CN116715676A - 一种靶向RORγ的新型小分子荧光探针及其制备方法与应用 - Google Patents
一种靶向RORγ的新型小分子荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种靶向RORγ的新型小分子荧光探针及其制备方法与应用,本发明设计的小分子荧光探针可与维甲酸受体相关孤儿受体γ变构位点结合,可作为荧光探针用于细胞成像或体内成像中的应用,从而可用于高通量筛选靶向RORγ的银屑病治疗药物,具有重要的应用价值。
Description
技术领域
本发明属于荧光探针技术领域,具体涉及可与维甲酸受体相关孤儿受体γ变构位点结合的新型荧光化合物及其作为荧光探针用于高通量筛选与成像的应用。
背景技术
银屑病俗称“牛皮癣”,是一种有特征鳞屑性红斑的复发性、慢性皮肤病。抑制白介素-17(IL-17)的生物制剂的成功证明了IL-17在银屑病中的重要作用(Lancet 2021,397,1301-1315),但是生物制剂较高的价格限制了更多患者从中受益。维甲酸受体相关孤儿受体(retinoic acid receptor-related orphan receptorγ,RORγ)属于转录因子核受体家族,是IL-17的主要调控蛋白,抑制RORγ是治疗银屑病的有效策略。与大部分核受体一样,在结构上RORγ由氮端结构域(NTD)、DNA结合结构域(DBD)、铰链区(Hinge)、配体结合结构域(LBD)与碳端结构域(CTD)组成。其中LBD是转录调节的关键结构域,其由十二个α-螺旋(H1-H12)组成,碳端的H12可招募共转录因子,促进DBD与靶基因结合调控下游基因表达(Int J Mol Sci 2020,21,5329-5346)。在核受体家族的LBD内部有一个保守的正构结合位点,内源性配体与该位点结合可稳固H12的结构,促进其与共转录因子结合,加强转录。尽管RORγ内源性配体目前尚未有定论,大量研究表明,利用外源性小分子作用于正构位点,可影响H12构象,从而调节其与共转录因子的结合能力,进而抑制或激活RORγ的转录活性。目前已有30多项RORγ正构抑制剂的项目进入过临床试验,但是由于疗效不够或安全性问题均宣告失败。这可能与正构口袋本身的高度保守性导致的脱靶效应有关。RORγ内部还有一变构口袋,其为RORγ独有,利用小分子靶向变构口袋,具有亚型选择性更好等优点(Nat.Commun.2015,6,8833-8843)。开发准确、稳定、可靠的RORγ变构位点活性测试方法有助于发现结构新颖的RORγ变构抑制剂,并指导其结构优化以获得活性更优的候选药物分子,为靶向RORγ的银屑病治疗药物的开发提供助力。
小分子荧光探针是药物化学中常用的辅助工具,广泛应用于活性测试与体内外成像研究。通过特定的连接方法将RORγ变构抑制剂MRL-871与异硫氰酸荧光素(FITC)相连,本发明公开了一系列可与RORγ变构位点结合的小分子荧光探针、其制备方法与在活性测试与体内外成像中的应用。
发明内容
一种靶向RORγ的新型小分子荧光探针,其包括如下通式(I)的化合物或其药学上可接受的盐:
在通式(I)中:
L代表L1L2/>
L3
L4L5/>
L6
L7L8/>
或L9
本发明还提供了所述RORγ小分子荧光探针的制备方法,包括以下步骤:
S1:将原料AI-1与一氧化碳及不同二胺类化合物在碱、催化剂催化条件下高温反应得到中间体AI-2-L1~3,7,8;
优选的,步骤S1中,所述反应中所使用的二胺类化合物为单叔丁氧羰基(Boc)保护的二胺;所使用的一氧化碳压力为1到3个大气压;所述反应选用的有机溶剂为N,N-二甲基甲酰胺(DMF)、1,4-二氧六环或四氢呋喃(THF)中的一种或几种;反应选用的碱为N,N-二异丙基乙胺(DIPEA)或三乙胺(TEA)中的一种或几种;反应选用催化剂为1,1'-双(二苯基膦基)二茂铁二氯化钯(Pd(dppf)Cl2)、醋酸钯(Pd(OAc)2)或四三苯基膦钯(Pd(PPh3)4)中的一种或几种;反应温度为80-120℃。
S2:将原料AI-1与二胺类化合物在碱、催化剂、配体条件下利用Buchwald-Hartwig反应偶联得到中间体AI-2-L4~6;
优选的,步骤S2中,所述Buchwald-Hartwig反应,选用的催化剂为Pd2(dba)3,Pd(OAc)2,Pd(Dppf)Cl2,选用的碱为N,N-二异丙基乙胺(DIPEA)或三乙胺(TEA)、碳酸铯(Cs2CO3),叔丁醇钾(t-BuOK),叔丁醇钠(t-BuONa)中的一种或几种,选用的配体为2-二环己基磷-2',4',6'-三异丙基联苯(XPhos)或1,1'-联萘-2,2'-双二苯膦(BINAP),选用溶剂为1,4-二氧六环。
S3:将原料AI-1与2-(7-辛炔-1-基)-1H-异吲哚-1,3-二酮在碱、催化剂条件下利用Sonogashira反应偶联得到中间体AI-L9;
优选的,步骤S3中,所述Sonogashira反应选用的催化剂为Pd2(dba)3,Pd(OAc)2,Pd(dppf)Cl2与CuI,选用的有机溶剂为N,N-二异丙基乙胺(DIPEA)或三乙胺(TEA)中的一种或几种。
S4:将原料AI-2-L1~8溶于二氯甲烷中,在酸性条件下室温反应,脱去叔丁氧羰基(Boc)得到化合物AI-3-L1~8;
优选的,步骤S4中,所述的酸性试剂为三氟乙酸或盐酸二氧六环溶液。
S5:将中间体AI-2-L9在乙醇中与水合肼回流反应,脱去保护基得到化合物AI-3-L9。
S6:将中间体AI-3-L1~9与异硫氰酸荧光素(FITC)在碱性条件下进行缩合反应,所得中间体直接在碱水溶液下水解脱去甲基,得到终产物P1~P9;
优选的,步骤S6中,缩合反应所使用的碱性条件为N,N-二异丙基乙胺(DIPEA)使用的溶剂为N,N-二甲基甲酰胺(DMF),反应条件为室温,氮气保护。所述碱水溶液为氢氧化锂水溶液。
本发明还提供了利用式(I)荧光探针测试其他化合物对RORγ变构位点的结合能力的测试方法,具体步骤如下:
S1:将式(I)的RORγ小分子荧光探针、包含RORγ配体结合结构域的蛋白与待测化合物以一定比例混合得待测体系;
S2:采用荧光偏振技术,通过多功能酶标仪测定待测体系的荧光偏振值,根据荧光偏振值确定待测化合物是否能与RORγ的变构位点结合。
有益效果:相对于现有技术,本发明提供的化合物具有荧光特性,对RORγ配体结合结构域变构结合位点有良好的特异性结合。将该化合物作为荧光偏振技术中的小分子荧光探针,通过竞争结合的方法,可实现对RORγ配体结合结构域变构结合位点的配体的筛选及配体亲和力的评价,为靶向RORγ配体结合结构域变构结合位点的药物发现与优化提供了便宜、快捷且稳定的活性测试方法。
附图说明
图1为荧光探针P8的荧光偏振信号随蛋白RORγ-LBD浓度变化的曲线图。
