CN115340511B - 荧光化合物、其制备方法及作为荧光探针的应用 - Google Patents
荧光化合物、其制备方法及作为荧光探针的应用 Download PDFInfo
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Abstract
本申请公开了一种荧光化合物、其制备方法及作为荧光探针的应用。包括一类以异氟尔酮为骨架的聚集诱导发光荧光分子。该类化合物在蛋白质维持完整三维结构时不发出荧光;当蛋白质错误折叠、变性并聚集时,分子可通过非共价键选择性高效结合,并在结合之后发出强烈荧光。所述荧光化合物的上述性质可通过非共价键识别探测活细胞内的聚集态蛋白质,且可用于活细胞原位荧光探测胞内蛋白质的聚集过程,为活细胞内原位探测聚集态蛋白质提供了便携、高效的方法。
Description
技术领域
本申请涉及一种荧光化合物、其制备方法及作为荧光探针的应用,属于荧光探针领域。
背景技术
蛋白质需要折叠成正确的三维结构才能获得生理功能。基因突变、外界压力、化学修饰以及年龄因素等都会导致蛋白质发生错误折叠和聚集。致病蛋白质分子的错误折叠、变性与聚集会导致多种人类疾病,包括神经-肌肉退行性疾病、代谢紊乱和心血管疾病等。蛋白质的聚集是一个复杂的、多步骤的、由液相到固相转变的过程,包括正确折叠态、未折叠态、错误折叠可溶低聚物态以及不可溶聚集态。根据聚集物的形貌特征和生化性质又可以分为淀粉样化聚集物和无定形聚集物。目前上述大部分疾病发病机理尚不清晰,其中一个主要原因是领域内缺乏精准实验工具在活体细胞中实时实地观测致病蛋白质的整个错误折叠过程,因而无法确定致病机理。此外,识别蛋白质错误折叠与聚集的荧光分子和相关检测方法也常用于搭建药物筛选平台,如热位移测定法(thermal shift assay)。因此,发展探测蛋白质聚集过程的方法具有广阔的应用前景。
目前识别蛋白质聚集的探针主要应用于淀粉样化蛋白,其原因是淀粉样蛋白具有明确的β-折叠堆积结构。一系列的化学探针被设计和开发,其中聚集诱导荧光探针的发现为化学家设计淀粉样蛋白识别探针提供了多样的化学结构和更好的生物兼容性。但是细胞内无定形蛋白质聚集物具有不确定的结构,增加了探针分子识别的难度。另一方面,如何实现在活细胞内选择性识别无定形蛋白质聚集物也是一个挑战。
现有能够探测细胞内蛋白质聚集态的方法包括:技术,该方法利用环境敏感分子,可以实现在缓冲液中和福尔马林固定细胞中探测聚集态蛋白质,该方法的缺点是无法实现活细胞内原位探测聚集态蛋白质。另一类方法是通过在环境敏感类分子上加装马来酸酐官能团,实现共价修饰细胞内蛋白质组。该方法的优点是可以在活细胞内探测聚集态蛋白质,缺点是对细胞内蛋白质组具有广谱性修饰,缺乏选择性。
针对活细胞原位探测蛋白质聚集态的荧光分子,领域内报道甚少。目前已报道的文献通过在聚集诱导荧光(AIE)类分子上加装马来酸酐来穿透活细胞的细胞膜(Angew.Chem.Int.Ed.2020,59,2–9),并在蛋白质发生聚集时,激活荧光。然而此类分子缺乏对聚集蛋白的特异性和选择性。上述方法的发光机理在于利用马来酸酐对半胱氨酸的反应活性普遍性的标记蛋白质组,当蛋白质组某一或某些蛋白发生聚集时激活各自标记的荧光分子,其他未聚集蛋白上的荧光分子部发出荧光。因此上述方法虽然解决了细胞穿透性的问题,但同时也失去了其胞内复杂环境下的选择性。
因此,发展可以穿透活细胞细胞膜且具有胞内聚集蛋白质选择性的荧光分子及其相关检测方法,对研究蛋白质聚集所导致的疾病有着重大的科学意义和临床价值。
发明内容
根据本申请的一个方面,提供一种荧光化合物,包括化合物I,该化合物I是一类以异氟尔酮为骨架的聚集诱导发光荧光分子。该类分子在蛋白质维持完整三维结构时不发出荧光;当蛋白质错误折叠、变性并聚集时,分子可通过非共价键选择性高效结合,并在结合之后发出强烈荧光。该荧光分子的上述性质可用于荧光方法探测活细胞内的聚集态蛋白质。该荧光分子通过非共价键识别聚集态蛋白质,且可用于活细胞原位荧光探测胞内蛋白质的聚集过程。
本申请所述的荧光化合物是基于异氟尔酮骨架设计的荧光探针。该类衍生物的荧光量子产率和荧光强度对外界微环境敏感。具备良好的生物兼容性和优异的荧光性质。其制备方法简单且原料廉价,可进行大规模量产。该荧光分子由荧光发光基团、非共价键结合聚集态蛋白基团、亲水嵌段组成。化合物Ⅰ中,非共价键结合基团为R1,亲水嵌合段为R2,分子结构中除了R1、R2之外的结构为发光基团。
所述荧光化合物选自具有式I所示化学式的化合物I中的至少一种;
式I中,Ar选自苯、萘、噻吩、呋喃、吡啶、吲哚、咔唑中的至少一种;
R1选自氨基、烷氧基、硝基、酯基和氢中的至少一种;
R2选自C1~C5的烷基、C1~C5的烷氧基、取代的C1~C5的烷基中的至少一种;
所述R2为取代的C1~C5的烷基时,取代基选自羟基、C6或C12的芳基中的至少一种;
所述R1为氨基时,R2选自C1~C5的烷基、C6芳基或取代的C1~C5的烷基中的至少一种。
R3选自丙二腈、丙二酸二乙酯、氰乙酸乙酯、氰乙酰胺、米氏酸或氧中的至少一种;
进一步地,所述荧光化合物选自具有以下化学式所示的化合物I中的至少一种。
可选地,所述化合物I的激发波长为430nm~560nm;发射波长为550nm~730nm。
本申请中,C1~C5指所包含的碳原子数。对所述“取代的C1~C5的烷基I”、“取代的C1~C5的烷基II”的碳原子数限定,是指烷基本身所含的碳原子数,而非取代后的碳原子数。如取代的C1~C5的烷基II,指碳原子数为1~5的烷基上,至少一个氢原子被取代基取代。
本申请中,“烷基”是由烷烃化合物分子上失去任意一个氢原子所形成的基团。所述烷烃化合物包括直链烷烃、支链烷烃、环烷烃、带有支链的环烷烃。
本申请中,所述“烷氧基”指其中R为烷基。
根据本申请的另一方面,还提供了一种上述化合物I的制备方法,至少包括如下步骤:
步骤(1):向化合物II中加入化合物III和溶剂I,在碱性或酸性条件下,反应I,得到中间产物I;
所述中间产物I选自具有式II所示化学式的化合物:
所述化合物II选自具有式II所示化学式的化合物:
所述化合物III选自丙二腈、丙二酸二乙酯、氰乙酸乙酯、氰乙酰胺、米氏酸中的至少一种;
步骤(2):将中间产物I与化合物IV和溶剂II混合,在碱性条件下,反应II,得到化合物I;
所述化合物IV选自具有式III所示化学式的化合物中的至少一种:
所述碱性条件下为含有碱性物质的反应环境;所述碱性物质选自哌啶、四氢吡咯、吗啉、哌嗪、吡啶、氢氧化钠、中的至少一种;所述碱性物质与化合物II的摩尔比为0.8~8:1;
所述酸性条件下为含有酸性物质的反应环境;所述酸性物质选四氯化钛、醋酸铵、醋酸中的至少一种;所述酸性物质与化合物II的摩尔比为1.5~6:1;
在所述步骤(1)中,反应I的条件为:
加热回流搅拌,温度为40~100℃;时间为12~30h;
所述化合物II和化合物III的摩尔比为1:0~2;
所述溶剂I选自甲醇、乙醇、四氢呋喃、甲苯、二氯甲烷中的至少一种;
在所述步骤(2)中,反应II的条件为:温度为80~100℃;时间为12~24h;
所述中间产物I和化合物IV的摩尔比为1:1~2;
所述溶剂II选自甲醇、乙醇、四氢呋喃、甲苯中的至少一种。
根据本申请的另一方面,还提供了一种荧光探针,包括上述化合物I、根据上述方法制备得到的化合物I中的至少一种。
根据本申请的另一方面,还提供了一种荧光探针的应用,其中使用了上述荧光化合物、根据上述方法制备得到的荧光化合物中的至少一种作为荧光探针。
本申请所述的荧光化合物是一类具有异氟尔酮骨架的聚集诱导发光的荧光分子。其次,此类分子的特殊性质使其可实现现有方法无法完成的多种生物医学的重要应用场景。
本申请还提供了上述荧光化合物、根据上述方法制备得到的荧光化合物中的至少一种在缓冲液中探测重组蛋白质聚集态的应用。
向缓冲液中加入0.1μmol/L~100μmol/L蛋白待测样本和5μmol/L~50μmol/L所述聚集诱导荧光探针,在25℃~37℃下进行孵育处理;孵育后,样本以间隔2℃,进行37℃~95℃温度梯度加热5min引发蛋白质聚集后进行实时荧光定量跟踪测量。
所述缓冲液的pH=4.0~8.5;
所述缓冲物质选自磷酸钠盐、磷酸钾盐、氨基丁三醇、氯化钠盐、氯化钾盐、乙酸钠中的至少一种;
所述磷酸钠盐、磷酸钾盐的浓度为1mmol/L~100mmol/L;
所述氨基丁三醇的浓度为1mmol/L~100mmol/L;
所述氯化钠盐、氯化钾盐的浓度为1mmol/L~500mmol/L;
所述乙酸钠的浓度为1mmol/L~500mmol/L。
进一步地,可以向缓冲液中加入0.1μmol/L~100μmol/L具有药物活性的小分子,将所测得的荧光曲线与所加入的具有药物活性的小分子的荧光曲线进行对比,可检测具有药物活性的小分子是否与目标蛋白发生结合,从而实现对目标蛋白的稳定作用。
本申请中的重组蛋白质的含义是:任一通过大肠杆菌、酵母菌、昆虫细胞或者哺乳动物细胞为载体,表达并纯化出的模型蛋白质均可称为重组蛋白质。
本申请中的蛋白质聚集态的含义是:加热或药物引发蛋白质错误折叠以及变性后的聚集的蛋白质。
本申请还提供了上述荧光化合物、根据上述方法制备得到的荧光化合物中的至少一种在荧光成像检测中的应用;
本申请所述荧光分子在活细胞中选择性结合蛋白质的聚集态,发出强烈荧光,用于荧光成像检测,具体包括:向活细胞中加入1nmol/L~1mmol/L的蛋白质聚集引发剂,同时引入0.1μmol/L~25μmol/L所述荧光探针在37℃孵化箱内原位孵育24h);待蛋白质发生聚集后,直接进行荧光成像,观测活细胞内的蛋白质聚集态的形貌和胞内位置。
所述蛋白质聚集引发剂选自坦螺旋霉素、氯喹、衣霉素、MG-132、普拉二烯内酯B、环己酰亚胺、亚砷酸钠中的至少一种。
本申请还提供了上述荧光化合物、根据上述方法制备得到的荧光化合物中的至少一种在筛选具有药物活性的小分子中的应用;
所述具有药物活性的小分子选自蛋白质的小分子抑制剂或者激活剂。
具体包括:向1μmol/L-1000μmol/L重组纯化蛋白中加入1nmol/L-100μmol/L荧光探针分子和10μmol/L-100μmol/L具有药物活性的小分子,在25℃-37℃下进行5min-30min孵育处理。通过温度梯度加热引发蛋白质聚集。加热的温度梯度方法为:(1)利用PCR或恒温加热装置在37℃-95℃之间每隔1℃-10℃中任意数值温度间隔,进行样本加热,然后分别读取荧光强度,绘制温度荧光曲线;(2)利用实时荧光PCR仪,从37℃到95℃每分钟1℃-10℃中任意数值温度递增,并实时记录荧光强度,绘制实时荧光曲线。通过比较导致蛋白聚集临界温度的变化,考察加入药物分子是否能够迁移荧光曲线,推导分子是否结合目标靶点蛋白。分子如果结合目标靶点蛋白,即证明小分子作用于蛋白质,可用于筛选目标蛋白的小分子抑制剂或者激活剂等具有药物活性的小分子。
所述蛋白质的激活剂或抑制剂选自甲氧苄啶、培美曲塞、二氟尼柳、乙胺嘧啶、Tafamidis中的至少一种。
本申请要解决的技术问题为利用荧光分子的环境敏感性和结构特异性,实现活细胞内对聚集态蛋白质有选择性的荧光识别和探测。在细胞内蛋白质具备正确的三维结构时不发出荧光,而当蛋白质发生错误折叠、变性并聚集时发出强烈荧光。并且该荧光探针的激活方式为非共价结合,避免共价结合广谱修饰蛋白质组所带来的细胞毒性和干扰。因此,该申请所述荧光探针可用于活细胞内非共价键探测聚集态的蛋白质分子。
本申请所述探测活细胞内蛋白质聚集态的荧光探针,其发光机理是,在蛋白质维持正确折叠态时,荧光分子内部的化学键在荧光激发态时可自由旋转,能量以热能形式释放,因此发生荧光淬灭。当蛋白质错误折叠、变性、聚集时,荧光分子与聚集态蛋白质发生快速的、有选择性的结合。由于分子与蛋白质聚集态的分子间相互作用,使得荧光分子激发态自由旋转的化学键受到禁阻能量以荧光形式释放,发出强烈荧光。同时,这类分子具有供电子-π键-吸电子(D-π-A)结构,激发后分子通过分子内电荷转移极化,对环境极性更加敏感。当蛋白质聚集时,疏水氨基酸残基暴露,使得荧光分子发出荧光。利用荧光强度的大幅跃升,探测活细胞内聚集态蛋白质的形成。
本申请中的荧光分子可发出红色荧光,此外,其发射光谱也覆盖了绿色发光区间,可同时使用两个发光光路,即绿色和红色。
