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BODIPY-based Ru(II) and Ir(III) organometallic complexes of avobenzone, a sunscreen material: Potent anticancer agents.
Journal of Inorganic Biochemistry 189 (2018) 17–29
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
BODIPY-based Ru(II) and Ir(III) organometallic complexes of avobenzone, a
sunscreen material: Potent anticancer agents
T
⁎
Gajendra Guptaa,d, , Shirisha Cherukommub, Gunda Srinivasb, Seon Woong Leea,
⁎
⁎⁎
Sung Hwan Munc, Jaehoon Jungc, Narayana Nageshb, , Chang Yeon Leea,d,
a
Department of Energy and Chemical Engineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea
CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India
c
Department of Chemistry, University of Ulsan, Ulsan 44610, Republic of Korea
d
Innovation Center for Chemical Engineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Avobenzone
Bodipy
Ruthenium complexes
Iridium complexes
Anticancer properties
The use of organic compounds with known medicinal properties in the synthesis of metal-based complexes is an
important alternative to improve the biological activity of metal-based drugs. The reaction of [M(arene)Cl2]2
(M = Ru, arene = p-cymene and M = Ir, arene = pentamethylcyclopentadienyl, cp*) with avobenzone (1-(4-tertbutylphenyl)-3-(4-methoxyphenyl)propane-1,3-dione, AVBH) and KOH in methanol leads to the formation of the
neutral complexes [Ru(p-cymene)(AVB)Cl] 1 and [Ir(cp*)(AVB)Cl] 2 (cp* = pentamethylcyclopentadienyl).
Subsequent reaction of 1 and 2 with pyridyl derivative-BODIPY ligands, BDP and BDPCC (BODIPY = boron
dipyrromethene, BDP = 4-dipyridine boron dipyrromethene, BDPCC = 4-ethynylpyridine boron dipyrromethene) in methanol gives a series of four new dicationic supramolecules: [Ru2(p-cymene)2(AVB)2BDP]
[2CF3SO3] 3, [Ir2(cp*)2(AVB)2BDP][2CF3SO3] 4, [Ru2(p-cymene)2(AVB)2BDPCC][2CF3SO3] 5 and [Ir2(cp*)2
(AVB)2BDPCC][2CF3SO3] 6. The synthesized complexes are fully characterized using multiple analytical techniques, including elemental analysis, 1H NMR, 13C NMR, 19F NMR (NMR = Nuclear Magnetic Resonance),
Infrared Radiation (IR), Electrospray Ionization-Mass Spectrometry (ESI-MS), Ultraviolet-visible (UV–Vis) and
fluorescence spectroscopy. The structures of these complexes are further rationalized using density functional
theory (DFT) calculations. The antiproliferative activity of the neutral and dinuclear cationic complexes is
evaluated in vitro in different human cancer cell lines. These complexes are found to be active against different
cancer cell lines with half maximal inhibitory concentration (IC50) values between 1 and 5 μM. Complexes 5 and
6 displayed the lowest IC50 values in all the cell lines studied. The activity of 5 and 6 is comparable to that of the
well-known chemotherapy drug doxorubicin. Detailed biophysical studies indicate that complexes 5 and 6 exhibit very good Deoxyribonucleic acid (DNA) binding properties, causing the unwinding of the double helix,
which is a probable reason for their high cytotoxicity.
1. Introduction
chemotherapy procedure, complexes based on metals other than platinum have emerged as a special research focus. Ruthenium complexes
have shown especially promising results due to the interesting properties of the ruthenium metal center [4]. Ruthenium complexes have
been shown to be less toxic than platinum based complexes, and appear
to work by a different mode of action [5]. The biological activity of
arene‑ruthenium based metal complexes is being studied by several
groups [6]. Two Ru-based anticancer drugs, imidazolium trans-[tetrachlorido(dimethylsulfoxide)(1H-imidazole)ruthenate(III)]
(NAMI-A)
and indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]
According to the World Health Organization, cancer is the second
largest cause of death in developed nations, and the estimated annual
cancer death toll will exceed 13 million by 2030 [1]. Metal-based drugs
are very important compounds in treating cancer, and platinum complexes in particular are widely used in cancer treatment [2]. Despite
their widespread use, platinum-based drugs have severe side effects
that are often as painful as the disease itself [3]. In order to avoid these
severe side effects, and to overcome drug resistance during the
⁎
Corresponding authors.
Correspondence to: C.Y. Lee, Innovation Center for Chemical Engineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic
of Korea.
E-mail addresses: gjngupt@gmail.com (G. Gupta), nagesh@ccmb.res.in (N. Nagesh), cylee@inu.ac.kr (C.Y. Lee).
⁎⁎
https://doi.org/10.1016/j.jinorgbio.2018.08.009
Received 17 March 2018; Received in revised form 8 August 2018; Accepted 9 August 2018
Available online 16 August 2018
0162-0134/ © 2018 Elsevier Inc. All rights reserved.
Journal of Inorganic Biochemistry 189 (2018) 17–29
G. Gupta et al.
as a yellow solid in high yield. The complexes were highly soluble in
common organic solvents, and were stable both in air and in solution.
The 1H NMR spectroscopy was initially used to confirm the formation of
these complexes. The 1H NMR spectra in CDCl3 showed all the required
protons which supported the formation of the expected structures and
the values are given in the experimental section. Electrospray ionization
(ESI) mass spectroscopy was further studied which displayed peaks
corresponding to the loss of the chloride ion attached to the metal.
Peaks were observed at 545.14 and 637.13 amu for complex 1 and 2
respectively (Fig. S1(a), (b)).
The reaction of mononuclear complex 1 with the BODIPY ligands
BDP and BDPCC in methanol under nitrogen at room temperature afforded dinuclear complexes 3 and 5, respectively (Scheme 2). Complexes 3 and 5 were obtained as their triflate salts, and were non-hygroscopic and stable in air and solution. The complxes were soluble in
common polar solvents such as dichloromethane, chloroform, methanol, ethanol, acetone and acetonitrile but were insoluble in hexane,
diethylether and petroleum ether. Complex 3 was red in color, while
complex 5 was dark pink. Both complexes were fully characterized
using multiple analytical methods. The 1H NMR spectra of both the
complexes displayed peaks similar to that of the free BODIPY ligands
and the mononuclear complex 1 (Fig. S2(a)). However, the peaks were
shifted downfield with respect to those of the free BODIPY ligands and
the mononuclear complex. This downfield shift in the proton signals
might have resulted from a change in the electron density on the metal
atoms after the chelation by the BODIPY ligands through their pyridine
nitrogen atoms to form the desired dinuclear metal complexes. They
showed two doublets at δ 8.51 and δ 7.15 and δ 8.48 and δ 7.31 which
belongs to the α and β protons of the pyridyl group of the BODIPY
ligands for complexes 3 and 5 respectively. The aromatic proton peaks
from the avobenzone ligand appeared between δ 7.90 and δ 6.94. The
spectra also contained doublets at around δ 7.79 and δ 5.68, which
corresponded to the aromatic CH protons of the p-cymene group. Beside
this they exhibited five singlets, a septet and a doublet between δ 3.85
and δ 1.24 corresponding to the methyl and methoxy protons of these
complexes. The observed 1H NMR peaks of these complexes were
consistent with the expected products. The ESI-MS spectroscopy was
carried out to further confirm the formation of the dinuclear complexes
3 and 5. Their ESI-MS spectra obtained in acetonitrile solution exhibited prominent peaks corresponding to the loss of two triflate anions
[M-2CF3SO3]2+. These peaks were centered at m/z 784.02 and 808.02
for complexes 3 and 5, respectively. The peaks were isotopically resolved, and matched well with its theoretical distributions (Fig. 1 and
Fig. S1(c), (e)).
