CN105503746A - 用于促进癌细胞凋亡的化合物、其医药组合物及其用途 - Google Patents
用于促进癌细胞凋亡的化合物、其医药组合物及其用途 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
- C07D239/72—Quinazolines; Hydrogenated quinazolines
- C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
- C07D239/94—Nitrogen atoms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
本发明公开了用于促进癌细胞凋亡的化合物、其医药组合物及其用途。具体地,本发明提供一种式(I)化合物或其盐,其中,m为2至7的整数,以及R独立选自由氢及C1-C20烷基所组成组的至少一者。该化合物用以促进癌细胞的细胞凋亡,以抑制癌细胞的生长。本发明进一步提供一种医药组合物,该医药组合物包含式(I)的化合物或其盐以及医药上可接受的载剂。本发明再进一步提供一种包含式(I)的化合物或其盐的用途,用于制造治疗癌症的药物。
Description
技术领域
本发明关于一种新颖化合物,尤其是,该化合物用于抑制癌细胞生长并可促进癌细胞凋亡。
背景技术
肺癌是现今高发病率与高致死率的癌症之一,而根据统计,肺癌是台湾最常见的癌症死因。肺癌主要可区分成小细胞肺癌(smallcelllungcarcinoma,简称SCLC)及非小细胞肺癌(non-smallcelllungcarcinoma,简称NSCLC),其中NSCLC约占肺癌病例的80%,SCLC则占20%。绝大数的NSCLC的病患观察到有上皮生长因子受体(epidermalgrowthfactorreceptor,简称EGFR)的大量表现,突变且异常增升的EGFR为新兴药物标靶蛋白之一。
艾瑞莎(亦称为吉非替尼(Gefitinib)或ZD1839),现已应用于在治疗非小细胞癌病人的临床用药,其会与ATP竞争EGFR的细胞内酪氨酸激酶(tyrosinekinase)区的受质结合位置,进而抑制酪氨酸激酶的自身磷酸化反应,进而抑制下游的讯息传导途径,是一种小分子的酪氨酸激酶抑制剂。
艾瑞莎的疗效与EGFR本身的突变有关,如在表现子19基因缺失(exon19)和第858氨基酸位点由原本的亮氨酸突变成精氨酸(L858R)时,会增加肿瘤对艾瑞莎的敏感性,治疗效果较原始未突变的EGFR更佳。目前的研究发现,艾瑞莎的抗药性与EGFR的第二个位点突变有关,临床上的数据指出50%对艾瑞莎产生抗性的非小细胞肺癌病人在EGFR的第790氨基酸产生突变,由原本的苏氨酸变异成蛋氨酸(T790M),T790M突变点位于EGFR与ATP结合位置上,其阻挡EGFR与其抑制子结合,甚至提高EGFR与ATP的结合能力而增强EGFR的活性。
发明内容
本发明提供一种式(I)化合物或其盐,
其中,m为2至7的整数,以及R独立选自由氢及C1-C20烷基所组成组的至少一者。
于本发明的一具体实施例中,m为2,R为C1-C13烷基。
于本发明的一具体实施例中,m为2,R为C12烷基。
于本发明的一具体实施例中,本发明的化合物用于促进癌细胞的细胞凋亡,用以抑制癌细胞的生长。优选地,该癌细胞选自由肺癌细胞、直肠癌细胞及膀胱癌细胞所组成组的至少一者。
于本发明的一具体实施例中,本发明的式(I)化合物用以抑制表皮生长因子受体(epidermalgrowthfactorreceptor,以下简称EGFR)蛋白激酶的活性,以促进癌细胞凋亡。
本发明进一步提供一种医药组合物,其包含如上述式(I)的化合物或其盐以及医药上可接受的载剂。
本发明再进一步提供一种包含本发明的医药组合物的用途,其用于制造治疗癌症的药物,其中,该癌症选自肺癌、直肠癌及膀胱癌所组成组的至少一者。
附图说明
图1显示不同浓度的艾瑞莎类似物(简称类似物)1、类似物2及艾瑞莎分别处理不同癌症细胞株24小时后的细胞存活率结果,(A)为RKO人类直肠癌细胞株,(B)为BFTC905人类膀胱癌细胞株;
图2显示类似物18相较于艾瑞莎对于促进不同人类肺癌细胞的细胞死亡更有效率;将不同人类肺癌细胞株以0至40μM的艾瑞莎或类似物18处理24小时,之后以MTT试验进行细胞存活率分析。(A)为A549人类肺腺癌细胞株(以下简称A549),(B)为H1299人类非小细胞肺癌细胞株(以下简称H1299),(C)CL3人类肺癌细胞株((以下简称CL3),其中(A)、(B)及(C)结果得自3个实验且该横杠表示平均值±S.E.,##p<0.01表示以艾瑞莎处理样品与以类似物18处理样品间的显著性差异;(D)为HFL1人类肺纤维母细胞株(以下简称HFL1),该结果得自6个实验且该横杠表示平均值±S.E.;
图3显示EGFR及磷酸化EGFR在不同人类肺癌细胞中的蛋白质表现;萃取自A549细胞株、H1299细胞株、CL3细胞株及A431人类表皮样癌(过分表现EGFR,其做为正控制组)的总体蛋白质,利用抗EGFR、抗磷酸化EGFR及抗肌动蛋白的抗体进行西方墨点法分析,并以肌动蛋白作为蛋白质充填控制组,该结果取自3个类似实验结果其中之1;
图4显示类似物18及艾瑞莎均抑制EGFR蛋白激酶的活性,(A)ATP转换至ADP的标准曲线,其与ADP的量具有正相关;(B)利用IVIS系统定量荧光强度的结果;(C)利用(A)的标准曲线计算蛋白激酶特定活性的结果;(D)以最大酶活性的%表现EGFR蛋白激酶活性,其中,以类似物18或艾瑞莎不存在时的蛋白激酶特定活性作为100%;上述结果得自3个实验且该横杠表示平均值±S.E.,*p<0.05、**p<0.01表示控制组(不含任何化合物者)与经类似物18或艾瑞莎处理样品间的显著性差异;
图5显示类似物18较艾瑞莎于人类肺癌细胞中引发细胞凋亡更具效率,A549细胞株以0至40μM的(A)类似物18或(B)艾瑞莎处理24小时后,利用膜联蛋白V-碘化丙啶(AnnexinV-PI(propidiumiodise,简称PI))染色及使用流式细胞仪分析测定被引发的细胞凋亡含量,该细胞群以AnnexinV+/PI-(右下图)及AnnexinV+/PI+表示者(右上图),分别表示细胞凋亡的早期及晚期,该结果取自3个类似实验的其中之一;
图6显示以CellQuest软体定量(A)早期细胞凋亡、(B)晚期细胞凋亡及(C)全部细胞凋亡的百分比,该横杠表示平均值±S.E.,*p<0.05、**p<0.01表示以控制组(不含任何化合物者)与以类似物18处理样品间的显著性差异,#p<0.05及##p<0.01表示以艾瑞莎处理样品与以类似物18处理样品间的显著性差异;
图7显示类似物18较艾瑞莎对于人类肺癌细胞的生长及细胞周期抑制更有效率,将A549细胞株以1×106细胞/p60培养皿的密度接种于60mm培养皿中培养18小时,再以30μM的类似物18或艾瑞莎处理24小时,待以药物处理完后,该细胞继续培养2天至6天,最后以血球计(hemocytometer)计算其细胞数,该结果得自3个实验且该横杠表示平均值±S.E.,*p<0.05表示以控制组(不含任何化合物者)与以艾瑞莎处理样品间的显著性差异,##p<0.01表示以控制组(不含任何化合物者)与以类似物18处理样品间的显著性差异;
图8显示将A549细胞株以0至40μM的(B)类似物18或(A)艾瑞莎处理24小时后,以流式细胞仪进行分析,该结果取自3个类似实验其中之一;
图9显示利用ModFitLT软体定量(A)G0/G1期、(B)S期、(C)G2/M期及(D)次-G1期的百分比,该结果得自3个实验且该横杠表示平均值±S.E.,*p<0.05、**p<0.01表示以控制组(不含任何化合物者)与以类似物18或艾瑞莎处理样品间的显著性差异,##p<0.01表示以类似物18处理样品与以艾瑞莎处理样品间的显著性差异;
图10显示类似物18较艾瑞莎于人类肺癌细胞中诱发凋亡蛋白酶3(caspase3)及多聚ADP核糖聚合酶(polyADPribosepolymerase,以下简称PARP)的蛋白质表现更有效率,(A)将A549细胞株以0至40μM的类似物18或艾瑞莎处理24小时后,进行蛋白质萃取并使用抗凋亡蛋白3、抗PARP及抗肌动蛋白的特定抗体进行西方墨点法分析,将肌动蛋白用于作为蛋白质充填控制组,该结果取自3个类似实验结果的其中之一;(B)及(C)以半量化法(semi-quantification)得出活化态凋亡蛋白3及切割态PARP的相对蛋白质强度,该结果得自3个实验且该横杠表示平均值±S.E.,*p<0.05表示以控制组(不含任何化合物者)与以类似物18处理样品间的显著性差异;
