Synthesis and Antitumor Activity Evaluation of Cyclometalated 2H-Indazole Ruthenium(II) and Iridium(III) Complexes

Article abstract of DOI:10.1002/cplu.202000516

In this work, a series of novel C?N cyclometalated 2H-indazole Ru(II) and Ir(III) complexes were synthesized, wherein chelating ligands with substituents like H, and isopropyl group in the R₄ position of the phenyl ring of the 2H-indazole chela

Full text of DOI:10.1002/cplu.202000516

A Multidisciplinary Journal Centering on Chemistry
Accepted Article
Title: Design, Synthesis and Antitumor Activity Evaluation of Novel
Cyclometalated 2H-Indazole Ru(II) and Ir(III) Complexes
Authors: Kaushik Chanda, Ramdas Nishanth Rao, Rajeeva Lochana
Panchangam, Venkatraman Manickam, and Musuvathi Motilal
Balamurali
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0
1/2020

ChemPlusChem
10.1002/cplu.202000516
Synthesis and Antitumor Activity Evaluation of Cyclometalated 2H-Indazole
Ruthenium(II) and Iridium(III) Complexes
[a]
[b]
[b]
Ramdas Nishanth Rao, Rajeeva Lochana Panchangam, Venkatraman Manickam,
[
c]
[a]
Musuvathi Motilal Balamurali,* and Kaushik Chanda*
[
a]
[
+
Ramdas Nishanth Rao, Prof. Kaushik Chanda Department of Chemistry, School of Advanced
Science, Vellore Institute of Technology, Vellore-632014, India.
b]
+
Rajeeva Lochana Panchangam, Prof. Venkatraman Manickam Department of Biosciences,
School of Biosciences and Technology, Vellore Institute of Technology, Vellore-632014, India.
[c]
Prof. Musuvathi Motilal Balamurali Chemistry Division, School of Advanced Sciences,
Vellore Institute of Technology, Chennai Campus, Chennai-600127, India
[+]
These authors contributed equally to this work.
Email: chandakaushik1@gmail.com
mmbala@gmail.com
Abstract: In this work, a series of novel C-N cyclometalated 2H-indazole Ru(II) and Ir(III)
complexes were synthesized wherein chelating ligands with substituents like H, and isopropyl
group in the R position of the phenyl ring of 2H-indazole chelating ligand are present. The
4
cytotoxicity of Ru(II) and Ir(III) complexes has been evaluated against different human cancer
cell lines (HeLa, MCF-7, and A549) in a concentration-dependent manner. The new iridium
complex with isopropyl substituent in the phenyl ring of 2H-indazole moiety showed good
cytotoxic activity against MCF-7 cells with IC50 value 3.5 µM. The complex also exhibited
cytotoxicity comparable to that of cisplatin. The ability of this compound inducing apoptosis was
tested by nuclear condensation, cell membrane blebbing and caspase 3/7 activation. Further, this
novel iridium complex is capable of inhibiting cancer cell migration when tested in MCF-7 cell
line. Subsequently, we have studied the DNA binding and protein binding ability of the newly
synthesized iridium complex.
1
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10.1002/cplu.202000516
Graphical Abstract
2
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Introduction
[
1-3]
[4,5]
The role of metal complexes in biological
and medical sciences
has been well established
for the past several decades. With the emergence of metallo-therapeutics several achievements
[
6,7]
were made in the field of medical research.
Interestingly, in the past two decades therapeutic
[
8,9]
stimuli-responsive (pH, enzyme, light, temperature, etc) metallo-drugs were reported.
Despite
the advancements in drug research and understanding of the complex nature, cancer still remains
as the major cause of death in human population. Though the serendipitous discovery of cisplatin
and platinum containing drugs like carboplatin, and oxaliplatin lead to the development of
[
10]
[11]
varieties of elemental anticancer drugs, they lack the much needed target specificity. Novel
approaches towards enhancing the efficacy of therapeutic treatments are being continuously
developed. Recent research outcomes on metal complexes have provided the unique ability to
alter pharmacology, efficacy and reduced side effects as cancer drugs. New therapeutic strategies
with these metal complexes lead to the bottom up approach for the rational design of novel
anticancer agents which can enhance the efficacy and tolerability. In tandem with several
platinum based metallodrugs, significant efforts have been dedicated to develop Ru, Ir, Os, Re,
[
12]
and Rh based metallodrugs. Several reports have shown that the in vivo antimetastatic activity
[
13]
of ruthenium complexes have paved way for the development of more nontoxic ruthenium(II)
[
14]
and iridium(III) complexes as angiogenesis inhibitors.
The anticancer activity can be
modulated uniquely through structural modifications in ligands around the metal center and has
been confirmed through the recently developed Pt and Ru anticancer compounds. Very good
anticancer activity was observed with the rationally designed cyclometalated Ru(II) and Ir(III)
[
15]
complexes against several cancer cell lines.
Recently, with the emergence of synthesised
small drug-like heterocyclic molecules and their use as potential chelating ligands for the
3
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10.1002/cplu.202000516
synthesis of metal complexes is well recognized as a tool for the generation of promising
metallodrugs with substantial anticancer property. The design concept of the currently
synthesized complexes has originated from the recognition of the biological role of core 2H-
[
16]
[17]
[18]
indazole moiety having antitumor,
HIV-protease inhibition,
anti-inflammatory,
and
[
19]
modulatory role on estrogen receptors. Interestingly, the 2H-indazole moiety ligands possess
the ability to target the drug resistant, epidermal growth factor receptor (EGFR) over expressing
[
20]
tumors, and are found in many best selling drugs such as pazopanib, and niraparib. Moreover,
2
H-indazole ligands also owe photo-physical property which makes them a promising candidate
[
21]
with inherent potential for theranostic applications.
Further, there are several reports in the
[
22]
[23]
literature on the application of Ru (II) and Ir (III) complexes in photodynamic therapies for
the treatment of cancer or certain types of infections. In addition, the core 2H-indazole can be
easily modified for the synthesis of diversely fused heterocycles with significant intellectual
appeals.
