Citation: Zhang Lei, Liu Lai, Zheng Chengyue, Wang Yang, Wang Jing, Yao Qizheng. Synthesis of Novel Indirubin Derivatives and Their Effects on the Proliferation, Cell Cycle and Apoptosis in Acute Myeloblastic Leukemia HL-60 Cells[J]. Chinese Journal of Organic Chemistry, ;2017, 37(6): 1523-1529. doi: 10.6023/cjoc201704018 shu

Synthesis of Novel Indirubin Derivatives and Their Effects on the Proliferation, Cell Cycle and Apoptosis in Acute Myeloblastic Leukemia HL-60 Cells

Zhang Lei ^a,*,, ,
Liu Lai ^a ,
Zheng Chengyue ^a ,
Wang Yang ^a ,
Wang Jing ^a,*,, ,
Yao Qizheng ^b

a.
School of Pharmacy, Zunyi Medical University, Zunyi 563003
b.
School of Pharmacy, China Pharmaceutical University, Nanjing 210009

Corresponding author: Zhang Lei, lzhang@zmc.edu.cn Wang Jing, wangjing@zmc.edu.cn
Received Date: 12 April 2017
Revised Date: 28 April 2017

Fund Project: the Undergraduate Training Programs for Innovation and Entrepreneurship of Zunyi Medical University [2014]5811the Department of Science and Technology of Guizhou Province [2014]7565the Discipline Construction Funding Medicinal Chemistrythe Undergraduate Training Programs for Innovation and Entrepreneurship of Zunyi Medical University [2014]2918the Department of Science and Technology of Guizhou Province [2014]7557the National Undergraduate Training Programs for Innovation and Entrepreneurship 201510661009the Priming Scientific Research Foundation for Doctoral Program of Zunyi Medical University F-631

Figures(7)

Acute myelogenous leukemia is a malignant disease of the hemopoietic tissue, which causes great harm to human health, and there is no therapeutic drugs with low toxicity and high efficiency. Indirubin is the active constituent of the traditional chinese medicine qingdai, which has potential anti-leukemia activity. However, poor water solubility and low bioavailability have limited its clinical treatment. To improve the water solubility and anti-leukemia activity of indirubin, hydrophilic amino side chain was linked to the indirubin, and five novel indirubin derivatives were synthesized, which were identified by HRMS, ¹H NMR and ¹³C NMR. Meanwhile, the effects of target molecules on the proliferation of acute myeloblastic leukemia HL-60 cells were evaluated using CCK-8 assay. The results showed that four derivatives displayed potent antiproliferative activity against HL-60 cells. Notably, N¹-(2-dimethylaminoethyl)indirubin (5a) exhibited the best anticacner activity with an IC₅₀ value of (3.564±0.211) μmol/L. Flow cytometry and Hoechst 33342 staining indicated that compound 5a could significantly trigger cell cycle arrest and induce apoptosis of HL-60 cells. Finally, compound 5a could regulate the levels of cell cycle arrest-and apoptosis-related proteins. Together, these findings revealed that compound 5a maybe be a promising lead candidate for the treatment of leukemia.
- indirubin,
- derivatives,
- anticancer activity,
- cell cycle,
- cell apoptosis,
- HL-60 leukemia cells
急性髓细胞白血病(acute myeloid leukemia, AML)是一种血液系统的恶性疾病^[1], 也是成人中最常见的急性白血病.目前, AML治疗的方法主要为化疗, 常见药物有阿糖胞苷、阿霉素、依托泊苷、全反式维甲酸和三氧化二砷等.虽然上述药物在一定程度上能够治疗AML, 但AML患者的预后较差, 10年生存率仍低于5%^[2], 此外, 临床研究显示, 化疗药物会产生较为严重的毒副作用^[3]以及耐药性^[4].因此, 研究新颖的低毒、高效AML药物十分必要.

