Synthesis and biological evaluation of novel 2-arylvinyl-substituted naphtho[2,3-d]imidazolium halide derivatives as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2017.12.008

Two series of novel 2-arylvinyl-naphtho[2,3-d]imidazol-3-ium iodide derivatives and 2-arylvinyl-naphtho[2,3-d]imidazol-3-ium bromide derivatives were designed and synthesized by the structural combination of YM155 with stilbenoids. All compounds were tested for anti-proliferative activity against PC-3, A375 and HeLa human cancer cell lines. Two of the compounds were selected for further investigation: 12b, which showed potent cytotoxicity against the three tested cell lines with IC₅₀ values in the range of 0.06–0.21 μM, and 7l, which displayed excellent selectivity for PC-3 cells with an IC₅₀ of only 22 nM. Western blot analysis results indicated that both 12b and 7l suppress the expression of Bcl-2 and Survivin proteins, which helps induce apoptosis. As determined by the percent of Annexin V-FITC-positive apoptotic cells, 12b was not only significantly more effective than 7l at a concentration of 100 nM in PC-3 cells but also induced apoptosis in a dose-dependent manner with more potency than 7l at a concentration of 1000 nM in A375 cells. Therefore, compound 12b was chosen for further in-depth studies investigating the mechanism of apoptosis. The results showed that it could activate caspase-3, hydrolyze PARP, and even inactivate ERK. Moreover, 12b arrested A375 cells at S phase in a time-dependent and dose-dependent manner, while having a visible effect on microtubule dynamics. In addition, (E)-2-(2-(1H-indol-3-yl)vinyl)-1-benzyl-3-(2-methoxyethyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide (12b) exhibited significant antitumor activity when evaluated in a subcutaneous solid tumor model. Our study reveals that 2-arylvinyl-substituted naphtho[2,3-d]imidazolium scaffolding is a promising new entity for the development of multi-target anticancer drugs.

Full text of DOI:10.1016/j.ejmech.2017.12.008

Accepted Manuscript
Synthesis and biological evaluation of novel 2-arylvinyl-substituted
naphtho[2,3-d]imidazolium halide derivatives as potent antitumor agents
Qingyun Wei, Ju Li, Feng Tang, Yin Yin, Yong Zhao, Qizheng Yao
PII:
S0223-5234(17)31014-0
10.1016/j.ejmech.2017.12.008
EJMECH 9986
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 April 2017
Revised Date: 2 December 2017
Accepted Date: 2 December 2017
Please cite this article as: Q. Wei, J. Li, F. Tang, Y. Yin, Y. Zhao, Q. Yao, Synthesis and biological
evaluation of novel 2-arylvinyl-substituted naphtho[2,3-d]imidazolium halide derivatives as potent
antitumor agents, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.008.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel 2-arylvinyl-substituted
naphtho[2,3-d]imidazolium halide derivatives as potent antitumor
agents
Qingyun Weia, Ju Lia, Feng Tangb, Yin Yinb, Yong Zhaob, **, Qizheng Yaoa,
*
a
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR
China
b
MtC Biopharma, Co., Ltd, Nanjing 210042, PR China
* Corresponding author. Tel./fax: +86 25 86634730
** Corresponding author. Tel./fax: +86 13952026874
E-mail address: qz_yao@163.com (Q. Yao), zhaoyong@mtcbiopharma.com (Y. Zhao)
This paper is dedicated to Prof. Wolfgang Pfleiderer for the occasion of his 90th birthday.
Abstract
Two series of novel 2-arylvinyl-naphtho[2,3-d]imidazol-3-ium iodide derivatives and
2-arylvinyl-naphtho[2,3-d]imidazol-3-ium bromide derivatives were designed and synthesized by
the structural combination of YM155 with stilbenoids. All compounds were tested for
anti-proliferative activity against PC-3, A375 and HeLa human cancer cell lines. Two of the
compounds were selected for further investigation: 12b, which showed potent cytotoxicity against
the three tested cell lines with IC50 values in the range of 0.06-0.21 µM, and 7l, which displayed
excellent selectivity for PC-3 cells with an IC50 of only 22 nM. Western blot analysis results
indicated that both 12b and 7l suppress the expression of Bcl-2 and Survivin proteins, which helps
induce apoptosis. As determined by the percent of Annexin V-FITC-positive apoptotic cells, 12b
was not only significantly more effective than 7l at a concentration of 100 nM in PC-3 cells but
also induced apoptosis in a dose-dependent manner with more potency than 7l at a concentration
of 1000 nM in A375 cells. Therefore, compound 12b was chosen for further in-depth studies
investigating the mechanism of apoptosis. The results showed that it could activate caspase-3,
hydrolyze PARP, and even inactivate ERK. Moreover, 12b arrested A375 cells at S phase in a
time-dependent and dose-dependent manner, while having a visible effect on microtubule
dynamics.
In
addition,
(E)-2-(2-(1H-indol-3-yl)vinyl)-1-benzyl-3-(2-methoxyethyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2
,3-d]imidazol-3-ium bromide (12b) exhibited significant antitumor activity when evaluated in a
subcutaneous solid tumor model. Our study reveals that 2-arylvinyl-substituted

ACCEPTED MANUSCRIPT
naphtho[2,3-d]imidazolium scaffolding is a promising new entity for the development of
multi-target anticancer drugs.
Keywords：naphth[2,3-d]imidazol-3-ium; arylvinyl; survivin inhibitor; antitumor drugs
1. Introduction
Survivin, a member of the Inhibitors of Apoptosis Protein (IAP) family, participates in
mitosis, apoptosis, and cellular stress response in mammalian cells [1]. Survivin is typically only
expressed during embryonic development and is absent in most normal and terminally
differentiated tissues. Several studies have shown that survivin is selectively over-expressed in
most human tumors and associated with multiple-drug resistance, increased tumor recurrence, and
shorter survival times, which make it an attractive target for cancer therapeutics. Targeted
therapies that seek to inhibit survivin expression have been utilized in an experimental setting;
these studies have used antisense oligonucleotides (LY2181308[2]) or siRNA (EZN-3042[3]),
vaccination strategies (EMD640744[4], DPX-0907[5]) and small molecule inhibitors (YM155[6],
FL118[7]), which have all yielded promising results to date. All three therapeutic strategies have
reached clinical trials, including the use of the small molecule inhibitor YM155 (Sepantronium
Bromide) for the treatment of solid tumors and lymphomas [8].
YM155,
a
novel small molecule inhibitor, belongs to the group of
4,9-dihydro-4,9-dioxo-1H-naphtho[2, 3-d] imidazolium halide derivative. It blocks the expression
of the survivin protein by directly inhibiting of survivin promoter [9, 10]. Preclinical studies show
that YM155 at nanomolar concentrations suppress survivin expression and inhibit the growth of
p53-deficient cancer cells. It was also demonstrated to have antitumor activity in human prostate,
pancreatic, and lung cancer xenografts in mice [6]. Researchers have found that the physiological
target of YM155 may be the RNA-binding protein interleukin enhancer-binding factor-3
(ILF3/NF110) or a zinc finger transcription factor that preferentially binds GC-rich DNA
sequences [11, 12]. YM155 is currently being investigated in a phase Ⅱ clinical trials in
combination with chemotherapy[13]. It has been used in combination with carboplatin and
paclitaxel to target NSCLC [14]. The combination of YM155 with docetaxel has also shown great
efficacy in making melanoma cells sensitive to anticancer drugs [15]. Along with the inhibition of
survivin, YM155 has also been shown to exert a synergistic anti-neoplastic effect in combination
with taxanes and platinum drugs in earlier studies.

