Novel polypyridyl ruthenium complexes acting as high affinity DNA intercalators, potent transcription inhibitors and antitumor reagents

Journal of Inorganic Biochemistry 185 (2018) 1–9

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Novel polypyridyl ruthenium complexes acting as high affinity DNA intercalators, potent transcription inhibitors and antitumor reagents

T

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Guo-Lan Maa, Xu-Dan Bia, Feng Gaoa, , Zheng Fenga, Dong-Chun Zhaoa, Feng-Jie Lina, Ru Yana, ⁎ ⁎ Dandan Liub, , Peng Liua, Jingbo Chena, Hongbin Zhanga, a

Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Functional Molecules Analysis and Biotransformation Key Laboratory of Universities in Yunnan Province, School of Chemical Science and Technology, Yunnan University, Kunming, Yunnan 650091, PR China b School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming, Yunnan 650500, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Ruthenium complex DNA binding Transcription inhibition Antitumor

Six novel polypyridyl ruthenium complexes with (E)-2-styryl-1H- imidazo[4,5-f][1,10]phenanthroline ligand and its analogues have been designed to enhance the DNA intercalation ability of their model compound [Ru (bpy)2(pip)]2+ (bpy = 2,2′-bipyridine, pip = 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline). As shown in the optimized geometry of the complexes, the introduction of styryl group not only extended the conjugated area of the intercalative ligand, but also retained the excellent planarity. These two merits have been proven to be beneficial for their DNA intercalation, thus greatly improved their inhibition activity towards DNA transcription by RNA polymerase and DNA topoisomerase, two enzymes closely related to both DNA and tumor cell growth. The relationships between the substituent group structures and the biological activities have also been investigated from energetic and electronic aspects by quantum chemistry calculations. Results from cell cytotoxicity and apoptosis assay testified that the styryl substituted ruthenium complexes possessed higher antitumor activity than [Ru(bpy)2(pip)]2+, as expected. As quantified in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the tumor cell death is caused mostly through apoptosis for Ru2 and Ru3, while non-apoptotic processes for Ru1, Ru4 and Ru5. In vitro fluorescence evaluation revealed that all complexes located mainly in cytoplasm, but the three complexes with high antiproliferative activity could enter nucleus. All complexes have shown apparent lower cytotoxicity towards normal human colon epithelial cell CCD-841-CON than the examined tumor cell lines.

1. Introduction Since polypyridyl Ru(II) complexes with dppz-type ligands (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) have been found to intercalate into adjacent DNA base pairs, and serving as luminescence switch of DNA [1–5], Ru(II) complexes have attracted a lot of attentions for their easily constructed rigid structures, rich photophysical properties, diverse binding modes of nucleic acids and promising antitumor activity [6–11]. Although the mechanism(s) of action for Ru(II) antitumor complexes remains to be elucidated, a series of polypyridyl Ru(II) complexes have exhibited good topoisomerase, telomerase and transcription inhibition activity and antitumor activity [12–17]. It is well established that the stabilization of DNA duplex by their intercalative ligand plays a very important role in their biological activity. DNA intercalative polypyridyl Ru(II) complexes based on dppz and pip are two ideal models for the develop of DNA binding reagents

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(Fig. 1). [Ru(bpy)2(pip)]2+ (Ru0, bpy = 2,2′-bipyridine, pip = 2phenyl-imidazo[4,5-f]-1,10-phenanthroline) and most of its derivatives have been found to be ideal DNA probes, due to their diverse DNAbinding modes, favored luminescence property and efficient DNA photocleavage effect [16–23]. However, most of the high activity complexes reported recently either have complicated synthesis routes or need high-cost materials, which restrained their further studies in animal experiments and pharmaceutical preparations. In this work, we designed a series of Ru0 analogues (Fig. 1) containing styryl group and developed a simple synthesis route to obtain gram scale of polypyridyl Ru(II) complexes within 8 h using low-cost cinnamaldehydes as starting material. The vinyl group not only expands the plane area of the intercalative ligand, but also keeps a fully conjugated structure of the ligand, giving a positive effect to the π–π stack with DNA base pairs. The DNA binding, DNA transcription (T7 RNA polymerase) inhibition, DNA topoisomerase inhibition, cell

Corresponding authors. E-mail addresses: [email protected] (F. Gao), [email protected] (D. Liu), [email protected] (H. Zhang).

https://doi.org/10.1016/j.jinorgbio.2018.04.019 Received 16 February 2018; Received in revised form 11 April 2018; Accepted 29 April 2018 Available online 02 May 2018 0162-0134/ © 2018 Elsevier Inc. All rights reserved.

Journal of Inorganic Biochemistry 185 (2018) 1–9

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[24–26]. (E)-Cinnamaldehyde, (E)-4-nitrocinnamaldehyde, ammonium hexafluorophosphate, (E,E)-2,4-hexadienal and (E)-4-dimethylaminocinnamaldehyde were purchased from Adamas-beta. (E)-4-methoxycinnamaldehyde was purchased from Acros. Ammonium acetate, glacial acetic acid, aqueous ammonia, ethylene glycol, alumina, acetonitrile, toluene and methanol were purchased from Greagent.

