【作者】 刘志红；

【导师】 孙晓莉； 药立波；

【作者基本信息】 第四军医大学 ， 药物化学， 2002， 硕士

【摘要】 表皮生长因子受体(epidermal growth factor receptor,EGFR)自分泌环途径在肿瘤发生发展的一系列过程(包括细胞增殖、凋亡、血管生成、转移速度)中起重要作用。EGFR在肿瘤中的关键作用引起了对EGFR信号转导途径抑制剂的大规模寻找。大量临床前及早期临床试验结果提示EGFR是肿瘤治疗的靶点。针对EGFR靶点目前有多种方法。最有临床开发前景的策略包括阻止配体结合的单克隆抗体和小分子的酪氨酸激酶抑制剂，它们抑制自动磷酸化及下游胞内信号。许多EGFR酪氨酸激酶抑制剂正在开发中。ZDl839和OSI-774是4-笨胺基喹唑啉类选择性的EGFR酪氨酸激酶抑制剂，目前它们正分别进行Ⅲ期及Ⅱ期临床研究。CI-1033是不可逆的选择性的4-苯胺基喹唑啉类EGFR酪氨酸激酶抑制剂，目前正进入Ⅰ期临床。用晚期病人进行的初步Ⅰ，Ⅱ期临床试验结果提示ZD1839和OSI-774对各类肿瘤病人有着较好的临床疗效。 为了进一步开发新的4-苯胺基喹唑啉类选择性的EGFR酪氨酸激酶抑制剂，我们做了以下工作： ①改进了用于制备4-苯胺基喹唑啉类的关键中间体4(3H)喹唑啉酮的Niementowski反应。方法是：在三氯氧磷存在下，以甲酰胺和邻氨基苯甲酸类化合物为原料，合成了5种喹唑啉酮化合物： 第四军医大学硕士学位论文 0－x－＃（an－喳哇咐酮（x＝wo。，of，sr，）D 1（a哺2－甲基）－0－磺酚胺基－v－ 氯什 IH卜喳哇琳酮。用改进后的 Niementowski反应，可使反应温度由 原来的 175℃以上降至 90习5℃，甚至能在冰浴条件下制备出化合物 l－（吱哺－2－甲基）－6－磺酚胺基－7－氯什 IH）－喳哩晰酮。 ②对改进的Niementowski反应历程进行了探讨。用N，N－H甲基 甲酚胺替代反应物中的甲酚胺，使反应停留在中间体阶段，用核磁共 振检测中间体，初步证明，改进后的反应历程与原Nie阳ntowski反应 历程一致。 ③从6－石基－4（3H厂哇哇晰酮出发，制备了一系列6－石基，6－ 氨基，6－肉桂酚胺基和个马来酚胺基取代的个苯胺基啥哇淋类化合 物。其中，6－肉桂酚胺基，6－马来酚胺基取代的个苯胺基喳哩琳类化 合物是运用不可逆EGFR酪氨酸激酶抑制剂原理设计的。 ④用EGFR过度表达的A431肿瘤细胞和永生型的正常的NIH3T3 细胞系作为模型，对所合成的化合物进行了初步筛选。结果，所有受 试的 4－苯胺基喳哇琳类化合物对 A4 31细胞均有抑制作用，且呈剂量 一效应关系。在其它基团保持不变的情况下，化合物对A43细胞的抑 制活性为取代的6－肉桂酚胺基乃－氨基巧－马来酚胺基乃正基。苯胺基 上的卤素取代对抑制活性的影响没有规律性。喳哩晰的6位为氨基取 代时，苯胺基上卤素取代对活性的影响为对位滨取代对 位涅取代乃－ 氯＊氟取代＞对位氯取代叶 位氯取代；啥哩晰的6位为马来酚胺基取 代时，苯胺基上卤素取代对活性的影响为3－氯＊氟取代＞对位滨取代＞ 对位氯取代＞间位滨取代。间位氯取代；喳哇晰的6位为肉桂酚胺基取 代时，苯胺基上卤素在间位和／或对位取代对 A43抑制活性的影响不 大；喳哇晰的6位为硝基取代时，苯胺基上卤素在间位和／或对位取代 对 A43抑制活性的影响也不大。此外，当睦哩淋的 6位为硝基取代时， 可观察到加药后不久，细胞内即开始显墨绿色。提示，6－硝基取代的 ·3－ 第四军医大学硕士学位论文 4苯胺基哇哩咐类化合物在胞内可能是硝基被还原为氨基而发生作用 的。 所有受试的个苯胺基喳哇咐类化合物对 N’－e3 T3的抑制活性均远 小于对 A43细胞的抑制活性。喳哩咐的 6位为氨基取代时，对 MH3T3 的IC；。大于对A43细胞的IC。。的十倍以上。喳哇琳的6位为马来酚’ 胺基取代时，对 NIH3T3的匝。大于对 A43细胞的K。的五倍以上。 哇哩晰的 6位为肉桂酚胺基取代时，对 MH3T3的 IC；。与对 A43细胞 的IC。。相差很小。特别是个＊－氯＊氟苯胺基）6－肉桂酚胺基哇哇琳和 个间澳苯胺基6－肉桂酚胺基嘻哩咐对 NIH3T3的u；。与对 A43细胞的 IC。。几乎接近，显示出较好的的选择性。更多还原

