CN111548360A - 一种二硫代氨基甲酸混价铜配合物及其制备和应用 - Google Patents
一种二硫代氨基甲酸混价铜配合物及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种二硫代氨基甲酸混价铜配合物及其制备和应用，该铜配合物化学式为[CuII 2(L)2(Br)2CuI(Br)]，其中HL为2‑（二（吡啶‑2‑基）亚甲基）肼甲基二硫代硫酸甲酯。该铜配合物制备方法简单，目标产物二硫代氨基甲酸混价铜配合物在可见光谱的绿色区域450‑650 nm中有明显的荧光，用作基于荧光的癌细胞成像，并用于制备癌细胞成像荧光探针。目标产物还具有良好的抗癌活性和抗癌细胞转移活性。特别地，本发明的目标产物可有诱导具有凋亡抗性的癌细胞，如胰腺癌细胞，铁死亡（一种非凋亡形式的细胞死亡），有效克服耐药性的发生，具有很好的应用价值。
Description
技术领域
本发明涉及铜配合物，具体是一种二硫代氨基甲酸混价铜配合物及其制备方法和应用。
背景技术
调查显示，在未来的十年中，胰腺癌将成为西方国家第二大导致癌症死亡的主要原因。与大多数癌症的生存率稳定增加相反，胰腺癌的进展缓慢，目前其5年生存率不到5％。手术切除仍是长期生存的唯一机会，但由于局部血管转移或浸润，不到五分之一的患者可以接受手术治疗。因此，胰腺癌，尤其是晚期胰腺癌的治疗很大程度上取决于全身化疗。早在1996年，吉西他滨单药治疗就被FDA批准为胰腺癌的治疗标准，但其总体5年生存率仅为6.7％，仍然是所有类型癌症中生存最低的之一。目前，已将两种具有中等临床活性的联合方案，即FOLFIRINOX(一种治疗组合，包括奥沙利铂，5-氟尿嘧啶，亚叶酸和伊立替康)和nab-紫杉醇-吉西他滨作为胰腺癌患者的治疗选择，但它们具有明显的毒性特征，例如中性粒细胞减少症。因此，迫切需要有效的抗胰腺癌化学疗法来提高患者的生存率和生活质量。
然而，胰腺癌细胞主要是由KRAS癌基因的突变驱动的，并且对细胞凋亡具有很强的抵抗力。因此，可以通过非凋亡性细胞死亡途径(例如铁死亡)杀死胰腺癌细胞的化合物的合成可能具有重要意义。铁死亡是一种公认的可控制细胞死亡的形式，与肿瘤的发生，发展和治疗密切相关。就诱导因子，形态学特征和调节途径而言，铁死亡与细胞凋亡，坏死和自噬明显不同。铁死亡主要表现为过度的氧化应激和膜脂质过氧化，最终导致细胞死亡。有趣的是，过表达致癌性RAS(KRAS，NRAS，HRAS)基因的细胞，例如具有突变KRAS基因的胰腺癌细胞，对铁死亡更为敏感。因此，能够诱导胰腺癌细胞非凋亡性细胞死亡的化学疗法，例如铁死亡，值得进一步的研究。本申请公开了一个可以诱导铁死亡的荧光铜(I/II)配合物及其制备方法，表征和基于细胞的研究和应用。
发明内容
本发明提供一种可以与铜离子螯合的配体2-(二(吡啶-2-基)亚甲基)肼甲基二硫代硫酸甲酯，利用该配体，合成了一种二硫代氨基甲酸混价铜配合物，并公开其制备方法和应用。
实现本发明目的的技术方案如下：
一种二硫代氨基甲酸混价铜配合物，它的化学式为[CuII 2(L)2(Br)2CuI(Br)]，其中HL为2-(二(吡啶-2-基)亚甲基)肼甲基二硫代硫酸甲酯；Cu金属与配体配位，HL上的氢会脱去，配体变成阴离子，所以化学式中用L表示；
该二硫代氨基甲酸混价铜配合物的结构式如下：
X射线单晶衍射显示，二硫代氨基甲酸混价铜配合物属于单斜晶系晶，空间群C2/c；存在三个Br原子，三个Cu原子和两个单负三齿L配体；Cu1或Cu1i(i＝-x，y，1.5-z)中心显示+2氧化态，并采用在基面上包含溴化物阴离子和负三齿L配体的方平面配位几何结构；Cu2原子具有+1氧化态，并处于三角平面几何形状，被一个Br阴离子和来自两个L配体的两个N原子(N3和N3i)包围。
