Design, synthesis and biological evaluation of naphthalenebenzimidizole platinum (II) complexes as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2019.112033

A serial of naphthalenebenzimidizole-Pt complexes 1–6 were designed and synthesized as antitumor agents. In vitro antitumor assay results showed that complexes 1–6 exhibited moderate to high antiproliferative activity against Hela, HepG2, SKOV-3, NCI–H460, BEL-7404 and A549 cancer cell lines, while they displayed obvious sensitivity and selectivity against SMMC-7721 and U251 cell lines and low toxicity against normal HL-7702 cells, in comparison with cisplatin. In vivo antitumor assay results indicated that complex 1 and 5 exhibited important in vivo antiproliferative activity in the NCI-460 and SMMC-7721 models, in comparison with cisplatin, respectively. Complexes 1 and 5 exhibited better antiproliferative activity against A549CDDP and SKOV3CDDP cell lines than cisplatin, with IC₅₀ values of 6.98 ± 0.47 μM, 5.62 ± 0.88 μM and 13.13 ± 2.11 μM, 5.30 ± 0.33 μM, respectively, while they displayed potential antiproliferation against A549 and SKOV3 cell lines, with IC₅₀ values of 7.32 ± 0.51 μM, 5.19 ± 0.49 μM and 14.92 ± 0.11 μM, 12.19 ± 0.92 μM, indicating the introduction of naphthalenebenzimidizole into platinum-metal system may overcome the resistance. Mechanistic studies showed that the representative complexes 1 and 5 exerted the antitumor effect mainly by the obvious covalent binding with DNA and the upregulation of the expression level of intracellular topo I, showing different action mechanism from cisplatin.

Full text of DOI:10.1016/j.ejmech.2019.112033

Journal Pre-proof
Design, synthesis and biological evaluation of naphthalenebenzimidizole platinum (II)
complexes as potential antitumor agents
Gui-Bin Liang, Yan-Cheng Yu, Jian-Hua Wei, Wen-Bin Kuang, Zhen-Feng Chen, Ye
Zhang
PII:
S0223-5234(19)31191-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.112033
EJMECH 112033
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 September 2019
Revised Date: 31 December 2019
Accepted Date: 31 December 2019
Please cite this article as: G.-B. Liang, Y.-C. Yu, J.-H. Wei, W.-B. Kuang, Z.-F. Chen, Y. Zhang, Design,
synthesis and biological evaluation of naphthalenebenzimidizole platinum (II) complexes as potential
antitumor agents, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2019.112033.
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Graphical abstract
R1
11
12
R2
10
9
13
N
8
O
N
1: R1 = R2 =H
2: R1 = R2 =Cl
Pt(DMSO)Cl2
7
3: R1 = R2 =CH3
4. R1 = Cl, R2 =H
5. R1 = F, R2 =H
6. R1 = H, R2 =CF3
6
5
1
2
4
3
1-6
5: IC50=2.36 µM
1 / 1

Design,
synthesis
and
biological
evaluation
of
naphthalenebenzimidizole platinum (II) complexes as potential
antitumor agents
Gui-Bin Lianga,b†, Yan-Cheng Yua,b,c†, Jian-Hua Weia,b, Wen-Bin Kuanga,b, Zhen-Feng Chena,*,
Ye Zhanga,b,d,
*
a School of Pharmacy, Guilin Medical University, Guilin 541004, China
b State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School
of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
c Jiangsu Key Laboratory of Drug Design and Optimization and Department of Chemistry, China
Pharmaceutical University, Nanjing 211198, China
d
Department of Chemistry & Pharmaceutical Science, Guilin Normal College, Xinyi Road 15,
Guangxi 541001, China
† These authors contributed equally to this work.
* Corresponding author. State Key Laboratory for Chemistry and Molecular Engineering of
Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin
541004, China; School of Pharmacy, Guilin Medical University, Guilin 541004, China.
E-mail addresses: chenzf@gxnu.edu.cn (Z.-F Chen), zhangye81@126.com (Y. Zhang)
1 / 20

Abstract
A serial of naphthalenebenzimidizole-Pt complexes 1–6 were designed and synthesized as
antitumor agents. In vitro antitumor assay results showed that complexes 1–6 exhibited moderate
to high antiproliferative activity against Hela, HepG2, SKOV-3, NCI-H460, BEL-7404 and A549
cancer cell lines, while they displayed obvious sensitivity and selectivity against SMMC-7721 and
U251 cell lines and low toxicity against normal HL-7702 cells, in comparison with cisplatin. In
vivo antitumor assay results indicated that complex 1 and 5 exhibited important in vivo
antiproliferative activity in the NCI-460 and SMMC-7721 models, in comparison with cisplatin,
respectively. Complexes 1 and 5 exhibited better antiproliferative activity against A549CDDP and
SKOV3CDDP cell lines than cisplatin, with IC50 values of 6.98±0.47µM, 5.62±0.88µM and
13.13±2.11µM, 5.30±0.33µM, respectively, while they displayed potential antiproliferation
against A549 and SKOV3 cell lines, with IC50 values of 7.32±0.51µM, 5.19±0.49µM and
14.92±0.11µM, 12.19±0.92µM, indicating the introduction of naphthalenebenzimidizole into
platinum-metal system may overcome the resistance. Mechanistic studies showed that the
representative complexes 1 and 5 exerted the antitumor effect mainly by the obvious covalent
binding with DNA and the upregulation of the expression level of intracellular topo I, showing
different action mechanism from cisplatin.
Keywords: naphthalenebenzimidizole-Pt complexes; antitumor activity; action mechanism;
covalent DNA binding; topo I inhibition
2 / 20