【Abstract】 The epidermal growth factor receptor (EGFR) autocrine pathway contributes to a number of processes important to cancer development and progression,including cell proliferation,apoptosis,angiogenesis,and metastatic speed.The critical role the EGFR plays in cancer has led to an extensive search for selective inhibitors of the EGFR signaling pathway.The results of a large body of preclinical studies and the early clinical trials thus far conducted suggest that targeting the EGFR could represent a significant contribution to cancer therapy. A variety of different approaches are currently being used to target the EGFR.The most promising strategies in clinical development include monoclonal antibodies to prevent ligand binding and small molecule inhibitors of the tyrosine kinase enzymatic activity to inhibit autophosphorylation and downstream intracellular signaling. A number of small molecule inhibitors of the EGFR tyrosine kinase enzymatic activity is in development.ZD1839 and OSI-774 are 4-anilinoquinazoline selective inhibitors of tyrosine kinase of EGFR,which are currently in Pase III and Phase II development,respectively. CI-1033 is an 4-anilinoquinazoline irreversible selective inhibitor of tyrosine kinase of EGFR that is currently in Phase I development.Preliminary results from Phase I and II trials in patients with advanced disease demonstrate that ZD1839 and OSI-774 have promising-5-clinical efficacy in patients with a variety of tumor types.In a further development of new 4-anilinoquinazoline selective inhibitors of tyrosine kinase of EGFR,we have done some work as down list:?An improved Niementowski reaction which was applied in preparing4(3H)quinazolinone------the critical intermediate of 4-anilinoquinazolines has beenstudied. Method: Five quinazolinone compounds, such as 6-X-4(3H)-quinazoinone ( X=NO2,Cl,Br J) and 1 -furfuryl-6-sulfarnide-7-chloro-4( 1 H)-quinazolinone(5),were synthesized by reaction of anthranilic acids with formamide accompanied by titrating with phosphoryl chloride. Results: When the improved Niementowski reaction was applied ,the reaction could go along well within 90-95癈. Compound 5 could even be prepared on the ice-bath condition.But the original Niementowski reaction needs the reaction temperature more than 175癈.(2) The course of the improved Niementowski reaction has been studied. Method: We selected N,N-dimethyl formamide as the reaction material instead of original reagent 梖ormamide in order to make the reaction halt in the intermediate phase and used NMR to identify the intermediate. Results: The course of the improved Niementowski reaction is similar to that of original Niementowski reaction.(D Using 6-niro-4(3H)-quinazolinone as the initial reagent, we synthesized a series of 4-anilinoquinazolines substituted with 6-nitro, 6-amino, 6-cinnamido or 6-malemido side chains. Among these compounds, 4-anilinoquinazolines substitued with 6-cinnamido or 6-malemido were designed by using principle of irreversible inhibitor of epidermal growth factor receptor .?The synthesized 4- anilinoquinazoline compounds has been rudimentarily screened by using A431 tumor cell line which overexpresses epidermal growth factor receptor and eternal normal NIH3T3 cell line as screening model. Method: A431 cells were plated out in Fi2 medium containing 10% fetal bovine serum at a-6-concentration of 104 cells/well in 96 well plates ,then cultured in the CO2 culturing tank.After 24h,they were given various concentrtions of 4-anilinoquinazoline compounds according to the plotting area on the plate.The cells growing status was observed through invert microscope everyday.The cultures would have been incubated for 4 days at 37癈 with 5% CO2.On day 5,each well was added 5mg/mL MTT and incubated continually for 4 h..Then,the upper solution of the wells was discarded gently.After this,every well was added 150 u L DMSO.Finally,the plate was surged for lOmin on Sunrise ELISA instrument.The OD values of each well was also detected at 570nm vs controlling更多还原