Fluorescent iridium(iii) coumarin-salicylaldehyde Schiff base compounds as lysosome-targeted antitumor agents

Article abstract of DOI:10.1039/d0dt00627k

Six fluorescent half-sandwich iridium(iii) coumarin-salicylaldehyde Schiff base (O^N) compounds ([(?⁵-Cp*)Ir(O^N)Cl]) were prepared and characterized. The introduction of a coumarin unit increased the antitumor activity (IC₅₀: 9.9 ± 0.1 μM-40.7 ± 12.9 μM) of these compounds, the best of which was nearly two times that of clinical cisplatin. The results of laser confocal microscopy demonstrated that these compounds possessed an energy-dependent cellular uptake mechanism, accumulated in the lysosomes (Pearson co-localization coefficient: ~0.7), damaged the integrity of the lysosomes, and induced apoptosis. The compounds could also decrease the mitochondrial membrane potential, catalyze the oxidation of the coenzyme (nicotinamide-adenine dinucleotide) and improve the levels of the intracellular reactive oxygen species, following an antitumor mechanism of oxidation. Additionally, these compounds could block the metastasis of tumor cells. Above all, these iridium(iii) compounds show potential as antitumor agents with dual functions: lysosomal damage and anti-metastasis.

Full text of DOI:10.1039/d0dt00627k

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DOI: 10.1039/D0DT00627K.
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14 March 2017
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Fluorescent Iridium(III) Coumarin-salicylaldehyde Schiff Base Compounds as
Lysosome-Targeted antitumor agents
Cong Liu, Xicheng Liu*, Xingxing Ge, Qinghui Wang, Lei Zhang, Wenjing Shang, Yue Zhang,
XiangAi Yuan, Laijin Tian, Zhe Liu*, Jinmao You
Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of
Life-Organic Analysis and Key Laboratory of Pharmaceutical Intermediates and Analysis of
Natural Medicine, School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu
273165, China.
*Corresponding authors (E-mail): chemlxc@163.com (X. C. Liu); liuzheqd@163.com (Z. Liu)
Abstract: Six fluorescent half-sandwich iridium(III) coumarin-salicylaldehyde Schiff
base (O^N) compounds ([(η5-Cp*)Ir(O^N)Cl]) were prepared and characterized. The
introduction of coumarin unit increased the antitumor activity (IC50: 9.9±0.1 μM ~
40.7±12.9 μM) of these compounds, the best of which was nearly two times that of
clinical cisplatin. Laser confocal shows compounds possessed an energy-dependent
cellular uptake mechanism, accumulated in lysosomes (Pearson co-localization
coefficient: ~0.7), damaged the integrity of lysosomes, and induce apoptosis.
Compounds can decrease the mitochondrial membrane potential, catalyze the
oxidation of coenzyme (nicotinamide-adenine dinucleotid) and improve the levels of
intracellular reactive oxygen species, follow an antitumor mechanism of oxidation.
Additionally, these compounds could block the metastasis of tumor cells. Above all,
these iridium(III) compounds are potential antitumor agents with dual functions:
lysosomal damage and anti-metastasis.
Keywords: Iridium(III) compounds; Antitumor; Schiff base; Coumarin
1. Introduction
Lysosomes, an organelle presenting in eukaryotic cells, have a significant feature
in an acidic environment (pH: 4.5~5.5), which are conducive to maintain the activity
of a large number of lysosome hydrolases1,2. Lysosomes involved in many important
physiological processes, including cell metabolism, autophagy, migration, apoptosis
etc.3-5 Compared with normal cells, tumor cells have high lysosomal content, poor
stability, and high cathepsin activity. Recent studies have demonstrated that weakly
basic antitumor drugs could effectively accumulate in the lysosome, lead to partial
lysosomal membrane permeabilization (LMP)6. Once the integrity of lysosomal
membrane is compromised, enzymes in the lysosome can cause apoptosis and
disintegration by hydrolyzing biomacromolecules7. Therefore, lysosomes may be
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potential targets for selective killing of tumor cells, and the development of lysosome
targeting drugs is expected to become a novel antitumor drug.
Because of the unique antitumor mechanisms, such as catalyzing the cellular
oxidation-reduction (REDOX) change (increasing the levels of reactive oxygen
species), inhibitor of various proteins (Vascular endothelial growth factor receptor 3
(VEGF R3), B-cell lymphoma-2 (Bcl-2), etc.), organometallic iridium(III) (IrIII)
compounds are expected to be the preferred alternative to platinum-based antitumor
drugs8-13.The general structural formula of half-sandwich IrIII compounds can be
described as [(η5-Cp*)Ir(L^L)Z]0/+, where Cp* is an electron-rich cyclopentadiene
group and its derivatives, Z is leaving group (usually chloride (Cl)), and L^L is a
variety of chelating ligands14-16. Rational adjustment and control of the peripheral
organic ligands had achieved out-standing antitumor effect in the development of
these compounds15. Among these, on account of the easy modification of L^L
chelating ligands, a mass of organic small molecular groups containing free electron
pairs, including carbazole17, triphenylamine18, rhodamine19, ferrocene20, had been
introduced into half-sanwich IrIII compounds, used as lysosome targeting antitumor
drugs, and achieved desired results. However, half-sandwich IrIII compounds are
difficult to form conjugate structures as effective as cyclometalated iridium(III)
compounds, and those with favorable luminescent properties are rarely reported. All
of these hinder the further study of their target and antitumor mechanism.
Coumarin derivatives, existing in a variety of plants (Tonka bean, sweet clover
grass, licorice, etc.), have antifungicidal21-23, antitumor24 and anticoagulant activities25.
Photophysics and photochemistry of coumarin derivatives are also important and have
served as dye lasers26. In recent years, various possible approaches, e.g., cell cycle
arrest, angiogenesis, telomerase, mitosis and regulation of reactive oxygen species
(ROS), have been reported for coumarins derivatives to treat cancer27,28. Meanwhile,
coumarin has the capacity to modulate multiple cellular signaling pathways, making it
useful for selective antitumor therapy29,30. Schiff base (-RC=N-), obtained by the
condensation of amine (hydrazone, thiosemicarbazones, shrinkage amine, aminoacids,
heterocyclic etc.) and aldehydes/ketones, possesses good coordination ability with
transition metals and has exhibited unique medicinal effects in antiviral, antibacterial
and antitumor studies31-33. And even, Schiff base-based compounds have been used as
models for biologically important species34-36. In view of the promising applications
of coumarin derivatives in the field of antitumor, Schiff base (O^N) chelating ligands
were obtained by condensation with salicylaldehyde, further reacted with the dimer of
iridium(III) and constructed half-sandwich iridium(III) Schiff base compounds ([(η5-
Cp*)Ir(O^N)Cl], Ir1-Ir6 compounds, Figure 1). MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) assay indicated that these compounds showed
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better anticancer activity than coumarin-appended Schiff base pro-ligands (L1 and L2)
and half-sandwich iridium(III) salicylaldehyde-aniline Schiff base compound (Ir7,
Scheme S1). In addition, we investigated the mechanism of antitumor and evaluated
the intracellular imaging properties due to the favorable fluorescence characteristics
of these compounds.
Figure 1. Structures of designed IrIII compounds.