1. Introduction.
Cisplatin (CDDP) has been approved as anticancer drugs by Food and Drug Administration
(FDA) and widely used for the treatment of a variety of cancers including testicular, ovarian, lung,
cervical, and bladder carcinomas in the clinical[1,2]. However, CDDP has been also found obvious
clinical drawbacks containing drug resistance, side effects and high toxicity in the clinical
applications[3,4]. Therefore, development of novel and more effective platinum-metal antitumor
drugs for overcoming the above mentioned shortcomings have greatly attracted bioinorganic
chemists’ interest and become a hot research topic in bioinorganic chemistry. To overcome these
shortcomings, a great deal of effort has been devoted to the design and synthesis of novel and
effective platinum based drugs in the past two decades[3-5]. The classic strategy has been also tried
to introduce the functional and potent antiproliferative active ligands into platinum-based
compounds and proved to be an effective method[3,4]
.
Naphthalimides, which generally contain a coplanar, generally л-deficient aromatic system and
basic side chains, are known as potential DNA and topoisomerase(topo) targeted antitumor agents
and exhibit potential antiproliferative activity against several cancers lines including breast,
hepatocellular, bladder and glioma carcinomas[6-16]. As one of the most widely studied
naphthalimides, amonafide exhibits excellent antitumor activity in clinical phase II breast cancer
trials and exerts the antitumor effect via binding DNA with intercalation and poisoning
Topoisomerase[10,17-19], indicating that screening for antitumor agents based on the active
pharmacal core naphthalimide may be a reasonable and feasible strategy, though it finally fails in
clinical phase III trials[10,17]. So the naphthalimide derivatives naphthalenebenzimidizoles were
herein selected as active ligands to introduce into platinum-based compounds to screening for
high-efficiency and low-toxic new antitumor complexes. Since that both of naphthalimide
derivatives[10,17] and platinum-based compounds[3,4] usually exhibit potential antiproliferative
activity, it is expected that the combination of naphthalimide core and platinum-metal system may
lead to better antitumor activity. In addition, naphthalimide aromatic moiety with multiple binding
sites is able to coordinate with various kinds of biologically important species such as DNA,
proteins, enzymes, biothiols and reactive oxygen species (ROS) etc[9]. Moreover, multiple
mechanisms have been proposed to improve the cytotoxicy and cellular resistance of cisplatin and
its analogues in preclinical models[3-5,20,21]. Furthermore, previous work had proved that
3 / 20

constructing complexes that displayed different DNA-binding modes was one of three viable
options in the design of new platinum drugs[22]. It was thus to expect that the introduction of
naphthalimides ligands into platinum-metal system may lead to the change of the action
mechanism including the DNA-binding modes and even multiple mechanisms, which may
consequently result to the improvement of the side effects and drug resistance. So a series of
naphthalenebenzimidizole-platinum (II) complexes were designed and synthesized in the present
work (Scheme 1). However, to the best of our knowledge, naphthalenebenzimidizole-platinum (II)
complexes have not been described in the literature. The in vitro and in vivo antitumor activities
and in vitro cytotoxity against normal cell lines, as well as the in vitro antiproliferative activity
against CDDP-resistant strain cell line were also evaluated. Furthermore, the covalent
DNA-binding and topo I inhibition assays were carried out to evaluate the action mechanism.
R1
R1
11
12
R2
R2
10
9
R1
R2
NH2
NH2
13
N
8
O
N
N
O
N
O
O
O
Pt(DMSO)Cl2
cis-Pt (DMSO)2Cl2
b
7
6
5
1
2
a
4
3
L
1-6
L1: R1 = R2 =H
L2: R1 = R2 =Cl
L3: R1 = R2 =CH3
L4: R1 =Cl, R2 = H
L5: R1 =F, R2 = H
L6: R1 = H, R2 =CF3
1: R1 = R2 =H
2: R1 = R2 =Cl
3: R1 = R2 =CH3
4: R1 =Cl, R2 = H
5: R1 =F, R2 = H
6: R1 = H, R2 =CF3
Scheme 1. General synthetic route of naphthalenebenzimidizole platinum (II) complexes 1–6. (a)
glacial acetic acid, 120 oC, 5h; (b) chloroform/methanol, 60 oC, 6h.
2. Results and Discussion
2.1. Synthesis
The ligands (naphthalenebenzimidizoles) L1‒L6 and complexes (1–6) were synthesized as
described in Scheme 1. The ligands L1‒L6 used in this study was conveniently synthesized in
moderate
yields
by
the
combination
of
1,8-naphthalic
anhydride
with
o
o-phenylenediamine/substituted o-phenylenediamine at the 120 C for 5h, in the presence of
glacial acetic acid, respectively, according to the previous literature[23,24]
.
The
naphthalenebenzimidizole platinum (II) complexes 1–6 were then prepared in moderate yields by
4 / 20

