CN112979671B - 济源冬凌草甲素-萘酰亚胺类荧光探针及其制备方法和应用 - Google Patents
济源冬凌草甲素-萘酰亚胺类荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及天然产物及药物化学领域，公开了一类新型的含1,2,3‑三氮唑基的济源冬凌草甲素‑萘酰亚胺荧光探针及其制备方法和应用。其制备方法：以济源冬凌草甲素为起始原料，在不影响其活性中心的前提下，通过点击反应将不同链长的济源冬凌草甲素炔基衍生物和萘酰亚胺叠氮衍生物连接起来，获得一类新型的含1,2,3‑三氮唑基的济源冬凌草甲素‑萘酰亚胺荧光探针。该类荧光探针可以用于济源冬凌草甲素的抗肿瘤机制研究，特别是涉及这类荧光探针在济源冬凌草甲素作用靶细胞和活体动物中的分布和应用。同时，此类化合物还显著提高了济源冬凌草甲素对食道癌、胰腺癌、胃癌、肝癌、前列腺癌等多种细胞的抗肿瘤活性。其具有如下通式：
Description
技术领域
本发明涉及天然产物及药物化学领域，具体涉及一类新型的含1,2,3-三氮唑基的济源冬凌草甲素-萘酰亚胺荧光化合物及其制备方法和应用。
背景技术
恶性肿瘤是威胁人类健康与生命的重大疾病之一，具有很高的发病率和死亡率。随着生命科学的发展，越来越多的天然结构抗肿瘤化合物被人们发现并研发成为药物。这些天然产物具有很好的抗肿瘤活性，但是它们的抗肿瘤作用机制常常是研发的难点。近年来，荧光成像是一种广泛应用于从分子到完整生物体的多层面细胞事件和相关表型可视化的策略。设计天然产物-荧光探针复合物，以其为工具探索天然化合物在靶细胞或动物中的分布具有非常重要的理论意义和广泛的应用前景(1.Green,J.C.；Jiang,Y.；He,L.；Xu,Y.；Sun,D.；Keoprasert,T.；Nelson,C.；Oh,U.；Lesnefsky,E.J.；Kellogg,G.E.；Chen,Q.；Zhang,S.,Characterization and Discovery of a Selective Small-MoleculeModulator of Mitochondrial Complex I Targeting a Unique Binding Site.Journalof Medicinal Chemistry 2020,63(20),11819-11830；2.
M.；Rimpelová,S.；
E.；
P.；Ruml,T.,Tailor-Made Fluorescent Trilobolide To Study ItsBiological Relevance.Journal of Medicinal Chemistry 2014,57(19),7947-7954.；3.
S.；Janiszewski,T.；Kaiserman,D.；Modrzycka,S.；Snipas,S.J.；Salvesen,G.；
M.；Bird,P.I.；Kasperkiewicz,P.,Detection of Active Granzyme A in NK92 Cellswith Fluorescent Activity-Based Probe.Journal of Medicinal Chemistry 2020,63(6),3359-3369.；4.Xu,S.；Luo,S.；Yao,H.；Cai,H.；Miao,X.；Wu,F.；Yang,D.-H.；Wu,X.；Xie,W.；Yao,H.；Chen,Z.-S.；Xu,J.,Probing the Anticancer Action of Oridonin withFluorescent Analogues:Visualizing Subcellular Localization toMitochondria.Journal of Medicinal Chemistry 2016,59(10),5022-5034.)。
冬凌草学名碎米桠，为唇形科香茶菜属植物，在我国黄河流域具有较为广泛的分布。其中，济源冬凌草是河南省济源市特产，2006年原国家质检总局批准对“济源冬凌草”实施地理标志产品保护，2019年11月济源冬凌草入选中国农业品牌目录。济源冬凌草作为一种作用广泛的、抗肿瘤效果显著的、毒副作用小的天然活性产物，具有良好的应用前景和价值。济源冬凌草甲素是从冬凌草中提取的一种对映-贝壳杉烯型二萜类化合物，研究表明，它对食道癌、胰腺癌、胃癌、乳腺癌、前列腺癌，原发性肝癌等具有良好的抗肿瘤活性(5.Ke,Y.；Hu,T.-X.；Huo,J.-F.；Yan,J.-K.；Wang,J.-Y.；Yang,R.-H.；Xie,H.；Liu,Y.；Wang,N.；Zheng,Z.-J.；Sun,Y.-X.；Wang,C.；Du,J.；Liu,H.-M.,Synthesis and in vitrobiological evaluation of novel derivatives of Flexicaulin A condensation withamino acid trifluoroacetate.European Journal of Medicinal Chemistry 2019,182,111645.；6.Ke,Y.；Liang,J.-J.；Hou,R.-J.；Li,M.-M.；Zhao,L.-F.；Wang,W.；Liu,Y.；Xie,H.；Yang,R.-H.；Hu,T.-X.；Wang,J.-Y.；Liu,H.-M.,Synthesis and biologicalevaluation of novel Jiyuan Oridonin A-1,2,3-triazole-azole derivatives asantiproliferative agents.European Journal of Medicinal Chemistry 2018,157,1249-1263.；7.Ke,Y.；Wang,W.；Zhao,L.-F.；Liang,J.-J.；Liu,Y.；Zhang,X.；Feng,K.；Liu,H.-M.,Design,synthesis and biological mechanisms research on 1,2,3-triazole derivatives of Jiyuan Oridonin A.Bioorganic&Medicinal Chemistry2018,26(17),4761-4773.)。
然而令人遗憾的是，以冬凌草甲素、济源冬凌草甲素等为代表的对映-贝壳杉烯型二萜类化合物的抗肿瘤机制和作用靶点尚未完全阐明，它们的抗肿瘤活性也有待优化。针对这些不足，本发明人首先通过对荧光光学性质的探索和筛查，确定其基本的荧光分子骨架。其次采用激光共聚焦实验，明确济源冬凌草甲素在靶细胞中的定位和分布。最后，经过SDS-PAGE凝胶成像和多重荧光Western Blot实验，确定济源冬凌草甲素的作用靶点。同时，还对合成的荧光化合物进行了体外抗肿瘤活性评价、细胞周期和凋亡、活性氧等机制实验。这些实验方法和思路对进一步研究天然产物尚未明确其靶点的抗癌药物，开发自主知识产权的创新药物具有重要意义和指导意义。
发明内容
本发明的目的在于提供一类新型的含1,2,3-三氮唑基的济源冬凌草甲素-萘酰亚胺类化合物；通过对济源冬凌草甲素的结构改造，提高此类化合物的抗肿瘤活性。
另一目的在于提供此类化合物作为以抗肿瘤机制和作用靶点尚不明确的天然产物在细胞定位及成像中的应用。
为实现本发明目的，本发明将济源冬凌草甲素和萘酰亚胺荧光发色团进行三氮唑反应得到了系列荧光化合物。
本发明所述一类新型的含1,2,3-三氮唑基的济源冬凌草甲素-萘酰亚胺类荧光探针具体通式如下：
其中R为哌啶基；
X＝-C-，-CH2O-；
m＝0-6；
n＝1-8；
p＝0-6。
进一步优选R为哌啶基；X＝-C-，-CH2O-；m＝0-3；n＝1-5；p＝0-4。
所述的荧光探针是一种双功能的分子，其有济源冬凌草甲素的骨架，保留了它们的抗肿瘤活性。通过细胞活性实验发现还提高了它们潜在的抑制作用，并具有良好的抗肿瘤活性和细胞定位作用。
上述的一类新型的含1,2,3-三氮唑基的济源冬凌草甲素-萘酰亚胺类荧光探针的制备方法如下：
(1)首先，称量4-溴-1,8-萘二甲酸酐和哌啶反应，得到4-哌啶基的萘二甲酸酐化合物。然后在回流条件下，与各种直链的氨基醇进行脱水缩合，得到的中间体在PBr3或者TsCl反应条件下，生成溴代产物或者对甲苯磺酰氯保护的产物。最后在DMF和NaN3作用下，进行亲核取代反应，得到了叠氮基取代的萘酰亚胺衍生物。
(2)济源冬凌草甲素在对甲苯磺酸的催化下，与各种炔醇进行20位羟基的醚化反应，得到济源冬凌草甲素炔基衍生物。
(3)将步骤1中的叠氮基取代的萘酰亚胺衍生物与步骤2中济源冬凌草甲素炔基衍生物在CuSO4/VcNa的催化下得到1,2,3-三氮唑基的济源冬凌草甲素-萘酰亚胺类荧光探针。
其合成路线如下：
步骤a：4-溴-1,8-萘二甲酸酐与哌啶在乙二醇单甲醚为溶剂，回流条件下，进行反应得到化合物2。
步骤b：将化合物2溶解于乙醇中，回流条件下，加入各种的氨基醇化合物，发生脱水缩合反应，柱色谱纯化可得中间体3或者6。
步骤c：中间体3先溶解于三氯甲烷(氯仿)中，在冰浴条件下，加入PBr3(三溴化磷)进行搅拌，随后回流温度下，进行溴代反应，柱色谱纯化得到中间体4。
步骤d：化合物6溶解于DCM(二氯甲烷)中，DABCO(三乙烯二胺)作为缚酸剂和催化剂，随后加入TsCl(对甲苯磺酰氯)进行羟基保护，得到中间体7。
步骤e：将中间体4或者中间体7溶于DMF(N,N-二甲基甲酰胺)中，加入叠氮钠(NaN3)，进行取代反应可得中间体5或者中间体8。
步骤f：将原料济源冬凌草甲素溶于THF(四氢呋喃)中，对甲苯磺酸为催化剂，与各种不同链长的炔醇进行20位的醚化反应，柱层析纯化得到中间体10。
步骤g：中间体10与化合物5或化合物8，在THF/H2O为混合溶剂，CuSO4/VcNa的催化下，得到1,2,3-三氮唑基的济源冬凌草甲素-萘酰亚胺类荧光探针。
本发明的创新点及优点：
1、提供了一种具有萘酰亚胺荧光发色基团与济源冬凌草甲素活性骨架的荧光探针，通过点击反应(Click)合成新型的1,2,3-三氮唑基的济源冬凌草甲素-萘酰亚胺荧光探针，避免了因荧光发色团和天然产物形成不稳定的官能团键(酯键，酰胺键)所导致的荧光淬灭现象。
2、该类萘酰亚胺荧光发色团具有较大的斯托克斯位移(Stokes shift)，高摩尔系数，低毒性和良好的稳定性等。同时该探针是一种双功能的分子，对胃癌细胞、胰腺癌、前列腺癌、食管癌等具有良好的抗肿瘤活性，兼具治疗与作用靶点研究的用途，可用于肿瘤细胞成像，药物作用机制研究，分子靶点确定或活体动物成像等。其制备方法反应条件温和，工艺简单，具有潜在的应用价值。
