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Lapachol in the Design of a New Ruthenium(II)-Diphosphine Complex as a Promising Anticancer Metallodrug.
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Lapachol and lawsone in the design of new ruthenium(II)diphosphine complexes as promising anticancer metallodrugs
Katia M. Oliveira, João Honorato, Felipe C. Demidoff, Mario
S. Schultz, Chaquip D. Netto, Marcia R. Cominetti, Rodrigo S.
Correa, Alzir A. Batista
PII:
S0162-0134(20)30317-2
DOI:
https://doi.org/10.1016/j.jinorgbio.2020.111289
Reference:
JIB 111289
To appear in:
Journal of Inorganic Biochemistry
Received date:
29 July 2020
Revised date:
8 October 2020
Accepted date:
17 October 2020
Please cite this article as: K.M. Oliveira, J. Honorato, F.C. Demidoff, et al., Lapachol
and lawsone in the design of new ruthenium(II)-diphosphine complexes as promising
anticancer metallodrugs, Journal of Inorganic Biochemistry (2018), https://doi.org/
10.1016/j.jinorgbio.2020.111289
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© 2018 Published by Elsevier.
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Lapachol and Lawsone in the Design of New Ruthenium(II)-Diphosphine
Complexes as Promising Anticancer Metallodrugs
Katia M. Oliveiraa,d,*, João Honoratoa, Felipe C. Demidoffb, Mario S. Schultzb, Chaquip D.
Nettob, Marcia R. Cominettic, Rodrigo S. Corread, Alzir A. Batistaa,*
a
Departamento de Química, Universidade Federal de São Carlos (UFSCar), CP 676, CEP
13565-905, São Carlos - SP, Brazil
b
Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Campus Aloísio
c
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Teixeira, CEP 27930-560, Macaé-RJ, Brazil
Departamento de Gerontologia, Universidade Federal de São Carlos (UFSCar), CP 676,
CEP 13565-905, São Carlos – SP, Brazil
Departamento de Química, ICEB, Universidade Federal de Ouro Preto (UFOP), CEP
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35400-000, Ouro Preto – MG, Brazil
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d
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* Corresponding authors: e-mail: kmoliveiraq@gmail.com (Katia M. Oliveira) and
daab@ufscar.br (Alzir A. Batista). Tel.: +55 16 33518285; Fax: +55 16 33518350.
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Abstract
The development of metal complexes containing natural products is a remarkable
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strategy to develop new anticancer candidates. Thus, we report here on the preparation of two
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new Ru(II)/diphosphine complexes containing Lapachol (Lap) and Lawsone (Law): (1)
[Ru(Lap)(dppm)2]PF6
and
(2)
[Ru(Law)(dppm)2]PF6,
where
dppm
=
bis(diphenylphosphino)methane. The complexes were synthetized and fully characterized by
elemental analyses, molar conductivity, UV-Vis, IR, 31P{1H}, 1H and 13C NMR, and the
crystal structure of the complex (1) was determined by X-ray diffraction. Complexes (1) and
(2) showed high in vitro cytotoxicity against four cancer cells (MDA-MB-231, MCF-7, A549
and DU-145), with IC50 values in the micromolar range (0.03 to 2.70 M). Importantly,
complexes (1) and (2) were more active than the cisplatin, the drug used as a reference in the
cytotoxic assays. Moreover, complex (1) showed high selectivity to triple-negative breast
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cancer cells (MDA-MB-231). Studies of the mechanism of action in MDA-MB-231 cancer
cells showed that complex (1) inhibit cell migration, colony formation, and induces cell cycle
arrest and apoptosis by activation of the mitochondrial pathway through the loss of
mitochondrial membrane potential (m). Furthermore, the complex (1) induces ROS
generation in MDA-MB-231 cells, which can cause DNA damage. Finally, complexes (1) and
(2) interact with DNA by minor grooves and show a moderate interaction with BSA, with the
involvement of hydrophobic interactions. Essentially, Ru(II)/diphosphine-naphthoquinone
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complexes have remarkable cytotoxic effects with high selectivity to triple-negative breast
cancer (MDA-MB-231) and could be promising anticancer candidates for cancer treatment.
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Keywords: Ruthenium complexes, antitumor, naphthoquinones, breast cancer, apoptosis,
ROS generation
Introduction
Over the last few decades, the development of anticancer agents containing metal has
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grown considerably, especially after the discovery of the cisplatin anticancer properties[1,2].
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Since cisplatin was clinically approved for cancer treatment by the FDA in 1978, a large
number of cisplatin analogs have been developed. Among them, carboplatin and oxaliplatin,
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have shown to be extremely successful as anticancer drugs[3–5]. Despite their effectiveness,
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platinum compounds showed clinical harms, as its toxicity leads to several undesired side
effects, as well as provokes drug resistance. In addition, its limited solubility in aqueous
solutions hampers its extensive use[6]. Thus, many research groups have focused on the
development of more effective and less toxic new metal-based drugs. Within this framework,
the development of new ruthenium-based compounds has emerged as a promising choice for
the design of new anticancer drugs[7–11].
Ruthenium is an attractive alternative to platinum compounds due to its rich synthetic
and coordination chemistry, the possibility to adopt different oxidation states, mainly II and
III, in physiological conditions. Moreover, ruthenium compounds have presented distinct
mechanisms of action from those of platinum, with low systemic toxicity[6,12].
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The first works involving ruthenium complexes were developed by M. J. Clarke and
co-workers in the 1980s, who investigated compounds such as fac-[RuCl3(NH3)3] and cis[RuCl2(NH3)4]Cl[13]. After that, several reports of ruthenium complexes as anticancer agents
were published[8,14]. To date, only three Ru-based compounds reached clinical trials as
candidates for anticancer drugs: NAMI-A, KP1019, and NKP1339, the sodium salt analog of
KP1019. NAMI-A acts as a potent antimetastatic drug, whereas KP1019 prevents the growth
of platinum-resistant colorectal cancers[15]. It seems that these compounds have a
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multifactorial and multitarget mode of action, however their mechanism of action is still
largely unclear[15]. It is also worth mentioning that the NAMI-A compound was already
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ruled out[16]. Furthermore, KP1019 has completed phase I trials and NPK1339 is in clinical
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development[17,18].
More recently, the Ru(II) polypyridyl complex called TLD-1433, entered phase II
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clinical trial for Non-Muscle Invasive Bladder Cancer (NMIBC), for photodynamic therapy
death[19,20].
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(PDT). TLD1433 acts as a photosensitizer and produces singlet oxygen to cause cancer cell
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The incorporation of ligands that can increase lipophilicity, and consequently, the
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cellular uptake and intracellular distribution is an interesting strategy for the development of
potential metallodrugs. In this sense, natural products, presenting known biological activities,
emerge as a promising class of compounds that can act as ligands. Recently, we have used
this strategy and many classes of natural products have been chosen to obtain active
ruthenium complexes[21–26].
Due to the favorable results observed for ruthenium complexes containing natural
products on cancer cells, in this work, we designed new Ru(II)/diphosphine complexes
containing Lapachol (Lap) and Lawsone (Law) as ligands. Lapachol (2-hydroxy-3-(3methylbut-2-en-1-yl)naphthalene-1,4-dione) and Lawsone (2-hydroxy-1,4-naphthoquinone)
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belong to the naphthoquinone class, which can be of natural or synthetic origin. These
naphthoquinones exhibited antibacterial, antiparasitic and anticancer biological properties,
among others[27,28].
Usually, Lap and Law can act as O,O-bidentate anionic ligands forming stable metal
complexes. Ruthenium complexes containing these naphthoquinones showed promising
activities against several cancer cell lines, confirming the synergistic effect of the
combination of metal and bioactive ligand[22,23,29].
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In order to investigate the influence of these naphthoquinones in a complex containing
two diphosphine as ligands, we report here on the syntheses, characterization, and biological
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activities of two new ruthenium(II)/diphosphine-naphthoquinone complexes on different
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tumor and non-tumor cell lines.
Materials and methods
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Materials
All reagents and chemicals used were from analytical or pure grade, and the solvents
using
Schlenk
techniques.
The
RuCl3.nH2O,
KPF6,
Lawsone
(Law),
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conditions
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were purified based on appropriate drying agents. The synthesis was performed under inert
bis(diphenylphosphino)methane (dppm), Calf-Thymus DNA (CT-DNA), pBR322 plasmid
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DNA from E. coli, Bovine Serum Albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) and 2’,7’-dichlorofluorescin diacetate (H2DCFDA) were
purchased from Sigma-Aldrich® MERCK (St. Louis, MO, USA) and used as received.
Lapachol (Lap) was obtained according to the procedure described in the literature[30].
Instrumentation
FT-IR spectra were acquired as KBr pellets on a Bomem-Michelson FT-MB-102
spectrophotometer, in the range of 4000 and 200 cm. UV-Vis spectra were recorded on a
Hewlett Packard diode array - 8452A spectrophotometer. Elemental analyses for C and H
were determined using an CHNS Element Analyzer (Thermo Scientific, Fisons EA 1108
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model). Molar conductivity was obtained using a MeterLab CDM2300 at room temperature.
Electrochemical experiments were performed using a Bioanalytical Systems Inc, model BAS100B/W at room temperature in CH2Cl2 containing 0.10 M of tetrabutylammonium
perchlorate (Bu4NOH). The reference electrode used was Ag/AgCl (0.10 M Bu4NOH in
CH2Cl2) in a Luggin capillary probe and the working and auxiliary electrodes were stationary
platinum foils. 31P{1H} (162 MHz), 1H (400 MHz) and 13C{1H} (100.62 MHz) NMR spectra
were recorded on a 9.4 T Bruker Avance III 400 MHz spectrometer using a 5 mm internal
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diameter indirect probe with Automatic Tuning Matching.
