Synthesis and cytotoxicity of some biurets against human breast cancer T47D cell line

Article abstract of DOI:10.1016/j.bmcl.2010.07.137

Design, synthesis and cytotoxicity of several known and novel biurets against human breast cancer T47D cell line in comparison to doxorubicin are described. Biurets incorporating 2-methyl quinoline-4-yl and benzo[d]thiazol-2-ylthio moieties showed higher cytotoxicity and decreased cell viability in a concentration- and time-dependent manner.

Full text of DOI:10.1016/j.bmcl.2010.07.137

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5772–5775
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxicity of some biurets against human breast cancer T47D
cell line
a,d
Shamileh Fouladdel a, Ali Khalaj b, Neda Adibpour c, , Ebrahim Azizi
*
a Molecular Research Lab, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
c Department of Medicinal Chemistry, School of Pharmacy, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran
d Department of Medical Biotechnology, School of Advanced Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Design, synthesis and cytotoxicity of several known and novel biurets against human breast cancer T47D
cell line in comparison to doxorubicin are described. Biurets incorporating 2-methyl quinoline-4-yl and
benzo[d]thiazol-2-ylthio moieties showed higher cytotoxicity and decreased cell viability in a concentra-
tion- and time-dependent manner.
Received 21 January 2010
Revised 18 July 2010
Accepted 31 July 2010
Available online 6 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Biurets
Cytotoxicity
T47D cancer cell line
Several 4-azolylalkylquinoline derivatives I1,2 and 1-azolylal-
kyl-4(1H) quinolones II1,3 (Fig. 1), have shown cytotoxicity compa-
rable or superior to adriamycin against various cancer cell lines.
While in the preliminary experiments 1-azolylalkyl-4(1H) quino-
lones II inhibited topoisomerases I, and II and did not bind to
DNA,1 further studies ruled out involvement of these enzymes in
their activity.3 In contrast by using flexible ligand docking tech-
nique it has been shown4 that the most probable mode of action
of 4-azolylalkylquinoline derivatives I are binding to DNA via inter-
calation of quinoline moiety between CG base pairs with linker
chain and azole moiety binding to minor groove.
nyl)azolylalkanamide III by exchange of –CH2– with –NH– group
have shown cytotoxicity comparable or higher than several antitu-
mor agents against human breast cancer T47D cell line.6 These re-
sults prompted us to investigate the effect of further bioisosteric
exchange of –CH2– groups of urea IV with –CO and –NH– groups
through the synthesis of compounds having biuret functionality
Since compounds of general formula I and II were ineffective
in vivo which was attributed to their high lipophilicity,1 efforts
were made to reduce the lipophilicity of compounds by synthesiz-
ing water soluble phenol and phenoxy acetic acid derivatives and
changing the nature of the chain between azolylalkyl and quinolyl
portions of the molecules.5 While resulting data for effects of
reducing lipophilicity on in vitro cytotoxicity were inconsistent,
of the synthesised compounds, N-(4-quinolyl)azolylalkanamide5
III which can be considered as bioisoster of the 4-azolylalkylquin-
olines I by exchange of the –CH2– groups of the chain with –NH
and –CO groups showed impressive in vitro and moderate in vivo
cytotoxic activity.
Additionally, several 1-(2-methylquinolin-4-yl)-3-azolyl urea
IV (Fig. 1) which could be considered as bioisoster of N-(4-quinoli-
Figure 1. Chemical structures of 4-azolylalkylquinolines I, 1-azolylalkyl-4(1H)
quinolones II, N-(4-quinolinyl)azolylalkanamide III and 1-(2-methylquinolin-4-yl)-
3-azolyl urea IV.
* Corresponding author. Tel.: +98 611 3738378; fax: +98 611 3738381.
E-mail address: n.adibpour@ajums.ac.ir (N. Adibpour).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.137

