CN109293673A - 一类脯氨酰羟化酶小分子荧光探针及其制备方法 - Google Patents
一类脯氨酰羟化酶小分子荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明涉及化学生物学领域。具体涉及一类基于脯氨酰羟化酶抑制剂设计的小分子荧光探针(I),该类探针的结构模块包括:脯氨酰羟化酶结合基团、连接链和荧光报告基团。本发明还公开了该类探针的其制备方法。本发明的脯氨酰羟化酶小分子荧光探针可用于脯氨酰羟化酶抑制剂的高通量筛选,指导脯氨酰羟化酶抑制剂的发现及结构优化。以此类结构的探针作为工具分子,可以快速、准确的确证小分子与靶标蛋白的结合情况。
Description
技术领域
本发明涉及化学生物学领域。具体涉及一类基于脯氨酰羟化酶抑制剂设计的小分子荧光探针,其制备方法,以及在测定化合物对脯氨酰羟化酶的抑制活性中的应用,可用于脯氨酰羟化酶抑制剂的高通量筛选,同时可指导脯氨酰羟化酶抑制剂的发现及结构优化,适应于指导发现治疗贫血症、缺血性疾病和肾损伤等与脯氨酰羟化酶密切相关疾病的小分子药物。
背景技术
肾性贫血作为慢性肾病患者最常见的并发症之一,严重降低慢性肾病患者的生活质量,还会导致心血管疾病发病率及死亡率升高。肾性贫血的主要诱因是缺氧诱导因子(HIF)表达下降,导致肾脏产生和分泌促红细胞生成素(Erythropoietin,EPO)不足。EPO的表达受缺氧诱导因子(HIF调控),而脯氨酰羟化酶(ProlylHydroxylase,PHD)又是HIF的关键负调控因子。因此,抑制PHD酶的生理功能,抑制PHD酶对HIF-α的羟基化作用,从而使得HIF-α逃脱被降解的命运,从而可内源性上调EPO等蛋白,有望治疗包括肾性贫血在内的缺血性疾病。近年来,PHD抑制剂是治疗肾性贫血最有前景的手段(MedChemComm 2016,7,1271-1284)。近十多年科学家们一直致力于开发一种可以治疗贫血的PHD小分子抑制剂。到目前为止,处于临床III期研究中的PHD抑制剂包括FG-4592(vadadustat)、AKB-6548(vadadustat)和GSK1278863(daprodustat)。体外抑制活性测试方法以及高通量筛选方法对PHD小分子抑制剂的发展至关重要。准确、稳定、可靠的活性测试方法有助于发现结构新颖的先导化合物,指导先导化合物的结构优化以获得活性更优的候选药物分子,为靶向PHD的肾性贫血等缺血性疾病的治疗药物的开发奠定基础。
目前已有的PHD活性测定方法主要包含两大类。一类是基于PHD酶催化活性的测试方法。这类方法主要原理是,PHD酶在辅因子Fe2+及2-酮戊二酸(2-OG)条件下,可羟基化HIF肽段底物,生成羟基化的HIF肽段产物,同时消耗辅因子2-OG。因此,通过检测羟基化HIF肽段产物的相对含量,或者通过检测辅因子2-OG相对消耗量,即可知道PHD酶催化反应的进程情况。那么加入化合物后,通过羟基化HIF肽段产物相对含量或辅因子2-OG相对含量的变化,即可知道PHD酶催化反应被抑制的程度,即可得到待测化合物对PHD酶的抑制率。按照对羟基化HIF肽段及2-OG检测手段的不同,又包括AlphaScreen法(Journal of MedicinalChemistry 2012,55,2945-2959.),MALDI-TOF质谱法(Journal of Medicinal Chemistry2009,52,2799–2805)以及邻苯二胺法(AnalyticalBiochemistry 2005,336,125-131.)。这些方法都是基于PHD酶的催化反应,因此检测得到的抑制率受反应时间影响较大。由于要保证酶的催化活性,需要保证Fe2+在体系中不被氧化,因此往往还需要在测试体系中加入大量的抗氧化剂,而且往往需要使用较大量的酶。此外,AlphaScreen法检测羟基化的HIF肽段,还需要额外加入抗体、beads doner及acceptor,整个检测体系非常复杂;MALDI-TOF质谱法需要用昂贵的质谱仪器;邻苯二胺法需要依赖于邻苯二胺与2-OG进一步发生化学反应转变为荧光物质从而检测2-OG的消耗量。这些方法由于影响测试稳定性的因素过多,测试稳定性往往会受到影响,缺乏灵敏性和容易出现假阳性,不太适用于高通量筛选。
另一类是基于PHD酶亲和力的测试方法。目前主要包括核磁共振法(Journal ofMedicinal Chemistry 2013,56,547-555)和HIF肽段荧光探针的荧光偏振法(ACSMedicinal Chemistry Letters 2015,6,1236-1240)。核磁共振法(Journal of MedicinalChemistry 2013,56,547-555),采用13C标记的辅因子2-OG与PHD酶共孵育,辅因子可进入PHD酶催化口袋与之结合。而加入抑制剂分子后,2-OG可被竞争性地挤出PHD酶结合口袋,呈游离状态。在核磁共振下,2-OG与PHD结合状态及游离状态的信号值是不同的,从而可计算获得结合状态与游离状态的比例,进而获得待测化合物对PHD酶的抑制率。然而,该方法需要依赖昂贵的核磁共振仪器,而且在核磁管中检测往往只能单通道进行,操作繁琐耗时长,不适用于高通量筛选。HIF肽段荧光探针的荧光偏振法(ACS Medicinal ChemistryLetters 2015,6,1236-1240)是我们在2015年开发的方法。其原理是,采用HIF肽段作为与PHD酶结合的亲和力基团,在HIF肽段末端标记上FITC荧光基团,利用荧光偏振技术,当HIF荧光肽段与PHD酶结合时,荧光偏振信号增强,而HIF荧光肽段与PHD酶分离呈游离状态时,荧光偏振信号减弱。因此,当在HIF荧光肽段及PHD酶体系中加入待测化合物后,通过检测荧光偏振信号值即可获得化合物对PHD酶的抑制剂率。该方法测试体系克服了前述方法无法高通量进行的问题,可用于PHD抑制剂的高通量筛选。但是该方法还是存在一些缺陷,比如该体系依赖于2-OG,因为没有2-OG,HIF荧光肽段则与PHD酶的结合力大幅下降;此外,体系中的内源性辅因子Fe2+被替换为了Mn2+,以避免HIF荧光肽段被羟基化丧失对PHD酶的结合力。因此,该测试体系依然较为复杂,而且采用Mn2+并不能真正反映出化合物与内源性含Fe2 +的PHD酶的结合情况。
