Synthesis and Structure of Arene Ru(II) NΛO-Chelating Complexes: In Vitro Cytotoxicity and Cancer Cell Death Mechanism

Article abstract of DOI:10.1021/acs.organomet.0c00092

A panel of six new structurally related organometallic arene Ru(II) complexes of general composition [(η6-benzene)Ru(L)Cl] (1-3) and [(η6-p-cymene)Ru(L)Cl] (4-6) (L = dimethylaminobenzhydrazones) have been designed and synthesized in search of new rutheni

Full text of DOI:10.1021/acs.organomet.0c00092

pubs.acs.org/Organometallics
Article
Synthesis and Structure of Arene Ru(II) N∧O‑Chelating Complexes: In
Vitro Cytotoxicity and Cancer Cell Death Mechanism
Sundarraman Balaji, Mohamed Kasim Mohamed Subarkhan, Rengan Ramesh,* Hangxiang Wang,
and David Semeril
Cite This: https://dx.doi.org/10.1021/acs.organomet.0c00092
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: A panel of six new structurally related organo-
metallic arene Ru(II) complexes of general composition [(η6-
benzene)Ru(L)Cl] (1−3) and [(η6-p-cymene)Ru(L)Cl] (4−6) (L
= dimethylaminobenzhydrazones) have been designed and
synthesized in search of new ruthenium anticancer drugs. The
identities of the synthesized complexes have been well-established
by elemental analysis and various spectral (FT-IR, UV−vis, NMR,
and HR-MS) methods. The solid-state molecular structures of the
ruthenium complexes were determined with the help of X-ray
crystallography and confirms the presence of a pseudo-octahedral
geometry around ruthenium. Furthermore, cytotoxicity of the complexes has been unveiled with the aid of MTT assay against A549
(lung carcinoma), LoVo (colon adenocarcinoma), HuH-7 (hepato cellular carcinoma) along with the noncancerous 16HBE (human
lung bronchial epithelium) cells and compared with the effect of the standard drug cisplatin. Interestingly, complexes 4, 5, and 6
which contain a p-cymene moiety induce a remarkable decrease of cell viability against all the cancer cells tested. The capacity
corresponding to the inhibition of A549 cells proliferation was analyzed by 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and
indicated a notable effect of p-cymene counterparts 4, 5, and 6 over cisplatin. Further studies such as AO-EB (acridine orange−
ethidium bromide) staining, flow cytometry, and Western blot analyses on cell death mechanism signified that the cytotoxicity was
associated with apoptosis in cancer cells. This clearly suggests that p-cymene-capped Ru(II) complexes are also one of the propitious
cancer therapeutic candidates and are worthy of further investigations.
clinical trial.10 Moreover, other ruthenium drugs KP1339,11
INTRODUCTION
■
TLD-1433,12 RM175,13 RAPTA-C,14 and RAED15 are under
GLOBOCAN estimates 36 types of cancers in 185 countries
(pre)clinical studies (Figure 1). Low-spin d6 arene ruthenium
based on the incidence, mortality and prevalence in the year
complexes have attracted great interest in cancer metal-
2019. Nowadays, chemotherapy is the most widely used cancer
lotherapeutics due to the structural diversity in ancillary ligands
treatment among radio, immune, hormone, and gene
and easy accessibility on the hydrophobic nature of arene by
therapies.1 Though many platinum-based anticancer drugs
substitutions.16 Recently, many ruthenium complexes ap-
are known, most of them are associated with adverse effects.2
Owing to this unsatisfactory treatment of platinum-based drugs
in the scenario of undesirable side effects, scientists search non-
platinum-metal-based drugs with target specificity, less toxicity
pended arene moiety were reported as potent anticancer
agents.17 Intense investigations were focused on ligands such
as arene, phosphine, and other multidentate ligands containing
oxygen, nitrogen, and sulfur.18−21
toward healthy cells, and potent activity against a wide
Schiff bases can coordinate to metal centers and exhibit
assortment of cancers.3 In the exploration of novel anticancer
different coordination modes which lead to the production of
drugs, great interest has been raised on ruthenium complexes,
many mono- and binuclear complexes with diverse stereo-
and these are being tested on several cancer cells.4−6
chemical properties.22 Metal hydrazone complexes can have a
Ruthenium complexes are considered one of the best
wide range of structures with different coordination numbers,
alternatives to their platinum counterparts by following
rationales such as effectivity toward some cisplatin-resistant
cancers,7 and high selectivity toward cancer cells/targets, as
Received: February 11, 2020
well as having minimal side effects and being mimetic of iron in
binding targets.8 Ruthenium-based drug KP1019 entered phase
I clinical trial, and its further development is limited due to low
solubility.9 Yet another drug, NAMI-A, succeeded in phase I
clinical studies and showed only limited efficacy in phase II
https://dx.doi.org/10.1021/acs.organomet.0c00092
© XXXX American Chemical Society
Organometallics XXXX, XXX, XXX−XXX
A

Organometallics
pubs.acs.org/Organometallics
Article
synthesis, antiproliferative activity, and cancer cell death
mechanism of arene Ru(II) complexes featuring benzhydra-
zones.34 In the present work, we report the synthesis and
structural chracterisation of arene Ru(II) complexes containing
4-dimethylamino benzhydrazones. Furthermore, the cytotox-
icity of the complexes toward the A549, LoVo, HuH-7 and
noncancerous 16HBE cells has been examined and the cancer
cell death mechanism was investigated by various biochemical
assays.
RESULTS AND DISCUSSION
■
Three hydrazone ligand derivatives have been easily obtained
in good yields from the reaction of 4-(dimethylamino)-
benzaldehyde with substituted benzhydrazides in an equi-
molar ratio. Metalation was accomplished by reacting the
ruthenium(II) arene precursors [(η6-arene)RuCl(μ-Cl)]2
(arene = benzene or p-cymene) and hydrazones in a 0.5:1
molar ratio respectively with 1 equiv of Et3N base (Scheme 1).
Scheme 1. Synthesis of Arene Ru(II) Complexes
Figure 1. Examples of some ruthenium-based anticancer com-
plexes.20,29−33
geometries, and accessible redox states.23 Furthermore,
ruthenium hydrazone complexes exhibit a broad spectrum of
significant biological activities as anticancer, antitumor,
antifungal, antibacterial, antimicrobial, antioxidant, herbicidal,
insecticidal, and plant stimulant properties, as well as being
present in natural oxygen carriers.19,24−26 In addition, the
compounds possessing a p-dimethylaminophenyl moiety
exhibit antinociceptive and analgesic activities.27,28
Pettinari et al. found that the Ru(II)−arene-acylpyrazolona-
to complex exhibits efficient cytotoxicityonvarious cancer cell
lines (HeLa, MCF-7, HepG2, and HCT-116; IC50 = 13−30
μM).29 Recently, the same authors have reported antiprolifer-
ative activity of half-sandwich Ru(II) complexes with N,O- or
N,N-pyrazolone-based hydrazones ligands against MCF-10A,
MCF-7, HeLa, and HCT116 cancer cell lines (IC50 = 10−175
μM).20 The synthesis and antiproliferative activity of RuII(η6-
arene) compounds carrying bioactive flavonol ligands have
been reported by Hartinger et al. (CH1, SW480, A549, 5637,
LCLC-103H, and DAN-G; IC50 = 0.86−19 μM).30 Wei Su et
al. have described the DNA binding properties and anticancer
activity of arene ruthenium(II) complexes bearing ketone-N4-
substituted thiosemicarbazones (SGC-7901, BEL-7404, and
HEK-293T; IC50 = 10−17 μM).31 Furthermore, Castonguay
and his co-workers have reported the synthesis of novel
ruthenium half-sandwich complexes containing (N,O)-bound
Schiff base ligands with moderate anticancer activity (A2780,
SH-SY5Y, MCF-7, T47D, and MCF-12A; IC50 = 8.9−59.3
μM).32 Sadler and his co-workers have reported the solution
behavior and antiproliferative activity of arene ruthenium
thiosemicarbazone complexes against A2780, A2780 Cis,
A549, HCT116, and PC3 cancer cell lines (IC50 = 0.36−
1.64 μM).33 Recently, our research group has explored the
Figure 2. Chemical structures of arene Ru(II) benzhydrazone
complexes 1−6.
