CN114957219B - 一种靶向降解Gli1蛋白的降解剂及其制备方法和应用 - Google Patents
一种靶向降解Gli1蛋白的降解剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种靶向降解Gli1蛋白的降解剂及其制备方法和应用，该靶向降解Gli1蛋白的降解剂包括Gli1降解剂化合物或其药学上可接受的盐、异构体、外消旋体、水合物或前体药物；Gli1降解剂化合物由式(I)所示结构与降解招募子通过共价连接得到，降解招募子为可招募泛素化的分子。本发明制备的靶向降解Gli1蛋白的降解剂不但能够一定程度上解决已有的Hh通路中SMO拮抗剂耐药性问题，还能够开发出新型Hh/Gli1调节剂，研究Gli1蛋白被化学敲低所带来的生理学效应，为治疗Hh/Gli1介导的癌症提供新的方法。
Description
技术领域
本发明涉及化学医药技术领域，尤其涉及一种靶向降解Gli1蛋白的降解剂及其制备方法和应用。
背景技术
Hedgehog(Hh)信号传导是人体生长发育的必需途径，在胚胎发育和组织维持更新中起重要作用。通过无序点突变或其他机制激活Hh信号传导通路与各种实体和血液恶性肿瘤的发生和进展密切相关。精准的Hh浓度以及信号持续时间对于我们体内许多组织的正确发育，规格和模式至关重要，并且在发育过程中未能执行适当的Hh信号转导与严重的胚胎畸形(如全脑畸形)有关。Hh信号传导途径，一般在伤口愈合和组织修复期间起作用，正常时在成人中处于休眠状态。然而，Hh信号通路的异常激活与不同类型的癌症的发展和进展有关：包括基底细胞癌(BCC)，横纹肌肉瘤(RMS)，髓母细胞瘤(MB)，胶质细胞瘤，黑色素瘤，白血病，淋巴瘤和食道、肺、胃、胰腺、肝细胞、结直肠、前列腺和卵巢癌。
虽然经典的Hh/Gli1通路拮抗剂已被批准用于临床(Vismodegib、Sonidegib和Glasdegib)，但这些药物主要靶向该通路上游中G蛋白偶联受体Smoothened(SMO)，对由Gli1激活的下游机制引起的癌症无效，并且SMO易于突变导致获得性耐药(如人SMO的473残基(D473H)的错义突变，它会干扰vismodegib与SMO的结合，降低治疗效果)。而Gli1小分子调节剂如：GANT58、GANT6和三氧化二砷等直接或间接调节Gli1活性的抑制剂正在开发中，其中许多已在依赖Gli1的癌症动物模型中显示出初步的治疗前景。Gli1作为Hh通路的靶基因和转录调节因子，无论发生Gli1的经典激活还是非经典激活，它都是最有希望成为广谱癌症的治疗干预靶标。但Gli1作为转录因子，蛋白表面光滑，很少有能与小分子结合的口袋，现有的Gli1小分子抑制剂效能低，成药性不足。因此，仍需寻找一种新的靶向Gli1的小分子降解剂。
靶向蛋白降解(Targeted protein degradation,TPD)，是通过设计的双功能小分子或者人为地对目标蛋白(protein of interest,POI)进行修饰，达到诱导POI通过蛋白酶体系统降解的技术，主要包括蛋白降解靶向双功能分子(Proteolysis-targetingchimeras,PROTAC)、疏水性标签(Hydrophobic tagging,HyT)和N-端(N-end)规则。PROTACs分子能同时招募POI和E3泛素酶，进而形成三元复合物后诱导POI被泛素化，最后被蛋白酶体降解。HyT能够模拟细胞内未成功折叠蛋白，会被伴侣蛋白或热休克蛋白识别修复，而修复失败的会被送到蛋白酶体降解。N-端规则是采用单个氨基酸模仿蛋白质末端氨基酸减少蛋白质半衰期。相比较于传统的小分子抑制剂通过结合于靶蛋白的活性位点抑制靶蛋白的功能的作用机制，靶向蛋白降解可直接将靶蛋白降解，从而达到疾病治疗的目的。由于其对靶向蛋白小分子配体的亲和力要求不高，且只需要很低浓度便能发挥蛋白降解功能，使得此技术能很好地克服现有药物的严重耐药性问题，并且可针对难以靶向的蛋白如：转录因子、磷酸酶和细胞支架蛋白等。目前尚未有将靶向蛋白降解策略引入到Hedgehog信号通路调节剂的设计合成中的报道。
发明内容
为解决上述技术问题，本发明的目的是提供一种通过靶向蛋白降解技术设计的靶向降解Gli1蛋白的降解剂，该Gli1小分子降解剂不但能够一定程度上解决已有的Hh通路中SMO拮抗剂耐药性问题，还能够开发出新型Hh/Gli1调节剂，研究Gli1蛋白被化学敲低所带来的生理学效应，为治疗Hh/Gli1介导的癌症提供新的方法。
本发明的一种靶向降解Gli1蛋白的降解剂，包括Gli1降解剂化合物或其药学上可接受的盐、异构体、外消旋体、水合物或前体药物；所述Gli1降解剂化合物由式(I)所示结构与降解招募子通过共价连接得到，其中，
R1选自—OCH3、—N(CH3)2；
所述降解招募子为可招募泛素化的分子。
进一步地，降解招募子选自CRBN配体、VHL配体、MDM2配体或疏水标签，CRBN配体、VHL配体、MDM2配体和疏水标签的结构分别如式(II)-(V)所示，
进一步地，所述共价连接为以非线性链、脂肪族链、芳香链或杂芳环结构链作为连接臂进行连接，连接臂可选自如下结构中的一种：
其中，n为1-10的整数。
进一步地，所述Gli1降解剂化合物选自如下结构中的一种或几种：
本发明中，根据Gli1降解剂化合物的制备方法，将上述Gli1降解剂化合物分为系列I Gli1降解剂化合物、系列II Gli1降解剂化合物、系列III Gli1降解剂化合物、系列IVGli1降解剂化合物。其中，系列I Gli1降解剂化合物中，连接臂为n＝1-10的烷基或者烷氧基链，降解招募子为上述CRBN配体；系列II Gli1降解剂化合物中，连接臂为n＝1-10的烷基或者烷氧基链，降解招募子为上述VHL配体；系列III Gli1降解剂化合物中，连接臂为n＝1-10的烷基或者烷氧基链，降解招募子为上述MDM2配体；系列IV Gli1降解剂化合物中，连接臂为n＝1-10的烷基或者烷氧基链，降解招募子为上述HyT配体。
进一步地，Gli1降解剂化合物可以含有不对称或手性中心，因此可以以不同立体异构形式存在。本发明化合物的所有立体异构形式，包括但不限于非对映异构体、对映异构体和阻转异构体以及他们的混合物(如外消旋物)。
进一步地，Gli1降解剂化合物还可以以不同互变异构形式存在，属于“互变异构体”或“互变异构形式”是指经由低能垒相互转化的不同能量的结构异构体。
进一步地，药学上可接受的盐包括与下列酸形成的加成盐：盐酸、氢溴酸、硫酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘二磺酸、乙酸、丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、富马酸、草酸、酒石酸、苯甲酸等。盐酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、三氟乙酸、马来酸、苯磺酸、琥珀酸以及类似的已知可以接受的酸成盐。
进一步地，前体药物是Gli1降解剂化合物的衍生物，它们自身可能具有较弱的活性甚至没有活性，但是在给药后，在生理条件下(例如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。
本发明的Gli1降解剂化合物的制备方法包括以下步骤：
S1：化合物A与一端含有离去基团(卤素(如Br)或者对甲苯磺酰基)另一端含有保护基的Linker(连接臂)在碳酸钾、碘化钾条件下于乙腈反应后再脱去保护基得到化合物B；
S2：化合物B通过酰胺缩合剂或者亲核取代与降解招募子反应得到化合物C；
S3：化合物C与(3-(((苄氧基)羰基)氨基)丙基)(2-(二甲氨基)苄基)氨基甲酸叔丁酯或(3-氨基丙基)(4-甲氧基苄基)氨基甲酸叔丁酯通过还原胺化反应最终脱去保护基，得到所述Gli1降解剂化合物；
其中，R1和n的定义如上所述，合成路线示意图如下所示：
本发明的靶向降解Gli1蛋白的降解剂可用于制备Hh/Gli1调节剂。
本发明还提供了一种抗肿瘤药物，该抗肿瘤药物中包括上述靶向降解Gli1蛋白的降解剂。
进一步地，所述肿瘤为实体肿瘤或血液系统恶性肿瘤，包括但不限于多发性骨髓瘤、胃癌、肺癌、乳腺癌、食管癌、结肠癌、髓母细胞瘤、急性粒细胞白血病、慢性白血病、前列腺癌、肝细胞瘤、肾细胞瘤、宫颈癌、皮肤癌、卵巢癌、结肠癌、神经胶质瘤、甲状腺癌、胰腺癌等。
本发明可以上述Gli1降解剂化合物及其药学上可接受的盐、水合物作为活性成份，与药学上可接受的赋形剂混合制备成组合物，并制备成临床上可接受的剂型，上述赋形剂是指可用于药学领域的稀释剂、辅助剂或载体。上述剂型是指临床上常用的注射剂、片剂、胶囊剂等。
进一步地，所述抗肿瘤药物中，靶向降解Gli1蛋白的降解剂可作为唯一的抗肿瘤药物单独使用，也可以与肿瘤治疗药物联合使用，用于治疗或预防肿瘤。
借由上述方案，本发明至少具有以下优点：
1、本发明提供了一种靶向降解Gli1蛋白的降解剂，将靶向蛋白降解策略首次引入到Hedgehog信号通路调节剂的设计合成中。
2、鉴于Gli1作为转录因子难以用小分子抑制剂靶向干预并且现有抑制剂活性较弱，因此设计开发靶向Gli1的小分子降解剂，不但能够一定程度上解决已有的Hh通路中SMO拮抗剂耐药性问题，还能够开发出新型Hh/Gli1调节剂，研究Gli1蛋白被化学敲低所带来的生理学效应，为治疗Hh/Gli1介导的癌症提供新的方法。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细说明如后。
附图说明
图1为化合物II-4，IV-5处理过HT29细胞24h后Gli1蛋白含量变化图；
图2为化合物II-4，IV-5处理的HT29细胞的流式细胞实验结果；
图3为qPCR测试化合物II-4和IV-5对耐药细胞模型MEFSUFU-/-细胞中Hh靶基因的影响。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
本发明中，化合物的结构通过核磁共振(1H-NMR)和/或质谱(MS)来确定。NMR测定是用ACF-300BRUK型核磁共振仪，测定溶剂为氘代氯仿(CDCl3)或氘代二甲亚砜(DMSO-D6)，TMS为内标。MS的测定用HP1100型质谱仪。柱层析采用200-300目硅胶(青岛海洋化工厂生产)。在Waters SQ Detector 2上进行了电喷雾电离(ESI)质谱(MS)分析。通过WatersACQUITY UPLC使用反相色谱柱(BEH，C18，1.7μm，2.1×50mm2)测定的所有最终化合物的纯度，溶剂A的溶剂梯度(H2O含0.1％TFA)和溶剂B(含0.1％TFA的CH3CN)。使用SCW X-4B显微熔点仪测量所有熔点。
实施例1
制备4-((1-(4-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)丁酰基)哌啶-4-基)氨基)-2-(2,6-二氧哌啶-3-基)异二氢吲哚-1,3-二酮(I-1)，其结构式如下：
通过下式i、ii、iii所示反应合成化合物I-1。
步骤如下：
(i)对羟基苯甲醛(1.5g,12.28mmol,1eq)和碳酸钾(2.55g,18.42mmol,1.5eq)室温下在5mL乙腈中搅拌30分钟后，加入4-溴丁酸甲酯(2.22g,12.28mmol,1eq)70℃反应5小时后，TLC监测反应结束后，用DCM萃取后，无水硫酸钠干燥后减压旋去溶剂后，加入5mL纯净水和5mL甲醇以及氢氧化锂(2.21g,50mmol,5eq)，rt反应过夜后，旋去大部分溶剂后，用盐酸调pH到3左右后，抽滤得白色固体2.1g，产率80％。
1H NMR(400MHz,DMSO-d6)δ12.18(s,1H),9.86(s,1H),7.89–7.81(m,2H),7.16–7.07(m,2H),4.10(t,J＝6.4Hz,2H),2.39(t,J＝7.3Hz,2H),1.97(p,J＝6.9Hz,2H).m.p.＞200℃.
