CN114249782B - 一种选择性破坏衰老细胞溶酶体的前药及其制备方法与应用 - Google Patents
一种选择性破坏衰老细胞溶酶体的前药及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种选择性破坏衰老细胞溶酶体的前药及其制备方法与应用，其结构式如式Ⅰ所示：
本发明首次提出通过靶向衰老细胞的溶酶体来选择性清除衰老细胞。药物的溶酶体靶向基团使药物更多的富集在衰老细胞中；在衰老细胞高表达的β半乳糖苷酶作用下释放药物；药物发挥溶酶体破坏作用，破坏易损的衰老细胞溶酶体，最终诱导衰老细胞凋亡。由于衰老细胞溶酶体具有普适性，因此溶酶体靶向senolytics前药具有广谱特点，能够选择性清除不同来源，不同刺激产生的衰老细胞。本发明的前药体外能够选择性破坏不同刺激下衰老的正常和癌细胞溶酶体，并诱导细胞凋亡；体内能够通过清除小鼠肝脏和肾脏的衰老细胞，恢复由阿霉素诱导的小鼠肝脏和肾脏的受损。
Description
技术领域
本发明属于有机药物合成技术领域，具体涉及一种选择性破坏衰老细胞溶酶体的前药及其制备方法与应用。
背景技术
细胞衰老是一种由外部或内部压力引起的细胞周期停滞的永久性状态，越来越被认为是导致衰老和各种与年龄有关的疾病的主要机制。此外，衰老细胞(SnCs)可持续分泌一系列称为衰老相关分泌表型(SASP)的因子，这些因子可以负面调节邻近的非SnCs，导致与许多疾病的进展、治疗抵抗和不良预后相关的发病机制。因此，开发能够有效且特异性地消除SnCs的药物已成为学术和临床领域的研究前沿。针对此问题，目前已经开发了一系列通过调节相关信号通路选择性杀死SnCs的化合物，称之为“senolytics”，相应的清除衰老细胞的治疗策略被称为“senotherapy”。目前已有的senolytics主要通过筛选靶向衰老细胞相关信号通路(例如抗凋亡，蛋白激酶和转录因子)的小分子来实现对衰老细胞的选择性清除。然而，目前还没有senolytics进入临床使用，其面临的最大挑战是细胞衰老的多方面、动态和高度异质性特征，尤其是当细胞受到不同刺激或压力时，经常导致senolytics在杀死SnCs方面表现出不同的的药物活性，甚至失效。因此，开发具有高效力、强特异性和广谱活性的senolytics仍然具有挑战性。
溶酶体作为细胞的“自杀袋”，负责细胞废物的降解和回收，感知和控制细胞能量稳态和新陈代谢。与不同SnCs之间的遗传信号变化不同，溶酶体中生物发生和分解代谢程序的广泛重塑是一个主要标志，在不同状态和不同来源的细胞衰老中通常共享。主要变化包括：(i)溶酶体的数量和质量显着增加；(ii)衰老相关酶的过度激活和积累，尤其是β-半乳糖苷酶(SA-β-gal)；(iii)化学环境变化导致降解能力功能下降、脂质成分改变和未消化物质积累；(iv)与其他细胞器的协同相互作用，如溶酶体-线粒体串扰，加剧衰老表型。这些显着的改变极大地促进了细胞衰老并加速了疾病的组织重塑和病理生物学。更重要的是，它们还使SnCs的溶酶体比正常溶酶体更脆弱，更容易破裂。因此，SnCs功能失调的溶酶体代表了一种广谱且有前途的替代药理学靶点。然而，具有高特异性的溶酶体破坏的senolytics目前未有报道。
发明内容
本发明的目的是提供一种以衰老细胞易损的溶酶体为靶点，可以实现体外和体内实现衰老细胞的特异性清除的选择性破坏衰老细胞溶酶体的前药及其制备方法与应用。
本发明这种选择性破坏衰老细胞溶酶体的前药的结构式，如式Ⅰ所示：
本发明这种选择性破坏衰老细胞溶酶体的前药的制备方法，包括以下步骤：
1)化合物5的合成：将鞘氨醇和中间体4加入到溶剂中，接着加入N,N-二异丙基乙胺(DIPEA)，再接着将反应混合物在室温和氮气气氛下进行搅拌反应，反应完毕后，反应液用去离子水稀释并用CH2Cl2萃取，合并有机层并用无水Na2SO4干燥，然后进行过滤和减压浓缩，并将混合物进行纯化，得到化合物5；
2)化合物6的合成：将步骤1)中的化合物5、中间体L2、CuSO4·5H2O和抗坏血酸钠置于圆底烧瓶中，接着在氩气气氛下，将有机溶剂和ddH2O注入圆底烧瓶中，再接着在室温下进行反应，反应完毕后，除去溶剂后，将粗产物直接通过快速色谱纯化，得到化合物6；
3)前药I的合成：将步骤2)中的化合物6溶解在溶剂中，接着在0℃下加入CH3ONa并在0℃下搅拌一段时间，搅拌完毕后将溶液用amberlite IRC50进行中和，在接着通过棉垫过滤并蒸发溶剂，然后将粗物质通过硅胶柱色谱纯化，得到前药I；
所述步骤1)中，鞘氨醇和中间体4的摩尔比为(3～4):(2～3)，溶剂为四氢呋喃和二甲基亚砜中的一种，中间体4和N,N-二异丙基乙胺摩尔体积比为(2～3)mmol:(0.5～1)mL，反应时间为10～14h。
所述步骤2)中，化合物5、中间体L2、CuSO4·5H2O和抗坏血酸钠的摩尔比为(1～2):(2～3):(0.3～0.5):(4～6)，有机溶剂为N,N-二甲基甲酰胺和甲苯中一种，有机溶剂与ddH2O的体积比为(4～6):1，反应时间为1～3h。
所述步骤3)中，溶剂为乙醇和甲醇中的一种，化合物6与溶剂的摩尔体积比为(0.07～0.12)mmol：20mL，化合物6与CH3ONa的(0.07～0.12):(1.2～1.8)，一段时间为15～25min，中和时间为5～15min。
所述的中间体L2的制备方法，包括以下步骤：将化合物L1加入到溶剂中，接着向其中加入叠氮化钠，然后将反应液在设定温度下进行反应，反应完毕后，反应液用H2O稀释并用CH2Cl2萃取；除去有机层的溶剂后，得到中间体L2；
所述的溶剂为N,N-二甲基甲酰胺和甲苯中一种，化合物L1与溶剂的摩尔体积比为(2～3)mmol:(5～15)mL，化合物L1与叠氮化钠的摩尔比为(2～3):(5～7)；设定温度为70～90℃，反应时间为18～30h。
所述的中间体4的制备方法，包括以下步骤：
S1将含有炔丙基溴的甲苯溶液滴加到含铝和催化量HgCl2的无水THF溶液中，在设定温度和氮气气氛下，进行第一段反应，反应完毕后，将反应液冷却至0℃，再接着向其中滴加含有化合物1的溶液，滴加完毕后，在搅拌条件下进行第二段反应，得到粗物质，将粗物质进行水解、萃取、浓缩和纯化后，得到化合物2；
S2将含有Ag2CO3和HMTTA(1,1,4,7,10,10-六甲基三亚乙基四胺)的无水CH3CN溶液在室温下搅拌设定时间，接着在0℃下，将化合物2和固体1-溴-(2,3,4,6-O-四乙酰基)-β-D-吡喃半乳糖苷加入上述溶液，然后并且在室温下搅拌混合物直到起始材料化合物2通过TLC分析消失，此后，用水淬灭反应，然后用乙酸乙酯萃取，得到粗产物，对粗产物进行干燥、浓缩和出纯化后，得到化合物3；
