CN110862403A - 喜树碱-羟基乙酸-去甲斑蝥素结合物及其应用 - Google Patents
喜树碱-羟基乙酸-去甲斑蝥素结合物及其应用 Download PDFInfo
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Abstract
本发明提供了喜树碱‑羟基乙酸‑去甲斑蝥素结合物I:
Description
技术领域
本发明属于新药设计与合成领域,具体涉及一类新型的喜树碱-羟基乙酸-去甲斑蝥素结合物及其抗肿瘤应用。
背景技术
喜树碱是1958年Monroe Ewald和Mansukh Canal发现的一类抗癌喹啉类生物碱,具有广泛的抗癌活性,主要用于结肠癌、卵巢癌、肝癌、骨癌及白血病等多种癌症的治疗。喜树碱是通过它的内酯结构与DNA拓扑异构酶I结合达到抗肿瘤作用。去甲斑蝥素(NCTD)是由斑蝥素去掉分子中2,3-甲基所得到的化合物,它是我国优先人工合成的新型抗癌药物。具有低毒性,而且还有升高白细胞功能。已有研究表明,NCTD可以保护肝细胞免受脂多糖(LPS)导致的损伤;保护肾小管质纤维化;还对人身体有良好的免疫调节功能。所以用去甲斑蝥素作先导物来进行结构修饰为本课题研究方向。
随着癌症死亡率与发病率不断增加,已成为我国十分注重的问题之一。细胞毒类药物仍是目前肿瘤治疗药物的主体,因为它作用于肿瘤细胞同时也作用寻常细胞,对实体瘤疗效不佳、副作用大、有耐药性,考虑到我国肿瘤的发病形势,研发高效、低毒、没耐药的肿瘤药物是非常重要。天然药物及其衍生物是抗肿瘤药物的重要来源之一,通过对天然药物进行结构修饰,仍然是发现临床候选化合物的最佳手段。当前,喜树碱是研究最广泛的天然产品之一,已经有三个喜树碱类化合物被批准用以一些恶性肿瘤的临床医治(伊立替康,拓扑替康和贝洛替康)。另外,喜树碱水溶性差以及在人体生理环境下不稳定的缺点依然存在,在临床中使用时会使人体免疫功能受损。而去甲斑蝥素有免疫调节功能,还会使白细胞增多的特点。经人们对喜树碱结构的研究发现抗肿瘤活性必须基团是E环上的内酯环,但内脂环,易水解,使其失去活性,喜树碱的20位羟基与具有羧酸基团的分子的酯化反应是常用的稳定内酯环的方法,因为在生理条件下其可以断开连接,释放具有内酯结构的喜树碱。
因此本课题以喜树碱作为合成原料利用羟基乙酸为结合中间体,把喜树碱,去甲斑蝥素衍生物链接起来,开发出具有新型抗肿瘤功效的喜树碱-20位酯衍生物,对其进行活性测定及筛选。从而达到一药治多病,一药有多效的目的,既能有喜树碱良好的抗肿瘤功效,又能增加其升高白细胞,提高免疫力的作用,具有较好的开发前景及运用途径。
为了寻找药效更好的抗癌药物候选者,本发明设计了将喜树碱的20位-羟基和去甲斑蝥素通过羟基乙酸连接在一起的结构独特的喜树碱-羟基乙酸-去甲斑蝥素结合物,并设计合成方法高收率制备了目标衍生物。
发明内容
本发明提供了一种新型喜树碱-羟基乙酸-去甲斑蝥素结合物I:
式I中,环上的虚线代表该位置上的键为饱和键或不饱和双键;R1选自C1-C6的烷基、取代烷基、环烷基、苄基或取代苄基;R2选自氢或卤素。
一种优选的实施方式,式I的R1选自式I的R选自C1-C4的烷基、环烷基或苄基;更优选的,式I的R1甲基、乙基、丙基、丁基、环丙基或苄基。
一种优选的实施方式,式I中所述卤素选自氟、氯或溴;优选溴。
另一方面,本发明提供了喜树碱-羟基乙酸-去甲斑蝥素结合物I的合成方法,包括以下步骤:1)喜树碱与苄氧基乙酸在偶联剂和有机碱存在下通过酯化反应得到化合物II;2)化合物II通过催化氢化反应脱除苄基得到化合物III;3)与去甲斑蝥素单酸酯IV在偶联剂和有机碱存在下通过酯化反应得到喜树碱-羟基乙酸-去甲斑蝥素结合物I,合成路线见以下:
其中,合成路线中式I与式IV中R1及R2的选取基团保持一致。
一种优选的实施方式,步骤1)中所述偶联剂选自EDCI、DCC或DIC;步骤1)中所述有机碱选自三乙胺、二异丙基胺、DMAP或DABCO;所述溶剂选自二氯甲烷或氯仿。
一种优选的实施方式,步骤2)中催化氢化反应的催化剂选自Pd/C,Pd(OH)2/C或Pt/C。
一种优选的实施方式,步骤3)中所述偶联剂选自EDCI、DCC或DIC;步骤1)中所述有机碱选自三乙胺、二异丙基胺、DMAP或DABCO;所述溶剂选自二氯甲烷或氯仿。
在上述合成路线中,反应溶剂可依据反应对温度、溶剂极性的需求,从N,N-二甲基甲酰胺、二甲基亚砜、二氯甲烷、氯仿、四氢呋喃或异丙醚中选取。
