Ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes induced necroptosis in MGC-803 cells.

{"full_text": " Metallomics, 14, 2022, mfac072\n https://doi.org/10.1093/mtomcs/mfac072\n Advance access publication date: 23 September 2022\n Paper\n\n\n\nUrsolic acid-piperazine-dithiocarbamate ruthenium(II)\npolypyridyl complexes induced necroptosis in\nMGC-803 cells\nHong Jiang 1 ,\u2021 , Jian-Hua Wei 1 ,2 ,3 , \u2217,\u2021 , Cui-Yan Lin 1 ,\u2021\n , Gui-Bin Liang1 , Rui-Jie He 4\n , Ri-Zhen Huang 1 ,\u2217\n , Xian-Li Ma 1\n ,\nGuo-Bao Huang 2 and Ye Zhang 1 ,2 ,3 , \u2217\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\n1\n Guangxi Engineering Research Center for Pharmaceutical Molecular Screening and Druggability Evaluation, School of Pharmacy, Guilin Medical University, Guilin\n541199, China, 2 Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, College of Chemistry and Food Science, Yulin Normal University,\nYulin 537000, China, 3 State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal\nUniversity, Guilin 541004, China and 4 Guangxi Key Laboratory of Plant Functional Phytochemicals and Sustainable Utilization, Guangxi Institute of Botany,\nGuangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China\n\u2217\n Correspondence: 1 Zhiyuan Road, Lingui District, School of Pharmacy, Guilin Medical University, Guilin 541199, Guangxi, P.R. China. E-mail:\nweijh1124@glmc.edu.cn (Jian-Hua Wei); rzhuang1783@163.com (Ri-Zhen Huang); zhangye81@126.com (Ye Zhang)\n\u2021\n These authors contributed equally to this work.\nElectronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx000 00x.\n\n\n\nAbstract\nThree ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes Ru1\u2013Ru3 were designed and synthesized for eval-\nuating antitumor activity. All the complexes exhibited high in vitro cytotoxicity against MGC-803, T24, HepG2, CNE2, MDA-MB-231,\nMCF-7, A549, and A549/DDP cell lines. Ru1, Ru2, and Ru3 were 11, 8 and 10 times, respectively, more active than cisplatin against\nA549/DDP. An in vivo study on MGC-803 xenograft mouse models demonstrated that representative Ru2 exhibited an effective in-\nhibitory effect on tumor growth, showing stronger antitumor activity than cisplatin. Biological investigations suggested that Ru2\nentered MGC-803 cells by a clathrin-mediated endocytic pathway, initially localizing in the lysosomes and subsequently escaping and\nlocalizing in the mitochondria. Mitochondrial swelling resulted in vacuolization, which induced vacuolation-associated cell death\nand necroptosis with the formation of necrosomes (RIP1\u2013RIP3) and the uptake of propidium iodide. These results demonstrate that\nthe potential of Ru2 as a chemotherapeutic agent to kill cancer cells via a dual mechanism represents an alternative way to eradicate\napoptosis-resistant forms of cancer.\n\nKeywords: ursolic acid-piperazine-dithiocarbamate, ruthenium (II) polypyridyl complexes, antitumor activity, action mechanism,\nvacuolation-associated cell death\n\n\nGraphical abstract\n\n\n\n\nOne ursolic acid-piperazine-dithiocarbamate ruthenium (II) polypyridyl complex (Ru2) as a potential chemotherapeutic agent was\ndeveloped. Ru2 exhibited significant anticancer activities in vitro and vivo. Biological investigation suggested that it entered MGC-803\ncells by a clathrin-mediated endocytic pathway, initially localizing in the lysosomes and subsequently escaping and localizing in the\nmitochondria. Mitochondrial swelling results in vacuolization, which induced vacuolation-associated cell death and necroptosis.\n\n\n\nIntroduction pathways have been explored and proven to be a feasible strat-\n egy for killing apoptotic-resistant tumor cells. As a form of pro-\nDrug resistance, which involves increments in drug efflux and\n grammed cell death featured by necrotic cell death morphology\ndecreased DNA repair ability,1 and alterations in apoptotic path-\n and autophagy activation, necroptosis represents a novel non-\nways, is still recognized as a great challenge in chemotherapy.2\u20134\n apoptotic cell death pathway and has been proven to play a sig-\nSince many therapeutic agents exert their antitumor effects by\n nificant role in multiple pathologies, including carcinoma, inflam-\ninducing intrinsic apoptosis,5 alternative nonapoptotic cell death\n mation, and sepsis.6 Necroptosis is now forged as a promising\n\n\nReceived: May 1, 2022. Accepted: August 31, 2022\n\u00a9 The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com\n\f2 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nScheme 1 Synthetic routes of Ru1\u2013Ru3. Reagents and conditions: (a) Ac2 O/DMAP/pyridine, r.t; (b) i: ClCOCOCl/CH2 Cl2 , r.t; ii: Et3 N/CH2 Cl2 , piperazine;\n(c) 4 mol/l NaOH aq, THF/MeOH, r.t; (d) CS2 , NaOH, r.t; (e) Dichlorobis(2,2-bipyridine) ruthenium(II) (A)/Dichlorobis(1,10-phenanthroline) ruthenium(II)\n(B)/Dichlorobis(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) (C), 65\u00b0C.\n\n\n\ntherapeutic form for cancer therapy.7 Therefore, the identifica- derivatives with high efficiency and low toxicity. Dithiocarbamato\ntion of necroptosis inducers/inhibitors has drawn special atten- is considered as a special coordination group for antitumor\ntion and may create an evolution of new chemotherapy interven- complexes, and some dithiocarbamato ruthenium complexes\ntions. Necroptosis has been proven to implicate the formation have exhibited potent antiproliferative activity.23 In our previous\nof receptor-interacting protein (RIP) kinases: RIP1\u2013RIP3 complex, work, we have synthesized and evaluated some dehydroabietyl\nwhich could mediate oxidative stress and lead to the generation piperazine dithiocarbamate ruthenium(II) polypyridyl complexes\nof mitochondrial reactive oxygen species (ROS), and consequently, as potential antitumor agents,24 by the combination of dehydroa-\ncause the induction of bioenergetic-related cell death.8\u201310 bietic acid (a TCM active ingredient), piperazine, dithiocarbamato\n Since the discovery of platinum antitumor complexes, includ- and ruthenium(II) polypyridyl.