Figure 1. The design of Novel Cyclometalated Indazole Ru(II) and Ir(III) Complexes
The figure 1 shows the readily available C-N site in indazole moiety for cyclometalation to
construct organometallic complexes. Gratifyingly, the easy derivatization of the phenyl ring on
both sides of the C-N functionality can be achieved for the structure activity relationship (SAR).
In continuation of our research to develop newer synthetic methodologies for the construction of
4
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10.1002/cplu.202000516
[
24]
heterocycles and subsequent bioactivity evaluation,
we propose to synthesize an array of
organometallic ruthenium and iridium complexes having 2H-indazole as major backbone. With
the aim of increasing the available lead metallo-drugs having cost-effective, wide spectrum anti-
cancer therapeutic roles, we believe that organometallic complexes with these polyvalent ligands
are completely innovative in the bibliography, and the observed results from anticancer studies
leaves a new footprint for designing metal drugs with diverse functions.
Results and Discussion
At the onset of our study, the synthesis of substituted 2H-indazole 3 ligand which could be
achieved by the simultaneous formation of C-N, and N-N bonds through the one-pot synthetic
transformation. The synthetic methodology was initiated by the Cu
catalyzed cyclization reaction between 2-bromobenzaldehyde 1, NaN and aromatic amine 2 and
,10-phenanthroline as ligand in DMSO solvent to obtain substituted 2H-indazole 3 derivatives
2
O rhombic dodecahedra
3
1
[
24c]
as shown in Scheme 1.
Scheme 1. Cu(I) catalyzed synthesis of key 2H-indazole ligands.
1
13
The synthesized ligands 3 were characterized by H, C NMR, Mass and IR spectroscopy. With
the key ligands in our hands, we attempted to synthesize the organometallic complexes with
ruthenium and iridium metals. The synthesis of ruthenium and iridium cyclometalated complexes
6
5
using [RuCl
2
(η -p-cymene)]
2
and [(η -C
5
Me
5
)IrCl
2
2
] with key 2H-indazole ligands 3 is shown in
Scheme 2. The synthesis of ruthenium cyclometalated complexes 4a-b was achieved by treating
5
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6
ligands 3a-b with [RuCl
2
(η -p-cymene)]
2
and sodium acetate in dichloromethane at room
temperature for 20 h in 72-75% yield in Scheme 2.
Scheme 2. Synthesis of 2H-indazole contained cyclometalated Ru(II) and Ir(III) complexes.
5
Similarly, half sandwich iridium(III) complexes 5a-b were obtained from [(η -C
5
Me
5
2 2
)IrCl ]
using similar synthetic conditions for 20 h in 82-85% yields. Accordingly, the table 1
demonstrated the series of complexes with yields. The structures of all complexes were
1
13
unequivocally confirmed by H, C NMR and ESI-MS while their high degree of purity was
determined by HRMS analysis.
Table1. List of synthesized cyclometalated complexes of Ru(II) and Ir(III).
Complex
R1
-H
Metal Complex
[(6-p-cymene)RuCl2]2
[(6-p-cymene)RuCl2]2
[(5-C Me )IrCl ]
Yield
72 %
75 %
82 %
85 %
4a
4b
5a
5b
-CH(CH3)2
-H
5
5
2 2
-CH(CH3)2
[(5-C Me )IrCl ]
5
5
2 2
6
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1
In the H NMR spectra of 4a-b, the disappearance of one aromatic proton and introduction of
four doublets at 5.1-5.9 ppm, a singlet at 2.0-2.1 ppm, and two doublets at 0.6-0.8 ppm for six
protons corresponding to p-cymene confirms the formation of 2H-indazole contained ruthenium
1
complexes. Likewise, for half-sandwich iridium (III) complexes 5a-b, H NMR spectrum
displayed a singlet at 1.7 ppm for fifteen protons corresponding to pentamethylcyclopentadienyl
(
C
5
Me ) moiety.
5
In order to evaluate any synthesized molecule as a potent drug candidate for it to exhibits its
functions under physiological conditions, it is always important to analyze the electronic
properties, environment dependent charge redistribution and dipolar interactions. The stock
solutions (5 mM) of all complexes 4a-4b, and 5a-5b were prepared in methanol. The working
concentration of all complexes was chosen as 5 µM in different solvents such as CH
2
Cl DMF,
2
ethyl acetate and methanol of varying polarity. The electronic absorption or emission spectrum in
water could not be recorded because of their poor solubility and the respective electronic
spectrum are depicted in Figure S1 (see supporting information). The absorption spectra were
recorded in all the above solvents in the range of 220 nm to 500 nm as shown in figure 2. The
long wavelength absorption band of 4a and 4b compounds was centered ~280 nm and 330 nm
and that of 5a and 5b compounds were centered ~280 nm. It can be observed from the absorption
spectrum that for all the complexes the π - π* transition band is in the range of ~250–360 nm and
the charge transfer band in the range of ~360–500 nm under aqueous environment.
7
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0
0
0
0
.020
.015
.010
.005
0.04
0.03
0.02
0.01
0.00
0.000
250
300
350
400
450
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
4
a
4b
0.10
0.08
0.06
0.04
0.02
0.00
0
.030
.025
.020
.015
.010
.005
0
0
0
0
0
0.000
250
300
350
400
450
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
5
a
5b
Figure 2. Absorption spectra depicting the observed solvatochromism of 4a-b and 5a-b in
different solvents - ●, CH Cl ; O, DMF; ▲, Ethylacetate and Δ, Methanol and ____ , Water.
