CN110563679B - 一种倍半萜内酯类化合物及其制备方法和在制备防治鼻咽癌的药物中的应用 - Google Patents
一种倍半萜内酯类化合物及其制备方法和在制备防治鼻咽癌的药物中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及化合物及医药技术领域,特别涉及一种倍半萜内酯类化合物及其制备方法和在制备防治鼻咽癌的药物中的应用。
背景技术
鼻咽癌(Nasopharyngeal carcinoma,NPC)是华南以及东南亚地区常见的头颈部肿瘤,其中广东的发病率最高,所以又称“广东癌”。鼻咽癌的发生与EB病毒(Epstein-BarrVirus,EBV)感染、环境因素和遗传因素相关。尽管以顺铂为基础的同期放化疗是鼻咽癌的标准治疗方案,由于鼻咽癌发病位置较隐蔽,超过2/3的鼻咽癌患者在就诊时已经处于局部区域晚期,具有易转移和复发的特点,而顺铂引起的严重副作用使患者治疗依从性差。鼻咽癌是一种高度化学敏感性肿瘤,到目前为止,临床上并没有特异性抗鼻咽癌的药物,均为广谱抗癌药物,包括顺铂、环磷酰胺、氮芥和5-氟尿嘧啶等,所以人们亟需高效低毒的抗鼻咽癌药物。
发明内容
本发明的目的在于克服目前临床上无特异性抗鼻咽癌的药物,且现有为广谱抗癌药物存在严重副作用的缺陷或不足,提供一种半萜内酯类化合物。本发明提供的半萜内酯类化合物具有较高的抗鼻咽癌活性,绝大多数化合物的IC50均在10.0μM以下,尤其地,化合物XF2-30抗鼻咽癌活性的IC50达到0.40μM。对该化合物的药效评价显示其抑制鼻咽癌细胞增殖、促进癌细胞凋亡、诱导自噬,并且对鼻咽癌细胞的迁移和侵袭均具有明显的抑制作用。在鼻咽癌裸鼠移植瘤实验中,7.5mg/kg剂量下的抑制率达到53%,与阳性对照顺铂(5mg/kg)相当,体内安全性实验也确认了化合物的安全性,可应用于制备防治鼻咽癌的药物。
本发明的另一目的在于提供上述倍半萜内酯类化合物的制备方法。
本发明的再一目的在于提供上述倍半萜内酯类化合物在制备防治鼻咽癌的药物中的应用。
为了实现本发明的上述目的,本发明提供了如下技术方案:
一种倍半萜内酯类化合物,其结构式如式(I)所示:
其中,R为饱和或不饱和烷基、胺基、卤基、羟基、酯基、苯基、取代苯基或杂环基。
本发明的发明人研究发现,倍半萜内酯部分具有潜在的抗鼻咽癌活性,通过构效关系分析发现,此类化合物的C-6位酯基取代能显著提高化合物的活性,故本发明对倍半萜内酯中化合量较大的麦角内酯Ergolide的C-6位酯基进行合理取代,得到的倍半萜内酯类化合物高效低毒,类药性优异。
本发明提供的倍半萜内酯类化合物具有较高的抗鼻咽癌活性,绝大多数化合物的IC50均在10.0μM以下,尤其地,化合物XF2-30抗鼻咽癌活性的IC50达到0.40μM。对该化合物的药效评价显示其抑制鼻咽癌细胞增殖、促进癌细胞凋亡、诱导自噬,并且对鼻咽癌细胞的迁移和侵袭均具有明显的抑制作用。在鼻咽癌裸鼠移植瘤实验中,7.5mg/kg剂量下的抑制率达到53%,与阳性对照顺铂(5mg/kg)相当,体内安全性实验也确认了化合物的安全性,可应用于制备防治鼻咽癌的药物。
优选地,R为氢、C1~C3的烷基、C1~C3的烯基、苯基、五元或六元杂环基、取代苯基或苯并稠环基,其中,取代苯基中取代基为C1~C3的烷基、三氟甲基、氰基或卤素。
更优选地,R为如下基团中的一种:
最优选地,R为如下基团中的一种:
以上基团取代的倍半萜内酯类化合物的IC50均在10.0μM以下。
本发明提供了上述化合物的制备方法,包括如下步骤:将麦角内酯Ergolide与酸类化合物RCOOH反应即得所述倍半萜内酯类化合物。
麦角内酯Ergolide可购买得到。
另外麦角内酯Ergolide也可从通过如下途径获得。
本专利团队前期从菊科植物旋覆花中分离得到了麦角内酯Ergolide。
优选地,Ergolide通过如下过程制备得到:将旋覆花干燥花序用乙醇水溶液浸提,减压浓缩得粗提物;将粗提物混悬,萃取,减压浓缩,过色谱柱洗脱,层析后即得所述麦角内酯Ergolide。
更为优选地,Ergolide通过如下过程制备得到:将旋覆花干燥花序用85~98%乙醇水溶液(体积分数),在室温下浸提3次,每次48小时,将提取液减压浓缩得粗提物;将粗提物混悬于水中,用乙酸乙酯萃取,减压浓缩得乙酸乙酯部位,经100~200目的硅胶柱色谱,然后用石油醚/乙酸乙酯按照梯度洗脱,梯度洗脱中石油醚/乙酸乙酯的体积比依次为9:1,8:2,2:1,1:2,0:1,然后反复柱即得所述麦角内酯Ergolide。
所述倍半萜内酯类化合物在制备防治鼻咽癌的药物中的应用也在本发明的保护范围内。
所述倍半萜内酯类化合物药学上可接受的载体或辅料在制备防治鼻咽癌的药物中的应用也在本发明的保护范围内。
一种用于防治鼻咽癌的药物,所述药物中含有上述倍半萜内酯类化合物或药学上可接受的载体或辅料。
本发明的化合物式(I)可用于制备防治鼻咽癌的药物。
本发明化合物用作药物时,可以直接使用,或者以药物组合物的形式使用。
优选地,所述药物中倍半萜内酯类化合物或药学上可接受的载体或辅料的质量分数为0.1~99%。
更为优选地,所述药物中倍半萜内酯类化合物或药学上可接受的载体或辅料的质量分数为0.5~90%。
其余组分可为药物学上可接受的,对人和动物无毒和惰性的可药用载体和/或赋形剂。
所述的药用载体或赋形剂是一种或多种固体、半固体和液体稀释剂、填充剂以及药物制品辅剂。
优选地,填充剂为玉米淀粉、葡萄糖、甘露醇、山黎醇,二氧化硅、微晶纤维素、羧甲基淀粉钠、复合淀粉或预胶化淀粉的一种或几种。
本发明的药物以单位体重服用量的形式使用。本发明的药物可经注射(静注、肌注)和口服两种形式给药。
本发明化合物的施用量可根据用药途径、患者的年龄、体重、所治疗的疾病的类型和严重程度等变化,其日剂量可以是0.01~10mg/kg体重,优选0.1~5mg/kg体重。可以一次或多次施用。
相对于现有技术,本发明具有如下的优点及效果:
本发明提供的倍半萜内酯类化合物对鼻咽癌具有明显的防治效果,绝大多数化合物的IC50均在10.0μM以下,尤其地,化合物XF2-30抗鼻咽癌活性的IC50达到0.40μM。对该化合物的药效评价显示其抑制鼻咽癌细胞增殖、促进癌细胞凋亡、诱导自噬,并且对鼻咽癌细胞的迁移和侵袭均具有明显的抑制作用。在鼻咽癌裸鼠移植瘤实验中,7.5mg/kg剂量下的抑制率达到53%,与阳性对照顺铂(5mg/kg)相当,体内安全性实验也确认了化合物的安全性,可应用于制备防治鼻咽癌的药物。
附图说明
图1分离所得化合物结构XF-1~XF-18;
图3为化合物XF-6的ECD光谱;
图4为化合物对四种鼻咽癌细胞细胞毒性测定和抑制克隆形成结果图;A:XF2-30对四种鼻咽癌细胞细胞毒性测定;B:XF2-30对四种鼻咽癌细胞抑制克隆形成结果图;
图5为化合物对HONE1细胞凋亡影响结果图;A:XF2-30使HONE1阻滞在G2/M期;B:XF2-30下调G2/M期相关蛋白;
图6为化合物对HONE1细胞凋亡影响结果图;A:Hoechst33342染色观察凋亡细胞形态;B:Annexin V-FITC凋亡结果图;C:凋亡关键调节蛋白表达调控;
图7为化合物对HONE1细胞迁移和侵袭影响结果图;A:XF2-30抑制HONE1细胞划痕愈合;B:XF2-30抑制HONE1细胞穿过Transwell膜孔;
图8为化合物对细胞自噬影响结果图;A-C:XF2-30促进GFP-LC3-Hela/A549细胞绿色荧光斑点数增加;D:透射电镜观察下XF2-30诱导HONE1细胞自噬体生成;E-G:自噬标记蛋白以及基因表达调控;
图9~12为化合物抑制裸鼠移植瘤结果图;A:裸鼠分组情况;B:化合物处理后剥离肿瘤图;C:裸鼠肿瘤体积;D:裸鼠肿瘤重量记录;E:裸鼠体重记录;F:裸鼠治疗周期生存率记录;G:小鼠心肝脾肺肾H&E切片染色结果;H:肿瘤组织免疫组化结果。
具体实施方式
以下结合实施例和附图进一步解释本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,本发明所用试剂和材料均为市购。
实施例1
从旋覆花(Euphorbia antiquorum L.)中制备化合物XF-1~XF-18。
旋覆花干燥花序,用95%乙醇水室温浸提3次,每次72小时,将提取液减压浓缩得粗提物。将粗提物混悬于水中,用乙酸乙酯萃取,减压浓缩得乙酸乙酯部位,经硅胶柱色谱(100~200目),用石油醚/乙酸乙酯梯度洗脱(9:1,8:2,2:1,1:2,0:1),TLC检测后分为3个组分(A-C)。将B部分(54g)经反相柱层析,用甲醇/水(50%-100%)洗脱,得到七个组分Fr.B1-7。Fr.B2经Sephadex LH-20,用二氯甲烷/甲醇(1:1)洗脱,得化合物XF-1(120mg),XF-2(2.3g),XF-3(50mg),XF-4(20mg)。Fr.B3通过HPLC分离得到XF-5(9mg),XF-6(4mg),XF-7(9mg),XF-9(15mg),XF-17(6mg)。Fr.B4经硅胶柱层析,用石油醚/乙酸乙酯洗脱,得到4个组分,再进行制备液相分离,得到XF-10(7mg),XF-13(11mg),XF-14(12mg),XF-16(6mg),XF-18(4mg)。Fr.B5经过多次分离,得化合物XF-8(10mg),XF-11(13mg),XF-12(8mg),XF-15(11mg)。化合物结构见图1,化合物XF-6的碳氢远程相关HMBC谱如图2,ECD光谱如图3。
实施例2
Ergolide衍生物XF2-1~XF2-30的制备
合成路线a:在室温条件下,将化合物Ergolide(XF2,10mg,0.03mmol)和催化当量的10%Pd/C,溶解在2mL甲醇中,在H2条件下,搅拌1h。低分辨质谱监测反应完全,移除装置,用微孔滤膜过滤除掉Pd/C,直接减压浓缩,得白色固体XF2-1(10mg,100%)。
b:将化合物Ergolide(50mg,0.17mmol)溶解在四氢呋喃(2mL)中,冰浴搅拌,加入5e.q溶解在1mL乙醇/水(1/1)的氢氧化锂(20mg,0.85mmol),TLC监测反应,待反应完全,旋干加适量饱和食盐水,冰浴滴加浓盐酸,析出白色固体Carpesiolin(XF-1,37mg,85%)
