CN114213490B - 一类三环核苷酸类似物及其合成方法和应用 - Google Patents
一类三环核苷酸类似物及其合成方法和应用 Download PDFInfo
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Abstract
本发明属于化学合成领域,具体地,涉及式(I)、(II)、(III)和(IV)所示的三环核苷酸类似物及其合成方法和应用。所述三环核苷酸类似物的结构式如式(I)、(II)、(III)和(IV):式(I)、(II)、(III)和(IV)化合物是一类含有三元氮杂稠环骨架的化合物。该合成使用对环境相对友好的AcOH作为溶剂,Ac2O为添加剂,1,1,3,3‑四甲氧基丙烷连接鸟苷或2’‑脱氧鸟苷上的亚氨基并发生环合,从而构建了一系列结构新颖的三环核苷酸类似物。实验过程操作简单、方便、收率高、化学选择性好,能高效地构建了一系列结构新颖的三环核苷酸类似物,为此类化合物的合成提供了新的方法与思路。
Description
技术领域
本发明属于化学合成领域,具体地,涉及式(I)、(II)、(III)和(IV)所示的三环核苷酸类似物及其合成方法和应用。
背景技术
核苷酸(nucleotide,NT)是构成生命遗传信息的基础物质,由嘌呤碱基或嘧啶碱基、核糖或脱氧核糖以及磷酸三种物质组成,是合成生物大分子核糖核酸(ribonucleicacid,RNA)及脱氧核糖核酸(deoxyribonucleic acid,DNA)的基本组成单位。根据磷酸化程度的不同,可将核苷酸分为3种:核苷单磷酸、双磷酸和三磷酸。RNA中主要有四种类型的核苷三磷酸:腺嘌呤核苷酸(ATP)、鸟嘌呤核苷酸(GTP)、胞嘧啶核苷酸(CTP)和尿嘧啶核苷酸(UTP),DNA中主要有四种类型脱氧核苷三磷酸:腺嘌呤脱氧核苷酸(dATP)、鸟嘌呤脱氧核苷酸(dGTP)、胞嘧啶脱氧核苷酸(dCTP)和胸腺嘧啶脱氧核苷酸(dTMP),它们是由各自相应的核苷酸在细胞激酶的作用下进一步磷酸化而成。
核苷酸在体内分布广泛,体内的核苷酸主要由机体细胞自身合成,细胞中主要以5′-核苷酸形式存在。体内核苷酸的合成存在两种合成途径—从头合成(denovosynthesis)和补救合成(salvage synthesis),从头合成途径的合成原料有磷酸核糖、氨基酸、一碳单位及CO2等,经过一系列酶促反应合成核苷酸。补救合成途径中,体内核苷酸降解产生的游离嘌呤/嘧啶碱基或核苷被重新利用以合成核苷酸。
通常,内源性核苷酸通过细胞膜上特定的载体进入细胞,随后在细胞内激酶的作用下逐步磷酸化,生成具有生物活性的核苷三磷酸类似物,这些活化后的核苷酸类似物可竞争性抑制病毒编码的DNA或RNA聚合酶,抑制细胞的生长。一般情况下,未经修饰的核苷酸类似物在生理条件下带负电而显示出极性,使其不易穿过细胞膜,在体内易受磷酸酶的降解而脱磷酸,为改善和解决上述问题,研究人员开发了多种合成方法,如通过在核苷酸类似物上修饰亲脂性基团后,以确保其不易被血液中的磷酸酶降解,同时能模仿核苷酸的生理学性质,参与到生物体内的代谢过程,嵌入到DNA及RNA中阻断其延伸复制,抑制细胞的生长和增殖,最终实现高效的抗肿瘤作用。
发明内容
本发明的目的在于提供一类三环核苷酸类似物及其合成方法和应用。为了实现上述目的,本发明的技术方案之一是:一类三环核苷酸类似物,其结构式如下列式(I)、(II)、(III)或(IV)所示:
其中,Ph是指苯基;
式(I):R1是苯氧基,R2是苯氧基或叉丙基保护羧基的丙氨酸基;
式(II):R1是乙酰氧基或氢;
式(IV):R1是苯氧基时,R2是苯氧基或叉丙基保护羧基的丙氨酸基;
上述化合物的三环骨架为平面型刚性结构。
具体的,所述三环核苷酸类似物的结构式如下列化合物7a、8a、9a、9b、10、11或12所示:
其中,Ph是指苯基。
本发明的技术方案之二是:三环核苷酸类似物的合成方法,具体包括以下步骤:(1)以化合物3a或4a和1,1,3,3-四甲氧基丙烷(TMOP)为原料,Ac2O为添加剂,溶剂为AcOH,平行反应仪90℃反应2h,得到化合物7a或8a。1,1,3,3-四甲氧基丙烷(TMOP)连接鸟苷上的亚氨基并发生环合,从而构建了一系列结构新颖的三环核苷酸类似物。
以化合物3a或4a为原料合成的反应式如路线1所示:
路线1
其中,R1=R2=苯氧基,是化合物3a;R1=苯氧基,R2=叉丙基保护羧基的丙氨酸基,是化合物4a。
反应条件:TMOP(1.2equiv),Ac2O(10.0equiv),AcOH,90℃,2h.
进一步,步骤(1):化合物3a或4a:TMOP:添加剂:溶剂的用量关系为1mmol:1.2mmol:10mmol:4mL。
(2)以化合物6a或6b和1,1,3,3-四甲氧基丙烷(TMOP)为原料,Ac2O为添加剂,溶剂为AcOH,平行反应仪90℃反应1h,得到化合物9a或9b。1,1,3,3-四甲氧基丙烷(TMOP)连接脱氧鸟苷上的亚氨基并发生环合,从而构建了一系列结构新颖的三环核苷酸类似物。
其中,以化合物6a为原料合成的反应式如路线2所示:
路线2
反应条件:TMOP(1.2equiv),Ac2O(10.0equiv),AcOH,90℃,1h.
