CN111153817B - 苯氧基-n-苯基苯胺衍生物及其应用 - Google Patents
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Abstract
Description
技术领域
本发明涉及苯氧基-N-苯基苯胺衍生物及其应用,属于新型结直肠癌药物技术领域。
背景技术
在全球范围内,结直肠癌(CRC)是癌症死亡的第二大主要原因,在女性常见癌症中排行第二,在男性常见癌症中排行第三。据统计,2018年全球大肠癌患者超过630万例,新诊断出180万例,导致880792人死亡。世界经济论坛估计,2010年与大肠癌相关的检查和治疗成本约为316亿美元,CRC对患者和社会造成了巨大的经济负担。尽管目前有一些CRC治疗方法,比如化学疗法,手术,放疗和分子靶向疗法等,但这些治疗方法的临床预后仍然不尽人意,特别是对于CRC转移的患者,治疗效果并不显著。此外,肿瘤异质性还将导致人体对化学疗法和靶向疗法产生抗性反应。目前用于结直肠癌患者的一线药物,如伊立替康,5-氟尿嘧啶及其衍生的前药,铂类药物等,不但临床疗效差,而且患者预后不好,会产生严重耐药性反应等缺陷。因此,需要寻找新型的有效药物来进行CRC治疗。
c-Myc是Myc原癌基因家族的成员,在CRC中常见的遗传异常是c-Myc的表达升高,在转基因小鼠实验研究中证明了c-Myc表达在CRC中的重要性,通过下调c-Myc可以抑制肠息肉,这是结肠癌过程中最重要的病变。因此,通过c-Myc的表达可以帮助抑制肿瘤的发生和发展。目前,现有的c-Myc抑制剂的作用类型主要包括:抑制Myc-Max蛋白质-蛋白质相互作用,例如10058-F4、10074-G5,SF-4-017,NUCC-0176242;抑制Myc-Max蛋白质-DNA结合相互作用,例如Mycro 3,KJ Pyr 9;与c-Myc G-quadruplex(G4)稳定剂的结合,例如茚三异喹啉5,IZCZ-3等。现有的c-Myc抑制剂的结构式如图1所示,目前尚无临床上可用的c-Myc抑制剂药物。而且,许多已知的c-Myc抑制剂由于体内疗效低或抗肿瘤活性差而不适用于临床研究,目前急需开发更多结构新颖的c-Myc抑制剂。所以,研究探索具有新型骨架的c-Myc小分子抑制剂显得尤为重要。
发明内容
针对以上缺陷,本发明解决的技术提供一些具有c-Myc抑制活性的新化合物。
本发明苯氧基-N-苯基苯胺衍生物的结构式为式Ⅰ所示:
其中,R1为氢或C1-C3烷氧基;R2为氢或C1-C3烷氧基;R3为氢或C1-C3烷氧基;R4为氢或C1-C3烷氧基;
优选的,苯氧基-N-苯基苯胺衍生物的结构式为式Ⅱ或式Ⅲ所示:
优选的,R1为氢或C1-C3烷氧基;R2为氢或C1-C3烷氧基;R3为氢或C1-C3烷氧基;R4为氢或C1-C3烷氧基;R5为R6为氢、甲基、羟基、甲氧基、氟、氯或溴。优选的,R1为氢或甲氧基;R2为氢或甲氧基;R3为氢或甲氧基;R4为氢或甲氧基;R5为R6为氢、甲基、羟基、甲氧基、氟、氯或溴。
更优选的,R1为氢或C1-C3烷氧基;R2为氢或C1-C3烷氧基;R3为氢或C1-C3烷氧基;R4为氢或C1-C3烷氧基;R5为作为优选方案,R1为氢或甲氧基;R2为氢或甲氧基;R3为氢或甲氧基;R4为氢或甲氧基;R5为
优选的,R1为氢或C1-C3烷氧基;R2为氢或C1-C3烷氧基;R3为氢或C1-C3烷氧基;R4为氢或C1-C3烷氧基;R5为R7为氢或甲氧基。优选的,R1为氢或甲氧基;R2为氢或甲氧基;R3为氢或甲氧基;R4为氢或甲氧基;R5为R7为氢或甲氧基。
本发明还提供本发明所述苯氧基-N-苯基苯胺衍生物在制备治疗结直肠癌药物中的应用。
本发明的化合物,对结直肠癌具有一定的疗效,可用于制备结直肠癌药物中。
本发明还提供本发明所述的苯氧基-N-苯基苯胺衍生物在制备c-Myc抑制剂中的应用。
本发明苯氧基-N-苯基苯胺衍生物,可用作c-Myc抑制剂使用。
本发明还提供本发明所述苯氧基-N-苯基苯胺衍生物的异构体、可药用的盐以及水合物。
本发明还提供本发明所述苯氧基-N-苯基苯胺衍生物的异构体、可药用的盐以及水合物在制备治疗结直肠癌药物中的应用。
本发明还提供本发明所述苯氧基-N-苯基苯胺衍生物的异构体、可药用的盐以及水合物在制备c-Myc抑制剂中的应用。
本发明还提供一种药物组合物,该药物组合物由有效成分和药学上可接受的辅料组成,所述有效成分包含治疗有效量的本发明所述的苯氧基-N-苯基苯胺衍生物或其异构体或可药用的盐或水合物。
与现有技术相比,本发明具有如下有益效果:
本发明设计合成了一系列新型苯氧基-N-苯基苯胺衍生物,该化合物可作为c-Myc抑制剂使用,对结直肠癌细胞的增殖有较好的抑制作用,为结直肠癌药物提供了新的选择。
附图说明
图1为一些现有的c-Myc抑制剂的结构式。
图2为本发明化合物42对CRC细胞和正常肝细胞LO2活力的影响。其中,A为化合物42对HT29,HCT15,HCT116,DLD-1的IC50值,B为化合物42、5-氟尿嘧啶和顺铂对LO2的毒性。相对于DMSO,**P<0.01,***P<0.001。
图3为本发明化合物42与c-Myc/Max的结合模式,其中,A为化合物42与c-Myc/Max的关键氨基酸残基的结合模式;B为c-Myc/Max二聚体表面中化合物42的结合模式;C为与Myc-Max-DNA的化合物42的复合物的计算机结合模式。
图4为用不同浓度的化合物42处理15天后检测到的HT29细胞和HCT15细胞的菌落簇。
图5为化合物42的流式细胞分析结果,其中,A为HT29和HCT15细胞用不同浓度的化合物42处理24h后的细胞周期分布分析;B为用一定浓度的化合物42处理细胞24h,的细胞凋亡水平以及凋亡细胞分析。相对于DMSO,*P<0.05,**P<0.01;***P<0.001。
图6为通过蛋白质印迹确定的化合物42对c-Myc表达的影响。
图7为本发明化合物42在HT29肿瘤异种移植模型中的体内实验结果。其中,A为化合物42处理对雌性BALB/c裸鼠中HT29异种移植肿瘤的生长抑制作用;B为30天治疗期间每组中小鼠的平均体重;C为治疗结束时各组小鼠的肿瘤重量。相对于DMSO,**P<0.01;***P<0.001。
具体实施方式
本发明苯氧基-N-苯基苯胺衍生物的结构式为式Ⅰ所示:
其中,R1为氢或C1-C3烷氧基;R2为氢或C1-C3烷氧基;R3为氢或C1-C3烷氧基;R4为氢或C1-C3烷氧基;
本发明C1-C3烷氧基为碳原子数为1-3的烷氧基,比如甲氧基、乙氧基、丙氧基等。
c-Myc蛋白是内在无序(ID)蛋白的成员,其特征是缺乏稳定的结构和广泛的骨架柔性,这是药物发现的一个相关但具有挑战性的目标,而为了寻求新型c-Myc抑制剂,c-Myc-Max相互作用抑制剂对临床研究最有价值。对现有的小分子抑制剂的核心结构进行研究,发现大多数抑制剂都包含长分子结构,因为c-Myc-Max二聚体界面是左旋,平行和四螺旋束,每个单体由由环隔开的两个R螺旋组成。据此,本发明通过结合核心结构设计了由三个苯环组成的长分子,即式Ⅰ所示的苯氧基-N-苯基苯胺衍生物。
该化合物设计为:
研究表明,R5基团的选择,Ⅱ区与Ⅰ区的连接,以及Ⅰ区与Ⅲ区的连接均可能形成对生物活性的巨大贡献。
作为其中一种实施方式,Ⅱ区与Ⅰ区的连接点为C-3位,即结构式为式Ⅱ所示的化合物:
作为另一种实施方式,Ⅱ区与Ⅰ区的连接点为C-4位,即结构式为式Ⅲ所示的化合物:
更优选的,R1为氢或C1-C3烷氧基;R2为氢或C1-C3烷氧基;R3为氢或C1-C3烷氧基;R4为氢或C1-C3烷氧基;R5为作为优选方案,R1为氢或甲氧基;R2为氢或甲氧基;R3为氢或甲氧基;R4为氢或甲氧基;R5为
作为另一种优选的实施方式,R1为氢或C1-C3烷氧基;R2为氢或C1-C3烷氧基;R3为氢或C1-C3烷氧基;R4为氢或C1-C3烷氧基;R5为R7为氢或甲氧基。优选的,R1为氢或甲氧基;R2为氢或甲氧基;R3为氢或甲氧基;R4为氢或甲氧基;R5为R7为氢或甲氧基。
以下为本发明优选的一些具体化合物的结构式。
本发明苯氧基-N-苯基苯胺衍生物的制备方法,可采用常规的方法,具体的制备方法详见实施例1。
本发明苯氧基-N-苯基苯胺衍生物,可用于制备结直肠癌药物,对结直肠癌具有一定的疗效。
本发明苯氧基-N-苯基苯胺衍生物,可用作c-Myc抑制剂使用。
