Synthesis and in vitro biological evaluation of three 4′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine iridium(III) complexes as new telomerase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.10.035

There iridium(III) complexes, [Ir(3-MeO-Phtpy)Cl₃] (1), [Ir(2-MeO-Phtpy)Cl₃] (2) and [Ir(4-MeO-Phtpy)Cl₃] (3) with 4′-(3-methoxyphenyl)-2,2′:6′,2″-terpyridine (3-MeO-Phtpy), 4′-(2-methoxyphenyl)-2,2′:6′,2″-terpyridine (2-M

Full text of DOI:10.1016/j.ejmech.2017.10.035

Accepted Manuscript
Synthesis and in vitro biological evaluation of three 4′-(4-methoxyphenyl)-2,2′:6′,2″-
terpyridine iridium(III) complexes as new telomerase inhibitors
Qi-Pin Qin, Ting Meng, Ming-Xiong Tan, Yan-Cheng Liu, Xu-Jian Luo, Bi-Qun Zou,
Hong Liang
PII:
S0223-5234(17)30827-9
10.1016/j.ejmech.2017.10.035
EJMECH 9827
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 July 2017
Revised Date: 29 September 2017
Accepted Date: 12 October 2017
Please cite this article as: Q.-P. Qin, T. Meng, M.-X. Tan, Y.-C. Liu, X.-J. Luo, B.-Q. Zou, H. Liang,
Synthesis and in vitro biological evaluation of three 4′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine iridium(III)
complexes as new telomerase inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.035.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and in vitro biological evaluation of three
4
′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine
iridium(III)
complexes as new telomerase inhibitors
a,c,1
a,c,1
a,c,*
c
Qi-Pin Qin
, Ting Meng
, Ming-Xiong Tan
, Yan-Cheng Liu , Xu-Jian Luo
a,c,*
*,b
c
,
Bi-Qun Zou , Hong Liang
1
–3 directly targeted c-myc promoter elements and inhibited the telomerase activity.
In addition, 1–3 may trigger cell apoptosis via a mitochondrial dysfunction pathway.

ACCEPTED MANUSCRIPT
Synthesis and in vitro biological evaluation of three
4
′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine
iridium(III)
complexes as new telomerase inhibitors
a,c,1
a,c,1
a,c,*
c
Qi-Pin Qin
, Ting Meng
, Ming-Xiong Tan
, Yan-Cheng Liu , Xu-Jian Luo
a,c,*
*,b
c
,
Bi-Qun Zou , Hong Liang
a
Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, College of
Chemistry and Food Science, Yulin Normal University, 1303 Jiaoyudong Road, Yulin 537000, PR
China.
b
Department of Chemistry, Guilin Normal College, 15 Xinyi Road, Gulin 541001, PR China.
c
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road, Guilin 541004,
PR China.
∗
Correspondence to:
E-mail: mxtan2018@126.com (M.-X. Tan), 2006315llxxjj@163.com (X.-J. Luo),
zoubiqun@163.com (B.-Q. Zou)
Tel: +86-775-2623650
1
These authors contributed equally to this work.
1

ACCEPTED MANUSCRIPT
Abstract
There iridium(III) complexes, [Ir(3-MeO-Phtpy)Cl ] (1), [Ir(2-MeO-Phtpy)Cl ] (2)
3
3
and [Ir(4-MeO-Phtpy)Cl ] (3) with 4′-(3-methoxyphenyl)-2,2′:6′,2″-terpyridine
3
(3-MeO-Phtpy), 4′-(2-methoxyphenyl)-2,2′:6′,2″-terpyridine (2-MeO-Phtpy) and
4
′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine (4-MeO-Phtpy) as ligands, respectively,
were synthesized and evaluated for their antiproliferative activities. In these
complexes, the iridium(III) center adopts a six-coordinate distorted octahedral
geometry. Among them, complex 1 exhibited the most potent activity, with IC50
values of 3.19−27.77 µM against four cancer cell lines (BEL-7404, Hep-G2,
NCI-H460 and MGC80-3 cells). Cellular mechanism studies suggested that
complexes 1–3 directly targeted c-myc promoter elements and inhibited the
telomerase activity. In addition, complexes 1–3 may trigger cell apoptosis via a
mitochondrial dysfunction pathway. We postulated that the difference in the in vitro
antitumor activities of complexes 1–3 is mainly dependent on the position of the
methoxy group on the phenyl ring of the iridium ligand.
Keywords
4
′-substituted-2,2′:6′,2′′-terpyridine, iridium(III) complexes, cell apoptosis, antitumor
activity, telomerase activity
2

ACCEPTED MANUSCRIPT
1
. Introduction
Since G-quadruplex (G4) DNA is widely located in the chromosomal telomeric
sequences as well as regulatory regions of oncogenes (such as KRAS, c-myc, HRAS,
bcl-2, c-KIT and VEGF) [1–11], it is considered as an important regulator of a wide
range of biological processes including translation, replication and splicing [1, 10–12].
The presence of G4 DNA in human cells has been firmly established using antibody
[
1, 10–13], thereby providing the basis for the elucidation of its functions in normal
and disease states. Therefore, rational design of small molecule ligands to selectively
interact and stabilize G4 DNA has been identified as a promising strategy for the
development of anti-cancer drugs selective towards tumor cells against normal cells
[
1,14].
The c-myc oncoprotein is a transcription factor that plays essential roles in controlling
cell proliferation, growth, and apoptosis as well as in regulating carcinogenic
progression [1,10,11,14]. Deregulation of c-myc expression is associated with human
tumors and cancers [1,10,11,14]. Thus, c-myc is an important drug target in oncology
[
1,10,11,14,15]. Strategies that aim to target c-myc both in the gene and protein levels,
including those that interfere with c-myc synthesis, stability, and transcriptional
activity, have emerged as effective anti-cancer therapies [1,10,11,14,15].
Recent studies have demonstrated that metal-terpyridine complexes with Zn(II),
Cu(II), Pd(II), and Pt(II) ion centres can act as effective G4 ligands [16–26]. For
example, Ang et al. designed four dinuclear Pt(II)-terpyridine complexes, which were
shown as efficient G4 ligands [24]. A dinuclear Cu(II)-terpyridine complex was
shown to effectively bind to G4 DNA and stabilize the antiparallel topology; its
selectivity for G4 DNA is 100-fold higher than that for duplex DNA [1,25,26].
3