General procedure to obtain Ru(II)/diphosphine-naphthoquinone complexes
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Firstly, the precursor complex cis-[RuCl2(dppm)2] was obtained according to what
was described by Chatt and Sullivan[31,32]. The naphthoquinone (Lap or Law) (0.14 mmol)
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was added in methanol solution with 50 L of triethylamine (Et3N). After 30 minutes, the
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precursor complex cis-[RuCl2(dppm)2] (0.11 mmol, 100 mg) and KPF6 (0.21 mmol, 39 mg)
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were added and the reaction was kept under argon atmosphere and reflux for 18 h.
Afterwards, the volume of the solution was reduced, and the precipitate formed was collected
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by filtration. The solid was washed with water, ethyl ether, and dried under vacuum.
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(1) [Ru(Lap)(dppm)2]PF6. Dark green solid. Yield: 93 mg (70 %). Elemental analysis for
[RuC65H57F6O3P5]: exp. (calc): C, 61.74 (62.15) and H, 4.80 (4.57)%. ΛM = 54.6 Ω−1 cm2
mol−1, in 1.0 mM CH2Cl2 solution. IR (cm-1): v(C1=O) 1545, v(C4=O) 1612, v(C2O) 1101,
v(P-F) 841 and 557, v(Ru-N) 480 v(Ru-O) 517. Molar extinction coefficient (ε(𝛌, nm), M-1 cm1
): ε(258 nm): 4.74 × 104, ε(365 nm): 6.31 × 103, ε(448 nm): 3.77 × 103, ε(615 nm): 4.49 × 103. 31P{1H}
NMR (162 MHz, CH2Cl2, 298 K): (ppm) 5.61 (2P, m), 10.61 and 12.42 (1P, 2m), 16.50
and 18.30 (1P, 2m), 144 (1P, hept, PF6, JPF = 711 Hz). 1H NMR (400 MHz, DMSO-d6,
298 K): (ppm) 7.9 – 6.3 (m, 44H, an overlap of aromatic protons of phenyl groups of dppm
(20 H) and Lap (4H)), 5.4 (m, 1H, Lap), 5.2 (m, 2H, dppm), 4.2 (m, 2H, dppm), 3.2 – 3.0 (m,
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2H, Lap), 1.8 – 1.6 (m, 6H, 2CH3 of Lap). 13C{1H} NMR (125.74 MHz, DMSO-d6, 298 K):
(ppm) 195.5 (C1=O), 181.1 (C4=O), 168.9 (C2O).
(2) [Ru(Law)(dppm)2]PF6. Dark blue solid. Yield: 97 mg (77%). Elemental analysis for
[RuC60H49F6O3P5]: exp. (calc): C, 61.14 (60.66), H, 4.73 (4.20)%. ΛM = 48.2 Ω−1 cm2 mol−1,
in 1.0 mM CH2Cl2 solution. IR (cm): v(C1=O) 1554, v(C4=O) 1604, v(C2O) 1097, v(P-F)
840 and 559, v(Ru-N) 485 v(Ru-O) 520. Molar extinction coefficient (ε(𝛌, nm), M-1 cm-1): ε(258
4
3
3
3 31
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nm): 4.59 × 10 , ε(357 nm): 6.54 × 10 , ε(450 nm): 3.64 × 10 , ε(585 nm): 4.61 × 10 . P{ H} NMR
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(162 MHz, CH2Cl2, 298 K): (ppm) 5.93 (2P, m), 11.17 and 13.02 (1P, 2m), 15.39 and
17.17 (1P, 2m), 144 (1P, hept, PF6, JPF = 711 Hz). 1H NMR (400 MHz, DMSO-d6, 298
-p
K): (ppm) 7.8 – 6.3 (m, 44H, an overlap of aromatic protons of phenyl groups of dppm (20
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H) and Law (4H), 5.90 (s, 1H, Law), 5.4 – 5.0 (m, 2H, dppm), 4.2 – 4.1 (m, 2H, dppm);
C{1H} NMR (125.74 MHz, DMSO-d6, 298 K): (ppm) 196.20 (C1=O), 182.24 (C4=O) and
171.54 (C2
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X-ray crystallography
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Green single crystal of complex (1) was obtained from slow evaporation of a
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dichloromethane/ethyl ether solution. Room temperature X-ray crystallographic analysis was
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carried out using an automatic Rigaku XtaLAB mini II diffractometer (graphite
monochromator) with Mo-Kα radiation (λ = 0.71073 Å), in which intensities were collected
(ω scans with θ and κ-offsets), integrated and scaled with CrysAlisPro[33]. The structure was
solved by the direct method using SHELXS-97 and refined using the software SHELXL97[34]. All non-hydrogen atoms of the complex, counter-ion and solvent were located and
refined with anisotropic thermal parameters. The hydrogen atoms of aromatic C–H and CH2
methylene groups of dichloromethane, dppm and lapachol were positioned and refined with
fixed displacement parameters [Uiso(H) = 1.2 Ueq(Csp2)] using a riding model with bond
lengths of 0.93 and 0.97 Å, respectively. Finally, C-H of methyl were located from an
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electron-density difference and refined as riding on their parent atoms with Uiso(H) values of
1.5Ueq(Csp3) and C-H distance of 0.96 Å Molecular representations were generated using the
MERCURY 4.0[35] program. The summary of crystal data collections and structure
refinement parameters are presented in Table S1 (supplementary information) and copies of
the data can be obtained, free of charge, on the CCDC (Cambridge Crystallographic Data
Centre) under deposition number 2003981.
DNA interaction studies
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Agarose gel electrophoresis. The pBR322 DNA (38 M) was mixed with the
ruthenium complexes in different molar ratios ([complexes]/[DNA]): 0.05 to 2) and the
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solutions were incubated for 18 h at 37 C. The samples were electrophoresed in agarose gel
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using a Tris-acetate-EDTA buffer (0.45 M Tris-HCl, 0.45 M acetic acid, 10 mM EDTA, pH
7.4). Ethidium bromide was used for staining. The bands were visualized and registered using
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a ChemiDoc MP imager (BioRad Laboratories, Hercules, CA, USA).
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Circular Dichroism. Circular Dichroism (CD) spectrum of CT-DNA was obtained
from the CT-DNA solution prepared in a Tris-HCl buffer (4.5 mM Tris-HCl, 0.5 mM Tris-
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base and 50 mM NaCl, pH = 7.4). The experiment was performed by keeping the CT-DNA
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concentration constant at 50 M and varying the concentration of the ruthenium complexes to
obtain the molar ratios ([complexes]/[DNA]) of 0.05 to 0.4. The samples were prepared in a
Tris-HCl buffer containing 10% of DMSO, and incubated for 18h at 37 C. The CD spectra
were recorded using a spectropolarimeter JASCO J720 in the range of 240 to 350 nm at 298
K, with a constant nitrogen flush. Each spectrum was performed using a circular quartz
cuvette with 1cm path length.
Competitive DNA-Hoechst assay. The Hoechst 33258 fluorophore can intercalate the
minor grooves of CT-DNA and shows an emission spectrum at 475 nm. The experiment was
conducted using a Hoechst solution (2.7 M) and CT-DNA (125 M), which was titrated
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with an increasing amounts of ruthenium complexes (0 – 100 M) in Tris-HCl buffer
containing 10% DMSO. The fluorescence spectra were registered in the range of 370 to 700
nm with an excitation wavelength of 343 nm using a Synergy/H1-Biotek fluorimeter at 37 C.
Protein binding studies
The protein binding studies were performed by fluorescence quenching using Bovine
Serum Albumin (BSA, 2.5 M) in a Tris-HCl buffer. The stock solutions of the ruthenium
complexes were prepared in DMSO, and then incubated with BSA in different concentrations
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(0 - 90 M). The fluorescence spectra were recorded at 298 and 310 K, with and excitation
wavelength of BSA at λex = 280 nm and emission at λem = 340 nm, using a fluorimeter
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SpectraMax M3. The analyses were carried out in triplicate and the results were analyzed
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F0/F = 1+ kqη0[Q] = 1 + Ksv[Q] (1)
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using the Stern-Volmer Equation[36]:
where F0 and F are the fluorescence intensity of BSA in the absence and presence of
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complexes, respectively, Ksv is the Stern-Volmer constant, [Q] is the concentration of
complexes, kq is the biomolecular quenching constant and η0 is the average lifetime of BSA
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without the quencher (~ 108 s)[36].
equation (2)[36]:
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The binding constant (Kb) and number of binding site (n) were obtained using
log[(F0-F)/F] = logKb + nlog[Q] (2)
Additionally, thermodynamic parameters such as enthalpy (∆H°), entropy (∆S°) and
Gibbs energy (∆G°) were determined, using equations (3) and (4)[37]:
ln(Kb1/Kb2) = (1/T1 – 1/T2) × ∆H/R (3)
ΔG° = RTlnKb = ΔH° TΔS° (4)
MTT assay
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The effects of the ruthenium complexes, the ligands Lapachol, Lawsone and the
cisplatin on the cell lines viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay[38].
