CN106699738A - 一类含甲硝唑吡唑类骨架的抗菌化合物的设计、合成及生物活性评价 - Google Patents
一类含甲硝唑吡唑类骨架的抗菌化合物的设计、合成及生物活性评价 Download PDFInfo
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- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
本发明公开了一类含甲硝唑吡唑类骨架的衍生物、其制备方法及应用,所述由甲硝唑合成的衍生物的结构如式所示:其中,R1选自:-H、2-CH3、3-CH3、4-CH3、4-OCH3、2-F、3-F、4-F、4-Cl、4-I;R2选自:-H、4-CH3、4-Cl。本发明对两株格兰仕阳性菌:枯草芽孢杆菌(Bacillus subtilis ATCC 6633)、金黄色葡萄球菌(Staphylococcus aureus ATCC 6538)以及两株格兰仕阴性菌:大肠杆菌(Escherichia coliATCC 35218)、铜绿假单胞菌(PseudomonasaeruginosaATCC 13525)有很好的抑制作用,尤其是对格兰仕阴性菌。因此本发明的含甲硝唑吡唑类骨架的衍生物可以应用于制备抗菌药物。
Description
技术领域
本发明属于药物化学领域,尤其涉及一类含甲硝唑吡唑类骨架的衍生物、其制备方法及应用。
发明简介
细菌感染在很长一段时间内一直都无情的威胁着人类健康,而且消耗着大量的时间和财力。目前,细菌对已有抗生素的抗性和新的治疗药物的短缺已经成为了普遍和棘手的临床问题,迫切需要对此解决。在许多天然产物和人工合成的化合物中普遍具有吡唑基团,其在预防疾病方面有不错的效果,包括抗菌,抗肿瘤,抗炎,抗抑郁等。硝基咪唑类衍生物同样具有广阔的生物活性,尤其是在抗菌方面。作为衍生物之一的甲硝唑目前已经是市场上常见的抗感染的临床用药。近年来,研究人员对于改造甲硝唑上的羟基充满了兴趣。在我们以前的工作中设计、合成了一些可以抗菌效果很好的吡唑类衍生物。研究得知其发挥疗效主要归功于含有吡唑的二芳基杂环。对此,我们对其结构进行了一些优化改造,引入了甲硝唑来取代其中的吗啉。实验证明设计合成的新的化合物具有不错的生物学效应。
发明内容
本发明的目的在于提供一类含甲硝唑吡唑类骨架的抗菌化合物的制备方法及其在抗菌药物中的应用。
技术方案:
一类含甲硝唑吡唑类骨架的衍生物,其结构如式所示:
其中,R1选自:-H、2-CH3、3-CH3、4-CH3、4-OCH3、2-F、3-F、4-F、4-Cl、4-I
R2选自:-H、4-CH3、4-Cl
制备方法包括如下步骤:
步骤1.回流条件下,向反应容器中依次加入草酸二甲酯和结构如式A所示的化合物,在含有甲醇钠的无水甲醇中反应。TLC跟踪反应,充分反应后,得到结构如式B所示的化合物。
步骤2.向反应容器中依次加入结构如式B所示的化合物以及结构如式C所示的化合物,稀盐酸作催化,在无水甲醇体系中回流,TLC跟踪反应,充分反应后,得到结构如式D所示的化合物。
步骤3.向反应容器中加入结构如式D所示的化合物,在搅拌的情况下加入氢氧化钾,并滴加蒸馏水使其充分反应,得到结构如式E所示的化合物。
步骤4.在二氯甲烷反应体系中依次加入结构如式E所示的化合物、EDC、HOBT、DMAP活化一段时间,再缓慢加入甲硝唑,于45℃反应一段时间,TLC跟踪反应,得到目标终产物,即结构如式E所示的化合物。
式A~F中,R1选自-H、2-CH3、3-CH3、4-CH3、4-OCH3、2-F、3-F、4-F、4-Cl、4-I;R2选自-H、4-CH3、4-Cl。
本发明对两株格兰仕阳性菌:枯草芽孢杆菌(Bacillus subtilis ATCC 6633)、金黄色葡萄球菌(Staphylococcus aureus ATCC 6538)以及两株格兰仕阴性菌:大肠杆菌(Escherichia coliATCC 35218)、铜绿假单胞菌(PseudomonasaeruginosaATCC 13525)有很好的抑制作用,尤其是对格兰仕阴性菌。因此本发明的含甲硝唑吡唑类骨架的衍生物可以应用于制备抗菌药物。