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As the body of data in clinical research grows, novel survivin antagonists are expected to have
broad activity in various tumor types, tolerable side-effects, and synergistic efficacy with
conventional anticancer treatment [1]. As we gain more understanding of the pathogenesis of
human cancers and the interrelated signaling pathways involved in tumor cells, single-target
inhibitors have proven to be insufficient in achieving the desired therapeutic effect. In this regard,
our interest is focused on developing a new compound by utilizing the structural properties of
YM155 in combination with other traditional anticancer drugs.
Stilbene derivatives, including combretastatin and resveratrol, are found in many natural
products with diverse applications and biological activities. Combretastatin binds to colchicine
sites and act as a tumor vascular-disrupting agent by inhibiting tubulin polymerization to block
tumor growth [16, 17]. Several derivatives of combretastatin A-4 (CA-4) have entered clinical
trials. It is well-known that tubulin polymerization inhibitors that behave similar to CA-4 require
two hydrophobic rings in the Z-configuration within the linking bridge to bind to the active site to
elicit a response [18-21]. Resveratrol, a polyphenolic stilbene, has long been an important staple in
Chinese and Japanese folk medicine [22]. Numerous studies have shown the great potential of
resveratrol as a potent chemotherapeutic agent in diverse human diseases, including a wide variety
of cancers [23].
In the present study, two series of novel 2-arylvinyl-substituted naphtho[2,3-d]imidazolium
halide derivatives have been designed, synthesized and evaluated for antitumor activity by
anti-proliferative assay in several human cancer cell lines. The current work in this study provides
a
basis for further structural modification and transformation of potential
naphthoquinone-imidazolium compounds.
2. Results and Discussion
2.1 Chemistry
Two groups of target compounds containing quaternary ammonium salts as the key
pharmacophore were prepared: the first set included those with iodized salt (7a-7q, Schemes 1),
and the second consisted of those with bromide salts (12a-12g, Schemes 2).
The first group of compounds was prepared as shown in Scheme 1. Amination of the starting
material 1 (2,3-dichloro-1,4-naphthoquinone) with ammonia in refluxing ethanol led to the
formation of 2-amino-3-chloro-1,4-naphthoquinone (2). Then, N-acylation of 2 with acetic
anhydride in the presence of concentrated H2SO4 gave rise to 2-acetamido-3-chloro-1,
4-naphthoquinone (3). The required 1-substituted-2-methylnaphth[2,3-d]imidazole-4,9-diones 5a
and 5b were synthesized in two steps from 3 and the suitable fatty primary amines via the
corresponding 2-(acylamino)-3-(alkylamino)-1,4-naphthoquinones 4a and 4b, respectively.
Reaction of 5 with ethyl iodide gave 2-methyl-3-ethyl-naphtho[2,3-d]imidazol-3-ium iodide
derivatives (6a-6b), according to referenced processes [24, 25]. The resulting compound 6 was
allowed to react with 2 eq. of various aromatic aldehydes in dioxane to yield the corresponding 2-
arylvinyl-naphtho[2,3-d]imidazol-3-ium iodide derivatives 7a-7l as the final products.

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Scheme 1. Synthesis of compounds 7a-l. Reagents and conditions: a) NH3, CH3CH2OH; b) Ac2O; c) R1NH2,
ethanol, 50°C, 30 min; d) NaOH (2N), ethanol, reflux, 30 min; e) ethyl iodide, 2-methoxyethanol, reflux, 4 h; f)
R-CHO, pyridine, dioxane, reflux, 2 h.
As shown in Scheme 2, compounds 12a-12g were prepared following procedures found in
cited literature [26]. First, 1 was reacted with 2-methoxyethylamine in a solution of DMF to
produce 2-((2-methoxyethyl) amino)-3-chloro-1,4-naphthoquinone (8). The resulting product from
N-acylation of compound 8 by acetic anhydride was subsequently reacted with benzylamine or
3-methoxybenzylamine to produce 2-benzylamino or 2-(3-methyloxybenzyl)amino derivatives
(10a-10b). Cyclization of 10 in a solution of HBr and 2-methoxyethanol mixture gave rise to
2-methyl-naphtho[2,3-d]imidazol-3-ium bromide derivatives (11a-11b). Finally, compounds 11
was allowed to reacted with 2 eq. of various aromatic aldehydes in dioxane to yield the final
product as 2-arylvinyl-naphtho[2,3-d]imidazol-3-ium bromide derivatives 12a-12g.

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Scheme 2. Synthesis of compounds 12a-g. Reagents and conditions: a)2-methoxyethylamine, triethylamine, DMF;
b) Ac2O, 50°C, 1 h; c) benzylamine or 3-methoxybenzylamine, ethanol; d) HBr, 2-methoxyethanol, reflux, 2 h; e)
R-CHO, pyridine, dioxane, reflux, 2 h.
2.2 Biological activities
2.2.1 In vitro anti-proliferative activity
The in vitro cytotoxicity of synthesized compounds was evaluated by determining the
corresponding IC50 levels against three human cancer cell lines — PC-3 (prostate), A375
(melanoma) and HeLa (cervix) — compared to the efficacy of YM155 (Table 1).
For the first series of 2-styryl-naphtho[2,3-d]imidazol-3-ium iodide derivatives 7a-7l,
compound 7l, which contains N1-isopropyl and C2-methylstyryl groups, exhibited better
cytotoxicity against PC-3 cells than the other compounds. Meanwhile, compound 7i, which
contains N1-isopropyl and C2-3-indolyl groups, showed better cytotoxicity against A375 cells.
The presence of N1-cyclopropyl or N1-isopropyl had little influence on the cytotoxic activity
against all three cancer lines (7d vs 7j; 7i vs 7f).
For the second series of 2-styryl-naphtho[2,3-d]imidazol-3-ium bromide derivatives 12a-12g,
compound 12b, which contains N1-benzyl and C2-(indol-3-yl)vinyl groups, exerted the most
cytotoxic activity against all three cancer cell lines. Compounds containing a benzyl group
expressed better cytotoxicity compared to those with a 3-methoxybenzyl group (12b vs 12e; 12d
vs 12g).

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Overall, compounds containing a C2-(indol-3-yl)vinyl group showed better cytotoxicity
compared to those C2-substitued-phenylvinyl group (7f among 7a-7f; 7i among 7g-7k; 12b
among 12a-12d; 12e among 12e-12g). Moreover, the cytotoxicity of 12b, which is added
C2-(indol-3-yl)vinyl group on the base of 11a, was better than 11a on A375 and HeLa cell lines.
The results indicated that the addition of indole ring improves the antiproliferative activity of these
derivatives.
Taken together, with IC50 values of 0.128 ± 0.017, 0.212 ± 0.029, and 0.059 ± 0.011 µM
against PC-3, A375, and HeLa cells, respectively, the results identified 12b as exhibiting the
strongest anticancer activities against all three tested cancer cell lines. Meanwhile, 7l showed
superior cell proliferation inhibition of the PC-3 cell line compared to the other two cancer cell
lines tested. The mean IC50 value for the PC-3 cell line was 0.022 µM while it was 1.068 µM and
1.12 µM for the A375 and HeLa cell line, respectively (Table 1). Therefore, we selected two
compounds for further studies on their mode of action, 7l with the highest selectively at PC-3 cell
lines, and 12b with excellent anti-proliferative effects against all three of human cancer cell lines.
Table 1. Inhibitory effect of the new naphtho[2,3-d]imidazolium halide compounds on in vitro cell growth.
IC50 (µM)a
compd
7a
X
I
R
R1
R3
PC-3
A375
HeLa
6.54 ± 0.062
13.5 ± 0.200
>30
C6H5
cPr
CH3CH2
0.038 ± 0.001
0.066 ± 0.006
0.143 ± 0.011
0.038 ± 0.000
0.028 ± 0.002
0.052 ± 0.003
0.039 ± 0.001
0.140 ± 0.016
0.071 ± 0.002
0.090 ± 0.003
0.072 ± 0.007
0.022 ± 0.001
0.212 ± 0.004
0.128 ± 0.017
0.022 ± 0.006
0.100 ± 0.004
0.215 ± 0.010
0.291 ± 0.030
0.222 ± 0.003
0.073 ± 0.003
1.313 ± 0.081
8.000 ± 0.859
25.993 ± 3.151
13.913 ± 1.631
5.357 ± 0.520
0.891 ± 0.108
1.429 ± 0.406
2.940 ± 1.704
0.553 ± 0.016
6.579 ± 0.195
5.580 ± 0.613
1.068 ± 0.069
0.551 ± 0.037
0.212 ± 0.029
0.536 ± 0.066
0.791 ± 0.023
0.353 ± 0.028
1.004 ± 0.107
1.118 ± 0.060
1.141 ± 0.140
I
3,4-(OCH3)2-C6H3
3,4,5-(OCH3)3-C6H2
4-OH,3-OCH3-C6H3
3-NO2-C6H4
3-indolyl
cPr
CH3CH2
7b
I
cPr
CH3CH2
7c
I
cPr
CH3CH2
18.5 ± 0.340
11.3 ± 0.340
2.05 ± 0.026
9.93 ± 0.265
13.0 ± 0.000
6.12 ± 0.025
>30
7d
I
cPr
CH3CH2
7e
I
cPr
CH3CH2
7f
I
4-NO2-C6H4
3-OH-C6H4
iPr
CH3CH2
7g
I
iPr
iPr
CH3CH2
7h
I
3-indolyl
CH3CH2
7i
I
4-OH,3-OCH3-C6H3
4-OH-C6H4
iPr
CH3CH2
7j
I
iPr
CH3CH2
>30
7k
I
4-CH3-C6H4
C6H5(CH=CH)
3-indolyl
iPr
CH3CH2
1.12 ± 0.270
1.28 ± 0.072
0.059 ± 0.011
1.13 ± 0.145
4.86 ± 0.230
1.37 ± 0.036
7.50 ± 0.495
7.33 ± 0.096
10.014 ± 0.263
7l
Br
Br
C6H5-CH2
C6H5-CH2
C6H5-CH2
C6H5-CH2
(3-OCH3-C6H4)-CH2
CH3O(CH2)2
CH3O(CH2)2
CH3O(CH2)2
CH3O(CH2)2
CH3O(CH2)2
CH3O(CH2)2
CH3O(CH2)2
CH3O(CH2)2
12a
12b
12c
12d
12e
12f
12g
11a
Br 4-OH,3-OCH3-C6H3
Br
Br
3-OH-C6H4
3-indolyl
Br 3,4,5-(OCH3)3-C6H2 (3-OCH3-C6H4)-CH2
Br
Br
3-OH-C6H4
none
(3-OCH3-C6H4)-CH2
C6H5-CH2