2.2. Syntheses and characterizations 2.2.1. Synthese of [Ru(bpy)2(sip)](PF6)2·2H2O (Ru1, sip = (E)-2-styryl1H-imida-zo[4,5-f][1,10]phenanthroline) A mixture of 1,10-phenanthroline-5,6-dione (0.530 g, 2.50 mmol), (E)-cinnamaldehyde (0.462 g, 3.50 mmol), ammonium acetate (3.88 g, 50.3 mmol) and glacial acetic acid (15 mL) was refluxed for 2 h. After cooling down, 40 mL water was added to the reaction mixture. The ligand sip precipitated as an orange solid by dropwise addition of concentrated aqueous ammonia. The solid was filtered, washed with 50 mL water and dried in vacuo. The obtained ligand (0.622 g, 1.92 mmol) was stirred with cis-[Ru(bpy)2]Cl2·2H2O (0.898 g, 1.73 mmol, 0.9 eq.), 2 mL water and 18 mL ethylene glycol under argon at 120 °C for 4 h. The dark red solution was cooled to room temperature and poured into water (80 mL). The excess ligand was removed by filtration. Upon addition of NH4PF6 (1 g, 6.2 mmol) in water (5 mL) to the filtrate, the complex Ru1 precipitated as red solid, which was isolated by suction filter, washed by water and dried in vacuo. The crude product was purified by flash column chromatography on alumina (200–300 mesh) with acetonitrile/toluene (2:1) as eluent, followed by recrystallization from acetonitrile/ether. Yield: 1.140 g, 68%. Anal. calc. for C41H30F12N8P2Ru·2H2O: C, 46.38; H, 3.23; N, 10.55. Found: C, 46.21; H, 3.48; N, 10.28%. 1H NMR (300 MHz, DMSO‑d6): δ 9.01 (d, J = 8.0 Hz, 2H), 8.87 (d, J = 8.5 Hz, 2H), 8.84 (d, J = 8.6 Hz, 2H), 8.23 (t, J = 7.8 Hz, 2H), 8.12 (d, J = 7.6 Hz, 2H), 8.07 (t, J = 4.7 Hz, 3H), 7.94 (d, J = 5.5 Hz, 1H), 7.91 (d, J = 5.4 Hz, 1H), 7.85 (d, J = 5.0 Hz, 3H), 7.79 (d, J = 6.7 Hz, 2H), 7.61 (q, 4H), 7.49 (q, 3H), 7.43 (t, J = 3.4 Hz, 1H), 7.35 (t, J = 6.5 Hz, 2H). ESI-FTMS (acetonitrile) m/z calc. for C41H30N8Ru ([M–2PF6]2+): 368.0813; Found: 368.0812.

2+

2.2.2. [Ru(bpy)2(nsip)](PF6)2·2H2O (Ru2, nsip = (E)-2-(4-nitrostyryl)1H-imidazo[4,5-f][1,10]phenanthroline) This compound was synthesized according to the same procedure as Ru1, using (E)-4-nitrocinnamaldehyde instead. Yield: 1.370 g, 81%. Anal. calc. for C41H29F12N9O2P2Ru·2H2O: C, 44.49; H, 3.01; N, 11.39. Found: C, 44.31; H, 3.29; N, 11.13%. 1H NMR (300 MHz, DMSO‑d6): δ 8.96 (d, J = 8.2 Hz, 2H), 8.84 (d, J = 10.4 Hz, 2H), 8.81 (d, J = 9.1 Hz, 2H), 8.19 (t, 4H), 8.09 (d, J = 7.8 Hz, 2H), 8.03 (d, J = 5.6 Hz, 2H), 7.98 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 5.4 Hz, 1H), 7.87 (d, J = 5.3 Hz, 1H), 7.82 (d, J = 4.9 Hz, 3H), 7.64 (s, 1H), 7.59 (d, J = 5.5 Hz, 2H), 7.55 (d, J = 6.7 Hz, 2H), 7.32 (t, J = 5.6 Hz, 2H). ESI-FTMS (acetonitrile) m/z calc. for C41H29O2N9Ru ([M–2PF6]2+): 390.5738; Found: 390.5741.

2+

Fig. 1. Structure of the complexes [Ru(bpy)2(dppz)] , [Ru(bpy)2(pip)] (Ru0), and novel complexes Ru1–Ru6 designed in this work.

cytotoxicity, apoptosis and cellular localization assay of the compounds have been carried out to explore their antitumor activities and mechanism. Quantum chemistry calculations based on density functional theory (DFT) have been performed to obtain the molecular conformations, energetic and electronic properties of the complexes, by which to discuss their roles in the biological activities. 2. Experimental 2.1. Materials and methods

2.2.3. [Ru(bpy)2(osip)](PF6)2·2H2O (Ru3, osip = (E)-2-(4-methoxystyryl)1H-imidazo[4,5-f][1,10]phenanthroline) This compound was synthesized according to the same procedure as Ru1, using (E)-4-methoxycinnamaldehyde instead. Yield: 1.070 g, 63%. Anal. calc. for C42H32F12N8OP2Ru·2H2O: C, 46.20; H, 3.32; N, 10.26. Found: C, 45.96; H, 3.59; N, 10.04%. 1H NMR (300 MHz, DMSO‑d6): δ 8.99 (d, J = 8.2 Hz, 2H), 8.88 (d, J = 8.4 Hz, 2H), 8.84 (d, J = 8.4 Hz, 2H), 8.22 (t, J = 7.9 Hz, 2H), 8.11 (t, J = 7.9, 2H), 8.07 (t, J = 5.7, 2H), 7.90 (m, 4H), 7.72 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 5.3 Hz, 2H), 7.59 (d, J = 6.7 Hz, 2H), 7.36(t, J = 6.6 Hz, 2H), 7.29 (s, 1H), 7.24 (s, 1H), 7.01 (d, J = 8.7 Hz, 2H), 3.81 (s, 3H). ESI-FTMS (acetonitrile) m/z calc. for C42H32ON8Ru ([M–2PF6]2+): 383.0866; Found: 383.0869.

1

H NMR spectra were recorded on a Bruker Avance 300 spectrometer at 300 MHz. All chemical shifts are given relative to tetramethylsilane (TMS). Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental analyzer. Electron Spray ionization mass spectra (ESI-MS) were recorded on AB QSTAR Pulsar mass spectrometer or Agilent LC/MSD TOF mass spectrometer. UV–Vis spectra were recorded on a Shimadzu SolidSpec-3700 spectrophotometer equipped with a temperature controller accessory and circulating water system. The compounds 1,10-phenanthroline-5,6-dione, cis-[Ru (bpy)2Cl2]·2H2O and cis-[Ru(dip)2Cl2]·2H2O (dip = 4,7-diphenyl-1,10phenanthroline) were synthesized according to the literature methods 2

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incubation for an additional 4 h. The medium was replaced with 150 μL of DMSO to dissolve the formed formazan salt. The color intensity of the formazan solution, which reflects the cell growth condition, was measured at 570 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA).

