CN107235894B - 具有抗耐药菌活性的季铵查尔酮衍生物、其制备方法及应用 - Google Patents

具有抗耐药菌活性的季铵查尔酮衍生物、其制备方法及应用 Download PDF

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CN107235894B
CN107235894B CN201710474526.7A CN201710474526A CN107235894B CN 107235894 B CN107235894 B CN 107235894B CN 201710474526 A CN201710474526 A CN 201710474526A CN 107235894 B CN107235894 B CN 107235894B
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张恩
秦上尚
楚文超
白鹏燕
杨兆青
崔得运
化永刚
王铭铭
王亚娜
刘宏民
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Abstract

本发明属于药物化学领域，公开了具有抗耐药菌活性的季铵查尔酮衍生物及其合成方法和应用。其结构式如下，本发明通过体外抗菌活性实验和红细胞溶血实验证明，该系列大部分化合物对革兰氏阳性菌金黄色葡萄球菌和大肠粪肠球菌，革兰氏阴性菌大肠埃希菌和铜绿假单胞菌均表现出良好的抑菌效果和选择性。选择的部分化合物对包括甲氧西林耐药的金黄色葡萄球菌（MRSA）、万古霉素耐药的肠球菌（VRE）、产碳青霉烯酶的肠杆菌科细菌（CRE）和携带NDM基因的耐药菌(NDM)在内的多种“超级细菌”也表现出优异的抗菌活性，其中对MRSA表现出极好的活性。体外红细胞毒性实验也显示该系列化合物具有较小的红细胞毒性，该系列化合物可以作为新的抗菌候选药物。

Description

具有抗耐药菌活性的季铵查尔酮衍生物、其制备方法及应用
技术领域
本发明属于药物化学技术领域，公开了具有抗耐药菌活性的季铵查尔酮衍生物、它们的制备方法及其作为一类新型的抗耐药菌药物的应用。
背景技术
20世纪30年代世界上首个抗生素-青霉素问世，给无数病人和外科医生带来福音。随着科技的发展，加之人们对抗生素认知上的错误，加速了抗生素滥用，导致细菌获得性耐药问题日益严重，耐药菌的比例逐年上升。据2016上半年度卫生部全国细菌耐药监测结果显示，全国30所医院细菌耐药性试验显示MRSA对75％的常见抗菌药物具有耐药性，对青霉素和苯唑西林的耐药性达到100％。继青霉素之后科研人员又相继发现合成多种抗生素，包括青霉素类、头孢菌素类、β-内酰胺酶抑制剂、氨基糖苷类等，但均表现出不同程度的耐药性。而万古霉素作为对抗革兰阳性菌的“最后一道防线”，也出现了耐药倾向(Science,2008,321,356-361.)。因此研发一种新型具有抗耐药性药物成为目前的迫切需要。
1981年瑞典科学家Boman首次发现，通过用蜡状芽孢杆菌诱导天蚕后产生了一种具有抗菌活性的多肽类物质-天蚕素(Nature,198l,292:246-248.)。随后又发现多种抗菌肽如magainins(FEBs Letters,1989,259,103-106.)等。科学家通过分析多种天然抗菌肽，发现抗菌肽通常由12-50个氨基酸残基组成，相对分子质量小，带有疏水基团，通过疏水基团破坏细菌膜完整性，从而达到杀死细菌的目的。由于其带正电荷，能够与细菌膜表面的阴离子结合增强与细菌膜的结合能力，然而由于天然抗菌肽生产成本高，不容易批量生产，体内易被降解，部分抗菌肽毒性大，选择性弱。因此，有不少研究人员期望根据天然抗菌肽的两亲性结构特征，合成抗菌肽模拟物来克服上述缺点。
天然产物及其衍生物一直为药物的研究和开发提供了巨大的资源库。查尔酮由于含有天然α,β-不饱和羰基结构，具有较大的柔性，能与不同的受体结合，因此具有广泛的生物活性，如：抗肿瘤、抑制和清除氧自由基、抗菌、抗病毒、抗溃疡和解痉等生物活性。据文献报道，查耳酮结构里的α,β-不饱和羰基能够与细菌内的亲核基团，如蛋白质中的巯基进行共轭加成而致使细菌死亡(Eur.J.Med.Chem,2010,45,5739.)。同时，由于查尔酮结构单元具有较好的柔性，作为底物，查尔酮就可与多种活性基团相结合，研究人员开始关注查尔酮的结构改造，他们试图通过结构修饰来开发具有优异抗菌活性的查尔酮类衍生物(J.Med.Chem,2005,48,2667.)。因此，我们期待通过查尔酮结合抗菌肽两亲性结构特征合成一系列新型具有抗耐药菌活性的化合物，并通过体外活性实验来验证其抗菌活性。
发明内容
基于上述，本发明目的之一是提供一类新型的具有抗耐药菌活性的季铵查尔酮衍生物；目的之二是提供该类化合物的制备方法；目的之三是提供该类化合物在制备抗耐药菌活性药物方面的应用。
为实现本发明，合成季铵查尔酮衍生物路线如下：
Figure BDA0001327480530000021
所述具有抗菌活性的季铵查尔酮衍生物结构通式如下：
Figure BDA0001327480530000022
本发明中采用改变烷烃链长度以及不同取代的芳香环对查尔酮衍生物进行修饰，通过改变两个活性位置，分析不同活性位置对活性影响大小。
1)Ar采用三种芳香环，吡啶环或卤素取代的吡啶环(f1-f6)，苯环或卤素、甲氧基、乙氧基、硝基、三氟甲基、正丁基取代的苯环或萘环(f7-f25)，含氧或硫的五元杂环(f26-f28)。
