CN112812584A - 一种含氮杂环丁烷螺环结构的荧光染料及其制备方法和应用 - Google Patents
一种含氮杂环丁烷螺环结构的荧光染料及其制备方法和应用 Download PDFInfo
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- CN112812584A CN112812584A CN201911123394.9A CN201911123394A CN112812584A CN 112812584 A CN112812584 A CN 112812584A CN 201911123394 A CN201911123394 A CN 201911123394A CN 112812584 A CN112812584 A CN 112812584A
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- spiro
- azetidine
- aryl
- heteroaryl
- alkyl
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
本发明属于荧光分析技术领域,涉及一种含四元螺环结构的荧光染料,制备方法和应用。具体涉及一种含氮杂环丁烷螺环结构的荧光染料,及其制备方法和应用。本发明的含氮杂环丁烷螺环结构的荧光染料,具有如下(Ⅰ)或(Ⅱ)的骨架结构,本发明的含氮杂环丁烷螺环结构的荧光染料具有优异光学特性、良好光稳定性、良好生物相容性、本发明还提供了具额外化学连结位点的新型含氮杂环丁烷螺环结构荧光染料,制备方法和在生物荧光分析上的应用。所述荧光分子显示出良好的水溶性,光稳定性,高荧光量子产率,可用于荧光标记,生物成像和开发新的荧光探针。
Description
技术领域
本发明属于荧光分析技术领域,涉及一种含四元螺环结构的荧光染料,制备方法和应用。具体涉及一种含氮杂环丁烷螺环结构的荧光染料,及其制备方法和应用。
背景技术
现有技术公开了荧光分析技术具有高的灵敏度、可视化强、操作便利、可以实时检测细胞及生物体等优点,已被广泛应用于离子、自由基、分子、多肽、酶,甚至还包括温度、pH等的识别;小分子荧光探针在医学、生物学、环境科学等多学科中得到了广泛的应用,用以分析检测和生物标记等。
荧光染料作为小分子荧光探针的重要组成部分,其光物理及理化性质具有重要意义,业内衡量一个荧光染料的性能主要考虑以下几个要素:(1)吸收和发射光的波长,具有较长的吸收和发射波长可以避免生物体系自发荧光干扰,提高穿透性,在生物成像领域具有明显优势;(2)斯托克斯位移,较大的斯托克斯位移可以更好的避免仪器激发光散射对样品检测所带来的干扰,提高数据采集的真实性;(3)荧光量子产率,较高的荧光量子产率有利于信号采集和识别,提高检测的灵敏度;(4)稳定性,包括荧光染料的化学稳定性、光稳定性、酸碱稳定性等,高稳定性的染料可以减少信号干扰,提高对检测物质响应信号的准确性;(5)生物相容性,包括水溶性、细胞透膜性以及细胞毒性等,是衡量能否在生物体系应用的一个重要因素。
随着生命科学的不断深入,生命体内各种活性物质的检测已是研究的热点,研究实践显示,在检测物质的选择性、灵敏度、检测速度以及细胞和活体的应用等方面对小分子荧光探针也提出了更高的要求,然而,现有的荧光染料对单分子和活细胞成像尚存在缺乏足够的亮度和光稳定性的缺陷。有研究公开了,扭曲分子内电荷转移(TICT)是荧光分子中主要的非辐射失活途径之一,在TICT状态下,二烷基氨基供体在光激发作用下使荧光团骨架产生90°左右的扭曲,形成一种无辐射、高活性的化学物种,因此,为了避免TICT的形成,研究者采用了各种改造策略,如Dylight、CF和Alexa染料等商业上可用的荧光团通过使氨基取代基刚性化而进行了复杂的结构修饰(J.Histochem.Cytochem.,1999,47,1179);宋相志等人使用了体积庞大的7-氮杂环[2.2.1]庚烷取代罗丹明染料中常用的二甲氨基取代基(J.Am.Chem.Soc.,2008,130,17652),这种结构修饰大大提高了罗丹明染料的量子产率和光稳定性,但是其水溶性变差;Lavis等人将萘酰亚胺、吖啶类化合物、吩噁嗪类、香豆素、罗丹明、氧杂罗丹明、碳代罗丹明和硅代罗丹明等几类荧光染料中N,N-二烷基氨基取代基替换为氮杂环丁烷,显示氮杂环丁烷取代显著提高了染料的亮度(Nat.Methods,2015,12,244),这种简单的结构修饰保留了母体荧光团的理化特性,如细胞透膜性等,为荧光染料结构修饰改善光学性能提供了很好的证明,然而,研究显示,所述氮杂环丁烷取代的荧光染料的光稳定性仍然不高(J.Am.Chem.Soc.,2019,141,981),因此,有必要进一步改善其光稳定性。
氮杂环丁烷螺环结构是一种特殊的小分子四元杂环螺环,由于螺环结构缓解了四元单环的电子密度,增强了稳定性,并且螺环的引入也可以在亲脂性、亲水性、空间构象等多维度调控分子的性质,在药物研究中逐渐受到关注,但目前基于氮杂环丁烷螺环结构的荧光染料还未见有报道。
由于氮杂环丁烷较大的环张力可能具有一定的不稳定性,所以在结构上有进行结构修饰的必要性。其次作为一个好的荧光团,应该具备易衍生化成为各种荧光探针的结构特征,最好具有多个化学连结位点。另外具备良好的水溶性以及透膜性,使其能够应用到细胞及生物体内检测中也是优秀染料所希望具有的特性。
基于现有技术的基础与现状,本申请的发明人拟提供具有优异光学特性、较好稳定性、良好生物相容性、并具额外化学修饰位点的新型荧光染料,涉及一种含四元螺环结构的荧光染料。
发明内容
本发明的目的在于,为克服现有技术的缺陷与不足,提供一类具有优异光学特性、良好光稳定性、良好生物相容性、并具额外化学连结位点的新型含四元螺环结构的荧光染料,具体涉及含氮杂环丁烷螺环结构荧光染料,其制备方法和在生物荧光分析中的应用。本发明的该荧光分子显示出良好的水溶性,光稳定性,高荧光量子产率,可用于荧光标记,高清生物成像和开发新的荧光探针。
本发明的的目的通过下述技术方案实现:
本发明提供了一种含氮杂环丁烷螺环结构的荧光染料,其具有如下骨架结构(Ⅰ)或者(Ⅱ):
其中:取代基R1,R2各自独立为H、卤素、羟基、C1-C18烷氧基、巯基、C1-C18烷硫基、氨基、C1-C18烷基、C1-C18不饱和烷基、C3-C8饱和杂环、C3-C8环烷基、芳基、杂芳基、酰基、酯基、羧基、磷酸基、磺酸基;所述卤素为F、Cl、Br;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基;
优选地,取代基R1,R2各自独立为H、卤素、羟基、C1-C6烷氧基、C1-C6烷基、羧基、磷酸基、磺酸基;所述卤素为F、Cl、Br;
进一步优选地,取代基R1,R2各自独立为H、卤素、C1-C4的直链或支链烷基;所述卤素为F、Cl;
m=0、1、2、3、4;n=0、1、2、3、4;且m+n=2、3、4、5;
X为C,或N,或O,或S,或S=O,或O=S=O,其中C和N原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、乙酰基、叔丁氧羰基、苄基、芳基等;
包含X的环上碳原子上可以有取代基,如甲基、乙基、羟基、氨基、羧基、氟等;
所述的含氮杂环丁烷螺环结构包括但不限于以下结构:
所述的荧光团包括但不限于以下各种骨架结构:
(1)包括萘酰亚胺类荧光染料(Ⅲ)或(Ⅳ):
其中:取代基R3,R6各自独立为H、卤素、氨基、芳基、杂芳基、磺酸基、醛基;取代基R4,R5各自独立为H、卤素、芳基、杂芳基;取代基R8为H、C1-C16直链或支链的饱和及不饱和烷基链、C1-C16直链或支链聚乙二醇链、C1-C16的多肽、C1-C16含羧基烷基链、C1-C16含酯基或酰胺结构烷基链、C1-C16含芳香环的烷基链、芳基、杂芳基等;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基等;
优选地,上述含氮杂环丁烷螺环结构萘酰亚胺类荧光染料包括但不限于以下结构:
进一步优选地,上述含氮杂环丁烷螺环结构萘酰亚胺类荧光染料包括以下结构:
(2)包括香豆素类荧光染料(Ⅴ)或(Ⅵ):
其中:取代基R3,R4,R5各自独立为H、卤素、氨基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基、磺酸基;取代基R6为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基、羧基、酯基、醛基、硝基、氰基、三氟甲基;取代基R7为H、卤素、硝基、C1-C6不饱和烷基、C1-C6取代的烯基、取代的芳烯基、芳基、杂芳基、氰基、醛基、酰基、酯基、叠氮基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基等;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
Z为N,或O,或S;
优选地,上述含氮杂环丁烷螺环结构香豆素类荧光染料包括但不限于以下结构:
进一步优选地,上述含氮杂环丁烷螺环结构香豆素类荧光染料包括以下结构:
(3)包括咕吨类荧光染料(Ⅶ)或(Ⅷ):
其中:取代基R3,R4,R5,R6,R7,R8各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R10为H、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、C3-C8杂环烷基、四元螺环结构;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R6和R8,或者R4和R7可以成环;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
Z独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上可以有不同取代基,如甲基、乙基、芳基等;
优选地,上述含氮杂环丁烷螺环结构咕吨类荧光染料包括但不限于以下结构:
进一步优选地,上述含氮杂环丁烷螺环结构咕吨类荧光染料包括以下结构:
(4)包括Rhodol类荧光染料(Ⅸ)或(Ⅹ):
其中:取代基R3,R4,R5,R6,R7,R8各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R6和R8,或者R4和R7可以成环;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
Z独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上可以有不同取代基,如甲基、乙基、芳基等;
优选地,上述含氮杂环丁烷螺环结构Rhodol类荧光染料包括但不限于以下结构:
(5)包括萘罗丹明类荧光染料(Ⅺ)或(Ⅻ):
其中:取代基R3,R4,R5,R6,R7,R8,R10,R11各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
Z独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上可以有不同取代基,如甲基、乙基、芳基等;
优选地,上述含氮杂环丁烷螺环结构萘罗丹明类荧光染料包括但不限于以下结构:
(6)包括三苯基吡喃类荧光染料(XIII)或(XIV):
其中:取代基R3,R4,R5,R6,R7,R8各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R10为H、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、C3-C8杂环烷基、四元螺环结构;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
优选地,上述含氮杂环丁烷螺环结构三苯基吡喃类类荧光染料包括但不限于以下结构:
(7)苯并呋咱类荧光染料(XV)或(XVI):
其中:取代基R3,R4各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基;取代基R5为卤素、芳基、杂芳基、氰基、硝基、醛基、酰基、酯基、磺酸基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C,或N,或O,或S,或Se,或Te,其中C原子上可以有不同取代基,如甲基、乙基、环烷基、羟基、氨基、羧基、酯基、芳基等;
优选地,上述含氮杂环丁烷螺环结构苯并呋咱类荧光染料包括但不限于以下结构:
(8)氟硼二吡咯(BODIPY)类荧光染料(XVII),或(XVIII),或(XIX),或(XX):