附图说明
图1为本发明化合物I-39荧光探针对HGC-27细胞染色实验结果；
图2为本发明化合物I-39荧光探针荧光共定位实验结果。
具体实施方式
通过以下具体实例进一步说明本发明，但应注意本发明的范围并不受这些实施例的任何限制。
实施例1化合物2的制备
称取4-溴-1,8-萘二甲酸酐(1g,1eq)置于50mL的茄形烧瓶，加入20mL的乙二醇单甲醚，随后再加入665μL的哌啶(2eq)，在125℃回流温度下反应4-5h，TLC检测反应，冷却至室温后，加冰水沉淀析出，过滤，用水洗涤三次，柱层析分离纯化，最后再用无水乙醇重结晶，可得765mg的黄色针状晶体化合物2，产率75％。1H NMR(400MHz,CDCl3)δ8.55(d,J＝7.3Hz,1H),8.45(m,2H),7.76–7.63(m,1H),7.18(d,J＝8.2Hz,1H),3.38–3.22(m,4H),1.97–1.85(m,4H),1.76(m,2H).13C NMR(101MHz,CDCl3)δ161.49,160.68,158.41,135.00,133.14,132.44,132.20,126.14,125.60,119.28,114.95,110.84,54.39,26.12,24.27.HR-MS(ESI):Calculated for C17H16NO3[M+H]+:282.1130,found 282.1121。
实施例2中间体3或者6系列的制备
称取一定量的化合物2(1eq)加入20mL的无水乙醇，然后加入氨基乙醇或二甘醇胺(2eq)回流1-2h即可反应完全，随后将反应液倒入冰水中，将得到沉淀进行200-300目硅胶柱层析分离纯化，乙酸乙酯重结晶，可得黄色固体中间体3或者6系列，产率50％-70％。
中间体3，黄色固体：1H NMR(400MHz,CDCl3)δ8.57(d,J＝7.3Hz,1H),8.49(d,J＝8.1Hz,1H),8.40(d,J＝8.5Hz,1H),7.67(dd,J＝8.3,7.4Hz,1H),7.17(d,J＝8.1Hz,1H),4.51–4.40(m,2H),4.02–3.91(m,2H),3.32–3.18(m,4H),1.95–1.83(m,4H),1.77–1.68(m,2H).13C NMR(101MHz,CDCl3)δ165.55,165.12,157.69,133.11,131.39,131.03,130.09,126.22,125.38,122.83,115.45,114.75,62.24,54.56,42.77,26.22,24.35.HR-MS(ESI):Calculated for C19H21N2O3[M+H]+:325.1552,found 325.1543。
中间体6，黄色固体：1H NMR(400MHz,CDCl3)δ8.55(d,J＝7.2Hz,1H),8.48(d,J＝8.1Hz,1H),8.37(d,J＝8.4Hz,1H),7.65(dd,J＝8.2,7.5Hz,1H),7.15(d,J＝8.1Hz,1H),4.42(t,J＝5.7Hz,2H),3.84(t,J＝5.7Hz,2H),3.68(m,4H),3.28–3.14(m,4H),2.56(s,1H),1.93–1.82(m,4H),1.79–1.65(m,2H).13C NMR(101MHz,CDCl3)δ164.88,164.38,157.46,132.92,131.22,130.79,130.00,126.19,125.34,122.90,115.62,114.71,72.25,68.55,61.85,54.53,39.31,26.21,24.34.HR-MS(ESI):Calculated for C21H25N2O4[M+H]+:369.1814,found 369.1804。
实施例3中间体4系列的制备
先将中间体3(1eq)先溶解于三氯甲烷中，在冰浴条件下，加入PBr3(3eq)搅拌30min后，在61℃回流下反应2-3h，随后加入饱和K2CO3直至没有气泡产生，二氯甲烷萃取5次，饱和食盐水洗涤3次，合并有机相，无水MgSO4干燥，过滤，浓缩，柱色谱纯化，得到黄色固体中间体4，产率25％-55％。1H NMR(400MHz,CDCl3)δ8.58(d,J＝7.3Hz,1H),8.50(d,J＝8.1Hz,1H),8.41(d,J＝8.4Hz,1H),7.68(dd,J＝8.3,7.4Hz,1H),7.18(d,J＝8.1Hz,1H),4.59(t,J＝7.2Hz,2H),3.79–3.56(m,2H),3.32–3.14(m,4H),2.01–1.82(m,4H),1.80–1.66(m,2H).13C NMR(101MHz,CDCl3)δ164.35,163.79,157.60,133.01,131.31,130.98,130.03,126.23,125.37,122.70,115.34,114.74,54.53,41.05,27.97,26.20,24.32.HR-MS(ESI):Calculated for C19H20BrN2O2[M+H]+:387.0708,found 387.0715。
实施例4中间体7系列的制备
称取中间体6(1eq)先溶解于二氯甲烷中，随后加入TABCO(2eq)和TsCl(1.5eq)，0℃条件下反应过夜，加入饱和食盐水，用二氯甲烷萃取3次，饱和食盐水洗涤3次，收集有机相，柱色谱分离纯化，得到黄色固体中间体7系列，产率50％-60％。1H NMR(400MHz,CDCl3)δ8.49(d,J＝7.4Hz,1H),8.43(d,J＝8.1Hz,1H),8.34(d,J＝8.4Hz,1H),7.73(d,J＝7.7Hz,2H),7.68–7.59(m,1H),7.27(d,J＝8.1Hz,2H),7.14(d,J＝8.1Hz,1H),4.32(t,J＝6.1Hz,2H),4.13(m,2H),3.78–3.67(m,4H),3.27–3.15(m,4H),2.39(s,3H),1.87(m,4H),1.72(m,2H).13C NMR(101MHz,CDCl3)δ164.48,163.95,157.35,144.66,132.98,132.71,131.03,130.71,129.92,129.75,127.91,126.17,125.30,122.86,115.59,114.66,69.35,68.10,67.99,54.51,38.62,26.20,24.32,21.60.HR-MS(ESI):Calculated for C28H31N2O6S[M+H]+:523.1903,found 523.1894。
实施例5中间体5或者8系列的制备
称取中间体4或者7(1eq)溶于DMF，随后加入NaN3(3eq)，55℃下回流2-3h，用乙酸乙酯和饱和食盐水萃取，柱色谱分离纯化，得到黄色固体中间体5或者8系列，产率70％-80％。
中间体5，黄色固体：1H NMR(400MHz,CDCl3)δ8.59(d,J＝7.3Hz,1H),8.51(d,J＝8.1Hz,1H),8.41(d,J＝8.4Hz,1H),7.68(dd,J＝8.3,7.4Hz,1H),7.18(d,J＝8.1Hz,1H),4.43(t,J＝6.4Hz,2H),3.65(t,J＝6.3Hz,2H),3.30–3.18(m,4H),1.93–1.83(m,4H),1.73(m,2H).13C NMR(101MHz,CDCl3)δ164.63,164.07,157.60,133.03,131.35,130.99,130.12,126.30,125.40,122.77,115.46,114.78,54.57,48.97,38.67,26.22,24.34.HR-MS(ESI):Calculated for C19H20N5O2[M+H]+:350.1617,found 350.1607。
中间体8，黄色固体：1H NMR(400MHz,CDCl3)δ8.57(d,J＝7.3Hz,1H),8.52–8.45(m,1H),8.39(d,J＝8.4Hz,1H),7.67(dd,J＝8.4,7.3Hz,1H),7.17(d,J＝8.1Hz,1H),4.44(t,J＝6.1Hz,2H),3.84(t,J＝6.1Hz,2H),3.77–3.68(m,2H),3.35(t,J＝5.1Hz,2H),3.26–3.17(m,4H),1.93–1.83(m,4H),1.80–1.68(m,2H).13C NMR(101MHz,CDCl3)δ164.68,164.16,157.33,132.77,131.13,130.71,130.05,126.28,125.36,123.04,115.85,114.73,69.50,68.14,54.57,50.79,38.86,26.22,24.35.HR-MS(ESI):Calculated for C21H24N5O3[M+H]+:394.1879,found394.1869。
实施例6中间体10系列的制备
将化合物9(1eq)溶于无水THF中，随后加入3-丁炔-1-醇(5eq)，最后加入对甲苯磺酸(0.15eq)催化，室温反应过夜，加入饱和NaHCO3进行猝灭，旋去多余的THF，乙酸乙酯萃取3次，收集有机相，过柱纯化，可得白色固体中间体10，产率50％-60％。1H NMR(400MHz,DMSO)δ5.78(s,1H),5.36(s,1H),5.15(s,1H),5.04(d,J＝2.7Hz,1H),4.64(s,1H),4.55–4.42(m,1H),4.15(d,J＝7.1Hz,1H),3.96(d,J＝3.2Hz,1H),3.80(m,1H),3.50–3.37(m,1H),2.84(t,J＝2.4Hz,1H),2.78(d,J＝9.0Hz,1H),2.70–2.56(m,2H),2.44(m,2H),2.01(d,J＝14.8Hz,1H),1.61–1.51(m,1H),1.41(m,2H),1.34(m,2H),1.26(m,1H),1.18(m,2H),1.09(m,1H),0.91(s,3H),0.81(s,3H).13C NMR(101MHz,DMSO)δ205.84,152.34,115.81,99.05,81.92,71.70,69.56,65.27,65.12,62.98,57.21,56.26,48.35,42.48,41.81,40.33,39.30,33.60,32.58,29.83,24.16,20.60,19.44,17.71.