S. Fouladdel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5772–5775
5773
Scheme 1. Synthesis of carbamates 2c, d, ureas 3a–d, allophanates 4a, c, d and biurets 6a–r. Reagents and conditions: (i) bis(trimethylsilyl)acetamide, DCM; (ii) heat
(60–70 °C), 5–6 h; (iii) pyridine,16 h, rt; (iv) K2CO3, DCM, reflux, 16 h.
in the chain between two rings. While analgesic,7 anti-inflamma-
tory,8,9 hypoglycemic activity10 and inhibition of the gastric acid
secretion11 for several biurets have been described, the antitumor
activity of these compounds have not been reported and this is the
first report on their cytotoxicity against a cancer cell line. This
paper describes synthesis and cytotoxicity of N,N0-diphenyl, N-phe-
nyl-N0-alkylphenyl, and N,N0-bis alkylphenyl biurets and analogous
compounds by replacing one phenyl group with 2-methylquino-
line-4-yl, benzo[d]thiazol-2-ylthio and (1-phenyl-1H-tetrazol-5-yl)
thio moieties. The rational for selection of the first two heterocyclic
moieties were reports on the antitumor activity of 2-methyl-4-
aminoquinoline,12 and benzo[d]thiazole-2-thiol13 due to DNA
intercalation and for 1-phenyl-(1H)-tetrazol was that it is a bioiso-
ster of 1-phenyl triazole14 which was one of the most effective
azole of 4-azolylalkylquinolines I. In addition these heterocycles
were azole moieties of 1-(2-methylquinolin-4-yl)-3-azolyl urea
IV which have shown6 cytotoxicity against human breast cancer
T47D cell line. Biurets 6a–r were prepared (Scheme 1) by the reac-
tion of allophanates 4a–d with amines 5a–g.15 Preparation of
biurets 6a16 and 6f17 under similar conditions, 6e by the reaction
of benzylamine with carbonyl-isocyanate-isothiocyanate18 and
6o by the reaction of nitrobiuret with 3-phenylpropylamine6 have
been reported previously. Phenyl allophanates 4a–d were prepared
by the reported method for the preparation of 4a from the reaction
of phenyl chloroformate with ureas 3a–d in dichloromethane in
the presence of pyridine.19 Of these compounds preparation of
4b by the reaction of benzylamine and phenoxycarbonylisocyanate
has also been described.20 The known ureas 3a,b were prepared by
the reaction of aniline hydrochloride with urea21 and benzylamine
with potassium isocyanate,22 respectively. Ureas 3c,d were
Table 1
In vitro cytotoxicity of biuret 6a–r and urea IV
a
Compound name
R1
R2
IC50
(
lM)
Survivalb (%)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
IV
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
3-Phenylpropyl
2-Methyl-quinoline-4-yl
3-(Benzo[d]thiazol-2-ylthio)propyl
Phenylmethyl
60
82.35
75.23
67.25
64.1
91.3
75.97
88.5
50
35
35
75
55
70
75
25
50
25
50
10
45
70
20
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
2-Phenylethyl
2-Phenylethyl
2-Phenylethyl
3-Phenylpropyl
3-Phenylpropyl
3-Phenylpropyl
3-Phenylpropyl
—
2-Phenylethyl
3-Phenylpropyl
2-(Pyridin-2-yl)ethyl
2-Methyl-quinoline-4-yl
3-(1-Phenyl-1H-tetrazol-5-ylthio)propyl
3-(Benzo[d]thiazol-2-ylthio)propyl
2-Phenylethyl
2-Methyl-quinoline-4-yl
3-(Benzo[d]thiazol-2-ylthio)propyl
3-Phenylpropyl
2-Methyl-quinoline-4-yl
3-(Benzo[d]thiazol-2-ylthio)propyl
3-(1-Phenyl-1H-tetrazol-5-ylthio)propyl
—
79.22
49.8
73.62
54.32
78.76
28.15
79.82
84.53
45.72
74.92
67.86
—
60
55
1006
Doxorubicin
—
—
0.25
50c
a
b
c
IC50 of compounds was determined after 2 days exposure using MTT assay.
Percent survival of T47D cells following exposure to 25 M concentration of compounds was determined after 2 days exposure using MTT assay.
Percent survival of T47D cells following exposure to 0.25 M concentration of doxorubicin was determined after 2 days exposure using MTT assay.
l
l

