CN101879158A青蒿素衍生物的新应用---防治肥胖病的|New application of artemisinin derivant
Entry posted by Roger in Artemisinin Patent｜青蒿素专利
55 views
The invention discloses an application of arteannuin derivant with the structure disclosed in I,II,III, IV and V for preparing medicines for preventing and treating adiposity, wherein n=0-1; R1 is selected from C1-C3 alkyl; and the R2 is selected from aryl or aromatic heterocyclic ring substituted by nitrogenous atom. The medicine prepared from the arteannuin organisms is taken as the medicine for resisting adipose differentiation and can play a very important role in treating the diseases.
本发明公开了具有以下式Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ结构的青蒿素衍生物在制备防治肥胖的药物中的应用，其中：n＝0～1；R1选自C1～C3烷基；R2选自芳基或含氮原子取代的芳香杂环。由所述青蒿素类生物制备的药物作为抗脂肪分化的药物，将会在这些疾病的治疗中发挥重要作用。
青蒿素衍生物的新应用
其中，二氢青蒿素、青蒿素苯甲酸酯具有所有检测过的青蒿素衍生物中最强的抑制3T3-L1细胞分化的效果。其次是青蒿琥酯，拥有仅次于前两者的抑制3T3-L1细胞分化的效果。这3个化合物都能够在10μM的水平完全抑制3T3-L1细胞的分化。
技术领域
本发明涉及青蒿素衍生物在制药中的新应用，属于化学生物学和细胞生物学领域。
背景技术
青蒿素是从黄花蒿中提取的含有过氧基团的倍半萜内酯药物。青蒿素类抗疟药物的发现是全球抗疟药物发展史上继奎宁之后的又一里程碑，它是我国发现的第一个植物化学药品，也是中国唯一被世界卫生组织认可的、可按合成药研究标准开发的中药。30多年来，青蒿素独特的分子结构和突出的生物活性深深吸引着科学家们的眼球。随着医药科技的发展，经过药理作用和临床应用证明，青蒿素衍生物被广泛应用于治疗疟疾，癌症，急性感染及高热病和皮肤病等疾病中。其中临床研究较多的青蒿素衍生物有二氢青蒿素、蒿甲醚、蒿乙醚和青蒿琥酯等。目前尚未见有关青蒿素衍生物抑制脂肪细胞分化的文献报道。
肥胖是指机体进食热量多于身体消耗量，造成脂肪的过渡积累与脂肪组织的过量扩增，使体重超过标准体重的20％以上，并伴有头昏乏力、神疲气短的一类病症，是高血压病、糖尿病、高血脂、冠心病、脑血管病的危险因素。当今，肥胖已成为全球蔓延速度最快、最严重的公共卫生问题之一。近年来，随着我国人民生活水平的提高，在城市人口中肥胖人数增加了，而且不仅发生在老年人，还发生在儿童、妇女，各个年龄组身体肥胖的人数都在增加，减肥已经成为人们的热点话题。引起肥胖症的病因有很多，其中主要与遗传和环境因素有密切关系。环境因素是指长期食入高脂、高热量食物，再加上体力活动减少，心理障碍等因素的作用下，引起体脂调控网络的神经内分泌调节紊乱。
因此，预防和控制肥胖病的发生已成为医学上十分关注的问题。目前，常用的减肥药物大致分为三类：一、控制食欲的药物，此类药物有诸多不良反应，如失眠、口干、便秘、心悸和高血压；二、增加能量消耗的药物；三、抑制肠道吸收的药物。尽管现在有许多研究已深入到基因药物水平，但目前仍然没有找到一种真正有效的、能长期使用的减肥药物，研究出更为理想的有效的方法和制剂用于肥胖病的诊断和治疗仍然具有深远的意义。
3T3-L1是一个衍生自3T3细胞(一个建立于1962年的标准成纤维细胞的细胞系)的，在生物研究中被应用于研究脂肪组织的细胞系。3T3-L1细胞拥有类似成纤维细胞的形态，但是在适当条件的诱导下，能够分化并形成脂肪细胞的表型。3T3-L1细胞分化成熟后的脂肪细胞形态，胞内甘油三酯的合成和积累相对于分化前的成纤维细胞状态都大大提高，这时候的3T3-L1细胞呈现一种图章戒指的脂肪细胞形态。这些细胞对调节脂肪生成和脂肪降解的激素和药物都敏感，例如肾上腺素，去甲肾上腺素，胰岛素等。
因为3T3-L1细胞的这些特性，该细胞系被广泛应用于脂肪分化和代谢疾病机理的研究，是国际公认的体外脂肪分化的细胞系模型。对该模型的研究已经帮助阐明了一个在脂肪细胞末端分化中最为主要的调控脂肪分化的转录级联信号转导，包括了一系列转录因子、调控蛋白和效应蛋白。
油红O染色技术是一种常用的脂类染色法，其优点是染色步骤简便，染色结果肯定。染色后脂肪呈鲜红色，细胞核呈蓝色，间质无色。油红O染出的红色与脂肪细胞内积累形成的油滴的量成正比，而后者与脂肪细胞分化程度的高低成正比，所以通过该技术，既可以定性观察判断，亦可以定量检测比较脂肪细胞分化程度的高低。
黄花蒿广泛存在于世界各地，但青蒿素含量较高的黄花蒿仅存在于我国重庆东部、福建、广西、海南部分地区，属于我国独有的药物资源。近几十年，青蒿素衍生物的研究主要集中在抗疟疾和抗肿瘤方面，将其应用到减肥药的研究却很罕见。
发明内容
本发明的目的是为了完善青蒿素衍生物在新领域里的应用，即提供一种蒿素衍生物的新应用。
具体技术方案如下：
具有以下式Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ结构的青蒿素衍生物在制备防治肥胖的药物中的应用，
其中：
n＝0～1；R1选自C1～C3烷基；R2选自芳基或含氮原子取代的芳香杂环。
优选地，所述青蒿素衍生物为具有以下结构式中的任一种
本发明的目的是开发青蒿素衍生物在减肥药物中的作用，对其抑制细胞脂肪分化进行了详细研究。
本发明具有以下优点：
(1)青蒿素及其衍生物是传统的抗疟疾药物，一直以来用于治疗疟疾，本发明的特点是首次提出青蒿素的部分衍生物具有抑制脂肪细胞分化的作用，首次将它们与代谢类的疾病联系起来，并首次提出它们作为应用于肥胖及肥胖相关疾病如心血管疾病的药物的潜在可能性。与肥胖相关的代谢类疾病在当今社会是人类健康的重大威胁，由所述青蒿素类生物制备的药物作为抗脂肪分化的药物，将会在这些疾病的治疗中发挥重要作用。
(2)本发明比较了不同青蒿素衍生物之间抑制脂肪细胞分化能力的强弱，得出了初步的构效关系，为进一步优化获得活性更好的青蒿素衍生物提供了依据。
附图说明
图1.为青蒿素系列衍生物对脂肪细胞分化的抑制作用，横坐标为药物使用剂量(浓度，以μM表示)，纵坐标为相对分化率(加药处理实验组油红染色OD值/完全分化对照组油红染色OD值)；
图2.为选取的抑制3T3-L1细胞分化效果最具有代表性的5个化合物的油红染色总体效果；其中，图2.1为化合物2浓度梯度影响3T3-L1细胞分化的油红染色总体效果；
图2.2为化合物4浓度梯度影响3T3-L1细胞分化的油红染色总体效果；
图2.3为化合物3浓度梯度影响3T3-L1细胞分化的油红染色总体效果；
图2.4为化合物5浓度梯度影响3T3-L1细胞分化的油红染色总体效果；
图2.5为化合物9浓度梯度影响3T3-L1细胞分化的油红染色总体效果；
图3为诱导3T3-L1细胞分化的流程阶段示意图。
具体实施方式
本发明所述具有以下式Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ结构的青蒿素衍生物，主要为具有以下结构式的化合物：
(a)所述具有式Ⅰ结构的化合物为：
(b)所述具有式Ⅱ结构的化合物为：
(c)所述具有式Ⅲ结构的化合物为：
(d)所述具有式Ⅳ结构的化合物为：
(e)所述具有式Ⅴ结构的化合物为：
为使本发明更加容易理解，下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明目的，而不用于限制本发明范围。
本领域的技术人员，可以从以下描述或现有技术，得到所述具有所述式Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ结构的青蒿素衍生物。
实施例1：脱氧青蒿素的合成
200mg(0.7087mmol)青蒿素溶于10mL THF，原料溶解后，加入547mg(20.2688mmol)Al粉和7.23g(30.4032mmol)NiCl2·6H2O，4小时后反应完毕，旋干溶剂，用乙酸乙酯溶解旋干物，抽滤，用乙酸乙酯淋洗滤渣。滤液经无水Na2SO4干燥，浓缩后柱层析(PE∶EA＝16∶1)得到72mg目标产物，收率38％。
1H NMR(CDCl3，400MHz)：δ＝5.68(s，1H)，3.19-3.16(m，1H)，2.02-1.58(m，6H)，1.51(s，3H)，1.28-1.03(m，11H).