青黛是一种常见的中药材, 为蓼科植物蓼蓝(Polygonum tinctorium Ait)、爵床科植物马蓝(Baphicacanthus cusiu (Ness) Bremek)或十字花科植物菘蓝(Isatis indigotica Fort.)的茎叶经炮制制得的干燥粉末、颗粒或团块, 具有清热解毒、清肝泻火和凉血止血等功效, 主要用于治疗吐血、小儿惊痫、热毒痈疮、斑疹、恶疮和湿疹等病症.现代医学研究发现, 青黛是中成药当归龙荟丸中抗白血病的主要成分^[5].近年来的研究还表明, 双吲哚类生物碱靛玉红(Indirubin, 图 1)是青黛的有效成分^[6], 其具有抗真菌、抗病毒、抗炎和治疗银屑病等多种药理活性^[7~10].同时, 靛玉红还具有潜在的抗肿瘤作用^{[11, 12]}.

图 1 靛玉红结构 Figure Figure1. Structure of indirubin

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虽然靛玉红显示出重要的生物活性, 但它具有一个大π共轭体系的平面刚性骨架, 因此其水溶性差, 生物利用度低, 这些缺点极大地限制其临床应用^[13].例如, Hsuan等^[14]研究发现, 靛玉红对人急性髓系白血病HL-60细胞具有一定的抑制活性, 但活性较差, LC₅₀值为98.1 μg/L.为了进一步提高靛玉红的水溶性和抗肿瘤活性, 人们对其结构进行了大量的修饰和改造工作, 如将肟基、多元醇、糖基和胺基片段等亲水性结构引入到靛玉红分子中, 发现了一些水溶性强及抗肿瘤活性显著的靛玉红衍生物.例如, Hoessel等^[15]发现, 3'-肟基靛玉红(a, 图 2)能够通过阻滞细胞周期而抑制多种肿瘤细胞株的增殖, 如人淋巴细胞瘤Jurkat细胞、人乳腺癌MCF-7细胞和人急性髓系白血病HL-60细胞等.该课题组^[16]又将亲水性的二醇片段连接到6-溴-3'-肟基靛玉红的肟羟基上, 得到了化合物b (图 2), 其能够显著抑制人神经母细胞瘤SH-SY5Y细胞的增殖, IC₅₀值为0.94 μmol/L. Song等^[17]研究发现, 3'-肟基靛玉红的5-二苯乙酰胺衍生物(c, 图 2)具有较强的抗肿瘤活性, 对多种白血病细胞株均有较强的抑制活性, 其中对HL-60细胞的抑制作用较为显著, IC₅₀值为(1.99±0.01) μmol/L.分子机制研究显示, 化合物c的抗肿瘤活性与诱导细胞凋亡和阻滞细胞周期相关. Libnow等^[18]将多种糖基片段引入到靛玉红的N¹位, 如L-鼠李糖、D-葡萄糖、D-半乳糖和D-甘露糖.体外抗肿瘤活性研究显示, 靛玉红的L-鼠李糖衍生物(d, 图 2)能够选择性抑制MCF-7细胞的增殖, IC₅₀值为(0.67±0.08) μmol/L. Cheng等^[19]合成了多种靛玉红的亲水性衍生物, 其中N-甲基哌嗪衍生物e (图 2)具有较强的水溶性, 并且对人肺癌LXFL529L细胞具有强烈的抑制活性, IC₅₀值为0.54 μmol/L. Wang等^[20]将多种亲水性胺基侧链引入到5-氟靛玉红分子中, 细胞毒活性结果显示, 该系列衍生物能够抑制多种肿瘤细胞株的增殖, 其中化合物f (图 2)对HL-60细胞具有较强的抑制作用, IC₅₀值为1.7 μmol/L, 强于阳性药舒尼替尼.近年来, 笔者^[21~23]也报道过靛玉红-3'-肟、7-氮杂-3'-肟基靛玉红以及7, 7'-氮杂(-3'-肟基)靛玉红等衍生物的合成及其抗肿瘤活性研究.

图 2 已报道的靛玉红类化合物 Figure Figure2. Reported indirubin derivatives

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为了提高靛玉红的水溶性, 进一步增强其抗白血病功效, 本文在靛玉红结构中引入亲水性的胺基侧链, 设计并合成了五个结构新颖的靛玉红衍生物, 其结构经HRMS、¹H NMR和¹³C NMR确证.同时, 采用CCK-8法测试目标产物对HL-60细胞的体外抑制活性.此外, 本文还利用流式细胞术、Hoechst 33342染色和Western blot方法初步研究目标产物的作用机制.合成路线见Scheme 1.

