Design, synthesis and structure-activity relationships of antiproliferative 1,3-disubstituted urea derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.04.011

Twenty-four new 1,3-disubstituted urea derivatives (compounds 1-24) were synthesized and reported for the first time. The antiproliferative activities of these compounds were evaluated against a panel of one human liver cell line (L02) and two human tumor

Full text of DOI:10.1016/j.ejmech.2008.04.011

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 453e459
http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and structureeactivity relationships of
antiproliferative 1,3-disubstituted urea derivatives
*
Huan-Qiu Li, Tao-Tao Zhu, Tao Yan, Yin Luo, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P.R. China
Received 21 December 2007; received in revised form 7 April 2008; accepted 19 April 2008
Available online 30 April 2008
Abstract
Twenty-four new 1,3-disubstituted urea derivatives (compounds 1e24) were synthesized and reported for the first time. The antiproliferative
activities of these compounds were evaluated against a panel of one human liver cell line (L02) and two human tumor cell lines (KB and K562)
by applying the MTT colorimetric assay. The series of 1,3-disubstituted urea derivatives show good antiproliferative activity against human can-
cer cell lines (KB and K562) and no antiproliferative activity against liver cell line (L02). The potent in vitro antiproliferative activity of these
derivatives and their selectivity for L02 are quite important points for an anticancer drug candidate with fewer side effects. Structureeactivity
relationships were also discussed based on the obtained experimental data. The hydroxyl groups on the phenyl ring reduced the antiproliferative
activities of 1,3-disubstituted urea derivatives. The OH groups could be responsible for a reduction in the permeability of the cell membrane.
Generally, an aromatic ring on N-3 seems to be in favor of enhancing the inhibitory activity, compounds introduced a nitro group substituent at
C-3 position on the aromatic ring approved to generally decrease activity.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Urea derivatives; Antiproliferative; K562 cell line; KB cell line; Morpholine ring; SAR
1. Introduction
in these receptors can thus result in loss of growth control and
the unregulated cell proliferation associated with malignancy
[4,5]. Therefore, selective interruption of signal transduction
in tumor cells by specific inhibitors of PTK activity has recently
emerged as a major new approach for the design of tumor-
specific drugs [6,7]. Urea derivatives are synthesized largely
in recent years and have become of particular interest to chem-
ists and biologists because of their wide range of biological ac-
tivities, such as anticonvulsant activity [8], colchicine-blinding
antagonist [9] and CXCR3 antagonist [10]. Some of these
compounds exhibit cell growth inhibition (GI50) on numerous
cancer cell lines and have developed various chemoresistance
mechanisms, and to be potent inhibitors of the PTK activity of
a number of trans-membrane growth factor receptors and
cellular oncogene products, particularly epidermal growth fac-
tor receptor (EGFR) [11]. We previously investigated the effect
of a series of urea-like compounds on the human-leukemia
K562 cell line and found that incorporating a heterocycle, espe-
cially a morpholine ring can highly improve their activity [12].
Despite progress made in discovering new compounds with
antiangiogenic activity, this research field is still considered
one of the most promising ways to treat diseases characterized
by abnormal angiogenesis including tumors [1]. Protein tyro-
sine kinases (PTK) have been intensively investigated because
of their role in the transduction of proliferative signals in mam-
malian cells. Many trans-membrane growth factor receptors
possess intracellular PTK activity, with initiation of this activity
following external binding of a growth factor being the first step
in the cellular signal transduction pathway which controls
mitogenesis and cell proliferation [2,3]. The over-expression
or inappropriate expression of normal or mutant PTK activity
* Corresponding author. State Key Laboratory of Pharmaceutical Biotech-
nology, Nanjing University, Nanjing 210093, P.R. China. Tel.: þ86 25 8359
2572; fax: þ86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.011

454
H.-Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 453e459
On the basis of the above work, we designed and prepared 24
new 1,3-disubstituted urea compounds (1e24), all of the com-
pounds were assayed for their antiproliferative activities against
a panel of one human liver cell line (L02) and two human tumor
cell lines (KB and K562) by applying the MTT colorimetric as-
say. The neck carcinoma cell line KB are overexpressing EGFR,
demonstrating EGF-independent cell growth, so the antiproli-
ferative activity against KB was evaluated. Although in general
leukemia cell lines K562 was not overexpressing EGFR, the
antiproliferative activity against K562 was also evaluated for
the possibility of another cellular target than EGFR for future
research.
Table 2. Etoposide was used as reference compounds. The
results show that 1,3-disubstituted urea derivatives inhibited
the growth of human cancer cell lines well, generally, the com-
pounds 1e24 illustrated in Table 1 showed better activity com-
pared with the 1-substituted urea derivatives reported before,
indicating that an aromatic ring on N-3 seems to be in favor
of enhancing the inhibitory activity. Against K562 and KB,
compound 2 was found to show the most potent in vitro activ-
ity (with IC50 of 0.27 and 0.9 mM, respectively) among the
new compounds tested, and was comparable with the positive
control etoposide (IC50 of 1.27 and 0.42 mM, respectively).
