齐墩果酸衍生物的合成及抗肿瘤活性研究

引用本文

MENG Yan-qiu, FENG Chu-qiao, ZHANG Liang-feng, ZHANG Meng, ZHAO Na. Synthesis and anti-tumor activity of oleanolic acid derivatives[J]. Acta Pharm Sin, 2015, 50(4): 469-474. 复制到剪切板

孟艳秋, 冯楚侨, 张良锋, 张萌, 赵娜. 齐墩果酸衍生物的合成及抗肿瘤活性研究[J]. 药学学报, 2015, 50(4): 469-474. 复制到剪切板

齐墩果酸衍生物的合成及抗肿瘤活性研究

孟艳秋 , 冯楚侨, 张良锋, 张萌, 赵娜

沈阳化工大学制药工程教研室, 辽宁沈阳 110142

收稿日期: 2014-11-05;修回日期: 2014-11-20.

基金项目：国家自然科学基金资助项目(21372156);辽宁省教育厅高等学校优秀人才支持计划资助项目(LR2013017);沈阳市科技计划资助项目(F13-316-1-47).

* 通讯作者：Tel: 86-24-89383903, Fax:86-24-89383760, E-mail:mengyanqiu@hotmail.com

摘要：以天然产物齐墩果酸为起始原料,对其C-3 位羟基、C-12 位双键、C-28 位羧基进行结构改造,合成了13 个新的齐墩果酸衍生物.目标化合物结构经MS、¹H NMR 及元素分析确证.采用MTT 法,选用高表达人癌细胞(SGC7901、MCF-7 和A549)对目标化合物进行初步的体外抗肿瘤活性研究.结果表明,所测化合物对3种肿瘤细胞的抑制活性均明显强于齐墩果酸,其中化合物II₂和II₃对MCF-7和A549细胞表现出很强的抑制活性,明显高于已上市药物吉非替尼,值得进一步研究.

关键词：齐墩果酸五环三萜类化合物抗肿瘤活性

Synthesis and anti-tumor activity of oleanolic acid derivatives

MENG Yan-qiu , FENG Chu-qiao, ZHANG Liang-feng, ZHANG Meng, ZHAO Na

Department of Pharmaceutical Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China

Abstract: Thirteen novel oleanolic acid (OA) derivatives were designed and synthesized with modification at positions of C-3, C-12 and C-28 of OA. Their structures were confirmed by MS, ¹H NMR and elemental analysis. Their in vitro cytotoxicities against various cancer cell lines (SGC7901, MCF-7 and A549) were evaluated by MTT assay. The results indicated that the tested derivatives were found to have stronger cell growth inhibitory activity than OA. Among them, compounds II₂ and II₃ showed more potent cytotoxicity on MCF-7 and A549 tumor cells than gefitinib (positive control). They are worthy to be studied further.

Key words: oleanolic acid pentacyclic triterpenoid anti-tumor activity

齐墩果酸 (oleanolic acid,OA) 又名士当归酸,是从女贞子、青叶胆等天然植物中分离得到的一种具有齐墩果烷型骨架的五环三萜类天然产物^[1]。研究表明,齐墩果酸具有多种重要的生物活性,如: 保肝、抗炎、抗肿瘤、降血糖及抗溃疡等作用^[2,3,4,5,6]。Finlay等^[7]将齐墩果酸C-3位羟基氧化成酮羰基得到3-氧代齐墩果酸衍生物,通过对前列腺细胞NRP152抑制增长作用的研究,表明齐墩果酸及其3-氧代产物的IC₅₀均大于5.0 μmol·L^-1。Kashiwada等^[8,9]将齐墩果酸C-3位羟基酰化、C-12位双键还原加氢,得到的3-烷酰氧基、C-12位饱和的齐墩果酸衍生物,具有很强的抗HIV作用。在五环三萜类化合物结构修饰的过程中,对羧基的修饰主要为羧基成酯和成酰胺的反应^{[10,11,12,13,14]}。本文以齐墩果酸 (OA) 为先导化合物,在本课题组前期对五环三萜类化合物结构改造以及构效关系研究的基础上^{[15,16,17,18,19]},保留五环三萜骨架,对其C-3、C-12、C-28位进行结构修饰,合成了13个新的齐墩果酸衍生物,并对化合物进行了体外抗肿瘤活性的研究。

