CN112079782A - 辛弗林唑类衍生物及其制备方法和应用 - Google Patents

辛弗林唑类衍生物及其制备方法和应用 Download PDF

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CN112079782A
CN112079782A CN202011027565.0A CN202011027565A CN112079782A CN 112079782 A CN112079782 A CN 112079782A CN 202011027565 A CN202011027565 A CN 202011027565A CN 112079782 A CN112079782 A CN 112079782A
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synephrine
alkyl
azole derivative
citrus
methyl
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CN112079782B (zh
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杨大成
罗鹏
李文粒
周传武
范莉
张世木
胡军华
唐雪梅
周围
吴玉珠
李洋
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Mianyang Diao Pharmaceutical Co ltd
Southwest University
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Southwest University
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Abstract

本发明公开了辛弗林唑类衍生物及其制备方法和应用，属于药物合成技术领域。辛弗林唑类衍生物的结构式如下。体外抗真菌活性测试结果表明，大部分化合物抑制毕赤酵母菌的MIC值为0.128mg/mL，个别化合物的MIC值低至0.064mg/mL，接近阳性对照药物氟康唑，强于所测试的其它抗菌药物。抗柑橘病菌生物活性测试结果表明，大多数化合物对柑橘胶孢炭疽病菌和柑橘褐斑病菌都具有抑制活性，个别化合物与阳性对照咪鲜胺的抑制活性相近，且未体现出抗药性；目标化合物对柑橘溃疡病的抑制活性均强于辛弗林。这些结果显示辛弗林唑类衍生物在抗真菌和抗柑橘病菌领域具有潜在的应用前景。
Figure DDA0002702565490000011

Description

辛弗林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，具体涉及辛弗林唑类衍生物及其制备方法和应用。
背景技术
辛弗林(synephrine)，是一种存在于芸香科柑橘属植物中的生物碱，其结构与肾上腺素及麻黄碱相似且具有类似的生物活性。辛弗林主要表现为肾上腺素α受体兴奋剂作用，对心脏β受体也有一定的兴奋作用，能促进血管收缩、提高心血输出量、升高血压、扩张支气管和气管；能促进新陈代谢、燃烧脂肪，从而具有减肥功效。中医临床上，辛弗林主要用于治疗支气管哮喘及低血压、虚脱、休克、体位性低血压，以及治疗食积不化、化痰除痞、胃下垂等病症；辛弗林注射液也用于抢救各种休克、心力衰竭病人以及治疗胃和十二指肠溃疡等病症；在中轻度抑郁症治疗、血糖调节等方面，辛弗林也取得了良好疗效。由于毒副作用，麻黄碱近年来受到一些国家禁用，但辛弗林副作用小且可用作麻黄碱的替代品，因此市场前景看好。受限于辛弗林的提取效率不高、反应位点复杂难控，目前对辛弗林的研究以化学合成方法、提取方法改进及其分析测试方面为主，对辛弗林衍生物的合成及生物活性研究较少，影响了辛弗林的发展前景。我国作为柑橘生产大国，每年产出的柑橘皮渣超过1000万吨，含有辛弗林5.5万吨以上，但绝大部分资源被浪费，因此研究开发以辛弗林为母核的衍生物及其生物活性，具有重要的理论意义和显著的经济价值。
抗生素挽救了成千上万人的生命。但抗生素的长期使用及滥用，导致细菌耐药性加速发展。除了临床合理使用抗生素外，开发新的抗生素及新的抗菌治疗方法，已成为药物研究工作者及制药企业的艰巨任务。
真菌是细菌的一种，能引起动植物和人的各种疾病。不同真菌可以通过不同的方式致病，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、拉诺康唑、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。其危害从柑橘叶、枝以及柑橘果实均有涉及，不及时治疗，病害加重，除了引起植被生长外，还严重危害柑橘生产及经济效益。柑橘病菌系分化复杂、发病率高、传播快、寄主范围广，所以如何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。开发新型抗柑橘病菌药物迫在眉睫。
发明内容
有鉴于此，本发明的目的在于提供辛弗林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所示的辛弗林唑类衍生物，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：
Figure BDA0002702565470000021
式Ⅰ中，
Y选自：
Figure BDA0002702565470000022
Figure BDA0002702565470000023
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或C1-C3烷基；
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为H或C1-C3烷基；
L选自：-(CH2)n-、-CO(CH2)nCO-、
Figure BDA0002702565470000031
n选自2、3或4；
X选自：烷酰基或磺酰基。
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；
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为甲基；
L选自：-(CH2)n-或
Figure BDA0002702565470000032
n选自2、3或4；
X为-R10R11；
R10选自：-CO-或-SO2-；
R11选自：C1-C3烷基、C1-C3羟烷基、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。
优选的，所述式Ⅰ中，
Y选自：
Figure BDA0002702565470000033
Figure BDA0002702565470000034
L选自：-(CH2)n-或
Figure BDA0002702565470000035
n选自3或4；
X为-R10R11；
R10选自：-CO-或-SO2-；
R11选自：C1-C3烷基、C1-C3羟烷基、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。
优选的，所述式Ⅰ中，
Y选自：
Figure BDA0002702565470000041
Figure BDA0002702565470000042
L选自：-(CH2)n-，n选自3或4；
X为-R10R11；
R10选自：-CO-；
R11选自：-CH3。
优选的，式Ⅰ所示的辛弗林唑类衍生物为以下化合物中的任一种：
Figure BDA0002702565470000043
Figure BDA0002702565470000051
Figure BDA0002702565470000061
优选的，式Ⅰ所示的辛弗林唑类衍生物为以下化合物中的任一种：TM3-6，TM3-7，TM3-10，TM3-12，TM3-14，TM3-15，TM5-2，TM5-11。
2、上述辛弗林唑类衍生物的制备方法，包括以下步骤：
将辛弗林进行胺基酰化，制得中间体IM1；
Figure BDA0002702565470000062
将中间体IM1与linker试剂反应，制得中间体IM2；
