CN116178125A - 一种四氢姜黄素衍生物及其制备方法和应用 - Google Patents

一种四氢姜黄素衍生物及其制备方法和应用 Download PDF

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CN116178125A
CN116178125A CN202211697624.4A CN202211697624A CN116178125A CN 116178125 A CN116178125 A CN 116178125A CN 202211697624 A CN202211697624 A CN 202211697624A CN 116178125 A CN116178125 A CN 116178125A
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tetrahydrocurcumin
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cancer
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朱全红
陈奕欣
段美涛
朱泳妍
段澳
徐晓田
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Southern Medical University
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Abstract

本发明属于生物医药技术领域，具体涉及一种四氢姜黄素衍生物及其制备方法和应用。该四氢姜黄素衍生物具有α,β‑不饱和羰基结构，并且通过实验证明，对多种癌细胞都具有显著的抑制增殖的活性，且效果均优于四氢姜黄素本身；并且通过动物实验证明，本发明的四氢姜黄素衍生物可以显著抑制结肠癌HCT116细胞动物模型的肿瘤生长，其效果与阳性对照5‑FU相当，但长期毒性明显低于5‑FU，安全性更高。另外的，发明四氢姜黄素衍生物的合成路线简单，后处理方便，产率较好，成本低，适于大规模产业化生产。

Description

一种四氢姜黄素衍生物及其制备方法和应用
技术领域
本发明属于生物医药技术领域。更具体地，涉及一种四氢姜黄素衍生物及其制备方法和应用。
背景技术
癌症当前已经成为危害人类生命健康的重要杀手。据世界卫生组织(WHO)预测，到2023年，全球每年新增癌症患者将达到2200万例，并且与癌症相关的死亡病例将达到1300万例。目前对于大多数癌症来说，化疗仍是目前治疗的主要手段之一，但化疗药物严重的毒副作用和多药耐药等缺点极大地限制了其临床应用。因此，发现更多高效杀伤肿瘤细胞同时毒副作用小的药物，已成为当今抗肿瘤药物研发亟待解决的重要问题。
天然活性化合物在抗癌药物的发现和开发中起着主导作用，在迄今为止批准的上市药物中，很大一部分药物来自天然化合物或其衍生物，如紫杉醇、羟基喜树碱、长春新碱等。姜黄素(Curcumin，CUR)是从姜黄根茎中提取的一种金黄色物质，其有效的抗癌作用已被广泛报道；但是，由于化学不稳定性、吸收不良、快速代谢和快速消除，姜黄素的生物利用度较差，造成临床应用受到限制。
因此，迫切需要提供更多具有抗肿瘤效果的活性化合物，以提供更多的临床抗肿瘤用药选择。
发明内容
本发明要解决的技术问题是克服现有缺少临床可选抗肿瘤化合物的缺陷和不足，提供一种四氢姜黄素衍生物。
本发明的目的是提供所述四氢姜黄素衍生物的制备方法。
本发明另一目的是提供所述四氢姜黄素衍生物的应用。
本发明上述目的通过以下技术方案实现：
四氢姜黄素(THC)是姜黄素的活性代谢物，具有更好的化学稳定性和生物利用度，基于此，本发明提供了一种四氢姜黄素衍生物，所述四氢姜黄素衍生物具有式(I)结构：
Figure BDA0004024081470000021
其中，R选自苯基或取代苯基、杂芳基或取代杂芳基、稠环杂芳基或取代稠环杂芳基中的一种；
所述取代的取代基为一个或多个，所述取代基选自羟基、硝基、卤素、C1～5烷氧基、卤代C1～5烷氧基、C1～5烷基、卤代C1～5烷基中的一种或多种。
优选地，所述R选自苯基或取代苯基、杂芳基、稠环杂芳基中的一种；
所述取代的取代基为一个或多个，所述取代基选自羟基、硝基、卤素、C1～5烷氧基、卤代C1～5烷氧基、卤代C1～5烷基中的一种或多种。
更优选地，所述R选自苯基或取代苯基、吡啶基、噻吩基、苯并噻吩基、吡咯基、苯并吡咯基、呋喃基、苯并呋喃基、噁唑基、苯并噁唑基、噻唑基、苯并噻唑基、咪唑基、苯并咪唑基、吡唑基、苯并吡唑基、嘧啶基、哒嗪基、吡嗪基、吡喃基、吲哚基、喹啉基、异喹啉基、嘌呤基中的一种；
所述取代的取代基为一个或多个，所述取代基选自羟基、硝基、卤素、C1～5烷氧基、卤代C1～5烷氧基、卤代C1～5烷基中的一种或多种。
具体的，所述R选自以下任一结构：
Figure BDA0004024081470000022
另外的，本发明还提供了所述四氢姜黄素衍生物的制备方法，合成路线如下：
Figure BDA0004024081470000031
具体包括以下步骤：
将四氢姜黄素与式(II)化合物溶于非质子有机溶剂中，加入缩合剂和酸性试剂作为催化体系，25～140℃反应完全，后处理，即得。
进一步地，所述缩合剂选自哌啶、吡啶、六氢吡啶、三乙胺、二甲胺、环己胺、苯胺、乙二胺、二异丙胺、氟化钾、磷酸铝、磷酸氢二铵、氢氧化钠、碳酸钠、四氯化钛和三乙胺中的一种或多种。
更进一步地，所述酸性试剂选自乙酸、甲酸、碳酸、氢氟酸中的一种或多种。
进一步地，所述非质子性有机溶剂选自甲苯、苯、乙醚、氯仿、四氯化碳、二甲基亚砜、N,N-二甲基甲酰胺、丙酮、乙腈、1,3-二甲基-2-咪唑啉酮中的一种或多种。
更进一步地，所述反应通过TLC监控反应时间。
进一步地，所述后处理包括以下步骤：反应结束后，用水洗涤去除缩合剂和酸性试剂，干燥，真空蒸发有机层得到粗产物，粗产物用石油醚/乙酸乙酯(V/V，1:1)通过硅胶柱色谱纯化。
另外的，本发明还要求保护所述四氢姜黄素衍生物在制备抗肿瘤药物中的应用。