c:取化合物Ergolide(20mg,0.08mmol)和2e.q溴化铜(36mg,0.16mmol)置于反应瓶中,加入2mL四氢呋喃,40℃中搅拌2h。TLC监测反应。反应液用微孔滤膜过滤,然后混合物直接减压浓缩,硅胶薄层制备板分离纯化(二氯甲烷/丙酮=30/1)得到相应的产物XF2-2(8mg,27%)。
d:XF2-3~XF2-4:取Carpesiolin(10mg,0.04mmol)和3e.q CuCN(10mg,0.12mmol),抽真空,加入1mL甲苯,取相应的酰氯(0.06mmol)加入1mL甲苯中稀释,缓慢滴加入反应瓶中,50℃中搅拌过夜。TLC监测反应。然后混合物用硅藻土过滤,并用乙酸乙酯洗涤,合并滤液减压浓缩。薄层制备色谱分离纯化(石油醚/乙酸乙酯=7/3)得到相应的产物XF2-3和XF2-4。
XF2-5:将化合物Carpesiolin(10mg,0.04mmol)和40e.q三乙胺(210μL,1.60mmol)以及6e.q DMAP(30mg,0.24mmol),抽真空,加入1mL干燥的二氯甲烷,冰浴搅拌0.5h。取苯甲酰氯15e.q(70μL,0.60mmol)于1mL二氯甲烷中溶解,缓慢滴加入反应瓶中,1h后撤去冰浴,室温搅拌过夜。TLC监测反应,反应结束后,混合物直接在旋转蒸发仪减压蒸馏去除溶剂,硅胶柱层析分离纯化(二氯甲烷/丙酮=200/1)得到相应的产物XF2-5(7mg,50%)
XF2-6-XF2-30:将化合物Carpesiolin(10mg,0.04mmol)和4e.q三乙胺(21μL,0.16mmol)以及2e.q 2,4,6-三氯苯甲酰氯(13μL,0.08mmol),抽真空,加入1mL甲苯,室温搅拌反应1-3h。取相应的酸2e.q(0.08mmol)和DMAP 2e.q(10mg,0.08mmol)加入1mL甲苯中溶解,缓慢滴加入反应瓶中,50-70℃中搅拌过夜。TLC监测反应。反应液直接减压浓缩,去除溶剂,薄层制备色谱分离纯化(石油醚/乙酸乙酯=1/1)得到相应的产物
XF2-6-XF2-30。
化合物合成路线如下所示:
实施例3
按实施例1和2的方法先制得式(I),按常规加注射用水,精滤,灌封灭菌制成注射液。
实施例4
按实施例1和2的方法先制得式(I),将其溶于无菌注射用水中,搅拌使溶,用无菌抽滤漏斗过滤。再无菌精滤,分装于2安瓿中,低温冷冻干燥后无菌熔封得粉针剂。
实施例5
按实施例1和2的方法先制得式(I),按其与赋形剂(例如淀粉浆)重量比为5:1的比例加入赋形剂,制粒压片。
实施例6
按实施例1和2的方法先制得式(I),按其与赋形剂(例如聚乙二醇400)重量比为5:1的比例加入赋形剂,制成胶囊。
实施例7
按实施例1和2的方法先制得式(I),按其与赋形剂(例如吐温80)重量比为3:1的比例加入赋形剂,制成胶囊。
实施例8
本发明化合物式(I)对抗鼻咽癌活性测定
一、细胞的培养
(1)细胞复苏。从液氮罐中取出冻存的CNE1、CNE2、HONE1、SUNE1细胞,立即放入37℃水浴晃动在1min内迅速溶解,将溶解的细胞转移到装有含5ml 10%FBS的RPMI 1640培养基的离心管中,800rpm 3min离心,弃去培养基,加入适量培养基吹打混匀,转移至培养皿中并在培养条件为37℃、5%CO2培养箱中培养,隔天换液,每三天传代一次。
(2)细胞传代。观察细胞生长至约80%汇合时,弃去培养液后,用PBS洗去血清与漂浮的细胞,后加入1ml 0.25%含EDTA胰蛋白酶消化约3min,观察当80%的细胞变圆后立即加入含10%FBS培养基终止消化,用移液枪轻轻吹打细胞并转移至5mL离心管中,800rpm3min离心,小心弃上清。加入适量培养基,轻轻吹打使细胞混匀,以1:3传代。
(3)细胞冻存。将处于对数期生长期的80%汇合细胞,用0.25%含EDTA的胰酶消化,800rpm 3min离心收集细胞,弃上清液,用含10%DMSO、90%血清配制的冻存液吹打混匀,细胞计数,调整密度为1×106个/ml转至无菌冻存管,密封标记,放入程序梯度降温盒中,置于-80℃冻存过夜,随后转入液氮罐。
二、MTT实验方法测定细胞毒性
1、实验方法
将状态良好的CNE1、CNE2、SUNE1、HONE1细胞弃原培养基,用PBS洗两次,加入1mL0.25%EDTA的胰酶消化细胞,以800rpm离心3min收集细胞沉淀,重悬与完全培养基中,使用细胞计数板计数,以3000个细胞/孔的接种密度,把每种细胞悬浮液接种于96孔板(100μL/孔),置于37℃、5%CO2培养箱中培养24小时。细胞贴壁后,弃原培养基,用含不同浓度的化合物替换原培养基,空白为不含细胞的培养基,对照为不含药物的细胞。每孔100μL,每组设置三个复孔。将96孔板置于培养基中分别培养24小时、48小时、72小时。培养结束后,加入20μL(5mg/mL)的MTT溶液,继续培养4小时,随后小心吸去上清,加入150μL的DMSO,振荡10分钟后,用酶标仪在492nm波长下测定其OD值。抑制率计算公式为:1-(加药组OD-空白组OD)/(对照组OD-空白组OD)*100%。
2、实验结果
合成所得化合物的抑制活性如表1所示。
表1合成所得化合物及其在四种鼻咽癌上的细胞活性
a表示化合物对对四种鼻咽癌细胞的半数抑制浓度;b该化合物无抑制活性;c顺铂作为阳性对照。
结果如表1所示,合成所得化合物中共有26个化合物的在四种鼻咽癌细胞上IC50均小于10μM,其中化合物XF2-30对四种鼻咽癌细胞系均具有最好的抑制效果,其在HONE1细胞上的抑制效果比阳性药顺铂强20倍。结合图4-A显示,化合物XF2-30对四种鼻咽癌细胞的抑制活性具有明显浓度时间依赖性,而对HONE1细胞的抑制作用最为明显,因此,选取XF2-30在HONE1鼻咽癌细胞进一步研究。
三、抑制增殖实验
1、克隆形成实验方法
将处于对数生长期的HONE1细胞经胰蛋白酶消化后,以500个细胞每孔接种于6孔板,加入不同浓度的化合物XF2-30(0、0.5、1、2μM)孵育7天,每两天换一次药,培养结束后用4%多聚甲醛固定细胞,再用1%结晶紫染色30分钟,室温风干,计集落数,每50个细胞为一个群落。
2、实验结果
如图4-B所示,化合物XF2-30能明显抑制四种鼻咽癌细胞的克隆形成,在HONE1细胞上1μM浓度下没有克隆形成,说明其在较低浓度下已具有明显抑制鼻咽癌细胞增殖的作用。
四、细胞周期检测
1、实验方法
为了探讨化合物XF2-30对细胞周期的影响,我们用流式细胞仪检测了化合物XF2-30对HONE1细胞周期的调控情况。
流式细胞仪检测细胞周期实验方法。4×105/孔的HONE1细胞种于6孔板中培养24h。化合物XF2-30以不同的浓度(0、1、2、4μM)处理HONE1细胞24h,随后胰酶消化,离心收集细胞。用冰冻的70%乙醇在4℃下固定过夜,随后用冷PBS缓冲液洗涤,然后用细胞周期和凋亡试剂盒在37℃避光染30分钟。处理好的样品用贝克曼流式细胞仪分析,每个样品收集15000细胞。结果分析在Modfit软件进行。
周期相关蛋白利用免疫印迹的方法检测。不同XF2-30浓度处理24h的HONE1细胞,首先经RIPA裂解液在冰上裂解,然后用细胞刮子收取总蛋白,再用BCA蛋白定量试剂盒测定总的蛋白含量。蛋白经变性后用12%的SDS-PAGE胶电泳分离,切离浓缩胶,将分离胶上的蛋白电转至PVDF膜上;随后电转后的PVDF膜用封闭液(5%脱脂牛奶)封闭2小时;加入相应的一抗(β-tubulin,CyclinB1,Cdc25c,and Cdc2抗体),在4℃摇床孵育过夜,用TBST洗膜3次,每次10分钟;加入相应的二抗室温孵育1h,用TBST洗膜3次,每次10分钟;最后,加入化学发光液在显影仪中显影,最后用Image J软件对蛋白条带进行灰度分析。
2、实验结果
结果如图5所示,从图5-A可知,对照组和实验组(1、2、4μM)相比,实验组能明显抑制细胞周期阻滞在G2/M期,G0/G1和S期的细胞数目相应的减少。
细胞周期是由细胞周期蛋白(Cyclins)和细胞周期依赖性蛋白激酶(Cdks)共同调节。G2/M期的调控主要与Cdc25c、Cdc2激酶、CyclinB1蛋白有关。Cdc2/CyclinB1复合物主要是负责细胞周期从G2期过渡到M期,然而Cdc2/CyclinB1复合物活性由Cdc25C蛋白激活。Cdc25C蛋白活性是细胞通过G2/M检测点的限速步骤。随后我们用免疫印迹检测了G2/M检测点关键调节蛋白的表达。图5-B所示,XF2-30处理HONE1细胞24h后,细胞周期蛋白CyclinB1、细胞周期依赖性蛋白激酶Cdc2、Cdc25C的蛋白表达水平都减少,这与流式检测到细胞周期G2/M期阻滞结果一致。
五、细胞凋亡检测
1、实验方法
为了进一步弄清化合物XF2-30的抗肿瘤作用机制,我们探讨了化合物XF2-30对HONE1细胞凋亡的影响。凋亡实验我们采用Hoechst 33342染色、Annexin V-FITC/PI流式检测以及Western Blot三个实验同时验证。采用Hoechst 33342主要是从形态上验证凋亡细胞的存在,Annexin V-FITC/PI流式检测凋亡细胞发生的比例,而Western Blot主要是检测与凋亡相关的关键蛋白表达,从分子水平验证凋亡的发生。
凋亡细胞形态使用Hoechst 33342染色法测定:15万/孔的HONE1细胞铺板于6孔板中,培养24小时使其贴壁生长。设置空白组和实验组(1μM、2μM、4μM)继续培养24h,4%多聚甲醛室温固定1h;加入冷的PBS,洗涤3次,每次1mL;加入0.5mL Hoechst 33342室温染色15min;细胞成像系统拍照记录。
凋亡细胞比例测定使用用Annexin V-FITC/PI试剂盒检测。HONE1细胞株分别在化合物XF2-30(0、1、2、4μM)浓度的下培养24h,用含0.25%EDTA的胰酶消化收集细胞,随后用冰PBS冲洗以除去残留的胰酶。然后用试剂盒里面的缓冲液100μL重悬细胞,分别加入5μL的Annexin V-FITC和10μLPI溶液,在室温下孵育15分钟,1h内上流式细胞仪检测细胞凋亡的荧光信号。每个样品收集15000细胞。结果分析在FlowJo VX软件进行。