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YM155 Br
none
Pyrazine
CH3O(CH2)2
0.005 ± 0.001
0.015 ± 0.003
0.137 ± 0.013
aIC50 values are the working concentrations (µM) of the tested compounds required to inhibit tumor cell
proliferation by 50% after a 48 h treatment; values represent the mean obtained from the dose response curves of
three independent experiments.
2.2.2 Compound 12b and 7l suppress the protein expression of Survivin
To investigate whether compound 12b and 7l exert their cytotoxicity activity by inhibiting the
expression of survivin and other IAP family proteins, A375 cells were treated with these
compounds at different concentration for 48 h. As shown in Figure 1, both compounds 12b and 7l
suppressed survivin protein expression and stimulated the proteolytic cleavage of procaspase-3. In
addition, the anti-apoptotic Bcl-2 protein expression was also decreased.
Figure 1. Effect of 12b and 7l on apoptosis-related protein expression levels in A375 cells. Cells were incubated
with either 12b or 7l, harvested at 48 h and whole cell lysates were assayed by immunoblotting. GAPDH was used
as a loading control.
2.2.3 Compounds 12b and 7l induce apoptosis.
To determine whether these compounds inhibit tumor cell proliferation through the induction
of apoptosis, Annexin V and PI (propidium iodide) dual staining analysis was performed by flow
cytometry. After being treated with compound 12b or 7l at different concentrations (1, 10, or 100
nM), apoptotic PC-3 cell populations were detected and quantified as the mean ± S.D. of three
independent experiments (Figure 2A).
As depicted in Figure 2B, A375 cells treated with compound 12b or 7l showed an
accumulation of Annexin V-positive cells in a concentration-dependent manner compared with
vehicle-treated controls. The percent of apoptotic cells in the 12b-treated group was significantly
higher than that of the 7l-treated group at each concentration level.
These results demonstrate that compound 12b significantly increased cellular apoptosis in a
concentration-dependent manner far better than 7l, not only PC-3 cell but also A375 cell. Given

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that anti-proliferative and apoptosis inducing activity of 12b better than 7l, compound 12b was
employed for the further study.
Figure 2. Flow cytometry analysis of apoptosis after treatment PC-3 cell (A) and A375 cell (B) with 12b and 7l at
the indicated concentrations after 24 h. Vehicle was the negative control group, and cells treated with YM155 (100
nM) was the positive control group. Representative flow cytometric histograms of apoptotic cells (left), and the
percent of cells found in the different regions of the biparametric histograms (right) are shown. (Mean ± S.D., n=3;
*p<0.05, ** p<0.01).
2.2.4 Effect of Compound 12b on other related proteins expression.

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To explore the underlying mechanism of apoptosis that is being induced by compound 12b in
more detail, its effect on other apoptosis-related protein expression levels pertaining to caspase
activity was examined. The cleaved form of PARP protein was detected upon exposure to
compound 12b. Similarly, the activity of caspase-3 and caspase-7 was upregulated (Figure 3A and
3B), and the expression of p53 was also affected after treatment with compound 12b. Moreover,
the activation of ERK as indicated by the presence of the tyrosine-phosphorylated form p-ERK
was also observed after treatment with a high dose of 12b. Collectively, the data reveals that 12b
induces apoptosis through both the activation of caspase and inactivation of the ERK pathway.
A
B
600
500
400
300
200
100
0
**
*
0.00
111.11 333.33 1000.00
Concentration (nM)
Figure 3. (A) Expression of other apoptosis-related proteins in the induction of apoptosis in A375 cells. A375 cells
were incubated with 12b at four different concentrations, harvested at 48 h and cell lysates were assayed by
immunoblotting for the cleavage of PARP, and expression of p-ERK and total ERK. GAPDH were used as a
loading control. (B) Caspase enzyme activity was measured after incubation with 12b for 24 h according to the
Caspase-Glo 3/7 assay kit instructions (Mean ± S.D., n=3; *p<0.05, ** p<0.01).
2.2.5 Compounds 12b effects cell cycle distribution.
To further evaluate the effects of compound 12b on cell growth, cell cycle distribution was
determined by flow cytometric analysis of total DNA staining by PI. As shown in Figure 4, with
each increase in either the concentration or the exposure time of compound 12b, the population of

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cells accumulated in S phase gradually increased. After 24 h of treatment with 12b, the percent of
S phase arrested cells increased from 23.62% (vehicle) to 48.06% at 100 nM and 66.08% at the
highest concentration (400 nM). In addition, when treated with 12b at 200 nM, the fraction of
cells arrested at S phase was 33.04% and >58.19% after 12 h and 48 h, respectively. The ability of
compound 12b to arrest A375 cells in S/G2 phase was inferred from these results.
A
B
C
G0/G1
S
G2/M
G0/G1
S
G2/M
100
50
0
100
50
0
0
100
200
400
0
12
24
48
Treated Conc. (nM)
Treated Time (Hrs)
Figure 4. Compound 12b affects the cell cycle distribution in A375 cells. (A) A375 cells were treatment with 12b
at various concentrations for 24 h and at 200 nM for various time points, respectively. (B, C) After treatment, cells
were collected and the DNA content analyzed by flow cytometer.
2.2.6 Effects of compound 12b on the microtubule network.
To get insight into the mechanism of growth inhibition induced by 12b, we investigated the
morphology of microtubules and cytoskeleton by immunofluorescence staining. As shown in
Figure 5, while the control group of A375 cells was spread out with a well-organized microtubule
network, the treated group of cells (Paclitaxel group, 300 nM of the 12b group) was shrunk with
improper microtubule bundling; of note, there was no obvious difference observed following
incubation with YM155. Furthermore, as highlighted by their rounded morphology, some cells had
detached in the presence of either Paclitaxel or the high concentration of 12b due to the heavy
impact of these agents on microtubule depolymerization and cytoskeleton disruption. The result

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suggests that tubulin might be a potential target of the antitumor activity of compound 12b
attributable to the arylvinyl-substituent introduced in the structure[27].
Figure 5. Effect of compound 12b on the microtubule network of A375 cells. (200×). Cells that were either
untreated (control) or treated with compound 12b at various concentrations for 24 h were stained with α-tubulin
and counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Microtubules and unassembled tubulin are shown
in green. DNA, stained with DAPI, is shown in blue. Arrows indicate the roundish cells. Paclitaxel, as a common
tubulin inhibitor, is a positive control group.
2.2.7 In vivo antitumor activity of compound 12b.
To evaluate the in vivo antitumor activity of compound 12b, a subcutaneous solid tumor model
were established by injecting Ehrlich’s ascites carcinoma (EAC) cells into the thigh of ICR mice
[28] [29] Forty-two mice were randomly assigned to six groups 24 h after subcutaneous tumor
inoculation. The mice were injected intraperitoneally once a day for 10 consecutive days with the
following: the test groups received a dose of either 0.06 mg/kg of 12b, 0.2 mg/kg of 12b, 0.6

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mg/kg of 12b, or 0.2 mg/kg of YM155; the positive control group received 30 mg/kg
cyclophosphamide (CTX); the vehicle control group received the solvent formula only. The last
day the tumors were stripped from mice (Figures 6A).
As shown in Figures 6B, the group receiving the high dose (0.6 mg/kg) of compound 12b had
a notable reduction in tumor progression (53.12%) when compared with the group receiving
administration of the vehicle only. Moreover, compound 12b inhibited tumor growth in a
dose-dependent manner. Those receiving either YM155 or anticancer drug CTX had a 52.26% and
48.87% tumor reduction, respectively. Hematoxylin and Eosin (HE)-stained tumor (Figures 6C)
showed that the high dose (0.6 mg/kg) of 12b induce lymphocytes infiltrating into tumors, and
surrounding tumor tissue necrosis.
To assess the toxicity of the compound, the weight of important organs was measured and
divided by the body weight of each mouse to represent the organ index. Compared with the
untreated control group, no remarkable index change was observed in the compound 12b-treated
groups (Figure 6D). In the CTX group, however, the spleen index and thymus index decreased
shapely. Therefore, the data indicated that compound 12b is effective against solid tumor growth
with less side-effect than CTX.
A

