Synthesis and biological evaluation of substituted 3-(2′-benzimidazolyl)coumarin platinum(II) complexes as new telomerase inhibitors

Article abstract of DOI:10.1016/j.jinorgbio.2018.09.004

Eight new platinum(II) complexes Pt1–Pt8 with substituted 3?(2′?benzimidazolyl) coumarins were successfully synthesized and characterized by single crystal X-ray diffraction analysis, nuclear magnetic resonance spectroscopy (NMR), electrospray ionization-mass spectrometry (ESI-MS), infrared spectrophotometry (IR) and elemental analysis. Crystallographic data of these Pt1–Pt8 complexes showed that the Pt(II) has distorted four-coordinated square planar geometry. Pt1–Pt8 were found to display high cytotoxic activity in vitro against the cisplatin-resistant SK-OV-3/DDP cancer cells with a low IC₅₀ from 1.01–10.32 μM, but low cytotoxicity on the normal HL-7702 cells. Further studies revealed that Pt1–Pt3 induced apoptosis in SK-OV-3/DDP cancer cells via mitochondria dysfunction signaling pathways. Our findings also indicated that Pt1 was a telomerase inhibitor targeting c-myc promoter elements.

Full text of DOI:10.1016/j.jinorgbio.2018.09.004

Accepted Manuscript
Synthesis and biological evaluation of substituted 3-(2′-
benzimidazolyl)coumarin platinum(II) complexes as new
telomerase inhibitors
Ting Meng, Qi-Pin Qin, Zhen-Rui Wang, Li-Ting Peng, Hua-
Hong Zou, Zhen-Yuan Gan, Ming-Xiong Tan, Kai Wang, Fu-Pei
Liang
PII:
DOI:
Reference:
S0162-0134(18)30245-9
doi:10.1016/j.jinorgbio.2018.09.004
JIB 10559
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
27 April 2018
8 September 2018
9 September 2018
Please cite this article as: Ting Meng, Qi-Pin Qin, Zhen-Rui Wang, Li-Ting Peng, Hua-
Hong Zou, Zhen-Yuan Gan, Ming-Xiong Tan, Kai Wang, Fu-Pei Liang , Synthesis and
biological evaluation of substituted 3-(2′-benzimidazolyl)coumarin platinum(II)
complexes as new telomerase inhibitors. Jib (2018), doi:10.1016/j.jinorgbio.2018.09.004
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ACCEPTED MANUSCRIPT
Synthesis
and
biological
evaluation
of
substituted
3-(2′-benzimidazolyl)coumarin platinum(II) complexes as new
telomerase inhibitors
a
a,*
Ting Meng , Qi-Pin Qin a,b,*, Zhen-Rui Wang a, Li-Ting Peng a, Hua-Hong Zou
,
Zhen-Yuan Gan a, Ming-Xiong Tan b,*, Kai Wang c, Fu-Pei Liang a,c,*
a 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. E-mail: gxnuchem@foxmail.com (H.-H. Zou); fliangoffce@yahoo.com (F.-P. Liang).
b Guangxi Key Lab of Agricultural Resources Chemistry and Biotechnology, College of Chemistry
and Food Science, Yulin Normal University, 1303 Jiaoyudong Road, Yulin 537000, PR China.
qpqin2018@126.com (Q.-P. Qin); mxtan2018@126.com (M.-X. Tan).
c
Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials,
College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
1

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Abstract
Eight new platinum(II) complexes Pt1−Pt8 with substituted 3-(2′-benzimidazolyl)
coumarins were successfully synthesized and characterized by single crystal X-ray
diffraction analysis, nuclear magnetic resonance spectroscopy (NMR), electrospray
ionization-mass spectrometry (ESI-MS), infrared spectrophotometry (IR) and
elemental analysis. Crystallographic data of these Pt1−Pt8 complexes showed that the
Pt(II) has distorted four-coordinated square planar geometry. Pt1−Pt8 were found to
display high cytotoxic activity in vitro against the cisplatin-resistant SK-OV-3/DDP
cancer cells with a low IC50 from 1.01−10.32 μM, but low cytotoxicity on the normal
HL-7702 cells. Further studies revealed that Pt1−Pt3 induced apoptosis in
SK-OV-3/DDP cancer cells via mitochondria dysfunction signaling pathways. Our
findings also indicated that Pt1 was a telomerase inhibitor targeting c-myc promoter
elements.
Keywords: 3-(2′-benzimidazolyl)coumarins, platinum(II) complexes, cell apoptosis,
telomerase activity, mitochondrial dysfunction
2

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1. Introduction
In the last few decades, Pt-based drugs such as cisplatin, carboplatin and oxaliplatin
have been developed for the treatment of various cancers [1, 2]. However, Pt-based
anticancer drugs exhibit drug resistance and severe side effects in the therapeutic
process. This fact has motivated researchers to search for more effective, less toxic,
and target-specific platinum-based anticancer drugs [3−6]. For example, Lippard
investigated trans-[Pt(NH3)2(phenanthridine)Cl]NO3 displays significant antitumor
properties, with a different spectrum of activity than that of classic bifunctional
cross-linking agents like cisplatin [7−11]. Hambley and co-authors reported that
trans-[Pt(OH)2(ox)(en)] (ox= OAc, en= 1,2-ethylenediamine) more cytotoxic against
human A2780 tumor cells (human ovarian cancer cell line) than against DLD-1
cancer cells (human colon cancer cell line) [12−16]. Farrell suggested that
[Pt(dien)L]2+ (dien= N'-[2-(dimethylamino)ethyl]-N,N-dimethylethylenediamine, L=
nucleobase or nucleoside) gave especially strong π–π stacking interactions with
tryptophan and the tryptophan-containing C-terminal zinc finger (ZF) of the HIV
nucleocapsid protein NCp7 [17−20]. Collins and Wheate demonstrated that
cisplatin@CB[7] and trans-[{PtCl(NH3)2}2(m-NH2(CH2)8NH2)]2+ (CT008) is just as
effective on A2780 tumor cells and a delivery mechanism for controlled slow release
of large dinuclear ruthenium(II) complexes, respectively [21−23].
