Novel NHC-coordinated ruthenium(II) arene complexes achieve synergistic efficacy as safe and effective anticancer therapeutics.

European Journal of Medicinal Chemistry 203 (2020) 112605 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech Research paper Novel NHC-coordinated ruthenium(II) arene complexes achieve synergistic efﬁcacy as safe and effective anticancer therapeutics Chao Chen a, b, Chang Xu a, Tongyu Li a, Siming Lu c, Fangzhou Luo a, Hangxiang Wang a, * a The First Afﬁliated Hospital, Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, School of Medicine, Zhejiang University, Hangzhou, 310003, PR China b College of Life Sciences, Huzhou University, Huzhou, 313000, PR China c Department of Laboratory Medicine, The First Afﬁliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, PR China a r t i c l e i n f o a b s t r a c t Article history: Received 31 March 2020 Received in revised form 16 June 2020 Accepted 18 June 2020 Available online 12 July 2020 There is an urgent need for more effective, less toxic cancer therapy agents. Motivated by this need, we synthesized a small panel of N-heterocyclic carbene (NHC)-coordinated ruthenium(II) arene complexes Ru1eRu6 with the formula [Ru(p-cymene)(L)Cl]PF6 (L ¼ NHC ligand with varying substituents). Cellbased in vitro studies revealed that despite the structural similarity, Ru1eRu6 exhibited distinct cytotoxic activities against cancer cells. In particular, Ru4 and Ru6, which bear n-octyl and pentamethylbenzyl motifs, respectively, were the most active at inducing apoptosis. In human ovarian A2780 cancer cells, Ru4 and Ru6 showed the highest cytotoxicities with IC50 values of 2.74 ± 0.15 mM and 1.98 ± 0.10 mM, respectively, and they were approximately 2-fold more potent than cisplatin (IC50 ¼ 5.55 ± 0.37 mM). In addition to the cell killing capacity, inhibition of cell migration was validated by using these two optimized complexes. Mechanistic studies revealed that Ru4 and Ru6 complexes induced apoptosis in a caspase-dependent manner, primarily through intracellular reactive oxygen species (ROS) overproduction and cell cycle arrest at G1 phase. Furthermore, in a preclinical metastatic model of A2780 tumor xenograft, administration of Ru4 and Ru6 (20 mmol/kg) resulted in a marked inhibition of tumor progression and metastasis. Finally, a substantially alleviated systemic toxicity was observed for both complexes in comparison with cisplatin in animals. Overall, this study greatly increases our understanding of NHC-coordinated Ru(II) arene metallodrugs, aiding further investigation of their therapeutic potential in the treatment of metastatic cancers. © 2020 Elsevier Masson SAS. All rights reserved. Keywords: N-heterocyclic carbene Ruthenium(II) complex Metallodrugs Anticancer activity Anti-Metastasis 1. Introduction Cancer remains one of the deadliest diseases worldwide, and it constantly evolves to resist and adapt to therapeutic insults [1]. Since the discovery of cisplatin [2], platinum-based chemotherapeutic agents have been widely used as standard-of-care drugs for the treatment of many types of cancer in the clinic [3]. However, dose-limiting side effects (e.g., nephrotoxicity and hepatotoxicity) and intrinsic or acquired drug resistance greatly limit the efﬁcacy of platinum drugs [4]. These intrinsic drawbacks stress the need to develop more effective and less toxic anticancer therapies. In line with this need, a variety of nonplatinum metal complexes have * Corresponding author. The First Afﬁliated Hospital, School of Medicine, Zhejiang University, 79, Qingchun Road, Hangzhou, 310003, China. E-mail address: wanghx@zju.edu.cn (H. Wang). https://doi.org/10.1016/j.ejmech.2020.112605 0223-5234/© 2020 Elsevier Masson SAS. All rights reserved. been designed and investigated, some of which have entered clinical trials [5]. Among various types of metallodrugs, ruthenium compounds have attracted signiﬁcant interest as alternative anticancer drug candidates [6]. Ruthenium possesses similar physiochemical properties to the iron family in the periodic table, which confers the ability to bind various biological macromolecules with less impact on normal cells. More importantly, ruthenium complexes also have low systemic toxicity [7,8], high antitumor activity, and anti-metastatic or anti-angiogenic properties [9e11]. For example, low-toxic ruthenium complexes [imiH]trans-[Ru(Nimi)(S-dmso)Cl4] (NAMI-A) [12] and [Na]trans-[Ru(N-ind)2Cl4] (NKP1339) [13] effectively inhibit tumor metastasis and angiogenesis, and both of them have entered clinical trials. Unfortunately, metallodrugs that can be used as successfully as platinum drugs are still lacking [14]. In the continuing search for effective anticancer ruthenium-based agents, half-sandwich ruthenium(II) (Ru(II)) arene complexes 2 C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 coordinated with various coligands have attracted a surge of attention [15]. In these complexes, the arene ligands are relatively inert towards substitutions, which stabilizes the low oxidation state of Ru(II) complexes under physiological conditions. Consequently, Ru(II) arene complexes can be adjusted to have high hydrophobicity and are also kinetically similar to platinum(II) complexes [16]. Serving as coligands, mono- or bidentate ligands containing different donor atoms have been