Rhodium(iii) complexes with isoquinoline derivatives as potential anticancer agents: in vitro and in vivo activity studies.

View Article Online Dalton View Journal Transactions An international journal of inorganic chemistry Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: T. Khan, N. S. Gul, X. Lu, R. Kumar, M. I. Choudhary, H. Liang and Z. Chen, Dalton Trans., 2019, DOI: 10.1039/C9DT01951K. This is an Accepted Manuscript, which has been through the Volume 46 Number 10 Dalton 1 P 4 a g M e a s r c 3 h 0 7 2 3 0 - 1 3 7 412 Royal Society of Chemistry peer review process and has been accepted for publication. Transactions Accepted Manuscripts are published online shortly after acceptance, An international journal of inorganic chemistry rsc.li/dalton before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. 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In no event ISSN 0306-0012 shall the Royal Society of Chemistry be held responsible for any errors CDNan o- O dHuM eg tetlM ea llrs u U o r Wc N eyn .I cy CSllit A ce c Tp act hI ai O o erbn n N e s en ( teX a sH l.t +a, bXil i=z eSd, Spea,r Teen)t sulfenyl, selenenyl, or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/dalton Page 1 of 17 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE t p i r Rhodium(III) complexes with isoquinoline derivatives as potential c anticancer agents: in vitro and in vivo activity studies s u Taj-Malook Khan1, Noor Shad Gul1, Xing Lu1, Rajesh Kumar1,2, Muhammad Iqbal Choudhary2, Hong n Liang1,*, and Zhen-Feng Chen1,* a M Two rhodium complexes Rh1 and Rh2 with isoquinoline derivatives were synthesized and characterized. Both complexes displayed strong anticancer activity against various cancer cells and low cytotoxicity against non-cancer cells. d These complexes triggered apoptosis via mitochondrial dysfunction that increased the levels of ROS and Ca2+ and e released cytochrome C which ultimately activated caspases and the apoptosis pathway. The different biological t p activities of Rh1 and Rh2 could be associated with the presence of methoxy substituents on the ligands. In vivo studies e showed that Rh1 effectively inhibited tumor growth in a T-24 xenograft mouse model with less adverse effect than c cisplatin. Overall, Rh1 and Rh2 induced apoptosis via mitochondrial pathways and could be developed as effective c anticancer agents. A s n based complexes possessing different mechanisms of action and o Introduction improved anticancer activity and safety profile than cisplatin.6 i t The serendipitous discovery and successful development of cisplatin c It is well recognized that the metabolic behavior of cancer cells a in cancer therapy has opened an avenue for researchers to discover is different from that of normal cells and that mitochondria are the s more potent metal complexes with improved therapeutic efficacy. A n organelle at the center of these metabolic pathways.7,8 The a plethora of platinum complexes has been synthesized and studied as mitochondria of cancerous cell undergo major changes, including r T potential therapeutic agents for different kinds of cancer.1 Among depolarization of the mitochondrial membrane potential and n these platinum complexes, carboplatin and oxaliplatin have been increased generation of reactive oxygen species (ROS).9 Thus, o used worldwide for the treatment of different types of cancers.2 The targeting mitochondrial metabolic pathways by metal complexes has t l primary mechanism of action of platinum complexes is to disrupt the a become a main focus in ongoing metallodrug research.10 In fact, a D replication and transcription of DNA.3 However, the use of handful of metal complexes that directly target the mitochondria are platinum-based metallodrugs in cancer therapy is limited by their in various phases of in vitro and in vivo investigation. These lack of specificity in targeting cancerous cells over healthy cells, complexes disrupt the mitochondrial membrane potential by which leads to severe side effects.4,5 To overcome these drawbacks, inducing apoptosis and cell death.11 Transition metal complexes tremendous efforts have been focused on discovering non-platinum other than platinum offer an excellent opportunity to develop This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1 Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 2 of 17 ARTICLE Journal Name anticancer drugs with interesting coordination chemistry, variable Recently rhodium complexes of camphor-derived oxidation states, novel structural features, and useful chemical and bis(pyrazolylpyridine) and terpyridine ligand were synthesized, physical characteristics.12 Among these non-platinum complexes, which showed a high affinity towards ct-DNA and BSA (bovine rhodium(ІІІ) and iridium(ІІІ) complexes have captured the attention serum albumin). The molecular docking studies showed that all these of the researchers, due to their unique chemical and biological complexes showed potential for DNA intercalation and affinity for properties.13 They were found to have different modes of action and the minor groove of DNA. In vitro cytotoxicity showed that Rh(ІІІ) t p cellular targets against cancer cells as compared to the traditional complex with a camphor-derived ligand having four methyl group is i platinum-based metallodrugs.14,15 However, there are still too few more