Benzoylurea derivatives as a novel class of antimitotic agents: Synthesis, anticancer activity, and structure-activity relationships

Article abstract of DOI:10.1021/jm070890u

Forty-six new compounds were synthesized on the basis of our knowledge of the 3-haloacylamino benzoylurea (HBU) series. Structure-activity relationship (SAR) analysis indicates that (i) the configuration of the chiral center in 1 (JIMB01) is not indispensable for the activity, (ii) the phenyl ring is essential, and (iii) a substitution at the 6-position of the phenyl ring with a halogen enhances the activity. Among the analogues, 11e and 14b bearing 6-fluoro substitution showed potent activities against nine human tumor cell lines, including CEM (leukemia), Daudi (lymphoma), MCF-7 (breast cancer), Bel-7402 (hepatoma), DU-145 (prostate cancer), PC-3 (prostate cancer), DND-1A(melanoma), LOVO (colon cancer), and MIA Paca (pancreatic cancer) with IC₅₀ values between 0.01 and 0.30 μM. 14b inhibited human hepatocarcinoma by 86% in volume in nude mice. The mechanism of 14b is to inhibit microtubule assembly, followed by the M-phase arrest, bcl-2 inactivation, and then apoptosis. We consider 14b promising for further anticancer investigation.

Full text of DOI:10.1021/jm070890u

3094
J. Med. Chem. 2008, 51, 3094–3103
Benzoylurea Derivatives as a Novel Class of Antimitotic Agents: Synthesis, Anticancer Activity,
and Structure-Activity Relationships
Dan-Qing Song,†,‡ Yan Wang,†,‡ Lian-Zong Wu,† Peng Yang,† Yue-Ming Wang,† Li-Mei Gao,† Yan Li,§ Jing-Rong Qu,§
Yong-Hong Wang,† Ying-Hong Li,† Na-Na Du,† Yan-Xing Han,† Zhi-Ping Zhang,† and Jian-Dong Jiang*,†
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China, and
DiVision of Hematology and Oncology, Box 1131, Mount Sinai School of Medicine, New York, New York 10029
ReceiVed July 24, 2007
Forty-six new compounds were synthesized on the basis of our knowledge of the 3-haloacylamino benzoylurea
(HBU) series. Structure-activity relationship (SAR) analysis indicates that (i) the configuration of the chiral
center in 1 (JIMB01) is not indispensable for the activity, (ii) the phenyl ring is essential, and (iii) a substitution
at the 6-position of the phenyl ring with a halogen enhances the activity. Among the analogues, 11e and
14b bearing 6-fluoro substitution showed potent activities against nine human tumor cell lines, including
CEM (leukemia), Daudi (lymphoma), MCF-7 (breast cancer), Bel-7402 (hepatoma), DU-145 (prostate cancer),
PC-3 (prostate cancer), DND-1A(melanoma), LOVO (colon cancer), and MIA Paca (pancreatic cancer)
with IC50 values between 0.01 and 0.30 µM. 14b inhibited human hepatocarcinoma by 86% in volume in
nude mice. The mechanism of 14b is to inhibit microtubule assembly, followed by the M-phase arrest,
bcl-2 inactivation, and then apoptosis. We consider 14b promising for further anticancer investigation.
Introduction
Microtubule (MTa) dynamics have been identified as a
rational site to interfere with the dividing of cancer cells. Drug
agents that target cellular MT dynamics arrest cancer cell cycle
at the M-phase followed by apoptosis. Drugs in this class have
been widely used for cancer patients with great clinical success
and are continuing to be important chemotherapeutic agents.1–3
Figure 1. Structure of the lead compounds.
The commonly used tubulin-active anticancer drugs are pacli-
1 (JIMB01),13 2 (BAABU),12 and 3 (IAABU),11 which interfere
taxel and vinca alkaloids. These drugs, however, have three
with the assembly of mitotic spindle microtubules from the free
major limitations.4,5 First of all, these drugs cause neurotoxic
tubulin pool in tumor cells, block the cell cycle at the M-phase,
side effects in patients.6 Second, they are difficult for de novo
cause apoptotic cell death through promoting bcl-2 phospho-
synthesis because the compounds are natural products existing
rylation, and show therapeutic efficacy in nude mice bearing
as large molecular structures. Third, they are all substrates of
human hepatocarcinoma.10–13 In contrast to the tubulin-active
P-glycoprotein and induce multidrug resistance (MDR), which
anticancer drugs that have been used in the clinic, these
is the main cause for the failure of clinical cancer chemo-
compounds have simple and small chemical structures, and that
therapy.7 Therefore, searching for drugable small tubulin ligands
provides advantages in chemical synthesis and opportunities of
with novel chemical structures or antimitotic agents with novel
formulation for oral administration. In preliminary SAR analysis,
modes of action has become an active field of investigation in
we have identified the essential structural and functional
medicinal chemistry.8,9 For instance, combretastain A-4 (CA4),
requirements for the anticancer activity.10 The results indicated
a new antimitotic agent binding at colchicine pocket on tubulin
that the benzoylurea group is a key for good anticancer activity;
with a smaller molecule, has entered into phase II clinical trail.1b
the iodoacetylamino, bromoacetylamino, or bromopropiony-
The 3-haloacylamino benzoylureas (HBUs) analogues were
lamino chain at the 3-position (meta position in relationship to
designed and synthesized in our group.10–13 Several compounds
the benzoylurea group) plays a significant role in regulating the
have been proved to be tubulin ligands that inhibit the
action. Their cytotoxicity in tumor cells was ranked in an order
polymerization of tubulin. The primary mechanism of these
of the nature of halogens: I > Br > Cl > F.10
compounds is to bind at the region of the colchicine binding
Benzoylurea derivatives constitute a class of antiproliferative
site on ꢀ-tubulin.11a The compounds have shown anticancer
agents, among which the N-substituted benzoylurea derivatives
activity in vitro and in vivo and become a new family of tubulin
have been reported to have antitumor activities.14–16 Therefore,
ligand leads.11–13 The representative compounds (Figure 1) are
the chemical and biological features of the 3-HBU derivatives
provoked our strong interest for a further SAR study. In this
study, we retain the benzoylurea group at the 1-position of the
aromatic ring and focus the study on the configuration of the
asymmetric center in the compound 1 structure, alteration of
the aromatic ring, and substitutions on the positions of aromatic
ring. On the basis of this strategy, new 3-HBU derivatives were
designed and synthesized.
Here, we describe the synthesis, in vitro evaluation, SAR
analysis, in vivo anticancer efficacy, and the primary mode of
* To whom correspondence should be addressed. Phone: 86-10-
63165290. Fax: 86-10-63017302. E-mail: jiang.jdong@163.com.
†
Chinese Academy of Medical Sciences and Peking Union Medical
College.
‡
These authors made equal contribution to this work.
Mount Sinai School of Medicine.
§
a Abbreviations: HBU, haloacylamino benzoylurea; MT, microtubule;
MDR, multidrug resistance; SAR, structure-activity relationship; DMA,
N,N-dimethylacetamide; DMF, N,N-dimethylformamide; DTT, dithiolth-
reitol; ECL, enhanced chemiluminescence; PBS, phosphate buffered saline.
10.1021/jm070890u CCC: $40.75
2008 American Chemical Society
Published on Web 05/06/2008

Benzoylurea DeriVatiVes as Antimitotic Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3095
Table 1. Antiproliferative Activity of 3-HBUs in CEM Leukemia Cells
Table 2. Antiproliferative Activity of 3-HBUs in CEM Leukemia Cells:
Heterocyclic Ring
a
compd
X
X1
R
R1
formula
IC50 (µM)
a
compd
R1
X
R
method
formula
IC50 (µM)
18a
18b
18c
19a
19b
O
O
O
S
Br CH3 2-CONHCONH2 C9H10BrN3O4
9.44 ( 2.63
4.05 ( 0.33
16.62 ( 4.44
Br
Cl
H
H
2-CONHCONH2 C8H8BrN3O4
2-CONHCONH2 C8H8ClN3O4
1
H
H
H
2-CH3
4-CH3
6-CH3
4-F
6-F
4-Cl
6- Cl
6-Br
4-OCH3
5-COOCH3 Br CH3
Br CH3
H
2-CH3
4-CH3
6-CH3
4-F
6-F
4-Cl
6-Cl
6-Br
4-OCH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
Br CH3
A
A
A
A
A
B
B
B
A
B
B
A
B
A
A
A
A
B
B
B
A
B
B
A
B
A
B
B
A
A
A
A
A
A
B
B
A
B
A
B
A
B
B
B
C11H12BrN3O3
C11H12BrN3O3
C11H12BrN3O3
C12H14BrN3O3
C12H14BrN3O3
C12H14BrN3O3
C11H11BrFN3O3 3.13 ( 0.15
C11H11BrFN3O3 0.52 ( 0.04
C11H11BrClN3O3 9.80 ( 0.68
C11H11BrClN3O3 2.55 ( 0.12
C11H11Br2N3O3 7.63 ( 0.52
C12H14BrN3O4
C13H14BrN3O5
C11H11BrN4O5
C10H10BrN3O3
C11H12BrN3O3
C11H12BrN3O3
C11H12BrN3O3
C10H9BrFN3O3
C10H9BrFN3O3
1.47 ( 0.09
1.47 ( 0.12
1.47 ( 0.16
0.48 ( 0.08
3.36 ( 0.13
>20
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
Br CH3 3-CONHCONH2 C9H10BrSN3O3 3.94 ( 0.18
S
Br 3-CONHCONH2 C8H8BrSN3O3 1.13 ( 0.08
H
a IC50: compound concentration required to inhibit CEM leukemia cell
proliferation by 50% after 72 h of treatment.