(KP1019), which were developed by Sava and Keppler, respectively, are
already in clinical trials, which further indicates the promising future of
metal-based complexes [7]. Since the discovery of these two important
ruthenium metal complexes, many groups have been actively working
on the biological applications of transition metal complexes [8]. The
structure and properties of cyclopentadienyl iridium complexes are
often considered to be similar to those of their arene ruthenium analogues. Although, the biological side of iridium complexes are not as
well-studied as those of ruthenium analogues, several half-sandwich
iridium complexes have shown greater potency than cisplatin against
various cancer cell lines [9].
An important strategy to improve the biological activity is by employing organic ligands with known biological properties to develop
new metal complexes [10]. In these direction, herein, we combine a
highly fluorescent BODIPY (boron dipyrromethene) ligand with
avobenzone (1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)propane-1,3dione, AVBH), an oil soluble compound used as sunscreen agent and in
cosmetic products, to develop new dicationic ruthenium and iridium
supramolecules. Recent studies have shown that AVBH has promising
antiproliferative activity against different human cancer cell lines [11].
Additionally, the dye BODIPY has been vividly studied for more than
three decades due to its excellent photo-physical and chemical properties. The BODIPY-based ligands were chosen on the basis of their
important applications in cancer therapy due to their absorption and
emission properties, which helps in biological labeling experiments for
the detection of tagged entities in and around biological cells [12]. To
combine the advantages of AVBH and BODIPY ligands, we designed
new dicationic ruthenium (II) and iridium (III) based supramolecules by
incorporating dipyridyl BODIPY ligands in ruthenium and iridium
AVBH complexes. The biological activities of these supramolecules
were investigated and the BDPCC (4-ethynylpyridine boron dipyrromethene) based ruthenium and iridium molecules were found to have
more potent anticancer properties than the BDP (4-dipyridine boron
dipyrromethene) containing supramolecules.
2. Results and discussion
2.1. Synthesis and characterization
The neutral mononuclear organometallic complexes 1 and 2 were
synthesized by the reaction of [Ru(p-cymene)Cl2]2 and [Ir(cp*)Cl2]2
dimers with avobenzone in the presence of KOH in methanol (Scheme
1). Further details of the syntheses of these complexes are provided in
experimental section. Both complexes were neutral and were obtained
Scheme 1. Synthetic scheme for mononuclear complexes 1 and 2. (p-cym = para-cymene).
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G. Gupta et al.
Scheme 2. Synthetic scheme for dinuclear ruthenium complexes 3 and 5.
Similarly, the mononuclear iridium complex 2 was reacted with a
half-equivalent of BDP or BDPCC in methanol to afford the cationic
dinuclear complexes 4 and 6, respectively, which were isolated as their
triflate salts (Scheme 3). Compound 4 was red in color, while 6 was
dark pink. The complexes were non-hygroscopic and stable in air and in
solution. They were soluble in common polar solvents such as dichloromethane, chloroform, methanol, ethanol, acetone and acetonitrile but were insoluble in hexane, diethyether and petroleum ether.
Both the complexes were fully characterized by different analytical
methods. Like the ruthenium derivatives, the 1H NMR spectra of complexes 4 and 6 were similar to those of the starting mononuclear
complex 2 and the BODIPY ligands (Fig. S2(b)). The peaks were shifted
downfield as compared to those of the free ligands. They displayed two
doublets, at δ 8.53 and δ 7.32, and δ 8.46 and δ 7.48, which corresponded to the α and β protons of the pyridyl group of the BODIPY
ligands for complexes 4 and 6, respectively. Beside this, four doublets
and a singlet between 7.97 and 6.97 ppm corresponding to the aromatic
peaks of avobenzone protons are also observed. The spectra of complexes 4 and 6 also exhibited a singlet corresponding to the pentamethylcyclopentadienyl group at 1.59 and 1.61 ppm, respectively. The
observed peaks were consistent with the expected products; detailed
assignments of the peaks are provided in the experimental section. The
ESI-MS spectra of complexes 4 and 6 in acetonitrile solution exhibited
peaks corresponding to the loss of two triflate anions. These peaks were
centered at m/z 876.12 and 900.15 for complexes 4 and 6, respectively
(Fig. S1(d), (f)).
The UV–Vis and fluorescence properties of these complexes were
studied in methanol and dichloromethane solution. In methanol, the free
BODIPY ligands gave rise to absorption peaks at approximately 517 nm
and 555 nm for BDP and BDPCC, respectively. However, a small red shift
of about 5 nm was observed when dichloromethane was used as the
solvent rather than methanol. The visible region of the spectra of the BDP
Fig. 1. ESI-MS spectra of the dinuclear complexes 3 and 5 in acetonitrile. Theoretical (blue) and experimental (black) isotopic distributions are shown for the peaks
m/z = [3-2OTf]2+ and m/z = [5-2OTf]2+. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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G. Gupta et al.
Scheme 3. Synthetic scheme for the iridium dinuclear complexes 4 and 6.