Ruthenium hydrazone complexes 1−6 (Figure 2) of the
general formula [(η6-arene)Ru(L)Cl], where L= substituted
dimethylaminobenzhydrazone derivatives, were collected in
good yields. The presence of Et3N in the reaction mixture
facilitates the amido-imidol tautomerization for easy coordi-
nation of imidolate oxygen. All complexes were found to be
stable in air and were readily dissolved in most organic solvents
including acetonitrile, EtOH, MeOH, CHCl3, DMF, DMSO,
and so on. The formation new ruthenium complexes was
B
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
authenticated with the aid of analytical and various spectral
techniques.
516.01 [M + H]+, 476.09 [M − Cl]+, 538.11 [M + H]+, 572.08
[M + H]+, and 568.12 [M + H]+, respectively, confirmingthe
presence of the expected moiety in the solution phase. Hence,
the mass spectral data are in good concord with the molecular
formula proposed for all the complexes.
X-ray Crystallographic Studies. Two of the complexes, 1
and 4, were isolated as single crystals by the slow evaporation
of the 1:2 CH2Cl2/petroleum ether mixture at room
temperature, and their molecular structures were studied
with the aid of crystallographic technique. Crystal data and
refinement parameters were given in Table S1. The selected
bond lengths and bond angles were gathered in Table S2.
From the unit cell parameters, it was obvious that the crystal
system of complex 1 was orthorhombic, belonging to the Pbca
space group, and 4 was monoclinic with the P21/c space group.
The ORTEP views of the complexes 1 and 4 were shown as
thermal ellipsoids in Figures 3 and 4 respectively, which clearly
The νN−H’s of the free ligands show bands in the region of
3242−3427 cm−1. Furthermore, the ligands displayed νCHN
and νCO IR absorptions in the regions of 1546−1601 cm−1
and 1653−1661 cm−1, respectively, indicated that the ligand
exists in its amide form (Figures S1−S9). Upon complexation,
the νN−H and νCO bands disappeared, which indicated that
the ligand underwent amido-imidol tautomerization and
subsequent coordination in its imidol form. This was further
supported by appearance of a C−O band around 1340−1370
cm−1. Coordination of the azomethine nitrogen to the
ruthenium metal center was corroborated by a decrease of
νCHN absorption frequency in the region of 1500−1530
cm−1.
Furthermore, 1H NMR spectra of the complexes support the
binding nature of the ligand to metal ion (Figures S19−S24).
Multiplets observed in the region of 6.68−8.10 ppm were due
to the aromatic protons of benzhydrazone ligands. The
appearance of a sharp singlet in the region of 8.80−8.88
ppm has been assigned to the azomethine proton. This signal
slightly shifted from a lower to a higher frequency during
complexation, which revealed that azomethine nitrogen is one
of the coordinating atoms to ruthenium center. The
disappearance of signal due to −NH protons of the free
ligands in the region of 10.29−11.55 ppm indicated amido-
imidol tautomerism and subsequent coordination of the
imidolate oxygen atom. Hence, spectral data clearly indicated
that the ligands bind to the ruthenium ion through the
coordination of nitrogen of azomethine group and the
imidolate oxygen. Furthermore, the aromatic protons of
benzene moiety in complexes 1−3 displayed a singlet in
region of 5.37−5.44 ppm. The p-cymene ring protons were
observed as doublets at 4.68−5.43 ppm for complexes 4−6. In
addition, a doublet of doublet in the region of 1.11−1.15 ppm
was appeared due to isopropyl methyl protons of the p-
cymene, and the −CH of isopropyl group was displayed as a
septet in the region of 2.29−2.67 ppm. Complexes 3 and 6
exhibited methoxy signals of the benzhydrazone ring as singlets
around 3.80 ppm. 13C{H} NMR of the complexes showed
resonances in the expected region (Figures S25−S30). A
higher frequency shift (160.3−161.4 ppm) obtained for all the
complexes due to imine carbon (−CN−) in comparison
with free ligands (148.0−149.8 ppm) indicated the coordina-
tion of the imine nitrogen to the ruthenium ion. Furthermore,
shifting of the signal from a lower frequency of 161.6−164.3
ppm to a higher frequency of 171.9−173.3 ppm due to the
reduction of the bond order (−NC−O) upon complexation
when compared to free ligands (−NH−CO).
Figure 3. ORTEP view of the complex 1 with 30% probability. All the
hydrogen atoms were omitted for clarity. Bond angles around Ru(II)
ion: O(1)−Ru(1)−N(2) = 76.74(9)°, O(1)−Ru(1)−Cl(1) =
85.99(6)°, N(2)−Ru(1)−Cl(1) = 88.79(7)°. Bond lengths: N(2)−
Ru(1) = 2.112(3)Å, O(1)−Ru(1) = 2.060(2)Å, Cl(1)−Ru(1) =
2.4116(9)Å.
Figure 4. ORTEP view of the complex 4 with 30% probability. All the
hydrogen atoms were omitted for clarity. Bond angles around Ru(I)
ion: O(1)−Ru(1)−N(1) = 76.73(12)°, O(1)−Ru(1)−Cl(1) =
84.89(8)°, N(1)−Ru(1)−Cl(1) = 85.88(10)°. Bond lengths:
N(1)−Ru(1) = 2.096(3)Å, O(1)−Ru(1) = 2.054(2)Å, Cl(1)−
Ru(1) = 2.4028(11)Å.
The absorption spectra of the complexes were monitored in
acetonitrile in the range of 800−200 nm at room temperature
and showed three bands with absorption maxima in the region
of 230−389 nm. Two ligand based transitions such as π−π*
and n−π* were observed around 230−294 nm. Bands with
medium intensity which appeared in the region of 383−389
nm are ascribed to MLCT transitions (Figures S31−S36). The
outline of the absorption spectra of all the complexes looks
similar to those of other reported octahedral arene ruthenium-
(II) complexes.26c,20
Furthermore, HR-MS analysis was performed under positive
ion ESI-MS mode in acetonitrile. The mass spectral data
confirmed the composition of synthesized complexes 1−6
(Figures S37−S44) displayed peaks at m/z 446.08 [M − Cl]+,
indicated that ruthenium was coordinated by the ligand via the
imine nitrogen and imidolate oxygen with the formation of a
five-member chelate ring with bite angles of 76.73(12)−
76.74(9)° in a piano-stool geometry. While the seat of the
piano stool was made by the arene ring, the N∧O bidentate
benzhydrazone and Cl ligands fashioned the three legs of the
stool. Ru−centroid distances fall in the range 1.656−1.677 Å,
and this is in agreement with already reported Ru(II) arene
C
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
Table 1. In Vitro Cytotoxic Effect of Arene Ru(II) Benzhydrazone Complexes after 72 h of Incubation (IC50 SD, μM) and
a
Their Calculated Partition Coefficients (log P)
A549 (SI)
LoVo (SI)
HuH-7 (SI)
16HBE
log P
complex 1
complex 2
complex 3
complex 4
complex 5
complex 6
cisplatin
12.63 0.49 (2.17)
13.85 0.77 (2.04)
9.92 0.98 (2.20)
7.15 0.78 (5.02)
7.73 0.58 (3.23)
6.52 0.69 (3.36)
8.72 0.74 (2.05)
25.04 1.83 (1.09)
21.91 1.55 (1.29)
17.88 1.03 (1.22)
8.21 0.86 (4.38)
6.92 0.61 (3.61)
5.62 0.49 (3.89)
10.95 0.67 (1.63)
19.74 1.12 (1.39)
22.14 1.37 (1.28)
12.78 0.51 (1.71)
7.43 0.48 (4.83)
8.74 0.68 (2.86)
5.37 0.56 (4.08)
10.68 0.68 (1.67)
27.52 1.16
28. 37 2.16
21.88 1.39
35.96 0.57
25.04 0.81
21.91 0.71
17.88 1.12
1.68 0.06
1.94 0.07
1.48 0.01
1.28 0.03
1.31 0.05
1.19 0.02
−1.95 0.09
a
Selectivity index (SI) is defined as IC50 ratio of non-cancerous cells (16HBE) to cancerous cells (A549, LoVo and HuH-7).
complexes.20,35 The σ-bond lengths of three legs of the stool,
Ru−N, Ru−O, and Ru−Cl, and the corresponding bond
angles correspond with other structurally related benzene and
p-cymene ruthenium complexes encompassing similar piano-
stool geometry.36
Stability Study. It is essential to study the stability of metal
complexes in aqueous media for drug development in clinical
investigations. The stability of ruthenium complexes in
aqueous solution was determined using UV−visible spectro-
photometry at various time intervals. The complexes were
taken in a solution of 1% dimethyl sulfoxide in phosphate
buffer at pH 7.4, and the representative spectra were given in
Figure S43. It has been observed from the spectral diagram
that there was no significant change over 72 h intervals,
evidencing the stability of the complexes and their fate under
physiological conditions; hence, they were subjected to further
studies.
