2-Substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,9-diones: Design, synthesis and antiproliferative activity

Article abstract of DOI:10.1016/j.bmcl.2018.06.007

Thirty-six of novel compounds 2-substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,9-diones, bearing a N-(2-morpholinoethyl) group and a 2-substituted imidazole segment on a naphthoquinone skeleton, were designed, synthesized and tested as anticancer agents. Cytotoxicity was evaluated in vitro against three human cancer cell lines: human breast carcinoma cell line (MCF-7), human cervical carcinoma cell line (Hela), and human lung carcinoma cell line (A549); and one normal cell line: mouse fibroblast cell line (L929). Among them, the compound 2-(3-chloro-4-methoxyphenyl)-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,9-dione showed good antiproliferative activity against MCF-7, Hela and A549 (IC₅₀ values are equal to 10.6 μM, 8.3 μM and 4.3 μM respectively) and low cytotoxicity to L929 (IC₅₀ value is equal to 67.3 μM).

Full text of DOI:10.1016/j.bmcl.2018.06.007

Accepted Manuscript
2-Substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d] imidazole-4,9-diones:
design, synthesis and antiproliferative activity
Zhanxiong Liu, Zhenfeng Zhang, Wanbin Zhang, Deyue Yan
PII:
S0960-894X(18)30492-X
https://doi.org/10.1016/j.bmcl.2018.06.007
BMCL 25888
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 March 2018
30 May 2018
2 June 2018
Please cite this article as: Liu, Z., Zhang, Z., Zhang, W., Yan, D., 2-Substituted-1-(2-morpholinoethyl)-1H-
naphtho[2,3-d] imidazole-4,9-diones: design, synthesis and antiproliferative activity, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.007
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2-Substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]
imidazole-4,9-diones: design, synthesis and antiproliferative
activity
Zhanxiong Liua, Zhenfeng Zhangb, Wanbin Zhangab*, Deyue Yana*
a
Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of
b
Chemistry and Chemical Engineering, and School of Pharmacy, Shanghai Jiao Tong
University, 800 Dongchuan Road, Shanghai 200240, P. R. China
Abstract:
Thirty-six
of
novel
compounds
2-substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,9-diones, bearing
a N-(2-morpholinoethyl) group and a 2-substituted imidazole segment on a
naphthoquinone skeleton, were designed, synthesized and tested as anticancer agents.
Cytotoxicity was evaluated in vitro against three human cancer cell lines: human
breast carcinoma cell line (MCF-7), human cervical carcinoma cell line (Hela), and
human lung carcinoma cell line (A549); and one normal cell line: mouse fibroblast
cell
line
(L929).
Among
them,
the
compound
2-(3-chloro-4-methoxyphenyl)-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,
9-dione showed good antiproliferative activity against MCF-7, Hela and A549 (IC50
values are equal to 10.6 µM, 8.3 µM and 4.3 µM respectively) and low cytotoxicity to
L929 (IC50 value is equal to 67.3 µM).
Keywords: naphthoquinone; imidazole; 2-morpholinoethyl; antiproliferative;
anticancer agent.
*Corresponding authors. E-mail addresses: wanbin@sjtu.edu.cn (W. Zhang);
dyyan@sjtu.edu.cn (D. Yan)

The quinone structural motif, especially naphthoquinone, is a very important
framework and pharmacophore in a number of chemotherapy agents, such as
Doxorubicin, Mitoxantrone and Alkannin, as well as in other biologically active
natural products.[1] Around ten years ago, 2-morpholino ethylamino-substituted
naphthoquinones[2] were designed rationally as inhibitors for the dual specificity
protein hosphatase CDC25, which is considered to be a potential target for anticancer
agents (Figure 1).[3-6] The above mentioned CDC25 inhibitors showed good
antiproliferative activity in vitro. However, no experimental data was reported
concerning their selectivity, which in addition to their activity is also a key criterion
for anticancer agents. Recently, a 2-substituted imidazole segment has been appended
to the naphthoquinone framework and the resulting compounds have shown increased
selectivity[7] while retaining high activity.[7-11] Inspired by these previous works, new
types
of
molecules,
2-substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,9-diones,
possessing both a N-(morpholinoethyl) group and a 2-substituted imidazole segment
on a naphthoquinone skeleton, were developed with the aim of achieving high
antiproliferative activity as well as good selectivity. To the best of our knowledge,
such species have not been reported thus far.
Figure 1. The design of new anticancer agents.
Most
of
the
target
compounds,
were
2-substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,9-diones,
prepared in two steps (Scheme 1, eq. a). In the first step, the
2-substuted-1H-naphtho[2,3-d]imidazole-4,9-diones were obtained after a one-step

