基于c-Met靶点的抗肿瘤药物的设计与合成研究
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摘要
c-Met酪氨酸激酶是肝细胞生长因子高亲和性配体,c-Met广泛表达于多种人体正常组织,但在肺癌、结肠癌、肝癌、直肠癌、胃癌、卵巢癌、肾癌、神经胶质瘤、黑素瘤、乳腺癌、前列腺癌等肿瘤组织中呈现出异常的高表达、突变或活性改变。在多种肿瘤组织中发现HGF/c-Met信号通路异常活化-这种异常活化参与并调控这些肿瘤的发生、发展或转移。由于c-Met是导致肿瘤形成及转移的许多通路的交叉点,以c-Met为靶标可相对较容易地实现对许多通路的同时干扰,因而,c-Met是抗肿瘤转移治疗的一个极有希望的靶点,已成为抗癌药物研究的热点领域之一。目前,已经发现了许多能够阻断HGF/c-Met信号传导途径的化合物,其中小分子c-Met激酶抑制剂已经成为研究的重点。
本课题以Pfizer开发的1-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪类化合物PF-04217903、Janssen开发的3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪类化合物JNJ-38877605、SGX公司的三唑哒嗪类化合物SGX-523以及课题组前期研究的2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类、螺[吲哚啉-3,4’-哌啶]-2-酮类专利化合物为先导化合物,基于激酶的"DFG-in"构象的晶体结构和I型抑制剂的药效团模型,结合三唑类I型c-Met抑制剂的构效关系,总结了国际知名药企对该类化合物的优化改造经验,通过生物电子等排体原理、拼合原理及分子对接等手段设计合成了一系列新化合物采用定向合成和其它化合物制备技术,合成这些化合物。然后对合成的衍生物进行活性评价,筛选出活性更好的化合物,采用分子对接对设计的化合物与c-Met结合情况进行了初步的分析和探讨,结合化合物的活性情况,进行了初步构效关系分析,为开发具有临床应用价值的c-Met酪氨酸激酶抑制剂奠定基础。
项目主要研究工作及结果如下：
1、将先导化合物中的1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环通过生物电子等排体原理用3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤、吲哚唑环替代,设计并合成3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤、吲哚唑系列的化合物：(1)3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶类化合物,在三唑[4,5-d]嘧啶环的5位分别引入1-甲基-1H-吡唑、1-(2-四氢-2H-吡喃-2-氧乙基)-1H-吡唑、1-(4-羟基环己基)-1H-吡唑1-[(1R,4R)-4-(正丁基二甲基硅氧基)环己基]-1H-吡唑和1-((1R,4R)-4-羟基环己基)-1H-吡唑得到化合物6-((5-(1-甲基-1H-吡唑-4-基)-3H-[1,2,3]三唑[4,5-d]嘧啶-3-基)甲基)喹啉WXY001,6-(5-(2-(1-(2-四氢-2H-吡喃-2-基氧乙基)-1H-吡唑-4-基)-3H-[1,2,3]三唑[4,5-d]嘧啶-3-基)甲基)喹啉WXY002,2-(4-(3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基)-1H-吡唑-1-基)乙醇WXY003,6-((5-(1-((1R,4R)-4-(叔丁基二甲基硅氧基)环己基)-1H-吡唑-4-y1)-3H-[1,2,3]三唑[4,5-d]嘧啶-3-基)甲基)喹啉WXY004,(1R,4R)-4-[4-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基]-1H-吡唑-1-基]环己醇WXY005。此类目标化合物是通过1位取代的4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)吡唑与5-氯-3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶进行Suzuki耦合反应得到。(2)7-(喹啉-6-基甲基)嘌呤类化合物,在嘌呤环的2位分别引入1-甲基-1H-吡唑、1-(2-四氢-2H-吡喃-2-氧乙基)-1H-吡唑、1-(4-羟基环己基)-1H-吡唑1-[(1R,4R)-4-(正丁基二甲基硅氧基)环己基]-1H-吡唑和1-((1R,4R)-4-羟基环己基)-1H-吡唑得到化合物6-[[2-(1-甲基-1H吡唑-4-基)-9H-嘌呤-9-基]甲基]喹啉WXY006,6-[[2-[1-(2-四氢-2H-吡喃-2-基氧乙基)1H-吡唑-4-基]-9H-嘌呤-9-基]甲基]喹啉WXY007,2-[4-[9-(6-喹啉基甲基)-9H-嘌呤-2-基]-1H吡唑-1-基]乙醇WXY008,6-((2-(1-((1R,4R)-4-(叔丁基二甲基硅氧基)环己基)-1H-吡唑-4-基)-9H-嘌吟-9-基)甲基)喹啉WXY009,(1R,4R)-4-[4-[9-(喹啉基-6-甲基)-9H-嘌呤-2-基]-1H-吡唑-1-基]环己醇WXY010。该类化合物以2-氯-N4-(喹啉-6-基-甲基)嘧啶-4,5-二胺为原料,与原甲酸三甲酯在对甲苯磺酸一水合物催化下环合后再与1位取代的4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)吡唑进行Suzuki耦合反应得到。(3)1(2)-(喹啉-6-基甲基)吲哚唑类化合物在吲哚唑6位引入1-甲基-1H吡唑、1-(2-四氢-2H-吡喃-2-氧乙基)-1H-吡唑、1-(4-羟基环己基)-1H-吡唑1-[(1R,4R)-4-(正丁基二甲基硅氧基)环己基]-1H-吡唑和1-((1R,4R)-4-羟基环己基)-1H-吡唑得到化合物6-[[6-(1-甲基-1H-吡唑-4-基)-1H-吲唑-1-基]甲基]喹啉WXY011,6-[[6-[1-(2-四氢-2H-吡喃-2-基氧乙基)-1H-吡唑-4-基]吲唑-1-基]甲基]喹啉WXY012,2-[4-[1-(喹啉-6-甲基)-1H吲唑-6-基]-1H-吡唑-1-基]乙醇WXY013,6-((6-(1-((1R,4R)-4-(叔丁二甲硅氧基)环己基)-1H-吡唑-4-基)-1H-吲唑-1-基)甲基)喹啉WXY014,(1R,4R)-[4-[4-[1-(喹啉-6-甲基)-1H-吲唑-6-基]-1H-吡唑-1-基]环己醇WXY015,6-[[6-(1-甲基-1H-吡唑-4-基)-2H-吲唑-2-基]甲基]喹啉WXY016,6-[[6-[1-(2-四氢-2H-吡喃-2-基氧乙基)-1H-吡唑-4-基]-2H-吲唑-2-基]甲基]喹啉WXY017,2-[4-[1-(喹啉-6-甲基)-1H-吲唑-6-基]-1H-吡唑-1-基]乙醇WXY018,6-((6-(1-((1R,4R)-4-(叔丁二甲硅氧基)环己基)-1H吡唑1-4-基)-2H-吲唑-2-基)甲基)喹啉WXY019,(1R,4R)-[4-[4-[2-(喹啉-6-甲基)-2H-吲唑-6-基]-1H-吡唑-1-基]环己醇WXY020。该类化合物以1位取代的4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)吡唑为原料,与商业可提供的6-溴-1H-吲哚唑发生Suzuki耦合反应,然后再与6-氯甲基喹啉发生亲电取代反应得到吲哚唑环1位取代的化合物及其对应的互变异构体吲哚唑环2位取代的化合物。MS和H'-NMR确认化合物的结构,对化合物进行c-Met生化半抑制浓度(IC50)实验,实验结果显示,3H-[1,2,3]三唑[4,5-d]嘧啶和嘌呤(咪唑并嘧啶)系列化合物活性较差,吲哚唑系列中大部分化合物对c-Met激酶有较强的抑制作用,除化合物WXY016和WXY019的IC50值超过10μM外,其余都在0.9μM以下。
2、保留先导化合物中的1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环、连接臂(亚甲基)、喹啉环三个部分不变,将课题组前期研究中的专利化合物2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类化合物中的2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚结构片段引入到该类化合物中替换吡嗪环或哒嗪环上的取代基,得到新型的含1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环的2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类化合物8-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚-2-甲酸叔丁酯WXY021,8-[3-(喹啉-6-基甲基)-3H-[1,2,3三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚WXY022,8-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚-2-甲醛WXY023,8-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢吡啶[4,3-b]吲哚-2-甲酰胺WXY024,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚-2-甲酸叔丁酯WXY025,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚WXY026,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚-2-甲醛WXY027,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢吡啶[4,3-b]吲哚-2-甲酰胺WXY028。8-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)-1,3,4,5-四氢吡啶并[4,3-b]吲哚-2-甲酸叔丁酯与6-[(5-溴三唑[4,5-d]嘧啶-3-基)甲基]喹啉和6-[(6-氯-[1,2,4]三唑[3,4-f]哒嗪-3-基)甲基]喹啉发生Suzuki耦合反应得到WXY021和WXY022,然后再与甲酸及1-(3-二甲基氨基丙基)-3-乙基碳化二亚胺盐酸盐发生甲酰化反应得到WXY023和WXY027,化合物WXY021和WXY022也与三甲基硅基异氰酸酯发生氨甲酰化反应得到WXY024,WXY028,同时得到化合物WXY025,WXY026。MS和H1-NMR确认化合物的结构,对化合物进行c-Met生化半抑制浓度(IC50)实验,实验结果显示,该类化合物对c-Met激酶有很强的抑制作用,化合物WXY022和WXY023的IC50值分别达到了0.0145和0.0995gM,其它化合物大部分在1gM以下。
3、保留先导化合物中的1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环以及其生物电子等排体3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤环、连接臂(亚甲基)、喹啉环三个部分不变,将课题组前期研究中的专利化合物螺环化合物中的螺[吲哚啉-3,4’-哌啶]-2-酮结构片段引入到该类化合物中替换吡嗪环、哒嗪环、嘧啶环上的取代基,结合前期通过分子对接得到的结果,得到新型的含1H-[1,2,3]三唑[4,5-b]吡嗪、3H-[1,2,3]三唑[4,3-b]哒嗪环、3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤环的螺[吲哚啉-3,4'-哌啶]-2-酮类化合物1’-甲基-5-[1-(6-喹啉基甲基)-1H-[1,2,3]三唑[4,5-b]吡嗪-6-基]螺[吲哚啉-3,4’-哌啶]-2-酮WXY029、1’-甲基-6-[1-(6-喹啉基甲基)-1H-[1,2,3]三唑[4,5-b]吡嗪-6-基]螺[吲哚啉-3,4’-哌啶]-2-酮WXY030、1’-甲基-5-(3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪-6-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY031、1’-甲基-6-(3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪-6-基)-螺[吲哚啉-3,4’-哌啶]-2-酮WXY032、1’-甲基-5-(3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基)-螺[吲哚啉-3,4’-哌啶]-2-酮WXY033、1’-甲基-6-(3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY034、1’-甲基-5-(7-(喹啉-6-基甲基)-嘌呤-2-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY035、1’-甲基-6-(7-(喹啉-6-基甲基)-嘌呤-2-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY036,该类化合物由5位和6位硼酸吡呐醇酯与溴取代的1-(6-喹啉基甲基)三唑[4,5-b]吡嗪、3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪、3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶、7-(喹啉-6-基甲基)-嘌呤发生Suzuki耦合反应得到。MS和H1-NMR确认化合物的结构,对化合物进行c-Met生化半抑制浓度(ICso)实验,实验结果显示,该类化合物对c-Met激酶有很强的抑制作用,化合物WXY029、WXY030、WXY031、WXY032对c-Met的抑制作用较化合物WXY033、WXY034、WXY035、WXY036作用强,化合物WXY029、WXY030较化合物WXY031、WXY032化合物作用强,WXY030的IC50值达到了0.0145μM较化合物WXY029(IC50=0.227μM)强,WXY032(IC50=0.22μM)较化合物WXY021(IC50=0.28μM)强,这表明含1H-[1,2,3]三唑[4,5-b]吡嗪、3H-[1,2,3]三唑[4,3-b]哒嗪的螺[吲哚啉-3,4'-哌啶]-2-酮化合物较含3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤环的螺[吲哚啉-3,4'-哌啶]-2-酮化合物有作用强,含有1H-[1,2,3]三唑[4,5-b]吡嗪的螺[吲哚啉-3,4’-哌啶]-2-酮化合物较含有3H-[1,2,3]三唑[4,3-b]哒嗪的螺[吲哚啉-3,4’-哌啶]-2-酮化合物作用强,螺[吲哚啉-3,4'-哌啶]-2-酮的6位取代较5为取代作用强。
4、对分子水平对c-Met酶有强抑制作用的吲哚唑、2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚、螺[吲哚啉-3,4'-哌啶]-2-酮类化合物进行MKN45细胞中c-Met半抑制浓度(IC50)实验,实验结果表明,大部分化合物对细胞中c-Met有较强的抑制作用,部分化合物的ICso值在10μM以下。
5、对五大类合成的36个化合物进行了分子对接研究,分子对接结果显示,所有化合物与c-Met位点均采取“U”型结合模式,具备Ⅰ型c-Met抑制剂的典型特征,喹啉环上的氮与Met1160主链羰基形成氢键,喹啉环C-8与Pro1158羰基上的氧形成一个非经典的C-H-O=C氢键。这种作用与其他ATP-竞争性酶作用相似,稠合环(1H-[1,2,3]三唑[4,5-b]吡嗪环、3H-[1,2,3]三唑[4,3-b]哒嗪环、3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤、吲哚唑环)在Tyr1230和Met1211之间通过π-π堆积作用形成一个夹心结构,喹啉环和稠环之间的连接臂(亚甲基)刚好与Leu1157,Arg1086和Tyr1230或11109,Lys1110,A1a1226和Va11092组成的疏水口袋嵌合,该部分主要是影响稠合环的π-π堆积作用,在所有稠环中,以三唑吡嗪环效果最好,因为三唑哒嗪环作为一个缺电子母核在Tyr1230和Met1211之间通过π-π堆积作用形成一个夹心结构,三唑吡嗪环上的两个氮原子与Asp1222骨架上的NH形成两个氢键,因而与只能形成一个氢键的其它稠环相比其有较高的亲和力和较低的IC50值,而且与Asp1222间的氢键因其具有较低能量可以稳定该类化合物的构型。另外螺环上的哌啶环主要指向u型折叠构型的亲水表面,而该表面远离铰链区。因为在491个酪氨酸激酶中仅仅有c-Met,Axl和Mer三个激酶有两个氨基酸存在,来自Met1211和Tyr1230的疏水作用增加了该类化合物的选择性,螺环5位或六位取代的亲水稠环延伸到结合口袋,与WXY030相比,因螺环的张力存在使得WXY029不能与活性位点匹配,从而导致构型的改变以及增加N原子和Asn1167间的距离而不能形成氢键,因此WXY029的活性较WXY030低,同样的原因可以解释WXY031、WXY033、WXY035活性较WXY032、WXY034、 WXY036低。
综上所述,本课题主要针对c-Met设计了118个信化合物,合成出了36个新化合物,未见文献报道,此外还合成了17个关键中间体。所有目标化合物合成中都用到了Suzuki耦合反应,该反应在在DMF/H2O的混合溶剂中、Pd (PPh3)4催化下进行获得较高的收率,得到的的目标化合物以吲哚唑类、2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类、螺[吲哚啉-3,4’-哌啶]-2-酮类化合物有较好的抗肿瘤活性,WXY030、WXY022和WXY023对c-Met生化半抑制浓度(ICso)达到了10nM,显示强的抗肿瘤活性,对分子水平抑制作用强的化合物进行细胞研究显示部分化合物对MKN45细胞中c-Met的抑制作用达到了10μM。分子对接研究证实和解释了这些化合物的活性。为开发具有临床应用价值的c-Met酪氨酸激酶抑制剂奠定基础。
