CN103242232A - 吡唑苯胺类化合物的制备及其在抗癌治疗中的应用 - Google Patents
吡唑苯胺类化合物的制备及其在抗癌治疗中的应用 Download PDFInfo
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Abstract
一类吡唑苯胺类衍生物,其特征是它有如下通式:
Description
技术领域
本发明涉及吡唑苯胺类化合物及其制备方法与抗肿瘤药物。
背景技术
癌症是一种严重威胁人类健康的疾病,临床治愈率很低。癌症是由于细胞的异常增殖引起的,和调控细胞周期相关基因、蛋白的突变和失活密切相关。因此阻断异常的细胞周期对于肿瘤的治疗是一种非常重要的手段。依赖于细胞周期蛋白激酶(Cyclin-dependent-kinase,CDK)是细胞周期进程中一种非常重要的蛋白。CDK一般由两个亚基组成:催化亚基和调控亚基。其中CDK2是最上游也是最重要的一种依赖于细胞周期蛋白激酶,它与细胞周期蛋白A和E结合,激活下游底物丝氨酸和酪氨酸位点的磷酸化,从而在细胞周期G1/S期的转化中起重要的调控作用,近期研究发现,CDK2在G2期中也有调控作用。CDK2在细胞周期和增殖中的重要作用,使其成为肿瘤以及其它细胞异常增殖相关疾病的重要靶点。
几乎所有的CDK抑制剂都是通过竞争性结合ATP结合位点从而抑制其催化活性。吡唑环抗CDK2的活性最早发现于具有CDK2抑制作用的天然产物Purvalanol B中,目前已经成为目前CDK2抑制药物中的重要骨架之一,可以有效的与CDK2中ATP结合位点作用。苯胺类化合物也具有很好的ATP竞争抑制活性,广泛的存在于ATP竞争性抑制剂中。本文中,我们合成了不同取代基的吡唑醛,通过醛基亲核加成、还原修饰不同取代基的苯胺,形成了一系列的未经报道的新化合物。这些新化合物,可能体现出吡唑醛和苯胺在CDK2抑制和抗肿瘤中的协同性。我们测试了这批化合物的生物活性,并发现其对人源宫颈癌Hela细胞具有良好的抑制作用,且能抑制CDK2,引起细胞凋亡。
发明内容
本发明的目的在于提供一类新型吡唑苯胺类化合物以及它们的制备方法与用途。
本发明的技术方案如下:
1.一类吡唑苯胺衍生物,其特征是它有如下通式:
结构式中R1为:4-氟、氯、溴、甲基、甲氧基。
R2为:4-氟、氯、溴、甲基。
2.一种制备上述的吡唑苯胺衍生物的方法,它由下列步骤组成:
步骤1.将取代基苯乙酮(20mmol)、盐酸苯肼(25mmol)、无水乙酸钠(30mmol)分别溶于乙醇,磁力搅拌,50-60℃反应3h(TLC检测反应进行程度)。反应完毕后将反应液旋干,乙酸乙酯和水萃取2-3次,旋干有机层,得固体。
步骤2.先将DMF(4ml)和POCl3(5ml)混合冰浴30min,然后将步骤1所得固体溶于3ml DMF滴加至上述混合液中,逐渐转入常温至70-80℃反应5h,冷却至常温后,倒入冰水中,用50%NaOH调pH至7-8,抽滤并水洗3遍,烘干后得固体。
步骤3.将步骤2所得产物(1mmol)溶于乙醇(20ml),加入取代基苯胺(1mmol),60℃反应5h(TLC检测反应进行程度)。反应完毕后将反应液旋干,四氢呋喃(15ml)溶解后加入NaBH4(2mmol)。待气泡消失后冰浴滴加I2的四氢呋喃溶液。30min后转入常温至40-50℃反应12h(TLC检测)。反应完毕后旋干反应液,乙酸乙酯和水萃取2-3次,无水Na2SO4干燥,旋干有机层,再减压蒸干溶剂即得到固体物。重结晶或过柱纯化固体物可得到纯净的化合物1-16。
本发明的吡唑苯胺类化合物具有抑制人源宫颈癌Hela细胞增殖的作用。因此本发明的吡唑苯胺类化合物可做潜在的抗肿瘤药物。
具体实施方式
实施例一:4-氟-N-((3-(4-氟苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物1)的制备
