CN113444085B - 一种具有克服顺铂耐药的抗肿瘤化合物及其制备与应用 - Google Patents
一种具有克服顺铂耐药的抗肿瘤化合物及其制备与应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及有效克服顺铂耐药的抗肿瘤化合物,具体涉及将具有蛋白激酶2(CK2)抑制活性的化合物键合到已知具有抗肿瘤活性的铂(II)化合物中,得到能够有效克服顺铂耐药的新型抗肿瘤化合物;本发明还涉及该类化合物的制备方法及应用。
背景技术
自20世纪60年代发现顺铂具有抗肿瘤活性以来,已有顺铂、卡铂和奥沙利铂等铂类药物上市,它们被广泛应用于临床。经过大量研究,一般认为顺铂类抗肿瘤药物的作用机制是铂(II)化合物与癌细胞DNA上的鸟嘌呤N7位靶点结合,形成DNA交联加合物,限制DNA复制从而抑制癌细胞的增殖。顺铂在目前所有已知的铂类抗肿瘤药物中综合效果是最好的,不仅抗肿瘤活性强,而且具有广谱的抗肿瘤活性,可作为治疗头颈部癌、卵巢癌、食道癌、膀胱癌和肺癌等癌症的一线药物。然而,顺铂在临床应用中也出现了明显的缺陷,一是具有较强的毒副作用,如肾毒性、神经毒性、耳毒性和胃肠道毒性等,限制了顺铂的给药剂量;二是在***过程中易产生耐药,降低药物的抗肿瘤效果。研究发现,在耐药癌细胞中存在着显著的DNA修复现象,这是因为癌细胞可以激活自身的DNA损伤修复机制进行修复,从而对DNA损伤药物产生耐药,降低顺铂类药物的抗肿瘤效果。因此DNA损伤修复被认为是影响以DNA为作用靶点化疗疗效的重要因素。
酪蛋白激酶2(casein kinaseⅡ,CK2),是一种多效且高度保守的第二信使非依赖性丝氨酸/苏氨酸蛋白激酶,广泛存在于真核细胞的细胞质及细胞核中。大量证据表明CK2在多种细胞活动中具有非常重要的作用。CK2的磷酸化底物超过300种,在细胞生长、增殖、凋亡和癌变的过程中发挥重要作用。它调节多条抗凋亡通路及前导信号级联,包括PI3K/AKT信号通路,Wnt信号级联,NF-κB转录和DNA损伤应答。研究表明CK2参与细胞G1/S期、G2/M期的调节,过表达的CK2可以抑制由抗癌药物引起的程序性死亡。实验证明,多种耐药癌细胞系均存在CK2过度表达的现象,而抑制CK2能够促进细胞凋亡并增加肿瘤细胞对抗癌药物和凋亡刺激的敏感度。
综上,在顺铂耐药癌细胞中存在着显著的DNA修复现象,而耐药癌细胞中CK2的过表达是造成顺铂耐药的主要原因之一。因此将具有抑制CK2活性的结构单元引入到具有抗肿瘤活性的铂(II)化合物中,借助其对耐药癌细胞中DNA损伤修复的抑制,克服顺铂药物耐药并降低毒性是十分有益的。
发明内容
技术问题:鉴于顺铂在临床应用中存在严重的毒性和耐药性,本发明所要解决的技术问题在于利用具有抑制蛋白激酶2活性的化合物能够抑制DNA损伤修复的特性,与已知含有功能基团的铂(II)化合物结合,设计合成一种新的铂(II)化合物,获得克服顺铂耐药且高效低毒的抗肿瘤药物,并且提供这种化合物的制备方法及其在抗肿瘤中的应用。
技术方案:本发明所述的具有克服顺铂耐药的抗肿瘤化合物,其结构如式Ⅰ所示的化合物1或化合物2,
式I中的化合物1由式II所示的反应式制备,
式II所示的反应式制备,具体步骤如下:在反应瓶中加入将等摩尔量的化合物3与化合物4悬浮于无水甲醇中,滴加乙酸,反应液在30-60℃下避光反应48-72小时,然后冷至室温,过滤,滤饼用无水甲醇冲洗三次,得黄色固体产物-化合物1。
式I中的化合物2由式III所示的反应式制备,
式Ⅲ所示的反应式制备,具体步骤如下:在反应瓶中加入将等摩尔量的化合物3与化合物5悬浮于无水甲醇中,滴加乙酸,反应液在30-60℃下避光反应48-72小时,然后冷至室温,过滤,滤饼用无水甲醇冲洗三次,得黄色固体产物-化合物2。
应用式II和式Ⅲ中所示的化合物3,其结构如下所示,