优选地，所述肿瘤包括结肠癌、宫颈癌、肺癌、肝癌、乳腺癌、前列腺癌、卵巢癌、膀胱癌、胃癌、胰腺癌和食管癌。
本发明具有以下有益效果：
本发明的四氢姜黄素衍生物具有α,β-不饱和羰基结构，并且通过实验证明，对多种癌细胞都具有显著的抑制增殖的活性，且效果均优于四氢姜黄素本身；并且通过动物实验证明，本发明的四氢姜黄素衍生物可以显著抑制结肠癌HCT116细胞动物模型的肿瘤生长，其效果与阳性对照5-FU相当，但长期毒性明显低于5-FU，安全性更高。另外的，发明四氢姜黄素衍生物的合成路线简单，后处理方便，产率较好，成本低，适于大规模产业化生产。
附图说明
图1为实验例2中化合物THC-5对HCT116细胞的细胞集落图。
图2为实验例2中化合物THC-5对HCT116细胞的细胞周期实验结果，A为流式细胞术统计DNA含量数据图；B为细胞周期的统计分析图，每组数据表示为平均值±标准差(n＝3)，与对照组相比，*P<0.05，**P<0.01，***P<0.001。
图3为实验例2中化合物THC-5对HCT116细胞的细胞凋亡实验结果：A为流式细胞术统计HCT116细胞凋亡图；B为细胞凋亡的统计分析图，每组数据表示为平均值±标准差(n＝3)，与对照组相比，*P<0.05，**P<0.01，***P<0.001。
图4为实验例2中化合物THC-5对HCT116细胞的蛋白免疫印迹实验结果：A为监测细胞凋亡蛋白Bcl-2和Bax在不同浓度的THC-5处理下的表达的蛋白免疫印迹图，其中Actin作为内参；B为细胞凋亡蛋白表达的统计分析图。
图5是为实验例3中化合物THC-5体内抗结肠癌药效实验中剥离的裸鼠肿瘤图像(A)、剥离的裸鼠肿瘤重量数据统计图(B)、肿瘤体积变化数据统计图(C)和裸鼠的平均体重数据统计图(D)。
图6为实验例3中化合物THC-5体内抗结肠癌药效实验肿瘤组织中Ki67的免疫组织化学染色图。
图7为实验例3中化合物THC-5体内抗结肠癌药效实验肿瘤组织中裸鼠组织脏器的HE染色图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，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。
本发明根据以下合成路线合成表1所示化合物：
Figure BDA0004024081470000041
表1四氢姜黄素衍生物结构
Figure BDA0004024081470000042
Figure BDA0004024081470000051
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(4-羟基亚苄基)庚烷-3,5-二酮(THC-1)的制备具体包括以下步骤：
称取的四氢姜黄素(300mg，0.806mmol)、4-羟基苯甲醛(196mg，1.612mmol)置于甲苯(20mL)中，加入哌啶(4μL)和乙酸(3.7μL)作为催化剂，在140℃下搅拌反应；当反应完成时(通过TLC监测)，用水(20mL)萃取以除去哌啶和乙酸，有机层用无水Na2SO4干燥，在真空下蒸发以获得粗产物，粗产物用石油醚/乙酸乙酯(V/V，1:1)通过硅胶柱色谱纯化，得到终产物1,7-双(4-羟基-3-甲氧基苯基)-4-(4-羟基亚苄基)庚烷-3,5-二酮(THC-1)。淡黄色粉末，产率62.43％。
1H NMR(500MHz,DMSO-d6)δ10.21(s,1H),8.69(d,J＝5.3Hz,2H),7.59(s,1H),7.19(d,J＝8.7Hz,2H),6.81(d,J＝1.9Hz,1H),6.77–6.72(m,2H),6.71(d,J＝2.0Hz,1H),6.68–6.60(m,3H),6.54(dd,J＝8.0,2.0Hz,1H),3.73(s,3H),3.70(s,3H),3.05(t,J＝7.6Hz,2H),2.75(d,J＝7.3Hz,6H).13C NMR(126MHz,DMSO-D6)δ207.31,198.68,160.04,147.41,144.69,144.65,139.71,138.53,132.24,131.82,131.40,123.71,120.51,120.46,115.95,115.28,112.72,112.52,55.55,55.49,45.09,39.94,29.49,28.31.HRMS(ESI)calculatedfor C28H28O7[M+H]+477.1835,found 477.1906.
实施例2一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(3-羟基亚苄基)庚烷-3,5-二酮(THC-2)的制备与实施例1不同之处在于，用3-羟基苯甲醛替换4-羟基苯甲醛，用三乙胺替换哌啶，其余参数及操作参考实施例1。得到化合物THC-2，淡黄色粉末，产率72.79％。
1H NMR(500MHz,DMSO-d6)δ9.76(s,1H),8.68(d,J＝2.6Hz,2H),7.65(s,1H),7.17(t,J＝7.7Hz,1H),6.86–6.81(m,3H),6.76(d,J＝7.8Hz,1H),6.68–6.64(m,2H),6.62(dd,J＝8.0,2.7Hz,2H),6.49(dd,J＝8.0,2.0Hz,1H),3.74(s,3H),3.69(s,3H),3.08(t,J＝7.5Hz,2H),2.77–2.67(m,6H).13C NMR(151MHz,DMSO)δ207.10,199.31,158.06,147.87,145.15,145.09,141.95,140.04,134.80,132.15,131.75,130.46,121.13,120.96,120.71,118.14,116.44,115.74,115.73,113.18,112.76,56.01,55.91,45.69,40.51,29.76,28.89.HRMS(ESI)calculated for C28H28O7[M+H]+477.1835,found 477.1912.