2
2
The photophysical properties of the compounds in the excited state were investigated by steady
state fluorescence emission spectra by exciting at 300 nm. The concentration of the samples was
-1
kept identical at 5 µM throughout the experiments. A red shift of 347, 597, 450 and 677 cm
respectively in complexes 4a, 4b, 5a, and 5b, when solvent polarity is increased from
dichloromethane to dimethyl formamide. This could be due to the induced electronic charge
redistribution upon excitation, thereby increasing the dipolar nature of the complexes. Moreover,
8
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as the protic nature increases, the hydrogen bond forming capacity between the complex and the
solvents increases that could stabilize them further. The luminescent properties as evaluated from
the relative quantum yield were found to vary with the polarity of solvents. The values were
found to increase with increasing polarity and protic nature of the solvents. This could be due to
the fact that non polar interactions could induce non-radiative decay process as shown in figure
3
. The variations observed in the quantum yield in all the solvents are shown in table 2.
120
60
80
40
4
0
20
0
0
350
400
450
500
550
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)
4
a
4b
120
90
90
60
60
30
30
0
0
350
400
450
500
550
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)
5
a
5b
Figure 3. Fluorescence emission spectra depicting the observed solvatochromism of 4a – b and
5
a – b in different solvents - ●, CH
2
Cl
2
; O, DMF; ▲, Ethylacetate and Δ, Methanol and ____ ,
Water.
9
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Table 2. Absorption and fluorescence emission spectral characteristics of 4a-b and 5a-b
complexes in different solvents.
ꢁ
ꢀ
ꢃꢁ ꢂ(ꢄꢅꢆꢇꢈꢉꢊꢋꢇ
ꢌꢄ
ꢀ
[a]
Compound Solvents
)
(ꢍ )
ꢁꢂ ꢌꢄ
Ethylacetate
263 (sh), 275 (3.81), 286 (3.85), 322 (3.49), 368
sh)
386 (0.41)
(
CH
DMF
Methanol
2
Cl
2
262 (sh), 275 (3.81), 286 (3.85), 324 (3.38), 368 (sh) 377 (0.09)
263 (sh), 275 (3.78), 286 (3.82), 323 (3.49), 367 (sh) 382 (0.22)
262 (sh), 274 (3.82), 285 (3.83), 318 (3.53), 368 (sh) 396 (0.53)
4
a
Water (pH 7.0)
Ethylacetate
273, 286 (-)
-
265 (sh), 280 (3.53), 288 (sh), 321 (sh), 362 (sh)
359 (0.20)
CH Cl
2 2
261 (sh), 280 (3.33), 292 (3.32), 330 (2.83), 375 (sh) 362 (0.01)
4b
5a
5b
DMF
Methanol
Water (pH 7.0)
Ethylacetate
266 (sh), 281 (3.49), 290 (sh), 320 (sh), 365 (sh)
252 (3.32), 282 (sh), 288 (3.38), 320 (sh), 368 (sh)
282 (-)
285 (4.15), 339 (sh)
276 (sh), 302 (4.10)
288 (4.13), 339 (sh)
259 (sh), 273 (4.18), 293 (4.19)
300 (-)
292 (3.59), 308 (sh), 339 (sh), 374 (sh)
280 (sh), 302 (3.29)
285 (sh), 302 (3.37), 340 (sh), 375 (sh)
266 (sh), 282 (sh), 296 (3.75)
-
370 (0.10)
367 (0.71)
-
349 (0.15)
362 (0.14)
368 (0.14)
367 (0.16)
-
349 (0.35)
360 (0.24)
369 (0.27)
351 (0.41)
-
2
CH Cl
2
DMF
Methanol
Water (pH 7.0)
Ethylacetate
2
CH Cl
2
DMF
Methanol
Water (pH 7.0)
[
a] Quantum yield were determined using quinine sulphate as the standard (ꢍ = 0.55 in water)
ꢎꢏ
Subsequently, we turned our attention for the potential use of these complexes 4a-b, and 5a-b as
therapeutic agents which can be assessed by their stability under ambient biological conditions of
cells. Complexes 4a-b, and 5a-b were investigated for aqueous stability in water by UV-Vis
spectroscopy for a period of 24 hours. Significant hypochromic shift was observed for 4a and 4b
while 5a and 5b displayed slight hypochromic shift as shown in figure 4. As reported for other
[
25]
ruthenium complexes, the observed shift could be due to the formation of aqua complex.
10
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4
4
4
4
a - 0 hours
a + NaCl - 0 hours
a - 24 hours
4
b - 0 hours
0
0
0
0
.12
4
4
4
b + NaCl - 0 hours
b - 24 hours
b + NaCl - 24 hours
0
0
.04
.02
a + NaCl - 24 hours
.08
.04
.00
0.00
2
50
300
350
400
450
2
50
300
350
400
450
Wavelength (nm)
Wavelength (nm)
4a
4b
5
5
5
5
a - 0 hours
a + NaCl - 0 hours
a - 24 hours
5b - 0 hours
5b + NaCl - 0 hours
5b - 24 hours
0
.3
.2
.1
.0
0
0
0
.08
a + NaCl - 24 hours
5b + NaCl - 24 hours
0
0
0
.04
.00
2
50
300
350
400
450
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
5a
5b
Figure 4: Stability of complexes 4a, 4b, 5a and 5b in presence and absence of 150 mM NaCl in
0 mM phosphate buffer, pH 7.2 and 5 % DMSO in a time interval of 0 and 24 hours.
1
The variation in the absorption spectra was also monitored in the presence of 1 mM GSH in 10
mM phosphate buffer, pH 7.2, under similar conditions. Significant changes were observed in
the absorption spectra of 4a, 4b, 5a and 5b after an incubation period of 24 hours. Though the
above results suggest that the complexes bind effectively with GSH, further investigations on its
[
26]
ability to interact with glutathione transferases will shed light on the above discussions. The
small hypochromic shift observed after 24 hours indicates that the complexes are stabilized by its
ability to bind to GSH. The influence of NaCl on the above observed hypochromism was also
investigated. In the absence of GSH, NaCl displayed negligible effect, whereas in the presence of
11
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10.1002/cplu.202000516
GSH, the hypochromism was suppressed significantly in the presence of 150 Mm NaCl for 5a
and 5b specifically after a stipulated time period as mentioned in the figure 4 and figure 5. This
indicates that under physiological conditions of high chloride ion concentrations the complexes
5
a and 5b maintain better stability when compared with 4a and 4b in the presence of GSH as
indicated in figure 5. Therefore under physiological conditions where the concentration of
chloride ions is significantly low, these complexes can react with intracellular nucleophiles and
hence can be considered as a scope for potent therapeutic agents.