本发明还提供如通式Ⅰ所示苯氧基-N-苯基苯胺衍生物的异构体、可药用的盐以及水合物。其中,可药用的盐包括但不限于通式Ⅰ化合物与无机酸如盐酸、硫酸、磷酸、亚磷酸、氢溴酸和硝酸所成的盐以及与各种有机酸,如苹果酸、马来酸、柠檬酸、延胡索酸、酒石酸、琥珀酸、乙酸、乳酸、对甲苯磺酸、甲磺酸、棕榈酸等所成的盐。本发明中的一些化合物可能用水或各种有机溶剂结晶或重结晶,在这种情况下,可能形成各种溶剂化物。本发明包括那些化学计量的溶剂化物,包括水合物,也包括在用低压升华干燥法制备时形成的包含可变量水的化合物。
本发明还提供本发明所述苯氧基-N-苯基苯胺衍生物的异构体、可药用的盐以及水合物在制备治疗结直肠癌药物中的应用。
本发明还提供本发明所述苯氧基-N-苯基苯胺衍生物的异构体、可药用的盐以及水合物在制备c-Myc抑制剂中的应用。
本发明的苯氧基-N-苯基苯胺衍生物或其异构体或可药用的盐或水合物可以单独使用,也可与可药用的载体或赋形剂一起以药物组合物的形式使用,当以药物组合物的形式使用时,通常将治疗有效量的本发明化合物或其可药用盐或水合物以及一种或多种可药用载体或稀释剂结合制成适当的施用形式或剂量形式。因此,本发明还提供了药物组合物,它包含治疗有效量的本发明所述的苯氧基-N-苯基苯胺衍生物、其所有可能的异构体或其可药用的盐或水合物以及至少一种可药用的载体。
本发明化合物的药用组合物,可以以下方面的任意方式施与:口服、喷雾吸入、直肠给药、鼻腔给药、***给药、局部给药、非肠道给药如皮下、静脉、肌内、腹膜内、销内、心室内、胸骨内或颅内注射或输入,或借助一种外植的储器用药,其中优选口服、肌注、腹膜内或静脉内用药方式。
本发明化合物或含有它的药物组合物可以单位剂量形式给药。给药剂型可以是液体剂型、固体剂型。液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、温悬剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、温悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂、包合物、埋植剂、贴剂、擦剂等。
本发明的药物组合物中还可以含有常用的载体,这里所述可药用载体包括但不局限于:离子交换剂,氧化铝,硬脂酸铝,卵磷脂,血清蛋白如人血清蛋白,缓冲物质如磷酸盐,甘油,山梨酸,山梨酸钾,饱和植物脂肪酸的部分甘油酯混合物,水,盐或电解质,如硫酸鱼精蛋白,磷酸氢二纳,磷酸氢钾,氧化纳,锌盐,胶态氧化硅,三硅酸镁,聚乙烯吡咯烷酮,纤维素物质,聚乙二醇,羧甲基纤维素纳,聚丙烯酸酯,蜂蜡,羊毛酯等。载体在药物组合物中的含量(重量比)可以是1~98%,通常大约占到80%。为方便起见,局部麻醉剂,防腐剂,缓冲剂等可直接溶于载体中。
下面结合实施例对本发明的具体实施方式做进一步的描述,并不因此将本发明限制在所述的实施例范围之中。
实施例1化合物的制备
本发明苯氧基-N-苯基苯胺衍生物,按照以下工艺合成设计:
其中,化合物7-38的合成流程如下:
试剂和条件:(ⅰ)1-氟-3,5-二甲氧基苯,POCl3,DMF,0℃至60℃,4h,产率62.30%;(ⅱ)3-氨基苯酚,溴苯,t-BuONa,氯(2-二环己基膦基-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯)[2-(2-氨基乙基苯基)]钯(II)(BrettPhos Palladacycle),1,4-二恶烷,90℃,2h,产率86.01%;(ⅲ)3-(苯氨基)苯酚,K2CO3,DMSO,100℃,3h,产率73%-85%;(ⅳ)NaBH(AcO)3,AcOH,DMF,80℃,过夜。产率45%-60%。
化合物3(4-氟-2,6-二甲氧基苯甲醛)的合成:在氩气氛下,将1-氟-3,5-二甲氧基苯(化合物1)(10g,64.06mmol)加入无水DMF(20mL)中,0℃搅拌20min,在15min内加入POCl3(11.77g,76.87mmol),将反应混合物在室温下搅拌30min,然后在60℃下加热4h。反应完成,冷却至室温,倒入冰水(400mL)中。用4N NaOH将水相调节至pH=9-10,用乙酸乙酯(2×400mL)萃取两次,合并有机相,并用盐水洗涤,经无水MgSO4干燥。通过真空旋转蒸发除去有机层,通过柱色谱法纯化,得到7.35g白色固体产物。产率62.30%。1H NMR(400MHz,CDCl3)δ10.44(s,1H),6.77(s,2H),3.91(s,6H).ESI-MS:mass calcd for[M+H]+(C9H9FO3)185.05;found m/z,185.06.
化合物5(3-(苯氨基)苯酚)的合成:3-氨基苯酚(5g,45.82mmol)和溴苯(8.63g,54.98mmol),叔丁醇钠(1.32g,137.46mmol),BrettPhos Palladacycle(182.78mg,0.23mmol,0.5mmol%)加入25mL无水1,4-二恶烷中,于氩气氛下,90℃搅拌,搅拌2h。通过TLC监测反应完成。将反应混合物真空浓缩,加入500mL水,用乙酸乙酯(2×400mL)萃取两次,合并有机相,用盐水洗涤,用无水MgSO4干燥。将有机层真空浓缩并通过硅胶色谱法纯化,得到4.31g棕色固体。产率86.01%。1H NMR(400MHz,CDCl3)δ7.28(dd,J=7.0,1.5Hz,1H),7.25(s,1H),7.14–7.06(m,3H),6.95(dd,J=10.5,4.2Hz,1H),6.64–6.58(m,1H),6.56(t,J=2.2Hz,1H),6.37(ddd,J=8.0,2.3,0.7Hz,1H),5.68(s,1H),4.71(s,1H).ESI-MS:mass calcd for[M+H]+(C12H11NO)186.08;found m/z,186.09.
化合物6a-6d的合成通用方法A:在无水DMSO(15mL)中,在氩气气氛下,向3-(苯氨基)苯酚(3g,16.20mmol)和K2CO3(6.72g,48.59mmol)的溶液中,搅拌加入具有不同取代基的4-氟苯甲醛(16.20mmol)。在100℃下放置3h,,并用薄层硅胶板(TLC)检查反应是否完成。反应完成后用冷水(100mL)淬灭反应,用DCM萃取,用无水MgSO4干燥,真空浓缩,得到粗产物,并通过硅胶色谱纯化,得到所需化合物。
其中,化合物6a:按照上述方法A,由化合物5和4-氟-2,6-二甲氧基苯甲醛制备而成。产率85.21%,棕色固体。1H NMR(400MHz,CDCl3)δ10.30(s,1H),7.25–7.20(m,2H),7.17(s,1H),7.07–7.02(m,2H),6.92(t,J=7.4Hz,1H),6.80(ddd,J=8.1,2.2,0.8Hz,1H),6.73(t,J=2.2Hz,1H),6.53(ddd,J=8.1,2.3,0.8Hz,1H),6.10(s,2H),3.74(s,6H).ESI-MS:mass calcd for[M+H]+(C21H19NO4)350.13;found m/z,350.23.
化合物6b:按照上述方法A,由化合物5和4-氟-2-甲氧基苯甲醛制备而成。产率82.35%,白色固体。1H NMR(400MHz,CDCl3)δ10.31(s,1H),7.77(d,J=8.6Hz,1H),7.30–7.23(m,3H),7.09(d,J=7.6Hz,2H),6.96(t,J=7.4Hz,1H),6.87(dd,J=8.1,1.6Hz,1H),6.78(t,J=2.1Hz,1H),6.62–6.52(m,3H),5.90(s,1H),3.85(s,3H).ESI-MS:mass calcdfor[M+H]+(C20H17NO3)320.12;found m/z,320.13.
化合物6c:按照上述方法A,由化合物5和4-氟-3-甲氧基苯甲醛制备而成。产率78.82%,无色油状。1H NMR(400MHz,CDCl3)1H NMR(400MHz,CDCl3)δ9.89(s,1H),7.51(d,J=1.7Hz,1H),7.39(dd,J=8.2,1.8Hz,1H),7.29–7.22(m,3H),7.08(d,J=7.8Hz,2H),6.97(dd,J=15.6,7.8Hz,2H),6.83(dd,J=8.1,2.0Hz,1H),6.77(t,J=2.1Hz,1H),6.56(dd,J=8.1,2.2Hz,1H),3.96(s,3H).ESI-MS:mass calcd for[M+H]+(C20H17NO3)320.12;foundm/z,320.12.