式II和式Ⅲ中所示的化合物3由式IV所示的反应式制备,
按照式Ⅳ所示的反应式制备,具体步骤如下:在装有甲醇的反应瓶中加入1当量的化合物6,室温下缓慢滴加2-10当量的水合肼,反应液加热回流24-72小时至反应完全,然后冷至室温,析出大量固体,过滤,滤饼用冰甲醇冲洗三次,得亮黄色固体产物-化合物3。
式II和式Ⅲ中所示的化合物4和5是已知的具有良好抗肿瘤活性的铂(II)化合物,按本发明人前期专利披露的方法制备(发明专利号:ZL201210422936.4;US9227991B2;EP2913335;日本特许第6159818号);式IV中所示的化合物6和已知靶向CK2抗肿瘤药物(CX-4945)均按文献报道的方法制备(J.Med.Chem.,2011,54,635-54),具体是以3-溴异烟酸为起始原料,经酯化反应后与2-氨基-4-甲酯基苯基硼酸盐酸盐发生Suzuki偶联反应得到中间体5,6-二氢苯并[c][2,6]萘啶-8-羧酸甲酯,然后与三氯氧磷发生氯代反应,再与间氯苯胺发生取代反应得到化合物6,然后将其水解得到CX-4945。反应路线如式V所示:
有益效果:
(一)体外CK2活性
采用已知CK2抑制剂CX-4945作为阳性对照,对所制备的化合物1、2和3测定了其体外对CK2的抑制活性,实验数据及结果见表1。结果表明,在CX-4945结构上引入肼基基团获得的化合物3可有效抑制CK2的活性,且活性优于其母体化合物。通过化合物3制备的化合物1和2对CK2的抑制活性相当,其中化合物1的活性还略好于CX-4945,这表明本发明所设计制备的铂(II)化合物具有有效抑制CK2的能力。
(二)体外细胞毒活性
用所制备的化合物1、2和3对人喉癌细胞Hep2、鼻咽癌细胞CNE2、胃癌细胞SGC-7901、结肠癌细胞HCT-116、乳腺癌细胞MCF-7、***细胞SiHa、***癌细胞PC-3、膀胱癌细胞T24、非小细胞肺癌细胞A549及其顺铂耐药癌细胞A549/CDDP、卵巢癌细胞A2780及其顺铂耐药癌细胞A2780/CDDP以及正常肝细胞LO2进行了体外细胞毒活性评价,CX-4945、顺铂和化合物4和5作为阳性对照,化合物在不同浓度下对肿瘤细胞生长抑制的IC50数据见表2。化合物1在测试的各种癌细胞系中,尤其是对已知CK2高表达的癌细胞系如T24、PC-3和HCT-116等的活性均优于CX-4945及其母体化合物3,且与其另一母体化合物4相当,但化合物1对正常肝细胞的毒性低于阳性对照药物。值得注意的是,尽管化合物1对顺铂敏感癌细胞A549和A2780的抑制能力稍低于顺铂,但其对顺铂耐药癌细胞A549/CDDP和A2780/CDDP均有显著抑制作用,抗癌活性显著优于顺铂,耐药因子最低达到0.71(化合物对顺铂耐药癌细胞的IC50值与对顺铂敏感癌细胞的IC50值的比值)。与之相比,化合物2虽然对于A549癌细胞的IC50值略低于化合物1,但其在耐药癌细胞A549/CDDP中的效果要明显弱于化合物1,耐药因子达到1.52。鉴于化合物1及其母体化合物3具有优于CX-4945抑制CK2的活性,同时化合物1对顺铂耐药癌细胞A549/CDDP和A2780/CDDP也表现出较好的抑制作用,对化合物1进行了后续研究。
(三)体外诱导DNA损伤与抑制DNA修复
通过Western Blot试验检测了顺铂敏感A549和A2780癌细胞与对应的顺铂耐药癌细胞A549/CDDP和A2780/CDDP中CK2的表达水平。从图1可知,与敏感癌细胞相比,耐药癌细胞中CK2的表达出现了明显的上调,分别是敏感癌细胞的1.74倍和1.96倍,这与DNA修复增强造成的癌细胞耐药性增加相吻合。进一步采用Western Blot试验考察了所设计化合物对DNA损伤修复相关蛋白表达的影响。如图2所示,在顺铂敏感癌细胞A549和A2780与顺铂耐药癌细胞A549/CDDP和A2780/CDDP中,与DNA修复相关的蛋白如PARP1的表达出现显著下调,与此同时DNA双链断裂的标志物γ-H2A.X的表达显著上调。这些结果表明,本发明所设计合成的化合物1不仅可以通过抑制CK2过表达所导致的DNA修复增强来克服耐药癌细胞的耐药性,而且还可以进一步上调DNA的损伤水平来实现更好的抗肿瘤活性。由于耐药癌细胞中CK2表达水平较敏感癌细胞更高,化合物1在耐药癌细胞中表现出更强的抑制活性。