其中:取代基R3,R4,R6各自独立为H、卤素、巯基、氨基、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、磺酸基、羧基、酰基、酯基、醛基、氰基;取代基R5为H、卤素、羟基、巯基、氨基、C1-C6烷氧基、C1-C6烷硫基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R7,R8各自独立为H、卤素、巯基、氨基、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、磺酸基、羧基、酰基、酯基、醛基、氰基、氨基、C1-C6含氮烷烃、C3-C8含氮环烷烃、含氮四元螺环结构;取代基R9,R10各自独立为F、C1-C16烷氧基、C1-C6烷基、C1-C6不饱和烷基、杂芳基、羧基、氰基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R6和R7可以成环;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
优选地,上述含氮杂环丁烷螺环结构氟硼二吡咯类荧光染料包括但不限于以下结构:
(9)二甲氨基萘酮类荧光染料(XXI)或(XXII):
其中:取代基R3,R4,R5,R6,R7各自独立为H、卤素、C1-C6烷氧基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R8为H、羟基、氨基、C1-C6烷氧基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、C1-C6取代的烯基、取代的芳烯基、芳基、杂芳基、三氟甲基;取代基R9为H、卤素、芳基、杂芳基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
优选地,上述含氮杂环丁烷螺环结构二甲氨基萘酮类荧光染料包括但不限于以下结构:
(10)氧杂蒽类荧光染料(XXIII)或(XXIV):
其中:取代基R3,R4,R6各自独立为H、卤素、C1-C6烷氧基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R5为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R7,R8,R9各自独立为H、芳基、杂芳基、羧基、酯基、醛基、氰基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上可以有不同取代基,如甲基、乙基、芳基等;
Z为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
优选地,上述含氮杂环丁烷螺环结构氧杂蒽类荧光染料包括但不限于以下结构:
上述荧光团骨架进行螺环结构修饰后,与二甲胺或者氮杂环丁烷取代衍生物相比较,最大吸收和发射波长均变化不大,通过四元螺环修饰的荧光团均保持或提高了荧光量子产率(如表1–表9所示);除此之外,氮杂环丁烷螺环衍生物与氮杂环丁烷衍生物相比较,引入了含氮螺环后,溶解度均有较大的提高。
上述的含氮杂环丁烷螺环结构荧光染料,当X为氮原子时,提供了新的连结位点,可以连结包括但不限于氨基酸、蛋白、糖蛋白、脂蛋白、受体、抗体或其片段、抗原、适配体、多糖、寡糖、核苷、核苷酸、低聚核苷酸、核酸、药物、抑制剂、激素、营养物、代谢物、生长因子、脂质、聚合物、聚合微粒、细胞、病毒、酶的底物等;可以连结线粒体靶向基团(如三苯基膦基团等)、溶酶体靶向基团(如吗啉基团、二甲氨基等)、内质网靶向基团(如五氟苯甲酰基、对甲苯磺酰基等)等,用于靶向细胞器的荧光探针(如线粒体,溶酶体,内质网,高尔基体的成像);可以连结蛋白标记结构(如SNAP-tag,Halo-tag等)、抗体连结结构、肿瘤靶向结构(如连结生物素,叶酸等),用于细胞高清显微镜成像和肿瘤靶向成像;可以连结三线态淬灭结构,用来增强荧光团的光稳定性;可以连结点击化学反应结构(如叠氮基、炔基等),用于点击化学(Click)等;其特征是它具有如下结构(XXV),或(XXVI),或(XXVII),或(XXVIII):
其中:所述氮杂环丁烷螺环结构荧光染料提供的新连结位点,可直接与功能结构(Functional Structure)相连(XXV或XXVI);所述氮杂环丁烷螺环结构荧光染料提供的新连结位点,可间接地通过连结子(Linker)与功能结构相连(XXVII或XXVIII);
其中:连接子(Linker)可以为C1-C16直链或支链的饱和及不饱和烷基链、C1-C16直链或支链聚乙二醇链、C1-C16的多肽、C1-C16含羧基烷基链、C1-C16含酯基或酰胺结构烷基链、C1-C16含芳香环的烷基链;
功能结构(Functional Structure)包括但不限于氨基酸、蛋白、糖蛋白、脂蛋白、受体、抗体或其片段、抗原、适配体、多糖、寡糖、核苷、核苷酸、低聚核苷酸、核酸、药物、抑制剂、激素、营养物、代谢物、生长因子、脂质、聚合物、聚合微粒、细胞、病毒、酶的底物等;包括但不限于线粒体靶向基团(如三苯基膦基团等)、溶酶体靶向基团(如吗啉基团、二甲氨基等)、内质网靶向基团(如五氟苯甲酰基、对甲苯磺酰基等)、蛋白标记结构(如SNAP-tag,Halo-tag等)、抗体连结结构、肿瘤靶向结构(如连结生物素,叶酸等)、三线态淬灭结构、点击化学反应结构(如叠氮基、炔基等)等:
本发明具有如下优点:
(1)所述的含氮杂环丁烷螺环结构荧光染料提供了新的连结位点,可用于构建靶向细胞器的荧光探针(如线粒体,溶酶体,内质网,高尔基体的成像),其特征是它包括但不限于以下结构:
(2)所述的含氮杂环丁烷螺环结构荧光染料提供了新的连结位点,可用于细胞内的蛋白标记以及高清显微镜成像(如SNAP-tag,Halo-tag等),其特征是它包括但不限于以下结构:
(3)所述的含氮杂环丁烷螺环结构荧光染料提供了新的连结位点,可用于肿瘤靶向成像(如连结生物素,叶酸等),其特征是它包括但不限于以下结构:
(4)所述的含氮杂环丁烷螺环结构荧光染料提供了新的连结位点,可用于点击化学(Click)或者缩合反应便捷连结其它功能团等,其特征是它包括但不限于以下结构:
(5)所述的含氮杂环丁烷螺环结构荧光染料提供了新的连结位点,可用于连结三重态淬灭基团来增强荧光团的光稳定性,其特征是它包括但不限于以下结构:
(6)所述的含氮杂环丁烷螺环结构荧光染料提供了新的连结位点,可用于构建荧光共振能量转移(FRET)探针,其特征是它具有以下结构(XXIX),或(XXX),或(XXXI),或(XXXII):
其中:所述氮杂环丁烷螺环结构荧光染料提供的新连结位点,可直接与另一个合适荧光团相连而设计成基于FRET的荧光探针(XXIX或XXX);所述氮杂环丁烷螺环结构荧光染料提供的新连结位点,可间接地通过连结子与另一个合适荧光团相连而设计成基于FRET的荧光探针(XXXI或XXXII);
连接子(Linker)可以为C1-C6直链或支链的饱和及不饱和烷基链、C1-C6直链或支链烷氧链、C1-C6含羧基烷基链、C1-C6的多肽、C1-C6含酯基或酰胺结构烷基链、C1-C6含芳香环的烷基链;
荧光团2(Fluorophore 2)包括但不限于为以下染料:所述的含氮杂环丁烷螺环结构荧光染料、萘酮类染料、香豆素染料、荧光素类染料、罗丹明类染料、BODIPY类染料、菁染料、苯并呋咱类染料、氧杂蒽类染料等;
所述的含氮杂环丁烷螺环结构荧光染料提供了新的连结位点,可用于构建荧光共振能量转移(FRET)探针,其特征是它包括但不限于以下结构:
附图说明
附图是用来提供对本发明的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本发明,但并不构成对本发明的限制;其中:
图1:染料JF549与染料RhN-05的光稳定性,其中测试溶液为含10%乙醇和0.1%三氟乙酸的去离子水;
图2:探针P1和P2对谷胱甘肽响应,其中激发波长为460nm,探针浓度为10μM;
图3:探针P1和P2对谷胱甘肽响应荧光增强倍数对比;
图4:探针FF600-Mito细胞成像图,其中红色图像为探针FF600-Mito,绿色图像为Mito-Track Green,探针FF600-Mito浓度为10μM;
图5:探针FF600-Lyso细胞成像图,其中红色图像为探针FF600-Lyso,绿色图像为Lyso-Track Green,探针FF600-Lyso浓度为10μM;
图6:探针FF600-ER细胞成像图。其中红色图像为探针FF600-ER,绿色图像为ER-Track Green,探针FF600-ER浓度为10μM;
图7:探针FRET-01与次硝酸(HNO)的响应荧光光谱,其中探针FRET-01浓度为10μM;
图8:探针FRET-01细胞成像图,探针浓度为10μM。
具体实施方式
为更好地理解本发明,下面结合附图和实施例对本发明作进一步的说明,有必要指出的是以下实施例不能解释为对发明保护范围的限制,该领域的技术熟练人员根据上述发明内容对本发明作出的一些非本质的改进和调整,仍应属于本发明的保护范围。
实施例1制备化合物Nap-02
将Nap-00(100mg,0.3mmol)和氮杂环丁烷盐酸盐(94mg,1.0mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物Nap-02(59mg,产率:64%)。1H NMR(400MHz,DMSO-d6)δ=8.39(dd,J=1.0,7.3Hz,1H),8.34(dd,J=1.0,8.4Hz,1H),8.18(d,J=8.6Hz,1H),7.58(dd,J=7.3,8.4Hz,1H),6.43(d,J=8.6Hz,1H),4.47(t,J=7.5Hz,4H),4.04-3.96(m,2H),2.50-2.42(m,2H),1.63-1.53(m,2H),1.39-1.29(m,2H),0.92(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO-d6)δ=164.11,163.27,152.70,133.23,131.19,131.12,130.33,124.32,122.12,120.59,108.73,106.50,55.59,30.25,20.31,16.94,14.21.MS(ESI):calc'd forC19H20N2O2[M+H]+309.2,measured 309.0;HRMS(ESI):calc'd for[M+H]+309.15975,measured 309.16024.。
实施例2制备化合物Nap-03
将Nap-00(100mg,0.3mmol)和2-氧杂-6-氮杂-螺[3,3]庚烷草酸盐(72mg,0.5mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物Nap-03(49mg,产率:47%)。1H NMR(400MHz,DMSO-d6)δ=8.40(ddd,J=0.9,7.9,18.0Hz,2H),8.23(d,J=8.4Hz,1H),7.64(dd,J=7.4,8.4Hz,1H),6.53(d,J=8.6Hz,1H),4.79(s,4H),4.67(s,4H),4.07-3.97(m,2H),2.04-1.94(m,1H),1.67-1.51(m,2H),1.39-1.29(m,2H),0.91(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO-d6)δ=164.12,163.32,152.47,133.23,131.26,131.06,130.27,130.10,124.62,122.27,120.88,109.38,107.22,80.14,64.47,39.47,39.12,35.58,30.24,29.49,29.19,29.14,29.04,25.57,20.30,14.41,14.21.MS(ESI):calc'd for C21H22N2O3[M+H]+351.2,measured 351.0;HRMS(ESI):calc'd for[M+H]+351.17032,measured 351.16999.。
实施例3制备化合物Nap-04
将Nap-00(100mg,0.3mmol)和6-氧杂-1-氮杂-螺[3,3]庚烷草酸盐(72mg,0.5mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物Nap-04(45mg,产率:43%)。1H NMR(400MHz,Methanol-d4)δ=8.60(d,J=8.4Hz,1H),8.50(d,J=7.3Hz,1H),8.38(d,J=8.7Hz,1H),7.59(dd,J=7.5,8.6Hz,1H),7.49(d,J=8.7Hz,1H),5.47(d,J=8.9Hz,2H),4.70(t,J=7.3Hz,2H),4.17-4.05(m,2H),3.35(s,2H),2.76(t,J=7.3Hz,2H),1.75-1.64(m,2H),1.50-1.40(m,2H),1.01-0.94(m,3H).13C NMR(101MHz,Methanol-d4)δ=164.75,164.17,150.02,137.22,133.09,131.07,131.00,130.50,123.59,122.01,121.27,108.89,106.57,79.34,73.82,52.73,39.46,29.92,28.35,19.96,12.80.MS(ESI):calc'd for C21H22N2O3[M+H]+351.2,measured 351.1;HRMS(ESI):calc'd for[M+H]+351.17032,measured351.17069.。
