CN112094279B - 对氨基水杨酸双氢青蒿素类衍生物及其制备方法和应用 - Google Patents
对氨基水杨酸双氢青蒿素类衍生物及其制备方法和应用 Download PDFInfo
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- CN112094279B CN112094279B CN202011027532.6A CN202011027532A CN112094279B CN 112094279 B CN112094279 B CN 112094279B CN 202011027532 A CN202011027532 A CN 202011027532A CN 112094279 B CN112094279 B CN 112094279B
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- Prior art keywords
- dihydroartemisinin
- aminosalicylic acid
- alkyl
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- derivatives
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- -1 P-aminosalicylic acid dihydroartemisinin derivative Chemical class 0.000 title claims abstract description 67
- 229960004909 aminosalicylic acid Drugs 0.000 title claims abstract description 46
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- 229930016266 dihydroartemisinin Natural products 0.000 claims abstract description 10
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- 125000006273 (C1-C3) alkyl group Chemical group 0.000 claims description 18
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 15
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- 229910052736 halogen Inorganic materials 0.000 claims description 9
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Abstract
本发明公开了对氨基水杨酸双氢青蒿素类衍生物及其制备方法和应用，属于药物合成技术领域。对氨基水杨酸双氢青蒿素类衍生物的结构式如下。体外抗细菌活性测定结果表明，目标化合物的整体抑菌活性较好；体外抗真菌活性测定结果表明，目标化合物对毕赤酵母菌的抑菌活性整体很好；抗柑橘病菌活性测定结果表明，化合物对柑橘胶孢炭疽菌、柑橘褐斑病菌和柑橘溃疡病菌均有抑制活性；抗耻垢分枝杆菌活性测定结果表明，中间体IM1'和IM2'的的抑制活性均很好。从而证明了对氨基水杨酸双氢青蒿素类衍生物及其中间体在抗细菌、抗真菌、抗柑橘病菌和抗结核领域具有潜在的应用前景。
Description
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双氢青蒿素类衍生物及其制备方法和应用。
背景技术
对氨基水杨酸(简称PAS)是最早发现的结核病治疗小分子药物，也被用于治疗炎症性肠病。PAS氨基衍生物可分为三大类。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(PZA)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)。第二类是PAS腙类衍生物，将异烟肼(INH)与PAS反应制备了含有腙基的化合物，实验结果显示，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。
双氢青蒿素(DHA)作为青蒿素的最重要衍生物，不仅比青蒿素的溶解度、生物利用度和生物活性更好，而且还有半缩醛羟基，该半缩醛羟基为进一步修饰提供了关键的反应位点，同时可以保留母体结构。从DHA开始，成功地设计和合成了许多DHA衍生物，它们具有出色的抗疟疾效力或其他潜在活性，包括抗结核、抗癌、抗过敏、抗菌增强、抗蠕虫病、抗巨细胞病毒、抗病毒和抗HIV的作用等。
结核病是由结核分枝杆菌引起的可导致人类死亡的慢性传染病，致死率仅次于艾滋病(HIV)。尽管抗结核药物已经有10多种，但2018年全球依然新增1000多万结核病患者，结核病仍然是威胁人类健康的重大传染病。
目前的结核病治疗需要多种药物的组合用药，药物敏感性结核病(DS-TB)的疗程为6个月，大多数广泛耐药结核病(XDR-TB)患者可能需要接受多达8种抗生素的治疗，疗程一般为9～20个月，有的甚至更长，导致患者依从性差和产生耐药性的几率增加，或者治疗结束时的临床结果不如人意。在全球范围内，最新的数据显示，DS-TB的治疗成功率为85％，耐多药结核病(MDR-TB)为56％，XDR-TB为39％。结核病治疗的主要挑战是药物疗程的持续时间和复杂性，两者都会影响依从性；此外，抗结核药物存在毒副作用，尤其是用于治疗耐药结核病的药物；再则，缺乏或有限的用于第二线治疗的儿科药物制剂。抗结核药物和抗逆转录病毒疗法之间的药物相互作用以及药物累积毒性增加了免疫重建性炎症综合症的风险，使艾滋病-结核共患(HIV-TB)、糖尿病-结核共患(DM-TB)以及M/XDR-TB患者的治疗更加复杂，治疗用药受限、治疗难度很大，导致了全球恐慌，以致WHO在1993和1998年两度宣布结核病全球紧急状态。全球迫切需要更有效、患者负担得起、无毒且能缩短治疗时间的治疗药物。
真菌能引起动植物和人的各种疾病。不同真菌通过不同方式致病，可以分为以下几种：(1)致病性真菌感染：由外源性真菌引起，如皮肤癣病菌；(2)条件致病性真菌感染：由内源性真菌引起，如白色念珠菌等；(3)真菌超敏反应性疾病：吸入或食入菌丝或孢子引起荨麻疹、哮喘等；(4)真菌性中毒症：食用含真菌毒素的霉变粮食所致；(5)真菌毒素：与肿瘤发生有关。常用于治疗真菌病的抗真菌剂，已知唑类抗真菌剂(卢立康唑、联苯苄唑、酮康唑、咪康唑、伊曲康唑、克霉唑、奈替康唑、奥昔康唑、噻康唑、氯康唑、奥莫康唑、硫康唑及其盐等)、苄胺类抗真菌剂(布替奈芬及其盐等)、烯丙胺类抗真菌剂(特比萘酚及其盐等)、吗啉类抗真菌剂(阿莫罗芬及其盐等)、硫代氨基甲酸类抗真菌剂(利拉萘酯、托萘酯、托西拉酯等)，及抗生素类(制霉菌素、曲古霉素、拟青霉素、干蠕孢菌素、硝吡咯菌素、两性霉素等)等，但这些抗菌药物的蓄积毒性较强，常常引起肝肾损伤、消化道刺激、头晕、过敏等，所以寻找作用机理独特的新型抗菌药物成为当今药物研发的热点之一。