如今,运用小分子荧光探针作为辅助工具,多种小分子与生物大分子的作用模式被确定,这些信息对合理药物设计具有十分重要的意义。比如新的药物靶点的发现,或者对一个已知蛋白的功能做新的阐述,这对于分子水平阐明疾病的发生、发展和治疗尤其重要。以小分子荧光探针作为辅助工具,这些靶点可被发展为高效的高通量筛选模型,并在短时间内随机筛选大量的活性化合物,发现活性更高的小分子化合物作为先导物用于进一步的药物研发。
发明内容
为了突破目前PHD酶抑制活性测试方法的局限性,采用我们报道的三氮唑吡啶类PHD抑制剂(Journal Medincal Chemistry 2018,61,5332-5349)作为PHD酶的结合片段,通过进一步引入荧光基团,得到一类基于PHD酶抑制剂设计的小分子荧光探针,基于本发明小分子荧光探针建立的活性测定方法,测试体系简单,保留内源性Fe2+,无需加入其它辅因子,受时间、溶剂影响小,可用于脯氨酰羟化酶抑制剂的高通量筛选,同时可指导脯氨酰羟化酶抑制剂的发现及结构优化,适应于指导发现治疗贫血症、缺血性疾病和肾损伤等与脯氨酰羟化酶密切相关疾病的小分子药物。
本发明的探针分子包括三个功能部位:脯氨酰羟化酶抑制剂A、连接链L和荧光报告基团B,具体结构如下:
其中荧光报告基团包括但不仅限于罗丹明衍生物类荧光片段,优选异硫氰酸荧光素(FITC)。
本发明的L优选4-10个原子长度的连接链,该连接链主链原子选自碳、氮或氧原子,
L可连接在X的邻位或间位。
X代表卤素原子。优选氯原子。
n优选1或2。
L优选:
本发明优选下列部分化合物:
本发明的小分子荧光探针可用下列方法制备:
其中X、L、n的定义同前。
下面是本发明部分化合物的生物学实验及结果如下:
半数效应浓度(EC50)检测;
探针分子要能应用于PHD酶的活性检测,首先需要保证探针分子与PHD能稳定结合,具有较好亲和力。我们通过荧光偏振法(affinity-based fluorescence polarizationassay,FP assay)来检测荧光探针对于PHD的结合亲和力,用半数效应浓度(EC50)来表征其亲和力强弱。PHD酶包含3中亚型,分别为PHD1、PHD2、PHD3,其中PHD2亚型为体内分别最为广泛的亚型,也是治疗肾性贫血等缺血性疾病的主要靶向亚型。我们首先考察了实施例探针分子对PHD2酶的亲和力。将PHD2蛋白40nM,荧光探针初浓度100μM三倍稀释,每个探针设置2个复孔,11个浓度梯度,于384孔黑板(#3575,Corning)中4℃共孵育1h。384孔黑板用多功能酶标仪(SpectraMaxiD)进行数据读取,以溶剂为对照。测试结果表明此类荧光探针的活性都保持在nM水平,如表1所示。因此,本发明中的探针分子均具有较好的PHD2酶结合能力。
表1本发明部分化合物的半数效应浓度
本发明以Ⅰ-3探针为例说明EC50计算过程。通过酶标仪读取的荧光偏振数值和对应的Ⅰ-3探针的浓度如表2所示,阴性对照代表只加40nMPHD2蛋白。当探针和蛋白结合时,荧光偏振值(mp)变小。△mp=阴性对照mp-均值mp,导入数据于Graphpad prism 7.0得出Ⅰ-3探针EC50。
表2不同探针浓度下的探针分子与PHD2蛋白结合的荧光偏振数值
将代表性实施例探针分子分别与PHD酶另外两种亚型(PHD1和PHD3)共孵育,具体操作和探针EC50的计算方式同前述一样,得到探针分子与PHD1和PHD3的结合情况,其EC50如表3所示:
表3荧光探针对PHD1、PHD3酶结合的EC50值
由表1和表3可见,本发明的荧光探针对PHD酶三种亚型都具有较强的结合能力,所以本发明的荧光探针可以用于PHD各亚型的抑制活性测定。其中荧光探针Ⅰ-3对PHD2活性较优,EC50值为35.2±0.11nM。
以探针Ⅰ-3为工具分子,说明基于荧光偏振建立PHD小分子抑制剂高通量筛选方法:
探针Ⅰ-3最适浓度的确定;
以PBS为溶媒,设定探针Ⅰ-3浓度范围为100μM-10nM,当探针浓度越高时,荧光偏振值越低,当荧光偏振值达到下平台的临界值时对应的浓度就是探针的最适浓度。通过荧光偏振仪读取数据,实验数据于Graphpad prism 7.0分析,表明探针Ⅰ-3偏振光的优选浓度为20-200nM。
探针Ⅰ-3和PHD蛋白作用最适缓冲体系;
接着对缓冲体系PBS进一步研究。探针Ⅰ-3浓度为30nM,PHD1-3初始浓度25μM三倍稀释,十个浓度梯度。将探针Ⅰ-3分别和PHD1-3蛋白于384黑板一起孵育1h,用多功能酶标仪进行数据读取,以PBS的孔为空白对照,只加30nM探针Ⅰ-3的孔为阴性对照。△mp=测试值mp-阴性mp,将△mp数值和蛋白浓度数值导入Graphpad prism 7.0分析。结果表明探针Ⅰ-3在不加外源性金属螯合离子和内源性配体2-OG时,就可得到理想的响应值,探针Ⅰ-3与PHD2酶的结合亲和力曲线如图1所示。
图1的实验中无需额外加入金属离子以及辅因子2-OG。经过Graphpad prism 7.0分析可以得出荧光探针Ⅰ-3对PHD2的EC50,说明探针Ⅰ-3对PHD2酶具有较好的亲和力及预期结合效果,且曲线拟合很好(R2≥99.0%),表明实验结果真实可信。
建立的高通量筛选方案测试已报道阳性药的对脯氨酰羟化酶的抑制IC50值;
以PBS为缓冲体系,探针Ⅰ-3浓度为30nM,PHD2蛋白浓度40nM,将阳性药FG-4592和FG-2216初始浓度100μM,三倍稀释,十个浓度梯度,两个复孔,分别与PHD2蛋白和探针Ⅰ-3于384孔板4℃孵育2h,酶标仪读取荧光偏振值,以只加探针缓冲液为空白对照,探针和PHD2蛋白为阴性对照。
以FG-4592为例说明此高通量方法化合物IC50的计算过程,酶标仪读取两个复孔的荧光偏振值如表4所示,通过公式%抑制率=100*(1-(实测值-空白)/(阴性值-空白)),得出具体浓度所对应的抑制率。将所得数据导入Graphpad prism 7.0分析拟合得IC50值(图2)。