实施例4制备化合物Nap-05
将Nap-00(100mg,0.3mmol)和2-硫-6-氮杂螺[3.3]庚烷草酸盐(64mg,0.4mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物Nap-05(38mg,产率:34%)。1H NMR(400MHz,Chloroform-d)δ=8.55(dd,J=1.0,7.3Hz,1H),8.40(d,J=8.3Hz,1H),8.21(dd,J=0.9,8.5Hz,1H),7.55(dd,J=7.5,8.4Hz,1H),6.44(d,J=8.4Hz,1H),4.49(s,4H),4.20-4.10(m,2H),3.51(s,4H),1.78-1.74(m,1H),1.72-1.71(m,1H),1.76-1.68(m,1H),1.50-1.37(m,2H),0.97(t,J=7.3Hz,3H).13C NMR(101MHz,Chloroform-d)δ=164.60,164.02,151.98,133.05,131.18,130.37,129.57,124.15,123.00,121.27,111.58,107.16,68.36,43.67,40.02,37.06,30.28,29.70,20.43,13.87.MS(ESI):calc'd for C21H22N2O2S[M+H]+367.1,measured 367.1;HRMS(ESI):calc'd for[M+H]+367.14748,measured 367.14800.。
实施例5制备化合物Nap-06
将间氯过氧苯甲酸(9.5mg,0.054mmol)加入到Nap-05(20mg,0.054mmol)的二氯甲烷(5mL)中,室温反应1小时,LC-MS检测反应已经完全,将溶剂旋干,残留物用反相制备色谱纯化得到产物Nap-06(7mg,产率:34%)。1H NMR(400MHz,Chloroform-d)δ=8.58(d,J=7.0Hz,1H),8.43(d,J=8.3Hz,1H),8.15(d,J=8.3Hz,1H),7.58(t,J=7.9Hz,1H),6.48(d,J=8.4Hz,1H),4.59-4.46(m,4H),4.21-4.11(m,2H),4.03(br d,J=12.2Hz,2H),3.55(brd,J=12.2Hz,2H),1.75-1.68(m,2H),1.47-1.42(m,2H),0.97(t,J=7.3Hz,3H).13C NMR(101MHz,Chloroform-d)δ=164.50,163.98,151.64,132.91,131.30,130.24,129.24,124.61,123.19,121.33,112.70,107.58,66.66,62.17,40.09,30.75,30.27,20.42,13.87.MS(ESI):calc'd for C21H22N2O3S[M+H]+383.1,measured 383.1;HRMS(ESI):calc'dfor[M+H]+383.14239,measured 383.14336.。
实施例6
化合物Nap-07的制备
将Nap-00(100mg,0.3mmol)和2,6-二氮杂螺[3.3]庚烷-2-甲酸叔丁酯草酸盐(64mg,0.4mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,冷至室温后反应体系用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Nap-07(28mg,产率:27%)。1H NMR(400MHz,Methanol-d4)δ=8.57-8.47(m,2H),8.34(d,J=8.6Hz,1H),7.68(dd,J=7.6,8.2Hz,1H),6.88(d,J=8.6Hz,1H),4.15-3.98(m,6H),3.87(s,2H),3.71(s,2H),1.71-1.58(m,2H),1.46-1.36(m,2H),0.99(t,J=7.4Hz,3H).13CNMR(101MHz,Methanol-d4)δ=164.63,164.18,151.34,134.23,130.94,129.67,127.71,124.54,122.22,120.60,109.46,103.86,63.11,50.13,45.18,43.67,39.44,29.94,19.96,12.81.MS(ESI):calc'd for C21H23N3O2[M+H]+350.2,measured 350.2;HRMS(ESI):calc'dfor[M+H]+350.18630,measured 350.18574.。
实施例7
化合物Nap-08的制备
将Nap-00(100mg,0.3mmol)和1,6-二氮杂螺[3.3]庚烷-6-甲酸叔丁酯草酸盐(97mg,0.4mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全冷至室温后反应体系用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Nap-08(29mg,产率:28%)。1H NMR(400MHz,Methanol-d4)δ=8.60(d,J=8.7Hz,1H),8.52(d,J=7.3Hz,1H),8.41(d,J=8.6Hz,1H),7.64(dd,J=7.5,8.6Hz,1H),7.11(d,J=8.6Hz,1H),5.17(d,J=13.4Hz,2H),4.75(t,J=7.3Hz,2H),4.29(d,J=13.3Hz,2H),4.17-4.10(m,2H),2.94(t,J=7.2Hz,2H),1.70-1.64(m,2H),1.46-1.40(m,2H),1.01-0.96(m,3H).13C NMR(101MHz,Methanol-d4)δ=164.50,164.05,148.80,132.40,131.18,130.82,130.18,124.26,122.23,121.87,110.80,106.83,69.08,55.62,52.93,39.52,30.86,29.88,19.95,12.80.MS(ESI):calc'd for C21H23N3O2[M+H]+350.2,measured 350.2;HRMS(ESI):calc'd for[M+H]+350.18630,measured 350.18562.。
实施例8
化合物Nap-09的制备
将Nap-00(100mg,0.3mmol)和2,6-二氮杂螺[3.4]辛烷-6-甲酸叔丁酯(70mg,0.33mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,冷至室温后反应体系用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Nap-09(35mg,产率:32%)。1HNMR(400MHz,Methanol-d4)δ=8.44(dd,J=0.9,7.3Hz,1H),8.36-8.29(m,1H),8.25(d,J=8.4Hz,1H),7.57(dd,J=7.5,8.4Hz,1H),6.50(d,J=8.6Hz,1H),4.61-4.47(m,4H),4.14-4.04(m,2H),3.66(s,2H),3.46(t,J=7.3Hz,2H),2.48(t,J=7.3Hz,2H),1.74-1.60(m,2H),1.48-1.38(m,2H),0.99(t,J=7.4Hz,3H).13C NMR(101MHz,Methanol-d4)δ=164.59,164.07,152.43,132.87,130.86,130.34,130.09,123.86,122.03,120.98,109.96,106.82,63.67,53.19,48.30,48.09,47.88,44.58,40.71,39.49,34.95,29.92,19.96,12.80.MS(ESI):calc'd for C22H25N3O2[M+H]+364.2,measured364.2;HRMS(ESI):calc'd for[M+H]+364.20195,measured 364.20140。
实施例9
化合物Nap-10的制备
将Nap-00(100mg,0.3mmol)和1,6-二氮杂螺[3.4]辛烷-6-甲酸叔丁酯(85mg,0.4mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,冷至室温后反应体系用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Nap-10(33mg,产率:31%)。1H NMR(400MHz,Methanol-d4)δ=8.59-8.50(m,3H),8.41(d,J=8.6Hz,1H),7.69(dd,J=7.5,8.3Hz,1H),6.88(d,J=8.7Hz,1H),4.15(br d,J=7.6Hz,2H),4.09(s,4H),3.70(t,J=7.1Hz,2H),2.72(br t,J=7.1Hz,2H),1.72-1.67(m,2H),1.48-1.43(m,2H),1.03-0.99(m,3H).MS(ESI):calc'd for C22H25N3O2[M+H]+364.2,measured 364.1。
实施例10
化合物Nap-11的制备
将Nap-00(100mg,0.3mmol)和2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(75mg,0.33mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,冷至室温后反应体系用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Nap-11(50mg,产率:44%)。1H NMR(400MHz,Methanol-d4)δ=8.34(dd,J=0.9,7.3Hz,1H),8.30-8.23(m,1H),8.14(d,J=8.4Hz,1H),7.48(dd,J=7.4,8.4Hz,1H),6.39(d,J=8.6Hz,1H),4.32(s,4H),4.09-3.98(m,2H),2.26-2.16(m,4H),1.69-1.59(m,2H),1.46-1.35(m,2H),0.98(t,J=7.3Hz,3H).13C NMR(101MHz,Methanol-d4)δ=164.54,163.98,152.42,132.87,130.78,130.48,130.08,123.53,121.78,120.68,109.09,106.27,63.78,48.10,47.89,41.12,39.48,33.07,31.49,29.92,19.98,12.82.MS(ESI):calc'd for C23H27N3O2[M+H]+378.2,measured 378.2;HRMS(ESI):calc'd for[M+H]+378.21760,measured378.21675.。
实施例11
化合物Nap-12的制备
将Nap-00(100mg,0.3mmol)和1,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(90mg,0.4mmol)溶于三乙胺(0.2mL)和DMSO(3mL)中,反应加热到120℃过夜,LC-MS检测反应已经完全,冷至室温后反应体系用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Nap-12(25mg,产率:22%)。1H NMR(400MHz,Methanol-d4)δ=8.56-8.48(m,2H),8.37(d,J=8.6Hz,1H),7.66(t,J=7.9Hz,1H),6.83(d,J=8.6Hz,1H),4.18-4.09(m,2H),3.68-3.58(m,4H),3.37-3.32(m,4H),2.58(br t,J=6.9Hz,2H),2.46(br s,2H),1.74-1.61(m,2H),1.46-1.37(m,2H),1.03-0.97(m,3H).13C NMR(101MHz,Methanol-d4)δ=164.37,134.87,134.50,130.88,127.76,124.26,122.17,115.56,103.75,41.66,40.80,40.70,39.41,34.98,29.98,24.39,19.96,12.81.MS(ESI):calc'd for C23H27N3O2[M+H]+378.2,measured378.2;HRMS(ESI):calc'd for[M+H]+378.21760,measured 378.21734.。
实施例12
化合物Cou-03的制备