o
the treatment of L1‒L6 with cis-Pt (DMSO)2Cl2 at the 60 C for 10h, in the presence of the
mixture of chloroform/methanol (v/v=1:1), respectively. The structures of complexes 1–6 were
1
determined by H NMR spectroscopy, high-resolution mass spectrometry (HR-MS), elemental
analysis, and single-crystal X-ray diffraction analysis (Part 1 in supplmentary materials).
Fig. 1. The crystal structures of complexes 1‒6.
2.2. Crystal structures of complexes 1–6
The crystal structures of complexes 1‒6 were determined by the X-ray diffraction and the
crystallographic data were summarized in the Supporting Information (Tables S2-1‒S2-12). As
shown in Fig. 1, the ligands (L1‒L6) and cis-Pt (DMSO)Cl2 formed as monodentate coordination
complexes, respectively. The Pt-N bond lengths for complexes 1‒6 were found to be 2.041(10)Å,
2.063(15)Å, 2.056(10)Å, 2.041(8)Å, 2.052(9) Å and 2.029(8) Å, respectively, which were within
the normal range.
2.3. Stability and solubility studies of complexes 1–6
The stability of complexes 1–6 in 10 mM Tris-HCl buffer solution (Tris, 80%DMSO, pH 7.4)
was monitored by the high performance liquid chromatography (HPLC) assay, according to the
literature[25]. As shown in Fig. S2-1(in supplmentary materials), for complexes 1–6, there were
5 / 20

chiefly two peaks (one for DMSO and the other for complexes 1–6 in the HPLC spectra for 0h,
24h and 48h, and these peaks in HPLC showed no obvious change within these periods. The
results indicated that these six complexes were stable in DMSO under the physiological
conditions.
2.4. In vitro antiproliferative activity
The in vitro antiproliferative activity of ligands L1−L6 and complexes 1−6 were evaluated
against the Hela (human cervical cancer cells), HepG2 (human liver cancer cells), SKOV3 (human
ovarian cancer cells), NCI-H460 (human large cell lung cancer cells), BEL-7404 (Human liver
cancer cells), A549 (human lung cancer cells), SMMC-7721 (human liver cancer cells), U251
(human glioma cells) and HL-7702 (human liver normal cells) cell lines, using the
methylthiazoltetrazolium (MTT) assay[25,26]. For comparison, cisplatin was used as positive
control.
Table 1. αIC50 values of complexes 1−6 towards the selected tumor cell lines.
IC50（µM）
Entry
Hela
HepG-2
NCI-H460
BEL-7404
>100
SMMC-7721
>100
U251
>100
87.97±1.32 20.86±0.37 13.31±0.82
L1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
L2
>100
>100
89.31±0.48
>100
L3
>100
>100
L4
18.73±.084 11.58±0.37
52.05±1.56 >100
>100
>100
90.09±0.56
>100
L5
>100
>100
L6
9.24±1.35 18.17±1.26 17.98±0.42
78.30±1.32
>100
6.85±1.41
5.46±0.76
4.92±1.55
4.99±1.64
2.36±1.44
7.33±0.89
27.43±0.35
11.61±1.19
19.00±0.39
9.80±0.84
11.44±0.57
8.37±0.94
8.75±1.34
40.6±0.77
1
>100
>100
>100
>100
>100
2
9.37±0.72 17.01±0.67
41.99±0.76
>100
3
>100
>100
>100
4
24.87±0.37 13.62±0.72
>100
>100
5
6
19.09±0.13 16.33±0.98
>100
9.45±0.25 10.28±0.77 20.36 ± 0.50 10.08 ±0.52
Cisplatin
αIC50 values are presented as mean ± SD (standard error of the mean) from three repeating experiments.
As shown in Table 1 and Table S3-1, the complexes 1−6 exhibited obvious sensitivity and
selectivity against SMMC-7721 and U251 cell lines, while they displayed lower cytotoxicity on
the HL-7702 normal cell line, compared with cisplatin, indicating that complexes 1−6 may be
good candidates for antitumor agents. By the comparison of the antiproliferative activity of L1
6 / 20

