium ethoxide, and sodium methoxide.
  • common bases such as sodium hydroxide, potassium hydroxide, aqueous ammonia, sodium ethoxide, and sodium methoxide.
  • the heating temperature is 30-50 ° C, and the reaction time is 2-5 h.
  • the molar ratio of diflavonoid to copper ion in the solution is 2-2.5:1.
  • the solvent used for recrystallization is dimethyl sulfoxide, and the drying method is freeze drying, low temperature vacuum drying, or the like.
  • the flavonoid-copper complex of the safflower was synthesized for the first time, and the antitumor activity of the Ame-Cu complex was studied by MTT method.
  • the results showed that the Ame-Cu complex inhibited liver cancer.
  • the ability of cells (HepG2) and cervical cancer cells (HeLa) is stronger than that of Ame itself.
  • UV-visible absorption spectroscopy, fluorescence spectroscopy and viscosity methods indicate that the mechanism of anti-tumor activity of Ame-Cu complexes may be complexes and embedded insert DNA. Causes apoptosis.
  • the pyrogallol autooxidation method and ABTS method showed that the Ame-Cu complex has stronger free radical scavenging ability than Ame itself, indicating that the antioxidant activity of the complex is stronger than that of Ame; it is beneficial to further develop biflavonoids and provide a basis for new drug research. It is conducive to the development of human health.
  • Figure 1 is an infrared spectrum of the Ame and Ame-Cu complexes
  • Figure 3 is an Ame mass spectrum
  • Figure 4 is a mass spectrum of the Ame-Cu complex
  • Figure 5 is a graph showing the results of inhibition of HegG2 cells by Ame and Ame-Cu complexes
  • Figure 6 is a graph showing the results of inhibition of Hela cells by Ame and Ame-Cu complexes
  • Figure 7 is a graph showing the effect of fDNA on the ultraviolet-visible absorption spectrum of Ame
  • Figure 8 is a graph showing the effect of fDNA on the ultraviolet-visible absorption spectrum of the Ame-Cu complex
  • Figure 9 is a graph showing the effect of Ame on the fluorescence emission spectrum of the fDNA-EB system
  • Figure 10 is a graph showing the effect of Ame-Cu complex on the fluorescence emission spectrum of the fDNA-EB system
  • Figure 11 is a graph showing the effect of the Ame and Ame-Cu complexes on the viscosity of the fDNA solution
  • Figure 12 is a graph showing the effect of Ame on the auto-oxidation rate of pyrogallol
  • Figure 13 is a graph showing the effect of the Ame-Cu complex on the autooxidation rate of pyrogallol
  • Figure 14 is a graph showing the results of Ame versus ABTS + ⁇ free scavenging ability
  • Figure 15 is a graph showing the results of the Ame-Cu complex versus ABTS + ⁇ free scavenging ability.
  • the absorption here is narrowed, and the peak shape at 3407 cm -1 becomes sharp, indicating that the intramolecular hydrogen bond is destroyed after the formation of the complex;
  • the strong peak at 1657 cm -1 in Ame What is caused by the stretching vibration of the carbonyl group is the characteristic absorption peak of the carbonyl group.
  • the absorption peak shifts to the low wave number and moves to 1630 cm -1 , indicating that the carbonyl group participates in the coordination; the complex is between 622 cm -1 .
  • the UV-visible absorption spectra of Ame and Ame-Cu complexes are shown in Figure 2.
  • Ame has two characteristic absorption peaks at 337 nm (band I) and 270 nm (band II), which is the characteristic absorption of flavonoids.
  • I and band II correspond to the ultraviolet absorption of the cinnamate system and the benzoyl system, respectively, which are caused by the transition of ⁇ - ⁇ *.
  • the Ame-Cu complex produced an absorption platform between 375-450 nm, which was caused by red shift of the band I, indicating that the cinnamic acid system participated in the coordination.
  • the position of the band II did not change significantly, the absorption intensity decreased relatively, and at 298 nm. New absorption peaks have also been generated.
  • Figure 3 is a mass spectrum of Ame in positive ion mode with the excimer ion peak m/z 539.0920 assigned to [Ame+H] + .
