CN114106027B - 一种氟硼荧光染料-四嗪类荧光探针及其制备方法和用途 - Google Patents
一种氟硼荧光染料-四嗪类荧光探针及其制备方法和用途 Download PDFInfo
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- CN114106027B CN114106027B CN202111327864.0A CN202111327864A CN114106027B CN 114106027 B CN114106027 B CN 114106027B CN 202111327864 A CN202111327864 A CN 202111327864A CN 114106027 B CN114106027 B CN 114106027B
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
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Abstract
本发明提供了式I所示的化合物或其盐、水合物;具有优异光学性能,具体表现为高量子产率、高摩尔消光系数和大斯托克斯位移,生物正交反应后大荧光增强倍数高,具有很好的作为细胞组织活体示踪的荧光探针的潜力。进一步引入卤素取代基修饰,可提升单态氧产率,得到能够克服非特异光毒性的光动力荧光探针,在光动力治疗中有潜在应用价值。
Description
技术领域
本发明属于化学合成领域,具体涉及一种氟硼荧光染料-四嗪类荧光探针及其制备方法和用途。
背景技术
荧光成像技术在药学和临床诊断学,分子生物学和生物化学,材料化学和分析化学等不同领域中发挥着重要的作用。该技术在对活细胞内物质检测和成像时有显著的优势,不仅有高灵敏度和高特异性,同时还具有操作简便,对细胞创伤低等优点。目前,该领域的成像技术进步显著,然而探针的开发不足成为了探索细胞及生物体内成像的限制因素。
目前,开发用于体内成像的反应型荧光探针的问题在于,如何实现智能传感器反应(生理条件下选择性检测目标分析物)、快速反应动力学(实时检测生物过程)、信号增强(减少背景干扰和高灵敏度)、以及从环境敏感的荧光信号中提取可靠的数据。生物正交反应指在活体细胞或组织中能够在不干扰生物自身生化反应的条件下可以进行的化学反应,这种反应生物兼容性好、选择性优越、反应快速,基于生物正交反应构建的“开-关”型荧光探针,可以较好地解决上述体内成像的反应型荧光探针开发的问题。
四嗪的生物正交反应具有较高的反应特异性和较好的反应动力学,能够选择性地标记以及追踪体内和体外的细胞和生物分子,已经广泛应用于荧光探针的开发。四嗪可作为荧光淬灭基团,当其吸收光谱与荧光染料的发射光谱有一定重叠且两者相隔距离适宜时,可通过荧光共振能量转移(FRET)原理,实现对荧光的调控,还可通过跨键能量转移原理(TBET),对荧光起到淬灭作用,并且可通过分子内电荷转移(intramolecular charge-transfer,ICT)原理,实现对远红\近红外荧光染料的淬灭。而通过四嗪的生物正交反应再实现荧光的开启,不需要洗涤步骤,即可进行高信噪比和高时空分辨率的活细胞活体荧光成像。
氟硼荧光染料(BODIPY)是荧光染料领域的研究热点。自首次合成以来已经被广泛用于荧光标记、生物成像以及光动力治疗等领域。BODIPY具有优异的光学性质:良好的光稳定性和化学稳定性、相对高的摩尔消光系数(通常ε>80000M-1·cm-1)和量子产率(通常ΦF>0.50)等。因此,以BODIPY作为荧光染料,结合四嗪作为荧光淬灭基团制备荧光探针吸引了研究人员的关注。但是,因为在BODIPY骨架的不同位点引入修饰不同基团,对BODIPY的性能会出现不同程度的影响,因此,在对BODIPY骨架荧光染料进行四嗪连接修饰时,修饰位点、连接方式的不同将会直接影响荧光染料本身和最终制得的荧光探针的性能,而现有报道的BODIPY-四嗪类荧光探针的光学性能往往有多种不足。
例如,Devaraj等人将Carlson等人通过烷基链将3-(4-苄基氨基)-1,2,4,5-四嗪连接修饰于荧光染料BODIPY FL上,制得了如下结构的探针,但其与亲二烯体反应后荧光增强仅仅只有15倍(Devaraj NK,Hilderbrand S,Upadhyay R,et al.Bioorthogonal turn-on probes for imaging small molecules inside living cells[J].Angew Chem IntEd,2010,49:2869-2872)。
Carlson等人则在BODIPY染料的C-8位通过共轭苯环结构连接修饰了四嗪结构:
虽然其与亲二烯体反应后荧光增强高达900倍,但是修饰后的基团对BODIPY染料斯托克斯位移、吸收波长、量子产率等关键光学性能的影响却难以考证(Carlson JCT,Meimetis LG,Hilderbrand SA,et al.BODIPY tetrazine derivatives as superbrightbioorthogonal turn-on probes[J].Angew Chem Int Ed,2013,52:6917-6920.)。
