Synthesis and structure-activity relationships of N-benzyl-N-(X-2-hydroxybenzyl)-N′-phenylureas and thioureas as antitumor agents

Article abstract of DOI:10.1016/j.bmc.2009.10.054

Two series of novel N-benzyl-N-(X-2-hydroxybenzyl)-N′-phenylureas and thioureas (1a-18a; 1b-18b) as potential EGFR and HER-2 kinase inhibitors have been discovered. These compounds displayed good EGFR and HER-2 inhibitory activity and the SARs are also been studied. Especially compound 7b demonstrated significant EGFR and HER-2 inhibitory activity (IC₅₀ = 0.08 μM for EGFR and IC₅₀ = 0.35 μM for HER-2). Docking simulation was performed to position compound 7b into the EGFR active site to determine the probable binding conformation and antiproliferative assay results indicating that these series of urea and thioureas own high antiproliferative activity against MCF-7. Above all, thiourea 7b would be a potential anticancer agent deserves further research.

Full text of DOI:10.1016/j.bmc.2009.10.054

Bioorganic & Medicinal Chemistry 18 (2010) 305–313
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of N-benzyl-N-
(X-2-hydroxybenzyl)-N0-phenylureas and thioureas as antitumor agents
*
*
Huan-Qiu Li, Tao Yan, Ying Yang, Lei Shi, Chang-Fang Zhou , Hai-Liang Zhu
State Key Laboratory of Pollution Control and Resource Reuse, State Key Laboratory of Pharmaceutical
Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Two series of novel N-benzyl-N-(X-2-hydroxybenzyl)-N0-phenylureas and thioureas (1a–18a; 1b–18b) as
potential EGFR and HER-2 kinase inhibitors have been discovered. These compounds displayed good
EGFR and HER-2 inhibitory activity and the SARs are also been studied. Especially compound 7b demon-
Article history:
Received 9 September 2009
Revised 27 October 2009
Accepted 27 October 2009
Available online 31 October 2009
strated significant EGFR and HER-2 inhibitory activity (IC50 = 0.08 lM for EGFR and IC50 = 0.35 lM for
HER-2). Docking simulation was performed to position compound 7b into the EGFR active site to deter-
mine the probable binding conformation and antiproliferative assay results indicating that these series of
urea and thioureas own high antiproliferative activity against MCF-7. Above all, thiourea 7b would be a
potential anticancer agent deserves further research.
Keywords:
EGFR
HER-2
Structure–activity relationship
Urea
Ó 2009 Elsevier Ltd. All rights reserved.
Thiourea
Anticancer agents
1. Introduction
events in a ligand-independent manner.4–6 The role of EGFR and
HER-2 has been most thoroughly studied in breast cancer, where
Receptor protein tyrosine kinases play a key role in signal trans-
duction pathways that regulate cell division and differentiation.
Upon binding to their specific extracellular growth factors, recep-
tor tyrosine kinases undergo dimerization and autophosphoryla-
tion, initiating a cascade of downstream signaling events. Many
of the growth factor receptor proteins have intracellulardomains
that function as tyrosine kinases, and it is with this that they effect
signaling. The interaction of growth factors with these receptors is
a necessary event in normal regulation of cell growth. However,
under certain conditions, as a result of overexpression, mutation,
or coexpression of the ligand and the receptor, these receptors
can become hyperactivated; the result of this is uncontrolled cell
proliferation.1 Among the growth factor receptor kinases that have
been identified as being important in cancer is epidermal growth
factor receptor (EGFR) kinase (also known as erb-B1 or HER-1)
and the related human epidermal growth factor receptor HER-2
(also known as erbB-2). Deregulation of growth-factor signaling
due to hyperactivation of the ErbB receptors (primarily EGFR and
HER-2) is seen in several cancer types.2,3 Activation of EGFR may
be because of overexpression, mutations resulting in constitutive
activation, or autocrine expression of ligand. In contrast, activation
of HER-2 occurs mainly by overexpression, which leads to sponta-
neous homodimerization and activation of downstream signaling
it is overexpressed in 25–30% of cases and is correlated with a poor
prognosis. Overexpression occurs primarily as a result of gene
amplification. EGFR and HER-2 overexpression is also seen in ovar-
ian cancer,7 lung cancer (especially lung adenocarcinomas),8–10
and in hormone-refractory prostate cancer.11 Compounds that in-