S3在0℃下，将吡啶加入到含有化合物3和对硝基苯基氯甲酸酯的无水CH2Cl2溶液中，接着将混合物在室温搅拌直至通过TLC监测反应完成，再接着用H2O稀释并用CH2Cl2萃取，然后经过干燥、浓缩和纯化后，得到化合物4；
所述S1步骤中，含有炔丙基溴的甲苯溶液中质量百分含量为70～90％；炔丙基溴、铝、HgCl2和化合物1的摩尔比为(20～30):(20～30):(0.3～0.4):(3～4)；设定温度为50～70℃，第一段反应时间为7～9h；含有化合物1的溶液为含有化合物1的THF溶液；第二段反应时间为5～7h。
所述S2步骤中，Ag2CO3、HMTTA、化合物2和1-溴-(2,3,4,6-O-四乙酰基)-β-D-吡喃半乳糖苷的摩尔比为(3～4):(0.5～1.0):(0.5～1.5):(1.5～2.5)，设定时间为1.5～2.5h。
所述步骤S3中，化合物3和对硝基苯基氯甲酸酯的摩尔比为(0.5～1.0):(1.5～2.5)，化合物3与吡啶的摩尔体积比为(0.5～1.0)mmol:(0.2～0.3)mL，化合物3与CH2Cl2的摩尔体积比为(0.5～1.0)mmol:(8～12)mL。
本发明的原理：首次合成了能够选择性破坏衰老细胞溶酶体并诱导衰老细胞凋亡的senolytics前药。该前药主要由三部分组成：第一部分破坏溶酶体的分子鞘氨醇：
鞘氨醇可在溶酶体内质子化并滞留，进而破坏溶酶体膜；第二部分为β-半乳糖苷酶响应的自消解linker：/>
第三部分为溶酶体靶向分子：/>
这三部分最终组成了溶酶体靶向senolytics前药(Lyso-Sphβ-gal)：/>
其具体的作用原理图如图1所示，该senolytics前药首先可通过溶酶体靶向分子富集在细胞内溶酶体中，由于衰老细胞溶酶体含量明显升高，因此药物能够更多的富集在衰老细胞内，增加了单位衰老细胞的药物富集量。随后，药物在衰老细胞溶酶体中高表达的β半乳糖苷酶(称作衰老相关的β半乳糖苷酶，senescence-associatedβ-galactosidase(SA-β-gal))作用下水解，自消解linker发生重排，最终释放出完整的鞘氨醇。由于水解前鞘氨醇的氨基被乙酰化，无法发生质子化。一旦鞘氨醇被完整释放，其氨基部分可在溶酶体的酸性环境下质子化并滞留在溶酶体内，加上长的烷基链的插膜作用，使其具有破坏溶酶体膜的功能。衰老细胞溶酶体被选择性破坏后，内部包括组织蛋白酶在内的多种水解酶释放到细胞质，引发溶酶体介导的细胞凋亡，从而实现对衰老细胞的选择性清除。
本发明的有益效果：本发明首次提出通过靶向衰老细胞的溶酶体来选择性清除衰老细胞。衰老细胞溶酶体具有以下特点：数量和体积增加；内部β半乳糖苷酶含量及活性增加；溶酶体膜更易被破坏。针对上述特点，本发明设计了溶酶体靶向senolytics前药。药物的溶酶体靶向基团使药物更多的富集在衰老细胞中；在衰老细胞高表达的β半乳糖苷酶作用下释放药物；药物发挥溶酶体破坏作用，破坏易损的衰老细胞溶酶体，最终诱导衰老细胞凋亡。由于衰老细胞溶酶体的以上特点具有普适性，因此溶酶体靶向senolytics前药具有广谱特点，能够选择性清除不同来源，不同刺激产生的衰老细胞。从效果上而言，本发明的前药体外能够选择性破坏不同刺激下衰老的正常和癌细胞溶酶体，并诱导细胞凋亡；体内能够通过清除小鼠肝脏和肾脏的衰老细胞，恢复由阿霉素诱导的小鼠肝脏和肾脏的受损。
附图说明
图1本发明的溶酶体靶向前药的作用原理图；
图2实施例1的合成路线图；
图3对比例1中的合成路线图；
图4对比例2中的合成路线图；
图5对比例3中的合成路线图；
图6对比例4中的合成路线图；
图7实施例2中衰老细胞溶酶体含量增加的实验验证图；
图8实施例2中溶酶体靶向senolytics前药在体外的酶选择性的实验结果图；
图9实施例2中溶酶体共定位的实验结果图；
图10实施例2中溶酶体靶向senolytics前药选择性破坏衰老细胞溶酶体的实验效果图；
图11实施例2中溶酶体靶向senolytics前药体外选择性清除衰老细胞的实验效果图；
图12实施例2中溶酶体靶向senolytics前药对衰老相关器官功能障碍的体内保护的效果图。
具体实施方式
实施例1
合成了溶酶体靶向β半乳糖苷酶可断裂senolytics前药Ⅰ(Lyso-Sphβ-gal)，其合成路线可见图1；
步骤Ⅰ：化合物L2的合成：将叠氮化钠(0.39g,6mmol)添加到4-(3-溴丙基)吗啉(L1,0.25g,2.42mmol)在DMF(10mL)中的溶液中，并将混合物在80℃下搅拌24h，然后用H2O稀释并用CH2Cl2萃取。除去有机层的溶剂后，无需进一步纯化即可获得纯产物化合物L2(0.16g，78％)。
化合物L2的核磁数据如下：1HNMR(400MHz,Chloroform-d)δ3.69(t,J＝4.1Hz,4H),3.34(t,J＝6.8Hz,2H),2.40(d,J＝7.7Hz,6H),1.75(p,J＝6.9Hz,2H).13CNMR(101MHz,Chloroform-d)δ66.92,55.61,53.80,53.62,53.48,49.44,25.91.
步骤Ⅱ:化合物2的合成：将80％炔丙基溴的甲苯溶液(2.6mL，24mmol)滴加到含铝(648mg，24mmol)和催化量HgCl2(0.38mmol)的30mL无水THF溶液中，反应混合物在60°搅拌并在氮气氛下保持8小时。冷却至0℃后，滴加6mL化合物1(650mg，3.84mmol)溶于THF的溶液，在搅拌下进行反应，TLC监测。反应完成后，混合物用1NHCl水解，产物用乙酸乙酯萃取。此后，合并的有机层用无水Na2SO4干燥，然后过滤并真空浓缩。所得粗产物通过硅胶柱色谱纯化，得到黄色油状物。将产物重新溶解在CH2Cl2中并用1NNaOH洗涤后，合并的水层用浓HCl酸化并用氯仿萃取，然后用无水Na2SO4干燥，过滤，减压浓缩。最后，粗物质通过硅胶柱层析(石油醚/乙酸乙酯：4/1)纯化，得到化合物2，为深棕色油状物(700mg，88％产率)。
化合物2的核磁数据如下：1HNMR(400MHz,Chloroform-d)δ10.53(s,1H),8.14(s,1H),7.64(d,J＝8.7Hz,1H),7.15(d,J＝8.7Hz,1H),4.87(t,J＝6.2Hz,1H),2.64(m,3H),2.10(m,1H).13CNMR(101MHz,Chloroform-d)δ154.56,135.21,135.04,133.25,122.28,120.07,79.67,71.80,70.77,29.27.
