Synthesis and biological evaluation of naphthoquinone phenacylimidazolium derivatives

Article abstract of DOI:10.1016/j.bmcl.2021.127977

In order to expand structural diversity and improve antitumor efficiency, forty new naphthoquinone phenacylimidazolium derivatives were designed, synthesized and evaluated. Good synthetic yields were obtained under mild conditions using easily available starting materials. Cytotoxicity of these compounds was evaluated in vitro against a panel of human tumor cell lines: human breast carcinoma cell lines (MCF-7), human cervical carcinoma cell lines (HeLa), and human lung carcinoma cell lines (A549). Among them, the optimal compound 7m showed splendid antiproliferative activity with low to 50 nM IC₅₀ values against MCF-7 and excellent selectivity of 256-fold compared with the normal cell lines L929. Compound 7m induced apoptosis in a dose-dependent manner. Further mechanism experiments showed that compound 7m dramatically inhibited the expression of survivin and activated the pro-apoptotic protein caspase-3. Our results indicated that the structural modification on the 1,3-substituents of naphthoquinone imidazoliums without 2-substituent is also promising to obtain new antitumor compounds.

Full text of DOI:10.1016/j.bmcl.2021.127977

Bioorg. Med. Chem. Lett. 41 (2021) 127977
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of naphthoquinone
phenacylimidazolium derivatives
,
,b
Jing Yuana, Zhanxiong Liua, Zhenfeng Zhangb *, Deyue Yana, Wanbin Zhanga
a Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, Frontier Science Center for Transformative Molecules, School of Chemistry and Chemical
Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China
b School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In order to expand structural diversity and improve antitumor efficiency, forty new naphthoquinone phenacy-
limidazolium derivatives were designed, synthesized and evaluated. Good synthetic yields were obtained under
mild conditions using easily available starting materials. Cytotoxicity of these compounds was evaluated in vitro
against a panel of human tumor cell lines: human breast carcinoma cell lines (MCF-7), human cervical carcinoma
cell lines (HeLa), and human lung carcinoma cell lines (A549). Among them, the optimal compound 7m showed
splendid antiproliferative activity with low to 50 nM IC50 values against MCF-7 and excellent selectivity of 256-
fold compared with the normal cell lines L929. Compound 7m induced apoptosis in a dose-dependent manner.
Further mechanism experiments showed that compound 7m dramatically inhibited the expression of survivin
and activated the pro-apoptotic protein caspase-3. Our results indicated that the structural modification on the
1,3-substituents of naphthoquinone imidazoliums without 2-substituent is also promising to obtain new anti-
tumor compounds.
Naphthoquinone
Imidazolium
Phenacyl
Antitumor
Antiproliferative
Quinones, being a common and classic skeleton, have been found in
numerous biological molecules and pharmaceutical compounds.1 For
instance, Doxorubicin as an anthraquinone and Mitomycin C as a ben-
zoquinone are well-known clinically used chemotherapeutics with
strong antitumor effect, but their side-effects and resistances cannot be
ignored.2,3 These made scientists committed to the synthesis of related
derivatives with different targets and mechanisms. YM155, containing a
naphthoquinone fused 1,2,3-trisubstituted imidazole structure, has
entered clinical trials to treat lung tumor, lymphoma, melanoma, and
prostate.4–6 Owing to its novel structure and excellent activity, in recent
years a series of YM155 analogs have been designed and synthesized as
antitumor drugs via DNA damage and/or survivin inhibition, such as
Go’s and Yao’s works (Fig. 1).7–10 To achieve both high activity and
selectivity, the design and synthesis of novel naphthoquinone imidaz-
oles with other substituting types is still highly desired.
Doxorubicin) and 16-fold selective ratio (better than Doxorubicin) for
the optimized compound. In order to further expand the diversity of this
kind of molecules, herein we designed and synthesized a new type of
naphthoquinone imidazoles with 1,3-disubstituents. Different from the
widely screened compounds with 1,2,3-trisubstituents, 1,3-disubstituted
ones without 2-substituent were almost untouched but expected to give
better performances. Furthermore, a 3-phenacyl group was introduced
inspired by the previously reported works.13–18 Gratifyingly, a more
promising molecule from forty tested compounds was selected with an
improved IC50 value of 50 nM against breast carcinoma cells and
selectivity of 256-fold.
Following an optimized reaction condition (see Supporting Infor-
mation for detail), twelve target compounds 1-12a possessing a 3-para-
nitro-phenacyl substituent were smoothly synthesized via a simple
nucleophilic substitution from the previously reported naphthoquinone
imidazoles bearing diverse alkyl and aryl substituents at 1-position
(Scheme 1).11,12 Other twenty-eight desired compounds 1/7b-o
bearing a methyl and isobutyl group at 1-position and different acyl
methylene substituents at 3-position were obtained in high yields by the
Previously, our group has developed a series of naphthoquinones
bearing a 1-monosubstituted or 1,2-disubstituted imidazole segment for
potential anticancer drugs.11,12 The in-vitro antiproliferative evaluation
recorded the IC50 values of 4.3–10.6 µM (a little worse than
* Corresponding author.
E-mail address: zhenfeng@sjtu.edu.cn (Z. Zhang).
https://doi.org/10.1016/j.bmcl.2021.127977
Received 2 February 2021; Accepted 14 March 2021
Available online 22 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