实施例3一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(4-甲氧基苄基)庚烷-3,5-二酮(THC-3)的制备与实施例1不同之处在于，用4-甲氧基苯甲醛替换4-羟基苯甲醛，用乙腈替换甲苯，用氢氧化钠替换哌啶，温度为85℃，其余参数及操作参考实施例1。得到化合物THC-3，淡黄色粉末，产率31.00％。
1H NMR(500MHz,Chloroform-d)δ7.40(s,1H),7.14(d,J＝8.9Hz,2H),6.84(d,J＝7.9Hz,1H),6.78(d,J＝8.0Hz,1H),6.75(d,J＝8.9Hz,2H),6.71(d,J＝1.9Hz,1H),6.69(dd,J＝7.9,2.0Hz,1H),6.67(d,J＝2.1Hz,1H),6.61(dd,J＝8.1,2.0Hz,1H),3.86(s,3H),3.81(s,3H),3.81(s,3H),2.96(td,J＝6.5,2.3Hz,2H),2.89(td,J＝7.4,6.9,3.6Hz,4H),2.79(t,J＝6.9Hz,2H).13C NMR(126MHz,CHLOROFORM-D)δ207.42,198.02,161.70,146.58,146.45,144.11,143.97,140.02,139.59,133.02,132.71,131.87,125.34,121.32,120.99,114.52,114.36,111.55,111.32,56.02,55.97,55.50,45.94,40.76,30.12,28.98.
实施例4一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(4-乙基亚苄基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-4)的制备与实施例1不同之处在于，用4-乙基苯甲醛替换4-羟基苯甲醛，用二氯甲烷替换甲苯，温度为25℃，其余参数及操作参考实施例1。得到化合物THC-4，黄色油状物，产率36.91％。
1H NMR(400MHz,Chloroform-d)δ7.45(s,1H),7.12(q,J＝8.9,7.6Hz,4H),6.84(d,J＝8.0Hz,1H),6.77(d,J＝8.1Hz,1H),6.70(d,J＝13.4Hz,2H),6.64(d,J＝2.3Hz,1H),6.57(d,J＝8.0Hz,1H),5.55(d,J＝8.7Hz,2H),3.87(s,3H),3.81(s,3H),2.95(d,J＝7.2Hz,2H),2.88(q,J＝7.2,6.7Hz,4H),2.75(q,J＝7.2,5.8Hz,2H),2.64(q,J＝7.7Hz,2H),1.26–1.20(m,3H).13C NMR(101MHz,CDCl3)δ207.14,198.09,147.60,146.60,146.46,144.15,144.00,141.35,139.83,132.96,132.64,130.35,130.00,128.61,121.23,121.01,114.53,114.35,111.45,111.34,56.04,55.95,45.98,40.90,30.08,29.06,28.84,15.20.HRMS(ESI)calculated for C30H32O6[M+H]+489.2199,found 489.2259.HRMS(ESI)calculated for C29H30O7[M+H]+491.1992,found 491.2058.
实施例5一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(4-氟亚苄基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-5)的制备与实施例1不同之处在于，用4-氟苯甲醛替换4-羟基苯甲醛，其余参数及操作参考实施例1。得到化合物THC-5，黄色油状物，产率84.46％。
1H NMR(600MHz,Chloroform-d)δ7.40(s,1H),7.21–7.16(m,2H),6.98–6.92(m,2H),6.84(d,J＝8.0Hz,1H),6.78(d,J＝8.0Hz,1H),6.71(d,J＝1.9Hz,1H),6.69(dd,J＝8.0,2.0Hz,1H),6.62(d,J＝2.0Hz,1H),6.59(dd,J＝8.1,2.0Hz,1H),3.87(s,3H),3.81(s,3H),2.95(ddd,J＝7.9,6.5,1.7Hz,2H),2.90(ddd,J＝8.6,6.5,1.7Hz,2H),2.85(t,J＝7.2Hz,2H),2.76–2.73(m,2H).13C NMR(151MHz,CDCl3)δ206.73,197.89,164.77,163.09,146.59,146.47,144.19,144.07,142.09,138.30,132.83,132.43,131.89,131.83,129.20,129.18,121.25,121.00,116.37,116.22,114.53,114.37,111.39,111.31,77.37,77.16,76.95,56.04,55.96,46.05,40.99,30.02,28.97,0.12.HRMS(ESI)calculated forC28H27FO6[M+H]+479.1792,found 479.1862.
实施例6一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(4-氯亚苄基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-6)的制备与实施例1不同之处在于，用4-氯苯甲醛替换4-羟基苯甲醛，用吡啶替换哌啶，用甲酸替换乙酸，其余参数及操作参考实施例1。得到化合物THC-6，黄色油状物，产率80.68％。
1H NMR(600MHz,Chloroform-d)δ7.39(s,1H),7.24–7.21(m,2H),7.13–7.10(m,2H),6.84(d,J＝8.0Hz,1H),6.80–6.77(m,1H),6.71(d,J＝2.0Hz,1H),6.69(dd,J＝8.0,2.0Hz,1H),6.59(d,J＝7.4Hz,2H),5.51(s,2H),3.87(s,3H),3.81(s,3H),2.95(ddd,J＝7.9,6.5,1.7Hz,2H),2.92–2.88(m,2H),2.85(t,J＝7.2Hz,2H),2.75–2.71(m,2H).13C NMR(151MHz,CDCl3)δ206.53,197.82,146.60,146.49,144.20,144.10,142.70,138.10,136.79,132.78,132.36,131.45,130.89,129.33,121.27,121.00,114.53,114.37,111.33,111.31,56.04,55.95,46.07,41.06,29.99,28.95,0.12.HRMS(ESI)calculated forC28H27ClO6[M+H]+495.1496,found495.1569.
