CN113004368B - 熊果酸嘧啶酰胺类衍生物及其制备方法和应用 - Google Patents
熊果酸嘧啶酰胺类衍生物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了熊果酸嘧啶酰胺类衍生物及其制备方法和应用,属于有机合成和药物化学技术领域。果酸嘧啶酰胺类衍生物结构式如式Ⅰ所示:
Description
技术领域
本发明属于有机合成和药物化学技术领域,具体涉及熊果酸嘧啶酰胺类衍生物及其制备方法和应用。
背景技术
肿瘤有恶性和良性两种之分,恶性肿瘤是指细胞已经失去自我控制而威胁个人生命的一种常见疾病,也就是所谓的癌症。由于多种原因都可能诱导癌症的发生,并且对癌症的治疗很困难,因此给人类的健康带来了巨大的威胁。癌症的攻克已成为当今医学及化学工作者必须面对的一项艰巨而有意义的工作。在临床治疗肿瘤的过程中,主要是采取综合的治疗方法,化学药物治疗是阻滞细胞生长,抑制或直接杀死癌细胞的一种手段,采用化学药物对癌细胞进行压制,治疗各种肿瘤疾病是一种快速高效的方法,当然也被归纳为目前在临床医疗方面缺一不可的一种治疗手段。
绝大多数新的抗肿瘤药物的发现与合成,都是先从天然资源下手,寻找天然资源中具有抗肿瘤等药物活性的成分,将它们分析研究后作为合成最终产物先导化合物,对它们进行结构分析及修饰改性,进而得到新型的能高效治疗肿瘤的药物。熊果酸作为一种天然药物,在自然界中分布十分广泛,价格便宜同时易得,因此极具有广阔的研究与发展前景。
氮杂环化合物因具有良好的生物活性,广泛应用于医药、农药等领域,对人类的健康发挥着重要的作用。嘧啶类衍生物作为一类重要的氮杂环化合物,广泛存在于人体及生物体内。嘧啶及其衍生物因具有抗肿瘤、抗病毒、杀菌、除草、杀虫等生物活性,且高效、低毒,被药物化学家和生物学家持续关注,其中氨基嘧啶更是目前聚焦的热点。熊果酸的羧基是活泼结构,能够发生甲酯化、酰胺化等反应,酰胺基团是抗肿瘤药物的常见基团,通过对羧基进行酰胺化有可能增强其生物活性。因此,如果在熊果酸分子上引入嘧啶基团,再对其羧基进行进一步衍生化,引入不同酰胺基团;对合成的系列衍生物进行抗肿瘤活性测试,如获得抗肿瘤活性较好的新型熊果酸嘧啶酰胺类衍生物,对于研发新型抗肿瘤药物将具有重要的化学和生物学的意义。
发明内容
针对现有技术中存在的问题,本发明要解决的一个技术问题在于提供熊果酸嘧啶酰胺类衍生物,该化合物具有抗肿瘤活性,具有开发抗肿瘤药物的潜在价值。本发明要解决的另一个技术问题在于提供熊果酸嘧啶酰胺类衍生物的制备方法,通过熊果酸A环上的C-3位羟基氧化为羰基后与不同取代基的苯甲醛反应得到亚苄基类熊果酸衍生物,再通过与不同的胺在DCC/HOBt作用下反应生成酰胺基团,然后熊果酸酰胺衍生物再在碱性环境下与盐酸胍反应得到相应的熊果酸嘧啶酰胺衍生物。本发明要解决的技术问题还有一个在于提供熊果酸嘧啶酰胺类衍生物在制备治疗肿瘤的药物中的应用,药理学实验表明,熊果酸嘧啶酰胺类杂环衍生物对人肺癌细胞A549、人乳腺癌细胞MCF-7、人子宫颈癌细胞HeLa和人肝癌细胞株Hep G2有显著的抑制作用。
为了解决上述问题,本发明所采用的技术方案如下:
熊果酸嘧啶酰胺类衍生物,结构式如式Ⅰ所示:
上述熊果酸嘧啶酰胺类衍生物的制备方法,包括以下步骤:
(1)熊果酸经过琼斯试剂氧化反应得到3-氧化熊果酸,结构式如式II所示:
(2)不同取代基的苯甲醛在KOH/Ethanol的作用下与3-氧化熊果酸进行ClaisenSchmidt缩合反应得到相应的含有不同取代基的亚苄基类熊果酸,结构式如式III所示:
(3)不同取代基的亚苄基类熊果酸在DCC/HOBt的作用下得到相应的亚苄基类熊果酸的酰胺化合物,结构式如式IV所示:
(4)不同取代基的亚苄基类熊果酸的酰胺化合物与盐酸胍和叔丁醇钾在碱性条件反应得到相应的熊果酸嘧啶酰胺衍生物,结构式如式Ⅰ所示。
所述熊果酸嘧啶酰胺类衍生物的制备方法,在步骤(1)中,向反应容器中加入4.6mmol熊果酸和250mL丙酮,搅拌溶解,在冰水中搅拌反应10~20min,缓慢滴加1.87mL琼斯试剂并升至室温,搅拌反应4~6h后,加入90mL异丙醇搅拌反应25~35min,反应结束后滤去沉淀收集滤液,滤液减压浓缩、重结晶,得到3-氧化熊果酸。
所述熊果酸嘧啶酰胺类衍生物的制备方法,在步骤(2)中,称取相对于3-氧化熊果酸的1.1当量的不同取代基苯甲醛于反应容器中,并加入20mL无水乙醇作为溶剂,并称取0.5g氢氧化钾,配制成质量分数为2.5%的氢氧化钾乙醇溶液,搅拌溶解,然后再加入0.1g的3-氧化熊果酸,充分反应后,先减压浓缩除去溶剂,然后用乙酸乙酯溶解并用饱和碳酸氢钠水溶液萃取三次,再用饱和盐水萃取三次,之后用无水硫酸钠除去水分,减压浓缩除去有机溶剂,分离纯化后得到亚苄基类熊果酸III。
所述熊果酸嘧啶酰胺类衍生物的制备方法,所述不同取代基苯甲醛分别为苯甲醛、4-氟代苯甲醛、3-氟代苯甲醛、2-氟代苯甲醛、4-氯代苯甲醛、4-溴代苯甲醛、4-甲基苯甲醛、3-甲基苯甲醛、2-甲基苯甲醛或4-甲氧基苯甲醛。
所述熊果酸嘧啶酰胺类衍生物的制备方法,在步骤(3)中,将0.05mmol的亚苄基类熊果酸III溶于2mL二氯甲烷中,将0.07mmol的HOBt和0.07mmol的DCC加入反应体系中,室温搅拌反应25~35min后,加入0.08mmol胺类化合物;室温搅拌过夜;反应结束后过滤掉沉淀,滤液减压浓缩后用乙腈溶解3~5℃放置过夜,进一步过滤掉沉淀,减压浓缩除去溶剂,用二氯甲烷/甲醇梯度洗脱,合并产物组分,减压浓缩,去除溶剂,分别制得纯亚苄基类熊果酸酰胺化合物IV。