以OA为原料,将C-3位羟基与对氯苯甲酸或呋喃甲酸反应成酯,同时C-28位羧基与相应的醇反应成酯,得到3个I系列3-O-对氯苯甲酰 (或呋喃甲酰)-齐墩果烷型-12-烯-28-羧酸酯类化合物。C-12位双键被间氯过氧苯甲酸氧化生成环氧化合物,然后在酸性条件下水解开环引入羟基,同时C-28位羧基与醇反应成酯,得到3个II系列3β,12α,13β-三羟基齐墩果烷-28-羧酸酯类化合物。将C-3位羟基氧化成羰基,然后与盐酸羟胺反应成肟,再与烟酸反应,同时将C-28位羧基与醇反应成酯或与胺反应成酰胺,得到7个III系列3-(N-烟酰氧基)亚氨基-齐墩果烷型-12-烯-28-羧酸酯类化合物。目标化合物的合成见合成路线1。

结果与讨论 1 化学合成

以齐墩果酸 (OA) 为起始原料,经过酰化、氧化、水解、酯化或酰胺化等反应得到13个目标化合物,目标化合物的结构经¹H NMR、元素分析和MS确定,均为新化合物,数据见表 1和表 2。

Table 1 MS and elemental analysis data of target compounds

Compd.	ESI-MS (m/z)	Elemental analysis/Found (Calcd.)
Compd.	ESI-MS (m/z)	C	H	N	O
I₁	624.3 [M+H]⁺	75.38 (75.15)	8.63 (8.89)	-	10.51 (10.27)
I₂	579.6 [M+H]⁺	76.81 (76.78)	9.37 (9.25)	-	14.61 (14.52)
I₃	593.7 [M+H]⁺	76.78 (76.99)	9.47 (9.52)	-	13.27 (13.49)
II₁	547.9 [M+H]⁺	74.91 (74.68)	10.35 (10.69)	-	14.34 (14.63)
II₂	561.5 [M+H]⁺	74.83 (74.95)	10.62 (10.78)	-	14.18 (14.26)
II₃	561.8 [M+H]⁺	75.12 (74.95)	10.89 (10.78)	-	14.52 (14.26)
III₁	603.2 [M+H]⁺	75.76 (75.71)	9.21 (9.03)	4.82 (4.65)	10.60 (10.62)
III₂	665.3 [M+H]⁺	77.42 (77.67)	8.33 (8.49)	4.40 (4.21)	9.87 (9.62)
III₃	616.7 [M+H]⁺	75.68 (75.93)	9.40 (9.15)	4.37 (4.54)	10.53 (10.37)
III₄	631.4 [M+H]⁺	76.38 (76.15)	9.45 (9.27)	4.10 (4.44)	10.32 (10.14)
III₅	645.5 [M+H]⁺	76.52 (76.36)	9.53 (9.38)	4.49 (4.34)	9.75 (9.92)
III₆	625.2 [M+H]⁺	74.70 (74.96)	8.57 (8.39)	8.51 (8.97)	7.70 (7.68)
III₇	650.7 [M+H]⁺	77.80 (77.62)	8.85 (8.53)	6.73 (6.47)	7.16 (7.39)

