CN109705095B - 一种含三氮唑环的稠环对苯醌类cdc25蛋白磷酸酶抑制剂及其制备方法和应用 - Google Patents
一种含三氮唑环的稠环对苯醌类cdc25蛋白磷酸酶抑制剂及其制备方法和应用 Download PDFInfo
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- CN109705095B CN109705095B CN201910136934.0A CN201910136934A CN109705095B CN 109705095 B CN109705095 B CN 109705095B CN 201910136934 A CN201910136934 A CN 201910136934A CN 109705095 B CN109705095 B CN 109705095B
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Abstract
本发明涉及含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂及其制备方法和应用。所述化合物具有式I所示的结构。本发明还涉及含有式I结构化合物的药物组合物。本发明还提供上述化合物以及含有一个或多个此类化合物的组合物在制备抗肿瘤药物中的应用。
Description
技术领域
本发明属于有机化合物合成与医药应用技术领域,具体涉及一种含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂及其制备方法和应用。
背景技术
当前,癌症依然是严重威胁人类健康和生存的重大疾病。近年来,伴随着分子生物学的迅速发展,人们对癌症发生与发展的分子水平机制有了更深刻的认识,促使抗肿瘤药物研究正从传统的细胞毒性药物转向基于靶点或机制的多环节作用的新型抗肿瘤药物。尽管每种癌症的发病原因及症状有所不同,但根本原因都是由于细胞周期调控基因改变导致细胞周期紊乱,进而造成肿瘤细胞恶性增殖所致。而细胞周期依赖性蛋白激酶(Cyclin-dependent kinases,CDK)及其调节亚基细胞周期蛋白(Cyclin)在细胞周期进程中起着关键作用。CDK的活性受到不同残基的磷酸化和去磷酸化的调节。而细胞分裂周期蛋白25(cell division cyclin 25,CDC25)由细胞周期中必需的Cdc25基因所编码,是一种双特异性蛋白磷酸酶,包含CDC25A、CDC25B、CDC25C三种亚型,该酶通过使这些残基去磷酸化以激活CDK,在真核生物的细胞有丝分裂中起重要调控作用,是细胞周期关键因子之一。因而,聚焦CDC25蛋白磷酸酶,研发新型高效的小分子抗肿瘤抑制剂已经成为当前肿瘤治疗领域的新方向。
发明内容
针对现有技术的不足,本发明提供了一种含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂及其制备方法,本发明还提供上述化合物作为CDC25蛋白磷酸酶抑制剂的活性筛选结果及其应用。
本发明的技术方案如下:
1.含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂
一种含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂,或其药学上可接受的盐,酯或前药,具有通式I所示的结构:
其中,
n为1或者2;
X为:N或者CH;
Y为:CH3,CH2OH或者卤素;
R为:苯基或吡啶基;或卤素,CH3,SO2NH2,SO2CH3,CONH2,NO2,CN,NH2,CF3,NHCH3,OH,COOH,CH2OH,CO2Me,OCH3,NHCOCH3取代的苯基,取代基为邻、间、对位单取代或多取代;或CONR2;五元杂环、六元杂环;
根据本发明优选的,所述的五元杂环、六元杂环优选为含氮五元杂环或者含氮六元杂环;
根据本发明进一步优选的,含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂是下列化合物之一:
本发明中所述的“药学上可接受的盐”是指在可靠的医药评价范围内,化合物的盐类适于与人或较低等动物的组织相接触而无不适当的毒性、刺激及过敏反应等,具有相当合理的收益与风险比例,通常是水或油可溶的或可分散的,并可有效地用于其预期的用途。包括药学上可接受的酸加成盐和药学上可接受的碱加成盐,在这里是可做预期的用途并与式I化合物的化学性质相容的。适宜的盐的列表参见S.M.Birge等,J.Pharm.Sci.,1977,66,1-19页。