柑橘溃疡病分布广泛，可危害几十种芸香科植物，是影响世界柑橘生产的重大检疫性病害。其危害对柑橘叶、枝以及果实，典型症状是形成溃疡斑，不及时治疗，病害加重，将严重危害柑橘生产及其经济效益。柑橘溃疡病菌菌系分化复杂、发病率高、传播快、寄主范围广，所以防治柑橘病一直是一个世界性难题，目前尚无一种方法可以根治。生产时常用波尔多液等含有金属铜离子的混合液体进行杀菌，需多次大量喷洒使用，既可能加速耐药性的产生，还会对土壤、其他益生菌产生毒害。开发新型抗柑橘病药物迫在眉睫。
发明内容
有鉴于此，本发明的目的在于提供对氨基水杨酸双氢青蒿素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双氢青蒿素类衍生物，其消旋体、立体异构体、互变异构体、氮氧化合物或药学上可接受的盐：
式Ⅰ中，R选自：
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；L1选自：-(CH2)n+1-、-(CH2)nCO-或-CO(CH2)nCO-，n为1，2，3或4；L2选自：-(CH2)m-，m为2，3，4或5；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；L1选自：-(CH2)n+1-或-(CH2)nCO-，n为1，2，3或4；L2选自：-(CH2)m-，m为2，3或4；X选自：C1-C3烷基。
优选的，所述式Ⅰ中，R选自：
L1选自：-(CH2)nCO-，n为1，2，3或4；L2选自：-(CH2)m-，m为2，3或4；X选自：乙基。
优选的，式Ⅰ所示的对氨基水杨酸双氢青蒿素类衍生物为以下化合物中的任一种：
2、上述对氨基水杨酸双氢青蒿素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'；
将中间体IM1'与linker试剂反应，制得中间体IM2'；
将中间体IM2'与唑偶联，制得TM4；
将双氢青蒿素和与linker试剂反应，制得中间体IM4；
将中间体IM4和TM4偶联，制得目标分子对氨基水杨酸双氢青蒿素类衍生物；
式中，R，L1，X和L2的定义与上述的对氨基水杨酸双氢青蒿素类衍生物结构式中R，L1，X和L2的定义相同；R14和R15独立的选自为卤素。
优选的，所述对氨基水杨酸双氢青蒿素类衍生物的制备方法，包括以下步骤：
A、将对氨基水杨酸在酸作用下与醇反应，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linker试剂在有机溶剂中、碱作用下进行反应，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、四氢呋喃或乙醚；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、碱作用下偶联，制得TM4；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。
D、将双氢青蒿素和linker试剂在有机溶剂、三氟化硼作用下，制得中间体IM4；所述有机溶剂为乙醚；
E、将IM4和TM4在有机溶剂、碱作用下偶联，制得对氨基水杨酸双氢青蒿素类衍生物TM7；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、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、上述制备方法制得的中间体IM2'，即
4、上述制备方法制得的中间体IM2'，即
在抗结核药物中的应用。
5、上述对氨基水杨酸双氢青蒿素类衍生物在抗细菌药物中的应用。
6、上述对氨基水杨酸双氢青蒿素类衍生物在抗真菌药物中的应用。
优选的，所述对氨基水杨酸双氢青蒿素类衍生物在抗毕赤酵母菌药物中的应用。
7、上述对氨基水杨酸双氢青蒿素类衍生物在抗柑橘病菌药物中的应用。
优选的，所述对氨基水杨酸双氢青蒿素类衍生物在抗柑橘胶孢炭疽病菌药物中的应用。
除另有说明外，本发明中的术语“消旋体”是指由等量对映体构成的光学不活性的有机物。“立体异构体”是指原子组成及键接相同而原子在三维空间排列上不同的分子。“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。“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双氢青蒿素类衍生物，以对氨基水杨酸为母核，对其氨基、羧基和酚羟基进行合理修饰，构建了一类结构新颖的对氨基水杨酸双氢青蒿素类衍生物，产物的化学结构经1H NMR,13C NMR和HR MS确认；
2)化合物体外抗细菌活性测定结果表明，TM7系列化合物抗菌活性整体好于母体PAS、中间体IM1'-1以及前体TM4-1系列化合物，MIC值低至2μg/mL。比较而言，TM7系列化合物对金黄色葡萄球菌的抑制活性最好，有6个分子MIC<64μg/mL，其中3个分子MIC＝16μg/mL、1个分子(TM7-10)MIC＝2μg/mL。此外，中间体IM2'-1抑制金黄色葡萄球菌、藤黄微球菌、沙门氏菌的能力都很强，甚至强于某些氟喹诺酮药物。这些结果表明，对氨基水杨酸双氢青蒿素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)化合物体外抗真菌活性测定结果表明，目标化合物TM7系列对毕赤酵母菌的抑菌活性整体很好，强于前体TM4-1系列化合物、母体PAS、中间体IM1'-1及IM2'-1；培养24h，测试的14个TM7系列化合物中有12个分子的MIC值＝4μg/mL，与阳性对照氟康唑的MIC相同，显示非常强的抗菌活性；中间体IM2'-1对毕赤酵母菌的抑制活性达到64μg/mL，强于母核PAS。这些数据证明，对氨基水杨酸双氢青蒿素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)化合物经过抗柑橘病菌活性测定结果表明，所测试化合物对柑橘胶孢炭疽病菌及柑橘褐斑病菌均有一定的抑制活性：对柑橘胶孢炭疽菌，在1μg/mL测试浓度下，12个TM7系列化合物对柑橘胶孢炭疽菌的抑制率超过阳性药物咪鲜胺的50％，有4个分子的活性超过咪鲜胺的80％；在4μg/mL测试下，12个TM7系列化合物的抑制率超过阳性药物咪鲜胺的50％，其中TM7-12的活性接近咪鲜胺的90％。对柑橘褐斑病菌，中间体IM2'-1的抑制率(64.29％)高于阳性对照咪鲜胺(50％)，TM7-2的抑制率(50％)与咪鲜胺相同。