实施例7通式I化合物的制备
分别称取炔基化的济源冬凌草甲素(1eq)和叠氮化的萘酰亚胺荧光发色团(1.2eq)于THF(4mL)中，随后在依次加入VcNa(0.8eq)和CuSO4(0.4eq)，最后再缓慢滴加1mL的水，于66℃避光反应2-3h，冷却至室温，过滤除去金属不溶物，旋去多余的THF，用二氯甲烷萃取3次，饱和食盐水洗涤3次，合并有机相，无水MgSO4干燥，过滤，浓缩，柱色谱纯化，得到黄色固体通式I化合物，产率70％-80％。
合成步骤同实例7，得黄色固体I-1。1H NMR(400MHz,DMSO)δ8.42(dd,J＝7.8,3.6Hz,2H),8.34(d,J＝8.1Hz,1H),7.90(s,1H),7.85–7.73(m,1H),7.30(d,J＝8.2Hz,1H),5.77(s,1H),5.35(s,1H),5.07(s,1H),4.96(d,J＝21.0Hz,1H),4.65(t,J＝6.1Hz,2H),4.51–4.40(m,2H),4.34(m,2H),4.13(d,J＝7.0Hz,1H),3.94(m,1H),3.92–3.85(m,1H),3.54–3.44(m,1H),3.26–3.13(m,4H),2.84(dd,J＝14.2,7.7Hz,2H),2.73(d,J＝9.1Hz,1H),2.63(t,J＝12.9Hz,1H),1.91(d,J＝8.0Hz,1H),1.83(m,4H),1.67(m,2H),1.58–1.49(m,1H),1.36(d,J＝12.0Hz,1H),1.29(m,3H),1.25–1.16(m,2H),1.16–1.10(m,2H),1.05(m,1H),0.88(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.80,163.49,162.89,156.85,152.38,132.36,130.76,130.67,129.29,125.81,125.45,124.50,122.79,122.28,115.89,114.91,114.71,99.28,69.52,66.90,65.14,63.04,57.29,56.21,53.94,48.37,46.96,42.50,41.80,40.38,33.64,32.65,29.99,26.27,25.68,24.25,23.82,20.69,17.67.HR-MS(ESI):Calculated for C43H52N5O7[M+H]+:750.3867,found 750.3854。
合成步骤同实例7，得黄色固体I-2。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.2Hz,1H),8.40(d,J＝8.6Hz,1H),8.37(d,J＝8.2Hz,1H),7.89(s,1H),7.83–7.75(m,1H),7.30(d,J＝8.2Hz,1H),5.76(s,1H),5.32(s,1H),5.11(s,1H),4.99(s,1H),4.42(t,J＝7.2Hz,2H),4.34(m,2H),4.11(t,J＝6.9Hz,3H),3.99–3.89(m,2H),3.55(dt,J＝13.4,6.7Hz,1H),3.19(m,4H),2.86(t,J＝6.4Hz,2H),2.72(d,J＝9.0Hz,1H),2.63(t,J＝12.7Hz,1H),2.25–2.16(m,2H),1.91(d,J＝9.2Hz,1H),1.82(m,4H),1.66(m,2H),1.58–1.50(m,1H),1.35(d,J＝13.1Hz,1H),1.28(m,3H),1.25–1.19(m,2H),1.15(m,2H),1.02(m,1H),0.88(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.70,163.14,156.74,152.37,143.96,132.25,130.57,129.23,125.76,125.41,122.53,122.20,115.84,115.02,114.87,99.32,69.52,66.79,65.13,63.03,57.30,56.23,53.94,48.37,47.47,42.51,41.79,40.36,37.07,33.63,32.64,29.96,28.52,26.30,25.68,24.24,23.82,21.70,20.68,17.69,13.86.HR-MS(ESI):Calculated for C44H54N5O7[M+H]+:764.4023,found 764.4019。
合成步骤同实例7，得黄色固体I-3。1H NMR(400MHz,DMSO)δ8.43(d,J＝7.1Hz,1H),8.36(m,2H),7.85(s,1H),7.77(t,J＝7.8Hz,1H),7.27(d,J＝8.2Hz,1H),5.77(s,1H),5.33(s,1H),5.07(s,1H),5.01(d,J＝2.6Hz,1H),4.42–4.30(m,4H),4.14–4.09(m,1H),4.06(t,J＝6.8Hz,2H),3.97(m,2H),3.61–3.48(m,1H),3.18(m,4H),2.88(m,2H),2.73(d,J＝9.0Hz,1H),2.62(t,J＝12.6Hz,1H),1.85(m,7H),1.64(m,4H),1.53(m,1H),1.30–1.19(m,6H),1.16–1.08(m,2H),1.00–0.90(m,1H),0.84(s,3H),0.76(s,3H).13C NMR(101MHz,DMSO)δ205.74,163.61,163.06,156.69,152.35,132.25,130.56,129.15,125.74,125.40,124.50,122.41,122.25,115.85,114.91,114.84,99.27,69.53,66.73,65.13,63.03,57.29,56.19,53.93,48.94,48.32,42.51,41.79,40.28,38.73,33.57,32.60,30.91,29.93,27.40,26.31,25.68,24.70,24.23,23.81,22.02,20.61,17.66,13.91.HR-MS(ESI):Calculated for C45H56N5O7[M+H]+:778.4180,found 778.4173。
合成步骤同实例7，得黄色固体I-4。1H NMR(400MHz,DMSO)δ8.44(m,1H),8.41–8.33(m,2H),7.84(s,1H),7.82–7.72(m,1H),7.28(t,J＝7.2Hz,1H),5.78(s,1H),5.36(s,1H),5.11(s,1H),5.07–4.94(m,1H),4.33(m,4H),4.13(d,J＝6.9Hz,1H),4.05–3.93(m,4H),3.56(m,1H),3.19(m,4H),2.92–2.83(m,2H),2.74(d,J＝8.9Hz,1H),2.64(t,J＝12.6Hz,1H),1.88(m,3H),1.82(m,4H),1.66(m,4H),1.59–1.49(m,1H),1.41–1.33(m,2H),1.30(m,4H),1.23(m,2H),1.19–1.11(m,2H),1.04(m,1H),0.87(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.53,162.98,156.68,152.36,144.07,132.23,130.54,129.13,125.76,125.41,122.45,122.03,115.88,114.96,114.87,99.31,69.53,66.79,65.14,63.07,57.31,56.22,53.93,49.04,48.36,42.52,41.81,40.37,33.63,32.64,30.92,29.98,29.43,27.00,26.31,25.68,24.25,23.81,23.41,22.02,20.67,17.71,13.92.HR-MS(ESI):Calculated for C46H58N5O7[M+H]+:792.4336,found 792.4332。
合成步骤同实例7，得黄色固体I-5。1H NMR(400MHz,DMSO)δ8.43(d,J＝7.2Hz,1H),8.37(d,J＝8.5Hz,1H),8.35(d,J＝8.2Hz,1H),7.85(s,1H),7.78(t,J＝7.9Hz,1H),7.27(d,J＝8.2Hz,1H),5.79(s,1H),5.36(s,1H),5.10(s,1H),5.01(d,J＝2.8Hz,1H),4.34(dt,J＝14.1,9.6Hz,4H),4.10(d,J＝7.0Hz,1H),4.03–3.94(m,4H),3.56(dt,J＝13.3,6.7Hz,1H),3.18(m,4H),2.93–2.85(m,2H),2.74(d,J＝9.1Hz,1H),2.63(t,J＝12.7Hz,1H),1.89(d,J＝8.3Hz,1H),1.81(m,6H),1.66(m,2H),1.59(m,2H),1.57–1.49(m,1H),1.34(m,2H),1.32–1.18(m,8H),1.15–1.08(m,2H),0.98(m,1H),0.86(s,3H),0.75(s,3H).13CNMR(101MHz,DMSO)δ205.73,163.51,162.97,156.65,152.35,144.11,132.20,130.52,129.12,125.74,125.41,122.47,122.00,115.86,114.98,114.85,99.29,69.51,66.77,65.13,63.06,57.30,56.18,53.93,49.14,48.31,42.51,41.80,40.31,33.58,32.60,30.92,29.97,29.57,27.38,26.32,25.93,25.68,25.62,24.23,23.82,22.02,20.64,17.69,13.91.HR-MS(ESI):Calculated for C47H60N5O7[M+H]+:806.4493,found 806.4484。
合成步骤同实例7，得黄色固体I-6。1H NMR(400MHz,DMSO)δ8.40(d,J＝7.7Hz,2H),8.32(d,J＝8.1Hz,1H),7.88(s,1H),7.79(t,J＝7.9Hz,1H),7.29(d,J＝8.2Hz,1H),5.80(s,1H),5.37(s,1H),5.10(s,1H),5.08(d,J＝2.8Hz,1H),4.65(t,J＝5.9Hz,2H),4.56(s,1H),4.53–4.41(m,3H),4.25(d,J＝7.0Hz,1H),3.98(d,J＝3.0Hz,1H),3.74(dt,J＝9.8,6.0Hz,1H),3.25–3.15(m,4H),2.83–2.77(m,1H),2.70–2.56(m,4H),2.02(d,J＝10.0Hz,1H),1.87–1.76(m,6H),1.67(m,2H),1.60–1.50(m,1H),1.40(d,J＝12.8Hz,2H),1.36(m,2H),1.32–1.23(m,2H),1.22–1.15(m,2H),1.13–1.01(m,1H),0.88(s,3H),0.81(s,3H).13C NMR(101MHz,DMSO)δ205.79,163.44,162.84,156.82,152.31,132.31,130.73,130.62,129.25,125.77,125.42,122.47,122.23,116.00,114.88,114.67,99.17,69.64,66.38,65.06,63.15,57.32,56.30,53.94,48.44,47.02,42.56,41.93,40.40,39.38,33.66,32.67,30.93,30.07,29.45,25.68,24.29,23.82,22.03,21.81,20.67,17.76,13.93.HR-MS(ESI):Calculated for C44H54N5O7[M+H]+:764.4023,found 764.4014。
合成步骤同实例7，得黄色固体I-7。1H NMR(400MHz,DMSO)δ8.44(d,J＝7.1Hz,1H),8.40–8.37(m,1H),8.35(d,J＝8.1Hz,1H),7.88(s,1H),7.82–7.75(m,1H),7.29(d,J＝8.2Hz,1H),5.80(s,1H),5.34(s,1H),5.07(m,2H),4.55(d,J＝9.3Hz,1H),4.50(dt,J＝16.3,8.0Hz,1H),4.42(t,J＝7.1Hz,2H),4.25(d,J＝7.0Hz,1H),4.08(t,J＝6.9Hz,2H),3.98(d,J＝3.1Hz,1H),3.86–3.73(m,1H),3.23–3.14(m,4H),2.81(d,J＝9.1Hz,1H),2.70–2.57(m,4H),2.24–2.15(m,2H),2.02(d,J＝12.1Hz,1H),1.82(m,6H),1.67(m,2H),1.60–1.49(m,1H),1.38(m,4H),1.32–1.23(m,2H),1.19(m,2H),1.11–0.99(m,1H),0.87(s,3H),0.78(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.66,163.10,156.71,152.32,132.22,130.54,129.19,125.74,125.38,122.50,121.86,115.99,115.00,114.83,99.24,69.63,66.63,65.07,63.14,57.31,56.27,53.94,48.40,47.44,42.55,41.93,40.37,39.37,37.07,33.63,32.63,30.05,29.28,28.46,25.67,24.27,23.81,21.91,20.66,17.78.HR-MS(ESI):Calculated for C45H56N5O7[M+H]+:778.4180,found 778.4161。