Figure BDA0002702565470000063
将中间体IM2与唑偶联，制得辛弗林唑类衍生物；
Figure BDA0002702565470000064
式中，X、Y和L的定义与上述的辛弗林唑类衍生物结构式中X、Y和L的定义相同；IM2中的Z为卤素。
优选的，包括以下步骤：
1)将辛弗林与醋酸酐在溶剂中反应，制得中间体IM1；所述溶剂为水；
2)将中间体IM1与linker试剂在有机溶剂，碱作用下偶联，制得中间体IM2；所述linker试剂为1,4-二溴丁烷或1,3-二溴丙烷；所述有机溶剂为二甲基甲酰胺、丙酮或丁酮；所述碱为碳酸钾；
3)将中间体IM2与唑在有机溶剂、碱作用下偶联，制得辛弗林唑类衍生物；所述有机溶剂为二甲基甲酰胺；所述碱为碳酸钾。
3、上述制备方法制得的中间体IM1和IM2，或其消旋体、立体异构体、药学上可接受的盐。
4、上述辛弗林唑类衍生物在抗真菌药物中的应用。
优选的，所述辛弗林唑类衍生物在抗毕赤酵母菌药物中的应用。
5、上述辛弗林唑类衍生物在抗柑橘病菌药物中的应用。
优选的，所述辛弗林唑类衍生物在抗柑橘胶孢炭疽菌药物中的应用。
优选的，所述辛弗林唑类衍生物在抗柑橘褐斑病菌药物中的应用。
优选的，所述辛弗林唑类衍生物在抗柑橘溃疡病菌药物中的应用。
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)本发明提供的辛弗林唑类衍生物，是以辛弗林为母核，对其氨基和酚羟基进行合理修饰而构建的一类结构新颖的辛弗林唑类衍生物，所得衍生物的化学结构经1H NMR，13C NMR和HR MS确认；
2)对毕赤酵母菌的抑制活性测试结果表明，大部分化合物都具有抑制活性。其中，TM3系列的MIC值低于0.128mg/mL的有11个，TM3-12、TM3-14抑制浓度低至0.064mg/mL，接近阳性对照药物氟康唑，强于所测试的其它抗菌药物；TM5系列的MIC值低于0.128mg/mL的有10个，均远低于辛弗林的MIC值0.256mg/mL，证明了辛弗林唑类衍生物在抗真菌领域具有潜在的应用前景；
3)经过抗柑橘病菌活性测试，发现本发明提供的辛弗林唑类衍生物对柑橘胶孢炭疽病菌显示出一定的抑制活性，有4个目标化合物在4μg/mL浓度下的抑制率≥40％，与阳性对照咪鲜胺的抑制活性相近。目标化合物对柑橘褐斑病菌均显示出一定的抑制活性，TM3-12和TM3-15的抑制活性超过阳性对照的80％且未体现出抗药性。目标化合物对柑橘溃疡病的抑制活性均强于辛弗林，绝大多数分子的活性强于阳性对照药物诺氟沙星。从而证明了辛弗林唑类衍生物在抗柑橘病菌领域具有潜在的应用前景。
附图说明
图1为本发明的辛弗林唑类衍生物的高活性化合物TM3-15复筛孔板图。
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
辛弗林，N,N-二异丙基乙胺，吡唑，咪唑，2-甲基咪唑，4-甲基咪唑，1H-四氮唑，5-甲基四氮唑，甲巯基四氮唑，2-巯基苯并噻唑，2-巯基苯并咪唑，2-巯基-5-甲基-1,3,4-噻二唑，1-苯基-5-巯基四氮唑，诺氟沙星，加替沙星，苯并咪唑，1,2,4-三氮唑，6-甲氧基-2-巯基苯并噻唑，1,2-二溴乙烷，1,3-二溴丙烷，1,4-二溴丁烷，4,6-二氯嘧啶，Boc2O，乙酸酐，碳酸钾，以及其余试剂均为市售化学纯或分析纯产品,反应溶剂经过干燥处理，其余试剂直接使用。
核磁共振仪(AV-600,TMS为内标)；高分辨质谱仪(HR ESI,Q TOF)；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)。
二、辛弗林唑类衍生物的制备
1、中间体IM1-1的合成
Figure BDA0002702565470000081
向反应瓶中依次加入原料辛弗林50mmol、H2O 10mL，室温下搅拌，移至冰水浴中，搅拌半小时，缓慢滴加醋酸酐6.1mL(约55mmol)。滴毕，搅拌半小时撤去冰水浴，遮光持续搅拌反应。薄层色谱(TLC)监测反应进程。反应结束后，加入冰冷饱和Na2CO3溶液至反应液不再有气体放出(pH约为4-6)，搅拌均匀后移入冰柜。冷却0.5h后，抽滤，干燥，得到中间体IM1-1(白色粉末状固体)9.217g，收率为88.2％。
2、中间体IM2-1的合成
Figure BDA0002702565470000082
向反应瓶中依次加入N-乙酰化辛弗林IM1-1 20mmol、N,N-二甲基甲酰胺(DMF)2mL、碳酸钾30mmol，室温下搅拌，移至45℃油浴锅中，搅拌半小时，加入1,4-二溴丁烷40mmol，遮光恒温搅拌反应，TLC监测反应进程至反应结束后，依次加入15mL冰冷饱和Na2CO3溶液和50mL二氯甲烷(DCM)，搅拌，分液，TLC监测下适当反萃，饱和氯化钠20mL×3洗涤，无水硫酸钠干燥，旋蒸除去溶剂，柱层析PE:EA＝2:1～1:4(v/v)，得中间体IM2-1(白色粉末状固体)4.569g，收率为65.8％。
3、中间体IM2-2的合成
Figure BDA0002702565470000091
向反应瓶中依次加入N-乙酰化辛弗林IM1-1 20mmol、DMF 2mL、碳酸钾30mmol，室温下搅拌，移至40℃油浴锅中，搅拌半小时，加入1,3-二溴丙烷30mmol，遮光恒温搅拌反应，TLC监测反应进程至反应结束后，依次加入15mL冰冷饱和Na2CO3溶液和50mL DCM搅拌，分液，TLC监测下，适当反萃，饱和氯化钠40mL×3洗涤，无水硫酸钠干燥，旋蒸除去溶剂，柱层析PE:EA＝2:1～1:4(v/v)，得中间体IM2-2(白色粉末状固体)4.325g，收率为65.5％。
4、辛弗林唑类衍生物TM3的合成
Figure BDA0002702565470000092
向反应瓶中依次加入唑、DMF，搅拌溶解，再加入碾细干燥的K2CO3，油浴45℃-50℃下搅拌，加入IM2-1，同温继续搅拌，TLC监测反应进程，当IM2-1点基本耗尽时，停止反应。冷却至室温，依次加入10mL冰冷饱和Na2CO3溶液和45mL DCM搅拌，分液，饱和氯化钠30mL×3洗涤，无水硫酸钠干燥2h，旋蒸除去溶剂，柱层析纯化(DCM/CH3OH＝200/1～80/1)，收集洗脱液，减压蒸干；TLC检查纯度，室温敞放一天，真空干燥，低温保存。得目标化合物TM3。实验条件及结果如表1所示。
表1目标化合物TM3系列合成的条件及结果
Figure BDA0002702565470000093
Figure BDA0002702565470000101
表1中数据显示，唑参与反应的收率在63.4％～96.1％之间，5种产物的收率超过90％。5、辛弗林唑类衍生物TM5的合成
Figure BDA0002702565470000102
向反应瓶中依次加入唑、DMF搅拌溶解，得到澄清液，再加入碾细的干燥K2CO3，油浴45℃-55℃下搅拌，加入IM2-2，TLC监测反应进程，当IM2-2基本耗尽时，停止反应。冷却至室温，依次加入冰冷饱和Na2CO3溶液15mL和DCM 45mL，搅拌，分液，TLC监测下适当反萃，饱和氯化钠溶液30mL×3洗涤，无水硫酸钠干燥2h，旋蒸除去溶剂，柱层析纯化(PE/EA＝3/1～1/3或DCM/CH3OH＝200/1～80/1,v/v)，收集洗脱液，减压蒸干；TLC检查纯度，室温敞放一天，真空干燥，低温保存，得目标化合物TM5。实验条件及结果如表2所示。
表2目标化合物TM5系列合成的条件及结果
Figure BDA0002702565470000111
从表2可知，大多数分子的收率都较好，收率最高者达到90.3％。
6、产物结构表征数据
1)中间体波谱数据表征
Figure BDA0002702565470000121
(R)-N-(2-Hydroxy-2-(4-hydroxyphenyl)ethyl)-N-methylacetamide(IM1)1HNMR(600MHz,DMSO-d6)δ9.45(s,1H,H-1),7.12(dd,J＝23.9,8.4Hz,2H,H-2),6.71(t,J＝8.6Hz,2H,H-3),5.28(d,J＝127.6Hz,1H,H-4),4.63(brs,1H,H-5),3.45–3.34(m,1H,H-6),3.29–3.19(m,1H,H-7),2.84(d,J＝49.4Hz,3H,H-8),1.90(d,J＝55.0Hz,3H,H-9).
