CN108314691A - Tspo荧光显像探针及其合成方法与应用 - Google Patents
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Abstract
本发明涉及TSPO荧光显像探针及其合成方法与应用,TSPO荧光显像探针分子结构如下:其中,R1与R2为单独的直链烷烃类取代基,R1为C1到C10的直链烷烃类取代基,R2为C1或C2的直链烷烃类取代基;R3与R4为结构相同的直链烷烃类取代基团,长度为C1到C10;n=2‑10;该分子结构式中Signaling Agent为信号基团。所述TSPO荧光显像探针可作为体外的TSPO检测剂或体内的影像检测剂。
Description
技术领域
本发明涉及小分子荧光探针的结构以及合成方法与应用,尤其是涉及多种TSPO荧光显像探针及其合成方法与应用。
背景技术
转位蛋白(TSPO:Translocator Protein)是一个10kDa的线粒体膜蛋白,它与VDAC与ANT组成了线粒体的转运通道(MPTP),并在类固醇转运,类固醇激素合成,细胞增殖与凋亡等多种生物过程中拥有重要的作用。在人体中,TSPO表达于很多健康组织中,且其表达水平因不同组织而异。例如,在肾上腺、松果腺、唾液腺、性腺等多种腺体组织中,TSPO的表达水平比较高,在肾脏与心脏的表达水平居中,在脑部与肝脏部位的表达水平相对较低。在老年痴呆症、帕金森症、亨廷顿症、多发性硬化等神经退行性疾病,及乳腺癌、前列腺癌、口腔癌、直肠癌、肝癌与神经胶质瘤等多种癌症中TSPO的表达量均高于正常组织。
在老年痴呆症、帕金森症、亨廷顿症、多发性硬化等神经退行性系统疾病中,TSPO是一种良好的诊断与治疗的生物标志物,被广泛应用于评估神经退行性疾病中的炎症,胶质增生与疾病的进展,对于退行性疾病的探测与疗效评估都有非常重要的价值。在神经胶质瘤的研究中,研究人员也发现TSPO的表达量不仅与疾病的进展以及病人的低存活率有直接的关系,同时也与癌症的转移能力存在正相关性。此外,先前的研究显示TSPO在神经胶质瘤与肿瘤细胞株中有非常高的表达,但是在正常脑部的表达量非常低,表明了TSPO也具有作为神经胶质瘤标志物的巨大潜力。因此小分子TSPO荧光探针的研发对于神经系统疾病的监测与疗效评估,细胞学实验都有着非常重要的意义。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供多种新型的TSPO荧光显像探针及其合成方法与应用。
本发明的目的可以通过以下技术方案来实现:
TSPO荧光显像探针,其分子结构如下:
R1与R2为单独的直链烷烃类取代基,R1为C1到C10的直链烷烃类取代基,R2为C1或C2的直链烷烃类取代基;R3与R4为结构相同的直链烷烃类取代基团,长度为C1到C10;n=2-10;此化合物中R2基团的长度不能大于2个碳链的长度;该分子结构式中Signaling Agent为信号基团。
据前期对TSPO小分子探针的研发成果显示,小分子荧光探针的框架中的R1与R2分子基团对其结合力影响较大,而R3与R4对其小分子的影响较小(Tang et al,Journal ofmedicinal chemistry 56(8),3429.Tang et al,Molecular Imaging and Biology 16(6),813.Tang et al,Molecular Imaging and Biology(2016).Tang et al,TetrahedronLetters 51(35),4595)。
根据以往的研究结果,本发明设计并合成了多种小分子荧光探针化合物。其中一些探针化合物拥有非常高的TSPO结合性能。
TSPO荧光显像探针分子结构式中信号基团为荧光染料,结构如下任一结构所示:
以上小分子荧光基团均为已有产品。这些小分子荧光基团均可作为探针的信号基团接入到TSPO荧光显像探针的分子框架中,并根据不同的需求与探测方法选择相应的荧光探针分子。
信号基团为以上6种荧光染料时的此类化合物荧光探针主要运用于多种荧光的分子成像,以及多种外科手术的治疗方法中。主要探测方法为近红外光谱的探测手段。其实用领域主要集中于探测在肿瘤及神经系统疾病中过度表达的转位蛋白(TSPO)。
进一步的,一些优选的TSPO荧光显像探针分子,其分子结构如下:
这些TSPO小分子的探针在体外的亲和试验中均表现出了良好的TSPO亲和性能(Ki<1.0nM),展现出了比先前报道的小分子更为强的结合能力,展现了其做为TSPO的探针能力与TSPO过量表达疾病的诊断能力。
