CN108434132A - 苯并吡喃并呋喃类化合物在制备调控热休克转录因子药物中的应用 - Google Patents
苯并吡喃并呋喃类化合物在制备调控热休克转录因子药物中的应用 Download PDFInfo
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Abstract
本发明涉及医药化合物的技术领域，公开了一种苯并吡喃并呋喃类化合物在制备调控热休克转录因子药物中的应用；以及在制备增强热休克转录因子HSF‑1的转录激活能力药物的应用；在制备提高热休克转录因子HSF‑1的下游分子伴侣HSP70蛋白的表达药物中的应用；在制备延长线虫的寿命和改善健康状态药物中的应用。本发明所述苯并吡喃并呋喃类化合物可增加热休克转录因子HSF‑1的转录激活能力，提高下游分子伴侣HSP70蛋白的表达，显著延长线虫的寿命和健康状态，可以用于制备相关药物，其中，化合物15效果最佳。
Description
技术领域
本发明涉及医药化合物的技术领域，更具体的，涉及一种苯并吡喃并呋喃类化合物在制备调控热休克转录因子药物中的应用。
背景技术
苯并吡喃并呋喃类化合物是南海红树林真菌Xylaria sp. 2508的代谢产物xyloketal B的一系列合成衍生物，其化学结构如下：
Xyloketal B 类化合物已被发现在氧化损伤以及心血管疾病的治疗中具有良好的活性，因此作为衍生物的苯并吡喃并呋喃化合物可能能被用作心血管疾病治疗药物的活性成分。
发明内容
本发明解决的技术问题是提供一种苯并吡喃并呋喃类化合物在制备调控热休克转录因子药物中的应用。
本发明通过以下技术方案予以实现：
一种苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，所述苯并吡喃并呋喃类化合物为：
优选地，所述苯并吡喃并呋喃类化合物在制备增强热休克转录因子HSF-1的转录激活能力药物的应用。
优选地，所述苯并吡喃并呋喃类化合物在制备提高热休克转录因子HSF-1的下游分子伴侣HSP70蛋白的表达药物中的应用。
优选地，所述苯并吡喃并呋喃类化合物在制备延长线虫的寿命和改善健康状态药物中的应用。
优选地，所述线虫为野生型的N2，hsf-1突变的PS3551 [hsf-1(sy441)I]或Aβ模型的CL4176 [smg-1(cc546)I]中的一种或多种。
优选地，所述苯并吡喃并呋喃类化合物为化合物1、化合物2、化合物4~8，化合物11，化合物14~19、化合物21、化合物23、化合物24和化合物26；
优选地，所述苯并吡喃并呋喃类化合物为化合物15。
与现有技术相比，本发明的有益效果：
本发明所述苯并吡喃并呋喃类化合物可增加热休克转录因子HSF-1的转录激活能力，提高下游分子伴侣HSP70蛋白的表达，显著延长线虫的寿命和健康状态，可以用于制备相关药物，其中，化合物15效果最佳。
附图说明
图1为苯并吡喃并呋喃类化合物(100 μM)对于热刺激损伤下线虫的保护作用。所有实验均重复至少三次，数据的表达为 means ± SEM。与对照组相比的显著性差异：* p <0.05; ** p < 0.01; *** p <0.005; **** p < 0.001。
图2为苯并吡喃并呋喃类化合物(300 μM)对于热刺激损伤下线虫的保护作用。A.7个苯并吡喃并呋喃类化合物作用下药物组线虫的生存曲线。 B. 7个苯并吡喃并呋喃类化合物(300 μM)作用下药物组线虫的平均存活时间。所有实验均重复至少三次，数据的表达为 means ± SEM。与对照组相比的显著性差异：* p < 0.001。
图3为化合物15对于N2线虫寿命的保护作用。A. 化合物15(300μM)和DMSO作用下线虫的寿命曲线。 B. 化合物15(300 μM)和DMSO作用下的线虫的平均寿命。药物组线虫的平均寿命为22.77 ± 0.31 d，而对照组的为20.41 ± 0.27 d。所有实验至少重复3次，数据表达形式为means ± SEM，显著性差异：* p < 0.001。
图4为化合物15对于线虫 healthspan 的保护作用。A. 药物组和对照组线虫吞咽(pumping)能力随着衰老的变化。B. 药物组和对照组线虫摆动(bending)能力随着衰老的变化。 C. 线虫吞咽能力处于健康状态的时期在整个生命周期中的比例。D. 线虫摆动能力处于健康状态的时期在整个生命周期中的比例。所有实验至少重复3次，数据的表达方式为means ± SEM，显著性差异：* p < 0.05; ** p < 0.01; *** p <0.005; **** p <0.001。
图5为化合物15对PS3551线虫的耐热性和寿命的作用。A. 药物组与对照组的PS3551线虫在热刺激实验中的生存曲线。B. 药物组与对照组的PS3551线虫在热刺激实验中的平均存活时间。C. 药物组与对照组的PS3551线虫的寿命曲线。 D. 药物组与对照组的PS3551线虫的平均寿命。
图6为化合物15对HSP70表达量的作用。A. Western blot 结果，β-actin作为内参蛋白。B. 药物组与对照组处理后线虫HSP70的表达量。Western blot至少重复3次，显著性差异：* p < 0.05。