实施例2：脱氧二氢青蒿素的合成
500mg(1.7596mmol)二氢青蒿素溶于5mL DCM，加入45mg 10％Pd/C，氢气保护下，40℃过夜反应完毕，过滤除去Pd/C，旋干溶剂，浓缩后柱层析(PE∶EA＝8∶1)得到202mg目标产物，收率43％。
1H NMR(CDCl3，400MHz)：δ＝5.34(s，1H)，4.78(t，J＝6.8Hz，1H)，2.83(d，J＝7.2Hz，1H)，2.40-0.79(m，21H).
实施例3：二氢青蒿素的合成
10g(35.4321mmol)青蒿素溶于500mL甲醇，搅拌降温至0-5℃，1小时内分批加入10g(265.7408mmol)NaBH4。保持该温度继续搅拌1.5小时后，往反应瓶中缓慢滴加15.5mLHAc调节pH＝7，逐渐析出大量白色固体。旋干大部分溶剂，加入10mL冷水，室温下搅拌15分钟，抽滤，用冷H2O∶MeOH＝2∶1洗涤滤饼，收集滤饼，干燥后得到8.0g目标产物，收率80％。
1H NMR(CDCl3，400MHz)：δ＝5.60(s，1H)，5.28(s，1H)，2.85(s，1H)，2.61(s，1H)，2.41-0.89(m，20H).
实施例4：β-蒿甲醚的合成
150mg(0.5275mmol)二氢青蒿素溶于1.0mL MeOH和2mL DCM混合溶剂中，加热至45℃，氩气保护下，迅速注入0.01mL(0.0559mmol)BF3·Et2O，在此温度下反应1小时，反应结束后，用醋酸钠水溶液洗涤反应液，有机层经无水MgSO4干燥后，浓缩即得到β-蒿甲醚和α-蒿甲醚的混合物，将混合物溶解在适量正己烷中，在-20℃保存36小时，收集白色晶体，并用冷正己烷淋洗，干燥后得到107mg纯β-蒿甲醚，收率65％。
1H NMR(CDCl3，400MHz)：δ＝5.38(s，1H)，4.68(d，J＝3.2Hz，1H)，3.42(s，3H)，2.64-0.85(m，21H).
实施例5：青蒿素苯甲酸酯的合成
氩气保护下，3.386g(11.9158mmol)二氢青蒿素溶于40mL DCM，加入6.1mL(76.2613mmol)吡啶，搅拌降温至0℃，加入2.2mL(19.0653mmol)苯甲酰氯，保持该温度下反应15分钟，撤去冰浴升至室温，继续反应16小时完毕，加入7％柠檬酸水溶液终止反应，旋干DCM，加入乙酸乙酯溶解旋干物，依次用7％柠檬酸水溶液、饱和NaHCO3溶液、饱和NaCl溶液洗涤有机层，有机层经无水Na2SO4干燥，浓缩后柱层析(PE∶EA＝16∶1)得到4.07g目标产物，收率88％。
1H NMR(CDCl3，400MHz)：δ＝8.12(d，J＝7.2Hz，2H)，7.59-7.55(m，1H)，7.44(t，J＝8.0Hz，2H)，6.01(d，J＝9.6Hz，1H)，5.53(s，1H)，2.76-0.92(m，21H).
实施例6：β-烯丙基青蒿素的合成
反应瓶中加入少量4
MS、105mg(0.7790mmol)无水ZnCl2，Ar保护下，注入3mL无水DCM和0.5mL(3.1160mmol)烯丙基三甲基硅烷，搅拌降温至0℃。
称252mg(0.6492mmol)青蒿素苯甲酸酯溶解在3mL无水二氯乙烷中，将溶液滴入上述反应液中，保持0℃反应1小时，撤去冰浴，升至室温反应3小时，加入适量乙酸乙酯稀释反应体系，依次用7％柠檬酸水溶液、饱和NaHCO3溶液、饱和NaCl溶液洗涤有机层，有机层经无水Na2SO4干燥，浓缩后柱层析(PE∶EA＝30∶1)，得到114mg目标产物，收率66％。
1H NMR(CDCl3，400MHz)：δ＝5.92(m，1H)，5.32(s，1H)，5.14-5.04(m，2H)，4.30(m，1H)，2.69-0.87(m，23H).
实施例7：β-乙酸青蒿素的合成
100mg(0.3245mmol)β-烯丙基青蒿素溶于4mL CCl4-CH3CN-H2O(V∶V∶V＝1∶1∶2)混合溶剂中，依次加入347mg(1.6225mmol)NaIO4和2mg(0.0097mmol)RuCl3，室温过夜反应。旋干溶剂，加入乙酸乙酯溶解旋干物，依次用饱和NaHSO3、饱和NaHCO3溶液、饱和NaCl溶液洗涤有机层，有机层经无水Na2SO4干燥，浓缩后柱层析(DCM∶MeOH＝30∶1)，得到92mg目标产物，收率87％。
1H NMR(CDCl3，400MHz)：δ＝5.37(s，1H)，4.86(m，1H)，2.71-0.88(m，24H).
实施例8：D-Biotin修饰青蒿素的合成
Reagents and conditions：(a)Boc2O，Et3N，THF，R.T.，15h；(b)D-biotin，EDCI，HOBt，(i-Pr)2NEt，DMF，R.T.，10h；(c)TFA/CH2Cl2，0℃～R.T.，9h；(d)EDCI，HOBt，(i-Pr)2NEt，DMF，R.T.，11h.
2的合成：
2.035g(13.7360mmol)溶于18mL THF中，加人0.63mL(4.5767mmol)NEt3，搅拌降温至0℃。
1g(4.5767mmol)Boc2O溶于9mL THF中，混合均匀后，逐滴滴入上述反应液中，滴加完毕后，自然升至室温反应15小时。反应完毕后，旋干溶剂，加水稀释后，用DCM萃取，有机层用饱和NaCl溶液洗涤，无水Na2SO4干燥，浓缩后柱层析(DCM∶MeOH＝5∶1)，得到906mg目标产物，收率80％。
1H NMR(CDCl3，400MHz)：δ＝5.18(s，1H)，3.56(s，4H)，3.50-3.45(m，4H)，3.30-3.25(m，2H)，2.82(t，J＝5.6Hz，2H)，1.53(s，2H)，1.38(s，9H).
3的合成：
氩气保护下，318mg(1.3mmol)D-Biotin、249mg (1.3mmol，1.3equiv.)EDCI、176mg(1.3mmol，1.3equiv.)HOBt溶于4mL干燥DMF，加入0.23mL(1.4mmol，1.4equiv.)DIPEA，室温搅拌。
248mg(1.0mmol)2溶于3mL干燥DMF中，将溶液注入上述反应瓶中，室温反应10小时。反应完毕后，往反应瓶中加入NH4Cl饱和溶液，氯仿萃取，有机层经NH4Cl饱和溶液、水、饱和NaCl溶液洗涤，无水Na2SO4干燥，浓缩后柱层析(DCM∶MeOH＝20∶1)，得到358mg目标产物，收率76％。
1H NMR(CDCl3，400MHz)：δ＝6.67(s，1H)，6.15(s，1H)，5.52(s，1H)，5.15(s，1H)，4.45-4.40(m，1H)，4.25-4.20(m，1H)，3.52(s，4H)，3.49-3.45(m，4H)，3.37-3.34(m，2H)，3.23-3.21(m，2H)，3.10-3.04(m，1H)，2.14(t，J＝7.6Hz，2H)，1.70-1.50(4H)，1.36(s，12H).
4的合成：
将118mg 3溶于3mL二氯甲烷中，搅拌降温至0℃，缓慢滴加1.0mL TFA，加完后撤去冰浴，自然升至室温反应9小时，反应完毕后，直接减压蒸馏除去溶剂，真空干燥得到粗产物(按100％计算，应得到93mg粗产物)4，未经分离直接用于下一步反应。
1H NMR(MeOD，400MHz)：δ＝4.52-4.49(m，1H)，4.32-4.29(m，1H)，3.72-3.65(m，6H)，3.56(t，J＝5.6Hz，2H)，3.39-3.35(m，2H)，3.22-3.20(m，1H)，3.14-3.11(m，2H)，2.95-2.91(dd，J＝12.8Hz，4.8Hz，1H)，2.71(d，J＝12.8Hz，1H)，2.22(t，J＝7.6Hz，2H)，1.71-1.60(m，4H)，1.47-1.43(m，2H).
D-Biotin修饰青蒿素的合成：
氩气保护下，74mg(0.2269mmol)羧酸青蒿素、57mg(0.2949mmol)EDCI、40mg(0.2949mmol)HOBt溶于3mL无水DMF，注入0.24mL(1.4293mmol)DIPEA，室温搅拌。
称93mg(0.2496mmol)4溶于2mL干燥DMF，将溶液注入上述反应液中，室温过夜反应。反应完毕后，加入DCM稀释反应液，依次用NH4Cl饱和溶液、水、饱和NaCl溶液洗涤，无水Na2SO4干燥，浓缩后柱层析(DCM∶MeOH＝10∶1)，得到89mg目标产物，收率57％。
1H NMR(CDCl3，400MHz)：δ＝7.24(s，1H)，δ6.85(s，1H)，δ6.30(s，1H)，δ5.41(s，1H)，δ5.35(s，1H)，δ4.75-4.71(m，1H)，δ4.52-4.50(m，1H)，δ4.32-4.29(m，1H)，δ3.61-3.40(m，12H)，δ3.15-3.16(m，1H)，δ2.90-0.88(m，33H).