具体实施方式
在某个具体的实施例中,本发明的制备过程和相关产物的结构式如下所述:
一种制备上述含甲硝唑吡唑类骨架的抗菌化合物的方法,它包括以下步骤:
步骤i.将草酸二甲酯(16mmol)和一系列取代的苯乙酮(8mmol)溶于20ml无水甲醇中配成溶液备用,缓慢地向溶有甲醇钠(16mmol)的甲醇(5mL)溶液中滴加配制好的溶液,在回流条件下搅拌5小时。待反应液冷却到室温倒至50mL的蒸馏水中,滴加稀盐酸(1M)到反应溶液至pH=3、4。并用乙酸乙酯(100mL)洗三次,取有机相用无水硫酸钠除水,并用混有2∶1的乙醇-石油醚溶液洗,真空干燥得到粗产物1a-1j。
步骤ii.将化合物1a-1j(1mmol)和不同取代的苯肼(2mmol)溶于12mL的无水甲醇中,搅拌条件下滴加1mL稀盐酸催化,回流条件下搅拌反应6小时,待反应液冷却至室温,用饱和食盐水、乙酸乙酯(各100mL)洗三次,柱层析提纯得到化合物2a-2y。
步骤iii.向干净的圆底烧瓶中加入甲醇溶液10ml,再加入合成的化合物2a-2y(0.5mmol),KOH(1.75mmol),再逐滴加入蒸馏水(1mL)。回流搅拌2小时,TLC跟踪反应,反应结束后,反应液倒进150mL蒸馏水并用稀盐酸(1M)调pH至3或4,乙酸乙酯萃取得到化合物3a-3y。
步骤iv.将化合物3(0.4mmol)、EDC(0.48mmol)、HOBT(0.48mmol)、DMAP(0.2mmol)依次溶于10mL的二氯甲烷中,45℃活化半小时后添加甲硝唑(0.52mmol)至反应液中,过夜。反应结束后,饱和食盐水和二氯甲烷(各50mL)洗三次,通过柱层析或者用乙醇重结晶得到目标化合物4a-4y。
实施例一:
2-(2,5-二甲基-1H-咪唑-1-基)乙基-5-(4-甲氧苯基)-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯(4a)的制备
向圆底烧瓶中加入20ml无水甲醇溶解草酸二甲酯(16mmol),4-甲氧基苯乙酮(8mmol),配成的溶液缓慢地加入溶有甲醇钠(16mmol)的甲醇(5mL)溶液中,在回流条件下搅拌5小时。反应液冷却到室温倒至50mL的蒸馏水中,滴加稀盐酸(1M)到反应溶液至pH=3、4。并用乙酸乙酯(100mL)洗三次,取有机相用无水硫酸钠除水,并用混有2∶1的乙醇-石油醚溶液洗再真空干燥得到粗产物1a。将上述合成的化合物1a(1mmol)和4-甲基苯肼(2mmol)溶于12mL的无水甲醇中,搅拌条件下滴加1mL稀盐酸催化,回流条件下搅拌反应6小时,待反应液冷却至室温,用饱和食盐水、乙酸乙酯(各100mL)洗三次,柱层析提纯得到化合物2a。向干净的圆底烧瓶中加入甲醇溶液10ml,再加入合成的化合物2a(0.5mmol),KOH(1.75mmol),再逐滴加入蒸馏水(1mL)。回流搅拌2小时,TLC跟踪反应,反应结束后,反应液倒至150mL蒸馏水并用稀盐酸(1M)调pH至3或4,乙酸乙酯萃取得到化合物3a。将合成好的化合物3a(0.4mmol)、EDC(0.48mmol)、HOBT(0.48mmol)、DMAP(0.2mmol)依次溶于10mL的二氯甲烷中,45℃活化半小时后添加甲硝唑(0.52mmol)至反应液中,过夜。反应结束后,饱和食盐水和二氯甲烷(各50mL)洗三次,通过柱层析或者用乙醇重结晶得到目标化合物4a。得淡黄色粉末,产率72.2%,m.p.167-168℃.1H NMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.28-7.16(m,6H,ArH),6.95-6.92(m,3H,ArH),4.72(t,J=2.4Hz,2H,CH2),4.66(t,J=2.3Hz,2H,CH2),3.76(s,3H,OCH3),3.34(s,3H,CH3),2.54(s,3H,CH3).MS(EI):461.48(C24H23N5O5,[M]+).
