秋水仙碱的氨(胺)解反应及其衍生物体外抗癌活性研究

秋水仙碱(Colchicine,1)用于治疗痛风乳腺癌、皮肤癌、白血病、何杰金氏病等[1,2],但其毒性过大限制了它的应用[3].因此,对1结构修饰寻求高效低毒的衍生物引起了人们的兴趣.构效研究表明:秋水仙碱的C-10位的取代基对其活性、毒性影响较大,C-10位被氨解或甲胺解后,活性提高,而毒性降低 [4～6].     

Synthesis and evaluation of nitrate derivatives of colchicine as anticancer agents

To search for more potent antitumor agent,a series of novel nitric oxide-donating colchicine(Col) derivatives(6a-f,8a and b) were synthesized by coupling nitrates with N-methyl colchiceinamide.Their cytotoxicity against four human cancer cell lines in vitro were evaluated by MTT assay.It was found that many of the derivatives displayed significant activity,particularly,compounds 6c,8a and 8b showed more potent cytotoxic activities than Col.     

秋水仙碱与人血清白蛋白相互作用的光谱学和电化学研究

用荧光光谱、紫外吸收光谱和电化学方法研究了在近似生理条件下秋水仙碱与人血清白蛋白的相互作用.研究表明,秋水仙碱对人血清白蛋白的荧光猝灭主要是静态猝灭过程,秋水仙碱使人血清白蛋白的构象发生变化.与此同时,在pH=7.4、含0.05%吐温-20的磷酸盐缓冲液中,秋水仙碱在玻碳电极上出现一不可逆的氧化峰,加入人血清白蛋白后秋水仙碱的氧化峰电位正移,峰电流下降.此外,利用光谱学方法和电化学方法测定的秋水仙碱与人血清白蛋白相互作用的结合常数和结合位点数吻合.     

Discovery of Novel Diarylamide N-Containing Heterocyclic Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer Activity

Tubulin has been regarded as an attractive and successful molecular target in cancer therapy and drug discovery. Vicinal diaryl is a simple scaffold found in many colchicine site tubulin inhibitors, which is also an important pharmacophoric point of tubulin binding and anti-cancer activity. As the continuation of our research work on colchicine binding site tubulin inhibitors, we designed and synthesized a series of diarylamide N-containing heterocyclic derivatives by the combination of vicinal diaryl core and N-containing heterocyclic skeletons into one hybrid though proper linkers. Among of these compounds, compound 15b containing a 5-methoxyindole group exhibited the most potent inhibitory activity against the tested three human cancer cell lines (MGC-803, PC-3 and EC-109) with IC₅₀ values of 1.56 μM, 3.56 μM and 14.5 μM, respectively. Besides, the SARs of these compounds were preliminarily studied and summarized. The most active compound 15b produced the inhibition of tubulin polymerization in a dose-dependent manner and caused microtubule network disruption in MGC-803 cells. Therefore, compound 15b was identified as a novel tubulin polymerization inhibitor targeting the colchicine binding site. In addition, the results of molecular docking also suggested compound 15b could tightly bind into the colchicine binding site of β-tubulin.     

秋水仙碱水解产物与某些酸性磺酞类染料相互作用的 共振瑞利散射光谱及其分析应用

在pH 2.9～4.6 Britton-Robinson (BR)缓冲溶液中, 秋水仙碱的水解产物(H-COL)能与溴酚蓝(BPB)、溴甲酚绿(BCG)、溴百里酚蓝(BTB)和百里酚蓝(TB)等酸性磺酞类染料(ASPD)反应形成1∶1的离子缔合物, 此时将引起共振瑞利散射(RRS)的急剧增强, 并产生新的RRS光谱. 秋水仙碱水解产物与溴酚蓝、溴甲酚绿、溴百里酚蓝和百里酚蓝形成离子缔合物的最大散射波长分别位于327, 311, 305和306 nm处. 散射增强(ΔI)与秋水仙碱浓度在一定范围内成正比, 不同体系对于秋水仙碱的检出限(3σ)分别为12.3, 15.1, 16.4和20.0 ng•mL－1 (TB). 研究了适宜的反应条件, 考察了共存物质的影响, 表明方法有较好的选择性. 基于秋水仙碱水解产物与酸性磺酞类染料离子缔合物的反应, 发展了一种较灵敏, 且简便、快捷测定秋水仙碱的新方法. 方法用于片剂、黄花、血清和尿样中秋水仙碱的测定, 获得了满意的结果.     

秋水仙碱分析方法的研究概述

针对秋水仙碱作为治疗急性痛风性关节炎药物的应用,综述了国内外秋水仙碱测量方法研究进展,重点介绍了伏安法、高效液相色谱法、高效液相色谱-质谱联用法等的研究情况.     