2.2.4. [Ru(bpy)2(dsip)](PF6)2·2H2O (Ru4, dsip = (E)-2-(4-dimethylaminostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline) This compound was synthesized according to the same procedure as Ru1, using (E)-4-dimethylaminocinnamaldehyde instead. Yield: 1.130 g, 66%. Anal. calc. for C43H35F12N9P2Ru·2H2O: C, 48.32; H, 3.30; N, 11.79. Found: C, 48.06; H, 3.57; N, 11.52%. 1H NMR (300 MHz, DMSO‑d6): δ 14.04 (s, 1H), 8.88 (d, J = 8.3 Hz, 3H), 8.85 (d, J = 8.4 Hz, 3H), 8.23 (d, J = 6.9 Hz, 2H), 8.19 (d, J = 5.3, 2H), 8.11 (t, 4H), 8.04 (d, J = 4.8 Hz, 3H), 7.61 (d, J = 5.8 Hz, 2H), 7.58 (d, J = 7.4, 2H), 7.58 (d, 4H), 7.34 (t, J = 6.5 Hz, 3H), 3.00 (s, 6H). ESI-FTMS (acetonitrile) m/z calc. for C43H35N9Ru ([M–2PF6]2+): 389.6024; Found: 389.6027.

2.6. Apoptosis assay HCT116 cells were seeded in six-well plates at a density of 2 × 105 cells per well and kept overnight at 37 °C humidified incubator with 5% CO2. The next day, cells were treated with 40 μM of compounds for 72 h. Cells were then collected and stained with Annexin V-FITC (fluorescein isothiocyanate) according to manufacturer's instruction. The apoptosis assay was performed by using BD FACSCalibur flow cytometry.

2.2.5. [Ru(bpy)2(pdip)](PF6)2·2H2O (Ru5, pdip = 2-((1E,3E)-penta-1,3dien-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) This compound was synthesized according to the same procedure as Ru1, using (E,E)-2,4-hexadienal instead. Yield: 1.060 g, 67%. Anal. calc. for C38H30F12N8P2Ru·2H2O: C, 44.50; H, 3.34; N, 10.92. Found: C, 44.26; H, 3.61; N, 10.64%. 1H NMR (300 MHz, DMSO‑d6): δ 9.02 (d, J = 1.1 Hz, 1H), 8.95 (d, J = 1.1 Hz, 1H), 8.87 (d, J = 8.1 Hz, 3H), 8.83 (d, J = 8.2 Hz, 3H), 8.19 (m, 4H), 8.10 (t, J = 7.7 Hz, 3H), 8.03 (d, J = 4.7 Hz, 1H), 7.95 (m, 3H), 7.83 (d, J = 5.0 Hz, 3H), 7.58 (m, 5H), 7.33 (t, J = 6.1 Hz, 3H). ESI-FTMS (acetonitrile) m/z calc. for C38H30N8Ru ([M–2PF6]2+): 350.0813; Found: 350.0819.

2.7. In vitro fluorescence evaluation Cellular localization of the compounds was assessed by confocal laser scanning microscope. HeLa cells were grown on 8 Chamber Glass Slide (Thermo Fisher Scientific, USA) at a density of 6 × 104 cells/mL and incubated for 2 h with the compounds at 40 μM. The cells were washed with PBS (phosphate buffer saline) twice and fixed in 4% formaldehyde solution (10% formaldehyde in 90% PBS) for 15 min. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). The slides were visualized under an Olympus FluoView 1000 confocal microscope (Olympus, Japan).

2.2.6. [Ru(dip)2(dsip)](PF6)2·2H2O (Ru6) This compound was synthesized according to the same procedure as Ru4, using cis-[Ru(dip)2]Cl2·2H2O instead. The column chromatography used methanol/acetonitrile (1:2) as eluent. Yield: 1.240 g, 64%. Anal. calc. for C71H51F12N9P2Ru·2H2O: C, 58.52; H, 3.80; N, 8.65. Found: C, 58.26; H, 4.02; N, 8.38%. 1H NMR (300 MHz, DMSO‑d6): δ 8.39 (d, J = 8.3 Hz, 2H), 8.30–8.27 (m, 8H), 8.16 (d, J = 5.1 Hz, 2H), 7.91 (t, 4H), 7.90 (d, J = 4.8 Hz, 2H), 7.85 (d, J = 4.8 Hz, 2H), 7.80–7.67 (m, 20H), 7.65 (d, J = 6.0, 2H), 6.77 (d, J = 8.4 Hz, 2H), 3.00 (s, 6H). ESI-FTMS (acetonitrile) m/z calc. for C71H51N9Ru ([M–2PF6]2+): 565.6655; Found: 565.6659.

2.8. Topoisomerase inhibition assay DNA Topoisomerase I (Topo) was purchased from Invitrogen. The reaction mixture (20 μL) contained 50 mM Tris HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 0.5 mM DTT (dithiothreitol), 0.1 mM EDTA, 30 μg/ mL BSA, 0.1 μg pBR322 DNA, 2 Unit Topo, and different concentration of Ru(II) complexes. The reaction mixtures were incubated at 37 °C for 30 min. The samples were electrophoresed through 1% agarose (Sangon) in TBE at 90 V for 30 min. The gel was stained with 4S Red Plus Nucleic Acid Stain (Sangon), photographed and quantified on a Bio-Rad gel imaging system. The concentrations of the Ru(II) complex that prevented 50% of the supercoiled DNA from being converted into relaxed DNA (IC50) were calculated by the midpoint concentration for Ru(II) complex induced DNA unwinding.

2.3. DNA binding experiments Calf thymus DNA (CT-DNA) was obtained from the Sigma Company. The preparation of DNA stock solution, determination of the DNA concentration and DNA-binding experiments of the Ru(II) complexes, including absorption titration, calculation of intrinsic binding constants Kb, viscosity measurements, and thermal denaturation studies were performed as described previously [14].

2.9. In vitro transcription inhibition assay The pGEM template DNA produces transcripts that are 1065 and 2346 bases in length with T7 RNA polymerase (T7 RiboMAX Express Large Scale RNA Production System, Promega). The transcription reaction was conducted for 30 min at 37 °C in the presence of MgCl2 and NTPs in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 7.5). The samples were analysed by electrophoresis for 1 h at 70 V on a 1% agarose gel in TBE buffer (89 mM Tris-borate acid, 2 mM EDTA, pH 8.3). The gel was stained and photographed as above. The concentrations of the Ru(II) complexes that prevented 50% of the template DNA from being transcribed to RNA (IC50) was determined from interpolation of plots of percent inhibition as a function of increasing complex concentration.