5774
S. Fouladdel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5772–5775
prepared by the reaction of carbamates 2c,d with concentrated
ammonia.23 Preparation of urea 3d24 by a similar method for the
preparation of 3b through the reaction 2-phenylethylamine with
sodium isocyanate has been previously described. Phenyl
carbamates 2c,d were synthesized by the reaction of amines with
phenyl chloroformate in dichloromethane in the presence of
bis(trimethylsilyl)acetamide.25 Amines 5a–d,g are commercial
and 3-(benzo[d]thiazol-2-ylthio)propan-1-amine 5e26 was pre-
pared by the reaction of the corresponding benzo[d]thiazole-2-
thiol with bromopropylammonium bromide salt and 5f was
prepared by a similar method. Mp’s of the known compounds
which were in agreement with the literature values and physico-
chemical data for the novel compounds which were consistent
with their structures are presented in the Supplementary data.
Initially the cytotoxicity of biurets 6a–r against human breast
cancer T47D cells by MTT assay27 after 2 days of exposure was
evaluated at concentration of 250 nM which doxorubicin inhibited
50% cell viability.27 However, the synthesized compounds at this
concentration and even up to 1
lM showed no significant cytotox-
icity and as a result concentration of 25
l
M was used for evalua-
tion and comparison of cytotoxicity of these compounds with
doxorubicin at concentration of 250 nM.
Results for each compound as the percentage of growth of the
treated cells in comparison to untreated cells are shown in Table 1.
The most active compounds were biurets 6m, 6p and 6i bearing 2-
methylquinoline-4-yl moiety which exhibited 28.15%, 45.72% and
49.8% of survival, respectively. Next to these compounds, biuret 6k
bearing benzo[d]thiazol-2-ylthio showed higher activity similar to
6i. Replacement of the benzo[d]thiazol-2-ylthio with (1-phenyl-
1H-tetrazol-5-yl)thio (compounds 6r and 6j) reduced the activity.
Among biurets without heterocyclic rings, the most active com-
pounds were biuret 6b and 6o and in general, percentage of the
growth of cells treated with symmetrical biurets 6a, 6e, 6l and 6p
were higher than those of other compounds of this study which were
unsymmetrical. The calculated IC50 values of all tested compounds
after two days exposure showed that the order of the cytotoxicity
from highest to lowest were 6m > 6p > 6i = 6k > 6c = 6d > 6n >
6b = 6j = 6l > 6f = 6r > 6a = 6q > 6g = 6o > 6e = 6h.
Figure 3. Time-dependency of cytotoxic activity of selected compounds 6c, d, i, k,
m, p. Data are mean SE of three separate experiments performed as triplicate
using MTT assay.
On the basis of the preliminary results, compounds 6m, 6p, 6i,
6k, 6c and 6d which showed highest cytotoxicity were selected
for further studies to determine the concentration-dependency at
three concentrations of 5, 10 and 25 lM and also time-dependency
after 2, 3 and 4 days exposure to T47D cells. These compounds
decreased cell viability at different concentrations in a concentra-
tion- and time-dependent manner (Figs. 2 and 3). The highest cyto-
toxicity after 2 days exposure was exhibited by compound 6m at
concentration of 10
l
M followed by 6p at concentration of 20
M that were significantly
lM and 6i and 6k at concentration of 25
l
(p <0.001) different from control RPMI. The cytotoxicity of 6m after
4 days exposure was even greater than growth inhibitory effect of
doxorubicin at concentration of 250 nM (Fig. 3).
Preliminary results show that 6m and 6p the most active com-
pound of this study are also cytotoxic on human colorectal HT-29
cells and were not significantly cytotoxic on NIH 3T3 cell line
where at concentration of 10
(unpublished data).
lM inhibited only 10% of cell viability
From the results of this study it appears that further exploration
of biurets 6 by incorporation of azoles of the most active 4-
azolylalkylquinolines and/or 4-oxoquinoline instead of 2-methyl-
quinoline might lead to compounds with both in vivo and
in vitro activity.
This approach is currently underway and results will be re-
ported in the course of time.
Acknowledgments
The authors thank the office of Vice-chancellor for Research and
Medicinal Chemistry Research Center of Tehran University of Med-
ical Sciences for the financial support of this investigation.
Supplementary data
Figure 2. Concentration-dependency of cytotoxic activity of selected compounds
6c, d, i, k, m, p. Data are mean SE of three separate experiments performed as
triplicate using MTT assay after 2 days exposure.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.137.

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