Benzylidene 2-aminoimidazolones derivatives: Synthesis and in vitro evaluation of anti-tumor carcinoma activity

Article abstract of DOI:10.1248/cpb.c13-00340

A series of benzylidene 2-aminoimidazolones derivatives were synthesized. Most compounds displayed strong inhibitory activity on the proliferation of human HepG2 cells in vitro. The active compounds were further evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against five human cancer cell lines in vitro. Compound 2b exhibited the strongest antitumor activities with IC₅₀ values ranging from 12.87-17.10 μM which were nearly 1-3.5 fold less than that of 5-FU (IC₅₀=18.39-56.12 μM) in vitro. Furthermore, compound 2b could induce SMMC-7721 cell apoptosis in a dose-dependent manner. Therefore, our novel findings may provide a new framework for the design of new benzylidene 2-aminoimidazolones derivatives for the treatment of cancer.

Full text of DOI:10.1248/cpb.c13-00340

October 2013
Note
Chem. Pharm. Bull. 61(10) 1081–1084 (2013)
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Benzylidene 2-Aminoimidazolones Derivatives: Synthesis and in Vitro
Evaluation of Anti-tumor Carcinoma Activity
Yong Ling,a Zhi-Qiang Wang,a,b You-An Xiao,a,c Chenyu Zhu,a Liucen Shen,a Xue-Min Wang,a
,a
Yi Hui,a and Xin-Yang Wang*
b
a Medical College, Nantong University; Nantong 226001, P.R. China: Department of Natural Medicinal Chemistry,
c
China Pharmaceutical University; Nanjing 210009, P.R. China: and Anhui Key Laboratory of Modernized Chinese
Material Medical, Anhui University of Traditional Chinese Medicine; Hefei 230031, P. R. China.
Received May 4, 2013; accepted July 6, 2013
A series of benzylidene 2-aminoimidazolones derivatives were synthesized. Most compounds displayed
strong inhibitory activity on the proliferation of human HepG2 cells in vitro. The active compounds were
further evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against five
human cancer cell lines in vitro. Compound 2b exhibited the strongest antitumor activities with IC50 values
ranging from 12.87–17.10µM which were nearly 1–3.5 fold less than that of 5-FU (IC50=18.39–56.12 µM) in
vitro. Furthermore, compound 2b could induce SMMC-7721 cell apoptosis in a dose-dependent manner.
Therefore, our novel findings may provide a new framework for the design of new benzylidene 2-aminoimid-
azolones derivatives for the treatment of cancer.
Key words synthesis; 2-aminoimidazolone derivative; anti-tumor agent; inhibitory activity; cell apoptosis
Cancer has been reported to become the leading cause of on the proliferation of human cancer cell lines and cell apo-
mortality worldwide in 2010 and cancer cases are expected to ptosis effect were carried out in vitro with expectation to find
double by 2020.1) Resourceful novel and structurally diverse better antitumor agents.
metabolites from marine organisms, in particular those com-
ing from sponges, have proved to display a wide variety of
Results and Discussion
Chemistry The synthetic route of 2-aminoimidazolone
biological activities including antihistamine activity,2) anti-
inflammation,3) neuroprotective activity,4,5) antitumor,6) anti- derivatives 1a–k, 2a–k was outlined in Chart 1. The starting
oxidant,7) etc. For example (see Fig. 1), marine natural prod- material glycine 3 was treated with NH4SCN at the present of
uct Dispacamide, which was isolated from Carriban Agelas acetic anhydride (Ac2O) to give 1-acetyl-2-thioxoimidazolidin-
sponges, shows a potent antihistamine activity,2) and Leucet- 4-one 4 by cyclization under microwave condition. The inter-
tamine B, from the sponge Leucetta microraphis HAECKEL mediate 4 was reacted with other commercially available ma-
(alcarea class) of the Argulpelu Reef in Palau, exhibits to play terial 4-hydroxybenzaldehyde 5a or vanillin 5b to form 6a or
a role as mediator of inflammation.3) Polyandrocarpamines A, 6b by condensation according to the literature.6,8) Compounds
extracted from a Fijian ascidian,8) has been shown to have di- 6a,b was oxidized by tert-butylhydroperoxide (TBHP) to
verse pharmacological activities.6,9,10) Many recent reports and obtain the important intermediate 7a,b. Then corresponding
reviews have also highlighted that Polyandrocarpamines A amine, alkane amines or alcohol amines were introduced onto
exhibit potent antitumor activity.6,11,12) Phorbatopsins A which the sulfonic group of compounds 7a,b to obtain the targeted
was isolated from the Mediterranean marine sponge Phorbas compounds 1a–k, 2a–k. The structures of the target com-
topsent exhibited antioxidant activity.7) Interestingly, these pounds were shown in Chart 1 and confirmed by of MS, IR,
marine natural products share a common skeletal structure 1H-NMR spectra and elemental analysis (EA).
that is 2-aminoimidazolone core. Both natural and synthetic
In Vitro Biological Assessment The inhibitory activity of
2-aminoimidazolone-containing compounds were identified target compounds 1a‒k, 2a–k on Hep G2 which was incubat-
to possess anticancer activities.13,14) However, their antitumor ed with 50µM of each test compound for 48h, was primarily
activities are limited. Therefore, development of new benzyl- screened and evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-
idene 2-aminoimidazolones derivatives with potent antitumor
activity should be of great significance.
For the further improvement of their antitumor activities,
different substituted alkane amines and alcohol amines were
selected to make further modifications on the 2-aminoimid-
azolone core by ammonification. Furthermore, with the con-
sideration whether the methoxy group on benzene ring could
confer to pharmacological activity, benzylidene 2-aminoimid-
azolones derivatives were designed via the conjugation of
2-aminoimidazolone core with vanillin or 4-hydroxybenzalde-
hyde. Herein, 22 target compounds 1a–k, 2a–k were synthe-
sized and their biological evaluation for inhibitory activities
The authors declare no conflict of interest.
Fig. 1. The Structures of Marine Natural Products
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: wangxinyang@ntu.edu.cn

