CN116410249A - β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药及其制备方法与用途 - Google Patents
β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药及其制备方法与用途 Download PDFInfo
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Abstract
本发明属于生物医药领域,涉及肿瘤靶向治疗,公开了如式I、II所示的β‑葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药。本发明所述的前药较于母体药物体内最大耐受剂量高,无心脏毒性,能够有效地抑制体内肿瘤的生长且对主要器官无明显影响,有望成为新的抗肿瘤药物。本发明还公开了所述的衍生物在制备抗肿瘤药物中的应用。
Description
技术领域
本发明属于生物医药领域,具体涉及β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药及其制备方法与应用。
背景技术
癌症又称恶性肿瘤,其形成和发展的内在机制十分复杂。据世界卫生组织(WTO)统计,全世界每年约有690万人死于癌症,预计到2035年全球癌症死亡人数将达到2400万。同时,全球每年因癌症带来的损失高达数千亿美元。因此,肿瘤已经对人类的健康和社会的进步构成了严重的危害。
目前,肿瘤的治疗方法主要有三种:手术治疗、放射治疗和化学治疗。化学治疗最为常见。尽管已有多种抗癌药物用于临床,但由于肿瘤细胞极易产生耐药性以及化疗药物毒副作用大等缺点,使得现有化疗药物难以满足临床治疗的需求。因此,寻求高效、低毒、靶向性强、生物利用度高的新型抗肿瘤药物具有重要的意义。
微管对于快速分裂的肿瘤细胞具有重要作用,已成为抗肿瘤药物开发的重要靶点之一,紫杉醇、长春碱等作用于微管的药物已成为临床中常用的化疗药物。与其他微管蛋白结合位点的药物相比,作用于秋水仙碱位点的化合物具有抗耐药、防止新生血管形成和破坏现有血管的优势。天然产物Combretastatin A-4(CA-4)通过结合于秋水仙碱位点抑制微管蛋白聚集表现出增殖抑制作用。然而,较差的代谢稳定性和溶解性限制了其进一步的临床应用。将CA-4结构中的双键替换为杂环、β-内酰胺等片段获得了一系列抗肿瘤作用较佳的CA-4衍生物,且代谢稳定性和水溶性均得到改善。然而,此类微管蛋白聚集抑制剂仍存在肿瘤组织选择性较差,难以直接应用于临床。因此,提高化合物的肿瘤特异性是亟待解决的问题。
研究表明,肿瘤组织内特异性或过表达的酶可用于将低毒或无毒前药转化为活性分子,从而获得更好的肿瘤组织选择性。β-葡萄糖醛酸苷酶在多种恶性肿瘤内高表达,并由炎症细胞分泌到肿瘤组织微环境中,而在健康组织中,其活性仅限于细胞溶酶体内。葡萄糖醛酸偶联物可视为β-葡萄糖醛酸苷酶的底物,葡萄糖醛酸片段的亲水性限制了细胞被动摄取和正常细胞溶酶体内β-葡萄糖醛酸酶的激活。因此,β-葡萄糖醛酸苷酶响应型前药可特异性作用于肿瘤细胞而不影响正常细胞。
目前肿瘤特异性给药已成为抗肿瘤药物开发的重要内容之一,有关微管蛋白聚集抑制剂β-葡萄糖醛酸苷酶响应性前药的研究较少。
发明内容
本发明的目的是采用前药策略,以手性二芳基-β-内酰胺化合物和2-氨基-5-羟基-6-甲氧基-3-(3,4,5-三甲氧基苯甲酰基)苯并呋喃为母体化合物,通过取代或未取代的对羟基苄基连接链引入β-葡萄糖醛酸结构单元,旨在寻找肿瘤组织特异性高、对正常组织无毒或低毒的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药。
本发明的目的是通过以下技术方案实现的:
如结构式I、II所示的:β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药
其中,R1选自氢、硝基;R2选自氢、羟基、氟。
具体地讲,通式I、II所示的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药优选自下列化合物18-1、18-2、18-3和8:
本发明的另一个目的是提供所述的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药的制备方法,包括:以乙腈为反应溶剂,式1所示化合物与取代或未取代的对羟基苯甲醛反应生成式2所示化合物;以四氢呋喃为反应溶剂,硼氢化钠为还原剂,式2所示化合物被还原为式3所示化合物;以二氯甲烷和四氢呋喃为反应溶剂,式2所示化合物与三苯基膦和四溴化碳反应得到式4所示化合物.