ACCEPTED MANUSCRIPT
Bertrand et al. synthesized a range of Cu(II)-terpyridine complexes which displayed
better affinity and selectivity for G4 DNA than similar complexes reported previously
[
1,16]. Nonetheless, little is known about the iridium(III) complexes with terpyridine
and its derivatives as ligands and detailed studies on their molecular mechanisms of
action are still lacking [27–31].
In this work, there iridium(III) complexes, [Ir(3-MeO-Phtpy)Cl3] (1),
[
Ir(2-MeO-Phtpy)Cl3]
(2)
′-(3-methoxyphenyl)-2,2′:6′,2″-terpyridine
′-(2-methoxyphenyl)-2,2′:6′,2″-terpyridine
and
[Ir(4-MeO-Phtpy)Cl3]
(3)
with
4
4
4
(3-MeO-Phtpy),
and
(2-MeO-Phtpy)
′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine (4-MeO-Phtpy) as ligands, respectively,
were synthesized and characterized by ESI-MS, IR spectroscopy, UV-vis spectroscopy,
elemental analysis, single-crystal X-ray diffraction analysis, and NMR spectroscopy.
In addition, the effects of these complexes on cell proliferation and apoptosis were
evaluated.
2
2
. Results and discussion
.1 Synthesis and characterization
Synthesis
of
4′-(3-methoxyphenyl)-2,2′:6′,2″-terpyridine
(3-MeO-Phtpy),
and
4
4
′-(2-methoxyphenyl)-2,2′:6′,2″-terpyridine
(2-MeO-Phtpy)
′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine (4-MeO-Phtpy) was performed as
described previously (Scheme 1) [27–33]. The three terpyridine ligands were
individually incubated with IrCl in CH OH and DMSO (v:v= 25:1) at 80 °C for 48 h
3
3
to obtain the corresponding iridium(III) metal complexes 1−3. Their structures were
characterized by spectroscopic and analytical methods, including ESI-MS, IR
spectroscopy, UV-vis spectroscopy, elemental analysis, single-crystal X-ray
4

ACCEPTED MANUSCRIPT
diffraction analysis, as well as NMR spectroscopy (Figs. S1−S18).
[Scheme 1]
2
.2 Crystal structures of complexes 1−3
The X-ray crystallographic data for complexes 1−3 are summarized in the Supporting
Information (Tables S1−S9). In these complexes, the iridium(III) center is
six-coordinate (three nitrogen ligands from the terpyridine moiety plus three chloride
ligands) with a distorted octahedral geometry (Figs. 1−3). The Ir−N bond lengths are
in the range of 1.948−2.043, 1.938−2.060 and 1.938−2.060 Å, respectively, which are
within the normal range.
[Fig. 1]
[Fig. 2]
[Fig. 3]
2
.3 Stability and solubility studies of complexes 1−3 in TBS and distilled water
–5
The stability of complexes 1−3 (3.0×10 M) in 10 mM Tris-HCl buffer solution
TBS), pH 7.35 was monitored by following the changes in the UV-vis, the
(
conductivity experiment and ESI-MS spectra (Figs. S12, S15, S18−S20). The UV-vis
spectra of the three complexes showed that they were stable in TBS for 48 h at room
temperature. ESI-MS characterization of these complexes revealed the most abundant
III
+
ions at m/z = 680.1 for complexes 1 ([Ir + (3-MeO-Phtpy) + 2Cl + DMSO] ), m/z =
III
−
6
52.1 for complexes 2 ([Ir + (2-MeO-Phtpy) + 3Cl + H O − H] ), and m/z = 680.1
2
III
+
for complexes 3 ([Ir + (4-MeO-Phtpy) + 2Cl + DMSO] ). After 48-h incubation in
TBS, there was no change in the m/z values of the most abundant ions, confirming
that complexes 1−3 were stable under this condition. Moreover, the conductivity
experiment was thus carried out for further assessment. As shown in Fig. S20, after
the stock solution of complexes 1−3 and cisplatin at their IC concentrations were
5
0
5

ACCEPTED MANUSCRIPT
diluted by cellular lysate (Hep-G2 cells) and was monitored immediately, the
conductivity of cisplatin was observed to increase rapidly, comparing with three Ir(III)
complexes 1−3 solution. Finally, the conductivity value of the cellular lysate (Hep-G2
cells) of complexes 1−3 and cisplatin boosted about 2.3, 2.4, 2.5 and 2.9 times in 48 h,
respectively, therefore, it could be concluded that Ir(III) complexes 1−3 was more
stable for 48 h in cellular lysate (Hep-G2 cells) than that of cisplatin, which further
supported the stability analysis results of UV-Vis spectroscopy and ESI-MS. Taken
together, three new Ir(III) complexes 1−3 during in the required time could retain a
coordinated geometry with a positive charge under physiological conditions.
In addition, the solubility of three Ir(III) complexes 1−3 was investigated
gravimetrically in distilled water by UV-vis spectroscopy [34], as shown in Fig. S21.
The solubility of Ir(III) complexes 1−3 were over 0.16, 0.08 and 0.07 g/L in distilled
water at room temperature, respectively.
2
.4 In vitro cytotoxicity
To analyze the in vitro antitumor activity of 3-MeO-Phtpy, 2-MeO-Phtpy,
-MeO-Phtpy, cisplatin, IrCl and complexes 1−3, we evaluated their cytotoxic effects
4
3
on the BEL-7404, Hep-G2, NCI-H460 and MGC80-3 tumor cell lines using the
normal human HL-7702 cells as a control. The MTT colorimetric assay was
performed to estimate the IC values of the compounds after a 48-h incubation of the
5
0
cells with these compounds. As shown in Table 1 and S10, complexes 1−3 exhibited
lower IC values (IC = 3.19−38.27 µM) against all the tested cancer cells than the
5
0
50
corresponding IC values of 3-MeO-Phtpy, 2-MeO-Phtpy, 4-MeO-Phtpy and IrCl .
5
0
3
Notably, in the Hep-G2 cells, complexes 1−3 showed higher cytotoxicity compared to
cisplatin. Among these three complexes, complex 1 exhibited the highest cytotoxicity
against all the tested cell lines, followed by complex 2 and then complex 3. All three
6

ACCEPTED MANUSCRIPT
complexes displayed higher cytotoxicity against the tumor cell lines compared to the
normal HL-7702 cell line. As shown in Table 1, complex 1 exhibited a remarkably
high selectivity index factor [36,37]. The high selectivity for the tumor cells rendered
complex 1 to be a promising lead anti-tumor compound.
[Table 1]
2
.5 Cellular uptake and distribution of complexes 1−3 in the Hep-G2 cells
Cellular uptake and distribution of complexes 1−3 in the Hep-G2 cells was measured
using ICP-MS after a 24 h exposure to the complexes at their IC concentrations. As
5
0
shown in Fig. 4A, the cellular uptake level of complex 1 was determined to be 6.843 ±
6
0
1
.224 nmol Ir / 10 cells, higher than the levels of complex 2 (5.027 ± 0.107 nmol Ir /
6
6
0 cells), complex 3 (3.945 ± 0.209 nmol Ir / 10 cells), and cisplatin (4.025 ± 0.154
6
nmol Pt / 10 cells). Thus, the high cytotoxicity of complex 1 might be caused by its
high cellular uptake level. The cellular distribution of complexes 1−3 in the Hep-G2
cells was determined using the methods reported previously [38−41]. As shown in Fig.
4
B, complexes 1−3 were accumulated in the nuclear fraction and the mitochondrial
fraction, whereas cisplatin was mainly accumulated in the mitochondrial fraction. The
cellular distribution of these compounds is likely related to the apoptotic pathways
that they activated [38−41].
[Fig. 4]
2
.6 Effects of complexes 1−3 on the expression of c-myc and hTERT in the
Hep-G2 cells
Recent findings have unambiguously demonstrated that the expression level of
hTERT is a key regulator of the telomerase activity [12,13]. The expression of hTERT
is regulated by several factors. For example, c-myc is shown to induce the expression
of hTERT, whereas the stimulating protein 1 (Sp1) suppresses it [42,43]. Herein, we
7