步骤Ⅲ：化合物3a的合成：将含有Ag2CO3(1g,3.7mmol)和HMTTA(0.19mL,0.7mmol)的40mL无水CH3CN溶液在室温(rt)下搅拌2小时，然后在0℃条件下，将化合物2(207mg,1mmol)和固体1-溴-(2,3,4,6-O-四乙酰基)-β-D-吡喃半乳糖苷(822mg,2mmol)加入上述溶液中，并且在室温下搅拌混合物直到起始材料化合物2通过TLC分析消失。此后，用水淬灭反应，然后用乙酸乙酯萃取。接着，将合并的有机层用无水Na2SO4干燥，过滤并减压浓缩。最后，将所获得的粗物质加载到去活硅胶柱上并通过快速色谱法(石油醚/乙酸乙酯：3/1、2/1、1/1)纯化以产生化合物3a(528mg，75％产量)为无色固体。
化合物3a的核磁谱图数据如下：1HNMR(400MHz,Chloroform-d)δ7.87(d,J＝9.4Hz,1H),7.58(t,J＝9.4Hz,1H),7.36(d,J＝8.6Hz,1H),5.50(dd,J＝21.3,5.8Hz,2H),5.14–5.05(m,2H),4.92(t,J＝5.8Hz,1H),4.26(d,J＝7.1Hz,1H),4.18(dd,J＝11.3,6.1Hz,1H),4.10(d,J＝6.3Hz,1H),2.68–2.64(m,2H),2.20(s,3H),2.14(s,4H),2.08(s,3H),2.03(s,3H).13CNMR(101MHz,Chloroform-d)δ170.32,170.19,170.13,169.42,148.69,148.68,141.23,141.16,138.71,138.67,130.99,130.94,122.65,122.59,119.77,119.67,100.76,79.49,71.95,71.43,70.67,70.56,67.89,66.74,61.81,61.34,29.42,20.80,20.64,20.62,20.54.
步骤Ⅳ：化合物4a的合成：在0℃下将吡啶(0.23mL，2.5当量)加入到含有化合物3a(453mg，0.84mmol)和对硝基苯基氯甲酸酯(338mg，2mmol)的无水CH2Cl2溶液(10mL)中。将混合物在室温搅拌直至通过TLC监测反应完成，然后用H2O稀释并用CH2Cl2萃取。有机相用Na2SO4干燥。真空浓缩后，混合物直接通过硅胶柱色谱纯化(石油醚/乙酸乙酯3/1，3/2)，得到化合物4a，为无色固体(506mg，86％产率)。
化合物4a的核磁谱图数据如下：1HNMR(400MHz,Chloroform-d)δ8.27(d,J＝8.3Hz,2H),7.93(s,1H),7.64(d,J＝8.4Hz,1H),7.39(dd,J＝14.0,8.9Hz,3H),5.83(s,1H),5.55(t,J＝9.0Hz,1H),5.48(s,1H),5.14(s,2H),4.27(dd,J＝11.4,6.8Hz,1H),4.15(dt,J＝20.5,6.4Hz,2H),3.01–2.77(m,2H),2.19(s,3H),2.13(s,4H),2.07(s,3H),2.02(s,3H).13CNMR(101MHz,Chloroform-d)δ170.25,170.12,170.07,169.28,155.15,151.47,149.65,145.57,141.16,133.23,131.95,125.34,123.58,121.65,119.61,100.51,72.46,71.52,70.46,67.81,66.69,61.32,26.22,20.63,20.60,20.53.
步骤Ⅴ：化合物5a的合成：将DIPEA(0.83mL)加入含有鞘氨醇(100mg，0.33mmol)和化合物4a(175mg，0.25mmol)的20mL无水THF溶液中，并将反应混合物在室温、氮气气氛下搅拌12小时。此后，将反应用H2O稀释并用CH2Cl2萃取。合并的有机层用无水Na2SO4干燥，然后过滤并减压浓缩。最后，混合物直接通过硅胶柱纯化，得到化合物5a(163mg，76％产率)。
化合物5a的核磁谱图数据如下：1HNMR(400MHz,Chloroform-d)δ7.87(s,1H),7.56(d,J＝8.6Hz,1H),7.36(d,J＝8.6Hz,1H),5.80–5.75(m,2H),5.55(m,2H),5.49(d,J＝3.4Hz,1H),5.15–5.08(m,2H),4.37–4.31(m,1H),4.29–4.24(m,1H),4.18(m,1H),4.11(m,1H),3.99(s,1H),3.71(m,1H),3.62(m,1H),2.78(d,J＝6.4Hz,1H),2.45(s,1H),2.20(s,3H),2.14(s,3H),2.08(s,4H),2.03(s,3H),0.91–0.87(m,3H).13CNMR(101MHz,Chloroform-d)δ170.35,170.20,170.13,169.42,149.08,131.92,131.84,128.53,123.13,119.46,100.60,78.49,74.75,72.63,71.85,71.44,70.55,67.86,66.73,62.10,61.34,55.56,32.28,31.91,29.67,29.64,29.61,29.47,29.34,29.21,29.10,29.05,26.52,22.68,20.66,20.63,20.55,14.11.
步骤Ⅵ：化合物6a的合成。将化合物5a(100mg,0.116mmol)、化合物L2(39.44mg,0.232mmol)、CuSO4·5H2O(10mg,0.04mmol)和抗坏血酸钠(100mg,0.5mmol)置于100mL圆底烧瓶中在氩气气氛下。然后将DMF(20mL)和ddH2O(4mL)注入混合物中。使反应在室温下进行2小时。除去溶剂后，粗产物直接通过快速色谱纯化，得到化合物6a(86mg，72％产率)。
化合物6a的核磁谱图数据如下：1HNMR(400MHz,DMSO-d6)δ7.83(s,1H),7.75(s,1H),7.60(d,J＝9.0Hz,1H),7.33(d,J＝8.8Hz,1H),6.92(d,J＝8.8Hz,1H),5.82(t,J＝6.5Hz,1H),5.56(d,J＝7.6Hz,2H),5.36(s,2H),5.26–5.20(m,2H),4.83–4.77(m,1H),4.51–4.43(m,2H),4.30(t,J＝7.0Hz,2H),4.12(d,J＝6.2Hz,2H),3.90(d,J＝6.3Hz,1H),3.56(d,J＝4.8Hz,4H),3.49–3.40(m,2H),3.17(s,2H),2.28(s,4H),2.14(m,4H),2.06–1.92(m,12H),1.90(m,2H),1.23(m,22H),0.87–0.83(m,3H).13CNMR(101MHz,DMSO-d6)δ170.37,170.30,169.96,169.30,155.53,148.06,142.14,140.42,131.44,123.76,117.88,72.98,71.22,70.39,68.14,67.55,66.64,61.71,58.07,55.08,53.62,47.79,31.75,29.51,29.47,29.38,29.23,29.16,29.09,27.13,22.54,20.96,20.82,20.77,20.74,14.40.
步骤Ⅶ：前药Ⅰ的合成：将化合物6a(100mg,0.096mmol)溶解在20mL CH3OH中，然后在0℃下加入CH3ONa(83mg,1.53mmol)。在0℃下再搅拌20分钟后，将溶液用amberliteIRC50中和10分钟，并通过棉垫过滤。然后蒸发溶剂，所得粗物质通过硅胶柱色谱纯化以产生前药Ⅰ(66mg，75％产率)，记作Lyso-Sphβ-gal。
前药Ⅰ的核磁谱图数据如下：Q1HNMR(400MHz,DMSO-d6)δ8.23(d,J＝3.4Hz,1H),7.86(d,J＝3.3Hz,1H),7.70(dd,J＝5.3,2.2Hz,1H),7.52(d,J＝8.8Hz,1H),7.34(d,J＝8.8Hz,1H),5.82(dt,J＝13.2,6.5Hz,1H),5.51(dq,J＝14.1,7.3,6.6Hz,1H),5.38(m,1H),4.99(dd,J＝7.7,4.8Hz,1H),4.68–4.55(m,5H),4.35–4.25(m,4H),3.90(q,J＝6.4,5.8Hz,1H),3.72(d,J＝3.1Hz,1H),3.62(q,J＝5.5Hz,1H),3.56(q,J＝4.7Hz,6H),3.52–3.45(m,2H),3.45–3.37(m,2H),3.34(dt,J＝9.5,5.7Hz,1H),3.20–3.14(m,2H),2.31(d,J＝4.9Hz,4H),2.19(d,J＝5.6Hz,2H),1.93(q,J＝7.1Hz,2H),1.23(d,J＝5.0Hz,22H),0.85(t,J＝6.6Hz,3H).13CNMR(101MHz,DMSO-d6)δ155.59,149.45,142.33,140.11,134.82,132.36,131.41,123.79,122.73,117.21,101.52,76.22,73.84,72.82,71.89,70.49,68.38,66.61,58.05,53.61,47.81,31.75,29.53,29.48,29.36,29.24,29.17,29.10,28.96,27.11,22.55,14.40.