(四)体内活性
采用顺铂敏感的卵巢癌细胞A2780与顺铂耐药的卵巢癌细胞A2780/CDDP的裸鼠异种移植瘤模型,分别测试了化合物1不同剂量的体内抗肿瘤活性,顺铂为阳性对照。
如表3、图3和图4所示,在顺铂敏感的卵巢癌细胞A2780移植瘤的动物模型中,顺铂的抗肿瘤效果较强,抑瘤率为67.30%。当给予动物相当于顺铂(4mg/kg)等摩尔剂量的化合物1(10mg/kg)时,抑瘤率为73.05%,高于顺铂;当化合物1的给药剂量达到20mg/kg时,抑瘤率达到89.65%;当化合物1的给药剂量增加为40mg/kg时,抑瘤率高达96.61%,说明与化合物1具有很强的体内抗肿瘤活性,呈剂量依赖性。由图5可知,小鼠体重变化表明化合物1对受试动物体重几乎没有影响,具有良好的耐受性。
如表4、图6和图7所示,在顺铂耐药卵巢癌癌细胞A2780/CDDP移植瘤的动物模型中,顺铂的抗肿瘤效果很弱,肿瘤体积及最终肿瘤的重量与不进行治疗的对照组相比相差不大,最终抑瘤率仅为10.97%。当给予动物相当于顺铂(4mg/kg)等摩尔剂量的化合物1(10mg/kg)时,抑瘤率达到67.94%,显著优于顺铂;当进一步提高化合物1的给药剂量为20mg/kg时,抑瘤率可到80.43%;当化合物1的给药剂量增加为40mg/kg时,抑瘤率高达95.32%,说明与化合物1具有很强的体内抗肿瘤活性,呈剂量依赖性。由图8可知,小鼠体重变化表明化合物1对受试动物体重几乎没有影响,具有良好的耐受性。
综上,本发明的化合物对癌细胞具有良好的抑制作用,特别是对顺铂耐药的癌细胞具有优良的抑制作用。体内试验结果表明,化合物1不仅对顺铂敏感的肿瘤而且对顺铂耐药的肿瘤都具有很强的抑制作用,且毒性较小,具有高效低毒的特点,可用于制备抗肿瘤药物。
附图说明
图1a是肺癌细胞A549与顺铂耐药肺癌细胞A549/CDDP、卵巢癌细胞A2780与顺铂耐药卵巢癌细胞A2780/CDDP中的CK2表达;
图1b是肺癌细胞A549与顺铂耐药肺癌细胞A549/CDDP、卵巢癌细胞A2780与顺铂耐药卵巢癌细胞A2780/CDDP中CK2表达的定量数值。
图2a是受试样品对肺癌细胞A549中DNA损伤修复相关蛋白的表达;
图2b是受试样品对顺铂耐药肺癌细胞A549/CDDP中DNA损伤修复相关蛋白的表达;
图2c是受试样品对卵巢癌细胞A2780中DNA损伤修复相关蛋白的表达;
图2d是受试样品对顺铂耐药卵巢癌细胞A2780/CDDP中DNA损伤修复相关蛋白的表达。
图3.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤生长体积变化的影响。
图4.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植肿瘤瘤重的影响。
图5.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤裸鼠体重的影响。
图6.受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤生长体积变化的影响。
图7.受试样品对顺铂耐药卵巢癌细胞A2780/CDDPP裸鼠异种移植肿瘤瘤重的影响。
图8.受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤裸鼠体重的影响。
具体实施方式
下面结合实施例对本发明做进一步详细说明,但本发明的保护范围不仅限于这些实施例。
