Synthesis and activity evaluation of phenylurea derivatives as potent antitumor agents

Article abstract of DOI:10.1016/j.bmc.2009.04.022

We have discovered several tubulin-active compounds in our previous studies. In the establishment of a compound library of small molecule weight tubulin ligands, 14 new N-3-haloacylaminophenyl-N′-(alkyl/aryl) urea analogs were designed and synthesized. The structure-activity relationship (SAR) analysis revealed that (i) the order of anticancer potency for the 3-haloacylamino chain was following -CH₂Br > -CHBrCH₃; (ii) the N′-substituent moiety was not essential for the anticancer activity, and a proper alkyl substitution might enhance the anticancer activity. Among these analogs, the compounds 16j bearing bromoacetyl at the N′-end exhibited a potent activity against eight human tumor cell lines, including CEM (leukemia), Daudi (lymphoma), MCF-7 (breast cancer), Bel-7402 (hepatoma), DU-145 (prostate cancer), DND-1A (melanoma), LOVO (colon cancer) and MIA Paca (pancreatic cancer), with the IC₅₀ values between 0.38 and 4.07 μM. Interestingly, compound 16j killed cancer cells with a mechanism independent of the tubulin-based mechanism, indicating a significant change of the action mode after the structure modification.

Full text of DOI:10.1016/j.bmc.2009.04.022

Bioorganic & Medicinal Chemistry 17 (2009) 3873–3878
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and activity evaluation of phenylurea derivatives as potent
antitumor agents
Dan-Qing Song , Na-Na Du , Yue-Ming Wang, Wei-Ying He, En-Zhu Jiang, Shi-Xiang Cheng,
*
Yan-Xiang Wang, Ying-Hong Li, Yu-Ping Wang, Xin Li, Jian-Dong Jiang
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We have discovered several tubulin-active compounds in our previous studies. In the establishment of a
compound library of small molecule weight tubulin ligands, 14 new N-3-haloacylaminophenyl-N0-(alkyl/
aryl) urea analogs were designed and synthesized. The structure–activity relationship (SAR) analysis
revealed that (i) the order of anticancer potency for the 3-haloacylamino chain was following –
CH2Br > –CHBrCH3; (ii) the N0-substituent moiety was not essential for the anticancer activity, and a
proper alkyl substitution might enhance the anticancer activity. Among these analogs, the compounds
16j bearing bromoacetyl at the N0-end exhibited a potent activity against eight human tumor cell lines,
including CEM (leukemia), Daudi (lymphoma), MCF-7 (breast cancer), Bel-7402 (hepatoma), DU-145
(prostate cancer), DND-1A (melanoma), LOVO (colon cancer) and MIA Paca (pancreatic cancer), with
Received 3 March 2009
Revised 11 April 2009
Accepted 14 April 2009
Available online 17 April 2009
Keywords:
Phenylurea
Anticancer
Structure–activity relationship
Tubulin
the IC50 values between 0.38 and 4.07 lM. Interestingly, compound 16j killed cancer cells with a mech-
anism independent of the tubulin-based mechanism, indicating a significant change of the action mode
Mitotic arrest
after the structure modification.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
microtubule assembly through alkylation of Cys239 residue of
b-tubulin near the colchicine binding site, and shows potent anti-
3-Haloacylamino benzoylurea (3-HBU) analogs are a novel class
of antimitotic agents described in our previous reports. The struc-
ture–activity relationship (SAR) was elucidated in CEM leukemic
cells for their antiproliferative activity.1,2 Among these analogs,
JIMB01 (1), BAABu (2) and IAABu (3) are the representative com-
pounds (Fig. 1) that inhibit tubulin polymerization, block cell cycle
at the M-phase, cause apoptotic cell death through promoting bcl-
2 phosphorylation and show promising therapeutic efficacy in
nude mice bearing human tumors.2–5 According to the previous
studies, the haloacylamino chain at the 3-position of the aromatic
ring is considered to be of significant importance in regulating the
action, and its cytotoxicity in tumor cells was ranked in an order of
–CH2I > –CH2Br > –CHBrCH3 > –CH2Cl.1,2,6 Furthermore, introduc-
tion of suitable aryl at the N0-end of the formylurea chain at the
1-position could retain or improve the anticancer activity, but
change the mechanism from mitotic arrest to that different from
the parent compounds.6
cancer activity.7,8 SAR analysis showed that an exocyclic urea and
a 2-chloroethyl moiety were required to ensure significant cyto-
toxicity. Especially, the N0-2-chloroethyl moiety is prerequisite
for the alkylation of b-tubulin.7,8
In the establishment of a chemical library of small molecule
tubulin ligands, we have designed a group of compounds based
upon the principle of conjugating the two pharmacophores of the
HBUs and CEUs (5, Fig. 1), with an anticipation that the obtained
hybrids could possess powerful antimitotic activity. In this struc-
ture design, we retain the haloacylamino chain at the 3-position
of the HBUs, and replace the formylurea chain with the 2-chloro-
ethylurea chain of the CEUs. Therefore, a new class of N-3-haloa-
cylaminophenyl-N0-2-chloroethyl urea analogs (16a–c) were
designed and synthesized.
