云南红豆杉抗肿瘤活性成分的研究

云南红豆杉(Taxus yunnanensis Cheng et L.K.Fu)树皮的乙醇提取物显示较强的抗肿瘤活性,从中分离得到8个紫杉烷类二萜及其生物碱。经光谱分析和化学反应鉴定7个已知物为taxinine E(1),taxinine J(2),1-acetoxy-5-deacetvl baccatin Ⅰ(4),baccatin Ⅲ(5),taxol(6),cephalomannine(7)和7-xylosyl-10-deacetyl taxol(8)。化合物3命名为云南红豆杉甲素(ymlnanxane),为一新的紫杉烷二萜化合物,其结构为taxa-4(20),11-diene-2α,5α,10β,14β-tetraol-2α,5α,10β-triacetate-14β-αmethyl-β-hydroxyl butyrate,并通过X-射线单晶衍射予以证实.经体外筛迭化合物3具有抗肿瘤活性。     

紫杉烷类化合物 SINENXAN A 的结构修饰及其衍生物的构效关系研究

为寻找新的综合性能好的紫杉烷类化合物,对sinenxanA(SIA)进行结构修饰。在SIA及其修饰物的C₁₄位引入不同的的侧链,共合成新的紫杉醇类似物21个,对其中的15个化合物进行了3种癌细胞的体外抗肿瘤活性测定,这些化合物的活性与紫杉醇比较,相差甚远。本文初步讨论了SIA衍生物的构效关系。     

β-榄香烯氨基酸衍生物的合成及抗肿瘤活性

目的设计合成β-榄香烯氨基酸衍生物,并进行体外、体内抗肿瘤活性研究。方法采用烯丙位的氯代反应合成β-榄香烯氯代物,再与氨基酸甲酯反应合成目标化合物。采用磺酰罗丹明B(SRB)染色法测定目标化合物体外对肿瘤细胞增殖的抑制作用,以小鼠腋下移植瘤为模型进行体内抗肿瘤试验,考察化合物5h对Lew is肺癌LL/2、肝癌H22模型的抑制作用。结果共合成15个β-榄香烯氨基酸和β-榄香烯氨基酸甲酯衍生物,其中12个化合物未见文献报道,合成化合物的结构经红外光谱、核磁共振氢谱和质谱确证。大部分目标化合物对人癌细胞HL-60、HeLa、SGC-7901的IC50值低于β-榄香烯。体内试验结果显示,化合物5h对Lewis肺癌LL/2、肝癌H22的生长有显著抑制作用。结论在β-榄香烯结构中引入氨基酸或氨基酸甲酯结构片段有利于提高此类化合物的抗肿瘤活性。     

黑乳海参皂苷nobiliside B及单乙酰化物体外抗肿瘤活性研究

目的研究黑乳海参中化合物nobiliside B及单乙酰化衍生物体外抗肿瘤活性及溶血作用。方法no-biliside B溶于吡啶,冰浴中与醋酐反应15 min,室温反应5h,应用多种色谱技术对反应产物分离纯化,根据化合物的理化性质和波谱数据鉴定其结构。采用磺酰罗单明B法,测定化合物的体外抗肿瘤活性。结果确定nobiliside B单乙酰化衍生物结构为:3-O-[6″-乙酰氧基-β-D-吡喃葡萄糖-(1(3)-4′-O-磺酸钠-β-D-吡喃木糖]-海参烷-9-烯22,25-环氧-3β,17α-二醇(2);活性测试结果表明,化合物2在保持抗肿瘤活性的同时,溶血活性显著降低。结论通过结构修饰,得到1个活性强,毒副作用小的新化合物。为进一步研发海参皂苷类化合物抗肿瘤新药提供了试验依据。     

转筋草的抗肿瘤迁移化学成分研究

目的研究转筋草Pachysandra terminalis中抗肿瘤迁移活性成分。方法综合运用硅胶柱色谱、凝胶柱色谱、HPLC制备色谱等多种色谱法对转筋草提取物中的活性部位进行分离纯化;采用NMR等波谱学方法鉴定化合物结构;通过趋化实验和伤口愈合实验评价单体化合物的抗肿瘤迁移活性。结果从转筋草中分离得到6个化合物,分别为粉蕊黄杨二醇A(1)、(+)-(20S)-20-dimethylamino-3-(3α-methylsenecioylamino)-5α-pregn-12β-ol(2)、2,4-二羟基苯乙酮(3)、香草酸(4)、epipachysamine D(5)、β-谷甾醇(6)。抗肿瘤迁移实验结果表明:化合物2、5在浓度为10μg·m L-1时趋化抑制率分别为74.1%、82.5%。结论化合物1⁴均为首次从该植物中分离得到;化合物2、5有抗肿瘤迁移活性。     