更重要的是，高活性分子TM7-5和TM7-12对柑橘褐斑病菌及胶孢炭疽病菌未体现出抗药性。对柑橘溃疡病菌，在1.6μg/mL测试浓度下，TM7系列目标分子的抑制活性高于35％的分子有5个，在0.64μg/mL测试浓度下，抑制率高于35％的分子有4个，其中TM7-1～TM7-3的活性强于诺氟沙星。这些结果证明，对氨基水杨酸双氢青蒿素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)经过抗耻垢分枝杆菌活性测定结果表明，中间体IM1'-1的MIC值均为0.19μg/mL，IM2'-1的MIC值为0.38μg/mL。从而证明了对氨基水杨酸双氢青蒿素类衍生物的中间体在抗结核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、1,2,4-三唑、2,4-二甲基吡唑、1H-四唑、5-甲基四唑、4,6-二甲基-2-巯基嘧啶、1-甲基-5-巯基四唑、1-苯基-5-巯基四唑、2-巯基-5-甲基-1,3,4-噻二唑(AR)；2-氨基-5-巯基-1,3,4-噻二唑(97.5％)；浓硫酸、乙醇、碳酸氢钠、碳酸钾；双氢青蒿素(AR)；3-溴-1-丙醇(≥97％)；无水乙醚、三氟化硼-乙醚(BF3·Et2O)(AR)；其余试剂均为市售化学纯或分析纯产品。
核磁共振仪(AV-600,600MHz,TMS为内标)；高分辨质谱仪(Varian 7.0T)；熔点测定仪(X-6)；自动旋光仪(WZZ-2S)；紫外分析仪(ZF-1)；旋转蒸发仪(RE-2000)；磁力搅拌低温恒温水槽。
二、对氨基水杨酸双氢青蒿素类衍生物的制备
1、中间体IM1'-1的合成
向反应瓶中加入PAS 20mmol、无水乙醇25mL，室温搅拌。冰浴，滴加浓硫酸50mmol，滴毕，80℃油浴回流反应，薄层层析法(TLC)监测直至反应结束。冰浴冷却，饱和碳酸钠(Na2CO3)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，柱层析，真空干燥后称重，得中间体IM1'-1 2.32g，收率为64％。
2、中间体IM2'-1的合成
向反应瓶中加入IM1'-1 5mmol、DCM 5mL、NaHCO3 12.5mmol；冰浴冷却，滴加氯乙酰氯10mmol。滴毕，冰浴(3℃)下持续反应，TLC跟踪监测至反应结束。停止搅拌，加入10mL冰冷的饱和食盐水使固体溶解，用2N HCl溶液调节pH在4-5，搅拌均匀后移入分液漏斗，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，柱层析得纯品，干燥，称重，得中间体IM2'-1 1.16g，收率为90％。
3、化合物TM4-1的制备
向反应瓶中依次加入唑、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℃水浴搅拌反应，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)，滤饼干燥得粗品，柱层析(PE/EA＝10:1-1:3,v/v)得纯品。当加入的唑带巯基时，加入THF做有机溶剂，柱层析前先用石油醚(PE)多次分散，其余条件及操作与上述相同。纯品真空干燥，称重，得目标化合物TM4-1。实验条件及结果如表1。
表1制备TM4-1的实验条件及结果
4、中间体IM4-1的制备
向反应瓶中加入DHA 1equiv、乙醚适量、3-溴-1-丙醇1.2equiv，-15℃条件下滴加BF3·Et2O液1.6equiv，-15℃继续反应，TLC监测反应进度。反应完全后，加入饱和碳酸氢钠水溶液、DCM，萃取分离，TLC监测水相无产品，有机相用无水硫酸钠干燥，旋蒸，得油状粗品。加入10mL石油醚后冷藏，抽滤。将滤液旋干后柱层析(PE/EA＝50:1,v/v)，加入6mL石油醚冷藏析晶，抽滤，滤饼合并，真空干燥，得中间体IM4-1。实验条件及结果如表2。
表2中间体IM4-1实验条件及结果
5、对氨基水杨酸双氢青蒿素类衍生物TM7的制备
向反应瓶中分别加入TM4-1、IM4-1、K2CO3、DMF，控温搅拌反应，TLC监测反应进程。反应结束后，加入15mL水和20mL EA，搅拌均匀，静置分液。EA多次萃取至水相无产品为止，并与之前的有机相合并。有机相用饱和NaHCO3溶液(10mL×1)洗涤、饱和食盐水洗涤(10mL×1)，无水硫酸钠干燥，旋蒸得粗品，柱层析(PE/EA＝10:1-5:1,v/v)，旋蒸，纯品真空干燥，称重，得目标化合物TM7。实验条件及结果如表3所示。
表3化合物TM7合成的实验条件及结果
6、TM7产物结构表征如下：
TM7-1:Ethyl 4-(2-(1H-imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate Colorless oil；
1H NMR(600MHz,DMSO-d6)δ10.57(s,1H,H-5),7.75(d,J＝8.6Hz,1H,H-3),7.63(s,1H,H-24),7.54(s,1H,H-26),7.15(s,1H,H-6),7.12(dd,J＝8.6Hz,1.3Hz,1H,H-4),6.90(s,1H,H-25),5.07(s,1H,H-22),4.93(s,2H,H-23),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,2H,H-2),4.06～4.00(m,2H,H-7),3.43～3.36(m,2H,H-9),2.10(td,J＝14.1,3.7Hz,1H,H-11),2.00(dt,J＝14.1,5.6Hz,2H,H-8),1.93～1.87(m,3H,H-16and H-20),1.67～1.61(m,1H,H-19),1.52(dt,J＝13.2,6.1Hz,2H,H-14 and H-15),1.31(t,J＝7.1Hz,3H,H-1),1.25(s,3H,H-21),1.21～1.16(m,2H,H-14 and H-15),1.13～1.09(m,1H,H-13),1.06(d,J＝7.0Hz,1H,H-18),0.97(dd,J＝11.2,4.8Hz,1H,H-19),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ172.40,166.81,165.39,159.53,144.13,138.78,132.97,128.42,121.12,114.61,110.74,103.51,100.81,87.15,80.83,64.95,62.95,60.60,55.34,52.38,49.74,44.24,36.61,34.39,30.98,28.83,26.06,24.51,21.49,20.63,14.59,13.19.LC MS calcd for C32H43N3O9,[M+H]+614.3,found 614.3.