\ning cisplatin, carboplatin, and oxaliplatin, a great deal of effort Recently, some ruthenium polypyridyl complexes have been\nhas been devoted to developing metal antitumor complexes evaluated as anticancer agents, including as photosensitizers in\nwith various physicochemical, biological, and pharmacological photodynamic therapy, most of which induce tumor cell death\ndiversities. Among the numerous metal antitumor complexes, through apoptosis.25\u201327 However, as mentioned above, drug re-\nruthenium complexes have attracted considerable attention be- sistance involves the alteration in apoptotic pathways. It is ex-\ncause of their good coordinative ability, relatively low toxicity, and tremely difficult to treat cancer with apoptotic resistance. And\npotential antitumor virtue.11 As an important form of ruthenium it is of great interest to design ruthenium polypyridyl complexes\nantitumor complexes, ruthenium(II) polypyridyl complexes have with high anticancer activity and non-apoptotic mechanisms.\nexhibited attractive antitumor properties and exerted antitumor Based on the above research background, we designed and intro-\neffects by multiple action mechanisms, including DNA damage, duced the piperazine and dithiocarbamato groups into the UA-\ntopoisomerase (topo I or/and II) inhibition, apoptosis induction, active core to offer a ligand, which was then coordinated with\nand cell cycle arrest.12\u201314 Small structural modifications to ruthenium polypyridyl intermediated to offer [Ru(bpy)2 (L)]Cl Ru1,\nruthenium(II) polypyridyl complexes could have a significant [Ru(phen)2 (L)]Cl Ru2, and [Ru(DIP)2 (L)]Cl Ru3, respectively. To the\ninfluence on their antitumor effect and mechanism. best of our knowledge, the design, synthesis, antitumor activity\n Traditional Chinese medicine (TCM) is an indispensable part and mechanisms evaluation of UA-piperazine-dithiocarbamate\nof Chinese culture and has been proven to play an important role ruthenium polypyridyl complexes have not been described in\nin antitumor therapy.15 As a well-known TCM ingredient, ursolic the literature. Our experimental results revealed that Ru2 ex-\nacid (UA) and its derivatives have been demonstrated to display hibited significant anticancer activities in vitro and vivo, as well\nattractive antitumor activity.16\u201319 A recent study has indicated as effectively inhibited the proliferation of cisplatin-resistant\nthat UA could exert antitumor activity through a necroptosis cells A549/DDP. Notably, Ru2 induced MGC-803 cancer cell death\nmechanism.20 The potential antitumor effect of UA and its mainly through necroptosis.\nderivatives encouraged us to design, synthesize and evaluate\nUA derivatives as antitumor agents. In addition, piperazine is\nconsidered as a lysosome-targeting privileged scaffold in antitu- Results and discussion\nmor drug discovery and has widely involved in many therapeutic Chemistry\nagents, while lysosomes are important organelles in animal cells The ligand UA-piperazine-dithiocarbamate (L) and its three com-\nand play a crucial role in many cellular processes.21 The special plexes Ru1\u2013Ru3 were synthesized as outlined in Scheme 1.\nvirtue of piperazine prompted us to introduce it to UA skeleton to Firstly, acetyl UA (AUA) was synthesized by the acetylation of\nscreen for antitumor agents. Moreover, since the antitumor effect UA with the acetic anhydride/pyridine/4-dimethylaminopyridine\nand mechanism of the old alcohol-aversion drug disulfiram has (DMAP) acetyl system, according to our previous work.19 Sec-\nbeen elucidated,22 a great deal of attention has been devoted to ondly, AUA was treated with oxalyl chloride to offer acetyl\nthe design, synthesis and evaluation dithiocarbamato antitumor UA chloride, which was then reacted with piperazine in\n\f Paper | 3\n\n\nTable 1 IC50 (\u03bcM) values of the compounds toward different cell lines after 48-h incubation\n\n IC50 (\u03bcM)\n\nCompd. MGC-803 T24 HepG2 CNE2 MDA-MB-231 MCF-7\n\n Ru1 0.96 \u00b1 0.01 1.77 \u00b1 0.03 2.88 \u00b1 0.12 1.34 \u00b1 0.21 2.41 \u00b1 0.37 1.16 \u00b1 0.34\n Ru2 0.73 \u00b1 0.12 0.99 \u00b1 0.62 2.09 \u00b1 0.89 2.43 \u00b1 0.36 1.95 \u00b1 0.11 3.25 \u00b1 0.50\n Ru3 10.35 \u00b1 0.11 2.65 \u00b1 0.37 41.22 \u00b1 0.29 >50 5.46 \u00b1 0.32 14.63 \u00b1 0.12\n L 19.61 \u00b1 0.36 11.16 \u00b1 0.89 8.68 \u00b1 0.10 >50 12.10 \u00b1 0.61 10.46 \u00b1 0.27\n A 15.51 \u00b1 0.16 9.16 \u00b1 0.16 29.03 \u00b1 0.63 15.34 \u00b1 0.49 8.39 \u00b1 0.02 8.94 \u00b1 0.46\n B >50 >50 >50 >50 >50 >50\n C >50 >50 >50 >50 >50 >50\ncisplatin 9.99 \u00b1 0.13 14.74 \u00b1 0.18 12.94 \u00b1 0.75 4.14 \u00b1 0.23 >50 21.51 \u00b1 0.58\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 1 In vivo antitumor activity of Ru2 in MGC-803 xenograft. (A) Ru2 (at 6 and 12 mg/kg doses), cisplatin (at 2 mg/kg dose), or vehicle (5% DMSO in\nsaline, v/v) was administered by tail vein injection to inhibit tumor growth. Tumor growth was monitored by the mean tumor volume (mm3) \u00b1SD\n(n = 4) and calculated as the relative tumor increment rate (T/C, %). (B) Photographs of the harvested tumors from the mice. (C) Body weight change in\nthe mice treated with Ru2. (D) Tumor weight of the mice. The tumors were collected from the mice on day 27 (**P < 0.01).