凋亡蛋白检测利用Western Blot检测。不同XF2-30浓度处理24h的HONE1细胞,首先经RIPA裂解液在冰上裂解,然后用细胞刮子收取总蛋白,再用BCA蛋白定量试剂盒测定总的蛋白含量。蛋白经变性后用12%的SDS-PAGE胶电泳分离,切离浓缩胶,将分离胶上的蛋白电转至PVDF膜上;随后电转后的PVDF膜用封闭液(5%脱脂牛奶)封闭2小时;加入相应的一抗(β-tubulin,Caspase-3,PARP,抗体),在4℃摇床孵育过夜,用TBST洗膜3次,每次10分钟;加入相应的二抗室温孵育1h,用TBST洗膜3次,每次10分钟;最后,加入化学发光液在显影仪中显影,最后用Image J软件对蛋白条带进行灰度分析。
2、实验结果
结果如图6所示,从图6-A可知,白色标记的为凋亡细胞(凋亡小体、核边缘化、细胞核固缩)。图6-B流式检测结果表明,凋亡细胞数目与化合物XF2-30的浓度呈剂量依赖性关系,对照组和实验组诱导细胞凋亡的比例分别为16.93%、22.29%、68.30%、81.00%。Caspase-3和PARP是凋亡程序执行过程中最关键的凋亡蛋白,从图6-C可知,化合物XF2-30激活了caspase-3,导致cleaved caspase-3的表达,也导致全长的PARP切割产生了89KD的cleaved-PARP。以上结果表明:化合物XF2-30通过诱导细胞凋亡,发挥抗肿瘤作用,并且对肿瘤细胞凋亡的影响呈浓度依赖性关系。
六、细胞迁移侵袭检测
1、实验方法
细胞划痕实验方法。将处于对数生长期的HONE1细胞接种于6孔板中,待细胞贴壁后,用100μL枪头在六孔板中央平滑的绘制一条直线,用PBS洗三遍,去掉划痕处划下来的悬浮细胞,更换含有1%FBS的不同浓度的XF2-30(0、0.5、1、2μM)培养基,放入培养箱中培养,观察0h、24h、48h HONE1细胞的迁移情况,并拍照记录细胞同一位置的迁移状态。最后用Image J软件计算划痕处空白面积。
Transwell测定细胞迁移和侵袭实验方法。细胞迁移和侵袭能力都是用8μm孔径大小的Transwell小室测试的。相比于迁移实验,侵袭实验在Tanswell的多孔滤膜上铺了一层基质胶(0.5mg/mL)。100μL HONE1细胞(4×105/mL)培养在含有不浓度的XF2-30(0、0.5、1、2μM)的RPMI 1640培养基(无血清)的Transwell上室,下室是600μL完全培养基(10%FBS)。培养24h后,细胞先用4%的多聚甲醛固定,然后用0.1%的结晶紫染色10分钟。随后用棉签轻轻擦掉上室染色的细胞,室温风干,再置于相差显微镜下观察与拍照。
2、实验结果
实验结果如图7所示:图7-A可知,XF2-30能明显抑制HONE1细胞划痕愈合的能力,仅0.5μM已能抑制划痕愈合,并呈明显的浓度依赖性。为了能够准确定量XF2-30抑制HONE1细胞转移的能力,随后利用Transwell小室研究了XF2-30对细胞的迁移能力的影响,然后在Transwell滤膜上铺层基质胶模拟人体的基底膜,探讨XF2-30是否能够分解基质膜,实现肿瘤的侵袭。XF2-30处理组和对照组的细胞,用结晶紫染色后,用棉签擦去上室染色细胞,剩下的染色细胞就是迁移和入侵的细胞。图7-B,我们可以看出:不管是在迁移实验还是侵袭实验,XF2-30处理组,穿过Tanswell膜的细胞明显减少,且呈浓度依赖性关系。实验结果表明,XF2-30能够抑制细胞的迁移和侵袭,加上前面实验证明XF2-30能够诱导细胞凋亡,表明XF2-30不仅是个能够杀死肿瘤细胞而且能够抑制肿瘤转移的潜在活性抗癌化合物。
七、细胞自噬检测
1、实验方法
GFP-LC3单荧光指示体系检测细胞自噬活性实验方法。利用LC3在自噬形成过程中发生聚集的原理:无自噬时,GFP-LC3融合蛋白弥散在胞浆中;自噬形成时,GFP-LC3融合蛋白转位至自噬体膜,在荧光显微镜下形成多个明亮的绿色荧光斑点,一个斑点相当于一个自噬体,可以通过计数来评价自噬活性的高低。在稳转的GFP-LC3A549和Hela细胞加入含有不浓度的XF2-30(0、1、2、4μM)培养24小时,在荧光显微镜下拍照观察并计算细胞中绿色荧光点数。
Western Blot实验方法。不同XF2-30浓度处理24h的HONE1细胞,首先经RIPA裂解液在冰上裂解,然后用细胞刮子收取总蛋白,再用BCA蛋白定量试剂盒测定总的蛋白含量。蛋白经变性后用12%的SDS-PAGE胶电泳分离,切离浓缩胶,将分离胶上的蛋白电转至PVDF膜上;随后电转后的PVDF膜用封闭液(5%脱脂牛奶)封闭2小时;加入相应的一抗(β-tubulin,P62,LC3B抗体),在4℃摇床孵育过夜,用TBST洗膜3次,每次10分钟;加入相应的二抗室温孵育1h,用TBST洗膜3次,每次10分钟;最后,加入化学发光液在显影仪中显影,最后用Image J软件对蛋白条带进行灰度分析。
PCR实验方法。利用Trizol法提取HONE1细胞总RNA,随后使用TaKaRa逆转录试剂盒进行反转录制备cDNA。采用SYBR Green染色,以β-actin为内部参照,以逆转录的cDNA为模板,每组基因设置3个复孔,数据分析采用Ct值2-△△Ct相对定量法,计算各浓度组自噬相关基因(P62和LC3B)相对表达量。
计算公式=2-[Ct(实验组)-Ct(内参组)]-[Ct(样品组目的基因)-Ct(内参组)]。
引物序列如下:
LC3,(forward:5′-GATACAAGGGTGAGAAGCAG-3′,reverse:5′-CGTTCACCAACAGGAAGAAG-3′);
P62(forward:
5′-CCAGGACAGCGAGAGGAAGG-3′,reverse:′-CTCACTTGGATTACAGGCGTAGG-3′);
β-actin
(forward:5′-CTCTTCCAGCCTTCCTTCCT-3′,reverse:5′-TGTTGGCGTACAGGTCTTTG-3′)。
透射电镜观察细胞自噬。收集6孔板中预先用化合物XF2-30处理过的HONE1细胞,在2.5%戊二醛(Solarbio,P1126)中固定24h,然后通过分级乙醇脱水并包埋。将超薄切片染色并安装在铜网格上。随后,用JEM-1400电子显微镜对样品进行拍照记录结果。
2、实验结果
实验结果如图8所示:图8A-C,XF2-30能明显使GFP-LC3-Hela和A549细胞的绿色荧光斑点数增加,并呈浓度依赖性,说明其具有能诱导细胞自噬的能力。进一步的,图8-D,经过XF2-30处理的HONE1细胞在透射电镜下能观测到自噬体的形成(红色箭头所示)。在分子水平上也进一步验证了自噬的发生,图8E-G,q-PCR和Western Blot实验显示XF2-30能明显上调自噬的分子标志物LC3的表达水平,LC3II/LC3I增加,说明了自噬小体的增加,同时P62表达水平减少,说明自噬活性增强。实验验证了XF2-30能诱导细胞自噬。
八、体内裸鼠移植瘤实验
1、实验方法
取对数生长期的HONE1细胞经0.25%胰酶消化后用无血清培养液离心洗涤2次,并用无血清培养液调整细胞浓度为1×105个/mL,0.4%台盼蓝染色法活细胞计数>98%。无菌条件下用带6号针头的注射器抽取0.1mL细胞悬液接种于经75%酒精消毒的裸鼠背部靠右侧腋窝皮下。接种裸鼠皮下肿瘤体积达到约50mm3,将荷瘤裸鼠随机分成5组,分别为溶媒对照(5%DMF,2%吐温80,93%生理盐水溶液);低剂量组(7.5mg/kg XF2-30);高剂量组(15mg/kg XF2-30);母体化合物组(15mg/kg XF1);阳性对照组(顺铂5mg/kg)。每3天腹腔注射一次,持续14天。;顺铂为每周一次腹腔注射给药,给药体积为0.1mL/10g。各组10只,均为雄性,实验为时2周。治疗过程中,每2-3天测量1次各组裸鼠的体重和瘤体大小,用游标卡尺测量瘤体最长径(a)与最短径(b),计算肿瘤体积(V)。观察至给药14天,根据需要取血和心肝脾肺肾脏器,分析相关指标,最后颈椎脱臼致死,取瘤组织,称瘤重。绘制生长曲线,计算抑瘤率。
2、实验结果
实验结果如图9~12所示:图9~12中的A~F,化合物XF2-30(15mg/kg)与空白溶媒相比具有明显抑制肿瘤体积的作用,且肿瘤重量相对减少了70.8%,优于母体化合物XF1(15mg/kg)45.8%以及阳性对照(顺铂5mg/kg)56.5%。虽然化合物XF2-30在高剂量(15mg/kg)时带来轻微的体重下降的副作用,但在治疗周期内没有引起裸鼠的死亡,结合图9~12中的G,对裸鼠的心肝脾肺肾的H&E染色也辅助说明化合物具有一定的安全性,没有引起相关的器官损伤。
图9~12中的H对裸鼠的肿瘤的免疫组化结果显示,XF2-30能使凋亡的关键调节分子cleaved-caspase3,自噬的标志物LC3的表达增加,P62的表达减少,这与细胞分子水平上免疫印迹的结果相一致。综上,化合物XF2-30是一个相对安全的并具有较好抑制鼻咽癌生长的活性化合物,有潜在的成药性。
新化合物的结构解析
白色固体,[α]20 D+6.0(c 0.07,MeOH)。高分辨质谱给出加钠离子峰m/z 331.1526[M+Na]+,提示该化合物分子式为C17H24O5。IR图谱中显示羟基(3359cm-1)的信号。如图1~3所示,NMR谱中的特征信号δH 6.20(1H,d,J=3.5Hz),5.92(1H,d,J=3.5Hz)和δC 162.1,121.9,174.8表明该化合物中存在1个典型的α,β-不饱和内酯结构。
通过分析化合物的核磁数据,发现其与化合物
(1S,2R,5R,6S,7S,8S,10R)-6-Hydroxy-2-methoxy-4-oxopseudoguai-11(13)-en-12,8-olide(XF-5)的核磁数据十分相似,除了C-2位的取代基由乙氧基(δC 64.5,15.5)代替了甲氧基(δC 56.6)。在二维碳氢远程相关HMBC谱中,根据H-2(δH 4.06)与C-OEt(δC64.5)的相关,以及在1H-1H COSY谱中可以观察到CH3-CH2O-的自旋耦合片段,可证实该化合物的二维结构。化合物XF-6的相对构型通过NOESY谱确定。NOESY谱中H3-14与H-1,H-2和H-9α相关,H-7与H-1和H-9α相关,表明CH3-14和H-1,H-2,H-7位于同一朝向,定为α朝向。最后通过计算化合物的ECD和实验值对比确定了化合物的绝对构型为1S,2R,5R,6S,7S,8S,10R。
新化合物的物理常数:
Inuloid A(XF-6):白色固体;[α]D 20+6.0(c 0.07,MeOH);UV(MeOH)λmax(logε)203(3.21)nm;ECD(MeOH)λmax(Δε)226(4.27),246(-0.62),296(1.67)nm;IRνmax 3359,2923,2854,1766,1465,1263,1087,800cm-1;1H NMR(CDCl3,500MHz):δH 6.20(1H,d,J=3.5Hz,H-13a),5.92(1H,d,J=3.1Hz,H-13b),4.42(1H,ddd,J=11.9,10.2,2.8Hz,H-8),4.06(1H,m,H-8),3.95(1H,d,J=8.5Hz,H-6),3.49(1H,m,H-1'),3.24(1H,m,H-1'),2.83(1H,td,J=9.6,8.5,4.0Hz,H-7),2.64(1H,d,J=18.6Hz,H-3a),2.49(1H,m,H-9a),2.24(1H,d,J=4.8Hz,H-3b),2.20(2H,overlap,H-1,10),1.43(1H,m,H-9b),1.25(3H,s,H-15),1.16(6H,overlap,H-14,2');and 13C NMR(CDCl3,100MHz):δC 223.1(C-4),169.7(C-12),139.1(C-11),121.6(C-13),76.5(C-6),76.2(C-8),76.0(C-2),64.5(CH2-OEt),57.0(C-5),52.2(C-7),49.7(C-1),44.5(C-3),44.0(C-9),26.4(C-10),22.3(C-15),19.8(C-14),15.5(CH3-OEt);HRESIMS m/z 331.1526[M+Na]+(calcd for C17H24O5,331.1516)。
(3R,3aR,4S,4aR,8R,9aS)-3,4a,8-trimethyl-2,5-dioxododecahydroazuleno[6,5-b]furan-4-ylacetate(XF2-1).1H NMR(500MHz,CDCl3):δH 5.33(1H,d,J=8.5Hz),4.47(1H,ddd,J=11.6,10.4,3.0Hz),2.62(1H,dq,J=11.6,7.0Hz),2.47(1H,ddd,J=13.2,4.7,3.0Hz),2.42–2.34(1H,m),2.24(1H,ddd,J=12.0,10.5,5.7Hz),2.18–2.08(3H,m),1.95(3H,s),1.84(1H,tdd,J=11.3,6.6,4.8Hz),1.49–1.31(2H,m),1.17(3H,d,J=7.0Hz),1.09(3H,s),1.08(1H,s);13C NMR(125MHz,CDCl3):δC 218.6,177.7,169.6,76.6,75.9,55.7,55.1,46.5,44.6,42.4,38.4,30.0,24.6,21.1,20.2,19.5,15.0;HRESIMS:m/zcalcd for C17H24O5[M+Na]+,331.1516;found 331.1513。
(3aR,4S,4aR,8R,9aS)-6-bromo-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl acetate(XF2-2)1H NMR(400MHz,CDCl3):δH6.22(1H,d,J=3.5Hz),5.80(1H,d,J=3.1Hz),5.52(1H,d,J=8.1Hz),4.48(1H,td,J=11.7,11.2,2.8Hz),4.30(1H,d,J=6.2Hz),3.13(1H,ddt,J=11.2,7.5,3.4Hz),2.70(1H,td,J=11.6,5.7Hz),2.52(1H,dt,J=13.2,3.6Hz),2.38(1H,dd,J=15.1,5.7Hz),2.07(3H,s),2.05–1.97(1H,m),1.89(1H,dq,J=11.5,6.0Hz),1.59–1.51(1H,m),1.14(3H,s),1.09(3H,d,J=6.5Hz);13C NMR(100MHz,CDCl3):δC 211.1,169.7,169.1,137.2,122.4,76.1,73.3,57.2,52.4,45.1,44.3,43.9,35.3,29.4,21.5,20.0,19.3;HRESIMS:m/z calcdfor C17H21O5Br[M+Na]+,407.0465;found 407.0464。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl 3-methylbut-2-enoate(XF2-3)Whitesolid,1H NMR(400MHz,CDCl3):δH 6.20(1H,d,J=3.5Hz),5.87(1H,d,J=3.2Hz),5.53(1H,d,J=7.8Hz),5.50(1H,p,J=1.3Hz),4.51(1H,ddd,J=11.8,10.3,2.8Hz),3.06(1H,ddt,J=10.9,7.7,3.4Hz),2.50(1H,ddd,J=13.2,4.5,2.8Hz),2.41–2.33(1H,m),2.33–2.26(1H,m),2.20(3H,d,J=1.2Hz),2.17(1H,dd,J=6.6,1.5Hz),2.14–2.08(1H,m),1.88(3H,d,J=1.4Hz),1.87–1.80(0H,m),1.56–1.45(1H,m),1.44–1.37(1H,m),1.11(3H,d,J=6.5Hz),1.08(3H,s);13C NMR(100MHz,CDCl3):δC 218.4,169.4,164.8,159.6,137.4,122.4,115.1,76.5,73.5,56.3,52.9,46.9,44.5,38.2,30.1,27.8,24.7,20.6,20.1,18.6;HRESIMS:m/z calcd for C20H26O5[M+Na]+,369.1672;found 369.1664。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-but-2-enoate(XF2-4)1H NMR(400MHz,CDCl3):δH 7.00(1H,dd,J=15.5,6.9Hz),6.20(1H,d,J=3.5Hz),5.85(1H,d,J=3.2Hz),5.72(1H,dd,J=15.5,1.7Hz),5.56(1H,d,J=7.8Hz),4.51(1H,ddd,J=11.8,10.2,2.7Hz),3.08(1H,ddt,J=10.9,7.1,3.3Hz),2.51(1H,ddd,J=13.2,4.5,2.7Hz),2.41–2.34(1H,m),2.31(1H,dt,J=10.7,6.1Hz),2.21–2.15(1H,m),2.15–2.06(1H,m),1.87(3H,dd,J=6.9,1.7Hz),1.86–1.79(0H,m),1.51(1H,dt,J=13.5,12.0Hz),1.46–1.40(1H,m),1.12(3H,d,J=6.5Hz),1.09(3H,s);13C NMR(100MHz,CDCl3):δC 218.1,169.3,164.8,146.5,137.3,122.5,122.1,76.4,74.4,56.2,52.8,47.0,44.5,38.2,30.2,24.7,20.1,18.7,18.3;HRESIMS:m/z calcd for C19H24O5[M+Na]+,355.1516;found 355.1517。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl benzoate(XF2-5)1H NMR(400MHz,CDCl3):δH 8.04–7.93(2H,m),7.67–7.50(1H,m),7.43(2H,dd,J=8.4,7.2Hz),6.21(1H,d,J=3.5Hz),5.92(1H,d,J=3.1Hz),5.78(1H,d,J=7.7Hz),4.57(1H,ddd,J=11.7,10.2,2.8Hz),3.22(1H,ddt,J=10.9,7.2,3.3Hz),2.56(1H,ddd,J=13.2,4.5,2.7Hz),2.43(1H,ddd,J=12.2,10.7,6.0Hz),2.34(1H,dd,J=18.3,7.6Hz),2.23(1H,ddd,J=12.2,9.0,5.8Hz),2.15–2.05(1H,m),1.98–1.87(1H,m),1.61–1.55(1H,m),1.47(1H,dd,J=12.4,7.7Hz),1.16(3H,d,J=6.6Hz);13C NMR(100MHz,CDCl3):δC 218.5,169.8,165.5,137.9,134.1,130.3,129.3,123.1,77.0,75.9,57.0,53.6,47.8,45.1,38.7,30.9,25.4,20.7,19.3;HRESIMS:m/z calcd for C22H24O5[M+Na]+,391.1516;found 391.1529。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl 3,4,5-trifluorobenzoate(XF2-6)1H NMR(500MHz,CDCl3):δH7.59(2H,t,J=7.4Hz),6.22(1H,d,J=3.5Hz),5.81(1H,d,J=3.5Hz),5.75(1H,dd,J=8.1,2.7Hz),4.55(1H,t,J=11.1Hz),3.19(1H,ddt,J=11.3,7.7,3.6Hz),2.56(1H,dd,J=13.8,3.7Hz),2.37(2H,dd,J=18.6,8.5Hz),2.24(1H,q,J=10.1,7.0Hz),2.06(1H,dt,J=20.1,10.9Hz),1.97–1.86(1H,m),1.64–1.55(2H,m),1.49(1H,ddd,J=18.9,15.7,10.4Hz),1.17(3H,d,J=6.7Hz),1.14(3H,s);13C NMR(125MHz,CDCl3):δC218.2,168.9,162.4,152.3(1C,m),150.2(1C,dd,J=10.1,3.2Hz),137.2,125.6(1C,dd,J=7.0,2.6Hz),122.4,114.4(1C,d,J=5.6Hz),114.3(1C,d,J=5.6Hz),76.5,76.2,56.5,52.6,47.1,44.4,37.9,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C22H21O5F3[M+Na]+,445.1233;found 445.1239。