本发明的一种具有克服顺铂耐药的抗肿瘤化合物其结构如式Ⅰ所示的化合物1或化合物2,
(一)化合物的制备
下面实施例中所有试剂均为分析纯。化合物结构表征所用的核磁共振数据由Bruker ARX-600核磁共振仪测定,内标为TMS;高分辨质谱(LS-MS-ESI)采用Agilent6224TOF LC/MS测定。
本发明中化合物4和化合物5的合成参考之前发表的专利方法(发明专利ZL201210422936.4)制备,经核磁氢谱验证。
化合物4:1H NMR(600MHz,DMSO-d6)δ4.21(s,6H),3.72(s,4H)ppm.
化合物5:1H NMR(600MHz,DMSO-d6)δ5.99(d,J=7.9Hz,2H),5.27(t,J=9.6Hz,2H),3.77-3.66(m,4H),2.08-2.01(m,2H),1.81(d,J=12.7Hz,2H),1.45(d,J=8.3Hz,2H),1.21(dd,J=19.9,11.0Hz,2H),1.06-0.96(m,2H)ppm.
本发明中,化合物6和CX-4945参考文献(J.Med.Chem.2011,54,635-654)所报道的方法制备,经核磁氢谱验证。
化合物6:1H NMR(600MHz,DMSO-d6)δ10.09(s,1H),9.91(s,1H),8.94(d,J=5.6Hz,1H),8.79(d,J=8.5Hz,1H),8.77(d,J=5.6Hz,1H),8.34(t,J=1.9Hz,1H),8.17(dd,J=6.3,1.4Hz,1H),7.90(dd,J=8.4,1.7Hz,1H),7.42(t,J=8.1Hz,1H),7.12(dd,J=7.9,1.4Hz,1H),3.92(s,1H)ppm.
CX-4945:1H NMR(600MHz,DMSO-d6)δ12.04(s,1H),10.30(s,1H),10.16(s,1H),9.08(d,J=5.8Hz,1H),9.00-8.97(m,1H),8.94(d,J=8.5Hz,1H),8.31-8.28(m,2H),8.12(d,J=8.2Hz,1H),8.03(dd,J=8.4,1.5Hz,1H),7.45(t,J=8.1Hz,1H),7.17(d,J=7.9Hz,1H)ppm.
实施例一:化合物1的制备
在50mL反应瓶中将等摩尔量的化合物3与化合物4(2mmol),悬浮于30mL无水甲醇中,加入2-3滴乙酸,30-60℃下避光反应48-72h,TLC监测至化合物3完全反应。冷却过滤,滤饼用30mL(3×10mL)甲醇冲洗,得黄色固体产物,收率96.4%。
1H NMR(600MHz,DMSO-d6)δ11.15(s,1H),10.19(s,1H),9.67(s,1H),8.99(d,J=4.2Hz,1H),8.86(d,J=7.7Hz,1H),8.58(d,J=3.8Hz,1H),8.26(s,1H),8.23(s,1H),8.14(d,J=7.2Hz,1H),7.93(d,J=7.3Hz,1H),7.45(t,J=7.5Hz,1H),7.14(d,J=6.9Hz,1H),4.26(s,6H),3.74(s,4H)ppm.13C NMR(150MHz,DMSO-d6)δ176.45,176.23,163.28,159.72,150.55,148.18,147.80,143.63,142.33,135.12,133.28,130.62,127.50,126.91,124.23,123.84,122.80,122.69,121.73,120.64,119.68,116.83,56.40,49.60,46.96ppm.HR-MS(m/z)(ESI):calcd for C25H23ClN7O5Pt[M+H]+:731.1091;found:731.1353.