TM7-2:Ethyl 4-(2-(5-methyl-1H-imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate Colorless oil；
1H NMR(600MHz,DMSO-d6)δ10.61(s,1H,H-5),7.76(d,J＝8.7Hz,1H,H-3),7.55(d,J＝1.3Hz,1H,H-6),7.36(d,J＝1.9Hz,1H,H-24),7.06(d,J＝3.4Hz,1H,H-4),6.74(s,1H,H-25),5.04(s,1H,H-22),4.69(d,J＝3.3Hz,1H,H-10),4.34(q,J＝7.1Hz,2H,H-23),4.25～4.20(m,2H,H-2),4.15～4.06(m,2H,H-7),3.41～3.35(m,2H,H-9),2.37～2.30(m,1H,H-11),2.23(s,3H,H-26),2.10～1.94(m,4H,H-8 and H-14and H-15),1.64～1.47(m,4H,H-14 and H-15 and H-16 and H-20),1.32(d,J＝3.5Hz,3H,H-1),1.24(s,3H,H-21),1.20～1.08(m,3H,H-19 and H-20),1.02～0.93(m,2H,H-13and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.64(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ172.41,169.12,166.87,165.38,161.82,159.54,145.41,131.35,126.30,121.49,110.93,106.77,103.49,87.12,80.82,61.57,60.60,55.35,52.36,49.18,44.22,36.59,34.37,33.60,30.98,26.06,24.53,21.50,20.64,19.36,14.54,13.19,12.94.LC MScalcd for C33H45N3O9,[M+H]+628.3,found 628.3.
TM7-3:Ethyl 4-(2-(4-methyl-1H-imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.101.3～102.7℃；
1H NMR(600MHz,DMSO-d6)δ10.55(s,1H,H-5),7.75(d,J＝8.4Hz,1H,H-3),7.54(d,J＝1.5Hz,1H,H-6),7.48(s,1H,H-24),7.11(d,J＝8.6Hz,1H,H-4),6.83(d,J＝5.4Hz,1H,H-26),5.07(s,1H,H-22),4.69(d,J＝3.1Hz,1H,H-10),4.23(q,J＝13.6,6.5Hz,2H,H-2),4.16～4.06(m,2H,H-23),4.06～3.99(m,2H,H-7),3.44～3.34(m,2H,H-9),2.40～2.29(m,1H,H-11),2.08(s,3H,H-25),2.04～1.86(m,4H,H-8and H-14 and H-15),1.67～1.46(m,4H,H-14 and H-15 and H-16 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28～1.26(m,1H,H-19),1.25(s,3H,H-21),1.21～0.91(m,4H,H-13and H-18 and H-19 and H-20),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.4Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ166.92,165.39,159.52,144.15,137.97,136.79,132.96,117.30,114.60,110.74,103.60,103.50,100.81,87.16,80.83,67.91,64.95,60.59,52.38,49.75,44.24,36.70,36.53,34.38,30.98,28.84,26.06,24.52,20.61,14.59,14.02,13.19,9.08.LC MS calcd for C33H45N3O9,[M+H]+628.3,found 628.3.
TM7-4:Ethyl 4-(2-(1H-pyrazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.93.8～95.2℃；
1H NMR(600MHz,DMSO-d6)δ10.59(s,1H,H-5),7.77(d,J＝2.1Hz,1H,H-26),7.77(d,J＝8.5Hz,1H,H-3),7.55(d,J＝1.5Hz,1H,H-6),7.47(d,J＝1.4Hz,1H,H-24),7.12(dd,J＝8.6Hz,1.6Hz,1H,H-4),6.29(t,J＝2.0Hz,1H,H-25),5.76(s,2H,H-23),5.05(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.14～4.07(m,1H,H-9),4.06～3.97(m,2H,H-7),3.44～3.35(m,1H,H-9),2.39～2.31(m,1H,H-11),2.14～1.58(m,6H,H-8 and H-14 and H-15 and H-20),1.56～1.44(m,2H,H-14 and H-15),1.32(t,J＝7.1Hz,3H,H-1),1.27(d,J＝7.0Hz,1H,H-16),1.25(s,3H,H-21),1.21～0.92(m,4H,H-19 and H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ166.57,165.39,159.53,144.12,139.55,132.97,132.06,114.62,110.77,105.78,103.61,103.50,100.79,87.14,80.84,64.93,62.90,60.59,55.35,54.98,52.40,44.24,36.69,36.54,34.38,30.98,28.82,26.07,24.51,20.63,14.59,13.20.LC MS calcd for C32H43N3O9,[M+H]+614.3,found614.3.
TM7-5:Ethyl 4-(2-(1H-benzo[d]imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.112.4～114.3℃；
1H NMR(600MHz,DMSO-d6)δ10.76(s,1H,H-5),8.24(s,1H,H-28),7.77(d,J＝8.6Hz,1H,H-3),7.68(d,J＝7.3Hz,1H,H-24),7.56(d,J＝1.3Hz,1H,H-6),7.50(d,J＝7.4Hz,1H,H-27),7.26～7.20(m,2H,H-25 and H-26),7.13(dd,J＝8.6Hz,1.5Hz,1H,H-4),5.05(s,1H,H-22),4.68(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.13～4.05(m,1H,H-9),4.06～3.96(m,3H,H-7 and H-9),2.39～2.30(m,1H,H-11),2.16～1.93(m,5H,H-8 and H-14 and H-15 and H-20),1.67～1.43(m,3H,H-14 andH-15 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28～1.25(m,1H,H-16),1.24(s,3H,H-21),1.18(t,J＝7.1Hz,2H,H-19),1.12～1.05(m,1H,H-13),0.98～0.92(m,1H,H-18),0.78(d,J＝7.3Hz,3H,H-12),0.63(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ170.76,166.56,165.38,159.55,145.44,144.08,143.70,134.87,132.99,122.84,122.01,119.86,114.66,110.78,110.69,103.50,100.79,87.14,80.82,64.95,62.92,60.60,60.20,55.35,52.37,47.89,44.22,36.60,34.36,30.97,28.80,26.06,24.50,20.63,14.56,13.17.LC MScalcd for C36H45N3O9,[M+H]+664.3,found 664.3.