(R)-N-(2-(4-(4-Bromobutoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(IM2-1)1HNMR(600MHz,DMSO-d6)δ7.24(dd,J＝26.6,8.6Hz,2H,H-1),6.91–6.86(m,2H,H-2),5.36(dd,J＝122.8,3.9Hz,1H,H-3),4.69(brs,1H,H-4),3.98(dd,J＝10.7,6.3Hz,2H,H-5),3.60(t,J＝6.7Hz,2H,H-6),3.46–3.35(m,1H,H-7),3.32–3.21(m,1H,H-8),2.85(d,J＝52.5Hz,3H,H-9),1.99–1.80(m,7H,H-10,H-11and H-12).
2)目标分子的波谱数据表征
Figure BDA0002702565470000122
(R)-N-(2-(4-(4-(1H-Imidazol-1-yl)butoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM3-1),white solid，m.p.66.0-67.1℃；
Figure BDA0002702565470000123
Figure BDA0002702565470000124
7.24(dd,J＝26.9,8.4Hz,2H,H-2),7.18(s,1H,H-3),6.88(dd,J＝11.3,6.2Hz,3H,H-4andH-5),5.49(s,1H,H-6),4.67(brs,1H,H-7),4.02(t,J＝7.0Hz,2H,H-8),3.95(dd,J＝7.5,5.0Hz,2H,H-9),3.46–3.36(m,1H,H-10),3.30–3.21(m,1H,H-11),2.85(d,J＝53.0Hz,3H,H-12),1.91(d,J＝50.3Hz,3H,H-13),1.84(dd,J＝14.9,7.4Hz,2H,H-14),1.66–1.59(m,2H,H-15).13C NMR(151MHz,DMSO-d6)δ(170.46,170.43),158.28,137.69,136.30,135.84,128.86,127.61,119.68,(114.59,114.51),(70.96,70.50),67.41,58.38,46.13,38.06,34.05,27.84,26.27,21.78.HR MS calcd for C18H25N3O3[M+H]+332.1969,found332.1975.
(R)-N-(2-(4-(4-(1H-Pyrazol-1-yl)butoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM3-2),white oil；
Figure BDA0002702565470000125
7.43(s,1H,H-2),7.24(dd,J＝25.9,8.4Hz,2H,H-3),6.87(t,J＝9.0Hz,2H,H-4),6.22(s,1H,H-5),4.69(brs,1H,H-6),4.16(t,J＝7.0Hz,2H,H-7),3.96–3.91(m,2H,H-8),3.57(t,J＝5.8Hz,1H,H-9),3.47–3.37(m,1H,H-10),3.31–3.22(m,1H,H-11),2.77(d,J＝45.0Hz,3H,H-12),1.94-1.98(m,5H,H-13),1.68–1.59(m,2H,H-14).13C NMR(151MHz,DMSO-d6)δ172.38,(170.47,170.43),162.76,158.31,138.88,135.81,130.11,(127.60,127.49),(114.59,114.50),105.32,(70.98,70.52),67.42,58.39,55.97,51.21,38.05,34.04,27.18,21.48.HR MS calcd for C18H25N3O3[M+H]+332.1969,found 332.1969.
Figure BDA0002702565470000131
(R)-N-(2-Hydroxy-2-(4-(4-(2-methyl-1H-imidazol-1-yl)butoxy)phenyl)ethyl)-N-methylacetam ide(TM3-3),yellow oil；
Figure BDA0002702565470000132
Figure BDA0002702565470000133
7.04(s,1H,H-2),6.88(t,J＝8.9 Hz,2H,H-3),6.71(s,1H,H-4),5.38(dd,J＝123.9,3.6Hz,1H,H-5),4.71(brs,1H,H-6),3.96(t,J＝6.1Hz,2H,H-7),3.92(t,J＝7.1Hz,2H,H-8),3.46–3.36(m,1H,H-9),3.29–3.21(m,1H,H-10),2.85(d,J＝53.0Hz,3H,H-11),2.27(s,3H,H-12),1.91(d,J＝52.1Hz,3H,H-13),1.82–1.75(m,2H,H-14),1.70–1.64(m,2H,H-15).13C NMR(151MHz,DMSO-d6)δ(170.45,170.42),(158.30,158.13),143.99,135.86,(127.62,127.51),126.71,119.94,(114.59,114.51),70.50,67.50,58.38,45.33,34.05,27.44,21.77,13.08.HR MS calcd for C19H27N3O3[M+H]+346.2125,found 346.2120.
(R)-N-(2-Hydroxy-2-(4-(4-(4-methyl-1H-imidazol-1-yl)butoxy)phenyl)ethyl)-N-methylacetamide(TM3-4),yellow oil；
Figure BDA0002702565470000134
Figure BDA0002702565470000135
7.24(dd,J＝26.9,8.4Hz,2H,H-2),6.92–6.81(m,3H,H-3),5.38(dd,J＝123.1,4.2Hz,1H,H-4),4.68(s,1H,H-5),4.05–3.88(m,4H,H-6),3.46–3.36(m,1H,H-7),3.29–3.21(m,1H,H-8),2.85(d,J＝53.5Hz,3H,H-9),2.01(dd,J＝117.9,52.5Hz,6H,H-10),1.85–1.75(m,2H,H-11),1.70–1.57(m,2H,H-12).13C NMR(151MHz,DMSO-d6)δ170.45,158.29,136.86,135.84,127.61,127.50,126.54,115.94,(114.59,114.51),70.96,70.50,67.42,58.38,46.02,34.05,27.78,26.28,21.77,14.14.HR MS calcd forC19H27N3O3[M+H]+346.2125,found 346.2121.