化合物6d:按照上述方法A,由化合物5和4-氟苯甲醛制备而成。产率73.55%,无色油状。1H NMR(400MHz,CDCl3)δ9.92(s,1H),7.88–7.82(m,2H),7.31–7.25(m,3H),7.09(ddd,J=7.9,4.8,1.8Hz,4H),6.97(dd,J=10.5,4.2Hz,1H),6.90–6.85(m,1H),6.78(t,J=2.2Hz,1H),6.59(ddd,J=8.1,2.2,0.7Hz,1H),5.79(s,1H).ESI-MS:mass calcd for[M+H]+(C19H15NO2)290.11;found m/z,290.12.
化合物7-38的合成通用方法B:在无水DMF(2mL)中,在氩气氛下,加入化合物6a-6d(0.30mmol),三乙酰氧基硼氢化钠(127mg,0.60mmol),不同的胺(0.36mmol)以及乙酸(0.03mmol),80℃搅拌过夜,并用薄层硅胶板(TLC)检查反应是否完成。反应完成后用冷水(100mL)淬灭反应,用乙酸乙酯萃取,用无水MgSO4干燥,真空浓缩,得到粗产物,并通过快速柱色谱法纯化,得到所需化合物。
化合物7的合成:按照上述方法B,由化合物6a和盐酸甲胺制备而成。产率52.50%,棕色固体。1H NMR(400MHz,CDCl3)δ7.28(d,J=7.8Hz,2H),7.22(t,J=8.1Hz,1H),7.10(d,J=7.9Hz,2H),6.96(t,J=7.3Hz,1H),6.84(d,J=8.1Hz,1H),6.73(s,1H),6.56(d,J=7.9Hz,1H),6.23(s,2H),4.21(s,2H),3.81(s,6H),2.54(s,3H).13C NMR(101MHz,CDCl3)δ160.42,160.03,157.26,145.23,142.27,130.47,129.41,121.76,118.68,112.52,111.12,107.78,101.09,94.70,56.01,40.47,31.19.ESI-MS:mass calcd for[M+H]+(C22H24N2O3)365.18;found m/z,365.19.
化合物8的合成:按照上述方法B,由化合物6a和环丙胺制备而成。产率61.05%,棕色固体。1H NMR(400MHz,CDCl3)δ7.25(d,J=7.2Hz,2H),7.19(t,J=8.1Hz,1H),7.09(d,J=7.6Hz,2H),6.95(t,J=7.4Hz,1H),6.80(dd,J=8.0,2.0Hz,1H),6.71(t,J=2.2Hz,1H),6.54(dd,J=8.1,2.2Hz,1H),6.26(s,2H),5.81(s,1H),4.02(s,2H),3.77(s,6H),2.20(tt,J=7.2,3.7Hz,1H),0.71(m,2H),0.52(q,J=6.6Hz,2H).13C NMR(101MHz,CDCl3)δ159.82,145.11,142.36,130.39,129.38,121.64,118.56,112.16,110.77,107.44,95.18,55.92,40.47,29.37,4.40.ESI-MS:mass calcd for[M+H]+(C24H26N2O3)391.19;found m/z,391.20.
化合物9的合成:按照上述方法B,由化合物6a和2-丙胺制备而成。产率52.36%,棕色固体。1H NMR(400MHz,CDCl3)δ7.29(s,2H),7.25(s,1H),7.20(t,J=8.1Hz,1H),7.10(d,J=7.7Hz,2H),6.95(t,J=7.3Hz,1H),6.82(d,J=8.1Hz,1H),6.70(d,J=2.0Hz,1H),6.55(dd,J=8.1,1.7Hz,1H),6.25(s,2H),5.92(s,1H),4.02(s,2H),3.77(s,6H),2.95(dt,J=12.9,6.4Hz,1H),1.23(d,J=6.4Hz,6H).13C NMR(101MHz,CDCl3)δ159.57,157.93,145.09,142.36,130.38,129.38,121.64,118.55,112.05,110.67,107.33,95.42,55.88,46.94,40.29,37.81,23.17.ESI-MS:mass calcd for[M+H]+(C24H28N2O3)393.21;found m/z,393.21.
化合物10的合成:按照上述方法B,由化合物6a和二甲胺制备而成。产率45.77%,棕色固体。1H NMR(400MHz,CDCl3)δ7.31–7.27(m,2H),7.23(d,J=8.1Hz,1H),7.11(d,J=7.6Hz,2H),6.97(t,J=7.3Hz,1H),6.86(dd,J=8.1,1.9Hz,1H),6.76(t,J=2.2Hz,1H),6.58(dd,J=8.0,2.1Hz,1H),6.25(s,2H),5.89(s,1H),4.22(s,2H),3.81(s,6H),2.72(s,6H).13C NMR(101MHz,CDCl3)δ161.35,160.49,156.76,145.32,142.18,130.58,129.43,121.90,118.78,112.95,111.44,108.02,99.54,94.50,56.04,49.03,42.40,29.70.ESI-MS:mass calcd for[M+H]+(C23H26N2O3)379.19;found m/z,379.20.
化合物11的合成:按照上述方法B,由化合物6a和2,2'-氮杂二基双(乙烷-1-醇)制备而成。产率56.15%,黄色油状。1H NMR(400MHz,CDCl3)δ7.29(d,J=7.8Hz,1H),7.23(d,J=8.1Hz,1H),7.11(d,J=7.9Hz,1H),6.97(t,J=7.3Hz,1H),6.86(dd,J=8.2,1.5Hz,1H),6.76(s,1H),6.58(dd,J=7.9,1.6Hz,1H),6.25(s,1H),5.88(s,1H),4.35(s,1H),3.99(s,2H),3.81(s,3H),3.28(s,2H).13C NMR(101MHz,CDCl3)δ160.02,130.58,129.41,121.84,118.76,112.88,111.44,108.05,94.65,56.15,56.10,55.35,47.58.ESI-MS:mass calcdfor[M+H]+(C23H26N2O3)379.19;found m/z,379.20.ESI-MS:mass calcd for[M+H]+(C25H30N2O5)439.22;found m/z,439.23.
化合物12的合成:按照上述方法B,由化合物6a和N-(2-氨基乙基)乙酰胺制备而成。产率61.42%,棕色油状。1H NMR(400MHz,CDCl3)δ7.27(d,J=7.0Hz,2H),7.20(t,J=8.1Hz,1H),7.13–7.08(m,2H),7.00(s,1H),6.94(t,J=7.3Hz,1H),6.83(dd,J=8.1,1.5Hz,1H),6.64(t,J=2.2Hz,1H),6.55(dd,J=8.1,1.6Hz,1H),6.26(s,2H),6.08(s,1H),3.92(s,2H),3.77(s,6H),3.39(d,J=5.3Hz,2H),2.84–2.73(m,2H),1.97(s,3H).13C NMR(101MHz,CDCl3)δ170.65,159.51,158.60,158.02,145.15,142.43,130.37,129.36,121.57,118.51,111.85,110.59,107.22,95.44,55.88,46.94,40.29,37.81,23.17.ESI-MS:mass calcd for[M+H]+(C25H29N3O4)436.22;found m/z,436.23.
化合物13的合成:按照上述方法B,由化合物6a和2-(哌嗪-1-基)乙-1-醇制备而成。产率53.18%,棕色油状。1H NMR(400MHz,CDCl3)δ7.28(dd,J=7.0,1.4Hz,2H),7.23(t,J=8.1Hz,1H),7.14–7.08(m,2H),6.96(t,J=7.4Hz,1H),6.84(dd,J=8.1,1.5Hz,1H),6.75(t,J=2.2Hz,1H),6.57(dd,J=8.1,1.6Hz,1H),6.25(s,2H),5.88(s,1H),4.08(s,2H),3.76(s,6H),3.70–3.65(m,2H),2.93(s,7H),2.72–2.66(m,2H).13C NMR(101MHz,CDCl3)δ160.54,157.23,145.23,142.25,130.49,129.41,121.81,118.69,112.62,111.18,107.79,99.99,94.89,59.16,57.64,55.93,50.55,48.17.ESI-MS:mass calcd for[M+H]+(C27H33N3O4)464.25;found m/z,464.26.
化合物14的合成:按照上述方法B,由化合物6a和1-乙基哌嗪制备而成。产率62.33%,棕色固体。1H NMR(400MHz,CDCl3)δ7.29(s,1H),7.25(s,1H),7.20(t,J=8.1Hz,1H),7.10(d,J=8.0Hz,2H),6.95(t,J=7.3Hz,1H),6.81(d,J=8.1Hz,1H),6.71(s,1H),6.55(dd,J=8.1,1.9Hz,1H),6.24(s,2H),5.88(s,1H),3.84(s,2H),3.73(s,6H),2.72(d,J=46.0Hz,8H),2.50–2.44(m,2H),1.10(t,J=7.1Hz,3H).13C NMR(101MHz,CDCl3)δ160.30,158.01,145.04,142.40,130.34,129.38,121.61,118.55,112.07,110.73,107.38,95.41,55.82,52.10,51.92,51.50,48.48,11.67.ESI-MS:mass calcd for[M+H]+(C27H33N3O3)448.25;found m/z,448.25.