Previous reports have shown that telomerase is overexpressed in 85−90% of the
human tumor cells but has undetectable activity in most of the human normal somatic
cells [24, 25]. Thus, the design of Pt-based drugs to interfere with or target telomerase
activity in cancer cells is a promising approach to discover potential anticancer agents
[26]. For example, Mao et al. reported that flexible trinuclear Pt(II) complexes are
effective and selective G4 binders and good telomerase inhibitors [27]. Yan and
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co-authors reported the high binding selectivity and affinity (∼107 dm3·mol-1) of this
dipyridophenazine (dppz) Pt(II) complex toward the G-quadruplex, as well as its
nanomolar potency against telomerase (TelIC50= 760 nM). Vilar et al. investigated
mono-substituted phenanthroline platinum(II) square planar complexes and showed
that they induce a high degree of quadruplex DNA stabilization and inhibit telomerase
activity [28].
Coumarin and its derivatives are important naturally occurring products and they
have wide-range applications as agrochemicals and drugs such as anticancer,
antituberculosis, anti-HIV, anti-inflammatory, anti-Alzheimer's, anti-influenza,
antiviral and antimicrobial agents [29,30]. Nevertheless, the metal complexes of
coumarin and its derivatives are rarely explored as telomerase inhibitors [31−34]. On
the basis of the previous studies, in order to exploit new Pt(II) complexes with
different coordination mode and more extended planar coumarin ligand, by means of
combining the potential of Pt(II) complexes as anticancer agents and the significant
bioactivity of alkaloids, herein, we firstly reported that the Pt(II) complexes
[Pt(MeOBC)(DMSO)Cl2] (Pt1), [Pt(OHBC)(DMSO)Cl2] (Pt2), [Pt(BC)(DMSO)Cl2]
(Pt3),
[Pt(FBC)(DMSO)Cl2]
(Pt4),
[Pt(BrBC)(DMSO)Cl2]
(Pt7)
(Pt5),
and
[Pt(FFBC)(DMSO)Cl2]
[Pt(BrBrBC)(DMSO)Cl2]
(Pt6),
[Pt(ClClBC)(DMSO)Cl2]
(Pt8)
with
eight
ligands
3-(2′-benzimidazolyl)-8-methoxycoumarin
-8-hydroxycoumarin (OHBC),
3-(2′-benzimidazolyl)-7-fluorocoumarin
(MeOBC),
3-(2′-benzimidazolyl)
(BC),
3-(2′-benzimidazolyl)-7-
3-(2′-benzimidazolyl)coumarin
(FBC),
bromocoumarin (BrBC), 3-(2′-benzimidazolyl)-6,8-difluorocoumarin (FFBC),
3-(2′-benzimidazolyl)-6,8-dichlorocoumarin (ClClBC) and 3-(2′-benzimidazolyl)
-6,8-dibromocoumarin (BrBrBC) were synthesized and fully characterized. By
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assessing their cytotoxicity against HeLa (human sarcoma HeLa cancer cell line),
Hep-G2 (human hepatocellular carcinoma cell line), SK-OV-3/DDP (human
cisplatin-resistant SK-OV-3 cell line), SK-OV-3 (human ovarian cancer cell line)
tumor cells and human normal HL-7702 cells (human normal hepatocytes cell line),
we proposed a possible antitumor mechanism.
2. Results and Discussion
2.1 Synthesis and Characterization
As shown in Scheme 1, the eight ligands MeOBC, OHBC, BC, FBC, BrBC,
FFBC, ClClBC and BrBrBC were synthesized according to the reported procedures
[35,36].
After
synthesizing
these
ligands,
the
Pt(II)
complexes
[Pt(MeOBC)(DMSO)Cl2] (Pt1), [Pt(OHBC)(DMSO)Cl2] (Pt2), [Pt(BC)(DMSO)Cl2]
(Pt3),
[Pt(FBC)(DMSO)Cl2]
(Pt6),
(Pt4),
[Pt(BrBC)(DMSO)Cl2]
(Pt7)
(Pt5),
[Pt(FFBC)(DMSO)Cl2]
[Pt(ClClBC)(DMSO)Cl2]
and
[Pt(BrBrBC)(DMSO)Cl2] (Pt8) were synthesized by the direct reaction of MeOBC,
OHBC, BC, FBC, BrBC, FFBC, ClClBC and BrBrBC with cis-Pt(DMSO)2Cl2,
respectively, in a mixture of CH3OH and DMSO at 90 ℃ for 48 h. These ligands and
their Pt(II) complexes were characterized by single crystal X-ray diffraction analysis,
1H NMR, ultraviolet-visible (UV-Vis), ESI-MS, IR and elemental analysis (Fig.