反应温度可依据反应类型适当选取。
反应时间可通过薄层层析TLC、高效液相色谱法HPLC或LC-MS液相质谱联用等监控手段追踪反应情况得出。
本申请文本中,常见的化学试剂或溶剂缩写包括:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(缩写EDCI)、N,N-二环己基碳二亚胺(缩写DCC)、N,N-二异丙基碳二亚胺(缩写DIC)、4-二甲氨基吡啶(缩写DMAP)、1,4-二氮杂二环[2.2.2]辛烷(缩写DABCO)、从N,N-二甲基甲酰胺(缩写DMF)、二甲基亚砜(缩写DMSO)、二氯甲烷(缩写DCM)等。
活性测试证明,本发明设计并合成得到喜树碱-羟基乙酸-去甲斑蝥素结合物I具有很好的抗肝癌效果。因此,第三方面,本发明提供了喜树碱-羟基乙酸-去甲斑蝥素结合物I用于制备抗肿瘤药物的用途;优选地,用于制备抗肝癌、胃癌、结肠癌和胰腺癌药物的用途。
本发明的有益之处在于:本发明提供了喜树碱-羟基乙酸-去甲斑蝥素结合物I,该衍生物整合了喜树碱与去甲斑蝥素衍生物两类活性药物片段,为新型双靶点肿瘤抑制剂。经活性测试证明该喜树碱-羟基乙酸-去甲斑蝥素结合物具有良好的抗肿瘤效果,尤其是肝癌、胃癌、结肠癌和胰腺癌活性高。此外,本发明制备喜树碱-羟基乙酸-去甲斑蝥素结合物I的方法,原料易得,成本低廉,合成反应目标产物收率高;易于制备得到。
具体实施方式
以下将通过具体实施例进一步阐述本发明,但并不用于限制本发明的保护范围。在不脱离本发明构思的前提下,本领域技术人员可对权利要求的各参数或条件做出的改进或组合,这些改进或组合也应视为本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。本发明中使用的溶剂及试剂来自国药集团上海试剂公司。除特别说明外,所用试剂均为化学纯。
实施例1、制备喜树碱-羟基乙酸中间体III
(1).制备化合物II
将喜树碱(1g,3.45mmol)和CH2Cl2(35ml)投入圆底烧瓶中,在常温下搅拌,得黄色悬浮液,依次缓慢加入DMAP(500mg,4.1mmol,1.2eq),苄氧乙酸(1mL,6.02mmol,1.74eq)和DIPC(5mL,39.68mmol,13eq),混合物继续在常温下搅拌反应。TLC点板追踪,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应12h后停止,过滤,滤饼干燥得目标产物II(1.08g,产率100%),为黄色固体,Rf=0.6(CH2Cl2:CH3OH=97:3)。产物直接投入下一步反应,无需进一步纯化。
(2).制备化合物III
取反应物II(200mg,0.4m mol)投入圆底瓶中,加入CH3OH 10ml,再加入10%Pd/C(50mg,0.5mmol);反应瓶抽真空后,通入H2并在30℃室温下搅拌反应24时,TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止,过滤除去催化剂,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物III(116mg,产率72%)。M.p.159-160℃,Rf=0.3(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.69(s,1H),8.14(dd,J=8Hz,8Hz,2H),7.85(t,J=8Hz,1H),7.70(t,J=8Hz,1H),7.08(s,1H),5.49(s,1H),5.29(s,1H),4.33(d,J=16Hz,1H),4.16(d,J=16Hz,1H),3.42(q,J=8Hz,2H),2.07-2.17(m,2H),0.95(dt,J=8Hz,8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=172.17,167.69,157.19,156.98,152.77,148.29,146.41,145.81,132.06,130.89,130.27,129.33,128.99,128.77,128.76,128.43,128.25,128.24,128.21,128.17,119.26,95.43,76.29,66.65,59.78,50.70,30.56,23.73,7.99.