实施例7一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(4-溴亚苄基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-7)的制备与实施例1不同之处在于，用4-溴苯甲醛替换4-羟基苯甲醛，用乙二胺替换哌啶，用碳酸替换乙酸，其余参数及操作参考实施例1。得到化合物THC-7，黄色油状物，产率67.15％。
1H NMR(600MHz,Chloroform-d)δ7.39(s,1H),7.38–7.36(m,2H),7.06–7.02(m,2H),6.84(d,J＝8.0Hz,1H),6.79(d,J＝8.5Hz,1H),6.70(d,J＝2.0Hz,1H),6.69(dd,J＝8.0,2.0Hz,1H),6.60–6.57(m,2H),5.51(s,2H),3.87(s,3H),3.81(s,3H),2.96–2.92(m,2H),2.91–2.87(m,2H),2.84(t,J＝7.2Hz,2H),2.72(t,J＝7.0Hz,2H).13C NMR(151MHz,CDCl3)δ206.49,197.81,146.59,146.49,144.19,144.10,142.79,138.16,132.76,132.34,132.30,131.88,131.03,125.18,121.28,121.00,114.53,114.37,111.31,111.30,56.04,55.97,46.07,41.07,29.98,28.94.HRMS(ESI)calculated for C28H27BrO6[M+H]+539.0991and 541.0971,found 539.1059and 541.1041.
实施例8一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(4-(三氟甲基)亚苄基)庚烷-3,5-二酮(THC-8)的制备与实施例1不同之处在于，用4-三氟甲基苯甲醛替换4-羟基苯甲醛，用三乙胺替换哌啶，用氢氟酸替换乙酸，其余参数及操作参考实施例1。得到化合物THC-8，黄色油状物，产率93.28％。
1H NMR(600MHz,Chloroform-d)δ7.62(d,J＝7.5Hz,1H),7.56(s,1H),7.44(s,1H),7.37–7.31(m,2H),6.84(d,J＝8.0Hz,1H),6.76(d,J＝8.0Hz,1H),6.71(d,J＝1.9Hz,1H),6.69(dd,J＝8.0,2.0Hz,1H),6.59(d,J＝2.0Hz,1H),6.55(dd,J＝8.0,2.0Hz,1H),5.50(d,J＝18.5Hz,2H),3.87(s,3H),3.80(s,3H),2.99–2.95(m,2H),2.90–2.92(m,2H),2.84(t,J＝7.3Hz,2H),2.73(t,J＝7.2Hz,2H).13CNMR(151MHz,CDCl3)δ206.19,197.83,146.62,146.49,144.25,144.08,143.87,137.50,133.87,132.65,132.29,132.27,131.69,131.48,129.70,126.97,126.95,126.93,126.53,126.50,124.59,122.78,121.09,121.03,114.56,114.40,111.30,111.22,56.04,55.92,46.22,41.11,29.99,28.97.HRMS(ESI)calculated for C29H27F3O6[M+H]+529.1760,found 529.1830.
实施例9一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(4-(三氟甲氧基)亚苄基)庚烷-3,5-二酮(THC-9)的制备与实施例1不同之处在于，用4-三氟甲氧基苯甲醛替换4-羟基苯甲醛，用氟化钾替换哌啶，其余参数及操作参考实施例1。得到化合物THC-9，黄色油状物，产率45.79％。
1H NMR(400MHz,Chloroform-d)δ7.40(s,1H),7.21(d,J＝7.4Hz,2H),7.09(d,J＝6.4Hz,2H),6.83(d,J＝8.7Hz,1H),6.77(d,J＝8.7Hz,1H),6.69(d,J＝10.3Hz,2H),6.63(d,J＝3.2Hz,1H),6.58(d,J＝8.3Hz,1H),5.61(s,2H),3.86(s,3H),3.80(s,3H),2.95(q,J＝4.4,3.8Hz,2H),2.93–2.81(m,4H),2.75(t,J＝5.6Hz,2H).13C NMR(101MHz,CDCl3)δ206.44,197.90,150.53,146.62,146.49,144.19,144.09,142.85,137.70,132.72,132.32,131.68,131.32,121.23,120.99,114.56,114.40,111.43,111.33,56.00,55.89,46.05,40.99,29.95,28.88.HRMS(ESI)calculated for C29H27F3O7[M+H]+545.1709,found545.1771.
实施例10一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(4-硝基亚苄基)庚烷-3,5-二酮(THC-10)的制备与实施例1不同之处在于，用4-硝基苯甲醛替换4-羟基苯甲醛，用丙酮替换甲苯，用苯胺替换哌啶，温度为60℃，其余参数及操作参考实施例1。得到化合物THC-10，橙色油状物，产率83.64％。
1H NMR(400MHz,Chloroform-d)δ8.13(d,J＝7.0Hz,2H),7.52(s,1H),7.38(d,J＝7.3Hz,2H),6.89(dd,J＝28.8,8.1Hz,2H),6.82–6.72(m,2H),6.66(d,J＝6.5Hz,2H),5.79(s,2H),3.96(s,3H),3.85(s,3H),3.13–2.96(m,4H),2.96–2.77(m,4H).13C NMR(101MHz,CDCl3)δ205.57,197.75,148.25,146.63,146.50,145.19,144.20,144.18,139.16,136.39,132.48,132.03,130.10,123.90,121.27,120.94,114.58,114.42,111.36,56.00,55.88,46.08,41.09,29.85,28.62.HRMS(ESI)calculated for C28H27NO8[M+H]+506.1737,found506.1804.