由图2可见,经过Graphpad prism 7.0分析可以得出FG-4592对PHD2的IC50,且曲线拟合很好(R2≥99.0%),FG-4592活性测试也体现出了预期的效果,且与已报道的活性数据相近,表明实验结果真实可信。
表4不同浓度下的化合物FG-4592在高通量筛选方案中的荧光偏振值及抑制率
浓度(μM) | 复孔1 | 复孔2 | 均值mp | 抑制率% |
100 | 299.96 | 292.98 | 296.47 | 108.2 |
33.33 | 316.70 | 319.12 | 317.91 | 99.8 |
11.11 | 353.14 | 362.50 | 357.82 | 69.26 |
3.70 | 380.76 | 350.31 | 365.53 | 63.49 |
1.23 | 370.19 | 380.23 | 375.02 | 61.31 |
0.41 | 399.64 | 401.39 | 400.52 | 44.18 |
0.14 | 420.91 | 424.36 | 422.63 | 29.81 |
0.045 | 426.20 | 430.30 | 428.10 | 23.47 |
0.015 | 426.27 | 466.44 | 446.36 | 5.67 |
0.005 | 468.00 | 446.02 | 461.00 | 0.661 |
空白对照 | 300.82 | 299.91 | 299.96 | |
阴性对照 | 465.24 | 464.87 | 465.06 |
同上述IC50计算过程,得FG-2216的IC50值。IC50测试结果如表5所示:
表5采用本发明方法测定的IC50值以及文献报道方法测定的IC50值
阳性药IC50测试结果与已报道得活性数据相近,且与已报道IC50值呈一定得相关性,确证了此高通量筛选的准确性和可靠性。
高通量筛选方法的稳定性和溶剂耐受性考察:
DMSO耐受性;
二甲基亚砜(DMSO)是一种常用的溶剂,多用于测试化合物的溶解。因此,基于探针Ⅰ-3和PHD2蛋白作用的荧光偏振测定应该能够耐受低体积的DMSO。在进行探针Ⅰ-3和PHD2蛋白结合试验时,结合体系中最大含有1.00%DMSO(v/v)。基于此,进一步测试了1%-30%(v/v)的DMSO对竞争结合实验的影响。实验结果表明,当DMSO(v/v)含量增加至30%时,荧光偏振测定的探针和蛋白结合亲和力依然相当稳定。MSO(v/v)占比为0%、1%、2%、3%、4%、6%、8%、10%、16%和30%时,用GraphPad Prism 7.0分析,当DMSO(V/V)≤30%,对探针蛋白结合几乎没有影响,表明这个方法溶剂耐受性良好,适用于小分子的高通量筛选。这种高通量筛选方法具有良好的DMSO耐受性。
实验稳定性Z’因子考察;
Z'因子统计实验,根据30次实验中高信号组(探针Ⅰ-3结合PHD2)和低信号组(仅探针Ⅰ-3)的偏振光,并且在不同的时间段重复这个实验2次,分析此高通量筛选方法的稳定性。统计得Z'因子为0.82,确证了该方法有较好的稳定性。
将30次实验荧光偏振值用GraphPad Prism 7.0统计分析,利用公式Z’=1-3(σb-σf)/|μb-μf|,(其中σb和σf分别是高信号组和低信号组的标准偏差),得出Z’因子为0.82,表明此方案可以用于药物的高通量筛选,具有廉价,稳定,快速,高效等优点。
本发明以荧光探针Ⅰ-3作为工具分子,建立一种体外酶水平活性检测方法。同时通过测试已报道的小分子酶亲和活力,验证了此方法的适用性和准确性。至此,基于这一类荧光探针建立一种稳定,快速以及廉价的高通量筛选方法,用于PHD小分子抑制剂的精准筛选,高效准确获得活性较优的先导化合物。
附图说明
图1是荧光探针Ⅰ-3与PHD2孵育后荧光偏振值(mp)与荧光探针Ⅰ-3浓度的关系图
图2是阳性药FG-4592对PHD2靶标抑制率的示意图
图3是荧光探针Ⅰ-3与PHD2酶结合的DMSO溶剂耐受性考察
图4是实验稳定性Z’因子的考察
具体实施方式
实施例1
荧光探针Ⅰ-1的制备
(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯(0.706g,2.0mmol),(5-乙炔基-3-羟基吡啶甲酰基)甘氨酸(0.440g,2.0mmol),四丁基氟化铵(1.0mol/L的THF溶液,1.56g,6.0mmol),碘化亚铜(0.023g,0.12mmol)和N,N-二异丙基乙胺(1.0mL)溶于MeOH(8mL)中,在N2保护下将混合物在80℃搅拌5.5小时,过滤反应混合物并浓缩。通过硅胶柱色谱(洗脱液:在石油醚中30-50%EtOAc)纯化产物,得到白色固体(5-(1-(2-(2-((3-((叔丁氧基羰基)氨基)丙基)氨基)-4-氟苯氧基)乙基)-1H-1,2,3-三唑-4-基)-3-羟基吡啶甲酰基)甘氨酸0.620g;将所得中间体全部溶于无水CH2Cl2(5mL)中,TFA(1mL)逐滴加入至反应液中,在室温下搅拌3小时后,将反应混合物真空浓缩。固体混合物溶于水,在冰浴中冷却,用3M的NaOH溶液(5mL)调PH至10左右,并用CH2Cl2(3×5mL)萃取。合并的有机相用饱和NaCl水(5mL)洗涤两次,无水Na2SO4干燥,过滤,真空浓缩,得到目标中间体5-(1-(2-(2-((3-氨基丙基)氨基)-4-氟苯氧基)乙基)-1H-1,2,3-三唑-4-基)-3-羟基吡啶甲酰基)甘氨酸0.52g;将所得中间体全部全部溶于无水DMF(2mL)中,缓慢加入荧光素FITC(389.0mg,1.0mmol)。