hibit the kinase activity of EGFR and/or HER-2 after binding of its
cognate ligand are of potential interest as new therapeutic antitu-
mor agents.12,13
The urea derivatives such as N-nitrosoureas, benzoylureas, thio-
ureas represent one of the generally most useful classes of antican-
cer agents, with
a wide range of activities against various
leukemias and solid tumors. The urea and the thiourea derivatives
play important role in anticancer agents because of their good
inhibitory activity against Receptor tyrosine kinases (RTKs), pro-
tein tyrosine kinases (PTKs), and NADH oxidase, which play critical
roles in many aspects of tumorigenesis.14–16 For example, pyrazol-
opyrimidine ureas bound to the ATP binding site of KDR kinase, the
amino thienopyrimidine core mimics the adenine of ATP in its
interaction with the hinge region of KDR.17 Hydrogen bonds are
formed between the exocyclic amino group of pyrazolopyrimidine
ureas and the backbone carbonyl of Glu 917 and the proximal ring
nitrogen and the backbone N–H of Cys 919. Our interest in this area
is to design and synthesize diverse active urea and thiourea deriv-
atives for antitumor agents.18–20 Since the tyrosine phosphoryla-
tion event catalyzed by EGFR or HER-2 propagates the signal for
cell division and since deregulation of these kinases has been
* Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.054

306
H.-Q. Li et al. / Bioorg. Med. Chem. 18 (2010) 305–313
associated with disease, an inhibitor of this event may have poten-
tial therapeutic value. Since these two receptor kinases have a high
sequence homology in their catalytic domains, in continuation of
our earlier studies, we now first describe the synthesis and the
SAR of two series of N-benzyl-N-(X-2-hydroxybenzyl)-N0-phenylu-
reas and thioureas. These compounds are potent inhibitors of EGFR
and HER-2, docking simulations were performed using the X-ray
crystallographic structure of the EGFR in complex with an inhibitor
to explore the binding modes of these compounds at the active
site.
condensed with phenylisocyanate or phenylisothiocyanate in chlo-
roform as the solvent, affording the target compounds after 2–8 h
for urea and thiouurea derivatives with electron-withdrawing sub-
stituent groups (1a–12a and 1b–12b), and after 4 h for the elec-
tron-donating substituted derivatives (13a–18a and 13b–18b).
The reactions were monitored by thin layer chromatography
(TLC) and the products were purified by recrystallization in etha-
nol. The yields were in the range of 55–95% for ureas and 47–
88% for the thioureas. All the ureas and thioureas 1a–18a and
1b–18b were synthesized for the first time and all compounds
were fully characterized by 1H NMR, ESI MS and elemental
analysis.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity and molecular modeling
The synthesized N-benzyl-N-(X-2-hydroxybenzyl)-N0-phenylu-
reas and thioureas were evaluated for their ability to inhibit the
autophosphorylation of EGFR and HER-2 kinases using a solid-
phase ELISA assay. The results were summarized in Table 1. For
the given compounds, we observed that the IC50 value for inhibi-
tion of HER-2 kinase is, in general, higher than that observed for
EGFR kinase but have the same trends. This is possibly due, in part,
to the fact that in the enzyme assays, we used higher concentration
of the purified HER-2 kinase than EGFR kinase. It is evident that
there is also a reasonable correlation between the EGFR and
HER-2 inhibitory activities, thus, this is not surprising in view of
The route adapted for the synthesis of N-benzyl-N-(X-2-
hydroxybenzyl)-N0-phenylureas and thioureas is outlined in
Schemes 1 and 2. In our previous study, salicylaldehyde derivatives
with halo-atoms in the R1 and R2 positions of aromatic ring showed
potent biological activities, and the activity of methyl group in the
R1 and R2 positions was low.21 So we choose the salicylaldehyde
with H, Cl, Br, and Me groups at R1 and R2 positions to prepare
the corresponding Schiff bases. Without further purification, the
secondary amines (1–18) were obtained from the corresponding
Schiff bases after the reduction reactions with sodium borohydride
in 60–90% yield.22 In the next step, the secondary amines were
Scheme 1. Synthesis of compounds 1a–12a and 1b–12b. Reagents and conditions: (A) ethanol, rt, 2 h; (B) ethanol, NaBH4, reflux, 3–6 h; (C) phenylisocyanate, chloroform,
reflux, 2–4 h; (D) phenylisothiocyanate, chloroform, reflux, 2–4 h.

