CN115057860B - 一种erk抑制剂及其制药用途 - Google Patents
一种erk抑制剂及其制药用途 Download PDFInfo
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Abstract
本发明提供了一种ERK抑制剂及其制药用途,属于制药领域。本发明提供的式I所示化合物对ERK1/2激酶具有良好的抑制效果,对结直肠癌细胞系表现出有效的抑制活性,能够降低结直肠癌细胞细胞中ERK1/2和p90RSK蛋白在细胞内的磷酸化水平,能够诱导结肠癌细胞凋亡。本发明化合物能够用来制备ERK抑制剂,以及预防和/或治疗与ERK活性相关的疾病的药物,应用前景广阔。
Description
技术领域
本发明属于制药领域,具体涉及一种ERK抑制剂及其制药用途。
背景技术
丝裂原活化蛋白激酶(MAPK)级联是调节多种细胞过程的关键信号通路,在肿瘤细胞增殖、分化、存活、迁移和凋亡等生理过程中起着至关重要的作用。细胞外信号调节激酶(ERK)是在80年代末发现的一类丝氨酸、苏氨酸蛋白激酶,其在正常和病理条件下均可调节细胞信号传导。ERK的表达对人体发育过程至关重要,它们的过度活化在肿瘤细胞的生存和发展中起着至关重要的作用。RAS-RAF-MEK-ERK信号级联是MAPK最经典的通路,有大量研究表明,该信号通路中蛋白激酶的持续失调或异常激活会诱发癌症、炎症、发育障碍和神经***等多种疾病。因此,ERK/MAPK通路相关的蛋白激酶已成为重要的药物靶点之一。
经过几十年坚持不懈的努力,针对MAPK通路BRAF、RAS和MEK等上游靶点的小分子抑制剂已经陆续被开发出来,有些甚至已被FDA批准上市,如维罗非尼(vemurafenib)、达拉非尼(dabrafenib)、曲美替尼(Trametinib)等。虽然这些药物在临床试验中也相对表现出了优异的抗肿瘤活性,但是,在患者临床治疗几个月后,也不可避免的产生了激酶突变引起的耐药性问题,这严重限制了此类药物进一步的发展。研究发现,MAPK通路上游RAS、BRAF相关激酶发生突变,导致末端激酶ERK1/2在该途径中的重新激活是BRAF和MEK抑制剂获得性耐药的关键事件。由于ERK位于MAPK信号通路的末端,很少发生突变,且ERK只能被MEK激活,因此抑制ERK1/2的活性可以阻断该通路的病理作用。临床前研究表明,ERK抑制剂可以克服肿瘤细胞对上游激酶抑制剂诱导的获得性耐药,表现出了显著的抗肿瘤细胞增殖作用,并且可以逆转上游通路突变引起的MAPK通路异常激活。
近年来,人们设计合成了许多ERK抑制剂,并被证明具有优良的ERK抑制活性和抗肿瘤细胞增殖作用。Ulixertinib(BVD-523)是第一个进入临床阶段的可逆型ATP竞争性小分子ERK1/2激酶抑制剂,其对ERK1/2的Ki值分别为0.3和0.04nmol/L。研究表明,Ulixertinib可抑制BRAF突变的黑色素瘤细胞(A375)和结直肠癌细胞(Colo205)的增殖,同时也在体内K-RAS突变的胰腺癌异种移植肿瘤动物模型中显示出良好的抗肿瘤活性和低毒性。LY3214996对ERK1/2的IC50值为5nmol/L,单独口服给药LY3214996不仅能显著抑制体内肿瘤细胞的生长,而且在BRAF或NRAS突变黑色素瘤、BRAF或KRAS突变的结直肠癌、肺癌和胰腺癌异种移植瘤患者中也显示出了良好的耐受性。SCH772984是一种ATP竞争性ERK1/2抑制剂,其对ERK1/2的IC50值分别为4和1nmol/L,在BRAFV600E突变的人体黑色素瘤细胞系LOX中,SCH772984不仅结合无活性的ERK1/2以阻断激酶的磷酸化激活,而且还促进了活性激酶的去磷酸化。此外,SCH772984在KRAS或BRAF突变的几种异种移植瘤模型中也表现出了良好的抗肿瘤细胞增殖作用。
尽管前期已有多个ERK抑制剂进入临床研究,但是这些抑制剂有相当一部分因为毒性太大、成药性差或其它原因而终止了临床研究,目前还没有ERK抑制剂被批准上市。因此,开发出更多新型的ERK抑制剂,对推动ERK抑制剂早日批准上市具有重要意义。
发明内容
本发明的目的在于提供一种新的ERK抑制剂及其制药用途。
本发明提供了一种化合物、其药学上可接受的盐、其立体异构体,所述化合物的结构如式I所示:
其中,Rb为氢,Rc为未被取代或被Rd取代的苯基;Rd选自C1-3烷基、羟基、羧基、NReRf;Re、Rf各自独立地选自氢、C1-3烷基;
或者,Rb与Rc连接成环;
Y1选自CH、N,Y2选自CH、N;
m选自1、2或3;
Ra各自独立地选自C1-5烷基、卤素取代的C1-5烷基、NRgRh、未被取代或被取代基取代的3~6元饱和环烷基、未被取代或被取代基取代的3~6元饱和杂环基;所述取代基选自C1-5烷基;或者两个相邻的Ra连接形成苯环;
Rg、Rh各自独立地选自氢、C1-5烷基。
进一步地,所述化合物的结构如式II所示:
其中,Rd选自C1-3烷基、羟基、羧基、NReRf;
Re、Rf各自独立地选自氢、C1-3烷基;
Y1选自CH、N,Y2选自CH、N;
m选自1、2或3;
Ra各自独立地选自C1-3烷基、卤素取代的C1-3烷基、NRgRh、未被取代或被取代基取代的5~6元饱和环烷基、未被取代或被取代基取代的5~6元饱和杂环基;所述取代基选自C1-3烷基;
Rg、Rh各自独立地选自氢、C1-3烷基。
进一步地,所述化合物的结构如式III-1或式III-2所示:
其中,Rd选自C1-3烷基、羟基、羧基、NReRf;
Re、Rf各自独立地选自氢、C1-3烷基;
Y1为CH,Y2为N;或者Y2为CH,Y1为N;
Ra1、Ra2各自独立地选自氢、C1-3烷基、卤素取代的C1-3烷基、NRgRh、且Ra1、Ra2不同时为氢;
Rg、Rh各自独立地选自氢、C1-3烷基;
Ri选自氢、C1-3烷基。
进一步地,所述化合物的结构如式IV所示:
其中,Y1选自CH、N,Y2选自CH、N;
m选自1、2或3;
Ra各自独立地选自C1-3烷基、卤素取代的C1-3烷基、NRgRh、未被取代或被取代基取代的5~6元饱和环烷基、未被取代或被取代基取代的5~6元饱和杂环基;所述取代基选自C1-3烷基;
Rg、Rh各自独立地选自氢、C1-3烷基。
进一步地,所述化合物的结构如式V-1或式V-2所示:
其中,Y1为CH,Y2为N;或者Y2为CH,Y1为N;
Ra1、Ra2各自独立地选自氢、C1-3烷基、卤素取代的C1-3烷基、NRgRh、且Ra1、Ra2不同时为氢;
Rg、Rh各自独立地选自氢、C1-3烷基;
Ri选自氢、C1-3烷基。
进一步地,所述化合物选自:
本发明还提供了一种药物,它是以上述化合物、其药学上可接受的盐、其立体异构体为活性成分,加上药学上可接受的辅料制备而成的制剂。
本发明还提供了上述化合物、其药学上可接受的盐、其立体异构体在制备ERK抑制剂中的用途。
进一步地,所述ERK抑制剂为ERK1抑制剂和/或ERK2抑制剂。
进一步地,所述ERK抑制剂为预防和/或治疗与ERK活性相关的疾病的药物;
优选地,所述与ERK活性相关的疾病为癌症;所述癌症优选为结直肠癌、肺癌、胰腺癌、黑素瘤、急性骨髓系白血病、胶质母细胞瘤。
实验结果表明,本发明提供的化合物对ERK1/2激酶具有良好的抑制效果。其中,化合物13l的抑制效果最佳,1μM下对ERK1、ERK2激酶的抑制率高达62.41%、65.96%,对ERK1、ERK2激酶的IC50值分别为1.69μM、0.87μM。本发明化合物能够用来制备ERK1/2激酶抑制剂。
本领域技术人员公知的,包括结直肠癌、肺癌、胰腺癌、黑素瘤、急性骨髓系白血病、胶质母细胞瘤等疾病在内的多种疾病与ERK1/2激酶活性有关,靶向抑制ERK1/2激酶活性是治疗结直肠癌、肺癌、胰腺癌、黑素瘤、急性骨髓系白血病、胶质母细胞瘤等疾病的有效手段。因此,本发明提供的化合物能够用来制备预防和/或治疗与ERK1/2激酶活性相关的疾病的药物。
本发明也通过实验证明,化合物13l对两种人结直肠癌细胞系均表现出有效的抑制活性,其对SW-620和HCT-116细胞的IC50值分别为1.16μM、0.59μM。蛋白质印迹分析表明,化合物13l以浓度依赖性的方式降低HCT116细胞中ERK1/2(Thr202、Tyr204)和p90RSK(Thr359、Ser363)蛋白在细胞内的磷酸化水平。最后,本发明还进一步证实了化合物13l能够诱导HCT116人结肠癌细胞的凋亡,经过1μM的化合物13l处理后,HCT116细胞凋亡率可达23.7%。
综上,本发明提供的化合物能够用于制备ERK1/2激酶抑制剂,以及预防和/或治疗与ERK1/2激酶活性相关的疾病的药物,应用前景广阔。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1:化合物13l对ERK1/2激酶的抑制曲线。
图2:化合物13l和SCH772984对结肠癌细胞的增殖抑制作用。***表示P<0.001。
图3:化合物13l对HCT116细胞中ERK1/2和RSK磷酸化水平的影响。