\n\n\n\n\ndichloromethane to provide the AUA piperazine (AUAP) in the The structures of ligand L and its three complexes Ru1\u2013Ru3\npresence of trimethylamine. Thirdly, AUAP was hydrolyzed with were then identified by 1D and 2D NMR (1 H NMR, 13 C NMR, 1 H-1 H\n4 mol/l NaOH aqueous solution to offer the important in- COSY NMR), high-resolution mass spectrometry (HR-MS), and/or\ntermediate compound UA piperazine (UAP) in the presence elemental analysis (EA). For the ligand L, in 1 H NMR, the chem-\nof the mixture of tetrahydrofuran and methanol. UAP was ical shift (\u03b4) in 5.09 ppm was attributed to the hydrogen in the\nthen treated with carbon disulfide in dichloromethane to sup- olefin (12-H) moiety. The broad peaks at \u03b4 in the range of 4.35\u2013\nply the target ligand UAP-dithiocarbamate (L) in good yield in 3.51 ppm were ascribed to four methylene hydrogens in the piper-\nthe presence of sodium hydroxide. Finally, three UA-piperazine- azine group, while \u03b4 at 0.6\u20131.04 ppm was attributed to seven\ndithiocarbamate ruthenium(II) polypyridyl target complexes methyl hydrogens (23-CH3 , 24-CH3 , 25-CH3 , 26-CH3 , 27-CH3 , 29-\nRu1\u2013Ru3 were obtained in moderate yield by the treatment of L CH3 , and 30-CH3 ) in the UA skeleton. In the 13 C NMR spectra, \u03b4 at\nwith di(2,2\u2019-bipyridine, bpy) ruthenium(II) dichloride (A), di(1,10- 194.45 and 174.97 ppm was attributed to the carbons in the thio-\nphenanthroline, phen) ruthenium(II) dichloride (B), and di(4, 7- carbonyl (C=S) and carbonyl (28-C=O) groups, respectively, while\ndiphenyl-1,10-phenanthroline, DIP) ruthenium dichloride (C) at \u03b4 at 124.85 (12-C) and 139.00 ppm (13-C) was attributed to the dou-\n65\u00b0C in methanol, respectively. ble bond carbon in the olefin moiety. In HR-MS, the mass peaks at\n\f4 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 2 (A) Intracellular ruthenium concentrations in whole cells for Ru1-Ru3(\u03bcg/mg protein, ). (B) Cellular fractionsn for Ru1 and Ru2 (ng/cell).\n\n\n\n\n645.3597 and 599.3714 m/z were ascribed to [L + Na]+ and [L-Na]\u2013 , As shown in Table 1, the complexes Ru1\u2013Ru3 exhibited bet-\nrespectively, accurately confirming the structure of the ligand L. ter in vitro antiproliferative activity than the ligand, UA and\n For complexes Ru1\u2013Ru3, in 1 H NMR, the chemical shift (\u03b4) in the corresponding ruthenium polypyridyl chloride intermediate A\u2013\nrange of 7.31\u201310.02 ppm was contributed by the aromatic hydro- C, respectively. These indicated that introducing UA-piperazine-\ngens in the polypyridyl ruthenium moiety, while all the hydrogen dithiocarbamate into ruthenium polypyridyl intermediate could\npeaks were similar to that of ligand L, indicating that ligand L had improve the antitumor activity. Notably, Ru1 and Ru2 exhib-\na coordination reaction with polypyridyl ruthenium. In 13 C NMR ited better antiproliferative activity than cisplatin on all the se-\nspectra, \u03b4 around 212 and 173 ppm were ascribed to the carbons in lected cancer cell lines, and the IC50 values were in the range of\nthe thiocarbonyl and carbonyl groups, respectively, while \u03b4 in the 0.73 \u00b1 0.12 to 3.25 \u00b1 0.50 \u03bcM. It is also important to note that\nrange of 123\u2212158 ppm was attributed to the aromatic carbon in Ru3 displayed better inhibitory activity than cisplatin against T24,\nthe polypyridyl moiety group and the double bond carbon in the MDA-MB-231, and MCF-7 cells with IC50 values of 2.65 \u00b1 0.37,\nolefin moiety, confirming the coordination effect of ligand L with 5.46 \u00b1 0.32 and 14.63 \u00b1 0.12 \u03bcM, respectively.\npolypyridyl ruthenium. By comparing the in vitro antiproliferative activity of three\n In HR-MS, the clean mass peaks at 1013.4136, 1061.4137, and complexes Ru1\u2013Ru3, it was found that the nature of the an-\n1365.5375 m/z were ascribed to [C55 H71 N6 O2 RuS2 ]+ (complex cillary ligands plays an important role in the antitumor activ-\nRu1), [C59 H71 N6 O2 RuS2 ]+ (complex Ru2), and [C83 H87 N6 O2 RuS2 ]+ ity of these complexes. When the complex incorporated two an-\n(complex Ru3), accurately verifying the structure of complexes cillary 1,10-phenanthroline ligands (Ru2), it had the lowest IC50\nRu1\u2013Ru3. values in the MGC-803, T24, HepG2, and MDA-MB-231 cell lines.\n To investigate the purity of Ru1\u2013Ru3, ultraperformance liq- In the CNE2 and MCF-7 cell lines, the influence of the ancil-\nuid chromatography (UPLC) assays were carried out with the iso- lary ligand on the antitumor activity of the complexes could\npropanol/methanol/water mobile phase. As shown in Figs. S2-1 to be listed as follows: 2,2\u2019-bipyridine > 1,10-phenanthroline > 4,7-\nS2-3, the purity of complexes Ru1\u2013Ru3 was higher than 95%. In diphenyl-1,10-phenanthroline. Our results may provide an ex-\naddition, Ru1\u2013Ru3 were stable in the mixed solution of methanol ample of tuning the selectivity of ruthenium complexes to dif-\nand cell culture medium after 24-h incubation (Figs. S3-1 to S3-3). ferent cancer cells by modifying the structure of the ancillary\n In EA, the data of C, H, and N atoms were consistent with that ligands.\nin NMR and HR-MS, well proving the structures of Ru1\u2013Ru3. Based To evaluate the resistance of complexes Ru1\u2013Ru3 to cisplatin,\non the 1D and 2D NMR, HR-MS, EA, and UPLC results, the chemical the MTT assay was carried out on A549 and its cisplatin-resistant\nstructures of the complexes Ru1\u2013Ru3 were well characterized and cell line A549/DDP (Table S1). Three complexes exhibited signifi-\nidentified. cant antitumor activity toward A549 and A549/DDP, and it is note-\n worthy that complexes Ru1\u2013Ru3 displayed significant inhibition\nAntiproliferative activity to A549/DDP with IC50 values of 2.66 \u00b1 0.27, 3.95 \u00b1 0.33, and\nIn vitro antiproliferative activity 2.97 \u00b1 0.14 \u03bcM, respectively, which were about 10-fold more po-\n tent than cisplatin (30.52 \u00b1 0.17 \u03bcM). These results indicate that\nTo investigate the in vitro antiproliferative activity of ruthe-\n complexes could overcome the cisplatin resistance probably with\nnium polypyridyl chloride intermediate A\u2013C, the ligand, and\n a different anticancer mechanism.