2)对部分化合物的烷烃链长短进行改变，n＝3,7,11,13,17。
优选：n＝3，7，Ar选吡啶环或氟、溴单取代的吡啶环；苯环或氟、氯、甲氧基、乙氧基、硝基、三氟甲基、正丁基单取代或双取代的苯环或萘环；含氧或硫的五元不饱和杂环。
具体合成目标化合物如下：
Figure BDA0001327480530000031
Figure BDA0001327480530000041
具体通过如下步骤实现：
1、化合物a与化合物b1-b22发生经典的Claisen-Schmidt反应，反应条件是在氢氧化钠的作用下，以乙醇/水为溶剂，常温下进行反应得到化合物c1-c22。
2、化合物c1-c22与氯乙酰氯在弱碱K2CO3和无水丙酮条件下，常温下进行反应得到一系列目标化合物d1-d22。
3、化合物d1-d22与N,N-二甲基烷烃胺在乙腈中，85℃高温高压反应釜中进行反应，得到一系列目标化合物f1-f28。
本发明所述新型的季铵查尔酮衍生物对革兰氏阳性菌、革兰氏阴性菌均表现出很好的活性，尤其对革兰氏阳性菌金黄色葡萄球菌。其中化合物f14，f18，f27的MIC(0.5μg/mL)表现出很好的活性，同时部分化合物对多株耐药菌MRSA、VRE、NDM、KPC也能表现出良好的抑菌效果，甚至优于阳性对照药万古霉素，并且多个化合物与阳性对照药美罗培南几乎有相当的抗菌活性。该类化合物对革兰氏阳性菌活性明显优于革兰氏阴性菌，说明化合物具有一定选择性；化合物f1-f6活性相对较弱，本发明中含吡啶环衍生物抗菌效果较其他两种芳香环活性较差；对苯环化合物f7-f25抗菌活性和HC50数据分析，可以发现氟原子取代位置对活性存在影响，同时氟原子数量增加会导致红细胞溶血性增加；红细胞溶血实验结果证明季铵查尔酮衍生物红细胞毒性较小。因此，本发明提供的此类新型查尔酮衍生物有望作为新的抗菌候选药物进行深入的研究，并对解决目前全球面临耐药菌日益严重的问题有重要意义。
具体实施方式
下面结合具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明要求保护的范围。
合成化合物表征使用的仪器：NMR谱使用瑞典Bruker DPX-400型超导核磁共振仪测定，TMS为内标；高分辨质谱使用Waters-Micromass公司Q-Tof质谱仪测定。
实施例1化合物f1-f28的制备
(1)化合物c1-c22的制备
取氢氧化钠(110.97mg，2.77mmol)于单口圆底烧瓶(200mL)中，加入水(25mL)室温磁力搅拌下溶解；然后将化合物a对氨基苯乙酮(300.00mg，2.22mmol)和化合物b1-b22(2.26mmol)加入锥形瓶(100mL)内，并加入无水乙醇(25mL)超声至该体系澄清，然后将该澄清溶液恒压滴加(1滴/秒)到上述搅拌的氢氧化钠溶液的单口瓶内，滴加完毕后室温下继续反应，此时体系棕黄色澄清。约6h后，TLC(PE：EA＝1:1)检测显示反应完全。停止反应，将反应体系倒入冰水(50mL)中，立即析出大量黄色固体，抽滤，滤饼水洗至中性，真空干燥，得到化合物c1-c22。采用石油醚，乙酸乙酯体系，硅胶柱层析对化合物进行纯化。
(2)化合物d1-d22的制备
取上述化合物c1-c22(754.36mmol)和碳酸钾(125.11mg，905.23mmol)于单口圆底烧瓶(5mL)中，然后加入丙酮(1.35mL)，塞上翻口橡胶塞室温下搅拌；然后用1mL注射器通过橡胶塞向反应体系注入化合物氯乙酰氯(68.16μl，905.23mmol)，体系立即发生浑浊。约0.5h后，TLC(PE：EA＝1:1)检测，显示反应完全，冰水(3mL)淬灭反应，搅拌10min后，将体系抽滤，滤饼冰水洗至中性，真空干燥得到化合物d1-d22。
(3)化合物f1-f28的制备
取上述化合物d1-d22(528.73mmol)于高温高压反应釜(10mL)中，然后加入无水乙腈(3mL)将化合物d1-d22溶解，移液枪取N,N-二甲基烷烃胺(1.59mmol)后加入磁子，拧紧高温高压反应釜盖，放入油浴锅中，85℃条件下磁力搅拌24h；TLC(PE：EA＝1:1)检测，显示反应完全，将反应体系转移到10mL圆底烧瓶中，减压旋蒸体系中溶液至原体积1/10左右，停止旋蒸，取下圆底烧瓶，加入过量乙醚，静置一会待有固体析出，将体系抽滤，滤饼乙醚洗涤2-3次，真空干燥得到化合物f1-f28。
f1：产物为褐色固体，产率为50％。
1H NMR(400MHz,DMSO)δ11.97(s,1H),8.68(d,J＝6.0Hz,2H),8.23(d,J＝8.8Hz,2H),8.18(d,J＝15.7Hz,1H),7.92(d,J＝8.8Hz,2H),7.85(d,J＝6.0Hz,2H),7.68(d,J＝15.7Hz,1H),4.55(s,2H),3.62–3.48(m,2H),3.30(s,6H),1.84–1.70(m,2H),1.40–1.26(m,2H),0.94(t,J＝7.4Hz,3H).13C NMR(101MHz,DMSO)δ187.56,162.75,150.31,142.66,141.83,140.64,132.71,130.09,126.28,122.48,119.16,64.82,62.39,51.21,23.87,19.13,13.42.HR-MS(ESI)Calcd for C22H28ClN3O2[M-Cl]+:366.2176,found:366.2180.