TM7-6:Ethyl 4-(2-(1H-1,2,4-triazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.104.7～106.1℃；
1H NMR(600MHz,DMSO-d6)δ10.69(s,1H,H-5),8.55(s,1H,H-25),8.00(s,1H,H-24),7.77(d,J＝8.6Hz,1H,H-3),7.53(d,J＝1.4Hz,1H,H-6),7.12(dd,J＝8.6Hz,1.6Hz,1H,H-4),5.17(s,2H,H-23),5.05(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.13～4.08(m,1H,H-9),4.05～4.00(m,2H,H-7),3.42～3.36(m,1H,H-9),2.39～2.30(m,1H,H-11),2.14～1.87(m,5H,H-8and H-14and H-15 and H-20),1.67～1.44(m,3H,H-14 and H-15 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28–1.26(m,1H,H-16),1.25(s,3H,H-21),1.20–0.91(m,4H,H-13 and H-18 andH-19),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ165.59,165.37,159.52,151.88,146.07,143.91,133.00,114.80,110.81,103.67,103.50,100.80,87.14,80.83,64.97,62.92,60.62,52.39,52.33,44.24,36.69,36.53,34.38,30.98,28.81,26.06,24.52,24.48,20.62,14.58,13.19.HR MS calcd forC31H42N4O9,[M+H]+615.3025,found 615.3040.
TM7-7:Ethyl 4-(2-(2H-tetrazol-2-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.112.3～114.7℃；
1H NMR(600MHz,DMSO-d6)δ10.83(s,1H,H-5),9.42(s,1H,H-24),7.77(d,J＝8.5Hz,1H,H-3),7.51(d,J＝1.4Hz,1H,H-6),7.11(dd,J＝8.6Hz,1.6Hz,1H,H-4),5.06(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.12～4.06(m,2H,H-23),4.05～4.00(m,2H,H-7),3.41～3.36(m,2H,H-9),2.39～2.28(m,1H,H-11),2.14～1.84(m,5H,H-8 and H-14 and H-15 and H-20),1.68～1.42(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.29～1.26(m,1H,H-19),1.25(s,1H,H-21),1.20～0.92(m,4H,H-13 and H-14 and H-15 and H-18),0.79(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ165.36,164.52,159.51,145.69,143.68,133.04,114.99,110.86,103.73,103.51,100.81,87.15,80.83,65.01,62.94,60.65,55.35,52.38,50.60,44.23,36.71,36.52,34.39,30.97,28.81,26.07,24.50,20.62,14.58,13.19.HR MS calcd for C30H41N5O9,[M+Na]+638.2796,found638.2788.
TM7-8:Ethyl 4-(2-(5-methyl-2H-tetrazol-2-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.108.6～109.9℃；
1H NMR(600MHz,DMSO-d6)δ10.85(s,1H,H-5),7.78(d,J＝8.5Hz,1H,H-3),7.51(d,J＝1.4Hz,1H,H-6),7.11(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.68(d,J＝3.2Hz,1H,H-10),4.23(q,J＝6.8Hz,2H,H-2),4.13～4.08(m,2H,H-23),4.05～3.99(m,2H,H-7),3.42～3.35(m,2H,H-9),2.50(s,3H,H-24),2.37～2.32(m,1H,H-11),2.13～1.86(m,5H,H-8 and H-14 and H-15 and H-20),1.66～1.45(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28～1.25(m,1H,H-19),1.24(s,3H,H-21),1.20～0.92(m,4H,H-13 and H-14 and H-15 and H-18),0.79(d,J＝7.3Hz,3H,H-12),0.64(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ165.35,164.44,159.51,153.89,143.62,133.04,115.03,110.92,103.78,103.50,100.79,87.13,80.82,65.02,62.91,60.65,52.37,49.76,44.22,36.68,36.52,34.37,30.98,28.80,26.06,24.53,24.48,20.62,14.58,13.18,8.75(s).HR MS calcd for C31H43N5O9,[M+Na]+652.2953,found 652.2942.
TM7-9:Ethyl 4-(2-((4,6-dimethylpyrimidin-2-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate Colorless oil；
1H NMR(600MHz,DMSO-d6)δ10.49(s,1H,H-5),7.74(d,JH4-H3＝8.6Hz,1H,H-3),7.53(d,J＝1.5Hz,1H,H-6),7.14(dd,J＝8.6Hz,1.7Hz,1H,H-4),6.97(s,1H,H-25),5.05(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.22(q,J＝7.1Hz,2H,H-2),4.06(s,2H,H-23),4.04～4.00(m,2H,H-7),3.58～3.33(m,2H,H-9),2.32(s,6H,H-24 and H-26),2.23～2.16(m,1H,H-11),2.14～2.05(m,2H,H-8),2.04～1.96(m,2H,H-14 and H-15),1.92～1.87(m,1H,H-20),1.65～1.44(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),1.17～1.06(m,3H,H-14 and H-15 and H-19),0.97～0.83(m,2H,H-13and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.61(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ169.64,167.59,167.43(3C),165.41,159.55,144.57(s),132.92,116.55,114.27,110.65,103.46(d,J＝8.4Hz),100.76,87.13,80.82,64.88,62.86,60.55,52.37,44.23,36.63,36.53,36.05,34.36,30.98,28.80,26.06,24.50,23.77(3C),20.60,14.59,13.19(s).HR MS calcd for C35H47N3O9S,[M+H]+686.3106,found686.3096.