2.4. Quantum chemistry calculation The DFT calculations were carried out with the Gaussian 09 program package [27] using Becke's three-parameter hybrid functional (B3LYP) method and LanL2DZ basis set (a double-zeta basis set containing effective core potential). The full geometry optimization computations were carried out. The stability of the optimized conformation of the complexes was confirmed by the frequency analysis, which shows no imaginary frequency for each energy minimum. 2.5. Cell cytotoxicity determination

3. Results and discussion

Human cervical cancer cell line HeLa, human colon cancer cell line HCT116, human breast cancer cell line MDA-MB-231, human melanoma cell line A375 and normal human colon epithelial cell CCD-841CON were seeded in 96-well tissue culture plates for 24 h and then incubated with the test compounds at different concentrations for 48 h. After incubation, the medium was aspirated and 20 μL of the MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/mL) in culture medium was added to each well, followed by

3.1. DNA binding study 3.1.1. Absorption spectra titration The absorption spectra of these polypyridyl ruthenium(II) complexes in the absence and presence of increasing concentrations of CTDNA are shown in Fig. 2 (for Ru1) and S1 (for Ru2–Ru5). Typical 3

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Fig. 2. Absorption spectra of Ru1 in 5 mM Tris-HCl and 50 mM NaCl buffer (pH 7.0) in the presence of increasing amounts of CT-DNA. Arrows indicate the change in absorbance upon increasing the DNA concentration. Inset: curve fitting that gave the value of intrinsic binding constants Kb. Table: Kb values for the Ru(II) complexes Ru1–Ru5 in this work, together with the model compound Ru0.

Fig. 3. The melting curves of CT-DNA (100 μM) at 260 nm in the absence and the presence of Ru complexes (10 μM). The values of DNA melting point change (ΔTm) are shown in the parenthesis (inset).

hypochromic effect (22–48%) and red shift (4–8 nm) in the MLCT (metal to ligand charge transfer) and IL (interligand) bands have been observed. The spectral characteristics suggest that there are strong interactions between the complexes and DNA so that the π → π* transition on the ligand is significantly perturbed. To compare quantitatively the DNA binding strength of these complexes, their intrinsic binding constants Kb with CT-DNA at 25 °C are determined from the decay of the absorbance at MLCT peaks with increasing concentration of DNA (Fig. 2). Sip compound Ru1 exhibited 5-fold higher binding constant than pip compound Ru0 [18], suggesting that the introduction of vinyl group has a positive effect on the π–π stack between the ligand and DNA base pairs. The vinyl group not only expands the plane area of the intercalative ligand, but also keeps a fully conjugated structure of the ligand. Ru2, with an additional nitro group, has higher DNA binding ability than Ru1, while Ru3, with a methoxy group, has lower DNA binding ability. It suggests that an electron-withdrawing group on the intercalative ligand is more favourable to the DNA binding than electron-donating group. Similar Kb changes have been observed in the pNO2 (7.3–8.2 × 105 M−1) and p-OMe (4.6–5.0 × 105 M−1) substituent of Ru0 [28,29]. Surprisingly, Ru4, with an electron-donating dimethylamino group, binds DNA much more tightly than does Ru1, Ru2 or Ru3. The underlying mechanism will be discussed in the latter quantum chemistry calculation study. Ru5 is designed to reduce the phenyl group in ligand sip to “half”, to explore if this ligand can stick itself into DNA base pairs like a needle. Both spectral change and calculated Kb value suggests that Ru5 binds to DNA with similar affinity to Ru0. As shown in previous studies, when Ru0 is further functionalized by p-phenyl (7.1 × 104 M−1), p-thiophenyl (2.7 × 105 M−1), p-phenoxyl (4.1 × 104 M−1) or p-(N-piperidinyl) (2.5 × 105 M−1) group, the DNA binding ability will decrease [30–33]. The reason is proposed to be the loss of planarity, as the introduced groups are not coplanar with the phenyl group of pip ligand. On the contrary, increases in the DNA binding ability of Ru0 have been achieved by introducing p-(N-carbazole) (8.2 × 105 M−1) and p-(2-benzo[d]thiazole) (2.3 × 106 M−1) groups, because these two compounds reserved planarity while extending the conjugated structure [34,35]. However, both the material accessibility and the complicated synthesis route restrained the largescale preparation and further studies in medicinal chemistry. Ru1–Ru4 with similar or even higher DNA binding abilities developed in this work could be prepared by one step ligand synthesis from the commonly used 1,10-phenanthroline-5,6-dione and low-cost cinnamaldehydes, followed by the reaction with cis-[Ru(bpy)2Cl2]·2H2O

directly without any purification of the ligands. The complexes could be prepared and purified in gram scale within 8 h, facilitating its use in animal experiments and pharmaceutical preparations in the further. 3.1.2. DNA thermal denaturation The intercalation of nature or synthesized intercalators generally results in a considerable increase in the melting temperature (Tm) of DNA by stabilizing the double-stranded DNA and preventing it from dissociating to single strands [36]. Here we examine the denaturation of free and Ru(II) complexes bound CT-DNA, to evaluate the DNA binding abilities of their complexes from another aspect besides absorption spectra titration. The denaturation curves of CT-DNA in the absence and presence of Ru1–Ru5 are shown in Fig. 3, together with Ru0. The increases of melting temperature (ΔTm) are shown in parenthesis (inset). With the addition of complexes, the Tm of CT-DNA increased remarkably. The large values of ΔTm of these compounds support a strong DNA binding through intercalation mode. The values of ΔTm follow an order of Ru4 > Ru2 > Ru1 > Ru3 > Ru0 > Ru5, which is in accordance with the result of absorption spectra titration. 3.1.3. Viscosity study Viscosity measurements that are sensitive to change in length of the DNA strands are regarded as the least ambiguous and the most critical tests of a binding model in solution in the absence of crystallographic or NMR structural data [37]. To clarify the mode of the interaction between the complexes and DNA, viscosity measurements were carried out. The effects of Ru1–Ru5, together with Ru0, on the viscosity of DNA are shown in Fig. 4. Upon increasing the concentration of the complex, the relative viscosity of DNA solutions increased steadily. It can be concluded that the complexes bind DNA via a classical intercalation model, and the increase in the viscosity of the DNA solution is a result of the DNA helix lengthening, as base pairs are separated to accommodate the intercalating ligands. The increased degree of viscosity depending on its DNA binding strength follow the order of Ru4 > Ru2 > Ru1 > Ru3 > Ru0 > Ru5, which is consistent with that observed in spectral titrations and DNA thermal denaturation. 3.2. Quantum chemistry calculation Electronic structure calculations on density functional theory were 4