实施例11一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(吡啶-4-亚甲基)庚烷-3,5-二酮(THC-11)的制备与实施例1不同之处在于，用吡啶-4-甲醛替换4-羟基苯甲醛，用N,N-二甲基甲酰胺替换甲苯，用磷酸氢二铵替换哌啶，其余参数及操作参考实施例1。得到化合物THC-11，黄色油状物，产率25.21％。
1H NMR(400MHz,Chloroform-d)δ8.50(s,2H),7.30(s,1H),7.01(s,2H),6.83(d,J＝6.9Hz,1H),6.77(d,J＝6.7Hz,1H),6.68(d,J＝4.2Hz,2H),6.62–6.51(m,2H),3.86(s,3H),3.79(s,3H),3.00–2.92(m,2H),2.90(d,J＝5.7Hz,2H),2.82(d,J＝6.3Hz,2H),2.72(d,J＝7.7Hz,2H).13C NMR(101MHz,CDCl3)δ205.40,197.72,150.34,146.71,146.63,145.75,144.33,144.25,140.62,135.98,132.46,132.06,123.13,121.21,120.99,114.67,114.58,111.46,111.38,56.04,55.97,46.14,41.21,29.87,28.80.HRMS(ESI)calculatedfor C27H27NO6[M+H]+462.1838,found 462.1904.
实施例12一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物1,7-双(4-羟基-3-甲氧基苯基)-4-(噻吩-3-亚甲基)庚烷-3,5-二酮(THC-12)的制备与实施例1不同之处在于，用噻吩-3-甲醛替换4-羟基苯甲醛，用二异丙胺替换哌啶，其余参数及操作参考实施例1。得到化合物THC-12，黄色油状物，产率78.23％。
1H NMR(500MHz,Chloroform-d)δ7.38(s,1H),7.33–7.30(m,1H),7.23(dd,J＝5.1,2.9Hz,1H),6.86(dd,J＝5.1,1.4Hz,1H),6.84(d,J＝7.9Hz,1H),6.80(d,J＝8.0Hz,1H),6.72–6.67(m,3H),6.64(dd,J＝8.0,2.0Hz,1H),5.56(s,2H),3.86(s,3H),3.83(s,3H),2.96(ddd,J＝8.0,6.6,1.7Hz,2H),2.93–2.91(m,2H),2.91–2.89(m,2H),2.88–2.87(m,1H),2.87–2.84(m,1H).13C NMR(126MHz,CHLOROFORM-D)δ207.04,198.33,146.58,146.49,144.14,144.04,140.48,134.72,132.88,132.75,132.61,130.44,127.76,127.17,121.31,120.97,114.52,114.41,111.54,111.30,56.01,45.94,40.56,30.05,28.93.HRMS(ESI)calculated for C26H26O6S[M+H]+467.1450,found 467.1525.
实施例13一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(苯并噻吩-3-亚甲基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-13)的制备与实施例1不同之处在于，用苯并噻吩-3-甲醛替换4-羟基苯甲醛，用四氯化钛和三乙胺替换哌啶，其余参数及操作参考实施例1。得到化合物THC-13，黄色固体，产率30.19％。
1H NMR(400MHz,Chloroform-d)δ7.85(d,J＝6.8Hz,1H),7.78(d,J＝6.9Hz,1H),7.72(s,1H),7.44(t,J＝8.7Hz,2H),7.27(s,1H),6.86(d,J＝7.9Hz,1H),6.73(d,J＝11.2Hz,3H),6.58(d,J＝11.1Hz,2H),3.87(s,3H),3.75(s,3H),3.03(d,J＝5.6Hz,2H),3.00–2.91(m,2H),2.90–2.83(m,2H),2.81(d,J＝6.1Hz,2H).13C NMR(101MHz,CDCl3)δ206.89,197.74,146.59,146.45,144.15,144.00,142.46,139.68,137.87,132.75,132.32,129.98,129.95,128.45,125.36,125.02,122.94,121.32,121.24,120.96,114.55,114.41,111.43,111.31,55.95,55.85,45.52,40.90,30.07,28.99.HRMS(ESI)calculated forC30H28O6S[M+H]+517.1607,found517.1675.
实施例14一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-((1H-吡咯-3-基)亚甲基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-14)的制备与实施例1不同之处在于，用1H-吡咯-3-甲醛替换4-羟基苯甲醛，用环己胺替换哌啶，其余参数及操作参考实施例1。得到化合物THC-14，棕色固体，产率52.69％。
1H NMR(600MHz,DMSO-d6)δ11.40(s,1H),8.66(s,2H),7.60(s,1H),7.22(dt,J＝3.2,1.8Hz,1H),6.81(dd,J＝5.1,2.3Hz,2H),6.75(d,J＝2.0Hz,1H),6.65(ddd,J＝7.2,6.5,0.8Hz,2H),6.62(dd,J＝8.1,1.9Hz,1H),6.56(dd,J＝8.0,2.0Hz,1H),5.94(q,J＝2.2Hz,1H),3.73(s,3H),3.72(s,3H),2.98(t,J＝7.7Hz,2H),2.84–2.80(m,2H),2.80–2.76(m,2H),2.74(t,J＝7.6Hz,2H).13C NMR(151MHz,DMSO)δ207.57,198.22,147.40,147.38,144.63,144.59,135.25,135.12,131.99,131.58,125.64,120.98,120.44,120.31,117.43,115.30,115.26,112.68,112.46,107.75,55.53,55.51,44.86,38.79,29.69,28.45.HRMS(ESI)calculated for C26H27NO6[M+H]+450.1838,found 450.1904.