incorporated to achieve high stability and concurrently, enhance pharmacologic potency [17e20]. Meanwhile, the size and hydrophobicity of arene substituents and the bulky structures of coligands also affect the biological activity of Ru(II) arene complexes [21e25]. Therefore, the structural variability makes these halfsandwich scaffold complexes ideal for construction of pharmacologically active agents. Additionally, unlike platinum-based drugs that exert cytotoxicity primarily through DNA damage, Ru(II) arene complexes usually show multiple mechanisms on cancer cells [26e29] and may potentially address cisplatin resistance by combining synergistic effects [30]. In the ongoing exploration of Ru(II) arene complexes, we constructed novel Ru(II) arene complexes that were coordinated with N-heterocyclic carbene (NHC) ligands and explored their efﬁcacy as metallodrugs against cancer cells. NHC is a class of ligands with strong coordination ability, which makes them sufﬁciently stable under harsh physiological conditions [31]. The structure of NHC ligands can vary widely for metallodrug optimization. Hartinger and Liu et al. have described some pyridyl-NHC [32] and imineNHC [33] coordination Ru(II) arene complexes in which the introduction of different substituents on the NHC ligands produced metal complexes with extremely different anticancer activities. These results suggest that substituent variation of the NHC ligands can be used to generate effective Ru(II) arene complexes. In particular, by exploiting the NHC ligands with biologically active heterocyclic groups, the anticancer synergy of designed metallodrugs could be anticipated by targeting different intracellular biomolecules. In this study, a small panel of benzothiazole-functionalized NHCeRu(II) arene complexes (i.e., Ru1eRu6) with altering substituents were synthesized. The substituents with the planar benzothiazole group may facilitate the intercalation of metallodrugs with DNA through p-p stacking, which potentially increases the drug activity. In vitro cytotoxicity studies showed that Ru4 with noctyl and Ru6 with pentamethylbenzyl displayed the most potent cytotoxicity. Compared with clinically used cisplatin at the same doses, Ru4 and Ru6 were less toxic in animals. In a preclinical metastatic model of human ovarian A2780 xenograft, administration of Ru4 and Ru6 resulted in a marked inhibition of tumor progression and metastasis. These data provide compelling evidence that these novel NHCeRu(II) arene complexes have great therapeutic potential for the treatment of cancers with metastases. Scheme 1. Synthetic scheme for benzothiazole functionalized N-heterocyclic carbene (NHC) ligands and the corresponding RueNHC complexes (Ru1eRu6). acetonitrile conveniently yielded NHCeRu(II) arene complexes (Ru1eRu6). The structures of NHCeRu(II) arene complexes were fully characterized by NMR spectra (e.g., 1H and 13C NMR) and elemental analysis. In the NMR spectra, disappearance of the imidazolium C2eH protons at approximately 10.0 ppm and concurrent appearance of the p-cymene protons conﬁrmed the formation of pcymene-coordinated NHCeRu(II) complexes. Furthermore, the typical downﬁeld carbon signal of Ru-carbene at approximately 188 ppm also veriﬁed the formation of the desired ﬁnal metal complexes. Additionally, as a representative of these RueNHC complexes, the structure of Ru2 was further conﬁrmed by X-ray single-crystal diffraction analysis. A single crystal of Ru2 was obtained by slow diffusion of ether into its acetonitrile solution. As expected, Ru2 adopts a typical three-legged piano stool structure (Fig. 1). The center Ru ion is hex-coordinated with one h6-coordinated p-cymene group, one carbon atom of imidazolylidene ligand, one nitrogen atom of benzothiazole, and one chloride. Similar to other thiazolefunctionalized NHC compounds, the sulfur atom on the thiazole ring is away from the coordinated center. The bond angles between the three piano legs NeRueC (76.09 ), CeRueCl (84.27 ) and NeRueCl (87.37 ) in Ru2 are all within the normal range of similar ruthenium-NHC complexes with a p-cymene group. Due to the strong coordination ability of imidazolylidene, the bond length of RueC (2.030 Å) is shorter than that of RueN (2.131 Å) and RueCl (2.384 Å). It is worth noting that the rings of the imidazolylidene and the benzothiazole in Ru2 are approximately coplanar with the small dihedral angle of 11.05 . 2. Results and discussion 2.1. Synthesis and characterization of N-heterocyclic carbene (NHC)-functionalized Ru(II) arene complexes Incorporating functional substituents into the NHC moiety is an effective way to construct intriguingly organometallic complexes, and this approach effectively modulates the biological activity of metallodrugs [34]. Inspired by this rationale, we herein designed and synthesized a small library of NHC-functionalized Ru(II) arene complexes bearing a rigid, planar benzothiazole motif (Scheme 1). The NHC precursors (HL(PF6), L1-L6) were constructed by reacting 2-chlorobenzothiazole with various N-substituted imidazole derivatives. Subsequently, the reaction of the imidazole salts HL(PF6) (L1-L6) with Ag2O followed by reaction with [Ru(p-cymene)Cl2]2 in Fig. 1. ORTEP drawing of Ru2 showing atomic numbering scheme at 50% probability ellipsoids. Selected bond lengths (Å) and angles(deg): Ru(1)eC(8) 