上述结构式所示混价铜配合物的制备方法如下：
将CuBr2加入到含有等摩尔2-(二(吡啶-2-基)亚甲基)肼甲基二硫代硫酸甲酯配体的乙醇溶液中，40-65℃回流3-6小时，然后将混合溶液冷却至室温，静置、析晶，收集晶体，即得到所述的二硫代氨基甲酸混价铜配合物。
所述CuBr2与2-(二(吡啶-2-基)亚甲基)肼甲基二硫代硫酸甲酯的摩尔比为1:1；
所述2-(二(吡啶-2-基)亚甲基)肼甲基二硫代硫酸甲酯与乙醇溶液的配比为1mmol:10mmol；
制备时，可按上述配比的倍数添加原料。
本发明的另一目的在于，提供所述的二硫代氨基甲酸混价铜配合物在制备细胞成像荧光探针中的应用，特别是癌细胞成像，研究表明上述配合物可富集于胰腺癌细胞(ASPC-1)的线粒体中。
本发明的另一目的在于，提供所述的二硫代氨基甲酸混价铜配合物在制备抗癌细胞(如胰腺癌细胞)转移药物方面的应用。
本发明的另一目的在于，提供所述的二硫代氨基甲酸混价铜配合物在制备诱导具有凋亡抗性的癌细胞(如胰腺癌细胞)铁死亡药物方面的应用。
本发明的优点：
(1)通过简单的实验步骤和实验条件，高效合成二硫代氨基甲酸配体及二硫代氨基甲酸混价铜配合物；
(2)尽管具有d9电子排列的Cu(II)是顺磁性的并且可以淬灭相邻荧光团的荧光，但是二硫代氨基甲酸混价铜配合物在可见光谱的绿色区域(约450-650nm)显示出明显的荧光。在相同浓度下，二硫代氨基甲酸混价铜配合物的荧光强度明显比二硫代氨基甲酸HL配体的高；
(3)本发明所述二硫代氨基甲酸混价铜配合物可同时用于细胞成像和癌症的治疗；
(4)本发明所述二硫代氨基甲酸混价铜配合物可以诱导胰腺癌细胞铁死亡，这对治疗具有凋亡抗性的肿瘤有指导意义。
附图说明
图1为本发明二硫代氨基甲酸混价铜配合物的晶体结构示意图，为了简洁，H原子全部删除(对称代码i＝–x,y,1.5–z)。
图2(A)为本发明二硫代氨基甲酸配体及二硫代氨基甲酸混价铜配合物的紫外吸收图；
图2(B)为本发明二硫代氨基甲酸配体及二硫代氨基甲酸混价铜配合物的荧光图(405nm激发)。
图3(A)为用二硫代氨基甲酸混价铜配合物与和远红色核染料DRAQ5共孵育的ASPC-1细胞的显微镜图像；
图3(B)为用二硫代氨基甲酸混价铜配合物与线粒体染料MitoTracker Deep RedFM共孵育的ASPC-1细胞的显微镜图像。
图4(A)为通过伤口愈合测定，本发明所述二硫代氨基甲酸混价铜配合物对ASPC-1细胞的抗迁移作用，比例尺：500μm；
图4(B)为在本发明所述二硫代氨基甲酸混价铜配合物的作用下，在ASPC-1细胞侵袭测定开始后24小时胶原凝胶底部的典型视图。
图5为本发明所述二硫代氨基甲酸混价铜配合物抗肿瘤作用的蛋白质组学分析；
图5(A)GO功能分类的差异累积蛋白；
图5(B)GO富集分析差异表达的蛋白质；
图5(C)差异表达蛋白的途径分析基于KEGG数据库。
图6(A)在暴露于本发明所述二硫代氨基甲酸混价铜配合物24小时后，使用BODIPY-C11测量ASPC-1细胞中细胞脂质ROS的水平；
图6(B)在48小时的温育后，在不存在和存在铁死亡抑制剂Liproxstatin-1(2μM)的情况下，本发明所述二硫代氨基甲酸混价铜配合物对ASPC-1细胞的体外细胞毒性；
图6(C)在全人类血液中本发明所述二硫代氨基甲酸混价铜配合物的稳定性，显示其半衰期为1.4小时。
图7为用指定浓度的本发明所述二硫代氨基甲酸混价铜配合物处理3D ASPC-1肿瘤球体的形态学变化，比例尺：500μm。
具体实施方式
下面结合具体实施例进一步详细说明本发明技术方案。
3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)，肼碳二硫代甲酯，二(吡啶-2-基)甲酮和CuBr2购自Sigma-Aldrich。所有其他试剂和溶剂均从商业来源获得，并且无需进一步纯化即可使用。