实施例15一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-((1H-吲哚-3-基)亚甲基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-15)的制备与实施例1不同之处在于，用1H-吲哚-3-基甲醛替换4-羟基苯甲醛，用二甲基亚砜替换甲苯，其余参数及操作参考实施例1。得到化合物THC-15，黄色油状物，产率17.41％。
1H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.77(s,1H),7.66(d,J＝7.4Hz,1H),7.37(d,J＝7.7Hz,1H),7.24(s,1H),6.89(s,1H),6.85(dd,J＝7.8,2.9Hz,1H),6.82–6.70(m,3H),6.70–6.60(m,2H),5.60(s,2H),3.87(s,3H),3.73(s,3H),3.05(d,J＝6.8Hz,2H),2.94(d,J＝13.5Hz,6H).13C NMR(101MHz,CDCl3)δ208.17,198.09,146.64,146.50,144.12,143.73,137.15,135.92,133.21,133.18,131.75,128.00,127.35,123.58,121.61,121.06,118.17,114.57,114.29,111.99,111.88,111.39,110.12,56.03,55.99,45.16,40.25,30.51,29.10.HRMS(ESI)calculated for C30H29NO6[M+H]+500.1995,found500.2066.
实施例16一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(呋喃-2-亚甲基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-16)的制备与实施例1不同之处在于，用呋喃-2-甲醛替换4-羟基苯甲醛，用磷酸铝替换哌啶，其余参数及操作参考实施例1。得到化合物THC-16，黄色油状物，产率79.84％。
1H NMR(600MHz,Chloroform-d)δ7.34(d,J＝1.7Hz,1H),7.14(s,1H),6.82(dd,J＝11.4,8.0Hz,2H),6.76(d,J＝1.9Hz,1H),6.71–6.66(m,3H),6.63(d,J＝3.5Hz,1H),6.44(dd,J＝3.5,1.8Hz,1H),3.86(s,3H),3.85(s,3H),2.99–2.96(m,2H),2.96–2.93(m,2H),2.92–2.89(m,2H),2.89–2.86(m,2H).13C NMR(151MHz,CDCl3)δ205.64,197.38,149.02,146.60,146.57,146.45,144.14,143.94,137.83,133.09,132.87,124.77,121.18,120.96,118.40,114.49,114.32,113.08,111.56,111.28,56.02,45.88,40.59,30.02,28.98.HRMS(ESI)calculated for C26H26O7[M+H]+451.1679,found 451.1749.
实施例17一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(呋喃-3-亚甲基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-17)的制备与实施例1不同之处在于，用呋喃-3-甲醛替换4-羟基苯甲醛，用乙二胺替换哌啶，其余参数及操作参考实施例1。得到化合物THC-17，橙色油状物，产率80.11％。
1H NMR(400MHz,Chloroform-d)δ7.57(s,1H),7.33(s,1H),7.23(s,1H),6.85–6.75(m,2H),6.74–6.62(m,4H),6.15(s,1H),3.83(d,J＝4.3Hz,6H),2.91(p,J＝8.5,7.3Hz,8H).13C NMR(101MHz,CDCl3)δ206.66,198.19,146.59,146.51,146.41,144.66,144.14,144.02,140.61,132.81,132.58,129.90,121.14,120.92,120.19,114.52,114.39,111.43,111.30,109.36,55.98,45.84,40.29,30.00,28.98.HRMS(ESI)calculated forC26H26O7[M+H]+451.1679,found 451.1751.
实施例18一种四氢姜黄素衍生物的制备
四氢姜黄素衍生物4-(苯并呋喃-2-亚甲基)-1,7-双(4-羟基-3-甲氧基苯基)庚烷-3,5-二酮(THC-18)的制备与实施例1不同之处在于，用苯并呋喃-2-甲醛替换4-羟基苯甲醛，用二异丙胺替换哌啶，其余参数及操作参考实施例1。得到化合物THC-18，黄色粉末，产率22.77％。
1H NMR(500MHz,Chloroform-d)δ7.58(d,J＝7.9Hz,1H),7.40–7.34(m,2H),7.28(s,1H),7.25(dd,J＝7.2,2.4Hz,1H),6.95(s,1H),6.83(dd,J＝18.4,8.0Hz,2H),6.73(d,J＝2.0Hz,1H),6.71(d,J＝2.0Hz,1H),6.71–6.70(m,1H),6.69–6.68(m,1H),5.56(s,2H),3.87(s,3H),3.79(s,3H),3.10–3.05(m,2H),3.05–3.00(m,2H),2.98–2.94(m,2H),2.93–2.88(m,2H).13C NMR(126MHz,CHLOROFORM-D)δ205.46,197.23,156.23,150.39,146.60,146.54,144.17,144.02,140.22,132.89,132.76,128.06,127.43,125.06,123.94,122.29,121.12,120.97,114.52,114.43,114.39,111.59,111.33,111.29,56.02,55.94,46.25,40.89,29.95,29.26.HRMS(ESI)calculated for C30H28O7[M+H]+501.1835,found501.1908.