氩气下,将化合物Cou-00(100mg,0.325mmol),2-氧杂-6-氮杂-螺[3,3]庚烷草酸盐(94mg,0.650mmol),Pd2(dba)3(59mg,0.065mmol),X-Phos(48mg,0.100mmol)和碳酸铯(326mg,1.00mmol)溶于二氧六环(5mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物Cou-03(39mg,产率:47%)。1H NMR(400MHz,Methanol-d4)δ=7.54(d,J=8.8Hz,1H),6.45(dd,J=2.2,8.7Hz,1H),6.26(d,J=2.2Hz,1H),5.99-5.94(m,1H),3.76(s,8H),2.41-2.37(m,3H).13C NMR(101MHz,Methanol-d4)δ=163.07,155.47,155.03,154.14,125.64,109.84,108.00,107.57,96.02,63.45,54.57,41.09,17.19.MS(ESI):calc'd for C15H15NO3[M+H]+258.1,measured 258.0;HRMS(ESI):calc'd for[M+H]+258.11247,measured258.11223.。
实施例13
化合物Cou-04的制备
氩气下,将化合物Cou-00(200mg,0.650mmol),6-氧杂-1-氮杂-螺[3,3]庚烷草酸盐(112mg,0.780mmol),Pd2(dba)3(118mg,0.130mmol),X-Phos(96mg,0.200mmol)和碳酸铯(652mg,2.00mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物Cou-04(88mg,产率:53%)。1H NMR(400MHz,DMSO-d6)δ=7.63(d,J=8.7Hz,1H),6.74(br d,J=8.3Hz,1H),6.55(s,1H),6.03(d,J=1.1Hz,1H),5.09(d,J=8.4Hz,2H),4.75(d,J=8.2Hz,2H),3.76(t,J=7.1Hz,2H),2.55(t,J=7.1Hz,2H),2.36(d,J=1.1Hz,3H).13C NMR(101MHz,DMSO-d6)δ=160.89,155.76,154.20,150.33,127.06,110.40,109.16,108.81,97.47,79.77,70.60,46.62,27.25,18.53.MS(ESI):calc'd forC15H15NO3[M+H]+258.1,measured 258.1;HRMS(ESI):calc'd for[M+H]+258.11247,measured258.11221.。
实施例14
化合物Cou-05的制备
氩气下,将化合物Cou-00(200mg,0.650mmol),2-硫-6-氮杂螺[3.3]庚烷草酸盐(156mg,1.30mmol),Pd2(dba)3(118mg,0.130mmol),X-Phos(96mg,0.200mmol)和碳酸铯(652mg,2.00mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物Cou-05(57mg,产率:32%)。1H NMR(400MHz,DMSO-d6)δ=7.53(d,J=8.7Hz,1H),6.42(dd,J=2.2,8.7Hz,1H),6.30(d,J=2.2Hz,1H),6.00(d,J=1.0Hz,1H),4.01(s,4H),3.41(s,4H),2.34(d,J=1.0Hz,3H).13C NMR(101MHz,DMSO-d6)δ=160.93,155.48,154.19,153.96,126.59,110.35,109.08,108.87,97.49,64.83,55.38,36.65,18.56.MS(ESI):calc'd for C15H15NO2S[M+H]+274.1,measured 274.0;HRMS(ESI):calc'dfor[M+H]+274.08963,measured274.08938.。
实施例15
化合物Cou-06的制备
氩气下,将化合物Cou-00(154mg,0.50mmol),2,6-二氮杂螺[3.3]庚烷-2-甲酸叔丁酯草酸盐(145mg,0.6mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(71mg,0.20mmol)和碳酸铯(652mg,2.0mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Cou-05(48mg,产率:37%)。1H NMR(400MHz,DMSO-d6)δ=7.54(d,J=8.7Hz,1H),6.44(dd,J=2.3,8.7Hz,1H),6.34(d,J=2.1Hz,1H),6.01(d,J=1.0Hz,1H),4.18(br s,4H),4.12(s,4H),2.33(d,J=1.0Hz,3H).13C NMR(101MHz,DMSO-d6)δ=160.91,155.46,154.19,153.64,126.60,110.44,109.19,108.81,97.51,61.19,55.27,36.27,18.56.MS(ESI):calc'd for C15H16N2O2[M+H]+257.1,measured 257.1;HRMS(ESI):calc'd for[M+H]+257.12845,measured257.12805.。
实施例16
化合物Cou-07的制备
氩气下,将化合物Cou-00(154mg,0.50mmol),2,6-二氮杂螺[3.4]辛烷-6-甲酸叔丁酯(127mg,0.6mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(71mg,0.20mmol)和碳酸铯(489mg,1.5mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Cou-07(63mg,产率:47%)。1H NMR(400MHz,Methanol-d4)δ=7.58(d,J=8.7Hz,1H),6.50(dd,J=2.2,8.7Hz,1H),6.32(d,J=2.3Hz,1H),6.02(d,J=1.1Hz,1H),4.09-3.98(m,4H),3.54(s,2H),3.40(t,J=7.3Hz,2H),2.43-2.33(m,5H).13C NMR(101MHz,Methanol-d4)δ=162.73,155.27,154.83,153.82,125.77,110.74,108.49,97.08,60.21,53.20,48.30,48.09,47.88,47.66,44.47,40.40,34.78,17.18.MS(ESI):calc'd for C16H18N2O2[M+H]+271.1,measured 271.1;HRMS(ESI):calc'd for[M+H]+271.1441,measured 271.14416.。
实施例17
化合物C-08的制备
氩气下,将化合物Cou-00(154mg,0.50mmol),2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(113mg,0.50mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(71mg,0.20mmol)和碳酸铯(489mg,1.5mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物Cou-08(56mg,产率:39%)。1H NMR(400MHz,DMSO-d6)δ=7.55(d,J=8.7Hz,1H),6.40(dd,J=2.2,8.7Hz,1H),6.25(d,J=2.2Hz,1H),5.99(d,J=1.1Hz,1H),3.75(s,4H),3.14-3.03(m,4H),2.33(d,J=1.0Hz,3H),1.99-1.90(m,4H).13C NMR(101MHz,DMSO-d6)δ=160.96,155.55,154.21,154.02,126.66,110.10,108.89,108.46,96.91,60.95,41.17,33.29,31.91,18.55.MS(ESI):calc'd for C17H20N2O2[M+H]+285.2,measured 285.1;HRMS(ESI):calc'd for[M+H]+285.15975,measured 285.15964.。
实施例18
化合物NBD-03的制备
常温下,将化合物4-氯-7-硝基苯并-2-氧杂-1,3-二唑(100mg,0.50mmol)和三乙胺(0.2mL)溶解于乙醇(5mL)中,将2-氧杂-6-氮杂-螺[3,3]庚烷草酸盐(86mg,0.6mmol)加入反应体系室温搅拌4小时,LC-MS检测反应已经完全,将溶液旋干,残留物用反相制备色谱纯化得到产物NBD-03(84mg,产率:64%)。1H NMR(400MHz,DMSO-d6)δ=8.49(d,J=8.9Hz,1H),6.08(d,J=8.9Hz,1H),4.95(br s,2H),4.79(br s,4H),4.61(br s,2H).13C NMR(101MHz,DMSO-d6)δ=205.10,145.29,144.97,144.25,136.94,120.07,100.40,79.58,39.47.MS(ESI):calc'd for C11H10N4O4[M+H]+263.1,measured 263.0;HRMS(ESI):calc'dfor[M+H]+263.07748,measured 263.07602.。
实施例19
化合物NBD-04的制备
常温下,将化合物4-氯-7-硝基苯并-2-氧杂-1,3-二唑(100mg,0.50mmol)和三乙胺(0.2mL)溶解于乙醇(5mL)中,将6-氧杂-1-氮杂-螺[3,3]庚烷草酸盐(86mg,0.6mmol)加入反应体系室温搅拌4小时,LC-MS检测反应已经完全,将溶液旋干,残留物用反相制备色谱纯化得到产物NBD-04(67mg,产率:51%)。1H NMR(400MHz,DMSO-d6)δ=8.61(br d,J=8.7Hz,1H),6.85-6.70(m,1H),5.22(br d,J=7.3Hz,2H),4.88(br d,J=7.6Hz,2H),4.55(br s,2H),2.74(br s,2H),2.49(td,J=1.8,3.6Hz,24H).MS(ESI):calc'd for C17H20N2O2[M+H]+285.2,measured 285.1.。
实施例20
化合物NBD-05的制备
常温下,将化合物4-氯-7-硝基苯并-2-氧杂-1,3-二唑(100mg,0.50mmol)和三乙胺(0.2mL)溶解于乙醇(5mL)中,将2,6-二氮杂螺[3.3]庚烷-2-甲酸叔丁酯草酸盐(136mg,0.6mmol)加入反应体系室温搅拌4小时,LC-MS检测反应已经完全,将溶液旋干得到中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物NBD-05(63mg,产率:48%)。1H NMR(400MHz,DMSO-d6)δ=8.72(br s,1H),8.48(d,J=8.9Hz,1H),6.12(d,J=9.0Hz,1H),4.94(br s,2H),4.58(br s,2H),4.26(br s,4H).13C NMR(101MHz,DMSO-d6)δ=144.91,144.87,144.21,136.92,120.33,100.51,54.85,37.05.MS(ESI):calc'd for C11H11N5O3[M+H]+262.1,measured 262.0;HRMS(ESI):calc'd for[M+H]+290.12477,measured 290.12540.。
实施例21
化合物NBD-06的制备
常温下,将化合物4-氯-7-硝基苯并-2-氧杂-1,3-二唑(100mg,0.50mmol)和三乙胺(0.2mL)溶解于乙醇(5mL)中,将2,6-二氮杂螺[3.4]辛烷-6-甲酸叔丁酯(127mg,0.6mmol)加入反应体系室温搅拌4小时,LC-MS检测反应已经完全,将溶液旋干得到中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物NBD-06(63mg,产率:46%)。1H NMR(400MHz,Methanol-d4)δ=8.27-8.20(m,1H),5.85(d,J=8.8Hz,1H),4.82-4.65(m,2H),4.60-4.32(m,2H),3.73(s,2H),3.47(t,J=7.3Hz,2H),2.52(t,J=7.3Hz,2H).13C NMR(101MHz,Methanol-d4)δ=144.59,144.14,143.69,135.64,120.80,99.23,53.00,44.52,41.23,34.73.MS(ESI):calc'd for C12H13N5O3[M+H]+276.1,measured276.0;HRMS(ESI):calc'd for[M+H]+276.10912,measured276.10957.。
