CN112028833B - 对氨基水杨酸唑类衍生物及其制备方法和应用 - Google Patents

对氨基水杨酸唑类衍生物及其制备方法和应用 Download PDF

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CN112028833B
CN112028833B CN202011025656.0A CN202011025656A CN112028833B CN 112028833 B CN112028833 B CN 112028833B CN 202011025656 A CN202011025656 A CN 202011025656A CN 112028833 B CN112028833 B CN 112028833B
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aminosalicylic acid
azole derivative
acid azole
bacteria
molecules
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CN112028833A (zh
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杨大成
潘建芳
范莉
唐雪梅
任艳会
徐兴然
胡军华
周围
徐志刚
吴玉珠
李洋
韩海燕
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Southwest University
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Abstract

本发明公开了对氨基水杨酸唑类衍生物及其制备方法和应用，属于药物合成技术领域。对氨基水杨酸唑类衍生物的结构式如下。抗菌活性测定结果表明，本发明的对氨基水杨酸唑类衍生物对常见致病菌抑制活性整体很好，部分分子对毕赤酵母菌的抑菌活性强于或相当于阳性药物氟康唑，所有分子对柑橘胶孢炭疽病菌的抑制活性较好、部分分子与阳性对照咪鲜胺的抑制活性相当或更强，多数分子对柑橘溃疡病菌具有良好的抑制作用。抗肿瘤细胞活性测定结果显示，本发明的对氨基水杨酸唑类衍生物对三种肿瘤细胞均有抑制作用。本发明的对氨基水杨酸唑类衍生物在抗细菌、抗真菌、抗柑橘病菌和抗肿瘤领域具有潜在的应用前景。

Description

对氨基水杨酸唑类衍生物及其制备方法和应用
技术领域
本发明涉及药物合成技术领域，具体涉及对氨基水杨酸唑类衍生物及其制备方法和应用。
背景技术
对氨基水杨酸(para-aminosalicylic acid，PAS)，为一种用于治疗结核病的抗菌药。其也被用于治疗炎症性肠病，通过抑制NF-κB及清除自由基而发挥作用。主要用于治疗结核病。
PAS氨基衍生物分为三大类。第一类是PAS席夫碱衍生物。研究结果表明，所合成的目标化合物，对耻垢分枝杆菌和牛型分枝杆菌的抑菌浓度均低于PAS；吡嗪酰胺和PAS的席夫碱衍生物，对H37Rv抑制活性强于PZA(MIC分别为3.13和6μg/mL)，同时存在缓释的情况。第二类是PAS腙类衍生物，将INH与PAS反应制备了含有腙基的化合物，试验结果显示，均比阳性对照(MICINH＝1μg/mL、MIC环丙沙星＝1.5μg/mL和MIC诺氟沙星＝10μg/mL)好。第三类，在氨基上接一些其他官能团的衍生物，这些分子也显示很好的生物活性。
含有唑类杂环的化合物在农药、医药、染料等领域应用广泛。许多上市药物中含有不同类型的唑类结构单元，仅2018年top 200中就有16种药物分子含唑类片段。唑类杂环化合物表现出各种生物活性，例如抗菌、抗疟疾、抗真菌、抗HIV、抗炎和抗结核等。唑类衍生物在药物化学领域受到持续关注，它们的许多衍生物不仅是上市药物，高活性的分子也不断涌现，如抗菌的Tazobactum、抗HIV的TSAO以及抗癌的CAI。但是未见PAS与唑类连接的衍生物及其生物活性的报道。
发明内容
有鉴于此，本发明的目的在于提供对氨基水杨酸唑类衍生物及其制备方法和应用。
经研究，本发明提供以下技术方案：
1、式Ⅰ所示的对氨基水杨酸唑类衍生物，其氮氧化合物、硫氧化物或药学上可接受的盐：
Figure GDA0003640403630000011
式Ⅰ中，R选自：
Figure GDA0003640403630000012
Figure GDA0003640403630000021
R1和R2独立地选自为H或C1-C3烷基；R3和R4独立地选自为H或C1-C3烷基；R5为H或C1-C3烷基；R6为H或C1-C3烷基；R7为H、C1-C3烷基或氨基；R8为H、C1-C3烷基、取代或未取代苯基，所述苯基上的取代基为一个或多个，独立选自卤素、羟基、氨基或C1-C3烷基；R9和R10独立地选自为H或C1-C3烷基；R11为-O-、-S-或-NH-；R12和R13独立地选自为H、卤素或烷酰基；L选自：-(CH2)n+1-、-(CH2)nCO-或-CO(CH2)nCO-，n为1，2，3或4；X选自：C1-C6烷基。
优选的，所述式Ⅰ中，R1和R2独立地选自为H或甲基；R3和R4独立地选自为H或甲基；R5为H或甲基；R6为H或甲基；R7为甲基或氨基；R8为甲基、取代或未取代苯基，所述苯基上的取代基为一个或多个，独立选自卤素、羟基、氨基或C1-C3烷基；R9和R10均为甲基；R11为-O-、-S-或-NH-；R12和R13独立地选自为H、卤素或乙酰基；L选自：-(CH2)n+1-或-(CH2)nCO-，n为1，2，3或4；X选自：C1-C3烷基。
优选的，所述式Ⅰ中，
R选自：
Figure GDA0003640403630000022
Figure GDA0003640403630000023
L选自：-(CH2)nCO-，n为1、2，3或4；X选自：甲基或乙基。
优选的，式Ⅰ所示的对氨基水杨酸唑类衍生物为以下化合物中的任一种：
Figure GDA0003640403630000024
Figure GDA0003640403630000031
Figure GDA0003640403630000041
优选的，式Ⅰ所示的对氨基水杨酸唑类衍生物为以下化合物中的任一种：TM1-2，TM1-4，TM1-5，TM1-6，TM1-8，TM1-9，TM1-10，TM1-11，TM1-12，TM1-14，TM4-1，TM4-2，TM4-4，TM4-7，TM4-8，TM4-9，TM4-10，TM4-11，TM4-12，TM4-13，TM4-14，TM4-15和TM4-17。
2、上述对氨基水杨酸唑类衍生物的制备方法，包括以下步骤：
将对氨基水杨酸羧基进行酯化，制得中间体IM1；
Figure GDA0003640403630000042
将中间体IM1与linker试剂反应，制得中间体IM2；
Figure GDA0003640403630000043
将中间体IM2与唑偶联，制得目标分子对氨基水杨酸唑类衍生物；
Figure GDA0003640403630000044
式中，X、L和R的定义与上述的对氨基水杨酸唑类衍生物结构式中X、L和R的定义相同；IM2中的Z为卤素。
优选的，所述对氨基水杨酸唑类衍生物的制备方法，包括以下步骤：
A、在酸作用下，将对氨基水杨酸与醇反应，制得中间体IM1；所述醇为甲醇或乙醇；所述酸为硫酸；
B、将中间体IM1与氯乙酰氯在有机溶剂中、碱作用下进行氨基酰化，制得中间体IM2；所述有机溶剂为二氯甲烷、氯仿、丙酮、乙酸乙酯、四氢呋喃或***；所述碱为碳酸钾、三乙胺或碳酸氢钠；
C、将中间体IM2与唑在有机溶剂中、碱作用下偶联制得对氨基水杨酸唑类衍生物；所述有机溶剂为二氯甲烷、氯仿、乙腈、四氢呋喃或N,N-二甲基甲酰胺；所述碱为碳酸氢钠、三乙胺、氢氧化钠、甲醇钠或碳酸钾。
更优选的，所述步骤B中，有机溶剂为二氯甲烷或氯仿，碱为碳酸氢钠。