所述熊果酸嘧啶酰胺类衍生物的制备方法,所述胺类化合物分别为3-二甲氨基-1-丙胺、N,N-二乙基-1,3-丙二胺、3-(二丁胺)丙胺、N-(3-氨丙基)吗啉、1-(3-氨丙基)-4-甲基哌嗪、1-(3-氨基丙基)吡咯烷或1-(3-氨丙基)-2-甲基哌啶。
所述熊果酸嘧啶酰胺类衍生物的制备方法,在步骤(4)中,将0.001mol亚苄基类熊果酸酰胺化合物IV溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷甲醇体系,所述二氯甲烷与甲醇的体积比为100:1,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I。
上述熊果酸嘧啶酰胺类衍生物在制备治疗肿瘤药物中的应用。
所述熊果酸嘧啶酰胺类衍生物在制备治疗肿瘤药物中的应用,所述肿瘤为人肺癌细胞A549、人乳腺癌细胞MCF-7、人子宫颈癌细胞HeLa和人肝癌细胞株Hep G2。
有益效果:与现有的技术相比,本发明的优点包括:
(1)本发明是通过熊果酸A环上的C-3位羟基氧化为羰基后与不同取代基的苯甲醛反应得到亚苄基类熊果酸衍生物,再通过与不同的胺在DCC/HOBt作用下反应生成酰胺基团,然后熊果酸酰胺衍生物再在碱性环境下与盐酸胍反应得到相应的熊果酸嘧啶酰胺衍生物。该类衍生物的结构较为新颖,国内外未见报道。具有开发抗肿瘤药物的潜在价值。
(2)本发明具有抗肿瘤活性,药理学实验表明,熊果酸嘧啶酰胺类杂环衍生物对人肺癌细胞A549、人乳腺癌细胞MCF-7、人子宫颈癌细胞HeLa和人肝癌细胞株Hep G2有显著的抑制作用,其中化合物I-b的体外抗肿瘤活性最好。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。
一种具有通式(I)所示结构的熊果酸嘧啶酰胺类衍生物:
熊果酸嘧啶酰胺类衍生物的I-a至I-p的制备方法,包括如下步骤:
(1)熊果酸经过琼斯试剂氧化反应得到3-氧化熊果酸,结构式如式II所示:
(2)不同取代基的苯甲醛在KOH/Ethanol的作用下与3-氧化熊果酸进行ClaisenSchmidt缩合反应得到相应的含有不同取代基的亚苄基类熊果酸,结构式如式III所示:
(3)不同取代基的亚苄基类熊果酸III在DCC/HOBt的作用下得到相应的亚苄基类熊果酸的酰胺化合物,结构式如式IV所示:
(4)不同取代基的亚苄基类熊果酸的酰胺化合物与盐酸胍在叔丁醇钾碱性条件反应得到相应的熊果酸嘧啶酰胺衍生物,结构式如式Ⅰ所示。
实施例1
熊果酸嘧啶酰胺(I-a)的合成,包括以下步骤:
(1)在500mL的圆底烧瓶中加入4.6mmol的熊果酸和250mL丙酮,搅拌溶解后在冰水中搅拌反应15min,缓慢滴加1.87mL琼斯试剂并升至室温搅拌反应5h后,加入90mL异丙醇搅拌反应30min,反应结束后滤去沉淀收集滤液,滤液减压浓缩得到的浅黄绿色粘稠状固体用甲醇重结晶得到的白色针状晶体,制得3-氧化熊果酸(II)(1.2g,65.6%);
(2)称取1.1当量(相对于3-氧化熊果酸)的苯甲醛于圆底烧瓶中,加入20mL无水乙醇作为溶剂,并称取催化剂0.5g氢氧化钾,配制成质量分数为2.5%的氢氧化钾乙醇溶液,待其充分搅拌溶解之后,加入0.1g的3-氧化熊果酸,反应过程中用TLC法检测反应进度;待其充分反应之后,先减压浓缩除去溶剂,然后用乙酸乙酯溶解并用饱和碳酸氢钠水溶液萃取三次,再用饱和盐水萃取三次,之后用无水硫酸钠除去水分,减压浓缩除去有机溶剂,分离纯化后得到亚苄基类熊果酸杂环衍生物III-a;
(3)将0.05mmol化合物III-a溶于2mL二氯甲烷中,加入0.07mmol的HOBt(1-羟基苯并三氮唑)和0.07mmol的DCC(二环己基碳二亚胺),室温搅拌反应30min,然后将0.08mmol的3-二甲氨基-1-丙胺添加进去;室温搅拌过夜;反应结束后过滤掉沉淀,滤液减压浓缩后用乙腈溶解4℃放置过夜,进一步过滤掉沉淀,减压浓缩除去溶剂,用二氯甲烷/甲醇梯度洗脱(二氯甲烷和甲醇的体积比为20:1),合并产物组分,减压浓缩,去除溶剂,分别制得纯亚苄基类熊果酸酰胺化合物IV-a;
(4)将0.001mol亚苄基类熊果酸酰胺化合物IV-a溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷甲醇体系,所述二氯甲烷与甲醇的体积比为100:1,合并产物组分减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-a,得率72%。