Partition Coefficient Determination (Lipophilicity).
The lipophilicity is an important factor to determine the cell
membrane permeation capacity of the complexes and is
studied in terms of the partition coefficient (log P). It is based
on the solubility and distribution of the complexes in an n-
octanol/water system. From the calculated log P values of
complexes 1−6 (Table 1), it was observed that the complexes
are likely to differ in their lipophilicity which might be due to
the variation in the different arene moieties and the nature of
the substituent in the ligands. Furthermore, complexes 4−6
which comprise p-cymene moiety showed higher lipophilicity
than the rest of the benzene complexes suggested that the
substituents on arene ring enhanced the lipophilicity. The
observed log P values for the complexes and cisplatin are in
agreement with the reported ruthenium complexes.37
In Vitro Cytotoxicity by MTT Assay. To check the
potency of the complexes in retarding the growth of cancer
cells, MTT assay was carried out with A549 (lung carcinoma),
LoVo (colon adenocarcinoma), HuH-7 (hepato cellular
carcinoma), and noncancerous 16HBE (human lung bronchial
epithelium) cells over 72 h (Figure S44). No inhibition of the
cell growth was observed by the free ligands or ruthenium
precursor even up to 100 μM. Hence, chelation plays an
important role in the observed cytotoxicity of the complexes.
Most of the complexes exhibit potent cytotoxicity against the
tested cancer cells (Table 1). All the complexes showed a
superior inhibitory effect on the growth of A549 lung cancer
cells among the three cancer cells screened. Complexes 3 and 6
comprising methoxy substituent have some marked effect on
inhibition of cancer cell growth. Notably, complexes 4−6
outperformed the anticancer activity of cisplatin and 2-fold
enhanced activity was found against LoVo and HuH cells in
case of complex 6.20,38,39 When scrutinizing the results, it was
found that the complexes containing a p-cymene moiety
displayed a significant effect when compared to complexes 1−3
which contain a benzene moiety, implying the less hydro-
phobic interaction of Ru(II)−cymene complexes 4−6 with the
cell membrane.26 This observation highlighted the impact of
arene moiety and electron-donating nature of the ligand in
antiproliferation. Advantageously, the cancer cell growth
inhibition capability of complexes 4−6 was significantly high
when compared to other arene ruthenium complexes found in
the literature as evidenced by the low IC50 values.40,41 Despite
this potency, the complexes were much less toxic toward the
noncancerous 16HBE (human lung bronchial epithelium)
cells, and the IC50 values were higher than those of cisplatin
(Table 1). Expediently, the complexes exhibited better activity
against the tested cells than other reported arene Ru(II)
complexes containing different chelating ligands.27 On the
basis of the results, we have selected complexes 4−6 for further
investigations.
Antiproliferative Activity by EdU Assay. Lung cancer
(A549) is the leading cause of death worldwide. Therefore, we
focused our interest to study the anticancer activity of
complexes 4−6 against the A549 cell line. Furthermore, all
the biochemical assay were performed for potent complexes
4−6 against A549 cancer cells. Since the complexes exhibit a
higher cytotoxicity at IC50 concentration in a 72 h incubation
period, we wish to perform all the biochemical assays with a
minimal concentration (3.5 μM) for a 24 h incubation period
in order to predict the apoptosis ratio of the particular cells.
Furthermore, 5-ethynyl-2-deoxyuridine (EdU) incorporation
assay was performed to evaluate the effect of potent Ru(II) p-
cymene complexes 4−6 and cisplatin on prohibiting the cancer
cell proliferation. One of the most proper ways to detect
changes in cell proliferation is estimation of DNA synthesis.
EdU is a thymidine analog comprising an alkyne terminal motif
that subsumes only into the newly synthesized DNA of
proliferating cancer cells when it is added to the growth media
treated with the test compounds. After that, the synthesis of
new DNA was quantified as green fluorescence by a copper-
imparted click reaction between the fluorescent azide and the
alkyne terminal of EdU. The percentage of EdU-stained cells
was calculated on the basis of five randomly selected fields for
each group after treatment with complexes 4−6 and cisplatin.
After the treatment of the complexes, it has been observed that
the percentage of cell proliferation was significantly decreased
(Figure 5). It is inferred from the results that the complexes
notably arrest A549 cell growth. Complex 6 exhibited higher
antiproliferation which might be due to the presence of
electron-donating nature of methoxy group.42
Apoptosis Induction by Dual Acridine Orange−
Ethidium Bromide (AO-EB) Fluorescent Staining. Dual
D
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
cytometry result reveals that the cells are mainly in late
apoptosis (Figure 7a,b), and complex 6 was more potent than
Figure 5. (a) Click-iTEdU assay for determining the proliferation of
A549 cells. The cells were treated with complexes 4−6 and cisplatin
(3.5 μM) for 24 h. (b) Quantification of cell proliferation.
AO-EB staining is a qualitative method to comprehend
apoptosis and categorize live, early apoptotic, late apoptotic,
and necrotic cells by documenting cell nuclear morphological
changes by fluorescent images. AO can permeate the intact
membrane of normal and early apoptotic cells and bind DNA,
then fluorescing green, which is uniform in the case of the
former case and as patches in the latter case due to differences
in chromatin condensation. In contrast, EB is permeable only
to the injured membranes of late apoptotic and dead cells and
fluoresces as bright orange patches by binding with DNA
fragments or apoptotic moieties and as uniform orange
fluorescence in necrotic cells which have nuclear morphologies
of viable cells. AO-EB-stained A549 cells were incubated with
complexes 4−6. As shown in Figure 6, the appearance of
Figure 7. (a) Alexa Fluor 488 Annexin V/propidium iodide (PI)
double-staining assay on A549 cells with complexes 4−6 and cisplatin.
(b) Quantification of apoptotic cell ratio.
cisplatin with regard to inducing apoptosis. Furthermore, the
flow cytometry analysis was performed at different concen-
trations of complex 6. The cell death population in the
quadrant represents cells undergoing apoptosis, which
increased from 9% to 11 and 23% for 1, 2, and 3 μM complex
respectively (Figure S45).45 Recently, Subarkhan et al. recently
reported the tetranuclear arene Ru(II) complexes induce late
apoptosis on A549 cancer cells.40a
Cell Cycle Analysis by Flow Cytometry. Cell cycle
progresses via different phases, namely, G0/G1 (Gap phase
where cell grows and prepares for duplication), S (synthesis
phase where DNA replicates), and G2/Mitosis (cell prepares
to divide and results in two new daughter cells). The
effectiveness of an anticancer drug depends on its ability to
halt cancer cell division by cell cycle arrest in the aforesaid
phases.40a,46 Thence, complexes 4−6 were subjected to FACS
to evaluate its influence on deregulating the cell cycle of A549.
The percentage of A549 cells after treatment at 3.5 μM
complexes 4−6 and cisplatin for 24 h in G0/G1 phase was
decreased from 65.5% (untreated cells) to 14.5, 14.1, 13.6, and
15.2%, respectively (Figure 8). Complexes 4−6 elicited a
strong S phase arrest in cells, accounting for 83.3, 86.7, and
87.3% of the cell population, respectively (untreated cells,
Figure 6. (a) AO-EB staining of A549 cells (3.5 μM, equiv
concentrations) for 24 h. (b) Cell population in the microscopic
field in AO-EB staining.
reddish orange fluorescence with fragmented chromatin after
A549 cells were treated with complexes 4−6 and cisplatin
suggests that complexes 4−6 largely induced apoptosis in A549
cells.43
Apoptosis Evaluation by Flow Cytometry. Apoptosis is
the process of programmed cell death. The apoptosis-inducing
property of arene Ru(II) complexes 4−6 was analyzed by flow
cytometry using Alexa Fluor 488 Annexin V combined with
propidium iodide (PI) staining assay in A549 cells. The cells at
different stages, i.e., live cells, early apoptotic cells, and late
apoptotic cells, were quantified in Annexin V−/PI−, Annexin
V+/PI−, and Annexin V+/PI+ quadrants, respectively, using the
fluorescence-activated cell sorting (FACS) methodology.44
The ability of complexes 4−6 to induce apoptosis in the cancer
cells was evaluated by flow cytometry. The A549 cells were
treated with complexes 4−6 and cisplatin at 24 h. The flow
Figure 8. (a) FACS analysis on A549. (b) Distribution of the cell
cycle.
