CN113105431B - 一种蛋白水解靶向嵌合体及其应用 - Google Patents
一种蛋白水解靶向嵌合体及其应用 Download PDFInfo
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Abstract
本发明提供了一种蛋白水解靶向嵌合体及其应用。该技术方案基于BMS‑37小分子开发了新型PROTAC降解剂化合物21a。这是基于结合PD‑L1蛋白的细胞外结构域的配体,降解膜蛋白的实例。能够有效降解各种恶性肿瘤细胞中的PD‑L1。从体内研究的结果可以看出,化合物21a处理后可以显著降低肿瘤内PD‑L1的水平,促进CD8+T细胞的浸润并显著抑制小鼠结直肠癌MC‑38细胞的生长。该PROTAC分子有希望作为癌症免疫疗法的新颖和替代策略之一。
Description
技术领域
本发明涉及化学技术领域,进一步涉及化学合成技术及药物化学技术,具体涉及一种蛋白水解靶向嵌合体及其应用。
背景技术
免疫检查点是指那些抑制CTL激活(cytotoxic T lymphocyte,细胞毒性T淋巴细胞,又称杀伤性T细胞)的相互作用。这些检查点是免疫反应的自然设计,它充当CTL的“制动器”,允许CTL激活一段时间,但不允许反应无限期地进行,这种机制是免疫反应的众多调节机制之一。免疫检查点治疗引起越来越多的关注,抑制程序性细胞死亡受体1(PD-1)/程序性细胞死亡配体1(PD-L1)相互作用是最有希望的策略之一。
众多临床试验证明靶向PD-1/PD-L1的免疫检查点阻断抗体药物可以治疗癌症。并且应用免疫疗法治疗多种恶性实体瘤可以在体内产生长久的抗瘤反应,长期有效缓解病情。目前,存在一种抗CTLA4单克隆抗体(ipilimumab)和几种PD-1/PD-L1单克隆抗体(例如,抗PD-1:nivolumab,pembrolizumab和cemiplimab;抗PD-L1:atezolizumab,avelumab和durvalumab)已被美国食品药品监督管理局(FDA)批准,另外其他一些抗体药物也正在临床试验中。这些PD-1/PD-L1抗体被批准直接用于转移性黑色素瘤,非小细胞肺癌(NSCLC),肾细胞癌和其他不同类型的癌症。此外,从一些临床前和临床结果中可以看出,PD-1/PD-L1抗体与其他疗法的结合可以显著提高疗效和应答率。
最近,报道了一系列靶向PD-1/PD-L1相互作用的小分子,即大环肽,肽,拟肽和非肽小分子。其中的一些小分子抑制剂目前正处于临床试验阶段(例如:CA-170,CA-327和BMS-986189在临床试验Ⅰ期),这为靶向PD-1/PD-L1相互作用将提供互补的和潜在的协同疗法。
Craig.Crews最早提出了PROTAC(蛋白水解靶向嵌合体)概念。PROTAC是通过适当的Linker缀合的异双功能小分子。它们可以将靶蛋白拖至接近E3连接酶的位置,从而导致蛋白酶体降解靶蛋白。到目前为止,PROTAC技术已成为化学降解特定蛋白质以治疗肿瘤的重要工具。例如AR,MDM2,CDK6,CDK9,BRD,BET,ALK,PARP-1等,已经开发成为PROTAC分子。但是,关于GPCR降解的报道非常有限(例如,FLT3)。值得注意的是,大多数报道的PROTAC都是通过与靶蛋白胞内结构域结合的配体结构设计的,然而几乎没有报道发现与PD-L1的胞内结构域结合的配体。因此这成为设计靶向PD-L1的PROTAC分子的重要挑战。
发明内容
本发明旨在针对该靶点结构独特导致的设计缺陷,提供一种基于结合细胞外结构域的配体合成的蛋白水解靶向嵌合体。
本发明要解决的另一技术问题是,如何开发一种对结肠癌具有显著疗效的新型PROTAC分子。
为实现以上技术目的,本发明采用以下技术方案:
蛋白水解靶向嵌合体,其分子结构如式(I)和/或式(II)所示:
式(I)中,Linker为任一化学上可行的连接结构。
式(II)中,Linker为任一化学上可行的连接结构。
作为优选,Linker为饱和脂肪链、不饱和脂肪链或脂肪酸链。
作为优选,该蛋白水解靶向嵌合体的分子结构如式(Ia)所示:
式(Ia)中,n=4、5、6、7、8、或9。
作为优选,该蛋白水解靶向嵌合体的分子结构如式(Ib)所示:
式(Ib)中,n=2、3或4。
作为优选,该蛋白水解靶向嵌合体的分子结构如式(Ic)所示:
式(Ic)中,m=1时,n=2、3、5或6;m=2时,n=3、4或6。
作为优选,该蛋白水解靶向嵌合体的分子结构如式(IIa)所示:
式(IIa)中,n=2、3或4。
在以上技术方案的基础上,本发明进一步提供了上述蛋白水解靶向嵌合体在制备用于刺激肿瘤区域CD8+T细胞浸润药物中的应用。
在以上技术方案的基础上,本发明进一步提供了上述蛋白水解靶向嵌合体在制备癌症治疗药物中的应用。
作为优选,所述癌症为结肠癌。
在以上技术方案的基础上,本发明进一步提供了一种药物组合物,包含药学上可接受的赋形剂或载体,以及上述式(I)和/或(II)所示的化合物或其药学上可接受的盐。
与现有技术相比,本发明具有以下有益效果:
本发明合成并筛选了一种新的化合物,基于BMS-37小分子开发了新型PROTAC降解剂化合物。基于结合PD-L1蛋白的细胞外结构域的配体,降解膜蛋白,可以以剂量依赖性和时间依赖性的方式有效诱导各种癌细胞系中PD-L1的降解。可显著下调PD-L1蛋白的水平,促进CD8+T细胞的侵袭,通过体内试验表现出有效抑制抗结肠癌的生长作用。
附图说明
图1为PD-L1的细胞更新及其降解途径示意图;
图2为合成的PROTAC分子降解PD-L1蛋白作用的筛选
图3为本发明化合物21a的1H NMR光谱图;
图4为本发明化合物21a的13C NMR光谱图;
图5为本发明化合物21a依赖于细胞内蛋白酶体降解各种细胞系中的PD-L1蛋白试验结果图;
图6为本发明化合物21a降解细胞质中的PD-L1蛋白试验结果图;
图7为本发明化合物21a对MC-38肿瘤生长延迟和体内回归的影响试验结果图。
具体实施方式
以下将对本发明的具体实施方式进行详细描述。为了避免过多不必要的细节,在以下实施例中对属于公知的结构或功能将不进行详细描述。以下实施例中所使用的近似性语言可用于定量表述,表明在不改变基本功能的情况下可允许数量有一定的变动。除有定义外,以下实施例中所用的技术和科学术语具有与本发明所属领域技术人员普遍理解的相同含义。
1、PROTAC分子设计与化合物21a的合成
大多数报道的PROTAC都是通过与靶蛋白胞内结构域结合的配体结构设计的。然而几乎没有报道发现与PD-L1的胞内结构域结合的配体。发明人前期调研发现PD-L1蛋白能够不断循环并不断地从细胞质到细胞膜实现自我更新。并且细胞质中的PD-L1共有两种来源——新产生的和通过内吞作用从膜上“回收”的。因此,发明人设想基于结合细胞外结构域的配体合成和筛选出PROTAC分子。正如希望的,筛选出的PROTAC分子能够有效降解细胞质中的新产生或“循环”至胞内的PD-L1。这可能会阻碍PD-L1在膜上的更新,导致整个细胞以及细胞膜上的PD-L1蛋白水平均显著降低。(图1)发明人相信设想的成功将为设计PROTAC小分子降解膜蛋白提供可行的策略。
发明人发现一个名为“BMS-37”的二芳基醚小分子可以与PD-L1的胞外域结合,最初由于其与PD-L1的高度结合力而成为配体。通过使用与各种类型的接头连接的CRBN配体,设计了聚焦的二芳基醚类似物库。总体上,制备了18个PROTAC分子。经过仔细筛选,化合物21a被证明是降解各种恶性肿瘤细胞中的PD-L1的最有效的化合物。从体内研究的结果可以看出,化合物21a处理后可以显著降低肿瘤内PD-L1的水平,促进CD8+T细胞的浸润并显著抑制小鼠结直肠癌MC-38细胞的生长。该PROTAC分子有希望作为癌症免疫疗法的新颖和替代策略之一。
化合物11a-11f的合成路线如下(n=4,5,6,7,8或9):
反应试剂和条件:a Phenylboronic acid,Pd(dppf)Cl2.CH2Cl2,2M NaHCO3,Toluene/EtOH/H2O,98%;b PPh3,DIAD,无水THF 0℃,36%;c AcONa,HOAc回流,80%;dDIPEA,DMF,80℃,56%;e 1)TFA,CH2Cl2,rt;2)NaBH(OAc)3,DCE,rt,48-66%。
中间体3的合成:
向(3-溴-2-甲基苯基)甲醇(1.00g,4.96mmol)在C2H5OH(3.30mL)和甲苯(10mL)中的溶液中加入苯基硼酸(1.21g,9.92mmol)和Pd(dppf)Cl2.CH2Cl2(20.20mg,0.025mmol)和2M NaHCO3(10mL)。将混合物用氩气置换3次并回流3小时。然后将混合物用EtOAc(100mL)稀释,并用饱和NaCl溶液(50mL)洗涤,经Na2SO4干燥,在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(石油醚:乙酸乙酯=12:1至7:1),得到化合物3(1.07g,98%),为白色固体。Mp:74-76℃;IR(KBr):3350,3049,1600,1465,1050,755cm-1.1H NMR(400MHz,CDCl3)δ7.47–7.20(m,8H),4.78(s,2H),2.25(s,3H).13C NMR(101MHz,CDCl3)δ142.9,142.1,139.2,133.6,129.5,128.1,126.8,125.6,77.1,76.7,64.1,15.9.HRMS(ESI)calculated forC14H14NaO+[M+Na]+:221.0937,found.221.0939.