2.030(4), Ru(1)eN(1) 2.131(3), Ru(1)eCl(1) 2.384(12), Ru(1)eC(19) 2.174(4), Ru(1)eC(17) 2.182(4), Ru(1)e C(20) 2.191(4), Ru(1)eC(18) 2.212(4), Ru(1)eC(16) 2.271(4), Ru(1)eC(15) 2.285(4), C(8)-Ru(1)-N(1) 76.09(14), C(8)-Ru(1)-Cl(1) 84.27(11), N(1)-Ru(1)-Cl(1) 87.37(9). C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 2.2. Examination of in vitro cytotoxicity Having these compounds in hand, we ﬁrst evaluated their in vitro cytotoxic potentials against six distinct human cancer cell lines, including nonsmall cell lung cancer A549 and HT-29, colon cancer HCT-116 and LoVo, cervical cancer HeLa, and ovarian cancer A2780 cells. The clinically used cisplatin was included as a control. As shown in Table 1 and Fig. S1, although these RueNHC complexes are structurally similar, their cytotoxicity varies greatly. Ru1eRu3 complexes with short alkyl substituents (methyl, isopropyl, and butyl, respectively) showed negligible activities with IC50 values larger than 100 mM in all tested cancer cells. However, the Ru4 complex bearing n-octyl substituent was effective against cancer cells, especially in HT-29 and A2780 cells with IC50 values of 8.51 ± 0.69 and 2.74 ± 0.15 mM, respectively. Obviously, the length of the alkyl substituents had a signiﬁcant effect on the cytotoxicity of these RueNHC complexes. Additionally, the potency of Ru5 and Ru6 complexes was modulated by the peripheral substituent groups. Ru5 with a benzyl group was less active in A549 cells and showed moderate activity in HCT-116, LoVo, and HeLa cells. In sharp contrast, Ru6 bearing a pentamethylbenzyl group exhibited distinguishingly high cytotoxicity against all cancer cells, even greater than that of cisplatin in A2780 cells (e.g., IC50 value was approximately 2.5-fold lower than cisplatin). Collectively, the order of in vitro cytotoxicity for these complexes is Ru6 > Ru4 > Ru5 > Ru1, Ru2, Ru3. These results provided an implication that small modiﬁcations the substituent of metal complexes greatly affect their biological activity. A previous study indicated that lipophilicity of metallodrugs plays a vital role in their antitumor activities [35]. Therefore, we determined the oil-water distribution coefﬁcients (log Po/w) of these complexes using the "shaking ﬂask" method. As shown in Table 1, the log Po/w values of for these complexes ranged from 0.51 to 0.92. Of note, the Ru4 and Ru6 complexes showed high lipophilicity, which makes both compounds more likely to penetrate the cell membrane. To further elucidate the substituent-activity relationship, cellular uptake of complexes was measured with Ru1, Ru2, Ru4, and Ru6 as model complexes. After 3 or 6 h of incubation, the metal concentrations in A2780 cells were analyzed by inductively coupled plasma mass (ICP-MS) spectrometry. As shown in Fig. S2, compared with the Ru1 and Ru2 treatment, exposure of cells to the Ru4 and Ru6 complexes resulted in signiﬁcantly higher accumulation in A2780 cells. Collectively, these results provide evidence that the superior cytotoxicity of Ru4 and Ru6 is associated with substituents of longer alkyl and polysubstituted aryl moieties, which eventually increase the lipophilicity of the entire NHCeRu(II) arene complex, cellular uptake and thereby, cytotoxic potency. Upon system administration, the complexes will encounter serum proteins and noncovalent interactions occur, which may reduce the drug activity. Therefore, we monitored the UVevis spectral changes of the Ru4 and Ru6 complexes when incubated in fetal bovine serum (FBS) with 10% (v/v) dimethyl sulfoxide 3 (DMSO). As a result, the spectra of both complexes showed negligible changes after incubation for two days, indicating that they are potentially stable during systemic circulation (Fig. S3). Additionally, we incubated the two complexes with mouse serum at 37 C for two days and further tested the cytotoxic activity in A2780 cells by the MTT assay. The results suggested that pre-incubation with mouse serum did not compromise their activity (IC50 ¼ 3.24 ± 0.17 mM for Ru4 and 2.64 ± 0.13 mM for Ru6) (Fig. S4). Thus, these data showed that the Ru4 and Ru6 complexes maintain stability under complicated biological conditions. 2.3. Anti-proliferation activity Having identiﬁed that Ru4 and Ru6 are promising chemotherapeutic agents, we further investigated their anti-proliferation activity in A2780 cells using the EdU (5-ethynyl-20 -deoxyuridine) incorporation assay. EdU is an alkyne-tagged thymine nucleoside derivative that can be incorporated into replicating DNA during cell proliferation. Subsequently, the EdU moiety can be labeled with a green ﬂuorescent dye by a click reaction and used to detect DNA replication activity. Compared with the untreated cells in which the average proliferation ratio was 53%, cisplatin (2 mM) slightly decreased the proliferation ratio to 49% (Fig. 2). After treatment with Ru4 or Ru6 (2 mM) for 24 h, the average proliferation ratio of A2780 cells signiﬁcantly decreased to 38% and 37%, respectively. Furthermore, when the drug concentrations increased to 4 mM, the proliferation ratios dramatically decreased to 15% and 16% for Ru4and Ru6-treated cells, respectively, which were signiﬁcantly lower than those for 4 mM cisplatin-treated cells (34%). These results showed that these Ru(II) arene metallodrugs effectively impede cancer cell proliferation in a dose-dependent manner. 