Discovery of novel 1,5-benzodiazepine-2,4-dione derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2014.06.041

A series of novel 1,5-benzodiazepine-2,4-dione derivatives with C-6 amide substituents were designed and synthesized using three-component reactions. The preliminary assays showed that most of them displayed moderate to good antitumor activities against human lung carcinoma (A549), human breast epithelial carcinoma (MCF-7), human colon carcinoma (HCT116), human cervical carcinoma (Hela) and Lewis lung carcinoma (2LL). Exhilaratingly, the activity level of 6m rivaled that of 5-Fluorouracil (5-Fu) against MCF-7 cell lines, which might be used as novel lead scaffold for potential anticancer development.

Full text of DOI:10.1016/j.bmcl.2014.06.041

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3948–3951
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 1,5-benzodiazepine-2,4-dione derivatives
as potential anticancer agents
⇑
Yinbo Chen, Vanminh Le, Xiaoyong Xu, Xusheng Shao, Jianwen Liu, Zhong Li
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,5-benzodiazepine-2,4-dione derivatives with C-6 amide substituents were designed
and synthesized using three-component reactions. The preliminary assays showed that most of them dis-
played moderate to good antitumor activities against human lung carcinoma (A549), human breast epi-
thelial carcinoma (MCF-7), human colon carcinoma (HCT116), human cervical carcinoma (Hela) and
Lewis lung carcinoma (2LL). Exhilaratingly, the activity level of 6m rivaled that of 5-Fluorouracil (5-Fu)
against MCF-7 cell lines, which might be used as novel lead scaffold for potential anticancer development.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 25 March 2014
Revised 30 May 2014
Accepted 13 June 2014
Available online 25 June 2014
Keywords:
1,5-Benzodiazepine-2,4-dione
Anthranilic diamide
Conformational restriction
Synthesis
Antitumor activity
Nowadays, cancer has been one of the worldwide serious dis-
eases that represent a real crisis for threatening human health
and life.1–3 Apart from the use of surgical treatment and irradia-
tion, chemotherapy still remains the backbone of treatment
against cancer.4,5 However, one of the major hurdles in cancer che-
motherapy is attributed to the prevalence of the drug and multi-
drug resistance.6–8 Therefore, considerable efforts have been
made on the design and the discovery of new anticancer agents
focusing on the neotype chemical entity for the successful treat-
ment of cancer.9–11
Anthranilic diamide scaffolds are privileged structures in
medicinal chemistry and also serve as versatile building blocks
for the construction of therapeutic leads. They are usually embed-
ded in many bioactive compounds (Fig. 1, A–D), which elicit a wide
range of biological potencies including antitumor,12,13 insecti-
cidal,14,15 and antiviral,16 etc. This gives us a great impetus to the
search for potential pharmacologically active compounds carrying
anthranilic diamide unit. The compounds containing the amide
group exist as a mixture of atropisomers in solution due to
restricted rotations at the amide (CO–N) and aryl–carbonyl (Ar–
CO) bonds.17 The conformational influence of amides may result
in the difference of activity and selectivity.18,19
group through formation of five- to seven-membered ring is the
common approach for conformational restriction.23–25 Our
research group has been interested in the molecular design using
conformational restriction strategy.26,27 Thus, locking one amide
group of anthranilic diamide unit arouses our interest. Also, it is
well documented that several synthetic approaches can make it.
For example, 1,5-benzodiazepine-2,4-dione scaffold28 provides a
feasible method for locking the amide group into phenyl ring con-
densed with a seven-membered heterocycle (Fig. 2).
Furthermore, 1,5-benzodiazepine-2,4-dione core is a highly
efficient pharmacophore and presents in a number of bioactive
R2
Br
N
H
N
H
N
H
N
O
O
O
R1
H
N
H
N
H
N
N
R2
O
N
R1
Cl
O
O
C
O
R
B
A
O
R
N
HO
O
O
O
N
H
N
NH
In recent years, conformational restriction is a promising strat-
egy in drug discovery and makes an ideal platform for structural
modification and derivatization.20–22 Rigidification of a functional
H
N
NH
R
N
N
O
Cl
O O
O
F
D
E
F
⇑
Corresponding author. Tel.: +86 21 64253540; fax: +86 21 64252603.
E-mail address: lizhong@ecust.edu.cn (Z. Li).