Table 1 MS and elemental analysis data of target compounds

Table 2 ¹H NMR data of target compounds

Compd.	¹H NMR (CDCl₃,500 MHz)
I₁	δ 7.76-7.40 (m,4H,Ph-H),5.32 (t,J = 7.8 Hz,1H,H-12),4.10 (m,2H,COOCH₂),2.86 (m,1H,H-3),2.70 (t,J = 7.0 Hz,1H,H-18),1.98 (t,J = 11.7 Hz,3H,COOCH₂CH₃),1.78,1.64,1.48 (s,9H,CH₃×3),1.20,1.13 (s,6H,CH₃×2),1.08 (t,J = 7.5 Hz,1H,H-9),0.96 (s,3H,CH₃),0.91 (t,J = 7.5 Hz,1H,H-5),0.87 (s,3H,CH₃)
I₂	δ 7.65-7.13 (m,3H,Ar-H),5.57 (m,1H,H-12),3.86 (m,2H,COOCH₂),3.45 (t,J = 12.0 Hz,1H,H-3),2.67 (t,J = 7.2 Hz,1H,H-18),2.01 (s,3H,COOCH₂CH₃),1.86,1.71,1.52 (s,9H,CH₃×3),1.38,1.25,1.09 (s,9H,CH₃×3),1.05 (t,J = 7.5 Hz,1H,H-9),0.86 (t,J = 7.0 Hz,1H,H-5),0.75 (s,H,CH₃)
I₃	δ 7.61-7.10 (m,3H,Ar-H),5.43 (t,J = 7.8 Hz,1H,H-12),4.21 (t,J = 12.0 Hz,2H,COOCH₂),3.37 (t,J = 12.0 Hz,1H,H-3),2.59 (t,J = 7.0 Hz,1H,H-18),2.00 (m,2H,COOCH₂CH₂CH₃),1.85 (t,J = 11.7 Hz,3H,COOCH₂CH₂CH₃),1.63,1.48,1.30 (s,9H,CH₃×3),1.19 (s,3H,CH₃),1.10 (m,1H,H-9),1.03,0.89 (s,6H,CH₃×2),0.81 (t,J = 7.0 Hz,1H,H-5),0.72 (s,3H,CH₃)
II₁	δ 5.21 (br s,1H,12-OH),4.93 (br s,1H,13-OH),4.65 (br s,1H,3-OH),4.17 (t,J = 7.0 Hz,2H,OCH₂(CH₂)₂CH₃),3.25 (m,1H,H-3),1.89,1.70 (m,3H,OCH₂(CH₂)₂CH₃),1.68 (t,J = 7.5 Hz,1H,H-18),1.56 (t,J = 7.2 Hz,3H,O(CH₂)₂CH₃),1.45,1.28,1.13 (s,9H,CH₃×3),1.06 (s,3H,CH₃),0.98,0.93 (t,J = 7.3 Hz,2H,H-5/9),0.91,0.80,0.72 (s,9H,CH₃×9)
II₂	δ 5.20 (br s,1H,12-OH),4.91 (br s,1H,13-OH),4.66 (br s,1H,3-OH),4.12 (t,J = 7.0 Hz,2H,OCH₂(CH₂)₃CH₃),3.28 (m,1H,H-3),1.90,1.83,1.64 (m,6H,OCH₂(CH₂)₃CH₃),1.60 (t,J = 7.5 Hz,1H,H-18),1.51 (t,J = 6.5 Hz,3H,O(CH₂)₃CH₃),1.47,1.32,1.25 (s,9H,CH₃×3),1.09 (s,3H,CH₃),0.97 (m,2H,H-5/9),0.92,0.83,0.75 (s,9H,CH₃×3)
II₃	δ 5.20 (br s,1H,12-OH),4.92 (br s,1H,13-OH),4.65 (br s,1H,3-OH),4.15 (t,J = 7.5 Hz,2H,OCH₂CH₂CH(CH₃)₂),3.26 (m,1H,H-3),1.85 (m,2H,OCH₂CH₂CH(CH₃)₂),1.78 (t,J = 7.2 Hz,1H,H-18),1.73 (m,1H,OCH₂CH₂CH(CH₃)₂),1.60,1.47 (d,J = 7.0 Hz,6H,OCH₂CH₂CH(CH₃)₂),1.39,1.30,1.21 (s,9H,CH₃×3),1.13 (s,3H,CH₃),0.98 (m,2H,H-5/9),0.95,0.80,0.71 (s,9H,CH₃×3)