实施例9：
称50mg(0.1288mmol)青蒿素苯甲酸酯和适量4
MS于反应瓶中，氩气保护下，注入2mL重蒸DCM溶解，降温至0℃，注入0.02mL(0.1546mmol)TMSCl，保持该温度反应1小时后升至室温过夜反应完毕。旋干溶剂，加入乙酸乙酯溶解旋干物，依次用饱和NaHCO3溶液、饱和NaCl溶液洗涤，有机层经无水Na2SO4干燥，浓缩后柱层析(PE∶EA＝8∶1)，得到33mg目标产物，收率96％。
1H NMR(CDCl3，400MHz)：δ＝6.19(s，1H)，5.54(s，1H)，2.44-0.99(m，20H).
实施例10：
氩气保护下，50mg(0.1865mmol)脱氧二氢青蒿素溶于3mL DCM中，降温至0℃，注入0.03mL(0.3729mmol)乙酰氯，逐渐升至室温过夜反应完毕。反应液经水洗涤，有机层经无水Na2SO4干燥，浓缩柱层析(PE∶EA＝16∶1)，得到作为副产物的消旋脱氧二氢青蒿素30mg，收率64％。
1H NMR(CDCl3，400MHz)：δ＝6.04(s，1H)，5.47(s，1H)，2.04-1.21(m，20H).
实施例11：
100mg(0.3517mmol)二氢青蒿素溶于5mL DCE，加人73mg(0.4220mmol)2-喹喔啉羧酸搅拌片刻后，加入5mL(0.0422mmol)DMAP，搅拌降温至0℃，加入80mg(0.3868mmol)DCC，搅拌5分钟后升至室温，16小时后停止反应，过滤反应液，滤液经水、饱和NaCl溶液洗涤，有机层经无水Na2SO4干燥后，浓缩柱层析，得到120mg目标产物，收率78％。
1H NMR(CDCl3，400MHz)：δ＝9.57(s，1H)，8.31(d，J＝8.0Hz，1H)，8.17(d，J＝8.0Hz，1H)，7.91-7.86(m，2H)，6.03-6.00(m，2H)，5.64(s，1H)，2.90-0.95(m，21H).
实施例12：
按照如实例12所述，100mg(0.3517mmol)二氢青蒿素与63mg(0.4220mmol)对醛基苯甲酸反应，柱层析得到116mg目标产物，收率79％。
1H NMR(CDCl3，400MHz)：δ＝10.10(s，1H)，8.27(d，J＝8.0Hz，2H)，7.96(d，J＝8.0Hz，2H)，6.03-6.00(m，1H)，5.54(s，1H)，2.90-0.95(m，21H).
实施例13：
按照如实例12所述，100mg(0.3517mmol)二氢青蒿素与133mg(0.4220mmol)Boc-3-(2-萘基)-L-丙氨酸反应，柱层析得到151mg目标产物，收率76％。
1H NMR(CDCl3，400MHz)：δ＝7.86-7.83(m，1H)，7.80-7.74(m，3H)，7.47-7.38(m，1H)，6.05-6.00(m，1H)，5.54(s，1H)，2.83-0.85(m，34H).
实施例14：
按照如实例12所述，100mg(0.3517mmol)二氢青蒿素与76mg(0.4220mmol)对硝基苯乙酸反应，柱层析得到123mg目标产物，收率78％。
1H NMR(CDCl3，400MHz)：δ＝8.18(d，J＝8.0Hz，2H)，7.48(d，J＝12.0Hz，2H)，6.03-6.00(m，1H)，5.44(s，1H)，3.82(s，2H)，3.82(s，2H)，2.65-0.68(m，23H).
实施例15：
氩气保护下，100mg(0.3517mmol)二氢青蒿素与0.08mL(0.5627mmol)对甲氧基苯甲酰氯溶于5mL DCM中，降温至0℃，注入0.2mL(2.2509mmol)吡啶，搅拌15分钟后，升至室温过夜反应完毕，用7％柠檬酸水溶液淬灭反应，乙酸乙酯萃取，再经饱和NaHCO3、饱和NaCl溶液洗涤，有机层经无水Na2SO4干燥，浓缩后柱层析，得到119mg目标产物，收率81％。
1H NMR(CDCl3，400MHz)：δ＝8.06(d，J＝12Hz，2H)，6.91(d，J＝12Hz，2H)，5.98(m，1H)，5.51(s，1H)，3.864(s，3H)，2.81-0.85(m，21H).
实施例16：
按照实例15所述，100mg(0.3517mmol)二氢青蒿素与104mg(0.5627mmol)对硝基苯甲酰氯反应，柱层析得到81mg目标产物，收率53％。
1H NMR(CDCl3，400MHz)：δ＝8.28(s，4H)，6.05-6.00(m，1H)，5.55(s，1H)，2.83-0.85(m，22H).
实施例17：青蒿琥酯的合成
室温条件下，250mg(0.9mmol)二氢青蒿素溶于25mL干燥DCM中，加入202mg(2mmol)丁二酸酐，降温至0～5℃，加入107mg(0.9mmol)DMAP，保持该温度反应0.5小时，自然升至室温反应1小时。反应结束后，加入5mL水，用10％稀盐酸调至pH＝3，有机层经水洗后，用无水MgSO4干燥，减压除去溶剂，得到317mg目标产物，收率94％。
1H NMR(CDCl3，400MHz)：δ＝5.74(d，J＝8.0Hz，1H)，5.39(s，1H)，2.72-2.59(m，4H)，2.53-2.48(m，1H)，2.36-2.28.(m，1H)，2.01-1.96(m，1H)，1.87-1.82(m，1H)，1.76-1.66(m，2H)，1.60-1.55(m，1H)，1.45-1.21(m，8H)，1.01-0.95(m，1H)，0.92(d，J＝8.0Hz，3H)，0.80(d，J＝4.0Hz，3H).
实施例18：青蒿素衍生物对3T3-L1细胞分化的影响
3T3-L1细胞的培养
3T3-L1细胞在37℃，5％CO2培养环境中贴壁生长，使用加入10％FBS(Hyclone胎牛血清)的DMEM高糖培养基(HyClone)。可根据细胞生长状况，2～3天换一次液。如果需要持续多次传代，并需要在多次传代之后细胞仍然具有良好的分化能力，那么就要避免让细胞长的太满(＜70％)，一般长到60％～70％就要传代分细胞了。细胞可以以稀到1∶15的比例传代分盘，但是通常以1∶10或者更高的比例传代分盘，以实验需要为准。
3T3-L1细胞诱导分化的培养基的配制
诱导培养基分为两种，MDI诱导培养基和胰岛素培养基，分别用于分化不同阶段，是由DMEM培养基加上适当量的FBS、地塞米松、胰岛素和IBMX配成。
IBMX溶液：将IBMX溶于0.5N的KOH溶液中，IBMX终浓度为0.0115g/ml，0.22μm针头过滤器过滤除菌，-20℃保存。
胰岛素储存溶液：将胰岛素溶于0.02M的HCl中至终浓度167uM(1mg/ml)，0.22μm针头过滤器过滤除菌，-20℃长期保存，4℃短期保存。
地塞米松储存溶液：冷冻储存液：10mM地塞米松溶于无水乙醇，-20℃保存；工作储存液：将冷冻储存液用PBS稀释至1mM，过滤除菌，储存于4℃。
MDI诱导培养基(现配现用，10ml/10cm培养皿，5ml/6cm培养皿)：含有10％FBS的DMEM高糖培养基再加入：1∶100的IBMX溶液，1∶1000胰岛素储存液，1∶1000地塞米松工作储存液。
胰岛素培养基(现配现用，10ml/10cm培养皿，5ml/6cm培养皿)：含有10％FBS的DMEM高糖培养基再加入：1∶1000胰岛素储存液。
诱导未分化的3T3-L1细胞(成纤维细胞状态)向成熟脂肪细胞分化：
如图3所示：
1.在含有10％FBS的DMEM高糖培养基中将3T3-L1前脂肪细胞培养至接合状态。
2.接合之后再养2天(此时为分化第0天)以MDI诱导培养基刺激细胞(换去原有的完全培养基，加入新配制的MDI诱导培养基)。在未来2天中会注意到细胞形态发生显著的变化(变得更加纺锤形)。
3.加入MDI诱导2天之后(分化第2天)，换去原有的MDI诱导培养基，加入新配制的胰岛素培养基。培养基会开始变得更加粘稠，因为细胞合成的游离脂肪酸被大量的分泌到培养基中。
4.两天之后(分化第4天)，将培养基换成普通的完全培养基(含有10％FBS的DMEM高糖培养基)，之后每2天换一次液(含有10％FBS的DMEM高糖培养基)。通常在分化第8天的时候达到分化完全的状态。
药物编号1至9分别代表的化合物结构式依次为：
油红O染色数据的分析和处理
油红O染色所用试剂的配制：
油红O储存液：油红O(AMRESCO，FW 408.51，Em(513nm))0.7g溶于200ml异丙醇，过夜颠倒振荡混匀，0.2μm滤膜过滤后储存在4℃。
油红O工作液：6份油红O储存液加上4份dH2O，混匀后室温静置20分钟，0.2μm滤膜过滤。
油红O染色：
1.吸去绝大部分培养基。
2.加入10％福尔马林(溶于PBS中)，室温孵育5分钟。
3.吸去10％福尔马林，加入同样体积的新鲜福尔马林(未稀释的37％甲醛溶液)，孵育至少1小时，甚至更久。注：细胞在福尔马林中可以保持好几天，然后再染色。将培养板四周用封口膜密封以防止甲醛挥发蒸干，并以铝箔纸包裹以避光。
4.用小枪头吸去所有福尔马林。
5.以60％异丙醇(溶于dH2O中)清洗培养孔。
6.让细胞培养孔彻底干燥。
7.加入油红O工作液，染色10分钟(加入时不要碰到孔壁)。
8.移去所有油红染液，立即加入dH2O，用水洗4遍(可直接在自来水龙头下冲洗)。
9.根据实验需要拍照。
10.移去所有的水，让培养孔完全干燥。
11.加入100％异丙醇洗脱油红O，孵育10分钟(可以更久)。
12.反复吹吸含有油红的异丙醇几次，保证所以油红都溶进溶液中。
13.将含有油红的异丙醇转移至EP管或者ELISA板的孔中。
14.在500nm波长处测量OD值，读数时间为0.5秒。