Figure 1. Representative derivatives with anthranilic diamide and 1,5-benzodiaz-
epine-2,4-dione scaffolds.
http://dx.doi.org/10.1016/j.bmcl.2014.06.041
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Y. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3948–3951
3949
Table 1
H
N
O
O
H
N
The chemical structure of compounds 6a–t
O
H
N
R
H
N
O
O
O
No
Compds
Substituents
R
R1
H
N
N
H
O
NH
1
2
3
4
5
6
6a
6b
6c
6d
6e
6f
Isopropyl
t-Butyl
Cyclopropyl
Benzyl
N
H
R2
Conformational
restriction
O
Figure 2. Design strategy of anthranilic diamide derivatives with conformational
3-(Trifluoromethyl)benzyl
3-Chloro-4-methylbenzyl
3-Chloro-4-fluorobenzyl
4-Methylbenzyl
2,5-Difluorobenzyl
2,3,6-Trifluorobenzyl
Pyridin-2-ylmethyl
Phenyl
3-(Trifluoromethyl)phenyl
3-Chlorophenyl
2-Fluorophenyl
4-(Trifluoromethyl)phenyl
2,3-Difluorophenyl
2,4-Difluorophenyl
5-chloro-2-Fluorophenyl
2,3,4-Trifluorophenyl
restriction.
7
8
9
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
compounds. They were developed as anxiolytic agents (Fig. 1,
E)29,30 and cholecystokinin-B receptor antagonists (Fig. 1, F).31
Inspired by the biological diversity described above, we intended
to investigate whether there would be some new beneficial
properties if 1,5-benzodiazepine-2,4-dione moiety hybridized
anthranilic diamide core structure.
Enlightened by all of the descriptions above, we utilized
anthranilic diamide scaffold as a key prototype structure unit
and introduced the 1,5-benzodiazepine-2,4-dione scaffold with
dual effects of locking the amide group and hybridizing its versatil-
ity. Herein we described the molecular design, synthesis and initial
findings on antitumor activity.
10
11
12
13
14
15
16
17
18
19
20
6s
6t
lung carcinoma (2LL), were assayed by the standard MTT method33
using 5-FU (5-Fluorouracil) as a positive control. The cancer cells
were preincubated for 48 h. The results expressed as IC50 were
summarized in Table 2.
All of the prepared compounds were screened against A549 cell
lines, and most of them exhibited moderate to good antitumor
activity. Firstly, the lower activity of compounds 6a–c derived from
alkyl groups than the corresponding benzylated and phenylated
counterparts 6d–t implied that the antitumor activity might be
influenced by the size or electronic effect of the substituents. Com-
pound 6m showed the most potent activity with IC50 value of
The synthetic route employed for the synthesis of the target
compounds 6a–t was outlined in Scheme 1. The commercially
available 2-amino-3-nitrobenzoic acid 1 was selected as the start-
ing material, which was transferred to the corresponding benzam-
ides by the general condensation reaction. When the R was alkyl or
benzyl amines, the treatment of 2-amino-3-nitrobenzoic acid with
amines in the presence of EDCI and HOBt afforded the intermedi-
ates 2a–k in 68–76% yields; when the R was anilines, 1 was firstly
converted to the corresponding acid chloride and then coupled
with anilines using DIPEA as acid acceptor to obtain the intermedi-
ates 2l–t in 57–75% yields. The catalytic hydrogenation of interme-
diates 2a–t with 15% Pd/C in MeOH afforded the key diamide
derivatives 3a–t in high yields, which were used for the next
reaction without further purification. The benzyl isocyanide 5
was synthesized following the literature method.32 In the final
step, a one-pot three-component reaction using diamide deriva-
tives 3a–t, Meldrum’s acid 4 and benzyl isocyanide 5 in 1,2-dichlo-
roethane at reflux under Ar produced the target compounds 6a–t
in 25–45% yields after column chromatography or recrystalliza-
tion. The chemical structures of the synthesized compounds 6a–t
were summarized in Table 1. Spectral data for all synthesized com-
pounds were given in the Supplementary material.
27.8 lg/mL. Introducing electron-withdrawing groups, electron-
donating groups, or halogen atoms at different positions did not
contribute to great improvement of the activity. Moreover, com-
pounds 6h and 6p were inactive, which indicated that the 4-posi-
tion was unfavorable to the activity. And, replacement of phenyl
group in 6d with pyridine group to generate analogue 6k resulted
in a big drop of potency. Maybe the pyridine unit was an ineffective
Table 2
Growth inhibitiona effect of the synthesized compounds 6a–t on 5 tumor cell lines
Compds A549 cells MCF-7 cells HCT116 cells Hela cells 2LL cells
The in vitro antitumor activities of the synthesized compounds
6a–t against five cancer cell lines, including human lung carcinoma
(A549), human breast epithelial carcinoma (MCF-7), human colon
carcinoma (HCT116), human cervical carcinoma (Hela) and Lewis
IC50 (lg/mL)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
>100
>100
>100
n.t.b
n.t.
n.t.
91.2 0.8
36.1 0.2
87.0 0.2
68.2 0.4
n.t.
74.8 0.3
n.t.
n.t.
21.3 2.6
10.0 1.3
11.2 1.4
n.t.
n.t.
21.0 1.8
n.t.