对比例1
合成非溶酶体靶向β半乳糖苷酶可断裂senolytics前药7a(Sphβ-gal)，合成路线可见图3：
化合物7a的合成：将实施1中合成的化合物5a(100mg，0.116mmol)溶解在20mLCH3OH中，然后在0℃下加入CH3ONa(83mg，1.53mmol)。在0℃下再搅拌20分钟后，将溶液用amberliteIRC50中和10分钟，并通过棉垫过滤。然后蒸发溶剂，所得粗物质通过硅胶柱色谱纯化以产生前药7a(58mg，72％产率)，记作Sphβ-ga。
前药7a核磁谱图数据如下：1HNMR(400MHz,DMSO-d6)δ7.86(dd,J＝7.5,2.2Hz,1H),7.64(dt,J＝8.6,2.7Hz,1H),7.41(dd,J＝8.9,3.3Hz,1H),5.66(dt,J＝12.0,5.9Hz,1H),5.54(tt,J＝17.3,6.8Hz,1H),5.39(ddd,J＝28.4,15.4,6.7Hz,1H),5.13(t,J＝4.9Hz,1H),5.03(t,J＝7.1Hz,1H),4.88(d,J＝5.9Hz,1H),4.78(dd,J＝17.1,5.2Hz,1H),4.67(q,J＝5.6Hz,1H),4.59(d,J＝4.4Hz,1H),4.49–4.33(m,1H),4.09(q,J＝5.3Hz,1H),3.88(q,J＝6.3Hz,1H),3.71m,1H),3.66–3.61(m,1H),3.57(dd,J＝4.8,2.1Hz,1H),3.52–3.48(m,1H),3.42–3.40(m,1H),3.18(d,J＝5.3Hz,4H),2.82(q,J＝2.4Hz,1H),2.76–2.74(m,1H),1.97(m,1H),1.24(s,22H),0.86(m,3H).13CNMR(101MHz,DMSO-d6)δ155.44,149.59,140.18,134.11,134.01,132.45,131.55,131.49,131.42,122.98,117.16,101.61,80.18,76.28,74.08,73.82,72.00,71.83,70.46,68.49,60.93,60.77,58.18,56.50,49.06,32.16,31.98,31.76,29.53,29.49,29.42,29.23,29.17,29.10,22.55,14.41.
对比例2
与实施例1的合成步骤基本相同，只是在步骤Ⅲ将1-溴-(2,3,4,6-O-四乙酰基)-β-D-吡喃半乳糖苷替换成不可断裂的1-溴-(2,3,4,6-O-四乙酰基)-β-D-吡喃葡萄糖苷，具体的合成路线可见图4，相对应的合成物质分别为：化合物3b、4b、5b、6b和前药Ⅱ(Lyso-Sphglu)。
化合物3b产率：81％，核磁谱图数据如下：。1HNMR(400MHz,Chloroform-d)δ7.85(d,J＝7.6Hz,1H),7.57(t,J＝8.5Hz,1H),7.32(d,J＝8.6Hz,1H),5.28(d,J＝6.2Hz,2H),5.15(dd,J＝20.1,6.7Hz,2H),4.89(d,J＝5.4Hz,1H),4.24(d,J＝6.1Hz,2H),3.93–3.85(m,1H),3.11(s,1H),2.63(d,J＝6.0Hz,2H),2.10(s,3H),2.08(s,4H),2.04(s,3H),2.03(s,3H).13CNMR(101MHz,Chloroform-d)δ170.58,170.24,169.44,169.41,148.55,141.13,141.08,138.99,138.96,131.15,131.12,122.69,122.64,119.70,119.63,100.10,90.05,79.61,79.59,72.30,72.27,71.85,71.14,70.66,70.50,68.11,61.79,29.32,20.67,20.55,20.52.
化合物4b产率：73％，核磁谱图数据如下：1HNMR(400MHz,Chloroform-d)δ8.29(d,J＝8.4Hz,2H),7.95(s,1H),7.65(d,J＝8.7Hz,1H),7.44–7.37(m,3H),5.83(t,J＝6.6Hz,1H),5.32(d,J＝5.8Hz,1H),5.26–5.16(m,2H),4.27(s,2H),3.91(d,J＝9.5Hz,1H),2.92(dd,J＝13.1,6.2Hz,2H),2.14(s,3H),2.11(s,4H),2.07(s,6H).13CNMR(101MHz,Chloroform-d)δ170.44,170.19,169.31,169.26,155.14,151.47,149.58,145.60,141.22,133.40,131.99,125.36,123.51,121.65,119.80,99.97,72.49,72.43,72.20,70.47,68.04,61.72,26.26,20.68,20.57,20.53.
化合物5b产率：69％，核磁谱图数据如下：1HNMR(400MHz,Chloroform-d)δ7.88(d,J＝2.6Hz,1H),7.57(d,J＝8.7Hz,1H),7.35(dd,J＝8.8,3.0Hz,1H),5.78(s,2H),5.55(d,J＝14.0Hz,1H),5.32(d,J＝5.7Hz,2H),5.15(d,J＝5.3Hz,1H),4.27(s,2H),3.99(d,J＝9.9Hz,1H),3.90(d,J＝9.3Hz,1H),3.63(s,1H),2.80(d,J＝17.8Hz,2H),2.51(s,1H),2.14(s,3H),2.11(s,4H),2.07(s,3H),2.06(s,3H),1.71(s,2H),1.28(s,22H),0.90(t,J＝6.1Hz,3H).13CNMR(101MHz,Chloroform-d)δ170.56,170.21,169.36,148.98,128.49,126.18,123.35,123.16,119.71,115.61,100.04,78.44,74.83,74.81,72.65,72.35,72.31,71.87,71.84,70.53,68.08,62.13,61.74,55.51,32.27,32.22,31.91,29.67,29.65,29.61,29.47,29.35,29.21,29.19,29.09,29.05,26.51,22.68,20.70,20.57,20.55,14.11.
化合物6b，产率：81％，核磁谱图数据如下：1HNMR(400MHz,DMSO-d6)δ7.84(s,1H),7.75(s,1H),7.60(d,J＝8.6Hz,1H),7.36(d,J＝8.6Hz,1H),6.92(d,J＝8.7Hz,1H),5.86(s,1H),5.62(d,J＝7.5Hz,1H),5.43–5.34(m,2H),5.05(dt,J＝19.3,9.1Hz,3H),4.33(s,4H),4.20–4.08(m,4H),3.84(dd,J＝17.4,8.5Hz,3H),3.61(s,4H),3.51(s,1H),3.45(s,1H),3.33(s,1H),3.18(s,2H),2.40–2.32(m,2H),2.02(s,3H),2.01(s,7H),1.96(s,3H),1.73(s,1H),1.23(s,22H),0.85(s,3H).13CNMR(101MHz,DMSO-d6)δ170.38,169.98,169.70,169.13,155.50,148.10,142.38,140.33,136.81,131.34,123.79,117.96,98.44,73.48,72.68,72.13,71.78,71.56,70.54,70.30,68.27,65.99,61.99,60.96,58.04,54.80,53.16,47.62,31.75,29.53,29.16,26.50,22.55,20.91,14.39.
