CN104031055B - 一种可用作Wnt信号途径激活剂的化合物及其制备与应用 - Google Patents
一种可用作Wnt信号途径激活剂的化合物及其制备与应用 Download PDFInfo
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Abstract
本发明提供一种用作Wnt信号途径激活剂的化合物及其制备与应用。本发明的化合物为式（I）或式（II）的化合物。本发明的化合物可用于制备调节经典Wnt信号途径的药物并可用于研究经典Wnt信号途径。本发明也为筛选调节Wnt信号途径的药物、预防和治疗经典Wnt信号途径相关疾病的药物、研究Wnt信号途径在干细胞中的应用提供了新的思路。
Description
技术领域
本发明涉及可用作Wnt信号途径激活剂的化合物及其制备与应用。
背景技术
Wnt信号转导途径是一类在生物体进化过程中高度保守的信号转导途径,调节控制着众多生命活动过程。在动物体早期发育中,Wnt信号决定背腹轴的形成、胚层建立、体节分化、组织或器官形成等一系列重要事件，并直接控制着增殖、分化、极化、凋亡与抗凋亡等细胞的命运[Clevers,H.and R.Nusse,Wnt/beta-Catenin Signaling and Disease.Cell,2012.149(6):p.1192-205]。Wnt蛋白的十几个成员通过与细胞膜上不同的受体作用参与了不同的信号转导途径。这些途径主要分为依赖于β-catenin/TCF转录复合物的经典Wnt信号转导途径（Wnt/β-catenin途径）及不依赖于β-catenin/TCF转录复合物的非经典Wnt信号转导途径（Wnt/Ca2+途径和Wnt/JNK途径）[Barker,N.and H.Clevers,Mining the Wntpathway for cancer therapeutics.Nature reviews.Drug discovery,2006.5(12):p.997-1014]。
对于Wnt/β-catenin信号转导途径的过程，我们可以从两个状态来理解。当这条途径没有被激活时，细胞质内的转录激活子β-catenin蛋白会被“降解复合物”所降解。在这个“降解复合物”中，Axin和APC（adenomatous polyposis coli）作为骨架蛋白，它们结合了能够对β-catenin进行磷酸化修饰的激酶CK1α（casein-kinase1α）和GSK3（glycogensynthase kinase3）[Liu,C.,et al.,Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.Cell,2002.108(6):p.837-47]。这两种激酶依次对β-catenin蛋白的N端的特定位点进行磷酸化修饰，使得磷酸化的β-catenin能够被泛素化E3连接酶β-Trcp识别，并对其继续进行泛素化进而被蛋白酶体降解。这样细胞质及核内的β-catenin始终维持在一个较低的水平，从而使得转录抑制因子结合转录因子Tcf/Lef（T-cell factor/lymphoid enhancer factor），阻碍了Wnt/β-catenin信号转导途径下游的目的基因的表达。当Wnt/β-catenin信号转导途径被激活时，即Wnt蛋白结合了七次跨膜受体Frizzled（Fz）和它的共受体LRP5/6（low-density lipoprotein receptor-relatedprotein5/6）。膜上Wnt-Fz-LRP5/6复合物的形成导致了骨架蛋白Dishevelled（Dvl）和七次跨膜受体Fz的细胞膜内的区域结合进而促使了LRP5/6的磷酸化。受体LRP5/6的磷酸化进一步招募了Axin所结合的复合物（GSK3-Axin-CK1）和其磷酸化的细胞膜内的区域结合[Zeng,X.,et al.,A dual-kinase mechanism for Wnt co-receptor phosphorylation andactivation.Nature,2005.438(7069):p.873-7.Mao,J.,et al.,Low-densitylipoprotein receptor-related protein-5binds to Axin and regulates thecanonical Wnt signaling pathway.Molecular cell,2001.7(4):p.801-9.]，导致了LRP5/6被多重磷酸化从而会招募更多的细胞质内的GSK3-Axin-CK1结合到受体LRP5/6细胞膜内的区域。这样就使得细胞质中结合β-catenin的降解复合物的关键成员不断减少，进而β-catenin不被磷酸化也即不被泛素化降解。细胞质内的β-catenin就会不断的累积并进一步运输到细胞核中与转录因子Tcf/Lef结合，激活Wnt/β-catenin信号转导途径下游的目的基因的表达。
由于Wnt/β-catenin信号途径与众多的癌症和疾病有关系，所以调节失控的Wnt/β-catenin信号途径可以作为治疗与Wnt/β-catenin信号途径相关疾病的一个很好手段，但是现阶段的研究还没有什么明显的进展。例如，阿尔茨海默病的病变过程伴随着Wnt/β-catenin信号转导途径的异常失活。与阿尔茨海默病紧密相关的早老素蛋白能够与-catenin和GSK3形成复合体[Kang,D.E.,et al.,Presenilin couples the pairedphosphorylation of beta-catenin independent of axin:implications for beta-catenin activation in tumorigenesis.Cell,2002.110(6):p.751-62]。所以针对该信号途径的分子靶向靶向治疗有望为与其相关疾病提供新的有效途径。另外，在干细胞中Wnt信号转导途径也起着维持干细胞多能性的作用，所以调节该信号途径有望为干细胞的应用提供新的有效方法。