III₁	δ 9.25 (s,1H,Py-H),8.81-8.34 (m,2H,Py-H),7.43 (d,J = 6.5 Hz,1H,Py-H),5.30 (m,1H,H-12),4.11 (m,2H,COOCH₂),1.96 (t,J = 9.3 Hz,3H,COOCH₂CH₃),1.77 (t,J = 7.2 Hz,1H,H-18),1.53,1.25,1.07 (s,9H,CH₃×3),1.01 (t,J = 6.5 Hz,1H,H-5),0.94 (s,3H,CH₃),0.89 (t,J = 7.0 Hz,1H,H-9),0.85,0.72,0.61 (s,9H,CH₃×3)
III₂	δ 9.27 (s,1H,Py-H),8.83-8.34 (m,2H,Py-H),7.45 (d,J = 7.0 Hz,1H,Py-H),7.31-7.02 (m,5H,Ph-H),5.30 (m,1H,H-12),4.80 (s,2H,PhCH₂),1.85 (s,3H,CH₃),1.75 (t,J = 7.0 Hz,1H,H-18),1.64,1.32,1.16,(s,9H,CH₃×3),1.09 (t,J = 6.5 Hz,1H,H-5),1.01,0.94 (s,6H,CH₃×2),0.86 (t,J = 7.3 Hz,1H,H-9),0.78 (s,3H,CH₃)
III₃	δ 9.25 (s,1H,Py-H),8.82-8.33 (m,2H,Py-H),7.45 (d,J = 7.0 Hz,1H,Py-H),5.31 (t,1H,J = 6.5 Hz,H-12),4.97 (m,1H,OCH(CH₃)₂),1.96,1.83 (d,J = 8.5 Hz,6H,OCH(CH₃)₂),1.77 (t,J = 7.0 Hz,1H,H-18),1.34,1.20 (s,6H,CH₃×2),1.12,1.10 (s,6H,CH₃×2),1.05 (t,J = 6.5 Hz,1H,H-5),0.96,0.93 (s,6H,CH₃×2),0.85 (t,J = 7.5 Hz,1H,H-9),0.80 (s,3H,CH₃)
III₄	δ 9.24 (s,1H,Py-H),8.80-8.34 (m,2H,Py-H),7.44 (t,J = 5.2 Hz,1H,Py-H),5.30 (m,1H,H-12),3.81 (d,J = 6.5 Hz,2H,OCH₂CH(CH₃)₂),1.87 (m,1H,OCH₂CH(CH₃)₂),1.79,1.68 (d,J = 8.5 Hz,6H,OCH(CH₃)₂),1.60 (m,1H,H-18),1.31,1.19,1.10 (s,9H,CH₃×3),1.02 (t,J = 7.0 Hz,1H,H-5),0.98,0.91 (s,6H,CH₃×2),0.88 (t,J = 7.0 Hz,1H,H-9),0.83,0.75 (s,6H,CH₃×2)
III₅	δ 9.24 (s,1H,Py-H),8.80-8.34 (m,2H,Py-H),7.44 (t,J = 5.0 Hz,1H,Py-H),5.28 (s,1H,H-12),4.03 (t,J = 7.0 Hz,2H,OCH₂(CH₂)₃CH₃),1.92,1.87,1.70 (m,6H,OCH₂(CH₂)₃CH₃),1.68 (t,J = 8.0 Hz,3H,O(CH₂)₄CH₃),1.63 (m,1H,H-18),1.45,1.30,1.15,1.07 (s,12H,CH₃×4),0.98 (t,J = 7.0 Hz,1H,H-5),0.89 (s,3H,CH₃),0.82 (t,J = 7.0 Hz,1H,H-9),0.73,0.65 (s,6H,CH₃×2)
III₆	δ 9.25 (s,1H,Py-H),8.81-8.29 (m,3H,Py-H),7.56-7.07 (m,3H,Ar-H),5.38 (t,J = 6.5 Hz,H-12),1.80 (t,J = 7.3 Hz,1H,H-18),1.31,1.20,1.14 (s,9H,CH₃×3),1.10 (m,1H,H-5),0.98,0.86 (s,6H,CH₃×2),0.82 (t,J = 7.0 Hz,1H,H-9),0.73,0.69 (s,6H,CH₃×2)
III₇	δ 9.24 (s,1H,Py-H),8.78-8.29 (m,3H,Py-H),7.66-7.22 (m,5H,Ar-H),7.06 (br s,1H,NH),5.42 (s,1H,H-12),1.77 (t,J = 7.0 Hz,1H,H-18),1.28,1.13,0.06 (s,9H,CH₃×3),0.97 (t,J = 7.2 Hz,1H,H-5),0.93,0.86 (s,6H,CH₃×2),0.80 (t,J = 6.5 Hz,1H,H-9),0.74,0.66 (s,6H,CH₃×2)