其中,R1选自氢,硝基;
以N,N-二甲基甲酰胺为反应溶剂,碳酸钾为缚酸剂,式4所示化合物与式5所示化合物生成式6所示化合物,接着经过两步酯解得到式8所示化合物。
式10所示化合物由式9所示化合物经过硝基化得到,与异香兰素反应得到式11所示化合物;式11所示化合物与丙炔酸甲酯反应生成式12所示化合物,再经还原得到式13所示化合物;非对映异构体式15所示化合物是由式13所示化合物经过与Boc-L-脯氨酸酯化拆分获得;以无水二氯甲烷为溶剂,式15所示化合物与三氟化二乙氨基硫反应生成式16所示化合物。
以N,N-二甲基甲酰胺为反应溶剂,碳酸钾为缚酸剂,式4所示化合物与式15所示化合物或式16所示化合物生成式17所示化合物,经过两步酯解得到式18所示化合物。
其中,R1选自氢,硝基;R2选氢、羟基,氟。
本发明的另一个目的是提供一种药物组合物,它以本发明所述的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药为有效成分,辅以药学上可接受的载体,制成任何药学上可接受的剂型。所述的剂型选自片剂、胶囊、滴丸、颗粒、粉剂、锭剂、水性或油性悬浮剂、注射剂。
本发明的另一个目的是提供所述的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药或以所述的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药为有效成分的药物组合物在制备抗肿瘤药物中的用途。
本发明β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药的制备方法反应条件温和,所用试剂低毒,原料易得,后处理方便,产率较高。药理实验研究表明,本发明前药在磷酸缓冲盐溶液中具有良好的稳定性,在β-葡萄糖醛酸苷酶作用下能够释放出活性分子,其对β-葡萄糖醛酸苷酶预处理和未处理的肿瘤细胞具有优异的选择性。研究表明,前药体内毒性低,抗肿瘤活性佳,且无心脏毒性。因此,本发明化合物具有制备高效低毒抗肿瘤药物的应用价值。
附图说明
图1为前药18-1、18-2、18-3和8在磷酸缓冲盐溶液中稳定性以及β-葡萄糖醛酸苷酶存在下水解情况。
图2为前药18-1对三阴性乳腺癌MDA-MB-231细胞裸鼠异种移植瘤生长抑制作用;图2A为小鼠给药治疗后不同药物组肿瘤体积变化;图2B为实验终止时,不同药物组肿瘤重量;图2C为实验终止时,不同药物组小鼠体重;图2D为实验终止时,不同药物组小鼠体内肿瘤剥离情况。
图3.小鼠肝、脾、肾、肿瘤组织H&E染色结果。
图4.化合物15和前药18-1对小鼠心电图的影响。
具体实施方案
为进一步阐明本发明的技术方案,下面列举一系列实施例。这些实施例是例证性的,不应当理解为对本发明的限制。
实施例1:葡糖苷酸前药8的合成
步骤A:发明按如下路线合成化合物4
反应试剂与条件:(a)Ag2O,CH3CN,rt,18h;(b)NaBH4,THF,0℃,4h;(c)CBr4,PPh3,CH2Cl2,THF,rt,12h.
1.1(2S,3R,4S,5S,6S)-2-(4-甲酰基苯氧基)-6-(甲氧羰基)-2H-四氢吡喃-3,4,5-三乙酸酯(化合物2)的合成
依次向圆底烧瓶中加入(2R,3R,4S,5S,6S)-2-溴-6-(甲氧羰基)-2H-四氢吡喃-3,4,5-三乙酸酯(化合物1)(1.1g,2.7mmol)、4-羟基苯甲醛(0.37g,3.05mmol)、氧化银(1.6g,6.9mmol)和乙腈(30mL),避光25℃反应18小时。反应完成,反应液经硅藻土过滤,乙酸乙酯洗涤,真空浓缩,乙酸乙酯重新溶解,依次用饱和碳酸氢钠和饱和食盐水洗涤,无水硫酸钠干燥。浓缩后经硅胶柱层析(乙酸乙酯/石油醚=1:4)纯化,得到白色固体800mg,产率68%。1HNMR(400MHz,DMSO-d6)δ9.90(s,1H),7.90(d,J=7.7Hz,2H),7.18(d,J=7.9Hz,2H),5.84(d,J=7.6Hz,1H),5.47(t,J=9.6Hz,1H),5.14(t,J=8.7Hz,1H),5.07(t,J=9.7Hz,1H),4.75(d,J=9.7Hz,1H),3.61(s,3H),2.04-1.96(m,9H).13C NMR(151MHz,DMSO-d6)δ191.34,169.35,169.14,168.85,166.79,160.42,131.65,131.20,116.26,96.00,70.86,70.77,70.17,68.66,52.44,20.11.