实施例22
化合物NBD-07的制备
常温下,将化合物4-氯-7-硝基苯并-2-氧杂-1,3-二唑(100mg,0.50mmol)和三乙胺(0.2mL)溶解于乙醇(5mL)中,将2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(136mg,0.6mmol)加入反应体系室温搅拌4小时,LC-MS检测反应已经完全,将溶液旋干得到中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物NBD-07(53mg,产率:37%)。1H NMR(400MHz,DMSO-d6)δ=8.58(br s,1H),8.56-8.54(m,1H),8.50(d,J=9.0Hz,1H),6.05(d,J=9.0Hz,1H),4.58(br s,2H),4.20(br s,2H),3.12(brt,J=5.1Hz,4H),2.15-1.94(m,4H).13C NMR(101MHz,DMSO-d6)δ=145.83,144.98,144.26,136.96,120.11,100.30,65.45,62.06,34.30,31.29.MS(ESI):calc'd for C13H15N5O3[M+H]+290.1,measured 290.0;HRMS(ESI):calc'd for[M+H]+290.12477,measured290.12487.。
实施例23
化合物RhN-01的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2-氧杂-6-氮杂-螺[3,3]庚烷草酸盐(144mg,1.00mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物RhN-01(112mg,产率:45%)。1H NMR(400MHz,Chloroform-d)δ8.00(d,J=7.4Hz,1H),7.62(dt,J=19.9,7.3Hz,2H),7.15(d,J=7.4Hz,1H),6.57(d,J=8.5Hz,2H),6.22(d,J=2.4Hz,2H),6.10(dd,J=8.7,2.4Hz,2H),4.84(s,8H),4.05(s,8H).13C NMR(151MHz,Chloroform-d)δ169.61,153.18,152.59,134.67,129.40,128.96,127.33,124.85,123.94,108.52,107.97,98.23,81.11,61.41,38.99.。
实施例24
化合物RhN-02的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),6-氧杂-1-氮杂-螺[3,3]庚烷草酸盐(144mg,1.00mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物RhN-02(54mg,产率:22%)。1H NMR(400MHz,Methanol-d4)δ=8.22-8.14(m,1H),7.77-7.70(m,2H),7.36-7.28(m,1H),7.13(d,J=9.0Hz,2H),7.00-6.84(m,4H),5.22(d,J=8.6Hz,4H),4.96-4.90(m,4H),3.99(t,J=7.3Hz,4H),2.67(t,J=7.3Hz,4H).13CNMR(101MHz,DMSO-d6)δ=168.69,153.68,153.56,150.54,150.46,135.29,130.53,130.01,128.02,127.99,126.38,125.98,125.88,125.83,119.57,109.92,109.47,109.33,109.26,109.21,97.52,70.78,70.67,46.78,27.25.MS(ESI):calc'd for C30H27N2O5[M]+495.2,measured 495.1;HRMS(ESI):calc'd for[M]+495.19145,measured495.19056.。
实施例25
化合物RhN-03的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2,6-二氮杂螺[3.3]庚烷-2-甲酸叔丁酯草酸盐(242mg,1.00mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体产物直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物RhN-03(69mg,产率:28%)。1H NMR(400MHz,Methanol-d4)δ=8.34(dd,J=1.2,7.7Hz,1H),7.90-7.76(m,2H),7.38(dd,J=1.0,7.4Hz,1H),7.12(d,J=9.0Hz,2H),6.69-6.60(m,4H),4.50(s,8H),4.38(s,8H).13C NMR(101MHz,Methanol-d4)δ=166.55,161.76,161.42,161.12,161.08,157.53,156.18,133.95,132.51,131.18,131.13,130.77,130.12,129.94,118.24,115.35,114.09,112.60,94.41,60.56,54.98,36.10.MS(ESI):calc'd forC30H29N4O3[M]+493.2,measured 493.2;HRMS(ESI):calc'd for[M]+493.22342,measured493.22317.。
实施例26
化合物RhN-04的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2,6-二氮杂螺[3.4]辛烷-6-甲酸叔丁酯(212mg,1.00mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体产物直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物RhN-04(49mg,产率:19%)。1H NMR(400MHz,Methanol-d4)δ=8.30(dd,J=1.2,7.8Hz,1H),7.86-7.73(m,2H),7.36(dd,J=1.2,7.4Hz,1H),7.10(d,J=9.2Hz,2H),6.67-6.57(m,4H),4.37-4.21(m,8H),3.58(s,4H),3.39(t,J=7.3Hz,4H),2.38(t,J=7.3Hz,4H).13C NMR(101MHz,Methanol-d4)δ=166.58,161.74,161.40,161.20,157.56,156.62,133.95,132.52,131.18,131.13,130.81,130.13,129.95,114.17,112.69,94.53,60.20,52.93,44.42,40.30,34.86.MS(ESI):calc'd for C32H33N4O3[M]+521.3,measured 521.1;HRMS(ESI):calc'd for[M]+521.25472,measured 521.25411.。
实施例27
化合物RhN-05的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(226mg,1.00mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体产物直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(1mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物RhN-05(57mg,产率:21%)。1H NMR(400MHz,Methanol-d4)δ=8.34(dd,J=1.2,7.8Hz,1H),7.91-7.75(m,2H),7.39(dd,J=1.1,7.5Hz,1H),7.12(d,J=9.2Hz,2H),6.73-6.61(m,4H),4.11(s,8H),3.29-3.19(m,8H),2.19-2.05(m,8H).13C NMR(101MHz,Methanol-d4)δ=166.56,161.30,161.01,157.54,156.92,133.95,132.49,131.19,131.07,130.82,130.10,129.98,114.02,112.55,94.28,60.20,40.98,32.92,31.17.MS(ESI):calc'd forC34H37N4O3[M]+549.3,measured 549.2;HRMS(ESI):calc'd for[M]+549.28602,measured549.28617.。
实施例28
化合物RhO-03的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2-氧杂-6-氮杂-螺[3,3]庚烷草酸盐(144mg,0.50mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体A直接用于下步反应。将中间体A溶解于甲醇/水(10/2mL)中,然后加入氢氧化钠(46mg,2mmol)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物RhO-03(60mg,产率:29%)。1H NMR(600MHz,DMSO-d6)δ8.01–7.95(m,1H),7.78(td,J=7.5,1.2Hz,1H),7.70(td,J=7.5,0.9Hz,1H),7.22(d,J=7.8Hz,1H),6.66(d,J=1.9Hz,1H),6.53(d,J=2.4Hz,2H),6.49(d,J=8.6Hz,1H),6.29(d,J=2.3Hz,1H),6.19(dd,J=8.6,2.3Hz,1H),4.70(s,4H),4.02(s,4H).13C NMR(151MHz,DMSO-d6)δ168.78,159.44,152.94,152.52,151.95,151.78,135.54,130.04,129.68,129.04,128.54,126.31,124.58,123.99,112.49,109.77,108.52,107.21,102.20,97.75,79.87,60.92,38.37.。
实施例29
化合物RhO-04的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2,6-二氮杂螺[3.3]庚烷-2-甲酸叔丁酯草酸盐(121mg,0.50mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体A直接用于下步反应。将中间体A溶解于甲醇/水(10/2mL)中,然后加入氢氧化钠(46mg,2mmol)室温搅拌3小时,LC-MS检测反应已经完全,将反应体系用1N稀盐酸调至中性后将溶剂旋干,得到粗产品中间体B直接用于下步反应。将中间体B溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物RhO-04(53mg,产率:26%)。1H NMR(400MHz,Methanol-d4)δ=8.33(dd,J=1.2,7.6Hz,1H),7.91-7.77(m,2H),7.39(dd,J=1.2,7.4Hz,1H),7.18(dd,J=7.3,9.2Hz,2H),7.07(d,J=2.3Hz,1H),6.95(dd,J=2.3,9.0Hz,1H),6.74(dd,J=2.1,9.3Hz,1H),6.62(d,J=2.2Hz,1H),4.53(s,4H),4.40(s,4H).13C NMR(101MHz,Methanol-d4)δ=167.06,166.93,158.15,156.88,156.59,135.37,132.84,131.66,131.05,130.64,130.43,130.33,129.38,116.81,115.25,114.81,114.20,102.00,94.58,60.94,54.87,36.06.MS(ESI):calc'd for C25H20N2O4[M+H]+413.1,measured413.1;HRMS(ESI):calc'd for[M+H]+413.14958,measured 413.14911.。
实施例30