\nits complexes Ru1\u2013Ru3, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-\n Based on the significant antiproliferative activity on selected\ndiphenyl-2-H-tetrazolium bromide (MTT) assay was carried out\n cell lines, Ru1 and Ru2 were chosen to further study the an-\nagainst several cancer cell lines, including human gastric cancer\n tiproliferative activity against MGC-803 multicellular spheroids.\ncells MGC-803, human bladder cancer cells T24, human liver\n As shown in Fig. S4, treatment with Ru1 and Ru2 dramatically\ncancer cells HepG2, human nasopharyngeal cancer cells CNE2,\n decreased the density of MGC-803 cancer cells, indicating the\nhuman breast cancer cells MDA-MB-231 and MCF-7, human\n promising antitumor activity.\nhepatoma cellA549. Cisplatin was used as the positive control.\n\f Paper | 5\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 3 Electron microscopy images of MGC-803cells treated with DMSO (control) or 2 \u03bcM Ru2 for 24 h.\n\n\n\n\nIn vivo antiproliferative activity As shown in Fig. 2A, the order for the dose of the three ruthenium\nThe antitumoral activity of Ru2 was then assessed for 27 days at complexes in whole cells was listed as follows: Ru1 > Ru2 > Ru3,\ntwo doses (6 mg/kg and 12 mg/kg) by tail vein injection once every which did not correlate and was even completely contrary to\n3 days. Specific pathogen-free BALB/c nude mice (both male and the trends observed in the logPo/w assay. It has been reported\nfemale) were divided randomly into four groups (n = 4), i.e. the ve- that the cellular uptake efficiencies of six classical coordinated\nhicle control group, the low-dose and high-dose administration of (N-N) ruthenium polypyridyl complexes are positively corre-\nRu2 groups, and the positive control groups. The dose of cisplatin lated with their lipophilicities.28 Here, we discover the incon-\nwas limited at 2 mg/kg owing to its severe toxicity. sistency between lipophilicity and cellular uptake, which may\n As illustrated in Fig. 1A\u2013D, the average tumor volume was be explained by introducing the UA-piperazine-dithiocarbamate\n318 mm3 for the control group and 207 mm3 and 105 mm3 for the ligand.\nlow-dose and high-dose administration of Ru2, which were 34% Combined with the MTT results, we could conclude that the\nand 67% lower than the control group. Ru2 with an inhibition ra- sharply elevated cellular levels of ruthenium complexes might be\ntio of 64.8% (P < 0.001) at high doses exhibited higher suppression one of the important reasons for the remarkable antiproliferative\nof tumor growth than cisplatin (47.3%, P < 0.001). This was despite activity of Ru1 and Ru2. Further investigation (Fig. 2B) indicated\nit displaying comparable in vivo toxicity with cisplatin, which was that the order for the proportion of ruthenium Ru1 and Ru2 in\ninvestigated by monitoring the weight loss of all the tested nude cellular fractions was membrane > cytoplasm > nuclear > cy-\nmice. These results indicated that Ru2 might be a good candidate toskeleton, implying that these two complexes mainly entered\nfor an antitumor agent. and stayed in the cell membrane.\n\n\n ICP-MS for the uptake mechanism of Ru2 in MGC-803 cells\nAction mechanism investigation Ru2 exhibited the best antiproliferative effect among the three\nLogP value and cellular accumulation of complexes complexes and was selected as the representative compound\nThe ability of the Ru(II) complexes to penetrate cell membranes for investigating possible cellular entry pathways. The aforemen-\nis generally attributed to their lipophilicity. Here, the lipophilic- tioned results implied that the order of the complexes accumu-\nity of the Ru(II) complexes was examined by determining the oc- lating in the cell was astonishingly inconsistent with the order\ntanol/water partition coefficients (logPo/w ) using the shake-flask of their lipophilicity. As mentioned in Poynton\u2019s 2017 review, the\nmethod. The results (Table S2) indicated that Ru1 exhibited the lipophilicity of complexes plays a very important role in pas-\nbest hydrophilicity with a negative logPo/w value of \u20130.98 109, Ru2 sive diffusion, which is the main mechanism of uptake for metal\ndisplayed moderate lipophilicity with a logPo/w of 0.428 897 and complexes.29 Thus, this inconsistency may illustrate that ruthe-\nRu3 showed the highest lipophilicity with a logPo/w of 1.49 152. nium complexes synthesized here may enter cells by facilitated\nIt is noted that increasing the aromatic surface area of the diffusion, active transport, or endocytosis rather than passive\npolypyridyl ligands resulted in increased lipophilicity of the com- diffusion.\nplexes, and the order of logPo/w for these three complexes was Supporting this hypothesis, MGC-803 cells were treated\nRu1 < Ru2 < Ru3. with various conditions and inhibitors: 37\u00b0C (control group),\n The intracellular ruthenium concentrations in whole cells and 4\u00b0C (free diffusion group), D-deoxy-glucose and oligomycin\ncellular fractions of the complexes Ru1\u2013Ru3 were then investi- (metabolic inhibited group), chloroquine (endosome acidity\ngated by inductively coupled plasma mass spectrometry (ICP-MS). (endocytosis) inhibition group), and NH4 Cl (endosome-lysosome\n\f6 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 4 MGC-803 cells were incubated with five inhibitors and were pretreated for 30 min in the presence or absence of specific inhibitors prior to\naddition of 2 \u03bcM Ru2. Phase-contrast images were taken after 24 h.\n\n\n\n\nfusion (endocytosis) inhibition group). The cells were treated with that clathrin-mediated endocytosis was the main mechanism by\nRu2 for 1 h at 4\u00b0C or 2 h at 37\u00b0C. For the several well-documented which Ru2 entered the cells.