图2为利用P8测试已报道RORγ变构抑制剂MRL003、MRL058、MRL871与RORγ正构抑制剂GSK2981278对RORγ变构位点亲和力。
图3为在高表达RORγ的细胞株A375中荧光探针P8的成像实验结果。
图4为在野生型72hpf斑马鱼中荧光探针P8的成像实验结果。
图5为探针P8的1H NMR图。
具体实施方式
下面结合实施例进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照本领域常规条件或按照制造厂商建议的条件;所使用的原料、试剂等,如无特殊说明,均为可从常规市场等商业途径得到的原料和试剂。本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
实施例1探针P1的合成
向AI-1(400mg,0.75mmol,1.0equiv.)、N-Boc-1,6-己二胺(484.3mg,2.24mmol,3.0equiv.)、1,1'-双(二苯基膦基)二茂铁]二氯化钯(109.5mg,0.15mmol,0.2equiv.)、三乙胺(228.15mg,2.24mmol,3.0equiv.)的混合体系中加DMF(4mL)。使用一氧化碳置换反应体系气体三次,80℃搅拌反应过夜。TLC检测反应完全,反应加入水(5mL)淬灭,所得混合物通过硅藻土垫层过滤,使用乙酸乙酯萃取(3×20mL)。合并的有机层用饱和食盐水(5mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸去溶剂,残余物用硅胶柱层析纯化得252mg无色透明固体AI-2-L1;收率48.2%。1H NMR(400MHz,Chloroform-d)δ9.00(s,1H),8.21-8.07(m,4H),7.91(d,J=8.2Hz,2H),7.75(t,J=7.8Hz,2H),7.64(t,J=8.0Hz,1H),6.65(s,1H),4.58(s,1H),3.97(s,3H),3.56(q,J=6.7Hz,2H),3.17(d,J=5.8Hz,2H),1.72(q,J=7.3Hz,2H),1.61(s,8H),1.55(s,2H).MS(ESI):m/z calcd for C35H36ClF3N4O6[M+H]+=700.2275,found 700.2。
向AI-2-L1(252mg,0.42mmol,1.0equiv.)的二氯甲烷(15mL)溶液中,加入三氟乙酸(30mL),在氮气保护状态下,室温反应1.5小时。TLC检测反应完全,减压蒸去溶剂得到AI-3-L1粗品,无需纯化直接用于下一步反应。
将AI-3-L1粗品溶于无水DMF(2mL)中,加入异硫氰酸荧光素(85.6mg,0.22mmol),二异丙基乙胺(28.43mg,0.22mmol)。加入完毕后,避光氮气保护,室温反应过夜。TLC检测反应完毕,反应混合物加水淬灭,用乙酸乙酯提取反应液(3×10mL)。合并的有机层用饱和食盐水(5mL)洗涤并用无水硫酸镁干燥。过滤后蒸去溶剂得到中间体粗品,将中间体粗品溶于THF(4.00mL)溶液中,加入LiOH(24mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护反应3小时。TLC检测反应完毕,将四氢呋喃减压除去后,调节溶液pH至5~6。使用乙酸乙酯进行萃取(3×10mL),合并有机相用硅胶柱层析纯化得到终产物P1,为橙黄色固体(161.3mg,74.5%)。1H NMR(500MHz,DMSO-d6)δ12.25(s,1H),10.45(s,1H),9.05(t,J=5.1Hz,1H),9.00(s,1H),8.40(s,1H),8.30(d,J=8.4Hz,1H),8.11(d,J=8.4Hz,1H),8.04(d,J=7.9Hz,3H),8.00(d,J=8.0Hz,1H),7.94(d,J=7.9Hz,1H),7.87(t,J=8.1Hz,1H),7.78(d,J=7.9Hz,2H),7.05(d,J=8.3Hz,1H),6.65(d,J=8.9Hz,2H),6.53(s,2H),6.46(d,J=8.7Hz,2H),3.49(d,J=4.8Hz,2H),3.36(d,J=6.0Hz,2H),1.65-1.57(m,4H),1.41(s,4H).HRMS(ESI):m/z calcd.for C50H37ClF3N5O9S[M+H]+=975.1953,found974.2005.HPLC:tR=14.16min,95.74%纯度。
实施例2探针P2的合成
向AI-1(300mg,0.56mmol,1.0equiv.)、N-Boc-1,7-庚二胺(258mg,1.12mmol,2.0equiv.)、1,1'-双(二苯基膦基)二茂铁]二氯化钯(82.1mg,0.11mmol,0.2equiv.)、三乙胺(171.1mg,1.68mmol,3.0equiv.)的混合体系中加DMF(4mL)。使用一氧化碳置换反应体系气体三次,80℃搅拌反应过夜。TLC检测反应完全,反应混合物加水淬灭(5mL)后,通过硅藻土垫层过滤,使用乙酸乙酯提取滤液(3×10mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后蒸去溶剂,残余物用硅胶柱层析纯化得200mg无色透明固体AI-2-L2;收率50.03%。1H NMR(400MHz,DMSO-d6)δ9.00(s,1H),8.94(t,J=5.6Hz,1H),8.34(d,J=8.4Hz,1H),8.13(d,J=8.3Hz,2H),8.11-8.09(m,1H),8.06(d,J=8.2Hz,1H),8.03(s,1H),8.01-7.98(m,2H),6.74(s,1H),3.90(s,3H),3.35(s,2H),2.91(q,J=6.6Hz,2H),1.59(s,2H),1.39(s,2H),1.36(s,9H),1.30-1.22(m,6H).MS(ESI)m/z calcd.forC36H38ClF3N4O6[M+H]+=714.2432,found 714.2。
向AI-2-L2(258.0mg,0.35mmol,1.0equiv.)的DCM(15mL)溶液中,加入三氟乙酸(3mL),在氮气保护状态下,室温反应1.5小时。TLC检测反应完全,反应混合物蒸去溶剂得到AI-3-L2粗品,无需纯化直接用于下一步反应。