E
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
27.9%). Furthermore, dose-dependent cell cycle analysis was
carried out for complex 6 and indicated the increase of
apoptotic activity (Figure S46). Thus, the enhancement of the
S phase arrest of cancer cells might result in apoptosis by
disrupting the cell cycle.
against LoVo and HuH-7 cells with low IC50 values 5.62 and
5.37 μM, respectively. The EdU incorporation assay confirmed
that all the complexes inhibit cancer cell proliferation via
minimal DNA synthesis. The cell membrane blebbing
associated with apoptosis induced by the complexes was
established by dual AO-EB fluorescent staining assay. Flow
cytometry analyses confirmed that complexes 4−6 induce
apoptosis in A549 cells, and the complexes have been shown to
arrest A549 cell cycle in S phase. Furthermore, the results of
Western blot analysis established that complex 6 is able to
induce mitochondria-mediated apoptosis in A549 cells.
Overall, the complexes presented herein exhibit considerably
improved biological activity even at low concentrations.
Therefore, this genre of complexes will be taken for further
in vivo studies.
Western Blot Analysis. Western blot technique was
employed to authenticate the exact pathway behind the cancer
cell death activity of the complex candidate. It is well-known
that apoptotic proteins like BAX and antiapoptotic protein
BCL-2 play a key role during the cell cycle.47 BCL-2 is an
intracellular membrane-associated oncogene responsible for
extending cellular survival, which further confirmed that cell
death occurred via apoptosis. BAX administers the release of
cytochrome c from mitochondria to cytosol resulting in an
increase of cytosolic cytochrome c. The expression levels of
BAX and BCL-2 proteins were analyzed in the control and
A549 cells treated with complex 6 (1, 2, and 3 μM). As
portrayed in Figure 9, the diminishing expression level of the
EXPERIMENTAL SECTION
■
Materials, methods, and crystal data collection were given in the
Supporting Information (section S2).
Synthesis of Dimethylaminobenzoylhydrazone Ligands.
The p-dimethylaminobenzaldehyde benzhydrazone ligands were
synthesized by stirring an ethanolic mixture (25 mL) of 4-substituded
benzhydrazides (0.1 mol) and p-dimethylaminobenzaldehyde (0.1
mol) at ambient temperature for 6 h. The completion of reaction
resulted the formation of solid benzhydrazone ligands and which was
collected by filtration, washed with ethanol then dried under vacuum.
Analytically pure compounds were procured from recrystallization
from methanol. NMR spectral data for the ligands were given in
Supporting Information (Figures S13−S18).
Synthesis of Arene Ru(II) Benzhydrazone Complexes. The
[(η6-arene)RuCl2]2 (arene: benzene or p-cymene) (0.5 mmol)
precursor (1 mmol), N,N-dimethylaminobenzhydrazone ligand, and
(1 mmol) triethylamine in 20 mL of benzene were combined and
stirred for 5 h at ambient temperature. The progression of reaction
was monitored through thin-layer chromatography, and column
chromatographic separations of the reaction mixture afforded
complexes 1−6 in 70:30% of hexane/ethyl acetate eluent.
Figure 9. Western blot of BAX and BCL-2 proteins on A549 cells by
complex 6.
BCL-2 protein indicated the programmed cell death associated
with complex 6. Furthermore, the level of proapoptotic protein
BAX in the cancer cells were exceptionally increased on the
treatment with the complex. On the whole, the treatment of
A549 cells with the complex induced the downregulation of
BCL-2 and upregulation of Bax and thus confirmed that the
complex 6 is a potential cancer cell apoptotic promoter. Upon
increasing the concentration of complex 6 (1, 2, and 3 μM) the
upregulation of BAX was increased, and the downregulation of
BCL-2 decreased suggesting the dose-dependent cancer cell
apoptotic activity of complex 6. Furthermore, BAX displays 3-
fold increase and BCL-2 shows a 0.3-fold decrease on
treatment with 3 μM concentration.34a,36b
[Ru(L1)Cl(η6-C6H6)] (1). Brown solid. Yield = 75%; mp: 185 °C
(with decomposition). Calcd: C22H22N3OClRu: C, 54.22; H, 4.64; N,
8.72%. Found: C, 54.25; H, 4.61; N, 8.74%. IR (KBr, cm−1): 1512
3
ν(CN−NC), 1362 ν(C−O). UV−vis (CH CN): λmax, nm (εmax dm
3
mol−1 cm−1) 383 (1733), 292 (6342), 230 (14950). H NMR (400
1
MHz, CDCl3) (ppm): 8.88 (s, 1H, HCN), 8.08 (m, H3, H5, H12,
H16), 7.35 (m, H13, H14, H15), 6.77 (d, 3J = 8 Hz, H2, H6), 3.08 (s,
1
6H, N(CH3)2). 13C{ H} NMR (100 MHz, CDCl3, δ, ppm): 173.3
(C−O), 160.9 (CN), 151.7 (C−N(CH3)2), 132.3 (ArC14), 132.1
(ArC11), 130.1 (ArC13C15), 128.6 (ArC3C5), 127.8 (ArC12C16), 121.7
(ArC4), 111.1 (ArC2,C6), 84.3 (CH of benzene), 40.1 (N(CH3)2), ESI-
MS: m/ z 446.0804 [M − Cl]+ (calcd m/z 481.0494).
[Ru(L2)Cl(η6-C6H6)] (2). Brown solid. Yield = 78%, mp: 189 °C
(with decomposition). Calcd: C22H21N3Cl2ORu: C, 51.22; H, 4.17;
N, 8.17%. Found: C, 51.27; H, 4.11; N, 8.15%. IR (KBr, cm−1): 1530
ν(CN−NC), 1366 ν(C−O). UV−vis (CH3CN): λmax, nm (εmax dm3
CONCLUSIONS
■
The present investigation highlights the synthesis of a new set
of arene ruthenium(II) complexes bearing N,N-dimethylami-
nobenzhydrazone. Analytical, IR, UV−vis, NMR, and HR-MS
methods have been used to establish the composition of the
complexes. X-ray crystallographic study of complexes con-
firmed the bidentate chelation of benzhydrazone ligands and
revealed the existence of pseudo-octahedral geometry around
the ruthenium ion. MTT assay was utilized to evaluate in vitro
cancer cell growth inhibition capacity of the complexes against
A549 (lung carcinoma), LoVo (colon adenocarcinoma), HuH-
7 (hepato cellular carcinoma) along with the noncancerous
16HBE (human lung bronchial epithelium) cells and
compared with the effect of the standard drug cisplatin. p-
Cymene complexes 4−6 outperformed benzene complexes 1−
3 and cisplatin in all the cancer cells tested. In particular, 2-fold
enhanced activity of complex 6 over that of cisplatin was found
1
mol−1 cm−1): 389 (2654), 291 (5510), 233 (10700). H NMR (400
MHz, CDCl3) (ppm): 8.80 (s, 1H, HCN), 8.00 (t, 3J = 16 Hz, H3,
3
3
H5, H12, H16), 7.23 (d, J = 8 Hz, H13, H15), 6.70 (d, J = 8 Hz,
H2, H6), 5.38 (s, 6H, CH-benzene), 3.01 (s, 6H, N(CH3)2). 13C{1H}
NMR (100 MHz, CDCl3, δ, ppm): 172.2 (C−O), 161.4 (CN),
151.8 (C−N(CH3)2), 136.2 (ArC14), 132.2 (ArC12,C16), 130.0
(ArC13,C15), 128.1 (ArC3,C5), 128.0 (ArC11) 121.4 (ArC4), 111.1
(ArC2,C6) 84.3 (CH of benzene), 40.1 (N(CH3)2), ESI-MS: m/z
516.0173 [M + H]+ (calcd m/z 515.0105).