Yellow powder solid;M.p.196~198℃;Yield:72%;1H NMR(400MHz,CDCl3):δ0.71(s,3H),0.76(s,3H),0.82(d,J=6.5Hz,3H),0.88(brs,3H),1.06(s,3H),1.20(s,3H),1.22(m,1H),1.24(s,3H),1.25~1.90(m,20H),2.10(d,J=15.7Hz,1H),2.14(s,6H),2.26(m,2H),2.52(d,J=15.3Hz,1H),2.98(m,1H),3.38(m,1H),5.15(brs,1H),5.17(brs,2H),6.98(t,J=4.5Hz,1H),7.37(m,1H),7.39(m,4H);13C NMR(100MHz,CDCl3):δ14.76,16.64,17.36,20.16,21.18,23.25,23.32,23.79,24.57,25.95,27.74,30.86,31.53,32.10,35.98,37.49,39.11,39.16,39.39,39.43,39.61,41.78,42.35,45.53,46.07,47.46,52.83,53.65,58.65,114.21,125.22,128.11,128.43,128.51,138.94,139.18,161.11,166.79,174.38,177.47;HRMS(ESI):m/z[M+H]+calcd for C43H64N5O:666.5104;found:666.5111.
实施例2
熊果酸嘧啶酰胺(I-b)的合成
以4-氟代苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-b,将0.001mol化合物IV-b溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-b,得率76%。
Yellow powder solid;M.p.194~196℃;Yield:76%;1H NMR(600MHz,CDCl3):δ0.75(s,3H),0.80(s,3H),0.88(d,J=6.4Hz,3H),0.95(brs,3H),1.10(m,1H),1.12(s,3H),1.26(s,3H),1.28(m,1H),1.29(s,3H),1.30~1.99(m,19H),2.14(d,J=15.4Hz,1H),2.29(s,6H),2.42(m,2H),2.53(d,J=15.3Hz,1H),3.06(m,1H),3.40(m,1H),4.87(brs,2H),5.25(t,J=3.7Hz,1H),6.90(t,J=4.8Hz,1H),7.15(t,J=8.6Hz,2H),7.45(m,2H);13C NMR(150MHz,CDCl3):δ14.95,16.83,17.49,20.35,21.35,23.47,23.49,23.98,24.78,25.91,27.93,31.03,31.74,32.27,36.22,37.69,38.72,39.26,39.62,39.71,39.85,42.09,42.61,45.22,45.79,47.80,52.99,53.82,58.09,114.76,115.44(d,J=21.4Hz),125.47,130.69(d,J=8.2Hz),135.07(d,J=3.0Hz),139.48,161.09,163.08(d,J=246.7Hz),165.94,175.08,178.19;HRMS(ESI):m/z[M+H]+calcd for C43H63FN5O:684.5010;found:684.5017.
实施例3
熊果酸嘧啶酰胺(I-c)的合成
以3-氟代苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-c,将0.001mol化合物IV-c溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-c,得率72%。
Yellow powder solid;M.p.211~213℃;Yield:72%;1H NMR(600MHz,CDCl3):δ0.77(s,3H),0.81(s,3H),0.89(d,J=6.4Hz,3H),0.95(brs,3H),1.09(m,1H),1.12(s,3H),1.26(s,3H),1.28(m,1H),1.30(s,3H),1.30~2.00(m,19H),2.13(d,J=15.4Hz,1H),2.27(s,6H),2.40(m,2H),2.53(d,J=15.4Hz,1H),3.06(m,1H),3.43(m,1H),4.86(brs,2H),5.25(t,J=3.4Hz,1H),6.99(t,J=5.6Hz,1H),7.12(td,J=8.5,2.0Hz,1H),7.18(dt,J=9.5,2.3Hz,1H),7.23(d,J=7.7Hz,1H),7.43(td,J=8.0,5.8Hz,1H);13C NMR(100MHz,CDCl3):δ15.00,16.88,17.55,20.37,21.36,23.48,23.51,24.01,24.78,25.71,27.97,31.06,31.74,32.27,36.24,37.76,39.25,39.26,39.63,39.73,39.85,41.90,42.59,44.99,45.75,47.80,52.98,53.75,57.80,114.77,115.73(d,J=19.8Hz),115.88(d,J=18.4Hz),124.49(d,J=2.8Hz),125.46,130.04(d,J=8.4Hz),139.42,141.31(d,J=7.3Hz),161.14,162.70(d,J=241.0Hz),165.72,175.17,178.16;HRMS(ESI):m/z[M+H]+calcd for C43H63FN5O:684.5010;found:684.5005.