将AI-3-L2粗品溶于无水DMF(2mL)中,加入异硫氰酸荧光素(107.00mg,0.22mmol),二异丙基乙胺(28.43mg,0.22mmol)。加入完毕后,避光氮气保护,室温反应过夜。反应混合物加水后,用乙酸乙酯提取反应液(3×10mL)。合并的有机层用盐水洗涤并用无水硫酸镁干燥。过滤后蒸去溶剂得到粗品,将粗品溶于四氢呋喃(4mL)中,加入氢氧化锂(24mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护反应3小时。TLC检测反应完全后,减压蒸除四氢呋喃后,调节溶液pH至5~6。使用乙酸乙酯进行萃取(3×10mL),合并有机相使用硅胶柱层析纯化得到终产物P2,为橙黄色固体(87.1mg,56.6%)。1H NMR(400MHz,DMSO-d6)δ12.01(s,1H),10.22(s,1H),8.99(d,J=5.8Hz,2H),8.37(s,1H),8.30(d,J=8.4Hz,1H),8.09(dd,J=8.5,1.2Hz,1H),8.04(d,J=8.0Hz,3H),7.99(d,J=7.9Hz,1H),7.92(d,J=9.6Hz,1H),7.87(t,J=8.1Hz,1H),7.78(d,J=8.2Hz,2H),7.04(d,J=8.3Hz,1H),6.66(d,J=8.9Hz,2H),6.50(s,2H),6.44(d,J=8.9Hz,2H),3.48(d,J=5.0Hz,2H),3.35(q,J=6.5Hz,2H),1.66-1.55(m,4H),1.38(s,6H).HRMS(ESI):m/z calcd.forC51H39ClF3N5O9S[M+H]+=989.2109,found 988.2172.HPLC:tR=12.085min,99.08%纯度。
实施例3探针P3的合成
向AI-1(300mg,0.56mmol,1.0equiv.)、N-Boc-1,8-辛二胺(242mg,1.12mmol,2.0equiv.)、1,1'-双(二苯基膦基)二茂铁]二氯化钯(82.1mg,0.11mmol,0.2equiv.)、三乙胺(171.1mg,1.68mmol,3.0equiv.)的混合体系中加入N,N-二甲基甲酰胺(4mL)。使用一氧化碳置换反应体系三次,80℃搅拌反应过夜。TLC检测反应完全,反应混合物加水(10mL)淬灭后通过硅藻土垫层过滤,滤液使用乙酸乙酯萃取(3×10mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸除溶剂,残余物用硅胶柱层析纯化得270.0mg白色固体AI-3-L3,收率66.21%。1H NMR(400MHz,DMSO)δ9.01(s,1H),8.94(t,J=5.4Hz,1H),8.33(d,J=8.5Hz,1H),8.11(t,J=6.4Hz,3H),8.05(d,J=8.1Hz,1H),8.03-7.95(m,3H),7.89(t,J=8.0Hz,1H),6.72(s,1H),3.89(s,3H),3.34(s,2H),2.92-2.87(m,2H),1.64-1.55(m,2H),1.39(s,2H),1.36(s,9H),1.25(dd,J=30.1,8.6Hz,8H).MS(ESI)m/z calcd.for C37H40ClF3N4O6[M+H]+=728.2588,found 728.2。
向AI-3-L3(200.0mg,0.28mmol)的二氯甲烷(15mL)溶液中,加入三氟乙酸(3mL),在氮气保护状态下,室温反应1.5小时。TLC检测反应完全,反应混合物蒸去溶剂得到AI-3-L3粗品。
将AI-3-L3粗品溶于无水DMF(2mL)中,加入异硫氰酸荧光素(95.4mg,0.25mmol)、二异丙基乙胺(57.64mg,0.45mmol)。加入完毕后,避光氮气保护,室温反应过夜。反应混合物加水淬灭,使用乙酸乙酯(10mL)萃取三次。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸去溶剂得到粗品,将粗品溶于THF(4mL)中,加入氢氧化锂(24.0mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护,室温反应3.0小时。TLC检测反应完全后,减压蒸除四氢呋喃,调节溶液pH至5~6。使用乙酸乙酯进行萃取(3×10mL),合并有机相用硅胶柱层析纯化得到终产物P3,为橙黄色固体(41.8mg,30.1%)。1H NMR(400MHz,DMSO-d6)δ11.94(s,1H),10.13(s,1H),8.98(s,2H),8.36(s,1H),8.30(d,J=8.0Hz,1H),8.09(s,1H),8.04(d,J=7.6Hz,2H),8.00(d,J=7.9Hz,1H),7.93-7.86(m,1H),7.78(d,J=7.6Hz,1H),7.04(d,J=8.1Hz,1H),6.66(d,J=8.6Hz,1H),6.48(s,1H),6.43(d,J=8.5Hz,1H),3.48(s,2H),3.34(s,2H),1.58(s,4H),1.29(d,J=45.8Hz,8H).MS(ESI):m/z calcd.for C52H41ClF3N5O9S[M+H]+=1003.2266,found 1002.2318.HPLC:tR=12.370min,99.05%纯度。
实施例4探针P4的合成
向AI-1(300.0mg,0.56mmol,1.0equiv.)、N-Boc-1,6-己二胺(241.1mg,1.12mmol,2.0equiv.)、醋酸钯(25.2mg,0.11mmol,0.2equiv.)、碳酸铯(363.8mg,1.11mmol,2.0equiv.)、1,1'-联萘-2,2'-双二苯膦(69.6mg,0.11mmol,0.2equiv.)的混合体系中加1,4-二氧六环(20mL)。在氮气保护条件下,回流反应过夜。TLC检测反应完全,反应加水(10mL)淬灭,通过硅藻土垫层过滤,滤液使用乙酸乙酯萃取(3×10mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后蒸去溶剂,残余物用硅胶柱层析纯化得白色固体AI-2-L4(203.0mg,54.1%)。1H NMR(500MHz,DMSO-d6)δ8.06(d,J=8.4Hz,2H),8.00(d,J=8.1Hz,1H),7.95(d,J=8.0Hz,1H),7.89(d,J=8.4Hz,2H),7.85-7.80(m,2H),7.55(d,J=1.5Hz,1H),6.93(dd,J=8.9,1.9Hz,1H),6.79(t,J=5.5Hz,1H),6.74(t,J=5.0Hz,1H),3.87(s,3H),3.15(q,J=6.6Hz,2H),2.94-2.90(m,2H),1.64(p,J=7.0Hz,2H),1.44-1.38(m,4H),1.37(s,9H),1.34-1.31(m,2H).MS(ESI):m/z calcd for C34H36ClF3N4O5[M+H]+=672.2,found 672.4。