实施例二:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1,5-二-对甲苯基-1H-吡唑-3-羧酸乙酯(4b)的制备
制备方法参考实施例一。得淡黄色粉末,产率67.8%,m.p.179-180℃.1HNMR(DMSO-d6,400MHz)δ:8.04(s,1H,ArH),7.26(d,J=8.2Hz,2H,ArH),7.20-7.14(m,4H,ArH),7.12(d,J=8.1Hz,2H,ArH),6.97(s,1H,ArH),4.72(t,J=4.6Hz,2H,CH2),4.65(s,J=4.7Hz,2H,CH2),2.53(s,3H,CH3),2.34(s,3H,CH3),2.29(s,3H,CH3).MS(EI):445.48(C24H23N5O4,[M]+).
实施例三:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(4-氟苯基)-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯(4c)的制备
制备方法参考实施例一。得淡黄色粉末,产率82.9%,m.p.168-169℃.1HNMR(DMSO-d6,400MHz)δ:8.04(s,1H,ArH),7.44(d,J=8.5Hz,2H,ArH),7.27(dd,J=8.2,6.4Hz,4H,ArH),7.19(d,J=8.3Hz,2H,ArH),7.07(s,1H,ArH),4.72(t,J=4.9Hz,2H,CH2),4.65(t,J=4.2Hz,2H,CH2),2.53(s,3H,CH3),2.35(s,3H,CH3).MS(EI):449.44(C23H20FN5O4,[M]+).
实施例四:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(4-氯苯基)-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯(4d)的制备
制备方法参考实施例一。得淡黄色粉末,产率79.6%,m.p.172-173℃.1HNMR(DMSO-d6,400MHz)δ:8.04(s,1H,ArH),7.44(d,J=8.0Hz,2H,ArH),7.32-7.16(m,6H,ArH),7.07(s,1H,ArH),4.69(dd,J=21.9,3.8Hz,4H,CH2),2.53(s,3H,CH3),2.35(s,3H,CH3).MS(EI):465.89(C23H20ClN5O4,[M]+).
实施例五:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(4-碘苯基)-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯(4e)的制备
制备方法参考实施例一。得淡黄色粉末,产率74.8%,m.p.192-193℃.1HNMR(DMSO-d6,400MHz)δ:8.03(s,1H,ArH),7.73(d,J=8.2Hz,2H,ArH),7.27(d,J=8.1Hz,2H,ArH),7.19(d,J=8.1Hz,2H,ArH),7.07-6.98(m,3H,ArH),4.68(dd,J=22.6,4.7Hz,4H,CH2),2.53(s,3H,CH3),2.35(s,3H,CH3).MS(EI):557.35(C23H20IN5O4,[M]+).
实施例六:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(4-甲氧基苯基)-1-苯基-1H-吡唑-3-羧酸乙酯(4f)的制备
制备方法参考实施例一。得淡黄色粉末,产率68.3%,m.p.142-143℃.1HNMR(DMSO-d6,400MHz)δ:8.04(s,1H,ArH),7.46(d,J=6.3Hz,3H,ArH),7.31(d,J=7.6Hz,2H,ArH),7.16(d,J=8.5Hz,2H,ArH),7.00-6.85(m,3H,ArH),4.69(dd,J=24.6,4.7Hz,4H,CH2),3.75(s,3H,OCH3),2.54(s,3H,CH3).MS(EI):447.45(C23H21N5O5,[M]+).
实施例七:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-苯基-5-(对甲苯基)-1H-吡唑-3-羧酸乙酯(4g)的制备
制备方法参考实施例一。得淡黄色粉末,产率71.6%,m.p.163-164℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.46(d,J=5.9Hz,3H,ArH),7.36-7.26(m,2H,ArH),7.14(dd,J=18.5,8.0Hz,4H,ArH),7.00(s,1H,ArH),4.69(dd,J=22.9,4.7Hz,4H,CH2),2.54(s,3H,CH3),2.29(s,3H,CH3).MS(EI):431.45(C23H21N5O4,[M]+).