[Ru(L3)Cl(η6-C6H6)] (3). Brown solid. Yield = 81%, mp: 202 °C
(with decomposition). Calcd: C23H24N3O2ClRu: C, 61.32; H, 4.08;
N, 4.93%. Found: C, 61.34 H, 4.04; N, 4.95%. IR (KBr, cm−1): 1500
ν(CN−NC), 1363 ν(C−O). UV−vis (CH3CN): λmax, nm (εmax dm3
1
mol−1 cm−1): 387 (2114), 293 (5649), 233 (10611). H NMR (400
MHz, CDCl3) (ppm): 8.85 (s, 1H, HCN), 8.05 (m, H3, H5, H12,
F
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
H16), 6.85 (d, 3J = 12 Hz, H13, H15), 6.76 (d, 3J = 12 Hz, H2, H6),
5.42 (s, 6H, CH−benzene), 3.07 (s, 6H, N(CH3)2), 3.82 (s, 3H,
OCH3). 13C{1H} NMR (100 MHz, CDCl3, δ, ppm): 173.2 (C−O),
162.0 (CN), 160.1 (ArC14), 151.6 (C−N(CH3)2), 132.2
(ArC12,C16), 130.3 (ArC3,C5), 124.7 (ArC4), 122.0 (ArC11), 113.1
(ArC13,C15), 111.1 (ArC2,C6), 84.2 (CH of benzene), 55.3 (OCH3) 40.1
(N(CH3)2). ESI-MS: m/z 476.0908 [M − Cl]+ (calcd m/z
511.0600).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.organomet.0c00092.
■
sı
Complete details and procedures for chemistry and
biology experiments. Characterization data for ligands,
tables for crystallographic data, refinement parameters,
selected bond lengths and bond angles. Figures
illustrating the FT-IR, NMR, UV−vis, HR-MS, stability
studies and MTT results of the new compounds (PDF)
[Ru(L1)Cl(η6-p-cymene)] (4). Red-brown solid. Yield = 85%, mp:
210 °C (with decomposition). Calcd: C26H30N3OClRu: C, 64.47; H,
5.24; N, 4.70%. Found: C, 64.49; H, 5.26; N, 4.68%. IR (KBr, cm−1):
1516 ν(CN−NC), 1361 ν(C−O). UV−vis (CH3CN): λmax, nm (εmax
1
dm3 mol−1 cm−1) 387 (2513), 294 (6140), 231 (11460). H NMR
Accession Codes
CCDC 1565996 and 1833167 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
(400 MHz, CDCl3) (ppm): 8.88 (s, 1H, HCN), 8.06 (m, H3, H5,
H12, H16), 7.34 (m, H13, H14, H15), 6.76 (d, 3J = 12 Hz, H2, H6),
5.43 (d, J = 6 Hz, 1H, p-cymene-H), 5.34 (d, J = 6 Hz, 1H, p-
cymene−H), 4.96 (d, J = 6.4 Hz, 1H, p-cymene−H), 4.72 (d, J = 6
Hz, 1H, p-cymene−H), (3.07, s, 6H, −N(CH3)2), 2.67 (m, 1H, p-
cym CH(CH3)2), 2.30 (s, 3H, p-cymene-CCH3), 1.13 (dd, J = 12.4
Hz, 6H, p-cymene−CH(CH3)2). 13C{1H}NMR (100 MHz, CDCl3,
δ, ppm): 173.1 (C−O), 160.3 (CN), 151.5 (C−N(CH3)2), 132.4
(ArC14), 132.3 (ArC11), 130.0 (ArC13C15), 128.6 (ArC3C5), 127.8
(ArC12C16), 121.7 (ArC4), 111.1 (ArC2,C6), 101.4 and 101.1 (quaternary
carbons of p-cymene), 80.9, 81.3, 82.1, 84.6 (Ar carbons of p-
cymene), 40.1 (N(CH3)2), 30.9 (CH, p-cymene), 22.6, 22.0 (2CH3,
p-cymene), 18.9 (CH3, p-cymene), ESI-MS: m/z 538.1190 (M +
H)+(calcd m/z 537.1120).
AUTHOR INFORMATION
Corresponding Author
■
Rengan Ramesh − Centre for Organometallic Chemistry, School
of Chemistry, Bharathidasan University, Tiruchirappalli 620
024, India; orcid.org/0000-0002-0257-8792;
Email: ramesh_bdu@yahoo.com
[Ru(L2)Cl(η6-p-cymene)] (5). Red-brown solid. Yield = 89%, mp:
205 °C (with decomposition). Calcd: C26H29N3Cl2ORu: C, 61.15; H,
4.49; N, 4.46%. Found: C, 61.13; H, 4.53; N, 4.46%. IR (KBr, cm−1):
1519 ν(CN−NC), 1340 ν(C−O). UV−vis (CH3CN): λmax, nm (εmax
Authors
Sundarraman Balaji − Centre for Organometallic Chemistry,
School of Chemistry, Bharathidasan University, Tiruchirappalli
620 024, India
1
dm3 mol−1 cm−1) 388 (1457), 287 (5564), 236 (10096). H NMR
Mohamed Kasim Mohamed Subarkhan − The First Affiliated
Hospital, Key Laboratory of Combined Multi-Organ
Transplantation, Ministry of Public Health, School of Medicine,
Zhejiang University, Hangzhou 310003, PR China
Hangxiang Wang − The First Affiliated Hospital, Key
Laboratory of Combined Multi-Organ Transplantation,
Ministry of Public Health, School of Medicine, Zhejiang
University, Hangzhou 310003, PR China; orcid.org/0000-
0001-6370-9728
(400 MHz, CDCl3) (ppm): 8.85 (s, 1H, HCN), 8.02 (m, H3, H5,
3
3
H12, H16), 7.28 (d, J = 8 Hz, H13, H15), 6.75 (d, J = 8 Hz, H2,
H6), 5.42 (d, J = 5.6 Hz, 1H, p-cymene−H), 5.33 (d, J = 5.6 Hz, 1H,
p-cymene−H), 4.96 (d, J = 5.6 Hz, 1H, p-cymene−H), 4.72 (d, J =
5.6 Hz, 1H, p-cymene−H), (3.07, s, 6H, −N(CH3)2), 2.64 (m, 1H, p-
cymene−CH(CH3)2), 2.30 (s, 3H, p-cymene−CCH3), 1.11−1.13
(dd, J = 12 Hz, 6H, p-cymene−CH(CH3)2). 13C{1H} NMR (100
MHz, CDCl3, δ, ppm): 171.9 (C−O), 160.6 (CN), 151.6 (C−
N(CH3)2), 136.0 (ArC14), 132.3 (ArC12,C16), 131.0 (ArC13,C15), 130.0
(ArC3,C5), 128.0 (ArC11) 122.0 (ArC4), 111.1 (ArC2,C6), 101.5 and
101.1 (quaternary carbons of p-cymene), 84.5, 82.1, 81.3, 80.9 (Ar
carbons of p-cymene), 40.1 (N(CH3)2), 30.9 (CH, p-cymene), 22.6,
21.9 (2CH3, p-cymene), 18.9 (CH3, p-cymene). ESI-MS: m/z
572.0800 (M + H)+(calcd m/z 571.0712).
David Semeril − Laboratoire de Chimie Inorganique et Catalyse,
Institut de Chimie, UMR 7177, CNRS, Universite de
Strasbourg, Strasbourg 67008, France
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.organomet.0c00092
[Ru(L3)Cl(η6-p-cymene)] (6). Red-brown solid. Yield = 90%, mp:
196 °C (with decomposition). Calcd: C27H32N3O2ClRu: C, 63.50; H,
5.01; N, 4.49%. Found: C, 63.48; H, 5.01; N, 4.48%. IR (KBr, cm−1):
1508 ν(CN−NC), 1370 ν(C−O). UV−vis (CH3CN): λmax, nm (εmax
Notes
The authors declare no competing financial interest.
1
dm3 mol−1 cm−1) 385 (1222), 290 (5975), 234 (16589). H NMR
ACKNOWLEDGMENTS
(400 MHz, CDCl3) (ppm): 8.86 (s, 1H, HCN), 8.03 (d, 3J = 8 Hz,
■
3
3
The authors thank DST−FIST, New Delhi, for HR-MS and
NMR facilities at the School of Chemistry, Bharathidasan
University, Tiruchirappalli. M.S.K. gratefully acknowledges the
Zhejiang Province Fund (No. 519000-X81801) for the
financial support.
H3, H5, H12, H16), 6.84 (d, J = 8 Hz, H13, H15), 6.76 (d, J = 8
Hz, H2, H6), 5.42 (d, J = 6 Hz, 1H, p-cymene−H), 5.33 (d, J = 5.6
Hz, 1H, p-cymene−H), 4.95 (d, J = 6 Hz, 1H, p-cymene−H), 4.69 (d,
J = 6 Hz, 1H, p-cymene−H), (3.07, s, 6H, −N(CH3)2), 3.81 (s, 3H,
OCH3), 2.67 (m, 1H, p-cymene−CH(CH3)2), 2.29 (s, 3H, p-
cymene−CCH3), 1.15 (dd, J = 13.2 Hz, 6H, p-cymene−CH(CH3)2).
