Triphenylamine/carbazole-modified ruthenium(ii) Schiff base compounds: Synthesis, biological activity and organelle targeting

Article abstract of DOI:10.1039/d0dt01547d

Four triphenylamine/carbazole-modified half-sandwich ruthenium(ii) compounds [(η6-p-cymene)Ru(N/O^N)Cl]0/+ with Schiff base chelating ligands (N/O^N) are synthesized and characterized. The introduction of Schiff base units effectively increases the antitumor activity of these compounds (IC50: 1.70 ± 0.56-17.75 ± 3.10 μM), which, meanwhile, can inhibit the metastasis of tumor cells effectively. These compounds follow an energy-dependent cellular uptake mechanism, mainly accumulate in lysosomes to destroy their integrity, and then eventually promote apoptosis. In addition, these compounds can induce an increase of intracellular reactive oxygen species (ROS) levels and provide an antitumor mechanism of oxidation, which is confirmed by the decrease of mitochondrial membrane potential (MMP) and the catalytic oxidation of the coenzyme nicotinamide-adenine dinucleotide (NADH). All these indicate that these ruthenium(ii) compounds are expected to be dual-functional antitumor agents: anti-metastasis and lysosomal damage.

Full text of DOI:10.1039/d0dt01547d

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Triphenylamine/carbazole-modified ruthenium(II)
Schiff base compounds: synthesis, biological
activity and organelle targeting†
Cite this: DOI: 10.1039/d0dt01547d
Shujiao Chen,a Xicheng Liu, *a Jie Huang,*b Xingxing Ge,a Qinghui Wang,a
a
Meimei Yao,a Yue Shao,a Tong Liu,a Xiang-Ai Yuan,a Laijin Tiana and Zhe Liu
*
Four triphenylamine/carbazole-modified half-sandwich ruthenium(II) compounds [(η6-p-cymene)Ru
(N/O^N)Cl]0/+ with Schiff base chelating ligands (N/O^N) are synthesized and characterized. The intro-
duction of Schiff base units effectively increases the antitumor activity of these compounds (IC50: 1.70
0.56–17.75
3.10 μM), which, meanwhile, can inhibit the metastasis of tumor cells effectively. These
compounds follow an energy-dependent cellular uptake mechanism, mainly accumulate in lysosomes to
destroy their integrity, and then eventually promote apoptosis. In addition, these compounds can induce
an increase of intracellular reactive oxygen species (ROS) levels and provide an antitumor mechanism of
oxidation, which is confirmed by the decrease of mitochondrial membrane potential (MMP) and the cata-
lytic oxidation of the coenzyme nicotinamide-adenine dinucleotide (NADH). All these indicate that these
ruthenium(II) compounds are expected to be dual-functional antitumor agents: anti-metastasis and lyso-
somal damage.
Received 28th April 2020,
Accepted 5th June 2020
DOI: 10.1039/d0dt01547d
rsc.li/dalton
limited by their remarkable drug resistance and side effects;
thus, numerous transition metal antitumor compounds with
Introduction
Lysosomes (pH = 3.5–5.5), containing more than 60 acid different mechanisms of action have been constantly devel-
hydrolases, are capable of degrading proteins, nucleic acid, oped and reported.7–13 Besides, ruthenium(II) (RuII)-based
polysaccharides and other biological macromolecules.1 After compounds such as NAMI-A, KP1019, and NKP1339 have
breaking up, cathepsins and other hydrolases in lysosomes shown promising therapeutic properties (Fig. 1), and have
will leak into the cytoplasm, which may digest the whole cell entered the stage of clinical research.14–17 Half-sandwich RuII
and promote apoptosis through a process known as lysosomal compounds, generally expressed as [(η6-p-cymene)Ru(X^Y)
membrane permeabilization (LMP).2,3 Because of their exten- Cl]0/+ (X^Y = bidentate ligands), have seen a surge of interest
sive involvement in various aspects of cell death, lysosomes because of their favorable stability and outstanding antitumor
are emerging as attractive targets for selective killing of tumor activity. Studies have showed that the type and the size of X^Y
cells.4 And the development of lysosome-targeting drugs is bidentate ligands could effectively influence the lipid solubility
expected to become a novel antitumor therapy.
and antitumor activity of these compounds, and among these,
Metallodrugs play a crucial role in the treatment of various a large number of weakly basic groups containing free electron
cancers in clinical chemotherapy, and among them, the pairs (such as triphenylamine,18 hydroxyquinoline,19 imine-N-
largest representatives are cisplatin and its derivatives.5,6 heterocyclic carbene,20 and benzimidazoles21) were introduced
Despite their great success, platinum-based drugs are still into X^Y bidentate ligands, coordinated with ruthenium(II)
ions, and lysosome-targeted antitumor agents were con-
structed, which achieved the expected success.
aInstitute of Anticancer Agents Development and Theranostic Application, The Key
Schiff base mainly refers to a series of organic compounds
Laboratory of Life-Organic Analysis and Key Laboratory of Pharmaceutical
containing an imine or a methylenimine-characteristic group
Intermediates and Analysis of Natural Medicine, School of Chemistry and Chemical
(–RCvN–). Usually, Schiff bases are formed by the conden-
sation of an amine and
reactive carbonyl group.22
Interestingly, because of their unique structures, Schiff bases
and their metal compounds have unique antiviral, antibacter-
ial and antitumor applications.23–27 Triphenylamine (TPA) and
N-phenylcarbazole (PhCz) are the classical hole-transport and
Engineering, Qufu Normal University, Qufu 273165, China.