15.空白对照使用100％异丙醇，染色本底对照使用来自按照上述步骤染色的没养细胞的空培养孔的异丙醇。
表1.油红O染色中各种试剂的用量
| 细胞培养板 | 福尔马林 | 60％异丙醇 | 油红O | 100％异丙醇 |
| 24孔板 | 500μl | 500μl | 200μl | 750μl |
| 12孔板 | 1ml | 1ml | 400μl | 1.5ml |
| 6孔板 | 2.4ml | 2.4ml | 1ml | 3.6ml |
青蒿素衍生物对3T3-L1细胞的分化的影响
将固体青蒿素(图1.1、3-7号化合物)溶解于DMSO中，配制成一系列梯度浓度的溶液，本实验中该浓度梯度包括5个水平：1mM、5mM、10mM、30mM、50mM。二氢青蒿素(化合物2)浓度梯度为：0.1mM、0.5mM、1mM、5mM、10mM、30mM、50mM；青蒿琥酯(化合物9)的浓度梯度为：0.05mM、0.1mM、0.5mM、1mM、5mM、10mM、30mM、50mM。
如图3所示，依照上述所描述的诱导未分化的3T3-L1细胞(成纤维细胞状态)向成熟脂肪细胞分化的操作流程，在分化第0天的时候，向已经达到接合状态但尚未分化的3T3-L1细胞加入诱导分化的MDI培养基。加入青蒿素药物的处理操作为，在MDI培养基中按照1/1000的比例预先加入如上所述配置成的青蒿素系列梯度浓度的溶液，混匀之后再将培养基加入细胞培养孔。1/1000的比例，使得化合物1、3-7药物的终浓度变为1μM、5μM、10μM、30μM、50μM，也就是本实验实际使用的药物剂量。同时需要设计一个处理，在培养基中加入1/1000的DMSO作为溶剂对照，也就是药物浓度为0μM的处理。
依照上述所描述的细胞培养条件培养经过处理的3T3-L1细胞2天，依照上述所描述的诱导未分化的3T3-L1细胞(成纤维细胞状态)向成熟脂肪细胞分化的操作流程，在分化第2天加入胰岛素培养基，并用如上所述同样的方法和剂量，加入系列梯度浓度的青蒿素(同样的细胞培养孔加入同一个浓度的同一种药物)。
依照上述描述，对完成分化的3T3-L1细胞进行油红O染色，拍照，测吸光度，收集数据，整理，做图，比较，结果见图1。
图1中这9个化合物每个浓度的数据均来源于3次不同批次的重复实验得到的平均值，误差由3次重复实验之间的SE值表示。
药物加入之后，会抑制或是促进或是不显著影响3T3-L1细胞脂肪分化(对于青蒿素系列药物，本发明关注的是抑制)。本发明以细胞脂肪分化的程度来表示药效。细胞分化的程度可以在实验完成后用油红染色法染色后，测OD值得以定量检测。分化程度越高，油滴积累越多，油红染色越深，OD值越高。
本发明的分化实验使用24孔细胞培养板来做，每个24孔板会设计不加药的几个孔做对照，也就是可以完全分化的细胞的对照。
每个药物在特定浓度的药效，是通过相应的OD值比上同一个24孔细胞培养板上的完全分化的对照的OD值得到(相对分化程度)。这样，不同培养板得出的数据就可以相互比较。
如图1所示，如果是完全分化，得到的值(相对分化程度)就是1(100％)；分化受到抑制，值就小于1(0～1之间)。分化抑制程度越高，值越小，药效越好。
结果如图1所示，青蒿素本身(图1.1号化合物)抑制3T3-L1细胞分化的效果很差，浓度在50μM的水平，3T3-L1细胞相对分化程度能达到93.3％；在10μM的水平，相对分化程度能达到94.8％。
结果见图1，图2.1，二氢青蒿素(图1.2号化合物)抑制3T3-L1细胞分化的效果为所有已经被测试过的青蒿素衍生物中最好的之一，在10μM的水平已经能完全抑制3T3-L1细胞的分化，相对分化程度只有24.5％。
结果见图1，图2.3，该化合物(图1.3号化合物)抑制3T3-L1细胞分化的效果一般，在10μM的水平，3T3-L1细胞的相对分化程度有62.5％。
结果见图1，图2.2，青蒿素苯甲酸酯(图1.4号化合物)抑制3T3-L1细胞分化的效果为所有已经被测试过的青蒿素衍生物中最好的之一，在10μM的水平已经能完全抑制3T3-L1细胞的分化，相对分化程度只有27.3％。
结果见图1，图2.4，烯丙基青蒿素(图1.5号化合物)抑制3T3-L1细胞分化的效果一般，在10μM的水平，3T3-L1细胞的相对分化程度有58.1％。
结果见图1，乙酸基青蒿素(图1.6号化合物)抑制3T3-L1细胞分化的效果较差，在10μM的水平，3T3-L1细胞的相对分化程度有81.3％。
结果见图1，青蒿素-生物素(图1.7号化合物)抑制3T3-L1细胞分化的效果很差，在10μM的水平，3T3-L1细胞的相对分化程度有91.6％。
结果见图1，去氧二氢青蒿素(图1.8号化合物)几乎没有抑制3T3-L1细胞分化的效果，在10μM的水平，3T3-L1细胞的相对分化程度能达到98.8％。
结果见图1，图2.5，青蒿琥酯(图1.9号化合物)抑制3T3-L1细胞分化的效果为所有已经被测试过的青蒿素衍生物中最好的之一，在10μM的水平已经能完全抑制3T3-L1细胞的分化，相对分化程度只有31.5％。
1.具有以下式Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ结构的青蒿素衍生物在制备防治肥胖的药物中的应用，
其中：
n＝0～1；R1选自C1～C3烷基；R2选自芳基或含氮原子取代的芳香杂环。
2.根据权利要求1所述的应用，其特征是：所述青蒿素衍生物为具有以下结构式中的任一种：
3.根据权利要求1所述的应用，其特征是：所述青蒿素衍生物为具有以下结构式中的任一种：
Description
The new application of artemisinin derivative
Wherein, dihydroartemisinine, arteannuin benzoate have the effect of inhibition 3T3-L1 cell differentiation the strongest in all artemisinin derivatives that detected.Next is an artesunate, has the effect of the inhibition 3T3-L1 cell differentiation that is only second to the above two.These 3 chemical compounds can both suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M.
Technical field
The present invention relates to the new application of artemisinin derivative in pharmacy, belong to chemical biology and cytobiology field.
Background technology
Arteannuin is the sesquiterpene lactones medicine that contains peroxy-radical that extracts from Herba Artemisiae annuae.The discovery of artemisinin-based antimalarial drug thing is the another milestone after quinine on the global antimalarial agent development history, it is first phytochemistry medicine that China finds, also be China unique that approved by World Health Organization (WHO), can be by the Chinese medicine of synthetic drug research standard exploitation.Over more than 30 year, arteannuin distinctive molecular structure and outstanding biological activity are attracting the eyeball of scientists deeply.Along with the development of medical sci-tech, prove that through pharmacological action and clinical practice artemisinin derivative is widely used in treating malaria, cancer is in the diseases such as actute infection and hyperpyrexia disease and dermatosis.Wherein the more artemisinin derivative of clinical research has dihydroartemisinine, Artemether, arteether and artesunate etc.Have not yet to see the bibliographical information that relevant artemisinin derivative suppresses the adipose cell differentiation.