本发明所述的TSPO荧光显像探针的合成方法,本发明运用与文献报告相似的合成方法(Tang et al,Tetrahedron Letters 51(35),4595,Tang et al,Journal ofmedicinal chemistry 56(8),3429,Tang et al,Molecular Imaging and Biology(2016)),先合成了多种化合物6(如图1所示)。在这个反应过程中,主要应用了微波合成的方法(MAOS),此种方法能够极大的争强合成效率以及缩短合成时间。运用合成的化合物6,在其酚羟基端加入了一个长直链的烷基取代物,并合成了化合物7,并经过两步反应生成了带有游离氨基的化合物9。最后一步的反应是将化合物9与荧光或染料小分子(带有异硫氰酸基)反应生成新型的荧光探针TDW-F-1,TDW-F-2,TDW-F-3。合成的具体路线图如附图1所示,图1中黑点代表signaling agent。
本发明中所述的TSPO荧光显像探针可以作为体外的TSPO检测剂,应用于体外细胞的共聚焦显像,如细胞学实验,组织与血液检测等,以及可以作为体内的影像检测剂,用于体内的影像检测,如各种TSPO过量表达的炎症,肿瘤等。
与现有技术相比,本发明具有以下优点及有益效果:
较之于以前的TSPO荧光探针,本发明探针拥有更为新颖的分子结构,更为良好的TSPO亲和力。能够更加准确的探测TSPO在体内的位置,为诊断以及体内影像提供了良好的病理学依据。
附图说明
图1为TSPO荧光显像探针的合成路线图;
图2为TDW-F-1的合成路线;
图3为TDW-F-2的合成路线;
图4为TDW-F-3的合成路线;
图5为竞争性结合实验结果,A)TDW-F-1.B)TDW-F-2.C)TDW-F-3;
图6为荧光探针TDW-F-1用于C6细胞的TSPO显像结果;
图7为荧光探针TDW-F-1在体内的分布情况。
具体实施方式
本发明所述的TSPO荧光显像探针的合成方法,包括以下步骤:
运用与文献报告相似的合成方法(Tang et al,Tetrahedron Letters 51(35),4595,Tang et al,Journal of medicinal chemistry 56(8),3429,Tang et al,Molecular Imaging and Biology(2016)),合成了多种化合物6(如图1所示)。在这个反应过程中,主要应用了微波合成的方法(MAOS),能够很好提升反应的速度与产率。运用合成的化合物6,在其酚羟基端加入了一个长直链的烷基取代物,并合成了化合物7,并经过两步反应生成了带有游离氨基的化合物9.最后一步的反应是将化合物9与荧光或染料小分子(带有异硫氰酸基)反应生成新型的荧光探针TDW-F-1,TDW-F-2,TDW-F-3。
不同种化合物6可由微波反应生成(具体方法见Tang et al,TetrahedronLetters 51(35),4595,Tang et al,Journal of medicinal chemistry 56(8),3429,Tanget al,Molecular Imaging and Biology(2016))。
下面结合附图和具体实施例对本发明进行详细说明。
实施例1、TDW-F-1的合成方法(如图2):
(1)化合物7-1的合成
将6-1(40mg,0.109mmol)溶解于5mL无水的四氢呋喃中,随后在冰浴的条件下加入NaH(7.9mg,0.327mmol),反应30分钟后加入octane-1,8-diyl bis(4-methylbenzenesulfonate)(148mg,0.327mmol),然后将反应体系放置于微波反应体系中在120℃的条件下加热30min.并用质谱检测反应进度。当反应结束后,用0.5M HCl(50mL)进行中和,并用二氯甲烷(50mL*3)进行萃取。然后用柱层析进行分离(分离条件:DCM/MeOH=95/5(V/V)),最后得到化合物7-1(黄色固体,50mg,70%产率)。
(2)化合物9-1的合成
将化合物7-1(50mg,0.076mmol)与potassium 1,3-dioxoisoindolin-2-ide(28mg,0.15mmol)加入到3mL DMF中,待其溶解后,放置于微波反应器中进行加热(140℃,30min),并用质谱检测反应进度,待反应结束后,将反应物蒸干,并加入4mL乙醇与0.5mLhydrazine,微波加热60分钟后(180℃),用质谱进行检测,待反应结束后,用HPLC进行纯化,最后得到白色产物30mg(产率:80%)。