更优选的，所述步骤C中，有机溶剂为N,N-二甲基甲酰胺，碱为碳酸钾。
3、上述对氨基水杨酸唑类衍生物在抗细菌药物中的应用。
4、上述对氨基水杨酸唑类衍生物在抗真菌药物中的应用。
5、上述对氨基水杨酸唑类衍生物在抗肿瘤药物中的应用。
6、上述对氨基水杨酸唑类衍生物在抗柑橘病菌药物中的应用。
除另有说明外，本发明中的术语“氮氧化物”是指三级氮连接氧原子形成+N-O-结构单元的有机物；“硫氧化物”指前体硫醚氧化所得的亚砜或砜；“药学上可接受的盐”可以是酸性盐，也可以是碱性盐，例如无机酸盐、有机酸盐、无机碱盐或有机碱盐。
术语“C1-C3烷基”指具有1-3个碳原子的直链或支链饱和一价烃基，例如甲基、乙基、丙基和异丙基。
术语“卤素”指F、Cl、Br和I。
本发明的有益效果在于：
1)本发明提供的对氨基水杨酸唑类衍生物，以对氨基水杨酸为母核，对其氨基和羧基进行合理修饰，构建了一类结构新颖的对氨基水杨酸唑类衍生物，产物的化学结构经1H NMR，13C NMR和HR MS确认；
2)体外抗细菌活性测定结果表明，化合物的整体抑菌活性很好。其中，对金黄色葡萄球菌，9个化合物的MIC值为32μg/mL，中间体IM2-1和IM2-2的MIC值分别为25.6μg/mL、12.8μg/mL，强于PAS及其甲酯乙酯(MIC值>256μg/mL)。对大肠杆菌，化合物TM4-11、TM4-12和TM4-13的MIC值分别为64μg/mL、32μg/mL和64μg/mL，强于PAS及其中间体(MIC值≥256μg/mL)。对藤黄微球菌，化合物TM4-9和TM4-11的MIC值均为16μg/mL，中间体IM2-2的MIC值为3.2μg/mL，强于PAS及其甲酯乙酯(MIC值>256μg/mL)。对沙门氏菌，中间体IM2-2的MIC值为0.8μg/mL，远强于PAS及其甲酯乙酯(MIC值>256μg/mL)。从而证明了对氨基水杨酸唑类衍生物及其中间体在抗细菌领域具有潜在的应用前景；
3)本发明提供的对氨基水杨酸唑类衍生物对毕赤酵母菌的抑菌活性很好，其中有4个分子的MIC≤4μg/mL，与阳性对照药物氟康唑的MIC值相同，显示它们对毕赤酵母菌株的抑制活性强于或相当于氟康唑；中间体IM2-1和IM2-2对毕此酵母菌的抑制活性达到64μg/mL，强于母核PAS。从而证明了对氨基水杨酸唑类衍生物及其中间体在抗真菌领域具有潜在的应用前景；
4)本发明提供的对氨基水杨酸唑类衍生物在5μM浓度下对3种癌细胞均有抑制作用。针对结肠癌细胞HCT116，化合物TM1-2、TM4-4、IM2-1和IM2-2的抑制率超过50％，中间体IM2-2的抑制率达到60％。针对结肠癌细胞SW620，大多数化合物均强于母核PAS(8％)。针对***癌细胞PC3，化合物TM4-3、TM4-5和IM2-2的抑制率高于50％，中间体IM2-2的抑制率达到78％，63％的化合物对***癌细胞PC3的抑制作用强于母核PAS(5％)。证明了对氨基水杨酸唑类衍生物及其中间体在抗肿瘤领域具有潜在的应用前景；
5)本发明提供的对氨基水杨酸唑类衍生物，在1μg/mL和4μg/mL浓度下，对柑橘胶孢炭疽病菌，达到阳性药物70％抑制率的分子数分别为8个和3个；对柑橘褐斑病菌，达到阳性药物70％抑制率的分子数分别为1个和3个；目标化合物TM1-2及中间体IM2-2对柑橘褐斑病菌的抑制活性很好，分别与阳性对照咪鲜胺的抑制活性相同或更强。同时，TM1-2、TM1-5、TM4-10和IM2-2未体现出抗药性。另外，大多数目标化合物对柑橘溃疡病菌具有良好的抑制作用。证明了对氨基水杨酸唑类衍生物及其中间体在抗柑橘病菌领域具有潜在的应用前景。
具体实施方式
下面结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一、主要试剂和仪器
对氨基水杨酸、氯乙酰氯、二氯甲烷、N,N-二甲基甲酰胺、咪唑、2-甲基咪唑、4-甲基咪唑、吡唑、苯并咪唑、1,2,4-三氮唑、2,4-二甲基吡唑、1H-四氮唑、5-甲基四氮唑、4,6-二甲基-2-巯基嘧啶、1-甲基-5-巯基四氮唑、1-苯基-5-巯基四氮唑、2-巯基-5-甲基-1,3,4-噻二唑；浓硫酸、甲醇、碳酸氢钠、碳酸钾；2-氨基-5-巯基-1,3,4-噻二唑；无水乙醇；前述均为分析纯试剂。其余试剂均为市售化学纯或分析纯产品。
核磁共振仪(AV-600,TMS为内标,DMSO-d6为溶剂，1H NMR为600MHz，13C NMR为151MHz)；高分辨质谱仪(Varian 7.0T)；熔点测定仪(X-6)；自动旋光仪(WZZ-2S)；紫外-可见分光光度计(ZF-1)；旋转蒸发仪(RE-2000)。
二、对氨基水杨酸唑类衍生物的制备
1、中间体IM1-1的合成
Figure GDA0003640403630000071
向反应瓶中加入PAS 1.53g(10mmol)、甲醇25mL，室温搅拌。冰浴，滴加浓硫酸1.3mL(25mmol)，滴毕，油浴回流反应，薄层层析法(TLC)监测直至反应结束。冰浴冷却，碳酸钠溶液调节pH在7-8，冷藏，抽滤，滤饼用冰水洗涤。滤液用二氯甲烷(DCM)萃取(3×30mL)，收集有机相，饱和NaCl溶液洗涤。无水硫酸钠干燥，旋蒸，并与滤饼合并，真空干燥后，柱层析，得中间体IM1-1(白色固体)1.096g，收率为65％。
2、中间体IM2-1的合成
Figure GDA0003640403630000072
向反应瓶中加入IM1-1 5mmol(0.766g)、DCM 5mL、NaHCO3 12.5mmol(1.1g)，冰浴冷却，滴加氯乙酰氯10mmol(0.75mL)。滴毕，冰浴下持续反应，TLC跟踪监测至反应结束。停止搅拌，加入10mL冰冷的饱和食盐水，用2N HCl溶液调节pH在4-5，移入分液漏斗，乙酸乙酯(EA)萃取两次，合并有机相，饱和食盐水洗涤，无水Na2SO4干燥，旋蒸除去溶剂，柱层析(PE(石油醚):EA＝10:1--5:1,v/v)，得纯品中间体IM2-1为1.03g，收率为83％。
3、对氨基水杨酸唑类衍生物TM1的合成
Figure GDA0003640403630000073
向反应瓶中依次加入唑(RH)、N,N-二甲基甲酰胺(DMF)、K2CO3室温搅拌，加入中间体IM2-1，并转移至45℃水浴搅拌，TLC跟踪监测至反应结束。停止搅拌，加入冰冷的饱和NaCl溶液，2N HCl溶液调节pH 6-7，冷藏。抽滤，滤饼用饱和食盐水洗涤(10mL×1)、冰水洗涤(5mL×1)，真空干燥后得粗品，柱层析(DCM:CH3OH＝50:1-5:1,v/v)、薄层层析和/或溶剂分散得目标化合物TM1。实验条件及结果如表1所示。
表1制备TM1的实验条件及结果
Figure GDA0003640403630000081
4、TM1产物结构表征数据如下：
Figure GDA0003640403630000091
TM1-1:Methyl 4-(2-(1H-imidazol-1-yl)acetamido)-2-hydroxybenzoateWhitesolid；m.p.184.0～186.3℃；1H NMRδ10.64(s,1H,H-5),10.60(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.63(s,1H,H-8),7.36(d,J＝1.3Hz,1H,H-4),7.17(s,1H,H-10),7.09(dd,J＝8.7Hz,1.4Hz,1H,H-2),6.90(s,1H,H-9),4.94(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.38,167.06,161.64,145.45,138.83,131.44,128.44,121.20,110.91,108.35,106.75,52.70,49.74.HRMS calcd for C13H13N3O4,[M+H]+276.0979,found 276.0976.