These compounds containing a morpholine ring (1, 2, 6, 7,
9, 10, 13, 14, 18, 19, 22, 23) exhibited the most potent in vitro
activities in vitro compared to the rest, implying that a morpho-
line ring can improve highly the antiproliferative activity on
the K562 and KB cell line. The effects of substituents on
the phenyl ring were investigated, when a nitro group placed
on the phenyl ring, derivatives exhibited most potent in vitro
activity, especially substituted on C-3 position. Against all
two human cancer cell lines, compounds 18 and 19 were
less active than 1 and 2, respectively. This result indicates
that the hydroxyl groups on the phenyl ring reduced the anti-
proliferative activities of 1,3-disubstituted urea derivatives.
The OH groups on phenyl ring lead to the decrease of their
permeability of the cell membrane, which may be the reason
of decreased biological activity of the corresponding deriva-
tives [13]. Other evidences are also found in Table 2 such as
compounds 20 and 21.
2. Results and discussion
Compounds 1e24 were prepared by the synthetic route
illustrated in Scheme 1. Since introducing a morpholine ring
can improve highly the cell growth inhibitory activity on the
human-leukemia K562 cell line according to our previous re-
port, to further optimize this activity, 12 compounds reported
in this paper contain a morpholine ring. In the first step, con-
densation reaction of aldehydes with various amines containing
functional groups (morpholine, furfurylamine, etc.), which are
cheap and available easily in market afforded the schiff bases.
Reduction of the latter with NaBH4 afforded the corresponding
saturated alkyl amines, to which dry DMF in SOCl2 was added
and refluxed for 4 h and excessive SOCl2 was evaporated under
reduced pressure. The residue was dissolved in dichlorome-
thane, various amines obtained in the first step dissolved in
dry pyridine and CH2Cl2 were added. As a result, 24 1,3-
disubstituted urea compounds were synthesized, which were
all first reported. All the compounds were purified by silica
gel column and identified by elemental C, H, N analysis,
NMR and MS and displayed in Table 1.
The in vitro antiproliferative activity of the synthesized
1,3-disubstituted urea derivatives was studied on a panel of
one human liver cell line (L02) and two human tumor cell
lines (KB, K562) by applying the MTT colorimetric assay.
Compounds were tested over a range of concentrations from
0.1 to100 mg/mL, and the calculated IC50 values, that is, the
concentration (mM) of a compound able to cause 50% of
cell death with respect to the control culture, are reported in
Moreover, compounds (1, 2, 6, 7, 9, 10, 13, 14, 18, 19, 22,
23) with a morpholine ring and etoposide were also evaluated
for a one human normal cell line (L02). The results (Table 3)
showed that these 1,3-disubstituted urea derivatives with
potent in vitro antiproliferative activity scarcely inhibited
normal cell lines, while etoposide showed no selectivity to in-
hibition growth of the three cell lines (L02, K562 and KB).
3. Experimental part
3.1. General
All chemicals and reagents used in current study were of
analytical grade. TLC was run on the silica gel coated
O
Cl
N
H
O
S
c
O
S
O
S
+
H
N
N
O
Cl
O
Cl
Cl
Cl
R1
R3
Cl
R2
R
N
N
d
O
R1
R1
R1
1-24
R2
R
R2
R
R2
CHO
b
N
a
N
+
R
NH2
H
R3
R3
R3
Scheme 1. General synthesis of urea compounds 1e24. Reagents and conditions: (a) ethanol, reflux, 1 h; (b) NaBH4, ethanol, 50 ꢀC, 2e3 h; (c) dry CH2Cl2, 70 ꢀC,
4 h; (d) dry CH2Cl2, dry pyridine, 60 ꢀC, 5e6 h.

H.-Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 453e459
455
Table 1
Physical properties of 1,3-disubstituted urea compounds
R1
R3
R2
R
N
N
O
Compound
R
R1
R2
R3
Formula
M.p. (ꢀC)
Yield (%)
51
1
NO2
H
H
H
H
H
H
C17H26N4O4
121e122
N
O
2
3
O
NO2
NO2
C16H24N4O4
C15H17N3O4
124e126
135e137
48
48
N
O
O
N
O
4
NO2
H
H
C18H26N4O5
Brown oil
44
O
5
6
7
Cl
Cl
Cl
H
H
H
OH
OH
OH
C15H17ClN2O3
C16H24ClN3O3
C17H26ClN3O3
Yellow oil
Yellow oil
Yellow oil
57
52
45
O
O
N
N
N
O
8
Cl
H
OH
C18H26ClN3O4
Yellow oil
42
O
9
O
O
H
H
OH
OH
H
H
C16H25N3O3
C17H27N3O3
144e146
55
41
N
N
10
Yellow oil
O
11
12
H
H
OH
OH
H
H
C15H18N2O3
C18H27N3O4
Yellow oil