S1−S48). Furthermore, Pt1−Pt8 were demonstrated to be stable for 48 h in TBS (10
mM Tris-HCl buffer containing 1% DMSO, pH = 7.35) by UV-Vis spectroscopy (Fig.
S41).
[Scheme 1]
2.2 Crystal structures of Pt1−Pt8
As shown in Figs. 1 and S42−S48, the Pt(II) centers of Pt(II) complexes Pt1−Pt8
adopt four coordinated square planar geometry and are surrounded by one substituted
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3-(2′-benzimidazolyl)coumarin (MeOBC, OHBC, BC, FBC, BrBC, FFBC, ClClBC or
BrBrBC), two chloride and one DMSO. The selected crystallographic information of
the Pt(II) complexes Pt1−Pt8 are summarized in Tables S1−S20. The Pt–N, Pt–S and
Pt–Cl distances are in the ranges of 2.0370 to 2.0640 Å, 2.2111 to 2.2239 Å and
2.2856 to 2.3102 Å, respectively (Tables S1−S20), which are within the normal range.
[Fig. 1]
2.3 In vitro cell cytotoxicity
The in vitro cytotoxicity of cis-Pt(DMSO)2Cl2, cisplatin, the ligands MeOBC, OHBC,
BC, FBC, BrBC, FFBC, ClClBC and BrBrBC, as well as the Pt(II) complexes
Pt1−Pt8, against a panel of human HeLa, Hep-G2, SK-OV-3/DDP, SK-OV-3 tumor
cell
lines
and
normal
HL-7702
cells
were
evaluated
by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Tables 1
and S21). Against the selected cancer cell lines, the inhibitory rates of Pt1−Pt8 were
higher than that of MeOBC, OHBC, BC, FBC, BrBC, FFBC, ClClBC, BrBrBC and
cis-Pt(DMSO)2Cl2, respectively (Table S21). As reported in Table 1, Pt1 showed the
highest cytotoxicity against all the selected cancer cell lines and the cytotoxicity of
metal complexes followed the order of Pt1>Pt2>Pt4>Pt5>Pt6>Pt7>Pt8>Pt3. Such
observed different antiproliferative effects can be due to the influence of the electronic
effect of the methoxy, hydroxyl and halogen substituted group of MeOBC, OHBC,
BC, FBC, BrBC, FFBC, ClClBC and BrBrBC in the Pt(II) complexes Pt1−Pt8 [37].
Importantly, Pt1−Pt8 showed overall higher in vitro cytotoxicity than that of cisplatin
towards human SK-OV-3/DDP tumor cells, with IC50 values from 1.01–10.32 µM.
The SK-OV-3/DDP cell line was the most sensitive to Pt1−Pt8. Furthermore, the Pt(II)
complexes Pt1−Pt8 displayed low cytotoxicity towards normal HL-7702 cells, which
suggested their potential cytotoxic selectivity for SK-OV-3/DDP tumor cells. In
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addition, Pt1 (1.0 µM), about 43.0% of the SK-OV-3/DDP tumor cells underwent cell
apoptotic phase, whereas in the case of cisplatin (70.0 µM), Pt2 (3.0 µM) and Pt3
(10.0 µM) treated cells, only 8.2%, 19.1% and 11.2% underwent apoptosis,
respectively (Figs. 8 and S50). Therefore, Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0
µM) were selected for the in vitro antitumor mechanism assays in SK-OV-3/DDP
tumor cells for 24 h.
[Table 1]
2.4 TRAP-silver staining assay
It has been reported that inhibition of telomerase activity by Pt-based drugs may lead
to continuous telomere shortening followed by growth arrest and cell apoptosis in the
cancer cells [39,40]. Here, we compared the ability of Pt1 (1.0 µM), Pt2 (3.0 µM),
Pt3 (10.0 µM), Pt4 (3.0 µM) and Pt5 (10.0 µM) to inhibit the telomerase activity in
the SK-OV-3/DDP cancer cells by the telomeric repeat amplification protocol
(TRAP)-silver staining assay. As shown in Fig. 2, the Pt(II) complex Pt1 (1.0 µM)
was shown to be highly active in the inhibition of the telomerase activity with an
inhibitory rate of 52.28%, and that by Pt2 (3.0 µM), Pt3 (10.0 µM), Pt4 (3.0 µM) and
Pt5 (10.0 µM) were 39.57%, 35.90%, 14.55% and 11.82%, respectively.