化合物15的合成:按照上述方法B,由化合物6a和金刚烷胺制备而成。产率41.27%,黄色固体。1H NMR(400MHz,CDCl3)δ7.28(s,1H),7.24(s,1H),7.21(d,J=8.1Hz,1H),7.12(d,J=7.7Hz,2H),6.94(t,J=7.3Hz,1H),6.85(d,J=8.1Hz,1H),6.69(s,1H),6.55(dd,J=8.0,1.6Hz,1H),6.23(d,J=8.1Hz,2H),4.16(s,2H),3.82(s,6H),2.13(s,3H),2.00(s,6H),1.64(s,6H).13C NMR(101MHz,CDCl3)δ159.91,159.42,157.66,145.22,142.36,130.38,129.34,121.57,118.62,111.91,110.82,107.57,102.98,95.45,58.30,56.16,38.68,35.58,33.64,29.11.ESI-MS:mass calcd for[M+H]+(C31H36N2O3)485.27;found m/z,485.28.
化合物16的合成:按照上述方法B,由化合物6a和NaBH4(2mg,0.057mmol)在甲醇中室温搅拌2min制备而成。反应后真空浓缩,得到粗产物,并通过硅胶色谱纯化,得到化合物16。产率65.25%,无色油状。1H NMR(400MHz,CDCl3)δ7.28–7.24(m,2H),7.19(t,J=8.1Hz,1H),7.08(d,J=7.6Hz,2H),6.96(d,J=7.3Hz,1H),6.79(dd,J=8.0,1.9Hz,1H),6.72(t,J=2.2Hz,1H),6.54(dd,J=8.1,1.8Hz,1H),6.26(s,2H),5.78(s,1H),4.73(s,2H),3.77(s,6H).ESI-MS:mass calcd for[M+H]+(C21H21NO4)352.15;found m/z,352.16.
化合物17的合成:按照上述方法B,由化合物6a和苯甲胺制备而成。产率58.02%,棕色固体。1H NMR(400MHz,CDCl3)δ7.31(dt,J=9.7,4.2Hz,5H),7.23(d,J=6.4Hz,2H),7.19(t,J=8.1Hz,1H),7.08(d,J=7.8Hz,2H),6.94(t,J=7.3Hz,1H),6.78(dd,J=8.0,1.6Hz,1H),6.72(t,J=2.1Hz,1H),6.54(dd,J=8.1,2.0Hz,1H),6.26(s,2H),5.75(s,1H),3.84(s,2H),3.76(s,2H),3.73(s,6H).13C NMR(101MHz,CDCl3)δ159.45,158.45,157.27,144.94,142.43,140.86,130.30,129.39,128.20,126.67,121.62,118.50,111.91,111.74,110.49,107.19,95.62,55.73,53.12,41.05.ESI-MS:mass calcd for[M+H]+(C28H28N2O3)441.21;found m/z,441.21.
化合物18的合成:按照上述方法B,由化合物6a和(4-氟苯基)甲胺制备而成。产率53.72%,黄色固体。1H NMR(400MHz,CDCl3)δ7.32–7.23(m,4H),7.18(t,J=8.1Hz,1H),7.07(d,J=7.7Hz,2H),7.02–6.91(m,3H),6.77(dd,J=8.1,1.4Hz,1H),6.71(t,J=2.0Hz,1H),6.53(dd,J=8.1,1.7Hz,1H),6.25(s,2H),5.80(s,1H),3.82(s,2H),3.72(s,8H).13C NMR(101MHz,CDCl3)δ163.03,160.61,159.44,158.39,157.37,144.99,142.46,136.61,136.58,130.32,129.75,129.67,129.59,129.39,121.60,121.25,118.50,115.01,114.80,111.97,111.59,110.50,107.23,95.59,55.73,52.33,40.92.ESI-MS:mass calcd for[M+H]+(C28H27FN2O3)459.20;found m/z,459.21.
化合物19的合成:按照上述方法B,由化合物6a和(4-氯苯基)甲胺制备而成。产率58.87%,棕色固体。1H NMR(400MHz,CDCl3)δ7.27–7.22(m,6H),7.19(d,J=8.1Hz,1H),7.10–7.05(m,2H),6.93(t,J=7.4Hz,1H),6.80–6.75(m,1H),6.71(t,J=2.2Hz,1H),6.56–6.51(m,1H),6.25(s,2H),5.80(s,1H),3.81(s,2H),3.72(s,8H).13C NMR(101MHz,CDCl3)δ159.44,158.34,157.46,144.99,142.45,139.26,132.34,130.33,129.58,129.39,128.28,121.61,118.51,112.01,111.35,110.53,107.26,95.54,55.73,52.25,40.86.ESI-MS:masscalcd for[M+H]+(C28H27ClN2O3)475.17;found m/z,475.17.
化合物20的合成:按照上述方法B,由化合物6a和(4-溴苯基)甲胺制备而成。产率61.25%,棕色固体。1H NMR(400MHz,CDCl3)δ7.42(d,J=8.3Hz,2H),7.26–7.17(m,5H),7.08(d,J=7.6Hz,2H),6.94(t,J=7.4Hz,1H),6.80–6.76(m,1H),6.72(t,J=2.2Hz,1H),6.56–6.52(m,1H),6.25(s,2H),5.77(s,1H),3.81(s,2H),3.72(d,J=6.5Hz,8H).13C NMR(101MHz,CDCl3)δ159.43,158.33,157.46,144.97,142.44,139.79,131.23,130.33,129.96,129.39,121.63,120.42,118.51,112.01,111.35,110.55,107.26,95.54,55.73,52.28,40.85.ESI-MS:mass calcd for[M+H]+(C28H27BrN2O3)519.12,521.12;found m/z,519.13,521.13.
化合物21的合成:按照上述方法B,由化合物6a和4-(氨基甲基)苯酚制备而成。产率48.73%,棕色油状.1H NMR(400MHz,CDCl3)δ7.26–7.22(m,2H),7.18(t,J=8.1Hz,1H),7.07(dd,J=8.5,1.0Hz,2H),7.02(d,J=8.4Hz,2H),6.93(t,J=7.3Hz,1H),6.81–6.77(m,1H),6.69(t,J=2.2Hz,1H),6.58–6.51(m,3H),6.23(s,2H),5.84(s,1H),3.89(s,2H),3.70(s,6H),3.62(s,2H).13C NMR(101MHz,CDCl3)δ159.46,158.21,157.75,156.24,145.01,142.45,130.33,129.97,129.71,129.37,121.60,118.51,115.76,111.97,110.61,110.16,107.34,95.46,55.74,52.09,40.74.ESI-MS:mass calcd for[M+H]+(C28H28N2O4)457.20;found m/z,457.21.
化合物22的合成:按照上述方法B,由化合物6a和(4-甲氧基苯基)甲胺制备而成。产率55.61%,棕色固体。1H NMR(400MHz,CDCl3)δ7.35(d,J=8.5Hz,2H),7.26(s,2H),7.20(t,J=8.1Hz,1H),7.10(d,J=7.7Hz,2H),6.95(t,J=7.3Hz,1H),6.88–6.83(m,3H),6.66(t,J=2.1Hz,1H),6.53(dd,J=8.0,1.9Hz,1H),6.20(s,2H),5.98(s,1H),4.07(s,2H),3.91(s,2H),3.77(s,3H),3.73(s,6H).13C NMR(101MHz,CDCl3)δ159.85,159.65,157.58,145.19,142.35,131.06,130.41,129.39,124.88,121.68,118.60,114.11,112.13,110.86,107.53,95.06,55.90,55.29,49.65,39.00.ESI-MS:mass calcd for[M+H]+(C29H30N2O4)471.22;found m/z,471.22.
化合物23的合成:按照上述方法B,由化合物6a和对甲苯甲胺制备而成。产率52.72%,棕色固体。1H NMR(400MHz,CDCl3)δ7.21–7.18(m,2H),7.15(dd,J=10.1,3.5Hz,3H),7.11(t,J=8.1Hz,1H),7.04(d,J=7.9Hz,2H),7.01(dd,J=8.5,1.0Hz,2H),6.86(dd,J=10.5,4.2Hz,1H),6.72(dd,J=8.1,1.5Hz,1H),6.64(t,J=2.2Hz,1H),6.48–6.44(m,1H),6.17(s,2H),5.74(s,1H),3.80(s,2H),3.68(s,2H),3.65(s,6H),2.25(s,3H).13C NMR(101MHz,CDCl3)δ159.49,158.27,157.70,145.00,142.43,136.62,136.36,130.31,129.38,129.00,128.39,121.61,118.52,111.97,110.55,110.29,107.28,95.50,55.75,52.35,40.73,21.11.ESI-MS:mass calcd for[M+H]+(C29H30N2O3)455.23;found m/z,455.24.
