CN117603286A - 一种溶酶体靶向的衰老细胞清除前药的制备及应用 - Google Patents
一种溶酶体靶向的衰老细胞清除前药的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种溶酶体靶向的衰老细胞清除前药的制备及应用,属于药物化学领域。本发明前药以衰老细胞的溶酶体为靶点,经衰老相关β‑半乳糖苷酶活化,可以实现衰老细胞的特异性清除。该前药对衰老细胞具有高度的选择性。本发明所述的β‑半乳糖苷酶响应且溶酶体靶向的前药,为式1化合物或其药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物,包括外消旋混合物。
Description
技术领域
本发明属于药物化学领域,具体涉及一种溶酶体靶向的衰老细胞清除前药的制备及应用。
背景技术
衰老是一个随时间推移生物体内各种损伤积累,机体生理功能逐渐退化的过程。衰老会引起大脑衰退、免疫力下降和多种衰老相关疾病,如特发性肺纤维化、动脉粥样硬化、神经退行性疾病、2型糖尿病等。
细胞衰老是生物体衰老的核心标志。细胞衰老是在压力或机体损伤状态下发生的一种生理过程,会对组织的再生能力产生负面影响,并可能导致炎症的发展。在衰老过程中,衰老细胞会大量积累在机体的组织中,影响正常细胞再生,最终损害机体健康。衰老细胞通过分泌不同的因子影响周围的细胞和组织,这些因子组合可能导致组织功能受损并导致年龄相关疾病的发生发展和癌症的恶化。研究表明,选择性消除衰老细胞可以预防或延缓组织功能障碍,并改善各种与衰老有关的疾病,对维持个体健康和预防疾病发生具有重要意义。
发明内容
本发明利用衰老细胞过表达的β-半乳糖苷酶(SA-β-gal),发展了一类溶酶体靶向的,经SA-β-Gal活化后释放细胞毒性药物,可以选择性清除衰老细胞的糖基前药。
本发明的第一个目的是提供一种具有式1所示结构的含碱性溶酶体靶向基团的半乳糖前药化合物,所述前药化合物可响应SA-β-gal释放细胞毒性药物;
式中,R为氢原子或者乙酰基;R1、R2独立地为氢原子或者溶酶体靶向基团-CH2NR3R4,且R1、R2不同时为氢原子;
其中,R3和R4为取代或未取代的C1-6的直链烷基、取代或未取代的C3-6的支链烷基;或-NR3R4选自取代或未取代的吡咯烷基、咪唑基、脯胺醇基、吗啉基、哌嗪基和哌啶基;所述取代的取代基选自卤素(F、Cl、Br、I)、羟基、C1-6烷氧基;
D为含有氨基的细胞毒性药物,所述细胞毒性药物选自5-氟尿嘧啶、吉西他滨、阿糖胞苷、5'-脱氧-5-氟胞苷。
具体地:R1为H、R2为-CH2NR3R4,或者,R1为-CH2NR3R4、R2为-CH2NR3R4。
优选:R为乙酰基;R1和R2独立地为氢原子或溶酶体靶向基团-CH2NR3R4,且R1、R2不同时为氢原子;其中,R3和R4为甲基、乙基,或-NR3R4选自取代或未取代的吡咯烷基、咪唑基、脯胺醇基、吗啉基、哌嗪基和哌啶基;D为含有氨基的细胞毒性药物,所述细胞毒性药物选自5-氟尿嘧啶、吉西他滨、阿糖胞苷、5'-脱氧-5-氟胞苷;相应结构如式2所示:
进一步优选:R1为溶酶体靶向基团-CH2NR3R4;其中,R3和R4为甲基、乙基,或-NR3R4选自取代或未取代的吡咯烷基、咪唑基、脯胺醇基、吗啉基、哌嗪基和哌啶基;D来源于吉西他滨;相应结构如式3所示:
更进一步优选:前药化合物A和B,相应的结构式如下所示:
在本发明的一种实施方式中,所述前药化合物的制备方法包括:
I)化合物i溶于乙醇中,在氮气保护下依次加入甲醛和吗啉,加热反应后得到化合物ii,化合物i、甲醛和吗啉的投料摩尔比为1:2.1:2.1,甲醛为37%的甲醛水溶液,反应温度为78℃,反应时间为12小时;
II)化合物ii溶于乙腈中,在氮气保护下加入四-O-乙酰基-β-溴代半乳糖和氧化银,避光下室温反应12小时得到化合物iii,化合物ii、四-O-乙酰基-β-溴代半乳糖和氧化银的投料摩尔比为1:1:3;
