Synthesis and antitumor evaluation of benzoylphenylurea analogs

Article abstract of DOI:10.1016/j.bmcl.2004.02.019

Novel series of benzoylphenylurea analogs 7-10 were prepared and evaluated for in vitro cytotoxic activity against a panel of eight different human cancer cell lines. A very interesting inhibition profile against BxPC3, Mia-Paca, and Hep2 cells with compo

Full text of DOI:10.1016/j.bmcl.2004.02.019

Bioorganic & Medicinal ChemistryLetters 14 (2004) 2213–2216
Synthesis and antitumor evaluation of benzoylphenylurea analogs
Hallur Gurulingappa, Maria L. Amador, Ming Zhao, Michelle A. Rudek,
Manuel Hidalgo and Saeed R. Khan*
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA
Received 14 January2004; revised 4 February2004; accepted 4 February2004
Abstract—Novel series of benzoylphenylurea analogs 7–10 were prepared and evaluated for in vitro cytotoxic activity against a
panel of eight different human cancer cell lines. A veryinteresting inhibition profile against BxPC3, Mia-Paca, and Hep2 cells with
compound 10 has been observed. Compounds 8 and 9 showed the significant cytotoxicity in Hep2 cells. All cell lines were resistant to
compound 7.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Pharmacokinetics studies have shown that compound 11
was metabolized to 6 with the cytotoxic activity similar
to parent compound.13 Previous studies have demon-
strated that benzoylphenylurea analogs are generally
poorlywater soluble thus making these compounds
difficult to formulate for administration. Due to the high
dosage (>400 mg/(kg d)) at which compound 2 showed
antitumor activitybyintraperitoneal administration and
lack of efficacybyoral administration, the utilityof such
compound as clinical therapeutic agents is limited.5 To
overcome this problem investigators have chemically
modified these compounds to enhance the solubilityin
water and to facilitate the oral absorption.6 In this
paper, we describe the synthesis and antitumor activity
of some new derivatives of benzoylphenylurea.
Antitumor agents that bind to tubulin and disrupt
microtubule dynamics have attracted considerable
attention. Two main groups of antimicrotubule agents
are used in the treatment of cancers. One type is taxol,
which promotes the assemblyof microtubules and
inhibits microtubule depolymerization.1;2 The other type
include vinca alkaloids and colchicines, which inhibit
tubulin polymerization and cause microtubule depoly-
merization.3;4 Benzoylphenylurea derivatives belong to
the second type and show high tumor activities.5;6 Pre-
vious studies have shown that benzoylphenylureas do
not compete either at the vinca or at the taxane binding
site on tubulin, but could potentiate inhibition of col-
chicine binding.7
Cl
Br
CONHCONH
N-[4-(5-Bromo-2-pyrimidinyloxy)-3-chlorophenyl]-N0-(2-
nitrobenzoyl)urea (1) administered orallyshows anti-
tumor activities.8;9 Furthermore, 1 is free from cross-
resistance to anyknown antitumor agents. 10 Its mode of
action is reported to be the inhibition of DNA poly-
merase.11;12 N-(2-Chlorobenzoyl)-N0-[3-chloro-4(5-tri-
N
O
N
N
NO
2
1
2
Cl
CF
CONHCONH
Cl
3
O
fluoromethyl-2-pyridyloxy)phenyl]urea (2) showed high
5
antitumor activityin vivo (P388 leukemia).
Studies
have demonstrated that N-[4-(5-bromo-2-pyrimidinyl-
oxy)-3-methylphenyl]-N0-(2-dimethylamino-benzoyl)urea
(11) significantlyinhibits tubulin polymerization in vitro.
2. Chemistry
The method employed for the synthesis of benzoylphen-
ylurea derivatives is outlined in Scheme 1. Condensa-
tion of 2-nitrobenzoylisocyanate (3) with 4-(5-bromo-
pyrimidin-2-yloxy)-3-methyl-phenylamine (4) following
reported procedure5 gave compound 5, which was
Keywords: Benzoylphenylurea analogs; Antitumor agents.