the (S)-(-)-enantiomer. Route A produced the compounds of
10a-d, 10h, 10k, 10m, 11a, 11b, 11f, 11i, 11k, 12a-d, 13a,
13b, 13e, 13g. The second one (route B) is the synthetic route
using commercially available derivatives of m-nitrobenzoic acid
for an acylation reaction. In this chemical reaction SOCl2 was
used as an acylating reagent as well as solvent, and pyridine
was used as a catalyst. Then the resultant intermediate directly
reacted with urea to yield key intermediate 9. The nitro
compounds 9 were then reduced to anilines analogues 7 after
treatment in the conventional condition of Fe/HCl.17 Finally,
relevant anilines reacted with chloroacetyl chloride, bromoacetyl
bromide, and 2-bromopropionyl bromide using N,N-dimethy-
lacetamide (DMA) as solvent to yield desired products (10e-g,
10i, 10j, 10l, 11c–e, 11g, 11h, 11j, 11l, 11m, 13c, 13d, 13f,
13h). The iodo compounds (14a-c) were obtained from the
chloro intermediates, through an exchange reaction with NaI.
The synthetic method of heterocyclic ring compounds was
similar to that of route B (Scheme 2). The starting materials
5-nitrothiophene- 3-carboxylic acid or 5-nitro-2-furoic acid (15)
were commercially available. The desired products (18a-c, 19a,
19b) were obtained after four step reactions including acylation,
amidation, reduction, and amination.
>20
2.7 ( 1.50
1.58 ( 0.38
0.725 ( 0.06
1.71 ( 0.032
4.28 ( 0.16
4.76 ( 0.19
2.29 ( 0.09
<0.01
10m 5-NO2
2
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
C10H9BrClN3O3 2.91 ( 0.72
C10H9BrClN3O3 0.54 ( 0.04
C10H9Br2N3O3
C11H12BrN3O4
C12H12BrN3O5
C10H9BrN4O5
C11H12BrN3O4
C10H10BrN3O4
C12H14ClN3O3
C12H14ClN3O3
1.16 ( 0.14
1.88 ( 0.06
1.3 ( 0.20
1.53 ( 0.23
1.57 ( 0.32
>20
5-CO2CH3 Br
5-NO2
6-OCH3
Br
Br
Br
11m 4-OH
12a
12b
12c
12d
13a
13b
13c
13d
13e
13f
13g
13h
3
2-CH3
4-CH3
4-Cl
4-OCH3
2- CH3
4-CH3
4-F
6-F
4-Cl
6-Cl
4-OCH3
4-OH
H
6-CH3
6-F
Cl CH3
Cl CH3
Cl CH3
Cl CH3
>20
>20
C11H11Cl2N3O3 >20
C12H14ClN3O4
C11H12ClN3O3
C11H12ClN3O3
C10H9ClFN3O3
C10H9ClFN3O3
C10H9Cl2N3O3
C10H9Cl2N3O3
C11H12ClN3O4
C10H10ClN3O4
C10H10IN3O3
C11H12IN3O3
C10H9FIN3O3
C12H12IN3O5
>20
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
I
H
H
H
H
H
H
H
H
H
H
H
H
2.70 ( 0.21
3.36 ( 0.11
>20
7.68 ( 0.97
15.9 ( 1.38
2.44 ( 0.11
>20
>20
<0.01
20.0 ( 0.76
<0.01
1.91 ( 0.21
Results and Discussion
In Vitro Antiproliferative Activity and SAR. The antipro-
liferative activity of the 46 compounds in CEM leukemia cells
was closely associated with their structure, as shown in Tables
1 and 2. CEM leukemia cells were used for initial biological
screening because of their known rapid proliferation and high
sensitivity to standard anticancer agents. Out of the 46 com-
pounds, 22 exhibited good activity with IC50 values of <3.0
µM, among which 6 compounds (10i, 10l, 11d, 11f, 13a, 13f)
demonstrated IC50 values of 2.0-3.0 µM, 11 compounds (10a,
10b, 10m, 11a, 11h, 11i, 11j, 11k, 11l, 14c, 19b) of 1.0-2.0
µM, 3 compounds (10c, 10g, 11g) of 0.1-1.0 µM, and 2
compounds (11e, 14b) of <0.01 µM. The last five compounds
(10c, 10g, 11e, 11g, 14b) showed an anticancer activity in CEM
cells equal to or greater than the lead compounds did.
14a
14b
14c
I
I
I
5- CO2CH3
a IC50: drug concentration required to inhibit 50% of cell proliferation
after 72 h of treatment.
action of these new derivatives. Among the 3-HBUs, compound
14b was selected as a representative agent for anticancer and
mechanism investigation.
Chemistry
The 46 new 3-HBU derivatives were grouped as phenyl ring
compounds (Table 1) and heterocyclic ring compounds (Table
2) and synthesized as described in Schemes 1 and 2, respec-
tively. Scheme 1 includes two synthetic methods. The first one
(route A) uses commercially available derivatives of m-
aminobenzoic acid as starting materials, with the method
reported previously.10 In this reaction intermediate 7 reacted
with the (R)- or (S)-2-bromopropionyl chloride to produce the
final enantiomerically pure (R)-1 (10a) or (S)-1 (10b) with a
good yield. The two isomers exhibited identical NMR spectra
with opposite optical rotation, in which the (R)-form exhibiting
dextrorotation was the (R)-(+)-enantiomer and the antipodal was
The lead compound 1 was a racemic mixture; the SAR study
was first focused on the effect of (R)- or (S)-configuration of
the asymmetric center on the anticancer activity of 1 series. In
a comparison of the cytotoxic activities of the isomers (10a,
10b) of 1, we found that introduction of the chiral center
maintained the IC50 values at the 1.47 µM level, hinting that
the configuration of chiral center was not indispensable for the
activities of 1. Therefore, the derivatives with a propionylamino
chain were subsequently examined in the racemate but not the
(R)- or (S)-enantiomer. It was also found that the orders of
anticancer activity in this cell line appeared to be I > Br > Cl,
and iodoacetyl > bromoacetyl > bromopropionyl . chloro-

3096 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Scheme 1. Synthesis of the Phenyl Ring Compounds
Song et al.
Scheme 2. Synthesis of the Heterocyclic Ring Compounds
presence of the reducing agent DTT (containing two -SH
groups per molecule).11b Therefore, we believe that the -SH
group displaces the halide ion and then results in the formation
of a covalent bond between the ꢀ-tubulin and these types of
agents. This is consistent with the previous reports.19a,b,20
Next, the aromatic ring of 3-HBUs was modified with
substituents that varied in their size, electronic character, and
position. The obtained IC50 values showed that the position of
the substituent on the phenyl ring greatly affected the anticancer
activity. Compounds methylated at the 6-position (e.g., 10e, 14a)
were inactive. Compounds methylated at the 4-position were
tolerated (10d, 11b), and those at the 2-position were optimal
(10c). The antitumor activities of methylated compounds
decreased in the order of 2-CH3 > 4-CH3 > 6-CH3. In addition,
compounds 11i and 11l bearing OCH3 substituent at the 4- and
6-position, respectively, had a similar activity (i.e., 4-OCH3 ∼
6-OCH3), which was, however, lower than that of their lead
compound. In contrast, compounds bearing a halogen substituent
at the 6-position showed an enhanced activity compared to the
parent compounds. The introduction of an atom of fluorine at
the 6-position afforded the most potent derivatives of 11e and
14b with IC50 less than 0.01 µM for both. However, moving
the fluorine from the 6- to 4-position (11d) or replacing the
acetyl > chloropropionyl, consistent with the previous observa-
tions.10 The alkyl halides of the agents are strongly electro-
philic18 and are reactive groups that interact with nucleophiles,
the sulfhydryl (-SH) group present on the ꢀ-tubulin. We have
shown before that the inhibition of microtubule assembly by
such types of compounds could be significantly reduced in the

Benzoylurea DeriVatiVes as Antimitotic Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3097
Figure 2. SAR of 3-HBUs.