2.4. Anti-migration assay In addition to the anti-proliferation activity, the ability to inhibit cancer cell migration is important for chemotherapeutic drugs in the treatment of metastatic cancer. Previous studies have shown that ruthenium complexes with aromatic groups such as Ru(II)acylthiourea [36] and Ru(II)-pcymene-amino oxime [37] exhibit high inhibitory effects on the migration of cancer cells. Therefore, we next explored the anti-migration capability of these Ru(II) arene complexes in A2780 cancer cells using a wound-healing assay. For this purpose, monolayer cells plated in 12-well plates were scratched and washed with PBS. Then, the complexes at different concentrations in FBS-free medium were added to suppress cell migration. As shown in Fig. 3, the wound healing of A2780 cancer cells was markedly reduced after 24 h treatment with Ru(II) complexes (4 mM) compared to untreated cells and cisplatin-treated cells. For example, the mean wound closure ratio of A2780 was 33.3% in untreated cells, whereas treatment with Ru4 or Ru6 dramatically reduced the migration ratios to 11.7% and 8.7%, Table 1 IC50 values (mM) of Ru1eRu6 complexes and cisplatin (cis-Pt) against a small panel of human cancer cell lines after 48 h of drug exposure. The cell viability was determined by the MTT assay. The data are presented as the means ± SD (n ¼ 4). The log Po/w values were determined via the "shaking ﬂask" method against n-octanol/water (1:1, v/v) partition. Cell line Ru1 Ru2 Ru3 Ru4 Ru5 Ru6 cis-Pt A549 HT-29 HCT-116 LoVo Hela A2780 log PO/W >100 >100 >100 >100 >100 >100 0.52 >100 >100 >100 >100 >100 84.1 ± 5.24 0.51 >100 >100 >100 >100 >100 >100 0.54 24.6 ± 2.36 5.20 ± 0.31 25.6 ± 1.67 19.4 ± 0.58 39.1 ± 1.35 2.74 ± 0.15 0.83 >100 8.51 ± 0.69 43.5 ± 2.54 56.8 ± 4.78 55.5 ± 1.04 6.61 ± 0.37 0.77 19.6 ± 1.47 3.17 ± 0.22 12.8 ± 0.68 11.9 ± 0.31 23.0 ± 0.46 1.98 ± 0.10 0.92 16.6 ± 0.75 8.19 ± 0.35 6.74 ± 0.45 8.58 ± 0.36 10.3 ± 1.13 5.55 ± 0.37 e 4 C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 Fig. 2. Anti-proliferation activity of ruthenium complexes Ru4 and Ru6 and cisplatin (cis-Pt). A2780 cells were treated with drugs for 24 h and then analyzed by EdU incorporation assay. Proliferating cells were shown in green and bright blue, and quiescent, nondividing cells were shown in blue. The data are presented as the means ± SD; n > 5 regions with a total of 1500e2000 cells analyzed, ***p < 0.001. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the Web version of this article.) Fig. 3. Wound-healing assay performed on A2780 cells. Cells were treated with Ru4, Ru6, or cisplatin at concentrations of 2 or 4 mM for 24 h. The data are presented as the means ± SD (n ¼ 3), ***p < 0.001. C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 respectively. In contrast, cisplatin showed negligible activity at preventing cell migration. Additionally, a dose-dependent activity to inhibit migration of cancer cells was observed using both the Ru4 and Ru6 complex, which is consistent with results observed in antiproliferative assays. 2.5. NHCeRu(II) arene complexes induce apoptosis in cancer cells The results of cell-based in vitro experiments clearly evidenced that Ru4 and Ru6 had superior antitumor activities among others in cancer cells. To further explore anticancer activity, we investigated how these complexes evoked cell death. Morphological change of cells is a preliminary indicator of cells responding to cytotoxic agents. After exposing A2780 cells to Ru4, Ru6, or cisplatin for 24 h, the cells were stained with AO/EB dyes and observed under ﬂuorescence microscopy. AO penetrates all cells and emits green ﬂuorescence, whereas EB only passes through broken cell membranes and interacts with DNA, emitting orange ﬂuorescence [38]. As shown in Fig. 4, healthy A2780 cells showed a fusiform structure with green ﬂuorescence. Treatment with 4 mM cisplatin produced a negligible effect observed in AO/EB staining. This may be attributable to A2780 cells overcoming the cisplatin effect at low concentrations over a short duration by cell protection mechanisms. However, after 24 h of treatment with Ru4 or Ru6 at 4 mM, the cell morphology was rounded and cells displayed typical condensed orange chromatin. This image indicated that the cell protection mechanisms of A2780 had been disturbed under the action of Ru4 or Ru6, and EB had entered the cells. Furthermore, cell rounding is usually a sign of apoptosis. Thus, the AO/EB assay suggests that NHCeRu(II) arene complexes (e.g., Ru4 and Ru6) present here disrupt cell membrane integrity and evoke cell death. To further clarify the mode-of-action of these complexes, the Annexin V-FITC/PI apoptosis assay was conducted in A2780 cells and analyzed by ﬂow cytometry. Annexin V-FITC is a ﬂuorescently labeled phospholipid-binding protein, which can bind to phosphatidylserine in early apoptotic cells. PI cannot penetrate normal cells and early apoptotic cells, but it can penetrate late apoptotic and necrotic cells and stain the nucleus. Thus, the combined use of two dyes can distinguish cells at different stages. After treatment with cisplatin and Ru4 and Ru6 complexes (4 mM) for 48 h, the total 5 populations of early apoptotic cells increased to 37.4%, 78.2%, and 84.3%, respectively (Fig. 5a and 5b). In sharp contrast, there were few late apoptotic and necrotic cells (PI-positive) observed, with the populations less than 8.5% (Fig. 5a). We further performed Western blot analysis to verify the mechanism of Ru4 and Ru6 in inducing cell death. Treatment of Ru4 and Ru6 increased the expression of several key apoptosis-associated proteins [39] such as cleaved PARP (c-PARP), cleaved caspase-3 and caspase-9, which was consistent with observations in cells treated with cisplatin (Fig. 5c). Thus, our results clearly demonstrated that Ru4 and Ru6 exert their anticancer activity primarily via the apoptotic pathway. 