0
0
0
.20
.15
.10
0
0
0
0
.075
.050
.025
.000
0.05
0
.00
250
2
50
300
350
400
450
300
350
400
450
Wavelength (nm)
Wavelength (nm)
4a
4b
0
0
0
0
.3
.2
.1
.0
0
0
0
.3
.2
.1
0.0
2
80
320
360
400
280
320
360
400
Wavelength (nm)
Wavelength (nm)
5a
5b
12
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Figure 5: Stability of complexes 4a, 4b, 5a and 5b in presence and absence of 150 mM NaCl in
0 mM phosphate buffer, pH 7.2 with 1 mM GSH and 5 % DMSO in a time interval of 0 and 24
hours. _____ 0 hrs, 0 mM NaCl; - - - - 0 hrs, 150 mM NaCl; …… 24 hrs, 0 mM NaCl and ___..
4 hrs, 150 mM NaCl
1
2
After synthesizing metal complexes 4a-b, and 5a-b, it is necessary to determine the cellular
uptake behaviors of metal complexes which have been reported to be affected by many factors
[27]
such as lipophilicity, molecular size, and the substituent group.
Lipophilicity is basically
referred to as the partition coefficient of the compound in n-octanol/water (Po/w). The
[
28]
lipophilicity of metal complexes as calculated by the classical shake flask method
indicates
the following trend: 5b > 5a > 4a > 4b. Among the iridium and ruthenium complexes, the
iridium complexes 5b and 5a were moderately found to be more lipophilic. The possible reason
for this observation could be the charge localization and the polarizability induced by the
chelating ligands attached to the metal center. Increased polarizability could lead to increased
hydrophilicity. Therefore, it could be inferred that ruthenium complexes 4a-b are more polarized
than iridium complexes 5a-b. The in vitro cytotoxicity of synthesized metal complexes 4a-b, 5a-
b and cisplatin was assessed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide
(
(
MTT) assay against different human cancer cell lines representing of human cervical cancer
HeLa), human breast adenocarcinoma (MCF-7), and human lung carcinoma (A549) respectively
in a concentration-dependent manner and the values were compared when the ligands alone were
used as control. The results of IC50 values are summarized in Table 3. Commercially available
metallo drug cisplatin was used as a positive control. Among all the metal complexes evaluated,
the compound 5b showed good cytotoxicity against the tested cancer cell lines with the IC50
concentrations of 3.9 µM (HeLa), 4.5 µM (A549), 3.58 µM (MCF-7). Interestingly from Table 3,
13
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it is also clear the compound 5b showed stronger cytotoxicity at lower concentrations when
compared to positive control cisplatin. Our earlier observations, as discussed above have shown
that 5b has higher stability in aqueous medium and higher lipophilicity compared to other
complexes. Not limited to both or either of these could be responsible for the observed
cytotoxicity. Moreover in order to evaluate the cytotoxicity of 5b in the presence of light,
phototoxicity experiment was carried out for 5b against MCF-7 cell lines (see supporting
information). The results revealed that the variation in the values of % cell viability was
negligible when cytotoxicity was tested in light (IC50 = 3.55 µM) and dark (3.62 µM). Based on
the above results, compound 5b with an effective IC50 value of 3.5 µM was considered for
further biological characterization using MCF-7 cell lines.
Table 3. Cytotoxicity of ligands and metal complexes 4a-b, and 5a-b expressed as IC50 values
[
a]
[
µM] at 24 h.
[
b]
[c]
[d]
Compound
HeLa
A549
MCF-7
3
3
4
4
5
5
a
b
a
b
a
b
94.6 ± 0.24
33.4 ± 1.09
> 100
>100
> 100
35.3 ± 0.60
54.6 ± 0.90
21.9 ± 1.05
10.7 ± 0.11
4.5 ± 0.05
7.0 ± 0.69
32.9 ± 0.81
36.4 ± 1.05
8.2 ± 0.42
8.8 ± 0.50
3.5 ± 0.05
10.0 ± 1.00
8.9 ± 0.43
6.0 ± 0.79
3.9 ± 0.36
3.5 ± 0.48
Cisplatin
[a] 50% inhibitory concentration values are an average of three individual experiments.
[
b] Cervical cancer. [c] Lung cancer. [d] Breast cancer.
Further, to establish the cytotoxicity of iridium complex 5b using cellular morphological
changes, MCF-7 cell lines were incubated with 3.5 µM of compound 5b for 24 h. The
microscopic results indicated clear morphological alterations with the formation of rounded cells,
with apoptotic morphology and decreased cell density due to cell death and the same is depicted
in figure 6.
14
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Figure 6. Iridium complex 5b induced morphological changes in MCF-7 cell lines. (a) MCF-7
cells without treatment showing healthy morphology, (b) MCF-7 cells treated with complex 5b
at 3.5 µM showing altered morphology.
It is well established that cancer cells have the tendency for uncontrolled cell growth and
proliferation, which results in the formation of colony like intensive growth under in vitro
[
29]
growth set up.
To establish the anti-proliferative effects of complex 5b, we evaluated its
interfering effect on colony formation in MCF-7 cells. The results showed that complex 5b
inhibited colony formation and significantly reduced the colony number and size in MCF-7 cells
at IC50 value as shown in figure 7A(b) whereas control cells developed large number of colonies
characterizing the proliferating cancerous cells as depicted in figure 7A(a).
15
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Figure 7. Iridium complex 5b inhibited cell survival and colony formation in MCF-7 cell lines.