The cell lines were the human breast MCF-7 (ATCC: HTB-22) and MDA-MB-231
(ATCC: HTB-26), human prostate DU-145 (ATCC: HTB-81), human lung A549 (ATCC:
CCL-185) and non-tumor breast cells, MCF-10A (ATCC: CRL-10317). The MDA-MB-231
and A549 cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM)
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supplemented with 10% FBS. MCF-7 and DU-145 cells were cultivated in Roswell Park
Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum (FBS).
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MCF-10A cells were maintained in DMEM/F12 medium containing horse serum (HS) 5%,
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EGF (0.02 mg mL-1), hydrocortisone (0.05 mg mL-1) and insulin (0.01 mg mL-1). All the
media contained penicillin (100 UI mL-1), streptomycin (100 mg mL-1) and L-glutamine (2
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mM).
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MDA-MB-231, MCF-7, A549, DU-145 and MCF-10A cells (1.5 × 104 cells/well)
were seeded in 96-well plates in 100 L of media and were allowed to attach overnight in a
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CO2 incubator at 37 ºC. The compounds were added in different concentrations in triplicate
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and incubated for 48 h. The percentage of DMSO used in the experiments was fixed at 0.5%.
The control cells were treated only with 0.5% of DMSO. Afterwards, 40 L of MTT (1 mg
mL-1) solution was added to each well and the plates were incubated for 4 h at 37 C. Finally,
100 L of isopropanol was added to each well to dissolve the purple formazan crystals. The
absorbance at 540 nm was determined using a microplate reader (Bio-Tek instruments). The
IC50 values were determined using the GraphPad Prism software.
Morphology assay
MDA-MB-231 and MCF-10A cells (1 × 105 cells/well) were seeded in 12-well plates
and incubated for approximately 24 h in a CO2 incubator at 37 C. After that, the complex (1)
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was added in different concentrations (0.01 to 15 M). Cells were examined using an inverted
optical microscope (Nikon, T5100) with an amplification of 100×. Images of cell morphology
were recorded at 0, 24 and 48 h of incubation with ruthenium complex. Control cells were
treated with 0.5 % DMSO.
Colony formation assay
MDA-MB-231 (300 cells/plate) were seeded into 6 cm Petri dishes and incubated for
approximately 24 h in a CO2 incubator at 37 C. Complex (1) was added in different
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concentrations (0.03 to 1 M) and incubated for 48 h. Next, the medium was removed and a
fresh medium without complex was added and cells were incubated for 10 days. Afterwards,
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cells were fixed using methanol and acetic acid (3:1) for 15 min and stained with crystal
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violet solution (1% crystal violet, 20% ethanol) for 30 min. The plates were washed in water
Software.
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Wound healing assay
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to remove extra dye. The colonies (> 50 cells) were quantified and measured using ImageJ
To evaluate the ability of complex (1) to inhibit cell migration, MDA-MB-231 cells (1
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× 105 cells/wells) were seeded in 12-well plates and incubated at 37 ºC in 5% CO2 up to 80%
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of confluence. A straight scratch was performed on the plate using a sterile pipette tip. The
removed cells were washed to eliminate debris and a new medium with different
concentrations of complex (1) (0.1, 0.5 and 1 M) was added. Migrating cells were
photographed using an inverted microscope at 0, 8 and 24 h. The closure area of migrating
cells was quantified using ImageJ software, as follows: Wound healing area (%) = [cell-free
area (0 h) – cell-free area (24 h)]/cell-free area (0 h) × 100.
Cell migration assay
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MDA-MB-231 cells (0.5 × 105 cells/well) in serum-free medium were seeded on the
upper chamber of Boyden transwell chambers. Complex (1) was added in different
concentrations, below the IC50 for cytotoxic assay after 24 h (0.1, 0.5 and 1 M). Medium
with 10% of FBS was added to the bottom chamber to act as a chemoattractant in the positive
control of migration. For the negative control of cell migration, serum-free medium was
added to the lower chamber. The transwell chambers were incubated at 37 C and 5% CO2 for
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24 h. After that, the cells that migrated were fixed with methanol for 10 min, stained with
toluidine blue, and washed with distilled water. Images of the membrane were registered
using an inverted microscope (Nikon Eclipe TS100) coupled to a camera (Moticam 1000 -
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1.3MP Live Resolution), and the migrated cells were quantified using ImageJ software. The
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data shows the mean of cells that migrated in two independent experiments SD.
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Cell death analysis
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Apoptosis/necrosis of cells treated with complex (1) was investigated using 7-AAD
annexin V and PE (BD Biosciences) staining. MDA-MB-231 cells (0.7 × 105 cells/well) were
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plated in 24-well plates, and after 24 h of incubation at 37 C and 5% CO2 were exposed to
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different concentrations of complex (1) (0.5 to 10 M) and incubated again for 24 h.
Thereafter, the cells were stained with 2.5 L of PE-Annexin-V and 2.5 L of 7-AAD, in the
dark for 15 min, according to the manufacturer’s directions. Analyses were performed using a
flow cytometer BD Accurri C6 Plus.
Cell cycle analysis
MDA-MB-231 cells (1 × 105 cells/well) were seeded in 12-well plates and incubated
overnight at 37 C and 5% CO2. Then, different concentrations of complex (1) (0.1 to 1.25
M) were added to the cells and incubated for 24 h. After the incubation period, the cells
were collected and fixed with 70% cold ethanol for 24 h. Posteriorly, the cells were
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centrifuged and incubated with 300 L of RNase A (0.2 mg mL) for 30 min at 37 C. Cells
were stained using hypothonic fluorochrome solution (propidium iodide 5 g mL, sodium
citrate 0.1% and Triton-X-100 0.1%) for 1 h. The samples were analyzed using a Biosciences
Accuri C6 BD, and 10,000 cells were counted in each sample. This assay was performed in
triplicate and the data were processed using C6 Plus Software.
Reactive oxygen species
The levels of intracellular ROS generation in MDA-MB-231 cells induced by complex
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(1) were measured using H2DCFDA (2’,7’-dichlorofluorescin diacetate). To do this, MDAMB-231 cells (6 × 105 cells/well) were seeded into 6-well plates and incubated overnight at
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37 C and 5% CO2. Then, the cells were treated with complex (1) (3 × IC50 of 24h) and with
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free Lap (10 M), for 4 h. H2O2 (10 M) was used as the positive control and as negative
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control, cells containing only 0.5% of DMSO. Next, cells were washed with PBS and
incubated with 10 M of H2DCFDA for 30 min at 37 C. Cells were washed using ice-cold
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PBS (3×) and harvested. The fluorescence analysis was carried out using a Synergy H1
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Hybrid Multi-Mode Microplate Fluorimeter at a λex = 400 nm and λem = 525 nm. Images were
registered using an Olympus BX50 fluorescence microscope.
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Mitochondrial membrane potential
Changes in the mitochondrial potential (m) of MDA-MB-231 cells after treatment
with complex (1) were investigated using a DBTM MitoScreen Kit with JC-1 (5’,5’,6,6’tetrachloro-1,1’,3,3’ tetraethylbenzimidazolcarbocyanine iodide) dye. Briefly, MDA-MB-231
cells (1 × 105) were seeded in 12-well plates and incubated overnight to allow cells to attach
prior to adding complex (1) at different concentrations (IC50 of 24 h and 2× IC50 of 24 h),
followed for additional incubation for 4 h. Cells were washed in ice-cold PBS, stained with
JC-1 in the dark, according to the manufacturer’s directions and the samples were analyzed
using a BD Accurri C6 Plus flow cytometer.
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Results and Discussion
Synthesis and Characterization
Ruthenium-naphthoquinone complexes identified as: (1) [Ru(Lap)(dppm)2]PF6 and (2)
[Ru(Law)(dppm)2]PF6
[Lap
=
Lapachol,
Law
=
Lawsone
and
dppm
=
bis(diphenylphosphine)methane)], were obtained from the precursor complex
cis-
[RuCl2(dppm)2], by substituting two chloride ligands and coordination of Lap or Law after
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of
deprotonation with triethylamine (Et3N), in methanol media (Scheme 1).
[Ru(Law)(dppm)2]PF6.
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Scheme 1. Synthetic route used to obtain complexes (1) [Ru(Lap)(dppm)2]PF6 and (2)
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Both complexes (1) and (2) were obtained as an air-stable solid, soluble in solvents
such as methanol, ethanol, dichloromethane, acetone and DMSO. On the other hand, the
compounds are insoluble in water, ethyl ether and hexane. The percentages of C and H
obtained by elemental analysis agree with the theoretical values. Moreover, the molar
conductivity result indicates that the complexes are cationic, with the PF6 as an anion.
The IR spectrum of the free Lap/Law show bands around 3354, 1643 and 1050 cm1,
assigned to v(O-H), v(C1=O) and v(C2-O), stretching vibrations, respectively. Upon
coordination of Lap or Law, the band assigned to the v(O-H) stretching vibration was not
observed, indicating the deprotonation and coordination by the oxygen as monoanionic.
Moreover, the v(C1=O) and v(C2-O) bands were shifted after coordination (Fig S1 and S2).
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For complex (1), these bands were observed at 1545 (C1=O) and 1101 (C2-O) cm1,
indicating the bidentate coordination of Lap by oxygen atoms[23]. In addition to those bands,
it was observed characteristic bands of a PF6 counterion were observed at 841 and 557 cm1,
which are in agreement with molar conductance, consistent with 1:1 electrolyte. For
complexes (1) and (2) containing the dppm, weak band corresponding to v(P-CH2) at around
660 cm1 was observed. Moreover, v(Ru-O) and v(Ru-N) were observed at 480 and 520 cm1,
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of
respectively[39].