Table 3. Antiproliferative Activities of 11e and 14b in Human Tumor
Cell Lines
fluorine with chlorine (11g) or bromine (11h) decreased the
anticancer activity. The anticancer activities of the halogenated
compounds decreased in the order of 6-F > 4-F, 6-Cl > 4-Cl
and 6-F > 6-Cl > 6-Br. From these results, we deduce that
substitution at the 6-position of the phenyl ring plays an
important role in the anticancer activity; a small substituent like
an atom of fluorine, an isosteric to the hydrogen atom, seemed
to have the highest activity among the substituents of the
aromatic ring moiety. Furthermore, compounds 10l, 10m, 11j,
and 11k bearing electron-withdrawing groups at the 5-position
(NO2, CO2CH3) had lower activity than that of the corresponding
parent compounds. The introduction of a hydroxyl group at the
4-position of compounds 11m and 13h completely abolished
the activity. Therefore, with the exception of fluorine the
substitution on the aromatic ring with electron-donating and
electron-withdrawing groups resulted in partial or complete loss
of the activity regardless of the position.
Furthermore, the modifications of the phenyl ring moiety were
explored, as shown in Table 2. In the evaluation of the activities
of heterocyclic ring compounds, we found that the thiophene
derivatives (19a, 19b) are anticancer agents with IC50 values
ranging from 1.13 to 3.94 µM in CEM cells. In contrast,
introduction of a furan ring, as that for compounds 18a-c,
resulted in a loss of cytotoxicity in cancer cells with elevated
IC50 values between 4.05 and 16.62 µM. Moreover, as previ-
ously noticed, compounds 18a-c in which the 4-position was
substituted were inactive. These data demonstrated that ana-
logues replaced by thiophene ring, a bioisosteric group to the
phenyl ring, possess a considerable potency although not as high
as that of the phenyl ring derivatives.
Thus, we have identified the essential structural and functional
requirements of 3-HBUs for the anticancer activity and sum-
marized the results in Figure 2. On the basis of the SAR analysis,
replacement of hydrogen with fluorine at the 6-position could
enhance the potency of the compounds; in contrast, the activity
was lost completely when the substituent was methyl group.
Compounds 11e and 14b bearing fluorine substituent at the
6-position showed the highest antitumor activity, and therefore,
both of them were selected for further evaluation.
In Vitro Anticancer Activity of 11e and 14b in Human
Cancer Cell Lines. The antiproliferative activity of compounds
11e and 14b was examined in nine human tumor cell lines
(Table 3). The IC50 values of 11e were in the range of 0.08-0.3
µM for solid tumor lines and less than 0.01 and 0.015 µM for
leukemia or lymphoma cells, respectively. Among the solid
tumors, the least sensitive cell line was DU-145 prostate cancer
(IC50 ) 0.30 µM), and the most sensitive cell line was MCF-7
breast cancer cells (IC50 ) 0.08 µM). Compound 11e showed
an enhanced activity compared to the lead compound 2. The
IC50 values of 14b were in the range 0.01-0.22 µM for solid
tumor lines and less than 0.01 µM for leukemia or lymphoma
cell lines. The least sensitive cell line was the DU-145 cells
(IC50 ) 0.22 µM), and the most sensitive cell lines was MCF-7
(IC50 < 0.01 µM), similar to that of 11e.
a
IC50 (µM)
cell line
humantumor
11e
<0.01
14b
CEM
Daudi
T-cell leukemia
B-cell lymphoma
breast cancer
hepatoma
prostate cancer
prostate cancer
melanoma
<0.01
0.015 ( 0.0058
0.08 ( 0.01
0.16 ( 0.01
0.30 ( 0.05
0.19 ( 0.023
0.11 ( 0.021
0.25 ( 0.04
0.19 ( 0.038
<0.01
MCF-7
Bel-7402
DU-145
PC-3
DND-1A
LOVO
MIA
<0.01
0.16 ( 0.02
0.22 ( 0.05
0.19 ( 0.028
0.02 ( 0.005
0.19 ( 0.03
0.054 ( 0.013
colon cancer
pancreas cancer
a IC50: drug concentration required to inhibit human tumor cells
proliferation by 50% after 72 h of treatment.
Since fluorine has a size and electronic properties similar to
those of hydrogen, it is often introduced as an isosteric to the
hydrogen atom. 11e and 14b bears fluorine substituent at the
6-position and might have anticancer bioavailability in vivo
superior to that of 2 and 3. Therefore, 11e and 14b entered into
animal experiments using human tumor-bearing nude mice.
Inhibition of Human Hepatocarcinoma by 11e and 14b
in Nude Mice. The anticancer efficacy of 11e and 14b was
shown in Figure 3. The drugs were given to the tumor-bearing
nude mice when the hepatoma (Bel-7402) tumor size on their
back was between 160 and 200 mm3. When the tumor volume
in the solvent-treated mice (control group) grew to 4300 mm3
on day 25 post-treatment, it was 2700 mm3 in the mice treated
with 5 mg/kg of 11e intraperitoneously (ip) every other day
(q2d), and 2050 mm3 in the group that received 10 mg/kg of
11e (ip, q2d) (Figure 3A). The tumor growth inhibition rate
was 37% and 52%, respectively. The average body weight of
the mice in the 11e treatment group (10 mg/kg) was 18.7 (
0.9 g before and 18.6 ( 0.9 g after the treatment.
As demonstrated in Figure 3B, when the average tumor
volume of untreated controls grew to 3000 mm3 on day 28 post-
treatment, the tumor volume of the mice treated with 14b at 5
mg/kg (ip, q2d) was 1150 mm3, showing 62% growth inhibition
in tumor volume. 14b at 10 mg/kg (ip, q2d) controlled the tumor
volume within 420 mm3, indicating an 86% inhibition of tumor
growth. Toxic effects of this regimen were not observed during
the treatment. The average body weight of the mice treated with
14b (10 mg/kg group) was 19.6 ( 0.7 g before and 21.6 (
0.9 g after the therapy. With respect to the parent compound
3,14b at equal molar concentration showed a higher tumor
inhibitory effect in vivo (57% inhibition by 3 vs 86% by 14b).
Since compound 14b contains a fluorine substituent at the
6-position, it could be at least part of the explanation that 14b
possesses a longer half-time in vivo with respect to their parent
compounds. In addition, the LD50 of 14b (ip) in the nude mice
was 38.8 mg/kg, higher than that of compound 3 (LD50, 31 mg/
kg).10
Mode of Action of 14b. We then turned our attention to the
mechanism of 14b to learn whether the chemical modification

3098 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Song et al.
Figure 3. Anticancer efficacy of 11e (A) and14b (B) in human hepatocarcinoma (Bel-7402) in nude mice. The nude mice were sc implanted with
Bel-7402 cells, and the treatments were initiated when the average tumor volume was about 200 mm3 (see Experimental Section). Presented in each
time point is the mean of tumor volume (n ) 5).
Figure 5. Metaphase block by 14b. CEM cells were treated with 14b
at 0.014 µM for 24 h. Cells were then harvested on slides for
morphology observation: (A) untreated; (B) 14b treated (Giemsa,
X400).
mitotic arrest, i.e., the disappearance of nuclear membrane and
disorientation and loose dispersion of metaphase chromosomes
in the cytoplasm (Figure 5), indicating that the cell cycle arrest
by 14b occurred at M but not G2 phase. Bcl-2 expression was
also examined because it is a cellular factor to inhibit apoptosis.
As shown in Figure 6, bcl-2 protein was inactivated by
phosphorylation in the cells treated with 14b. These results
suggest that compound 14b can arrest the tumor cell cycle at
Figure 4. Cell cycle arrest at the M-phase by 14b. CEM cells were
incubated without (control) or with 14b (0.22 µM) for 4, 12, or 24 h.
Cells were then analyzed for their cell cycle distribution using flow
the M-phase and induce apoptotic cell death by promoting bcl-2
phosphorylation.
Next, we accessed the direct effect of 14b on microtubule
assembly in a cell-free system using purified tubulin. 14b
significantly inhibited the process of microtubule assembly
(Figure 7) but had no effect on the disassembly process (not
shown). The result was consistent with the activity of the lead
compounds 3, 2, and 1. A significant inhibition of microtubule
assembly was observed even when the concentration of 14b
was as low as 1 µM. The IC50 value of 14b in the assay was
0.71 µM. Well-known tubulin-active drugs were used as
references in the experiment system. An inhibitory effect on
microtubule assembly was detected in vincristine but not in
cytometry. A significant M-phase accumulation appeared between 12
and 24 h post-treatment.
retains the mode of action of its parent compound. Flow
cytometric analysis of the DNA profile in the CEM cells (Figure
4) showed that 14b treatment (0.22 µM) produced a major shift
of the cell population from G0/G1 to G2/M, revealing a
significant accumulation of cells in the G2/M-phase. CEM cells
treated with 11e exhibited a similar cell cycle distribution (data
not shown). To further define the cell cycle arrest, morphology
examination was done. CEM leukemic cells treated with 14b
(0.014 µM) for 24 h displayed the characteristic features of

Benzoylurea DeriVatiVes as Antimitotic Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3099
Figure 6. Bcl-2 phosphorylation by 14b. CEM cells were treated with 14b at different concentrations (0.014-0.27 µM) for 12 h (A) and 24 h (B).
Cells were harvested, and the cellular proteins were extracted for Western blot analysis, Bcl-2-p, phosphorylated bcl-2.