化合物24的合成:按照上述方法B,由化合物6a和苯并[d][1,3]二恶唑-5-基甲胺制备而成。产率58.36%,棕色固体。1H NMR(400MHz,CDCl3)δ7.28(d,J=8.5Hz,2H),7.20(t,J=8.1Hz,1H),7.10(d,J=7.6Hz,2H),6.99(s,1H),6.95(t,J=7.4Hz,1H),6.90(d,J=7.9Hz,1H),6.84(dd,J=8.1,1.6Hz,1H),6.74(d,J=7.9Hz,1H),6.67(t,J=2.2Hz,1H),6.52(dd,J=8.0,1.7Hz,1H),6.19(s,2H),5.92(s,2H),4.09(s,2H),3.90(s,2H),3.75(s,6H).13C NMR(101MHz,CDCl3)δ160.08,159.67,157.35,148.17,147.94,145.21,142.31,130.44,129.41,125.31,123.86,121.74,118.64,112.32,111.01,110.18,108.43,107.69,102.21,101.31,94.87,55.95,49.76,38.85.ESI-MS:mass calcd for[M+H]+(C29H28N2O5)485.20;found m/z,485.20.
化合物25的合成:按照上述方法B,由化合物6a和(R)-1-苯基乙-1-胺制备而成。产率51.83%,棕色固体。1H NMR(400MHz,CDCl3)δ7.51–7.45(m,2H),7.32(ddd,J=9.4,6.6,1.9Hz,4H),7.24(t,J=1.9Hz,1H),7.17(t,J=8.1Hz,1H),7.09(dd,J=8.5,1.0Hz,2H),6.95(t,J=7.3Hz,1H),6.81(dd,J=8.1,1.5Hz,1H),6.67(t,J=2.2Hz,1H),6.50(dd,J=8.1,1.6Hz,1H),6.16(s,2H),5.92(s,1H),3.98(d,J=6.7Hz,1H),3.97–3.92(m,2H),3.71(s,6H),1.64(d,J=6.8Hz,3H).13C NMR(101MHz,CDCl3)δ159.64,159.27,157.75,145.10,142.40,130.33,129.39,128.70,128.30,127.57,121.62,118.54,112.10,110.75,107.49,95.04,57.98,55.80,38.52,21.10.ESI-MS:mass calcd for[M+H]+(C29H30N2O3)455.23;found m/z,455.23.
化合物26的合成:按照上述方法B,由化合物6a和(R)-1-(4-甲氧基苯基)乙-1-胺制备而成。产率50.07%,棕色固体。1H NMR(400MHz,CDCl3)δ7.30–7.25(m,4H),7.18(t,J=8.1Hz,1H),7.09(d,J=7.7Hz,2H),6.94(t,J=7.3Hz,1H),6.86(d,J=8.6Hz,2H),6.79(dd,J=8.1,1.4Hz,1H),6.69(t,J=2.1Hz,1H),6.53(dd,J=8.1,1.6Hz,1H),6.23(s,2H),5.79(s,1H),3.80(s,3H),3.71(s,6H),3.69(s,2H),1.34(d,J=6.6Hz,3H).13C NMR(101MHz,CDCl3)δ159.40,158.47,158.46,157.24,144.94,142.46,130.28,129.38,127.86,121.59,118.49,113.57,111.81,110.47,107.16,95.65,56.83,55.68,55.28,39.50,24.26.ESI-MS:mass calcd for[M+H]+(C30H32N2O4)485.24;found m/z,485.25.
化合物27的合成:按照上述方法B,由化合物6a和2-苯基乙胺制备而成。产率61.09%,棕色固体。1H NMR(400MHz,CDCl3)δ7.28(d,J=4.6Hz,3H),7.22(dd,J=7.6,2.9Hz,2H),7.17–7.12(m,2H),7.09(d,J=7.6Hz,2H),6.96(t,J=7.4Hz,1H),6.83(dd,J=8.1,1.5Hz,1H),6.71(t,J=2.2Hz,1H),6.53(dd,J=8.1,1.7Hz,1H),6.18(s,2H),5.85(s,1H),4.19(s,2H),3.65(s,6H),3.11(d,J=6.2Hz,2H),3.06(d,J=6.2Hz,2H).13C NMR(101MHz,CDCl3)δ159.92,159.62,157.36,145.21,142.24,136.87,130.48,129.42,128.92,128.90,127.05,121.83,118.69,112.52,110.96,107.59,94.94,55.88,47.27,40.17,32.62.ESI-MS:mass calcd for[M+H]+(C29H30N2O3)455.23;found m/z,455.24.
化合物28的合成:按照上述方法B,由化合物6a和2-(4-氯苯基)乙烷-1-胺制备而成。产率52.36%,棕色固体。1H NMR(400MHz,CDCl3)δ7.29(s,2H),7.25(d,J=2.5Hz,2H),7.21(d,J=8.1Hz,1H),7.09(dd,J=7.5,5.3Hz,4H),6.96(t,J=7.3Hz,1H),6.83(dd,J=8.1,2.1Hz,1H),6.71(t,J=2.1Hz,1H),6.53(dd,J=8.1,2.2Hz,1H),6.19(s,2H),5.86(s,1H),4.17(s,2H),3.67(s,6H),3.07(d,J=6.4Hz,2H),3.02(d,J=6.4Hz,2H).13C NMR(101MHz,CDCl3)δ160.43,159.67,157.11,145.27,142.19,134.94,133.15,130.55,130.30,129.44,129.10,121.89,112.72,111.03,107.69,94.81,55.95,46.78,40.05,31.53.ESI-MS:mass calcd for[M+H]+(C29H29ClN2O3)489.19;found m/z,489.20.
化合物29的合成:按照上述方法B,由化合物6a和2-(4-氟苯基)乙-1-胺制备而成。产率48.28%,黄色固体。1H NMR(400MHz,CDCl3)δ7.25(t,J=7.9Hz,2H),7.20–7.06(m,5H),6.99–6.90(m,3H),6.77(dd,J=8.0,1.5Hz,1H),6.70(t,J=2.0Hz,1H),6.52(dd,J=8.1,1.7Hz,1H),6.23(s,2H),5.81(s,1H),3.81(s,2H),3.67(s,6H),2.80(dd,J=11.0,5.3Hz,4H).13C NMR(101MHz,CDCl3)δ162.60,160.18,159.34,158.41,157.28,144.98,142.46,136.02,135.99,130.30,130.14,130.07,129.38,121.59,118.49,115.12,114.92,111.94,111.39,110.42,107.17,95.56,55.66,50.15,41.15,35.31.ESI-MS:mass calcd for[M+H]+(C29H29FN2O3)473.22;found m/z,473.23.
化合物30的合成:按照上述方法B,由化合物6a和2-(对甲苯基)乙-1-胺制备而成。产率55.37%,黄色油状。1H NMR(400MHz,CDCl3)1H NMR(400MHz,CDCl3)δ7.26(s,1H),7.23(d,J=5.4Hz,1H),7.17(t,J=8.1Hz,1H),7.10–7.03(m,6H),6.93(t,J=7.3Hz,1H),6.77(dd,J=8.0,1.6Hz,1H),6.70(t,J=2.2Hz,1H),6.52(dd,J=8.1,1.8Hz,1H),6.22(s,2H),5.89(s,1H),3.86(s,2H),3.65(s,6H),2.83(dd,J=10.6,5.3Hz,4H),2.29(s,3H).13C NMR(101MHz,CDCl3)δ159.43,158.29,157.67,145.06,142.48,136.73,135.53,130.31,129.38,129.09,128.68,121.58,118.52,111.98,110.47,110.08,107.24,95.47,55.68,49.79,40.94,35.14,21.02.ESI-MS:mass calcd for[M+H]+(C30H32N2O3)469.24;foundm/z,469.25.
化合物31的合成:按照上述方法B,由化合物6a和4-(2-氨基乙基)苯酚制备而成。产率51.03%,棕色油状。1H NMR(400MHz,MeOD)δ7.21(td,J=8.2,4.6Hz,3H),7.11–7.04(m,4H),6.89–6.84(m,2H),6.77(s,1H),6.74(dd,J=4.4,2.2Hz,2H),6.51–6.47(m,1H),6.36(s,2H),4.21(s,2H),3.79(s,6H),3.17–3.12(m,2H),2.94–2.87(m,2H).13C NMR(101MHz,CDCl3)δ165.12,163.81,160.91,160.36,150.01,146.90,134.04,133.31,132.73,130.79,124.57,121.86,119.31,116.20,114.04,110.78,105.23,98.17,59.12,43.41,34.69.ESI-MS:mass calcd for[M+H]+(C29H30N2O4)471.22;found m/z,471.23.
化合物32的合成:按照上述方法B,由化合物6a和2-(4-甲氧基苯基)乙-1-胺制备而成。产率58.95%,棕色固体。1H NMR(400MHz,CDCl3)δ7.25(t,J=7.9Hz,2H),7.18(t,J=8.1Hz,1H),7.08(dd,J=8.5,2.1Hz,4H),6.94(t,J=7.3Hz,1H),6.85–6.76(m,3H),6.70(d,J=1.9Hz,1H),6.52(dd,J=8.1,2.1Hz,1H),6.22(s,2H),5.85(s,1H),3.84(s,2H),3.77(s,3H),3.67(s,6H),2.83(d,J=5.8Hz,2H),2.79(d,J=6.0Hz,2H).13C NMR(101MHz,CDCl3)δ160.25,159.73,158.67,157.23,145.26,142.26,130.48,129.92,129.41,128.46,121.78,118.68,114.29,112.52,111.00,107.67,101.40,94.81,55.91,55.29,47.02,39.72,31.23.ESI-MS:mass calcd for[M+H]+(C30H32N2O4)485.24;found m/z,484.25.