以下介绍一种具有式I-1所示的化学式的化合物I的制备方法:
S1.将异氟尔酮、化合物III和哌啶在有机溶剂中混合,加热回流搅拌,反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用有机溶剂重结晶,得棕色固体
S2.将上述步骤所得产物与化合物IV在有机溶剂中混合,加入少量哌啶后加热。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终化合物I。
在其中一优选实例中,S1回流时间为12~24小时。
在其中一优选实例中,S2加热温度为80~100℃,加热时间为12~24小时。
在其中一优选实例中,异氟尔酮与化合物III的摩尔比为1:1~5。
在其中一优选实例中,S1所得产物与化合物IV的摩尔比为1:1~5。
在其中一优选实例中,S1所得产物与加入的哌啶摩尔比为1:0.01~1。
在其中一优选实例中,S1反应有机溶剂为乙醇,重结晶溶剂为乙醇。
在其中一优选实例中,S2反应有机溶剂为乙醇,萃取溶剂为二氯甲烷,柱色谱层析所用溶剂为石油醚与乙酸乙酯混合溶剂,乙酸乙酯和石油醚的体积比为1:1~10。
本申请的有益效果包括但不限于:
(1)本申请提供的荧光化合物可以作为荧光探针与聚集态蛋白质发生非共价结合,具体地,整个分子结构与聚集态蛋白质作为一个整体发生非共价键结合,其中分子的R1、R2基团对结合强度起到重要作用;
(2)本申请提供的荧光化合物可以作为荧光探针实现在胞内复杂生物环境下特异性的结合聚集态蛋白质;
(3)本申请提供的荧光化合物可以作为荧光探针在活细胞内通过荧光成像方法探测蛋白质聚集态的形貌和位置。
本申请以异氟尔酮为骨架设计合成了一系列荧光化合物,可作为聚集诱导发光的分子探针,该类荧光化合物在蛋白质维持完整的三维结构时不发出荧光;当蛋白质错误折叠、变性并聚集时,荧光化合物可通过非共价键选择性高效结合并发出强烈荧光。该荧光化合物的上述性质可用于荧光方法探测活细胞内的聚集态蛋白质。该申请为活细胞原为内探测聚集态蛋白质提供了便携、高效的方法。
附图说明
图1为实施例25中荧光分子缓冲液中对聚集态蛋白质的荧光光谱扫描。
图2为实施例26中基于荧光分子的热位移测定法测定大肠杆菌二氢叶酸还原酶结合抗生素甲氧苄胺嘧啶后的稳定性提升。
图3为实施例27中基于荧光分子的荧光显微镜成像效果;在人胚胎肾细胞293细胞系(HEK293)中加入热休克蛋白90抑制剂17AAG,通过本申请提供的荧光探针广谱识别聚集态蛋白质在活细胞中聚集的位置;其中,图(a)说明抑制剂17AAG诱导蛋白沉淀,荧光探针分子在共聚焦显微镜下可以识别蛋白质聚集态;图(b)是将荧光图片与明场图片叠加,以展示蛋白质聚集态位置分布,聚集态蛋白质分布在细胞质周围。
图4为实施例27中基于荧光分子的荧光显微镜成像效果;在人胚胎肾细胞293细胞系(HEK293)中加入蛋白酶体抑制剂MG132,通过本申请提供的荧光探针广谱识别聚集态蛋白质在活细胞中聚集的位置;其中,图(a)说明抑制剂MG132诱导蛋白沉淀,荧光探针分子在共聚焦显微镜下可以识别蛋白质聚集态;图(b)是将荧光图片与明场图片叠加,以展示蛋白质聚集态位置分布,聚集态蛋白质分布在细胞核周围。
图5为实施例27中基于荧光分子的荧光显微镜成像效果;在人胚胎肾细胞293细胞系(HEK293)中加入剪切因子抑制剂Pladienolide B,通过本申请提供的荧光探针广谱识别聚集态蛋白质在活细胞中聚集的位置;其中,图(a)说明抑制剂Pladienolide B诱导蛋白沉淀,荧光探针分子在共聚焦显微镜下可以识别蛋白质聚集态;图(b)是将荧光图片与明场图片叠加,以展示蛋白质聚集态位置分布,聚集态蛋白质分布在细胞核周围。
图6为实施例27中基于荧光分子的荧光显微镜成像效果。在人宫颈癌细胞系(HeLa)中加入热休克蛋白90抑制剂17AAG,通过本申请提供的荧光探针广谱识别聚集态蛋白质在活细胞中聚集的位置;其中,图(a)说明抑制剂17AAG诱导蛋白沉淀,荧光探针分子在共聚焦显微镜下可以识别蛋白质聚集态;图(b)是将荧光图片与明场图片叠加,以展示蛋白质聚集态位置分布,聚集态蛋白质分布在细胞质和细胞核周围。
图7为实施例27中基于荧光分子的荧光显微镜成像效果。在人宫颈癌细胞系(HeLa)中加入蛋白酶体抑制剂MG132,通过本申请提供的荧光探针广谱识别聚集态蛋白质在活细胞中聚集的位置;其中,图(a)说明抑制剂MG132诱导蛋白沉淀,荧光探针分子在共聚焦显微镜下可以识别蛋白质聚集态;图(b)是将荧光图片与明场图片叠加,以展示蛋白质聚集态位置分布,聚集态蛋白质分布在细胞质和细胞核周围。
图8为实施例27中基于荧光分子的荧光显微镜成像效果。在人宫颈癌细胞系(HeLa)中加入剪切因子抑制剂Pladienolide B,通过本申请提供的荧光探针广谱识别聚集态蛋白质在活细胞中聚集的位置;其中,图(a)说明抑制剂Pladienolide B诱导蛋白沉淀,荧光探针分子在共聚焦显微镜下可以识别蛋白质聚集态;图(b)是将荧光图片与明场图片叠加,以展示蛋白质聚集态位置分布,聚集态蛋白质分布在细胞核周围。
图9为图3附图的灰度图。
图10为图4附图的灰度图。
图11为图5附图的灰度图。
图12为图6附图的灰度图。
图13为图7附图的灰度图。
图14为图8附图的灰度图。
具体实施方式
以下结合具体实施例来进一步说明本申请,但实施例并不对本申请做任何形式的限定。在不背离本发明精神和实质的情况下,对本申请方法、步骤或条件所作的简单修改或替换,均属于本申请的范围;若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
如无特殊说明,本申请所用原料和试剂均来自商业购买,未经处理直接使用,所用仪器设备采用厂家推荐的方案和参数。
本申请实施例中的核磁数据采用核磁共振波谱仪Bruker AVANCE III 400MHz进行;
本申请实施例使用的共聚焦荧光显微镜型号为Olympus FV1000FluoViewTMConfocal microscope。