中间体5的合成:
向3(1.00g,5.05mmol)的无水THF(50mL)溶液中加入4-羟基-2,6-二甲氧基苯甲醛(1.19g,6.56mmol)和三苯基膦(2.51mg,9.59mmol)。在冰下缓慢滴加DIAD(1.94,9.59mmol)。将混合物在室温回流22小时,然后将混合物真空浓缩。将残余物通过硅胶上的柱色谱法纯化(石油醚:乙酸乙酯=5:1至4:1),得到化合物5(699mg,36%),为白色固体。Mp:161.5-162℃;IR(KBr):3020,2928,1657,1613,1610,1585,1430,1160cm-1.1H NMR(400MHz,CDCl3)δ10.38(s,1H),7.44–7.25(m,8H),6.21(s,2H),5.16(s,2H),3.89(s,6H),2.28(s,3H).13C NMR(101MHz,CDCl3)δ187.8,165.5,164.2,143.3,141.8,134.2,130.7,129.4,128.5,128.2,127.1,125.8,91.1,69.6,56.1,16.3.HRMS(ESI)calculated for C23H22NaO4 +[M+Na]+:385.1410,found.385.1409.
中间体8的合成:
向9-氟邻苯二甲酸酐6(3.30g,20.0mmol)的CH3COOH(100mL)溶液中加入3-氨基哌啶-2,6-二酮7(3.30g,20.0mmol)。将混合物回流12小时。然后将混合物用EtOAc(200mL)稀释,并用饱和HCl溶液(1N,50mL)洗涤,经Na2SO4干燥,在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=50:1),得到化合物8(4.78g,80%),为白色固体。Mp:289℃;IR(KBr):3180,1725,1706,739.6cm-1.1H NMR(400MHz,DMSO-d6)δ11.17(s,1H),7.95(ddd,J=8.3,7.3,4.5Hz,1H),7.89–7.65(m,2H),5.17(dd,J=12.9,5.4Hz,1H),2.97–2.76(m,1H),2.63–2.49(m,2H),2.18–1.95(m,1H).13C NMR(100MHz,DMSO-d6)δ172.8,169.7,166.2,164.0,158.1,155.5,138.1,133.5,123.0,120.1,49.1,30.9,21.9.HRMS(ESI)calculated for C13H9FN2NaO4+[M+Na]+:299.0438,found.299.0436.
中间体10a-f的合成(n=4,5,6,7,8或9):
以10a为例,向8(500mg,1.81mmol)的DMF(6mL)溶液中加入N-Boc-丁二胺9a(606mg,2.70mmol)。逐滴添加DIPEA(466mg,3.62mmol)。将混合物回流12小时,然后将混合物用EtOAc(30mL)稀释,用饱和NaCl溶液(15mL)洗涤,经Na2SO4干燥并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=15:1至12:1),得到化合物10a(338mg,40%),为深绿色荧光粘性液体化合物。IR(KBr):3338,3231,3106,2931,2868,1692,1541,1440,1326,1258,1193,876,628cm-1.1H NMR(400MHz,CDCl3)δ8.33(s,1H),7.48(dd,J=8.6,7.1Hz,1H),7.08(d,J=7.0Hz,1H),6.88(d,J=8.6Hz,1H),6.23(t,J=5.8Hz,1H),4.95–4.84(m,1H),4.62(s,1H),3.29(q,J=6.7Hz,2H),3.16(t,J=6.7Hz,2H),2.90–2.70(m,3H),2.18–2.05(m,1H),1.71–1.66(m,2H),1.63–1.56(m,2H),1.43(s,9H).13C NMR(100MHz,CDCl3)δ171.3,169.6,168.5,167.7,156.1,146.9,136.2,132.5,116.7,111.6,110.0,79.4,48.9,42.3,40.1,31.5,28.5,28.5,28.5,27.6,26.5,22.9.HRMS(ESI)calculated for C22H28N4NaO6 +[M+Na]+:467.1901,found467.1900.
与10a所述方法相同,从8和9b(N-Boc-戊二胺)得到化合物10b。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=16:1至12:1),产率30%,为深绿色荧光粘性液体化合物。IR(KBr):3330,3220,3108,2936,2872,1691,1544,1445,1396,1255,1196,880,605cm-1.1HNMR(400MHz,CDCl3)δ8.39–8.23(m,1H),7.48(dd,J=8.6,7.1Hz,1H),7.08(d,J=7.1Hz,1H),6.86(d,J=8.6Hz,1H),6.22(t,J=5.7Hz,1H),4.98–4.84(m,1H),4.58(s,1H),3.25(q,J=7.0Hz,2H),3.20–3.04(m,2H),2.91–2.69(m,3H),2.12(ddt,J=9.5,4.5,2.8Hz,1H),1.71–1.65(m,2H),1.53(t,J=7.7Hz,2H),1.43(s,11H).13C NMR(100MHz,CDCl3)δ171.0,169.3,168.3,167.4,155.8,146.7,135.9,132.3,116.4,111.2,109.7,79.0,48.7,42.3,40.2,31.2,29.7,28.7,28.2,28.2,28.2,23.9,22.6.HRMS(ESI)calculated forC23H30N4NaO6 +[M+Na]+:481.2057,found481.2059.
与10a所述方法相同,从8和9c(N-Boc-己二胺)得到化合物10c。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=16:1至12:1),产率40%,为深绿色荧光粘性液体化合物。IR(KBr):3350,3240,3118,2930,2872,1620,1545,1443,1392,1256,1196,882,603cm-1.1HNMR(400MHz,CDCl3)δ8.44(s,1H),7.47(dd,J=8.6,7.0Hz,1H),7.07(d,J=7.1Hz,1H),6.86(d,J=8.6Hz,1H),6.22(t,J=5.6Hz,1H),4.94–4.84(m,1H),4.57(s,1H),3.24(td,J=7.1,5.7Hz,2H),3.09(t,J=6.7Hz,2H),2.91–2.68(m,3H),2.16–2.07(m,1H),1.64(q,J=7.4Hz,2H),1.50–1.34(m,15H).13C NMR(100MHz,CDCl3)δ171.7,169.9,168.4,168.1,156.4,147.4,136.5,132.9,117.1,111.8,110.2,79.6,49.3,43.0,40.9,31.8,30.4,29.6,28.9,28.9,28.9,27.0,26.9,23.2.HRMS(ESI)calculated for C24H32N4NaO6 +[M+Na]+:495.2214,found.495.2216.
与10a所述方法相同,从8和9d(N-Boc-庚二胺)得到化合物10d。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=16:1至12:1),产率40%,为深绿色荧光粘性液体化合物。IR(KBr):3089,2926,2856,2848,1630,1541,1448,1382,1248,1188,876,601cm-1.1H NMR(400MHz,CDCl3)δ8.42(s,1H),7.47(dd,J=8.6,7.0Hz,1H),7.07(d,J=7.1Hz,1H),6.86(d,J=8.6Hz,1H),6.22(t,J=5.7Hz,1H),4.91(dd,J=12.2,5.6Hz,1H),4.58(s,1H),3.24(q,J=7.0Hz,2H),3.09(t,J=3.7Hz,2H),2.89–2.68(m,3H),2.11(ddt,J=9.5,4.5,2.8Hz,1H),1.63(d,J=7.6Hz,2H),1.48–1.30(m,17H).13C NMR(100MHz,CDCl3)δ171.3,169.6,168.6,167.7,156.1,147.1,136.2,132.6,116.8,111.5,110.0,79.2,49.0,42.7,40.7,31.5,30.1,29.2,29.0,28.6,28.6,28.6,26.9,26.8,22.9.HRMS(ESI)calculatedfor C25H34N4NaO6 +[M+Na]+:509.2370,found 509.2369.
与10a所述方法相同,从8和9e(N-Boc-辛二胺)得到化合物10e。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=16:1至12:1),产率45%,为深绿色荧光粘性液体化合物。IR(KBr):3084,2925,2858,2847,1631,1542,1448,1383,1241,1186,878,601cm-1.1H NMR(400MHz,CDCl3)δ8.18(s,1H),7.48(t,J=7.9Hz,1H),7.08(d,J=7.1Hz,1H),6.87(d,J=8.6Hz,1H),6.23(t,J=5.6Hz,1H),4.91(dd,J=12.1,5.4Hz,1H),4.53(s,1H),3.25(q,J=6.7Hz,2H),3.09(t,J=6.8Hz,2H),2.96–2.65(m,3H),2.16–2.09(m,1H),1.65(dd,J=12.7,5.5Hz,3H),1.44(s,12H),1.32(s,6H).13C NMR(100MHz,CDCl3)δ171.2,169.7,168.6,167.8,147.2,136.2,132.7,116.8,111.5,110.0,79.2,49.0,42.8,40.8,31.7,31.6,30.2,29.3,29.3,28.6,28.6,28.6,27.0,26.8,23.0,22.8.HRMS(ESI)calculated forC26H36N4NaO6 +[M+Na]+:523.2527,found 523.2529.
与10a所述方法相同,从8和9f(N-Boc-壬二胺)得到化合物10f。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=16:1至13:1),产率50%,为深绿色荧光粘性液体化合物。IR(KBr):3082,2915,2868,2845,1633,1545,1465,1387,1281,1188,875,605cm-1.1H NMR(400MHz,CDCl3)δ8.14(s,1H),7.49(dd,J=8.5,7.1Hz,1H),7.09(d,J=7.1Hz,1H),6.88(d,J=8.5Hz,1H),6.23(t,J=5.6Hz,1H),4.92(dd,J=12.1,5.3Hz,1H),4.53(s,1H),3.26(q,J=6.6Hz,2H),3.10(q,J=6.7Hz,2H),2.82(m,3H),2.13(ddd,J=12.4,5.7,3.2Hz,1H),1.65(dd,J=13.5,6.1Hz,3H),1.37(d,J=56.9Hz,21H).13C NMR(101MHz,CDCl3)δ22.8,26.8,26.9,28.5,29.2,29.4,30.1,31.4,42.6,48.9,111.4,116.7,132.5,136.1,147.0,167.7,168.4,169.5,171.0.HRMS(ESI)calculated for C27H38N4NaO6 +[M+Na]+:537.2683,found.537.2686.