* Corresponding author. Tel.: +1-410-614-0200; fax: +1-410-614-8397;
e-mail: khansa@jhmi.edu
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.019

2214
H. Gurulingappa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2213–2216
CONHCONH
Me
O
Br
O
N
CONCO
N
i
N
N
H2N
Me
Br
NO2
NO2
5
3
4
ii
Me
CONHCONH
NH2
Me
Br
Me
Br
CONCONH
N
N
O
N
O
N
iii
NHMe
6
7
v
Me
Me
O
Br
CONCONH
N
CONHCONH
NHMe
Me
O
Br
N
N
NH2
8
N
10
iv
Me
O
Br
CONHCONH
NMe2
N
Me
CONCONH
Me
Me
O
Br
N
N
11
N
NHCONCO
H2N
9
Scheme 1. Reagents and conditions: (i) 1,4-Dioxane, rt, 5 h; (ii) Fe, AcOH, 80 ꢁC, 2 h; (iii) CH3I, K2CO3, rt, 24 h; (iv) Silica, CH2Cl2, rt, 10 h; (v) 37%
HCHO, NaBH3CN, AcOH, CH3CN, rt, 1 h.
reduced with Fe and AcOH to give aniline derivative 6.
Reaction of 6 with CH3I in presence of K2CO3 gave
mixture of 7 and 8, which were purified byHPLC in 18%
and 72% yield, respectively. Initial attempts to purify
these compounds byconventional column chromato-
graphyover silica gel resulted in to compound 9 as
major product along with minor quantityof 7, 8, and
other unidentified compounds. Formation of 9 from 8
was confirmed bystirring 8 with silica gel, which gave 9
in 42% yield. Methylation of free amino group in 6
following reported method14 gave mixture of 10 and
11,15 which were purified byHPLC. The proportion of
10 and 11 varied with the reaction time. Stirring at room
temperature for 1 h followed byusual work up gave 10
(62% yield) and 11 (20%). As the reaction time
increased, the formation of 11 increased. Work up after
5 h gave only 11 in 82% yield. All the compounds were
characterized by 1H NMR, 13C NMR, and LC–MS
studies.16–19
Johns Hopkins. Of these, SNU-308 was derived from
gallbladder carcinoma, HuCCT-1 was derived from
intrahepatic cholangiocarcinoma, BxPC3, Mia-Paca,
and Panc-1 were derived from pancreatic cancers, A431
was derived from vulvar epithelial carcinoma, and Hep2
and HN006 were derived from squamous cell carcinoma
of the head and neck. Cells were grown in monolayer
culture in different media cell cultures were maintained
at 37 ꢁC in a humified atmosphere of 5% CO2 in air.
Stock solutions were prepared for each compound in
dimethyl sulfoxide (DMSO) and stored at )20 ꢁC.
In vitro drug sensibilitywas assessed bythe tetrazolium
(MTT) dye conversion.20
4. Results and discussions
We first tested the susceptibilityof the different cell lines
to the known compounds 11 and its metabolite 6. These
results were used as standard to compare activitywith
the BPU analogs. The susceptibilityto these compounds
3. Biology
in our panel of human cancer cell lines was determined
21
byusing an MTT assay.
We explored concentrations
A panel of eight different human cancer cell lines from
different origins was used for this study. The cell lines
included: SNU-308 cell line obtained from the Korean
Cell Line Bank (Seoul, Korea) HuCCT-1 cell line
obtained from the Health Science Research Resources
Bank (Japan), BxPC3, Panc-1, Mia-Paca, A431, Hep2
cell lines were purchased from the American Tissue
Culture Collection (Manassas, VA), and HN006 was
obtained from Dr. David SidranskyÕs laboratoryat
of the compounds ranging from 10 nM to 10 lM. These
results are shown in Figure 1. All the cell lines in the
panel, except HuCCT-1 were susceptible to 11, with IC50
being 1 lM (Fig. 1a). In case of 6 (Fig. 1b), we observed
higher activitywith IC 50 in the nano molar range for all
the cell lines except for HuCCT-1.