effective and cytotoxic than having three methyl group and r c Rh(ІІІ) complexes being studied. terpyridine against HCT-116 cancer cell line23. Cyclometalated s u The biological activity of metal complexes can be increased by iridium(ІІІ) complexes with diimine ligand exhibited high anticancer n combination with cytotoxic ligands and engagement with multiple activity against a panel of cancer cells including HepG2, A549, a M cellular targets.16 Additionally, ligand substitution can further A549R, HeLa, PC3, HeLaρ0 and HLF with IC values higher than 50 improve the cytotoxicity of rhodium complexes. For example, the cisplatin. These complexes were accumulated in mitochondria. As a d e trichlorido complex of Rh(ІІІ) bonded with a 5,6-dimethyl result, it is affecting the function of mitochondria in term of ATP t p substituted ligand of phenanthroline demonstrated 18-times higher depletion, metabolic disturbance and respiration inhibition which e anticancer activity than the non-substituted phenanthroline. This ultimately induced apoptotic cell death and mitophagy. Further c c molecule induced toxicity by targeting the mitochondria and mechanistic studies revealed that these complexes are mainly A triggering apoptosis in Jurkat leukemia cells, along with an increase targeting the mtDNA and mitochondrial genome which may be the s in reactive oxygen species (ROS).17 Similarly, rhodium complexes target of metallodrugs to circumvent the resistance of cisplatin24. n o that possess novel benzimidazole chelating ligands show excellent Half sandwich iridium(ІІІ)-NHC complexes exhibited better i anti-cancer activities against A2780, HT29, T47D, and A2780 CisR anticancer activity than cisplatin against A549 and HeLa cancer cell t c cancer cell lines.18 Some Rh(ІІІ) complexes target plasmid DNA as line. Cytotoxic activity was correlated with the number of phenyl a s an intercalator by binding to and disrupting regions close to the base groups attached to the central metal ion. The mechanistic studies of n pair mismatches.19 Rhodium butyrate complexes inhibit the these complexes showed that these complexes converted NADH to a r synthesis of DNA by arresting the cell cycle at S phase.20 NHC- NAD+ by capturing the hydrogen from the molecule that producing T rhodium complexes trigger apoptosis via dysfunction of ROS which led to the decrease of mitochondrial membrane potential n o mitochondrial membrane potential accompanied by oxidative and lysosomal damage and ultimately induced apoptotic cell death25. t l damage of DNA and cell cycle arrest. In addition, NHC-rhodium Similarly, rhodium metalloinsertors complexes bind to DNA base a D complexes show selective cellular targeting ability, thioredoxin pair mismatches and kill the cells which are deficient mismatch reductase inhibition, and antimetastatic properties in mammalian repair than mismatch repair proficient counterpart at low cells and E. coli.21 Similarly, a cyclometaleted rhodium complex concentrations.26 Iridium complexes with aroylthiourea ligands exhibited cytotoxicity by inhibiting angiogenesis and essential signal proved to be not less than cisplatin in chemo-sensitivity against transduction pathways.22 HCT-116 and ARPE-19 cancer cell lines.27 2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K Page 3 of 17 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE Coordination of a biologically active ligand with the transition Scheme 1 Synthesis of rhodium complexes. Chemical reagents (a). metal core may form complexes with unique biological and physical SOCl , benzene (24 h at room temperature), yield: 70-90% (b). 2 properties.28 Therefore, the selection of appropriate ligands to POCl , CH CN (12 h at 80°C) yield: 60-80% (c). SnCl .2H O, EtOAc 3 3 2 2 modulate the cytotoxic properties of the complexes is crucial. In this (12-24 h at 90°C) yield: 50-60% (d). La, Rh(DMSO)Cl MeOH, 3, respect, isoquinoline is an excellent choice because of its various acetonitrile(1:3), (72h at 80°C) yield: 7080% (e) Lb, Rh(DMSO)Cl , 3 biological and pharmacological properties, including anti- MeOH, acetonitrile(1:3), (72h at 80°C) yield: 8090%. t inflammatory and anticancer activity.29 Many isoquinoline metal p i complexes demonstrate different cytotoxic mechanisms of action Synthesis and crystal structure of Rh1 and Rh2 r c and cellular targets compared to that of cisplatin.30,31 Two new rhodium(III) complexes were synthesized by mixing La or s u In this paper, we report the synthesis and characterization of two Lb with Rh(DMSO)Cl in methanol and acetonitrile (1:3) in a sealed 3 n new rhodium complexes with isoquinoline as a ligand. We also glass tube. The structures of these complexes were characterized by a M studied their in vitro and in vivo anticancer activities, and propose a 1H, 13C-NMR, ESI-MS (Figures S1S6, supporting information), possible anticancer mechanism. Elemental analysis, and was further confirmed by single-crystal x- d e ray diffraction analysis. Summary of the crystallographic data and t Results and discussion p refinement details are available in Table S1. Selected bond lengths e Synthesis of Ligands and angles are listed in Table S2 (supporting information). c c To prepare the ligands, 6,7-dimethoxy-1-(2-aminophenyl)-3,4- A Structural features dihydroisoquinoline (La) and 6-methoxy-1-(-2-aminophenyl)-3,4- In Rh1 and