Lipophilicity-antiproliferative activity relationship study leads to the preparation of a ruthenium(II) arene complex with considerable in vitro cytotoxicity against cancer cells and a lower in vivo toxicity in zebrafish embryos than clinically approved cis-platin

Article abstract of DOI:10.1016/j.ejmech.2017.03.029

Ru(II)-arene complexes are attracting increasing attention due to their considerable antitumoral activity. However, it is difficult to clearly establish a direct relationship between their structure and antiproliferative activity, as substantial structural changes might not only affect their anticancer activity but also tightly control their activation site(s) and/or their biological target(s). Herein, we describe the synthesis and characterization of four ruthenium(II) arene complexes bearing bidentate N,O-donor Schiff-base ligands ([Ru(η⁶-benzene)(N-O)Cl]) that display a significantly distinct antiproliferative activity against cancer cells, despite their close structural similarity. Furthermore, we suggest there is a link between their respective antiproliferative activity and their lipophilicity, as the latter affects their ability to accumulate into cancer cells. This lipophilicity-cytotoxicity relationship was exploited to design another structurally related ruthenium complex with a much higher antiproliferative activity (IC₅₀ > 25.0 μM) against three different human cancer cell lines. Whereas this complex shows a slightly lower activity than that of clinically approved cis-platin against the same human cancer cell lines, it displays a lower toxicity in zebrafish (Danio rerio) embryos at concentrations up to 20 μM.

Full text of DOI:10.1016/j.ejmech.2017.03.029

Accepted Manuscript
Lipophilicity-antiproliferative activity relationship study leads to the preparation of a
ruthenium(II) arene complex with considerable in vitro cytotoxicity against cancer cells
and a lower in vivo toxicity in zebrafish embryos than clinically approved cis-platin
MohammadMehdi Haghdoost, Golara Golbaghi, Myriam Létourneau, Shunmoogum
A. Patten, Annie Castonguay
PII:
S0223-5234(17)30187-3
10.1016/j.ejmech.2017.03.029
EJMECH 9289
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2017
Revised Date: 13 March 2017
Accepted Date: 15 March 2017
Please cite this article as: M. Haghdoost, G. Golbaghi, M. Létourneau, S.A. Patten, A. Castonguay,
Lipophilicity-antiproliferative activity relationship study leads to the preparation of a ruthenium(II)
arene complex with considerable in vitro cytotoxicity against cancer cells and a lower in vivo toxicity in
zebrafish embryos than clinically approved cis-platin, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.03.029.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Lipophilicity-Antiproliferative Activity Relationship Study Leads to the
Preparation of a Ruthenium(II) Arene Complex with Considerable In Vitro
Cytotoxicity Against Cancer Cells and a Lower In Vivo Toxicity in Zebrafish
Embryos than Clinically Approved Cis-Platin
MohammadMehdi Haghdoost, Golara Golbaghi, Myriam Létourneau, Shunmoogum A.
Patten and Annie Castonguay*
INRS - Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Laval, Quebec, H7V 1B7,
Canada
Abstract
Ru(II)-arene complexes are attracting increasing attention due to their considerable antitumoral activity.
However, it is difficult to clearly establish a direct relationship between their structure and antiproliferative
activity, as substantial structural changes might not only affect their anticancer activity but also tightly
control their activation site(s) and/or their biological target(s). Herein, we describe the synthesis and
characterization of four ruthenium(II) arene complexes bearing bidentate N,O-donor Schiff-base ligands
([Ru(η6-benzene)(N-O)Cl]) that display a significantly distinct antiproliferative activity against cancer cells,
despite their close structural similarity. Furthermore, we suggest there is a link between their respective
antiproliferative activity and their lipophilicity, as the latter affects their ability to accumulate into cancer
cells. This lipophilicity-cytotoxicity relationship was exploited to design another structurally related
ruthenium complex with a much higher antiproliferative activity (IC50 ˂ 25.0 µM) against three different
human cancer cell lines. Whereas this complex shows a slightly lower activity than that of clinically
approved cis-platin against the same human cancer cell lines, it displays a lower toxicity in zebrafish
(Daniorerio) embryos at concentrations up to 20 µM.
Keywords: Ruthenium complexes, Antiproliferative activity, Lipophilicity, Cancer, In vivo toxicity,
Zebrafish1.
Abbreviations: Cis-platin, cis-diamminedichloroplatinum(II); NAMI-A, Imidazolium-trans-tetrachloro(dimethylsulfoxide) imidazole ruthenium(III); RAPTA,
([Ru(η6-arene)Cl2(1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane)]; PTA, 1,3,5-Triaza-7-phosphaadamantane; hpf, hours post fertilization; MTS, 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium bromide; KH2O, rate constant of the hydrolysis reaction; LC50,
median lethal concentration; HR-ESI-MS, high-resolution electrospray ionisation mass spectrometry; ICP-MS, inductively coupled plasma mass
spectrometry; FBS, fetal bovine serum; CCD, charge coupled device; ANOVA, analysis of variance; EtOH, ethanol.
1

ACCEPTED MANUSCRIPT
Introduction
Despite their numerous limitations, platinum-based chemotherapeutics are still nowadays involved in
approximately 70% of all cancer treatments [1]. The nephrotoxicity, neurotoxicity and cancer cell
resistance they induce have prompted researchers to seek for anticancer compounds based on other
metals [2]. As some ruthenium complexes were found to exhibit considerable activity against cis-platin
resistant cell lines[3], induce a lower occurrence of side effects in comparison to platinum-based
therapeutics [4, 5] and display their antiproliferative activity via various mechanisms, ruthenium-based
complexes hold promise to replace platinum-based drugs as a first line cancer treatment [6].
The antiproliferative activity of various ruthenium complexes against cancer cells was previously studied
as a function of their intrinsic properties, as for instance their ability to bind to DNA (ex: NAMI-A) [7, 8], to
human serum albumin [9] and to cyclin-dependent kinases (CDKs) [10, 11], their ligand hydrolysis rate
(ex: RAPTA and Keppler-type complexes) [12-14], their lipophilicity [15-18], their water solubility [19], and
their redox potential [20, 21]. However, results collected from one study to another are often inconsistent.
As structural changes might not only affect their anticancer activity but also tightly control the nature of
their activation site(s) and/or their affinity towards cellular target biomolecule(s) [22], the absence of
convincing data on well-defined mechanisms of action of ruthenium compounds [23, 24] prevents the
establishment of reliable structure-activity relationships (SARs) [25-31]. For instance, it was recently
shown that substituting the chloro ligand in complex [(η6-p-cymene)Ru(ethylene-diamine)Cl]PF6 with
1,3,5-triaza-7-phosphaadamantane (PTA) alters the preferential target of the complex from chromatic
DNA to histone proteins [32]. Thus, establishing any reliable structure-antiproliferative activity relationship
requires the use of ruthenium complexes with very similar structures in order to limit possible shifts in their
mode(s) of action throughout the series.
Herein, we report the synthesis and characterization of a series of structurally similar Ru(II) complexes
bearing bidentate N,O-donor Shiff-base ligands, as well as the assessment of their in vitro
antiproliferative activity against three different human cancer cell lines, notably MCF-7 (breast), SH-SY5Y
(neuroblastoma) and A2780 (ovarian). In addition, we report an in vivo toxicity assessment of the most
promising ruthenium compound of the series in zebrafish embryos, and compare the results to that of cis-
platin, a metal-based chemotherapeutic widely used in clinics.
Results and discussion
Complex synthesis and characterization
To assess the influence of structurally similar Ru(II) arene complexes of different lipophilicity on cancer
cell antiproliferative activity, N,O-donor ligands bearing furyl pentyl, phenyl and cyclohexyl substituents
were synthesized in high yields (≥80%) by the Schiff base condensation of 2-hydroxy-1-naphthaldehyde
with different amines (1a: furyl; 1b: pentyl; 1c: phenyl; 1d: cyclohexyl). Ru(II) arene complexes (2a-d)
2

ACCEPTED MANUSCRIPT
were obtained from the reaction of [Ru(benzene)Cl2]2 dimer with two equivalents of the corresponding
N,O-donor ligands in refluxing ethanol (Scheme 1). In the case of complex 2c, a more satisfactory yield
was obtained when four equivalents of ligand were used. Although to a different extent, all complexes are
soluble in chloroform, dichloromethane, acetone and dimethylsulfoxide (DMSO), less soluble in alcohols,
and insoluble in ethers and aliphatic solvents. In addition, complexes are fairly soluble in water in the
presence of minimum volume of DMSO (less than 1% v/v), a mixture commonly used for the preparation
of stock solutions for biological assays. All complexes are stable enough to be handled in air in the solid
state, but are unstable when kept in solution. NMR studies showed that the nature of all complexes
remains unchanged after one or two days in CDCl3 solutions (in air), but that complete decomposition
(most likely due to oxidation) occurs in the course of approximatively one week (color change from red to
green and broadening of NMR peaks). Their decomposition rate is much slower in DMSO and acetonitrile
than chlorinated solvents, which can be explained by the low solubility of oxygen in these solvents [33].
Scheme 1. Synthetic route to complexes 2a-d.
Complexes were fully characterized by NMR spectroscopy as well as high resolution and high accuracy
mass spectrometry (ESI-MS). Single crystals suitable for X-ray diffraction analysis were grown by the
slow evaporation of ethanol or methanol/dichloromethane solutions of the complexes. The solid-state
structure of all complexes (Fig. 1) confirmed their pseudooctahedral three-legged piano stool geometry in
which the arene ring and the 1a-d ligands are respectively coordinated to the ruthenium center via a η6-
and η2-coordination mode. Bond distance and angle values around the metal are within the expected
range for that type of complex and are not significantly different throughout the series (Table 1).
3

ACCEPTED MANUSCRIPT
Fig. 1. ORTEP diagrams of complexes 2a-d showing thermal ellipsoids at the 30% probability level.
4