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2.0
1.6
1.2
0.8
0.4
0.0
B
*
*
*
C
D

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Figure 6. Inhibition of subcutaneous solid tumor growth in vivo by compound 12b and YM155. (A) Picture of the
stripping tumor from mice. (B) The effect of 12b and YM155 on tumor weight was significantly different
compared with the vehicle control by the t-test (Mean ± S.D., n=7; *, P < 0.05). (C) Histological appearance of the
tumor. (D) Effect of 12b against spleen and thymus index (*, P < 0.05). CTX, a common clinical agent, was used
as a positive control of the study design.
3. Conclusion
Two novel series of 2-arylvinyl-substituted naphtho[2,3-d]imidazolium derivatives have been
successfully synthesized and screened for their antitumor activity against three human cancer cell
lines. The result revealed that although most of the new compounds show good activity with IC50
values below 10 µM, compound 12b displayed the strongest overall activity with IC50 values of
0.128 ± 0.017, 0.212 ± 0.029, and 0.059 ± 0.011 µM against PC-3, A375, and HeLa cell lines,
respectively. 7l showed the strongest activity against PC-3 cells with an IC50 value of only 0.022
µM. Additionally, both 7l and 12b exhibited potent suppression of Survivin protein expression.
Furthermore, the rate at which apoptosis was induced in PC-3 or A375 cells was higher after
treatment for 24 h with 12b than with 7l. Compound 12b was found to induce apoptosis, arrest
cell cycle progression at S phase, and lead to the depolymerization of microtubules and disruption
of the cytoskeleton in A375 cells. The underlying mechanism for this anticancer activity was
associated with a decrease in the expression of anti-apoptosis protein Bcl-2, hydrolysis of PARP,
inactivation of ERK, and activation of caspase-3 and caspase-7. In vivo tests demonstrated that
compound 12b provides effective antitumor activities with low immunotoxicity. Overall, these
results show that compound 12b, a structural modification from YM155, will lead to the
promising development of new anticancer drugs. Studies on the properties of PK
(pharmacokinetic) / PD (pharmacodynamic) and Toxicology are already in progress.
4. Experimental protocols
4.1. General
Melting points were determined in open capillaries using the MEL-TEMP II Melting Point
apparatus and are uncorrected values. Electrospray ionization (ESI) mass spectrometry data were
measured on an HP 1100 IC/MSD and reported as m/z. Elemental analyses were obtained using
the Elementar Vario EL III. NMR spectra were recorded with a BRUKER AV spectrometer at 300
MHz for 1H NMR and 100 MHz for 13C NMR. The chemical shifts are reported in parts per
million (ppm), and the coupling constants (J) are expressed in hertz (Hz) using trimethylsilane as
the internal standard. Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet
(q), quintet, broad (br) or multiplet (m). Reactions were monitored by analytical thin layer
chromatography, and the products were visualized by UV light.
2,3-Dichloro-1,4-naphthoquinone was purchased from TCI (Shanghai) Development Co., Ltd.
Known compounds 2 and 3 (see Scheme 1) were prepared from appropriate
2,3-dichloro-1,4-naphthoquinone according to previously published procedures [25]. The
synthesis of 9 (see Scheme 2) was carried out according to Ref. [30]. All solvents were distilled
before they were used, and all other reagents were obtained from commercial suppliers and were
of analytical grade. Silica gel (100-200 mesh) used in column chromatography was provided by
Tsingtao Marine Chemistry Co. Ltd..

ACCEPTED MANUSCRIPT
4.2. Synthesis
4.2.1. Experimental procedures for Scheme 1
4.2.1.1 Synthesis of compounds 4a-4b
General procedure: To a solution of 2-acetamido-3-chloro-1,4-naphthoquinone (3) (8 mmol) in
ethanol (20 mL) was added dropwise the appropriate primary amines (2 equivalents). The reaction
solution color turned from yellow to red. The mixture was stirred at 50°C for 30 min, and then
cool to room temperature. The resulting precipitated solid was filtered, washed with cooled
ethanol. The crude product was purified through recrystallization from ethanol to afford 4a-4b.
2-(Acylamino)-3-(cyclopropylamino)-1,4-naphthoquinones (4a). According to the general
procedure, compound 3 was treated with cyclopropylamine, and purification by recrystallization
from ethanol gave compound 4a.
1
Yield: 87.1%; orange red powder; mp: 140-141 °C; H NMR (CDCl3, 400MHz) δ: 8.01-8.09(m,
2H), 7.59-7.72(m, 2H), 7.20(s, 1H), 6.23(s, 1H), 2.95(s, 1H), 2.23(s, 3H), 0.78(s, 2H), 0.66(s, 2H);
ESI-MS: m/z 271.0 [M+H]+, C15H14N2O3 (MW=270.1).
2-(Acylamino)-3-(isopropylamino)-1,4-naphthoquinones (4b). According to the general procedure,
compound 3 was treated with isopropylamine, and purification by recrystallization from ethanol
gave compound 4b.
1
Yield: 81.6%; red powder; mp: 149-151 °C; H NMR (DMSO-d6, 400MHz) δ: 9.08(s, 1H),
7.96-8.01(m, 2H), 7.73-7.87(m, 2H), 6.43(d, J=8.4Hz, 1H), 4.09-4.14(m, 1H), 2.03(s, 3H), 1.19(d,
J=6.2Hz, 6H); ESI-MS: m/z 273.0 [M+H]+, C15H16N2O3 (MW=272.1).
4.2.1.2 Synthesis of compounds 5a-5b
General procedure: To a solution of compounds 4a-4b (10 mmol) in ethanol (50 mL) was added
2N NaOH (5 mL). The reaction mixture was heated at reflux for 30 min, then diluted with hot
water, and added 2N HCl (5 mL). After cooling to room temperature, yellow needle crystals
formed and was filtered. The crude product was purified through recrystallization from ethanol to
give 5a-5b.
1-Cyclopropyl-2-methylnaphth[2,3-d]imidazole-4,9-diones (5a).
1
Yield: 87.3%; yellow solid; mp: 210-212 °C; H NMR (CDCl3, 400MHz) δ: 8.13-8.15(m, 2H),
7.69-7.73(m, 2H), 3.34-3.38(m, 1H), 2.65(s, 3H), 1.36-1.42(m, 2H), 1.02-1.06(m, 2H); ESI-MS:
m/z 253.0 [M+H]+, C15H12N2O2 (MW=252.1).
1-Isopropyl-2-methylnaphth[2,3-d]imidazole-4,9-diones (5b).

ACCEPTED MANUSCRIPT
1
Yield: 82.7%; yellow solid; mp: 240-241 °C; H NMR (CDCl3, 400MHz) δ: 8.22-8.15(m, 2H),
7.69-7.74(m, 2H), 5.18(br, 1H), 2.65(s, 3H), 1.66(s, 3H), 1.64(s, 3H); ESI-MS: m/z 255.0 [M+H]+,
C15H14N2O2 (MW=254.1).
4.2.1.3 Synthesis of compounds 6a-6b
General procedure: To a solution of compounds 5a-5b (10 mmol) in 2-methoxyethanol (20mL)
was added ethyl iodide (1.5 equivalents). The reaction mixture was heated at reflux for 4 h. After
cooling, the red precipitate was filtered, washed with ethanol. The crude product was purified
through recrystallization from methanol to give 6a-6b as orange powder.
1-Cyclopropyl-2-methyl-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-ium
iodide
(6a).
Yield: 88.2%; orange powder; mp: 242-243 °C; 1H NMR (DMSO-d6, 400MHz) δ: 8.195-8.203 (m,
2H), 7.995-8.014(m, 2H), 4.62(m, 2H) 3.66-3.68(m, 1H), 2.92(s, 3H), 1.43(t, J=7.0Hz, 3H),
+
1.41(d, 2H), 1.21(s, 2H); ESI-MS: m/z 281.1 [M-I]+,C17H17IN2O2 (MW=408.2) , C17H17N2O2
(MW=281.1).
1-Isopropyl-2-methyl-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-ium iodide (6b).
1
Yield: 85.9%; orange powder; mp: 233-234 °C; H NMR (DMSO-d6, 400MHz) δ: 8.19-8.23(m,
2H), 8.00-8.03(m, 2H), 5.44(br, 1H), 4.69-4.70(m, 2H), 3.00(s, 3H), 1.66(d, J=6.8Hz, 6H), 1.44(t,
J=7.0Hz, 3H); ESI-MS: m/z 283.1 [M-I]+ ,C17H19IN2O2 (MW=410.2) , C17H19N2O2+ (MW=283.1).
4.2.1.4 Synthesis of compounds 7a-7l
General procedure: A mixture of compounds 6a (1 mmol), aromatic aldehydes (2 equivalents), 8
mL of dioxane, and 0.4 mL of pyridine was refluxed for 2 h. After cooling to room temperature,
the precipitate was filtered off, washed with ethanol, and dried. The crude product was purified
through recrystallization from methanol to give 7a-7f as orange powder.
Compounds 7g-7l were synthesized from 6b using the same method.
(E)-1-Cyclopropyl-2-styryl-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-ium iodide
(7a).
1
Yield: 91.7%; orange powder; mp: 216-217 °C; H NMR(DMSO-d6, 300 MHz) δ: 8.22(s, 2H),
8.13-8.30(m, 5H), 7.57(d, J=15Hz, 2H), 7.50-7.65(m, 2H), 4.80(s, 1H), 4.10-4.27(m, 2H),
+
1.37-1.43(m, 4H), 1.12(s, 3H); ESI-MS: m/z 369.1 [M-I]+, C24H21N2O2 (MW=369.1); Anal.calcd
for C24H21IN2O2·0.15HI (515.25): C56.00, H4.15, N5.27; Found: C55.92, H4.14, N5.43.
(E)-1-Cyclopropyl-2-(3,4-dimethoxystyryl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidaz
ol-3-ium iodide (7b).