Fig. 2. Absorption (left) spectra of BDP, BDPCC, and metal complexes 1–6 in methanol, and the emission (right) spectra of BDP, BDPCC, and metal complexes 3–6 in
methanol (Excitation wavelength was 480 nm). Photographic images (inset) of BDP, BDPCC, and metal complexes 1–6 in methanol under UV-light (365 nm).
based supramolecules 3 and 4 was dominated by intense peaks similar to
those of free BDP. These peaks, which were centered at approximately
520 nm, were assigned to the π-π* transition arising from the BDP ligand
present in these complexes. Similarly, the spectra of supramolecules 5
and 6 were dominated by intense peaks centered at 557 nm, arising from
the BDPCC ligands present in these complexes. Other lower intensity
peaks centered between 380 nm and 390 nm were also observed; these
may arise from a combination of intra/intermolecular π-π* transitions
mixed with MLCT (metal-to-ligand charge-transfer) transitions. The luminescence spectra of the BODIPY ligands and their complexes were also
investigated in methanol and dichloromethane solution. The ligands and
the complexes were emissive in solution, and their spectra exhibited
emission bands centered at 532 nm and 575 nm, arising from the BDP
and BDPCC groups present in these complexes, respectively. These
complexes gave rise to similar absorption spectra, in agreement with the
UV–visible spectrum of the free BODIPY ligands. The absorption and
emission spectra are shown in Fig. 2.
not successful. Therefore, in order to gain insight into the molecular
structures of the BODIPY-based organometallic complexes 3–6, we
carried out a computational study based on density functional theory
(DFT). The structures of the complexes were optimized using the
ωB97X-D functional [13a] and 6-31G(d) basis set implemented in the
Gaussian09 software package [13b], which were recently applied to
describe the formation of complicated Ru(II) organometallic complexes,
Borromean ring [13c] and Solomon link [13d]. For the Ru and Ir atoms,
the LanL2DZ effective pseudo-potential and corresponding basis set
were used for all the calculations [13e]. The polarizable continuum
model (PCM) using the integral equation formalism variant (IEFPCM)
was employed to reflect the influence of the solvent medium used in the
syntheses of these complexes (i.e., for methanol, ε = 32.613) [13f].
The local minimum structures of Ru(II) complexes 3 and 5 are
presented in Fig. 3. The relative energies of the local minimum structures are within only ~1 kcal/mol (Table S1). For the case of Ir(III)
complexes 4 and 6, nearly identical results were observed (Table S1 and
Fig. S4(a)). Therefore, our computational results suggest that the relative stability of the isolated organometallic complexes did not depend
strongly on the orientation of the side functional groups and could not
be determined at this level of DFT. The influence of the orientation of
2.2. DFT calculations
Our repeated efforts to grow single crystals of these complexes were
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G. Gupta et al.
Fig. 3. Optimized structures of the cis- and trans-forms of the dinuclear organometallic complexes 3 and 5. (Ru, green; F, sky-blue; O, red; N, blue; C, gray; B, orange;
H atoms are omitted for clarity.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) of the cis-forms of 3 and 5.
presence of ethynyl group reduced the HL gap. Therefore, our computational results indicated that the optical properties of organometallic
complexes containing BODIPY-based ligand can be finely tuned by
adjusting the length of π-conjugated network around the BODIPY ligand.
the side functional groups on the frontier electronic structures of Ru(II)
complexes was also insignificant, because the highest occupied and
lowest unoccupied molecular orbitals (HOMO and LUMO) were mainly
distributed on the BDP and BDPCC ligands for 3 and 5, respectively, as
shown in Fig. 4. Not only the HOMO and LUMO for the trans-form of 3
and 5 are similar to those of their cis-form (Fig. S4(b)) but also Ir(III)
complexes exhibited the identical spatial distributions of HOMO and
LUMO (Fig. S4(c), S4(d)). Based on our experimental and theoretical
studies, we ruled out the formation of any isomers in our complexes.
The computationally obtained frontier molecular orbitals provided a
reasonable insight into the optical properties of organometallic complexes. The energy gaps between HOMO and LUMO (HL gaps) of 3 and
4 with BDP and 5 and 6 with BDPCC were ~6.25 and ~6.01 eV, respectively (Table S1). These values were consistent with the absorption
spectra which were dominated by the intramolecular π-π* transition
(Fig. 2). The extension of the π-conjugated network of BDP due to the
2.3. Biological studies
2.3.1. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assays
Considering the increasing importance of ruthenium and iridium
complexes as anticancer agents, the anticancer activity of free avobenzone and complexes 1–6 was studied against the human cancer cell
lines A549 (lung cancer), HeLa (cervical cancer) and MCF-7 (breast
cancer) as well as a non-malignant NIH T3T cells (mouse embryo fibroblast) cell line. They exhibited moderate to high activity, ranging
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G. Gupta et al.
2.5. Fluorescence titration studies
Table 1
In vitro antiproliferative activity (IC50 in μM) of free AVBH, complexes 1–6, and
doxorubicin on cancerous (A549, Hela, MCF-7) and non-cancerous (NIH 3 T3)
cell lines.
Complexes
NIH 3T3
A549
Hela
MCF-7
AVBH
1
2
3
4
5
6
Doxorubicin
3.0 ± 0.3
3.0 ± 0.4
4.0 ± 0.6
4.0 ± 0.7
5.0 ± 1.4
2.0 ± 0.5
1.0 ± 0.8
1.7 ± 0.2
5.0 ± 0.8
4.0 ± 0.6
5.0 ± 0.8
4.0 ± 0.5
4.0 ± 0.4
3.0 ± 0.7
3.0 ± 0.4
1.5 ± 0.6
4.0 ± 0.8
3.0 ± 0.4
4.0 ± 0.6
4.0 ± 0.7
4.0 ± 0.6
3.0 ± 0.7
2.0 ± 0.3
1.4 ± 0.4
3.0 ± 0.5
4.0 ± 0.6
4.0 ± 0.7
5.0 ± 0.8
3.0 ± 0.4
1.8 ± 0.6
1.3 ± 0.3
1.0 ± 0.9
Since the supramolecules 5 and 6 were fluorescent, their interaction
with CT-DNA could be monitored using fluorescence spectroscopy. The
interaction of CT-DNA with 5 and 6 gave rise to an emission peak at
around 601 nm that exhibited hyperchromicity. As an interacting
fluorescent molecule approaches the DNA bases, the exchange of
fluorescent energy from the fluorescent molecule and adjacent DNA
bases increases the intensity of the fluorescence emission signal [15].
The only way in which 5 and 6 could approach the DNA bases is by
insertion between two stacked base pairs. Therefore, these results indicated that the supramolecules 5 and 6 may intercalate into the bases
of CT-DNA. The fluorescence spectra obtained during the interaction of
CT-DNA with supramolecules 5 and 6 are shown in Fig. 6.
from 1 to 5 μM. The results are summarized in Table 1. Among the
tested compounds, ruthenium complex 5 and iridium complex 6 featuring the BDPCC ligand exhibited the highest cytotoxic activity against
MCF-7, with IC50 values of 1.8 ± 0.6 and 1.3 ± 0.3 μM, respectively.
The extent of toxicity in non-malignant NIH T3T cells however, was
more or less comparable with the other cell lines, thus ruling out the
possibility of any selectivity towards cancer cell killing. In contrast, the
control molecule doxorubicin showed the lowest cytotoxicity in all
cells. The free BODIPY ligands were previously found to be non-toxic
[9f,12a,12g].