实施例4
熊果酸嘧啶酰胺(I-d)的合成
以2-氟代苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-d,将0.001mol化合物IV-d溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-d,得率76%。
Yellow powder solid;M.p.231~233℃;Yield:76%;1H NMR(600MHz,CDCl3):δ0.79(s,3H),0.80(s,3H),0.84(d,J=6.4Hz,3H),0.92(brs,3H),1.05(m,1H),1.07(s,3H),1.24(s,3H),1.26(m,1H),1.28(s,3H),1.30~2.10(m,20H),2.20(s,6H),2.32(m,3H),3.03(m,1H),3.42(m,1H),5.00(brs,2H),5.20(t,J=3.0Hz,1H),6.98(t,J=4.7Hz,1H),7.13(t,J=8.9Hz,1H),7.23(t,J=7.5Hz,1H),7.31(t,J=6.8Hz,1H),7.39(m,1H);13C NMR(150MHz,CDCl3):δ14.94,16.82,17.49,20.28,21.31,23.34,23.43,23.94,24.75,26.13,27.90,31.02,31.54,32.27,35.96,37.65,39.27,39.55,39.61,39.67,39.76,40.62,42.54,45.52,45.65,47.64,52.99,53.85,58.80,115.87(d,J=21.4Hz),116.35,124.52(d,J=3.4Hz),125.35,127.00(d,J=16.3Hz),130.51(d,J=7.7Hz),130.58(d,J=3.6Hz),139.43,161.23,163.59(d,J=242.9Hz),165.90,174.42,177.64;HRMS(ESI):m/z[M+H]+calcd for C43H63FN5O:684.5010;found:684.5013.
实施例5
熊果酸嘧啶酰胺(I-e)的合成
以4-氯代苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-e,将0.001mol化合物IV-e溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-e,得率71%。
Yellow powder solid;M.p.217~319℃;Yield:71%;1H NMR(600MHz,CDCl3):δ0.73(s,3H),0.78(s,3H),0.86(d,J=6.4Hz,3H),0.92(brs,3H),1.05(m,1H),1.09(s,3H),1.23(s,3H),1.25(m,1H),1.26(s,3H),1.30~2.00(m,19H),2.12(d,J=15.5Hz,1H),2.22(s,6H),2.34(m,2H),2.49(d,J=15.4Hz,1H),3.02(m,1H),3.40(m,1H),4,99(brs,2H),5.22(t,J=3.5Hz,1H),7.01(t,J=4.6Hz,1H),7.38~7.42(m,4H);13C NMR(150MHz,CDCl3):δ14.90,16.77,17.49,20.28,21.30,23.42,23.44,23.92,24.71,25.98,27.88,30.99,31.67,32.20,36.15,37.64,39.12,39.24,39.54,39.62,39.77,42.00,42.52,45.50,45.72,47.65,52.91,53.79,58.58,114.53,125.30,128.52,130.18,134.71,137.48,139.38,161.17,165.67,174.97,177.76;HRMS(ESI):m/z[M+H]+calcd forC43H63ClN5O:700.4714;found:700.4711.
实施例6
熊果酸嘧啶酰胺(I-f)的合成
以4-溴代苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-f,将0.001mol化合物IV-f溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-f,得率69%。
Yellow powder solid;M.p.211~213℃;Yield:69%;1H NMR(600MHz,CDCl3):δ0.75(s,3H),0.81(s,3H),0.88(d,J=6.4Hz,3H),0.94(brs,3H),1.08(m,1H),1.11(s,3H),1.25(s,3H),1.27(m,1H),1.29(s,3H),1.30~2.00(m,19H),2.14(d,J=15.4Hz,1H),2.21(s,6H),2.33(m,2H),2.51(d,J=15.3Hz,1H),3.03(m,1H),3.43(m,1H),4.92(brs,2H),5.24(t,J=3.5Hz,1H),7.02(t,J=4.9Hz,1H),7.35(d,J=8.4Hz,2H),7.58(d,J=8.5Hz,2H);13C NMR(150MHz,CDCl3):δ14.96,16.82,17.57,20.35,21.36,23.47,23.50,23.98,24.77,26.15,27.94,31.05,31.73,32.27,36.21,37.69,39.33,39.37,39.61,39.69,39.85,42.06,42.59,45.73,45.79,47.71,52.97,53.90,58.87,114.63,123.10,125.34,130.52,131.54,138.02,139.49,161.20,165.76,175.09,177.74;HRMS(ESI):m/z[M+H]+calcd for C43H63BrN5O:744.4209;found:744.4213.
实施例7
熊果酸嘧啶酰胺(I-g)的合成
以4-甲基苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-g,将0.001mol化合物IV-g溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-g,得率78%。
Yellow powder solid;M.p.200~202℃;Yield:78%;1H NMR(400MHz,CDCl3):δ0.71(s,3H),0.76(s,3H),0.83(d,J=6.4Hz,3H),0.89(brs,3H),1.06(s,3H),1.20(s,3H),1.24(s,3H),1.25~1.90(m,21H),2.11(d,J=17.2Hz,1H),2.16(s,6H),2.28(m,2H),2.35(s,3H),2.55(d,J=15.4Hz,1H),2.99(m,1H),3.37(m,1H),5.08(brs,2H),5.18(brs,1H),6.98(t,J=4.1Hz,1H),7.20(d,J=7.8Hz,2H),7.31(d,J=7.8Hz,2H);13C NMR(100MHz,CDCl3):δ14.78,16.68,17.38,20.19,21.20,21.34,23.33,23.35,23.80,24.61,25.95,27.77,30.88,31.56,32.13,36.03,37.53,39.11,39.13,39.43,39.64,41.91,42.39,45.49,45.60,46.01,47.51,52.85,53.68,58.57,114.31,125.32,128.56,128.83,136.06,138.38,139.23,161.10,166.83,174.29,177.61;HRMS(ESI):m/z[M+H]+calcd forC44H66N5O:680.5260;found:680.5251.