实施例2、制备中间体IV
按照论文(题为“喜树碱去甲斑蝥素酯的合成及初步体外抗肿瘤活性评价”,中国新药杂志,2018,27(8):99-107)公开的合成方法,可以容易地合成中间体IV:顺丁烯二酸酐与呋喃在***反应通[4+2]环加成反应立体选择性地得到5-烯去甲斑蝥素1;化合物1在Pd/C催化作用下,加氢得到去甲斑蝥素2;同时,化合物1在氯仿条件下,通过液溴加成以较高收率得到5,6-二溴去甲斑蝥素3;化合物1、2和3在三乙胺碱催化作用下,醇解以较高收率得到相应的5-烯去甲斑蝥素单酸单酯的侧链IVa-i(参考以下合成路线)。
实施例3、制备喜树碱-羟基乙酸-去甲斑蝥素结合物I
(1).制备化合物Ia
取反应物III(100mg,0.25mmol)、反应物IVa(200mg,1.01mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)置于50ml封管,在50℃油浴中搅拌反应。TLC点板跟踪反应,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应6h停止,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Ia(108mg,74%).M.p.128-129℃,Rf=0.41(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.66(s,1H),8.11(dd,J=8Hz,8Hz,2H),7.83(t,J=8Hz,1H),7.68(t,J=8Hz,1H),7.15(d,J=8Hz,1H),6.42(s,2H),5.26(s,1H),5.09(d,J=12Hz,1H),5.03(dd,J=4Hz,4Hz,1H),4.90(s,1H),3.56-3.64(m,4H),3.44(s,3H),2.84(dd,J=8Hz,8Hz,2H),2.15(q,J=8Hz,2H),0.92(t,J=8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=171.87,171.84,171.04,171.03,167.23,167.18,157.20,156.91,152.75,148.31,145.21,136.80,131.89,130.83,130.21,129.32,128.95,128.40,128.12,119.34,96.21,80.50,80.20,77.20,77.17,66.74,52.13,52.00,50.67,46.68,46.34,23.68,7.93.IR(KBr)ν(cm-1)=3442,2924,2853,1750,1720,1668,1618,1457,1401,1380,1221,1175,838,764,732.HRMS(ESI-TOF):587.1690[M+1].
(2).制备化合物Ib
取反应物III(100mg,0.25mmol)、反应物IVb(200mg,0.94mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)投于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止后,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Ib(120mg,81%)。M.p.135-136℃,Rf=0.52(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.67(s,1H),8.12(dd,J=8Hz,8Hz,2H),7.84(t,J=8Hz,1H),7.69(t,J=8Hz,1H),7.16(d,J=8Hz,1H),6.41(d,J=8Hz,1H),5.27(s,1H),5.29(s,1H),5.05(dd,J=20Hz,8Hz,2H),4.81(dd,J=20Hz,20Hz,1H),3.81-4.02(m,2H),3.56-3.64(m,4H),2.83(dd,J=8Hz,8Hz,2H),2.14(q,J=8Hz,2H),0.90(t,J=8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=171.30,171.00,167.24,157.21,156.92,152.79,148.32,146.52,145.21,137.24,136.79,132.04,130.85,130.18,129.27,128.97,128.40,128.13,119.21,95.63,80.23,77.29,66.70,60.68,50.70,46.22,30.64,23.72,14.17,7.93.IR(KBr)ν(cm-1)=3441,3060,2982,2940,1754,1668,1620,1561,1501,1457,1403,1232,1157,1049,811,784.HRMS(ESI-TOF):601.1847[M+1].