图11显示类似物18较艾瑞莎对于抑制人类肺癌细胞中的生存素(survivin)的基因及蛋白质表现更有效率,(A)将A549细胞株以0至40μM的类似物18或艾瑞莎处理24小时后,根据使用者手册萃取出细胞的总RNA,以半定量逆转录聚合酶连锁反应(reversetranscriptionpolymerasechainreaction,以下简称RT-PCR)分析mRNA的表现,以GAPDH作为内部控制组,该结果取自3个相似实验结果的其中之一;(B)该横杠表示平均值±S.E.,#p<0.05表示以类似物18处理样品与以艾瑞莎处理样品间的显著性差异;(C)将A549细胞株以0至40μM的类似物18或艾瑞莎处理24小时后,使用抗生存素及抗肌动蛋白的特定抗体进行西方墨点法以分析总蛋白萃取物,该结果取自3个相似实验结果的其中之一;(D)以半定量化法自西方墨点法得出生存素的相对蛋白质强度,该结果得自3个实验且该横杠表示平均值±S.E.,*p<0.05表示以控制组(不含任何化合物者)与以类似物18或艾瑞莎处理样品间的显著性差异,#p<0.05表示以类似物18处理样品与以艾瑞莎处理样品间的显著性差异;
图12显示在肺癌细胞中以GFP-生存素表现载体转染的生存素过度表现,可抑制类似物18所引发的细胞死亡,(A)将总蛋白萃取物进行西方墨点法分析;(B)在转染及以类似物18处理后,以MTT试验进行细胞存活率分析,该结果得自3个实验且该横杠表示平均值±S.E.,**p<0.01表示控制组的转染与生存素组的转染或以类似物18处理的生存素组转染样品间的显著性差异,##p<0.01表示以类似物18处理的样品及控制组间的显著性差异;
图13显示类似物18可在异体移植人类肺癌细胞的裸鼠中抑制肿瘤大小及生存素蛋白质的表现,(A)每4天测量一次肿瘤的体积,该结果得自8只裸鼠且该横杠表示平均值±S.E.,***p<0.001表示控制组(不含任何化合物者)与以类似物18处理样品间的显著性差异;(B)在以药物处理后的第16天牺牲裸鼠并取出肿瘤;(C)将得自各组的异体肿瘤组织均质化并使用抗生存素及抗肌动蛋白的特定抗体进行西方墨点法以分析总裂解物,该结果得自3个相似实验结果的其中之一,以半定量化法自西方墨点法得出生存素的相对蛋白质强度,该结果得自3个实验。
具体实施方式
以下通过特定的具体实施例说明本发明的实施方式,该领域技术人员可由本说明书所揭示的内容轻易地了解本发明的优点及功效。本发明也可通过其它不同的实施方式加以施行或应用,本说明书中的各项细节亦可基于不同观点与应用,在不悖离本发明所揭示的精神下赋予不同的修饰与变更。
本发明提供一种式(I)化合物或其盐,
其中,m为2至7的整数,以及R独立选自由氢及C1-C20烷基所组成组的至少一者。
于本发明的一具体实施例中,优选地,m为2,R为C1-C20烷基。最佳者,m为2,R为C12烷基。
于本发明的一具体实施例中,本发明的化合物为4-(4-(3-(4-(3-氯-4-氟苯胺基)-7-甲氧喹唑啉-6-氧基)丙基)哌嗪-1-基)-4-氧代丁酸十二酯(类似物18)。
于本发明的一具体实施例中,本发明的化合物用于促进癌细胞的细胞凋亡,用以抑制癌细胞的生长。较佳者,该癌细胞选自由肺癌细胞、直肠癌细胞及膀胱癌细胞所组成组的至少一者。
于本发明的一具体实施例中,本发明的式(I)化合物用以抑制EGFR(epidermalgrowthfactorreceptor)蛋白激酶的活性,以促进癌细胞凋亡。
于本发明的一具体实施例中,本发明的化合物用以促进癌细胞中凋亡蛋白酶3(caspase3)的活化及多聚ADP核糖聚合酶(polyADPribosepolymerase)的切割,并抑制生存素(survivin)的蛋白质及基因表现,以促进癌细胞凋亡。
本发明进一步提供一种医药组合物,其包含本发明的式(I)的化合物或其盐以及医药上可接受的载剂。
本发明化合物具有低毒性及可通过与药理上可接受的载剂等混合而呈医药组合物用于哺乳动物(例如:人类、小鼠、大鼠、兔、狗、猫、牛、马、猪、猴)。
作为药药上可接受的载剂,可使用传统上用作为配方材料的各种有机或无机载剂物质。该等被合并作为用于固体配方的赋形剂、润滑剂、结合剂及崩解剂,用于液体配方的溶剂、溶解剂、悬浮剂、等渗剂、缓冲液、舒缓剂,及其类似者,以及如需要时可添加的配方添加剂(例如:防腐剂、抗氧化剂、着色剂、甜味剂、及其类似物)。
该医药组合物的剂型的实例包括口服制剂(例如:锭剂(包括糖衣锭、膜衣锭、舌下含锭、口服崩解锭)、胶囊(包括软胶囊、微胶囊)、颗粒、粉末、片剂、糖浆、乳液、悬浮液、薄膜(例如:口服可崩解薄膜)及其类似物;以及肠外试剂(例如:注射液(例如:皮下注射液、静脉注射液、肌肉注射液、腹腔注射液、点滴注射液)、丸剂、鼻制剂、肺制剂(吸入剂)及其类似物。
本发明再进一步提供一种包含如上述式(I)的化合物或其盐的用途,用于制造治疗癌症的药物,其中,该癌症选自由肺癌、直肠癌及膀胱癌所组成组的至少一者。
用于本发明的说明书中,术语「艾瑞莎(Iresa)」指治疗肺癌药物的商品名,其学名为吉非替尼(Gefitinib),其为表皮细胞生长因子受体的抑制剂。
用于本发明的说明书中,术语「艾瑞莎类似物(analogues)」指一系列吉非替尼的哌嗪类似物,其中,在吉非替尼的吗啉基经多种的哌嗪基所取代。
实施例
细胞株与细胞培养
RKO为人类直肠癌细胞株;BFTC905为人类膀胱癌细胞株;A549细胞株(ATCC编号:CCL-185)为衍生自58岁的男性白种人(Caucasian)的肺腺癌细胞(含有野生型p53者);H1299细胞株为人类非小细胞肺癌(non-smallcelllungcarcinoma)细胞株(无法表现p53蛋白质);CL3由国立台湾大学杨泮池博士所提供;HFL-1(ACTT编号CCL-153)是衍生自白种胎儿的正常肺纤维母细胞;A431细胞株是衍生自85岁女性的表皮样癌(epidermoidcarcinoma)(过分表现EGFR者)细胞株。BFTC905、A549、H1299及CL3细胞株均以RPMI-1640培养基培养(Gibco,LifeTechnologies,GrandIsland,NY,USA);RKI、HFL1及A431细胞株均以DMEM培养基培养(Gibco,LifeTechnologies,GrandIsland,NY,USA)。完全培养基含有10%胎牛血清(fetalbovineserum,以下简称FBS)、100单位/毫升(U/mL)青霉素、100微克/毫升(μg/mL)链霉素以及碳酸氢钠。所有细胞株均培养于37℃的5%CO2的潮湿培养箱(310/Thermo,FormaScientific,Inc.,Marietta,OH)。
统计分析
各实验至少重复3次,数据以学生t检定(Student’sttest)分析;与多组比较上,以变异数分析(analysisofvariance)测试分析数据。以p值<0.05认定为统计上显著差异。
实施例1艾瑞莎类似物的合成
1.艾瑞莎类似物(简称类似物)1及类似物2的合成:
(1).化合物1:4-甲氧基-4-氧代丁酸(步骤1)
将丁二酸酐(1公克(g),10毫摩尔(mmol))与干甲醇(20毫升(mL),500mmol)混合后,以回流方式(reflux)加热并剧烈地搅拌2.5小时。在减压下移除多余的甲醇后,以水将剩余物溶解。接着将该溶液以二氯甲烷萃取后,再以MgSO4进行干燥,蒸发后获得产率为68%的化合物1(0.9g,6.81mmol)。
1HNMR(400MHz,CDCl3,δ):10.89(b,1H),3.70(s,3H),2.71–2.61(m,4H);13CNMR(100.6MHz,CDCl3,δ):178.1,172.6,51.9,28.8,28.6;IR(KBr):3028,2957,1736,1690,1175,1003cm–1;MSm/z:132.0(M+,0.1),114.1(11.2),101.0(100.0),73.1(20.9),59.1(17.2),55.0(41.8);HRMS-EI(m/z):[M]+calcdforC5H8O4,132.0423;found,132.0424.