化合物RhO-05的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(113mg,0.50mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体产物A直接用于下步反应。将中间体A溶解于甲醇/水(10/2mL)中,然后加入氢氧化钠(46mg,2mmol)室温搅拌3小时,LC-MS检测反应已经完全,将反应体系用1N稀盐酸调至中性后将溶剂旋干,得到粗产品中间体B直接用于下步反应。将中间体B溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物RhO-05(46mg,产率:21%)。1H NMR(400MHz,Methanol-d4)δ=8.31(dd,J=1.0,7.7Hz,1H),7.90-7.79(m,2H),7.39(dd,J=0.9,7.4Hz,1H),7.18-7.04(m,3H),6.92(dd,J=2.3,9.0Hz,1H),6.77-6.66(m,2H),4.15(s,4H),3.28-3.23(m,4H),2.18-2.11(m,4H).MS(ESI):calc'd for C27H24N2O4[M+H]+441.2,measured441.1;HRMS(ESI):calc'dfor[M+H]+441.18088,measured441.18141.。
实施例31
化合物RhO-06的制备
氩气下,将化合物RhN-00(300mg,0.50mmol),2,6-二氮杂螺[3.4]辛烷-6-甲酸叔丁酯(106mg,0.50mmol),Pd2(dba)3(92mg,0.10mmol),X-Phos(72mg,0.15mmol)和碳酸铯(489mg,1.50mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(40mL)和水(40mL)萃取,有机相浓缩后得到粗产品中间体产物A直接用于下步反应。将中间体A溶解于甲醇/水(10/2mL)中,然后加入氢氧化钠(46mg,2mmol)室温搅拌3小时,LC-MS检测反应已经完全,将反应体系用1N稀盐酸调至中性后将溶剂旋干,得到粗产品中间体B直接用于下步反应。将中间体B溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,将反应溶剂旋干后用反相制备色谱纯化得到产物RhO-06(38mg,产率:18%)。1H NMR(400MHz,Methanol-d4)δ=8.29(dd,J=0.9,7.8Hz,1H),7.89-7.77(m,2H),7.38(dd,J=0.8,7.4Hz,1H),7.18-7.03(m,3H),6.91(dd,J=2.2,8.9Hz,1H),6.75-6.64(m,2H),4.40-4.27(m,4H),3.61(s,2H),3.42(t,J=7.3Hz,2H),2.42(t,J=7.3Hz,2H).13C NMR(101MHz,DMSO-d6)δ=168.74,159.06,158.72,154.33,153.79(br dd,J=4.4,91.7Hz,1C),153.20,135.47,130.59,130.12,129.94,129.69,127.66,126.38,126.28,125.62,118.31,113.97,111.18,111.11,102.65,97.72,61.27,53.26,44.57,35.29.MS(ESI):calc'd for C26H22N2O4[M+H]+427.2,measured 427.1;HRMS(ESI):calc'd for[M+H]+427.16523,measured427.16535.。
实施例32
化合物Oxa-02的制备
氩气下,将化合物Oxa-00(50mg,0.15mmol),氮杂环丁烷盐酸盐(39mg,0.42mmol),Pd2(dba)3(20mg,0.02mmol),X-Phos(40mg,0.08mmol)和碳酸铯(200mg,0.61mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物Oxa-02(22mg,产率:58%)。1H NMR(600MHz,Chloroform-d)δ7.56(d,J=8.7Hz,1H),7.40(d,J=9.7Hz,1H),6.79(dd,J=9.7,2.0Hz,1H),6.40(dd,J=8.8,2.3Hz,1H),6.30(d,J=2.0Hz,1H),6.16(d,J=2.4Hz,1H),4.14(t,J=7.4Hz,4H),2.53(p,J=7.5Hz,2H).13C NMR(151MHz,Chloroform-d)δ186.12,153.91,150.36,146.81,141.33,134.37,132.27,131.90,126.88,109.27,106.04,94.79,51.48,16.13.。
实施例33
化合物Oxa-03的制备
氩气下,将化合物Oxa-00(50mg,0.15mmol),6-氧杂-1-氮杂-螺[3,3]庚烷草酸盐(60mg,0.42mmol),Pd2(dba)3(20mg,0.02mmol),X-Phos(40mg,0.08mmol)和碳酸铯(200mg,0.61mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物Oxa-03(25mg,产率:57%)。1H NMR(600MHz,Chloroform-d)δ7.56(d,J=8.7Hz,1H),7.37(d,J=9.7Hz,1H),6.77(dd,J=9.8,2.1Hz,1H),6.38(dd,J=8.8,2.5Hz,1H),6.27(d,J=2.1Hz,1H),6.17(d,J=2.5Hz,1H),4.89(s,4H),4.24(s,4H).13C NMR(151MHz,Chloroform-d)δ186.17,153.15,150.19,146.53,142.35,134.46,132.74,131.91,127.07,109.48,106.24,95.71,80.76,60.91,38.74.。
实施例34
化合物Oxa-04的制备
氩气下,将化合物Oxa-00(50mg,0.15mmol),2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(95mg,0.42mmol),Pd2(dba)3(20mg,0.02mmol),X-Phos(40mg,0.08mmol)和碳酸铯(200mg,0.61mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,得到粗产品中间体直接用于下步反应。将中间体溶解于二氯甲烷(10mL)中,然后加入三氟醋酸(2mL)室温搅拌3小时,LC-MS检测反应已经完全,有机相浓缩后用反相制备色谱纯化得到产物Oxa-04(20mg,产率:42%)。1H NMR(400MHz,DMSO-d6)δ7.59(d,J=8.8Hz,1H),7.44(d,J=9.6Hz,1H),6.64(d,J=9.7Hz,1H),6.52(d,J=8.8Hz,1H),6.35(s,1H),6.16(s,1H),3.86(s,4H),2.92(s,4H),1.86(s,4H).13C NMR(151MHz,DMSO-d6)δ184.59,153.85,149.96,146.26,140.05,134.40,131.73,131.47,126.35,109.99,105.00,94.85,60.63,41.29,33.23,32.51.。
实施例35
化合物DAN-02的制备
氩气下,将化合物DAN-00(95mg,0.3mmol),氮杂环丁烷盐酸盐(39mg,0.42mmol),Pd2(dba)3(20mg,0.02mmol),X-Phos(40mg,0.08mmol)和碳酸铯(200mg,0.61mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物DAN-02(50mg,产率:74%)。1H NMR(400MHz,Chloroform-d)δ8.32(d,J=1.9Hz,1H),7.92(dd,J=8.8,1.9Hz,1H),7.78(d,J=8.7Hz,1H),7.61(d,J=8.7Hz,1H),6.81(dd,J=8.9,2.3Hz,1H),6.61(d,J=2.3Hz,1H),4.04(t,J=7.2Hz,4H),2.67(d,J=3.1Hz,3H),2.45(p,J=7.3Hz,2H).13C NMR(151MHz,Chloroform-d)δ197.68,151.42,137.44,130.88,130.80,130.64,125.88,125.58,124.70,114.82,103.94,52.05,26.39,16.76.。
实施例36
化合物DAN-03的制备
氩气下,将化合物DAN-00(95mg,0.3mmol),6-氧杂-1-氮杂-螺[3,3]庚烷草酸盐(60mg,0.42mmol),Pd2(dba)3(20mg,0.02mmol),X-Phos(40mg,0.08mmol)和碳酸铯(200mg,0.61mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物DAN-03(45mg,产率:56%)。1H NMR(400MHz,Chloroform-d)δ8.33(s,1H),7.94(d,J=8.4Hz,1H),7.80(d,J=8.6Hz,1H),7.63(d,J=8.5Hz,1H),6.81(dd,J=9.0,2.5Hz,1H),6.64(d,J=2.4Hz,1H),4.89(d,J=2.6Hz,4H),4.18(d,J=2.5Hz,4H),2.67(d,J=2.4Hz,3H).13C NMR(151MHz,Chloroform-d)δ197.00,149.80,136.58,130.70,130.32,129.89,125.40,125.37,124.22,114.31,104.16,80.51,60.74,38.42,25.78.。
实施例37
化合物DAN-04的制备
氩气下,将化合物DAN-00(95mg,0.3mmol),2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(95mg,0.42mmol),Pd2(dba)3(20mg,0.02mmol),X-Phos(40mg,0.08mmol)和碳酸铯(200mg,0.61mmol)溶于二氧六环(10mL)中,温度升至100℃反应过夜,LC-MS检测反应已经完全,将反应溶剂旋干,残留物用二氯甲烷(20mL)和水(20mL)萃取,有机相浓缩后用反相制备色谱纯化得到产物DAN-04(60mg,产率:68%)。1H NMR(400MHz,Methanol-d4)δ8.30(d,J=1.8Hz,1H),7.80–7.71(m,2H),7.52(d,J=8.7Hz,1H),6.80(dd,J=8.9,2.3Hz,1H),6.60(d,J=2.3Hz,1H),3.74(s,4H),3.14(t,J=5.9Hz,4H),2.55(s,3H),2.00(t,J=5.9Hz,4H).13C NMR(100MHz,Methanol-d4)δ200.28,152.63,139.00,132.27,132.07,131.92,127.25,127.03,125.35,116.11,105.31,61.87,42.58,34.27,33.11,26.43.。
实施例38
化合物BDP-A2的制备
将BDP-A0(20mg,0.07mmol)和氮杂环丁烷盐酸盐(26mg,0.28mmol)溶于三乙胺(0.1mL)和乙腈(3mL)中,室温反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物BDP-A2(14mg,产率:67%)。1H NMR(400MHz,Chloroform-d)δ6.88(d,J=4.6Hz,1H),6.60(s,1H),5.77(d,J=4.6Hz,1H),4.49(t,J=7.8Hz,4H),2.50–2.36(m,7H),2.12(s,3H),1.06(t,J=7.6Hz,3H).13C NMR(150MHz,Chloroform-d)δ158.99,142.57,133.74,132.23,128.96,128.49,127.97,113.69,108.06,53.64,17.45,16.23,15.10,11.86,9.25.。
实施例39
化合物BDP-A3的制备
将BDP-A0(20mg,0.07mmol)和2-氧杂-6-氮杂-螺[3,3]庚烷草酸盐(40mg,0.28mmol)溶于三乙胺(0.1mL)和乙腈(3mL)中,室温反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物BDP-A3(15mg,产率:62.5%)。1H NMR(400MHz,Chloroform-d)δ6.89(d,J=4.5Hz,1H),6.66(s,1H),5.75(d,J=4.5Hz,1H),4.86(s,4H),4.62(s,4H),2.41(d,J=15.8Hz,5H),2.13(s,3H),1.06(t,J=7.7Hz,3H).13C NMR(151MHz,Chloroform-d)δ158.35,144.21,133.30,132.11,129.89,129.36,128.62,114.86,107.39,80.71,62.78,58.49,38.73,18.45,17.42,15.01,11.95,9.27.。
实施例40
