CN116143843A - 一种具有fto抑制活性的金属配合物及其制法和应用 - Google Patents
一种具有fto抑制活性的金属配合物及其制法和应用 Download PDFInfo
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- CN116143843A CN116143843A CN202310142825.6A CN202310142825A CN116143843A CN 116143843 A CN116143843 A CN 116143843A CN 202310142825 A CN202310142825 A CN 202310142825A CN 116143843 A CN116143843 A CN 116143843A
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- metal complex
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- metal
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- 229940055695 pancreatin Drugs 0.000 description 1
- WQIQNKQYEUMPBM-UHFFFAOYSA-N pentamethylcyclopentadiene Chemical compound CC1C(C)=C(C)C(C)=C1C WQIQNKQYEUMPBM-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了一种具有FTO抑制活性的金属配合物,还公开了上述具有FTO抑制活性的金属配合物的制备方法和应用。本发明金属配合物将采用炔基修饰的FTO抑制剂与含叠氮基团的金属配合物前体通过CuAAC反应得到,金属配合物具有良好的抗癌活性和FTO抑制活性,一方面能够克服单独FTO抑制剂存在的靶标选择性差的问题,另一方面能够克服单纯的金属配合物(前体)作为抗肿瘤药物或抗肿瘤药物组分时存在毒副作用大的问题。
Description
技术领域
本发明涉及一种具有FTO抑制活性的金属配合物,还涉及上述金属配合物的制备方法和应用。
技术背景
癌症是目前威胁人类生命最严重的疾病之一。目前临床使用的多数铂类及其它药物(如顺铂、卡铂、奥沙利铂等)都具有毒副作用大、选择性差以及耐药性等缺点。随着对抗癌药物在肿瘤细胞中作用机制的深入研究,近年来已出现不少具有广阔应用前景的金属抗肿瘤药物。目前,芳基钌、铱、铑及锇等金属配合物因其具有高活性,选择性,多靶点,低耐药性等优点,被认为是最有前景的金属抗癌药物。目前国内外很多专家如英国华威大学的Peter Sadler教授、瑞士洛桑联邦理工学院的Paul Dyson教授以及奥地利维也纳大学的Bernhard Keppler教授等人在这方面都有深入研究。
2007年英国科学家在与Ⅱ型糖尿病相关的单核苷酸多态性(SNP)研究中发现了和肥胖相关的FTO基因(Fat mass and obesity-associated protein),FTO基因编码蛋白是AlkB家族中的一员,是依赖于二价铁Fe2+与2-氧戊二酸(2OG)的加氧酶,具有催化核苷酸的去甲基化作用。相关研究表明FTO在急性髓性白血病的发生过程中发挥了癌基因的作用,并清楚揭示出FTO介导的mRNA上的m6A修饰参与了肿瘤的发生。2012年上海药物所的研究人员筛选出大黄酸等3个化合物能抑制FTO的活性,其中大黄酸的抑制效果最好,更适合作为候选药物分子。2014年美国科学家在研究抗癫痫药物时意外发现所得到的化学物不能有效抑制靶点蛋白PHD2,却能有效抑制其2OG加氧酶家族的FTO,而且IC50值和广谱性2OG酶抑制剂NOG接近,强于之前报道的大黄酸。但目前报道的FTO抑制剂大部分由于靶标选择性差或药代动力学差而不适合临床应用。
发明内容
发明目的:本发明目的旨在提供一种具有FTO抑制活性的金属配合物,该金属配合物将采用炔基修饰的FTO抑制剂与含叠氮基团的金属配合物前体通过CuAAC反应得到,金属配合物具有良好的抗癌活性和FTO抑制活性,一方面能够克服单独FTO抑制剂存在的靶标选择性差的问题,另一方面能够克服单纯的金属配合物(前体)作为抗肿瘤药物或抗肿瘤药物组分时存在毒副作用大的问题。
技术方案:本发明所述的具有FTO抑制活性的金属配合物,所述配合物的结构通式如下所示:
上述具有FTO抑制活性的金属配合物的制备方法,包括如下步骤:
(1)制备含叠氮基团的金属配合物前体:在惰性气氛下,将金属钌的二聚体、金属铱的二聚体、金属铑的二聚体或金属锇的二聚体溶解在甲醇中,与螯合配体进行配位反应,反应后减压旋蒸除去溶剂,加入饱和阴离子盐的甲醇溶液,置于低温下冷冻,冷冻后离心收集固体,通过柱色谱分离对粗产物提纯,得到金属配合物前体;
(2)制备炔基修饰的有机活性分子配体:在惰性气氛下,将有机活性分子、炔基化合物和催化剂置于有机溶剂中反应,反应结束后,旋蒸除去溶剂,得到粗产物经过柱层析纯化后,得到有机活性分子配体;
(3)在惰性气氛下,将金属配合物前体、有机活性分子配体和催化剂在有机溶剂中进行CuAAC反应,反应结束后,旋蒸除去溶剂,粗产物经色谱柱纯化,得到金属配合物。