实施例八:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(4-氟苯基)-1-苯基-1H-吡唑-3-羧酸乙酯(4h)的制备
制备方法参考实施例一。得淡黄色粉末,产率70.9%,m.p.160-161℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.50-7.42(m,5H,ArH),7.34-7.29(m,2H,ArH),7.26(d,J=8.5Hz,2H,ArH),7.10(s,1H,ArH),4.69(dd,J=20.1,4.8Hz,4H,CH2),2.54(s,3H,CH3).MS(EI):435.42(C22H18FN5O4,[M]+).
实施例九:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(4-氯苯基)-1-苯基-1H-吡唑-3-羧酸乙酯(4i)的制备
制备方法参考实施例一。得淡黄色粉末,产率77.6%,m.p.164-165℃.1HNMR(DMSO-d6,400MHz)δ:8.04(s,1H,ArH),7.50-7.42(m,5H,ArH),7.32(d,J=7.8Hz,2H,ArH),7.26(d,J=8.4Hz,2H,ArH),7.10(s,1H,ArH),4.69(dd,J=20.0,4.8Hz,4H,CH2),2.54(s,3H,CH3).MS(EI):451.87(C22H18ClN5O4,[M]+).
实施例十:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(4-碘苯基)-1-苯基-1H-吡唑-3-羧酸乙酯(4j)的制备
制备方法参考实施例一。得淡黄色粉末,产率75.3%,m.p.152-153℃.1HNMR(DMSO-d6,400MHz)δ:8.04(s,1H,ArH),7.73(d,J=8.0Hz,2H,ArH),7.47(s,3H,ArH),7.32(d,J=5.1Hz,2H,ArH),7.10-6.98(m,3H,ArH),4.69(dd,J=20.7,3.9Hz,4H,CH2),2.53(s,3H,CH3).MS(EI):543.32(C22H18IN5O4,[M]+).
实施例十一:
2-(2-甲基-5-硝基-1H-咪唑-1-基)乙基-1-(4-氯苯基)-5-(4-甲氧基苯基)-1H-吡唑-3-羧酸乙酯(4k)的制备
制备方法参考实施例一。得淡黄色粉末,产率78.8%,171-172℃.1H NMR(CDCl3,400MHz)δ:7.98(s,1H,ArH),7.34(d,J=8.8Hz,2H,ArH),7.26(d,J=3.5Hz,2H,ArH),7.12(d,J=8.7Hz,2H,ArH),6.87(t,3H,ArH),4.73(s,4H,CH2),3.82(s,3H,OCH3),2.60(s,3H,CH3).MS(EI):481.89(C23H20ClN5O5,[M]+).
实施例十二:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-(对甲苯基)-1H-吡唑-3-羧酸乙酯(4l)的制备
制备方法参考实施例一。得淡黄色粉末,产率65.8%,m.p.185-186℃.1HNMR(CDCl3,400MHz)δ:7.98(s,1H,ArH),7.34(d,J=8.8Hz,2H,ArH),7.26(d,J=4.4Hz,2H,ArH),7.15(d,J=8.0Hz,2H,ArH),7.08(d,J=8.1Hz,2H,ArH),6.93(s,1H,ArH),4.73(s,4H,CH2),2.60(s,3H,CH3),2.36(s,3H,CH3).MS(EI):465.89(C23H20ClN5O4,[M]+).
实施例十三:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-(4-氟苯基)-1H-吡唑-3-羧酸乙酯(4m)的制备
制备方法参考实施例一。得淡黄色粉末,产率71.9%,m.p.190-191℃.1HNMR(CDCl3,400MHz)δ:7.98(s,1H,ArH),7.39-7.31(m,4H,ArH),7.26(d,J=9.9Hz,2H,ArH),7.13(d,J=8.5Hz,2H,ArH),6.96(s,1H,ArH),4.74(s,4H,CH2),2.60(s,3H,CH3).MS(EI):469.86(C22H17ClFN5O4,[M]+).
实施例十四:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1,5-双(4-氯苯基)-1H-吡唑-3-羧酸乙酯(4n)的制备
制备方法参考实施例一。得淡黄色粉末,产率80.2%,m.p.192-193℃.1HNMR(CDCl3,400MHz)δ:7.98(s,1H,ArH),7.39-7.31(m,4H,ArH),7.23(d,J=8.7Hz,2H,ArH),7.13(d,J=8.5Hz,2H,ArH),6.96(s,1H,ArH),4.74(s,4H,CH2),2.60(s,3H,CH3).MS(EI):486.31(C22H17Cl2N5O4,[M]+).