ACCEPTED MANUSCRIPT
1
Yield: 65.9%; orange red powder; mp: 250 °C; H NMR(DMSO-d6, 300 MHz) δ: 8.19-8.22(m,
2H), 7.98-8.02 (m, 2H), 7.94(d, J=15Hz, 1H), 7.49-7.59(m, 2H), 7.35(d, J=15Hz, 1H), 7.14(d,
J=6Hz, 1H), 4.80-4.84(m, 2H), 3.87(s, 1H), 1.38-1.48(m, 4H), 1.08(s, 3H); ESI-MS: m/z 429.2
[M-I]+, C26H25IN2O4 (MW=429.2); Anal.calcd for C26H25IN2O4·0.06HI (563.75): C55.35, H4.23,
+
N4.83; Found: C55.36, H4.48, N4.97.
(E)-1-Cyclopropyl-2-(3,4,5-trimethoxystyryl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imi
dazol-3-ium iodide (7c).
Yield: 55.9%; red brown powder; mp: 235-236°C; 1H NMR(DMSO-d6, 300 MHz) δ: 8.02-8.22(m,
2H), 8.00-8.03(m, 2H), 7.92(d, J=18Hz, 1H), 7.44(d, J=18Hz, 1H), 7.30(s, 2H), 4.80-4.83(m, 2H),
4.27(s, 1H), 3.89(s, 6H), 3.76(s, 3H), 1.39-1.46(m, 4H), 1.09(s, 3H); 13C-NMR (DMSO-d6,
100MHz) δ: 175.29, 174.96, 173.44, 173.38, 153.28, 150.75, 150.06, 149.33, 140.76, 135.34,
135.01, 133.11, 132.05, 131.40, 130.28, 129.64, 126.92, 106.79, 105.74, 105.20, 60.25, 56.38,
+
56.34, 42.84, 34.74, 31.36, 11.38; ESI-MS: m/z 459.2 [M-I]+, C27H27IN2O5 (MW=459.2);
Anal.calcd for C27H27IN2O5·0.11HI (600.18): C53.93, H4.39, N4.40; Found: C54.00, H4.55,
N4.67.
(E)-1-Cyclopropyl-2-(4-hydroxy-3-methoxystyryl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d
]imidazol-3-ium iodide (7d).
1
Yield: 75.3%; orange powder; mp: 260°C; H NMR(DMSO-d6, 300 MHz) δ: 10.00(s, 1H),
8.20-8.22(m, 2H), 8.01-8.03(m, 2H), 7.93(d, J=18Hz, 1H), 7.56(s, 1H), 7.39-7.45(m, 1H), 7.28(d,
J=18Hz, 1H), 6.93(d, J=6Hz, 1H), 4.79-4.81(m, 2H), 4.04-4.06(m, 1H), 3.90(s, 3H), 1.38-1.48(m,
+
4H), 1.08(s, 3H); ESI-MS: m/z 415.2 [M-I]+, C25H23N2O4 (MW=415.2); Anal.calcd for
C25H23IN2O4·0.09HI (553.62): C54.15, H3.93, N4.87；Found: C54.21, H4.20, N5.06.
(E)-1-Cyclopropyl-2-(3-nitrostyry)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-iu
m iodide (7e).
Yield: 90.6%; orange powder; mp: 236-238°C; 1H NMR(DMSO-d6, 300 MHz) δ: 8.20-8.24(m,
2H), 8.06(d, J=15Hz, 1H), 8.01-8.03(m, 3H), 7.56(d, J=15Hz, 1H), 7.57-7.58(m, 3H),
4.78-4.81(m, 2H), 4.11-4.15(m, 1H), 1.36-1.45(m, 4H), 0.90-0.95(m, 3H); 13C-NMR (DMSO-d6,
100 MHz) δ: 174.91, 148.48, 146.38, 146.03, 135.81, 135.77, 135.40, 135.06, 134.51, 131.99,
130.66, 126.99, 126.80, 125.58, 123.35, 110.06, 109.56, 66.33, 43.11, 34.87, 31.37, 14.69, 11.28,
+
11.07; ESI-MS: m/z 414.1 [M-I]+, C24H20N3O4 (MW=414.1); Anal.calcd for C24H20IN3O4·0.12HI
(556.39): C51.78, H3.45, N7.24; Found: C51.78, H3.64, N7.55.

ACCEPTED MANUSCRIPT
(E)-1-Cyclopropyl-2-(2-(1H-indol-3-yl)vinyl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imi
dazol-3-ium iodide (7f).
1
Yield: 94.3%; red powder; mp: 329°C; H NMR(DMSO-d6, 300 MHz) δ: 12.01(s, 1H), 8.29(d,
J=18Hz, 1H), 8.208.22(m, 2H), 8.14-8.15(m,2H), 8.00-8.00(m, 2H), 7.55-7.58(m, 1H),
7.29-7.31(m, 2H), 7.10(d, J=18Hz, 1H), 4.79-4.81(m, 2H), 4.08-4.13(m, 1H), 1.39-1.52(m, 4H),
1.10(s, H3H); 13C-NMR (DMSO-d6, 100 MHz) δ: 175.08, 173.47, 150.96, 143.51, 137.47, 135.17,
134.83, 133.68, 132.72, 131.93, 131.49, 129.54, 126.78, 126.61, 124.95, 123.24, 121.69, 120.01,
112.95, 112.78, 97.90, 66.33, 42.45, 31.14, 14.29, 11.54; ESI-MS: m/z 408.2 [M-I]+,
+
C26H22IN3O2 (MW=408.2); Anal.calcd for C26H22IN3O2·0.05HI(541.47): C57.60, H3.72, N7.62;
Found: C57.64, H4.10, N7.76.
(E)-1-Isopropyl-2-(4-nitrostyry)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-ium
iodide (7g).
1
Yield: 83.1%; yellow powder; mp: 208°C; HNMR(DMSO-d6, 300 MHz) δ: 8.38-8.42(m, 2H),
8.17-8.28(m, 4H), 7.99-8.06(m, 2H), 7.63-7.75(m, 2H), 5.33-5.38(m, 1H), 4.68-4.71(m, 2H),
1.64-1.70(m, 6H), 1.41-1.45(m, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ: 174.90, 174.08, 148.49,
148.19, 144.56, 139.79, 135.50, 135.09, 131.88, 131.48, 131.09, 129.88, 127.37, 126.78, 124.03,
111.81, 66.33, 54.42, 52.75, 43.94, 42.73, 20.08, 19.61, 14.63; ESI-MS: m/z 416.1 [M-I]+,
+
C24H20N3O4 (MW=416.1); Anal.calcd for C24H22IN3O4·0.08HI (553.09): C51.96, H4.06, N7.11;
Found: C51.95, H4.01, N7.57.
(E)-1-Isopropyl-2-(3-hydroxystyryl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-iu
m iodide (7h).
1
Yield: 75.8%; orange powder; mp: 234-235°C; H NMR(DMSO-d6, 300 MHz) δ: 9.79(s, 1H),
8.03-8.20(m, 4H), 7.35-7.51(m, 4H), 7.30(d, J=15Hz, 2H), 6.95(d, J=6Hz, 1H), 5.30-5.32(m, 1H),
4.66-4.68(m, 2H), 1.68(d, J=6Hz, 6H), 1.46-1.51(m, 3H); ESI-MS: m/z 387.2 [M-I]+,
+
C24H23N2O3 (MW=387.2); Anal.calcd for C24H23IN2O3·0.05HI (520.47): C55.96, H4.21, N5.00;
Found: C55.58, H4.48, N5.40.
(E)-1-Isopropyl-2-(2-(1H-indol-3-yl)vinyl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidaz
ol-3-ium iodide (7i).
1
Yield: 91.6%; red brown powder; mp: 230°C; HNMR(DMSO-d6, 300 MHz) δ: 12.10(s,1H),
8.11-8.27(m, 4H), 8.01-8.08(m, 2H)，7.83(d, J=16.5Hz, 1H), 7.54-7.57(m, 1H), 7.25-7.32(m, 2H),
7.05(d, J=16.5Hz, 1H), 5.34-5.38(m, 1H), 4.70-4.72(m, 2H), 1.72(d, J=6Hz, 6H), 1.51-1.55(m,
3H); 13C-NMR (DMSO-d6, 100 MHz) δ: 175.03, 174.21, 141.19, 137.36, 135.34, 135.09, 133.01,
127.22, 126.44, 124.73, 123.04, 121.40, 119.92, 112.66, 112.03, 98.74, 66.33, 53.97, 43.63, 39.74,