  • Figure 4a is a mass spectrum of the Ame-Cu complex. It can be seen from the figure that the main excimer ion peak of the Ame-Cu complex is an ion peak with two positive charges, and each has a positive charge, respectively m/ Z678.0256 and m/z 756.0403.
  • Figure 4b is an isotope mass spectrum of the ion peak m/z 678.0256.
  • the isotope peak of the ion peak m/z 678.0256 is m/z 679.0284, m/z 680.0243, m/z 681.0268, m/z 682.0297, adjacent
  • the molecular weights of the ion peaks differed by 1.028, 0.9959, 1.0025, and 1.0029, respectively, confirming that the ion peak carries a positive charge.
  • the solvent of the complex is DMSO
  • DMSO contains oxygen atom and sulfur atom, has strong coordination ability, and is difficult to ionize, so the complex may contain DMSO, and it is speculated that m/z 678.0256 belongs to [Cu(Ame- H) (DMSO)] + , the elemental composition is C 32 H 23 O 11 CuS, which is consistent with the possible elemental composition of m/z 678.0256 (Fig. 4c).
  • [Cu(Ame-H)(DMSO)] + was simulated by mass spectrometry software to obtain an analog mass spectrum, as shown in Figure 4d.
  • the isotope ion peaks of [Cu(Ame-H)(DMSO)] + are m/z 678.0252, m/z 679.0285, m/z 680.0234, m/z 681.0267, m/z 682.0301, and m, respectively.
  • the isotope mass spectrum peak matching of /z 678.0256 is very high. Therefore, it can be confirmed that the ion corresponding to the ion peak m/z 678.0256 is [Cu(Ame-H)(DMSO)] + .
  • the ion corresponding to the ion peak m/z 826.0636 (Fig.
  • the ions [Cu(Ame-H)(DMSO)] + and [Cu(Ame-H)(DMSO) 2 ] + are similar in structure, and the difference is only that they contain different amounts of DMSO molecules because of the ion [Cu(Ame -H)(DMSO) 2 ] + Loss of one DMSO under the influence of the instrument voltage, therefore, the structure of the complex ion is [Cu(Ame-H)(DMSO) 2 ] + .
  • the metal salt in the test is a nitrate or a hydrochloride
  • the complex contains nitrate or chloride ions. Therefore, the structural formula of the Ame-Cu complex is as follows:
  • X is NO 3 - or Cl - .
  • the antitumor activity of Ame and Ame-Cu complexes was studied by MTT method. The process is as follows:
  • HepG2 HeLa cell strain suspension was inoculated into a 96-well culture plate, 100 ⁇ L per well, (1 ⁇ 10 5 / mL), 37 ° C, cultured in a 5% CO 2 incubator for 24 h;
  • IR is the inhibition rate
  • OD 0 is the absorbance of the control group
  • OD 1 is the absorbance of the sample group
  • Xm is lg (the maximum dose)
  • I is lg (the maximum dose / adjacent dose)
  • P is the positive reaction rate.
  • Pm is the maximum positive reaction rate
  • Pn is the minimum positive reaction rate.
  • Blank sample removal ABTS + ⁇ Free radical ion capacity determination At 25 ° C, 2.9 mL of ABTS + ⁇ free radical ion working solution was added to a 10 mL sample tube, and 100 ⁇ L of DMSO was added. After 5 min of reaction, the UV-visible spectrum was measured, and The absorption intensity A 0 at 730 nm was recorded.
  • Sample removal ABTS + ⁇ Free radical ion capacity determination At 25 ° C, add 2.9ml of ABTS + ⁇ free radical ion working solution in 10mL sample tube, and add 100 ⁇ L of different concentration of sample DMSO solution, after 5min reaction, measure UV- The spectrum was visible and the absorption intensity A 1 at 730 nm was recorded.
  • the ABTS + ⁇ radical ion scavenging rate is calculated according to Equation 4.
  • the Ame and Ame-Cu complexes have a concentration-dependent ability to scavenge ABTS + ⁇ free radicals. Within a certain range, the clearance rate is linear with the concentration.