(2).化合物2:4-(4-苄基哌嗪-1-基)-4-氧代丁酸甲酯(步骤2)
将丁二酸单甲酯(0.35g,2.65mmol)及亚硫酰氯(0.22mL,2.9mmol)的5mL苯溶液回流1.5小时,再以蒸馏法(distillation)将大部分的亚硫酰氯及苯去除。将该混合物冷却至室温并于真空下干燥得到粗3-氯羰基-丙酸甲酯。通过套管将3-氯羰基-丙酸甲酯(0.5g,2mmol)的二氯甲烷(5mL)溶液加入含有1-苄基哌嗪(0.5g,2.84mmol)的二氯甲烷圆形烧瓶中,随后添加吡啶(0.65mL,8mmol)。将所得溶液在室温下搅拌过夜,最后加水使其淬灭(quench)。加入2M的NaOH溶液使该pH成碱性(pH9),接着以二氯甲烷萃取且以MgSO4干燥及蒸发,获得粗的残留物。再将该残留物以管柱层析法纯化,并以乙酸乙酯/己烷(1:2.3,1:1,3:1)洗脱(elute)后获得产率为34%的化合物2(0.26g,0.90mmol)。
1HNMR(400MHz,CDCl3,δ):7.32–7.29(m,4H),7.28–7.27(m,1H),3.69(s,3H),3.63–3.60(t,J=5.1Hz,2H),3.51(s,2H),3.49–3.47(t,J=5.1Hz,2H),2.68–2.59(m,4H),2.45–2.39(m,4H);13CNMR(100.6MHz,CDCl3,δ):173.7,169.5,137.6,129.1,128.3,127.3,62.9,52.9,52.7,51.8,45.3,41.8,29.1,27.9;IR(KBr):2949,1736,1646,1438,1226,1165,998,744cm–1;MSm/z:290.1(M+,14.0),259.1(16.4),146.1(48.7),134.1(21.5),91.1(100.0);HRMS-EI(m/z):[M]+针对C16H22N2O3的计算值,290.1630;实测值,290.1634.
(3).化合物3:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸甲酯(步骤3)
将化合物2(0.21g,0.72mmol)及含有10%钯碳催化剂(palladiumoncarbon)(22mg,10重量%(wt%))的20mL甲醇的混合物置入巴氏容器(Parrglassvessel)中,并小心地以氢气换气三次。最后将该巴氏容器以60psi氢气充填后,机械式摇动12小时。反应完成后,将该反应混合物以硅藻土垫过滤并以过量的甲醇冲洗。减压下浓缩该过滤物后,得到产率为66%的4-氧代-4-哌嗪-1-基-丁酸甲酯(0.095g,0.48mmol)。将4-氧代-4-哌嗪-1-基-丁酸甲酯溶于10mL的四氢呋喃中,再加入三乙胺(0.08mL,0.57mmol)及1-溴-3-氯丙烷(0.057mL,0.57mmol),接着将该溶液搅拌过夜,最后加水使其淬灭。将所得溶液以乙酸乙酯萃取,再以MgSO4干燥,蒸发以获得粗残留物。接着将此残留物以管柱层析法(Al2O3)纯化,并以乙酸乙酯洗脱后获得产率为18%的化合物3(0.024g,0.087mmol)。
1HNMR(400MHz,CDCl3,δ):3.67(s,3H),3.60–3.57(t,J=6.3Hz,4H),3.48–3.45(t,J=5.1Hz,2H),2.66–2.58(m,4H),2.49–2.46(t,J=7.1Hz,2H),2.43–2.41(t,J=5.0Hz,2H),2.39–2.36(t,J=5.1Hz,2H),1.95–1.88(m,2H);13CNMR(100.6MHz,CDCl3,δ):173.6,169.5,55.1,53.3,52.8,51.8,45.2,43.0,41.7,29.7,29.0,27.9;IR(KBr):2950,2814,1736,1647,1438,1369,1227,1168cm–1;MSm/z:276.1(M+,8.6),245.1(28.2),213.1(100.0),132.1(15.4);HRMS-EI(m/z):[M]+C12H21ClN2O3计算值,276.1241;实测值276.1242.
(4).化合物4:4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-醇(步骤4)
将6-(芐氧基)-N-(3-氯-4-氟苯基)-7-甲氧基喹唑啉-4-胺(0.15g,0.37mmol)及含有10%钯碳催化剂(palladiumoncarbon)(25mg,10wt%)的20mL甲醇混合物置入巴氏容器中,并小心地以氢气冲洗三次。最后将该巴氏容器以60psi氢气充填后,机械式摇动24小时。反应完成后,将该反应混合物以硅藻土垫过滤并以过量的甲醇冲洗。减压下浓缩该过滤物以得到产率为82%的化合物4(0.096g,0.3mmol)。
(5).类似物1:4-(4-(3-(4-(3-氯-4-氟苯胺基)-7-甲氧喹唑啉-6-基氧基)基丙基)哌嗪-1-基)-4-氧代丁酸甲酯(步骤5)
先将化合物4(20mg,0.063mmol)溶解于0.4mL的N,N-二甲基甲酰胺(以下简称DMF)中,再加入碳酸钾(17mg,0.125mmol)及化合物3(17mg,0.063mmol),并在90℃下加热过夜。再将该反应混合混合物冷却至室温并加水使其淬灭。将所得溶液以乙酸乙酯萃取,再以MgSO4干燥,接着浓缩后获得粗残留物。最后将该残留物以管柱层析法纯化且以乙酸乙酯洗脱后获得产率为57%的类似物1(0.023g,0.041mmol)。
1HNMR(400MHz,CDCl3,δ):8.65(s,1H),7.89–7.86(dd,J=6.4,2.5Hz,1H),7.74(b,1H),7.56–7.53(m,1H),7.24–7.23(m,2H),7.20–7.06(m,1H),4.18–4.15(t,J=6.5Hz,2H),3.97(s,3H),3.68(s,3H),3.63(b,2H),3.51(b,2H),3.66–2.59(m,6H),2.51(b,2H),2.46(b,2H),2.12–2.07(m,4H);13CNMR(100.6MHz,CDCl3,δ):173.8,169.6,160.8,158.4,156.7,155.1,153.6,148.9,147.3,134.6,127.3,124.2,115.8,115.6,109.0,107.7,101.2,67.4,56.2,54.7,53.2,52.7,51.9,45.1,41.6,29.7,29.0,27.9,26.3;IR(KBr):3322,2949,2893,2838,1747,1644,1633,1579,1502,1472,1433,1220,1172,1005,839cm–1;MSm/z:559.2(M+,2.9),525.2(20.1),494.2(23.1),381.1(21.0),297.1(28.2),285.1(32.3),241.1(28.5),213.1(100.0),99.1(33.2),70.1(29.7);HRMS-EI(m/z):[M]+C27H31ClFN5O5计算值559.1998;实测值559.2007.