室温反应5小时后停止反应,将反应混合物滴加到3mL冰水中,有黄色固体析出,过滤并收集固体,得目标产物探针Ⅰ-1 0.641g,三步收率37.1%,Rf:0.11(甲醇:乙酸乙酯=2:5),m.p.156.7-157.2℃,该化合物的1HNMR(500MHz,DMSO-d6)δ9.39(s,1H),8.62(d,J=1.3Hz,1H),8.41(s,1H),8.20(s,1H),7.72(dd,J=7.5,2.0Hz,1H),7.52(d,J=7.3Hz,1H),7.36(d,J=7.5Hz,1H),7.25(dd,J=4.4,3.1Hz,2H),7.00(s,2H),6.75(dd,J=7.6,5.7Hz,1H),6.65–6.55(m,4H),6.54–6.44(m,2H),6.26(dd,J=8.9,2.0Hz,1H),5.22(dd,J=12.4,11.0Hz,1H),4.87(dt,J=12.4,2.0Hz,1H),4.71(dd,J=12.2,11.0Hz,1H),4.55–4.39(m,3H),4.22(td,J=12.4,3.2Hz,1H),4.12(d,J=12.2Hz,1H),3.76–3.62(m,3H),1.95–1.83(m,1H),1.56–1.44(m,1H),13CNMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,153.94,151.93,146.85,146.41,142.66,139.43,137.31,136.03,133.05,125.11,124.76,122.68,122.46,118.46,117.21,114.89,112.07,111.91,111.87,109.68,102.60,102.44,101.08,85.16,65.24,48.40,41.81,41.26,41.16,28.47.,HRMS(ESI):found862.2186(C42H35FN8O10S,[M+H]+,requires 862.2181),HPLC(85:15=甲醇:水):tR=3.314min,97.45%。
实施例2
荧光探针Ⅰ-2的制备
制备方法同实施例1,用(7-((2-(3-叠氮基丙氧基)-5-氯苯基)氨基)庚基)氨基甲酸叔丁酯(0.878g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.524g,三步收率27.6%,Rf:0.11(甲醇:乙酸乙酯=2:5),m.p.159.7-160.1℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.10(s,1H),8.87(d,J=1.3Hz,1H),8.34(s,1H),8.20(s,1H),7.75–7.69(m,2H),7.65(d,J=2.0Hz,1H),7.27–7.19(m,2H),7.14(dd,J=10.2,7.4Hz,2H),7.01(dd,J=7.5,2.0Hz,1H),7.00(s,2H),6.78–6.70(m,2H),6.64–6.57(m,2H),6.50(d,J=2.1Hz,1H),6.34(dd,J=7.5,2.0Hz,1H),4.55(td,J=12.4,1.9Hz,1H),4.40(s,1H),4.23(d,J=12.3Hz,1H),4.14(d,J=12.4Hz,1H),3.91(ddt,J=20.2,12.2,2.9Hz,2H),3.69–3.57(m,1H),3.40(dd,J=12.4,10.9Hz,1H),3.28(td,J=12.0,1.2Hz,1H),2.90(td,J=12.2,4.2Hz,1H),2.29–2.18(m,1H),2.04–1.54(m,6H),1.53–1.43(m,2H),1.44–1.25(m,2H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,153.94,151.93,147.37,146.85,146.41,142.66,139.43,137.94,137.31,136.03,133.05,130.99,125.11,124.76,123.74,122.68,122.46,118.46,117.21,116.15,114.70,111.87,109.68,101.08,85.16,68.55,47.58,44.41,43.19,41.81,30.00,29.80,28.90,28.39,28.26,26.91.,HRMS(ESI):found948.2670(C47H45ClN8O10S,[M+H]+,requires 948.2668),HPLC(85:15=甲醇:水):tR=3.26min,97.88%。
实施例3
荧光探针Ⅰ-3的制备
制备方法同实施例1,用(4-(2-(2-叠氮基乙氧基)-5-碘苯氧基)丁基)氨基甲酸叔丁酯(0.950g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.586g,三步收率29.8%,Rf:0.19(甲醇:乙酸乙酯=1:2),m.p.161.7-162.8℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.59(s,1H),8.99(d,J=1.3Hz,1H),8.57(s,1H),8.20(s,1H),7.72(dd,J=7.5,2.0Hz,1H),7.68–7.61(m,3H),7.42–7.34(m,2H),7.30(s,1H),7.16–7.10(m,1H),7.00(s,2H),6.85(dd,J=19.8,7.5Hz,2H),6.52(dd,J=7.4,1.9Hz,1H),6.38(s,1H),5.09–4.96(m,2H),4.79–4.65(m,2H),4.58(ddt,J=12.5,4.8,1.4Hz,1H),4.36(ddd,J=12.6,11.4,4.0Hz,1H),4.17–4.08(m,2H),4.01–3.89(m,2H),2.32(qt,J=12.4,3.3Hz,1H),2.21–2.10(m,1H),2.06–1.95(m,1H),1.81(dd,J=12.2,8.6,Hz,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,153.94,151.93,150.97,149.24,146.85,146.41,142.66,139.43,137.31,136.03,133.05,130.57,125.11,124.76,124.23,122.68,122.46,118.46,117.63,117.21,111.87,109.68,101.08,85.16,83.52,69.40,65.24,48.40,44.41,41.81,26.85,26.66.,HRMS(ESI):found985.1240(C43H36IN7O11S,[M+H]+,requires985.1238),HPLC(85:15=甲醇:水):tR=3.46min,98.12%。