因此,进一步通过恰当的修饰制备新的综合光学性能优异的BODIPY-四嗪类荧光探针,进一步丰富荧光探针的类型,推动器在临床诊断检测中的应用,具有重要意义。
发明内容
本发明的目的在于提供一种光学性能优异的BODIPY-四嗪类荧光探针。
本发明提供了式I所示的化合物或其盐、水合物:
其中R1为或C1~C3的烷基;Ra为C1~C3的烷基;
R2为H或C1~C3的烷基;L为L’为无或苯环,n为0~2的整数;
R3为C1~C3的烷基或Rd、Re分别独立选自/>R’为C1~C3的烷基或/>p为1~4的整数;
R4为H或卤素。
进一步地,上述R1为或甲基;
R2为H或甲基;L为L’为无或苯环,n为1或2;
R3为甲基或Rd、Re为/>R’为/>p为2~3的整数;
R4为H或I。
更进一步地,上述L为
R3为甲基或
进一步地,上述的化合物具有式II所示结构:
优选地,其中R1为
更优选地,其中R2为甲基和/或R3为甲基;
更进一步地,上述化合物具有如下结构:
进一步地,上述化合物具有式III所示结构:
优选地,所述R3为更进一步地,上述化合物具有如下结构:
本发明还提供了上述的化合物的制备方法,R1为所述制备方法包括如下步骤:
(1)以化合物A为原料,与化合物B在室温下反应制得化合物C;
(2)化合物C与化合物D在有机碱作用下室温反应得到化合物E即为所述化合物;
或化合物E和卤素单体、高卤酸在50~70℃反应制得化合物E’即为所述化合物;
或化合物E或化合物E’与化合物F在催化剂作用下于100~120℃反应制得化合物G即为所述化合物;
反应式如下:
其中,X为卤素;
或R1为C1~C3烷基,所述制备方法包括如下步骤:
(a)以化合物A为原料,与化合物B’在有机碱作用下室温反应制得化合物C’即为所述化合物。
优选地,所述有机碱为三乙胺,所述催化剂为哌啶和冰醋酸。
本发明还提供了上述式II的化合物在细胞成像试剂中的应用,优选地,所述细胞成像试剂是荧光探针。
本发明还提供了上述式III的化合物在光动力治疗用试剂中的应用。
本发明的有益效果:本发明通过对BODIPY荧光染料的C-8位进行酰基化修饰,并进一步结合四嗪结构,制得了具有优异的光学性能的荧光探针,具体表现为高量子产率、大斯托克斯位移,四嗪能够有效淬灭BODIPY的荧光并且能够在生物正交反应后获得大荧光增强倍数,具有很好的作为细胞组织活体示踪的荧光探针的潜力。进一步引入卤素取代基修饰,可增加分子单重态至三重态的系间穿越能力,从而提升单态氧产率,得到能够克服非特异光毒性的光动力荧光探针,在光动力治疗中有潜在应用价值。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为BODIPY-四嗪荧光探针的培养基稳定性:(a)荧光探针F14-F18的培养基稳定性;(b)光动力荧光探针F19和F20的培养基稳定性;F17的监测波长为455nm,F18的监测波长为660nm,其余探针监测波长为520nm,所有实验均重复三遍。
图2为抗体预定位细胞成像示意图。
图3为探针F14用于抗体预定位细胞成像图:(a)实验组:100nM TCO-曲妥珠单抗工作液与5μM探针F14;(b)对照组:100nM曲妥珠单抗与5μM探针F14;Ex:514nm,Em:521-650nm,Scale bar:50μm。
图4为BODIPY-四嗪光动力荧光探针的单态氧检测实验:(a)可见光探针F19及其生物正交产物随光照时间延长于414nm处的吸光度值变化,DMF中Φ△RB=0.39;(b)近红外探针F20及其生物正交产物随光照时间延长于414nm处的吸光度值变化,乙醇中Φ△MB=0.49。
图5为探针的光动力活性实验:(a)不同浓度探针F20在660nm激光(光照强度7.5J/cm2)照射下于A549细胞上的光毒性和暗毒性;(b)BCN-TPP的化学结构。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品或通过说明书记载的合成方式自行制备所得。
本发明实施例的实验得到国家自然科学基金(21801178)和中央高校基本科研业务费专项资金(2019SCU12025,四川大学专职博士后基金)的支持。
实施例1、本发明化合物的合成
1、本发明化合物F14的合成:
四嗪B9的合成:
将四嗪T16(480mg,2mmol)加入到100mL反应瓶内,加入26mL的DCM和三氟乙酸酐(26mL,190mmol)。室温(25℃)下搅拌1h,经TLC确认反应完全后,加入饱和碳酸氢钠溶液淬灭反应,DCM(20mL×3)萃取,有机相用饱和食盐水洗(30mL×1),无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得红色液体化合物B9(200mg),收率:72%。
1H NMR(400MHz,MeOD)δ3.68(t,J=6.3Hz,2H),3.61(t,J=6.4Hz,2H),3.02(s,3H).