with 1, L2 with 2, L3 with 3, L4 with 4, L5 with 5, L6 with 6, respectively, it could be found that
the complexes 1−6 exhibited increased antiproliferative activity than their corresponding ligands
against the selected cancer cell lines, implying that synergistic effect of complexes could improve
the antiproliferative activity. In the Hela line assay, complexes 1 and 2 exhibited equal
antiproliferation to cisplatin (IC50 = 9.45±0.25 µM), with IC50 values of 9.24±1.35 and 9.37±0.7
µM, respectively. In the SKOV-3 and A549 cell line assay (Table S3-1), complex 6 showed better
antiproliferative activity than cisplatin (IC50 =16.32±1.37µM and 6.37±1.03µM ), with IC50 of
12.19±0.92 µM and 5.19±0.49µM, respectively. In the NCI-H460 cell line assay, complexes 1 and
6 exhibited higher antiproliferation than cisplatin (IC50 = 20.36±0.50 µM), with IC50 of 17.98±0.42
and 13.62±0.72 µM, respectively. It was important noting that complexes 1−6 exhibited better
antiproliferation than cisplatin against SMMC-7721 and U251 cell lines, with IC50 values in the
range of 2.36−11.61 µM, indicating that the introduction of naphthalenebenzimidizole ligands into
platinum-metal system may lead to sensitivity and selectivity against SMMC-7721 and U251 cell
lines.
The structure-activity relationship study for complexes 1−6 was then performed. The
substituents in benzimidazole moiety were found to have important and irregular influence on
their antiproliferative activity. By the comparison of complex 1 with 2, 3 and 6, respectively, it
could be found that the introduction of two methyl, two chlorine and trifluoromethyl groups in
benzimidazole moiety increased the cytotoxity against the normal cell line HL-7702, implying that
the introduction of substituents in 12-position (R2) may increase the toxicity. In addition, by the
comparison of complex 1 with 4, 1 and 5, respectively, it could be concluded that the introduction
of fluorine and trifluoromethyl groups in benzimidazole moiety could increase the
antiproliferation against SMMC-7721 and U251 cell lines. Moreover, by the comparison of
complex 1 with 3 and 4, respectively, it should be found that the introduction of one chlorine and
two methyl groups in moiety may decrease the scope of antiproliferation.
2.5. In vivo antiproliferative activity
In vivo antiproliferation assay was performed to further evaluate the antitumor activity of
complex 1 and 5. The NCI-460 and SMMC-7721 xenograft models were chosen in this study, and
cisplatin was used as the positive control. The SPF BALB/c nude mice were randomly divided
7 / 20

into three groups including the vehicle control group, the experimental group, and the positive
control group.
As shown in Fig. 2, complex 1 at the dose of 4 mg/kg exhibited important inhibition on
NCI-460 tumour growth, and the relative tumor increment rates (T/C) were found to be 55.9%,
based on the tumor volume. In comparison, cisplatin at the 2 mg/kg dose showed a lower T/C
value of 29.9%. The tumors were harvested and weighted on day 14, and the inhibitory rates on
the growth of tumor weight were then determined. As shown in Fig. 2C, complex 1 (at the 4
mg/kg dose) displayed obvious antitumor activity in the NCI-460 model with an inhibitory rate of
39.7%, lower than that of cisplatin (63.2%, P < 0.01). Further investigation showed that no
significant change in body weight and no other adverse effects were observed among the mice
treated with complex 1, suggesting that 1 exhibited no significant toxicity to the mice within the
13-day period of treatment (Fig. 2B).
Fig. 2. In vivo anticancer activity of 1 in the NCI-460 xenograft model. (A) Effect of 1 (at the
doses of 4 mg/kg), cisplatin (at the dose of 2 mg/kg), or vehicle (5% DMSO in saline, v/v) on
tumor growth. Tumor growth was monitored by the mean tumor volume (mm3) ± SD (n = 6) and
calculated as the relative tumor increment rate (T/C, %). (B) Body weight change of the mice
treated with 1. (C) Tumor weight of the mice. The tumors were collected in the mice at day 14.
8 / 20

The “**” signs represent p < 0.01 (versus the vehicle control group). (D) Photographs of the
harvested tumors from the mice.
Fig. S3-1 (in supplmentary materials) showed that complex 5 exhibited evident inhibition on
SMMC-7721 tumour growth, with the relative tumor increment rates (T/C) of 20.99% (at 10
mg/kg dose) and 46.24% (at 4 mg/kg dose). In comparison, T/C value of cisplatin at the 2 mg/kg
dose was found to be 41.31%. It was important to note that complex 5 (at the 10 mg/kg dose)
exhibited potent antitumor activity in the SMMC-7721 model with the inhibitory rate of 64.63%,
much higher than that of cisplatin (35.85%, P < 0.05). In addition, no significant changes in body
weight were observed among the mice treated with complex 5. This result showed that complex 5
may be good candidate for antitumor drug.
2.6. Drug resistance trait experiment
According to the MTT results in Table 1 and Table S3-1, complexes 1 and 5 showed the most
potential antiproliferation sensitivity and selectivity activity against these cancer cell lines and
lower cytotoxicity on the HL-7702 normal cell line. Therefore, complexes 1 and 5 were selected
as representative compounds for further studies. In order to investigate the drug resistance of
representative complexes 1 and 5, the antiproliferative activity of the complexes 1 and 5 were
evaluated against the cisplatin-resistant strain A549CDDP and SKOV3CDDP cell lines by
MTT[26], using cisplatin as positive control.
As shown in Table S3-1, the IC50 of cisplatin against A549 and SKOV3 cell lines were found to
be 6.37±1.03µM and 16.32±1.37µM, while that of cisplatin against A549CDDP and
SKOV3CDDP cell lines was determined to be 23.01±0.46µM and 53.68±1.85 (more than three
times of that in A549 and SKOV3 assay), indicating that cisplatin exhibited obvious and serious
drug resistance. It was worth noting that the IC50 of complexes 1 and 5 against A549CDDP and
SKOV3CDDP were similar with (or lower than) that against A549 and SKOV3, respectively,
demonstrating that complexes 1 and 5 exhibited no obvious drug resistance. The antitumor
mechanisms of complexes 1 and 5 were then investigated.
2.7 Action mechanism
2.7.1. Covalent DNA binding assayed by HR-MS
9 / 20

