CN105541955B - 23‑羟基白桦酸荧光探针、制备及其在细胞定位与摄取中的用途 - Google Patents
23‑羟基白桦酸荧光探针、制备及其在细胞定位与摄取中的用途 Download PDFInfo
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Abstract
本发明涉及一类新型的23‑羟基白桦酸荧光衍生物的有机合成和药物化学领域,具体涉及将香豆素荧光发色团连接到23‑羟基白桦酸骨架上获得的23‑羟基白桦酸荧光探针。本发明还公开了这些23‑羟基白桦酸荧光衍生物用于抗肿瘤机制研究及其对癌症治疗的潜在用途,特别涉及这类荧光探针在检测23‑羟基白桦酸作用靶细胞定位和摄取中的应用。
Description
技术领域
本发明涉及有机合成和药物化学领域,具体涉及一类新型的抗肿瘤活性天然产物23-羟基白桦酸荧光探针的制备,特别涉及这类荧光探针用于检测23-羟基白桦酸在靶细胞定位与摄取中的应用。
背景技术
随着经济的发展,肿瘤已经成为威胁人类健康的头号杀手。几个世纪以来科学家们一直致力于肿瘤的研究,针对各类肿瘤疾病的药物也随之被研发出来。临床数据表明80%的抗肿瘤药物都来源于天然产物。天然产物对抗肿瘤药物的发展起到了重要的作用。尽管天然产物在肿瘤治疗中取得了成功,但是大部分天然产物的抗肿瘤机制仍不明确,这限制了天然产物的进一步发展,也对多药耐药带来了挑战。因此,对天然产物抗肿瘤机制的研究迫在眉睫。
五环三萜是自然界里含量最丰富的萜类之一,由于其生物活性多样,一直备受科学家们的关注。其中23-羟基白桦酸(23-HBA)作为羽扇烷型天然产物,是从中药白头翁中分离得到的白桦酸类似物,也是中药白头翁的主要活性成分之一,在抗肿瘤,抗HIV等方面有着良好的应用前景。其对黑色素瘤细胞、神经外胚层肿瘤细胞、口腔上皮癌细胞、恶性脑瘤细胞、白血病细胞等都具有较好的抑制或杀伤作用。除此之外,23-HBA对抗肿瘤药物还有协同增敏活性,在较低浓度下即能显著提高肿瘤细胞对多种化疗药物的敏感度,增加抗肿瘤药物在细胞内的蓄积。由于其活性好,毒性低,相信随着对白桦酸类化合物研究的不断深入,此类化合物有可能开发成为抗肿瘤药物应用于临床。
然而,23-HBA的抗肿瘤分子机制尚未完全阐明,细胞内的作用靶点与作用模式也不明确,相关分子作用机制有待探索。故通过一定的技术手段,探明23-HBA在靶细胞作用中的亚细胞定位及摄取,将有助于阐明其作用靶点和作用机制。
当今创新药物的发现越来越依赖于靶点的发现及靶点与活性化合物作用模式的确定,荧光标记技术在细胞水平由于具有高灵敏度和可定量分析的特点,已成为阐明药用机制的重要手段,是研究天然产物作用模式、结合位点等生物学相关性质的有力工具。近年来,荧光标记技术已广泛用于亚细胞定位、靶点与活性化合物作用方式等相关领域的研究。
发明内容
本发明的目的在于提供一种23-HBA荧光探针和其在细胞内定位及摄取中的应用。本发明公开了一种23-HBA荧光探针,其含有荧光发色基团和23-HBA骨架结构,如通式(I)所示:
其中R表示-NH(CH2)n-,n=1-5;
-NH(CH2)nNHC(O)(CH2)2-,n=1-5;
-O(CH2CH2O)nC(O)(CH2)2-,n=1-5;
-NH(CH2)O-;-O(CH2)NH-;-NH(CH2CH2O)2CH2-。
本发明中所述的23-HBA荧光探针,最优选是R为-NH(CH2)nNHC(O)(CH2)2-,n=2。