III)将化合物iii溶于乙醇,在氮气保护下加入硼氢化钠,反应后得到化合物iv,化合物iii和硼氢化钠的投料摩尔比为1:2,反应温度为0℃,反应时间为3小时;
IV)将化合物iv溶于乙腈中,在氮气保护下加入对硝基苯基氯甲酸酯,在室温下反应3小时,得到化合物v,化合物iv和对硝基苯基氯甲酸酯的投料摩尔比为1:2;
V)将用叔丁基二甲基硅烷(TBS)保护的吉西他滨TBSGem溶于四氢呋喃中,依次加入双(三甲基硅烷基)氨基锂(LiHMDS)和化合物v反应后得到化合物vi,TBSGem、LiHMDS和化合物v的投料摩尔比为1:1.1:0.9,投料温度为-78℃,之后室温反应30min;
VI)将化合物vi溶于四氢呋喃中,在氮气保护下加入四丁基氟化铵(TBFA),反应后得到化合物vii,化合物vi和TBFA的投料摩尔比为1:2.5,反应温度为0℃,反应时间为2小时;
VII)将化合物vii溶于乙腈中,在氮气保护下加入吗啉或二甲胺,反应后分别得到前药A和B,化合物vii和吗啉或二甲胺的投料比为1:2.2,反应温度为78℃,反应时间为3小时。
本发明还提供上述半乳糖前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物;具体包括外消旋混合物。
本发明还提供上述半乳糖前药化合物或上述半乳糖前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物在制备抗衰老药物中的应用。
本发明还提供含有上述半乳糖前药化合物或上述半乳糖前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物的组合物。
所述组合物或制剂为:
(1)用于制备选择性清除SA-β-gal高表达的衰老细胞的组合物或制剂;
(2)用于制备改善或治疗衰老相关疾病的组合物或制剂。
所述组合物为药物组合、膳食补充剂、食品组合物或者保健品组合物。
本发明还提供一种抗衰老药物,所述药物中含有上述半乳糖前药化合物或上述半乳糖前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物,以及药学上可接受的载体、赋形剂或稀释剂。
所述药学上可接受的载体选自微囊、微球、纳米粒和脂质体。
所述药物的制剂包括多种临床药物剂型,具体可选自注射液、注射用冻干粉针、混悬剂、植入剂、栓塞剂、胶囊剂、片剂、丸剂和口服液。
细胞衰老过程中,衰老相关的β-半乳糖苷酶(SA-β-gal)、P21和P53等水平随衰老程度加剧而显著增加,故常被用做衰老细胞检测的生物标志物。SA-β-gal可特异性地水解不同底物的β-半乳糖苷键,是最经典的衰老细胞生物标志物之一,已被广泛应用于衰老细胞的检测和治疗。SA-β-Gal是一种在溶酶体中分泌并富集的外切糖苷酶,将SA-β-Gal响应的前药靶向到溶酶体中,进一步提高药物对衰老细胞的选择性,发挥更好的衰老细胞清除效果,减少对正常细胞的毒副作用。本发明在SA-β-Gal响应的衰老细胞清除前药结构中引入碱性溶酶体靶向基团,制备了一类新型溶酶体靶向的衰老细胞清除前药。活性测试表明,与不含溶酶体靶向基团的前药相比,本发明制备的溶酶体靶向前药对衰老细胞的选择性显著提高。
与现有技术相比,本发明具有如下优点及有益效果:
(1)本发明提供的衰老细胞清除前药是未曾报道过的新类型化合物。
(2)该前药中碱性基团的引入增加了其溶酶体靶向能力。
(3)与已报道的衰老细胞清除前药相比,本发明前药提高了对清除衰老细胞选择性,其中优选化合物前药A对于衰老细胞的选择性指数为对照药物SSK1(CN114341148A中的最优活性化合物)的2.3~2.6倍。
附图说明
图1是前药化合物A的核磁氢谱图。
图2是前药化合物A的核磁碳谱图。
图3是前药化合物A的质谱图。
图4是前药化合物B的核磁氢谱图。
图5是前药化合物B的核磁碳谱图。
图6是前药化合物B的质谱图。