化合物B10的合成:
将B1(53.2mg,0.2mmol)和B9(56mg,0.4mmol)加入到10mL反应管内,氩气保护下,加入2mL的乙腈;再将三乙胺(56μL,0.4mmol)滴加到反应液中;然后在室温(25℃)下搅拌20h,经TLC确认反应完全后,浓缩,采用硅胶柱层析纯化后,得黄色固体化合物B10(20mg),收率:28%。
1H NMR(400MHz,CDCl3)δ7.13(s,1H),6.99(d,J=3.4Hz,2H),6.19(d,J=3.6Hz,2H),4.18(dt,J=10.8,5.4Hz,2H),3.77(t,J=6.1Hz,2H),3.10(s,3H),2.56(s,6H).
13C NMR(101MHz,CDCl3)δ167.51,166.07,146.85,144.31,129.02,129.00,128.71,128.68,122.17,114.28,43.37,32.23,20.20,13.12.
HRMS[M–H]–m/z calcd.[C16H17BF2N7]–356.1612,found 356.1622.
本发明化合物F14的合成:
将B10(9mg,0.025mmol)加入到10mL反应瓶内,氩气保护下,加入0.5mL的DCM。在冰浴下滴加DIPEA(12.5μL,0.075mmol),乙酰氯(4μL,0.05mmol)。然后在室温(25℃)下搅拌24h,经TLC确认后,浓缩,采用硅胶柱层析纯化后,得橙红色固体化合物F14(4mg),收率:40%。
1H NMR(400MHz,CDCl3)δ7.17(d,J=3.7Hz,2H),6.36(d,J=4.0Hz,2H),4.34(t,J=6.3Hz,2H),3.57(t,J=6.4Hz,2H),3.04(s,3H),2.64(s,6H),1.92(s,3H).
13C NMR(101MHz,CDCl3)δ170.26,167.59,165.09,160.26,133.69,133.60,130.04,129.94,129.75,127.74,120.91,120.86,49.45,34.37,22.70,22.07,15.07,14.13.
HRMS[M+H]+m/z calcd.for[C18H21BF2N7O]+400.1863,found 400.1863.
2、本发明化合物F15、F16、F18的合成:
化合物B12、B13、B14、F15、F18根据路线一合成,化合物B15、B16、B17、F16根据路线二合成。
化合物B11的合成:
化合物B11根据参考文献[77]合成,具体操作如下:将4-氰基苯甲胺(1g,7.6mmol)加入到200mL反应瓶内,氩气保护下,加入75mL的DCM和TEA(1.6mL,11.4mmol)。将二碳酸二叔丁酯(1.9mL,8.3mmol)溶于5mL的DCM中,在冰浴下滴加进反应液中。然后在室温(25℃)下搅拌20h,经TLC确认反应完全后,浓缩,加入乙醚稀释反应液,盐酸(1M)洗涤萃取,有机相用饱和碳酸氢钠溶液洗(30mL×1),饱和食盐水洗(30mL×1),无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得无色液体化合物B11(1.6g),收率:91%。
1H NMR(400MHz,CDCl3)δ7.62(d,J=8.2Hz,2H),7.39(d,J=8.2Hz,2H),5.02(dd,J=14.0,8.4Hz,1H),4.37(d,J=5.7Hz,2H),1.46(s,9H).
化合物B12的合成:
将B11(696mg,3mmol),乙腈(1.58mL,30mmol),3-巯基丙酸(261μL,3mmol)加入50mL反应瓶内;氩气保护下,在冰浴搅拌下,加入水合肼(9.3mL,150mmol),然后在60℃油浴锅里搅拌反应16h;将反应液倒入亚硝酸钠(4.2g,60mmol)冰水溶液中,在0℃搅拌下,缓慢加入盐酸(1M)至溶液pH为3–4,然后继续搅拌5min。用二氯甲烷萃取水相(50mL×3),饱和食盐水洗涤有机相,无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得红色液体化合物B12(342mg),收率:38%。
1H NMR(400MHz,CDCl3)δ8.56(d,J=8.3Hz,2H),7.50(d,J=8.2Hz,2H),4.98(s,1H),4.44(d,J=5.6Hz,2H),3.09(s,3H),1.48(d,J=3.5Hz,9H).