(3).制备化合物Ic
取反应物III(100mg,0.25mmol)、反应物IVc(200mg,0.73mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)投于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止后,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Ic(114mg,70%)。M.p.125-126℃,Rf=0.51(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.58(s,1H),8.10(dd,J=8Hz,8Hz,2H),7.82(t,J=8Hz,1H),7.66(t,J=8Hz,1H),7.19-7.31(m,4H),7.14(d,J=8Hz,2H),7.02(d,J=2Hz,1H),6.41(s,1H),4.93-5.21(m,4H),4.75(dd,J=20Hz,12Hz,2H),3.60(q,J=8Hz,2H),2.88(dd,J=8Hz,8Hz,1H),2.80(d,J=8Hz,1H),0.91(d,J=8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=171.34,171.29,171.19,170.93,170.87,167.35,167.12,157.22,155.88,152.60,148.29,146.40,154.31,130.81,128.77,128.68,128.54,128.50,119.19,95.59,80.52,80.30,77.24,66.52,66.44,60.68,45.42,41.09,40.58,40.16,39.74,39.53,30.57,7.95.IR(KBr)ν(cm-1)=3432,2921,2854,1753,1663,1618,1560,1402,1154,1058,997,760,723,699.HRMS(ESI-TOF):663.2001[M+1].
(4).制备化合物Id
取反应物III(100mg,0.25mmol)、反应物IVd(200mg,1.00mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)投于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止后,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Id(120mg,83%)。M.p.197-198℃,Rf=0.5(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.36(s,1H),8.20(d,J=8Hz,1H),7.90(d,J=8Hz,1H),7.80(t,J=8Hz,1H),7.63(t,J=8Hz,1H),7.23(d,J=2Hz,1H),5.64(dd,J=20Hz,4Hz,1H),5.37(d,J=20Hz,1H),5.24(s,1H),4.85-4.90(m,1H),4.93(d,J=8Hz,1H),4.72(dd,J=16Hz,16Hz,1H),4.16(d,J=2Hz,2H),3.81(dd,J=8Hz,8Hz,1H),3.51-3.60(s,3H),3.00(dd,J=12Hz,4Hz,2H),2.93(d,J=8Hz,2H),2.28(dd,J=8Hz,8Hz,1H),2.16(dd,J=8Hz,8Hz,1H),1.69-1.77(m,2H),1.45(dd,J=8Hz,8Hz,2H),0.95(t,J=8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=171.26,171.23,170.26,170.21,166.97,166.84,166.75,166.69,157.22,156.99,152.19,152.13,148.80,148.77,146.49,146.40,145.08,144.91,131.19,130.67,130.63,129.62,128.38,128.33,128.14,128.12,128.04,120.20,96.04,78.52,78.44,78.42,78.37,76.86,76.82,67.20,67.12,60.65,60.39,52.17,52.04,51.68,51.45,49.94,42.08,31.80,31.76,29.67,29.00,28.98,28.94,28.85,23.47,22.67,14.11,7.50.IR(KBr)ν(cm-1)=3442,2952,2923,2853,1753,1712,1666,1619,1560,1501,1456,1402,1297,1230,1150,1057,1003,816,787,723.HRMS(ESI-TOF):589.1848[M+1].