ACCEPTED MANUSCRIPT
Table 1. Selected bond lengths (Å) and angles (deg) of complexes 2a-d.
2a
2b
2c
2da
bonds
Ru1-O1
Ru1-N1
2.049(2)
2.088(2)
2.4395(6)
1.680
2.057(2)
2.078(2)
2.4451(7)
1.664
2.058(1)
2.083(1)
2.4234(5)
1.668
2.070(4), 2.059(5)
2.091(9), 2.089(8)
2.427(2), 2.425(2)
1.653, 1.671
Ru1-Cl1
Ru1-areneb
angles
O1-Ru1-N1
O1-Ru1-Cl1
N1-Ru1-Cl1
85.91(8)
88.11(6)
84.00(6)
86.86(8)
86.58(6)
85.30(6)
85.15(4)
86.61(3)
82.94(3)
86.2(2), 86.7(2)
87.7(2), 85.2(2)
85.0(2), 84.4(2)
a Two molecules per asymmetric unit.
b Distance between the Ru atom and the benzene ring center.
In vitro Antiproliferative activity
To evaluate if structurally similar complexes 2a-d display a significantly distinct antiproliferative activity,
cellular mitochondrial metabolic activity was measured in the presence of the complexes, respectively,
using the MTS assay. For this study, three human cancerous cell lines from distinct tissues were used,
specifically MCF-7 (breast adenocarcinoma), SH-SY5Y (neuroblastoma) and A2780 (ovarian carcinoma).
Cancerous cells were incubated with various concentrations of the complexes for 48 h at 37 °C. Careful
analysis of concentration-response results (Fig. 2) allowed the determination of an IC50 value for each
complex (Table 2). Despite their structural similarities, complexes 2a-d display significantly different
antiproliferative activities. For instance, complex 2a (R = furan) shows no apparent (or very low)
cytotoxicity against the three cell lines used whereas complex 2d (R = cyclohexyl) IC50 value is the lowest
of the series. Complexes 2b and 2c also exhibit distinct activities, complex 2b (R = pentyl) being more
active against cancer cells than 2c (R = phenyl). As expected, the overall sensitivity of the cancer cell
lines used in this study is considerably different (A2780˃SH-SY5Y˃MCF-7) [34]. Nevertheless, it is
interesting to note that the antiproliferative activity of 2a-d follows the same trend (2d ˃ 2b ˃ 2c ˃ 2a) for
each of the three cell lines.
5

ACCEPTED MANUSCRIPT
Fig. 2. Effect of complexes 2a-d concentration on the mitochondrial metabolic activity of SH-SY5Y, MCF-
7, and A2780 cancer cells. Error bars represent standard error of the mean (n=5).
6

ACCEPTED MANUSCRIPT
Table 2. IC50 values of complexes 2a-d against MCF-7, SH-SY5Y and A2780 cancer cells.
IC50 (ꢀM)a
MCF-7
˃150
SH-SY5Y
˃150
A2780
2a
2b
2c
2d
134.8 (±4.0)
40.1 (±6.2)
69.0 (±3.4)
30.4 (±2.0)
108.2 (±4.0)
132.1 (±4.9)
88.7 (±5.9)
91.7 (±3.4)
126.7 (±14.1)
67.1 (±0.7)
a
Inhibitory activity was determined by exposure of cell lines to each complex for 48 h, and expressed as
the concentration required to inhibit cell metabolic activity by 50% (IC50). Values in parentheses
correspond to the standard deviation of 2-4 independent experiments.
Hydrolysis
It is well-known that hydrolysis plays a critical role in the anticancer activity of various compounds [35-41].
As anticancer agents are usually introduced into the blood stream, it is of high importance to assess their
rate of hydrolysis as well as the stability of their hydrolyzed version in aqueous media [42]. Although
some Ru(II)-arene complexes were found to resist hydrolysis [36, 37], most of the compounds reported
undergo rapid aquation, in some cases even faster than that of cis-platin [38]. For instance, it is believed
that hydrolysis positively affects the anticancer activity of Ru [13, 43] and Pt complexes [44] by activating
DNA binding. The reactivity of complexes 2a-d with water was then investigated to evaluate i) if they
undergo hydrolysis, ii) if their rate of aquation varies despite their small structural differences, and iii) if the
timescale of the hydrolysis process could be responsible for their distinct antiproliferative activities (Fig. 2,
Table 2). When dissolved in phosphate buffer (1% DMSO), UV-Vis spectra of all four complexes
displayed significant time-dependent changes with isosbestic points in the region 280-500 nm, suggesting
the occurrence of a hydrolysis reaction (Fig. S1) [45]. The nature of the hydrolysis product formed from
complex 2a was supported by ESI-MS. In 0.1 M phosphate buffer, the peak corresponding to the initial
chloro complex 2a, [Ru(η6-benzene)(1a)Cl], observed at m/z 487.99 (pseudomolecular ion [M+Na]+),
disappeared at the expense of a peak assigned to the expected aqua cationic complex [Ru(η6-
benzene)(1a)(H2O)]+, observed at m/z 447.5 (Fig. S2). It is noteworthy that the presence of the aqua
species [Ru(η6-benzene)(1a)(H2O)]+ was not observed in the ESI-MS spectrum of 2a when anhydrous
methanol was used for sample preparation. In contrast to some previous reports [10, 36, 46], there was
no evidence of the formation of ruthenium dimer [Ru(η6-benzene)2(OH)3]+, even 24 hours after sample
7

ACCEPTED MANUSCRIPT
preparation, indicating the considerable stability of the aqua cationic species [Ru(η6-benzene)(1a)(H2O)]+.
Pseudo first-order rate constants (KH2O) and reaction half-lives (t1/2) were determined for the hydrolysis of
complexes 2a-d (Table 3). Rate constants observed for the hydrolysis of complexes 2a-d were found to
be different from one complex to another (2a > 2d > 2b > 2c), reaction half-lives varying from
approximately 4 to 75 min, the slowest rate observed in this series being two times faster than the
hydrolysis rate of cis-platin under the same conditions (half-life of 144 min) [47]. From this study, we
conclude that the aquation process of 2a-d is overall much faster than the time scale of the in vitro
cytotoxicity experiments performed to assess their IC50 values (48 h), ruling out any effect on those
previously determined antiproliferative activities.
Table 3. Information on the hydrolysis of complexes 2a-d (0.1 M phosphate buffer pH=7, 1% DMSO).
Isosbestic
points (nm)
Complex
λmax (nm)a
KH2O (10-5 s-1)b
t1/2 (min)
2a
2b
2c
2d
332
320
310
304
316
277.83(±15.07)
52.25(±3.15)
15.22(±1.35)
124.1(±5.80)
4.16
22.11
75.91
9.31
366, 338
367, 339
375, 337,439
a Selected wavelength for kinetic study (corresponding to the maximum change in UV-Vis spectrum).
b
Standard deviations are indicated between parentheses. Linear regression equations used to
established pseudo first-order kinetic constants displayed coefficient values (R2) ≥0.995.
Lipophilicity vs in vitro antiproliferative activity and cellular uptake
Due to the nature of cell membranes, passive diffusion of therapeutics is easier for compounds with a
higher lipophilic character [48]. Lipophilicity, commonly expressed as log P (the partition coefficient of a
compound in two immiscible solvents such as water and octanol), plays an important role in the biological
activity of various molecules. Previous reports on platinum [49, 50], osmium(II) [51] and ruthenium [17,
30] complexes have demonstrated a correlation between lipophilicity and complex accumulation inside
cells. As the improved cellular uptake of biologically active compounds can lead to more active drug
candidates, we were interested in evaluating the extent at which we could exploit this intrinsic property.
As complexes 2a-d undergo hydrolysis of the chloro ligand in aqueous media (vide supra), their
respective log P value could not be experimentally determined. As an alternative, the lipophilic character
of each of the four complexes was estimated from the calculated log P values for their free ligand using
the program ALOGSP 2.1 [52, 53], with the assumption that more lipophilic ligands would give rise to
8

ACCEPTED MANUSCRIPT
more lipophilic complexes [54]. This strategy was previously exploited to assess the lipophilicity of some
platinum anticancer compounds [54-56] and a Ru(II)-arene complex [31]. Moreover, the relative cellular
accumulation of ruthenium in SH-SY5Y cancer cells was assessed by ICP-MS after 48 h. Results are
presented in Fig. 3.
Fig. 3. Relation between relative lipophilicity (free ligand), cellular uptake (amount of ruthenium in SH-
SY5 cells after 48 h) and antiproliferative activity (IC50 values) of complexes 2a-d against the same cell
line. Error bars shown in the graph represent the standard deviation.
These results emphasize the importance of lipophilicity in designing suitable anticancer ruthenium drugs,
as they demonstrate that the more lipophilic a compound is, the more it accumulates into cells, and the
higher is its cytotoxicity. In addition, these results suggest that lipophilicity can be used, although to a
limited extent, as a tool to predict the antiproliferative activity for this class of compound, and that the
structural changes from one compound to another are small enough to prevent any shift in their
antiproliferative mode(s) of action. In contrast to corresponding Ru complexes, however, the cytotoxicity
of free ligands 1a-d was found unrelated to their lipophilicity, as a rather similar antiproliferative behavior
was observed for all of them (Fig. S3).
In vitro antimigratory activity
It is required for ruthenium complexes to partially internalize into cancerous cells for their cytotoxicity to be
displayed. Importantly, the portion that remains outside the cells can also play an important role on
cancer cells fate, such as their ability to spread [57]. As cell migration assays are routinely used as
screening tools to assess the antimetastatic properties of various compounds [58], we were interested in
evaluating the effect of 2a-d on cancer cell migration using a wound healing assay (scratch assay) [59],
and to compare results with the effect displayed by NAMI-A, an antimetastatic ruthenium complex that
reached clinical trials [60]. Unlike 2a-d, NAMI-A is a Ru(III) complex that displays no apparent
antiproliferative activity against various cancer cells. For instance, we determined IC50 values higher than
9