化合物B13的合成:
将B12(180mg,0.6mmol)加入到50mL反应瓶内,加入8mL的DCM和三氟乙酸酐(8mL,58mmol)。室温(25℃)下搅拌0.5h,经TLC确认反应完全后,加入饱和碳酸氢钠溶液淬灭反应,DCM(20mL×3)萃取,有机相用饱和食盐水洗(30mL×1),无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得红色液体化合物B13(89mg),收率:74%。
1H NMR(400MHz,MeOD)δ8.63(d,J=8.3Hz,2H),7.71(d,J=8.3Hz,2H),4.26(s,2H),3.05(s,3H).
13C NMR(101MHz,MeOD)δ167.69,163.53,137.37,132.98,129.41,128.05,42.53,19.71.
化合物B14的合成:
将B1(144mg,0.54mmol)和B13(120mg,0.6mmol)加入到10mL反应管内,氩气保护下,加入2mL的乙腈;再将三乙胺(90μL,0.65mmol)滴加到反应液中;然后在60℃油浴锅里搅拌3h,经TLC确认反应完全后,浓缩,采用硅胶柱层析纯化后,得黄色固体化合物B14(108mg),收率:47%。
1H NMR(400MHz,CDCl3)δ8.65(d,J=8.3Hz,2H),7.59(d,J=8.3Hz,2H),6.92(d,J=3.9Hz,2H),6.32(t,J=5.3Hz,1H),6.19(d,J=3.9Hz,2H),4.93(d,J=5.6Hz,2H),3.12(s,3H),2.58(s,6H).
13C NMR(101MHz,CDCl3)δ167.59,163.61,145.64,139.98,132.34,128.96,128.37,123.15,115.46,50.92,21.23,14.20.
HRMS[M+Na]+m/z calcd.for[C21H20BF2N7Na]+442.1734,found 442.1739.
化合物F15的合成:
将化合物B14(100mg,0.24mmol)加入到10mL反应瓶内,氩气保护下,加入3mL的DCM。在冰浴下滴加DIPEA(119μL,0.72mmol),乙酰氯(34μL,0.48mmol)。然后在室温(25℃)下搅拌39h,经TLC确认后,浓缩,采用硅胶柱层析纯化后,得橙红色固体化合物F15(101mg),收率:87%。
1H NMR(400MHz,CDCl3)δ8.50(d,J=8.3Hz,2H),7.53(d,J=8.3Hz,2H),6.58(d,J=4.2Hz,2H),6.20(d,J=4.2Hz,2H),5.01(s,2H),3.10(s,3H),2.61(s,6H),2.05(s,3H).
13C NMR(101MHz,CDCl3)δ170.11,167.33,163.83,160.08,141.28,138.68,133.60,131.55,130.68,128.09,127.45,120.55,54.21,22.35,21.17,15.07.
HRMS[M+Na]+m/z calcd.for[C23H22BF2N7NaO]+484.1839,found 484.1842.
化合物F18的合成:
将化合物F15(35mg,0.076mmol)和化合物7(98mg,0.228mmol)加入到10mL反应管内,氩气保护下,加入2mL的DMF。在冰浴下滴加哌啶(22μL,0.38mmol),冰醋酸(35μL,0.38mmol)。然后在室温(25℃)下搅拌3h,经TLC确认反应完全后,浓缩,采用硅胶柱层析纯化后,得绿色油状化合物F18(40mg),收率:51%。
1H NMR(400MHz,CDCl3)δ8.51(d,J=8.2Hz,2H),7.62–7.48(m,4H),7.29(s,3H),7.24(s,1H),7.13(s,2H),6.95(d,J=8.5Hz,2H),6.81(d,J=4.5Hz,2H),6.61(d,J=4.5Hz,2H),5.03(s,2H),4.24(dt,J=9.7,5.0Hz,8H),3.90(t,J=4.7Hz,8H),3.77(dd,J=9.8,4.8Hz,8H),3.74–3.59(m,16H),3.55(ddd,J=9.3,5.6,3.7Hz,8H),3.37(d,J=10.2Hz,12H),3.10(s,3H),2.08(s,3H).
13C NMR(101MHz,CDCl3)δ170.64,167.28,163.85,156.42,150.72,148.90,141.41,138.33,135.25,134.27,131.48,130.76,129.80,128.09,126.09,121.94,117.22,117.12,114.44,114.14,71.90,70.84,70.65,70.49,69.75,69.60,69.18,68.66,59.00,58.97,50.67,22.28,21.13.