In addition, as the trimethoxyphenyl group (6, Fig. 2) is
essential for the antiproliferative activity of colchicine which is
a well-known antimitotic agent,11 this active group was used
to replace the 2-chloroethyl moiety at the N0-end, from which
two analogs (16m, 16n) were then created. In addition, a phenyl
group was also employed as substituent at the same position
(16l). Accordingly, three new diarylurea derivatives (16l–n) were
generated.
N-Aryl-N0-2-(chloroethyl)urea (CEU) analogs are another new
class of antimitotic agents.7–10 Among the CEU analogs, N-(4-tert-
butylphenyl)-N0-(2-chloroethyl)urea (4, Fig. 1) disrupts the
* Corresponding author. Tel.: +86 10 63188423; fax: +86 10 63017302.
E-mail address: jiang.jdong@163.com (J.-D. Jiang).
These authors made equal contribution to this work.
Next, the structure–activity relationship (SAR) study for the
anticancer effect of the substituents at the N0-end was carried
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.022

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D.-Q. Song et al. / Bioorg. Med. Chem. 17 (2009) 3873–3878
NH
CHXR1
NH
CHXR1
NH
NH
Cl
O
O
O
R1
N
H NH
O
NHCONH2
Cl
O
1
, X=Br, R1=CH3,
4,
tert
-butyl
R1=
5
2
, X=Br, R1=H,
3
, X=I, R1=H,
Figure 1. Chemical structures of compounds 1–5.
2. Chemistry
O
H3CO
H3CO
CH3
The 14 new N0-alkyl/arylphenylurea analogs were synthesized
as described in Scheme 1, that includes three synthetic methods
(route A, B and C) according to the different starting materials.
The preparation of nitro compound 14 is the key step for all of
the methods. The route A for 16a–c uses commercially available
m-nitroaniline (7) and 2-chloroethyl isocyanate (8) as starting
materials, with a standard preparation procedure of N,N-unsym-
metrical ureas,7 followed by a reduction and amidation reaction1,2
for 16a–c. In the reductive reaction, nitro compound 14 was
reduced to aniline analogs 15 under conventional conditions using
Pd/C-hydrogenation in a good yield.
NH
OCH3
C
O
OCH3
6,
Colchicine
Figure 2. Chemical structure of chochicine.
out. Instead of the 2-chloroethyl moiety, different aliphatic acyl
substituents such as formyl (16h, 16i), acetyl (16f, 16g), bromoace-
tyl (16j), bromopropionyl (16k) and 3-chloropropionyl (16d, 16e)
with sizes bigger or smaller than that of 2-chloroethyl were used,
and accordingly eight N-3-haloacylamino phenyl-N0-acylurea ana-
logs were synthesized.
The desired products 16d–i (route B) were prepared according
to the standard procedure reported previously.1,6,12 The starting
materials were the commercial available alkylamide (9, 11), which
reacted with oxalyl chloride in refluxing methylene chloride and
converted into the acyl isocyanates (10, 12). The phenylureas
16d–i were obtained through condensation of 10 or 12 with
Route A
Route B
NO2
O
O
Cl
2C
H
b
+
Cl
N
8
O
N
12
O
NH2
11
NH2
7
a
c
Route C
Route B
NO2
NO2
O
O
d
b
c
R3
R3
N
10
NH2
O
HN
NH
R2
N
O
9
O
13
14 (a-n)
e or f
NHCOCHXR1
NH2
g
or g, h
HN
NH
R2
HN
NH
R2
O
O
15
16 (a-n)
Scheme 1. Synthetic routes of phenylurea analogs 16a–n. Reagents and conditions for the chemical synthesis: (a) DMF, 12 h; (b) (COCl)2, CH2ClCH2Cl, reflux, 4 h;
(c) m-nitroaniline, CH3CN, 12 h; (d) NH3/relevant aniline, DMF, 12 h; (e) Pd/C, EtOH, 4 h; (f) Fe/HCl, 95%EtOH, reflux, 4 h; (g) CH3CHR1COX, DMA, 2 h; (h) NaI, DMA, 2 h.

D.-Q. Song et al. / Bioorg. Med. Chem. 17 (2009) 3873–3878
3875
Table 2
m-nitroaniline, then reduction with Fe/HCl as reducing agent and
amidation using similar conditions to that reported previously.6,7
In the course of condensation of 11 with oxalyl chloride, the hydro-
chloride produced in the reaction underwent 1,4-conjugate addi-
tion, and gave the product 3-chloropropionyl isocyanate (12).