实施例1
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与96.1mg糠醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.49(s,1H),6.93–6.79(m,3H),6.55(d,J=3.4Hz,1H),6.48(m,1H),2.59(s,2H),2.40(s,2H),1.06(s,6H).13C-NMR(100MHz,CDCl3)δ169.1,153.6,152.33,144.7,127.4,123.8,123.5,113.7,113.6,112.9,112.7,43.1,39.1,32.1,28.1.HRMS(m/z)Anal.Calc’d for C17H17N2O(M+H)+:265.1335,Found(M+H)+:265.1336.
本实施例的合成路线如下:
实施例2
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与214.2mg 3-吡啶甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ8.70(m,1H),8.56(m,1H),7.86(m,1H),7.34(m,1H),7.03(m,2H),6.87(m,1H),2.61(s,2H),2.47(s,2H),1.08(s,6H).13C-NMR(100MHz,CDCl3)δ169.1,152.8,150.3,149.3,133.6,132.8,131.7,131.3,124.7,124.1,113.3,112.5,80.1,43.1,39.2,32.2,28.1.HRMS(m/z)Anal.Calc’d forC18H17N3K(M+K)+:314.1054,Found(M+K)+:314.1060.
本实施例的合成路线如下:
实施例3
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与112.1mg 2-噻吩甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.37(d,J=5.1Hz,1H),7.19(m,2H),7.06(m,1H),6.78(m,2H),2.59(s,2H),2.42(s,2H),1.07(s,6H).13C-NMR(100MHz,CDCl3)δ169.1,153.6,141.6,129.9,129.7,128.6,128.4,128.2,123.3,113.7,112.9,78.5,43.1,39.2,32.1,28.1.HRMS(m/z)Anal.Calc’d for C17H20N3S(M+NH4)+:298.1372,Found(M+NH4)+:298.1377.
本实施例的合成路线如下:
实施例4
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与156.2mg 1-萘甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ8.19–8.11(m,1H),7.95–7.84(m,3H),7.79(d,J=7.2Hz,1H),7.64–7.47(m,3H),7.09(d,J=15.7Hz,1H),6.90(m,1H),2.64(s,2H),2.62(s,2H),1.14(s,6H).13C-NMR(100MHz,CDCl3)δ169.3,153.9,133.9,133.6,133.0,131.8,131.4,130.3,129.1,126.9,126.4,125.8,124.8,124.0,123.1,113.6,112.8,43.1,39.5,32.2,28.2.HRMS(m/z)Anal.Calc’d for C23H21N2(M+H)+:325.1699,Found(M+H)+:325.1699.
本实施例的合成路线如下:
实施例5
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与318.4mg 1-甲基吲哚-3-甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.95(d,J=7.6Hz,1H),7.36(m,2H),7.34(m,1H),7.32(m,1H),7.30(s,1H),7.00(m,1H),6.79(s,1H),3.84(s,3H),2.59(s,2H),2.50(s,2H),1.09(s,6H).13C-NMR(100MHz,CDCl3)δ169.4,155.9,138.3,132.3,131.3,126.0,124.9,123.4,121.6,120.9,120.6,114.5,114.0,113.7,110.3,75.3,43.1,39.1,33.5,32.1,28.2.HRMS(m/z)Anal.Calc’d for C22H22N3(M+H)+:328.1808,Found(M+H)+:328.1809.
本实施例的合成路线如下:
实施例6
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与155.2mg 5-(二甲氨基)-2-噻吩甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.10(d,J=15.2Hz,1H),6.96(m,1H),6.64(m,1H),6.34(d,J=H).13C-NMR(100MHz,CDCl3)δ168.6,162.6,155.3,134.3,132.1,126.3,122.0,120.1,115.0,114.1,103.6,43.1,42.45,39.4,32.1,29.9,29.5,28.2.HRMS(m/z)Anal.Calc’d for C19H22N3S(M+H)+:324.1529,Found(M+H)+:324.1518.