向AI-2-L4(203.0mg,0.3mmol)的二氯甲烷(15mL)溶液中,加入三氟乙酸(3mL),在氮气保护状态下,室温反应1.5小时。TLC检测反应完全,反应混合物减压蒸去溶剂得到AI-3-L4粗品。该化合物无需纯化直接用于下一步反应。
将AI-3-L4粗品溶于无水N,N-二甲基甲酰胺(2mL)中,加入异硫氰酸荧光素(79.0mg,0.2mmol),二异丙基乙胺(52.5mg,0.41mmol)。加入完毕后,避光氮气保护,室温反应过夜。反应加水(10mL)淬灭后,用乙酸乙酯萃取(3×20mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸去溶剂得到中间体粗品,将粗品溶于四氢呋喃(4mL)中,加入氢氧化锂(24.0mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护,室温反应3.0小时。TLC检测反应完全后,减压蒸除四氢呋喃,调节溶液pH至5~6。使用乙酸乙酯萃取(3×10mL),合并有机相用硅胶柱层析纯化得到终产物P4,为橙黄色固体(51.1mg,41.2%)。1H NMR(400MHz,DMSO-d6)δ11.68(s,1H),9.97(s,1H),8.20(s,1H),7.98(dd,J=7.9,4.5Hz,3H),7.94(d,J=8.0Hz,1H),7.87(d,J=7.2Hz,1H),7.81(dd,J=10.5,8.9Hz,2H),7.66(d,J=8.2Hz,2H),6.95(d,J=8.3Hz,1H),6.92(d,J=9.0Hz,1H),6.76(s,1H),6.70(d,J=9.1Hz,2H),6.20(d,J=9.5Hz,2H),6.17(s,2H),3.51(d,J=6.1Hz,2H),3.17(d,J=3.1Hz,2H),1.64-1.59(m,2H),1.49(s,2H),1.43(d,J=6.6Hz,2H),1.24(s,2H).HRMS(ESI):m/z calcd.for C49H37ClF3N5O8S[M+H]+=947.2003,found 946.2001.HPLC:tR=12.384min,96.7%纯度。
实施例5探针P5的合成
向AI-1(400.0mg,0.75mmol,1.0equiv.)、N-Boc-1,7-庚二胺(343.3mg,1.5mmol,2.0equiv.)、醋酸钯(33.6mg,0.15mmol,0.2equiv.)、碳酸铯(489.00mg,1.50mmol,2.00equiv.)、1,1'-联萘-2,2'-双二苯膦(124.5mg,0.15mmol,0.2equiv.)的混合体系中加1,4-二氧六环(20mL)。在氮气保护下回流反应过夜。TLC检测反应完全,反应加水(15mL)淬灭,随后通过硅藻土垫层过滤,使用乙酸乙酯萃取滤液(3×20mL)。合并的有机层用饱和食盐水(15mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸除溶剂,残余物用硅胶柱层析纯化得白色固体AI-2-L5(200.0mg,46.6%)。1H NMR(400MHz,DMSO-d6)δ8.07(d,J=8.5Hz,2H),8.00(d,J=8.1Hz,1H),7.95(d,J=7.9Hz,1H),7.89(d,J=8.5Hz,2H),7.84(d,J=8.7Hz,2H),7.55(d,J=1.8Hz,1H),6.93(dd,J=8.9,2.0Hz,1H),6.77-6.70(m,2H),3.88(s,3H),3.16(q,J=6.7Hz,2H),2.91(d,J=6.0Hz,2H),1.64(p,J=7.0Hz,2H),1.53(dt,J=14.6,7.1Hz,2H),1.41(s,2H),1.37(s,10H),1.30(d,J=5.0Hz,2H),1.26(d,J=3.8Hz,2H).MS(ESI):m/z calcd for C35H38ClF3N4O5[M+H]+=686.2,found 686.3。
向AI-2-L5(240.0mg,0.35mmol)的二氯甲烷(15mL)溶液中,加入三氟乙酸(3mL),在氮气保护下,反应1.5小时。TLC检测反应完全,反应混合物减压蒸去溶剂得到AI-3-L5粗品。该化合物直接用于下一步反应无需纯化。
将AI-3-L5粗品溶于无水N,N-二甲基甲酰胺(2mL)中,加入异硫氰酸荧光素(135.7mg,0.35mmol)与二异丙基乙胺(90.04mg,0.70mmol)。加入完毕后,避光氮气保护,室温反应过夜。反应加水(10mL)淬灭后,用乙酸乙酯萃取(3×20mL)。合并的有机层用饱和食盐水(15mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸除溶剂得到中间体粗品,将粗品溶于四氢呋喃(4mL)中,加入氢氧化锂(24mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护,室温反应3.0小时。TLC检测反应完全后,减压蒸除四氢呋喃,调节溶液pH至5~6。使用乙酸乙酯进行萃取(3×10mL),合并的有机相使用硅胶柱层析纯化得到终产物P5为橙黄色固体(25.6mg,32.0%)。1H NMR(400MHz,DMSO-d6)δ12.06(s,1H),10.28(s,1H),8.32(s,1H),7.99(d,J=8.0Hz,3H),7.94(d,J=8.1Hz,1H),7.90(s,1H),7.81(t,J=8.4Hz,2H),7.66(d,J=6.7Hz,2H),7.55(s,1H),7.01(d,J=8.3Hz,1H),6.96(d,J=8.2Hz,1H),6.91(d,J=8.7Hz,1H),6.71(s,1H),6.65(t,J=9.0Hz,2H),6.44(d,J=14.3Hz,2H),6.38(s,2H),3.48(s,2H),3.16(d,J=5.2Hz,2H),1.68(d,J=6.7Hz,2H),1.60(s,2H),1.46(s,2H),1.38(s,2H),1.24(s,2H).MS(ESI):m/z calcd.for C50H39ClF3N5O8S[M+H]+=961.2160,found961.0580.HPLC:tR=12.403min,96.6%纯度。
实施例6探针P6的合成