The desired products 16j–n were prepared as shown in route C
with the commercial available m-nitrophenyl isocyanate (13) as
starting material. Condensation of 13 with 3,4,5-trimethoxylani-
line, aniline or NH3 in DMF provided the nitro compounds 14,
which were converted into 16j–n by reduction and amidation reac-
tions as previously reported.6,7
Antiproliferative activities of 16j in human tumor cell lines
a
Cell line
Human tumor
IC50 (lM)
Daudi
MCF-7
Bel-7402
DU-145
PC-3
DND-1A
LOVO
MIA
B-cell lymphoma
Breast cancer
hepatoma
Prostate cancer
Prostate cancer
Melanoma
0.38 0.05
0.59 0.06
4.07 0.20
1.28 0.08
>100
0.51 0.08
1.99 0.18
2.81 0.36
Colon cancer
Pancreas cancer
a
IC50: drug concentration required to inhibit 50% of cell proliferation after 72 h of
treatment. Each experiment was repeated three times.
3. Results and discussion
The human CEM leukemic cells were used for initial biological
screening because of their rapid proliferation and high sensitivity
to anticancer agents. The antiproliferative activity of the 14 com-
pounds was closely associated with their structures, as shown in
Table 1. Out of the 14 compounds, 6 compounds (16a, 16c, 16h,
16j, 16l and 16m) exhibited potent activity with IC50 values of
anticancer activity with IC50 values of 0.85 and 3.02 lM, respec-
tively. Substitution with groups bigger than the size of chloroethyl
afforded comparable activity. The compounds (16d, 16e) bearing
N0-3-chloropropionyl exhibited anticancer IC50 values of 1.90 and
2.05
lM, respectively. The compounds with bromoacetyl (16j) or
bromopropionyl (16k) at the same position showed good activity
61.0
l
M. The anticancer potency of the 3-bromoacylamino chain
with IC50 values of 0.56 lM for 16j and 1.34 lM for 16k. These data
followed the order of bromoacetyl > bromopropionyl, consistent
suggest that N0-2-chloroethyl was not essential for the anticancer
activity, and proper N0-acyl such as N0-formyl (16h) and N0-bromo-
acetyl (16j) afforded promising activity against cancer.
with our previous observation on 3-HBUs.1,2,6
The compounds 16a and 16c bearing the active 2-chloroethyl at
N0-end showed a potent activity with IC50 values of 0.77 and
To confirm the observation, more modifications were made at
this position. The compound (16m) possessing an active group of
N0-trimethoxyphenyl provided good activity with IC50 value of
0.10 l
M, respectively. Replacing N0-2-chloroethyl with smaller
substituents, we obtained compound 16h bearing formyl and 16f
bearing acetyl at the N0-end; these two compounds showed an
1.0
lM. Replacing trimethoxyphenyl with phenyl, compound 16l
showed an IC50 of 0.86
l
M. Therefore, we concluded that the group
at the N0-end is replaceable, and could be used to optimize the
compounds.
Table 1
Structures and anticancer activity of the phenylureas in the CEM cells
As 16j showed a potent activity in leukemia, the antiprolifera-
tive activity of this compound was further examined in eight solid
or liquid human tumor cell lines (Table 2). It appears that 16j was a
potent killer for lymphoma (Daudi), breast cancer (MCF-7) and
melanoma (DND-1) with IC50 values in the range of 0.38ꢀ
NHCOCHXR1
O
R2
NH
R2
NH
0.59
lM. The least sensitive one was PC-3 prostate cancer cells
a
Compound
R1
X
IC50 (lM)
with an IC50 > 100
lM. Human colon cancer (LOVO), prostate
1b
1.47 0.09
0.725 0.06
0.77 0.09
22.7 5.15
0.10 0.026
1.90 0.14
2.05 0.06
3.20 0.68
10.56 0.07
0.85 0.06
10.2 0.13
0.56 0.06
1.34 0.26
cancer (DU-145), pancreatic cancer (MIA paca) and hepatoma
(Bel-7402) showed moderate degree of susceptibility to the com-
pound. Figure 3 shows the killing effect on the cancer cells by
16j. The kinetics of the cell death demonstrates a dose-dependent
killing of the CEM leukemia cells in 16j treatment.
After the activity evaluation we extend our work to the mecha-
nism investigation. To learn whether or not these analogs would
still block cell cycle progress at the M-phase as their parent com-
pounds do, flow cytometric analysis was done with the individual
compound 16c, 16h and 16j (Fig. 4) using 1 as the reference. The
cell population accumulated on the left hand side of the G1/G0
peak in the samples treated with compound 1, 16c, 16h or 16j
2b
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
H
CH3
H
H
CH3
H
CH3
H
CH3
H
Br
Br
I
–CH2CH2Cl
–CH2CH2Cl
–CH2CH2Cl
–COCH2CH2Cl
–COCH2CH2Cl
–COCH3
–COCH3
–CHO
–CHO
–COCH2Br
–COCHBrCH3
Br
Br
Br
Br
Br
Br
Br
Br
CH3
16l
H
Br
0.86 0.29
100
75
50
25
0
H CO
3
16m
H
Br
1.00 0.068
H CO
3
OCH
3
H CO
3
16n
CH3
Br
3.99 0.022
H CO
3
-2
-1
0
1
2
Drug Concentration (µmol/L, log)
OCH
3
a
IC50: drug concentration required to inhibit 50% of cell proliferation after 72 h of
treatment. Each experiment was repeated three times.