2. Results and discussion
Coumarin-appended half-sandwich structural IrIII compounds ([(η5-
Cp*)Ir(O^N)Cl]) were obtained by the reaction of [(η5-Cpx)IrCl2]2 (Dimer 1), [(η5-
Cpxph)IrCl2]2 (Dimer 2) or [(η5-Cpxbiph)IrCl2]2 (Dimer 3) with coumarin-
salicylaldehyde Schiff base (O^N) chelating pro-ligands (L1 and L2), and chlorine
(Cl) is the leaving group (Scheme 1). L1 and L2 were obtained by the condensation
reaction of 7-amino-coumarin and salicylaldehyde37-39. Ir1-Ir6 compounds are new
synthesized and obtained in good yields (~80%) (Figure 1), and fully characterized by
Fourier transform-infrared spectroscopy (FT-IR), nuclear magnetic resonance
spectrum (1H NMR, 13C NMR and 19F NMR), mass spectrum (MS) and elemental
analysis (Figures S1-S5). The peak shift of C=O (1720-1750 cm-1) and C=N (1600-
1640 cm-1) stretching vibration absorption was estimated by FT-IR. Deuterated
1
reagent CDCl3 (7.26 ppm) was used as a solvent to test the H NMR spectra of Ir1-
Ir6. Methyl hydrogen atoms of cyclopentadiene (η5-Cp*) are shown in the range of
1.0 to 2.0 ppm, while the ones on coumarin (Ir1-Ir3) are shown in 2.3 to 2.5 ppm.
Hydrogen atoms on the aromatic nucleus (coumarin and benzene) are in the range of
6.0 to 8.5 ppm. In addition, these compounds used a variety of solvents during the
synthesis and purification, including dichloromethane (5.30 ppm), methanol (3.32
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1
ppm), n-hexane (0.88 ppm), which showed some residual spectra in H NMR. The
methyl carbon atoms of Cp* are shown in 7.72~9.16 ppm, and which on coumarin is
13
at 17.70~18.91 ppm in C NMR spectra. The carbon atoms of the benzene ring and
coumarin are shown in the range of 111.40~167.64 ppm. Fluorine of trifluoromethyl
unit was shown in ~64.70 ppm in 19F NMR spectra. The data of ESI-MS are
consistent with the results of theoretical calculations (loss of chlorine (Cl)).
Scheme 1. Synthesis process of half-sandwich IrIII compounds.
Through the slow diffusion of n-hexane into [(η5-Cpx)Ir(L1)Cl] (Ir1) and [(η5-
Cpx)Ir(L2)Cl] (Ir4) saturated dichloromethane solution, single crystals suitable for X-
ray diffraction analysis were obtained. Crystal structures are illustrated in Figure 2
and the crystallographic data are listed in Tables S1 and S2. Ir1 and Ir4 show the
expected geometry of the half-sandwich "three-legged piano stool". The coumarin
unit at the end presents a conventional planar configuration, despite the benzene ring
(P1) attached to it has a relatively large angle (30.65° and 24.15° for Ir1 and Ir4,
respectively). Angles between P1 and Schiff base unit (-C=N-) are 10.56° and 10.89°.
The smaller angles ensure the favorable coplanar property of L1 and L2, and further
improve the electron donor ability. Additionally, the length of Ir-Cl is the longest
around the central iridium(III) ion (Table 1), which provides the basis for further
hydrolysis and exhibiting antitumor activity of these compounds40.
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Figure 2. Single crystal structures of Ir1 and Ir4 drawn with 50% thermal ellipsoidal probability
(Hydrogen atoms have been omitted for clarity).
Table 1. Distance around central iridium and selected angles for Ir1 and Ir4.
Distance (Å)
Ir-N1 Ir-O1
θ(°)
P1-C=N
Bond Order
Ir-Cl1
Compound
Ir-Cp
1.774
1.772
Ir-Cl1
P1-P2
30.65
24.15
Ir1
Ir4
2.100 2.072 2.416
2.107 2.081 2.409
10.56
10.89
0.5493
0.5505
Stability of organometallic compounds in solution effectively affects their
biological activity. The stability of Ir1-Ir6 in a mixture of 20% DMSO/80% H2O (v/v)
was monitored by ultraviolet-visible (UV-vis) spectra at 298K (Figure S6). As shown,
no significant changes in absorbance were observed within 8 h. This conclusion
indicates that compounds exhibit fairly stability, which lays a foundation for the
subsequent biological property test.
2.1 Cell cytotoxicity assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was
used to determine the antitumor activity of these IrIII compounds against human lung
cancer cells (A549) and human cervical cancer cells (HeLa) after 24 h exposure, and
cisplatin was utilized as a control. The IC50 values (concentration when 50% cell
growth was inhibited) are listed in Table 2. As shown, compared with cisplatin and
coumarin-unattached half-sandwich IrIII compound (Ir7, Scheme S1), these
compounds (Ir1-Ir6) exhibit potential antitumor activity with the IC50 values ranging
from 9.9 ± 0.1 µM to 40.7 ± 12.9 µM, and the best of which (Ir2) is almost 2 times
that of cisplatin (widely used in clinical studies) under the same conditions, which
indicate that the introduction of coumarin is benefical to the improvement of
antitumor activity for these IrIII compounds. Additionally, compared with the simple
bipyridine-based IrIII compounds11, the activity (Ir1-Ir6) was still higher suggesting
that the introduction of Schiff base was effective for antitumor activity. Compounds
containing Cpxph ligand (Ir2 and Ir5, Figure 1) show better antitumor activity than
those containing Cp* unit (Ir1 and Ir4) because of the enhancing lipid solubility of
Cpxph ligand. However, the introduction of Cpxbiph ligand (larger organic molecule)
improves the lipid solubility of Ir3 and Ir6, more importantly, the water solubility of
these two compounds is further reduced. Above all, Cpxph ligand based compounds
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(Ir2 and Ir5) showed the best antitumor activity. The oil-water distribution
coefficients were determined, and the values were 0.12, 0.45 and 1.21 for Ir1, Ir2 and
Ir3, respectively, which indicates that the appropriate increase of lipid solubility is
beneficial to the improvement of antitumor activity for these compounds.
Further, compounds containing methyl-substituted coumarin unit (Ir1, Ir2 and
Ir3) show better antitumor ability than the corresponding trifluoromethyl-modified
analogues (Ir4, Ir5 and Ir6), respectively. To understand these, quantum chemical
computation was used to assess after comprehensively considering the crystal
structures of Ir1 and Ir4. The Wiberg bond order of Ir-Cl were obtained by density
functional theory calculation at the B3LYP//6-311G(d,p) (C, H, O, N, Cl)/LANL2TZf
(Ir) level with the values of 0.5493 and 0.5505 for Ir1 and Ir4 (Table 1)41,42
,
respectively. Obviously, the introduction of trifluoromethyl group with strong
electron-withdrawing property decreases the electron donor capacity of Schiff base
ligand (L2), increases the Wiberg bond order of Ir-Cl to some extent, then weakens
the cytotoxicity of Ir4-Ir6.
Table 2. IC50 values of Ir1-Ir7 and cisplatin against A549 and Hela cells over 24 h.
IC50 (μM)
Compound
A549 cells
30.9±1.6
12.0±0.1
13.1±0.8
39.6±1.0
24.2±4.2
33.4±0.7
﹥100
Hela cells
36.0±10.4
9.9±0.1
Ir1
Ir2
Ir3
10.8±0.1
40.7±12.9
22.1±0.5
39.2±2.5
﹥100
Ir4
Ir5
Ir6
Ir7
Cisplatin
21.3±1.7
7.5±0.2
2.2 Cell localization and cellular uptake assay
Photophysical properties of Ir1-Ir6 were measured by UV-vis and
photoluminescence (PL) spectra. As shown in Figure S7, Ir1-Ir6 exhibit almost the
same photophysical properties, which have a strong intraligand absorption band (π-π*)
at around 250-300 nm and a weaker metal-to-ligand charge-transfer (MLCT)
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transition band at around 300-400 nm. Electron cloud distribution of the frontier
molecular orbitals (Figure S8) shows that the HOMOs of Ir1 and Ir4 are mainly
localized on Ir, Cl and salicylaldehyde unit, and the LUMOs are mainly localized on
the O^N chelating ligands. This conclusion indicates that the introduction of coumarin
unit effectively improve the conjugate performance of O^N ancillary ligands, and
which have been confirmed through crystal data of Ir1 and Ir4 (The whole O^N
ligand exhibit a slight angle, Figure 2). Meanwhile, the calculated values were found
to show the similar variation trend with the UV-vis spectrum data (Table S3).