合成步骤同实例7，得黄色固体I-8。1H NMR(400MHz,DMSO)δ8.44(d,J＝6.9Hz,1H),8.39–8.33(m,2H),7.85(s,1H),7.81–7.74(m,1H),7.28(d,J＝8.2Hz,1H),5.80(s,1H),5.34(s,1H),5.17–4.98(m,2H),4.59–4.45(m,2H),4.36(t,J＝6.9Hz,2H),4.25(d,J＝7.0Hz,1H),4.05(t,J＝7.0Hz,2H),3.97(d,J＝3.1Hz,1H),3.82–3.73(m,1H),3.22–3.15(m,4H),2.80(d,J＝9.0Hz,1H),2.72–2.66(m,2H),2.66–2.56(m,2H),2.01(d,J＝10.9Hz,1H),1.90–1.83(m,5H),1.82(m,3H),1.70–1.64(m,2H),1.63–1.58(m,2H),1.57–1.48(m,1H),1.34(m,4H),1.30–1.22(m,2H),1.16(m,2H),1.03(m,1H),0.85(s,3H),0.77(s,3H).13CNMR(101MHz,DMSO)δ205.76,163.60,163.05,156.69,152.32,146.21,132.25,130.56,129.15,125.76,125.39,122.43,121.81,115.98,114.92,114.86,99.23,69.61,66.66,65.06,63.13,57.30,56.24,53.93,48.88,48.39,42.55,41.92,40.35,39.36,38.73,33.60,32.62,30.04,29.28,27.42,25.67,24.69,24.26,23.81,21.93,20.63,17.76.HR-MS(ESI):Calculated for C46H58N5O7[M+H]+:792.4336,found 792.4329。
合成步骤同实例7，得黄色固体I-9。1H NMR(400MHz,DMSO)δ8.48–8.43(m,1H),8.42–8.38(m,1H),8.37(d,J＝8.1Hz,1H),7.82(s,1H),7.80(m,1H),7.30(d,J＝8.2Hz,1H),5.79(s,1H),5.37(s,1H),5.10(s,2H),4.61–4.44(m,2H),4.29(m,3H),4.00(m,3H),3.82–3.73(m,1H),3.23–3.17(m,4H),2.81(d,J＝9.1Hz,1H),2.71–2.64(m,3H),2.61(m,1H),2.02(d,J＝12.4Hz,1H),1.90–1.82(m,6H),1.81(m,2H),1.66(m,2H),1.65–1.60(m,2H),1.59–1.51(m,1H),1.43–1.35(m,4H),1.31–1.22(m,4H),1.21–1.15(m,2H),1.11–1.02(m,1H),0.93–0.88(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.55,163.00,156.70,152.32,146.22,132.26,130.58,129.14,125.80,125.42,122.47,121.66,116.01,114.96,114.90,99.24,69.62,66.68,65.08,63.15,57.31,56.27,53.94,48.98,48.41,42.55,41.93,40.38,39.38,33.65,32.65,30.06,29.39,29.32,26.95,25.68,24.27,23.82,23.35,21.93,20.69,17.79.HR-MS(ESI):Calculated for C47H60N5O7[M+H]+:806.4493,found 806.4480。
合成步骤同实例7，得黄色固体I-10。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.2Hz,1H),8.38(m,2H),7.84(s,1H),7.79(m,1H),7.29(d,J＝8.2Hz,1H),5.79(s,1H),5.34(s,1H),5.14–5.02(m,2H),4.58–4.44(m,2H),4.29(t,J＝7.5Hz,2H),4.24(d,J＝7.0Hz,1H),4.04–3.94(m,3H),3.83–3.69(m,1H),3.23–3.16(m,4H),2.83–2.77(m,1H),2.65(m,4H),2.00(d,J＝11.8Hz,1H),1.86(m,5H),1.81–1.77(m,3H),1.66(m,2H),1.62–1.57(m,2H),1.56–1.48(m,1H),1.35(m,6H),1.30–1.22(m,4H),1.19–1.12(m,2H),1.08–0.99(m,1H),0.85(s,3H),0.75(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.52,162.98,156.68,152.31,146.19,132.23,130.55,129.13,125.79,125.42,122.48,121.66,115.99,114.99,114.89,99.23,69.61,66.63,65.06,63.14,57.29,56.25,53.93,49.09,48.38,42.55,41.91,40.34,39.36,33.60,32.59,30.04,29.55,29.27,27.39,25.91,25.68,25.60,24.25,23.81,21.90,20.64,17.76.HR-MS(ESI):Calculated for C48H62N5O7[M+H]+:820.4649,found 820.4636。
合成步骤同实例7，得黄色固体I-11。1H NMR(400MHz,DMSO)δ8.40(m,2H),8.32(d,J＝8.1Hz,1H),7.86(s,1H),7.79(t,J＝7.9Hz,1H),7.30(d,J＝8.2Hz,1H),5.79(s,1H),5.35(s,1H),5.07(m,2H),4.65(t,J＝5.9Hz,2H),4.54(s,1H),4.50–4.39(m,3H),4.18(d,J＝7.2Hz,1H),3.98(d,J＝3.1Hz,1H),3.81–3.67(m,1H),3.21(m,4H),2.77(d,J＝9.0Hz,1H),2.70–2.53(m,4H),1.98(d,J＝11.8Hz,1H),1.82(m,4H),1.67(m,2H),1.56(m,5H),1.43–1.31(m,4H),1.30–1.21(m,2H),1.17(m,2H),1.07(m,1H),0.91(s,3H),0.82(s,3H).13C NMR(101MHz,DMSO)δ205.80,163.44,162.83,156.83,152.33,146.55,132.33,130.73,130.63,129.26,125.78,125.42,122.34,122.25,115.99,114.90,114.67,99.12,69.64,66.84,65.05,63.10,57.29,56.29,53.93,48.43,46.96,42.56,41.91,40.40,39.36,33.66,32.68,30.04,28.45,25.77,25.68,24.38,24.30,23.82,20.73,17.78.HR-MS(ESI):Calculated for C45H56N5O7[M+H]+:778.4180,found 778.4167。
合成步骤同实例7，得黄色固体I-12。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.3Hz,1H),8.40(d,J＝7.7Hz,1H),8.37(d,J＝8.1Hz,1H),7.86(s,1H),7.83–7.75(m,1H),7.30(d,J＝8.2Hz,1H),5.78(s,1H),5.36(s,1H),5.18–4.97(m,2H),4.53(s,1H),4.43(m,3H),4.19(d,J＝6.7Hz,1H),4.09(t,J＝6.9Hz,2H),3.97(d,J＝3.1Hz,1H),3.82–3.68(m,1H),3.25–3.14(m,4H),2.77(d,J＝8.7Hz,1H),2.68–2.53(m,4H),2.26–2.13(m,2H),1.97(d,J＝11.9Hz,1H),1.82(m,4H),1.70–1.60(m,4H),1.55(m,3H),1.35(m,4H),1.29–1.21(m,2H),1.15(m,2H),1.09–0.98(m,1H),0.88(s,3H),0.76(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.68,163.13,156.72,152.32,146.54,132.24,130.56,129.22,125.77,125.41,122.54,121.71,115.96,115.03,114.86,99.08,69.63,66.86,65.04,63.09,57.29,56.28,53.95,48.41,47.44,42.55,41.89,40.37,37.08,33.63,32.64,30.03,28.76,28.48,25.73,25.68,24.57,24.27,23.81,20.69,17.75.HR-MS(ESI):Calculated for C46H58N5O7[M+H]+:792.4336,found 792.4323。
合成步骤同实例7，得黄色固体I-13。1H NMR(400MHz,DMSO)δ8.47–8.43(m,1H),8.41–8.34(m,2H),7.83(s,1H),7.82–7.76(m,1H),7.29(d,J＝8.2Hz,1H),5.79(s,1H),5.36(s,1H),5.08(m,2H),4.53(s,1H),4.44(m,1H),4.36(t,J＝6.9Hz,2H),4.19(d,J＝7.0Hz,1H),4.06(t,J＝7.0Hz,2H),3.97(d,J＝3.1Hz,1H),3.83–3.70(m,1H),3.24–3.15(m,4H),2.77(d,J＝9.0Hz,1H),2.60(m,4H),1.97(d,J＝10.3Hz,1H),1.84(m,6H),1.59(m,9H),1.41–1.20(m,6H),1.19–1.10(m,2H),1.07–0.96(m,1H),0.87(s,3H),0.79(s,3H).13CNMR(101MHz,DMSO)δ205.77,163.63,163.08,156.72,152.32,146.64,132.28,130.59,129.18,125.78,125.42,122.46,121.68,115.98,114.94,114.89,99.09,69.63,66.85,65.05,63.08,57.29,56.27,53.94,48.85,48.40,42.56,41.90,40.36,38.73,33.62,32.65,30.02,28.76,27.45,25.75,25.68,24.70,24.57,24.27,23.81,20.69,17.74.
HR-MS(ESI):Calculated for C47H60N5O7[M+H]+:806.4493,found 806.4479。
合成步骤同实例7，得黄色固体I-14。1H NMR(400MHz,DMSO)δ8.44(d,J＝7.2Hz,1H),8.41–8.30(m,2H),7.81(s,1H),7.78(m,1H),7.28(d,J＝8.2Hz,1H),5.79(s,1H),5.36(s,1H),5.15–5.02(m,2H),4.54(s,1H),4.52–4.41(m,1H),4.31(t,J＝7.0Hz,2H),4.18(d,J＝7.2Hz,1H),4.00(m,3H),3.77(m,1H),3.23–3.14(m,4H),2.78(d,J＝8.9Hz,1H),2.70–2.54(m,4H),1.98(d,J＝11.5Hz,1H),1.86(m,6H),1.72–1.60(m,6H),1.55(m,3H),1.44–1.32(m,4H),1.32–1.28(m,2H),1.28–1.21(m,2H),1.21–1.13(m,2H),1.11–1.00(m,1H),0.93–0.87(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.52,162.98,156.68,152.32,132.23,130.54,129.12,125.76,125.40,122.44,121.53,115.97,114.94,114.86,99.12,69.63,66.87,65.06,63.11,57.30,56.29,53.93,48.97,48.41,42.58,41.91,40.38,39.36,33.64,32.65,30.04,29.42,28.79,26.96,25.78,25.68,24.57,24.29,23.81,23.35,20.70,17.77.HR-MS(ESI):Calculated for C48H62N5O7[M+H]+:820.4649,found 820.4635。
合成步骤同实例7，得黄色固体I-15。1H NMR(400MHz,DMSO)δ8.47–8.42(m,1H),8.40–8.33(m,2H),7.83(s,1H),7.78(m,1H),7.28(d,J＝8.2Hz,1H),5.77(s,1H),5.36(s,1H),5.15–5.00(m,2H),4.53(s,1H),4.52–4.40(m,1H),4.30(t,J＝7.0Hz,2H),4.18(d,J＝6.8Hz,1H),3.99(m,3H),3.83–3.71(m,1H),3.23–3.13(m,4H),2.77(d,J＝9.1Hz,1H),2.71–2.53(m,4H),1.97(d,J＝9.8Hz,1H),1.85–1.74(m,6H),1.71–1.63(m,4H),1.63–1.50(m,5H),1.29(m,10H),1.18–1.07(m,2H),1.05–0.95(m,1H),0.86(s,3H),0.75(s,3H).13CNMR(101MHz,DMSO)δ205.74,163.52,162.98,156.66,152.31,146.67,132.22,130.54,129.13,125.76,125.42,122.48,121.50,115.96,114.98,114.87,99.11,69.62,66.87,65.04,63.09,57.28,56.25,53.93,49.08,48.38,42.57,41.90,40.35,33.60,32.62,30.01,29.57,28.78,27.37,25.89,25.79,25.68,25.57,24.59,24.27,23.81,20.67,17.75.HR-MS(ESI):Calculated for C49H63N5NaO7[M+Na]+:856.4625,found 856.4613。