前药Ⅱ，产率：77％，核磁谱图数据如下：1HNMR(400MHz,DMSO-d6)δ8.23(s,1H),7.88(s,1H),7.70(dd,J＝7.9,2.1Hz,1H),7.52(dt,J＝8.8,2.5Hz,1H),7.35(d,J＝8.8Hz,1H),5.81(q,J＝7.0,6.5Hz,1H),5.74(d,J＝2.0Hz,1H),5.51(td,J＝14.4,13.7,7.3Hz,1H),5.43–5.32(m,1H),5.05(t,J＝6.6Hz,1H),4.69(s,5H),4.31(td,J＝7.0,2.8Hz,2H),3.71–3.64(m,1H),3.57(t,J＝4.6Hz,4H),3.53–3.44(m,2H),3.41–3.33(m,2H),3.28–3.21(m,2H),3.17(q,J＝6.9,5.7Hz,2H),2.32(t,J＝4.8Hz,4H),2.21(t,J＝6.9Hz,2H),1.98–1.83(m,4H),1.22(d,J＝4.3Hz,22H),0.85(t,J＝6.6Hz,3H).13CNMR(101MHz,DMSO-d6)δ164.77,155.59,149.30,142.21,140.01,134.75,132.39,131.53,131.35,123.81,122.86,117.16,100.73,77.67,77.10,73.50,73.04,71.87,69.84,66.54,60.93,60.83,58.06,55.34,55.11,53.56,47.81,32.17,31.76,29.53,29.39,29.25,29.17,27.06,22.55,14.38.
对比例3
为了方便观察，将鞘氨醇替换成带有荧光的香豆素类似物，合成了β半乳糖苷酶可断裂荧光分子(荧光前药Ⅲ，Lyso-Coumβ-gal)和非溶酶体靶向β半乳糖苷酶可断裂荧光分子(7C，Coumβ-gal)。合成路线可见图5：
步骤Ⅰ：化合物c2的合成，产率68％；核磁谱图数据如下:1HNMR(400MHz,DMSO-d6)δ8.57(s,1H),7.63(d,J＝9.0Hz,1H),6.78(dd,J＝9.0,2.4Hz,1H),6.56(d,J＝2.3Hz,1H),3.48(q,J＝7.0Hz,4H),1.14(t,J＝7.0Hz,6H).13CNMR(101MHz,DMSO-d6)δ164.94,160.07,158.32,153.35,149.81,132.27,110.51,107.85,107.70,96.39,44.86,12.78.
步骤Ⅱ：化合物c3的合成，产率82％；核磁谱图数据如下:1HNMR(400MHz,DMSO-d6)δ8.64(s,2H),7.67(d,J＝8.9Hz,1H),6.86–6.70(m,2H),6.60(s,1H),3.48(q,J＝6.3Hz,4H),3.33(s,4H),1.36(s,11H),1.28(s,4H),1.14(t,J＝6.7Hz,6H).13CNMR(101MHz,DMSO-d6)δ162.25,157.63,156.02,152.83,148.04,131.97,110.56,110.00,108.12,96.32,77.72,44.78,29.86,29.58,28.73,26.65,26.45,12.77.
步骤Ⅲ：化合物c4的合成，产率72％；核磁谱图数据如下:1HNMR(400MHz,DMSO-d6)δ8.64(s,1H),7.81(s,1H),7.67(d,J＝8.9Hz,1H),6.80(d,J＝8.9Hz,1H),6.60(s,1H),3.48(d,J＝6.8Hz,2H),3.35(s,4H),2.78(t,J＝6.8Hz,2H),1.56–1.48(m,3H),1.33(s,3H),1.14(t,J＝6.7Hz,6H).13CNMR(101MHz,DMSO-d6)δ162.55,157.64,152.86,148.06,131.99,110.59,109.94,108.11,96.31,44.79,39.20,39.16,29.39,27.35,25.90,12.77.
步骤Ⅵ：化合物5c的合成：向化合物4a(702mg，1mmol)和化合物c4(414mg，1.2mmol)在10mL无水DCM中的溶液中加入三乙胺(0.33mL，2.5当量)。搅拌过夜后，溶液用饱和NaCl溶液洗涤。除去溶剂后，粗品经柱层析纯化得到化合物5c(745mg，82％产率)。
化合物5c核磁谱图数据如下:1H1HNMR(400MHz,Chloroform-d)δ8.91(m,1H),8.68(s,1H),8.15–8.11(m,1H),7.85(d,J＝2.1Hz,1H),7.54(d,J＝8.6Hz,1H),7.43(d,J＝9.0Hz,1H),7.34(d,J＝8.6Hz,1H),6.96(d,J＝9.2Hz,1H),6.66(dd,J＝9.0,2.4Hz,1H),6.50(s,1H),5.76(s,1H),5.58–5.51(m,1H),5.48(s,1H),5.14(s,1H),5.11(d,J＝4.3Hz,1H),5.05(t,J＝5.7Hz,1H),4.25(dd,J＝11.2,6.9Hz,1H),4.19(d,J＝5.8Hz,1H),4.17–4.10(m,2H),3.46(m,6H),3.22–3.10(m,2H),2.74(s,2H),2.19(s,3H),2.13(s,3H),2.07(s,3H),2.05(s,1H),2.02(s,3H),1.62(s,2H),1.51(s,2H),1.38(s,4H),1.25(m,6H).13CNMR(101MHz,Chloroform-d)δ170.30,170.20,170.09,169.42,163.49,163.21,162.81,157.63,152.68,149.05,148.09,131.19,126.11,119.39,115.70,110.08,109.90,108.36,96.54,71.69,71.39,70.58,67.89,66.77,61.35,60.42,45.08,40.96,39.48,29.58,29.32,26.52,26.45,26.20,20.63,20.61,20.54,14.17,12.41.
步骤Ⅴ：化合物6c的合成。将化合物5c(100mg,0.11mmol)、化合物L2(39.44mg,0.23mmol)、CuSO4·5H2O(10mg,0.04mmol)和抗坏血酸钠(100mg,0.5mmol)置于100mL圆底在氩气气氛下烧瓶。然后将DMF(20mL)和ddH2O(4mL)注入混合物中。将混合物在室温搅拌2小时。除去溶剂后，粗产物直接通过快速色谱纯化，得到化合物6c(83mg，70％产率)。1HNMR(400MHz,Chloroform-d)δ8.81(d,J＝5.9Hz,1H),8.68(d,J＝1.6Hz,1H),7.48(td,J＝8.5,2.2Hz,1H),7.42(dd,J＝9.0,2.0Hz,1H),7.31(dd,J＝8.1,4.5Hz,2H),6.65(dd,J＝8.9,2.4Hz,1H),6.49(d,J＝2.4Hz,1H),5.53(m,1H),5.47(q,J＝1.4Hz,1H),5.12(m3H),4.37(m,2H),4.27–4.13(m,2H),4.10(m,1H),3.69(m,4H),3.45(m,6H),3.31–3.15(m,2H),3.12(m,2H),2.39(m,4H),2.29(td,J＝6.8,2.3Hz,2H),2.18(s,3H),2.11(s,3H),2.06(d,J＝2.2Hz,3H),2.01–2.00(m,3H),1.64–1.57(m,2H),1.49(t,J＝6.9Hz,2H),1.38–1.36(m,2H),1.23(m,6H).13CNMR(101MHz,Chloroform-d)δ170.25,170.15,169.36,163.14,162.80,157.59,155.23,152.55,148.00,131.11,110.28,109.99,108.37,100.64,96.53,71.46,70.56,67.84,66.89,66.78,61.36,54.97,53.53,53.44,48.06,45.06,40.83,39.23,29.67,29.53,29.38,27.04,26.32,26.11,20.66,20.63,20.61,20.53,12.41.