Figure 7. Effect of 14b on the microtubule assembly process. Free ꢀ-tubulin in reaction buffer was incubated with GTP and Mg2+ at room
temperature for the polymerization in the absence (control) or presence of 14b (1, 5, 25, 50, or 100 µM) or paclitaxel (20 µM) or vinblastine (20
µM). Microtubule assembly was determined every 1.5 min by OD at 340 nm. Each point represents the mean of two independent experiments.
paclitaxel, suggesting that the mode of action of 14b is similar
to that of vincristine, as well as colchicine.
prevents the formation of mitotic spindles, suspends the cell
cycle at M-phase, and eventually leads the tumor cells to
apoptosis by promoting bcl-2 phosphorylation. Monotherapy
with compound 14b showed a strong anticancer effect against
human hepatocarcinoma in nude mice with over 86% tumor
growth inhibition. The activity was higher than that of the parent
compound 3. Taken together, the 3-HBU derivatives constitute
a class of the anticancer tubulin ligands, and particularly,
compound 14b merits further investigation.
As M-phase arrest is a phenotype of tubulin disruption, it
was examined to learn the relationship between chemical
structure and anti-tubulin activity. As shown in Figure 8,
compounds 14b and 11e with 6-F substitution caused M-phase
arrest by 37.6% and 31.3%, respectively, after 12 h of treatment;
however, replacing the fluorine with chlorine (11g) or bromine
(11h) or moving the fluorine from the 6- to 4-position (11d)
greatly decreased M-phase accumulation. The results are
consistent with the results from the IC50 assay.
Experimental Section
Chemistry. Melting points (mp) were obtained with YRT-3
melting point apparatus and are uncorrected. 1H nuclear magnetic
resonance spectra were performed on a Varian Inova 400 MHz
spectrometer (Varian, San Francisco, CA) in either DMSO-d6 or
CD3OD (as internal standard on a δ scale). Infrared (IR) spectra
were recorded on a Nicolet IMPACT-400 spectrometer (Thermo
Electron Corporation, Madison WI). FAB and EI mass spectra were
recorded on an Autospec UItima-TOF mass spectrometer (Micro-
mass UK Ltd., Manchester, U.K.). For the sake of consistency, the
positions of the substituents on the phenyl ring of the study
compounds are designated according to the orientation presented
in Figure 2.
Method A (for 10a-d, 10h, 10k, 10m, 11a, 11b, 11f, 11i,
11k, 12a-d, 13a, 13b, 13e, 13g). The parent intermediates (5-7)
were synthesized according to the procedure previously described.10
2-Methyl-3-(2′-chloropropionamido)benzoylurea (12a). To a
stirred solution of the 2-methyl-3-aminobenzoylurea (386 mg, 2.0
mmol) in 5 mL of DMA, 2-chloropropionyl chloride (1.0 mL, 10.2
mmol) was added dropwise. The reaction mixture was stirred at
Conclusions
In continuation of our study on 3-HBU derivatives, we have
synthesized 46 new analogues defined through variations of the
three regions of the core structure. The SAR information
revealed that (i) the configuration of asymmetric center present
in 1 has no influence the antiproliferative activity and the
configuration of 1 does not appear to be essential, (ii) the
anticancer activity of the HBU largely depends on how active
the halide ion is as a leaving group and therefore is ranked in
the order of -CH2I > -CH2Br > (()-CHBrCH3 ) (R)-
CHBrCH3 ) (S)-CHBrCH3 . -CH2Cl > -CHClCH3, (iii) a
phenyl ring system is essential for the anticancer activity and
replacing phenyl ring with thiophene or furan reduced the
activity, and (iv) a substitution at the 6-position of the phenyl
ring with an atom of fluorine enhances the anticancer activity.
Mechanism studies show that compound 14b interferes with
the microtubule dynamics selectively at the assembly phase,

3100 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Song et al.
2.4, 8.4 Hz, 1H), 8.22 (d, J ) 2.4 Hz, 1H). IR (KBr): γ 3390,
3310, 3232 (NH), 1716, 1680, 1664 (CdO) cm-1. Anal.
(C11H11BrClN3O3) C, H, N.
4-Methoxy-3-(2′-bromopropionamido)benzoylurea (10k). The
title compound was obtained from 4-methoxy-3-aminobenzoylurea
and 2-bromopropionyl bromide using a procedure similar to that
of compound 12a. Yield: 54%. White solid, mp 222-224 °C. MS
m/z 344 (M + 1). 1H NMR (CD3OD): δ 1.79 (d, J ) 6.0 Hz, 3H),
3.93 (s, 3H), 4.79 (q, J ) 6.0 Hz, 1H), 7.10 (d, J ) 8.7 Hz, 1H),
7.71 (dd, J ) 1.8, 8.7 Hz, 1H), 8.48 (d, J ) 1.8 Hz, 1H). IR (KBr):
γ
3377, 3215 (NH), 1709, 1674 (CdO) cm-1
. Anal.
(C12H14BrN3O4) C, H, N.
5-Nitro-3-(2′-bromopropionamido)benzoylurea (10m). The
title compound was obtained from 5-nitro-3-aminobenzoylurea and
2-bromopropionyl bromide using a procedure similar to that of
compound 12a. Yield: 43%. White solid, mp 190-193 °C. MS
Figure 8. Relationship between chemical structure and anti-tubulin
activity of the study compounds. CEM cells were treated with the
compounds at their IC50 for 12 h. The cells were then collected for
cell cycle analysis as described in Biological Methods.
1
m/z 359 (M + 1). H NMR (DMSO-d6): δ 1.78 (d, J ) 6.8 Hz,
3H), 4.70 (q, J ) 6.8 Hz, 1H), 7.48, 7.89 (s, s, 2H), 8.44 (s, 1H),
8.53 (s, 1H), 8.77 (s, 1H), 10.97 (s, 1H), 10.99 (s, 1H). IR (KBr):
γ 3471, 3354, 3327 (NH), 1768, 1707, 1684 (CdO) cm-1. Anal.
(C11H11BrN4O5) C, H, N.
2-Methyl-3-(bromoacetamido)benzoylurea (11a). The title
compound was obtained from 2-methyl-3-aminobenzoylurea and
bromoacetyl bromide using a procedure similar to that for com-
pound 12a. Yield: 52%. White solid, mp 242-244 °C. MS m/z
313 (M+). 1H NMR (CD3OD): δ 2.25 (s, 3H), 4.00 (s, 2H),
7.26-7.30 (m, 2H), 7.40-7.44 (m, 1H). IR (KBr): γ 3411, 3309
(NH), 1697, 1682, 1662 (CdO) cm-1. Anal. (C11H12BrN3O3) C,
H, N.
room temperature for 2 h and poured into ice-water and extracted
with ethyl acetate. The organic layer was washed with saturated
brine, dried over Na2SO4, and evaporated. The crude product was
purified by flash chromatography over silica gel, affording the title
compound (256 mg, 60%) as a white solid, mp 244-246 °C. MS
1
m/z 283 (M+). H NMR (CD3OD): δ 1.68 (d, J ) 6.6 Hz, 3H),
2.23 (s, 3H), 4.63 (q, J ) 6.6 Hz, 1H), 7.23-7.30 (m, 2H),
7.37-7.40 (m, 1H). IR (KBr): γ 3383, 3311, 3242 (NH), 1712,
1664 (CdO) cm-1. Anal. (C12H14ClN3O3) C, H, N.
(R)-(+)-3-(2′-Bromopropionamido)benzoylurea (10a). The title
compound was obtained from 3-aminobenzoylurea and (R)-(+)-2-
bromopropionyl chloride with a procedure similar to that for
compound 12a. Yield: 62%. White solid, mp 221-223 °C. [R]D
4-Methyl-3-(bromoacetamido)benzoylurea (11b). The title
compound was obtained from 4-methyl-3-aminobenzoylurea and
bromoacetyl bromide using a procedure similar to that for com-
pound 12a. Yield: 54%. White solid, mp 220-222 °C. MS m/z
1
+20.87 (c 0.2, DMF); MS m/z 314 (M + H). H NMR (DMSO-
d6): δ 1.75 (d, J ) 6.8 Hz, 3H), 4.69 (q, J ) 6.8 Hz, 1H), 7.44 (t,
1H), 7.67 (d, J ) 7.6 Hz, 1H), 7.80 (d, J ) 8.4 Hz, 1H), 7.38, 7.98
(s, s, 2H), 8.13 (s, 1H), 10.50 (s, 1H), 10.54 (s, 1H). IR (KBr): γ
3383, 3360, 3280 (NH), 1720, 1666 (CdO) cm-1. Anal.
(C11H12BrN3O3) C, H, N.
1
313 (M+). H NMR (CD3OD): δ 2.29 (s, 3H), 4.01 (s, 2H), 7.35
(d, J ) 7.5 Hz, 1H), 7.66 (dd, J ) 1.5, 7.5 Hz, 1H), 7.85 (d, J )
1.5 Hz, 1H). IR (KBr): γ 3367, 3263 (NH), 1732, 1680, 1664
(CdO) cm-1. Anal. (C11H12BrN3O3) C, H, N.