发明内容
本发明的目的在于提供新的Wnt信号途径激活剂及其制备与应用。
本发明首先提供了一种化合物，其结构式为：
或
以下将结构式为I的化合物称为化合物I，结构式为II的化合物称为化合物II，结构式为III的中间体称为中间体III，以此类推。
本发明进一步提供了化合物I的制备方法，其制备路线为：
包括下列步骤：
1）以石蒜碱（Lycorine）为起始原料经通过N-甲基化与霍夫曼降解制备中间体III并分离产物；
2）使中间体III氢化还原获得产物化合物I。
本发明进一步提供了化合物II的制备方法，其制备路线为：
包括下列步骤：
1）以石蒜碱（Lycorine）为起始原料经N-甲基化与霍夫曼降解制备中间体III并分离产物；
2）使中间体III氢化还原获得化合物I并分离产物；
3）化合物I通过降解二氧亚甲基制备中间体IV并分离产物；
4）使中间体IV与溴丙炔进行烷基化反应制备中间体V并分离产物；
5）将中间体V与化合物VI通过Click反应获得产物化合物II。
其中，化合物VI的制备路线为：
包括下列步骤：
1）以生物素（Biotin）为起始原料经缩合反应制备中间体VII并分离产物；
2）使中间体VII与化合物VIII类酯交换反应获得化合物VI并分离产物。
其中，化合物VIII的制备路线为：
为以2-溴乙胺氢溴酸盐（2-Bromoethylamine hydrobromide）为起始原料与叠氮钠（sodium azide）亲核取代制备化合物VIII并分离产物。
本发明还提供了所述化合物I和化合物II在制备经典Wnt信号途径激动剂中的用途。
进一步的，所述经典Wnt信号途径激动剂能激活经典Wnt信号途径的报告基因或目的基因的表达活性。
本发明还提供了所述化合物I和化合物II在制备药物中的应用，所述药物选自以下任一：
1）预防或治疗由经典Wnt信号途径异常失活所引起的疾病或失调的药物；
2）需要特异性激活经典Wnt信号途径的疾病或失调的药物；
3）干细胞数目扩增药物。
此处的“疾病或失调”包括任何可从本发明的处理获益的病症。其包括但不局限于各种慢性和急性失调或疾病。在一个较佳的实施方案中，所述疾病或失调是老年痴呆症、风湿性关节炎或骨质疏松症。在另一个较佳的实施方案中，所述疾病或失调包括造血干细胞移植。
进一步的，所述药物选自以下任一：老年痴呆症药物、风湿性关节炎药物、骨质疏松症药物、造血干细胞移植药物、干细胞多能性维持药物或斑马鱼发育调控药物。
本发明进一步提供了一种用于预防或治疗由经典Wnt信号途径异常失活引起的或用于需要特异性激活经典Wnt信号途径的疾病或失调的药物组合物：含有治疗有效量的化合物I或化合物II以及在药学上可接受的赋形剂。
术语“包含”指“含有”和“由﹍构成”，例如“包含”X的组合物可以完全由X构成，或者可以含有X之外的物质，例如X-Y。本文所用的术语“治疗有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。该效果例如可通过化学标记或抗原水平来检测。治疗效果也包括生理性症状的减轻。对于某一对象的精确有效剂量取决于该对象的体型和健康状况，病症的性质或程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的症状而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。
所述药学上可接受的赋形剂（或运载体）指用于治疗剂给药的赋形剂，其本身及其用量不应诱导接受该组合物的个体产生有害的抗体，且给药后没有过分的毒性。药学上可接受的赋形剂通常包括无毒的固体、半固体或液体填充剂、稀释剂、包裹材料或任何常规类型的制剂辅助物。合适的赋形剂包括但不仅限于水、葡萄糖、甘油、盐水、乙醇或其组合。所述赋形剂还包含有其他试剂如湿润剂或乳化剂、pH缓冲剂或增强制剂效力的佐剂。其他材料如抗氧化剂、保湿剂、粘度稳定剂和类似试剂可根据需要添加。脂质体也包括在药学上可接受的赋形剂的定义中。
通常，可将药物组合物制成可注射剂，例如液体溶液或悬浮液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
本发明的化合物可用于制备调节经典Wnt信号途径的药物并可用于研究经典Wnt信号途径。本发明也为筛选调节Wnt信号途径的药物、预防和治疗经典Wnt信号途径相关疾病的药物、研究Wnt信号途径在干细胞中的应用提供了新的思路。
附图说明
图1：化合物I对经典Wnt信号途径报告基因TOPFlash的活性的影响结果：
HLY78：化合物I
DMSO：二甲基亚砜
在HEK293T细胞中转染TOPFlash报告基因18小时后加入特定浓度的HLY78和溶剂对照DMSO的Wnt条件培养基或对照培养基。6小时后收集细胞检测报告基因活性。
结果表明：通过哺乳动物细胞实验报告基因筛选系统发现化合物I（图中表示为HLY78）能够以一种依赖于受体的形式去激活经典Wnt信号途径报告基因TOPFlash的活性。
图2：化合物I对经典Wnt信号途径靶基因mRNA水平的影响结果：
HLY78：化合物I
DMSO：二甲基亚砜
AXIN2/GAPDH：看家基因GAPDH的mRNA水平表达量标准化AXIN2的mRNA水平
DKK1/GAPDH：看家基因GAPDH的mRNA水平表达量标准化DKK1的mRNA水平
HEK293T细胞加入特定浓度的HLY78和溶剂对照DMSO的Wnt条件培养基或对照培养基。6小时后收集细胞作RT-PCR检测靶基因mRNA水平。
结果说明：化合物I能够以一种依赖于受体的形式去激活经典Wnt信号途径靶基因mRNA水平。
图3：化合物I对β-catenin的蛋白水平的影响结果
HLY78：化合物I
DMSO：二甲基亚砜
Ctr：对照
α-β-catenin：用β-catenin的特异性抗体检测蛋白；
α-β-Actin：用β-Actin的特异性抗体检测蛋白；
α-SP1：用SP1的特异性抗体检测蛋白；
