CN1151209A - 用双-二阳离子芳基呋喃荧光检测核酸及细胞骨架成分的方法 - Google Patents
用双-二阳离子芳基呋喃荧光检测核酸及细胞骨架成分的方法 Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
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- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
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Abstract
本发明公开了一种荧光检测核酸的方法。该方法包括将该核酸和双-二阳离子芳基呋喃化合物如:2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂卓-2-基)苯基]呋喃;2,5-双{[4-(N-异丙基)脒基]苯基}呋喃及其生理上可接受的盐相接触,然后将该核酸暴露于一定频率的光中以诱发该化合物的荧光。本发明还公开了一种荧光检测细胞骨架成分的方法以及新的双—二阳离子芳基呋喃化合物。
Description
本发明是在来自国家健康研究所的编号为U01-AI-3363的政府资助下完成的。政府对本发明持有某些权利。
本发明涉及结合并检测核酸和细胞骨架成分的方法。更具体地说,本发明涉及使用双-二阳离子芳基呋喃化合物荧光检测核酸和细胞骨架成分的方法。
很多种样品的分析依赖于一种染料的荧光特性。当一个分子被一定波长的光激发后回复到非激发态(稳定态)而放射出波长更长的光时便产生了荧光。激发光和发射光由于波长不同,可用滤光器彼此区分开来。荧光已通过光学显微术被应用于观测某些分子(及其结构)很多年了,它也应用于诸如流式血细胞计数等其它分析技术。
在荧光分析中使用的荧光探针可分为两大类,即那些共价标记其它探针(通常为抗体)的探针以及那些其分布或荧光反映出它们的环境和细胞特定特征的探针。在后一类中,那些特异性地结合于核酸或细胞骨架结构或组分的荧光化合物特别重要。
已知各种各样的荧光探针,例如丙锭和乙锭类染料已被应用。然而,这些化合物和脱氧核糖核酸(DNA)以及双链核糖核酸(RNA)都能结合。因此,如果要检测DNA,那么必需除去RNA。
4′,6-二脒基-2-苯基吲哚(DAPI)也作为DNA染料广泛地应用于细胞学,核酸的生物物理分析以及流式血细胞计数。DAPI优选结合于富含AT的DNA序列小沟,但它也嵌入GC和混杂GC/AT的序列,更重要的是它还和RNA结合。W.D.Wilson,等人,“The Effects of Ligand Structure on Binding Mode of Unfused Aro-matic Cations with DNA”in Molecular Basis of Specificity in NucleicAcid-Drug Interactions,Klumar Academic Publishers,Amsterdam(1990),pps.331-353;W.D.Wilson,等人,Biochemistry 32,4098-4104(1993)。DAPI也和诸如微管蛋白等其它细胞组分结合,导致RNA及微管蛋白被染色。
过去已经合成许多芳基联脒用作抗原生生物制剂,B.P.Das andD.W.Boykin,J.Med,Chem.20,531-536(1977)。象DAPI一样,2,5-双(4-脒基苯基)呋喃也优选结合于富含AT的DNA序列中的DNA小沟,但也相互作用于GC和混杂GC/AT序列,W.D.Wilson,等人.(1990),Supra;W.D.Wilson,等人(1993),Supra。
另一个应用于荧光分析的染料是双-苯酰亚胺Hoechst 33258。Loontiens等提出,作为一紧密相关的分子,Hoechst 33258在DNA序列大沟中通过自缔合复合物与富含GC的序列结合。F.G.Loon-tiens,et al,Biochemistry29,9029-9039(1990)。此染料也和RNA有明显的结合。Wilson,et al.(1993),Supra。因此,同DAPI以及2,5-双(4-脒基苯基)呋喃一样,Hoechst 33258也会与非DNA成分明显染色。
其中:R1和R2各自独立选自H,低级烷基,环烷基,芳基,羟基烷基,氨基烷基或者烷基氨基烷基,或者R1和R2共同代表一个C2到C10的亚烷基或R1和R2一起为:
其中:n是从1到3的数字,R10是H或者-CONHR11NR15R16,其中R15和R16是各自独立选自H和低级烷基;R3是H,低级烷基,环烷基,芳基,羟基烷基,氨基烷基或者烷基氨基烷基;
A是杂环芳基团,选自如下一组基团:
其中R4,R5和R6各自独立选自H,低级烷基,卤素,氧烷基,氧芳基,或者氧芳基烷基;
R12是H,低级烷基,羟基,氨基烷基或者烷基氨基烷基。
其中:n是从1到3的数字,R10是H或者-CONHR11NR15R16,其中R15和R16各自独立选自H和低级烷基;并且R3,R4和R5每一个是H。在本发明另一个优选实施方案中,R1,R3,R4和R5为H,而R2则为低级烷基。
本发明的第二个方面是荧光检测核酸的方法。此方法包括,将根据式(I)的化合物与核酸相接触,且暴露核酸于光中以诱发式(I)化合物的荧光。
本发明的第三方面是在含有DNA和RNA的核酸混合物中选择性地对DNA进行荧光检测的方法。此方法包括步骤(a):将根据式(I)的化合物与核酸混合物相接触;步骤(b)暴露核酸混合物于光中以诱发式(I)化合物的荧光。
本发明的另一方面是荧光检测微管结构的方法。此方法包括,将根据式(I)的化合物与微管结构相接触,且暴露微管结构于光中以诱发式(I)化合物的荧光。
本发明还有一个方面就是在一细胞中同时荧光检测第一细胞结构和第二细胞结构的方法,这里所说的第一细胞结构和第二细胞结构是不同的。此方法包括(a)将细胞与第一荧光化合物及第二荧光化合物相接触。第一荧光化合物和第二荧光化合物彼此存在着结构上的不同,但每一荧光化合物都有根据式(I)的结构。第一荧光化合物选择性地结合于第一结构,而第二化合物则选择性地结合于第二结构。并且,第一和第二荧光化合物具有不同的荧光发射光谱。当细胞与第一和第二荧光化合物接触后,(b)将细胞暴露于光中以诱发第一和第二荧光化合物的荧光,从而使第一细胞结构和第二细胞结构以不同的荧光发射光谱发射荧光。
本发明还有另一方面,是为荧光检测细胞结构而提供的试剂盒。试剂盒包括(a)根据式(I)的化合物,(b)一种充足量的溶剂,以便当化合物和含有核酸的样品相接触时能形成被式(I)的化合物标记的核酸的混合物。
本发明进一步的方面是包括含有式(I),(Ia)或(Ib)化合物或其生理上可接受的盐作为试剂盒中的成分的试剂盒。
对本发明上述的以及其它的目的和方面在下面叙述中有详细解释。
这里公开了在荧光检测核酸方法中有用的化合物。该化合物包括如下式(I)所示的化合物:
其中n是从1到3的数字,R10是H或者-CONHR11NR15R16,这里的R15和R16各自独立选自H和低级烷基。优选地,R10是H。R10可以位于环中C-2,C-3,C-4或C-5的任何位置。优选地,R10位于C-3位置。
这里的n和R10如上所述。
在另一优选实施方案中,R1和R2一同代表C2到C10的直链饱和亚烷基。更优选地,R1和R2一同代表C2到C5的直链饱和亚烷基。
在本发明再一优选实施方案中,R1为H,而R2则为低级烷基,优选异丙基。
R3可选自H,低级烷基,环烷基,芳基,氨基烷基或烷基氨基烷基。优选地,R3是H或如式-R14OH的烷基羟基,这里的R14为低级烷基。优选地,R14为-(CH2)2-。
前述的代表A的基团可被R4和R5邻、间、对位取代。
R4可选自H,低级烷基,卤素,氧烷基,氧芳基或氧芳基烷基。优选R4为H或者低级烷基。
R5可选自H,低级烷基,卤素,氧烷基,氧芳基或氧芳基烷基。优选地,R5为H。
R12可选自H,低级烷基,羟基,氨基烷基或烷基氨基烷基。
在一优选实施方案中,A为
这里R4和R5如上述定义,但优选地,R4为H,且R5为OCH3或O(C6H4)R,这里R为H或低级烷基。更优选地,R为低级烷基,优选为甲基。