f2：产物为棕色固体，产率为90％。
1H NMR(400MHz,DMSO)δ11.42(s,1H),8.68(d,J＝4.6Hz,2H),8.23(d,J＝8.7Hz,2H),8.16(d,J＝15.7Hz,1H),7.85(d,J＝8.3Hz,4H),7.68(d,J＝15.6Hz,1H),4.42(s,2H),3.57–3.49(m,2H),3.27(s,6H),1.75(s,2H),1.32–1.23(m,10H),0.85(t,J＝6.7Hz,3H).13CNMR(101MHz,DMSO)δ187.50,162.77,150.31,142.72,141.81,140.64,132.66,130.08,126.22,122.49,119.11,64.88,62.32,51.23,31.11,28.37,25.67,21.92,13.90.HR-MS(ESI)Calcd for C26H36ClN3O2[M-Cl]+:422.2802,found:422.2809.
f3：产物为棕色固体，产率为52％。
1H NMR(400MHz,DMSO)δ11.77(d,J＝16.4Hz,1H),9.04(s,1H),8.69–8.56(m,1H),8.37(d,J＝8.0Hz,1H),8.23(d,J＝8.7Hz,2H),8.10(d,J＝15.7Hz,1H),7.89(d,J＝8.5Hz,2H),7.77(d,J＝15.7Hz,1H),7.51(dd,J＝7.8,4.8Hz,1H),4.50(s,2H),3.63–3.45(m,2H),3.28(s,6H),1.77(s,2H),1.45–1.16(m,10H),0.84(d,J＝7.0Hz,3H).13C NMR(101MHz,DMSO)δ187.36,162.73,150.93,150.32,142.52,140.15,135.09,132.90,130.54,129.97,123.88,123.74,119.09,64.89,62.31,51.23,31.11,28.37,25.66,21.92,13.90.HR-MS(ESI)Calcd for C26H36ClN3O2[M-Cl]+:422.2802,found:422.2805.
f4：产物为棕色固体，产率为86％。
1H NMR(400MHz,DMSO)δ12.11(s,1H),8.69(d,J＝2.8Hz,1H),8.35–8.02(m,3H),7.92(d,J＝11.9Hz,4H),7.71(d,J＝15.4Hz,1H),7.43(s,1H),4.59(s,2H),3.54(s,2H),3.30(s,6H),1.77(s,2H),1.23(d,J＝22.9Hz,10H),0.81(s,3H).13C NMR(101MHz,DMSO)δ188.33,163.25,153.29,150.48,143.19,143.07,137.66,133.39,130.33,125.45,125.41,125.26,119.70,65.36,62.81,51.73,31.61,28.87(d,J＝2.2Hz),26.18,22.49,22.35,14.39.HR-MS(ESI)Calcd for C26H36ClN3O2[M-Cl]+:422.2802,found:422.2806.
f5：产物为黄色固体，产率为81％。
1H NMR(400MHz,DMSO)δ11.76(s,1H),8.14(d,J＝8.8Hz,2H),8.08(d,J＝15.4Hz,1H),7.97(d,J＝7.4Hz,1H),7.91–7.88(m,2H),7.86(d,J＝7.7Hz,1H),7.70(d,J＝7.8Hz,1H),7.64(d,J＝15.4Hz,1H),4.49(s,2H),3.53(dd,J＝10.2,6.6Hz,2H),3.28(s,6H),1.76(s,2H),1.31–1.21(m,10H),0.84(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)δ187.65,162.76,154.23,142.63,141.64,140.74,140.45,132.71,129.96,128.98,126.25,124.35,119.23,64.90,62.28,51.24,31.12,28.37,25.66,22.00,21.83,13.91.HR-MS(ESI)Calcdfor C26H35BrClN3O2[M-Cl]+:500.1907,found:500.1914.
f6：产物为棕色固体，产率为85％。
1H NMR(400MHz,CDCl3)δ11.86(s,1H),8.49(d,J＝3.8Hz,1H),8.16(d,J＝15.3Hz,1H),8.02(dd,J＝20.4,11.9Hz,3H),7.93(d,J＝8.3Hz,2H),7.46(t,J＝9.0Hz,1H),7.37–7.28(m,1H),4.93(s,2H),3.75–3.63(m,2H),3.48(s,6H),1.81(s,2H),1.27(d,J＝42.5Hz,10H),0.84(t,J＝6.2Hz,3H).13C NMR(101MHz,CDCl3)δ188.61,162.01,158.48(d,J＝264.4Hz),145.72(d,J＝5.1Hz),142.15,141.83(d,J＝11.0Hz),134.44,133.81,129.92,126.55(d,J＝4.0Hz),125.87(d,J＝4.1Hz),123.84(d,J＝19.4Hz),119.76,65.90,63.58,52.12,31.52,28.95,26.15,22.87,22.49,14.00.HR-MS(ESI)Calcd for C26H35ClFN3O2[M-Cl]+:440.2708,found:440.2715.
f7：产物为浅黄色固体，产率为78％。
1H NMR(400MHz,DMSO)δ12.21(s,1H),8.24(d,J＝8.0Hz,2H),8.04–7.88(m,5H),7.77(d,J＝15.5Hz,1H),7.48(s,3H),4.64(s,2H),3.58(s,2H),3.34(s,6H),1.79(s,2H),1.25(d,J＝22.6Hz,10H),0.83(d,J＝6.1Hz,3H).13C NMR(101MHz,DMSO)δ187.61,162.71,143.60,142.37,134.70,133.16,130.54,129.84,128.88,121.89,119.09,64.88,62.32,51.24,31.12,28.37,25.67,22.00,21.83,13.91.HR-MS(ESI)Calcd for C27H37ClN2O2[M-Cl]+:421.2850,found:421.2856.