2.6. Circular dichroism studies
The UV–visible and fluorescence spectroscopy studies indicated that
complexes 5 and 6 may possibly intercalate into CT-DNA. In order to
further understand the effect of complexes 5 and 6 on the conformation
of CT-DNA, circular dichroism (CD) studies were performed. The CTDNA showed positive and negative CD bands at around 270 nm and
240 nm, respectively. Upon the addition of 15 μM of 5 and 6 to CTDNA, the positive CD band intensity decreased, indicating unwinding of
CT-DNA [16]. Further, upon doubling the concentration of 5 and 6 (to a
1:2 ratio), the positive band intensity decreased further, suggesting that
at higher concentrations of 5 and 6, the melting of CT-DNA increased. A
greater decrease in the intensity of CD band was noted for complex 6
than for complex 5. These results indicate that the interactions of
complexes 5 and 6 with CT- DNA caused the CT- DNA structure to
unwind gradually. The CD spectra of supramolecules 5 and 6 with CTDNA are shown in Fig. 7.
2.4. DNA binding studies
2.4.1. UV–visible spectroscopic study
To determine how these small molecules interact with DNA, detailed binding assays were carried out using UV–visible spectroscopy.
Upon the addition of Calf-Thymus-DNA (CT-DNA) to solutions of
complexes 5 and 6, the characteristic peak observed at 572 nm exhibited concentration-dependent hyperchromic shift (Fig. 5). No isosbestic point was observed upon the interaction of complexes 5 or 6
with the CT-DNA. The hyperchromicity of the absorption band upon the
addition of CT-DNA indicated that supramolecules 5 and 6 interacted
with the CT-DNA and damaged its double helical structure [14]. Based
on the absorbance values obtained, the 5/6 CT-DNA dissociation
constants (Kd) was calculated. The Kd values obtained for the interaction of 5 and 6 with CT-DNA were 180 μM and 132 μM, respectively.
The results indicated that CT-DNA interacted more strongly with the
ruthenium complex 5 than with the iridium complex 6. Nevertheless,
UV–visible titration studies alone were not sufficient to fully characterize the CT-DNA-5/6 interaction. To determine the binding mode
and the extend of the interaction between CT-DNA and 5/6, additional
spectroscopic studies were carried out.
2.7. Flow cytometry experiments
2.7.1. Cell cycle assay
The results obtained from the MTT assays indicated that among the
complexes studied, complexes 5 and 6 exhibited the highest cytotoxicity in the studied cancer cell lines. To better understand the role of
these complexes in cells and to identify a possible cause for the higher
cytotoxicity and efficacy of complexes 5 and 6 in the cancer cell lines,
cell cycle assays and apoptotic assays were performed using flow cytometry in the presence and absence of the complexes at their respective IC50 values. The results of these experiments suggested that
complexes 5 and 6 inhibit the cell cycle at the subG1 stage. It has been
previously reported that subG1 phase arrest can enhance cytotoxicity
and induce apoptosis in cells [17]. Among the complexes studied, the
greatest subG1 phase inhibition values were observed for 5 μM and
1 μM of complex 6 (14.67% and 11.67%, respectively), followed by
Fig. 5. UV–vis absorption spectra indicating the
binding interaction between complexes 5 (A), 6
(B) and CT DNA. UV–vis spectra of complexes 5
and 6 (2.5 μM) obtained upon the addition with
increments of 0.05 μM CT DNA are shown. (A)
UV–vis spectra obtained when CT DNA in
100 mM TE (pH 7.0) was added to complex 5 at
20 °C. (B) UV–vis spectra obtained when CT DNA
in 100 mM TE (pH 7.0) was added to complex 6
at 20 °C. The arrows indicate the direction in
which the absorption peak moves after interaction of complexes 5 and 6 with CT DNA.
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G. Gupta et al.
Fig. 6. Fluorescence spectroscopic titration of complexes 5 (A), 6 (B) with CT DNA. Fluorescence spectra of complexes 5 and 6 (10 μM) is recorded on the addition
with increments of 0.005 μM CT DNA are shown. (A) Fluorescence spectra obtained when the CT DNA in 100 mM TE (pH 7.0) was added to 10 μM of complex 5 at
20 °C. (B) Fluorescence spectra obtained when the CT DNA in 100 mM TE (pH 7.0) was added to 10 μM of complex 6 at 20 °C. The arrows indicate the direction in
which the fluorescence peak moves upon addition of CT DNA to the solution of complexes 5 and 6.
complex 5 (10.08%), while the untreated MCF-7 cells showed a subG1
phase inhibition of only 5.10%. These results confirmed that both
complexes induced cytotoxicity followed by apoptosis in the cancer cell
lines through subG1 phase arrest. The details of the cell cycle assays are
presented in Fig. 8 and documented in Table 2.
condensation [18]. Several apoptotic cells with condensed and fragmented DNA were observed upon treating the cells with 1 μM of complexes 5 and 6. When the concentration of 5 and 6 was further increased
to 5 μM, the number of apoptotic cells also increased. A significant increase in the percentage of cells with condensed and fragmented DNA
(apoptotic cells) occurred when the concentrations of 5 and 6 were
increased from 1 μM to 5 μM (Fig. 10). The cells with condensed DNA
can be seen as bright blue spots in the microscopy images. They are
marked with white arrows in Fig. 10. Several previous studies emphasized that the majority of synthetic small molecules, such as doxorubicin [19], mitoxantrone [20], acridine orange [21], exhibit anticancer activity through dsDNA intercalation [22]. We speculate that the
possible intercalation of 5 and 6 with dsDNA is a possible reason for the
potential anticancer activity of 5 and 6.
2.7.2. Apoptosis assay
From the apoptosis assays, it was clear that 5 μM of complex 6 had a
higher potential to induce apoptosis (11.86%) in MCF-7 cells than a
similar concentration of complex 5 (7.81%). The results also indicated
that complex 6 (at 1 μM) was effective at inducing a higher proportion
of apoptotic cells [6.45% late apoptotic cells (Annexin-V+/PI+) and
2.62% early apoptotic cells (Annexin-V+/PI−)] in MCF-7 cells. Upon
treating the cells with a higher concentration of complex 6 (5 μM) a
higher percentage of apoptotic cells were observed [7.65% late apoptotic cells (Annexin-V+/PI+) and 4.21% early apoptotic cells
(Annexin-V+/PI−)]. However, when MCF-7 cells were treated with
5 μM complex 5, fewer apoptotic cells were observed (7.81%) than with
complex 6, indicating the greater potential of complex 6 to induce
apoptosis in human breast cancer cells. The results obtained from the
apoptosis assay supported those of the MTT assay. The percentages of
cell population in viable, necrotic, and early or late apoptotic phases are
shown in Table 3. The results from the apoptosis assay for untreated
MCF-7 cells and cells treated with 1 μM and 5 μM of complexes 5 and 6
are depicted in Fig. 9.