咔啉类衍生物的合成及抗肿瘤活性研究

目的合成咔啉类衍生物并研究其抗肿瘤活性。方法以α-咔啉、β-咔啉以及1,2,3,4-四氢-β-咔啉-3-羧酸为原料,经过多步反应分别得到6-取代α-咔啉类、6-取代β-咔啉类以及3-取代β-咔啉类衍生物,用MTT法考察目标化合物对肿瘤细胞的抑制作用。结果合成了15个新的咔啉类衍生物,结构经过1H NMR和ESI-M S确证。结论 MTT法测试所得的目标化合物对2种受试细胞株均具有一定的抗肿瘤活性,部分化合物显示出与阳性对照紫杉醇相当或更佳的肿瘤细胞抑制活性。     

1-吲哚基取代β-咔啉生物碱及其衍生物的合成和初步抗肿瘤活性

目的设计合成eudistomin U及其6位甲氧基/溴取代衍生物和5′位溴取代衍生物并测试其抗肿瘤活性。方法以吲哚-3-甲醛或5-溴-吲哚-3-甲醛和色胺或取代色胺为原料先缩合,再通过Pictet-Spengler反应环合得到四氢-β-咔啉,然后用DDQ脱氢芳构化得到目标化合物。结果合成了eudistomin U及其6位甲氧基/溴取代衍生物和5′位溴取代衍生物,利用核磁共振、质谱、高分辨质谱确认结构。结论合成了海洋生物碱eudistomin U及其一系列衍生物,初步体外抗肿瘤试验结果表明它们均具有一定的抗肿瘤活性。     

4-烷硫基-4-脱氧-4′-去甲表鬼臼毒素的合成和抗肿瘤活性

对4’-去甲表鬼臼毒素的C_4位进行化学修饰,合成和筛选了10个4-烷硫基-4-脱氧-4’-去甲表鬼臼毒素衍生物以进一步研究C_4位不同的原子和取代基与活性之间的关系及寻找结构简单、活性更强的抗肿瘤新药。4’-去甲表鬼臼毒素与硫醇在三氟化硼·乙醚或三氟乙酸存在下生成相应的硫醚,也可用硫醇与4β-溴-4-脱氧-4’-去甲表鬼臼毒素反应生成相应的硫醚。在体外筛选中,化合物10和12抑制L1210白血病细胞的活性与依托泊甙相当或更强,化合物9,10,12和15抑制KB细胞的活性与依托泊甙相当或更强。     

3-取代苯基苯并吡喃酮类化合物 的设计合成及抗肿瘤活性

目的 在7-甲氧基或7-羟基苯并吡喃酮的3位引入各种取代苯基,以发现抗肿瘤活性更强的异黄酮类化合物。方法 以丹皮酚和甲酸乙酯为原料,经多步反应制得关键中间体3-碘-7-甲氧基苯并吡喃酮(5),再经Suzuki coupling反应制得目标化合物,通过1H-NMR、MS和IR方法确定目标化合物的结构,部分化合物还进行了13C-NMR测定。选择人结肠癌细胞株HCT116和人肝癌细胞株7721为试验瘤株,以姜黄素和大豆异黄酮为阳性对照测定体外抗肿瘤活性。结果 设计合成的20个新目标化合物均有一定的体外抗肿瘤活性,其中化合物6, 9, 16和19的活性较好,与对照品姜黄素的IC50值相当, 明显优于对照品大豆异黄酮的IC50值。结论 可以通过引入不同的3-取代苯基改变异黄酮类化合物的抗肿瘤活性;在这类化合物的3位苯基上引入甲基、甲氧基或三氟甲基体积较小的基团似乎有利于其抗肿瘤活性。 关键词：化学合成; 苯并吡喃酮; Suzuki coupling偶联反应; 抗肿瘤活性     