ACCEPTED MANUSCRIPT
150 ꢀM for NAMI-A against MCF-7, SH-SY5Y and A2780 cells. Its lack of cytotoxicity to various cancer
cells is related to the fact that it cannot efficiently enter cells, whereas interactions with proteins on the cell
surface or collagens of the extracellular matrix are suggested mechanisms for its antimetastatic behavior
[61]. NAMI-A was recently found to be cytotoxic to some leukemia cell lines, but its activity was attributed
to the selective blockage of KCa 3.1 channels [62]. Cell migration assay was performed by evaluating the
extent of wound closure after incubation of human ovarian A2780 cells in the presence of 10 µM of the
compounds. Interestingly, all complexes (2a-d and NAMI-A) similarly inhibited cells mobility by 50-57%
(Fig. 4). The fact that the observed results are independent on complex lipophilicity (and consequently
cellular uptake) is consistent with the occurrence of an extracellular process.
Fig. 4. Migration inhibition assessment of A2780 cells after exposure to 10 µM of 2a-d over 24 h. Error
bars shown in the graph represent the standard deviation (n=5).
Rational design of a complex with superior in vitro antiproliferative activity
From the lipophilicity-antiproliferative activity trend we previously observed, we undertook the rational
design of a new ruthenium complex with expected higher antiproliferative activity. It is well documented
that the incorporation of halogen atoms increases the lipophilic character of molecules and as a result,
improves their penetration of lipid membranes [63]. Lipophilicity calculations were then conducted on
10

ACCEPTED MANUSCRIPT
various similar Schiff base ligands bearing chloro atoms. Ligand 1e (log P = 6.21) bearing a phenyl ring
with three chloro substituents was selected to undertake the synthesis of a new Ru arene complex with
expected higher antiproliferative activity. Reaction of ligand 1e with [Ru(benzene)Cl2]2 dimer in refluxing
1
ethanol failed to afford the desired product, as no conversion was observed by H NMR even after five
days. By substituting the solvent to acetonitrile and by adding potassium carbonate to the reaction
mixture, complex 2e could be isolated in 55% yield (Scheme 2). The complex was fully characterized
using NMR, HR-ESI-MS, as well as single crystal X-ray crystallography (Fig. 5).
Scheme 2. Synthetic route to complex 2e.
Fig. 5. ORTEP diagram of complex 2e showing thermal ellipsoids at the 30% probability level.
11

ACCEPTED MANUSCRIPT
As predicted, complex 2e exhibits a significantly higher antiproliferative activity than 2a-d. This complex
inhibits the metabolic activity of MCF-7, SH-SY5Y and A2780 cells with IC50 values ranging from 12.6 to
23.0 µM (Table 4), which is nearly 2-3 fold lower than 2d (Table 2), the most active complex of the initial
series, and slightly less active than that corresponding to the antineoplastic drug cis-platin (IC50 values
ranging from 1.3 to 27 µM). It is noteworthy that complex 2e is also markedly more cytotoxic to all three
cell lines than its corresponding free ligand 1e, emphasizing the importance of the ruthenium center on
the antiproliferative activity of this complex (Fig. S3).
Table 4. IC50 values of ligand 1e, complexes 2e and cis-platin against SH-SY5Y, MCF-7 and A2780
cancer cell lines.
IC50 (ꢀM)a
MCF-7
˃50
SH-SY5Y
˃50
A2780
˃50
1e
2e
23.0 (±2.0)
27.2 (±2.3)
16.1 (±3.5)
4.1 (±0.1)
12.6 (±1.0)
1.3 (±0.3)
cis-platin
a
Inhibitory activity was determined by exposure of cell lines to the complex for 48 h and expressed as a
concentration required to inhibit the cell metabolic activity by 50% (IC50). Values in parentheses represent
the standard deviation of two to four independent experiments.
In addition, ICP-MS analyses showed that higher amounts of ruthenium were found in SH-SY5Y cells
(6.92 ± 2.04 ng Ru/106 cells) when exposed to 4 µM of 2e for 48 h under the conditions previously used
for complexes 2a-d, confirming that the enhanced cytotoxicity of the complex is, as expected, closely
linked to its higher cellular accumulation into cancer cells.
In vivo toxicity assessment using zebrafish model
In recent years, Zebrafish (Daniorerio) have been considered as a suitable animal model for phenotype-
based drug discovery due to their high homology with human DNA, rapid development, high fertility and
transparency [64]. It also enables high-quality in vivo validation of drug targets and is a very useful
bioassay tool for toxicological studies [65, 66], Nevertheless, reports of the effects of Ru(II) complexes on
the development of zebrafish embryos are still scarce [67, 68]. The in vivo toxicity of 2e and cis-platin, a
widely used anticancer agent, was assessed on the development of zebrafish embryos. More specifically,
the lethality and hatching rates of zebrafish embryos exposed to 0-40 µM of both compounds were
determined at 24, 48, 72 and 96 hours post fertilization (hpf) (Fig. 6 and 7), after which their respective
median lethal concentration (LC50) was calculated.
12

ACCEPTED MANUSCRIPT
After 96 hpf exposure of zebrafish to 10 and 20 µM of both compounds, ruthenium complex 2e was found
to be less toxic than cis-platin (the difference being less significant when lower concentrations were
used). This is illustrated by its LC50 value of (20.0 ± 1.8 µM), which is significantly higher (P < 0.05) than
that of cis-platin (11.3 ± 3.0 µM) (LC50 values were calculated from results reported in Fig. 6, after 96 hpf).
It is noteworthy that 40 µM of compound 2e led to the death of all embryos after 24 hpf, whereas the
same concentration of cis-platin also led to a very high lethality rate (90%), but after a longer exposure
(96 hpf). At this concentration, both compounds completely prevented hatching to occur. However, when
treated with concentrations from 2.5-20 µM of compound 2e, approximately 100% hatching was reached
after 72 and 96 hpf (similar to the values recorded for the control), whereas hatching was severely
affected upon treatment of cis-platin. For instance, hatching completely prevented when concentrations of
10 and 20 µM of cis-platin were used, and significantly lowered (40%) when exposed to a 5 µM
concentration of the drug (Fig. 7). Hatching success is a sensitive end-point of the zebrafish embryo
toxicity tests [69, 70]. Delayed hatching might be a result of retarded embryo development, as previously
reported [71], but a failure in hatching usually leads to the lethal outcome [70]. Altogether our findings
suggest that the ruthenium complex 2e is less toxic than cis-platin.
13

ACCEPTED MANUSCRIPT
Fig. 6. Lethality rate of zebrafish embryos exposed to various concentrations of 2e and cis-platin over 4
days post-fertilization (96 hpf). Data are expressed as means ± standard deviation from three
independent experiments (a total of sixty embryos).
14

ACCEPTED MANUSCRIPT
Fig. 7. Effects of 2e and cis-platin on the hatching success on developing zebrafish embryos. Hatching
rates were assessed at five different concentrations over 4 days post-fertilization (96 hpf) and data are
expressed as means ± standard deviation from three independent experiments (a total of sixty embryos).
Gross morphological phenotypes of zebrafish exposed to 2e and cis-platin were also assessed. None of
tested compounds showed adverse morphological defects compared to controls at 24 hpf (Fig. 8).
Following three days exposed to 10µM of 2e and cis-platin, a few embryos displayed developmental
defects such as hearth edema (swelling caused by accumulation of watery fluid) and smaller eyes (Fig.
8). However, 2e was significantly less toxic than cis-platin, with 7.4 ± 1.8% zebrafish treated with 2e
displaying some of these developmental defects, compared to 25.6 ± 2.4% of zebrafish treated with cis-
platin.
15

ACCEPTED MANUSCRIPT
Fig. 8. Gross morphological phenotypes in zebrafish embryos at 72 hpf: untreated (left), treated with 10
µM of 2e (middle) and cis-platin (right).
Conclusion
From this study of a series of structurally similar Ru(II)-arene complexes (2a-d), we observed that an
increase in their N,O-donor ligand lipophilicity facilitates their cellular uptake, resulting in an enhancement
of their cytotoxicity. The lipophilicity - antiproliferative activity correlation we established was then
exploited to design a new Ru(II)-arene complex (2e) with enhanced cytotoxicity. This work clearly
demonstrates that the lipophilicity of carrier ligands may serve as a primary criterion for preliminary
predictions of cytotoxicity in a series of similar metallic complexes. Furthermore, the low toxicity of 2e
compared to that of clinically approved drug cis-platin on the development of zebrafish embryos opens
the door to further in vivo investigations with this type of ruthenium complex.
Experimental section
General Remarks. All chemicals were used without purification as purchased from commercial sources
(mainly from Fluka and Sigma-Aldrich). The [Ru(benzene)Cl2]2 dimer and NAMI-A were prepared
according to literature procedures. [72, 73]. The complexation reactions were performed under inert
atmosphere of N2 using Schlenk techniques. CellTiter 96 Aqueous One Solution Cell Proliferation Assay
(MTS reagent) was purchased from Promega Corp. (Madison, WI). A Flexstation 3 multi-mode
microplate reader (Molecular Device) was used to record the absorbance at 490 nm. Cell suspensions
were counted with a Bright-Line Hemacytometer from Hausser Scientific. NMR spectra (1H, 13C{1H},
COSY, HSQC and HMQC) were recorded on 300 and 400 MHz Varian spectrometers (25 ºC). Chemical
shifts (δ) are reported in ppm (and were referenced to solvent residual peaks) and coupling constants (J)
are reported in Hz. UV-Vis spectra were obtained on a Carry 300 Bio UV-Vis spectrometer. The purity
(≥95%) of all complexes was confirmed by elemental analyses (Laboratoire d’Analyse Élémentaire,
Département de Chimie, Université de Montréal). HPLC-MS analyses were performed with a Waters
2795 separations module coupled to a Waters Micromass Quattro Premier XE tandem quadrupole mass
spectrometer. High-resolution and high accuracy mass spectra (HR-ESI-MS) were obtained using an
16