将4-氟苯乙酮(20mmol)、盐酸苯肼(25mmol)、无水乙酸钠(30mmol)分别溶于乙醇,磁力搅拌,50-60℃反应3h(TLC检测反应进行程度)。反应完毕后将反应液旋干,乙酸乙酯和水萃取2-3次,旋干有机层,得固体。将DMF和POCl3(5ml)混合冰浴30min,然后将上述固体溶于4ml DMF并滴加至上述混合液中,逐渐转入常温至70-80℃反应5h。冷却至常温后,倒入冰水中,用50%NaOH调pH至7-8,抽滤并水洗3遍,烘干后得固体。取1mmol所得固体溶于乙醇(20ml),加入4-氟苯胺(1mmol),60℃反应5h(TLC检测反应进行程度)。反应完毕后将反应液旋干,四氢呋喃(15ml)溶解后加入NaBH4(2mmol)。待气泡消失后冰浴滴加I2的四氢呋喃溶液。30min后转入常温至40-50℃反应12h(TLC检测)。反应完毕后旋干反应液,乙酸乙酯和水萃取2-3次,无水Na2SO4干燥,旋干有机层,再减压蒸干溶剂即得到固体物。重结晶或过柱纯化固体物可得到白色晶体状目标化合物。产率83%,mp:116-117℃;1H NMR(300MHz,DMSO-d6,δppm):4.18(d,J=5.13Hz,2H);5.86(s,1H);6.63-6.67(m,2H);6.92(t,J=8.86Hz,2H);7.25-7.34(m,3H);7.50(t,J=7.88Hz,2H);7.84(t,J=8.33Hz,4H);8.56(s,1H).MS(ESI):362.5(C22H18F2N3,[M+H]+).Anal.Calcd for C22H17F2N3:C,73.12;H,4.74;N,11.63;Found:C,73.17;H,4.77;N,11.50%.
实施例二:4-氯-N-((3-(4-氟苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物2)的制备
制备方法同实施例一。以4-氯苯胺代替例一中的4-氟苯胺。得到白色晶体状目标化合物。产率73%,mp:136-137℃;1H NMR(300MHz,DMSO-d6,δppm):4.19(d,J=4.95Hz,2H);6.16(t,J=4.85Hz,1H);6.66(d,J=8.94Hz,2H);7.10(d,J=8.79Hz,2H);7.25-7.34(m,3H);7.50(t,J=7.86Hz,2H);7.79-7.87(m,4H);8.56(s,1H).MS(ESI):378.3(C22H18ClFN3,[M+H]+).Anal.Calcd for C22H17ClFN3:C,69.93;H,4.53;N,11.12;Found:C,69.87;H,4.55;N,11.21%.
实施例三:4-溴-N-((3-(4-氟苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物3)的制备
制备方法同实施例一。以4-溴苯胺代替例一中的4-氟苯胺。得到白色晶体状目标化合物。产率80%,mp:145-147℃;1H NMR(300MHz,DMSO-d6,δppm):4.19(d,J=4.77Hz,2H);6.19(t,J=4.85Hz,1H);6.62(d,J=8.79Hz,2H);7.24(d,J=6.93Hz,2H);7.28-7.33(m,3H);7.50(t,J=7.96Hz,2H);7.79-7.87(m,4H);8.56(s,1H).MS(ESI):423.8(C22H18BrFN3,[M+H]+).Anal.Calcd for C22H17BrFN3:C,62.57;H,4.06;N,9.95;Found:C,62.66;H,4.11;N,9.81%.
实施例四:4-甲基-N-((3-(4-氟苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物4)的制备
制备方法同实施例一。以4-甲基苯胺代替例一中的4-氟苯胺。得到白色晶体状目标化合物。产率77%,mp:102-104℃;1H NMR(300MHz,DMSO-d6,δppm):2.16(s,3H);4.20(s,2H);5.68(s,1H);6.60(d,J=4.92Hz,2H);6.91(d,J=4.95Hz,2H);7.27-7.37(m,3H);7.51(t,J=4.77Hz,2H);7.84-7.88(m,4H);8.55(s,1H).MS(ESI):358.8(C23H21FN3,[M+H]+).Anal.Calcd for C23H20FN3:C,77.29;H,5.64;N,11.76;Found:C,77.25;H,5.66;N,11.83%.