Yellow oil
51
40
N
O
O
O
O
13
14
H
H
NO2
NO2
H
H
C16H24N4O4
C17H26N4O4
Yellow oil
Brown oil
47
53
N
N
O
O
15
16
H
H
NO2
NO2
H
H
C15H17N3O4
C18H26N4O5
118e120
48
42
N
O
Yellow oil
O
17
18
19
NO2
NO2
NO2
H
H
H
OH
OH
OH
C15H17N3O5
C16H24N4O5
C17H26N4O5
Yellow oil
Yellow oil
Yellow oil
51
53
45
O
O
N
N
N
O
20
NO2
H
OH
C18H26N4O6
Yellow oil
41
O
(continued on next page)

456
H.-Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 453e459
Table 1 (continued)
Compound
R
R1
Br
R2
H
R3
Formula
M.p. (ꢀC)
Yellow oil
Yield (%)
55
O
21
22
23
OH
C15H17BrN2O3
O
O
Br
Br
H
H
OH
OH
C16H24BrN3O3
C17H26BrN3O3
Yellow oil
Yellow oil
51
41
N
N
24
Br
H
OH
C18H26BrN3O4
Yellow oil
40
aluminum sheets (silica gel 60 GF254, E. Merk, Germany) and
visualized in UV light (254 nm). All the NMR spectra were
recorded on a Bruker DRX 500 model Spectrometer in either
The mixture of CH2Cl2 (15 ml) dry DMF (3 ml, 40 mmol)
and SOCl2 (7 ml, 0.10 mol) was stirred to reflux at 70 ꢀC for
4 h and cooled. The solvents and excess SOCl2 were then
removed under reduced pressure. The residue dissolved in
CH2Cl2 (15 ml) was added dry pyridine (4 ml) and various
amines (40 mmol). The reaction mixture was stirred at
50e60 ꢀC for 5e6 h, and then added to 20 ml ice-water,
organic layer was separated, and the aqueous layer was
extracted with ethyl acetate (2 ꢁ 10 ml). The organic layer
was combined and washed with saturated NaHCO3, dried
with anhydrous Na2SO4 for 0.5 h and concentrated under vac-
uum; ultimately the residue was purified by silica gel column
(eluent EtOAc / petroleum ether, 1:2 - 2:1).
1
(d6) DMSO or CDCl3. Chemical shifts (d) for H-NMR spec-
tra are reported in parts per million to residual solvent protons.
Melting points were measured on a Boetius micro-melting
point apparatus. The ESI-MS spectra were recorded on a Mar-
iner System 5304 Mass spectrometer. Elemental analyses were
performed on a CHN-O-Rapid instrument.
3.2. Compounds
Aldehyde was dissolved in 25 ml of ethanol, and amine was
added to the solution. The reaction mixture was refluxed for
1 h, and then 0.05 mmol of NaBH4 was added to the reaction
solution slowly, and stirred under 50 ꢀC for 2e3 h. The mix-
ture was evaporated under vacuum, and dissolved in EtOAc
(30 ml). The solution was washed with 20 ml water twice,
dried over anhydrous sodium sulfate, and evaporated. Purifica-
tion by silica gel afforded pure products.
3.2.1. 1,1-Dimethyl-3-(3-morpholinopropyl)-3-
(3-nitrobenzyl)urea (1)
Light brown solid. 3.57 g (51%). M.p. 121e122 ꢀC.
1H-NMR (500 MHz, DMSO-d6, dppm): 1.77 (m, 2H); 2.83
(s, 6H); 3.20 (t, J ¼ 5.2, 2H); 3.26 (t, J ¼ 4.5, 4H); 3.44
(t, J ¼ 4.3, 2H); 3.52 (t, J ¼ 4.1, 4H); 4.23 (s, 2 H); 7.72
(t, J ¼ 7.8, 1H); 8.14 (d, J ¼ 7.5, 1H); 8.27 (d, J ¼ 8.0, 1H);
8.48 (s, 1H). MS (ESI): 350.2 ([M þ H]þ). Anal. Calcd. for
C17H26N4O4: C, 58.27; H, 7.48; N, 15.99%. Found: C,
58.34; H, 7.35; N, 15.30%.
Table 2
Antiproliferative activity against K562 and KB cell line of compounds 1-24
Compound IC50 (mM)
Compound IC50 (mM)
KB
K562
KB
K562
1
0.17 ꢂ 0.06 0.42 ꢂ 0.15 13
0.09 ꢂ 0.38 0.27 ꢂ 0.13 14
23.8 ꢂ 12.1 40.7 ꢂ 25.2 15
15.6 ꢂ 5.4 24.9 ꢂ 10.1 16
49.5 ꢂ 21.8 71.3 ꢂ 36.7 17
2.24 ꢂ 0.94 3.88 ꢂ 1.35 18
1.75 ꢂ 0.59 2.92 ꢂ 1.14 19
25.7 ꢂ 13.4 33.5 ꢂ 12.8 20
4.25 ꢂ 1.96 11.8 ꢂ 4.9 21
2.88 ꢂ 1.41 7.5 ꢂ 1.9 22
52.1 ꢂ 21.2 75.1 ꢂ 34.1 23
48.6 ꢂ 20.7 67.2 ꢂ 33.6 24
0.75 ꢂ 0.33 2.24 ꢂ 0.95
0.52 ꢂ 0.26 1.20 ꢂ 0.46
17.5 ꢂ 8.6 35.4 ꢂ 11.6
16.4 ꢂ 8.2 26.0 ꢂ 9.8
20.8 ꢂ 8.5 28.6 ꢂ 10.1
4.8 ꢂ 2.3 10.8 ꢂ 3.7
2
Table 3
Antiproliferative activity against human normal cell line L02 of compounds
3
4
Compound
IC50 (mM)
Compound
IC50 (mM)
5
6
L 02
L 02
7
3.3 ꢂ 1.4
8.5 ꢂ 3.8
1
134 ꢂ 35
155 ꢂ 43
82 ꢂ 17
81 ꢂ 14
81 ꢂ 16
109 ꢂ 34
13
14
18
19
22
23
65 ꢂ 17
85 ꢂ 19
26 ꢂ 7
8
24.2 ꢂ 8.4 38.4 ꢂ 17.0
32.8 ꢂ 12.0 40.5 ꢂ 14.5
2
9
6
10
11
12
7.3 ꢂ 3.4
13.5 ꢂ 5.5
7
125 ꢂ 34
21 ꢂ 4
9.0 ꢂ 3.5 18.6 ꢂ 6.5
9
10
24.6 ꢂ 10.3 41.8 ꢂ 14.8
86 ꢂ 23
Etoposidea IC50/L02: 13 ꢂ 3
Etoposidea IC50/KB: 0.42 ꢂ 0.21
Used as a positive control. The data represent the mean of three experi-
ments performed in triplicate and are expressed as means ꢂ SD. The IC50
value is the concentration at which 50% survival of cells was observed.