Rh2, one aminophenyl dihydroisoquinoline, one s dihydroisoquinoline (Lb), a Bischler-Napieralski synthesis procedure n dimethyl sulfoxide, and three chloride atoms are coordinated with was followed as reported in the literature.32 The only difference in o Rh(III) to give a six-coordinated distorted octahedral geometry i the structure of the ligands is the number of methoxy groups t c (Figures 1, 2). The coordination of N1 and N2 atoms of bidentate attached to the benzene ring of isoquinoline where La has methoxy a aminophenyl dihydroisoquinoline ligand with Rh atom is responsible s groups at C6 and C7, whereas Lb has only one at C6. n for the formation of a six-membered ring (N1-N2/C1/C6/C7) in a a half-chair conformation. The RhN2 [Rh1N2 = 2.100 Å, Rh2N2 r T = 2.085 Å] and RhS1 [Rh1S1 = 2.28 Å, Rh2S1 = 2.265 Å] are n assembled perpendicular to each other, and three chloride atoms o t [Rh1Cl1 = 2.33 Å, Rh1Cl2 = 2.35 Å, Rh1Cl3 = 2.34 Å, l a Rh2Cl1 = 2.37 Å, Rh2Cl2 = 2.35 Å, Rh2Cl3 = 2.34 Å] and N1 D [Rh1N1 = 2.066 Å, Rh2N1 = 2.089 Å] of the bidentate aminophenyl dihydroisoquinoline ligands are located on the basal positions of the square. Rings C1C6 and C8C13 of aminophenyl dihydroisoquinoline are both planar, having the dihedral angles of This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3 Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 4 of 17 ARTICLE Journal Name 57.4(3), and 60.11(15) between them. Ring bonds in complexes Rh1 and Rh2. In Rh1, an N(1)H(1B)O (3) (N2/C7/C8/C13/C14/C15) exists in a boat conformation [(Q) = intra-molecular interaction was found to be the strongest one, having 0.490(6) Å, = 114.1(7) °,  = 82.9(7) °, (Q) = 0.505(3) Å,  = a bond length of 2.33Å. Other interactions are summarized in Table 66.4(3) °,  = 264.9(3) ° for Rh1, and Rh2, respectively]. In Rh1, S3 (supporting information). Similar to Rh1, among all hydrogen disorder at C16 was resolved using the 50% site occupancy factor bonds in Rh2 involved in unit cell packing, N(1)H(1A)O(2) was and application of the EADP constrain. Torsion angles of found to be the strongest one having a bond length of 2.36Å. Other t p C15/N2/Rh/Cl2, Cl1/Rh/S/O, Cl2/Rh/S/O, Cl3/Rh/S/O in Rh1, and interactions are summarized in Table S4 (supporting information). i r Rh2 were found to be 36.7°, 90.7°, 179.4°, 89.1°, 52.2°, 88.7°, C(18)H(18C)Cl(1) in Rh1, and C(16)H(16B)Cl(2) in Rh2 are c s 179.6°, and 89.5°, respectively. The larger dihedral angles are responsible for the chain elongation in a zig-zag manner along the b- u attributed to the steric hindrance around Rh(III).33 axis. NHCl intermolecular interactions are responsible for the S 8 n a ring motif in both complexes. (Figures S7, S8, supporting M information). d e Hirshfeld surface analysis t A two-dimensional Hirshfeld surface36 was generated for both p e complexes, showing bright red spots that represent the location of c atoms with the potential to form the hydrogen bonds (Figure S9). c A The brightness of these red spots indicates the potential strength of s the hydrogen bonds. Figure 3 shows the Hirshfeld surface interacting Fig. 1 ORTEP view of the complex Rh1 n with neighboring molecules. 2D fingerprint plots revealed the o i contributions of HH at its maximum because the overall molecular t c surface has its hydrogen atoms more exposed towards the a s neighboring molecules. The ClH contact was found to be the n strongest one in both complexes as it was confirmed by the spikes a r extending towards the bottom having 19.3%, and 20.6% in T complexes Rh1, and Rh2 respectively. Other important contacts are n o CH, and OH having 14.2%, 13.5% for CH, 12.5%, and 11.6%, t Fig. 2 ORTEP view of the complex Rh2 l respectively37 (Figures S10, S11, supporting information). a Supramolecular features D Intra- and intermolecular interactions exert significant influence on the obtained structures, their physical properties34, and their biological activities.35 Topological analysis of hydrogen bonding reveals the presence of conventional and non-conventional hydrogen 4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K Page 5 of 17 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE methoxy groups and their electron donating effects. This hypothesis is supported by the analysis of the electrostatic potential surface38 mapped over the Hirshfeld surface, which reveals more potential sites available in Rh1 than in Rh2 (Figure: S11, Supporting information). It was evident from the IC values in Table 1 that Rh1 50 and Rh2 showed a significantly increased cytotoxicity as compared t p Fig. 3 Interaction with Hirshfeld surface for complexes Rh1 and Rh2 with the corresponding ligand and rhodium(III) salt towards the T-24 i cells. Against T-24, Rh1 and Rh2 exhibited higher cytoxicity than r c Stability of complexes in PBS solution cisplatin did. In the case of normal human liver cells (HL-7702), the s Under physiological conditions, the stability of Rh1 and Rh2 were u cytotoxicity of Rh1 are higher than Rh2, corresponding ligands and n investigated in PBS (phosphate buffer saline) containing 1% DMSO, rhodium(III) salts. Against BEL-7704, Rh1 was more toxic than a pH 7.4 at room temperature for 0, 4, 12 and 24 h by UV-Vis M Rh2, corresponding ligands, rhodium(III) salt and cisplatin. Also, the spectroscopy. Further stability and purity studies were carried out