向AI-1(400.0mg,0.75mmol,1.0equiv.)、N-Boc-1,8-辛二胺(365.2mg,1.5mmol,2.0equiv.)、醋酸钯(33.6mg,0.15mmol,0.2equiv.)、碳酸铯(489.0mg,1.5mmol,2.0equiv.)、1,1'-联萘-2,2'-双二苯膦(124.5mg,0.15mmol,0.2equiv.)的混合体系中加1,4-二氧六环(20mL)。在氮气保护下回流反应过夜。TLC检测反应完全,反应加水(10mL)淬灭,随后通过硅藻土垫层过滤,乙酸乙酯萃取(3×15mL)。合并的有机层用饱和食盐水洗涤并用无水硫酸镁干燥。过滤后减压蒸去溶剂,残余物用硅胶柱层析纯化得白色固体AI-2-L6(210.0mg,40.1%)。1H NMR(400MHz,DMSO-d6)δ8.09-8.05(m,2H),8.00(d,J=8.1Hz,1H),7.95(d,J=7.9Hz,1H),7.91-7.87(m,2H),7.83(dd,J=8.6,7.2Hz,2H),7.54(d,J=2.0Hz,1H),6.93(dd,J=8.9,2.1Hz,1H),6.72(t,J=4.3Hz,2H),3.88(s,3H),3.15(q,J=6.6Hz,2H),2.90(q,J=6.5Hz,2H),1.64(q,J=7.2Hz,2H),1.37(s,2H),1.37(s,9H),1.31-1.23(m,8H).MS(ESI):m/z calcd.for C36H40ClF3N4O5[M+H]+=700.2,found 700.2。
向AI-2-L6(210.0mg,0.3mmol)的二氯甲烷(15mL)溶液中,加入三氟乙酸(3mL),在氮气保护状态下,反应1.5小时。TLC检测反应完全,反应混合物减压蒸去溶剂得到AI-3-L6粗品。该化合物无需纯化直接进行下一步反应。
将AI-3-L6粗品溶于无水N,N-二甲基甲酰胺(2mL)中,加入异硫氰酸荧光素(121.80mg,0.31mmol),二异丙基乙胺(80.8mg,0.63mmol)。加入完毕后,避光氮气保护,室温反应过夜。反应混合物加水(10mL)淬灭后,用乙酸乙酯提取反应液。合并的有机层用饱和食盐水(15mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸去溶剂得到中间体粗品,将粗品溶于四氢呋喃(4mL)中,加入氢氧化锂(24.0mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护反应3.0小时。TLC检测反应完全后,减压蒸除四氢呋喃,调节溶液pH至5~6。使用乙酸乙酯进行萃取(3×15mL),合并有机相用硅胶柱层析纯化得到终产物P6,为橙黄色固体(71.0mg,40.0%)。1H NMR(400MHz,DMSO-d6)δ11.98(s,1H),10.20(s,1H),8.30(s,1H),7.98(t,J=7.3Hz,3H),7.94(d,J=8.0Hz,1H),7.90(d,J=7.9Hz,1H),7.81(t,J=8.5Hz,2H),7.66(d,J=8.2Hz,2H),7.55(d,J=1.6Hz,1H),7.01(d,J=8.3Hz,1H),6.90(dd,J=9.0,1.8Hz,1H),6.70(s,1H),6.67(d,J=8.9Hz,2H),6.41(d,J=14.3Hz,2H),6.36(d,J=8.8Hz,2H),3.47(d,J=5.1Hz,2H),3.19-3.12(m,2H),1.68-1.64(m,2H),1.59(d,J=6.5Hz,2H),1.45(s,2H),1.36(s,4H),1.24(s,2H).MS(ESI):m/z calcd.forC51H41ClF3N5O8S[M+H]+=975.2316,found 974.2340.HPLC:tR=13.014min,96.9%纯度。
实施例7探针P7的合成
向AI-1(300mg,0.56mmol,1.0equiv.)、N-Boc-2,2’-(亚乙二氧基)二乙胺(417.0mg,1.68mmol,3.0equiv.)、1,1'-双(二苯基膦基)二茂铁]二氯化钯(82.1mg,0.11mmol,0.2equiv.)、三乙胺(171.1mg,1.68mmol,3.0equiv.)的混合体系中加入N,N-二甲基甲酰胺(4mL)。使用一氧化碳置换反应体系三次,80℃搅拌反应过夜。TLC检测反应完全,反应加入水(10mL)淬灭反应,随后通过硅藻土垫层过滤,滤液使用乙酸乙酯萃取(3×10mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后减压蒸去溶剂,残余物使用硅胶柱层析纯化得270.0mg白色固体AI-2-L7,收率46.3%。1H NMR(500MHz,DMSO-d6)δ9.04(dd,J=11.2,5.5Hz,2H),8.35(d,J=8.4Hz,1H),8.12(dd,J=8.4,2.0Hz,3H),8.06(d,J=8.1Hz,1H),8.01(dd,J=7.9,6.2Hz,3H),7.89(t,J=8.1Hz,1H),6.74(s,1H),3.89(s,3H),3.62(t,J=5.9Hz,2H),3.58(dd,J=5.7,3.4Hz,2H),3.52(dt,J=11.7,5.7Hz,4H),3.39(t,J=6.1Hz,2H),3.06(dd,J=11.6,5.8Hz,2H),1.35(s,9H).MS(ESI):m/z calcd.for C37H40ClF3N4O6[M+H]+=732.2,found 732.1。
向AI-2-L7(110.0mg,0.17mmol)的二氯甲烷(15mL)溶液中,加入三氟乙酸(3mL),在氮气保护状态下,室温反应1.5小时。TLC检测反应完全,反应混合物减压蒸去溶剂得到AI-3-L7粗品。
将AI-3-L7粗品溶于无水DMF(2mL)中,加入异硫氰酸荧光素(75.0mg,0.19mmol,1.1equiv.),二异丙基乙胺(45.0mg,0.35mmol,2.0equiv.)。加入完毕后,避光氮气保护,室温反应过夜。TLC检测反应完全后,反应混合物加水(10mL)淬灭,随后用乙酸乙酯萃取(3×10mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥,过滤后滤液减压蒸除溶剂得到粗品。将粗品溶于四氢呋喃(4mL)中,加入氢氧化锂(24mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护室温反应3小时。TLC检测反应完全后,减压蒸除四氢呋喃,调节溶液pH至5~6。所得混合物使用乙酸乙酯萃取(3×10mL),合并的有机相使用硅胶柱层析纯化得到终产物P7,为橙黄色固体(41.0mg,68.8%)。1H NMR(400MHz,DMSO-d6)δ12.21(s,1H),10.31(s,1H),9.15(s,1H),9.02(s,1H),8.43(s,1H),8.31(d,J=8.4Hz,1H),8.12(d,J=8.6Hz,1H),8.08-8.02(m,2H),7.99(d,J=7.9Hz,1H),7.89(dt,J=16.0,8.0Hz,2H),7.79(d,J=7.9Hz,1H),7.07(d,J=8.3Hz,1H),6.67-6.57(m,3H),6.50(d,J=8.7Hz,1H),3.65(d,J=6.0Hz,2H),3.56-3.48(m,2H),1.79(s,8H).HRMS(ESI):m/z calcd.forC50H37ClF3N5O9S[M+H]+=1007.1851,found 1006.1876.HPLC:tR=11.214min,96.4%纯度。