Figure 3. Dose-dependent killing of CEM cells by 16j. CEM cells were treated with
16j at different concentrations for 24 h and the cell death was measured using MTT
assay as described in Section 5.
b
See Figure 1.

3876
D.-Q. Song et al. / Bioorg. Med. Chem. 17 (2009) 3873–3878
G1/G0
G2/M
S
S G2/M
G1/G0
0
40
80
120
160
200
0
40
80
120
160
200
Channels (FL2-A)
Channels (FL2-A)
Control
Compound 1
G1/G0
G1/G0
G1/G0
S
S
SG2/M
G2/M
80
G2/M
0
40
80
120
160
200
0
40
120
160
200
0
40
80
120
160
200
Channels (FL2-A)
Channels (FL2-A)
Channels (FL2-A)
16c
16h
16j
Figure 4. Cell cycle analysis. CEM cells were untreated or treated with 1 (3.2 lM), 16c (10 lM), 16h (3.3 lM) and 16j (6.4 lM) respectively, for 18 h at 37 °C. Cell cycle was
analyzed as described in Section 5.5.
represents the dead cells with DNA degradation, featuring apopto-
tic cells death, and the % of the dead cells are in Table 3. Compound
1 arrested CEM cells in the G2/M phase as expected, 16c, 16h and
16j caused no G2/M phase arrest in CEM cells, and appeared to kill
the CEM cells through G1/G0 suspension. To validate the observa-
tion, we accessed the direct effect of 16c, 16h and 16j on microtu-
bule assembly in a cell-free system. As shown in Figure 5, different
from the parent compound 1, none of three analog compounds
inhibited microtubule assembly. The results indicate that the
modifications made on 16c, 16h and 16j compounds have changed
the anticancer mechanism from mitotic arrest to the one different
from their parent compounds.
mode of action independent of tubulin-based mechanism. The SAR
analysis showed that the anticancer potency of the 3-haloacylami-
no chain followed the order of –CH2Br > –CHBrCH3. The substitu-
ents at the N0-end play a role for chemical regulation of the
anticancer activity, and suitable aliphatic substituents might
improve the activity. Of the study compounds, compound 16j
exhibited good anti-proliferative activity in a variety of in vitro cell
culture experiments using cell lines derived from human liquid
and solid tumors, and a mechanism different from its parent
compounds. The in vivo anticancer activity and detailed mecha-
nism pathway of the compound are currently under investigation
in our laboratory.
4. Conclusions
0 min
0.600
0.500
0.400
0.300
0.200
Taken together, we consider that N-3-haloacylaminophenyl-N0-
2-alkylureas are a class of promising antiproliferative agents with a
30 min
Table 3
Apoptotic cell death by study compoundsa
0.100
0.000
G0/G1
S
G2/M
Apoptosis (%)
Control
Compound 1
16c
16h
16j
Control
1
16j
16h
16c
34.5
2.0
48.3
33.5
34.1
54.6
25.3
13.7
12.9
30.1
9.4
63.2
13.2
2.0
1.5
9.5
24.8
52.6
33.2
Figure 5. Compounds 16c, 16h and 16j showed no inhibitory effect on microtubule
polymerization. The OD at 340 nm was taken as a proxy measurement of the degree
of microtubule polymerization. Microtubule polymerization was determined by
measuring OD value (340 nm) at 0 min and 30 min after initiation of the assembly
2.6
reaction. The sample was treated with the solvent (control), compound 1 (3.2
16c (10 M), 16h (3.3 M) and 16j (6.4 M), respectively. Each column represents
the mean SD of three independent assays.
lM),
a
The cell cycle distribution and apoptotic cell death was determined using flow
cytometry (see Section 5).
l
l
l

D.-Q. Song et al. / Bioorg. Med. Chem. 17 (2009) 3873–3878
3877
for 4 h and concentrated under vacuum to give an oil. To a stirring
solution of the oil in CH3CN (40 ml) was added m-nitroaniline
(1.39 g, 10 mmol). The reaction mixture was stirred for 12 hrs at
rt and concentrated under vacuum. The residue was stirred in
water (60 ml) for 30 min. The mixture was filtered, and the solid
was washed with water and dried to give nitro compound (13),
which was used for the next step without purification. Using pro-
cedures previous reported,5,6 the title compounds were obtained
after two steps reactions including reduction (Fe/HCl as a reductive
agent) and acylamidation.