cytotoxic effect of Rh1 was better than Rh2, corresponding ligands d and confirmed by HPLC. The spectral data showed that both Rh1 e and rhodium(III) salt against HeLa cells, but it was worse than t and Rh2 are stable at the given conditions in a DMSO stock solution p cisplatin. In comparison with corresponding ligands and rhodium(III) e which was 2000 µM (Figures S12S15, supporting information). salt, rhodium complexes did not show significant cytotoxicity c c towards MGC80-3. Against A-549 cell lines, Rh2 exhibited better Assessment of in vitro cytotoxicity by MTT A cytotoxicity than Rh1, corresponding ligands and rhodium(III) salt. The in vitro cytotoxicity of rhodium(III) complexes and the s Although the rhodium(III) complexes displayed an improved corresponding ligands were assessed against different tumor cell n o potency compared to ligands and rhodium(III) salt to most of the lines including BEL-7704, MGC80-3, HeLa, A-549, T-24, SK-OV- i tested cells, but to SK-OV-3 cell lines, it was exceptional. In case of t 3, and HL-7702 (human normal liver cells) by the MTT assay. c SK-OV-3, the rhodium(III) salt exhibited better cytotoxicity than a Cisplatin and the corresponding rhodium(III) salt were taken as the s rhodium complexess and corresponding ligands. In a word, viewing control. Except SK-OV-3, to the tested cell lines, synergistic effects n from the Table 1, it was found that T-24 was the best cell lines a were observed upon coordination of the isoquinoline ligand to the r exhibited anticancer activity in response to the rhodium complexes. T rhodium core. Except to A-549, the cytotoxic effect of Rh1 is higher n in comparison with Rh2, which may be due to the number of o Table 1. IC (μM) values of Rh1 and Rh2, on selected tumor cells for 48 h 50 t Compound HL-7702 T-24 BEL-7704 HeLa MGC80-3 A-549 SK-OV-3 l a D La 20.04±0.7 61.3±0.4 95.03±0.3 46.3±0.5 ﹥100 ﹥100 ﹥100 Lb 95.0±0.3 56.02±0.5 75.02±0.5 95.04±0.4 86.04±0.5 74.01±0.5 41.4±0.3 Rh(DMSO)Cl 85.0±0.5 48.06±0.6 74.05±0.4 61.02±0.2 67.9±0.5 92.02±0.2 25.4±0.2 3 Rh1 35.0±0.9 4.08±0.3 18.04±0.3 22.02±0.6 62.05±0.6 75.1±0.9 44.02±0.2 This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5 Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 6 of 17 ARTICLE Journal Name Rh2 53.02±0.3 10.02±0.8 70.03±0.5 59.03±0.1 65.7±0.4 30.4±0.5 ﹥100 cisplatin 20.6±0.9 27.02±0.3 24.03±0.6 9.5±0.8 8.02±0.8 19.9±0.5 22.05±0.3 Fig. 4 Cellular uptake and distribution of Rh1, Rh2 and cisplatin Cellular uptake and distribution of rhodium in cancer cells contents in different parts of T-24 cell line measured by ICP-MS. The cellular uptake can affect the biological activity of a drug.39 The Control was provided with 1% DMSO. Rh1, Rh2 (10 μM) of t uptake and distribution of rhodium complexes in various parts of the complexes were treated for 24 h at 37°C, respectively. Cisplatin (2 p cell, including cytoskeletal, cytosolic, membrane/particulate, and i µM) was taken as a positive control. *P < 0.05, **p < 0.01 and ***P r c nuclear fractions were examined in T-24 cells treated with Rh1, Rh2 < 0.001 determined by ANOVA. s and cisplatin (treated control) for 24 h using the inductively coupled u n Cell cycle arrest plasma mass spectroscopy (Figure 4). The accumulation of Rh1 and a The cell cycle plays an important role in cell division and consists of Rh2 was higher than untreated and treated control in the M a sequential series of events within the cell. Cell cycle arrest is often membrane/particulate fraction (4377, 2745 ng), cytoskeletal fraction d (2084, 602 ng), and cytosolic fraction (1941, 1553 ng) respectively. observed in treatments against cancer and other diseases and is e t closely related to the induction of cell apoptosis and cell death.41,42 In addition, the concentration of Rh1 is almost double of the Rh2 in p To explore the arresting effect of Rh1 (2.0 4.0, 8.0 μM), Rh2 (5.0, e the membrane fraction. In case of cisplatin (treated control) there has c 10.0, 20.0 μM) and cisplatin (2 µM) on cell cycle progression, T-24 been no significant accumulation of platinum in any part of cell. The c A cells were exposed for 24 h and analyzed by flow cytometry (Figure distribution of different concentrations of rhodium in different parts S16, supporting information). A dose-dependent increase in cell s of cells could be associated with the cytotoxicity and various cellular n and apoptotic pathways involving various mechanisms of action.40 population in S phase was observed in Rh1 treated cells (48.6%, o Due to the uptake and accumulation of rhodium in T-24 cells, a 56.9% and 71.3%) versus the control (G1: 51.6%, G2: 10.1%, S: i t c 38.3%). Although there is no significant effect of cisplatin (treated detailed investigation of Rh1 and Rh2 was conducted to explore the a possible mechanism of action of the complexes. control) on cell cycle arrest was observed. Whereas in the case of s n Rh2 treated cells (22.7%, 32.5%, and 39.4%), an increase of cells in a G2 phase occurred as compared with the control (G1: 51.6%, G2: r T 10.1%, S: 38.3%). From the above result, it was concluded that Rh1 n cause cell cycle arrest in S phase whereas Rh2 in G2 phase (Figure o 5). t l a D 6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K Page 7 of 17 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE cisplatin (treated control) had no significant effect