ACCEPTED MANUSCRIPT
examined the effects of complexes 1−3 on the expression of c-myc and hTERT in the
Hep-G2 cells. We found that treatment with complexes 1−3 for 24 h downregulated
the expression of c-myc and hTERT genes and proteins in the Hep-G2 cells (Figs. 5
and 6). Notably, complex 1 displayed the most significant effect, followed by
complex 2 and complex 3.
[
[
Fig. 5]
Fig. 6]
2
.7 Complexes 1−3 inhibited the telomerase activity
Recent studies have demonstrated that the telomerase activity is elevated in
immortalized cell lines, germinal cells, and in 85-90% of human cancer specimens,
thereby supporting the role of telomerase in cell immortalization and in cancer/tumor
progression [42,43]. To investigate the effects of complexes 1−3 on the telomerase
activity in the Hep-G2 cells, the TRAP assay (TRAP-silver staining assay) was
performed on the Hep-G2 cells treated with complexes 1−3. As shown in Figs. 7 and
S22, complexes 1−3 significantly inhibited the telomerase activity in the Hep-G2 cells.
Again, complex 1 showed the highest inhibitory activity, followed by complex 2 and
complex 3.
[Fig. 7]
2
.8 Transfection assay
To probe whether complexes 1−3 directly regulated the c-myc promoter in the
Hep-G2 cells, the cells were transfected by the enhanced green fluorescent protein
(
EGFP) plasmid or the c-myc plasmid (including c-myc G4 DNA promoter) was then
examined by the luciferase reporter gene, using the methods reported previously
34,38,40,44,45]. Compared with the untreated group, the Hep-G2 cells with the
[
EGFP plasmid transfection emitted green fluorescence (Fig. 8A), indicating that the
8

ACCEPTED MANUSCRIPT
transfection was successful. In the Hep-G2 cells transfected with the c-myc plasmid,
treatment with complexes 1−3 on the Hep-G2 cells induced a pronounced decrease of
the fluorescence signal in the luciferase assay, suggesting that complexes 1−3 directly
targeted the c-myc promoter (Fig. 8B). Complex 1 caused the most significant
decrease of the fluorescence signal (from 100.0% to ca. 20.3%).
[Fig. 8]
2
.9 Mitochondrial membrane potential (∆ψ)
Mitochondria plays a crucial role in the induction and control of apoptosis through the
release of proapoptotic factors such as cytochrome c (cyt c) [46,47]. As shown in Fig.
4
, complexes 1−3 were accumulated in the mitochondrial fraction and the nuclear
fraction. Therefore, alterations in the mitochondrial membrane potential (∆ψ) were
measured in the Hep-G2 cells using an inverted fluorescence microscope after
staining the cells with the JC-1 cationic dye (Fig. 9). JC-1 can aggregate on intact
mitochondria with normal ∆ψ values and form red fluorescence as a result of the
aggregation. When the mitochondria membrane is depolarized and an abnormal ∆ψ is
present, JC-1 remains as monomers and emits green fluorescence. Treatment with
complexes 1−3 at their IC concentrations caused a decrease in the intensity of the
50
red fluorescence signal and an increase in the intensity of the green fluorescence
signal. This result indicates the cytotoxicity of complexes 1−3 is associated with a
mitochondrial pathway [46,47].
[Fig. 9]
2
.10 Effects of complexes 1-3 on intracellular ROS generation
Many anticancer agents have been shown to induce the generation of reactive oxygen
species (ROS) [48,49], which are considered as key mediators of apoptotic signaling.
We investigated the effects of complexes 1−3 in inducing the production of ROS in
9

ACCEPTED MANUSCRIPT
the Hep-G2 cells. The production of ROS was examined by using an oxidant-sensitive
fluorescent probe, 2′,7′-dichlorofluorescindiacetate (DCFDA). After treatment with
complexes 1−3 at their IC concentrations for 24 h, the level of ROS in the Hep-G2
50
cells was significantly increased, as shown in Fig. 10. The results demonstrated that
complexes 1−3 induced the generation of ROS. Among these three complexes,
complex 1 induced the highest level of ROS.
[Fig. 10]
2
.11 Effects of complexes 1−3 on the expression of apoptosis related proteins in
the Hep-G2 cells
To further elucidate the molecular mechanisms involved in the cytotoxicity of
complexes 1−3, we investigated the effects of these three complexes on the expression
of proteins important for mitochondria-mediated apoptosis. As shown in Fig. 11, we
found that complexes 1−3 were able to decrease the expression level of bcl-2 in the
Hep-G2 cells. In contrast, the expression of p53, bax, cyt c, and apaf-1 was increased
after the treatment with complexes 1−3. These proteins are involved in the induction
of mitochondrial membrane depolarization and cell apoptosis [48−50]. Accordingly, a
recent study has demonstrated that DNA damage in mitochondrial possibly through
increased ROS generation and activation of a DNA damage response [51−58].
Moreover, 53BP1 plays a potential role in DNA damage responses and binds to the
tumor suppressor protein p53 [39,59]. Thus, to verify the effects of Ir(III) complexes
1
−3 on mitochondrial fraction in Hep-G2 cells, we carried out Western blot analyses
on 53BP1 protein in Hep-G2 cells and showed the results in Fig. 23. Compared with
the control group cells, significant increases of 53BP1 level on mitochondrial fraction
in Hep-G2 cells were observed, suggesting that Ir(III) complexes 1−3 significantly
induced DNA damages on mitochondrial fraction in BEL-7404 cells [51−59].
1
0

ACCEPTED MANUSCRIPT
[Fig. 11]
2
.12 Activation of caspase-3 and caspase-9
The activation of caspase-3 and caspase-9 plays an important role in the process of
programmed cell death and apoptosis [49,60]. We monitored the activation of
caspase-3 and caspase-9 in the Hep-G2 cells upon the treatment with complexes 1−3
for 24 h. Compared to the untreated cells, the treated cells showed activation of
caspase-3 and caspase-9 (Fig. 12), suggesting that complexes 1−3 induced cell death
via apoptosis in the Hep-G2 cells [49,60]. The cells treated with complex 1 showed
the highest levels of activated caspase-3 and caspase-9.
[Fig. 12]
2
.13 Cell apoptosis
As demonstrated in the sections above, complexes 1−3 directly regulated c-myc
promoter, inhibited the expression of c-myc and hTERT, disrupted the mitochondrial
function, and ultimately led to cell apoptosis [34,38,40,48−50,60−62]. The apoptotic
effects of complexes 1−3 were evaluated by the Annexin V FITC and PI dual staining
assay, which can examine the occurrence of phosphatidylserine externalization and
determine whether this is caused by physiological apoptosis or nonspecific necrosis
[
34,38,40,48−50,60−62]. The Hep-G2 cells were treated with complexes 1−3 at their
IC concentrations for 24 h. As shown in Fig. 13, the result of the staining assay
5
0
confirmed that the treatment with the complexes caused apoptosis in the Hep-G2 cells.
Complex 1 induced the highest level of cell apoptosis, followed by complex 2 and
complex 3.
[Fig. 13]
3
. Conclusion
1
1