这里的术语“低级烷基”指的是C1到C4直链或支链烷基,比如甲基,乙基,丙基,丁基,异丙基,仲丁基和叔丁基。这里的术语“环烷基”是指C3到C6的环状烷基,比如环丙基,环丁基,环戊基和环己基。这里的术语“芳基”是指C3到C10的环状芳基比如苯基,萘基及其类似物,并包括诸如甲苯基的取代芳基。这里的术语“羟基烷基”是指从C1到C4的直链或支链羟基取代的烷基,比如,-CH2OH,-(CH2)2OH,等等。这里的术语“氨基烷基”是指C1到C4的直链或支链氨基取代的烷基,其中的术语“氨基”是指NR′R″基团,这里的R′和R″独立选自H或如上述定义的低级烷基,比如:-NH2,-NHCH3,-N(CH3)2等等。这里的术语“氧烷基”是指C1到C4的氧取代烷基,如:-OCH3,而术语“氧芳基”则为C3到C10的氧取代的环状芳基。
本发明方法中优选的且代表根据式(I)的新的化合物的化合物包括,但不局限于:2,5-双(4-脒基苯基)呋喃;2,5-双[4-(4,5-二氢-1H-咪唑基-2-基)苯基]呋喃;2,5-双[4-(1,4,5,6-四氢嘧啶-2-基)苯基]呋喃;2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂卓-2-基)苯基]呋喃;2,5-双[4-(N-异丙基脒基)苯基]呋喃;以及它们生理上可接受的盐。
本发明的方法是将在其基本上纯的溶液中或者在一含有核酸的生物样品中的核酸与上述式(I)的化合物接触或混合,然后暴露该生物样品于光中以诱发式(I)化合物的荧光。暴露生物样品于光中以诱发式(I)化合物的荧光的步骤可以分析样品,即,检测其中所含的核酸组分的存在。本发明的方法可应用于诸如光学显微术,流式血细胞计数,染色体组型分析,核酸检测和定量,电泳和其它类似的技术中。
本发明的化合物可结合样品中的核酸,从而适合于染色任何怀疑含有核酸的生物标本。这里所述的术语“核酸”是指脱氧核糖核酸(DNA)和核糖核酸(RNA)两种。任何核酸均可被染色,包括染色体和染色体以外的核酸(比如:质粒,RNA病毒等等)。从而,通过荧光检测与其相连的化合物用本领域已知的检测样品核酸的技术可以测定存在于生物样品中的核酸,这些技术包括光学显微术,共焦光学显微术,流式血细胞计数以及其它类似的技术。
按照本发明,适合染色的典型样品的例子是以生物学流体或生物学组织形式获得的样品。本领域技术人员懂得,可以从能获得有用的诊断信息的任何生物来源中抽提样品。合适的生物学流体包括,但并不局限于,血液,唾液,尿液,奶液,淋巴液和口、鼻及支气管的粘膜。合适的组织样品包括,但不局限于,组织活检样品,比如来自肾脏,肝脏,淋巴结或其它器官和皮肤以及其它软组织样品(如肌肉)。从被检者取出的试样可直接染色,或者从试样进行组织生长培养。合适的生物学试样的选择以及合适的培养技术对本领域熟练技术人员来说是显而易见的。样品也可为待测核酸的基本上纯的溶液,即从如上述的试样中分离的胞内组分的溶液。
可从动物和植物物种中收集样品。动物物种包括人类和非人类物种(如:狗,猫,鼠,牛,马,羊,猴等等)。其它类型的细胞,比如细菌,真菌,原生动物以及其它单细胞生物,也可按照本发明来处理。因此,可用的细胞包括真核的和原核的。
一般地,细胞在合适的条件下和按照式(I)的荧光化合物保温时被染色。例如,根据式(I)的化合物的染色可按照本领域已知的标准的DAPI染色方案而完成,如O.Miller的Principles and Practices inMedical Genetics(Longman Group Limited,New York 1983)以及D.Silvonen的ACT Cytogenetic Lab Manual(University of California,San Francisco 1980)中所述。另外,可以使用标准的双苯酰亚胺染色方案,这也是本领域技术人员已知的。参见,如Human Cytogenet-ics,Volume l,Constitutional Analysis,pg113(D.Rooney and B.Czepulkowski,Eds.,IRL Press at Oxford University Press)。
将样品和一定量的式(I)的化合物接触或混合以便使化合物和样品中的核酸相结合。由于一个式(I)的化合物分子通常结合四个碱基对双链核酸,所以可以使用任何足以产生可检测信号的量。因此,式(I)化合物与待测靶的比例或比率可能约为1∶4,1∶8,1∶16,1∶32或更高,这取决于靶是如何被检测的其它方面。若需要,过量的化合物可在染色后被选择性洗掉。正如熟练的操作者所知的,当样品是如上述的基本上纯的核酸溶液时,分析技术中就不需要洗涤了,分析时使用诸如荧光计等仪器。
可以水溶液形式提供化合物,且接触步骤可只简单地将样品浸入溶液中来进行。但这里不需象所报道的DAPI必需的那样将溶液贮存于黑暗中以保持化合物的稳定。这一溶液的最小终浓度可为约0.01微摩尔/升(μm),虽然通常此溶液的终浓度可能介于0.01和5,10或者25微摩尔/升,或者更高值之间。样品和化合物间接触时间取决于接触技术而可能有很大的变化,但一般介于约30秒和两小时之间,当样品浸入如上述的浓度的溶液中时优选地介于约1到15分钟之间。
样品也可通过使用荧光染料的组合而染色。因此,在不同波长下,根据式(I)的化合物的荧光以及其它化合物的荧光可被同时检测。正如熟练技术人员所了解的那样,当使用不只一个标记时,应注意选择具有最大的发射波长或光谱不至重叠的荧光染料。而且,使用荧光能量转移技术,能选择放大荧光染料的组合的荧光信号。同样,正如熟练技术人员所了解的那样,当使用不只一种荧光染料时,应注意选择那些不相互发生化学反应或者不和根据式(I)的化合物发生化学反应的荧光染料。
在本发明方法的应用中,修饰式(I)的化合物以去除DNA结合亲合力而不去除可能结合其它物质的方便的结合位点可能有益。式(I)化合物可能连接的合适的物质包括DNA和RNA的寡核苷酸。式(I)化合物的这类结合物可能有益于反义试剂,杂交研究的探针,DNA-蛋白质复合物的条带移位分析,或在荧光DNA测序和PCR应用中的引物。而且,式(I)化合物可联接原位标记研究中的许多试剂。典型的试剂包括免疫组化用的抗体和测量体积用的大小标记分子。为了构成测量酶活性的底物,式(I)化合物也可联接试剂,此时荧光随着酶活的变化失去或获得,比如:蛋白酶,磷酸酶,激酶,抗生素失活,核酸酶以及碳水化合物。结合的式(I)化合物可能是也可能不是在原位非结合而产生荧光。换句话说,结合的式(I)化合物可能尽管结合但保持荧光,或者可能从结合物裂解而发射荧光。
本发明的暴露步骤可通过适于诱发式(I)化合物产生可检测信号的已知的技术而进行。总的说来,这将包括暴露生物样品于一定频率的可诱发式(I)化合物产生荧光的(如:210-380nm)紫外光中。唯一限制条件是此光频率和光强度不破坏待测的细胞内成分,如核酸。
正如熟练技术人员所能理解的,选作诱发化合物产生荧光的波长即“最大激发”是本领域已知,即被一分子吸收并且激发该分子达到一更高电子态的波长。在本发明中如上述的激发波长在紫外光波段中。当该分子从较高回到较低电子态时便发射出一种可见辐射,如:荧光,此波长即为“最大发射”。这正是本发明中所检测的荧光。化合物所发射的可检测信号可用本领域已知的技术检测,比如用肉眼观察,用电子设备检测产生的波长或者用其它类似技术手段。
有益地利用滤光器,荧光的波长可被完全从激发光的波长中去除从而使两波长得以很好地分离。例如,滤过的光可用于在输入一侧选择所需激发光波长,和/或用于选择合适范围的发射波长用以测定荧光的峰值或最大值。
本发明的另一方面涉及结合在一样品中细胞的骨架成分并且测检它们的存在的方法。在本发明方法的这一方面,按上述式(I)的一化合物或其生理上可接受的盐与在一基本上纯的溶液或者包含细胞骨架成分的生物样品中的细胞骨架成分接触或混合。如上述,混合式(I)的化合物和细胞骨架成分后,将样品暴露于一定频率的光中,该频率诱发式(I)化合物产生荧光,从而用于分析样品,即检测包含于其中的细胞骨架成分的存在。本发明的这一方面应用于诸如光学显微术,其中包括共焦光学显微术,生化分析以及其它相近的技术中。本发明方法的这一方面的详细描述可参照上述有关荧光检测核酸的部分。