实施例二:化合物2的制备
用化合物3和化合物5参照实施例一所示方法制备,得黄色固体产物,收率95.2%。
1H NMR(600MHz,DMSO-d6)δ10.18(s,1H),10.09(s,1H),9.68(s,1H),8.99(d,J=5.5Hz,1H),8.84(d,J=8.4Hz,1H),8.59(d,J=5.4Hz,1H),8.35(s,1H),8.23(s,1H),8.09(d,J=8.1Hz,1H),7.93(d,J=8.2Hz,1H),7.45(t,J=8.0Hz,1H),7.15(d,J=7.7Hz,1H),6.01(d,J=8.0Hz,2H),5.30(m,2H),3.70-3.79(m,4H),2.07(s,2H),1.83(d,J=11.9Hz,2H),1.44-1.46(m,2H),1.22(d,J=8.8Hz,2H),1.02(t,J=9.5Hz,2H)ppm.13C NMR(150MHz,DMSO-d6)δ206.07,176.03,165.83,150.48,148.15,147.72,143.76,142.39,134.71,133.28,130.54,127.52,126.26,124.19,123.01,122.86,122.62,121.48,120.56,119.57,116.82,62.57,56.35,46.86,31.96,24.50ppm.HR-MS(m/z)(ESI):calcd forC31H31ClN7O5Pt[M+H]+:811.1717;found:811.1158.
实施例三:化合物3的制备
于50mL单口瓶中将化合物6(1.09g,3mmol)溶于20mL甲醇中,室温下缓慢滴加水合肼(6-30mmol),加热回流24-72h至完全反应。冷却,析出大量固体,抽滤,滤饼用3×10mL冰甲醇冲洗,得亮黄色固体产物1.05g,收率97.1%。1H NMR(600MHz,DMSO-d6)δ10.14(s,1H),10.08(s,1H),9.63(s,1H),8.95(d,J=6.0Hz,1H),8.80(d,J=12.0Hz,1H),8.55(d,J=6.0Hz,1H),8.33(t,J=1.8Hz,1H),8.21(d,J=1.6Hz,1H),8.07(d,J=6.0Hz,1H),7.91(dd,J=12.0,1.6Hz,1H),7.42(t,J=6.0Hz,1H),7.12(dd,J=8.0,1.4Hz,1H),4.63(s,2H)ppm.13C NMR(150MHz,DMSO-d6)δ165.77,150.47,148.13,147.69,143.76,142.40,134.73,133.35,130.52,127.52,126.26,124.17,123.00,122.83,122.60,121.47,120.57,119.56,116.79ppm.HR-MS(m/z)(ESI):calcd for C19H14ClN5O[M+H]+:364.0965;found:364.0967.