ACCEPTED MANUSCRIPT
In this study, there iridium(III) complexes with terpyridine ligands were synthesized
and characterized by different spectroscopic and analytic techniques. These
complexes exhibited higher cytotoxicity against different cell lines compared to the
terpyridine ligands. More importantly, they showed selective cytotoxicity against the
tumor cell lines. In vitro assays with the Hep-G2 tumor cells revealed that complexes
1
–3 acted as a telomerase inhibitor by directly targeting the c-myc promoter elements.
Moreover, complexes 1–3 triggered the Hep-G2 cell apoptosis via a mitochondrial
dysfunction pathway, which caused ROS generation, disruption of ∆ψ,
downregulation of the expression of bcl-2 and upregulation the expression of p53, bax,
cyt c, 53BP1 and apaf-1. It should be noted that complex 1 showed the highest
cytotoxicity among the three complexes. The high cytotoxicity of complex 1 was
postulated to be related to the substitution position of the methoxy group on the
terpyridine moiety. The 3-MeO substitution corresponded to highest level of cellular
uptake. Taken together, complex 1 is an excellent lead compound for the development
of new telomerase inhibitors.
4
4
4
. Experimental methods
.1 Synthesis
.1.1 Synthesis and characterization of the terpyridine ligands
Synthesis
of
4′-(3-methoxyphenyl)-2,2′:6′,2″-terpyridine
(3-MeO-Phtpy),
and
4
4
′-(2-methoxyphenyl)-2,2′:6′,2″-terpyridine
(2-MeO-Phtpy)
′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine (4-MeO-Phtpy) were performed using the
methods described previously [27–33]. 2-Acetylpyridine (2.813 g, 23.2 mmol, 2 eq.)
was added to 2-methoxybenzaldehyde, 3-methoxybenzaldehyde or
-methoxybenzaldehyde (11.6 mmol, 1 eq.) dissolved in 50 mL ethanol. KOH pellets
4
1
2

ACCEPTED MANUSCRIPT
(46.5 mmol, 4 eq.) were added to this solution. The reaction mixture was stirred at
room temperature for 10 min. NH (40 mL, 25% aq.) was slowly added to the reaction
3
o
mixture. After a 24-h incubation at 34 C, 5 mL of 25% aq. NH was added to the
3
reaction mixture again. The flask containing the reaction mixture was cooled to
-20 °C. The obtained white precipitate in the flask was isolated through filtration and
washed with cold ethanol. We further purified the each product using recrystallization
in ethanol-H O. After recrystallization, each product was recovered by filtration,
2
washed with cold ethanol and petroleum ether, and dried under high vacuum for 24 h
(Scheme 1).
+
Characterization of 3-MeO-Phtpy. Yield: 45.62%. ESI-MS: m/z = 701.4 for [2M+Na] ,
+
1
m/z = 362.1 for [M+Na] . H NMR (600 MHz, DMSO-d ): δ 8.68 (s, 2H), 8.55 (d, J =
6
7
1
1
.6 Hz, 4H), 7.94 (s, 2H), 7.44 (s, 2H), 7.41 (s, 1H), 7.34 (s, 1H), 7.28 (s, 1H), 7.03 (s,
H), 3.80 (s, 3H). Elemental analysis: calcd (%) for C H N O: C 77.86, H 5.05, N
2
2
17
3
2.38; found: C 77.81, H 5.10, N 12.35.
+
Characterization of 2-MeO-Phtpy. Yield: 45.62%. ESI-MS: m/z = 701.4 for [2M+Na] ,
+
1
m/z = 362.1 for [M+Na] . H NMR (600 MHz, DMSO-d ): δ 8.71 (s, 2H), 8.56 (s,
6
2
H), 8.00 (s, 2H), 7.86 (d, J = 8.1 Hz, 2H), 7.78 (s, 2H), 7.25 (s, 1H), 7.11 (s, 1H),
6
.99 (s, 1H), 6.91 (s, 1H), 3.81 (s, 3H). Elemental analysis: calcd (%) for C H N O:
22
17
3
C 77.86, H 5.05, N 12.38; found: C 77.84, H 5.08, N 12.34.
+
Characterization of 4-MeO-Phtpy. Yield: 45.62%. ESI-MS: m/z = 701.4 for [2M+Na] ,
+
1
m/z = 362.1 for [M+Na] . H NMR (600 MHz, DMSO-d ): δ 8.77 (s, 2H), 8.66 (s,
6
4
H), 7.96 (d, J = 93.6 Hz, 4H), 7.53 (s, 2H), 7.13 (s, 2H), 3.86 (s, 3H). Elemental
analysis: calcd (%) for C H N O: C 77.86, H 5.05, N 12.38; found: C 77.89, H 5.07,
2
2
17
3
N 12.33.
.1.2 Synthesis and characterization of complexes 1−3
4
1
3

ACCEPTED MANUSCRIPT
IrCl (0.0299 g, 0.1 mmol) was mixed with 3-MeO-Phtpy, 2-MeO-Phtpy or
3
4
-MeO-Phtpy (0.0339 g, 0.1 mmol), 0.20 mL DMSO and 0.50 mL CH OH in a Pyrex
3
tube. The Pyrex tube containing the reaction mixture was then placed in liquid N2,
vacuumed and sealed. The reaction was initiated by heating the reaction mixture to 80
°
C. After two days of incubation at 80 °C, the resulting brown-black crystals were
isolated and characterized by different spectroscopic and analytic techniques
1
Characterization of complex 1. Yield: 92.20%. H NMR (600 MHz, DMSO-d ): δ
6
8
1
3
1
.87 (s, 4H), 8.75 (s, 2H), 8.32 (s, 2H), 7.78 (s, 2H), 7.52 (s, 2H), 7.47 (s, 1H), 7.10 (s,
+
H), 3.87 (s, 3H). ESI-MS: m/z = 680.1 for [M−Cl+DMSO] . IR (KBr): 3913, 3887,
782, 3697, 3658, 3432, 3065, 2942, 2839, 1603, 1547, 1473, 1406, 1352, 1265, 1214,
−
1
164, 1100, 1028, 904, 849, 787, 731, 688, 651, 517, 461 cm . Elemental analysis:
calcd (%) for C H Cl IrN O: C 41.42, H 2.69, N 6.59; found: C 41.39, H 2.65, N
2
2
17
3
3
6
.60.
1
Characterization of complex 2. Yield: 85.30%. H NMR (600 MHz, DMSO-d ): δ
6
8
.87 (d, J = 6.9 Hz, 4H), 8.66 (s, 2H), 8.33 (s, 2H), 7.78 (s, 2H), 7.58 (s, 1H), 7.50 (s,
−
1
H), 7.22 (s, 1H), 7.15 (s, 1H), 3.84 (s, 3H). ESI-MS: m/z = 652.1 for [M+H O−H] .
2
IR (KBr): 3913, 3887, 3783, 3695, 3658, 3432, 3069, 2973, 2842, 1601, 1542, 1496,
469, 1410, 1295, 1247, 1161, 1130, 1098, 1051, 1014, 889, 843, 791, 760, 655, 636
1
−
1
cm . Elemental analysis: calcd (%) for C H Cl IrN O: C 41.42, H 2.69, N 6.59;
2
2
17
3
3
found: C 41.35, H 2.73, N 6.57.
Characterization of complex 3. Yield: 80.90%. H NMR (600 MHz, DMSO-d ): δ
1
6
8
2
3
1
.89 (d, J = 7.8 Hz, 2H), 8.86 (s, 2H), 8.73 (s, 2H), 8.34 (s, 2H), 7.95 (s, 2H), 7.79 (s,
+
H), 7.11 (s, 2H), 3.83 (s, 3H). ESI-MS: m/z = 680.1 for [M−Cl+DMSO] . IR (KBr):
957, 3913, 3783, 3696, 3659, 3633, 3574, 3459, 3067, 2957, 2836, 1560, 1519, 1464,
435, 1409, 1304, 1241, 1189, 1121, 1026, 889, 829, 790, 759, 726, 653, 601, 519,
1
4