本实施例的合成路线如下:
实施例7
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与334.9mg N-乙基咔唑-3-甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ8.25(m,1H),8.12(d,J=7.7Hz,1H),7.68(m,1H),7.51(m,1H),7.46–7.40(m,2H),7.33–7.27(m,2H),7.06(m,1H),6.85(m,1H),4.39(q,J=7.2Hz,2H),2.60(m,2H),2.53(m,2H),1.46(t,J=7.2Hz,3H),1.10(s,6H).13C-NMR(100MHz,CDCl3)δ169.4,154.9,141.1,140.6,138.8,127.0,126.5,126.4,125.5,123.7,122.9,122.3,120.7,120.7,119.9,114.5,113.3,109.2,109.1,43.0,39.3,37.9,32.1,28.2,14.0.HRMS(m/z)Anal.Calc’d for C27H26N3(M+H)+:392.2121,Found(M+H)+:392.2121.
本实施例的合成路线如下:
实施例8
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与199.3mg 4-二甲氨基-1-萘醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ8.29–8.23(m,1H),8.13(d,J=8.1Hz,1H),7.85(d,J=15.8Hz,1H),7.76(d,J=8.0Hz,1H),7.61–7.50(m,2H),7.13–6.99(m,2H),6.87(s,1H),2.96(s,6H),2.63(s,2H),2.59(s,2H),1.13(s,6H).13C-NMR(100MHz,CDCl3)δ169.4,154.5,153.2,133.8,132.8,129.6,128.5,126.9,126.8,125.4,125.4,125.3,123.4,123.1,113.8,45.1,43.1,39.5,32.2,28.2.HRMS(m/z)Anal.Calc’dfor C25H26N3(M+H)+:368.2121,Found(M+H)+:368.2126.
本实施例的合成路线如下:
实施例9
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与226.8mg 4-硝基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ8.25(m,2H),7.65(m,2H),7.16–7.02(m,2H),6.94(s,1H),2.63(s,2H),2.48(s,2H),1.10(s,6H).13C-NMR(100MHz,CDCl3)δ168.9,152.4,147.9,142.0,133.8,133.3,128.0,125.7,124.4,113.1,112.4,80.8,43.0,39.2,32.2,28.1.HRMS(m/z)Anal.Calc’d for C19H17N3O2 M+:319.1315,Found M+:319.1309.
本实施例的合成路线如下:
实施例10
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与164.2mg对甲酰基苯甲酸甲酯和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ8.05(d,J=8.3Hz,2H),7.57(d,J=8.1Hz,2H),7.07(s,2H),6.89(s,1H),3.93(s,3H),2.62(s,2H),2.48(s,2H),1.09(s,6H).13C-NMR(100MHz,CDCl3)δ169.1,166.6,153.1,1340.0,135.6,131.5,130.8,130.3,127.4,124.8,113.3,112.6,79.9,52.4,43.1,39.3,32.2,28.1.HRMS(m/z)Anal.Calc’d for C21H21N2O2(M+H)+:333.1598,Found(M+H)+:333.1578.
本实施例的合成路线如下:
实施例11
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与106.1mg苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.51(m,2H),7.44–7.30(m,3H),7.11–6.94(m,2H),6.85(s,1H),2.61(s,2H),2.48(s,2H),1.09(s,6H).HRMS(m/z)Anal.Calc’d for C19H19N2(M+H)+:275.1543,Found(M+H)+:275.1546.
本实施例的合成路线如下:
实施例12
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与149.8mg 4-甲氧基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,DMSO-d6)δ7.67(m,2H),7.28(s,2H),6.98(m,2H),6.84(s,1H),3.80(s,3H),2.61(s,2H),2.54(s,2H),1.02(s,6H).HRMS(m/z)Anal.Calc’d for C20H21N2O(M+H)+:305.1648,Found(M+H)+:305.1659.
本实施例的合成路线如下:
实施例13
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与121.1mg 4-氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.40(m,2H),6.95–6.79(m,2H),6.79–6.64(m,3H),3.03(s,2H),2.39(s,2H),1.03(s,6H).13C-NMR(100MHz,CDCl3)δ164.4,152.9,151.0,135.4,128.9,125.4,124.7,124.2,112.1,40.9,40.2,38.9,31.4,28.4.HRMS(m/z)Anal.Calc’d for C19H20N3(M+H)+:290.1652,Found(M+H)+:290.1657.
本实施例的合成路线如下:
实施例14
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与273.3mg 4-二苯氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.68(s,1H),7.35–7.18(m,7H),7.12–6.95(m,8H),3.98(s,2H),3.50(s,2H),1.69(s,6H).13C-NMR(100MHz,CDCl3)δ181.6,174.5,149.3,146.9,131.3,131.1,129.6,127.1,125.6,125.2,124.3,121.6,50.3,49.7,26.3,25.6,24.2.HRMS(m/z)Anal.Calc’d for C31H27N3K(M+K)+:480.1837,Found(M+K)+:480.1809.
本实施例的合成路线如下:
实施例15
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与164.1mg 4-二甲氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.46(m,2H),7.02(m,1H),6.85(m,3H),6.78(m,1H),3.07(s,6H),2.58(s,2H),2.46(s,2H),1.07(s,6H).HRMS(m/z)Anal.Calc’d for C21H24N3(M+H)+:318.1965,Found(M+H)+:318.1965.
本实施例的合成路线如下:
实施例16
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与177.3mg 4-二乙氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.38(d,J=8.7Hz,2H),7.05–6.74(m,2H),6.73(s,1H),6.65(d,J=8.7Hz,2H),3.41(q,J=7.1Hz,4H),2.55(s,2H),2.44(s,2H),1.20(t,J=7.0Hz,6H),1.06(s,6H).13C-NMR(100MHz,CDCl3)δ169.3,155.6,149.3,138.5,129.9,123.8,122.9,121.2,111.7,44.6,43.1,39.4,32.1,28.2,12.8.HRMS(m/z)Anal.Calc’d for C23H28N3(M+H)+:346.2278,Found(M+H)+:346.2287.