实施例十五:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-(4-碘苯基)-1H-吡唑-3-羧酸乙酯(4o)的制备
制备方法参考实施例一。得黄色粉末,产率76.9%,m.p.219-220℃.1HNMR(CDCl3,400MHz)δ:7.98(s,1H,ArH),7.69(d,J=8.4Hz,2H,ArH),7.37(d,J=8.8Hz,2H,ArH),7.23(d,J=8.8Hz,2H,ArH),6.99-6.90(m,3H,ArH),4.74(s,4H,CH2),2.60(s,3H,CH3).MS(EI):577.76(C22H17ClIN5O4,[M]+).
实施例十六:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-苯基-5-(邻甲苯基)-1H-吡唑-3-羧酸乙酯(4p)的制备
制备方法参考实施例一。得淡黄色粉末,产率66.5%,m.p.172-173℃.1HNMR(CDCl3,400MHz)δ:7.99(s,1H,ArH),7.34-7.27(m,4H,ArH),7.25-7.21(m,2H,ArH),7.19(d,J=7.4Hz,3H,ArH),4.75(s,4H,CH2),2.65(s,3H,CH3).MS(EI):431.45(C23H21N5O4,[M]+).
实施例十七:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-(邻甲苯基)-1H-吡唑-3-羧酸乙酯(4q)的制备
制备方法参考实施例一。得淡黄色粉末,产率78.1%,m.p.153-154℃.1HNMR(CDCl3,400MHz)δ:7.99(s,1H,ArH),7.36-7.24(m,4H,ArH),7.21(d,J=7.5Hz,2H,ArH),7.19-7.15(m,3H,ArH),4.74(s,4H,CH2),2.63(s,3H,CH3),1.98(s,3H,CH3).MS(EI):465.89(C23H20ClN5O4,[M]+).
实施例十八:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1,5-二苯基-1H-吡唑-3-羧酸乙酯(4r)的制备
制备方法参考实施例一。得淡黄色粉末,产率69.7%,m.p.159-160℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.48-7.44(m,3H,ArH),7.38-7.29(m,5H,ArH),7.26-7.22(m,2H,ArH),7.05(s,1H,ArH),4.69(dd,J=21.2,4.8Hz,4H,CH2),2.54(s,3H,CH3).MS(EI):417.43(C22H19N5O4,[M]+).
实施例十九:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-苯基-1H-吡唑-3-羧酸乙酯(4s)的制备
制备方法参考实施例一。得淡黄色粉末,产率75.1%,m.p.169-170℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.54(d,J=6.9Hz,2H,ArH),7.42-7.24(m,7H,ArH),7.06(s,1H,ArH),4.69(d,J=23.4Hz,4H,CH2),2.54(s,3H,CH3).MS(EI):451.87(C22H18ClN5O4,[M]+).
实施例二十:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(2-氟苯基)-1-苯基-1H-吡唑-3-羧酸乙酯(4t)的制备
制备方法参考实施例一。得淡黄色粉末,产率69.8%,m.p.147-148℃.1HNMR(DMSO-d6,400MHz)δ8.05(s,1H,ArH),7.54-7.36(m,5H,ArH),7.31-7.22(m,4H,ArH),7.07(s,1H,ArH),4.76-4.71(m,2H,CH2),4.69-4.64(m,2H,CH2),2.55(s,3H,CH3).MS(EI):435.42(C22H18FN5O4,[M]+).
实施例二十一:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-(2-氟苯基)-1H-吡唑-3-羧酸乙酯(4u)的制备
制备方法参考实施例一。得淡黄色粉末,产率79.4%,m.p.188-189℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.52(d,J=8.7Hz,3H,ArH),7.43(t,J=6.9Hz,1H,ArH),7.35-7.25(m,4H,ArH),7.09(s,1H,ArH),4.70(dd,J=20.9,4.8Hz,4H,CH2),2.55(s,3H,CH3).MS(EI):469.86(C22H17ClFN5O4,[M]+).