其中,步骤(1)中,所述金属钌的二聚体为对伞花烃二氯化钌(Ⅱ)二聚体(购于上海麦克林生化科技股份有限公司,CAS:52462-29-0),金属铱的二聚体为二氯(五甲基环戊二烯基)合铱(Ⅲ)二聚体(购于上海麦克林生化科技股份有限公司,CAS:12354-84-6),金属铑的二聚体为二氯(五甲基环戊二烯基)合铑(Ⅲ)二聚体(购于上海麦克林生化科技股份有限公司,CAS:12354-85-7),金属锇的二聚体为对伞花烃二氯化锇(Ⅱ)二聚体(购于aablocks公司,CAS:78615-08-4)。
其中,步骤(1)中,金属钌的二聚体与螯合配体的摩尔比为8~9:1;金属铱的二聚体与螯合配体的摩尔比为13~14:1;金属铑的二聚体与螯合配体的摩尔比为12~13:1;金属锇的二聚体与螯合配体的摩尔比为12~13:1。
其中,步骤(1)中,所述螯合配体为4-叠氮甲基-4'-甲基-2,2'-联吡啶,4-叠氮甲基-4'-甲基-2,2'-联吡啶采用文献“Artificial photosynthesis dendrimersintegrating light-harvesting,electron delivery and hydrogen production”中所报道的方法制备而成。
其中,步骤(1)中,冷冻温度为-20~0℃,冷冻时间为8~14h。
其中,步骤(2)中,所述炔基化合物为溴丙炔、炔丙胺或戊炔酸;催化剂为碳酸钾或催化剂为N-羟基苯并三唑和N,N-二异丙基乙胺按摩尔比82~83:135的组合物。
其中,步骤(2)中,有机活性分子与炔基化合物的摩尔比为0.1~4:1。
其中,步骤(3)中,催化剂为五水硫酸铜和抗坏血酸钠按摩尔比3~3.5:4的组合物。
其中,步骤(3)中,金属配合物前体与有机活性分子配体的反应摩尔比为3~25.5:1。
上述具有FTO抑制活性的金属配合物在用于制备抗肿瘤药物、抗肿瘤药物组分、FTO抑制药物和FTO抑制药物组分方面的应用。
本发明金属配合物合成过程的反应机理为:
其中:M为Ru、Ir、Rh或Os;
Y为有机活性分子配体中的炔烃基团。
本发明芳基/茂基金属配合物是以具有抗FTO活性的分子作为炔端,以芳基/茂基金属作为叠氮端,通过CuAAC反应得到,本发明芳基/茂基金属配合物具有良好的脂溶性,可以提高癌细胞产生的活性氧,诱导线粒体膜电位的产生,阻滞细胞周期。
有益效果:本发明将具有抗肿瘤、抗菌、抗炎功效的FTO抑制剂进行特定基团修饰并与茂基、芳基金属配位,从而获得具有良好抑制肥胖基因FTO活性、抗癌活性、抗菌活性、抗炎活性的配合物,因此本发明金属配合物具有靶向作用的抗癌、抗菌、抗炎及抑制FTO去甲基化酶活性。
附图说明
图1为配合物FTO-2-Ru/Ir/Rh在细胞中的细胞定位图;
图2为配合物FTO-2-Ru/Ir/Rh以及有机活性分子配体与有机活性分子诱导细胞凋亡图;
图3为配合物FTO-2-Ru/Ir/Rh以及有机活性分子配体与有机活性分子促进细胞内活性氧生成的共聚焦荧光成像图和流式细胞图;
图4为配合物FTO-2-Ru/Ir/Rh以及有机活性分子配体与有机活性分子诱导线粒体膜电位下降的流式细胞图和共聚焦荧光成像图;
图5为配合物FTO-2-Ru/Ir/Rh以及有机活性分子配体与有机活性分子对细胞周期阻滞影响图。
图6为配合物FTO-2-Ru/Ir/Rh以及有机活性分子配体与有机活性分子对FTO活性抑制的示意图。
具体实施方式
以下结合具体实施例对本发明的技术方案做进一步说明。
实施例1
本发明金属配合物FTO-2-Ru的制备方法,包括以下步骤:
步骤1,将6-(5-甲基吡嗪-2-基)萘-2-醇(FTO-2)(0.13g,0.5mmol)、溴丙炔(0.53g,4.5mmol)和碳酸钾(2.05g,15.2mmol)溶解于DMF(N,N-二甲基甲酰胺)中,于80℃下搅拌12h,加水,乙酸乙酯萃取,无水硫酸钠干燥,旋蒸除去溶剂得到粗产物,粗产物通过柱层析方法分离纯化,得到有机化合物FTO-2-alkyne,产率为85%。1H NMR(400MHz,DMSO-d6)δ9.06(s,2H),8.25(d,J=1.9Hz,1H),7.93(dd,J=8.8,6.7Hz,2H),7.86(dd,J=8.6,1.9Hz,1H),7.45(d,J=2.6Hz,1H),7.26(dd,J=9.0,2.6Hz,1H),4.95(d,J=2.4Hz,2H),3.99(s,3H),3.64(t,J=2.4Hz,1H).
上述FTO-2采用文献“Broad spectrum anti-cancer compounds for treatmentof cancer”中所报道的合成方法制备得到。
步骤2,在常温下,将桥联配体4-叠氮甲基-4'-甲基-2,2'-联吡啶(0.9g,4mmol)与对伞花烃二氯化钌(Ⅱ)二聚体(1.96g,3.2mmol)在甲醇溶液中反应48h,旋蒸除去溶剂,加入饱和的(NH4)PF6的甲醇溶液,置于冰箱中于-20℃下冷冻过夜,冷冻后离心收集固体,通过柱色谱分离技术对粗产物提纯,得到纯品,产率为85%,命名为Ru-N3。1H NMR(400MHz,DMSO-d6)δ9.50(d,J=5.8Hz,1H),9.36(d,J=5.8Hz,1H),8.60–8.57(m,2H),7.74(dd,J=6.0,1.7Hz,1H),7.67–7.63(m,1H),6.22–6.19(m,2H),5.98–5.94(m,2H),4.88(s,2H),2.58(s,3H),2.57–2.52(m,1H),2.18(s,3H),0.94(dd,J=7.0,2.0Hz,6H).