13C{1H} NMR (100 MHz, CDCl3, δ, ppm): 173.0 (C−O), 161.2
(CN), 159.6 (ArC14), 151.4 (C−N(CH3)2), 132.2 (ArC12,C16),
130.3 (ArC3,C5), 125.0 (ArC4), 122.3 (ArC11), 113.1 (ArC13,C15), 111.1
(ArC2,C6), 101.4 and 101.0 (quaternary carbons of p-cymene), 84.6,
82.1, 81.4, 80.9 (Ar carbons of p-cymene), 55.3 (OCH3), 40.2
(N(CH3)2), 30.9 (CH, p-cymene), 22.6, 22.0 (2CH3, p-cymene), 18.9
(CH3, p-cymene). ESI-MS: m/z 568.1295 (M + H)+ (calcd m/z
567.1226).
REFERENCES
■
(1) Kelland, L. The resurgence of platinum-based cancer chemo-
therapy. Nat. Rev. Cancer 2007, 7, 573−584.
(2) Judson, I.; Cerny, T.; Epelbaum, R.; Dunlop, D.; Smyth, J.;
Schaefer, B.; Roelvink, M.; Kaplan, S.; Hanauske, A. Phase II trial of
the oral platinum complex JM216 in non-small-cell lung cancer: An
EORTC early clinical studies group investigation. Annals of Oncology
1997, 8, 604−606.
G
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
(3) Koepf-Maier, P.; Koepf, H. Non-platinum group metal antitumor
agents. History, current status, and perspectives. Chem. Rev. 1987, 87,
1137−1152.
(4) (a) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Combination of
Ru(II) complexes and light: new frontiers in cancer therapy. Chem.
Sci. 2015, 6, 2660−2686. (b) Mari, C.; Gasser, G. Lightening up
Ruthenium Complexes to Fight Cancer? Chimia 2015, 69, 176−181.
(c) Kostova, I. Ruthenium Complexes as Anticancer Agents. Curr.
Med. Chem. 2006, 13, 1085−1107.
(5) Reisner, E.; Arion, V. B.; Keppler, B. K.; Pombeiro, A. J. L.
Electron-transfer activated metal-based anticancer drugs. Inorg. Chim.
Acta 2008, 361, 1569−1583.
(6) Biersack, B. Anticancer Activity and Modes of Action of (arene)
ruthenium(II) Complexes Coordinated to C-, N-, and O-ligands.
Mini-Rev. Med. Chem. 2016, 16, 804−814.
(7) Bergamo, A.; Gaiddon, C.; Schellens, J. H. M.; Beijnen, J. H.;
Sava, G. Approaching tumour therapy beyond platinum drugs: Status
of the art and perspectives of ruthenium drug candidates. J. Inorg.
Biochem. 2012, 106, 90−99.
(8) Bergamo, A.; Sava, G. Ruthenium anticancer compounds: myths
and realities of the emerging metal-based drugs. Dalton Trans. 2011,
40, 7817−7823.
(9) (a) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast,
B.; Zorbas, H.; Keppler, B. K. From bench to bedside - preclinical and
early clinical development of the anticancer agent indazolium trans-
[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J.
Inorg. Biochem. 2006, 100, 891−904. (b) Hartinger, C. G.; Jakupec,
M. A.; Zorbas-Seifried, S.; Groessl, M.; Egger, A.; Berger, W.; Zorbas,
H.; Dyson, P. J.; Keppler, B. K. KP1019, A New Redox-Active
Anticancer Agent - Preclinical Development and Results of a Clinical
Phase I Study in Tumor Patients. Chem. Biodiversity 2008, 5, 2140−
2155.
(10) (a) Bergamo, A.; Messori, L.; Piccioli, F.; Cocchietto, M.; Sava,
G. Biological role of adduct formation of the ruthenium(III) complex
NAMI-A with serum albumin and serum transferrin, Invest. Invest.
New Drugs 2003, 21, 401−411. (b) Alessio, E.; Mestroni, G.;
Bergamo, A.; Sava, G. Ruthenium Antimetastatic Agents. Curr. Top.
Med. Chem. 2004, 4, 1525−1535. (c) Alessio, E. 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.
Treatment of Tumors. Coord. Chem. Rev. 2016, 306, 86−114.
(d) Morris, R. E.; Aird, R. E.; del Socorro Murdoch, P.; Chen, H.;
Cummings, J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.;
Jodrell, D. I.; Sadler, P. J. Inhibition of Cancer Cell Growth by
Ruthenium(II) Arene Complexes. J. Med. Chem. 2001, 44, 3616−
3621. (e) Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.;
Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, C. H.; Ang, W. H.; Droge,
P.; Rothlisberger, U.; Dyson, P. J.; Davey, C. A. Ligand substitutions
between ruthenium-cymene compounds can control protein versus
DNA targeting and anticancer activity. Nat. Commun. 2014, 5, 3462.
(f) Berndsen, R. H.; Weiss, A.; Abdul, U. K.; Wong, T. J.; Meraldi, P.;
Griffioen, A. W.; Dyson, P. J.; Nowak-Sliwinska, P. Combination of
ruthenium(II)-arene complex [Ru(η6-p-cymene)Cl2(pta)] (RAP-
TAC) and the epidermal growth factor receptor inhibitor erlotinib
results in efficient angiostatic and antitumor activity. Sci. Rep. 2017, 7,
43005.
(17) (a) Martínez-Pena, F.; Infante-Tadeo, S.; Habtemariam, A.;
Pizarro, A. M. Reversible pH-Responsive Behavior of Ruthenium(II)
Arene Complexes with Tethered Carboxylate. Inorg. Chem. 2018, 57,
5657−5668. (b) Zhao, J.; Zhang, D.; Hua, W.; Li, W.; Xu, G.; Gou, S.
Anticancer Activity of Bifunctional Organometallic Ru(II) Arene
Complexes Containing a 7-Hydroxycoumarin Group. Organometallics
̃
́
2018, 37, 441−447. (c) Chen, F.; Romero-Canelon, I.; Soldevila-
Barreda, J. J.; Song, J.-I.; Coverdale, J. P. C.; Clarkson, G. J.;
Kasparkova, J.; Habtemariam, A.; Wills, M.; Brabec, V.; Sadler, P. J.
Transfer Hydrogenation and Antiproliferative Activity of Tethered
Half-Sandwich Organoruthenium Catalysts. Organometallics 2018, 37,
1555−1566. (d) Li, J. J.; Tian, Z.; Ge, X.; Xu, Z.; Feng, Y.; Liu, Z.
Design, synthesis, and evaluation of fluorine and Naphthyridinee
Based half-sandwich organoiridium/ruthenium complexes with
bioimaging and anticancer activity. Eur. J. Med. Chem. 2019, 163,
830−839. (e) Ma, W.; Guo, L.; Tian, Z.; Zhang, S.; He, X.; Li, J.;
Yang, Y.; Liu, Z. Rhodamine-modified Fluorescent Half-sandwich
Iridium and Ruthenium Complexes: Potential Application as
Bioimaging and Anticancer Agents. Dalton Trans. 2019, 48, 4788−
4793.
̌
́
́
́
́
(18) Grguric-Sipka, S.; Ivanovic, I.; Rakic, G.; Todorovic, N.;
̌
́
́
̌
́
Gligorijevic, N.; Radulovic, S.; Arion, V. B.; Keppler, B. K.; Tesic, Z.
L. Ruthenium(II)-arene complexes with functionalized pyridines:
Synthesis, characterization and cytotoxic activity. Eur. J. Med. Chem.
2010, 45, 1051−1058.
(11) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen,
Z. S. The development of anticancer ruthenium(II) complexes: from
single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017,
46, 5771−5804.
(19) Martínez-Pena, F.; Infante-Tadeo, S.; Habtemariam, A.;
̃
Pizarro, A. M. Reversible pH-Responsive Behavior of Ruthenium(II)
Arene Complexes with Tethered Carboxylate. Inorg. Chem. 2018, 57,
5657−5668.
(12) Smithen, D. A.; Yin, H.; Beh, M. H. R.; Hetu, M.; Cameron, T.