H.-Q. Li et al. / Bioorg. Med. Chem. 18 (2010) 305–313
307
Scheme 2. Synthesis of compounds 13a–18a and 13b–18b. Reagents and conditions: (A) ethanol, 60 °C, 4 h; (B) ethanol, NaBH4, reflux, 8–10 h; (C) phenylisocyanate,
chloroform, reflux, 4–6 h; (D) phenylisothiocyanate, chloroform, reflux, 4–6 h.
0.35
l
M for HER-2), and comparable to the positive control erloti-
Table 1
Inhibition (IC50) of EGFR and HER-2 kinases and inhibition (IC50) of cell proliferation
nib (MIC IC50 = 0.02
lM). In the all ureas and thioureas, thioureas
R1
R2
R3
Enzyme assays IC50
(
lM)
MCF-7 IC50 (lM)
(1b–18b) displayed potent inhibitory activity than ureas (1a–18a),
it suggests that the external N–H of the thiourea has a better
hydrogen bond interaction with the protein than the urea.
Structure–activity relationships in these new compounds dem-
onstrated that compounds containing a R1 and/or R2 = Br or Cl are
consistently more potent than those with a methyl group. This
trend was observed in all the series of compounds used herein
whether they were ureas or thioureas. Many compounds with
R1 = R2 or R1 = H (e.g., 1b and 5b, 2b and 4b) shown IC50 in the
Compd
EGFR
HER-2
1a
2a
3a
4a
5a
6a
7a
8a
H
H
H
Cl
Br
Me
H
H
H
Cl
Br
Me
H
H
H
Cl
Br
Me
H
H
H
Cl
Br
Me
H
H
H
Cl
Br
Me
H
H
H
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
Me
Cl
Br
F
F
F
F
F
F
OH
OH
OH
OH
OH
OH
—
—
—
—
—
—
F
F
F
F
F
F
OH
OH
OH
OH
OH
OH
—
—
—
—
—
1.98
2.24
21.71
4.15
4.68
27.35
0.96
1.07
7.51
1.86
1.12
9.26
32.12
36.88
>50
3.45
5.17
38.23
9.64
12.42
>50
2.27
2.66
18.43
2.21
4.53
16.12
>50
1.1 0.3
1.4 0.5
5.5 2.3
2.8 1.3
3.6 1.4
6.6 2.7
0.19 0.04
0.25 0.06
5.3 2.6
1.1 0.4
1.32 0.46
8.5 3.3
9.3 2.7
9.1 2.9
52 24
1 lM range, indicating little tolerance of bulkiness in the hydro-
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
15b
16b
17b
18b
Erlotinib
phobic pocket of the ATP site of EGFR (and, presumably, HER-2).
Hydroxyl derivatives at R3 position are more potent then corre-
sponding fluorine derivatives, so the increased lipophilicity of fluo-
rine derivatives at R3 position may be detrimental to the inhibitory
activity. Ureas and thioureas with alkyl group at N-2 (13a–18a and
>50
>50
>50
>50
30
35 13
26
9
13b–18b) displayed weaker inhibitory activity (most >50 lM) than
>50
>50
others, we surmised that the alkyl chain group in the urea and
thioureas led to decrease in EGFR and HER-2 inhibitory activity
due to the ability of the alkyl chain group to impose a torsion angle
between the plane of the aromatic urea bicycle and that of the ure-
ido function of the resulting molecules. This may lead to a less pla-
nar structure, a geometry that may hinder its binding in the ATP
pocket of EGFR (and, presumably, HER-2).
To help understand the SARs observed at the EGFR and guide
further SAR studies, molecular docking of the most potent inhibitor
7b into ATP binding site of EGFR kinase was performed on the
binding model based on the EGFR complex structure (1M17.pdb).