上述桥联配体4-叠氮甲基-4'-甲基-2,2'-联吡啶采用文献“Artificialphotosynthesis dendrimers integrating light-harvesting,electron delivery andhydrogen production”中所报道的合成方法制备得到,具体为:
取4,4'-二甲基-2,2'-联吡啶(8.0g,4.3mmol)和1,4-二氧六环(50~250mL)配置悬浮液,再加入SeO2(8.0g,7.1mmol)加热回流48h,过滤得滤液,滤液减压除去溶剂得固体物质;将固体物质溶解在氯仿中,过滤,将滤液减压除去得到固体物质,重复三次以上步骤最终得到粗产物;将硼氢化钠(2.8g)溶解于氢氧化钠溶液(0.2mol,20mL)中,滴加至溶解有粗产物的甲醇悬浮液(50mL)中,冷却搅拌1h后减压除去甲醇,再加入80mL的饱和Na2CO3溶液稀释,萃取干燥有机相,再蒸发溶剂,通过色谱柱法纯化以得到4-羟甲基-4'-甲基-2,2'-联吡啶;将4-羟甲基-4'-甲基-2,2'-联吡啶(3.2g,5.3mmol)溶解在HBr(48%,40mL)中,再加入浓硫酸(10mL)加热回流过夜,冷却后调节pH至8,用氯仿萃取直至有机层无色,取有机层干燥除去氯仿,得到4-溴甲基-4'-甲基-2,2'-联吡啶;将4-溴甲基-4'-甲基-2,2'-联吡啶(2.8g,10.7mmol)和NaN3(1.2g,18.5mmol)溶解在N,N-二甲基甲酰胺的水溶液(55mL,v/v=10:1)中70℃搅拌12h,除去溶剂后得粗产物,用CH2Cl2萃取,萃取得到的有机层用水洗涤,干燥后除去溶剂,通过色谱柱法纯化得到4-叠氮甲基-4'-甲基-2,2'-联吡啶。1H NMR(400MHz,DMSO-d6)δ8.68(dd,J=4.9,0.8Hz,1H),8.55(dd,J=5.0,0.8Hz,1H),8.38(dd,J=1.7,0.9Hz,1H),8.26(dt,J=1.7,0.9Hz,1H),7.44–7.41(m,1H),7.31(ddd,J=5.0,1.8,0.8Hz,1H),4.70(s,2H),2.42(d,J=0.7Hz,3H).
步骤3,在氩气氛围下,将步骤1的FTO-2-alkyne(0.52g,1.8mmol)、Ru-N3(1.49g,3mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物FTO-2-Ru,产率为83.3%。1HNMR(400MHz,DMSO-d6)δ9.50(d,J=5.9Hz,1H),9.37(d,J=5.8Hz,1H),9.06(s,2H),8.64(s,1H),8.54(s,1H),8.48(s,1H),8.25(s,1H),7.97–7.90(m,2H),7.89–7.83(m,1H),7.66(d,J=5.9Hz,1H),7.58(d,J=2.5Hz,1H),7.49(d,J=5.9Hz,1H),7.26(dd,J=8.9,2.5Hz,1H),6.19(dd,J=14.8,6.1Hz,2H),6.00–5.91(m,4H),5.35(s,2H),3.99(s,3H),2.58(s,3H),2.55(s,1H),2.16(s,3H),0.94(t,J=7.0Hz,6H).
实施例2
本发明金属配合物FTO-2-Ir的制备方法,包括以下步骤:
步骤1,在常温下,将桥联配体4-叠氮甲基-4'-甲基-2,2'-联吡啶(0.9g,4mmol)与二氯(五甲基环戊二烯基)合铱(Ⅲ)二聚体(2.54g,3.2mmol)在甲醇溶液中反应10h,旋蒸除去溶剂,加入饱和的(NH4)PF6的甲醇溶液,置于冰箱中于-20℃下冷冻过夜,冷冻后离心收集固体,通过柱色谱分离技术对粗产物提纯,得到纯品,产率为87.6%,命名为Ir-N3。1H NMR(400MHz,DMSO-d6)δ8.95(d,J=5.8Hz,1H),8.81(d,J=5.8Hz,1H),8.72(t,J=2.3Hz,2H),7.79(dd,J=6.0,1.7Hz,1H),7.70(dd,J=6.0,1.7Hz,1H),4.94(s,2H),2.64(s,3H),1.65(s,15H).
步骤2,在氩气氛围下,将实施例1步骤1的FTO-2-alkyne(0.52g,1.8mmol)、Ir-N3(2.17g,3.0mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物FTO-2-Ir,产率为85.3%。1H NMR(400MHz,DMSO-d6)δ9.06(s,2H),8.95(d,J=5.9Hz,1H),8.84–8.78(m,2H),8.62(s,1H),8.55(s,1H),8.25(d,J=1.8Hz,1H),7.96–7.91(m,2H),7.86(dd,J=8.6,1.9Hz,1H),7.71(d,J=5.8Hz,1H),7.59(d,J=2.6Hz,1H),7.49(dd,J=5.9,1.8Hz,1H),7.27(dd,J=8.9,2.5Hz,1H),6.00(s,2H),5.36(s,2H),3.99(s,3H),2.64(s,3H),1.64(s,15H).