图4:化合物13l对HCT116细胞的凋亡分析。
图5:化合物13l诱导HCT116细胞凋亡的凋亡率。
具体实施方式
如果文中没有特别说明,本发明实验中所用到的试剂和原料均从试剂公司购买,所有试剂纯度均为分析纯或色谱纯,未经进一步处理,均可直接使用。
实施例1:合成目标化合物13a-l、14a-b、15a-e
合成路线:
1.中间体8的合成
中间体8的合成步骤:以N-Boc-3-氰基-4-吡咯烷酮为起始原料来合成系列二的化合物。首先,将化合物7(N-Boc-3-氰基-4-吡咯烷酮,(21.00g,100mmol))和盐酸肼(10.30g,150mmol)加入到500mL反应瓶中,接下来再加入无水乙醇(250mL)将其溶解,然后,将反应瓶置于60℃下加热回流3小时。通过TLC对反应进行监测,等到反应彻底结束后,减压浓缩,除去溶剂,随后在搅拌下缓慢加入饱和NaHCO3水溶液,调pH至7~8,EA萃取(3×300mL),收集上层溶液,加入适量的无水Na2SO4干燥,再次减压浓缩,残余物用乙酸乙酯进行重结晶,减压抽滤,收集滤饼并烘干后即得白色固体8,中间体8不经纯化即可用于下一步。
2.中间体9的合成
中间体9的合成步骤:在干燥的500mL圆底烧瓶中,依次加入中间体8(3-氨基-5-(叔丁基)-吡咯并[3,4-C]吡唑,(11.20g,50mmol))、DIEA(34.80mL,200mmol)、THF(250mL),然后将上述混合物置于0℃下搅拌5分钟,随后在0℃下用滴液漏斗缓慢滴加由无水THF(120mL)稀释的氯甲酸乙酯(4.80mL,50mmol)的混合溶液,此过程要2小时,等反应滴加完后再将混合溶液转移至常温下搅拌反应12小时。通过TLC监测,待反应完全后,减压浓缩,用乙酸乙酯和饱和NaCl水溶液萃取(3×250mL)残余物,收集上层溶液,加入适量的无水Na2SO4干燥,真空减压浓缩后即得白色固体9,使用硅胶色谱柱(石油醚/乙酸乙酯=4/1~2/1)进行纯化处理,即可得纯净的中间体9。
3.中间体10的合成
中间体10a、10b、10d的合成步骤:在250mL反应瓶中,依次加入中间体9(5-(叔丁基)-3-氨基-4,6-二氢吡咯并[3,4-c]吡唑-1-羧酸乙酯,(14.80g,50mmol))、DIEA(34.80mL,200mmol)、THF(100mL),随后在0℃搅拌下加入相对应的酰氯(75mmol),并持续搅拌1小时,然后转移至室温下持续搅拌12小时。TLC检测,等到反应彻底结束后,减压浓缩,用饱和NaHCO3将残余物调pH至7~8,再用乙酸乙酯萃取(3×250mL),收集上层溶液,加适量无水Na2SO4进行干燥,减压浓缩,使用硅胶色谱柱(石油醚/乙酸乙酯=1/1)进行纯化后,即得纯净的中间体10。
中间体10c的合成步骤:在250mL反应瓶中,依次加入中间体9(5-(叔丁基)-3-氨基-4,6-二氢吡咯并[3,4-c]吡唑-1-羧酸乙酯,(14.80g,50mmol))、DIEA(34.80mL,200mmol)、THF(100mL),随后在0℃搅拌下加入相对应的酰氯(75mmol),并持续搅拌1小时,随后将反应混合物升温至45℃搅拌12小时,此过程中会形成沉淀。TLC检测,待反应完全后,减压抽滤,并用THF清洗3次,收集滤饼并烘干后即得粗产品10c,粗产品10c不经纯化即可用于下一步。
4.中间体12的合成
中间体11a-d的合成步骤:首先用DCM(100mL)将中间体10(30mmol)溶解在250mL反应瓶中,然后在0℃搅拌下滴加含有4N HCl的1,4-二氧六环溶液(30mL),随后将反应物转移至常温下持续搅拌12小时,此过程中会不断生成沉淀。通过TLC对反应进行监测,等到反应彻底结束后,减压抽滤,并用二氯甲烷清洗3次,收集滤饼并烘干后即得粗产品11,粗产品11不经纯化即可用于下一步。
中间体12的合成步骤:首先,将中间体11(5mmol)、DIEA(1.70mL,10mmol)、DCM(30mL)依次加入100mL反应瓶中,随后将上述混合物于0℃搅拌下缓慢加入相对应的酰氯(7.50mmol)并持续搅拌1小时,然后将反应瓶转移至常温搅拌下反应10小时。TLC检测,等到反应彻底结束后,减压浓缩,用饱和NaHCO3调pH至7~8,再用乙酸乙酯萃取(3×50mL),收集上层溶液,加适量无水Na2SO4进行干燥,真空减压浓缩,使用硅胶色谱柱(石油醚/乙酸乙酯=1/1)进行纯化后,即得纯净的中间体12。
5.目标化合物13a-1的合成
目标化合物13a-l的合成步骤:将中间体12(2mmol)加入干燥的50mL圆底烧瓶中,然后加入MeOH(20mL)将其溶解,随后在搅拌下加入LiOH(80mg,2mmol),30分钟后,通过TLC检测,等反应完全后,减压浓缩,使用硅胶色谱柱纯化(二氯甲烷/甲醇=9/1)后即得白色固体13,接下来,再经甲醇重结晶后即得纯净的目标化合物13。
按照上述方法合成得到目标化合物13a-l。
6.目标化合物14a-b的合成
目标化合物14a-b的合成步骤:首先,用1,4-二氧六环溶液(15mL)将中间体13(2mmol)溶解在50mL圆底烧瓶中,随后在搅拌下滴加1N的LiOH(1mL)溶液,接下来,将此反应混合物转移至室温下搅拌反应3小时。TLC检测,等反应彻底结束后,用1N的HCl溶液调pH至4~5,此过程中会有沉淀生成,减压抽滤,并用水清洗3次,收集滤饼并烘干后即得白色固体14,随后,用甲醇对粗产品进行重结晶,减压抽滤,收集滤饼,待烘干后即得纯净的目标化合物14。
7.目标化合物15a-e的合成
目标化合物15a-e的合成步骤:首先,将中间体13(2mmol)、NH4Cl(0.13g,2.40mmol)、EtOH(20mL)、H2O(5mL)、DMF(2mL)、THF(6mL)、铁粉(0.56g,10mmol)、冰醋酸(3~4滴)依次加入到100mL反应瓶中。随后将反应混合物转移至80℃下搅拌回流8小时,过程中溶液由黄色悬浊液转至浅棕色悬浊液。通过TLC检测,等到反应结束后,趁热过滤除去剩余的铁粉,减压浓缩,用饱和NaHCO3调pH至7~8,随后用乙酸乙酯萃取(3×50mL),收集上层溶液,加适量无水Na2SO4进行干燥,减压浓缩,最后使用硅胶色谱柱进行纯化(二氯甲烷/甲醇=9/1)后即得纯净的目标化合物15。按照上述方法合成得到目标化合物15a-e。
实施例2:合成目标化合物19a-g
合成路线:
1.中间体16的合成
中间体16的合成步骤:将中间体9(5-(叔丁基)-3-氨基-4,6-二氢吡咯并[3,4-c]吡唑-1-羧酸乙酯,(14.80g,50mmol))加入干燥的250mL两颈烧瓶中,然后加入超干的DMF(100mL)将其溶解,随后在N2保护下缓慢滴加氯乙基异氰酸酯(12.80mL,150mmol),接下来将上述混合物在冰浴下搅拌5分钟,随后将此反应混合物转移至常温下搅拌反应3小时,此过程中会有沉淀产生。TLC检测,待反应结束后,将上述反应物趁热倒入500mL冰水中,随后减压抽滤,并用水洗涤3次,收集滤饼并烘干备用。接下来将烘干后的粗产品、叔丁醇钾(5.05g,45mmol)和THF(150mL)依次加入到250mL的两口瓶中,然后在N2保护下将上述反应混合物在常温下搅拌反应2小时,此过程中会有固体析出。TLC检测,待反应结束后,减压抽滤,用THF清洗3次,收集滤饼,烘干后即得白色固体16,最后通过硅胶色谱柱(石油醚/乙酸乙酯=1/1)纯化即得纯净的中间体16。
2.中间体18a-g的合成
中间体17的合成步骤:首先,用DCM(150mL)将中间体16(5-(叔丁基)1-乙基3-(2-氧咪唑-1-基)-4,6-二氢吡咯[3,4-c]吡唑-1,5-二羧酸酯,(16.43g,45mmol))溶解在250mL反应瓶中,然后在0℃搅拌下滴加含有4N HCl的1,4-二氧六环溶液(30mL),随后将反应物转移至常温下持续搅拌12小时,此过程中会不断生成沉淀。通过TLC对反应进行监测,等到反应彻底结束后,减压抽滤,并用二氯甲烷清洗3次,收集滤饼并烘干后即得白色固体17,粗产品17不经纯化即可用于下一步。
中间体18a-g的合成步骤:首先,将中间体17(3-(2-氧咪唑烷-1-基)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯,(0.79g,3mmol))、DIEA(1.02mL,6mmol)、PyBop(1.72g,3.3mmol)和DCM(30mL)依次加入到100mL圆底烧瓶中,随后在冰浴下缓慢加入相对应的酸(4.50mmol),然后,将上述混合物转移至0℃下搅拌反应1小时,随后再转移至常温下搅拌反应10小时,通过TLC进行监测,等到反应彻底结束后,减压浓缩,用饱和NaHCO3将残余物调pH至7~8,随后用乙酸乙酯进行萃取(3×50mL),收集上层溶液,加适量无水Na2SO4进行干燥,再次减压浓缩,最后通过硅胶色谱柱进行纯化(二氯甲烷/甲醇=30/1)后即得纯净的目标化合物18。
按照上述方法合成得到中间体18a-g。
3.目标化合物19a-g的合成