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl 4-(trifluoromethyl)benzoate(XF2-7)1HNMR(400MHz,CDCl3):δH 8.07(2H,d,J=8.0Hz),7.70(2H,d,J=8.2Hz),6.22(1H,d,J=3.5Hz),5.87(1H,d,J=3.2Hz),5.80(1H,d,J=7.8Hz),4.57(1H,ddd,J=11.7,10.2,2.7Hz),3.23(1H,ddt,J=10.9,7.8,3.3Hz),2.57(1H,ddd,J=13.3,4.5,2.7Hz),2.46–2.40(1H,m),2.36(1H,dd,J=18.6,7.6Hz),2.24(1H,ddd,J=12.7,9.0,6.1Hz),2.07(1H,ddd,J=18.6,12.7,9.1Hz),1.93(1H,tdd,J=11.1,6.5,4.6Hz),1.61–1.56(1H,m),1.49(1H,dd,J=12.6,7.7Hz),1.17(3H,d,J=6.6Hz),1.15(3H,s);13C NMR(100MHz,CDCl3):δC218.1,169.0,163.9,137.2,135.2,134.8,132.9,130.1,125.8(1C,q,J=3.7Hz),122.5,76.2,76.1,56.5,52.7,47.2,44.4,38.0,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd forC23H23O5F3[M+Na]+,459.1390;found 453.1399。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl 4-cyanobenzoate(XF2-8)1H NMR(500MHz,CDCl3):δH 8.05(2H,d,J=8.4Hz),7.74(2H,d,J=8.3Hz),6.22(1H,d,J=3.4Hz),5.84(1H,d,J=3.1Hz),5.79(1H,d,J=7.8Hz),4.57(1H,ddd,J=12.5,10.4,2.7Hz),3.21(1H,ddt,J=11.0,7.3,3.3Hz),2.57(1H,ddd,J=13.3,4.6,2.7Hz),2.42–2.37(1H,m),2.37–2.33(1H,m),2.27–2.20(1H,m),2.05(1H,ddd,J=18.8,12.8,9.1Hz),1.92(1H,ddt,J=13.4,7.6,3.9Hz),1.58(1H,q,J=12.3Hz),1.53–1.45(1H,m),1.17(3H,d,J=6.6Hz),1.15(3H,s);13C NMR(125MHz,CDCl3):δC 218.2,169.0,163.5,137.2,133.5,132.5,130.2,122.4,117.9,117.0,76.3,76.2,56.5,52.7,47.2,44.4,37.9,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C23H23NO5[M+Na]+,416.1468;found 416.1468。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl nicotinate(XF2-9).1H NMR(400MHz,CDCl3):δH 9.10(1H,d,J=1.8Hz),8.78(1H,dd,J=4.9,1.8Hz),8.27(1H,dt,J=8.0,2.0Hz),7.41(1H,ddd,J=8.0,4.9,0.9Hz),6.23(1H,d,J=3.4Hz),5.90(1H,d,J=3.2Hz),5.81(1H,d,J=7.7Hz),4.57(1H,ddd,J=11.7,10.2,2.7Hz),3.22(1H,ddt,J=10.8,7.1,3.3Hz),2.57(1H,ddd,J=13.2,4.5,2.7Hz),2.46–2.39(1H,m),2.39–2.32(1H,m),2.24(1H,ddd,J=12.7,9.3,5.9Hz),2.15–2.07(1H,m),1.97–1.87(1H,m),1.61–1.56(1H,m),1.52–1.45(1H,m),1.17(3H,d,J=6.6Hz);13C NMR(100MHz,CDCl3):δC 218.3,169.0,163.8,154.0,150.6,137.5,137.2,125.8,123.7,122.4,76.3,75.9,56.5,52.8,47.2,44.5,38.0,30.3,24.7,20.2,18.6;HRESIMS:m/z calcd for C21H23NO5[M+H]+,370.1649;found 370.1665。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl thiazole-5-carboxylate(XF2-10).1HNMR(500 MHz,CDCl3):δH 8.95(1H,s),8.47(1H,s),6.26–6.22(1H,m),5.91(1H,t,J=2.3Hz),5.71(1H,dd,J=7.8,1.6Hz),4.70–4.41(1H,m),3.19(1H,ddt,J=8.0,5.4,2.9Hz),2.55(1H,ddt,J=13.4,4.4,2.0Hz),2.42–2.37(1H,m),2.34(1H,d,J=13.7Hz),2.23(1H,dt,J=13.8,7.3Hz),2.16–2.07(1H,m),1.91(1H,tt,J=11.8,5.9Hz),1.57(2H,q,J=12.8Hz),1.48(1H,tt,J=12.5,6.1Hz),1.16(4H,d,J=6.6Hz),1.13(1H,s);
13C NMR(125MHz,CDCl3):δC 218.2,169.0,159.7,158.6,149.4,137.1,129.2,122.5,76.4,76.2,56.4,52.6,47.1,44.4,38.0,30.2,24.7,20.1,18.7;HRESIMS:m/zcalcd for C19H21NO5S[M+Na]+,398.1033;found 398.1052。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl thiophene-2-carboxylate(XF2-11).1HNMR(500MHz,CDCl3):δH 7.81(1H,dd,J=3.8,1.3Hz),7.55(1H,dd,J=5.0,1.3Hz),7.10(1H,dd,J=5.0,3.8Hz),6.23(1H,d,J=3.5Hz),5.97(1H,d,J=3.2Hz),5.68(1H,d,J=7.7Hz),4.55(1H,ddd,J=11.7,10.2,2.7Hz),3.19(1H,ddt,J=10.9,7.0,3.3Hz),2.54(1H,ddd,J=13.2,4.6,2.8Hz),2.45–2.37(1H,m),2.37–2.30(1H,m),2.27–2.20(1H,m),2.20–2.11(1H,m),1.91(1H,tq,J=11.0,5.9,5.3Hz),1.64–1.52(1H,m),1.46(1H,ddd,J=11.8,7.7,3.9Hz),1.16(3H,d,J=6.6Hz),1.12(3H,s);13C NMR(125MHz,CDCl3):δC 218.0,169.2,160.5,137.2,134.5,132.9,128.2,122.6,76.3,75.7,56.3,52.8,47.1,44.5,38.1,30.2,24.8,20.1,18.6;HRESIMS:m/z calcd for C20H22O5S[M+Na]+,397.1080;found397.1095。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl furan-2-carboxylate(XF2-12).1H NMR(500MHz,CDCl3):δH 7.54(1H,d,J=1.6Hz),7.17(1H,d,J=3.5Hz),6.50(1H,d,J=1.8Hz),6.23(1H,d,J=3.5Hz),5.96(1H,d,J=3.1Hz),5.69(1H,d,J=7.7Hz),4.54(1H,ddd,J=12.4,10.4,2.7Hz),3.19(1H,ddt,J=10.9,7.3,3.3Hz),2.54(1H,ddd,J=13.3,4.5,2.8Hz),2.45–2.39(1H,m),2.39–2.32(1H,m),2.22(1H,dt,J=13.6,4.1Hz),2.19–2.12(1H,m),1.95–1.85(1H,m),1.59–1.52(1H,m),1.51–1.41(1H,m),1.15(3H,d,J=6.6Hz),1.12(3H,s);13C NMR(125MHz,CDCl3):δC 218.1,169.2,157.1,146.8,144.3,137.2,122.6,119.0,112.2,76.3,75.5,56.4,52.8,46.9,44.4,38.1,30.2,24.7,20.1,18.5;HRESIMS:m/z calcd for C20H22O6[M+Na]+,381.1309;found 381.1307。