CN113287620B - 1H-吡唑并[3,4-d]嘧啶类化合物在制备禾谷镰刀菌杀菌剂中的应用 - Google Patents
1H-吡唑并[3,4-d]嘧啶类化合物在制备禾谷镰刀菌杀菌剂中的应用 Download PDFInfo
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Abstract
本发明公开了1H‑吡唑并[3,4‑d]嘧啶类化合物或其农学上可接受的盐或其立体异构体在制备禾谷镰刀菌杀菌剂中的应用,属于杀菌剂领域。所述1H‑吡唑并[3,4‑d]嘧啶类化合物的结构式如式(Ⅰ)所示。本发明首次公开该类化合物具有抑制禾谷镰刀菌的促分裂原活化蛋白激酶(FgGpmk1)磷酸化方面的活性,通过抑制FgGpmk1磷酸化而发挥抗菌作用,同时能够抑制病原菌毒素的产生,且对农作物具有较好的安全性,因此,本发明所提供的化合物可用于禾谷镰刀菌侵染引起的作物赤霉病防治中,尤其针对小麦赤霉病,该类化合物作为杀菌剂具有良好的开发前景。
Description
技术领域
本发明涉及杀菌剂领域,具体涉及1H-吡唑并[3,4-d]嘧啶类化合物的真菌防治用途,特别涉及在防治赤霉病中的应用。
背景技术
小麦是世界三大粮食作物之一。在我国,小麦是第二大粮食作物,小麦的产量和品质安全直接关系到国计民生。由禾谷镰刀菌(Fusarium graminearium)导致的小麦赤霉病,严重威胁我国乃至世界小麦生产安全。小麦赤霉病是一种毁灭性的真菌性植物病害,严重时导致粮食大幅减产甚至绝收。小麦赤霉病在世界范围内分布广泛、且发生频率高,多发于温暖潮湿或半潮湿地区,气候湿润多雨的温带地区受害更为多发与严重。在我国,小麦赤霉病多发生于长江中下游、华南和东北地区,严重发病的年份产量损失可达20%-40%。近年来,小麦赤霉病高发地区不断增加,包括山东、河南、河北等小麦主产区。
小麦赤霉病除了导致粮食减产外,病原菌所产生的毒素,包括脱氧雪腐镰刀菌烯醇(DON)具有很强的细胞毒性并且干扰免疫系统,能引起人、畜的多种疾病,包括发热、呕吐、腹泻等。由于缺乏抗性品种,化学防治目前依旧是世界上防治小麦赤霉病的主要手段。目前防控小麦赤霉病主要施用三大类药剂,包括苯并咪唑类杀菌剂、甾醇生物合成抑制剂类杀菌剂和氰基丙烯酸酯类杀菌剂。其中,苯并咪唑类杀菌剂、甾醇生物合成抑制剂类杀菌剂对小麦赤霉病虽有较好防治效果,但并不能抑制病原菌毒素的产生。相反,部分苯并咪唑类杀菌剂、甾醇生物合成抑制剂类杀菌剂会刺激毒素的生成。而氰基丙烯酸酯类杀菌剂既能较好地抑制病原菌的生长,同时抑制其毒素的产生,为理想的杀菌剂。然而,耐药性的发生,导致多种类型的杀菌剂效益降低。因此,开发新型的杀菌剂,并且能够抑制毒素的生成,是防治小麦赤霉病关键科学问题,具有重要的社会和经济价值。
丝裂原活化蛋白激酶(MAPK)信号通路在真核细胞中进化保守,并通过磷酸化水平调节细胞的生长,分化和应激反应。丝裂原活化蛋白激酶的磷酸化水平与其激酶活性密切相关。在几种植物病原性真菌中,已发现多种丝裂原活化蛋白激酶与其致病性相关,致病性取决于其磷酸化水平。禾谷镰刀菌中的丝裂原活化蛋白激酶FgGpmk1对禾谷镰刀菌的毒力和渗透性具有关键的作用,是控制小麦赤霉病的关键靶标。因此,挖掘能够抑制FgGpmk1磷酸化的小分子杀菌剂具有开发前景。
发明内容
本发明的目的在于提供一种能够抑制FgGpmk1磷酸化的小分子杀菌剂,将其应用到小麦赤霉病的防治当中。
为实现上述目的,本发明采用如下技术方案:
本发明提供了一种1H-吡唑并[3,4-d]嘧啶类化合物,其结构式如式(Ⅰ)所示,
其中,
R1为哌嗪-1-基或者N-取代的哌嗪-1-基,所述哌嗪-1-基上N-取代的取代基为烷基、链上含杂原子的烷基、链上含芳基的烷基、脂肪酰基、芳基酰基、芳基上含给电子取代基的芳基酰基、芳基、芳基上含吸电子取代基的芳基、嘧啶基上含给电子取代基的嘧啶-2-基或吡啶-2-基;