HEK293T细胞加入特定浓度的HLY78和溶剂对照DMSO。1小时后加入含有相同药物浓度的Wnt条件培养液或对照条件培养液。6小时后收集细胞分离细胞质和细胞核检测β-catenin的蛋白水平。其中β-actin作为细胞质标记物，SP1作为细胞核标记物。
结果表明：化合物I能够以一种依赖于受体的形式去影响β-catenin的蛋白水平。
图4：药物对经典Wnt信号途径报告基因TOPFlash的活性的影响结果
HLY78：化合物I
HLY179：化合物II
DMSO：二甲基亚砜
在HEK293T细胞中转染TOPFlash报告基因17小时后加入10μM的HLY179,生物素,HLY78和溶剂对照DMSO的Wnt条件培养基或对照培养基。6小时后收集细胞检测报告基因活性。结果表明：化合物I和II能够以一种依赖于受体的形式去激活经典Wnt信号途径报告基因TOPFlash的活性。
图5：化合物I作用斑马鱼造血干细胞后，对表现造血干细胞的标记物runx1/cmyb的mRNA水平表达的原位染色试验结果图。
40/45：总数45条鱼中有40条如图示；
23/32：总数32条鱼中有23条如图示；
在斑马鱼三体节时加入80μM的HLY78和溶剂对照DMSO。24小时后收集斑马鱼胚胎做原位染色实验，检测造血干细胞的标记物runx1/cmyb的mRNA水平表达。
结果表明：化合物I增强斑马鱼造血干细胞的发育。
具体实施方式
发明人经过深入研究，首次发现化合物I和化合物II能够以一种依赖于受体的形式去激活经典Wnt信号途径。由于经典Wnt信号途径与多种疾病，例如老年痴呆症,风湿性关节炎、骨质疏松症以及调控造血干细胞的发育有关，因此发现化合物I和化合物II能够以一种依赖于受体的形式去激活经典Wnt信号途径为开发新的药物来治疗这些疾病提供了新的思路。
下面结合本发明实施例来进一步说明本发明的实质性内容，但不以此来限定本发明。应理解，这些实施例仅为了说明本发明而非限制本发明的范围。实施例所采用的具体实验方法和实验条件是本领域常规方法和条件，或制造厂商所推荐的方法和条件。除非另有定义，文中所用的专业与科学用语与本领域技术人员所知的相同。
以下化合物和中间体通过液相色谱-质谱（LC-MS）和核磁共振（NMR）表征，在制备所述化合物中使用的起始物质和试剂可以从供应商购得或者通过本领域技术人员已知的方法制备。以下一般性的合成路线仅仅举例说明了通过其可合成本发明化合物的方法，并且对于已经参考本发明公开内容的本领域技术人员，所述合成路线的多种修改是可以做出来的并且是得到启示的。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等
MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold SpringHarbor Laboratory Press，1989and Third edition，2001；Ausubel等，CURRENTPROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodicupdates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1
1.实验材料
1)质粒
TOPFlash质粒从Millipore的公司购买。
GFP质粒从Millipore的公司购买。
2)抗体
β-actin（sc-47778）的抗体购自Santa Cruz公司；SP1（S9809）的抗体购自Sigma公司；β-catenin（610154）的抗体购自BD Biosci.Pharmingen公司。
3)试剂
蛋白酶抑制剂（完全蛋白酶抑制剂混合片剂）和NP-40购自Roche公司；Lipofectamine和PLUS转染试剂购自Invitrogen公司；
Wnt3a条件培养基从Wnt3a细胞株（CRL-2647，购自美国ATCC细胞库）制备：采用加了10%小牛血清的DMEM细胞培养基培养Wnt3a细胞4天，然后离心收集上清培养基。
对照条件培养基从L细胞株（CRL-2648，购自美国ATCC细胞库）制备：采用加了10%小牛血清的DMEM细胞培养基培养L细胞4天，然后离心收集上清培养基。
4)化合物的制备：
合成路线：
a.以Lycorine为原料制备中间体III：5-methyl-4-vinyl-5,6-dihydro-[1,3]dioxolo[4,5-j]phenanthridine
石蒜碱（Lycorine）（28.7mg,0.1mmol）溶解解至N，N-二甲基甲酰胺（DMF）（5mL）中，加入碘甲烷（MeI）（17.0mg，0.12mmol），在50℃下搅拌反应12h,减压蒸馏出溶剂后直接用于下一步反应；在氮气保护条件下，将上步制得的化合物(30.2mg,0.1mmol)溶剂于叔丁醇（t-BuOH）(5mL)中，加入叔丁醇钾（t-BuOK）(30mg)后回流反应4h,反应结束后加入氯化铵溶液，然后加入乙醚萃取2次（2×10ml），接着用氯化铵液洗涤2次（2×10ml），用饱和食盐水洗涤2次（2×10ml）,有机层用饱和食盐水洗后用无水硫酸镁干燥，减压移除易挥发的溶剂后用硅胶色谱柱纯化（乙酸乙酯/石油醚200:1，Rf=0.23），得到中间体III，21.2mg,得率80%。
NMR、MS分析确认产物：
1H NMR(400MHz,CDCl3)δ:7.58(d,J=7.7Hz,1H),7.47(d,J=7.7Hz,1H),7.26(dt,J=10.7,7.1Hz,2H),7.16(t,J=7.7Hz,1H),6.72(s,1H),5.99(s,2H),5.75(d,J=17.8Hz,1H),5.32(d,J=11.1Hz,1H),4.03(s,2H),2.51(s,3H);13C NMR(100MHz,CDCl3)δ:147.4(C),145.1(C),133.4(CH),133.2(C),129.2(C),126.4(C),125.8(C),124.9(CH),124.3(CH),122.7(CH),114.3(CH2),107.1(CH),103.6(CH),100.9(CH2),54.8(CH2),41.5(CH),EIMS m/z:266[M+H]+.