图7是正常增殖细胞和衰老细胞的SA-β-Gal染色结果。
图8是吉西他滨对增殖细胞和衰老细胞的细胞毒性结果。
图9是SSK1对增殖细胞和衰老细胞的细胞毒性结果。
图10是前药化合物A对增殖细胞和衰老细胞的细胞毒性结果。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。实施例中,所用试剂均为市购。
实施例1:前药A的合成
I)化合物ii的合成:
具体操作:将化合物i(5.00g,40.94mmol)溶于50mL乙醇中,在氮气保护下依次加入37%的甲醛水溶液(6.41mL,85.97mol)和吗啉(7.46mL,85.97mmol),在78℃反应12小时。薄层色谱(TLC)检测反应结束后,浓缩反应液并通过柱层析纯化(乙酸乙酯:石油醚=1:9)得到化合物ii为白色固体(8.00g,产率为61.0%)。1H NMR(600MHz,CDCl3)δ9.82(s,1H),7.72(dd,J=8.3,2.1Hz,1H),7.58(dd,J=2.1,1.0Hz,1H),6.93(d,J=8.3Hz,1H),3.80(s,2H),3.77(s,4H),3.70(t,J=4.7Hz,1H),2.60(s,4H).13C NMR(151MHz,CDCl3)δ190.65,163.85,132.38,130.31,128.87,121.14,116.73,66.64,61.41,52.83.
II)化合物iii的合成:
具体操作:
a)将β-半乳糖五乙酸酯(5.00g,12.80mmol)溶于干燥的二氯甲烷(80mL)中,在氮气保护和0℃下搅拌,向反应体系中加入含33%氢溴酸的醋酸溶液(7.2mL,41.20mmol),在室温下反应2.5小时。当TLC显示反应结束后,有机相依次用冰水(2×20mL)、冷的饱和碳酸氢钠溶液(2×20mL)和饱和食盐水(2×20mL)洗涤。有机相用无水硫酸钠干燥,过滤并浓缩得到四-O-乙酰基-β-溴代半乳糖,直接用于下一步反应。
b)将化合物ii(3.79g,11.84mmol)置于250mL反应瓶中,并在氮气保护下依次加入无水乙腈(30mL)和分子筛。用20mL无水乙腈溶解四-O-乙酰基-β-溴代半乳糖(2.62g,11.84mmol),并将其加入反应瓶中,再加入氧化银(8.23g,35.52mmol),避光下室温反应12小时。当TLC显示反应结束后,用硅藻土过滤除去固体残渣。滤液旋干后通过柱层析纯化分离提纯产物(乙酸乙酯:石油醚=3:2),得到化合物iii为白色固体(6.16g,产率为80%)
1H NMR(400MHz,CDCl3)δ9.98(s,1H),7.90(s,2H),5.70(d,J=8.0Hz,1H),5.51(dd,J=10.5,8.0Hz,1H),5.40(dd,J=3.4,1.2Hz,1H),5.03(dd,J=10.5,3.5Hz,1H),4.04(dd,J=6.9,2.3Hz,2H),3.81-3.75(m,3H),3.74-3.67(m,8H),3.43(d,J=13.9Hz,2H),2.50(q,J=6.7,5.8Hz,8H),2.22(s,3H),2.16(s,3H),2.02(s,3H),1.91(s,3H).13C NMR(151MHz,CDCl3)δ170.29,170.26,170.21,170.15,170.11,169.11,156.07,152.38,137.27,135.73,131.51,128.61,127.89,106.72,102.45,72.24,71.34,71.00,70.86,69.89,69.41,67.13,66.96,66.45,64.74,64.28,61.75,60.47,58.49,57.20,53.61,53.46,52.91,46.11,21.17,20.79,20.77,20.73,20.71,20.58,20.55,10.58,7.94.