  • the present invention is drawn to the concentration of the clearance (c) is a graph, to yield the corresponding linear equations, and calculates the maximum inhibition concentration (IC 50 value), and Ame Ame-Cu complex clearance radical ABTS + ⁇ 50 values of IC They were 20.703 and 6.310 ⁇ mol ⁇ L -1 , respectively. It can be seen that the ability of Ame-Cu complex to scavenge ABTS + ⁇ free radicals is significantly stronger than that of Ame.
  • the flavonoid-copper complex of the safflower was synthesized for the first time, and its anti-tumor and anti-oxidation activities were studied. It was found that the anti-tumor and anti-oxidation activities of the complex were stronger than the flavonoid itself, and the flavonoids were opened.
  • the research work of complexes provides an important reference value for the development of new drugs.

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Abstract

本发明公开了双黄酮类-铜配合物及其制备方法和应用。本发明以穗花杉双黄酮为配体，铜离子为中心离子，经反应得到穗花杉双黄酮-铜配合物，用红外光谱、紫外-可见吸收光谱和高分辨质谱对配合物的结构进行表征。同时，本发明研究了穗花杉双黄酮-铜配合物的抗肿瘤和抗氧化活性。MTT法表明穗花杉双黄酮-铜配合物具有较好的抗肿瘤活性，且抗肿瘤活性强于穗花杉双黄酮；邻苯三酚自氧化法、ABTS法均表明穗花杉双黄酮-铜配合物的抗氧化活性强于穗花杉双黄酮本身。双黄酮类-铜配合物的形成，提高了穗花杉双黄酮抗肿瘤、抗氧化活性，有望用于药物的开发。

Description

双黄酮-铜配合物及其制备方法和应用 技术领域
本发明属于有机合成、医药领域，具体涉及一种双黄酮-铜配合物及其制备方法和应用。
背景技术
双黄酮类化合物是裸子植物特有的化学成分，例如银杏、卷柏等，具有抗氧化、抗炎、抗病毒、抗肿瘤等生物活性。其中，穗花杉双黄酮(Amentoflavone，Ame)是双黄酮类化合物中较为常见的一种，其结构式如下所示：
Figure PCTCN2018076736-appb-000001
Sun等研究发现穗花杉双黄酮通过激活hPPARγ来提高抗癌基因的表达，从而达到抑制乳腺癌细胞和宫颈癌细胞的效果[Lee E.,Shin S.,Lee J.etal.Cytotoxic activities of amentoflavone against human breast and cervical cancers are mediated by increasing of pten expression levels due to peroxisome proliferator-activated receptorγactivation[J].Bulletin of the Korean Chemical Society,2012,33(7):2219-2223.]。