f8：产物为棕色固体，产率为56％。
1H NMR(400MHz,DMSO)δ12.13(s,1H),8.59(d,J＝15.3Hz,1H),8.28(dd,J＝12.4,6.3Hz,4H),8.06(dd,J＝21.3,6.5Hz,3H),7.97(d,J＝8.5Hz,2H),7.70–7.59(m,3H),4.61(s,2H),3.61–3.51(m,2H),3.32(s,6H),1.79(s,2H),1.25(dd,J＝14.9,8.4Hz,10H),0.84(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)δ187.54,162.73,142.46,139.47,133.34,133.12,131.32,131.17,130.77,129.92,128.78,127.20,126.27,125.66,124.38,122.93,119.14,64.87,62.34,51.23,31.11,28.37,25.67,22.00,21.85,13.90.HR-MS(ESI)Calcdfor C31H39ClN2O2[M-Cl]+:471.3006,found:471.3013.
f9：产物为白色固体，产率为93％。
1H NMR(400MHz,DMSO)δ11.87(s,1H),8.19(s,2H),7.90(d,J＝18.9Hz,5H),7.71(d,J＝14.1Hz,1H),7.52(s,2H),4.50(s,2H),3.51(s,2H),3.26(s,6H),1.74(s,2H),1.25(s,10H),0.82(s,3H).13C NMR(101MHz,DMSO)δ187.51,162.69,142.37,142.12,135.00,133.69,133.10,130.52,129.89,128.92,122.65,119.12,64.93,62.33,51.26,31.11,28.35,25.66,21.98,21.83,13.89.HR-MS(ESI)Calcd for C27H36Cl2N2O2[M-Cl]+:455.2460,found:455.2464.
f10：产物为浅黄色固体，产率为67％。
1H NMR(400MHz,DMSO)δ11.99(s,1H),8.24(d,J＝8.6Hz,2H),8.11–8.02(m,2H),7.92(d,J＝8.6Hz,2H),7.84(d,J＝6.1Hz,1H),7.72(d,J＝15.6Hz,1H),7.53–7.46(m,2H),4.56(s,2H),3.60–3.50(m,2H),3.30(s,6H),1.78(s,2H),1.31–1.19(m,10H),0.84(t,J＝6.6Hz,3H).13C NMR(101MHz,DMSO)δ187.94,163.23,143.00,142.37,137.47,134.27,133.46,131.16,130.56,130.48,128.38,123.89,119.56,65.38,62.80,51.73,31.62,28.87,26.17,22.50,22.33,14.41.HR-MS(ESI)Calcd for C27H36Cl2N2O2[M-Cl]+:455.2460,found:455.2463.
f11：产物为白色固体，产率为83％。
1H NMR(400MHz,DMSO)δ12.04(s,1H),8.22(d,J＝8.5Hz,2H),8.05–7.88(m,5H),7.75(d,J＝15.6Hz,1H),7.32(t,J＝8.6Hz,2H),4.57(s,2H),3.65–3.48(m,2H),3.31(s,6H),1.78(s,2H),1.36–1.16(m,10H),0.84(t,J＝6.4Hz,3H).13C NMR(101MHz,DMSO)δ187.50,163.13(d,J＝250.07Hz),162.70,142.36,133.13,131.39(d,J＝3.0Hz),131.19(d,J＝8.6Hz),129.84,121.78,119.07,115.89(d,J＝21.7Hz),64.88,62.31,51.23,31.11,28.36,25.66,22.00,21.83,13.90.HR-MS(ESI)Calcd for C27H36ClFN2O2[M-Cl]+:439.2755,found:439.2762.
f12：产物为浅黄色固体，产率为58％。
1H NMR(400MHz,DMSO)δ12.09(s,1H),8.24(d,J＝8.7Hz,2H),8.05(d,J＝15.6Hz,1H),7.91(dd,J＝23.1,9.5Hz,3H),7.80–7.65(m,2H),7.52(dd,J＝14.2,7.9Hz,1H),7.30(td,J＝8.6,2.3Hz,1H),4.59(s,2H),3.56(dd,J＝10.2,6.5Hz,2H),3.31(s,6H),1.79(s,2H),1.32–1.18(m,10H),0.84(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)δ187.49,162.73,162.56(d,J＝244.62),142.51,142.09(d,J＝2.7Hz),137.29(d,J＝8.1Hz),132.97,130.80(d,J＝8.4Hz),129.94,125.53(d,J＝2.4Hz),123.33,119.07,117.16(d,J＝21.4Hz),114.62(d,J＝22.0Hz),64.87,62.32,51.23,31.11,28.36,25.67,22.00,21.84,13.89.HR-MS(ESI)Calcd for C27H36ClFN2O2[M-Cl]+:439.2755,found:439.2763.
f13：产物为浅黄色固体，产率为76％。
1H NMR(400MHz,DMSO)δ12.06(s,1H),8.32–8.09(m,3H),8.02(d,J＝15.7Hz,1H),7.93(d,J＝8.5Hz,2H),7.84(d,J＝15.7Hz,1H),7.54(dd,J＝13.2,6.7Hz,1H),7.34(dd,J＝13.0,6.2Hz,2H),4.58(s,2H),3.66–3.50(m,2H),3.31(s,6H),1.77(d,J＝7.4Hz,2H),1.33(dd,J＝14.4,7.2Hz,2H),0.94(t,J＝7.3Hz,3H).13C NMR(101MHz,DMSO)δ187.44,162.73,160.87(d,J＝251.3Hz),142.53,134.71(d,J＝4.2Hz),132.89,132.59(d,J＝8.6Hz),129.92,129.09,124.93(d,J＝3.4Hz),124.01(d,J＝4.0Hz),122.32(d,J＝11.2Hz),119.14,116.05(d,J＝21.6Hz),64.77,62.35,51.18,23.87,19.13,13.43.HR-MS(ESI)Calcd for C23H28ClFN2O2[M-Cl]+:383.2129,found:383.2135.