2.7.4. Mitochondrial outer membrane permeability
Binding studies so far, indicate that complexes 5 and 6 are capable
of interacting with DNA. Furthermore, flow cytometry and fluorescence
microscopy experiments, exhibit their potential to induce apoptosis
among MCF-7 cells. Therefore, it becomes interesting to understand
their effects on mitochondria, as thus have an insight on their mechanistic side. Alteration in mitochondrial outer membrane permeability is assessed using Mitotracker red staining. The MCF-7 cells before and after the treatment with complexes 5 and 6 are labeled with
Mitotracker red. It is evidenced that mitochondria were evenly distributed in the cytosol of untreated cells (see Fig. 11), where about
10–15 mitochondria were seen in the cytosol. Moreover, the untreated
MCF-7 cells (control) showed freely distributed mitochondria in the
cytosol. However, upon treatment with 5 μM of the complexes, aggregations of mitochondria were observed (the aggregated
2.7.3. Fluorescence microscopy study
Induction of apoptosis among cancer cells occurs due to multiple
causes. In the majority of cases, the primary feature observed upon the
onset of apoptosis is the fragmentation of nuclear DNA and chromatin
Fig. 7. Circular dichroism studies illustrating the changes in the CT-DNA conformation upon interaction with complexes 5 (left) and 6 (right). (A) CD spectra
obtained with the CT-DNA in 100 mM TE (pH 7.0) at 20 °C and upon addition of 1:1 and 1:2 ratios of CT-DNA and complex 5 (B) CD spectra obtained with the CTDNA in 100 mM TE (pH 7.0) at 20 °C and upon addition of 1:1 and 1:2 ratios of CT-DNA and complex 6. CD spectra were averaged over three scans. The arrows
indicate the direction of movement of CD peaks upon addition of complexes 5 and 6.
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G. Gupta et al.
Fig. 8. Cell cycle studies indicating the effect of complexes 5 and 6 on cell cycle progression (A) MCF-7 cells without the addition of any complex (control MCF-7
cells) (B) MCF-7 cells treated with 1 μM of complex 5 for 48 h (C) MCF-7 cells treated with 5 μM complex of 5 for 48 h (D) MCF-7 cells treated with 1 μM of complex 6
for 48 h (E) MCF-7 cells treated with 5 μM of complex 6 for 48 h.
which in turn triggered the cascade pathway to apoptosis. From Fig. 11,
it was clear that mitochondria were getting bundled with complexes 5
and 6, indicating the onset of apoptosis in MCF-7 cells.
Table 2
Data obtained from cell cycle histograms indicating the percentage of cells in
each phase of the cell cycle before and after treatment with complexes 5 and 6.
S. No.
Complexes
Sub G1
G0/G1
G2/M
S
1
2
3
4
5
Control
5 (1 μM)
6 (1 μM)
5 (5 μM)
6 (5 μM)
05.10%
09.28%
11.67%
10.08%
14.67%
70.47%
57.78%
22.84%
67.74%
64.55%
10.72%
08.81%
10.95%
07.01%
07.88%
13.63%
18.90%
54.10%
12.19%
12.48%
3. Conclusions
In conclusion, we have designed new ruthenium and iridium metal
complexes based on the sunscreen agent avobenzone and fluorescent
BODIPY ligands. The complexes were fully characterized using multiple
analytical techniques, and their structures were further rationalized by
density functional theory studies. In light of the growing interest of the
use of metal-based complexes in biological systems, the anticancer
properties of the synthesized complexes were studied in detail. The
BDPCC based complexes 5 and 6 were found to be more cytotoxic than
mitochondria are marked with white arrows). The aggregation of mitochondria was reported to occur, when cells enter into apoptosis [23],
and accordingly leading to the release of cytochrome c into the cytosol,
24
Journal of Inorganic Biochemistry 189 (2018) 17–29
G. Gupta et al.
the BDP compounds 3 and 4, with their activity comparable to that of
the chemotherapy drug doxorubicin. These complexes were also found
to bind strongly with genomic DNA causing the unwinding of the
double helix, where complex 6 showed a comparatively better activity.
Complex 6, also showed better affinity to instigate apoptosis in breast
cancer cells than 5, which was further confirmed by Annexin V/PI
apoptosis and fluorescent microscopy studies. To further understand its
mechanism, in-depth studies of these complexes in animal models are
being performed in our laboratory.
Table 3
Data obtained from dot plots indicating the percentage of viable and apoptotic
cells before and after treatment with complexes 5 and 6.
S. No.
1
2
3
4
5
Samples
Control
5 (1 μM)
5 (5 μM)
6 (1 μM)
6 (5 μM)
Percentage of
Viable Cells (LL)
99.84
96.01
88.08
90.18
81.24
Percentage of
Necrotic Cells (UL)
00.02
00.05
04.11
00.75
07.09
Percentage of
apoptotic cells
UR
LR
00.07
00.18
05.08
06.45
07.65
00.07
03.76
02.73
02.62
04.21
4. Experimental section
4.1. General information
LL: lower left quadrant of the dot plot; UL: upper right quadrant of the dot plot;
LR: lower right quadrant of the dot plot; UR: upper right quadrant of the dot
plot.
The dipyridine bodipy ligands were synthesized by our previously
reported methods.9f, 12g The starting dimers [(η 6-p-cymRu)Cl2]2 and
Fig. 9. Annexin V-FITC vs propidium iodide plots (dot plots) obtained from the gated cells show the population of cells corresponding to viable, early, and late
apoptotic and necrotic cells. (A) MCF-7 cells without the addition of any complex (control MCF-7 cells) (B) MCF-7 cells treated with 1 μM of complex 5 for 24 h (C)
MCF-7 cells treated with 5 μM of complex 5 for 24 h (D) MCF-7 cells treated with 1 μM of complex 6 for 24 h (E) MCF-7 cells treated with 5 μM of complex 6 for 24 h.