(二)抑制CK2活性的测试
实验方法:采用CK2酶联免疫分析(ELISA)试剂盒进行检测。首先取标准品蛋白稀释至合适浓度后,加入包被纯化的CK2捕获抗体的微孔板中,再加入已稀释至合适浓度梯度的待测化合物,并设置不加药的对照组进行对比。放入孵箱孵育1h,使药物有效抑制蛋白与抗体的结合后,将微孔板中的液体弃去,加入200μL已稀释好的洗涤液,静置30s后弃去并重复洗涤5次。加入100μL的HRP标记的酶标试剂,孵箱孵育30min,使其形成抗体-抗原-酶标抗体复合物。再将微孔板中的液体弃去,加入200μL已稀释好的洗涤液,静置30s后弃去,并重复洗涤5次。接下来每孔先加入显色剂A 50μL,再加入显色剂B 50μL,轻轻震荡混匀,37℃避光显色15min,使底物TMB在HRP酶的催化下转化成蓝色。最终加入50μL终止液,使TMB在酸的作用下转化成最终的黄色,颜色的深浅和CK2的活性呈正相关。在15min以内,通过酶标仪在450nm波长依序测量各孔的吸光度(OD值),最终得到待测化合物相对应的CK2抑制活性。
采用上述方法对化合物1、2和3抑制CK2的活性进行了测试,以CX-4945作为阳性对照,结果见表1。
表1.化合物对CK2的抑制活性
(三)体外细胞毒活性测试
实验方法:采用MTT方法对本发明所制备的化合物进行了细胞毒活性测试。取对数生长期的细胞计数,接种于96孔培养板内,每孔约104个细胞,于37℃,5%CO2孵箱培养24h至细胞贴壁后给药,分别设给药组和不给药的对照组。对照组更换新鲜培养基,待测的化合物4和5及顺铂用5%的葡萄糖水溶液溶解,其它化合物用DMSO配制成母液,临用前用对应的细胞培养基稀释成合适的浓度梯度加入给药组,每个浓度设3个平行组。加药后于37℃,5%CO2孵箱培养72h,加10μL浓度为5mg/mL的MTT,37℃孵育4h后,弃去上清,加入130μL的DMSO溶解甲瓒。用酶标仪在490nm波长下测定每孔的OD值,并计算IC50值。
采用上述方法,用所制备的化合物对人喉癌细胞Hep2、鼻咽癌细胞CNE2、胃癌细胞SGC-7901、结肠癌细胞HCT-116、乳腺癌细胞MCF-7、***细胞SiHa、***癌细胞PC-3、膀胱癌细胞T24、非小细胞肺癌A549及其顺铂耐药癌细胞A549/CDDP、卵巢癌细胞A2780与顺铂耐药癌细胞A2780/CDDP以及正常肝细胞LO2进行了体外抗肿瘤活性评价,CX-4945作为阳性对照。观察化合物在不同浓度下对肿瘤细胞生长的抑制情况,计算其IC50值来评价药物的细胞毒活性,结果见表2。
表2.化合物对所测细胞的细胞毒活性
*.nd:没有测定。
(四)体外诱导DNA损伤及抑制DNA修复的测试
实验方法:采用Western Blot试验对本发明所涉及的化合物进行了检测。取对数生长期的细胞计数,接种于6孔培养板内,每孔约105个细胞。于37℃,5%CO2孵箱培养24h至细胞贴壁后给药,分别设给药组和不给药的对照组。对照组更换新鲜培养基,待测的化合物4用5%的葡萄糖水溶液溶解,其它化合物用DMSO配制成母液,临用前用相对应的细胞培养基稀释成合适的浓度加入给药组,加药后于37℃,5%CO2孵箱培养24h,将孔中的细胞消化,离心收集至离心管中并用PBS洗涤后,向细胞中滴加适量裂解液,于冰盒中裂解细胞2h。10000rpm离心20~30min,取上清液待用。用BCA蛋白试剂盒(Thermo,Waltham,MA)测定蛋白含量。
用去离子水清洗玻璃板,晾干。再将洗净的玻璃板安装在凝胶架上并检漏。再分别配制好10%的分离胶和15%的积层胶。先用向玻璃板中加入分离胶至1/2界面处,再沿着玻璃板加入去离子水封胶,直至水满溢出。等待20min分离胶固化之后,倒掉去离子水,吸干。接着加入积层胶至玻璃板上界面,***梳子等待30min后积层胶凝固。将玻璃板取下,用纯净水冲洗干净转移放入电泳槽中,加入少许缓冲液进行检漏,再向电泳槽中缓慢倒入缓冲液至满。缓慢拔除梳子,然后用受试样品依次加入到各个孔内,用60V电压进行电泳。电泳结束后将胶板取出计算条带的剪切区域。再进行转膜操作,将剪切的条带放入半干转膜缓冲液中,将电泳仪电压调至25V,室温下转膜50min。转膜完成后,从半干转膜仪中移出条带,放入丽春红溶液中进行染色,观察转膜是否成功,再放入去离子水中清洗,接着用PBS洗涤条带3次,每次8min。将条带置于培养皿中,注入30mL牛奶液(用PBST溶),于37℃孵箱中,振荡1h后用PBST洗涤3次,每次8min。向封口膜中注入一抗,封装,置于4℃冰箱中振荡过夜。弃去一抗,将膜转移至PBST溶液中振荡清洗3次,每次8min。加入二抗,封装,室温振荡孵育1h。弃去二抗,PBST溶液振荡清洗3次,每次8min,再用PBS洗涤一次,5min后将条带取出置于干净的玻璃板上待用。配制AB发光液(A:B=1:1),滴加于条带上,作用约10s左右,去除多余的发光液后盖上薄膜,并将胶片覆于薄膜上,用手按压70s,置于预热好的曝光机中曝光。胶片洗出,观察条带。该过程需要避光操作。