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Table 1 The calculated lowest unoccupied molecular orbital (LUMO) energy levels and charge populations of Ru1–Ru5. Compound

ELUMO (a.u.)a

CRub

CLc

Ru1 Ru2 Ru3 Ru4 Ru5

−0.2712 −0.2762 −0.2687 −0.2642 −0.2709

0.8918 0.8920 0.8915 0.8910 0.8927

0.4115 0.3972 0.4200 0.4352 0.6879

a

LUMO energy level of the Ru complexes. Charge on Ru atom. c Charge populated on the intercalative ligand (Sum of the charges on each atom of L [39]). b

low ELUMO and high CL than Ru4, although it shows the lowest DNA binding ability in the experiments. This could be explained by the dienyl group cannot stably stack with adjacent DNA base pairs as the phenyl group does. However, it is still interesting to explore other biological activities of Ru5 with such good energetic and electronic properties. 3.3. DNA transcription inhibition The inhibition of transcription was examined by the amount of the RNA produced by T7 RNA polymerase during the transcription reaction of pGEM template DNA at different concentrations of the complexes, keeping the concentrations of all other components constant. As shown in Fig. S3, the produced mRNA decreased relative to the control lane as the concentration of complex increased. The IC50 values of Ru1–Ru5 (Table 2) are quite comparable to those of a series of DNA intercalative dirhodium(II) complexes [40–43] and some polypyridyl Ru(II) complexes recently reported by us [14,43–45]. Ru4 has 7-fold higher transcription inhibition activity than [Ru(bpy)2(dppz)]2+ (IC50 = 4.2 μM). The activities of Ru1–Ru3 are also higher than that of [Ru(bpy)2(dppz)]2+. Although Ru5 has the lowest activity among these five complexes, the activity is still higher than most reported DNA-intercalative Ru(II) complexes [14,44–46].

Fig. 4. Effect of increasing amounts of Ru complexes on the relative viscosity of CT-DNA at 28 ± 0.1 °C. The total concentration of DNA is 100 μM.

3.4. Topoisomerase inhibition Topoisomerase I (TopoI) makes transient breaks in single strand of DNA molecule allowing the passage of the other DNA strand through the gap. DNA intercalative Ru(II) complexes can inhibit the activity of TopoI as catalytic inhibitors or poisons. The results of concentrationdepended TopoI inhibition assay of Ru1–Ru5 are shown in Fig. S4. The negatively supercoiled plasmid DNA can be entirely relaxed by TopoI in the absence of the Ru(II) complexes. As the concentration of the complexes increased, the amount of the relaxed DNA decreased gradually. The IC50 values are shown in Table 2. The TopoI inhibition activity of Ru1 (IC50 = 2.8 μM) is higher than classical organic TopoI inhibitors camptothecin (IC50 = 17 μM), topostatin (IC50 = 17 μM) and Hoechst 33,258 (IC50 = 30 μM), and most polypyridyl Ru(II) complexes reported recently [16,17,44–46]. Ru2–Ru4 also have good inhibition activity, with IC50 < 20 μM. Under the examined concentrations, Ru5

Fig. 5. Optimized geometry of Ru1 by DFT calculation (B3LYP/LanL2DZ). The numbers represent dihedral angles marked with zigzag patterns.

conducted on the cations [Ru(bpy)2(L)]2+ to assess the effects of the ligands L on the molecular orbital energy levels of their complexes Ru1–Ru5 with which to understand how their DNA binding abilities are modulated by the ligand structures. As shown in the optimized geometry of Ru1 (Fig. 5), the illustrated dihedral angles are around 180°. Similar low torsions are observed in the structure of Ru2–Ru5 (Fig. S2). It is suggested that the excellent planarity of these ligands renders their complexes high DNA binding abilities. In the π-π interaction between DNA and [Ru(bpy)2(L)]2+, DNA base pairs are electron donors and the complexes are electron acceptors (through L) because the highest occupied molecular orbital (HOMO) energy level of DNA is relative high [38]. The calculated lowest unoccupied molecular orbital (LUMO) energy levels of Ru1–Ru5 are listed in Table 1. It is predictable that a lower energy level of LUMO should aid the π-π interaction between the intercalative ligands and adjacent DNA base pairs. For Ru1–Ru3, the values of ELUMO follow an order of Ru2 < Ru1 < Ru3, which is consistent with the experimental results. For Ru4, although ELUMO is slightly higher than Ru1–Ru3, the positive charges locate more on its ligand nsip (CL, Table 1), facilizing its interaction with electron-rich DNA base pairs. Surprisingly, Ru5 has both

Table 2 Inhibitory activity data (IC50) of Ru1–Ru5 towards T7 DNA polymerase (T7) and DNA topoisomerase I (Topo).

5

Compound

IC50 (T7, μM)

IC50 (TopoI, μM)

Ru1 Ru2 Ru3 Ru4 Ru5

3.2 2.0 2.4 0.6 6.4

2.8 10.0 6.0 16.0 > 70

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Table 3 Cytotoxicities (IC50, μM) of Ru1–Ru5 and cisplatin towards human cancer and normal cell lines by MTT method. Compound

HCT116

MDA-MB-231

A375

Hela

CCD-841-CON

Ru1 Ru2 Ru3 Ru4 Ru5 Cisplatin

11.41 ± 1.28 19.68 ± 1.07 22.26 ± 0.87 9.69 ± 0.23 17.94 ± 1.59 5.83 ± 0.61

36.62 ± 3.91 53.89 ± 4.25 62.21 ± 4.58 24.05 ± 1.92 35.57 ± 2.90 5.75 ± 0.16

21.88 ± 0.86 53.89 ± 4.25 62.21 ± 4.58 17.14 ± 0.18 34.73 ± 2.57 5.49 ± 0.28

21.12 ± 0.98 23.77 ± 0.91 24.34 ± 2.20 21.12 ± 1.59 48.63 ± 4.92 7.92 ± 0.22

208.1 ± 8.9 229.5 ± 9.6 206.1 ± 8.2 196.3 ± 7.4 243.4 ± 8.3 8.23 ± 0.26

Fig. 6. Apoptosis of HCT116 cells treated with 40 μM of compounds Ru1–Ru5 and DMSO for 72 h.