其中,以化合物6b为原料合成的反应式如路线3所示:
路线3
反应条件:TMOP(1.2equiv),Ac2O(10.0equiv),AcOH,90℃,1h.
进一步,步骤(1):化合物6a或6b:TMOP:添加剂:溶剂的用量关系为1mmol:1.2mmol:10mmol:4mL。
进一步,为了得到更多的三环核苷酸类似物,还包括步骤(3):将步骤(1)所得产物通过加入三氟乙酸(TFA)脱保护,将丙叉基保护的双羟基水解成羟基,即得;具体为:
以步骤(1)所得产物化合物7a或化合物8a为原料,溶剂为三氟乙酸,在常温下反应1h,即得化合物10或11。
进一步,步骤(3):化合物7a或化合物8a:溶剂的用量关系为1mmol:10mL。
以化合物7a或化合物8a为原料合成的反应式如路线4所示:
路线4
反应条件:TFA,rt,1h.
进一步,为了得到更多的三环核苷酸类似物,还包括步骤(4):通过加入三乙胺(Et3N)水解,将乙酰氧基水解成羟基,即得;具体为:
将步骤(2)所得产物化合物9a为原料,Et3N为添加剂,溶剂为甲醇,在常温下反应12h,即得化合物12。
进一步,步骤(4):化合物9a:添加剂:溶剂的用量关系为1mmol:11mmol:10mL。
以化合物9a为原料合成的反应式如路线5所示:
路线5
反应条件:Et3N(11.0equiv),MeOH(1mL),rt,12.0h.
为了确保产物的品质,步骤(1)-(5)还包括对反应结束后所得反应液进行后处理的步骤,
步骤(1):浓缩反应液,通过柱层析分离纯化,分离出目标产物;其中:
分离化合物7a所用洗脱剂为DCM:MeOH=8:1(v/v)的二氯甲烷和甲醇混合液;分离化合物8a所用洗脱剂为DCM:MeOH=15:1(v/v)的二氯甲烷和甲醇混合液;
步骤(2):浓缩反应液,以DCM:MeOH=15:1(v/v)的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物;
步骤(3)、(4):浓缩反应液,以DCM:MeOH=10:1(v/v)的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物。
进一步,步骤(1)中化合物3a或4a的制备方法,包括以下步骤:
S1.将鸟苷溶于丙酮中,再向其中加入对甲苯磺酸一水合物(PTSA)和2,2-二甲氧基丙烷,常温下,反应12h,得到化合物2。
以鸟苷(即化合物1)为原料合成的反应式如路线Y1所示:
路线Y1
进一步,步骤S1:鸟苷:PTSA:2,2-二甲氧基丙烷:溶剂的用量关系为1mmol:1mmol:25mmol:(1-5)mL。
S2.将化合物2溶于DCM和DMF的混合溶剂中(其中DCM和DMF的体积比为1:1),在0℃下向体系中加入氯磷酸二苯酯和三乙胺,常温下反应24h,得到化合物3a。
以化合物2为原料合成的反应式如路线Y2所示:
路线Y2
进一步,步骤S2:化合物2:氯磷酸二苯酯:三乙胺:混合溶剂的的用量关系为1mmol:2mmol:5mmol:(1-5)mL。
S3.将化合物2溶于THF中,接着缓慢向体系中加入1.0mol/L叔丁基氯化镁的THF溶液,搅拌,再缓慢向体系中加入N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯,常温下反应18h,得到化合物4a。
以化合物2为原料合成的反应式如路线Y3所示:
路线Y3
进一步,步骤S3:化合物2:叔丁基氯化镁:N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯:溶剂的用量关系为1mmol:2.1mmol:1.2mmol:(2-10)mL。
进一步,步骤(2)、(3)中化合物6a或6b的制备方法,包括以下步骤:
S4.将2’-脱氧鸟苷溶于吡啶中,接着缓慢向体系中加入1.0mol/L叔丁基氯化镁的THF溶液,搅拌,再缓慢向体系中加入N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯,常温下反应18h,得到化合物6a或6b。
以2’-脱氧鸟苷(即化合物5)为原料合成的反应式如路线Y4所示:
路线Y4
进一步,步骤S4:化合物5:叔丁基氯化镁:N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯:溶剂的的用量关系为1mmol:2.1mmol:1.2mmol:(2-10)mL。
为了确保产物的品质,步骤S1-S4还包括对反应结束后所得反应液进行后处理的步骤,
步骤S1:浓缩反应液,再加一定量的水和碳酸氢钠固体继续搅拌2小时,产生大量沉淀,于是将沉淀通过抽滤瓶抽滤并干燥,即得目标产物。
步骤S2:浓缩反应液,再将反应液用乙酸乙酯和水萃取,通过无水硫酸钠干燥,收集并浓缩有机相,以DCM:MeOH=10:1(v/v)的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物;
步骤S3:用甲醇淬灭反应,浓缩反应液,以DCM:MeOH=10:1(v/v)的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物;
步骤S4:用甲醇淬灭反应,浓缩反应液,以DCM:MeOH=10:1(v/v)的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物。
本发明的技术方案之三是:上述三环核苷酸类似物或上述方法合成得到的三环核苷酸类似物在制备抗肿瘤药物中的应用,优选在MCF-7(人乳腺癌细胞)、HGC-27(人未分化胃癌细胞)、A549(人非小细胞肺癌细胞)中的应用。
优选的,化合物8a、9a、9b、10、11或12或上述方法合成得到的化合物8a、9a、9b、10、11或12在MCF-7(人乳腺癌细胞)、HGC-27(人未分化胃癌细胞)、A549(人非小细胞肺癌细胞)中的应用;更优选的,化合物11或12或上述方法合成得到的化合物11或12在MCF-7(人乳腺癌细胞)、HGC-27(人未分化胃癌细胞)、A549(人非小细胞肺癌细胞)中的应用。