目标化合物19a-g的合成步骤:将中间体18(2mmol)加入干燥的50mL圆底烧瓶中,然后加入MeOH(20mL)将其溶解,随后在搅拌下加入LiOH(80mg,2mmol),30分钟后,通过TLC检测,等反应完全后,减压浓缩,使用硅胶色谱柱纯化(二氯甲烷/甲醇=9/1)后即得白色固体19,接下来,再经甲醇重结晶后即得纯净的目标化合物19。
按照上述方法合成得到中间体19a-g。
以下为上述中间体化合物和目标化合物的结构和表征数据。
1.中间体化合物的结构和表征数据
表1.中间体化合物的结构
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3-氨基-5-(叔丁基)-吡咯并[3,4-C]吡唑(8).白色粉末,产率:38.2%.1HNMR(400MHz,DMSO-d6)δ(ppm):11.19(s,1H),4.99(s,2H),4.17(d,J=9.6Hz,2H),4.12(m,2H),1.44(s,9H).
5-(叔丁基)-3-氨基-4,6-二氢吡咯并[3,4-c]吡唑-1-羧酸乙酯(9).白色粉末,产率:88.5%.1H NMR(400MHz,DMSO-d6)δ(ppm):5.68(s,2H),4.46(d,J=3.2Hz,2H),4.27(q,J=7.2Hz,2H),4.17(d,J=3.2Hz,2H),1.44(s,9H),1.28(t,J=7.2Hz,3H).
5-(叔丁基)-1-乙基-3-(4-(甲氧基羰基)苯甲酰氨基)-4,6-二氢吡咯并[3,4-c]吡唑-1,5-羧酸乙酯(10a).白色粉末,产率:72.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.86(d,J=2.0Hz,1H),8.16(d,J=8.0Hz,2H),8.07(d,J=8.0Hz,2H),4.55(s,2H),4.49(s,2H),4.42(q,J=7.6Hz,2H),3.90(s,3H),1.54(s,9H),1.35(t,J=7.6Hz,3H).
5-(叔丁基)-1-乙基-3-(4-羟基苯甲酰胺)-4,6-二氢吡咯并[3,4-c]吡唑-1,5-羧酸乙酯(10b).白色粉末,产率:76.5%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.86(s,1H),8.35(d,J=8.8Hz,2H),8.26(d,J=8.8Hz,2H),4.55(s,2H),4.49(s,2H),4.43(q,J=7.2Hz,2H),1.49(s,9H),1.36(t,J=7.2Hz,3H).
5-(叔丁基)-1-乙基-3-(4-(二甲氨基)苯甲酰氨基)-4,6-二氢吡咯并[3,4-c]吡唑-1,5-羧酸乙酯(10c).棕色粉末,产率:68.7%.1H NMR(400MHz,Chloroform-d)δ(ppm):8.65(s,1H),7.74(d,J=8.8Hz,2H),6.68(d,J=8.8Hz,2H),4.73(s,2H),4.72(s,2H),4.46(q,J=7.2Hz,2H),3.05(s,6H),1.52(s,9H),1.42(t,J=7.2Hz,3H).
3-(4-硝基苯甲酰胺)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(10d).黄色粉末,产率:83.4%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.01(s,1H),8.34(d,J=8.8Hz,2H),8.26(d,J=8.8Hz,2H),4.55(s,2H),4.49(s,2H),4.42(q,J=7.2Hz,2H),1.55(s,9H),1.36(t,J=7.2Hz,3H).
3-(4-羟基苯甲酰氨基)-5-(4-甲基苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12a).白色粉末,产率:45.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.84(s,1H),10.23(s,1H),7.92(dd,J=8.4Hz,2H),7.53(d,J=7.6Hz,2H),7.34(d,J=7.6Hz,2H),6.88(d,J=8.4Hz,2H),4.71(s,2H),4.56(s,2H),4.44(q,J=7.2Hz,2H),2.40(s,3H),1.38(t,J=7.2Hz,3H).
3-(4-羟基苯甲酰氨基)-5-(4-(三氟甲基)苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12b).白色粉末,产率:54.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.84(s,1H),10.15(s,1H),7.91(d,J=8.4Hz,2H),7.86(d,J=7.6Hz,2H),7.83(d,J=7.6Hz,2H),6.85(d,J=8.4Hz,2H),4.74(s,2H),4.54(s,2H),4.42(q,J=7.2Hz,2H),1.29(t,J=7.2Hz,3H).
3-(4-(二甲基氨基)苯甲酰氨基)-5-(4-甲基苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12c).白色粉末,产率:55.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.87(s,1H),7.93(d,J=8.8Hz,2H),7.64(d,J=8.0Hz,2H),7.36(t,J=8.0Hz,2H),6.81(d,J=8.8Hz,2H),4.67(s,2H),4.56(s,2H),4.43(q,J=7.2Hz,2H),2.99(s,6H),2.35(s,3H),1.37(t,J=7.2Hz,3H).
3-(4-(二甲氨基)苯甲酰氨基)-5-(4-(三氟甲基)苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12d).白色粉末,产率:49.0%.1H NMR(400MHz,Chloroform-d)δ(ppm):9.52(s,1H),8.23(d,J=9.2Hz,2H),7.74(d,J=3.6Hz,2H),7.71(d,J=3.6Hz,2H),6.66(d,J=9.2Hz,2H),5.06(s,2H),4.87(s,2H),4.55(q,J=7.2Hz,2H),3.05(s,6H),1.49(t,J=7.2Hz,3H).
3-(4-(二甲氨基)苯甲酰氨基)-5-(4-(二甲氨基)苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12e).白色粉末,产率:51.4%.1H NMR(400MHz,)δ(ppm):11.69(s,1H),8.28(d,J=9.6Hz,2H),7.97(d,J=8.8Hz,2H),7.86(m,2H),7.76(d,J=8.8Hz,2H),5.29(s,2H),4.99(s,2H),4.42(q,J=7.2Hz,2H),3.52(dd,J=9.2Hz,12H),1.36(t,J=7.2Hz,3H).
3-(4-(二甲胺基)苯甲酰)-5-(3-(二甲胺基)苯甲酰)-5,6-二氢吡咯[3,4-c]吡唑-1(4氢)-羧酸乙酯(12f).白色粉末,产率:58.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.06(s,1H),7.88(d,J=8.4Hz,2H),7.28(t,J=7.4Hz,1H),6.84(s,1H),6.82(m,1H),6.81(s,1H),6.67(d,J=8.4Hz,2H),4.80(m,2H),4.64(s,2H),4.43(q,J=7.2Hz,2H),2.99(d,J=12.4Hz,6H),2.93(s,6H),1.37(t,J=7.2Hz,3H).
5-(1-萘酰)-3-(4-(二甲胺基)苯甲酰胺)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(12g).白色粉末,产率:49.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.03(s,1H),8.31(s,1H),8.04(d,J=7.6Hz,2H),7.90(s,1H),7.78(d,J=7.8Hz,1H),7.66–7.61(m,1H),7.60(s,1H),7.59(s,1H),7.58(t,J=2.0Hz,1H),6.68(d,J=7.6Hz,2H),4.97(s,2H),4.41(s,2H),4.43(q,J=7.2Hz,2H),2.98(s,6H),1.27(d,J=7.2Hz,3H).