ACCEPTED MANUSCRIPT
+
39.53, 39.32, 20.10, 14.52; ESI-MS: m/z 410.2 [M-I]+,C26H24N3O2 (MW=410.2); Anal.calcd for
C26H24IN3O2·0.02HI (539.65): C57.83, H4.19, N7.53; Found: C57.80, H4.48, N7.78.
(E)-1-Isopropyl-2-(4-hydroxy-3-methoxystyryl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]i
midazol-3-ium iodide (7j).
1
Yield: 67.8%; orange powder; mp: 225-226°C; H NMR(DMSO-d6, 300 MHz) δ: 9.90(s, 1H),
8.20-8.24(m,2H), 8.01-8.03(m,2H), 7.53(s, 1H), 7.36(t, J=18Hz, 2H)，7.22-7.36(m, 1H), 6.91(d,
J=9Hz, 1H), 5.28-5.32(m, 1H), 4.66-4.68(m, 2H), 3.89(s, 3H), 1.63-1.70(m, 6H), 1.43-1.51 (m,
3H); 13C-NMR (DMSO-d6, 100 MHz) δ: 174.97, 174.13, 150.31, 149.56, 148.09, 147.35, 135.42,
135.18, 131.85, 131.79, 131.34, 131.14, 127.29, 126.70, 125.28, 123.86, 115.64, 111.86, 103.09,
56.04, 54.10, 43.72, 39.74, 20.06, 14.59; ESI-MS: m/z 417.2 [M-I]+,C25H25N2O4 (MW=417.2);
+
Anal.calcd for C25H25IN2O4·0,.02HI (546.64): C54.80, H4.18, N4.82; Found: C54.87, H4.61,
N5.12.
(E)-1-Isopropyl-2-(4-hydroxystyryl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-iu
m iodide (7k).
1
Yield: 75.8%; yellow powder; mp: 235°C; HNMR(DMSO-d6, 300 MHz) δ: 10.24(s, 1H),
8.02-8.20(m, 4H), 7.78(d, J=9Hz, 2H), 7.40(d, J=15Hz, 2H), 6.91(d, J=9Hz, 2H), 5.30(s, 1H),
4.66-4.68(m, 2H), 1.67(d, J=9Hz, 6H), 1.47-1.51(m, 3H); ESI-MS: m/z 387.2
+
[M-I]+,C24H23N2O3 (MW=387.2); Anal.calcd for C24H23IN2O3·C4H8O2 (601.93): C55.73, H4.73,
N4.20; Found: C55.82, H5.19, N4.65.
(E)-1-Isopropyl-2-(4-methylstyryl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-iu
m iodide (7l).
1
Yield: 68.1%; orange powder; mp: 210-212°C; H NMR(DMSO-d6, 300 MHz) δ: 8.21-8.26(m,
2H), 8.02-8.05(m, 2H), 7.92-7.93(m, 2H), 7.52-7.59(m,2H), 7.49(t, J=15Hz, 2H),
5.33-5.35(m,1H), 4.67-4.70(m, 2H), 1.68(d, J=6Hz, 6H), 1.48-1.52(m, 3H); ESI-MS: m/z 385.2
+
[M-I]+,C25H25N2O2 (MW=385.2); Anal.calcd for C25H25IN2O2·0.5H2O (521.09): C53.35, H5.05,
N5.57; Found: C53.60, H4.72, N5.49.
4.2.2. Experimental procedures for Scheme 2
4.2.2.1 2-Chloro-3-((2-methoxyethyl)amino)-1,4-naphoquinone (8).
To an ice-cold stirred solution of compounds 1 (10 mmol) and triethylamine (1 mL) in DMF (20
mL) was added dropwise 2-methoxyethylamine (12.5 mmol). After the mixture color turned from
light yellow to red, the reaction continued at room temperature for 1h, then poured into 300 mL
water to form a orange red precipitate. The resulting precipitated solid was filtered, washed with
cooled water, and dried.

ACCEPTED MANUSCRIPT
1
Yield: 96.1%; orange powder; mp: 82-83°C; H NMR (DMSO-d6, 400MHz) δ: 7.98(m, 2H),
7.75-7.87(m, 2H), 7.25(s, 1H), 3.92-3.94(m, 2H), 3.57(t, J = 5.7Hz, 2H), 3.29-3.38(s, 3H);
ESI-MS: m/z 266.1 [M+H]+, C13H12ClNO3 (MW=265.1).
4.2.2.2 2-Chloro-3-((2-methoxyethyl)acetylamino)-1,4-naphoquinone (9).
This compound was prepared using a modification of the procedure described by Mastsuhisa [30].
A few drops of concentrated sulfuric acid was added to a solution of compound 8 (10 mmol) in
acetic anhydride (3 mL), and the mixture was stirred at 50°C for 1 h. By adding ethanol (3 mL) to
the reaction solution, excess acetic anhydride was esterified. After cooling, ethyl acetate was
added to the reaction solution and the mixture was washed with water and brine before being dried
over anhydrous sodium sulfate. The solvent was evaporated and the residue was crystallized from
diethyl ether to give 9.
4.2.2.3 Synthesis of compounds 10a-10b
To a solution of compound 9 (10 mmol) in ethanol (20 mL) was added 22.5 mmol benzylamine ,
then the mixture color turned from yellow to red. The reaction solution continued for 1h at room
temperature, and then concentrated to dryness under reduced pressure. The crude material was
purified on a silica gel column with the gradient eluent to yield compounds 10a. The elution was
petroleum ether/ethyl acetate (v/v from 10:1 to 2:1, based on the polarity of final products).
2-benzylamino-3-((2-methoxyethyl)acetylamino)-1,4-naphoquinone (10a).
1HNMR (DMSO-d6, 400MHz) δ: 8.12-8.14 (m, H), 8.07-8.09 (m, H), 7.75-7.78 (m, H), 7.65-7.68
(m, H), 7.32-7.39 (m, 4H), 7.27-7.30 (m, H), 6.58 (br, H), 4.80-4.82 (m, H), 4.57-4.60 (m, H),
3.69-3.74 (m, H), 3.42-3.47(m, H), 3.14-3.25 (m, 3H), 1.80-2.04 (m, 3H), 1.5-1.8 (m, 2H);
ESI-MS: m/z 378.2 [M]+.
To a solution of compound 9 (10 mmol) in ethanol (20 mL) was added 22.5 mmol
3-methoxybenzylamine, then the mixture color turned from yellow to red. The reaction solution
continued for 1h at room temperature, and then concentrated to dryness under reduced pressure.
The intermediate 10b was obtained.
4.2.2.4 Synthesis of compounds 11a-11b
Hydrogen bromide (1 mL) was added to a solution of compound 10a (10 mmol) in
2-methoxyethanol (20 mL), and the mixture was refluxed for 2 h. The reaction solution was
cooled and the precipitate was filtered, wash and dried. The crude product was purified through
recrystallization from methanol to give 11a as yellow powder.