Table 2 ¹H NMR data of target compounds

2 抗肿瘤活性测试

以吉非替尼为阳性对照药,采用MTT法测试目标化合物体外抗肿瘤活性,活性数据见表 3。

Table 3 Inhibitory activity of the target compounds on the SGC7901,MCF-7 and A549 cells proliferation. *The concentration of the target compounds is 1×10^-5 mol·L^-1 and process- time is 72 h; nt: Not tested

Compd.	Inhibition rate*/%			IC₅₀/μmol·L^-1
Compd.	SGC7901	MCF-7	A549	SGC7901	MCF-7	A549
OA	12.6	69.3	22.0	>50	5.62	30.6
I₁	18.3	nt	28.8	41.8	nt	17.2
I₂	5.04	nt	7.95	>50	nt	>50
I₃	17.3	nt	12.4	46.2	nt	>50
II₁	14.6	21.5	35.7	>50	35.1	8.40
II₂	16.7	32.7	42.4	47.3	26.5	7.13
II₃	20.1	38.2	53.8	39.2	20.8	6.85
III₁	18.2	nt	nt	42.7	nt	nt
III₂	21.9	nt	nt	36.1	nt	nt
III₃	25.3	20.1	30.4	29.5	40.2	9.71
III₄	nt	nt	39.5	nt	nt	8.20
III₅	nt	nt	49.7	nt	nt	7.75
III₆	nt	13.7	24.5	nt	>50	18.4
III₇	nt	14.5	20.1	nt	>50	32.1
Gefitinib	43.4	21.6	30.3	10.5	38.8	28.7
Doxorubicin	84.1	81.4	61.0	0.24	0.26	0.57

结果表明: 所测试的目标化合物对SGC7901、MCF-7和A549三种细胞生长均具有一定的抑制作用,其中化合物II₂和II₃对SGC7901、MCF-7和A549三种细胞表现出很好的抑制活性,II₂对SGC7901、MCF-7和A549细胞的抑制率分别为16.7%、32.7% 和42.4%; II₃对SGC7901、MCF-7和A549细胞的抑制率分别为20.1%、38.2% 和53.8%,明显高于母体化合物OA,对MCF-7和A549细胞的抑制活性高于临床常用药物吉非替尼。化合物III₅对A549细胞的抑制率为49.7%,高于OA和吉非替尼。III₃对SGC7901和A549细胞的抑制率分别为25.3% 和30.4%,高于母体化合物OA,对A549细胞的抑制活性与吉非替尼相当。可以看出,在C-3位羟基酯化,C-12位双键氧化,同时在C-28位羧基成酯可以提高其抗肿瘤活性。