步骤Ⅵ：荧光前药Ⅲ的合成。将化合物6c(100mg,0.092mmol)溶解在20mL CH3OH中，然后在0℃下加入CH3ONa(83mg,1.53mmol)。将混合物在室温和0℃搅拌20分钟，用amberliteIRC50中和，并通过棉垫过滤。然后蒸发溶剂，所得粗物质通过硅胶柱色谱纯化以产生荧光前药Ⅲ(66mg，80％产率)，命名为Lyso-Coumβ-gal。
荧光前药Ⅲ的核磁谱图数据为：1HNMR(400MHz,DMSO-d6)δ8.65(s,1H),8.63(s,1H),8.20(s,1H),7.80(s,1H),7.72(d,J＝1.9Hz,1H),7.68(d,J＝9.0Hz,1H),7.54(d,J＝8.8Hz,1H),7.37(d,J＝8.6Hz,1H),7.30(s,1H),6.81(d,J＝11.4Hz,1H),6.61(s,1H),5.84(s,1H),5.75(s,1H),5.01(d,J＝7.6Hz,1H),4.31(s,2H),3.70(s,1H),3.63(s,2H),3.56(s,6H),3.48(d,J＝6.9Hz,6H),3.27(s,2H),3.17(s,2H),2.93(s,2H),2.28(s,2H),2.16(s,2H),1.94–1.88(m,2H),1.50–1.45(m,2H),1.35(s,2H),1.26(s,4H),1.14(m,6H).13CNMR(101MHz,DMSO-d6)δ162.52,162.26,157.64,155.65,152.86,149.49,148.06,142.56,140.11,131.99,122.79,110.59,110.00,108.13,101.51,96.33,76.26,73.81,70.47,68.42,66.64,60.71,55.37,55.07,53.61,47.78,44.79,39.24,29.66,29.54,27.13,26.60,26.34,12.78.
步骤Ⅶ：化合物7c的合成。将化合物5c(100mg，0.116mmol)溶解在20mL CH3OH中，然后在0℃下加入CH3ONa(83mg，1.53mmol)。在0℃下再搅拌20分钟后，将溶液用amberliteIRC50中和10分钟，并通过棉垫过滤。然后蒸发溶剂，所得粗物质通过硅胶柱色谱纯化以产生化合物7c(63mg，78％产率),命名为Coumβ-gal。
化合物7c的核磁谱图数据为：1HNMR(400MHz,DMSO-d6)δ8.65(s,1H),8.62(s,1H),7.85(d,J＝2.1Hz,1H),7.67(d,J＝9.0Hz,1H),7.63(d,J＝8.8Hz,1H),7.43(d,J＝7.9Hz,1H),6.80(dd,J＝9.0,2.3Hz,1H),6.61(d,J＝2.1Hz,1H),5.75(s,1H),5.68(t,J＝6.0Hz,1H),5.14(s,1H),5.05(d,J＝7.6Hz,1H),4.59(s,1H),3.71(s,1H),3.65(t,J＝6.1Hz,1H),3.58(s,1H),3.55(s,1H),3.48(q,J＝6.8Hz,4H),3.42(d,J＝2.8Hz,1H),3.29(s,2H),2.96(h,J＝6.6Hz,2H),2.84(t,J＝2.5Hz,1H),2.77(dd,J＝6.5,2.6Hz,2H),1.53–1.45(m,2H),1.43–1.36(m,2H),1.28(s,4H),1.14(t,J＝7.0Hz,6H).13CNMR(101MHz,DMSO-d6)δ162.52,162.26,157.64,155.50,152.85,149.63,148.06,140.15,132.32,131.99,122.87,117.30,110.58,110.01,108.13,101.55,96.33,80.47,76.29,74.02,73.81,70.47,68.49,60.78,55.36,44.79,39.70,39.25,29.63,29.54,26.60,26.36,26.06,12.78.对比例4
β半乳糖苷酶不可断裂组荧光分子前药Ⅴ(Lyso-Coumglu)，合成路线如图6所示：
将对比例3中的化合物4a换成对比例2中化合物4b。
化合物5d，产率：76％，核磁谱图数据如下：1HNMR(400MHz,Chloroform-d)δ8.65(s,1H),8.52(s,1H),7.86(s,3H),7.71(s,1H),7.42(s,1H),7.29(s,1H),7.23(s,1H),6.52(d,J＝8.8Hz,1H),6.34(s,1H),5.61(s,1H),5.17(s,1H),4.20–4.06(m,2H),3.87(s,1H),3.33(s,2H),2.82(s,4H),2.73(s,4H),1.95(s,3H),1.93(s,4H),1.90(s,3H),1.89(s,3H),1.41(dd,J＝27.0,7.1Hz,3H),1.27–1.21(m,3H),1.09(t,J＝6.9Hz,6H).13CNMR(101MHz,Chloroform-d)δ170.26,169.90,169.23,169.09,162.94,162.41,157.44,155.07,152.45,148.78,147.79,140.67,135.73,132.03,130.99,123.12,122.83,118.95,110.02,108.16,99.64,96.30,78.74,72.22,72.05,70.40,67.98,61.66,44.91,40.79,39.23,36.33,31.24,29.46,26.14,20.36,12.29.
化合物7d，产率：85％，核磁谱图数据如下：1HNMR(400MHz,Chloroform-d)δ8.80(s,1H),8.67(s,1H),7.69(s,1H),7.48(t,J＝7.6Hz,1H),7.41(d,J＝8.9Hz,1H),7.36(d,J＝10.8Hz,1H),6.64(dd,J＝9.0,2.3Hz,1H),6.48(d,J＝2.1Hz,1H),5.93–5.87(m,1H),5.34(s,1H),5.28(s,1H),5.19–5.09(m,2H),4.38(s,2H),4.31–4.17(m,2H),3.90(s,2H),3.70(s,4H),3.44(p,J＝6.7Hz,6H),3.24(d,J＝19.5Hz,2H),3.10(s,2H),2.44(s,4H),2.34(s,2H),2.09(s,3H),2.08(s,3H),2.04(s,3H),2.02(s,3H),1.63–1.56(m,2H),1.50–1.44(m,2H),1.36(s,2H),1.23(t,J＝7.1Hz,6H).13CNMR(101MHz,Chloroform-d)δ170.48,170.13,169.34,169.31,163.18,162.80,157.58,155.25,152.56,148.71,148.02,141.15,140.93,136.78,136.69,132.21,131.67,131.12,123.18,122.51,119.68,110.18,108.35,100.08,96.50,73.42,72.32,70.43,68.05,66.60,61.72,54.90,53.38,48.00,45.05,40.83,39.24,32.94,29.51,26.11,20.69,12.41.
荧光分子前药Ⅴ(Lyso-Coumglu)，产率：71％，核磁谱图数据如下：1HNMR(400MHz,DMSO-d6)δ8.64(s,1H),7.83–7.79(m,1H),7.72(t,J＝2.1Hz,1H),7.67(d,J＝9.0Hz,1H),7.55–7.50(m,1H),7.41–7.35(m,1H),7.33–7.27(m,1H),6.80(dd,J＝9.0,2.4Hz,1H),6.61(d,J＝2.3Hz,1H),5.84(td,J＝6.7,2.8Hz,1H),5.75(s,2H),5.08(d,J＝7.1Hz,1H),4.31(t,J＝6.9Hz,3H),3.68(m,4H),3.55(t,J＝4.6Hz,4H),3.49(d,J＝2.4Hz,4H),3.42–3.38(m,1H),3.30–3.26(m,2H),3.25(d,J＝6.6Hz,1H),3.19–3.13(m,3H),2.28(t,J＝4.7Hz,4H),2.19–2.14(m,2H),1.99(s,1H),1.90(m,2H),1.51–1.45(m,2H),1.40–1.34(m,2H),1.27(s,2H),1.16m,8H).13CNMR(101MHz,DMSO-d6)δ162.52,162.26,157.64,155.64,152.85,149.36,148.05,140.02,132.28,131.98,123.60,110.58,109.99,108.12,100.72,96.33,77.68,77.10,73.50,69.87,66.62,60.97,60.21,55.36,55.08,53.60,47.79,44.79,39.24,32.72,29.66,29.54,27.12,26.59,26.34,21.22,14.55,12.77.