1.2(2S,3R,4S,5S,6S)-2-(4-羟甲基苯氧基)-6-(甲氧羰基)-2H-四氢吡喃-3,4,5-三乙酸三酯(化合物3)的合成
化合物2(590mg,1.3mmol)和硼氢化钠(56mg,1.4mmol)溶于干燥四氢呋喃(10mL),0℃搅拌30分钟。反应完成,反应液用饱和氯化铵溶液(75mL)淬灭,并以二氯甲烷(3×25mL)和乙酸乙酯(3×25mL)萃取。合并有机相,无水硫酸钠干燥,浓缩,硅胶柱层析纯化(乙酸乙酯/石油醚=1:1),得到白色固体450mg,产率85%。1H NMR(400MHz,DMSO-d6)δ7.24(d,J=7.8Hz,2H),6.92(d,J=8.0Hz,2H),5.61(d,J=7.8Hz,1H),5.45(t,J=9.6Hz,1H),5.18-4.97(m,3H),4.68(d,J=9.9Hz,1H),4.41(d,J=5.1Hz,2H),4.01(q,J=7.0Hz,1H),3.61(s,3H).13C NMR(151MHz,DMSO-d6)δ169.36,169.14,168.88,166.95,154.93,137.12,127.77,115.94,97.09,70.92,70.76,70.39,68.85,62.18,52.41,20.12.
1.3(2S,3R,4S,5S,6S)-2-(4-(溴乙基)苯氧基)-6-(甲氧羰基)-2H-四氢吡喃-3,4,5-三乙酸三酯(化合物4)的合成
化合物3(260mg,0.59mmol)溶于二氯甲烷-四氢呋喃混合溶液(v:v=1:1)中,加入三苯基膦(386mg,1.48mmol),搅拌3min后加入四溴化碳(488mg,1.48mmol),氮气保护下室温搅拌。反应完成后,乙酸乙酯稀释,经硅胶柱层析纯化(乙酸乙酯/石油醚=1:4),得白色固体290mg,产率98%。1H NMR(400MHz,CDCl3)δ7.32(d,J=8.5Hz,2H),6.95(d,J=8.5Hz,2H),5.39-5.23(m,3H),5.14(d,J=7.1Hz,1H),4.47(s,2H),4.18(d,J=8.9Hz,1H),3.72(s,3H),2.04(s,9H).13C NMR(151MHz,DMSO-d6)δ169.34,169.12,168.85,166.88,155.88,132.60,130.76,116.28,96.63,70.87,70.78,70.28,68.78,52.43,34.13,20.14.
步骤B:发明按如下路线合成目标化合物8
合成试剂和条件:(d)K2CO3,DMF,N2,rt,8h;(e)MeONa,MeOH,N2,0℃,1h;(f)1NNaOH,acetone,0℃,2h.
1.4化合物6的合成
化合物5(75mg,0.2mmol)、化合物4(100mg,0.2mmol)和碳酸钾(55mg,0.4mmol)溶于干燥N,N-二甲基甲酰胺(5mL)中,室温搅拌12小时。反应完成,加入水(20mL),乙酸乙酯(10mL×3)萃取。有机相饱和食盐水洗涤,无水硫酸钠干燥,过滤。滤液浓缩,经硅胶经柱层析(乙酸乙酯/石油醚=1.2:1)得白色固体128mg,产率81%。1H NMR(400MHz,CDCl3)δ8.02(s,1H),7.42(d,J=8.0Hz,1H),7.09(s,2H),6.99(d,J=5.9Hz,4H),6.69(q,J=8.7Hz,1H),5.38-5.33(m,2H),5.29(dd,J=7.0,2.9Hz,1H),5.17(s,2H),4.20(d,J=8.0Hz,1H),3.86(d,J=4.0Hz,9H),2.96(s,3H),2.89(s,3H),2.06(s,9H).13C NMR(151MHz,DMSO-d6)δ187.21,169.35,169.12,168.86,166.92,166.72,162.12,155.83,152.51,147.35,140.77,139.10,136.29,132.11,131.91,129.61,121.34,115.98,111.74,109.31,104.40,96.82,91.55,73.59,70.88,70.79,70.35,68.82,60.02,56.57,55.81,52.43,35.61,30.60,20.14.