或,氨基或者N-取代的氨基,所述氨基氮上的取代基为烷基、链上含脂杂环取代基的烷基、链上含芳杂环取代基的烷基、芳基、芳基上含脂杂环取代基的芳基、芳基上含吸电子取代基的芳基或芳基上含给电子取代基的芳基;
或,哌啶-1-基或者哌啶环上含取代基的哌啶-1-基,所述哌啶-1-基上的取代基为氨甲酰基或者N-烷基取代的氨甲酰基;
或,腙基或者含取代基的腙基,所述腙基上的取代基为芳基或芳基上含给电子取代基的芳基;
或,肼基或者含取代基的肼基,所述肼基上的取代基为脂肪酰基;
或,巯基或者S-取代的硫醚基,所述硫醚基的硫上的取代基为烷基;
R2为烷基或者N-取代的氨甲酰烷基,所述氨甲酰烷基氮上的取代基为链上含杂原子的烷基、环烷基或含吸电子取代基的芳基;
或,芳基或者芳基上含取代基的芳基,所述芳基上的取代基为给电子取代基或吸电子取代基。
优选的,所述给电子取代基为甲基、甲氧基或羟基;所述吸电子取代基为卤素、硝基或三氟甲基;所述脂杂环取代基为吗啉基;所述芳杂环取代基为咪唑基。
本发明研究表明,所述1H-吡唑并[3,4-d]嘧啶类化合物的关键靶标之一是禾谷镰刀菌(Fusarium graminearium)的促分裂原活化蛋白激酶(FgGpmk1),能够抑制FgGpmk1磷酸化而发挥抗菌作用。因此,本发明提供了所述1H-吡唑并[3,4-d]嘧啶类化合物或其农学上可接受的盐或其立体异构体在制备禾谷镰刀菌(Fusarium graminearium)杀菌剂中的应用。
作为优选,R1为以下取代基的任意一种:
R2为以下取代基中任意一种:
更为优选,所述1H-吡唑并[3,4-d]嘧啶类化合物的结构式为:
本发明研究表明,上述化合物能够显著抑制禾谷镰刀菌中FgGpmk1磷酸化,减少毒素小体的生成,降低禾谷镰刀菌中DON含量,既能抑制病原菌的生长,同时抑制其毒素的产生,为理想的杀菌剂。
所述禾谷镰刀菌杀菌剂中含有0.01%-99.99%重量百分比的所述的1H-吡唑并[3,4-d]嘧啶类化合物或其农学上可接受的盐或其立体异构体的一种或多种。
所述禾谷镰刀菌杀菌剂的制剂形式可以为但不限于微乳剂、水剂、粉剂、悬浮剂、乳油、种衣剂。
本发明提供的禾谷镰刀菌杀菌剂对作物无毒性作用,可将其应用于防治禾谷镰刀菌侵染引起的作物赤霉病,尤其是用于防治小麦赤霉病。
具体的,所述应用包括:于小麦扬花期至盛花期,将所述禾谷镰刀菌杀菌剂施用于小麦麦穗。
本发明具备的有益效果:
本发明首次公开1H-吡唑并[3,4-d]嘧啶类化合物具有抑制禾谷镰刀菌的促分裂原活化蛋白激酶(FgGpmk1)磷酸化方面的活性,通过抑制FgGpmk1磷酸化而发挥抗菌作用,同时能够抑制病原菌毒素的产生,且对农作物具有较好的安全性,因此,本发明所提供的化合物可用于禾谷镰刀菌侵染引起的作物赤霉病防治当中,尤其针对小麦赤霉病,该类化合物作为杀菌剂具有良好的开发前景。
附图说明
图1为化合物1和31对禾谷镰刀菌分生孢子萌发的抑制效果。
图2为化合物1和31对禾谷镰刀菌FgGpmk1磷酸化水平的蛋白质印迹分析。
图3为化合物31对禾谷镰刀菌毒素小体生成的影响。
图4为化合物31对禾谷镰刀菌DON生成的影响。
图5为化合物31对小麦幼苗的影响。
具体实施方式
以下结合具体实施例对本发明作进一步说明。具体实施例的举例说明,不应被理解为对本发明范围的限制。此外,在阅读本发明所阐述的内容后,本领域技术人员所做的相关修改或改动,应理解为等价形式修改或改动,同样为本发明所述的权利范围内。
实施例中所使用的实验技术与方法如无特殊说明,均为常规方法。所用的材料、试剂等,如无特殊说明,均可从商业途径购得。
实施例1
一、本实施例从商业库筛选出33个1H-吡唑并[3,4-d]嘧啶类候选化合物,如表1所示。
表1