目标化合物11a-11f的合成(n=4,5,6,7,8或9):
将10a(400mg,0.900mmol)用TFA(2mL)的CH2Cl2(8mL)溶液在室温处理1h。将混合物在减压下浓缩得到深绿色荧光粘稠液体化合物的中间体化合物,将其直接添加至5(296mg,0.900mmol)的无水DCE(18mL)溶液中。然后在室温下将得到的混合物加入NaBH(OAc)3(152mg,2.43mmol)。将反应混合物用EtOAc(50mL)稀释,并用饱和NaCl溶液(25mL)洗涤,经Na2SO4干燥,在减压下浓缩,得到呈绿色荧光粉末的化合物11a(423mg,66%)。Mp:122.1-123.5℃;IR(KBr):3020,2855,2360,1696,1540,1407,1262,813,797cm-1.1H NMR(400MHz,CDCl3)δ7.45–7.25(m,9H),7.03(d,J=7.1Hz,1H),6.82(d,J=8.5Hz,1H),6.22(s,3H),5.05(s,2H),4.89(s,1H),4.17(s,2H),3.82(s,6H),3.24(d,J=6.8Hz,2H),2.91–2.61(m,5H),2.25(s,3H),2.08(d,J=10.2Hz,1H),1.92(s,2H),1.67(q,J=7.6Hz,2H).13C NMR(101MHz,CDCl3)δ171.2,168.4,161.8,159.8,146.5,130.4,129.2,128.3,128.0,126.8,125.6,116.5,111.5,109.8,99.3,90.9,69.2,55.7,48.7,45.1,39.2,31.3,26.5,23.2,22.6,16.1.HRMS(ESI)calculated for C40H42N4NaO7+[M+Na]+:713.2945,found713.2948.
与11a所述的方法相同,由10b和5得到目标化合物11b(59%),为绿色荧光粉末。Mp:121.6-122.1℃;IR(KBr):3010,2856,2360,1696,1540,1407,1262,815,797cm-1.1HNMR(400MHz,CDCl3)δ7.44–7.21(m,9H),7.05(d,J=7.0Hz,1H),6.84(d,J=8.6Hz,1H),6.21(d,J=15.4Hz,3H),5.06(s,2H),4.88(s,1H),4.18(s,2H),3.83(s,6H),3.22(q,J=6.6Hz,2H),2.93–2.66(m,5H),2.25(s,3H),2.16–2.06(m,1H),1.87(s,2H),1.63(t,J=7.5Hz,2H),1.41(d,J=7.8Hz,2H).13C NMR(101MHz,CDCl3)δ171.0,169.4,168.4,161.9,159.8,146.7,143.8,136.1,134.5,130.4,129.3,128.2,128.1,126.9,125.6,116.9,111.7,109.8,99.2,91.0,69.5,55.8,48.8,39.2,31.3,28.6,25.3,24.0,22.7,16.2.HRMS(ESI)calculated for C41H44N4NaO7+[M+Na]+:727.3102,found 727.3100.
与11a所述的方法相同,由10c和5得到目标化合物11c(57%),为绿色荧光粉末。Mp:124.5-126.1℃;IR(KBr):3185,3020,2360,1696,1540,1407,1262,813,797cm-1.1HNMR(400MHz,CDCl3)δ7.37(m,8H),7.04(d,J=7.1Hz,1H),6.84(d,J=8.6Hz,1H),6.21(d,J=13.4Hz,3H),5.07(s,2H),4.88(dd,J=12.0,5.3Hz,1H),4.19(s,2H),3.85(s,6H),3.21(q,J=6.6Hz,2H),2.78(m,5H),2.25(s,3H),2.10(m,1H),1.89(s,2H),1.62(p,J=6.9Hz,2H),1.37(t,J=7.3Hz,4H).13C NMR(100MHz,CDCl3)δ171.15,169.49,168.48,167.63,161.85,159.95,146.90,143.14,141.78,136.17,134.62,134.55,130.53,129.37,128.35,128.15,126.98,125.73,116.73,111.43,99.46,91.06,69.41,55.90,48.89,45.31,42.46,39.13,31.42,28.98,26.60,26.39,25.63,22.80,16.28.HRMS(ESI)calculated forC42H46N4NaO7+[M+Na]+:741.3258,found 741.3257.
与11a所述的方法相同,由10d和5得到目标化合物11d(65%),为绿色荧光粉末。Mp:122.1-123.5℃;IR(KBr):2935,2855,2360,1698,1615,1419,1408,797,747cm-1.1HNMR(400MHz,CDCl3)δ7.48–7.25(m,9H),7.06(d,J=7.1Hz,1H),6.85(d,J=8.5Hz,1H),6.23(s,2H),6.20(t,J=6.3Hz,1H),5.08(s,2H),4.90(s,1H),4.21(s,2H),3.85(s,6H),3.22(d,J=6.4Hz,2H),3.00–2.55(m,5H),2.26(s,3H),2.15–2.05(m,1H),1.86(s,2H),1.60(d,J=7.4Hz,2H),1.31(dd,J=7.9,4.0Hz,6H).13C NMR(100MHz,CDCl3)δ170.7,161.4,159.4,146.5,142.6,135.6,134.1,134.0,131.9,130.0,128.8,127.8,127.6,126.5,125.2,116.2,110.9,98.9,90.5,68.9,55.4,48.4,42.1,38.6,30.5,28.0,28.2,26.2,26.2,25.0,22.2,15.8.HRMS(ESI)calculated for C43H48N4NaO7+[M+Na]+:755.3415,found 755.3414.
与11a所述的方法相同,由10e和5得到目标化合物11e(47%),为绿色荧光粉末。Mp:128.5-129.8℃;IR(KBr):2929,2854,2359,2342,1558,1547,1229,1173,762,719cm-1.1HNMR(400MHz,CDCl3)δ7.51–7.26(m,10H),7.06(dd,J=7.1,1.4Hz,1H),6.86(dd,J=8.6,1.4Hz,1H),6.23(d,J=1.5Hz,3H),5.07(d,J=1.5Hz,2H),4.93–4.76(m,1H),4.20(s,2H),3.83(d,J=1.5Hz,6H),3.23(d,J=6.4Hz,2H),2.89–2.65(m,5H),2.26(d,J=1.5Hz,3H),2.18–2.07(m,1H),1.87–1.77(m,2H),1.61(t,J=7.1Hz,2H),1.40–1.20(m,10H).13CNMR(101MHz,CDCl3)δ171.1,168.6,167.6,159.9,146.9,143.1,136.1,134.5,134.5,130.5,129.3,128.3,128.1,126.9,125.7,116.7,111.3,99.3,91.0,69.4,55.8,48.9,39.2,31.4,29.0,28.9,26.7,26.6,25.6,22.8,16.2.HRMS(ESI)calculated forC44H50N4NaO7+[M+Na]+:769.3571,found 769.3574.
与11a所述的方法相同,由10f和5得到目标化合物11f(59%),为绿色荧光粉末。Mp:130.4-131.9℃;IR(KBr):2965,2860,2360,2343,1696,1598,1324,1264,813,750cm-1.1H NMR(400MHz,CDCl3)δ7.51–7.34(m,5H),7.34–7.26(m,4H),7.06(d,J=7.1Hz,1H),6.86(d,J=8.5Hz,1H),6.23(d,J=1.1Hz,2H),6.21(d,J=6.0Hz,1H),5.08(s,2H),4.98–4.80(m,1H),4.21(s,2H),3.84(d,J=1.1Hz,6H),3.23(d,J=6.4Hz,2H),2.95–2.65(m,5H),2.26(d,J=1.1Hz,3H),2.15–2.04(m,1H),1.83(d,J=9.7Hz,2H),1.68–1.56(m,2H),1.41–1.34(m,2H),1.27(d,J=7.8Hz,10H).13C NMR(101MHz,CDCl3)δ170.6,169.0,161.4,159.5,146.5,141.3,135.7,134.0,128.9,127.8,127.7,126.5,125.2,116.2,110.8,99.0,90.6,68.9,55.4,48.4,42.0,31.0,28.6,28.5,28.4,28.3,26.3,26.3,25.2,22.3,15.8.HRMS(ESI)calculated for C45H52N4NaO7+[M+Na]+:783.3728,found 783.3731.
化合物18a-c和21a-c的合成路线如下(n=2,3或4):
反应试剂和条件:a EDCI,DIPEA,MeCN,HOBt,rt,82-87%;b 1)TFA,CH2Cl2,rt 2)NaBH(OAc)3,DCE,5,rt 48-66%;c AcONa,HOAc,reflux 85%;d Pd(PPh3)2Cl2,DIPEA,15a-c K2CO3,CuI,DMF,80℃47%-49%;
中间体14a-c的合成(n=2,3或4)
以14a为例,向N-Boc-乙二胺13(4.00g,24.9mmol)在CH3CN(150mL)中的溶液中添加戊炔酸12a(3.00g,30.5mmol),EDCI(7.30g,38.0mmol),HoBt(5.16g,38.0mmol))和DIPEA(12.6mL),将混合物在室温搅拌18小时,然后将混合物用EtOAc(500mL)稀释,并用饱和NaHCO3水溶液(200mL)和1N HCl(150mL)洗涤,经Na2SO4干燥,并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=60:1),得到化合物14a(5.41g,83%),为白色固体。Mp:83.5-85.0℃;IR(KBr):3301,3296,2980,1654,1550,1284,860,635cm-1.1H NMR(400MHz,CDCl3)δ6.36(s,1H),4.97(s,1H),3.38(td,J=6.5,5.7,4.4Hz,2H),3.28(q,J=5.8Hz,2H),2.53(td,J=6.9,2.5Hz,2H),2.40(t,J=7.0Hz,2H),2.01(s,1H),1.44(s,9H).13C NMR(100MHz,CDCl3)δ171.70,157.05,83.10,69.42,40.89,40.26,35.46,28.49,15.05.HRMS(ESI)calculated for C12H20N2NaO3 +[M+Na]+:263.1366,found 263.1368.