(1)2-(二(吡啶-2-基)亚甲基)肼甲基二硫代硫酸甲酯配体(HL)的合成与表征：
通过在MeOH中回流肼碳二硫代甲酯(0.61g，5mmol)和二(吡啶-2-基)甲酮(0.92g，5mmol)合成配体2-(二(吡啶-2-基)亚甲基)肼基二硫代甲基酯(HL)，得到黄色溶液。通过在4℃下缓慢蒸发溶剂可生成HL的黄色发光晶体，产量1.09g(76％)。计算值C13H12N4S2(288.39):C,54.14；H,4.19和N,19.43。实测值:C,60.62；H,5.43和N,21.52.1H NMR(400MHz,DMSO-d6)δ14.92(s,1H),8.92–8.74(m,1H),8.69–8.50(m,1H),8.07–7.85(m,3H),7.69–7.56(m,2H),7.51(d,J＝0.6Hz,1H),2.56(s,3H).13C NMR(101MHz,DMSO-d6)δ201.01,154.65,150.53,148.70,148.42,144.20,137.84,137.45,127.61,125.29,124.44和16.96ppm。
(2)二硫代氨基甲酸混价铜配合物([CuII 2(L)2(Br)2CuI(Br)])的合成和结构表征：
将CuBr2(5mmol)添加到含有HL配体(5mmol)的乙醇(50mmol)溶液中，65℃回流6小时，然后将混合溶液冷却至室温并过滤，通过缓慢蒸发滤液溶剂而获得的块状二硫代氨基甲酸混价铜配合物，产率:68％。
计算值C26H22Br3Cu3N8S4(1005.11):C,31.07；H,2.21和N,11.15。
实测值:C,31.11；H,2.33and N,11.07。IR(KBr,cm-1):2361w,1588m,1543w,1403vs,1331s,1306m,1208m,1087s,994s,911m,793m,747w,651w,604w,523w。
单晶结构表征：在具有Mo-Kα
源的Bruker SMART Apex II CCD衍射仪上室温收集数据用。结构通过直接方法求解，并使用SHELXTL 5.1版进行结构优化。热椭圆体图用于所有非氢原子。[CuII 2(L)2(Br)2CuI(Br)]的选择晶体参数和键合参数列于表1和表2中。
表1.二硫代氨基甲酸混价铜配合物的晶体数据。
表2.二硫代氨基甲酸混价铜配合物的键长
和键角(°)。
X射线单晶衍射显示，二硫代氨基甲酸混价铜配合物属于单斜晶系晶，空间群C2/c。如图1所示，存在三个Br原子，三个Cu原子和两个单负三齿L配体。Cu1或Cu1i(i＝-x，y，1.5-z)中心显示+2氧化态，并采用在基面上包含溴化物阴离子和负三齿L配体的方平面配位几何结构。Cu 2原子具有+1氧化态，并处于三角平面几何形状，被一个Br阴离子和来自两个L配体的两个N原子(N3和N3i)包围。
(3)二硫代氨基甲酸混价铜配合物的紫外吸收和荧光光谱实验：
图2A显示了HL及其二硫代氨基甲酸混价铜配合物的吸收光谱，这些光谱在H2O(10％DMSO，v/v)中测定。HL配体在250-700范围内表现出两个强烈的吸收带(约280nm和346nm)，它们被分配给n→π*和π→π*跃迁。配合物的UV-vis吸收光谱显示出在约412nm的低能量吸收带。这是金属配体电荷转移(MLCT)的特征。在415nm激发后，在含10％DMSO(v/v)的水中测量这些二硫代氨基甲酸混价铜配合物和HL配体的荧光光谱(图2B)。铜是顺磁性的(d9电子排列)，然后可以淬灭相邻荧光团的荧光。有趣的是，硫代氨基甲酸混价铜配合物的荧光强度分别比HL配体的荧光强度高26.6。硫代氨基甲酸混价铜配合物中一价铜的存在可能在其突出的荧光强度中起重要作用。值得注意的是，硫代氨基甲酸混价铜配合物和HL配体在可见光谱的绿色区域(450-650nm)均显示荧光，可用于基于荧光的生物成像。