ACCEPTED MANUSCRIPT
−
1
4
6
4
61, 430 cm . Elemental analysis: calcd (%) for C H Cl IrN O: C 41.42, H 2.69, N
22 17 3 3
.59; found: C 41.37, H 2.71, N 6.56.
.2 Instrumentation, materials and other experimental methods
The instrumentation, materials, and methods of different in vitro assays and
experiments, including the MTT assay, determination of the cellular uptake and
distribution of complexes 1−3, the JC-1 staining assay, detection of ROS generation,
cell apoptosis analysis, western blot, RT-PCR, and the transfection assay, were
described in Ng and Chen previously published work [34,37–40,63]. The TRAP-silver
staining assay was performed using methods reported previously [62,64].
Supporting Information
Vendor codes for 3-MeO-Phtpy, 2-MeO-Phtpy, 4-MeO-Phtpy and complexes 1−3,
inhibitory rates (%) against the selected five human cell lines, as well as ESI-MS,
1
UV-vis, H NMR, IR and crystallographic data. In addition, CCDC No. 1561943,
1
561944 and 1561950 for iridium(III) complexes 1−3 contains the supplementary
crystallographic data for this paper.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (No.
2
1261025, 21761033), the Key Foundation Project of Colleges and Universities in
Guangxi (No. ZD2014108), the Innovative Team & Outstanding Talent Program of
Colleges and Universities in Guangxi (2014-49), IRT_16R15, the project of guibei
characteristic medicine resources research center of Guangxi Province (KYA201703),
the basic skills improvement project for the young and middle-aged teachers in
Guangxi colleges and universities (KY2016YB595) as well as the State Key
1
5

ACCEPTED MANUSCRIPT
Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
Ministry of Science and Technology of China (CMEMR2017-B17).
References
References
[
1] Q. Cao, Y. Li, E. Freisinger, P. Z. Qin, R.K.O. Sigel, Z.-W. Mao, G-quadruplex
DNA targeted metal complexes acting as potential anticancer drugs, Inorg. Chem.
Front. 4 (2017) 10−32.
[
2] T.-M. Ou, Y.-J. Lu, J.-H. Tan, Z.-S. Huang, K.-Y. Wong, L.-Q. Gu, G-quadruplexes:
targets in anticancer drug design, ChemMedChem 3 (2008) 690−713.
3] A. Siddiqui-Jain, C.L. Grand, D.J. Bearss, L.H. Hurley, Direct evidence for a
[
G-quadruplex in a promoter region and its targeting with a small molecule to repress
c-MYC transcription, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 11593−11598.
[
4] S. Cogoi, L.E. Xodo, G-quadruplex formation within the promoter of the KRAS
proto-oncogene and its effect on transcription, Nucleic Acids Res. 34 (2006)
536−2549.
5] A. Membrino, S. Cogoi, E.B. Pedersen, L.E. Xodo, G4-DNA formation in the
2
[
HRAS promoter and rational design of decoy oligonucleotides for cancer therapy,
PloS one 6 (2001) e24421.
[
6] S. Rankin, A.P. Reszka, J. Huppert, M. Zloh, G.N. Parkinson, A.K. Todd, S.
Ladame, S. Balasubramanian, S.Neidle, Putative DNA quadruplex formation within
the human c-kit oncogene, J. Am. Chem. Soc. 127 (2005) 10584−10589.
[
7] D. Sun, W.-J. Liu, K. Guo, J.J. Rusche, S. Ebbinghaus, V. Gokhale, L.H. Hurley,
The proximal promoter region of the human vascular endothelial growth factor gene
has a G-quadruplex structure that can be targeted by G-quadruplex-interactive agents,
1
6

ACCEPTED MANUSCRIPT
Mol. Cancer Ther. 7 (2008) 880−889.
8] D. Sun, K. Guo, Y.J. Shin, Evidence of the formation of G-quadruplex structures
[
in the promoter region of the human vascular endothelial growth factor gene, Nucleic
Acids Res. 39 (2010) 1256−1265.
[
9] S. Cogoi, A.E. Shchekotikhin, A. Membrino, Y.B. Sinkevich, L.E. Xodo,
Guanidino anthrathiophenediones as G-quadruplex binders: uptake, intracellular
localization, and anti-Harvey-Ras gene activity in bladder cancer cells, J. Med. Chem.
5
6 (2013) 2764−2778.
[
10] Y. Jiang, A.-C. Chen, G.-T. Kuang, S.-K. Wang, T.-M. Ou, J.-H. Tan, D. Li, Z.-S.
Huang, Design, synthesis and biological evaluation of 4-anilinoquinazoline
derivatives as new c-myc G-quadruplex ligands, Eur. J. Med. Chem. 122 (2016)
2
64−279.
[
11] H.-Y. Liu, A.-C. Chen, Q.-K. Yin, Z. Li, S.-M. Huang, G. Du, J.-H. He, L.-P. Zan,
S.-K. Wang, Y.-H. Xu, J.-H. Tan, T.-M. Ou, D. Li, L.-Q. Gu, Z.-S. Huang, New
disubstituted quindoline derivatives inhibiting Burkitt’s lymphoma cell proliferation
by impeding c-MYC transcription, J. Med. Chem. 60 (2017) 5438–5454.
[
12] D. Rhodes, H.J. Lipps, G-quadruplexes and their regulatory roles in biology,
Nucleic Acids Res. 43 (2015) 8627−8637.
13] G. Biffi,D. Tannahill, J. McCafferty, S. Balasubramanian, Quantitative
visualization of DNA G-quadruplex structures in human cells, Nat. Chem. 5 (2013)
82−186.
14] S. Pelengaris, M. Khan, G.I. Evan, Suppression of Myc-induced apoptosis in β
[
1
[
cells exposes multiple oncogenic properties of Myc and triggers carcinogenic
progression, Cell 109 (2002) 321−334.
[
15] L. Soucek, J. Whitfield, C.P. Martins, A.J. Finch, D.J. Murphy, N.M. Sodir, A.N.
1
7