f14：产物为黄色固体，产率为84％。
1H NMR(400MHz,CDCl3)δ12.03(s,1H),7.97(dd,J＝19.1,8.6Hz,4H),7.89(d,J＝15.9Hz,1H),7.66(d,J＝7.4Hz,1H),7.62(d,J＝15.7Hz,1H),7.44–7.32(m,1H),7.20(t,J＝7.5Hz,1H),7.16–7.05(m,1H),4.95(s,2H),3.75–3.57(m,2H),3.44(d,J＝9.6Hz,6H),1.83(s,2H),1.30(d,J＝43.4Hz,10H),0.86(t,J＝6.4Hz,3H).13C NMR(101MHz,CDCl3)δ188.99,161.71,141.79,137.19,134.27,131.77(d,J＝8.8Hz),129.74,124.48,124.39,123.08(d,J＝11.2Hz),119.84,116.27(d,J＝21.9Hz),66.50,63.90,52.10,31.54,28.96,26.17,22.91,22.52,14.01.HR-MS(ESI)Calcd for C27H36ClFN2O2[M-Cl]+:439.2755,found:439.2764.
f15：产物为黄色固体，产率为77％。
1H NMR(400MHz,DMSO)δ12.05(d,J＝21.5Hz,1H),8.20(d,J＝8.6Hz,2H),8.15(t,J＝7.7Hz,1H),8.01(d,J＝15.7Hz,1H),7.93(d,J＝8.0Hz,2H),7.84(d,J＝15.7Hz,1H),7.53(dd,J＝13.5,6.9Hz,1H),7.34(dd,J＝13.0,5.5Hz,2H),4.57(s,2H),3.66–3.48(m,2H),3.30(s,6H),1.78(s,2H),1.30–1.17(m,18H),0.83(t,J＝6.4Hz,3H).13C NMR(101MHz,DMSO)δ187.29,162.73,160.87(d,J＝251.4Hz),142.58,134.63(d,J＝4.1Hz),132.86,132.46(d,J＝8.7Hz),129.81,129.06,124.83(d,J＝3.1Hz),123.86(d,J＝3.9Hz),122.32(d,J＝11.2Hz),119.09,115.95(d,J＝21.7Hz),64.72,62.33,51.29,31.27,29.03,28.94,28.76,28.72,28.39,25.68,22.05,21.86,13.83.HR-MS(ESI)Calcd for C31H44ClFN2O2[M-Cl]+:495.3381,found:495.3388.
f16：产物为浅黄色固体，产率为79％。
1H NMR(400MHz,DMSO)δ11.85(s,1H),8.19(d,J＝8.8Hz,2H),8.13(t,J＝7.5Hz,1H),8.00(d,J＝15.7Hz,1H),7.89(d,J＝8.7Hz,2H),7.83(d,J＝15.7Hz,1H),7.53(dd,J＝13.1,6.4Hz,1H),7.33(dd,J＝13.2,5.7Hz,2H),4.51(s,2H),3.53(dd,J＝10.1,6.6Hz,2H),3.28(s,6H),1.76(s,2H),1.29–1.16(m,22H),0.83(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)δ187.24,162.72,160.88(d,J＝251.5Hz),142.57,134.63(d,J＝4.1Hz),132.86,132.42(d,J＝8.7Hz),129.80,129.03,124.80(d,J＝3.1Hz),123.82(d,J＝3.8Hz),122.33(d,J＝11.2Hz),119.07,115.92(d,J＝21.7Hz),64.67,62.31,51.30,31.28,29.10,29.05,28.96,28.78,28.73,28.40,25.68,22.06,21.86,13.82.HR-MS(ESI)Calcd forC33H48ClFN2O2[M-Cl]+:523.3694,found:523.3697.
f17：产物为浅黄色固体，产率为78％。
1H NMR(400MHz,DMSO)δ11.81(s,1H),8.19(d,J＝8.8Hz,2H),8.13(t,J＝7.5Hz,1H),8.00(d,J＝15.7Hz,1H),7.89(d,J＝8.7Hz,2H),7.84(d,J＝15.8Hz,1H),7.53(dd,J＝13.1,6.5Hz,1H),7.33(dd,J＝13.2,5.5Hz,2H),4.51(s,2H),3.53(dd,J＝10.1,6.6Hz,2H),3.29(s,6H),1.76(s,2H),1.35–1.12(m,32H),0.84(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)δ186.21,161.60,159.75(d,J＝251.3Hz),141.38,133.57(d,J＝4.2Hz),131.76,131.43(d,J＝8.8Hz),128.76,127.94,123.77(d,J＝3.2Hz),122.79(d,J＝3.8Hz),121.19(d,J＝11.2Hz),117.97,114.90(d,J＝21.5Hz),63.53,61.10,50.23,30.13,27.92,27.90,27.85,27.80,27.55,27.19,24.46,20.93,20.63,12.77.HR-MS(ESI)Calcd forC37H56ClFN2O2[M-Cl]+:579.4320,found:579.4323.
f18：产物为黄色固体，产率为58％。
1H NMR(400MHz,DMSO)δ12.10(s,1H),8.21(d,J＝8.8Hz,2H),8.05(d,J＝15.7Hz,1H),7.98(d,J＝7.6Hz,1H),7.94(d,J＝8.8Hz,2H),7.80(d,J＝15.7Hz,1H),7.54(dd,J＝17.2,8.3Hz,1H),7.33(dd,J＝12.8,7.7Hz,1H),4.59(s,2H),3.56(dd,J＝10.2,6.5Hz,2H),3.31(s,6H),1.79(s,2H),1.32–1.21(m,10H),0.84(t,J＝6.8Hz,3H).13C NMR(101MHz,CDCl3)δ192.52,168.00,156.43(d,J＝12.7Hz),155.05(d,J＝13.4Hz),153.99(d,J＝12.6Hz),152.54(d,J＝13.2Hz),147.92,138.64(t,J＝3.6Hz),137.95,135.21,130.64(d,J＝4.1Hz),130.36(d,J＝5.3Hz),130.30(d,J＝3.0Hz),129.93(d,J＝8.2Hz),129.49(d,J＝2.6Hz),124.39,124.19,70.17,67.61,56.49,36.36,33.60,30.93,27.24,27.10,19.12.HR-MS(ESI)Calcd for C27H35ClF2N2O2[M-Cl]+:457.2661,found:457.2666.