(4)二硫代氨基甲酸混价铜配合物体外抗胰腺癌活性研究：
人胰腺癌细胞(BxPc-3,ASPC-1和PANC-1)和人正常胰腺导管上皮细胞(HPDE6-C7)在含有10％FBS(胎牛血清)和1％链霉素/青霉素的DMEM中培养(37℃和5％CO2)。使用常用MTT法测定法测量目二硫代氨基甲酸混价铜配合物的细胞毒性作用。
如观察到的IC50值所揭示的，HL配体的IC50值明显高于二硫代氨基甲酸混价铜配合物的IC50值，因此表明HL配体的抗癌活性明显受到与铜金属配位的影响。值得注意的是，二硫代氨基甲酸混价铜配合物显示出明显的体外抗胰腺癌活性，IC50值在亚微摩尔范围内(0.41至0.74μM)，显着低于用顺铂测定的值。对于BxPc-3,ASPC-1和PANC-1细胞，二硫代氨基甲酸混价铜配合物的IC50值分别比顺铂的低72.6-，163.9-，和72.8-倍。
表3.二硫代氨基甲酸混价铜配合物抑制人类癌细胞系生长的IC50值(μM)。
(5)二硫代氨基甲酸混价铜配合物细胞成像研究：
基于二硫代氨基甲酸混价铜配合物的荧光性质，通过Zeiss LSM510共焦显微镜使用油镜(63×)对铜配合物进行细胞成像。简而言之，预先在37℃和5％CO2条件下用不同的染料(DRAQ5和MitoTracker Deep Red FM)处理ASPC-1细胞，然后用二硫代氨基甲酸混价铜配合物(25μM)孵育。二硫代氨基甲酸混价铜配合物，DRAQ5，MitoTracker Deep Red FM在405、638和638nm处独立激发，并分别在510、690和665nm处检测到发射。
结果如图3A所示，与ASPC-1细胞一起在37℃孵育后，DRAQ5在638nm处显示红色荧光，二硫代氨基甲酸混价铜配合物在405nm处产生绿色荧光的点状图案。与二硫代氨基甲酸混价铜配合物和DRAQ5共同孵育后，DRAQ5的红色荧光未与二硫代氨基甲酸混价铜配合物的绿色荧光重叠，表明铜复合物未在核内定位(图3A)。线粒体作为重要的细胞器，参与重要的过程，例如细胞能量供应，细胞分化和细胞信息传递，并已成为抗癌剂日益理想的靶标。因此，我们接下来使用MitoTracker Deep Red FM染料研究了该配合物是否与线粒体共定位。
如图3B所示，使用MitoTracker Deep Red FM进行的共定位研究导致绿色和红色合并形成黄色，这表明二硫代氨基甲酸混价铜配合物在线粒体区域中积累。靶向线粒体具有显着的优势，即：①作为真核细胞的动力源，线粒体功能障碍可以阻止癌细胞的快速生长，甚至导致癌细胞死亡。②位于线粒体中的抗癌药可以避免核切除修复(NER)机制，该机制可以恢复DNA-药物加合物；③位于肿瘤细胞中的线粒体比正常细胞中的线粒体更易发生线粒体功能障碍。因此，考虑到线粒体在癌细胞中的重要作用，这可以被认为是有意义的结果。
(6)二硫代氨基甲酸混价铜配合物的抗胰腺癌细胞转移作用测定：
目前，手术切除胰腺肿瘤为长期生存提供了唯一机会，但是由于局部血管浸润或转移，只有五分之一的患者可以通过手术治疗。因此，我们研究了二硫代氨基甲酸混价铜配合物对ASPC-1细胞中细胞迁移和侵袭的影响。通过伤口愈合试验和侵袭试验研究了二硫代氨基甲酸混价铜配合物在ASPC-1细胞系中的抗转移研究。对于伤口愈合测定，将ASPC-1细胞接种在六孔板中，然后培养直至细胞生长到约90％汇合。使用无菌移液器吸头在单层细胞中产生伤口间隙。