Chen等研究发现穗花杉双黄酮通过抑制因子NF-kappaB的活性，阻碍血管的生成和癌细胞的新陈代谢，达到抑制肿瘤细胞生长的目的[Chen J.H.,Chen W.L.,Liu Y.C.Amentoflavone induces anti-angiogenic and anti-metastatic effects through suppression of NF-kappa B activation in MCF-7cells[J].Anticancer Research,2015,35(12):6685-6693.]。Lee等研究发现，穗花杉双黄酮可以抑制由紫外辐射引起的金属蛋白酶的表达，从而起到抗氧化、防辐射的作用[Lee C.W.,Na Y.,Park N.,etal.Amentoflavone inhibits UVB-induced matrix metalloproteinase-1expression through the modulation of AP-1mponents in normal human fibroblasts[J].Applied Biochemistry and Biotechnology,2012,166:1137-1147.]。Zhang等研究发现穗花杉 双黄酮和银杏黄素具有一定的抗氧化活性，去除DPPH自由基的能力较强[Zhang Y.P.,Shi S.Y.,Wang Y.X.,etal.Target-guided isolation and purification of antioxidants from Selaginella sinensis by offline coupling of DPPH-HPLC and HSCCC experiments[J].Journal of Chromatography B,2011,879:191-196.]。Li等研究表明，穗花杉双黄酮具有抗氧化活性，可有效清除OH -·，O 2 -·，DPPH·，ABTS +·等自由基，并可以保护DNA免受OH -·引起的氧化损伤[Li X.C.,Wang L.,Han W.J.,etal.Amentoflavone protects against hydroxyl radical-induced DNA damage via antioxidant mechanism[J].Turkish Journal of Biochemistry-Turk Biyokimya Dergisi,2014,39(1):30-36.]。
中药配位化学表明，微量元素和有机化合物反应生成的配合物存在着配合平衡，所以可以呈现出原来成分的生物活性；又由于微量元素间、有机成分间、配合物间以及它们相互间的协同和拮抗作用可以减弱或增强原有各成分的生物活性，也可能产生新的生物活性[曹治权.中药药效的物质基础和作用机理研究新思路(一)-中药中化学物种形态和生物活性关系的研究[J].上海中医药大学学报,2000,14(1):36-39.]。例如，Zhou等研究发现槲皮素稀土配合物清除O 2 -·的能力均强于槲皮素，槲皮素稀土配合物可以抑制多种肿瘤，且抗肿瘤活性均强于槲皮素，其中配合物对膀胱肿瘤细胞具有较强的抑制作用，而槲皮素无此作用[Zhou J.,Wang L.F.,Wang J.Y.,etal.Synthesis,characterization,antioxidative and antitumor activities of solid quercetin rare earth(III)complexes[J].Journal of Inorganic Biochemistry,2001,83:41-48.]。
然而，到目前为止，有关双黄酮配合物的合成及其生物活性的研究未见报道。
发明内容
发明目的：针对现有技术中存在的不足，本发明的目的是提供一种穗花杉双黄酮-铜配合物，满足抗肿瘤和抗氧化活性药物的使用需求。本发明的另一目的是提供一种上述双黄酮-铜配合物的制备方法。本发明还有一目的是提供双黄酮-铜配合物的应用。
技术方案：为实现上述发明目的，本发明的技术方案为：
双黄酮-铜配合物，结构式如下：
Figure PCTCN2018076736-appb-000002
X为NO 3 -或Cl -。
一种制备所述双黄酮-铜配合物的方法：将铜盐用醇溶解后加入到双黄酮的醇溶液中，控制pH为5-7，加热搅拌，反应2-5h，有沉淀产生，将沉淀过滤，用醇和水洗涤后用二甲亚砜作为溶剂重结晶，干燥，得双黄酮-铜配合物。