The c-Met RTK family is the only known high-affinity receptor for hepatocyte growth factor (HGF), also known as scatter factor (SF), which is produced by stromal and mesenchymal cells and acts primarily on c-Met-expressing epithelial cells in an endocrine and/or paracrine fashion. c-Met and HGF are widely expressed in a variety of tissues, and their expression is normally confined to cells of epithelial and mesenchymal origin, c-Met is expressed by most carcinomas and its elevated expression relative to normal tissue has been detected in a number of cancers including lung, breast, colorectal, prostate, pancreatic, head and neck, gastric, hepatocellular, ovarian, renal, glioma, melanoma, and a number of sarcomas. c-Met expression has been reported to be up-regulated in tumors by a number of epigenetic mechanisms including tumor secreted growth factors, tumor hypoxia, and activation of other oncogenes.In addition, c-Met gene amplification and subsequent overexpression has been reported in gastric cancer, colorectal cancer, and glioma. Activation of the HGF/c-Met signaling pathway has been shown to lead to a wide array of cellular responses including proliferation, survival, angiogenesis, wound healing, tissue regeneration, scattering, motility, invasion and branching morphogenesis. Since c-Met is at the crossing of many roads leading to tumorigenesis and metastasis, targeting this receptor could be a relatively simple way to interfere with many pathways simultaneously. HGF/c-Met signaling pathway has come into the spotlight as a promising therapeutic target for inhibiting tumor growth and has become one of the leading molecular targets in cancer. Currently various strategies are currently in development to disrupt the HGF-Met signal transduction pathway, in which small molecular inhibitors have been a particularly active field.
1-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b] pyrazine of Pfizer's PF-04217903,3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine of Janssen's JNJ-33377605and3H-[1,2,4]triazolo[4,3-b]pyridazin of SGX's SGX-523are potent and selective c-Met inhibitors. Based on the crystal structure of the c-Met kinase "DFG-in" conformation, type I kinase inhibitors pharmacophore, structure-activity relationships of triazoles type I c-Met inhibitors and experience of optimization and modification on these compounds, this study designed a series of novel compounds using PF-04217903, JNJ-33377605, SGX-523and our previous patented compounds spiro[indoline-3,4'-piperidine]-2-ones and2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indoles as leading compouds by means of biological isostere principle, combination principles and molecular docking, then synthesized these compounds through directional synthesis and other method of chemical synthesis. We investigated their biological activities on cellular level to obtain more active compounds, analyzed and discussed binding mode of compounds and c-Met kinase through molecular docking experiments and discussed preliminary structure-activity relationship to lay the foundation for developing c-Met inhibitors with clinical application value.The main contents are summarized as follows:1. we designed and synthesized structurally relevant novel compounds by replacing1H-[1,2,3]triazolo[4,5-b]pyrazine and3H-[1,2,4]triazolo[4,3-b]pyridazin of leading compounds with3H-[1,2,3]triazolo[4,5-d]pyrimidine,7H-purine and indazole moiety:(1)3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidines WXY001, WXY002, WXY003, WXY004, WXY005were designed by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-7H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1 H-pyrazole and1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazole to the5-position of3H-[1,2,3]triazolo[4,5-d]pyrimidine. The target compounds were prepared by the reaction of1-position substituted4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazoles with5-chloro-3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine via Suzuki coupling reaction.(2)7-(quinolin-6-ylmethyl)-purines WXY006, WXY007, WXY008, WXY009, WXY010were designed by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1H-pyrazole and1-((1R,4R)-4-hy droxy-cyclohexyl)-1H-pyrazole to the2-position of purines, These compounds were synthesized using2-chloro-N4-(quinolin-6-ylmethyl)-4,5-pyrimidinediamino as material via cyclization reaction and Suzuki coupling reaction.(3)1(2)-(quinolin-6-ylmethyl)-1H-indazoles WXY011, WXY012, WXY013, WXY014, WXY015,WXY016,WXY017,WXY018, WXY019, WXY020can be obtained by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethy1)-1H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy) cyclohexyl]-1H-pyrazole and1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazole to the6-position of indazoles, The4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole groups reacted with6-bromo-1H-indazole to obtain6-(1-methyl-pyrazol-3-yl)-1H-indazole,6-[1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole, and6-{1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclo hexyl]-1H-pyrazol-4-yl}-1H-indazole respectively via Suzuki coupling reaction, then reacted with commercially available6-chloromethylquinoline to give1-(quinolin-6-ylmethyl)-1H-indazoles their isomers2-(quinolin-6-ylmethyl)-1H-indazoles as a result of two isomeric forms of indazole. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that the3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo [4,5-d]pyrimidine and7-(quinolin-6-ylmethyl)-purines exhibited lower inhibitory effect while1(2)-(quinolin-6-ylmethy1)-1H-indazoles exhibited significant inhibitory effect on c-Met with IC50<0.9μM in in TR-FRET-based assay except WXY016and WXY019with IC50>10μM.
2.Keeping1H-[1,2,3]triazolo[4,5-b] pyrazine or3H-[1,2,4]triazolo[4,3-b]pyridazine, methylenelink, quinoline of the lead compounds unchanged, the substituents of the pyrazine/pyridazine were replaced by the structure2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole to obtain novel2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles containing1H-[1,2,3]triazolo[4,5-b] pyrazine and3H-[1,2,4]triazolo[4,3-b]pyridazine WXY021, WXY022, WXY023, WXY024, WXY025, WXY026, WXY027, WXY028, these compounds were prepared as followed:The reaction of tert-butyl8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol e-2-carboxylate with6-bromo-l-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b]pyrazine and5-chloro-3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine, respectively, gave WXY021and WXY022via Suzuki coupling reaction. Then, WXY021and WXY022were transformed to WXY023, WXY027and WXY024, WXY028by formylation and carbanoylation, and obtained WXY025, WXY026concurrently. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that these compounds exhibited potent inhibition on c-Met, in which WXY022and WXY023had stronger with IC50values of0.0145μM and0.0995μM in TR-FRET-based assay than others with IC50<1μM.
3.Keeping1H-[1,2,3]triazolo[4,5-b]pyrazine,3H-[1,2,4]triazolo[4,3-b]pyridazine,3H-[1,2,3]triazolo[4,5-d]pyrimidine,7H-purine, methylenelink and quinoline unchanged, the substituents of the pyrazine, pyridazine and7H-purine were replaced by the structure spiro[indoline-3,4'-piperidine]-2-one of our previous patented compounds to obtain novel spiro[indoline-3,4'-piperidine]-2-ones WXY029, WXY030, WXY031, WXY032, WXY033, WXY034, WXY035and WXY028, which were prepared by reaction of the pinacol borane esters of5and6position of spiro[indoline-3,4'-piperidine]-2-one with bromine substituted1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine, 3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine,3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine and7-(quinolin-6-ylmethyl)-purine via Suzuki coupling. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that these compounds exhibited potent inhibition on c-Met, in which WXY029, WXY030, WXY031, WXY032had stronger than WXY033, WXY034, WXY035,WXY036, and WXY029、WXY030stronger than WXY031, WXY032, WXY030had stronger with IC50values of0.0147μM in TR-FRET-based assay than WXY029with IC50values of0.227μM and WXY032(IC50=0.22μM) stronger than WXY021(IC50=0.28μM). These results suggested that spiro [indoline-3,4'-piperidine]-2-ones containing1-(quinolin-6-ylmethy1)-1H-[1,2,3] triazolo[4,5-b]pyrazine and3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine were more effect on c-Met than others, of which1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo [4,5-b]pyrazine was stronger. Moreover,6position substituted spiro [indoline-3,4'-piperidine]-2-ones presented more effective activity than5position substituted compounds.
4.For the strong inhibition on c-Met kinase, the compounds1(2)-(quinolin-6-ylmethyl)-1H-indazoles,2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles and spiro [indoline-3,4'-piperidine]-2-ones were evaluated in cell-based assay. The results showed that some target compounds exhibited significant inhibitory effect on c-Met with IC5o<10μM in cell-based assay.
