Ru(II)‑p‑Cymene Complexes of Furoylthiourea Ligands for Anticancer Applications against Breast Cancer Cells

Half-sandwich Ru(II) complexes containing nitro-substituted furoylthiourea ligands, bearing the general formula [(η6-p-cymene)RuCl2(L)] (1–6) and [(η6-p-cymene)RuCl(L)(PPh3)]+ (7–-12), have been synthesized and characterized. In contrast to the spectroscopic data which revealed monodentate coordination of the ligands to the Ru(II) ion via a “S” atom, single crystal X-ray structures revealed an unusual bidentate N, S coordination with the metal center forming a four-membered ring. Interaction studies by absorption, emission, and viscosity measurements revealed intercalation of the Ru(II) complexes with calf thymus (CT) DNA. The complexes showed good interactions with bovine serum albumin (BSA) as well. Further, their cytotoxicity was explored exclusively against breast cancer cells, namely, MCF-7, T47-D, and MDA-MB-231, wherein all of the complexes were found to display more pronounced activity than their ligand counterparts. Complexes 7–12 bearing triphenylphosphine displayed significant cytotoxicity, among which complex 12 showed IC50 values of 0.6 ± 0.9, 0.1 ± 0.8, and 0.1 ± 0.2 μM against MCF-7, T47-D, and MDA-MB-231 cell lines, respectively. The most active complexes were tested for their mode of cell death through staining assays, which confirmed apoptosis. The upregulation of apoptotic inducing and downregulation of apoptotic suppressing proteins as inferred from the western blot analysis also corroborated the apoptotic mode of cell death. The active complexes effectively generated reactive oxygen species (ROS) in MDA-MB-231 cells as analyzed from the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining. Finally, in vivo studies of the highly active complexes (6 and 12) were performed on the mice model. Histological analyses revealed that treatment with these complexes at high doses of up to 8 mg/kg did not induce any visible damage to the tested organs.本研究合成并表征了一系列含硝基取代糠酰硫脲（nitro-substituted furoylthiourea）配体的半夹心型Ru(II)配合物（half-sandwich Ru(II) complexes），其通式分别为[(η⁶-对伞花烃(p-cymene))RuCl₂(L)]（1~6）与[(η⁶-对伞花烃(p-cymene))RuCl(L)(三苯基膦(PPh₃))]⁺（7~12）。光谱数据显示配体通过“S”原子与Ru(II)离子形成单齿配位，但单晶X射线衍射结构却揭示了一种非常规的双齿N、S配位模式，金属中心与配体形成了四元环结构。通过吸收光谱、发射光谱及黏度测定开展的相互作用研究表明，该类Ru(II)配合物可与小牛胸腺DNA（calf thymus DNA, CT DNA）发生插入相互作用；同时，这类配合物也可与牛血清白蛋白（bovine serum albumin, BSA）产生良好的结合作用。进一步针对三类乳腺癌细胞——MCF-7、T47-D及MDA-MB-231，探究了该类配合物的细胞毒性。结果显示，所有配合物的抗肿瘤活性均优于其对应配体。其中带有三苯基膦取代基的配合物7~12展现出更为显著的细胞毒性，尤以配合物12的活性最为突出：其对MCF-7、T47-D与MDA-MB-231细胞的半数抑制浓度（IC₅₀）分别为0.6 ± 0.9、0.1 ± 0.8及0.1 ± 0.2 μM。选取活性最优的配合物开展细胞死亡模式探究，染色实验证实其诱导细胞发生凋亡。蛋白质印迹分析结果显示，促凋亡蛋白表达上调、抗凋亡蛋白表达下调，进一步佐证了该类配合物通过凋亡途径诱导细胞死亡。通过2′,7′-二氯荧光素二乙酸酯（2′,7′-dichlorofluorescein diacetate, DCFH-DA）染色分析发现，活性配合物可在MDA-MB-231细胞中有效产生活性氧（reactive oxygen species, ROS）。最终选取活性优异的配合物6与12，构建小鼠模型开展体内研究。组织病理学分析结果表明，即便以最高8 mg/kg的高剂量给药，该类配合物也未对受试小鼠的脏器造成可见损伤。