III)化合物iv的合成:
具体操作:将化合物iii(3.58g,5.50mmol)置于250mL反应瓶中,在0℃氮气保护下依次加入乙醇(50mL)、硅胶(3g)和硼氢化钠(416.13mg,11.00mmol),0℃下反应3小时。当TLC监测反应结束后,加入60mL水淬灭反应,用二氯甲烷(4×60mL)萃取有机相。合并有机相用无水硫酸钠干燥,过滤并浓缩有机相。通过柱层析纯化(乙酸乙酯:石油醚:甲醇=30:20:1)得到化合物iv(3.19g,产率为88.7%)为无色油状液体。
1H NMR(400MHz,CDCl3)δ7.43(d,J=2.2Hz,1H),7.23(dd,J=8.4,2.2Hz,1H),7.03(d,J=8.4Hz,1H),5.53(dd,J=10.5,7.9Hz,1H),5.46(d,J=3.5Hz,1H),5.12(dd,J=10.5,3.4Hz,1H),5.04(d,J=7.9Hz,1H),4.64(s,2H),4.28-4.11(m,2H),4.06(t,J=6.6Hz,1H),3.72(t,J=4.7Hz,4H),3.52(d,J=2.4Hz,2H),2.89(s,1H),2.51(q,J=4.5Hz,4H),2.20(d,J=0.8Hz,3H),2.08(dd,J=9.2,0.8Hz,6H),2.02(d,J=0.8Hz,3H).13C NMR(151MHz,CDCl3)δ191.56,170.18,170.15,170.08,169.06,157.81,133.03,131.35,102.40,71.11,70.85,69.26,67.08,66.84,60.46,56.98,53.50,29.78,29.70,29.60,29.53,29.32,27.22,21.12,20.73,20.57,20.51,14.12.
IV)化合物v的合成:
具体操作:将化合物iv(124.00mg,0.19mmol)溶于干燥的二氯甲烷(3mL)中,在氮气保护和0℃下搅拌,向反应体系中加入吡啶(0.04mL)和氯甲酸对硝基苯酯(75.00mg,0.37mmol),在室温下反应3小时。当TLC显示反应结束后,加入饱和碳酸氢钠溶液(10mL)终止反应,用二氯甲烷(3×10mL)萃取有机相。有机相用无水硫酸钠干燥,过滤并浓缩有机相。通过柱层析纯化(乙酸乙酯:石油醚=1:4)得到化合物v(107.47mg,产率为78.9%)为无色油状液体。
V)化合物vi的合成:
具体操作:将用叔丁基二甲基硅烷保护的吉西他滨(TBSGem,78.68mg,0.16mmol)溶于干燥的四氢呋喃(1mL)中,在氮气保护和-78℃的条件下,加入LiHMDS的四氢呋喃溶液(1.0M,0.10mL,0.18mmol),搅拌10min。用2mL干燥的四氢呋喃溶解化合物v(107.47mg,0.15mmol),并将其加入反应体系中,逐渐升至室温下继续反应30min。当TLC显示反应结束后,40℃下浓缩反应液,通过柱层析纯化(甲醇:二氯甲烷=2:100)得到化合物vi(128.29mg,产率为80.0%)为白色固体。
1H NMR(400MHz,MeOD)δ8.34(d,J=7.7Hz,1H),7.92(d,J=2.2Hz,1H),7.72(dd,J=8.7,2.2Hz,1H),7.49(d,J=8.6Hz,1H),7.34(d,J=7.7Hz,1H),6.31-6.23(m,1H),5.49(dd,J=3.4,1.2Hz,1H),5.46-5.34(m,2H),5.28(d,J=6.6Hz,3H),4.40-4.26(m,2H),4.23(d,J=6.5Hz,2H),3.98(tdd,J=6.6,5.8,2.4Hz,2H),3.86-3.79(m,1H),2.20(s,3H),2.10(s,3H),2.07(s,3H),1.99(s,3H).13C NMR(101MHz,MeOD)δ170.59,170.53,169.99,169.84,163.95,156.01,153.15,148.86,144.35,141.01,133.21,131.57,124.33,122.52,118.42,99.64,95.62,85.43,81.47,71.59,70.62,69.01,68.73,68.65,68.15,67.19,65.51,61.19,58.87,19.24,19.19,19.06.