2.6. Mechanistic studies of NHCeRu(II) arene complexes against cancer cells Next, the mode of action using the Ru4 and Ru6 complexes against cancer cells was investigated. Previous studies have demonstrated that the main cytotoxic activity of metal complexes against cancer cells is caused by overproduction of intracellular reactive oxygen species (ROS) [40]. Therefore, we ﬁrst measured ROS production in A2780 cells using a ROS indicator, 6-carboxy20 ,70 -dichlorodihydroﬂuorescein diacetate (DCFH-DA). Upon uptake by cells, DCFH-DA can be hydrolyzed and oxidized to produce green ﬂuorescence in the presence of ROS. Thus, the ﬂuorescence emission correlates with the level of ROS in cells. As shown in Fig. 6a, after treatment with Ru4 or Ru6 at 4 mM for 12 h, A2780 cells displayed a higher green ﬂuorescence signal than untreated cells and cisplatin-treated cells. The ﬂow cytometry analysis also showed that cells treated with the metal complexes overproduced ROS, resulting in increased ﬂuorescence intensity (Fig. 6b). As is well known, mitochondria are the main organelle for ROS production. During mitochondrial respiration, a small number of electrons leak out of the mitochondrial electron transport chain and encounter O2 to generate ROS. Therefore, overproduction of ROS indicates that mitochondrial respiration has been disrupted. To conﬁrm the role of mitochondria in ROS production, we used a well-established mitochondria indicator, JC-1 (5,50 ,6,60 -tetrachloro1,10,3,30 -tetraethylbenzimidazolyl carbocyanine iodide), to study the changes of mitochondrial membrane potential in A2780 cells. As shown in Fig. 6c, the JC-1 dye labeled the mitochondrial matrix Fig. 4. Representative images of AO/EB dual staining of untreated, Ru4, Ru6, and cisplatin (4 mM) A2780 cells after 24 h of treatment. 6 C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 Fig. 5. (a, b) Apoptosis in response to treatment with Ru4, Ru6, and cisplatin (4 mM, 48 h) was determined by Annexin V/PI double-staining assay. (c) Western blot analysis to examine the expression of apoptosis-associated proteins in A2780 cells after treatment with Ru4, Ru6, and cisplatin (4 mM) for 24 h. of untreated normal cells and formed orange-emitting aggregates. When the mitochondrial membrane potential is destroyed, JC-1 dye will not be able to accumulate in the mitochondria and emit green ﬂuorescence. As expected, after 24 h of treatment with Ru4 or Ru6, A2780 cells emitted green ﬂuorescence with reduced orange ﬂuorescence, suggesting mitochondrial dysfunction induced by the NHCeRu(II) arene complexes. This result was further supported by ﬂow cytometry analysis (Fig. 6d). Treatment of A2780 cells with Ru complexes signiﬁcantly increased the green ﬂuorescence ratio of JC-1 from 7.64% (untreated cells) to 44.41% (Ru4-treated cells) and 50.63% (Ru6-treated cells), respectively. However, the effect of cisplatin is very minor, as green ﬂuorescence signal only increased to 18.48% after 24 h treatment with the same drug concentration. Considering these results, we speculate on the potential mode of action causing cell death. Negatively charged mitochondria could be the major target of Ru4 and Ru6 in cells. Upon uptake by cancer cells, the cationic moiety of the complexes could interact with mitochondria, and then depolarize the mitochondrial membrane potential. This could further disrupt cell respiration and produce excess ROS. Eventually, oxidative stress could cause signiﬁcant damage to various intracellular (bio)molecules, thereby affecting their normal biochemical functions and leading to apoptosis. As shown in previous studies [39,41], cell cycle arrest is another major cause of apoptosis. We therefore examined the effect of Ru4 and Ru6 on cycle distribution of A2780 cells, and cisplatin was used as a control. After 24 h of treatment with Ru4, Ru6, and cisplatin at 4 mM, cells were harvested and analyzed by ﬂow cytometry (Fig. 7). As expected, the untreated cells showed a normal cycle distribution, with the mean population percentages of G1 (50.88%), S (28.68%) and G2/M (20.43%). The cisplatin-treated cells showed a slightly decreased population in G1 (43.71%), increased population in S (36.66%), and approximately equal population in G2/M (19.62%). This could be explained by cisplatin exerting its anticancer activity mainly through DNA interactions (blocking in S phase). Ru4 and Ru6 showed G1 phase arrest activity. After exposure to Ru4 or Ru6 for 24 h, A2780 cells displayed a signiﬁcant increase in G1 phase, with mean population percentages of 70.16% and 74.74%, respectively (Fig. 7). G1 phase is the presynthesis phase of DNA. When cells are arrested in this phase, progression from G1 to S and G2/M phase slows down and eventually leads to cell swelling and apoptosis. Moreover, G1 phase arrest of these Ru(II) complexes indicates that interaction with DNA is not the main anticancer mechanism of Ru4 and Ru6, although they had multiple modes of action in the cells. Hence, the distinct mechanism endowed by these complexes makes them useful anticancer drug candidates for the treatment of cancers with cisplatin resistance. 