所用双黄酮为穗花杉双黄酮，但不局限于穗花杉双黄酮，泛指具有5-OH和4-C＝O或5″-OH和4″-C＝O的双黄酮类化合物。
所用铜盐为硝酸铜、氯化铜等醇溶性铜盐。
所用溶剂为包括乙醇、甲醇及不同浓度的甲醇、乙醇水溶液等。
用碱的醇溶液调节pH值，所用碱包括氢氧化钠、氢氧化钾、氨水、乙醇钠、甲醇钠等常用碱类。
反应时，加热温度为30-50℃，反应时间为2-5h。
溶液中双黄酮与铜离子的摩尔比为2-2.5：1。
重结晶所用溶剂为二甲亚砜，干燥方法为冷冻干燥、低温真空干燥等。
所述双黄酮-铜配合物在制备抗肿瘤药物和/或抗氧化药物中的应用。
有益效果：与现有技术相比，本发明首次合成得到了穗花杉双黄酮-铜配合物，采用MTT法研究了Ame-Cu配合物的抗肿瘤活性，结果表明，Ame-Cu配合物抑制肝癌细胞(HepG2)和宫颈癌细胞(HeLa)的能力强于Ame本身，紫外-可见吸收光谱、荧光光谱和粘度法表明Ame-Cu配合物抗肿瘤活性的机理可能是配合物与嵌入式插入DNA，引起细胞凋亡。邻苯三酚自氧化法、ABTS法显示Ame-Cu配合物清除自由基能力强于Ame本身，说明配合物的抗氧化活性强于Ame；有利于进一步开发双黄酮类化合物，为新药研究提供依据，有利于 人类健康事业的发展。
附图说明
图1是Ame和Ame-Cu配合物的红外光谱谱图；
图2是Ame和Ame-Cu配合物的紫外-可见吸收光谱谱图；
图3是Ame质谱图；
图4是Ame-Cu配合物的质谱图；
图5是Ame和Ame-Cu配合物对HepG2细胞的抑制作用结果图；
图6是Ame和Ame-Cu配合物对HeLa细胞的抑制作用结果图；
图7是fDNA对Ame紫外-可见吸收光谱的影响结果图；
图8是fDNA对Ame-Cu配合物紫外-可见吸收光谱的影响结果图；
图9是Ame对fDNA-EB体系荧光发射光谱的影响结果图；
图10是Ame-Cu配合物对fDNA-EB体系荧光发射光谱的影响结果图；
图11是Ame和Ame-Cu配合物对fDNA溶液粘度的影响结果图；
图12是Ame对邻苯三酚自氧化速率的影响结果图；
图13是Ame-Cu配合物对邻苯三酚自氧化速率的影响结果图；
图14是Ame对ABTS +·自由清除能力结果图；
图15是Ame-Cu配合物对ABTS +·自由清除能力结果图。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
实施例1
精确称取53.8mg Ame于圆底烧瓶中，用5mL乙醇溶解，精确称量三水合硝酸铜12.1mg，用5mL乙醇溶解，将硝酸铜溶液滴加到Ame溶液中，向反应溶液中滴加乙醇-氨水(V/V,3:1)溶液，调节pH为6，保持30℃反应4-5h，产生沉淀，过滤，依次用乙醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例2
精确称取53.8mg Ame于圆底烧瓶中，用5mL90％的乙醇溶解，精确称量三水合硝酸铜12.1mg，用5mL90％的乙醇溶解，将硝酸铜溶液滴加到Ame溶液中，向反应溶液中滴加乙醇-乙醇钠溶液，调节pH为5，保持30℃反应4-5h，产生沉淀，过滤，依次用乙醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例3
精确称取53.8mg Ame于圆底烧瓶中，用5mL甲醇溶解，精确称量三水合硝酸铜12.1mg，用5mL甲醇溶解，将硝酸铜溶液滴加到Ame溶液中，向反应溶液中滴加甲醇-甲醇钠溶液，调节pH为7，保持40℃反应3-4h，产生沉淀，过滤，依次用甲醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例4
精确称取53.8mg Ame于圆底烧瓶中，用5mL85％的甲醇溶解，精确称量三水合硝酸铜12.1mg，用5mL85％的甲醇溶解，将硝酸铜溶液滴加到Ame溶液中，向反应溶液中滴加甲醇-甲醇钠溶液，调节pH为7，保持50℃反应2-3h，产生沉淀，过滤，依次用甲醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例5
精确称取53.8mg Ame于圆底烧瓶中，用5mL乙醇溶解，精确称量二水合氯化铜8.5mg，用5mL乙醇溶解，将氯化铜溶液滴加到Ame溶液中，向反应溶液中滴加乙醇-氨水(V/V,3:1)溶液，调节pH为6，保持30℃反应4-5h，产生沉淀，过滤，依次用乙醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例6