5-(2-萘酰)-3-(4-(二甲胺基)苯甲酰胺)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(12h).白色粉末,产率:61.8%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.07(s,1H),8.87(d,J=4.4Hz,1H),8.32(d,J=8.8Hz,2H),8.05(d,J=4.4Hz,1H),8.03(d,J=4.4Hz,1H),8.00(d,J=4.4Hz,1H),7.83(d,J=8.8Hz,1H),7.68(d,J=8.4Hz,1H),7.64–7.59(m,1H),6.69(d,J=8.8Hz,2H),4.93(s,2H),4.74(s,2H),4.45(q,J=7.2Hz,2H),2.98(s,6H),1.38(t,J=7.2Hz,3H).
3-(4-(二甲胺基)苯甲酰胺)-5-异烟酰-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(12i).白色粉末,产率:66.7%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.87(s,1H),8.79(d,J=4.8Hz,2H),7.95(d,J=5.6Hz,2H),7.63(d,J=5.6Hz,2H),6.72(d,J=4.8Hz,2H),4.77(s,2H),4.54(s,2H),4.43(q,J=7.2Hz,2H),2.99(s,6H),1.36(t,J=7.2Hz,3H).
3-(4-(二甲胺基)苯甲酰胺)-5-烟酰-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(12j).白色粉末,产率:55.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.15(s,1H),9.08(dd,J=6.0,2.0Hz,1H),8.93(d,J=5.6Hz,1H),8.56(t,J=8.8Hz,1H),8.02–7.93(m,2H),7.89(d,J=8.8Hz,1H),6.76(d,J=8.8Hz,2H),4.90(s,2H),4.83–4.75(m,2H),4.34(t,J=7.2Hz,2H),3.00(s,6H),1.37(t,J=7.2Hz,3H).
3-(4-(二甲氨基)苯甲酰胺基)-5-(4-(4-甲基哌嗪-1-基)苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12k).白色粉末,产率:46.5%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.70(d,J=27.2Hz,1H),7.86(d,J=8.8Hz,2H),7.54(d,J=8.8Hz,2H),7.06(d,J=8.8Hz,2H),6.81(d,J=8.8Hz,2H),4.91(s,2H),4.68(s,2H),4.28(t,J=7.2Hz,2H),4.04(d,J=12.0Hz,2H),3.52(d,J=12.0Hz,2H),3.21(t,J=12.0Hz,2H),3.05(t,J=12.0Hz,2H),3.02(s,6H),2.91(s,3H),1.29(t,J=7.2Hz,3H).
3-(4-(二甲氨基)苯甲酰胺基)-5-(3-(4-甲基哌嗪-1-基)苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12l).白色粉末,产率:55.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.68(s,1H),7.93(d,J=8.8Hz,2H),7.36(q,J=7.6Hz,1H),7.24(d,J=7.2Hz,2H),7.08(d,J=7.6Hz,2H),6.98(d,J=7.2Hz,1H),6.81(d,J=8.8Hz,2H),4.68(m,2H),4.57(s,2H),4.44(q,J=7.2Hz,2H),3.21(s,4H),2.99(s,6H),2.49(s,4H),2.26(s,3H),1.38(t,J=7.2Hz,3H).
4-((1-(乙氧羰基)-5-(4-甲基苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)氨基甲酰基)苯甲酸(12m).白色粉末,产率:49.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.75(s,1H),8.07(d,J=7.2Hz,2H),7.96(d,J=8.4Hz,2H),7.49(d,J=8.4Hz,2H),7.30(d,J=7.2Hz,2H),4.84(m,2H),4.69(s,2H),4.44(q,J=7.2Hz,2H),3.88(s,3H),2.38(s,3H),1.38(t,J=7.2Hz,3H).
4-((1-(乙氧基羰基)-5-(4-(三氟甲基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)氨基甲酰基)苯甲酸(12n).白色粉末,产率:71.5%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.75(s,1H),8.09(d,J=8.8Hz,2H),8.01(d,J=8.0Hz,2H),7.88(d,J=8.0Hz,2H),7.84(d,J=8.8Hz,2H),4.86(m,2H),4.68(s,2H),4.45(q,J=7.2Hz,2H),3.88(s,3H),1.38(t,J=7.2Hz,3H).
5-(4-甲基苯甲酰基)-3-(4-硝基苯甲酰氨基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12o).白色粉末,产率:62.0%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.94(s,1H),7.77(d,J=8.8Hz,2H),7.48(d,J=8.0Hz,2H),7.30(d,J=8.0Hz,2H),6.54(d,J=8.8Hz,2H),5.82(s,2H),4.70(s,2H),4.42(q,J=7.2Hz,2H),2.37(s,3H),1.36(t,J=7.2Hz,3H).
3-(4-硝基苯甲酰氨基)-5-(4-(三氟甲基)苯甲酰基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12p).白色粉末,产率:49.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.54(s,1H),7.83(d,J=8.4Hz,2H),7.69(d,J=8.0Hz,2H),7.61(d,J=8.0Hz,2H),6.49(d,J=8.4Hz,2H),4.66(m,2H),4.52(s,2H),4.38(q,J=7.2Hz,2H),1.32(t,J=7.2Hz,3H).
5-(3-(二甲氨基)苯甲酰基)-3-(4-硝基苯甲酰氨基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-甲酸乙酯(12q).白色粉末,产率:57.7%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.43(s,1H),7.73(d,J=8.0Hz,2H),7.28(m,1H),6.85(d,J=7.6Hz,1H),6.80(s,1H),6.76(d,J=7.6Hz,1H),6.52(d,J=8.0Hz,2H),4.69(s,2H),4.48(s,2H),4.43(q,J=7.2Hz,2H),2.91(s,6H),1.38(t,J=7.2Hz,3H).
3-(4-(二甲胺基)苯甲酰胺)-5-烟酰-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(12x).白色粉末,产率:55.8%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.91(s,1H),8.72(t,J=6.4Hz,2H),8.32(d,J=8.4Hz,2H),8.27–8.12(m,2H),7.58(dd,J=8.4,5.6Hz,2H),4.85(s,2H),4.72(m,2H),4.40(d,J=7.2Hz,2H),1.37(t,J=7.2Hz,3H).
5-烟酰-3-(4-硝基苯甲酰胺)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-羧酸乙酯(12y).白色粉末,产率:49.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.87(s,1H),9.19(s,1H),8.98(dd,J=5.6,1.6Hz,1H),8.66(d,J=8.0Hz,1H),8.34(d,J=8.8Hz,2H),8.27(t,J=8.8Hz,2H),7.91(dd,J=8.0,5.2Hz,1H),4.88(m,2H),4.55(s,2H),4.43(q,J=7.2Hz,2H),1.36(t,J=7.2Hz,3H).
N-(5-(4-甲基苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)-4-硝基苯甲酰胺(13o).白色粉末,产率:57.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.28(s,1H),10.36(s,1H),7.75(d,J=8.8Hz,2H),7.53(d,J=8.0Hz,2H),7.32(t,J=8.0Hz,2H),6.61(d,J=8.8Hz,2H),4.68(s,2H),4.56(s,2H),2.37(s,3H).
4-硝基-N-(5-(4-(三氟甲基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13p).白色粉末,产率:67.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.46(s,1H),10.48(s,1H),7.91(d,J=8.4Hz,2H),7.86(d,J=8.4Hz,2H),7.72(d,J=8.4Hz,2H),6.63(d,J=8.4Hz,2H),4.71(m,2H),4.54(s,2H).
N-(5-(3-(二甲氨基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)-4-硝基苯甲酰胺(13q).白色粉末,产率:71.5%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.39(s,1H),10.48(s,1H),7.76(d,J=8.0Hz,2H),7.28(m,1H),6.84(d,J=7.6Hz,1H),6.82(s,1H),6.77(d,J=7.6Hz,1H),6.59(d,J=8.0Hz,2H),4.67(s,2H),4.52(s,2H),2.94(s,6H).
N-(5-异烟酰-1,4,5,6-四氢吡咯[3,4-c]吡唑-3-基)-4-硝基苯酰胺(13x).白色粉末,产率:59.8%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.61(s,1H),11.42(s,1H),8.72(d,J=6.4Hz,2H),8.32(d,J=8.4Hz,2H),8.27–8.12(m,2H),7.58(dd,J=8.4,5.6Hz,2H),4.73(m,2H),4.58(s,2H).
N-(5-烟酰基-1,4,5,6-四氢吡咯-[3,4-c]吡唑-3-基)-4-硝基苯酰胺(13y).白色粉末,产率:64.0%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.50(s,1H),11.33(s,1H),8.81(dd,J=7.6,2.0Hz,1H),8.69(t,J=4.4Hz,1H),8.36(d,J=8.4Hz,1H),8.30(d,J=8.4Hz,1H),8.25(d,J=8.4Hz,1H),8.16(d,J=8.4Hz,1H),8.05(t,J=8.4Hz,1H),7.53(dd,J=9.2,4.5Hz,1H),4.75(m,2H),4.65(s,2H).