25
Journal of Inorganic Biochemistry 189 (2018) 17–29
G. Gupta et al.
Fig. 10. Confocal microscopy images showing variation in the morphology of cells among normal and apoptosis-induced MCF-7 cells upon staining with Hoechst
33258 and MCF-7 cells. MCF-7 cells were treated with complexes 5 and 6 for 48 h (A) Untreated MCF-7 cells (control) do not show chromatin condensation (B) MCF7 cells treated with 5 μM of complex 5 (C) MCF-7 cells treated with 5 μM of complex 6. The condensed chromatin after Hoechst 33258 labeling can be seen as a bright
blue spot under the fluorescence microscope. The white arrows point to the apoptotic cells that are bright blue in color. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
z = 545.14 [M − Cl]+. IR (KBr pellets): ν = 2960 (m, CeH); 1608,
1588, 1528 (s, C]O, C]C). Anal. calcd for C30H35O3ClRu·CH2Cl2: C,
55.99; H, 5.61. Found: C, 55.69; H, 5.44. 1H NMR (400 MHz,
Acetone‑d6): δ = 8.00 (d, 2H, J = 8 Hz, Ha), 7.93 (d, 2H, J = 8 Hz,
Hb), 7.47 (d, 2H, J = 8 Hz, Hc), 6.97 (d, 2H, J = 8 Hz, Hd), 6.47 (s, 1H,
He), 5.70 (d, 2H, J = 8 Hz, Hcym), 5.39 (d, 2H, J = 8 Hz, Hcym), 3.85 (s,
3H, OCH3, AVB), 3.00 (m, 1H, CH(CH3)2), 2.28 (s, 3H, CH3,cym), 1.41 (d,
6H, J = 4 Hz, CH(CH3)2), 1.33 (s, 9H, (CH3)3, AVB). 13C{1H}-NMR
(100 MHz, Acetone‑d6): δ = 180.19, 180.00, 161.78, 153.92, 136.62,
131.50, 129.01, 126.90, 125.09, 113.32, 98.79, 96.97, 91.36, 83.59,
79.19, 54.91, 34.41, 30.48, 21.75, 17.10.
[(η 5-C5Me5Ir)Cl2]2 were purchased from Aldrich and avobenzone was
purchased from TCI Korea and used as received. Dry solvents were
purchased from Aldrich and all other reagents were commercially
available and used without further purification. The 1H, 13C{1H} and
19
F NMR spectra were recorded on an Agilent 400-MR spectrometer
using the residual protonated solvent as internal standard. Infrared
spectra were recorded on a Bruker/Hyperion 2000 FTIR spectrometer
using KBr pellets. UV–visible absorption spectra were recorded on a
Thermo Scientific Genesys 10S UV–Vis spectrometer. Fluorescence
spectra were recorded on a Scinco FluoroMate FS-2 fluorescence spectrometer. ESI-MS were recorded on a Waters SynaptG2Si spectrometer
at KBSI, South Korea. Elemental analysis was recorded on an Elemental
Analyzer (Elementar Analysensysteme GmbH, Germany) at KBSI,
Korea.
4.2.2. Complex 2
4.2.2.1. Yellow solid. Yield 175 mg (76%). ESI-MS (CH3CN): m/
z = 637.13 [M − Cl]+. IR (KBr pellets): ν = 2966 (m, CeH); 1580,
1525 (s, C]O, C]C). Anal. calcd for C30H35O3ClIr: C, 53.60; H, 5.40.
Found: C, 53.29; H, 5.43. 1H NMR (400 MHz, Acetone‑d6): δ = 8.04 (d,
2H, J = 8 Hz, Ha), 7.97 (d, 2H, J = 8 Hz, Hb), 7.48 (d, 2H, J = 8 Hz,
Hc), 6.98 (d, 2H, J = 8 Hz, Hd), 6.58 (s, 1H, He), 3.86 (s, 3H, OCH3,
13
C{1H}-NMR
AVB), 1.66 (s, 15H, cp*), 1.33 (s, 9H, (CH3)3, AVB).
(100 MHz, Acetone‑d6): δ = 179.48, 178.78, 162.92, 152.24, 135.44,
131.99, 129.66, 127.60, 126.02, 114.40, 93.56, 84.15, 55.74, 35.40,
31.36, 8.84.
Synthesis of complexes 3 and 5: Compound 1 (30 mg, 0.051 mmol)
was dissolved in methanol (20 mL) and AgCF3SO3 (15 mg, 0.058 mmol)
was added to the solution. The reaction mixture was stirred for 1 h at
room temperature and filtered to remove AgCl formed. BODIPY
(12.5 mg, 0.26 mmol for BDP, 3 and 13.7 mg, 0.26 mmol for BDPCC, 5)
was then added to the filtrate and dry nitrogen was bubbled for
10–15 min. The resulting mixture was then stirred for 24 h at room
4.2. General synthesis
Synthesis of complex 1 and 2: Complex 1 was synthesized by following a recently reported method [11a]. KOH (21 mg, 0.374 mmol)
was added to a solution of avobenzone (116 mg, 0.374 mmol) dissolved
in dry methanol (10 mL). The mixture was stirred for around 1 h at
room temperature followed by the addition of [(η 6-p-cymRu)Cl2]2
(104 mg, 0.169 mmol, for complex 1) and [(η 5-C5Me5Ir)Cl2]2 (135 mg,
0.169 mmol, for complex 2). The mixture was stirred for 24 h at room
temperature. The orange or yellow precipitate formed was centrifuged
and washed several times with hexane and recrystallized in dichloromethane. Then the solid was dried under vacuum.
4.2.1. Complex 1
4.2.1.1. Orange solid. Yield 156 mg (79%). ESI-MS (CH3CN): m/
Fig. 11. Mitochondrial aggregation among MCF-7 cells before and after the treatment with 5 μM of complexes 5 and 6 (white arrows show aggregation of mitochondria), A-mitochondria before complex treatment, B-mitochondria after treatment with complex 5 and C-mitochondria after treatment with complex 6.
26
Journal of Inorganic Biochemistry 189 (2018) 17–29
G. Gupta et al.
FC–F), −145.35 (q, 2F, FB–F).
temperature. The solution was reduced to 3 mL and an excess of hexane
was slowly layered and kept in refrigerator. The precipitate formed was
collected by centrifugation, washed several times with hexane, recrystallized in chloroform and dried in vacuum.
Complex 3: Red solid. Yield 35 mg (73%). ESI-MS (CH3CN): m/
z = 784.02 [M − 2CF3SO3]2+. IR (KBr pellets): ν = 2968 (m, CeH);
1598, 1523 (s, C]O, C]C); 1249 (s, CF3). Anal. calcd for
C91H95O12Ru2N4BF8S2·5.5CHCl3: C, 45.95; H, 4.02; N, 2.22. Found: C,
46.35; H, 4.03; N, 2.29. 1H NMR (400 MHz, CDCl3): δ = 8.51 (d, 4H,
J = 4 Hz, Hα), 7.89 (d, 4H, J = 8 Hz, Ha), 7.81 (d, 4H, J = 8 Hz, Hb),
7.45 (d, J = 8, 4H, Hc, + 3H, HPh), 7.24 (m, 2H, HPh), 7.15 (d, 4H,
J = 8 Hz, Hβ), 6.94 (d, 4H, J = 8 Hz, Hd), 6.29 (s, 2H, He), 5.79 (d, 4H,
J = 4 Hz, Hcym), 5.68 (d, 4H, J = 8 Hz, Hcym), 3.85 (s, 6H, OCH3, AVB),
2.87 (sept, 2H, J = 8 Hz, CH(CH3)2), 2.43 (s, 6H, CH3,BDP), 2.19 (s, 6H,
CH3,cym), 1.32 (br, 12H, CH(CH3)2) + 18H, (CH3)3, AVB), 1.24 (s, 6H,
CH3,BDP). 13C{1H}-NMR (100 MHz, Acetone‑d6): δ = 180.44, 180.42,
162.55, 154.73, 151.68, 150.58, 144.07, 134.83, 133.65, 132.92,
131.07, 129.60, 129.19, 129.11, 129.09, 129.00, 128.98, 127.35,
126.75, 126.14, 124.99, 113.31, 102.36, 98.60, 91.61, 83.77, 81.84,
54.54, 34.14, 30.20, 29.98, 21.19, 16.26, 11.72. 19F NMR (376 MHz,
CDCl3): δ = −78.12 (s, 6F, FC-F), −145.48 (q, 2F, FB-F).