实施例2结果与分析
一、证明衰老细胞溶酶体含量增加
使用LysoTracker作为指标分析增殖和衰老细胞的溶酶体含量。简而言之，根据制造商的说明，将增殖或各种衰老细胞接种在12孔板(1×105个细胞/培养皿)中并用LysoTrackerGreen染色。用DPBS洗涤两次后，消化细胞，在488nm激发下通过Blu-FL-1通道进行流式细胞术分析。正常细胞BJ分别通过复制型，阿霉素(DOX)，过氧化氢，D-半乳糖刺激衰老，以增殖状态(proliferation)为对照；癌细胞A549分别通过阿霉素(DOX)，过氧化氢，D-半乳糖刺激衰老，以增殖状态(proliferation)为对照。如图7A，B所示，衰老后的细胞(BJ和A547)中LysoTracker荧光信号强度相对于增殖状态细胞明显增加，证明溶酶体含量明显上升。
二、溶酶体靶向senolytics前药在体外的酶选择性
为了便于观测，选用Lyso-Coumβ-gal(对比例3制备)来探究senolytics前药在体外的酶选择性，以Lyso-Coumglu(对比例3制备)作为对照。
分别将Lyso-Coumβ-gal和Lyso-Coumglu(20μM)与β-半乳糖苷酶(10U/μmol，≥500单位/mg)/溶菌酶(10U/μmol)/组织蛋白酶B(10U/μmol)/还原酶(10U/μmol)/半胱氨酸(1mM)/谷胱甘肽(1mM)/H2O2(1mM)在PBS中，在37度下孵育2小时。通过用HPLC监测香豆素类似物c4在430nm处的吸收变化来检测水解过程。如图8所示，完整的Lyso-Coumβ-gal和Lyso-Coumglu出峰时间为19.7min。Lyso-Coumβ-gal只能够响应β-半乳糖苷酶，释放出香豆素类似物c4，出峰时间为变为18.5min。而对照组Lyso-Coumglu对以上物质都不响应。证明Lyso-Coumβ-gal能够在体外被β-半乳糖苷酶特异性水解，并释放出完整的香豆素类似物。
三、溶酶体共定位
为了评估溶酶体靶向senolytics前药的溶酶体靶向能力，使用荧光香豆素类似物作为报告单位，并探索葡萄糖作为连接子构建分子前药(Lyso-Coumglu)，以排除分子通过被β-半乳糖苷酶水解后释放染料引起的干扰。将细胞接种在共聚焦培养皿中(1×105个细胞/培养皿)，并与Lyso-Coumglu(10μM)一起孵育30分钟。用DPBS洗涤两次后，根据制造商的说明，用LysoTracker Red/MitoTracker Red/ERTracker Red/GolgiTracker Red染色细胞。通过共聚焦荧光成像测量溶酶体共定位(Ex/Em波长：Lyso-Coumglu为405/450nm；细胞器跟踪器为543/590nm)。如图9A所示，以BJ复制型衰老为例，Lyso-Coumglu能够与溶酶体共定位，而不与其他细胞器共定位。此外，Lyso-Coumglu还能与不同刺激诱导的衰老细胞溶酶体发生共定位(图9B)。
四、溶酶体靶向senolytics前药选择性破坏衰老细胞溶酶体
吖啶橙(AO)染色用于研究溶酶体靶向senolytics前药对衰老细胞的溶酶体破坏。AO是一种溶酶体碱，在酸性环境下质子化后可以作为异染荧光团。在488nm激发下，AO在细胞质中以去质子化单体的形式发出绿色荧光(530nm)，而在完整溶酶体中质子化并发出橙色荧光(620nm)。一旦溶酶体膜受损，质子化的AO会泄漏到细胞质中，导致溶酶体中橙色荧光减弱。在这方面，增殖或衰老细胞首先在含有AO(2μg/mL)的新鲜培养基中孵育15分钟，然后用游离鞘氨醇、Lyso-Sphβ-gal(实施例1制备)或Lyso-Sphglu(15μM，对比例1制备)处理指定时间期间。用DPBS洗涤两次后，用胰蛋白酶消化细胞并转移到流动管中。AO的橙色荧光通过Blu-FL-3通道通过流式细胞术测定。AO橙色荧光降低的细胞被定义为苍白细胞(M1区域)，它们被单独用AO处理的细胞门控。如图10所示，当添加游离鞘氨醇(红色：0h，蓝色：1h，橙色：2h)时，细胞内橙色荧光的快速有效衰减，表明溶酶体膜通透化(lysosomal membranepermeabilization,LMP)程度增加。然而，游离鞘氨醇未能将衰老与非衰老细胞区分开来。对于不可切割的Lyso-Sphglu(红色：0h，蓝色：2h，橙色：4h)，衰老与非衰老细胞都没有表现出显著的橙色荧光下降，表明大多数溶酶体在用Lyso-Sphglu处理后是完整的，主要是由于鞘氨醇无法释放。相反，橙色荧光在Lyso-Sphβ-gal(红色：0h，蓝色：2h，橙色：4h)处理的衰老细胞中几乎消失，而非衰老细胞中荧光几乎没有减弱，表明Lyso-Sphβ-gal介导的溶酶体损伤是在衰老细胞中特异性触发的。
五、.溶酶体靶向senolytics前药体外选择性清除衰老细胞
通过CCK-8测定溶酶体靶向senolytics前药的抗衰老活性。增殖或衰老细胞接种在96孔板(5×103个细胞/孔)中并培养24小时。然后将培养基更换为100μL含有不同浓度Lyso-Sphβ-gal(实施例1制备)、Lyso-Sphglu(对比例2制备)或Sphβ-gal(对比例1制备)(2.5、5、10、15、20和25μM)的培养基，并培养细胞24小时。根据制造商的说明，使用CCK-8测定法评估细胞活力。结果如图11A，B所示，Lyso-Sphβ-gal能够选择性清除不同刺激衰老的BJ细胞和A549细胞，而对增殖细胞影响较小；相反，Lyso-Sphglu则对衰老和增殖细胞都没有明显毒性。为了进一步验证溶酶体靶向功能，以BJ复制型衰老细胞为例，测试了Lyso-Sphβ-gal与Sphβ-gal的毒性。结果如图11C，D所示，含有溶酶体靶向的Lyso-Sphβ-gal对衰老细胞的毒性更强，选择性也更好。
六、溶酶体靶向senolytics前药对衰老相关器官功能障碍的体内保护1.模型构建与药物处理
为了探究溶酶体靶向senolytics前药在活体水平的药物效果，构建了动物器官衰老模型。为此，6-8周龄的健康雌性C57BL/6J小鼠每周两次腹膜内注射DOX(10毫克/千克)(第0天和第7天注射)。另一组小鼠注射PBS并用作对照。经过两次注射，小鼠肝脏肾脏功能受损并产生衰老细胞。
从第10天开始，DOX预处理的小鼠每隔一天腹膜内注射PBS、Lyso-Sphβ-gal(15毫克/千克)或Lyso-Sphglu(15毫克/千克)，共两周。在第0天和第24天记录每只小鼠的体重。在第24天处死小鼠，收集血液和器官用于血液分析和组织学分析。每组5只老鼠，随机分配。
2.免疫荧光
从上述正常和衰老小鼠中收获的肝脏和肾脏被切片(10μm)用于IL-6和LMNB1的免疫荧光染色。简而言之，首先将组织切片在含有5％牛血清白蛋白的PBST(PBS，0.3％TritonX-100)缓冲液中封闭2小时，然后与相应的一抗(1:1000)在4℃下孵育过夜。PBS洗涤3次(每次5分钟)后，加入二抗(1:500)，室温再孵育2小时。细胞核用DAPI染色。最后，使用共聚焦激光扫描显微镜(NikonTI-E+A1SI，日本)对切片进行成像。
3.SA-β-gal染色