HRMS[M+Na]+m/z calcd.for[C65H86BF2N7NaO17]+1308.6034,found 1308.6031.
化合物B15的合成:
将B11(1g,4.3mmol),醋酸甲脒(4472mg,43mmol),三氟甲烷磺酸锌(313mg,0.86mmol)加入20mL反应瓶内;氩气保护下,在冰浴搅拌下,加入无水肼(6.8mL,215mmol),然后在30℃油浴锅里搅拌反应36h;将反应液倒入亚硝酸钠(6.1g,87mmol)冰水溶液中,在0℃搅拌下,缓慢加入盐酸(1M)至溶液pH为3–4,然后继续搅拌5min。用二氯甲烷萃取水相(50mL×3),饱和食盐水洗涤有机相,无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得红色液体化合物B15(454mg),收率:37%。
1H NMR(400MHz,CDCl3)δ8.60(d,J=8.4Hz,2H),7.53(d,J=8.3Hz,2H),4.45(d,J=5.7Hz,2H),1.49(s,9H).
化合物B16的合成:
将B15(200mg,0.7mmol)加入到50mL反应瓶内,加入8.5mL的DCM和三氟乙酸酐(8.5mL,61mmol)。室温(25℃)下搅拌1h,经TLC确认反应完全后,加入饱和碳酸氢钠溶液淬灭反应,DCM(20mL×3)萃取,饱和食盐水洗涤有机相(30mL×1),无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得红色液体化合物B16(100mg),收率:76%。
1H NMR(400MHz,MeOD)δ8.67(d,J=8.4Hz,2H),7.73(d,J=8.3Hz,2H),4.27(s,2H).
化合物B17的合成:
氩气保护下,将B1(26.6mg,0.1mmol)和B16(22.4mg,0.12mmol)加入到10mL反应管内,加入1mL的乙腈;再将三乙胺(21μL,0.15mmol)滴加到反应液中;然后在室温(25℃)下搅拌4h,经TLC确认反应完全后,浓缩,采用硅胶柱层析纯化后,得黄色固体化合物B17(17mg),收率:42%。
1H NMR(400MHz,CDCl3)δ8.71(d,J=8.3Hz,2H),7.62(d,J=8.3Hz,2H),6.93(d,J=3.8Hz,2H),6.20(d,J=3.9Hz,2H),4.97(d,J=5.7Hz,2H),2.59(s,6H).
HRMS[M–H]–m/z.for[C20H17BF2N7]–404.1612,found 404.1621.
化合物F16的合成:
将B17(10mg,0.025mmol)加入到10mL反应瓶内,氩气保护下,加入0.6mL的DCM。在冰浴下滴加DIPEA(12.4μL,0.075mmol),乙酰氯(3.6μL,0.05mmol)。然后在室温(25℃)下搅拌15h,经TLC确认后,浓缩,采用硅胶柱层析纯化后,得橙红色固体化合物F16(10mg),收率:89%。
1H NMR(400MHz,CDCl3)δ8.54(d,J=8.4Hz,2H),7.56(d,J=8.4Hz,2H),6.59(d,J=4.1Hz,2H),6.20(d,J=4.2Hz,2H),5.02(s,2H),2.61(s,6H),2.05(s,3H).
HRMS[M–H]–m/z.for[C22H19BF2N7O]–446.1718,found 446.1725.
化合物B18的合成:
将3-甲胺基丙腈(3g,35.7mmol)加入到200mL反应瓶内,氩气保护下,加入30mL的DCM和TEA(7.4mL,53.6mmol)。将二碳酸二叔丁酯(12.3mL,53.6mmol)溶于5mL的DCM中,在冰浴下滴加进反应液中。然后在室温(25℃)下搅拌18h,经TLC确认反应完全后,浓缩,加入乙醚稀释反应液,盐酸(1M)洗涤萃取,有机相用饱和碳酸氢钠溶液洗(30mL×1),饱和食盐水洗(30mL×1),无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得无色液体化合物B18(5.5g),收率:84%。
1H NMR(400MHz,CDCl3)δ3.51(t,J=6.6Hz,2H),2.97(s,3H),2.60(s,2H),1.47(s,9H).