这里所用的术语“细胞骨架成分”是指包括细胞结构框架的蛋白质纤维。本发明特别优选地应用于作为细胞骨架成分的微管结构,其对本方法特别敏感。正如熟练的技术人员所认识到的,微管蛋白是微管的主要蛋白。因此,本发明的这一方面能提供用于检测形成微管结构的微管蛋白聚合物的破裂的技术。
对光学显微术来说,在引入本发明的化合物(染料)前样品优选地被固定在一固体支持物上。任何固体支持物均可使用,典型的固体支持物如显微镜载玻片,反应孔的壁面,试管,比色池以及珠。固体支持物可由本领域技术人员已知的适宜物质制成,包括玻璃,聚苯乙烯,聚乙烯,聚丙烯以及交联多糖。优选地,样品固定于一玻璃制显微镜载玻片上。样品可通过任何合适的步骤固定于固体支持物上,例如空气干燥或者化学或热处理,但这种步骤不能干扰随后的样品观察。优选地,通过一能用光学显微镜观察的方法固定于载玻片上。
对光学显微术来说,样品可通过如上述的按照本领域已知的DAPI或双-苯酰亚胺的标准操作步骤而用根据式(I)的化合物来染色。有益地,样品在染色后通过洗涤除去过量的式(I)化合物以除去背景干扰和提高样品的分辨率。通过将样品浸入化合物水溶液中使样品和其量足以结合核酸或细胞骨架成分的式(I)化合物接触而开始染色步骤。式(I)化合物的量以及接触时间如上述。
那些已备好的样品载玻片可用已知荧光技术比如荧光显微术而得以分析。例如,样品可用一装有诸如汞灯或氙灯的紫外光源和合适的滤器的照相显微镜来观察,并且用常规技术照像。细胞在作为激发光源的紫外灯下照着,必须能产生激发本发明荧光化合物的特定波长。
本发明的方法也能与流式血细胞计数等其它技术一起使用。流式血细胞计数是当颗粒或细胞在液流中流动而一个接一个通过检测点时对其快速计数的技术。样品制备的目的是为了产生分散的以特定方式着色的颗粒的悬浮液,以使其通过该系统时不致破坏液流的平滑或阻塞管道或口。
按照已知的流式血细胞计数技术制备样品。为了染色样品,将样品和式(I)化合物按照上述的接触浓度和时间进行接触。使样品与式(I)化合物接触后,注射或将样品分散到流式血细胞计数系统的液流中以产生分散性颗粒悬浮液。正如熟练技术人员能理解的,体液,如包含个体细胞的血液,可直接在流体血细胞计数器上染色并操作,从而测定出所研究的大分子的绝对细胞含量。可直接制备固体组织,如通过简单的切碎和研磨器官,然后再用筛分以及密度梯度离心。其它组织则要用酶消化。
为了将随机的三维悬浮液中的颗粒一个接一个地分布到光柱空间交叉的特定位置,一般将样品悬浮液注射到有液体流过的封闭通路的中心。细胞流过有光照的检测点。对于光学显微术,光是激发光源,且光源必须能产生可用于激发本发明荧光化合物的特定波长。通过照射光柱的细胞所产生的发散光和荧光被光检测仪所收集,这种光检测仪能将光脉冲转变成电信号。进一步的电子和计算机处理便产生图表显示和统计学分析结果。
本发明中所使用的化合物可按照本领域技术人员已知的技术制备(例如参见B.P.Das and D.W.Boykin,J.Med,Chem.20,531-536(1977),其全部公开内容作为参考文献并入本发明)。可按照如下实施例1-4及6-8中例证的技术制备,其有关变化对那些熟练的技术人员来说是显而易见的。而且,这些化合物可用一稍作改进的B.P.Das and D.W.Boykin的方法进行合成,并在如下实施例5中得以例证。
大体上说,式(I)化合物是按如下反应路线所示而进行制备的:(a)按R.E.Lutz等J.Am,Chem.Soc.56,2698(1934)所述步骤由1,4-二酮(1)进行环化脱水呋喃化生成2,5-双(4-溴苯基)呋喃(2);(b)2,5-双(4-溴苯基)呋喃(2)用Cu2(CN)2腈化作用生成相应的双腈即2,5-双-(4-氰基苯基)呋喃(3);(c)将双腈(3)通过转化成中间体亚氨酸酯(imidate ester)并将中间体和氨或者诸如1,2-乙二胺、1,3-丙二胺等合适的二胺反应转变成式(I)的双-二阳离子芳基呋喃(4),这在下面的实施例中将得以证实。步骤(a)和(b)更为详尽地描述于实施例1,而步骤c则更为详尽地描述于实施例2-8。
另外,上述步骤(c)可用一热分解步骤代替,这里的双腈(3)是通过将二胺盐,比如胺的氢氯化物,直接与双腈加热融化而转化成式(I)化合物,如下述实施例5所例证。这种替代步骤仅限于制备R1和R2同出自一环状部分的式(I)化合物。
正如指出的那样,在本发明中使用的化合物可以其生理上可接受的盐(如:那些不致使样品过分破裂从而使化合物不能被检测的盐)存在。这些盐类包括:海绿石,乳酸盐,乙酸盐,酒石酸盐,柠檬酸盐,马来酸盐,富马酸盐,磷酸盐,硼酸盐,硝酸盐,硫酸盐以及盐酸盐。一般来说,本发明的盐可通过将两当量的脒碱性化合物和所需的酸在溶液中反应而制得。反应完成后,通过加入合适量的此盐不能溶解于其中的溶剂使盐从溶液中结晶。
作为荧光染料的式(I)化合物的光谱学特征和效应跟DAPI以及Hoechst 33258相似。然而,按照式(I)的化合物能高度选择性地和双链DNA紧密结合,却基本上和RNA没有结合。例如:2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂卓-2-基)苯基]呋喃基本上没有可检测的与RNA的亲和性。并且,式(I)化合物发射出一波长稍微不同于DAPI的蓝色,这样在观察时便比使用DAPI减少了光散射从而使图解更加详尽。
本发明以一个试剂盒的形式对使用者更为有利。典型地,一个染色试剂盒包括足量的试剂以完成前述的步骤,即按照式(I)的化合物。各种试剂应制成诸如晶体粉末或水溶剂等适合长期贮存的形式。并且,除此之外试剂盒一般包括,贮存试剂的瓶子,混合用容器以及一份描述以上各步骤的说明书。试剂也可能包括那些诸如缓冲试剂及类似的各种辅助试剂。如果需要,试剂盒还可以进一步包含在试验中减少背景干扰的试剂,对照试剂,完成试验的仪器及相关设备。
本发明将通过如下非限制性实施例得以进一步阐明,其中“g”表示克,“mg”表示毫克,“μg”表示微克,“mmol”表示毫摩尔,“h”表示小时,“ml”表示微升,“M”表示摩尔浓度,“mM”表示毫摩尔浓度,“μM”表示微摩尔浓度,“UV”表示紫外光,“HCl”表示氯化氢,“mp”表示熔点,“HCN”表示氢氰酸以及“℃”表示摄氏温度。除非另外指明,否则有关按照式(I)的化合物的合成试验细节参照B.P.Das andD.W.Boykin,J.Med,Chem.20,531-536(1977)中阐明的合成步骤。
实施例1
制备前体化合物
2,5-双(对-溴苯基)呋喃。
使用了本领域已知的文献方法从溴苯和反式丁烯二酰氯制备反式-双-对-溴苯甲酰基乙烯。J.B.Conant和R.E.Lutz,J.Am,Chem.Soc.47,881(1925)。用Zn-HOAc将乙烯化合物还原制备1,4-双-对-溴苯基-1,4-丁二酮。E.Campaigne and W.D.Foye,J.Org,Chem,17,1405(1952)。将饱和的1,4二酮(7.9克,0.02摩尔)悬浮于80毫升AC2O中,混合物加热至回流。加入(4-5滴)浓H2SO4且继续回流五分钟。将溶液倾入冰水中(1升),充分搅拌,过滤:粗产品7克(93%)。用乙酸重结晶得到5.6克(75%)产品,熔点198-199℃(文献(R.E.Lutz and W.M.Eisner,J.Am.Chem.Soc.56,2698(1934)熔点200-201℃)
2,5-双(对-氰基苯基)呋喃。
将7.5克(0.02摩尔)2,5-双-(4-溴苯基)呋喃和4克(0.045摩尔)Cu(CN)在45毫升喹啉中的混合物回流两小时。将混合物倾入300毫升稀盐酸溶液中(注意:有HCN放出),过滤。所得固体用水、稀氢氧化钠、稀盐酸、再用水洗涤。将固体双腈溶解于丙酮中,过滤以除去无机残渣,经过氧化铝短柱以除去痕量铜盐。由于铜盐被带入双脒中就很难从中纯化,所以铜盐必需除去。一个方便的检测铜盐存在方法是火焰实验。蒸镏氧化铝柱的洗脱液并用乙醇重结晶得到3.5克(65%),熔点294-295℃。
实施例2
制备2,5-双(4-脒基苯基)呋喃二氢氯化物