(ii)中间体酸(61mg，0.339mmol,1eq)和HATU(193mg，509mmol,1.5eq)以及DIPEA(156mmL,0.849mmol,2.5eq)加入到25mL反应瓶后，加入3mL DMF室温搅拌5分钟后，再加入中间体I-1a(132mg,0.339mmol,1eq)，室温反应12小时后，往反应液中加入50mL EA后，有机层分别用饱和碳酸氢钠、氯化铵以及饱和氯化钠溶液洗涤后，无水硫酸钠干燥后，残留物通过硅胶柱色谱纯化(二氯甲烷：甲醇＝10:1)得到白色固体80mg，产率43％，
1H NMR(400MHz,Chloroform-d)δ9.91(s,1H),8.11(s,1H),7.90–7.85(m,2H),7.49(dd,J＝8.5,7.1Hz,1H),7.12–7.01(m,3H),6.87(d,J＝8.5Hz,1H),6.52(s,1H),6.21(t,J＝5.6Hz,1H),4.91(dd,J＝12.2,5.3Hz,1H),4.57(s,2H),3.36(q,J＝7.2,6.1Hz,2H),3.25(q,J＝6.9,5.6Hz,2H),2.93–2.68(m,3H),2.18–2.09(m,1H),1.66(dd,J＝14.7,7.3Hz,2H),1.59–1.51(m,2H),1.46–1.35(m,4H).m.p.＞200℃.
(iii)将上一步得到的醛中间体(80mg,0.145mmol,1eq)和(3-氨基丙基)(2-(二甲氨基)苄基)氨基甲酸叔丁酯(50mg，0.145mmol,1eq)一起加入到10mL DCE中，滴加几滴醋酸和分子筛后，室温搅拌5分钟后，加入三乙酰氧基硼氢化钠(77mg,0.363mmol,3eq)室温反应12小时后，抽滤反应液后，减压旋去溶剂后，加入三氟醋酸1mL和二氯甲烷1mL后，室温反应2小时后，TLC监测反应结束后，加入碳酸氢钠溶液后，DCM/甲醇萃取后，无水硫酸钠干燥后，残留物通过硅胶柱色谱纯化(二氯甲烷：甲醇＝10:1)得到无色油状物40mg，产率40％
1H NMR(400MHz,DMSO-d6)δ7.60(dd,J＝8.6,7.1Hz,1H),7.34(dd,J＝7.6,1.7Hz,1H),7.25–7.16(m,5H),7.09–7.04(m,2H),6.98(td,J＝7.4,1.3Hz,1H),6.89–6.83(m,2H),6.25(d,J＝8.4Hz,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.32(d,J＝13.1Hz,1H),3.97(t,J＝6.5Hz,2H),3.85(td,J＝11.5,9.0,3.5Hz,4H),3.75(s,2H),3.64(s,2H),3.23–3.15(m,2H),2.95–2.75(m,3H),2.60(s,11H),2.48–2.43(m,1H),2.06–1.88(m,4H),1.61(p,J＝6.9Hz,2H).UPLC-MS(ESI)for C41H52N7O6[M+H]+calculated 738.39,obtained 738.05.
实施例2
制备4-((1-(6-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)己酰基)哌啶-4-基)氨基)-2-(2,6-二氧哌啶-3-基)异二氢吲哚-1,3-二酮(I-2)，其结构式如下：
参照实施例1中的方法制备目标化合物，产物为淡黄色油状物32mg，产率21％。
1H NMR(400MHz,Chloroform-d)δ7.48(dd,J＝8.5,7.2Hz,1H),7.28–7.24(m,1H),7.23–7.14(m,3H),7.09(dd,J＝7.2,2.2Hz,2H),7.00(td,J＝7.4,1.3Hz,1H),6.89(d,J＝8.5Hz,1H),6.81(d,J＝8.2Hz,2H),6.24(d,J＝8.0Hz,1H),4.87(dd,J＝12.2,5.2Hz,1H),4.42(d,J＝13.6Hz,1H),3.94(t,J＝6.4Hz,2H),3.88–3.78(m,6H),3.68(s,2H),3.22(t,J＝12.1Hz,1H),2.94(ddd,J＝13.9,10.9,3.2Hz,1H),2.80(ddd,J＝17.9,10.4,3.1Hz,2H),2.75–2.64(m,10H),2.41–2.33(m,2H),2.09–2.04(m,2H),1.79(p,J＝7.0Hz,2H),1.76–1.66(m,4H),1.57–1.43(m,4H).UPLC-MS(ESI)for C43H56N7O6[M+H]+calculated 766.42,obtained 766.01.
实施例3
制备4-((1-(8-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)辛酰基)哌啶-4-基)氨基)-2-(2,6-二氧哌啶-3-基)异二氢吲哚-1,3-二酮(I-3)，其结构式如下：
参照实施例1中的方法制备目标化合物，产物为淡黄色油状物26mg，产率18％。
1H NMR(400MHz,Chloroform-d)δ7.49(dd,J＝8.5,7.1Hz,1H),7.30–7.26(m,1H),7.24–7.14(m,3H),7.13–7.06(m,2H),7.01(td,J＝7.4,1.3Hz,1H),6.90(d,J＝8.5Hz,1H),6.84–6.79(m,2H),6.24(d,J＝7.9Hz,1H),4.88(dd,J＝12.1,5.2Hz,1H),4.43(d,J＝13.6Hz,1H),3.93(t,J＝6.5Hz,2H),3.85(s,2H),3.69(s,2H),3.53–3.38(m,4H),3.23(ddd,J＝14.0,10.9,2.9Hz,1H),2.95(ddd,J＝13.7,10.9,2.9Hz,1H),2.88–2.80(m,1H),2.80–2.71(m,1H),2.71–2.64(m,10H),2.40–2.31(m,2H),2.13–2.02(m,2H),1.82–1.69(m,4H),1.63(q,J＝7.3Hz,2H),1.54–1.43(m,4H),1.41–1.35(m,4H).13C NMR(101MHz,DMSO-d6)δ172.79,171.96,170.51,170.04,169.03,167.18,159.08,145.30,136.36,132.21,131.73,130.85,125.55,123.53,121.33,117.77,114.38,110.88,109.34,67.50,54.94,49.39,48.55,46.64,45.39,44.30,43.64,43.35,32.27,31.37,30.97,28.75,28.61,25.42,24.82,22.19,21.09,13.95.UPLC-MS(ESI)for C45H60N7O6[M+H]+calculated794.45,obtained 794.30.