The electronic spectra of complexes (1) and (2) presented characteristic bands of
intraligand transitions IL (π → π*) around 260 and 290 nm of the dppm and Lap/Law ligands.
-p
In addition, bands around 400 and 600 nm can be assigned to metal-to-ligands charge transfer
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(MLCT), from Ru(dπ) to the ligand (π*) and also, n → π* transitions of quinone carbonyl
lP
groups (Fig S3)[21–23].
The electrochemical behavior of complexes (1) and (2) have been studied in CH2Cl2
na
by cyclic voltammetry. This study is useful to determine the changes in the electronic density
of ruthenium(II) upon coordination of Lap or Law. The precursor complex cis-
ur
[RuCl2(dppm)2] exhibits an oxidation process referring to RuII/RuIII at 1043 mV. After the
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coordination of Lap or Law, the oxidation processes RuII/RuIII increased considerably (Fig.
S4-S5). For complex (1) it was observed a positive shift of potential for RuII/RuIII couple after
coordination of Lap to 1373 mV, while in complex (2) the potential presents the value of
1435 mV. The presence of the prenyl group in the lapachol molecule can contribute to
electron donation to ruthenium, justifying the lower potential of complex (1) compared to
complex (2). In addition, the higher oxidation potentials observed for complexes (1) and (2)
compared with the precursor are due to the replacement of two chloride ligands, which are
good donors of electronic density by oxygen atoms, poor donors. These results confirm a
greater stability of both complexes after the coordination of the Lap or Law ligands. The same
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behavior was observed for complexes containing Lap or Law, in the previous work of our
research group[23].
The 31P{1H} NMR spectra of complexes (1) and (2) showed a typical ABMX spin
system. For complex (1), a set of signals in the region of 10.61, 12.42, 16.50 and 18.30
ppm is equivalent to the two phosphorus trans positioned to the two other phosphorus atoms.
In 5.61 ppm, a multiplet is observed that refers to the two phosphorus trans positioned to the
oxygen atoms of the Lap (Fig. S6). Complexes (1) and (2) showed downfield shifts in
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positions of the phosphorus signals as compared to the precursor complex (cis[RuCl2(dppm)2]: δ 0.65 and 26.17 ppm (3JP-P = 35.6 Hz)). This is attributed to the exchange
-p
of two chlorido ligands (ζ and π-donor) and coordination of Lap or Law to the ruthenium by
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oxygens that unshielded the phosphorus atoms. The same behavior was observed for the
ruthenium complex containing mercaptopyrimidine as ligands, observed in earlier studies[40].
lP
Furthermore, a septet around -144 ppm due to the PF6 anion was observed confirming the
1
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presence of the counterion in complexes (1) and (2).
H and 13C NMR spectra of complexes (1) and (2) exhibited signals consistent with
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the structures proposed by other techniques (Fig. S7 – S11). The 1H NMR spectrum of
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complex (1), for example, showed a set of signs in the region of 7.87 to 6.33 ppm, which are
equivalent to 44H of the aromatic groups of dppm and Lap ligands. Signals referring to six
hydrogen atoms of the two CH3 groups of Lap were observed at 1.76 – 1.61 ppm (Fig. S7).
Furthermore, the most informative result provided by the 13C NMR spectra of complexes (1)
and (2) are related to the C1=O and C2O carbon atoms. These signals were observed around
195.5 (C1=O) and 168.9 (C2O) ppm, in complex (1). On the other hand, in the free Lap,
these signals are observed at around 184.8 (C1=O) and 152.9 (C2O) ppm. The displacement
can be attributed to the effect of the coordination of oxygen atoms to the metal, changing the
electronic density.
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The molecular structure of complex (1) was obtained by single-crystal X-ray
crystallography (Figure 1). Complex (1) crystallizes in the non-centrosymmetric monoclinic
Pn space group, with lattice parameters: a = 15.2069(3) Å; b = 12.0838(2) Å; c = 16.9390(3)
Å; β = 93.999(2)°; Z = 2, in which the asymmetric unit consists of one molecule of cationic
complex, one counterion PF6 and one dichloromethane as solvate (Table S1). As previously
confirmed by the other characterizations, the structure of complex (1) is stabilized by three
chelate ligands, with two dppm forming a tensioned four-membered ring [with P-Ru-P chelate
ro
of
bite angle of 71.40(5) - 71.66(5)°] and lapachol with a five-membered assuming a bite angle
of 75.50(14)°. The structure of complex (1) presents a distorted octahedral geometry, which is
-p
comparable with other ruthenium/phosphine analogs with lapachol[21,23,41]. Analyzing the
re
bond lengths, the Ru1-O1 [2.139(4) Å] and Ru1-O2 [2.136(4) Å] bonds are quite similar
because both oxygen atoms are trans to phosphorus of dppm. Similarly, due to the same
lP
stereochemistry, the Ru-P1 [2.2967(14) Å] and Ru-P3 [2.3028(13) Å] separation is close,
na
while Ru-P2 [2.3649(13) Å] and Ru-P4 [2.3439(14) Å] distances present almost the same
values. As can be seen, both Ru-P2 and Ru-P4 lengths are larger than the Ru-P1/Ru-P3
ur
separation, because of higher trans influence when phosphorus is trans to phosphorus,
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compared with phosphorus is trans to oxygen[42].
The lapachol molecule coordinated to metal is composed by a naphthoquinone ring
almost planar and one no coplanar prenyl chain. The C-O bonds occurring in the lapachol free
of metal present the values of 1.226(2), 1.348(2), and 1.225(2) Å for C1-O1, C2-O2, and C4O4, respectively[43]. After coordination, the C1-O1, C2-O2, and C4-O4 distances adopt the
values of 1.234(7), 1.295(7), and 1.233(8) Å, respectively, in which the main change is
observed for the C2-O2 bond length that is slightly shortened as a result of deprotonation and
coordination.
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A detailed analysis of intermolecular contacts of the molecule was carried out. The
molecule is well-ordered by hydrophobic regions formed mainly by C-H of diphosphines and
prenyl able to be involved in van der Waals contacts (Figure S12A). Only around the C4-O4
carbonyl group displays capability to form polar contacts, such as hydrogen bonds with
macromolecules[44,45]. Besides van der Waals contacts, the crystal packing is stabilized by
weak hydrogen bonds connecting the complex/counter-ion and complex/solvent (Figure
S12B). These intermolecular interactions display an important role to stabilize the molecular
ro
of
conformation and crystal self-assembly of Ru(II)-based complexes[46]. In the complex (1),
intermolecular interactions involving the O4 atom C1s-H1sa of the (CH2) group of the solvent
-p
and C413-H413…Cl2s present the separation of 2.391 Å and 2.774 Å, respectively.
re
Moreover, one interaction between C1s-H1sb of solvent and F5 atom of counter-ion [distance
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na
lP
of 2.474 Å] contributes to keep the guest solvent caged.
Figure 1. Crystal structure representation of the complex (1) at 30% probability thermal
ellipsoids. For the sake of clarity, the molecules of PF6 and CH2Cl2 were omitted.
Biological investigation
Cytotoxicity assay
The cytotoxic effects of ruthenium complexes (1) and (2) were evaluated by MTT
assay against four cancerous cell lines, including triple-negative breast (MDA-MB-231),
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hormone-dependent breast (MCF-7), lung (A549) and prostate (DU-145). Moreover, one nontumor cell line from the epithelial breast tissue was tested, (MCF-10A). The IC50 and
selective index (SI) for complexes (1), (2), precursor complex cis-[RuCl2(dppm)2] and
cisplatin, after 48 h of incubation, are summarized in Table 1. All ruthenium complexes
showed cytotoxicity activity with IC50 values lower than those of cisplatin and the free
ligands Lap and Law. In general, the cytotoxicity of the complex containing Lap (1) and Law
(2) was similar. Although the IC50 values of complexes (1) and (2) and complex precursor are
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close, one interesting observation refers to the high selectivity (20 times more active) of
complex (1) to the triple-negative breast cancer (MDA-MB-231) compared to non-tumor
-p
breast cells (MCF-10A). This result confirms that the coordination of the Lap or Law to
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ruthenium, forming (1) and (2), increase the cytotoxicity effect and their selectivity.
Moreover, complex (1) was 27-fold cytotoxic than cisplatin against MDA-MB-231 cells.
lP
Complexes (1) and (2) showed similar or better cytotoxic activity than complexes previously
na
reported by our research group[23].
Given that complex (1) showed high selectivity for tumor MDA-MB-231 cells, it was
ur
selected for further investigations related to better understanding its mechanism of action. It is
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worth mentioning that before carrying out biological experiments, the stability of complex (1)
in DMSO was studied using the 31P{1H} NMR assay, and it was observed that the complex is
stable in this solvent after 72 h (Fig. S13).