ACCEPTED MANUSCRIPT
Exactive Orbitrap spectrometer from ThermoFisher Scientific (Department of Chemistry, McGill
University). A Perkin Elmer Nexion 300X ICP mass spectrometer was used for the determination of
ruthenium in samples (Department of Chemistry, Université de Montréal). Diffraction measurements were
performed on a SMART APEX II diffractometer equipped with a CCD detector, an Incoatec IMuS source
(Cu) and a Quazar MX mirror (2a, 2b and 2d) or a Bruker Venture diffractometer (a liquid Ga Metal Jet
source) equipped with a Photon 100 CMOS detector, a Helios MX optics and a Kappa goniometer (2c
and 2e) (Department of Chemistry, Université de Montréal). All statistical analyses were done using the
GraphPad Prism 6.01 software. Outliers were identified and removed from the analysis using the Rout
method (Q < 5%). ANOVA analysis was used for testing the significance of the difference between the
means and a p-value <0.05 was considered statistically significant.
General procedure for the synthesis of 1a, 1b and 1d. A round-bottomed flask was charged with 2-
hydroxy-1-naphthaldehyde and the corresponding amine (1-1.1 equivalent) in dry methanol (10 mL). The
mixture was stirred under reflux for 5 h. Volatiles were then removed in vacuo to give ligand 1 in an
analytically pure form.
Ligand 1a. 2-hydroxy-1-naphthaldehyde (861 mg, 5 mmol) and furfurylamine (534 µL, 5 mmol). Yield
1
3
73%. H NMR (300 MHz, CDCl3) δH, ppm: 8.91 (1H, s, N=CH), 7.90 (1H, d, JH,H = 8.2 Hz), 7.71 (1H, d,
3
3
ArHnaphthalene, JH,H = 9.0 Hz), 7.65 (1H, d, ArHnaphthalene, JH,H = 8.2 Hz), 7.45 (1H, m, ArHnaphthalene), 7.42
3
(1H, m, ArHfuran), 7.27 (1H, m, ArHnaphthalene), 6.98 (1H, d, ArHnaphthalene, JH,H = 9.0 Hz), 6.34-6.38 (m, 2H,
ArHfuran), 4.78 (s, 2H, N-CH2); 13C{1H} NMR (75 MHz, CDCl3) δC, ppm: 172.7 (1C, Cnaphthalene), 159.4 (1C,
C=N), 150.1 (1C, Cfuran), 142.9 (1C, Cnaphthalene), 136.6 (1C, Cnaphthalene), 133.4 (1C, Cnaphthalene), 129.2 (1C,
Cnaphthalene), 127.9 (1C, Cfuran), 126.7 (1C, Cnaphthalene), 123.4 (1C, Cnaphthalene), 123.0 (1C, Cnaphthalene), 118.3
(1C, Cnaphthalene), 110.6 (1C, Cfuran), 108.5 (1C, Cfuran), 107.5 (1C, Cnaphthalene), 50.7 (1C, CH2); HR-ESI-MS
(+) m/z: found 252.10 [M+H]+ (calc. 252.10).
Ligand 1b. 2-hydroxy-1-naphthaldehyde (652 mg, 3.79 mmol) and hexylamine (550 µL, 4.18 mmol). Yield
1
3
85%. H NMR (300 MHz, CDCl3) δH, ppm: 8.65 (1H, s, N=CH), 7.82 (1H, d, ArH, JH,H = 7.32), 7.65 (1H,
3
3
d, ArH, JH,H = 9.32), 7.58 (1H, d, ArH, JH,H = 7.82), 7.41 (1H, m, ArH), 7.20 (1H, m, ArH), 6.90 (1H, d,
3
3
ArH, JH,H = 9.35), 3.57 (2H, t, N-CH2, 3JH,H = 7.07), 1.72 (2H, quint, N-CH2-CH2, JH,H = 7.71), 1.47-1.27
(6H, m, CH2), 0.89 (3H, m, CH3); 13C{1H} NMR (75 MHz, CDCl3) δC, ppm: 177.59 (1C, CAr), 157.37 (1C,
C=N), 137.34 (1C, CAr), 133.93 (1C, CAr), 129.22 (1C, CAr), 127.89 (1C, CAr), 126.02 (1C, CAr), 125.49
(1C, CAr), 122.52 (1C, CAr), 117.58 (1C, CAr), 106.25 (1C, CAr), 52.71 (1C, N-CH2), 31.37 (1C, N-CH2-
CH2), 30.66 (1C, CH2), 26.43 (1C, CH2), 22.50 (1C, CH2), 14.02 (1C, CH3); HR-ESI-MS (+) m/z: found
256.17 [M+H]+ (calc. 256.17), 278.15 [M+Na]+ (calc. 278.15), 511.33 [2M+H]+ (calc. 511.33), 533.31
[2M+Na]+ (calc. 533.31).
Ligand 1d. 2-hydroxy-1-naphthaldehyde (631 mg, 3.66 mmol) and cyclohexanemethylamine (500 µL,
3.84 mmol). Yield 90%. 1H NMR (300 MHz, CDCl3) δH, ppm: 8.60 (1H, s, N=CH), 7.82 (1H, d, , ArH, 3JH,H
17

ACCEPTED MANUSCRIPT
3
= 8.21), 7.66 (1H, d, ArH , 3JH,H = 9.38, ), 7.59 (1H, d, ArH, JH,H = 7.82), 7.41 (1H, m, ArH), 7.21 (1H, m,
3
3
ArH), 6.90 (1H, d, ArH, JH,H = 9.38), 3.43 (2H, t, N-CH2, JH,H = 5.86), 1.88-1.60 (6H, m, cyclohexane),
1.36-0.97 (5H, m, cyclohexane); 13C{1H} NMR (75 MHz, CDCl3) δC, ppm: 177.64 (1C, CAr), 157.81 (1C,
C=N), 137.71 (1C, CAr), 133.89 (1C, CAr), 129.27 (1C, CAr), 128.00 (1C, CAr), 126.05 (1C, CAr), 125.37
(1C, CAr), 122.64 (1C, CAr), 117.60 (1C, CAr), 106.07 (1C, CAr), 59.15 (1C, N-CH2), four peaks were found
at 38.85, 30.66, 26.43 and 22.50 ppm correspond to 6C of cyclohexene group; HR-ESI-MS(+) m/z: found
268.17 [M+H]+ (calc. 268.17), 290.15 [M+Na]+ (calc. 290.15), 535.33 [2M+H]+ (calc. 535.33) 557.31
[2M+Na]+ (calc. 557.31).
Synthesis of 1c. A round-bottomed flask was charged with 2-hydroxy-1-naphthaldehyde (646 mg, 3.75
mmol) and benzylamine (408 µL, 3.75 mmol) in dry methanol (10 mL). The mixture was stirred under
reflux for 2.5 h. After cooling, the solvent was evaporated under reduced pressure. The yellow solid was
1
washed with cooled diethyl ether and dried under vacuum. Yield 76%. H NMR (300 MHz, CDCl3) δH,
ppm: 8.86 (1H, s, N=CH), 7.87 (1H, d, ArH, 3JH,H = 8.21), 7.70 (1H, d, ArH, 3JH,H = 9.38), 7.63 (1H, d, ArH,
3
3JH,H = 8.21), 7.44 (1H, m, ArH), 7.41-7.29 (5H, m, ArH), 7.25 (1H, m, ArH), 6.97 (1H, d, ArH, JH,H
=
9.38), 4.81 (2H, s, N-CH2); 13C{1H} NMR (75 MHz, CDCl3) δC, ppm: 174.54 (1C, CAr), 158.57 (1C, C=N),
136.94 (1C, CAr), 136.88 (1C, CAr), 133.60 (1C, CAr), 129.26 (1C, CAr), 128.95 and 127.93 (5C, CAr),
127.57 (1C, CAr), 126.50 (1C, CAr), 124.18 (1C, CAr), 122.89 (1C, CAr), 118.05 (1C, CAr), 107.15 (1C, CAr),
52.63 (1C, N-CH2,); HR-ESI-MS m/z (+): found 262.12 [M+H]+ (calc. 262.12), 284.10 [M+Na]+ (calc.
284.10), 545.22 [2M+Na]+ (calc. 545.22).
Synthesis of 1e. A round-bottomed flask was charged with a solution of 2-hydroxy-1-naphthaldehyde
(500 mg, 2.90 mmol) in dry ethanol (10 mL). The mixture was stirred under reflux until the aldehyde was
completely dissolved, and 2,4,5-trichloroaniline (600 mg, 3.05 mmol) was added to the solution. Following
addition of the amine, the mixture was kept under reflux for 3 h, cooled, and filtered. The obtained solid
1
was washed with cold ethanol and diethyl ether and dried in air. Yield 82%. H NMR (300 MHz, CDCl3)
3
3
δH, ppm: 9.40 (1H, s, N=CH), 8.15 (1H, d, ArHnaphthalene, JH,H = 8.60), 7.87 (1H, d, ArHnaphthalene, JH,H
=
=
=
3
3
8.99), 7.76 (1H, d, ArHnaphthalene, JH,H = 7.82), 7.59 (1H, s, ArHbenzene), 7.57 (1H, t, ArHnaphthalene, JH,H
3
3
7.42, ), 7.46 (1H, s, ArHbenzene), 7.39 (1H, t, ArHnaphthalene, JH,H = 7.42), 7.16 (1H, d, ArHnaphthalene, JH,H
9.38); 13C{1H} NMR (75 MHz, CDCl3) δC, ppm: 167.26 (1C, CAr), 166.68 (1C, CAr), 157.71 (1C, C=N),
143.49 (1C, CAr), 137.23 (1C, CAr), 132.76 (1C, CAr), 131.99 (1C, CAr), 131.25 (1C, CAr), 130.35 (1C, CAr),
129.51 (1C, CAr), 128.46 (1C, CAr), 127.71 (1C, CAr), 124.10 (1C, CAr), 120.57 (1C, CAr), 120.06 (1C, CAr),
119.16 (1C, CAr), 109.27 (1C, CAr); HR-ESI-MS m/z (-): 347.97 [M-H]- (calc. 347.97).
General procedure for the synthesis of 2a, 2b and 2d. [Ru(benzene)Cl2]2 and the corresponding N,O-
ligand 1 (2 equivalent) were stirred in ethanol (15 mL) under reflux for 6 h. The mixture was allowed to
cool to room temperature and was filtered. Red-orange crystals were obtained upon slow evaporation of
the filtrate at room temperature. Crystals were washed with deoxygenated diethyl ether and dried under
18