4-Chloro-3-(bromoacetamido)benzoylurea (11f). The title
compound was obtained from 4-chloro-3-aminobenzoylurea and
bromoacetyl bromide using a procedure similar to that for com-
pound 12a. Yield: 59%. White solid, mp 230-232 °C. MS m/z
(S)-(-)3-(2′-Bromopropionamido)benzoylurea (10b). The title
compound was obtained from 3-aminobenzoylurea and (S)-(-)-2-
bromopropionyl chloride, using a procedure similar to that for
compound 12a. Yield: 62%. White solid, mp 221-223 °C. [R]D
1
333.5 (M+). H NMR (CD3OD): δ 4.07 (s, 2H), 7.57 (d, J ) 8.4
1
-22.46 (c 0.2, DMF). MS m/z 314 (M + H). H NMR (DMSO-
Hz, 1H), 7.66-7.70 (m, 1H), 8.27 (d, J ) 2.1 Hz, 1H). IR (KBr):
γ 3369, 3346, 3319 (NH), 1705, 1676 (CdO) cm -1. Anal.
(C10H9BrClN3O3) C, H, N.
d6): δ 1.75 (d, J ) 6.8 Hz, 3H), 4.69 (q, J ) 6.8 Hz, 1H), 7.44 (m,
1H), 7.67 (d, J ) 7.6 Hz, 1H), 7.80 (d, J ) 8.4 Hz, 1H), 7.38, 7.98
(s, s, 2H), 8.13 (s, 1H), 10.50 (s, 1H), 10.54 (s, 1H). IR (KBr): γ
3384, 3370, 3287 (NH), 1712, 1674 (CdO) cm-1. Anal.
(C11H12BrN3O3) C, H, N.
4-Methoxy-3-(bromoacetamido)benzoylurea (11i). The title
compound was obtained from 4-methoxy-3-aminobenzoylurea and
bromoacetyl bromide using a procedure similar to that for com-
pound 12a. Yield: 46%. White solid, mp 224-226 °C. MS m/z
2-Methyl-3-(2′-bromopropionamido)benzoylurea (10c). The
title compound was obtained from 2-methyl-3-aminobenzoylurea
and 2-bromopropionyl bromide using a procedure similar to that
for compound 12a. Yield: 60%. White solid, mp 240-242 °C. MS
1
330 (M + 1). H NMR (CD3OD): δ 3.94 (s, 3H), 4.07 (s, 2H),
7.10 (d, J ) 8.4 Hz, 1H), 7.70 (dd, J ) 2.1, 8.4 Hz, 1H), 8.50 (d,
1
m/z 327 (M+). H NMR (CD3OD): δ 1.80 (d, J ) 6.6 Hz, 3H),
J ) 2.1 Hz, 1H). IR (KBr): γ 3383, 3228 (NH), 1745, 1684, 1670
(CdO) cm -1. Anal. (C11H12BrN3O ) C, H, N.
4
2.24 (s, 3H), 4.67 (q, J ) 6.6 Hz, 1H), 7.24-7.31 (m, 2H),
7.38-7.41 (m, 1H). IR (KBr): γ 3383, 3311, 3242 (NH), 1712,
1664, 1626 (CdO) cm-1. Anal. (C12H14BrN3O3) C, H, N.
4-Methyl-3-(2′-bromopropionamido)benzoylurea (10d). The
title compound was obtained from 4-methyl-3-aminobenzoylurea
and 2-bromopropionyl bromide using a procedure similar to that
of compound 12a. Yield: 56%. White solid, mp 217-218 °C. MS
m/z 327 (M+). 1H NMR (DMSO-d6): δ 1.75 (d, J ) 6.8 Hz, 3H),
2.18 (s, 3H, CH3), 4.80 (q, J ) 6.8 Hz, 1H,), 7.23-7.28 (m, 2H),
7.40-7.43 (m, 1H), 7.37, 7.86 (s, s, 2H), 9.82 (s, 1H), 10.57 (s,
1H). IR (KBr): γ 3386, 3278 (NH), 1766, 1687 (CdO) cm-1. Anal.
(C12H14BrN3O3) C, H, N.
5-Nitro-3-(bromoacetamido)benzoylurea (11k). The title com-
pound was obtained from 5-nitro-3-aminobenzoylurea and bro-
moacetyl bromide using a procedure similar to that for compound
12a. Yield: 42%. White solid, mp 178-182 °C. MS m/z 345 (M
1
+ 1). H NMR (DMSO-d6): δ 4.10 (s, 2H), 7.48, 7.89 (s, s, 2H),
8.41 (s, 1H), 8.53 (s, 1H), 8.75 (s, 1H), 10.97 (s, 1H), 11.04 (s,
1H). IR (KBr): γ 3491, 3352, 3325 (NH), 1716, 1670, 1624 (CdO)
cm -1. Anal. (C10H9BrN4O ) C, H, N.
5
4-Methyl-3-(2′-chloropropionamido)benzoylurea (12b). The
title compound was obtained from 4-methyl-3-aminobenzoylurea
and 2-chloropropionyl chloride using a procedure similar to that
for compound 12a. Yield: 52%. White solid, mp 232-234 °C. MS
m/z 284 (M + 1). 1H NMR (CD3OD): δ 1.68 (d, J ) 6.6 Hz, 3H),
2.23 (s, 3H), 4.63 (q, J ) 6.6 Hz, 1H), 7.26 (d, J ) 7.8 Hz, 2H),
7.38 (dd, J ) 2.1, 7.8 Hz, 1H), 7.83 (d, J ) 2.1 Hz, 1H). IR (KBr):
γ 3458, 3278, 2983 (NH), 1705, 1670, 1665 (CdO) cm-1. Anal.
(C12H14ClN3O3) C, H, N.
4-Chloro-3-(2′-bromopropionamido)benzoylurea (10h). The
title compound was obtained from 4-chloro-3-aminobenzoylurea
and 2-bromopropionyl bromide using a procedure similar to that
of compound 12a. Yield: 47%. White solid, mp 228-230 °C. MS
m/z 348 (M + 1). 1H NMR (CD3OD): δ 1.81 (d, J ) 6.6 Hz, 3H),
4.78 (q, J ) 6.6 Hz, 1H), 7.56 (d, J ) 8.4 Hz, 1H), 7.64 (dd, J )

Benzoylurea DeriVatiVes as Antimitotic Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3101
4-Chloro-3-(2′-chloropropionamido)benzoylurea (12c). The
title compound was obtained from 4-chloro-3-aminobenzoylurea
and 2-chloropropionyl chloride using a procedure similar to that
for compound 12a. Yield: 52%. White solid, mp 230-231 °C. MS
m/z 304 (M + 1). 1H NMR (CD3OD): δ 1.69 (d, J ) 6.6 Hz, 3H),
4.73 (q, J ) 6.6 Hz, 1H), 7.58 (d, J ) 8.4 Hz, 1H), 7.70 (dd, J )
2.1, 8.4 Hz, 1H), 8.26 (d, J ) 2.1 Hz, 1H). IR (KBr): γ 3375,
3350, 3236 (NH), 1710, 1690, 1666 (CdO) cm -1. Anal.
(C11H11Cl2N3O3) C, H, N.
6-Methyl-3-(2′-bromopropionamido)benzoylurea (10e). Yield:
38%. White solid, mp 210-212 °C. MS m/z 328 (M + 1). 1H NMR
(DMSO-d6 ): δ 1.74 (d, J ) 6.8 Hz, 3H), 2.28 (s, 3H), 4.68 (q, J )
6.8 Hz, 1H), 7.22 (d, J ) 8.4 Hz, 1H), 7.36, 7.84 (s, s, 2H), 7.57 (dd,
J ) 2.0, 8.4 Hz, 1H), 7.68 (d, J ) 2.0 Hz, 1H), 10.58 (s, 1H), 10.69
(s, 1H). IR (KBr): γ 3375, 3325, 3219 (NH), 1703, 1682, 1666 (CdO)
cm -1. Anal. (C12H14BrN3O3) C, H, N.
4-Fluoro-3-(2′-bromopropionamido)benzoylurea (10f). Yield:
1
42%. White solid, mp 226-228 °C. MS m/z 331 (M+). H NMR
4-Methoxy-3-(2′-chloropropionamido)benzoylurea (12d). The
title compound was obtained from 4-methoxy-3-aminobenzoylurea
and 2-chloropropionyl chloride using a procedure similar to that
for compound 12a. Yield: 54%. White solid, mp 226-227 °C. MS
m/z 300 (M + 1). 1H NMR (CD3OD): δ 1.66 (d, J ) 6.6 Hz, 3H),
3.95 (s, 3H), 4.75 (q, J ) 6.6 Hz, 1H), 7.11 (d, J ) 8.7 Hz, 1H),
7.72 (dd, J ) 2.4, 8.7 Hz, 1H), 8.50 (d, J ) 2.4 Hz, 1H). IR (KBr):
γ 3377, 3215 (NH), 1709, 1674 (CdO) cm -1. Anal. (C12H14ClN3O
4) C, H, N.