实施例8
熊果酸嘧啶酰胺(I-h)的合成
以3-甲基苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-h,将0.001mol化合物IV-h溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-h,得率74%。
Yellow powder solid;M.p.197~199℃;Yield:74%;1H NMR(600MHz,CDCl3):δ0.76(s,3H),0.80(s,3H),0.85(d,J=6.2Hz,3H),0.93(s,3H),1.07(m,1H),1.10(s,3H),1.24(s,3H),1.26(m,1H),1.28(s,3H),1.30~1.99(m,19H),2.11(d,J=15.4Hz,1H),2.21(s,6H),2.33(m,2H),2.40(s,3H),2.57(d,J=15.4Hz,1H),3.03(m,1H),3.42(m,1H),4.95(brs,2H),5.21(brs,1H),6.99(t,J=4.5Hz,1H),7.20(m,2H),7.26(s,1H),7.31(t,J=7.5Hz,1H);13C NMR(150MHz,CDCl3):δ14.94,16.81,17.47,20.32,21.32,21.63,23.42,23.46,23.93,24.76,25.07,27.91,31.01,31.67,32.26,36.16,37.65,39.23,39.27,39.57,39.59,39.79,41.85,42.55,45.61,45.69,47.66,53.00,53.84,58.72,114.65,125.43,125.64,127.99,129.22,129.36,138.18,139.09,139.38,161.10,167.30,174.51,177.73;HRMS(ESI):m/z[M+H]+calcd for C44H66N5O:680.5260;found:680.5265.
实施例9
熊果酸嘧啶酰胺(I-i)的合成
以2-甲基苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-i,将0.001mol化合物IV-i溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-i,得率64%。
Yellow powder solid;M.p.192~194℃;Yield:64%;1H NMR(600MHz,CDCl3):δ0.77(s,3H),0.79(brs,6H),0.88(brs,3H),1.02(s,3H),1.03(m,1H),1.23(s,3H),1.25(s,3H),1.27(m,1H),1.30~1.95(m,19H),2.07(s,3H),2.17(s,6H),2.17(d,J=15.4Hz,1H),2.29(m,2H),2.56(d,J=15.4Hz,1H),3.00(m,1H),3.39(m,1H),5.08(brs,2H),5.15(brs,1H),6.97(t,J=4.5Hz,1H),7.04(m,1H),7.19~7.25(m,3H);13C NMR(150MHz,CDCl3):δ14.97,16.76,17.42,19.44,20.20,21.23,23.20,23.33,23.76,24.65,26.09,27.81,30.94,31.50,32.20,35.85,37.56,39.18,39.25,39.45,39.55,39.68,42.31,42.45,45.42,45.63,47.56,52.90,53.80,58.76,115.06,125.26,125.49,125.82,127.79,128.13,130.27,138.64,139.31,161.09,168.13,174.20,177.53;HRMS(ESI):m/z[M+H]+calcd for C44H66N5O:680.5260;found:680.5253.
实施例10
熊果酸嘧啶酰胺(I-j)的合成
以4-甲氧基苯甲醛、3-二甲氨基-1-丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-j,将0.001mol化合物IV-j溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-j,得率75%。
Yellow powder solid;M.p.195~197℃;Yield:75%;1H NMR(600MHz,CDCl3):δ0.74(s,3H),0.80(s,3H),0.88(d,J=6.4Hz,3H),0.94(brs,3H),1.08(m,1H),1.11(s,3H),1.24(s,3H),1.28(s,3H),1.30~2.00(m,20H),2.16(d,J=15.4Hz,1H),2.21(s,6H),2.33(m,2H),2.60(d,J=15.4Hz,1H),3.03(m,1H),3.43(m,1H),3.86(s,3H),4.87(brs,2H),5.23(t,J=3.5Hz,1H),6.97(d,J=8.8Hz,2H),7.00(t,J=5.1Hz,1H),7.44(d,J=8.8Hz,2H);13C NMR(150MHz,CDCl3):δ14.92,16.83,17.52,20.36,21.35,23.50,23.51,23.95,24.79,26.14,27.94,31.05,31.73,32.28,36.21,37.69,39.10,39.31,39.53,39.60,39.83,42.28,42.58,45.67,45.82,47.69,53.00,53.88,55.47,58.81,113.71,114.61,125.46,130.31,131.58,139.42,160.01,161.15,166.60,174.48,177.72;HRMS(ESI):m/z[M+H]+calcd for C44H66N5O2:696.5210;found:696.5218.
实施例11
熊果酸嘧啶酰胺(I-k)的合成
以4-氟代苯甲醛、N,N-二乙基-1,3-丙二胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-k,将0.001mol化合物IV-k溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-k,得率85%。
Yellow powder solid;M.p.205~207℃;Yield:85%;1H NMR(600MHz,CDCl3):δ0.75(s,3H),0.81(s,3H),0.88(d,J=6.4Hz,3H),0.95(brs,3H),1.02(t,J=7.1Hz,6H),1.09(m,1H),1.11(s,3H),1.25(s,3H),1.28(m,1H),1.29(s,3H),1.30~2.02(m,19H),2.14(d,J=15.4Hz,1H),2.47(t,J=6.5Hz,2H),2.50~2.56(m,5H),3.07(m,1H),3.38(m,1H),4.88(brs,2H),5.25(t,J=3.3Hz,1H),6.68(t,J=4.8Hz,1H),7.14(t,J=8.6Hz,2H),7.46(m,2H);13C NMR(150MHz,CDCl3):δ11.31,14.95,16.88,17.45,20.36,21.38,23.49,23.51,23.98,24.80,26.14,28.00,31.05,31.75,32.30,36.24,37.63,39.17,39.22,39.62,39.68,39.84,42.13,42.61,45.82,46.82,47.65,51.70,53.00,53.76,114.71,115.40(d,J=21.3Hz),125.38,130.71(d,J=8.2Hz),135.18(d,J=3.0Hz),139.60,161.16,163.05(d,J=246.8Hz),166.00,174.92,177.70;HRMS(ESI):m/z[M+H]+calcd for C45H67FN5O:712.5323;found:712.5328.