\ninhibitor groups, the cells were incubated for 30 min with the cor-\nresponding inhibitor before treatment with 10 \u03bcM Ru2 for 2 h. As\nshown in Fig. S5, compared with the control group, significantly Transmission electron microscopy for cell morphology\ndecreased Ru levels were found at 4\u00b0C in the D-deoxy-glucose The transmission electron microscope assay was performed on\n(oligomycin), chloroquine, and NH4 Cl groups, confirming the MGC-803 cells to investigate changes in cell morphology. Remark-\nrole of energy-dependent transport and the possibility of en- ably, treating MGC-803 cells with Ru2 led to an accumulation of\ndocytosis, since these processes are responsible for the uptake cytoplasmic vacuoles, as shown in Fig. 3. It is an interesting ex-\nof certain types of metal complexes.30 , 31 To further determine perimental phenomenon for ruthenium polypyridyl complex to\nthe possible pathways of endocytosis, the specific inhibitors of induce tumor cell death. In 2017, Sun and co-workers discovered\nchlorpromazine (clathrin-mediated endocytosis inhibition group) an UA-derived small molecule that could cause the accumulation\nand nystatin (caveolae-mediated endocytosis inhibition group) of vacuoles derived from macropinosomes.32 We wonder whether\nwere also employed to treat MGC-803 cells. The results displayed the mechanism of vacuolation induced by Ru2 was associated\nthat chlorpromazine exhibited a significant inhibitory effect on with the compound reported in Sun\u2019s research and due to the\nthe cellular uptake of Ru2 instead of nystatin. This confirmed presence of the UA-piperazine-dithiocarbamate ligand.\n\f Paper | 7\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 5 MGC-803 cells were incubated with chlorpromazine or nystatin alone and were pretreated for 30 min in the presence or absence of two\ninhibitors prior to addition of 2 \u03bcM Ru2. Phase-contrast images were taken after 12 h.\n\n\nIntracellular vesicle inhibition investigation with siRNA could modulate clathrin function. To support this hy-\nIn considering the possible origins of the vacuole formation pothesis, chlorpromazine pretreatment and knocking down CHC\nin MGC-803 cells, pharmacological treatments were carried with specific siRNAs were used in the Ru2-induced vacuolization\nout with inhibitors: bafilomycin A1 (Baf-A), a specific inhibitor assay.\nof the vacuolar-type H+ -ATPase, which could block clathrin- As shown in Fig. 5, compared to the treatment with Ru2\nindependent endocytosis, like micropinocytosis; 5- (N-ethyl-N- alone, pretreatment with chlorpromazine reduced the vacuoles\nisopropyl) amiloride (EIPA), an inhibitor of the Na+ /H+ exchanger in MGC-803 cells after a further 24-h incubation, while nys-\nthat blocks macropinocytosis and phagocytosis; 3-methyladenine tatin did not decrease the vacuoles. To further confirm the reg-\n(3-MA), which blocks the formation of autophagosomes by inhibit- ulatory role of clathrin, disordered and longitudinal clathrin\ning class III PI3K.33 The dynamin inhibitor dynasore (DY) and actin siRNAs were employed to treat MGC-803 cells in the pres-\ninhibitor cytochalasin D (CD) were also selected for investigating ence and absence of Ru2. The results (Fig. 6) suggested that\nthe pathways involved in the formation of intracellular vesicles treatment with Ru2 (1.5 and 2 \u03bcM) and disordered clathrin\nafter treatment with Ru2. siRNA with Ru2 (NC-Ru2) induced vacuolization in the MGC-\n As shown in Fig. 4, in comparison with the treatment of Ru2 803 cells. In contrast, siCHC treatment alone or co-incubation\nalone, pre-incubation of MGC-803 cells with DY led to the dis- with siCHC and Ru2 lessened the vacuoles in MGC-803 cells,\nappearance of intracellular vesicles along with inhibition of cell implying that clathrin-mediated endocytosis was required for\ndeath, which was consistent with the cell growth inhibition rate Ru2 internalization and played an important role in cytoplasmic\nresults (Fig. S6). When CD, 3-MA, and Baf-A were added before Ru2 vacuolation.\ntreatment, there was an obvious diminution of vacuoles, but cell\nviability was not blocked. Preincubation cells with EIPA neither Assay for lysosomal membrane permeabilization\nblocked the formation of vesicles nor inhibited cell viability. These\n It is known that lysosomes are the final destination of endocytosis.\nresults demonstrated that the cytoplasmic vacuoles induced by\n Ru2 was found to be internalized into cells via clathrin-mediated\nRu2 were dynamin dependent, which was the critical regulator\n endocytosis and deservedly accumulated in lysosomes. It is worth\nof clathrin-mediated endocytosis.34 Furthermore, we could con-\n noting that the content of ruthenium in lysosomes diminished\nclude that the vacuolization induced by Ru2 was different from\n with prolonged incubation of Ru2 with MGC-803 cells (Fig. S7),\nthat induced by UA derivatives reported by Sun,32 and the inhibi-\n which suggested that Ru2 could undergo lysosome escape. This\ntion of Ru2 internalization by DY resulted in the suppression of\n could be proven by the lysosome assay with acridine orange (AO)\ncytoplasmic vacuolation.\n staining. AO emits red fluorescence in acidic lysosomes and green\n fluorescence in the cytosol and nucleus. As shown in Fig. 7, treat-\nClathrin-mediated endocytosis and siCHC2 RNA ment with Ru2 significantly decreased the mean red fluorescence\nClathrin-mediated endocytosis or caveolin-mediated endocytosis in the cells compared with the control cells since the stacked AO\nis the main form of endocytosis. Our ICP-MS assay (Fig. S5) ex- (red fluorescence) accumulated in the lysosomes. The result indi-\nhibited that Ru2 entered the cells mainly by clathrin-mediated cated that the lysosome integrity was damaged after treatment\nendocytosis. Prompted by the data showing that chlorpromazine with Ru2, with consequent leakage of the AO into the cytosol and\ncould decrease the level of Ru, we asked whether the formation nucleus to stain the DNA or RNA (green fluorescence). In addi-\nof cytoplasmic vacuoles could be blocked by chlorpromazine and tion, the results confirmed that Ru2 could escape from lysosome\nwhether downregulating clathrin heavy chain (CHC) expression digestion.