与14a所述的方法相同,由12b(己炔酸)和13得到目标化合物14b。在硅胶上进行柱色谱纯化(二氯甲烷:甲醇=60:1),得到化合物14b(80%),为白色固体。Mp:81.2-82.0℃;IR(KBr):3341,3294,2981,1656,1557,1286,861,637cm-1.1H NMR(400MHz,CDCl3)δ6.18(s,1H),4.91(s,1H),3.31(dq,J=37.2,5.5Hz,4H),2.27(m,4H),1.96(t,J=2.7Hz,1H),1.85(p,J=7.1Hz,2H),1.44(s,9H).13C NMR(101MHz,CDCl3)δ172.8,83.4,69.1,40.6,40.3,40.3,35.0,28.3,24.1,17.8.HRMS(ESI)calculated for C13H22N2NaO3 +[M+Na]+:277.1522found 277.1520.
与14a所述的方法相同,由12c(庚炔酸)和13得到目标化合物14c。在硅胶上进行柱色谱纯化(二氯甲烷:甲醇=60:1),得到化合物14c(75%),为白色固体。Mp:80.1-80.9℃;IR(KBr):3341,3294,2981,1656,1557,1286,861,637cm-1.1H NMR(400MHz,CDCl3)δ6.40(s,1H),5.08(s,1H),3.31(dq,J=36.7,5.8Hz,4H),2.21(t,J=7.1Hz,4H),1.95(q,J=2.4Hz,1H),1.75(p,J=7.6Hz,2H),1.56(p,J=7.3Hz,2H).13C NMR(100MHz,CDCl3)δ173.3,156.9,84.0,79.6,68.6,40.6,40.2,36.0,28.3,27.9,24.7,18.1.HRMS(ESI)calculated forC14H24N2NaO3 +[M+Na]+:291.1679,found 291.1678.
中间体15a-c的合成(n=2,3或4):
以15a为例,用Et3N将14a(1.18g,8.45mmol)在CH2Cl2(30mL)中的溶液调节至pH5-6。在室温下将化合物5(1.50g,4.14mmol)加入溶液中4小时。然后将混合物用EtOAc(100mL)稀释并用饱和NaCl溶液(50mL)洗涤,经Na2SO4干燥并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=60:1),得到化合物15a(3.44g,80%),为白色固体。Mp:87.1-88.2℃;IR(KBr):3058,2929,2840,1716,1610,1499,1197,1142,745,703cm-1.1H NMR(400MHz,CDCl3)δ8.86(s,2H),8.00(t,J=5.9Hz,1H),7.42(m,3H),7.37(m,1H),7.31(m,2H),7.28(d,J=3.3Hz,1H),6.22(s,2H),5.06(s,2H),4.14(s,2H),3.82(s,6H),3.50(d,J=4.9Hz,2H),3.04(t,J=5.0Hz,2H),2.45(m,2H),2.40(d,J=6.4Hz,2H),2.26(s,3H),1.91(t,J=2.5Hz,1H).13C NMR(100MHz,CDCl3)δ172.4,162.3,159.9,143.2,134.6,130.6,129.4,128.4,128.2,127.0,125.8,99.0,91.1,69.5,69.0,55.9,46.9,40.3,36.0,34.7,16.3,14.7.HRMS(ESI)calculated for C30H34N2NaO4 +[M+Na]+:509.2411,found 509.2412.
与15a所述的方法相同,从5和14b得到目标化合物15b。在硅胶上柱色谱纯化(二氯甲烷:甲醇=60:1),得到化合物15b(75%),为白色固体。Mp:89.5-90.1℃;IR(KBr):3058,2929,2840,1718,1627,1501,1197,1144,748,705cm-1.1H NMR(400MHz,CDCl3)δ8.00(t,J=5.9Hz,1H),7.35(m,8H),6.22(s,2H),5.06(s,2H),4.15(s,2H),3.84(s,6H),3.52(q,J=5.2Hz,2H),2.99(m,2H),2.33(t,J=7.5Hz,2H),2.25(s,3H),2.20(td,J=7.1,2.7Hz,2H),1.93(t,J=2.6Hz,1H),1.82(p,J=7.2Hz,2H).13C NMR(101MHz,CDCl3)δ173.4,162.1,159.7,143.1,141.8,134.5,130.5,129.3,128.3,128.1,126.9,125.7,98.8,91.0,69.4,69.0,55.8,46.9,40.0,35.8,34.8,24.2,18.0,16.2.HRMS(ESI)calculated forC31H36N2NaO4 +[M+Na]+:523.2567,found 523.2566.
与15a所述的方法相同,从5和14c得到目标化合物15c。在硅胶上柱色谱纯化(二氯甲烷:甲醇=60:1),得到化合物15c(78%),为白色固体。Mp:88.4-89.3℃;IR(KBr):3028,2924,2830,1719,1628,1506,1199,1144,758,709cm-1.1H NMR(400MHz,CDCl3)δ7.82(t,J=5.9Hz,1H),7.35(m,8H),6.22(s,2H),5.06(s,2H),4.14(s,2H),3.81(s,6H),3.49(q,J=5.5Hz,2H),3.03(t,J=4.9Hz,2H),2.25(s,3H),2.17(m,4H),1.91(t,J=2.7Hz,1H),1.69(p,J=7.5Hz,2H),1.51(m,2H).13C NMR(101MHz,CDCl3)δ174.1,162.2,159.8,134.5,130.5,129.4,128.3,128.1,127.0,125.7,99.0,91.1,69.4,68.5,55.8,46.9,40.1,35.9,35.5,28.0,24.6,18.2,16.2.HRMS(ESI)calculated for C32H38N2NaO4 +[M+Na]+:537.2724,found 537.2726.
中间体17的合成:
向16(4.54g,20.0mmol)的CH3COOH(100mL)溶液中加入7(2.20g,20.0mmol)。将混合物在120℃下回流12小时。然后将混合物用EtOAc(200mL)稀释并用饱和HCl溶液(50mL)洗涤,经Na2SO4干燥并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=50:1),得到化合物17(5.89g,82%),为白色固体。Mp>250℃(decom.);IR(KBr):3195,1724,1706.7,739.4cm-1.1H NMR(400MHz,DMSO-d6)δ11.14(s,1H),8.20–8.04(m,2H),7.86(d,J=7.9Hz,1H),5.17(dd,J=12.8,5.4Hz,1H),2.89(ddd,J=17.0,13.8,5.4Hz,1H),2.66–2.51(m,2H),2.07(ddq,J=10.5,5.4,2.9,2.5Hz,1H).13C NMR(100MHz,DMSO-d6)δ172.7,169.6,166.4,165.8,137.6,133.1,130.1,128.5,126.4,125.2,49.2,30.9,21.9.HRMS(ESI)calculated for C13H9BrN2NaO4 +[M+Na]+:358.9638,found 358.9639.
目标化合物18a-18c的合成(n=2,3或4):
以18a为例,向17(0.21g,0.62mmol)在DMF(3mL)中的溶液中添加15a(0.30g,0.62mmol),Pd(PPh3)2Cl2(43mg,0.060mmol),CuI(11mg,0.060mmol)和DIPEA(1mL)。将混合物用氩气替换三次,并在50℃下搅拌12h。然后将混合物用EtOAc(15mL)稀释,并用饱和NaCl溶液(5mL)洗涤,经Na2SO4干燥,在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=20:1至5:1),得到化合物18a(0.31g,66%),为白色固体。Mp:145.7-147.1℃;IR(KBr):2925,2849,1717,1636,1595,1420,1377,1141,1113,732,707cm-1.1H NMR(400MHz,CDCl3)δ7.95(s,1H),7.76–7.72(m,1H),7.66–7.60(m,2H),7.44–7.26(m,9H),6.19(d,J=1.3Hz,2H),5.06(s,3H),4.16(d,J=3.5Hz,2H),3.82(s,6H),3.63(d,J=5.5Hz,2H),2.99(t,J=5.4Hz,2H),2.80(q,J=7.4Hz,5H),2.59(t,J=7.0Hz,2H),2.25(d,J=1.3Hz,3H),2.16–2.11(m,1H).13C NMR(100MHz,CDCl3)δ171.8,171.0,168.0,166.9,166.8,162.4,160.0,143.3,141.9,137.5,134.7,132.0,130.8,130.7,130.0,129.5,128.5,128.3,127.1,126.7,125.9,123.7,98.8,94.8,91.3,69.6,56.1,49.6,46.9,40.3,35.9,35.0,31.5,29.8,29.8,29.5,22.8,22.7,16.4,15.9.HRMS(ESI)calculated forC43H42N4NaO8+[M+Na]+:765.2895,found 765.2897.
与18a所述的方法相同,从17和15b得到目标化合物18b。用硅胶柱色谱纯化(二氯甲烷:甲醇=20:1至5:1),得到化合物18b(59%),为白色固体。Mp:146.8-147.9℃;IR(KBr):2926,2850,1777,1636,1595,1420,1387,1140,1113,732,707cm-1.1H NMR(400MHz,CDCl3)δ8.12(d,J=6.3Hz,1H),7.83(d,J=1.1Hz,1H),7.72(s,2H),7.35(m,8H),6.21(s,2H),5.05(s,2H),4.94(m,1H),4.19(s,2H),3.84(s,6H),3.61(q,J=5.7Hz,2H),2.82(m,5H),2.46(dt,J=17.2,7.3Hz,4H),2.24(s,3H),2.11(ddd,J=10.0,6.3,2.4Hz,1H),1.94(p,J=7.3Hz,2H).13C NMR(101MHz,CDCl3)δ173.14,170.72,167.82,166.70,162.17,159.76,143.12,141.74,137.44,134.48,130.56,129.34,128.40,128.13,126.96,126.62,125.71,123.59,98.65,91.07,79.93,69.43,55.90,49.37,46.81,40.11,35.74,35.16,31.37,29.70,24.27,22.57,19.21,16.26.HRMS(ESI)calculated for C44H44N4NaO8+[M+Na]+:779.3051,found 779.3050.