实施例12
熊果酸嘧啶酰胺(I-l)的合成
以4-氟代苯甲醛、3-(二丁胺)丙胺为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-l,将0.001mol化合物IV-l溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-l,得率82%。
Yellow powder solid;M.p.307~309℃;Yield:82%;1H NMR(600MHz,CDCl3):δ0.75(s,3H),0.80(s,3H),0.87(d,J=6.3Hz,3H),0.88(t,J=7.3Hz,6H),0.94(brs,3H),1.08(m,1H),1.11(s,3H),1.24(s,3H),1.26(m,5H),1.28(s,3H),1.33(d,J=12.6Hz,1H),1.35~2.05(m,22H),2.12(d,J=15.4Hz,1H),2.40(t,J=7.6Hz,4H),2.45(t,J=6.7Hz,2H),2.52(d,J=15.4Hz,1H),3.07(m,1H),3.34(m,1H),4.94(brs,2H),5.25(t,J=3.4Hz,1H),6.47(t,J=4.9Hz,1H),7.13(t,J=8.7Hz,2H),7.44(dd,J=8.7,5.5Hz,2H);13C NMR(150MHz,CDCl3):δ14.17,14.94,16.88,17.41,20.32,20.88,21.33,23.43,23.46,23.95,24.79,26.27,28.00,28.73,31.01,31.70,32.28,36.20,37.55,38.76,39.14,39.59,39.66,39.82,42.03,42.61,45.76,47.66,52.48,52.99,53.77,53.81,114.63,115.35(d,J=21.4Hz),125.36,130.64(d,J=8.2Hz),135.12(d,J=3.3Hz),139.65,161.16,163.00(d,J=246.8Hz),166.00,174.87,177.73;HRMS(ESI):m/z[M+H]+calcd for C49H75FN5O:768.5949;found:768.5958.
实施例13
熊果酸嘧啶酰胺(I-m)的合成
以4-氟代苯甲醛、N-(3-氨丙基)吗啉为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-m,将0.001mol化合物IV-m溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-m,得率45%。
Yellow powder solid;M.p.199~201℃;Yield:45%;1H NMR(600MHz,CDCl3):δ0.75(s,3H),0.80(s,3H),0.89(d,J=6.4Hz,3H),0.95(brs,3H),1.10(m,1H),1.12(s,3H),1.25(s,3H),1.27(m,1H),1.29(s,3H),1.30~2.01(m,19H),2.14(d,J=15.4Hz,1H),2.38(t,J=5.8Hz,2H),2.44(brs,4H),2.53(d,J=15.4Hz,1H),3.06(m,1H),3.40(m,1H),3.72(t,J=4.2Hz,4H),4.89(brs,2H),5.25(t,J=3.1Hz,1H),6.40(t,J=4.3Hz,1H),7.14(t,J=8.6Hz,2H),7.46(m,2H);13C NMR(150MHz,CDCl3):δ14.97,16.90,17.46,20.34,21.35,23.47,23.55,23.98,24.87,25.45,27.95,31.00,31.74,32.27,36.23,37.63,38.74,39.22,39.62,39.68,39.87,42.12,42.67,45.78,47.75,52.98,53.92,53.97,57.62,66.93,114.64,115.40(d,J=21.2Hz),125.35,130.72(d,J=8.2Hz),135.14(d,J=3.5Hz),139.83,161.16,163.05(d,J=246.8Hz),166.00,174.89,177.86;HRMS(ESI):m/z[M+H]+calcd for C45H65FN5O2:726.5115;found:726.5121.
实施例14
熊果酸嘧啶酰胺(I-n)的合成
以4-氟代苯甲醛、1-(3-氨丙基)-4-甲基哌嗪为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-n,将0.001mol化合物IV-n溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-n,得率79%。
Yellow powder solid;M.p.201~203℃;Yield:79%;1H NMR(600MHz,CDCl3):δ0.71(s,3H),0.76(s,3H),0.86(d,J=6.4Hz,3H),0.90(d,J=6.1Hz,3H),1.05(m,1H),1.08(s,3H),1.20(s,3H),1.22(m,1H),1.24(s,3H),1.30~2.00(m,19H),2.10(d,J=15.4Hz,1H),2.23(s,3H),2.33(m,2H),2.41(brs,8H),2.48(d,J=15.4Hz,1H),3.01(m,1H),3.35(m,1H),5.07(brs,2H),5.21(t,J=3.6Hz,1H),6.48(t,J=4.9Hz,1H),7.09(t,J=8.6Hz,2H),7.42(dd,J=8.5,5.5Hz,2H);13C NMR(150MHz,CDCl3):δ14.83,16.76,17.32,20.22,21.23,23.40,23.43,23.86,24.63,25.49,27.82,30.88,31.61,32.14,36.09,37.47,39.03,39.05,39.48,39.54,39.73,41.97,42.48,45.65,46.03,47.55,52.86,53.39,53.72,55.08,57.44,114.32,115.22(d,J=21.3Hz),125.19,130.59(d,J=8.1Hz),135.03(d,J=3.2Hz),139.63,161.14,162.87(d,J=246.8Hz),165.81,174.70,177.54;HRMS(ESI):m/z[M+H]+calcd for C46H68FN6O:739.5432;found:739.5428.