实施例3
本发明金属配合物FTO-2-Rh的制备方法,包括以下步骤:
步骤1,在常温下,将桥联配体4-叠氮甲基-4'-甲基-2,2'-联吡啶(0.9g,4mmol)与二氯(五甲基环戊二烯基)合铑(Ⅲ)二聚体(2.26g,3.2mmol)在甲醇溶液中反应10h,旋蒸除去溶剂,加入饱和的(NH4)PF6的甲醇溶液,置于冰箱中于-20℃下冷冻过夜,冷冻后离心收集固体,通过柱色谱分离技术对粗产物提纯,得到纯品,产率为78.5%,命名为Rh-N3。1H NMR(400MHz,DMSO-d6)δ8.95(d,J=5.7Hz,1H),8.81(d,J=5.7Hz,1H),8.62(t,J=1.9Hz,2H),7.82(dd,J=5.8,1.7Hz,1H),7.75–7.69(m,1H),4.89(s,2H),2.59(s,3H),1.66(s,15H).
步骤2,在氩气氛围下,将实施例1步骤1的FTO-2-alkyne(0.52g,1.8mmol)、Rh-N3(1.91g,3mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物FTO-2-Rh,产率为86.5%。1H NMR(400MHz,DMSO-d6)δ9.06(s,2H),8.95(d,J=5.8Hz,1H),8.82(d,J=5.7Hz,1H),8.72(s,1H),8.54(d,J=4.8Hz,2H),8.25(s,1H),7.98–7.91(m,2H),7.88–7.84(m,1H),7.73(d,J=5.8Hz,1H),7.59(d,J=2.5Hz,1H),7.52(d,J=5.8Hz,1H),7.27(dd,J=8.9,2.4Hz,1H),5.96(s,2H),5.36(s,2H),3.99(d,J=1.1Hz,3H),2.59(s,3H),1.65(s,15H).
实施例4
本发明金属配合物FTO-2-Os的制备方法,包括以下步骤:
步骤1,在常温下,将桥联配体4-叠氮甲基-4'-甲基-2,2'-联吡啶(0.9g,.4mmol)与对伞花烃二氯化锇(Ⅱ)二聚体(2.25g,3.2mmol)在甲醇溶液中反应10h,旋蒸除去溶剂,加入饱和的(NH4)PF6的甲醇溶液,置于冰箱中于-20℃下冷冻过夜,冷冻后离心收集固体,通过柱色谱分离技术对粗产物提纯,得到纯品,产率为86.3%,命名为Os-N3。1H NMR(400MHz,CD3OD)δ9.21(d,J=5.9Hz,1H),9.02(d,J=5.8Hz,1H),8.33(d,J=19.1Hz,2H),7.52(ddd,J=44.9,5.9,1.4Hz,2H),6.31-6.15(m,2H),5.74-5.59(m,2H),4.81(s,2H),2.58(m,4H),2.23(s,3H),1.11(dd,J=6.9,1.0Hz,6H).
步骤2,在氩气氛围下,将实施例1步骤1的FTO-2-alkyne(0.52g,1.8mmol)、Os-N3(1.76g,3mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物FTO-2-Os,产率为84.2%。1H NMR(400MHz,DMSO-d6)δ9.52(d,J=5.9Hz,1H),9.38(d,J=5.8Hz,1H),9.07(s,2H),8.63(s,1H),8.52(s,1H),8.46(s,1H),8.26(s,1H),7.97–7.91(m,2H),7.89–7.85(m,1H),7.63(d,J=5.9Hz,1H),7.57(d,J=2.5Hz,1H),7.45(d,J=5.9Hz,1H),7.26(dd,J=8.9,2.5Hz,1H),6.18(dd,J=14.8,6.1Hz,2H),6.02–5.90(m,4H),5.32(s,2H),3.95(s,3H),2.59(s,3H),2.56(s,1H),2.26(s,3H),1.14(t,J=7.0Hz,6H).
实施例5
本发明金属配合物FTO-4-Ru的制备方法,包括以下步骤:
步骤1,在氩气氛围下,将戊炔酸(29.4mg,0.3mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(57.5mg,0.3mmol)和三乙胺(50μL)溶解在DMF中,冰浴条件下反应30min,将N-羟基苯并三唑(32.4mg,0.24mmol)溶解于DMF中并滴加到上述混合物中,于冰浴条件下反应15min,再向混合溶液中滴加6-(5-甲氧基吡啶-3-基)苯并噻唑-2-胺(FTO-4)(129.2mg,0.5mmol),使反应物料升温至室温,搅拌过夜后,除去有机溶剂,经过柱层析纯化得到白色固体FTO-4-alkyne,产率65.6%。1H NMR(400MHz,DMSO-d6)δ8.91(s,2H),8.09(d,J=1.8Hz,1H),7.61(dd,J=8.3,1.8Hz,1H),7.45(d,J=8.3Hz,1H),3.96(s,3H),2.35=4(td,J=7.1,2.7Hz,4H),2.05(s,1H).