TM1-2:Methyl 2-hydroxy-4-(2-(2-methyl-1H-imidazol-1-yl)acetamido)benzoateWhite solid；m.p.188.6～191.0℃；1H NMRδ10.65(s,1H,H-5),10.61(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.8Hz,1H,H-4),7.08(dd,J＝8.7Hz,1.8Hz,1H,H-2),6.84(s,1H,H-10),6.74(s,1H,H-9),4.84(s,2H,H-7),3.86(s,3H,H-6),2.18(s,3H,H-8).13C NMRδ169.68,168.23,164.36,144.2,143.01,131.52,126.94,119.19,114.11,108.42,106.21,51.69,49.61,13.27.HR MS calcd for C14H15N3O4,[M+H]+290.1135,found290.1131.
TM1-3:Methyl 2-hydroxy-4-(2-(4-methyl-1H-imidazol-1-yl)acetamido)benzoateWhite solid；m.p.184.8～186.9℃；1H NMRδ10.64(s,1H,H-5),10.56(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.48(s,1H,H-8),7.36(d,J＝1.8Hz,1H,H-4),7.08(dd,J＝8.7Hz,1.8Hz,1H,H-2),6.84(s,1H,H-10),4.84(s,2H,H-7),3.86(s,3H,H-6),2.08(s,3H,H-9).13C NMRδ169.38,167.17,161.64,145.46,138.03,136.78,131.43,117.36,110.91,108.32,106.74,52.70,49.72,14.05.HR MS calcd for C14H15N3O4,[M+H]+290.1135,found290.1133.
Figure GDA0003640403630000092
TM1-4:Methyl 4-(2-(1H-benzo[d]imidazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.227.5～229.3℃；1H NMRδ10.78(s,2H,H-3 and H-5),8.22(s,1H,H-8),7.67(t,J＝8.5Hz,2H,H-9 and H-12),7.54(d,J＝7.8Hz,1H,H-1),7.24(s,1H,H-4),7.20(d,J＝6.3Hz,2H,H-10 and H-11),6.94(d,J＝8.6Hz,1H,H-2),5.20(s,2H,H-7),3.80(s,3H,H-6).13C NMRδ169.27,166.72,163.01,145.48,145.30,143.66,134.92,131.58,122.87,121.99,119.82,110.81,109.97,108.75,107.52,52.45,47.87.HRMS calcd for C17H15N3O4,[M+H]+326.1135,found 326.1147.
TM1-5:Methyl 4-(2-(1H-pyrazol-1-yl)acetamido)-2-hydroxybenzoateWhitesolid；m.p.203.1.0～206.3℃；1H NMRδ10.69(s,1H,H-5),10.51(s,1H,H-3),7.80(d,J＝8.5Hz,1H,H-1),7.76(s,1H,H-10),7.54(d,J＝1.7Hz,1H,H-4),7.47(s,1H,H-9),7.23(dd,J＝8.7Hz,1.7Hz,1H,H-2),6.29(s,1H,H-9),4.82(s,2H,H-7),3.87(s,3H,H-6),H-8.13CNMRδ169.13,166.78,161.82,145.41,139.56,132.10,131.34,110.93,108.40,106.76,105.83,61.57,54.98.HR MS calcd for C13H13N3O4,[M+H]+276.0979,found 276.0977.
TM1-6:Methyl 4-(2-(3,5-dimethyl-1H-pyrazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.260.3～262.4℃；1H NMRδ10.66(s,1H,H-5),10.47(s,1H,H-3),7.80(d,J＝8.5Hz,1H,H-1),7.54(d,J＝1.7Hz,1H,H-4),7.23(dd,J＝8.7Hz,1.7Hz,1H,H-2),5.82(s,1H,H-8),4.82(s,2H,H-7),3.87(s,3H,H-6),2.69(s,3H,H-9),2.17(s,3H,H-10).13C NMRδ169.47,166.92,161.34,145.84,142.59,137.32,129.37,113.56,104.29,104.15,102.84,61.28,54.18,14.71,13.50.HR MS calcd for C15H17N3O4,[M+H]+304.1292,found 304.1294.
Figure GDA0003640403630000101
TM1-7:Methyl 4-(2-(1H-1,2,4-triazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.220.8～222.9℃；1H NMRδ10.71(s,1H,H-5),10.65(s,1H,H-3),8.57(s,1H,H-8),8.01(s,1H,H-9),7.76(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.6Hz,1H,H-2),5.19(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.35,165.82,161.62,151.88,146.10,145.19,131.47,110.97,108.57,106.88,52.70,52.36.HRMS calcd for C12H12N4O4,[M+H]+277.0931,found 277.0938.
TM1-8:Methyl 4-(2-(1H-tetrazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.187.8～189.9℃；1H NMRδ10.90(s,1H,H-5),10.64(s,1H,H-3),9.05(s,1H,H-8),7.77(d,J＝8.7Hz,1H,H-1),7.33(s,1H,H-4),7.09(d,J＝8.7Hz,1H,H-2),5.77(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.27,164.14,161.55,153.89,144.88,131.56,111.04,108.87,107.03,55.57,52.74.HR MS calcd for C11H11N5O4,[M+H]+278.0884,found 278.0881.
TM1-9:Methyl 4-(2-(2H-tetrazol-2-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.207.3～209.8℃；1H NMRδ10.91(s,1H,H-5),10.63(s,1H,H-3),9.43(s,1H,H-8),7.77(d,J＝8.7Hz,1H,H-1),7.34(s,1H,H-4),7.10(d,J＝8.7Hz,1H,H-2),5.54(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.29,164.76,161.56,145.70,145.00,131.54,110.99,108.76,106.96,52.72,50.66.HR MS calcd for C11H11N5O4,[M+H]+278.0884,found 278.0882.
Figure GDA0003640403630000111
TM1-10:Methyl 2-hydroxy-4-(2-(5-methyl-1H-tetrazol-1-yl)acetamido)benzoate White solid；m.p.189.0～190.8℃；1H NMRδ10.86(s,1H,H-5),10.64(s,1H,H-3),7.77(d,J＝8.7Hz,1H,H-1),7.34(d,J＝1.3Hz,1H,H-4),7.08(dd,J＝8.7Hz,1.4Hz,1H,H-2),5.66(s,2H,H-7),3.87(s,3H,H-6),2.49(s,3H,H-8).13C NMRδ169.29,164.23,162.91,161.56,144.91,131.53,111.03,108.82,107.01,55.45,52.73,10.83.HR MScalcd for C12H13N5O4,[M+H]+292.1040,found 292.1039.