ACCEPTED MANUSCRIPT
steam of nitrogen. The obtained crystals were found to be suitable for single-crystal X-ray diffraction
studies. Crystals were stored under inert atmosphere at room temperature.
1
Complex 2a. [Ru(benzene)Cl2]2 (95 mg, 0.2 mmol) and 1a (99 mg, 0.4 mmol). Yield 20%. H NMR (400
MHz, CDCl3) δH, ppm: 8.42 (1H, s, N=CH), 7.68-7.61 (4H, m, 3ArHnaphthalene+1ArHfuran), 7.41 (1H, t,
3
ArHnaphthalene, JH,H = 7.82), 7.22-7.17 (2H, m, ArHnaphthalene), 6.56-6.53 (2H, m, ArHfuran), 5.42 (1H, d, CH2,
3JH,H = 15.24), 5.38 (6H, s, η6-benzene), 4.93 (1H, d, CH2, 3JH,H = 15.24); 13C{1H} NMR (100 MHz, CDCl3)
δC, ppm: 169.42 (1C, CAr), 160.70 (1C, C=N), 150.78 (1C), 143.00 (1C, CAr), 135.10 (1C, CAr), 134.20
(1C, CAr), 128.84 (1C, CAr), 127.44 (1C, CAr), 126.56 (1C, CAr), 124.82 (1C, CAr), 122.19 (1C, CAr), 118.71
(1C, CAr), 113.05 (1C, CAr), 111.44 (1C, Cfuran), 110.41 (1C, Cfuran), 83.84 (6C, η6-benzene,), 62.27 (1C,
CH2); HR-ESI-MS m/z (+): found 953.00 [2M+Na]+ (calc. 953.00), 895.04 [2M-Cl]+ (calc. 895.04), 487.99
[M+Na]+ (calc. 487.99), 430.03 [M-Cl]+ (calc. 430.03); Anal. Calcd (%) for C22H18ClNO2Ru: C 56.84, H
3.90, N 3.01; Found: C 57.48, H 4.02, N 3.14;
Complex 2b. [Ru(benzene)Cl2]2 (90 mg, 0.18 mmol) and 1b (95 mg, 0.38 mmol). Yield 33%. 1H NMR (400
3
MHz, CDCl3) δH, ppm: 8.40 (1H, s, N=CH), 7.68 (1H, d, ArH, JH,H = 8.60), 7.60-7.58 (2H, m, ArH), 7.38
3
(1H, t, ArH, JH,H = 7.36), 7.18-7.14 (2H, m, ArH), 5.59 (6H, s, η6-benzene), 4.19 (2H, m, N-CH2), 2.02
3
(2H, m, N-CH2-CH2), 1.52-1.31 (6H, m, CH2), 0.93 (3H, t, CH3, JH,H = 7.03); 13C{1H} NMR (100 MHz,
CDCl3) δC, ppm: 167.13 (1C, CAr,), 158.23 (1C, C=N), 134.85 (1C, CAr), 134.30 (1C, CAr), 128.85 (1C,
CAr), 127.21 (1C, CAr), 126.55 (1C, CAr), 124.66 (1C, CAr), 121.85 (1C, CAr), 118.50 (1C, CAr), 111.95 (1C,
CAr), 84.03 (6C, η6-benzene), 68.94 (1C, N-CH2), 31.71 (1C, CH2), 31.45 (1C, N-CH2-CH2), 26.83 (1C,
CH2), 22.67 (1C, CH2), 14.10 (1C, CH3); HR-ESI-MS m/z (+): found 961.13 [2M+Na]+ (calc. 961.13),
492.06 [M+Na]+ (calc. 492.06), 434.10 [M-Cl]+ (calc. 434.10); Anal. Calcd (%) for C23H26ClNORu: C 58.90,
H 5.59, N 2.99; Found: C 58.55, H 5.74, N 2.89.
Complex 2d. [Ru(benzene)Cl2]2 (95 mg, 0.2 mmol) and 1d (105 mg, 0.4 mmol). Yield 26%. 1H NMR (400
3
MHz, CDCl3) δH, ppm: 8.26 (1H, s, N=CH), 7.63 (1H, d, ArH, JH,H = 8.21), 7.60-7.54 (2H, m, ArH), 7.36
3
3
(1H, t, ArH, JH,H = 8.21), 7.17-7.11 (2H, m, ArH), 5.57 (6H, s, η6-benzene), 4.13 (1H, N-CH2, dd, JH,H1
=
12.55, 3JH,H2= 5.58), 3.68 (1H, dd, N-CH2, 3JH,H1= 12.55, 3JH,H2= 8.37), 2.24 (1H, m, N-CH2-CH), 2.02-1.79
(4H, m, cyclohexane), 1.44-1.06 (6H, m, cyclohexane); 13C{1H} NMR (100 MHz, CDCl3) δC, ppm: 166.65
(1C, CAr), 158.58 (1C, C=N), 134.95 (1C, CAr), 134.43 (1C, CAr), 128.84 (1C, CAr), 127.24 (1C, CAr),
126.57 (1C, CAr), 124.53 (1C, CAr), 121.80 (1C, CAr), 118.53 (1C, CAr), 111.11 (1C, CAr), 84.20 (6C, η6-
benzene), 76.61 (1C, N-CH2), 37.59 (1C, N-CH2-CH), 31.31 (1C, cyclohexane), 31.14 (1C, cyclohexane),
26.48 (1C, cyclohexane), 26.26 (1C, cyclohexane), 25.70 (1C, cyclohexane); HR-ESI-MS m/z (+): found
937.21 [2M+2Na-H]+ (calc. 937.18), 504.06 [M+Na]+ (calc. 504.06), 446.10 [M-Cl]+ (calc. 446.10); Anal.
Calcd (%) for C24H26ClNORu: C 59.93, H 5.45, N 2.91; Found: C 59.72, H 5.77, N 2.88.
Synthesis of 2c. 95 mg of [Ru(benzene)Cl2]2 (0.2 mmol) and 206 mg 1c (0.79 mmol) were suspended in
dry degassed ethanol (15 mL) and refluxed for 5 h under N2. After cooling to room temperature, a yellow-
19

ACCEPTED MANUSCRIPT
brown solid formed which was filtered off and washed with deoxygenated ethanol and diethyl ether.
Crystals suitable for single crystal X-ray analysis were grown by slow evaporation of MeOH/CH2Cl2
1
solution of complex. Yield 61%. H NMR (400 MHz, CDCl3) δH, ppm: 8.43 (1H, s, N=CH), 7.63-7.56 (5H,
3
m, ArH), 7.50 (2H, t, ArH, JH,H = 7.03), 7.46-7.36 (2H, m, ArH), 7.22-7.17 (2H, m, ArH), 5.48 (1H, d, N-
3
CH2, JH,H = 14.88), 5.29 (6H, s, η6-benzene), 5.19 (1H, d, N-CH2, J= 14.88); 13C{1H} NMR (100 MHz,
CDCl3) δC, ppm: 168.77 (CAr), 160.43 (1C, C=N), 137.87 (CAr), 135.12 (CAr), 134.27 (CAr), 129.22 (CAr),
128.90 (CAr), 128.90 (CAr), 128.64 (CAr), 128.48 (CAr), 127.42 (CAr), 126.60 (CAr), 124.72 (CAr), 122.11
(CAr), 118.62 (CAr), 112.28 (CAr), 83.49 (6C, η6-benzene), 70.73 (1C, N-CH2); HR-ESI-MS m/z (+): found
973.04 [2M+Na]+ (calc. 973.04), 915.08 [2M-Cl]+ (calc. 915.08), 498.01 [M+Na]+ (calc. 498.01), 440.05
[M-Cl]+ (calc. 440.05); Anal. Calcd (%) for C24H20ClNORu: C 60.69, H 4.24, N 2.95; Found: C 60.13, H
4.37, N 2.92;
Synthesis of 2e. A solution of 50 mg [Ru(benzene)Cl2]2 (0.1 mmol), 74 mg 1e (0.21 mmol) and 31 mg
K2CO3 (0.22 mmol) in dry acetonitrile (10 mL) was bubbled with dry nitrogen for more than 15 min, and
then refluxed for 6 hours under inert atmosphere. The precipitate was collected by filtration and washed
with ethyl acetate and diethyl ether to remove unreacted ligand. Dichloromethane was added to dissolve
the product and the solution was then passed through Celite. The solvent was removed in vacuo to yield
2e as a red powder. Crystals suitable for single crystal X-ray analysis were obtained by slow evaporation
of a MeOH/CH2Cl2 solution of the complex. Yield 55%. 1H NMR (400 MHz, CD2Cl2) δH, ppm: 8.34 (1H, s,
3
N=CH), 8.26 (1H, s, ArHbenzene), 7.76 (1H, s, ArHbenzene), 7.69 (1H, d, ArHnaphtalene, JH,H = 9.38), 7.63 (1H,
d, ArHnaphtalene, 3JH,H = 8.60), 7.60 (1H, d, ArHnaphtalene, 3JH,H = 8.21), 7.36 (1H, m, ArHnaphtalene), 7.19 (1H, m,
3
1
ArHnaphtalene), 7.08 (1H, d, ArHnaphtalene, JH,H = 9.38), 5.40 (6H, s, η6-benzene); H NMR (400 MHz, CDCl3)
δH, ppm: 8.33 (1H, s, N=CH), 8.32 (1H, s, ArHbenzene), 7.70 (1H, s, ArHbenzene), 7.65 (1H, d, ArHnaphtalene, J =
3JH,H = 8.60), 7.55 (1H, d, ArHnaphtalene 3JH,H = 7.03), 7.32 (1H, t,
9.38), 7.60 (1H, d, ArHnaphtalene, ,
3
ArHnaphtalene, JH,H = 7.34), 7.18-7.12 (2H, m, ArHnaphtalene), 5.39 (6H, s, η6-benzene); 13C{1H} NMR (100
MHz, CD2Cl2) δC, ppm: 167.37 (1C, CAr), 160.54 (1C, C=N), 154.41 (1C, CAr), 137.15 (1C, CAr), 134.55
(1C, CAr), 131.67 (1C, CAr), 131.15 (1C, CAr), 130.85 (1C, CAr), 129.41 (1C, CAr), 128.93 (1C, CAr), 127.86
(1C, CAr), 126.47 (1C, CAr), 126.22 (1C, CAr), 124.59 (1C, CAr), 122.46 (1C, CAr), 118.39 (1C, CAr), 86.61
(6C, η6-benzene); HR-ESI-MS m/z (+): found 1092.82 [2M-Cl]+ (calc. 1092.82), 585.88 [M+Na]+ (calc.
585.88), 527.92 [M-Cl]+ (calc. 527.92); Anal. Calcd (%) for C23H15Cl4NORu: C 48.96, H 2.68, N 2.48;
Found: C 48.27, H 2.67, N 2.43.
X-ray crystallographic studies. Cell refinement and data reduction were done using APEX2. Absorption
corrections were applied using SADABS. Structures were solved by direct methods using SHELXS-97
and refined on F2 by full-matrix least squares using SHELXL-97 or SHELXL-2014. All non-hydrogen
atoms were refined anisotropically, whereas hydrogen atoms were refined isotropic on calculated
positions using a riding model [74-76].
20