5-(叔丁基)1-乙基3-(2-氧咪唑-1-基)-4,6-二氢吡咯[3,4-c]吡唑-1,5-二羧酸酯(16).白色粉末,产率:76.5%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.35(s,1H),4.43(s,2H),4.38(s,2H),4.19(t,J=7.2Hz,2H),3.86(d,J=8.4Hz,2H),3.79(d,J=8.4Hz,2H),1.46(s,9H),1.25(t,J=7.2Hz,3H).
3-(2-氧咪唑烷-1-基)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(17).白色粉末,产率:85.9%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.27(s,1H),10.74(s,2H),4.35(s,2H),4.30(s,2H),4.20(q,J=7.2Hz,2H),3.87(d,J=8.4Hz,2H),3.80(d,J=8.4Hz,2H),1.25(t,J=7.2Hz,3H).
5-(4-甲基苯甲酰)-3-(2-氧咪唑烷-1-基)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(18a).白色粉末,产率:44.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.36(s,1H),7.48(d,J=7.6Hz,2H),7.27(t,J=7.6Hz,2H),4.68(s,2H),4.56(s,2H),4.15(q,J=7.2Hz,2H),3.83(s,2H),3.80–3.72(m,2H),2.37(s,3H),1.20(t,J=7.2Hz,3H).
3-(2-氧咪唑-1-基)-5-(4-(三氟甲基)苯甲酰)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(18b).白色粉末,产率:51.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.39(s,1H),7.90–7.83(m,2H),7.79(d,J=8.0Hz,2H),4.71(s,2H),4.55(s,2H),4.15(q,J=7.2Hz,2H),3.82(d,J=4.0Hz,2H),3.81–3.72(m,2H),1.19(t,J=7.2Hz,3H).
5-(3-(二甲胺基)苯甲酰)-3-(2-氧咪唑-1-基)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(18c).白色粉末,产率:49.0%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.29(s,1H),7.21(t,J=7.6Hz,1H),6.78(d,J=2.4Hz,1H),6.72(s,1H),6.62(d,J=7.6Hz,1H),4.58(s,2H),4.46(s,2H),4.44(q,J=7.2Hz,2H),3.71(d,J=8.0Hz,2H),3.32(t,J=8.0Hz,2H),2.88(s,6H),1.35(t,J=7.2Hz,3H).
5-异烟酰-3-(2-氧基咪唑-1-基)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(18d).白色粉末,产率:42.7%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.39(s,1H),8.78(d,J=2.4Hz,1H),8.73–8.64(m,1H),8.03(t,J=9.6Hz,1H),7.51(t,J=12.0Hz,1H),4.71(s,2H),4.61(s,2H),4.15(q,J=7.2Hz,2H),3.83(d,J=7.6Hz,2H),3.82–3.72(m,2H),1.20(t,J=7.2Hz,3H).
5-烟酰基-3-(2-氧代咪唑烷-1-基)-5,6-二氢吡咯并[3,4-c]吡唑-1(4H)-羧酸乙酯(18e).白色粉末,产率:55.4%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.28(s,1H),9.16(d,J=4.4Hz,1H),8.97(s,1H),8.72(d,J=7.6Hz,1H),8.06(s,1H),4.72(s,2H),4.64(s,2H),4.22(q,J=7.2Hz,2H),3.81(d,J=8.8Hz,2H),3.42(d,J=8.8Hz,2H),1.24(t,J=7.2Hz,3H).
5-(4-(4-甲基哌嗪-1-基)苯甲酰)-3-(2-氧咪唑-1-基)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(18f).白色粉末,产率:50.1%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.40(s,1H),7.52(d,J=8.0Hz,2H),7.06(t,J=8.0Hz,2H),4.70(s,2H),4.65(s,2H),4.17(d,J=7.2Hz,2H),3.86(d,J=6.4Hz,2H),3.84–3.71(m,4H),3.44(d,J=6.4Hz,2H),2.84(s,4H),2.56(s,3H),1.21(t,J=7.2Hz,3H).
5-(3-(4-甲基哌嗪-1-基)苯甲酰)-3-(2-氧咪唑-1-基)-5,6-二氢吡咯[3,4-c]吡唑-1(4H)-羧酸乙酯(18g).白色粉末,产率:44.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.40(s,1H),7.35(q,J=7.6Hz,1H),7.16(d,J=11.6Hz,1H),7.11(d,J=8.4Hz,1H),7.03(d,J=7.6Hz,1H),4.68(s,2H),4.54(s,2H),4.15(q,J=7.2Hz,2H),3.77(d,J=8.4Hz,2H),3.40(d,J=8.4Hz,2H),2.85(s,4H),2.54(s,4H),2.28(s,3H),1.20(t,J=7.2Hz,3H).
2.目标化合物的结构和表征数据
表2.目标化合物的结构
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4-羟基-N-(5-(4-甲基苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13a).白色粉末,熔点:280.9~283.7℃,产率:63.2%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.31(s,1H),10.67(s,1H),10.23(s,1H),7.87(d,J=8.4Hz,2H),7.49(d,J=7.6Hz,2H),7.29(d,J=7.6Hz,2H),6.82(d,J=8.4Hz,2H),4.67(s,2H),4.56(s,2H),2.37(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):170.17,164.36,161.39,139.88,134.37,130.39,130.34(2C),129.39(2C),129.27,127.45,127.27(2C),115.48,115.41(2C),50.87,45.55,21.40.HRMS(ESI)+calculated for C20H18N4NaO3,[M+Na]+:m/z 385.1266,found:385.1271.
4-羟基-N-(5-(4-(三氟甲基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13b).白色粉末,熔点:241.6~243.8℃,产率:68.4%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.35(s,1H),10.66(s,1H),10.12(s,1H),7.95–7.85(m,2H),7.84(d,J=7.6Hz,2H),7.81(d,J=7.6Hz,2H),6.83(d,J=8.0Hz,2H),4.71(s,2H),4.54(s,2H).HRMS(ESI)+calculated for C20H15F3N4NaO3,[M+Na]+:m/z 439.0986,found:439.0988.
4-(二甲氨基)-N-(5-(4-甲基苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13c).白色粉末,熔点:260.8~265.1℃,产率:78.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.21(s,1H),10.52(s,1H),7.87(d,J=8.8Hz,2H),7.49(d,J=7.6Hz,2H),7.28(d,J=7.6Hz,2H),6.72(d,J=8.8Hz,2H),4.67(s,2H),4.56(s,2H),2.99(s,6H),2.37(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):170.20,164.51,152.99,139.90,134.35,129.76,129.71(2C),129.41(2C),129.29,127.44,127.26(2C),119.75,111.16(2C),50.88,48.16(2C),45.55,21.40.HRMS(ESI)+calculated for C22H24N5O2,[M+H]+:m/z390.1917,found:390.1925.
4-(二甲氨基)-N-(5-(4-(三氟甲基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13d).白色粉末,熔点:303.4~305.3℃,产率:69.5%.1HNMR(400MHz,DMSO-d6)δ(ppm):12.25(s,1H),10.56(s,1H),7.90(d,J=8.4Hz,2H),7.84(d,J=8.0Hz,2H),7.82(d,J=8.0Hz,2H),6.73(d,J=8.4Hz,2H),4.73(d,J=28.4Hz,2H),4.56(s,2H),2.99(d,J=14.0Hz,6H).13C NMR(100MHz,DMSO-d6)δ(ppm):168.67,168.47,164.51,152.97,141.25,130.51,129.78,129.73(2C),128.23(2C),128.10(2C),125.84,123.06,111.21,111.12(2C),50.97,48.16(2C),45.65.HRMS(ESI)+calculated for C22H20F3N5NaO2,[M+Na]+:m/z 466.1454,found:466.1461.
4-(二甲氨基)-N-(5-(4-(二甲氨基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13e).白色粉末,熔点:306.8~310.2℃,产率:63.9%.1HNMR(400MHz,TFA-d6)δ(ppm):11.69(s,1H),11.61(s,1H),8.21(d,J=9.6Hz,2H),7.97(d,J=8.8Hz,2H),7.92–7.83(m,2H),7.79(d,J=9.6Hz,2H),5.25(s,2H),4.99(s,2H),3.48(s,12H).HRMS(ESI)+calculated for C23H26N6NaO2,[M+Na]+:m/z 441.2014,found:441.2009.
4-(二甲氨基)-N-(5-(3-(二甲氨基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13f).白色粉末,熔点:294.6~297.8℃,产率:71.3%.1HNMR(400MHz,DMSO-d6)δ(ppm):12.22(s,1H),10.49(s,1H),7.86(d,J=8.4Hz,2H),7.26(q,J=7.2Hz,1H),6.83(d,J=7.6Hz,1H),6.80(s,1H),6.78(d,J=7.6Hz,1H),6.69(d,J=8.4Hz,2H),4.66(s,2H),4.53(s,2H),2.99(s,6H),2.93(s,6H).HRMS(ESI)+calculated forC23H26N6NaO2,[M+Na]+:m/z 441.1999,found:441.2009.