TM7-10:Ethyl 4-(2-((1-methyl-1H-tetrazol-5-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.109.4～110.7℃；
1H NMR(600MHz,DMSO-d6)δ10.65(s,1H,H-5),7.75(d,J＝8.6Hz,1H,H-3),7.48(d,J＝1.5Hz,1H,H-6),7.12(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.32(s,2H,H-23),4.22(q,J＝7.1Hz,2H,H-2),4.12～4.08(m,2H,H-7),3.98(s,3H,H-24),3.56～3.41(m,2H,H-9),2.38～2.32(m,1H,H-11),2.14～2.04(m,2H,H-8),2.02(s,2H,H-14 and H-15),1.92～1.88(m,1H,H-20),1.65～1.43(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),1.20～1.14(m,1H,H-19),1.12～1.07(m,2H,H-14 and H-15),0.98～0.84(m,2H,H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.62(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ170.78,166.03,165.38,159.51,153.72,144.12,132.99,114.69,110.77,103.49,100.78,87.14,80.84,68.12,65.99,63.69,60.61,52.39,44.24,38.18,36.67,34.12,30.98,28.81,26.07,24.51,21.14,20.60,15.48,14.58,13.20.LC MS calcdfor C31H43N5O9S,[M+H]+662.3,found 662.3.
TM7-11:Ethyl 4-(2-((1-phenyl-1H-tetrazol-5-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propox-y)benzoate White solid；m.p.117.4～119.0℃；
1H NMR(600MHz,DMSO-d6)δ10.70(s,1H,H-5),7.76(d,J＝8.6Hz,1H,H-3),7.71～7.65(m,5H,H-24 and H-25and H-26 and H-27 and H-28),7.51(d,J＝1.5Hz,1H,H-6),7.12(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.43(s,2H,H-23),4.23(q,J＝7.1Hz,2H,H-2),4.07～3.99(m,2H,H-7),3.47～3.36(m,2H,H-9),2.39～2.29(m,1H,H-11),2.15～1.95(m,4H,H-8 and H-14 and H-15),1.93～1.85(m,1H,H-20),1.66～1.57(m,1H,H-16),1.57～1.44(m,2H,H-14and H-15),1.21～1.14(m,1H,H-19),1.12～1.04(m,2H,H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),0.99～0.88(m,2H,H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.62(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ165.80,165.38,159.52,154.32,144.12,133.51,132.98,131.15,130.56(3C),124.84(2C),114.69,110.77,103.49,100.79,87.14,80.83,68.12,64.97,63.69,62.92,60.60,52.39,44.24,38.26,36.60,34.33,30.98,28.82,26.07,24.51,20.60,14.59,13.19.LC MS calcdfor C36H45N5O9S,[M+H]+724.3,found 724.3.
TM7-12:Ethyl 4-(2-((5-methyl-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.108.7～109.9℃；
1H NMR(600MHz,DMSO-d6)δ10.63(s,1H,H-5),7.75(d,J＝8.6Hz,1H,H-3),7.51(d,J＝1.5Hz,1H,H-6),7.13(dd,J＝8.6Hz,J＝1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.29(s,2H,H-23),4.22(q,J＝7.1Hz,2H,H-2),4.06～4.01(m,2H,H-7),3.40～3.36(m,2H,H-9),2.67(s,3H,H-24),2.39～2.30(m,1H,H-11),2.14～2.05(m,2H,H-19 and H-20),2.01～1.97(m,2H,H-8),1.93～1.87(m,1H,H-20),1.67～1.57(m,1H,H-16),1.56～1.45(m,2H,H-14 and H-15),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),1.20～1.15(m,1H,H-19),1.13～1.02(m,2H,H-14 and H-15),0.99～0.84(m,2H,H-18 and H-13),0.80(d,J＝7.3Hz,3H,H-12),0.62(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ166.30,166.13,165.38,164.63,159.52,144.22,132.97,114.62,110.75,103.49(2C),100.78,87.13,80.83,68.12,64.95,62.90,60.60,55.35,52.39,44.24,38.65,36.60,34.35,30.98,28.81,26.07,24.52,20.61,15.64,14.59,13.21.HR MS calcd for C32H43N3O9S2,[M+Na]+700.2333,found700.2334.
TM7-13:Ethyl 4-(2-((5-amino-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.113.4～114.7℃；
1H NMR(600MHz,DMSO-d6)δ10.52(s,1H,H-5),7.75(d,J＝8.5Hz,1H,H-3),7.51(d,J＝1.4Hz,1H,H-6),7.30(s,2H,H-24),7.12(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.22(q,J＝7.1Hz,2H,H-2),4.14～4.04(m,2H,H-7)，4.03(s,2H,H-23),3.41～3.31(m,2H,H-9),2.39～2.31(m,1H,H-20),2.15～2.05(m,1H,H-19),2.10(td,J＝14.1,3.7Hz,1H,H-11),2.05～1.97(m,2H,H-8),1.93～1.87(m,1H,H-16),1.66～1.59(m,1H,H-20),1.57～1.47(m,2H,H-14 and H-15),1.31(t,J＝7.1Hz,3H,H-1),1.30～1.23(m,2H,H-14 and H-15),1.24(s,3H,H-21),1.22～1.16(m,1H,H-19),1.13～1.02(m,1H,H-13),0.98～0.91(m,1H,H-18),0.81(d,J＝7.3Hz,3H,H-12),0.63(d,J＝6.3Hz,3H,H-17).13C NMR(151MHz,DMSO-d6)δ170.40,166.87,165.40,159.52,149.67,144.28,132.95,114.57,110.74,103.49(2C),100.78,87.14,80.85,64.94,62.91,60.59,52.41,44.25,39.25,36.67,36.54,34.34,30.99,28.82,26.07,24.55,24.49,20.63,14.59,13.22.LC MS calcd for C31H42N4O9S2,[M+H]+679.25,found 679.30.