1.5化合物7的合成
化合物6(128mg,0.16mmol)溶于无水甲醇中(5mL),0℃搅拌,加入30%甲醇钠的甲醇溶液(28μL),缓慢升至室温,反应2小时。反应完成,向体系中加入5μL乙酸淬灭,溶液浓缩,硅胶柱层析(二氯甲烷/甲醇=25:1)得白色固体83mg,产率70%。1H NMR(400MHz,CDCl3)δ7.79(s,1H),7.58(d,J=9.0Hz,1H),7.43(d,J=7.8Hz,2H),7.08-7.00(m,3H),6.88(d,J=8.6Hz,1H),5.21(s,2H),4.93(s,1H),3.99(d,J=11.0Hz,1H),3.89(s,6H),3.85(s,6H),3.80(d,J=3.5Hz,3H),3.73(s,3H).13C NMR(151MHz,DMSO-d6)δ189.32,169.06,164.17,156.48,155.56,151.90,148.38,142.02,139.45,135.08,131.86,131.00,129.43,123.88,115.65,113.98,109.72,106.22,100.49,99.59,75.38,74.96,73.78,72.73,71.23,59.90,56.69,55.68,51.75.
1.6(2S,3S,4S,5R,6S)-6-(4-(((2-氨基-6-甲氧基-3-(3,4,5-三甲氧基苯甲酰基)苯并呋喃-7-基)氧基)甲基)苯氧基)-3,4,5-三羟基-2H-四氢吡喃-2-羧酸(8)的合成
化合物7(83mg,0.12mmol)溶于丙酮(5mL),0℃搅拌,加入1mL NaOH(1M)溶液。反应完成后,用盐酸(1M)中和至中性。反应液浓缩,冷冻干燥,固体甲醇溶解,过滤。滤液浓缩得棕色固体70mg,产率86%。1H NMR(600MHz,CD3OD)δ7.49(d,J=8.6Hz,2H),7.12(d,J=8.6Hz,2H),7.07(d,J=6.8Hz,3H),6.53(d,J=8.9Hz,1H),4.99(s,2H),3.91(s,6H),3.82(s,3H),3.80(s,3H),3.54(dt,J=4.6,1.1Hz,3H),3.33(q,J=1.6Hz,2H).13C NMR(151MHz,CD3OD)δ176.76,174.42,156.98,151.62,150.88,148.65,137.41,136.97,136.50,131.55,129.33,129.25,125.82,121.80,118.79,115.45,104.94,102.59,100.46,75.69,74.35,73.37,72.64,71.44,59.15,54.57.ESI-HRMS(m/z):calcd.for C32H33NO14[M-H]-,654.1828;found,654.1860.。
实施例2:葡糖苷酸前药18-1的合成
步骤A:发明按如下路线合成化合物15
(3R,4R)-1-(3,4,5-三甲氧基苯基)-3-羟甲基-4-(3-羟基-4-甲氧基苯基)氮杂环丁烷-2-酮(化合物15)
合成试剂和条件:(g)HNO3,AcOH,0℃-rt,12h;(h)Isovanillin,Zn,AcOH,MeOH,N2,0℃-rt,18h;(i)Methyl propiolate,CuI,Et3N,DMF,0℃-rt,overnight;(j)NaBH4,DryTHF,0℃-rt,10h;(k)Boc-L-Proline,EDCI,DMAP,Et3N,CH2Cl2,rt,overnight;(l)NaOH,MeOH,rt,4h.
2.1化合物10的合成
1L的三口烧瓶中加入乙酸(400mL)和浓硝酸(160mL),冰浴冷却后,分批加入3,4,5-三甲氧基苯甲酸(化合物9,100g,0.47mol),室温反应18小时。反应完成,将反应液倒入1L水中,过滤,滤饼水洗白色固体80g,产率80%。产物直接用于下一步反应,无需进一步纯化。
2.2化合物11的合成
化合物10(30g,0.14mol)、异香草醛(21g,0.14mol)和锌粉(9.8g,0.15mol)溶于甲醇(300mL),0℃氮气保护下搅拌30分钟,缓慢加入乙酸,加料结束继续0℃反应2小时,缓慢升至室温继续反应4小时。反应结束,过滤,滤液浓缩,加入乙醇析出固体,过滤出固体为化合物11(15g,粗产率32%)。产物直接用于下一步反应,无需进一步纯化。
2.3化合物12的合成
在氮气保护下,将丙炔酸甲酯(1.07mL,12mmol)和三乙胺(1.67mL,12mmol)溶于干燥N,N-二甲基甲酰胺(30mL),0℃搅拌15min。加入碘化亚铜(2.28g,12mmol),0℃搅拌30min。然后将化合物11(4g,12mmol)加入混合液中,缓慢升至室温,继续反应12小时。反应结束,加入水(50mL),乙酸乙酯(20mL×3)萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,过滤,滤液浓缩,经硅胶柱层析(二氯甲烷/甲醇=100:1)得白色固体2.5g,产率49%。1HNMR(600MHz,DMSO-d6)δ9.13(s,1H),6.97(dd,J=8.3,1.8Hz,1H),6.93(d,J=8.3Hz,1H),6.90(d,J=1.7Hz,1H),6.57(s,2H),5.32(d,J=2.4Hz,1H),4.24(d,J=2.5Hz,1H),3.76(s,3H),3.75(s,3H),3.66(s,6H),3.58(s,3H).13C NMR(151MHz,DMSO-d6)δ166.75,159.03,152.97,148.01,146.70,133.95,132.61,128.25,118.17,113.34,112.03,94.75,61.69,59.92,56.77,55.60,55.42,52.41.