4-酰硫基-4-脱氧-4-去甲表鬼臼毒素衍生物的合成和抗肿瘤活性

为研究4’-去甲表鬼臼毒素的C_4位不同原子及不同取代基类型同活性之间的关系,寻找活性高、毒性低的抗肿瘤新药,合成了11个4-酰硫基-4-脱氧-4’-去甲表鬼臼毒素,衍生物并进行了抗肿瘤活性试验。4-巯基-4-脱氧-4’-去甲表鬼臼毒素和不同的羧酸在二乙氧基磷酰氰酯存在下得相应的硫酯。该类化合物在L1210白血病细胞和KB细胞的体外抑制试验中普遍表现出抑制活性。化合物10的活性与依托泊甙相当。其余活性比依托泊甙差。与相应的C_4位酰氨基的4’-去甲表鬼臼毒素衍生物相比,活性明显弱。提示氮取代的衍生物活性更好。     

新型14β-侧链紫杉醇衍生物的合成及构效关系研究

以生物合成得到的紫杉烷 sinenxan A为起始原料,合成一系列新的紫杉醇衍生物,以寻找高效低毒、抗瘤谱广、综合性能好的新一代紫杉醇类抗癌药,并进行构效关系研究。从半合成的紫杉烷中间体7出发,分别经5步和6步反应成功地合成了4位羟基和4位乙酸酯两类共8个新的14β 侧链紫杉醇衍生物,2位基团为苯甲酸酯、间氯苯甲酸酯、正戊酸酯和苯乙酸酯。将目标化合物连同已合成的2个14β-侧链紫杉醇衍生物进行了微管聚合试验和体外肿瘤细胞抑制试验。所有化合物在浓度为10μmol·L^-1时对微管无作用。在体外肿瘤细胞抑制试验中,大部分化合物显示边缘细胞毒活性。14β-侧链紫杉醇衍生物的构效关系与13α-侧链紫杉醇衍生物有所不同,2位脂肪酸酯与2位芳香酸酯活性相当,表明2位基团的改变对活性无明显影响。4位羟基衍生物的活性好于4位乙酸酯。     

PEG-PE/phosphatidylcholine mixed immunomicelles specifically deliver encapsulated taxol to tumor cells of different origin and promote their efficient killing

Mixed micelles were prepared from poly(ethyleneglycol)-distearyl phosphoethanolamine (PEG2000-PE) and egg phosphatidylcholine. The micelles were covalently modified with the nucleosome-specific monoclonal antibody 2C5 known to recognize and bind a variety of tumor cells via their surface-bound nucleosomes. Covalent attachment of 2C5 antibody was performed via a micelle-incorporated PEG-PE with the distal terminus of the PEG block activated with p-nitrophenylcarbonyl group (pNP-PEG-PE). Micelle surface-attached 2C5 antibody maintained its specific activity. 2C5-targeted immunomicelles were able to carry more than 3 wt% of taxol. Taxol-loaded immunomicelles specifically recognized tumor cell lines of several types. The cytotoxicity of 2C5-targeted taxol-loaded immunomicelles in a cell culture model was much higher when compared with free taxol or taxol in non-targeted micelles.     

PEG-PE/Phosphatidylcholine Mixed Immunomicelles Specifically Deliver Encapsulated Taxol to Tumor Cells of Different Origin and Promote their Efficient Killing

Mixed micelles were prepared from poly(ethyleneglycol)-distearyl phosphoethanolamine (PEG 2000 -PE) and egg phosphatidylcholine. The micelles were covalently modified with the nucleosome-specific monoclonal antibody 2C5 known to recognize and bind a variety of tumor cells via their surface-bound nucleosomes. Covalent attachment of 2C5 antibody was performed via a micelle-incorporated PEG-PE with the distal terminus of the PEG block activated with p -nitrophenylcarbonyl group (pNP-PEG-PE). Micelle surface-attached 2C5 antibody maintained its specific activity. 2C5-targeted immunomicelles were able to carry more than 3 wt% of taxol. Taxol-loaded immunomicelles specifically recognized tumor cell lines of several types. The cytotoxicity of 2C5-targeted taxol-loaded immunomicelles in a cell culture model was much higher when compared with free taxol or taxol in non-targeted micelles.     