Additionally, compared with Ir1-Ir3, an obvious red shift phenomenon (~20 nm) is
reflected in Ir4-Ir6 because of the smaller angles between P1 and P2 (Table 1). This
conclusion is more obvious in the fluorescence spectrum (Figure S7b), Ir4-Ir6 exhibit
the longer emission spectra (474 nm) than Ir1-Ir3 (422 nm).
Owning to suitable fluorescence characteristics, the subcellular localization in
A549 cells for these compounds was determined by laser confocal microscopy. Lyso
Tracker Red DND-99 (LTRD) and Mito Tracker Deep Red (MTDR) were employed
as the lysosomes and mitochondria fluorescence probes, respectively43,44. As shown in
Figure 3, Ir2 and Ir5 could effectively accumulate in lysosomal tissues with the
Pearson’s colocalization coefficient (PCC) of 0.64 and 0.72 over 1 h. However, the
PCC for mitochondrion-targeted is 0.22 and 0.18, respectively. This result indicates
that these compounds are mainly target lysosomes in vivo. Additionally, these
compounds did not cause abnormal cell death immediately, which made it convenient
to track changes in lysosomal morphology timely.
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Figure 3. Intercellular localization of Ir2 and Ir5 were determined by confocal microscopy. A549
cells were incubated with Ir2 or Ir5 (10.0 μM) for 1 h at 37 °C, then incubated with MTDR (500
nM) or LTDR (75 nM) for 20 min. Compounds were excited at 405 nm and collected at 430-580
nm. LTDR was excited at 594 nm and collected at 630 ± 30 nm. MTDR was excited at 644 nm
and collected at 690 ± 30 nm.
Lysosomes, containing more than 60 acid hydrolases, are organelles for the
decomposition of proteins, nucleic acids, polysaccharides and other biological
macromolecules. When breaking up, lysosomes release hydrolases that digest the
whole cell and promote its apoptosis45. Acridine orange (AO) is a potent probe
(red/green fluorescence in lysosome/cytoplasm and nucleus, respectively) for
determining the lysosomal integrity of tumor cells46. A549 cells were incubated with
Ir2 and Ir5 (10 μM) over 6 h, then stained with AO (5 μM, 15 min). As shown in
Figure 4, a significant decrease for red fluorescence was shown when A549 cells
hatched in Ir2 and Ir5 (1.0 × IC50) for 6 h, and obvious lysosomal damage was found
in the concentration of 3.0 × IC50. The results confirmed that these IrIII compounds
could mainly accumulate in lysosome, cause lysosomal damage and ultimately lead to
cell death.
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Figure 4. Lysosomal disruption in A549 cells after treated with Ir2 and Ir5 for 6 h at 310 K, then
stained by acridine orange (AO, 5 μM) for 15 min. Emission was collected at 510±20 nm (green)
and 625±20 nm (red) by excitation at 488 nm.
Good intracellular uptake mechanism is favorable to the improvement of drug
activity, non-energy-depended mechanisms (including passive transport and free
diffusion) and energy-depended mechanisms (including active transport and
endocytosis) are two main cellular uptake mechanisms for organometallic drugs47.
Then, A549 cells were treated with Ir2 and Ir5 at 277 K and 310 K for 2 h, and
hatched
with
chloroquine
(endocytosis
modulator,
50
μM)
and
chlorocyanochlorophenyl (CCCP, metabolic inhibitor, 50 μM) to ascertain the cellular
uptake mechanisms (Figure S9). As shown, except for cells hatched at 4 °C, others
show favorable cellular uptake for Ir2 and Ir5, which indicate that these IrIII
compounds enter cells primarily followed an energy-dependent pathways.
2.3 Antitumor mechanism study
Apoptosis refers to the autonomous and orderly death of cells controlled by
genes in order to maintain internal environment stability, and most transition metal
anticancer drugs can clear tumor cells by inducing apoptosis of cancer cells48. In order
to investigate whether these IrIII compounds could induce apoptosis, A549 cells were
stained by annexin V-FITC/Propidium Iodide (PI) and treated with Ir2 and Ir5 at the
concentrations of 1.0× IC50, 2.0× IC50 and 3.0×IC50 for 24 h, and then analyzed by
flow cytometry (Figure 5 and Tables S4-S5). As shown, Ir2 and Ir5 induced
apoptosis in a dose-dependent manner, mainly in the late stage. The percentage of late
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apoptosis cells increased from 8.37% and 8.31% to 26.46% and 32.52% when the
concentration changed from 1.0×IC50 to 3.0×IC50 for Ir2 and Ir5, respectively.
Meanwhile, 95.29% of A549 cells survived for the control under the same conditions.
These results indicated these compounds could lead to cell death through apoptosis.
Figure 5. Apoptosis analysis of A549 cells after 24 h of exposure to Ir2 and Ir5 obtained by flow
cytometry using Annexin V-FITC/PI staining.
Generally, IrIII compounds can cause a decline in function by disrupting the cell
cycle and exert their anticancer effects12. To investigate the effect of these compounds
on cell cycle arrest, A549 cells (stained by PI) exposed to Ir2 and Ir5 for 24 h at the
concentration of 0.25 × IC50, 0.5 × IC50, 1.0 × IC50 and analyzed by flow cytometry.
As shown in Figure S10 and Tables S6-S7, when the concentration changed from 0.25
× IC50 to 1.0 × IC50, there were almost no change happened to A549 cells at S phase,
G0/G1 phase, and G2/M phase. This result indicates these compounds are not effective
at blocking the cell cycle. Additionally, mitochondria, the source of energy or ATP
(adenosine triphosphate) in cells, play a unique role in the process of apoptosis49. The
decrease of mitochondrial membrane potential (ΔΨm) is a vital signal in the early
apoptotic process, and accompanied by the production of reactive oxygen species50.
JC-1 (5,5,6,6′-tetrachloro-1,1′,3,3′ tetraethyl benzimidazolyl carbocyanine iodide), an
ideal fluorescent probe for detecting ΔΨm (the high and low ΔΨm would show red and
green fluorescence), was utilized to clarify whether apoptosis induced through
destroying mitochondrial homeostasis and estimated by flow cytometry51. As shown
in Figure 6 and Tables S8-S9, a significant dose-dependent decline of ΔΨm occurs in
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A549 cells induced by Ir2 and Ir5, the population of mitochondrial membrane
depolarized cells increased by 28.47% and 35.24% for Ir2 and Ir5 when the
concentration changed from 0.25 × IC50 to 2.0 × IC50, respectively. It's not hard to see
that although these compounds do not target mitochondria, widespread lysosomal
damage confirmed by laser confocal testing results in the release of cathepsins and
other hydrolases from the lysosomal lumen to the cytosol, which directly lead to the
mitochondrial dysfunction, and eventually induce apoptosis.
Figure 6. Changes in ΔΨm of A549 cells induced by Ir2 and Ir5. Data are quoted as mean ± SD
of two replicates.
Reactive oxygen species (ROS) are mainly produced and stored in the
mitochondria, and the increase of intracellular ROS levels may induce apoptosis52.
Using oxidant-sensitive DCFH-DA (2,7-dichlorodi-hydrofluorescein diacetate) as a
fluorescent probe, flow cytometry was used to monitor the ROS levels of A549 cells
at the concentration of 0.25×IC50 and 0.5×IC50 (Ir2 and Ir5) after 24 h exposure. As
shown in Figure 7, when the concentrations of Ir2 and Ir5 increased from 0.25×IC50
to 0.5×IC50, the intracellular ROS levels in A549 cells showed a dose-dependent
increase. When the concentration was 0.5×IC50, compared with the negative control,
the intracellular ROS levels showed a 1.742/1.103-fold increases for Ir2/Ir5,
respectively.