上述FTO-4采用文献“Broad spectrum anti-cancer compounds for treatmentof cancer”中所报道的合成方法制备得到。
步骤2,在氩气氛围下,将步骤1制得的FTO-4-alkyne(0.61g,1.8mmol)、Ru-N3(1.49g,3mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物FTO-4-Ru,产率为75.2%。1H NMR(400MHz,DMSO-d6)δ9.50(d,J=5.9Hz,1H),9.08(s,2H),8.91(s,2H),8.63(s,1H),8.52(s,1H),8.23(s,1H),8.09(d,J=1.8Hz,1H),8.02(dd,J=8.0,1.7Hz,1H),7.98–7.90(m,2H),7.89–7.2(m,1H),7.64(d,J=5.9Hz,1H),7.61(dd,J=8.3,1.8Hz,1H),7.45(d,J=8.3Hz,1H),7.42(d,J=8.3Hz,1H),7.31–7.24(m,2H),7.18(d,J=2.1Hz,2H),6.94(s,1H),6.61(d,J=2.4Hz,1H),6.78(ddd,J=8.2,7.1,1.1Hz,1H),6.00–5.93(m,4H),5.34(s,2H),3.96(s,3H),2.78(td,J=7.1,2.7Hz,4H),2.54(s,1H),2.43(d,J=0.7Hz,3H),2.35=4(td,J=7.1,2.7Hz,4H),2.13(s,3H),2.05(s,1H),0.95(t,J=7.0Hz,6H).
实施例6
本发明金属配合物FL1-Ru的制备方法,包括以下步骤:
步骤1,在氩气氛围下,在氩气氛围下,将戊炔酸(29.4mg,0.3mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(57.5mg,0.3mmol)和三乙胺(50μL)溶解在DMF中,冰浴条件下反应30min,将N-羟基苯并三唑(32.4mg,0.24mmol)溶解于DMF中并滴加到上述混合物中,于冰浴条件下反应15min,再向混合物中加入5-氨基荧光素(FL1)(0.42g,1.2mmol),使反应物料升温至室温,搅拌过夜后,除去有机溶剂,经过柱层析纯化,得到白色固体FL1-alkyne,产率69.5%。1H NMR(400MHz,Methanol-d4)δ7.16(d,J=2.1Hz,2H),7.10(d,J=2.2Hz,1H),7.08(d,J=2.2Hz,1H),6.93(s,1H),6.91(s,1H),6.67(s,1H),6.61(d,J=2.4Hz,1H),6.58(d,J=2.4Hz,1H),2.63(td,J=7.1,2.7Hz,4H),2.05(s,1H).
步骤2,在氩气氛围下,将步骤1制得的FL1-alkyne(0.77g,1.8mmol)、Ru-N3(1.49g,3mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物FL1-Ru,产率为85.2%。1H NMR(400MHz,DMSO-d6)δ9.50(d,J=5.9Hz,1H),9.08(s,2H),8.63(s,1H),8.52(s,1H),8.23(s,1H),8.02(dd,J=8.0,1.7Hz,1H),7.98–7.90(m,2H),7.89–7.2(m,1H),7.64(d,J=5.9Hz,1H),7.42(d,J=8.3Hz,1H),7.31–7.24(m,2H),7.18(d,J=2.1Hz,2H),6.94(s,1H),6.61(d,J=2.4Hz,1H),6.78(ddd,J=8.2,7.1,1.1Hz,1H),6.00–5.93(m,4H),5.34(s,2H),2.78(td,J=7.1,2.7Hz,4H),2.54(s,1H),2.43(d,J=0.7Hz,3H),2.13(s,3H),0.96(t,J=7.0Hz,6H).
实施例7
本发明金属配合物FL2-Ru的制备方法,包括以下步骤:
步骤1,在氩气氛围下,将2',7'-二氯荧光素(FL2)(0.48g,1.2mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(57.5mg,0.3mmol)和三乙胺(50μL)溶解在DMF中,冰浴条件下反应30min,将N-羟基苯并三唑(32.4mg,0.24mmol)溶解于DMF中并滴加到上述混合物中,于冰浴条件下反应15min,再向混合物中滴加炔丙胺(82.6mg,1.5mmol),使反应物料升温至室温过夜,搅拌过夜后,除去有机溶剂,经过柱层析纯化,得到白色固体FL2-alkyne,产率77.8%。1H NMR(400MHz,DMSO-d6)δ11.38(s,2H),8.04–8.00(m,1H),7.83(td,J=7.5,1.2Hz,1H),7.76(td,J=7.5,1.0Hz,1H),7.35(d,J=7.7Hz,1H),7.07(s,2H),6.63(s,2H),4.88(s,2H),2.05(s,1H).
步骤2,在氩气氛围下,将步骤1制得的FL2-alkyne(0.79g,1.8mmol)、Ru-N3(1.49g,3mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物FL2-Ru,产率78.6%。1H NMR(400MHz,DMSO-d6)δ11.38(s,2H),9.50(d,J=5.9Hz,1H),8.63(s,1H),8.09(d,J=1.8Hz,1H),8.04–8.00(m,1H),7.98–7.90(m,2H),7.64(d,J=5.9Hz,1H),7.61(dd,J=8.3,1.8Hz,1H),7.31–7.24(m,2H),7.07(s,2H),6.00–5.93(m,4H),4.88(s,2H),2.78(td,J=7.1,2.7Hz,4H),2.54(s,1H),2.43(d,J=0.7Hz,3H),2.13(s,3H),2.05(s,1H),0.95(t,J=7.0Hz,6H).