将于100毫升二恶烷和25毫升无水乙醇的混合液中的2,5-双(4-氰基苯基)呋喃(3克,0.011摩尔)(如例1所述制备),在5℃用干盐酸气饱和。将溶液置于压力瓶中并振摇3天(室温)。将中间产品即沉淀的黄色固体亚氨酸酯氢氯化物过滤,室温真空干燥过夜。亚氨酸酯氢氯化物的红外光谱没有腈的吸收,并且不用进一步鉴定可以直接使用。将亚氨酸酯氢氯化物(3.5克)于100毫升无水乙醇中的悬浮液在5℃用无水氨气饱和。悬浮液(压力瓶)置室温振摇3天。将反应混合物过滤,将固体干燥并溶解在温无水乙醇中(约1.5升)。将溶液在5℃用无水氯化氢酸化,在室温下真空浓缩获得2.5克(60%)的黄色晶体。从无水乙醇重结晶得到熔点为400-401℃(分解)。
实施例3制备2,5-双-[4-(4,5-二氢-1H-咪唑-2-基)苯基]呋喃
将如实施例2所述合成的亚氨酸酯氢氯化物中间体2.1克(0.005摩尔)和0.6克(0.01摩尔)1,2-乙二胺于50毫升无水乙醇中的溶液回流加热过夜。将所得的固体过滤并从无水氯化氢饱和的无水乙醇中重结晶,得到2,5-双[4-(2-咪唑啉基)苯基]呋喃1.9克(90%),熔点409-410℃(分解)。
实施例4
制备2,5-双-[4-(1,4,5,6-四氢
-嘧啶-2-基)苯基]呋喃
用一与实施例3相似的方法,将如实施例2所述合成的亚氨酸酯氢氯化物与1,3-丙二胺反应得到产品(90%):2,5-双[4-(1,4,5,6-四氢-嘧啶-2-基)苯基]呋喃,熔点430-431℃(分解)。
实施例5
制备2,5-双[4-(4,5-二氢-1H-咪唑
-2-基)苯基]呋喃二氢氯化物二水合物
这个化合物通过另一个方法从2,5-双(4-氰基苯基)呋喃而获得。这个反应使用了二腈(0.5克,1.9毫摩尔),1,2-乙二胺二氢氯化物(4.9克,37毫摩尔)以及1,2-乙二胺(2.5毫升,37毫摩尔)。将二腈和1,2-乙二胺二氢氯化物以及1,2-乙二胺的混合物维持在300-310℃,十分钟,然后溶解在热水中。冷却使黄色晶体析出。化合物在沸水中重结晶。产品:208毫克(24%)。
TLC(CHCl3∶CH3OH∶25%NH4OH=11∶4∶1,one spot),
mp>360℃. 分析计算:C22H20N4O·2H2O
(465.37):C,56.78;H,5.60;N,12.04.实测值:C,
56.69;H,5.63;N,12.07. 1H-NMR(DMSO-d6,TMS),δ4.01
(s,8H),7.45(s,2H),8.08(d,4H,J=8.3Hz),8.15(d,
4H,J=8.3Hz),8.15(d,4H,J=8.3Hz),10.50(brs,4H).
13C-NMR(DMSO-d6,TMS),δ45.5,113.2,121.6,125.3,
130.1,135.8,153.4,165.8. IR(KBr):v3412,3123,
2971,1608,1580,1491,1367,1287,1033,850,745,
673. MS,m/z:356(free base).
实施例6
制备2,5-双[4-(4,5,6,7-四氢-1H-
1,3-二氮杂卓-2-基)苯基]呋喃二氢氯化物半七水化物
将双-甲氧基乙醇亚氨酸酯(1克,0.002摩尔)和1,4-二氨基丁烷(0.5克,0.0057摩尔)于10毫升的1,2-二甲氧基乙烷中的溶液回流两天。于真空中去除溶剂,加入水。沉淀物过滤,用水洗涤,真空箱中烘干(熔点大于300℃)。双-甲氧乙醇亚氨酸酯和1,4-二氨基丁烷的游离碱反应产物通过甲醇中的盐酸而转变成氢氯化物(熔点大于300℃)。滤液用2N氢氧化钠中和,获得另一部分的游离碱。游离碱总收率为51%。
分析计算:C26H28N4O·2HCl·3.5H2O(548.50):C,56.93;
H,6.80;N,10.22. 实测值:C,56.99;H,6.80;N,
10.26. 1H-NMR(DMSO-d6),δ2.02(s,8H),3.71(s,8H),
7.38(s,2H),7.86(d,4H,J=8.3Hz),8.04(d,4H,
J=8.3Hz),9.77(s,4H). 13C-NMR(
D2O(CH3)3SiCH2CH2CO2Na),δ28.0,47.0,113.9,126.5,129.7,
131.2,137.0,154.5,167.1. IR(KBr),v687,747,814,
930,1331,1364,1459,1597,3008,3164. MS(EI),m/z
(% rel.int.)412(100)(free base),384(23),354(9),
340(13),298(11),284(23).
实施例7
制备2,5-双{[4-(N-异丙基)-咪基]苯基}呋喃
将干燥的异丙胺(0.47克,0.008摩尔)加入按实施例2所述的亚氨酸酯于45ml无水乙醇的悬浮液中(1.3克,0.003摩尔)。半小时内,亚氨酸酯溶解且亚氨酸酯和异丙胺的混合物着色。大约三小时后,有白色固体沉淀;在室温搅浆过夜。减压去除溶剂,用水稀释,过滤并用水洗涤。固体干燥后,在乙醇和乙醚的混合物中重结晶,得到白色固体:0.9克(78%);熔点233-4℃,
1H NMR(DMSO-
d6)/60℃)7.79(brs,8H),7.11(s,2H),6.25(br,4H,
3.81(br,2H),1.14(d,6H,J=5.9). 13C NMR(DMSO-
d6/60℃)152.4,142.0,136.6,130.4,126.8,122.8,
108.7,43.5,22.8.
实施例8
制备2,5-双[4-(N-异丙基)咪基)苯基]呋喃二氢氯化物
将0.78克(0.002摩尔)按实施例7制备的游离碱溶解在10毫升的无水乙醇中,用氯化氢饱和的乙醇10毫升处理,保温2小时。混合物体积减少到5毫升。加入20毫升无水乙醚产生明黄色沉淀,过滤,用3×5毫升无水乙醚洗涤且在65℃真空干燥2小时,得到产品0.8克(87%)。熔点276-7℃(分解)。红外光谱(KBr)。
1H NMR(DMSO-d6)9.72(s,1H)
9.69(s,1H),9.57(s,2H),9.24(s,2H),8.06(d,4H,
J=8.1),7.86(d,4H,J=8.1),7.42(s,2H),4.14(s,2H,
J=6.6),1.29(d,12H,J=6.6). 13C NMR(DMSO-d6)161.1,
152.3,133.6,129.2,127.7,123.5,111.3,45.1,21.1.
分析计算:C24H28N4O·2HCl·1.25 H2O:
C,59.57;H,6.79;N,11.57. 实测值:C,60.00;H,6.80;
N,11.52.
实施例9
制备2,5-双[(4-(4,5-二氢-1H-咪唑
-2-基)苯基)]-3-(4-甲苯氧基)呋喃
1-(4-甲苯氧基)-1,2-双(4-溴苯甲酰基)乙烯。
在1,2-二溴-1,2-二(4-溴苯甲酰基)乙烷(11.1克,0.02摩尔)于35毫升四氢呋喃的溶液中加入4-甲基酚钠(通过将0.92克(0.04摩尔)的钠和4.32克(0.04摩尔)4-甲基苯酚在30毫升的四氢呋喃中回流4-5小时而制得)的悬浮液。将黄色混合物回流2-3小时(随后薄层色谱),然后减压去除四氢呋喃。用水处理残渣,过滤固体,水洗,干燥(Na2SO4),溶于氯仿。氯仿溶液流过一硅石柱(用2-5%溶于己烷的乙醚洗脱)。产物为灰白色晶状固体,4.95克(50%),熔点137-8℃。
IR
(KBr)3087,3035,2868,1687,1646,1587,1572,1557,
1502,1399,1364,1194,1068,1009,971,876,815,772,
526. 1H NMR(CDCl3/35℃)7.92(d,2H,J=8.8),7.65(d,
2H,J=8.8),7.55(d,2H,J=8.8),7.48(d,2H,J=8.8),
7.27(d,2H,J=8.3),7.11(d,2H,J=8.3),6.32(s,1H),
2.4(s,3H). 13C NMR(CDCl3/35℃)189.4,187.6,168.4,
150.9,136.6,136.0,133.4,132.3,131.8,130.9,130.3,
129.6,129.2,128.2,120.6,101.8,20.95.MS m/e500
(M+).