(5).制备化合物Ie
取反应物III(0.25mmol)、反应物IVe(200mg,0.93mmol)、EDCI(100mg,1.04mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)投于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Ie(121mg,82%)。M.p.141-143℃,Rf=0.52(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.66(s,1H),8.14(d,J=8Hz,1H),8.09(d,J=8Hz,1H),7.82(t,J=8Hz,1H),7.67(t,J=8Hz,1H),7.13(t,J=8Hz,1H),5.27(s,2H),4.93(dd,J=8Hz,8Hz,1H),4.77(dd,J=8Hz,8Hz,1H),4.54-4.71(m,2H),3.76-4.0(m,2H),3.13(d,J=8Hz,1H),3.02(d,J=8Hz,1H),2.08-2.18(m,1H),1.39-1.55(m,4H),1.1(dt,J=8Hz,8Hz,3H),0.90(t,J=8Hz,3H),0.8(q,J=8Hz,2H).13C NMR(100MHz,DMSO-d6)δ=170.88,170.54,167.16,157.22,156.91,152.71,148.37,146.47,146.27,132.01,131.95,130.80,130.75,129.35,128.94,128.68,128.22,128.15,128.09,128.07,119.30,95.55,79.57,78.40,78.31,78.13,77.12,76.73,72.67,66.71,60.88,60.57,60.48,60.40,52.31,51.58,51.48,51.30,50.62,41.08,39.96,39.75,30.69,29.48,28.87,28.75,23.71,22.53,14.43,14.28,14.14,7.96.IR(KBr)ν(cm-1)=3448,3061,2957,2823,1753,1664,1616,1566,1501,1458,1401,1296,1261,1232,1191,1142,1085,1056,801,724,560.HRMS(ESI-TOF):603.1986[M+1].
(6).制备化合物If
取反应物III(100mg,0.25mmol)、反应物IVf(200mg,0.72mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)投于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止后,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物If(145mg,89%)。M.p.127-129℃,Rf=0.53(DCM:CH3OH=97:3).1H NMR(400MHz,CDCl3)δ=8.23(s,1H),7.90(dd,J=8Hz,8Hz,1H),7.80(t,J=8Hz,1H),7.64(t,J=8Hz,1H),7.21-7.31(m,5H),7.15(t,J=8Hz,1H),6.94(d,J=2Hz,1H),5.66(t,J=16Hz,1H),5.26(dd,J=4Hz,4Hz,1H),5.03(s,1H),4.95(s,1H),4.89(d,J=8Hz,1H),4.87(d,J=8Hz,2H),4.72(d,J=4Hz,1H),4.65(d,J=4Hz,1H),3.59(t,J=8Hz,1H),3.08(d,J=8Hz,1H),2.96(d,J=8Hz,1H),2.1-2.31(m,2H),1.65-1.81(m,4H),0.98(q,J=8Hz,3H).13C NMR(100MHz,CDCl3)δ=174.70,172.15,170.80,170.63,170.15,166.93,166.88,166.80,166.75,157.32,157.25,152.10,151.86,148.73,148.67,146.43,146.32,145.81,145.23,135.54,135.50,135.15,131.28,131.05,130.76,130.72,129.62,129.58,128.66,128.59,128.55,128.51,128.49,128.39,128.36,128.34,128.28,128.26,128.20,128.17,128.15,128.10,129.09,128.06,120.15,119.66,96.41,96.33,80.70,78.49,77.85,67.12,67.06,66.90,66.84,66.71,60.53,60.18,52.13,51.83,51.68,51.37,51.06,50.90,50.07,49.94,31.80,31.58,29.68,29.19,28.95,28.85,25.77,22.68,14.12,7.56,7.50.IR(KBr)ν(cm-1)=3445,2954,2882,1752,1661,1616,1499,1455,1385,1351,1295,1175,1054,1001,929,752,699,593.HRMS(ESI-TOF):600.1997[M+1].
(7).制备化合物Ig
取反应物III(100mg,0.25mmol)、反应物IVg(200mg,0.56mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)投于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Ig(138mg,75%)。M.p.137-138℃,Rf=0.42(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.39(s,1H),8.23(dd,J=8Hz,8Hz,1H),7.93(d,J=8Hz,1H),7.82(t,J=8Hz,1H),7.66(t,J=8Hz,1H),7.33(d,J=8Hz,1H),5.68(t,J=16Hz,1H),5.40(dd,J=16Hz,8Hz,1H),5.28(s,2H),4.81-5.03(m,2H),4.57-4.68(m,2H),4.29(s,3H),3.68-3.75(m,2H),2.14-2.31(m,2H),1.11-1.14(m,2H),0.97(t,J=8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=176.03,164.39,147.04,143.63,140.24,131.60,126.93,126.83,126.76,126.04,125.52,124.37,123.58,123.37,123.28,123.25,123.22,123.02,122.98,122.96,122.87,122.83,115.11,109.99,90.74,68.12,62.00,61.39,44.77,26.60,24.48,8.90,2.23.IR(KBr)ν(cm-1)=3446,2936,2854,1753,1664,1617,1565,1500,1455,1437,1402,1285,1232,1165,1056,995,947,899,761,723.