图式1 目标产物的合成 Scheme1. Synthesis of target compounds

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1   结果与讨论

1.1   目标化合物的合成

在室温条件下, 原料靛红1和吲哚乙酸酯2反应制得中间体3. 3与氯代胺基盐酸盐4在碳酸铯条件下制备得到目标产物5a、5b和5d; 在氢化钠条件下制备得到目标产物5c和5e, 合成路线如Scheme 1所示.目标产物的结构经HRMS、¹H NMR和¹³C NMR确证.

1.2   目标化合物的体外抗肿瘤活性

利用CCK-8法研究五个目标产物对HL-60细胞的抗增殖活性, 靛玉红(Indirubin)和依托泊苷(Etoposide)为阳性对照药, 活性数据如表 1所示.经过72 h共同孵化后, 四个化合物显示出较强的抗肿瘤活性, 如5a、5b、5c和5e, 其IC₅₀值分别为(3.564±0.211)、(11.536±0.44)、(4.446±0.459) 和(8.046±0.339) μmol/L.而阳性对照靛玉红没有显示出抗HL-60细胞增殖的作用, 这可能与靛玉红的水溶性较差有关, 其IC₅₀值大于100 μmol/L.依托泊苷是一种临床上用于治疗白血病的药物, 对HL-60细胞具有较强的抑制作用, IC₅₀值为(1.163±0.025) μmol/L.目标产物5a的抗肿瘤活性显著强于母体靛玉红, 与阳性药依托泊苷相当.上述研究表明, 在靛玉红N¹位引入亲水性的胺基侧链, 能够不同程度地增强化合物的抗肿瘤活性.

表 1 目标分子对HL-60细胞的体外细胞毒活性 Table 1. Cytotoxic effects of target molecules against HL-60 cells in vitro

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初步的构效关系研究显示, 将亲水性的胺基侧链连接到靛玉红N¹位有利于提高化合物的抗肿瘤活性.其中, 引入链状胺基链时, 二甲氨基衍生物的抗肿瘤活性优于二乙氨基; 引入环状胺基侧链时, 连接臂为两个碳的吡咯烷和三个碳的哌啶优于两个碳的哌啶.整体上, 引入的胺基链具有较少亲脂性原子或基团时, 化合物的抗肿瘤活性更显著, 如化合物5a和5c.

1.3   化合物5a对HL-60细胞周期的影响

靛玉红是一种作用于细胞周期蛋白激酶的天然产物^[24], 为了进一步研究本文目标产物的抗肿瘤机制, 我们利用流式细胞术检测目标产物5a对HL-60细胞周期的影响.如图 3所示, 化合物5a与HL-60细胞孵化48 h后, 与对照组相比, 实验组HL-60细胞的周期分布发生显著变化.在5 μmol/L浓度下, 化合物5a能够阻滞HL-60细胞周期在S期; 但随着浓度增加到10 μmol/L, 化合物5a将HL-60细胞的周期阻滞在G1期.上述实验结果表明, 目标产物5a能够影响HL-60细胞周期的分布, 并且对周期的影响与药物浓度相关.

图 3 化合物5a对HL-60细胞周期的影响 Figure Figure3. Effects of compound 5a on the cell cycle of HL-60 cells

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1.4   化合物5a对HL-60细胞凋亡的影响

凋亡调控异常在肿瘤发生、发展过程中起到关键作用^[25], 多种抗肿瘤药物能够通过诱导凋亡的方式而产生抗肿瘤活性^[26].为了探索目标产物5a对凋亡的影响, 本文利用Annexin V-FITC/PI双染法研究5a对HL-60细胞凋亡的调节作用.如图 4所示, 作用48 h后, 5和10 μmol/L的化合物5a分别能够诱导HL-60细胞产生55.37%和71.09%的凋亡率, 而对照组细胞的凋亡率为6.65%.上述研究表明, 化合物5a能够浓度依赖性地诱导HL-60细胞凋亡.