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl 5-nitrofuran-2-carboxylate(XF2-13).1H NMR(400MHz,CDCl3):δH 7.34(2H,d,J=1.2Hz),6.25(1H,d,J=3.4Hz),5.90(1H,d,J=3.0Hz),5.70(1H,d,J=7.7Hz),4.54(1H,td,J=11.9,11.1,2.6Hz),3.23(1H,ddt,J=11.1,7.5,3.4Hz),2.55(1H,dt,J=13.5,3.5Hz),2.47–2.35(2H,m),2.31–2.19(2H,m),1.91(1H,dp,J=11.2,5.8Hz),1.59(1H,q,J=12.2Hz),1.52–1.41(1H,m),1.17(3H,d,J=6.5Hz),1.13(3H,s);13C NMR(100MHz,CDCl3):δC 219.0,168.9,156.0,144.4,137.1,122.4,120.0,111.8,77.5,76.2,56.6,52.3,46.9,44.4,38.0,30.2,24.7,20.1,18.6;HRESIMS:m/z calcd for C20H21NO8[M+Na]+,426.1159;found 426.1171。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl 2-phenylthiazole-4-carboxylate(XF2-14).1H NMR(400MHz,CDCl3):δH 8.10(1H,s),7.87(2H,dd,J=6.6,3.0Hz),7.43–7.32(3H,m),6.18(1H,d,J=3.5Hz),5.98(1H,d,J=3.1Hz),5.69(1H,d,J=7.7Hz),4.51(1H,ddd,J=12.1,10.4,2.7Hz),3.24(1H,ddt,J=10.8,7.2,3.4Hz),2.50(1H,dd,J=4.6,2.9Hz),2.47–2.42(1H,m),2.28(1H,dd,J=5.2,3.5Hz),2.26–2.21(1H,m),2.21–2.13(1H,m),1.84(1H,dq,J=11.4,5.3,4.4Hz),1.53(1H,d,J=12.5Hz),1.45–1.40(1H,m),1.10(3H,d,J=6.5Hz),1.08(3H,s);13C NMR(100MHz,CDCl3):δC 218.5,169.2,169.0,160.3,147.5,137.2,133.0,130.9,129.2,128.3,127.0,122.7,76.4,76.3,56.5,52.6,46.9,44.4,38.2,30.2,24.8,20.1,18.6;HRESIMS:m/z calcd for C25H25NO5S[M+H]+,452.1526;found452.1542。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl 6-chlorobenzo[b]thiophene-2-carboxylate(XF2-15).1H NMR(500MHz,CDCl3):δH 7.97(1H,d,J=7.9Hz),7.80(1H,d,J=7.9Hz),7.54(1H,t,J=7.4Hz),7.50(1H,t,J=7.5Hz),6.26(1H,d,J=1.9Hz),6.00(1H,d,J=2.1Hz),5.78(1H,dd,J=7.8,1.5Hz),4.65–4.48(1H,m),3.25(1H,td,J=9.2,8.1,4.2Hz),2.56(1H,ddt,J=13.4,4.3,1.9Hz),2.44(1H,td,J=11.4,5.4Hz),2.35(1H,d,J=7.1Hz),2.33–2.27(1H,m),2.27–2.19(1H,m),1.91(1H,dh,J=11.8,6.1Hz),1.60(2H,q,J=12.3Hz),1.54–1.42(1H,m),1.17(3H,d,J=6.5Hz);13C NMR(125MHz,CDCl3):δC 218.4,169.1,159.9,138.9,137.2,137.1,128.7,128.2,125.8,125.3,124.1,122.8,122.7,76.4,76.3,56.6,52.8,47.1,44.4,37.9,30.3,24.8,20.2,18.6;HRESIMS:m/z calcd forC24H23O5SCl[M+Na]+,481.0847;found 481.0865。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl quinoline-2-carboxylate(XF2-16).1HNMR(400MHz,CDCl3):δH 8.29(1H,dd,J=8.6,0.8Hz),8.19(1H,dd,J=8.6,1.1Hz),8.14(1H,d,J=8.5Hz),7.87(1H,dd,J=8.1,1.4Hz),7.77(1H,ddd,J=8.4,6.8,1.5Hz),7.65(1H,ddd,J=8.1,6.8,1.2Hz),6.27(1H,d,J=3.5Hz),6.09(1H,d,J=3.2Hz),5.84(1H,d,J=7.7Hz),4.61(1H,ddd,J=11.7,10.2,2.7Hz),3.46–3.35(1H,m),2.64(1H,ddd,J=12.3,10.7,6.1Hz),2.56(1H,ddd,J=13.2,4.5,2.7Hz),2.43(1H,ddd,J=18.3,12.9,8.8Hz),2.36–2.30(1H,m),2.30–2.21(1H,m),2.00–1.86(1H,m),1.71–1.57(2H,m),1.48(1H,qd,J=12.7,7.5Hz),1.18(3H,d,J=6.5Hz),1.16(3H,s);13C NMR(100MHz,CDCl3):δC 218.8,169.3,164.5,147.7,147.7,137.3,137.3,130.8,130.3,129.5,128.8,127.7,122.7,121.3,77.0,76.5,56.6,52.5,46.8,44.3,38.2,30.2,24.9,20.1,18.6;HRESIMS:m/zcalcd for C25H25NO5[M+Na]+,442.1625;found 442.1643。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl quinoxaline-2-carboxylate(XF2-17).1HNMR(400MHz,CDCl3):δH 9.48(1H,s),8.23–8.19(1H,m),8.17(1H,dd,J=8.2,1.6Hz),7.90(1H,ddd,J=8.4,6.9,1.7Hz),7.85(1H,ddd,J=8.4,6.9,1.6Hz),6.27(1H,d,J=3.5Hz),6.04(1H,d,J=3.1Hz),5.87(1H,d,J=7.8Hz),4.60(1H,ddd,J=11.7,10.2,2.7Hz),3.37(1H,ddt,J=10.9,7.7,3.3Hz),2.64–2.58(1H,m),2.56(1H,td,J=5.4,4.8,2.6Hz),2.36–2.32(1H,m),2.31(1H,d,J=3.2Hz),2.29–2.19(1H,m),1.94(1H,dtd,J=12.6,6.5,3.3Hz),1.67–1.58(1H,m),1.50(1H,ddd,J=13.0,10.3,2.5Hz),1.18(3H,d,J=6.6Hz),1.17(3H,s);13C NMR(100MHz,CDCl3):δC 218.9,169.1,163.3,145.2,143.9,142.2,141.6,137.2,132.6,131.1,130.7,129.5,122.6,77.3,76.3,56.6,52.4,46.9,44.3,38.1,30.2,24.8,20.1,18.6;HRESIMS:m/z calcd for C24H24N2O5[M+Na]+,443.1577;found 443.1584。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl cinnamate(XF2-18).1H NMR(400MHz,CDCl3):δH 7.69(1H,d,J=16.0Hz),7.53–7.48(2H,m),7.39(2H,d,J=2.2Hz),7.38(1H,d,J=1.7Hz),6.30(1H,d,J=16.0Hz),6.22(1H,d,J=3.5Hz),5.89(1H,d,J=3.1Hz),5.65(1H,d,J=7.8Hz),4.54(1H,ddd,J=11.7,10.2,2.7Hz),3.15(1H,ddt,J=10.9,7.7,3.3Hz),2.54(1H,ddd,J=13.2,4.5,2.8Hz),2.44–2.39(1H,m),2.36(1H,td,J=7.5,6.7,3.8Hz),2.19(1H,m),2.18–2.10(1H,m),1.89(1H,tdd,J=11.1,6.5,4.6Hz),1.58–1.52(1H,m),1.50–1.42(1H,m),1.14(3H,d,J=6.5Hz),1.12(3H,s);13C NMR(100MHz,CDCl3):δC218.0,169.3,165.4,146.4,137.3,134.2,130.8,129.0,128.4,122.5,117.2,76.4,74.8,56.3,52.9,47.0,44.5,38.2,30.2,24.7,20.2,18.7;HRESIMS:m/z calcd for C24H26O5[M+Na]+,417.1672;found 417.1683。