N-(5-(1-萘甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)-4-(二甲氨基)苯甲酰胺(13g).白色粉末,熔点:310.4~313.3℃,产率:64.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.26(s,1H),10.45(s,1H),8.09–7.99(m,2H),7.93(s,1H),7.87(d,J=3.6Hz,1H),7.72(s,1H),7.66–7.61(m,1H),7.60(s,1H),7.59(s,1H),7.58(t,J=2.4Hz,1H),6.69(d,J=5.6Hz,2H),4.85(s,2H),4.27(s,2H),2.98(s,6H).HRMS(ESI)+calculated forC25H23N5NaO2,[M+Na]+:m/z448.1738,found:448.1744.
N-(5-(2-萘甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)-4-(二甲氨基)苯甲酰胺(13h).白色粉末,熔点:313.1~315.7℃,产率:52.9%.1H NMR(400MHz,TFA-d6)δ(ppm):11.86(s,1H),11.71(s,1H),8.14(d,J=4.4Hz,1H),8.07(d,J=8.4Hz,1H),7.98(d,J=8.4Hz,1H),7.93(d,J=8.8Hz,2H),7.84(d,J=4.4Hz,1H),7.76(d,J=8.8Hz,1H),7.68(d,J=8.8Hz,1H),7.64–7.59(m,1H),7.41(d,J=8.8Hz,2H),5.57–5.12(m,2H),4.97(s,2H),3.27(s,6H).HRMS(ESI)+calculated for C25H23N5NaO2,[M+Na]+:m/z 448.1749,found:448.1744.
4-(二甲氨基)-N-(5-异烟酰基-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13i).白色粉末,熔点:303.1~305.8℃,产率:66.2%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.11(s,1H),10.52(s,1H),8.73(d,J=4.8Hz,2H),7.86(d,J=5.6Hz,2H),7.56(d,J=5.6Hz,2H),6.69(d,J=4.8Hz,2H),4.70(s,2H),4.54(s,2H),2.99(s,6H).13C NMR(100MHz,DMSO-d6)δ(ppm):165.15,164.37,152.46,152.09,147.03,143.61(2C),143.45,129.87,129.76(2C),125.23,125.14(2C),112.38(2C),50.11,45.76,40.48(2C).HRMS(ESI)+calculated for C20H20N6NaO2,[M+Na]+:m/z 399.1539,found:399.154.
4-(二甲氨基)-N-(5-烟酰基-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13j).白色粉末,熔点:251.3~257.6℃,产率:55.8%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.25(s,1H),10.53(s,1H),8.81(dd,J=6.0,2.4Hz,1H),8.69(t,J=5.6Hz,1H),8.03(d,J=8.0Hz,1H),7.96–7.77(m,2H),7.52(dd,J=12.0,4.8Hz,1H),6.72(d,J=8.4Hz,2H),4.70(d,J=24.4Hz,2H),4.61(s,2H),2.99(d,J=12.4Hz,6H).13C NMR(100MHz,DMSO-d6)δ(ppm):167.81,164.53,153.01,151.09,148.04,135.14,135.09,133.03,129.78,129.73(2C),124.06,123.89,111.23,111.15(2C),50.97,48.16(2C),45.55.HRMS(ESI)+calculated for C20H21N6O2,[M+H]+:m/z 377.1715,found:377.1721.
4-(二甲氨基)-N-(5-(4-(4-甲基哌嗪-1-基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13k).白色粉末,熔点:315.8~318.1℃,产率:51.8%.1H NMR(400MHz,DMSO-d6)δ(ppm):10.70(s,1H),9.88(s,1H),7.90(d,J=8.8Hz,2H),7.57(d,J=8.8Hz,2H),7.09(d,J=8.8Hz,2H),6.80(d,J=8.8Hz,2H),4.87(s,2H),4.48(s,2H),4.00(d,J=12.0Hz,2H),3.56(d,J=12.0Hz,2H),3.19(t,J=12.0Hz,2H),3.09(t,J=12.0Hz,2H),3.02(s,6H),2.89(s,3H).HRMS(ESI)+calculated for C26H32N7O2,[M+H]+:m/z474.2606,found:474.2612.
4-(二甲氨基)-N-(5-(3-(4-甲基哌嗪-1-基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(13l).白色粉末,熔点:272.3~277.8℃,产率:67.4%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.15(s,1H),10.51(s,1H),7.87(d,J=8.6Hz,2H),7.29(q,J=7.6Hz,1H),7.08(s,1H),7.03(dd,J=6.0,2.8Hz,1H),6.93(d,J=7.2Hz,1H),6.72(d,J=8.6Hz,2H),4.65(s,2H),4.53(s,2H),3.18(s,4H),2.99(s,6H),2.45(s,4H),2.22(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):170.33,170.06,164.42,153.00,150.26,138.36,129.77(2C),129.73,119.05,117.93,117.04,116.08,114.25,111.22,111.15(2C),53.24(2C),46.45(2C),45.51,43.49(2C),40.10,40.08.HRMS(ESI)+calculated for C26H32N7O2,[M+H]+:m/z 474.2603,found:474.2612.
4-((5-(4-甲基苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)氨基甲酰基)苯甲酸(14a).白色粉末,熔点:316.6~318.4℃,产率:80.7%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.85(s,2H),11.19(s,1H),8.14(d,J=8.0Hz,1H),8.06(d,J=4.0Hz,1H),8.03(s,1H),8.01(s,1H),7.49(dd,J=8.0Hz,2H),7.29(d,J=8.0Hz,2H),4.70(s,2H),4.60(s,2H),2.37(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):167.10,163.84,139.90,137.31,134.31,134.03,129.74,129.66(2C),129.38(2C),129.27,128.57,128.51(2C),127.45,127.29(2C),50.88,45.56,21.40.HRMS(ESI)+calculated for C21H18N4NaO4,[M+Na]+:m/z413.1216,found:413.122.
4-((5-(4-(三氟甲基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)氨基甲酰基)苯甲酸(14b).白色粉末,熔点:296.5~298.1℃,产率:85.9%.1H NMR(400MHz,DMSO-d6)δ(ppm):11.71(s,1H),11.95(s,1H),11.40(s,1H),8.19(d,J=8.2Hz,1H),8.09(d,J=5.2Hz,1H),8.07(d,J=5.2Hz,1H),8.01(d,J=8.2Hz,1H),7.88(d,J=8.2Hz,2H),7.83(d,J=8.2Hz,2H),4.75(s,2H),4.59(s,2H).13C NMR(100MHz,DMSO-d6)δ(ppm):168.66,167.04,163.83,141.16,137.31,137.16,134.08,129.74,129.66(2C),129.38(2C),128.61,128.54(2C),128.25,128.12(2C),125.98,50.62,45.66.HRMS(ESI)+calculated for C21H15F3N4NaO4,[M+Na]+:m/z 467.0934,found:467.0938.
4-氨基-N-(5-(4-甲基苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(15a).白色粉末,熔点:180.6~188.3℃,产率:55.9%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.18(s,1H),10.32(s,1H),7.71(d,J=8.4Hz,2H),7.49(d,J=8.0Hz,2H),7.28(t,J=8.0Hz,2H),6.53(s,2H),5.77(d,J=8.4Hz,2H),4.68(s,2H),4.56(s,2H),2.37(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):170.15,164.59,152.88,139.85,134.41,130.01,129.96(2C),129.39(2C),129.28,127.46,127.26(2C),113.06,113.00(2C),55.35,45.55,21.41.HRMS(ESI)+calculated for C20H19N5NaO2,[M+Na]+:m/z 384.1424,found:384.1431.
4-氨基-N-(5-(4-(三氟甲基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(15b).白色粉末,熔点:303.2~305.7℃,产率:49.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.21(s,1H),10.43(s,1H),7.87(d,J=8.4Hz,2H),7.80(d,J=8.4Hz,2H),7.66(d,J=8.4Hz,2H),6.57(d,J=8.4Hz,2H),5.80(s,2H),4.69(s,2H),4.52(s,2H).13CNMR(100MHz,DMSO-d6)δ(ppm):168.67,168.47,164.60,141.28,130.02,129.97(2C),128.23,128.09(2C),125.97(2C),125.82,125.78,123.06,113.06,112.99(2C),55.35,45.66.HRMS(ESI)+calculated for C20H16F3N5NaO2,[M+Na]+:m/z 438.1152,found:438.1148.
4-氨基-N-(5-(3-(二甲氨基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(15c).白色粉末,熔点:175.6~178.3℃,产率:43.5%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.21(s,1H),10.41(s,1H),7.71(d,J=8.4Hz,2H),7.31–7.22(m,1H),6.82(d,J=7.6Hz,1H),6.80(s,1H),6.77(d,J=7.6Hz,1H),6.56(d,J=8.4Hz,2H),5.77(s,2H),4.67(s,2H),4.51(s,2H),2.93(s,6H).13C NMR(100MHz,DMSO-d6)δ(ppm):170.85,164.58,150.69,150.65,138.15,130.04,129.99(2C),129.52,129.40,114.28,113.68,113.08,113.02(2C),110.77,110.51,50.97,48.16(2C),45.49.HRMS(ESI)+calculated forC21H22N6NaO2,[M+Na]+:m/z 391.1871,found:391.1877.