TM1-11:Methyl 4-(2-((4,6-dimethylpyrimidin-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.184.5～186.7℃；1H NMRδ10.70(s,1H,H-5),10.54(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.34(d,J＝1.6Hz,1H,H-4),7.08(dd,J＝8.8Hz,1.6Hz,1H,H-2),6.96(s,1H,H-8),4.05(s,2H,H-7),3.87(s,3H,H-6),2.31(s,6H,H-9 andH-10).13C NMRδ169.65,169.19,167.87,167.43(2C),161.86,145.88,131.23,116.54,110.84,108.04,106.52,61.52,36.13,23.76(2C).HR MS calcd for C16H17N3O4S,[M+H]+348.1013,found 348.1016.
TM1-12:Methyl 2-hydroxy-4-(2-((1-methyl-1H-tetrazol-5-yl)thio)acetamido)benzoateWhite solid；m.p.262.1～264.4℃；1H NMRδ10.66(brs,2H,H-3 andH-5),7.75(d,J＝8.7Hz,1H,H-1),7.34(s,1H,H-4),7.06(d,J＝8.2Hz,1H,H-2),4.33(s,2H,H-7),3.99(s,3H,H-6),3.87(s,3H,H-8).13C NMRδ169.34,166.24,161.81,153.74,145.37,131.44,110.77,108.52,106.87,52.65,38.28,34.15.HR MS calcd forC12H13N5O4S,[M+H]+324.0761,found 324.0758.
Figure GDA0003640403630000112
TM1-13:Methyl 2-hydroxy-4-(2-((1-phenyl-1H-tetrazol-5-yl)thio)acetamido)benzoateWhite solid；m.p.224.9～226.6℃；1H NMRδ10.66(s,2H,H-5 and H-3),7.69(m,6H,H-1,H-8～H-12),7.18(s,1H,H-4),6.90(d,1H,H-2),4.41(s,2H,H-7),3.80(s,3H,H-6).13C NMRδ169.20,165.85,163.79,154.42,145.29,133.54,131.61,131.14,130.55(2C),124.93(2C),109.37,108.92,107.91,52.29,38.44.HR MS calcd forC17H15N5O4S,[M+H]+386.0918,found 386.0921.
TM1-14:Methyl 2-hydroxy-4-(2-((5-methyl-1,3,4-thiadiazol-2-yl)thio)acetamido)benzoate White solid；m.p.191.7～193.0℃；1H NMRδ10.63(s,1H,H-5),8.57(s,1H,H-3),7.67(d,J＝8.4Hz,1H,H-1),7.20(s,1H,H-4),6.93(d,J＝8.4Hz,1H,H-2),4.28(s,2H,H-7),3.80(s,3H,H-6),2.67(s,3H,H-8).13C NMRδ169.32,166.46,166.22,164.68,162.34,145.45,131.46,110.40,108.58,107.12,52.55,38.73,15.63.HR MScalcd for C13H13N3O4S2,[M+H]+340.0420,found 340.0417.
TM1-15:Methyl 4-(2-((5-amino-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.200.3～202.9℃；1H NMRδ10.63(s,1H,H-5),10.55(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.5Hz,1H,H-4),7.32(s,2H,H-8),7.06(dd,J＝8.7Hz,1.6Hz,1H,H-2),4.02(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.38,167.10,162.79(2C),161.62,145.54,131.38,110.93,108.36,106.74,52.69,36.24.HR MScalcd for C12H12N4O4S2,[M+H]+341.0373,found 341.0362.
5、中间体IM1-2的合成
Figure GDA0003640403630000121
向反应瓶中加入PAS 20mmol、无水乙醇25mL，室温搅拌。冰浴，缓慢滴加浓硫酸50mmol，滴毕，80℃油浴回流反应，TLC监测直至反应结束。冰浴冷却，饱和碳酸钠调节pH在7-8，冷藏，抽滤，滤饼用冰水洗涤。滤液用DCM萃取(3×30mL)，收集有机相，饱和NaCl溶液洗涤。无水硫酸钠干燥，旋蒸，并与滤饼合并，真空干燥后，柱层析，旋蒸，称重，得中间体IM1-2(白色固体)2.32g，收率为64％。
6、中间体IM2-2的合成
Figure GDA0003640403630000122
向反应瓶中加入IM1-2 5mmol、DCM 5mL、NaHCO3 12.5mmol；冰浴冷却，滴加氯乙酰氯10mmol。冰浴下持续反应，TLC跟踪监测至反应结束。停止搅拌，加入10mL冰冷的饱和食盐水，用2N HCl溶液调节pH在4-5，搅拌均匀后移入分液漏斗，EA萃取两次，合并有机相，饱和食盐水洗涤，无水Na2SO4干燥，旋蒸除去溶剂，柱层析得纯品，干燥，称重，得中间体IM2-2为1.16g，收率为90％。
7、对氨基水杨酸唑类衍生物TM4的合成
Figure GDA0003640403630000131
向反应瓶中依次加入唑(RH)、DMF、K2CO3室温搅拌，加入中间体IM2-2，并转移至45℃水浴搅拌，TLC跟踪监测至反应结束。停止搅拌，加入冰冷的饱和NaCl溶液，2N HCl溶液调节pH 6-7，冷藏。抽滤，滤饼用饱和食盐水洗涤(10mL×1)、冰水洗涤(5mL×1)，真空干燥后得粗品，柱层析(PE/EA＝10:1-1:3,v/v)。当唑为巯基唑时，DMF改为四氢呋喃(THF)，柱层析前先用石油醚多次分散，其余条件或操作与上述非巯基唑相同。干燥，得目标化合物TM4。实验条件及结果如表2所示。
表2制备TM4的实验条件及结果
Figure GDA0003640403630000132
Figure GDA0003640403630000141
8、TM4产物结构表征数据如下：
Figure GDA0003640403630000142
TM4-1:Ethyl 4-(2-(1H-imidazol-1-yl)acetamido)-2-hydroxybenzoate Whitesolid；m.p.206.7～207.9℃；1H NMRδ10.72(s,1H,H-5),10.60(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.63(s,1H,H-11),7.36(d,J＝1.7Hz,1H,H-4),7.17(s,1H,H-9),7.10(dd,J＝8.7Hz,1.8Hz,1H,H-2),6.90(s,1H,H-10),4.94(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.12,167.05,161.82,145.46,138.82,131.35,128.45,121.18,110.91,108.38,106.74,61.56,49.74,14.50.HR MS calcd forC14H15N3O4,[M+H]+290.1135,found 290.1135.
TM4-2:Ethyl 2-hydroxy-4-(2-(2-methyl-1H-imidazol-1-yl)acetamido)benzoate White solid；m.p.203.4～205.7℃；1H NMRδ10.72(s,1H,H-5),10.61(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.7Hz,1H,H-4),7.10(dd,J＝8.7Hz,1.8Hz,1H,H-2),7.05(s,1H,H-9),6.73(s,1H,H-10),4.85(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),2.23(s,3H,H-11),1.33(t,J＝6.9Hz,3H,H-7).13C NMRδ169.12,166.91,161.82,145.41,144.47,131.34,126.51,121.45,110.94,108.41,106.79,61.56,49.18,14.50,13.01.HRMS calcd for C15H17N3O4,[M+H]+304.1292,found 304.1293.
TM4-3:Ethyl 2-hydroxy-4-(2-(4-methyl-1H-imidazol-1-yl)acetamido)benzoate White solid；m.p.207.3～209.9℃；1H NMRδ10.71(s,1H,H-5),10.56(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.48(s,1H,H-11),7.36(d,J＝1.6Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.6Hz,1H,H-2),6.84(s,1H,H-9),4.84(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),2.08(s,3H,H-11),1.33(t,J＝6.9Hz,3H,H-7).13C NMRδ169.13,167.15,161.82,145.47,138.02,136.79,131.33,117.36,110.91,108.35,106.73,61.56,49.74,14.50,14.04.HRMS calcd for C15H17N3O4,[M+H]+304.1292,found 304.1289.