2,5-双(4-溴苯基)-3-(对-甲苯氧基)呋喃。
将5.0克(0.01摩尔)的1-(4-甲苯氧基)-1,2-双-(4-溴苯甲酰基)乙烯于10毫升三氯化磷中的溶液回流加热3-4小时(接着进行薄层色谱分析)。过量的三氯化磷蒸馏除去,残渣用冰/水研制(放热反应)。用二氯甲烷(75毫升)提取溶液,二氯甲烷层用饱和碳酸氢钠溶液洗涤,水洗,干燥(Na2SO4)。减压去除溶剂。残留固体用乙醚∶己烷(2∶8至1∶1)作洗脱剂通过硅胶进行色谱分析。获得灰白色晶态固体2.78克(56%),熔点为92-3℃。
IR(KBr)2923,2851,
1560,1506,1467,1390,1209,1072,1066,945,825,
707,486. 1H NMR(CDCl3/35℃)7.69(d,2H,J=8.8),
7.46-7.43(m,6H),7.12(d,2H,J=8.3),7.0(d,2H,
J=8.3),6.47(s,1H),2.31(s,3H). 13C NMR
(CDCl3/135℃)150.8,150.1,142.8,139.3,133.0,131.9,
131.7,130.3,129.1,128.6,125.1,125.0,121.8,120.5,
117.1,102.7,20.6.MS m/e484(M+).
2,5-双(4-氰基苯基)-3-(4-甲苯氧基)呋喃。
将如上述制备的二溴化合物(2.5克,0.0051摩尔)和氰化亚铜(1.81克,0.02摩尔)在8毫升无水N-甲基-2-吡咯烷酮中的混合物在氮气中200℃下加热约2.5小时(随后进行薄层色谱分析),冷却并倾入200毫升的水中。过滤沉淀固体,重新悬浮于100毫升水中,加入100毫升10%的氰化钠,混合物搅拌3-4小时。过滤固体,水洗,并放入索克斯累特回流提取器中(Soxlate device)用丙酮回流约24小时。减小丙酮提取物体积,过一短的中性氧化铝柱,洗脱液蒸发后,用三氯甲烷∶乙醚(2∶8)重结晶得到黄色结晶固体1.2克(62%),熔点198-9℃。
IR(KBr)3067,2223,
1618,1303,1505,1402,1220,1169,1008,926,840,
820,668,546cm-1.1H NMR(CDCl3/35℃)7.98(d,2H,
J=8.8),7.75(d,2H,8.3),7.68(d,2H,J=8.8),7.65
(d,2H,J=8.8),7.19(d,2H,J=8.3),7.05(d,2H,
J=8.3),6.66(s,1H),2.36(s,3H).13C NMR
(CDCl3/35℃ )154.3,150.3,145.8,139.1,134.0,133.6,
133.3,132.7,132.6,130.5,124.2,123.8,119.0,118.6,
117.8,111.5,110.0,104.5,20.7. 分析计算:
C25H16N2O2:C,79.76;H,4.28;N,7.44;实测值:C,79.68;
H,4.31;N,7.39. MS m/e376(M+).
2,5-双[(4-(4,5-二氢-1H-咪唑-2-基)苯基)]-3-(4-甲苯氧基)呋喃。
将如上述制备的双腈[1克(0.0026摩尔)]置于已在0℃用无水氯化氢饱和的20毫升无水乙醇和50毫升无水二恶烷中。室温搅拌此混合物四天。粘稠的黄色沉淀形成,加入100毫升无水乙醚,过滤固体,用100毫升无水乙醚洗涤,于25℃真空干燥5小时得0.78克(66%)亚氨酸酯氢氯化物。将亚氨酸酯重新悬浮于25毫升无水乙醇中,和0.31克(0.0053摩尔)1,2-乙二胺缓缓回流加热12小时。蒸馏除去过量乙醇,残留物用水处理,用1M的氢氧化钠碱化处理(搅拌且冷冻)。过滤黄色沉淀,用水洗涤,干燥并从沸乙醇中重结晶,得0.6克产品(74%),熔点156-7℃。
IR(KBr)3218,2927,2862,1609,1506,1398,1218,1105,987,84 8,669cm-1.1H NMR(DMSO-d6/50℃)7.94-7.84(m,8H),7.21(d,2H,J=8.3),7.12(s,1H),7.08(d,2H,J=8.79),3.63(s,4H),3.62(s,4H),2,28(s,3H). 13CNMR(DMSO-d6/50℃)163.0,162.9,154.3,150.4,142.8,139.0,132.4,130.8,130.4,130.1,129.7,128.5,127.5,127.4,123.2,122.6,116.5,104.0,49.3,49.2,19.9.MS m/e462(M+).
将10毫升乙醇氯化氢中的游离碱[0.5克(0.001摩尔)]于回流加热3小时,加入稀释的50毫升无水乙醚中。过滤所得黄色沉淀,用无水乙醚洗涤且在80℃真空干燥24小时,得产品0.48克(90%),熔点>300℃。
分析计算:C29H26N4O2·2HCl:C,
65.04;H,5.27;N,10.46.实测值:C,64.83;H,4.99;N.
10.22.IR(KBr)3422,3235,2964,2775,1609,1506,
1370,1289,1206,848,667cm-1.1H NMR(DMSO-
d6/D2O/TSP/60℃)7.98-7.86(m,8H),7.19(d,2H,
J=8.79),7.09(s,1H),7.03(d,2H,J=8.3),3.88(s,
4H),3.76(s,4H),2.24(s,3H).13C NMR(DMSO-
d6/D2O/TSP/60℃)165.3,165.3,154.7,151.2,145.7,
139.5,134.3,134.2,135.1,131.2,129.6,129.5,124.8,
124.1,123.3,121.6,117.7,106.0,45.8,45.6,20.7.
实施例10
制备2,5-双[4-(2-四氢嘧啶基)苯基]
3-(4-甲苯氧基)呋喃
置于30毫升无水乙醇中的亚氨酸酯(1.08克,0.002摩尔)和新蒸馏的1,3-二氨基丙烷(0.43克,0.006摩尔)的混合物在搅拌下轻微回流(防止蒸气)12小时。减压去除过量乙醇,残留物用50毫升蒸馏水滴定。在冷却和搅拌下将混合物用1M的氢氧化钠(pH10)碱化;过滤沉淀的游离碱,水洗、干燥,在热乙醇中重结晶得0.80克(81.6%)产品;熔点190-191℃。
IR(KBr):3267,2931,2858,
1609,1505,1369,1216,846,666cm-1.1H NMR
(DMSO-d6/50℃)7.88-7.78(m,8H),7.2(d,2H,J=8.8),
7.12(s,1H),7.07(d,2H,J=8.8),3.38(t,8H,J=5.1),
2.28(s,3H),1.75(tt,4H,J=5.1):13C NMR(DMSO-
d6/50℃)154.4,153.8,153.4,150.5,142.8,139.0,
134.5,132.9,132.4,130.6,130.3,130.3,126.8,126.7,
123.1,122.5,116.6,104.1,41.0,40.8,20.0,19.8;MS
m/e490(M+).
用10毫升乙醇盐酸处理置于5毫升无水乙醇中的0.5克(0.001摩尔)游离碱的悬浮液且轻微回流加热2小时。加入50毫升无水乙醚得到黄色沉淀,过滤,用无水乙醚洗涤,于60℃真空干燥12小时。得0.46克黄色固体(82%)。熔点>320℃。
IR
(KBr):3423,3117,3002,1638,1609,1507,1375,1315,
1202,846,669cm-1;1H NM(DMSO-d6/D2O/TSP/65℃)8.12
(d,2H,J=7.8),8.08(d,2H,J=7.3),7.88(d,4H,
J=8.3),7.32(d,2H,J=8.3),7.22(s,1H),7.16(d,2H,
J=8.3),3.6(br m,8H),2.37(s,3H),2.1(br m,4H).