A) The colony formation of (a) Control, (b) complex 5b at 3.5 µM concentration, B) Histogram
representing the corresponding quantification of colony numbers against concentration. All the
images are representative of three independent experiments and data reported as mean ± SD;
significant at the *p < 0.005.
Apoptosis, a programmed cell death is the major cell death principle which is a preferred mode
for any cancer treatment options. Activation of executive enzymes, caspase-3 and -7, is a critical
step during any apoptotic cell death process. The activated caspases will act on many
downstream targets like nuclear, mitochondrial and cytoskeletal proteins which lead to ordered
[
30]
dismantling of cancer cells.
To check the effect of iridium complex 5b on activation of
caspase 3/7 in MCF-7 cell lines, we used an active caspase 3/7 reactive probe. The nucleic acid
binding dye present in this reagent emits green fluorescence upon binding to cellular DNA, this
process is inhibited in healthy cells through the inhibitory DEVD peptide. However, in the
apoptotic cells, the activated caspase 3/7 cleave the inhibitory DEVD peptide and allow the
fluorescent dye to bind DNA and emit green fluorescence. With our experiments, it is clearly
evident that there is no green fluorescence observed in control cells as shown in figure 8a,
whereas cells in figure 8b showed bright green fluorescence upon treatment with complex 5b
confirming the apoptotically activated caspase 3/7. Further, to observe the nuclear level
morphological changes associated with apoptosis, like nuclear fragmentation, chromatin
condensation were analyzed by DAPI staining in iridium complex 5b treated MCF-7 cells. The
DAPI is a nucleus specific fluorescent dye which efficiently binds to nucleic acid. In the
apoptotic cells due to DNA fragmentation and chromatin condensation, the DAPI will emit
bright blue fluorescence while non-apoptotic cells appear with regular blue color nucleus. Figure
16
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8
c demonstrated that control cells do not exhibit any apoptotic cell morphology and the nucleus
was intact, whereas the figure 8d showed MCF-7 cells treated with 3.5 µM of iridium complex
b exhibited typical nuclear fragmentation and chromatin condensation, which are observed in
5
majority of the cells and are represented by arrows. Phenotypically, cellular membrane blebbing
is one of the distinct features of apoptosis. During apoptosis due to the changes in cellular
cytoskeleton, the membrane bulges outwards as blebs, and these blebs later separates as
apoptotic bodies. To check the formation of apoptotic blebs, and confirm the dying morphology,
MCF-7 cells upon treatment with complex 5b were observed under the scanning electron
microscope. The figure 8f showed changes in the cell surface with the formation of apoptotic
blebs after treatment with IC50 concentration of complex 5b whereas in control cells no apoptotic
morphology is seen and are with typical smooth surface as in figure 8e.
17
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Figure 8. Iridium complex 5b induced cellular apoptosis in MCF-7 cell lines: (a) & (b)
Representing the caspase 3/7 activity, (c) & (d) Representing nuclear fragmentation by DAPI
staining. The arrow marks indicate the fragmented nuclei induced by complex 5b, (e) & (f)
indicates the SEM images of MCF-7 cells showing apoptotic morphology. The images are
representative of three independent experiments.
It is well known that the mortality risk of cancer patients increases mainly after spreading of
cancer cells from the primary tumor site to secondary sites. Cellular migration and invasion are
the crucial steps of metastasis, which play the important role of spreading cancer to distant
[
31]
organs. To check the ability of iridium complex 5b to inhibit in vitro cell migration, wound
healing assay was performed. The results with MCF-7 cells demonstrated that the complex 5b,
when treated at its IC50 concentration
MCF-7 cells as depicted in figure 9A(d) when compared to the control cells as shown in figure
A(c).
,
have significantly inhibited the migratory potential of
9
Figure 9. Iridium complex 5b inhibited the migration of MCF-7 cell lines: A) Representative
th
images of iridium complex 5b inhibiting migration of MCF-7 cells. (a & b) 0 -hour images of
th
control and complex 5b treatment groups. (c & d) 24 -hour images of control and complex 5b
treatment groups. The lines represent the borders of the wound. B) graph representing the
18
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10.1002/cplu.202000516
percentage of wound closure at 24 h. Data represented is reported as mean ± SD with
significance at the **p < 0.005 versus control.
It is always important to study the interaction of anticancer metallodrugs with biomolecules such
as DNA, and proteins as they play a vital role in the biodistribution, mechanism of action, and
[
32]
enhanced toxicity.
investigation on its interaction with calf thymus DNA (ct-DNA) and human serum albumin
HSA). The binding interaction of complexes with ct-DNA was investigated by following a
Based on the above observations, herein we have chosen 5b for further
(
competitive binding experiment in the presence of ethidium bromide and the mode of interaction
was evaluated. Here eithidium bromide (EtBr) was not only used as a fluorescent probe but also
as a competing agent to bind ct-DNA in the presence of complex 5b. EtBr a planar cationic dye
with phenanthridine ring is used as a fluorescence probe for native DNA. The intense
fluorescence emission observed from EtBr in the presence of ct-DNA is due to strong interaction
between adjacent DNA base pairs via intercalative binding. The remarkable decrease in
fluorescence intensity of EtBr-ct-DNA with increasing concentration (0-100 µM) of the
quencher 5b was observed and is shown in Figure 10. The fluorescence intensity of the EtBr
bound ct-DNA was quenched as its interaction with the 5b replace the DNA bound to EtBr. The
quenching constants of metal complexes were analyzed by following Stern-Volmer equation:
I
0
/ I = 1+Ksv [Q]………………………………….(1)
and I are the emission intensities in the absence and presence of quencher,
respectively. Ksv is the linear Stern-Volmer quenching constant, and [Q] is the
concentration of quencher in equation 1. The Ksv value was obtained from slope of I
Here I
0
0
/
I vs. [Q]. The quenching plot depicts that the quenching of EtBr bound to ct-DNA by
metal complexes are in good agreement with the Stern-Volmer equation. The quenching
19
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10.1002/cplu.202000516
-
1
constant values (Ksv) were found to be 13.2 µM . The apparent binding constant (Kapp
)
values for metal complexes were obtained from the following equation 2:
K
EB [EB] = Kapp [complex]…………………….(2)
7
-1
Here KEB = 1.0 × 10 M , [EtBr] = 5 µM and [complex] is the complex concentration at
5
0 % reduction in the fluorescence intensity of EtBr. The quenching constant Kapp value
-
1
of 5b was found to be 25.3 µM . These results suggest that the complex 5b bind with ct-
DNA via intercalation mode and binding constant values are less while comparing with
other classical intercalators.