Figure BDA0002702565470000136
(R)-N-(2-(4-(4-(1H-Benzo[d]imidazol-1-yl)butoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM3-6),white oil；
Figure BDA0002702565470000137
Figure BDA0002702565470000138
7.64(dd,J＝19.3,8.0Hz,2H,H-2),7.23(dt,J＝15.5,6.2Hz,4H,H-3),6.87(t,J＝9.1Hz,2H,H-4),5.38(dd,J＝122.2,4.1Hz,1H,H-5),4.69(brs,1H,H-6),4.32(t,J＝7.0Hz,2H,H-7),3.97(t,J＝4.7Hz,2H,H-8),3.46–3.36(m,1H,H-9),3.30–3.20(m,1H,H-10),2.85(d,J＝49.1Hz,3H,H-11),1.99–1.93(m,3H,H-12),1.89(d,J＝26.6Hz,2H,H-13),1.72–1.63(m,2H,H-14).13C NMR(151MHz,DMSO-d6)δ170.43,158.29,144.45,134.31,127.61,127.50,122.69,121.86,119.93,114.61,110.85,70.97,70.52,67.45,58.38,55.97,44.30,38.05,34.05,26.71,26.46,21.76.HR MS calcd for C22H27N3O3[M+H]+382.2125,found382.2119.
(R)-N-(2-(4-(4-(1H-1,2,4-Triazol-1-yl)butoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM3-7),white oil；
Figure BDA0002702565470000141
Figure BDA0002702565470000142
7.96(s,1H,H-2),7.24(dd,J＝26.4,7.7Hz,2H,H-3),6.88(t,J＝8.6Hz,2H,H-4),5.36(d,J＝122.6Hz,1H,H-5),4.70(brs,1H,H-6),4.25(t,J＝6.5Hz,2H,H-7),3.96(s,2H,H-8),3.48–3.34(m,1H,H-9),3.29-3.23(m,1H,H-10),2.85(d,J＝52.0Hz,3H,H-11),1.98–1.84(m,5H,H-12),1.69–1.60(m,2H,H-13).13C NMR(151MHz,DMSO-d6)δ170.46,158.27,158.10,151.82,144.41,135.85,127.61,127.50,114.60,70.51,67.31,58.38,55.96,48.76,34.04,26.61,21.77.HR MS calcd for C17H24N4O3[M+H]+333.1921,found 333.1919.
Figure BDA0002702565470000143
(R)-N-(2-(4-(4-(1H-Tetrazol-1-yl)butoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM3-8),white solid，m.p.60.3-62.0℃；
Figure BDA0002702565470000144
Figure BDA0002702565470000145
7.25(dd,J＝27.0,8.4Hz,2H,H-2),6.88(t,J＝9.1Hz,2H,H-3),5.38(dd,J＝122.3,4.3Hz,1H,H-4),4.79(t,J＝7.0Hz,2H,H-5),4.69(brs,1H,H-6),3.98(dd,J＝10.0,6.1Hz,2H,H-7),3.46–3.36(m,1H,H-8),3.30–3.21(m,1H,H-9),2.85(d,J＝50.8Hz,3H,H-10),2.12–2.03(m,2H,H-11),1.91(d,J＝49.8Hz,3H,H-12),1.73–1.66(m,2H,H-13).13C NMR(151MHz,DMSO-d6)δ170.43,158.22,153.66,136.34,135.89,127.62,114.59,70.96,67.14,58.38,55.96,52.65,34.04,26.12,26.08,21.77.HR MS calcd for C16H23N5O3[M+H]+334.1874,found 334.1871.
(R)-N-(2-(4-(4-(2H-Tetrazol-2-yl)butoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM3-9),white solid,m.p.63.1-64.1℃；
Figure BDA0002702565470000146
Figure BDA0002702565470000147
7.24(dd,J＝27.0,8.5Hz,2H,H-2),6.88(t,J＝9.0Hz,2H,H-3),5.37(dd,J＝122.3,4.3Hz,1H,H-4),4.69(brs,1H,H-5),4.54(t,J＝7.1Hz,2H,H-6),4.00–3.94(m,2H,H-7),3.46–3.36(m,1H,H-8),3.29–3.21(m,1H,H-9),2.85(d,J＝53.2Hz,3H,H-10),2.04–1.97(m,2H,H-11),1.91(d,J＝50.4Hz,3H,H-12),1.73–1.65(m,2H,H-13).13C NMR(151MHz,DMSO-d6)δ170.42,158.23,144.35,135.91,127.62,127.51,114.60,114.52,70.50,67.20,58.38,55.96,47.73,34.05,26.08,21.77.HR MS calcd for C16H23N5O3[M+H]+334.1874,found334.1875.
Figure BDA0002702565470000151
(R)-N-(2-Hydroxy-2-(4-(4-(5-methyl-1H-tetrazol-1-yl)butoxy)phenyl)ethyl)-N-methylacetamide(TM3-10),white solid，m.p.67.1-67.7℃；
Figure BDA0002702565470000152
Figure BDA0002702565470000153
6.91–6.84(m,2H,H-2),5.37(dd,J＝122.7,4.2Hz,1H,H-3),4.68(t,J＝7.0Hz,3H,H-4),3.98(t,J＝5.2Hz,2H,H-5),3.46–3.36(m,1H,H-6),3.30–3.20(m,1H,H-7),2.85(d,J＝52.0Hz,3H,H-8),2.45(s,3H,H-9),2.10–2.00(m,2H,H-10),1.91(d,J＝50.3Hz,3H,H-12),1.73–1.66(m,2H,H-11).13C NMR(151MHz,DMSO-d6)δ170.46,162.64,158.23,136.34,127.61,114.59,70.97,67.16,58.39,55.97,52.46,34.04,26.11,22.23,10.87.HR MS calcd for C17H25N5O3[M+H]+348.2030,found 348.2036.
(R)-N-(2-Hydroxy-2-(4-(4-(5-methyl-2H-tetrazol-2-yl)butoxy)phenyl)ethyl)-N-methylacetamide(TM3-11),white oil；
Figure BDA0002702565470000154
Figure BDA0002702565470000155
6.89(t,J＝9.1Hz,2H,H-2),5.37(dd,J＝122.1,4.3Hz,1H,H-3),4.68(brs,1H,H-4),4.40(t,J＝7.2Hz,2H,H-5),4.02–3.94(m,2H,H-6),3.45–3.35(m,1H,H-7),3.30–3.21(m,1H,H-8),2.85(d,J＝53.0Hz,3H,H-9),2.53(s,3H,H-10),2.00–1.92(m,3H,H-11),1.87(s,2H,H-12),1.75–1.67(m,2H,H-13).13C NMR(151MHz,DMSO-d6)δ170.43,152.41,135.90,127.63,127.51,114.60,114.52,70.50,67.28,58.38,55.35,46.57,38.06,34.05,26.11,26.07,8.71.HR MS calcd for C17H25N5O3[M+H]+348.2030,found 348.2036.
Figure BDA0002702565470000156
(R)-N-(2-Hydroxy-2-(4-(4-((1-methyl-1H-tetrazol-5-yl)thio)butoxy)phenyl)ethyl)-N-methylacetamide(TM3-12),white oil；
Figure BDA0002702565470000157
Figure BDA0002702565470000158
6.88(t,J＝8.6Hz,2H,H-2),5.36(d,J＝122.7Hz,1H,H-3),4.68(s,1H,H-4),3.99(s,2H,H-5),3.93(s,3H,H-6),3.48–3.39(m,1H,H-7),3.31-3.23(m,1H,H-8),2.85(d,J＝52.3Hz,3H,H-9),1.89(t,J＝31.2Hz,7H,H-10).13C NMR(151MHz,DMSO-d6)δ170.42,158.28,154.16,135.86,(127.62,127.51),(114.62,114.53,)(70.97,70.51),(67.38,67.33),58.38,39.66,38.06,(34.05,33.98),33.02,27.96,26.29,22.23,21.77.HR MS calcd for C17H25N5O3S[M+H]+380.1751,found 380.1746.