白花前胡甲素联用低浓度紫杉醇对卵巢癌细胞紫杉醇耐药株A2780/TAX的作用研究

目的: 探讨白花前胡甲素(praeruptorin A, PA)增强卵巢癌A2780/TAX细胞对紫杉醇化疗敏感性的作用及机制,为卵巢癌的减毒增敏治疗提供新思路。方法: 体外培养A2780/TAX细胞,分组(对照组、紫杉醇组、PA组、PA+紫杉醇组)干预;噻唑蓝溴化四唑(MTT)法检测细胞活力;乳酸脱氢酶(LDH)细胞毒性检测法检测细胞毒性;克隆形成实验评价细胞增殖能力;流式细胞术Annexin V-FITC/ PI双染法检测细胞凋亡情况;蛋白免疫印迹实验检测Bax、Bcl-2、Cleaved Caspase 3、Caspase 3、Cleaved PARP、PARP及 MMP9、MMP2的蛋白表达水平;划痕实验检测细胞迁移情况。结果: PA与低浓度紫杉醇联用显著抑制A2780/TAX细胞活力和增殖能力;流式细胞术Annexin V-FITC/ PI双染结果显示,联用组的细胞凋亡率显著高于紫杉醇单用组;蛋白免疫印迹结果显示,与对照组、紫杉醇组相比,PA与低浓度紫杉醇联用能显著降低Bcl-2、Caspase 3、PARP、MMP9、MMP2蛋白表达量,提高Bax、Cleaved Caspase 3和Cleaved PARP蛋白水平;划痕实验结果显示,PA与低浓度紫杉醇联用能抑制A2780/TAX细胞迁移。结论: PA与低浓度紫杉醇联用可抑制A2780/TAX细胞活力和增殖能力,诱导细胞凋亡,并通过下调MMP9和MMP2表达抑制细胞迁移。     

A semisynthetic cabazitaxel analogue,C1 overcomes P-gp-meditaed multidrug resistance in non-small-cell lung cancer

OBJECTIVE To investigate the effect of C1 against P-glycoprotein(P-gp)-meditaed multidrug resistance(MDR) in non-small-cell lung cancer cells. METHODS Human non-small-cell lung cancer(NSCLC) cell lines A549, H1299, H460, normal lung cell lines MRC5,taxol-resistant daughter line A549/TR and cisplatin-resistant daughter A549/CR were used to achieve this objective. Cell proliferation was analyzed by cytotoxicity assay,P-gp ATPase activity in vitro was detected by P-gp-Glo?Assay System, P-gp activity in vivo was determined by FACS analyses of intracellular accumulation of Rh123 in A549/TR and A549/CR cells, anti-tumor effect of C1 and taxol were evaluated by parental and drug-resistant cell xenograft of nude mice. RESULTS Cytotoxicity assay results showed that C1 potently inhibits cell proliferation in NSCLC cell lines but not in normal lung cells. However,C1 was more efficacious than taxol in the A549/TR-and A549/CR-treated cells that overproduced P-gp. The drugefflux function of P-gp depends on the ATP hydrolysis that reflects ATPase activity. Taxol, the most widely used taxoid in NSCLC treatment, could inhibit P-gp ATPase activity at lower concentrations, but stimulated it at higher concentrations. In contrast, C1 could significantly inhibit P-gp ATPase activity in a concentration-dependent manner.When the drug-resistant cells were treated in the presence of C1 at 0.1, 0.2 and 0.4 μmol·L-1, the intracellular accumulation of rhodamine 123 was higher than that in A549/TR and A549/CR treated with taxol at the same concentrations, respectively. These results indicated that C1 can reduce drug efflux through inhibiting the transmembrane pumping function of P-gp but not decreasing the expression of P-gp protein. We next compared the efficacy of TM2 and taxol in vivo using A549, A549-Taxol,and A549-CDDP xenograft models. In parental and drugresistant cell xenograft, C1 could significantly inhibit tumor growth in a dose-dependent manner. The TGI for the group receiving docetaxel treatment(8.5 mg · kg~(-1)) was 46.06% and 87.88% for the C1-treated group(10 mg·kg~(-1))in A549 xenografts. The maximal TGI of C1 was 80.4% in A549/TR xenografts, and 54.5% in A549/CR xenografts. Importantly, C1 produced a significant inhibition of tumor growth in drug-resistant cell xenografts, compared with taxol at the same dosing schedule and frequency.Additionally, C1 did not cause reduced body weight of the host mice or other side effects such as hair loss, mortality,and lethargy. CONCLUSION C1 overcomes P-gp-mediated MDR by directly inhibiting its transport function.     