IC50/K562: 1.27 ꢂ 0.45
a
a
Used as a positive control. The data represent the mean of three experi-
ments performed in triplicate and are expressed as means ꢂ SD. The IC50
value is the concentration at which 50% survival of cells was observed.

H.-Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 453e459
457
3.2.2. 1,1-Dimethyl-3-(2-morpholinoethyl)-3-
(3-nitrobenzyl)urea (2)
([M þ H]þ). Anal. Calcd. for C17H26ClN3O3: C, 57.38; H,
7.36; N, 11.81%. Found: C, 57.25; H, 7.42; N, 11.90%.
Light brown solid. 3.22 g (48%). M.p. 124e126 ꢀC.
1H-NMR (500 MHz, DMSO-d6, dppm): 2.61 (s, 6H); 2.82 (t,
J ¼ 5.6, 2H); 3.21 (t, J ¼ 4.2, 4H); 3.24 (t, J ¼ 4.6, 2H); 3.50
(t, J ¼ 4.7, 4H); 4.28 (s, 2H); 7.73 (t, J ¼ 7.9, 1H); 8.14 (d,
J ¼ 7.5, 1H); 8.28 (d, J ¼ 7.0, 1H); 8.50 (s, 1H). MS (ESI):
336.18 ([M þ H]þ). Anal. Calcd. for C16H24N4O4: C, 57.13;
H, 7.19; N, 16.66%. Found: C, 57.24; H, 7.08; N, 16.62%.
3.2.8. Ethyl 4-(1-(5-chloro-2-hydroxybenzyl)-3,
3-dimethylureido)piperidine-1-carboxylate (8)
1
Yellow oil. 3.22 g (42%). H-NMR (500 MHz, DMSO-d6,
dppm): 1.28 (t, J ¼ 3.8, 3H); 1.72 (m, 4H); 2.80 (s, 6H);
3.13 (m, 4H); 3.46 (m, 1H); 4.17 (q, J ¼ 7.0, 2H); 4.29 (s,
2H); 6.92 (d, J ¼ 8.3, 1H); 7.28 (d, J ¼ 7.8, 1H); 7.43 (s,
1H) 8.36 (s, 1H). MS (ESI): 383.2 ([M þ H]þ). Anal. Calcd.
for C18H26ClN3O4: C, 56.32; H, 6.83; N, 10.95%. Found: C,
56.46; H, 6.56; N, 11.23%.
3.2.3. 1-(Furan-2-ylmethyl)-3,3-dimethyl-1-
(3-nitrobenzyl)urea (3)
1
Brown solid. 3.22 g (48%). M.p. 135e137 ꢀC. H-NMR
3.2.9. 1-(4-Hydroxybenzyl)-3,3-dimethyl-1-
(2-morpholinoethyl)urea (9)
(500 MHz, DMSO-d6, dppm): 2.91 (s, 6H); 4.25 (s, 2H);
4.84 (s, 2H); 6.27 (d, J ¼ 2.4, 1H); 6.42 (t, J ¼ 1.8, 1H);
7.51 (d, J ¼ 2.6, 1H); 7.59 (t, J ¼ 7.8, 1H); 8.11 (d, J ¼ 7.6,
1H); 8.28 (d, J ¼ 7.5, 1H); 8.39 (s, 1H). MS (ESI): 303.1
([M þ H]þ). Anal. Calcd. for C15H17N3O4: C, 59.40; H,
5.65; N, 13.85%. Found: C, 59.60; H, 5.82; N, 13.68%.
1
Yellow solid. 3.38 g (55%). M.p. 144e146 ꢀC. H-NMR
(500 MHz, DMSO-d6, dppm): 2.55 (t, J ¼ 5.0, 4H); 2.94
(s, 6H); 3.08 (t, J ¼ 5.2, 2H); 3.26 (t, J ¼ 4.6, 2H); 3.61
(t, J ¼ 4.0, 4H); 4.49 (s, 2H); 6.75 (d, J ¼ 8.5, 2H); 7.52
(d, J ¼ 8.5, 2H) 8.16 (s, 1H). MS (ESI): 307.2 ([M þ H]þ).
Anal. Calcd. for C16H25N3O3: C, 62.52; H, 8.20; N, 13.67%.