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(4-fluorophenyl)acrylate(XF2-19).1H NMR(500MHz,CDCl3):δH 7.66(1H,d,J=15.9Hz),7.54–7.45(2H,m),7.07(2H,t,J=8.6Hz),6.23(1H,d,J=6.1Hz),6.21(1H,d,J=6.3Hz),5.88(1H,d,J=3.2Hz),5.64(1H,d,J=7.8Hz),4.53(1H,ddd,J=11.7,10.2,2.7Hz),3.13(1H,ddt,J=10.9,7.8,3.4Hz),2.53(1H,ddd,J=13.2,4.6,2.7Hz),2.37(2H,tdd,J=15.1,8.9,3.7Hz),2.25–2.11(2H,m),1.89(1H,tdd,J=11.2,6.5,4.7Hz),1.59–1.48(1H,m),1.48–1.40(1H,m),1.14(3H,d,J=6.6Hz),1.11(3H,s);13C NMR(125MHz,CDCl3):δC 218.1,169.2,165.3,163.2,145.1,137.3,130.5(1C,d,J=3.2Hz),130.3,130.2,122.5,116.9,116.3,116.1,76.4,74.9,56.3,52.8,47.0,44.5,38.2,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C24H25O5F[M+Na]+,435.1578;found 435.1590。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(4-chlorophenyl)acrylate(XF2-20).1H NMR(500MHz,CDCl3):δH 7.64(1H,d,J=16.0Hz),7.46–7.41(2H,m),7.38–7.33(2H,m),6.27(1H,d,J=16.0Hz),6.22(1H,d,J=3.5Hz),5.87(1H,d,J=3.1Hz),5.65(1H,d,J=7.8Hz),4.53(1H,ddd,J=11.6,10.1,2.7Hz),3.13(1H,ddt,J=11.0,7.4,3.3Hz),2.54(1H,ddd,J=13.3,4.6,2.8Hz),2.37(2H,tdd,J=16.6,8.8,4.2Hz),2.25–2.12(2H,m),1.89(1H,tt,J=11.3,5.3Hz),1.59–1.51(1H,m),1.50–1.41(1H,m),1.14(3H,d,J=6.5Hz),1.12(3H,s);13C NMR(125MHz,CDCl3):δC 218.1,169.2,165.2,144.9,137.3,136.7,132.7,129.5,129.4,122.5,117.8,76.3,75.0,56.3,52.8,47.0,44.5,38.2,30.2,24.7,20.2,18.7;HRESIMS:m/z calcd for C24H25O5Cl[M+Na]+,151.1283;found 451.1299。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(4-bromophenyl)acrylate(XF2-21).1H NMR(400MHz,CDCl3):δH 7.63(1H,d,J=16.0Hz),7.51(2H,d,J=8.5Hz),7.36(2H,d,J=8.5Hz),6.28(1H,d,J=15.9Hz),6.22(1H,d,J=3.5Hz),5.87(1H,d,J=3.2Hz),5.64(1H,d,J=7.8Hz),4.53(1H,ddd,J=11.7,10.2,2.7Hz),3.13(1H,ddt,J=10.9,7.8,3.3Hz),2.53(1H,ddd,J=13.2,4.5,2.7Hz),2.43–2.36(1H,m),2.36–2.30(1H,m),2.26–2.19(1H,m),2.18–2.11(1H,m),1.88(1H,tq,J=11.8,6.5,6.0Hz),1.59–1.49(1H,m),1.48–1.42(1H,m),1.14(3H,d,J=6.5Hz),1.11(3H,s);13C NMR(100MHz,CDCl3):δC 218.2,169.2,165.2,145.0,137.3,133.1,132.3,129.7,125.1,122.5,117.9,76.3,75.0,56.3,52.8,47.0,44.5,38.1,30.2,24.7,20.2,18.7;HRESIMS:m/z calcd for C24H25O5Br[M+Na]+,495.0778;found 495.0778。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(4-(trifluoromethyl)phenyl)acrylate(XF2-22).1H NMR(500MHz,CDCl3):δH 7.71(1H,d,J=16.0Hz),7.64(2H,d,J=8.2Hz),7.60(2H,d,J=8.3Hz),6.36(1H,d,J=16.0Hz),6.22(1H,d,J=3.4Hz),5.87(1H,d,J=3.1Hz),5.66(1H,d,J=7.8Hz),4.53(1H,td,J=11.1,2.6Hz),3.14(1H,ddt,J=10.9,7.2,3.4Hz),2.54(1H,dt,J=13.3,3.6Hz),2.44–2.33(2H,m),2.25–2.11(2H,m),1.89(1H,m),1.54(1H,d,J=12.4Hz),1.50–1.40(1H,m),1.14(3H,d,J=6.6Hz);13C NMR(125MHz,CDCl3):δC 218.2,169.2,164.9,144.5,137.5,137.3,128.5,126.0(1C,d,J=3.8Hz),122.4,119.8,76.3,75.2,56.4,52.8,47.0,44.5,38.1,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C25H25O5F3[M+Na]+,485.1546;found 485.1547。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(3-(trifluoromethyl)phenyl)acrylate(XF2-23).1H NMR(500MHz,CDCl3):δH 7.75(1H,s),7.71(1H,dd,J=16.1,2.5Hz),7.65(2H,dd,J=12.8,8.3Hz),7.52(1H,td,J=7.9,2.3Hz),6.37(1H,dd,J=15.9,2.5Hz),6.22(1H,d,J=3.4Hz),5.87(1H,d,J=3.5Hz),5.66(1H,dd,J=8.0,2.5Hz),4.54(1H,t,J=11.0Hz),3.14(1H,ddt,J=10.8,7.5,3.5Hz),2.54(1H,dd,J=13.4,3.6Hz),2.45–2.32(2H,m),2.26–2.11(2H,m),1.89(1H,dp,J=12.3,6.2,5.8Hz),1.60–1.51(1H,m),1.50–1.43(1H,m),1.17–1.13(3H,m),1.12(3H,s);13C NMR(125MHz,CDCl3):δC218.2,169.2,164.9,144.5,137.3,135.0,131.6,129.6,127.1(1C,d,J=3.8Hz),124.7(1C,d,J=3.7Hz),122.4,119.2,76.3,75.1,56.4,52.8,47.0,44.5,38.1,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C25H25O5F3[M+Na]+,485.1546;found 485.1556。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(2-(trifluoromethyl)phenyl)acrylate(XF2-24).1H NMR(500MHz,CDCl3):δH 7.99(1H,d,J=15.9Hz),7.70(2H,t,J=8.3Hz),7.57(1H,t,J=8.1Hz),7.50(1H,d,J=7.9Hz),6.33(1H,dd,J=15.8,2.8Hz),6.24(1H,d,J=3.4Hz),5.90(1H,d,J=3.1Hz),5.65(1H,dd,J=8.2,2.6Hz),4.54(1H,t,J=11.2Hz),3.15(1H,ddd,J=11.3,7.4,3.5Hz),2.54(1H,dd,J=13.7,3.7Hz),2.38(2H,ddt,J=24.8,14.5,7.1Hz),2.19(2H,tq,J=20.2,10.7,9.6Hz),1.90(1H,q,J=9.5,8.4Hz),1.61–1.51(1H,m),1.51–1.42(1H,m),1.15(3H,d,J=6.1Hz),1.12(3H,s),;13C NMR(125MHz,CDCl3):δC 218.2,169.2,164.3,140.9,137.3,132.3,130.1,128.0,126.4(1C,d,J=5.5Hz),122.5,121.7,76.4,75.2,56.4,52.8,47.1,44.4,37.9,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C25H25O5F3[M+Na]+,485.1546;found 485.1556。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(3,4-dimethoxyphenyl)acrylate(XF2-25).1H NMR(400MHz,CDCl3):δH 7.63(1H,d,J=15.9Hz),7.09(1H,dd,J=8.3,2.0Hz),7.01(1H,d,J=2.0Hz),6.86(1H,d,J=8.3Hz),6.22(1H,d,J=3.5Hz),6.16(1H,d,J=15.8Hz),5.89(1H,d,J=3.1Hz),5.65(1H,d,J=7.8Hz),4.53(1H,ddd,J=11.7,10.2,2.7Hz),3.91(3H,s),3.91(4H,s),3.14(1H,ddt,J=10.9,7.5,3.3Hz),2.53(1H,ddd,J=13.2,4.6,2.7Hz),2.44–2.29(2H,m),2.27–2.19(1H,m),2.19–2.12(1H,m),1.89(1H,tt,J=11.0,5.1Hz),1.59–1.51(1H,m),1.50–1.41(1H,m),1.14(3H,d,J=6.5Hz),1.12(3H,s);13C NMR(100MHz,CDCl3):δC 218.0,169.3,165.6,151.5,149.3,146.3,137.3,127.2,123.1,122.5,114.8,111.1,109.8,76.4,74.6,56.3,56.1,56.0,52.9,47.0,44.5,38.2,30.2,24.7,20.2,18.8;HRESIMS:m/z calcd for C26H30O7[M+Na]+,477.1884;found477.1890。