本发明涉及的“eqviv”、“当量”含义相同,均是指物质的量当量。
本发明涉及的“常温”、“rt”含义相同,均是指温度范围为25–30℃。
与现有技术相比,本发明的优点和有益效果在于:
本发明提供了一类结构新颖的三环核苷酸类似物的合成方法。目前只报道了侧链含磷酸酯的三环亚乙烯基类似物,且合成路径长,合成方法复杂,目标化合物的收率低,产量少,操作复杂。本方法使用对环境相对友好的AcOH作为溶剂,Ac2O为添加剂,实验过程操作简单,方便,收率高,化学选择性好,能高效地构建了一系列结构新颖的三环核苷酸类似物,为此类化合物的合成提供了新的方法与思路。基于所述合成方法,本发明合成了一类结构新颖的三环核苷酸类似物。
附图说明
图1为化合物9f诱导MCF-7细胞48h和72h凋亡细胞定量分析图
具体实施方式
以下具体实施例仅用于详细说明本发明的具体实施方式,并不限制本发明的权利要求书请求保护的范围。
TMOP指1,1,3,3-tetramethoxypropane(1,1,3,3-四甲氧基丙烷)(98%,伊诺凯);Ac2O指Acetic anhydride(乙酸酐)(>98.5%,国药集团化学试剂有限公司);AcOH指Aceticacid(乙酸)(99.5%,阿拉丁);2,2-二甲氧基丙烷(99%,安耐吉);PTSA(对甲苯磺酸一水合物)(98%,Acros);NaHCO3(>99.8%,阿拉丁);Acetone(丙酮)(99.5%+,国药集团化学试剂有限公司);Et3N(三乙胺)(>99.0%,国药集团化学试剂有限公司);DCM指Dichloromethane(二氯甲烷)(>99.5%,GENERAL-REAGENT);DMF指N,N-Dimethylformamide(N,N-二甲基甲酰胺)(99.9%,阿拉丁);氯磷酸二苯酯(98%,毕得医药);吡啶(99.5%+,国药集团化学试剂有限公司);1.0mol/L叔丁基氯化镁的THF溶液(阿拉丁),N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯(98%,毕得医药),TFA指Trifluoroacetic acid(三氟乙酸)(99.9%,伊诺凯)。鸟苷(99%,Adamas);2’-脱氧鸟苷(99%,Aladdin)。
平行反应仪:联华玻璃仪器(ETS-D5);旋转蒸发仪:EYELA(OSB-2100);真空隔膜泵:WELCH(115046)。
原料的合成:
化合物2的制备:
操作如下:将化合物1(鸟苷534.5mg,2.0mmol)溶解在丙酮(5mL)中。加入对甲苯磺酸一水合物(380.4mg,2.0mmol)和2,2-二甲氧基丙烷(6.1mL,50.0mmol),并将反应混合物在室温下搅拌12h。将反应混合物蒸发浓缩,然后溶解在H2O(2mL)中。小心地分三次加入固体NaHCO3(168.0mg,2.0mmol)并将溶液搅拌2小时。加入饱和NaHCO3溶液(4mL)再搅拌2小时。过滤悬浮液,产物用H2O(2×100mL)洗涤,得到白色固体2(552.7mg,85%)。White solid,mp:260–262℃.1HNMR(DMSO-d6,500MHz)δ10.66(s,1H),7.87(s,1H),6.62(s,2H),5.91(d,J=2.5Hz,1H),5.18(dd,J=2.5and 6.0Hz,1H),5.11(s,1H),4.97(dd,J=3.0and 6.0Hz,1H),4.11–4.15(m,1H),3.54-3.51(m,2H),1.51(s,3H),1.31(s,3H)。13C{1H}NMR(125MHz,DMSO-d6)157.3,154.2,151.2,136.4,117.2,113.5,88.9,87.1,84.0,81.7,62.1,27.5,25.7.HRMS(ESI)m/z:[M+H]+Calcd for C13H18N5O5324.1308,Found 324.1307.IR(KBr)v(cm-1):3427,3322,3206,2729,1717,1629,1375,1213,1071,861.
化合物3a的制备:
操作如下:将化合物2(32.3mg,0.1mmol)溶解在装有0.5mLDCM和0.5mL DMF的25mL圆底烧瓶中,在0℃下依次加入氯磷酸二苯酯(56.3mg,0.2mmol)和三乙胺(50.6mg,0.5mmol),将反应混合物在0℃至常温下搅拌24小时。真空浓缩反应混合物,将产物溶解在乙酸乙酯中,有机相用水洗涤,无水Na2SO4干燥和减压除去有机相得到粗产物,再将粗产物直接通过柱层析分离纯化,以DCM:MeOH=10:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物3a(13.7mg,25%yield).White solid,mp:124–126℃.1HNMR(500MHz,DMSO-d6)δ12.43(s,1H),9.80(s,1H),7.36–7.30(m,2H),7.23(d,J=8.4Hz,3H),7.19–7.16(m,5H),7.00–6.96(m,1H),6.41(s,1H),5.50(s,1H),5.42–5.36(m,1H),5.05(t,J=9.9Hz,1H),4.86(d,J=6.1Hz,1H),4.68(dd,J=10.6and 5.4Hz,1H),4.43(dd,J=8.9and 4.1Hz,1H),1.66(s,3H),1.40(s,3H).13C{1H}NMR(125MHz,DMSO-d6)δ157.1,153.9,150.9,150.3(dd,J=6.5and3.7Hz),137.0,130.4,130.1,124.0,121.5–121.3(m),120.8,120.3,117.8,94.7,86.9,83.4(d,J=7.9Hz),81.6,66.8(d,J=5.3Hz),26.8,26.5.HRMS(ESI)m/z:[M+H]+Calcd for C25H27N5O8P 556.1597,Found 556.1584.IR(KBr)v(cm-1):2730,1720,1654,1388,1245,1056.