图 4 化合物5a对HL-60细胞凋亡的影响 Figure Figure4. Effects of compound 5a on the apoptosis of HL-60 cells

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1.5   Hoechst 33342染色

通过Hoechst 33342染色法, 进一步验证化合物5a对HL-60细胞凋亡的诱导作用.实验结果如图 5所示, 孵化24 h后, 化合物5a能够明显减少HL-60细胞的数量.同时, HL-60细胞出现皱缩、染色质固缩、裂解和凋亡小体等现象.并且, 化合物5a的浓度越高, 上述凋亡现象越显著. Hoechst 33342染色研究进一步证明, 化合物5a能够显著诱导HL-60细胞凋亡.

图 5 Hoechst 33342染色 Figure Figure5. Hoechst 33342 staining

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1.6   化合物5a对HL-60细胞中周期和凋亡相关蛋白表达的影响

在前面的研究中, 化合物5a能够诱导HL-60细胞周期阻滞和凋亡, 因此我们进一步探索化合物5a对HL-60细胞中周期和凋亡相关蛋白(CDK1、CDK2和Cleaved Caspase-3) 表达的影响. HL-60细胞与化合物5a共同孵化48 h后, 利用蛋白质免疫印迹法(Western Blot)检测细胞中相关蛋白的表达, 结果如图 6所示.在5 μmol/L浓度下, 化合物5a对CDK1的表达没有影响, 但能够下调CDK2的表达; 而在10 μmol/L浓度下, 化合物5a能够抑制CDK1和CDK2的的表达.上述研究结果说明, 化合物5a对HL-60细胞中CDK1和CDK2表达的调控受到浓度的影响, 这也与前文的研究结果一致, 即目标产物5a能够影响HL-60细胞周期的分布, 并且对周期的影响与药物浓度相关.

图 6 化合物5a对HL-60细胞中CDK1, CDK2和活化Caspase-3表达的影响 Figure Figure6. Effects of compound 5a on the expressions of CDK1, CDK2 and Cleaved Caspase-3 in HL-60 cells

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此外, 图 6还表明, 随着化合物5a浓度的增加, 其对HL-60细胞中Cleaved Caspase-3的上调作用越显著, 这也与流式细胞术检测凋亡的结果一致.

2   结论

为了进一步提高天然产物靛玉红的水溶性和抗白血病细胞增殖活性, 设计并合成了五个结构新颖的靛玉红衍生物. CCK-8法实验结果显示, 四个目标产物在体外能够有效抑制HL-60细胞的增殖, 强于母体靛玉红.其中, 化合物5a的抗肿瘤活性最为显著, IC₅₀值为(3.564±0.211) μmol/L.初步的分子机制研究表明, 通过调节周期和凋亡相关蛋白的表达, 化合物5a能够显著诱导HL-60细胞周期阻滞和凋亡, 从而发挥抗肿瘤活性.

3   实验部分

3.1   仪器与试剂

核磁共振仪为Agilent-NMR-vnmrs 400型(CDCl₃为溶剂), 质谱仪为AgilentAccurate-Mass-Q-TOF-MS 6520型(HRMS), 熔点为SGWX-4显微熔点仪.薄层层析板和200~300目硅胶购自青岛海洋化工厂, 所用试剂为分析纯.

3.2   实验方法

中间体3的合成参照文献[27].

3.2.3   CCK-8法测定目标产物的体外抗肿瘤活性

以靛玉红和依托泊苷为阳性药, 利用CCK-8法测试目标产物分子对HL-60细胞的体外抑制活性. HL-60细胞接种于96孔板中, 孵化24 h.根据组别设置加入含有测试药物的培养基, 并设立溶媒组、阴性对照组和阳性对照组.药物作用72 h后, 每孔加入10 μL CCK-8溶液, 再孵化3 h.用酶标仪在450 nm处测定每孔的吸光度值(OD), 并计算抑制率.实验结果用SPSS17.0计算得到半数抑制浓度值(IC₅₀).

3.2.7   蛋白质免疫印迹

HL-60细胞与不同浓度5a共同孵化48 h后, 提取细胞中总蛋白, 方法同文献[28, 29].蛋白经变性处理后, 于质量分数为12%的SDS-PAGE凝胶电泳分离转移到NC膜膜上, 加入质量分数为5%的脱脂奶粉的封闭液封闭2 h, 再加入CDK1、CDK2或Cleaved Capase-3一抗于4 ℃下孵化过夜. TBST洗涤三次, 每次10 min, 用相应的二抗室温孵化2 h, TBST洗涤三次, 每次10 min.最后, 用ECL化学发光试剂检测蛋白质印迹, 并用G-BOX ChemiXR5凝胶成像分析系统成像.