5. Experimental
5.1. Chemical methods
Melting point (mp) was obtained with YRT-3 melting point appa-
ratus and uncorrected. 1H NMR spectra was performed on a Varian
Inova 400 MHz spectrometer (Varian, San Francisco, CA) in DMSO-
d6, with Me4Si as the internal standard. ESI high-resolution mass
spectra (HSMS) were recorded on an Autospec UItima-TOF mass
spectrometer (Micromass UK Ltd, Manchester, UK). Purity estima-
tion was done with TLC (thin layer chromatography), and only those
with purity around 95% were tested for biological activity.
5.3.1. N-(3-Bromoacetamido)phenyl-N0-3-chloropropionylurea
(16d)
Solvent and reagent abbreviations used are DMF = dimethyl-
formamide, DMA = dimethylacetamide, DMSO = dimethylsulfoxide.
Using the previous procedure, yield: 8%. Mp 193.8ꢀ194.7 °C. 1H
NMR: d 2.91 (t, J = 6.0, 6.4 Hz, 2H), 3.85 (t, J = 6.0, 6.4 Hz, 2H), 4.02
(s, 2H), 7.15 (d, J = 8.0, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz,
5.2. General procedure for the synthesis of compounds 16aꢀc
1H), 7.87 (s, 1H), 10.42 (s, 1H), 10.46 (s, 1H), 10.82 (s, 1H); IR (KBr)
c
(route A)
3297, 3133 (NH), 1719, 1671, 1608 (C@O) cmꢀ1; HRMS: calcd for
C12H13BrClN3O3 (M+Na+H)+ 384.9805, found 384.9794.
To a solutionof m-nitroaniline (2.76 g, 20 mmol)in EtOAc (20 ml)
and EtOH (20 ml) was added dropwise 2-chloroethyl isocyanate
(2.5 ml, 29 mmol) with stirring. The mixture was stirred at room
temperature (rt) for 12 h, and the precipitated product was filtered,
washed with ether and dried to give the nitro compound (14). The
nitro compound (2.0 g, 8.2 mmol) was dissolved in absolute ethanol
(60 ml), and to the solution was added 10% Pd/C (0.2 g). After stirring
at rt for 6 h under a hydrogen atmosphere, the catalyst was removed
by filtration, and filtrate was concentrated under reduced pressure,
soaked in ether, filtered and dried to give white solid 15, which
was used for the next step without purification.
5.3.2. N-(3-b-Bromopropionamido)phenyl-N0-3-
chloropropionylurea (16e)
Using the previous procedure, yield: 15%. Mp 198.7ꢀ199.4 °C.
1H NMR: d 1.74 (d, J = 6.4 Hz, 3H), 2.92 (t, J = 6.0, 6.4 Hz, 2H),
3.85 (t, J = 6.0, 6.4 Hz, 2H), 4.69 (q, J = 6.8, 6.4 Hz, 1H), 7.14 (d,
J = 8.0 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.90
(s, 1H), 10.37, 10.46, 10.83 (s, s, s, 3H); IR (KBr)
c 3291, 3132
(NH), 1720, 1690, 1666, 1607 (C@O) cmꢀ1; HRMS: calcd for
C13H15BrClN3O3 (M+Na+H)+ 398.9961, found 398.9968.
By use of a procedure previously described,5,6 the title com-
pounds were obtained from 14 after its reaction respectively with
chloroacetyl chloride, bromoacetyl bromide, or 2-bromopropionyl
bromide in DMA. The residue is purified by recrystallization from
ethanol.
5.3.3. N-(3-Bromoacetamido)phenyl-N0-acetylurea (16f)
Using the previous procedure, yield: 28%. White solid, mp
199.7ꢀ201.4 °C. 1H NMR: d 2.49 (s, 3H), 3.31 (s, 2H), 7.14 (d,
J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.82
(s, 1H), 10.41, 10.53, 10.66 (s, s, s, 3H); IR (KBr)
c
3259, 3140
(NH), 1714, 1662, 1609 (C@O) cmꢀ1
;
HRMS: calcd for
5.2.1. N-(3-Bromoacetamido)phenyl-N0-2-chloroethylurea (16a)
Using the previous procedure, yield: 65%. White needle, mp
155ꢀ157 °C. 1H NMR: d 3.40 (q, J = 6.0 Hz, 2H), 3.65 (t, J = 6.0 Hz,
2H), 4.00 (s, 2H), 6.34 (t, J = 4.4, 4.8 Hz, 1H), 7.10ꢀ7.15 (m, 3H),
C11H12BrN3O3 (M+Na)+ 335.9960, found 335.9955.