\n\f8 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 6 Cytoplasmic vacuoles originated from mitochondria. (A) Cells were stained with 150 nM Mito-Tracker probe for 15 min after treatment with\n2.0 \u03bcM Ru2 for 12 h. Fluorescent and phase-contrast images were obtained by laser confocal microscopy. Scale bar, 50 \u03bcm. (B) Transmission electron\nmicroscopy images of microstructural changes in the mitochondria of MGC-803 cells incubated with 0, 0.5 and 2 \u03bcM Ru2 for 24 h.\n\n\n\nDilation of mitochondria is the inducement of cytoplasmic tion with the endoplasmic reticulum-tracker under the same ex-\nvacuolation perimental condition (Fig. S8). Dilated mitochondria were further\nMitochondria play central roles in energy production and var- confirmed by transmission electron microscopy (Fig. 8B), which\nious metabolic activities. Therefore, it is an ideal target for clearly showed the formation of cytoplasmic vacuoles after treat-\ntherapeutic agents. One key characteristic of this organelle ment with Ru2 at 0.5 and 2.0 \u03bcM for 24 h. The vacuoles are\nis the extremely negative membrane potential caused by the the double-membrane organelles. Together, these observations\nproton gradient across the mitochondrial inner membrane.35 demonstrated that vacuoles were the result of dilated and swollen\nConsidering this high negative potential of the outer membrane, mitochondria induced by Ru2.\nwe hypothesized that Ru2 initially localized in the lysosomes\nby energy-dependent endocytosis and subsequently escaped Cell death mode investigation\nand localized in the mitochondria due to its intrinsic cationic The mode of cell death induced by antitumor agents is of great\nlipophilicity. significance for investigating their mechanism of action. To de-\n As mentioned in Fig. S6 and Fig. 4, treating MGC-803 cells with termine the mode of cell death induced by Ru2, necrostatin-1 (a\nRu2 and dynasore/chlorpromazine could simultaneously sup- necroptosis inhibitor), leupeptin (a lysosome pathway-associated\npress cytoplasmic vacuoles and cell death suggesting that the cell death inhibitor), Z-VAD-FMK (an apoptosis inhibitor), cy-\nformation of vacuoles was pivotal for Ru2-induced cytotoxicity. cloheximide (a paraptosis inhibitor), and 3-methyladenine (an\nTo explore cytoplasmic vacuolation, mitochondria were labeled autophagy inhibitor) were employed with Ru2 to treat MGC-803\nwith Mito-Tracker Red (MTR) (Fig. 8A). Integrated, compact, retic- cells. As shown in Fig. S9, the cell growth inhibition rate at 1.5 \u03bcM\nulate shapes were observed around the nucleus in the control Ru2 was significantly reduced in the presence of necrostatin-1\ngroup. In contrast, dilated and swollen mitochondria appeared and leupeptin, indicating that death of the MGC-803 cells was by\nin the Ru2-treated cells. However, there was no similar observa- necroptosis and related to lysosomes.\n\f Paper | 9\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 7 Fluorescence images of MGC-803 cells loaded with acridine orange.\n\n\n\n\nFig. 8 Cytoplasmic vacuoles originated from mitochondria. (A) Cells were stained with 150 nM Mito-Tracker probe for 15 min after treatment with\n2.0 \u03bcM Ru2 for 12 h. Fluorescent and phase-contrast images were obtained by laser confocal microscopy. Scale bar, 50 \u03bcm. (B) Transmission electron\nmicroscopy images of microstructural changes in the mitochondria of MGC-803 cells incubated with 0, 0.5 and 2 \u03bcM Ru2 for 24 h.\n\n\n\n\nIntracellular ROS nucleic acid stains, while PI is membrane impermeant and\nMitochondria are the main source of ROS production. Ru2 could generally excluded from viable cells. In view of the different char-\ninduce dilation of mitochondria. Moreover, mitochondrial ROS are acteristics of these two dyes, PI is used for identifying dead cells\nknown to be involved in necroptosis.36 With these facts in mind, probably undergoing necroptosis. As shown in Fig. 9, the results\nthe commercial ROS fluorescent probe 6-carboxy-2\u0003 ,7\u0003 -dichlorodi- indicated that treatment with Ru2 led to a decrease in the blue\nhydrofluorescein diacetate (DCFH-DA) was employed to evaluate fluorescence in Hoechst 33258 staining and an increase in the red\nthe intracellular generation of ROS. As shown in Fig. S10, MGC-803 fluorescence in PI staining implying the occurrence of necroptosis.\ncells treated with Ru2 (2 \u03bcM for 12 h) exhibited stronger green flu-\norescence than the untreated control cells (in black), confirming RIP1\u2013RIP3 complex\nthe intracellular ROS overproduction. Necroptosis is the best-characterized programmed necrosis and\n modulated in a receptor-interacting protein kinase 1 (RIPK1)- and\nHoechst 33258/propidium iodide double staining assay 3 (RIPK3)-dependent manner. RIPK1, RIPK3, and their substrate,\nCells encountering necroptosis usually exhibit classical necrosis- mixed-lineage kinase domain-like protein (MLKL), function as piv-\nlike morphological characteristics, including cell swelling and otal proteins in executing necroptosis and compose a necrosome\nmembrane rupture.37\u201339 To further investigate whether treatment complex.8\u201310 And the formation of functional necrosomes can be\nwith Ru2 could induce necroptosis, the Hoechst 33258/propidium promoted by ROS production.40 As mentioned above, necrostatin-\niodide (PI) double staining assay was performed with MGC-803 1, a potent RIP1 kinase inhibitor, attenuated the cell growth in-\ncells. The blue fluorescent Hoechst dyes are cell-permeable hibition rate at 1.5 \u03bcM Ru2 treatment in the mode of cell death\n\f10 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 9 Fluorescence microscopy images of MGC-803 cells untreated and treated with Ru2 (2 \u03bcM for 24 h), then stained with Hoechst 33258 and PI.