实验例1细胞活性检测(MTT)
1、实验方法：
取对数生长期、生长状态良好的细胞(结肠癌细胞HCT116、宫颈癌细胞Hela、肺癌细胞A549、肝癌细胞HepG2)，以5×103个/孔，接入96孔板，同时设空白组，37℃培养24h(在细胞孔周围内加入100μL无菌PBS)；用不同浓度的四氢姜黄素衍生物处理72h后，每孔加入10μL 5mg/ml的MTT溶液，37℃培养4h后，弃上清液，加入150μL DMSO溶液，振荡10min，用酶标仪在490nm下测定各孔OD值，计算抑制率，得到IC50。
2、实验结果：
实验结果参见表2。
表2四氢姜黄素衍生物对肿瘤细胞的抗增殖活性
Figure BDA0004024081470000141
Figure BDA0004024081470000151
由表可见，通过MTT法，将一系列四氢姜黄素衍生物对四种癌细胞进行细胞活性测定，结果显示这些衍生物对四种肿瘤细胞均具有较好的抑制增殖效果，其中对HCT116细胞的抑制作用效果最显著，并且化合物THC-5的作用效果最好。因此后续以化合物THC-5为例进行相应的活性实验，其余化合物效果与化合物THC-5接近。
实验例2化合物THC-5对HCT116细胞进一步机制研究
1、集落形成实验
取对数生长期、生长状态良好的HCT116细胞，以1×103个/孔，接入6孔板，同时设空白组(Control)和阳性对照组(5-氟尿嘧啶，5-FU，12μM)，37℃培养24h；用不同浓度(3μM、6μM、9μM)的四氢姜黄素衍生物化合物THC-5处理48h后，用含有10％ FBS的完全新鲜培养基替换，再培养7天；用PBS洗涤集落三次，并用4％多聚甲醛固定30分钟，用0.1％结晶紫溶液染色集落30分钟，然后用PBS清洗3至5次；最后，通过Image J软件对可见菌落进行拍照和计数。
结果参见图1，由图可见，化合物THC-5可以抑制菌落形成，并且随着化合物THC-5浓度的增加，癌细胞的菌落数显著减少，当THC-5的浓度达到9μM时，肿瘤细胞生长几乎被完全抑制，甚至效果比阳性对照5-FU更好。以上结果表明，化合物THC-5显著抑制HCT116细胞生长。
2、细胞周期实验
取对数生长期、生长状态良好的HCT116细胞，以2×105个/孔，接入6孔板，同时设空白组(Control)和阳性对照组(5-氟尿嘧啶，5-FU，12μM)，37℃培养24h；用不同浓度(3μM、6μM、9μM)的四氢姜黄素衍生物化合物THC-5处理48h后，收集细胞并在预冷PBS中洗涤，悬浮在冰冷的70％乙醇中，并在4℃下储存过夜；固定后，在室温下用PBS洗涤细胞；最后，将细胞与PI/RNase染色缓冲液在37℃下在黑暗中孵育30分钟，通过流式细胞术评估不同组细胞中的DNA含量。
结果参见图2，由图可见，化合物THC-5对细胞周期具有阻滞作用；用化合物THC-5处理48h后，S期细胞百分比从空白组的22.85％显著增加到56.98％，呈现剂量依赖性积累。这些结果表明，化合物THC-5可以诱导HCT116细胞周期阻滞在S期。
3、细胞凋亡实验
取对数生长期、生长状态良好的HCT116细胞，以2×105个/孔，接入6孔板，同时设空白组(Control)和阳性对照组(5-氟尿嘧啶，5-FU，12μM)，37℃培养24h；用不同浓度(3μM、6μM、9μM)的四氢姜黄素衍生物化合物THC-5处理48h后，收集细胞，用PBS洗涤两次，再悬浮在0.5mL结合缓冲液中；膜联蛋白V-FITC(5μL)和碘化丙啶PI(5μL)将混合物在室温下在黑暗中反应15分钟，通过流式细胞仪分析测量凋亡细胞。
结果参见图3，由图可见，化合物THC-5具有诱导细胞凋亡的作用；随化合物THC-5的浓度从3μM增加到9μM时，晚期凋亡的细胞比例从空白组的5.73％显著增加到19.30％和70.30％；用浓度为3μM、6μM、9μM的化合物THC-5处理48h后，HCT116细胞总凋亡细胞(早期+晚期)的百分比分别为22.14％、33.21％、76.92％，而空白组中仅含有6.68％的凋亡细胞。这些结果表明，化合物THC-5可以通过诱导细胞凋亡发挥抗肿瘤作用。
4、蛋白免疫印迹实验
取对数生长期、生长状态良好的HCT116细胞，以2×105个/孔，接入6孔板，同时设空白组，37℃培养24h；用不同浓度(3μM、6μM、9μM)的四氢姜黄素衍生物处理48h；收集细胞，冰上提取蛋白，测定蛋白浓度；电泳分离，转膜，封闭后，加入不同抗体，4℃孵育过夜。HRP标记羊抗兔抗体作为二抗，曝光，实验重复三次，记录相关数据。
结果如图4所示，由图4(A、B)可见，与空白组相比，化合物THC-5可以显著影响一些Bcl-2家族蛋白的表达，尤其是上调促凋亡蛋白Bax的表达，下调抗凋亡蛋白Bcl-2的表达，且具有剂量依赖性。这些结果表明，THC-5可以通过调节凋亡相关蛋白来诱导细胞凋亡。
以上实验表明，所选化合物THC-5作为一种新型结构的先导物，可以有效抑制结肠癌HCT116细胞的增殖、阻滞细胞S期、诱导细胞凋亡，进而抑制细胞生长。
实验例3化合物THC-5体内抗结肠癌药效实验
1、裸鼠抗肿瘤实验