imidazole ring formation from 1[12] and the corresponding aldehydes or acids under an
atmosphere of air.[13-15] In the second step, the desired compounds were obtained via
the substitution of 2-substuted-1H-naphtho[2,3-d]imidazole-4,9-diones with
4-(2-iodoethyl)morpholine. The compounds 8n and 9q were obtained by other
methods differing in the imidazole ring formation step. Compound 8n was produced
from 1 and cyclobutanecarbonyl chloride via amidation, imidazole ring formation and
substitution (eq. b), while 9q was prepared from the known compound 4[6] and
pyrazin-2-ylmethanamine via amination, imidazole ring formation and substitution
(eq. c).
Scheme 1. Synthetic routes for the target compounds.
The desired products bearing 2-alkyl, 2-alkenyl, and 2-aryl substituents on the
imidazole ring were obtained in good yields (Figure 2). It should be noted that the
2-alkyl group of 8k and 8l are present in the natural material
3-(4-isopropylphenyl)isobutyraldehyde
and
the
medical
intermediate
5-methylfuran-2-propionaldehyde. The diverse aryl residues at the 2-position of 9c-q
were inspired by the listed anticancer drugs Gefitinib, Tasigna, Sorafenib and
Lapatinib.

Figure 2. The tested compounds bearing an N-(2-morpholinoethyl) group.
In order to study the effects of the morpholino group, a number of additional
compounds 11-16 were synthesized via methylation of 8g, 9a, 9f, 9g, 9m, and 9n
(Scheme 2).
Scheme 2. Synthesis of methylated compounds 11-16.
The structures of the target compounds were confirmed by the single crystal

diffraction of 3f (CCDC 1827317), 9q (CCDC 1827318) and 15 (CCDC 1827319)
(Figure 3). These data are important for mechanistic biological studies.
Figure 3. Molecular structure of 3f, 9q and 15 and hydrogen atom labelling scheme.
According to the modified method that our group previously reported,[16-18] an MTT
assay was utilized to evaluate the antiproliferative activities and selectivity of the
target compounds against MCF-7, Hela, A549, and L929 cells (Table 1). For the
species bearing alkyl groups at the 2-position, most of these exhibited approximately
100 µM IC50 values for the four tested cell lines MCF-7, Hela, A549 and L929
(entries 1-15). 8f provided moderate selectivity between cancer cell A549 and normal
cell L929 (entry 6). Compound 8g possessing an electron-withdrawing CF3 group at
the 2-position showed better antiproliferative activity than the others, but
unsatisfactory selectivity (entry 7). Another compound 8m bearing a 2-substituted
cyclopropyl group also showed moderate antiproliferative activity (entry 13).
Compound 8p bearing a more electronegative 2-alkenyl substituent gave increased
activity (entry 16). Most of the 2-aryl-substituted compounds 9a-r displayed IC50
values below 100 µM (entries 17-34). Among them, 9c and 9m displayed good
antiproliferative activity to A549 and a low cytotoxicity to L929 (entries 19 and 29).
With regards to 9f, an excellent balance was maintained between cytotoxicity and
selectivity. Against MCF-7, Hela and A549, the IC50 values were 10.6 µM, 8.3 µM,
4.3 µM respectively, while very low toxicity was observed for the non-neoplastic cell
L929 with an IC50 of 67.3 µM (entry 22). The non-quinone compounds 10a and 10b
were also tested and showed almost no cytotoxicity to all the cells (entries 35 and 36).
The positive control, WDP1263,[5] showed a good tolerance to both cancer cells and
normal cell (entry 37), while, doxorubicin hydrochloride, showed good anticancer
activity but low selectivity (entry 37).
Table 1 Cytotoxicity of
2-substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,9-diones.
Cell Type (IC50a, µM)
Entry
No.