13C NMR(DMSO-d6/D2O/TSP/65℃):159.5,154.8,151.1,
145.1,140.9,139.6,134.1,133.9,133.5,133.2,128.7,
128.5,127.2,124.8,117.6,105.9,41.5,41.4,20.6,
18.2. 分析计算:C31H30N4O2·2HCl.C,66.06;
H,5.36;N,9.94. 实测值:C,65.91;H,5.21;N,9.88.
实施例11
制备2,5-双[4-(2-咪唑啉基)苯基]-3-甲氧基-呋喃
1,2-双(4-溴苯甲酰基)-1-甲氧基乙烷。
在1,2-二溴-1,2-二(4-溴苯甲酰基)乙烷(11.1克,0.02摩尔)于150ml无水甲醇的溶液中加入于甲醇中的甲醇钠(0.92克钠于50毫升甲醇中)溶液。回流黄棕色混合物1-1.5小时。蒸馏除去溶剂,残留物悬浮于水中,用100毫升氯仿萃取混合物。用水洗涤氯仿萃取物,干燥(Na2SO4)且浓缩。用无水甲醇-乙醚(3∶1)滴定所得残留物得灰白色结晶固体,6.6克(78%),熔点153-154℃。
IR
(KBr):3106,3062,2932,1689,1649,1583,1556,1403,
1223,1202,1182,1086,1010,1000,857,814,738,618,
472cm-1.1H(DMSO-d6/40℃):7.95(d,2H,J=7.8),7.77
(4H,J=8.8),7.72(d,2H,J=7.8),6.89(s,1H),4.03
(s,3H).13C(DMSO-d6/40℃):189.9,187.2,168.8,
139.9,135.9,133.1,132.2,131.8,130.3,128.1,127.4,
98.6,58.5.MS m/e424(M+).
2,5-双-[4-溴苯基]-3-甲氧基-呋喃。
将如上述制备的甲氧基乙烷溶解于5毫升的三氯化磷中,并且回流加热3小时。蒸馏除去过量的三氯化磷。用冰和水处理使残留物形成粘块。用氯仿萃取混合物,以水洗涤有机层,干燥(Na2SO4)并用以己烷∶乙醚(4∶1到2∶1)作为洗脱剂进行硅胶柱色谱纯化,获得产率为62%的灰白色固体;熔点112-113℃[文献:熔点113℃;R.E.Lutz,J.Am.Chem.Soc.51,3008(1929)]。
IR(KBr)3062,2908,
2877,1617,490,1391,1211,1160,1099,1073,1034,
1006,925,827,787.1H NMR(CDC13)7.69(d,2H,
J=8.8),7.67.5(m,4H),7.47(d,2H,J=8.8),6.64(s,
1H),3.9(s,3H).13C NMR(CDCI3)149.7,147.5,135.5,
131.9,131.5,129.4,129.3,125.0,124.5,121.5,119.3,
98.6,58.6.MS m/e 408(M+).
2,5-双(4-氰基苯基)-3-甲氧呋喃。
将于10毫升无水N-甲基-2-吡咯烷酮中的2,5-双(溴苯基)-3-甲氧基呋喃(4.08克,0.01摩尔)和氰化亚铜(3.09克,0.035摩尔)的混合物在氮气中于约200℃加热2.5小时。冷却并将混合物注入200毫升水中,过滤沉淀出的黄棕色固体,用水充分洗涤。将固体重新悬浮于50毫升水和100毫升10%的氰化钠中,搅拌2小时。过滤稀浆,水洗涤,干燥并悬浮于250毫升丙酮中且经过中性氧化铝柱。用丙酮洗脱得到黄色固体。在三氯甲烷∶乙醚(1∶1)中重结晶得到产物(1.8克,60%),熔点为257-258℃。
IR(KBr)3128,
2223,1608,1599,1501,1409,1174,1163,1027,924,
836,815,651,537cm-1.1H NMR(DMSO/45℃)8.03(d,
2H,J=8.3),7.95(d,2H,J=8.79),7.91(d,2H,J=8.3),
7.85(d,2H,J=8.79),7.62(s,1H),4.0(s,3H).13C
NMR(DMSO/45℃)150.0,149.8,134.6,133.4,133.2,
132.7,132.5,124.0,122.7,118.9,118.5,110.0,107.6,
102.4,59.0.MS m/e 300(M+). 分析计算:
C19H12N2O(300.31):C,75.98;H,4.03;N,9.33;实测值:
C,76.02;H,4.04;N,9.36.
2,5-双[4-(2-咪唑啉基)苯基]-3-甲氧基-呋喃。
将如上述制备的双腈(0.9克,0.003摩尔)悬浮在70毫升无水乙醇中,于0-5℃用无水氯化氢饱和且在无水环境中搅拌3-4天。混合物用200毫升无水乙醚稀释,过滤黄色酰胺酯,用无水乙醚洗涤,将固体在真空干燥5-6小时得1.2克产物(86%)。将所得固体重新悬浮于30毫升无水乙醇中并与0.46克(0.008摩尔)的无水1,2-乙二胺轻轻回流12小时。蒸馏去除溶剂。残留物用50毫升冷水悬浮并用1M的氢氧化钠碱化。过滤得黄色沉淀,水洗,干燥。在乙醇-乙醚的混合物中重结晶得到0.74克产物(75%),熔点186-187℃(分解)。 IR(KBr)3444,3245,
2931,2857,1601,1512,1397,1366,1277,1162,1104,
1031,926,842,743,670cm-1.1H(DMSO-d6/60℃)
7.93-7.86(m,8H),7.32(s,1H),3.98(s,3H),3.69(s,
4H),3.67(s,4H).13C NMR(DMSO-d6/60℃:163.3,
163.1,150.0,148.6,138.3,134.7,131.9,131.3,128.9,
127.6,126.1,123.0,121.9,100.6,58.7,49.0,48.5.
MS m/e386(M+).
将0.58克游离碱(0.0015摩尔)溶解于10毫升的热乙醇中并用10毫升饱和乙醇氯化氢处理。将混合物回流加热30分钟。在真空中将体积减小到5-6毫升。所得混合物用60毫升无水乙醚稀释。过滤得黄色晶状固体,用无水乙醚洗涤且在60℃真空干燥12小时得0.62克产物(83%),熔点189-190℃(分解)。
IR(KBr):3422,3128,2975,1599,1510,1405,1363,
1285,1207,1028,845,666cm-1.1H(D2O/TSP/50℃)
7.52-7.43(m,8H),6.87(s,1H),3.92(s,3H),3.86(s,
8H).13C(D2O/TSP/50℃)167.2,153.1,152.4,137.6,
137.6,137.2,130.9,130.7,126.5,125.4,122.1,119.8,
104.2,61.5,47.0,46.9. 分析计算:
C23H22N4O2-0.5H2O-2HCl:C,58.97;H,5.38;N,11.96.
实测值:C,59.16;H,5.35;N,11.80.
实施例12
制备2,5-双[4(N-环-丙基脒基)苯基呋喃
将在35毫升无水乙醇中的亚氨酸酯(1.3克,0.003摩尔)和环丙胺(0.43克,0.0075摩尔)的混合物搅拌过夜,真空去除溶剂并加入水形成黄色溶液。在冷却和搅动下用1M的氢氧化钠碱化。过滤所得固体,用水洗涤并干燥,将固体溶于氯仿,用硫酸钠干燥,去除溶剂。从乙醚∶三氯甲烷(5∶1)中重结晶残留物得淡黄色固体0.8克(70.9%),熔点185-186℃(分解)。
IR(KBr):3464,3320,
3080,1610,1510,1364,1022,848,791cm-1.1H NMR
(CDC13):7.71(br s,8H),6.78(s,2H),5.3(v br,4H),
2.6(br m,2H),0.87-0.81(m,4H),0.67-0.62(m,4H).
13C NRM(CDC13+DMSO-d6):159.6,152.2,134.8,130.7,
126.4,122.6,107.7,25.7,6.04.MS m/e388(M+).
将游离碱(0.6克,0.0015摩尔)悬浮于3毫升无水乙醇中并用6毫升乙醇氯化氢处理,于65℃缓缓加热1小时。用50毫升无水乙醚稀释黄色溶液,过滤,用无水乙醚洗涤,75℃真空干燥12小时。得黄色固体0.55克(80%),熔点>310℃(分解)。
IR(KBr):3369,3181,3037,
1665,1607,1502,1032,782,674cm-1.1H NMR(DMSO-d6):
10.24(s,2H),9.86(s,2H),9.27(s,2H),8.06(d,4H,
J=7.94),7.95(d,4H,J=8.54),7.42(s,2H),2.87(br
m,2H),1.09-0.85(m,8H).13C NMR(DMSO-d6):163.9,
152.3,133.7,129.1,126.6,123.5,111.3,24.7,6.5.