Figure GDA0003640403630000151
TM4-4:Ethyl 4-(2-(1H-benzo[d]imidazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.246.1～248.1℃；1H NMRδ10.77(s,1H,H-5),10.71(s,1H,H-3),8.24(s,1H,H-9),7.77(d,J＝8.7Hz,1H,H-1),7.68(d,J＝7.8Hz,1H,H-13),7.55(d,J＝7.9Hz,1H,H-10),7.36(d,J＝1.8Hz,1H,H-4),7.28–7.17(m,2H,H-11 and H-12),7.12(dd,J＝8.8Hz,1.8Hz,1H,H-2),5.22(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.32(t,J＝7.1Hz,3H,H-7).13C NMRδ169.11,166.82,161.82,145.47,145.41,143.67,134.94,131.38,122.88,122.00,119.84,110.95,110.81,108.43,106.78,61.57,47.86,14.50.HR MS calcd for C18H17N3O4,[M+H]+340.1292,found 340.1291.
TM4-5:Ethyl 4-(2-(1H-pyrazol-1-yl)acetamido)-2-hydroxybenzoate Whitesolid；m.p.221.9～223.7℃；1H NMRδ10.72(s,1H,H-5),10.61(s,1H,H-3),7.76(d,J＝8.7Hz,2H,H-1 and H-9),7.47(s,1H,H-11),7.36(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,J＝1.5Hz,1H,H-2),6.29(s,1H,H-10),5.06(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.13,166.78,161.82,145.41,139.56,132.10,131.34,110.93,108.40,106.76,105.83,61.57,54.98,14.50.HR MS calcd forC14H15N3O4,[M+H]+290.1135,found 290.1133.
TM4-6:Ethyl 4-(2-(1H-1,2,4-triazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.273.5～275.8℃；1H NMRδ10.71(s,2H,H-5 and H-3),8.56(s,1H,H-9),8.01(s,1H,H-10),7.77(d,J＝8.7Hz,1H,H-1),7.35(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.7Hz,1H,H-2),5.18(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.08,165.82,161.79,151.88,146.09,145.20,131.40,110.96,108.59,106.85,61.58,52.35,14.50.HR MS calcd for C13H14N4O4,[M+H]+291.1088,found291.1087.
Figure GDA0003640403630000161
TM4-7:Ethyl 4-(2-(1H-tetrazol-1-yl)acetamido)-2-hydroxybenzoate Whitesolid；m.p.245.7～247.1℃；1H NMRδ10.90(s,1H,H-5),10.71(s,1H,H-3),9.05(s,1H,H-9),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.09(d,J＝8.7Hz,1.7Hz,1H,H-2),5.77(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.02,164.13,161.74,153.88,144.89,131.47,111.02,108.87,107.00,61.61,55.57,14.49.HRMS calcd for C12H13N5O4,[M+H]+292.1040,found 292.1040.
TM4-8:Ethyl 4-(2-(2H-tetrazol-2-yl)acetamido)-2-hydroxybenzoate Whitesolid；m.p.255.1～256.8℃；1H NMRδ10.78(s,2H,H-5 and H-3),9.42(s,1H,H-9),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.09(d,J＝8.7Hz,1.7Hz,1H,H-2),5.53(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.03,164.76,161.75,145.70,144.99,131.48,110.97,108.79,106.93,61.60,50.66,14.50.HRMS calcd for C12H13N5O4,[M+H]+292.1040,found 292.1042.
TM4-9:Ethyl 2-hydroxy-4-(2-(5-methyl-1H-tetrazol-1-yl)acetamido)benzoateWhite solid；m.p.203.1～205.8℃；1H NMRδ10.86(s,1H,H-5),10.71(s,1H,H-3),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.08(d,J＝8.7Hz,1.7Hz,1H,H-2),5.65(s,2H,H-8),4.34(d,J＝7.1Hz,2H,H-6),2.49(s,3H,H-9),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.03,164.22,162.91,161.74,144.91,131.46,111.02,108.85,106.98,61.61,55.45,14.50,10.84.HR MS calcd for C13H15N5O4,[M+H]+306.1197,found306.1195.
Figure GDA0003640403630000162
TM4-10:Ethyl 2-hydroxy-4-(2-(5-methyl-2H-tetrazol-2-yl)acetamido)benzoateWhite solid；m.p.216.3～218.8℃；1H NMRδ10.85(s,1H,H-5),10.71(s,1H,H-3),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.8Hz,1H,H-2),5.43(s,2H,H-8),4.34(d,J＝7.0Hz,2H,H-6),2.51(s,3H,H-9),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.02,164.64,161.74,153.95,144.91,131.46,111.02,108.83,107.00,61.61,49.78,14.50,8.76.HR MS calcd for C13H15N5O4,[M+H]+306.1197,found306.1196.
TM4-11:Ethyl 4-(2-((4,6-dimethylpyrimidin-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.279.3～281.2℃；1H NMRδ10.71(s,1H,H-5),10.55(s,1H,H-3),7.74(d,J＝8.7Hz,1H,H-1),7.37(d,J＝1.6Hz,1H,H-4),7.09(dd,J＝8.8Hz,1.6Hz,1H,H-2),6.96(s,1H,H-10),4.33(q,J＝7.1Hz,2H,H-6),4.05(s,2H,H-8),2.31(s,6H,H-9 and H-11),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.65,169.19,167.87,167.43(2C),161.86,145.88,131.23,116.54,110.84,108.04,106.52,61.52,36.13,23.76(2C),14.51.HR MS calcd for C17H19N3O4S,[M+H]+362.1169,found 362.1165.
TM4-12:Ethyl 2-hydroxy-4-(2-((1-methyl-1H-tetrazol-5-yl)thio)acetamido)benzoate White solid；m.p.241.7～243.3℃；1H NMRδ10.78(s,2H,H-5 andH-3),7.74(d,J＝8.7Hz,1H,H-1),7.34(s,1H,H-4),7.05(d,J＝8.3Hz,1H,H-2),4.35～4.31(m,4H,H-8 and H-6),3.99(s,3H,H-9),1.32(t,J＝7.1Hz,3H,H-7).13C NMRδ169.08,166.27,153.75,145.41,131.34(2C),110.73,108.50,106.85,61.51,38.24,34.15,14.51.HR MS calcd for C13H15N5O4S,[M+H]+338.0918,found 338.0920.
Figure GDA0003640403630000171
TM4-13:Ethyl 2-hydroxy-4-(2-((1-phenyl-1H-tetrazol-5-yl)thio)acetamido)benzoateWhite solid；m.p.214.3～215.9℃；1H NMRδ10.74(s,2H,H-5 and H-3),7.75(d,J＝8.7Hz,1H,H-1),7.72–7.67(m,5H,H-9、H-10、H-11、H-12 and H-13),7.34(d,J＝1.5Hz,H-4),7.06(dd,J＝8.6Hz,1.5Hz,1H,H-2),4.43(s,2H,H-8),4.33(q,J＝7.1Hz,2H,H-6),1.32(t,J＝7.1Hz,3H,H-7).13C NMRδ169.09,166.00,162.00,154.38,145.40,133.51,131.36,131.16,130.56(2C),124.90(2C),110.76,108.51,106.84,61.52,38.35,14.51.HR MS calcd for C18H17N5O4S,[M+H]+400.1074,found 400.1074.
TM4-14:Ethyl 2-hydroxy-4-(2-((5-methyl-1,3,4-thiadiazol-2-yl)thio)acetamido)benzoate White solid；m.p.218.1～219.9℃；1H NMRδ10.71(s,1H,H-5),10.65(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.37(d,J＝1.5Hz,H-4),7.08(dd,J＝8.7Hz,1.5Hz,1H,H-2),4.34(q,J＝7.1Hz,2H,H-6),4.30(s,2H,H-8),2.68(s,3H,H-9),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.11,166.51,166.23,164.62,161.81,145.50,131.33,110.88,108.39,106.70,61.57,38.67,15.64,14.51.HR MS calcd forC14H15N3O4S2,[M+H]+353.0504,found 353.0501.