化合物Cou-P2的制备
将Cou-00(177mg,1.0mmol),EDCI(286mg,1.5mmol),DMAP(184mg,1.5mmol)和4-吡啶乙酸盐酸盐(259mg,1.5mmol)溶于三乙胺(0.4mL)和二氯甲烷(10mL)中,室温反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物Cou-P2(150mg,产率:54%)。1HNMR(400MHz,Chloroform-d)δ8.63(d,J=5.2Hz,2H),7.86(s,1H),7.68(d,J=5.2Hz,2H),7.35(d,J=8.4Hz,1H),6.37–6.29(m,1H),6.22(s,1H),4.05(t,J=7.4Hz,4H),2.48(p,J=7.4Hz,2H).13C NMR(151MHz,Chloroform-d)δ160.59,156.28,154.15,149.84,143.24,142.20,129.48,122.32,117.75,109.54,108.30,96.03,51.53,16.35.。
实施例41
化合物Cou-P3的制备
将Cou-01(219mg,1.0mmol),EDCI(286mg,1.5mmol),DMAP(184mg,1.5mmol)和4-吡啶乙酸盐酸盐(259mg,1.5mmol)溶于三乙胺(0.4mL)和二氯甲烷(10mL)中,室温反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物Cou-P3(160mg,产率:50%)。1HNMR(400MHz,Chloroform-d)δ8.64(d,J=5.2Hz,2H),7.86(s,1H),7.66(d,J=5.2Hz,2H),7.37(d,J=8.5Hz,1H),6.35(d,J=8.5Hz,1H),6.26(s,1H),4.88(s,4H),4.19(s,4H).13CNMR(151MHz,Chloroform-d)δ160.37,156.10,153.35,149.91,143.00,142.01,129.57,122.36,118.73,110.19,108.68,96.86,80.88,60.95,38.84.。
实施例42
化合物BDP-B2的制备
将BDP-B0(30mg,0.12mmol)和化合物R1(24mg,0.15mmol)溶于甲苯(10mL)中,加入哌啶(0.1mL)和乙酸(0.1mL),110℃反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物BDP-B2(16mg,产率:35.3%)。1H NMR(400MHz,Chloroform-d)δ7.59(s,1H),7.50(d,J=9.4Hz,4H),7.04(s,1H),6.82(d,J=3.8Hz,1H),6.40(d,J=7.0Hz,3H),3.99(t,J=7.3Hz,4H),2.71(q,J=7.5Hz,2H),2.42(p,J=7.3Hz,2H),2.20(s,3H),1.22(d,J=7.5Hz,3H).13C NMR(151MHz,Chloroform-d)δ158.14,152.78,141.18,140.93,137.55,136.68,134.82,132.82,129.52,125.40,123.52,120.44,115.37,114.35,110.89,51.88,18.87,16.64,13.72,9.21.。
实施例43
化合物BDP-B3的制备
将BDP-B0(30mg,0.12mmol)和化合物R2(30mg,0.15mmol)溶于甲苯(10mL)中,加入哌啶(0.1mL)和乙酸(0.1mL),110℃反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物BDP-B3(30mg,产率:69.3%)。1H NMR(400MHz,Chloroform-d)δ7.79–7.42(m,5H),7.07(s,1H),6.84(d,J=3.8Hz,1H),6.42(d,J=8.2Hz,3H),4.87(s,5H),4.12(s,4H),2.71(q,J=7.6Hz,2H),2.21(s,3H),1.28–1.18(m,5H).13C NMR(151MHz,Chloroform-d)δ190.44,157.78,151.79,141.02,140.59,137.43,137.14,134.75,132.91,129.42,126.33,124.01,120.96,115.59,115.04,111.45,110.38,81.14,80.98,61.25,60.86,38.97,29.32,18.83,13.73,9.22.。
实施例44
化合物DCI-02的制备
将DCI-00(100mg,0.54mmol)和化合物R1(63mg,0.39mmol)溶于甲苯(10mL)中,加入哌啶(0.1mL)和乙酸(0.1mL),110℃反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物DCI-02(98mg,产率:56%)。1H NMR(600MHz,Chloroform-d)δ7.40–7.36(m,2H),7.01(d,J=15.9Hz,1H),6.80(d,J=15.9Hz,1H),6.75(s,1H),6.41–6.37(m,2H),3.98(t,J=7.3Hz,4H),2.57(s,2H),2.45(s,2H),2.41(q,J=7.3Hz,2H),1.06(s,6H).13CNMR(151MHz,Chloroform-d)δ169.19,155.17,152.66,138.27,129.25,124.41(d,J=15.0Hz),121.51,114.18,113.40,110.99,75.96,51.84,43.04,39.29,32.00,28.06,16.64.。
实施例45
化合物DCI-03的制备
将DCI-00(100mg,0.54mmol)和化合物R2(80mg,0.39mmol)溶于甲苯(10mL)中,加入哌啶(0.1mL)和乙酸(0.1mL),110℃反应,LC-MS检测反应已经完全,将反应液用反相制备色谱纯化得到产物DCI-03(67mg,产率:46.5%)。1H NMR(600MHz,Chloroform-d)δ7.39(dt,J=7.7,4.1Hz,2H),7.00(d,J=15.6Hz,1H),6.87–6.78(m,1H),6.77–6.71(m,1H),6.47–6.33(m,2H),4.86(dt,J=5.1,2.4Hz,4H),4.11(dt,J=5.0,2.3Hz,4H),2.58(dt,J=4.9,2.3Hz,2H),2.44(d,J=4.7Hz,2H),1.06(dt,J=5.1,2.3Hz,6H).13C NMR(151MHz,Chloroform-d)δ169.25,154.90,151.70,137.80,129.17,125.28,125.12,121.88,114.04,113.28,111.56,81.10,61.22,43.00,39.24,38.96,32.01,28.05.。
实施例46
N,N-二甲氨基取代的萘酰亚胺(Nap-01)作为重要的环境敏感性荧光染料,由于其在水中极低的荧光量子产率(λmax=435nm,ε=8900M-1cm-1,Φ<0.01;表1),限制了其的广泛应用。将其N,N-二甲氨基替换为氮杂环丁烷和氮杂环丁烷螺环结构,得到了一系列新的荧光染料(Nap-02–Nap-12)。这些新的荧光染料在乙醇和水中都具有较高的荧光量子产率,且远远高于其母体结构Nap-01(如表1所示)。除此之外,氮杂环丁烷螺环结构修饰的荧光染料表现出良好的透膜性和溶解性。
表1萘酰亚胺类荧光染料光物理及理化性质
实施例47
四甲基罗丹明(TMR)作为重要的荧光染料(λmax=549nm,ε=82800M-1cm-1,Φ=0.41;表2),广泛应用于荧光标记与生物成像。Lavis等人将其N,N-二甲氨基替换为氮杂环丁烷结构,得到新荧光染料JF549的荧光量子产率明显提高。将其二甲氨基替换为氮杂环丁烷螺环结构,得到了一系列新的荧光染料(RhN-01–RhN-05)。这些新的荧光染料在水中都具有较高的荧光量子产率,且远远高于其母体结构TMR(如表2所示)。除此之外,氮杂环丁烷螺环结构修饰的荧光染料表现出良好的溶解性,以及优良的光稳定性(如图1所示)。
表2罗丹明类荧光染料光物理及理化性质
实施例48
将Rhodol染料二甲氨基替换为氮杂环丁烷螺环结构,得到了一系列新的荧光染料(RhO-03–RhO-06)。这些新的荧光染料在水中都具有较高的荧光量子产率,且远远高于其母体结构RhO-01(如表3所示)。除此之外,氮杂环丁烷螺环结构修饰的荧光染料表现出良好的溶解性。
表3 Rhodol类荧光染料光物理及理化性质
实施例49
将香豆素(Cou-01)的二甲氨基替换为氮杂环丁烷螺环结构,得到了一系列新的荧光染料(Cou-03–Cou-08)。这些新的荧光染料在水中都具有较高的荧光量子产率,且远远高于其母体结构Cou-01(如表4所示)。除此之外,氮杂环丁烷螺环结构修饰的荧光染料表现出良好的溶解性。
表4香豆素类荧光染料光物理及理化性质
实施例50
将7-硝基苯并呋咱(NBD-01)的二甲氨基替换为氮杂环丁烷螺环结构,得到了一系列新的荧光染料(NBD-03–NBD-07)。这些新的荧光染料在水中都具有较高的荧光量子产率,且高于其母体结构NBD-01(如表5所示)。除此之外,氮杂环丁烷螺环结构修饰的荧光染料表现出良好的溶解性。
表57-硝基苯并呋咱类荧光染料光物理及理化性质
实施例51
将Oxa-01的二甲氨基替换为氮杂环丁烷螺环结构,得到了一系列新的荧光染料(Oxa-02–Oxa-04)。这些新的荧光染料在水中都具有较高的荧光量子产率,且远远高于其母体结构Oxa-01(如表6所示)。
表6 Oxazine类荧光染料光物理性质
实施例52
将二甲氨基萘酮(DAN-01)的二甲氨基替换为氮杂环丁烷螺环结构,得到了一系列新的荧光染料(DAN-02–DAN-04)。这些新的荧光染料在水中都具有较高的荧光量子产率,且远远高于其母体结构DAN-01(如表7所示)。
表7 Acedan类荧光染料光物理性质
实施例53
将BDP-A1的二甲氨基替换为氮杂环丁烷螺环结构,得到了新的荧光染料(BDP-A2–BDP-A3)。这些新的荧光染料在水中都具有较高的荧光量子产率,且远远高于其母体结构BDP-A1(如表8所示)。
表8 BODIPY类荧光染料光物理性质
实施例54
将Cou-P1的二甲氨基替换为氮杂环丁烷螺环结构,得到了新的荧光染料(Cou-P2–Cou-P3)。这些新的荧光染料在水中都具有较高的荧光量子产率,且远远高于其母体结构Cou-P1(如表9所示)。
表9吡啶香豆素类荧光染料光物理性质
实施例55
将BDP-B1的二甲氨基替换为氮杂环丁烷螺环结构,得到了新的荧光染料(BDP-B2–BDP-B2)。这些新的荧光染料在乙醇中荧光量子产率较低,相差较小(如表10所示)。
表10 BODIPY类荧光染料光物理性质
实施例56
将DCI-01的二甲氨基替换为氮杂环丁烷螺环结构,得到了新的荧光染料(DCI-02–DCI-03)。这些新的荧光染料在乙醇中荧光量子产率较低,相差较小(如表11所示)。
表11二氰基异氟尔酮类荧光染料光物理性质
实施例57
将BDP-C1的二甲氨基替换为氮杂环丁烷螺环结构,得到了新的荧光染料(BDP-C2–BDP-C3)。这些新的荧光染料在二氯甲烷中荧光量子产率较低,相差较小(如表12所示)。
表12 BODIPY类荧光染料光物理性质
实施例58
将Nap-01和Nap-04分别设计为检测生物硫醇的荧光探针P1和P2。探针P1与谷胱甘肽反应后荧光增强6.4倍;探针P2与谷胱甘肽反应后荧光增强19.5倍(如图2所示);探针P2的检测效果明显优探针P1(如图3所示)。
实施例59
将Oxa-04(FF600)连接上不同的细胞器靶向基团,得到三个探针FF600-Mito,FF600-Lyso和FF600-ER。它们的细胞成像Pearson’s系数分别为0.85,0.80,和0.74,表明这些探针都有良好的细胞器靶向能力(如图4-6所示)。
实施例60
将染料RhO-05连接上识别基团和FRET供体,设计成为检测次硝酸(HNO)的探针FRET-01。此探针能够有效地形成FRET效应(如图7所示),能够作为检测次硝酸的比率型探针,并且成功地应用于细胞成像(如图8所示)。
Claims (32)
1.如下骨架结构(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,
其中:取代基R1,R2各自独立为H、卤素、羟基、C1-C18烷氧基、巯基、C1-C18烷硫基、氨基、C1-C18烷基、C1-C18不饱和烷基、C3-C8饱和杂环、C3-C8环烷基、芳基、杂芳基、酰基、酯基、羧基、磷酸基、磺酸基;所述卤素为F、Cl、Br;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基;
m=0或1或2或3或4;n=0或1或2或3或4;且m+n=2、3、4、5;