三、对氨基水杨酸双氢青蒿素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，先以含5％吐温80的DMSO溶液配成母液，浓度为3.2mg/mL，再吸取200μL母液，用肉汤稀释至500μL，得浓度为1.28μg/μL的待测液。
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(即5mL肉汤/5μL菌)。
操作：在96孔板每一列的第一孔加入空白肉汤60μL，其余孔加入空白肉汤50μL；在每一列的第一孔加入待测液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，再充分吹打使之混匀后吸取50μL加入第3孔，照此重复直至第八孔，第八孔吸取50μL弃去；此时每孔待测物浓度从上到下依次为512,256,128,64,32,16,8,4μg/mL。每一块板的最后两列作为对照，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浓度从上到下依次为256,128,64,32,16,8,4,2μg/mL。
将接种好的96孔板放入37℃恒温培养箱培养20-24h，观察孔内细菌生长情况。确定生长对照孔的细菌正常生长、阴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。
对氨基水杨酸双氢青蒿素类衍生物及中间体的体外抑菌活性测试结果如表4所示。
表4化合物对6种致病菌的抑制活性(MIC,μg/mL)
分析表4可知：母体PAS、中间体IM1'-1几乎没有抗菌能力；TM4-1系列化合物对六种菌株的活性整体不好，活性最好分子的MIC值为16μg/mL；TM7系列化合物抗菌活性整体好于TM4-1系列化合物，MIC值低至2μg/mL。比较而言，TM7系列化合物对金黄色葡萄球菌的抑制活性最好，有6个分子MIC<64μg/mL，其中3个分子MIC＝16μg/mL、1个分子(TM7-10)MIC＝2μg/mL。此外，中间体IM2'-1抑制金黄色葡萄球菌、藤黄微球菌、沙门氏菌的能力都很强，甚至强于某些氟喹诺酮药物。这些结果表明，对氨基水杨酸双氢青蒿素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、体外抗真菌活性测定
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)。具体操作如下：
(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的溶液，封口膜封口后，冰柜避光保存。对于部分难溶的化合物使用DMSO/吐温-80＝200/1(v/v)以增加溶解度，吐温-80为助溶剂。
(2)待测液的配制：用适宜的溶剂及稀释剂将待测物配制成浓度为3.2mg/mL的储备液，再吸取320μL储备液，加入沙氏培养基至总体积为0.5mL，其浓度为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、第二孔加入待测液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1024,512,256,128,64,32,16,8μg/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浓度从高至低(从上至下)依次为512,256,128,64,32,16,8,4μg/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24、30h。培养完成后取出，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。
对氨基水杨酸双氢青蒿素类衍生物及中间体的体外抑真菌活性测试结果如表5所示。
表5化合物对毕赤酵母菌的抑制活性(MIC,μg/mL)
分析表5数据可知，目标化合物TM7系列对毕赤酵母菌的抑菌活性整体很好，强于前体TM4-1系列化合物、母体PAS、中间体IM1'-1及IM2'-1；培养24h，测试的14个TM7系列化合物，其MIC值在4～64μg/mL之间，其中有12个分子的MIC值＝4μg/mL，与阳性对照氟康唑的MIC相同，显示非常强的抗菌活性；中间体IM2'-1对毕赤酵母菌的抑制活性达到64μg/mL，强于母核PAS。这些数据证明，对氨基水杨酸双氢青蒿素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、抗柑橘病菌生物活性测定(初筛)
(1)药剂母液的配制：用适宜的溶剂及稀释剂将药剂母液稀释至所需的浓度(样品质量为1.0mg，先配成药剂母液1.0mg/1mL＝1.0mg/mL。每种药剂设置2个稀释浓度，0.001mg/mL(即稀释1000倍)和0.004mg/mL(即稀释250倍))。
(2)操作：药剂培养基配制：稀释1000倍的药剂培养基配制：取5μL药剂与5mL热PDA培养基在10mL离心管中充分混匀；稀释250倍的药剂培养基配制：取20μL药剂与4980μL热PDA培养基在10mL离心管中充分混匀。对照组：以不加药剂的PDA培养基和加入咪鲜胺的药剂培养基(稀释1000和稀释250倍)作为对照。接菌：将配置好的药剂培养基倒入24孔板内，每株菌每种药每个浓度倒一个孔，并做好编号的标记。挑取28℃培养7d的菌株的菌丝，接种于每孔正中央。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：将不同药剂抑制率同咪鲜胺药剂的抑制率进行比较。
复筛
初筛获得的高活性分子TM7-4、TM7-5、TM7-12和TM7-14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。TM7-5和IM2'-1对柑橘褐斑病菌Alternaria alternata菌株Al.6进行复筛，获得毒力方程。
操作：药剂梯度稀释：设置6个稀释梯度：0.01、0.004、0.002、0.001、0.0005、0.00025mg/mL，即稀释100、250、500、1000、2000、4000倍。药剂培养基配制：分别取50、20、10、5、2.5、1.25μL的药剂母液与5mL热PDA培养基在10mL离心管中充分混匀。将药剂培养基倒入孔内，每种药剂每个梯度重复4次。以PDA培养基与咪鲜胺作为对照组。接菌：挑取28℃培养7d的菌株的菌丝，接种于每孔正中央。培养：将24孔板放于2 8℃，光照16h培养箱内培养48h。测量：十字交叉法测量菌落直径。计算：使用农药室内生物测定数据处理系统(PBT数据处理系统)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。