(R)-N-(2-(4-(4-(Benzo[d]thiazol-2-ylthio)butoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM3-13),white solid，m.p.122.1-122.9℃；
Figure BDA0002702565470000161
Figure BDA0002702565470000162
7.84(d,J＝8.0Hz,1H,H-2),7.46(t,J＝7.5Hz,1H,H-3),7.36(t,J＝7.5Hz,1H,H-4),7.24(dd,J＝25.9,8.5Hz,2H,H-5),6.89(t,J＝9.2Hz,2H,H-6),5.38(dd,J＝122.3,4.2Hz,1H,H-7),4.69(d,J＝32.8Hz,1H,H-8),4.01(dd,J＝9.9,5.8Hz,2H,H-9),3.48–3.36(m,3H,H-10and H-11),3.29-3.22(m,1H,H-12),2.85(d,J＝49.4Hz,3H,H-13),1.94(d,J＝22.7Hz,3H,H-14),1.89(d,J＝13.5Hz,4H,H-15and H-16).13C NMR(151MHz,DMSO-d6)δ170.45,167.13,158.31,158.14,153.28,135.03,127.61,127.50,126.81,124.88,122.19,121.58,114.64,114.55,70.97,67.40,67.35,58.39,34.05,33.13,28.20,26.19,21.77.HR MScalcd for C22H26N2O3S2[M+H]+431.1458,found 431.1453.
Figure BDA0002702565470000163
(R)-N-(2-Hydroxy-2-(4-(4-((5-methyl-1,3,4-thiadiazol-2-yl)thio)butoxy)phenyl)ethyl)-N-methyl acetamide(TM3-14),white solid，m.p.59.8-61.8℃；
Figure BDA0002702565470000164
Figure BDA0002702565470000165
6.88(t,J＝9.3Hz,2H,H-2),5.37(dd,J＝122.3,4.3Hz,1H,H-3),4.68(brs,1H,H-4),4.03–3.94(m,2H,H-5),3.46–3.34(m,2H,H-6),3.32-3.22(m,2H,H-6),2.85(d,J＝51.7Hz,3H,H-7),2.68(s,3H,H-8),1.99–1.80(m,7H,H-9).13C NMR(151MHz,DMSO-d6)δ170.42,165.73,165.45,158.28,135.85,127.62,127.50,114.62,114.54,70.97,70.51,(67.36,67.32),58.38,34.05,34.00,28.10,26.13,21.78.HR MS calcd for C18H25N3O3S2[M+H]+396.1410,found 396.1402.
(R)-N-(2-Hydroxy-2-(4-(4-((1-phenyl-1H-tetrazol-5-yl)thio)butoxy)phenyl)ethyl)-N-methylacetamide(TM3-15),white oil；
Figure BDA0002702565470000166
Figure BDA0002702565470000167
7.24(dd,J＝26.7,8.5Hz,2H,H-2),6.92–6.84(m,2H,H-3),5.37(dd,J＝122.5,4.3Hz,1H,H-4),4.67(brs,1H,H-5),3.98(dd,J＝10.3,5.9Hz,2H,H-6),3.48–3.35(m,3H,H-7),3.25(dd,J＝17.1,12.2Hz,1H,H-8),2.85(d,J＝51.4Hz,3H,H-9),2.01–1.80(m,7H,H-10).13C NMR(151MHz,DMSO-d6)δ170.42,158.27,154.84,135.85,133.61,131.06,130.46,127.61,127.50,125.09,114.62,114.53,70.51,67.37,67.32,58.38,55.97,38.06,33.04,28.02,26.08,21.77.HR MS calcd for C22H27N5O3S[M+H]+442.1907,found 442.1903.
Figure BDA0002702565470000168
(R)-N-(2-(4-(3-(1H-Imidazol-1-yl)propoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM5-1),yellow oil；
Figure BDA0002702565470000171
Figure BDA0002702565470000172
7.25(dd,J＝26.9,8.4Hz,2H,H-2),7.19(s,1H,H-3),6.89(t,J＝9.2Hz,3H,H-4and H-5),5.41(d,J＝122.5Hz,1H,H-6),4.70(brs,1H,H-7),4.13(t,J＝6.9Hz,2H,H-8),3.88(t,J＝5.9Hz,2H,H-9),3.47–3.42(m,1H,H-10),3.30–3.21(m,1H,H-10),2.85(d,J＝52.5Hz,3H,H-11),2.15(p,J＝6.4Hz,2H,H-12),1.91(d,J＝52.0Hz,3H,H-13).13C NMR(151MHz,DMSO-d6)δ170.48,158.13,137.79,136.53,128.86,127.66,119.82,114.65,70.96,64.96,58.37,55.95,43.44,34.05,30.68,22.23.HR MS calcd for C17H23N3O3[M+H]+318.1812,found 318.1812.
(R)-N-(2-(4-(3-(1H-Pyrazol-1-yl)propoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM5-2),yellow oil；
Figure BDA0002702565470000173
Figure BDA0002702565470000174
7.44(s,1H,H-2),7.25(dd,J＝26.8,8.5Hz,2H,H-3),6.88(t,J＝9.2Hz,2H,H-4),6.22(s,1H,H-5),5.40(dd,J＝122.1,4.2Hz,1H,H-6),4.69(brs,1H,H-7),4.27(t,J＝6.8Hz,2H,H-8),3.90(t,J＝6.0Hz,2H,H-9),3.47–3.37(m,1H,H-10),3.30–3.20(m,1H,H-11),2.85(d,J＝50.8Hz,3H,H-12),2.20(p,J＝6.4Hz,2H,H-13),1.91(d,J＝49.9Hz,3H,H-14).13C NMR(151MHz,DMSO-d6)δ170.43,158.19,139.09,136.00,130.33,(127.64,127.54),114.63,105.43,70.51,65.14,58.38,55.96,34.05,30.19,21.77.HR MS calcd for C17H23N3O3[M+H]+318.1812,found 318.1810.
Figure BDA0002702565470000175
(R)-N-(2-Hydroxy-2-(4-(3-(2-methyl-1H-imidazol-1-yl)propoxy)phenyl)ethyl)-N-methylacetamide(TM5-3),yellow oil；
Figure BDA0002702565470000176
Figure BDA0002702565470000177
7.03(s,1H,H-2),6.89(t,J＝8.8Hz,2H,H-3),6.72(s,1H,H-4),5.42(d,J＝112.5Hz,1H,H-5),4.69(brs,1H,H-6),4.03(t,J＝6.8Hz,2H,H-7),3.87(t,J＝5.9Hz,2H,H-8),3.47–3.36(m,1H,H-9),3.30–3.22(m,1H,H-10),2.85(d,J＝50.3Hz,3H,H-11),2.24(s,3H,H-12),2.13–2.04(m,2H,H-13),1.88(d,J＝32.3Hz,3H,H-14).13C NMR(151MHz,DMSO-d6)δ170.42,158.05,144.22,136.08,127.68,127.57,126.86,119.85,114.59,114.51,70.49,64.62,58.36,55.95,34.06,30.27,21.74,12.89.HR MScalcd for C18H25N3O3[M+H]+332.1969,found 332.1971.