本实施例的合成路线如下:
实施例17
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与201.3mg 4-二甲氨基-1-萘醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.03(s,2H),6.93(d,J=15.8Hz,1H),6.79(d,J=15.9Hz,1H),6.74(s,1H),3.29(t,J=5.7Hz,4H),2.80(t,J=6.6Hz,4H),2.57(s,2H),2.43(s,2H),2.09(m,4H),1.06(s,6H).13C-NMR(100MHz,CDCl3)δ169.1,155.8,144.9,138.9,127.4,123.3,122.7,121.4,120.8,114.7,74.6,50.1,43.1,39.4,32.1,28.2,27.8,21.7.HRMS(m/z)Anal.Calc’d for C22H28N3(M+H)+:370.2278,Found(M+H)+:370.2277.
本实施例的合成路线如下:
实施例18
(1)将0.45mL异氟尔酮、447.6mg 4-二甲氨基苯甲醛和240mg氢氧化钠在15mL乙醇溶液中40℃搅拌12h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.40(d,J=8.7Hz,2H),6.97–6.70(m,2H),6.68(d,J=8.6Hz,2H),6.01(s,1H),3.01(s,6H),2.47(s,2H),2.30(s,2H),1.10(s,6H).13C-NMR(100MHz,CDCl3)δ200.3,156.1,151.1,135.7,128.8,125.2,124.9,124.11,112.2,51.6,40.3,39.2,33.4,28.7.HRMS(m/z)Anal.Calc’d for C18H23NOK(M+K)+:308.1411,Found(M+K)+:308.1407.
本实施例的合成路线如下:
实施例19
(1)将3.5mL四氯化钛加入40mL四氢呋喃溶液中,并冷却至0℃。然后将混有2.2mL异氟尔酮和2.3mL丙二酸二乙酯的10mL四氢呋喃溶液缓慢滴加至上述冷却溶液中,滴加完毕后,再缓慢加入6mL吡啶,室温搅拌12h。反应结束后,加入乙酸乙酯萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,得黄色油状物。
(2)取上述所得黄色油状物560.8mg,与298.4mg 4-二甲氨基苯甲醛和催化量哌啶于10mL N,N-二甲基甲酰胺中加热至100℃搅拌过夜。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.32–7.23(m,2H),6.92(s,1H),6.68(m,2H),6.62–6.55(m,2H),4.19(m,4H),2.90(s,6H),2.47–2.39(m,2H),2.23(s,2H),1.24(q,J=6.1Hz,6H),0.95(s,6H).13C-NMR(100MHz,CDCl3)δ151.7,150.4,147.3,132.1,128.1,126.7,124.8,123.4,120.3,112.2,60.7,60.6,41.5,40.2,38.8,31.0,28.4,14.2,14.1.HRMS(m/z)Anal.Calc’d for C25H33NO4K(M+K)+:450.2041,Found(M+K)+:450.2042.
本实施例的合成路线如下:
实施例20
(1)将3.0mL异氟尔酮、2.3mL氰乙酸乙酯、231mg醋酸铵和343μL醋酸加入80mL甲苯中,加热至90℃回流脱水搅拌24h。反应结束后冷却至室温,加入乙酸乙酯萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,得黄色油状物。
(2)取上述所得黄色油状物466.3mg,与298.4mg 4-二甲氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌过夜。反应结束后冷却至室温,有红色固体析出,抽滤,无水乙醇洗涤,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.40(d,J=8.5Hz,2H),6.98–6.81(m,3H),6.69(d,J=8.5Hz,2H),4.26(q,J=7.1Hz,2H),3.03(s,6H),2.98(s,2H),2.40(s,2H),1.35(t,J=7.1Hz,3H),1.04(s,6H).13C-NMR(100MHz,CDCl3)δ166.2,152.7,150.2,134.9,128.1,124.5,123.7,123.2,111.2,96.7,60.3,40.1,39.3,38.0,30.5,27.5,13.3.HRMS(m/z)Anal.Calc’d for C23H29N2O2(M+H)+:365.2224,Found(M+H)+:365.2233.
本实施例的合成路线如下:
实施例21
(1)将1.5mL异氟尔酮、2.8g氰乙酰胺、2.3g醋酸铵和1.9mL醋酸加入20mL甲苯中,加热至60℃搅拌24h。反应结束后冷却至室温,加入乙酸乙酯萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,得灰白色固体。
(2)取上述所得黄色油状物612.8mg,与447.6mg 4-二甲氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌过夜。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.40(m,2H),6.95–6.79(m,2H),6.73(m,2H),6.68(s,1H),3.03(s,8H),2.39(s,2H),1.03(s,6H).13C-NMR(100MHz,CDCl3)δ163.9,150.1,134.4,128.8,125.1,122.8,112.0,40.8,39.8,39.5,38.1,30.9,27.9.HRMS(m/z)Anal.Calc’d for C23H28O2(M+H)+:336.2070,Found(M+H)+:336.2088.
本实施例的合成路线如下:
实施例22
(1)将4.6mL四氯化钛加入50mL四氢呋喃溶液中,并冷却至0℃。然后将混有3.0mL异氟尔酮和2.9g米氏酸的10mL四氢呋喃溶液缓慢滴加至上述冷却溶液中,滴加完毕后,再缓慢加入8mL吡啶,室温搅拌12h。反应结束后,有黄色固体析出,抽滤,四氢呋喃洗涤,得黄色固体。
(2)取上述所得黄色固体396.5mg,与223.8mg 4-二甲氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌过夜。反应结束后冷却至室温,加入乙酸乙酯萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.91(s,1H),7.42(d,J=8.7Hz,2H),7.06–6.87(m,2H),6.69(d,J=8.7Hz,2H),3.04(s,6H),2.98(s,2H),2.46(s,2H),2.17(s,2H),1.73(s,6H),1.05(s,6H).13C-NMR(100MHz,CDCl3)δ168.0,162.8,157.2,151.4,144.5,137.1,130.2,129.4,126.4,125.0,124.2,122.6,112.2,112.0,108.4,103.0,43.8,40.3,40.3,39.2,32.0,29.8,28.5,27.1.HRMS(m/z)Anal.Calc’d forC24H30NO4(M+H)+:396.2169,Found(M+H)+:396.2158.