实施例4
荧光探针Ⅰ-4的制备
制备方法同实施例1,用((2-(3-(2-(2-叠氮基乙氧基)-5-氯苯氧基)丙氧基)乙基)氨基甲酸叔丁酯(0.830g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.416g,三步收率22.4%,Rf:0.20(甲醇:乙酸乙酯=1:2),m.p.154.7-155.8℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.14(s,1H),9.01(d,J=1.1Hz,1H),8.69(s,1H),8.20(s,1H),7.72(dd,J=7.5,2.0Hz,1H),7.63(dd,J=17.9,1.6Hz,2H),7.56(d,J=7.5Hz,1H),7.27–7.17(m,3H),7.02–6.94(m,4H),6.63–6.54(m,2H),6.40(d,J=6.4Hz,2H),4.89(d,J=12.5Hz,1H),4.82(dd,J=12.3,9.6,Hz,1H),4.75–4.64(m,2H),4.58(dd,J=25.4,12.2,Hz,2H),4.32–4.14(m,3H),4.00(dd,J=12.4,2.3Hz,2H),3.69(dtd,J=42.7,12.3,2.4Hz,2H),3.24(ddd,J=12.5,3.6,2.2Hz,1H),2.20(qt,J=12.0,3.6Hz,1H),2.03(dd,J=15.8,12.2,Hz,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,153.94,151.93,150.39,149.80,146.85,146.41,142.66,139.43,137.31,136.03,133.05,128.71,125.11,124.76,122.68,122.46,121.40,118.46,117.21,116.83,116.44,111.87,109.68,101.08,85.16,70.46,68.30,67.24,65.24,48.40,44.22,41.81,29.87.,HRMS(ESI):found923.1991(C44H38ClN7O12S,[M+H]+,requires923.1988),HPLC(80:20=甲醇:水):tR=3.21min,97.51%。
实施例5
荧光探针Ⅰ-5的制备
制备方法同实施例1,用((2-(2-((2-(2-叠氮基乙氧基)-5-氯苯基)氨基)乙氧基)乙基)氨基甲酸叔丁酯(0.798g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.406g,三步收率22.3%,Rf:0.20(甲醇:乙酸乙酯=1:2),m.p.154.9-156.3℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.04(s,1H),8.85(d,J=1.1Hz,1H),8.57(s,1H),8.20(s,1H),7.75–7.63(m,4H),7.26(d,J=7.4Hz,1H),7.19(d,J=7.5Hz,1H),7.01(d,J=13.9Hz,3H),6.79(dd,J=7.5,2.0Hz,1H),6.64–6.55(m,3H),6.51(d,J=7.5Hz,1H),6.40(dd,J=7.5,2.0Hz,1H),4.84–4.71(m,3H),4.61(ddd,J=12.5,3.4,1.2Hz,1H),4.58(s,1H),4.55–4.44(m,2H),4.19(td,J=12.2,2.9Hz,1H),4.04–3.94(m,2H),3.87(ddd,J=12.2,2.3,1.2Hz,1H),3.77(ddd,J=12.2,3.0,0.9Hz,1H),3.72–3.56(m,2H),3.52(td,J=12.1,2.3Hz,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,153.94,151.93,147.37,146.85,146.41,142.66,139.43,137.94,137.31,136.03,133.05,130.99,125.11,124.76,123.74,122.68,122.46,118.46,117.21,116.15,114.70,111.87,109.68,101.08,85.16,70.46,65.24,48.40,44.22,42.44,41.81.,HRMS(ESI):found908.1994(C43H37ClN8O11S,[M+H]+,requires 908.1991),HPLC(80:20=甲醇:水):tR=3.56min,96.41%。
实施例6
荧光探针Ⅰ-6的制备