The cytotoxicity of compounds 7, 8, 9, and 10 in panel
of human cancer cell lines was determined using the

H. Gurulingappa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2213–2216
2215
HuCCT-1 (BIL)
SNU-308 (BIL)
HN006 (HN)
Hep2 (HN)
140%
120%
100%
80%
60%
40%
20%
0%
HuCCT-1 (BIL)
SNU-308 (BIL)
HN006 (HN)
Hep2 (HN)
A431
140%
120%
100%
80%
60%
40%
20%
0%
A431
MiaPaca (PANC)
Panc-1(PANC)
BxPC3 (PANC)
MiaPaca (PANC)
Panc-1(PANC)
BxPC3 (PANC)
0mM
1 mM
0 mM
0,000001 mM
1 mM
10 mM
10 mM
0,001 mM
0,001 mM
(a)
Drug Concentrations
0,000001 mM
(a)
Drug Concentrations
140%
120%
100%
80%
60%
40%
20%
0%
HuCCT-1 (BIL)
SNU-308 (BIL)
HN006 (HN)
Hep2 (HN)
140%
120%
100%
80%
60%
40%
20%
0%
HuCCT-1 (BIL)
SNU-308 (BIL)
HN006 (HN)
Hep2 (HN)
A431
MiaPaca (PANC)
Panc-1(PANC)
BxPC3 (PANC)
A431
MiaPaca (PANC)
Panc-1(PANC)
BxPC3 (PANC)
0 mM
1 mM
10 mM
0,001 mM
0mM
1 mM
10 mM
0,000001 mM
0,001 mM
(b)
Drug Concentrations
(b)
Drug concentrations
0,000001 mM
Figure 3. MTT assayin a panel of eight human-derived cancer cell
lines from different origins. Relative growth after exposure to
increasing concentrations of compounds 9 (3a) and 7 (3b).
Figure 1. MTT assayin a panel of eight human-derived cancer cell
lines from different origins. Relative growth after exposure to
increasing concentrations of compounds 11 (1a) and 6 (1b). Name of
the cells is shown in the legend next to the figure.
(Figs. 2b and 3a). However, all cell lines were resistant
to compound 7 (Fig. 3b). Collectively, our finding with
panel of human cancer cell lines stronglysuggest that
the structural modifications; that is methyl substitution
on nitrogen flanked bytwo carbonyls ( 7 and 8) render
them less cytotoxic than the parent molecules 10 and 6.
same MTT assay. The results are summarized in Figures
2 and 3. Cells exhibit verydifferent sensitivities to the
compounds. Compound 10 was effective at inhibiting
the growth of cell lines BxPC3, Mia-Paca, and Hep2 cell
lines (IC50 1 lM) (Fig. 2a). Compounds 8 and 9 showed
the significant cytotoxicity in Hep2 cells (IC50 5 lM)
In summary, we have synthesized four new benzoyl-
phenylurea analogs and evaluated against eight different
human cancer cell lines. A veryinteresting inhibition
profile against BxPC3, Mia-Paca, and Hep2 cells with
compound 10 has been observed. Compounds 8 and 9
showed the significant cytotoxicity in Hep2 cells. All cell
lines were resistant to compound 7. This is specially
interesting in some of these cell lines, like Hep2, that are
known to be resistant to classic cytotoxic drugs, and
some targeted agents.22
120%
HuCCT-1
100%
SNU-308
HN006
Hep2
80%
60%
40%
20%
0%
A431
MiaPaca
Panc-1
BxPC3
0 mM
1 mM
10 mM
0,001 mM
0,000001 mM
(a)
Acknowledgements
Drug Concentrations
160%
140%
120%
100%
80%
60%
40%
20%
0%
We thank Sharyn Baker and Yelena Zabelina for helpful
discussions. Supported bygrant from Commonwealth
Foundation for Cancer Research to M.A.R. and M.H.,
and an intramural grant from the Maryland Cigarette
Restitution Fund to S.R.K.