Table 1. IC50 (M) values of ruthenium complexes and cisplatin, toward tumor and nontumor cell lines
Compounds
IC50 (µM); 48 h
(1)
(2)
*(a)
Cisplatin
Lap and Law
A549
(0.063 ± 0.018)
(0.03 ± 0.01)
(0.35 ± 0.07)
(11.54 ± 1.19)
>100
DU-145
(0.11 ± 0.05)
(0.06 ± 0.02)
(1.17 ± 0.01)
(2.00 ± 0.47)
>100
MCF-7
(2.03 ± 0.26)
(1.15 ± 0.19)
(1.17 ± 0.21)
(13.98 ± 2.02)
>100
MDA-MB-231
(0.13 ± 0.01)
(0.09 ± 0.02)
(1.10 ± 0.09)
(2.43 ± 0.20)
>100
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MCF-10A
(2.70 ± 0.50)
(0.55 ± 0.07)
(3.11 ± 0.04)
(29.45 ± 0.85)
>100
#
SI
1
1.33
0.48
2.66
2.11
--
#
2
20.77
6.11
2.88
12.12
--
SI
*cis-[RuCl2(dppm)2], #SI = Selectivity Index = SI1 = IC50MCF-10A/IC50MCF-7 e SI2 = IC50 MCF-10A/IC50
MDA-MB-231
The MDA-MB-231 and MCF-10A cells were incubated with complex (1) in different
concentrations and changes in the morphology of these cells have been recorded over time
(Figure 2). After 48 h of incubation, MDA-MB-231 and MCF-10A cells treated with 0.13 M
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of
and 2.7 M (IC50 concentration), respectively, showed alterations in the morphology and a
reduced cell number. At 5 and 15 M, morphology of tumor cells has been completely
-p
changed, exhibiting loss of adhesion, shrinkage and fragmentation. On the other hand, in the
re
non-tumor cells these morphological changes were subtler and only pronounced when
complex (1) was incubated at 15 M for 24 and 48 h. These observations are in accordance
lP
with the results of cytotoxicity activity and corroborate with the higher selectivity of complex
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(1) to MDA-MB-231 cells.
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Figure 2. Effects of complex (1) on cell morphology of (A) MDA-MB-231 and (B) MCF10A cells, after exposure of 0, 24 and 48 h.
The ability of a single cell to grow in a colony, after being exposed to complex (1),
was investigated based on the clonogenic assay. The cells exposed to 0.13 (IC 50
concentration), 0.3 and 1 M demonstrated a significant decrease in the number and size of
colonies, when compared to the negative control (Figure 3). In fact, at 0.3 and 1 M was not
na
lP
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-p
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of
possible to observe colony formation.
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Figure 3. Representative images of clonogenic assay showing MDA-MB-231 cells treated
with complex (1) and graphs of quantifications of colony number and size. These experiments
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were representative of three independent assays. Significance at the * p < 0.01 and ** p <
0.0001 using ANOVA followed by Tukey’s test.
Metastatic dissemination gives rise to new tumors in other regions of the body.
Briefly, this process involves different steps, such as migration, invasion, transport through
the circulatory system, and growth into a secondary organ. Metastases are responsible for
90% of the deaths, and for this reason, it is important to control this process, to ensure an
effective cancer treatment[47]. Herein, we evaluated the effects of complex (1) on the
migration of MDA-MB-231 cells, which are known for their notable invasive properties. To
do this, the wound healing and transwell assays were used. Complex (1) was capable to
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inhibit cell migration at concentrations of 0.5 and 1 M after 24 h of incubation (Figure 4 A).
The wound closure reduced from 73% to 19%, for control and complex (1) at 1 M,
respectively. Furthermore, we also investigated the ability of complex (1) inhibit the cell
migration through the basement membrane (physical barrier) using a Boyden-Chamber assay.
After 24 h of incubation, complex (1) effectively inhibited the cell migration (Figure 4 B) at
0.5 and 1 µM, concentrations that are lower than IC50 at 24 h (IC50 = 1.27 0.10 µM), when
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of
compared to the control. This is in accordance with wound-healing results, and clearly
confirms that complex (1) is capable of acting as an inhibitor of cell migration at non-
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lP
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-p
cytotoxic concentrations.
Figure 4. Effects of complex (1) on MDA-MB-231 cell migration. (A) Representative images
of cells after treatment with the indicated concentrations of complex (1) taken at 0, 8 and 24 h
using an inverted microscope (4×). (B) Quantitative analysis of the cell migration after
treatment with the indicated concentrations of complex (1), by measuring the cell wounded
region using ImageJ software. (C) Representative images of cell migration through the
membranes examined using Boyden Chambers by using the inverted microscope (10×). (D)
Percentage of migrating cells after 24 h of incubation with complex (1). Values were
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expressed as means SD in triplicates. Significance at the indicated p, using ANOVA
followed by the Tukey’s test.
The effects of complex (1) on the cell cycle distribution were evaluated in MDA-MB231 cells using flow cytometry analysis. To do this, the cells were exposed to complex (1) in
different concentrations (0.1, 0.5 and 1.25 µM) for 24 h. Complex (1) caused alterations in
cell cycle distribution with increased percentage of cells at Sub-G1 and G1 phases, whereas
this percentage decreased at S and G2 phases (Fig. S14). The increase in cells at the Sub-G1
ro
of
phase is an indicative of apoptosis[48].
To prove that this type of cell death occurs when MDA-MB-231 cells were treated
with different concentrations (0.5 – 10 µM) of complex (1), flow cytometry analysis using
-p
PE-Annexin-V and 7- AAD detection kit was performed. The percentage of apoptotic cells
re
increases considerably with an increase of the concentration of the complex (1), compared to
lP
untreated control (Figure 5). The percentage of cells in apoptosis was 23.2% and 86.2%, when
incubated with 1.25 µM (IC50 at 24 h = 1.27 0.10 µM) and 10 µM, respectively, showing
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ur
na
that the complex (1) induces tumor cell death via an apoptotic pathway.
Figure 5. Effects of complex (1) on MDA-MB-231 cell apoptosis. (A) Representative
overlay dot plot from apoptosis analysis of MDA-MB-231 cell after treatment with complex
(1) for 24 h by flow cytometry using annexin V-PE vs 7AAD staining. (B) Quantitative
analysis of apoptotic cells after treatment with the indicated concentrations of complex (1).
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Data are represented as mean SD of three replicates. Significance at the indicated p, using
ANOVA followed by Tukey’s test.
Mitochondrial membrane potential
Mitochondria is an important organelle in cellular metabolism and is able to activate
the extrinsic and intrinsic apoptotic pathways[49], acting as a target for cell death.
Compounds that have mitochondria as targets are promising in anticancer drug development.
The mitochondrial membrane potential (m) is an indicator of the its regulation[50].
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An important probe used to investigate the m is JC-1, which exhibits a red
fluorescence in normal conditions of mitochondria, and green fluorescence when this
organelle is perturbed[51].
-p
As shown in Figure 6, a considerable increase in bright green fluorescence (R2)
re
indicates the loss of m induced by complex (1), suggesting that this complex promotes
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lP
mitochondrial dysfunction probably leading to cell apoptosis.
Figure 6. Effects of complex (1) on the m of MDA-MB-231 cells. (A) Analysis of the
m of MDA-MB-231 cells after 4h of treatment with complex (1) at 1.27 and 2.54 µM by
flow cytometry. (B) Quantitative analysis of the m of MDA-MB-231 cells. Data are
represented as mean SD of three replicates. Significance at the indicated p, using ANOVA
followed by Tukey’s test.
Reactive Oxygen Species (ROS)
Several studies have shown that the cytotoxic effects of naphthoquinones are mediated
via ROS generation[52]. ROS can be responsible for many physiological processes, for
example, activating tyrosine kinases and acting as mediators in signal transduction.
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Nevertheless, ROS can also induce oxidative stress and trigger the activation of apoptotic cell
signaling[53,54].
Thus, experiments were performed to determine whether complex (1) can generate
ROS in MDA-MB-231 cells. The analysis was carried out monitoring intracellular ROS
levels using H2DCFDA (2’,7’-dichlorofluorescein diacetate) after treatment of the cells with
complex (1) (2 × IC50 of 24 h) for 4h. The H2O2 was used as a positive control. Moreover, the
free Lap was analyzed. Fluorescence measurements were carried out to analyze the
ro
of
intracellular ROS generation. The green fluorescence observed indicate the formation of ROS
in the analyzed cells, but a stronger effect was observed in the cells treated with complex (1)
-p
(Figure 7). Thus, complex (1) can disturb the cellular redox and the ROS generated can lead
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lP
re
the cell death by apoptosis.
Figure 7. Effects of complex (1) on ROS generation in MDA-MB-231 cells. (A)
Representative pictures of ROS generation in MDA-MB-231 cells after treatment with H2O2
(10 µM), Lap (10 µM) and complex (1) (2.54 µM), detected by H2DCFDA staining and (B)
Fluorescence signal detected for the cells in the presence of the compounds.
DNA-interaction studies
Modification in the DNA structure may result in conformational changes that can
modify and affect the message of the genetic sequence. Thus, irreversible alterations can
influence the development of tumor cells, and induce cell death. One of the ways reported for
the action of cisplatin is the ability of binding to the N7 of guanine of DNA, and blocking cell
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division, causing cell death. Therefore, DNA is an exploring target when it comes to
developing drugs to treat cancer[55,56].
The interaction of ruthenium complexes (1) and (2) with DNA was investigated by
circular dichroism, analysis of DNA mobility and competitive DNA fluorescence studies.
Each of these techniques provides information about the type of interaction that occurs
between the ruthenium complexes and the DNA molecule.
DNA shows two bands in the circular dichroism (CD) spectra, a positive band at 275
ro
of
nm and a negative band at 245 nm, due to the base stacking interaction and right-handed
helicity, respectively. Compounds that can interact with DNA may cause conformational
-p
changes at the CD spectra. The interaction of ruthenium complexes with DNA was monitored
re
by incubating different molar ratios of complexes and CT-DNA. The complex (1)
demonstrated a weak interaction with DNA, given no significant changes were observed in
lP
the intensity of the ellipticity of CT-DNA spectra (Figure 8A). This result indicates that
na
complex (1) cannot make significant conformational changes in the CT-DNA structure.