实施例4
制备4-((1-(10-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)癸酰基)哌啶-4-基)氨基)-2-(2,6-二氧哌啶-3-基)异二氢吲哚-1,3-二酮(I-4)，其结构式如下：
参照实施例1中的方法制备目标化合物，产物为黄色油状物28mg，产率19％。
1H NMR(400MHz,Chloroform-d)δ7.49(dd,J＝8.5,7.1Hz,1H),7.30–7.24(m,1H),7.24–7.14(m,3H),7.12–7.07(m,2H),7.01(td,J＝7.4,1.3Hz,1H),6.90(d,J＝8.5Hz,1H),6.85–6.79(m,2H),6.24(d,J＝7.9Hz,1H),4.88(ddd,J＝12.2,5.5,2.1Hz,1H),4.42(d,J＝13.3Hz,1H),3.92(t,J＝6.5Hz,2H),3.85(s,2H),3.69(s,2H),3.54–3.39(m,4H),3.22(ddd,J＝13.9,10.8,2.8Hz,1H),2.95(ddd,J＝13.8,10.8,3.0Hz,1H),2.89–2.81(m,1H),2.77(dd,J＝12.4,3.9Hz,1H),2.71–2.64(m,10H),2.33(td,J＝7.4,2.8Hz,2H),2.13–2.04(m,2H),1.81–1.69(m,4H),1.61(q,J＝7.3Hz,2H),1.55–1.40(m,4H),1.38–1.30(m,8H).13CNMR(101MHz,DMSO-d6)δ172.79,170.52,170.04,169.03,167.19,159.08,145.30,136.36,132.94,132.21,131.74,130.94,125.47,123.53,121.42,117.78,114.37,110.89,109.34,67.51,54.94,49.38,48.57,48.54,46.77,45.36,44.34,43.65,43.35,34.19,32.30,32.09,31.38,30.95,28.92,28.85,28.80,28.75,28.63,25.50,24.89,22.19,22.14,22.05,13.96.UPLC-MS(ESI)for C47H64N7O6[M+H]+calculated 822.48,obtained 822.60.
实施例5
制备4-((2-(2-(4-(((3-((2(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙氧基)乙基)氨基)-2-(2,6-二氧代哌啶-3-基)异二氢吲哚-1,3-二酮(I-5)，其结构式如下：
通过下式i、ii、iii所示反应合成化合物I-5。
步骤如下：
(i)对羟基苯甲醛(1.5g,12.28mmol,1eq)和碳酸钾(2.55g,18.42mmol,1.5eq)室温下在5mL乙腈中搅拌30分钟后，加入(2-(2-溴乙氧基)乙基)氨基甲酸叔丁酯(3.56g,12.28mmol,1eq)70℃反应5小时后，TLC监测反应结束后，用DCM萃取后，无水硫酸钠干燥后减压旋去溶剂后，加入2mL三氟醋酸和2mL二氯甲烷，rt反应过夜后，旋去大部分溶剂后可直接用于下一步反应，产率70％。
(ii)中间体胺(250mg，0.25mmol,1eq)DIPEA(320mmL,0.65mmol,2.3eq)加入到25mL反应瓶后，加入3mL DMF，最后加入4-氟沙利度胺(128mg，0.21mmol,0.8eq)氮气保护下，90℃反应12小时后，往反应液中加入50mL EA后，有机层分别用饱和碳酸氢钠、氯化铵以及饱和氯化钠溶液洗涤后，无水硫酸钠干燥后，残留物通过硅胶柱色谱纯化(二氯甲烷：甲醇＝10:1)得到黄色油状物152mg，产率35％。
1H NMR(400MHz,Chloroform-d)δ9.86(s,1H),7.80(d,J＝8.8Hz,2H),7.46(dd,J＝8.6,7.1Hz,1H),7.08(dd,J＝7.2,0.6Hz,1H),7.03–6.98(m,2H),6.91(d,J＝8.5Hz,1H),6.49(t,J＝5.7Hz,1H),4.92–4.85(m,1H),4.25–4.20(m,2H),3.90–3.86(m,2H),3.79(t,J＝5.3Hz,2H),3.49(q,J＝5.5Hz,2H),2.77–2.69(m,2H),2.14–2.05(m,2H).m.p.＞200℃.
(iii)将上一步得到的醛中间体(152mg,0.2mmol,1eq)和(3-氨基丙基)(2-(二甲氨基)苄基)氨基甲酸叔丁酯(75mg，0.245mmol,1eq)一起加入到10mL DCE中，滴加几滴醋酸和分子筛后，室温搅拌5分钟后，加入三乙酰氧基硼氢化钠(120mg,0.45mmol,2eq)室温反应12小时后，抽滤反应液后，减压旋去溶剂后，加入三氟醋酸1mL和二氯甲烷1mL后，室温反应2小时后，TLC监测反应结束后，加入碳酸氢钠溶液后，DCM/甲醇萃取后，无水硫酸钠干燥后，残留物通过硅胶柱色谱纯化(二氯甲烷：甲醇＝10:1)得到无色油状物52mg，产率25％
1H NMR(400MHz,Chloroform-d)δ7.45(dd,J＝8.6,7.1Hz,1H),7.28–7.24(m,1H),7.24–7.14(m,3H),7.12–7.05(m,1H),7.01(td,J＝7.4,1.3Hz,1H),6.92–6.82(m,3H),6.54(t,J＝5.7Hz,1H),4.84(dd,J＝12.4,5.3Hz,1H),4.15–4.11(m,2H),3.85(d,J＝3.9Hz,4H),3.78(dt,J＝10.4,5.6Hz,4H),3.69(d,J＝1.7Hz,2H),3.46(q,J＝5.4Hz,2H),2.84–2.71(m,3H),2.70(s,0H),2.69–2.62(m,10H),2.09–2.01(m,1H),1.73(p,J＝6.8Hz,2H).UPLC-MS(ESI)for C36H45N6O6[M+H]+calculated 657.33obtained 657.20.
实施例6
制备4-((2-(2-(2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙氧基)乙氧基)乙基)氨基)-2-(2,6-二氧哌啶-3-基)异二氢吲哚-1,3-二酮(I-6)，其结构式如下：
参照实施例5中的方法制备目标化合物，产物为黄色油状物25mg，产率23％。
1H NMR(400MHz,Chloroform-d)δ7.46(dd,J＝8.6,7.1Hz,1H),7.25(d,J＝4.4Hz,1H),7.22(dd,J＝7.6,1.8Hz,1H),7.16(d,J＝8.6Hz,2H),7.13–7.09(m,1H),7.07(d,J＝7.0Hz,1H),7.02(td,J＝7.4,1.3Hz,1H),6.89(d,J＝8.5Hz,1H),6.83(d,J＝8.6Hz,2H),6.50(t,J＝5.7Hz,1H),4.83(dd,J＝12.3,5.3Hz,1H),4.19–4.06(m,2H),3.89(s,2H),3.88–3.83(m,2H),3.76–3.72(m,4H),3.70(q,J＝3.5,2.8Hz,4H),3.62(dd,J＝8.8,3.7Hz,4H),3.48–3.42(m,2H),2.83–2.75(m,2H),2.71(td,J＝6.6,4.3Hz,4H),2.65(s,6H),2.63–2.47(m,1H),2.09–1.97(m,1H),1.76(p,J＝6.6Hz,2H).UPLC-MS(ESI)for C38H49N6O7[M+H]+calculated 701.36obtained 701.10.
实施例7
制备4-((2-(2-(2-(2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙氧基)乙氧基)乙氧基)乙基)氨基)-2-(2,6-二氧哌啶-3-基)异二氢吲哚-1,3-二酮(I-7)，其结构式如下：
参照实施例5中的方法制备目标化合物，产物为黄色油状物50mg，产率26％。
1H NMR(400MHz,Chloroform-d)δ7.46(dd,J＝8.6,7.1Hz,1H),7.30–7.26(m,2H),7.24–7.15(m,3H),7.11–7.05(m,2H),7.01(td,J＝7.4,1.3Hz,1H),6.90(d,J＝8.5Hz,1H),6.83(d,J＝8.6Hz,2H),6.48(t,J＝5.7Hz,1H),4.86(dd,J＝12.2,5.4Hz,1H),4.12–4.07(m,2H),3.87–3.81(m,4H),3.73–3.65(m,12H),3.45(q,J＝5.6Hz,2H),2.87–2.73(m,3H),2.72–2.64(m,10H),2.11–2.02(m,1H),1.73(p,J＝6.8Hz,2H).UPLC-MS(ESI)for C40H53N6O8[M+H]+calculated 745.38,obtained 746.10.
实施例8
制备4-((1-(2-(2-(2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙氧基)乙氧基)乙酰基)哌啶-4-基)氨基)-2-(2,6-二氧代哌啶-3-基)异二氢吲哚-1,3-二酮(I-8)，其结构式如下：
参照实施例5中的方法制备目标化合物，产物为黄色油状物25mg，产率23％。
1H NMR(400MHz,Chloroform-d)δ7.45(dd,J＝8.6,7.1Hz,1H),7.24(dd,J＝7.7,1.7Hz,1H),7.20(dd,J＝7.7,1.7Hz,1H),7.15(ddd,J＝8.8,6.4,2.0Hz,2H),7.10–7.04(m,2H),6.99(td,J＝7.4,1.3Hz,1H),6.86–6.78(m,3H),6.21(d,J＝7.8Hz,1H),4.84(dd,J＝12.1,5.3Hz,1H),4.30(dd,J＝17.7,6.4Hz,1H),4.25–4.16(m,2H),4.10–4.00(m,6H),3.84(s,2H),3.82(dd,J＝5.7,4.0Hz,2H),3.71(dq,J＝6.9,4.5,4.1Hz,4H),3.65(d,J＝2.3Hz,2H),3.24–3.15(m,1H),3.02–2.92(m,1H),2.81–2.59(m,12H),2.07–1.96(m,3H),1.72(p,J＝6.8Hz,2H),1.46(ddd,J＝23.5,17.4,10.1Hz,2H).UPLC-MS(ESI)for C43H56N7O8[M+H]+calculated 798.42,obtained 798.12.