所述荧光探针含有23-HBA骨架,保留了23-HBA的抗肿瘤活性,能够发挥癌症治疗作用。进一步的肿瘤细胞实验证明,所述探针能够抑制肿瘤细胞的生长,具有潜在的抗肿瘤功效。
上述的23-HBA荧光探针的制备方法概述如下:
(1)在室温下,将香豆素荧光化合物与N-羟基琥珀酰亚胺在EDCI的作用下反应得香豆素活性酯b;(2)将得到的活性酯b与N-Boc-乙二胺混合,加入缩合剂反应得到相应的N-Boc胺产物c;(3)将得到的N-Boc胺产物c与三氟醋酸混合脱除保护基得相应的胺化合物d;(4)将得到的胺化合物d与酸酐在DMAP作用下得到酸衍生物e;(5)将步骤(4)中得到的酸衍生物e与23-羟基-28苄基白桦酸在草酰氯存在下反应得到23-羟基-28苄基白桦酸荧光探针衍生物f;(6)将得到的荧光探针衍生物在H2,Pd/C的作用下脱去苄基得到23-HBA荧光探针衍生物g。
其合成路线如下:
作为本发明制备方法的一个优选方案步骤(2)使用的是香豆素酸活性酯而非直接用香豆素酸,反应时间大大缩短,基本没有副产物,极大提高目标化合物的收率。
本发明的另一个优选方案,步骤(5)先用草酰氯将化合物e变成酰氯,再与23-羟基-28-苄基白桦酸反应。因为23位的羟基虽然是伯羟基,但由于位阻,反应活性不是很高,此方法提高了目标化合物的产率,而且极大缩短了反应时间。
本发明的另一个优选方案,步骤(6)中的反应条件是50℃反应,且加入0.5mL的甲醇,此方法加快了反应的进程,缩短了反应时间,优于传统溶剂只用四氢呋喃,且在室温下反应的方法。
本发明还提供了所述23-HBA荧光探针用于23-HBA抗肿瘤机制研究的用途,包括细胞摄取和亚细胞定位的检测,内容如下:
一.23-HBA荧光探针定性和定量的细胞摄取研究
a.通过荧光分光光度计,将一系列入射光(200-467nm)扫描10μM的23-HBA荧光探针,确定最大激发波长为415nm,最大发射波长为475nm。
b.将肿瘤细胞用浓度为2μM、4μM、10μM、20μM的23-HBA荧光探针分别处理15min、30min、1h、2h、4h和24h,得到荧光探针处理的细胞。在415nm激发光下,用荧光显微镜观察细胞摄取情况。
c.将肿瘤细胞用浓度为10μM的23-HBA荧光探针处理5min、15min、30min、60min、120min和240min,得到荧光探针处理的靶细胞,用流式细胞仪,定量研究细胞摄取情况。
二.23-HBA荧光探针共定位研究
a.根据细胞摄取的结果,将目标细胞用浓度为4μM的23-HBA荧光探针处理1小时,得到荧光探针处理的细胞;
b.将步骤a中得到的细胞加入细胞器专属染料探针,得到细胞器探针标记的靶细胞;
c.将步骤b中所得到的细胞在激光共聚焦显微镜下观察23-HBA荧光探针与靶细胞器探针的荧光分布情况,根据荧光的重叠情况确定23-HBA在靶细胞中的定位。
本发明中的方法可以用于检测哺乳动物的任何细胞,优选的细胞为肿瘤细胞。
本发明中的细胞器探针可以根据检测的细胞器的不同,选择细胞生物学上可以接受的任何探针,所优选的细胞器探针为细胞膜荧光探针、线粒体荧光探针或者是细胞核荧光探针。更为优选的是线粒体红色荧光探针( Deep Red FM)。
试验结果证实,该发明所述的23-HBA荧光探针能快速地进入到细胞中,并高选择性地标记线粒体,表明线粒体是23-HBA的主要聚集位点。