(R)-N-(2-Hydroxy-2-(4-(3-(4-methyl-1H-imidazol-1-yl)propoxy)phenyl)ethyl)-N-methyl-acetamide(TM5-4),yellow oil；
Figure BDA0002702565470000178
Figure BDA0002702565470000179
7.27(d,J＝8.4Hz,2H,H-2),6.88(dd,J＝18.2,8.4Hz,3H,H-3 and H-4),5.41(dd,J＝122.9,2.7Hz,1H,H-5),4.71(brs,1H,H-6),4.04(t,J＝6.9Hz,2H,H-7),3.87(t,J＝5.8Hz,2H,H-8),3.45-3.39(m,1H,H-9),3.31–3.19(m,1H,H-10),2.85(d,J＝52.1Hz,3H,H-11),2.15–2.09(m,2H,H-12),2.06(s,3H,H-13),1.91(d,J＝52.4Hz,3H,H-14).13C NMR(151MHz,DMSO-d6)δ170.44,158.14,137.26,136.96,136.07,127.65,116.04,114.66,70.49,64.97,58.37,55.96,34.05,30.69,30.66,22.23,21.76,14.12.HR MS calcd for C18H25N3O3[M+H]+332.1969,found 332.1966.
Figure BDA0002702565470000181
(R)-N-(2-(4-(3-(1H-Benzo[d]imidazol-1-yl)propoxy)phenyl)-2-hydroxyethyl)-N-methyl-acetamide(TM5-6),yellow oil；
Figure BDA0002702565470000182
Figure BDA0002702565470000183
7.62(dd,J＝33.8,7.7Hz,2H,H-2),7.30–7.14(m,4H,H-3),6.88(t,J＝8.8Hz,2H,H-4),5.39(dd,J＝121.9,4.2Hz,1H,H-5),4.68(brs,1H,H-6),4.43(t,J＝6.7Hz,2H,H-7),3.91(t,J＝5.9Hz,2H,H-8),3.55–3.36(m,1H,H-9),3.31–3.17(m,1H,H-10),2.85(d,J＝47.4Hz,3H,H-11),2.31–2.14(m,2H,H-12),1.91(d,J＝52.8Hz,3H,H-13).13C NMR(151MHz,DMSO-d6)δ170.47,158.08,157.91,144.53,142.34,136.54,136.09,134.32,127.64,122.73,121.89,119.94,114.66,110.72,70.96,70.52,65.06,58.38,55.97,34.07,29.55,21.75.HR MScalcd for C21H25N3O3[M+H]+368.1969,found 368.1961.
(R)-N-(2-(4-(3-(1H-1,2,4-Triazol-1-yl)propoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM5-7),yellow oil；
Figure BDA0002702565470000184
Figure BDA0002702565470000185
7.98(s,1H,H-2),7.25(dd,J＝27.2,8.5Hz,2H,H-3),6.88(t,J＝9.1Hz,2H,H-4),5.40(dd,J＝121.9,4.2Hz,1H,H-5),4.68(brs,1H,H-6),4.35(t,J＝6.9Hz,2H,H-7),3.94(t,J＝5.9Hz,2H,H-8),3.48–3.38(m,1H,H-9),3.30–3.21(m,1H,H-10),2.85(d,J＝52.5Hz,3H,H-11),2.23(p,J＝6.4Hz,2H,H-12),1.92(d,J＝49.4Hz,3H,H-13).13C NMR(151MHz,DMSO-d6)δ170.44,158.10,151.89,144.58,136.53,(127.64,127.53),(114.64,114.55),70.96,65.07,58.37,55.96,34.05,29.56,21.77.HR MS calcd for C16H22N4O3[M+H]+319.1765,found 319.1757.
Figure BDA0002702565470000186
(R)-N-(2-(4-(3-(1H-Tetrazol-1-yl)propoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM5-8),white solid，m.p.92.8-93.3℃；
Figure BDA0002702565470000187
Figure BDA0002702565470000188
7.25(dd,J＝26.9,8.3Hz,2H,H-2),6.86(t,J＝8.0Hz,2H,H-3),5.39(dd,J＝122.4,4.3Hz,1H,H-4),4.88(t,J＝6.8Hz,1H,H-6),4.77(t,J＝6.8Hz,1H,H-7),4.70(brs,1H,H-5),4.04–3.95(m,2H,H-7),3.47–3.36(m,1H,H-8),3.30–3.21(m,1H,H-9),2.85(d,J＝52.0Hz,3H,H-10),2.35(d,J＝32.1Hz,2H,H-11),1.92(d,J＝49.4Hz,3H,H-12).13C NMR(151MHz,DMSO-d6)δ170.43,162.68,157.98,153.68,136.12,127.63,127.53,114.61,114.52,70.95,70.49,64.83,64.79,58.37,55.96,34.04,29.06,29.03,29.01,22.23,21.77.HR MS calcdfor C15H21N5O3[M+H]+320.1717,found 320.1712.
(R)-N-(2-(4-(3-(2H-Tetrazol-2-yl)propoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM5-9),white solid,m.p.148.2-149.6℃；
Figure BDA0002702565470000191
Figure BDA0002702565470000192
7.25(dd,J＝27.2,8.5Hz,2H,H-2),6.86(t,J＝8.8Hz,2H,H-3),5.39(dd,J＝122.1,4.3Hz,1H,H-4),4.70(brs,1H,H-5),4.64(t,J＝6.9Hz,2H,H-6),3.99(t,J＝5.8Hz,2H,H-7),3.47–3.36(m,1H,H-8),3.31–3.21(m,1H,H-9),2.85(d,J＝53.6Hz,3H,H-10),2.36–2.27(m,2H,H-11),1.92(d,J＝48.5Hz,3H,H-12).13C NMR(151MHz,DMSO-d6)δ170.47,157.97,144.49,136.60,127.63,114.61,70.48,65.05,58.37,55.95,34.04,29.36,21.78.HR MS calcdfor C15H21N5O3[M+H]+320.1717,found 320.1713.
Figure BDA0002702565470000193
(R)-N-(2-Hydroxy-2-(4-(3-(5-methyl-1H-tetrazol-1-yl)propoxy)phenyl)ethyl)-N-methylacetamide(TM5-10),white solid，m.p.101.3-102.2℃；
Figure BDA0002702565470000194
Figure BDA0002702565470000195
6.86(t,J＝9.0Hz,2H,H-2),5.39(dd,J＝122.5,4.3Hz,1H,H-3),4.77(t,J＝6.8Hz,2H,H-4),4.73–4.66(m,1H,H-5),4.03–3.95(m,2H,H-6),3.47–3.36(m,1H,H-7),3.29–3.22(m,1H,H-8),2.85(d,J＝52.2Hz,3H,H-9),2.45(s,3H,H-10),2.38–2.29(m,2H,H-11),1.92(d,J＝49.7Hz,3H,H-12).13C NMR(151MHz,DMSO-d6)δ170.43,162.68,157.98,136.12,127.63,114.61,70.95,64.83,58.37,55.96,34.04,29.03,22.23,10.89.HR MS calcd for C16H23N5O3[M+H]+334.1874,found334.1872.