VI)化合物vii的合成:
具体操作:将化合物vi(128.29mg,0.12mmol)加入干燥的四氢呋喃(1mL)中,在0℃氮气保护下加入TBFA(0.32ml,0.30mmol),并在0℃下反应3小时。TLC监测反应终止后,用二氯甲烷(4×10mL)萃取有机相。合并有机相用无水硫酸钠干燥,过滤并浓缩有机相。通过柱层析纯化(甲醇:二氯甲烷=4:100)得到化合物vii(84.06mg,产率为83.3%)为白色固体。
1H NMR(600MHz,CDCl3)δ8.10(s,1H),7.50(s,2H),7.24(s,1H),6.29(s,1H),5.54(ddd,J=10.5,8.0,1.0Hz,1H),5.41(dd,J=3.4,1.2Hz,1H),5.22-5.15(m,2H),5.11-5.07(m,1H),5.00(d,J=8.0Hz,1H),4.74(d,J=11.9Hz,2H),4.67(d,J=11.9Hz,2H),4.35(q,J=10.3Hz,1H),4.17-4.08(m,2H),4.03(t,J=12.2Hz,2H),3.91(dd,J=11.7,2.0Hz,1H),3.87-3.80(m,1H),2.23(s,3H),2.19(s,3H),2.02(s,3H),1.95(d,J=0.9Hz,3H),1.63(s,3H),1.25(s,1H).13CNMR(151MHz,CDCl3)δ170.54,170.32,170.21,169.50,163.25,155.35,152.24,151.32,133.24,132.50,131.29,129.89,124.06,122.34,102.78,96.08,81.49,71.07,70.98,69.11,66.83,60.73,59.69,40.50,29.78,29.32,27.22,20.90,20.75,20.58,20.57.
VII)前药化合物A的合成:
具体操作:将化合物vii(84.06mg,0.10mmol)溶于干燥的乙腈中,向反应体系中加入N,N-二异丙基乙胺(0.03mL)和吗啉(0.05mL,0.22mmol),在室温下反应3小时。当TLC显示反应结束后,加入饱和碳酸氢钠溶液(10mL)终止反应,用二氯甲烷(3×10mL)萃取有机相。有机相用无水硫酸钠干燥,过滤并浓缩有机相。通过柱层析纯化(乙酸乙酯:石油醚=7:13)得到前药化合物A(75.35mg,产率为91.4%)为白色固体。
1H NMR(600MHz,CDCl3)δ8.13(d,J=7.7Hz,1H),7.36(d,J=2.8Hz,2H),6.18(s,1H),5.56(d,J=8.1Hz,1H),5.47(d,J=2.5Hz,1H),5.39(dd,J=3.3,1.5Hz,1H),5.20-5.12(m,2H),5.05-5.02(m,1H),4.46(s,1H),4.08-3.97(m,5H),3.92-3.87(m,1H),3.77(d,J=9.2Hz,2H),3.69(d,J=5.7Hz,8H),3.40-3.35(m,2H),2.58(q,J=7.2Hz,5H),2.52-2.44(m,8H),2.23-2.20(m,3H),2.16-2.14(m,3H),2.02-2.00(m,3H),1.92-1.90(m,3H),1.05(td,J=7.2,1.6Hz,7H).13C NMR(151MHz,CDCl3)δ170.35,170.28,170.15,169.19,163.03,153.22,144.80,131.81,131.31,130.11,122.46,120.73,102.39,81.57,70.94,70.84,69.36,68.81,67.72,67.02,66.90,60.43,59.53,56.98,53.40,46.12,46.09,21.18,20.75,20.59,20.53,11.14,11.07.MS(m·z-1):940.5[M-H]-.