5.3.4. N-(3-b-Bromopropionamido)phenyl-N0-acetylurea (16g)
Using the previous procedure, yield: 29%. White solid, mp
214.6ꢀ216.3 °C. 1H NMR: d 1.73 (d, J = 6.8 Hz, 3H), 2.49 (s, 3H),
4.68 (q, J = 6.8 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz,
1H), 7.38 (d, J = 8.4 Hz, 1H), 7.85 (s, 1H), 10.35, 10.54, 10.67 (s, s, s,
7.71 (s, 1H), 8.70 (s, 1H), 10.30 (s, 1H); IR (KBr)
c 3319, 3242
(NH), 1660, 1633 (C@O) cmꢀ1; HRMS: calcd for C11H13BrClN3O2
(M+Na)+ 355.9777, found 355.9761.
c ;
3251, 3145 (NH), 1714, 1663, 1608 (C@O) cmꢀ1
5.2.2. N-(3-b-Bromopropionamido)phenyl-N0-2-chloroethylurea
(16b)
3H); IR (KBr)
HRMS: calcd for C12H14BrN3O3 (M+Na) 350.0116, found 350.0096.
Using the previous procedure, yield: 66%. White needle, mp
171ꢀ173 °C. 1H NMR: d 1.74 (d, J = 6.4 Hz, 3H), 3.43 (q, J = 6.0 Hz,
2H), 3.66 (t, J = 6.0 Hz, 2H), 4.70 (q, J = 6.8, 6.4 Hz, 1H), 6.37 (t,
J = 5.6, 6.0 Hz, 1H), 7.09ꢀ7.21 (m, 3H), 7.75 (s, 1H), 8.70 (s, 1H),
5.3.5. N-(3-Bromoacetamido)phenyl-N0-formylurea (16h)
Using the previous procedure, yield: 16%. White solid, mp
214.6ꢀ216.7 °C. 1H NMR: d 4.02, 4.24 (s, s 2H), 7.27 (m, 1H), 7.43
(m, 2H), 7.97 (s, 1H), 8.17 (s, 1H), 8.26, 10.36, 10.56 (s, s, s, 3H);
10.26 (s, 1H); IR (KBr)
c ;
3317, 3240 (NH), 1660, 1633 (C@O) cmꢀ1
IR (KBr)
c
3296 (NH), 1714, 1667, 1609 (C@O) cmꢀ1; HRMS: calcd
HRMS:calcd forC12H15BrClN3O2 (M+Na)+ 369.9934, found 369.9903.
for C10H10BrN3O3 (M+Na)+ 321.9803, found 321.9823.
5.2.3. N-(3-Iodoacetamido)phenyl-N0-2-chloroethylurea (16c)
Using the previous procedure, yield: 65%. White needle, mp
164ꢀ166 °C (dec). 1H NMR: d 3.42 (q, J = 6.0 Hz, 2H), 3.66 (t,
J = 6.0 Hz, 6.4 Hz, 2H), 3.81 (s, 2H), 6.35 (m, 1H), 7.11ꢀ7.15 (m,
5.3.6. N-(3-b-Bromopropionamido)phenyl-N0-formylurea (16i)
Using the previous procedure, yield: 16%. White solid, mp
191ꢀ193 °C. 1H NMR: d 1.73 (d, J = 6.4 Hz, 3H), 4.71 (q, J = 6.4 Hz,
1H), 7.27 (m, 1H), 7.43 (m, 2H), 7.97 (s, 1H), 8.13 (s, 1H), 8.25,
3H), 7.70 (s, 1H), 8.70 (s, 1H), 10.25 (s, 1H); IR (KBr)
c 3319,
3240 (NH), 1651, 1640 (C@O) cmꢀ1; HRMS: calcd for C11H13ClI-
10.36, 10.56 (s, s, s, 3H); IR (KBr)
c 3390, 3302 (NH), 1668, 1605
(C@O) cmꢀ1; HRMS calcd for C11H12BrN3O3 (M+H)+ 314.0140,
N3O2 (M+Na)+ 403.9639, found 403.9611.
found 314.0127.
5.3. General procedure for the synthesis of compounds 16dꢀk
5.4. General procedure for the synthesis of compounds 16jꢀn
To a solution of relevant acylamide (9, 11, 10 mmol) in anhy-
drous dichloroethane (40 ml) was added oxalyl chloride (1.3 ml,
15 mmol) dropwise. The reaction mixture was heated to reflux
To a solution of m-nitrophenyl isocyanate (0.66 g, 4.0 mmol) in
DMF (10 ml) was added relevant aniline or ammonia (4.0 mmol)

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D.-Q. Song et al. / Bioorg. Med. Chem. 17 (2009) 3873–3878
with stirring at rt. The reaction mixture was stirred at rt for 6–8 h,
then poured into ice water. The precipitated product was collected
by filtration and washed with water, then dried to give nitro com-
pound (13). Using procedures previous reported,5,6 the title com-
pounds were obtained after two steps reactions, including
reduction (with Fe/HCl as reducing agent) and acylamidation.
5.5.2. Anticancer activity in vitro
Cells were seeded into 96-well plates (Falcon, CA) with 1 ꢁ 105
cells in 250 lL per well, followed by treatment with test com-
pounds at concentrations between 0.001 and 100 lM for 72 h at
37 °C. Cell viability was assessed with the 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). The IC50
value was defined as the drug concentration killing 50% of the cells
in comparison with the untreated controls, and calculated with
non-linear regression analysis. The IC50 values were determined
in duplicates, and each experiment was repeated three times under
identical conditions.