具体实施方式
本发明可以结合以下具体实施例进一步解释和阐明，但具体实施例并不对本发明有任何形式的限定。
实施例1 苯并吡喃并呋喃类化合物在延长线虫的寿命和健康状态药物中的应用
（一）材料与方法
1. 药物：化合物1-26, 溶于DMSO，储备液浓度100 mM，常温保存备用。
2. 线虫培养
2.1 线虫品系及预处理
实验所用到线虫品系为野生型的N2，hsf-1突变的PS3551 [hsf-1(sy441)I]以及Aβ模型的CL4176 [smg-1(cc546)I]，三种品系的线虫均使用线虫生长培养基，以大肠杆菌OP50作为食物进行培养。N2和PS3551线虫在20℃培养箱培养，而CL4176线虫在15℃培养箱中培养。药物组化合物与对照组DMSO预先溶解于大肠杆菌OP50菌液中，DMSO的终浓度不大于0.3%。
在实验前，线虫需要进行同步化以确保线虫的生存状态一致。挑取5~10条成虫到干净的NGM培养基上数小时以产出足够数量的卵。用无菌水冲洗培养基，并转移到15 mL EP管中，加无菌水至3.5 mL，再加入0.5 mL 5mol/L NaOH与1.5 mL NaClO，振荡裂解30 s，静置2 min，重复振荡5次至裂解充分。1300 rpm 离心1 min，弃上清，加无菌水至5 mL，重复一次，将卵吸取转移到干净的线虫培养基上。
2.2 热应激实验
当同步化后的线虫长大至L1幼虫期后，将其转移到含有相应化合物的实验组或对照组培养基上，分别培养至成虫第二天。各组随机挑选50条成虫至干净的培养基上，并将培养基从20℃培养箱转移到37℃培养箱中开始热应激实验。每小时检查并记录线虫的存活和死亡数量。死亡的定义是指用挑虫针轻轻触碰线虫身体没有反应，或者线虫停止摄入食物。
2.3 寿命实验和healthspan实验
在寿命实验中，分别将至少50条L1 期幼虫转移到干净的药物组或对照组培养基上，此后每隔1天将线虫转移到新的培养基上。以同步化的当天作为生存曲线上的第0天，每天统计线虫的存活，死亡以及丢失的数量。死亡定义为用挑虫针轻轻触碰线虫身体没有反应。将粘连到培养基壁上或外阴破裂或成为囊状线虫的线虫定义为丢失。
在healthspan实验中，分别将至少100条L1期幼虫转移到干净的药物组或对照组培养基上，此后每隔1天将线虫转移到新的培养基上，以同步化的当天作为生存曲线上的第0天。每一次摆动(bending)或者吞咽(pumping)实验中至少选择各组的10条线虫进行测试。在摆动实验中，将线虫转移到每孔含有100 μL M9缓冲液的96孔板上，休息2 min待线虫状态恢复后开始计算30 s内线虫摆动的次数。摆动的定义是以线虫的中部身体区域为中心的旋转，完成一次摆动指线虫的横向弯曲方向的完全改变。每隔两天进行一次摆动的统计。在吞咽实验中，将线虫转移到干净的培养基上，休息5 min待线虫状态恢复。在解剖显微镜下用视频软件录下每条线虫10 s的吞咽次数，并在慢速播放下准确计数线虫的吞咽次数。每隔一天统计一次吞咽能力。持续统计摆动和吞咽至该项能力降低一半的时间点。
2.4 CL4176 瘫痪实验
在瘫痪实验中，同步化后的线虫卵在15℃培养箱中培养至L1期后，将幼虫转移到药物组或对照组干净的培养基上。继续培养到同步化后36 h，将线虫从15℃培养箱转移到26℃培养箱中培养36 h。每隔2小时统计线虫瘫痪的数量。 瘫痪定义为线虫只能移动其头部或者完全不能移动。
2.5 Western blot
收集足够的成虫第二天的N2，转移到37℃培养箱培养1h 后用无菌水洗3次以除去线虫身体上的OP50。加入裂解液破裂线虫身体，用低温超声法进行彻底裂解，4℃低温12000 rpm离心30 min，移去上清液后，加入SDS上样缓冲液煮沸10 min。将得到的蛋白样品在10%SDS/PAGE 电泳胶上进行电泳，用半干法转膜到PVDF膜上，然后开始进行western blot印迹。
使用5%牛奶的TBST封闭条带1 h，用TBST洗三次，每次10 min。加入适量一抗，摇床摇匀1 h后，4℃摇床过夜。过夜后，用TBST清洗三次，再加入对应的二抗，摇床摇匀1 h后，TBST洗三次，然后就行发光与曝光。
实验组与对照组的一抗分别为Abcam的鼠单克隆抗HSP70[N27F3-4]抗体(ab47454)和 CST 的β-actin (13E5)兔单克隆抗体(#4970)，稀释比例均为1:1000。实验组的二抗是Santa Cruz Biotechnology 的goat anti-mouse lgG-HRP (Cruz Marker)抗体(sc-2031)，而对照组抗体为CST的anti-rabbit lgG HRP-linked抗体(#7074)，稀释比例均为1:2000。所有抗体均在5%脱脂奶粉的TBST中稀释。使用ImageQuant Las4000mini(General Electric of Japan)进行检测。
2.6 统计分析
本实验使用SPSS 21.0软件进行统计分析。寿命曲线通过Kaplan-Meier统计方法进行分析，化合物药效评价通过以标准的卡方检验为基础的时序检验进行。使用方差分析检验healthspan中同一时间点各组化合物间的差异。使用t检验分析各组化合物间healthspan的差异以及western的结果。