[Fig. 2]
2.5 Cellular uptake
Previous studies have suggested that the biological activity of Pt-based drugs was
strongly dependent on the accumulation in tumor cells [41−48]. Because of the weak
inhibitory activity of Pt4 (3.0 µM) and Pt5 (10.0 µM) in telomerase activity assay,
only Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) were studied. The cellular uptake
assay for Pt1 (1.0 µM), Pt2 (3.0 µM), Pt3 (10.0 µM) and cisplatin (15.0 µM) were
conducted in SK-OV-3/DDP tumor cells, and the accumulation of platinum (Pt) was
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examined by inductively coupled plasma mass spectrometry (ICP-MS) assay, which
were performed as Sadler and Lippard et al reported [41−48]. Notably, the uptake of
Pt1 (6.52±0.26 nmol of Pt/106 cells) appeared to increase steadily over the course of
24 h, and its concentration was higher than that of Pt detected in the SK-OV-3/DDP
cells treated with Pt2 ((5.18±0.12 nmol of Pt)/106 cells), Pt3 ((4.35±0.05 nmol of
Pt)/106 cells) and cisplatin ((4.11±0.18 nmol of Pt)/106 cells) under the same
condition (Fig. 3).
[Fig. 3]
2.6 Effects of Pt1−Pt3 on c-myc and hTERT transcription and expression
Western blot, Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay and
transfection were performed [49−52] to investigate whether the Pt(II) complexes Pt1
(1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) could affect the transcription and
expression of the oncoprotein c-myc and human telomerase reverse transcriptase gene
(hTERT), consequently inhibiting the telomerase activity. Firstly, we explored the
effects of Pt(II) complexes Pt1 (1.0 µM), Pt2 (3.0 µM), Pt3 (10.0 µM) on the mRNA
and protein levels of c-myc and hTERT. As shown in Figs. 4A-C, Western blot and
RT-PCR analysis revealed that Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM),
especially Pt1 (1.0 µM), reduced the mRNA and protein levels of c-myc and hTERT
in the SK-OV-3/DDP tumor cells. Furthermore, after successful transfection of
enhanced green fluorescent protein (EGFP) and c-myc plasmid vector, Pt1 (1.0 µM)
exhibited a remarkable decrease in the emission of the bright green fluorescence of a
luciferase reporter (Figs. 4D and E). The inhibition of the c-myc promoter in the
SK-OV-3/DDP cells was in the following order: Pt1>Pt2>Pt3.
[Fig. 4]
2.7 Pt1−Pt3 increase levels of mitochondrial Ca2+ fluctuation and triggers ROS
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generation
Studies suggested that increased levels of mitochondrial Ca2+ stimulate mitochondrial
reactive oxygen species (ROS) formation, which causes the opening of mPTP
(mitochondrial permeability transition pore) and the complete collapse of
mitochondria membrane potential (MMP) [53−55]. To determine whether Pt1 (1.0
µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) induce increase of mitochondrial Ca2+ and
trigger ROS generation in SK-OV-3/DDP tumor cells, FACS analysis of
mitochondrial Ca2+ fluctuation and ROS generation were carried out. As shown in
Figs. 5A and B, treatments of SK-OV-3/DDP tumor cells with Pt1 (1.0 µM), Pt2 (3.0
µM) and Pt3 (10.0 µM) for 24 h resulted in a pronounced increase of ROS level and
Ca2+ fluctuation in the following order: Pt1>Pt2>Pt3. The results suggested that
treatments with Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) led to an increase in
ROS production, which may represent a critical step in the Pt1 (1.0 µM), Pt2 (3.0 µM)
and Pt3 (10.0 µM) induced apoptosis in human SK-OV-3/DDP tumor cells [53−55].
[Fig. 5]
2.8 Pt1−Pt3 trigger apoptosis via the mitochondrial pathway
To investigate the effects of Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) on
mitochondrial
function,
we
first
measured
MMP
using
the
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine (JC-1) staining
[53−55]. JC-1 is a fluorescent dye that accumulates selectively in mitochondria
depending on the membrane potential. As shown in Fig. 6, exposure of SK-OV-3/DDP
tumor cells to Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) for 24 h could cause
disruption of MMP. In addition, flow cytometry and Western blot analysis showed that
treating cells with Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) resulted in
accumulated cytochrome c, decrease of bcl-2 level and increases of capase-3, bax,
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apaf-1 and caspase-9 levels (Figs. 7 and S49). All these results demonstrated that Pt1
(1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) could induce a mitochondrion-mediated
apoptotic pathway in SK-OV-3/DDP tumor cells [53−55].
[Fig. 6]
[Fig. 7]
2.9 Induction of apoptosis by Pt1−Pt3 in SK-OV-3/DDP cells
We determined whether Pt1−Pt3-mediated loss of SK-OV-3/DDP tumor cell
viability was due to the induction of cell apoptosis [56, 57]. After exposure to Pt1 (1.0
µM), about 43.0% of the SK-OV-3/DDP tumor cells underwent cell apoptotic phase,
whereas in the case of Pt2 (3.0 µM) and Pt3 (10.0 µM) treated cells, 19.1% and 11.2%
underwent apoptosis, respectively (Fig. 8). However, cisplatin (70.0 µM) did not
significantly induce SK-OV-3/DDP tumor cell apoptosis (Q2+Q3, 8.17%) under the
same conditions (Fig. S50). Therefore, Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM)
were selected for the in vitro antitumor mechanism assays in SK-OV-3/DDP tumor
cells for 24 h.
[Fig. 8]
Structure-Activity Relationships
Based on the above described results, the cytotoxic mechanism studies of
different substituted 3-(2′-benzimidazolyl) coumarins ligands (MeOBC, OHBC, BC,
FBC, BrBC, FFBC, ClClBC and BrBrBC) and eight new platinum(II) complexes
Pt1−Pt8, certain SAR (structure-activity relationships) trends emerged among the
different groups substituted in substituted 3-(2′-benzimidazolyl) coumarins ligands.