(6).类似物2:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸甲酯酸(步骤6)
将含有类似物1(0.08g,0.143mmol)及氢氧化锂(9mg,0.214mmol)的8mL甲醇溶液于60℃下加热过夜。接着,将该反应混合物冷却至室温,再进行蒸发后,得到粗产物。该粗产物再以水溶解后,利用乙酸乙酯进行萃取,以MgSO4进行干燥,最后蒸发,得到产率为68%的类似物2(53mg,0.097mmol)。
1HNMR(400MHz,MeOD,δ):8.46(s,1H),8.02–7.99(dd,J=6.7,2.4Hz,1H),7.74(s,1H),7.71–7.66(m,1H),7.30–7.25(t,J=8.9Hz,2H),7.19–7.15(m,1H),4.30–4.27(t,J=5.9Hz,2H),4.01(s,3H),3.66–3.63(t,J=4.9Hz,4H),3.33–3.31(m,2H),2.75–2.72(t,J=7.2Hz,2H),2.69–2.66(m,4H),2.61–2.58(m,4H),2.19–2.14(m,2H);13CNMR(100.6MHz,CDCl3,δ):174.4,169.9,156.5,155.0,154.8,153.1,152.4,148.8,147.5,137.3,124.0,122.9,122.8,119.3,119.1,117.1,116.9,109.2,107.8,102.3,67.6,56.4,54.9,53.5,53.1,45.1,41.6,40.6,29.5,27.9,26.6;IR(KBr):3387,2963,2812,1723,1646,1625,1584,1533,1499,1476,1427,1238,854cm–1;MSm/z:546.0(M+,50.9),512.0(16.1),389.0(13.7),320.0(26.8),307.0(30.1),227.0(36.1),199.0(28.3),154.0(99.9),136.0(100.0),90.0(80.5),78.0(76.5);HRMS-FAB(m/z):[M+1]+C26H30ClFN5O5计算值,546.1920;实测值546.1930.
2.类似物3至7的合成:
(1).化合物5:1-(3-氯丙基)-哌嗪·盐酸盐(步骤7)
将1-(3-氯丙基)-4-(叔丁氧羰基)-哌嗪(4.0g,15.2mmol)以溶于乙酸乙酯的盐酸处理后得到1-(3-氯丙基)-哌嗪·盐酸盐(3.17g,13.5mmol),产率89%。
1HNMR(400MHz,D2O,δ):3.63–3.54(m,9H),3.39(s,1H),3.40–3.35(m,2H),2.21–2.14(m,2H).13CNMR(100.6MHz,D2O,δ):54.8,48.6,41.1,40.7,26.2.IR(KBr):3356,3001,1443,1301,1160,1084cm-1.MSm/z:162.1(M+,13.6),120.1(100.0),99.1(79.5),70.1(29.2),56.1(49.6).HRMS-EI(m/z):[M]+计算值,C7H15ClN2,162.0924;实测值,162.0930.
(2).化合物6至10:(步骤8)
将含有单烷基酸(碳数分别为3至7)(1.2eq)及亚硫酰氯(1.4eq)于5mL苯的溶液回流(reflux)3小时。接着,将大部分的亚硫酰氯及苯以蒸馏方式去除,将该混合物冷却至室温后,真空下干燥以获得粗氯羰基-烷酸甲酯(烷酸的碳数分别为3至7)。将化合物5置入圆形烧瓶,再以套管(cannula)将含有该氯羰基-烷酸甲酯(烷酸的碳数分别为3至7)的5mL二氯甲烷溶液加入其中,接着加入吡啶(3.5eq)。将所得的溶液在室温下搅拌过夜,再加入水使其淬灭。将该溶液以乙酸乙酯萃取,再以MgSO4干燥,接着蒸发以获得粗残留物,将该残留物以管柱层析法(Al2O3)纯化,并以乙酸乙酯/己烷(1:15)洗脱后分别获得化合物6至10。
化合物6:5-(4-(3-氯丙基)哌嗪-1-基)-5-氧代戊酸甲酯
产率:35%,1HNMR(400MHz,CDCl3,δ):3.65(s,3H),3.60–3.57(t,J=6.5Hz,4H),3.46–3.43(t,J=4.9Hz,2H),2.49–2.46(t,J=7.0Hz,2H),2.46–2.33(m,8H),1.96–1.92(m,4H);13CNMR(100.6MHz,CDCl3,δ):173.8,170.6,55.1,53.4,52.8,51.5,45.4,43.0,41.5,33.2,32.1,30.0,20.4;IR(KBr):2923,2853,1735,1647,1457,1373,1105cm–1;MSm/z:290.2(M+,6.8),259.2(27.3),227.2(100.0),132.1(75.2),99.1(79.6),70.1(39.8),55.1(50.5);HRMS-EI(m/z):[M]+C13H23ClN2O3计算值,290.1397;实测值290.1391.
化合物7:6-(4-(3-氯丙基)哌嗪-1-基)-6-氧代己酸甲酯
产率:56%,1HNMR(400MHz,CDCl3,δ):3.64(s,3H),3.60–3.58(t,J=6.4Hz,4H),3.45–3.42(t,J=4.9Hz,2H),2.50–2.46(t,J=7.0Hz,2H),2.46–2.36(m,4H),2.34–2.29(m,4H),1.96–1.89(m,2H),1.67–1.63(m,4H);13CNMR(100.6MHz,CDCl3,δ):173.9,171.0,55.1,53.5,52.8,51.5,45.5,42.9,41.5,33.8,32.8,29.7,24.7.IR(KBr):2949,1735,1645,1436,1249,1004cm–1;MSm/z:304.2(M+,6.2),273.1(25.1),241.1(100.0),132.1(96.0),99.1(41.7),70.1(27.8),55.1(55.6);HRMS-EI(m/z):[M]+C14H25ClN2O3计算值,304.1554;实测值304.1558.
化合物8:7-(4-(3-氯丙基)哌嗪-1-基)-7-氧代庚酸甲酯
产率:60%,1HNMR(400MHz,CDCl3,δ):3.64(s,3H),3.60–3.57(t,J=6.3Hz,4H),3.44–3.42(t,J=4.6Hz,2H),2.50–2.46(t,J=6.9Hz,2H),2.38–2.36(m,4H),2.31–2.27(m,4H),1.95–1.89(m,2H),1.67–1.61(m,4H),1.38–1.30(m,2H);13CNMR(100.6MHz,CDCl3,δ):174.1,171.3,55.1,53.5,52.8,51.5,45.5,43.0,41.5,33.9,33.0,29.7,28.9,24.9,24.7;IR(KBr):2947,1736,1644,1435,1174,1004cm–1.MSm/z:318.2(M+,4.5),287.2(22.4),255.2(100.0),132.1(90.2),99.1(70.5),70.1(23.7),55.1(27.9;HRMS-EI(m/z):[M]+C15H27ClN2O3计算值,318.1710;实测值318.1718.
化合物9:8-(4-(3-氯丙基)哌嗪-1-基)-8-氧代辛酸甲酯
产率:67%,1HNMR(400MHz,CDCl3,δ):3.61(s,3H),3.61–3.57(m,4H),3.42–3.40(t,J=4.9Hz,2H),2.47–2.43(m,2H),2.39–2.33(m,4H),2.27–2.23(t,J=7.5Hz,4H),1.93–1.87(m,4H),1.59–1.54(m,4H),1.30–1.29(b,4H);13CNMR(100.6MHz,CDCl3,δ):174.1,171.4,55.1,53.5,52.8,51.4,45.5,43.0,41.4,33.9,33.0,29.7,29.0,28.9,25.0,24.7;IR(KBr):2934,2858,1737,1645,1462,1435,1370,1173,1004cm–1;MSm/z:332.2(M+,7.5),301.2(20.4),269.2(100.0),132.1(99.5),120.1(34.8),99.1(32.2),70.1(11.4),55.1(14.1);HRMS-EI(m/z):[M]+C16H29ClN2O3计算值,332.1867;实测值332.1880.
化合物10:9-(4-(3-氯丙基)哌嗪-1-基)-9-氧代壬酸甲酯
产率:63%,1HNMR(400MHz,CDCl3,δ):3.64(s,3H),3.60–3.57(m,4H),3.45–3.42(t,J=4.8Hz,2H),2.50–2.46(t,J=7.0Hz,2H),2.42–2.36(m,4H),2.29–2.26(m,4H),1.96–89(m,2H),1.61–1.57(m,4H),1.30(b,6H);13CNMR(100.6MHz,CDCl3,δ):174.2,171.5,55.2,53.5,52.9,51.4,45.6,42.9,41.5,34.0,33.2,29.7,29.2,29.0,28.9,25.2,24.9;IR(KBr):2932,2856,1737,1645,1435,1004cm–1;MSm/z:346.2(M+,0.5),313.2(53.4),284.1(54.3),269.1(26.9),191.2(100.0),132.1(99.5),120.1(10.2),99.1(12.3),55.1(90.6);HRMS-EI(m/z):[M]+C17H31ClN2O3计算值,346.2023;实测值346.2031.