The binding model of compound 7b and EGFR is depicted in Figure
1. In the binding model, thiourea 7b is nicely bound to the region
with the urea N–H groups of 7b project toward the side chain car-
bonyl group of Leu 768, with the external N–H forming a more
optimal H-bond interaction; the hydroxyl group of 7b also forms
hydrogen bond with amino hydrogen of Gln767, while the corre-
sponding urea 7a can not bound nicely. There is only carbonyl
>50
>50
8
0.67
0.98
8.54
1.34
1.76
19.41
0.08
0.12
3.46
0.56
0.87
5.83
18.12
22.04
43.21
24.65
31.94
>50
1.41
1.87
15.66
6.49
6.73
37.82
0.35
0.66
7.13
1.23
2.24
9.47
32.54
39.51
>50
0.52 0.45
0.73 0.6
3.8 1.3
1.1 0.4
1.7 0.5
10.5 4.4
0.03 0.01
0.06 0.04
1.2 0.6
0.22 0.21
0.36 0.32
2.6 1.0
7.4 2.6
14.2 5.1
23.6 7.41
12.8 3.7
16.7 6.3
34.1 11.1
Cl
Br
Me
39.78
>50
>50
Me
—
0.02
0.12
0.01 0.003
group bond with the amine group of Lys828 and a p-cation inter-
the high sequence homology of the catalytic domains of these two
kinases.
A number of synthesized compounds displayed potent EGFR
and HER-2 inhibitory activity. N-(5-Chloro-2-hydroxybenzyl)-N-
(4-hydroxybenzyl)-N0-phenylthiourea (7b) showed the most
action between benzyl ring of compound 7a and Lys828 (Fig. 2).
These were supported by EGFR potency difference between ureas
(1a–18a) and thioureas (1b–18b). The modeling also suggested
that there is a
pound 7b and Lys828,
order of magnitude as hydrogen bonds or salt bridges and play an
p
-cation interaction between R3-benzyl ring of com-
p-cation interaction energies are of the same
potent inhibitory activity (IC50 = 0.08 lM for EGFR and IC50 =

308
H.-Q. Li et al. / Bioorg. Med. Chem. 18 (2010) 305–313
strated significant efficacy in tumor growth inhibition and dis-
played favorable EGFR and HER-2 inhibitory activity.
3. Conclusion
Two series of N-benzyl-N-(X-2-hydroxybenzyl)-N0-phenylureas
and thioureas that may function as inhibitors of EGFR and HER-2 ki-
nases have been prepared, a number of synthesized compounds dis-
played potent EGFR and HER-2 inhibitory. Extensive SAR studies led
to the conclusion that compounds containing a R1 and/or R2 = Br or
Cl are consistently more potent than those with a methyl group,
the alkyl chain group in the urea and thioureas led to decrease in
EGFR and HER-2 inhibitory activity. The EGFR molecular docking
model suggested that the urea N–H groups of thiourea project
toward carbonyl group of Leu 768,
p-cation interaction between
R3-benzyl ring and Lys828 may an important role in stabilizing the
three dimensional structure of a protein. The decrease of activity
caused by the replacement of the terminal aryl group with alkyl
group (13a–18a and 13b–18b) is probably due to the effect of an
inferior interaction of the alkyl groups with the hydrophobic region.
Antiproliferative assay results indicating that these series of urea
and thioureas own high inhibitory activity against MCF-7, in partic-
ular, thiourea 7b has demonstrated significant EGFR inhibitory
activity and antiproliferative activity as a potential anticancer agent.
Figure 1. Molecular docking modeling of compounds 7b with EGFR kinase: The N–
H groups of 7b project toward the side chain carbonyl group of Leu 768, the
hydroxyl group of 7b also forms hydrogen bond with amino hydrogen of Gln767,
and there is a
Lys828.
p
-cation interaction between R3-benzyl ring of compound 7b and
4. Experimental
4.1. Chemistry general
All the NMR spectra were recorded on a Bruker DRX 500 or DPX
300 model Spectrometer in DMSO-d6. Chemical shifts (d) for 1H
NMR spectra were 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 Mariner
System 5304 Mass spectrometer. All chemicals and reagents used
in current study were of analytical grade. TLC was run on the silica
gel coated aluminum sheets (Silica Gel 60 GF254, E. Merk, Germany)
and visualized in UV light (254 nm).
4.2. General procedure for amines preparations (1–18)
Figure 2. Molecular docking modeling of compounds 7a with EGFR kinase.
To a solution of R1 and R2 substituted salicylaldehyde (2 mmol)
and the primary substituted amines (2 mmol) were added, in eth-
anol. The mixture was refluxed, with stirring and the water was
collected in a Dean–Stark apparatus. The reaction was monitored
by TLC. The products were filtered and recrystallized in ethanol.
Without further purification, to an ethanolic solution of synthe-
sized Schiff bases (2 mmol), NaBH4 (2 mmol) was slowly added
in an ice bath with stirring. The mixture was refluxed for 3–6 h,
then the solvent was evaporated and water (2 mL) was added.