on the induction of apoptosis. Therefore, both complexes can induce apoptosis in the T-24 cell line significantly (Figure 6). t p i r c s u n a M Fig. 5 Effect of rhodium complexes and cisplatin (treated control), d on the arrest of the cell cycle of T-24 cells. Mean population (%) of e t T-24 cells in different phases of the cell cycle by 24 h treatment p e compared with control. Presented data are the mean ± SD obtained Fig. 6 Effect of rhodium complexes and cisplatin (treated control), c from three independent experiments. *P < 0.05, **p < 0.01 and on the induction of apoptosis in T-24 cell. Mean population (%) of c A ***P < 0.001 determined by ANOVA. cells in early and late apoptosis after the 24 h treatment with Rh1, s Rh2 and cisplatin compared with untreated control. Data were n Induction of apoptosis presented as the mean ± SD acquired from three independent o Apoptosis is a conserved process that regulates the growth of a i experiments. *P < 0.05, **p < 0.01 and ***P < 0.001 determined by t multicellular organism by eliminating unwanted and abnormal cells c ANOVA. a through a controlled sequence of molecular events. In cancer cells, s n the process of apoptosis is dysregulated so that it could be exploited Measurement of reactive oxygen species (ROS) generation a as an anti-cancer target.43-45 In order to determine the ability of Rh1 Reactive oxygen species cause oxidative damage to the cell and play r T (2.0, 4.0, 8.0 μM), Rh2 (5.0, 10.0, 20.0 μM) and cisplatin (2 µM) to a crucial role in cellular biological function and signaling.46 The n induce apoptotic cell death, T-24 cells were treated with various mitochondrion is the primary source of production of ROS, and the o concentrations of rhodium complexes and treated control for 24 h, unnecessary production of ROS is considered a warning feature t l a stained with annexin V and propidium iodide, and analyzed by flow leading to mitochondrial damage, genetic instability, and ultimately D cytometry (Figure S17, supporting information). The result showed a apoptotic cell death.47 Therefore, ROS targeting is accepted as a dose-dependent increase in the population of early apoptotic cells viable strategy for the treatment of cancer.48 ROS production upon from 0.8% in control to 70.2% at the maximum concentration of 24 h treatment with Rh1 (4 μM), Rh2 (10 μM) and cisplatin (2 µM) Rh1. Late apoptosis was observed in the case of Rh2 from 0.5% in in T-24 was determined using flow cytometry. Cisplatin was taken control to 41.4% at the highest concentration. In comparison, as positive control. To confirm whether the apoptosis induced by the This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7 Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 8 of 17 ARTICLE Journal Name complexes and cisplatin (treated control) related to the level of ROS, and analyzed by flow cytometry. As shown in Figure 8, the level of a fluorescent marker was used (Figure 7). It was shown that a high intracellular calcium in the negative control was lower than in level of ROS was generated by Rh1, whereas Rh2 considerably complex-treated cells at maximum concentrations of Rh1(4 μM) and elevated the level of ROS and cisplatin have an insignificant effect. Rh2 (10 μM) and cisplatin (2 µM). It was noteworthy that the level It is well known that the induction of cytotoxicity depends on the of calcium in Rh1 treated cells was higher as compared to those with level of ROS generation.49,50 Therefore, it is suggested that ROS- Rh2 and cisplatin (treated control). t p induced apoptosis was activated by exposure to the rhodium i complexes and thereby damaging the function of the mitochondria. r c s u n a M d e t p e c c A Fig. 8 Flow cytometric investigation of Ca2+ in T-24 cells treated with s Fig. 7 Flow cytometric exploration of reactive oxygen species Rh1, Rh2 and cisplatin (treated control) as compared with n produced by the treatment of Rh1, Rh2 and cisplatin (treated o untreated control. i control) compared with untreated control. t c Assessment of changes in mitochondrial membrane potential a Mitochondria play a major role in the induction of apoptosis and are s Fluctuation of intracellular Ca2+ n regarded as a new target for antitumor drugs.53 The membrane For regular cellular activity, a tightly controlled regulation of a potential of the mitochondria is tightly-controlled and well-regulated r intracellular levels of calcium is essential and associated with cell T in the normal cells.54,55 Any damage to the membrane potential can proliferation and apoptosis.51 It is considered that cancer cells n activate apoptotic pathways that ultimately lead to apoptotic death o possess a remarkably high level of calcium either due to their by the release of pro-apoptotic factors.56 To evaluate apoptosis t malfunctioning mitochondria, which can preserve high levels of l a caused by rhodium complexes and cisplatin, T-24 cells were treated D calcium or due to their intemperate influx of extracellular calcium. for 24 h and analyzed for depolarization of membrane potential by This may also be linked to cell apoptosis due to mitochondrial flow cytometry using a fluorescent dye JC-1. As depicted from the damage and utilized for determining the efficacy of anticancer Figure S18 (supporting information), in both complexes, a dose- drugs.52 To examine the effect