f19：产物为浅黄色固体，产率为75％。
1H NMR(400MHz,DMSO)δ12.11(s,1H),8.38–8.10(m,3H),8.01(d,J＝15.7Hz,1H),7.93(d,J＝8.4Hz,2H),7.78(d,J＝15.7Hz,1H),7.33(dt,J＝16.5,9.1Hz,2H),4.59(s,2H),3.66–3.51(m,2H),3.31(s,6H),1.79(s,2H),1.31–1.17(m,10H),0.83(d,J＝6.7Hz,3H).13C NMR(101MHz,DMSO)δ187.34,164.76(d,J＝12.7Hz),162.73,162.42(d,J＝12.7Hz),162.26(d,J＝12.9Hz),159.90(d,J＝12.6Hz),142.54,133.75(d,J＝1.9Hz),132.87,130.71(dd,J＝10.0,3.6Hz),129.87,123.76,119.26(d,J＝3.8Hz),119.13,112.58(d,J＝3.4Hz),112.37(d,J＝3.5Hz),104.55(t,J＝26.1Hz),64.90,62.35,51.25,31.10,28.35(d,J＝1.9Hz),25.67,21.98,21.84,13.87.HR-MS(ESI)Calcd forC27H35ClF2N2O2[M-Cl]+:457.2661,found:457.2668.
f20：产物为黄色固体，产率为73％。
1H NMR(400MHz,DMSO)δ11.98(s,1H),8.82(s,1H),8.29(d,J＝7.3Hz,1H),8.21(d,J＝8.3Hz,2H),8.13(d,J＝15.8Hz,1H),7.94(dd,J＝23.0,12.0Hz,3H),7.32(d,J＝9.2Hz,1H),4.56(s,2H),3.55(s,2H),3.30(s,6H),1.78(s,2H),1.27(d,J＝23.0Hz,11H),0.84(d,J＝6.6Hz,3H).13C NMR(101MHz,DMSO)δ187.48,162.73,162.04,142.49,140.85,135.86,132.99,129.92,127.17,124.31,123.97,123.73,119.10,113.01,65.26,64.91,62.33,51.25,31.11,28.36,25.67,21.99,21.83,14.31,13.89.HR-MS(ESI)Calcd forC29H40ClN3O5[M-Cl]+:510.2962,found:510.3000.
f21：产物为棕色固体，产率为95％。
1H NMR(400MHz,DMSO)δ11.98(s,1H),8.09(d,J＝8.7Hz,2H),7.90(dd,J＝17.4,12.4Hz,3H),7.69(d,J＝16.0Hz,1H),7.63–7.50(m,1H),7.27(t,J＝8.8Hz,2H),4.55(s,2H),3.60–3.49(m,2H),3.30(s,6H),1.78(s,2H),1.31–1.21(m,10H),0.84(t,J＝6.7Hz,3H).13C NMR(101MHz,DMSO)δ187.53,162.76,161.06(d,J＝253.6Hz),142.62,132.63,132.47(d,J＝11.1Hz),129.80,128.91,127.31(t,J＝7.5Hz),119.25,112.47(d,J＝5.0Hz),112.20(d,J＝9.4Hz),111.93(d,J＝15.3Hz),64.87,62.27,51.24,31.12,28.36,25.66,22.00,21.83,13.89.HR-MS(ESI)Calcd forC27H35ClF2N2O2[M-Cl]+:457.2661,found:457.2668.
f22：产物为棕色固体，产率为87％。
1H NMR(400MHz,DMSO)δ11.84(s,1H),8.19(d,J＝8.5Hz,2H),8.00–7.85(m,3H),7.81(d,J＝8.2Hz,2H),7.72(d,J＝15.5Hz,1H),7.48(d,J＝8.2Hz,2H),4.52(s,2H),3.73–3.42(m,2H),3.28(s,6H),1.77(s,2H),1.35–1.17(m,19H),0.85(t,J＝6.6Hz,3H).13C NMR(101MHz,DMSO)δ187.65,162.67,153.54,143.57,142.22,133.32,132.01,129.77,128.70,125.70,121.11,119.12,64.93,62.33,51.26,34.63,31.11,30.88,28.35,25.66,21.99,21.83,13.90.HR-MS(ESI)Calcd for C31H45ClN2O2[M-Cl]+:477.3476,found:477.3482.
f23：产物为浅黄色固体，产率为83％。
1H NMR(400MHz,DMSO)δ12.13(s,1H),8.38(d,J＝7.7Hz,1H),8.25(d,J＝8.0Hz,2H),8.08(d,J＝15.3Hz,1H),7.97(t,J＝11.2Hz,3H),7.83(dd,J＝16.5,7.8Hz,2H),7.69(t,J＝7.4Hz,1H),4.61(s,2H),3.56(d,J＝7.3Hz,2H),3.33(s,6H),1.79(s,2H),1.26(d,J＝23.9Hz,10H),0.83(d,J＝6.7Hz,3H).13C NMR(101MHz,CDCl3)δ192.40,167.64,147.64,143.05,138.29,138.01,137.67,135.13,134.98,134.71(d,J＝10.7Hz),133.65,133.26(d,J＝8.2Hz),132.92,131.07(d,J＝5.3Hz),130.99,130.52,127.80,124.33,70.29,67.63,56.38,36.37,33.68,31.00,27.27,27.23,19.05.HR-MS(ESI)Calcd forC28H36ClF3N2O2[M-Cl]+:489.2723,found:489.2728.