实施例2:前药化合物B的合成
I)~VI)步与实施例1相同。
VII)前药化合物B的合成:
具体操作:将化合物vii(84.06mg,0.10mmol)溶于干燥乙腈中,向反应体系中加入N,N-二异丙基乙胺(0.03mL)和二甲胺(0.03mL,0.22mmol),在室温下反应3小时。当TLC显示反应结束后,加入饱和碳酸氢钠溶液(10mL)终止反应,用二氯甲烷(3×10mL)萃取有机相。有机相用无水硫酸钠干燥,过滤并浓缩有机相。通过柱层析纯化(乙酸乙酯:石油醚=7:13)得到前药化合物B(63.25mg,产率为73.8%)为白色固体。
1H NMR(400MHz,CDCl3)δ8.19(d,J=7.7Hz,1H),7.41(s,2H),6.20(t,J=7.0Hz,1H),5.48(dd,J=10.4,7.9Hz,1H),5.37(d,J=3.5Hz,1H),5.31-5.28(m,1H),5.16(s,2H),5.05(dd,J=10.4,3.4Hz,1H),4.45(q,J=11.3Hz,1H),4.01(s,2H),3.97(d,J=10.9Hz,2H),3.88(d,J=12.1Hz,1H),3.79(d,J=14.6Hz,2H),3.74(d,J=7.0Hz,1H),3.49(d,J=14.3Hz,2H),2.62-2.45(m,11H),2.18(s,3H),2.13(s,3H),2.00(s,3H),1.88(s,3H),1.02(td,J=7.2,1.7Hz,16H).13C NMR(101MHz,CDCl3)δ170.36,170.24,170.19,169.30,163.05,155.08,152.43,144.73,133.52,131.41,129.27,125.07,122.48,119.89,101.97,101.81,96.02,81.51,71.12,71.01,70.78,70.63,69.52,69.39,67.80,67.05,66.89,60.62,59.38,51.80,46.93,46.80,46.66,46.20,46.07,29.68,21.06,20.99,20.70,20.61,20.58,20.55,20.48,11.53,11.42.MS(m·z-1):912.6[M-H]-.
实施例3
衰老细胞株构建:
阿霉素(Doxorubicin hydrochloride,Dox)诱导的衰老细胞:准备A549和MDA-MB-231细胞,胰酶消化后传代至T25板中,于37℃、含5% CO2的细胞培养箱中培养24小时。保证细胞贴壁良好后,加入含100nmol·L-1Dox的新鲜培养基,置于培养箱中继续培养48小时即可。阿霉素诱导的A549细胞和MDA-MB-231细胞分别命名为A549/DOX和MDA-MB-231/DOX。
双氧水(H2O2)诱导的衰老细胞:准备A549和MDA-MB-231细胞,胰酶消化后传代至T25板中,于37℃、含5% CO2的细胞培养箱中培养24小时。保证细胞贴壁良好后,加入含100μmol·L-1H2O2的新鲜培养基,置于培养箱中继续培养72小时即可。过氧化氢诱导的A549细胞和MDA-MB-231细胞分别命名为A549/H2O2和MDA-MB-231/H2O2。
使用SA-β-Gal染色试剂盒检测正常增殖细胞和衰老细胞内的SA-β-Gal活性水平。显微镜观察显示,与正常细胞相比,经DOX或H2O2诱导后的细胞内蓝绿色范围显著增加(附图7),说明细胞内SA-β-Gal活性升高,衰老细胞株诱导成功。
实施例4
溶酶体靶向的前药A选择性清除衰老细胞:
通过MTT比色法测定前药A的衰老细胞清除活性,以SSK1和吉西他滨为对照化合物。正常增殖或衰老细胞接种在96孔板,正常增殖和衰老细胞的铺板密度分别为4000/孔和8000/孔。待细胞贴壁后,将培养基更换为100μL含有不同浓度药物的培养基。细胞给药孵育48小时后,将原培养基吸出,用PBS洗去残留后加入含10% MTT试剂的新鲜培养基,继续在37℃含5% CO2的细胞培养箱中孵育2到3小时,用多功能酶标仪检测其在490nm处的吸光值,根据公式计算出细胞存活率:
其中,OD+为加有MTT试剂、细胞和药物的孔的吸光值,ODcontrol为仅加有MTT试剂的孔的吸光值,OD-为仅加有MTT试剂和细胞的孔的吸光度。
实验结果见附图8-10,前药A能够选择性清除不同刺激衰老的A549细胞和MDA-MB-231细胞,而对正常增殖细胞影响较小,说明前药A具有选择性杀伤衰老细胞的作用。进一步分析实验结果,计算药物对不同细胞的IC50和衰老细胞清除的选择指数Senolytic index(SI),结果如表1和表2所示。
表1药物对不同细胞的IC50(μM)
表2药物对不同衰老细胞的SI值a
aSI值为药物对正常增殖细胞的IC50值与衰老细胞IC50值的比值。
由表1-2可知,前药A对不同刺激衰老的A549细胞和MDA-MB-231细胞的SI均大于SSK1。例如,前药A对A549/DOX细胞的SI值为50.3,而SSK1对A549/DOX细胞的SI为21.4;前药A对MDA-MB-231/DOX细胞的SI值为35.4,而SSK1对A549/DOX细胞的SI为15.0,前药A对于衰老细胞的选择性指数为SSK1的2.3到2.6倍。综上,含有溶酶体靶向基团的前药A可选择性杀伤衰老细胞,且衰老细胞选择性优于SSK1。
以上所提供的实施例并非用以限制本发明所涵盖的范围,所描述的步骤也不是用以限制其执行顺序。本领域技术人员结合现有公知常识对本发明做显而易见的改进,亦落入本发明权利要求书所界定的保护范围之内。
Claims (10)
1.一种SA-β-gal响应且溶酶体靶向清除衰老细胞的前药化合物,其结构如式1所示:
式中,R为氢原子或者乙酰基;R1、R2独立地为氢原子或者溶酶体靶向基团-CH2NR3R4,且R1、R2不同时为氢原子;
其中,R3和R4为取代或未取代的C1-6的直链烷基、取代或未取代的C3-6的支链烷基;或-NR3R4选自取代或未取代的吡咯烷基、咪唑基、脯胺醇基、吗啉基、哌嗪基和哌啶基;所述取代的取代基选自卤素、羟基、C1-6烷氧基;
D为含有氨基的细胞毒性药物,所述细胞毒性药物选自5-氟尿嘧啶、吉西他滨、阿糖胞苷、5'-脱氧-5-氟胞苷。
2.根据权利要求1所述的前药化合物,其特征在于,所述前药化合物包括前药化合物A和前药化合物B:
3.一种制备权利要求1或2所述的化合物的方法,其特征在于,所述方法是将化合物i与吗啉反应得到化合物ii,化合物ii的酚羟基经四-O-乙酰基-β-溴代半乳糖取代和硼氢化钠还原后,与氯甲酸对硝基苯酯形成活性中间体,再与吉西他滨偶联,最后引入不同的溶酶体靶向基团反应得到的;其中化合物i的结构为化合物ii的结构为/>
4.一类权利要求1或2所述前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物。
5.权利要求1-2任一项所述前药化合物或权利要求4所述的前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物在制备抗衰老药物中的应用。
6.一种组合物,其特征在于,含有权利要求1-2任一项所述前药化合物或权利要求4所述的前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物。
7.根据权利要求6所述的组合物,其特征在于,为药物组合、膳食补充剂、食品组合物或者保健品组合物。
8.一种抗衰老药物,其特征在于,含有权利要求1-2任一项所述前药化合物或权利要求4所述的前药化合物的药学上可接受的盐、溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物,以及药学上可接受的载体、赋形剂或稀释剂。
9.根据权利要求8所述的抗衰老药物,其特征在于,所述药学上可接受的载体选自微囊、微球、纳米粒和脂质体。
10.根据权利要求8所述的抗衰老药物,其特征在于,所述药物的制剂选自注射液、注射用冻干粉针、混悬剂、植入剂、栓塞剂、胶囊剂、片剂、丸剂和口服液。
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