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Figure 7. (a) Effect of Ir2 and Ir5 on intracellular ROS levels in A549 lung cancer cells for 24 h;
(b) The histograms of ROS levels to A549 cells hatched by Ir2 and Ir5 (concentrations = 0.25,
0.5× IC50). Data was averaged for three replicate experiments ± SD.
The reduced state of nicotinamide adenine dinucleotide (NADH) is a control
marker in the energy production chain of organisms53. Ru(II), Rh(III), Os(II) and
Ir(III) compounds have been reported which could catalyse the change of
intracytoplasmic NADH to NAD+ (Oxidation state of NADH, loss of hydrogen
catalyzed by organometallic compounds), lead to the accumulation of reactive oxygen
species (1O2), and follow the antitumor mechanism of oxidation54. UV-visible
spectrophotometry was utilized to investigate the interaction between these
compounds and NADH in 10% MeOH/90% H2O (v/v) at 298 k after different time
intervals. As shown in Figures 8 and S11, with the increase of Ir1-Ir6, the absorbance
at 339 nm (the maximum absorbance of NADH) and 259 nm (the maximum
absorbance of NAD+) had been significantly weakened and strengthened55. These
changes can be well reflected through the turnover numbers (TONs, Figure 4b). Ir2
show the highest TONs, which indicates that Ir2 has the best biocatalytic
performance among these compounds. Additionally, TONs of Ir2 and Ir5 (η5-Cpxph
coordination) are obviously higher than that of the corresponding
methyl/trifluoromethyl-appended analogues (Ir1, Ir3 and Ir4, Ir6), and these results
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are consistent with those of the MTT assay, indicating that the larger the TONs, the
better the antitumor activity. Above all, widespread lysosomal damage led to
apoptosis, which could be further demonstrated by the decrease in mitochondrial
membrane potential, the increase of intracellular ROS (which was also confirmed by
catalyzing the oxidation of NADH), and demonstrated an antitumor mechanism of
oxidation.
Figure 8. (a) UV–vis spectra of NADH (100 μM) with Ir2 (1 μM) in 10% MeOH/90% H2O (v:v)
over 8 h at 298 K. The arrows show the changes over time; (b) TONs of Ir1-Ir6.
2.4 Antimetastatic assay
Tumor metastasis inhibition is an effective pathway for tumor treatment56.
Wound healing assay was utilized on A549 cells to assess the antimetastatic
properties of these compounds57. As shown in Figure 9, there are no tumor cells in the
intermediate region between the two blue lines ("wound"), and R0 and R1 represent
the distance between the A549 cells on either side with or without targeting
compounds. After incubation for 24 h, Ir2 and Ir5 (0.25 × IC50) showed the
characteristic of inhibiting cell migration, and the wound closure ratios (WCRs = (R0-
R1)/R0 × 100%) were 5.33% and 11.78%, respectively. Compared with the control
group (WCR= 30.39%), the smaller WCRs indicate that A549 cells rarely migrate to
the intermediate wound area after the addition of Ir2 and Ir5, and these compounds
possess a certain possibility of inhibiting the migration of tumor cells. Additionally,
Ir2 performed better than Ir5, which is consistent with the results of the MTT assay
(Ir2 to Ir5 has better antitumor activity).
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Figure 9. (a) Wound healing assay against A549 cells incubated with Ir2 and Ir5 (0.25 × IC50)
after 24 h. Scale bar: 100 μM. (b) Histograms of wound healing assay after 24 h. Data are
quoted as mean ± SD of three replicates.
Conclusion
In this paper, six coumarin-appended fluorescent half-sandwich IrIII Schiff base
compounds were obtained and characterized. Compounds showed favorable stability
in the biological environment and laid a foundation for biological performance test.
Subtle structural adjustment has a significant effect on the cytotoxicity: Properly
regulating and controling the number of phenyl groups in cyclopentadiene ligand and
electron donor capacity of Schiff base ligands (Replacing trifluoromethyl with methyl)
were beneficial to the improvement of antitumor activity for these compounds.
Benefitting from the suitable fluorescence properties, laser scanning confocal imaging
demonstrated that the cellular uptake of these compounds followed an energy-
dependent mechanism, effectively accumulated in lysosomes and destroyed the
structural integrity of acid lysosomes, further led to the decrease in mitochondrial
membrane potential, which could be demonstrated by the increase of intracellular
reactive oxygen species and the catalytic acceleration of the oxidation of NADH.
Additionally, these compounds could effectively block the metastasis of tumor cells.
In summary, coumarin-modified half-sandwich iridium(III) compounds can induce
apoptosis and show potential applications value in organometallic antitumor drugs.
3. Experimental section
3.1. General information
Iridium trichloride hydrate (IrCl3·3H2O), 1,2,3,4,5-pentamethyl-cyclopentadiene
(95%), 2,3,4,5-tetramethyl-2-cyclopentenone (95%), phenyl magnesium bromide (3.0
M in diethyl ether), 4-bromobiphenyl, butyllithium solution (1.6 M in hexane),
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salicylaldehyde, 7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin,
formic acid, aniline, crystalline sodium acetate and all kinds of organic solvents
(dichloromethane, n-hexane, methanol, tetrahydrofuran, methylbenzene etc.) were
purchased from Rhea biotechnology co. LTD. For the biological experiments, DMEM
medium, fetal bovine serum, penicillin/streptomycin mixture and trypsin/EDTA were
purchased from Sangon Biotech. A549 (lung cancer cells), Hela (cervical cancer cell)
and BEAS-2B (human normal lung epithelial cells) were obtained from Shanghai
Institute of Biochemistry and Cell Biology (SIBCB). The dimer of iridium (Dimer 1-3)
were prepared according to literature procedures12.
NMR spectra were obtained on a Bruker DPX 500 spectrometers instrument,
with the chemical shifts reported in ppm using tetramethylsilane (TMS) as an internal
standard. Mass spectrum (MS) was measured on a Waters Q-TOF MicroTM mass
spectrometer. Elemental analysis was performed on a VarioMICRO CHNOS
elemental analyzer. The quantum chemical data were calculated using a Inspur's
enterprise server. UV-vis spectroscopy was performed on a PERSEE TU-1901 UV
spectrometer. Fluorescence spectra were collected by a Hitachi F-4600 fluorescence
spectrophotometer, with a 400 V voltage and 5 nm slit width for both excitation and
emission. Induction of apoptosis, cell cycle and mitochondrial membrane potential
(ΔΨm) determination was carried out by an ACEA Novocyte2040R flow cytometry.
Viability assay (MTT) was measured using a Perlong DNM-9606 microplate reader at
an absorbance of 570 nm. Cell uptake and cellular localization were carried out on a
Carl Zeiss AG */LSM/880NLO two photon laser scanning microscope.
3.2. Synthesis of coumarin-salicylaldehyde Schiff base ligands (L)
0.12 g (1.0 mmol) of salicylaldehyde, 0.18 g (1.0 mmol) 7-amino-4-
methylcoumarin, anhydrous methanol (40 mL) and a drop of formic acid were added
to a 200 mL round bottom flask and refluxed for 12 h until 7-amino-4-
methylcoumarin was consumed completely (monitored by thin-layer chromatography).
Most of the solvent were concentrated in vacuum and kept at 253 K for 12 h, filtered
and washed with cold methanol and diethyl ether. The 1H NMR (500 MHz), 13C NMR
(126 MHz) and ESI-MS are shown in Figures S12-S14. The data were listed as
follows: Yield: 0.23 g (81%). 1H NMR (500 MHz, CDCl3): δ 8.65 (s, 1H), 7.64 (d, J
= 8.3 Hz, 1H), 7.43 (t, J = 7.8 Hz, 2H), 7.23 – 7.19 (m, 2H), 7.09 – 6.95 (m, 3H), 6.28
(d, J = 0.9 Hz, 1H), 2.46 (d, J = 1.0 Hz, 3H).13C NMR (126 MHz, CDCl3): δ 164.51,
161.29, 160.67, 154.46, 152.02, 151.60, 134.11, 132.88, 125.64, 119.44, 118.86,
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118.55, 118.26, 117.43, 114.52, 108.78, 18.72. ESI-MS (m/z): Calcd for C17H12O3N:
278.1; Found: 278.1 [M-H]-. Elem anal. Calcd for C17H13O3N: C, 73.11; H, 4.69; N,
5.02; O, 17.19%. Found: C, 73.31; H, 4.79; N, 4.95; O, 17.11%.