Further to study the nature of interaction between HSA and the metal complexes,
electronic spectrum of 5b was recorded. HSA is the highly abundant blood serum proteins
which serve as carrier for a wide range of ligands, drugs, and metal ions. The quenching
effect was observed by titrating fixed amount of protein against varying concentration of
complex 5b. The decrease in the emission intensity of HSA with increasing amounts of
5
b indicates the type of interaction between protein and complex. As no shift in the
absorption spectra is observed, it indicates a static quenching rather than dynamic
quenching.
Mainly the presence of amino acids phenylalanine, tyrosine and tryptophan are the
responsible for the observed fluorescence in HSA. Out of these three, tryptophan plays a
major role in the intrinsic fluorescence of HSA. Fluorescence spectral titrations were
carried out in the wave length range of 285-500 nm upon excitation at 280 nm. The effect
of 5b on fluorescence spectra of protein are shown in Figure 11. The increasing
concentration of metal complexes (0 - 100 µM) shows a significant quenching of
20
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10.1002/cplu.202000516
fluorescence intensity at 345 nm, accompanied by a red shift. The observed shift could be
due to the binding of metal complexes with one of the active sites of the protein.
In order to investigate the nature of the binding site, competitive titration was performed
in the presence of 5b along with warfarin (specifically binds to Site I) and dancyl glycine
(
specifically binds to Site II). The quenching of HSA fluorescence in the presence of
warfarin indicates that 5b binds to site I of HSA and results are shown in Figure 11.
The fluorescence quenching constant values are described by the Stern-Volmer equation
as given above.
If small molecules bind independently to a set of equivalent sites on a macromolecule, the
equilibrium between free and bound molecules is represented by the Scatchard equation:
log [(I
is the binding constant of the complex with HSA and n is the number of binding
sites. Employing the plot of log [(I - I)/ I] versus log [Q], the number of binding sites (n)
and the binding constant (K ) values could be obtained. The quenching constant (K ),
binding constant (K ) and number of binding sites (n) for the interaction of the complexes
0
- I)/ I] = log K
b
+ n log [Q]
Here K
b
0
b
q
b
6
-1
6
-1
with HSA are 0.0377 x 10 M , 0.046 x 10 M , and 1 respectively. The results indicated
that in complex 5b, only one binding site is available for interaction with HSA.
21
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10.1002/cplu.202000516
0
.5
2
2
2
2
1
1
1
1
1
0
.6
c = 0.9716070497
KSV = 13292090.38
log K = 7.404505581
n = 1.040933842
r ² = 0.9955268946
.4
1
5
0
5
0
0.0
0.5
1.0
.2 r ² = 0.9954054288
.0
.8
.6
.4
.2
.0
.8
-
-
1
-1.5
-2.0
-
9.0
-8.5
-8.0
-7.5
-7.0
-6.5
0.0
3.0e-8
6.0e-8
9.0e-8
1.2e-7
5
40 560 580 600 620 640 660 680
log Q
[
5b] (M)
Wavelength (nm)
a
b
c
Figure 10. Fluorescence spectral titration showing the competitive quenching EtBr:DNA fluorescence by 5b. (b) Stern-
Volmer quenching plot for the titration of 5b against EtBr:DNA complex (c) Scatchard plot
22
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10.1002/cplu.202000516
5
4
3
2
1
60
40
20
0
c = 0.9753
Ksv 0.0377 M
r ² = 0.9912
n log KA = -1.3893
n = 1.0388
r ² = 0.9910
0
=
-1
-2
-3
1.50
0
-
-0.75
0.00
0.75
1.50
375
450
525
0
20
40
60
80
100
log[1/[complex]-[HSA](F -F)/F ]
Wavelength (nm)
[5b] (M)
0
0
a
b
c
0
6
4
2
0
150 M
0
0
0
350
400
450
500
550
Wavelength (nm)
d
Figure 11. (a) The binding of 5b with HSA as indicated by the fluorescence emission spectra. (b) The linear plot showing
the Stern-volmer quenching effect (c) The scatchard plot for binding of 5b with HSA. (d) Identification of binding site on
HSA by the competition reaction of 5b with warfarin specific for site I.
23
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The chemical nuclease activity of iridium complex 5b was investigated using pBR322 plasmid
DNA in 50 mM Tris-HCl buffer (pH = 7.2). Depending on the ability of the metal complexes to
cleave the supercoiled DNA, the ratio of the three forms (supercoiled, nicked circular and open
linear) will vary and can be identified from their electrophoretic mobility on agarose gel. Figure
1
2 depicts the results of agarose gel electrophoretic separations of various forms of pBR322
DNA in the presence of metal complex 5b. As shown in Figure 12, in the presence of complexes,
the supercoiled DNA is gradually converted into nicked circular and linear DNA depending on
the cleaving ability. In addition, the oxidative cleavage of DNA in the presence of hydrogen
peroxide is also studied by gel electrophoresis and is shown in Figure 12. It could be observed
that in presence of hydrogen peroxide, the cleavage ability of 5b was enhanced considerably as
compared with other complexes.