(3)化合物10-1的合成
将化合物9-1(30mg,0.061mmol)与荧光素(24mg,0.061mmol)放置于2mL甲醇中,在室温条件下进行过夜搅拌。用质谱进行反应进度检测,待反应结束后,用HPLC进行纯化,得到反应产物50mg(产率90%)。
实施例2 TDW-F-2的合成方法(如图3):
(1)化合物7-2的合成
将6-2(40mg,0.109mmol)溶解于5mL无水的四氢呋喃中,随后在冰浴的条件下加入NaH(7.9mg,0.327mmol),反应30分钟后加入octane-1,8-diyl bis(4-methylbenzenesulfonate)(148mg,0.327mmol),然后将反应体系放置于微波反应体系中在120℃的条件下加热30min.并用质谱检测反应进度。当反应结束后,用0.5M HCl(50mL)进行中和,并用二氯甲烷(50mL*3)进行萃取。然后用柱层析进行分离(分离条件:DCM/MeOH=95/5(V/V)),最后得到化合物7-1(黄色固体,52mg,74%产率)。
(2)化合物9-2的合成
将化合物7-2(50mg,0.076mmol)与potassium 1,3-dioxoisoindolin-2-ide(28mg,0.15mmol)加入到3mL DMF中,待其溶解后,放置于微波反应器中进行加热(140℃,30min),并用质谱检测反应进度,待反应结束后,将反应物蒸干,并加入4mL乙醇与0.5mLhydrazine,微波加热60分钟后(180℃),用质谱进行检测,待反应结束后,用HPLC进行纯化,最后得到白色产物30mg(产率:80%)。
(3)化合物10-2的合成
将化合物9-2(30mg,0.061mmol)与荧光素(24mg,0.061mmol)放置于2mL甲醇中,在室温条件下进行过夜搅拌。用质谱进行反应进度检测,待反应结束后,用HPLC进行纯化,得到反应产物50mg(产率90%)。
实施例3 TDW-F-3的合成方法(如图4):
(1)化合物7-3的合成
将6-3(50mg,0.127mmol)溶解于5mL无水的四氢呋喃中,随后在冰浴的条件下加入NaH(9.1mg,0.380mmol),反应30分钟后加入octane-1,8-diyl bis(4-methylbenzenesulfonate)(173mg,0.380mmol),然后将反应体系放置于微波反应体系中在120℃的条件下加热30min.并用质谱检测反应进度。当反应结束后,用0.5M HCl(50mL)进行中和,并用二氯甲烷(50mL*3)进行萃取。然后用柱层析进行分离(分离条件:DCM/MeOH=95/5(V/V)),最后得到化合物7-1(黄色固体,65mg,75%产率)。
(2)化合物9-3的合成
将化合物7-3(65mg,0.096mmol)与potassium 1,3-dioxoisoindolin-2-ide(37.2mg,0.2mmol)加入到3mL DMF中,待其溶解后,放置于微波反应器中进行加热(140℃,30min),并用质谱检测反应进度,待反应结束后,将反应物蒸干,并加入4mL乙醇与0.5mLhydrazine,微波加热60分钟后(180℃),用质谱进行检测,待反应结束后,用HPLC进行纯化,最后得到白色产物40mg(产率:80%)。
(3)化合物10-3的合成
将化合物9-3(40mg,0.077mmol)与荧光素(30mg,0.077mmol)放置于2mL甲醇中,在室温条件下进行过夜搅拌。用质谱进行反应进度检测,待反应结束后,用HPLC进行纯化,得到反应产物63mg(产率90%)。
实施例4 荧光探针亲和性的测定
新型荧光探针的亲和性试验是通过对[3H]PK11195的竞争性试验获得的,在这个过程中,提取大鼠肾脏的细胞膜,并保存在-20℃的环境中。在放射性竞争实验中,相应的细胞膜会被融化,并悬浮于测试溶液中(Tris-HCl 50mM,pH 7.4),运用Bradford方法测定溶液中的蛋白浓度。在接下来的竞争性实验中,将含有蛋白30微克的细胞膜与不同浓度的[3H]PK11195在0℃下共同孵育90分钟。[3H]PK11195的浓度梯度是0.001nM到10nM,终体积为500微升。在孵育结束后,将样品通过真空过滤收集在GF/C滤膜上,并用测试溶液清洗3遍。滤膜的放射性通过液体闪射计数器进行测定(TopCount,PerkinElmer)。实验数据及曲线分析通过GraphPad Prism进行分析。结果如图5所示,图5中A、B、C表示化合物TDW-F-1、TDW-F-2、TDW-F-3的竞争性结合实验数据。TDW-F-1,TDW-F-2与TDW-F-3的Ki值分别为6.2pM,9.8pM与5.0pM,显示了非常强的体外TSPO亲和特性。