5. Molecular docking showed that all the compounds assumed a'U-shaped'binding mode, which is the typical character of type I c-Met inhibitors. The quinoline moiety engages the MET hinge region with a single canonical hydrogen bond between the quinoline nitrogen and backbone carbonyl of Met1178and a nonclassic C-H-O=C hydrogen bond between quinoline C8and the carbonyl oxygen of Pro1176, which was highly characteristic of all compounds bound to ATP binding site in kinase domains. Fused-rings1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine,3-(quinolin-6 ylmethyl-3H-[1.2,4]triazolo[4,3-b]pyridazine,3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine,7-(quinolin-6-ylmethyl)-purine and indazoles were swiched in between Tyr1230and Met1211through a π-π stacking interaction. The methylenelink could better match the sub hydrophobic pocket composed of Leul157, Arg1086and Tyr1230, or Va11109, Lys1110, Ala1226and Va11092and enhanced the π-π interaction, of which triazolopyridine was the best because triazolopyridazine, as an electron deficient group, was sandwiched in between Tyr1230and Metl211through a π-π stacking interaction. In addition, the hydrogen bond was important to the binding affinity and two nitrogen atoms of the triazolopyridine formed two hydrogen bonds with the back bone NH of Asp1222so it had higher affinity and lower IC50value.The spirocyclic pyridine ring also pointed out towards the solvent surface after "U-shape" folding, however, the solvent surface was far away from the hinge region. The hydrophobic interactions from Met1211and Tyr1230would contribute to the selectivity since the two amino acid were conserved among only three(c-Met, Axl, Mer) of the491kinases. The methylene connecting quinoline and triazolopyridazine was folded up to fit the subhydrophobic pocket composed by Leu1157, Arg1086and Tyr1230. The adjacent hydrophilic ring,5or6substituent of spiro [indoline-3,4'-piperidine]-2-ones, is extended to the binding pocket. Comparing to WXY030, WXY029could not match to the active site because of the tension from spire. It leaded to the conformation changes and larger distance between N from N-CH3-pyridine and the residue Asn1167, and could not generate hydrogen bond (shown in Fig.5). Therefore, WXY029had the lower affinity and the lower activity. Similar reasons explained why the analogues WXY032, WXY034, WXY036had more powerful activity than the analogues WXY031, WXY033, WXY035.
In conclusion, we designed118new compounds,36of which were synthesized. Meanwhile,17key intermediates were also synthesized. All the target compounds were prepared via Suzuki coupling reaction. This coupling reaction with pinacol borane esters gave good yields under basic conditions using DMF/H2O as a solvent and Pd (PPh3)4as a catalyst. Indazoles,2.3.4.5-tetrahydro-1H-pyrido[4,3-b]indoles and spiro[indoline-3,4'- piperidine]-2-ones had the best inhibitory effect, in which IC50values of WXY030, WXY022and WXY023reached lOnM in TR-FRET-based assay and IC50values of some compounds reached1OμM in cell-based assay. Molecular docking experiments verified and explained the results.The study lay the foundation for developing c-Met inhibitors with clinical application value.
本课题以Pfizer开发的1-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪类化合物PF-04217903、Janssen开发的3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪类化合物JNJ-38877605、SGX公司的三唑哒嗪类化合物SGX-523以及课题组前期研究的2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类、螺[吲哚啉-3,4’-哌啶]-2-酮类专利化合物为先导化合物,基于激酶的"DFG-in"构象的晶体结构和I型抑制剂的药效团模型,结合三唑类I型c-Met抑制剂的构效关系,总结了国际知名药企对该类化合物的优化改造经验,通过生物电子等排体原理、拼合原理及分子对接等手段设计合成了一系列新化合物采用定向合成和其它化合物制备技术,合成这些化合物。然后对合成的衍生物进行活性评价,筛选出活性更好的化合物,采用分子对接对设计的化合物与c-Met结合情况进行了初步的分析和探讨,结合化合物的活性情况,进行了初步构效关系分析,为开发具有临床应用价值的c-Met酪氨酸激酶抑制剂奠定基础。
项目主要研究工作及结果如下：
1、将先导化合物中的1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环通过生物电子等排体原理用3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤、吲哚唑环替代,设计并合成3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤、吲哚唑系列的化合物：(1)3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶类化合物,在三唑[4,5-d]嘧啶环的5位分别引入1-甲基-1H-吡唑、1-(2-四氢-2H-吡喃-2-氧乙基)-1H-吡唑、1-(4-羟基环己基)-1H-吡唑1-[(1R,4R)-4-(正丁基二甲基硅氧基)环己基]-1H-吡唑和1-((1R,4R)-4-羟基环己基)-1H-吡唑得到化合物6-((5-(1-甲基-1H-吡唑-4-基)-3H-[1,2,3]三唑[4,5-d]嘧啶-3-基)甲基)喹啉WXY001,6-(5-(2-(1-(2-四氢-2H-吡喃-2-基氧乙基)-1H-吡唑-4-基)-3H-[1,2,3]三唑[4,5-d]嘧啶-3-基)甲基)喹啉WXY002,2-(4-(3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基)-1H-吡唑-1-基)乙醇WXY003,6-((5-(1-((1R,4R)-4-(叔丁基二甲基硅氧基)环己基)-1H-吡唑-4-y1)-3H-[1,2,3]三唑[4,5-d]嘧啶-3-基)甲基)喹啉WXY004,(1R,4R)-4-[4-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基]-1H-吡唑-1-基]环己醇WXY005。此类目标化合物是通过1位取代的4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)吡唑与5-氯-3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶进行Suzuki耦合反应得到。(2)7-(喹啉-6-基甲基)嘌呤类化合物,在嘌呤环的2位分别引入1-甲基-1H-吡唑、1-(2-四氢-2H-吡喃-2-氧乙基)-1H-吡唑、1-(4-羟基环己基)-1H-吡唑1-[(1R,4R)-4-(正丁基二甲基硅氧基)环己基]-1H-吡唑和1-((1R,4R)-4-羟基环己基)-1H-吡唑得到化合物6-[[2-(1-甲基-1H吡唑-4-基)-9H-嘌呤-9-基]甲基]喹啉WXY006,6-[[2-[1-(2-四氢-2H-吡喃-2-基氧乙基)1H-吡唑-4-基]-9H-嘌呤-9-基]甲基]喹啉WXY007,2-[4-[9-(6-喹啉基甲基)-9H-嘌呤-2-基]-1H吡唑-1-基]乙醇WXY008,6-((2-(1-((1R,4R)-4-(叔丁基二甲基硅氧基)环己基)-1H-吡唑-4-基)-9H-嘌吟-9-基)甲基)喹啉WXY009,(1R,4R)-4-[4-[9-(喹啉基-6-甲基)-9H-嘌呤-2-基]-1H-吡唑-1-基]环己醇WXY010。该类化合物以2-氯-N4-(喹啉-6-基-甲基)嘧啶-4,5-二胺为原料,与原甲酸三甲酯在对甲苯磺酸一水合物催化下环合后再与1位取代的4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)吡唑进行Suzuki耦合反应得到。(3)1(2)-(喹啉-6-基甲基)吲哚唑类化合物在吲哚唑6位引入1-甲基-1H吡唑、1-(2-四氢-2H-吡喃-2-氧乙基)-1H-吡唑、1-(4-羟基环己基)-1H-吡唑1-[(1R,4R)-4-(正丁基二甲基硅氧基)环己基]-1H-吡唑和1-((1R,4R)-4-羟基环己基)-1H-吡唑得到化合物6-[[6-(1-甲基-1H-吡唑-4-基)-1H-吲唑-1-基]甲基]喹啉WXY011,6-[[6-[1-(2-四氢-2H-吡喃-2-基氧乙基)-1H-吡唑-4-基]吲唑-1-基]甲基]喹啉WXY012,2-[4-[1-(喹啉-6-甲基)-1H吲唑-6-基]-1H-吡唑-1-基]乙醇WXY013,6-((6-(1-((1R,4R)-4-(叔丁二甲硅氧基)环己基)-1H-吡唑-4-基)-1H-吲唑-1-基)甲基)喹啉WXY014,(1R,4R)-[4-[4-[1-(喹啉-6-甲基)-1H-吲唑-6-基]-1H-吡唑-1-基]环己醇WXY015,6-[[6-(1-甲基-1H-吡唑-4-基)-2H-吲唑-2-基]甲基]喹啉WXY016,6-[[6-[1-(2-四氢-2H-吡喃-2-基氧乙基)-1H-吡唑-4-基]-2H-吲唑-2-基]甲基]喹啉WXY017,2-[4-[1-(喹啉-6-甲基)-1H-吲唑-6-基]-1H-吡唑-1-基]乙醇WXY018,6-((6-(1-((1R,4R)-4-(叔丁二甲硅氧基)环己基)-1H吡唑1-4-基)-2H-吲唑-2-基)甲基)喹啉WXY019,(1R,4R)-[4-[4-[2-(喹啉-6-甲基)-2H-吲唑-6-基]-1H-吡唑-1-基]环己醇WXY020。该类化合物以1位取代的4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)吡唑为原料,与商业可提供的6-溴-1H-吲哚唑发生Suzuki耦合反应,然后再与6-氯甲基喹啉发生亲电取代反应得到吲哚唑环1位取代的化合物及其对应的互变异构体吲哚唑环2位取代的化合物。MS和H'-NMR确认化合物的结构,对化合物进行c-Met生化半抑制浓度(IC50)实验,实验结果显示,3H-[1,2,3]三唑[4,5-d]嘧啶和嘌呤(咪唑并嘧啶)系列化合物活性较差,吲哚唑系列中大部分化合物对c-Met激酶有较强的抑制作用,除化合物WXY016和WXY019的IC50值超过10μM外,其余都在0.9μM以下。
2、保留先导化合物中的1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环、连接臂(亚甲基)、喹啉环三个部分不变,将课题组前期研究中的专利化合物2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类化合物中的2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚结构片段引入到该类化合物中替换吡嗪环或哒嗪环上的取代基,得到新型的含1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环的2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类化合物8-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚-2-甲酸叔丁酯WXY021,8-[3-(喹啉-6-基甲基)-3H-[1,2,3三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚WXY022,8-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚-2-甲醛WXY023,8-[3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-b]吡嗪-5-基]-2,3,4,5-四氢吡啶[4,3-b]吲哚-2-甲酰胺WXY024,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚-2-甲酸叔丁酯WXY025,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚WXY026,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚-2-甲醛WXY027,8-[3-(6-喹啉基甲基)-[1,2,4]三唑[4,3-b]哒嗪-6-基]-2,3,4,5-四氢吡啶[4,3-b]吲哚-2-甲酰胺WXY028。8-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)-1,3,4,5-四氢吡啶并[4,3-b]吲哚-2-甲酸叔丁酯与6-[(5-溴三唑[4,5-d]嘧啶-3-基)甲基]喹啉和6-[(6-氯-[1,2,4]三唑[3,4-f]哒嗪-3-基)甲基]喹啉发生Suzuki耦合反应得到WXY021和WXY022,然后再与甲酸及1-(3-二甲基氨基丙基)-3-乙基碳化二亚胺盐酸盐发生甲酰化反应得到WXY023和WXY027,化合物WXY021和WXY022也与三甲基硅基异氰酸酯发生氨甲酰化反应得到WXY024,WXY028,同时得到化合物WXY025,WXY026。MS和H1-NMR确认化合物的结构,对化合物进行c-Met生化半抑制浓度(IC50)实验,实验结果显示,该类化合物对c-Met激酶有很强的抑制作用,化合物WXY022和WXY023的IC50值分别达到了0.0145和0.0995gM,其它化合物大部分在1gM以下。