实施例五:N-((3-(4-氯苯基)-1-苯基-1H-4-吡唑基)甲基)-4-氟苯胺(化合物5)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮。得到白色晶体状目标化合物。产率81%,mp:132-134℃;1H NMR(300MHz,DMSO-d6,δppm):4.19(d,J=4.77Hz,2H);6.15(s,1H);6.66(d,J=8.76Hz,2H);7.10(d,J=8.79Hz,2H);7.23-7.32(m,3H);7.49(t,J=7.86Hz,2H);7.66(d,J=8.04Hz,2H);7.85(d,J=7.86Hz,2H);8.53(s,1H).MS(ESI):379.5(C22H18ClFN3,[M+H]+).Anal.Calcd for C22H17ClFN3:C,69.93;H,4.53;N,11.12;Found:C,69.85;H,4.51;N,11.19%.
实施例六:4-氯-N-((3-(4-氯苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物6)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮,4-氯苯胺代替例一中的4-氟苯胺。得到黄色晶体状目标化合物。产率71%,mp:131-133℃;1HNMR(300MHz,DMSO-d6,δppm):4.20(s,2H);6.16(s,1H);6.68(d,J=5.31Hz,2H);7.02(d,J=5.22Hz,2H);7.12(d,J=5.31Hz,2H);7.30(t,J=4.40Hz,2H);7.73(d,J=5.22Hz,2H);7.86(d,J=4.95Hz,2H);8.54(s,1H).MS(ESI):395.9(C22H18Cl2N3,[M+H]+).Anal.Calcd for C22H17Cl2N3:C,67.01;H,4.35;N,10.66;Found:C,67.08;H,4.31;N,10.61%.
实施例七:4-溴-N-((3-(4-氯苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物7)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮,4-溴苯胺代替例一中的4-氟苯胺。得到棕褐色晶体状目标化合物。产率77%,mp:114-115℃;1H NMR(300MHz,DMSO-d6,δppm):4.41(s,2H);6.86(d,J=7.68Hz,4H);7.20(d,J=7.86Hz,3H);7.31(s,1H);7.44(t,J=7.77Hz,4H);7.72(d,J=7.68Hz,2H);8.26(s,1H).MS(ESI):440.3(C22H18BrClN3,[M+H]+).Anal.Calcd for C22H17BrClN3:C,60.22;H,3.91;N,9.58;Found:C,60.10;H,3.93;N,9.64%.
实施例八:N-((3-(4-氯苯基)-1-苯基-1H-4-吡唑基)甲基)-4-甲基苯胺(化合物8)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮,4-甲基苯胺代替例一中的4-氟苯胺。得到黄色晶体状目标化合物。产率85%,mp:103-104℃;1H NMR(300MHz,DMSO-d6,δppm):2.15(s,3H);4.20(d,J=4.77Hz,2H);5.70(s,1H);6.59(d,J=8.40Hz,2H);6.91(d,J=8.40Hz,2H);7.32(t,J=7.31Hz,1H);7.49(d,J=3.66Hz,4H);7.51-7.88(m,4H);8.57(s,1H).MS(ESI):375.5(C23H21ClN33,[M+H]+).Anal.Calcd for C23H20ClN3:C,73.89;H,5.39;N,11.24;Found:C,73.77;H,5.36;N,11.31%.
实施例九:N-((3-(4-溴苯基)-1-苯基-1H-4-吡唑基)甲基)-4-氟苯胺(化合物9)的制备
制备方法同实施例一。以4-溴苯乙酮代替例一中的4-氟苯乙酮。得到黄色晶体状目标化合物。产率77%,mp:116-117℃;1H NMR(300MHz,DMSO-d6,δppm):4.20(s,2H);5.68(s,1H);6.60(d,J=4.92Hz,2H);6.91(d,J=4.95Hz,2H);7.27-7.37(m,3H);7.51(t,J=4.77Hz,2H);7.84-7.88(m,4H);8.55(s,1H).MS(ESI):422.8(C22H18BrFN3,[M+H]+).Anal.Calcd for C22H17BrFN3:C,62.57;H,4.06;N,9.95;Found:C,62.36;H,4.09;N,10.13%.