Complex 5: Red solid. Yield 25 mg (58%). ESI-MS (CH3CN): m/
z = 808.02 [M − 2CF3SO3]2+. IR (KBr pellets): ν = 2966 (m, CeH);
2205 (m, C^C); 1611, 1517 (s, C]O, C]C); 1249 (s, CF3). Anal. calcd
for C95H95O12Ru2N4BF8S2·5CHCl3: C, 47.84; H, 4.01; N, 2.23. Found: C,
48.10; H, 4.05; N, 2.23. 1H NMR (400 MHz, CDCl3): δ = 8.48 (d, 4H,
J = 4 Hz, Hα), 7.90 (d, 4H, J = 8 Hz, Ha), 7.81 (d, 4H, J = 8 Hz, Hb),
7.51 (m, 3H, HPh), 7.46 (d, 4H, J = 8 Hz, Hc), 7.31 (d, 4H, J = 4 Hz,
Hβ), 7.20 (m, 2H, HPh), 6.94 (d, 4H, J = 8 Hz, Hd), 6.39 (s, 2H, He), 5.81
(d, 2H, J = 8 Hz, Hcym), 5.76 (d, 2H, J = 4 Hz, Hcym), 5.68 (d, 4H,
J = 8 Hz, Hcym), 3.85 (s, 6H, OCH3, AVB), 2.84 (sept, 2H, J = 8 Hz, CH
(CH3)2), 2.59 (s, 6H, CH3, BDP), 2.18 (s, 6H, CH3, cym), 1.48 (s, 6H, CH3,
13
C{1H}-NMR
BDP), 1.32 (m, 12H, CH(CH3)2, + 18H, (CH3)3, AVB).
(100 MHz, Acetone‑d6): δ = 180.81, 180.76, 163.03, 155.20, 152.13,
146.08, 135.19, 134.11, 133.53, 130.04, 129.86, 129.62, 129.48,
127.77, 127.67, 127.24, 126.82, 125.41, 113.79, 102.98, 99.30, 93.16,
91.98, 84.36, 84.30, 82.30, 55.05, 34.61, 30.44, 21.65, 16.75, 13.42,
12.72. 19F NMR (376 MHz, CDCl3): δ = −78.09 (s, 6F, FC–F), −145.99
(q, 2F, FB–F).
Synthesis of complexes 4 and 6: Compound 2 (30 mg, 0.045 mmol)
was dissolved in methanol (20 mL) and AgCF3SO3 (13 mg, 0.050 mmol)
was added to the solution. The reaction mixture was stirred for 1 h at
room temperature and filtered to remove AgCl formed. BODIPY
(10.7 mg, 0.022 mmol for BDP, 4 and 11.8 mg, 0.023 mmol for BDPCC,
6) was then added to the filtrate and dry nitrogen was bubbled for
10–15 min. The resulting mixture was then stirred for 24 h at room
temperature. The solution was reduced to 3 mL and an excess of hexane
was slowly layered and kept in refrigerator. The precipitate formed was
collected by centrifugation, washed several times with hexane, recrystallized in chloroform and dried in vacuum.
4.2.4. Complex 6
4.2.4.1. Dark pink solid. Yield 27 mg (57%). ESI-MS (CH3CN): m/
z = 900.15 [M − 2CF3SO3]2+. IR (KBr pellets): ν = 2966 (m, CeH);
2202 (m, C^C); 1605, 1518 (s, C]O, C]C); 1258 (s, CF3). Anal. calcd
for C95H97O12Ir2N4BF8S2·CHCl3: C, 52.00; H, 4.45; N, 2.53. Found: C,
51.95; H, 4.52; N, 2.56. 1H NMR (400 MHz, CDCl3): δ = 8.46 (d, 4H,
J = 8 Hz, Hα), 7.96 (d, 4H, J = 8 Hz, Ha), 7.87 (d, 4H, J = 8 Hz, Hb),
7.48 (m, 11H, HPh + Hc + Hβ), 7.21 (m, 2H, HPh), 6.97 (d, 4H,
J = 8 Hz, Hd), 6.42 (s, 2H, He), 3.86 (s, 6H, OCH3, AVB), 2.61 (s, 6H,
CH3, BDP), 1.61 (s, 30H, cp*), 1.44 (s, 6H, CH3, BDP), 1.33 (s, 18H,
(CH3)3, AVB). 13C{1H}-NMR (100 MHz, Acetone‑d6): δ = 179.14,
179.10, 163.11, 155.36, 150.61, 150.57, 135.37, 129.64, 129.47,
127.87, 127.67, 127.23, 127.19, 125.59, 125.31, 113.96, 93.39,
86.20, 55.08, 34.67, 30.42, 12.75, 12.72, 7.61. 19F NMR (376 MHz,
CDCl3): δ = −78.08 (s, 6F, FC–F), −145.94 (q, 2F, FB–F).
4.3. Biological experiments
4.3.1. MTT assay
Mouse embryo fibroblast cell line (NIH 3T3) and cancer cells (MCF7, HeLa, A549) were purchased from American Type Culture Collection
(Rockville, MD) and National Centre for Cell Sciences, India respectively. They were cultured according to the supplier's recommendations. To study the cytotoxicity of the synthesized complexes, after 80%
confluence, cells were trypsinized with 0.1% trypsin-EDTA and harvested by centrifugation at 500 x g. Serial dilutions of cells were made
from 1 × 106 to 1 × 103 cells per ml. The cells were seeded in triplicate
in a 96 well plate. The suspended cells were treated with 1, 3, 5, 7,
10 μM of complexes 5 and 6 for 24 h duration. Since doxorubicin was
known as potential anticancer drug, the fibroblast and tumor cells were
treated with doxorubicin, in the same concentration range
(1 μM–10 μM) were considered as control. The cell viability was determined by measuring the ability of cells to transform MTT to a purple
colored formazan dye. The absorbance of samples at 570 nm was
measured using a UV–Visible spectrophotometer. Percentage of viable
cells was calculated by using the formula given below.