合成步骤同实例7，得黄色固体I-16。1H NMR(400MHz,DMSO)δ8.40(m,2H),8.32(d,J＝8.1Hz,1H),7.85(s,1H),7.79(t,J＝7.9Hz,1H),7.30(d,J＝8.2Hz,1H),5.79(s,1H),5.34(s,1H),5.07(m,2H),4.64(t,J＝5.9Hz,2H),4.54(s,1H),4.50–4.41(m,3H),4.18(d,J＝7.2Hz,1H),3.98(d,J＝3.0Hz,1H),3.72(m,1H),3.24–3.16(m,4H),2.77(d,J＝9.2Hz,1H),2.60(m,4H),1.98(d,J＝10.1Hz,1H),1.82(m,4H),1.67(m,2H),1.59–1.48(m,5H),1.37(m,4H),1.33–1.22(m,4H),1.17(m,2H),1.07(m,1H),0.90(s,3H),0.81(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.43,162.84,156.83,152.33,146.65,132.31,130.73,130.61,129.26,125.77,125.44,122.30,122.26,116.01,114.89,114.70,99.07,69.64,67.10,65.05,63.12,57.32,56.29,53.94,48.42,46.92,42.57,41.92,40.40,33.66,32.67,30.04,29.03,28.70,25.68,25.05,24.89,24.29,23.82,20.73,17.78.HR-MS(ESI):Calculated for C46H58N5O7[M+H]+:792.4336,found 792.4327。
合成步骤同实例7，得黄色固体I-17。1H NMR(400MHz,DMSO)δ8.45(d,J＝6.7Hz,1H),8.39(m,2H),7.85(s,1H),7.83–7.75(m,1H),7.30(d,J＝8.2Hz,1H),5.74(s,1H),5.36(s,1H),5.07(m,2H),4.52(s,1H),4.48–4.28(m,3H),4.11(m,3H),3.96(d,J＝3.1Hz,1H),3.74(m,1H),3.24–3.12(m,4H),2.78(d,J＝9.0Hz,1H),2.70–2.52(m,4H),2.24–2.14(m,2H),1.95(d,J＝11.9Hz,1H),1.83(m,4H),1.66(m,2H),1.62–1.49(m,5H),1.35(m,6H),1.27–1.21(m,2H),1.17–1.10(m,2H),1.04(m,1H),0.88(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.75,163.70,163.15,156.72,152.32,146.57,132.24,130.56,129.25,125.76,125.42,122.58,121.69,115.98,115.07,114.86,99.08,69.60,67.10,65.03,63.09,57.29,56.23,53.95,48.37,47.44,42.55,41.88,40.36,37.10,33.63,32.65,30.02,29.06,28.59,28.43,25.68,25.35,25.00,24.28,23.82,20.69,17.75.HR-MS(ESI):Calculated for C47H60N5O7[M+H]+:806.4493,found 806.4479。
合成步骤同实例7，得黄色固体I-18。1H NMR(400MHz,DMSO)δ8.45(d,J＝6.8Hz,1H),8.38(m,2H),7.83(s,1H),7.82–7.76(m,1H),7.30(d,J＝8.2Hz,1H),5.79(s,1H),5.37(s,1H),5.16–5.01(m,2H),4.53(s,1H),4.51–4.42(m,1H),4.34(t,J＝6.9Hz,2H),4.19(d,J＝6.8Hz,1H),4.06(t,J＝7.0Hz,2H),3.97(d,J＝3.1Hz,1H),3.74(m,1H),3.23–3.11(m,4H),2.78(d,J＝8.8Hz,1H),2.68–2.55(m,4H),1.97(d,J＝9.7Hz,1H),1.88–1.77(m,6H),1.63(m,5H),1.59–1.50(m,4H),1.39(m,3H),1.34–1.29(m,3H),1.25(m,2H),1.15(m,2H),1.04(m,1H),0.87(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.63,163.09,156.73,152.33,146.70,132.28,130.59,129.19,125.79,125.43,122.48,121.66,115.99,114.96,114.90,99.07,69.62,67.06,65.03,63.10,57.30,56.26,53.94,48.84,48.39,42.57,41.91,40.37,38.74,33.63,32.65,30.02,29.02,28.63,27.42,25.68,25.40,25.04,24.70,24.28,23.81,20.70,17.75.HR-MS(ESI):Calculated for C48H62N5O7[M+H]+:820.4649,found 820.4637。
合成步骤同实例7，得黄色固体I-19。1H NMR(400MHz,DMSO)δ8.44(d,J＝7.2Hz,1H),8.37(m,2H),7.81(s,1H),7.78(d,J＝8.3Hz,1H),7.29(d,J＝8.2Hz,1H),5.79(s,1H),5.35(s,1H),5.07(m,2H),4.54(s,1H),4.47(d,J＝8.0Hz,1H),4.30(t,J＝7.0Hz,2H),4.20(s,1H),3.99(m,3H),3.75(m,1H),3.23–3.14(m,4H),2.78(d,J＝9.2Hz,1H),2.61(m,4H),1.98(d,J＝10.0Hz,1H),1.84(m,6H),1.63(m,5H),1.59–1.51(m,4H),1.42–1.32(m,6H),1.30–1.21(m,4H),1.20–1.12(m,2H),1.05(m,1H),0.89(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.53,162.99,156.68,152.33,132.23,130.55,129.13,125.77,125.42,122.46,121.54,115.98,114.96,114.88,99.08,69.63,67.09,65.03,63.11,57.31,56.28,53.94,48.94,48.40,42.57,41.92,40.38,33.63,32.64,30.03,29.39,29.03,28.66,26.95,25.68,25.40,25.03,24.28,23.82,23.35,20.69,17.76.HR-MS(ESI):Calculated for C49H64N5O7[M+H]+:834.4806,found 834.4797。
合成步骤同实例7，得黄色固体I-20。1H NMR(400MHz,DMSO)δ8.45(d,J＝6.7Hz,1H),8.41–8.34(m,2H),7.83(s,1H),7.82–7.76(m,1H),7.31(d,J＝8.4Hz,1H),5.79(s,1H),5.36(s,1H),5.06(m,2H),4.53(s,1H),4.51–4.41(m,1H),4.28(t,J＝7.0Hz,2H),4.19(d,J＝7.0Hz,1H),4.03–3.94(m,3H),3.74(m,1H),3.23–3.14(m,4H),2.78(d,J＝9.0Hz,1H),2.68–2.53(m,4H),1.96(d,J＝9.8Hz,1H),1.86–1.76(m,6H),1.59(m,9H),1.42–1.33(m,6H),1.32–1.26(m,4H),1.24(m,2H),1.15(m,2H),1.04(m,1H),0.86(s,3H),0.78(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.53,162.99,156.68,152.33,146.71,132.23,130.55,129.15,125.79,125.44,122.50,121.49,115.97,115.01,114.90,99.06,69.62,67.07,65.03,63.10,57.30,56.26,53.94,49.06,48.39,42.56,41.91,40.37,33.62,32.63,30.03,29.54,29.01,28.66,27.37,25.88,25.68,25.58,25.41,25.05,24.27,23.82,20.68,17.74.HR-MS(ESI):Calculated for C50H65N5NaO7[M+Na]+:870.4782,found870.4771。
合成步骤同实例7，得黄色固体I-21。1H NMR(400MHz,DMSO)δ8.40(m,2H),8.31(d,J＝8.1Hz,1H),7.85(s,1H),7.82–7.73(m,1H),7.29(d,J＝8.2Hz,1H),5.79(s,1H),5.36(s,1H),5.15–5.00(m,2H),4.64(t,J＝5.8Hz,2H),4.53(s,1H),4.44(m,3H),4.21(d,J＝6.9Hz,1H),3.98(d,J＝3.1Hz,1H),3.74(m,1H),3.23–3.13(m,4H),2.79(d,J＝9.0Hz,1H),2.70–2.52(m,4H),1.99(d,J＝10.9Hz,1H),1.82(m,4H),1.66(m,2H),1.53(m,5H),1.35(m,6H),1.30–1.22(m,4H),1.17(m,2H),1.08(m,1H),0.90(s,3H),0.81(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.41,162.82,156.82,152.33,146.69,132.29,130.71,130.59,129.25,125.76,125.43,122.29,122.25,115.99,114.88,114.69,99.10,69.65,67.19,65.04,63.11,57.33,56.29,53.94,48.42,46.92,42.55,41.95,40.40,39.36,33.66,32.67,30.04,29.22,29.00,27.93,25.68,25.49,24.77,24.30,23.82,20.72,17.79.
HR-MS(ESI):Calculated for C47H60N5O7[M+H]+:806.4493,found 806.4474。
合成步骤同实例7，得黄色固体I-22。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.2Hz,1H),8.38(m,2H),7.85(s,1H),7.83–7.74(m,1H),7.30(d,J＝8.2Hz,1H),5.78(s,1H),5.35(s,1H),5.06(m,2H),4.53(s,1H),4.51–4.35(m,3H),4.20(d,J＝6.1Hz,1H),4.08(t,J＝6.9Hz,2H),3.96(d,J＝3.2Hz,1H),3.74(m,1H),3.23–3.13(m,4H),2.78(d,J＝9.0Hz,1H),2.69–2.53(m,4H),2.24–2.14(m,2H),1.97(d,J＝10.0Hz,1H),1.82(m,4H),1.67(m,2H),1.54(m,5H),1.35(m,8H),1.25(m,2H),1.15(m,2H),1.05(m,1H),0.87(s,3H),0.76(s,3H).
13C NMR(101MHz,DMSO)δ205.77,163.68,163.14,156.71,152.32,146.66,132.23,130.55,129.23,125.77,125.42,122.57,121.67,115.97,115.06,114.86,99.08,69.64,67.13,65.02,63.09,57.32,56.27,53.95,48.40,47.43,42.54,41.94,40.37,37.11,33.63,32.64,30.03,29.21,28.91,28.43,28.18,25.68,25.48,24.88,24.27,23.82,20.68,17.75.HR-MS(ESI):Calculated for C48H62N5O7[M+H]+:820.4649,found 820.4636。
合成步骤同实例7，得黄色固体I-23。1H NMR(400MHz,DMSO)δ8.46(d,J＝7.2Hz,1H),8.39(m,2H),7.83(s,1H),7.79(m,1H),7.31(d,J＝8.2Hz,1H),5.78(s,1H),5.36(s,1H),5.07(m,2H),4.51(s,1H),4.46(m,1H),4.34(t,J＝7.0Hz,2H),4.21(s,1H),4.06(t,J＝7.1Hz,2H),3.96(d,J＝3.0Hz,1H),3.72(m,1H),3.23–3.17(m,4H),2.77(d,J＝8.8Hz,1H),2.69–2.52(m,4H),1.97(d,J＝12.3Hz,1H),1.89–1.77(m,6H),1.70–1.62(m,2H),1.62–1.54(m,4H),1.54–1.44(m,3H),1.32(m,8H),1.27–1.21(m,2H),1.15(m,2H),1.05(m,1H),0.90(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.64,163.10,156.74,152.32,146.74,132.29,130.61,129.20,125.82,125.44,122.49,121.68,116.01,114.97,114.92,99.08,69.64,67.07,65.05,63.09,57.31,56.26,53.95,48.78,48.26,42.54,41.94,40.38,38.73,33.64,32.66,30.02,29.20,28.94,28.17,27.44,25.68,25.47,24.88,24.70,23.81,20.70,17.76.HR-MS(ESI):Calculated for C49H64N5O7[M+H]+:834.4806,found 834.4795。