E-mail: chemlxc@163.com, liuzheqd@163.com
bQingdao University of Science and Technology, Qingdao 266061, China.
E-mail: huangjiesci@163.com
a
†Electronic supplementary information (ESI) available. CCDC 1993083. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
D0DT01547D
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Fig. 1 Structures of reported and target half-sandwich RuII compounds.
luminescent materials that are widely used in the organic reaction of the dimer of RuII ([(η6-p-cymene)RuCl2]2) and Schiff
photoelectric field.28,29 Importantly, studies have shown that base pro-ligands (L1–L4) with good yield (70%–85%). Among
−
TPA/PhCz-based compounds endowed with good lumine- these, Ru1–Ru3 were isolated as hexafluorophosphate (PF6
)
salt, and sodium acetate (NaAc) was used as the base to
remove hydrogen from the phenolic hydroxyl group (L4) and
Ru4 was obtained. Fourier transform infrared spectroscopy
scence performance have favorable applications in the fields of
biological probes and antitumor drug targeting.18 Above all, in
this study, a series of lysosome-targeted TPA/PhCz-appended
half-sandwich RuII Schiff base antitumor compounds (Fig. 1)
were prepared and characterized by infrared (IR) spectroscopy
(IR), nuclear magnetic resonance (NMR) spectroscopy, mass
spectrometry (MS), elemental analysis and X-ray single crystal
diffraction. The antitumor activity of these compounds was
determined by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay towards A549 (human
lung cancer) and HeLa (human cervical cancer) cells, and the
ability of anti-metastasis towards the A549 cells was also evalu-
ated. The antitumor, cellular uptake, intracellular localisation
and transport mechanisms were exploited by using flow cyto-
metry and laser confocal technology. The TPA/PhCz-appended
half-sandwich RuII Schiff base compounds are expected to be
used as antitumor agents for further evaluation.
1
(FT-IR, Fig. S1†), H NMR (Fig. S2†), 13C NMR (Fig. S3†), elec-
trospray ionization-mass spectrometry (ESI-MS, Fig. S4†),
elemental analysis, and X-ray crystallography (Fig. 2) were used
to obtain the composition and structures of Ru1–Ru4.
According to the FT-IR data, the significant vibration ranges of
CvN, CvC and C–N were distributed at 1595–1637,
1430–1493 and 1231–1333 cm−1
, respectively. Deuterated
solvent CDCl3 (7.26 ppm) or DMSO (2.50 ppm) was used as the
1
solvent for determining the H NMR spectra. The distribution
of methyl hydrogen atoms on the benzene ring ranged from
1.03 to 2.70 ppm, while the hydrogen atoms on the aromatic
hydrocarbons (benzene and pyridine) ranged from 4.5 to
9.5 ppm. In addition, a variety of solvents were used during
the experiment, of which dichloromethane (5.30 and
13
5.76 ppm) showed some residues in the 1H NMR spectra.
The C NMR spectra showed that the carbon atoms of benzene
and pyridine were in the 80.61 to 164.62 ppm range, and the
distribution of the methyl group was 17.54 to 22.85 ppm.
Results and discussion
Schiff base pro-ligands (L1–L4) were synthesized by the con- The results of ESI-MS were consistent with the theoretical
−
densation of amino-substituted TPA/PhCz and aromatic alde- values of calculation (loss of PF6 for Ru1–Ru3 and loss of Cl−
hyde (Scheme 1). Target RuII compounds were obtained by the
for Ru4).
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Scheme 1 Synthesis process of Schiff base pro-ligands (L1–L4) and target RuII compounds.
trum, were obtained in CH2Cl2 at 298 K. As shown in Fig. S5,†
Ru1–Ru4 (20.0 μM) showed similar broad and weak band
maxima at ca. 340 and 520 nm, respectively. Interestingly, com-
pared with other compounds, the maximum emission of Ru3
was significantly red-shifted (∼35 nm), which indicated that
the introduction of the quinolyl group resulted in better conju-
gation of Ru3. Meanwhile, the absolute fluorescence quantum
yields of Ru1–Ru4 in CH2Cl2 solution are 0.41%, 0.33%, 0.35%
and 0.45%, respectively. The suitable luminescence properties
provide appropriate conditions for further study of the anti-
cancer mechanism. The stability of organometallic com-
pounds in solution was inseparable from their biological
activity.31 Therefore, the stability of Ru1–Ru4 in 60% DMSO-
d6/40% PBS (pH ≈ 7.2, prepared from D2O) solution (v/v) was
−
Fig. 2 X-ray crystal structure of Ru2 (hydrogen atoms and PF6 have
been omitted for clarity).