Found: C, 62.46; H, 8.36; N, 13.53%.
3.2.4. Ethyl 4-(3,3-dimethyl-1-(3-nitrobenzyl)ureido)
piperidine-1-carboxylate (4)
Brown oil. 3.33 g (44%). H-NMR (500 MHz, DMSO-d6,
1
3.2.10. 1-(4-Hydroxybenzyl)-3,3-dimethyl-1-
(3-morpholinopropyl)urea (10)
dppm): 1.26 (t, J ¼ 3.7, 3H); 1.74 (m, 4H); 2.85 (s, 6H);
3.18 (m, 4H); 3.49 (m, 1 H); 4.15 (q, J ¼ 7.0, 2H); 4.26 (s,
2H); 7.75 (t, J ¼ 7.8, 1H); 8.12 (d, J ¼ 7.8, 1H); 8.26 (d,
J ¼ 7.5, 1H); 8.52 (s, 1H). MS (ESI): 378.2 ([M þ H]þ).
Anal. Calcd. for C18H26N4O5: C, 57.13; H, 6.93; N, 14.81%.
Found: C, 57.42; H, 6.86; N, 14.73%.
Light yellow oil. 2.63 g (41%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 1.67 (m, 2H); 2.76 (t, J ¼ 5.4, 2H); 2.81
(s, 6H); 3.02 (t, J ¼ 4.6, 4H); 3.26 (t, J ¼ 4.7, 2H); 3.44 (t,
J ¼ 4.0, 4H); 4.28 (s, 2H); 6.73 (d, J ¼ 8.4, 2H); 7.52 (d,
J ¼ 8.8, 2H) 8.15 (s, 1H). MS (ESI): 321.2 ([M þ H]þ).
Anal. Calcd. for C17H27N3O3: C, 63.53; H, 8.47; N, 13.07%.
Found: C, 63.67; H, 8.42; N, 12.98%.
3.2.5. 1-(5-Chloro-2-hydroxybenzyl)-1-
(furan-2-ylmethyl)-3,3-dimethylurea (5)
1
Yellow oil. 3.51 g (57%). H-NMR (500 MHz, DMSO-d6,
3.2.11. 1-(Furan-2-ylmethyl)-1-(4-hydroxybenzyl)-3,
3-dimethylurea (11)
dppm): 2.82 (s, 6H); 4.27 (s, 2H); 4.54 (s, 2H); 6.27 (d,
J ¼ 2.3, 1H); 6.39 (t, J ¼ 1.7, 1H); 6.73 (d, J ¼ 2.4, 1H);
7.08 (m, 1H); 7.10 (d, J ¼ 7.8, 1H); 7.17 (s, 1H). MS (ESI):
308.1 ([M þ H]þ). Anal. Calcd. for C15H17ClN2O3: C,
58.35; H, 5.55; N, 9.07%. Found: C, 58.50; H, 5.62; N, 8.89%.
1
Yellow oil. 2.80 g (51%). H-NMR (500 MHz, DMSO-d6,
dppm): 2.84 (s, 6H); 4.26 (s, 2H); 4.57 (s, 2H); 6.26 (d,
J ¼ 2.2, 1H); 6.35 (t, J ¼ 1.9, 1H); 6.72 (d, J ¼ 2.4, 1H);
6.85 (d, J ¼ 8.4, 2H); 7.55 (d, J ¼ 8.8, 2H) 8.05 (s, 1H). MS
(ESI): 274.1 ([M þ H]þ). Anal. Calcd. for C15H18N2O3: C,
65.68; H, 6.61; N, 10.21%. Found: C, 65.50; H, 6.72; N,
10.29%.
3.2.6. 1-(5-chloro-2-hydroxybenzyl)-3,3-dimethyl-1-
(2-morpholinoethyl)urea (6)
Light yellow oil. 3.55 g (52%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 2.62 (t, J ¼ 5.2, 2H); 2.94 (s, 6H); 3.16
(t, J ¼ 4.8, 4H); 3.26 (t, J ¼ 4.2, 2H); 3.52 (t, J ¼ 4.2, 4H);
4.36 (s, 2H); 6.88 (d, J ¼ 8.8, 1H); 7.32 (d, J ¼ 8.2, 1H);
7.52 (s, 1H) 8.53 (s, 1H). MS (ESI): 341.1 ([M þ H]þ).
Anal. Calcd. for C16H24ClN3O3: C, 56.22; H, 7.08; N,
12.29%. Found: C, 56.40; H, 7.12; N, 12.09%.
3.2.12. Ethyl 4-(1-(4-hydroxybenzyl)-3,3-dimethylureido)
piperidine-1-carboxylate (12)
Yellow oil. 2.79 g (40%). H-NMR (500 MHz, DMSO-d6,
1
dppm): 1.26 (t, J ¼ 3.7, 3H); 1.74 (m, 4H); 2.85 (s, 6H);
3.15 (m, 4H); 3.43 (m, 1H); 4.18 (q, J ¼ 5.2, 2H); 4.33 (s,
2H); 6.83 (d, J ¼ 8.5, 2H); 7.56 (d, J ¼ 8.9, 2H) 8.16 (s,
1H). MS (ESI): 349.2 ([M þ H]þ). Anal. Calcd. for
C18H27N3O4: C, 61.87; H, 7.79; N, 12.03%. Found: C,
61.95; H, 7.86; N, 11.68%.