本发明具有以下有益效果:提供了一种具有香豆素荧光发色基团与23-HBA结构骨架的荧光探针,该探针具有较好的抗肿瘤作用,兼有治疗与作用靶点研究的用途;本发明提供了这类荧光探针的制备方法,条件温和,工艺简单;本发明运用荧光显微镜和流式细胞仪提供了在不同浓度和不同时间点靶细胞对23-HBA荧光探针摄取情况;本发明同时也公开了运用激光共聚焦显微镜来检测不同靶细胞中23-HBA荧光探针细胞分布的情况,为进一步研究23-HBA细胞内作用靶点和作用机制奠定了基础。
附图说明:
图1:23-HBA荧光标记探针g对多种细胞株的杀伤作用。
图2:23-HBA荧光标记探针g与B16F10细胞孵育后不同时间点细胞内的染色情况:
A图为荧光显微镜定性检测:a图化合物浓度为2μM;b图为浓度4μM;c图为浓度10μM;d图为浓度20μM;B、C图为流式定量检测:化合物g的浓度为10μM,检测时间为5min,15min,30min,60min,120min,240min。
图3:23-HBA荧光探针与线粒体专属荧光染料探针在B16F10细胞内的共定位染色图:
a图为放大60倍的图像:左图表示23-HBA荧光探针的染色图,中间图为两部分的叠加图,右图为线粒体专属荧光染料探针的染色图。
b图为放大40倍的图像:左图表示23-HBA荧光探针的染色图,中间图为两部分的叠加图,右图为线粒体专属荧光染料探针的染色图。
具体实施方式
实施例1
23-HBA荧光探针的合成
将化合物a(2g,7.7mmol)与N-羟基琥珀酰亚胺(1.3g,11.5mmol)及EDCI(1.8g,9.2mmol)溶于二氯甲烷中(20.0mL),室温搅拌4h,浓缩后加入水,二氯甲烷提取(30mL×3),合并有机层,水洗,饱和食盐水洗,无水Na2SO4干燥,浓缩后柱层析(CH2Cl2/CH3OH 100∶1,v/v)得到目标化合物b(2g,72.9%)。1H-NMR(CDCl3,300MHz)δ:ppm 1.26(6H,t,J=7.1Hz),2.90(4H,s),3.48(4H,q,J=7.1Hz),6.45(1H,d,J=1.9Hz,Ar-H),6.65(1H,dd,J=9.0Hz,J=2.2Hz,Ar-H),7.38(1H,d,J=9.0Hz,Ar-H),8.59(1H,s,Ar-H)。
将化合物b(700mg,1.95mmol)与N-Boc-乙二胺(0.4mL)及DIPEA(1.4mL)溶于二氯甲烷中(20.0mL),室温搅拌3h,浓缩后加入水,二氯甲烷提取(30mL×3),合并有机层,水洗,饱和食盐水洗,无水Na2SO4干燥,浓缩后柱层析(CH2Cl2/CH3OH 80∶1,v/v)得到目标化合物c(700mg,88.8%)。
将化合物c(700mg)溶于二氯甲烷中(15.0mL),并向溶液中加入三氟醋酸(3.0mL),室温搅拌2h,浓缩后加入10%的氢氧化钠溶液,二氯甲烷提取(30mL×3),合并有机层,水洗,饱和食盐水洗,无水Na2SO4干燥,浓缩后柱层析(CH2Cl2/CH3OH 30∶1,v/v)得到目标化合物d(450mg,80.1%)。
将化合物d(500mg,1.65mmol)与丁二酸酐(247mg,2.47mmol)溶于二氯甲烷中(20.0mL),加入催化量DMAP,室温搅拌过夜,浓缩后加入水,二氯甲烷提取(30mL×3),合并有机层,水洗,饱和食盐水洗,无水Na2SO4干燥,浓缩后柱层析(CH2Cl2/CH3OH 20∶1,v/v)得到目标化合物e(600mg,90%)。