Scheme 1 Synthetic routes of target compounds

3 小结

实验结果说明,齐墩果酸C-3位羟基成酯所引入基团的结构与C-28位羧基所连接酯或胺的结构对抑制活性具有较大影响。C-12位双键氧化水解引入羟基,可能提高其抗肿瘤活性,这对进一步开展齐墩果酸的结构改造和抗肿瘤活性研究具有一定的指导意义。

实验部分

Büchi B-540熔点测定仪 (温度未校正); BrukerARX-500型核磁共振分析仪,CDCl₃为溶剂,TMS为内标; 热电-菲尼根LCQ型质谱仪; SPECTRA MAX plus型酶标仪; XDS-1B倒置显微镜; TGL-16B低速台式离心机。齐墩果酸(质量分数 > 98%) 购于西安小草天然产物有限公司; 薄层色谱硅胶GF254 (100～200目,青岛海洋化工厂),显色剂为10% 硫酸乙醇溶液; RPMI-1640培养基 (含10% 胎牛血清,100 u·mL^-1青霉素,100 μg·mL^-1链霉素)、溴化四氮唑盐 (MTT)、胰蛋白酶 (Trypsin) 和标准胎牛血清 (FBS)、SGC7901细胞、MCF-7细胞和A549细胞由沈阳药科大学药理教研室提供。所有试剂均为分析纯或化学纯。

1 化学合成 1.1 3β,12α,13β-三羟基-齐墩果烷-28-酸 (OA-2)

将OA (100 mg,0.219 mmol) 与间氯过氧苯甲酸 (74.8 mg,0.433 mmol) 溶于10 mL二氯甲烷中,室温搅拌过夜。减压蒸除溶剂,加入10 mL水,用二氯甲烷萃取 (10 mL×2)。合并有机相,用饱和碳酸氢钠水溶液洗涤2次,然后水洗至中性,无水硫酸镁干燥,减压蒸除溶剂,得到白色固体。将其溶于10 mL氯仿中,加入2 mL浓盐酸,室温搅拌6 h。加入10 mL水,用氯仿萃取 (10 mL×3),合并有机相,用饱和碳酸氢钠水溶液洗涤3次,然后水洗至中性,无水硫酸镁干燥,过滤,加压蒸除溶剂。粗品经柱色谱纯化,洗脱剂为石油醚-乙酸乙酯=5∶1,得到白色固体68.1 mg,产率63.4%,mp 153.2～154.6 ℃; ESI-MS (m/z): 491.3 [M+H]⁺。

1.2 3-氧代-齐墩果烷型-12-烯-28-羧酸 (OA-3)

参照文献^[15]方法制备3-氧代-齐墩果烷型-12-烯-28-羧酸(OA-3),白色固体90.5 mg,产率90.5%,mp 220.3～222.1 ℃; ESI-MS (m/z): 455.1 [M+H]⁺。

1.3 3-肟基-齐墩果烷型-12-烯-28-羧酸 (OA-4)

参照文献^[15]方法制备3-肟基-齐墩果烷型-12-烯-28-羧酸 (OA-4),白色固体88.1 mg,产率87.3%,mp 188.4～189.8 ℃; ESI-MS (m/z): 470.5 [M+H]⁺。

1.4 3-[N-烟酰氧基]亚氨基-齐墩果烷型-12-烯-28-羧酸 (OA-5)

参照文献^[15]方法通过烟酸 (163 mg,1.32 mmol) 制备3-[N-烟酰氧基]亚氨基-齐墩果烷型- 12-烯-28-羧酸(OA-5),白色粉末状固体60.7 mg,产率62.1%,mp 144.6～146.6 ℃; ESI-MS (m/z): 575.3 [M+H]⁺。

1.5 3-O-对氯苯甲酰-齐墩果烷型-12-烯-28-乙酯 (I₁)