3、保留先导化合物中的1H-[1,2,3]三唑[4,5-b]吡嗪或3H-[1,2,3]三唑[4,3-b]哒嗪环以及其生物电子等排体3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤环、连接臂(亚甲基)、喹啉环三个部分不变,将课题组前期研究中的专利化合物螺环化合物中的螺[吲哚啉-3,4’-哌啶]-2-酮结构片段引入到该类化合物中替换吡嗪环、哒嗪环、嘧啶环上的取代基,结合前期通过分子对接得到的结果,得到新型的含1H-[1,2,3]三唑[4,5-b]吡嗪、3H-[1,2,3]三唑[4,3-b]哒嗪环、3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤环的螺[吲哚啉-3,4'-哌啶]-2-酮类化合物1’-甲基-5-[1-(6-喹啉基甲基)-1H-[1,2,3]三唑[4,5-b]吡嗪-6-基]螺[吲哚啉-3,4’-哌啶]-2-酮WXY029、1’-甲基-6-[1-(6-喹啉基甲基)-1H-[1,2,3]三唑[4,5-b]吡嗪-6-基]螺[吲哚啉-3,4’-哌啶]-2-酮WXY030、1’-甲基-5-(3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪-6-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY031、1’-甲基-6-(3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪-6-基)-螺[吲哚啉-3,4’-哌啶]-2-酮WXY032、1’-甲基-5-(3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基)-螺[吲哚啉-3,4’-哌啶]-2-酮WXY033、1’-甲基-6-(3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶-5-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY034、1’-甲基-5-(7-(喹啉-6-基甲基)-嘌呤-2-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY035、1’-甲基-6-(7-(喹啉-6-基甲基)-嘌呤-2-基)-螺[吲哚啉-3,4'-哌啶]-2-酮WXY036,该类化合物由5位和6位硼酸吡呐醇酯与溴取代的1-(6-喹啉基甲基)三唑[4,5-b]吡嗪、3-(喹啉-6-基甲基)-3H-[1,2,4]三唑[4,3-b]哒嗪、3-(喹啉-6-基甲基)-3H-[1,2,3]三唑[4,5-d]嘧啶、7-(喹啉-6-基甲基)-嘌呤发生Suzuki耦合反应得到。MS和H1-NMR确认化合物的结构,对化合物进行c-Met生化半抑制浓度(ICso)实验,实验结果显示,该类化合物对c-Met激酶有很强的抑制作用,化合物WXY029、WXY030、WXY031、WXY032对c-Met的抑制作用较化合物WXY033、WXY034、WXY035、WXY036作用强,化合物WXY029、WXY030较化合物WXY031、WXY032化合物作用强,WXY030的IC50值达到了0.0145μM较化合物WXY029(IC50=0.227μM)强,WXY032(IC50=0.22μM)较化合物WXY021(IC50=0.28μM)强,这表明含1H-[1,2,3]三唑[4,5-b]吡嗪、3H-[1,2,3]三唑[4,3-b]哒嗪的螺[吲哚啉-3,4'-哌啶]-2-酮化合物较含3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤环的螺[吲哚啉-3,4'-哌啶]-2-酮化合物有作用强,含有1H-[1,2,3]三唑[4,5-b]吡嗪的螺[吲哚啉-3,4’-哌啶]-2-酮化合物较含有3H-[1,2,3]三唑[4,3-b]哒嗪的螺[吲哚啉-3,4’-哌啶]-2-酮化合物作用强,螺[吲哚啉-3,4'-哌啶]-2-酮的6位取代较5为取代作用强。
4、对分子水平对c-Met酶有强抑制作用的吲哚唑、2,3,4,5-四氢-1H-吡啶[4,3-b]吲哚、螺[吲哚啉-3,4'-哌啶]-2-酮类化合物进行MKN45细胞中c-Met半抑制浓度(IC50)实验,实验结果表明,大部分化合物对细胞中c-Met有较强的抑制作用,部分化合物的ICso值在10μM以下。
5、对五大类合成的36个化合物进行了分子对接研究,分子对接结果显示,所有化合物与c-Met位点均采取“U”型结合模式,具备Ⅰ型c-Met抑制剂的典型特征,喹啉环上的氮与Met1160主链羰基形成氢键,喹啉环C-8与Pro1158羰基上的氧形成一个非经典的C-H-O=C氢键。这种作用与其他ATP-竞争性酶作用相似,稠合环(1H-[1,2,3]三唑[4,5-b]吡嗪环、3H-[1,2,3]三唑[4,3-b]哒嗪环、3H-[1,2,3]三唑[4,5-d]嘧啶、嘌呤、吲哚唑环)在Tyr1230和Met1211之间通过π-π堆积作用形成一个夹心结构,喹啉环和稠环之间的连接臂(亚甲基)刚好与Leu1157,Arg1086和Tyr1230或11109,Lys1110,A1a1226和Va11092组成的疏水口袋嵌合,该部分主要是影响稠合环的π-π堆积作用,在所有稠环中,以三唑吡嗪环效果最好,因为三唑哒嗪环作为一个缺电子母核在Tyr1230和Met1211之间通过π-π堆积作用形成一个夹心结构,三唑吡嗪环上的两个氮原子与Asp1222骨架上的NH形成两个氢键,因而与只能形成一个氢键的其它稠环相比其有较高的亲和力和较低的IC50值,而且与Asp1222间的氢键因其具有较低能量可以稳定该类化合物的构型。另外螺环上的哌啶环主要指向u型折叠构型的亲水表面,而该表面远离铰链区。因为在491个酪氨酸激酶中仅仅有c-Met,Axl和Mer三个激酶有两个氨基酸存在,来自Met1211和Tyr1230的疏水作用增加了该类化合物的选择性,螺环5位或六位取代的亲水稠环延伸到结合口袋,与WXY030相比,因螺环的张力存在使得WXY029不能与活性位点匹配,从而导致构型的改变以及增加N原子和Asn1167间的距离而不能形成氢键,因此WXY029的活性较WXY030低,同样的原因可以解释WXY031、WXY033、WXY035活性较WXY032、WXY034、 WXY036低。
综上所述,本课题主要针对c-Met设计了118个信化合物,合成出了36个新化合物,未见文献报道,此外还合成了17个关键中间体。所有目标化合物合成中都用到了Suzuki耦合反应,该反应在在DMF/H2O的混合溶剂中、Pd (PPh3)4催化下进行获得较高的收率,得到的的目标化合物以吲哚唑类、2,3,4,5-四氢-1H-吡啶并[4,3-b]吲哚类、螺[吲哚啉-3,4’-哌啶]-2-酮类化合物有较好的抗肿瘤活性,WXY030、WXY022和WXY023对c-Met生化半抑制浓度(ICso)达到了10nM,显示强的抗肿瘤活性,对分子水平抑制作用强的化合物进行细胞研究显示部分化合物对MKN45细胞中c-Met的抑制作用达到了10μM。分子对接研究证实和解释了这些化合物的活性。为开发具有临床应用价值的c-Met酪氨酸激酶抑制剂奠定基础。
The c-Met RTK family is the only known high-affinity receptor for hepatocyte growth factor (HGF), also known as scatter factor (SF), which is produced by stromal and mesenchymal cells and acts primarily on c-Met-expressing epithelial cells in an endocrine and/or paracrine fashion. c-Met and HGF are widely expressed in a variety of tissues, and their expression is normally confined to cells of epithelial and mesenchymal origin, c-Met is expressed by most carcinomas and its elevated expression relative to normal tissue has been detected in a number of cancers including lung, breast, colorectal, prostate, pancreatic, head and neck, gastric, hepatocellular, ovarian, renal, glioma, melanoma, and a number of sarcomas. c-Met expression has been reported to be up-regulated in tumors by a number of epigenetic mechanisms including tumor secreted growth factors, tumor hypoxia, and activation of other oncogenes.In addition, c-Met gene amplification and subsequent overexpression has been reported in gastric cancer, colorectal cancer, and glioma. Activation of the HGF/c-Met signaling pathway has been shown to lead to a wide array of cellular responses including proliferation, survival, angiogenesis, wound healing, tissue regeneration, scattering, motility, invasion and branching morphogenesis. Since c-Met is at the crossing of many roads leading to tumorigenesis and metastasis, targeting this receptor could be a relatively simple way to interfere with many pathways simultaneously. HGF/c-Met signaling pathway has come into the spotlight as a promising therapeutic target for inhibiting tumor growth and has become one of the leading molecular targets in cancer. Currently various strategies are currently in development to disrupt the HGF-Met signal transduction pathway, in which small molecular inhibitors have been a particularly active field.
1-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b] pyrazine of Pfizer's PF-04217903,3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine of Janssen's JNJ-33377605and3H-[1,2,4]triazolo[4,3-b]pyridazin of SGX's SGX-523are potent and selective c-Met inhibitors. Based on the crystal structure of the c-Met kinase "DFG-in" conformation, type I kinase inhibitors pharmacophore, structure-activity relationships of triazoles type I c-Met inhibitors and experience of optimization and modification on these compounds, this study designed a series of novel compounds using PF-04217903, JNJ-33377605, SGX-523and our previous patented compounds spiro[indoline-3,4'-piperidine]-2-ones and2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indoles as leading compouds by means of biological isostere principle, combination principles and molecular docking, then synthesized these compounds through directional synthesis and other method of chemical synthesis. We investigated their biological activities on cellular level to obtain more active compounds, analyzed and discussed binding mode of compounds and c-Met kinase through molecular docking experiments and discussed preliminary structure-activity relationship to lay the foundation for developing c-Met inhibitors with clinical application value.The main contents are summarized as follows:1. we designed and synthesized structurally relevant novel compounds by replacing1H-[1,2,3]triazolo[4,5-b]pyrazine and3H-[1,2,4]triazolo[4,3-b]pyridazin of leading compounds with3H-[1,2,3]triazolo[4,5-d]pyrimidine,7H-purine and indazole moiety:(1)3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidines WXY001, WXY002, WXY003, WXY004, WXY005were designed by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-7H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1 H-pyrazole and1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazole to the5-position of3H-[1,2,3]triazolo[4,5-d]pyrimidine. The target compounds were prepared by the reaction of1-position substituted4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazoles with5-chloro-3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine via Suzuki coupling reaction.(2)7-(quinolin-6-ylmethyl)-purines WXY006, WXY007, WXY008, WXY009, WXY010were designed by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1H-pyrazole and1-((1R,4R)-4-hy droxy-cyclohexyl)-1H-pyrazole to the2-position of purines, These compounds were synthesized using2-chloro-N4-(quinolin-6-ylmethyl)-4,5-pyrimidinediamino as material via cyclization reaction and Suzuki coupling reaction.(3)1(2)-(quinolin-6-ylmethyl)-1H-indazoles WXY011, WXY012, WXY013, WXY014, WXY015,WXY016,WXY017,WXY018, WXY019, WXY020can be obtained by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethy1)-1H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy) cyclohexyl]-1H-pyrazole and1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazole to the6-position of indazoles, The4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole groups reacted with6-bromo-1H-indazole to obtain6-(1-methyl-pyrazol-3-yl)-1H-indazole,6-[1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole, and6-{1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclo hexyl]-1H-pyrazol-4-yl}-1H-indazole respectively via Suzuki coupling reaction, then reacted with commercially available6-chloromethylquinoline to give1-(quinolin-6-ylmethyl)-1H-indazoles their isomers2-(quinolin-6-ylmethyl)-1H-indazoles as a result of two isomeric forms of indazole. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that the3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo [4,5-d]pyrimidine and7-(quinolin-6-ylmethyl)-purines exhibited lower inhibitory effect while1(2)-(quinolin-6-ylmethy1)-1H-indazoles exhibited significant inhibitory effect on c-Met with IC50<0.9μM in in TR-FRET-based assay except WXY016and WXY019with IC50>10μM.