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Reagents and conditions: (a) NH4SCN, Ac2O, AcOH, reflux, 2h. (b) NaOAc, AcOH, reflux, 5h. (c) TBHP, MeOH, rt, 2h. (d) Alkane amines or alcohol amines, MeOH,
rt, 10h.
Chart 1
Fig. 2. The Inhibitory Activity of Target Compounds 1a k, 2a–k (50 µM) on Proliferation of Hep G2 Cells
diphenyltetrazolium bromide (MTT) assay in vitro, and using potent compared to that against the other tumor cells. Espe-
5-fluorouracil (5-FU) as positive control. The results showed cially, compounds 2b exhibited the strongest antitumor activi-
in Fig. 2 illustrated the inhibitory effects of target compounds ties with IC50 values ranging from 12.87–17.10µM which was
on the proliferation of Hep G2. Twelve out of the twenty 1–3.5 fold less than that of 5-FU (IC50=18.39–56.12 µM).
two tested compounds exhibited inhibitory effects that were
For determining whether the most potent compound 2b
similar or superior to 5-FU. Especially, compounds 1b, 1j, 2b was due to cell apoptosis, apoptosis assay employing human
and 2k (percent inhibition over 90%) displayed more potent hepatocellular carcinoma cells (SMMC-7721) was further as-
inhibition than 5-FU. Therefore, ten compounds (list in Table sessed to determine the effect of 2b on cell apoptosis. The
1) possessing strong inhibition on the proliferation of human SMMC-7721 cells were incubated with vehicle alone or with
Hep G2 cells were selected for the further investigation of 2b at 6.25, 12.5 or 25µM final concentrations. The percentages
the inhibitory activity against five human cancer cell lines of apoptotic cells were determined by flow cytometry analysis.
in vitro, such as SMMC-7721, HepG2, MCF-7, SW480 and As shown in Fig. 3, in the untreated group, the frequency of
SGC-7901. Their IC50 values are presented in Table 1. All the SMMC-7721 cell apoptosis was unobvious. In contrast, the
tested compounds showed remarkable antitumor activities in frequency of the cell apoptosis showed an upward trend in
a low micromolar range (except 2a) with IC50 12.87–39.51 µM. 2b-treated SMMC-7721 cell with the dose increased. Low
In addition, the antitumor activities of each active compound concentration (6.25µM) of 2b only induced 25.9% apoptosis
against SMMC-7721 and SGC-7901 cells showed much more activity on SMMC-7721 cells. Following treatment with 25µM