将OA (50.0 mg,0.109 mmol) 和无水碳酸钾 (30.0 mg,0.217 mmol) 加入到5 mL DMF中,缓慢滴加3滴溴乙烷,室温下反应,TLC监测终点,反应完毕后,加入5 mL水,用乙酸乙酯萃取3次,合并有机相,无水硫酸镁干燥,减压蒸除溶剂得白色固体。将其溶解于5 mL二氯甲烷溶液中,加入少量DMAP、DCC和对氯苯甲酸 (20 mg,0.128 mmol),室温搅拌,TLC监测反应终点,反应完毕后,浓缩得到粗品,经硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (10∶1),得白色固体40.7 mg,产率59.7%,mp 146.3～147.5 ℃。

1.6 3-O-呋喃甲酰-齐墩果烷型-12-烯-28-乙酯 (I₂)

按照化合物I₁的合成方法,与呋喃甲酸 (23 mg,0.205 mmol) 在室温条件下反应,粗品经硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (12∶1),得白色固体44.8 mg,产率70.7%,mp 172.6～174.1 ℃。

1.7 3-O-呋喃甲酰-齐墩果烷型-12-烯-28-正丙酯 (I₃)

按照化合物I₁的合成方法,将得到的呋喃甲酰化合物与溴代正丙烷在室温下反应,粗品经硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (10∶1),得白色固体36.5 mg,产率56.2%,mp 167.0～169.3 ℃。

1.8 3β,12α,13β-三羟基-齐墩果烷-28-正丁酯 (II₁)

将OA-2 (100 mg,0.204 mmol) 和无水碳酸钾 (60 mg,0.434 mmol) 加入到5 mL DMF中,缓慢滴加3滴溴正丁烷,室温反应5 h。浓缩,加入5 mL水,用乙酸乙酯萃取 (4 mL×3),合并有机相,饱和NaCl水洗3次,无水硫酸镁干燥,抽滤,减压蒸除溶剂,粗品经硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (7∶1),得白色晶体58.7 mg,产率52.7%,mp 186.0～196.7 ℃。

1.9 3β,12α,13β-三羟基-齐墩果烷-28-正戊酯 (II₂)

按照制备II₁的方法,由中间体OA-2 (100 mg,0.204 mmol) 与1 mL溴正戊烷反应,经硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (8∶1),得白色晶体60.2 mg,产率52.7%,mp 146.6～152.5 ℃。

1.10 3β,12α,13β-三羟基-齐墩果烷-28-异戊酯 (II₃)

按照制备II₁的方法,由中间体OA-2 (100 mg,0.204 mmol) 与1 mL溴异戊烷反应,经硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (6∶1),得白色晶体59.8 mg,产率52.3%,mp 161.1～165.7 ℃。

1.11 3-(N-烟酰氧基)亚氨基-齐墩果烷型-12-烯-28-羧酸乙酯 (III₁)

将OA-5 (100 mg,0.174 mmol) 和无水碳酸钾 (57.0 mg,0.412 mmol) 加入到5 mL DMF中,缓慢滴加3滴溴乙烷,室温反应5 h。浓缩,然后加入5 mL水,用乙酸乙酯萃取3次,合并有机相,饱和NaCl水洗3次,无水硫酸镁干燥,抽滤,浓缩,得白色固体,粗品用硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (7∶1),得白色粉末状固体74.3 mg,产率72.5%,mp 80.6～83.8 ℃。

1.12 3-(N-烟酰氧基)亚氨基-齐墩果烷型-12-烯-28-羧酸苄酯 (III₂)

按照化合物III₁的合成方法,由化合物OA-5 (100 mg,0.174 mmol) 与溴化苄 (116 mg,0.678 mmol) 反应得到粗品,用硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (7∶1),得白色粉末状固体42.1 mg,产率37.2%,mp 83.7～86.7 ℃。

1.13 3-(N-烟酰氧基)亚氨基-齐墩果烷型-12-烯-28-羧酸异丙酯 (III₃)