L2 was synthesized by 7-amino-4-trifluoromethylcoumarin (0.23 g, 1.0 mmol)
and salicylaldehyde (0.12 g, 1.0 mmol) using the same method as L1.Yield: 0.28 g
1
(84%). H NMR (500 MHz, CDCl3): δ 8.66 (s, 1H), 7.78 (dd, J = 9.0, 1.6 Hz, 1H),
7.58 – 7.48 (m, 1H), 7.46 (dd, J = 10.9, 4.6 Hz, 2H), 7.06 (d, J = 8.6 Hz, 1H), 7.05 –
6.96 (m, 2H), 6.78 (s, 1H), 6.65 – 6.54 (m, 1H). 13C NMR (126 MHz, CDCl3): δ
165.35, 161.38, 158.77, 155.37, 152.87, 151.22, 134.56, 133.07, 126.43, 119.59,
119.04, 118.72, 117.56, 115.12, 115.08, 111.92, 109.51. ESI-MS (m/z): Calcd for
C17H9O3NF3: 332.1; Found: 332.1 [M-H]-. Elem anal. Calcd for C17H10O3NF3: C,
61.27; H, 3.02; N, 4.20; O, 14.40%. Found: C, 61.42; H, 3.18; N, 4.12; O, 14.30%.
3.3. Synthesis of [(η5-Cp*)Ir(O^N)Cl] (Ir1-Ir6)
The general synthesis method is as follows: A solution of dimer of iridium
(Dimer 1/Dimer 2/Dimer 3, 40 mg/46 mg/54 mg, 0.050 mmol), sodium acetate (33
mg, 0.40 mmol) and coumarin-salicylaldehyde Schiff base ligands (L1/L2, 28 mg/34
mg, 0.10 mmol) in methanol (40 mL) resulting suspension was stirred at ambient
temperature overnight. Distilled under reduced pressure, the final compounds were
obtained by slow diffusion of n-hexane into a saturated dichloromethane solution of
the crude product. The FT-IR (KBr), 1H NMR (500 MHz,), 13C NMR (126 MHz), 19F
NMR (471 MHz), ESI-MS, and elementary analysis of Ir1-Ir6 are shown in Figures
S1-S5. The data were listed as follows:
[(η5-Cpx)Ir(L1)Cl] (Ir1) Yield: 27 mg (83%). Selected FT-IR (KBr): 2990 (C-H),
1
1722 (C=O), 1600 (C=N), 1380 (C-N), 1130 (C-O). H NMR (500 MHz, CDCl3): δ
8.03 (s, 1H, HC=N), 7.96 (dd, J = 8.5, 2.1 Hz, 1H, Ar-H), 7.62 (d, J = 8.5 Hz, 1H, Ar-
H), 7.47 (d, J = 2.1 Hz, 1H, Ar-H), 7.38 (ddd, J = 8.6, 6.8, 1.8 Hz, 1H, Ar-H), 7.12
(dd, J = 7.9, 1.6 Hz, 1H, Ar-H), 6.97 (d, J = 8.6 Hz, 1H, Ar-H), 6.50 – 6.44 (m, 1H,
Ar-H), 6.30 (d, J = 1.1 Hz, 1H, Ar-H), 2.47 (d, J = 1.0 Hz, 3H, Ar-CH3), 1.35 (s, 15H,
13
Cp-CH3). C NMR (126 MHz, CDCl3): δ 164.58 (HC=N), 161.51, 160.62, 157.98,
153.33, 151.95, 136.09, 134.91, 124.66, 123.69, 122.67, 120.27, 118.82, 115.52,
115.18, 112.63, 85.98 (Cp-C), 18.91 (Ar-CH3), 8.69 (Cp-CH3). ESI-MS (m/z): Calcd
for C27H27O3NIr: 606.1; Found: 606.2 [M-Cl]+. Elem anal. Calcd for C27H27O3NClIr:
C, 50.58; H, 4.24; N, 2.18; O, 7.49%. Found: C, 50.79; H, 4.34; N, 2.11; O, 7.42%.
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5
xph
[(η -Cp )Ir(L1)Cl] (Ir2) Yield: 29 mg (84%). Selected FT-IR (KBr): 2980 (C-H),
1
1730 (C=O), 1600 (C=N), 1380 (C-N), 1150 (C-O). H NMR (500 MHz, CDCl3): δ
8.00 (s, 1H, HC=N), 7.79 (dd, J = 8.5, 2.0 Hz, 1H, Ar-H), 7.47 – 7.43 (m, 1H, Ar-H),
7.38 (ddd, J = 8.6, 6.9, 1.7 Hz, 1H, Ar-H), 7.31 (ddd, J = 8.6, 5.0, 3.1 Hz, 1H, Ar-H),
7.25 (d, J = 4.6 Hz, 5H, Ar-H), 7.13 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 6.96 (d, J = 8.6
Hz, 1H, Ar-H), 6.48 (t, J = 7.3 Hz, 1H, Ar-H), 6.23 (s, 1H, Ar-H), 2.40 (s, 3H, Ar-
CH3), 1.58 (s, 3H, Cp-CH3), 1.55 (d, J = 5.6 Hz, 6H, Cp-CH3), 1.44 (s, 3H, Cp-CH3).
13C NMR (126 MHz, CDCl3): δ 163.22 (HC=N), 160.51, 159.42, 156.84, 152.07,
150.68, 135.19, 133.79, 129.91, 129.09, 127.60, 127.16, 123.36, 122.54, 121.42,
118.91, 117.55, 114.54, 113.99, 111.40, 95.95 (Cp-C), 90.63 (Cp-C), 84.57 (Cp-C),
82.05 (Cp-C), 80.22 (Cp-C), 17.70 (Ar-CH3), 7.98 (Cp-CH3), 7.85 (Cp-CH3), 7.75
(Cp-CH3), 7.72 (Cp-CH3). ESI-MS (m/z): Calcd for C32H29O3NIr: 668.2; Found:
668.3 [M-Cl]+. Elem anal. Calcd for C32H29O3NClIr: C, 54.65; H, 4.16; N, 1.99; O,
6.82%. Found: C, 54.83; H, 4.36; N, 1.88; O, 6.74%.
[(η5-Cpxbiph)Ir(L1)Cl] (Ir3) Yield: 31 mg (79%). Selected FT-IR (KBr): 2980 (C-H),
1
1720 (C=O), 1640 (C=N), 1390 (C-N), 1060 (C-O). H NMR (500 MHz, CDCl3): δ
8.03 (s, 1H, HC=N), 7.83 (dd, J = 8.5, 2.0 Hz, 1H, Ar-H), 7.59 (d, J = 7.4 Hz, 2H, Ar-
H), 7.50 (d, J = 8.2 Hz, 2H, Ar-H), 7.47 – 7.43 (m, 3H, Ar-H), 7.43 – 7.39 (m, 1H,
Ar-H), 7.39 – 7.35 (m, 3H, Ar-H), 7.32 (d, J = 2.0 Hz, 1H, Ar-H), 7.15 (dd, J = 7.8,
1.2 Hz, 1H, Ar-H), 6.99 (d, J = 8.6 Hz, 1H, Ar-H), 6.51 (t, J = 7.3 Hz, 1H, Ar-H),
6.23 (s, 1H, Ar-H), 2.38 (s, 3H, Ar-CH3), 1.58 (s, 6H, Cp-CH3), 1.56 (s, 3H, Cp-CH3),
1.51 (s, 3H, Cp-CH3). 13C NMR (126 MHz, CDCl3): δ 164.29 (HC=N), 161.57,
160.42, 157.83, 153.12, 151.67, 140.94, 140.27, 136.23, 134.82, 130.53, 129.99,
128.88, 127.63, 127.27, 127.01, 124.41, 123.61, 122.52, 119.97, 118.60, 115.60,
115.04, 112.43, 96.68 (Cp-C), 92.31 (Cp-C), 85.09 (Cp-C), 83.54 (Cp-C), 80.68 (Cp-
C), 18.70 (Ar-CH3), 9.01 (Cp-CH3), 8.98 (Cp-CH3), 8.96 (Cp-CH3), 8.79 (Cp-CH3).