of Rh1, Rh2 and cisplatin (treated dependent increase took place where the number of cells with control) on calcium homeostasis in T-24 cell, the cells were treated disrupted mitochondrial membrane increased from 1.4% in control 8 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K Page 9 of 17 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE to 51.2% in Rh1 treated cells, and 36.1 % in Rh2 treated cells. compared with the control. Similarly, the expression levels of Whereas cisplatin has no significant effect compared to control. The activated caspase-8 were increased from 1.3% to 30.2% and 17.4% results demonstrated that apoptosis induced by rhodium complexes respectively, and to 56.4% and 29.4% respectively for caspase-9, was through a mitochondrial-mediated pathway, although other compared with the controls whereas the treatment of cisplatin pathways may also be involved (Figure 9). (treated control) has minimal effect but not significant, in the activation of caspases. The activation of t p caspases gave evidence that rhodium complexes especially Rh1 was i an efficient activator of caspases and induced cell apoptosis by r c triggering caspase-3/8/9 in T-24 cells. s u n a M d e t p e Fig. 9 Graph bar represented depolarization (%) of T-24 cells at a c c different concentration of Rh1, Rh2 and cisplatin (treated control) A for 24h. Data are displayed as a mean ± SD of three independent s Fig. 10 Determination of activated caspases; -3, -8, -9 in T-24 cells n experiments. *P < 0.05, **p < 0.01 and ***P < 0.001 determined by o treated with Rh1, Rh2 and treated control (cisplatin) as compared ANOVA. i t with untreated control. The determination was performed in c Caspase 3/8/9 activation assay. a triplicate, and one representative experiment was displayed. s Caspases are the most critical apoptosis-related proteins, and they n Western blotting of apoptosis-related proteins play an essential role in the mitochondrial-mediated pathway. It is a r well-considered that upon the disruption of mitochondrial A direct relationship exists between Bcl-2 family proteins and T membrane, cytochrome C is released which consequently causes a apoptosis. There are two classes: anti-apoptotic Bcl-2, Bcl-xl, and n o cascade of caspase activation, which includes caspase 3 and caspase proapoptotic Bax, Bak and Bim. In the generation of a death signal, t 9.57,58 Flow cytometry was performed to measure the activation level l the expression of proteins from both classes is of high a of caspase-3, -8, and -9 and to better understand the anticancer D importance.59,60 By Western blot, the expression level of these mechanisms of Rh1 and Rh2 in the induction of apoptosis via the proteins could be measured upon the treatment of T-24 cells with mitochondrial-mediated pathway in T-24 cells. Figure 10 shows that Rh1 and Rh2. It is shown in Figure 11 that Bax, and Bak were the levels of caspase-3 upon exposure to Rh1 (4 μM) and Rh2 (10 upregulated, and Bcl-2 and Bcl-xl were downregulated. μM) increased from 1.4% to 61.3% and 32.3%, respectively, This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 9 Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 10 of 17 ARTICLE Journal Name cyclin A, cyclin B, and cyclin D decreased, and significantly increased the level of chk1 and chk2. The increase in the level of these proteins may be a result of cell cycle arrest at S-phase and G2 phase. Further examination of Cdks binding protein confirmed the upregulation of proteins including p21, p27, and p53 upon treatment with rhodium complexes. These proteins inhibit the activity of cdk2 t p and consequently cause cell cycle arrest at S and G2 phases. All of i these results are consistent with the flow cytometry analysis r c regarding the cell cycle arrest. s u n a M d e t p e c c A s n o i t c a s n Fig. 11 (A) Expression levels of apoptosis-related proteins of T-24 a r cells for 24 h treatment. (B) (C) Western blotting bands were T quantified with Image J (three independent experiments). *P < 0.05 n o and **P < 0.01 determined by ANOVA. t l a Western blotting of cell cycle related proteins D Cell cycle proteins, such as cdc25 A, cyclin A, cyclin D, cyclin B, chk1, chk2, and cdks, are involved in controlling the cell cycle.61,62 Therefore, we evaluated the expression of these proteins upon the treatment of T-24 cells with Rh1 and Rh2 for 24 h by Western blotting. As shown in Figure 12, the expression level of cdc25 A, 10 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K Page 11 of 17 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE blotting band with Image J (three independent trials). *P < 0.05 and **P < 0.01 determined by ANOVA. In vivo investigation of rhodium complexes in a tumor xenograft model of T-24 To further investigate the in vivo anticancer activity of rhodium t p complexes, Rh1 was used in a mouse model bearing T-24 i r xenografts. The mice were randomly divided into the negative c control group, Rh1 treated, and positive control groups. Two groups s u of mice were treated intraperitoneally (IP) with Rh1 at a high dose of n (15.0 mg/kg) and a lower dose of (7.5 mg/kg) daily for 15 days after a M the volume of tumor was 90110 mm3. The control group was given d 5% DMSO in saline (V/V), whereas the cisplatin group was given e 2mg/kg of cisplatin and was used as the reference. A