Obesity is meant that body feed heat is more than the health consumption, cause the transition accumulation of fat and the excessive amplification of fatty tissue, make body weight more than 20% of body weight that is above standard, and the class disease weak with giddy, that Mental fatigue is breathed hard, be the risk factor of hypertension, diabetes, hyperlipidemia, coronary heart disease, cerebrovascular.Now, fat one of fast, the most serious public health problem of global rate of propagation that become.In recent years, along with the raising of China's living standards of the people, fat number has increased in urban population, and do not occur over just the old people, also occur in child, women, the number of each age group body obesity is all increasing, and fat-reducing has become people's much-talked-about topic.The cause of disease that causes obesity has a lot, and wherein main and h and E factor has substantial connection.Environmental factors is meant eats high fat, high heat food for a long time, adds physical exertion and reduces, and under the effect of factors such as mental maladjustment, causes that the neuroendocrine of body fat regulated and control network is regulated disorderly.
Therefore, the generation of prevention and controlling obesity disease has become the problem of medically very paying close attention to.At present, slimming medicine commonly used roughly is divided three classes: one, the medicine of control appetite, and this type of medicine has many untoward reaction, as insomnia, xerostomia, constipation, cardiopalmus and hypertension; Two, increase the medicine of energy expenditure; Three, the medicine that suppresses intestinal absorption.Although there are many researchs to be deep into the genomic medicine level now, but still do not find at present a kind of real effectively, slimming medicine that can life-time service, work out diagnosis and the treatment that even more ideal effective method and preparation be used for obesity and still have profound significance.
3T3-L1 be one derived from 3T3 cell (the fibroblastic cell line of standard that builds on 1962), in biological study, be applied to studying the cell line of fatty tissue.The 3T3-L1 cell has similar fibroblastic form, but under the inducing of felicity condition, can break up and form the phenotype of adipose cell.Adipose cell form after the 3T3-L1 cell differentiation maturation, the synthetic and accumulation of the intracellular glycerol three esters fibroblast state preceding with respect to differentiation all improves greatly, and 3T3-L1 cell at this time presents a kind of adipose cell form of signet ring.These cells are all responsive to hormone and the medicine of regulating lipogenesis and fat acid decomposition, epinephrine for example, norepinephrine, insulin etc.
Because these characteristics of 3T3-L1 cell, this cell line are widely used in the research of fat differentiation and metabolic disease mechanism, be the cell line model of internationally recognized external fat differentiation.The cascade signal of transcribing that this Study of model has been helped to illustrate a main regulation and control fat differentiation in the terminal differentiation of adipose cell is transduceed, and has comprised a series of transcription factor, modulin and effect protein.
The oil red O stain technology is a kind of lipid staining commonly used, and its advantage is that staining procedure is easy, and coloration result is sure.Dyeing back fat is cerise, and it is blue that nucleus is, and a matter is colourless.The redness that oil red O dyes is directly proportional with the amount of the oil droplet that the interior accumulation of adipose cell forms, and the latter is directly proportional with the height of adipose cell differentiation degree, so by this technology, both can judge by qualitative observation, can also detection by quantitative the height of adipose cell differentiation degree relatively.
Herba Artemisiae annuae extensively is present in all over the world, but the higher Herba Artemisiae annuae of artemislnin content exists only in east, China Chongqing, Fujian, Guangxi, some areas, Hainan, belongs to the exclusive drug resource of China.Nearly decades, the research of artemisinin derivative mainly concentrates on malaria and anti-tumor aspect, and the research that applies it to appetrol is but very rare.
Summary of the invention
The objective of the invention is promptly provides a kind of new application of artemisin derivant in order to improve the application of artemisinin derivative in frontier.
Concrete technical scheme is as follows:
The application of artemisinin derivative in the fat medicine of preparation control with following formula I, II, III, IV, V structure,
Wherein:
N=0～1; R 1Be selected from C 1～C 3Alkyl; R 2Be selected from the aromatic heterocycle that aryl or nitrogen atom replace.
Preferably, described artemisinin derivative is to have in the following structural formula any
The objective of the invention is to develop the effect of artemisinin derivative in slimming medicine, it is suppressed the cellular fat differentiation study in great detail.
The present invention has the following advantages:
(1) arteannuin and derivant thereof are traditional anti-malaria medicaments, be used for the treatment of malaria all the time, characteristics of the present invention are that the part derivant that proposes arteannuin first has the effect that suppresses the adipose cell differentiation, disease with them and metabolism class connects first, and proposes their first as the potential probability that is applied to the medicine of obesity and obesity-related disease such as cardiovascular disease.The metabolism class disease relevant with obesity is the significant threat of human health in current society, by the medicine of the biological medicine for preparing of described artemisine as the differentiation of lipotropism fat, will play a significant role in these treatment of diseases.
(2) the present invention has compared the power that suppresses the adipose cell differentiation capability between the different artemisinin derivatives, has drawn preliminary structure activity relationship, provides foundation for further optimizing the active better artemisinin derivative of acquisition.
Description of drawings
Fig. 1. be the inhibitory action of arteannuin series derivates to the adipose cell differentiation, abscissa is a drug use dosage (concentration, represent with μ M), vertical coordinate is relative differentiation rate (dosing handle experimental group oil red dyeing OD value/break up fully matched group oil red dye OD value);
Fig. 2. be the oil red dyeing general effect of 5 the most representative chemical compounds of the inhibition 3T3-L1 cell differentiation effect of choosing; Wherein, Fig. 2 .1 is the oil red dyeing general effect that chemical compound 2 Concentraton gradient influence the 3T3-L1 cell differentiation;
Fig. 2 .2 is the oil red dyeing general effect that chemical compound 4 Concentraton gradient influence the 3T3-L1 cell differentiation;
Fig. 2 .3 is the oil red dyeing general effect that chemical compound 3 Concentraton gradient influence the 3T3-L1 cell differentiation;
Fig. 2 .4 is the oil red dyeing general effect that chemical compound 5 Concentraton gradient influence the 3T3-L1 cell differentiation;
Fig. 2 .5 is the oil red dyeing general effect that chemical compound 9 Concentraton gradient influence the 3T3-L1 cell differentiation;
Fig. 3 is the flow process stage sketch map of inducing the 3T3-L1 cell differentiation.
The specific embodiment
Artemisinin derivative with following formula I, II, III, IV, V structure of the present invention is mainly the chemical compound with following structural formula:
(a) described chemical compound with formula I structure is:
(b) described chemical compound with formula II structure is:
(c) described chemical compound with formula III structure is:
(d) described chemical compound with formula IV structure is:
(e) described chemical compound with formula V structure is:
For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used for illustration purpose, and be not used in the restriction scope of the invention.
Those skilled in the art can obtain described artemisinin derivative with described formula I, II, III, IV, V structure from following description or prior art.
Embodiment 1: deoxidation arteannuin synthetic
200mg (0.7087mmol) arteannuin is dissolved in 10mL THF, behind the material dissolution, adds 547mg (20.2688mmol) Al powder and 7.23g (30.4032mmol) NiCl 26H 2O, afterreaction finished in 4 hours, was spin-dried for solvent, was spin-dried for thing with acetic acid ethyl dissolution, and sucking filtration is with ethyl acetate drip washing filtering residue.Filtrate is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=16: 1) obtain the 72mg target product, yield 38%.
1H?NMR(CDCl 3，400MHz)：δ＝5.68(s，1H)，3.19-3.16(m，1H)，2.02-1.58(m，6H)，1.51(s，3H)，1.28-1.03(m，11H).
Embodiment 2: deoxidation dihydroartemisinine synthetic
500mg (1.7596mmol) dihydroartemisinine is dissolved in 5mL DCM, adds 45mg 10%Pd/C, under the hydrogen shield; 40 ℃ of reaction overnight finish, and remove by filter Pd/C, are spin-dried for solvent; concentrate back column chromatography (PE: EA=8: 1) obtain the 202mg target product, yield 43%.
1H?NMR(CDCl 3，400MHz)：δ＝5.34(s，1H)，4.78(t，J＝6.8Hz，1H)，2.83(d，J＝7.2Hz，1H)，2.40-0.79(m，21H).
Embodiment 3: dihydroartemisinine synthetic
10g (35.4321mmol) arteannuin is dissolved in 500mL methanol, stirs and is cooled to 0-5 ℃, adds 10g (265.7408mmol) NaBH in 1 hour in batches 4Keep this temperature to continue to stir after 1.5 hours, in reaction bulb, slowly drip 15.5mLHAc adjusting pH=7, separate out a large amount of white solids gradually.Be spin-dried for most of solvent, add 10mL cold water, stirred 15 minutes under the room temperature, sucking filtration is used cold H 2O: MeOH=2: 1 washing leaching cake, collect filter cake, obtain the 8.0g target product after the drying, yield 80%.
1H?NMR(CDCl 3，400MHz)：δ＝5.60(s，1H)，5.28(s，1H)，2.85(s，1H)，2.61(s，1H)，2.41-0.89(m，20H).