b.从中间体III进一步出发制备化合物I：N-甲基-4-乙基-5，6-二氢-8，9-二氧亚甲基-菲啶（4-ethyl-5-methyl-5,6-dihydro-[1,3]dioxolo[4,5-j]phenanthridine）
在氮气保护条件下，中间体III（26.5mg,0.1mmol）溶解在四氢呋喃（THF）/H2O（5mL，4:1）中，然后加入对甲苯磺酰肼（TsNHNH2）(37.2mg,0.2mmol)回流反应4h，冷却至室温然后加入醋酸钠（NaOAc）（73.8mg,0.3mmol）反应结束后加入氯化铵溶液，然后加入乙醚萃取2次（2×10ml），接着用氯化铵液洗涤2次（2×10ml），用饱含食盐水洗涤2次（2×10ml）,有机层用饱和食盐水洗后用无水硫酸镁干燥，减压移除易挥发的溶剂后用硅胶色谱柱纯化（乙酸乙酯/石油醚200:1，Rf=0.21），得到化合物I。黄色固体26.8mg，得率95%。产物m.p.（熔点）179-180°C。
NMR、MS分析确认产物：
1H NMR(400MHz,MeOD)δ:7.51(t,J=8.4Hz,1H),7.28(d,J=6.4Hz,2H),7.20(t,J=7.9Hz,2H),6.75(s,1H),6.01(s,1H),4.01(s,2H),2.83(q,J=7.5Hz,2H),2.50(s,3H),1.32(dd,J=15.7,8.2Hz,3H)；13C NMR(100MHz,CDCl3)δ:147.3(C),147.1(C),145.4(C),139.4(C),129.3(C),127.7(CH),126.6(C),126.3(C),124.6(CH),121.0(CH),107.2(CH),103.7(CH),100.9(CH2),55.2(CH2),41.02(CH),23.1(CH2),14.8(CH3),HREIMS m/z217.1261[M]+(calcd for C17H17NO2,267.1259).
c.从化合物I出发制备中间体IV:4-ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diol
-78℃条件下，在化合物I(26.7mg,0.1mmol)溶解到二氯甲烷（CH2Cl2）（2mL）中，加入三溴化硼（BBr3）（49.4mg,0.2mmol）反应2h，反应结束后加入碳酸氢钠溶液，然后加入乙醚萃取2次（2×10ml），接着用碳酸氢钠溶液洗涤2次（2×10ml），用饱和食盐水洗涤2次（2×10ml）,有机层用饱和食盐水洗后用无水硫酸镁干燥，减压移除易挥发的溶剂后用硅胶色谱柱纯化（乙酸乙酯/石油醚1:1，Rf=0.22），得到中间体IV，黄色固体17.9mg，得率70%。
NMR、MS分析确认产物：
1H NMR(400MHz,CDCl3)δ:7.46(d,J=7.1Hz,1H),7.27(d,J=2.7Hz,1H),7.17-7.11(m,2H),6.75(s,1H),3.96(s,2H),2.79(q,J=7.5Hz,2H),2.47(s,3H),1.28(dd,J=14.7,7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ:143.8(C),143.0(C),139.4(C),129.0(C),127.6(CH),125.7(C),125.5(C),124.7(CH),120.9(CH),113.8(CH),113.4(C),110.4(CH),54.6(CH2),41.2(CH3),23.1(CH2),14.8(CH3);EIMS m/z:256[M+H]+.