化合物B19的合成:
将B18(800mg,4.35mmol),无水乙腈(1.58mL,30mmol),三氟甲烷磺酸镍(776mg,2.2mmol)加入50mL反应瓶内;氩气保护下,在冰浴搅拌下,加入无水肼(5mL,157mmol),然后在42℃油浴锅里搅拌反应18h;将反应液倒入亚硝酸钠(6.1g,87mmol)冰水溶液中,在0℃搅拌下,缓慢加入盐酸(1M)至溶液pH为3–4,然后继续搅拌5min。用二氯甲烷萃取水相(50mL×3),饱和食盐水洗涤有机相,无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得红色液体化合物B19(150mg),收率:14%。
1H NMR(400MHz,CDCl3)δ3.51(t,J=6.6Hz,2H),2.97(s,3H),2.59(d,J=13.9Hz,2H),1.47(s,9H),1.34(s,3H).
13C NMR(101MHz,CDCl3)δ167.94,155.46,154.86,80.62,80.37,45.36,35.74,34.78,28.34,28.27,21.09,16.66.
HRMS[M+H]+m/z calcd.for[C11H20N5O2]+254.1612,found 254.1610.
化合物B20的合成:
将B19(130mg,0.5mmol)加入到50mL反应瓶内,加入6.5mL的DCM和三氟乙酸酐(6.5mL,47mmol)。室温(25℃)下搅拌0.5h,经TLC确认反应完全后,加入饱和碳酸氢钠溶液淬灭反应,DCM(20mL×3)萃取,饱和食盐水洗涤有机相(30mL×1),无水Na2SO4干燥后,过滤浓缩,采用硅胶柱层析纯化后,得红色液体化合物B20(50mg),收率:64%。
1H NMR(400MHz,CDCl3)δ3.84(t,J=6.5Hz,2H),3.64(q,J=6.9Hz,2H),3.05(s,3H),2.76(s,3H).
3、化合物F17的合成:
氩气保护下,将B1(10.4mg,0.04mmol)和B20(5mg,0.033mmol)加入到10mL反应管内,加入0.5mL的乙腈;再将三乙胺(7μL,0.05mmol)滴加到反应液中;然后在室温(25℃)下搅拌2h,经TLC确认反应完全后,浓缩,采用硅胶柱层析纯化后,得黄色固体化合物F17(5.5mg),收率:45%。
1H NMR(400MHz,CDCl3)δ6.98(d,J=4.0Hz,2H),6.20(d,J=4.0Hz,2H),4.53(t,J=7.1Hz,2H),3.80(t,J=7.1Hz,2H),3.63(s,3H),3.01(s,3H),2.55(s,6H).
13C NMR(101MHz,CDCl3)δ168.11,166.96,151.27,148.56,126.02,123.97,115.76,56.68,44.84,33.15,31.63,21.12,14.39.
HRMS[M+Na]+m/z calcd.for[C17H20BF2N7Na]+394.1734,found 394.1732.
4、化合物F19的合成:
将F15(20mg,0.04mmol)和碘(24mg,0.09mmol)加入到20mL反应瓶内,氩气保护下,加入5mL的乙醇;将高碘酸(15mg,0.08mmol)溶于160μL的水中,滴加进反应液中。然后在60℃油浴锅里搅拌5h,经TLC确认反应完全后,浓缩,采用硅胶柱层析纯化后,得黄色固体化合物F19(18mg),收率:59%。
1H NMR(400MHz,CDCl3)δ8.53(d,J=8.3Hz,2H),7.50(d,J=8.3Hz,2H),6.77(s,2H),4.99(s,2H),3.11(s,3H),2.61(s,6H),2.04(s,3H).
HRMS[M+Na]+m/z calcd.for[C23H20BF2I2N7NaO]+735.9772,found 735.9772.
5、化合物F20的合成:
将化合物F19(10mg,0.014mmol)和C9(18mg,0.042mmol)加入到10mL反应管内,氩气保护下,加入0.5mL的DMF。在冰浴下滴加哌啶(6μL,0.07mmol),冰醋酸(4μL,0.07mmol)。然后在室温(25℃)下搅拌1h,经TLC确认反应完全后,浓缩,采用硅胶柱层析纯化后,得绿色油状化合物F20(4mg),收率:19%。
1H NMR(400MHz,CDCl3)δ8.55(d,J=8.3Hz,2H),8.10(d,J=16.5Hz,2H),7.54(d,J=8.3Hz,2H),7.45(d,J=16.5Hz,2H),7.29(dd,J=8.5,1.8Hz,2H),7.14(d,J=1.8Hz,2H),6.96(d,J=8.5Hz,2H),6.83(s,2H),5.00(s,2H),4.23(dd,J=9.8,4.7Hz,8H),3.90(dt,J=8.1,4.1Hz,8H),3.76(dd,J=5.5,3.8Hz,8H),3.70–3.62(m,16H),3.54(ddd,J=11.2,5.7,3.7Hz,8H),3.37(d,J=9.7Hz,12H),3.11(s,3H),2.07(s,3H).