实施例十:N-((3-(4-溴苯基)-1-苯基-1H-4-吡唑基)甲基)-4-氯苯胺(化合物10)的制备
制备方法同实施例一。以4-溴苯乙酮代替例一中的4-氟苯乙酮,4-氯苯胺代替例一中的4-氟苯胺。得到黄褐色晶体状目标化合物。产率85%,mp:114-115℃;1H NMR(300MHz,DMSO-d6,δppm):4.23(s,2H);6.17(s,1H);6.68(d,J=5.22Hz,2H);7.12(d,J=5.22Hz,2H);7.33(t,J=4.38Hz,1H);7.52(t,J=4.76Hz,2H);7.65(d,J=5.13Hz,2H);7.76(d,J=5.04Hz,2H);7.87(d,J=4.65Hz,2H);8.58(s,1H).MS(ESI):438.4(C22H18BrClN3,[M+H]+).Anal.Calcd for C22H17BrClN3:C,60.22;H,3.91;N,9.58;Found:C,60.36;H,3.96;N,9.43%.
实施例十一:4-溴-N-((3-(4-溴苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物11)的制备
制备方法同实施例一。以4-溴苯乙酮代替例一中的4-氟苯乙酮,4-溴苯胺代替例一中的4-氟苯胺。得到棕色晶体状目标化合物。产率68%,mp:127-128℃;1HNMR(300MHz,DMSO-d6,δppm):4.22(s,2H);6.17(s,1H);6.63(d,J=8.76Hz,2H);7.22(d,J=8.94Hz,2H);7.32(t,J=7.41Hz,1H);7.52(t,J=7.95Hz,2H);7.65(d,J=8.61Hz,2H);7.75(d,J=8.40Hz,2H);7.87(d,J=8.40Hz,2H);8.56(s,1H).MS(ESI):484.1(C22H18Br2N3,[M+H]+).Anal.Calcd for C22H17Br2N3:C,54.68;H,3.55;N,8.70;Found:C,54.56;H,3.59;N,8.87%.
实施例十二:N-((3-(4-溴苯基)-1-苯基-1H-4-吡唑基)甲基)-4-甲基苯胺(化合物12)的制备
制备方法同实施例一。以4-溴苯乙酮代替例一中的4-氟苯乙酮,4-甲基苯胺代替例一中的4-氟苯胺。得到白色晶体状目标化合物。产率70%,mp:105-106℃;1H NMR(300MHz,DMSO-d6,δppm):2.15(s,3H);4.20(d,J=4.95Hz,2H);5.70(s,1H);6.59(d,J=8.25Hz,2H);6.91(d,J=8.04Hz,2H);7.32(t,J=7.31Hz,1H);7.51(t,J=7.77Hz,2H);7.64(d,J=8.22Hz,2H);7.77(d,J=8.43Hz,2H);7.87(d,J=8.04Hz,2H);8.57(s,1H).MS(ESI):420.7(C23H21BrN33,[M+H]+).Anal.Calcd forC23H20BrN3:C,66.04;H,4.82;N,10.04;Found:C,66.13;H,4.79;N,9.95%.
实施例十三:4-氟-N-((3-(4-甲氧基苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物13)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮。得到白色晶体状目标化合物。产率76%,mp:111-113℃;1H NMR(300MHz,DMSO-d6,δppm):3.77(s,3H);4.16(d,J=4.92Hz,2H);5.84(t,J=5.03Hz,1H);6.62-6.67(m,2H);6.92(t,J=8.95Hz,2H);7.00(d,J=8.76Hz,2H);7.28(t,J=7.41Hz,1H);7.49(t,J=7.95Hz,2H);7.73(d,J=8.79Hz,2H);7.84(d,J=7.68Hz,2H);8.52(s,1H).MS(ESI):372.9(C23H21FN3O,[M+H]+).Anal.Calcd for C23H20FN3O:C,73.98;H,5.40;N,11.25;Found:C,73.85;H,5.44;N,11.33%.