合成步骤同实例7，得黄色固体I-24。1H NMR(400MHz,DMSO)δ8.46(d,J＝6.8Hz,1H),8.39(m,2H),7.83–7.76(m,2H),7.31(d,J＝8.2Hz,1H),5.78(s,1H),5.36(s,1H),5.07(m,2H),4.53(s,1H),4.47(d,J＝8.0Hz,1H),4.29(t,J＝7.0Hz,2H),4.20(s,1H),4.06–3.93(m,3H),3.74(m,1H),3.24–3.16(m,4H),2.79(d,J＝9.1Hz,1H),2.70–2.53(m,4H),1.98(d,J＝10.3Hz,1H),1.89–1.78(m,6H),1.70–1.61(m,4H),1.59–1.47(m,5H),1.36(m,6H),1.32–1.22(m,6H),1.16(m,2H),1.06(m,1H),0.87(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.55,163.01,156.71,152.32,132.26,130.63,130.58,129.16,125.81,125.44,122.48,121.52,115.98,114.98,114.91,99.09,69.64,67.14,65.03,63.10,57.32,56.28,53.95,48.93,48.41,42.54,41.95,40.38,33.64,32.65,30.03,29.39,29.22,28.95,28.20,26.94,25.68,25.46,24.88,24.28,23.82,23.34,20.70,17.77.HR-MS(ESI):Calculated for C50H66N5O7[M+H]+:848.4962,found 848.4952。
合成步骤同实例7，得黄色固体I-25。1H NMR(400MHz,DMSO)δ8.46(d,J＝7.2Hz,1H),8.39(m,2H),7.83(s,1H),7.79(m,1H),7.31(d,J＝8.2Hz,1H),5.78(s,1H),5.36(s,1H),5.09–4.94(m,2H),4.53(s,1H),4.50–4.42(m,1H),4.28(t,J＝7.1Hz,2H),4.20(d,J＝7.1Hz,1H),4.04–3.94(m,3H),3.79–3.68(m,1H),3.21(m,4H),2.77(d,J＝8.8Hz,1H),2.59(m,4H),1.97(d,J＝9.6Hz,1H),1.87–1.75(m,6H),1.66(m,2H),1.63–1.55(m,4H),1.51(m,3H),1.34(m,8H),1.30–1.22(m,6H),1.19–1.11(m,2H),1.04(m,1H),0.87(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.54,163.00,156.70,152.32,146.76,132.25,130.56,129.16,125.81,125.45,122.51,121.50,115.97,115.01,114.91,99.08,69.63,67.12,65.03,63.09,57.32,56.27,53.94,49.03,48.40,42.54,41.95,40.38,33.67,33.63,32.64,30.92,30.04,29.54,29.20,28.98,28.19,27.37,25.86,25.68,25.56,25.46,24.90,24.28,23.76,22.00,20.69,17.76.HR-MS(ESI):Calculated for C51H68N5O7[M+H]+:862.5119,found 862.5108。
合成步骤同实例7，得黄色固体I-26。1H NMR(400MHz,DMSO)δ8.44(m,1H),8.42–8.34(m,2H),7.79(m,1H),7.71(s,1H),7.29(d,J＝8.2Hz,1H),5.77(s,1H),5.35(s,1H),5.06(d,J＝10.4Hz,1H),4.97(d,J＝2.8Hz,1H),4.45(t,J＝5.2Hz,2H),4.37–4.28(m,2H),4.25–4.18(m,2H),4.10(d,J＝7.0Hz,1H),3.95(d,J＝3.0Hz,1H),3.93–3.86(m,1H),3.85–3.79(m,2H),3.67(t,J＝6.0Hz,2H),3.53–3.44(m,1H),3.23–3.16(m,4H),2.76–2.57(m,4H),1.89(d,J＝7.6Hz,1H),1.82(m,4H),1.67(m,2H),1.58–1.48(m,1H),1.35(d,J＝13.1Hz,1H),1.28(m,3H),1.21(m,2H),1.16–1.09(m,2H),0.99(m,1H),0.86(s,3H),0.78(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.60,163.03,156.75,152.37,143.93,132.32,130.63,129.17,125.76,125.41,122.51,122.35,115.88,114.87,114.82,99.22,69.50,68.37,67.07,66.71,65.12,63.04,57.29,56.20,53.94,49.27,48.36,42.51,41.77,40.36,38.36,33.61,32.64,29.95,26.12,25.68,24.24,23.82,20.66,17.69.HR-MS(ESI):Calculated for C45H56N5O8[M+H]+:794.4129,found 794.4116。
合成步骤同实例7，得黄色固体I-27。1H NMR(400MHz,DMSO)δ8.46–8.41(m,1H),8.41–8.33(m,2H),7.79(dd,J＝8.3,7.4Hz,1H),7.65(s,1H),7.29(d,J＝8.2Hz,1H),5.80(s,1H),5.37(s,1H),5.08(m,2H),4.54(s,1H),4.51–4.38(m,3H),4.22(m,3H),3.98(d,J＝3.0Hz,1H),3.81(t,J＝5.1Hz,2H),3.70(m,3H),3.22–3.14(m,4H),2.81(d,J＝9.0Hz,1H),2.71–2.55(m,2H),2.50–2.42(m,2H),2.01(d,J＝12.1Hz,1H),1.83(m,4H),1.75–1.62(m,4H),1.59–1.50(m,1H),1.45–1.33(m,4H),1.33–1.21(m,2H),1.21–1.14(m,2H),1.10–1.01(m,1H),0.88(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.75,163.58,163.02,156.75,152.31,146.06,132.32,130.63,129.16,125.77,125.40,122.34,122.04,116.00,114.88,114.80,99.19,69.63,68.44,67.06,66.62,65.08,63.15,57.31,56.28,53.94,49.24,48.41,42.57,41.92,40.38,39.37,38.45,33.63,32.64,30.03,29.21,25.69,24.28,23.82,21.76,20.67,17.79.HR-MS(ESI):Calculated for C46H58N5O8[M+H]+:808.4285,found 808.4269。
合成步骤同实例7，得黄色固体I-28。1H NMR(400MHz,DMSO)δ8.46(m,1H),8.43–8.34(m,2H),7.80(dd,J＝8.4,7.4Hz,1H),7.62(s,1H),7.30(d,J＝8.2Hz,1H),5.77(s,1H),5.36(s,1H),5.07(m,2H),4.52(s,1H),4.47–4.38(m,3H),4.22(t,J＝6.0Hz,2H),4.18(d,J＝7.1Hz,1H),3.97(d,J＝3.0Hz,1H),3.80(t,J＝5.1Hz,2H),3.76–3.70(m,1H),3.66(t,J＝6.0Hz,2H),3.24–3.17(m,4H),2.78(d,J＝8.9Hz,1H),2.69–2.54(m,2H),2.43–2.34(m,2H),1.97(d,J＝9.9Hz,1H),1.83(m,4H),1.67(m,2H),1.58–1.45(m,5H),1.42–1.31(m,4H),1.31–1.21(m,2H),1.16(m,2H),1.04(m,1H),0.90(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.61,163.04,156.77,152.32,146.46,132.35,130.66,129.20,125.80,125.43,122.38,121.95,116.00,114.90,114.83,99.10,69.63,68.44,67.06,66.86,65.05,63.11,57.29,56.29,53.95,49.23,48.41,42.57,41.89,40.38,38.45,33.65,32.66,30.03,28.77,25.73,25.69,24.40,24.29,23.82,20.71,17.77.HR-MS(ESI):Calculated for C47H60N5O8[M+H]+:822.4442,found 822.4426。
合成步骤同实例7，得黄色固体I-29。1H NMR(400MHz,DMSO)δ8.48–8.44(m,1H),8.39(m,2H),7.81(dd,J＝8.3,7.4Hz,1H),7.62(s,1H),7.30(d,J＝8.2Hz,1H),5.78(s,1H),5.35(s,1H),5.12–5.01(m,2H),4.52(s,1H),4.50–4.38(m,3H),4.25–4.15(m,3H),3.98(d,J＝3.1Hz,1H),3.80(t,J＝5.0Hz,2H),3.73(m,1H),3.67(t,J＝5.9Hz,2H),3.24–3.16(m,4H),2.79(d,J＝9.2Hz,1H),2.70–2.56(m,2H),2.35(t,J＝7.5Hz,2H),1.98(d,J＝8.4Hz,1H),1.83(m,4H),1.68(m,2H),1.54–1.43(m,5H),1.32(m,4H),1.25(m,4H),1.20–1.15(m,2H),1.09–1.01(m,1H),0.88(s,3H),0.81(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.60,163.03,156.76,152.32,146.49,132.32,130.64,129.19,125.78,125.43,122.37,121.91,115.99,114.88,114.83,99.06,69.63,68.44,67.08,65.04,63.12,57.31,56.27,53.95,49.20,48.40,42.57,41.90,40.38,38.49,33.64,32.65,30.03,29.01,28.58,27.64,25.69,25.32,24.82,24.28,23.82,20.68,17.76.HR-MS(ESI):Calculated forC48H62N5O8[M+H]+:836.4598,found 836.4582。
合成步骤同实例7，得黄色固体I-30。1H NMR(400MHz,DMSO)δ8.46(d,J＝7.2Hz,1H),8.40(m,2H),7.86–7.76(m,1H),7.59(s,1H),7.31(d,J＝8.2Hz,1H),5.77(s,1H),5.36(s,1H),5.07(m,2H),4.53(s,1H),4.43(m,3H),4.22(t,J＝5.9Hz,3H),3.97(d,J＝3.2Hz,1H),3.78(t,J＝5.0Hz,2H),3.73(m,1H),3.66(t,J＝5.9Hz,2H),3.24–3.17(m,4H),2.79(d,J＝9.0Hz,1H),2.68–2.55(m,2H),2.31(t,J＝7.6Hz,2H),1.98(d,J＝9.8Hz,1H),1.83(m,4H),1.67(m,2H),1.58–1.46(m,3H),1.43–1.37(m,3H),1.36–1.21(m,9H),1.16(m,2H),1.06(m,1H),0.89(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.62,163.05,156.78,152.32,146.56,132.35,130.66,129.21,125.82,125.45,122.39,121.87,116.00,114.91,114.85,99.09,69.65,68.45,67.15,67.09,65.04,63.11,57.33,56.29,53.96,49.20,48.41,42.55,41.95,40.39,38.51,33.65,32.66,30.04,29.23,28.90,28.22,25.69,25.48,24.70,24.29,23.83,20.70,17.77.HR-MS(ESI):Calculated for C49H64N5O8[M+H]+:850.4755,found 850.4776。
合成步骤同实例7，得黄色固体I-31。1H NMR(400MHz,DMSO)δ8.43(d,J＝7.2Hz,1H),8.39–8.32(m,2H),7.93(s,1H),7.78(dd,J＝8.3,7.4Hz,1H),7.27(d,J＝8.2Hz,1H),5.77(s,1H),5.36(s,1H),5.14(s,1H),5.06(d,J＝2.8Hz,1H),4.59(s,1H),4.50(t,J＝5.1Hz,2H),4.45(m,1H),4.39(d,J＝5.4Hz,1H),4.21(t,J＝6.1Hz,2H),4.14(d,J＝7.0Hz,1H),3.98(d,J＝3.1Hz,1H),3.85(t,J＝5.1Hz,2H),3.81(m,1H),3.67(t,J＝6.1Hz,2H),3.56(t,J＝4.8Hz,2H),3.51–3.44(m,1H),3.24–3.15(m,4H),2.76(d,J＝9.1Hz,1H),2.69–2.53(m,2H),1.98(d,J＝11.9Hz,1H),1.82(m,4H),1.67(m,2H),1.58–1.49(m,1H),1.40–1.28(m,4H),1.28–1.21(m,2H),1.15(m,2H),1.02(m,1H),0.86(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.85,163.56,162.99,156.72,152.40,143.75,132.30,130.60,129.14,125.74,125.37,124.01,122.32,115.86,114.84,114.78,99.18,69.52,69.16,68.31,67.06,66.12,65.13,63.33,63.11,57.29,56.26,53.93,49.37,48.38,42.58,41.81,40.38,38.34,33.63,32.61,29.98,25.68,24.26,23.82,20.62,17.72.HR-MS(ESI):Calculated for C46H58N5O9[M+H]+:824.4235,found 824.4218。