ESI-MS (m/z): Calcd for C38H33O3NIr: 744.2, Found: 744.3 [M-Cl]+. Elem anal.
Calcd for C38H33O3NClIr: C, 58.56; H, 4.27; N, 1.80; O, 6.16%. Found: C, 58.86; H,
4.39; N, 1.72; O, 6.06%.
[(η5-Cpx)Ir(L2)Cl] (Ir4) Yield: 28 mg (80%). Selected FT-IR (KBr): 2980 (C-H),
1750 (C=O), 1600 (C=N), 1390 (C-N), 1270 (C-F), 1150 (C-O). 1H NMR (500 MHz,
CDCl3): δ 8.03 (s, 1H, HC=N), 7.91 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 7.76 (dd, J = 8.6,
1.2 Hz, 1H, Ar-H), 7.71 (d, J = 2.1 Hz, 1H, Ar-H), 7.39 (ddd, J = 8.6, 6.9, 1.7 Hz, 1H,
Ar-H), 7.11 (dd, J = 7.9, 1.5 Hz, 1H, Ar-H), 6.97 (d, J = 8.7 Hz, 1H, Ar-H), 6.80 (s,
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13
1H, Ar-H), 6.49 – 6.45 (m, 1H, Ar-H), 1.35 (s, 15H, Cp-CH3). C NMR (126 MHz,
CDCl3): δ 164.93 (HC=N), 161.66, 158.89, 158.67, 154.30, 136.45, 134.97, 125.41,
123.93, 123.16, 120.24, 115.96, 115.71, 113.80, 112.28, 86.06 (Cp-C), 8.74 (Cp-CH3).
19F NMR (471 MHz, CDCl3): δ -64.68 (s). ESI-MS (m/z): Calcd for C27H24O3NF3Ir:
660.1; Found: 660.2 [M-Cl]+. Elem anal. Calcd for C27H24O3NF3ClIr: C, 46.65; H,
3.48; N, 2.01; O, 6.90%. Found: C, 46.88; H, 3.59; N, 1.94; O, 6.82%.
[(η5-Cpxph)Ir(L2)Cl] (Ir5) Yield: 30 mg (79%). Selected FT-IR (KBr): 3060 (C-H),
1730 (C=O), 1600 (C=N), 1440 (C-N), 1280 (C-F), 1140 (C-O). 1H NMR (500 MHz,
CDCl3): δ 8.00 (s, 1H, HC=N), 7.74 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 7.59 – 7.56 (m,
1H, Ar-H), 7.48 (d, J = 2.0 Hz, 1H, Ar-H), 7.41 (ddd, J = 8.6, 6.9, 1.7 Hz, 1H, Ar-H),
7.30 – 7.26 (m, 5H, Ar-H), 7.14 (dd, J = 8.0, 1.4 Hz, 1H, Ar-H), 6.98 (d, J = 8.5 Hz,
1H, Ar-H), 6.74 (s, 1H, Ar-H), 6.50 (dd, J = 10.8, 3.9 Hz, 1H, Ar-H), 1.59 (s, 3H, Cp-
CH3), 1.57 (d, J = 2.5 Hz, 6H, Cp-CH3), 1.45 (s, 3H, Cp-CH3). 13C NMR (126 MHz,
CDCl3): δ 167.64 (HC=N), 164.66, 161.77, 158.68, 154.12, 136.65, 134.98, 130.91,
130.20, 128.78, 128.37, 125.29, 123.89, 123.06, 120.04, 116.15 – 115.72, 113.62,
112.14, 96.89 (Cp-C), 91.78 (Cp-C), 86.25 (Cp-C), 83.05 (Cp-C), 81.27 (Cp-C), 9.16
(Cp-CH3), 9.09 (Cp-CH3), 9.06 (Cp-CH3), 8.91 (Cp-CH3). 19F NMR (471 MHz,
CDCl3): δ -64.73 (s). ESI-MS (m/z): Calcd for C32H26O3NF3Ir: 722.2; Found: 722.3
[M-Cl]+. Elem anal. Calcd for C32H26O3NF3ClIr: C, 50.76; H, 3.46; N, 1.85; O, 6.34%.
Found: C, 50.96; H, 3.59; N, 1.76; O, 6.27%.
[(η5-Cpxbiph)Ir(L2)Cl] (Ir6) Yield: 32 mg (76%). Selected FT-IR (KBr): 3060 (C-H),
1740 (C=O), 1600 (C=N), 1440 (C-N), 1280 (C-F), 1140 (C-O). 1H NMR (500 MHz,
CDCl3): δ 8.01 (s, 1H, HC=N), 7.77 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 7.60 – 7.56 (m,
3H, Ar-H), 7.53 (d, J = 2.0 Hz, 1H, Ar-H), 7.49 (d, J = 8.3 Hz, 2H, Ar-H), 7.45 (t, J =
7.6 Hz, 3H, Ar-H), 7.36 (t, J = 7.1 Hz, 3H, Ar-H), 7.14 (dd, J = 7.9, 1.5 Hz, 1H, Ar-
H), 7.00 (d, J = 8.6 Hz, 1H, Ar-H), 6.69 (s, 1H, Ar-H), 6.53 – 6.49 (m, 1H, Ar-H),
13
1.60 (s, 3H, Cp-CH3), 1.57 (s, 6H, Cp-CH3), 1.52 (s, 3H, Cp-CH3). C NMR (126
MHz, CDCl3): δ 163.91 (HC=N), 161.58, 160.23, 152.46, 152.03, 142.88, 135.97,
134.61, 131.11, 130.16, 128.62, 128.17, 127.99, 123.96, 123.47, 119.98, 118.21,
117.61, 116.54, 115.51, 96.61 (Cp-C), 90.98 (Cp-C), 86.83 (Cp-C), 82.61 (Cp-C),
19
81.35 (Cp-C), 9.10 (Cp-CH3), 8.91 (Cp-CH3), 8.81 (Cp-CH3). F NMR (471 MHz,
CDCl3): δ -64.75 (s). ESI-MS (m/z): Calcd for C38H30O3NF3Ir: 798.2; Found: 798.3
[M-Cl]+. Elem anal. Calcd for C38H30O3NF3ClIr: C, 54.77; H, 3.63; N, 1.68; O, 5.76%.
Found: C, 55.89; H, 3.83; N, 1.59; O, 5.68%.
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Acknowledgments
We thank the University Research Development Program of Shandong Province (J18KA082),
the Student’s Platform for Innovation and Entrepreneurship Training Program
(S201910446034), the National Natural Science Foundation of China (21671118), the
Taishan Scholars Program and High Performance Computing Center of Qufu Normal
University for support.
Supporting Information
Details of the experimental section, Scheme S1, Figures S1−S16, and Tables S1−S9.
Accession Codes
CCDC 1970999 and 1971001 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: +441223 336033.
Conflicts of interest
The authors declare no competing financial interest.
References
1. M. Savini, Q. Zhao and M. Wang, Lysosomes: Signaling hubs for metabolic sensing and
longevity, Trends Cell Biol., 2019, 29, 876-887.
2. C. L. Minchew and V. V. Didenko, Dual detection of nucleolytic and proteolytic markers of
lysosomal cell death: DNase II-type breaks and cathepsin D, Methods Mol. Biol., 2017, 1554,
229-236.