化合物4a的制备:
操作如下:将化合物2(161.7mg,0.5mmol)溶解在装有3mLTHF的25mL的圆底烧瓶中,并缓慢加入1.0mol/L叔丁基氯化镁的THF溶液(1.05mL,1.05mmol),在室温下滴加3分钟。将白色悬浮液在该温度下搅拌30分钟,然后加入N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯(271.98mg,0.6mmol),持续3分钟。在该温度下搅拌混合物18小时。反应混合物用甲醇(1mL)淬灭,将反应液在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=10:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物白色固体4a(97.4mg,33%yield).White solid,mp:101–136℃.1H NMR(500MHz,DMSO-d6)δ12.54(s,1H),10.73(s,1H),7.83(s,1H),7.35–7.31(m,2H),7.18–7.13(m,3H),6.55(s,1H),6.03–5.98(m,2H),5.16(dd,J=6.0and 2.0Hz,1H),5.12(dd,J=6.4and 2.8Hz,1H),4.79(d,J=6.4Hz,1H),4.31–4.22(m,2H),4.04–3.99(m,1H),3.80–3.69(m,1H),1.49(s,3H),1.29(s,3H),1.15(d,J=6.8Hz,3H),1.09(dd,J=6.0and 1.5Hz,6H).13C NMR(125MHz,DMSO-d6)δ173.3(d,J=4.6Hz),157.4,154.4,151.3(d,J=6.8Hz),151.0,136.9,130.3,125.3,120.8(d,J=4.6Hz),117.7,113.9,89.2,85.8(d,J=8.4Hz),84.5,81.8,68.6,66.4,50.4,27.7,25.9,22.1(d,J=3.8Hz),20.3(d,J=6.1Hz).HRMS(ESI)m/z:[M+H]+Calcd for C25H34N6O9P593.2125,Found 593.2160.IR(KBr)v(cm-1):2960,2830,1524,1443,1310.
化合物6a和6b的制备:
操作如下:将化合物5(2’-脱氧鸟苷267.2mg,1.0mmol)溶解在装有3mL吡啶的25mL的圆底烧瓶中,并缓慢加入1.0mol/L叔丁基氯化镁的THF溶液(2.1mL,2.1mmol),在室温下滴加3分钟。将白色悬浮液在该温度下搅拌30分钟,然后加入N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯(544.0mg,1.2mmol),持续3分钟。在该温度下搅拌混合物18小时。反应混合物用甲醇(1mL)淬灭,将反应液在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=10:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物白色固体6a(24.1mg,9%)和白色固体6b(76.9mg,19%).6a:White solid,mp:130–132℃.1HNMR(500MHz,DMSO-d6)δ10.77(s,1H),7.85(s,1H),7.36(t,J=7.9Hz,2H),7.21(d,J=8.5Hz,2H),7.17(t,J=7.4Hz,1H),6.60(s,2H),6.14(dd,J=7.7and 6.3Hz,1H),6.02(dd,J=12.9and 10.1Hz,1H),5.47(d,J=3.9Hz,1H),4.88–4.80(m,1H),4.42–4.33(m,1H),4.24–4.20(m,2H),4.07–4.02(m,1H),4.00–3.96(m,1H),3.83–3.72(m,1H),2.26–2.20(m,1H),1.20(d,J=7.1Hz,3H),1.13(dd,J=6.2and 2.0Hz,6H).13C{1H}NMR(125MHz,DMSO-d6)δ171.5(d,J=7.6Hz),157.1,153.9,150.5(d,J=2.5Hz),137.0,130.3,130.1,121.3,120.4(t,J=4.8Hz),117.8,92.5,89.6–88.4(m),70.7,69.5,63.7(d,J=5.1Hz),46.9,40.4,21.9,21.6,19.1.HRMS(ESI)m/z:[M+H]+Calcd for C22H30N6O8P537.1863,Found537.1851.IR(KBr)v(cm-1):2988,1582,1521,1484,1376,1210.
6b:White solid,mp:93–95℃.1H NMR(500MHz,CDCl3)δ12.66(s,1H),10.46(s,1H),7.48(s,1H),7.18(t,J=7.8Hz,2H),7.10(t,J=8.3Hz,3H),7.08–6.99(m,5H),6.87(t,J=7.3Hz,1H),6.38–6.27(m,1H),6.22–6.14(m,1H),5.47(t,J=11.5Hz,1H),5.32–5.21(m,2H),5.03(t,J=9.3Hz,1H),4.86–4.73(m,2H),4.52(t,J=7.9Hz,1H),4.04–3.86(m,1H),3.80–3.66(m,1H),2.92–2.77(m,2H),1.27(d,J=6.2Hz,3H),1.18(dd,J=15.7and7.0Hz,6H),1.09(dd,J=10.1and 6.3Hz,6H),1.04(d,J=6.3Hz,3H).13C{1H}NMR(125MHz,DMSO-d6)δ172.5(d,J=7.5Hz),171.5(d,J=7.2Hz),157.1,153.9,150.6(d,J=2.2Hz),137.0,130.5,130.1,121.5,121.3,120.2(t,J=4.6Hz),117.8,92.3,86.8–86.6(m),71.6(d,J=5.0Hz),69.9,69.5,63.5,46.9,46.3,37.5,21.9,21.6,19.3,19.1.HRMS(ESI)m/z:[M+H]+Calcd for C34H46N7O12P2806.2680,Found 806.2674.IR(KBr)v(cm-1):2981,1583,1542,1472,1334,1215,1156.