In order to gain more insight into the nature of the interactions between ruthenium
ur
complexes and DNA, the gel electrophoresis of pBR322 was performed to investigate the
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influence of complexes on the mobility of this plasmid. The pBR322 showed three different
bands related with the shapes of the DNA, a band namely supercoiled form (SC), circular
form (OC) and the linear form (L) (Figure 8B). The intensity of the bands decreases (or even
disappeared) when the concentration of the ruthenium complex (1) increased (Figure 8B, lines
a, b and c). These results suggest that the ruthenium complex (1) interacts with DNA
covalently or by minor groove, promoting the expulsion of ethidium bromide from the DNA
structure, preventing the visualization of the bands.
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Figure 8. Interaction of complex (1) with DNA molecule. (A) Circular dichroism spectra of
lP
CT-DNA incubated with complex (1) at different molar ratios (Ri) = [complex]/[CT-DNA] =
0.05, 0.1, 0.2, 0.3 and 0.4, (B) Agarose gel electrophoresis image of pBR322 plasmid DNA
incubated for 18 h at 37 °C with complex (1). Lane 1 (M): molecular weight marker, lane 2
na
(DNA): pure pBR322 with DMSO; lane 3 - 9 (a - g) = Ri = 0.05 to 2 and (C) Emission
spectra of HoechstCT-DNA (2.7 M; 125 M) in the absence and presence of increasing
ur
amounts of complex (1) (0 – 100 M).
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To further investigate whether the complex (1) may be covalently interacting with
DNA, a solution of complex (1) was prepared with guanosine, and monitoring was carried out
by 31P{1H} NMR. The 31P{1H} NMR spectra (Fig. S15) showed no alteration of signals of
complex (1), indicating that it is not able to form a strong association with the DNA structure,
then rejecting the possibility of covalent interaction.
To confirm the hypothesis of interaction by minor groove, a competitive binding study
with Hoechst 33258 was used. This fluorophore is able to interact with DNA by a minor
groove and the formed Hoechst-DNA adduct emits a high fluorescence at 450 nm, when
excited at 343 nm. If the compounds interact with the DNA through the minor grooves, it will
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compete with Hoechst, replacing it from the DNA structure and so, the fluorescence intensity
will decrease[57]. A considerable decrease in fluorescence intensity of Hoechst-DNA adduct
was performed detected after increases in the ruthenium complex (1) concentration (Figure
8C). Overall, these results indicate that complexes (1) and (2) interact with the DNA by minor
grooves. Previous research findings showed the same behavior for ruthenium complexes
containing lawsone as ligand, as well as for ruthenium-arene complexes[25,58,59].
Protein binding studies
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Albumin is the protein responsible for transporting endogenous and exogenous
substances, such as anticancer drugs, through the body. Thus, when it comes to drug
-p
development, the investigators have been studying the interaction of the drug candidates with
the albumin, because this protein may be the carrier of the drug[60,61].
re
Bovine serum albumin (BSA) is structurally homologous with Human Serum Albumin
lP
(HSA), and for that reason, is extensively used as the model protein binding studies. BSA
na
exhibits intrinsic fluorescence mainly due to the presence of tryptophan residue. The
fluorescence of BSA provides significant information about the structure, dynamics and
ur
protein folding, thus any change in the fluorescence will indicate changes in its structure.
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Fluorescence quenching is related to the process that causes a decrease in the fluorescence
intensity after the incubation with a fluorophore (ruthenium complexes, in this case)[36].
The BSA-binding experiment was performed by incubating the BSA with ruthenium
complexes (1) and (2) in different concentrations, at 298 and 310 K, monitoring the
fluorescence intensity. Complexes (1) and (2) acted as quenchers since successive increases
of complexes concentration promoted decreases in the intensity of BSA fluorescence (Figure
9A), evidencing that the complexes interact with the BSA, changing its structure. The
interactions can occur by static or dynamic mechanism, or a combination of both. To
distinguish these mechanisms, the fluorescence quenching was studied at different
temperatures, and analyzed by the Stern-Volmer constant (Ksv) showing its increase as the
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temperatures rise (Table 2). Dynamic quenching occurs when the fluorophore and the
quencher (ruthenium complexes) come into contact with the transient excited state, and static
quenching, indicating the formation of fluorophore-quencher complexes in the ground state.
Thus, the increase in the temperature favors dynamic quenching, because this mechanism
depends on the diffusion, therefore high temperatures result in high diffusion coefficient, and
consequently, the Ksv constant must increase when the temperature rises. On the other hand,
static quenching is disadvantaged with the increase in temperature, resulting in a low value of
ro
of
Ksv constant[36,62]. Therefore, as the Ksv values increase with the temperature, the dynamic
mechanism may be involved. On the other hand, the kq values obtained are higher than the
-p
pure dynamic quenching (kq = 2.0 1010 M s), indicating that the interaction between
re
ruthenium complexes (1) and (2) with BSA occurs by both a dynamic and static quenching
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lP
mechanism.
Figure 9. Interaction of complex (1) with BSA. (A) Fluorescence spectra of BSA (2.5 M) in
the absence and presence of increasing concentrations of complex (1), (B) Stern-Volmer and
(C) log(F0-F/F) vs. log[Q] plots, at 310 K.
The values of the binding constant (Kb) and number of binding sites (n), listed in
Table 2, reveals that the complexes show a moderate interaction with BSA, where Kb values
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compare to other ruthenium complexes found in the literature[58,63,64]. The n values found
were approximately 1, indicating that the interaction involves one site in the protein.
Table 2. The values of Ksv (M-1), kq (M-1 s-1), Kb (M-1) and n for interaction of BSA and
complexes (1) and (2)
1
2
T (K)
Ksv (104)
kq (1012)
Kb (104)
n
298
2.21± 0.24
2.21
8.55 ± 0.38
1.16
310
2.28 ± 0.14
2.28
9.33 ± 0.83
1.15
298
2.15 ± 0.10
2.15
2.27 ± 0.37
1.18
310
2.34 ± 0.11
2.34
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of
Complexes
2.41 ± 0.20
1.07
-p
To gain further insight regarding the type of force involved in the interaction between the
ruthenium complexes and BSA, some thermodynamic parameters were determined (enthalpy
re
change, entropy change, and Gibbs free energy change). Table 3 presents the thermodynamic
lP
parameters for the system analyzed. The positive values of enthalpy and entropy changes
na
indicate the involvement of hydrophobic interactions between BSA and complexes (1) and
(2). Furthermore, the negative values of Gibbs free energy suggest that this interaction is
ur
spontaneous[65].
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Table 3. Thermodynamic parameters for interaction of BSA and complexes (1) and (2)
Complexes
1
2
T(K)
G (kJ mol-1)
298
28.13
310
29.49
298
24.99
310
25.85
H (kJ mol-1)
S (J mol-1 K-1)
113.12
5.58
113.13
96.71
3.83
95.74
Conclusions
In this paper, two new ruthenium(II) complexes containing diphosphine (dppm =
bis(diphenylphosphine)methane, (1) Lapachol and (2) Lawsone as ligands were synthesized
and fully characterized by elemental analyses, molar conductivity, UV-Vis, IR, 31P{1H}, 1H
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and 13C NMR. Crystal structure determination was possible for complex (1), confirming the
proposed composition. Complex (1) showed a 20-fold higher activity to the triple-negative
breast cancer (MDA-MB-231) when compared to non-tumor breast (MCF-10A). Moreover,
complex (1) showed the ability to inhibit cell migration and colony formation and to induce
cell cycle arrest and apoptosis by activating the mitochondrial apoptosis pathway through
ROS induction and loss of mitochondrial membrane potential (m) in MDA-MB-231 cells.
DNA binding studies suggest that complexes (1) and (2) interact with DNA by minor
ro
of
grooves. The interactions with BSA occurs by dynamic and static mechanism in a
spontaneous process with the involvement of hydrophobic interactions. On the basis of the
-p
reported results, the ruthenium(II)/diphosphine-naphthoquinone can be further studied using
re
in vivo models, as they represent promising candidates for anticancer applications.
There are no conflicts to declare.
Acknowledgements
lP
Conflicts of interest
na
The authors are grateful for the financial support provided by the Brazilian Agencies of
ur
Research: CAPES, CNPq and FAPESP. K. M. Oliveira would like to thank FAPESP for a
research fellowship (grant number 2014/04147-9), and Alzir A. Batista for a research grant
Jo
(2016/16312-0). R. S. Correa would like to thank CNPq for the financial support (project
403588/2016-2 and 308370/2017-1). We would like to thank Dr. Eduardo Ernesto Castellano
from IFSC-USP who provided the X-ray diffractometer for the crystallographic
measurements.
References
[1]
[2]
[3]
[4]
C.C. Konkankit, S.C. Marker, K.M. Knopf, J.J. Wilson, Anticancer activity of
complexes of the third row transition metals, rhenium, osmium, and iridium, Dalt.
Trans. 47 (2018) 9934–9974.
B. Rosenberg, Platinum Complexes for the Treatment of Cancer, Interdiscip. Sci. Rev.
3 (1978) 134–147.
B. Rosenberg, L. Van Camp, J.E. Trosko, V.H. Mansour, Platinum compounds: a new
class of potent antitumour agents, Nature. 222 (1969) 385–386.