Such observed different in vitro antitumor activity and antitumor mechanism can be
due to the influence of the electronic effect of the methoxy, hydroxyl and halogen
substituted group of MeOBC, OHBC, BC, FBC, BrBC, FFBC, ClClBC and BrBrBC
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in the Pt(II) complexes Pt1−Pt8 [37].
i) The in vitro antitumor activity was follow the order of
MeOBC>OHBC>FBC>BrBC>FFBC>ClClBC>BrBrBC>BC
(or
Pt1>Pt2>Pt4>Pt5>Pt6>Pt7>Pt8>Pt3).
ii) The telomerase activity was follow the order of MeOBC>OHBC>FBC>BrBC>
BC (or Pt1>Pt2>Pt4>Pt5>Pt3).
iii) RT-PCR and Western blot of c-myc and hTERT, transfection and cell
apoptosis, Pt1−Pt3 increase levels of mitochondrial Ca2+ fluctuation and triggers ROS
generation, Pt1−Pt3 caused mitochondrial dysfunction in SK-OV-3/DDP tumor cells
as well as cellular uptake ability follow the order of MeOBC>OHBC>BC (or
Pt1>Pt2>Pt3).
3. Conclusions
Eight
[Pt(OHBC)(DMSO)Cl2] (Pt2), [Pt(BC)(DMSO)Cl2] (Pt3), [Pt(FBC)(DMSO)Cl2]
(Pt4), [Pt(BrBC)(DMSO)Cl2] (Pt5), [Pt(FFBC)(DMSO)Cl2] (Pt6),
new
Pt(II)
complexes
[Pt(MeOBC)(DMSO)Cl2]
(Pt1),
[Pt(ClClBC)(DMSO)Cl2] (Pt7) and [Pt(BrBrBC)(DMSO)Cl2] (Pt8) have been
synthesized and fully characterized. Pt1 exhibited selective cytotoxicity to
SK-OV-3/DDP tumor cells, and it showed higher cytotoxicity than that of Pt2−Pt8
and the corresponding substituted 3-(2′-benzimidazolyl)coumarins. Additionally, the
Pt(II) complexes Pt1−Pt3 increased the levels of ROS and mitochondrial Ca2+
fluctuation, and induced the decrease of MMP, consequently causing mitochondrial
dysfunction. Various experiments showed that Pt1 was a telomerase inhibitor
targeting c-myc promoter elements.
4. Experimental
4.1 Synthesis
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4.1.1 Synthesis of ligands
Synthesis of MeOBC, OHBC and BC ligands
The ligands MeOBC, OHBC and BC were synthesized according to the reported
procedures [35,36].
Synthesis of FBC, BrBC, FFBC, ClClBC and BrBrBC ligands
2-cyanomethylbenzimidazole
4-fluoro-2-hydroxybenzaldehyde,
3,5-difluorosalicylaldehyde,
(0.01mol)
and
4-bromo-2-hydroxybenaldehyde,
3,5-dichlorosalicylaldehyde or
0.01
mol
3,5-dibromosalicylaldehyde were added to ethanol (30 mL). Piperidine (0.1 mL) was
then added and the mixture stirred at 55 ℃ for 24 h. The yellow precipitate which
gradually separated out of the solution was collected and heated under reflux in 2.0%
aqueous hydrochloric acid solution (150 mL) for 6.0 h. The mixture was cooled and
excess
sodium
acetate
added.
The
resulting
brown
product
of
3-(2′-benzimidazolyl)-7-fluorocoumarin
3-(2′-benzimidazolyl)-7-bromocoumarin
(FBC),
(BrBC),
(FFBC),
and
3-(2′-benzimidazolyl)-6,8-difluorocoumarin
3-(2′-benzimidazolyl)-6,8-dichlorocoumarin
(ClClBC)
3-(2′-benzimidazolyl)-6,8-dibromocoumarin (BrBrBC) were suitable for structural
characterization.
Characterization of FBC. Yield: 82.56%. ESI-MS m/z: 281.0 [M+H]+. Elemental
analysis calcd (%) for C16H9FN2O2: C 68.57, H 3.24, N 10.00; found: C 68.65, H 3.31,
N 9.89. 1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.09 (dd, J = 8.7, 6.4 Hz, 1H),
7.69 (dd, J = 6.1, 3.2 Hz, 2H), 7.55 (dd, J = 9.6, 2.5 Hz, 1H), 7.37 (td, J = 8.7, 2.5 Hz,
1H), 7.27 (dd, J = 6.1, 3.2 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 166.25, 163.74,
159.33, 155.10, 154.96, 145.91, 142.78, 132.33, 132.22, 123.33, 116.58, 116.55,
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115.99, 115.55, 113.85, 113.62, 104.58, 104.32, 40.63, 40.42, 40.21, 40.00, 39.79,
39.58, 39.38, 25.09.