实施例1
化合物7a的制备:
操作如下:将化合物3a(55.5mg,0.1mmol),Ac2O(95uL,1.0mmol)、AcOH(0.4mL)和TMOP(20uL,0.12mmol)依次加入25mL玻璃密封管中,用塞子密封,将反应混合物放在平行反应仪中90℃下搅拌2小时。反应完后,冷却至室温,先将反应液转移至25mL的圆底烧瓶中,在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=8:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物7a(56.1mg,86%yield).Yellow solid,mp:94–96℃.1HNMR(500MHz,CDCl3)δ9.41(d,J=6.1Hz,1H),8.97–8.90(d,J=1.1Hz,1H),8.04(s,1H),7.29–7.22(m,5H),7.14–7.10(m,5H),7.08(dd,J=7.0and 3.7Hz,1H),6.24(d,J=2.3Hz,1H),5.25(dd,J=6.0and 2.2Hz,1H),5.08(dd,J=6.0and2.7Hz,1H),4.62–4.53(m,2H),4.52(s,1H),1.62(s,3H),1.36(s,3H).13C{1H}NMR(125MHz,CDCl3)δ161.3,157.8,153.9,150.6(dd,J=6.5and 3.7Hz),149.5,146.3,137.2,133.5,130.1,125.8,122.6,121.2–121.0(m),115.3,110.0,105.3,90.6,86.1,84.8(d,J=7.9Hz),84.2,81.0,68.0(d,J=5.3Hz),29.4,26.8,26.0.HRMS(ESI)m/z:[M+H]+Calcd for C28H27N5O8P 592.1597,Found592.1590.IR(KBr)v(cm-1):1725,1489,1218,1189,1073,1025,954.
实施例2
化合物8a的制备:
操作如下:将化合物4a(59.3mg,0.1mmol),Ac2O(95uL,1.0mmol)、AcOH(0.4mL)和TMOP(20uL,0.12mmol)依次加入25mL玻璃密封管中,用塞子密封,将反应混合物放在平行反应仪中90℃下搅拌2小时。反应完后,冷却至室温,先将反应液转移至25mL的圆底烧瓶中,在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=15:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物8a(54.7mg,87%yield).Yellow solid,mp:73–75℃.1HNMR(500MHz,CDCl3)δ9.42(dd,J=7.1and 2.0Hz,1H),9.00–8.91(m,1H),8.08(s,1H),7.18(t,J=7.8Hz,2H),7.10–7.07(m,3H),7.05(t,J=7.5Hz,1H),6.18(d,J=3.1Hz,1H),5.39(dd,J=6.3and 3.1Hz,1H),5.17(dd,J=6.3and 3.1Hz,1H),4.97–4.89(m,1H),4.88–4.45(m,1H),4.45–4.40(m,1H),4.38–4.33(m,2H),4.05–3.96(m,1H),1.61(s,3H),1.36(s,3H),1.32(d,J=7.0Hz,3H),1.17(d,J=6.3Hz,6H).13C{1H}NMR(125MHz,CDCl3)δ173.5(d,J=8.1Hz),161.4,152.8,150.9(d,J=6.8Hz),149.7,149.3,141.4,137.7,129.7,125.0,120.3(d,J=4.6Hz),119.4,115.1,110.0,90.7,84.6(d,J=8.3Hz),83.8,81.0,69.0,65.9(d,J=5.5Hz),50.2(d,J=2.6Hz),26.9,25.00,21.3(d,J=7.3Hz),20.8(d,J=4.5Hz).HRMS(ESI)m/z:[M+H]+Calcd for C28H34N6O9P 629.2125,Found 629.2120.IR(KBr)v(cm-1):2983,1731,1575,1533,1490,1456.
实施例3
化合物9a的制备:
操作如下:将化合物6a(53.6mg,0.1mmol),Ac2O(95uL,1.0mmol)、AcOH(0.4mL)和TMOP(20uL,0.12mmol)依次加入25mL玻璃密封管中,用塞子密封,将反应混合物放在平行反应仪中90℃下搅拌1小时。反应完后,冷却至室温,先将反应液转移至25mL的圆底烧瓶中,在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=15:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物9a(20.3mg,33%yield).Yellow solid,mp:85–87℃.1HNMR(500MHz,CDCl3)δ9.45(dd,J=7.2and 2.3Hz,1H),8.99(dd,J=3.8and 2.3Hz,1H),8.20(s,1H),7.26–7.20(m,5H),7.13(dd,J=7.2and 3.8Hz,1H),6.44(t,J=7.0Hz,1H),5.37–5.31(m,1H),5.06–4.92(m,2H),4.57–4.50(m,1H),4.48–4.43(m,2H),4.42–4.36(m,1H),2.94–2.86(m,1H),2.69–2.63(m,1H),2.02(s,3H),1.36(d,J=7.1Hz,3H),1.19(d,J=6.3Hz,6H).13C{1H}NMR(125MHz,CDCl3)δ171.6(d,J=7.7Hz),173.4(d,J=7.4Hz),161.5,152.7,151.9(d,J=2.6Hz),149.2,140.9,137.5,129.8,125.1,120.5(t,J=4.8Hz),119.3,109.7,84.5,84.1–83.7(m),77.0(d,J=5.5Hz),69.1,65.6(d,J=5.1Hz),50.3(d,J=1.8Hz),50.2,38.5(d,J=4.2Hz),29.3,21.3,21.3–21.2(m),20.5(d,J=5.2Hz).HRMS(ESI)m/z:[M+H]+Calcd for C27H32N6O9P 615.1968,Found 615.1959.IR(KBr)v(cm-1):2987,1742,1601,1554,1498,1458,1325,1226.