本发明中所述的“前药”是指药学上可接受的衍生物,以便这些衍生物所得的生物转换产物是如式I化合物所定义的活性药物。
2.含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂的制备方法
含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂的制备方法,步骤包括:以取代稠环对苯醌化合物1为起始原料,碳酸钾做缚酸剂,在乙醇溶液中与丙炔胺发生亲核取代反应,得到炔基片段中间体2;然后中间体2与叠氮取代基发生一价铜催化的叠氮-末端炔环加成反应(CuAAC Click)反应得到目标产物I。
试剂及条件:(i)丙炔胺,碳酸钾,乙醇,室温;(ii)维生素C钠,五水硫酸铜,体积比1:1的四氢呋喃/水,叠氮取代基,室温;
n,X,Y,R同上述通式I所示;
所述的叠氮取代基为:邻甲基苄基叠氮、间甲基苄基叠氮、对甲基苄基叠氮、邻氯苄基叠氮、间氯苄基叠氮、对氯苄基叠氮、邻溴苄基叠氮、间溴苄基叠氮、对溴苄基叠氮、邻氟苄基叠氮、间氟苄基叠氮、对氟苄基叠氮、2,4-二氟苄基叠氮、3,4-二氟苄基叠氮、邻氰基苄基叠氮、间氰基苄基叠氮、对氰基苄基叠氮、邻硝基苄基叠氮、间硝基苄基叠氮、对硝基苄基叠氮、邻甲氧基苄基叠氮、间甲氧基苄基叠氮、对甲氧基苄基叠氮、对甲磺酰基苄基叠氮、间甲磺酰基苄基叠氮、邻甲磺酰基苄基叠氮、对磺酰胺基苄基叠氮、间磺酰胺基苄基叠氮、邻磺酰胺基苄基叠氮、对甲酰胺基苄基叠氮、间甲酰胺基苄基叠氮、邻甲酰胺基苄基叠氮苄、2-叠氮基-1-(吡咯烷-1-基)乙-1-酮、2-叠氮基-N-环丙基乙酰胺、2-叠氮基-1-吗啉-1-酮。
根据本发明优选的,含有三唑的稠环对苯醌类化合物Ⅰ制备方法,具体步骤如下:
(1)将起始原料1,丙炔胺,碳酸钾混合于10mL的乙醇溶液中,室温搅拌10~12h,TLC检测;随后,减压除去溶剂乙醇,再向反应液中加入适量的水,用乙酸乙酯萃取3次,每次10mL;合并有机层,饱和氯化钠溶液洗涤3次,每次30mL,无水硫酸钠干燥;过滤,减压蒸干溶剂;硅胶柱色谱分离,得到中间体2;
(2)将炔基片段2与不同的叠氮取代基加入到体积比1:1的THF/H2O混合溶液中,然后向此混合溶液中加入VcNa和CuSO4.5H2O,室温搅拌6-12h,TLC检测;随后,向反应液中加入适量的水,用乙酸乙酯萃取3次,每次10mL;合并有机层,饱和氯化钠溶液洗涤3次,每次30mL,无水硫酸钠干燥;过滤,减压蒸干溶剂;硅胶柱色谱分离;乙酸乙酯/石油醚或者二氯甲烷/正己烷重结晶得到目标化合物含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂Ⅰ。
本发明所述的室温为20-30℃。
3.含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂的抗HIV-1活性及应用
本发明对按照上述方法合成的部分含三氮唑环的稠环对苯醌类衍生物进行了酶水平及细胞水平的抗肿瘤活性测试,以文献报道的CDC25抑制剂NSC663284和/或紫杉醇(PXL)作为阳性对照。
目标化合物A1,A2,A3的CDC25酶活抑制结果如表1所示,除A1对CDC25B的抑制效果(IC50=2.79μM)略弱于阳性对照NSC 663284(IC50=1.36μM)外,化合物A1对CDC25A和CDC25C及A2、A3对CDC25A、CDC25B、CDC25C的抑制效果均与阳性对照NSC663284相当或较好。其中,化合物A3对CDC25C的抑制效果(IC50=0.09μM)尤为突出,约是阳性对照NSC663284抑制效果(IC50=0.76μM)的9倍。
目标化合物A1,A2,A3的抗肿瘤细胞活性结果如表2所示,整体来看,与阳性对照紫杉醇(PXL)和NSC663284相比,目标化合物A1,A2,A3对于腺癌人类肺泡基底上皮细胞(A549),人口腔表皮样癌细胞(KB),具有耐药性的人口腔上皮癌细胞(KB-VIN)和人乳腺癌细胞(MCF-7/MDA-MB-231)的抑制效果相当或较弱。其中,较为突出的则是目标化合物A3对于KB-VIN肿瘤细胞具有较好的抑制效果(IC50=6.810μM),与阳性对照PXL(IC50=3145.828nM)和NSC663284(IC50=4.874μM)对于KB-VIN肿瘤细胞的抑制效果相当,具有进一步开发的价值。
本发明的含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂可作为小分子抗肿瘤抑制剂应用。具体地说,作为A549、KB、KB-VIN、MCF-7或者MDA-MB-231抑制剂用于制备抗肿瘤药物。