制备方法同实施例1,用(2-((2-((5-(2-叠氮基乙氧基)-2-氯苯基)氨基)乙基)氨基)乙基)氨基甲酸叔丁酯(0.790g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.394g,三步收率22.0%,Rf:0.19(甲醇:乙酸乙酯=1:2),m.p.155.9-156.2℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.01(d,J=1.3Hz,1H),8.85(s,1H),8.59(s,1H),8.20(s,1H),7.86(d,J=1.3Hz,1H),7.72(dd,J=7.5,2.0Hz,1H),7.55(d,J=7.5Hz,1H),7.23(d,J=7.4Hz,1H),7.13(d,J=7.5Hz,1H),7.07(d,J=7.5Hz,1H),7.00(s,2H),6.71(s,1H),6.65(d,J=1.9Hz,2H),6.53(dd,J=7.4,1.9Hz,1H),6.36(dt,J=4.7,2.3Hz,2H),6.26(dd,J=7.5,2.0Hz,1H),4.97–4.79(m,2H),4.72–4.55(m,4H),4.04–3.94(m,2H),3.65–3.52(m,2H),3.06(dtd,J=13.3,11.9,4.0Hz,2H),2.87(ddd,J=12.5,3.6,1.7Hz,1H),2.76(td,J=12.2,2.5Hz,1H),2.52(td,J=12.0,2.4Hz,1H),1.90(s,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,157.12,153.94,151.93,146.85,146.41,142.66,139.43,137.31,136.03,133.05,130.82,125.11,124.76,122.68,122.46,120.82,118.46,117.21,111.87,110.29,109.68,104.63,101.08,85.16,65.14,49.26,49.21,48.40,42.36,42.14,41.81.,HRMS(ESI):found907.2152(C43H38ClN9O10S,[M+H]+,requires 907.2151),HPLC(80:20=甲醇:水):tR=3.25min,97.11%。
实施例7
荧光探针Ⅰ-7的制备
制备方法同实施例1,用(2-(2-(2-叠氮基乙氧基)-5-氯苯甲酰氨基)乙基)氨基甲酸叔丁酯(0.800g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.352g,三步收率18.9%,Rf:0.20(甲醇:乙酸乙酯=1:2),m.p.157.4-159.1℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.00–8.93(m,2H),8.51(s,1H),8.20(s,1H),8.05(d,J=2.0Hz,1H),7.72(dd,J=7.5,2.0Hz,1H),7.65(t,J=1.9Hz,2H),7.64–7.52(m,2H),7.24(d,J=7.5Hz,1H),7.18(d,J=7.5Hz,1H),7.11(d,J=7.5Hz,1H),7.00(s,2H),6.75(s,1H),6.63–6.51(m,3H),5.28(dt,J=12.1,1.7Hz,1H),5.10–4.94(m,2H),4.90–4.77(m,2H),4.33–4.23(m,2H),4.17(d,J=12.4Hz,1H),3.83(td,J=12.2,3.1Hz,1H),3.28(ddd,J=12.5,3.1,1.4Hz,1H).13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,165.87,158.76,156.58,153.94,151.93,146.85,146.41,142.66,139.43,137.31,136.03,134.67,133.05,129.75,125.11,124.86,124.76,123.74,122.68,122.46,118.46,117.21,116.18,111.87,109.68,101.08,85.16,65.24,48.40,42.28,41.81,39.08.,HRMS(ESI):found892.1680(C42H33ClN8O11S,[M+H]+,requires 892.1678),HPLC(80:20=甲醇:水):tR=3.22min,98.01%。
实施例8
荧光探针Ⅰ-8的制备
制备方法同实施例1,用((6-(2-(2-叠氮基乙氧基)-5-氯苯甲酰氨基)己基)氨基甲酸叔丁酯(0.880g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.365g,三步收率19.2%,Rf:0.18(甲醇:乙酸乙酯=1:2),m.p.165.4-166.9℃,该化合物的1H-NMR(500MHz,DMSO-d6)δ8.81(s,6H),8.76(s,6H),7.93(s,6H),7.66(s,6H),7.59(s,7H),7.55(s,4H),7.31(s,6H),7.13(s,12H),6.94(s,6H),6.79(d,J=5.0Hz,12H),6.72(d,J=2.0Hz,12H),6.49(d,J=5.0Hz,24H),5.77(s,6H),4.84(s,12H),3.67(s,9H),3.60(s,12H),3.30(s,8H),1.58(s,6H),1.48(s,8H),1.33(d,J=45.0Hz,29H),1.25(d,J=2.2Hz,1H),13C-NMR(126MHz,DMSO-d6)δ177.03,173.07,170.38,167.73,161.90,154.33,153.30,148.75,146.35,144.67,143.57,140.90,137.39,134.62,130.61,129.69,127.63,127.19,124.31,123.11,120.10,118.85,115.36,115.12,114.63,114.10,113.05,111.95,100.70,87.83,70.79,44.33,44.17,43.52,29.57,29.50,27.49.,HRMS(ESI):found 906.2191(C44H39ClN8O10S,[M+H]+,requires 906.2198),HPLC(85:15甲醇:水):tR=3.281min,98.78%。
实施例9
荧光探针Ⅰ-9的制备