与18a所述的方法相同,从17和15c得到目标化合物18c。用硅胶上进行柱色谱纯化(二氯甲烷:甲醇=10:1至5:1),得到化合物18c(57%),为白色固体。Mp:149.7-150.8℃;IR(KBr):2939,2843,1716,1656,1595,1420,1397,1141,1113,745,737cm-1.1H NMR(400MHz,CDCl3)δ8.12(t,J=5.8Hz,1H),7.74(m,3H),7.36(m,8H),6.21(s,2H),5.05(s,2H),4.95(dd,J=12.2,5.3Hz,1H),4.18(s,2H),3.85(s,7H),3.60(t,J=5.4Hz,2H),2.83(m,6H),2.46(t,J=7.0Hz,2H),2.31(t,J=7.4Hz,2H),2.25(s,3H),2.13(m,1H),1.79(p,J=7.4Hz,2H),1.63(d,J=15.5Hz,2H).13C NMR(100MHz,CDCl3)δ173.62,170.92,162.13,159.78,141.77,137.37,134.54,131.82,130.94,130.56,129.36,128.38,128.14,126.97,126.61,125.73,123.62,91.07,69.43,55.91,49.38,40.09,35.72,31.38,27.95,24.80,22.58,19.39,16.26.HRMS(ESI)calculated for C45H46N4NaO8+[M+Na]+:793.3208,found793.3209.
中间体20的合成:
向9-碘邻苯二甲酸酐19(2.00g,7.30mmol)的CH3COOH(100mL)溶液中加入3-氨基哌啶-2,6-二酮7(935.2mg,7.30mmol)。将混合物120℃回流6小时。然后将混合物用EtOAc(200mL)稀释,并用饱和HCl溶液(1N,50mL)洗涤,经Na2SO4干燥,在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=50:1),得到化合物20(2.24,80%),为白色固体。Mp:304–305℃;IR(KBr):3205,1726,1390,1202,737.9cm-1;1H NMR(400MHz,CDCl3)δ11.13(s,1H),8.27(d,J=7.9Hz,1H),7.92(d,J=7.3Hz,1H),7.58(t,J=7.6Hz,1H),5.16(dd,J=12.8,5.4Hz,1H),2.89(ddd,J=18.0,13.8,5.4Hz,1H),2.56(m,3H),2.08(m,1H)ppm.13C NMR(101MHz,CDCl3)δ177.94,174.94,171.24,170.63,150.69,140.90,138.48,137.04,128.45,95.61,54.42,44.70,36.15,27.07ppm.HRMS(ESI)calculated forC13H10IN2O4 +[M+H]+:384.9685,found.384.9663.
目标化合物21a-21c的合成(n=2,3或4):
以21a为例,向化合物20(0.14g,0.37mmol)在DMF(2mL)中的溶液中添加15a(0.18g,0.37mmol),Pd(PPh3)2Cl2(25mg,0.037mmol),CuI(7.1mg,0.037mmol))和DIPEA(1mL)。将混合物用氩气替换3次,并在50℃下搅拌12h。然后将混合物用EtOAc(15mL)稀释,并用饱和NaCl溶液(5mL)洗涤,经Na2SO4干燥并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=10:1至5:1),得到化合物21a(183mg,65%),为白色固体。Mp:142.1-143.5℃;IR(KBr):2920,2840,1720,1646,1597,1422,1397,1151,1113,740,737cm-1.1H NMR(400MHz,CDCl3)δ7.89(m,1H),7.75(dd,J=6.5,1.9Hz,1H),7.65(m,2H),7.36(m,9H),6.19(s,2H),5.06(s,3H),4.17(m,2H),3.81(s,6H),3.64(m,2H),3.02(t,J=5.3Hz,2H),2.79(ddt,J=18.1,16.6,6.3Hz,5H),2.58(t,J=6.9Hz,2H),2.25(s,3H),2.16(m,1H).13C NMR(100MHz,CDCl3)δ172.3,171.4,168.7,166.7,166.5,159.9,143.2,138.4,134.7,134.1,132.2,130.6,129.4,128.5,128.2,127.0,125.8,122.9,97.7,91.2,69.5,56.6,49.6,40.2,36.4,35.2,31.5,22.8,16.4,16.4.HRMS(ESI)calculated forC43H42N4NaO8+[M+Na]+:765.2895,found 762.2899.
与21a所述的方法相同,从20和15b得到目标化合物21b。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=10:1至5:1),得到化合物21b(47%),为白色固体。Mp:143.2-144.5℃;IR(KBr):3057,2939,1653,1595,1391,1260,1142,1024,745,730cm-1.1H NMR(400MHz,CDCl3)δ7.77(dt,J=7.1,1.2Hz,1H),7.66(m,3H),7.35(m,8H),6.21(s,2H),5.08(m,3H),4.17(m,2H),3.81(m,6H),3.66(m,2H),3.02(d,J=5.5Hz,2H),2.81(m,3H),2.53(m,4H),2.26(s,3H),2.17(m,1H),1.92(m,2H).13C NMR(101MHz,CDCl3)δ173.7,171.3,168.6,159.7,143.1,141.7,138.3,134.5,134.5,134.0,132.1,130.5,129.3,128.4,128.1,126.9,125.7,122.7,98.2,91.1,69.4,55.9,49.5,46.6,40.2,36.4,34.3,31.3,23.7,22.7,18.7,16.2.HRMS(ESI)calculated for C44H44N4NaO8+[M+Na]+:779.3051,found779.3050.
与21a所述的方法相同,从20和15c得到目标化合物21c。在硅胶上进行柱色谱纯化(二氯甲烷:甲醇=10:1至5:1),得到化合物21c(59%),为白色固体。Mp:141.9-143.1℃;IR(KBr):3020,2915,1645,1590,1381,1255,1139,1037,746,735cm-1.1H NMR(400MHz,CDCl3)δ7.97(s,1H),7.72(dd,J=7.4,3.3Hz,1H),7.62(m,2H),7.34(m,8H),6.20(t,J=2.5Hz,2H),5.05(d,J=3.4Hz,2H),4.97(m,1H),4.16(s,2H),3.83(m,6H),3.60(s,2H),2.97(d,J=5.8Hz,2H),2.77(dq,J=33.2,11.5,10.7Hz,3H),2.52(d,J=7.7Hz,2H),2.32(d,J=8.4Hz,2H),2.25(m,3H),2.07(d,J=8.4Hz,1H),1.82(q,J=8.0Hz,2H),1.67(m,2H).13C NMR(100MHz,CDCl3)δ171.35,168.56,166.54,162.03,159.80,143.09,141.80,138.38,134.61,134.55,133.84,132.17,130.49,129.36,128.38,128.13,126.94,125.70,122.47,99.38,99.09,91.06,69.40,55.91,49.27,46.84,40.10,35.92,35.78,31.39,27.75,24.77,22.67,19.62,16.25.HRMS(ESI)calculated for C45H46N4NaO8+[M+Na]+:793.3208,found 793.3206.
化合物30a-g的合成路线如下:(m=1时,n=2,3,5,或6;m=2时,n=3,4或6)
反应试剂和方法:a NaBH(OAc)3,DCE,rt 61-86%;b LiOH,H2O MeOH,80℃,93-95%;c AcONa,HOAc,reflux 85%;d tert-Butyl bromoacetate,KHCO3,KI,DMF,60℃,75%;e 1)TFA,CH2Cl2,rt 2)HATU,DIPEA,DMF,rt,77-80%;f 1)TFA,CH2Cl22)HATU,DIPEA,DMF,rt,41-48%
中间体23a-b的合成(m=1或2):
以23a为例,在85℃下向5(1.00g,2.76mmol)的CH2Cl2(20mL)溶液中添加22a(1.04g,8.28mmol)和NaBH(AcO)3(1.75g,8.28mmol)。然后将混合物用EtOAc(80mL)稀释,并用饱和NaCl溶液(20mL)洗涤,经Na2SO4干燥,在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=25∶1),得到化合物23a(1.06g,84%),为白色固体。Mp:82.1-82.9℃;IR(KBr):3338,2993,2942,2841,1737,1609,1594,1496,1485,1461,1386,1348,1330,1272,1200,1149,1112,1064,1032,969,898,849,804,764,701,646,615cm-1.1H NMR(400MHz,CDCl3)δ7.46–7.40(m,3H),7.38–7.35(m,1H),7.34–7.30(m,2H),7.29–7.25(m,2H),6.24(s,2H),5.09(s,2H),3.86(s,2H),3.81(s,6H),3.68(s,3H),3.39(s,2H),2.27(s,3H).13C NMR(101MHz,CDCl3)δ172.9,160.1,159.6,143.1,142.0,135.1,134.6,130.4,129.5,128.4,128.2,127.0,125.7,107.9,91.2,69.4,55.8,51.8,49.6,40.6,16.3.HRMS(ESI)calculated for C26H29NNaO5 +[M+Na]+:458.1938,found 458.1935.
与23a所述的方法相同,从5和22b得到目标化合物23b。在硅胶上进行柱色谱纯化(二氯甲烷:甲醇=10:1至5:1),得到化合物23b(79%),为白色固体。Mp:93.3-94.6℃;IR(KBr):3020,2941,2885,2840,2745,2679,2620,2487,2447,2356,1734,1613,1597,1578,1461,1382,1294,1240,1178,1139,1027,982,914,851,783,710,637,592cm-1.1H NMR(400MHz,CDCl3)δ7.41(d,J=7.2Hz,3H),7.36(d,J=7.0Hz,1H),7.33–7.29(m,2H),7.28–7.25(m,2H),6.25(s,2H),5.08(s,2H),4.22(s,2H),3.86(s,6H),3.66(s,3H),3.08–2.97(m,4H),2.26(s,3H).13C NMR(100MHz,CDCl3)δ172.3,161.9,159.8,143.1,141.8,134.6,134.6,130.5,129.4,128.4,128.1,126.9,125.7,99.7,91.2,69.4,55.9,52.1,41.6,40.1,30.1,16.3.HRMS(ESI)calculated for C27H31NNaO5 +[M+Na]+:472.2094,found 472.2091.