5.4.1. N-(3-Bromoacetamido)phenyl-N0-bromoacetylurea (16j)
Using the previous procedure, yield: 54%. White solid, mp
186ꢀ187 °C. 1H NMR: d 4.02 (s, 2H), 4.14 (s, 2H), 7.16 (d,
J = 8.8 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H),
7.86ꢀ7.87 (m, 1H), 10.20 (s, 1H), 10.45 (s, 1H), 11.00 (s, 1H); IR
(KBr)
c
3340, 3257 (NH), 1655 (C@O) cmꢀ1; HRMS: calcd for
5.5.3. Cell cycle analysis
C11H11Br2N3O3 (M+Na)+ 413.9065, found 413.9094.
Cell cycle was analyzed using a method reported previously.11
Briefly, the CEM cells were treated with 16c, 16h and 16j, respec-
tively, at 37 °C for 18 h. The cells were then re-suspended in 70%
ethanol for 18 h at 4 °C. The cells were treated with RNase A
5.4.2. N-(3-b-Bromopropionamido)phenyl-N0-2-
bromopropionylurea (16k)
Using the previous procedure, yield: 56%. White silod, mp
191ꢀ192 °C. 1H NMR: d 1.73 (d, J = 6.4 Hz, 6H), 4.69 (q, J = 6.8 Hz,
2H), 7.14ꢀ7.16 (m, 1H), 7.25ꢀ7.29 (m, 1H), 7.39ꢀ7.41 (m, 1H),
(50
l
g/ml, Sigma, MO) for 0.5 h at 37 °C, and exposed to propidium
iodide at a final concentration of 50
l
g/ml for 0.5 h at 4 °C. 104 cells
per sample were analyzed in a flow cytometer (BD FACS Calibur
System, San Jose, CA) and the cell cycle phase was calculated with
Cell Quest Pro software (version 2.0).
7.93 (s, 1H), 10.28 (s, 1H), 10.40 (s, 1H), 11.05 (s, 1H); IR (KBr)
c
3340, 3257 (NH), 1655, 1599 (C@O) cmꢀ1; HRMS: calcd for
C13H15Br2N3O3 (M+Na)+ 441.9378, found 441.9419.
5.5.4. Microtubule polymerization assays
5.4.3. N-(3-Bromoacetamido)phenyl-N0-phenylurea (16l)
Using the previous procedure, yield: 72%. White solid, mp
184ꢀ186 °C (dec). 1H NMR: d 4.03 (s, 2H), 6.95ꢀ6.99 (m, 1H),
7.16ꢀ7.30 (m, 5H), 7.44ꢀ7.46 (m, 2H), 7.81 (s, 1H), 8.60 (s, 1H),
Purified tubulin from calf brain (Sigma, St. Luis, MO) was used
to determine polymerization reaction.4 The OD at 340 nm was
taken as a proxy measurement of the degree of microtubule poly-
merization. 100
lL of b-tubulin (1 mg/ml) was mixed gently with
8.74 (s, 1H), 10.37 (s, 1H); IR (KBr)
c
3340, 3257 (NH), 1655
400 L of reaction buffer containing 0.1 M 2-[N-morpholino]eth-
l
(C@O) cmꢀ1; HRMS: calcd for C15H14BrN3O2 (M+Na)+ 370.0167,
found 370.0158.
ane sulfonic acid (MES), 1 mM EGTA, 0.5 mM MgCl2, 0.1 mM EDTA,
and 2.5 M glycerol. Then, the solvent (control), compound 1, 16c,
16h, or 16j was added to each sample cuvet. After adding GTP to
5.4.4. N-(3-Bromoacetamido)phenyl-N0-30,40,50-
each sample to a final concentration of 1 mM, the starting
trimethoxyphenylurea (16m)
(0 min) OD was measured as the baseline by a spectrophotometer
(UNIC UV-2100) at 340 nm. Assembly reaction was completed
within 30 min at room temperature.
Using the previous procedure, yield: 53%. White solid, mp
124ꢀ127 °C. 1H NMR: d 3.60 (s, 3H), 3.70 (s, 3H), 3.71 (s, 3H),
4.02 (s, 2H), 6.79 (s, 2H), 7.08 (d, J = 8.0 Hz, 1H), 7.20 (m, 1H),
7.26 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 8.54 (s, 1H), 8.67 (s, 1H),
Acknowledgment
10.37 (s, 1H); IR (KBr)
c 3307, 3221 (NH), 1761, 1668 (C@O)
cmꢀ1; HRMS: calcd for C18H20BrN3O5 (M+Na)+ 460.0484, found
460.0488.
This study was supported by the National Natural Science Foun-
dation of the PR China (Grant 30371673).