To better evaluate whether DNA is the potential target of complexes 1–6, the HR-MS with high
accuracy was used for determining the interaction of complexes with DNA. Previous work has
indicated that cisplatin exerted the antitumor effect mainly by covalent binding with the N-7 atom
of guanosine monophosphate (GMP) in DNA[5,27]. As shown in Fig. 3, cisplatin could be
hydrolyzed into mono- and diaqua- products under the physiological conditions, respectively. By
the nucleophilic substitution reaction of these mono- and diaqua- products with GMP,
respectively, monoadducts was subsequently obtained and finally to offer bisadduct[5,27]. So GMP
was herein selected as a DNA model to study the interaction between the complexes with DNA. In
this part, complexes 1 and 5 were chose as representative compounds.
H3N
H3N
OH2
Cl
H3N
H3N
GMP
Cl
Pt
Pt
physiology
H3N
H3N
GMP
GMP
H3N
H3N
Cl
Cl
intermediates
Pt
Pt
GMP
+
X
H3N
H3N
OH2
OH2
in H2O
H3N
H3N
GMP
OH2
Pt
Pt
mono- and diaqua products
monoadducts
bisadducts
Fig. 3. A variety of adducts generated by the interaction of cisplatin and guanosine
As shown in Fig. 4, there were mainly four peaks in the HR-MS of the mixture of complex 1
with GMP for 1h, 6h, 12h and 24h, respectively. The peaks at 596.0530 and 657.0394 were
attributed to the complexes of [1‒Cl+H2O]+ and [1‒Cl+DMSO]+, respectively, whereas the peaks
at 956.9932 and 1210.459 were attributed to that of [1‒Cl+GMP+H2O]+ and [1‒DMSO‒
2Cl+2GMP+H2O]+. The peak intensity of [1‒Cl+H2O]+ increased with time and stabilized in
about 12h. The peak intensity of [1‒Cl+DMSO]+ decreased with time and stabilized in about 12h.
However, the peaks of [1‒Cl+GMP+H2O]+ and [1‒DMSO‒2Cl+2GMP+H2O]+ occurred within 1h
and its intensity decreased with time. Evidently, there was an important competitive relationship
among these four complexes. In fact, due to the important departure ability of chlorine group,
complex 1 may covalently bond with H2O, DMSO and GMP. The covalent binding ability should
be in the order H2O > DMSO > GMP. These result demonstrated that complex 1 exhibited
obvious covalent binding ability with GMP to offer mono- and bis- adducts, similar with that of
cisplatin[5,27]
.
10 / 20

Fig. 4. HR-MS of the mixture of 1 with GMP for 1h, 6h, 12h and 24h.
The HR-MS of the mixture of complex 5 with GMP (Fig. S4-1, in supplmentary materials) for
1h, 6h, 12h and 24h was similar with that of complex 1. There were four main peaks at
614.0428[5‒Cl+H2O]+, 675.0298[5‒Cl+DMSO]+, 957.9574[5‒Cl+GMP]+ and 974.9836 [5‒
Cl+GMP+H2O]+ in the HR-MS, respectively, indicating that complex 5 displayed obvious
covalent binding ability with GMP to offer monoadducts, similar with that of complex 1 and
cisplatin[5,27]. Fig. S4-1 also demonstrated that the covalent binding ability order of complex 5 to
H2O, DMSO and GMP was similar with that of complex 1, namely H2O > DMSO > GMP.
2.6.2 Verification experiment of topoisomerase I target
11 / 20