Percentage of cell viability =
OD570 Sample
× 100
OD570 Control s
where the OD570 (sample) corresponds to absorbance obtained from the
wells treated with complexes and OD570 (control) represents the absorbance from the wells in which no complex was added.
4.3.2. UV–Visible spectroscopy
UV–Visible absorption spectra were recorded using ABI Lambda
Spectrophotometer (Waltham, MA, USA) at 25 °C. All the experiments
were carried out in polystyrene cuvettes to minimize binding of complexes to the surface of the cuvettes. 5.0 μM of complexes 5 and 6 was
prepared in DMSO and 5.0 μM of CT-DNA in 100 mM Tris buffer
(pH 7.0). Each complex was added to 1 ml Milli Q water taken in 1 cm
path length cuvette and absorption spectra were recorded in the range
of 300 nm to 400 nm after the subsequent additions of 5 μl CT-DNA. All
the solutions were freshly prepared before starting the experiment and
titration was carried out until saturation of absorbance.
4.2.3. Complex 4
4.2.3.1. Dark pink solid. Yield 21 mg (61%). ESI-MS (CH3CN): m/
z = 876.12 [M − 2CF3SO3]2+. IR (KBr pellets): ν = 2966 (m, CeH);
1608, 1531 (s, C]O, C]C); 1254 (s, CF3). Anal. calcd for
C91H97O12Ir2N4BF8S2·0.75CHCl3: C, 51.50; H, 4.60; N, 2.62. Found: C,
51.55; H, 4.72; N, 2.71. 1H NMR (400 MHz, CDCl3): δ = 8.53 (d, 4H,
J = 4 Hz, Hα), 7.97 (d, 4H, J = 12 Hz, Ha), 7.88 (d, 4H, J = 8 Hz, Hb),
7.47 (d, 3H + 4H, J = 8 Hz, HPh + Hc), 7.32 (d, 4H, J = 8 Hz, Hβ), 7.23
(b, 2H, HPh), 6.97 (d, 4H, J = 12 Hz, Hd), 6.45 (s, 2H, He), 3.85 (s, 6H,
OCH3, AVB), 2.47 (s, 6H, CH3, BDP), 1.59 (s, 30H, cp*), 1.32 (s, 18H,
(CH3)3, AVB), 1.28 (s, 6H, CH3, BDP). 13C{1H}-NMR (100 MHz,
Acetone‑d6): δ = 178.78, 178.75, 162.62, 154.84, 153.93, 150.09,
144.78, 140.33, 134.97, 129.22, 129.16, 129.01, 127.28, 126.74,
125.14, 122.58, 119.38, 113.48, 92.92, 85.77, 54.61, 34.25, 29.94,
12.21, 11.76, 7.05. 19F NMR (376 MHz, CDCl3): δ = −78.05 (s, 6F,
4.3.3. Fluorescence spectroscopic titrations
Fluorescence emission spectra were measured at 25 °C using a
Hitachi F4500 spectrofluorimeter (Maryland, USA) using a 1 cm path
length quartz cuvette. Quartz cuvettes was thoroughly washed with
distilled water and dilute nitric acid (~0.1 N) to minimize non-specific
binding of the molecules to the surface of the cuvette. Throughout the
fluorescence experiment, the concentration of the complexes was kept
constant (10 μM) and titrated with increasing concentrations of CTDNA (multiples of 0.005 μM). Fluorescence spectra were recorded after
27
Journal of Inorganic Biochemistry 189 (2018) 17–29
G. Gupta et al.
each addition of CT-DNA to the fluorescent cuvette. After each experiment, the quartz cuvettes was thoroughly washed with distilled
water and dilute nitric acid (~0.1 N) to remove traces of molecules
binding to the walls of quartz cuvette. Complexes 5 and 6 were excited
at 440 nm, and emission spectra for each titration were recorded sin the
wavelength range from starting from 550 nm to 615 nm. Each spectrum
was recorded three times and the average of three scans was taken.
the glass slides and observed using laser scanning confocal microscopy
(Leica TCS SP5, Heidelberg, Germany). The Mitotracker Red was excited using 543 nm laser source and the fluorescence emission signals
were collected at 570 nm to 635 nm.
Abbreviations
AVBH
(1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)propane-1,3dione
BDP
4-dipyridine boron dipyrromethene
BDPCC 4-ethynylpyridine boron dipyrromethene
p-cym
para-cymene
DFT
Density Functional Theory
MLCT
Metal-to-Ligand Charge-Transfer
PCM
Polarizable Continuum Model
IEFPCM Integral Equation Formalism Polarizable Continuum Model
CT-DNA Calf-Thymus Deoxyribonucleic Acid
4.3.4. Circular dichroism (CD) spectroscopy
Circular dichroism experiments were performed using a JASCO 815
CD Spectro polarimeter (Jasco, Tokyo, Japan). The solution of CT-DNA
was prepared in 100 mM Tri buffer. To 10 μM of CT-DNA, 10 and 20 μM
of complexes 5 and 6 were added. The CD spectra were recorded from
230 nm to 330 nm in 1 mm path length cuvette. The spectra were
averaged over 3 scans, which were recorded at 100 nm/min with a
response time of 1 s and a band width of 1 nm.
4.3.5. Cell cycle analysis by flow cytometry
In cancer cells, the cell cycle is often deregulated and undergo unscheduled cell divisions. Therefore, inhibition of cell cycle provides an
opportunity to identify a suitable molecule to treat proliferative diseases like cancer. For cell cycle assay, complexes 5 and 6 was chosen as
it was earlier shown that these complexes are cytotoxic (with the MTT
assay). This assay was performed to assess the effect of complexes 5 and
6 on different stages of the cell cycle. Flow cytometry experiments were
carried out by following the protocol reported earlier [9k]. As complexes 5 and 6 were showing maximum cytotoxic effect on MCF-7 cells,
they were incubated with 1.0 μM and 5.0 μM concentrations of 5 and 6
for 48 h. Untreated MCF-7 cells were used as control. Untreated and
treated cells were harvested, washed with phosphate buffered saline
(PBS), fixed in ice-cold 70% alcohol and stained with propidium iodide
(PI) (Sigma Aldrich). The cell cycle assay was performed using Becton
Dickinson FACSCaliber flow cytometer.
Acknowledgements
This research was supported by a Post-Doctoral Research Program
(2018) for GG through Incheon National University, Incheon, Republic
of Korea. This research was also supported by mid-career researcher
programs of the National Research Foundation of Korea funded by the
Ministry of Science, ICT & Future Planning (NRF-2016R1A2B4010376).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinorgbio.2018.08.009.
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28
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