（二） 实验结果
1. 苯并吡喃并呋喃类化合物对热刺激影响下线虫的保护作用
如图1~2所示，急性热刺激会造成线虫的损伤并最终导致线虫死亡。在100 μM(图1)，26个苯并吡喃并呋喃类化合物中，大部分化合物对于急性热刺激损伤下的线虫具有优秀的保护作用，其中共有18个化合物显示出显著的差异(p < 0.001)。在300μM下(图2)，7个活性最强的苯并吡喃并呋喃类化合物对于急性热刺激损伤下的线虫具有十分显著的保护作用(p< 0.001)，其药效也更为突出。其中，化合物15具有最强的保护效果，能够延长线虫的平均存活时间23.96 ± 1.42 %。这表明此类化合物能够增强线虫抵抗外界刺激的能力。
苯并吡喃并呋喃类化合物对于线虫寿命的保护作用
化合物15和对照组DMSO作用下线虫的生存曲线如图3所示。药物组线虫的平均寿命为22.77 ± 0.31 d，而对照组的平均寿命为20.41 ± 0.27 d。化合物15能够显著地延长线虫的平均寿命(p < 0.001)。
苯并吡喃并呋喃类化合物对于线虫healthspan的保护作用
图4A和B所示的是药物组线虫和对照组线虫在整个生命周期中摆动能力和吞咽能力的变化状况。图4C和D所示的是药物组和对照组线虫的healthspan在整个寿命周期中所占的比例。
可以明显看出，在化合物15保护下，药物组线虫的摆动能力和吞咽能力的下降趋势都被显著地延缓，随着衰老程度的加深，化合物15的效果也越来越明显。在摆动实验中，药物组的healthspan是17.73 ± 0.44 d，与对照组的14.57 ± 0.33 d相比，延长了22%(p < 0.01)。而在吞咽实验中，药物组线虫的healthspan是14.68 ± 0.13 d，与对照组(12.65 ± 0.45 d)相比延长了16%(p < 0.05)。因此，化合物15不仅能够延长线虫的寿命，还能够延长在整个生命周期中，线虫处于健康状态的时期。
苯并吡喃并呋喃类化合物延长线虫寿命和健康状态的机制研究
苯并吡喃并呋喃类化合物能够增强线虫的抵抗外界热刺激能力以及延长线虫的寿命。热休克转录因子HSF-1，同样在热休克应答以及调节线虫寿命上起着重要的作用。由此推测苯并吡喃并呋喃类化合物可能通过与HSF-1的作用来起到延长寿命的作用。PS3551线虫是hsf-1突变的线虫品系，不能够产生有功能的HSF蛋白。如图5所示，化合物15在PS3551线虫上并不能表现出如同在N2线虫上增强抵抗热刺激的能力以及延长寿命的效果，由此说明化合物15的延长线虫寿命和健康状态作用需要通过HSF-1来生效。
通过western blot来研究热休克蛋白HSP70的表达量变化。HSP70是HSF-1的下游蛋白，作为分子伴侣在蛋白质稳态中起着重要作用。从图6可知，与对照组相比，化合物15作用的线虫的HSP70的表达量上调了28%(p < 0.05)，预示着苯并吡喃并呋喃类化合物可能通过增强HSF-1的转录激活能力，提高HSP蛋白的表达量。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (7)
1.一种苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，所述苯并吡喃并呋喃类化合物为：
。
2.根据权利要求1所述苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，其特征在于，在制备增强热休克转录因子HSF-1的转录激活能力药物的应用。
3.根据权利要求2所述苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，其特征在于，在制备提高热休克转录因子HSF-1的下游分子伴侣HSP70蛋白的表达药物中的应用。
4.根据权利要求3所述苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，其特征在于，在制备延长线虫的寿命和改善健康状态药物中的应用。
5.根据权利要求4所述苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，其特征在于，所述线虫为野生型的N2，hsf-1突变的PS3551 [hsf-1 (sy441)I]或Aβ模型的CL4176 [smg-1(cc546)I]中的一种或多种。
6.根据权利要求3所述苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，其特征在于，所述苯并吡喃并呋喃类化合物为化合物1、化合物2、化合物4~8，化合物11，化合物14~19、化合物21、化合物23、化合物24和化合物26。
7.根据权利要求3所述苯并吡喃并呋喃类化合物在制备调控热休克转录因子转录激活能力药物中的应用，其特征在于，所述苯并吡喃并呋喃类化合物为化合物15。
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