HuCCT-1 (BIL)
SNU-308 (BIL)
HN006 (HN)
Hep2 (HN)
A431
MiaPaca (PANC)
Panc-1(PANC)
BxPC3 (PANC)
References and notes
0 mM
1 mM
10 mM
0,001 mM
0,000001 mM
(b)
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Figure 2. MTT assayin a panel of eight human-derived cancer cell
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2216
H. Gurulingappa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2213–2216
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(2H, m), 7.25 (1H, t, J ¼ 8 Hz), 7.11 (1H, d, J ¼ 9 Hz),
6.81 (1H, d, J ¼ 8 Hz), 6.65 (1H, t, J ¼ 8 Hz), 6.54 (2H,
br, s), 3.34 (3H, s), 2.22 (3H, s); 13C NMR (125 MHz,
DMSO-d6): d 169.45, 166.35, 159.23, 155.27, 153.34,
140.23, 134.75, 133.37, 130.36, 127.33, 122.45, 121.37,
119.53, 117.47, 115.21, 112.83, 28.32, 15.63; LC–MS m=z
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18. Compound 9: 1H NMR (400 MHz, DMSO-d6): d 11.45
(2H, br, s), 8.73 (2H, s), 7.93 (2H, d, J ¼ 8 Hz), 7.63–7.67
(2H, m), 7.16–7.21 (4H, m), 6.73 (1H, d, J ¼ 8:4 Hz), 6.44
(1H, s), 6.41 (1H, d, J ¼ 8:4 Hz), 4.94 (2H, br, s), 3.25 (6H,
s), 1.9 (3H, s); 13C NMR (125 MHz, DMSO-d6): d 164.38,
162.63, 160.62, 150.82, 146.87, 141.99, 139.77, 135.31,
130.15, 127.72, 122.88, 122.38, 116.24, 115.52, 114.14,
112.99, 112.65, 27.46, 16.48; LC–MS m=z 631 [M]þ, 633
[M+2]þ.
1
19. Compound 10: H NMR (400 MHz, DMSO-d6): d 10.78
(1H, br, s), 8.60 (2H, s), 7.75 (1H, d, J ¼ 8 Hz), 7.48–7.49
(2H, m), 7.26 (1H, t, J ¼ 8 Hz), 7.13 (1H, d, J ¼ 9 Hz),
6.80 (1H, d, J ¼ 8 Hz), 6.70 (1H, t, J ¼ 8 Hz), 6.36 (1H,
br, s), 2.98 (3H, s), 2.22 (3H, s); 13C NMR (125 MHz,
DMSO-d6): d 170.21, 167.52, 158.37, 155.32, 151.25,
141.63, 133.37, 132.82, 130.27, 127.93, 122.65, 120.78,
118.37, 117.43, 117.02, 112.83, 111.58, 33.57, 15.27; LC–
MS m=z 455 [M]þ, 457 [M+2]þ.
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21. MTT Assay: Cells were trypsinized, seeded at 5 · 103 cells/
well in 96-well plate and allowed to grow for 24 h before
the treatment with exponential increasing concentrations
of drugs in the presence of 10% FBS. After a 96 h period of
treatment, 20 lL of MTT solution (5 mg/mL in PBS)
(Sigma, St Louis, MO) were added to each well, and the
plates were then incubated for 3 h at 37 ꢁC. Medium was
then replaced with 100 lL of DMSO per well. Plates were
shaken and the optical densitywas measured at 570 nm
using a multiwell plate reader (Bio-Rad, Model 550, Bio-
Rad Inc., Hercules, CA). Each experiment was performed
in triplicate for each drug concentration and was carried
16. Compound 7: 1H NMR (400 MHz, DMSO-d6): d 11.31
(1H, br, s), 8.60 (2H, s), 7.60 (1H, d, J ¼ 8 Hz), 7.46–7.49
(2H, m), 7.26 (1H, t, J ¼ 8 Hz), 7.10 (1H, d, J ¼ 9 Hz),
6.78 (1H, d, J ¼ 8 Hz), 6.54 (1H, t, J ¼ 8 Hz), 6.34 (1H,
br, s), 3.32 (3H, s), 2.92 (3H, s), 2.21 (3H, s); 13C NMR
(125 MHz, DMSO-d6): d 168.23, 164.21, 158.71, 154.36,
152.42, 141.12, 133.63, 132.67, 130.55, 128.37, 122.36,
121.20, 118.67, 117.53, 114.54, 112.47, 33.74, 28.43, 14.58;
LC–MS m=z 469 [M]þ, 471 [M+2]þ.
out independentlyat least three times. The IC value was
50
defined as the concentration needed for a 50% reduction in
the absorbance calculated based on the survival curves.
Response to drug treatment was assessed bystandardizing
treatment groups to untreated controls.
17. Compound 8: 1H NMR (400 MHz, DMSO-d6): d 11.35
(1H, br, s), 8.59 (2H, s), 7.56 (1H, d, J ¼ 8 Hz), 7.47–7.49
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