3.2.7. 1-(5-Chloro-2-hydroxybenzyl)-3,3-dimethyl-
1-(3-morpholinopropyl)urea (7)
Light yellow oil. 3.20 g (45%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 1.76 (m, 2H); 2.83 (s, 6 H); 2.96 (t,
J ¼ 5.4, 2H); 3.22 (t, J ¼ 4.6, 4H); 3.36 (t, J ¼ 4.7, 2H); 3.54
(t, J ¼ 4.0, 4H); 4.34 (s, 2H); 6.87 (d, J ¼ 7.8, 1H); 7.32 (d,
J ¼ 7.6, 1H); 7.53 (s, 1H) 8.53 (s, 1H). MS (ESI): 355.2
3.2.13. 1,1-Dimethyl-3-(2-morpholinoethyl)-
3-(4-nitrobenzyl)urea (13)
Light yellow oil. 3.16 g (47%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 2.43 (t, J ¼ 5.2, 4H); 2.61 (t, J ¼ 6.5,

458
H.-Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 453e459
2H); 2.84 (s, 6H); 3.55 (t, J ¼ 4.9, 2H); 3.76 (t, J ¼ 6.5, 4H);
4.42 (s, 2H); 7.98 (d, J ¼ 9.0, 2H); 8.29 (d, J ¼ 9.0, 2H). MS
(ESI): 336.2 ([M þ H]þ). Anal. Calcd. for C16H24N4O4: C,
57.13; H, 7.19; N, 16.66%. Found: C, 57.42; H, 7.06; N,
16.53%.
3.2.19. 1-(2-Hydroxy-5-nitrobenzyl)-3,3-dimethyl-1-
(3-morpholinopropyl)urea (19)
Light yellow oil. 3.30 g (45%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 1.74 (m, 2H); 2.82 (s, 6H); 2.98 (t,
J ¼ 5.2, 2H); 3.24 (t, J ¼ 4.8, 4H); 3.38 (t, J ¼ 4.8, 2H); 3.56
(t, J ¼ 4.4, 4H); 4.36 (s, 2H); 6.92 (d, J ¼ 7.8, 1H); 7.46 (d,
J ¼ 7.6, 1H); 7.67 (s, 1H) 8.50 (s, 1H). MS (ESI): 366.2
([M þ H]þ). Anal. Calcd. for C17H26N4O5: C, 55.72; H,
7.15; N, 15.29%. Found: C, 55.65; H, 7.23; N, 15.36%.
3.2.14. 1,1-Dimethyl-3-(3-morpholinopropyl)-3-
(4-nitrobenzyl)urea (14)
1
Brown oil. 3.71 g (53%). H-NMR (500 MHz, DMSO-d6,
dppm): 1.70 (m, 2H); 2.38 (t, J ¼ 7.5, 2H); 2.67 (t, J ¼ 6.6,
4H); 2.88 (s, 6H); 3.28 (t, J ¼ 5.6, 2H); 3.64 (t, J ¼ 4.8,
4H); 4.32 (s, 2H); 7.49 (d, J ¼ 8.1, 2H); 8.16 (d, J ¼ 7.8,
2H). MS (ESI): 350.2 ([M þ H]þ). Anal. Calcd. for
C17H26N4O4: C, 58.27; H, 7.48; N, 15.99%. Found: C,
58.33; H, 7.28; N, 15.87%.
3.2.20. Ethyl 4-(1-(2-hydroxy-5-nitrobenzyl)-3,3-
dimethylureido)piperidine-1-carboxylate(20)
1
Yellow oil. 3.23 g (41%). H-NMR (500 MHz, DMSO-d6,
dppm): 1.26 (t, J ¼ 3.7, 3H); 1.74 (m, 4H); 2.82 (s, 6H);
3.14 (m, 4H); 3.48 (m, 1H); 4.18 (q, J ¼ 4.0, 2H); 4.32 (s,
2H); 6.94 (d, J ¼ 7.9, 1H); 7.56 (d, J ¼ 7.8, 1H); 7.89 (s,
1H) 8.52 (s, 1H). MS (ESI): 394.2 ([M þ H]þ). Anal. Calcd.
for C18H26N4O6: C, 54.81; H, 6.64; N, 14.20%. Found: C,
54.56; H, 6.78; N, 14.23%.
3.2.15. 1-(Furan-2-ylmethyl)-3,3-dimethyl-1-
(4-nitrobenzyl)urea (15)
1
Brown solid. 3.22 g (48%). M.p. 121e122 ꢀC. H-NMR
3.2.21. 1-(5-Bromo-2-hydroxybenzyl)-1-(furan-2-ylmethyl)-
3,3-dimethylurea (21)
(500 MHz, DMSO-d6, dppm): 2.88 (s, 6H); 4.28 (s, 2H);
4.85 (s, 2H); 6.35 (q, J ¼ 2.5, 1H); 6.46 (t, J ¼ 1.9, 1H);
7.41 (q, J ¼ 2.5, 1H); 7.94 (q, J ¼ 7.8, 2H); 8.27 (d, J ¼ 8.2,
2H). MS (ESI): 303.1 ([M þ H]þ). Anal. Calcd. for
C15H17N3O4: C, 59.40; H, 5.65; N, 13.85%. Found: C,
59.23; H, 5.87; N, 13.89%.