Figure 12. Agarose gel electrophoresis depicting the mobility of cleaved products of pBR322 by
complex 5b in the presence and absence of H
singlet oxygen radical scavenger). Lanes 1: DNA; 2: DNA + 5b; 3: DNA + 5b + H
5b + H + DMSO; 5: DNA + 5b + H + NaN ; 6: DNA + H ; 7: DNA + H
DMSO; 8: DNA + H + NaN
2
O
2
, DMSO (hydroxyl radical scavenger) and NaN
3
(
2
O
2
; 4: DNA
+
2
O
2
2
O
2
3
2
O
2
2
2
O +
2
O
2
3
.
In order to investigate the cleavage mechanisms the reaction was carried out in the presence and
absence of hydroxyl radical scavenger (DMSO) and singlet oxygen quencher (NaN ). Inhibition
3
24
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1
of DNA cleavage was observed in the present of NaN
3
indicating that O
2
is involved in the
reaction. At the same time, when hydroxyl radical scavenger, DMSO is used the nuclease
activity was significantly diminished indicating the involvement of the hydroxyl radical in the
cleavage process. According to the results mentioned in figure 12, we propose the hypothesis
that the complexes examined here may be capable of promoting DNA cleavage through an
oxidative DNA damage pathway, in which the active oxygen species involved in the reaction are
1
.
singlet oxygen, ( O
2
) and hydroxyl radical ( OH) at varying extents.
Conclusion
In conclusion, we have successfully synthesized a series of C-N cyclometalated 2H-indazole
Ru(II) and Ir(III) complexes with substituents such as H, and isopropyl group in the R position
4
of the phenyl ring of 2H-indazole chelating ligand. The Ru(II) and Ir(III) complexes exhibit good
anticancer activity against Hela, A549, and MCF-7 cancer cell lines. The new iridium complex
5
b with isopropyl substituent in the phenyl ring of 2H-indazole moiety showed good activity
against MCF-7 cells and show apoptosis with increased caspase-3/7 activity. Further, the
complex 5b is capable of inhibiting cancer cell migration in MCF-7 cell line. Subsequently, the
iridium complex 5b bind to DNA via intercalation mode and bind to HSA at sites I.
Experimental Section
Chemistry
General Methods
Unless otherwise indicated all common reagents and solvents were used as obtained from
1
13
commercial suppliers without further purification. H NMR (400 MHz) and C NMR (100
25
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10.1002/cplu.202000516
MHz) were recorded on a Bruker DRX400 spectrometer. Chemical shifts are reported in ppm
relative to the internal solvent peak. Coupling constants, J, are given in Hz. Multiplicities of
peaks are given as: d (doublet), m (multiplet), s (singlet), and t (triplet). Mass spectra were
recorded on a Perkin Elmer Calrus 600 GC-MS spectrometer. High resolution mass spectra
(
HRMS) were recorded in ESI mode using Thermo Exactive LC-MS mass spectrometer. UV/vis
spectroscopy was carried out on a UV-2550, Shimadzu Corporation, and Kyoto, Japan. The
fluorescence spectra were recorded on Hitachi F-7000 FL spectrophotometer. Solvents were
6
5
dried by the usual methods. [(η -p-cymene)RuCl
2
]
2
, [(η -C
5
Me
5
)IrCl ] , sodium salt of calf
2
2
thymus DNA, ethidium bromide (EB), human serum albumin (HSA) were obtained from Sigma-
Aldrich (Bangalore, India); pBR322 plasmid DNA used in the studies were obtained from
Sigma-Aldrich (Bangalore, India). The synthesis of metal complexes 4a-b, and 5a-b was carried
[
12f]
out from previously reported literature method with a slight modification.
General Procedure for the Synthesis of substituted 2H-indazole Ru(II) complexes 4a-b.
In a round bottomed flask equipped with magnetic bar under nitrogen atmosphere, 2-phenyl-2H-
indazole (1 mmol) 3a was dissolved in a freshly distilled dichloromethane solution (5mL).
Sodium acetate (1.2 mmol) was added in to the flask at room temperature with constant stirring
6
followed by the addition of [(η -p-cymene)RuCl
2
2
] (0.5 mmol). The reaction mixture was stirred
at room temperature for 20h and the progress of reaction was monitored by TLC. After
completion of the complex formation, diethyl ether (10 mL) was added in the mixture. Reaction
mixture was stirred for 10 min to precipitate out the product. The crystalline product was filtered
through a fritted funnel and dried well. The yellow colored ruthenium complex 4a was obtained
1
in good yield with 72% yield. R
f
= 0.25 (40%EtOAc/n-hexane); H NMR (400 MHz, CDCl
3
) δ
8
.43 (s, 1H), 8.23 (d, J = 7.2 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.4 Hz,1H), 7.47-
26
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7
.43 (m, 2H), 7.20-7.16 (m, 2H), 7.05 (dd, J = 6.8, 1.6 Hz, 1H), 5.93 (d, J = 5.8 Hz, 1H), 5.74
(
d, J = 5.8 Hz, 1H), 5.41 (d, J = 5.8 Hz, 1H), 5.17 (d, J = 5.8 Hz, 1H), 2.28-2.23 (m,1H), 2.11 (s,
1
3
3
1
8
H), 0.88 (d, J = 6.90 Hz, 3H), 0.72 (d, J = 6.90 Hz, 3H); C NMR (100MHz, CDCl ) δ 148.5,
3
41.1, 140.5, 128.0, 127.2, 123.4, 122.8, 121.5, 118.4, 115.9, 113.1, 102.3, 99.1, 89.5, 89.3, 82.4,
+
1.0, 30.7, 22.5, 21.7, 18.9; MS (ESI, MS) 429 (M-Cl) ; HRMS (ESI, m/z) calcd for
C
23
H
23
2
N Ru: m/z 429.0905; Found 429.0903.