ACCEPTED MANUSCRIPT
Karnezis, L.B. Swigart, S. Nasi, G.I. Evan, Modelling Myc inhibition as a cancer
therapy, Nature 455 (2008) 679−683.
[
16] H. Bertrand, D. Monchaud, A. De Cian, R. Guillot, J.-L. Mergny, M.-P.
Teulade-Fichou, The importance of metal geometry in the recognition of
G-quadruplex-DNA by metal-terpyridine complexes, Org. Biomol. Chem. 5 (2007)
2
555−2559.
[
17] P. Wang, C.-H. Leung, D.-L. Ma, S.-C. Yan, C.-M. Che, Structure-based design
of platinum(II) complexes as c-myc oncogene down-regulators and luminescent
probes for G-quadruplex DNA, Chem. Eur. J. 16 (2010) 6900−6911.
[
18] S. Gama, I. Rodrigues, F. Mendes, I.C. Santos, E. Gabano, B. Klejevskaja, J.
Gonzalez-Garcia, M. Ravera, R. Vilar, A. Paulo, Anthracene-terpyridine metal
complexes as new G-quadruplex DNA binders, J. Inorg. Biochem. 160 (2016)
2
75−286.
19] C. Wei, L. Ren, N. Gao, Interactions of terpyridines and their Pt(II) complexes
with G-quadruplex DNAs and telomerase inhibition, Int. J. Biol. Macromol. 57 (2013)
−8.
20] E. Largy, F. Hamon, F. Rosu, V. Gabelica, E. De Pauw, A. Guédin, J.-L. Mergny,
[
1
[
M.-P. Teulade-Fichou, Tridentate N-donor palladium(II) complexes as efficient
coordinating quadruplex DNA binders, Chem. Eur. J. 17 (2011) 13274−13283.
[
21] K. Suntharalingam, A.J.P. White, R. Vilar, Synthesis, structural characterization,
and quadruplex DNA binding studies of platinum(II)-terpyridine complexes, Inorg.
Chem. 48 (2009) 9427−9435.
[
22] J.-T. Wang, Y. Li, J.-H. Tan, L.-N. Ji, Z.-W. Mao, Platinum(II)-triarylpyridines
complexes with electropositive pendants as efficient G-quadruplex binders, Dalton
Trans. 40 (2011) 564−566.
1
8

ACCEPTED MANUSCRIPT
[
23] C. Yu, K.H.Y. Chan, K.M.-C. Wong, V. W.-W. Yam, Nucleic acid-induced
self-assembly of a platinum(II) terpyridyl complex: detection of G-quadruplex
formation and nuclease activity, Chem. Commun. (2009) 3756−3758.
[
24] D.L. Ang, B.W. J. Harper, L. Cubo, O. Mendoza, R. Vilar, J. Aldrich-Wright,
Quadruplex DNA-stabilising dinuclear platinum(II) terpyridine complexes with
flexible linkers, Chem. Eur. J. 22 (2016) 2317−2325.
[
25] K. Suntharalingam, A.J.P. White, R. Vilar, Two metals are better than one:
investigations on the interactions between dinuclear metal complexes and quadruplex
DNA, Inorg. Chem. 49 (2010) 8371−8380.
[
26] V.S. Stafford, K. Suntharalingam, A. Shivalingam, A.J.P. White, D.J. Mann, R.
Vilar, Syntheses of polypyridyl metal complexes and studies of their interaction with
quadruplex DNA, Dalton Trans. 44 (2015) 3686−3700.
[
27] A. Jacques, A. Kirsch-De Mesmaeker, B. Elias, Selective DNA purine base
photooxidation by bis-terdentate iridium(III) polypyridyl and cyclometalated
complexes, Inorg. Chem. 53 (2014) 1507−1512.
[
28] B.N. Mongal, S. Naskar, Synthesis, characterization, electrochemical and
theoretical study of substituted phenyl-terpyridine and pyridine-quinoline based
mixed chelate ruthenium complexes, J. Coord. Chem. 70 (2017) 451−462.
[
29] W. Leslie, A.S. Batsanov, J.A.K. Howard, J.A.G. Williams, Cross-couplings in
the elaboration of luminescent bis-terpyridyl iridium complexes: the effect of
extended or inhibited conjugation on emission, Dalton Trans. (2004) 623−631.
[
30] M. Cavazzini, P. Pastorelli, S. Quici, F. Loiseau, S. Campagna, Two-color
luminescence from a tetranuclear Ir(III)/Ru(II) complex, Chem. Commun. (2005)
266−5268.
31] L.S. Natrajan,
5
[
A. Toulmin,
A. Chew, S.W. Magennis, Two-photon
1
9

ACCEPTED MANUSCRIPT
luminescence from polar bis-terpyridyl-stilbene derivatives of Ir(III) and Ru(II),
Dalton Trans. 39 (2010) 10837−10846.
[
32] J. Wang, G.S. Hanan, A facile route to sterically hindered and non-hindered
′-aryl-2, 2′: 6′, 2′′-terpyridines, Synlett 2005 (2005) 1251−1254.
33] Y. Li, M. Cheng, J. Hao, C. Wang, G. Jia, C. Li, Terpyridine-Cu(II) targeting
4
[
human telomeric DNA to produce highly stereospecific G-quadruplex DNA
metalloenzyme, Chem. Sci. 6 (2015) 5578−5585.
[
34] Z.-F. Chen, Q.-P. Qin, J.-L. Qin, Y.-C. Liu, K.-B. Huang, Y.-L. Li, T. Meng, G.-H.
Zhang, Y. Peng, X;-J. Luo, H. Liang, Stabilization of G-quadruplex DNA, inhibition
of telomerase activity and tumor cell apoptosis of organoplatinum(II) complexes with
oxoisoaporphine, J. Med. Chem. 58 (2015) 2159−2179.
[
35] R. Cao, J.-L. Jia, X.-C. Ma, M. Zhou, H. Fei, Membrane localized iridium(III)
complex induces endoplasmic reticulum stress and mitochondria-mediated apoptosis
in human cancer cells, J. Med. Chem. 56 (2013) 3636−3644.
[
36] H.-Y. Zhou, F.-Q. Dong, X.-L. Du, Z.-K. Zhou, H.-R. Huo, W.-H. Wang, H.-D.
Zhan, Y.-F. Dai, J. Meng, Y.-P. Sui, J. Li, F. Sui, Y.-H. Zhai, Antitumor activities of
biscoumarin and dihydropyran derivatives, Bioorg. Med. Chem. Lett. 26 (2016)
3
876–3880.
[
37] Q.-P. Qin, T. Meng, Z.-Z. Wei, C.-H. Zhang, Y.-C. Liu, H. Liang, Z.-F. Chen,
II
II
Synthesis, crystal structure, cytotoxicity, and mechanism of action of Zn , Mn , and
III
Fe complexes with 6-hydroxyloxoisoaporphine, Eur. J. Inorg. Chem. 2017 (2017)
1
824–1834.
[
38] J.-L. Qin, Q.-P. Qin, Z.-Z. Wei, C.-C. Yu, T. Meng, C.-X. Wu, Y.-L. Liang, H.
Liang, Z.-F. Chen, Stabilization of c-myc G-quadruplex DNA, inhibition of
telomerase activity, disruption of mitochondrial functions and tumor cell apoptosis by
2
0