然后，将细胞碎片用冷PBS小心地除去，并在不包含或包含二硫代氨基甲酸混价铜配合物的RPMI-1640培养基中培养，该培养基含有0.8％的胎牛血清(以最大程度地减少测定期间ASPC-1细胞的增殖)，持续24h。对于侵袭试验操作如下：使用涂有Matrigel的24孔transwell在ASPC-1细胞中研究了侵袭试验，该孔可使细胞迁移通过聚碳酸酯滤膜(8.0μm孔)。简而言之，将75μL Matrigel工作溶液(1mg/ml)添加到上表面，并使其在37℃下固化一小时。将750μL含10％胎牛血清的RPMI-1640培养基添加至下孔，而将100μL密度为5×104个细胞/孔的细胞悬浮液(含或不含二硫代氨基甲酸混价铜配合物)铺在上层室中。温育24小时后，将插入物下表面上的粘附细胞固定在MeOH中，并用0.1％结晶紫染料染色。
如图4A所示，在不存在二硫代氨基甲酸混价铜配合物的情况下，细胞快速迁移并且间隙显着减小，而在存在二硫代氨基甲酸混价铜配合物的情况下观察到较少的迁移细胞和更窄的迁移距离。如图4B中的结果显示用二硫代氨基甲酸混价铜配合物处理后，ASPC-1细胞的侵袭被显着抑制。这些结果表明，ASPC-1抑制了ASPC-1细胞的迁移和侵袭能力。
(7)二硫代氨基甲酸混价铜配合物抗胰腺癌机理研究：
二硫代氨基甲酸混价铜配合物抗胰腺癌机理主要通过蛋白组学和脂质活性氧(ROS)研究。蛋白组学主要通过串联质量标签分析(Tandem mass tag analysis，TMT)。原始的MS/MS数据输入到Proteome DiscovererTM软件(2.1版)中进行数据处理。使用Mascot 2.6软件和Uniprot_HomoSapiens数据库(20386_20180905)对肽进行鉴定和定量。鉴定和定量分析参数设置如下：酶(胰蛋白酶)，最大错位切割数(2)，仪器(ESI-TRAP)，前体质量公差(±10ppm)，碎片质量公差(0.05Da)，使用平均前体质量(假)，来自Quan方法的修饰基团(TMT 10plex)，动态修饰(氧化和乙酰基)，静态修饰(氨基甲酰甲基)和数据库模式(诱饵)。为了进行蛋白质鉴定，将肽FDR(错误发现率)调整为≤0.01。差异表达蛋白的GO(基因本体论)注释来自Blast2GO数据库(www.geneontology.org)。GO注释根据分子功能，细胞区室和生物学过程等3个类别对差异表达的蛋白质进行了分类。差异表达蛋白的KEGG(基因和基因组京都百科全书)途径分析是使用KOALA(KEGG正交与链接注释)软件和在线KEGG分析数据库(www.genome.jp/kegg/pathway.html)进行的。KEGG途径富集和GO富集分析是基于Fisher的精确测试进行的。
ASPC-1细胞脂质ROS生成通过BODIPY-C11脂质探针(Thermo Fisher Scientific，美国)测量。将ASPC-1细胞以指定的浓度暴露于二硫代氨基甲酸混价铜配合物。24小时后，洗涤细胞，并在37℃下用BODIPY-C11(5μM)标记15分钟。在流式细胞仪(FACS-can，BectionDickinson)上使用同时用于还原染料和氧化染料的通道测量脂质ROS。
蛋白组学实验分析结果如图5。经过质量验证后，定量了6262种蛋白质。其中，根据以下标准：P<0.05，比率≥1.5(上调)或比率≤0.667(下调)，在二硫代氨基甲酸混价铜配合物处理组和对照组之间获得了157种差异累积的蛋白质。在这些改变的蛋白质中，有113个蛋白质被上调，而44个蛋白质被下调。为了了解差异表达蛋白的特性和功能，这些蛋白根据基因本体论(GO)术语进行注释的，该术语基于三类：细胞成分，生物学过程和分子功能。差异表达的蛋白质的GO分析显示在图5A中。根据GO术语中>40％的基因百分比，细胞成分分析显示，大多数表达改变的蛋白质分别属于膜部分，细胞部分，细胞器部分，含蛋白质的复合物，细胞器和膜。生物过程分析表明，这些类别主要涉及免疫系统过程，定位，多细胞生物过程，细胞过程，细胞成分组织或生物发生，对刺激的反应，生物调节，多生物过程和代谢过程。有关分子功能的GO分析表明差异表达的蛋白质主要参与结合和催化活性。