辅助材料(Supporting Information)目标产物的¹H NMR, ¹³C NMR, HRMS谱图.这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.

3.2.5   细胞凋亡检测

将HL-60细胞接种到6孔板中, 孵化24 h.再根据组别设置, 加入不同浓度的5a培养基, 并设立阴性对照组.药物作用48 h后, 加入500 μL Binding Buffer, 再分别加入5 μL Annexin V-APC以及5 μL 7-AAD, 放置15 min.最后, 用流式细胞仪检测细胞凋亡情况.

3.2.2   目标产物5c和5e的合成

将0.38 mmoL中间体3和0.44 mmoL 4加入到10 mL圆底烧瓶中, 分批加入1.9 mmoL氢化钠, 在N₂保护下60 ℃搅拌反应2~3 h. TLC监测反应完全后, 加入40 mL饱和氯化铵溶液淬灭反应, 二氯甲烷萃取三次, 水洗两次, 饱和氯化钠溶液洗涤一次, 无水硫酸镁干燥过夜.抽滤, 减压除去溶剂, 得到的粗品用200~300目硅胶柱层析[洗脱剂: V(二氯甲烷):V(甲醇)＝50:1]纯化, 得到目标产物5c和5e.

N¹-[2-(1-吡咯烷)乙基]靛玉红(5c):紫色固体, 产率69%. m.p. 181~182 ℃; ¹H NMR (CDCl₃, 400 MHz) δ: 10.51 (s, 1H), 8.86 (d, J＝7.6 Hz, 1H), 7.70 (d, J＝7.2 Hz, 1H), 7.46 (t, J＝7.2 Hz, 1H), 7.27 (t, J＝7.2 Hz, 1H), 7.09 (t, J＝7.6 Hz, 1H), 6.99~6.89 (m, 3H), 3.96 (t, J＝7.6 Hz, 2H), 2.77 (t, J＝7.2 Hz, 2H), 2.64 (s, 4H), 1.81 (s, 4H); ¹³C NMR (CDCl₃, 100 MHz) δ: 188.21, 170.47, 151.56, 141.14, 139.23, 136.83, 129.12, 125.56, 125.18, 122.54, 121.52, 121.22, 119.98, 111.82, 107.99, 106.49, 54.35, 53.21, 39.15, 23.54; HRMS calcd for C₂₂H₂₂N₃O₂[M+ H]⁺ 360.1707, found 360.1707.

N¹-[3-(1-哌啶)丙基]靛玉红(5e):紫色固体, 产率72%. m.p. 130~132 ℃; ¹H NMR (CDCl₃, 400 MHz) δ: 10.52 (s, 1H), 8.87 (d, J＝6.4 Hz, 1H), 7.73 (d, J＝5.6 Hz, 1H), 7.49 (s, 1H), 7.29 (d, J＝6.0 Hz, 1H), 7.11 (s, 1H), 7.01~6.95 (m, 3H), 3.88 (s, 2H), 2.50 (s, 4H), 2.02 (s, 2H), 1.66 (s, 4H), 1.46 (s, 2H), 1.25 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ: 188.27, 170.68, 151.59, 141.24, 139.25, 136.89, 129.20, 125.50, 125.23, 122.58, 121.57, 121.14, 120.01, 111.85, 108.20, 106.54, 56.08, 54.35, 38.04, 29.69, 25.26, 24.76, 23.92; HRMS calcd for C₂₄H₂₆N₃O₂[M+H]⁺ 388.2020, found 388.2021.

3.2.6   Hoechst 33342染色

HL-60细胞与不同浓度5a共同孵化24 h后, 收集细胞, 并浸入4%的多聚甲醛固定液中0.5 h.再加入Hoechst 33342染色液, 室温放置10 min后, 用荧光显微镜观察细胞情况.