实施例4
化合物9b的制备:
操作如下:将化合物6b(80.6mg,0.1mmol),Ac2O(95uL,1.0mmol)、AcOH(0.4mL)和TMOP(20uL,0.12mmol)依次加入25mL玻璃密封管中,用塞子密封,将反应混合物放在平行反应仪中90℃下搅拌1小时。反应完后,冷却至室温,先将反应液转移至25mL的圆底烧瓶中,在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=15:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物9b(28.6mg,34%yield).Yellow solid,mp:70–72℃.1HNMR(500MHz,CDCl3)δ9.42(dd,J=7.2and 2.3Hz,1H),8.95(dd,J=3.8and 2.3Hz,1H),8.12(s,1H),7.32(t,J=7.9Hz,2H),7.26–7.23(m,2H),7.20–7.11(m,5H),7.08(dd,J=7.2and 3.8Hz,1H),7.03(t,J=7.0Hz,1H),6.44(dd,J=8.5and 5.6Hz,1H),5.37–5.31(m,1H),5.06–4.92(m,2H),4.57–4.50(m,1H),4.48–4.43(m,2H),4.42–4.36(m,1H),4.08–3.96(m,3H),2.94–2.86(m,1H),2.69–2.63(m,1H),1.41–1.38(m,3H),1.36(d,J=7.1Hz,3H),1.23(t,J=6.8Hz,6H),1.18(d,J=6.3Hz,6H).13C{1H}NMR(125MHz,CDCl3)δ173.6(d,J=7.6Hz),173.4(d,J=7.3Hz),161.2,152.8,150.9(d,J=2.2Hz),150.9(d,J=2.5Hz),149.9,149.2,140.8,137.7,130.1,129.7,125.4,125.1,120.5(t,J=4.6Hz),119.2,109.9,84.4,84.1–83.7(m),77.1(d,J=5.2Hz),69.3,69.0,65.5(d,J=5.2Hz),50.3(d,J=1.7Hz),50.2,38.4(d,J=4.0Hz),29.1,29.2,21.3,21.3–21.2(m),20.8(d,J=4.5Hz),20.5(d,J=5.1Hz).HRMS(ESI)m/z:[M+H]+Calcd for C37H46N7O12P2842.2680,Found842.2672.IR(KBr)v(cm-1):2982,1732,1591,1533,1490,1455,1374,1318,1211,1153.
实施例5
化合物10的制备:
操作如下:将化合物7a(59.2mg,0.1mmol)和TFA(1mL)加入10mL反应瓶中,将反应混合物在常温下搅拌1.0h。反应完后,先将反应液转移至25mL的圆底烧瓶中,在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=10:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物10(45.2mg,82%yield).Yellow solid,mp:110–112℃.1H NMR(500MHz,CDCl3)δ9.32(d,J=6.2Hz,1H),8.00(s,1H),7.35–7.25(m,5H),7.18–7.14(m,5H),7.18(dd,J=7.0and 3.7Hz,1H),6.20(d,J=2.4Hz,1H),5.31(dd,J=6.1and 2.3Hz,1H),5.01(dd,J=6.0and 2.7Hz,1H),4.75(s,1H),4.50–4.48(m,2H),4.32(s,1H),4.20–4.18(m,1H),4.12–4.10(m,1H).13C{1H}NMR(125MHz,CDCl3)δ163.8,157.3,153.0,150.6(dd,J=6.5and 3.7Hz),144.5,137.0,132.8,130.5,130.1,121.3,120.2–120.0(m),117.8,106.2,96.3,86.8(d,J=7.9Hz),73.9,70.4,68.0(d,J=5.3Hz).HRMS(ESI)m/z:[M+H]+Calcd for C25H23N5O8P 552.1284,Found 552.1279.IR(KBr)v(cm-1):1710,1456,1200,1170,1050,1006.
实施例6
化合物11的制备:
操作如下:将化合物8a(62.9mg,0.1mmol),TFA(1mL)加入10mL反应瓶中,将反应混合物在常温下搅拌1.0h。反应完后,先将反应液转移至25mL的圆底烧瓶中,在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=10:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物11(50.0mg,85%yield).Yellow solid,mp:85–87℃.1H NMR(500MHz,CDCl3)δ9.38(dd,J=7.1and 2.0Hz,1H),8.99–8.95(m,1H),8.02(s,1H),7.20(t,J=7.8Hz,2H),7.15–7.10(m,3H),7.00(t,J=7.5Hz,1H),6.16(d,J=3.2Hz,1H),5.53(dd,J=6.3and 3.1Hz,1H),5.17(dd,J=6.3and 3.1Hz,1H),4.95–4.90(m,1H),4.78–4.72(m,1H),4.55–4.50(m,2H),4.45–4.40(m,1H),4.37–4.33(m,1H),4.30–4.25(m,1H),3.58–3.54(m,1H),1.29(d,J=7.0Hz,3H),1.17(d,J=6.3Hz,6H).13C{1H}NMR(125MHz,CDCl3)δ175.3(d,J=8.1Hz),173.0,169.5,160.3,154.0,150.2(d,J=6.8Hz),142.3,138.5,130.1,129.6,125.1,120.8(d,J=4.6Hz),116.5,110.3,85.3,73.6(d,J=8.3Hz),70.4,69.1,64.2(d,J=5.5Hz),49.5(d,J=2.6Hz),21.8,21.6,19.1.HRMS(ESI)m/z:[M+H]+Calcd forC25H30N6O9P 589.1812,Found589.1806.IR(KBr)v(cm-1):2983,1701,1526,1510,1460.
实施例7
化合物12的制备:
操作如下:将化合物9a(61.5mg,0.1mmol),Et3N(150uL,1.1mmol)和MeOH(1mL)依次加入10mL反应瓶中,将反应混合物在常温下搅拌12h。反应完后,先将反应液转移至25mL的圆底烧瓶中,在30℃的水浴锅中通过旋转蒸发仪在真空隔膜泵中蒸发浓缩反应液5min,反应液旋干后直接通过柱层析分离纯化,以DCM:MeOH=10:1(v/v)作为洗脱剂,分离出目标产物,然后再将目标产物蒸发浓缩成固体,得到产物12(47.5mg,83%yield).Yellowsolid,mp:92–94℃.1HNMR(500MHz,CDCl3)δ9.30(dd,J=7.1and 2.0Hz,1H),8.96–8.90(m,1H),7.86(s,1H),7.45–7.40(m,2H),7.26–7.20(m,3H),7.05(t,J=7.5Hz,1H),6.28(d,J=3.2Hz,1H),5.95(dd,J=6.3and 3.1Hz,1H),4.93(dd,J=6.3and 3.1Hz,1H),4.70–4.65(m,2H),4.55–4.50(m,2H),4.45–4.41(m,1H),4.35–4.30(m,1H),4.29–4.22(m,1H),3.55–3.50(m,1H),1.29(d,J=7.0Hz,3H),1.17(d,J=6.3Hz,6H).13C{1H}NMR(125MHz,CDCl3)δ173.6(d,J=7.7Hz),171.5(d,J=7.4Hz),165.5,153.1,151.5(d,J=2.6Hz),149.8,140.2,137.6,130.8,126.1,120.6(t,J=4.8Hz),119.5,109.8,86.2,83.5–82.4(m),70.1,66.6(d,J=5.1Hz),52.4(d,J=1.8Hz),51.3,39.5(d,J=4.2Hz),29.9,22.3–22.0(m),20.50(d,J=5.2Hz).HRMS(ESI)m/z:[M+H]+Calcd for C25H30N6O8P 573.1863,Found573.1856.IR(KBr)v(cm-1):2983,1701,1526,1510,1460.