J. Zhao, S. Li, X. Wang, G. Xu, S. Gou, Dinuclear Organoruthenium Complexes
30
Journal Pre-proof
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
ro
of
[11]
-p
[10]
re
[9]
lP
[8]
na
[7]
ur
[6]
Jo
[5]
Exhibiting Antiproliferative Activity through DNA Damage and a Reactive-OxygenSpecies-Mediated Endoplasmic Reticulum Stress Pathway, Inorg. Chem. 58 (2019)
2208–2217.
R.G. Kenny, C.J. Marmion, Toward Multi-Targeted Platinum and Ruthenium Drugs—
A New Paradigm in Cancer Drug Treatment Regimens?, Chem. Rev. 119 (2019) 1058–
1137.
M.A. Jakupec, M. Galanski, V.B. Arion, C.G. Hartinger, B.K. Keppler, Antitumour
metal compounds: more than theme and variations, Dalt. Trans. (2008) 183–194.
S.M. Meier-Menches, C. Gerner, W. Berger, C.G. Hartinger, B.K. Keppler, Structure–
activity relationships for ruthenium and osmium anticancer agents – towards clinical
development, Chem. Soc. Rev. 47 (2018) 909–928.
S. Thota, D.A. Rodrigues, D.C. Crans, E.J. Barreiro, Ru ( II ) Compounds : NextGeneration Anticancer Metallotherapeutics ?, J. Med. Chem. 61 (2018) 5805–5821.
A. Levina, A. Mitra, P.A. Lay, Recent developments in ruthenium anticancer drugs,
Metallomics. 1 (2009) 458–470.
F.E. Poynton, S.A. Bright, S. Blasco, D.C. Williams, J.M. Kelly, T. Gunnlaugsson, The
development of ruthenium(II) polypyridyl complexes and conjugates for in vitro
cellular and in vivo applications, Chem. Soc. Rev. 46 (2017) 7706–7756.
C.G. Hartinger, P.J. Dyson, Bioorganometallic chemistry—from teaching paradigms to
medicinal applications, Chem. Soc. Rev. 38 (2009) 391–401.
A. Rilak Simović, R. Masnikosa, I. Bratsos, E. Alessio, Chemistry and reactivity of
ruthenium(II) complexes: DNA/protein binding mode and anticancer activity are
related to the complex structure, Coord. Chem. Rev. 398 (2019) 113011.
M. Abid, F.S. and A. Azam, Ruthenium Complexes: An Emerging Ground to the
Development of Metallopharmaceuticals for Cancer Therapy, Mini-Reviews Med.
Chem. 16 (2016) 772–786.
D.A. Smithen, H. Yin, M.H.R. Beh, M. Hetu, T.S. Cameron, S.A. McFarland, A.
Thompson, Synthesis and Photobiological Activity of Ru(II) Dyads Derived from
Pyrrole-2-carboxylate Thionoesters, Inorg. Chem. 56 (2017) 4121–4132.
E. Alessio, L. Messori, NAMI-A and KP1019/1339, Two Iconic Ruthenium Anticancer
Drug Candidates Face-to-Face: A Case Story in Medicinal Inorganic Chemistry,
Molecules. 24 (2019) 1995.
E. Alessio, Thirty Years of the Drug Candidate NAMI-A and the Myths in the Field of
Ruthenium Anticancer Compounds: A Personal Perspective, Eur. J. Inorg. Chem. 2017
(2017) 1549–1560.
R. Trondl, P. Heffeter, C.R. Kowol, M.A. Jakupec, W. Berger, B.K. Keppler, NKP1339, the first ruthenium-based anticancer drug on the edge to clinical application,
Chem. Sci. 5 (2014) 2925–2932.
P. Chellan, P.J. Sadler, Enhancing the Activity of Drugs by Conjugation to
Organometallic Fragments, Chem. Eur. J. 26 (2020) 8676.
F. Qu, S. Park, K. Martinez, J.L. Gray, F.S. Thowfeik, J.A. Lundeen, A.E. Kuhn, D.J.
Charboneau, D.L. Gerlach, M.M. Lockart, J.A. Law, K.L. Jernigan, N. Chambers, M.
Zeller, N.A. Piro, W.S. Kassel, R.H. Schmehl, J.J. Paul, E.J. Merino, Y. Kim, E.T.
Papish, Ruthenium Complexes are pH-Activated Metallo Prodrugs (pHAMPs) with
Light-Triggered Selective Toxicity Toward Cancer Cells, Inorg. Chem. 56 (2017)
7519–7532.
S. Monro, K.L. Colón, H. Yin, J. Roque, P. Konda, S. Gujar, R.P. Thummel, L. Lilge,
C.G. Cameron, S.A. McFarland, Transition Metal Complexes and Photodynamic
Therapy from a Tumor-Centered Approach: Challenges, Opportunities, and Highlights
from the Development of TLD1433, Chem. Rev. 119 (2019) 797–828.
31
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
[21] K.M. Oliveira, R.S. Corrêa, M.I.F. Barbosa, J. Ellena, M.R. Cominetti, A.A. Batista,
Ruthenium(II)/triphenylphosphine complexes: An effective way to improve the
cytotoxicity of lapachol, Polyhedron. 130 (2017) 108–114.
[22] K.M. Oliveira, L.-D. Liany, R.S. Corrêa, V.M. Deflon, M.R. Cominetti, A.A. Batista,
Selective Ru(II)/lawsone complexes inhibiting tumor cell growth by apoptosis, J.
Inorg. Biochem. 176 (2017) 66–76.
[23] K. Oliveira, E.J. Peterson, M. Carroccia, M.R. Cominetti, V.M. Deflon, N.P. Farrell,
A.A. Batista, R.S. Correa, Ru(II)-naphthoquinone complexes with high selectivity for
triple negative breast cancer, Dalt. Trans. (2020). doi:10.1039/D0DT01091J.
[24] A.E. Graminha, J. Honorato, L.L. Dulcey, L.R. Godoy, M.F. Barbosa, M.R. Cominetti,
A.C. Menezes, A.A. Batista, Evaluation of the biological potential of ruthenium(II)
complexes with cinnamic acid, J. Inorg. Biochem. 206 (2020) 111021.
[25] R.A. De Grandis, P.W. da S. dos Santos, K.M. de Oliveira, A.R.T. Machado, A.F.
Aissa, A.A. Batista, L.M.G. Antunes, F.R. Pavan, Novel lawsone-containing
ruthenium(II) complexes: Synthesis, characterization and anticancer activity on 2D and
3D spheroid models of prostate cancer cells, Bioorg. Chem. 85 (2019) 455–468.
[26] M.A. Naves, A.E. Graminha, L.C. Vegas, L. Luna-Dulcey, J. Honorato, A.C.S.
Menezes, A.A. Batista, M.R. Cominetti, Transport of the Ruthenium Complex
[Ru(GA)(dppe)2]PF6 into Triple-Negative Breast Cancer Cells Is Facilitated by
Transferrin Receptors, Mol. Pharm. 16 (2019) 1167–1183.
[27] L.-J. Huang, F.-C. Chang, K.-H. Lee, J.-P. Wang, C.-M. Teng, S.-C. Kuo, Synthesis
and antiplatelet, antiinflammatory, and antiallergic activities of substituted 3-chloro5,8-dimethoxy-1,4-naphthoquinone and related compounds, Bioorg. Med. Chem. 6
(1998) 2261–2269.
[28] K.W. Wellington, Understanding cancer and the anticancer activities of
naphthoquinones – a review, RSC Adv. 5 (2015) 20309–20338.
[29] M. Kubanik, W. Kandioller, K. Kim, R.F. Anderson, E. Klapproth, M.A. Jakupec, A.
Roller, T. Söhnel, B.K. Keppler, C.G. Hartinger, Towards targeting anticancer drugs:
ruthenium(II)–arene complexes with biologically active naphthoquinone-derived ligand
systems, Dalt. Trans. 45 (2016) 13091–13103.
[30] M.B.P. Moreira, D. R. M.; de Sá, M. S.; Macedo, T. S.; Reys, J. R. M.; Santana, A. E.
G.; Silva, T. L.; Maia, G. L. A.; Barbosa-Filho, J. M.; Camara, C. A.; Silva, T. M. S.;
Silva, K. N.; Guimaraes, E. T.; Santos, R. R.; Goulart, M. O. F.; Soares, Evaluation of
Naphthoquinones Identified the Acetylated Isolapachol as a Potent and Selective
Antiplasmodium Agent, J Enzym. Inhib Chem. 30 (2014) 615–621.
[31] B.P. Sullivan, T.J. Meyer, Comparisons of the Physical and Chemical Properties of
Isomeric Pairs. 2. Photochemical, Thermal, and Electrochemical Cis-Trans
Isomerizations of M(Ph2PCH2PPh2)2Cl2 (M = RuII, OsII), Inorg. Chem. 21 (1982)
1037–1040.
[32] S. Complexes, S. Complexes, T. Phosphines, Some Complexes of Tertiary Phosphines
with Ruthenium(II) and Osmium(II), Proc. Chem. Soc. 153 (1960) 896–904.
[33] CrysAlis Pro and CrysAlis RED, (2015).
https://www.rigaku.com/products/smc/crysalis.
[34] G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Crystallogr. Sect. C
Struct. Chem. 71 (2015) 3–8.
[35] C.F. Macrae, I. Sovago, S.J. Cottrell, P.T.A. Galek, P. McCabe, E. Pidcock, M.
Platings, G.P. Shields, J.S. Stevens, M. Towler, P.A. Wood, Mercury 4.0: from
visualization to analysis, design and prediction, J. Appl. Crystallogr. 53 (2020) 226–
235.