Compound 4b was synthesized using similar procedure for synthesis of compound 4a. Yield:
1
7
5%; pale yellow solid; R
f
= 0.27 (40%EtOAc/n-hexane); H NMR (400 MHz, CDCl
3
) δ 8.34
(
s, 1H), 8.01 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 6.8 Hz, 1H),
.36-7.32 (m, 1H), 7.14 -7.10 (m, 1H), 6.89 (d, J = 8.0 Hz, 1H), 5.83 (d, J = 5.8 Hz, 1H), 5.69 (d,
J = 5.8 Hz, 1H), 5.29 (d, J = 5.8 Hz, 1H), 5.09 (d, J = 5.8 Hz, 1H), 2.97-2.90 (m,1H), 2.26-2.19
7
(
m, 1H), 2.03 (s, 3H), 1.29-1.26 (m, 6H), 0.85 (d, J = 6.90 Hz, 3H), 0.68 (d, J = 6.90 Hz, 3H);
1
3
C NMR (100MHz, CDCl ) δ 147.4, 146. 138.2, 137.7, 127.4, 126.8, 121.6, 120.3, 116.9, 114.9,
3
1
4
11.7, 101.1, 98.4, 88.7, 88.0, 80.9, 79.9, 33.2, 29.7, 28.6, 23.6, 22.9, 21.5, 20.6; MS (ESI, MS)
+
71 (M-Cl) ; HRMS (ESI, m/z) calcd for C26
H
29
2
N Ru: m/z 471.1374; Found 471.1341.
In a round bottomed flask equipped with magnetic bar under nitrogen atmosphere, 2-phenyl-2H-
indazole (1 mmol) 3a was dissolved in a freshly distilled dichloromethane solution (5mL).
Sodium acetate (1.2 mmol) was added in to the flask at room temperature with constant stirring
5
followed by the addition of [(η -C
5
Me
5
)IrCl
2
2
] (0.5 mmol). The reaction mixture was stirred at
room temperature for 20 h and the progress of reaction was monitored by TLC. After completion
of the complex formation, dichloromethane was distilled under reduced pressure and diethyl
ether (10 mL) was added in the mixture. Reaction mixture was stirred for 10 min to precipitate
out the product. The crystalline product was filtered through a fritted funnel and dried well. The
27
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orange colored iridium complex 5a was obtained in good yield with 82% yield. R
f
= 0.26 (40%
) δ 8.37 (s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.72-
.66 (m, 2H), 7.52 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.20-7.16 (m, 2H), 7.07 (dd, J =
1
EtOAc/n-hexane); H NMR (400 MHz, CDCl
3
7
8
1
1
3
.0, 1.2 Hz, 1H), 1.76 (s, 15H); C NMR (100MHz, CDCl
3
) δ 147.7, 146.9, 136.9, 128.4, 128.3,
+
22.9, 122.7, 121.6, 119.1, 115.2, 112.4, 88.5, 9.6; MS (ESI, MS) 521 (M-Cl) ; HRMS (ESI,
m/z) calcd for C23
H
25ClIrN : m/z 557.1336; Found 557.1349.
2
Compound 5b was synthesized using similar procedure for synthesis of compound 5a. Yield:
1
8
1
7
5%; Pale yellow solid; R
f
= 0.34 (40%EtOAc/n-hexane); H NMR (400 MHz, CDCl
3
) δ 8.69 (s,
H), 7.72 (d, J = 7.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.29-7.27 (m, 2H), 7.19-7.15 (m, 1H),
1
3
.05 (dd, J = 7.6, 1.2 Hz, 1H), 3.02-2.95 (m, 1H), 1.4 (s, 15H), 1.30 (d, J = 7.2 Hz, 6H); C
NMR (100MHz, CDCl
3
) δ 148.4, 147.5, 146.7, 140.3, 135.1, 128.1, 122.5, 121.5, 118.6, 115.1,
+
1
12.1, 88.4, 34.1, 24.7, 23.7, 9.6; MS (ESI, MS) 564 (M-Cl) ; HRMS (ESI, m/z) calcd for
C
26
H
31IrN : m/z 564.2116; Found 564.2114.
2
Biology
Chemicals and reagents
Tissue culture plates, Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS),
trypsin-EDTA, and all the other cell culture products were purchased from Himedia (Mumbai,
India). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased
from Himedia (Mumbai,India). Cisplatin was purchased from Sigma Aldrich (Bangalore, India),
Cell Event Caspase-3/7 Green Ready Probes Reagent was purchased from Thermo Fisher
scientific (Bangalore, India).
Cell lines and cell culture conditions
MCF-7 (human breast adenocarcinoma cells), HeLa (human cervical cancer cells), A549 (human
lung carcinoma) cell lines were obtained from National center for cell sciences (NCCS) Pune,
28
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10.1002/cplu.202000516
India. All the cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL
penicillin, 100 µg/ml streptomycin and 2.5 µg/ml Amphotericin B. Cell were maintained in a
humidified incubator at 37 °C with 5% CO atmosphere.
2
Cell Viability Assay
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to
check the cytotoxicity of metal complexes on three different cancer cell lines MCF-7, A549, and
HeLa. Briefly, 5000 cell/well were seeded in 96 well plates and incubated for 24 h, and then
treated with different concentrations of complexes 4a-b, and 5a-b for 24h. The spent medium
was removed and cells were incubated with 25 µl of MTT solution (5mg/ml in 1XPBS) for 4 h.
At the end of incubation purple-colored formazan formed was dissolved by adding 100 µl of
DMSO. The absorbance was measured at 490 nm by using ELISA plate reader (BioTek-
ELx800) and percentage viability calculated by plotting percentage viability against the
concentration. The final concentration of DMSO does not exceed more than 0.25% in all the
experiments.
Colony formation assay
Colony formation assay was performed to analyze the anti-proliferative effect of compound 5b
on MCF-7 cells. Cells were seeded at a density of 1000 cells/well in 6 well tissue culture plates
and left for attachment at 37 °C. Then cells were treated with 3.5 µM of 5b and control cells
were left without treatment. Cells were allowed to form colonies for 14 days, spent media was
removed and cells were washed thrice with 1X PBS. Colonies were fixed with 4%
paraformaldehyde for 10 min and stained with 0.5% crystal violet for 20 min. The number of
colonies was counted both in the treated and untreated samples.
29
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