Taxol metabolism. Isolation and identification of three major metabolites of taxol in rat bile

The elimination of nonradioactive taxol in bile and urine was investigated in the rat after administration via the caudal vein (10 mg/kg). As in humans, no metabolites of taxol were detected by HPLC in rat urine, and only 10% of the injected taxol was recovered in urine over a 24-hr period. In contrast, 11.5% and 29% of the injected taxol was recovered in rat bile as unchanged taxol and metabolites, respectively. Among the nine taxol metabolites detected by HPLC, the side chain at C13, which is required for pharmacological activity, had been removed in only one minor metabolite, baccatin III. The chemical structures of the two major hydroxylated metabolites were determined by mass spectrometry (fast atom bombardment and desorption chemical ionization) and 1H-NMR spectroscopy. One was a taxol derivative hydroxylated on the phenyl group at C3' of the side chain at C13, while the other corresponded to a taxol derivative hydroxylated in the m-position on the benzoate of the side chain at C2. Although these two major taxol metabolites were as active as taxol in preventing cold microtubule disassembly, they were, respectively, 9 and 39 times less cytotoxic as taxol on in vitro L1210 leukemia growth. These results show for the first time that there is a significant hepatic metabolism of taxol.     

紫杉烷二萜类化合物精细立体结构研究

抗肿瘤药物紫杉醇(taxol)是一类新型的纺锤体毒药物。已有的关于紫杉醇及其类似物的构效关系研究表明其分子结构中六元A环的C-13侧链与C₄-C₅-C₂₀四元氧环对活性有重要贡献。本文研究了紫杉烷二萜类化合物中5/7/6三环骨架C-4取代方式不同的化合物(4个)及6/8/6三环骨架C-4取代方式不同的3类化合物(10个)的晶体结构,阐述了晶态下紫杉烷二萜类化合物因C-4取代方式不同对分子立体结构的影响,从晶体学角度给出6/8/6/4骨架与抗癌活性的关系。     

Silencing Op18/stathmin by RNA Interference Promotes the Sensitivity of Nasopharyngeal Carcinoma Cells to Taxol and High‐Grade Differentiation of Xenografted Tumours in Nude Mice

Nasopharyngeal carcinoma (NPC) is a refractory tumour, and chemotherapy is one of the primary treatment modalities. Oncoprotein 18 (Op18)/stathmin is a conserved small cytosolic phosphoprotein and highly expressed in tumours, which plays a vital role in maintaining the malignant phenotype of tumours. Taxol is a clinically widely used chemotherapeutic agent for a broad range of taxol‐resistant tumours. This study showed that Op18/stathmin silencing by RNA interference (RNAi) combined taxol cooperatively improved cellular apoptosis in CNE1 cells mainly via initiating endogenous death receptor pathway, impaired the capabilities of cellular proliferation and cellular migration and down‐regulated the half maximal inhibitory concentration (IC₅₀) of taxol, meanwhile decreased the expression of the upstream extracellular regulated kinase 1 (ERK1) in vitro. Evidence also showed that taxol cytotoxicity was markedly augmented for Op18/stathmin RNAi in other NPC cells. In vivo animal experiments have demonstrated that early combination of Op18/stathmin silencing and taxol evidently inhibited tumourigenicity of CNE1 cells and growth of xenografted tumours in nude mice. Remarkably, silencing Op18/stathmin by RNAi still promoted transformation of late‐stage CNE1 cells in NPC‐xenografted tumours from moderately to highly differentiated and inhibited the pleiotropic cytokine interleukin‐10 (IL‐10) autocrine by transplanted tumours. These findings suggest that silencing Op18/stathmin by RNAi promotes chemosensitization of NPC to taxol and reverses malignant phenotypes of NPC, which provides a new clue for treating drug‐resistant tumours.     

Suppression of multidrug resistance by migrastatin

Migrastatin (MGS) is a Streptomyces metabolite that inhibits cancer cell migration. In this study, we found that MGS also enhanced the cytotoxicity of vinblastine, vincristine, and taxol in P-glycoprotein-overexpressing VJ-300 cells and P388/VCR cells. Furthermore, MGS increased the intracellular concentration of labeled vinblastine, vincristine, and taxol in both VJ-300 cells and P388/VCR cells. P-glycoprotein was photolabeled with [3H]azidopine, but this photolabeling was significantly inhibited in the presence of MGS. These results indicated that MGS directly interacts with and inhibits P-glycoprotein, thereby sensitizing drug-resistant cells to anticancer drugs.