实施例十四:4-氯-N-((3-(4-甲氧基苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物14)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮,4-氯苯胺代替例一中的4-氟苯胺。得到橙黄色晶体状目标化合物。产率71%,mp:131-133℃;1H NMR(300MHz,DMSO-d6,δppm):3.79(s,3H);4.20(s,2H);6.16(s,1H);6.68(d,J=5.31Hz,2H);7.02(d,J=5.22Hz,2H);7.12(d,J=5.31Hz,2H);7.30(t,J=4.40Hz,1H);7.50(t,J=4.71Hz,2H);7.73(d,J=5.22Hz,2H);7.86(d,J=4.95Hz,2H);8.54(s,1H).MS(ESI):391.9(C23H21ClN3O,[M+H]+).Anal.Calcd forC23H20ClN3O:C,70.85;H,5.17;N,10.78;Found:C,70.68;H,5.21;N,10.92%.
实施例十五:4-溴-N-((3-(4-甲氧基苯基)-1-苯基-1H-4-吡唑基)甲基)苯胺(化合物15)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮,4-溴苯胺代替例一中的4-氟苯胺。得到黄色晶体状目标化合物。产率80%,mp:145-147℃;1H NMR(300MHz,DMSO-d6,δppm):4.19(d,J=4.77Hz,2H);6.19(t,J=4.85Hz,1H);6.62(d,J=8.79Hz,2H);7.24(d,J=6.93Hz,2H);7.28-7.33(m,3H);7.50(t,J=7.96Hz,2H);7.79-7.87(m,4H);8.56(s,1H).MS(ESI):423.8(C22H18BrFN3,[M+H]+).Anal.Calcd for C22H17BrFN3:C,62.57;H,4.06;N,9.95;Found:C,62.66;H,4.11;N,9.81%.
实施例十六:N-((3-(4-甲氧基苯基)-1-苯基-1H-4-吡唑基)甲基)-4-甲基苯胺(化合物16)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮,4-甲基苯胺代替例一中的4-氟苯胺。得到黄色晶体状目标化合物。产率81%,mp:107-108℃;1H NMR(300MHz,DMSO-d6,δppm):2.15(s,3H);3.78(s,3H);4.17(d,J=5.13Hz,2H);5.68(s,1H);6.59(d,J=8.22Hz,2H);6.91(d,J=8.04Hz,2H);7.01(d,J=8.76Hz,2H);7.29(t,J=7.32Hz,1H);7.50(t,J=7.86Hz,2H);7.75(d,J=8.79Hz,2H);7.85(d,J=7.86Hz,2H);8.51(s,1H).MS(ESI):369.1(C24H24N3O,[M+H]+).Anal.Calcd for C24H23N3O:C,78.02;H,6.27;N,11.37;Found:C,78.11;H,6.32;N,11.26%.