合成步骤同实例7，得黄色固体I-32。1H NMR(400MHz,DMSO)δ8.44(d,J＝7.2Hz,1H),8.37(m,2H),7.80(s,1H),7.79–7.74(m,1H),7.27(d,J＝8.2Hz,1H),5.77(s,1H),5.35(s,1H),5.09(s,1H),4.99(d,J＝2.8Hz,1H),4.41(t,J＝5.3Hz,2H),4.37–4.31(m,2H),4.21(t,J＝6.4Hz,2H),4.11(d,J＝7.0Hz,1H),3.99–3.92(m,2H),3.75(t,J＝5.3Hz,2H),3.63(t,J＝6.4Hz,2H),3.58–3.53(m,3H),3.53–3.47(m,2H),3.23–3.15(m,4H),2.87(t,J＝6.7Hz,2H),2.73(d,J＝9.1Hz,1H),2.60(m,1H),1.90(d,J＝8.4Hz,1H),1.82(m,4H),1.66(m,2H),1.57–1.48(m,1H),1.34(d,J＝12.9Hz,1H),1.28(m,3H),1.22(m,2H),1.13(m,2H),1.05–0.97(m,1H),0.86(s,3H),0.77(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.57,163.00,156.72,152.36,143.95,132.28,130.60,129.15,125.76,125.38,122.53,122.37,115.87,114.85,114.84,99.21,69.53,69.48,69.37,68.74,67.02,66.74,65.13,63.04,57.29,56.20,53.92,49.15,48.35,42.51,41.78,40.35,38.46,33.60,32.62,29.96,26.23,25.68,24.23,23.82,20.62,17.68.HR-MS(ESI):Calculated for C47H60N5O9[M+H]+:838.4391,found 838.4374。
合成步骤同实例7，得黄色固体I-33。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.2Hz,1H),8.39(m,2H),7.79(m,1H),7.76(s,1H),7.29(d,J＝8.2Hz,1H),5.79(s,1H),5.36(s,1H),5.07(m,2H),4.55(s,1H),4.49(q,J＝8.4Hz,1H),4.38(t,J＝5.2Hz,2H),4.21(t,J＝6.4Hz,3H),3.97(d,J＝3.0Hz,1H),3.79–3.71(m,3H),3.63(t,J＝6.4Hz,2H),3.54(m,2H),3.51–3.46(m,2H),3.23–3.15(m,4H),2.80(d,J＝9.0Hz,1H),2.70–2.56(m,4H),2.00(d,J＝11.3Hz,1H),1.84(m,6H),1.67(m,2H),1.58–1.49(m,1H),1.41–1.32(m,4H),1.30–1.21(m,2H),1.16(m,2H),1.06(m,1H),0.86(s,3H),0.77(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.59,163.02,156.76,152.32,146.15,132.31,130.63,129.18,125.80,125.41,122.40,122.12,115.99,114.89,114.86,99.23,69.63,69.47,69.41,68.74,67.02,66.72,65.07,63.14,57.30,56.27,53.93,49.13,48.40,42.56,41.92,40.37,39.37,38.48,33.63,32.64,30.03,29.34,25.68,24.27,23.82,21.92,20.67,17.76.HR-MS(ESI):Calculated for C48H62N5O9[M+H]+:852.4548,found 852.4523。
合成步骤同实例7，得黄色固体I-34。1H NMR(400MHz,DMSO)δ8.47(d,J＝7.2Hz,1H),8.40(m,2H),7.80(dd,J＝8.3,7.4Hz,1H),7.74(s,1H),7.29(d,J＝8.8Hz,1H),5.78(s,1H),5.36(s,1H),5.07(m,2H),4.52(s,1H),4.45(m,1H),4.38(t,J＝5.2Hz,2H),4.24–4.16(m,3H),3.99–3.93(m,1H),3.75(m,3H),3.62(t,J＝6.4Hz,2H),3.53(m,2H),3.51–3.46(m,2H),3.25–3.16(m,4H),2.77(m,1H),2.64–2.54(m,4H),1.96(d,J＝9.2Hz,1H),1.82(m,4H),1.67–1.61(m,3H),1.54(m,4H),1.32(m,4H),1.23(m,2H),1.15(m,2H),1.03(m,1H),0.86(s,3H),0.78(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.61,163.04,156.78,152.32,146.54,132.33,130.65,129.21,125.82,125.44,122.43,122.04,115.98,114.92,114.88,99.10,69.63,69.49,69.41,68.75,67.02,66.87,65.04,63.10,57.29,56.27,53.94,49.12,48.40,42.56,41.90,40.37,38.48,33.67,33.63,32.65,30.03,28.82,25.79,25.68,24.92,24.55,24.27,23.82,20.73,20.67,17.75,17.44.HR-MS(ESI):Calculated for C49H64N5O9[M+H]+:866.4704,found 866.4687。
合成步骤同实例7，得黄色固体I-35。1H NMR(400MHz,DMSO)δ8.46(d,J＝7.2Hz,1H),8.39(m,2H),7.85–7.76(m,1H),7.73(s,1H),7.30(d,J＝8.2Hz,1H),5.78(s,1H),5.36(s,1H),5.12–5.04(m,2H),4.52(s,1H),4.44(m,1H),4.37(t,J＝5.2Hz,2H),4.20(dd,J＝13.1,6.7Hz,3H),3.96(d,J＝3.1Hz,1H),3.76–3.69(m,3H),3.62(t,J＝6.4Hz,2H),3.53(m,2H),3.51–3.46(m,2H),3.22–3.14(m,4H),2.78(d,J＝8.9Hz,1H),2.67–2.52(m,4H),1.96(d,J＝9.5Hz,1H),1.82(m,4H),1.67(m,2H),1.61–1.48(m,5H),1.33(m,6H),1.24(m,2H),1.15(m,2H),1.05(m,1H),0.87(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.78,163.60,163.03,156.78,152.33,146.59,132.32,130.64,129.20,125.82,125.43,122.42,122.00,116.00,114.91,114.88,99.05,69.63,69.50,69.41,68.73,67.05,67.03,65.03,63.10,57.30,56.28,53.94,49.11,48.40,42.56,41.91,40.38,38.48,33.63,32.65,30.02,29.01,28.68,25.68,25.40,24.99,24.28,23.82,20.68,17.75.HR-MS(ESI):Calculated for C50H66N5O9[M+H]+:880.4861,found 880.4844。
合成步骤同实例7，得黄色固体I-36。1H NMR(400MHz,DMSO)δ8.46(d,J＝7.2Hz,1H),8.39(m,2H),7.79(dd,J＝8.4,7.4Hz,1H),7.73(s,1H),7.29(d,J＝8.2Hz,1H),5.77(s,1H),5.36(s,1H),5.09–5.03(m,2H),4.53(s,1H),4.45(m,1H),4.37(t,J＝5.2Hz,2H),4.21(t,J＝6.8Hz,3H),3.97(d,J＝3.0Hz,1H),3.76–3.68(m,3H),3.62(t,J＝6.4Hz,2H),3.53(m,2H),3.49(m,2H),3.22–3.15(m,4H),2.79(d,J＝9.0Hz,1H),2.68–2.52(m,4H),1.97(d,J＝9.4Hz,1H),1.82(m,4H),1.67(m,2H),1.58–1.44(m,5H),1.40–1.28(m,8H),1.24(m,2H),1.16(m,2H),1.05(m,1H),0.87(s,3H),0.78(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.58,163.02,156.76,152.32,146.66,132.31,130.62,129.18,125.79,125.42,122.40,122.00,115.98,114.89,114.86,99.09,69.64,69.51,69.42,68.74,67.15,67.02,65.03,63.10,57.32,56.27,53.93,49.10,48.40,42.55,41.95,40.38,38.47,33.63,32.65,30.03,29.22,29.00,28.25,25.68,25.47,24.86,24.28,23.82,20.68,17.76.HR-MS(ESI):Calculated for C51H68N5O9[M+H]+:894.5017,found 894.5002。
合成步骤同实例7，得黄色固体I-37。1H NMR(400MHz,DMSO)δ8.44(d,J＝7.2Hz,1H),8.37(m,2H),7.99(s,1H),7.78(dd,J＝8.4,7.4Hz,1H),7.28(d,J＝8.2Hz,1H),5.77(s,1H),5.35(s,1H),5.14(s,1H),5.05(d,J＝2.7Hz,1H),4.59(s,1H),4.53(d,J＝4.9Hz,1H),4.49–4.41(m,3H),4.21(t,J＝6.4Hz,2H),4.15(d,J＝7.0Hz,1H),3.97(d,J＝3.0Hz,1H),3.82(m,1H),3.77(t,J＝5.2Hz,2H),3.61(m,4H),3.51(m,5H),3.22–3.13(m,4H),2.75(d,J＝9.1Hz,1H),2.68–2.53(m,2H),1.98(d,J＝11.7Hz,1H),1.82(m,4H),1.66(m,2H),1.56–1.48(m,1H),1.39–1.28(m,4H),1.28–1.20(m,2H),1.18–1.11(m,2H),1.02(m,1H),0.85(s,3H),0.77(s,3H).13C NMR(101MHz,DMSO)δ205.85,163.57,163.00,156.73,152.40,143.82,132.29,130.61,129.15,125.77,125.39,124.05,122.37,115.86,114.87,114.84,99.18,69.53,69.47,69.39,69.20,68.67,67.02,66.14,65.13,63.46,63.10,57.29,56.27,53.92,49.25,48.38,42.57,41.82,40.38,38.46,33.62,32.61,29.99,25.68,24.25,23.82,20.60,17.71.HR-MS(ESI):Calculated for C48H62N5O10[M+H]+:868.4497,found 868.4479。
合成步骤同实例7，得黄色固体I-38。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.2Hz,1H),8.38(m,2H),7.82(s,1H),7.79(dd,J＝8.3,7.4Hz,1H),7.29(d,J＝8.2Hz,1H),5.77(s,1H),5.35(s,1H),5.09(s,1H),4.98(d,J＝2.8Hz,1H),4.44(t,J＝5.3Hz,2H),4.33(m,2H),4.22(t,J＝6.4Hz,2H),4.11(d,J＝7.1Hz,1H),4.00–3.92(m,2H),3.74(t,J＝5.3Hz,2H),3.64(t,J＝6.4Hz,2H),3.59–3.51(m,3H),3.48–3.39(m,6H),3.23–3.16(m,4H),2.87(dd,J＝14.4,7.7Hz,2H),2.73(d,J＝9.1Hz,1H),2.59(m,1H),1.90(d,J＝8.8Hz,1H),1.82(m,4H),1.67(m,2H),1.56–1.47(m,1H),1.36(d,J＝14.0Hz,1H),1.32–1.26(m,3H),1.22(m,2H),1.14(m,2H),1.06–0.97(m,1H),0.85(s,3H),0.77(s,3H).13C NMR(101MHz,DMSO)δ205.79,163.58,163.01,156.74,152.37,143.93,132.30,130.62,129.17,125.79,125.41,122.57,122.41,115.88,114.88,99.22,69.61,69.56,69.53,69.48,68.71,66.98,66.76,65.13,63.05,57.29,56.22,53.93,49.17,48.37,42.51,41.79,40.36,38.50,33.61,32.62,29.97,26.25,25.68,24.23,23.82,20.63,17.68.HR-MS(ESI):Calculated forC49H64N5O10[M+H]+:882.4653,found 882.4635。