not fully in accordance with its DNA binding and enzyme inhibiting ability, suggesting there might be other potential targets in these tumor cells for this compound. All Ru(II) complexes in this study have high antiproliferative activity towards HCT116 cell line. To the best of our knowledge, no antitumor study has been reported on [Ru(bpy)2L]2+ type DNA intercalative complexes towards this cell line. Considering all types of ruthenium containing compounds, only very recently, Bezerra's group has reported the antitumor activity of two types of non-DNA intercalative Ru(II)/diphosphine/diimine complexes towards HCT116 [48,49]. The mechanism is suggested to be apoptosis through caspasedependent and mitochondrial intrinsic pathway. Therefore, apoptosis assay and cellular localization study were carried out below. Cell cytotoxicity towards a normal cell line (human colon epithelial cell CCD-841-CON) was tested for Ru1–Ru5. All the complexes showed very low toxicity towards this normal cell line (Table 3), suggesting they have good potential as antitumor drugs. In summary, depending on this cytotoxicity screen, the Ru(II) complexes Ru1–Ru5 have moderate antitumor activity towards four different tumor cell lines and very low toxicity towards normal cell line CCD-841-CON. The dimethylamino substituted compound Ru4 has the highest antiproliferative activity.

did not exhibit any TopoI inhibition activity, indicating TopoI is not a biological target for this complex. 3.5. Antitumor study 3.5.1. Cell cytotoxicity determination All complexes were tested for their cytotoxicity against four different cell lines (human cervical cancer HeLa, human colon cancer HCT116, human breast cancer MDA-MB-231 and human melanoma A375). The complexes induced significant cell death in a dose-dependent manner. The IC50 data of Ru1–Ru5, together with cisplatin (positive control) are shown in Table 3. Cisplatin has a IC50 value of 7.92 ± 0.22 μM for Hela cell in this study, comparable to the value of 5.9 ± 1.2 μM, reported by another group [47]. Ru1–Ru4 have very similar cytotoxicities to Hela cell, with IC50 values from 21.12 to 24.34 μM, higher than most derivatives of Ru0 recently reported, such as [Ru(bpy)2depip]2+ (IC50 = 44.3 ± 6.3 μM) [23]. For the other three cell lines, Ru4 showed the highest antiproliferative activity, which is attributed to its highest DNA binding ability and transcription inhibition activity. Ru5 has a lower inhibition activity towards Hela cell (IC50 = 48.63 ± 4.92 μM) than Ru1–Ru4, but higher inhibition activity towards the other three cell lines than Ru2 and Ru3. This result is 6

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Fig. 7. Cellular localization of Ru1–Ru5 visualized by confocal laser scanning microscope (Nuclei were counterstained with DAPI).

control) shows that all these complexes induce apoptosis (Fig. 6). The HCT116 cells treated with Ru1 show 21.1% of cells in apoptotic stage. Compared with the results from MTT assay stated above, Ru1 is an antitumor compound with both apoptosis-inducing and antiproliferative activity. In contrast, treatment of Ru2–Ru4 shows an apoptotic cell population of 42.1%, 54.1% and 26.5%, respectively, which are all higher than that of Ru1. It is evident that, although Ru2 and Ru3 show lower antiproliferative activities than Ru1 and Ru4 in MTT assay, they have much higher apoptosis-inducing activity than

3.5.2. Apoptosis assay To further explore the antitumor activity and mechanism of these complexes, Annexin V FITC staining and flow cytometric analysis were performed. One of the early events of apoptosis is the translocation of phosphatidyl serine (PS) from the inner leaflet of the plasma membrane to the outer leaflet. This event exposes the PS to the outer cellular environment which can be detected by FITC labelled Annexin V which has high affinity to PS [50]. Treatment of HCT116 cells with Ru1–Ru5 (DMSO as negative 7

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Ru1 and Ru4. For Ru5, however, only 12.0% apoptotic cell population is observed, suggesting a low apoptosis-inducing activity. Considering together with the cell cytotoxicity results above, all complexes have good antitumor activities. The tumor cell death is caused mostly through apoptosis for Ru2 and Ru3, while non-apoptotic processes for Ru1, Ru4 and Ru5.

the normal cell line, suggesting they have good potential as antitumor drugs. Abbreviations bpy pip dppz dip sip nsip osip

2,2′-bipyridine 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline dipyrido[3,2-a:2′,3′-c]phenazine 4,7-diphenyl-1,10-phenanthroline (E)-2-styryl-1H-imida-zo[4,5-f][1,10]phenanthroline (E)-2-(4-nitrostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline (E)-2-(4-methoxystyryl)-1H-imidazo[4,5-f][1,10]phenanthroline dsip (E)-2-(4-dimethyl-aminostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline pdip 2-((1E,3E)-penta-1,3-dien-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline depip 2-(4-(diethoxymethyl)-1H-imidazo[4,5-f][1,10]phenanthroline CT-DNA calf thymus DNA MTT 2-(4,5-Dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazol-3-ium HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid DMSO dimethyl sulfoxide DTT dithiothreitol FITC fluorescein isothiocyanate DAPI 4′,6-diamidino-2-phenylindole PBS phosphate buffer saline PS phosphatidyl serine DFT density functional theory HOMO highest occupied molecular orbital LUMO lowest unoccupied molecular orbital