Characterization of BrBC. Yield: 79.52%. ESI-MS m/z: 339.2 [M-H]−. Elemental
analysis calcd (%) for C16H9BrN2O2: C 56.33, H 2.66, N 8.21; found: C 56.25, H 2.60,
N 8.13. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 7.95 (d, J = 1.8 Hz, 1H), 7.92
(d, J = 8.4 Hz, 1H), 7.81 (dd, J = 6.1, 3.2 Hz, 2H), 7.72 (dd, J = 8.3, 1.8 Hz, 1H), 7.44
13
(dd, J = 6.1, 3.2 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 158.40, 154.27, 144.83,
144.55, 131.73, 129.15, 127.77, 125.17, 120.03, 118.24, 115.51, 40.63, 40.42, 40.21,
40.00, 39.79, 39.58, 39.37.
Characterization of FFBC. Yield: 81.42%. ESI-MS m/z: 299.0 [M+H]+. Elemental
analysis calcd (%) for C16H8F2N2O2: C 64.43, H 2.70, N 9.39; found: C 64.52, H 2.77,
N 9.25. 1H NMR (400 MHz, DMSO-d6) δ 12.59 (s, 1H), 9.13 (d, J = 1.2 Hz, 1H), 7.85
13
– 7.73 (m, 2H), 7.69 (dd, J = 5.8, 3.4 Hz, 2H), 7.28 – 7.22 (m, 2H). C NMR (101
MHz, DMSO-d6) δ 158.32, 145.55, 141.05, 121.61, 121.58, 121.49, 121.47, 119.28,
113.45, 110.90, 110.86, 110.65, 110.62, 108.60, 108.39, 108.31, 108.10, 40.62, 40.41,
40.20, 39.99, 39.78, 39.58, 39.37.
Characterization of ClClBC. Yield: 75.01%. ESI-MS m/z: 330.9 [M+H]+.
Elemental analysis calcd (%) for C16H8Cl2N2O2: C 58.03, H 2.43, N 8.46; found: C
58.10, H 2.39, N 8.39. 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.13 (d, J = 2.4
Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H), 7.72 (dd, J = 6.1, 3.2 Hz, 2H), 7.29 (dd, J = 6.1, 3.2
13
Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 158.40, 148.20, 145.26, 141.36, 132.32,
129.21, 128.04, 123.63, 121.95, 121.32, 40.62, 40.42, 40.21, 40.00, 39.79, 39.58,
39.37, -15.55.
Characterization of BrBrBC. Yield: 79.63%. ESI-MS m/z: 418.9 [M-H]−. Elemental
analysis calcd (%) for C16H8Br2N2O2: C 45.75, H 1.92, N 6.67; found: C 45.70, H
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2.00, N 6.64. 1H NMR (400 MHz, DMSO-d6) δ 9.14, 8.27, 8.26, 8.25, 8.25, 8.24, 8.23,
8.03, 8.03, 8.02, 8.02, 7.77, 7.76, 7.73, 7.72, 7.72, 7.71, 7.70, 7.32, 7.31, 7.30, 7.29,
7.28, 7.28, 7.26, 7.21, 7.21, 2.52, 2.51, 2.51, 2.50, 2.50. 13C NMR (101 MHz,
DMSO-d6) δ 158.45, 149.69, 145.13, 141.55, 137.72, 131.55, 123.77, 122.28, 117.24,
110.64, 40.62, 40.42, 40.21, 40.00, 39.79, 39.58, 39.37.
4.1.2 Synthesis of Pt1−Pt8
The Pt(II) complexes [Pt(MeOBC)(DMSO)Cl2] (Pt1), [Pt(OHBC)(DMSO)Cl2] (Pt2),
[Pt(BC)(DMSO)Cl2] (Pt3), [Pt(FBC)(DMSO)Cl2] (Pt4), [Pt(BrBC)(DMSO)Cl2]
(Pt5), [Pt(FFBC)(DMSO)Cl2] (Pt6), [Pt(ClClBC)(DMSO)Cl2] (Pt7) and
[Pt(BrBrBC)(DMSO)Cl2] (Pt8) were prepared by treating MeOBC, OHBC, BC, FBC,
BrBC, FFBC, ClClBC or BrBrBC (0.10 mmol) with cis-Pt(DMSO)2Cl2 (0.10 mmol)
in methanol (2.0 mL) and DMSO (0.5mL) at 90 ℃ for 48 h, respectively. The yellow
block crystals suitable for X-ray diffraction analysis were harvested.
Characterization of Pt1. Yield: 91.53%. ESI-MS m/z: 635.1 [M-H]−. Elemental
analysis calcd (%) for C19H18Cl2N2O4PtS: C 35.86, H 2.85, N 4.40; found: C 35.80, H
2.94, N 4.43. IR (KBr): 3311, 3024, 2924, 1713, 1604, 1577, 1467, 1436, 1272, 1121,
1104, 1032, 953, 778, 754, 537, 438 cm−1.
Characterization of Pt2. Yield: 80.69%. ESI-MS m/z: 621.0 [M-H]−. Elemental
analysis calcd (%) for C18H16Cl2N2O4PtS: C 34.74, H 2.59, N 4.50; found: C 34.65, H
2.67, N 4.43. IR (KBr): 3325, 3005, 1702, 1606, 1577, 1515, 1435, 1290, 1142, 1092,
1018, 976, 750, 730, 543, 510, 438 cm−1.