3. P. R. Pryor, Chapter eight-analyzing lysosomes in live cells, Method Enzymol., 2012, 505, 145-
157.
4. P. Saftig and J. Klumperman, Lysosome biogenesis and lysosomal membrane proteins:
Trafficking meets function, Nat. Rev. Mol. Cell Biol., 2009, 10, 623-635.
5. M. E. Guicciardi, M. Leist and G. J. Gores, Lysosomes in cell death, Oncogene, 2004, 23,
2881-2890.
6. J. P. Luzio and P. R. Pryor, Lysosomes: Fusion and function, Nat. Rev. Mol. Cell Biol., 2007, 8,
622-632.
19

Dalton Transactions
Page 20 of 24
View Article Online
7. A. Serrano-Puebla and P. Boya, Lysosomal membrane permeabilization in cell deDaOthI::10N.1e0w39/D0DT00627K
evidence and implications for health and disease, Ann. N. Y. Acad. Sci., 2016, 1371, 30-44.
8. L. He, Y. Li, C. Tan, R. Ye, M. Chen, J. Cao, L. Ji and Z. Mao, Cyclometalated iridium(III)
complexes as lysosome-targeted photodynamic anticancer and real-time tracking agents, Chem.
Sci., 2015, 6, 5409-5418.
9. N. Cutillas, G. S. Yellol, C. Haro, C. Vicente, V. Rodríguez and J. Ruiz, Anticancer
cyclometalated complexes of platinum group metals and gold, Coord. Chem. Rev., 2013, 257,
2784-2797.
10. Y. Li, B. Liu, X. Lu, M. Li, L. Ji and Z. Mao, Cyclometalated iridium(III) N-heterocyclic
carbene complexes as potential mitochondrial anticancer and photodynamic agents, Dalton
Trans., 2017, 46, 11363-11371.
11. S. Chen, X. Liu, X. Ge, Q. Wang, Y. Xie, Y. Hao, Y. Zhang, L. Zhang, W. Shang and Z. Liu,
Lysosome-targeted iridium(III) compounds with pyridine-triphenylamine Schiff base ligands:
Syntheses, antitumor application and mechanism, Inorg. Chem. Front., 2020, 7, 91-100.
12. Z. Liu and P. J. Sadler, Organoiridium complexes: Anticancer agents and catalysts, Acc. Chem.
Res., 2014, 47, 1174-1185.
13. N. P. E. Barry and P. J. Sadler, Dicarba-closo-dodecarborane-containing half-sandwich
complexes of ruthenium, osmium, rhodium and iridium: Biological relevance and synthetic
strategies, Chem. Soc. Rev., 2012, 41, 3264-3279.
14. X. Liu, X. He, X. Zhang, Y. Wang, J. Liu, X. Hao, Y. Zhang, X. Yuan, L. Tian and Z. Liu,
Synthesis and antineoplastic applications of TPE appended organometallic iridium(III)
complexes, ChemBioChem, 2019, 20, 2767-2776.
15. L. K. McKenzie, I. V. Sazanovich, E. Baggaley, M. Bonneau, V. Guerchais, J. A. G. Williams,
J. A. Weinstein and H. E. Bryant, Metal complexes for two-photon photodynamic therapy: A
cyclometallated iridium complex induces two-photon photosensitization of cancer cells under
near-IR light, Chem. Eur. J., 2017, 23, 234-238.
16. S. J. Lucas, R. M. Lord, R. L. Wilson, R. M. Phillips, V. Sridharan and P. C. McGowan,
Synthesis of iridium and ruthenium complexes with (N,N), (N,O) and (O,O) coordinating
bidentate ligands as potential anti-cancer agents, Dalton Trans., 2012, 41, 13800-13802.
17. S. Chen, X. Liu, Z. Tian, X. Ge, H. Hao, Y. Hao, Y. Zhang, Y. Xie, L. Tian and Z. Liu,
Triphenylamine and carbazole-modified iridium(III) 2-phenylpyridine complexes: Synthesis,
anticaner application and targeted research, Appl. Organomet. Chem., 2019, 33, (e5053).
18. X. He, X. Liu, Y. Tang, J. Du, M. Tian, Z. Xu, X. Liu and Z. Liu, Half-sandwich iridium(III)
complexes with triphenylamine-substituted dipyridine frameworks and bioactivity applications,
Dyes Pigments, 2019, 160, 217-226.
19. W. Ma, Z. Tian, S. Zhang, X. He, J. Li, X. Xia, X. Chen and Z. Liu, Lysosome targeted drugs:
Rhodamine B modified N^N-chelating ligands for half-sandwich iridium(III) anticancer
complexes, Inorg. Chem. Front., 2018, 5, 2587-2597.
20

Page 21 of 24
Dalton Transactions
View Article Online
20. X. Ge, S. Chen, X. Liu, Q. Wang, L. Gao, C. Zhao, L. Zhang, M. Shao, X. Yuan, L.DTOiI:a1n0.a10n3d9/D0DT00627K
Z. Liu, Ferrocene-appended iridium(III) complexes: Configuration regulation, anticancer
application, and mechanism research, Inorg. Chem., 2019, 58, 14175-14184.
21. T. Patonay, G. Litkei, R. Bognar, J. Erdei and C. Miszti, Synthesis, antibacterial and antifungal
activity of 4-hydroxycoumarin derivatives, analogues of novobiocin, Die Pharmazie, 1984, 39,
84-91.
22. B. M. W. Ouahouo, A. G. B. Azebaze, M. Meyer, B. Bodo, Z. T. Fomum and A. E. Nkengfack,
Cytotoxic and antimicrobial coumarins from mammea africana, Ann. Trop. Med. Parasit.,
2004, 98, 733-739.
23. M. D. Camacho, J. D. Phillipson, S. L. Croft, V. Yardley and P. N. Solis, In vitro antiprotozoal
and cytotoxic activities of some alkaloids, quinones, flavonoids, and coumarins, Planta. Med.,
2004, 70, 70-72.
24. J. Koshy, V. G. Das, S. Balabaskaran, S. W. Ng and N. Wahab, High in-vitro antitumour
activity of triphenyltin coumarin 3-carboxylate and its coordination complexes with
monodentate oxygen donor ligands against the epstein barr virus (EBV)-DNA positive raji and
the P-388 murine leukaemia cell lines, and evidence for the suppression by organotin of the
early antigen complex in the EBV lytic cycle, Met.-Based Drugs, 2000, 7, 245-251.
25. J. W. Suttle, Warfarin and vitamin K, Clin. Cardiol., 1990, 13, 16-18.
26. K. V. Basavarajappa, Y. A. Nayaka, R. O. Yathisha and P. Manjunatha, Synthesis,
characterization, optical, electrochemical and current-voltage characteristics of coumarin dyes,
J. Fluoresc., 2019, 29, 1201-1211.
27. B. Thati, A. Noble, B. S. Creaven, M. Walsh, M. McCann, M. Devereux, K. Kavanaghe and D.
A. Egan, Role of cell cycle events and apoptosis in mediating the anti-cancer activity of a
silver(I) complex of 4-hydroxy-3-nitro-coumarin-bis(phenanthroline) in human malignant
cancer cells, Eur. J. Pharmacol., 2009, 602, 203-214.
28. A. H. Bedair, N. A. El-Hady, M. S. A. El-Latif, A. H. Fakery and A. M. El-Agrody, 4-
hydroxycoumarin in heterocyclic synthesis: Part III. synthesis of some new pyrano[2,3-
d]pyrimidine, 2-substituted[1,2,4]triazolo[1,5-c]pyrimidine and pyrimido[1,6-b][1,2,4]triazine
derivatives, II Farmaco, 2000, 55, 708-714.
29. G. J. Finn, E. Kenealy, B. S. Creaven and D. A. Egan, In vitro cytotoxic potential and
mechanism of action of selected coumarins, using human renal cell lines, Cancer Lett., 2002,
183, 61-68.