实施例9
制备4-((1-(2-(2-(2-(2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧)乙氧基)乙氧基)乙氧基)乙酰基)哌啶-4-基)氨基)-2-(2,6-二氧哌啶-3-基)异二氢吲哚-1,3-二酮(I-9)，其结构式如下：
参照实施例5中的方法制备目标化合物，产物为黄色油状物21mg，产率20％。
1H NMR(400MHz,Chloroform-d)δ7.46(dd,J＝8.5,7.1Hz,1H),7.28–7.23(m,1H),7.20(td,J＝7.7,1.7Hz,1H),7.17–7.13(m,2H),7.11–7.05(m,2H),7.00(td,J＝7.4,1.3Hz,1H),6.86(d,J＝8.6Hz,1H),6.81(d,J＝8.4Hz,2H),6.22(d,J＝7.9Hz,1H),4.85(dd,J＝12.1,5.2Hz,1H),4.37–4.28(m,1H),4.20(d,J＝9.5Hz,2H),4.07(t,J＝4.9Hz,2H),3.96–3.86(m,4H),3.84(s,2H),3.81(t,J＝4.9Hz,2H),3.72–3.63(m,10H),3.20(t,J＝12.3Hz,1H),3.01–2.92(m,1H),2.84–2.61(m,12H),2.09–1.99(m,3H),1.72(p,J＝6.8Hz,2H),1.59–1.40(m,2H).UPLC-MS(ESI)for C45H60N7O9[M+H]+calculated 842.45,obtained 842.00.
实施例10
制备4-((1-(14-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)-3,6,9,12-四氧基哌啶-4-基)氨基)-2-(2,6-二氧哌啶-3-基)异吲哚-1,3-二酮(I-10)，其结构式如下：
参照实施例5中的方法制备目标化合物，产物为黄色油状物20mg，产率19％。
1H NMR(400MHz,Chloroform-d)δ7.46(dd,J＝8.6,7.1Hz,1H),7.27–7.23(m,1H),7.21–7.13(m,3H),7.07(d,J＝7.2Hz,2H),6.99(td,J＝7.4,1.3Hz,1H),6.87(d,J＝8.6Hz,1H),6.84–6.78(m,2H),6.22(d,J＝7.9Hz,1H),4.88–4.81(m,1H),4.32(d,J＝13.5Hz,1H),4.19(d,J＝8.3Hz,2H),4.07(dd,J＝5.7,4.0Hz,2H),3.81(d,J＝6.5Hz,4H),3.75(d,J＝5.7Hz,2H),3.71–3.60(m,14H),3.20(t,J＝12.2Hz,1H),2.96(t,J＝11.7Hz,1H),2.83–2.59(m,12H),2.09–2.00(m,3H),1.70(p,J＝6.8Hz,2H),1.58–1.40(m,2H).UPLC-MS(ESI)for C47H64N7O10[M+H]+calculated 886.47,obtained 886.20.
实施例11
制备(2S,4R)-1-((S)-2-(4-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)丁酰氨基)-3,3-二甲基丁酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-1)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可)，产物为黄色油状物20mg，产率21％。
1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),7.44(t,J＝6.0Hz,1H),7.37–7.25(m,6H),7.23–7.13(m,3H),7.09(d,J＝7.9Hz,1H),7.04–6.98(m,1H),6.79(d,J＝8.4Hz,2H),6.37(d,J＝8.9Hz,1H),4.62(t,J＝7.9Hz,1H),4.52(dd,J＝15.8,7.4Hz,2H),4.47–4.43(m,1H),4.31(dd,J＝15.1,5.3Hz,1H),3.99–3.89(m,3H),3.82(s,2H),3.66(s,2H),3.59(dd,J＝11.2,3.8Hz,1H),2.65(s,6H),2.49(s,3H),2.45–2.29(m,4H),2.11–1.98(m,4H),1.69(p,J＝6.9Hz,2H),1.46(d,J＝7.0Hz,3H)0.91(s,9H).UPLC-MS(ESI)forC46H64N7O5S[M+H+]calculated 826.46obtained 826.10.
实施例12
制备(2S,4R)-1-((S)-2-(6-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)六氨基)-3,3-二甲基丁酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-2)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可)，产物为黄色油状物20mg，产率16％。
1H NMR(400MHz,Chloroform-d)δ8.65(s,1H),7.45(dd,J＝7.9,2.8Hz,1H),7.41–7.33(m,4H),7.27(d,J＝8.5Hz,1H),7.23–7.15(m,3H),7.09(dd,J＝8.0,1.3Hz,1H),7.01(td,J＝7.4,1.3Hz,1H),6.83–6.78(m,2H),6.26(dd,J＝9.0,4.3Hz,1H),5.16–4.98(m,1H),4.66(t,J＝7.8Hz,1H),4.58(d,J＝9.0Hz,1H),4.48–4.43(m,1H),4.01(d,J＝11.5Hz,1H),3.91(t,J＝6.3Hz,2H),3.83(s,2H),3.67(s,2H),3.58(dd,J＝11.3,3.8Hz,1H),2.70–2.62(m,10H),2.51(s,3H),2.50–2.37(m,2H),2.19(t,J＝7.5Hz,2H),2.07–1.98(m,1H),1.80–1.60(m,6H),1.47(d,J＝7.0Hz,3H),1.02(s,9H).UPLC-MS(ESI)for C48H66N7O5S[M+H]+calculated 854.49obtained 855.20.
实施例13
制备(2S,4R)-1-((S)-2-(8-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)辛酰胺基)-3,3-二甲基丁酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-3)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可)，产物为黄色油状物19mg，产率15％。
1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),7.45(d,J＝7.9Hz,1H),7.41–7.33(m,4H),7.29–7.25(m,1H),7.24–7.15(m,3H),7.09(dd,J＝8.0,1.3Hz,1H),7.01(td,J＝7.4,1.3Hz,1H),6.83–6.78(m,2H),6.23(d,J＝8.9Hz,1H),5.08(p,J＝7.0Hz,1H),4.67(t,J＝7.9Hz,1H),4.58(d,J＝8.9Hz,1H),4.49–4.43(m,1H),4.05–3.99(m,1H),3.91(t,J＝6.5Hz,2H),3.84(s,2H),3.68(s,2H),3.57(dd,J＝11.3,3.8Hz,1H),2.69–2.62(m,10H),2.51(s,3H),2.42(ddd,J＝13.0,7.8,4.7Hz,1H),2.16(t,J＝7.6Hz,2H),2.03(td,J＝9.1,8.1,3.9Hz,1H),1.78–1.67(m,4H),1.65–1.54(m,2H),1.47(d,J＝6.9Hz,3H),1.45–1.39(m,2H),1.36–1.29(m,4H),1.03(s,9H).13C NMR(101MHz,DMSO-d6)δ172.02,170.60,169.58,159.10,151.48,144.66,131.69,131.11,129.66,128.79,126.37,123.49,120.27,114.40,68.71,67.49,58.52,56.32,47.68,45.71,45.05,43.96,43.28,37.72,35.17,34.85,34.17,30.93,28.58,28.46,26.43,25.38,24.76,22.41,22.04,15.96,13.94.UPLC-MS(ESI)for C50H72N7O5S[M+H]+calculated 882.52obtained 882.30.
实施例14
制备(2S,4R)-1-((S)-2-(10-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)癸酰胺)-3,3-二甲基丁酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-4)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可)，产物为黄色油状物22mg，产率16％。
1H NMR(400MHz,Chloroform-d)δ8.67(s,1H),7.46(d,J＝7.9Hz,1H),7.43–7.34(m,4H),7.30–7.26(m,1H),7.25–7.16(m,3H),7.10(dd,J＝8.0,1.3Hz,1H),7.02(td,J＝7.4,1.3Hz,1H),6.86–6.80(m,2H),6.21(d,J＝8.9Hz,1H),5.09(p,J＝7.0Hz,1H),4.69(t,J＝7.9Hz,1H),4.58(d,J＝8.9Hz,1H),4.50–4.44(m,1H),4.04(dt,J＝11.5,1.9Hz,1H),3.93(t,J＝6.5Hz,2H),3.84(s,2H),3.69(s,2H),3.58(dd,J＝11.3,3.8Hz,1H),2.71–2.63(m,10H),2.53(s,3H),2.51–2.41(m,1H),2.16(t,J＝7.6Hz,2H),2.08–1.99(m,1H),1.80–1.68(m,4H),1.65–1.53(m,2H),1.48(d,J＝7.0Hz,3H),1.46–1.39(m,2H),1.35–1.24(m,8H),1.04(s,9H).13C NMR(101MHz,CDCl3)δ173.73,172.01,169.97,158.27,152.77,150.43,148.55,143.30,134.09,132.00,131.66,130.96,129.85,129.64,129.43,127.91,126.56,123.52,119.51,114.52,77.36,69.85,68.04,58.77,57.41,56.80,53.41,50.37,48.90,48.02,47.86,45.06,36.63,35.79,35.39,29.78,29.40,29.31,29.26,26.60,26.04,25.69,22.27,16.17.UPLC-MS(ESI)for C52H76N7O5S[M+H]+calculated910.56obtained 911.25.