ACCEPTED MANUSCRIPT
Cell culture. Human MCF-7 breast carcinoma, human SH-SY5Y neuroblastoma and human A2780
ovarian carcinoma were obtained from ATCC (American Type Culture Collection) and Sigma-Aldrich. The
MCF-7 and A2780 cells were routinely grown in a Roswell Park Memorial Institute medium (RPMI 1640)
supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. The complete growth
media for SH-SY5Y was an equal volume mixture of minimum essential media and Ham's F12 nutrient
mixture supplemented with sodium pyruvate (50 mg/L), penicillin-streptomycin and 15% FBS. All
components of culture media were purchased from Gibco. Both cell lines were grown as adherent
monolayers in a humidified atmosphere of 5% CO2-95% air at 37 °C. These cells were passaged twice a
week at ca. 70–80% confluency by harvesting with trypsin:EDTA and seeding at a 1:4 to 1:10 ratio into 75
cm2 flasks. Cells were used up to 10 passages after thawing.
MTS assays. Cell culture treated 96-well plates (Sarstedt) were used to seed 5,000 (A2780), 10,000
(MCF-7) or 30,000 (SH-SY5Y) cells per well. The plates were pre-incubated with drug-free medium at 37
°C (5% CO 2) for 24 h before adding of compounds. Stock solutions of the test compounds in DMSO (20
mM) were diluted in complete culture medium to give final concentrations in the range of 1-150 ꢀM
(maximum DMSO content did not exceed 0.75%). Media was removed from wells and 100 ꢀL aliquots of
these dilutions were added to each well for a drug exposure period of 48 h. Then, 20 µL of MTS reagent
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium bromide) was
added to each well. The optical density, directly proportional to the cells metabolic activity, was quantified
at 490 nm using a multiwell plate reader and the antiproliferative activity was calculated by comparison to
positive (25% DMSO) and negative controls (untreated wells). IC50 values, as the concentration that
inhibits cells metabolic activity by 50%, were determined from concentration-effect curves by interpolation.
This assay was carried out in two to four independent sets of experiments, each experiment with 4-6
replicates per concentration level. DMSO at concentrations lower than 0.8% was found to be non-
cytotoxic to cells. IC50 values for cis-platin (Sigma-Aldrich, pharmaceutical secondary standard) were also
determined by the same procedure.
Hydrolysis. 7.5 ꢀL of stock solution of the complexes in DMSO (20 mM) was added directly to 992.5 ꢀL
phosphate buffer (0.1 M, pH=7) in a UV-Vis cell to give final complex concentration of 150 µM.
Absorbance was monitored using UV-Vis spectroscopy at 5 min intervals over 2 h at 297 K. Plots of the
change in absorbance (at the wavelength corresponding to the maximum change in UV-Vis spectra)
versus time were fitted to the equation for pseudo first-order kinetics using the GraphPad Prism 6.01
software. The reaction rate constant (KH2O) and half-life (t1/2) were determined by the software and
expressed as mean ± standard deviation of three independent experiments. The same solution was used
for ESI-MS studies.
Wound healing assay. Human A2780 ovarian carcinoma cells were seeded at density of 600,000
cells/well into 24-well plates (Sarstedt) and grown to confluence. The cell monolayer was scraped in a
straight line with a p200 pipet tip to create a scratch. Cells were washed twice with 250 µL of the full
21

ACCEPTED MANUSCRIPT
growth medium to remove the debris and smooth the edges, then the medium was replaced by 500 µL
fresh RPMI-1640 medium supplemented with 0.5% FBS serum containing 10 µM ruthenium complexes.
The RPMI-1640 medium containing 0.5% FBS and 0.05% DMSO (vehicle) was used as a control. Images
showing migration of the treated and untreated cells into the wound area at 0 h and 24 h incubations were
captured by a Nikon Eclipse Ti microscope (equipped with Nikon DS-Ri2 camera) at 10× magnification.
Changes in wound area were analyzed using ImageJ software and the MRI wound healing tool plugin
(NIH, Bethesda, MD) and computed into percentage of control (means ± standard deviation; n = 5) using
untreated wells at 100%.
Cell uptake measurements. SH-SY5Y cells were seeded in 6-well plates (600,000 cells per well) and
incubated for 24 hours. Freshly prepared stock solutions of the complexes in DMSO (20 mM) were diluted
in complete culture medium to give final concentration of 4 ꢀM. A concentration much lower than the IC50
values was chosen to maintain nearly complete cell survival. 2 mL of these dilutions were added to each
well and the incubation was continued for 48 h. Drug containing media were removed by suction and cells
were washed two times with 1.5 mL phosphate-buffered saline (PBS). Then, cells were trypsinized and
resuspended in 800 ꢀL of growth media. . The number of cells in each sample was counted and cell
pellets were collected by centrifugation (3,000X g for 10 min at room temperature). Each pellet was
digested for 3 days at room temperature in HNO3 (70%, Sigma-Aldrich) and the resulting solutions were
diluted to 25 mL using Milli-Q water (final concentration of 2.8% nitric acid). The amount of Ru cellular
uptake was determined by ICP-MS. The experiment was carried out as duplicates.
Toxicity against zebrafish embryos. Wild type zebrafish (Daniorerio) embryos were raised at 28.5 °C
and staged as previously described [77]. Embryos at 4-cell stage were exposed to different
concentrations of 2e or cis-platin (2.5, 5, 10, 20, 40 ꢀM) prepared by diluting the stock solution of each
compound in the fish medium. Ano-treatment control was also included. Experiments were performed in
triplicates and a total of sixty embryos have been used per each treatment. The mortality, gross
morphology and hatching rates of the embryos in each system were observed every 24 h for a period of
96 h under a stereo microscope (Olympus SZX12). All protocols were carried out in compliance with
guidelines described by the Canadian Council for Animal Care (CCAC) and our local animal care
committee.
Supplementary information
Supplementary information is available free of charge via the Internet at http://pubs.acs.org and includes
all UV-Vis and MS data related to the hydrolysis section (Fig. S1-S2), as well the IC50 value
corresponding to each ligand 1a-e against human A2780 cancer cells (Fig. S3). CCDC reference
numbers 1529850-1529854 contain the supplementary crystallographic data for the X-ray studies
reported in this paper (for complexes 2a-e). This data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, e-mail:
deposit@ ccdc.cam.ac.uk.
22

ACCEPTED MANUSCRIPT
Acknowledgments
This work was supported by INRS University, Fonds de Recherche en Santé du Québec (Établissement
de Jeunes Chercheurs), the Canadian Foundation for Innovation (John Evans Leaders Fund) and the
Armand-Frappier Foundation of INRS University (Banque Nationale Scholarship to G.G.). We would like
to thank Prof. David Chatenet (INRS) and Prof. Isabelle Plante (INRS) for providing the human cancer
cell lines used in this study, Prof. Chatenet and Prof. Alain Fournier for allowing access to their cell
culture facility, and Prof. Géraldine Delbès for allowing the use of her inverted microscope. Finally, we
also thank Gabriel Desrosier-Grégoire for his help with the in vivo toxicity screens.
References
[1] G. Mangiapia, G. Vitiello, C. Irace, R. Santamaria, A. Colonna, R. Angelico, A. Radulescu, G. D'Errico,
D. Montesarchio, L. Paduano, Anticancer cationic ruthenium nanovectors: from rational molecular design
to cellular uptake and bioactivity, Biomacromolecules 14 (2013) 2549-2560.
[2] P.C. Bruijnincx, P.J. Sadler, New trends for metal complexes with anticancer activity, Curr. Opin.
Chem. Biol. 12 (2008) 197-206.
[3] R.E. Aird, J. Cummings, A.A. Ritchie, M. Muir, R.E. Morris, H. Chen, P.J. Sadler, D.I. Jodrell, In vitro
and in vivo activity and cross resistance profiles of novel ruthenium (II) organometallic arene complexes in
human ovarian cancer, Br. J. Cancer 86 (2002) 1652-1657.
[4] C.G. Hartinger, M.A. Jakupec, S. Zorbas-Seifried, M. Groessl, A. Egger, W. Berger, H. Zorbas, P.J.
Dyson, B.K. Keppler, KP1019, a new redox-active anticancer agent – preclinical development and results
of a clinical phase I study in tumor patients, Chem. Biodiversity 5 (2008) 2140-2155.
[5] J.M. Rademaker-Lakhai, D. van den Bongard, D. Pluim, J.H. Beijnen, J.H.M. Schellens, A phase I and
pharmacological study with imidazolium-trans-dmso-imidazole-tetrachlororuthenate, a novel ruthenium
anticancer agent, Clin. Cancer Res. 10 (2004) 3717-3727.
[6] S. Leijen, S.A. Burgers, P. Baas, D. Pluim, M. Tibben, E. van Werkhoven, E. Alessio, G. Sava, J.H.
Beijnen, J.H. Schellens, Phase I/II study with ruthenium compound NAMI-A and gemcitabine in patients
with non-small cell lung cancer after first line therapy, Invest. New Drugs 33 (2015) 201-214.
[7] V. Brabec, O. Novakova, DNA binding mode of ruthenium complexes and relationship to tumor cell
toxicity, Drug Resist. Update 9 (2006) 111-122.
[8] D. Pluim, R.C. van Waardenburg, J.H. Beijnen, J.H. Schellens, Cytotoxicity of the organic ruthenium
anticancer drug Nami-A is correlated with DNA binding in four different human tumor cell lines, Cancer
Chemoth. Pharm. 54 (2004) 71-78.
[9] Y. Zhang, A. Ho, J. Yue, L. Kong, Z. Zhou, X. Wu, F. Yang, H. Liang, Structural basis and anticancer
properties of ruthenium-based drug complexed with human serum albumin, Eur. J. Med. Chem. 86 (2014)
449-455.
23