30. G. J. Finn, B. Creaven and D. A. Egan, Study of the in vitro cytotoxic potential of natural and
synthetic coumarin derivatives using human normal and neoplastic skin cell lines, Melanoma
Res., 2001, 11, 461-467.
31. M. A. Malik, O. A. Dar, P. Gull, M. Y. Wani and A. A. Hashmi, Heterocyclic Schiff base
transition metal complexes in antimicrobial and anticancer chemotherapy, Med. Chem.
Commun., 2018, 9, 409-436.
21

Dalton Transactions
Page 22 of 24
View Article Online
DOI: 10.1039/D0DT00627K
32. A. Prakash and D. Adhikari, Application of Schiff bases and their metal complexes-a review,
Int. J. Chem. Tech. Res., 2011, 3, 1891-1896.
33. W. H. Mahmoud, R. G. Deghadi and G. G. Mohamed, Novel Schiff base ligand and its metal
complexes with some transition elements. Synthesis, spectroscopic, thermal analysis,
antimicrobial and in vitro anticancer activity, Appl. Organomet. Chem., 2016, 30, 221-230.
34. X. Qiao, Z. Ma, C. Xie, F. Xue, Y. Zhang, J. Xu, Z. Qiang, J. Lou, G. Chen and S. Yan, Study
on potential antitumor mechanism of a novel Schiff base copper(II) complex: Synthesis,
crystal structure, DNA binding, cytotoxicity and apoptosis induction activity, J. Inorg.
Biochem., 2011, 105, 728-737.
35. S. R. Collinson and D. E. Fenton, Metal complexes of bibracchial Schiff base macrocycles,
Coord. Chem. Rev., 1996, 148, 19-40.
36. M. Nath and P. K. Saini, Chemistry and applications of organotin(IV) complexes of Schiff
bases, Dalton Trans., 2011, 40, 7077-7121.
37. L. Wang, H. Li and D. Cao, A new photoresponsive coumarin-derived Schiff base:
Chemosensor selectively for Al3+ and Fe3+ and fluorescence "turn-on" under room light,
Sensor. Actuat. B-Chem., 2013, 181, 749-755.
38. L. S. Guinesi and E. T. G. Cavalheiro, Influence of some reactional parameters on the
substitution degree of biopolymeric Schiff bases prepared from chitosan and salicylaldehyde,
Carbohydr. Polym., 2006, 65, 557-561.
39. A. A. El-Sherif and M. S. Aljahdali,Review: Protonation, complex-formation equilibria, and
metal-ligand interaction of salicylaldehyde Schiff bases, J. Coord. Chem., 2013, 66, 3423-
3468.
40. Z. Liu, I. Romero-Caneln, B. Qamar, J. M. Hearn, A. Habtemariam, N. P. E. Barry, A. M.
Pizarro, G. J. Clarkson and P. J. Sadler, The potent oxidant anticancer activity of
organoiridium catalysts, Angew. Chem. Int. Ed., 2014, 53, 3941-3946.
41. O. Edfors, M. Sandell, J. J. van de Beek, S. K. Wilson and P. O. Börjesson, Analysis of DFT-
based channel estimators for OFDM, Wireless Pers. Commun., 2000, 12, 55-70.
42. B. G. Yang, Z. G. Cao and K. B. Letaief, Analysis of low-complexity windowed DFT-based
MMSE channel estimator for OFDM systems, IEEE Trans. Commun., 2001, 49, 1977-1987.
43. S. Daum, M. S. V. Reshetnikov, M. Sisa, T. Dumych, M. D. Lootsik, R. Bilyy, E. Bila, C.
Janko, C. Alexiou, M. Herrmann, L. Sellner and A. Mokhir, Lysosome-targeting amplifiers of
reactive oxygen species as anticancer prodrugs, Angew. Chem. Int. Ed., 2017, 56, 15545-
15549.
44. J. Yang, J. Zhao, Q. Cao, L. Hao, D. Zhou, Z. Gan, L. Ji and Z. Mao, Simultaneously inducing
and tracking cancer cell metabolism repression by mitochondria-immobilized rhenium(I)
complex, ACS Appl. Mater. Interfaces, 2017, 9, 13900-13912.
45. K. Kågedal, M. Zhao, I. Svensson and U. T. Brunk, Sphingosine-induced apoptosis is
dependent on lysosomal proteases, Biochem. J., 2001, 15, 335-343.
22

Page 23 of 24
Dalton Transactions
View Article Online
DOI: 10.1039/D0DT00627K
46. G. Kroemer and L. Galluzzi, Lysosome-targeting agents in cancer therapy, Oncotarget, 2017,
8, 112168-112169.
47. C. Li, M. Yu, Y. Sun, Y. Wu, C. Huang and F. Li, A nonemissive iridium(III) complex that
specifically lights-up the nuclei of living cells, J. Am. Chem. Soc., 2011, 133, 11231-11239.
48. V. Novohradsky, L. Zerzankova, J. Stepankova, A. Kisova, H. Kostrhunova, Z. Liu, P. J.
Sadler, J. Kasparkova and V. Brabec, A dual-targeting, apoptosis-inducing organometallic
half-sandwich iridium anticancer complex, Metallomics, 2014, 6, 1491-1501.
49. C. Ledderose, Y. Bao, M. Lidicky, J. Zipperle, L. L. Li, K. Strasser, N. I. Shapiro and W. G.
Junger, Mitochondria are gate-keepers of T cell function by producing the ATP that drives
purinergic signaling, J. Biol. Chem., 2014, 289, 25936-25945.
50. T. Chen, Y. Liu, W. Zheng, J. Liu and Y. Wong, Ruthenium polypyridyl complexes that
induce mitochondria-mediated apoptosis in cancer cells, Inorg. Chem., 2010, 49, 6366-
6368.
51. H. Antonicka, K. Choquet, Z. Y. Lin, A. C. Gingras, C. L. Kleinman and E. A. Shoubridge, A
pseudouridine synthase module is essential for mitochondrial protein synthesis and cell
viability, Embo. Rep., 2017, 18, 28-38.
52. I. Romero-Canelon, M. Mos and P. J. Sadler, Enhancement of selectivity of an organometallic
anticancer agent by redox modulation, J. Med. Chem., 2015, 58, 7874-7880.
53. A. F. Santidrian, A. Matsuno-Yagi, M. Ritland, B. B. Seo, S. E. LeBoeuf, L. J. Gay, T. Yagi
and B. Felding-Habermann, Mitochondrial complex I activity and NAD(+)/NADH balance
regulate breast cancer progression, J. Clin. Invest., 2013, 123, 1068-1081.
54. S. Betanzos-Lara, Z. Liu, A. Habtemariam, A. M. Pizarro, B. Qamar and P. J. Sadler,
Organometallic ruthenium and iridium transfer-hydrogenation catalysts using coenzyme
NADH as a cofactor, Angew. Chem. Int. Ed., 2012, 51, 3897-3900.
55. Z. Liu, R. J. Deeth, J. S. Butler, A. Habtemariam, M. E. Newton and P. J. Sadler, Reduction of
quinones by NADH catalyzed by organoiridium complexes, Angew. Chem. Int. Ed., 2013, 52,
4194-4197.
56. F. Wang, M. Chen, Y. Lin, H. Zhang, C. Tan, L. Ji and Z. Mao, Dual functions of
cyclometalated iridium(III) complexes: Antimetastasis and lysosome-damaged
photodynamic therapy, ACS Appl. Mater. Interfaces, 2017, 9, 42471-42481.
57. Q. He and J. Shi, MSN anti-cancer nanomedicines: Chemotherapy enhancement, overcoming
of drug resistance, and metastasis inhibition, Adv. Mater., 2014, 26, 391-411.
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Graphical Abstract
Fluorescent iridium (III) coumarin-salicylaldehyde Schiff base antitumor compounds
change the ROS and ΔΨm, induce lysosomal damage, lead to apoptosis.