2.4化合物13的合成
化合物12(2.5g,6mmol)和硼氢化钠(342mg,9mmol)溶于干燥四氢呋喃(30mL),氮气保护下室温反应12小时。反应完成,反应液用饱和氯化铵溶液(75mL)淬灭,乙酸乙酯(40mL×3)萃取,有机相无水硫酸钠干燥,滤液浓缩,经硅胶柱层析(乙酸乙酯/石油醚=1:1)纯化得白色固体2.0g,产率86%。1H NMR(600MHz,DMSO-d6)δ9.09(s,1H),6.92(d,J=8.3Hz,1H),6.88(dd,J=8.2,1.6Hz,1H),6.84-6.81(m,1H),6.54(s,2H),5.76(s,1H),5.10(t,J=4.9Hz,1H),4.95(d,J=2.0Hz,1H),3.83-3.78(m,1H),3.75(s,3H),3.64(s,6H),3.57(s,3H),3.22-3.19(m,1H).13C NMR(151MHz,DMSO-d6)δ165.57,152.91,147.49,146.70,133.50,133.39,130.33,117.56,112.95,112.17,94.26,61.59,59.94,56.91,56.45,55.54.
2.5化合物15的合成
化合物13(1.16g,3mmol)溶于二氯甲烷(50mL),加入Boc-L-脯氨酸(1.93g,9mmol)、EDCI(1.73g,9mmol)、DMAP(5.2mg,1.35mmol)和Et3N(1.25mL,9mmol),室温反应过夜。反应结束,加入二氯甲烷(50mL)稀释,饱和食盐水洗涤,无水硫酸钠干燥,过滤,滤液浓缩。对映体混合物经柱层析分离(乙酸乙酯/石油醚=1:3),然后在氢氧化钠甲醇溶液中酯解得白色固体500mg,产率43%。=15.90(c 1.00,CHCl3).1H NMR(600MHz,DMSO-d6)δ9.07(s,1H),6.91(d,J=8.3Hz,1H),6.86(dd,J=8.2,1.8Hz,1H),6.81(d,J=1.6Hz,1H),6.52(s,2H),5.74(s,1H),5.08(t,J=5.0Hz,1H),4.94(d,J=2.1Hz,1H),3.82-3.76(m,1H),3.73(s,3H),3.62(s,6H),3.56(s,3H),3.19(q,J=3.8Hz,1H).13C NMR(151MHz,DMSO-d6)δ165.57,152.92,147.49,146.71,133.50,133.40,130.33,117.56,112.96,112.17,94.26,61.59,59.93,56.91,56.46,55.54,55.45.
步骤B:发明按如下路线合成目标化合物18-1:
合成试剂:(n)K2CO3,DMF,N2,rt,8h;(o)1)MeONa,MeOH,N2,0℃,1h;2)NaOH,Acetone,0℃,2h.
2.6化合物17-1的合成
化合物4(110mg,0.22mmol)溶于N,N-二甲基甲酰胺(1mL),依次加入碳酸钾(54mg,0.4mmol)和化合物15(40mg,0.1mmol),氮气保护室温反应18小时。反应结束,加入水(10mL),乙酸乙酯(10mL×3)萃取,有机相饱和食盐水洗涤,无水硫酸钠干燥,滤液浓酸,经硅胶柱层析(乙酸乙酯/石油醚=1:1)分离得到白色固体131.8mg,收率74%。1HNMR(600MHz,DMSO-d6)δ7.95(s,1H),7.38(d,J=8.6Hz,1H),7.20(s,1H),7.03(d,J=8.3Hz,1H),6.98(dd,J=8.3,4.3Hz,3H),6.53(s,2H),5.87(s,1H),5.66(d,J=8.0Hz,1H),5.47(t,J=9.6Hz,1H),5.12-5.09(m,1H),5.09-5.04(m,1H),5.02-4.99(m,1H),4.71(d,J=9.9Hz,1H),3.74(s,2H),3.68(s,1H),3.64(s,3H),3.62(s,1H),3.61(d,J=4.6Hz,6H),3.56(s,2H),3.46(s,1H),2.89(s,2H),2.73(s,2H),2.02(s,3H),2.01(s,3H),2.00(s,3H).13C NMR(150MHz,DMSO-d6)δ169.40,169.12,168.86,166.91,165.67,162.12,155.75,152.89,148.95,147.80,133.52,133.35,131.47,129.70,129.52,116.00,112.09,112.00,96.80,94.25,70.91,70.78,70.31,69.30,68.82,61.44,59.92,56.77,56.57,55.50,55.42,55.26,52.42,35.61,30.60,20.06.