实施例15
制备(2S,4R)-1-((S)-2-(2-(2-(2-(4-((((3((2(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙氧基)乙氧基)乙酰胺)-3,3-二甲基丁酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-5)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可)，产物为黄色油状物45mg，产率15％。
1H NMR(400MHz,Chloroform-d)δ8.65(s,1H),7.53(d,J＝7.7Hz,1H),7.40–7.31(m,5H),7.28–7.24(m,1H),7.21(dd,J＝7.6,1.7Hz,1H),7.17(d,J＝8.7Hz,2H),7.09(dd,J＝8.0,1.3Hz,1H),7.00(td,J＝7.4,1.3Hz,1H),6.83(d,J＝8.6Hz,2H),5.05(p,J＝7.0Hz,1H),4.68(t,J＝7.8Hz,1H),4.56(d,J＝8.9Hz,1H),4.43(dd,J＝4.3,2.3Hz,1H),4.11(ddd,J＝6.8,3.7,2.1Hz,2H),4.03–3.89(m,3H),3.84(d,J＝5.8Hz,4H),3.75–3.64(m,7H),3.57(dd,J＝11.2,3.9Hz,1H),2.71–2.62(m,10H),2.50(s,3H),2.39(ddd,J＝12.8,7.7,4.8Hz,1H),2.06–1.96(m,1H),1.71(p,J＝6.8Hz,2H),1.45(d,J＝6.9Hz,3H),1.03(s,9H).UPLC-MS(ESI)for C48H68N7O7S[M+H]+calculated 886.48,obtained 887.14.
实施例16
制备(2S,4R)-1-((S)-2-(叔丁基)-14-(4-(((3-((2-(2-(二甲氨基)苄基)氨基)丙烷(氨基)甲基)基)苯氧基)-4-氧代-6,9,12-三氧杂-3-氮杂二癸酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-6)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可)，产物为黄色油状物43mg，产率16％。
1H NMR(400MHz,Chloroform-d)δ8.64(s,1H),7.49(d,J＝7.7Hz,1H),7.39–7.31(m,5H),7.31–7.25(m,1H),7.21(dd,J＝7.6,1.7Hz,1H),7.19–7.14(m,2H),7.09(dd,J＝8.0,1.3Hz,1H),7.00(td,J＝7.4,1.3Hz,1H),6.83(d,J＝8.6Hz,2H),5.05(p,J＝7.0Hz,1H),4.68(t,J＝7.7Hz,1H),4.56(d,J＝8.9Hz,1H),4.43(dt,J＝4.5,2.2Hz,1H),4.09(dd,J＝5.8,4.0Hz,2H),3.99–3.88(m,3H),3.85(s,2H),3.83–3.79(m,2H),3.73–3.63(m,10H),3.57(dd,J＝11.0,3.9Hz,1H),2.71–2.63(m,11H),2.50(s,3H),2.45–2.36(m,1H),2.04–1.95(m,1H),1.73(p,J＝6.7Hz,2H),1.45(d,J＝6.9Hz,3H),1.03(s,9H).UPLC-MS(ESI)forC50H72N7O8S[M+H]+calculated 930.51,obtained 931.21.
实施例17
制备(2S,4R)-1-((S)-2-(叔丁基)-17-(4-(((((3-((2-(2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)-4-氧代-6,9,12,15-四氧代-3-氮杂十七烷)-4-羟基-N-((S)-1-(4-(4-(4-甲基-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-7)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可)，产物为黄色油状物40mg，产率18％。
1H NMR(400MHz,Chloroform-d)δ8.65(s,1H),7.50(d,J＝7.8Hz,1H),7.40–7.32(m,5H),7.31–7.24(m,2H),7.24–7.14(m,3H),7.09(dd,J＝8.0,1.3Hz,1H),7.01(td,J＝7.4,1.3Hz,1H),6.82(d,J＝8.6Hz,2H),5.06(p,J＝7.1Hz,1H),4.69(t,J＝7.8Hz,1H),4.56(d,J＝8.8Hz,1H),4.43(dq,J＝4.4,2.3Hz,1H),4.08(dd,J＝5.7,4.1Hz,2H),3.95(q,J＝15.7Hz,3H),3.85(s,2H),3.82(dd,J＝5.7,4.0Hz,2H),3.72–3.62(m,14H),3.57(dd,J＝10.9,3.7Hz,1H),2.71–2.63(m,10H),2.50(s,3H),2.41(ddd,J＝12.7,7.4,4.8Hz,1H),2.01(ddd,J＝13.3,7.1,4.2Hz,1H),1.73(p,J＝6.7Hz,2H),1.46(d,J＝6.9Hz,3H),1.03(s,9H).UPLC-MS(ESI)for C52H76N7O9S[M+H]+calculated 974.53,obtained 975.24.
实施例18
制备(2S,4R)-1-((S)-2-(8-(4-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯基)哌嗪-1-基)辛酰胺基)-3,3-二甲基丁酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-8)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可，反应起始原料4-羟基苯酚替换成4-(哌嗪-1-基)苯甲醛盐酸盐)，产物为黄色油状物40mg，产率23％。
1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),7.46–7.34(m,5H),7.19(dd,J＝18.4,8.0Hz,3H),7.10(d,J＝8.0Hz,1H),7.02(t,J＝7.3Hz,1H),6.90–6.82(m,2H),6.21(d,J＝8.9Hz,1H),5.08(p,J＝7.1Hz,1H),4.70(t,J＝7.9Hz,1H),4.60(d,J＝8.9Hz,1H),4.47(s,1H),4.05(d,J＝11.4Hz,1H),3.86(s,2H),3.69(s,2H),3.57(dd,J＝11.3,3.7Hz,1H),3.17(t,J＝4.9Hz,4H),2.73–2.67(m,8H),2.61–2.56(m,4H),2.52(s,3H),2.49–2.42(m,1H),2.35(t,J＝7.8Hz,2H),2.19(td,J＝7.3,5.2Hz,2H),2.08–1.99(m,1H),1.74(p,J＝6.7Hz,2H),1.60(q,J＝7.3Hz,2H),1.54–1.50(m,2H),1.47(d,J＝6.9Hz,3H),1.35–1.24(m,7H),1.03(s,9H).UPLC-MS(ESI)for C54H80N9O4S[M+H]+calculated 950.60,obtained950.50.
实施例19
制备(2S,4R)-1-((S)-2-(10-(4-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯基)哌嗪-1-基)癸酰胺)-3,3-二甲基丁酰基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(II-9)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成VHL配体即可，反应起始原料4-羟基苯酚替换成4-(哌嗪-1-基)苯甲醛盐酸盐)，产物为黄色油状物36mg，产率25％。
1H NMR(400MHz,Chloroform-d)δ8.64(s,1H),7.50(d,J＝7.9Hz,1H),7.40–7.32(m,4H),7.25–7.12(m,5H),7.07(d,J＝8.0Hz,1H),6.99(t,J＝7.4Hz,1H),6.84(d,J＝8.3Hz,2H),6.29(d,J＝9.0Hz,1H),5.07(p,J＝7.0Hz,1H),4.64(t,J＝7.9Hz,1H),4.58(d,J＝9.0Hz,1H),4.44–4.36(m,1H),3.94(d,J＝11.2Hz,1H),3.81(s,3H),3.65(s,2H),3.56(dd,J＝11.2,3.9Hz,1H),3.15(t,J＝5.0Hz,4H),2.65(d,J＝6.6Hz,12H),2.56(t,J＝5.0Hz,4H),2.49(s,3H),2.38–2.30(m,2H),2.13(t,J＝7.6Hz,2H),2.00(dd,J＝13.7,7.9Hz,1H),1.69(p,J＝7.2Hz,2H),1.62–1.52(m,4H),1.46(d,J＝6.9Hz,3H),1.26(d,J＝5.9Hz,10H),1.01(s,9H).13C NMR(101MHz,CDCl3)δ173.52,171.99,169.92,152.75,150.57,150.36,148.50,143.28,133.42,131.62,130.90,130.28,129.93,129.58,129.20,128.05,126.51,123.58,119.54,116.01,69.74,58.84,58.74,57.34,56.71,53.50,53.31,53.13,50.16,49.22,48.84,48.10,47.86,45.15,45.03,36.59,35.80,35.42,29.50,29.39,29.25,29.00,27.58,26.89,26.57,25.67,22.26,16.14.UPLC-MS(ESI)forC56H84N9O4S[M+H]+calculated 978.63,obtained 978.50.
实施例20
制备N-(2-(2-(4-(4,5-双(4-氯苯基)-2-(2-异丙氧基-4-甲氧基苯基))-4,5-二氢-1H-咪唑-1-羰基)哌嗪-1-基)乙氧基)乙基)-4-(4-(((3-((4-甲氧基苄基)氨基)丙基)氨基)甲基)苯氧基)丁酰胺(III-1)，其结构式如下：
通过下式i、ii、iii所示反应合成化合物III-1。
步骤如下：
(i)MDM2配体(0.5g,6.53mmol,1eq)和碳酸钾(1.2g,9.42mmol,1.5eq)室温下在5mL乙腈中搅拌30分钟后，加入(2-(2-溴乙氧基)乙基)氨基甲酸叔丁酯(3.56g,12.28mmol,1eq)70℃反应5小时后，TLC监测反应结束后，用DCM萃取后，无水硫酸钠干燥后减压旋去溶剂后，加入自制的乙酸乙酯盐酸溶液10mL，rt反应过夜后，减压旋去溶剂剩余固体可以直接用于下一步反应，产率70％。
1H NMR(400MHz,Chloroform-d)δ7.49(d,J＝8.4Hz,1H),7.11–7.05(m,2H),7.05–7.00(m,2H),6.98–6.92(m,2H),6.89–6.82(m,2H),6.55–6.48(m,2H),5.60(d,J＝9.8Hz,1H),5.44(d,J＝9.8Hz,1H),4.61(hept,J＝6.0Hz,1H),3.84(s,3H),3.46(q,J＝5.7Hz,4H),3.30–3.23(m,2H),3.21–3.12(m,4H),2.43–2.35(m,2H),2.13–1.99(m,4H),1.43–1.34(m,15H).m.p.＞200℃.