合成步骤同实例7，得黄色固体I-39。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.2Hz,1H),8.38(m,2H),7.81–7.75(m,2H),7.29(d,J＝8.2Hz,1H),5.77(s,1H),5.37(s,1H),5.13–5.01(m,2H),4.56(s,1H),4.52–4.46(m,1H),4.42(t,J＝5.2Hz,2H),4.25–4.18(m,3H),3.97(d,J＝3.2Hz,1H),3.81–3.76(m,1H),3.73(t,J＝5.2Hz,2H),3.64(t,J＝6.4Hz,2H),3.57–3.50(m,2H),3.47–3.38(m,6H),3.23–3.15(m,4H),2.80(d,J＝9.0Hz,1H),2.72–2.56(m,4H),2.01(d,J＝11.7Hz,1H),1.88–1.78(m,6H),1.67(m,2H),1.58–1.49(m,1H),1.35(m,4H),1.31–1.21(m,2H),1.16(m,2H),1.05(m,1H),0.87(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.77,163.58,163.01,156.74,152.32,146.15,132.30,130.62,129.17,125.79,125.41,122.41,122.15,115.99,114.88,99.23,69.62,69.59,69.46,68.71,66.98,66.73,65.08,63.15,57.31,56.27,53.93,49.15,48.40,42.56,41.92,40.37,39.37,38.51,33.63,32.64,30.04,29.36,25.68,24.28,23.82,21.95,20.67,17.77.HR-MS(ESI):Calculated for C50H66N5O10[M+H]+:896.4810,found 896.4792。
合成步骤同实例7，得黄色固体I-40。1H NMR(400MHz,DMSO)δ8.43(d,J＝7.2Hz,1H),8.36(m,2H),7.81–7.73(m,2H),7.27(d,J＝8.2Hz,1H),5.78(s,1H),5.36(s,1H),5.19–5.00(m,2H),4.54(s,1H),4.45(m,3H),4.21(m,3H),3.98(d,J＝3.0Hz,1H),3.81–3.71(m,3H),3.64(t,J＝6.4Hz,2H),3.54(m,2H),3.48–3.40(m,6H),3.22–3.15(m,4H),2.78(d,J＝9.0Hz,1H),2.68–2.54(m,4H),1.98(d,J＝10.1Hz,1H),1.82(m,4H),1.65(m,4H),1.55(m,3H),1.41–1.21(m,6H),1.16(m,2H),1.09–1.00(m,1H),0.87(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.55,162.98,156.71,152.33,146.54,132.26,130.58,129.14,125.74,125.38,122.37,122.07,115.96,114.84,99.11,69.62,69.60,69.58,69.48,68.73,66.98,66.87,65.06,63.10,57.29,56.27,53.92,49.15,48.40,42.57,41.90,40.37,39.34,38.49,33.62,32.64,30.03,28.82,25.81,25.68,24.57,24.28,23.82,20.67,17.76.HR-MS(ESI):Calculated for C51H68N5O10[M+H]+:910.4966,found 910.4951。
合成步骤同实例7，得黄色固体I-41。1H NMR(400MHz,DMSO)δ8.43(d,J＝7.2Hz,1H),8.36(m,2H),7.81–7.74(m,2H),7.26(d,J＝8.2Hz,1H),5.79(s,1H),5.37(s,1H),5.11–5.04(m,2H),4.54(s,1H),4.44(m,3H),4.25–4.17(m,3H),3.98(d,J＝3.0Hz,1H),3.77–3.70(m,3H),3.64(t,J＝6.4Hz,2H),3.57–3.50(m,2H),3.48–3.38(m,6H),3.21–3.14(m,4H),2.80(d,J＝9.0Hz,1H),2.69–2.54(m,4H),1.97(d,J＝8.8Hz,1H),1.82(m,4H),1.70–1.63(m,2H),1.62–1.49(m,5H),1.42–1.31(m,6H),1.30–1.21(m,2H),1.16(m,2H),1.08–1.00(m,1H),0.88(s,3H),0.79(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.54,162.97,156.70,152.34,146.59,132.26,130.57,129.14,125.73,125.37,122.36,122.03,115.97,114.84,99.07,69.62,69.60,69.47,68.71,67.06,66.98,65.04,63.11,57.31,56.27,53.92,49.13,48.40,42.58,41.91,40.38,39.33,38.49,33.62,32.64,30.02,29.03,28.70,25.68,25.41,25.03,24.28,23.82,20.68,17.76.HR-MS(ESI):Calculatedfor C52H70N5O10[M+H]+:924.5123,found 924.5102。
合成步骤同实例7，得黄色固体I-42。1H NMR(400MHz,DMSO)δ8.44(d,J＝7.2Hz,1H),8.37(m,2H),7.81–7.74(m,2H),7.28(d,J＝8.2Hz,1H),5.79(s,1H),5.37(s,1H),5.08(m,2H),4.54(s,1H),4.51–4.45(m,1H),4.42(t,J＝5.2Hz,2H),4.22(t,J＝6.4Hz,3H),3.98(d,J＝3.1Hz,1H),3.73(m,3H),3.64(t,J＝6.4Hz,2H),3.56–3.51(m,2H),3.47–3.41(m,6H),3.20(m,4H),2.80(d,J＝9.0Hz,1H),2.69–2.55(m,4H),1.98(d,J＝9.5Hz,1H),1.82(m,4H),1.67(m,2H),1.60–1.47(m,5H),1.39–1.22(m,10H),1.20–1.13(m,2H),1.06(m,1H),0.89(s,3H),0.80(s,3H).13C NMR(101MHz,DMSO)δ205.76,163.56,162.99,156.72,152.33,146.67,132.27,130.59,129.15,125.75,125.39,122.38,122.04,115.97,114.86,99.09,69.61,69.59,69.47,68.71,67.15,66.99,65.04,63.10,57.32,56.27,53.93,49.12,48.40,42.55,41.95,40.38,39.34,38.49,33.63,32.65,30.03,29.23,29.01,28.26,25.68,25.49,24.90,24.28,23.82,20.68,17.77.HR-MS(ESI):Calculated forC53H72N5O10[M+H]+:938.5279,found 938.5254。
合成步骤同实例7，得黄色固体I-43。1H NMR(400MHz,DMSO)δ8.45(d,J＝7.3Hz,1H),8.38(m,2H),8.01(s,1H),7.79(dd,J＝8.4,7.4Hz,1H),7.29(d,J＝8.2Hz,1H),5.77(s,1H),5.35(s,1H),5.14(s,1H),5.05(d,J＝2.8Hz,1H),4.59(s,1H),4.54(d,J＝5.0Hz,1H),4.51–4.43(m,3H),4.22(t,J＝6.4Hz,2H),4.15(d,J＝7.1Hz,1H),3.97(d,J＝3.0Hz,1H),3.86–3.80(m,1H),3.75(t,J＝5.3Hz,2H),3.62(m,4H),3.55–3.51(m,2H),3.48(m,1H),3.47–3.41(m,6H),3.24–3.14(m,4H),2.75(d,J＝9.1Hz,1H),2.68–2.52(m,2H),1.98(d,J＝12.0Hz,1H),1.82(m,4H),1.67(m,2H),1.57–1.49(m,1H),1.34(m,4H),1.28–1.20(m,2H),1.15(m,2H),1.02(m,1H),0.85(s,3H),0.78(s,3H).13C NMR(101MHz,DMSO)δ205.86,163.58,163.01,156.74,152.39,143.83,132.30,130.62,129.17,125.79,125.40,124.07,122.40,115.87,114.87,99.19,69.61,69.57,69.53,69.46,69.21,68.64,66.97,66.15,65.13,63.48,63.11,57.29,56.28,53.93,49.27,48.40,42.57,41.83,40.39,39.34,38.51,33.63,32.62,29.99,25.68,24.26,23.82,20.62,17.71.HR-MS(ESI):Calculated for C50H66N5O11[M+H]+:912.4759,found 912.4746。
实施例8本发明所合成的化合物对五种肿瘤细胞(MGC，SW1990，PC3，TE1，HGC)的抗肿瘤活性如下：没有特别说明，所述含量均为质量百分含量。
实验方法
选处于对数生长期的肿瘤细胞消化离心并重悬计数，稀释细胞悬液制成浓度为5×104/mL的细胞悬液，在96孔板中每孔加入100μL细胞悬液使每孔有5000个细胞，外周孔加入PBS液。将96孔板放置于体积百分含量5％CO2，37℃细胞培养箱中培养24小时待细胞完全贴壁后，弃掉旧培养基，设不加药只加培养基的阴性对照组，用完全培养基稀释药物至所需系列浓度,96孔板的每孔加入150μL相应药物浓度的含药培养基，每个浓度设3组复孔。将加完药的96孔板置于体积百分含量5％CO2，37℃细胞培养箱中培养24小时。24小时后将96孔板取出，弃含药旧培养基，每孔加入150μL 10％三氯乙酸溶液，放入4℃冰箱固定两个小时。固定完成后取出，弃液，每孔每次加入300μL超纯水，洗涤三次，室温干燥一小时甚至更久。待其干燥后每孔加入200μL 0.4％SRB溶液进行染色，平板震荡20min，时间到后弃掉染色液，用1％乙酸洗涤三次，室温干燥透。干燥后每孔加入200μL 10mM Tris(pH＝10.5)溶液平板震荡20min使其充分溶解。本实验使用酶标仪在560nm处读出每孔的吸光度值,计算抑制率，并根据抑制率算得IC50值。本实验平行三次，得到三次的平均值和标准差。其结果如表1。
表1济源冬凌草荧光探针的抗肿瘤增殖活性评价结果
JOA为济源冬凌草甲素(化合物9)：作为阳性对照
从上表可以看出，本发明所设计合成的济源冬凌草甲素-萘二酰亚胺类荧光化合物均具有良好的抗肿瘤活性，优于先导化合物济源冬凌草甲素，可以应用于人胃癌细胞、胰腺癌、前列腺癌、食管癌细胞的抗增殖活性研究，同时在保证活性前提下，可以选择活性较好的化合物进行后续的生物学机制探索和研究，具有广泛的研究价值和良好的应用前景。
实施例9荧光探针I-39对HGC-27细胞染色实验
实验方法
选取对数生长期的HGC-27细胞接种到放置在6孔板中的玻璃盖玻片上,每孔50000个细胞,待24小时细胞贴壁后加入用培养基配制的本发明化合物I-39(1μmol/L)在细胞培养箱中孵育不同的时间点15min,30min,1h,2h,3h,4h。在孵育相应的时间后,细胞用多聚甲醛固定10分钟,用PBS洗涤后将玻璃盖玻片取出用于荧光成像。在荧光显微镜下观察细胞着色部位,荧光分布及亮度变化。其结果如图1所示。
从图1可以看出，随着时间的变化，发现在4h时，其荧光强度最强。这表明荧光探针I-39对孵育时间呈现依赖性。
实施例10荧光探针I-39荧光共定位研究
实验方法
选取对数生长期的HGC-27细胞接种到放置在6孔板中的玻璃盖玻片上,每孔50000个细胞,待24小时细胞贴壁后加入用培养基配制的本发明化合物I-39(1μmol/L)在细胞培养箱中孵育1h。I-39孵育后用PBS洗涤两次,加入配置好的线粒体红色荧光探针MitoTracker Red CMXROS工作液，在细胞培养箱中孵育40min。孵育完成后用PBS洗涤细胞并用4％多聚甲醛室温固定10分钟,用PBS洗涤后加入即用型DAPI溶液，染色10min。DAPI染色结束用PBS洗涤，将玻璃盖玻片取出用于荧光成像。成像结果由共聚焦显微镜(尼康type108)获得。I-39荧光探针的激发波长采用413nm,发射波长采用533nm,线粒体专属染色体的激发波长采用579nm,发射波长采用599nm。先用明镜显微镜寻找到细胞视野,然后切换到激光共聚焦视野,进行断层扫描寻找最佳切片图像,其结果如图2所示。
从图2可知，荧光探针I-39在细胞内呈现明显的绿色荧光，表明该探针可以用于细胞染色以及共定位研究。研究发现，荧光探针I-39和线粒体专属染料(mito-tracker)有着很大程度的重叠，而和细胞核专属染料(DAPI)几乎没有重叠。这表明该探针主要定位在细胞线粒体。
Claims (5)
1.济源冬凌草甲素-萘酰亚胺类荧光化合物，其特征在于，具有通式Ⅰ所示结构：
其中R为哌啶基；
X＝-CH2-，-CH2O-；
m＝0-6；
n＝1-8；
p＝0-6。
2.如权利要求1所述的济源冬凌草甲素-萘酰亚胺类荧光化合物，其特征在于：通式I中：R为哌啶基；X＝-CH2-，-CH2O-；m＝0-3；n＝1-5；p＝0-4。
3.如权利要求2所述的济源冬凌草甲素-萘酰亚胺类荧光化合物，其特征在于：选如下化合物：
4.如权利要求1、2或3其中之一所述的济源冬凌草甲素-萘酰亚胺类荧光化合物在制备抗肿瘤药物中的应用，其特征在于，以其为活性成分，制备治疗胃癌细胞、胰腺癌、前列腺癌或食管癌药物。
5.如权利要求1、2或3其中之一所述的济源冬凌草甲素-萘酰亚胺类荧光化合物在制备肿瘤细胞成像，药物作用机制研究，分子靶点确定或活体动物成像荧光探针中的应用。
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