实施例15
熊果酸嘧啶酰胺(I-o)的合成
以4-氟代苯甲醛、1-(3-氨基丙基)吡咯烷为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-o,将0.001mol化合物IV-o溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-o,得率82%。
Yellow powder solid;M.p.207~209℃;Yield:82%;1H NMR(600MHz,CDCl3):δ0.74(s,3H),0.79(s,3H),0.87(d,J=6.4Hz,3H),0.94(brs,3H),1.07(m,1H),1.10(s,3H),1.24(s,3H),1.27(m,1H),1.28(s,3H),1.30~1.77(m,16H),1.78(t,J=4.9Hz,4H),1.86(m,1H),1.92(d,J=10.9Hz,1H),1.97(td,J=13.4,4.1Hz,1H),2.13(d,J=15.4Hz,1H),2.45~2.58(m,7H),3.03(m,1H),3.46(m,1H),4.94(brs,2H),5.21(brs,1H),7.08(t,J=4.3Hz,1H),7.13(t,J=8.6Hz,2H),7.45(dd,J=8.5,5.5Hz,2H);13C NMR(150MHz,CDCl3):δ14.92,16.82,17.40,20.33,21.34,23.50,23.55,23.58(2C),23.95,24.67,27.12,27.93,30.99,31.71,32.26,36.19,37.73,39.29,39.59,39.64(2C),39.77,42.09,42.51,45.77,47.58,52.97,53.79,54.42(2C),55.79,114.62,115.35(d,J=21.3Hz,2C),125.26,130.69(d,J=8.2Hz,2C),135.13(d,J=3.3Hz),139.46,161.14,163.00(d,J=246.6Hz),165.93,174.89,177.62;HRMS(ESI):m/z[M+H]+calcd for C45H65FN5O:710.5166;found:710.5172.
实施例16
熊果酸嘧啶酰胺(I-p)的合成
以4-氟代苯甲醛、1-(3-氨丙基)-2-甲基哌啶为原料,采用上述方法制备亚苄基类熊果酸酰胺化合物IV-p,将0.001mol化合物IV-p溶于10mL的叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷/甲醇梯度洗脱(200:1-100:1),合并产物组分,减压浓缩,去除溶剂,制得纯化合物熊果酸嘧啶酰胺类杂环衍生物I-p,得率82%。
Yellow powder solid;M.p.307~309℃;Yield:82%;1H NMR(600MHz,CDCl3):δ0.74(s,3H),0.79(s,3H),0.88(d,J=6.1Hz,3H),0.94(brs,3H),1.08(m,1H),1.11(s,3H),1.13(d,J=5.4Hz,3H),1.24(s,3H),1.28(s,3H),1.30~2.05(m,26H),2.13(d,J=15.1Hz,1H),2.21(m,1H),2.36(m,1H),2.51(m,1H),2.53(d,J=15.2Hz,1H),2.83(brs,1H),2.95(brs,1H),3.07(m,1H),3.35(m,1H),4.91(brs,2H),5.28(brs,1H),6.65(brs,1H),7.13(t,J=8.3Hz,2H),7.45(m,2H);13C NMR(150MHz,CDCl3):δ14.94,16.82,16.88,17.39,20.34,21.36,23.43,23.46,23.49,23.52,23.96,24.70,27.93,28.00,31.03,31.72,32.29,36.21,37.55,37.58,39.03,39.08,39.60,39.65,39.80,42.09,42.56,45.78,47.63,52.98,53.55,53.61,53.97,56.95,114.66,115.38(d,J=21.3Hz),125.41,130.68(d,J=8.2Hz),135.13(d,J=3.4Hz),139.48,161.14,163.02(d,J=246.8Hz),166.00,174.89,177.84;HRMS(ESI):m/z[M+H]+calcd for C47H69FN5O:738.5479;found:738.5483.
实施例17
体外抗肿瘤活性筛选
筛选细胞株为:人肺癌细胞A549、人乳腺癌细胞MCF-7、人子宫颈癌细胞HeLa和人肝癌细胞株Hep G2。
实验方法:
取对数生长期状态良好的细胞,胰蛋白酶消化,制成5×104细胞/mL的悬液。将细胞悬液移入96孔培养板,每孔100μL,置37℃、5%CO2条件下培养24h。
将受试衍生物用DMSO(二甲基亚砜)配制成一定浓度的母液,再用DMEM培养基将衍生物母液稀释成不同作用浓度的稀释液。移去旧培养基,加入不同浓度的含药无血清DMEM培养基或1640培养基,每孔100μL。另设空白对照组和阳性对照依托泊苷(VP-16)对照组。药物作用72h后,于每孔再加入MTT溶液(5mg/mL)10μL,继续温育4h。