f24：产物为浅黄色固体，产率为66％。
1H NMR(400MHz,DMSO)δ12.08(s,1H),8.21(d,J＝8.7Hz,2H),7.99–7.84(m,3H),7.80(d,J＝8.0Hz,2H),7.72(d,J＝15.6Hz,1H),7.29(d,J＝8.0Hz,2H),4.59(s,2H),3.56(dd,J＝10.0,6.6Hz,2H),3.31(s,6H),2.36(s,3H),1.78(s,2H),1.25(dd,J＝14.9,8.2Hz,10H),0.84(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)δ187.55,162.69,143.65,142.30,140.58,133.26,131.98,129.75,129.50,128.87,120.80,119.07,64.86,62.32,51.23,31.12,28.37,25.68,22.00,21.84,21.06,13.90.HR-MS(ESI)Calcd for C28H39ClN2O2[M-Cl]+:435.3006,found:435.3013.
f25：产物为浅黄色固体，产率为85％。
1H NMR(400MHz,DMSO)δ12.00(s,1H),8.20(d,J＝8.7Hz,2H),7.97–7.79(m,5H),7.72(d,J＝15.5Hz,1H),7.03(d,J＝8.7Hz,2H),4.57(s,2H),3.83(s,3H),3.55(dd,J＝10.2,6.5Hz,2H),3.30(s,6H),1.78(s,2H),1.25(dd,J＝14.8,7.9Hz,10H),0.84(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)δ187.46,162.66,161.29,143.58,142.15,133.44,130.73,129.67,127.34,119.34,119.05,114.37,64.87,62.31,55.35,51.23,31.12,28.36,25.67,22.00,21.83,13.90.HR-MS(ESI)Calcd for C28H39ClN2O3[M-Cl]+:451.2955,found:451.2963.
f26：产物为红褐色固体，产率为70％。
1H NMR(400MHz,DMSO)δ11.99(s,1H),8.11(s,2H),7.92(d,J＝9.9Hz,3H),7.57(s,2H),7.12(s,1H),6.71(s,1H),4.55(s,2H),3.54(s,2H),3.29(s,6H),1.77(s,2H),1.28(s,10H),0.84(s,3H).13C NMR(101MHz,DMSO)δ187.03,162.69,151.15,146.14,142.27,133.10,130.13,129.59,119.16,118.50,116.96,113.10,64.88,62.29,51.23,31.11,28.36,25.66,21.99,21.82,13.90.HR-MS(ESI)Calcd for C25H35ClN2O3[M-Cl]+:411.2642,found:411.2649.
f27：产物为浅黄色固体，产率为78％。
1H NMR(400MHz,DMSO)δ11.75(s,1H),8.13(d,J＝8.6Hz,2H),7.91(d,J＝15.3Hz,1H),7.87(d,J＝8.6Hz,2H),7.80(d,J＝5.0Hz,1H),7.70(d,J＝3.4Hz,1H),7.57(d,J＝15.3Hz,1H),7.23–7.17(m,1H),4.49(s,2H),3.58–3.49(m,2H),3.28(s,6H),1.76(s,2H),1.31–1.21(m,10H),0.84(t,J＝6.7Hz,3H).13C NMR(101MHz,DMSO)δ187.09,162.67,142.20,139.73,136.40,133.12,132.78,130.38,129.69,128.70,120.14,119.14,64.90,62.26,51.25,31.11,28.35,25.65,21.99,21.82,13.91.HR-MS(ESI)Calcd forC25H35ClN2O2S[M-Cl]+:427.2414,found:427.2417.
f28：产物为棕色固体，产率为65％。
1H NMR(400MHz,DMSO)δ8.13(d,J＝8.8Hz,2H),7.90(t,J＝11.9Hz,3H),7.80(d,J＝5.0Hz,1H),7.70(d,J＝3.5Hz,1H),7.57(d,J＝15.3Hz,1H),7.20(dd,J＝5.0,3.7Hz,1H),4.52(s,2H),3.61–3.49(m,2H),3.29(s,6H),1.81–1.70(m,2H),1.40–1.27(m,2H),0.94(t,J＝7.4Hz,3H).13C NMR(101MHz,DMSO)δ187.12,162.66,142.23,139.74,136.38,133.12,132.75,130.36,129.69,128.69,120.19,119.16,64.81,62.35,51.20,23.87,19.13,13.43.HR-MS(ESI)Calcd for C21H27ClN2O2S[M-Cl]+:371.1788,found:371.1790.。
应用例1体外抗菌活性测试
1、实验方法
微量肉汤稀释法：
(1)抗菌药物贮存液制备：制备抗菌药物贮备液的浓度为2560μg/mL，溶解度低的抗菌药物可稍低于上述浓度。所需抗菌药物溶液量或粉剂量可公式进行计算。配制好的抗菌药物贮存液应贮存于-20℃以下环境，保存期不超过6个月。
(2)待测菌的制备：用接种环挑取过夜培养的MH(A)培养皿上的单菌落于MH(B)培养基中，校准为0.5麦氏比浊标准，约含菌数1×108CFU/mL，然后稀释100倍，即得到约含菌数1×106CFU/mL的菌液，备用。