实施例8
本发明金属配合物MA-Ru的制备方法,包括以下步骤:
步骤1,在氩气氛围下,将甲氯芬那酸(MA)(0.36g,1.2mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(57.5mg,0.3mmol)和三乙胺(50μL)溶解在DMF中,冰浴条件下反应30min,将N-羟基苯并三唑(32.4mg,0.24mmol)溶解于DMF中并滴加到上述混合物中,于冰浴条件下反应15min,再向混合溶液中滴加炔丙胺(82.6mg,1.5mmol),使反应物料升温至室温过夜,搅拌过夜后,除去有机溶剂,经过柱层析纯化,得到白色固体MA-alkyne,产率72.5%。1H NMR(400MHz,Methanol-d4)δ8.00(dd,J=8.0,1.7Hz,1H),7.41(d,J=8.3Hz,1H),7.31–7.23(m,2H),6.76(ddd,J=8.2,7.1,1.1Hz,1H),6.25(dd,J=8.4,1.0Hz,1H),4.88(s,2H),3.08(s,1H),2.43(d,J=0.7Hz,3H).
步骤2,在氩气氛围下,将步骤1制得的MA-alkyne(0.60g,1.8mmol)、Ru-N3(1.49g,3mmol)、五水硫酸铜(0.34g,1.50mmol)和抗坏血酸钠(0.40g,2.0mmol)溶解在DMF中搅拌10h,反应结束后,旋蒸除去溶剂,柱层析分离得到橙黄色产物MA-Ru,产率80.2%。1H NMR(400MHz,DMSO-d6))δ9.50(d,J=5.9Hz,1H),9.37(d,J=5.8Hz,1H),9.06(s,2H),8.64(s,1H),8.54(s,1H),8.48(s,1H),8.25(s,1H),8.00(dd,J=8.0,1.7Hz,1H),7.97–7.90(m,2H),7.89–7.83(m,1H),7.66(d,J=5.9Hz,1H),7.41(d,J=8.3Hz,1H),7.31–7.23(m,2H),6.76(ddd,J=8.2,7.1,1.1Hz,1H),6.25(dd,J=8.4,1.0Hz,1H),6.00–5.91(m,4H),5.35(s,2H),4.88(s,2H),3.08(s,1H),2.43(d,J=0.7Hz,3H),2.16(s,3H),0.95(t,J=7.0Hz,6H).
本发明金属配合物用于肿瘤细胞治疗方面的应用。
方法:MTT比色法、CCK-8比色法。本实验测定对人源癌细胞系如人乳腺癌细胞(MCF-7),小鼠来源的脑胶质瘤细胞(A261),人慢性髓系白血病细胞(K562),人急性早幼粒细胞白血病细胞(NB-4)和人正常肝细胞(LO2)在体外的抗癌活性。MCF-7,MDA-MB-231和LO2细胞在含有10%胎牛血清、1%青霉素-链霉素溶液的DMEM培养基中,K562,NB-4细胞在含有10%胎牛血清、1%青霉素-链霉素溶液的1640培养基中,于37℃,5%CO2细胞培养箱中培养。将细胞按照5000细胞/孔的初始密度接种到96孔细胞培养板中,培养24h后,加入不同浓度梯度的药物培养基,继续培养48h后,铺有MCF-7,A261和LO2细胞的孔板,每个孔中加入20μL的MTT水溶液(5mg/mL),继续孵育4h,移除培养基,加入150μL的DMSO,震荡至紫色结晶完全溶解,使用酶标仪(型号为Tecan Infinite M1000 Pro)晃动1分钟并读取490nm的吸光度。而铺有K562和NB-4细胞孔板,每个孔板中加入10μL CCK-8,继续孵育3h,直接使用酶标仪(型号为Tecan Infinite M1000 Pro)晃动1分钟并读取450nm的吸光度。
实施例1~3制得的FTO-2钌/铱/铑配合物的抗癌活性如表1所示。
表1为配合物FTO-2-Ru、FTO-2-Ir、FTO-2-Rh、FTO-2、FTO-2-alkyne及顺铂(CDDP)的IC50(μM)值
结果表明:有机活性分子FTO-2以及FTO-2-alkyne对几种细胞几乎没有活性(IC50>100μM),FTO-2-Ru对几种细胞表现出低毒性,但对NB-4细胞表现出极好的选择性,FTO-2-Ir和FTO-2-Rh对贴壁细胞表现出低毒性,但对白血病细胞具有很好的选择性,尤其是NB-4细胞,同时,对人正常肝细胞(LO2)活性很差,这表明基于FTO-2的芳基、茂基金属配合物对白血病细胞有很高的选择性。因此,FTO-2-Ru、FTO-2-Ir、FTO-2-Rh均有助于降低药物的副作用,且具有较好的抗癌活性。本发明配合物对白血病具有一定的选择性,可实现对白血病细胞的靶向性治疗。
实施例9
本发明实施例1~3制得的FTO-2-Ru/Ir/Rh配合物在细胞中的定位。
方法:将NB-4细胞接种到10cm培养皿中,培养24h后,分别加入含有FTO-2-Ru(3μM)、FTO-2-Ir(5μM)和FTO-2-Rh(5μM)的1640培养液继续培养12h。收集细胞并用PBS(4℃)洗涤2次。使用线粒体/细胞核制备试剂盒取出细胞核、线粒体和细胞质,然后每个样品中依次用浓硝酸(100μL,95℃)消化2h,过氧化氢(50μL,95℃)消化1h和浓盐酸(100μL,95℃)消化2h。冷却后用超纯水将所得液体稀释至2mL,通过ICP-MS测定样品中金属的含量,再用各个样品中的细胞数对细胞内金属含量进行归一化。