实施例8探针P8的合成
向AI-1(300.0mg,0.56mmol,1.0equiv.)、(6-(3-氨基丙酰胺基)己基)氨基甲酸叔丁酯(482.5mg,1.68mmol,3.0equiv.)、1,1'-双[(二苯基膦基)二茂铁]二氯化钯(82.1mg,0.11mmol,0.2equiv.)、三乙胺(171.1mg,1.68mmol,3.0equiv.)的混合体系中加N,N-二甲基甲酰胺(4mL)。使用一氧化碳置换反应体系中的气体三次,80℃搅拌反应过夜。TLC检测反应完全,反应混合物水(10mL)淬灭后,通过硅藻土垫层过滤,滤液使用乙酸乙酯(3×10mL)萃取。合并的有机层用饱和食盐水洗涤并用无水硫酸镁干燥。过滤后减压蒸去溶剂,残余物用硅胶柱层析纯化得白色固体AI-2-L8(270.0mg,56.44%)。1H NMR(400MHz,DMSO-d6)δ9.01(s,1H),8.96(t,J=5.6Hz,1H),8.34(d,J=8.4Hz,1H),8.15-8.09(m,3H),8.06(d,J=8.0Hz,1H),8.04-7.98(m,3H),7.89(t,J=8.4Hz,1H),7.81(t,J=5.4Hz,1H),6.70(s,1H),3.90(s,3H),3.11(q,J=7.0Hz,2H),3.04(q,J=6.1,5.6Hz,2H),3.03-2.96(m,2H),2.21(t,J=7.3Hz,2H),1.58(d,J=9.0Hz,2H),1.45-1.39(m,2H),1.36(s,9H),1.35(s,4H).MS(ESI):m/z calcd.for C38H41ClF3N5O7[M+H]+=771.2,found 771.0。
向AI-2-L8(100.0mg,0.15mmol)的二氯甲烷(15mL)溶液中,加入三氟乙酸(3mL),在氮气保护状态下,室温反应1.5小时。TLC检测反应完全,反应混合物减压蒸去溶剂得到AI-3-L8粗品。该化合物无需纯化直接用于下一步反应。
将AI-3-L8粗品溶于无水N,N-二甲基甲酰胺(2mL)中,加入异硫氰酸荧光素(58.0mg,0.15mmol)、二异丙基乙胺(38.50mg,0.30mmol)。加入完毕后,避光氮气保护,室温反应过夜。反应混合物加水(5mL)淬灭后,用乙酸乙酯提取反应液(3×10mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后蒸去溶剂得到中间体粗品,将粗品溶于四氢呋喃(4mL)溶液中,加入氢氧化锂(24mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护反应3小时。TLC检测反应完全后,减压蒸除四氢呋喃,调节溶液pH至5~6。使用乙酸乙酯进行萃取(3×10mL),合并有机相使用硅胶柱层析纯化得到终产物P8,为橙黄色终产物(30.2mg,38.0%)。1H NMR(500MHz,DMSO-d6)δ12.35(s,1H),10.50(s,1H),9.00(s,1H),8.99(d,J=2.8Hz,1H),8.42(s,1H),8.31(d,J=8.4Hz,1H),8.09(d,J=8.6Hz,1H),8.05(d,J=8.3Hz,2H),8.00(s,1H),7.93(d,J=8.4Hz,1H),7.91-7.84(m,1H),7.78(d,J=7.8Hz,1H),7.08(d,J=8.3Hz,1H),6.60(d,J=8.5Hz,3H),6.50(d,J=8.9Hz,2H),3.65(d,J=4.7Hz,2H),3.33(d,J=6.3Hz,2H),3.07(d,J=6.0Hz,2H),2.42(t,J=7.2Hz,2H),1.58(d,J=6.6Hz,2H),1.45(d,J=6.6Hz,2H),1.35(s,4H),1.24(s,2H).(图4)HRMS(ESI):m/zcalcd.for C53H42ClF3N6O10S[M+H]+=1046.2324,found 1045.2350.HPLC:tR=11.162min,95.7%纯度。
实施例9探针P9的合成
向AI-1(375.2mg,0.7mmol,1.2equiv.)、辛-7-炔-1-胺(148.8mg,0.6mmol,1.0equiv.)、醋酸钯(10.8mg,0.05mmol,0.04equiv.)、碘化亚铜(9.1mg,0.05mmol,0.04equiv.)、三苯基膦(37.6mg,0.14mmol,0.12equiv.)的混合体系中加入1,4-二氧六环(20.0mL)。使用氮气对体系进行保护,回流反应过夜。TLC检测反应完全,反应混合物加水(10mL)淬灭,通过硅藻土垫层过滤,使用乙酸乙酯萃取滤液(3×20mL)。合并的有机层用饱和食盐水(10mL)洗涤并用无水硫酸镁干燥。过滤后蒸去溶剂,残余物用硅胶柱层析纯化得白色固体AI-2-L9(250.0mg,46.9%)。1H NMR(400MHz,DMSO-d6)δ8.49(d,J=5.0Hz,1H),8.23(d,J=8.4Hz,1H),8.12(d,J=2.2Hz,1H),8.10(d,J=2.2Hz,1H),8.05(d,J=8.1Hz,1H),8.02(s,1H),7.99-7.95(m,2H),7.89(t,J=7.7Hz,1H),7.70(s,2H),7.64(dd,J=8.4,1.3Hz,1H),3.89(d,J=1.1Hz,3H),2.85-2.76(m,2H),2.54(dd,J=13.8,6.8Hz,2H),1.63(dt,J=17.0,8.1Hz,2H),1.58-1.51(m,2H),1.52-1.44(m,2H),1.40(dd,J=15.1,8.0Hz,2H),1.32(d,J=14.6Hz,2H).MS(ESI)m/z calcd.for C39H29ClF3N3O5[M+H]+=711.2,found711.0。