4-氨基-N-(5异烟酰基-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(15d).白色粉末,熔点:198.6~202.3℃,产率:39.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.15(s,1H),10.41(s,1H),8.72(t,J=6.8Hz,2H),7.71(d,J=8.4Hz,2H),7.61–7.53(m,2H),6.56(d,J=8.4Hz,2H),5.80(s,2H),4.67(s,2H),4.51(s,2H).13C NMR(100MHz,DMSO-d6)δ(ppm):165.87,164.35,150.68,145.24,144.89,130.00,129.93(2C),124.57,124.36(2C),117.41(2C),117.00,114.52(2C),50.30,45.78.HRMS(ESI)+calculated for C18H17N6O2,[M+H]+:m/z 349.1509,found:349.1508.
4-氨基-N-(5-烟酰-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)苯甲酰胺(15e).白色粉末,熔点:299.8~301.7℃,产率:54.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.50(s,1H),11.33(s,1H),8.81(dd,J=7.6,2.0Hz,1H),8.69(t,J=4.4Hz,1H),8.36(d,J=8.4Hz,1H),8.30(d,J=8.4Hz,1H),8.25(d,J=8.4Hz,1H),8.16(d,J=8.4Hz,1H),8.05(t,J=8.8Hz,1H),7.53(dd,J=9.2,4.4Hz,1H),6.35(s,2H),4.75(s,2H),4.65(s,2H).13C NMR(100MHz,DMSO-d6)δ(ppm):165.18,164.90,163.04,149.76,149.72,145.54,143.04,142.71,142.13,134.98,129.70(2C),126.90,126.81,123.80(2C),50.60,45.84.HRMS(ESI)+calculated for C18H16N6NaO2,[M+Na]+:m/z 371.1223,found:371.1227.
1-(5-(4-甲基苯甲酰)-1,4,5,6-四氢吡咯[3,4-c]吡唑-3-基)咪唑烷-2-酮(19a).白色粉末,熔点:315.3~318.1℃,产率:62.7%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.08(s,1H),7.46(d,J=7.6Hz,2H),7.26(d,J=7.6Hz,2H),6.85(s,1H),4.63(s,2H),4.51(s,2H),3.76(s,2H),3.38(d,J=8.0Hz,2H),2.36(d,J=8.0Hz,2H).HRMS(ESI)+calculated for C16H17N5NaO2,[M+Na]+:m/z 334.1268,found:334.1274.
1-(5-(4-(三氟甲基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)咪唑啉-2-酮(19b).白色粉末,熔点:313.5~317.2℃,产率:80.1%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.16(s,1H),7.84(t,J=8.0Hz,2H),7.77(d,J=8.0Hz,2H),6.89(s,1H),4.67(s,2H),4.49(s,2H),3.77(t,J=8.0Hz,2H),3.38(t,J=8.0Hz,2H).HRMS(ESI)+calculatedfor C16H14F3N5NaO2,[M+Na]+:m/z388.0985,found:388.0992.
1-(5-(3-(二甲氨基)苯甲酰基)-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基)咪唑啉-2-酮(19c).白色粉末,熔点:194.6~198.4℃,产率:77.9%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.07(s,1H),7.25(t,J=7.6Hz,1H),6.89(m,1H),6.81(d,J=2.4Hz,1H),6.78(s,1H),6.75(d,J=7.6Hz,1H),4.63(s,2H),4.51(s,2H),3.76(d,J=8.0Hz,2H),3.38(d,J=8.0Hz,2H),2.92(s,6H).13C NMR(100MHz,DMSO-d6)δ(ppm):170.68,170.51,150.63,138.06,137.91,129.42,114.50,114.43,113.80,110.74,110.66,45.21,43.99,40.52,40.49,37.95(2C).HRMS(ESI)+calculated for C17H21N6O2,[M+H]+:m/z 341.1711,found:341.1721.
1-(5-异烟酰基-1,4,5,6-四氢吡咯[3,4-c]吡唑-3-基)咪唑啉-2-酮(19d).白色粉末,熔点:314.2~316.5℃,产率:71.2%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.25(s,1H),9.08(d,J=5.6Hz,2H),8.21(d,J=5.6Hz,2H),7.01(m,1H),4.73(s,2H),4.54(s,2H),3.89–3.75(m,2H),3.41(t,J=8.2Hz,2H).HRMS(ESI)+calculated for C14H15N6O2,[M+H]+:m/z 299.1249,found:299.1251.
1-(5-烟酰基-1,4,5,6-四氢吡咯[3,4-c]吡唑-3-基)咪唑啉-2-酮(19e).白色粉末,熔点:303.2~305.8℃,产率:64.3%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.32(s,1H),9.19(t,J=7.6Hz,1H),9.00(s,1H),8.76(d,J=6.4Hz,1H),8.12(s,1H),6.94(s,1H),4.72(s,2H),4.64(s,2H),3.81(d,J=8.8Hz,2H),3.42(d,J=8.8Hz,2H).HRMS(ESI)+calculated for C14H15N6O2,[M+H]+:m/z299.1248,found:299.1251.
1-(5-(4-甲基哌嗪-1-基)苯甲酰)-1,4,5,6-四氢吡咯罗[3,4-c]吡唑-3-基)咪唑烷-2-酮(19f).白色粉末,熔点:272.7~279.3℃,产率:79.6%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.13(s,1H),7.48(d,J=8.0Hz,2H),6.97(d,J=8.0Hz,2H),6.81(s,1H),4.65(s,2H),4.60(s,2H),3.79(d,J=8.0Hz,2H),3.44(d,J=8.0Hz,2H),3.42–3.34(m,4H),2.56(s,4H),2.30(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):169.82,158.58,150.81,129.11,129.03(2C),127.51,118.63,115.69,115.09(2C),52.50(2C),51.11,45.43,45.17(2C),44.00,42.47,37.94.HRMS(ESI)+calculated for C20H26N7O2,[M+H]+:m/z 396.2139,found:396.2142.
1-(5-(3-(4-甲基哌嗪-1-基)苯甲酰)-1,4,5,6-四氢吡咯罗[3,4-c]吡唑-3-基)咪唑烷-2-酮(19g).白色粉末,熔点:216.5~220.4℃,产率:76.1%.1H NMR(400MHz,DMSO-d6)δ(ppm):12.07(s,1H),7.28(t,J=7.6Hz,1H),7.05(d,J=9.6Hz,1H),7.03(d,J=3.6Hz,1H),6.96–6.90(m,1H),6.85(m,1H),4.62(s,2H),4.50(s,2H),3.77(d,J=7.2Hz,2H),3.40(d,J=7.2Hz,2H),3.20(s,4H),2.54(s,4H),2.28(s,3H).13C NMR(100MHz,DMSO-d6)δ(ppm):170.36,170.18,158.64,151.14,138.09,137.97,129.52,117.33,117.25,116.85,113.83,65.38,54.73(2C),48.01(2C),45.81,43.99,37.93,15.63.