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1-benzyl-3-(2-methoxyethyl)-2-methyl-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imidazol-3-ium
bromide(11a).
Yield: 80.6%; orange yellow powder; mp: 199-201°C; 1HNMR (DMSO-d6, 400MHz) δ: 8.16-8.17
(m, 2H), 7.98-8.03 (m, 2H), 7.38-7.43 (m, 3H), 7.31-7.36 (m, 2H), 6.03 (s, 2H), 4.86 (t, J=5Hz,
2H), 3.80 (t, J=5Hz, 2H), 3.24(s, 3H), 2.84(s, 3H); ESI-MS: m/z 361.1 [M-Br]+.
Hydrogen bromide (1 mL) was added to a solution of intermediate 10b in 2-methoxyethanol (20
mL), and the mixture was refluxed for 2 h. The reaction solution was cooled and condensed to
afford crude 11b as brown liquid, which used immediately for the next step.
4.2.2.5 Synthesis of compounds 12a-12g
General procedure: A mixture of compound 11a (1 mmol), aromatic aldehydes (2 equivalents), 8
mL of dioxane, and 0.4 mL of pyridine was refluxed for 2 h. After allowing to cool to room
temperature, the precipitate was filtered off, washed with ethanol, and dried. The crude product
was purified through recrystallized from methanol to give 12a-12d as yellow powder.
Compounds 12e-12g were synthesized from 11b using the same method.
1-Benzyl-3-(2-methoxyethyl)-2-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)-4,9-dioxo-4,9-dihydro-1H-n
aphth[2,3-d]imidazol-3-ium bromide (12a).
1
Yield: 63.7%; yellow powder; mp: 202°C; H NMR(DMSO-d6, 300 MHz) δ: 8.02-8.22(m, 4H),
7.54-7.62(m, 3H), 7.38-7.42(m, 2H), 7.28-7.35(m, 5H), 7.06(d, J=15Hz, 2H), 6.08(s, 2H),
4.81-4.89(m, 2H), 3.93(d, J=21Hz, 2H), 3.28-3.30(m, 2H), 3.02(s, 3H); ESI-MS: m/z 475.2
+
[M-Br]+,C31H27N2O3 (MW=475.2); Anal.calcd for C31H27BrN2O3·0.25HBr (574.20): C64.09，
H4.86，N5.14; Found: C64.23, H4.74, N4.83.
(E)-1-Benzyl-3-(2-methoxyethyl)-2-(2-(1H-indol-3-yl)vinyl)-4,9-dioxo-4,9-dihydro-1H-naphth[2,3
-d]imidazol-3-ium bromide (12b).
1
Yield: 85.6%; orange powder; mp: 200-202°C; H NMR(DMSO-d6, 300 MHz) δ: 12.12(s, 1H),
8.00-8.20(m, 4H), 7.93-7.96(m, 2H), 7.77-7.80(m, 3H), 7.40-7.42(m, 5H), 7.23(t, J=15Hz, 2H),
6.15(s, 2H), 4.93-4.97(m, 2H) 3.95-4.01(m, 2H), 3.57(s, 3H); 13CNMR(75 MHz, DMSO-d6,) δ:
175.39, 151.22, 142.18, 137.84, 135.64, 134.92, 133.81, 132.24, 131.25, 130.98, 129.52, 128.71,
127.27, 126.89, 125.15, 123.69, 122.12, 119.88, 113.34, 113.06, 98.37, 70.34, 59.07, 50.95, 48.52,
+
40.75, 40.47, 39.91, 39.07; ESI-MS: m/z 488.2 [M-Br]+,C31H26N3O3 (MW=488.2); Anal.calcd for
C31H26BrN3O3·0.25HBr (587.20): C63.38, H4.97, N6.60; Found: C63.35, H4.69, N6.93.
(E)-1-Benzyl-3-(2-methoxyethyl)-2-(4-hydroxy-3-methoxystyryl)-4,9-dioxo-4,9-dihydro-1H-naphth
[2,3-d]imidazol-3-ium bromide (12c).

ACCEPTED MANUSCRIPT
1
Yield: 57.6%; brown powder; mp: 178-180°C; H NMR(DMSO-d6, 300 MHz) δ: 7.40-7.49(m,
9H), 7.32-7.34(m, 2H), 7.09(d, J=15Hz, 1H), 6.93(s, 1H), 6.51(d, J=15Hz, 1H), 3.87(s, 2H),
3.78-3.80(m, 2H), 3.75-3.76(m, 2H), 3.28(s, 3H), 3.25(s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ:
ESI-MS: m/z 495.2 [M-Br]+; Anal.calcd for C30H27BrN2O5·1.3H2O (597.40): C60.35, H4.72,
N5.93; Found: C60.47, H5.01, N5.68.
(E)-1-Benzyl-3-(2-methoxyethyl)-2-(3-hydroxystyryl)-4,9-dioxo-4,9-dihydro-1H-naphth[2,3-d]imid
azol-3-ium bromide (12d).
1
Yield: 72.9%; yellow powder; mp: 199-200°C; H NMR(DMSO-d6, 300 MHz) δ: 9.80(s, 1H),
8.00-8.20(m, 4H), 7.32-7.38(m, 5H), 7.27-7.30(m, 3H), 7.09(d, J=18Hz, 2H), 6.93(s, 1H), 3.87(s,
+
2H), 3.88(s, 2H), 3.57(s, 2H), 3.25(s, 3H); ESI-MS: m/z 465.2 [M-Br]+ ,C29H25N2O4 (MW=465.2);
Anal.calcd for C29H25BrN2O4·0.5H2O (553.20): C62.81, H4.82, N4.46; Found: C62.82, H4.73,
N4.68.
(E)-1-(3-Methoxybenzyl)-3-(2-methoxyethyl)-2-(2-(1H-indol-3-yl)vinyl)-4,9-dioxo-4,9-dihydro-1H
-naphth[2,3-d]imidazol-3-ium bromide (12e).
Yield: 86.8%; purple red powder; mp: 196-197°C; 1H NMR(DMSO-d6, 300 MHz) δ: 8.20-8.22(m,
2H), 8.00-8.02(m, 2H),7.94(m, 1H), 7.68(d, J=9Hz, 1H), 7.53(d, J=9Hz, 1H), 7.35(t, J=9Hz, 1H),
7.18-7.29(m, 2H), 6.93-6.98(m, 3H), 6.12(s, 2H), 4.96(s, 1H), 3.94-3.97(m, 2H), 3.71(s, 3H),
3.57(s,3H); 13C-NMR (DMSO-d6, 100 MHz) δ: 174.92, 159.76, 141.72, 137.39, 135.99, 133.31,
131.71, 130.28, 126.79, 124.73, 123.21, 121.59, 119.41, 118.39, 113.31, 112.86, 112.63, 112.51,
97.88, 69.88, 66.33, 58.58, 55.12, 50.29, 47.99, 40.16, 39.95, 39.74, 39.53, 39.32, 39.11, 38.91;
+
ESI-MS: m/z 518.2 [M-Br]+,C32H28N3O4 (MW=518.2); Anal.calcd for C32H28BrN3O4·0.6H2O
(607.80): C63.32, H5.02, N6.52; Found: C63.08, H4.83, N6.90.
(E)-1-(3-Methoxybenzyl)-3-(2-methoxyethyl)-2-(3,4,5-trimethoxystyryl)-4,9-dioxo-4,9-dihydro-1H
-naphth[2,3-d]imidazol-3-ium bromide (12f).
1
Yield: 50.9%; yellow powder; mp: 194-195°C; H NMR(DMSO-d6, 300 MHz) δ: 8.22-8.26(m,
2H), 8.00-8.03(m, 2H), 7.61(d, J=15Hz, 1H), 7.43(d, J=15Hz, 1H), 7.32-7.38(m, 1H), 7.03(s, 2H),
6.90-6.95(m, 3H), 6.15(s, 2H), 4.99(s, 2H), 3.86(s, 6H), 3.73(s, 3H), 3.31(s, 3H); ESI-MS: m/z
+
569.2 [M-Br]+,C33H33N2O7 (MW=569.2); Anal.calcd for C33H33BrN2O7·2H2O (684.20): C56.17,
H5.56, N4.72; Found: C56.17, H5.30, N4.36.
(E)-1-(3-Methoxybenzyl)-3-(2-methoxyethyl)-2-(3-hydroxystyryl)-4,9-dioxo-4,9-dihydro-1H-napht
h[2,3-d]imidazol-3-ium bromide (12g).
1
Yield: 61.5%; orange powder; mp: 204°C; H NMR(DMSO-d6, 300 MHz) δ: 9.80(s, 1H),
8.00-8.20(m, 4H), 7.53-7.57(m, 1H), 7.28-7.33(m, 3H), 7.09(d, J=15Hz, 2H), 6.90-6.94(m, 4H),
6.05(s, 2H), 4.94(s, 2H), 3.69(s, 3H), 3.25(s, 3H); ESI-MS: m/z 495.2