制备方法同实施例1,用(2-((2-(2-(2-叠氮基乙氧基)-5-溴苯甲酰氨基)乙基)氨基)乙基)氨基甲酸叔丁酯(0.860g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.325g,三步收率18.6%,Rf:0.16(甲醇:乙酸乙酯=1:2),m.p.170.1-172.4℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.39(s,1H),8.92(d,J=1.3Hz,1H),8.61(s,1H),8.20(s,1H),7.89(d,J=2.0Hz,1H),7.76–7.69(m,4H),7.57(d,J=7.5Hz,1H),7.24(d,J=7.5Hz,1H),7.18(d,J=7.3Hz,1H),7.06(d,J=7.5Hz,1H),7.00(s,2H),6.75(s,1H),6.63–6.54(m,2H),6.43–6.36(m,2H),4.94–4.86(m,2H),4.67–4.51(m,3H),4.21(d,J=3.1Hz,2H),4.20–4.09(m,1H),3.50(ddd,J=12.3,2.7,1.6Hz,1H),3.26(ddd,J=12.4,3.4,1.2Hz,1H),3.12–2.87(m,4H),1.38(s,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,165.87,158.76,156.51,153.94,151.93,146.85,146.41,142.66,139.43,137.31,136.03,134.08,133.82,133.05,125.11,124.76,122.68,122.46,118.46,117.21,117.11,111.87,109.68,101.08,85.16,65.24,49.21,48.40,46.68,42.14,41.81.,HRMS(ESI):found979.1596(C44H38BrN9O11S,[M+H]+,requires979.1595),HPLC(85:15甲醇:水):tR=3.356min,98.27%。
实施例10
荧光探针Ⅰ-10的制备
制备方法同实施例1,用((6-(2-(2-叠氮基乙氧基)-5-氯苯甲酰氨基)己基)氨基甲酸叔丁酯(0.880g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.365g,三步收率19.2%,Rf:0.18(甲醇:乙酸乙酯=1:2),m.p.165.4-166.9℃,该化合物的1H NMR(500MHz,DMSO-d6)δ8.98(d,J=1.3Hz,1H),8.76(d,J=19.4Hz,2H),8.29(d,J=2.0Hz,1H),8.20(s,1H),7.81(d,J=7.5Hz,1H),7.73(ddd,J=13.9,7.4,1.9Hz,2H),7.67(dd,J=15.1,1.7Hz,2H),7.59(d,J=7.5Hz,1H),7.16(dd,J=12.9,7.5Hz,2H),7.00(s,2H),6.81(s,1H),6.77–6.67(m,2H),6.43–6.35(m,2H),5.03–4.89(m,2H),4.88–4.65(m,4H),4.21(d,J=12.4Hz,1H),3.72–3.62(m,1H),3.28–3.02(m,6H),2.93(dtd,J=44.1,11.7,1.9Hz,2H),2.77(ddd,J=12.7,11.3,1.5Hz,1H),2.67(ddd,J=12.3,10.8,1.4Hz,1H),1.40(s,1H),1.17(s,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,165.87,158.76,156.51,153.94,151.93,146.85,146.41,142.66,139.43,137.31,136.03,134.08,133.82,133.05,125.11,124.76,122.68,122.46,118.46,117.21,117.11,111.87,109.68,101.08,85.16,65.24,49.21,48.40,46.73,46.68,42.14,41.81.,HRMS(ESI):found1022.2020(C46H43BrN10O11S,[M+H]+,requires 1022.2017),HPLC(85:15甲醇:水):tR=3.1450min,97.51%。
实施例11
荧光探针Ⅰ-11的制备
制备方法同实施例1,用(2-(2-(3-(2-(2-叠氮基乙氧基)-5-碘苯氧基)丙氧基)乙氧基)乙基)氨基甲酸叔丁酯(1.10g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.426g,三步收率21.0%,Rf:0.15(甲醇:乙酸乙酯=1:2),m.p.175.4-177.2℃,该化合物的1H NMR(500MHz,DMSO-d6)δ8.97(d,J=1.3Hz,1H),8.75(s,1H),8.68(s,1H),8.20(s,1H),7.85(d,J=1.1Hz,1H),7.72(dd,J=7.5,2.0Hz,1H),7.58(d,J=7.5Hz,1H),7.37(d,J=7.1Hz,1H),7.27(d,J=7.5Hz,1H),7.04(d,J=2.0Hz,1H),7.00(s,2H),6.94(dd,J=7.5,2.0Hz,1H),6.80(s,1H),6.75(d,J=7.5Hz,1H),6.64(d,J=2.0Hz,1H),6.60–6.52(m,3H),5.33–5.22(m,1H),4.73–4.62(m,2H),4.56–4.39(m,2H),4.32–4.21(m,2H),4.19–4.07(m,2H),4.10–3.99(m,4H),3.96–3.87(m,1H),3.81–3.70(m,1H),3.56(ddd,J=12.2,9.6,1.2Hz,1H),3.28–3.17(m,2H),2.09(qdd,J=12.2,5.8,3.1Hz,1H),1.81(dddt,J=12.1,10.2,3.0,1.6Hz,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,153.94,151.93,150.39,149.80,146.85,146.41,142.66,139.43,137.31,136.03,133.05,128.71,125.11,124.76,122.68,122.46,121.40,118.46,117.21,116.83,116.44,111.87,109.68,101.08,85.16,70.10,69.60,69.31,68.30,67.24,65.24,48.40,44.81,41.81,29.87.,HRMS(ESI):found1059.1600(C46H42IN7O13S,[M+H]+,requires 1059.1606),HPLC(85:15甲醇:水):tR=3.114min,98.26%。