2.6.2.1 Topoisomerase I assayed by gel electrophoresis
It is generally accepted that the main targets of naphthalimide derivative are DNA binding and
topoisomerase inhibition[6-16]. Moreover, naphthalimide exhibits a similar structure with the classic
topoisomerase I inhibitor camptothecin (Fig. 5). The interaction of complexes 1–6 with topo I was
thus assayed by gel electrophoresis.
R1
R2
O
N
O
O
OH
O
N
N
H3C
N
camptothecin
naphthalimides
Fig. 5. The chemical structures of naphthalimides and camptothecin.
As shown in Fig. S5-1 (in supplmentary materials), complexes 1–6 exhibited obvious in vitro
inhibition on topo I. It was worth noting that complexes 1 and 5 completely inhibited the catalytic
function of topo I (0.1U/L) at the concentration of 40µM, while complexes 2–4 gained that effect
at 80µM. It was well known that camptothecin was a classical topo I poison with IC50 values of
about 20µM[28]. By the comparison of the topo I inhibition concentrations values of camptothecin
(T, Fig. S5-1) with complexes 1 and 5 (20µM), respectively, it could be concluded that complexes
1 and 5 showed similar in vitro inhibition activity with camptothecin. These results indicated that
complexes 1 and 5 may exert the antitumor activity by the inhibition of topo I at molecular level.
It is well known that topoisomerase I inhibitors could be divided into topoisomerase I poisons
and suppressors. Suppressors prevent or reverse topoisomerase I-DNA complex formation, which
may lead to the upregulation topo I in cancer cells, while poisons act specifically at the level of the
topoisomerase I-DNA complex and stimulate DNA cleavage[29,30]. To further study whether
complexes 1 and 5 have important effect on the expression level of intracellular topo I, the
treatment of topo I in SMMC-7721 cells with complexes 1 and 5 were then investigated by
western blot assay. As shown in Fig. 6, the treatment of SMMC-7721 cells with complexes 1 and
5 lead to evident increment in the expression level of topo I, compared to the control, while
complex
5
could upregulate the expression level of intracellular topo
I
in
a
concentration-dependent manner. This result confirmed that in addition to covalent binding to
12 / 20

DNA, complexes 1 and 5 may also exert the antitumor activity by the cellular upregulation of topo
I, demonstrating that the antitumor mechanism of complexes 1 and 5 was indeed different from
that of cisplatin.
Fig. 6. The expression of topo I in SMMC-7721 cells with 1 and 5.
3. Conclusions
In summary, we designed and synthesized a serial of naphthalenebenzimidizole-Pt complexes
1–6 and evaluated their antiproliferative activity against eight cancer cell lines (Hela, HepG2,
SKOV3, NCI-H460, BEL-7404, SMMC-7721, U251, A549). We identified the six complexes
exhibiting obvious in vitro sensitivity and selectivity against SMMC-7721 and U251 cell lines and
low toxicity against normal HL-7702 cells, while complex 1 exhibited important in vivo
antiproliferative activity in the NCI-460 model. Drug resistance experiment showed that
representative complexes 1 and 5 exhibited no resistance on A549-CDDP, while cisplatin showed
evident and serious resistance. Mechanistic studies showed that complexes 1 and 5 exerted their
antitumor effect mainly by the covalent binding with DNA and the upregulation of the expression
level of intracellular topo I, indicating that their targets were different from that of cisplatin. This
work suggests that the introducing new ligands naphthalenebenzimidizole into platinum-metal
system may change the anti-tumor mechanisms and thus to improve the antipoliferative activity,
drug resistance and toxicity, well consistent with the previous literatures[31-34]
.
4. Experimental
4.1. Chemistry.
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All chemicals of reagent grade were commercially available and used without further
purification. NMR spectra were recorded on a BRUKER AVANCE AV 400/500
spectrometer using tetramethylsilane (TMS) as the internal standard. The mass spectra
were collected on a BRUKER ESQUIRE HCT spectrometer. The GelRed nucleic acid
stain was purchased from Biotium.
4.2 General synthetic procedure for complexes 1–6
The mixture of 1,8-naphthalene anhydride (1 mmol), substituented o-phenylenediamine
(1mmol) and 20 mL glacial acetic acid was reacted at 120oC for 6h, according to the
literature[23,24]. The solution was cooled to room temperature and then filtered to offer
yellow powders of
without further purification, while the powders of L4
silica column chromatography with hexane/ethylacetate (v/v=5:1) as the eluent. The
mixture of ligands L1–L6)(0.37mmol), cis-dichloride bis(dimethyl sulfoxide)
L(
L1–L6). It was important to noted that Ligands L1–L3 were then used
‒L6 needed to be further purified by
L(
platinum(II) (0.37mmol), 5mL methanol and 5mL dichloromethane was reacted at 60oC for
48h. The hot solution was filtered and cooled to the room temperature. The cooled solution
was filtered to offer yellow powder. Upon recrystallization from the mixed solution of
methanol and chloroform (V:V=1:1), yellow crystals of the complex was obtained.
1
: Yields 75.15%. 1H NMR (400 MHz, d-DMSO) δ 8.72 (d, J = 8.3 Hz, 1H, H-Ar), 8.68
(d, J = 7.3 Hz, 1H, H-Ar), 8.51 (d, J = 8.2 Hz, 1H, H-Ar), 8.45 – 8.39 (m, 1H, H-Ar), 8.37
(d, J = 8.3 Hz, 1H, H-Ar), 7.95 – 7.85 (m, 3H, H-Ar), 7.53 – 7.45 (m, 2H, H-Ar).
ESI-HRMS, calculated m/z for [M–Cl+DMSO]+: 657.0408, found: 657.0385. Anal.
Calc.(for C20H16Cl2N2O2PtS) C 39.10; H 2.62; N 4.56%,Found. C 39.41; H 2.57; N 4.32%.
1
2
: Yields 47.17%. H NMR (400 MHz, d-DMSO) δ 8.63 (dd, J = 13.3, 5.6 Hz, 2H,
H-Ar), 8.49 (d, J = 8.2 Hz, 1H, H-Ar), 8.47 – 8.25 (m, 2H, H-Ar), 8.01 – 7.73 (m, 2H,
H-Ar), 7.65 – 7.55 (m, 1H, H-Ar). ESI-HRMS, calculated m/z for [M+Cl]-: 716.8866,
found: 716.8864. Anal. Calc.(for C20H14Cl4N2O2PtS) C 35.16; H 2.07; N 4.10%,Found. C
35.14; H 2.09; N 4.12%.
1
3
: Yields 60.46%. H NMR (400 MHz, d-DMSO) δ 8.78 – 8.67 (m, 2H, H-Ar), 8.54 (d,
J = 7.3 Hz, 1H, H-Ar), 8.38 (d, J = 7.5 Hz, 1H, H-Ar), 8.24 (s, 1H, H-Ar), 8.00 – 7.88 (m,
2H, H-Ar), 7.66 (s, 1H, H-Ar), 2.43 (s, 3H, CH3), 2.39 (s, 3H, CH3). ESI-HRMS,
14 / 20