将化合物e(100mg,0.25mmol)溶于无水二氯甲烷中(20.0mL),加入催化量DMF和草酰氯(48mg,0.38mmol),室温搅拌10min,浓缩后加入无水二氯甲烷,并加入23-羟基-28-苄基白桦酸(112mg,0.20mmol)及三乙胺(38mg,0.38mmol),室温搅拌30min,浓缩后加入水,二氯甲烷提取(30mL×3),合并有机层,水洗,饱和食盐水洗,无水Na2SO4干燥,浓缩后柱层析(CH2Cl2/CH3OH 80∶1,v/v)得到目标化合物f(80mg,34.0%)。
将化合物f(80mg,0.08mmol)溶于THF/MeOH(10mL/0.5mL),加入10%的Pd/C,于1atm的H2下50度搅拌1h,抽滤,浓缩后柱层析(CH2Cl2/CH3OH 60∶1,v/v)得到目标化合物g(50mg,69.1%)。1H-NMR(CDCl3,300MHz)δ:ppm 0.63(3H,s),0.83(3H,s),0.89(3H,s),0.97(3H,s),1.68(3H,s),2.15-2.28(2H,m),2.51-2.56(2H,m),2.63-2.76(2H,m),3.01(1H,m,H-19),3.45-3.50(7H,m),3.58(2H,m),3.77(1H,d,J=11.3Hz,H-23a),4.17(1H,d,J=11.5Hz,H-23b),4.60,4.73(each 1H,s,H-29),6.49(1H,d,J=1.9Hz,Ar-H),6.66(1H,dd,J=8.9Hz,J=2.1Hz,Ar-H),6.95(1H,s),7.44(1H,d,J=9.0 Hz,Ar-H),8.67(1H,s,Ar-H),9.09(1H,m);13C NMR(CDCl3,75MHz)δ:11.9,12.4,12.7,14.7,16.0,16.6,18.1,19.3,20.9,25.4,26.3,29.7,29.8,30.6,31.1,32.2,34.0,37.0,38.3,38.5,39.2,40.6,40.8,42.1,42.4,45.1,46.9,48.2,49.2,50.5,56.3,67.2,72.1,96.5,108.3,109.4,109.6,110.1,131.3,148.4,150.5,152.8,157.7,162.7,164.8,171.9,173.4,181.0;ESI-MS m/z:858.4[M+H]+;HRMS(m/z)(ESI):calcd for C50H71N3O9[M+H]+:858.5263;found:858.5251.
实施例2
23-HBA荧光探针的抗肿瘤增殖活性
将处于对数生长期的肿瘤细胞消化、计数、制成浓度为5×104个/mL的细胞悬液,96孔板中每孔加入100μL细胞悬液(每孔5×103个细胞)。96孔板置于37℃,5%CO2培养箱中培养24小时后用完全培养基稀释药物至所需浓度,每孔加入100μL相应的含药培养基。96孔板置于37℃,5%CO2培养箱中培养72小时后将96孔板进行MTT染色;每孔加入20μL MTT(5mg/mL),在培养箱继续培养4小时;弃去培养基,每孔加入150μL DMSO溶解,摇床10分钟轻轻混匀;λ=490nm,酶标仪读出每孔的OD值,计算抑制率。实验结果(见图1)表明,本发明中的23-HBA荧光探针具有较好的抗肿瘤增殖效果,兼具肿瘤治疗作用。
实施例3
23-HBA荧光探针对B16F10细胞染色
选取对数生长期的B16F10细胞接种到放置在6孔板中的玻璃盖玻片上,每孔5×104个细胞,贴壁24小时后加入化合物g(2μM,4μM,10μM and 20μM in DMSO)在完全培养基中孵育不同的时间点(15min,30min,1h,2h,4h,24h)。在孵育相应的时间后,细胞用4%的多聚甲醛(in 1×PBS)固定10分钟,用PBS洗涤后就可以将玻璃盖玻片取出用于荧光成像。在荧光显微镜下观察细胞着色部位,荧光分布及亮度变化等,其结果见图2A。