精确称取53.8mg Ame于圆底烧瓶中，用5mL90％的乙醇溶解，精确称量二水合氯化铜8.5mg，用5mL90％的乙醇溶解，将氯化铜溶液滴加到Ame溶液中，向反应溶液中滴加乙醇-乙醇钠溶液，调节pH为5，保持30℃反应4-5h，产生沉淀，过滤，依次用乙醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例7
精确称取53.8mg Ame于圆底烧瓶中，用5mL甲醇溶解，精确称量二水合氯化铜8.5mg，用5mL甲醇溶解，将氯化铜溶液滴加到Ame溶液中，向反应溶液中滴加甲醇-甲醇钠溶液，调节pH为7，保持40℃反应3-4h，产生沉淀，过滤，依次用甲醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例8
精确称取53.8mg Ame于圆底烧瓶中，用5mL85％的甲醇溶解，精确称量二水合氯化铜8.5mg，用5mL85％的甲醇溶解，将氯化铜溶液滴加到Ame溶液中，向反应溶液中滴加甲醇-甲醇钠溶液，调节pH为7，保持50℃反应2-3h，产生 沉淀，过滤，依次用甲醇、水洗涤，DMSO重结晶，冷冻干燥得Ame-Cu配合物。
实施例9
对实施例1-8制备的产物进行表征，Ame和Ame-Cu配合物的红外光谱图如图1所示。由图可以看出，Ame在2800-3500cm -1有宽的吸收，这是缔和羟基伸缩振动峰，因为Ame分子中5-OH和4-C＝O之间，5″-OH和4″-C＝O之间形成了分子内氢键。而Ame-Cu配合物中此处的吸收均变窄，且在3407cm -1左右的峰形变得尖锐，说明形成配合物后，分子内氢键遭到破坏；Ame中1657cm -1处的强峰为羰基的伸缩振动引起的，是羰基的特征吸收峰，形成配合物后，此处吸收峰向低波数移动，移动到1630cm -1，说明羰基参与了配位；配合物在622cm -1之间产生一吸收峰，这是Cu-O伸缩振动引起的，是氧原子参与配位的有力证明。因此，可以推测，Ame中的羰基、羟基参与了配位，而最可能的配位点为5-OH，4-C＝O，5″-OH和4″-C＝O。
Ame和Ame-Cu配合物的紫外-可见吸收光谱如图2所示，Ame在337nm(带I)和270nm(带II)处有两个特征吸收峰，这是黄酮类化合物的特征吸收，带I和带II分别对应着桂皮酰系统和苯甲酰系统的紫外吸收，均为π-π*的跃迁引起的。Ame-Cu配合物在375-450nm之间产生吸收平台，为带I红移所致，说明桂皮酰系统参与了配位，带II虽然位置没有明显变化，但是吸收强度相对降低，且在298nm处还产生了新的吸收峰，这些现象表明共属于桂皮酰系统和苯甲酰系统的羰基参与了配位，配位后，共轭体系增大，电子跃迁所需要能量降低，π-π*更易发生，故带I发生红移。而4-C＝O更易发生n-π*跃迁，故在298nm处产生一新峰。可以推断，Cu 2+与Ame形成配合物的位点为5-OH，4-C＝O，5″-OH，4″-C＝O。
在正离子模式下，分别对Ame及Ame-Cu配合物作了质谱分析，并根据质谱模拟得到谱图上分子离子峰对应的离子结构式，并由此推断出Ame-Cu的结构。
图3为Ame在正离子模式的质谱图，准分子离子峰m/z 539.0920归属为[Ame+H] +。图4a为Ame-Cu配合物的质谱图，由图中可以看出，Ame-Cu配合物主要准分子离子峰为带两个正电荷的离子峰，且均带一个正电荷，分别为m/z678.0256和m/z 756.0403。图4b为离子峰m/z 678.0256的同位素质谱图，可以 看到离子峰m/z 678.0256的同位素峰为m/z 679.0284，m/z 680.0243，m/z681.0268，m/z 682.0297，相邻离子峰的分子量分别相差1.0028，0.9959，1.0025，1.0029，从而证实，该离子峰带一个正电荷。由红外光谱和紫外-可见光谱可知，Ame与Cu 2+形成配合物的配位点为5-OH，4-C＝O，5″-OH，4″-C＝O，由于重结晶和溶解配合物的溶剂为DMSO，DMSO含有氧原子和硫原子，具有强的的配位能力，且较难电离，故配合物中可能含有DMSO，由此推测m/z 678.0256归属为[Cu(Ame-H)(DMSO)] +，元素组成为C 32H 23O 11CuS，与m/z 678.0256可能的元素组成一致(图4c)。