HRMS[M+Na]+m/z calcd.for[C65H84BF2I2N7NaO17]+1560.3966,found 1560.3970.
以下通过实验例证明本发明的有益效果。
实验例1、本发明化合物荧光探针的光学性能
对本发明化合物的光学性能以及与亲二烯体反应后的光学性能(包括发射波长λex、吸收波长λem、摩尔消光系数ε、反应后荧光增强倍数Turn on、斯托克斯位移Δλ、荧光量子产率Φ),结果如表1所示
表1 BODIPY-四嗪荧光探针的光学性能[a]
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注:[a]所有数据均在探针与相应亲二烯体完全反应后于乙醇溶剂中测量;[b]L·mol-1·cm-1;[c]Fluorescence increase;[d]Stokes shift;[e]量子产率。
BCN:TCO:/>Sph:/>Cyp:/>
从表1可以看出,本发明的化合物均具有较高的量子产率、摩尔消光系数和斯托克斯位移。虽然C-8位N-酰基荧光探针F14、F15和F16相比C-8位N-烷基荧光探针F17:虽然斯托克斯位移小,但最大吸收和发射波长分别红移80nm和20nm左右,组织穿透力更好,更有利用用于生物体内成像;从生物正交反应后的荧光增强倍数来看,双烷基四嗪取代的探针F14具有相对最优的性能,其次是F15,再次是F16。而F18通过进一步的修饰,最大吸收波长到达近红外区域,是一种新型的近红外荧光探针,其生物正交反应后的荧光增强倍数也能达到1.2倍。
以上结果说明,本发明化合物的光学性能优异,具有很好的作为荧光探针使用的潜力,尤其是荧光探针F14,具有最优的综合性能。
实验例2、本发明化合物荧光探针的稳定性
能够生物应用的新型BODIPY-四嗪荧光探针均应具有较高的稳定性。于是为了进一步探索探针的生物应用潜能,我们检测了本发明BODIPY-四嗪荧光探针在体外含10%FBS的培养基中的稳定性。
实验结果如图1所示,荧光探针F14、F15、F17、F18和光动力荧光探针F20的培养基稳定性优异,24h时仍有90%以上的稳定性;荧光探针F16稳定性略差,在6h时稳定性为85%;光动力荧光探针F19稳定性相对更差,在6h时稳定性为39%,推测为探针F19与培养基中的存在的丝氨酸发生亲核取代反应,从而导致探针结构破坏。
总的来说,本发明的荧光探针F14、F15、F17、F18和F20稳定性好,具有潜在的应用能力。
实验例3、本发明化合物荧光探针反应动力学和细胞成像
综合BODIPY-四嗪荧光探针的光学性能和培养基稳定性结果,选择荧光增强倍数较高和培养基稳定性良好的探针F14做进一步的生物应用和验证。于是测定探针F14与4e-TCO发生生物正交反应的二级速率常数k2,结果发现探针F14与4e-TCO的二级动力学速率常数为208.5M-1S-1,可以满足细胞成像的应用需求。且F14相比于其它探针,与亲二烯体的反应动力学最快。
进一步采用F14进行抗体预定位细胞成像实验,由于SKOV3细胞表达过量的表皮生长因子受体(HER2),而曲妥珠单抗作为抗体能够选择性地与HER2结合。于是我们将TCO-NHS通过氨基修饰曲妥珠单抗得到TCO-曲妥珠单抗,将TCO-曲妥珠单抗与SKOV3细胞孵育后,再孵育探针F14,通过探针F14的四嗪基团与TCO发生生物正交反应,得到荧光开启的产物,从而进行抗体预定位细胞成像,示意图如图2所示。
实验结果见图3,当使用无TCO修饰的曲妥珠单抗进行细胞孵育后,加入探针F14,细胞无荧光开启信号(图3b);(2)当使用TCO修饰的曲妥珠单抗进行细胞孵育后,加入探针F14,细胞具有荧光开启信号(图3a)。实验证明本发明新型BODIPY-四嗪生物正交荧光探针F14用于抗体预定位细胞成像时具有明显的荧光信号增强,同时有良好光学性能以及生物兼容性,具有进一步生物应用的潜力。
实验例4、本发明化合物荧光探针的单态氧产率和光动力活性
本发明通过在2,6位引入卤素(碘)取代基,以增加分子单重态至三重态的系间穿越能力,构建了可提升单态氧产率的BODIPY-四嗪光动力荧光探针(F19和F20)。通过测量探针在溶剂中的单态氧产率,初步评价探针的光动力治疗潜力。