(ii)将上一步得到的中间体胺(100mg,0.576mmol,1eq)、4-(4-甲酰基苯氧基)丁酸(265mg,0.384mmol,0.7eq)以及HATU(220mg,0.576mmol,1eq)、DIPEA(173mg,1.34mmol,2eq)加入到3mL DMF中，室温反应5小时后，用50mL乙酸乙酯和水分液后，有机层分别用饱和柠檬酸和碳酸氢钠溶液最后用饱和氯化钠洗涤后，无水硫酸钠干燥后，残余物通过硅胶柱色谱法纯化得到淡黄白色固体150mg，产率46％。
1H NMR(400MHz,Chloroform-d)δ9.87(s,1H),7.81(d,J＝8.8Hz,2H),7.47(d,J＝8.5Hz,1H),7.07–6.95(m,6H),6.86–6.77(m,4H),6.56(dd,J＝8.5,2.1Hz,1H),6.39(s,1H),6.09(s,1H),5.67(d,J＝10.1Hz,1H),5.49(d,J＝10.1Hz,1H),4.58(hept,J＝12.6,12.1,6.6Hz,1H),4.08(t,J＝6.1Hz,2H),3.85(s,3H),3.47(dt,J＝9.8,5.1Hz,4H),3.42–3.35(m,2H),3.25–3.16(m,4H),2.55–2.48(m,2H),2.37(t,J＝7.2Hz,2H),2.33–2.21(m,4H),2.11(p,J＝6.6Hz,2H),1.37(d,J＝5.9Hz,3H),1.31(d,J＝6.0Hz,3H).m.p.＞200℃.
(iii)将上一步得到的醛中间体(150mg,0.177mmol,1eq)和(3-氨基丙基)(4-甲氧基苄基)氨基甲酸叔丁酯(52mg，0.177mmol,1eq)一起加入到10mL无水甲醇中，滴加几滴醋酸和分子筛后，室温搅拌5分钟后，加入氰基硼氢化钠(33mg,0.5mmol,3eq)室温反应12小时后，抽滤反应液后，减压旋去溶剂后，加入三氟醋酸1mL和二氯甲烷1mL后，室温反应2小时后，TLC监测反应结束后，加入碳酸氢钠溶液后，DCM/甲醇萃取后，无水硫酸钠干燥后，残留物通过硅胶柱色谱纯化得到无色油状物45mg，产率36％。
1H NMR(400MHz,Chloroform-d)δ7.48(d,J＝8.4Hz,1H),7.18(dd,J＝8.7,7.3Hz,4H),7.09–7.04(m,2H),7.01(d,J＝8.4Hz,2H),6.87–6.79(m,6H),6.54–6.47(m,2H),6.03(t,J＝5.2Hz,1H),5.58(d,J＝9.8Hz,1H),5.42(d,J＝9.8Hz,1H),4.59(hept,J＝6.1Hz,1H),3.96(t,J＝5.9Hz,2H),3.83(s,3H),3.78(s,3H),3.69(d,J＝5.0Hz,4H),3.47–3.37(m,6H),3.17–3.08(m,4H),2.68(td,J＝6.8,1.3Hz,4H),2.40–2.31(m,4H),2.13–2.03(m,6H),1.71(p,J＝6.8Hz,2H),1.39(d,J＝6.0Hz,3H),1.34(d,J＝6.0Hz,3H).UPLC-MS(ESI)for C56H70Cl2N7O7[M+H]+calculated 1022.46,obtained 1022.60.
实施例21
制备N-(3-(4-(4,5-双(4-氯苯基)-2-(2-异丙氧基-4-甲氧基苯基)-4,5-二氢-1H-咪唑-1-羰基)哌嗪-1-基)丙基)-4-(4-((((3-((4-甲氧基苄基)氨基)丙基)氨基)甲基)苯氧基)丁酰胺(III-2)，其结构式如下：
参照实施例20中的方法制备目标化合物，产物为黄色油状物49mg，产率40％。
1H NMR(400MHz,Chloroform-d)δ7.49(d,J＝8.4Hz,1H),7.18(dd,J＝10.3,7.7Hz,4H),7.10–7.05(m,2H),7.03–6.99(m,2H),6.96–6.91(m,2H),6.88–6.77(m,6H),6.55–6.47(m,2H),6.38(t,J＝5.5Hz,1H),5.57(d,J＝9.8Hz,1H),5.42(d,J＝9.8Hz,1H),4.59(hept,J＝6.0Hz,1H),3.95(t,J＝5.9Hz,2H),3.82(s,3H),3.78(s,3H),3.69(d,J＝4.6Hz,4H),3.27–3.18(m,2H),3.11(d,J＝5.0Hz,4H),2.68(t,J＝6.7Hz,4H),2.27(t,J＝7.2Hz,2H),2.20(t,J＝6.6Hz,2H),2.10–2.04(m,2H),1.96(d,J＝6.0Hz,4H),1.71(p,J＝6.7Hz,2H),1.53(p,J＝7.0Hz,2H),1.38(d,J＝6.0Hz,3H),1.33(d,J＝6.0Hz,3H).UPLC-MS(ESI)for C55H68Cl2N7O6[M+H]+calculated 992.45,obtained 992.50.
实施例22
制备N-(6-(4-(4,5-双(4-氯苯基)-2-(2-异丙氧基-4-甲氧基苯基)-4,5-二氢-1H-咪唑-1-羰基)哌嗪-1-基)己基)-4-(4-((((3-((4-甲氧基苄基)氨基)-丙基)氨基)甲基)苯氧基)丁酰胺(III-3)，其结构式如下：
参照实施例20中的方法制备目标化合物，产物为黄色油状物50mg，产率42％。
1H NMR(400MHz,Chloroform-d)δ7.46(d,J＝8.4Hz,1H),7.22–7.14(m,4H),7.10–7.04(m,2H),7.01(d,J＝8.4Hz,2H),6.97–6.91(m,2H),6.87–6.79(m,6H),6.54–6.47(m,2H),5.65(t,J＝5.7Hz,1H),5.59(d,J＝9.9Hz,1H),5.42(d,J＝9.8Hz,1H),4.60(hept,J＝6.1Hz,1H),3.96(t,J＝5.9Hz,2H),3.82(s,3H),3.78(s,3H),3.68(d,J＝3.4Hz,4H),3.22–3.08(m,6H),2.67(t,J＝6.8Hz,4H),2.33(t,J＝7.3Hz,2H),2.16–2.05(m,4H),2.00(d,J＝5.1Hz,4H),1.70(p,J＝6.8Hz,2H),1.39(d,J＝6.0Hz,3H),1.35(d,J＝6.0Hz,3H),1.32–1.22(m,8H).UPLC-MS(ESI)for C57H72Cl2N7O6[M+H]+calculated 1020.48,obtained1020.50.
实施例23
制备N-(金刚烷-1-基)-2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙酰胺(IV-1)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物35mg，产率11％。
1H NMR(400MHz,Chloroform-d)δ7.26(dd,J＝7.5,1.7Hz,1H),7.21–7.15(m,3H),7.06(dd,J＝8.0,1.3Hz,1H),6.99(td,J＝7.4,1.3Hz,1H),6.83–6.78(m,2H),3.95(t,J＝5.9Hz,2H),3.82(s,2H),3.68(s,2H),2.69–2.64(m,8H),2.08–1.99(m,5H),1.93(d,J＝3.1Hz,6H),1.70(p,J＝6.8Hz,2H),1.63(t,J＝2.2Hz,6H).UPLC-MS(ESI)for C31H44N4O2[M+H]+calculated 505.35,obtained 505.20.
实施例24
制备N-(金刚烷-1-基)-4-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)丁酰胺(IV-2)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物46mg，产率15％。
1H NMR(400MHz,Chloroform-d)δ7.26(dd,J＝7.5,1.7Hz,1H),7.21–7.15(m,3H),7.06(dd,J＝8.0,1.3Hz,1H),6.99(td,J＝7.4,1.3Hz,1H),6.83–6.78(m,2H),3.95(t,J＝5.9Hz,2H),3.82(s,2H),3.68(s,2H),2.69–2.64(m,8H),2.08–1.99(m,5H),1.93(d,J＝3.1Hz,6H),1.70(p,J＝6.8Hz,2H),1.63(t,J＝2.2Hz,6H).UPLC-MS(ESI)for C31H44N4O2[M+H]+calculated 505.35,obtained 505.20.
实施例25
制备2-(金刚烷-1-基)-N-(3-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)丙基)乙酰胺(IV-3)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物26mg，产率17％。
1H NMR(400MHz,Chloroform-d)δ7.29–7.24(m,1H),7.21–7.15(m,3H),7.07(dd,J＝8.0,1.3Hz,1H),6.99(td,J＝7.4,1.3Hz,1H),6.82–6.77(m,2H),3.98(t,J＝5.9Hz,2H),3.81(s,2H),3.67(s,2H),3.40(q,J＝6.3Hz,2H),2.69–2.61(m,10H),1.99–1.91(m,4H),1.88(s,2H),1.74–1.55(m,15H).UPLC-MS(ESI)for C34H51N4O2[M+H]+calculated 547.39,obtained 547.50.
实施例26
制备N-(2-(2-(4-(金刚烷-1-基)苯氧基)乙氧基)乙基)-2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙酰胺(IV-4)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物35mg，产率23％。
1H NMR(400MHz,Chloroform-d)δ7.50(dd,J＝7.6,1.8Hz,1H),7.25–7.15(m,4H),7.08–6.97(m,3H),6.83(dd,J＝25.8,8.7Hz,4H),4.45(s,2H),4.09–4.05(m,2H),3.80–3.76(m,2H),3.60–3.53(m,6H),3.24(s,2H),2.68(s,6H),2.56(t,J＝5.6Hz,4H),2.08(q,J＝3.2Hz,2H),1.90–1.66(m,15H).UPLC-MS(ESI)for C41H57N4O4[M+H]+calculated 669.43,obtained 670.01.