S.; McFarland, S. A.; Thompson, A. Synthesis and Photobiological
Activity of Ru(II) Dyads Derived from Pyrrole-2-carboxylate
Thionoesters. Inorg. Chem. 2017, 56, 4121−4132.
(13) Hayward, R. L.; Schornagel, Q. C.; Tente, R.; Macpherson, J.
S.; Aird, R. E.; Guichard, S.; Habtemariam, A.; Sadler, P.; Jodrell, D. I.
Investigation of the role of Bax, p21/Waf1 and p53 as determinants of
cellular responses in HCT116 colorectal cancer cells exposed to the
novel cytotoxic ruthenium(II) organometallic agent, RM175. Cancer
Chemother. Pharmacol. 2005, 55, 577−583.
(14) Chatterjee, S.; Kundu, S.; Bhattacharyya, A.; Hartinger, C. G.;
Dyson, P. J. The ruthenium(II)-arene compound RAPTA-C induces
apoptosis in EAC cells through mitochondrial and p53-JNK pathways.
JBIC, J. Biol. Inorg. Chem. 2008, 13, 1149−1155.
(15) Thota, S.; Rodrigues, D. A.; Crans, D. C.; Barreiro, E. J. Ru(II)
Compounds: Next-Generation Anticancer Metallotherapeutics. J.
Med. Chem. 2018, 61, 5805−5821.
(16) (a) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.;
Chen, Z.-S. The development of anticancer ruthenium(II) complexes:
from single molecule compounds to nanomaterials. Chem. Soc. Rev.
2017, 46, 5771−5804. (b) Zhang, P.; Sadler, P. J. Advances in the
design of organometallic anticancer complexes. J. Organomet. Chem.
2017, 839, 5−14. (c) Murray, B. S.; Babak, M. V.; Hartinger, C. G.;
Dyson, P. J. The Development of RAPTA Compounds for the
(20) Pettinari, R.; Marchetti, F.; Di Nicola, C.; Pettinari, C.;
Galindo, A.; Petrelli, R.; Cappellacci, L.; Cuccioloni, M.; Bonfili, L.;
Eleuteri, A. M.; Guedes da Silva, M. F. C.; Pombeiro, A. J. L. Ligand
Design for N, O- or N, N-Pyrazolone-Based Hydrazones Ruthenium-
(II)-Arene Complexes and Investigation of Their Anticancer Activity.
Inorg. Chem. 2018, 57, 14123−14133.
(21) Rahman, F.-U.; Bhatti, M. Z.; Ali, A.; Duong, H.-Q.; Zhang, Y.;
Ji, X.; Lin, Y.; Wang, H.; Li, Z.-T.; Zhang, D.-W. DimetallicRu(II)
arene complexes appended on bis-salicylaldimine induce cancer cell
death and suppress invasion via p53-dependent signaling. Eur. J. Med.
Chem. 2018, 157, 1480−1490.
(22) Yamada, S. Advancement in stereochemical aspects of Schiff
base metal complexes. Coord. Chem. Rev. 1999, 190−192, 537−555.
(23) Yan, Y. K.; Melchart, M.; Habtemariam, A.; Sadler, P. J.
Organometallic chemistry, biology and medicine: ruthenium arene
anticancer complexes. Chem. Commun. 2005, 4764−4776.
(24) Kandile, N. G.; Mohamed, M. I.; Zaky, H.; Mohamed, H. M.
Novel pyridazine derivatives: Synthesis and antimicrobial activity
evaluation. Eur. J. Med. Chem. 2009, 44, 1989−1996.
(25) Hruskova, K.; Kovarikova, P.; Bendova, P.; Haskova, P.;
Mackova, E.; Stariat, J.; Vavrova, A.; Vavrova, K.; Simunek, T.
Synthesis and Initial in Vitro Evaluations of Novel Antioxidant
Aroylhydrazone Iron Chelators with Increased Stability against
Plasma Hydrolysis. Chem. Res. Toxicol. 2011, 24, 290−302.
H
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
(26) (a) Leyva, L.; Sirlin, C.; Rubio, L.; Franco, C.; Lagadec, R. L.;
Spencer, J.; Bischoff, P.; Gaiddon, C.; Loeffler, J.-P.; Pfeffer, M.
Synthesis of Cycloruthenated Compounds as Potential Anticancer
Agents. Eur. J. Inorg. Chem. 2007, 2007, 3055−3066. (b) Fetzer, L.;
Boff, B.; Ali, M.; Xiangjun, M.; Collin, J.-P.; Sirlin, C.; Gaiddon, C.;
Pfeffer, M. Library of second-generation cycloruthenated compounds
and evaluation of their biological properties as potential anticancer
drugs: Passing the nanomolar barrier. Dalton Trans. 2011, 40, 8869−
8878. (c) Mohamed Kasim, M. S.; Sundar, S.; Rengan, R. Synthesis
and structure of new binuclear ruthenium(II) arene benzilbis-
(benzoylhydrazone) complexes: investigation on antiproliferative
activity and apoptosis induction. Inorg. Chem. Front. 2018, 5, 585−
596.
Synthesis, structures and catalytic transfer hydrogenation of ketones.
J. Organomet. Chem. 2013, 723, 26−35.
(37) (a) Gupta, R. K.; Sharma, G.; Pandey, R.; Kumar, A.; Koch, B.;
Li, P.-Z.; Xu, Q.; Pandey, D. S. DNA/protein binding, molecular
docking, and in vitro anticancer activity of some thioether-dipyrrinato
complexes. Inorg. Chem. 2013, 52, 13984−13996. (b) Gupta, R. K.;
Pandey, R.; Sharma, G.; Prasad, R.; Koch, B.; Srikrishna, S.; Li, P.-Z.;
Xu, Q.; Pandey, D. S. DNA Binding and Anti-Cancer Activity of
Redox-Active Heteroleptic Piano-Stool Ru(II), Rh(III), and Ir(III)
Complexes Containing 4(2-Methoxypyridyl)phenyldipyrromethene.
Inorg. Chem. 2013, 52, 3687−3698. (c) OECD. Guidelines for Testing
of Chemicals; OECD: Paris, France, 1995; Vol. 107.
(38) (a) Vajpayee, V.; Yang, Y. J.; Kang, S. C.; Kim, H.; Kim, I. S.;
Wang, M.; Stang, P. J.; Chi, K.-W. Hexanuclear self-assembled arene-
ruthenium nano-prismatic cages: potential anticancer agents. Chem.
Commun. 2011, 47, 5184−5186. (b) Kubanik, M.; Holtkamp, H.;
(27) (a) Ribeiro, I. G.; da Silva, K. C. M.; Parrini, S. C.; de Miranda,
A. L. P.; Fraga, C. A. M.; Barreiro, E. J. Synthesis and antinociceptive
properties of new structurally planned imidazo[1,2-a]pyridine 3-
acylarylhydrazone derivatives. Eur. J. Med. Chem. 1998, 33, 225−235.
̈
Sohnel, T.; Jamieson, S. M. F.; Hartinger, C. G. Impact of the
Halogen Substitution Pattern on the Biological Activity of Organo-
ruthenium 8-Hydroxyquinoline Anticancer Agents. Organometallics
2015, 34, 5658−5668. (c) Gupta, R. K.; Sharma, G.; Pandey, R.;
Kumar, A.; Koch, B.; Li, P.-Z.; Xu, Q.; Pandey, D. S. DNA/Protein
Binding, Molecular Docking, and in Vitro Anticancer Activity of Some
Thioether-Dipyrrinato Complexes. Inorg. Chem. 2013, 52, 13984−
13996. (d) Mukhopadhyay, S.; Gupta, R. K.; Paitandi, R. P.; Rana, N.
K.; Sharma, G.; Koch, B.; Rana, L. K.; Hundal, M. S.; Pandey, D. S.
Synthesis, Structure, DNA/Protein Binding, and Anticancer Activity
of Some Half-Sandwich Cyclometalated Rh(III) and Ir(III)
Complexes. Organometallics 2015, 34, 4491−4506.
́
(28) Lima, P. C.; Lima, L. M.; da Silva, K. C. M.; Leda, P. H. O.; de
Miranda, A. L. P.; Fraga, C. A. M.; Barreiro, E. J. Synthesis and
analgesic activity of novel N-acylarylhydrazones and isosters, derived
from natural safrole. Eur. J. Med. Chem. 2000, 35, 187−203.