化合物33的合成:按照上述方法B,由化合物6a和2-(2-甲氧基苯基)乙-1-胺制备而成。产率47.64%,棕色固体。1H NMR(400MHz,CDCl3)δ7.25(t,J=7.9Hz,2H),7.20–7.11(m,3H),7.08(d,J=7.6Hz,2H),6.94(t,J=7.3Hz,1H),6.88–6.76(m,3H),6.70(t,J=2.1Hz,1H),6.51(dd,J=8.1,1.7Hz,1H),6.22(s,2H),5.83(s,1H),3.87(s,2H),3.75(s,3H),3.67(s,6H),2.87(s,4H).13C NMR(101MHz,CDCl3)δ159.40,158.37,157.68,157.45,145.01,142.45,130.42,130.29,129.38,128.29,127.38,121.59,120.40,118.52,111.92,110.40,110.38,107.17,95.52,55.70,55.26,48.33,41.06,30.36.ESI-MS:mass calcdfor[M+H]+(C30H32N2O4)485.24;found m/z,484.25.
化合物34的合成:按照上述方法B,由化合物6a和2-(3-甲氧基苯基)乙-1-胺制备而成。产率52.11%,棕色固体。1H NMR(400MHz,CDCl3)δ7.24(t,J=7.9Hz,2H),7.17(t,J=8.0Hz,2H),7.07(d,J=7.7Hz,2H),6.93(t,J=7.3Hz,1H),6.81–6.67(m,5H),6.52(dd,J=8.1,1.8Hz,1H),6.22(s,2H),5.87(s,1H),3.83(s,2H),3.76(s,3H),3.66(s,6H),2.83(dd,J=15.7,5.8Hz,4H).13C NMR(101MHz,CDCl3)δ159.68,159.38,158.42,157.33,145.01,142.49,141.90,130.29,129.38,129.31,121.55,121.22,118.48,114.43,111.87,111.42,111.17,110.41,107.16,95.56,55.66,55.14,49.87,41.13,36.07.ESI-MS:mass calcdfor[M+H]+(C30H32N2O4)485.24;found m/z,484.25.
化合物35的合成:按照上述方法B,由化合物6a和3-(4-甲氧基苯基)丙-1-胺制备而成。产率55.61%,棕色油状。1H NMR(400MHz,CDCl3)δ7.28–7.22(m,2H),7.18(t,J=8.1Hz,1H),7.11–7.05(m,4H),6.93(t,J=7.3Hz,1H),6.83–6.77(m,3H),6.70(t,J=2.2Hz,1H),6.56–6.51(m,1H),6.24(s,2H),5.87(s,1H),3.83(s,2H),3.76(s,3H),3.73(s,6H),2.60(dt,J=24.7,7.5Hz,4H),1.88–1.77(m,2H).13C NMR(101MHz,CDCl3)δ159.47,158.32,157.72,157.59,145.03,142.49,134.26,130.31,129.38,129.29,121.56,118.50,113.75,111.94,110.60,110.49,107.26,95.52,55.77,55.27,48.19,40.99,32.66,31.33.ESI-MS:mass calcd for[M+H]+(C31H34N2O4)499.25;found m/z,499.26.
化合物36的合成:按照上述方法B,由化合物6b和2-(4-甲氧基苯基)乙-1-胺制备而成。产率50.09%,棕色固体。1H NMR(400MHz,CDCl3)δ7.24(dd,J=11.2,4.6Hz,2H),7.16(t,J=8.1Hz,1H),7.12(d,J=8.1Hz,1H),7.10–7.04(m,4H),6.92(t,J=7.3Hz,1H),6.84–6.79(m,2H),6.79–6.75(m,1H),6.69(t,J=2.2Hz,1H),6.56(d,J=2.2Hz,1H),6.51(ddd,J=7.6,5.6,2.0Hz,2H),5.83(s,1H),3.76(s,5H),3.67(s,3H),2.84(dd,J=10.9,4.2Hz,2H),2.77(dd,J=10.7,3.9Hz,2H).13C NMR(101MHz,CDCl3)δ158.71,158.44,158.06,157.29,144.99,142.49,131.96,130.64,130.31,129.69,129.38,122.74,121.54,118.50,113.90,111.96,110.61,110.32,107.37,102.46,55.34,55.29,50.33,48.72,35.08.ESI-MS:mass calcd for[M+H]+(C29H30N2O3)455.23;found m/z,455.23.
化合物37的合成:按照上述方法B,由化合物6c和2-(4-甲氧基苯基)乙-1-胺制备而成。产率55.62%,棕色油状。1H NMR(400MHz,CDCl3)δ7.24–7.20(m,2H),7.12(t,J=8.2Hz,3H),7.06–7.02(m,2H),6.96–6.88(m,3H),6.85–6.78(m,3H),6.71(dd,J=8.0,1.5Hz,1H),6.64(t,J=2.2Hz,1H),6.45(dd,J=7.9,2.0Hz,1H),5.76(s,1H),3.80(s,3H),3.76(s,5H),2.88(t,J=6.8Hz,2H),2.78(t,J=6.9Hz,2H).13C NMR(101MHz,CDCl3)δ159.21,158.13,151.46,144.70,143.78,142.69,136.86,131.86,130.09,129.68,129.32,121.28,121.08,120.63,118.30,113.98,112.67,111.45,109.28,106.14,56.02,55.29,53.54,50.62,35.21.ESI-MS:mass calcd for[M+H]+(C29H30N2O3)455.23;found m/z,455.24.
化合物38的合成:按照上述方法B,由化合物6d和2-(4-甲氧基苯基)乙-1-胺制备而成。产率52.36%,棕色油状。1H NMR(400MHz,CDCl3)δ7.25–7.19(m,4H),7.14(t,J=8.1Hz,1H),7.09(d,J=8.6Hz,2H),7.03(dd,J=8.5,1.0Hz,2H),6.98–6.93(m,2H),6.90(dd,J=10.5,4.2Hz,1H),6.84–6.79(m,2H),6.76–6.72(m,1H),6.67(t,J=2.2Hz,1H),6.51–6.47(m,1H),5.79(s,1H),3.74(s,3H),3.73(s,2H),2.85(dd,J=10.8,3.8Hz,2H),2.74(t,J=7.0Hz,2H).13C NMR(101MHz,CDCl3)δ158.64,158.12,156.01,145.01,142.56,135.30,132.06,130.35,129.71,129.54,129.42,121.53,119.11,118.52,114.01,111.96,110.70,107.49,55.31,53.32,50.76,35.41.ESI-MS:mass calcd for[M+H]+(C28H28N2O2)425.22;found m/z,425.23.
化合物42-45的合成流程如下:
试剂和条件:(ⅰ)3-氨基苯酚,溴苯,t-BuONa,氯(2-二环己基膦基-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯)[2-(2-氨基乙基苯基)]钯(II)(BrettPhosPalladacycle),1,4-二恶烷,90℃,2h。产率82.37%;(ⅱ)4-(苯氨基)苯酚,K2CO3,DMSO,100℃,3h。产率76.55%;(ⅲ)NaBH(AcO)3,AcOH,DMF,80℃,过夜。产率47%-56%。
化合物40的合成:按照与化合物5相同的步骤,由4-氨基苯酚和溴苯制备而成。产率82.37%,棕色固体。1H NMR(400MHz,CDCl3)δ7.24–7.18(m,2H),7.04–6.99(m,2H),6.93–6.86(m,2H),6.83(t,J=7.3Hz,1H),6.80–6.74(m,2H),5.46(s,1H),4.64(s,1H).ESI-MS:mass calcd for[M+H]+(C12H11NO)186.08;found m/z,186.09.
化合物41的合成:采用上述方法A,由化合物39和4-氟-2,6-二甲氧基苯甲醛制备而成。产率76.55%,无色油状。1H NMR(400MHz,CDCl3)δ10.32(s,1H),7.26–7.21(m,2H),7.07–7.02(m,4H),6.96(d,J=8.8Hz,2H),6.93(t,J=7.4Hz,1H),6.12(s,2H),3.72(s,6H).ESI-MS:mass calcd for[M+H]+(C21H19NO4)350.13;found m/z,350.14.
化合物42的合成:采用上述方法B,由化合物41和2-(4-甲氧基苯基)乙-1-胺制备而成。产率56.35%,棕色油状。1H NMR(400MHz,CDCl3)δ7.25(t,J=7.8Hz,2H),7.08(t,J=8.3Hz,4H),7.01(d,J=8.1Hz,2H),6.95(d,J=8.8Hz,2H),6.90(t,J=7.3Hz,1H),6.81(d,J=8.5Hz,2H),6.17(s,2H),5.68(s,1H),3.84(s,2H),3.77(s,3H),3.66(s,6H),2.81(dd,J=13.1,5.6Hz,4H).13C NMR(101MHz,CDCl3)δ159.38,158.75,157.99,150.95,143.82,138.93,132.05,129.69,129.40,120.63,120.28,120.09,116.99,113.82,109.89,94.39,55.66,55.28,49.97,41.03,34.86.ESI-MS:mass calcd for[M+H]+(C30H32N2O4)485.24;found m/z,485.25.