MCF- 7
>200
Hela
>200
A549
105
L929
118
1
2
8a
8b
8c
8d
8e
8f
179
95.9
113
123
150
17.9
123
>200
108
110
166
64.7
105
>200
58.3
70.1
30.1
116
106
86.9
10.6
144
95.1
166
84.9
37.4
107
106
80.3
>200
79.8
86.0
16.2
100
3
4
5
125
32.5
21.8
15.9
175
6
88.2
15.1
>200
180
7
8g
8h
8i
8
9
25.9
57.3
>200
72.8
36.0
73.0
76.4
76.9
24.5
16.6
23.6
70.0
84.5
4.3
157
10
11
12
13
14
15
16
17
18
19
20
21
22
8j
95.9
122
79.4
>200
96.2
40.0
84.9
80.3
120
8k
8l
88.2
61.2
99.3
>200
82.1
61.4
19.2
121
8m
8n
8o
8p
9a
9b
9c
9d
9e
9f
40.6
41.0
80.2
71.9
101
99.8
112
8.3
67.3

23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
37
9g
106
48.4
134
140
137
169
115
37.7
167
140
53.5
39.1
181
>200
>200
4.6
133
52.5
185
87
31.2
33.3
45.2
91.9
147
20.9
32.2
37.8
120
78.3
63.4
112
>200
158
8.5
131
44.3
72.5
111
177
175
122
118
44.6
116
49.6
121
187
>200
149
7.5
9h
9i
9j
119
9k
173
9l
191
9m
136
9n
105
9o
109
9p
153
9q
9r
79.1
79.2
>200
>200
>200
1.4
10a
10b
WDP1263
Doxorubicinb
aIC50 values are defined as the concentrations corresponding to 50% growth inhibition for 72 h by
b
MTT assay which were calculated from two or three independent experiments. Doxorubicin
hydrochloride.
Table 2 outlines the antiproliferative activity of methylated compounds 11-16.
Unfortunately, the antiproliferative activity of these compounds decreased
tremendously compared to their corresponding starting materials (entries 1-6). The
above experimental evidence illustrates the importance of the N-(2-morpholinoethyl)
group.

Table 2. Cytotoxicity (IC50) of methylated compounds 11-16.
Cell Type (IC50a, µM)
Entry
No.
MCF- 7
139
Hela
A549
L929
>200
1
2
3
4
5
6
11
12
13
14
15
16
116
93.5
184
>200
166
>200
97.5
166
>200
174
166
115
141
>200
>200
>200
133
133
160
141
106
106
aIC50 values are defined as the concentrations corresponding to 50% growth inhibition for 72 h.
In summary, thirty-six of novel biological compounds bearing
a
a
N-(2-morpholinoethyl) group,
a
2-substituted imidazole segment, and
naphthoquinone skeleton were designed, prepared, and evaluated as anticancer agents.
Among them,
2-(3-chloro-4-methoxyphenyl)-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]imidazole-4,
9-dione (9f, Figure 4) showed the most promising activity to MCF-7, Hela and A549,
as well as a low toxicity to normal cell line L929. The primary research revealed that
all three parts of the molecule, the N-(2-morpholinoethyl) group, imidazole segment,
and naphthoquinone skeleton are necessary for good activity and selectivity. Further
studies concerning the biological mechanism and the development of other new
naphthoquinone imidazole structure-based derivatives are ongoing.

Figure 4. The optimized active compound 9f.
Acknowledgments
This work was supported by National key research and development plan of P. R.
China (2016YFA0201500). We thank the Instrumental Analysis Center of Shanghai
Jiao Tong University.
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Graphical Abstract
2-Substituted-1-(2-morpholinoethyl)-1H-naphtho[2,3-d]
imidazole-4,9-diones: design, synthesis and antiproliferative
activity
Zhanxiong Liua, Zhenfeng Zhangb, Wanbin Zhangab*, Deyue Yana*

Highlights
1. 2-Substituted-1-(2-morpholinoethyl) naphthoquinone imidazoles as antitumor drugs.
2. Good cytotoxicity was observed against MCF-7, Hela and A549 cell lines.
3. Good selectivity was observed against L929 cell line.