实施例5 生物学活性及荧光显像的效果测定
荧光探针可应用于体外细胞的共聚焦显像,如图6所示,新型的荧光探针TDW-F-1可以用于C6细胞的TSPO显像。
C6胶质瘤细胞的线粒体拥有非常高的TSPO表达量,被广泛应用于TSPO的体内外检测评估中。在本实施例中,检测了C6胶质瘤细胞对新型探针TDW-F-1的吸收。通过本次检测,发现TDW-F-1选择性的聚集在C6细胞的线粒体中(图6A与图6B),但相同的C6细胞进行荧光探针没有选择性的吸收(图6C与图6D),这些充分验证了新型荧光探针TDW-F-1对TSPO的选择性。为了充分论证此新型TSPO荧光探针的选择性,本实施例进一步的检验了TDW-F-1在细胞器中的位置,通过运用TDW-F-1与MitoTracker Red染料对线粒体的标记,发现TDW-F-1的荧光区域(图6E)与MitoTracker Red的荧光区域重合(图6F),进一步证明了TDW-F-1能够选择性的标记C6细胞中的线粒体。
实施例6 活体动物成像与其在胰腺肿瘤中的诊断效果
在乳腺癌(MDA-AB-231)的皮下种植的小鼠中(右侧腋下),运用TDW-F-1探针对其进行荧光显像,并实时探测了荧光探针在体内的分布情况。在此实施例中,发现荧光探针TDW-F-1能够在乳腺癌的区域选择性的聚集,并在探针注射后20小时能够给出可靠的检验结果(图7A,7B,7C),探针注射后40小时会进行降解(图7D)。证明了其在体内靶向肿瘤组织的特异性。
以上实施例5、6是以荧光探针TDW-F-1为例进行的,根据荧光探针TDW-F-1、荧光探针TDW-F-2、荧光探针TDW-F-3的结构相似性,对荧光探针TDW-F-2、荧光探针TDW-F-3进行同样的实验,结果同样表明荧光探针TDW-F-2、荧光探针TDW-F-3具有与荧光探针TDW-F-1相同的生物学活性及荧光显像,同时具有在体内靶向肿瘤组织的特异性。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.TSPO荧光显像探针,其特征在于,其分子结构如下:
R1与R2为单独的直链烷烃类取代基,R1为C1到C10的直链烷烃类取代基,R2为C1或C2的直链烷烃类取代基;
R3与R4为结构相同的直链烷烃类取代基团,长度为C1到C10;
n=2-10;
该分子结构式中Signaling Agent为信号基团。
2.根据权利要求1所述的TSPO荧光显像探针,其特征在于,分子结构式中信号基团为荧光染料,结构如下任一结构所示:
3.根据权利要求1所述的TSPO荧光显像探针,其特征在于,其分子结构如下:
4.根据权利要求1所述的TSPO荧光显像探针,其特征在于,其分子结构如下:
5.根据权利要求1所述的TSPO荧光显像探针,其特征在于,其分子结构如下:
6.如权利要求1所述的TSPO荧光显像探针的合成方法,其特征在于,先合成化合物6,运用合成的化合物6,在其酚羟基端加入一个长直链的烷基取代物,并合成化合物7,经过两步反应生成带有游离氨基的化合物9,最后将化合物9与带有异硫氰酸基的荧光或染料小分子反应生成所述TSPO荧光显像探针;
化合物6、7、9结构式如下:
R1与R2为单独的直链烷烃类取代基,R1为C1到C10的直链烷烃类取代基,R2为C1或C2的直链烷烃类取代基;
R3与R4为结构相同的直链烷烃类取代基团,长度为C1到C10;
n=2-10。
7.根据权利要求6所述的TSPO荧光显像探针的合成方法,其特征在于,所述化合物6通过以下反应合成:
8.根据权利要求7所述的TSPO荧光显像探针的合成方法,其特征在于,所述化合物6合成应用了微波合成的方法。
9.如权利要求1所述的TSPO荧光显像探针的应用,其特征在于,所述TSPO荧光显像探针作为体外的TSPO检测剂的应用。
10.如权利要求1所述的TSPO荧光显像探针的应用,其特征在于,所述TSPO荧光显像探针作为体内的影像检测剂的应用。
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