2.Keeping1H-[1,2,3]triazolo[4,5-b] pyrazine or3H-[1,2,4]triazolo[4,3-b]pyridazine, methylenelink, quinoline of the lead compounds unchanged, the substituents of the pyrazine/pyridazine were replaced by the structure2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole to obtain novel2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles containing1H-[1,2,3]triazolo[4,5-b] pyrazine and3H-[1,2,4]triazolo[4,3-b]pyridazine WXY021, WXY022, WXY023, WXY024, WXY025, WXY026, WXY027, WXY028, these compounds were prepared as followed:The reaction of tert-butyl8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol e-2-carboxylate with6-bromo-l-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b]pyrazine and5-chloro-3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine, respectively, gave WXY021and WXY022via Suzuki coupling reaction. Then, WXY021and WXY022were transformed to WXY023, WXY027and WXY024, WXY028by formylation and carbanoylation, and obtained WXY025, WXY026concurrently. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that these compounds exhibited potent inhibition on c-Met, in which WXY022and WXY023had stronger with IC50values of0.0145μM and0.0995μM in TR-FRET-based assay than others with IC50<1μM.
3.Keeping1H-[1,2,3]triazolo[4,5-b]pyrazine,3H-[1,2,4]triazolo[4,3-b]pyridazine,3H-[1,2,3]triazolo[4,5-d]pyrimidine,7H-purine, methylenelink and quinoline unchanged, the substituents of the pyrazine, pyridazine and7H-purine were replaced by the structure spiro[indoline-3,4'-piperidine]-2-one of our previous patented compounds to obtain novel spiro[indoline-3,4'-piperidine]-2-ones WXY029, WXY030, WXY031, WXY032, WXY033, WXY034, WXY035and WXY028, which were prepared by reaction of the pinacol borane esters of5and6position of spiro[indoline-3,4'-piperidine]-2-one with bromine substituted1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine, 3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine,3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine and7-(quinolin-6-ylmethyl)-purine via Suzuki coupling. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that these compounds exhibited potent inhibition on c-Met, in which WXY029, WXY030, WXY031, WXY032had stronger than WXY033, WXY034, WXY035,WXY036, and WXY029、WXY030stronger than WXY031, WXY032, WXY030had stronger with IC50values of0.0147μM in TR-FRET-based assay than WXY029with IC50values of0.227μM and WXY032(IC50=0.22μM) stronger than WXY021(IC50=0.28μM). These results suggested that spiro [indoline-3,4'-piperidine]-2-ones containing1-(quinolin-6-ylmethy1)-1H-[1,2,3] triazolo[4,5-b]pyrazine and3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine were more effect on c-Met than others, of which1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo [4,5-b]pyrazine was stronger. Moreover,6position substituted spiro [indoline-3,4'-piperidine]-2-ones presented more effective activity than5position substituted compounds.
4.For the strong inhibition on c-Met kinase, the compounds1(2)-(quinolin-6-ylmethyl)-1H-indazoles,2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles and spiro [indoline-3,4'-piperidine]-2-ones were evaluated in cell-based assay. The results showed that some target compounds exhibited significant inhibitory effect on c-Met with IC5o<10μM in cell-based assay.
5. Molecular docking showed that all the compounds assumed a'U-shaped'binding mode, which is the typical character of type I c-Met inhibitors. The quinoline moiety engages the MET hinge region with a single canonical hydrogen bond between the quinoline nitrogen and backbone carbonyl of Met1178and a nonclassic C-H-O=C hydrogen bond between quinoline C8and the carbonyl oxygen of Pro1176, which was highly characteristic of all compounds bound to ATP binding site in kinase domains. Fused-rings1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine,3-(quinolin-6 ylmethyl-3H-[1.2,4]triazolo[4,3-b]pyridazine,3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine,7-(quinolin-6-ylmethyl)-purine and indazoles were swiched in between Tyr1230and Met1211through a π-π stacking interaction. The methylenelink could better match the sub hydrophobic pocket composed of Leul157, Arg1086and Tyr1230, or Va11109, Lys1110, Ala1226and Va11092and enhanced the π-π interaction, of which triazolopyridine was the best because triazolopyridazine, as an electron deficient group, was sandwiched in between Tyr1230and Metl211through a π-π stacking interaction. In addition, the hydrogen bond was important to the binding affinity and two nitrogen atoms of the triazolopyridine formed two hydrogen bonds with the back bone NH of Asp1222so it had higher affinity and lower IC50value.The spirocyclic pyridine ring also pointed out towards the solvent surface after "U-shape" folding, however, the solvent surface was far away from the hinge region. The hydrophobic interactions from Met1211and Tyr1230would contribute to the selectivity since the two amino acid were conserved among only three(c-Met, Axl, Mer) of the491kinases. The methylene connecting quinoline and triazolopyridazine was folded up to fit the subhydrophobic pocket composed by Leu1157, Arg1086and Tyr1230. The adjacent hydrophilic ring,5or6substituent of spiro [indoline-3,4'-piperidine]-2-ones, is extended to the binding pocket. Comparing to WXY030, WXY029could not match to the active site because of the tension from spire. It leaded to the conformation changes and larger distance between N from N-CH3-pyridine and the residue Asn1167, and could not generate hydrogen bond (shown in Fig.5). Therefore, WXY029had the lower affinity and the lower activity. Similar reasons explained why the analogues WXY032, WXY034, WXY036had more powerful activity than the analogues WXY031, WXY033, WXY035.
In conclusion, we designed118new compounds,36of which were synthesized. Meanwhile,17key intermediates were also synthesized. All the target compounds were prepared via Suzuki coupling reaction. This coupling reaction with pinacol borane esters gave good yields under basic conditions using DMF/H2O as a solvent and Pd (PPh3)4as a catalyst. Indazoles,2.3.4.5-tetrahydro-1H-pyrido[4,3-b]indoles and spiro[indoline-3,4'- piperidine]-2-ones had the best inhibitory effect, in which IC50values of WXY030, WXY022and WXY023reached lOnM in TR-FRET-based assay and IC50values of some compounds reached1OμM in cell-based assay. Molecular docking experiments verified and explained the results.The study lay the foundation for developing c-Met inhibitors with clinical application value.
引文
[1]Varmus H.The new era in cancer research [J]. Science,2006,312(5777): 1162-1165.
[2]Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia [J]. N Engl J Med, 2001,344:1031-1037.
[3]Durkin WJ. Cancer drug therapy [J]. Primary Care,1992,19(4):759-778.
[4]Burchenal JH, Murphy ML, Tan CT. Treatment of acute leukemia [J]. Pediatrics, 1956,18:643.
[5]Chabner BA, Roberts TG. Time line:Chem otherapy and thewar on cancer [J]. Nat Rev Cancer,2005,5(1):65-72.
[6]Kim JA. Targeted therapies for the treatment of cancer [J]. Am J Surg,2003, 186(3):264-268.
[7]李亚巍,冯波.肿瘤靶向药物的研究进展[J].吉林医药学院学报,2010,31(4)：223-226
[8]Saltz LB, Lenz HJ, Kindler HL, et al. Randomized hase Ⅱ trial of cetuximab, bevacizumab, and irinotecan compared with cetuximab and bevacizumab alone in irinotecan-refractory colorectal cancer:the BOND-2 study [J]. J Clin Oncol,2007, 25(29):4557-4561.
[9]Ciafra SA, Niola F, Wannenes F, et al. An anti-VEGF ribozyme embedded with in the adenoviral VAI sequence inhibits glioblastom a cell angiogenic potential in vitro [J]. J.Vase Res,2004,41(3):220-228.
[10]Robert LY, Jeff S. Recent advances in pathway-targeted cancer drug therapies emerging from cancer genome analysis [J]. Current Opinion in Genetics & Development 2012,22:45-49.
[11]Blumg-Jensen P, Hunter T. Oncogenic kinase signaling [J]. Nature,2001,41: 355-365.
[12]Daneey J, Sausville EA. Issues and progress with protein kinase inhibitors for cancer treatment [J]. Nat Rev Drug Discov,2003,2(4):296-313.
[13]Daniela SK, Richard AVE. Tyrosine kinases as targets for cancer therapy [J]. N Engl J Med,2005,353:172-187.
[14]Sharma PS, Sharma R, Tyagi T. Receptor tyrosine kinase inhibitors as potent weapons in war against cancers [J]. C urr Pharm Design,2009,15(7):758-776.
[15]Huang H, Jia Q, Ma JG. Discovering novel quercetin-3-O-aminoacid-esters as a new class of Src tyrosine kinase inhibitors [J]. Eur J Med Chem,2009,44(5): 1982-1988.
[16]Lin LG, Xie H, Li HL. Naturally occurring homoisoflavonoids function as potent protein tyrosinekinase inhibitors by c-Src-based high-throughput screening [J]. J Med Chem,2008,51(15):4419-4429.
[17]Li S, Schmitzk R, Jeffrey PD. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab [J]. Cancer Cel,2005,7 (4):301-311.
[18]Rochalima CM, Soares HP, Rzea LE. EGFR targeting of solid tumors [J]. Cancer Control,2007,14(3):295-304.
[19]Wu M, Rivkin A, Pham T. Panitumumab:human monoclonal antibody against epidermal growth factor receptors for the treatment of metastatic colorectal cancer [J]. Clin Ther,2008,30(1):14-30.
[20]Beaudry P, Nilsson M, Rioth M, et al. Potent antitum or effects of ZD6474 on neurob lastoma via dual targeting of tumor cells and tumor endothelium [J]. Mol Cancer The r,2008,7(2):418-424.
[21]Frampton JE. Lapatinib:a review of its use in the treatment of HER2-over expressing, trastuzum abrefractory, advanced or metastatic breast cancer [J]. Drugs,2009,69(15):2125-2148.
[22]Wissner A, Fraser HL, Ingalls CL, et al. Dual irreversible kinase inhibitors: quinazoline-based inhibitors incorporating two independent reactive centers with each targeting different cysteine residues in the kinase domains of EGFR and VEGFR-2[J]. Bioorg Med Chem,2007,15(11):3635-3648.
[23]Wong KK. HKI-272 in non-small cell lung cancer [J]. Clin Cancer Res,2007,13 (15):4593-4596.
[24]Goratybor J, Robak T. Targeted drugs in chronic myeloid leukemia [J]. Curr Med Chem,2008,15(29):3036-3051.
[25]Morphy R. Selectively nonselective kinase inhibition:striking the right balance [J]. J Med Chem,2010,53(4):1413-1437.
[26]Gang JA, Kurup S, Ihnat MA, et al. Synthesis and biological activity of N 4-phenylsubstituted-6-(2,4-dichorophenylmethyl)-7H-pyrrolo[2,3-d] pyrimidine-2, 4-diamines as vascular endo thelial growth factor receptor-2 inhibitors and antiangiogenic and antitumor agents[J]. Bioorg Med Chem,2010,18(10):3575-3587.
[27]Gu YH, Zhao W, Meng FY, et al. Sunitinib impairs the proliferation and function of human per-ipheral T cell and prevents T-cell mediated immune response in mice [J]. Clin Immuno,2010,135 (1):55-62.
[28]Kammasud N, Boonyarat C, Sanphanya K, et al.5-Substituted yrido [2,3-d] pyrimidine, an inhibitor against three receptor tyrosine kinases [J]. Bioorg Med Chem Lett,2009,19(3):745-750.
[29]Chaudhary NI, Roth GJ, Hilberg F, et al. Inhibition of PDGF, VEGF and FGF signa lling attenuates fibrosis [J]. Eur Respir J,2007,29(5):976-985.
[30]Coxon A, Bush T, Saffran D, e t al. Broad antitumor activity in breastcancer xengrafts by motesanib, a highly selective, oral inhibitor of vascular endothelial growth factor, plate let derived growth factor, and kit receptors [J]. Clin Cancer Res,2009,15(1):110-118.
[31]Rixe O, Bukowskir M, Michaelson MD, et al. Axitinib treatment in patients with cytokine-refractory meta static renal cell cancer:a phasell study [J]. Lancet Oncol, 2007,8(11):975-984.