分析计算:C24H24N4O-2HCl:Cal.C,63.02;H,
5.73;N,12.25.实测值:C,62.89;H,5.95;N,12.00.
实施例13
制备荧光检测的样品
化学药品:DAPI,Hoechst 33258和司替霉素,得自Sigma化学公司
样品来源。肠兰伯氏鞭毛虫(Giardia lamblia)用以前发表的常规方法培养(C.A.Bell,et al.,Agents and Chemother.37,2668-2673(1993)),冷却使之从培养管中释放,用HBSS-缓冲液(Hanks平衡的盐溶液,来自Sigma化学公司)洗涤,然后将生物体和染料保温五分钟,在显微镜装片前用HBSS-缓冲液洗一次。中期染色体涂布是用常规方法从人淋巴细胞制备而来的。所有的染料染色均按DA(司替霉素)/DAPI带C染色用的的常规步骤进行(O.M.Miller,Principles and practices in Medical Genetics(Longman Group Limit-ed,New York1983)and D.Silvonen,ACT Cytogenetic Lab manual(University of California,San Francisco(1980))。
具体地说,用DAPI染色时,载玻据报道片浸入一缓冲溶液即MacIlvaine′s缓冲液(pH7.5)中大约十分钟(MacIlvaine′s缓冲液的制备方法是已知的,即混合无水的柠檬酸溶液(0.2M,19.2克/升)和无水的磷酸氢二钠溶液(0.2M,28.4克/升))。将该载玻片用DAPI(0.2微克/毫升)染色约十分钟,然后用MacIlvaine′s缓冲液漂洗。除用0.02微克/毫升的化合物染色载玻片外,用式(I)化合物进行的染色步骤和上述步骤相同。
实施例14
检测核酸
将组织置于载玻片上的HBSS-缓冲液或甘油中,盖上一玻璃盖玻片。不需其它特殊物质。在一装备有紫外光学器件(360nm激发光及460nm发射光的滤光器)以及新氟(neofluor)透镜的Nikon照像显微镜下观察样品。图像可用Ektachrome 1600 ASA胶卷或Technical Pan 400 ASA黑白胶卷拍摄,暴光时间为0.1到5秒。
从照片上可显示出肠兰伯氏鞭毛虫的核用式(I)化合物染色和DAPI及Hoechst 33258染色的结果对比。作为高度特异性的DNA染料,2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂卓-2-基)苯基]呋喃无贾弟虫胞质背景染色,表明对RNA或细胞骨架成分没有结合发生。而对DAPI,Hoechst 33258以及2,5-双(4-脒基苯基)呋喃均观察到了明显的贾弟虫胞质着色。对DAPI和2,5-双(4-脒基苯基)呋喃来说,也有明显的微管或丝状结构着色。有报道说DAPI能染色微管蛋白和微管(D.Bonne,et al.,J.Biol.chem.260,2819-2825(1985)),但作者报告在完整成纤维细胞中未能观察到染色。这些照片表明在贾弟虫中,DAPI染色可能是微管蛋白的结构成分以及强烈染色核酸。
除了贾弟虫外,由于DAPI广泛应用于检测于染色体1,9,15,16和Y上产生C带的染色体AT富集区的标记,所以也对用式(I)化合物染色的人中期染色体与DAPI作了比较。(O.Miller,Principlesand Practices in Medical Genetics(Longman Group Limited,NewYork 1983))。呋喃染料2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂卓-2-基)苯基]呋喃;2,5-双{[4-(N-异丙基)脒基]苯基}呋喃以及2,5-双(4-脒基苯基)呋喃提供了与DAPI本质上相似的染色模式。呋喃染料发射出的兰色与DAPI发出的兰色波长略有不同,这导致了比用DAPI观察到的光散射较少,而这正是描绘更多细节的关键,而且对呋喃染料来说不需要特殊处理和/或储存方案。
二阳离子呋喃也可用于检测很多其它类型的组织,比如细菌,酵母,植物以及组织培养细胞。在每种情况下,由于它们的更深的蓝色的优势使之对DNA的检测灵敏度均比DAPI对DNA的检测灵敏度更高而且DNA的具体特性能被显示得更详细。
实施例15
核酸结合测定
除了式(Ia)和(Ib)化合物外,染料对核酸的结合亲和性以前已有报道(W.D.Wilson,et al.,Biochemistry 3,4098-4104(1993);W.D.Wilson,et al.,Molecular Basis of Specificity in Nucleic Acid-DrugInteractions(Kluwer Academic Publishers,Amsterdam 1990),pp.331-353)。对这些化合物的核酸结合参数象其它物质一样采用同样的方式确定。具体地说,核酸的结合参数使用Cary 219分光光度计联通一台AppleIIe微机以熔点Tm研究来确定的。Cary的温度由一台Haake PG20温度程序控制器来控制,这个温度程序控制器和一能在一分钟内将温度升高0.5℃的Haake A81冷却浴相连。在参比池内安装有一热敏电阻器用以监控温度。用RNA聚合物Poly(A)·poly(U)和对应的序列DNA poly(dA).poly(dT)进行熔点对比研究。将聚合物加入置体积减小1厘米路径长的石英池的1毫升缓冲液中,通过测量260纳米的吸收度定出浓度。一般地,实验在5×10-5M的DNA碱基对浓度以及化合物/碱基对的比率为0.6的情况下进行。通过化合物产生的Tm值的增加来进行化合物的比较(ΔTm=复合物的Tm-游离核酸的Tm)
表I比较了荧光染料2,5-双(4-脒基苯基)呋喃(DB75);2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂卓-2-基)苯基]呋喃(DB161);2,5-双{[4-(N-异丙基)脒基]苯基}呋喃(DB181)以及2,5-双[4-(1,4,5,6-四氢嘧啶-2-基)苯基]呋喃(DB103)对DNA和RNA的亲和性,并和化合物DAPI以及Hoechst 33258作比较。
表I
染料的结构及其由熔点温度测定的它们的核酸结合结构 ΔTm(A-U) ΔTm(dA-dT)DAPI 3.9 >25Hoechst 33258 17.5 >25DB75 5.7 24.6DB103 2.5 >25DB161 0 >26DB181 1.5 >26
实施例16
光谱测定
将有或无DNA或RNA存在下的染料在Shimadzu双-光束分光光度计上扫描出最大吸收值。在LKB荧光计上测定它们的荧光激发和发射最大值。最大吸收值、激发系数、最大激发以及最大发射如下述表II所示。
表II
化合物 | 吸收 | 荧光 | ||||||
(nm) | 最大激发(nm) | 最大发射(nm) | R1 | R2 | R3 | R4 | R5 | |
DB75 | 355 | 359 | 462 | H | H | H | H | H |
DB60 | 365 | 366 | 459 | -(CH2)2- | H | H | H | |
DB103 | 355 | 356 | 445 | -(CH2)3- | H | H | H | |
DB99 | 365 | 363 | 468 | H | H | H | CH3 | CH3 |
DB116 | 388 | 394 | 503 | -(CH2)2- | H | H | OCH3 | |
DB154 | 375 | 381 | 495 | -(CH2)2- | H | H | O-苯基-CH3 | |
DB155 | 354 | 354 | 471 | -(CH2)3- | H | H | O-苯基-CH3 | |
DB159 | 387 | 390 | 510 | 氨基乙基酰氨基苯并咪唑 | H | H | ||
DB160 | 375 | 385 | 467 | 六氢化苯并咪唑 | H | H | ||
DB161 | 354 | 354 | 470 | -(CH2)4- | H | H | H | |
DB180 | 356 | 356 | 469 | -(CH2)2- | -(CH2)2OH | H | H | |
DB181 | 354 | 356 | 454 | H | H | -CH(CH3)2 | H | H |
DB182 | 357 | 356 | 458 | H | H | -(CH2)3N(CH3)2 | H | H |
实施例17
细胞骨架成分的检测
样品的制备和观察如上述实施例9和实施例10所述,但用下述化合物DB99、DB154及DB155染色。照片显示出化合物DB99、DB154及DB155对细胞骨架成分如兰伯氏贾弟虫的微管或丝状结构染色。
前面的各实施例阐明了本发明,但不应理解为局限于此。本发明由下面的权利要求限定,这里也包含了与权利要求等同的内容。
Claims (34)
1.荧光检测核酸的方法,包括:
或者R1和R2一同代表C2到C10的亚烷基;
这里的n是从1到3的数字而R10是H或者—CONHR11NR15R16,其中R11是低级烷基,而R15和R16则各自独立选自H和低级烷基;