(DMSO-d6 ): δ 1.75 (d, J ) 6.4 Hz, 3H), 4.90 (q, J ) 6.4 Hz, 1H),
7.40-7.42 (m, 1H), 7.38, 7.98 (s, s, 2H), 7.82 (m, 1H), 8.49 (m, 1H),
10.28 (s, 1H), 10.61 (s, 1H). IR (KBr): γ 3392, 3359, 3282 (NH),
1718, 1689, 1664 (CdO) cm -1. Anal. (C11H11BrFN3O3) C, H, N.
6-Fluoro-3-(2′-bromopropionamido)benzoylurea (10g). Yield:
43%. White solid, mp 167-168 °C. MS m/z 332 (M + 1). 1H NMR
(DMSO-d6 ): δ 1.74 (d, J ) 6.4 Hz, 3H), 4.67 (q, J ) 6.4 Hz, 1H),
7.30 (t, 1H), 7.44 (s, 1H), 7.71-7.75 (m, 2H), 7.88 (dd, J ) 2.4, 2.8
Hz, 1H), 10.50 (s, 1H), 10.52 (s, 1H). IR (KBr): γ 3375, 3228, 3084
(NH), 1697, 1674, 1628 (CdO) cm -1. Anal. (C11H11BrFN3O3) C,
H, N.
2-Methyl-3-(chloroacetamido)benzoylurea (13a). The title
compound was obtained from 2-methyl-5-aminobenzoylurea and
chloroacetyl chloride using a procedure similar to that for compound
12a. Yield: 55%. Mp 246-248. MS m/z 269.5 (M+). 1H NMR
(CD3OD): δ 2.26 (s, 3H), 4.21 (s, 2H), 7.26-7.31 (m, 2H),
7.42-7.45 (m, 1H). IR (KBr): γ 3386, 3303, 3250 (NH), 1700,
1675, 1670 (CdO) cm -1. Anal. (C11H12ClN3O3) C, H, N.
4-Methyl-3-(chloroacetamido)benzoylurea (13b). The title
compound was obtained from 4-methyl-3-aminobenzoylurea and
chloroacetyl chloride using a procedure similar to that for compound
12a. Yield: 55%. White solid, mp 222-223 °C. MS m/z 270 (M
6-Chloro-3-(2′-bromopropionamido)benzoylurea (10i). Yield:
42%. White solid, mp 202-204 °C. MS m/z 348 (M + 1). 1H NMR
(DMSO-d6 ): δ 1.75 (d, J ) 6.8 Hz, 3H), 4.68 (q, J ) 6.8 Hz, 1H),
7.44, 7.70 (s, s, 2H), 7.47 (d, J) 2.4 Hz, 1H), 7.66 (dd, J ) 2.4, 8.8
Hz, 1H), 7.78 (d, J ) 8.8 Hz, 1H), 10.59 (s, 1H), 10.70 (s, 1H). IR
-1
(KBr): γ 3373, 3309, 3230, 3134 (NH), 1709, 1685 (CdO) cm
Anal. (C11H11BrClN3O3) C, H, N.
.
6-Bromo-3-(2′-bromopropionamido)benzoylurea (10j). Yield:
1
36%. White solid, mp 185.5-187 °C. MS m/z 414 (M + Na). H
1
+ 1). H NMR (CD3OD): δ 2.29 (s, 3H), 4.30 (s, 2H), 7.34 (d, J
NMR (DMSO-d6 ): δ 1.74 (d, J ) 6.8 Hz, 3H), 4.67 (q, J ) 6.8 Hz,
1H), 7.43, 7.69 (s, s, 2H), 7.57-7.62 (m, 2H), 7.74 (d, J) 2.4 Hz,
1H), 10.58 (s, 1H), 10.69 (s, 1H). IR (KBr): γ 3375, 3309 (NH), 1709,
1687 (CdO) cm -1. Anal. (C11H11Br2N3O3) C, H, N.
) 8.1 Hz, 1H), 7.66 (dd, J ) 1.8, 8.1 Hz, 1H), 7.87 (d, J ) 1.8
Hz, 1H). IR (KBr): γ 3361, 3317 (NH), 1730, 1705, 1676 (CdO)
cm -1. Anal. (C11H12ClN3O3) C, H, N.
4-Chloro-3-(chloroacetamido)benzoylurea (13e). The title
compound was obtained from 4-chloro-3-aminobenzoylurea and
chloroacetyl chloride using a procedure similar to that for compound
12a. Yield: 55%. White solid, mp 232-234 °C. MS m/z 290 (M
+ 1). 1H NMR (CD3OD): δ 4.28 (s, 2H), 7.58 (m, 1H), 7.66-7.77
(m, 1H), 8.32 (d, J ) 1.8 Hz, 1H). IR (KBr): γ 3367, 3317, 3207
(NH), 1701, 1676 (CdO) cm -1. Anal. (C10H9Cl2N3O3) C, H, N.
4-Methoxy-3-(chloroacetamido)benzoylurea (13g). The title
compound was obtained from 4-methoxy-3-aminobenzoylurea and
chloroacetyl chloride using a procedure similar to that for compound
12a. Yield: 50%. White solid, mp 230-231 °C. MS m/z 285 (M+).
1H NMR (CD3OD): δ 3.81 (s, 3H), 4.29 (s, 2H), 7.35 (d, J ) 8.1
Hz, 1H), 7.65-7.68 (m, 1H), 7.81 (d, J ) 1.8 Hz, 1H). IR (KBr):
γ 3458, 3388, 3271 (NH), 1705, 1670, 1650 (CdO) cm -1. Anal.
(C11H12ClN3O4) C, H, N.
Method B (for 10e-g, 10i, 10j, 10l, 11c-e, 11g, 11h, 11j,
11l, 11m, 13c, 13d, 13f, 13h, 14a-c, 18a-c, 19a, 19b). A solution
of relevant m-nitrobenzoic acid (8, 15, 0.01 mol, commercially
available) in SOCl2 (10 mL) was heated with reflux for 1.5 h. The
reaction mixture was concentrated under vacuum to give an oil.
To a stirred solution of the oil in toluene (50 mL) was added urea
(0.05 mol). The reaction mixture was stirred at 90-100 °C for 2 h
and concentrated under vacuum. The residue was stirred in water
(60 mL) at room temperature for 0.5 h. The reaction mixture was
filtered, and the solid was washed in water and dried to give the
nitro compound (9, 16),which was used for the next step without
purification. The nitro compound (2.2 mmol) was dissolved in a
mixture of 95% ethanol (20 mL) and 4 N HCl (0.5 mL), and to the
solution was added iron powder (6.6 mmol) portionwise over 20
min at 70-80 °C. After continued stirring at the same temperature
for 4 h, the reaction mixture was filtered immediately, and the filtrate
was concentrated under reduced pressure, soaked in ether, filtered,
and dried to give solid 7, 17, which was used for the next step
without purification.
5-Methyl Formate 3-(2′-Bromopropionamido)benzoylurea
(10l). Yield: 52%. White solid, mp 193-195 °C. MS m/z 372 (M +
1). 1H NMR (DMSO-d6 ): δ 1.76 (d, J ) 6.8 Hz, 3H), 3.88 (s, 3H),
4.68 (q, J ) 6.8 Hz, 1H), 7.41, 7.91 (s, s, 2H), 8.21 (s, 1H), 8.33 (s,
1H), 8.44 (s, 1H), 10.73 (s, 1H), 10.82 (s, 1H). IR (KBr): γ 3464,
3259 (NH), 1768, 1703 (CdO) cm -1. Anal. (C13H14BrN3O5) C, H,
N.
6-Methyl-3-(bromoacetamido)benzoylurea (11c). Yield: 41%.
1
White solid, mp 226.5-227.5 °C. MS m/z 314 (M + 1). H NMR
(DMSO-d6): δ 2.29 (s, 3H), 4.02 (s, 2H), 7.22 (d, J ) 8.4 Hz, 1H),
7.36, 7.84 (s, s, 2H), 7.55 (dd, J ) 2.4 Hz, 1H), 7.65 (d, J ) 2.4 Hz,
1H), 10.45 (s, 1H), 10.51 (s, 1H). IR (KBr): γ 3379, 3303, 3222 (NH),
1703, 1685, 1662 (CdO) cm -1. Anal. (C11H12BrN3O3) C, H, N.
4-Fluoro-3-(bromoacetamido)benzoylurea (11d). Yield: 47%.
White solid, mp 256-258 °C. MS m/z 317 (M+). 1H NMR (DMSO-
d6 ): δ 4.16 (s, 2H), 7.38-7.43 (m, 2H), 7.43, 7.80 (s, s, 2H), 8.51 (d,
J ) 6.0 Hz, 1H), 10.35 (s, 1H), 10.62 (s, 1H). IR (KBr): γ 3363,
3315, 3205, 3032 (NH), 1705, 1682 (CdO) cm -1. Anal.
(C10H9BrFN3O3) C, H, N.
6-Fluoro-3-(bromoacetamido)benzoylurea (11e). Yield: 38%.