ACCEPTED MANUSCRIPT
platinum(II) complex with 9-amino-oxoisoaporphine, Eur. J. Med. Chem. 124 (2016)
4
17−427.
39] Z.-F. Chen, Q.-P. Qin, J.-L. Qin, J. Zhou, Y.-L. Li, N. Li, Y.-C. Liu, H. Liang,
Water-soluble ruthenium(II) complexes with chiral
-(2,3-dihydroxypropyl)-formamide oxoaporphine (FOA): In vitro and in vivo
[
4
anticancer activity by stabilization of G-Quadruplex DNA, inhibition of telomerase
activity, and induction of tumor cell apoptosis, J. Med. Chem. 58 (2015) 4771−4789.
[
40] Q.-P. Qin, J.-L. Qin, T. Meng, W.-H. Lin, C.-H. Zhang, Z.-Z. Wei, J.-N. Chen,
Y.-C. Liu, H. Liang, Z.-F. Chen, High in vivo antitumor activity of cobalt
oxoisoaporphine complexes by targeting G-quadruplex DNA, telomerase and
disrupting mitochondrial functions, Eur. J. Med. Chem. 124 (2016) 380−392.
[
41] E. Schreiber, P. Matthias, M.M. Mueller, W. Schaffner, Rapid detection of
octamer binding proteins with ‘mini-extracts’ prepared from a small number of cells,
Nucleic Acids Res. 17 (1989) 6419.
[
42] J.C. Poole, L.G. Andrews, T.O. Tollefsbol, Activity, function, and gene regulation
of the catalytic subunit of telomerase (hTERT), Gene 269 (2001) 1−12.
43] H.-S. Huang, J.-F. Chiou, Y. Fong, C.-C. Hou, Y.-C. Lu, J.-Y. Wang, J.-W. Shih,
[
Y.-R. Pan, J.-J. Lin, Activation of human telomerase reverse transcriptase expression
by some new symmetrical bis-substituted derivatives of the anthraquinone, J. Med.
Chem. 46 (2003) 3300−3307.
[
44] M. Chalfie, Y. Tu, G. Euskirchen, W.W. Ward, D.C. Prasher, Green fluorescent
protein as a marker for gene expression, Science 263 (1994) 802−805.
45] T.-C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J.
[
Morin, B. Vogelstein, K.W. Kinzler, Identification of c-MYC as a target of the APC
pathway, Science 281 (1998) 1509−1512.
2
1

ACCEPTED MANUSCRIPT
[
46] A. Elkamhawy, J.-E. Park, A.H.E. Hassand, H. Ra, A.N. Pae, J. Lee, B.-G. Park,
B. Moon, H.-M. Park, E.J. Roh, Discovery of 1-(3-(benzyloxy)
pyridin-2-yl)-3-(2-(piperazin-1-yl) ethyl) urea: A new modulator for amyloid
beta-induced mitochondrial dysfunction, Eur. J. Med. Chem. 128 (2017) 56−69.
[
47] P. Sakthivel, A. Ilangovan, M.P. Kaushik, Natural product-inspired rational
design, synthesis and biological evaluation of 2, 3-dihydropyrano [2, 3-f] chromen-4
8H)-one based hybrids as potential mitochondrial apoptosis inducers, Eur. J. Med.
Chem. 122 (2016) 302−318.
48] V. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity
screening, Nat. Protoc. 1 (2006) 1112−1116.
49] D.Yugandhar, V.L. Nayak, S. Archana, K.C. Shekar, A.K. Srivastav, Design,
(
[
[
synthesis and anticancer properties of novel oxa/azaspiro [4, 5] trienones as potent
apoptosis inducers through mitochondrial disruption, Eur. J. Med. Chem. 101 (2015)
3
48−357.
[
50] S. Li, S. Zhang, X. Jin, X. Tan, J. Luo, X. Zhang, Y. Zhao, Singly protonated
dehydronorcantharidin silver coordination polymer induces apoptosis of lung cancer
cells via reactive oxygen species-mediated mitochondrial pathway, Eur. J. Med. Chem.
8
6 (2014) 1−11.
51] K.K. Singh, Mitochondria damage checkpoint in apoptosis and genome stability,
FEMS Yeast Res. 5 (2004) 127−132.
52] R. Wang, Y.-Y. Liu, X.-Y. Liu, S.-W. Jia, J. Zhao, D. Cui, L. Wang, Resveratrol
[
[
protects neurons and the myocardium by reducing oxidative stress and ameliorating
mitochondria damage in a cerebral ischemia rat model, Cell. Physiol. Biochem. 34
(2014) 854−864.
[
53] K.K. Singh, Mitochondria damage checkpoint, aging, and cancer, Ann. NY Acad.
2
2

ACCEPTED MANUSCRIPT
Sci. 1067 (2006) 182−190.
54] J.H. Santos, J.N. Meyer, B. Van Houten, Mitochondrial localization of telomerase
[
as a determinant for hydrogen peroxide-induced mitochondrial DNA damage and
apoptosis, Hum. Mol. Genet. 15 (2006) 1757−1768.
[
55] J.H. Santos, J.N. Meyer, M. Skorvaga, L.A. Annab, B. Van Houten,
Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage, Aging cell
(2004) 399−411.
56] J.F. Passos, G. Saretzki, T. von Zglinicki, DNA damage in telomeres and
mitochondria during cellular senescence: is there a connection?, Nucleic Acids Res.
5 (2007) 7505−7513.
57] S.Y. Park, B. Choi, H. Cheon, Y.K. Pak, M. Kulawiec, K.K. Singh, M.-S. Lee,
Cellular aging of mitochondrial DNA-depleted cells, Biochem. Bioph. Res. Commun.
25 (2004) 1399−1405.
58] H. Kimura, K. Shintani-Ishida, M. Nakajima, S. Liu, K. Matsumoto, K.-I.
3
[
3
[
3
[
Yoshida, Ischemic preconditioning or p38 MAP kinase inhibition attenuates
myocardial TNF α production and mitochondria damage in brief myocardial ischemia,
Life Sci. 78 (2006) 1901−1910.
[
59] A. Fradet-Turcotte, M.D. Canny, C. Escribano-Díaz, A. Orthwein, C.C.Y. Leung,
H. Huang, M.-C. Landry, J. Kitevski-LeBlanc, S.M. Noordermeer, F. Sicheri, D.
Durocher, 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark,
Nature 499 (2013) 50−54.
[
60] B. Chakravarti, M. Ranjani, J.A. Siddiqui, B. Hemant Kumar, S.M. Rajendran,
Prem P. Yadav, K. Rituraj, In vitro anti-breast cancer activity of ethanolic extract of
Wrightia tomentosa: role of pro-apoptotic effects of oleanolic acid and urosolic acid, J.
Ethnopharm. 142 (2012) 72−79.
2
3