1
Single crystals of [(η6-p-cymene)Ru(L2)Cl]PF6 (Ru2) were
obtained by the slow diffusion of n-hexane into a saturated di-
chloromethane solution. The crystal structure and crystallo-
graphic data are shown in Fig. 2, and Tables S1 and S2,†
respectively. Ru2 had the typical “three-legged piano stool”
half-sandwich configuration, and the terminal triphenylamine
group had a “propeller” shape (the angle between the benzene
rings was ∼69.6°). However, the angle between P1 (the pyridine
tested by H NMR spectroscopy at 37 °C. As shown in Fig. S6,†
there was no additional change in the 1H NMR spectra after
24 h, which confirmed that this series of compounds had
sufficient stability in the simulated biological solution and
could be used for further research on chemical reactivity and
biological activity.
Cytotoxicity assay
ring) and P2 (the benzene ring of triphenylamine) was only The antitumor activity of the target RuII compounds against
36.1°. Obviously, the smaller angle ensured that the coplanar the A549 (human lung cancer) and HeLa (human cervical
property of the Schiff base pro-ligand was good and further cancer) cells was determined by using the MTT (3-(4,5-di-
improved its electron donor ability. The distance between the methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
central ruthenium(II) ion and the center of the p-cymene ring The IC50 values (the concentration at which 50% of cell growth
was 1.686 Å, and the bond distances of Ru–N1, Ru–N2 and was inhibited) of Ru1–Ru4 after 24 h of treatment are shown
Ru–C1 were 2.094(9), 2.088(9) and 2.387(3) Å, respectively. The in Table 1. Obviously, compared with cisplatin (a widely used
longer distance was consistent with the conclusion that the clinical antitumor drug) and Ru5 (a bipyridine-based simple
metal-chlorine bond was the active center for half-sandwich half-sandwich RuII compound, Scheme S1†), Ru1–Ru4 showed
organometallic compounds.30
better antitumor activity against the A549 cell, which indicated
The photophysical properties of these compounds, includ- that the introduction of the Schiff base chelating ligands was
ing ultraviolet-visible (UV-vis) spectrum and fluorescence spec- beneficial for the improvement of antitumor activity. The
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Table 1 IC50 values of Ru1–Ru5 and cisplatin against A549, HeLa and
BEAS-2B cells
inhibitor, 50.0 μM) and chloroquine (endocytosis regulator,
50.0 μM) for 1 h to determine the cellular uptake mechanism
of these compounds (Fig. S7†). Obviously, there was a signifi-
cant change in the low temperature treatment (4 °C) compared
with the control groups (37 °C or CCCP/chloroquine treat-
ment), which indicated that the mechanism by which these
RuII compounds entered the cells followed an energy depen-
dent one.
In addition, the intracellular distribution of Ru3 was
detected through laser confocal microscopy because of the
appropriate targeted luminescence characteristics of these
compounds.35,36 The A549 cells were pretreated with Ru3
(10.0 μM) for 1 h, and then stained by Mito Tracker Deep Red
(MTDR, mitochondrial fluorescent probe, 500 nM) and Lyso
Tracker Deep Red (LTDR, lysosome fluorescent probe, 500 nM)
for 20 min, respectively. As shown in Fig. 3a, Ru3 could effec-
tively accumulate in the lysosomes with a Pearson correlation
coefficient (PCC) of 0.77; however, the value for mitochondria
was 0.10. These results indicated that these ruthenium(II) com-
pounds with the Schiff base chelating ligands were beneficial
for targeting acidic lysosomes (pH: 3.5–5.5). Interestingly,
these RuII compounds did not cause abnormal cell death
immediately, and could be utilized to track the intracellular
changes in lysosomes in real time.