[32]赵欣,郭长彬,周化蛋白酪氨酸激酶抑制剂的研究进展[J].中国药物化学杂志,2010,20(5)：442-449.
[33]Gherardi E, Sandin S, Petoukhv MV, et al. Structural basis of hepatocyte growth factor/scatter factor and MET signaling[J]. Proc Natl Acad Sci USA, 2006,103:4046-4051
[34]Peruzzi B, BottardoDP.Targeting the c-Met signaling pathway [J]. Clin Cancer Res 2006,12:3657-3660
[35]Oseph PE, George F. Vande W, Scott AB, et al. Novel therapeutic inhibitors of the c-Met signaling pathway in cancer [J]. Clin Cancer Res,2009,15:2207-2214.
[36]Liu X, YaoW, Newton RC, Scherle PA. Targeting the c-MET signaling pathway For cancer therapy [J].Expert Opin Invest Drugs,2008,17:997-1011
[37]Danilkovitch-Miagkova A, Zbar B. Dysregulation of Met receptor tyrosine kinase activity in invasive tumors [J]. J Clin Invest,2002,109:863-867
[38]Birchmeier CBW, Gherardi E, Vande WGF. Met, metastasis, motility and more [J]. Nat Rev Mol Cell Biol,2003,4:915-925
[39]Seiwert TY, JagadeeswaranR, FaoroL, et al.The MET receptor tyrosine kinase is a potential novel therapeutic target for head and neck squamous cell carcinoma [J]. Cancer Res,2009,69:3021-3031
[40]Bean J, Brennan C, Shih JY, et al. MET amplification occurs with or without T790 Mmutations in EGFR mutantlung tumors with acquired resistance to gefitinib or erlotinib [J]. Proc Natl Acad Sci USA,2007,104:20932-20937
[41]Smolen GA, Sordella R, Muir B, et al. Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752[J]. Proc Natl Acad Sci USA.2006,103:2316-2321
[42]Bachleitner-HofmannT, Sun MY, Chen CT, et al. HER kinase activation confers resistance to MET tyrosine kinase inhibition in MET oncogene-addicted gastric cancercells [J]. Mol Cancer Ther.2008,7:3499-3508
[43]Comoglio PM, Giordano S, Trusolino L. Drug development of MET inhibitors: targeting oncogene addiction and expedience [J]. Nat Rev Drug Discovery,2008, 7:504-516
[44]Jun HT, Sun J, Rex K, et al. AMG 102, A fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts [J]. Clin Cancer Res.2007, 13:6735-6742
[45]Martens T, Schmidt NO, Eckerich C, et al. A novel one-armed anti-c-Metantibody inhibits glioblastoma growth in vivo [J]. Clin Cancer Res, 2006,12:6144-6152
[46]Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G. Targeting MET in cancer:rationale and progress [J]. Nat Rev Cancer.2012,12(2):89-103
[47]Jung KH, Park BH, Hong SS. Progress in cancer therapy targeting c-Met signaling pathway [J]. Arch Pharm Res.2012,35(4):595-604.
[48]Liu X., Yao W., Newton R. C, Scherle P. A., Targeting the c-MET signaling pathway for cancer therapy [J]. Expert Opin. Investig. Drugs,2008,17: 997-1011.
[49]Trusolino L., Bertotti A., Comoglio PM, MET signalling:principles and functions in development, organ regeneration and cancer [J]. Nat. Rev. Mol. Cell Biol.,2010,11:834-848.
[50]Chirgadze DY. Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding [J]. Nature Struct. Biol.1999,6:72-79.
[51]Ultsch M, Lokker, NA, Godowski, PJ. et al. Crystal structure of the NK1 fragment of human hepatocyte growth factor at 2.0 A resolution [J]. Structure, 1998,6,1383-1393.
[52]Tolbert WD, Daugherty-Holtrop J, Gherardi E, et al. Structural basis for agonism and antagonism of hepatocyte growth factor[J]. Proc. Natl Acad. Sci. USA,2010, 107:13264-13269.
[53]Kirchhofer D. Yao X, Peek M, et al. Structural and functional basis of the serine protease-like hepatocyte growth factor β-chain in Met binding and signaling [J]. J. Biol. Chem.2004,279:39915-39924.
[54]Stamos J, Lazarus RA, Yao X, Kirchhofer D. Crystal structure of the HGF β-chain in complex with the Sema domain of the Met receptor [J]. EMBO J.2004, 23:2325-2335.
[55]Niemann HH. Structure of the human receptor tyrosine kinase met in complex with the listeria invasion protein In1B [J]. Cell,2007,130:235-246.
[56]Shia S. Conformational lability in serine protease active sites:structures of Hepatocyte Growth Factor Activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-IB [J]. J. Mol. Biol.2005,346:1335-1349.
[57]Li W. Wang BE, Moran P, et al. GanesanPegylated kunitz domain inhibitor suppresses hepsin-mediated invasive tumor growth and metastasis [J]. Cancer Res. 2009,69:8395-8402.
[58]Wu Y, Eigenbrot C, Liang WC, et al. Structural insight into distinct mechanisms of protease inhibition by antibodies [J]. Proc. Natl Acad. Sci. USA,2007, 104:19784-19789.
[59]Ganesan R, Eigenbrot C, Wu Y, et al. Unraveling the allosteric mechanism of serine protease inhibition by an antibody [J]. Structure,2009,17:1614-1624.
[60]Farady CJ, Sun J, Darragh MR, et al. The mechanism of inhibition of antibody-based inhibitors of membrane-type serine protease 1 (MT-SP1) [J]. J. Mol. Biol.2007,369:1041-1051.
[61]Cao B, Su Y, Oskarsson M, et al. Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models [J]. Proc. Natl Acad. Sci. USA 2001,98:7443-7448.
[62]Burgess TL, Sun J, Meyer S, et al. Biochemical characterization of AMG 102:a neutralizing, fully human monoclonal antibody to human and nonhuman primate hepatocyte growth factor [J]. Mol. Cancer Ther.2010,9:400-409.
[63]Jakubczak JL, LaRochelle WJ. Merlino G, et al. NK1, a natural splice variant of hepatocyte growth factor/scatter factor, is a partial agonist in vivo [J]. Mol. Cell. Biol.1998,18:1275-1283.
[64]Tolbert WD, Jennifer DH, Gherardi E, et al.. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist [J]. Proc. Natl Acad. Sci. USA. 2007,104:14592-14597.
[65]Youles M. Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist [J]. J. Mol. Biol.2008,377:616-622.
[66]Otsuka T, Jakubczak J, Vieira W, et al. Disassociation of met-mediated biological responses in vivo:the natural hepatocyte growth factor/scatter factor splice variant NK2 antagonizes growth but facilitates metastasis [J]. Mol. Cell. Biol. 2000,20:2055-2065.
[67]Wang W. Tsai J, Kumar A, et al. Structural characterization of autoinhibited c-Met kinase produced by coexpression in bacteria with phosphatase [J]. Proc. Natl Acad. Sci. USA,2006,103:3563-3568.
[68]Rickert KW, Ford RE, Guerin DJ, et al. Structural basis for se lective smallmolecule kinase inhibition of activated c-Met [J]. J. Biol.Chem,2011, 286:11218-11225.
[69]Buchanan SG, Jorg H, Patrick SL, et al. SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo [J]. Mol.Cancer Ther.2009,8:3181-3190.
[70]Timofeevski SL. K, Cui J, Zou HY, Zhu JX, et al.Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors [J]. Biochemistry,2009, 48:5339-5349.
[71]Yao D, Dietmar WS. In vitro cancer metastatic phenotype stimulated prostate- suppresses hepatocyte growth factor BMS-777607, a small-molecule Met kinase inhibitor, a selective and orally efficacious inhibitor of the Met kinase superfamily [J]. Mol Cancer Ther,2010,9:1554-1561.
[72]D'Angelo ND. Design, synthesis, and biological evaluation of potent c-Met inhibitors[J]. J Med Chem,2008,51:5766-5779.
[73]Munshi N, Jeay S, Li Y, et al. ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity [J]. Mol Cancer Ther,2010,9:1544-1553.
[74]Eathiraj S, Palma R, Volckova E, et al. Discovery of a novel mode of protein kinase inhibition characterized by the mechanism of inhibition of human mesenchymalepithelial transition factor (c-Met) protein autophosphorylation by ARQ 197[J]. J Biol Chem,2011,286:20666-20676.
[75]Moosa M, Gerald M, Li S, et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors [J]. Science,1997,76: 955-960.
[76]Nikolaus S, Stefan K, Marinai T, et al. Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a [J]. PNAs,2003,100(22):12654-12659.
[77]Wang X, Le P, Liang C, et al. Potent and selective inhibitors of the Met hepatocyte growth factor/scatter factor(HGF/SF)receptor tyrosine kinase block HGF/SF-induced tumor cell growth and invasion[J]. Mol CancerTher,2003, 2(11):1085-1092.
[78]武维斌,詹轶群,杨晓明.酪氨酸激酶类受体c-Met抑制剂研究进展.军事医学科学院院刊[J],2007,31(5)：477-480
[79]Zou HY, Li Q, Lee JH, et al. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms [J]. Cancer Res,2007,67: 4408-4417
[80]Bellon SF, Kaplan-Lefko P, Yang Y, et al.c-Met inhibitors with novel binding modeshow activity against several hereditarypapillary renal cell carcinoma-related mutations [J]. J Biol Chem,2008,283:2675-2683.
[81]Amgen Inc. Fused heterocyclic derivatives and methods of use [P]. WO08008539, 2008.
[82]AstraZeneca. Pyrazine derivatives [P].WO09007390,2009.
[83]AstraZeneca. Pyridine and pyrazine derivatives [P]. WO09053737,2009.
[84]AstraZeneca.2-Pyrazinylbenzimidazole derivatives as receptor tyrosine kinase inhibitors [P]. WO09024825,2009
[85]Bayer Schering Pharma Aktiengesellschaft. Dihydropyridinone derivatives useful as kinase inhibitors [P]. WO08071451,2008.
[86]Cephalon, Inc. Pyridopyrazines and derivatives thereof as Alk and c-Met inhibitors [P]. WO07130468,2007.
[87]Perera T, Lavrijssen T, Janssens B, et al. JNJ-38877605:a selective Met kinase inhibitor inducing regression of Met-driven tumor models [abstract 4837].99th Annual Meeting for American Association for Cancer Research (AACR),12-16 April 2008.
[88]San Diego, USA Pfizer Products, Inc. Triazolopyridazine derivatives [P]. WO07138472,2007.
[89]Pfizer Products, Inc. Triazolopyrazine derivatives useful as anti-cancer agents [P].WO07132308,2007.
[90]Zou H, Li Q, Joseph L, et al. PF-04217903, a novel selective c-Met kinase inhibitor with potent antitumor and anti-angiogenic properties in vitro and in vivo [abstract 4841].99th Annual Meeting for American Association for Cancer Research (AACR),12-16 April 2008
[91]Sharon D, Jason B, Thomas PM, et al.Species-specific metabolism of SGX523 by aldehyde oxidase and the toxicological implications [J]. Drug Metabolism and diaposition,2010,8:1277-1285.
[92]Schroeder GM, et al. Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluoropheny l)-4-ethoxy-1-(4-fluoropheny l)-2-oxo-1,2-dihy dropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily [J]. I Med. Chem.2009,52:1251-1254.
[93]Raeppel S, Claridge S, Saavedra O, etal. N-(3-fluoro-4-(2-ary lthieno [3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides:a novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors[J]. Bioorg Med Chem Lett,2009,19:1323-1328
[94]Arqule, Inc. Pyrrolidinone, pyrrolidine-2,5-dione, pyrrolidine and thiosuccinimide derivatives, compositions and methods for treating cancer [P]. WO09002806,2009.
[95]Arqule, Inc. Compositions and methods for treatment of cancer [P]. WO09002807,2009.
[96]Cui JR. Aminoheteroaryl compounds as protein kinase inhibitors [P]. WO076412, 2004.