3.5.3. In vitro fluorescence evaluation Fluorescence microscopy has been used to obtain further information on the exact cellular localization of Ru1–Ru5. As shown in Fig. 7, all complexes entered cell membrane very well, indicating by the apparent red areas. Most polypyridyl Ru(II) complexes have been reported to be mitochondria targeted and difficult to enter the nucleus [21,51]. Here, live-cell imaging showed that Ru1, Ru2 and Ru3 were not limited to the cytoplasm, where mitochondria located, but appeared in the nuclei regions of HeLa cells. From Fig. 7, there are obvious overlaps between the red fluorescence of Ru(II) complexes and the blue fluorescence of DAPI, a dye for nucleus. The hydrophobicity, increased by the introduction of vinyl group, and the size, not greatly increased by the substituent group, are two possible contributions to the nucleus entering. However, no Ru4 has been found in the nucleus. Some bright points have been found in the cytoplasm, indicating a great aggregation extent and excellent cellular uptake efficiency of Ru4. This suggests that although Ru4 has very high DNA-binding and enzyme inhibition activity, it binds to other targets in the cytoplasm before it enters the nucleus. Previous studies reported that Ru(II) complexes can induce cancer cell apoptosis by activating the mitochondrial intrinsic pathways [47,48,52]. Therefore, the high accumulation of Ru4 in the cytoplasm, where mitochondria located, might be an important factor for its higher antitumor activity than the other four complexes. 3.6. Attempts to further modification of Ru4

Acknowledgements [Ru(dip)2(dppz)]2+, containing two hydrophobic 4,7-diphenyl1,10-phenanthroline ligands, has been reported to have 25-fold higher cellular uptake efficiency than [Ru(bpy)2(dppz)]2+ [53]. Therefore, we further modified Ru4, which has the highest antitumor activity among the Ru(II) complexes in this study, with dip ligand. Regretfully, [Ru (dip)2(dsip)]2+ (Ru6) could hardly dissolve in water or aqueous solution of 10% DMSO. When incubated with Hela cells, Ru6 precipitated, as shown in the photograph taken under white light (Fig. S5), and no exact uptake amount of Ru6 could be obtained. At the same time, cell cytotoxicity and apoptosis assay were also attempted for Ru6, but still failed because its low solubility. Therefore, no further DNA binding and enzyme inhibition study has been carried out for this compound.

This work was supported by National Natural Science Foundation of China (21662039) and grant from the Program for Changjiang Scholars and Innovative Research Team in University (IRT17R94). F. Gao thanks High Performance Computing Center of Yunnan University for computational support. D. Liu thanks the support of grants from the Yunnan Provincial Science and Technology Department (2017FE468(-022)), Yunnan Provincial Department of Education (2017zzx196) and Kunming Medical University Hundred Talent Program (2018LDD01). H. Zhang thanks the support of grants from National Natural Science Foundation of China (U1702286 and 21332007). Appendix A. Supplementary data

4. Conclusions Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jinorgbio.2018.04.019.

Six novel polypyridyl Ru(II) complexes with styryl substituted pip ligand and its analogues have been designed to enhance the DNA intercalation ability of their model compound Ru0. It is proven that the introduction of styryl group retains excellent planarity while extends the conjugated area of the intercalative ligand. The designed complexes bind DNA much more tightly than Ru0 through intercalation. As a result, their inhibition activity towards DNA transcription by RNA polymerase is greatly improved. Also, they are good topoisomerase I inhibitors, except Ru5. Ru4 could inhibit DNA transcription with IC50 of 0.6 μM and Ru1 could inhibit Topoisomerase I with IC50 of 2.8 μM, both represent the top activities among reported Ru(II) complexes and organic drugs. Results from cell cytotoxicity and apoptosis assay testified that these complexes exhibited higher antitumor activity than Ru0, as expected. As quantified in the MTT assay, the tumor cell death is caused mostly through apoptosis for Ru2 and Ru3, while non-apoptotic processes for Ru1, Ru4 and Ru5. In vitro fluorescence evaluation revealed that all complexes located mainly in cytoplasm, but Ru1–Ru3 could enter nucleus. All the complexes showed very low cytotoxicity towards

References [1] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 112 (1990) 4960–4962. [2] A.E. Friedman, C.V. Kumar, N.J. Turro, J.K. Barton, Nucleic Acids Res. 19 (1991) 2595–2602. [3] R.M. Hartshorn, J.K. Barton, J. Am. Chem. Soc. 114 (1992) 5919–5925. [4] C.J. Cardin, J.M. Kelly, S.J. Quinn, Chem. Sci. 8 (2017) 4705–4723. [5] A.K.F. Martenssona, P. Lincoln, Phys. Chem. Chem. Phys. (2018), http://dx.doi. org/10.1039/C7CP03184J. [6] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777–2795. [7] B. Elias, A. Kirsch-De Mesmaeker, Coord. Chem. Rev. 250 (2006) 1627–1641. [8] M.J. Clarke, Coord. Chem. Rev. 236 (2003) 209–233. [9] L.N. Ji, X.H. Zou, J.G. Liu, Coord. Chem. Rev. 216–217 (2001) 513–536. [10] F. Gao, H. Chao, L.N. Ji, Chem. Biodivers. 5 (2008) 1962–1979. [11] L. Zeng, P. Gupta, Y. Chen, E. Wang, L.N. Ji, H. Chao, Z.S. Chen, Chem. Soc. Rev. 46 (2017) 5771–5804. [12] F. Gao, H. Chao, J.Q. Wang, Y.X. Yuan, B. Sun, Y.F. Wei, B. Peng, L.N. Ji, J. Biol. Inorg. Chem. 12 (2007) 1015–1027. [13] F. Gao, H. Chao, F. Zhou, X. Chen, Y.F. Wei, L.N. Ji, J. Inorg. Biochem. 102 (2008)

8

Journal of Inorganic Biochemistry 185 (2018) 1–9

G.-L. Ma et al.