R3是H,低级烷基,环烷基,芳基,羟基烷基,氨基烷基或者烷基氨基烷基;并且A是从如下一组基团中选择的一个杂环芳基:
这里的R4,R5及R6各自独立选自H,低级烷基,卤素,氧烷基、氧芳基、或氧芳基烷基;
R12是氢,低级烷基,羟基,氨基烷基或烷基氨基烷基;
(b)暴露所述的核酸于光中以诱发所述的式(I)化合物的荧光。
2.一种按权利要求1的方法,其中所述核酸为DNA。
3.一种按权利要求1的方法,其中所述核酸为RNA。
6.一种按照权利要求1的方法,其中A有结构式:
8.一种按照权利要求1的方法,其中A有结构式:
12.一种按照权利要求1的方法,其中A有结构式:
14.一种按照权利要求1的方法,其中R1和R2一同代表C2到C4的亚烷基,R3是H;
15.一种按照权利要求1的方法,其中R1和R2一同代表C4亚烷基,而R3为H。
16.一种按照权利要求1的方法,其中R1和R3是H而R2是低级烷基。
17.一种按照权利要求16的方法,其中R2是异丙基。
18.一种按照权利要求1的方法,其中所述的化合物选自下列一组物质:
2,5-双(4-脒基苯基)呋喃;
2,5-双[4-(4,5-二氢-1H-咪唑-2-基)苯基]呋喃;
2,5-双[4-(1,4,5,6-四氢嘧啶-2-基)苯基]呋喃;
2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂_-2-基)苯基]呋喃以及
2,5-双[4-(N-异丙基脒基)苯基]呋喃;和其生理上可接受的盐。
19.一种按照权利要求1的方法,其中所述的式(I)化合物是2,5-双[4-(4,5,6,7-四氢-1H-1,3-二氮杂_-2-基)苯基]呋喃或者其生理上可接受的盐。
20.一种按照权利要求1的方法,其中所述的式(I)化合物是2,5-双[4-(N-异丙基脒基)苯基]呋喃或其生理上可接受的盐。
22.一种具有式(I)的化合物或其生理上可接受的盐其中:
R1和R2一同代表C2直链饱和亚烷基;
R3是-R14OH,这里R14是低级烷基;
A是:
这里R4和R5各自是H。
23.一种按照权利要求22的化合物,其中R14是-(CH2)2-
25.一种按照权利要求24的化合物,其中R3,R4和R5是H。
26.一种具有式(I)的化合物或其生理上可接受的盐其中:
R1和R2是H;
R3是异丙基;A是:
其中R4和R5各自为H。
29.一种选择性地荧光检测含有DNA和RNA的核酸混合物中的DNA的方法,该方法包括:
(a)将所述核酸混合物与按照式(I)的化合物或其生理上可接受的盐接触其中:
R1和R2各自独立选自H,低级烷基,环烷基,芳基,羟基烷基或氨基烷基,
或者R1和R2一同代表C2到C10的亚烷基;
或者R1和R2一同为
其中n是从1到3的数字且R10是H或者-CONHR11NR15R16,其中的R11是低级烷基,R15和R16各自独立选自H和低级烷基;
这里R4,R5和R6各自独立选自H,低级烷基,卤素,氧烷基,氧芳基或者氧芳基烷基;
R12是卤素,低级烷基,羟基或氨基烷基;
(b)暴露所述的核酸混合物于光中以诱发所述式(I)化合物的荧光。
30.一种荧光检测微管结构的方法,包括:
R1和R2各自独立选自H,低级烷基,环烷基,芳基,羟基烷基或氨基烷基,
或者R1和R2一同代表C2到C10的亚烷基;
这里的n是从1到3的数字,而R10是H或-CONHR11NR15R16,其中R11是低级烷基,R15和R16各自独立选自H和低级烷基;
R3是H,低级烷基,环烷基,芳基,羟基烷基或者氨基烷基;并且
这里R4,R5和R6各自独立选自H,低级烷基,卤素,氧烷基,氧芳基或者氧芳基烷基;
R12是H,低级烷基,羟基或氨基烷基;(b)将所述的微管结构暴露于光中以诱发该式(I)化合物产生荧光。
31.按照权利要求30的一种方法,其中微管结构存在于一细胞骨架中。
32.一种同时荧光检测细胞中第一细胞结构和第二细胞结构的方法,这里所说的第一细胞结构和所说的第二细胞结构是不同的,该方法包括:
(a)将所述的细胞与第一荧光化合物及第二荧光化合物或其生理上可接受的盐接触,其中,所说的第一荧光化合物选择性地结合于所述的第一结构且第二荧光化合物则选择性地结合于所述的第二结构,其中,该第一荧光化合物和该第二荧光化合物具有不同的荧光发射光谱,其中,所述第二荧光化合物和所述第一荧光化合物具有结构上的差异,但其中每种荧光化合物都有根据式(I)的结构其中:
R1和R2各自独立选自H,低级烷基,环烷基,芳基,羟基烷基或氨基烷基,
或者R1和R2一同代表C2到C10的亚烷基;
或者R1和R2一同为
其中n是从1到3的数字且R10是H或者-CONHR11NR15R16,
其中R11是低级烷基,R15和R16是各自独立选自H和低级烷基;
R3是H,低级烷基,环烷基,芳基,羟基烷基或者氨基烷基;并且
这里的R4,R5和R6各自独立选自H,低级烷基,卤素,氧烷基,氧芳基,或者氧芳基烷基;
R12是氢,低级烷基,羟基或者氨基烷基;以及
(b)将该细胞暴露于光中以诱导该第一和第二荧光化合物的荧光从而使该第一细胞结构和该第二细胞结构在不同的荧光发射光谱发射荧光。
33.一种按照权利要求32的方法,其中所述的第一细胞结构和第二细胞结构选自DNA,RNA和微管结构。
34.一种荧光检测细胞结构的试剂盒,包括:
R1和R2各自独立选自H,低级烷基,环烷基,芳基,羟基烷基或氨基烷基,
或者R1和R2一同代表C2到C10的亚烷基;
或者R1和R2一同是
其中n是从1到3的数字,R10是H或-CONHR11NR15R16,
这里R11是低级烷基,R15和R16各自独立选自H和低级烷基;
R3是H,低级烷基,环烷基,芳基,羟基烷基或者氨基烷基;并且
其中R4,R5和R6各自独立选自H,低级烷基,卤素,氧烷基,氧芳基或者氧芳基烷基;和
(b)一种在式(I)化合物与含有核酸的样品接触时能形成该化合物标记的核酸混合物的足量的溶剂,该式(I)化合物在暴露于光中以诱发其发射荧光时能进行荧光检测。
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US20060105348A1 (en) * | 2004-11-15 | 2006-05-18 | Lee Jun E | Compositions and methods for the detection and discrimination of nucleic acids |
US20060275792A1 (en) * | 2004-11-15 | 2006-12-07 | Lee Jun E | Enhancement of nucleic acid amplification using double-stranded DNA binding proteins |
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US4407942A (en) * | 1981-01-29 | 1983-10-04 | Atomic Energy Of Canada Limited | Fluorescent detection of DNA damage |
US4555396A (en) * | 1982-12-22 | 1985-11-26 | Eastman Kodak Company | Use of pyrylium and thiapyrylium compounds as biological stains |
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1995
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- 1995-05-05 CN CN95193470A patent/CN1151209A/zh active Pending
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CN100334227C (zh) * | 1998-04-17 | 2007-08-29 | 里格尔制药公司 | 检测细胞参数变化和筛选小分子文库所用的多参数facs分析 |
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AU699223B2 (en) | 1998-11-26 |
CA2189125A1 (en) | 1995-11-16 |
EP0758453A1 (en) | 1997-02-19 |
HU9603078D0 (en) | 1997-01-28 |
JPH10501615A (ja) | 1998-02-10 |
WO1995030901A1 (en) | 1995-11-16 |
JP3098545B2 (ja) | 2000-10-16 |
US5594138A (en) | 1997-01-14 |
BR9507616A (pt) | 1997-08-19 |
US5723288A (en) | 1998-03-03 |
AU2436995A (en) | 1995-11-29 |
HUT76440A (en) | 1997-08-28 |
NZ285309A (en) | 1998-10-28 |
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