2.7(2S,3S,4S,5R,6S)-3,4,5-三羟基-6-(4-((5-((2R,3R)-3(羟甲基)-4-氧代-1-(3,4,5-三甲氧基苯基)氮杂环丁烷-2-基)-2甲氧基苯氧基)甲基)苯氧基)-2H-四氢吡喃-2-羧酸(18-1)的合成
化合物17-1(100mg,0.08mmol)溶于无水甲醇(5mL),0℃搅拌备用,加入30%甲醇钠的甲醇溶液(28μL),缓慢升至室温反应2小时,加入5μL醋酸淬灭,反应液减压浓缩,所得固体丙酮(5mL)溶解,冷却至0℃,加入1M NaOH(1mL)溶液,搅拌30min后,1M HCl(1mL)中和,减压条件下浓缩,冷冻干燥。固体甲醇溶解,过滤,滤液浓缩得到白色固体36.2mg,收率68%。1H NMR(600MHz,CD3OD)δ7.27(d,J=8.5Hz,2H),7.05(s,1H),7.04(d,J=4.4Hz,3H),7.01(d,J=7.9Hz,1H),6.57(s,2H),5.05(s,2H),4.99(d,J=2.1Hz,1H),4.94(d,J=7.0Hz,1H),3.99(dd,J=11.9,4.9Hz,1H),3.93(dd,J=11.9,3.7Hz,1H),3.85(s,3H),3.79(d,J=9.1Hz,1H),3.71(s,3H),3.67(s,6H),3.55-3.53(m,2H),3.37(s,1H),3.22-3.18(m,1H).13C NMR(150MHz,CD3OD)δ174.42,165.96,156.90,152.71,149.53,147.69,133.46,133.12,130.23,129.53,128.19,118.82,115.80,111.88,111.66,100.30,94.08,75.69,74.35,72.68,71.50,69.74,61.46,59.22,57.11,56.79,54.55,54.44.ESI-HRMS(m/z):calcd.for C33H37NO14[M-H]-,670.2141;found,670.2147.。
实施例3:葡糖苷酸前药18-2的合成
3.1化合物16的合成
合成试剂和条件:(m)DAST,Dry CH2Cl2,-78℃-rt,6h.
在25mL的Shlenck管中加入化合物15(200mg,0.51mmol)和干燥二氯甲烷(10mL),冷却到-78℃下搅拌2小时,加入DAST(16mg,1.0mmol),-78℃下反应2小时后缓慢升至室温继续反应12小时。反应完成,反应液浓缩,经硅胶柱层析(乙酸乙酯/石油醚=1:1)分离得白色固体98mg,产率50%。1H NMR(600MHz,DMSO-d6)δ9.13(s,1H),6.94(s,2H),6.88(s,1H),6.56(s,2H),5.05(d,J=2.3Hz,1H),4.84(dt,J=47.3,4.3Hz,2H),3.75(s,3H),3.65(s,6H),3.57(s,4H).13C NMR(151MHz,DMSO-d6)δ163.42(d,J=8.2Hz),152.94,147.73,146.74,133.68,133.03,129.38,117.83,113.06,112.12,94.57,79.27(d,J=165.7Hz),59.93,59.16(d,J=20.3Hz),56.26,55.58,55.45.
3.2(2S,3S,4S,5R,6S)-6-(4-((5-((2R,3S)-3-(氟甲基)-4-氧代-1-(3,4,5-三甲氧基苯基))氮杂环丁烷-2-基)-2-甲氧基苯氧基)甲基)苯氧基)-3,4,5-三羟基-2H-四氢吡喃-2-羧酸(18-2)的合成
合成试剂:(n)K2CO3,DMF,N2,rt,8h;(o)1)MeONa,MeOH,N2,0℃,1h;2)NaOH,Acetone,0℃,2h.