IC50 (μM)
Compounds
A549
HeLa
BEAS-2B
[(η6-p-cymene)Ru(L1)Cl]PF6 (Ru1)
[(η6-p-cymene)Ru(L2)Cl]PF6 (Ru2)
[(η6-p-cymene)Ru(L3)Cl]PF6 (Ru3)
[(η6-p-cymene)Ru(L4)Cl] (Ru4)
8.0 0.07 9.5 0.5 3.09 0.6
8.2 2.5 10.4 0.6 12.6 0.9
2.8 1.0
17.8 3.1
1.7 0.6 0.9 0.1
9.5 3.6 7.1 0.9
[(η6-p-cymene)Ru(Bipy)Cl]PF6 (Ru5) >100
>100
>100
Cisplatin
21.3 1.7
7.5 0.2 38.4 2.8
activity of Ru3, which showed the optimal activity among these
compounds, was about 8 times that of cisplatin under the
same conditions. Furthermore, the activity of Ru1–Ru3 and
the N^N ligand-based compounds effectively improved com-
pared to the O^N based one (Ru4). The triphenylamine and
N-phenylcarbazole groups of the Schiff base pro-ligands made
little difference to the antitumor activity of these compounds
(Ru1 and Ru2); however, the activity effectively improved with
the increase of benzene rings (Ru3). To understand these
workings, quantum chemical computation was applied for
evaluation after comprehensively considering the crystal struc-
ture of Ru2. The Wiberg bond order of Ru-Cl, the active center
of such compounds, was obtained by density functional theory
(DFT) calculations at the B3LYP/6-31G(d,p) (C, H, N, Cl)/SDD
(Ru) level. The values were 0.6860 and 0.6900 for Ru2 and Ru3,
respectively.32 In addition, the natural population analysis
(NPA) for the central ruthenium(II) ion and the chlorine ion
(the leaving group) was also obtained. The results of NPA
charge population for the chlorine ion were almost the same
with the values of −0.313 and −0.312 for Ru2 and Ru3, respect-
ively; however, those for the central ruthenium ion were
−0.002 and 0.003, respectively, which was mainly attributed to
the better electron donor capacity of L3 due to the introduction
of more phenyl groups in L3. Experiments show that the
log PO/W (partition coefficient in oil/water) values of Ru1–Ru5
were 0.42, 0.41, 0.75, 0.35 and 0.22, respectively, which further
confirmed that the increase of lipid solubility improved the
antitumor activity of these compounds. In addition, the cyto-
toxicity of Ru1–Ru4 towards human lung epithelial cells
(BEAS-2B) was further assessed. The results were not optimis-
tic, and no significant selectivity existed between normal cells
and tumour cells. However, favourable antitumor activity laid
the structural foundation for the further design and optimiz-
ation of such compounds.
Lysosomal damage, a process known as lysosomal mem-
brane permeabilization (LMP), could lead to the leak of hydro-
lases from the lysosomal lumen to the cytosol, thus inducing
apoptosis.37 Therefore, in order to further detect whether lyso-
somal damage was a pathway for apoptosis, acridine orange
(AO), an effective probe for acidic organelle with red or green
fluorescence representing the aggregation in lysosome or the
binding to RNA in the nucleus and cytoplasm,38 respectively,
Cellular uptake and localization
A good intracellular uptake mechanism is favorable for the
anticancer activity of drugs.33 The cellular uptake mechanism
typically falls into either of the two categories: “energy-depen- Fig. 3 (a) Cellular localization of Ru3: A549 cells were incubated with
Ru3, and then hatched with MTDR and LTDR. Ru3 was excited at
488 nm and collected at 550 30 nm. LTDR was excited at 594 nm and
collected at 630 30 nm. MTDR was excited at 543 nm and collected at
690 30 nm. (b) Lysosomal damage of the A549 cells incubated with
dent” mechanisms (including endocytosis and active trans-
port) or “non-energy-dependent” mechanisms (including free
diffusion and passive transport).34 In this study, A549 cells
were treated with Ru3 at 37 °C and 4 °C for 1 h, and then incu-
Ru3, and then stained by AO. Emission was collected at 510
20 nm
bated with chlorocyanochlorobenzene (CCCP, metabolic (green) and 625 20 nm (red) and excited at 488 nm.
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was used to detect the integrity of lysosomes in cells. As shown
in Fig. 3b, the A549 cells incubated with AO alone (the control)
showed an obvious red fluorescence in the lysosomes, which
confirmed the integrity of the intracellular lysosomal tissue.
However, the red fluorescence weakened quickly after it was
hatched with Ru3, increasing from 1.0× IC50 to 3.0× IC50. This
conclusion confirmed that the effective accumulation of these
RuII compounds in the lysosomes induced damage to the
integrity of acidic lysosomes (LMP).
Antitumor mechanism study
Apoptosis, an active process, autonomously regulated cell
death by genes.39 To assess whether these compounds might
induce apoptosis, the A549 cells were stained by annexin
V-FITC/PI (propidium iodide) and treated with Ru3 at the con-
Fig. 5 The loss of MMP induced by Ru3 and stained with JC-1 for
centrations of 1.0× IC50, 2.0× IC50 and 3.0× IC50 for 24 h and 30 min at 310 K. The red aggregates and green monomers are gated.