化合物43的合成:采用上述方法B,由化合物41和2-(3-甲氧基苯基)乙-1-胺制备而成。产率51.21%,棕色油状。1H NMR(400MHz,CDCl3)δ7.28–7.25(m,1H),7.24(dd,J=5.7,3.8Hz,1H),7.21–7.16(m,1H),7.09–7.05(m,2H),7.01(dd,J=8.6,1.0Hz,2H),6.98–6.93(m,2H),6.90(t,J=7.3Hz,1H),6.79–6.71(m,3H),6.17(s,2H),5.66(s,1H),3.85(s,2H),3.77(s,3H),3.65(d,J=7.9Hz,6H),2.85(dd,J=12.5,5.6Hz,4H).13C NMR(101MHz,CDCl3)δ161.02,159.93,159.71,149.71,143.49,139.68,138.95,129.77,129.42,121.13,120.95,120.90,119.69,117.32,114.23,112.53,93.70,55.82,55.22,47.29,40.00,32.87.ESI-MS:mass calcd for[M+H]+(C30H32N2O4)485.24;found m/z,485.25.
化合物44的合成:采用上述方法B,由化合物41和2-(2-甲氧基苯基)乙-1-胺制备而成。产率47.38%,棕色油状。1H NMR(400MHz,CDCl3)δ7.19(d,J=7.2Hz,1H),7.16(s,1H),7.12–7.05(m,2H),7.02–6.98(m,2H),6.94(dd,J=8.5,0.9Hz,2H),6.90–6.85(m,2H),6.84(d,J=7.3Hz,1H),6.80–6.77(m,1H),6.75(d,J=8.2Hz,1H),6.10(s,2H),5.60(s,1H),3.79(s,2H),3.70(s,3H),3.59(s,6H),2.79(s,4H).13C NMR(101MHz,CDCl3)δ160.92,159.60,157.50,149.69,143.45,139.75,131.18,129.42,128.49,125.30,120.93,120.86,120.83,119.65,117.36,110.48,93.75,55.82,55.20,45.68,40.21,28.44.ESI-MS:masscalcd for[M+H]+(C30H32N2O4)485.24;found m/z,485.25.
化合物45的合成:采用上述方法B,由化合物41和3-(4-甲氧基苯基)丙-1-胺制备而成。产率53.13%,棕色油状。1H NMR(400MHz,CDCl3)δ7.28(s,1H),7.24(s,1H),7.10–7.06(m,3H),7.05–7.01(m,3H),6.98–6.94(m,2H),6.90(dd,J=11.6,4.1Hz,1H),6.81–6.77(m,2H),6.18(s,2H),5.69(s,1H),3.94(s,2H),3.76(s,3H),3.73(s,6H),2.71–2.66(m,2H),2.59–2.54(m,2H),1.97–1.90(m,2H).13C NMR(101MHz,CDCl3)δ160.89,159.85,157.92,149.86,143.53,139.59,132.78,129.42,129.29,120.90,120.87,119.76,117.28,113.83,93.71,55.85,55.24,45.80,39.55,32.16,28.18.ESI-MS:mass calcd for[M+H]+(C31H34N2O4)499.25;found m/z,499.26.
实施例2化合物对肿瘤细胞的抑制活性
使用的肿瘤细胞株为人结直肠癌细胞株,包括HT29、HCT15、HCT116、DLD-1和SW620,以上肿瘤细胞株购自于美国ATCC公司。
采用MTT法进行肿瘤细胞抑制活性的评估。将细胞以2~4×103细胞/0.1mL/孔的细胞密度接种在96孔板中。培养24h后加入化合物,处理72小时后,向每个孔中添加20μLMTT溶液(5mg/mL),并在37℃下再孵育2~4h。弃去上清液,再向每个孔中加入150μLDMSO,充分摇晃溶解10~15min。使用Spectra MAX M5微孔板分光光度计(Molecular Devices,CA,USA)在570nm处测量96孔板的吸光度值(OD),从而计算得到每个目标化合物对应的肿瘤细胞的相对增殖抑制率,在由软件计算得到IC50值。其结果见表1。IC50值是至少三个独立实验的平均值,并由GraphPad Prism5软件计算。
表1
从表1可以看出,本发明的化合物7~38以及化合物42~45均可以抑制结直肠癌细胞的增殖,其中,化合物42的抑制活性最佳。
图2为本发明化合物42对肿瘤细胞(CRC细胞)和正常肝细胞LO2活力的影响。其中,A为化合物42对HT29,HCT15,HCT116,DLD-1的IC50值,B为化合物42、5-氟尿嘧啶和顺铂对LO2的毒性。数据均为三次重复试验所得。相对于DMSO,**表示P<0.01,***表示P<0.001。
将Ⅱ区与Ⅰ区的连接点改变为C-4位,所得化合物42对HT29的抗肿瘤活性,IC50值为0.32μm(如图2中的A所示),比化合物30高3倍,比阳性药物高31倍(顺铂:IC50=10.75μM,5-氟尿嘧啶:IC50=13.37μM)。通过MTT分析分析了化合物42对人正常肝细胞系(LO2)的毒性,结果如图2中的B所示,化合物42的细胞毒性显着低于阳性药物(顺铂和5-氟尿嘧啶),证明化合物42在体外具有良好的安全性。
实施例3化合物与c-Myc/MAX蛋白的相互作用
采用电泳迁移率实验(EMSA)来分析化合物与c-Myc/MAX蛋白的相互作用,具体方法如下:
1、c-Myc和Max的纯化
将含有b-HLH-Zip结构域的人c-Myc基因(残基353-439)和Max(S)(151个氨基酸残基)通过N端六组氨酸(His 6)标记蛋白的骨架载体pET151D,在重组大肠杆菌BL21DE3中表达。细菌的培养和蛋白质的纯化采用现有的方法,细菌在37℃,225rpm下培养过夜,再培养20h,在0.8mM异丙基-L-硫代-B-D-吡喃半乳糖苷(IPTG,Sigma)中表达蛋白质。然后,收获培养物并在缓冲液(8M尿素,100mM NaH2PO4和10mM Tris)中溶解,通过NTA-Ni-琼脂糖色谱柱(Qiagen,Inc.Chatsworth)纯化,并在存储缓冲液(TrisHCl 50mM,pH 6.5,150mM NaCl和30%甘油)中透析。通过使用Nanodrop定量蛋白质,并将其用于电泳迁移率测定(EMSA)。
2、电泳迁移率测定
按照现有文献所报道方法制备形成包含c-Myc/Max二聚体结合位点的双链DNA生物素化寡核苷酸(5-CACCCGGTCACGTGGCCTACAC-3,50nM)和纯化后的蛋白质复合物(Myc/Max复合物,50nM)。结合反应缓冲液包含10mM EDTA,1×PBS(pH 7.0),500mM KCl,30mM MgCl2、5%甘油,0.1%NP40、1.5mM DTT。10%DMSO。将化合物溶解在DMSO中进行测试,以DMSO作为对照。在室温下制备蛋白质相互作用复合物和20μM化合物1.5h,然后再加入DNA oligo0.5h,将样品加载至8%SDS胶中,缓冲液为10%甘油,0.5TBE溶液,通过Bio-Rad检测ImageJ并分析数据。其结果见表2。表2中所有数据为三次试验的平均值。
表2
化合物编号 | 相对抑制率(%)<sup>a</sup> | 化合物编号 | 相对抑制率(%)<sup>a</sup> |
7 | 2.5 | 26 | 36.1 |
8 | 1.7 | 27 | 45.1 |
9 | N/A | 28 | 43.7 |
10 | 3.2 | 29 | 40.5 |
11 | 6.1 | 30 | 46.2 |
12 | 10.2 | 31 | 51.7 |
13 | N/A | 32 | 75.1 |
14 | 8.9 | 33 | 67.4 |
15 | 12.6 | 34 | 60.8 |
16 | N/A | 35 | 63.4 |
17 | 26.4 | 36 | 42.1 |
18 | 21.2 | 37 | 42.3 |
19 | 25.7 | 38 | 35.6 |
20 | 29.3 | 42 | 83.2 |
21 | 31.8 | 43 | 65.2 |
22 | 48.7 | 44 | 57.3 |
23 | 32.4 | 45 | 70.6 |
24 | 42.1 | DMSO | 0 |
25 | 30.5 |