向AI-2-L9(270.0mg,0.38mmol)的N,N-二甲基甲酰胺(2mL)溶液中,加入异硫氰酸荧光素(67.0mg,0.17mmol),二异丙基乙胺(44.3mg,0.34mmol)。加入完毕后,避光氮气保护,室温反应过夜。反应混合物加水(10mL)淬灭后,用乙酸乙酯萃取(3×20mL)。合并的有机层用饱和食盐水洗涤并用无水硫酸镁干燥。过滤后蒸去溶剂得到粗品,将粗品溶于四氢呋喃(4mL)溶液中,加入氢氧化锂(24mg,1.0mmol,1.0equiv.)的水溶液(2mL)。避光氮气保护,室温反应3.0小时。TLC检测反应完全后,减压蒸除四氢呋喃,调节溶液pH至5~6。使用乙酸乙酯进行萃取(3×20mL),合并的有机相使用硅胶柱层析纯化得到终产物P9,为橙黄色固体(38.4mg,31.1%)。1H NMR(400MHz,DMSO-d6)δ11.42(s,1H),9.74(s,1H),8.49(s,1H),8.18(d,J=8.4Hz,1H),8.08(s,1H),8.04(d,J=8.0Hz,1H),7.99(d,J=7.6Hz,3H),7.88(d,J=7.8Hz,1H),7.80(d,J=8.1Hz,1H),7.72(d,J=8.0Hz,2H),7.63(dd,J=8.4,1.5Hz,1H),6.90(d,J=8.2Hz,1H),6.67(d,J=9.2Hz,3H),6.05(d,J=9.1Hz,2H),5.97(s,2H),2.56(d,J=7.1Hz,2H),2.03-1.98(m,2H),1.52(s,2H),1.42(d,J=7.3Hz,2H).HRMS(ESI):m/zcalcd.for C51H41ClF3N5O8S[M+H]+=956.1894,found 955.1955.HPLC:tR=8.524min,96.3%纯度。
实施例10本发明部分化合物的光学与生物学实验
1)荧光探针在Tris缓冲溶液中的光学性质
使用Tris缓冲溶液(20mM Tris,150mM NaCl,5mM DTT,pH=8)将以上实施例1~9制备得到的荧光探针P1~P9从母液(10mM)稀释至100μM。使用多功能酶标仪(SpectraMaxM5),设置激发波长为490nm,扫描待测探针在410~516nm范围下相应的荧光激发强度,获得探针的荧光激发光谱。使用多功能酶标仪(SpectraMax M5),设置发射波长(525nm),扫描待测探针在490~624nm下相应的荧光发射强度值,获得探针的荧光发射光谱。使用水配制1μM荧光素,并检测1μM荧光素的积分荧光强度与在激发波长(490nm)下的最大吸光度值。检测100μM的荧光探针的积分荧光强度与在激发波长下(490nm)的最大吸光度值。将所获各参数代入量子产率计算公式进行荧光探针溶液的荧光量子产率的计算计算公式为:
式中YU为待测末知样品的量子产率;YS为标准物质的荧光量子产率;FU、FS为待测样、标准物稀溶液的积分荧光强度;AU、AS为待测样、标准物对在激发波长的最大吸光度值。
实验结果如表1所示,本发明所述的小分子荧光探针均可在激发光照射下发射荧光,最大发射波长均为520nM左右。部分探针如P1与P8具有较高的荧光量子产率。
表1本发明荧光探针的最大激发、发射波长与荧光量子产率
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2)表面等离子体共振法检测探针与RORγ-LBD的亲和力
将RORγ-LBD(a.a.261-518)蛋白溶解于PBS缓冲溶液(pH=8,150mM NaCl,0.05%tween-20)。使用EDC(40mg/mL)活化SPR芯片(CM5)表面的羧基基团后,将RORγ-LBD蛋白固定在SR7000 GOLD SENSOR SLIDE传感器芯片(CM5,Biacore)上。蛋白固定完毕后使用NHS(5mg/mL)封闭芯片。将RORγ-LBD蛋白与不同浓度的荧光探针(250、125、62.5、31.25、15.625、7.81、3.91、1.95、0.977、0.488μM)在含有5% DMSO、0.05%tween20的PBS(1X)缓冲液中孵育。信号使用Reichert4SPR仪器记录并分析。
实验结果如表2所示,本发明所述的9种荧光探针均可与RORγ-LBD结合。探针P1、P2、P3、P8具有较好的RORγ-LBD亲和力。
表2表面等离子体共振法检测本发明荧光探针与RORγ-LBD的亲和力
化合物 | Kd a |
P1 | B |
P2 | B |
P3 | B |
P4 | C |
P5 | C |
P6 | C |
P7 | C |
P8 | A |
P9 | C |
aA:小于1μM;B:1μM至10μM;C:大于10μM,小于100μM。
实施例11利用荧光偏振技术与荧光探针P8测试化合物对RORγ变构位点的亲和力
荧光偏振(fluorescence polarization,FP)是荧光分子发出的光沿不同偏振轴具有不相等强度的现象,该现象目前被广泛用于检测大分子与小分子的结合,具有快捷、稳定、便宜等优点(Methods Appl.Fluoresc.2016,4,022001)。在本发明中,当荧光探针与RORγ-LBD蛋白结合时,其荧光偏振信号,毫偏值(millipolarization units,mP)相比未结合时会上升。此时,在该体系中加入与荧光探针竞争与RORγ-LBD变构位点结合的待测化合物,将导致mP下降,根据下降的程度判断待测化合物与RORγ-LBD的亲和力强弱。
以下是以探针P8为荧光探针测试化合物对RORγ变构位点抑制活性的实施例:
1)测试探针-蛋白曲线确定最佳蛋白工作浓度
在384孔黑板(#3575,Corning)的待测孔中加入20μL荧光探针P8(终浓度100nM),20μL的Tris缓冲溶液(20mM Tris,150mM NaCl PH=8.0,0.05%Tween-20)与20μL RORγ-LBD(a.a.261-518)蛋白。RORγ-LBD蛋白的最终工作浓度从100nM开始进行1.5倍稀释,每个浓度设置3个复孔,总共13个浓度梯度。加入完毕后,于4℃孵育0.5小时。随后使用多功能酶标仪(SpectraMax M5)的荧光偏振模块(ex:490nm,em:525nm)读取各孔的mP值,数据使用Graph pad Prism 7.0进行曲线拟合并作图分析(图1)。实验结果表明,当RORγ-LBD蛋白浓度为60nM时,荧光偏振信号达到上平台,因此选择使用60nM作为RORγ-LBD蛋白的工作浓度。
2)利用探针P8测试化合物与RORγ-LBD变构位点亲和力
本实施例测试了已报道的RORγ变构抑制剂MRL003、MRL058、MRL871与RORγ正构抑制剂GSK2981278,通过比较本方法测试的活性与文献报道的活性来验证本方法的可靠性。