参照化合物18-1的制法,以化合物16替代化合物15,最终得到化合物18-2为白色固体。1H NMR(600MHz,CD3OD)δ7.27(d,J=8.2Hz,2H),7.08-7.02(m,3H),7.03-6.98(m,2H),6.63(s,1H),6.57(s,1H),5.80(s,1H),5.52(s,1H),5.19(s,1H),5.03(s,2H),4.97-4.92(m,1H),3.84(s,3H),3.72(s,1H),3.71(d,J=2.6Hz,3H),3.69(s,3H),3.67(s,3H),3.58-3.50(m,3H),3.37(s,1H).13C NMR(151MHz,CD3OD)δ160.82,156.86,152.81,152.74,149.88,149.03,147.73,132.82,130.16,128.26,128.20,128.10,119.72,119.01,115.79,112.27,111.83,111.65,111.51,109.50,100.30,94.30,94.06,75.67,72.66,69.75,62.96,59.23,54.66,54.37.ESI-HRMS(m/z):calcd.for C33H36FNO13[M-H]-,672.2098;found,672.2097.。
实施例4:葡糖苷酸前药(2S,3S,4S,5R,6S)-3,4,5-三羟基-6-(4-((5-((2R,3R)-3-(羟甲基)-4-氧代-1-(3,4,5-三甲氧基苯基)氮杂环丁烷-2-基)-2-甲氧基苯氧基)甲基)-2-硝基苯氧基)-2H-四氢吡喃-2-羧酸(18-3)的合成
合成试剂与条件:(n)K2CO3,DMF,N2,rt,8h;(o)1)MeONa,MeOH,N2,0℃,1h;2)NaOH,Acetone,0℃,2h.
本发明中前药18-3合成方法同实施例2,用化合物4-1替代化合物4,得目标化合物18-3为白色固体。1H NMR(600MHz,D2O)δ7.59(s,1H),7.39(d,J=9.7Hz,1H),7.24(d,J=8.7Hz,1H),6.96(d,J=8.3Hz,1H),6.85(d,J=8.3Hz,1H),6.71(s,1H),6.13(s,2H),6.03(d,J=4.5Hz,1H),5.74(d,J=2.7Hz,1H),5.06-4.98(m,2H),4.83(s,1H),4.21(s,1H),4.05(s,1H),3.90(m,J=28.2,12.3,4.4Hz,3H),3.71(s,2H),3.49(s,3H),3.46(d,J=8.6Hz,1H),3.42(s,6H),3.27-3.22(m,1H).13C NMR(151MHz,D2O)δ167.28,151.77,148.52,147.98,145.98,143.24,138.26,132.94,132.73,132.23,131.61,128.73,123.54,120.69,119.06,111.58,110.45,106.61,97.14,94.15,67.59,67.13,63.98,59.99,57.37,56.59,55.10,54.88.ESI-HRMS(m/z):calcd.for C33H36N2O16[M-H]-,715.1992;found,715.1977.。
实施例5:酶水解实验
前药(1mg)溶于磷酸盐缓冲液(300μL)中,均匀分成两份,每份150μL,其中一份添加β-葡萄糖醛酸苷酶(0.6μL),另一份作为对照。37℃孵育,10分钟、30分钟、1小时、2小时各取40μL的溶液,加入冷甲醇(160μL),离心,取上清液,进行HPLC分析。所得结果如图1所示。
从图1显示表明,前药18-1在30分钟内逐渐被酶裂解,然后迅速自我消除,释放出活性药物15,2小时内原药释放量达94%。前药18-2在β-葡萄糖醛酸苷酶的存在下更容易水解,10分钟内几乎完全水解,但自毁片段消除所需时间超过前药18-1,2小时内未完全消除。前药18-3在2小时内药物分子释放量最少,2小时内前药8少量裂解。由上述结果可以得出结论,药物结构的微小变化会导致酶解和自消除速率的差异;前药18-1和18-2表现出良好的稳定性,在酶的作用下可恢复生物活性。
实施例6:体外肿瘤细胞增殖抑制能力测试