TM4-15:Ethyl 4-(2-((5-amino-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.237.1～238.8℃；1H NMRδ10.71(s,1H,H-5),10.54(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.37(d,J＝1.5Hz,H-4),7.32(s,2H,H-9),7.06(dd,J＝8.7Hz,1.5Hz,1H,H-2),4.34(q,J＝7.0Hz,2H,H-6),4.03(s,2H,H-8),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ170.48,169.12,167.09,161.81,149.61,145.55,131.30,110.89,108.35,106.70,61.56,39.29,14.51.HR MS calcd for C13H14N4O4S2,[M+H]+355.0529,found 355.0523.
Figure GDA0003640403630000181
TM4-16:Ethyl 4-(2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.249.4～251.7℃；1H NMRδ10.74(s,1H,H-5),10.71(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.74(d,J＝1.8Hz,1H,H-9),7.69(d,J＝8.7Hz,1H,H-11),7.38(d,J＝2.0Hz,1H,H-10),7.36(d,J＝1.5Hz,1H,H-4),7.10(dd,J＝8.7Hz,1.5Hz,1H,H-2),4.44(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.09,166.30,166.05,161.80,150.63,145.42,142.96,131.37,129.48,124.78,118.52,111.99,110.91,108.49,106.75,61.57,37.46,14.51.HR MS calcd forC18H15ClN2O5S,[M+H]+407.0463,found 407.0462.
TM4-17:Ethyl 2-hydroxy-4-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate White solid；m.p.245.3～247.2℃；1H NMRδ12.53(s,1H,H-9),10.81(s,1H,H-5),10.72(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.38(d,J＝1.8Hz,1H,H-10),7.36(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,J＝1.5Hz,1H,H-2),6.94(s,1H,H-13),6.79～6.74(m,1H,H-11),4.34(q,J＝7.1Hz,2H,H-6),4.26(s,2H,H-8),3.77(s,3H,H-12),1.33(t,J＝7.1Hz,3H,H-7).HR MS calcd for C19H19N3O5S,[M+H]+402.1118,found402.1118.
TM4-18:Ethyl 4-(2-((6-chlorobenzo[d]thiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.253.5～255.9℃；1H NMRδ10.74(s,1H,H-5),10.72(s,1H,H-3),8.18(d,J＝1.9Hz,1H,H-9),7.80(d,J＝8.7Hz,1H,H-11),7.76(d,J＝8.7Hz,1H,H-1),7.49(dd,J＝8.7,2.1Hz,1H,H-10),7.38(d,J＝1.8Hz,1H,H-4),7.11(dd,J＝8.7Hz,1.8Hz,1H,H-2),4.44(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.12,167.72,166.39,161.83,151.78,145.50,136.86,131.32,129.47,127.28,122.58,122.07,110.91,108.42,106.74,61.56,38.41,14.50.HR MScalcd for C18H15ClN2O4S2,[M+H]+423.0235,found 423.0237.
Figure GDA0003640403630000182
TM4-19:Ethyl 4-(2-((6-bromobenzo[d]thiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.241.7～243.8℃；1H NMRδ10.74(s,1H,H-5),10.72(s,1H,H-3),8.31(d,J＝1.8Hz,1H,H-9),7.76(d,J＝8.8Hz,1H,H-11),7.74(d,J＝8.7Hz,1H,H-1),7.61(dd,J＝8.7,1.8Hz,1H,H-10),7.38(d,J＝1.7Hz,1H,H-4),7.11(dd,J＝8.7Hz,1.8Hz,1H,H-2),4.44(s,2H,H-8),4.34(dd,J＝14.1,7.0Hz,2H,H-6),1.33(t,J＝7.0Hz,3H,H-7).13C NMRδ169.12,167.78,166.38,161.83,152.06,145.50,137.30,131.32,129.97,124.90,122.95,117.47,110.91,108.42,106.74,61.56,38.41,14.51.HR MScalcd for C18H15BrN2O4S2,[M+H]+:466.9729,found 466.9727.
TM4-20:Ethyl 2-hydroxy-4-(2-((6-methoxybenzo[d]thiazol-2-yl)thio)acetamido)benzoate White solid；m.p.236.4～238.8℃；1H NMRδ10.73(s,1H,H-5),10.71(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.72(d,J＝8.9Hz,1H,H-9),7.62(d,J＝2.5Hz,1H,H-12),7.39(d,J＝1.9Hz,1H,H-4),7.11(dd,J＝8.7Hz,2.0Hz,1H,H-2),7.06(dd,J＝8.9,2.6Hz,1H,H-11),4.39(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),3.81(s,3H,H-10),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.14,166.62,162.78,161.84,157.29,147.45,145.55,136.79,131.30,122.09,115.63,113.52,110.90,106.72,105.47,61.55,56.18,38.39,14.49.
三、对氨基水杨酸唑类衍生物的生物活性检测
1、体外抗细菌活性测定
采用微量肉汤稀释法，测定化合物抑制金黄色葡萄球菌(Staphyloccocus aureusATCC 25129)、藤黄微球菌(Micrococcus luteus)、大肠杆菌(Escherichia coli ATCC25922)、鲍曼不动杆菌(Acinetobacter baumannii ATCC 19606)、沙门氏菌(SalmonellaEnteritidis ATCC 13076)和铜绿假单胞菌(Pseudomonas aeruginosa ATCC 27853)的活性(MIC值)。