中间体24a-b的合成(m=1或2):
以24a为例,将LiOH在水(15mL)中的溶液在80℃下于23a(1.02g,2.34mmol)在MeOH(22mL)中的溶液中处理3小时。用1N HCl将混合物的pH调节至3。然后将混合物减压浓缩,得到化合物24a(935mg,90%),为白色固体。Mp:102.3-103.6℃.IR(KBr):3394,2941,2841,1612,1594,1498,1463,1420,1386,1320,1232,1198,1139,1045,1026,808,762,702cm-1.1HNMR(400MHz,DMSO-d6)δ7.51–7.42(m,3H),7.38(t,J=7.3Hz,1H),7.34–7.30(m,2H),7.28(d,J=7.7Hz,1H),7.23–7.17(m,1H),6.44(s,2H),5.20(s,2H),4.02(s,2H),3.81(s,6H),3.29(s,2H),2.21(s,3H).13C NMR(100MHz,DMSO-d6)δ167.5,161.4,159.6,142.2,141.4,135.3,134.1,129.8,129.2,128.4,128.3,127.0,125.6,99.9,91.5,68.8,56.0,47.3,40.1,16.0.HRMS(ESI)calculated for C25H27NNaO5 +[M+Na]+:444.1781,found 444.1782.
与24a所述的方法相同,从23b得到目标化合物24b。减压浓缩,得到化合物24b(90%),为白色固体。Mp:111.3-112.1℃;IR(KBr):3616,3300,2936,2856,1594,1500,1436,1418,1347,1313,1239,1197,1178,1138,1100,1029,975,810,765,739,703,635,593cm-1.1H NMR(400MHz,DMSO-d6)δ7.50–7.42(m,3H),7.37(t,J=7.3Hz,1H),7.34–7.29(m,2H),7.26(d,J=7.6Hz,1H),7.19(d,J=7.6Hz,1H),6.35(s,2H),5.14(s,2H),3.75(s,6H),3.60(s,2H),2.54(t,J=6.8Hz,2H),2.20(s,3H),2.05(t,J=6.6Hz,2H).13C NMR(101MHz,DMSO-d6)δ176.6,159.2,159.0,142.2,141.4,135.6,134.0,129.7,129.2,128.2,127.0,125.5,108.5,91.5,68.6,55.7,45.6,37.7,15.9.HRMS(ESI)calculated forC26H29NNaO5 +[M+Na]+:458.1938,found 458.1936.
中间体26的合成:
向9-羟基邻苯二甲酸酐25(2.00g,7.30mmol)的CH3COOH(100mL)溶液中加入3-氨基哌啶-2,6-二酮7(935.2mg,7.30mmol)。将混合物回流6小时。然后将混合物用EtOAc(200mL)稀释,并用饱和HCl溶液(1N,50mL)洗涤,经Na2SO4干燥,在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=50:1),得到化合物26(2.24,80%),为白色固体。M.P.243.5-244.9℃;IR(KBr):3285,2972,2897,1852,1771,1759,1572,1402,1360,1305,1259,1169,777,596cm-1.1H NMR(400MHz,DMSO-d6)δ11.16(d,J=43.1Hz,2H),7.73–7.56(m,1H),7.28(ddd,J=27.4,7.8,2.2Hz,2H),5.07(ddd,J=12.9,5.4,1.9Hz,1H),2.94–2.81(m,1H),2.58(d,J=16.5Hz,2H),2.02(dq,J=12.9,5.7,4.2Hz,1H).13C NMR(100MHz,DMSO-d6)δ172.9,170.1,167.1,165.9,155.5,136.4,133.2,123.6,114.4,114.3,48.7,31.0,22.1.HRMS(ESI)calculated for C13H10N2NaO5+[M+Na]+:297.0482,found297.0481.
中间体27的合成:
向26(3.00g,11.0mmol)的DMF(20mL)溶液中添加KI(182mg,1.10mmol),KHCO3(1.65g,16.5mmol)和溴乙酸叔丁酯(2.57g,13.2mmol)逐滴添加。将混合物在60℃下搅拌3小时。然后将混合物用EtOAc(80mL)稀释,并用饱和NaCl溶液(30mL)洗涤,经Na2SO4干燥并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(石油醚:乙酸乙酯=3:2),得到化合物27(3.39g,75%),为白色固体。Mp:121.3-122.9℃;IR(KBr):3190,3087,2979,2913,1834,1394,1304,1205,751cm-1.1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),7.80(td,J=8.6,2.6Hz,1H),7.48(dd,J=7.4,2.3Hz,1H),7.38(dd,J=8.6,2.3Hz,1H),5.14–5.06(m,1H),4.96(d,J=2.1Hz,2H),2.98–2.84(m,1H),2.66–2.51(m,2H),2.04(d,J=12.6Hz,1H),1.43(d,J=2.6Hz,9H).13C NMR(100MHz,DMSO-d6)δ172.6,169.8,167.0,166.6,165.0,155.0,136.7,133.2,120.0,116.4,115.8,81.9,65.6,48.8,30.9,27.6,27.6,27.6,21.9.HRMS(ESI)calculated for C19H20N2NaO7 +[M+Na]+:411.1163,found 411.1161.
中间体29a-e的合成(n=2,3,4,5或6):
以29a为例,在室温下将化合物27(3.16g,8.14mmol)加入到TFA(20mL)中2小时。将混合物减压浓缩,得到粗制中间体化合物,为白色固体。将上述粗中间体化合物(500mg,1.51mmol)溶于DMF(10mL)。向所得溶液中添加HATU(687mg,1.81mmol),DIPEA(584.00mg,4.52mmol)和28a(315mg,1.81mmol)。将混合物在室温搅拌2h。然后将混合物用EtOAc(50mL)稀释并用饱和NaCl溶液(15mL)洗涤,经Na2SO4干燥并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=25∶1),得到化合物29a(3.37g,83%),为白色固体。Mp:128.3-129.9℃;IR(KBr):3623,3573,3330,2976,2923,1614,1530,1440,1325,1200,1097,1055,939,820,785,644cm-1.1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),7.99(t,J=6.0Hz,1H),7.68(t,J=7.9Hz,1H),7.37(d,J=7.3Hz,1H),7.27(d,J=8.6Hz,1H),6.76(t,J=5.9Hz,1H),5.00(dd,J=12.9,5.6Hz,1H),4.65(s,2H),3.05(q,J=6.5Hz,2H),2.89(q,J=6.4Hz,2H),2.78(s,1H),2.50–2.33(m,2H),1.98–1.87(m,1H),1.23(s,9H).13C NMR(100MHz,DMSO-d6)δ172.9,170.0,167.1,166.8,165.5,155.7,155.2,137.0,133.1,120.5,116.8,116.1,77.8,67.6,48.9,39.1,38.7,31.0,28.3,28.3,28.3,22.1.HRMS(ESI)calculated for C22H26N4NaO8 +[M+Na]+:497.1643,found 497.1642.
与29a所述的方法相同,从27和28b得到目标化合物29b。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=25:1),得到化合物29b(80%),为白色固体。Mp:113.9-114.5℃;IR(KBr):3372,3082,2935,2864,1712,1613,1561,1504,1445,1395,1297,1098,744,603cm- 1.1H NMR(400MHz,DMSO-d6)δ11.13(s,1H),7.99(t,J=5.9Hz,1H),7.81(t,J=7.9Hz,1H),7.49(d,J=7.2Hz,1H),7.38(d,J=8.6Hz,1H),6.80(t,J=6.3Hz,1H),5.12(dd,J=13.1,5.6Hz,1H),4.77(s,2H),3.13(q,J=6.9Hz,2H),2.92(q,J=6.3,5.9Hz,3H),2.59(d,J=15.9Hz,2H),2.10–1.97(m,1H),1.53(t,J=7.0Hz,2H),1.36(d,J=1.7Hz,9H).13C NMR(100MHz,DMSO-d6)δ172.8,169.9,166.8,166.8,165.5,155.1,155.1,137.0,133.1,120.4,116.8,116.1,77.5,67.6,48.8,37.4,36.1,31.0,29.6,28.3,28.3,28.3,22.1.HRMS(ESI)calculated for C23H28N4NaO8 +[M+Na]+:511.1799,found 511.1797.
与29a所述的方法相同,从27和28c得到目标化合物29c。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=25:1),得到化合物29c(70%),为白色固体。Mp:92.9-93.8℃;IR(KBr):3365,3089,2975,2933,2360,1774,1712,1614,1539,1484,1442,1395,1365,1198,1120,1077,1055,1014,989,878,820,748,704,666cm-1.1H NMR(400MHz,DMSO-d6)δ11.12(s,1H),7.96(t,J=5.7Hz,1H),7.81(dd,J=8.5,7.3Hz,1H),7.49(d,J=7.2Hz,1H),7.38(d,J=8.6Hz,1H),6.78(t,J=5.7Hz,1H),5.12(dd,J=12.9,5.4Hz,1H),4.76(s,2H),3.13(q,J=6.3Hz,2H),2.94–2.84(m,3H),2.64–2.51(m,2H),2.09–1.98(m,1H),1.42–1.37(m,4H),1.36(s,9H).13C NMR(101MHz,DMSO-d6)δ173.24,170.3,167.2,167.1,165.9,156.0,155.5,137.4,133.5,120.9,117.3,116.5,79.6,77.8,68.1,49.3,38.6,31.4,28.7,27.4,26.9,22.5.HRMS(ESI)calculated for C24H30N4NaO8 +[M+Na]+:525.1956,found 525.1961
与29a所述的方法相同,从27和28d得到目标化合物29d。在硅胶上柱色谱纯化(二氯甲烷:甲醇=25:1),得到化合物29d(77%),为白色固体。Mp:87.8-89.2℃;IR(KBr):3373,3089,2931,2861,1711,1613,1563,1502,1442,1394,1297,1097,748,603cm-1.1H NMR(400MHz,CDCl3)δ9.20–8.77(m,1H),7.74(t,J=7.9Hz,1H),7.55(d,J=7.4Hz,2H),7.19(d,J=8.5Hz,1H),5.00(q,J=5.4Hz,1H),4.77–4.54(m,3H),3.45(dd,J=13.9,7.5Hz,1H),3.38–3.26(m,1H),3.13(dd,J=13.4,6.6Hz,2H),2.94–2.74(m,3H),2.23–2.10(m,1H),1.43(s,15H).13C NMR(100MHz,CDCl3)δ171.3,168.3,166.8,166.8,166.3,156.3,154.7,137.2,133.7,119.8,118.4,117.6,79.5,68.3,49.4,40.7,39.1,31.6,29.8,28.9,28.6,28.6,28.6,24.2,22.9.HRMS(ESI)calculated for C25H32N4NaO8 +[M+Na]+:539.2112,found 539.2111.