d.从中间体IV出发制备中间体V:4-ethyl-5-methyl-8,9-bis(prop-2-ynyloxy)-5,6-dihydrophenanthridine
在氮气保护条件下，中间体IV（25.5mg,0.1mmol）溶解在THF（5mL）中，然后加入氢化钠（NaH）(180mg,0.3mmol,40%)和丙炔溴(14mg,0.3mmol,80%)，室温下反应8小时，反应结束后加入氯化铵溶液，然后加入乙醚萃取2次（2×10ml），接着用氯化铵溶液洗涤2次（2×10ml），用饱和食盐水洗涤2次（2×10ml）,有机层用饱和食盐水洗后用无水硫酸镁干燥，减压移除易挥发的溶剂后用硅胶色谱柱纯化（乙酸乙酯/石油醚200:1，Rf=0.21），得到中间体V，黄色固体26.4mg，得率80%。
NMR、MS分析确认产物：
1H NMR(400MHz,CDCl3)δ1H NMR(400MHz,CDCl3)δ7.54(d,J=6.9Hz,1H),7.46(s,1H),7.23–7.12(m,2H),6.91(s,1H),4.83(s,2H),4.80(s,2H),4.02(s,2H),3.09–2.99(m,2H),2.86–2.74(m,2H),2.47(s,3H),1.29–1.17(m,3H);；13C NMR(100MHz,CDCl3)δ:147.4(C),146.8(C),145.6(C),139.5(C),128.9(C),127.9(CH),126.8(C),126.3(C),124.6(CH),121.0(CH),113.1(CH),110.5(CH),78.6(C),78.4(C),75.9(CH),75.8(CH),57.2(CH2),56.9(CH2),54.8(CH2),41.31(CH3),23.1(CH2),14.8(CH3);ESIMS m/z:330[M-H]-.
e.制备化合物VIII:2-Azidoethylamine
2-溴乙胺氢溴酸盐（2-Bromoethylamine hydrobromide）(500mg,2.44mmol)和叠氮钠（sodium azide）(475.9mg,7.32mmol)溶解到H2O(2mL)中，75°C下搅拌反应21h然后冷却至0°C，加入氢氧化钾（KOH）(800mg)和Et2O(2mL)，水层用乙醚萃取2次（2×10ml），有机层用饱和食盐水洗后用无水硫酸镁干燥，减压移除易挥发的溶剂后用硅胶色谱柱纯化（氯仿/甲醇20:1，Rf=0.21），得到化合物VIII，黄色液体171mg,1.99mmol,得率82%。
NMR、MS分析确认产物：
1H NMR(400MHz,CDCl3)δ:1.27(s,2H,NH2),2.80–2.84(m,2H,CH2N3),3.30(t,J=5.7Hz,2H,CH2NH2)；13C NMR(100MHz,CDCl3)δ:41.2(CH2NH2),54.6(CH2N3)。
f.制备中间体VII：2,5-dioxopyrrolidin-1-yl5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate
Biotin(24.4mg,0.1mmol)溶解至N，N-二甲基甲酰胺（DMF）（10mL）中,加入吡啶（Py）（2mL）和N,N'-二环己基碳酰亚胺（DCC）（41.2mg,0.2mmol）,反应半小时后加入N-羟基琥珀酰亚胺（N-Hydroxysuccinimide）(13.8mg,0.12mmol),反应24h后停止反应，过滤后滤液减压蒸馏移除DMF，接着用热异丙醇溶剂溶解，然后过滤，滤液在4℃冰箱中沉淀2天，最后过滤得到白色固体沉淀10.2mg,得率30%。
NMR、MS分析确认产物：
1H NMR(400MHz,[D6]DMSO)δ:6.42(s,1H,3-NH),6.36(s,1H,1-NH),4.27–4.32(m,1H,6a-H),4.11–4.16(m,1H,3a-H),3.06–3.12(m,1H,SCH),2.78–2.85(m,5H,CH2CH2(succinyl),SCH2),2.66(t,J=7.3Hz,2H,2'-H),2.57(d,J=11.4Hz,1H,SCH2),1.58–1.68(m,3H,3'-H,5'H),1.35–1.54(m,3H,4'-H,5'-H);13C NMR(100MHz,[D6]DMSO)δ:170.3(N(CO)2),168.9(CO2),162.7((HN)2CO),61.0(C-3a),59.2(C-6a),55.2(SCH),39.9(SCH2),30.0(C-2'),27.8(C-4'),27.6(C-5'),25.4(CH2CH2(succinyl)),24.3(C-3');EIMS m/z:342[M+H]+。
g.制备化合物VI：N-(2-azidoethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide
中间体VII（34.1mg,0.1mmol）溶解至DMF(2mL)中，加入Et3N（12.0mg,1.2mmol），然后加入化合物VIII（17.2mg,0.2mmol）在室温下反应24h,减压蒸馏移除DMF后用硅胶色谱柱纯化（氯仿/甲醇20:1，Rf=0.23），得到化合物VI，黄色固体21.8mg，得率70%。
NMR、MS分析确认产物：
1H NMR(400MHz,[D6]DMSO)δ:8.03(t,J=5.3Hz,1H,CONH),6.42(s,1H,3-NH),6.35(s,1H,1-NH),4.26–4.32(m,1H,6a-H),4.08–4.14(m,1H,3a-H),3.31(d,J=7.6Hz,2H,CH2N3),3.19–3.24(m,2H,CH2CH2N3),3.05–3.11(m,1H,SCH),2.80(dd,J=12.4,5.1Hz,1H,SCH2),2.56(d,J=12.9Hz,1H,SCH2),2.06(t,J=7.3Hz,2H,2'-H),1.55–1.65(m,1H,5'-H),1.39–1.55(m,3H,3'-H,5'-H),1.20–1.38(m,2H,4'-H);13C NMR(100MHz,[D6]DMSO)δ:172.4(CONH),162.7((HN)2CO),61.0(C-3a),59.2(C-6a),55.4(SCH),50.0(CH2N3),39.9(SCH2),38.1(CH2CH2N3),35.1(C-2'),28.2(C-4'),28.0(C-5'),25.2(C-3');HRESI+MS:m/z313.14419[M+H]+(calcd for C12H20N6O2S+H,313.14412).