dose-dependent t p tumor growth inhibition was observed as shown in Figure 13. After e 15 days of treatment, the mice were sacrificed, and the rate of tumor c c growth inhibition rate (IR%) was determined as 54.5% for Rh1 (15.0 A mg/kg) at a high dose treatment, and 37.6% for Rh1 at a low dose. s The tumor weight inhibition rate (IR%) of cisplatin was 54.5%, n o which was comparable with that of the high dose treatment with Rh1 i t (Figure 13 A, B). No apparent adverse effects were observed during c a the days of treatment in the Rh1-treated group, which exhibited s satisfactory results without substantial losses in body weight, n a whereas the cisplatin-treated group demonstrated some weight loss. r T These findings suggested that the safety profile of Rh1 is higher than that of cisplatin, and Rh1 could inhibit the growth of T-24 tumor in n o vivo comparably to cisplatin. t l a D Fig. 12 (A) Expression levels of cell cycle-related protein in T-24 cell treated with Rh1 and Rh2. (B) Quantification of the Western This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 11 Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 12 of 17 ARTICLE Journal Name (1.25200µM) to carried out different biological assays and spectroscopic experiments. The concentration of DMSO in stock solution was 100%, and the concentration of DMSO was 0.5% in the working solution. In the pharmacological studies, all compounds of high purity were used, which were more than 99%. All the assays kits including MTT, cell cycle evaluation (RNAse, PI), apoptosis t p detection (annexin V, PI), mitochondrial membrane potential i r detection (JC-1), activated caspases determination, ROS detection, c and Ca2+ were obtained from Bio-Vision and BD Biosciences. All s u antibodies were purchased from Abcam (USA). All cell lines were n provided by the Shanghai Institute of Biological Sciences. The a M xenograft mouse model of T-24 was purchased from Beijing Bioscience Co. Ltd. (Beijing, China) d Fig. 13 The in vivo antitumor activity of Rh1 in mice having T-24 e Instruments t tumor xenograft. (A) Effect on the growth of tumor by Rh1(7.5, 15.0 p A Bruker HCT mass spectrometer was used for acquiring the ESI- e mg/kg), cisplatin (2 mg/kg) or control (5% DMSO in saline v/v) in MS spectra. Bruker AVANCE NEO-500 NMR spectrometer was c tumor xenograft model. The tumor growth was measured by the c used for recording of NMR spectra. Bruker Smart APEX II was used A mean tumor volume (mm3) ±SD and calculated as the relative to collect the single-crystal X-ray data. TU-1901 ultraviolet s tumor increment rate (TC%). (B) The weight of the tumor was spectrometer was used to obtain the absorption spectra. HPLC n determined comparatively with control after the mice were o analysis was performed on an Elite P230ІІ (Dalian, China). MTT i sacrificed. *P < 0.05, *p < 0.01 and ***P < 0.001, p vs vehicle t assays were carried out on microplate reader M1000 of Tecan c control, determined by ANOVA. (C) Changes in body weight were Trading Co. Ltd., Shanghai, China. Analysis of cell cycle was a s recorded and exhibited in % values from the initial weight. (D) achieved by using a FACS Aria ІІ flow cytometer (BD Biosciences, n Photograph of the tumor-containing control group, rhodium- San Jose, USA). For confirmation and detection of protein a r treated and cisplatin-treated. expression, Western blot assays were run on an ECL Western T blotting system. n Experimental o Synthesis and characterization of La, Lb, Rh1, and Rh2 t l Materials a Synthesis of Ligands D All the reagents were purchased from Xilong Chemical Co., Ltd, La and Lb were synthesized according to procedures reported by Alfa Aesar, and Sigma Aldrich and used without further purification. Nussbaum and co-workers.63 DMSO was used for the dissolution of Rh1 and Rh2, to prepare a Synthesis of Rh1 and Rh2 stock solution of 2000µM. Furthermore, PBS (phosphate buffer A mixture of La, Lb (0.025 mmol) and Rh (DMSO)Cl 3 (0.05 mmol, saline) was used to prepare different working solution in the range of 0.010 g) in acetonitrile (0.15 mL) and methanol (0.45 mL) was 12 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K Page 13 of 17 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE placed in a thick Pyrex glass tube of 25 cm length and frozen in ORTEP366 was used to draw the 3D view of the molecule, and liquid nitrogen, exposed to vacuum, and sealed by fire assembly. MERCURY67 was used for visualizing the interactions and crystal Dark orange block crystals were harvested after three days of heating packing. Table S1 shows the crystallographic data of Rh1 and Rh2 at a constant temperature of 80 °C, appropriate for X-ray analysis. and S2 show selected bond length and angles. Hirshfeld surface analysis H, 13C-NMR data of Rh1 Hirshfeld surface analysis and its properties were determined using 1H NMR (600 MHz, (CD )SO) δ 7.41(td, J = 1.5, 8.0 Hz, 1H), 7.29 t 32 p standard parameters. A 2D Hirshfeld surface d was generated on norm (d, J = 7.0 Hz, 1H), 7.24 (t, J = 7.6 Hz, 2H), 7.14 (s, 1H), 6.74 (s, i r a surface scale of 0.3271.475 Å for Rh1, and 0.2651.295 Å for c 1H), 3.92 (s, 3H), 3.63 (s, 3H), 3.42(s, 6H), 2.88 (t, J = 7.0 Hz, 2 Rh2 by utilizing the Crystal Explorer Software package.68 2D s H), 2.53 (m, 2 H). 