Embodiment 4: β-Artemether synthetic
150mg (0.5275mmol) dihydroartemisinine is dissolved in 1.0mL MeOH and the 2mL DCM mixed solvent, is heated to 45 ℃, under the argon shield, injects 0.01mL (0.0559mmol) BF rapidly 3Et 2O, reaction is 1 hour under this temperature, and after reaction finished, with sodium acetate aqueous solution washing reaction liquid, organic layer was through anhydrous MgSO 4After the drying, concentrate the mixture that promptly obtains β-Artemether and α-Artemether, mixture is dissolved in an amount of normal hexane, preserved 36 hours, collect white crystal, and, obtain the pure β-Artemether of 107mg after the drying, yield 65% with cold normal hexane drip washing at-20 ℃.
1H?NMR(CDCl 3，400MHz)：δ＝5.38(s，1H)，4.68(d，J＝3.2Hz，1H)，3.42(s，3H)，2.64-0.85(m，21H).
Embodiment 5: arteannuin benzoate synthetic
Under the argon shield; 3.386g (11.9158mmol) dihydroartemisinine is dissolved in 40mL DCM, adds 6.1mL (76.2613mmol) pyridine, stirs and is cooled to 0 ℃; add 2.2mL (19.0653mmol) Benzenecarbonyl chloride.; keep reacting 15 minutes under this temperature, remove ice bath and rise to room temperature, continue reaction and finished in 16 hours; add 7% aqueous citric acid solution cessation reaction; be spin-dried for DCM, add acetic acid ethyl dissolution and be spin-dried for thing, use 7% aqueous citric acid solution, saturated NaHCO successively 3Solution, saturated NaCl solution washing organic layer, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=16: 1) obtain the 4.07g target product, yield 88%.
1H?NMR(CDCl 3，400MHz)：δ＝8.12(d，J＝7.2Hz，2H)，7.59-7.55(m，1H)，7.44(t，J＝8.0Hz，2H)，6.01(d，J＝9.6Hz，1H)，5.53(s，1H)，2.76-0.92(m，21H).
Embodiment 6: β-pi-allyl arteannuin synthetic
Add a small amount of 4 in the reaction bulb
The anhydrous ZnCl of MS, 105mg (0.7790mmol) 2, the Ar protection is injected anhydrous DCM of 3mL and 0.5mL (3.1160mmol) allyl trimethyl silane down, stirs and is cooled to 0 ℃.
Claim 252mg (0.6492mmol) arteannuin benzoate to be dissolved in the anhydrous dichloroethanes of 3mL, solution is splashed in the above-mentioned reactant liquor, keep 0 ℃ of reaction 1 hour, remove ice bath, rose to room temperature reaction 3 hours, add an amount of ethyl acetate diluting reaction system, use 7% aqueous citric acid solution, saturated NaHCO successively 3Solution, saturated NaCl solution washing organic layer, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=30: 1), obtain the 114mg target product, yield 66%.
1H?NMR(CDCl 3，400MHz)：δ＝5.92(m，1H)，5.32(s，1H)，5.14-5.04(m，2H)，4.30(m，1H)，2.69-0.87(m，23H).
Embodiment 7: β-acetic acid arteannuin synthetic
100mg (0.3245mmol) β-pi-allyl arteannuin is dissolved in 4mL CCl 4-CH 3CN-H 2In O (V: V: V=1: 1: the 2) mixed solvent, add 347mg (1.6225mmol) NaIO successively 4And 2mg (0.0097mmol) RuCl 3, the ambient temperature overnight reaction.Be spin-dried for solvent, add acetic acid ethyl dissolution and be spin-dried for thing, use saturated NaHSO successively 3, saturated NaHCO 3Solution, saturated NaCl solution washing organic layer, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=30: 1), obtain the 92mg target product, yield 87%.
1H?NMR(CDCl 3，400MHz)：δ＝5.37(s，1H)，4.86(m，1H)，2.71-0.88(m，24H).
Embodiment 8:D-Biotin modifies the synthetic of arteannuin
Reagents?and?conditions：(a)Boc 2O，Et3N，THF，R.T.，15h；(b)D-biotin，EDCI，HOBt，(i-Pr) 2NEt，DMF，R.T.，10h；(c)TFA/CH 2Cl 2，0℃～R.T.，9h；(d)EDCI，HOBt，(i-Pr) 2NEt，DMF，R.T.，11h.
2 synthesize:
2.035g (13.7360mmol) be dissolved among the 18mL THF, add people 0.63mL (4.5767mmol) NEt 3, stir and be cooled to 0 ℃.
1g (4.5767mmol) Boc 2O is dissolved among the 9mL THF, behind the mix homogeneously, dropwise splashes in the above-mentioned reactant liquor, after dropwising, rises to room temperature reaction naturally 15 hours.After reaction finishes, be spin-dried for solvent, behind the thin up, with DCM extraction, organic layer is with saturated NaCl solution washing, anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=5: 1), obtain the 906mg target product, yield 80%.
1H?NMR(CDCl 3，400MHz)：δ＝5.18(s，1H)，3.56(s，4H)，3.50-3.45(m，4H)，3.30-3.25(m，2H)，2.82(t，J＝5.6Hz，2H)，1.53(s，2H)，1.38(s，9H).
3 synthesize:
Under the argon shield, (1.3mmol, 1.3equiv.) (1.3mmol, 1.3equiv.) HOBt is dissolved in the dry DMF of 4mL, adds 0.23mL (1.4mmol, 1.4equiv.) DIPEA, stirring at room for EDCI, 176mg for 318mg (1.3mmol) D-Biotin, 249mg.
248mg (1.0mmol) 2 is dissolved among the dry DMF of 3mL, solution is injected above-mentioned reaction bulb, room temperature reaction 10 hours.After reaction finishes, in reaction bulb, add NH 4The Cl saturated solution, chloroform extraction, organic layer is through NH 4Cl saturated solution, water, saturated NaCl solution washing, anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=20: 1), obtain the 358mg target product, yield 76%.
1H?NMR(CDCl 3，400MHz)：δ＝6.67(s，1H)，6.15(s，1H)，5.52(s，1H)，5.15(s，1H)，4.45-4.40(m，1H)，4.25-4.20(m，1H)，3.52(s，4H)，3.49-3.45(m，4H)，3.37-3.34(m，2H)，3.23-3.21(m，2H)，3.10-3.04(m，1H)，2.14(t，J＝7.6Hz，2H)，1.70-1.50(4H)，1.36(s，12H).
4 synthesize:
118mg 3 is dissolved in the 3mL dichloromethane, stirring is cooled to 0 ℃, slowly drip 1.0mL TFA, add the recession deicing and bathe, rose to room temperature reaction naturally 9 hours, after reaction finishes, directly distilling under reduced pressure removes and desolvates, vacuum drying obtains crude product (by 100% calculating, should obtain the 93mg crude product) 4, is directly used in next step reaction without separation.
1H?NMR(MeOD，400MHz)：δ＝4.52-4.49(m，1H)，4.32-4.29(m，1H)，3.72-3.65(m，6H)，3.56(t，J＝5.6Hz，2H)，3.39-3.35(m，2H)，3.22-3.20(m，1H)，3.14-3.11(m，2H)，2.95-2.91(dd，J＝12.8Hz，4.8Hz，1H)，2.71(d，J＝12.8Hz，1H)，2.22(t，J＝7.6Hz，2H)，1.71-1.60(m，4H)，1.47-1.43(m，2H).
D-Biotin modifies the synthetic of arteannuin:
Under the argon shield, 74mg (0.2269mmol) carboxylic acid arteannuin, 57mg (0.2949mmol) EDCI, 40mg (0.2949mmol) HOBt are dissolved in the 3mL dry DMF, inject 0.24mL (1.4293mmol) DIPEA, stirring at room.
Claim 93mg (0.2496mmol) 4 to be dissolved in the dry DMF of 2mL, solution is injected above-mentioned reactant liquor, the ambient temperature overnight reaction.After reaction finishes, add the DCM dilute reaction solution, use NH successively 4Cl saturated solution, water, saturated NaCl solution washing, anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=10: 1), obtain the 89mg target product, yield 57%.
1H?NMR(CDCl 3，400MHz)：δ＝7.24(s，1H)，δ6.85(s，1H)，δ6.30(s，1H)，δ5.41(s，1H)，δ5.35(s，1H)，δ4.75-4.71(m，1H)，δ4.52-4.50(m，1H)，δ4.32-4.29(m，1H)，δ3.61-3.40(m，12H)，δ3.15-3.16(m，1H)，δ2.90-0.88(m，33H).
Embodiment 9:
Claim 50mg (0.1288mmol) arteannuin benzoate and an amount of 4
MS under the argon shield, injects 2mL and heavily steams the DCM dissolving in reaction bulb, is cooled to 0 ℃, injects 0.02mL (0.1546mmol) TMSCl, keeps this thermotonus to rise to the ambient temperature overnight reaction after 1 hour and finishes.Be spin-dried for solvent, add acetic acid ethyl dissolution and be spin-dried for thing, use saturated NaHCO successively 3Solution, saturated NaCl solution washing, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=8: 1), obtain the 33mg target product, yield 96%.