J. Yuan et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127977
Fig. 1. The design of novel anticancer agents.
similar synthetic method (Scheme 1).
shown in Fig. 2, compound 7m remarkably induced apoptosis of MCF-7
MTT assay was utilized to evaluate the synthesized compounds in
vitro against three human cancer cell lines: human breast carcinoma cell
lines (MCF-7), human cervical carcinoma cell lines (HeLa), and human
lung carcinoma cell lines (A549), and one normal cell line: mouse
fibroblast cells (L929). Good efficacy is necessary for a chemothera-
peutic drug, and safety is also a very important parameter. To our
delight, the compounds synthesized in this paper have enormous
lethality to cancer cells with almost no damage to the normal cells. The
activity and selectivity data of the compounds possessing different
substituents at 1- and 3-positions were displayed in Table 1 and Table 2.
Firstly, the compounds 1-12a bearing 3-phenacyl group and different 1-
substituents were tested. Compounds 4-5a and 10-12a with propargyl,
cyclopropyl, and aryl at 1-position showed lower activity than others
with simple alkyl groups. The compounds 1a and 7a possessing methyl
and isobutyl group at 1-position showed the best selectivity and the
highest activity respectively were chosen for the further studies. Sub-
stances of series 1 with 1-methyl exhibit good antitumor activity ranging
from low micromole to nanomole level and excellent selectivity to both
MCF-7 and A549, especially like 1i and 1m. The substances of series 7
with the 1-isobutyl group show good antiproliferative ability except 7c.
The IC50 values of 7d, 7f, 7i, 7l, and 7m reach a nanomole level to the
three tested cancer cells. What is worth mentioning is that 7i and 7m
were able to kill all the three cancer cells without obvious harm to
normal cells, giving a much better performance than the clinical drug
Doxorubicin. Considering both antiproliferation activity and selectivity,
compound 7m bearing 1-isobutyl and 3-(2-oxo-2-(thiophen-2-yl)ethyl)
groups was chosen as the optimal molecule.
cells, exhibited 52.12%, 73.64% and 91.17% apoptosis rate at concen-
tration of 0.02 μM, 0.1 μM and 0.2 μM, respectively.
We further investigated the effect of compounds 7m on cell cycle by
flow cytometry. The results indicated that compound 7m had remark-
ably arrested cells on S phase of cell cycle (Fig. 3). After 48 h of treat-
ment with 7m, the percent of S phase arrested cells increased from
24.4% of vehicle to 43.7% in a concentration-dependent manner.
To further elucidate the potential mechanism of compound 7m
induced apoptosis, western blotting was performed on apoptosis-related
markers, in which Actin was the internal reference. As shown in Fig. 4,
treatment of MCF-7 cells with compound 7m enhanced protein expres-
sion of cleavage caspase 3 at low concentrations, which carried out the
cell apoptosis procedure. Moreover, the expression of survivin was
significantly inhibited by compound 7m.
In summary, totally forty novel naphthoquinone phenacylimidazo-
lium derivatives were successfully synthesized and their antitumor ac-
tivities against three human cancer cell lines and the selectivity to a
normal cell line were screened. Among them, the optimal compound
7m, 1-isobutyl-4,9-dioxo-3-(2-oxo-2-(thiophen-3-yl)ethyl)-4,9-dihydro-
1H-naphtho[2,3-d]imidazol-3-ium bromide showed the excellent activ-
ity of 50 nM IC50 against MCF-7 cell line and 256-fold selectivity versus
normal cells. Furthermore, compound 7m was found to significantly
induce apoptosis. More importantly, compound 7m not only activated
pro-apoptotic protein caspase-3, but also dramatically inhibited the
expression of survivin. On balance, the results indicated that the struc-
tural modification on the 1,3-substituents of naphthoquinone imidazo-
liums without 2-substituent is also promising to obtain new antitumor
compounds. Further research and structural expansion are already
under study.
To evaluate whether compound 7m inhibits tumor proliferation by
inducing apoptosis, annexin V/PI staining assays were conducted. As
2