White solid. mp 214-215 °C. MS m/z 318 (M + 1). 1HNMR (DMSO-
d6 ): δ 4.03 (s, 2H), 7.29 (t, 1H), 7.44 (s, 1H), 7.68-7.72 (m, 2H),
7.85 (dd, J ) 2.8 Hz, 1H), 10.50 (s, 1H), 10.58 (s, 1H). IR (KBr): γ
3352, 3290, 3222 (NH), 1711, 1685, 1672 (CdO) cm -1. Anal.
(C10H9BrFN3O3) C, H, N.
6-Chloro-3-(bromoacetamido)benzoylurea (11g). Yield: 46%.
White solid, mp 224-226 °C. MS m/z 334 (M + 1). 1H NMR
(DMSO-d6 ): δ 4.04 (s, 2H), 7.46-7.49 (m, 2H), 7.62-7.65 (m,
1H), 7.70 (s, 1H), 7.74 (d, J )3.2 Hz, 1H), 10.66 (s, 1H), 10.72 (s,
1H). IR (KBr): γ 3383, 3317, 3251, 3134 (NH), 1711, 1678 (CdO)
cm -1. Anal. (C10H9BrClN3O3) C, H, N.
6-Bromo-3-(bromoacetamido)benzoylurea (11h). Yield: 46%.
1
Off-white solid, mp 213.5-215.5 °C. MS m/z 400 (M + Na). H
NMR (DMSO-d6): δ 4.04 (s, 2H), 7.43, 7.63 (s, s, 2H), 7.56 (dd,
J ) 2.4, 2.8 Hz, 1H), 7.61 (s, 1H), 7.72 (d, J ) 2.4 Hz, 1H), 10.64
(s, 1H), 10.69 (s, 1H). IR (KBr): γ 3379, 3296 (NH), 1702, 1689,
1678 (CdO) cm -1. Anal. (C10H9Br2N3O3) C, H, N.
By use of a procedure similar to method A, the title compound
was obtained from 7 or 17 after its reaction respectively with
chloroacetyl chloride, bromoacetyl bromide, or 2-bromopropionyl
bromide in DMA.

3102 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Song et al.
5-Methyl Formate 3-(Bromoacetamido)benzoylurea (11j).
Yield: 48%. White solid, mp 136-139 °C. MS m/z 358 (M + 1).
1H NMR (DMSO-d6): δ 3.89 (s, 3H), 4.07 (s, 2H), 7.42, 7.92 (s,s,
2H), 8.22 (s, 1H), 8.32 (s, 1H), 8.42 (s, 1H), 10.79 (s, 1H), 10.82
5-(2′-Bromopropionamido)-2-furoylurea (18a). Yield: 43%.
White solid, mp 184.3-185 °C. MS m/z 304 (M + 1). H NMR
1
(DMSO-d6 ): δ 1.72 (d, J )6.8 Hz, 3H), 4.65 (q, J ) 6.8 Hz, 1H),
6.41 (d, J ) 3.6 Hz, 1H), 7.28, 7.80 (s, s, 2H), 7.73 (d, J ) 3.6 Hz,
1H), 10.28 (s, 1H), 11.98 (s, 1H). IR (KBr): γ 3427, 3307, 3224 (NH),
1699, 1676 (CdO) cm -1. Anal. (C9H10BrN3O4 ·0.5H2O) C, H, N.
5-Bromoacetamido-2-furoylurea (18b). Yield: 43%. White solid,
(s, 1H). IR (KBr): γ 3429, 3329 (NH), 1718, 1685 (CdO) cm -1
.
Anal. (C12H12BrN3O5) C, H, N.
6-Methoxy-3-(bromoacetamido)benzoylurea (11l). Yield: 36%.
White solid, mp 203-205 °C. MS m/z 330 (M + 1). 1H NMR
(DMSO-d6 ): δ 3.86 (s, 3H), 4.01 (s, 2H), 7.19 (d, J ) 8.8 Hz,
1H), 7.43, 7.88 (s, s, 2H), 7.76 (d, J ) 8.8 Hz, 1H), 7.97 (s, 1H),
9.96 (s, 1H), 10.43 (s, 1H). IR (KBr): γ 3454, 3329, 3307 (NH),
1707, 1680 (CdO) cm -1. Anal. (C11H12BrN3O4) C, H, N.
4-Hydroxy-3-(bromoacetamido)benzoylurea (11m). Yield:
51%. Pale-yellow solid, mp 212-214 °C. MS m/z 316 (M + 1)+.
1H NMR (DMSO-d6): δ 4.20 (s, 2H), 6.90-6.92 (m, 1H), 7.28,
8.06 (s, s, 2H), 7.66-7.68 (m, 1H), 8.51 (m, 1H), 9.66 (s, 1H),
10.29 (s, 1H), 10.86 (s, 1H). IR (KBr): γ 3373, 3329, 3220 (NH),
1709, 1670 (CdO) cm -1. Anal. (C10H10BrN3O4) C, H, N.
4-Fluoro-3-(chloroacetamido)benzoylurea (13c). Yield: 48%.
White solid, mp 248-250 °C. MS m/z 273 (M+). 1H NMR
(DMSO-d6): δ 4.37 (s, 2H), 7.39-7.44 (m, 2H), 8.0 (s, 1H),
7.80-7.84 (m, 1H), 8.48-8.50 (m, 1H), 10.27 (s, 1H), 10.62 (s,
1H). IR (KBr): γ 3361, 3311, 3207, 3150 (NH), 1703, 1684 (CdO)
cm -1. Anal. (C10H9ClFN3O3) C, H, N.
1
mp 148-149 °C. MS m/z 290 (M + 1). H NMR (DMSO-d6 ): δ
4.08 (s, 2H), 6.40 (d, J ) 3.6 Hz, 1H), 7.29, 7.80 (s, s, 2H), 7.73 (d,
J ) 3.6 Hz, 1H), 10.28 (s, 1H), 12.00 (s, 1H). IR (KBr): γ 3321,
3248 (NH), 1715, 1670 (CdO) cm -1. Anal. (C8H8BrN3O4 ·0.5H2O)
C, H, N.
5-Chloroacetamido-2-furoylurea (18c). Yield: 48%. White solid,
mp 192.8-194 °C. MS m/z 246 (M + H)+. 1H NMR (DMSO-d6): δ
4.31 (s, 2H), 6.40 (d, J ) 3.6 Hz, 1H), 7.29, 7.80 (s, s, 2H), 7.73 (d,
J ) 3.6 Hz, 1H), 10.28 (s, 1H), 11.93 (s, 1H). IR (KBr): γ 3381,
3321, 3244 (NH), 1730, 1672 (CdO) cm -1. Anal. (C8H8ClN3-
O4 ·H2O) C, H, N.
5-(2′-Bromopropionamido)thiophene-3-carbonylurea (19a).
Yield: 51%. White solid, mp 216-220 °C. MS m/z 320 (M + 1). 1H
NMR (DMSO-d6 ): δ 1.76 (d, J ) 6.8 Hz, 3H), 4.70 (q, J ) 6.8 Hz,
1H), 7.32, 7.97 (s, s, 2H), 7.14 (d, J ) 1.2 Hz, 1H), 8.10 (s, 1H),
10.37 (s, 1H), 11.73 (s, 1H). IR (KBr): γ 3381, 3319, 3228 (NH),
1705, 1668 (CdO) cm -1. Anal. (C9H10BrSN3O3) C, H, N.
5-(Bromoacetamido)thiophene-3-carbonylurea (19b). Yield:
6-Fluoro-3-(chloroacetamido)benzoylurea (13d). Yield: 56%.
1
1
48%. White solid, mp 227-230 °C. MS m/z 306 (M + 1). H
White solid, mp 228.1-229 °C. MS m/z 274 (M + 1). H NMR
NMR (DMSO-d6): δ 4.08 (s, 2H), 7.32, 7.97 (s, s, 2H), 7.13 (s,
1H), 8.09 (s, 1H), 10.37 (s, 1H), 11.77 (s, 1H). IR (KBr): γ
3491, 3367, 3213 (NH), 1709, 1684,1633 (CdO) cm -1. Anal.
(C8H8BrSN3O3) C, H, N.
(DMSO-d6): δ 4.25 (s, 2H), 7.30 (t, 1H), 7.44 (s, 1H), 7.70-7.74
(m, 2H), 7.84-7.86 (m, 1H), 10.49 (s, 2H). IR (KBr): γ 3365,
3327, 3222 (NH), 1711, 1685, 1672 (CdO) cm -1. Anal.
(C10H9ClFN3O3) C, H, N.
Biological Methods. Tumor Cell Lines. The human tumor cell
lines CEM (leukemia), MCF-7 (breast cancer), DU-145 (prostate
cancer), PC-3 (prostate cancer), Lovo (colon cancer), Daudi (lym-
phoma), and MIA Paca (pancreatic cancer) were from the American
Tissue Culture Collection (ATCC, Rockville, MD). DND-1 melanoma
cells were from Dr. Ohnuma (Mount Sinai School of Medicine, New
York, NY). Bel-7402 hepatoma cell originating from Chinese human
hepatocarcinoma patients were provided by Cancer Institute, Chinese
Academy of Medical Sciences. All cells were cultivated in RPMI-
1640 supplemented with 10% heat inactivated fetal calf serum,
penicillin (150 µg/mL), and streptomycin (150 µg/mL) with 5% CO2
at 37 °C. Cells in exponential growth were used for experiments.