a通过实验组与对照组(DMSO)的比例计算的相对抑制。
N/A为未测定。
从表2可知,本发明化合物与c-Myc/MAX蛋白有不同的结合抑制率,而在这些分子中,化合物42的效果最佳,表明化合物42具有出色的c-Myc-Max/DNA复合物强大的破坏活性。结果与体外抗增殖活性相对应。
下面重点研究化合物42与c-Myc/MAX的可能结合模式。使用Myc/Max的蛋白晶体结构(PDB ID:1NKP)。如图3所示,阐明了预测的结合模式以及化合物42与Myc/Max的详细相互作用。化合物42可以紧密结合Myc-Max的基本/螺旋-环-螺旋/亮氨酸拉链(bHLHZ)结构域。可以清楚地看到,N-苯基苯胺(Ⅰ区)段的NH和Ala237形成氢键,N-苯基苯胺分别通过疏水特征与ILe242,arg226,His223相互作用。此外,Ⅱ区的两个甲氧基通过氢键与Arg913相互作用。更重要的是,Ⅱ区的NH和Arg913形成了一个额外的氢键。此外,Lys939,Lys918,Arg914中Ⅲ区的苯基分别形成疏水相互作用。此外,整个分子位于Myc-Max二聚体界面和蛋白质-DNA界面,这表明该分子与Myc-Max蛋白质的结合稳定性可能破坏c-Myc/Max/DNA复合物的形成。分子结合模式的所有特征均可预示化合物42的优异抗肿瘤活性。
实施例4化合物42的细胞克隆形成试验
为了测试用化合物42处理的细胞的存活率,将HT29细胞和HCT15细胞(500细胞/孔)铺在6孔板中,并在37℃下孵育过夜,然后加入指定剂量的化合物42(0-5.0μM),以DMSO作为对照)用新鲜培养基培养15天。当观察到细胞形成的集落时终止细胞培养,然后除去上清液并用PBS缓冲溶液洗涤两次,然后在弃去溶液之前将其用4%聚甲醛固定15分钟。用4%多聚甲醛固定菌落,并用0.5%结晶紫溶液染色15分钟,然后除去结晶紫溶液,并使用PBS缓冲溶液冲洗染色液。其结果如图4所示,图4中的数据来自三个独立的实验。处理的培养物中的菌落数表示为对照培养物的百分比。可见,在实验组中,HCT15细胞和HT29细胞的集落形成率显著下降。如图4所示,当42的浓度增加到1.25μM时,在HT29细胞中几乎没有观察到集落形成。相似的是,当HCT15细胞中42的浓度达到2.5μM时,几乎完全抑制了细胞集落的形成。平板菌落形成的结果与MTT测定的结果相对应。
实施例5化合物42诱导的细胞周期分布和细胞凋亡率
采用流式细胞仪(FCM)研究化合物42对细胞周期分布和细胞凋亡率的影响。将HT29细胞和HCT15细胞以每孔3×105细胞的量接种在6孔板中,并孵育24小时,然后用化合物42分别处理24小时。收集细胞,并在4℃下用冰冷的70%乙醇固定12小时,然后除去乙醇,并用冷PBS洗涤细胞。之后,使用胰蛋白酶消化将细胞从平板上分离,通过以1000rpm离心5min收集细胞。然后将细胞重悬于结合缓冲液中,并通过细胞周期检测试剂盒(keygentec,KGA512)和膜联蛋白V-FITC/PI双重标记(keygentec,KGA107)进行染色。使用流式细胞仪(ACEA NovoCyte Advanteon,艾森生物科学公司,美国)分析所得样品。结果见图5。
图5为化合物42的流式细胞分析结果,其中,A为HT29和HCT15细胞用不同浓度的化合物42处理24h后的细胞周期分布分析;B为用一定浓度的化合物42处理细胞24h,并通过FITC-Annexin V/PI评估细胞凋亡水平,并通过流式细胞术分析凋亡细胞。所有数据均来自三组平行实验,*相对于DMSO,P<0.05,**相对于DMSO,P<0.01;***与DMSO相比,P<0.001。
如图5中的A所示,在HT29和HCT15细胞中,受化合物42影响的细胞周期进程都明显停滞在G0/G1期,并且化合物42明显以剂量依赖的方式分化了细胞周期分布。本文中止的细胞周期的抑制抑制了这些细胞的增殖,这反映了它们破坏DNA与c-Myc-Max异二聚体结合的能力。
如图5中的B中所示,发现化合物42种以不同浓度以剂量依赖性方式显着影响HT29和HCT15细胞凋亡。HT29和HCT15细胞处理24h后浓度为10μM的化合物42,可明显引起细胞凋亡,预示着c-Myc对增殖细胞的抑制能力强。
实施例6化合物42的蛋白质印迹(Western blot)分析
采用Western blot分析化合物42抑制c-Myc表达的能力。具体Western blot方法为:在处理前将细胞接种在60×15mm的培养皿中过夜,然后在37℃下与指定剂量的化合物42孵育24小时,然后收集细胞沉淀并重悬于NP40裂解缓冲液(Beyotime)中,并加入额外的-蛋白酶体抑制剂PMSF和磷酸酶抑制剂Cocktail(Sigma)。全细胞蛋白裂解物在冰上孵育30分钟,并在12000rpm和4℃下离心20分钟。使用BCA ProteinAssay Kit(Solarbio PC0020)测定上清液。通过SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)分离样品,并转移至聚偏二氟乙烯膜(Millipore)。将膜与一级抗体在5%BSA/TBST缓冲液中于4℃孵育过夜,同时轻轻摇动。洗涤后,将膜与适当的过氧化物酶偶联的二抗(抗-IgG-HRP1:5000,CST)在室温下孵育1小时,然后再次洗涤,然后使用增强的ECL免疫印迹检测试剂(Millipore)观察蛋白条带通过使用成像仪(Abbkine)拍摄照片。抗体信息:c-Myc抗体(1/1000稀释,CST#9402),β-肌动蛋白(1/1000稀释,CST#4970)。
所有实验均独立重复3次,其结果表明化合物42降低了HT29细胞和HCT15细胞c-Myc蛋白的表达水平。如图6所示,很明显,化合物42对c-Myc蛋白的下调基本上是剂量依赖性的。结果表明,化合物42可能靶向HT29细胞和HCT15细胞中的c-Myc/Max,因此大概下调了它的表达。
实施例7化合物42的体内实验
动物研究是在四川大学实验动物管理委员会的批准下进行的。购买雌性BALB/c裸鼠(北京HFK生物科学有限公司,中国北京)。将HT29细胞(8×106)皮下注射到6-7周龄的雌性BALB/c小鼠中。一旦平均肿瘤体积增长到大约100mm3,将小鼠随机分为五组(每组n=5)。通过经口灌胃给药(10%DMSO,1%Tween-80和生理盐水),化合物42(40mg/kg,80mg/kg,120mg/kg溶于10%DMSO,1%Tween-80和生理盐水),5-氟尿嘧啶(30mg/kg溶于10%DMSO,1%Tween-80和生理盐水)。每两天测定一次肿瘤大小和体重。用游标卡尺测量肿瘤体积并计算为[0.5×最短直径2×最长直径]。使用以下公式计算肿瘤生长的抑制率:100×{1-[治疗组最终肿瘤体积-肿瘤体积初始]/[媒介物治疗组最终肿瘤体积-肿瘤体积初始]]}。其结果见图7。
图7为本发明化合物42在HT29肿瘤异种移植模型中的体内实验结果。其中,A为化合物42处理对雌性BALB/c裸鼠中HT29异种移植肿瘤的生长抑制作用;B为30天治疗期间每组中小鼠的平均体重;C为治疗结束时各组小鼠的肿瘤重量。**相对于DMSO,P<0.01;***与DMSO相比,P<0.001。
如图7所示,化合物42以剂量依赖性方式显著抑制肿瘤生长。在HT29异种移植模型中,分别以120mg/kg,80mg/kg,40mg/kg的剂量观察到肿瘤生长抑制(TGI)为71.58%,61.21%,55.17%。5-氟尿嘧啶以30mg/kg的剂量用作阳性对照,治疗10天后小鼠体重显着降低。相反,在30天内用42进行的治疗显示出优异的抗肿瘤功效,而不会引起明显的体重减轻和毒性。
综上,本发明所有的化合物,尤其是化合物42对于结肠癌肿瘤细胞表现出了优异的抗肿瘤活性,未来有望用于CRC治疗的新型药物开发中。
Claims (12)
2.根据权利要求1所述的苯氧基-N-苯基苯胺衍生物,其特征在于:R1为氢或甲氧基;R2为氢或甲氧基;R3为氢或甲氧基;R4为氢或甲氧基。
7.权利要求1~6任一项所述的苯氧基-N-苯基苯胺衍生物在制备治疗结直肠癌药物中的应用。
8.权利要求1~6任一项所述的苯氧基-N-苯基苯胺衍生物在制备c-Myc抑制剂中的应用。
9.权利要求1~6任一项所述的苯氧基-N-苯基苯胺衍生物的可药用的盐。
10.权利要求9所述的苯氧基-N-苯基苯胺衍生物的可药用的盐在制备治疗结直肠癌药物中的应用。
11.权利要求9所述的苯氧基-N-苯基苯胺衍生物的可药用的盐在制备c-Myc抑制剂中的应用。
12.一种药物组合物,其特征在于:由有效成分和药学上可接受的辅料组成,所述有效成分包含治疗有效量的权利要求1~6任一项所述的苯氧基-N-苯基苯胺衍生物或其可药用的盐。
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