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non‐radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine‐, vinblastine‐ and paclitaxel‐binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin‐binding site of 4‐substituted methoxylbenzoyl‐aryl‐thiazoles (SMART‐H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART‐H specifically and reversibly bound only to the colchicine‐binding site, but not to vinblastine‐ or paclitaxel sites. This new non‐radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.     

植物生物分子的电分析化学研究Ⅷ.秋水仙碱的微分脉冲极谱行为及分析应用

秋水仙碱是近年来新发现的有前途的抗癌植物有效成分。秋水仙碱的测定方法,如容量滴定法^[1]、电位滴定法^[2]、紫外分光光度法^[3]、荧光分析法^[4]和高效液相色谱法^[5]等,手续均不太简便。电化学分析研究秋水仙碱主要有阳极伏安法^[6]和交流极谱法^[7],前者因使用固态电极,重现性较差。后者用非水溶剂作介质,灵敏度不高。     

Polymorphism of Inclusion Complexes and Unsolvated Hosts. III. Dimorphism of the Alkaloid Colchicine Hydrate. The Structure of Colchicine Monohydrate

The alkaloid colchicine forms, in addition to the previously known dihydrate host–guest complex, a monohydrate complex. The crystal structure of the monohydrate was determined by direct methods and refined to a final R value of 0.046 for 1425 observed reflections. Crystal data are: orthorhombic, space group P2 12 12 1, a = 9.145(2) Å; b = 13.270(3) Å; c = 17.942(4) Å, V = 2177(1) Å3, Z= 4, Dx = 1.22 g cm-3, T = 293 K. The conformation of the molecule is practically identical with the conformation in the dihydrate inclusion complex. Water molecules show proton donor as well as proton acceptor properties and are hydrogen bonded with the three colchicine molecules giving rise to the three dimensional H-bonded network.     

Bicyclic isothioureas for conjugation with tubulin targeted anticancer agents

Two bicyclic annulated isothiourea derivatives were synthesized using as a key stage either the reaction of isothiocyanate halide with sodium sulfide or cyclization of unsaturated thiourea in the presence of bromine. X-ray molecular structure of N-[(3aSR,7aRS,Z)-hexahydro-1,3-benzothiazol-2(3H)-ylidene]glycine was determined. The conjugate of colchicine with [(3aR,5S,6aS)-2-(tert-butylamino)-3a,5,6,6a-tetrahydro-4H-cyclopenta[d]thiazol-5-yl]methanol obtained demonstrated pronounced cytotoxic effect on cancer cells.     

Inhibition and reversal of myogenic differentiation by purine-based microtubule assembly inhibitors

Using a muscle cell differentiation screen, we have identified myoseverin from a 2,6,9-trisubsituted purine library as a purine-based microtubule binding molecule [1]. Structure-activity relation studies of myoseverin identify positions N2 and N6 to be critical for inhibiting muscle differentiation. Inhibition of microtubule polymerization induced the reversion of terminally differentiated myotubes to mononucleated cells that were responsive to both growth and differentiation conditions, without any observable cytotoxicity. Comparison of myoseverin derivatives to taxol, vinblastine, nocodazole, and colchicine identify myoseverin's effect as being selectively reversible in addition to lacking the cytotoxic effects of these non-purine-based microtubule-disrupting molecules. Myoseverin, as a purine-based microtubule inhibitor, reverted terminal muscle-differentiated cells to a state that was responsive to environmental cues. These results suggest that myoseverin may have applications in muscle regeneration and stem cell differentiation.     

Dyscusins A-C, three new steroids from the leaves of Dysoxylum cumingianum

Three new steroids dyscusins A-C (1-3), including a stigmastane-type sterol and two pregnanes, together with two known steroids were isolated from the leaves of Dysoxylum cumingianum (Meliaceae). Their structures were elucidated on the basis of extensive spectroscopic analyses. In a cytotoxicity assay, compound 1 showed ten-fold enhanced cytotoxicity against multi-drug resistant cancer cells (KB-C2) in the presence of 2.5 μM colchicine as compared with the absence of colchicine. This notable finding indicated that 1 possessed a multi-drug resistant reversal effect.     

Simultaneous determination of colchicine and febuxostat in rat plasma: Application in a rat pharmacokinetic study

A selective, sensitive and rapid LC–MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d₆ as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C₁₈ column. The injection volume and flow rate were 5.0 μl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25–254 and 2.60–622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58–13.0 and 1.03–4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze–thaw cycles and long-term storage at −80 ° C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.     