2.7. In vivo systemic toxicity Intrigued by the in vitro potential including potent cytotoxic and anti-migration activity, we aimed to assess the antitumor activity of Ru4 and Ru6 in animals. Prior to therapeutic studies, the in vivo toxicity of both complexes was investigated in ICR mice. For this purpose, the compounds were dissolved in DMSO/saline (1:1, v/v) solution and intraperitoneally injected into healthy ICR mice (n ¼ 8 in each group) at different doses (10 and 20 mmol/kg). Cisplatin was also administered as a reference. Following ﬁve injections, the changes in body weight of these mice were recorded to assess the drug toxicity (Fig. 8a). When the body weight loss of the mice exceeds 20%, we consider the administered drugs highly toxic. Previous studies have shown that cisplatin can cause a signiﬁcant reduction in the body weight of mice [42]. Five injections of cisplatin at a dose of 10 mmol/kg caused six out of eight mice to lose more than 20% of their body weight in 15 days. Encouragingly, Ru4 and Ru6 complexes were well tolerated in animals at this dose. For instance, the mice treated with Ru4 or Ru6 at doses of 10 mmol/kg did not show body weight loss. We further increased the dose of the drugs to 20 mmol/kg for ﬁve intraperitoneal injections. As shown in Fig. 8a, the mice still showed considerable tolerance to the high doses of the Ru(II) complexes. Only two Ru4- and one Ru6-treated mice lost over 20% of body weight after ﬁve successive injections. In sharp contrast, all mice injected with cisplatin at a dose of 20 mmol/ kg lost more than 20% of their body weights within 15 days. These results provided compelling evidence that NHCeRu(II) arene C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 7 Fig. 6. (a) Representative images of DCFH-DA-stained A2780 cells after treatment with Ru4, Ru6, and cisplatin (4 mM) for 12 h. (b) Representative histograms displaying ROS production as determined by ﬂow cytometry analysis. The cells were treated with Ru4, Ru6, and cisplatin (4 mM) for 12 h. (c) Fluorescence microscopy images of JC-1 staining after A2780 cells were treated with Ru4, Ru6, and cisplatin (4 mM) of 24 h. (d) Changes in the mitochondrial membrane potential in A2780 cells after 24 h. 2.8. Therapeutic efﬁcacy in a preclinical metastatic model of human A2780 cancer Fig. 7. Flow cytometry analysis of cell cycle distribution. A2870 cancer cells were exposed to Ru4, Ru6, and cisplatin (4 mM) for 24 h. The data are presented as the means ± SD (n ¼ 3). complexes possess higher safety margins compared to the clinically used cisplatin agent. Though the mechanisms underlying the low toxicity remain unclear, we reasonably envision that stable coordination of ruthenium metal to NHC ligands contributes to the reduced toxicity of these Ru(II) complexes, which deserves further investigation. Encouraged by reduced drug toxicity in animals, we therefore evaluated the in vivo anticancer efﬁcacy of Ru(II) arene complexes in comparison with cisplatin in Balb/c nude mice bearing A2780 cell-derived ovarian cancer. Each mouse in treatment groups (n ¼ 8 in each group) was intraperitoneally injected with 200 mL of A2780 cells suspension (5 107 cells/mL in PBS). Starting the next day, we initiated therapy by intraperitoneally administering drugs in 200 mL of DMSO/saline (1:1, v/v) solution. Body weights were recorded during the experiment, and tumors and ovaries were collected for evaluation at the end of the study. To avoid potential toxicity, drug administration was reduced to three times on day 0, 3, and 6. Thus, the body weight of the mice treated with Ru4 and Ru6 decreased slightly in the initial days but immediately rebounded after cessation of treatment (Fig. 8b). However, cisplatin-treated mice showed substantial weight loss (above 20%). This result again conﬁrmed that at the same doses, Ru4 and Ru6 were less toxic than cisplatin in mice. Administration of Ru4 and Ru6 at the dose of 20 mmol/kg yielded mean tumor growth inhibition (TGI) rates of 65.2%, and 68.7%, respectively (Fig. 8a and 8b). Compared with cisplatin treatment with a TGI rate of 36.7%, Ru4 and Ru6 treatments showed superior antitumor efﬁcacy. More 8 C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 Fig. 8. (a) Survival of healthy ICR mice following intraperitoneal injection of saline, cisplatin, Ru4, and Ru6 for ﬁve successive times every other day. The mice were deﬁned as dead when body weight loss exceeded 20%. Arrows indicate intraperitoneal injections. (b) Body weights of nude mice were measured to evaluate drug toxicity. (c) Photographs of tumorbearing nude mice after treatments. (d) Total tumor weight excised from the mice after different treatments and in each group at the end of the study. (e, f) Representative images of tumors and ovaries. Healthy mice without tumor cell implantation were used as controls, and the data are presented as the means ± SD (n ¼ 8 in each group); **p < 0.01, and ***p < 0.001. signiﬁcantly, Ru4 and Ru6 suppressed