then analyzed by flow cytometry. As shown in Fig. 4 and
Table S3,† Ru3 could induce apoptosis in a concentration-
membrane potential (MMP), and the decrease of MMP was a
key indicator of cell apoptosis.41 JC-1 (5,5′,6,6′-tetrachloro-
1,1′,3,3′-tetraethylbenzimidazolyl phthalocyanine iodide), an
ideal fluorescent probe for detecting MMP, was used to reflect
the depolarization of MMP and was evaluated by flow cytome-
try. When MMP was high, JC-1 existed as a polymer and
showed a red fluorescence; in contrast, when it was in the
form of a monomer, it exhibited a green fluorescence. A sig-
nificant dose-dependent increase of green fluorescence was
demonstrated at the indicated concentrations (Fig. 5 and
Table S5†). Compared with the control groups (3.80%), the
population of mitochondrial membrane depolarized cells
(green) increased by 28.66% for Ru3 when the concentration
changed from 0. 5× IC50 to 3.0× IC50. It was not hard to see
that although these compounds did not target mitochondria,
widespread lysosomal damage confirmed by laser confocal
testing resulted in the release of cathepsins and other hydro-
lases from the lysosomal lumen to the cytosol, which directly
led to mitochondrial dysfunction, and eventually induced
apoptosis.
dependent manner. A total of 62.58% (early apoptosis + late
apoptosis) cells were in the apoptotic phase after being treated
with Ru3 at 3.0× IC50, however, which was 0.06% for the
control. This conclusion confirmed that these compounds
could effectively induce apoptosis. Generally, one pathway of
apoptosis was that the cell cycle was blocked, and the effect of
Ru3 on the cell cycle was assessed by flow cytometry.40 The
A549 cells were treated with Ru3 at different concentrations
(1.0× IC50, 2.0× IC50 and 3.0× IC50) for 24 h. When the concen-
tration of Ru3 changed from 1.0× IC50 to 3.0× IC50, however,
there was no significant change in the cell cycle (Fig. S8 and
Table S4†). This result indicated that the effect of these RuII
compounds on the cell cycle is negligible.
Mitochondria, the main energy producing sites of cells,
could store electrochemical potential energy in the inner mem-
brane and promote cell energy conversion.33 Meanwhile, asym-
metric distribution of proton and other ion concentrations on
both sides of the inner membrane could lead to mitochondrial
Generally, the accumulation of ROS in mitochondria (the
main sites of ROS storage and generation) could induce mito-
chondrial dysfunction, and eventually lead to apoptosis.42,43 In
this study, the ROS levels of the A549 cells were determined by
flow cytometry using DCFH-DA staining (dichlorofluorescein-
diacetate, an oxidant sensitive fluorescent probe) after treat-
ment with Ru3 for 24 h. As shown in Fig. 6, a dose-dependent
increase in intracellular ROS levels was found with the increase
of Ru3. At the concentration of 2.0× IC50, the level of ROS was
nearly 2.19 times of the negative control.
NADH (nicotinamide adenine dinucleotide), a reduced
coenzyme, widely existed in the cytoplasm and played an irre-
placeable role in regulating energy production in mitochon-
dria. Organometallic compounds might catalyze the conver-
sion of NADH to NAD+ (the oxidation state of NADH after it
loses hydrogen) accompanied by the production of ROS
(1O2).44,45 In this study, the interaction between these com-
pounds (1.0 μM) and NADH (100.0 μM) in 20% CH3OH/80%
Fig. 4 Apoptosis analysis chart of A549 cells after exposure to Ru3 at
37 °C for 24 h and stained by Annexin VFITC/PI.
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Fig. 6 (a) ROS levels in A549 cells treated with Ru3 at 1.0× IC50 and 2.0× IC50 for 24 h. (b) Histograms of the relative ROS levels. Data are quoted as
mean SD of three replicates.
Fig. 7 (a) UV-vis spectra of NADH (100.0 µM) after being reacted with Ru3 (1.0 µM) in 20% MeOH/80% H2O (v/v) at 298 K for 8 h. The arrows indi-
cate the changes over time. (b) TONs of Ru1–Ru4.
H2O (v/v) was investigated and monitored by UV-vis spec- tion spectra and photofluorescence (PL) spectra (Fig. 8 and
troscopy at 298 K for 8 h (Fig. 7 and S9†). A significant S10–S15†).
decrease and increase occurred at 339 nm (maximum UV-vis
Obviously, the UV-vis spectra showed a significant
absorbance of NADH) and 259 nm (maximum absorbance of reduction and red shift (∼4 nm) at 228 nm with the increase of
NAD+), respectively, which indicated the change of NADH to these compounds, which contributed to the disturbance of the
NAD+ with the addition of Ru1–Ru4. The catalytic effect could alpha-helix caused by RuII compounds and the influence of a
be clearly described through the turnover numbers (TONs) by polar solvent (water) on BSA, respectively.18 In addition, the
measuring the changes at 339 nm. As shown in Fig. 7b, Ru3 reduction at 278 nm meant the interaction between these com-
showed the highest TONs among these compounds. pounds and the amino acid residues of BSA (Fig. 8a).