\n\n\n\nexperiments. Therefore, immunoblotting was carried out to study of cytoplasmic vacuolation. Cell death pathway inhibition assays\nthe expression of RIP1, RIP3, and MLKL, as well as their phosphory- showed that Ru2-induced MGC-803 cell death was necroptosis\nlated proteins. The results (Fig. S11) indicated that treatment with and related to lysosomes. Furthermore, upregulation of the ex-\nRu2 in a series of concentrations (0, 0.5, 1, and 2 \u03bcM) for 24 h could pression levels of p-RIP1(Ser 166), p-RIP3(Ser 227), and p-MLKL (Ser\nupregulate the expressions of p-RIP1 Ser 166, p-RIP3, and p-MLKL. 358) was observed in western blot assays, indicating that Ru2-\nThese findings suggested that RIP1, RIP3, and MLKL were involved induced necroptosis was primarily modulated by the activation\nin the Ru2-induced necroptosis. In addition, the upregulation of of RIP1 and RIP3, which phosphorylated MLKL. In conclusion, Ru2\np- RIP3 at ser227 was confirmed by an immunofluorescence assay could act as a chemotherapeutic agent to kill cancer cells via\n(Fig. S12) in which the green fluorescence indicated the expression vacuolation-associated cell death and RIP1-dependent necropto-\nof p-RIP3 and the blue fluorescence located the nuclei. Compared sis. These dual mechanisms could be key factors for the high ac-\nwith the control, treatment with Ru2 (2 \u03bcM, 24 h) enhanced the tivity and overcoming cisplatin resistance of Ru2.\ngreen fluorescence. Together, our data showed that the formation\nof necrosomes containing RIP1 and RIP3 was a key regulator in\nRu2-induced necroptosis. Experimental\n Materials and instruments\nConclusion All chemicals in the synthetic experiments and the materials\n used for the biological assays were commercially available and\nIn this study, we designed and synthesized three UA-piperazine- used without further purification. The NMR spectra, HR-MS, HPLC,\ndithiocarbamate ruthenium(II) complexes Ru1 \u2212 Ru3 to screen and EA were measured on a BRUKER ESQUIRE HCT spectrome-\nas antitumor agents. MTT screening results showed that Ru1 ter, BRUKER ESQUIRE HCT spectrometer, shimazuLC-20A HPLC in-\nand Ru2 exhibited better antiproliferative effects than the clini- strument and PE2400II element analyzer, respectively.\ncally used cisplatin on all the selected cancer cell lines, including\nMGC-803, T24, HepG2, CNE2, MDA-MB-231, A549, and MCF-7 with\nIC50 values in the range of 0.73 \u00b1 0.12 to 3.25 \u00b1 0.50 \u03bcM. Ru3 Synthetic procedure of ligand (L) and its complexes Ru1\u2013Ru3\ndisplayed better inhibition than cisplatin against T24, MDA-MB- A mixture of UAP (10 mmol) and sodium hydroxide aqueous solu-\n231, and MCF-7 with IC50 values of 2.65 \u00b1 0.37, 5.46 \u00b1 0.32, and tion (10 mmol NaOH in 1 ml water) was stirred in 50 ml ethanol,\n14.63 \u00b1 0.12 \u03bcM, respectively. MGC-803 multicellular spheroids and excess carbon disulfide (100 mmol) was added dropwise\nand xenograft mouse models assays exhibited that Ru2 should be and reacted for 2 h. After the reaction, a white powder ligand\na promising effective antitumor. By the comparison of the antitu- (L) was obtained through vacuum evaporation and purified by\nmor activities of Ru1\u2013Ru3 with dehydroabietyl piperazine dithio- silica column chromatography using dichloromethane\u2013methanol\ncarbamate ruthenium(II) polypyridyl complexes in our previous (v: v = 250:3) solution as the eluent. The mixture of L (1 mmol),\nwork, respectively,24 it should be concluded that the replacement dipyridine ruthenium intermediate (1 mmol), tetramethyl ammo-\nof UA skeleton of dehydroabietyl group could lead to better anti- nium hydroxide aqueous solution (1 ml), and dichloromethane-\ntumor activity. In addition, Ru1\u2013Ru3 could overcome the cisplatin methanol solution (20 ml, volume ratio 2:1) was stirred and\nresistance by exhibiting antiproliferative activity on A549/DDP. We reacted at 65\u00b0C for 24 h. After the reaction, the red powder\nfound that representative Ru2 initially localized in the lysosomes complexes Ru1\u2013Ru3 were obtained through vacuum evaporation\nby energy-dependent endocytosis and subsequently escaped and and then purified by neutral alumina column chromatography\naccumulated in the mitochondria. This localization resulted in using dichloromethane\u2013methanol (v: v = 100:2) solution as the\nthe swelling and dilation of mitochondria, which was the origin eluent. The characterization data were listed as below.\n\f Paper | 11\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nFig. 10 Key HMBC (arrows) and 1 H-1 H COSY (bonds) correlations of Ru1\u2013Ru3.\n\n\n\n\n Ligand L: Yields 72.8%. 1 H NMR (500 MHz, DMSO-d6 ) \u03b4 5.30 (s, for: 645.3500, found: 645.3601; C35 H55 N2 NaO2 S2 [L-Na]\u2013 calcd for:\n1H, OH), 5.09 (s, 1H, 12-H), 4.35\u20133.51 (br, 9H, 3-H and 4 \u00d7 CH2 in 599.3705, found: 599.3714.\npiperazine), 3.04\u20132.94 (br, 1H), 2.35 (d, J = 10.5 Hz, 1H), 2.00 (dd, Complex Ru1: Yields 63.4%. 