Characterization of Pt3. Yield: 76.22%. ESI-MS m/z: 605.1 [M-H]−. Elemental
analysis calcd (%) for C18H16Cl2N2O3PtS: C 35.65, H 2.66, N 4.62; found: C 35.60, H
2.73, N 4.59. IR (KBr): 3328, 3302, 3005, 1712, 1605, 1572, 1434, 1319, 1140, 1106,
1024, 741, 440 cm−1.
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Characterization of Pt4. Yield: 70.88%. ESI-MS m/z: 623.0 [M-H]−. Elemental
analysis calcd (%) for C18H15Cl2FN2O3PtS: C 34.63, H 2.42, N 4.49; found: C 34.59,
H 2.49, N 4.40. IR (KBr): 3319, 3235, 3006, 1706, 1609, 1575, 1509, 1420, 1235,
1132, 1023, 775, 732, 618, 492, 437 cm−1.
Characterization of Pt5. Yield: 95.03%. ESI-MS m/z: 685.0 [M-H]−. Elemental
analysis calcd (%) for C18H15BrCl2N2O3PtS: C 31.55, H 2.21, N 4.09; found: C 31.50,
H 2.32, N 4.13. IR (KBr): 3393, 3288, 3021, 2925, 1720, 1594, 1400, 1314, 1133,
1062, 968, 727, 589, 438 cm−1.
Characterization of Pt6. Yield: 78.03%. ESI-MS m/z: 641.0 [M-H]−. Elemental
analysis calcd (%) for C18H14Cl2F2N2O3PtS: C 33.66, H 2.20, N 4.36; found: C 33.60,
H 2.29, N 4.30. IR (KBr): 3242, 3039, 1727, 1590, 1431, 1378, 1309, 1241, 1139,
1095, 969, 737, 677, 442 cm−1.
Characterization of Pt7. Yield: 83.56%. ESI-MS m/z: 675.0 [M-H]−. Elemental
analysis calcd (%) for C18H14Cl4N2O3PtS: C 32.02, H 2.09, N 4.15; found: C 31.89, H
2.00, N 4.26. IR (KBr): 3225, 1729, 1564, 1407, 1152, 1111, 1024, 992, 764, 737,
548, 434 cm−1.
Characterization of Pt8. Yield: 70.12%. ESI-MS m/z: 762.9 [M-H]−. Elemental
analysis calcd (%) for C18H14Br2Cl2N2O3PtS: C 28.29, H 1.85, N 3.67; found: C 28.20,
H 2.07, N 3.71. IR (KBr): 3206, 1727, 1614, 1555, 1448, 1401, 1247, 1150, 1110,
1023, 975, 935, 740, 528, 432 cm−1.
4.2 Materials and methods
The X-Ray crystallography structures of Pt1−Pt8 were solved by Sheldrick method
[58]. The in vitro antitumor mechanism assays of Pt1−Pt8 were according to the
reported procedures [59−64]. In addition, all the materials, instrumentation and the
detailed procedures for other experimental methods were described in supporting
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information.
Abbreviations
TBS
Tris-HCl buffer
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
reactive oxygen species
ROS
IC50
half maximal inhibitory concentration
mitochondrial permeability transition pore
mitochondria membrane potential
mPTP
MMP
SK-OV-3/DDP human cisplatin-resistant SK-OV-3 cells
cells
Hep-G2 cells
SK-OV-3 cells human ovarian cancer cells
HeLa cells human sarcoma HeLa cancer cells
HL-7702 cells human normal hepatocytes cells
human hepatocellular carcinoma cells
PI
propidium iodide
Δψ
JC-1
ox
mitochondrial membrane potential
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine
OAc
en
1,2-ethylenediamine
dien
N'-[2-(dimethylamino)ethyl]-N,N-dimethylethylenediamine
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Acknowledgement
This work was supported by the National Natural Science Foundation of China (Nos.
51572050, 21867017, 21601038, 21261025 and 21761033), the Natural Science
Foundation of Guangxi (Nos. 2018GXNSFBA138021, 2015GXNSFDA139007 and
2016GXNSFAA380085), Guangxi Key Laboratory of Electrochemical and
Magnetochemical Functional Materials (EMFM20162107), the Key Foundation
Project of Colleges and Universities in Guangxi (No. ZD2014108), the PhD Research
Startup Program of Yulin Normal University (No. G2017009) and the Innovative Team
& Outstanding Talent Program of Colleges and Universities in Guangxi (2014-49).
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Figure Captions:
Fig. 1. The molecular structures of the Pt1.
Fig. 2. TRAP assay performed in the presence of Pt1 (1.0 µM), Pt2 (3.0 µM), Pt3
(10.0 µM), Pt4 (3.0 µM) and Pt5 (10.0 µM) in SK-OV-3/DDP cancer cells,
respectively.
Fig. 3. ICP-MS data for SK-OV-3/DDP cancer cells incubated with Pt1 (1.0 µM), Pt2
(3.0 µM), Pt3 (10.0 µM) and cisplatin (15.0 µM), respectively.
Fig. 4. Induction of telomerase activity by Pt1 (1.0 µM), Pt2 (3.0 µM), Pt3 (10.0 µM)
via c-myc promoter elements activation. (A) The expression percentage of the hTERT
and c-myc genes after 24 h of incubation of SK-OV-3/DDP tumor cells with the Pt(II)
complexes Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) as determined by means of
the RT-qPCR assay. (B) Western blot analysis of hTERT and c-myc expression in the
SK-OV-3/DDP tumor cells with the Pt(II) complexes Pt1 (1.0 µM), Pt2 (3.0 µM) and
Pt3 (10.0 µM). (C) The whole cell extracts were prepared and analyzed by Western
blot analysis using antibodies against hTERT and c-myc proteins. The same blots
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were stripped and reprobed with a β-actin antibody to show equal protein loading.