h.制备化合物II：(R,S,S)-N,N'-(2,2'-(4,4'-(4-ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diyl)bis(oxy)bis(methylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(ethane-2,1-diyl))bis(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)
中间体V（33.1mg,0.1mmol）和化合物VI（68.6,0.22mmol）溶解至tBuOH（5mL）中，抗坏血酸钠（sodium ascorbate）（19.8mg,0.1mmol）和硫酸铜(1.8mg,0.01)加入水（2mL），然后把水溶液加入t-BuOH溶液中，50℃下搅拌反应12小时，减压蒸馏移除溶剂后用硅胶色谱柱纯化（氯仿/甲醇25:1，Rf=0.23），得到化合物12，黄色固体57.3mg，得率60%。m.p.196-197°C。
NMR、MS分析确认产物：
1H NMR(400MHz,MeOD)δ7.96(s,1H),7.95(s,1H),7.58–7.51(m,1H),7.46(s,1H),7.19–7.11(m,2H),6.97(s,1H),5.24(s,2H),5.21(s,2H),4.55–4.48(m,6H),4.46–4.39(m,2H),4.31–4.22(m,2H),4.02–3.94(m,2H),3.71–3.62(m,4H),3.35–3.26(m,4H),2.80–2.72(m,2H),2.43(s,3H),2.20–2.04(m,4H),1.75–1.46(m,8H),1.39–1.32(m,4H),1.28(t,J=7.1Hz,3H)；13C NMR(101MHz,CDCl3)δ176.42,170.23,165.98,163.95,153.09,152.51,152.04,149.87,149.69,147.94,147.35,147.29,144.84,143.41,143.20,142.15,138.90,128.20,127.50,126.39,126.01,125.20,124.26,123.96,120.57,119.87,110.56,108.66,92.87,89.43,62.94,62.41,61.37,59.65,55.06,54.05,41.63,40.49,39.58,38.68,34.89,27.76,27.51,24.85,22.55,14.04,10.24;HREIMS m/z955.4321[M]+(calcd forC46H61N13O6S2,955.4307).
2.实验方法
1)细胞培养
HEK293T，细胞在含10％胎牛血清的DMEM（Invitrogen）培养液中培养，37℃，CO2浓度5％。稳定分泌小鼠Wnt3a的L细胞株及对照株购自美国ATCC细胞库，该细胞在生长至大约70％密度时对其进行换液（含10％胎牛血清的DMEM），连续培养四天后，收集培养液并离心，经液氮速冻后-80℃长期保存。
2)细胞转染
HEK293T细胞在转染前24小时分盘，培养一天后进行转染。转染所用质粒转染试剂以24孔盘每孔的用量来计算：质粒总量为250ng/孔，如转染质粒量不足用lacZ质粒补足；质粒首先加入无血清的培养基（25μL/孔）中混匀，然后按照1μL/孔加入PLUS试剂（Invitrogen）混合，静置15分钟；按照1μL/孔的量将Lipofectamine（Invitrogen）脂质体加入无血清DMEM培养基（25μL/孔）中混匀，再与上述质粒和PLUS的混合溶液混合，静置15分钟；将细胞换入无血清DMEM培养基（200μL/孔），朝细胞中滴加入最终的质粒、PLUS和Lipofectamine的包装混合物，孵育3小时进行转染，过后用含10%胎牛血清的培养基中止转染反应。
3)报告基因测活
将用于测定报告基因活性的HEK293T细胞在转染前24小时分入24孔盘中，并以2）的方法进行转染。转染的各质粒量为，2.5ng/孔TOPFlash和2.5ng/孔作为内标的GFP质粒。余下用lacZ补齐250ng/孔。在转染后18小时加入加有小于等于20uM的小分子药物的Wnt3a条件培养基刺激6-8小时，用Boehringer Mannheim Luci-ferase Assay Kit裂解细胞（200μL/孔），用荧光计FL600（BIO-TEK Inc.Winooski，VT）测细胞裂解液中GFP蛋白的强度，作为细胞表达量的内标，然后用每孔20μL荧光素酶的底物，用Micro Lumate Plus（PerkinElmer Inc.Wellesley，MA）luminometer测定荧光素酶活性，活性越高表示Wnt报告基因表达量越高。最后用GFP的荧光量为内标修正荧光素酶的活性数据。同时以含有初筛药物的对照培养基进行平行试验作为对照，以及只加溶剂对照DMSO的Wnt3a条件培养基平行试验作为对照。
4)细胞内游离β-catenin检测
HEK293T细胞（6孔盘）假适当刺激后，弃去培养基，并置于冰上。用预冷的PBS收到EP管中，4℃3000rpm×5分钟离心后去掉PBS，用低渗缓冲液（10mMHEPES-KOH pH7.9，1.5mMMgCl2，1mM EDTA，10mM KCl，1mM焦磷酸，用前补加蛋白酶抑制剂，NaF和Na3VO4）120μL悬浮，冰上放置10分钟后用胰岛素针抽打6-8次。4℃3000rpm×5分钟离心，这一步离心得到的上清用于制备细胞浆组分，沉淀用于制备细胞核组分。细胞浆组分在100,000g4℃条件下超速离心1小时，最后超速离心得到的上清为细胞浆样品。细胞核组分用300μL低渗缓冲液洗涤，4℃3000rpm×5分钟离心，向沉淀中加入50μL高渗缓冲液（20mM HEPES pH7.9，420mM NaCl，0.2mM EDTA，25%甘油，1mM焦磷酸，用前补加蛋白酶抑制剂，NaF和Na3VO4），冰上放置30分钟后，100,000g4℃条件下超速离心1小时，最后超速离心得到的上清为核组分。经SDS-PAGE电泳后利用β-catenin特异性抗体进行Western Blot，检测细胞浆组分和核组分中的游离β-catenin。