实施例1-7制备的化合物对三种癌细胞的抗增殖活性
1.材料
样品:实施例1-7制备的化合物7a,8a,9a,9b,10,11,12。
仪器:电子天平(梅特勒-托利多仪器(上海)有限公司);SW-CJ-IFD型超净工作台(苏净安泰);BWS-12型恒温水槽(一恒科学仪器有限公司);L500型低速离心机(湘仪);Biotek酶标仪、GL-150型干式恒温器、VORTEX-5型涡旋振荡器:其林贝尔;流式细胞分选仪,BD公司;CO2恒温培养箱、96孔细胞培养板:Thermo公司;
试剂:DMEM培养基、胎牛血清:GIBCO公司;0.01mol/LpH 7.4PBS缓冲液干粉,索莱宝;胰蛋白酶消化液(Trypsin-EDTA)(生工生物工程);四甲基偶氮唑蓝(MTT)、二甲亚砜(DMSO)、阿糖胞苷:美国SIGMA公司;AnnexinV-FITC/PI凋亡检测试剂盒(BestBio)。
细胞:MCF-7(人乳腺癌细胞)、HGC-27(人未分化胃癌细胞)、A549(人非小细胞肺癌细胞)均来源于广州吉妮欧生物科技有限公司。
2.实验方法
(1)化合物7a,8a,9a,9b,10,11,12的溶解。
用电子天平称取一定质量的化合物固体粉末,用DMSO溶解为所需浓度。
(2)MTT溶液:用0.01mol/LpH7.4PBS溶解,配成5mg/mL溶液,0.22um滤膜过滤除菌,分装,避光4℃保存。
(3)DMEM完全培养基:含10%胎牛血清、1%双抗溶液和1%谷氨酰胺溶液的DMEM培养液,4℃保存备用。
(4)细胞培养
分别将MCF-7、HGC-27、A549培养于DMEM完全培养基,连续传代3次,保持对数生长期供实验用。
(5)抗肿瘤细胞毒活性实验
将处于对数生长期的细胞(MCF-7、HGC-27、A549)分别以每孔3000个细胞接种在96孔板中。细胞培养24小时后,弃去培养基,将配置的不同浓度的化合物7a,8a,9a,9b,10,11,12和阳性对照阿糖胞苷分别加入96孔板中,每个浓度设6个复孔。药物作用72h后,将5mg/mL的MTT加到每个孔中。在37℃二氧化碳培养箱中4小时后,吸出培养基。然后每孔加入200μLDMSO,振荡均匀。使用酶标仪在490nm波长下测量吸光度。采用SPSS 16.0软件计算抑制细胞生长50%所需的化合物浓度(IC50)。
(6)MCF-7细胞凋亡实验
将处于对数生长期的MCF-7细胞处理后接种在6孔板中,并用不同浓度(0、17、18、19、20、21μg/mL)的化合物11处理48小时和72小时。然后收集细胞并用AnnexinV-FITC/PI凋亡检测试剂盒进行后续处理,即将细胞重悬于磷酸盐缓冲液(0.01mol/LpH7.4)中,加5uL的AnnexinV/FITC溶液,然后将细胞在暗处孵育40分钟之后,再向其加入10uL的PI溶液,混匀即上机监测,用流式细胞仪(Becton,Dickinson and Company,NJ)分析样品,并使用Graphpad软件分析数据。
3.实验结果
(1)抗肿瘤细胞毒活性实验结果
表1为7种化合物作用于三种细胞中的IC50值。
表1
注:IC50(μg/mL):细胞活性被抑制一半所需的化合物浓度;阿糖胞苷用作阳性对照。
从表中可以看出:7种化合物对3种细胞均有一定程度地抗增殖活性,化合物8a,9a,9b,10,11,12分别对MCF-7细胞、HCG-27细胞、A549细胞的抗增殖活性较好,其中,化合物11对MCF-7细胞、HCG-27细胞和A549细胞的抗增殖活性最好,IC50值分别为(19.10±0.36)μg/mL、(17.36±0.21)μg/mL、(15.33±0.25)μg/mL,表明化合物11对MCF-7细胞、HCG-27细胞和A549细胞具有潜在的抗肿瘤活性。
(2)化合11诱导MCF-7细胞凋亡
肿瘤细胞的凋亡被认为是大多数抗癌药物杀死肿瘤细胞的主要方式。为了更好地表征化合物11诱导肿瘤细胞凋亡的能力,我们使用细胞凋亡检测试剂盒进行了双参数细胞荧光分析。图1为阳性对照药阿糖胞苷和化合物11分别诱导MCF-7细胞48h和72h的凋亡细胞定量分析图。不同浓度(0、17、18、19、20、21μg/mL)的化合物11分别处理MCF-7细胞48h和72h的凋亡率与对照组(Cytarabine,阿糖胞苷)的相应浓度(0、17、18、19、20、21μg/mL)相比,具有显著性差异,*P<0.5,**P<0.01。其中化合物11处理MCF-7细胞48h的抗增殖活性略低于72h的,其化合物11处理MCF-7细胞72h的IC50值为(19.10±0.36)μg/mL。同时,化合物11能够以浓度依赖和时间依赖的方式诱导MCF-7细胞凋亡。
从图1中看出随着化合物11的浓度从0增加至21ug/mL,MCF-7细胞的凋亡率在逐步升高,当化合物11的浓度从20ug/mL增加到21ug/mL时,MCF-7细胞的凋亡率趋于平缓,因此,当化合物11的浓度为20ug/mL时,处理MCF-7细胞48h和72h的凋亡率达到最大,分别为84.8%和87.3%;从图1中看出化合物11在同一浓度时,不同时间下,MCF-7细胞凋亡率也不同,且同一浓度时,化合物11处理MCF-7细胞72h的凋亡能力强于化合物11处理MCF-7细胞48h的。因此,化合物11能够以浓度依赖和时间依赖的方式诱导MCF-7细胞凋亡。
Claims (10)
1.一类三环核苷酸类似物,其特征在于,其结构式如下列化合物7a、8a、9a、9b、10、11或12所示:
其中,Ph是指苯基。