5.4.5. N-(3-b-Bromopropionamido)phenyl-N0-3,4,5-
trimethoxyphenylurea (16n)
References and notes
Using the previous procedure, yield: 52%. White solid, mp
187.2ꢀ191 °C. 1H NMR: d 1.74 (d, J = 6.8 Hz, 3H), 3.60 (s, 3H),
3.74 (s, 6H), 4.70 (q, J = 6.8 Hz, 1H), 6.79 (s, 2H), 7.05 (d,
J = 8.8 Hz, 1H), 7.18ꢀ7.22 (m, 1H), 7.29 (d, J = 8.8 Hz, 1H), 7.86
1. Song, D. Q.; Wang, Y.; Wu, L. Z.; Yang, P.; Wang, Y. M.; Gao, L. M.; Li, Y.; Qu, J. Q.;
Wang, Y. H.; Li, Y. H.; Du, N. N.; Han, Y. X.; Zhang, Z. P.; Jiang, J. D. J. Med. Chem.
2008, 51, 3094.
2. Jiang, J. D.; Roboz, J.; Imre, W.; Deng, L.; Ma, L. H.; Holland, J. F.; Bekesi, J. G.
Anti-Cancer Drug Des. 1998, 13, 735.
3. Jiang, J. D.; Davis, A. S.; Middleton, K.; Ling, Y. H.; Roman, P. S.; Holland, J. F.;
Bekesi, J. G. Cancer Res. 1998, 58, 5389.
4. Jiang, J. D.; Wang, Y.; Roboz, J.; Strauchen, J.; Holland, J. F.; Bekesi, J. G. Cancer
Res. 1998, 58, 2126.
(m, 1H), 8.55 (s, 1H), 8.66 (s, 1H), 10.31 (s, 1H); IR (KBr)
c 3365,
3317, 3280 (NH), 1704, 1684 (C@O) cmꢀ1; HRMS: calcd for
C19H22BrN3O5 (M+Na)+ 474.0640, found 474.0633.
5. Li, J. N.; Song, D. Q.; Lin, Y. H.; Hu, Q. Y.; Yin, L.; Bekesi, G.; Holland, J. F.; Jiang, J.
D. Biochem. Pharmacol. 2003, 65, 1691.
6. Song, D. Q.; Wang, Y. M.; Du, N. N.; He, W. Y.; Chen, K. L.; Wang, G. F.; Yang, P.;
Wu, L. Z.; Zhang, X. B.; Jiang, J. D. Bioorg. Med. Chem. Lett. 2009, 19, 755.
7. Mounetou, E.; Legault, J.; Lacroix, J.; Gaudreault, R. C. J. Med. Chem. 2001, 44,
694.
8. Mounetou, E.; Legault, J.; Lacroix, J.; Gaudreault, R. C. J. Med. Chem. 2003, 46,
5055.
9. Legault, J.; Gaulin, J. F.; Mounetou, E.; Bolduc, S.; Lacroix, J.; Poyet, P.;
Gaudreault, R. C. Cancer Res. 2000, 60, 985.
10. Petitclerc, E.; Deschesnes, R. G.; Cote, M. F.; Marquis, C.; Janvier, R.; Lacroix, J.;
Noirault, E. M.; Legault, J.; Mounetou, E.; Madelmont, J. C.; Gaudreault, R. C.
Cancer Res. 2004, 64, 4654.
11. (a) Andreu, J. M.; Timasheff, S. N. Biochemistry 1982, 21, 6465; (b) Bai, R.; Pei, X.
F.; Boye, O.; Getahan, Z.; Grover, S.; Bekisz, J.; Nguyen, N. Y.; Brossi, A.; Hamel,
E. J. Biol. Chem. 1996, 271, 12639; (c) Dumortier, C.; Gorbunoff, M. J.; Andreu, J.
M.; Engelbourghs, Y. Biochemistry 1996, 35, 4387.
5.5. Biological methods
5.5.1. Tumor cell lines
The human tumor cell lines CEM (leukemia), MCF-7 (breast can-
cer), DU-145 (prostate cancer), PC-3 (prostate cancer), LOVO (colon
cancer), Daudi (lymphoma) and MIA Paca (pancreatic cancer) were
from the American Type Culture Collection (ATCC, Manassas, VA,
USA). DND-1 melanoma cells were from Dr. Ohnuma (Mount Sinai
School of Medicine, New York, NY). Bel-7402 hepatoma cells were
from the Cancer Institute, Chinese Academy of Medical Sciences.
All of the cells were cultivated in the RPMI-1640 medium (Invitro-
gen) supplemented with 10% inactivated fetal calf serum, penicillin
12. Robert, J. W.; Stanford, B., Jr.; Mark, A. E.; Elizabeth, B. FS.; David, G. L.; Peter, H.
N.; Anthony, L. P. J. Med. Chem. 1991, 34, 1630.
(150
5% CO2 at 37 °C.
lg/ml) and streptomycin (150 lg/ml), in the atmosphere of at