实施例12
荧光探针Ⅰ-12的制备
制备方法同实施例1,用(2-(2-(2-(2-叠氮基乙氧基)-5-氯苯乙氧基)乙氧基)乙基)氨基甲酸叔丁酯(0.850g,2.0mmol)替换(3-((2-(2-叠氮基乙氧基)-5-氟苯基)氨基)丙基)氨基甲酸叔丁酯,得黄色固体0.321g,三步收率17.3%,Rf:0.18(甲醇:乙酸乙酯=1:2),m.p.172.7-174.8℃,该化合物的1H NMR(500MHz,DMSO-d6)δ9.11(s,1H),8.94(d,J=1.3Hz,1H),8.64(s,1H),8.20(s,1H),7.75–7.69(m,2H),7.67–7.57(m,2H),7.27(d,J=7.4Hz,1H),7.23–7.13(m,3H),7.00(s,2H),6.90(d,J=8.0Hz,1H),6.84(s,1H),6.64–6.56(m,2H),6.44(d,J=2.0Hz,1H),6.37(dd,J=7.5,2.0Hz,1H),5.13(ddd,J=12.4,11.4,4.2Hz,1H),4.90(d,J=12.4Hz,1H),4.78–4.60(m,3H),4.38(ddd,J=13.2,11.0,2.0Hz,1H),4.15–4.01(m,2H),3.76–3.54(m,4H),3.34–3.18(m,3H),2.91–2.82(m,2H),2.56–2.47(m,1H).,13C NMR(125MHz,DMSO-d6)δ179.64,172.72,170.00,168.45,158.76,156.11,153.94,151.93,146.85,146.41,142.66,139.43,137.31,136.03,133.05,130.23,130.16,128.73,128.63,125.11,124.76,122.68,122.46,118.46,117.21,115.59,111.87,109.68,101.08,85.16,70.10,70.06,69.87,69.60,65.24,48.40,44.81,41.81,33.73.,HRMS(ESI):found937.2141(C45H40ClN7O12S,[M+H]+,requires 937.2144),HPLC(80:20甲醇:水):tR=3.024min,97.18%。
实施例13
荧光探针EC50实验步骤:
荧光偏振实验在384孔平底黑色板(#3575,Corning)中进行,最终体积为90μL。最终的测定缓冲液含有40nMPHD2、PHD1和PHD3,以及探针Ⅰ-3,用来测试的384黑色板在室温下孵育2h,然后使用具有偏振滤光器和用于荧光素的光学模块SpectraMaxGeminiXS读板器读取数值(λex=485nM±25nM,λem=535nM±25nM)。GraphPad Prism 7.0分析标准误差。
实施例14
荧光探针Ⅰ-3的高通量筛选方法的建立:
鉴于PHD2(181-426)与全长PHD2具有相似的活性,利用PHD2(181-426)检测探针Ⅰ-3的结合强度,并且通过SpectraMaxGeminiXS(Molecular Devices,Sunnyvale)记录荧光测定结果,激发波长为485±25nM以及发射波长为535±25nM。最终的测定缓冲液含有40nMPHD2(181-426)、30nM探针Ⅰ-3和FG-2216及FG-4592,共孵育2h后酶标仪读取数值,处理数据,GraphPad Prism 7.0分析得化合物IC50。测试结果表明,与已报道的小分子活性几乎一致。
同上述操作一样,进一步考察了DMSO对此方法的影响和此高通量筛选方法的稳定性,,因此,荧光探针Ⅰ-3作为工具分子,通过荧光偏振(fluorescence polarization,FP)建立了一种PHD小分子高通量筛选方法,此方案可以用于药物的高通量筛选,具有廉价,稳定,快速,高效等特点。
Claims (9)
1.通式(I)的小分子荧光探针:
其中L代表4~10个原子长度的连接链,该连接链主链原子选自碳、氮或氧原子,L连接在X的邻位或间位;
B为荧光报告基团;
X代表卤素;
n代表1~3。
2.权利要求1的小分子荧光探针,其中荧光报告基团B为异硫氰酸荧光素。
3.权利要求1的小分子荧光探针,其中L代表
4.权利要求1的小分子荧光探针,其中X代表氯原子。
5.权利要求1的小分子荧光探针,其中n代表1或2。
6.权利要求1的小分子荧光探针的制备方法,当B为异硫氰酸荧光素时,包括:
其中X、L、n的定义同权利要求1。
7.权利要求1到5中任一项的小分子荧光探针用于测定化合物对脯氨酰羟化酶的抑制活性的用途。
8.一种脯氨酰羟化酶抑制活性的测试方法,包括:将待测化合物、权利要求1到5中任一项的小分子荧光探针和脯氨酰羟化酶,在缓冲体系中共孵育,在偏振光激发波长为485nm条件下,采用荧光偏振酶标仪记录发射波长为535nm条件下的偏振光信号值,计算即得。
9.权利要求8的测试方法,其中小分子荧光探针浓度为20-200纳摩尔,脯氨酰羟化酶浓度为20-200纳摩尔,孵育时间为1-24小时。
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