为了验证SA-β-gal在衰老细胞中的高表达，组织切片根据制造商的说明，使用衰老β-半乳糖苷酶染色试剂盒(碧云天)检测SA-β-gal活性。
4.器官功能的血液分析
为了测试分子前药的组织保护作用，通过监测相应的血液指标来评估肝肾功能。为此，通过以1200g离心10分钟从上述正常和衰老小鼠的血液中分离血清。尿素、天冬氨酸转氨酶(AST)和肌酐(CRE)的血清水平使用酶联免疫吸附测定(ELISA)进行定量。
结果分析：
在给予DOX的小鼠中组，组织学染色和免疫荧光分析显示肝脏和肾脏组织切片中有明显的衰老特征，包括SA-β-gal的积累、IL-6的分泌增加和LMNB1的丢失(图12A，B)。用Lyso-Sphβ-gal(实施例1)处理减轻了SA-β-gal和IL-6的升高，但增强了LMNB1的表达(图12C，D)。相反，接受Lyso-Sphglu(对比例2)的小鼠在这些指标上表现出微不足道的变化。
进一步对血清指标进行分析表明，DOX给药逐渐引起肝肾功能障碍，这反映在DOX注射后小鼠血清包括天冬氨酸转氨酶(AST)、尿素(Urea)和肌酐(CRE)在内的血浆指标水平升高。与正常小鼠相比，在用Lyso-Sphβ-gal处理后，这些指标的血浆浓度几乎降至正常水平。此外，与不可切割的Lyso-Sphglu相比，Lyso-Sphβ-gal可以抵消DOX诱导的体重减轻(图12E)。综上所述，我们的研究结果表明，溶酶体靶向senolytics前药具有预防和补救化疗引起的全身副作用的潜力。
Claims (10)
1.一种选择性破坏衰老细胞溶酶体的前药，其特征在于，其结构式如式Ⅰ所示：
2.根据权利要求1所述的选择性破坏衰老细胞溶酶体的前药的制备方法，包括以下步骤：
1)化合物5的合成：将鞘氨醇和中间体4加入到溶剂中，接着加入N,N-二异丙基乙胺，再接着将反应混合物在室温和氮气气氛下进行搅拌反应，反应完毕后，反应液用去离子水稀释并用CH2Cl2萃取，合并有机层并用无水Na2SO4干燥，然后进行过滤和减压浓缩，并将混合物进行纯化，得到化合物5；
2)化合物6的合成：将步骤1)中的化合物5、中间体L2、CuSO4·5H2O和抗坏血酸钠置于圆底烧瓶中，接着在氩气气氛下，将有机溶剂和ddH2O注入圆底烧瓶中，再接着在室温下进行反应，反应完毕后，除去溶剂后，将粗产物直接通过快速色谱纯化，得到化合物6；
3)前药I的合成：将步骤2)中的化合物6溶解在溶剂中，接着在0℃下加入CH3ONa并在0℃下搅拌一段时间，搅拌完毕后将溶液用amberlite IRC 50进行中和，在接着通过棉垫过滤并蒸发溶剂，然后将粗物质通过硅胶柱色谱纯化，得到前药I；
3.根据权利要求2所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述步骤1)中，鞘氨醇和中间体4的摩尔比为(3～4):(2～3)，溶剂为四氢呋喃和二甲基亚砜中的一种，中间体4和N,N-二异丙基乙胺摩尔体积比为(2～3)mmol:(0.5～1)mL，反应时间为10～14h。
4.根据权利要求2所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述步骤2)中，化合物5、中间体L2、CuSO4·5H2O和抗坏血酸钠的摩尔比为(1～2):(2～3):(0.3～0.5):(4～6)，有机溶剂为N,N-二甲基甲酰胺和甲苯中一种，有机溶剂与ddH2O的体积比为(4～6):1，反应时间为1～3h。
5.根据权利要求2所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述步骤3)中，溶剂为乙醇和甲醇中的一种，化合物6与溶剂的摩尔体积比为(0.07～0.12)mmol：20mL，化合物6与CH3ONa的(0.07～0.12):(1.2～1.8)，一段时间为15～25min，中和时间为5～15min。
6.根据权利要求2或4所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述的中间体L2的制备方法，包括以下步骤：将化合物L1加入到溶剂中，接着向其中加入叠氮化钠，然后将反应液在设定温度下进行反应，反应完毕后，反应液用H2O稀释并用CH2Cl2萃取；除去有机层的溶剂后，得到中间体L2；
7.根据权利要求6所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述的溶剂为N,N-二甲基甲酰胺和甲苯中一种，化合物L1与溶剂的摩尔体积比为(2～3)mmol:(5～15)mL，化合物L1与叠氮化钠的摩尔比为(2～3):(5～7)；设定温度为70～90℃，反应时间为18～30h。
8.根据权利要求2或3所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述的中间体4的制备方法，包括以下步骤：
S1将含有炔丙基溴的甲苯溶液滴加到含铝和催化量HgCl2的无水THF溶液中，在设定温度和氮气气氛下，进行第一段反应，反应完毕后，将反应液冷却至0℃，再接着向其中滴加含有化合物1的溶液，滴加完毕后，在搅拌条件下进行第二段反应，得到粗物质，将粗物质进行水解、萃取、浓缩和纯化后，得到化合物2；
S2将含有Ag2CO3和HMTTA的无水CH3CN溶液在室温下搅拌设定时间，接着在0℃下，将化合物2和固体1-溴-(2,3,4,6-O-四乙酰基)-β-D-吡喃半乳糖苷加入上述溶液，然后并且在室温下搅拌混合物直到起始材料化合物2通过TLC分析消失，此后，用水淬灭反应，然后用乙酸乙酯萃取，得到粗产物，对粗产物进行干燥、浓缩和出纯化后，得到化合物3；
S3在0℃下，将吡啶加入到含有化合物3和对硝基苯基氯甲酸酯的无水CH2Cl2溶液中，接着将混合物在室温搅拌直至通过TLC监测反应完成，再接着用H2O稀释并用CH2Cl2萃取，然后经过干燥、浓缩和纯化后，得到化合物4；
9.根据权利要求8所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述S1步骤中，含有炔丙基溴的甲苯溶液中质量百分含量为70～90％；炔丙基溴、铝、HgCl2和化合物1的摩尔比为(20～30):(20～30):(0.3～0.4):(3～4)；设定温度为50～70℃，第一段反应时间为7～9h；含有化合物1的溶液为含有化合物1的THF溶液；第二段反应时间为5～7h；所述S2步骤中，Ag2CO3、HMTTA、化合物2和1-溴-(2,3,4,6-O-四乙酰基)-β-D-吡喃半乳糖苷的摩尔比为(3～4):(0.5～1.0):(0.5～1.5):(1.5～2.5)，设定时间为1.5～2.5h。
10.根据权利要求8所述的选择性破坏衰老细胞溶酶体的前药的制备方法，其特征在于，所述步骤S3中，化合物3和对硝基苯基氯甲酸酯的摩尔比为(0.5～1.0):(1.5～2.5)，化合物3与吡啶的摩尔体积比为(0.5～1.0)mmol:(0.2～0.3)mL，化合物3与CH2Cl2的摩尔体积比为(0.5～1.0)mmol:(8～12)mL。
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