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(4-(dimethylamino)phenyl)acrylate(XF2-26).1H NMR(500MHz,CDCl3):δH 7.61(1H,d,J=15.8Hz),7.38(2H,d,J=8.5Hz),6.64(2H,d,J=8.4Hz),6.21(1H,d,J=3.5Hz),6.06(1H,d,J=15.7Hz),5.92(1H,d,J=3.1Hz),5.63(1H,d,J=7.7Hz),4.59–4.47(1H,m),3.14(1H,ddt,J=10.9,7.2,3.5Hz),3.02(6H,s),2.52(1H,dt,J=13.3,3.9Hz),2.43–2.33(2H,m),2.23–2.13(2H,m),1.89(1H,td,J=13.1,12.3,6.3Hz),1.58–1.50(1H,m),1.50–1.42(1H,m),1.13(3H,d,J=6.5Hz),1.10(3H,s);13C NMR(125MHz,CDCl3):δC 217.9,169.4,166.2,152.1,146.9,137.3,130.1,122.6,122.0,111.8,111.2,76.4,74.3,56.2,52.9,47.0,44.5,40.3,38.3,30.2,24.7,20.2,18.7;HRESIMS:m/z calcd for C26H31NO5[M+H]+,438.2275;found438.2281。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(thiophen-2-yl)acrylate(XF2-27).1H NMR(400MHz,CDCl3):δH 7.79(1H,d,J=15.6Hz),7.39(1H,d,J=5.1Hz),7.25(0H,s),7.05(1H,dd,J=5.0,3.7Hz),6.21(1H,d,J=3.5Hz),6.08(1H,d,J=15.6Hz),5.88(1H,d,J=3.1Hz),5.63(1H,d,J=7.8Hz),4.52(1H,ddd,J=12.6,10.3,2.7Hz),3.12(1H,ddt,J=10.9,7.1,3.3Hz),2.53(1H,ddd,J=13.2,4.6,2.8Hz),2.37(2H,dq,J=16.9,6.8,5.9Hz),2.24–2.14(2H,m),1.88(1H,tt,J=11.3,5.4Hz),1.53(1H,d,J=12.5Hz),1.49–1.41(1H,m),1.14(3H,d,J=6.5Hz),1.11(3H,s);13C NMR(100MHz,CDCl3):δC218.0,169.3,165.3,139.4,138.7,137.3,131.7,129.1,128.3,122.5,115.8,76.4,74.8,56.3,52.9,47.0,44.5,38.2,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C22H24O5S[M+Na]+,423.1237;found 423.1252。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(pyridin-4-yl)acrylate(XF2-28).1H NMR(400MHz,CDCl3):δH 8.72–8.53(2H,m),7.61(1H,d,J=16.0Hz),7.37–7.30(2H,m),6.45(1H,d,J=16.0Hz),6.22(1H,d,J=3.4Hz),5.85(1H,d,J=3.1Hz),5.65(1H,d,J=7.8Hz),4.53(1H,ddd,J=12.4,10.3,2.7Hz),3.13(1H,ddt,J=11.0,7.3,3.4Hz),2.54(1H,ddd,J=13.3,4.5,2.7Hz),2.45–2.30(2H,m),2.25–2.09(2H,m),1.88(1H,tq,J=11.6,6.3,5.9Hz),1.61–1.48(1H,m),1.49–1.40(1H,m),1.14(3H,d,J=6.6Hz),1.12(3H,s);13C NMR(100MHz,CDCl3):δC 218.2,169.1,164.6,150.7,143.4,141.4,137.3,122.3,122.0,121.9,76.3,75.4,56.4,52.8,47.0,44.5,38.1,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C23H25NO5[M+Na]+,396.1805;found 396.1817。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(furan-2-yl)acrylate(XF2-29).1HNMR(400MHz,CDCl3):δH 7.47(1H,d,J=1.7Hz),7.44(1H,d,J=15.7Hz),6.63(1H,d,J=3.4Hz),6.47(1H,dd,J=3.5,1.8Hz),6.21(1H,d,J=3.5Hz),6.17(1H,d,J=15.7Hz),5.88(1H,d,J=3.2Hz),5.62(1H,d,J=7.8Hz),4.57–4.45(1H,m),3.12(1H,ddt,J=10.8,7.1,3.3Hz),2.52(1H,ddd,J=13.2,4.5,2.8Hz),2.44–2.30(2H,m),2.24–2.09(2H,m),1.88(1H,tt,J=11.3,5.5Hz),1.53(1H,q,J=12.2Hz),1.44(1H,ddd,J=11.6,7.1,3.6Hz),1.13(3H,d,J=6.5Hz),1.10(3H,s);13C NMR(100MHz,CDCl3):δC 218.0,169.3,165.5,150.9,145.1,137.3,132.5,122.5,115.7,114.8,112.6,76.4,74.8,56.3,52.9,47.0,44.5,38.2,30.2,24.7,20.1,18.7;HRESIMS:m/z calcd for C22H24O6[M+Na]+,407.1465;found407.1475。
(3aR,4S,4aR,8R,9aS)-4a,8-dimethyl-3-methylene-2,5-dioxododecahydroazuleno[6,5-b]furan-4-yl(E)-3-(5-nitrofuran-2-yl)acrylate(XF2-30).1H NMR(400MHz,CDCl3):δH 7.45(1H,d,J=15.8Hz),7.34(1H,d,J=3.8Hz),6.78(1H,d,J=3.9Hz),6.51(1H,d,J=15.8Hz),6.21(1H,d,J=3.5Hz),5.83(1H,d,J=3.1Hz),5.63(1H,d,J=7.7Hz),4.58–4.46(1H,m),3.11(1H,ddt,J=10.9,7.4,3.5Hz),2.53(1H,dt,J=13.4,3.6Hz),2.45–2.30(2H,m),2.19(2H,dt,J=18.3,9.1Hz),1.88(1H,dq,J=11.1,5.8Hz),1.62–1.52(1H,m),1.51–1.40(1H,m),1.14(3H,d,J=6.6Hz),1.11(3H,s);13CNMR(100MHz,CDCl3):δC 218.4,169.1,164.3,152.1,137.3,130.3,122.2,121.6,116.2,113.0,76.3,75.6,56.4,52.8,47.0,44.5,38.0,30.2,24.6,20.1,18.6;HRESIMS:m/zcalcd for C22H23NO8[M+Na]+,452.1316;found 452.1332。
Claims (9)
2.一种权利要求1所述倍半萜内酯类化合物的制备方法,其特征在于,包括如下步骤:将麦角内酯Ergolide脱酯基成醇后,与酸类化合物RCOOH反应即得所述倍半萜内酯类化合物。
3.根据权利要求2所述制备方法,其特征在于,麦角内酯Ergolide通过如下过程制备得到:将旋覆花干燥花序用乙醇水溶液浸提,减压浓缩得粗提物;将粗提物混悬,萃取,减压浓缩,过色谱柱洗脱,层析后即得所述麦角内酯Ergolide。
4.根据权利要求2所述制备方法,其特征在于,所述麦角内酯Ergolide通过如下过程制备得到:将旋覆花干燥花序用85~98%乙醇水溶液,在室温下浸提3次,每次48小时,将提取液减压浓缩得粗提物;将粗提物混悬于水中,用乙酸乙酯萃取,减压浓缩得乙酸乙酯部位,经100~200目的硅胶柱色谱,然后用石油醚/乙酸乙酯按照梯度洗脱,梯度洗脱中石油醚/乙酸乙酯的体积比依次为9:1,8:2,2:1,1:2,0:1,然后反复柱即得所述麦角内酯Ergolide。
5.权利要求1所述倍半萜内酯类化合物在制备防治鼻咽癌的药物中的应用。
6.一种用于防治鼻咽癌的药物,其特征在于,所述药物中含有权利要求1所述倍半萜内酯类化合物。
7.根据权利要求6所述药物,其特征在于,所述药物中还含有药学上可接受的载体或辅料。
8.根据权利要求7所述药物,其特征在于,所述药物中倍半萜内酯类化合物或药学上可接受的载体或辅料的质量分数为0.1~99%。
9.根据权利要求6所述药物,其特征在于,所述药物的剂型为注射液、粉针剂、片剂或胶囊。
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