Obviously, the higher the value of TONs, the better the antitu- Synchronous fluorescence spectra showed that the fluo-
mor activity, and this conclusion was consistent with the vari- rescence intensity decreased significantly when adding this
ation tendency of the MTT assay.
series of compounds; however, a weak blue shift (∼2 nm)
appeared at Δλ = 60 nm and there was no change at Δλ =
15 nm, which indicated that the property of the tryptophan
Protein binding assay
Human serum albumin (HSA) is the most abundant carrier residues was affected by these RuII compounds (Fig. 8c and
protein in plasma, which could bind a variety of substrates d).47 In addition, the phenomenon of fluorescence quenching
including metal cations, hormones and the most therapeutic further confirmed that these compounds could interact with
drugs.46 Due to the advantages of abundance, remarkable BSA.48,49 With the increase of these compounds, the fluo-
binding properties and high similarity to the structure of HSA, rescence intensity of BSA at 352 nm decreased significantly
bovine serum albumin (BSA) was used as the model to replace (Fig. 8b). The quenching mechanism could be explained by
HSA to study the binding mechanism of organometallic com- the classic Stern–Volmer equation. The values of the quench-
pounds in plasma. In this study, the interaction between BSA ing rate constant (Kq) were 1.04 × 1013 to 2.34 × 1013 M−1 s−1
and these RuII compounds was established by UV-vis absorp- for Ru1–Ru4, respectively, which were nearly one order of mag-
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Fig. 8 (a) UV-vis spectra of BSA (10.0 μM) in Tris–HCl with the increase of Ru3 (0–10.0 μM). Inset: the wavelength of 250–300 nm. (b) Fluorescence
spectra of BSA (10.0 μM) with the increase of Ru3. Synchronous fluorescence spectra of BSA (10.0 μM) with the increase of Ru3 at Δλ = 60 nm (c)
and Δλ = 15 nm (d). The arrows show the changes with the increase of Ru3.
nitude higher than that of a pure dynamic quenching mecha- R1 represent the distance in the absence and presence of
nism (<2.0 × 1012 M−1 −1) and confirmed that the interaction target compounds (24 h), respectively. The wound closure
s
of these RuII compounds and BSA followed a static quenching ratio (WCR = (R0 − R1)/R0 × 100%) could visually express the
mechanism.50 Above all, BSA could be a good carrier for these anti-metastatic ability of these compounds. Obviously, the
RuII compounds in plasma.51–53
lower the wound closure ratio, the greater the transfer inhi-
bition ability of these compounds. After 24 h of culture with
the concentration of 0.25× IC50, the wound closure ratios of
Ru3 and Ru4 were 10.16 and 22.48 compared with the control
(WCR = 31.94), which indicated that these compounds were
inseparable from the migration of tumor cells and could well
inhibit the tumor metastasis. In addition, this conclusion
was consistent with the variation tendency of the MTT assay
(Ru3 showed better antitumor activity than Ru4), which indi-
cated that these RuII compounds possessed good antitumor
activity and anti-metastatic ability, and provided a basis for
further evaluation.
Anti-metastasis assay
Tumor metastasis, the process in which malignant tumor
cells continue to grow from the primary site through lym-
phatic channels or others, has often been the main cause of
terminal tumor treatment failure, following which metastasis
inhibition has become an effective means of treating
cancer.54,55 Wound closure assay has usually been used to
evaluate anti-metastatic ability intuitively. As shown in Fig. 9,
the intermediate regions between the two blue lines
(“wounds”) represent the absence of tumor cells, and R0 and
Fig. 9 The wound healing assay of the A549 cells of Ru3 and Ru4; the length of the red line (R0 and R1) represents the width of the wound.
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DNM-9606 microplate reader. Apoptosis, cell cycle and mito-
chondrial membrane potential (MMP) tests were all performed
Conclusion
In
summary,
four
triphenylamine/N-phenylcarbazole- on an ACEA Novocyte 2040R flow cytometer. A Carl Zeiss
appended half-sandwich RuII Schiff base compounds were pre- AG*/LSM/880NLO two-photon laser scanning microscope
pared and characterized, and they showed favorable antitumor completes the cellular uptake and localization of these
and antimetastatic potential. Subtle structural adjustments compounds.