metastasis of cancer cells to ovaries. The ovary in Ru(II) complex-treated mice had normal morphology; however, cancer cells clearly migrated to the ovaries and uterus in DMSO/saline-treated mice (Fig. 8f). Metastasis accounts for the majority of cancer-associated death in patients, and it remains a signiﬁcant therapeutic challenge. The compounds Fig. 9. Representative H&E staining of the major tissues (e.g., heart, liver, spleen, lung, and kidney) excised from the different treatment groups. C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 presented here not only suppressed the growth of bulky primary tumors but also reduced the metastatic burden to other organs. Therefore, such multifunctional agents could greatly beneﬁt the long-term survival of patients in future clinical use. Previous studies have suggested that nephrotoxicity and hepatotoxicity are the major side effects for the clinic use of the cisplatin drug [43]. Cases of pulmonary toxicity have also been reported [44]. We hypothesized that Ru4 and Ru6 were able to reduce organ toxicity in animals. Following administration of drugs, the main organs (e.g., heart, liver, spleen, lung, and kidney) from each group were excised and subjected to histological analysis (Fig. 9). In cisplatin-treated mice, we found that hepatocytes exhibited extensive inﬂammatory response, dissolution of cytoplasm, and necrosis. The livers of Ru4- and Ru6-treated mice presented the similar pathological characteristics with that of saline-treated mice. In kidneys, cisplatin treatment resulted in the collapse of glomeruli. Encouragingly, no nephrotoxicity was observed in Ru4- and Ru6treated mice. In cisplatin-treated mice, the lung displayed local inﬂammation with the diffused damage of pulmonary alveoli. Ru4and Ru6-treated mice showed similar pathology but less severe. Additionally, the morphology of other tissues (e.g., heart and spleen) in drug-treated mice are similar to that of the saline-treated mice. Together, these in vivo studies revealed that Ru4 and Ru6 can effectively inhibit tumor growth and metastasis and have good tolerability in mice, as evidenced by this metastatic A2780 tumor model. 3. Conclusion In summary, we synthesized and characterized a series of promising Ru(II) arene complexes (Ru1eRu6) bearing benzothiazolefunctionalized NHC ligands. The in vitro cell-based studies demonstrated that Ru4 and Ru6 complexes are potent anticancer agents across distinct cancer cell types. Given the intrinsic feature of NHCeRu(II) arene complexes in circumventing cisplatin resistance and their synergistic modes of action and low toxicity [31,33,45,46], these new NHCeRu(II) arene complexes may be of potential use in cancer patients with impaired renal function and deserve further investigation. 4. Experimental section 4.1. Materials and methods for synthesis of Ru(II) arene complexes 2-Chlorobenzothiazole and [(h6-p-cym)RuCl2]2 were purchased from TCI (Shanghai, China). All other chemicals were of reagentgrade quality, obtained from commercial sources and used as received. Chromatographic puriﬁcation was accomplished using ﬂash column chromatography on silica gel (neutral, Qingdao Haiyang Chemical Co., Ltd). 1H and 13C NMR spectra were recorded on a Bruker Avance-400 (400 MHz) spectrometer at 400 MHz for 1H and 100 MHz for 13C. Chemical shifts (d) are expressed in ppm downﬁeld to TMS at ¼ 0 ppm and coupling constants (J) are expressed in Hz. Elemental analyses were performed with a Flash EA 1112 from ThermoFinnigan. Crystal data collection and reduction were performed using the Oxford Diffraction CrysAlisPro software. The structure of Ru2 was solved by direct methods, and the nonhydrogen atoms were subjected to anisotropic reﬁnement by full-matrix least squares on F2 using the SHELXTXL package. Crystal data of Ru2 are in Table S1, and CCDC 1992990 contains the supplementary crystallographic data for this paper. 4.1.1. General procedure for synthesis of benzothiazolefunctionalized imidazolium salts HLPF6 (L1-L6) Imidazole derivatives (6 mmol) and 2-chlorobenzothiazole 9 (5 mmol) were mixed in 20 mL acetonitrile and heated overnight. The resulting solid was separated and dissolved in water, and then a saturated NH4PF6 aqueous solution was added. The resulting precipitate was obtained as white solid. Yield: 65e83%. NMR data and spectrum are shown in support information. 4.1.2. Synthesis of benzothiazole functionalized NHCe Ru(II)-arene complexes (Ru1eRu6) A mixture of respective imidazolium salt HLPF6 (0.5 mmol) and silver oxide (0.3 mmol) in 10 mL CH3CN was stirred at 50 C for 12 h. After cooling to room temperature, [Ru(p-cymene)Cl2]2 (0.25 mmol) was added to the mixture and stirred for another 6 h. The resulting orange solution was ﬁltered through Celite, concentrated under reduced pressure and puriﬁed by ﬂash column chromatography. [(p-cymene)Ru(L1)Cl](PF6) (Ru1). Orange solid, Yield: 236 mg (75%). 1H NMR (400 MHz, DMSO‑d6) d (ppm): 8.58, 7.91 (both d, J ¼ 2.0 Hz, imidazole CH, 2H), 8.38, 8.31 (both d, J ¼ 8.0 Hz, benzothiazole CH, 2H), 7.85, 7.70 (t, J ¼ 8.0 Hz, benzothiazole CH, 2H), 6.81, 6.63, 6.35, 5.87 (both d, J ¼ 6.0 Hz, pcym-H, 4H), 4.20 (s, CH3, 3H), 2.30-2.23 (m, p-cym CH(CH3)2, 1H), 2.16 (s, p-cym CH3, 3H), 0.81, 0.76 (both d, J ¼ 6.4 Hz, p-cym CH(CH3)2, 6H). 