1
Yellow oil. 3.87 g (55%). H-NMR (500 MHz, DMSO-d6,
dppm): 2.87 (s, 6H); 4.15 (s, 2H); 4.41 (s, 2H); 6.18 (d,
J ¼ 2.5, 1H); 6.38 (t, J ¼ 1.9, 1H); 6.93 (d, J ¼ 2.2, 1H);
7.18 (d, J ¼ 7.8, 1H); 7.40 (d, J ¼ 7.8, 1H); 7.62 (s, 1H);
8.82 (s, 1H). MS (ESI): 352.0 ([M þ H]þ). Anal. Calcd. for
C15H17BrN2O3: C, 51.01; H, 4.85; N, 7.93%. Found: C,
51.23; H, 4.72; N, 7.80%.
3.2.16. Ethyl 4-(3,3-dimethyl-1-(4-nitrobenzyl)ureido)
piperidine-1-carboxylate (16)
Yellow oil. 3.18 g (42%). H-NMR (500 MHz, DMSO-d6,
1
3.2.22. 1-(5-Bromo-2-hydroxybenzyl)-3,3-dimethyl-
1-(2-morpholinoethyl)urea (22)
dppm): 1.24 (t, J ¼ 3.6, 3H); 1.72 (m, 4H); 2.89 (s, 6H);
3.13 (m, 4H); 3.44 (m, 1H); 4.16 (q, J ¼ 5.6, 2H); 4.35 (s,
2H); 7.48 (d, J ¼ 8.0, 2H); 8.15 (d, J ¼ 7.9, 2H). MS (ESI):
378.2 ([M þ H]þ). Anal. Calcd. for C18H26N4O5: C, 57.13;
H, 6.93; N, 14.81%. Found: C, 57.55; H, 7.06; N, 14.68%.
Light yellow oil. 3.93 g (51%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 2.57 (t, J ¼ 5.2, 2H); 2.92 (s, 6H); 3.12
(t, J ¼ 4.6, 4H); 3.28 (t, J ¼ 4.8, 2H); 3.56 (t, J ¼ 4.3, 4H);
4.42 (s, 2H); 7.06 (d, J ¼ 7.5, 1H); 7.42 (d, J ¼ 7.6, 1H);
7.70 (s, 1H); 8.67 (s, 1H). MS (ESI): 385.1 ([M þ H]þ).
Anal. Calcd. for C16H24BrN3O3: C, 49.75; H, 6.26; N,
10.88%. Found: C, 49.49; H, 6.38; N, 10.96%.
3.2.17. 1-(Furan-2-ylmethyl)-1-(2-hydroxy-5-nitrobenzyl)-
3,3-dimethylurea (17)
1
Yellow oil. 3.25 g (51%). H-NMR (500 MHz, DMSO-d6,
3.2.23. 1-(5-Bromo-2-hydroxybenzyl)-3,3-dimethyl-
1-(3-morpholinopropyl)urea (23)
dppm): 2.86 (s, 6H); 4.26 (s, 2H); 4.56 (s, 2H); 6.28 (d,
J ¼ 2.2, 1H); 6.41 (t, J ¼ 1.9, 1H); 6.75 (d, J ¼ 2.1, 1H);
7.02 (m, 1H); 7.13 (d, J ¼ 7.8, 1H); 7.27 (s, 1H), 8.24 (s,
1H). MS (ESI): 319.1 ([M þ H]þ). Anal. Calc. for
C15H17N3O5: C, 56.42; H, 5.37; N, 13.16%. Found: C,
56.57; H, 5.42; N, 13.02%.
Light yellow oil. 3.27 g (41%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 1.68 (m, 2H); 2.86 (s, 6H); 2.89 (t,
J ¼ 4.8, 2H); 3.01 (t, J ¼ 4.4, 4H); 3.23 (t, J ¼ 4.6, 2H); 3.52
(t, J ¼ 4.2, 4H); 4.38 (s, 2H); 7.08 (d, J ¼ 7.6, 1H); 7.44 (d,
J ¼ 7.6, 1H); 7.72 (s, 1H); 8.66 (s, 1H). MS (ESI): 399.2
([M þ H]þ). Anal. Calcd. for C17H26BrN3O3: C, 51.01; H,
6.55; N, 10.50%. Found: C, 51.29; H, 6.41; N, 10.32%.
3.2.18. 1-(2-Hydroxy-5-nitrobenzyl)-3,3-dimethyl-1-
(2-morpholinoethyl)urea (18)
Light yellow oil. 3.73 g (53%). 1H-NMR (500 MHz,
DMSO-d6, dppm): 2.60 (t, J ¼ 4.2, 2H); 2.96 (s, 6H); 3.06
(t, J ¼ 4.9, 4H); 3.24 (t, J ¼ 4.8, 2H); 3.54 (t, J ¼ 4.6, 4H);
4.37 (s, 2H); 6.98 (d, J ¼ 8.9, 1H); 7.42 (d, J ¼ 8.0, 1H);
7.62 (s, 1H) 8.58 (s, 1H). MS (ESI): 352.2 ([M þ H]þ).