had a major impact on their cytotoxicity, and an appropriate
Synthesis of [(η6-p-cymene)Ru(N^N)Cl]PF6 (Ru1–Ru3)
increase in the number of phenyl groups belonging to the and [(η6-p-cymene)Ru(O^N)Cl] (Ru4). [(η6-p-cymene)RuCl2]2
Schiff base bidentate ligand was beneficial for improving the (Dimer: 48.9 mg, 0.08 mmol, 1 equiv.), Schiff base ligands L
antitumor activity of these RuII compounds. Laser confocal (0.16 mmol, 2 equiv.) and ammonium hexafluorophosphate
and flow cytometry confirmed that these compounds followed (130.4 mg, 0.7 mmol, 10 equiv.) or sodium acetate (108.8 mg,
an energy-dependent cellular uptake mechanism, accumulated 0.8 mmol, 10 equiv.) in methanol (50 mL) were stirred at room
in lysosomes, damaged the integrity of the lysosomes, and temperature (R.T.) overnight. The precipitate ammonium hexa-
eventually induced apoptosis. In addition, these compounds fluorophosphate was filtered through Celite, and the solvent
might transport through serum protein in plasma, and the was concentrated to about 5.0 mL under reduced pressure and
decrease of mitochondrial membrane potential (MMP) and maintained at 253 K for 12 h, filtered and washed with cold
catalytic oxidation of the coenzyme nicotinamide-adenine methanol and diethyl ether. The FT-IR, 1H NMR, 13C NMR,
dinucleotide (NADH) confirmed that these compounds could ESI-MS and elemental analysis data of Ru1–Ru4 are presented
increase the levels of intracellular reactive oxygen species in Fig. S1–S4.† The concrete data are listed as follows:
(ROS), which provided an antitumor mechanism of oxidation.
Above all, these RuII Schiff base compounds are potential anti- Selected FT-IR (KBr): 2974 (–CH3), 1603 (CvN), 1451 (CvC),
[(η6-p-cymene)Ru(L1)Cl]PF6 (Ru1). Yield: 103 mg (78%).
cancer agents and further evaluation is worth the effort.
1231 (C–N), 842 (CH). 1H NMR (500 MHz, DMSO) δ 9.62 (d, J =
5.4 Hz, 1H), 9.08 (s, 1H), 8.36–8.29 (m, 4H), 8.11 (d, J = 8.6 Hz,
2H), 7.93 (d, J = 8.7 Hz, 3H), 7.59 (d, J = 8.2 Hz, 2H), 7.52 (t, J =
7.6 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 6.19 (d, J = 6.3 Hz, 1H),
5.90–5.86 (m, 2H), 5.74 (d, J = 6.1 Hz, 1H), 2.61–2.58 (m, 1H),
2.23 (s, 3H), 1.04 (t, J = 6.3 Hz, 6H). 13C NMR (126 MHz,
Experimental section
General information
Ruthenium chloride hydrate, a-terpinene, sodium hydride, DMSO) δ 168.73, 156.55, 155.05, 150.73, 140.50, 140.36,
diphenylamine, 1-fluoro-4-nitrobenzene, lindlar catalyst, 138.44, 130.72, 129.50, 128.01, 126.89, 124.91, 123.49, 121.15,
hydrazine hydrate, 2-formylpyridine, 2-quinolinecarboxalde- 121.01, 110.36, 105.66, 104.42, 87.46, 86.68, 85.38, 85.30,
hyde, ammonium hexafluorophosphate, N,N-dimethyl- 31.05, 22.30, 22.14, 18.90. Elemental analysis: found: C, 53.70;
formamide, and salicylaldehyde were all purchased from Ruiya H, 5.02; N, 5.61%, calcd for C, 53.51; H, 4.09; N, 5.51%.
Biotechnology Co., Ltd. Fetal bovine serum was purchased ESI-MS (m/z): calcd for C38H41N3ClRu: 618.1; [M − PF6]+, found
from Sangon Biotech, A549 (lung cancer cells), HeLa (cervical 618.2.
cancer cells) and BEAS-2B (human normal lung epithelial
[(η6-p-cymene)Ru(L2)Cl]PF6 (Ru2). Yield: 112 mg (85%).
cells) were provided by the Shanghai Institute of Biochemistry Selected FT-IR (KBr): 2963 (–CH3), 1595 (CvN), 1493 (CvC),
1
and Cell Biology (SIBCB). Amino substituted triphenylamine/ 1333 (C–N), 840 (CH). H NMR (500 MHz, CDCl3) δ 9.29 (d, J =
N-phenylcarbazole, Schiff base chelating pro-ligands (L1–L4) 5.3 Hz, 1H), 8.36 (s, 1H), 7.99 (dt, J = 15.1, 7.4 Hz, 2H), 7.64 (d,
and the dimer of ruthenium(II) (Scheme S1†) were synthesized J = 8.9 Hz, 3H), 7.35 (t, J = 7.9 Hz, 4H), 7.17 (dd, J = 13.9, 7.5
according to the method described in the literature,30,56 and Hz, 6H), 7.09 (d, J = 8.9 Hz, 2H), 5.75 (d, J = 6.1 Hz, 1H), 5.49
the data are given in the ESI.†
(dd, J = 12.8, 6.1 Hz, 2H), 5.38 (d, J = 6.0 Hz, 1H), 2.68 (dt, J =
A Bruker DPX 500 spectrometer and LCQ Advantage MAX 13.8, 6.9 Hz, 1H), 2.18 (s, 3H), 1.15 (d, J = 6.9 Hz, 3H), 1.10 (d, J
mass spectrometer were utilized to obtain the nuclear mag- = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 161.94, 154.65,
netic resonance spectra and mass spectra of the intermediates 153.53, 149.01, 145.39, 143.71, 138.41, 128.77, 128.31, 127.79,
and target compounds. A VarioMICRO CHNOS elemental ana- 124.79, 123.78, 122.56, 119.70, 105.92, 102.25, 85.62, 85.19,
lyzer was used to obtain the elemental analysis data of these 85.14, 83.83, 30.10, 21.15, 20.88, 17.54. Elemental analysis:
compounds. UV-vis spectra and fluorescence spectra were found: C, 53.68; H, 4.40; N, 5.51%, calcd for C, 53.37; H, 4.35;
recorded by using a PERSEE TU-1901 ultraviolet spectrometer N, 5.49%. ESI-MS (m/z): calcd for C38H43N3ClRu 620.1;
and Hitachi F-4600 fluorescence spectrometer, respectively. [M − PF6]+, found 620.2.