吸去各孔内上清液,每孔加入DMSO 100μL,振荡5min,使结晶物充分溶解,酶标仪测定540nm处各孔的光吸收值(OD值),计算细胞的增殖抑制率:抑制率(%)=(1-用药组平均OD值/空白对照组平均OD值)×100%。应用SPSS16.0软件进行数据处理并计算癌细胞增殖的半数抑制浓度(IC50),结果见表1。表1为熊果酸嘧啶酰胺类衍生物对人肺癌细胞A549、人乳腺癌细胞MCF-7、人子宫颈癌细胞HeLa和人肝癌细胞株Hep G2以及人正常肝上皮细胞Ges-1的体外增殖抑制作用结果。化合物I-a~I-p的抗肿瘤活性结果如表1所示:
表1
如表1所示,所合成的熊果酸嘧啶酰胺衍生物对四种肿瘤细胞(HeLa、MCF-7、HepG2和A549)一种正常细胞Ges-1均表现出了不同程度的抑制作用,化合物I-d、I-e、I-h对四种癌细胞具有较强的细胞毒活性,化合物I-a、I-b、I-c、I-f、I-g、I-i、I-j、I-k也表现出了中等的抑制活性。其中,化合物I-a~I-n对HeLa细胞的IC50值比阳性对照依托泊苷低,化合物I-a~I-k以及I-n~I-p对MCF-7细胞的IC50值比阳性对照依托泊苷低,比阳性对照依托泊苷的抗肿瘤活性强。初步的构效关系分析表明,该系列化合物的嘧啶酰胺侧链结构对于抗肿瘤活性具有一定的影响。熊果酸嘧啶三碳二甲氨基酰胺侧链结构(I-a~I-j)活性要优于三碳杂环酰胺及三碳长链酰胺侧链的衍生物;而对于三碳二甲氨基酰胺来说,含对氟、对氯、间甲基的衍生物活性较好。以上结果表明此类化合物对于人宫颈癌细胞HeLa、乳腺癌细胞MCF-7具有显著的抑制作用,对肝癌细胞Hep G2和肺癌细胞A549抑制作用较差,具有开发抗癌药物的潜力。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。
Claims (10)
2.权利要求1所述熊果酸嘧啶酰胺类衍生物的制备方法,其特征在于,包括以下步骤:
(1)熊果酸经过琼斯试剂氧化反应得到3-氧化熊果酸,结构式如式II所示:
(2)不同取代基的苯甲醛在KOH/乙醇的作用下与3-氧化熊果酸进行Claisen Schmidt缩合反应得到相应的含有不同取代基的亚苄基类熊果酸,结构式如式III所示:
(3)不同取代基的亚苄基类熊果酸III在DCC/HOBt的作用下得到相应的亚苄基类熊果酸的酰胺化合物,结构式如式IV所示:
(4)不同取代基的亚苄基类熊果酸的酰胺化合物与盐酸胍和叔丁醇钾在碱性条件反应得到相应的熊果酸嘧啶酰胺衍生物,结构式如式Ⅰ所示。
3.根据权利要求2所述熊果酸嘧啶酰胺类衍生物的制备方法,其特征在于,在步骤(1)中,向反应容器中加入4.6mmol熊果酸和250mL丙酮,搅拌溶解,在冰水中搅拌反应10~20min,缓慢滴加1.87mL琼斯试剂并升至室温,搅拌反应4~6h后,加入90mL异丙醇搅拌反应25~35min,反应结束后滤去沉淀收集滤液,滤液减压浓缩、重结晶,得到3-氧化熊果酸。
4.根据权利要求2所述熊果酸嘧啶酰胺类衍生物的制备方法,其特征在于,在步骤(2)中,称取相对于3-氧化熊果酸的1.1当量的不同取代基苯甲醛于反应容器中,并加入20mL无水乙醇作为溶剂,并称取0.5g氢氧化钾,配制成质量分数为2.5%的氢氧化钾乙醇溶液,搅拌溶解,然后再加入0.1g的3-氧化熊果酸,充分反应后,先减压浓缩除去溶剂,然后用乙酸乙酯溶解并用饱和碳酸氢钠水溶液萃取三次,再用饱和盐水萃取三次,之后用无水硫酸钠除去水分,减压浓缩除去有机溶剂,分离纯化后得到亚苄基类熊果酸III。
5.根据权利要求2或4所述熊果酸嘧啶酰胺类衍生物的制备方法,其特征在于,所述不同取代基苯甲醛分别为苯甲醛、4-氟代苯甲醛、3-氟代苯甲醛、2-氟代苯甲醛、4-氯代苯甲醛、4-溴代苯甲醛、4-甲基苯甲醛、3-甲基苯甲醛、2-甲基苯甲醛或4-甲氧基苯甲醛。
6.根据权利要求2所述熊果酸嘧啶酰胺类衍生物的制备方法,其特征在于,在步骤(3)中,将0.05mmol亚苄基类熊果酸III溶于2mL二氯甲烷中,将0.07mmol的HOBt和0.07mmol的DCC加入反应体系中,室温搅拌反应25~35min后,加入0.08mmol胺类化合物;室温搅拌过夜;反应结束后过滤掉沉淀,滤液减压浓缩后用乙腈溶解3~5℃放置过夜,进一步过滤掉沉淀,减压浓缩除去溶剂,用二氯甲烷/甲醇梯度洗脱,合并产物组分,减压浓缩,去除溶剂,制得亚苄基类熊果酸酰胺化合物IV。
7.根据权利要求2或6所述熊果酸嘧啶酰胺类衍生物的制备方法,其特征在于,所述胺类化合物分别为3-二甲氨基-1-丙胺、N,N-二乙基-1,3-丙二胺、3-(二丁胺)丙胺、N-(3-氨丙基)吗啉、1-(3-氨丙基)-4-甲基哌嗪、1-(3-氨基丙基)吡咯烷或1-(3-氨丙基)-2-甲基哌啶。
8.根据权利要求2所述熊果酸嘧啶酰胺类衍生物的制备方法,其特征在于,在步骤(4)中,将0.001mol亚苄基类熊果酸酰胺化合物IV溶于10mL叔丁醇,将0.005mol叔丁醇钾、0.004mol盐酸胍依次缓慢溶于反应液内,缓慢升至85℃搅拌回流反应24h,反应结束后用乙酸乙酯萃取3次收集有机相,水洗3次、饱和碳酸氢钠溶液和浓盐水各洗一次后用无水硫酸钠除水,减压浓缩除去有机溶剂得到白色固体,用氧化铝柱对其进行分离纯化,溶剂选用二氯甲烷甲醇体系,所述二氯甲烷与甲醇的体积比为100:1,制得熊果酸嘧啶酰胺类杂环衍生物I。
9.权利要求1所述熊果酸嘧啶酰胺类衍生物在制备治疗肿瘤药物中的应用。
10.根据权利要求9所述熊果酸嘧啶酰胺类衍生物在制备治疗肿瘤药物中的应用,其特征在于,所述肿瘤为人肺癌细胞A549、人乳腺癌细胞MCF-7、人子宫颈癌细胞HeLa 和人肝癌细胞株Hep G2。
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