(R)-N-(2-Hydroxy-2-(4-(3-(5-methyl-2H-tetrazol-2-yl)propoxy)phenyl)ethyl)-N-methylacetamide(TM5-11),white solid，m.p.112.3-112.9℃；
Figure BDA0002702565470000196
Figure BDA0002702565470000197
6.86(t,J＝8.6Hz,2H,H-2),5.39(dd,J＝121.7,4.3Hz,1H,H-3),4.69(brs,1H,H-5),4.50(t,J＝6.8Hz,2H,H-4),3.97(t,J＝5.7Hz,2H,H-6),3.45-3.37(m,1H,H-7),3.30-3.25(m,1H,H-8),2.85(d,J＝51.5Hz,3H,H-9),2.49(s,3H,H-10),2.31–2.22(m,2H,H-11),1.91(d,J＝55.4Hz,3H,H-12).13C NMR(151MHz,DMSO-d6)δ170.43,157.91,152.59,136.15,127.67,114.55,70.94,64.82,58.36,55.94,34.06,28.98,22.23,8.65.HR MS calcd for C16H23N5O3[M+H]+334.1874,found334.1873.
Figure BDA0002702565470000201
(R)-N-(2-Hydroxy-2-(4-(3-((1-methyl-1H-tetrazol-5-yl)thio)propoxy)phenyl)ethyl)-N-ethylacetamide(TM5-12),white solid，m.p.94.5-95.2℃；
Figure BDA0002702565470000202
Figure BDA0002702565470000203
6.90(t,J＝9.4Hz,2H,H-2),5.38(dd,J＝122.3,4.3Hz,1H,H-3),4.70(s,1H,H-4),4.07(dd,J＝9.7,5.7Hz,2H,H-5),3.93(s,3H,H-6),3.46–3.37(m,3H,H-7and H-8),3.30–3.21(m,1H,H-8),2.85(d,J＝52.5Hz,3H,H-9),2.18(dd,J＝12.9,6.4Hz,2H,H-10),1.91(d,J＝50.2Hz,3H,H-11).13C NMR(151MHz,DMSO-d6)δ170.46,158.11,154.07,136.06,127.65,114.65,70.95,66.25,58.37,55.96,34.05,30.16,29.13,21.78.HR MS calcd forC16H23N5O3S[M+H]+366.1594,found 366.1590.
(R)-N-(2-(4-(3-(Benzo[d]thiazol-2-ylthio)propoxy)phenyl)-2-hydroxyethyl)-N-methylacetamide(TM5-13),white solid，m.p.98.5-99.6℃；
Figure BDA0002702565470000204
Figure BDA0002702565470000205
7.85(d,J＝8.0Hz,1H,H-2),7.47(t,J＝7.6Hz,1H,H-3),7.37(t,J＝7.6Hz,1H,H-4),7.26(dd,J＝26.8,8.5Hz,2H,H-5),6.93(t,J＝9.0Hz,2H,H-6),5.39(dd,J＝121.7,4.2Hz,1H,H-7),4.72(s,1H,H-8),4.11(dd,J＝10.2,5.8Hz,2H,H-9),3.52(t,J＝7.1Hz,2H,H-10),3.47–3.36(m,1H,H-11),3.31–3.20(m,1H,H-11),2.85(d,J＝49.2Hz,3H,H-12),2.29–2.17(m,2H,H-13),2.02–1.84(m,3H,H-14).13C NMR(151MHz,DMSO-d6)δ170.43,166.91,158.13,153.25,136.04,135.04,127.64,127.54,126.82,124.93,122.23,121.61,114.67,114.58,70.96,66.39,58.38,55.97,30.27,29.07,(22.25,21.79).HR MS calcd for C21H24N2O3S2[M+H]+417.1301,found 417.1290.
Figure BDA0002702565470000206
(R)-N-(2-Hydroxy-2-(4-(3-((5-methyl-1,3,4-thiadiazol-2-yl)thio)propoxy)phenyl)ethyl)-N-methyl-acetamide(TM5-14),white solid，m.p.89.9-91.5℃；
Figure BDA0002702565470000207
Figure BDA0002702565470000208
6.90(t,J＝9.4Hz,2H,H-2),5.40(dd,J＝121.5,3.9Hz,1H,H-3),4.69(brs,1H,H-4),4.07(dd,J＝10.3,5.6Hz,2H,H-5),3.47–3.37(m,3H,H-6and H-7),3.31–3.22(m,1H,H-8),2.83(t,J＝33.9Hz,3H,H-9),2.68(s,3H,H-10),2.20–2.12(m,2H,H-11),1.92(d,J＝49.9Hz,3H,H-12).13C NMR(151MHz,DMSO-d6)δ170.48,170.44,165.87,165.27,158.11,157.94,136.49,127.65,114.66,114.57,(70.95,70.50),(66.30,66.25),58.38,55.96,34.05,31.15,29.05,(22.23,21.77),15.65.HR MS calcd for C17H23N3O3S2[M+H]+382.1254,found382.1245.
(R)-N-(2-Hydroxy-2-(4-(3-((1-phenyl-1H-tetrazol-5-yl)thio)propoxy)phenyl)ethyl)-N-methylacetamide(TM5-15),white oil；
Figure BDA0002702565470000211
Figure BDA0002702565470000212
7.26(dd,J＝27.5,8.4Hz,2H,H-2),6.89(t,J＝9.4Hz,2H,H-3),5.40(dd,J＝121.9,4.2Hz,1H,H-4),4.70(brs,1H,H-5),4.18–4.01(m,2H,H-6),3.49(t,J＝7.0Hz,2H,H-7),3.46–3.36(m,1H,H-8),3.31–3.21(m,1H,H-9),2.85(d,J＝50.0Hz,3H,H-10),2.32–2.10(m,2H,H-11),1.90(d,J＝64.6Hz,3H,H-12).13C NMR(151MHz,DMSO-d6)δ170.46,170.43,158.10,157.93,154.75,136.07,133.60,131.07,130.46,127.64,127.53,125.07,114.66,114.57,70.96,66.34,58.38,55.97,30.23,28.93,(22.23,21.78).HR MS calcd for C21H25N5O3S[M+H]+428.1751,found 428.1744.