(8).制备化合物Ih
取反应物III(100mg,0.25mmol)、反应物IVh(200mg,0.54mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止后,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Ih(148mg,79%)。M.p.138-139℃,Rf=0.51(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.63(s,1H),8.10(dd,J=8Hz,8Hz,2H),7.83(t,J=8Hz,1H),7.68(t,J=8Hz,1H),7.14(dd,J=8Hz,2Hz,1H),5.23(d,J=4Hz,2H),4.94(t,J=8Hz,1H),4.83(dd,J=8Hz,8Hz,1H),4.69(dd,J=20Hz,4Hz,1H),4.51(dd,J=8Hz,8Hz,1H),3.98-4.06(m,2H),3.58-3.63(m,2H),3.47-3.53(m,1H),3.22(t,J=8Hz,1H),2.16(t,J=8Hz,2H),1.06-1.16(m,3H),0.91(t,J=8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=171.30,170.49,170.43,169.56,169.17,167.35,167.18,167.07,167.01,157.20,156.88,152.74,152.70,148.35,148.30,146.51,146.43,145.28,145.04,137.73,132.05,132.01,131.98,131.95,131.92,131.91,130.90,130.88,130.84,130.23,130.18,130.15,129.37,129.35,129.33,129.30,128.95,128.76,128.74,128.59,128.47,128.38,128.24,128.20,128.16,128.14,119.39,119.18,95.54,95.50,90.64,88.70,88.62,86.74,77.54,77.32,77.28,76.79,72.67,66.76,66.75,66.72,66.65,66.64,61.88,61.68,60.18,54.34,54.19,54.00,51.96,51.85,50.64,49.12,49.04,48.99,48.65,30.76,30.56,30.45,21.19,14.51,14.34,14.21,14.06,8.00.IR(KBr)ν(cm-1)=3443,2977,2935,1753,1664,1617,1565,1500,1545,1402,1294,1231,1167,1054,1013,947,888,761.HRMS(ESI-TOF):761.0163[M+1].
(9).制备化合物Ii
取反应物III(100mg,0.25mmol)、反应物Ii(200mg,0.46mmol)、EDCI(100mg,0.52mmol)、DMAP(20mg,0.16mmol)、CH2Cl2(15mL)投于50mL封管,在50℃油浴中搅拌反应6h。TLC点板,展开剂(CH2Cl2:CH3OH=97:3)在紫外灯254nm下观察。反应停止,用NaHCO3水溶液(7mL)于分液漏斗中对反应液进行洗涤,留下有机相,干燥15min,抽滤,将滤液旋干,粗产品采用洗脱剂(CH2Cl2:CH3OH=97:3)进行柱层析纯化得黄色固体产物Ii(162mg,80%)。M.p.123-124℃,Rf=0.53(DCM:CH3OH=97:3).1H NMR(400MHz,DMSO-d6)δ=8.33(s,1H),8.21(t,J=8Hz,1H),7.90(dd,J=8Hz,8Hz,1H),7.80(t,J=8Hz,1H),7.64(t,J=8Hz,1H),7.24-7.29(m,4H),7.16(t,J=8Hz,1H),6.94(d,J=8Hz,1H),5.63(t,J=8Hz,1H),5.34(dd,J=8Hz,8Hz,1H),5.18(d,J=4Hz,1H),5.04(d,J=8Hz,1H),4.95(d,J=8Hz,1H),4.85(d,J=8Hz,1H),4.75(d,J=8Hz,1H),4.70(d,J=8Hz,1H),4.24(dt,J=8Hz,1H),3.91(dd,J=8Hz,8Hz,1H),3.80(q,J=8Hz,1H),3.04(dd,J=40Hz,4Hz,1H),2.20-2.29(m,2H),2.09-2.15(m,2H),0.99(t,J=8Hz,3H).13C NMR(100MHz,DMSO-d6)δ=169.15,169.14,168.90,168.76,166.91,166.86,166.42,166.38,157.24,157.18,152.13,151.90,148.72,148.66,146.46,146.36,145.60,145.08,140.92,134.99,134.65,131.24,131.02,130.76,130.72,129.59,129.55,128.81,128.64,128.60,128.59,128.50,128.44,128.42,128.36,128.32,128.29,128.18,128.17,128.09,128.06,127.56,126.96,120.04,119.62,96.11,96.03,86.34,86.29,82.48,82.47,77.25,77.15,76.91,67.51,67.37,67.12,66.09,65.24,60.85,60.57,53.60,53.58,53.46,52.64,49.98,49.86,49.68,49.48,46.55,46.24,31.78,31.54,29.69,7.53.IR(KBr)ν(cm-1)=3451,2939,2884,1753,1664,1617,1562,1500,1455,1385,1349,1231,1165,1056,946,811,756.HRMS(ESI-TOF):823.0324[M+1].