与29a所述的方法相同,从27和28e得到目标化合物29e。在硅胶上柱色谱纯化(二氯甲烷:甲醇=25:1),得到化合物29e(73%),为白色固体。Mp:89.1-89.9℃;IR(KBr):3372,2974,2933,2859,1711,1613,1530,1484,1394,1198,1097,1076,787,603cm-1.1H NMR(400MHz,CDCl3)δ9.40(s,1H),7.74(t,J=7.9Hz,1H),7.57–7.45(m,2H),7.22(d,J=8.3Hz,1H),5.09–4.95(m,1H),4.80(t,J=6.5Hz,1H),4.65(s,2H),3.37(dp,J=20.1,6.7Hz,2H),3.10(dq,J=13.6,6.8Hz,2H),2.91–2.78(m,3H),2.15(d,J=7.3Hz,1H),1.66–1.55(m,2H),1.52–1.33(m,15H).13C NMR(100MHz,CDCl3)δ171.6,168.5,166.8,166.7,166.08,156.2,154.5,137.1,133.6,119.6,118.1,117.3,79.2,68.0,49.3,40.5,39.1,36.6,31.5,30.0,29.1,28.5,28.5,28.5,26.5,22.7.HRMS(ESI)calculated forC26H34N4NaO8 +[M+Na]+:553.2269,found 553.2268.
目标化合物30a-g的合成(m=1时,n=2,3,5,或6;m=2时,n=3,4或6):
在室温下将29a(0.61g,1.3mmol)加入TFA(5mL)和CH2Cl2(10mL)中1小时。将混合物减压浓缩,得到粗产物(88%),为白色固体。将上述粗制中间体溶于DMF(2.5mL)中,加入HATU(0.17g,0.43mmol),DIPEA(0.18mL,1.1mmol)和24a(0.15g,0.35mmol)。将混合物在室温搅拌3小时。将混合物用EtOAc(10mL)稀释,用饱和NH4Cl溶液(5mL)洗涤,经Na2SO4干燥并在减压下浓缩。将残余物通过硅胶上的柱色谱法纯化(二氯甲烷:甲醇=35:1),得到化合物30a(451mg,45%),为白色固体。Mp:131.4-132.1℃;IR(KBr):2961,2925,2853,1773,1716,1612,1558,1541,1498,1457,1419,1396,1262,1136,1099,1054,1021,845,763,728,703,558cm-1.1H NMR(400MHz,CDCl3)δ7.66(s,1H),7.57(s,1H),7.43–7.30(m,6H),7.21(s,2H),7.14(s,2H),6.17(s,1H),5.04(s,2H),4.93(s,1H),4.56(s,2H),4.04(s,2H),3.73(s,6H),3.57(s,2H),3.38(s,4H),2.71(s,3H),2.21(s,3H),2.04(s,1H).13C NMR(100MHz,CDCl3)δ170.2,167.6,166.8,165.6,165.4,159.8,158.5,153.7,142.1,140.8,136.0,133.8,133.5,132.4,129.4,128.4,127.3,127.1,125.9,124.6,119.3,117.2,116.5,90.3,76.2,68.3,67.5,54.8,48.3,40.2,37.9,30.2,28.7,21.6,15.2.HRMS(ESI)calculated forC42H43N5NaO10 +[M+Na]+:800.2902,found 800.2904.
与30a所述的方法相同,从29b和24a得到目标化合物30b。用硅胶柱色谱纯化(二氯甲烷:甲醇=35:1),得到化合物30b(48%),为白色固体。Mp:124.4-125.0℃.IR(KBr):2961,2925,2852,1773,1716,1609,1556,1541,1498,1457,1419,1396,1315,1252,1136,1097,1054,1021,845,763,728,703,663,558cm-1.1H NMR(400MHz,CDCl3)δ7.64(t,J=7.7Hz,2H),7.40(q,J=7.8,7.1Hz,4H),7.34(d,J=6.8Hz,1H),7.29(s,1H),7.27(s,1H),7.23(s,2H),7.21–7.15(m,2H),6.19(s,2H),5.06(s,2H),4.95(s,1H),4.58(s,2H),4.08(s,2H),3.76(s,6H),3.55(s,2H),3.23(s,4H),2.72(d,J=18.3Hz,3H),2.23(s,3H),2.07(d,J=17.5Hz,1H),1.69(d,J=12.6Hz,2H).13C NMR(100MHz,CDCl3)δ171.2,168.2,166.8,165.6,165.5,160.6,158.7,153.8,142.0,140.8,136.2,132.2,129.4,128.3,127.2,127.1,125.9,124.6,119.5,117.0,116.4,90.2,76.2,68.3,54.8,48.2,40.2,36.0,35.5,30.1,28.7,27.6,21.5,15.2.HRMS(ESI)calculated for C43H45N5NaO10 +[M+Na]+:814.3059,found 814.3063.
与30a所述的方法相同,从29d和24a得到目标化合物30c。用硅胶柱色谱纯化(二氯甲烷:甲醇=35:1),得到化合物30c(48%),为白色固体。Mp:120.4-121.7℃.IR(KBr):3405,3081,2940,2861,1773,1715,1613,1544,1499,1484,1456,1395,1262,1234,1197,1138,1054,1023,990,912,845,764,731,704,603cm-1.1H NMR(400MHz,CDCl3)δ7.68(t,J=7.2Hz,1H),7.57(s,1H),7.48(d,J=7.3Hz,1H),7.40(d,J=6.9Hz,3H),7.35(d,J=6.8Hz,1H),7.30(d,J=7.7Hz,2H),7.26(s,2H),7.19(d,J=8.4Hz,2H),6.22(s,2H),5.08(s,2H),4.98(d,J=10.7Hz,1H),4.57(s,2H),4.13(s,2H),3.80(s,6H),3.59(s,2H),3.37(dd,J=13.9,6.8Hz,1H),3.23(dd,J=17.4,9.7Hz,3H),2.76(dt,J=22.5,11.0Hz,3H),2.25(d,J=2.6Hz,3H),2.11(s,1H),1.54(q,J=9.7,8.7Hz,4H),1.41(dd,J=13.9,7.7Hz,3H).13CNMR(100MHz,CDCl3)δ172.3,168.8,167.2,166.7,166.2,161.8,159.7,154.5,143.0,141.8,137.1,134.7,134.5,133.3,130.4,129.4,128.3,128.1,126.9,125.7,120.0,118.0,117.3,91.1,77.2,69.4,68.1,55.9,49.2,48.2,41.2,39.7,38.8,31.2,28.4,23.7,22.6,16.2.HRMS(ESI)calculated for C45H49N5NaO10 +[M+Na]+:842.3372,found 842.3369.
与30a所述的方法相同,从29e和24a得到目标化合物30d。用硅胶柱色谱纯化(二氯甲烷:甲醇=35:1),得到化合物30d(44%),为白色固体。Mp:115.4-116.8℃.IR(KBr):3406,3082,2938,2859,1773,1715,1613,1549,1498,1484,1457,1420,1395,1263,1234,1197,1137,1023,912,845,763,748,704,602cm-1.1H NMR(400MHz,CDCl3)δ7.67(d,J=8.3Hz,1H),7.46(d,J=6.9Hz,2H),7.39(d,J=6.8Hz,3H),7.34(d,J=6.7Hz,1H),7.29(d,J=7.7Hz,2H),7.26(d,J=2.6Hz,2H),7.24(s,1H),7.19(d,J=7.5Hz,1H),7.13(s,1H),6.22(s,2H),5.07(s,2H),4.95(s,1H),4.58(s,2H),4.05(s,2H),3.79(s,6H),3.52(s,2H),3.21(s,4H),2.87–2.68(m,3H),2.24(s,3H),2.17–2.03(m,1H),1.49(d,J=24.7Hz,4H),1.28(d,J=22.7Hz,5H).13C NMR(100MHz,CDCl3)δ172.5,169.0,167.2,166.8,166.2,162.0,159.9,154.6,143.1,141.9,137.2,134.8,134.6,133.5,130.5,129.4,128.4,128.2,127.0,125.8,120.0,118.0,117.3,91.3,77.4,69.5,68.0,56.0,49.3,48.1,41.3,39.7,39.0,31.4,29.8,29.0,28.9,26.3,26.2,22.6,16.3.HRMS(ESI)calculated forC46H51N5NaO10 +[M+Na]+:856.3528,found 856.3531.
与30a所述的方法相同,从29b和24b得到目标化合物30e。用硅胶柱色谱纯化(二氯甲烷:甲醇=35:1),得到化合物30e(41%),为白色固体。Mp:130.4-131.2℃;IR(KBr):3357,3064,2937,1773,1714,1669,1612,1558,1484,1456,1420,1354,1324,1261,1233,1197,1136,1054,910,817,763,729,703,603cm-1.1H NMR(400MHz,CDCl3)δ7.96(t,J=5.8Hz,1H),7.76(t,J=5.9Hz,1H),7.62(dd,J=8.4,7.3Hz,1H),7.42(d,J=7.3Hz,1H),7.36–7.32(m,3H),7.31–7.26(m,1H),7.24(d,J=1.6Hz,1H),7.22(t,J=1.5Hz,1H),7.20(d,J=2.4Hz,1H),7.18(d,J=2.3Hz,1H),7.15(d,J=8.5Hz,1H),6.15(s,2H),5.01(s,2H),4.92(dd,J=12.0,5.7Hz,1H),4.60–4.57(m,2H),4.11(s,2H),3.75(s,6H),3.35(s,2H),3.28(tt,J=13.1,6.9Hz,2H),3.11(t,J=6.7Hz,2H),3.00(t,J=5.8Hz,2H),2.81(t,J=5.9Hz,2H),2.75–2.69(m,2H),2.69–2.60(m,1H),2.18(s,3H),2.07–1.98(m,1H),1.65(p,J=7.1Hz,2H).13C NMR(100MHz,CDCl3)δ171.9,171.6,169.0,167.4,166.8,166.3,162.0,159.9,154.9,143.2,141.8,137.1,134.7,134.6,133.5,130.5,129.4,128.4,128.2,127.0,125.7,120.4,118.2,117.4,99.4,91.2,77.4,69.5,68.7,56.0,50.5,49.4,43.2,40.4,36.5,31.4,30.8,28.8,22.7,16.3.HRMS(ESI)calculated for C44H47N5NaO10 +[M+Na]+:828.3215,found 828.3211.