calculated m/z for [M–Cl+DMSO]+: 685.0721, found: 685.0704. Anal. Calc.(for
C22H20Cl2N2O2PtS) C 41.13; H 3.14; N 4.36%,Found. C 41.33; H 3.17; N 4.35%.
1
4
: Yields 76.13%. H NMR (400 MHz, d-DMSO) δ 8.71 (dd, J = 14.3, 7.4 Hz, 2H,
H-Ar), 8.54 (d, J = 8.1 Hz, 1H, H-Ar), 8.49 – 8.31 (m, 2H, H-Ar), 8.11 – 7.77 (m, 3H,
H-Ar), 7.51 (d, J = 8.3 Hz, 1H, H-Ar). ESI-HRMS, calculated m/z for [M–Cl+DMSO]+:
691.0018, found: 690.9980. Anal. Calc.(for C20H15Cl3N2O2PtS) C 37.02; H 2.33; N
4.32%,Found. C 37.04; H 2.30; N 4.32%.
1
5
: Yields 72.14%. H NMR (400 MHz, d-DMSO) δ 8.63 (dd, J = 13.3, 5.6 Hz, 2H,
H-Ar), 8.49 (d, J = 8.2 Hz, 1H, H-Ar), 8.47 – 8.25 (m, 2H, H-Ar), 8.01 – 7.73 (m, 3H,
H-Ar), 7.65 – 7.55 (m, 1H, H-Ar). ESI-HRMS, calculated m/z for [M+Cl]-: 663.0125,
found: 663.0032. Anal. Calc.(for C20H15Cl2FN2O2PtS) C 37.99; H 2.39; N 4.43%, Found.
C 37.92; H 2.38; N 4.42%.
6
: Yields 53.14%. 1H NMR (400 MHz, d-DMSO) δ 8.77 – 8.48 (m, 4H, H-Ar), 8.40 (dd,
J = 8.2, 4.4 Hz, 1H, H-Ar), 8.21 – 7.85 (m, 3H, H-Ar), 7.80 (d, J = 8.4 Hz, 1H, H-Ar).
ESI-HRMS, calculated m/z for [M–Cl+CH3CH2OH]+: 691.0540, found: 690.9979. Anal.
Calc. (for C21H15Cl2F3N2O2PtS) C 36.96; H 2.22; N 4.11%,Found. C 37.01; H 2.23; N
4.08%.
4.3. In vitro antipoliferative activity
The cell lines were all obtained from the Institute of Biochemistry and Cell Biology,
China Academy of Sciences. They were cultured in a humidified, 5% CO2 atmosphere at
37°C and maintained in monolayer culture in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin and
100
mg/mL
penicillin.
Chemosensitivity
was
assessed
with
a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly,
exponentially growing cells were seeded into 96-well plates and treated with the indicated
concentrations of complexes for 48 h, and then 10 mL of MTT (10 mg/mL) was added.
After incubation for 4 h at 37oC, the purple formazan crystals (a reduced form of MTT)
generated in viable cells were dissolved by adding 100 µL DMSO to each well. The plates
were swirled gently for 10 min to dissolve the precipitate, and quantified by measuring the
optical density of the plates at 490 nm using a plate reader (TECAN infinite M1000). Each
15 / 20