(3).类似物3至7:(步骤9)
先以1mL的N,N-二甲基甲酰胺溶解化合物4(1eq)后,再加入碳酸钾(2eq)及自化合物6至10选一者加入,并在80℃下加热过夜。再将该反应混合物冷却至室温并加水使其淬灭。将所得的溶液以乙酸乙酯萃取,接着将所合并的萃取物以水及盐水(brine)清洗,再以MgSO4干燥,接着蒸发后以获得残留物。最后将该残留物以管柱层析法纯化后获得类似物3至7。
类似物3:5-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-5-氧代戊酸甲酯
产率:40%;1HNMR(400MHz,CDCl3,δ):9.50(s,1H),8.43(s,1H),7.83(s,1H),7.79–7.75(m,2H),7.25–7.20(t,J=8.3Hz,2H),4.20–4.17(t,J=6.1Hz,2H),3.94(s,3H),3.59(s,3H),3.45–3.42(m,4H),2.41(b,2H),2.36–2.30(m,6H),2.02–1.99(m,2H),1.76–1.69(m,2H);13CNMR(100.6MHz,CDCl3,δ):173.6,170.4,160.0,157.6,156.9,154.8,153.3,148.7,147.3,136.2,124.9,124.8,115.6,115.4,109.2,107.8,103.2,67.6,56.3,54.9,53.6,53.1,51.7,45.2,41.4,33.1,31.8,26.5,20.7;IR(KBr):3374,2949,1729,1624,1508,1428,1214,854cm–1;MSm/z:573.2(M+,6.3),539.2(19.6),508.2(23.2),381.2(35.6),297.1(55.8),285.1(87.3),227.1(100.0),213.1(57.0),99.1(95.7),70.1(85.8),55.0(47.0);HRMS-EI(m/z):[M]+C28H33ClFN5O5计算值,573.2154;实测值,573.2940.
类似物4:6-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-6-氧代己酸甲酯
产率:30%;1HNMR(400MHz,CDCl3,δ):8.62(s,1H),7.62–7.58(m,2H),7.37(b,1H),7.24(s,1H),7.12–7.07(m,2H),4.18–4.15(t,J=6.6Hz,2H),3.98(s,3H),3.65(s,3H),3.62–3.60(t,J=4.7Hz,2H),3.46–3.44(t,J=4.9Hz,2H),2.59–2.56(t,J=7.0Hz,2H),2.47–2.40(m,4H),2.35–2.29(m,4H),2.13–2.06(m,2H),1.66(b,4H);13CNMR(100.6MHz,CDCl3,δ):173.9,171.0,160.8,158.4,156.5,155.1,153.7,148.9,147.5,134.5,124.2,124.1,115.9,115.7,108.9,108.0,100.8,67.5,56.2,54.8,53.5,52.9,51.5,45.5,41.5,33.8,32.8,26.4,24.7,24.6;IR(KBr):3379,2949,1733,1623,1508,1430,1214cm–1;MSm/z:587.2(M+,0.9),551.2(14.3),522.2(23.4),381.1(33.5),355.1(19.5),297.1(53.2),285.0(100.0),269.1(56.4),241.1(80.4),99.0(73.4),70.0(56.2),55.0(44.9);HRMS-EI(m/z):[M]+C29H35ClFN5O5计算值,587.2311;实测值,587.2314.
类似物5:7-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-7-氧代庚酸甲酯
产率:33%;1HNMR(400MHz,CDCl3,δ):8.63(s,1H),7.63–7.59(m,2H),7.24(s,1H),7.12–7.08(m,3H),4.19–4.16(t,J=6.3Hz,2H),3.99(s,3H),3.66(s,3H),3.62(b,3H),3.46(b,3H),2.61–2.57(t,J=6.9Hz,2H),2.46–2.44(m,4H),2.33–2.28(m,4H),2.14–2.09(m,2H),1.68–1.60(m,4H),1.40–1.32(m,2H);13CNMR(100.6MHz,CDCl3,δ):174.1,171.3,160.9,158.4,156.5,155.2,153.7,148.9,147.6,134.5,124.2,124.1,115.9,115.7,108.8,108.1,100.7,67.6,56.2,54.8,53.5,52.9,51.5,45.5,41.5,33.9,32.9,28.9,26.4,24.9,24.7;IR(KBr):2927,1735,1624,1582,1508,1429,1214,732cm–1.
类似物6:8-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-8-氧代辛酸甲酯
产率:35%;1HNMR(400MHz,CDCl3,δ):8.63(s,1H),7.63–7.59(m,2H),7.25(s,1H),7.13–7.08(m,3H),4.21–4.18(t,J=6.5Hz,2H),4.00(s,3H),3.66(s,3H),3.64–3.61(t,J=5.1Hz,2H),3.48–3.45(t,J=4.7Hz,2H),2.61–2.58(t,J=6.9Hz,2H),2.48–2.43(m,4H),2.31–2.28(t,J=7.4Hz,4H),2.15–2.08(m,2H),1.66–1.60(m,4H),1.36–1.32(m,4H);13CNMR(100.6MHz,CDCl3,δ):174.2,171.5,160.9,158.4,156.5,155.2,153.7,148.9,147.6,134.5,124.2,124.1,115.9,115.7,108.8,108.1,100.7,67.6,56.2,54.8,53.5,52.9,51.5,45.6,41.5,33.9,33.1,29.1,28.9,26.4,25.1,24.8;IR(KBr):2931,1734,1623,1583,1508,1429,1244,1214,1141,1005,833cm–1.
类似物7:9-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-9-氧代壬酸甲酯
产率:31%;1HNMR(400MHz,CDCl3,δ):8.62(s,1H),7.63–7.59(m,2H),7.29(s,1H),7.24(s,1H),7.11–7.07(m,2H),4.19–4.16(t,J=6.3Hz,2H),3.99(s,3H),3.66(s,3H),3.63–3.61(t,J=4.7Hz,2H),3.47–3.45(t,J=4.7Hz,2H),2.60–2.57(t,J=6.9Hz,2H),2.48–2.42(m,4H),2.31–2.27(t,J=7.4Hz,4H),2.13–2.06(m,2H),1.62–1.59(m,4H),1.31(b,6H);13CNMR(100.6MHz,CDCl3,δ):174.2,171.6,160.8,158.4,156.5,155.2,153.7,148.9,147.6,134.5,124.2,124.1,115.9,115.7,108.9,108.1,100.9,67.6,56.2,54.8,53.5,52.9,51.4,45.6,41.5,34.0,33.2,29.2,29.0,28.9,26.5,25.2,24.9;IR(KBr):2927,1732,1628,1508,1265,739,704cm–1;MSm/z:629.4(M+,0.9),593.4(20.8),564.4(27.7),381.2(54.3),355.2(32.6),312.2(40.8),297.2(80.6),285.1(100.0),269.1(25.8),125.1(31.2),99.1(99.6),70.1(77.6),55.1(64.7);HRMS-EI(m/z):[M]+C32H41ClFN5O5计算值,629.2780;实测值,629.2789.