2.权利要求1所述的三环核苷酸类似物的合成方法,具体包括以下步骤:
(1)以化合物3a或4a和1,1,3,3-四甲氧基丙烷为原料,Ac2O为添加剂,溶剂为AcOH,90℃反应2h,得到化合物7a或8a;
所述化合物3a或4a的结构式如下:
其中,R1=R2=苯氧基,是化合物3a;R1=苯氧基,R2=叉丙基保护羧基的丙氨酸基,是化合物4a;
步骤(1):化合物3a或4a:1,1,3,3-四甲氧基丙烷:添加剂:溶剂的用量关系为1mmol:1.2mmol:10mmol:4mL;
(2)以化合物6a或6b和1,1,3,3-四甲氧基丙烷为原料,Ac2O为添加剂,溶剂为AcOH,90℃反应1h,得到化合物9a或9b;
步骤(2):化合物6a或6b:1,1,3,3-四甲氧基丙烷:添加剂:溶剂的用量关系为1mmol:1.2mmol:10mmol:4mL;
所述化合物6a或6b的结构式如下:
(3)以步骤(1)所得产物化合物7a或化合物8a为原料,溶剂为三氟乙酸,在常温下反应1h,即得化合物10或11;
步骤(3):化合物7a或化合物8a:溶剂的用量关系为1mmol:10mL;
(4)将步骤(2)所得产物化合物9a为原料,Et3N为添加剂,溶剂为甲醇,在常温下反应12h,即得化合物12;
步骤(4):化合物9a:添加剂:溶剂的用量关系为1mmol:11mmol:10mL。
3.根据权利要求2所述的合成方法,其特征在于,步骤(1)-(4)还包括对反应结束后所得反应液进行后处理的步骤:浓缩反应液,通过柱层析分离纯化。
4.根据权利要求3所述的合成方法,其特征在于,步骤(1)-(4)后处理步骤所用洗脱剂为:
步骤(1):分离化合物7a所用洗脱剂为体积比为8:1的二氯甲烷和甲醇混合液;分离化合物8a所用洗脱剂为体积比为15:1的二氯甲烷和甲醇混合液;
步骤(2):以体积比为15:1的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化;
步骤(3)、(4):以体积比为10:1的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化。
5.根据权利要求2-4任一所述的合成方法,其特征在于,所述步骤(1)中化合物3a或4a的制备方法,包括以下步骤:
S1.将鸟苷溶于丙酮中,再向其中加入对甲苯磺酸一水合物和2,2-二甲氧基丙烷,常温下,反应12h,得到化合物2;
步骤S1:鸟苷:对甲苯磺酸一水合物:2,2-二甲氧基丙烷的用量关系为1mmol:1mmol:25mmol;
S2.将化合物2溶于DCM和DMF按体积比1:1的混合溶剂中,在0℃下向体系中加入氯磷酸二苯酯和三乙胺,常温下反应24h,得到化合物3a;
步骤S2:化合物2:氯磷酸二苯酯:三乙胺的用量关系为1mmol:2mmol:5mmol;
S3.将化合物2溶于THF中,接着缓慢向体系中加入1.0mol/L叔丁基氯化镁的THF溶液,搅拌,再缓慢向体系中加入N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯,常温下反应18h,得到化合物4a;
步骤S3:化合物2:叔丁基氯化镁:N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯的用量关系为1mmol:2.1mmol:1.2mmol。
6.根据权利要求2-4任一所述的合成方法,其特征在于,所述步骤(2)中化合物6a或6b的制备方法,包括以下步骤:
S4.将2’-脱氧鸟苷溶于吡啶中,接着缓慢向体系中加入1.0mol/L叔丁基氯化镁的THF溶液,搅拌,再缓慢向体系中加入N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯,常温下反应18h,得到化合物6a或6b;
步骤S4:2’-脱氧鸟苷:叔丁基氯化镁:N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯的用量关系为1mmol:2.1mmol:1.2mmol。
7.根据权利要求5所述的合成方法,其特征在于,步骤S1-S3还包括对反应结束后所得反应液进行后处理的步骤,其中,
步骤S1:浓缩反应液,接着加一定量的水和碳酸氢钠固体继续搅拌2-3小时,产生大量沉淀,再将沉淀通过抽滤瓶抽滤并干燥,即得目标产物;
步骤S2:浓缩反应液,再将反应液用乙酸乙酯和水萃取,通过无水硫酸钠干燥,收集并浓缩有机相,以体积比为10:1的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物;
步骤S3:用甲醇淬灭反应,浓缩反应液,以体积比为10:1的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物。
8.根据权利要求6所述的合成方法,其特征在于,步骤S4还包括对反应结束后所得反应液进行后处理的步骤:用甲醇淬灭反应,浓缩反应液,以体积比为10:1的二氯甲烷和甲醇混合液为洗脱剂通过柱层析分离纯化,分离出目标产物。
9.权利要求1所述的三环核苷酸类似物或权利要求2-8任一所述的方法合成的三环核苷酸类似物在制备抗乳腺癌、胃癌或肺癌药物中的应用。
10.权利要求1所述的三环核苷酸类似物或权利要求2-8任一所述的方法合成的三环核苷酸类似物在制备抗MCF-7、HGC-27或A549的药物中的应用。
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