1 H NMR (500 MHz, DMSO-d6 ) \u03b4: 8.77\nJ = 69.7, 59.1 Hz, 6H), 1.57\u20131.13 (m, 14H), 1.03 (s, 3H, 27-CH3 ), 0.92 (d, J = 7.9 Hz, 2H, H-3\u0003 ,3\u0003\u0003 \u0003\u0003 ), 8.15 (t, J = 7.5 Hz, 2H, H- H-4\u0003 ,4\u0003\u0003 \u0003\u0003 ), 7.86\n(d, J = 5.1 Hz, 4H, 26-CH3 and CH), 0.88 (s, 3H, 25-CH3 ), 0.86\u20130.81 (m, (t, J = 7.6 Hz, 2H, H-5\u0003 ,5\u0003\u0003 \u0003\u0003 ), 9.51 (d, J = 4.9 Hz, 2H, H-6\u0003 ,6\u0003\u0003 \u0003\u0003 ), 8.67 (d,\n6H, 23-CH3 and 24-CH3 ), 0.66 (s, 3H, 29-CH3 ), 0.65 (s, 3H, 30-CH3 ) J = 7.9 Hz, 2H, H-3\u2019\u0003 , 3\u0003\u0003 \u0003 ), 7.91 (t, J = 7.6 Hz, 2H, H-4\u0003\u0003 ,4\u0003\u0003 \u0003 ), 7.31 (t,\nppm. 13 C NMR (126 MHz, DMSO-d6 ) \u03b4 194.45(C = S), 174.97(C = O), J = 7.6 Hz, 2H, H-5\u0003\u0003 ,5\u0003\u0003 \u0003 ), 7.66 (d, J = 4.8 Hz, 2H, H-6\u0003\u0003 ,6\u0003\u0003 \u0003 ), 3.00 (br\n139.00 (C = C), 124.85 (C = C), 77.30, 55.33, 51.40, 51.34, 49.50, 48.28, s, 1H, H-3), 4.34 (s, 1H, 3-OH), 1.82 (m, 4H, H-7a,11b,15a, 22b), 2.05\n47.57, 44.65, 44.64, 38.83, 38.70, 37.00, 33.95, 32.94, 30.49, 29.59, (m, 1H, H-11a), 5.06 (s, 1H, H-12), 2.32 (d, J = 10.4 Hz, 1H, H-18), 2.02\n29.35, 28.72, 27.47, 25.85, 23.37, 21.53, 18.45, 17.75, 16.80, 16.52, (s, 1H, H-22a), 1.20-1.44 (m, 19H), 0.89 (s, 6H, H-23,30), 0.66 (s, 3H,\n15.69 ppm. HRMS (m/z) (ESI): C35 H55 N2 NaO2 S2 [L + Na]+ calcd H-24), 0.64 (s, 3H, H-25), 0.83 (s, 6H, H-26,29), 1.02 (s, 3H, H-27), 3.84\n\f12 | Metallomics\n\n\n(br s, 4H, H-32,36), 3.58 (br s, 4H, H-33,35) ppm; 13 C NMR (125 MHz, Biological assays\nDMSO-d6 ) \u03b4: 36.5 (C-1), 27.0 (C-2), 76.8 (C-3), 38.2 (C-4), 54.8 (C-5), The materials, procedures, and instruments for the biological as-\n17.9 (C-6), 32.7 (C-7), 39.9 (C-8), 47.0 (C-9), 38.2 (C-10), 22.8 (C-11), says, including cell culture and transfections, electron microscopy,\n124.4 (C-12), 138.2 (C-13), 40.1 (C-14), 27.6 (C-15), 29.0 (C-16), 39.9 metal distribution, cellular uptake, lysosomal membrane perme-\n(C-17), 54.8 (C-18), 38.4 (C-19), 38.4 (C-20), 30.0 (C-21), 33.4 (C-22), abilization (acridine orange assay), intracellular ROS, immunoflu-\n28.2 (C-23), 16.1 (C-24), 15.2 (C-25), 16.4 (C-26), 23.6 (C-27), 174.6 orescence analysis, and western blot assays are depicted in the\n(C-28), 17.3 (C-29), 21.0 (C-30), 47.0 (C-32,36), 46.2 (C-33,35), 212.0 Supplementary Data (Part 1, \u2020 ESI).\n(C-37), 157.5 (C-2\u0003 ,2\u0003\u0003 \u0003\u0003 ), 123.6 (C-3\u0003 ,3\u0003\u0003 \u0003\u0003 ), 135.2 (C-4\u0003 ,4\u0003\u0003 \u0003\u0003 ), 127.0 (C-\n5\u0003 ,5\u0003\u0003 \u0003\u0003 ), 153.5 (C-6\u0003 ,6\u0003\u0003 \u0003\u0003 ), 157.8 (C-2\u0003\u0003 ,2\u0003\u0003 \u0003 ), 123.4 (C-3\u0003\u0003 ,3\u0003\u0003 \u0003 ), 135.9 (C-\n4\u0003\u0003 ,4\u0003\u0003 \u0003 ), 126.4 (C-5\u0003\u0003 ,5\u0003\u0003 \u0003 ), 151.0 (C-6\u0003\u0003 ,6\u0003\u0003 \u0003 ) ppm. The key HMBC and Supplementary material\n1\n H-1 H COSY correlations of Ru1 was shown in Fig. 10. Anal. Calcd.\n Supplementary data are available at Metallomics online.\nfor [C55 H71 N6 O2 RuS2 ]Cl: C, 62.98; H, 6.82; N, 8.01. Found: C, 62.91;\nH, 6.74; N, 8.10. HRMS (m/z) (ESI): (C55 H71 N6 O2 RuS2 + ) [M]+ calcd\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/10/mfac072/6712343 by guest on 12 May 2026\nfor: 1013.4123, found: 1013.4136. Funding\n Complex Ru2: Yields 64.1%. 1 H NMR (500 MHz, DMSO-d6 ) \u03b4: 8.81\n This study was supported by the Guangxi Natural Science\n(d, J = 8.2 Hz, 2H, H-4\u0003 ,4\u0003\u0003 \u0003\u0003 ), 8.26 (m, 2H, H-5\u0003 ,5\u0003\u0003 \u0003\u0003 ), 9.87 (d, J = 5.1 Hz,\n Foundation (Nos. 2020GXNSFFA325001, 2020GXNSFAA297167 and\n2H, H-6\u0003 ,6\u0003\u0003 \u0003\u0003 ), 8.49 (d, J = 8.1 Hz, 2H, H-4\u0003\u0003 ,4\u0003\u0003 \u0003 ), 7.52 (dd, J = 8.1, 5.3 Hz,\n 2016GXNSFAA380300), National Natural Science Foundation of\n2H, H-5\u0003\u0003 ,5\u0003\u0003 \u0003 ), 7.92 (d, J = 5.3 Hz, 2H, H-6\u0003\u0003 ,6\u0003\u0003 \u0003 ), 8.37 (d, J = 8.9 Hz,\n China (Nos. Nos. 82160657, 82104008 and 32060108), Guangxi New\n2H, H-3\u2019a,3\u0003\u0003 \u0003\u0003 a), 8.22 (m, 2H, H-3\u0003\u0003 a,3\u0003\u0003 \u0003 a), 3.00 (m, 1H, H-3), 4.34 (s,\n Century Ten, Hundred and Thousand Talents Project ([2017]42),\n1H, 3-OH), 1.82 (m, 4H, H-7a,11b,15a, 22b), 2.04 (m, 2H, H-11a,22a),\n Guangxi Science and Technology Base and Talents Program\n5.06 (s, 1H, H-12), 2.32 (d, J = 11.1 Hz, 1H, H-18), 1.20-1.44 (m,\n (AD20297059), Guangxi Key Laboratory of Agricultural Resources\n19H), 0.89 (s, 6H, H-23,30), 0.66 (s, 3H, H-24), 0.63 (s, 3H, H-25),\n Chemistry and Biotechnology (2019KF02), and State Key Labo-\n0.81 (s, 6H, H-26,29), 1.02 (s, 3H, H-27), 3.85 (br s, 4H, H-32,36), 3.59\n ratory for Chemistry and Molecular Engineering of Medicinal\n(br s, 4H, H-33,35) ppm; 13 C NMR (125 MHz, DMSO-d6) \u03b4: 36.5 (C-\n Resources (CMEMR2020-B05).\n1), 27.0 (C-2), 76.8 (C-3), 38.2 (C-4), 54.8 (C-5), 18.0 (C-6), 32.7 (C-7),\n40.0 (C-8), 48.0 (C-9), 38.2 (C-10), 22.9 (C-11), 124.4 (C-12), 138.6 (C-\n13), 40.1 (C-14), 27.6 (C-15), 29.0 (C-16), 40.0 (C-17), 54.8 (C-18), 38.4\n Data availability\n(C-19), 38.4 (C-20), 30.0 (C-21), 33.4 (C-22), 28.3 (C-23), 16.1 (C-24),\n15.2 (C-25), 16.4 (C-26), 24.0 (C-27), 174.6 (C-28), 17.3 (C-29), 21.0 (C- The data underlying this article will be shared on reasonable\n30), 47.0 (C-32,36), 46.2 (C-33,35), 212.2 (C-37), 148.5 (C-2\u0003 ,2\u0003\u0003 \u0003\u0003 ), 129.8 request to the corresponding author.\n(C-3\u0003 ,3\u0003\u0003 \u0003\u0003 ), 134.3 (C-4\u0003 ,4\u0003\u0003 \u0003\u0003 ), 126.0 (C-5\u0003 ,5\u0003\u0003 \u0003\u0003 ), 154.9 (C-6\u0003 ,6\u0003\u0003 \u0003\u0003 ), 148.1 (C-\n2\u0003\u0003 ,2\u0003\u0003 \u0003 ), 130.1 (C-3\u0003\u0003 ,3\u0003\u0003 \u0003 ), 134.8 (C-4\u0003\u0003 ,4\u0003\u0003 \u0003 ), 125.1 (C-5\u0003\u0003 ,5\u0003\u0003 \u0003 ), 152.0 (C-\n6\u0003\u0003 ,6\u0003\u0003 \u0003 ), 127.9 (C-3\u2019a,3\u0003\u0003 \u0003\u0003 a), 127.4 (C-3\u0003\u0003 a,3\u0003\u0003 \u0003 a) ppm. The key HMBC\n Conflicts of interest\nand 1 H-1 H COSY correlations of Ru2 was shown in Fig. 10. Anal. There are no conflicts to declare.\nCalcd. for [C59 H71 N6 O2 RuS2 ]Cl: C, 64.60; H, 6.52; N, 7.66. Found: C,\n64.53; H, 6.59; N, 7.74. HRMS (m/z) (ESI): (C59 H71 N6 O2 RuS2 + )[M]+\ncalcd for: 1061.4123, found:1061.4137. References\n Complex Ru3: Yields 64.1%. 1 H NMR (500 MHz, DMSO-d6 ) \u03b4: 1. G. Szakacs, J. K. Paterson, J. A. Ludwig, C. Booth-Genthe and M. M.\n8.26 (m, 2H, H-5\u0003 ,5\u0003\u0003 \u0003\u0003 ), 10.01 (d, J = 5.3 Hz, 2H, H-6\u0003 ,6\u0003\u0003 \u0003\u0003 ), 7.82 (m, Gottesman, Targeting multidrug resistance in cancer, Nat. 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