实施例3.溶解度实验
选择合成得到的化合物Ia以及母体化合物喜树碱喜树碱,溶于25℃的氯仿,溶解度结果列于表1.
表1.化合物Ia及喜树碱在25℃氯仿中的溶解度
化合物Ia在氯仿中的溶解度是喜树碱的数倍。可见,合成系列产物I在有机溶剂中的溶解度要比喜树碱好很多。
实施例4.生物活性测试实验
细胞株和溶剂
肿瘤细胞株:人肝癌细胞HEPG2,人胃癌细胞BGC803,人结肠癌细胞SW480,人胰腺癌细胞PANC-1细胞培养于含10%胎牛血清的RPMI1640中培养基
溶剂:二甲亚砜(简称为DMSO)。
MTT法检测细胞抗肿瘤活性实施方案
本试验以斑蝥素为阳性对照,DMSO溶剂为空白对照,进行了浓度为50μmol/mL喜树碱-羟基乙酸-去甲斑蝥素结合物对肝癌细胞HEPG2、人胃癌细胞BGC803、结肠癌细胞SW480和胰腺癌PANC-1四种肿瘤细胞的抑制活性测试。在药物处理72h后,用标准MTT测定法评估使用喜树碱-羟基乙酸-去甲斑蝥素结合物I的抑制率。
表2、喜树碱-羟基乙酸-去甲斑蝥素结合物I对于四种肿瘤细胞的抑制活性
atest solvent is DMSO.
通过两次偶联反应,将喜树碱、羟基乙酸和功能化去甲斑蝥素构建成结合物,并以较好的收率得到一系列新型喜树碱-羟基乙酸-去甲斑蝥素结合物I。测试了合成得到的结合物I对于癌细胞体外抑制活性,尤其是,对于人肝癌细胞HEPG2、胃癌细胞BGC803、结肠癌细胞SW480和胰腺癌PANC-1四种癌细胞系有较强的抑制活性;可将其用于制备相应抗肿瘤候选药物。
Claims (9)
2.根据权利要求1所述的喜树碱-羟基乙酸-去甲斑蝥素结合物I,其中,式I的R选自C1-C4的烷基、环烷基或苄基。
3.根据权利要求2所述的喜树碱-羟基乙酸-去甲斑蝥素结合物I,其中,式I的R选自甲基、乙基、丙基、丁基、环丙基或苄基。
5.根据权利要求4所述的喜树碱-羟基乙酸-去甲斑蝥素结合物I的合成方法,其中,步骤1)中所述偶联剂选自EDCI、DCC或DIC;步骤1)中所述有机碱选自三乙胺、二异丙基胺、DMAP或DABCO;所述溶剂选自二氯甲烷或氯仿。
6.根据权利要求4所述的喜树碱-羟基乙酸-去甲斑蝥素结合物I的合成方法,其中,步骤2)中催化氢化反应的催化剂选自Pd/C,Pd(OH)2/C或Pt/C。
7.根据权利要求4所述的喜树碱-羟基乙酸-去甲斑蝥素结合物I的合成方法,其中,步骤3)中所述偶联剂选自EDCI、DCC或DIC;步骤1)中所述有机碱选自三乙胺、二异丙基胺、DMAP或DABCO;所述溶剂选自二氯甲烷或氯仿。
8.根据权利要求1-3任意所述的喜树碱-羟基乙酸-去甲斑蝥素结合物I用于制备抗肿瘤药物的用途。
9.根据权利要求7所述的用途,其中,所述肿瘤选自肝癌、胃癌、结肠癌或胰腺癌。
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