(3)分别将抗菌药物贮备液母液(2560μg/mL)稀释10倍，得到浓度为256μg/mL的抗菌药物溶液。取无菌的96孔板，第一孔加入200μL的抗菌药物，第二至十孔分别加入100μL的MH肉汤培养基，从第一孔吸取100μL加入第二孔，混匀，再吸取100μL至第三孔，依次类推，第十孔吸取100μL弃去。此时各孔药物浓度依次为：256、128、64、32、16、8、4、2、1、0.5μg/mL，第十一孔加入200μL菌液(阳性对照)，第十二孔加入200μLMH(B)培养基(阴性对照)。
(4)然后在1至10孔各加入50μL之前备好的菌液，使每管最终菌液浓度约为5×105CFU/mL，第1孔至第11孔药物浓度分别为128、64、32、16、8、4、2、1、0.5、0.25μg/mL。将接种好的96孔板放置37℃培养箱进行培养，24h观察菌液生长情况。同时用标准株做质控。
(5)结果判断与解释：在读取和报告所测试菌株的MIC前，应检查生长对照管的细菌生长情况是否良好，同时还应检查接种物的传代培养情况以确定其是否污染，质控菌株的MIC值是否处于质控范围。以肉眼观察，药物最低浓度管无细菌生长者，即为受试菌的MIC。
应用例2体外红细胞溶血性实验
(1)实验材料：10mLEP管，96孔板，新鲜脱脂羊血。
(2)PBS缓冲液：500mL规格，氯化钠4g，氯化钾100mg，二水合磷酸二氢钠1.49g，无水磷酸二氢钾100mg，去离子水定容至490mL，调节PH7.2-7.4之间，灭菌，用10mL灭过菌的超纯水溶解900mg葡萄糖后加入PBS溶液中。
(3)质量百分含量5％红细胞悬浮液的制备：新鲜的脱纤维羊血冷冻于冰箱里，配置好的PBS缓冲液放置于37℃水浴锅中，即用即取。
取两支10mL EP管置于试管架，将37℃PBS取出水浴锅和冷藏的新鲜羊血一起喷酒精，放入超净台。用移液枪分别吸取5700微升PBS加入两支EP管中，再分别吸取300微升羊血，缓慢加入到PBS溶液中，盖盖子，上下缓慢颠倒混匀，放入离心机1500转、离心10min，取出EP管，小心吸取上清，移除上清。再重新分别加入5～7mL PBS溶液，上下缓慢颠倒混匀，放入离心1500转离心10min。如此反复操作，直至离心后上清液不再浑浊。最后一次离心过后，撇去上清液，红细胞沉积物留置待用。
取几支10mL EP管，放置试管架上，于每支EP管中加入5700μL的PBS(37℃)，然后依次加入300μL的红细胞沉积物。上下缓慢颠倒混匀，如此，便配置好质量百分含量5％的红细胞悬液。
(4)样品溶液的配置：用少量的DMSO溶解药物(DMSO终浓度不能大于0.5％)，并且用相同体积的DMSO做阴性对照。溶解药物后的DMSO用PBS稀释，此时这支EP管内的药物为初始药物(例如，第一孔浓度定为1000μg/mL，那么第一孔加入的50μL中药物的含量就是2mg，配置成2mg/50μL的溶液)。然后平行取九支1.5mL EP管置于试管架中，分别加入200μL的PBS(编号2号、3号、4号……10号)。所有药物都如此平行操作。最后，由初始药物EP管中吸取200μL的药品溶液加入2号EP管中，反复吹洗后吸取200μL到3号EP管中，反复吹洗……重复操作，直到10号EP管。如此，稀释好药物。
(5)铺板：取96孔板，写好实验编号，药品代码，日期。将移液枪调至150μL，将配置好的质量百分含量5％红细胞悬液上下轻缓颠倒混匀，依次吸取铺入96孔板中(6×10)。然后将配置好的药物对应加入96孔板中，一个药物三个复孔。加完后放置37℃恒温箱内孵育1h。
(6)后处理：将96孔板从恒温箱内取出，置于离心机内离心(3500rpm，5min)。离心完毕，每块板对应都取一块新的96孔板。标注和离心后的板子对照。然后对应地吸取100μL上清液(孔孔对应)。吸取完毕后，与酶标仪中测取OD值，分析数据，得到HC50。
实验结果：
表一：目标化合物f1-f28对革兰氏阴性及阳性敏感菌和耐药菌的MIC(μg/mL)
结果和体外红细胞溶血性HC50(μg/mL)结果
Figure BDA0001327480530000161
a:MRSA(耐甲氧西林的金黄色葡萄球菌),VRE(耐万古霉素的肠球菌),KPC(产KPC-2酶CRE),NDM(产NDM-1的CRE)；b:未测MIC；c:万古霉素；d:美罗培南
表二：部分化合物对10株无重复MRSA临床株的MIC(μg/mL)
Figure BDA0001327480530000171
a：耐甲氧西林的金黄色葡萄球菌
表三：部分化合物对12株无重复产NDM-1酶临床株MIC(μg/mL)
Figure BDA0001327480530000172
a:产NDM-1的CRE
表四：部分化合物对12株无重复产KPC-2酶临床株MIC(μg/mL)结果
Figure BDA0001327480530000181
a:产KPC-2酶CRE
表五：部分化合物对9株无重复VRE临床株的MIC(μg/mL)结果
Figure BDA0001327480530000182
a:耐万古霉素的肠球菌
由表一可见，所合成的化合物f1-f28中，大部分化合物针对革兰氏阳性菌金黄色葡萄球菌和大肠粪肠球菌，革兰氏阴性菌大肠埃希菌和铜绿假单胞菌的MIC(μg/mL)均表现出很好的活性，显示这类化合物具有优异的广谱抗菌活性；同时，其体外红细胞溶血性数据显示，具有较小的毒性，很好的选择性。
根据表一中MIC结果，可以得出f8，f9，f10，f11，f14，f19，f23，f24，f25，f26，f27活性相对较好，然后对这11个化合物测定对四种临床分离的耐药菌活性，结果见表二，三，四，五。可以看出这类化合物对于MRSA、VRE、产NDM-1和KPC-2酶的CRE临床株菌显示良好的抗菌活性。因此，可以得出结论：该类化合物具有较好的成药前景。

Claims (1)

1.具有抗耐药菌活性的季铵查尔酮衍生物，其特征在于，该化合物结构式如下：
Figure FDA0002309856060000011
Ar选
Figure FDA0002309856060000012
n＝7。
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