20.4 2.2
n.t.
12.1 0.3
n.t.
n.t.
n.t.
85.0 0.2
46.1 0.4
>100
49.9 0.1
n.t.
80.1 0.2
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
45.3 0.9
80.5 0.7
59.4 0.5
68.1 0.8
>100
43.1 1.0
97.7 2.0
>100
85.3 2.3
27.8 0.8
56.2 1.3
96.1 3.2
>100
42.0 1.1 48.5 0.8
8.1 0.3 30.5 1.0
16.7 1.2 58.3 0.9
21.1 1.2 66.8 1.0
H
N
O
HO
O
R
n.t.
n.t.
NH2
NO2
NH2
NO2
25.1 1.2 40.2 1.1
n.t.
n.t.
41.9 1.5 29.3 2.3
42.0 2.0 17.6 0.9
24.0 0.9 17.1 0.6
n.t.
n.t.
37.8 1.2 32.9 2.2
n.t. n.t.
35.4 1.6 19.3 1.1
n.t.
5.7 0.3
n.t.
n.t.
n.t.
2a-t
1
26.6 1.7
20.5 2.4
17.3 1.9
n.t.
n.t.
25.0 2.8
n.t.
21.3 2.6
n.t.
4.8 0.1
n.t.
n.t.
H
N
H
N
O
O
NC
R
80.4 0.7
>100
63.3 2.0
>100
H
N
O
O
R
O
O
NH2
NH2
6s
6t
5-Fu
O
O
NH
N
H
n.t.
2.9 0.8
O
4.1 0.6
6a-t
3a-t
5
4
a
As measured by the MTT assay after 48 h incubation.
n.t.: not tested.
b
Scheme 1. Protocol for synthesis of the target compounds.

3950
Y. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3948–3951
group for activity. The compound 6j and 6t showed relatively low
inhibitory activity, which implied that multi-substitution had a
negative effect on the activity.
as novel lead scaffold for potential anticancer development. Fur-
ther chemo-biological optimization on 1,5-benzodiazepine-2,4-
dione derivatives is well ongoing in our laboratory.
Furthermore, the majority of the compounds with relatively
higher inhibitory activity against A549 cell lines were selected
for further evaluation against other four cell lines. For MCF-7 cell
lines, the compounds bearing benzyl groups showed relatively
lower activity than that of phenylated counterparts. Notably, the
activity level of derivatives 6m and 6n rivaled that of 5-Fu. After
the comparison between active compounds, it indicated that intro-
duction of CF3 or Cl at the 3-position of phenyl group had a positive
effect on the activity. Also, the mono-substituted derivatives 6e,
6m and 6n had higher activity comparing with the non- or
multi-substituted compounds. For Hela cell lines, all tested com-
pounds showed good cytotoxic activity, with IC50 values ranging
Acknowledgements
This work was financially supported by the National Basic
Research Program of China (973 Program, 2010CB126100), the
National High Technology Research and Development Program of
China (863 Program, 2011AA10A207), and the National Natural
Science Foundation of China (21372079, 21272071). This work
was also partly supported by the National Key Technology R&D
Program of China (2011BAE06B01) and the Fundamental Research
Funds for the Central Universities.
from 8.1 to 42.0
the 3-position of benzyl group was essential for optimal antitumor
activity against Hela cell lines with IC50 value of 8.1 g/mL and its
lg/mL. Among them, compound 6e with CF3 at
Supplementary data
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06
.041.
activity was comparable to that of 5-Fu. Compared with non-
substituted compound 6d, di-substituted benzylated compounds
exhibited higher activity. However, the activity of the mono- and
di-substituted phenylated compounds did not greatly improve
compared with non-substituted compound 6l. For HCT116 and
2LL cell lines, the potency order of the tested compounds was sim-
ilar with that of compounds against MCF-7 cell lines. The most
active compound 6n was a slight decrease in activity of 5-Fu. The
preliminary antitumor assays indicated that the tested compounds
showed stronger activity against Hela than other four cancer cell
lines. Among them, compound 6e displayed excellent selectivity
for Hela cell lines.
In addition, the dose–response analysis of cell growth inhibition
activity for representative compounds 6e and 6m was displayed in
Fig. 3, which indicated that the cytotoxic effects on cell lines of tar-
get compounds showed concentration-dependent manner.
In conclusion, a series of novel 1,5-benzodiazepine-2,4-dione
derivatives possessing C-6 amide units were synthesized using
three-component reactions and screened for their cytotoxic activ-
ity against five human cell lines (A549, MCF-7, HCT116, Hela and
2LL) by MTT assay. Most of compounds showed moderate to good
antitumor activity and they exhibited stronger activity against
Hela than other four cancer cell lines. In particular, compound 6e
displayed good selectivity for Hela cell lines with IC50 value of
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