实验结果如图4和表2所示。可见光探针F19与BCN反应后的单态氧产率(Φ△=0.302)相比反应前(Φ△=0.187)增加了1.6倍;(2)近红外探针F20与BCN反应后单态氧产率(Φ△=0.200)相比反应前(Φ△=0.170)增加了1.2倍。
表2 BODIPY-四嗪光动力荧光探针的单态氧产率
注:[a]探针F19单态氧测量使用520nm激光器;[b]DMF中Φ△RB=0.39[86];[c]探针F20单态氧测量使用660nm激光器;[d]乙醇中Φ△MB=0.49。
以上结果证明本发明的荧光探针F19、F20具有用于光动力治疗的潜力。
为了进一步评估荧光探针的光动力活性,探索其在光照下产生的单态氧对细胞的杀伤作用。我们综合光动力荧光探针的稳定性和溶剂中的单态氧产率调控能力,选用探针F20做细胞毒性实验。利用光动力荧光探针在生物正交反应前后单态氧产率的不同,考察了在有或无光照条件下,荧光探针对细胞的光毒性和暗毒性作用。首先预先孵育靶向线粒体的BCN试剂,将BCN预靶向到线粒体上,再通过荧光探针上的四嗪基团与之发生生物正交反应,从而调控探针的单态氧产生能力。
实验结果如图5所示。当无光照时2.5μM的探针F20对细胞杀伤作用低,在有无BCN-TPP存在下,细胞存活率均在80%以上。而当探针F20的浓度增加为5μM,细胞的存活率降低为65%左右。在有光照时,2.5μM的探针F20在与BCN-TPP反应前后的细胞存活率分别为38%和27%,相差1.4倍。5μM的探针F20在与BCN-TPP反应前后的细胞存活率分别为17%和14%,相差1.2倍。这与探针F20在溶剂中的单态氧产率测量的结果一致(探针F20与BCN反应前后相差1.2倍)。
上述结果证明,本发明的光动力荧光探针F20在光照条件下可产生单态氧对细胞进行杀伤,正交反应前后细胞杀伤作用差距可达1.4倍,具有一定的用于光动力治疗的潜力。
综上,本发明通过对BODIPY荧光染料的C-8位进行酰基化修饰,并进一步结合四嗪结构,制得了具有优异的光学性能的荧光探针,具体表现为高量子产率、高摩尔消光系数和大斯托克斯位移,四嗪能够有效淬灭BODIPY的荧光并且能够在生物正交反应后获得大荧光增强倍数,具有很好的作为细胞组织活体示踪的荧光探针的潜力。进一步引入卤素取代基修饰,可增加分子单重态至三重态的系间穿越能力,从而提升单态氧产率,得到能够克服非特异光毒性的光动力荧光探针,在光动力治疗中有潜在应用价值。
Claims (10)
1.式I所示的化合物或其盐:
其中R1为或甲基;Ra为C1~C3的烷基;
R2为H或甲基;L为或/>n为1或2;
R3为甲基或
R4为H或I。
2.如权利要求1所述的化合物或其盐,其特征在于,所述化合物具有式II所示结构:
其中R1为R2为甲基,R3为甲基。
3.如权利要求1所述的化合物或其盐,其特征在于,所述化合物具有如下结构:
4.如权利要求1所述的化合物或其盐,其特征在于,所述化合物具有式III所示结构:
所述R3为甲基或为
5.如权利要求4所述的化合物或其盐,其特征在于,所述化合物具有如下结构:
6.权利要求1所述的化合物的制备方法,其特征在于,R1为所述制备方法包括如下步骤:
(1)以化合物A为原料,与化合物B在室温下反应制得化合物C;
(2)化合物C与化合物D在有机碱作用下室温反应得到化合物E即为所述化合物;
或化合物E和卤素单体、高卤酸在50~70℃反应制得化合物E’即为所述化合物;
或化合物E或化合物E’与化合物F在催化剂作用下于100~120℃反应制得化合物G即为所述化合物;
反应式如下:
其中,X为I,Rd、Re为R’为/>
或R1为C1~C3烷基,所述制备方法包括如下步骤:
(a)以化合物A为原料,与化合物B’在有机碱作用下室温反应制得化合物C’即为所述化合物。
7.如权利要求6所述的制备方法,其特征在于,所述有机碱为三乙胺,所述催化剂为哌啶和冰醋酸。
8.权利要求1~5任一项所述的化合物或其盐在制备细胞成像试剂中的应用。
9.如权利要求8所述的应用,其特征在于,所述细胞成像试剂是荧光探针。
10.权利要求4或5所述的化合物或其盐在制备光动力治疗用试剂中的应用。
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