取样品3.2mg，先以质量分数5％吐温80的DMSO液配成母液，浓度为3.2mg/mL，再吸取200μL母液，用肉汤稀释至500μL，浓度为1.28μg/μL，为待测液。
接种保存的菌株于普通液体培养基中，置于37℃恒温摇床活化培养17h。活化后用脑心浸液肉汤(BHI)培养基分别稀释成105CFU/mL的菌悬液备用。
操作：在96孔板每一列的第1孔加入空白肉汤60μL，其余孔加入空白肉汤50μL；在每一列的第1孔加配好的待测液(浓度为1.28μg/μL)40μL，然后对待测物进行二倍稀释，即，第一孔中加入待测液后用移液枪充分吹打(至少三次以上)使待测物与肉汤充分混匀，然后吸取50μL加入第2孔，再充分吹打使之与肉汤充分混匀，吸取50μL加入第3孔，照此重复直至第8孔，吸取50μL弃去；此时每孔待测物浓度从上到下依次为512,256,128,64,32,16,8,4(单位：μg/mL)。每一块板的最后两列作为对照，两列都不含待测物，一列作为细菌生长对照加入菌液，另一列作为阴性对照不加菌液。最后，在每列1-8孔中加入稀释好的菌液50μL，采取复孔测试，每块板测试5个化合物，此时每孔待测物浓度即最终待测物浓度从上到下依次为256,128,64,32,16,8,4,2(单位：μg/mL)。
将接种好的96孔板放入37℃恒温培养箱培养培养20-24h，观察孔内细菌生长情况。确定阴性对照孔的细菌正常生长、阳性对照孔无细菌生长。观察没有细菌生长的孔中的药物浓度作为该药物对该细菌的MIC值。
对氨基水杨酸唑类衍生物及中间体的体外抑菌活性测试结果，如表3所示。
表3化合物对6种致病菌的抑制活性(MIC值，μg/mL)
Figure GDA0003640403630000201
Figure GDA0003640403630000211
从表3中分析可知：PAS、PAS的甲酯(IM1-1)和乙酯(IM1-2)几乎没有抑制活性(MIC>256μg/mL)；目标化合物即对氨基水杨酸唑类衍生物的抑菌活性整体好于PAS、IM1-1和IM1-2。化合物对金黄色葡萄球菌的抑制活性好于其它菌株，有9个化合物的MIC值为32μg/mL，其中，乙酯类衍生物(TM4)有6个，甲酯类衍生物(TM1)有3个；中间体IM2-1和IM2-2对金黄色葡萄球菌的抑菌活性低至25.6μg/mL和12.8μg/mL。对大肠杆菌，化合物TM4-12的MIC值为32μg/mL。对藤黄微球菌，化合物TM4-9和TM4-11的MIC值为16μg/mL。特别地，中间体IM2-2对藤黄微球菌的MIC值为3.2μg/mL、对沙门氏菌MIC值为0.8μg/mL，抑菌活性很强。上述结果显示，对氨基水杨酸氯乙酰基衍生物、对氨基水杨酸唑类衍生物均具有抗细菌活性。从而证明了对氨基水杨酸唑类衍生物及其中间体在抗细菌领域具有潜在的应用前景。
2、体外抑制真菌活性测定
采用NCCLS推荐的微量肉汤稀释法，氟康唑为阳性对照药物，测定化合物抑制毕赤酵母菌的活性(MIC值)。
(1)样品溶液的制备
用万分之一天平在干燥室内精确称取样品3.2mg于2mL PE管中，移液枪向PE管中加入1mL DMSO，溶解为澄清透明液体，配制成3.2mg/mL的母液或储备液，封口膜封口后，冰柜避光保存。溶剂为DMSO，对于部分难溶的化合物使用DMSO/吐温-80＝200/1(v/v)为溶剂以增加溶解度，吐温-80为助溶剂。
(2)待测液的配制
根据待测物的效果或是含量及所需要的体积，计算出待测物所需的量，并准确称取所需的各种抗菌待测物，用适宜的溶剂及稀释剂将待测物稀释至所需的浓度。
待测液B配制：吸取320μL浓度为3.2mg/mL储备液，加入沙氏培养基180μL至总体积0.5mL，其稀释液浓度为2048μg/mL，即为待测液B。
(3)菌悬液的制备
接种保存的菌株于沙氏琼脂液体培养基中，置于30℃恒温摇床活化培养24h。活化后用蒸馏水洗涤琼脂表面菌落，后用沙氏培养基稀释成105CFU/mL的菌悬液备用。
(4)加样操作
无菌条件下，在96孔板每个孔加入沙氏培养基50μL；在第一排的第一孔、第二孔添加配好的待测液B 50μL，经过此二倍稀释后，浓度为1024μg/mL；第一孔、第二孔用移液枪充分吹打，使待测物与培养基充分混匀，然后吸取50μL加入第二排的第一孔、第二孔，再吹打使之与培养基充分混匀，照此重复直至第八排，第八排每孔吸取50μL弃去；此时每孔待测物浓度从高至低(从上至下)依次为1024,512,256,128,64,32,16,8(单位：μg/mL)；再在96孔板每孔中加入稀释好的菌液50μL，此时每孔待测物浓度即最终待测物浓度从高至低(从上至下)依次为512,256,128,64,32,16,8,4(单位：μg/mL)。
(5)培养和结果判定
将接种好的96孔板放入30℃恒温培养箱培养30h和48h。培养完成后，将96孔板从恒温箱中取出，观察孔内细菌生长情况。确定空白无药对照(阴性对照)孔的细菌正常生长和阳性对照(培养基+菌株+阳性药物)孔无细菌生长，所测试的结果才算正常。肉眼观察没有细菌生长的孔中的药物浓度作为该药物对该细菌的MIC。
对氨基水杨酸唑类衍生物及中间体的体外抑真菌活性测试结果，如表4所示。
表4毕赤酵母菌的MIC值测定结果(μg/mL)
Figure GDA0003640403630000221
Figure GDA0003640403630000231
从表4中可知，母核PAS、IM1-1和IM1-2的MIC>256μg/mL，对毕赤酵母菌几乎没有抑制活性；中间体IM2-1和IM2-2，对毕赤酵母菌的抑制活性达到64μg/mL，强于母核PAS。绝大多数目标化合物对毕赤酵母菌的抑制活性很好，强于母体PAS、PAS的甲酯、PAS的乙酯；培养24h，35个目标分子中，有30个分子的MIC值在4～256μg/mL之间，有28个分子的MIC值在4～128μg/mL之间，有16个分子的MIC值在4～64μg/mL之间，有10个分子的MIC值在4～32μg/mL之间，甚至有4个分子的MIC＝4μg/mL，与阳性对照药物氟康唑的MIC值相同，显示这4个分子对毕赤酵母菌株的抑制活性强于或相当于氟康唑，从而证明了对氨基水杨酸唑类衍生物及其中间体在抗真菌领域具有潜在的应用前景。
3、抗肿瘤生物活性测定
一系列小分子化合物在肿瘤细胞中的抑制率由MTT实验获得，具体步骤如下：
(1)处于对数生长期的肿瘤细胞悬液接种于96孔培养板(3×103细胞/孔)，每孔培养基总量200μL，于37℃、5％CO2细胞培养箱中培养过夜；
(2)待细胞完全贴壁后，用含10μM的小分子化合物的完全培养基更换之前的培养基，对照组添加等体积的DMSO完全培养基，连续培养48h；
(3)加入20μL MTT溶液后，继续放置在37℃、5％CO2细胞培养箱中孵育4h后，取出培养板，轻柔吸除上清，尽量避免吸取紫色结晶，再向每孔加入150μL DMSO溶液；
(4)将上述处理后的培养板置于摇床上，在室温下避光轻柔振摇10min，使得结晶紫尽量溶解均匀；
(5)将上述培养板置于酶标仪上，选择波长为570nm，读取并记录96孔板中对应孔中的吸光值(OD值)，进而统计获得小分子化合物在不同肿瘤细胞中的相应抑制率。5μM浓度下测定结果如表5所示。
表5化合物对肿瘤细胞的抑制活性结果
Figure GDA0003640403630000241
分析表5数据可知，对氨基水杨酸唑类衍生物在5μM浓度下对三种癌细胞均有抑制作用。针对结肠癌细胞HCT116，有4个化合物(TM1-2、TM4-4、IM2-1和IM2-2)的抑制率超过50％，中间体IM2-2的抑制率达到60％，远强于母体对氨基水杨酸、对氨基水杨酸甲酯及其乙酯。针对结肠癌细胞SW620，整体都有一定的抑制作用，大多数化合物均强于母核PAS(8％)。针对***癌细胞PC3，有3个化合物(TM4-3、TM4-5和IM2-2)的抑制率高于50％，中间体IM2-2的抑制率达到78％，63％的化合物对***癌细胞PC3的抑制作用强于母核PAS(5％)。证明了对氨基水杨酸唑类衍生物及其中间体在抗肿瘤领域具有潜在的应用前景。
4、抗柑橘真菌病菌生物活性测定
1)初筛
(1)待测物母液及稀释液的配制