实施例1的FTO-2-Ru配合物细胞内定位如图1A所示,将配合物FTO-2-Ru(3μM)与NB-4细胞共孵育12h后,提取细胞器,通过电感耦合等离子质谱ICP-MS测定不同细胞器中样品中金属Ru的含量,图中数据说明了配合物FTO-2-Ru主要分布在细胞核和线粒体中,部分分布于细胞质中。实施例2FTO-2-Ir配合物细胞内定位如图1B所示,将配合物FTO-2-Ir(5μM)与NB-4细胞共孵育12h后,提取细胞器,通过电感耦合等离子质谱ICP-MS测定不同细胞器中样品中金属Ir的含量,图中数据说明了配合物FTO-2-Ir主要分布于线粒体中。实施例3FTO-2-Rh配合物细胞内定位如图1C所示,将配合物FTO-2-Rh(5μM)与NB-4细胞共孵育12h后,提取细胞器,通过电感耦合等离子质谱ICP-MS测定不同细胞器中样品中金属Rh的含量,图中数据说明了配合物FTO-2-Rh主要分布于细胞核中。
结果表明:配合物FTO-2-Ru/Ir/Rh可以短时间内大量通过细胞膜被NB-4细胞摄取,FTO-2-Ru主要分布在细胞核和线粒体中,FTO-2-Ir主要分布于线粒体中,FTO-2-Rh主要分布于细胞核中。
实施例10
本发明实施例1~3制得的FTO-2-Ru/Ir/Rh诱导细胞凋亡。
方法:将NB-4细胞接种到六孔板中,生长18h,加入不同浓度的配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh。孵育24h,用PBS洗涤两次,收集细胞并将细胞重悬于500μL Bindingbuffer中,加入5μL Annexin V-FITC并混匀。5分钟后加入5μL Propidium Iodide混匀,避光孵育15分钟,BD FACSverse流式细胞仪分析样品,FlowJo7.6软件分析数据。
实施例1的配合物FTO-2-Ru、实施例2的FTO-2-Ir和实施例3的FTO-2-Rh诱导细胞凋亡如图2所示,给药24h后,不同浓度梯度下的配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh诱导NB-4细胞凋亡的流式图以及细胞的凋亡各期百分比图,由图中数据可以看出,配合物FTO-2-Ru不能诱导细胞凋亡,配合物FTO-2-Ir和FTO-2-Rh随着给药浓度增大,细胞逐渐发生凋亡。
结果表明:配合物FTO-2-Ru不能诱导细胞凋亡,配合物FTO-2-Ir和FTO-2-Rh均可不同程度诱导细胞发生凋亡。
实施例11
本发明实施例1~3制得的FTO-2-Ru/Ir/Rh促进细胞内活性氧生成;
方法:共聚焦显微镜检测ROS的生成:NB-4细胞接种在共聚焦比色皿中,37℃孵育12h。在与2IC50的FTO-2-Ru、FTO-2-Ir和FTO-2-Rh孵育24h后,用PBS洗涤两次。在37℃,将细胞暴露于荧光探针2',7'-二氯荧光素二乙酸酯(DCFH-DA,10μM)中20分钟。然后用PBS洗涤两次,并在激光共聚焦显微镜下对细胞进行成像。
流式细胞术检测ROS的生成:NB-4细胞接种在6孔板中,37℃孵育12h。在与2IC50的FTO-2-Ru、FTO-2-Ir和FTO-2-Rh孵育24h后,用PBS洗涤两次。在37℃,将细胞暴露于荧光探针2',7'-二氯荧光素二乙酸酯(DCFH-DA,10μM)中20分钟。PBS洗涤2次后收集细胞并用PBS重悬细胞。随后立即使用BD FACSverse流式细胞仪检测,激发波长为488nm,发射波长为510~540nm,使用FlowJo 7.6软件处理数据。
实施例1的配合物FTO-2-Ru、实施例2的FTO-2-Ir和实施例3的FTO-2-Rh均可不同程度促进活性氧生成,如图3所示,NB-4细胞分别与2IC50浓度的FTO-2-Ru、FTO-2-Ir和FTO-2-Rh共孵育24h后,通过流式细胞术和激光共聚焦检测配合物诱导的胞内活性氧产生。由图中数据可以看出,当加入配合物24h时,细胞内就有活性氧产生。
结果表明:FTO-2-Ru、FTO-2-Ir和FTO-2-Rh均可不同程度促进活性氧生成。
实施例12
本发明实施例1~3制得的FTO-2-Ru、FTO-2-Ir和FTO-2-Rh诱导线粒体膜电位下降;
方法:将NB-4细胞接种到6孔板中,生长12h,加入不同浓度梯度的配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh。孵育24h,吸出培养液,胰酶消化,收取细胞,PBS洗涤三次,随后加入配制好的JC-1工作液染色30min。用JC-1染色缓冲液洗涤两次,最后使用JC-1染色缓冲液重悬细胞,立即使用BD FACSverse流式细胞仪检测待测样品,设置两通道分别为FL1和FL2,使用FlowJo 7.6软件处理数据并进行分析。
实施例1的配合物FTO-2-Ru、实施例2的FTO-2-Ir和实施例3的FTO-2-Rh诱导线粒体膜电位下降如图4所示,NB-4细胞在不同浓度配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh中孵育24h后,细胞线粒体膜电位变化。可以看出随着药物浓度的增加,线粒体膜电位呈下降趋势,说明配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh均可不同程度下诱导NB-4细胞线粒体膜电位的下降。
结果表明:配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh均可不同程度下诱导线粒体膜电位下降。
实施例13
本发明实施例1~3制得的FTO-2-Ru、FTO-2-Ir和FTO-2-Rh细胞周期阻滞现象。