同时，采用质谱模拟软件对[Cu(Ame-H)(DMSO)] +进行模拟，得到模拟质谱图，如图4d所示。由图可以看出[Cu(Ame-H)(DMSO)] +的同位素离子峰分别为m/z 678.0252，m/z 679.0285，m/z 680.0234，m/z 681.0267，m/z 682.0301，与m/z 678.0256的同位素质谱峰匹配度很高，因此，可以证实，离子峰m/z 678.0256对应的离子为[Cu(Ame-H)(DMSO)] +。同理，离子峰m/z826.0636(图4e)对应的离子为[Cu(Ame-H)(DMSO) 2] +。综上所述，Ame与Cu 2+形成了1:1的配合物，红外光谱和紫外-可见光谱证实Ame与Cu 2+形成配合物的配位点为5-OH，4-C＝O，5″-OH和4″-C＝O，由于5″-OH的活性比5-OH的高，因此，Ame与Cu 2+形成的配合物的配位点为5″-OH和4″-C＝O可能性最大，故[Cu(Ame-H)(DMSO)] +和[Cu(Ame-H)(DMSO) 2] +最有可能的结构式如下所示：
Figure PCTCN2018076736-appb-000003
可以发现离子[Cu(Ame-H)(DMSO)] +和[Cu(Ame-H)(DMSO) 2] +结构相似，区别仅是含有不同数量的DMSO分子，其原因是离子[Cu(Ame-H)(DMSO) 2] +在仪器电压作用下失去一个DMSO，因此，配合物离子的结构为[Cu(Ame-H)(DMSO) 2] +。此外，由于试验中金属盐为硝酸盐或盐酸盐，故配合物中含有硝酸根或氯离子，因此，Ame-Cu配合物结构式如下所示：
Figure PCTCN2018076736-appb-000004
X为NO 3 -或Cl -。
实施例10
采用MTT法研究了Ame和Ame-Cu配合物的抗肿瘤活性，过程如下：
(1)将HepG2、HeLa细胞株悬浮液接种于96孔培养板，每孔加100μL，(1×10 5个/mL)，37℃，于5％CO 2培养箱中培养24h；
(2)培养24h后，弃上清液，加入100μL预先稀释好的样品，每个浓度设10个复孔，于5％CO 2培养箱中培养24h；同时设置对照孔(DMSO、细胞液、MTT)、调零孔(培养基、DMSO、MTT)；
(3)培养36h后，弃上清液，加入100μL含MTT(5mg/mL)的DMEM培养基，继续培养4h；
(4)4h后小心去除上清，每孔加200μL DMSO，在恒温振荡器中充分振荡15min，酶标仪595nm处测定吸光度值，通过OD值计算样品对HepG2、HeLa细胞的抑制率，运用改良Karber公式算出半数抑制浓度IC 50值。
Figure PCTCN2018076736-appb-000005
lgIC 50＝Xm-I(P-(3-Pm-Pn)/4)(公式2)
式中，IR为抑制率，OD 0为对照组的吸光度，OD 1为样品组的吸光度，Xm为lg(最大剂量)，I为lg(最大剂量/相邻剂量)，P为阳性反应率之和，Pm为最大阳性反应率，Pn为最小阳性反应率。
结果如图5-6所示。实验发现Ame-Cu配合物能有效抑制肝癌细胞HepG2和宫颈癌细胞HeLa的生长，IC 50值分别为5.638和5.247μmol·L -1，比Ame的 IC 50值小(Ame的IC 50值分别为13.633和8.040μmol·L -1)，说明Ame-Cu配合物抗肿瘤活性较好，且强于Ame。采用紫外-可见光谱法、荧光光谱法和粘度法研究了Ame和Ame-Cu配合物与鲱鱼精DNA(fDNA)的相互作用，以进一步揭示抗肿瘤活性的机理，所得图谱如图7-11所示，结果表明，Ame和Ame-Cu配合物与fDNA的相互作用为嵌插模式，且配合物与fDNA作用能力均强于Ame。由此可以推测，Ame及其配合物的抗肿瘤活性的机理可能是Ame或其配合物进入细胞内部，与DNA链发生嵌插作用，引起细胞凋亡。由于配合物与DNA的相互作用强于Ame，故其抗肿瘤活性也强于Ame。
实施例11
采用邻苯三酚自氧化法、ABTS法研究了Ame和Ame-Cu配合物清除自由基能力，步骤如下：
(1)邻苯三酚自氧化法