为了确定差异表达的蛋白质是否在某些功能类型中显着富集，我们通过GO分类进一步对差异表达的蛋白质进行了富集分析。如图5B中显示了具有P值的10个项中最丰富的GO项。大量富集的细胞组分GO术语主要与细胞外空间，免疫球蛋白复合物循环，质膜外侧，单体IgA免疫球蛋白复合物和分泌型二聚体IgA免疫球蛋白复合物相关。对于GO的生物学过程，差异表达的蛋白质在B细胞活化，吞噬作用识别，白细胞迁移，补体活化经典途径和其他生物细胞杀伤的正调控方面显着丰富。显着丰富的分子功能GO术语是抗原结合，免疫球蛋白受体结合，铜离子结合，抗氧化活性和花生四烯酸结合。
途径分析是更系统和全面地了解细胞的生物学过程，性状或疾病的机制以及药物作用机制的最直接和必要的方法。以已建立的根蛋白为背景，研究了显着富集的KEGG途径，可以确定受二硫代氨基甲酸混价铜配合物处理显着影响的信号转导和代谢途径。如图5C所示，10个最重要的途径是IL-17信号传导途径，类风湿性关节炎，慢性粒细胞白血病，体液切应力和动脉粥样硬化，铁死亡，MAPK信号传导途径，结肠直肠癌，炎症性肠病(IBD)，细胞因子-细胞因子受体相互作用和B细胞受体信号通路。其中，与ASPC-1细胞的增殖，分化，迁移和死亡相关的3种途径明显涉及铁死亡，MAPK信号通路和细胞因子-细胞因子受体的相互作用。
通过KEGG分析发现，铁死亡途径主要是由血红素加氧酶1(HMOX1)的显着上调(P＝3.2889E-20)诱导的。从流式细胞术分析获得了二硫代氨基甲酸混价铜配合物通过铁死亡途径发挥抗肿瘤作用的另一个证实，该分析用于评估脂质ROS的产生水平。从图6A可以看出，二硫代氨基甲酸混价铜配合物确实诱导了脂质ROS的产生。另外，我们在铁死亡抑制因子Liproxstatin-1的存在下进行了细胞毒性研究。与Liproxstatin-1共同孵育可降低二硫代氨基甲酸混价铜配合物对ASPC-1细胞的毒性(图6B)。综上所述，这些数据表明二硫代氨基甲酸混价铜配合物是铁死亡的诱导剂。该结果首次提供了证据，表明铜配合物可引起铁死亡。
(8)二硫代氨基甲酸混价铜配合物抑制肿瘤球生长能力和在全人类血液中的稳定性：
通过评估不同浓度的二硫代氨基甲酸混价铜配合物处理后肿瘤球体的大小来评估3D肿瘤球体的抑制能力。ASPC-1细胞在使用超低附着率的圆底96孔板(
Sigma-Aldrich)，每孔600个细胞培养。温育2天后，将所选的球体用0.1％DMSO(对照)或不同浓度的二硫代氨基甲酸混价铜配合物处理7天。
二硫代氨基甲酸混价铜配合物在全人类血液中的稳定性测定如下：将100μM的二硫代氨基甲酸混价铜配合物在2mL全血中与肝素锂在37℃下孵育。涡旋振荡5秒钟后，立即将200μL混合血液样品的等分试样转移至装有500μL辛醇的新试管中(0小时时间点)。剩余的1.8mL血液继续在37℃旋转孵育。然后将辛醇-血液混合物离心以分离各相。使用ICP-MS分析分离的辛醇提取物(包含未反应的配合物)。在不同的时间点(0、0.5、1、2、4、6和8h)，从孵育的全人类血液混合物中取出200μL等分试样，并如上所述提取。
图7显示了用不同浓度的二硫代氨基甲酸混价铜配合物处理后对3D ASPC-1肿瘤球的抑制作用。观察到，在对照组中，肿瘤球确实增加并且非常紧凑。相比之下，通过细胞膜破裂，表面崩解，细胞渗漏和3D结构损失评估的肿瘤球体在二硫代氨基甲酸混价铜配合物治疗组(0.4和0.8μM)中观察到，特别是对于0.8μM的治疗的肿瘤球体更明显。