5)Western Blot
配制合适浓度的SDS-PAGE胶，加入蛋白样品并进行电泳分离。经电转将蛋白质转到硝酸纤维膜上，硝酸纤维膜用封闭液（0.5%脱脂牛奶）封闭1小时，用TTBS洗涤3次，每次5分钟，再加对应的一抗（β-catenin、β-Actin或SP1的特异性抗体）室温孵育1小时（如果一抗为针对内源蛋白的抗体需要在4℃孵育过夜）。一抗处理过后用TTBS洗涤3次，每次5分钟，最后加入针对一抗物种的HRP二抗，室温孵育1小时，洗涤三次后，HRP偶联二抗免疫印迹的硝酸纤维素膜加入反应底物后用FujiFilm Las4000曝光扫描。
6)反转录PCR和实时定量PCR检测
利用TRIzol试剂（Invitrogen）将总RNA从细胞中抽提得到，再利用superscriptTMIII first strand sythesis system（Invitrogen）试剂盒将其反转录为cDNA。cDNA适当稀释后，使用Quantitative SYBR green PCR kit(Takara SYBR premix Ex Taq)试剂盒配制实时定量PCR反应体系。实时定量PCR使用的仪器是Rotor-gene RG-3000A apparatus(Corbett Research)。
用于Quantitative Real-Time PCR（qPCR）实验的相应基因的引物分别是：
human GAPDH:5’-GCACCACCAACTGCTTA-3’(SEQ ID NO:1)
5’-AGTAGAGGCAGGGATGA-3’(SEQ ID NO:2)；
human AXIN2:5’-AGTGTGAGGTCCACGGAAAC-3’(SEQ ID NO:3)
5’-CTTCACACTGCGATGCATTT-3’(SEQ ID NO:4)
human DKK1:5’-CTGCAAAAATGGAATATGTGT-3’(SEQ ID NO:5)
5’-CTTCTTGTCCTTTGGTGTGA-3’；(SEQ ID NO:6)
7)斑马鱼的品系和培养条件
Tüebingen strain的斑马鱼培养在28.5°C的水中。
8)斑马鱼的原位杂交实验
原位杂交是一种可以对组织内特定核酸进行定量定位的方法。在斑马鱼胚胎中通常所用的都是RNA探针，用来检测特定mRNA的表达。最常用的外缘标记物是地高辛（Digoxigenin，Roche），具体的原位杂交技术已经很成熟(Thisse,C.and B.Thisse,High-resolution in situ hybridization to whole-mount zebrafish embryos.Nat Protoc,2008.3(1):p.59-69.)。本实验中所用的探针是runx1和c-myb，它们都是标记斑马鱼造血干细胞的基因(North,T.E.,et al.,Prostaglandin E2regulates vertebratehaematopoietic stem cell homeostasis.Nature,2007.447(7147):p.1007-11.;Burns,C.E.,et al.,A genetic screen in zebrafish defines a hierarchical network ofpathways required for hematopoietic stem cell emergence.Blood,2009.113(23):p.5776-82.)。质粒PBSK-runx1和PBSK-cmyb分别被HindⅢ和BamhⅠ线性化，纯化模板后用T7体外转录酶（Ambion）合成相应的探针。原位杂交之后，所有的照片都是在室温下通过Olympus的DP71相机在Olympus的显微系统下拍摄（SZX16,10×或40×;Olympus）。
3.实验结果
1)化合物I能够以依赖于Wnt3a受体的形式激活经典Wnt信号途径的报告基因系统
发明人利用经典Wnt信号途径的报告基因系统筛选化合物。发现化合物I能够协同Wnt3a剂量依赖性的激活经典Wnt信号途径的报告基因系统（图1）；而在没有Wnt3a受体存在的情况下不影响经典Wnt信号途径的报告基因系统（图1）。
2)化合物I能够以依赖于Wnt3a受体的形式激活经典Wnt信号途径的内源靶基因的表达
为了进一步确认化合物I能够激活经典Wnt信号途径，发明人通过实时定量PCR检测经典Wnt信号途径的表达情况，发现化合物I能够协同Wnt3a剂量依赖性的激活经典Wnt信号途径的内源靶基因（AXIN2和DKK1）的表达（图2）；而在没有Wnt3a受体存在的情况下不影响经典Wnt信号途径的内源靶基因的表达（图2）。
3)化合物I能够以依赖于Wnt3a受体的形式提高细胞质和细胞核内的β-catenin积累
发明人通过细胞核和细胞质的分离实验发现了，化合物I确实能够提高经典Wnt信号途径的传递分子β-catenin在细胞质和细胞核内的积累，并且这种提高时依赖于Wnt3a受体（图3）。
4)化合物I、化合物II能够以依赖于Wnt3a受体的形式激活经典Wnt信号途径的报告基因系统
发明人利用经典Wnt信号途径的报告基因系统。发现化合物II能够协同Wnt3a剂量依赖性的激活经典Wnt信号途径的报告基因系统（图4）；而在没有Wnt3a受体存在的情况下不影响经典Wnt信号途径的报告基因系统（图4）。
5)化合物I能够提高斑马鱼中造血干细胞的形成