ACCEPTED MANUSCRIPT
[10] A. Kurzwernhart, W. Kandioller, S. Bachler, C. Bartel, S. Martic, M. Buczkowska, G. Muhlgassner,
M.A. Jakupec, H.B. Kraatz, P.J. Bednarski, V.B. Arion, D. Marko, B.K. Keppler, C.G. Hartinger, Structure-
activity relationships of targeted RuII(η6-p-cymene) anticancer complexes with flavonol-derived ligands, J.
Med. Chem. 55 (2012) 10512-10522.
[11] A. Kurzwernhart, W. Kandioller, E.A. Enyedy, M. Novak, M.A. Jakupec, B.K. Keppler, C.G. Hartinger,
3-Hydroxyflavones vs. 3-hydroxyquinolinones: Structure-activity relationships and stability studies on
Ru(II)(arene) anticancer complexes with biologically active ligands, Dalton Trans. 42 (2013) 6193-6202.
[12] P. Mura, F. Piccioli, G. Gabbiani, M. Camalli, L. Messori, Structure-function relationships within
keppler-type antitumor ruthenium(III) complexes: The case of 2-aminothiazolium[trans-tetrachlorobis(2-
aminothiazole)ruthenate(III)], Inorg. Chem. 44 (2005) 4897−4899.
[13] A.K. Renfrew, A.D. Phillips, A.E. Egger, C.G. Hartinger, S.S. Bosquain, A.A. Nazarov, B.K. Keppler,
L. Gonsalvi, M. Peruzzini, P.J. Dyson, Influence of structural variation on the anticancer activity of
RAPTA-type complexes: ptn versus pta, Organometallics 28 (2009) 1165-1172.
[14] I. Bratsos, D. Urankar, E. Zangrando, P. Genova-Kalou, J. Kosmrlj, E. Alessio, I. Turel, 1-(2-Picolyl)-
substituted 1,2,3-triazole as novel chelating ligand for the preparation of ruthenium complexes with
potential anticancer activity, Dalton Trans. 40 (2011) 5188-5199.
[15] C.G. Mendoza-Ferri, C.G. Hartinger, R.E. Eichinger, N. Stolyarova, K. Severin, M.A. Jakupec, A.A.
Nazarov, B.K. Keppler, Influence of the spacer length on the in vitro anticancer activity of dinuclear
ruthenium-arene compounds, Organometallics 27 (2008) 2405-2407.
[16] J. Mattsson, P. Govindaswamy, A.K. Renfrew, P.J. Dyson, P. Stepnicka, G. Süss-Fink, B. Therrien,
Synthesis, molecular structure, and anticancer activity of cationic arene ruthenium metallarectangles,
Organometallics 28 (2009) 4350-4357.
[17] H. Huang, P. Zhang, Y. Chen, L. Ji, H. Chao, Labile ruthenium(II) complexes with extended phenyl-
substituted terpyridyl ligands: synthesis, aquation and anticancer evaluation, Dalton Trans. 44 (2015)
15602-15610.
[18] G. Lv, L. Guo, L. Qiu, H. Yang, T. Wang, H. Liu, J. Lin, Lipophilicity-dependent ruthenium N-
heterocyclic carbene complexes as potential anticancer agents, Dalton Trans. 44 (2015) 7324-7331.
[19] M. Martinez-Alonso, N. Busto, F.A. Jalon, B.R. Manzano, J.M. Leal, A.M. Rodriguez, B. Garcia, G.
Espino, Derivation of structure-activity relationships from the anticancer properties of ruthenium(II) arene
complexes with 2-aryldiazole ligands, Inorg. Chem. 53 (2014) 11274-11288.
[20] E. Reisner, V.B. Arion, M.F.C. Guedes da Silva, R. Lichtenecker, A. Eichinger, B.K. Keppler, V.Y.
Kukushkin, A.J.L. Pombeiro, Tuning of redox potentials for the design of ruthenium anticancer drugs - an
electrochemical study of [trans-RuCl4L(dmso)]- and [trans-Rucl4L2]- complexes, where L= imidazole, 1,2,4-
triazole, Indazole, Inorg. Chem. 43 (2004) 7083-7093.
[21] M. Groessl, E. Reisner, C.G. Hartinger, R. Eichinger, O. Semenova, A.R. Timerbaev, M.A. Jakupec,
V.B. Arion, B.K. Keppler, Structure-activity relationships for NAMI-A-type complexes (HL)[trans-RuCl4L(s-
dmso)ruthenate(IIII)] (L = imidazole, indazole, 1,2,4-triazole, 4-amino-1,2,4-triazole, and 1-methyl-1,2,4-
24

ACCEPTED MANUSCRIPT
triazole): Aquation, redox properties, protein binding, and antiproliferative activity, J. Med. Chem. 50
(2007) 2185-2193.
[22] S. Sersen, J. Kljun, K. Kryeziu, R. Panchuk, B. Alte, W. Korner, P. Heffeter, W. Berger, I. Turel,
Structure-related mode-of-action differences of anticancer organoruthenium complexes with β-
diketonates, J. Med. Chem. 58 (2015) 3984-3996.
[23] A. Bergamo, G. Sava, Ruthenium anticancer compounds: myths and realities of the emerging metal-
based drugs, Dalton Trans. 40 (2011) 7817-7823.
[24] A.R. Timerbaev, Role of metallomic strategies in developing ruthenium anticancer drugs, TrAC,
Trends Anal. Chem. 80 (2016) 547-554.
[25] R. Mitra, A.G. Samuelson, Substitution-modulated anticancer activity of half-sandwich ruthenium(II)
complexes with heterocyclic ancillary ligands, Eur. J. Inorg. Chem. 2014 (2014) 3536-3546.
[26] A. Habtemariam, M. Melchart, R. Fernandez, S. Parsons, I.D.H. Oswald, A. Parkin, F.P.A. Fabbiani,
J.E. Davidson, A. Dawson, R.E. Aird, D.I. Jodrell, P.J. Sadler, Structure-activity relationships for cytotoxic
ruthenium(II) arene complexes containing N,N-, N,O-, and O,O-chelating ligands, J. Med. Chem. 49
(2006) 6858-6868.
[27] I. Romero-Canelon, L. Salassa, P.J. Sadler, The contrasting activity of iodido versus chlorido
ruthenium and osmium arene azo- and imino-pyridine anticancer complexes: Control of cell selectivity,
cross-resistance, p53 dependence, and apoptosis pathway, J. Med. Chem. 56 (2013) 1291-1300.
[28] E. Corral, A.C. Hotze, H. den Dulk, A. Leczkowska, A. Rodger, M.J. Hannon, J. Reedijk, Ruthenium
polypyridyl complexes and their modes of interaction with DNA: Is there a correlation between these
interactions and the antitumor activity of the compounds?, J. Biol. Inorg. Chem. 14 (2009) 439-448.
[29] J. Liu, W. Zheng, S. Shi, C. Tan, J. Chen, K. Zheng, L. Ji, Synthesis, antitumor activity and structure-
activity relationships of a series of Ru(II) complexes, J. Inorg. Biochem. 102 (2008) 193-202.
[30] H. Huang, P. Zhang, H. Chen, L. Ji, H. Chao, Comparison between polypyridyl and cyclometalated
ruthenium(II) complexes: anticancer activities against 2D and 3D cancer models, Chem. Eur. J. 21 (2015)
715-725.
[31] M. Kubanik, J.K.Y. Tu, T. Söhnel, M. Hejl, M.A. Jakupec, W. Kandioller, B.K. Keppler, C.G. Hartinger,
Expanding on the structural diversity of flavone-derived rutheniumII(ƞ6-arene) anticancer agents,
Metallodrugs 1 (2015) 24-33.
[32] Z. Adhireksan, G.E. Davey, P. Campomanes, M. Groessl, C.M. Clavel, H. Yu, A.A. Nazarov, C.H.
Yeo, W.H. Ang, P. Droge, U. Rothlisberger, P.J. Dyson, C.A. Davey, Ligand substitutions between
ruthenium-cymene compounds can control protein versus DNA targeting and anticancer activity, Nat.
Commun. 5 (2014) 3462-3474.
[33] I.B. Golovanov, S.M. Zhenodarova, Quantitative structure-property relationship: XXIII. Solubility of
oxygen in organic solvents, Russ. J. Gen. Chem. 75 (2005) 1795-1797.
[34] J. Barretina, G. Caponigro, N. Stransky, K. Venkatesan, A.A. Margolin, S. Kim, C.J. Wilson, J. Lehar,
G.V. Kryukov, D. Sonkin, A. Reddy, M. Liu, L. Murray, M.F. Berger, J.E. Monahan, P. Morais, J. Meltzer,
25