实施例27
制备N-(2-(2-(4-(金刚烷-1-基)苯氧基)乙氧基)乙基)-2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)甲基)苯氧基)乙酰胺(IV-5)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物120mg，产率32％。
1H NMR(400MHz,Chloroform-d)δ7.29–7.24(m,1H),7.21–7.14(m,3H),7.07(dd,J＝8.0,1.3Hz,1H),6.99(td,J＝7.4,1.3Hz,1H),6.83(d,J＝8.6Hz,2H),5.97(t,J＝5.8Hz,1H),4.09–4.04(m,2H),3.82(s,2H),3.80–3.75(m,2H),3.68(s,2H),3.58(t,J＝5.1Hz,2H),3.43(q,J＝5.3Hz,2H),2.69–2.62(m,10H),2.22(s,2H),1.95–1.88(m,3H),1.86(s,2H),1.70(p,J＝6.9Hz,2H),1.60–1.54(m,12H).13C NMR(101MHz,Chloroform-d)δ170.96,157.66,152.61,134.40,132.93,129.60,129.30,127.61,123.26,119.26,114.42,70.14,69.49,67.30,53.40,51.66,50.31,48.02,47.87,44.94,42.53,38.96,36.75,32.65,30.11,28.60.UPLC-MS(ESI)for C35H52N4O3[M+H]+calculated 577.40,obtained 577.95.
实施例28
制备2-(金刚烷-1-基)-N-(2-(2-(2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)-甲基)苯氧基)乙氧基)乙氧基)乙基)乙酰胺(IV-6)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物109mg，产率35％。
1H NMR(400MHz,Chloroform-d)δ7.26(dd,J＝7.5,1.7Hz,1H),7.17(d,J＝8.5Hz,2H),7.07(dd,J＝8.0,1.3Hz,1H),6.99(td,J＝7.4,1.3Hz,1H),6.82(d,J＝8.6Hz,2H),6.11(t,J＝5.6Hz,1H),4.10–4.06(m,2H),3.84–3.79(m,4H),3.69–3.65(m,4H),3.61(dt,J＝4.6,3.1Hz,2H),3.52(dd,J＝5.6,4.6Hz,2H),3.40(q,J＝5.3Hz,2H),2.69–2.62(m,10H),2.60(s,3H),1.93–1.89(m,3H),1.87(s,2H),1.72(p,J＝6.8Hz,2H),1.68–1.55(m,12H).13C NMR(101MHz,Chloroform-d)δ170.98,157.65,152.58,133.91,132.48,129.65,129.28,127.71,123.31,119.30,114.44,70.62,70.14,70.00,69.65,67.38,53.21,51.53,50.15,47.93,47.78,44.90,42.49,38.94,36.70,32.59,29.62,28.56.UPLC-MS(ESI)forC37H56N4O4[M+H]+calculated 621.43,obtained 621.20.
实施例29
制备2-(金刚烷-1-基)-N-(2-(2-(2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)-甲基)苯氧基)乙氧基)乙氧基)乙基)乙酰胺(IV-7)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物41mg，产率16％。
1H NMR(400MHz,Chloroform-d)δ7.29(d,J＝7.6Hz,2H),7.19(d,J＝8.6Hz,1H),7.17–7.13(m,1H),7.06(td,J＝7.4,1.3Hz,1H),6.87(d,J＝8.6Hz,2H),6.14(t,J＝5.8Hz,1H),4.15–4.12(m,2H),3.96(s,2H),3.87(dd,J＝5.7,4.0Hz,2H),3.77–3.73(m,4H),3.72–3.62(m,6H),3.56(t,J＝5.0Hz,2H),3.45(q,J＝5.2Hz,2H),2.77(q,J＝6.6Hz,4H),2.67(s,6H),1.97(t,J＝3.1Hz,3H),1.93(s,2H),1.81(p,J＝6.5Hz,2H),1.74–1.67(m,3H),1.64(dd,J＝9.3,2.5Hz,9H).13C NMR(101MHz,Chloroform-d)δ171.13,158.02,152.82,132.29,131.49,130.12,129.52,128.52,123.87,119.78,114.71,70.90,70.64,70.62,70.26,70.16,69.83,67.53,52.99,51.72,49.98,48.23,48.07,45.07,42.67,39.09,36.87,32.77,28.73,28.16.UPLC-MS(ESI)for C39H60N4O5[M+H]+calculated 665.46,obtained 665.89.
实施例30
制备2-(金刚烷-1-基)-N-(2-(2-(2-(4-(((3-((2-(二甲氨基)苄基)氨基)丙基)氨基)-甲基)苯氧基)乙氧基)乙氧基)乙基)乙酰胺(IV-8)，其结构式如下：
参照实施例1中的方法制备目标化合物(只需将CRBN配体替换成金刚烷乙酸或者金刚烷胺即可)，产物为黄色油状物70mg，产率55％。
1H NMR(400MHz,Chloroform-d)δ7.26(dd,J＝7.4,1.7Hz,1H),7.21–7.12(m,3H),7.06(dd,J＝8.1,1.3Hz,1H),6.98(td,J＝7.4,1.3Hz,1H),6.83(d,J＝8.7Hz,2H),6.16(t,J＝5.5Hz,1H),3.82(s,2H),3.66(s,2H),3.58(t,J＝5.6Hz,2H),3.50(dd,J＝5.6,4.6Hz,2H),3.41(q,J＝5.2Hz,2H),3.19–3.14(m,4H),2.70–2.57(m,16H),2.46(s,2H),1.93(t,J＝3.1Hz,2H),1.90(s,2H),1.72–1.55(m,15H).13C NMR(101MHz,Chloroform-d)δ171.00,152.64,150.25,134.26,131.33,129.67,129.02,127.68,123.32,119.32,115.99,69.84,68.17,57.82,53.54,53.37,51.65,50.28,49.15,48.01,47.82,44.97,42.60,38.94,36.78,32.70,29.91,28.64.UPLC-MS(ESI)for C39H61N6O2[M+H]+calculated 645.48,obtained 645.84.
实施例31化合物的细胞活性测试
测试化合物在结直肠癌细胞系HT29(该细胞系癌基因C-Myc、K-RAS、H-RAS、N-RAS、Myb等阳性导致Gli1过表达)，Gli1蛋白低水平肝细胞癌细胞系HepG2中的细胞活性，以GANT61为对照化合物。
化合物细胞水平的活性检测采用Cell Counting Kit-8简称CCK-8试剂，是一种基于WST-8(2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐)可以广泛应用于细胞增殖和细胞毒性的快速高灵敏度检测试剂。
具体步骤：取对数生长期的细胞平均加到96孔板中，细胞密度设置为每孔1×104个，加入100μL添加了10％胎牛血清的DMEM培养基，将96孔板置于37℃，5％CO2细胞培养箱中培养24小时，待细胞贴壁后，测试孔中加入200μL含药培养基，设3个复孔，8个浓度，药物起始浓度为20μM，依次三倍稀释，同时设立空白孔和对照孔，将96孔板置于37℃，5％CO2培养箱中培养72小时，将含药孔培养基吸出，并用预冷PBS洗涤一次，吸出PBS，每孔加入含10％CCK-8试剂的培养基100μL，将96孔板置于37℃，5％CO2培养箱中培养0.5～1小时，使用酶标仪在450nM波长处测定OD值，数值处理：抑制率％＝[(OD药物-OD空白)/(OD对照-OD空白)]×100，然后通过GraphPad以化合物浓度和相应的抑制率做S曲线拟合，得到化合物的IC50值。部分化合物的实验结果见表2：
表2优选化合物对HT29细胞抗增殖活性。
| 化合物 | HT29 IC50(μM) | 化合物 | HT29 IC50(μM) |
| I-1 | 3.119±1.557 | II-6 | 5.625±0.003 |
| I-2 | 1.436±0.558 | II-7 | 5.136±0.209 |
| I-3 | 0.442±0.093 | II-8 | 0.962±0.043 |
| I-4 | 0.389±0.042 | II-9 | 0.665±0.063 |
| I-5 | 1.590±0.315 | III-1 | 1.199±0.099 |
| I-6 | 3.965±0.883 | III-2 | 1.331±0.081 |
| I-7 | 5.038±0.709 | III-3 | 0.698±0.076 |
| I-8 | ＞20 | IV-1 | 0.792±0.194 |
| I-9 | ＞20 | IV-2 | 0.645±0.046 |
| I-10 | ＞20 | IV-3 | 0.486±0.025 |