本实施例的合成路线如下:
实施例23
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中90℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与179.2mg 4-(N-甲基-N-羟乙基)氨基苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.38(d,J=8.4Hz,2H),7.00–6.74(m,4H),6.71(s,1H),3.81(t,J=5.5Hz,2H),3.52(t,J=5.5Hz,2H),3.04(s,2H),2.51(s,2H),2.39(s,2H),1.19(s,3H),1.00(s,6H).13C-NMR(100MHz,CDCl3)δ169.4,155.4,151.0,138.1,129.6,124.5,124.0,121.5,114.3,113.5,112.3,75.8,60.2,54.7,43.1,39.3,39.1,32.1,28.1.HRMS(m/z)Anal.Calc’d for C24H28O2(M+H)+:348.2070,Found(M+H)+:348.2078.
本实施例的合成路线如下:
实施例24
(1)将6.0mL异氟尔酮、2.9g丙二腈和0.5mL的哌啶在40mL乙醇溶液中100℃下加热回流搅拌12h。反应结束后冷却至室温,将反应液倾入水中有固体析出,过滤。抽滤所得固体用乙醇重结晶,得2.1g棕色固体。
(2)取上述所得棕色固体186.3mg,与313.8mg 4-(N,N-双(2-羟乙基)氨基)苯甲醛和催化量哌啶于10mL乙醇中加热至85℃搅拌20h。反应结束后冷却至室温,加入二氯甲烷萃取,收集有机相成分浓缩,最后利用柱色谱层析法将其纯化,即得最终产物。
将最终产物使用核磁共振波谱仪进行测试,液相色谱高分辨飞行时间质谱Q-TOF6540进行结构表征和纯度测定,结果如下:1H-NMR(400MHz,CDCl3)δ7.42(m,2H),7.00(m,1H),6.88–6.74(m,3H),3.92(t,J=4.9Hz,4H),3.69(s,4H),2.58(s,2H),2.45(s,2H),1.07(s,6H).HRMS(m/z)Anal.Calc’d for C23H28N3O2(M+H)+:378.2716,Found(M+H)+:378.2175
本实施例的合成路线如下:
实施例25
荧光探针在缓冲液中通过荧光激活探测重组蛋白质聚集态的方法
对实施例1至实施例24中制备得到的仿生荧光探针进行在缓冲液中通过荧光激活探测重组蛋白质聚集态测试,具体操作步骤为:取实施例中制备的仿生荧光探针(25μM),与重组纯化的二氢叶酸还原酶(DHFR)(50μM)在酸性诱导聚集缓冲液(NaOAc 200mM,KCl100mM,通过冰醋酸酸化至pH=6.23)中37℃下进行孵育(5min)处理。引发蛋白质聚集的方法为加热在64℃下5min。蛋白质发生聚集后,利用荧光酶标仪TecanSpark进行定量的荧光强度测量,考察荧光激活强度。
以实施例15为典型代表,图1(a)为使用685nm作为发射波长,采集400-670nm的激发信号,图1(b)为使用537nm作为激发波长,采集552-850nm的发射信号。如图可看出,荧光分子在DHFR蛋白质未发生错误折叠与聚集时不发出荧光。当DHFR因为热导致聚集时,荧光分子与聚集态DHFR结合并发出强烈荧光,荧光增益为28倍。最大激发为548nm,最大发射为666nm。
实施例26
荧光探针在缓冲液中通过热转移荧光曲线测量药物分子与目标靶点蛋白的结合
对实施例1至实施例24中制备得到的仿生荧光探针进行通过热转移荧光曲线测量药物分子与目标靶点蛋白的结合,具体操作步骤为:取实施例中制备得到的荧光探针(1nM-100μM),与重组纯化的二氢叶酸还原酶(DHFR)(1μM-1000μM)在37℃下进行孵育(5min-30min)处理。通过温度梯度加热引发蛋白质聚集。加热的温度梯度方法为:(1)利用PCR或恒温加热装置在37℃~95℃之间每隔1℃~10℃中任意数值温度间隔,进行样本加热,然后分别读取荧光强度,绘制温度荧光曲线;(2)利用实时荧光PCR仪,从37℃到95℃每分钟1℃~10℃中任意数值温度递增,并实时记录荧光强度,绘制实时荧光曲线。通过比较导致蛋白聚集临界温度的变化,考察加入药物分子是否能够引起曲线迁移,推测分子是否与目标靶蛋白结合。如果分子结合目标靶点蛋白,即证明小分子作用于蛋白质,可用于筛选目标蛋白的小分子抑制剂或者激活剂等具有药物活性的小分子。
以实施例15为典型代表,结果如图2所示,由图2可看出,荧光分子在DHFR加热引发聚集时会发出荧光。加热温度上升,引发的聚集程度加剧,因此荧光强度逐步上升。当DHFR蛋白质结合甲氧苄啶TMP小分子时,稳定性上升,因此需要更高的温度引发其发生聚集并发出荧光。横轴为开尔文温度,纵轴为相对荧光强度。证明了TMP结合DHFR并稳定DHFR蛋白,TMP分子是一个已知的DHFR抑制剂,是常见的抗生素。因此用TMP与DHFR的强结合力,验证我们的方法可用于筛选其他蛋白质的抑制剂。
实施例27
荧光探针在活细胞中通过荧光成像探测药物导致蛋白质聚集态的方法
对实施例1至实施例24中制备得到的仿生荧光探针进行在活细胞中通过荧光成像探测药物导致蛋白质聚集态,具体操作步骤为:取实施例中制备的仿生荧光探针(1nM-50μM),放入人胚胎肾细胞293细胞系(HEK293)或人宫颈癌细胞系(HeLa)中加入热休克蛋白90抑制剂17AAG(1nM-50μM)、蛋白酶体抑制剂MG132(1nM-50μM)或者剪切因子抑制剂Pladienolide B(1nM-50μM)。12小时至72小时后,通过各自荧光显微镜识别聚集态蛋白质在活细胞中聚集的形貌和位置。