October 2013
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Table 1. The Inhibitory Activity (IC50, µM) of 1a–v against Five Human Cancer Cell Lines
In vitro inhibitory activity (IC50, µM)
Compound
SMMC-7721
MCF-7
Hep G2
SW480
SGC-7901
5-FU
1b
1j
37.84
17.76
19.62
28.06
33.48
12.87
27.46
25.02
16.65
14.12
18.39
25.03
22.18
35.02
>50
15.16
30.24
19.98
24.86
20.74
35.67
21.52
19.87
25.96
39.51
14.93
32.39
20.53
20.25
18.05
23.11
22.87
23.79
33.05
34.75
17.10
29.65
26.75
27.02
21.17
56.12
20.97
27.29
29.65
35.61
15.53
35.13
22.87
17.84
16.92
1k
2a
2b
2c
2f
2j
2k
Fig. 3. Analysis of 2b-Induced Cell Apoptosis in SMMC-7721 Cells
The SMMC-7721 cells were treated with 6.25, 12.5 or 25µM 2b for 48h. (A) Apoptosis was determined by Annexin V-FITC/PI staining in the SMMC-7721 cells by flow
cytometry. (B) Quantitative analysis of apoptotic cells. Data are representative flow cytometry charts that are illustrated as a mean S.E.M. of the percentages of apoptotic
cells from three duplicate experiments.
of 2b induced over 80% of SMMC-7721 cell apoptosis, which the compounds 2j and 2k linked with propylene glycol side
was significantly higher frequency of the cell apoptosis com- chain displayed strong anticancer activity. In addition, the ac-
pared to the control cells, and it revealed that the antitumor tive compounds displayed more potent inhibitory activity on
activity of 2b appeared to be concentration-dependent. How- SMMC-7721 cells than the other four cancer cells. However,
ever, the precise mechanisms underlying the apoptosis effect the precise SAR of these compounds with potent anticancer
of these compounds selectively to tumor cells remain to be activity should be further investigated.
further investigated.
Structure–Activity Relationships (SARs) Analysis of
Conclusion
structure–activity relationships revealed that in series of
In summary, 20 benzylidene 2-aminoimidazolones deriva-
target compounds 1a–e or 2a–e with different unsubstituted tives with various substituted alkane amines or alcohol amines
or substituted alkane amines, the compound 1b or 2b linked were synthesized. Their inhibitory activity against tumor cell
with methylamine possessed better anticancer activity com- lines of synthetic compounds was evaluated in vitro. Most
pared to compound 1a or 2a linked with unsubstituted amine. compounds displayed strong anticancer activity. Particularly
However, increasing the length of alkane did not contribute to compounds 2b and 2k had a great potency superior to 5-FU in
their activities in vitro while 1b or 2b showed the strongest these cancer cells. Furthermore, compound 2b with the stron-
anticancer activity among their respective series in vitro. In gest antitumor activity could induce cancer cell apoptosis in a
the other hand, the compounds 1f–k or 2f–k linked with al- dose-dependent manner. We are interested in further investi-
cohol amine substituents displayed slightly stronger anticancer gating the mechanisms of these 2-aminoimidazolone deriva-
activity compared to that with alkane amine substituents, and tives underlying the action of these compounds in inhibition
the anticancer activity was similarly decreased with increas- of human tumorigenesis.
ing the chain length of alcohol amines in vitro. Interestingly,

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Experimental
General chemistry methods, synthesis procedures, spectral
data, and bioassay methods are given in Supplemental infor-
mation.
Acknowledgments The work was supported by Applied
Research Projects of Nantong City (BK2012085) and the
Priority Academic Programs Development of Jiangsu Higher
Education Institutions (PAPD).
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