邻苯三酚自氧化速率V 0的测定：25℃下，在10mL样品管中加入2mL的Tris-HCl缓冲液(pH＝8.20)，并加入100μL DMSO作为对照，加入0.8mL蒸馏水后，再加入浓度为2mmol·L -1的邻苯三酚溶液0.2mL，混匀后倒入比色皿中，以纯水为空白，测定322nm处的吸光度，每10s记录一次A值，共反应4min。以t为横坐标，A为纵坐标进行线性回归，其直线斜率为V 0，测定三次，求平均值。
加入样品后邻苯三酚自氧化速率V 1的测定：25℃下，在10mL样品管中加入2mL的Tris-HCl缓冲液(pH＝8.2)，并加入100μL不同浓度的样品DMSO溶液，加入0.8mL蒸馏水后，再加入浓度为2mmol·L -1的邻苯三酚溶液0.2mL，混匀后倒入比色皿中，以双蒸水为空白，测定322nm处的吸光度，每10s记录一次A值，共反应4min。以t为横坐标，A为纵坐标进行线性回归，其直线斜率为V 1，测定三次，求平均值。根据公式3计算自由基清除率。
SR(％)＝(1-v 1/v 0)×100％(公式3)
(2)ABTS法
空白样清除ABTS +·自由基离子能力测定：25℃下，在10mL样品管中加入2.9mL的ABTS +·自由基离子工作液，并加入100μL DMSO，反应5min后，测量紫外-可见光谱，并记录730nm处的吸收强度A 0。
样品清除ABTS +·自由基离子能力测定：25℃下，在10mL样品管中加入2.9ml的ABTS +·自由基离子工作液，并加入100μL不同浓度的样品DMSO溶液，反应5min后，测量紫外-可见光谱，并记录730nm处的吸收强度A 1。根据公式4计算ABTS +·自由基离子清除率。
SR(％)＝(1-A 1/A 0)×100％(公式4)
如图12-15所示。邻苯三酚自氧化法结果表明Ame和Ame-Cu配合物清除O 2 -·自由基IC 50为23.273μmol·L -1和4.5683μmol·L -1，可以发现Ame-Cu配合物清除O 2 -·自由基的能力明显强于Ame。
Ame和Ame-Cu配合物对ABTS +·自由基的清除能力具有浓度依赖性，在一定的范围内，清除率与浓度呈线性关系。本发明绘制了清除率与浓度(c)的曲线，得到相应的线性方程，并计算得到最大半抑制浓度(IC 50值)，Ame和Ame-Cu配合物清除ABTS +·自由基的IC 50值分别为20.703和6.310μmol·L -1，可以看出，Ame-Cu配合物清除ABTS +·自由基的能力明显强于Ame。
本发明首次合成得到了穗花杉双黄酮-铜配合物，并对其抗肿瘤和抗氧化活性进行研究，发现，配合物的抗肿瘤、抗氧化活性均强于双黄酮本身，开辟了双黄酮类配合物的研究工作，为新药研发提供了重要的参考价值。

Claims (10)

  1. 双黄酮-铜配合物，其特征在于，结构式如下：
    Figure PCTCN2018076736-appb-100001
    X为NO 3 -或Cl -。
  2. 一种制备权利要求1所述双黄酮-铜配合物的方法，其特征在于：将铜盐用醇溶解后加入到双黄酮的醇溶液中，控制pH为5-7，加热搅拌，反应2-5h，有沉淀产生，将沉淀过滤，用醇和水洗涤后用二甲亚砜作为溶剂重结晶，干燥，得双黄酮-铜配合物。
  3. 根据权利要求2所述制备双黄酮-铜配合物的方法，其特征在于，所用双黄酮为穗花杉双黄酮。
  4. 根据权利要求2所述制备双黄酮-铜配合物的方法，其特征在于，所用铜盐为硝酸铜、氯化铜。
  5. 根据权利要求2所述制备双黄酮-铜配合物的方法，其特征在于，所用溶剂为甲醇、乙醇及它们的水溶液。
  6. 根据权利要求2所述制备双黄酮-铜配合物的方法，其特征在于，用碱的醇溶液调节pH值，所用碱包括氢氧化钠、氢氧化钾、氨水、乙醇钠、甲醇钠。
  7. 根据权利要求2所述制备双黄酮-铜配合物的方法，其特征在于，反应温度为30-50℃，反应时间为2-5h。
  8. 根据权利要求2所述制备双黄酮-铜配合物的方法，其特征在于，溶液中双黄酮与铜离子的摩尔比为2-2.5：1。
  9. 根据权利要求2所述制备双黄酮-铜配合物的方法，其特征在于，重结晶所用 溶剂为二甲亚砜，干燥方法为冷冻干燥、低温真空干燥。
  10. 权利要求1所述双黄酮-铜配合物在制备抗肿瘤药物和/或抗氧化药物中的应用。
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