3.类似物8至26的合成:
(1).化合物11至29:(步骤10)
将含有丁二酸酐(1eq)及干燥醇类(碳数分别为2至20)(1eq)的4mL甲苯溶液回流下加热2.5小时。减压下移除甲苯后,以水将残留物溶解。接着将此溶液以二氯甲烷萃取,再以MgSO4进行干燥,蒸发后得到单烷基丁二酸(烷基的碳数分别为2至20)。
将含有单烷基丁二酸(烷基的碳数分别为2至20)(1.2eq)及亚硫酰氯(1.4eq)的5mL苯溶液回流3小时,再以蒸馏方式除去大部分的亚硫酰氯及苯。将该混合物冷却至室温,真空干燥以获得粗氯羰基-烷基酯(烷基的碳数为2至20)。将化合物5(1eq)置于圆形烧瓶中,再以套管(cannula)将该氯羰基-烷基酯(烷基的碳数为2至20)的5mL二氯甲烷溶液加入其中,接着加入吡啶(1.4eq)。将该所得溶液在室温下搅拌过夜,再加入水使其淬灭。以乙酸乙酯萃取,再以MgSO4干燥,蒸发以获得残留物。将该残留物以管柱层析法(Al2O3)纯化,以乙酸乙酯/己烷(1:15)洗脱后得到化合物11至29(其物化数据详见于表1)。
化合物11:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸乙酯
化合物12:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸丙酯
化合物13:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸丁酯
化合物14:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸戊酯
化合物15:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸己酯
化合物16:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸庚酯
化合物17:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸辛酯
化合物18:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸壬酯
化合物19:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸癸酯
化合物20:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十一酯
化合物21:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十二酯
化合物22:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十三酯
化合物23:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十四酯
化合物24:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十五酯
化合物25:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十六酯
化合物26:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十七酯
化合物27:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十八酯
化合物28:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸十九酯
化合物29:4-(4-(3-氯丙基)哌嗪-1-基)-4-氧代丁酸二十酯
表1、化合物11至29的物化数据
(2).类似物8至26的合成:(步骤11)
将化合物4(1eq)溶于1mL二甲基甲酰胺中,分别加入化合物11至29(1eq)及碳酸钾(2eq),并在80℃下加热过夜,将该反应混合物冷却至室温后再加水使其淬灭,以乙烯乙酯萃取该所得溶液。将所合并的萃取物以水及盐水(brine)清洗后,以MgSO4干燥,蒸发以获得粗残留物,将该残留物以管柱层析法纯化后得到类似物8至26(其物化数据详见于表2)。
类似物8:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸乙酯
类似物9:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸丙酯
类似物10:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸丁酯
类似物11:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸戊酯
类似物12:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸己酯
类似物13:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸庚酯
类似物14:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸辛酯
类似物15:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸壬酯
类似物16:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸癸酯
类似物17:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十一酯
类似物18:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十二酯
类似物19:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十三酯
类似物20:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十四酯
类似物21:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十五酯
类似物22:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十六酯
类似物23:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十七酯
类似物24:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十八酯
类似物25:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十九酯
类似物26:4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸二十酯
表2、类似物8至26的物化数据
实施例2艾瑞莎类似物对于癌细胞的毒杀效果
首先,以本发明实施例1所合成的艾瑞莎类似物(简称类似物)处理RKO人类肠癌细胞株(以下简称RKO细胞株)、BFTC905人类膀胱癌细胞株(以下简称BFTC905细胞株)及A549人类肺腺癌细胞株(以下简称A549细胞株),以评估本发明所合成的艾瑞莎类似物的癌细胞毒杀作用。
将上述细胞株以1×104细胞/孔的密度接种于96-孔盘中培养16至20小时。接着,于RPMI-1640培养基中,以分别含有或不含有艾瑞莎或艾瑞莎类似物(包括本发明的类似物1至26)处理该细胞24小时。于药物处理后,以磷酸盐缓冲液(phosphate-bufferedsaline,以下简称PBS)洗涤细胞,并以新鲜的完全培养基RPMI-1640培养2天。之后,更换培养基且将该细胞与0.5mg/mL的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴盐(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide,以下简称MTT)(SigmaChemicalCo.,St.Louis,MO)于完全培养基中培养4小时。存活的细胞将MTT转化为甲簪(formazan),其溶解于二甲基亚砜(以下简称DMSO)时产生蓝紫色。使用读盘仪(platereader)(VERSAmax,MolecularDynamicsSunnyvale,CA)于565nm下测定甲簪强度。通过将各实验中该经处理细胞的吸收值除以控制组的吸收值而得出细胞存活率的相对百分比。
如图1(A)及(B)所示,在细胞存活率的结果中显示类似物1较艾瑞莎对于RKO细胞株及BFTC905细胞株具有显著的毒杀效果;然而,相较于艾瑞莎及类似物1,类似物2对于RKO细胞株则无毒杀效果,而对于BFTC905细胞株则仅有小量毒杀作用。因此,由上述结果确认类似物哌嗪基的末端应该连接酯基,以下以类似物1的结构为基础进一步设计合成类似物3至7及26至44。
如表3所示,类似物3至7的细胞存活率分析结果显示,增加类似物1中二个羧酸基间的碳链后(类似物3至7分别为增加1至5个碳链),相较于艾瑞莎,对于A549细胞株并不具有显著的毒杀效果。
如表3所示,类似物8至26的细胞存活率分析结果显示,增加类似物1的哌嗪基的末端碳链后(类似物8至26分别为增加1至19个碳链),相较于艾瑞莎,对于A549细胞株具有相似或显著的毒杀效果;其中又以类似物18的毒杀效果最佳,当以浓度为40μM的类似物18处理A549细胞株时,其毒杀效果增加15至20倍。因此确认艾瑞莎类似物18,4-(4-(3-(4-(3-氯-4-氟苯基胺基)-7-甲氧基喹唑啉-6-基氧基)丙基)哌嗪-1-基)-4-氧代丁酸十二酯,为一种新颖的抗肺癌化合物,以下称为类似物18,下述实施例并针对类似物18的抑制癌细胞机制做进一步的测试。