[97]Koenig M, Cui JR, Wei CC.lndolinone hydrorazides as c-Met inhibitors[P]. WO2005005378,2005.
[98]Vojkovsky T,'Koenig M, Cui JR.Tetracyclic compounds as c-Met inhibitors[P]. WO2005004808,2005.
[99]Zhang FJ, Vojkovsky T, Huang P. Triazolotriazine [P]. WO2005010005,2005.
[100]Cui JR, Botrous L.Arylmethyl triazolo and imidazopyrazines as e-Mel inhibitors[P]. WO2005004607,2005.
[101]Kung PE. Etherlinked heteroarylcompounds [P]. WO2005121125,2005.
[102]Cui JJ, Funk LA. Pyrazole-substituted aminoheleroaryi compounds as protein kinase inhibitors[P]. WO2006021881,2006.
[103]Cui JR, Funk LA, Kung PP, et al. Aminoheteroaryl compounds as protein tyrosinekinase inhibitors[P]. WO2006021886,2006.
[104]Harmange JC, Booker S. Compounds and methods of use [P]. WO2005073224, 2005.
[105]Arkadii VK, Stephen WC, Franck R.Inhibitors of VEGF receptor and HGF receptor signaling [P].US20060074056A1,2005.
[106]Saavedra OM, Claridge SW, Zhan L. Inhibitors of VEGF receptor signaling [P]. US20071004675 Al,2007.
[107]王海勇,基于c-Met和HDAC靶点的抗癌药物设计与合成研究[D].北京：中国人民解放军军事医学科学院,2007.
[108]John Porter, Simon Lumb, Richard JF. Discovery of 4-azaindoles as novel inhibitors of c-Met kinase [J]. Bioorganic & Medicinal Chemistry Letters,2009, 19:2780-2784.
[109]吴曙光,张嘉杰,吴少瑜,等.一种四氢吡啶并吲哚类化合物及其制备方法和应用[P].CN101857594A,2010.
[110]吴曙光,张嘉杰,吴少瑜,等.一种螺环化合物及其制备方法和应用[P].CN101851237A,2010.
[111]Bridoux A, Goossens L, Houssin R, J. P. Henichart, Synthesis of 8-substituted tetrahydro-y-carbolines[J]. J Heterocycl Chem,2006,43:571-578.
[112]Albany MRI, Guzzo P. (1-Azine)-substituted pyridoindoles as MCH antagonists [P]. WO2009089432,2009
[113]Discovery of small molecule c-Met inhibitors:evolution and profiles of clinical candidates [J]. Anti-Cancer Agents in Medicinal Chemistry,2010,10:7-27.
[114]Cui JJ, Tran-Dube M, Shen H, et al. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase[J]. J Med Chem,2011,18:6342-6363.
[115]Hiroki N, Takashi C, Kenji M, et al. Bioorg. Med. Chem. Lett.2010,20: 1405-1409
[116]Lu T, Alexander R, Connors RW, et al. Triazolopyridazines as tyrosine kinase modulators [P]. WO2007075567,2007
[2]Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia [J]. N Engl J Med, 2001,344:1031-1037.
[3]Durkin WJ. Cancer drug therapy [J]. Primary Care,1992,19(4):759-778.
[4]Burchenal JH, Murphy ML, Tan CT. Treatment of acute leukemia [J]. Pediatrics, 1956,18:643.
[5]Chabner BA, Roberts TG. Time line:Chem otherapy and thewar on cancer [J]. Nat Rev Cancer,2005,5(1):65-72.
[6]Kim JA. Targeted therapies for the treatment of cancer [J]. Am J Surg,2003, 186(3):264-268.
[7]李亚巍,冯波.肿瘤靶向药物的研究进展[J].吉林医药学院学报,2010,31(4)：223-226
[8]Saltz LB, Lenz HJ, Kindler HL, et al. Randomized hase Ⅱ trial of cetuximab, bevacizumab, and irinotecan compared with cetuximab and bevacizumab alone in irinotecan-refractory colorectal cancer:the BOND-2 study [J]. J Clin Oncol,2007, 25(29):4557-4561.
[9]Ciafra SA, Niola F, Wannenes F, et al. An anti-VEGF ribozyme embedded with in the adenoviral VAI sequence inhibits glioblastom a cell angiogenic potential in vitro [J]. J.Vase Res,2004,41(3):220-228.
[10]Robert LY, Jeff S. Recent advances in pathway-targeted cancer drug therapies emerging from cancer genome analysis [J]. Current Opinion in Genetics & Development 2012,22:45-49.
[11]Blumg-Jensen P, Hunter T. Oncogenic kinase signaling [J]. Nature,2001,41: 355-365.
[12]Daneey J, Sausville EA. Issues and progress with protein kinase inhibitors for cancer treatment [J]. Nat Rev Drug Discov,2003,2(4):296-313.
[13]Daniela SK, Richard AVE. Tyrosine kinases as targets for cancer therapy [J]. N Engl J Med,2005,353:172-187.
[14]Sharma PS, Sharma R, Tyagi T. Receptor tyrosine kinase inhibitors as potent weapons in war against cancers [J]. C urr Pharm Design,2009,15(7):758-776.
[15]Huang H, Jia Q, Ma JG. Discovering novel quercetin-3-O-aminoacid-esters as a new class of Src tyrosine kinase inhibitors [J]. Eur J Med Chem,2009,44(5): 1982-1988.
[16]Lin LG, Xie H, Li HL. Naturally occurring homoisoflavonoids function as potent protein tyrosinekinase inhibitors by c-Src-based high-throughput screening [J]. J Med Chem,2008,51(15):4419-4429.
[17]Li S, Schmitzk R, Jeffrey PD. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab [J]. Cancer Cel,2005,7 (4):301-311.
[18]Rochalima CM, Soares HP, Rzea LE. EGFR targeting of solid tumors [J]. Cancer Control,2007,14(3):295-304.
[19]Wu M, Rivkin A, Pham T. Panitumumab:human monoclonal antibody against epidermal growth factor receptors for the treatment of metastatic colorectal cancer [J]. Clin Ther,2008,30(1):14-30.
[20]Beaudry P, Nilsson M, Rioth M, et al. Potent antitum or effects of ZD6474 on neurob lastoma via dual targeting of tumor cells and tumor endothelium [J]. Mol Cancer The r,2008,7(2):418-424.
[21]Frampton JE. Lapatinib:a review of its use in the treatment of HER2-over expressing, trastuzum abrefractory, advanced or metastatic breast cancer [J]. Drugs,2009,69(15):2125-2148.
[22]Wissner A, Fraser HL, Ingalls CL, et al. Dual irreversible kinase inhibitors: quinazoline-based inhibitors incorporating two independent reactive centers with each targeting different cysteine residues in the kinase domains of EGFR and VEGFR-2[J]. Bioorg Med Chem,2007,15(11):3635-3648.
[23]Wong KK. HKI-272 in non-small cell lung cancer [J]. Clin Cancer Res,2007,13 (15):4593-4596.
[24]Goratybor J, Robak T. Targeted drugs in chronic myeloid leukemia [J]. Curr Med Chem,2008,15(29):3036-3051.
[25]Morphy R. Selectively nonselective kinase inhibition:striking the right balance [J]. J Med Chem,2010,53(4):1413-1437.
[26]Gang JA, Kurup S, Ihnat MA, et al. Synthesis and biological activity of N 4-phenylsubstituted-6-(2,4-dichorophenylmethyl)-7H-pyrrolo[2,3-d] pyrimidine-2, 4-diamines as vascular endo thelial growth factor receptor-2 inhibitors and antiangiogenic and antitumor agents[J]. Bioorg Med Chem,2010,18(10):3575-3587.
[27]Gu YH, Zhao W, Meng FY, et al. Sunitinib impairs the proliferation and function of human per-ipheral T cell and prevents T-cell mediated immune response in mice [J]. Clin Immuno,2010,135 (1):55-62.
[28]Kammasud N, Boonyarat C, Sanphanya K, et al.5-Substituted yrido [2,3-d] pyrimidine, an inhibitor against three receptor tyrosine kinases [J]. Bioorg Med Chem Lett,2009,19(3):745-750.
[29]Chaudhary NI, Roth GJ, Hilberg F, et al. Inhibition of PDGF, VEGF and FGF signa lling attenuates fibrosis [J]. Eur Respir J,2007,29(5):976-985.
[30]Coxon A, Bush T, Saffran D, e t al. Broad antitumor activity in breastcancer xengrafts by motesanib, a highly selective, oral inhibitor of vascular endothelial growth factor, plate let derived growth factor, and kit receptors [J]. Clin Cancer Res,2009,15(1):110-118.
[31]Rixe O, Bukowskir M, Michaelson MD, et al. Axitinib treatment in patients with cytokine-refractory meta static renal cell cancer:a phasell study [J]. Lancet Oncol, 2007,8(11):975-984.
[32]赵欣,郭长彬,周化蛋白酪氨酸激酶抑制剂的研究进展[J].中国药物化学杂志,2010,20(5)：442-449.
[33]Gherardi E, Sandin S, Petoukhv MV, et al. Structural basis of hepatocyte growth factor/scatter factor and MET signaling[J]. Proc Natl Acad Sci USA, 2006,103:4046-4051
[34]Peruzzi B, BottardoDP.Targeting the c-Met signaling pathway [J]. Clin Cancer Res 2006,12:3657-3660
[35]Oseph PE, George F. Vande W, Scott AB, et al. Novel therapeutic inhibitors of the c-Met signaling pathway in cancer [J]. Clin Cancer Res,2009,15:2207-2214.
[36]Liu X, YaoW, Newton RC, Scherle PA. Targeting the c-MET signaling pathway For cancer therapy [J].Expert Opin Invest Drugs,2008,17:997-1011
[37]Danilkovitch-Miagkova A, Zbar B. Dysregulation of Met receptor tyrosine kinase activity in invasive tumors [J]. J Clin Invest,2002,109:863-867
[38]Birchmeier CBW, Gherardi E, Vande WGF. Met, metastasis, motility and more [J]. Nat Rev Mol Cell Biol,2003,4:915-925
[39]Seiwert TY, JagadeeswaranR, FaoroL, et al.The MET receptor tyrosine kinase is a potential novel therapeutic target for head and neck squamous cell carcinoma [J]. Cancer Res,2009,69:3021-3031
[40]Bean J, Brennan C, Shih JY, et al. MET amplification occurs with or without T790 Mmutations in EGFR mutantlung tumors with acquired resistance to gefitinib or erlotinib [J]. Proc Natl Acad Sci USA,2007,104:20932-20937
[41]Smolen GA, Sordella R, Muir B, et al. Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752[J]. Proc Natl Acad Sci USA.2006,103:2316-2321
[42]Bachleitner-HofmannT, Sun MY, Chen CT, et al. HER kinase activation confers resistance to MET tyrosine kinase inhibition in MET oncogene-addicted gastric cancercells [J]. Mol Cancer Ther.2008,7:3499-3508
[43]Comoglio PM, Giordano S, Trusolino L. Drug development of MET inhibitors: targeting oncogene addiction and expedience [J]. Nat Rev Drug Discovery,2008, 7:504-516
[44]Jun HT, Sun J, Rex K, et al. AMG 102, A fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts [J]. Clin Cancer Res.2007, 13:6735-6742
[45]Martens T, Schmidt NO, Eckerich C, et al. A novel one-armed anti-c-Metantibody inhibits glioblastoma growth in vivo [J]. Clin Cancer Res, 2006,12:6144-6152
[46]Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G. Targeting MET in cancer:rationale and progress [J]. Nat Rev Cancer.2012,12(2):89-103
[47]Jung KH, Park BH, Hong SS. Progress in cancer therapy targeting c-Met signaling pathway [J]. Arch Pharm Res.2012,35(4):595-604.