ACCEPTED MANUSCRIPT
[
61] A. De Cian, G. Cristofari, P. Reichenbach, E. De Lemos, D. Monchaud, M.-P.
Teulade-Fichou, K. Shin-ya, L. Lacroix, J. Lingner, J.-L. Mergny, Reevaluation of
telomerase inhibition by quadruplex ligands and their mechanisms of action, PNAS
1
04 (2007) 17347−17352.
[
62] J.E. Reed, A.A. Arnal, S. Neidle, R. Vilar, Stabilization of G-quadruplex DNA
and inhibition of telomerase activity by square-planar nickel(II) complexes, J. Am.
Chem. Soc. 128 (2006) 5592−5993.
[
63] S.-T. Von, H.-L. Seng, H.-B. Lee, S.-W. Ng, Y. Kitamura, M. Chikira, C.-H. Ng,
DNA molecular recognition and cellular selectivity of anticancer metal(II) complexes
of ethylenediaminediacetate and phenanthroline: multiple targets, J. Biol. Inorg. Chem.
1
7 (2012) 57–69.
64] L. Xu, X. Chen, J. Wu, J. Wang, L. Ji, H. Chao, Dinuclear ruthenium(II)
complexes that induce and stabilise G-quadruplex DNA, Chem. Eur. J. 21 (2015)
008−4020.
[
4
Figure legends
Fig. 1. ORTEP drawing of complex 1. Thermal ellipsoids for non-hydrogen atoms are
drawn at a level of 30% probability.
Fig. 2. ORTEP drawing of complex 2.
Fig. 3. ORTEP drawing of complex 3.
Fig. 4. Ir or Pt contents in the Hep-G2 cancer cells (A) and different cellular fractions
(B). ICP-MS was used to determine the metal contents.
Fig. 5. RT-PCR analysis of the expression level of c-myc and hTERT in the Hep-G2
2
4

ACCEPTED MANUSCRIPT
cells treated with complexes 1−3 at their IC concentrations for 24 h.
5
0
Fig. 6. Western blot analysis of the expression level of c-myc and hTERT in the
Hep-G2 cells treated with complexes 1−3 at their IC concentrations for 24 h. (A)
5
0
C-myc and hTERT protein levels in Hep-G2 cells were analyzed by western blot. (B)
The whole-cell extracts were prepared and analyzed by Western blot analysis using
antibodies against c-myc and hTERT. The same blots were stripped and re-probed
with a β-actin antibody to show equal protein loading. Western blot bands from three
independent measurements were quantified with Image J. in (B), with the mean ± SD
(standard error of the mean).
Fig. 7. Inhibition of the telomerase activity by complexes 1−3 at their IC50
concentrations in the Hep-G2 cancer cells for 24 h.
Fig. 8. Transfection of the EGFP plasmid (A) and the c-myc plasmid (B) in the
Hep-G2 tumor cells. In (A), the cells were imaged using a Nikon Te2000 microscope
(magnification 200×). In (B), the transfected cells were treated complexes 1−3 at their
IC concentrations for 24 h. The expression level of c-myc was determined by a
5
0
multi-model plate reader with luciferase reporter gene assay kit.
Fig. 9. Fluorescence spectroscopic characterization of the Hep-G2 cells treated with
complexes 1−3 at their IC concentrations for 24 h. The treated cells were stained by
50
the JC-1 dye and imaged using a Nikon Te2000 microscope (magnification 200×).
Fig. 10. Determination of ROS generation in the Hep-G2 cells treated with complexes
1
−3 at their IC50 concentrations for 24 h. The treated cells were stained by an
oxidant-sensitive fluorescent probe, 2′,7′-dichlorofluorescindiacetate (DCFDA) and
imaged using a Nikon Te2000 microscope (magnification 200×).
Fig. 11. Western blot analysis of the effects of complexes 1−3 on the expression levels
of proteins involved in mitochondrion-initiated apoptosis. (A) The apoptosis related
2
5

ACCEPTED MANUSCRIPT
proteins protein levels in Hep-G2 cells were analyzed by western blot. (B) The
whole-cell extracts were prepared and analyzed by Western blot analysis using
antibodies against apoptosis related proteins. The same blots were stripped and
re-probed with a β-actin antibody to show equal protein loading. Western blot bands
from three independent measurements were quantified with Image J. in (B).
Fig. 12. Flow cytometry analysis of the effects of complexes 1−3 on the levels of
active caspase-3 and caspase-9 in the Hep-G2 cells.
Fig. 13. Apoptosis rate of the Hep-G2 cells. These Hep-G2 cells were the three Ir
complexes 1−3 at their IC concentrations for 24 h and stained with Annexin V-FITC
50
and PI, followed by flow cytometry analysis.
Scheme 1. Synthetic routes for three terpyridine ligands and the corresponding Ir
complex 1−3.
a
Table 1. IC50 values (µM) of 3-MeO-Phtpy, 2-MeO-Phtpy, 4-MeO-Phtpy, cisplatin,
and complexes 1−3 on the five selected human cell lines.
c
Compound
BEL-7404
Hep-G2
4.34±1.02 19.67±0.73 19.96±0.35 20.96±0.31
7.77±0.59 3.19±1.15 6.93±0.44 8.68±1.05
NCI-H460 MGC80-3
HL-7702
3
2
3
3
5
3
35.11±0.81 (1.78)
37.51±0.56 (11.76)
17.16±1.03 (0.65)
31.46±1.69 (4.75)
18.11±1.07 (0.64)
39.13±0.53 (2.59)
17.98±1.94 (0.96)
3
2
4
-MeO-Phtpy
1
6.94±0.80 26.40±1.45 24.73±1.10 23.02±1.44
1.29±1.30 6.63±1.01 10.42±1.39 14.69±2.12
6.78±0.65 28.43±0.39 33.78±1.76 35.71±0.92
8.27±0.43 15.08±0.62 27.91±0.93 22.09±0.71
-MeO-Phtpy
2
-MeO-Phtpy
3
b
Cisplatin
16.91±1.01 18.68±0.67 18.01±1.23 13.99±0.76
a
The IC values are presented as mean
±SD (standard error of the mean) from five or
5
0
b
six independent experiments. Cisplatin was prepared at a concentration of 1.0 mM in
2
6

ACCEPTED MANUSCRIPT
c
a 0.154 M NaCl solution [34,35]. The selectivity index factors, defined as IC50
(
HL-7702 cells)/IC (Hep-G2 tumor cells), are shown in parentheses.[36,37]
50
Fig. 1
2
7

ACCEPTED MANUSCRIPT
Fig. 2
2
8