J. Yuan et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127977
Scheme 1. Synthesis of desired compounds.
3

J. Yuan et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127977
Table 1
Table 2
Selective ratio of naphthoquinone phenacylimidazolium derivatives.
Cytotoxicity of naphthoquinone phenacylimidazolium derivatives.
Comp.
Cell Type (IC50
MCF-7
/μ
M)
Comp.
Selective ratio
L929/MCF-7
HeLa
A549
L929
L929/HeLa
L929/A549
1a
12.1
16.7
8.3
10.1
8.8
3.6
2.1
53.1
27.7
24.2
19.0
6.1
1a
4.39
1.66
2.92
0.46
0.10
3.23
2.00
3.04
2.19
0.52
1.99
0.97
2.86
2.32
80.8
1.81
16.5
24.5
2.61
53.5
2.90
2.84
9.71
24.4
3.61
0.57
5.79
2.40
5.19
6.56
7.42
8.57
3.46
21.9
14.2
22.5
26.6
256
5.26
3.15
2.88
1.10
0.27
4.55
1.70
3.23
5.29
1.53
0.59
<0.47
4.86
4.80
19.9
3.11
7.31
4.20
2.10
5.87
2.15
2.49
2.78
3.27
4.72
21.5
3.86
1.81
3.00
4.12
28.8
2.67
2.86
40.0
2.47
5.74
14.2
21.3
1.11
4.59
6.25
14.75
13.19
8.07
1.26
0.50
8.00
4.60
15.1
14.2
0.77
3.71
1.74
16.8
83.6
71.2
13.6
48.8
91.9
14.9
12.1
14.2
21.8
136
2a
2a
3a
8.4
3.0
3a
4a
40.9
60.4
6.2
17.2
22.2
4.4
15.1
12.3
2.5
4a
5a
5a
6a
20.0
4.6
6a
7a
2.3
2.7
1.0
7a
8a
8.9
8.4
1.8
27.1
41.3
31.0
24.5
46.5
36.9
41.8
29.9
19.0
11.7
14.7
19.3
18.2
14.2
20.7
13.6
22.9
27.4
4.3
8a
9a
18.9
59.1
12.3
47.9
12.9
18.0
0.37
10.5
0.71
0.60
7.4
7.8
2.9
9a
10a
11a
12a
1b
1c
20.3
41.7
>100
7.6
40.4
6.6
10a
11a
12a
1b
1c
26.7
2.2
8.7
0.50
0.42
1.4
1d
1e
1.5
1d
1e
6.1
1f
1.6
0.24
0.16
1.3
1f
1g
3.5
1g
1h
1i
9.2
1h
1i
0.34
4.9
3.1
1.5
1j
6.6
1.0
1j
1k
1l
7.3
8.3
0.95
0.10
0.30
2.4
1k
1l
1.4
4.9
1m
1n
0.94
7.6
7.0
1m
1n
1o
7b
7c
76.3
11.4
43.0
5.59
3.78
8.44
13.6
12.1
13.7
3.37
82.0
2.85
6.46
23.0
61.0
3.15
147
5.8
1o
7b
7c
7.6
0.20
4.2
0.10
2.9
2.8
16.2
133
2.7
55.4
0.52
2.7
73.6
0.90
4.3
35.2
0.32
1.3
7d
7e
7d
7e
17.7
2.3
7f
0.31
0.56
7.6
0.08
1.8
0.19
0.35
7.8
7f
7g
4.8
7g
7h
7i
9.2
26.3
16.4
9.4
7h
7i
0.75
0.66
0.69
0.32
0.05
10.4
65.6
4.6
0.41
3.8
0.20
3.3
7j
7j
7k
7l
2.7
2.4
15.5
8.5
7k
7l
0.60
0.60
9.4
0.37
0.21
3.3
7m
7n
7o
Doxorubicin
12.8
10.4
58.8
7.5
7m
7n
7o
Doxorubicin
1.00
0.90
1.63
12.8
1.2
0.40
8.5
0.88
4

J. Yuan et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127977
Fig. 2. Compound 7m induced apoptosis of MCF-7 cells with various concentrations for 48 h by flow cytometry.
Fig. 3. Compound 7m induced cell cycle S-phase arrest of MCF-7 cells with various concentrations for 48 h by flow cytometry.
Acknowledgments
This work was partially supported by the National Natural Sciences
Foundation of China [grant number 22071150]. We thank the Instru-
mental Analysis Center of Shanghai Jiao Tong University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127977.
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interests or personal relationships that could have appeared to influence
the work reported in this paper.
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