以实施例23为典型代表,结果如图3-8所示,图3为在人胚胎肾细胞293细胞系(HEK293)中加入热休克蛋白90抑制剂17AAG,图4为在人胚胎肾细胞293细胞系(HEK293)中加入蛋白酶体抑制剂MG132,图5为在人胚胎肾细胞293细胞系(HEK293)中加入剪切因子抑制剂Pladienolide B,图6为在人宫颈癌细胞系(HeLa)中加入热休克蛋白90抑制剂17AAG,图7为在人宫颈癌细胞系(HeLa)中加入蛋白酶体抑制剂MG132,图8为在人宫颈癌细胞系(HeLa)中加入剪切因子抑制剂Pladienolide B,通过本申请提供的荧光探针广谱识别聚集态蛋白质在活细胞中聚集的位置。最大激发为543nm,最大发射为610nm。
其中,各图图(a)说明抑制剂诱导蛋白沉淀,荧光探针分子在共聚焦显微镜下可以识别蛋白质聚集态;图(b)是将荧光图片与明场图片叠加,以展示蛋白质聚集态位置分布,聚集态蛋白质分布在细胞核或细胞质周围。
以上所述,仅是本申请的几个实施例,并非对本申请做任何形式的限制,虽然本申请以较佳实施例揭示如上,然而并非用以限制本申请,任何熟悉本专业的技术人员,在不脱离本申请技术方案的范围内,利用上述揭示的技术内容做出些许的变动或修饰均等同于等效实施案例,均属于技术方案范围内。
Claims (6)
1.一种荧光化合物在制备荧光探针中的应用,其特征在于,
所述荧光化合物选自具有以下化学式所示的化合物中的至少一种;
;
所述荧光探针包括在缓冲液中探测重组蛋白质聚集态中的应用、在活细胞内荧光成像检测中的应用、在筛选具有药物活性的小分子中的应用;
其中,所述在活细胞内荧光成像检测中的应用至少包括以下步骤:
向活细胞中加入1 nmol/L ~1 mmol/L的蛋白质聚集引发剂,同时引入0.1μmol/L ~25μmol/L聚集诱导荧光探针在37 ℃孵化箱内原位孵育24 h;待蛋白质发生聚集后,直接进行荧光成像,观测活细胞内的蛋白质聚集态的形貌和胞内位置;
所述蛋白质聚集引发剂选自坦螺旋霉素、氯喹、衣霉素、MG-132、普拉二烯内酯B、环己酰亚胺、亚砷酸钠中的至少一种;
其中,所述在筛选具有药物活性的小分子中的应用具体步骤包括:
向1μmol/L~1000μmol/L重组纯化蛋白中加入1nmol/L~100μmol/L荧光探针分子和10μmol/L-100μmol/L具有药物活性的小分子,在25 ~37/>下进行5 min~30 min孵育处理,通过温度梯度加热引发蛋白质聚集;加热的温度梯度方法为:(1)利用PCR或恒温加热装置在37 oC~95 oC之间每隔1/> ~10/>中任意数值温度间隔,进行样本加热,然后分别读取荧光强度,绘制温度荧光曲线;(2)利用实时荧光PCR仪,从37/>C到95/>每分钟1/>~10/>中任意数值温度递增,并实时记录荧光强度,绘制实时荧光曲线,可实现具有药物活性的小分子的筛选;
所述具有药物活性的小分子选自蛋白质的抑制剂或者激活剂。
2.根据权利要求1所述的应用,其特征在于,
所述荧光化合物通过如下步骤制备:
步骤(1):向化合物II中加入化合物III和溶剂I,在碱性或酸性条件下,反应I,得到中间产物I;
中间产物I
所述化合物II选自具有式II所示化学式的化合物:
式II;
所述化合物III选自丙二腈、丙二酸二乙酯、氰乙酸乙酯、氰乙酰胺、米氏酸中的至少一种;
步骤(2):将中间产物I与化合物IV和溶剂II混合,在碱性条件下,反应II,得到化合物I;
所述化合物IV选自具有式III所示化学式的化合物中的至少一种:
式III;
所述化合物I具有式I所示的结构:
式I;
式I中,Ar、R1、R2、R3取代基团与权利要求1中各化合物上的取代基团一一对应。
3.根据权利要求2所述的应用,其特征在于,
所述碱性条件下为含有碱性物质的反应环境;所述碱性物质选自哌啶、四氢吡咯、吗啉、哌嗪、吡啶、氢氧化钠中的至少一种;所述碱性物质与化合物II的摩尔比为0.8~8:1;
所述酸性条件下为含有酸性物质的反应环境;所述酸性物质选四氯化钛、醋酸铵、醋酸中的至少一种;所述酸性物质与化合物II的摩尔比为1.5~6:1;
在所述步骤(1)中,反应I的条件为:
加热回流搅拌,温度为40~100℃;时间为12~30h;
所述化合物II和化合物III的摩尔比为1:0~2;
所述溶剂I选自甲醇、乙醇、四氢呋喃、甲苯、二氯甲烷中的至少一种;
在所述步骤(2)中,反应II的条件为:温度为80~100℃;时间为12~24h;
所述中间产物I和化合物IV的摩尔比为1:1~2;
所述溶剂II选自甲醇、乙醇、四氢呋喃、甲苯中的至少一种。
4.根据权利要求1所述的应用,其特征在于,所述在缓冲液中探测重组蛋白质聚集态的应用至少包括以下步骤:
向缓冲液中加入0.1μmol/L~100μmol/L蛋白待测样本和5μmol/L~50μmol/L所述荧光探针,在25℃~37℃下进行孵育处理;孵育后,样本以间隔2 oC, 进行37℃~95℃温度梯度加热5 min引发蛋白质聚集后进行实时荧光定量跟踪测量;
向缓冲液中加入0.1μmol/L~100μmol/L具有药物活性的小分子,将所测得的荧光曲线与所加入的具有药物活性的小分子的荧光曲线进行对比,可检测具有药物活性的小分子是否与目标蛋白发生结合,从而实现对目标蛋白的稳定作用。
5.根据权利要求4所述的应用,其特征在于,
其中:所述缓冲液为缓冲物质的水溶液;
所述缓冲液的pH=4.0~8.5;
所述缓冲物质选自磷酸钠盐、磷酸钾盐、氨基丁三醇、氯化钠盐、氯化钾盐、乙酸钠中的至少一种;
所述磷酸钠盐、磷酸钾盐的浓度为1 mmol/L ~100 mmol/L;
所述氨基丁三醇的浓度为1 mmol/L ~100 mmol/L;
所述氯化钠盐、氯化钾盐的浓度为1 mmol/L ~500 mmol/L;
所述乙酸钠的浓度为1 mmol/L ~500 mmol/L。
6.根据权利要求1所述的应用,其特征在于,所述蛋白质的激活剂或抑制剂选自甲氧苄啶、培美曲塞、二氟尼柳、乙胺嘧啶、Tafamidis中的至少一种。
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