The fluorescence quantum yield was obtained by using an
[(η6-p-cymene)Ru(L3)Cl]PF6 (Ru3). Yield: 112 mg (80%).
Edinburgh FLS1000 steady/transient fluorescence spectro- Selected FT-IR (KBr): 2969 (–CH3), 1637 (CvN), 1430 (CvC),
1
meter. X-ray diffraction data were obtained on a Bruker Smart 1255 (C–N), 877 (CH). H NMR (500 MHz, CDCl3) δ 8.75–8.65
CCD area detector with graphite monochromatic MoKα radi- (m, 2H), 8.50 (d, J = 8.3 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.04
ation (λ = 0.71073). The high-performance computing system (d, J = 7.9 Hz, 1H), 8.02–7.97 (m, 1H), 7.93 (d, J = 8.9 Hz, 1H),
Inspur TS10 K was used to obtain the theoretical calculation 7.87–7.82 (m, 1H), 7.36 (t, J = 7.8 Hz, 3H), 7.26–7.25 (m, 4H),
data. The MTT test was performed with
a
Perlong 7.18 (dd, J = 15.3, 7.5 Hz, 6H), 7.12 (d, J = 8.8 Hz, 2H), 5.64
Dalton Trans.
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(d, J = 5.9 Hz, 1H), 5.46 (d, J = 5.9 Hz, 1H), 5.26 (d, J = 6.1 Hz,
1H), 2.62 (dt, J = 12.2, 6.0 Hz, 1H), 2.13 (s, 3H), 1.26 (s, 3H),
1.13 (d, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 162.78,
157.28, 151.56, 150.33, 147.03, 146.37, 145.17, 141.69, 131.63,
129.80, 129.41, 128.31, 125.93, 124.90, 123.77, 123.03, 121.78,
120.60, 107.63, 102.40, 87.16, 85.94, 85.69, 84.82, 30.90, 22.79,
21.34, 18.28. Elemental analysis: found: C, 68.88; H, 5.29; N,
6.95%, calcd for C, 68.10; H, 5.26; N, 6.27%. ESI-MS (m/z):
calcd for C42H45ClN3Ru 670.2; [M − PF6]+, found 670.4.
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[(η6-p-cymene)Ru(L4)Cl] (Ru4). Yield: 71 mg (70%). Selected
FT-IR (KBr): 2961 (–CH3), 1616 (CvN), 1456 (CvC), 1317
1
(C–N), 760 (CH). H NMR (500 MHz, CDCl3) δ 8.18 (d, J = 7.7
Hz, 2H), 7.94–7.88 (m, 3H), 7.67 (d, J = 8.5 Hz, 2H), 7.45 (t, J =
6.1 Hz, 4H), 7.35–7.31 (m, 2H), 7.28 (s, 1H), 7.03 (t, J = 8.6 Hz,
2H), 6.48 (t, J = 7.2 Hz, 1H), 5.40 (d, J = 6.0 Hz, 1H), 5.32 (d, J =
6.1 Hz, 1H), 5.18 (d, J = 5.7 Hz, 1H), 4.46 (d, J = 5.6 Hz, 1H),
2.68 (dt, J = 13.8, 6.9 Hz, 1H), 2.18 (s, 3H), 1.22 (d, J = 7.0 Hz,
3H), 1.18 (d, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
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109.49, 101.77, 98.16, 86.36, 83.49, 83.23, 80.61, 30.44, 22.85,
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Accession codes
CCDC 1993083 contain the supplementary crystallographic
data for this paper.†
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Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
We thank the University Research Development Program of
Shandong Province (J18KA082), the National Natural Science
Foundation of China (Grant No. 21671118), the Taishan
Scholars Program and the High Performance Computing
Center of Qufu Normal University for their support.
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