[48]Liu X., Yao W., Newton R. C, Scherle P. A., Targeting the c-MET signaling pathway for cancer therapy [J]. Expert Opin. Investig. Drugs,2008,17: 997-1011.
[49]Trusolino L., Bertotti A., Comoglio PM, MET signalling:principles and functions in development, organ regeneration and cancer [J]. Nat. Rev. Mol. Cell Biol.,2010,11:834-848.
[50]Chirgadze DY. Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding [J]. Nature Struct. Biol.1999,6:72-79.
[51]Ultsch M, Lokker, NA, Godowski, PJ. et al. Crystal structure of the NK1 fragment of human hepatocyte growth factor at 2.0 A resolution [J]. Structure, 1998,6,1383-1393.
[52]Tolbert WD, Daugherty-Holtrop J, Gherardi E, et al. Structural basis for agonism and antagonism of hepatocyte growth factor[J]. Proc. Natl Acad. Sci. USA,2010, 107:13264-13269.
[53]Kirchhofer D. Yao X, Peek M, et al. Structural and functional basis of the serine protease-like hepatocyte growth factor β-chain in Met binding and signaling [J]. J. Biol. Chem.2004,279:39915-39924.
[54]Stamos J, Lazarus RA, Yao X, Kirchhofer D. Crystal structure of the HGF β-chain in complex with the Sema domain of the Met receptor [J]. EMBO J.2004, 23:2325-2335.
[55]Niemann HH. Structure of the human receptor tyrosine kinase met in complex with the listeria invasion protein In1B [J]. Cell,2007,130:235-246.
[56]Shia S. Conformational lability in serine protease active sites:structures of Hepatocyte Growth Factor Activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-IB [J]. J. Mol. Biol.2005,346:1335-1349.
[57]Li W. Wang BE, Moran P, et al. GanesanPegylated kunitz domain inhibitor suppresses hepsin-mediated invasive tumor growth and metastasis [J]. Cancer Res. 2009,69:8395-8402.
[58]Wu Y, Eigenbrot C, Liang WC, et al. Structural insight into distinct mechanisms of protease inhibition by antibodies [J]. Proc. Natl Acad. Sci. USA,2007, 104:19784-19789.
[59]Ganesan R, Eigenbrot C, Wu Y, et al. Unraveling the allosteric mechanism of serine protease inhibition by an antibody [J]. Structure,2009,17:1614-1624.
[60]Farady CJ, Sun J, Darragh MR, et al. The mechanism of inhibition of antibody-based inhibitors of membrane-type serine protease 1 (MT-SP1) [J]. J. Mol. Biol.2007,369:1041-1051.
[61]Cao B, Su Y, Oskarsson M, et al. Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models [J]. Proc. Natl Acad. Sci. USA 2001,98:7443-7448.
[62]Burgess TL, Sun J, Meyer S, et al. Biochemical characterization of AMG 102:a neutralizing, fully human monoclonal antibody to human and nonhuman primate hepatocyte growth factor [J]. Mol. Cancer Ther.2010,9:400-409.
[63]Jakubczak JL, LaRochelle WJ. Merlino G, et al. NK1, a natural splice variant of hepatocyte growth factor/scatter factor, is a partial agonist in vivo [J]. Mol. Cell. Biol.1998,18:1275-1283.
[64]Tolbert WD, Jennifer DH, Gherardi E, et al.. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist [J]. Proc. Natl Acad. Sci. USA. 2007,104:14592-14597.
[65]Youles M. Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist [J]. J. Mol. Biol.2008,377:616-622.
[66]Otsuka T, Jakubczak J, Vieira W, et al. Disassociation of met-mediated biological responses in vivo:the natural hepatocyte growth factor/scatter factor splice variant NK2 antagonizes growth but facilitates metastasis [J]. Mol. Cell. Biol. 2000,20:2055-2065.
[67]Wang W. Tsai J, Kumar A, et al. Structural characterization of autoinhibited c-Met kinase produced by coexpression in bacteria with phosphatase [J]. Proc. Natl Acad. Sci. USA,2006,103:3563-3568.
[68]Rickert KW, Ford RE, Guerin DJ, et al. Structural basis for se lective smallmolecule kinase inhibition of activated c-Met [J]. J. Biol.Chem,2011, 286:11218-11225.
[69]Buchanan SG, Jorg H, Patrick SL, et al. SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo [J]. Mol.Cancer Ther.2009,8:3181-3190.
[70]Timofeevski SL. K, Cui J, Zou HY, Zhu JX, et al.Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors [J]. Biochemistry,2009, 48:5339-5349.
[71]Yao D, Dietmar WS. In vitro cancer metastatic phenotype stimulated prostate- suppresses hepatocyte growth factor BMS-777607, a small-molecule Met kinase inhibitor, a selective and orally efficacious inhibitor of the Met kinase superfamily [J]. Mol Cancer Ther,2010,9:1554-1561.
[72]D'Angelo ND. Design, synthesis, and biological evaluation of potent c-Met inhibitors[J]. J Med Chem,2008,51:5766-5779.
[73]Munshi N, Jeay S, Li Y, et al. ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity [J]. Mol Cancer Ther,2010,9:1544-1553.
[74]Eathiraj S, Palma R, Volckova E, et al. Discovery of a novel mode of protein kinase inhibition characterized by the mechanism of inhibition of human mesenchymalepithelial transition factor (c-Met) protein autophosphorylation by ARQ 197[J]. J Biol Chem,2011,286:20666-20676.
[75]Moosa M, Gerald M, Li S, et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors [J]. Science,1997,76: 955-960.
[76]Nikolaus S, Stefan K, Marinai T, et al. Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a [J]. PNAs,2003,100(22):12654-12659.
[77]Wang X, Le P, Liang C, et al. Potent and selective inhibitors of the Met hepatocyte growth factor/scatter factor(HGF/SF)receptor tyrosine kinase block HGF/SF-induced tumor cell growth and invasion[J]. Mol CancerTher,2003, 2(11):1085-1092.
[78]武维斌,詹轶群,杨晓明.酪氨酸激酶类受体c-Met抑制剂研究进展.军事医学科学院院刊[J],2007,31(5)：477-480
[79]Zou HY, Li Q, Lee JH, et al. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms [J]. Cancer Res,2007,67: 4408-4417
[80]Bellon SF, Kaplan-Lefko P, Yang Y, et al.c-Met inhibitors with novel binding modeshow activity against several hereditarypapillary renal cell carcinoma-related mutations [J]. J Biol Chem,2008,283:2675-2683.
[81]Amgen Inc. Fused heterocyclic derivatives and methods of use [P]. WO08008539, 2008.
[82]AstraZeneca. Pyrazine derivatives [P].WO09007390,2009.
[83]AstraZeneca. Pyridine and pyrazine derivatives [P]. WO09053737,2009.
[84]AstraZeneca.2-Pyrazinylbenzimidazole derivatives as receptor tyrosine kinase inhibitors [P]. WO09024825,2009
[85]Bayer Schering Pharma Aktiengesellschaft. Dihydropyridinone derivatives useful as kinase inhibitors [P]. WO08071451,2008.
[86]Cephalon, Inc. Pyridopyrazines and derivatives thereof as Alk and c-Met inhibitors [P]. WO07130468,2007.
[87]Perera T, Lavrijssen T, Janssens B, et al. JNJ-38877605:a selective Met kinase inhibitor inducing regression of Met-driven tumor models [abstract 4837].99th Annual Meeting for American Association for Cancer Research (AACR),12-16 April 2008.
[88]San Diego, USA Pfizer Products, Inc. Triazolopyridazine derivatives [P]. WO07138472,2007.
[89]Pfizer Products, Inc. Triazolopyrazine derivatives useful as anti-cancer agents [P].WO07132308,2007.
[90]Zou H, Li Q, Joseph L, et al. PF-04217903, a novel selective c-Met kinase inhibitor with potent antitumor and anti-angiogenic properties in vitro and in vivo [abstract 4841].99th Annual Meeting for American Association for Cancer Research (AACR),12-16 April 2008
[91]Sharon D, Jason B, Thomas PM, et al.Species-specific metabolism of SGX523 by aldehyde oxidase and the toxicological implications [J]. Drug Metabolism and diaposition,2010,8:1277-1285.
[92]Schroeder GM, et al. Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluoropheny l)-4-ethoxy-1-(4-fluoropheny l)-2-oxo-1,2-dihy dropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily [J]. I Med. Chem.2009,52:1251-1254.
[93]Raeppel S, Claridge S, Saavedra O, etal. N-(3-fluoro-4-(2-ary lthieno [3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides:a novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors[J]. Bioorg Med Chem Lett,2009,19:1323-1328
[94]Arqule, Inc. Pyrrolidinone, pyrrolidine-2,5-dione, pyrrolidine and thiosuccinimide derivatives, compositions and methods for treating cancer [P]. WO09002806,2009.
[95]Arqule, Inc. Compositions and methods for treatment of cancer [P]. WO09002807,2009.
[96]Cui JR. Aminoheteroaryl compounds as protein kinase inhibitors [P]. WO076412, 2004.
[97]Koenig M, Cui JR, Wei CC.lndolinone hydrorazides as c-Met inhibitors[P]. WO2005005378,2005.
[98]Vojkovsky T,'Koenig M, Cui JR.Tetracyclic compounds as c-Met inhibitors[P]. WO2005004808,2005.
[99]Zhang FJ, Vojkovsky T, Huang P. Triazolotriazine [P]. WO2005010005,2005.
[100]Cui JR, Botrous L.Arylmethyl triazolo and imidazopyrazines as e-Mel inhibitors[P]. WO2005004607,2005.
[101]Kung PE. Etherlinked heteroarylcompounds [P]. WO2005121125,2005.
[102]Cui JJ, Funk LA. Pyrazole-substituted aminoheleroaryi compounds as protein kinase inhibitors[P]. WO2006021881,2006.
[103]Cui JR, Funk LA, Kung PP, et al. Aminoheteroaryl compounds as protein tyrosinekinase inhibitors[P]. WO2006021886,2006.
[104]Harmange JC, Booker S. Compounds and methods of use [P]. WO2005073224, 2005.
[105]Arkadii VK, Stephen WC, Franck R.Inhibitors of VEGF receptor and HGF receptor signaling [P].US20060074056A1,2005.
[106]Saavedra OM, Claridge SW, Zhan L. Inhibitors of VEGF receptor signaling [P]. US20071004675 Al,2007.
[107]王海勇,基于c-Met和HDAC靶点的抗癌药物设计与合成研究[D].北京：中国人民解放军军事医学科学院,2007.
[108]John Porter, Simon Lumb, Richard JF. Discovery of 4-azaindoles as novel inhibitors of c-Met kinase [J]. Bioorganic & Medicinal Chemistry Letters,2009, 19:2780-2784.
[109]吴曙光,张嘉杰,吴少瑜,等.一种四氢吡啶并吲哚类化合物及其制备方法和应用[P].CN101857594A,2010.
[110]吴曙光,张嘉杰,吴少瑜,等.一种螺环化合物及其制备方法和应用[P].CN101851237A,2010.
[111]Bridoux A, Goossens L, Houssin R, J. P. Henichart, Synthesis of 8-substituted tetrahydro-y-carbolines[J]. J Heterocycl Chem,2006,43:571-578.
[112]Albany MRI, Guzzo P. (1-Azine)-substituted pyridoindoles as MCH antagonists [P]. WO2009089432,2009
[113]Discovery of small molecule c-Met inhibitors:evolution and profiles of clinical candidates [J]. Anti-Cancer Agents in Medicinal Chemistry,2010,10:7-27.
[114]Cui JJ, Tran-Dube M, Shen H, et al. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase[J]. J Med Chem,2011,18:6342-6363.
[115]Hiroki N, Takashi C, Kenji M, et al. Bioorg. Med. Chem. Lett.2010,20: 1405-1409
[116]Lu T, Alexander R, Connors RW, et al. Triazolopyridazines as tyrosine kinase modulators [P]. WO2007075567,2007