X为C或N或O或S或S=O或O=S=O,其中C和N原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、乙酰基、叔丁氧羰基、苄基、芳基;
包含X的环上碳原子上有取代基甲基、乙基、羟基、氨基、羧基或氟。
2.根据权利要求1所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,其中:取代基R1,R2各自独立为H、卤素、羟基、C1-C6烷氧基、C1-C6烷基、羧基、磷酸基、磺酸基;所述卤素为F、Cl、Br。
3.根据权利要求1所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,其中:取代基R1,R2各自独立为H、卤素、C1-C4的直链或支链烷基;所述卤素为F、Cl。
5.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料中,其荧光团(Fluorophore)包括萘酰亚胺类荧光染料(Ⅲ)或(Ⅳ):
其中:取代基R3,R6各自独立为H、卤素、氨基、芳基、杂芳基、磺酸基、醛基;取代基R4,R5各自独立为H、卤素、芳基、杂芳基;取代基R8为H、C1-C16直链或支链的饱和及不饱和烷基链、C1-C16直链或支链聚乙二醇链、C1-C16的多肽、C1-C16含羧基烷基链、C1-C16含酯基或酰胺结构烷基链、C1-C16含芳香环的烷基链、芳基、杂芳基等;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R3和R4,或者R5和R6可以成环。
7.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料中,其荧光团(Fluorophore)包括香豆素类荧光染料(Ⅴ)或(Ⅵ):
其中:取代基R3,R4,R5各自独立为H、卤素、氨基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基、磺酸基;取代基R6为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基、羧基、酯基、醛基、硝基、氰基、三氟甲基;取代基R7为H、卤素、硝基、C1-C6不饱和烷基、C1-C6取代的烯基、取代的芳烯基、芳基、杂芳基、氰基、醛基、酰基、酯基、叠氮基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基等;
Z为N或O或S。
9.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料中,其荧光团(Fluorophore)包括咕吨类荧光染料(Ⅶ)或(Ⅷ):
其中:取代基R3,R4,R5,R6,R7,R8各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R10为H、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、C3-C8杂环烷基、四元螺环结构;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R6和R8,或者R4和R7可以成环;
Y为C,或N,其中C原子上有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基;
Z独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上可以有不同取代基,如甲基、乙基、芳基。
11.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料,其荧光团(Fluorophore)包括Rhodol类荧光染料(Ⅸ)或(Ⅹ):
其中:取代基R3,R4,R5,R6,R7,R8各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R6和R8,或者R4和R7可以成环;
Y为C,或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基;
Z独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上可以有不同取代基,如甲基、乙基、芳基。
13.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料,其荧光团(Fluorophore)包括萘罗丹明类荧光染料(Ⅺ)或(Ⅻ):
其中:取代基R3,R4,R5,R6,R7,R8,R10,R11各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C或N,其中C原子上有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基;
Z独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上有不同取代基,如甲基、乙基、芳基。
15.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料,其荧光团(Fluorophore)包括三苯基吡喃类荧光染料(XIII)或(XIV):
其中:取代基R3,R4,R5,R6,R7,R8各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R9为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R10为H、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、C3-C8杂环烷基、四元螺环结构;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基。
17.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料,其荧光团(Fluorophore)包括苯并呋咱类荧光染料(XV)或(XVI):
其中:取代基R3,R4各自独立为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基;取代基R5为卤素、芳基、杂芳基、氰基、硝基、醛基、酰基、酯基、磺酸基、三氟甲基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R3和R4可以成环;
Y为C或N或O或S或Se或Te,其中C原子上可以有不同取代基,如甲基、乙基、环烷基、羟基、氨基、羧基、酯基、芳基。
19.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料,其荧光团(Fluorophore)包括氟硼二吡咯(BODIPY)类荧光染料(XVII),(XVIII),(XIX)或(XX):
其中:取代基R3,R4,R6各自独立为H、卤素、巯基、氨基、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、磺酸基、羧基、酰基、酯基、醛基、氰基;取代基R5为H、卤素、羟基、巯基、氨基、C1-C6烷氧基、C1-C6烷硫基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R7,R8各自独立为H、卤素、巯基、氨基、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、磺酸基、羧基、酰基、酯基、醛基、氰基、氨基、C1-C6含氮烷烃、C3-C8含氮环烷烃、含氮四元螺环结构;取代基R9,R10各自独立为F、C1-C16烷氧基、C1-C6烷基、C1-C6不饱和烷基、杂芳基、羧基、氰基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
R6和R7可以成环;
Y为C或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基。
21.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料,其荧光团(Fluorophore)包括二甲氨基萘酮类荧光染料(XXI)或(XXII):
其中:取代基R3,R4,R5,R6,R7各自独立为H、卤素、C1-C6烷氧基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R8为H、羟基、氨基、C1-C6烷氧基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、C1-C6取代的烯基、取代的芳烯基、芳基、杂芳基、三氟甲基;取代基R9为H、卤素、芳基、杂芳基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y为C或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基。
23.根据权利要求1–4所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,所述的含氮杂环丁烷螺环结构的荧光染料,其荧光团(Fluorophore)包括氧杂蒽类荧光染料(XXIII)或(XXIV):
其中:取代基R3,R4,R6各自独立为H、卤素、C1-C6烷氧基、C1-C6烷基、C1-C6不饱和烷基、C3-C8环烷基、芳基、杂芳基;取代基R5为H、卤素、C1-C6烷基、C1-C6不饱和烷基、芳基、杂芳基、羧基、酰基、酯基、醛基、硝基、氰基、叠氮基、三氟甲基;取代基R7,R8,R9各自独立为H、芳基、杂芳基、羧基、酯基、醛基、氰基;所述卤素为F、Cl、Br、I;所述芳基为苯基、萘基;杂芳基为噻吩基、呋喃基、吡啶基、嘧啶基、苯并噻唑基、苯并恶唑基、苯并咪唑基、吲哚基;
Y独立为C、N、O、S、S=O、O=S=O、Si、Se、Se=O、O=Se=O、Ge、Sn、Te、Te=O、O=Te=O、P,其中C、N、Si、Ge、Sn、P原子上可以有不同取代基,如甲基、乙基、芳基;
Z为C或N,其中C原子上可以有不同取代基,如甲基、乙基、羟基、氨基、羧基、酯基、芳基。
25.根据权利要求1–24所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料,其特征在于,当X为氮原子时,其新的连结位点结构为(XXV),或(XXVI),或(XXVII),或(XXVIII):
所述新的连结位点连结氨基酸、蛋白、糖蛋白、脂蛋白、受体、抗体或其片段、抗原、适配体、多糖、寡糖、核苷、核苷酸、低聚核苷酸、核酸、药物、抑制剂、激素、营养物、代谢物、生长因子、脂质、聚合物、聚合微粒、细胞、病毒、酶的底物;或,连结线粒体靶向基团、溶酶体靶向基团、内质网靶向基团,用于靶向细胞器的荧光探针;连结蛋白标记结构、抗体连结结构、肿瘤靶向结构,用于细胞高清显微镜成像和肿瘤靶向成像;连结三线态淬灭结构,用于增强荧光团的光稳定性;连结点击化学反应结构,用于点击化学(Click);
其中:所述的新连结位点,直接与功能结构(Functional Structure)相连(XXV或XXVI);所述的新连结位点间接地通过连结子(Linker)与功能结构相连(XXVII或XXVIII);
所述连接子(Linker)为C1-C16直链或支链的饱和及不饱和烷基链、C1-C16直链或支链聚乙二醇链、C1-C16的多肽、C1-C16含羧基烷基链、C1-C16含酯基或酰胺结构烷基链、C1-C16含芳香环的烷基链;
所述功能结构(Functional Structure)为氨基酸、蛋白、糖蛋白、脂蛋白、受体、抗体或其片段、抗原、适配体、多糖、寡糖、核苷、核苷酸、低聚核苷酸、核酸、药物、抑制剂、激素、营养物、代谢物、生长因子、脂质、聚合物、聚合微粒、细胞、病毒、酶的底物;或,线粒体靶向基团、溶酶体靶向基团、内质网靶向基团、蛋白标记结构、抗体连结结构、肿瘤靶向结构、三线态淬灭结构或点击化学反应结构。
31.权利要求1–25所述的(Ⅰ)或(Ⅱ)的含氮杂环丁烷螺环结构的荧光染料在用于备构建荧光共振能量转移(FRET)探针中的用途,其中,所述的新的连结位点结构为,
其中:所述的新连结位点,直接与另一个合适荧光团相连而设计成基于FRET的荧光探针(XXIX或XXX);所述的新连结位点,间接地通过连结子与另一个合适荧光团相连而设计成基于FRET的荧光探针(XXXI或XXXII);
所述的连接子(Linker)为C1-C6直链或支链的饱和及不饱和烷基链、C1-C6直链或支链烷氧链、C1-C6含羧基烷基链、C1-C6的多肽、C1-C6含酯基或酰胺结构烷基链、C1-C6含芳香环的烷基链;
所述的荧光团2(Fluorophore 2)为以下染料:权利要求1–22所述的含氮杂环丁烷螺环结构荧光染料、萘酮类染料、香豆素染料、荧光素类染料、罗丹明类染料、BODIPY类染料、菁染料、苯并呋咱类染料或氧杂蒽类染料。
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