Synthesis and Biological Evaluation of Novel Uracil and 5-Fluorouracil-1-yl Acetic Acid-Colchicine Conjugate

A series of novel uracil and 5-fluorouracil-1-yl-acetic acid-colchicine derivatives(6a-6n) was synthesized via coupling uracil and 5-fluorouracil(5-FU) with C-10 analogues of colchicine. The antitumor activities of the target compounds against human hepatocellular carcinoma(BEL7402) cells, human ovary carcinoma(A2780) cells, human lung adenocarcinoma(A549) cells and human breast carcinoma(MCF7) cells were tested in vitro, and the structure-activity relationship(SAR) of the compounds was also studied. The bioassay results demonstrate that most of the tested compounds display significant activity and particularly, compounds 6a, 6e, 6h and 6l show more potent cytotoxic activities than 5-fluorouracil and colchicine. The results show that the new derivatives of colchicine are potential suppressors on human cancer.     

秋水仙的化学成分

秋水仙碱（Ｃｏｌｃｈｉｃｉｎｅ,１）是从欧洲百合科植物秋水仙（ｃｏｌｃｈｉｃｕｍａｕｔｕｍｎａｌｅＬ．）中发现的重要生物碱［１,２］。国内生产秋水仙碱主要是云南省丽江山慈姑中提取［３］．但资源日益减少。自１９９４年以来,云南省昭通地区引种欧洲秋水仙植物成功,现已扩大规模载培。因此,研究其中的化学成分,对利用云南省生物资源、发展经济有着重要的意义。本文首次对云南省昭通地区引种欧洲百合科植物秋水仙花和球茎的化学成分进行了研究。从秋水仙干花中分离、鉴定出六个生物碱：秋水侧碱（１）,２－去甲秋水仙碱（２…     

An Optical-Fiber Sensor for Colchicine Using Photo-Polymerized N-Vinylcarbazole

An optical-fiber sensor for colchicine has been fabricated. It is based on the use of the fluorescent dye N-vinylcarbazole, which contains a vinyl unit making it photo-copolymerizable in the presence of a monomer, 1,2-cyclohexanediol diacrylate, on a quartz surface of an optode slide. Prior to the photo-polymerization process the quartz surface was modified with -(methacryloxy)propyl trimethoxysilane to introduce vinyl units. A dye-incorporating optode membrane tightly immobilized on the quartz surface was prepared in this way. The spectral characteristics of the optode for sensing colchicines were evaluated, and the response mechanism was discussed. The optode membrane has a relatively long lifetime and short response and recovering time. The linear range of determining colchicine is from 1.0×10^–6 to 4.0×10^–3molL^–1. The sensor was applied to determine the colchicine content in pharmaceutical tablets the results of which agreed with those obtained by the pharmacopoeia method.Received December 12, 2002; accepted March 25, 2003 Published online July 16, 2003     

Successive construction of cellulase hyperproducers of Trichoderma using hyperpolyploids

When the swollen conidia of Trichoderma reesei QM 6a are treated with 0.1% (w/v) colchicine solution, huge autopolyploid nuclei can be formed in those swollen conidia. When a mycelial mat derived from such a conidum is treated with a haploidizing reagent, benomyl, many fan-shaped sectors are produced from the colony, and cellulase hyperproducers are selected from conidia on the colony. When colchicine and benomyl treatments are repeated on cellulase hyperproducers, new hyperproducers can be constructed successively and systematically. Moreover, when conidia derived from autopolyploids are treated with ethylmethanesulfonate solution, another type of cellulase hyperproducers (polyploids) can be obtained.     

Ligands of the colchicine site of tubulin: A common pharmacophore and new structural classes

Structure-activity relationships for ligands of the colchicine site of tubulin were analyzed based on their common pharmacophore. The role of the elucidation of the three-dimensional structure of the colchicine site of tubulin on the development of studies aimed at the search for the ligands of this site is analyzed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 655–662, April, 2007.     

秋水仙碱立体异构体电子结构及圆二色谱性质的理论研究

采用量子化学密度泛函理论(DFT)在B3LYP/6-311++G二水平上对秋水仙碱四个立体异构体分子几何构型进行了优化,在优化的基础上进行了振动圆二色谱(VCD),紫外-可见光谱(UV-Vis)和电子圆二色谱(ECD)研究.为模拟真实条件,以水为溶剂,计算其对分子电子结构和光谱性质的影响.研究结果表明:秋水仙碱四个立体...     

Aziridine and hydroxy- and chloroethylamine derivatives of colchicine and their biological activity

With the aim of enhancing the cytostatic properties of the initial alkaloid, new aziridine and bis(chloroethyl)amine derivatives of colchicine have been synthesized by the direct interaction of colchicine with chloroethylamine hydrochloride and also via the mono- and diethanolamine derivatives. The structures of the compounds obtained have been studied by spectral methods. An increased radiomodifying and antitumoral activity and a decreased toxicity of the substances synthesized as compared with the initial colchicine has been shown. Results obtained in the National Cancer Institute of the USA from the study of the cytostatic activity of the bis(chloroethyl)amino derivative of 60 tumor lines are presented. Institute of Oncology and Radiology, Academy of Sciences of the Republic of Uzbekistan. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 782–789, November–December, 1998.