选取对数生长期的B16F10细胞接种到放置在6孔板中的玻璃盖玻片上,每孔5×105个细胞,贴壁24小时后加入10μM的化合物g在完全培养基中孵育不同的时间(5min,15min,30min,1h,2h,4h)。在孵育相应的时间后,用冷的PBS洗三次,在流式细胞仪下,定量检测细胞的荧光强度,其结果见图2B、2C。由图2可知,23-HBA荧光探针能够快速进入细胞中,并成时间和浓度依赖性。
实施例4
23-HBA荧光探针和线粒体红复染实验
选取对数生长期的B16F10细胞接种到放置在6孔板中的玻璃盖玻片上,每孔5×104个细胞,贴壁24小时后加入化合物g(4μM in DMSO)在完全培养基中孵育1个小时,在孵育相应的时间后,细胞用4%的多聚甲醛(in 1×PBS)固定10分钟,用PBS洗涤后就可以将玻璃盖玻片取出用于成像。成像结果由Zeiss LSM 780共聚焦显微镜(Zeiss,Germany)获得。23-HBA荧光探针的激发波长采用415nm,发射波长采用475nm,线粒体专属染色体的激发波长采用579nm,发射波长采用600±10nm。先用明镜显微镜寻找到细胞视野,然后切换到激光共聚焦视野,进行断层扫描寻找最佳切片图像,其结果如图3所示。由图可知,23-HBA荧光探针与线粒体专属荧光探针有着很好的重叠,说明23-HBA荧光探针主要定位在线粒体。
Claims (5)
1.通式(I)的23-羟基白桦酸(23-HBA)荧光探针衍生物:
其中R表示-NH(CH2)n-,n=1-5;
-NH(CH2)nNHC(O)(CH2)2-,n=1-5;
-O(CH2CH2O)nC(O)(CH2)2-,n=1-5;
-NH(CH2)O-;-O(CH2)NH-;-NH(CH2CH2O)2CH2-。
2.权利要求1所述的23-HBA荧光探针衍生物中药理活性较好的化合物有:
R为-NH(CH2)n-,n=3,5;
-NH(CH2)nNHC(O)(CH2)2-,n=2,4;
-O(CH2CH2O)nC(O)(CH2)2-,n=1,2,3;
-NH(CH2)O-;-O(CH2)NH-;-NH(CH2CH2O)2CH2-。
3.权利要求2所述的23-HBA荧光探针衍生物中根据药理活性最适合做进一步生物学研究的优选化合物:R为-NH(CH2)nNHC(O)(CH2)2-,n=2。
4.权利要求3所述的23-HBA荧光探针衍生物的制备方法,其中具体步骤如下:
(1)在室温下,将香豆素荧光探针与N-羟基琥珀酰亚胺混合,加入缩合剂反应得到相应的活性酯产物;
(2)将得到的活性酯产物与N-Boc-乙二胺混合在DIPEA作用下得到N-Boc-胺产物;
(3)将得到的N-Boc-胺产物与三氟醋酸混合后得相应的胺化合物;
(4)将得到的胺化合物与酸酐在DMAP作用下得到酸衍生物;
(5)将步骤(4)中得到的酸衍生物与23-羟基-28苄基白桦酸在草酰氯存在下反应得到23-羟基-28苄基白桦酸荧光探针衍生物;
(6)将得到的荧光探针衍生物在H2,Pd/C的作用下脱去苄基得到23-HBA荧光探针衍生物。
5.根据权利要求4所述荧光探针衍生物的制备方法,其特征在于:步骤(5)中使用草酰氯进行制备;步骤(6)中反应温度为50℃,溶剂为四氢呋喃和甲醇混合溶剂。
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