实施例十七:吡唑苯胺类化合物对肿瘤抑制活性的研究
采用MTT[3-(4,5)-双甲基-2-噻唑-(2,5)-苯基溴化四氮唑蓝]法来测定计算吡唑苯胺类化合物对人源宫颈癌细胞Hela半数抑制浓度(IC50)。
(1)培养液(每升)的配制:①悬浮细胞:RPMI-1640培养粉一袋(10.4g),新生牛血清100ml,青霉素溶液(20万U/ml)0.5ml,链霉素溶液(20万U/ml)0.5ml,加三蒸水溶解后,用5.6%的NaHCO3溶液调pH值至7.2-7.4,最后定容至1000ml。过滤灭菌。②贴壁细胞:同上,再加入NaHCO3 2.00g,HEPES 2.38g。
(2)D-Hanks缓冲液(每升)的配制:NaCl 8.00g,KCl 0.40g,Na2HPO4·12H2O 0.06g,KH2PO4 0.06g,NaHCO3 0.35g。高压灭菌。
(3)胰蛋白酶液的配制:利用D-Hanks缓冲液配成浓度为0.5%胰蛋白酶液。过滤除菌。
(4)实验药液的配制:将测试样品用少量的三蒸水溶解配成储备液,一般按实验最高浓度的10倍配制储备液。根据化合物溶解性不同,可用三蒸水直接溶解,或用少量DMSO助溶,再加三蒸水溶解。加药后的每孔细胞悬液中DMSO的终浓度一般不超过0.05%-0.1%。储备液保存于-20℃冰箱中备用。
(5)宫颈癌细胞Hela的培养:为贴壁生长细胞,常规培养于RPMI-1640培养液内(含10%小牛血清、100U/ml链霉素),置37℃、5%CO2培养箱中培养,每隔3-4天传代一次。传代时先弃去原培养液,再用D-Hanks缓冲液洗涤;然后用0.5%胰蛋白酶消化30秒左右,加入少量新鲜培养液终止消化;吹打,使贴壁细胞从培养瓶壁上脱落下来;移取适量至新鲜培养瓶中,再补充新鲜培养液至原体积(培养液体积约为培养瓶容量的1/10)。
(6)细胞孵育:取对数生长期的上述肿瘤细胞,调细胞悬液浓度为2×104个ml-1。在96孔培养板中每孔加细胞悬液100μl,置37℃,5%CO2培养箱中培养24h。24h后,分别按设计加入药液。
(7)加药:将测试药液按照最终浓度的浓度梯度分别加入到各个孔中,每个浓度设6个平行孔。实验分为药物试验组(分别加入不同浓度的测试药)、对照组(只加培养液和细胞,不加测试药)和空白组(只加培养液,不加细胞和测试药)。将加药后的96孔板置于37℃,5%CO2培养箱中培养48h。按照测试样品的方法测定。
(8)存活细胞的测定:在培养了48h后的96孔板中,每孔加MTT 200μl(用200μl PBS配成0.5mg/ml的MTT)。在37℃放置4h后,移去上清液。每孔加150μlDMSO 30min后利用自动酶标仪在570nm波长处检测各孔的光密度(OD值)。
半数抑制浓度(IC50)定义为当50%的肿瘤细胞存活时的药物浓度。根据测定的光密度(OD值),制作细胞生长抑制率的标准曲线,在标准曲线上求得其对应的药物浓度。
(9)CDK2活性抑制实验,用FlashPlate TM实验法检测。
表1.化合物1-16对Hela细胞增殖和CDK2的抑制作用
Claims (4)
2.一种制备上述的吡唑苯胺衍生物的方法,它由下列步骤组成:
步骤1.将取代基苯乙酮(20mmol)、盐酸苯肼(25mmol)、无水乙酸钠(30mmol)分别溶于乙醇,磁力搅拌,50-60℃反应3h(TLC点板检测反应)。反应完毕后将反应液旋干,乙酸乙酯和水萃取2-3次,旋干乙酸乙酯层,得到黄色固体。
步骤2.先将DMF(4ml)和POCl3(5ml)混合冰浴30min,然后将步骤1产物溶于5ml DMF滴加至上述混合液中,恢复常温继续搅拌30min,至70-80℃反应5h,冷却至常温后,倒入冰水中,用50%NaOH调PH至7-8,抽滤并水洗3遍,烘干后得固体。
步骤3.将步骤2所得产物(1mmol)溶于乙醇(20ml),加入不同取代基苯胺(1mmol),60℃反应5h(TLC检测反应进行程度)。反应完毕后将反应液旋干,四氢呋喃(15ml)溶解后加入NaBH4(2mmol)。待气泡消失后冰浴滴加I2的四氢呋喃溶液。30min后转入常温至40-50℃反应12h(TLC检测)。反应完毕后旋干反应液,乙酸乙酯和水萃取2-3次,无水Na2SO4干燥,旋干有机层,再减压蒸干溶剂即得到固体物。重结晶或过柱纯化固体物可得到纯净的化合物1-16。
3.根据权利要求2所述的吡唑苯胺衍生物的制备方法。
4.权利要求1所述的吡唑苯胺衍生物在制备抗肿瘤药物中的应用。
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