13C NMR (100 MHz, DMSO‑d6): d (ppm) 188.3 (RueC), 161.7, 147.4, 130.7, 129.3, 127.9, 127.3, 125.2, 121.8, 118.7, 89.7, 87.7, 38.6, 30.9, 22.5, 19.2. Anal. Calc. for C21H23ClF6N3PRuS: C, 39.97; H, 3.67; N, 6.66. Found: C, 39.86; H, 3.47; N, 6.83. [(p-cymene)Ru(L2)Cl](PF6) (Ru2). Orange solid, Yield: 247 mg (78%). 1H NMR (400 MHz, DMSO‑d6) d (ppm): 8.66, 8.18 (both d, J ¼ 2.4 Hz, imidazole CH, 2H), 8.38, 8.31 (both d, J ¼ 8.0 Hz, benzothiazole CH, 2H), 7.86, 7.70 (t, J ¼ 8.0 Hz, benzothiazole CH, 2H), 6.77, 6.42, 6.32, 5.87 (both d, J ¼ 6.0 Hz, pcym-H, 4H), 5.05-4.98 (m, s, CH(CH3)2, 1H), 2.28-2.24 (m, p-cym CH(CH3)2, 1H), 2.26 (s, p-cym CH3, 3H), 1.79, 1.45 (both d, J ¼ 6.8 Hz, p-cym CH(CH3)2, 6H), 0.83, 0.79 (both d, J ¼ 6.8 Hz, p-cym CH(CH3)2, 6H). 13C NMR (100 MHz, DMSO‑d6): d (ppm) 186.7 (RueC), 161.7, 147.5, 130.7, 129.3, 127.2, 125.2, 123.1, 121.8, 119.9, 89.6, 87.6, 54.4, 24.0, 22.8, 22.6, 22.4, 19.2. Anal. Calc. for C23H27ClF6N3PRuS: C, 41.92; H, 4.13; N, 6.38. Found: C, 42.06; H, 4.10; N, 6.35. [(p-cymene)Ru(L3)Cl](PF6) (Ru3). Orange solid, Yield: 212 mg (63%). 1H NMR (400 MHz, DMSO‑d6) d (ppm): 8.61, 8.00 (both d, J ¼ 2.4 Hz, imidazole CH, 2H), 8.38, 8.31 (both d, J ¼ 8.0 Hz, benzothiazole CH, 2H), 7.84, 7.70 (t, J ¼ 8.0 Hz, benzothiazole CH, 2H), 6.76, 6.56, 6.36, 5.80 (both d, J ¼ 6.0 Hz, pcym-H, 4H), 4.58-4.45 (m, CH2, 2H), 2.28-2.22 (m, p-cym CH(CH3)2, 1H), 2.14 (s, p-cym CH3, 3H), 1.97-1.90 (m, CH2, 2H), 1.45-1.30 (m, CH2, 2H), 0.95 (t, J ¼ 7.2 Hz, CH3, 3H), 0.81, 0.78 (both d, J ¼ 7.2 Hz, p-cym CH(CH3)2, 6H). 13C NMR (100 MHz, DMSO‑d6): d (ppm) 187.3 (RueC), 161.6, 147.5, 130.7, 129.3, 127.2, 126.6, 125.1, 121.8, 118.9, 89.7, 87.5, 51.4, 32.2, 30.9, 22.4, 19.7, 19.2, 14.1. Anal. Calc. for C24H29ClF6N3PRuS: C, 42.83; H, 4.34; N, 6.24. Found: C, 42.77; H, 4.25; N, 6.19. [(p-cymene)Ru(L4)Cl](PF6) (Ru4) Orange solid, Yield: 219 mg (60%). 1H NMR (400 MHz, DMSO‑d6) d (ppm): 8.60, 8.00 (both d, J ¼ 2.4 Hz, imidazole CH, 2H), 8.49, 8.33 (both d, J ¼ 8.0 Hz, benzothiazole CH, 2H), 7.84, 7.70 (t, J ¼ 8.0 Hz, benzothiazole CH, 2H), 6.76, 6.56, 6.36, 5.79 (both d, J ¼ 5.6 Hz, pcym-H, 4H), 4.55e4.45 (m, CH2, 2H), 2.26e2.23 (m, p-cym CH(CH3)2, 1H), 2.14 (s, p-cym CH3, 3H), 1.96e1.92 (m, CH2, 2H), 1.38e1.26 (m, CH2, 10H), 0.86 (t, J ¼ 7.2 Hz, CH3, 3H), 0.81, 0.78 (both d, J ¼ 6.8 Hz, p-cym CH(CH3)2, 6H). 13C NMR (100 MHz, DMSO‑d6): d (ppm) 187.3 (RueC), 161.6, 147.5, 130.7, 129.3, 127.2, 126.6, 125.2, 121.9, 119.0, 89.7, 87.5, 51.6, 31.7, 30.9, 30.2, 29.1, 29.0, 26.4, 22.6, 22.5, 19.2, 14.4. Anal. Calc. for C28H37ClF6N3PRuS: C, 46.12; H, 5.11; N, 5.76. Found: C, 45.98; H, 5.14; N, 5.82. [(p-cymene)Ru(L5)Cl](PF6) (Ru5) Orange solid, Yield: 251 mg (71%).1H NMR (400 MHz, DMSO‑d6) 10 C. Chen et al. / European Journal of Medicinal Chemistry 203 (2020) 112605 d (ppm): 8.63, 7.74 (both d, J ¼ 2.4 Hz, imidazole CH, 2H), 8.39, 8.33 (both d, J ¼ 8.0 Hz, benzothiazole CH, 2H), 7.86, 7.71 (t, J ¼ 8.0 Hz, benzothiazole CH, 2H), 7.51e7.40 (m, C6H5, 5H), 6.78 (d, J ¼ 6.0 Hz, p-cym-H, 1H), 6.36, (t, J ¼ 6.0 Hz, p-cym-H, 2H), 5.93 (d, J ¼ 6.0 Hz, p-cym-H, 1H), 5.76 (q, J ¼ 15.2 Hz CH2, 2H), 2.28e2.21 (m, p-cym CH(CH3)2, 1H), 2.16 (s, p-cym CH3, 3H), 0.80, 0.75 (both d, J ¼ 6.8 Hz, p-cym CH(CH3)2, 6H). 13C NMR (100 MHz, DMSO‑d6): d (ppm) 188.4 (RueC), 161.8, 147.4, 136.0, 130.8, 129.4, 129.3, 129.0, 128.9, 127.3, 126.4, 125.2, 121.8, 119.7, 89.7, 87.8, 55.0, 30.8, 22.4, 19.2, 15.6. Anal. Calc. for C27H27ClF6N3PRuS: C, 45.86; H, 3.85; N, 5.94. Found: C, 45.89; H, 3.74; N, 6.03. [(p-cymene)Ru(L6)Cl](PF6) (Ru6) Orange solid, Yield: 245 mg (63%).1H NMR (400 MHz, DMSO‑d6): d (ppm) 8.49, 6.95 (both d, J ¼ 2.4 Hz, imidazole CH, 2H), 8.37, 8.35 (both d, J ¼ 8.0 Hz, benzothiazole CH, 2H), 7.86, 7.70 (t, J ¼ 8.0 Hz, benzothiazole CH, 2H), 6.83, 6.79 (both d, J ¼ 6.0 Hz, p-cym-H, 2H), 6.54, 6.52, (both s, p-cym-H, 2H), 5.96, 5.67 (both d, J ¼ 14.4 Hz, CH2, 2H), 2.33e2.14 (m, p-cym CH(CH3)2 (1H) þ p-cym CH3 (3H) þ CH3 (15H)), 0.88, 0.84 (both s, p-cym CH(CH3)2, 6H). 13C NMR (100 MHz, DMSO‑d6): d (ppm) 187.6 (RueC), 161.6, 147.5, 136.3, 134.5, 133.3, 130.8, 129.3, 127.8, 127.3, 126.4, 125.2, 121.9, 119.0, 90.0, 87.3, 50.9, 31.0, 22.5, 19.2, 17.5, 17.1, 15.6. Anal. Calc. for C32H37ClF6N3PRuS: C, 49.45; H, 4.80; N, 5.41. Found: C, 50.06; H, 4.53; N, 5.55. 4.2. Cell lines and animals Cells were cultured in medium containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 mg/mL). Cells were maintained in a humid atmosphere at 37 C with 5% CO2. Fiveto six-week-old ICR and female Balb/c mice were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences. All experiments were performed under the guidance of the Zhejiang University Committee for Animal Use and Care. Animal experiments were approved by Zhejiang University Committee, and Zhejiang Experimental Animal Center provided aseptic conditions, autoclaved rodent diet and sterile water. For details of the cell and animals experiments, see supporting information. 4.3. Statistical analysis All of the quantitative data are presented as the means ± SD. The statistical signiﬁcance between the measurements was assessed using Student’s t-test. A p-value less than 0.05 was considered statistically signiﬁcant, whereas a p-value less than 0.01 was considered highly signiﬁcant. Declaration of competing interest The authors declare no potential conﬂicts of interest. 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