(29) Pettinari, R.; Pettinari, C.; Marchetti, F.; Skelton, B. W.; White,
A. H.; Bonfili, L.; Cuccioloni, M.; Mozzicafreddo, M.; Cecarini, V.;
Angeletti, M.; Nabissi, M.; Eleuteri, A. M. Arene-Ruthenium(II)
Acylpyrazolonato Complexes: Apoptosis-Promoting Effects on
Human Cancer Cells. J. Med. Chem. 2014, 57, 4532−4542.
̈
(30) Kurzwernhart, A.; Kandioller, W.; Bachler, S.; Bartel, C.;
Martic, S.; Buczkowska, M.; Muhlgassner, G.; Jakupec, M. A.; Kraatz,
̈
(39) Morris, R. E.; Aird, R. E.; del Socorro Murdoch, P.; Chen, H.;
Cummings, J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.;
Jodrell, D. I.; Sadler, P. J. Inhibition of Cancer Cell Growth by
Ruthenium(II) Arene Complexes. J. Med. Chem. 2001, 44, 3616−
3621.
H.-B.; Bednarski, P. J.; Arion, V. B.; Marko, D.; Keppler, B. K.;
Hartinger, C. G. Structure-Activity Relationships of Targeted
RuII(η6-p-Cymene) Anticancer Complexes with Flavonol-Derived
Ligands. J. Med. Chem. 2012, 55, 10512−10522.
(31) Su, W.; Qian, Q.; Li, P.; Lei, X.; Xiao, Q.; Huang, S.; Huang, C.;
Cui, J. Synthesis, Characterization, and Anticancer Activity of a Series
of Ketone-N4-Substituted Thiosemicarbazones and Their
Ruthenium(II) Arene Complexes. Inorg. Chem. 2013, 52, 12440−
12449.
(32) Haghdoost, M. M.; Guard, J.; Golbaghi, G.; Castonguay, A.
Anticancer Activity and Catalytic Potential of Ruthenium(II)-Arene
Complexes with N, O-Donor Ligands. Inorg. Chem. 2018, 57, 7558−
7567.
(40) (a) Mohamed Subarkhan, M. K.; Ren, L.; Xie, B.; Chen, C.;
Wang, Y.; Wang, H. Novel tetranuclear ruthenium(II) arene
complexes showing potent cytotoxic and antimetastatic activity as
well as low toxicity in vivo. Eur. J. Med. Chem. 2019, 179, 246−256.
̌
(b) Canovic, P.; Simovic, A.; Radisavljevic, S.; Bratsos, I.; Demitri, N.;
̌
̌
Mitrovic, M.; Zelen, I.; Bugarcic, Z. D. Impact of aromaticity on
anticancer activity of polypyridylruthenium(II) complexes: Synthesis,
structure, DNA/proteinbinding, lipophilicity and anticancer activity.
JBIC, J. Biol. Inorg. Chem. 2017, 22 (7), 1007−1028. (c) Habtemariam,
́
(33) Gatti, A.; Habtemariam, A.; Romero-Canelon, I.; Song, J.;
́
A.; Melchart, M.; Fernandez, R.; Parsons, S.; Oswald, I. D. H.; Parkin,
Heer, B.; Clarkson, G. J.; Rogolino, D.; Sadler, P. J.; Carcelli, M. Half-
Sandwich Arene Ruthenium(II) and Osmium(II) Thiosemicarbazone
Complexes: Solution Behavior and Antiproliferative Activity. Organo-
metallics 2018, 37, 891−899.
A.; Fabbiani, F. P. A.; Davidson, J. E.; Dawson, A.; Aird, R. E.; Jodrell,
D. I.; Sadler, P. J. Structure-Activity Relationships for Cytotoxic
Ruthenium(II) Arene Complexes Containing N, N-, N, O-, and O, O-
Chelating Ligands. J. Med. Chem. 2006, 49, 6858−6868.
(34) (a) Subarkhan, M. K. M.; Ramesh, R. Ruthenium(II) arene
complexes containing benzhydrazone ligands: synthesis, structure and
antiproliferative activity. Inorg. Chem. Front. 2016, 3, 1245−1255.
(b) Raj Kumar, R.; Ramesh, R.; Małecki, J. G. Synthesis and structure
of arene ruthenium(II) benzhydrazone complexes: Antiproliferative
activity, apoptosis induction and cell cycle analysis. J. Organomet.
Chem. 2018, 862, 95−104.
(35) Haghdoost, M.; Golbaghi, G.; Letourneau, M.; Patten, S. A.;
Castonguay, A. 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 cisplatin.
Eur. J. Med. Chem. 2017, 132, 282−293.
(36) (a) Mohan, N.; Mohamed Subarkhan, M. K.; Ramesh, R.
Synthesis, antiproliferative activity and apoptosis-promoting effects of
arene ruthenium(II) complexes with N, O chelating ligands. J.
Organomet. Chem. 2018, 859, 124−131. (b) Mohamed Subarkhan, M.
K.; Ramesh, R.; Liu, Y. Synthesis and molecular structure of arene
ruthenium(II) benzhydrazone complexes: impact of substitution at
the chelating ligand and arene moiety on antiproliferative activity.
New J. Chem. 2016, 40, 9813−9823. (c) Pandiarajan, D.; Ramesh, R.
Ruthenium(II) half-sandwich complexes containing thioamides:
(41) Ruiz, J.; Rodríguez, V.; Cutillas, N.; Espinosa, A.; Hannon, M. J.
A Potent Ruthenium(II) Antitumor Complex Bearing a Lipophilic
Levonorgestrel Group. Inorg. Chem. 2011, 50, 9164−9171.
́
(42) (a) Liu, Z.; Romero-Canelon, I.; Habtemariam, A.; Clarkson,
G. J.; Sadler, P. J. Potent Half-Sandwich Iridium(III) Anticancer
Complexes Containing C∧N-Chelated and Pyridine Ligands. Organo-
metallics 2014, 33, 5324−5333. (b) Mohan, N.; Muthumari, S.;
Ramesh, R. Synthesis, structure and anticancer activity of (η6-
benzene) ruthenium(II) complexes containing aroylhydrazone
ligands. J. Organomet. Chem. 2016, 807, 45−51.
(43) He, L.; Liao, S.-Y.; Tan, C.-P.; Ye, R.-R.; Xu, Y.-W.; Zhao, M.;
Ji, L.-N.; Mao, Z.-W. Ruthenium-Arene-β-Carboline Complexes as
Potent Inhibitors of Cyclin-Dependent Kinase 1: Synthesis,
Characterization and Anticancer Mechanism Studies. Chem. - Eur. J.
2013, 19, 12152−12160.
(44) Mohamed Subarkhan, M.; Prabhu, R. N.; Raj Kumar, R.;
Ramesh, R. Antiproliferative activity of cationic and neutral
thiosemicarbazone copper(II) complexes. RSC Adv. 2016, 6,
25082−25093.
(45) Wang, H.; Chen, J.; Xu, C.; Shi, L.; Tayier, M.; Zhou, J.; Zhang,
J.; Wu, J.; Ye, Z.; Fang, T.; Han, W. Cancer Nanomedicines Stabilized
by π-π Stacking between HeterodimericProdrugs Enable Exception-
I
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX

Organometallics
pubs.acs.org/Organometallics
Article
ally High Drug Loading Capacity and Safer Delivery of Drug
Combinations. Theranostics 2017, 7, 3638−3652.
(46) Wang, J.; Wang, H.; Li, J.; Liu, Z.; Xie, H.; Wei, X.; Lu, D.;
Zhuang, R.; Xu, X.; Zheng, S. iRGD-Decorated Polymeric Nano-
particles for the Efficient Delivery of Vandetanib to Hepatocellular
Carcinoma: Preparation and in Vitro and in Vivo Evaluation. ACS
Appl. Mater. Interfaces 2016, 8, 19228−19237.
(47) Yuan, Y.; Xu, L.; Dai, S.; Wang, M.; Wang, H. A facile
supramolecular approach to fabricate multifunctional upconversion
nanoparticles as a versatile platform for drug loading, in vivo delivery
and tumor imaging, A facile supramolecular approach to fabricate
multifunctional upconversion nanoparticles as a versatile platform for
drug loading, in vivo delivery and tumor imaging. J. Mater. Chem. B
2017, 5, 2425−2435.
J
https://dx.doi.org/10.1021/acs.organomet.0c00092
Organometallics XXXX, XXX, XXX−XXX