一种抗肿瘤药物组合物,包括本发明的含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂和一种或多种药学上可接受载体或赋形剂。
本发明提供了结构全新的含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂及其制备方法,本发明还提供了化合物CDC25酶活抑制和抗肿瘤活性筛选结果及其在抗病毒领域中的首次应用。经过试验证明,本发明的含三氮唑环的稠环对苯醌类衍生物可作为肿瘤抑制剂应用并具有较高的应用价值。具体地说,作为A549,KB,KB-VIN,MCF-7或者MDA-MB-231抑制剂用于制备抗肿瘤药物。
具体实施方式
通过下述实施例有助于理解本发明,但是不能限制本发明的内容。
实施例中所涉及的合成路线如下:
实施例1:6-氯-7-(丙-2-炔-1-基氨基)喹啉-5,8-二酮(4)的制备
将起始原料3(0.581mmol,1.0eq),丙炔胺(1.16mmol,2.0eq)和碳酸钾(0.42mmol,1.0eq)混合于10mL的国药乙醇溶液中,室温搅拌10~12h,TLC检测。随后,减压除去溶剂乙醇,再向反应液中加入适量的水,用乙酸乙酯萃取(3×10mL);合并有机层,饱和氯化钠溶液洗涤(3×30mL),无水硫酸钠干燥;过滤,减压蒸干溶剂;硅胶柱色谱分离,得到中间体6-氯-7-(丙-2-炔-1-基氨基)喹啉-5,8-二酮。红褐色固体,收率:82%,1H NMR(400MHz,DMSO-d6)δ8.93(dd,J=4.6,1.6Hz,1H),8.34(dd,J=7.9,1.6Hz,1H),7.97–7.73(m,2H),4.48(dd,J=6.7,2.4Hz,2H),3.28(s,1H).ESI-MS:m/z 244.93(M-H)-,C12H7ClN2O2(246.02)
实施例2:目标化合物A1-A3的制备
将炔基片段4(1.0eq)与不同的叠氮取代基(1.2eq)加入到8mL的THF/H2O(v:v=1:1)混合溶液中,然后向此混合溶液中加入VcNa(0.3eq)和CuSO4.5H2O(0.1eq),室温搅拌6-12h,TLC检测。随后,向反应液中加入适量的水,用乙酸乙酯萃取(3×10mL);合并有机层,饱和氯化钠溶液洗涤(3×30mL),无水硫酸钠干燥;过滤,减压蒸干溶剂;硅胶柱色谱分离;乙酸乙酯/石油醚或者二氯甲烷/正己烷重结晶得到目标化合物A1,A2,A3。
2-((4-(((6-氯-5,8-二氧代-5,8-二氢喹啉-7-基)氨基)甲基)-1H-1,2,3-三唑-1-基)甲基)苯甲腈(A1)红褐色固体,产率:67%,mp:154-156℃。1H NMR(400MHz,DMSO-d6)δ8.92(dd,J=4.6,1.6Hz,1H),8.34(dd,J=7.8,1.6Hz,1H),8.17(s,1H),8.02(s,1H),7.90(dd,J=7.7,1.3Hz,1H),7.81(dd,J=7.9,4.7Hz,1H),7.69(td,J=7.7,1.4Hz,1H),7.55(td,J=7.7,1.2Hz,1H),7.29(d,J=7.8Hz,1H),5.79(s,2H),5.19–4.91(m,2H).13C NMR(100MHz,DMSO)δ178.70,153.50,146.03,139.40,134.23,133.79,129.61,128.87,124.06,117.40,111.55,51.44.ESI-MS:427.19(M+Na)+,C20H13ClN6O2(404.08).
3-((4-(((6-氯-5,8-二氧代-5,8-二氢喹啉-7-基)氨基)甲基)-1H-1,2,3-三唑-1-基)甲基)苯甲腈(A2)红褐色固体,产率:74%,mp:175-177℃。1H NMR(400MHz,DMSO-d6)δ8.92(s,1H),8.34(d,J=7.7Hz,1H),8.18(s,1H),8.03(s,1H),7.79(d,J=15.0Hz,3H),7.59(s,2H),5.66(s,2H),5.03(s,2H).13C NMR(100MHz,DMSO)δ178.69,153.49,146.21,138.15,134.21,133.22,132.40,131.94,130.51,128.87,123.74,118.85,112.11,52.30.ESI-MS:427.22(M+Na)+,C20H13ClN6O2(404.08).