以下通过实验例证明本发明的有益效果。
实验例1:体外激酶活性评价
1.实验方法
利用ERK1/2(Phospho-T202/Y204)TR-FRET细胞检测试剂盒测定本发明目标化合物在1μM浓度下对ERK1/2激酶的抑制率。以已知的ERK1/2激酶抑制剂SCH772984作为阳性对照。
ERK1/2和ERK-TR-FRET细胞检测试剂盒均购自Biorbyt公司,检测方法按照试剂盒的检测手册进行细胞处理、细胞裂解、蛋白质检测的实验,它通过量化激酶反应后溶液中所剩余的ATP的量进而测量激酶活性。实验测定的发光信号与存在的ADP的量相关,并且与激酶活性的量相关。
进一步测定了低浓度下抑制活性较好的目标化合物13l在不同浓度下对ERK1/2的抑制作用,并使用GraphPad Prism软件进行非线性回归计分析来计算IC50值。IC50定义为抑制率达50%时所需的化合物浓度。
2.实验结果
表3.目标化合物在1μM下对ERK1/2激酶的抑制率
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表4.目标化合物在1μM下对ERK1/2激酶的抑制率
可以看出,本发明化合物在较低浓度(1μM下)对ERK1/2激酶具有良好的抑制效果。其中,化合物13l的抑制效果最佳,对ERK1、ERK2激酶的抑制率高达62.41%、65.96%。进一步计算出化合物13l对ERK1、ERK2激酶的IC50值分别为1.69μM、0.87μM(图1)。
上述实验结果表明,本发明化合物能够有效抑制ERK1/2激酶,可以作为ERK1/2激酶抑制剂。
实验例2:体外细胞增殖抑制实验
1.实验方法
本实验评估了化合物13l对两种携带了K-Ras激活突变的SW-620和HCT-116肿瘤细胞系的抗增殖活性。首先,将处于对数生长期的SW620和HCT116人结肠癌细胞用10%的PBS配制成细胞悬液,并以1×105个细胞/mL培养基的密度分别接种到96孔板中,每孔体积为100μL,随后转移至37℃下含5%CO2的培养箱中培养12h。接下来用DMSO溶解化合物13l,并用DMEM培养基处理,稀释成各种目标浓度梯度的溶液(0.3μM、0.5μM、1.0μM、5.0μM),接下来将不同浓度的待测化合物添加到每个孔中处理细胞,随后转移至37℃下含5%CO2的培养箱中孵育24-72h。接下来将配制的5mg/ml浓度的MTT溶液加入到上述孵育好细胞的96孔板中(20μL/孔),再次转移至37℃下含5%CO2的培养箱中孵育2-4h,然后弃去孔中培养基,并在每孔中加入150μL的DMSO溶液,随即用摇床震荡孔板直至结晶物充分溶解,最后使用酶标仪在570nm波长下测定各个孔的吸光度值,并取三个复孔测算抑制率。抑制率=(对照组-给药组)/对照组×100%。使用GraphPad Prism6分析数据,计算IC50值。
以已知的ERK1/2激酶抑制剂SCH772984作为阳性对照。
2.实验结果
表5.化合物13l对人结直肠癌细胞的IC50值
表5和图2数据表明,化合物13l对两种人结直肠癌细胞系均表现出有效的抑制活性,其对SW-620和HCT-116细胞的IC50值分别为1.16μM、0.59μM。
实验例3:蛋白免疫荧光印记分析
1.实验方法
(1)样品制备
将处于对数生长期的SW620和HCT116人结肠癌细胞分别以1×105细胞/孔的密度分布到6孔板上,在加入适量的DMEM培养基后,放置在37℃且含有5%CO2的培养箱中孵育12h。待细胞密度达到80%~90%后,分别以含有不同浓度(0.5μM、1.0μM)的化合物13l的培养基处理两种细胞24h。接下来用干净的刮刀收集细胞,随后加3ml 4℃预冷的PBS缓冲液清洗,1500rpm下离心5分钟后弃去上清液,并重复清洗操作两次以洗去培养液。接着在冰上用适量包含有蛋白酶抑制剂和磷酸酶抑制剂的裂解缓冲液来裂解清洗好的细胞,为使细胞充***解此过程需要经常来回摇动且应在冰上至少进行30分钟。在裂解完后,需要用枪将细胞碎片和裂解液转移到1.5ml离心管中,随后在4℃下提前预冷的离心机里以12000rpm离心10分钟。收集上清液后即可进行下一步的蛋白含量测定实验。
(2)蛋白含量测定
首先将A液与B液按照50:1的体积比配置BCA工作液2mL备用,然后使用PBS将蛋白样品浓度稀释成0.5mg/mL,接下来按照下表6进行梯度稀释,用以标准浓度曲线的绘制。
表6.标准浓度曲线梯度稀释表
编号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
蛋白标准稀释液(μL) | 0 | 2 | 4 | 6 | 8 | 12 | 16 | 20 |
PBS Diluent(μL) | 20 | 18 | 16 | 14 | 12 | 8 | 4 | 0 |
蛋白终浓度(mg/mL) | 0 | 0.05 | 0.10 | 0.15 | 0.20 | 0.30 | 0.40 | 0.50 |
接下来,将表中的样品各取20μL小心加入96孔板中,随即每个孔中再各加入200μLBCA工作液,在震荡均匀后,放37℃下使其显色15-30min。最后用酶标仪在570nm波长下测定各孔的吸光度值,利用空白对照绘制标准曲线,得回归方程,将测定管吸光值代入公式即可得到各组蛋白浓度。
(3)SDS-PAGE电泳
将清洗好的玻璃板用夹子夹紧并垂直的卡在配胶架上,按照检测蛋白配置10%的分离胶,随后加入TEMED并使其混合均匀,随后用移液枪沿玻璃夹缝缓慢灌胶,之后胶的上层加异丙醇封闭,使其凝固更快。室温下静置30分钟,在胶充分凝固后倒去液封的异丙醇,用蒸馏水清洗并擦干水分备用。加下来,按同样方法配置4%的浓缩胶,然后加入TEMED并充分混合,再次灌胶后,将梳子水平的***浓缩胶中,室温下再次静置30分钟,等到胶完全凝固后,缓慢将梳子拔出,清洗凝胶后,完整的将其放入电泳槽中,将测定蛋白含量后的样品分别加入加样孔中。最后,在80V下电泳3~4h,直至电泳结束即可进行下一步实验。
(4)转膜
首先,根据凝胶的大小剪6张滤纸和1张PVDF膜备用,然后用甲醇浸泡活化PVDF膜。接下来将凝胶缓慢从玻璃板中取出备用,待膜放入转移液后,将转膜夹黑色面朝上,并依次铺垫一层海绵垫、三层滤纸、凝胶、PVDF膜、三层滤纸和一层海绵垫,最后合上转膜夹红色面(此过程中应避免气泡)。最后将做好的上述转膜夹放入转膜槽中,待加满转移液后,在80V下转膜2h(此过程中,应在槽的另一边加少许冰块,以散去电转移时产生的热量)。
(5)免疫反应
首先,将上述PVDF膜用PBST缓冲液漂洗三次,然后转移至含有5%脱脂奶粉的PBST封闭液中室温下脱色1h。然后将封闭好的PVDF膜用PBST缓冲液再次漂洗三次,随后转移到含有适量一抗稀释液的杂交袋中,接着在摇床上室温下孵育1~2h后,用PBST缓冲液再次漂洗三次,同样方法加二抗稀释液再次室温下孵育1~2h。
(6)显色
将上述孵育好的PVDF膜用PBST缓冲液漂洗三次后,然后在膜蛋白面上均匀滴加显影液,1分钟后立即检测实验结果。
2.实验结果
检测经化合物13l处理后HCT116细胞凋亡相关蛋白的表达水平。如图3结果表明,化合物13l以浓度依赖性的方式降低HCT116细胞中ERK1/2(Thr202、Tyr204)和p90RSK(Thr359、Ser363)蛋白在细胞内的磷酸化水平。
实验例4:细胞凋亡实验
1.实验方法
本实验采用Annexin V-FITC/PI染色和Hoechst 33258染色的方法来测定细胞的凋亡率。首先,将处于对数生长期的HCT116人结肠癌细胞以1×105细胞/孔的密度分布到6孔板上,在加入适量的DMEM培养基后,放置在37℃且含有5%CO2的培养箱中孵育12h。待细胞密度达到80%~90%后,分别以含有不同浓度(0、0.5μM、1.0μM)的化合物13l的培养基处理HCT116细胞。24h后收获13l处理和未处理的细胞,随即用不含EDTA的胰蛋白酶进行消化处理细胞,在3000rpm下离心5分钟后,用预冷的PBS将细胞洗涤两次,随后弃去上清液,并选择收集1~5×105个细胞。接下来,在室温下加入100μL的结合缓冲液进行吹打混匀,随后再加入5μL的Annexin-VFITC和5μL的PI染液,在震荡混合均匀后,室温下避光孵育15分钟,最后上机用流式细胞仪对细胞进行观察和检测,并使用Flowjo软件分析结果。
2.实验结果
本实验通过流式细胞仪检测细胞的凋亡率从而来探索细胞的死亡原因。首先,用化合物13l处理的HCT116细胞经Hoechst 33258染色后,很容易观察到浓缩明亮的凋亡细胞核(如图4所示)。接下来,分别以0、0.5、1μM浓度的化合物13l处理HCT116细胞24h后,通过Annexin V-FITC/PI对细胞染色,随后上机用流式细胞仪对细胞进行观察和检测。细胞凋亡结果如图5所示,结果显示,化合物13l可以浓度依赖的方式诱导HCT116人结肠癌细胞的凋亡。在化合物13l的1μM浓度下,HCT116细胞的总凋亡率可达23.7%。进一步证实了化合物13l可诱导人结肠癌细胞凋亡。
综上,本发明提供了一种ERK抑制剂及其制药用途。本发明提供的化合物对ERK1/2激酶具有良好的抑制效果,对结直肠癌细胞系表现出有效的抑制活性,能够降低结直肠癌细胞细胞中ERK1/2(Thr202、Tyr204)和p90RSK(Thr359、Ser363)蛋白在细胞内的磷酸化水平,能够诱导结肠癌细胞凋亡。本发明化合物能够用来制备ERK1/2激酶抑制剂,以及预防和/或治疗与ERK1/2激酶活性相关的疾病的药物,应用前景广阔。
Claims (7)
1.一种化合物或其药学上可接受的盐,其特征在于:所述化合物为:
。
2.一种药物,其特征在于:它是以权利要求1所述化合物或其药学上可接受的盐为活性成分,加上药学上可接受的辅料制备而成的制剂。
3.权利要求1所述化合物或其药学上可接受的盐在制备ERK抑制剂中的用途。
4.根据权利要求3所述的用途,其特征在于:所述ERK抑制剂为ERK1抑制剂和/或ERK2抑制剂。
5.根据权利要求3或4所述的用途,其特征在于:所述ERK抑制剂为预防和/或治疗与ERK活性相关的疾病的药物。
6.根据权利要求5所述的用途,其特征在于:所述与ERK活性相关的疾病为癌症。
7.根据权利要求6所述的用途,其特征在于:所述癌症为结直肠癌、肺癌、胰腺癌、黑素瘤、急性骨髓系白血病、胶质母细胞瘤。
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