为了验证我们筛选到的化合物的确能够在生物体内起作用，我们选取了斑马鱼的造血干细胞系统。已有文献报道提高经典Wnt信号能够促进造血干细胞的形成。在我们的研究中，我们的确看到了化合物I能够显著性提高造血干细胞的标记物runx1/cmyb[Goessling,W.,et al.,Genetic interaction of PGE2and Wnt signaling regulatesdevelopmental specification of stem cells and regeneration.Cell,2009.136(6):p.1136-47]，也就是化合物I能够显著性提高造血干细胞的数量（图5）。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
Claims (9)
1.一种化合物，其结构式为：
2.化合物II的制备方法，其制备路线为：
具体包括下列步骤：
1)以石蒜碱为起始原料经N-甲基化与霍夫曼降解制备中间体III并分离产物；
2)使中间体III氢化还原获得化合物I并分离产物；
3)化合物I通过二氧亚甲基降解制备中间体IV并分离产物；
4)使中间体IV与溴丙炔经烷基化反应制备中间体V并分离产物；
5)将中间体V与化合物VI通过Click反应制备产物化合物II。
3.如权利要求2所述化合物II的制备方法，其特征在于，所述化合物VI的制备路线为：
4.如权利要求3所述化合物II的制备方法，其特征在于，其中，化合物VIII的制备路线为：
5.如权利要求1所述化合物在制备经典Wnt信号途径激动剂中的用途。
6.如权利要求5所述的用途，其特征在于，所述经典Wnt信号途径激动剂能激活经典Wnt信号途径的报告基因或目的基因的表达活性。
7.如权利要求1所述化合物在制备药物中的用途，所述药物选自以下任一：
1)预防或治疗由经典Wnt信号途径异常失活所引起的疾病或失调的药物；
2)需要特异性激活经典Wnt信号途径的疾病或失调的药物；
3)干细胞数目扩增药物。
8.如权利要求7所述化合物的用途，其特征在于，所述药物选自：老年痴呆症药物、风湿性关节炎药物、骨质疏松症药物、造血干细胞移植药物、干细胞数目扩增药物或斑马鱼发育调控药物。
9.一种用于预防或治疗由经典Wnt信号途径异常失活引起的或用于需要特异性激活经典Wnt信号途径的疾病或失调的药物组合物，含有治疗有效量的权利要求1所述化合物以及在药学上可接受的赋形剂。
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| EP1267616B1 (en) * | 2000-03-31 | 2007-08-08 | The General Hospital Corporation | Methods of increasing hair growth by wnt polypeptide |
| CN103145617A (zh) * | 2013-03-01 | 2013-06-12 | 中国科学院昆明植物研究所 | 菲啶类衍生物及其药物组合物和其制备方法与应用 |
-
2013
- 2013-03-07 CN CN201510896988.9A patent/CN105343073B/zh active Active
- 2013-03-07 CN CN201310073332.8A patent/CN104031055B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1267616B1 (en) * | 2000-03-31 | 2007-08-08 | The General Hospital Corporation | Methods of increasing hair growth by wnt polypeptide |
| CN103145617A (zh) * | 2013-03-01 | 2013-06-12 | 中国科学院昆明植物研究所 | 菲啶类衍生物及其药物组合物和其制备方法与应用 |
Non-Patent Citations (3)
| Title |
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| 4-Aryl-4-oxo-N-phenyl-2-aminylbutyramides as acetyl- and butyrylcholinesterase inhibitors. Preparation, anticholinesterase activity, docking study, and 3D structure–activity relationship based on molecular interaction fields;Maja D. Vitorovic-Todorovic，等;《Bioorganic & Medicinal Chemistry》;20091222;第18卷(第3期);第1181-1193页 * |
| Preparation of secolycorines against acetylcholinesterase;Shoei-Sheng Lee，等;《Bioorganic & Medicinal Chemistry》;20061018;第15卷(第2期);第1034-1043页 * |
| Synthesis and antiplasmodial activity of lycorine derivatives;Juan C. Cedrón，等;《Bioorganic & Medicinal Chemistry》;20100511;第18卷(第13期);第4694-4701页 * |
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| Publication number | Publication date |
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| CN104031055A (zh) | 2014-09-10 |
| CN105343073A (zh) | 2016-02-24 |
| CN105343073B (zh) | 2018-05-04 |
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