concentration was repeated in three wells and the same experimental conditions were
maintained for all testing procedures. The MTT assays were repeated three times for each
cell line.
4.4 In vivo antipoliferative activity
Nude mice received subcutaneous injection of 5×107 tumor cells in the right flank.
When the xenograft tumor growth to the volume about 1000 mm3, the mice were killed and
the tumor tissue were cut into small pieces at about 1.5 mm3 and then transplanted into the
right flank of male nude mice. When the average tumors reached the volumes of 100−150
mm3, the mice were randomly divided into solvent control and treatment groups (n = 6 (or
4) /group). The complexes at different doses (5% v/v DMSO/saline) were given twice a
day. Cisplatin was given to mice by ip administration at a dosage of 2 mg/kg/per 2 days
and used as a positive reference for comparison. Tumor size and body weight were
monitored every 2 days. The animals were finally sacrificed for humane reasons, and the
tumors were weighted and recorded. The tumor volumes were determined every 2 days by
measuring length (l) and width (w) and calculating with the formula of V = lw2/2 as
described elsewhere. Meanwhile, body weight of mice was measured and taken as a
parameter of systemic toxicity. The rate of tumor growth was calculated using a formula of
(1 − TWt/TWc) × 100, where TWt is the tumor weight of comlexes treated mice and the
TWc is the tumor weight of vehicle-treated animals.
4.5 DNA binding assay
Briefly, the Tris-NO3 (pH7.4) solution of GMP(4µM) and the DMSO solution of
complex
1/5 (2µM) were treated for 1h, 6h, 12h and 24h, respectively, and then mixtures
were diluted to 10 times with methanol and determined by high-resolution mass
spectrometry (BRUKER ESQUIREHCT)
.
4.6 Topo I inhibition[35]
Briefly, a mixture (20 µL) containing 0.25 µg of pBR322 DNA in relaxation buffer (10
mM Tris, 0.1 mM EDTA, 5 mM MgCl2, 50 mM KCl, plus 1 mM DTT, 0.01% bovine
serum albumin (BSA), pH 7.5) was incubated with 1 unit of calf thymus Topisomerase I
(topo I, from TaKaRa Biotechnology Co., Ltd., Dalian) in the absence or in the presence of
complexes at different concentrations, for 30 min at 37 °C. Topo I was preincubated with
16 / 20

the complex for 15 min at 37 °C prior to the addition of plasmid pBR322 DNA. The
reaction was terminated by the addition of 4 µL of 10 × loading buffer (0.9% sodium
dodecyl sulfate (SDS), 0.05% bromophenol blue and 50% glycerol). The sample was then
analyzed using a 0.8% agarose gel in TBE buffer. The gels were stained with gel red and
visualized in the electrophoresis gel documentation and analysis system.
2.8. Western Blot Assay[36]
Tumour cells were collected after treatment with complex (10 µM) for 12 h and then
lysed in ice-cold lysis buffer (1% sodium dodecyl sulfate in 25 mM pH 7.5 Tris–HCl, 4
mM EDTA, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin and
10 mg/mL soybean trypsin inhibitor). Whole-cell lysates were centrifuged at 12,000×g for
5 min. Thereafter, the protein concentration was determined with a bicinchoninic acid
protein assay kit (Beyotime Co, China). An aliquot of cell lysate (40–50 µg) was
fractionated by SDS-PAGE on 12% polyacrylamide gels for 2 h and transferred to
polyvinylidene difluoride membranes. After blocking with 5% non-fat dry milk in PBS-t
for 1 h at room temperature, the membranes were incubated with topo I (topo I, from
TaKaRa Biotechnology Co., Ltd., Dalian) overnight at 4oC, washed with tris-buffered
saline and Tween 20, and then incubated with horseradish peroxidase-conjugated
secondary antibodies for 1 h at room temperature. Proteins were detected by
electrochemiluminescence, Thermo Fisher Scientific, USA) and analysed by Image J
software.
4.9. Statistical Analysis
Data are expressed as mean ± SD for three different determinations. Statistical
significance was analyzed by one-way ANOVA. Mean separations were performed using
the least significant difference method. P<0.05was defined as statistically significant.
Acknowledgements. This study was supported by the National Natural Science
Foundation of China (No. 21501032), the Innovative Team & Outstanding Talent Program
of Colleges and Universities in Guangxi (2017-38), Guangxi Natural Science Foundation
(Nos. 2016GXNSFAA380300 and 2014GXNSFBA118050) and Guangxi New Century
Ten, Hundred and Thousand Talents Project ([2017]42).
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Highlights
ò
A serial of naphthalenebenzimidizole-Pt complexes 1–6 were designed and synthesized as
antitumor agents
ò
ò
In vitro cytotoxic and noncancerous screening for all the targeted compounds.
Complex 1 and 5 exhibited important inhibitory on tumor growth in NCI-H460 and
SMMC-7721 xenograft mouse models, respectively.
Complexes 1 and 5 showed obvious covalent binding with DNA.
Complexes 1 and 5 upregulated of the expression level of extracellular and intracellular topo
I.
ò
ò
1 / 1

Declaration of Interest Statement
We declare that we have no financial and personal relationships with other people or organizations
that can inappropriately influence our work, there is no professional or other personal interest of
any nature or kind in any product, service and/or company that could be construed as influencing
the position presented in, or the review of, the manuscript entitled “Design, synthesis and
biological evaluation of naphthalenebenzimidizole platinum (II) complexes as potential antitumor
agents”.
1 / 1