方法:将NB-4细胞接种在6孔细胞板中,在5%CO2的气氛中,在37℃下孵育18小时后,加入不同浓度梯度的配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh。在37℃下处理24小时后,收集细胞,用冷PBS洗涤两次,并在4℃下用70%乙醇固定过夜。固定后的混合物用PBS洗涤2次,用RNase A(100μg/mL)预处理10min后与碘化丙啶(PI,50μg/mL)孵育30min,用PBS洗涤2次,然后进行流式细胞仪分析以评估NB-4对细胞周期阻滞的影响。
实施例1的配合物FTO-2-Ru、实施例2的FTO-2-Ir和实施例3的FTO-2-Rh将细胞周期阻滞在G1/G2期如图5所示,给药24h后,不同浓度梯度下的配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh对NB-4细胞周期阻滞效果的流式图,由图中可以看出,配合物FTO-2-Ru不能阻滞细胞周期,随着配合物FTO-2-Ir和FTO-2-Rh浓度的增大,G1/G2期的细胞越来越多,由此可得出配合物FTO-2-Ir和FTO-2-Rh可将NB-4细胞阻滞在G1/G2期。
结果表明:配合物FTO-2-Ru不能阻滞细胞周期,FTO-2-Ir和FTO-2-Rh能够将细胞周期阻滞在G1/G2期。
实施例14
有机活性分子FTO-2、FTO-2-alkyne、配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh抑制FTO活性的应用。
方法:将0.1nmol含有m6A的ssDNA,0.1nmol的FTO,300μM的2OG,280μM的(NH4)2Fe(SO4)2,2mM的L-ascorbic acid以及100μM的FTO-2、FTO-2-alkyne、配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh溶于100μL的Tris-HCl(50mM,PH=7.5)缓冲溶液,室温孵育2h后,于90℃条件下煮5min后退火,加入20%的非还原PAGE,进行电泳跑样后使用核酸染料GelRed染色,比较条带强度,强度越强,抑制效果越明显。
FTO-2、FTO-2-alkyne、配合物FTO-2-Ru、FTO-2-Ir和FTO-2-Rh的FTO抑制活性如图6所示,由图中可以看出,FTO-2、FTO-2-alkyne无抑制活性,配合物FTO-2-Ru具有降解DNA的作用,FTO-2-Ir和FTO-2-Rh均具有FTO抑制活性。
结果表明:FTO-2-Ir和FTO-2-Rh具有FTO抑制活性。
Claims (10)
2.权利要求1所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于,包括如下步骤:
(1)制备含叠氮基团的金属配合物前体:在惰性气氛下,将金属钌的二聚体、金属铱的二聚体、金属铑的二聚体或金属锇的二聚体溶解在甲醇中,与螯合配体进行配位反应,反应后减压旋蒸除去溶剂,加入饱和阴离子盐的甲醇溶液,置于低温下冷冻,冷冻后离心收集固体,通过柱色谱分离对粗产物提纯,得到金属配合物前体;
(2)制备炔基修饰的有机活性分子配体:在惰性气氛下,将有机活性分子、炔基化合物和催化剂置于有机溶剂中反应,反应结束后,旋蒸除去溶剂,得到粗产物经过柱层析纯化后,得到有机活性分子配体;
(3)在惰性气氛下,将金属配合物前体、有机活性分子配体和催化剂在有机溶剂中反应,反应结束后,旋蒸除去溶剂,粗产物经色谱柱纯化,得到金属配合物。
3.根据权利要求2所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于:步骤(1)中,金属钌的二聚体与螯合配体的摩尔比为8~9:1;金属铱的二聚体与螯合配体的摩尔比为13~14:1;金属铑的二聚体与螯合配体的摩尔比为12~13:1;金属锇的二聚体与螯合配体的摩尔比为12~13:1。
4.根据权利要求2所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于:步骤(1)中,所述螯合配体为4-叠氮甲基-4'-甲基-2,2'-联吡啶。
5.根据权利要求2所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于:步骤(1)中,冷冻温度为-20~0℃,冷冻时间为8~14h。
6.根据权利要求2所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于:步骤(2)中,所述炔基化合物为溴丙炔、炔丙胺或戊炔酸;催化剂为碳酸钾或催化剂为N-羟基苯并三唑和N,N-二异丙基乙胺按摩尔比82~83:135的组合物。
7.根据权利要求2所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于:步骤(2)中,有机活性分子与炔基化合物的摩尔比为0.1~4:1。
8.根据权利要求2所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于:步骤(3)中,催化剂为五水硫酸铜和抗坏血酸钠按摩尔比3~3.5:4的组合物。
9.根据权利要求2所述的具有FTO抑制活性的金属配合物的制备方法,其特征在于:步骤(3)中,金属配合物前体与有机活性分子配体的反应摩尔比为3~25.5:1。
10.权利要求1所述的具有FTO抑制活性的金属配合物在用于制备抗肿瘤药物、抗肿瘤药物组分、FTO抑制药物和FTO抑制药物组分方面的应用。
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