实施例二十二:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-苯基-5-(间甲苯基)-1H-吡唑-3-羧酸乙酯(4v)的制备
制备方法参考实施例一。得淡黄色粉末,产率72.5%,m.p.106-107℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.48-7.29(m,5H,ArH),7.24-7.13(m,3H,ArH),7.03(s,1H,ArH),6.95(d,J=7.1Hz,1H,ArH),4.69(dd,J=21.2,4.7Hz,4H,CH2),2.54(s,3H,CH3),2.24(s,3H,CH3).MS(EI):431.45(C23H21N5O4,[M]+).
实施例二十三:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-(间甲苯基)-1H-吡唑-3-羧酸乙酯(4w)的制备
制备方法参考实施例一。得淡黄色粉末,产率73.2%,178-179℃.1H NMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.54(d,J=8.4Hz,2H,ArH),7.33(d,J=8.5Hz,2H,ArH),7.26-7.15(m,3H,ArH),7.03(s,1H,ArH),6.96(d,J=7.1Hz,1H,ArH),4.69(dd,J=21.1,4.5Hz,4H,CH2),2.54(s,3H,CH3),2.26(s,3H,CH3).MS(EI):465.89(C23H20ClN5O4,[M]+).
实施例二十四:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-5-(3-氟苯基)-1-苯基-1H-吡唑-3-羧酸乙酯(4x)的制备
制备方法参考实施例一。得淡黄色粉末,产率80.6%,m.p.123-124℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.51-7.36(m,4H,ArH),7.36-7.11(m,5H,ArH),7.04(d,J=7.8Hz,1H,ArH),4.69(dd,J=18.5,4.8Hz,4H,CH2),2.54(s,3H,CH3).MS(EI):435.42(C22H18FN5O4,[M]+).
实施例二十五:
2-(2-甲基-5-硝基-1H-咪唑-1-基)-乙基-1-(4-氯苯基)-5-(3-氟苯基)-1H-吡唑-3-羧酸乙酯(4y)的制备
制备方法参考实施例一。得淡黄色粉末,产率74.4%,m.p.165-166℃.1HNMR(DMSO-d6,400MHz)δ:8.05(s,1H,ArH),7.56(d,J=8.7Hz,2H,ArH),7.47-7.33(m,3H,ArH),7.30-7.12(m,3H,ArH),7.05(d,J=7.8Hz,1H,ArH),4.69(dd,J=18.5,4.8Hz,4H,CH2),2.54(s,3H,CH3).MS(EI):469.86(C22H17ClFN5O4,[M]+).
上述含甲硝唑吡唑类骨架的抗菌化合物的生物活性评价方法,包括体外抗菌活性检测和体外细胞毒性检测两个方面。具体说明如下:
实施例一:含甲硝唑吡唑类骨架衍生物的体外抗菌活性研究
采用MH稀释法来测定含甲硝唑吡唑类骨架的衍生物对两株格兰仕阳性菌:枯草芽孢杆菌(Bacillus subtilis ATCC 6633)、金黄色葡萄球菌(Staphylococcus aureus ATCC 6538)以及两株格兰仕阴性菌:大肠杆菌(Escherichia coliATCC 35218)、铜绿假单胞菌(PseudomonasaeruginosaATCC 13525)的最小抑菌浓度,即MIC(minimuminhibitory concentration)。
(1)培养基的配制:24gMH肉汤培养基溶于1000mL的蒸馏水中,灭菌备用。
(2)四种细菌株的活化:从实验室冻存库中取实验用菌分别接种到适量的上述培养基中,在37℃培养箱中培养过夜。
(3)四种细菌株的培养:将活化的菌株接种到适量的上述培养基中,在37℃培养箱中培养,通过测OD600值监测菌种的生长状态,当OD600=0.5左右时菌株处于生长对数期,取该时期细菌备用。
(4)药物存储液配制:取1mg的药物(包括合成的化合物、青霉素、氯霉素和甲硝唑)溶解在1mL的2%的DMSO中,配制成的药物存储液浓度为1mg/L,放在-20℃冰箱中保存备用。