| II-1 | 2.355±0.180 | IV-4 | 0.468±0.050 |
| II-2 | 2.224±0.357 | IV-5 | 0.462±0.046 |
| II-3 | 0.356±0.135 | IV-6 | 0.604±0.114 |
| II-4 | 0.211±0.038 | IV-7 | 0.697±0.054 |
| II-5 | 8.591±1.469 | IV-8 | 0.456±0.060 |
| GANT61 | 2.892±0.178 |
从表2中看出，靶向Gli1的双功能分子普遍比对照化合物GANT61在HT29细胞中抗增殖活性有所提高。发明人在HT29、MEF细胞中也通过WB等实验验证了本发明的靶向Gli1的双功能分子可降解Gli1靶蛋白。
表3优选化合物对Hh信号通路不活跃即Gli1蛋白低水平癌细胞系HepG2的抑制活性(72小时)
| 化合物 | HepG2 IC50(μM) |
| I-4 | ＞10 |
| II-4 | ＞10 |
| III-3 | ＞10 |
| IV-4 | ＞10 |
| GANT61 | ＞20 |
实验结果表明，相对于HT29细胞，双功能分子对Hh信号通路不活跃的HepG2细胞抑制增殖的作用是大大减弱的，且活性比小分子抑制剂更好，表明双功能分子对Gli1蛋白具有良好的选择性。
实施例32蛋白质印迹
采用WB的方法，对靶向Gli1双功能分子处理过的细胞(HT29或者MEFPTCH1-/-)中Gli1的量进行考察。
(1)实验方法：
细胞处理和收集：取适量密度的HT29细胞接种于6孔板中，12～24小时后待细胞贴壁，加入不同浓度的化合物作用12～24小时。吸掉培养基，冷的PBS洗三次，加入适量0.25％胰酶消化，离心收集细胞干，细胞干用预冷的PBS洗涤两次。
(2)细胞裂解：将上一步收集的细胞干加入RIPA裂解液和蛋白酶磷酸酶抑制剂混合物于冰上静置裂解30分钟。待裂解充分后，0℃，12000rpm离心10分钟取上清，使用BCA蛋白含量测定试剂盒测试上清中蛋白含量。然后加入4×SDS电泳上样缓冲液，置于金属浴中95℃加热10分钟使蛋白变性，放置-20℃冰箱保存，待检。
(3)SDS-PAGE凝胶电泳操作：
取适量蛋白样品(控制蛋白上样量在40～50μg，参阅文献和合作实验室的protocol得出)在4～12％浓度的聚丙烯酰胺凝胶进行SDS-PAGE电泳，电压90V跑浓缩胶，120V跑分离胶。
(4)转膜：
电泳结束后使用半干转移法将蛋白从凝胶上转移至PVDF膜上，使用恒流220mA，转膜2小时。
(5)免疫杂交和显色：
1)1×TBST洗膜2次，每次10分钟，室温摇床缓慢摇动。
2)将PVDF膜置于封闭缓冲液中，室温摇床缓慢摇动，封闭2小时。
3)用1×TBST缓冲液洗膜3次，每次10分钟。
4)将PVDF膜置于杂交袋中，加入合适浓度的一抗(Gli1:500～1000稀释，GAPDH或β-tubulin 1:5000稀释)。4℃摇床缓慢摇动，孵育一抗过夜。
5)次日，取出孵育过一抗的PVDF膜，1×TBST洗膜3次，每次10分钟。
6)将膜置于含相应二抗的杂交袋中，37℃置于摇床孵育1小时，使用TBST缓冲液洗膜
3次，每次10分钟。
7)显影：按照发光法进行显影。发光反应时将试剂盒中两种溶液按1:1比例混合，
(6)将PVDF膜浸没于溶液中，静置一段时间，上机检测。最后对条带进行分析，使用GAPDH作内参。实验结果见图1。
根据WB结果发现，靶向Gli蛋白降解剂分子能够有效降解细胞内部的Gli1，且化合物分子对于Gli1的降解能力呈现浓度相关性。证明了这些分子可以在细胞内实现诱导降解Gli1蛋白。
实施例33PI/Annexin V双染检测凋亡
磷脂酰丝氨酸(PS)是一种带负电荷的磷脂结构，正常情况下主要存在于细胞膜的内侧面，在细胞发生凋亡的时候，磷脂酰丝氨酸会转移到细胞膜外。Annexin V是一种钙离子依赖性磷脂结合蛋白，能与膜外磷脂酰丝氨酸高亲和力特异性结合。但PS转移到细胞膜外这一现象也可发生在细胞坏死中。这两种细胞死亡方式间的差别是在凋亡的初始阶段细胞膜是否完好，坏死细胞在其早期阶段细胞膜的完整性就被破坏了。Propidium iodide(PI)是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和死细胞，PI能够透过细胞膜与细胞核结合呈现红色。所以将Annexin V与PI匹配使用，可以将凋亡早期的细胞和晚
期的细胞以及死细胞区分开来。
实验方法
一、仪器参数调节
1.收集1×106-3×106个细胞，用预冷PBS离心洗涤两次，弃上清；
2.加入500μL Apoptosis Positive Control Solution重悬，置冰上孵育30分钟。
3.用预冷PBS离心洗涤，弃上清。
4.加入适量预冷1×Binding Buffer重悬，并加入数量相同且未经处理的活细胞与之混合。加入预冷1×Binding Buffer补充至1.5mL，等分成三管，其中一管为空白对照管、两管为单染管。
5.单染管分别加入5μL Annexin V-APC或10μL PI，室温避光孵育5分钟。
6.在流式细胞仪上，用空白管调节FSC、SSC和荧光通道的电压，并在此电压条件下，用单染管调节荧光通道的补偿。
二、样本检测
1.按常规方法在12孔板孵育HT29细胞，待细胞贴壁后，加入不同药物处理48小时；
2.用预冷PBS离心洗涤，收集1～10×105个细胞(包括培养上清中的细胞)。用双蒸水稀释5×Binding Buffer为1×工作液，取500μL 1×Binding Buffer重悬细胞。
3.每管加入5μL Annexin V-APC和10μL PI。
4.轻柔涡旋混匀后，室温避光孵育5分钟。
5.上机进行分析。
试验结果见图2。
从图2可以看出，在双变量的散点图上，左下象限代表活细胞；右下象限代表死细胞；左上象限表示晚期凋亡细胞；而右上象限代表早期凋亡的细胞。从图中我们可以明显看出：优选化合物II-4和IV-5 5μM就能促进HT29细胞的凋亡。
实施例34qPCR测试化合物II-4和IV-5对耐药细胞模型MEFSUFU-/-细胞中Hh靶基因的影响
实验方法
一、RNA提取
1.将适量的MEFPTCH1-/-和MEFSUFU-/-细胞加入适量的RNAiso Plus后匀浆；
2.室温静置5min后，12000rpm 4℃离心5min，吸取上清液至新的1.5mL离心管中；
3.加入1/5RNAiso Plus体积量的氯仿，震荡混合，室温静置5min后，12000rpm 4℃离心5min，吸取上清液至新的1.5mL离心管中；
4.加入0.5～1倍RNAiso Plus体积量的异丙醇，室温静置10min后，12000rpm 4℃离心10min；
5.用与RNAiso Plus等量的75％乙醇洗涤沉淀，7500rpm 4℃离心5min；
6.弃去上清保留沉淀，干燥后，溶解于适量的DEPC处理水中备用。
二、逆转录
1.基因组DNA去除
在RNase-free离心管中配制如下混合液
/>
用移液器轻轻吹打混匀，42℃ 2min。
2.配制逆转录反应体系
在第1步的反应管中加入5×HiScript III qRT
5×HiScript III qRT SuperMix 4μL
第1步的反应液 16μL
用移液器轻轻吹打混匀。
3.进行逆转录反应
37℃ 15min
85℃ 5sec
三、PCR上机反应
根据样品不同对应引物不同的原则，每组设置至少三个复孔，每孔体系如下：
上机，进行qRT-PCR反应，数据采用Graphpad软件处理。
试验结果见图3。
从图3可以看出，我们在MEFSUFU-/-细胞共孵育合成的降解剂，观察化合物对Hh通路靶基因mRNA水平的影响，通过加药共孵育48小时后，进行测试，在该细胞中GNAT61和两个降解剂均能减少Gli1 mRNA水平，但GANT61对PTCH2 mRNA水平基本无影响，而本发明的两个降解剂依然能显著减少PTCH2 mRNA水平。这个实验初步验证了本发明的Gli1降解剂对于SMO拮抗剂Vismodegib耐药的细胞系模型中依然具有抑制过度活跃的Hh信号通路的作用，这可能有助于解决SMO拮抗剂耐药性问题。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Claims (5)
1.一种靶向降解Gli1蛋白的降解剂，其特征在于：所述靶向降解Gli1蛋白的降解剂包括Gli1降解剂化合物或其药学上可接受的盐、外消旋体；所述Gli1降解剂化合物由式(I)所示结构与降解招募子通过共价连接得到，所述降解招募子为可招募泛素化的分子；
其中，R1选自—OCH3或—N(CH3)2；
所述降解招募子选自CRBN配体、VHL配体、MDM2配体或疏水标签，所述CRBN配体、VHL配体、MDM2配体和疏水标签的结构分别如式(II)-(V)所示：
所述共价连接为以非线性链、脂肪族链、芳香链或杂芳环结构链作为连接臂进行连接；
所述Gli1降解剂化合物选自如下结构中的一种：
2.权利要求1所述的靶向降解Gli1蛋白的降解剂的制备方法，其特征在于，包括以下步骤：
S1：化合物A与一端含有离去基团另一端含有保护基的连接臂反应后脱去保护基得到化合物B；
S2：化合物B通过酰胺缩合或亲核取代与降解招募子反应得到化合物C；
S3：化合物C与(3-(((苄氧基)羰基)氨基)丙基)(2-(二甲氨基)苄基)氨基甲酸叔丁酯或(3-氨基丙基)(4-甲氧基苄基)氨基甲酸叔丁酯通过还原胺化反应脱去保护基，得到所述靶向降解Gli1蛋白的降解剂；其中，化合物A为4-羟基苯甲醛，化合物B选自如下结构中的一种：
3.根据权利要求2所述的制备方法，其特征在于：所述离去基团为卤素或对甲苯磺酰基。
4.一种抗肿瘤药物，其特征在于：所述抗肿瘤药物包括权利要求1所述的靶向降解Gli1蛋白的降解剂。
5.权利要求1所述的靶向降解Gli1蛋白的降解剂在制备Hh/Gli1调节剂或抗肿瘤药物中的应用，其特征在于，所述Hh/Gli1调节剂或抗肿瘤药物用于治疗结肠癌。
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