CN112707921A - 一种酶抑制剂的荧光标记的合成 - Google Patents
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Abstract
本发明本发明将非甾体抗炎药吲哚美辛转与罗丹明B反应形成缀合物,该缀合物能够选择性和有效地抑制两种纯化蛋白中的COX‑2和完整的细胞。这些不会显着抑制COX‑1的结合物可在体内炎症性病变和表达COX‑2的肿瘤中对COX‑2进行光学成像。该研究用于优化来自多种NSAID或COX‑2选择性抑制剂(COXIB)的荧光缀合物。将缀合物评估为靶向COX‑2的成像剂,并在体外和体内验证活性分子作为靶向COX‑2的细胞和肿瘤制剂。这为我们提供了一种荧光标记的光学成像剂,该荧光剂经验证可用于靶向COX‑2的炎症和癌症体内成像。本发明以吲哚美辛为原料,经与己二胺缩合,所得产物再与罗丹明B进行缩合。本发明提供了一种操作简便、原料易得,杂质含量少、生产成本低的在COX‑2抑制剂吲哚美辛上引入荧光基团的合成方法,在保证其基本骨架结构的基础上引入荧光基团,该化合物可自发荧光,为其在细胞生物学中的示踪应用。
Description
技术领域
本发明属于药物化学领域
背景技术
叶酸是所有生物体生长的必需维生素。还原形式四氢叶酸是合成嘌呤,氨基酸,S-腺苷甲硫氨酸,胸苷单磷酸和甲酰基甲硫氨酸所需的碳转移反应的关键辅助因子。哺乳动物和高等真核生物依赖于饮食中的叶酸,而植物和大多数微生物是从头合成的。靶向叶酸合成途径的二氢蝶呤合酶(DHPS)的磺胺药物在数十年后仍在临床中使用,即使在今天,磺胺类药物仍与细菌二氢叶酸还原酶(DHFR)抑制剂共同使用,作为一种协同的广谱抗菌药物,可预防或治疗多种疾病和感染,包括疟疾,弓形虫脑炎,卡氏肺孢子虫肺炎,肺结核和金黄色葡萄球菌感染。
金黄色葡萄球菌的耐甲氧西林菌株(MRSA)(“超级细菌”)的出现突出了抗生素耐药性问题的严重性。最初仅限于医院环境,最近又传播到社区,并开始影响那些没有任何危险因素的人。寻找有效的未来治疗方法已引起人们对新型抗菌药物的替代酶标靶以及新型免疫策略的兴趣。有文献报道基于6-羟甲基-7,8-二氢蝶呤焦磷酸激酶(HPPK)抑制剂的开发,该酶是叶酸合成途径中位于DHPS之前的酶,负责催化焦磷酸酰基从结合镁的ATP辅助因子转移至6-羟甲基-7,8-二氢蝶呤(HMDP)。HPPK不是任何现有抗生素的已知靶标,没有相关的人类同系物,并且具有可开发可对当前和未来的SMX/TMP耐药病原体有效的抗菌药物的位点,目前发现8-巯基鸟嘌呤(8MG)是金黄色葡萄球菌HPPK(SaHPPK)的抑制剂。
如上所述,6-羟甲基-7,8-二氢蝶呤焦磷酸激酶(HPPK)抑制剂有着如此巨大的科研和生物医学潜力,但其作用机理并没有完全清晰,本发明在8-巯基鸟嘌呤结构的基础上引入罗丹明B荧光基团,使8-巯基鸟嘌呤可自发荧光,以便被示踪。
发明内容
本发明要解决的技术问题在于提供一种操作简便、原料易得,杂质含量少、生产成本低的8-巯基鸟嘌呤罗丹明合物的合成方法。
本发明提供的8-巯基鸟嘌呤罗丹明合物的技术方案如下:
罗丹明B酰肼的制备方法:
将罗丹明B(2g,4.2mmol)溶解于15mL乙醇的溶液中,随后加入过量的水合肼(2.5mL),然后将反应溶液回流直至粉红色消失。之后,将冷却的反应溶液倒入蒸馏水中,并用乙酸乙酯(3×30mL)萃取。合并的萃取物用无水硫酸钠干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱法进一步纯化,用石油醚/CH2Cl2=1∶1洗脱,得到罗丹明B酰肼(1.0g,2.2mmol),为白色固体。产率:52.4%。反应式如下:
在0℃下向溶解于15mL无水CHCl3中的罗丹明B酰肼(815mg,1.8mmol)的溶液中添加三乙胺(332μL,2.4mmol),并将反应溶液搅拌几分钟。然后,滴加2-氯乙酰氯(180μL,2.4mmol)于5mL无水CHCl3中的溶液。之后,将混合物加热至室温并搅拌4小时。然后将反应溶液倒入蒸馏水中,并用CH2Cl2(3×30mL)萃取。合并的有机层依次用饱和NaCl水溶液(50mL)洗涤,并用无水Na2SO4干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱进一步纯化,用石油醚/乙酸乙酯=3∶1洗脱,得到Rh-2(860mg,1.6mmol),为白色固体。产率:88.9%。反应式如下:
2.将罗丹明荧光基团引入6-羟甲基-7,8-二氢蝶呤焦磷酸激酶(HPPK)抑制剂8-巯基鸟嘌呤中,其具体合成方法为:
将Rh-2(530mg,1mmol)溶解于15m四氢呋喃中,随后加入过量的三乙胺(3mmol),再加入8-巯基鸟嘌呤,随后加热回流,反应过夜。之后,将冷却的反应溶液倒入蒸馏水中,并用乙酸乙酯(3×30mL)萃取。合并的萃取物用无水硫酸钠干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱法进一步纯化,用CH2Cl2/MeOH=20∶1洗脱,得到8-巯基鸟嘌呤罗丹明合物(366mg,0-54mmol),为白色固体。产率:54%。反应式如下:
3.上述的8-巯基鸟嘌呤罗丹明合物,其特征在于,该化合物可以自发荧光,应用于细胞生物学中的示踪。
具体实施方式
下面结合实施例对本发明作进一步说明,但本发明的保护范围并不限于此
实施例1
罗丹明B酰肼的合成:将罗丹明B(2g,4.2mmol)溶解于15mL乙醇的溶液中,随后加入过量的水合肼(2.5mL),然后将反应溶液回流直至粉红色消失。之后,将冷却的反应溶液倒入蒸馏水中,并用乙酸乙酯(3×30mL)萃取。合并的萃取物用无水硫酸钠干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱法进一步纯化,用石油醚/CH2Cl2=1∶1洗脱,得到罗丹明B酰肼(1.0g,2.2mmol),为白色固体。1H NMR(400MHz,CDCl3)δ7.94(d,J=8.0Hz,1H),7.51-7.38(m,2H),7.11(d,J=7.4Hz,1H),6.46(d,J=8.8Hz,2H),6.42(s,2H),6.29(d,J=8.8Hz,2H),3.61(s,2H),3.34(q,J=7.0Hz,8H),1.17(t,J=7.0Hz,12H)。
Rh-2的合成:在0℃下向溶解于15mL无水CHCl3中的罗丹明B酰肼(815mg,1.8mmol)的溶液中添加三乙胺(332μL,2.4mmol),并将反应溶液搅拌几分钟。然后,滴加2-氯乙酰氯(180μL,2.4mmol)于5mL无水CHCl3中的溶液。之后,将混合物加热至室温并搅拌4小时。然后将反应溶液倒入蒸馏水中,并用CH2Cl2(3×30mL)萃取。合并的有机层依次用饱和NaCl水溶液(50mL)洗涤,并用无水Na2SO4干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱进一步纯化,用石油醚/乙酸乙酯=3∶1洗脱,得到Rh-2(860mg,1.6mmol),为白色固体。产率:88.9%。1H NMR(400MHz,DMSO)δ9.92(s,1H),7.83(d,J=7.6Hz,1H),7.62-7.48(m,2H),7.02(d,J=6.7Hz,1H),6.55-6.45(m,2H),6.34(dd,J=7.2,2.3Hz,4H),4.00(s,2H),3.32(q,J=7.0Hz,8H),1.08(t,J=7.0Hz,12H)。