在对数期培养并收集HCT116细胞，对BALB/c裸鼠(4～5周龄)皮下注射悬浮在0.1mL PBS中的5×106细胞；当肿瘤生长至80～120m3时，将裸鼠随机分为五组，每组6只，用于HCT116异种移植模型。对于溶媒组(Vehicle)，小鼠腹腔注射3％ DMA/50％ PEG400/47％生理盐水；对于阳性对照组，小鼠腹腔注射25mg/kg 5-FU；对于不同剂量的药物组，分别向小鼠腹腔注射25mg/kg、50mg/kg和75mg/kg的化合物THC-5。每2天测量一次体重和肿瘤体积。用游标卡尺测定肿瘤体积，计算公式为：A×B2/2(A，长径；B，短径)。21天后，当处死小鼠时，剥离肿瘤并称重。
结果如图5所示，由图可见，化合物THC-5能显著抑制肿瘤生长：与5-FU诱导的77.15％的进展抑制相比，化合物THC-5不同浓度的肿瘤生长抑制率分别为30.76％、66.28％和85.12％；与载体组相比，化合物THC-5治疗组的小鼠体重没有显著下降，而5-FU组的有所下降；另外的，载体组肿瘤体积迅速增大，21天后达到924.50±207.74mm3，而化合物THC-5不同浓度组肿瘤体积缓慢增大，21天后分别达到727.17±183.20mm3、359.20±131.83mm3和185.40±99.93mm3。其中，化合物THC-5的75mg/kg组的抑制效果与阳性对照5-FU(200.50±80.29mm3)相比，效果更好。这些结果说明化合物THC-5可以显著抑制肿瘤的增长。
2、免疫组化
为了进一步确认化合物THC-5对体内肿瘤的影响，本发明对动物研究结束时收集的HCT116肿瘤组织用PBS洗涤两次以去除血液，然后用4％福尔马林固定，石蜡包埋后，用Ki67做免疫组化。
结果如图6所示，在化合物THC-5和5-FU处理的小鼠肿瘤切片中，细胞增殖标记物Ki67的染色明显减少，这说明THC-5能够有效抑制肿瘤在体内的的增殖及生长。
3、HE染色
当处死小鼠时，剥离主要组织脏器，包括心脏、肝脏、脾脏、肺和肾脏，用PBS洗涤两次以去除血液，然后用4％福尔马林固定，石蜡包埋并用HE染色。
在治疗期间，5-FU组的小鼠在第12天死亡1只，第18天死亡2只，而载体组和化合物THC-5的25mg/kg剂量未出现小鼠死亡情况，化合物THC-5的50mg/kg和75mg/kg在第18天分别死亡一只小鼠。
结果参见图7，通过器官组织(心、肝、脾、肺、肾)的组织学分析，可以体现药物对裸鼠初级器官的潜在细胞毒性。由图可见，与载体组相比，化合物THC-5在25mg/kg浓度下的组织未发现异常，而在50mg/kg和75mg/kg浓度下出现点状性或片状性肝坏死情况；此外，5-FU组同样出现肝坏死，且情况更加严重。这说明长期采用5-FU治疗会产生肝毒性导致小鼠死亡，化合物THC-5在50mg/kg和75mg/kg的浓度下也会导致小鼠肝损伤，但毒性程度不及5-FU。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.一种四氢姜黄素衍生物，其特征在于，所述四氢姜黄素衍生物具有式(I)结构：
Figure FDA0004024081460000011
其中，R选自苯基或取代苯基、杂芳基或取代杂芳基、稠环杂芳基或取代稠环杂芳基中的一种；
所述取代的取代基为一个或多个，所述取代基选自羟基、硝基、卤素、C1～5烷氧基、卤代C1～5烷氧基、C1～5烷基、卤代C1～5烷基中的一种或多种。
2.根据权利要求1所述四氢姜黄素衍生物，其特征在于，所述R选自苯基或取代苯基、杂芳基、稠环杂芳基中的一种；
所述取代的取代基为一个或多个，所述取代基选自羟基、硝基、卤素、C1～5烷氧基、卤代C1～5烷氧基、卤代C1～5烷基中的一种或多种。
3.根据权利要求2所述四氢姜黄素衍生物，其特征在于，所述R选自苯基或取代苯基、吡啶基、噻吩基、苯并噻吩基、吡咯基、苯并吡咯基、呋喃基、苯并呋喃基、噁唑基、苯并噁唑基、噻唑基、苯并噻唑基、咪唑基、苯并咪唑基、吡唑基、苯并吡唑基、嘧啶基、哒嗪基、吡嗪基、吡喃基、吲哚基、喹啉基、异喹啉基、嘌呤基中的一种；
所述取代的取代基为一个或多个，所述取代基选自羟基、硝基、卤素、C1～5烷氧基、卤代C1～5烷氧基、卤代C1～5烷基中的一种或多种。
4.根据权利要求3所述四氢姜黄素衍生物，其特征在于，所述R选自以下任一结构：
Figure FDA0004024081460000021
5.权利要求1～4任一所述四氢姜黄素衍生物的制备方法，其特征在于，合成路线如下：
Figure FDA0004024081460000022
具体包括以下步骤：
将四氢姜黄素与式(II)化合物溶于非质子有机溶剂中，加入缩合剂和酸性试剂作为催化体系，25～140℃反应完全，后处理，即得。
6.根据权利要求5所述制备方法，其特征在于，所述缩合剂选自哌啶、吡啶、六氢吡啶、三乙胺、二甲胺、环己胺、苯胺、乙二胺、二异丙胺、氟化钾、磷酸铝、磷酸氢二铵、氢氧化钠、碳酸钠、四氯化钛和三乙胺中的一种或多种。
7.根据权利要求5所述制备方法，其特征在于，所述酸性试剂选自乙酸、甲酸、碳酸、氢氟酸中的一种或多种。
8.根据权利要求5所述制备方法，其特征在于，所述非质子有机溶剂选自甲苯、苯、乙醚、氯仿、四氯化碳、二氯甲烷、二甲基亚砜、N,N-二甲基甲酰胺、丙酮、乙腈、1,3-二甲基-2-咪唑啉酮中的一种或多种。
9.权利要求1～4任一所述四氢姜黄素衍生物在制备抗肿瘤药物中的应用。
10.根据权利要求9所述应用，其特征在于，所述肿瘤包括结肠癌、宫颈癌、肺癌、肝癌、乳腺癌、前列腺癌、卵巢癌、膀胱癌、胃癌、胰腺癌和食管癌。
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