1H?NMR(CDCl 3，400MHz)：δ＝6.19(s，1H)，5.54(s，1H)，2.44-0.99(m，20H).
Embodiment 10:
Under the argon shield, 50mg (0.1865mmol) deoxidation dihydroartemisinine is dissolved among the 3mL DCM, is cooled to 0 ℃, injects 0.03mL (0.3729mmol) chloroacetic chloride, rises to the ambient temperature overnight reaction gradually and finishes.Reactant liquor is through water washing, and organic layer is through anhydrous Na 2SO 4Drying, evaporating column chromatography (PE: EA=16: 1), obtain racemization deoxidation dihydroartemisinine 30mg, yield 64% as by-product.
1H?NMR(CDCl 3，400MHz)：δ＝6.04(s，1H)，5.47(s，1H)，2.04-1.21(m，20H).
Embodiment 11:
100mg (0.3517mmol) dihydroartemisinine is dissolved in 5mL DCE, adding people 73mg (0.4220mmol) 2-quinoxaline carboxylic acid stirs moments later, add 5mL (0.0422mmol) DMAP, stirring is cooled to 0 ℃, adds 80mg (0.3868mmol) DCC, stirs and rises to room temperature after 5 minutes, stopped reaction after 16 hours, filtering reacting liquid, filtrate is through water, saturated NaCl solution washing, and organic layer is through anhydrous Na 2SO 4After the drying, the evaporating column chromatography obtains the 120mg target product, yield 78%.
1H?NMR(CDCl 3，400MHz)：δ＝9.57(s，1H)，8.31(d，J＝8.0Hz，1H)，8.17(d，J＝8.0Hz，1H)，7.91-7.86(m，2H)，6.03-6.00(m，2H)，5.64(s，1H)，2.90-0.95(m，21H).
Embodiment 12:
According to as described in example 12,100mg (0.3517mmol) dihydroartemisinine and the reaction of 63mg (0.4220mmol) terephthalaldehydic acid, column chromatography obtains the 116mg target product, yield 79%.
1H?NMR(CDCl 3，400MHz)：δ＝10.10(s，1H)，8.27(d，J＝8.0Hz，2H)，7.96(d，J＝8.0Hz，2H)，6.03-6.00(m，1H)，5.54(s，1H)，2.90-0.95(m，21H).
Embodiment 13:
According to as described in example 12,100mg (0.3517mmol) dihydroartemisinine and 133mg (0.4220mmol) Boc-3-(2-naphthyl)-L-alanine reaction, column chromatography obtains the 151mg target product, yield 76%.
1H?NMR(CDCl 3，400MHz)：δ＝7.86-7.83(m，1H)，7.80-7.74(m，3H)，7.47-7.38(m，1H)，6.05-6.00(m，1H)，5.54(s，1H)，2.83-0.85(m，34H).
Embodiment 14:
According to as described in example 12,100mg (0.3517mmol) dihydroartemisinine and the reaction of 76mg (0.4220mmol) paranitrophenylacetic acid, column chromatography obtains the 123mg target product, yield 78%.
1H?NMR(CDCl 3，400MHz)：δ＝8.18(d，J＝8.0Hz，2H)，7.48(d，J＝12.0Hz，2H)，6.03-6.00(m，1H)，5.44(s，1H)，3.82(s，2H)，3.82(s，2H)，2.65-0.68(m，23H).
Embodiment 15:
Under the argon shield; 100mg (0.3517mmol) dihydroartemisinine and 0.08mL (0.5627mmol) anisoyl chloride are dissolved among the 5mL DCM; be cooled to 0 ℃; inject 0.2mL (2.2509mmol) pyridine; stir after 15 minutes, rise to the ambient temperature overnight reaction and finish, react with 7% aqueous citric acid solution cancellation; ethyl acetate extraction is again through saturated NaHCO 3, saturated NaCl solution washing, organic layer is through anhydrous Na 2SO 4Drying concentrates the back column chromatography, obtains the 119mg target product, yield 81%.
1H?NMR(CDCl 3，400MHz)：δ＝8.06(d，J＝12Hz，2H)，6.91(d，J＝12Hz，2H)，5.98(m，1H)，5.51(s，1H)，3.864(s，3H)，2.81-0.85(m，21H).
Embodiment 16:
Described according to example 15,100mg (0.3517mmol) dihydroartemisinine and the reaction of 104mg (0.5627mmol) paranitrobenzoyl chloride, column chromatography obtains the 81mg target product, yield 53%.
1H?NMR(CDCl 3，400MHz)：δ＝8.28(s，4H)，6.05-6.00(m，1H)，5.55(s，1H)，2.83-0.85(m，22H).
Embodiment 17: artesunate synthetic
Under the room temperature condition, 250mg (0.9mmol) dihydroartemisinine is dissolved among the dry DCM of 25mL, adds 202mg (2mmol) succinic anhydride, be cooled to 0～5 ℃, add 107mg (0.9mmol) DMAP, kept this thermotonus 0.5 hour, rose to room temperature reaction naturally 1 hour.After reaction finishes, add 5mL water, transfer to pH=3 with 10% dilute hydrochloric acid, organic layer is used anhydrous MgSO after washing 4Drying, removal of solvent under reduced pressure obtains the 317mg target product, yield 94%.
1H?NMR(CDCl 3，400MHz)：δ＝5.74(d，J＝8.0Hz，1H)，5.39(s，1H)，2.72-2.59(m，4H)，2.53-2.48(m，1H)，2.36-2.28.(m，1H)，2.01-1.96(m，1H)，1.87-1.82(m，1H)，1.76-1.66(m，2H)，1.60-1.55(m，1H)，1.45-1.21(m，8H)，1.01-0.95(m，1H)，0.92(d，J＝8.0Hz，3H)，0.80(d，J＝4.0Hz，3H).
Embodiment 18: artemisinin derivative is to the influence of 3T3-L1 cell differentiation
The cultivation of 3T3-L1 cell
The 3T3-L1 cell is at 37 ℃, 5%CO 2Adherent growth in the culture environment is used the DMEM high glucose medium (HyClone) that adds 10%FBS (Hyclone hyclone).Can be according to cell growth condition, changed a not good liquor in 2～3 days.Continue if desired repeatedly to go down to posterity, and need still have good differentiation capability by cell after repeatedly go down to posterity, too full (＜70%) that will avoid so making cell long generally longly will go down to posterity the branch cell to 60%～70%.Cell can be with go down to posterity branch dish of rare ratio by 1: 15, but usually with 1: 10 or the higher ratio branch dish that goes down to posterity, need be as the criterion with experiment.
The culture medium preparation of 3T3-L1 cell induction differentiation
Inducing culture is divided into two kinds, and MDI inducing culture and insulin culture medium are respectively applied for different differentiation phases, is to add that by the DMEM culture medium FBS, dexamethasone, insulin and the IBMX of appropriate amount are made into.
IBMX solution: IBMX is dissolved in the KOH solution of 0.5N, the IBMX final concentration is 0.0115g/ml, 0.22 μ m syringe filters filtration sterilization ,-20 ℃ of preservations.
The insulin storage solutions: insulin is dissolved among the HCl of 0.02M to final concentration 167uM (1mg/ml), 0.22 μ m syringe filters filtration sterilization ,-20 ℃ of long preservation, 4 ℃ of short-terms are preserved.
Dexamethasone storage solutions: stored frozen liquid: the 10mM dexamethasone is dissolved in dehydrated alcohol ,-20 ℃ of preservations; The work storage liquid: stored frozen liquid is diluted to 1mM with PBS, and filtration sterilization is stored in 4 ℃.
MDI inducing culture (now with the current, 10ml/10cm culture dish, 5ml/6cm culture dish): the DMEM high glucose medium that contains 10%FBS adds again: 1: 100 IBMX solution, 1: 1000 insulin storage liquid, 1: 1000 dexamethasone work storage liquid.
Insulin culture medium (now with the current, 10ml/10cm culture dish, 5ml/6cm culture dish): the DMEM high glucose medium that contains 10%FBS adds again: 1: 1000 insulin storage liquid.
Induce undifferentiated 3T3-L1 cell (fibroblast state) to break up to mature fat cell:
As shown in Figure 3:
In containing the DMEM high glucose medium of 10%FBS with 3T3-L1 before adipose cell be cultured to engagement state.
2. support 2 days (being differentiation the 0th day this moment) after engaging again with MDI inducing culture irritation cell (change original complete medium, add the MDI inducing culture of new preparation).Can notice that in following 2 days cellular morphology takes place by significant change (spindle more becomes).
3. add MDI and induce (breaking up the 2nd day) after 2 days, change original MDI inducing culture, add the insulin culture medium of new preparation.Culture medium can begin the thickness more that becomes, because the synthetic free fatty of cell is by a large amount of being secreted in the culture medium.