按照化合物III₁的合成方法,由化合物OA-5 (100 mg,0.174 mmol) 与溴代异丙烷 (103 mg,0.682 mmol) 反应得到粗品,用硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (7∶1),得白色粉末状固体77.8 mg,产率74.2%,mp 90.8～94.6 ℃。

1.14 3-(N-烟酰氧基)亚氨基-齐墩果烷型-12-烯-28-羧酸异丁酯 (III₄)

按照化合物III₁的合成方法,由化合物OA-5 (100 mg,0.174 mmol) 与溴代异丁烷 (93.2 mg,0.680 mmol) 反应得到粗品,用硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (7∶1),得白色粉末状固体72.5 mg,产率67.6%,mp 84.1～86.5 ℃。

1.15 3-(N-烟酰氧基)亚氨基-齐墩果烷型-12-烯-28-羧酸正戊酯 (III₅)

按照化合物III₁的合成方法,由化合物OA-5 (100 mg,0.174 mmol) 与溴代正戊烷 (103 mg,0.682 mmol) 反应得到粗品,用硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (7∶1),得白色粉末状固体77.9 mg,产率71.1%,mp 80.5～83.7 ℃。

1.16 N-[3-(N-烟酰氧基)-齐墩果烷型-12-烯-28-羰基]咪唑 (III₆)

将OA-5 (100 mg,0.174 mmol) 溶解于10 mL二氯甲烷中,加入适量草酰氯,室温搅拌20 h,生成3-(N-烟酰氧基)亚氨基-齐墩果烷型-12-烯-28-酰氯,减压蒸除溶剂,加入2 mL环己烷,减压蒸除环己烷,反复操作2次。将得到的酰氯溶于5 mL二氯甲烷,加三乙胺调pH 9～10,搅拌5 min后,加入咪唑 (46.3 mg,0.680 mmol),室温下反应,TLC监测反应终点。反应结束后,减压蒸除溶剂,然后加入3 mL水,以2 mol·L^-1盐酸调pH 3～4,析出白色固体,抽滤,水洗滤饼至中性。得白色固体,粗品用硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (2∶1),得白色粉末状固体68.7 mg,产率64.7%,mp 158.1～160.2 ℃。

1.17 N-苯基-3-(N-烟酰氧基)亚氨基-齐墩果烷型- 12-烯-28-酰胺 (III₇)

按照化合物III₆的合成方法,由化合物OA-5 (100 mg,0.174 mmol) 与苯胺 (63.3 mg,0.680 mmol) 反应得到粗品,用硅胶柱色谱纯化,洗脱剂为石油醚-乙酸乙酯 (6∶1),得白色粉末状固体27.1 mg,产率24.5%,mp 106.8～108.7 ℃。

2 初步体外细胞毒活性测试

以阿霉素和吉非替尼为阳性对照药,采用MTT法对合成的目标化合物进行体外抗肿瘤活性测试,所选用的肿瘤细胞为人胃癌细胞 (SGC7901)、人乳腺癌细胞 (MCF-7) 和人非小细胞肺癌细胞 (A549)。对数生长期肿瘤细胞培养于96孔培养板内,每孔100 μL (大约含4 000个细胞),置37 ℃、5% CO₂温箱中培养,培养24 h后,给药组加入含有浓度为1×10^-5 mol·L^-1的测试物,至少设3个平行孔,阴性对照组加入与给药组等体积的溶剂,并设只加培养基的空白对照组,置37 ℃、5% CO₂温箱中培养,48 h后弃培养液,每孔加入MTT溶液 (5 mg·mL^-1) 20 μL,37 ℃孵育3 h,弃上清液,每孔加入100 μL DMSO溶解甲臜颗粒,轻度振荡溶解。在酶标仪波长490 nm条件下测定光密度值 (OD),计算所测化合物对细胞的抑制率和IC₅₀值。试验重复3次,结果取平均值。

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药学学报 2015, Vol. 50 Issue (4): 469-474	PDF