二、检测化合物1-33对禾谷镰刀菌分生孢子萌发的抑制活性
将2×104/孔的野生型禾谷镰刀菌菌株PH-1的分生孢子添加到含2.0mL/孔2%蔗糖水溶液的24孔板中。然后,将不同浓度的候选化合物添加到孔中。在25℃孵育3小时后,用计数器在显微镜下计算每个孔中萌发的分生孢子数,计算EC50,结果如表2所示。
表2.化合物1-33对禾谷镰刀菌分生孢子萌发抑制的半最大效应浓度(EC50)
由上表可知,化合物1-33对禾谷镰刀菌分生孢子萌发的抑制半最大效应浓度(EC50)均小于50μg·mL-1,具有较好的抑制效果,特别是化合物31,其EC50达到3.46±1.63μg·mL-1。菌落形态显示化合物31优于化合物1与对照组(二甲基亚砜,DMSO),劣于多菌灵组(图1)。
三、检测化合物对禾谷镰刀菌丝裂原活化蛋白激酶FgGpmk1磷酸化的抑制活性
将野生型禾谷镰刀菌菌株PH-1在含马铃薯葡萄糖肉汤培养基中于25℃孵育2天,随后加入化合物继续孵育6小时。收集菌丝,通过10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离得到的蛋白质,并转移至转移膜。用p44/42丝裂原活化蛋白激酶(MAPK)抗体9101S以1:1000稀释度检测磷酸化的FgGpmk1,用1:1000稀释度p44/42MAPK抗体9102S检测总的FgGpmk1。
如图2所示,化合物1和化合物31均能抑制FgGpmk1的磷酸化,而上市药物多菌灵则对FgGpmk1的磷酸化无影响,说明化合物1和化合物31均能抑制FgGpmk1的磷酸化而抑制禾谷镰刀菌的生长。此外,化合物31的抑制效果好于化合物1,说明抑制FgGpmk1磷酸化的能力是和分生孢子萌发的抑制活性相关的。
四、禾谷镰刀菌毒素小体检测
为了观察化合物对禾谷镰刀菌毒素小体生成的影响,使用Zeiss LSM780共聚焦显微镜检查培养在TBI(trichothecene biosynthesis induction medium)培养基的新鲜菌丝中的Tri1(DON合成酶Tri1)-GFP(绿色荧光蛋白)信号。
如图3所示,相比对照组(DMSO组),化合物31能够降低毒素小体的生成,而上市药物多菌灵并不能降低毒素小体的生成,反而促进其生成。
五、禾谷镰刀菌DON检测
野生型禾谷镰刀菌菌株PH-1在TBI培养基中于28℃下避光培养3天,加入化合物(多菌灵,1.4μg·mL-1,化合物31,15μg·mL-1)后孵育6小时,然后用三层滤纸过滤,滤液用于测定DON含量,滤纸上的菌丝烘干后称量干重作为内参。DON测定试剂(维赛科技,Wis008)盒检测DON含量。
如图4所示,化合物31能够显著降低禾谷镰刀菌DON含量,而上市药物多菌灵反而促进DON的生成。
六、化合物31安全性检测
小麦幼苗在25℃下浸泡在最终浓度为20μg/mL的化合物31的水中24小时,然后继续孵育7天观察麦苗生长状态。
如图5所示,化合物31组相比对照组(DMSO)并无明显区别,说明化合物31对小麦幼苗无明显毒性作用。
Claims (6)
2.如权利要求1所述的应用,其特征在于,所述禾谷镰刀菌杀菌剂中含有0.01%-99.99%重量百分比的所述的1H-吡唑并[3,4-d]嘧啶类化合物或其农学上可接受的盐的一种或多种。
3.如权利要求1所述的应用,其特征在于,所述禾谷镰刀菌杀菌剂的制剂形式为微乳剂、水剂、粉剂、悬浮剂、乳油或种衣剂。
4.如权利要求1所述的应用,其特征在于,所述禾谷镰刀菌杀菌剂用于防治作物赤霉病。
5.如权利要求4所述的应用,其特征在于,所述作物为小麦。
6.如权利要求5所述的应用,其特征在于,所述应用包括:于小麦扬花期至盛花期,将所述禾谷镰刀菌杀菌剂施用于小麦麦穗。
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