三、辛弗林唑类衍生物的生物活性检测
1、体外真菌抑制活性测定
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)。
具体为：(1)待测液B配制：吸取320μL储备液(浓度为3.2mg/mL＝3200μg/mL)，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。(2)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)。(3)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。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。
辛弗林唑类衍生物对真菌毕赤酵母菌的MIC时，均设有空白对照(培养基)、阴性对照(培养基+菌液)、阳性对照(培养基+菌液+阳性药物)，结果如表3所示。
表3化合物对毕赤酵母菌的抑制活性(MIC,μg/mL)
Figure BDA0002702565470000221
从表3中分析可知，空白对照与阴性对照对毕赤酵母菌的MIC值表现为>0.512mg/mL，6种抗菌药物的MIC值≥0.256mg/mL；辛弗林的MIC值为0.256mg/mL，大多数辛弗林含唑衍生物及其中间体的MIC值为0.128mg/mL，强于辛弗林和测试的抗菌药物，证明辛弗林含唑衍生物具有抗毕赤酵母菌的能力。其中，TM3系列的MIC值低于0.128mg/mL的有11个，占该系列分子总数的79％，其中TM3-12、TM3-14抑制浓度低至0.064mg/mL。TM5系列的MIC值低于0.128mg/mL的有10个，占该系列分子总数的71％。这些结果表明，辛弗林唑类衍生物具有抗真菌活性，在抗真菌领域具有潜在的应用前景。
2、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
待测物培养基配制：①稀释1000倍的待测物培养基配制:取5μL浓度为1μg/mL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倍的药剂培养基配制:取20μL浓度为4μg/mL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
经过初筛后，待测物TM3-12和TM3-15对柑橘褐斑病菌Alternaria alternata菌株Al.6活性很好，因而挑选出来进行复筛，获得回归方程。
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。化合物对柑橘胶孢炭疽病菌和柑橘褐斑病菌活性初筛结果见表4。
表4化合物对柑橘真菌的抑制活性(初筛结果)
Figure BDA0002702565470000241
Figure BDA0002702565470000251
从表4中化合物对柑橘胶孢炭疽病菌、柑橘褐斑病菌初筛结果发现：①目标化合物对柑橘胶孢炭疽病菌具有一定的抑制活性，有7个目标化合物在4μg/mL浓度下的抑制率≥33％，达到阳性对照物活性的50％以上；有4个目标化合物在4μg/mL浓度下的抑制率≥40％，达到阳性对照咪鲜胺抑制活性的70％。②目标化合物对柑橘褐斑病菌的抑制活性，在1μg/mL浓度下有5个分子的抑制活性超过阳性对照的50％，有3个分子的抑制活性达到阳性对照的70％；在4μg/mL浓度下，有7个分子的抑制活性超过阳性对照的50％，有2个分子的抑制活性超过阳性对照的80％。这些结果证明，辛弗林唑类衍生物对柑橘真菌病菌具有抗菌活性。
对高活性化合物TM3-12和TM3-15进行了柑橘褐斑病菌复筛，结果如表5和图1所示。
表5高活性化合物对柑橘褐斑病菌的抑制活性(复筛)
Figure BDA0002702565470000252
从表5中的测试结果表明，TM3-12和TM3-15未体现出抗药性，证明了其在抗柑橘病菌领域具有潜在的应用前景。
图1为高活性化合物TM3-15复筛孔板图。图1中复筛孔板为24孔板(4行6列)。左为正面图：每一行从左至右浓度依次为0.01、0.004、0.002、0.00、0.0005、0.00025(单位：mg/mL)，即稀释100、250、500、1000、2000、4000倍；每一列为相同浓度重复4次。右为反面图：每一行从右至左浓度依次为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。
3、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。在各2mL离心管中分别加入450μL柑桔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％)。每个样品每个浓度三次重复。结果如表6和表7所示。
表6部分化合物对柑橘溃疡病菌的抑制活性
Figure BDA0002702565470000261
表7部分化合物对柑橘溃疡病菌的抑制活性
Figure BDA0002702565470000262
表7显示，所测试辛弗林的唑类衍生物的活性全部都强于辛弗林。在1.6μg/mL测试浓度下，20个测试分子中，有12个分子的活性强于阳性对照药物诺氟沙星；0.64μg/mL浓度下，20个测试分子中，有18个分子的活性强于诺氟沙星。这些结果表明，所测试的大多数辛弗林唑类衍生物的抗柑橘溃疡病的活性强于诺氟沙星和辛弗林。
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 (10)

1.式Ⅰ所示的辛弗林唑类衍生物，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：
Figure FDA0002702565460000011
式Ⅰ中，
Y选自：
Figure FDA0002702565460000012
Figure FDA0002702565460000013
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或C1-C3烷基；
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为H或C1-C3烷基；
L选自：-(CH2)n-、-CO(CH2)nCO-、
Figure FDA0002702565460000014
n选自2、3或4；
X选自：烷酰基或磺酰基。
2.根据权利要求1所述辛弗林唑类衍生物，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；
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为甲基；
L选自：-(CH2)n-或
Figure FDA0002702565460000021
n选自2、3或4；
X为-R10R11；
R10选自：-CO-或-SO2-；
R11选自：C1-C3烷基、C1-C3羟烷基、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。
3.根据权利要求2所述辛弗林唑类衍生物，其特征在于，所述式Ⅰ中，
Y选自：
Figure FDA0002702565460000022
Figure FDA0002702565460000023
L选自：-(CH2)n-，n选自3或4；
X为-R10R11；
R10选自：-CO-；
R11选自：-CH3。
4.根据权利要求3所述辛弗林唑类衍生物，其特征在于，式Ⅰ所示的辛弗林唑类衍生物为以下化合物中的任一种：
Figure FDA0002702565460000024
Figure FDA0002702565460000031
Figure FDA0002702565460000041
5.根据权利要求4所述辛弗林唑类衍生物，其特征在于，式Ⅰ所示的辛弗林唑类衍生物为以下化合物中的任一种：TM3-6，TM3-7，TM3-10，TM3-12，TM3-14，TM3-15，TM5-2，TM5-11。
6.如权利要求1至权利要求5任一所述辛弗林唑类衍生物的制备方法，其特征在于，包括以下步骤：
将辛弗林进行胺基酰化，制得中间体IM1；
Figure FDA0002702565460000042
将中间体IM1与linker试剂反应，制得中间体IM2；
Figure FDA0002702565460000043
将中间体IM2与唑偶联，制得辛弗林唑类衍生物；
Figure FDA0002702565460000044
式中，X、Y和L的定义与权利要求1～4中任一所述的辛弗林唑类衍生物结构式中X、Y和L的定义相同；IM2中的Z为卤素。
7.根据权利要求6所述辛弗林唑类衍生物的制备方法，其特征在于，包括以下步骤：
1)将辛弗林与醋酸酐在溶剂中反应，制得中间体IM1；所述溶剂为水；
2)将中间体IM1与linker试剂在有机溶剂、碱作用下偶联，制得中间体IM2；所述linker试剂为1,4-二溴丁烷或1,3-二溴丙烷；所述有机溶剂为二甲基甲酰胺、丙酮或丁酮；所述碱为碳酸钾；
3)将中间体IM2与唑在有机溶剂、碱作用下偶联，制得辛弗林唑类衍生物；所述有机溶剂为二甲基甲酰胺；所述碱为碳酸钾。
8.如权利要求6所述制备方法制得的中间体IM1和IM2，或其消旋体、立体异构体、药学上可接受的盐。
9.如权利要求1至权利要求5任一所述辛弗林唑类衍生物在抗真菌药物中的应用。
10.如权利要求1至权利要求5任一所述辛弗林唑类衍生物在抗柑橘病菌药物中的应用。
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