[36] J.R. Lacowicz, Principles of Fluorescence Spectroscopy, Kluver Academic/Plenum
32
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
Publishers, New York, 1999.
[37] M. Ganeshpandian, R. Loganathan, E. Suresh, A. Riyasdeen, M.A. Akbarsha, M.
Palaniandavar, New ruthenium(ii) arene complexes of anthracenyl-appended
diazacycloalkanes: effect of ligand intercalation and hydrophobicity on DNA and
protein binding and cleavage and cytotoxicity., Dalton Trans. 43 (2014) 1203–19.
[38] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays, J. Immunol. Methods. 65 (1983) 55–63.
[39] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds,
6a edição, 2009.
[40] V.S. Velozo-Sá, L.R. Pereira, A.P. Lima, F. Mello-Andrade, M.R.M. Rezende, R.M.
Goveia, W.C. Pires, M.M. Silva, K.M. Oliveira, A.G. Ferreira, J. Ellena, V.M. Deflon,
C.K. Grisolia, A.A. Batista, E.P. Silveira-Lacerda, In vitro cytotoxicity and in vivo
zebrafish toxicity evaluation of Ru(II)/2-mercaptopyrimidine complexes, Dalt. Trans.
48 (2019) 6026–6039.
[41] M.I.F. Barbosa, R.S. Corrêa, K.M. De Oliveira, C. Rodrigues, J. Ellena, O.R.
Nascimento, V.P.C. Rocha, F.R. Nonato, T.S. Macedo, J.M. Barbosa-Filho, M.B.P.
Soares, A.A. Batista, Antiparasitic activities of novel ruthenium/lapachol complexes, J.
Inorg. Biochem. 136 (2014) 33–39.
[42] J.C.S. Lopes, J.L. Damasceno, P.F. Oliveira, D.C. Tavares, V.M. Deflon, N.P. Lopes,
M. Pivatto, A.A. Batista, P.I.S. Maia, G. Von Poelhsitz, Ruthenium(II) Complexes
Containing Anti-Inflammatory Drugs as Ligands: Synthesis, Characterization and in
vitro Cytotoxicity Activities on Cancer Cell Lines, J. Brazilian Chem. Soc. 26 (2015)
1838–1847.
[43] I.K. Larsen, L.A. Andersen, B.F. Pedersen, Structures of two crystalline modifications
of lapachol, Acta Crystallogr. Sect. C. 48 (1992) 2009–2013.
[44] R.S. Correa, L.M. Bomfim, K.M. Oliveira, D.R.M. Moreira, M.B.P. Soares, J. Ellena,
D.P. Bezerra, A.A. Batista, Ru(II) complexes containing uracil nucleobase analogs
with cytotoxicity against tumor cells, J. Inorg. Biochem. 198 (2019) 110751.
[45] R.S. Correa, V. Freire, M.I.F. Barbosa, D.P. Bezerra, L.M. Bomfim, D.R.M. Moreira,
M.B.P. Soares, J. Ellena, A.A. Batista, Ru(II)–thyminate complexes: new metallodrug
candidates against tumor cells, New J. Chem. 42 (2018) 6794–6802.
[46] R.S. Corrêa, J.P. Barolli, M.I.F. Barbosa, J. Ellena, A.A. Batista, The effect of guest
molecules on the conformation and molecular assembly of the fac-[RuCl3(NO)(dppb)]
complex, J. Mol. Struct. 1048 (2013) 11–17.
[47] P. Mehlen, A. Puisieux, Metastasis: A question of life or death, Nat. Rev. Cancer. 6
(2006) 449–458.
[48] X. Huang, H.D. Halicka, F. Traganos, T. Tanaka, A. Kurose, Z. Darzynkiewicz,
Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis,
Cell Prolif. 38 (2005) 223–243.
[49] J. Li, H. Chen, L. Zeng, T.W. Rees, K. Xiong, Y. Chen, L. Ji, H. Chao, Mitochondriatargeting cyclometalated iridium(III) complexes for tumor hypoxic imaging and
therapy, Inorg. Chem. Front. 6 (2019) 1003–1010.
[50] S. Wen, D. Zhu, P. Huang, Targeting cancer cell mitochondria as a therapeutic
approach, Future Med. Chem. 5 (2013) 53–67.
[51] S. Sakamuru, X. Li, M.S. Attene-Ramos, R. Huang, J. Lu, L. Shou, M. Shen, R.R.
Tice, C.P. Austin, M. Xia, Application of a homogenous membrane potential assay to
assess mitochondrial function., Physiol. Genomics. 44 (2012) 495–503.
[52] A.T. Dharmaraja, Role of Reactive Oxygen Species (ROS) in Therapeutics and Drug
Resistance in Cancer and Bacteria, J. Med. Chem. 60 (2017) 3221–3240.
[53] L. Zeng, Y. Chen, J. Liu, H. Huang, R. Guan, L. Ji, H. Chao, Ruthenium(II)
33
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
Complexes with 2-Phenylimidazo[4,5-f][1,10]phenanthroline Derivatives that Strongly
Combat Cisplatin-Resistant Tumor Cells, Sci Rep. 6 (2016) 19449.
[54] L.-N. Li, L. Wang, Y.-N. Cheng, Z.-Q. Cao, X.-K. Zhang, X.-L. Guo, Discovery and
Characterization of 4-Hydroxy-2-pyridone Derivative Sambutoxin as a Potent and
Promising Anticancer Drug Candidate: Activity and Molecular Mechanism, Mol.
Pharm. 15 (2018) 4898–4911.
[55] D. Shaloam, P.B. Tchounwou, Cisplatin in cancer therapy: Molecular mechanisms of
action, Eur. J. Pharmacol. 740 (2014) 364–378.
[56] A. Kellett, Z. Molphy, C. Slator, V. McKee, N.P. Farrell, Molecular methods for
assessment of non-covalent metallodrug–DNA interactions, Chem. Soc. Rev. 48 (2019)
971–988.
[57] N. Narayanaswamy, M. Kumar, S. Das, R. Sharma, P.K. Samanta, S.K. Pati, S.K.
Dhar, T.K. Kundu, T. Govindaraju, A thiazole coumarin (TC) turn-on fluorescence
probe for AT-base pair detection and multipurpose applications in different biological
systems, Sci. Rep. 4 (2014) 1–10.
[58] B.N. Cunha, L. Colina-Vegas, A.M. Plutín, R.G. Silveira, J. Honorato, K.M. Oliveira,
M.R. Cominetti, A.G. Ferreira, E.E. Castellano, A.A. Batista, Hydrolysis reaction
promotes changes in coordination mode of Ru(II)/acylthiourea organometallic
complexes with cytotoxicity against human lung tumor cell lines, J. Inorg. Biochem.
186 (2018) 147–156.
[59] L. Colina-Vegas, K.M. Oliveira, B.N. Cunha, M.R. Cominetti, M. Navarro, A.
Azevedo Batista, Anti-Proliferative and Anti-Migration Activity of Arene–
Ruthenium(II) Complexes with Azole Therapeutic Agents, Inorganics. 6 (2018) 132.
[60] W. Ma, Z. Tian, S. Zhang, X. He, J. Li, X. Xia, X. Chen, Z. Liu, Lysosome targeted
drugs: rhodamine B modified N^N-chelating ligands for half-sandwich iridium(iii)
anticancer complexes, Inorg. Chem. Front. 5 (2018) 2587–2597.
[61] J.J. Miller, L.M.F. Gomes, T. Storr, A. Casini, The Interaction of Metal Compounds
with Protein Targets: New Tools in Medicinal Chemistry and Chemical Biology,
Encycl. Inorg. Bioinorg. Chem. (2017).
[62] Y. Shu, M. Liu, S. Chen, X. Chen, J. Wang, New Insight into Molecular Interactions of
Imidazolium Ionic Liquids with Bovine Serum Albumin, J. Phys. Chem. B. 115 (2011)
12306–12314.
[63] R.S. Correa, K.M. De Oliveira, F.G. Delolo, A. Alvarez, R. Mocelo, A.M. Plutin, M.R.
Cominetti, E.E. Castellano, A.A. Batista, Ru(II)-based complexes with N-(acyl)-N’,N’(disubstituted)thiourea ligands: Synthesis, characterization, BSA- and DNA-binding
studies of new cytotoxic agents against lung and prostate tumour cells, J. Inorg.
Biochem. 150 (2015) 63–71.
[64] P. Mandal, B.K. Kundu, K. Vyas, V. Sabu, A. Helen, S.S. Dhankhar, C.M. Nagaraja,
D. Bhattacherjee, K.P. Bhabak, S. Mukhopadhyay, Ruthenium(II) arene NSAID
complexes: inhibition of cyclooxygenase and antiproliferative activity against cancer
cell lines, Dalt. Trans. 47 (2018) 517–527.
[65] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces
contributing to stability., Biochemistry. 20 (1981) 3096–3102.
Synopsis
The naphthoquinones Lapachol and Lawsone can form new ruthenium compounds with
promising anticancer properties.
Declaration of Interest Statement
There are no conflicts to declare.
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Highlights
Ruthenium-naphthoquinone compounds with promising anticancer properties.
Promising cytotoxicity against breast, lung and prostate cancer cells.
Exciting results of selectivity are reported against breast cancer cells.
Complex inhibits cell migration and colony formation of breast cancer cells.
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Complex induces apoptosis by activation of the mitochondrial pathway.
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