在384孔黑板(#3575,Corning)的待测孔中加入20μL荧光探针P8(终浓度100nM),与20μL RORγ-LBD(终浓度60nM)蛋白与20μL相应浓度的待测化合物。MRL-003浓度从33μM开始两倍倍稀释至1.52nM,MRL-058、MRL871与GSK2981278的测试浓度从10μM开始三倍稀释至0.057nM,每个浓度设置三个复孔。加入完毕后,4℃孵育0.5小时,随后使用多功能酶标仪(SpectraMax M5)的荧光偏振模块(ex:490nm,em:525nm)读取各孔的mP值,数据使用Graphpad Prism 7.0进行曲线拟合计算各化合物的IC50值(图2)。实验结果表明化合物MRL003、MRL058、MRL871的IC50分别为3635.0nM、90.8nM、2.0nM,结果与这三个化合物报道的活性数据相似,这说明利用本发明荧光探针P8的活性测试方法可较好地验证化合物是否具有RORγ-LBD变构位点亲和力。同时,已报道的靶向RORγ配体结合结构域正构位点的阳性药物GSK2981278使用该活性检测方法未检测出活性,这说明利用本发明荧光探针P8的活性测试方法不会受到RORγ正构抑制剂影响。
实施例12荧光探针P8用于RORγ高表达细胞的成像
选取高表达RORγ的A375细胞进行成像实验。将A375细胞接种于6孔板中,分为探针组与阳性药探针组两组,每组三个复孔。阳性药探针组加入25μM阳性化合物MRL871孵育过夜,探针组不处理。次日,吸弃培养基,各孔使用PBS洗涤(3×1mL)后,加入4%多聚甲醛固定液(1mL,P0099,Beyotime)室温固定15分钟。随后吸弃固定液,使用PBS洗涤(3×1mL)后,在各孔中均加入终浓度为10μM探针P8(1mL),室温孵育30分钟。随后,吸弃探针溶液,PBS洗涤(3×1mL),加入少量可覆盖孔底的DAPI细胞核染色液(C1005,Beyotime)室温染色3-5分钟。染色完毕后吸弃染色液,PBS洗涤(3×1mL)后,在Zeiss LSM 800(Carl Zeiss,Germany)荧光倒置显微镜下观察并记录荧光成像结果,相关荧光参数为:FITC(Ex:494nm/Em:518nm)、DAPI(Ex:359nm/Em:461nm)。实验结果(图3)表明的结果中可以看出,P8作为RORγ特异性的绿色荧光探针,可清晰的染色高表达RORγ的A375细胞。此外在阳性药探针组,RORγ变构抑制剂MRL871处理过的A375细胞中,荧光强度显著低于探针组,这也表明荧光探针P8在成像实验中具有较好的特异性。
实施例13荧光探针P8用于斑马鱼体内成像
收集AB野生型斑马鱼胚胎,在光照培养箱中饲养至72小时后,将斑马鱼置于6孔板中,随机分成空白对照组,探针组和阳性药探针组3组。每孔20条斑马鱼幼鱼,设立三个平行组。随后,在探针组加入终浓度为10μM的探针P8,阳性药探针组中同时加入终浓度为10μM探针P8与终浓度为25μM的阳性化合物MRL871,空白对照组不处理。室温避光孵育30分钟后,吸弃各孔的溶液并用PBS洗涤三遍。挑选各孔斑马鱼在体视荧光显微镜(Leica DFC17000T)下观察并记录成像结果。实验结果如图4所示,在72hpf的斑马鱼体内,其RORγ的表达主要集中于肝、肠等器官,在探针组中的斑马鱼肠道当中发出显著绿色荧光。此外使用P8与MRL871共同孵育的斑马鱼肠道中,绿色荧光显著降低。表明RORγ变构抑制剂MRL871可以与荧光探针P8竞争结合,与细胞成像实验结果一致,也说明了探针P8具有较好的RORγ特异性。
Claims (10)
1.一种靶向RORγ的新型小分子荧光探针,其包括如下通式(I)的化合物或其药学上可接受的盐:
在通式(I)中:
L代表L1L2/>
L3
L4L5/>
L6
L7L8/>
或L9
2.权利要求1所述的靶向RORγ的新型小分子荧光探针的制备方法,其特征在于,包括以下步骤:
S1:将原料AI-1与一氧化碳及不同二胺类化合物在碱、催化剂催化条件下高温反应得到中间体AI-2-L1~3,7,8;
S2:将原料AI-1与二胺类化合物在碱、催化剂、配体条件下利用Buchwald-Hartwig反应偶联得到中间体AI-2-L4~6;
S3:将原料AI-1与2-(7-辛炔-1-基)-1H-异吲哚-1,3-二酮在碱、催化剂条件下利用Sonogashira反应偶联得到中间体AI-L9;
S4:将原料AI-2-L1~8溶于二氯甲烷中,在酸性条件下室温反应,脱去叔丁氧羰基Boc得到化合物AI-3-L1~8;
S5:将中间体AI-2-L9在乙醇中与水合肼回流反应,脱去保护基得到化合物AI-3-L9;
S6:将中间体AI-3-L1~9与异硫氰酸荧光素(FITC)在碱性条件下进行缩合反应,所得中间体直接在碱水溶液下水解脱去甲基,得到终产物P1~P9;
。
3.根据权利要求2所述的靶向RORγ的新型小分子荧光探针的制备方法,其特征在于,步骤S1中,所述的二胺类化合物为单叔丁氧羰基Boc保护的二胺;所使用的一氧化碳压力为1到3个大气压;所述反应的有机溶剂为N,N-二甲基甲酰胺、1,4-二氧六环或四氢呋喃中的一种或几种;反应选用的碱为N,N-二异丙基乙胺或三乙胺中的一种或几种;反应选用催化剂为1,1'-双(二苯基膦基)二茂铁二氯化钯、醋酸钯或四三苯基膦钯中的一种或几种;反应温度为80-120℃。
4.根据权利要求2所述的靶向RORγ的新型小分子荧光探针的制备方法,其特征在于,所述的步骤S2中,所述Buchwald-Hartwig反应,选用的催化剂为Pd2(dba)3、Pd(OAc)2或Pd(Dppf)Cl2;选用的碱为N,N-二异丙基乙胺、三乙胺、碳酸铯、叔丁醇钾或叔丁醇钠中的一种或几种;选用的配体为2-二环己基磷-2',4',6'-三异丙基联苯或1,1'-联萘-2,2'-双二苯膦,选用溶剂为1,4-二氧六环。
5.根据权利要求2所述的靶向RORγ的新型小分子荧光探针的制备方法,其特征在于,所述的步骤S3中,所述Sonogashira反应选用的催化剂为Pd2(dba)3、Pd(OAc)2、Pd(dppf)Cl2或CuI,选用的有机溶剂为N,N-二异丙基乙胺或三乙胺中的一种或几种。
6.根据权利要求2所述的靶向RORγ的新型小分子荧光探针的制备方法,其特征在于,所述的步骤S4中,所述的酸性试剂为三氟乙酸或盐酸二氧六环溶液。
7.根据权利要求2所述的靶向RORγ的新型小分子荧光探针的制备方法,其特征在于,所述的步骤S6中,缩合反应所使用的碱性条件为N,N-二异丙基乙胺;使用的溶剂为N,N-二甲基甲酰胺;反应条件为室温,氮气保护;所述碱水溶液为氢氧化锂水溶液。
8.权利要求1所述的小分子荧光探针在测试化合物对RORγ亲和力中的应用。
9.小分子荧光探针在测试化合物对RORγ亲和力的方法,具体包括如下步骤:
(1)将通式(I)的RORγ小分子荧光探针,包含RORγ配体结合结构域的蛋白与待测化合物以一定比例混合得待测体系;
S2:采用荧光偏振技术,通过多功能酶标仪测定待测体系的荧光偏振值,根据荧光偏振值确定待测化合物是否能与RORγ的变构位点结合。
10.权利要求1所述的小分子荧光探针在细胞成像或体内成像中的应用。
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