ACCEPTED MANUSCRIPT
A. Korejwa, J. Jane-Valbuena, F.A. Mapa, J. Thibault, E. Bric-Furlong, P. Raman, A. Shipway, I.H.
Engels, J. Cheng, G.K. Yu, J. Yu, P. Aspesi, Jr., M. de Silva, K. Jagtap, M.D. Jones, L. Wang, C. Hatton,
E. Palescandolo, S. Gupta, S. Mahan, C. Sougnez, R.C. Onofrio, T. Liefeld, L. MacConaill, W. Winckler,
M. Reich, N. Li, J.P. Mesirov, S.B. Gabriel, G. Getz, K. Ardlie, V. Chan, V.E. Myer, B.L. Weber, J. Porter,
M. Warmuth, P. Finan, J.L. Harris, M. Meyerson, T.R. Golub, M.P. Morrissey, W.R. Sellers, R. Schlegel,
L.A. Garraway, The cancer cell Line encyclopedia enables predictive modelling of anticancer drug
sensitivity, Nature 483 (2012) 603-607.
[35] Y. Jung, S.J. Lippard, Direct cellular responses to platinum-induced DNA damage, Chem. Rev. 107
(2007) 1387-1407.
[36] W.H. Ang, E. Daldini, C. Scolaro, R. Scopelliti, L. Juillerat-Jeannerat, P.J. Dyson, Development of
organometallic ruthenium-arene anticancer drugs that resist hydrolysis, Inorg. Chem. 45 (2006) 9006-
9013.
[37] S.J. Dougan, M. Melchart, A. Habtemariam, S. Parsons, P.J. Sadler, Phenylazo-pyridine and
phenylazo-pyrazole chlorido ruthenium(II) arene complexes: Arene loss, aquation, and cancer cell
cytotoxicity, Inorg. Chem. 45 (2006) 10882-10894.
[38] F. Wang, H. Chen, S. Parsons, I.D. Oswald, J.E. Davidson, P.J. Sadler, Kinetics of aquation and
anation of ruthenium(II) arene anticancer complexes, acidity and X-ray structures of aqua adducts, Chem.
Eur. J. 9 (2003) 5810-5820.
[39] C. Scolaro, C.G. Hartinger, C.S. Allardyce, B.K. Keppler, P.J. Dyson, Hydrolysis study of the
bifunctional antitumour compound RAPTA-C, [Ru(η6-p-cymene)Cl2(pta)], J. Inorg. Biochem. 102 (2008)
1743-1748.
[40] C. Gossens, A. Dorcier, P.J. Dyson, U. Rothlisberger, pKa estimation of ruthenium(II)-arene pta
complexes and their hydrolysis products via a DFT/continuum electrostatics approach, Organometallics
26 (2007) 3969-3975.
[41] M. Groessl, C.G. Hartinger, P.J. Dyson, B.K. Keppler, CZE-ICP-MS as a tool for studying the
hydrolysis of ruthenium anticancer drug candidates and their reactivity towards the DNA model compound
dGMP, J. Inorg. Biochem. 102 (2008) 1060-1065.
[42] E. Melendez, Titanium complexes in cancer treatment, Cri. Rev. Oncol. Hemat. 42 (2002) 309-315.
[43] H. Chen, J.A. Parkinson, R.E. Morris, P.J. Sadler, Highly selective binding of organometallic
ruthenium ethylenediamine complexes to nucleic acids: novel recognition mechanisms, J. Am. Chem.
Soc. 125 (2003) 173-186.
[44] N.P. Johnson, J.D. Hoeschele, R.O. Rahn, Kinetic analysis of the in vitro binding of radioactive cis-
and trans-dichlorodiammineplatinum(II) to DNA, Chem.-Biol. Interact. 30 (1980) 151-169.
[45] S.E. Braslavsky, Glossary of terms used in photochemistry, 3rd edition (IUPAC Recommendations
2006), Pure Appl. Chem. 79 (2009) 1365-3075.
26

ACCEPTED MANUSCRIPT
[46] W. Kandioller, C.G. Hartinger, A.A. Nazarov, M.L. Kuznetsov, R.O. John, C. Bartel, M.A. Jakupec,
V.B. Arion, B.K. Keppler, From pyrone to thiopyrone ligands−rendering maltol-derived
ruthenium(II)−arene complexes that are anticancer active in vitro, Organometallics 28 (2009) 4249-4251.
[47] R.J. Knox, F. Friedlos, D.A. Lydall, J.J. Roberts, Mechanism of cytotoxicity of anticancer platinum
drugs: evidence that cis-diamminedichloroplatinum(II) and cis-diammine (1,1-cyclobutanedicarboxylato)
platinum(II) differ only in the kinetics of their interaction with DNA, Cancer Res. 46 (1986) 1972-1979.
[48] V. Pichler, S. Goschl, S.M. Meier, A. Roller, M.A. Jakupec, M. Galanski, B.K. Keppler, Bulky N(,N)-
(di)alkylethane-1,2-diamineplatinum(II) compounds as precursors for generating unsymmetrically
substituted platinum(IV) complexes, Inorg. Chem. 52 (2013) 8151-8162.
[49] A. Ghezzi, M. Aceto, C. Cassino, E. Gabano, D. Osella, Uptake of antitumor platinum(II)-complexes
by cancer cells, assayed by inductively coupled plasma mass spectrometry (ICP-MS), J. Inorg. Biochem.
98 (2004) 73-78.
[50] J.A. Platts, D.E. Hibbs, T.W. Hambley, M.D. Hall, Calculation of the hydrophobicity of platinum drugs,
J. Med. Chem. 44 (2001) 472-474.
[51] S.H. van Rijt, A. Mukherjee, A.M. Pizarro, P.J. Sadler, Cytotoxicity, hydrophobicity, uptake, and
distribution of osmium(II) anticancer complexes in ovarian cancer cells, J. Med. Chem. 53 (2010) 840-
849.
[52] I.V. Tetko, J. Gasteiger, R. Todeschini, A. Mauri, D. Livingstone, P. Ertl, V.A. Palyulin, E.V.
Radchenko, N.S. Zefirov, A.S. Makarenko, V.Y. Tanchuk, V.V. Prokopenko, Virtual computational
chemistry laboratory-design and description, J. Comput.-Aided Mol. Des. 19 (2005) 453-463.
[53] VCCLAB, Virtual Computational Chemistry Laboratory, http://www.vcclab.org, (2005).
[54] J.J. Wilson, S.J. Lippard, In vitro anticancer activity of cis-diammineplatinum(II) complexes with β-
diketonate leaving group ligands, J. Med. Chem. 55 (2012) 5326-5336.
[55] A. Zamora, V. Rodriguez, N. Cutillas, G.S. Yellol, A. Espinosa, K.G. Samper, M. Capdevila, O.
Palacios, J. Ruiz, New steroidal 7-azaindole platinum(II) antitumor complexes, J. Inorg. Biochem. 128
(2013) 48-56.
[56] A. Bakalova, R. Buyukliev, H. Varbanov, G. Momekov, Design, synthesis and comparative cytotoxic
investigation of platinum(II) complexes with some derivatives of 5-methyl-5-(4-pyridyl)hydantoin, Inorg.
Chim. Acta 423 (2014) 46-51.
[57] A. Bergamo, G. Sava, Linking the future of anticancer metal-complexes to the therapy of tumour
metastases, Chem. Soc. Rev. 44 (2015) 8818-8835.
[58] C.R. Justus, N. Leffler, M. Ruiz-Echevarria, L.V. Yang, In vitro cell migration and invasion assays, J.
Vis. Exp. (2014) e51046.
[59] C.C. Liang, A.Y. Park, J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for
analysis of cell migration in vitro, Nat. Protoc. 2 (2007) 329-333.
27

ACCEPTED MANUSCRIPT
[60] J.M. Rademaker-Lakhai, D. van den Bongard, D. Pluim, J.H. Beijnen, J.H.M. Schellens, A phase I
and pharmacological study with imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a novel
ruthenium anticancer agent, Clin. Cancer Res. 10 (2004) 3717-3727.
[61] 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. [Online early access]. DOI:
10.1002/ejic.201600986.
[62] S. Pillozzi, L. Gasparoli, M. Stefanini, M. Ristori, M. D'Amico, E. Alessio, F. Scaletti, A. Becchetti, A.
Arcangeli, L. Messori, NAMI-A is highly cytotoxic toward leukaemia cell lines: evidence of inhibition of
KCa 3.1 channels, Dalton Trans. 43 (2014) 12150-12155.
[63] G. Thomas, Medicinal Chemistry: An Introduction, Wiley, New York, 2000.
[64] C.A. MacRae, R.T. Peterson, Zebrafish as tools for drug discovery, Nat. Rev. Drug Discov. 14 (2015)
721-731.
[65] N. Mandrekar, N.L. Thakur, Significance of the zebrafish model in the discovery of bioactive
molecules from nature, Biotechnol. Lett. 31 (2008) 171-179.
[66] J.M. Spitsbergen, M.L. Kent, The state of the art of the zebrafish model for toxicology and toxicologic
pathology research-advantages and current limitations, Toxicol. Pathol. 31 (2003) 62-87.
[67] Q. Wu, K. Zheng, S. Liao, Y. Ding, Y. Li, W. Mei, Arene ruthenium(II) complexes as low-toxicity
inhibitor against the proliferation, migration, and invasion of MDA-MB-231 cells through binding and
stabilizing c-myc G-quadruplex DNA, Organometallics 35 (2016) 317-326.
[68] Y.H. Wang, C.C. Cheng, W.J. Lee, M.L. Chiou, C.W. Pai, C.C. Wen, W.L. Chen, Y.H. Chen, A novel
phenotype-based approach for systematically screening antiproliferation metallodrugs, Chemico-
biological interactions 182 (2009) 84-91.
[69] G.W. Suter, A.E. Rosen, E. Linder, D.F. Parkhurst, Endpoints for responses of fish to chronic toxic
exposures, Environ. Toxicol. Chem. 6 (1987) 793-809.
[70] L. Senerovic, M.D. Zivkovic, A. Veselinovic, A. Pavic, M.I. Djuran, S. Rajkovic, J. Nikodinovic-Runic,
Synthesis and evaluation of series of diazine-bridged dinuclear platinum(II) complexes through in vitro
toxicity and molecular modeling: correlation between structure and activity of Pt(II) complexes, J. Med.
Chem. 58 (2015) 1442-1451.
[71] R. Osterauer, N. Haus, B. Sures, H.R. Kohler, Uptake of platinum by zebrafish (Danio Rerio) and
ramshorn snail (Marisa Cornuarietis) and resulting effects on early embryogenesis, Chemosphere 77
(2009) 975-982.
[72] M.A. Bennett, A.K. Smith, Arene ruthenium(II) complexes formed by dehydrogenation of
cyclohexadienes with ruthenium(III) trichloride, J. Chem. Soc., Dalton Trans. (1974) 233-241.
[73] B.M. Blunden, A. Rawal, H. Lu, M.H. Stenzel, Superior chemotherapeutic benefits from the
ruthenium-based anti-metastatic drug NAMI-A through conjugation to polymeric micelles, Macromolecules
47 (2014) 1646-1655.
28