人乳腺癌细胞株(MDA-MB-231和MCF-7)、人卵巢癌细胞株(SKOV-3)和人宫颈癌细胞株(HeLa)分别以5×104/mL接种于96孔培养板,100μl/孔,96孔板周围用PBS液封,置于37℃、CO2培养箱内培养24小时。待药物完全贴壁后,换成含药物处理的培养液,设调零组,对照组和加药组。加药组分别加入前药18-1和18-2,设置加酶和不加酶两个组别,对照组加入原药15和16,每种药物均设置6个浓度梯度,设立3个复孔,继续培养48小时。培养结束,将MTT试剂加入到96孔板中,终浓度为10μl/孔,继续孵育4小时。吸除孔内培养基,每孔加入100μl DMSO,平板摇床振摇10min。酶联免疫检测仪在波长490nm或者540nm处检测每孔的吸光值,并按下列公式计算化合物对细胞的抑制率,3次初筛结果平均值为其最终抑制率。化合物的半数抑制浓度(IC50值)根据6个浓度的抑制率计算所得,3次重复实验结果为所测化合物的最终IC50值。活性结果如表1所示。
细胞抑制率%=[(空白对照OD值-给药组OD值)/空白对照组OD值]×100%
表1.化合物肿瘤细胞增殖抑制活性(IC50,nM)
由表1表明,在没有β-葡萄糖醛酸苷酶存在的情况下,前药18-1和18-2对测试细胞的存活率影响很小。然而,前药18-1和18-2对β-葡萄糖醛酸苷酶预处理的肿瘤细胞显示出显著的细胞毒功效(IC50=3.53-22.63nM),与相应的母体药物的活性相当。此外,根据QIC50值(QIC50=IC50(-Enz)/IC50(+Enz))大于5000可以发现,前药18-1和18-2的毒性明显低于母体药物。上述结果表明,前药18-1和18-2对正常组织和肿瘤组织具有潜在的选择性。
实施例7:药物小鼠的急性毒性研究
昆明小鼠(体重18-20g)随机分为7组,每组10只(雌雄各半)。各组分别静脉注射母体药物15(12.5、25、50、70、100mg/kg)、前药18-1(500mg/kg)和PBS。每天监测小鼠的死亡情况,并记录注射后14天内小鼠的死亡情况。
表2.化合物15和前药18-1的急性毒性
由表2可知,化合物15的LD50值约为65mg/kg,前药18-1的LD50值大于500mg/kg。表明,前药18-1毒性较低,为癌症治疗提供了一个更广阔的治疗窗。
实施例8:药物体内抗肿瘤活性研究
MDA-MB-231细胞(5×106/mL)皮下注射接种于6周龄Balb/C裸鼠,建立裸鼠移植瘤模型,SPF条件下自然生长。待裸鼠皮下MDA-MB-231移植瘤长至体积约100mm3,将裸鼠随机分成4组,每组6只,隔天分别静脉注射前药18-1(20mg/kg和40mg/kg)化合物15(3.75mg/kg)和空白对照,游标卡尺记录移植瘤生长大小。移植瘤体积按以下公式计算:肿瘤体积(mm3)=a×b2×0.52(a为最长的直径,b为最短的直径,0.52为经验系数)。当空白对照组移植瘤平均体积达到2000mm3后,眼眶取血进行血常规和血清肝指标测定,处死裸鼠,剥离瘤组织,剥离小鼠脏器进行H&E染色。称取瘤质量并计算抑瘤率:
抑瘤率=(1-实验组平均瘤质量/对照组平均瘤质量)×100%
药物对小鼠体内的肿瘤抑制结果如图2,H&E染色结果如图3,血常规结果如表3,肝指标结果如表4所示。
表3.化合物18-1和15给药的小鼠血常规检查
表4.化合物15和前药18-1的血清生化分析
从图2的结果可以看出,前药18-1剂量依赖性地抑制肿瘤生长。H&E染色显示(图3),静脉给药前药18-1和母体药物15,对小鼠主要器官无明显影响。此外,前药18-1治疗组(40mg/kg)和母体药物15治疗组(3.75mg/kg)的血常规指标和肝指标与对照组相比无显著差异(表3,表4)。
实施例9:化合物15和前药18-1的心脏毒性评价
6-8周龄的雌性裸鼠随机分为三组,每组3只,分别静脉注射前药18-1(20mg/kg和40mg/kg)、母体药物15(3.75mg/kg)和PBS(空白组)。连续给药5次,皮下植入电极,右前肢作为阴极,左前肢作为阳极,右后肢作为参考电极,测定其心电图,结果如图4所示,母体药物15诱导QRS波复合体异常(R波呈凹迹,呈双峰R波;QRS波时间变宽,ST波上升),QT间期延长,提示心肌梗死等可能的心脏疾病。相比之下,空白组和前药18-1的心电图无显著差异,表明前药18-1无明显心脏毒性。基于这些结果,我们得出结论,前药18-1在产生治疗作用时具有较高的体内安全性。
Claims (5)
3.以权利要求1或2所述的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药为有效成分的药物组合物。
4.权利要求1或2所述的β-葡萄糖醛酸苷酶响应型微管蛋白聚集抑制剂前药的药物组合物在制备抗肿瘤药物中的用途。
5.根据权利要求4所述的用途,其特征在于所述的肿瘤为乳腺癌、卵巢癌、宫颈癌、前列腺癌、肾癌、膀胱癌、卵巢癌、结直肠癌、胰腺癌、食管癌、肾癌、胃癌、肺癌、肝癌。
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