8-巯基鸟嘌呤罗丹明合物的合成:将Rh-2(530mg,1mmol)溶解于15m四氢呋喃中,随后加入过量的三乙胺(3mmol),再加入8-巯基鸟嘌呤,随后加热回流,反应过夜。之后,将冷却的反应溶液倒入蒸馏水中,并用乙酸乙酯(3×30mL)萃取。合并的萃取物用无水硫酸钠干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱法进一步纯化,用CH2Cl2/MeOH=20∶1洗脱,得到8-巯基鸟嘌呤罗丹明合物(366mg,0-54mmol),为白色固体。产率:54%。1H NMR(600MHz,DMSO-d6)δ10.50(s,1H),8.69(s,1H),7.82-7.76(m,1H),7.52-7.45(m,2H),7.01-6.95(m,1H),6.36(s,5H),6.04(s,2H),3.49(s,2H),3.34-3.24(m,7H),3.08(t,J=7.3Hz,2H),2.78(q,J=6.7Hz,2H),1.06(t,J=7.0Hz,12H)。
Claims (3)
1.罗丹明B酰肼的制备方法:
将罗丹明B(2g,4.2mmol)溶解于15mL乙醇的溶液中,随后加入过量的水合肼(2.5mL),然后将反应溶液回流直至粉红色消失。之后,将冷却的反应溶液倒入蒸馏水中,并用乙酸乙酯(3×30mL)萃取。合并的萃取物用无水硫酸钠于燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱法进一步纯化,用石油醚/CH2Cl2=1∶1洗脱,得到罗丹明B酰肼(1.0g,2.2mmol),为白色固体。产率:52.4%。反应式如下:
在0℃下向溶解于15mL无水CHCl3中的罗丹明B酰肼(815mg,1.8mmol)的溶液中添加三乙胺(332μL,2.4mmol),并将反应溶液搅拌几分钟。然后,滴加2-氯乙酰氯(180μL,2.4mmol)于5mL无水CHCl3中的溶液。之后,将混合物加热至室温并搅拌4小时。然后将反应溶液倒入蒸馏水中,并用CH2Cl2(3×30mL)萃取。合并的有机层依次用饱和NaCl水溶液(50mL)洗涤,并用无水Na2SO4干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱进一步纯化,用石油醚/乙酸乙酯=3∶1洗脱,得到Rh-2(860mg,1.6mmol),为白色固体。产率:88.9%。反应式如下:
2.根据权利要求1所述的Rh-2的合成方法,其特征在于,将罗丹明荧光基团引入6-羟甲基-7,8-二氢蝶呤焦磷酸激酶(HPPK)抑制剂8-巯基鸟嘌呤中,其具体合成方法为:
将Rh-2(530mg,1mmol)溶解于15m四氢呋喃中,随后加入过量的三乙胺(3mmol),再加入8-巯基鸟嘌呤,随后加热回流,反应过夜。之后,将冷却的反应溶液倒入蒸馏水中,并用乙酸乙酯(3×30mL)萃取。合并的萃取物用无水硫酸钠干燥。减压除去溶剂,并将残余物通过硅胶上的柱色谱法进一步纯化,用CH2Cl2/MeOH=20∶1洗脱,得到8-巯基鸟嘌呤罗丹明合物(366mg,0-54mmol),为白色固体。产率:54%。反应式如下:
3.根据权利要求2所述的8-巯基鸟嘌呤罗丹明合物的合成方法,其特征在于,该化合物可以自发荧光,应用于细胞生物学中的示踪。
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Application publication date: 20210427 |