用适宜的溶剂及稀释剂将待测物母液稀释至所需的浓度。样品质量为1.0mg，先配成待测物母液1.0mg/1mL＝1.0mg/mL；每种待测物设置2个稀释浓度，0.001mg/mL(即稀释1000倍，1μg/mL)和0.004mg/mL(即稀释250倍，4μg/mL)。
(2)操作
待测物培养基的配制：①稀释1000倍的待测物培养基的配制：取5μL浓度为1μg/mL的待测物稀释液与5mL热PDA培养基在10mL离心管中充分混匀；②稀释250倍的药剂培养基的配制：取20μL浓度为4μg/mL的待测物稀释液与4980μL热PDA培养基在10mL离心管中充分混匀。
对照组：以不加待测物的PDA培养基和加入咪鲜胺的培养基(稀释1000倍和稀释250倍)作为对照，分别为空白对照和阳性对照。接菌：将配置好的待测物培养基倒入24孔板内，每株菌每种待测物每个浓度倒一个孔。挑取28℃培养7d的菌株的菌丝，接种于每孔。培养：将24孔板放于28℃、光照16h的培养箱内培养48h。测量：运用十字交叉法测量菌落直径。计算：抑制率％＝(CK菌落直径值-测量菌落直径值)×100％/CK菌落直径值。筛选：将不同待测物抑制率同咪鲜胺的抑制率进行比较，获得初筛结果。
2)复筛
初筛获得的高活性分子TM1-5和TM4-10进行抑制柑橘胶孢炭疽病菌Colletotrichum gloeosporioides菌株Co.3的复筛，获得毒力方程。TM1-2和IM2-2对柑橘褐斑病菌Alternaria alternata菌株Al.6进行复筛，获得毒力方程。
第一步：待测物梯度稀释。设置6个稀释梯度，即0.01、0.004、0.002、0.001、0.0005、0.00025(单位：mg/mL)，即稀释100、250、500、1000、2000、4000倍。第二步：待测物培养基配制。分别取50、20、10、5、2.5、1.25μL的待测物稀释液与5mL热PDA培养基在10mL离心管中充分混匀。将待测物培养基倒入孔内，每种待测物每个梯度重复4次。以PDA培养基与咪鲜胺作为对照组(空白对照和阳性对照)。第三步：接菌。挑取28℃培养7d的菌株的菌丝，接种于每孔正中央。第四步：培养。将24孔板放于28℃、光照16h的培养箱内培养48h。第五步：测量。十字交叉法测量菌落直径。第六步：计算。使用农药室内生物测定数据处理***(PBT数据处理***)处理数据，获得回归方程、KD50、KD90、R、标准误差、卡平方值和95％置信的值。
初筛及复筛测定结果如表6、表7、表8和表9所示。
表6化合物对柑橘真菌病菌的抑制活性(初筛结果)
Figure GDA0003640403630000251
Figure GDA0003640403630000261
分析表6数据发现，所测试化合物对柑橘胶孢炭疽菌菌株、柑橘褐斑病菌菌株均有一定的抑制活性；在1μg/mL和4μg/mL浓度下，对柑橘胶孢炭疽菌，达到阳性药物50％抑制率的分子数分别为19个和18个，达到阳性药物70％抑制率的分子数分别为8个和3个；对柑橘褐斑病菌，达到阳性药物50％抑制率的分子数分别为4个和9个，达到阳性药物70％抑制率的分子数分别为1个和3个。特别值得一提的是，目标化合物TM1-2及中间体IM2-2对柑橘褐斑病菌的抑制活性，分别与阳性对照咪鲜胺的抑制活性相当或更强，显示了很好的开发潜力。
表7化合物对柑橘真菌病菌的抑制活性(初筛结果)
Figure GDA0003640403630000271
表7显示，8种沙星药物和PAS在测试浓度下对两种柑橘真菌都没有抑制活性；然而TM1-13，TM4-1，TM4-8却对柑橘胶孢炭疽菌菌株显示一定抑制活性，强于阳性对照，值得进一步开发。
表8高活性分子对柑橘胶孢炭疽病菌的抑制活性(复筛结果)
Figure GDA0003640403630000272
表9高活性分子对柑橘褐斑病菌的抑制活性(复筛结果)
Figure GDA0003640403630000273
分析表8和表9中数据可知，TM1-2、TM1-5、TM4-10和IM2-2未体现出抗药性，证明了对氨基水杨酸唑类衍生物及其中间体在抗柑橘病菌领域具有潜在的应用前景。
5、抗柑橘溃疡病菌生物活性测定
测定方法：称取1mg样品于50μL DMSO中溶解，用超纯水定容，获得不同浓度样品母液。取10μL母液于1mL超纯水(0.02％吐温)中作为样品溶液a，然后采用倍比稀释法依次配制不同浓度的样品溶液b，c，d等。
将已在PDA培养基上培养3d的溃疡病菌用5mL LB液体培养基洗下，加至195mL LB液体培养基中，振荡混匀备用。在2mL离心管中分别加入450μL柑橘溃疡病菌菌液和上述各不同浓度(a～d)样品溶液50μL，使得各混合菌液中样品最终浓度分别为A(1.6μg/mL)、B(0.64μg/mL)、C(0.5μg/mL)、D(0.1μg/mL)，28℃、200r·min-1恒温振荡培养14h后测定OD600下各混合菌液OD值并计算抑制率(抑制率％＝(OD空白-OD样品)/OD空白×100％)。每个样品每个浓度重复三次。对柑橘溃疡病菌的检测结果如表10和表11所示。
表10化合物对柑橘溃疡病菌的抑制活性
Figure GDA0003640403630000281
表11化合物对柑橘溃疡病菌的抑制活性
Figure GDA0003640403630000282
Figure GDA0003640403630000291
从表11数据可以看出，母体PAS在上述测试浓度下，其抑制率分别为9.44％和6.27％，活性很弱。而对氨基水杨酸唑类衍生物在上述测试浓度下，大多化合物的抑制率优于PAS。其中，在1.6μg/mL测试浓度下，目标分子的抑制活性高于30％的分子有7个，在0.64μg/mL测试浓度下，抑制率高于30％的分子有5个。从而证明对氨基水杨酸唑类衍生物对柑橘溃疡病菌具有良好的抑制作用。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (8)

1.式Ⅰ所示的对氨基水杨酸唑类衍生物或其药学上可接受的盐：
Figure FDA0003640403620000011
式Ⅰ中，
R选自：
Figure FDA0003640403620000012
Figure FDA0003640403620000013
L为-COCH2- ；
X为甲基或乙基。
2.根据权利要求1所述对氨基水杨酸唑类衍生物或其药学上可接受的盐，其特征在于，式Ⅰ所示的对氨基水杨酸唑类衍生物为以下化合物中的任一种：
Figure FDA0003640403620000014
Figure FDA0003640403620000021
Figure FDA0003640403620000031
3.根据权利要求2所述对氨基水杨酸唑类衍生物或其药学上可接受的盐，其特征在于，式Ⅰ所示的对氨基水杨酸唑类衍生物为以下化合物中的任一种：
Figure FDA0003640403620000032
Figure FDA0003640403620000041
4.权利要求1所述对氨基水杨酸唑类衍生物或其药学上可接受的盐的制备方法，其特征在于，包括以下步骤：
将对氨基水杨酸羧基进行酯化，制得中间体IM1；
Figure FDA0003640403620000042
将中间体IM1与linker试剂反应，制得中间体IM2；
Figure FDA0003640403620000043
将中间体IM2与唑偶联，制得目标分子对氨基水杨酸唑类衍生物；
Figure FDA0003640403620000051
式中，X、L和R的定义与权利要求1所述的对氨基水杨酸唑类衍生物结构式中X、L和R的定义相同；IM2中的Z为卤素。
5.权利要求1至权利要求3任一所述对氨基水杨酸唑类衍生物或其药学上可接受的盐在制备抗细菌药物中的应用。
6.权利要求1至权利要求3任一所述对氨基水杨酸唑类衍生物或其药学上可接受的盐在制备抗真菌药物中的应用。
7.权利要求1至权利要求3任一所述对氨基水杨酸唑类衍生物或其药学上可接受的盐在制备抗结肠癌和***癌药物中的应用。
8.权利要求1至权利要求3任一所述对氨基水杨酸唑类衍生物或其药学上可接受的盐在制备抗柑橘病菌药物中的应用。
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