由于人血中生物活性化合物的稳定性对于临床配方和体内作用方式都很重要，因此使用最近报道的方案(J.Am.Chem.Soc.2015,137,2967)研究了二硫代氨基甲酸混价铜配合物在人血中的稳定性。该方法利用辛醇从水溶液中提取疏水金属基复合物(二硫代氨基甲酸混价铜配合物log P＝1.21)的能力。将二硫代氨基甲酸混价铜配合物与新鲜的含肝素锂的全血在37℃下孵育，并在不同时间点用辛醇提取等分试样。使用ICP-MS记录辛醇萃取液(未反应的二硫代氨基甲酸混价铜配合物)中的铜含量。如图6C所示，二硫代氨基甲酸混价铜配合物在人血中的半衰期约为1.4小时，这比顺铂(t1/2＝21.6min)的半衰期更长。
Claims (6)
1.一种二硫代氨基甲酸混价铜配合物，其特征在于，该配合物的化学式为[CuII 2(L)2(Br)2CuI(Br)]，其中HL为2-（二（吡啶-2-基）亚甲基）肼甲基二硫代硫酸甲酯；
该配合物的结构式如下：
X射线单晶衍射显示，二硫代氨基甲酸混价铜配合物属于单斜晶系晶，空间群C2/c；存在三个Br原子，三个Cu原子和两个单负三齿L配体； Cu1或Cu1i，i = -x，y，1.5-z，中心显示+2氧化态，并采用在基面上包含溴化物阴离子和负三齿L配体的方平面配位几何结构； Cu2原子具有+1氧化态，并处于三角平面几何形状，被一个Br阴离子和来自两个L配体的两个N原子，N3和N3i包围。
2.如权利要求1 所述的二硫代氨基甲酸混价铜配合物的制备方法，其特征在于：
将CuBr2加入到含有等摩尔 2-（二（吡啶-2-基）亚甲基）肼甲基二硫代硫酸甲酯配体的乙醇溶液中，40-65℃回流3-6小时，然后将混合溶液冷却至室温，静置、析晶，收集晶体，即得到所述的二硫代氨基甲酸混价铜配合物。
3.如权利要求1 所述的二硫代氨基甲酸混价铜配合物的制备方法，其特征在于：
所述CuBr2与2-（二（吡啶-2-基）亚甲基）肼甲基二硫代硫酸甲酯的摩尔比为1:1；
所述2-（二（吡啶-2-基）亚甲基）肼甲基二硫代硫酸甲酯与乙醇溶液的配比为1 mmol:10 mmol。
4.如权利要求1所述的二硫代氨基甲酸混价铜配合物的应用，其特征在于：所述二硫代氨基甲酸混价铜配合物在可见光谱的绿色区域450-650 nm中有明显的荧光，用作基于荧光的癌细胞成像，并用于制备癌细胞成像荧光探针。
5.如权利要求1所述的二硫代氨基甲酸混价铜配合物的应用，其特征在于：所述二硫代氨基甲酸混价铜配合物富集于胰腺癌细胞的线粒体中，在制备抗胰腺癌细胞转移药物中的应用。
6.如权利要求1所述的二硫代氨基甲酸混价铜配合物的应用，其特征在于：所述二硫代氨基甲酸混价铜配合物在制备抗具有凋亡抗性的胰腺癌细胞、铁死亡药物中的应用。
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| CN114957294A (zh) * | 2022-07-08 | 2022-08-30 | 桂林医学院 | 一种n-杂环硫代氨基脲混价铜配合物的制备方法和应用 |
| CN115141217A (zh) * | 2022-07-08 | 2022-10-04 | 桂林医学院 | 二硫代氨基甲酯混价铜配合物及其热敏水凝胶的制备方法 |
| CN114957294B (zh) * | 2022-07-08 | 2023-12-08 | 桂林医学院 | 一种n-杂环硫代氨基脲混价铜配合物的制备方法和应用 |
| CN115141217B (zh) * | 2022-07-08 | 2024-02-02 | 桂林医学院 | 二硫代氨基甲酯混价铜配合物及其热敏水凝胶的制备方法 |
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