[30] L. Tan, H. Chao, Y. Zhou, L.N. Ji, Polyhedron 26 (2007) 3029–3036. [31] B. Pedras, R.M.F. Batista, L. Tormo, S.P.G. Costa, M.M.M. Raposo, G. Orellana, J.L. Capelo, C. Lodeiro, Inorg. Chim. Acta 381 (2012) 95–103. [32] L. Tan, H. Chao, H. Li, Y. Liu, B. Sun, W. Wei, L.N. Ji, J. Inorg. Biochem. 99 (2005) 513–520. [33] V.R. Kumar, P. Nagababu, G. Srinivas, M.R. Reddy, M.V. Rani, M. Ravi, S. Satyanarayana, J. Coord. Chem. 70 (2017) 3790–3809. [34] Y. Lu, L. Gao, M. Han, K. Wang, Eur. J. Inorg. Chem. (2006) 430–436. [35] B. Sun, J. Guan, L. Xu, B. Yu, L. Jiang, J. Kou, L. Wang, X. Ding, H. Chao, L.N. Ji, Inorg. Chem. 48 (2009) 4637–4639. [36] M.J. Neyhart, N. Grover, S. Smith, R.W.A. Kalsbeck, T.A. Fairly, M. Cory, H.H. Thorp, J. Am. Chem. Soc. 115 (1993) 4423–4428. [37] S. Satyanarayana, J.C. Daborusak, J.B. Chaires, Biochemistry 32 (1993) 2573–2584. [38] N. Kurita, K. Kobayashi, Comput. Chem. 24 (2000) 351–357. [39] L. Xu, S. Shi, J. Li, S. Liao, K. Zheng, L.N. Ji, Dalton Trans. (2008) 291–301. [40] P.K.L. Fu, P.M. Bradley, C. Turro, Inorg. Chem. 42 (2003) 878–884. [41] K. Sorasaenee, P.K.L. Fu, A.M. Angeles-Boza, K.R. Dunbar, C. Turro, Inorg. Chem. 42 (2003) 1267–1271. [42] H.T. Chifotides, P.K.L. Fu, K.R. Dunbar, C. Turro, Inorg. Chem. 43 (2004) 1175–1183. [43] J.D. Aguirre, H.T. Chifotides, A.M. Angeles-Boza, A. Chouai, C. Turro, K.R. Dunbar, Inorg. Chem. 48 (2009) 4435–4444. [44] X. Chen, F. Gao, Z.X. Zhou, W.Y. Yang, L.T. Guo, L.N. Ji, J. Inorg. Biochem. 104 (2010) 576–582. [45] X. Chen, F. Gao, W.Y. Yang, Z.X. Zhou, J.Q. Lin, L.-N. Ji, Chem. Biodivers. 10 (2013) 367–384. [46] X. Chen, F. Gao, W.Y. Yang, J. Sun, Z.X. Zhou, L.N. Ji, Inorg. Chim. Acta 378 (2011) 140–147. [47] D. Sun, Y. Liu, D. Liu, R. Zhang, X. Yang, J. Liu, Chem. Eur. J. 18 (2012) 4285–4295. [48] C.O. D'Sousa, J.H. Araujo, I.R.S. Baliza, R.B. Dias, F. Valverde, M.T.A. Vidal, C.B.S. Sales, C.A.G. Rocha, D.R.M. Moreira, M.B.P. Soares, A.A. Batista, D.P. Bezerra, Oncotarget 8 (2017) 104367–104392. [49] V.R. Silva, R.S. Correa, L. de Souza Santos, M.B.P. Soares, A.A. Batista, D.P. Bezerra, Sci. Rep. 8 (2018) 288. [50] B. Schutte, R. Nuydens, H. Geerts, F. Ramaekers, J. Neurosci. Methods 86 (1998) 63–69. [51] J. Wang, P. Zhang, C. Qian, X. Hou, L.N. Ji, H. Chao, J. Biol. Inorg. Chem. 19 (2014) 335–348. [52] X.J. Meng, M.L. Leyva, M. Jenny, I. Gross, S. Benosman, B. Fricker, Cancer Res. 69 (2009) 5458–5466. [53] C.A. Puckett, J.K. Barton, J. Am. Chem. Soc. 129 (2007) 46–47.

1050–1059. [14] F. Gao, X. Chen, J.Q. Wang, Y. Chen, H. Chao, L.N. Ji, Inorg. Chem. 48 (2009) 5599–5601. [15] Q. Yu, Y. Liu, J. Zhang, F. Yang, D. Sun, D. Liu, Y. Zhou, J. Liu, Metallomics 5 (2013) 222–231. [16] G. Liao, X. Chen, J. Wu, C. Qian, Y. Wang, L. N, H. Chao Ji, Dalton Trans. 44 (2015) 15145–15156. [17] K. Du, J. Liang, Y. Wang, J. Kou, C. Qian, L.N. Ji, H. Chao, Dalton Trans. 43 (2014) 17303–17316. [18] J.Z. Wu, B.H. Ye, L. Wang, L.N. Ji, J.Y. Zhou, R.H. Li, Z.Y. Zhou, J. Chem. Soc. Dalton Trans. (1997) 1395–1401. [19] H. Xu, K.C. Zheng, Y. Chen, Y.Z. Li, L.J. Lin, H. Li, P.X. Zhang, L.N. Ji, Dalton Trans. (2003) 2260–2268. [20] M.J. Han, L.H. Gao, K.Z. Wang, New J. Chem. 30 (2006) 208–214. [21] Q. Yu, Y. Liu, L. Xu, C. Zheng, F. Le, X. Qin, Y. Liu, J. Liu, Eur. J. Med. Chem. 82 (2014) 82–95. [22] S. Zhang, Q. Wu, H. Zhang, Q. Wang, X. Wang, W. Mei, X. Wu, W. Zheng, J. Inorg. Biochem. 176 (2017) 113–122. [23] R.K. Vuradi, K. Dandu, P.K. Yata, R.M. Vinoda, R.R. Mallepally, N. Chintakuntla, R. Ch, S.S. Thakur, C.M. Rao, S. Satyanarayana, New J. Chem. 42 (2018) 846–859. [24] M. Yamada, Y. Tanaka, Y. Yoshimato, S. Kuroda, I. Shimao, Bull. Chem. Soc. Jpn. 65 (1992) 1006–1011. [25] B.P. Sullivan, D.J. Salmon, T.J. Meyer, Inorg. Chem. 17 (1978) 3334–3341. [26] R. Caspar, C. Cordier, J.B. Waern, C. Guyard-Duhayon, M. Gruselle, P.L. Floch, H. Amouri, Inorg. Chem. 45 (2006) 4071–4078. [27] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Gaussian, Inc., Wallingford CT, 2009. [28] H. Xu, K.C. Zheng, H. Deng, L.J. Lin, Q.L. Zhang, L.N. Ji, New J. Chem. 27 (2003) 1255–1263. [29] S. Shi, J. Liu, J. Li, K.C. Zheng, C.P. Tan, L.M. Chen, L.N. Ji, Dalton Trans. (2005) 2038–2046.

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