13C NMR (150 MHz, (CD 3 ) 2 SO) δ 169.8, 152.8, u fingerprint plot has been generated to visualize the percentage n 146.6, 142.1, 134.3, 133.3, 131.7, 131.5, 125.2, 123.6, 121.6, 114.2, contribution of contacts towards the overall crystal packing. a 110.6, 56.3, 56.1, 51.8, 41.1, 40.8, 26.6. H-RMS(EI): Calcd for M Electrostatic potential surface was generated by the HF method C H ClN O RhS m/z 567.9628, found m/z 568.5673 for [M+H]+. 19 24 3 2 3 , utilizing the TONTO package incorporated in Crystal explorer. d Elemental analysis: calcd (%) for C H ClN O RhS: C, 40.05; H, 19 24 3 2 3 e Cell culture and other experimental methods t 4.25; N, 4.92; S, 5.63; found: C, 40.21; H, 3.57; N, 4.80; S, 5.62. p T-24, HeLa, MGC80-3, A-549, HL-7702, SK-OV-3, and BEL-7704 1H, 13C-NMR data of Rh2 e cells were purchased from Shanghai Cell Bank of Chinese Academy c 1H NMR (600 MHz, (CD)SO) δ 7.42 (m, 1H), 7.25-7.21 32 c of Sciences. Cell culture was carried out in Dulbecco’s Modified (overlapped, 4H), 7.07 (m, 1H), 7.00 (dd, J = 3.0, 11.0 Hz, 1H), 3.89 A Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). All (s, 3H), 3.42 (s, 6H), 2.94 (t, J = 8.5 Hz, 2 H), 2.55 (m, 2 H). 13C s assays, including MTT, cell cycle analysis, detection of apoptosis, in n NMR (150 MHz, (CD )SO) δ 169.9, 162.8, 142.4, 142.1, 133.2, 32 o vivo xenograft animal model, Western blotting was performed 133.0, 131.8, 131.4, 125.2, 123.6, 122.6, 112.7, 112.5, 56.1, 51.6, i t according to procedures reported by Wei et al.69 Flow cytometric c 41.1, 40.8, 27.4. H-RMS(EI): Calcd for C H ClN O RhS m/z 18 22 3 2 2 , a analysis of activated caspases, intracellular calcium ion and 537.9523, found m/z 538.9420 for [M+H]+. Elemental analysis: s measurement of reactive oxygen species were performed according n calcd (%) for C H ClN O RhS: C, 40.06; H, 4.11; N, 5.19; S, 18 22 3 2 2 to methods reported by Qin et al.70 The membrane potential of a 5.94; found: C, 40.5; H, 3.01; N, 5.22; S, 5.99. r mitochondria was detected according to methods reported by Hu et T al.71 The in vivo experimental procedure for mice was approved by n X-ray crystallography o the 181st Chinese People’s Liberation Army Hospital (Guilin, China) Crystals structures of Rh1 and Rh2 were collected on a Bruker t l and was conducted according to the guidelines provided by NIH for a SMART Apex ІІ CCD diffractometer equipped with a graphite D care and use of laboratory animals in research.72 All experimental monochromated Mo-Kα (λ=0.710 73 Å) at room temperature. details are shown in supplementary materials. Structures were solved using the direct method, Fourier transformations, and SHELXL97 program.64 All non-hydrogen Conclusions atoms were refined using anisotropic thermal parameters. PLATON65 was utilized for various crystal parameters calculations, This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 13 Please do not adjust margins .MP 92:93:3 9102/22/6 no ytisrevinU tnerT mahgnittoN yb dedaolnwoD .9102 enuJ 02 no dehsilbuP View Article Online DOI: 10.1039/C9DT01951K PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 14 of 17 ARTICLE Journal Name Two rhodium(ΙΙΙ) complexes Rh1 and Rh2 with isoquinoline Supporting Information: Vendor codes for Rh1 and Rh2 crystal data, HPLC derivatives as ligands were synthesized and characterized by various data, 1H-NMR, 13C-NMR data, ESI-MS data. The supplementary crystallographic data of this paper can be seen under the CCDC No. physical methods. Rh1 and Rh2 demonstrated considerable in vitro 1851232,1851233. anticancer activity against a variety of tumor cells and promising selectivity towards T-24 cell lines relative to cisplatin. To most of [1] M. S. Jeremić, H. Wadepohl, V. V. Kojić, D. S. Jakimov, R. Jelić, the tested cells, both complexes presented the synergistic effect in S. Popović, Z. D. Matović, P. Comba, RSC Adv., 2017, 7, t p cytotoxicity as compared with the rhodium(III) salt and free ligands. 52825296. i Rh1 and Rh2 generated their anticancer activity by arresting the cell [2] I. Ott, Coord. Chem. Rev., 2009, 253, 16701681. r c cycle at S-phase and inducing apoptosis via mitochondrial s [3] A. Basu, S. Krishnamurthy, J. Nucleic Acids., 2010, 10, 116. u dysfunction. Rh1 exhibited a more favourable in vivo safety profile [4] E. Wong, C. M. Giandomenico, Chem. Rev., 1999, 99, n than cisplatin, and the tumor inhibition ratios were comparable. Rh1 a 24512466. M is a potential leading compound for further development as an [5] M. A. Fuertes, C. Alonso, J. M. Pérez, Chem. Rev., 2003, 103, anticancer agent. d 645662. e Conflicts of interest [6] S. P. Fricker, Dalton Trans., 2007, 49034917. t p [7] R. A. Cairns, I. S. Harris, T. W. Mak, Nat. Rev. Cancer, 2011, 11, e The authors declare no conflict of interest. c 8595. c Acknowledgments A [8] R. J. De Berardinis, N. S. Chandel, Sci. Adv., 2016 2, 118. [9] S. Fulda, L. Galluzzi, G. Kroemer, Nat. Rev. Drug. Discov., 2010, s This work was supported by the National Natural Science n 9, 447464. Foundation of China (Grants 81473102, 21431001), IRT_16R15, o and Natural Science Foundation of Guangxi Province of China [10] S. E. Weinberg, N.S. Chandel, Nat. Chem. Biol., 2015, 11, 915. t i c (Grant 2016GXNSFGA380005) as well as “BAGUI Scholar” [11] D. van Straten, V. Mashayekhi, H. S. de Bruijn, S. Oliveira, D. a s program of Guangxi Province of China. J. Robinson, Cancers, 2017 9, 154. n [12] M. 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