与30a所述的方法相同,从29c和24b得到目标化合物30f。用硅胶柱色谱纯化(二氯甲烷:甲醇=35:1),得到化合物30f(41%),为白色固体。Mp:117.4-119.1℃;IR(KBr):3210,3064,2925,2852,1773,1714,1669,1612,1558,1499,1484,1456,1420,1395,1355,1298,1262,1233,1197,1136,1075,1023,817,763,748,703,603cm-1.1H NMR(400MHz,CDCl3)δ7.73(qd,J=7.6,4.3Hz,3H),7.52(d,J=7.3Hz,2H),7.44–7.40(m,3H),7.38–7.34(m,1H),7.32(d,J=1.7Hz,1H),7.30(t,J=1.5Hz,1H),7.28(s,1H),7.26(s,2H),7.21(d,J=8.4Hz,1H),6.23(s,2H),5.10(d,J=4.4Hz,2H),4.98(dd,J=11.9,5.7Hz,1H),4.64(q,J=14.3Hz,2H),4.16(s,2H),3.82(s,6H),3.47–3.40(m,1H),3.20(q,J=6.3,5.7Hz,3H),3.04(t,J=5.9Hz,2H),2.87(d,J=6.4Hz,2H),2.84–2.80(m,2H),2.78–2.70(m,1H),2.26(s,3H),2.10(tt,J=7.1,4.0Hz,1H),1.56(d,J=5.7Hz,4H).13C NMR(100MHz,CDCl3)δ171.8,171.6,169.0,167.1,166.8,166.5,161.9,159.9,155.0,143.2,141.9,137.2,134.8,134.7,133.6,130.6,129.5,128.5,128.3,127.1,125.8,120.6,118.5,117.7,100.0,91.3,77.4,69.6,68.9,56.0,49.4,43.2,40.5,39.2,39.0,31.5,31.0,29.8,26.6,26.6,22.8,16.4.HRMS(ESI)calculated for C45H49N5NaO10 +[M+Na]+:842.3372,found842.3377.
与30a所述的方法相同,从29e和24b得到目标化合物30g。在硅胶上进行柱色谱分离(二氯甲烷:甲醇=36:1),得到化合物30g(43%),为白色固体。Mp:108.2-109.7℃;IR(KBr):3413,3203,2937,2858,1774,1715,1660,1613,1550,1499,1464,1396,1355,1299,1233,1197,1136,1054,844,764,704,603,557cm-1.1H NMR(400MHz,CDCl3)δ7.73(dd,J=8.4,7.3Hz,1H),7.53(d,J=7.3Hz,2H),7.49(d,J=9.5Hz,1H),7.43(ddd,J=7.7,5.5,2.7Hz,4H),7.39–7.35(m,1H),7.33(d,J=1.7Hz,1H),7.31(d,J=1.4Hz,1H),7.27(d,J=6.5Hz,2H),7.21(s,1H),6.25(s,2H),5.20–5.08(m,2H),4.97–4.91(m,1H),4.62(s,2H),4.15(d,J=3.2Hz,2H),3.83(s,6H),3.50–3.44(m,1H),3.27(dp,J=19.8,6.2Hz,2H),3.11(d,J=6.2Hz,1H),3.05(q,J=5.0Hz,2H),2.87(dt,J=9.4,4.6Hz,1H),2.83–2.79(m,1H),2.77(d,J=3.9Hz,1H),2.74(s,1H),2.27(s,3H),2.13–2.02(m,2H),1.56(d,J=6.6Hz,2H),1.46(d,J=8.0Hz,3H),1.39–1.31(m,4H).13C NMR(100MHz,CDCl3)δ171.8,171.7,168.6,166.8,166.7,166.2,161.8,160.0,154.6,143.2,141.9,137.2,134.8,134.7,133.7,130.6,129.5,128.4,128.3,127.1,125.8,119.7,118.2,117.4,91.4,77.4,69.6,68.1,56.0,49.3,43.1,40.5,39.4,38.8,31.6,29.8,29.4,29.1,26.7,26.3,22.8,16.4ppm.HRMS(ESI)calculated for C47H53N5NaO10 +[M+Na]+:870.3685,found 870.3682.
其中化合物21a的1H NMR光谱和13C NMR光谱分别如图3、图4所示。
2、化合物21a性能试验
对于所有的PROTACs,用标准MTT法检测在四种肿瘤细胞系的抗增殖能力(如表1所示)。通过MTT分析结果,化合物30d,11d和11e表现出抑制癌细胞生长,可能对PD-L1的选择性较差。
表1. 18种PROTACs化合物对癌细胞系增殖的影响
IC50>100μmol/L意味着没有明显抑制增殖作用;IC50由三个独立实验测定。MTT法检测化合物在48小时后的细胞毒性作用。
随后,应用蛋白质免疫印迹法对所有的protac分子降解PD-L1的能力进行了评估,发明人在小鼠结直肠细胞系MC-38中进行了蛋白质免疫印迹分析。结果显示11d和21a这两种化合物具有明显降解癌细胞中PD-L1的作用(如图2所示)。
基于Western免疫印迹结果分析,发明人筛选出在体外降解PD-L1蛋白效果最好的两个化合物——11d和21a。但是,化合物11d具有强大的抗增殖活性,可能不适合进行后续生物学研究,最担心的是它可能在体内引起安全问题。于是,发明人在体外对正常细胞——人胚肾293T,人肝LO2和鼠成纤维细胞系NIH-3T3进行细胞增殖抑制测定(表2)。化合物11d在杀伤肿瘤细胞的同时也影响了正常细胞的生长,而化合物21a对这些正常细胞的杀伤作用明显很弱。此外,向C57BL/6小鼠尾静脉注射这两种化合物,单次剂量(50mg/kg,n=5)。注射化合物11d的所有小鼠在24小时内均死亡,表明化合物11d具有极高的毒性。然而注射化合物21a的小鼠全部耐受。从上述结果可以看出,化合物21a可能更安全,更适合进行深入研究。
表2化合物11d和21a对人正常细胞的细胞毒性作用
基于以上的实验结果,发明人选择化合物21a进行深入研究。在不同癌细胞系:人血液恶性肿瘤细胞(Skno-1,HL-60,Kasumi-1),人乳腺癌细胞MCF-7,小鼠膀胱癌细胞MB-49,人结肠癌细胞SW-480和人前列腺癌细胞PC-3分别检测PD-L1蛋白水平。结果显示这些细胞用21a处理48h之后,以浓度依赖性地显著诱导PD-L1蛋白降解(图5A)。为了验证化合物21a处理导致PD-L1蛋白降解是由于PROTAC介导的降解,故检测放线菌酮处理后的PD-L1蛋白水平。如图5B所示,放线菌酮在没有与21a协同作用的情况下也促进了PD-L1的降解,表明PD-L1蛋白水平的降低不是由于mRNA的减少。随后,为了验证化合物21a所诱导的PD-L1蛋白降解是由于蛋白酶体降解,将Skno-1、PC-3细胞用与沙利度胺,MG132、BMS-37预处理,发现用MG132预处理后PD-L1蛋白降解明显受阻(图5C)。结果表明化合物21a依赖于细胞内蛋白酶体降解PD-L1蛋白。
为了研究PD-L1蛋白是否在细胞质中被降解,进行了蛋白质印迹分析将细胞与5μM化合物21a一起培养指定的时间。用proteinExt Mammalian膜质蛋白提取试剂盒提取细胞质和细胞膜上的PD-L1蛋白,并用RIPA缓冲液提取总PD-L1蛋白,western blot检测PD-L1蛋白。如图6A所示,化合物21a以时间依赖性显著降解细胞质中的PD-L1蛋白,并且PD-L1在细胞膜上的减少位于细胞质的后面。与以上结果相符,PD-L1蛋白与荧光标记的化合物21a在细胞质中存在共定位的现象(图6B)。用5μM化合物21a处理细胞24小时。固定细胞,对PD-L1(红色)进行免疫染色,并通过共聚焦荧光显微镜进行分析,化合物21a在被激发光照射后发出绿色荧光。与发明人最初的设计一致。
之后发明人评估了化合物21a的体内抗肿瘤活性,发现对MC-38肿瘤生长的具有抑制作用。如图7A-D所示,尾静脉注射化合物21a的小鼠(15mg/kg),肿瘤生长被抑制。并且两组的体重没有显着差异。然后对肿瘤组织的免疫组织化学分析表明,用化合物21a治疗可以显著降低PD-L1水平,并促进CD8+T细胞在肿瘤组织中的浸润(图7E)。此外,在肿瘤组织中GzmB,IFN-y和Prf1的基因表达上调,这是CD8+T细胞发挥细胞毒性的关键介质(图7F)。以上结果表明,尾静脉注射化合物21a可以降解瘤内PD-L1蛋白的水平并明显抑制动物体内MC-38小鼠结直肠癌细胞的生长。图7(A)用MC38细胞皮下注射C57BL/6J小鼠。MC38异种移植肿瘤体积的变化。(B)在第12天称重肿瘤。(C)动物的体重。(D)空白组和化合物21a处理的小鼠中解剖的具有代表性的肿瘤图像。T-检验;平均值±SD,n=6,**P<0.001.(E)用PD-L1抗体和CD8抗体对MC38异种移植模型的肿瘤组织进行染色,然后拍照(100X)。(F)提取肿瘤组织中的总RNA并用实时荧光定量PCR分析细胞因子的含量。
综上所述,基于BMS-37小分子开发了新型PROTAC降解剂化合物21a。这是基于结合PD-L1蛋白的细胞外结构域的配体,降解膜蛋白的实例。正如从体外研究的结果所揭示,化合物21a可以以剂量依赖性和时间依赖性的方式有效诱导各种癌细胞系中PD-L1的降解。此外,根据体内研究,化合物21a的治疗可显著下调PD-L1蛋白的水平,促进CD8+T细胞的侵袭并抑制MC-38肿瘤生长。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,并不用以限制本发明。凡在本发明的申请范围内所做的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
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