双酰基单环β-内酰胺衍生物的体外抗肿瘤活性研究
Biacyl Substituted Monocycle β-LactamDerivative for Evaluation as Anticancer Agents

作者: 高海涛 :湖北医药学院药学院,湖北 十堰; 曾小华 :湖北省武当特色中药研究重点实验室及湖北医药学院医学化学研究所,湖北 十堰;

关键词: 双酰基取代单环β-内酰胺体外抗肿瘤活性研究Diacyl Substituted β-Lactam Antitumor

摘要:
目的:研究双酰基取代单环β-内酰胺类化合物对脑胶质瘤细胞(U87)、宫颈癌细胞(HeLa)、肝癌细胞(HepG2)3种肿瘤细胞和1种正常细胞间充质干细胞(MSC)的体外抗肿瘤活性。方法:采用MTT法测定了不同药物浓度对细胞的抑制作用,以半数抑制浓度(IC50)评价目标化合物的抗肿瘤活性。结果:实验结果表明双酰基取代单环β-内酰胺对U87、HeLa、HepG2和MSC正常细胞都具有一定的抑制作用,其中,化合物5a对HepG2细胞增殖的抑制活性最强,其IC50值为8.15 μmol/L。结论:初步的体外抗肿瘤活性测试显示双酰基取代单环β-内酰胺类化合物具有抑制肿瘤细胞的生长,表现出潜在的抗肿瘤活性。

Abstract: Objective: To study antitumor activities of diacyl substituted monocyclic β-Lactam derivatives against U87, HeLa and HepG2 human tumor cell lines and MSC human normal cell lines. Method: The antitumor activities of diacyl substituted monocyclic β-Lactam derivatives were screened using cis-platinum as positive contron by MTT method. Results: Among them 5a stood out as the most po-tent showing an IC50 of 8.15 μmol/L against human tumor cell lines (HepG2). Conclusion: Bioassay of the compounds indicated that diacyl substituted monocyclic β-Lactam derivatives showed potential antitumor activities, which these compounds can be established as lead molecules for developing novel antitumor drugs.

1. 引言

肿瘤对人类的健康和生存构成重大威胁,是世界面临的最重要的社会问题之一。目前,在临床上对于肿瘤的治疗手段主要是化疗方法,然而常用的抗肿瘤药普遍存在疗效低、毒副作用大、易产生多药耐药性等缺点 [1] [2] ,因此研究新型抗肿瘤新药是一项迫切且意义重大的任务。β-内酰胺作为一类重要的四元杂环骨架,是抗生素化合物的核心部分,具有杀菌能力强、毒性低和临床疗效好等优点。单环β-内酰胺是分子内含有吖丁啶2-酮结构的化合物,它除在抗细菌方面的应用外,还具有抗肿瘤、抗结核以及各种酶的抑制剂 [3] 。如单环β-内酰胺Combretastatin A-4类似物对乳腺癌细胞MCF-7和MDA-MB-231具有较强的细胞抑制活性(图1)。因此其合成方法也备受人们的关注 [4] [5] ,特别是多组分反应合成β-内酰胺。例如,通过β-氨基酸、醛和异腈 [6] [7] 或β-酮酸、胺和异腈 [8] [9] 的Ugi 4C-3CR)一步合成β-内酰胺,也有应用Passerini或Ugi的缩合中间体合成β-内酰胺 [10] [11] 等。但2-芳酰基-2-氨酰基单环β-内酰胺的抗肿瘤活性少见报道,这里我们在前期研究基础上 [12] ,采用MTT (3-(4, 5-二甲基噻唑-2) 2, 5-二苯基四氮唑溴盐)法研究新型双酰基取代的单环β-内酰胺抑制肿瘤细胞增殖活性。