3.2.1   目标产物5a, 5b和5d的合成

将0.38 mmoL中间体3, 0.44 mmoL 4, 0.95 mmoL碳酸铯加入到10 mL圆底烧瓶中, 加入5 mL N, N-二甲基甲酰胺(DMF)溶解, 25 ℃下搅拌4~5 h.薄层色谱(TLC)监测反应完全后, 加入40 mL水淬灭反应, 二氯甲烷萃取三次, 水洗两次, 饱和氯化钠溶液洗涤一次, 无水硫酸镁干燥过夜.抽滤, 减压除去溶剂, 得到的粗品用200~300目硅胶柱层析[洗脱剂: V(二氯甲烷):V(甲醇)＝50:1]纯化, 得到目标产物5a、5b和5d.

N¹-(2-二甲氨基乙基)靛玉红(5a):紫色固体, 产率64%. m.p. 167~169 ℃; ¹H NMR (CDCl₃, 400 MHz) δ: 10.52 (s, 1H), 8.86 (d, J＝6.0 Hz, 1H), 7.71 (d, J＝5.6 Hz, 1H), 7.47 (s, 1H), 7.28 (d, J＝6.4 Hz, 1H), 7.10 (s, 1H), 6.98~6.89 (m, 3H), 3.92 (s, 2H), 2.60 (s, 2H), 2.34 (s, 6H); ¹³C NMR (CDCl₃, 100 MHz) δ: 188.22, 170.55, 151.57, 141.12, 139.26, 136.84, 129.11, 125.59, 125.20, 122.56, 121.53, 121.26, 120.00, 111.82, 107.91, 106.49, 56.46, 45.67, 38.12; HRMS calcd for C₂₀H₂₀N₃O₂[M+ H]⁺ 334.1550, found 334.1550.

N¹-(2-乙氨基乙基)靛玉红(5b):紫色固体, 产率57%. m.p. 119~121 ℃; ¹H NMR (CDCl₃, 400 MHz) δ: 10.52 (s, 1H), 8.86 (d, J＝6.8 Hz, 1H), 7.70 (d, J＝6.0 Hz, 1H), 7.46 (s, 1H), 7.28 (d, J＝6.8 Hz, 1H), 7.09 (s, 1H), 6.97~6.88 (m, 3H), 3.89 (s, 2H), 2.73 (s, 2H), 2.64 (d, J＝6.4 Hz, 4H), 1.05 (s, 6H); ¹³C NMR (CDCl₃, 100 MHz) δ: 188.22, 170.51, 151.58, 141.27, 139.20, 136.82, 129.11, 125.55, 125.18, 122.49, 121.50, 121.21, 120.01, 111.81, 107.95, 106.54, 49.95, 47.54, 38.39, 12.00; HRMS calcd for C₂₂H₂₄N₃O₂[M+H]⁺ 362.1863, found 362.1865.

N¹-[2-(1-哌啶)乙基]靛玉红(5d):紫色固体, 产率60%. m.p. 204~206 ℃; ¹H NMR (CDCl₃, 400 MHz) δ: 10.52 (s, 1H), 8.86 (d, J＝7.6 Hz, 1H), 7.71 (d, J＝7.2 Hz, 1H), 7.47 (t, J＝7.6 Hz, 1H), 7.28 (t, J＝6.8 Hz, 1H), 7.10 (t, J＝7.2 Hz, 1H), 6.99~6.90 (m, 3H), 3.94 (t, J＝7.2 Hz, 2H), 2.61 (t, J＝6.8 Hz, 2H), 2.51 (s, 4H), 1.60 (s, 4H), 1.44 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ: 188.23, 170.48, 151.59, 141.26, 139.21, 136.84, 129.11, 125.53, 125.19, 122.51, 121.51, 121.21, 120.00, 111.83, 108.07, 106.56, 56.01, 54.78, 37.63, 25.90, 24.20; HRMS calcd for C₂₃H₂₄N₃O₂ [M+H]⁺ 374.1863, found 374.1862.

3.2.4   细胞周期检测

将HL-60细胞接种到6孔板中, 孵化24 h.再根据组别设置, 加入不同浓度的5a培养基, 并设立阴性对照组.作用48 h后, 收集细胞, 并进行离心, 再用体积分数为70%冰乙醇固定.加100 μL RNase A孵化0.5 h, 再加入400 μL PI进行染色.最后, 用流式细胞仪检测488 nm处荧光值.

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