(5)96孔培养板准备:取灭菌后的MH培养基加入到96孔培养板中,每行第一个孔150μL,剩余孔100μL。取50μL的药物存储液加入到第一个孔,吹打均匀后吸取100μL到第二个孔,吹打三次后吸取100μL到后一个孔,反复至第11个孔,吸100μL混有药物的培养基弃去,第12个孔设置为阴性对照。取对数期的细菌用培养基稀释到OD600=0.1加入到96孔培养板中(100μL/孔),在37℃培养箱中培养12-18h。
(6)抗菌活性检测:使用比浊法对已培养了12-18h后的96孔板进行统计,经过稀释之后第一个药物终浓度为125μg/mL,依次对半稀释。统计以在小孔内完全抑制细菌生长的最低药物浓度为MIC。当对照孔(即不含抗生素)内细菌明显生长试验才有意义。重复三次,统计MIC平均值,制成如下表:
从上述实验可知:本发明对两株格兰仕阳性菌:枯草芽孢杆菌(Bacillussubtilis ATCC 6633)、金黄色葡萄球菌(Staphylococcus aureus ATCC 6538)以及两株格兰仕阴性菌:大肠杆菌(Escherichia coliATCC 35218)、铜绿假单胞菌(Pseudomonasaeruginosa ATCC 13525)有很好的抑制作用,尤其是对格兰仕阴性菌。与阳性对照组相比,抑菌效果明显。
实施例二:含甲硝唑吡唑类骨架衍生物的体外抗菌活性研究
本发明测试了新合成化合物4a-4y对人肾上皮细胞(293T)的细胞毒性,每个化合物的毒性用抑制293T细胞存活率到50%时的浓度(CC50)来表示。
实验方法:
(1)药物存储液配制:取m=2mg左右的已合成化合物溶于200μL的2%DMSO中,放在-20℃保存备用。
(2)培养基准备:DMEM基础培养基(89%)+小牛血清(10%)+抗生素(1%)混合均匀,备用。
(3)用上述培养基培养人肾上皮细胞(293T)直至达到其对数生长期末细胞趋于融合,用细胞消化液消化分散细胞,用细胞培养液配制成1×104个/mL的细胞悬液。取96孔培养板,每孔中加入100μL的细胞悬液。轻轻水平转动培养板使细胞均匀地分散在皿孔的表面。置于含5%CO2细胞培养箱中,在37±0.5℃温度下培养12h。
(4)药物浓度配制:通过公式计算,吸取相对应体积的存储液到500μL的培养基中,配制药物浓度为200μg/mL。然后用培养基稀释浓度到100、50、25、10、5、1、0.1μg/mL等浓度梯度,各吸取100μL(×3)到各自的孔中。培养板放置在37±0.5℃,5%CO2的培养箱下进行培养48h。
(5)每个培养间期后,每孔加入MTT溶液20μL,置于含5%CO2培养箱中,在37±0.5℃温度下培养4h。
(6)弃去孔内液体,每孔分别加入200μL DMSO,将培养板放置10min,水平摇晃使孔内溶液颜色均匀。
(7)用酶标仪测定吸光度,波长采用570nm。
测得的CC50见下表所示:
从上述实验可知:本发明对人肾上皮细胞(293T)表现出了相当的细胞安全性,可以用于制取抗菌药物。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种等同变换,这些等同变换均属于本发明的保护范围。另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合。为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
Claims (2)
1.一类含甲硝唑吡唑类骨架的衍生物,其结构如式所示:
其中,R1选自:
-H、2-CH3、3-CH3、4-CH3、4-OCH3、2-F、3-F、4-F、4-Cl、4-I
R2选自:
-H、4-CH3、4-Cl。
2.制备方法包括如下步骤:
步骤1.回流条件下,向反应容器中依次加入草酸二甲酯和结构如式A所示的化合物,在含有甲醇钠的无水甲醇中反应。TLC跟踪反应,充分反应后,得到结构如式B所示的化合物。
步骤2.向反应容器中依次加入结构如式B所示的化合物以及结构如式C所示的化合物,稀盐酸作催化,在无水甲醇体系中回流,TLC跟踪反应,充分反应后,得到结构如式D所示的化合物。
步骤3.向反应容器中加入结构如式D所示的化合物,在搅拌的情况下加入氢氧化钾,并滴加蒸馏水使其充分反应,得到结构如式E所示的化合物。
步骤4.在二氯甲烷反应体系中依次加入结构如式E所示的化合物、EDC、HOBT、DMAP活化一段时间,再缓慢加入甲硝唑,于45℃反应一段时间,得到目标终产物,即结构如式E所示的化合物。
式A~F中,R1选自-H、2-CH3、3-CH3、4-CH3、4-OCH3、2-F、3-F、4-F、4-Cl、4-I;R2选自-H、4-CH3、4-Cl。
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