Figure 1. β-Lactam CA-4 analogues

图1. β-内酰胺Combretastatin A-4类似物

2. 实验材料

2.1. 实验材料

人脑胶质瘤细胞(U87)、宫颈癌细胞(HeLa)、肝癌细胞(HepG2)间充质干细胞(MSC)由湖北医药学院胚胎干细胞湖北省重点实验室研究所科研工作站传代保种;实验药物参考文献方法 [12] ,以伯胺1、芳基酮醛2、异腈3和α-溴乙酸4各个组分的结构变化合成结构多样性的2-芳酰基-2-氨基酰基吖丁啶-4-酮衍生物5,合成路线和药物结构见图2

Figure 2. Preparation of diacyl substituted β-Lactam derivatives 5

图2. 双酰基单环β-内酰胺衍生物5的制备

2.2. 方法

2.2.1. 细胞培养

脑胶质瘤细胞(U87)、宫颈癌细胞(HeLa)、肝癌细胞(HepG2)和间充质干细胞(MSC,正常细胞)用RPMI-1640培养液于CO2孵箱中 37 ℃ 、5% CO2饱和湿度下培养。贴壁细胞用0.25%胰蛋白酶消化传代。

2.2.2. MTT比色法

将处于对数生长期的细胞制成单细胞悬液,调整细胞浓度为5 × 104个/mL,接种于96孔培养板,每孔接种100 μL,加入测试药物5a-5 h (用培养液稀释10 μmol/L、30 μmol/L、50 μmol/L、100 μmol/L不同浓度),同时设阴性对照组(不加药)和阳性对照(顺铂),每组均设4个复孔,37℃培养24 h,弃上清后加MTT (5 mg/mL) 100 μL,弃去上清液,加DMSO 100 μL,在平板摇床上摇匀,用全自动酶联免疫检测仪于495 nm波长处测定吸光度(OD)值,求出IC50。整个试验操作都在湖北医药学院干细胞研究湖北省重点实验室完成。

抑制率(%) = [A495(阴性对照)-A495(加药组)]/A495(阴性对照) × 100

2.2.3. 结果与讨论

实验数据以c ± s表示,数据分析采用SPSS17.0统计软件进行。用Origin软件,通过半对数拟合直线求IC50值,结果见表1

Table 1. Antitumor activities of diacyl substituted β-Lactam derivatives 5 (IC50, μmol/L)

表1. 双酰基单环β-内酰胺衍生物5的抗肿瘤活性(IC50, μmol/L)

aPositive control.

根据测试数据显示双酰基取代单环β-内酰胺类化合物对脑胶质瘤细胞(U87)、宫颈癌细胞(HeLa)、肝癌细胞(HepG2)三中肿瘤细胞和一种正常细胞间充质干细胞(MSC)都能一定程度抑制细胞的生长,其中U87、HeLa表现出中等程度的细胞毒性,对HepG2肿瘤细胞表现较为突出,IC50值在8.15 μmol/L~36.76 μmol/L,基本与抗肿瘤药物顺铂活性一致(IC50值17.27 μmol/L),对间充质干细胞(MSC)正常细胞IC50值多数在40 μmol/L~50 μmol/L范围,表现出比HepG2细胞较低毒性。基本构效关系分析显示R1和R2为叔丁基或正丁基的活性较好(如:5a、5f),R1和R2为异丙基或环己基活性稍差一点(如:5c、5d),但R1为苯基,R2为环己基时,也表现较好的活性(5 h)。

3. 结论

本文在前期研究基础上,通过体外药物活性试验,采用MTT法测定了双酰基取代单环β-内酰胺类化合物分别对U87、HeLa、HepG2和正常细胞MSC生长的抑制活性,结果显示该类化合物具有潜在的抗肿瘤活性。此研究结果为单环β-内酰胺衍生物的抗肿瘤药物研究奠定了一定的研究基础,对于开发新的先导化合物具有重要意义。

NOTES

*通讯作者。

文章引用: 高海涛 , 曾小华 (2019) 双酰基单环β-内酰胺衍生物的体外抗肿瘤活性研究。 世界肿瘤研究, 9, 27-31. doi: 10.12677/WJCR.2019.91005

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