6-氯-7-(((1-(4-甲基苄基)-1H-1,2,3-三唑-4-基)甲基)氨基)喹啉-5,8-二酮(A3)红褐色固体,产率:57%,mp:163-164℃。1H NMR(400MHz,DMSO-d6)δ8.92(dd,J=4.7,1.6Hz,1H),8.34(dd,J=7.8,1.6Hz,1H),8.07(s,1H),8.00(s,1H),7.81(dd,J=7.9,4.7Hz,1H),7.16(d,J=2.5Hz,4H),5.50(s,2H),5.01(d,J=6.6Hz,2H),2.27(s,3H).13CNMR(100MHz,DMSO)δ178.68,153.47,145.98,137.86,134.20,133.57,129.68,128.85,128.28,123.31,53.06,21.14.ESI-MS:m/z 393.90(M+H)+,C20H16ClN5O2(393.10).
实施例3:目标化合物的CDC25酶活活性测试实验
实验材料:
待测化合物、CDC25蛋白磷酸酶试剂盒(CycLex Protein PhosphataseCdc25Combo Fluorometric Assay Kit,Abnova)、不同规格的微量加样器(10μL、20μL、100μL、200μL、1000μL)、酶标仪、三蒸水、阳性对照NSC 663284。
测试方法:
CDC25的酶活测定实验严格按照Abnova的商品说明书进行。首先将40μL“测试混合液”(30μL三蒸水、5μL 10X Assay Buffer、5μL 10X OMFP)加入96孔荧光板的待测孔中,然后向上述待测孔中加入5μL待测化合物的80%DMSO溶液,两者充分混合。接着,让蛋白磷酸酶CDC25A、CDC25B、CDC25C自然解冻,向加入待测化合物的孔中加入5μL CDC25A、CDC25B或CDC25C,引发反应,室温孵育5-8min后,立即在485nm、525nm波长下进行时长1h的检测,观察0min-60min过程中的酶活变化或者某时间点的待测化合物的荧光强度。测试过程中,需要设置“溶媒对照”(Vehicle Control,VC)孔和“无酶对照”(No Enzyme Control,NC)孔。“溶媒对照”孔中加入40μL“测试混合液”、5μL DMSO、5μL CDC25蛋白磷酸酶溶液;“无酶对照”孔中加入40μL“测试混合液”、5μL DMSO、5μL三蒸水。
结果计算:
根据上述公式分别计算出单个浓度下待测化合物对CDC25A、CDC25B、CDC25C的荧光强度。而不同浓度下的待测化合物对CDC25各亚型的荧光强度结合GraphPad Prism 5软件处理得到IC50值。
按照上述实验方法对合成的含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂进行了酶活水平的测试,结果如表1所示。
表1目标化合物A1、A2、A3的酶活测试结果
实施例4:目标化合物的体外抗肿瘤细胞活性测试实验
测试原理:
化合物体外抗肿瘤细胞活性筛选采用SRB检测方法(磺酰罗丹明B比色法)。磺酰罗丹明B(Sulforhodamine B,SRB),是一种粉红色阴离子染料,在酸性条件下可特异性地与细胞内组成蛋白质的碱性氨基酸结合,从而在490nm波长下产生吸收峰,而吸光值与细胞量成线性正相关,故可以用作细胞数的定量检测。
实验材料:
(1)SRB溶液:以1%乙酸稀释SRB粉至浓度为0.04%;
(2)洗脱液:乙酸用三蒸水配制至1%浓度;
(3)Tris base液:Tris base用单蒸水配制至10mM浓度,调节pH值为10.5;
(4)50%三氯乙酸(TCA):称取2.5gTCA加水定容至250ml,4℃棕色瓶或锡纸避光保存
(5)阳性对照紫杉醇(PXL,由美国北卡罗莱那大学药学院天然产物研究室李国雄教授提供)和NSC663284;待测化合物A1,A2,A3。
(6)肿瘤细胞(由美国北卡罗莱那大学药学院天然产物研究室李国雄教授提供):腺癌人类肺泡基底上皮细胞(A549),人口腔表皮样癌细胞(KB),具有耐药性的人口腔上皮癌细胞(KB-VIN),人乳腺癌细胞(MCF-7/MDA-MB-231)。
实验方法:
当癌细胞传代至对数生长期以后,调节至合适浓度(5×104/mL),接种于96孔板中(200μL/孔),于5%二氧化碳,37℃条件下孵育24h。孵育完毕,弃去96孔板中原有液体,随后向各孔中添加含有相应浓度的待测化合物或阳性药物的细胞培养液,每个浓度设3个复孔,同时设立无药细胞对照孔,继续在5%二氧化碳,37℃培养箱中培养72h。当待测化合物的作用时间结束时,每孔加入50μL 4℃预冷的TCA溶液(50%,w/v)固定细胞。静置5min后移入4℃冰箱中固定30min,取出用三蒸水冲洗5遍,室温晾干。待96孔板室温下晾干后,每孔加入100μL 0.04%的SRB染液,染色30min后倒掉染液,用1%乙酸冲洗4次,去除未结合的染料,室温晾干。最后,用150μL非缓冲Tris-base碱液(10mM,pH=10.5)溶解与细胞蛋白结合的染料,水平摇床上振荡20min,采用酶标仪测定515nm处的吸光度值(OD值)。(Ling Y,Yang QX,Teng Y N,et al.Development of novel amino-quinoline-5,8-dione derivativesas NAD(P)H:quinone oxidoreductase 1(NQO1)inhibitors with potentantiproliferative activities[J].Eur J Med Chem,2018,154:199-209;Lu Y,Li Y Q,Liu Y N,et al.Cytotoxic and potential anticancer constituents from the stemsof Schisandra pubescens[J].Pharm Biol,2013,51(9):1204-1207.)
结果计算:所获肿瘤细胞生长抑制率定义为药物对肿瘤细胞的体外抑制率。
不同浓度下的待测化合物对各肿瘤细胞的抑制率结合GraphPad Prism 5软件处理得到IC50值。按照上述实验方法对合成的含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂进行了细胞水平的测试,结果如表2所示。
表2目标化合物A1,A2,A3的抗肿瘤细胞活性测试结果
Claims (6)
3.如权利要求1所述的含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂的制备方法,步骤如下:以取代稠环对苯醌化合物1为起始原料,碳酸钾做缚酸剂,在乙醇溶液中与丙炔胺发生亲核取代反应,得到炔基片段中间体2;然后中间体2与叠氮取代基发生一价铜催化的叠氮-末端炔环加成反应(CuAACClick)反应得到目标产物I;
试剂及条件:(i)丙炔胺,碳酸钾,乙醇,室温;(ii)维生素C钠,五水硫酸铜,体积比1:1的四氢呋喃/水,叠氮取代基,室温;
其中,n、X、R如权利要求1所述,Y为Cl;
所述的叠氮取代基为:苄基叠氮、邻甲基苄基叠氮、间甲基苄基叠氮、对甲基苄基叠氮、邻氯苄基叠氮、间氯苄基叠氮、对氯苄基叠氮、邻溴苄基叠氮、间溴苄基叠氮、对溴苄基叠氮、邻氟苄基叠氮、间氟苄基叠氮、对氟苄基叠氮、邻氰基苄基叠氮、间氰基苄基叠氮、对氰基苄基叠氮。
4.如权利要求3所述的含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂的制备方法,步骤如下:
(1)将起始原料1,丙炔胺,碳酸钾混合于10mL的乙醇溶液中,室温搅拌10~12h,TLC检测;随后,减压除去溶剂乙醇,再向反应液中加入适量的水,用乙酸乙酯萃取3次,每次10mL;合并有机层,饱和氯化钠溶液洗涤3次,每次30mL,无水硫酸钠干燥;过滤,减压蒸干溶剂;硅胶柱色谱分离,得到中间体2;
(2)将炔基片段2与不同的叠氮取代基加入到体积比1:1的THF/H2O混合溶液中,然后向此混合溶液中加入VcNa和CuSO4·5H2O,室温搅拌6-12h,TLC检测;随后,向反应液中加入适量的水,用乙酸乙酯萃取3次,每次10mL;合并有机层,饱和氯化钠溶液洗涤3次,每次30mL,无水硫酸钠干燥;过滤,减压蒸干溶剂;硅胶柱色谱分离;乙酸乙酯/石油醚或者二氯甲烷/正己烷重结晶得到目标化合物含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂。
5.如权利要求1-2任一项所述含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂在制备抗肿瘤药物中的应用。
6.一种药物组合物,包含权利要求1-2任一项所述含三氮唑环的稠环对苯醌类CDC25蛋白磷酸酶抑制剂和一种或多种药学上可接受的载体或赋形剂。
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