Anticancer Activity in Vitro. Cells were distributed into 96-well
plates (Falcon, Oxnard, CA) with 1 × 105 cells and a total volume of
250 µL per well, followed by treatment with study compounds at
concentrations between 0.001 and 10 µM at 37 °C for 48 h. Cell
viability was assessed by Trypan blue. The IC50 value was defined as
a drug concentration killing 50% cells in comparison with untreated
controls and calculated by nonlinear regression analysis. The IC50
values were determined in duplicate, and each experiment was repeated
three times under identical conditions.
Morphology. Cell samples on slides were prepared in a Cytospin
centrifuge (LTP-C, Tianjin,China) at 700g for 5 min. Slides were air-
dried, fixed in methanol, and stained with Giemsa at room temperature
for 15 min. Cells in the mitotic arrest were recognized by the
disappearance of the nuclear membrane and the appearance of
chromosomes throughout the cytoplasm. The percentage of mitotic
cells was counted after 24 h of incubation using optical microscopy.
Apoptotic cells were identified using common criteria, i.e., cell
shrinkage, chromatin condensation, and fragmentation of the nucleus
into discrete masses.
6-Chloro-3-(chloroacetamido)benzoylurea (13f). Yield: 47%.
White solid, mp 234-236 °C. MS m/z 290 (M + 1). 1H NMR
(DMSO-d6): δ 4.27 (s, 2H), 7.46, 7.70 (s, s, 2H), 7.48 (d, J ) 8.7
Hz, 1H), 7.66 (dd, J ) 1.8, 8.7 Hz, 1H), 7.74 (d, J ) 1.8 Hz, 1H),
10.58 (s, 1H), 10.72 (s, 1H). IR (KBr): γ 3386, 3311, 3255, 3136
(NH), 1711, 1678 (CdO) cm -1. Anal. (C10H9Cl2N3O3) C, H, N.
4-Hydroxy-3-(chloroacetamido)benzoylurea (13h). Yield: 54%.
1
Pale-yellow solid, mp 220-223 °C. MS m/z 272 (M + H)+. H
NMR (DMSO-d6): δ 4.38 (s, 2H), 6.89-6.91 (m, 1H), 7.28, 8.00
(s, s, 2H2), 7.62-7.64 (m, 1H), 8.52 (m, 1H), 9.56 (s, 1H), 10.34
(s, 1H), 10.90 (s, 1H). IR (KBr): γ 3375, 3334, 3215 (NH), 1711,
1668 (CdO) cm -1. Anal. (C10H10ClN3O4) C, H, N.
6-Fluoro-3-(iodoacetamido)benzoylurea (14b). A mixture of
13d (300 mg, 1.1 mmol) and sodium iodine in 5 mL of DMA was
stirred at room temperature for 2 h. The reaction mixture was slowly
poured into the ice-water and extracted with ethyl acetate. The
organic layer was washed with saturated brine, dried over Na2SO4,
and evaporated. The crude product was purified by flash chroma-
tography over silica gel, affording the title compound as a white
1
solid. Yield: 45%. Mp 207-210 °C. MS m/z 388 (M + Na). H
NMR (DMSO-d6): δ 3.81, 4.25 (s, s 2H), 7.25-7.32 (m, 1H), 7.43
(s, 1H), 7.65-7.74 (m, 2H), 7.82-7.86 (m, 1H), 10.49 (s, 1H),
10.52 (s, 1H). IR (KBr): γ 3437, 3315, 3267 (NH), 1711, 1684,
1676 (CdO) cm -1. Anal. (C10H9FIN3O3) C, H, N.
6-Methyl-3-(iodoacetamido)benzoylurea (14a). The title com-
pound was obtained from the corresponding chloro intermediate
and NaI using a procedure similar to that for compound 14b. Yield:
40%. White solid, mp 227.1-228.4 °C. MS m/z 362 (M + 1). 1H
NMR (DMSO-d6): δ 2.27 (s, 3H), 3.30 (s, 2H), 7.20-7.23 (m,
1H), 7.36, 7.84 (s, s, 2H), 7.51-7.58 (m, 1H), 7.63-7.66 (m, 1H),
10.39 (s, 1H), 10.51 (s, 1H). IR (KBr): γ 3383, 3303, 3251 (NH),
1705, 1670 (CdO) cm -1. Anal. (C11H12IN3O3) C, H, N.
5-Methyl Formate 3-(Iodoacetamido)benzoylurea (14c). The
title compound was obtained from the corresponding chloro
intermediate and NaI using a procedure similar to that for compound
14b. Yield: 43%. White solid, mp 133-137 °C. MS m/z 406 (M
+ 1). 1H NMR (DMSO-d6): δ 3.88 (s, 3H), 4.29 (s, 1H), 7.41,
7.91 (s, s, 2H), 8.22 (s, 1H), 8.32 (s, 1H), 8.42 (s, 1H), 10.69 (s,
1H), 10.81 (s, 1H). IR (KBr): γ 3406, 3259 (NH), 1718, 1685
(CdO) cm -1. Anal. (C12H12IN3O5) C, H, N.
Bcl-2 Phosphorylation. Tumor cells were treated with different
concentrations of 14b for 12 h. Aliquots of cells were taken and lysed
with lysis buffer containing 50 mM Tris-HCl (pH 7.4), 0.1% Triton
X-100, 1% SDS, 250 mM NaCl, 15 mM MgCl2, 1 mM DTT, 2 mM
EDTA, 2 mM EGTA, 25 mM NaF, 1 mM PMSF, 10 mg/mL
leupeptin, and 10 mg/mL aprotinin. Equal amounts of lysate were
subjected to electrophoresis using 10% SDS-polyacrylamide gels. The
gels were blotted onto PVDF membranes (Millipore, CA) using a
semidry electrophoretic transfer system (Bio-Rad, CA). After blocking
with 5% nonfat milk in TBST buffer (20 mM Tris-HCl, 137 mM NaCl,

Benzoylurea DeriVatiVes as Antimitotic Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3103
1997, 8, 187–190.
0.05% Tween-20) at 37 °C for 1 h, Bcl-2 protein was probed with
anti-Bcl-2 monoclonal antibody (Calbiochem, CA), followed by goat
antimouse HRP. The signals were detected by enhanced chemilumi-
nescence (ECL) and exposure to X-film.
Cell Cycle Analysis. The cell cycle was analyzed by a method
reported previously.11 Briefly, suspensions of the CEM cells treated
with 14b were incubated at 37 °C for 48 h and were resuspended in
70% ethanol for at least 12 h at 4 °C. After being washed with PBS,
the cells were treated with RNase A (50 µg/mL, Sigma, Mo) at 37 °C
for 30 min and then exposed to propidium iodide at a final concentra-
tion of 50 µg/mL at 4 °C for 30 min. At least 104 cell events per
sample were analyzed in a flow cytometer (Beckman Coulter EPICS
XL, San Jose, CA) with EPICS software (version 2.0).
Microtubule Polymerization Assays. The HTS-tubulin polym-
erization assay kit (Cytoskeleton Inc., Denver, CO) was used to
measure polymerization, following the process recommended by the
vendor. G-PEM buffer (80 mM PIPES, pH 6.9, 1 mM EGTA, 5%
glycerol, 1 mM GTP) was used as the diluent, and 14b was added
into the ice-cold 96-well plate. Each well contain G-PEM buffer, 14b
at different concentrations, and ꢀ-tubulin at a concentration of 1 mg/
mL. The plate was shaken for 20 s and warmed to 37 °C, and the
absorbance was read at 340 nm every 1.5 min for 30 min.
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Anticancer Effect in Vivo. Balb/c nude mice (male, 16-20 g,
6-7 weeks old) were from the Experimental Animal Center, Chinese
Academy of Medical Sciences, and used for the human hepatocarci-
noma (Bel-7402) xenograft. Experimental mice received subcutaneous
injection of Bel-7402 at 5 × 106 cells per mouse on their backs. Twelve
days later, tumor-bearing nude mice were divided randomly into
solvent control and treatment cages with 5 mice per group. 11e or
14b at 5 or 10 mg/kg were given ip on day 12 postimplantation. The
regimens were continued with six injections at 2-day intervals (q2d).
Therapeutic response was monitored by measuring tumor volume every
week until the tumor volume of the controls reached a size greater
than 2500 mm3, the criterion for euthanatizing the animals.21 Two
perpendicular tumor measurements, width and length, were obtained
with calipers, followed by calculation using the following formula:
tumor volume ) (length)(width)2(0.52).22
Acknowledgment. The project was supported by the Na-
tional Natural Science Foundation of the P. R. China (Grant
30371673).
Supporting Information Available: Element analysis data of
new 3-haloacylaminobenzoylurea compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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