对氨基水杨酸双氢青蒿素类衍生物及中间体抗柑橘病菌的测定结果见表6～表9。
表6化合物对柑橘真菌病菌的抑制活性(初筛结果)
从表6数据可知，所测试化合物对柑橘胶孢炭疽菌Colletotrichumgloeosporioides菌株Co.3、柑橘褐斑病菌Alternaria alternata菌株Al.6均有一定的抑制活性：对柑橘胶孢炭疽菌，在1μg/mL测试浓度下，12个TM7系列化合物对柑橘胶孢炭疽菌的抑制率超过阳性药物咪鲜胺的50％，有4个分子的活性超过咪鲜胺的80％；在4μg/mL测试下，12个TM7系列化合物的抑制率超过阳性药物咪鲜胺的50％，其中TM7-12的活性接近咪鲜胺的90％。对柑橘褐斑病菌，中间体IM2'-1的抑制率(64.29％)高于阳性对照咪鲜胺(50％)，TM7-2的抑制率(50％)与咪鲜胺相同。
初筛获得的高活性分子TM4-1-10、TM7-4、TM7-5、TM7-12和TM7-14进行柑橘胶孢炭疽菌复筛；高活性分子IM2'-1、TM7-5进行柑橘褐斑病菌复筛。
表7高活性分子对柑橘胶孢炭疽病菌的抑制活性(复筛结果)
表8高活性分子对柑橘褐斑病菌的抑制活性(复筛结果)
从表6和表7中分析可知，TM7-5和TM7-12未体现出抗药性，证明了对氨基水杨酸双青蒿素衍生物在抗柑橘病菌领域具有潜在的应用前景。
表9化合物对柑橘溃疡病菌的抑制活性
从表9数据可以看出，母体PAS在上述测试浓度下，活性很弱。在1.6μg/mL测试浓度下，TM7系列目标分子的抑制活性高于35％的分子有5个，在0.64μg/mL测试浓度下，抑制率高于35％的分子有4个，TM7-1～TM7-3的活性强于诺氟沙星。
4、抗结核活性测定
试验菌株：耻垢分枝杆菌Mycolicibacterium smegmatis(strain ATCC 700084/mc(2)155)
测定耻垢分枝杆菌所用培养基为7H9培养基；所用试剂、工具均需提前灭菌；所有操作应在超净台的无菌条件下进行。
待测液的配制：准确称取待测样品10.0mg，用适宜的溶剂及稀释剂配成浓度为1.0μg/μL的待测液。取1.0μg/μL的待测液，用一次性过滤器(过滤直径为13～30mm)过滤得待测液C。
操作：在96孔板的第1列加入培养好的野生型的耻垢分枝杆菌菌液200μL菌液，第2-12列分别加入100μL菌液；第1列的首孔加入待测液C10μL，用移液枪充分吹打(至少3次以上，使待测物与菌液充分混匀)，然后吸取100μL加入第2列第一孔，充分吹打使之混匀，再从第2列第一孔吸取100μL加入第3列第一孔，照此重复直至第11列；第12列为100μL菌液的阴性对照。此时每孔待测物浓度从左到右依次为50,25,12.5,6.25,3.12,1.56,0.78,0.39,0.19,0.09,0.05μg/mL，每一块板的最后一列为阴性对照。将接种好的96孔板放入37℃恒温培养箱培养3d，观察孔内细菌生长情况。确定空白无药对照孔的耻垢分枝杆菌正常生长、阴性对照孔无菌生长。将肉眼观察到的没有耻垢分枝杆菌生长的孔中的药物浓度作为该药物对该细菌的MIC。每株菌做3个重复，如出现多处跳孔，则不应报告结果，需重复试验。将对氨基水杨酸双氢青蒿素类衍生物及中间体对耻垢分枝杆菌进行MIC的测定。测定过程均设有空白对照、阴性对照、阳性对照，结果如表10所示。
表10化合物对抗耻垢分枝杆菌的抑制活性(MIC值)
从表10中分析可知，中间体IM1'-1 MIC值均为0.19μg/mL；IM2'-1的MIC值为0.38μg/mL。从而证明了对氨基水杨酸双氢青蒿素类衍生物的中间体在抗结核领域具有潜在的应用前景。
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)
1.一种如式Ⅰ所示的对氨基水杨酸双氢青蒿素类衍生物，或其药学上可接受的盐：
式Ⅰ中，R选自：
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；
L1选自：-(CH2)n+1-、-(CH2)nCO-或-CO(CH2)nCO-，n为1，2，3或4；
L2选自：-(CH2)m-，m为2，3，4或5；X选自：C1-C6烷基。
2.根据权利要求1所述对氨基水杨酸双氢青蒿素类衍生物，其特征在于，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；
L1选自：-(CH2)n+1-或-(CH2)nCO-，n为1，2，3或4；
L2选自：-(CH2)m-，m为2，3或4；
X选自：C1-C3烷基。
3.根据权利要求2所述对氨基水杨酸双氢青蒿素类衍生物，其特征在于，所述式Ⅰ中，R选自：
L1选自：-(CH2)nCO-，n为1，2，3或4；
L2选自：-(CH2)m-，m为2，3或4；
X选自：乙基。
4.根据权利要求3所述对氨基水杨酸双氢青蒿素类衍生物，其特征在于，式Ⅰ所示的对氨基水杨酸双氢青蒿素类衍生物为以下化合物中的任一种：
5.权利要求1至权利要求4任一所述对氨基水杨酸双氢青蒿素类衍生物的制备方法，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'；
将中间体IM1'与linker试剂反应，制得中间体IM2'；
将中间体IM2'与唑偶联，制得TM4；
将双氢青蒿素和与linker试剂反应，制得中间体IM4；
将中间体IM4和TM4偶联，制得目标分子对氨基水杨酸双氢青蒿素类衍生物；
式中，R，L1，X和L2的定义与权利要求1～3中任一所述的对氨基水杨酸双氢青蒿素类衍生物结构式中R，L1，X和L2的定义相同；R14和R15独立地选自为卤素。
6.权利要求1至权利要求4任一所述对氨基水杨酸双氢青蒿素类衍生物在制备抗细菌药物中的应用。
7.权利要求1至权利要求4任一所述对氨基水杨酸双氢青蒿素类衍生物在制备抗真菌药物中的应用。
8.权利要求1至权利要求4任一所述对氨基水杨酸双氢青蒿素类衍生物在制备抗柑橘病菌药物中的应用。
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