4. (broke up the 4th day) after two days, change culture medium into common complete medium (the DMEM high glucose medium that contains 10%FBS), changed a not good liquor (the DMEM high glucose medium that contains 10%FBS) in per afterwards 2 days.Usually reach in the 8th day in differentiation and break up state completely.
Medicine numbering 1 to 9 structural formula of compound of representative respectively is followed successively by:
The analysis of oil red O stain data and processing
The preparation of oil red O stain agents useful for same:
Oil red O storage liquid: oil red O (AMRESCO, FW 408.51, Em (513nm)) 0.7g is dissolved in the 200ml isopropyl alcohol, spends the night and puts upside down the vibration mixing, is stored in 4 ℃ behind the 0.2 μ m membrane filtration.
Oil red O working solution: 6 parts of oil red O storage liquid add 4 parts of dH 2O, room temperature left standstill 20 minutes behind the mixing, 0.2 μ m membrane filtration.
Oil red O stain:
1. inhale and go most culture medium.
2. add 10% formalin (being dissolved among the PBS), incubated at room 5 minutes.
3. inhale and remove 10% formalin, add the fresh formalin (undiluted 37% formalin) of same volume, hatched at least 1 hour, in addition more of a specified duration.Annotate: cell can keep several days in formalin, and then dyeing.With around the culture plate with sealing film phonograph seal preventing formaldehyde volatilization evaporate to dryness, and wrap up with lucifuge with aluminium-foil paper.
4. inhale with the lancet head and remove all formalin.
5. (be dissolved in dH with 60% isopropyl alcohol 2Among the O) the cleaning culture hole.
6. allow cell culture hole finish-drying.
7. add oil red O working solution, dye 10 minutes (adding the fashionable hole wall that keeps off).
8. remove all oil red dye liquors, add dH immediately 2O washes with water 4 times (can directly wash under water tap).
9. take pictures according to the experiment needs.
10. remove all water, allow the culture hole bone dry.
11. add 100% isopropyl alcohol eluting oil red O, hatch 10 minutes (can be more of a specified duration).
12. repeatedly pressure-vaccum contain oil red isopropyl alcohol several times, so guarantee that oil red all dissolves in the solution.
13. will contain in the hole that the isopropyl alcohol of oil red is transferred to EP pipe or elisa plate.
14. measure the OD value at 500nm wavelength place, the reading duration is 0.5 second.
15. blank uses 100% isopropyl alcohol, dyeing background contrast is used from according to the painted isopropyl alcohol of the empty culture hole of foster cell of above-mentioned steps with not ing.
The consumption of all ingredients in table 1. oil red O stain
| Tissue Culture Plate | Formalin | 60% isopropyl alcohol | Oil red O | 100% isopropyl alcohol |
| 24 orifice plates | ??500μl | ??500μl | ??200μl | ??750μl |
| 12 orifice plates | ??1ml | ??1ml | ??400μl | ??1.5ml |
| 6 orifice plates | ??2.4ml | ??2.4ml | ??1ml | ??3.6ml |
Artemisinin derivative is to the influence of the differentiation of 3T3-L1 cell
Solid arteannuin (Fig. 1 .1,3-7 chemical compound) is dissolved among the DMSO, is mixed with the solution of a series of gradient concentrations, this Concentraton gradient comprises 5 level: 1mM, 5mM, 10mM, 30mM, 50mM in this experiment.Dihydroartemisinine (chemical compound 2) Concentraton gradient is: 0.1mM, 0.5mM, 1mM, 5mM, 10mM, 30mM, 50mM; The Concentraton gradient of artesunate (chemical compound 9) is: 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM, 10mM, 30mM, 50mM.
As shown in Figure 3, according to above-mentioned described operating process of inducing undifferentiated 3T3-L1 cell (fibroblast state) to the mature fat cell differentiation, in differentiation the 0th day, to reaching engagement state but still undifferentiated 3T3-L1 cell adds the MDI culture medium of inducing differentiation.The processing that adds the arteannuin medicine is operating as, and adds the solution of the arteannuin series gradient concentration that is configured to as mentioned above in the MDI culture medium in advance according to 1/1000 ratio, culture medium is added the cell culture hole after the mixing again.1/1000 ratio makes the final concentration of chemical compound 1,3-7 medicine become 1 μ M, 5 μ M, 10 μ M, 30 μ M, 50 μ M, the actual drug dose that uses of this experiment just.Need to design a processing simultaneously, the DMSO of adding 1/1000 is as solvent control in culture medium, and just drug level is the processing of 0 μ M.
Cultivated treated 3T3-L1 cell 2 days according to above-mentioned described cell culture condition, according to above-mentioned described operating process of inducing undifferentiated 3T3-L1 cell (fibroblast state) to the mature fat cell differentiation, added the insulin culture medium on the 2nd day in differentiation, and with same as mentioned above method and dosage, add the arteannuin (same cell culture hole adds same a kind of medicine of same concentration) of serial gradient concentration.
According to foregoing description, the 3T3-L1 cell of finishing differentiation is carried out oil red O stain, take pictures, survey absorbance, collect data, figure is in arrangement, relatively, the results are shown in Figure 1.
The data of these 9 each concentration of chemical compound all derive from the meansigma methods that the repeated experiments of 3 different batches obtains among Fig. 1, and error is by the SE value representation between 3 repeated experiments.
After medicine adds, can suppress or promote or not appreciable impact 3T3-L1 cellular fat differentiation (for the arteannuin medicine series, what the present invention paid close attention to is to suppress).The present invention represents drug effect with the degree of cellular fat differentiation.The degree of cell differentiation can be with after the dyeing of oil red staining after experiment is finished, and surveying OD is worth with detection by quantitative.Differentiation degree is high more, and the oil droplet accumulation is many more, and oil red dyeing is dark more, and the OD value is high more.
Analytical Chemical Experiment of the present invention uses 24 porocyte culture plates to do, and each 24 orifice plate can design several holes of not dosing and do contrast, just the contrast of the cell that can break up fully.
Each medicine is to obtain (differentiation degree relatively) by corresponding OD value than the OD value of the contrast of differentiation fully on the last same 24 porocyte culture plates in the drug effect of specific concentrations.Like this, the data that draw of different culture plate just can compare mutually.
As shown in Figure 1, if differentiation fully, the value that obtains (differentiation degree relatively) is exactly 1 (100%); Differentiation is suppressed, and value is just less than 1 (between 0～1).Differentiation inhibition degree is high more, is worth more for a short time, and drug effect is good more.
The result as shown in Figure 1, arteannuin itself (Fig. 1 .1 chemical compound) suppresses the poor effect of 3T3-L1 cell differentiation, concentration is in the level of 50 μ M, the relative differentiation degree of 3T3-L1 cell can reach 93.3%; In the level of 10 μ M, differentiation degree can reach 94.8% relatively.
The results are shown in Figure 1, Fig. 2 .1, the effect that dihydroartemisinine (Fig. 1 .2 chemical compound) suppresses the 3T3-L1 cell differentiation be that all are one of best in the artemisinin derivative of tested mistake, can suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M, and relative differentiation degree has only 24.5%.
The results are shown in Figure 1, Fig. 2 .3, the effect that this chemical compound (Fig. 1 .3 chemical compound) suppresses the 3T3-L1 cell differentiation is general, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 62.5%.
The results are shown in Figure 1, Fig. 2 .2, the effect that arteannuin benzoate (Fig. 1 .4 chemical compound) suppresses the 3T3-L1 cell differentiation is that all are one of best in the artemisinin derivative of tested mistake, can suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M, differentiation degree has only 27.3% relatively.
The results are shown in Figure 1, Fig. 2 .4, the effect that pi-allyl arteannuin (Fig. 1 .5 chemical compound) suppresses the 3T3-L1 cell differentiation is general, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 58.1%.
The results are shown in Figure 1, the effect of acetate arteannuin (Fig. 1 .6 chemical compound) inhibition 3T3-L1 cell differentiation is relatively poor, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 81.3%.
The results are shown in Figure 1, arteannuin-biotin (Fig. 1 .7 chemical compound) suppresses the poor effect of 3T3-L1 cell differentiation, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 91.6%.
The results are shown in Figure 1, deoxidation dihydroartemisinine (Fig. 1 .8 chemical compound) does not almost suppress the effect of 3T3-L1 cell differentiation, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell can reach 98.8%.
The results are shown in Figure 1, Fig. 2 .5, the effect that artesunate (Fig. 1 .9 chemical compound) suppresses the 3T3-L1 cell differentiation be that all are one of best in the artemisinin derivative of tested mistake, can suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M, and relative differentiation degree has only 31.5%.
Claims (3)
Hide Dependent
1. the application of artemisinin derivative in the fat medicine of preparation control that has following formula I, II, III, IV, V structure,
Wherein:
N=0～1; R 1Be selected from C 1～C 3Alkyl; R 2Be selected from the aromatic heterocycle that aryl or nitrogen atom replace.
2. application according to claim 1 is characterized in that: described artemisinin derivative is to have in the following structural formula any:
3. application according to claim 1 is characterized in that: described artemisinin derivative is to have in the following structural formula any:
0 Comments
Recommended Comments
There are no comments to display.