表3、在不同浓度下艾瑞莎及其类似物的细胞存活率结果
实施例3类似物18对于不同肺癌细胞株均具有毒杀效果
将A549人类肺腺癌细胞株(以下称为A549细胞株)、H1299人类非小细胞肺癌细胞株(以下称为H1299细胞株)、CL3人类肺癌细胞株(以下称为CL3细胞株)及HFL1人类肺纤维母细胞株HFL1(做为控制组)以1×104细胞/孔的密度接种于96-孔盘维持16至20小时。接着分别以0至40μM的类似物18或艾瑞莎在37℃下于RPMI-1640培养基处理细胞24小时。于药物处理后,以PBS洗涤细胞,并以完全培养基RPMI-1640培养2天。之后,更换培养基且将细胞与0.5mg/mL的MTT(SigmaChemicalCo.,St.Louis,MO)于完全培养基中培养4小时。使用读盘仪(VERSAmax,MolecularDynamicsSunnyvale,CA)于565nm下测定甲簪强度。通过将各实验中该经处理者的吸收值除以控制组的吸收值而得出细胞存活率的相对百分比。
如图2(A)、(B)及(C)所示,其结果显示类似物18较艾瑞莎对于降低A549、H1299及CL3细胞株的存活率上效果更佳。然而,根据图2(D)所示,类似物18对于HFL1人类正常肺纤维母细胞株的存活率效果与艾瑞莎相似。在类似物18的浓度为40μM时,其对于三种肺癌细胞株(包括A549、H1299及CL3)的毒杀效果大于99%,而对于正常肺组织细胞的存活率,类似物18及艾瑞莎均维持大于30%的细胞存活率。此结果表示,类似物18对肺癌细胞的毒杀效果较佳,对于正常肺组织细胞不会造成严重的毒杀效果。
实施例4类似物18可抑制肺癌细胞中EGFR蛋白激酶的活性
本发明测试类似物18是否通过抑制肺癌细胞的上皮生长因子受体(epidermalgrowthfactorreceptor,以下简称EGFR)蛋白激酶(proteinkinase)活性而造成其细胞存活率降低。
1.步骤1:检验不同人类肺癌细胞中EGFR的表现情形:
分别将A549细胞株、H1299细胞株、CL3细胞株及A431人类表皮样癌(以下称为A431细胞株,其过分表现EGFR者,做为正控制组)以pH7.6的冰冷却细胞萃取缓冲液(含有0.5mMDTT、0.2mMEDTA、20mMHEPES、2.5mMMgCl2、75mMNaCl、0.1mMNa3VO4、50mMNaF及0.1%TritonX-100)将细胞裂解,将蛋白酶抑制剂(含有1μg/ml抑肽酶(aprotinin)、0.5μg/ml亮肽素(leupeptin)及100μg/ml4-(2-胺基乙基)苯磺酰氟(4-(2-aminoethyl)benzenesulfonylfluoride))添加于该细胞悬浮液中,于4℃下震荡30分钟后,再以10,000rpm离心10分钟。通过BCA蛋白质试验套组(Pierce,Rockford,IL)测定蛋白质浓度。制备该总细胞蛋白质萃取物,于8至12%十二烷基硫酸钠-聚丙烯酰胺(sodiumdodecylsulfatepolyacrylamide,以下简称SDS)胶体上分离,且将蛋白质电泳转移至聚偏氟乙烯(polyvinylidenedifluoride)膜上,于4℃下,使用封闭缓冲液(将5%脱脂奶粉溶于含有50mMTris/HCl(pH8.0),2mMCaCl2,80mM氯化钠,0.05%Tween20及0.02%迭氮化钠的溶液中)封闭该聚偏氟乙烯膜过夜,然后,以初级抗体(primaryantibody)及随后的辣根过氧化物酶共轭(horseradishperoxidase-conjugated)的二级抗体依序杂交该膜,使用增强化学发光检测系统(enhancedchemiluminescencedetectionsystem,PerkinElmerLifeandAnalyticalSciences,Boston,MA)于X光胶片上观察到该蛋白质条带,使用特定抗体进行EGFR及磷酸化(tyr1068)EGFR的西方墨点法分析,为了确认蛋白质的均等充填及转移,将肌动蛋白(actin)作为蛋白质充填控制组。最后利用胶体数位化软体Un-Scan-Itgel(V5.1,SilkScientific,Inc.,Orem,UT)分析X光胶片上的蛋白质条带强度。
如图3所示,A549细胞株、H1299细胞株及CL3细胞株均会表现EGFR及磷酸化(Tyr1068)EGFR,其表示各肺癌细胞株中均具有活化态的EGFR蛋白激酶。
2.步骤2:测试类似物18抑制EGFR蛋白激酶的活性:
利用ADP-GloTM蛋白激酶试验(ADP-GloTMKinaseAssay)及EGFR蛋白激酶酶系统(EGFRKinaseEnyzmeSystem;Promega,Madison,WI),依照使用者手册进行操作。该蛋白激酶反应在pH7.5的反应缓冲液A、2mMMnCl2及2mMDTT中进行。步骤简述如下:
(1).在50μM的ATP/ADP范围中,制备ATP转换至ADP的标准曲线,如图4(A)所示,其与ADP的含量呈正相关;
(2).将下述反应组合物加入96-孔盘中:
5纳克(ng)活化态EGFR、0.25μg/μl的(Glu4,Tyr1)多肽物基质、反应缓冲液A(含有40mM的Tris-HCl(pH7.5),20mM的MgCl2及0.1mg/mL的BSA)以及浓度为20至100纳摩尔(nM)的类似物18或艾瑞莎;
(3).在30℃下加入50μM的ATP反应15分钟,以启动EGFR蛋白激酶反应;
(4).在室温下加入ADP-GloTM试剂反应40分钟,以中止EGFR蛋白激酶反应并移除剩余的ATP;
(5).在室温下加入蛋白激酶侦测试剂(KinaseDetectionReagent)作用30分钟,以进行ADP至ATP的转换作用;
(6).以IVIS系统(XenogenIVISSpectrum,CaliperLifeSciences)及光度计(ModulusSingleTube,TurnerBiosystems,Inc.,Sunnyvale,CA)观察及分析ADP转换至ATP时所发出的荧光;使用上述标准曲线测定在该多肽物基质有无中所产生的ADP含量,蛋白激酶特定活性的计算依照下列公式:
(ADP-空白组的ADP)(nmol)/反应时间(分钟)×蛋白激酶的重量(mg)。
如图4(B)、(C)及(D)所示,其结果显示以20至100nM的类似物18或艾瑞莎处理后,其荧光强度均下降,且经计算后的EGFR蛋白激酶特定活性(以nmol/分钟/mg表示者)及EGFR蛋白激酶活性(以%表示者,以0nM的类似物18或艾瑞莎处理时的蛋白激酶特定活性作为100%)均呈现依药物处理浓度的上升而呈下降的趋势。另外,以IVIS系统量测类似物18或艾瑞莎的IC50分别为130.1nM及56.3nM;以光度计量测类似物18或艾瑞莎的IC50分别为125.9nM及55.4nM。
(3).测试类似物18抑制EGFR蛋白激酶的专一性
以KinaseProfilerTM服务试验(MerckMillipore,Billerica,MA)进行类似物18对蛋白激酶专一性的测试,蛋白激酶活性剩余值(kinaseactivityremainingvalues,简称KAR值)的数值与该蛋白激酶的活性呈负相关,KAR值为50时表示抑制50%蛋白激酶活性时的浓度为10μM。
如表4所示,表4类似物18及艾瑞莎均为EGFR蛋白激酶的潜在抑制剂。
表4、以KinaseProfilerTM服务试验测试类似物18及艾瑞莎对不同蛋白激酶的专一性
实施例5类似物18引发肺癌细胞进行细胞凋亡的效果较艾瑞莎更佳
本实施例测试类似物18是否通过引发肺癌细胞的细胞凋亡而造成细胞存活率降低,且类似物18较艾瑞莎对于引起肺癌细胞凋亡是否具有较佳的效果。
通过膜联蛋白V-碘化丙啶(Annexin-propidiumiodise(以下简称PI))分析来测定经类似物18或艾瑞莎所引发的细胞凋亡含量,根据制造商手册,AnnexinV-PI染色套组(BioVision,MountainView,CA)用于检测通过与荧光素异氰酸酯(fluoresceinisocyanate,简称FITC)共轭的AnnexinV及PI培养的细胞。该细胞显示AnnexinV+/PI-及AnnexinV+/PI+,分别表示在细胞凋亡的早期及晚期。将A549细胞株以7×105细胞密度培养于60mm培养皿中16至20小时。分别以0至40μM的类似物18或艾瑞莎处理4小时后,以PBS洗涤该细胞。该细胞经胰蛋白酶消化且于1500rpm离心5分钟收集。此后,于室温及避光下,以500μl的AnnexinV-PI标记溶液(含有5μl的AnnexinV-FITC及5μl的PI于PBS中)培养该细胞5分钟。为了避免细胞凝集,将该细胞溶液经尼龙筛网膜(Becton-Dickinson,SanJose,CA)过滤。最后,通过流式细胞仪(FACSCalibur,BDBiosciences,Heidelberg,Germany)立即分析该样品,通过CellQuest软体(BDBiosciences)自最小10,000个细胞定量AnnexinV-PI染色细胞的百分比。
如图5(A)所示,在以10至40μM的类似物18或艾瑞莎处理后,A549细胞株的AnnexinV+/PI-及V+/PI+细胞数量均增加,表示正在进行细胞凋亡的细胞数增加;且如图6(A)、(B)及(C)所示,类似物18引发67.9%的细胞凋亡,而艾瑞莎仅引发19.6%的细胞凋亡。此结果表示,类似物18是通过更有效率地引发肺癌细胞的细胞凋亡而造成其细胞存活率降低。
实施例6类似物18抑制癌症细胞生长并增加肺癌细胞的次-G1期
本发明测试类似物18是否通过引发肺癌细胞进行细胞凋亡而抑制该细胞的生长,并进一步探讨类似物18对于肺癌细胞的细胞周期的影响。
(1).步骤1:测试类似物18对肺癌细胞生长的抑制:
将A549细胞株以1×106细胞密度接种于含有完全培养基的60mm培养皿中维持18小时,再以30μM的类似物18或艾瑞莎处理24小时。药物处理完后,以PBS洗涤该细胞,并置换新鲜的RPMI-1640完全培养基且培养2天至6天,最后以血球计(hemocytometer)计算细胞数。
如图7所示,类似物18及艾瑞莎均有抑制A549细胞株生长的功效,且类似物18的效果较艾瑞莎更为显著。
(2).步骤2:测试类似物18对肺癌细胞的细胞周期的影响:
将A549细胞株以7×105细胞密度接种于含有完全培养基的60mm培养皿中培养16至20小时,再分别以0至40μM的类似物18或艾瑞莎在37℃下处理24小时。药物处理完后,将收集细胞并以70%冰冷却乙醇在-20℃下固定过夜。接着,以1500rpm离心5分钟后,将沉淀物在37℃下以4μg/mL的PI溶液(含有1%TritonX-100及50μg/mL的RNase)避光处理30分钟。为了避免细胞凝集,将该细胞溶液经尼龙筛网膜(Becton-Dickinson,SanJose,CA)过滤。最后,通过流式细胞仪分析该样品,并通过ModFitLT软体(Ver.2.0,Becton-Dickinson)自10,000个细胞分析其位于不同细胞周期的百分比。
如图8(A)及(B)以及图9(A)至(D)所示,类似物18并未显著改变A549细胞株的G1、S及G2/M期的部分,但却显著地增加A549细胞株的次-G1期部分(sub-G1),且在以40μM的类似物18处理后,A549细胞株的次-G1部分被提升至66.7%;然而以40μM的艾瑞莎处理者,仅造成A549细胞株的G1部分从60.5%提升至76.1%。此结果表示,类似物18并未改变A549肺腺癌细胞株的细胞周期,次-G1期部分增加表示正在进行细胞凋亡的细胞数增加,亦即类似物18会促使A549细胞株进行细胞凋亡而抑制其生长,且效果较艾瑞莎显著。
实施例7类似物18促进肺癌细胞中凋亡蛋白酶3的活化及PARP的切割
本发明进一步测试类似物18可通过活化肺癌细胞中的凋亡蛋白酶3(caspase3)及促进多聚ADP核糖聚合酶(polyADPribosepolymerase,简称PARP)的切割,而造成其细胞凋亡。
将A549细胞株以1×106细胞密度接种于60mm含有完全培养基的培养皿培养16至20小时。接着分别以0至40μM的类似物18或艾瑞莎在37℃下处理细胞24小时。于药物处理后,以实施例4所提供的方法进行蛋白质萃取并使用特定抗体进行西方墨点法分析,为了确认蛋白质的均等充填及转移,将肌动蛋白用于作为蛋白质充填控制组。
如图10(A)所示,以10至40μM的类似物18处理可显著地引发A549细胞株的凋亡蛋白酶3转化成活化态,此外,类似物18并引发PARP蛋白的切割态;然而,艾瑞莎并未造成凋亡蛋白酶3及PARP的显著改变。如图10(B)及(C)所示,类似物18可显著地增加活化态凋亡蛋白酶3并促使PARP蛋白质的切割,而造成A549细胞株凋亡,相较于在A549细胞株中以艾瑞莎处理者,并无显著改变活化态凋亡蛋白酶3及PARP蛋白质的切割。
实施例8类似物18抑制肺癌细胞中生存素的蛋白质及基因表现
本发明测试类似物18通过抑制肺癌细胞中的生存素的基因及蛋白质表现而引发细胞凋亡。
将A549细胞株以1×106细胞密度接种于60mm含有完全培养基的培养皿培养16至20小时。接着分别以0至40μM的类似物18或艾瑞莎在37℃下处理细胞24小时。于药物处理后,以实施例4所提供的方法进行蛋白质萃取并使用特定抗体进行西方墨点法分析,为了确认蛋白质的均等充填及转移,将肌动蛋白作为蛋白质充填控制组。
另外,将A549细胞株以2×106细胞密度接种于60mm含有完全培养基的培养皿培养16至20小时。接着分别以0至40μM的类似物18或艾瑞莎处理细胞24小时。于药物处理后,利用ZRRNAMiniPrepTM(ZymoResearch,Irvine,CA),依照使用者手册,将细胞的RNA纯化,并以分光光度计测量RNA的浓度。接着,通过含oligo(dT)12-18引物(Invitrogen)的SuperScriptTMIII逆转录酶合成cDNA,以GAPDH作为内控制品而扩增各逆转录样品,DNA热循环机(Mastercyclergradient,Hamburg,Germany)进行逆转录聚合酶连锁反应RT-PCR是使用DNA热循环机(Mastercyclergradient,Hamburg,Germany)进行,该反应试剂如表5所示,其所使用的引物对如表6所示,其反应条件如下:以94℃反应2分钟进行初期变性,随后在94℃反应30秒、56℃反应30秒、72℃反应40秒,进行共30个循环,最后以72℃反应5分钟以进行扩增。的后,将RT-PCR产物以1.2%琼脂糖凝胶(agarosegel)电泳分离,并进行溴化乙锭染色后,以UV透照法(transillumination)观察,最后以DH27-S3相机(Medclub,Taoyuan,Taiwan)拍照保存。
为了更进一步确认类似物18抑制生存素而造成细胞凋亡的影响,依照使用者手册,使用LipofectamineTM2000(Invitrogen)将pCT-GFP2及pCT-GFP-sur8用于转染,该pCT-GFP-sur8构筑载体(会过度表现生存素),并以pCT-GFP2做为控制组(仅过度表现绿色荧光蛋白(greenfluorescentprotein,简称GFP))。将A549细胞株以2×106细胞密度接种于60mm培养皿培养16至20小时。接着,以20μg的pCT-GFP-sur8或pCT-GFP2做为控制组或生存素表现载体在无血清的培养基中,并置于37℃的CO2培养基中进行细胞转染(transfection)8小时。之后,再加入等量含有20%FBS的培养基作用24小时。于转染后,分别依照实施例2及实施例4的方法进行细胞存活率测试及西方墨点法分析。
表5、RT-PCR的反应试剂
表6、PCR所使用的引物
引物名称 | 引物序列 |
生存素正向引物 | 5’-GGCATGGGTGCCCCGACGTTG-3’ |
生存素反向引物 | 5’-CAGAGGCCTCAATCCATGGCA-3’ |
GAPDH正向引物 | 5’-CGGAGTCAACGGATTTGGTCGTAT-3’ |
GAPDH反向引物 | 5’-AGCCTTCTCCATGGTGGTGAAGAC-3’ |
如图11(A)至(D)所示,以类似物18处理A549细胞株,其生存素的基因表现明显地被抑制,且依类似物18处理浓度的增加而呈下降的趋势,而艾瑞莎并未显著地改变生存素的基因表现;此外,类似物18及艾瑞莎均显著抑制生存素的蛋白质表现。此结果表示,类似物18可显著地抑制生存素的基因表现,并造成A549细胞凋亡,且效果较艾瑞莎更佳。
如图12(A)所示,经pCT-GFP-sur8载体转染的A549细胞确实可利用针对GFP及生存素的特定抗体侦测到生存素-GFP融合蛋白质(43.5kDa),而控制组(pCT-GFP2载体)则仅表现GFP(27kDa),另外,A549细胞本身含有的生存素为16.5kDa。如图12(B)所示,利用pCT-GFP-sur8载体在A549细胞中过度表现生存素会增加该细胞的存活率,且转染pCT-GFP-sur8载体可抑制类似物18所引发的细胞凋亡,且其效率较转染pCT-GFP2者更佳。此结果表示,类似物18确实通过抑制生存素而引发肺癌细胞进行细胞凋亡。
实施例9类似物18抑制肺癌肿瘤的形成
本发明测试类似物18在体内实验可抑制小鼠中异体移植人类肺癌细胞的肿瘤的扩大。自BioLASCO(BioLASCO.,Ltd.,Taipei,Taiwan)获得BALB/cAnN.Cg-Foxn1nu/CrlNarl小鼠(三周龄大的公鼠),先进行2周的环境适应后,将2×106的A549细胞以皮下注射注入各小鼠的腹腔中。坚实的A549侧腹肿瘤于10天后形成,然后通过每间隔4天以皮下注射100μl玉米油(控制组小鼠)或30mg/kg的类似物18(实验组小鼠)三次。利用数显卡尺(digitalcaliper),每4天测量该肿瘤大小,并以下列公式计算:(长度)×(宽度)2×0.5。将小鼠牺牲后取出肿瘤,将该肿瘤均质化后且将总裂解物依照实施例4的方法进行西方墨点法分析。
如图13(A)及(B)所示,经注射类似物18的裸鼠的肿瘤大小显著地较控制组为小,详言之,开始每4天注射玉米油至第16天的裸鼠的肿瘤平均大小为406.52mm3,而注射类似物18的裸鼠的肿瘤平均大小为118.19mm3。此外,依图13(C)所示,经注射类似物18的小鼠,其肿瘤中的生存素的蛋白表现量较控制组减少约一半。此结果表示,类似物18可通过抑制生存素的表现而在异体移植人类肺癌细胞的小鼠中抑制肿瘤的扩大。
Claims (10)
1.一种式(I)化合物或其盐,
其特征在于,m为2至7的整数,以及R独立选自由氢及C1-C20烷基所组成组的至少一者。
2.如权利要求1所述的式(I)化合物或其盐,其特征在于,m为2以及R为C1-C13烷基。
3.如权利要求1所述的式(I)化合物或其盐,用以促进癌细胞的细胞凋亡,以抑制癌细胞的生长。
4.如权利要求3所述的式(I)化合物或其盐,其特征在于,所述癌细胞选自由肺癌细胞、直肠癌细胞及膀胱癌细胞所组成组的至少一者。
5.如权利要求1所述的式(I)化合物或其盐,用以抑制表皮生长因子蛋白激酶的活性。
6.一种医药组合物,其包含如权利要求1所述的式(I)化合物或其盐及医药上可接受的载剂。
7.如权利要求6所述的医药组合物,用以促进癌细胞的细胞凋亡,以抑制癌细胞的生长。
8.如权利要求6所述的医药组合物,其特征在于,所述癌细胞选自由肺癌细胞、直肠癌细胞及膀胱癌细胞所组成组的至少一者。
9.一种包含如权利要求6所述的医药组合物的用途,用于制造治疗癌症的药物。
10.如权利要求9所述的用途,其特征在于,所述癌症选自由肺癌、直肠癌及膀胱癌所组成组的至少一者。
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