Western blotting bands from three independent measurements were quantified with
Image J. in (C). Transfection of 2.0 mg EGFP (enhanced green fluorescent protein)
plasmid (D) and 2.0 mg c-myc plasmid vectors (E) in the SK-OV-3/DDP tumor cells
treated with the Pt(II) complexes Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) for
24.0 h, respectively, and these results was assessed by fluorescence microcopy or
Multimodel Plate Reader with luciferase reporter gene assay kit.
Fig. 5. (A) Intracellular ROS levels in SK-OV-3/DDP tumor cells exposed to the Pt(II)
complexes Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) for 24 h and the DCF
fluorescent intensity was determined by flow cytometric analysis, respectively. (B)
Effects of the Pt(II) complexes Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) on
Ca2+ activation level in SK-OV-3/DDP tumor cells.
Fig. 6. The Pt(II) complexes Pt1 (1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) induced
depletion of mitochondrial membrane potential (Δψm) in SK-OV-3/DDP tumor cells.
These cells were treated with the Pt(II) complexes Pt1 (1.0 µM), Pt2 (3.0 µM) and
Pt3 (10.0 µM) for 24 h and then analyzed by JC-1 flow cytometry.
Fig. 7. (A) The SK-OV-3/DDP tumor cells were treated with the Pt(II) complexes Pt1
(1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) for 24 h and the expression levels of the
apoptosis-related proteins were examined by Western blotting. (B) The whole cell
extracts were prepared and analyzed by Western blot analysis using antibodies against
the apoptosis related proteins. The same blots were stripped and reprobed with a
β-actin antibody to show equal protein loading. Western blotting bands from three
independent measurements were quantified with Image J. in (B).
Fig. 8. Apoptosis of SK-OV-3/DDP tumor cells induced by the Pt(II) complexes Pt1
(1.0 µM), Pt2 (3.0 µM) and Pt3 (10.0 µM) for 24 h, respectively.
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Scheme 1. Synthetic routes for substituted 3-(2′-benzimidazolyl)coumarins
platinum(II) complexes Pt1−Pt8. (a) piperidine, EtOH, 55℃ for 24 h; (b) 2.0% HCl,
reflux, 6.0 h; (c) cis-Pt(DMSO)2Cl2, CH3OH/DMSO (v/v=2.0 /0.5mL), 90℃ for 24 h.
Table 1. IC50a (µM) values of cisplatin, the ligands MeOBC, OHBC, BC, FBC, BrBC,
FFBC, ClClBC, BrBrBC, the Pt(II) complexes Pt1−Pt8 against the five human cells
for 48 h.
Compounds
MeOBC
Pt1
HeLa
Hep-G2
SK-OV-3/DDP
90.06 ± 0.41
1.01 ± 0.27
＞100
SK-OV-3
＞100
HL-7702
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
30.19 ± 0.63
5.26 ± 0.87 25.69 ± 0.46
＞100
7.21 ± 1.45 35.26 ± 1.43
19.89 ± 1.46
＞100
OHBC
Pt2
38.06 ± 1.03
3.26 ± 1.06
＞100
28.06 ± 1.27
＞100
＞100
＞100
＞100
＞100
BC
58.79 ± 0.56
40.02 ± 1.85
10.32 ± 0.66
＞100
86.36 ± 1.65
＞100
Pt3
FBC
10.23 ± 0.45 45.26 ± 1.06
＞100
16.02 ± 0.47 50.11 ± 1.44
＞100
25.12 ± 1.17 86.03 ± 0.55
5.15 ± 0.59
＞100
35.29 ± 0.77
＞100
Pt4
BrBC
Pt5
56.23 ± 1.21
8.02 ± 1.19
＞100
40.11 ± 1.17
＞100
FFBC
Pt6
68.16 ± 0.91
8.69 ± 0.83
＞100
57.56 ± 1.49
＞100
＞100
＞100
＞100
＞100
ClClBC
Pt7
78.02 ± 0.55
31.25 ± 1.52
86.34 ± 1.70
41.51 ± 1.02
9.16 ± 1.59
＞100
65.22 ± 0.61
＞100
BrBrBC
Pt8
9.86 ± 0.54
72.06 ±1.45
Cisplatinb 15.09 ± 1.19 12.06 ± 0.91
70.26 ± 1.93
17.12 ± 0.59 16.24 ± 0.63
24

ACCEPTED MANUSCRIPT
a IC50 values are presented as the mean ± SD (standard error of the mean) from five independent
experiments. b Cisplatin was dissolved at a concentration of 1.0 mM in 0.154 M NaCl [37, 38].
Fig. 1
25

ACCEPTED MANUSCRIPT
Fig. 2
26

ACCEPTED MANUSCRIPT
Fig. 3
Fig. 4
27

ACCEPTED MANUSCRIPT
Fig. 5
Fig. 6
28

ACCEPTED MANUSCRIPT
Fig. 7
Fig. 8
29