Anal. Calcd. for C16H24N4O5: C, 54.53; H, 6.86; N, 15.90%.
Found: C, 54.40; H, 6.98; N, 15.92%.
3.2.24. Ethyl 4-(1-(5-bromo-2-hydroxybenzyl)-3,3-
dimethylureido)piperidine-1-carboxylate (24)
1
Yellow oil. 3.41 g (40%). H-NMR (500 MHz, DMSO-d6,
dppm): 1.32 (t, J ¼ 3.6, 3H); 1.74 (m, 4H); 2.82 (s, 6H);
3.15 (m, 4H); 3.48 (m, 1H); 4.19 (q, J ¼ 4.0, 2H); 4.32 (s,
2H); 7.06 (d, J ¼ 7.6, 1H); 7.42 (d, J ¼ 7.6, 1H); 7.67 (s,
1H); 8.56 (s, 1H). MS (ESI): 427.1 ([M þ H]þ). Anal. Calcd.

H.-Q. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 453e459
459
for C18H26BrN3O4: C, 50.47; H, 6.12; N, 9.81%. Found: C,
50.78; H, 5.69; N, 9.77%.
antiproliferative toward human cancer cell lines; compounds
introduced a nitro group substituent at C-3 position on the phe-
nyl ring approved to generally decrease activity. It may be
concluded that compounds 1 and 2, possess promising
anticancer activity combined with low antiproliferative to
human normal cell line. These compounds are now undergoing
further lead optimization in our laboratory for development as
an anticancer agent.
3.3. Antiproliferative assay
The antiproliferative activity of 1e24 was determined
using a standard (MTT)-based colorimetric assay (Sigma),
using etoposide as reference drugs. Briefly, cell lines were
seeded at a density of 7 ꢁ 103 cells/well in 96-well microtiter
plates (Costar). After 24 h, exponentially growing cells were
exposed to the indicated compounds at final concentrations
ranging from 0.1 to 100 mg/mL. After 48 h, cell survival was
determined by the addition of an MTT solution (10 mL of
5 mg/mL MTT in PBS). After 4 h, 100 mL of 10% SDS in
0.01 N HCl was added, and the plates were incubated at
37 ꢀC for a further 18 h; optical absorbance was measured at
570 nm on an LX300 Epson Diagnostic microplate reader.
Survival ratios are expressed in percentages with respect to
untreated cells. IC50 values were determined from replicates
of six wells from at least two independent experiments.
Acknowledgments
The work was financed by a grant (Project 30772627) from
National Natural Science Foundation of China.
References
[1] M. Gourley, J.S. Williamson, Curr. Pharm. Des. 6 (2000) 417e439.
[2] A. Ullrich, J. Schlessinger, Cell 61 (1990) 203e212.
[3] S.R. Hubbard, J.H. Till, Annu. Rev. Biochem. 69 (2000) 373e398.
[4] M.J. Hayman, P.J. Enrietto, Cancer Cells 3 (1991) 302e307.
[5] I. Dikic, S. Giordano, Curr. Opin. Cell Biol. 15 (2003) 128e135.
[6] F.M. Uckun, C. Mao, Curr. Pharm. Des. 10 (2004) 1083e1091.
[7] P. Traxler, J. Green, H. Mett, U. Sequin, P. Furet, J. Med. Chem. 42
(1999) 1018e1026.
4. Conclusions
In approach of preparing new anticancer agents, we have
synthesized a series of analogues of 1,3-disubstituted urea
derivatives. For all the compounds synthesized, antiprolifera-
tive activity against one human normal cell line (L02) and
two tumor cell lines (K562 and KB) was determined. The
chemical modification of urease derivatives generated some
potent compounds, with IC50 values up to over 20-fold lower
comparable to those of the reference compounds etoposide.
The series of 1,3-disubstituted urea derivatives show good
antiproliferative activity against human cancer cell lines and
no antiproliferative activity against normal cell line (L02).
Compounds with a morpholine ring exhibited remarkable
[8] J.A. Shimshoni, M. Bialer, B. Wlodarczyk, R.H. Finnell, B. Yagen, J.
Med. Chem. 50 (2007) 6419e6427.
[9] J.S. Fortin, J. Lacroix, M. Desjardins, P. Michel, P.E. Alexandre,
C.G. Rene, Bioorg. Med. Chem. 15 (2007) 4456e4469.
[10] R.J. Watson, D.R. Allen, H.L. Birch, G.A. Chapman, A. Gayle,
F.C. Frances, L.A. Knight, D. Meier, K. Oliver, J.W.G. Meissner,
D.A. Owen, E.J. Thomas, N. Tremayne, S.C. Williams, Bioorg. Med.
Chem. Lett. 18 (2008) 147e151.
[11] P. Be0chard, J. Lacroix, P. Poyet, Eur. J. Med. Chem. 29 (1994) 963e966.
[12] P. Cao, X.F. Huang, D. Hui, H.M. Ge, H.Q. Li, B.F. Ruan, H.L. Zhu,
Chem. Biodiv. 4 (2007) 881e886.
[13] P. Krzysztof, K. P.JoannaPiotr, R. Jan, P. Wies1aw, Z.S. Oliwia, B. Jerzy,
S.S. Janusz, P.M. Aleksander, G. Grzegorz, Cancer Lett. 203 (2004)
59e65.