ACCEPTED MANUSCRIPT
+
[M-Br]+,C30H27N2O5 (MW=495.2); Anal.calcd for C30H27BrN2O5·0.18HBr (588.60): C60.74,
H4.68, N4.34; Found: C60.77, H4.62, N4.72.
4.3. Biological evaluation procedures
4.3.1. Drugs and drug treatments
Compound YM155 was used as is after purchasing from Zhongjiu Technology (Shanghai) Co. Ltd.
Paclitaxel (Sigma, T7191) was dissolved in DMSO to make a stock concentration of 70 µM.
Solutol® HS15, a nonionic solubilizer that consists of polyglycol mono- and di-esters of
12-hydroxystearic acid with approximately 30% of free polyethylene glycol, was purchased from
BASF AG (Ludwigshafen, Germany) for making the injection solutions.
4.3.2. Cell lines and cultures
A375 (human malignant melanoma cell line), PC-3 (human prostate carcinoma cell line),
Ramos (Human Burkitt's lymphoma cell line) and Hela (human cervical carcinoma cell line) were
purchased from ATCC. The cells were cultured in DMEM, F12K and RPMI 1640 medium (Gibco),
respectively, and supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100
U/mL), and streptomycin (100 µg/mL) in a humidified atmosphere of 5% CO2 at 37°C.
4.3.3. Anti-proliferative assays
Cells were seeded into 96-well plates (100 µL of media containing 3000-10000 cells per well)
and precultured for 1 day. Stock solutions (10 mM) of test compounds were prepared fresh by
dissolving in DMSO. These solutions were used to prepare 10 µM of working solution, along with
a 3-fold serial dilution in appropriate media. 100 µL of agent-containing media or control vehicle
was added to each well. After 48 h of incubation at 37°C in a humidified atmosphere of 5% CO2,
cytotoxicity was determined using the CellTiter 96 Aqueous One Solution Reagent (Promega,
USA) according to the manufacturer's instructions. The detection is based on the cellular
conversion of a tetrazolium salt into a soluble formazan product as a measure of proliferation. The
absorbance was measured at 492 nM by the Tecan Sunrise Microplate Reader (Tecan,
Switzerland). Cellular response curves and IC50 values were calculated from the data.
4.3.4. Apoptosis detection assays
A375 cells were grown in the absence or presence of different concentrations (10, 100 and 1,000
nM) of 12b for 24 h. For FACS analysis, cells were treated with either 12b or YM155 for 24 h and
then stained with Annexin V and propidium iodide using the Annexin V/propidium iodide
apoptosis kit per the manufacturer's instructions (Biouniquer, BU-AP0103). Data were acquired
using a FACSCalibur system (BD Biosciences) and analyzed by FlowJo software.
4.3.5. Western blot analysis

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A375 cells (1×105 cells/well) were seeded into 6-well culture plates. After treatment with 12b or
YM155 at the indicated concentration for 48 h, the media was removed and cells were
immediately lysed in RIPA Lysis Buffer (Beyotime, P0013B). The resulting lysates containing
equal amounts of protein were boiled in sodium dodecyl sulfate (SDS)-sample buffer for 5 min
before loading on a 12% SDS-polyacrylamide gel. Proteins were electro-transferred to a
polyvinylidene difluoride (PVDF) membrane and blocked in PBS containing 5% non-fat dry milk
for 1 h at room temperature. Primary antibodies used included a polyclonal antibody to Survivin
(Novus biologicals, NB500-201), caspase-3 (Cell Signaling Technology, 9662S), Bcl-2 (Cell
Signaling Technology, 2876), PARP (Beyotime, AP102), p-ERK (Cell Signaling Technology,
4695), and total-ERK (Cell Signaling Technology, 9101) as well as a monoclonal antibody to
GAPDH (Beyotime, AG019) at a 1:1000 dilution. Secondary antibodies used included anti-rabbit
IgG peroxidase conjugate (Sigma, A0545) and anti-mouse IgG peroxidase conjugate (Sigma,
A2554). Bands were visualized using the ECL system (PerkinElemer, NEL104001EA), and
pictures were taken using the Bio-Rad ChemiDoc XRS System.
4.3.6. Caspase activity assay
Cells were seeded at 8 × 103 cells/well and treated with the test compounds at 1000 nM.
Subsequently, caspase 3/7 activity was measured to gauge cell apoptosis as determined by the
Caspase-Glo 3/7 assay kit (Promega) following the manufacturer's instructions. Luminescence
was detected using a Tecan Infinite 200 microplate reader (Tecan, Switzerland).
4.3.7. Cell cycle arrest assay
Cell cycle arrest was analyzed by flow cytometry. Briefly, A375 cells (2×105 cells/well) were
seeded into 6-well plates and exposed to various concentrations of compounds 12b (0, 100, 200
and 400 nM). At appropriate intervals, treated and control cells were harvested by trypsinization
and centrifuged at 4°C. Cell pellets were washed twice with phosphate buffered saline (PBS) and
fixed in 70% ice-cold ethanol at -20°C until all time points had been collected for analysis. The
samples were concentrated by removing EtOH and staining the cellular DNA with PI/RNase
Staining Buffer (BD, 550825) for 30 min at 4°C in the dark. The cell cycle distribution was
detected using a FACScan Flow Cytometry (BD FACSCalibur) with 10,000 events per
determination, and the DNA content of the cells was analyzed using the Modfit LT program
(Verity Software, USA).
4.3.8. Immunofluorescence staining
All reagents for fixation, wash, and blocking steps were purchased from Beyotime (Beyotime).
A375 cells (8×103 cells/well) were maintained in black 96-well microplates (Corning, 3603) and
treated with DMSO, 12b, YM155 or Paclitaxel for 24 h at 37°C. Samples were fixed, blocked, and
permeabilized after being washed. Cells were subsequently incubated with monoclonal α-tubulin

ACCEPTED MANUSCRIPT
primary antibody (Beyotime, AT819) overnight, washed extensively, and incubated in
FITC-labeled secondary antibody (Invitrogen, 626511) for 3 h. Nuclei were labeled with DAPI.
Images of fluorescently labeled tubulin and nuclei were captured on an ImageXpress 5000A
automated cellular imaging and analysis system (Molecular Devices).
4.3.9. Antitumor evaluation in mice
Adult male ICR mice, 6 weeks old and weighing 20-25 g, were purchased from the Experimental
Animal Center of Yangzhou University. The mice were housed in polycarbonate cages under
standard laboratory conditions (24±1°C, 12 light/dark cycle) with food and water ad libitum. All
procedures used for animal experimentation were approved by the China Pharmaceutical
University Institute Animal Care and Use Committee. Preliminary experiments determined the
maximum tolerated dose of compound 12b to be 1.0 mg/kg in non-tumor-bearing animals. YM155
and 12b were freshly prepared daily using the formulation solvent containing saline, 4% N,
N-Dimethylacetamide (DMA), and 2% Solutol® HS15. Cyclophosphamide (CTX) was diluted
with only saline. All the solutions were injected intraperitoneally in a volume of 0.1 mL per 10 g
of body weight.
Ehrlich ascites carcinoma (EAC) is an undifferentiated carcinoma, originally hyperdiploid, and
has high transplantable capability, rapid proliferation, shorter life span and 100% malignancy.
EAC resembles human tumors that are most sensitive to chemotherapy because they are
undifferentiated and have a rapid growth rate [28]. A fixed number of viable EAC cells (1×106
cells/22 g b. wt) were implanted into the peritoneal cavity of each donor mouse. The tumor cells
were then withdrawn, diluted in sterile saline, counted and inoculated (5×106 cells/animal) to the
right thigh of experimental animals by subcutaneous (s.c.) injection for the development of solid
tumor [29].
The day of tumor implantation was considered day 0. On day 1, animals were randomly divided
into five groups (n=7 each) and administered with or without compound 12b by intraperitoneal
(i.p.) injection for 10 d: (1) Vehicle control group: only formulation; (2) CTX group: 30
mg/kg/day of CTX; (3) 12b-1 group: 0.06 mg/kg/day of 12b; (4) 12b-2 group: 0.2 mg/kg/day of
12b; and (5) 12b-3 group: 0.6 mg/kg/day of 12b. Mice with subcutaneous solid tumors were
weighed every day. At the end of the experiment, mice were sacrificed and their tumor masses and
important organs dissected and weighed.
4.3.10. Experimental design and data analysis
Dose-response curves were analyzed using the GraphPad PrismTM 5 software program to calculate
IC50 values using a three-parameter logistic equation. Data were expressed as the mean and
standard deviation. The statistical significance of the differences observed in the results of the
independent experiments was analyzed using the Student’s t-test. A p-value of <0.05 was
considered statistically significant.

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Synthesis and biological evaluation of novel 2-arylvinyl-substituted
naphtho[2,3-d]imidazolium halide derivatives as potent antitumor
agents
*
Qingyun Weia, Ju Lia, Feng Tangb, Yin Yinb, Yong Zhaob, **, Qizheng Yaoa,
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China
b MtC Biopharma, Co., Ltd, Nanjing 210042, PR China
Highlights
•
Two series of 2-arylvinyl-substituted naphtho[2,3-d]imidazolium derivatives were
designed and synthesized.
•
•
•
Compound 12b exhibited potent antiproliferative activity.
Compound 12b induced cell apoptosis and affected microtubule networking.
In vivo 12b inhibited subcutaneous solid tumor growth.