采用上述方法,首先测试了癌细胞A549和A2780与对应的顺铂耐药癌细胞A549/CDDP和A2780/CDDP中CK2的表达水平,实验结果见图1;其次测试了所涉及化合物对DNA损伤修复相关蛋白表达的影响,以顺铂为阳性对照,实验结果见图2。
(五)体内抗肿瘤活性测试
受试动物:BALB/c(nu/nu)裸鼠,体重16~18g,雌性,购自上海西普尔-必凯实验动物有限公司,饲养于SPF级饲养环境中,室内温度控制在23±2℃,自由饮食和摄水。动物总数20只。接种前适应性饲养7天。药物及试剂:顺铂用5%葡萄糖注射液超声溶解,化合物1用DMF溶解后,依次用Tween80和5%葡萄糖注射液稀释。组别及给药方案如下。模型对照组:尾静脉注射0.1mL 5%葡萄糖注射液,每周1次,连续4周。顺铂(4mg/kg):尾静脉注射0.1mL顺铂(剂量为4mg/kg),每周1次,连续4周。化合物1(10mg/kg):尾静脉注射0.1mL化合物1(剂量为10mg/kg),每周1次,连续4周。化合物1(20mg/kg):尾静脉注射0.1mL化合物1(剂量为20mg/kg),每周1次,连续4周。化合物1(40mg/kg):尾静脉注射0.1mL化合物1(剂量为40mg/kg),每周1次,连续4周。
实验方法:取对数生长期A2780或A2780/CDDP细胞,制备细胞悬液浓度为5×107cell/mL,裸鼠右腋下接种0.2mL/只,建立A2780或A2780/CDDP裸鼠移植瘤模型,裸小鼠移植瘤用游标卡尺测量移植瘤直径,待肿瘤生长至100mm3后将动物随机分组。使用测量瘤径的方法,动态观察被试物抗肿瘤的效应。肿瘤直径的测量次数为每3天测1次,同时每3天检查一次小鼠体重。4周后,注射水合氯醛处死所有小鼠,剖腹观察,摘除肿瘤,仔细剔除无关组织,用D-Hanks液洗涤2~3次,洗去血液,沥干水分保存,称取重量和测量体积。肿瘤体积(tumor volume,TV)的计算公式为:TV=1/2×a×b2,其中a、b分别表示长宽。
据测量的结果计算出相对肿瘤体积(relative tumor volume,RTV),计算公式为:RTV=Vt/V0,其中V0为分笼给药时(即d0)测量所得肿瘤体积,Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标:相对肿瘤增殖率T/C(%),计算公式如下:
其中,TRTV:治疗组RTV;CRTV:模型组RTV。
抗肿瘤活性的评价指标:肿瘤生长抑制率(%),计算公式如下:
均值用X±SD表示,组间分析用t检验进行统计学处理,应用SPSS(StaffsticalPackage for the Social Science)17.0对结果进行统计分析。
有关受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤生长的抑制作用结果见表3、图3和图4,受试动物的体重变化见图5。有关受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤生长的抑制作用结果见表4、图6和图7,受试动物的体重变化见图8。
表3.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤生长的抑制作用
表4.受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤生长的抑制作用
Claims (5)
5.一种如权利要求1所述的一类具有克服顺铂耐药的抗肿瘤化合物的应用,其特征在于,用于制备抗肿瘤药物。
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