The product extraction was carried out with CHCl3 (3 ꢀ 2 mL).
The organic layer was dried over Na2SO4, and after solvent evapo-
ration the residue was purified by flash column chromatography
(Silica Gel 60, 35–70 mesh) using CHCl3 as the eluent.
important role in stabilizing the three dimensional structure of a
protein.23
The phenyl group in 7b projects into a hydrophobic region,
which is comprised of the side chains of Pro 770, Lys 822 and
Met 769, that was important for the potent inhibitory activity of
7b, the decrease of activity caused by the replacement of the termi-
nal aryl group with alkyl group (13a–18a and 13b–18b) is proba-
bly due to the effect of an inferior interaction of the alkyl groups
with the hydrophobic region. Also, in these thiourea compounds,
replacement of a methyl group or a bromide at R2 position by a
chloride group resulted in the decrease of EGFR inhibitory activity.
This suggested that compounds with strong electron-drawing
groups on aromatic ring may benefit for the molecular binding be-
tween the compounds with EGFR.
The in vitro antiproliferative activity of the synthesized N-ben-
zyl-N-(X-2-hydroxybenzyl)-N0-phenylureas and thioureas was
studied on a panel of one human tumor cell line (MCF-7), which
overexpresses EGFR and, to a less extent HER-2. The compounds
which have potent inhibitory activity of EGFR and HER-2 displayed
high antiproliferative activity against MCF-7, indicating that these
series ureas and thioureas were potent inhibitor of EGFR and HER-
2 as antitumor agents. In particular, compounds 7b has demon-
4.2.1. 4-Chloro-2-((4-fluorobenzylamino)methyl)phenol (1)
Yellow oil. Yield: 84%; 1H NMR (DMSO-d6) d = 1.58 (s, 1H), 3.64
(s, 2H), 3.83 (s, 2H), 6.82 (d, J = 8.4 Hz, 1H), 7.20–7.32 (m, 6H),
13.72 (s, 1H). MS (ESI) C14H14ClFNO [M+H]+ 266.1. Anal. Calcd for
C14H13ClFNO: C, 63.28; H, 4.93; Cl, 13.34; F, 7.15; N, 5.27. Found:
C, 63.25; H, 4.92; Cl, 13.31; F, 7.14; N, 5.26.
4.2.2. 4-Bromo-2-((4-fluorobenzylamino)methyl)phenol (2)
Yellow oil. Yield: 81%; 1H NMR (DMSO-d6) d = 1.55 (s, 1H), 3.63
(s, 2H), 3.81 (s, 2H), 6.77 (d, J = 8.4 Hz, 1H), 7.15–7.28 (m, 6H),

H.-Q. Li et al. / Bioorg. Med. Chem. 18 (2010) 305–313
309
13.76 (s, 1H). MS (ESI) C14H14BrFNO [M+H]+ 310.0. Anal. Calcd for
C14H13BrFNO: C, 54.21; H, 4.22; Br, 25.76; F, 6.13; N, 4.52. Found:
C, 54.18; H, 4.21; Br, 25.72; F, 6.11; N, 4.49.
C14H13Br2NO2: C, 43.44; H, 3.39; Br, 41.29; N, 3.62. Found: C,
43.41; H, 3.32; Br, 41.24; N, 3.60.
4.2.12. 2-((4-Hydroxybenzylamino)methyl)-4,6-
dimethylphenol (12)
4.2.3. 2-((4-Fluorobenzylamino)methyl)-4-methylphenol (3)
Yellow oil. Yield: 93%; 1H NMR (DMSO-d6) d = 1.58 (s, 1H), 2.48
(s, 3H), 3.65 (s, 2H), 3.86 (s, 2H), 6.85 (s, 1H), 7.21–7.34 (m, 6H),
13.73 (s, 1H). MS (ESI) C15H17FNO [M+H]+ 246.1. Anal. Calcd for
C15H16FNO: C, 73.45; H, 6.57; F, 7.75; N, 5.71. Found: C, 73.41; H,
6.52; F, 7.73; N, 5.74.
Yellow oil. Yield: 84%; 1H NMR (DMSO-d6) d = 1.56 (s, 1H), 2.31
(s, 3H), 2.47 (s, 3H), 3.65 (s, 2H), 3.81 (s, 2H), 7.10–7.23 (m, 4H),
7.29–7.32 (m, 2H), 13.71 (s, 1H). MS (ESI) C16H20NO2 [M+H]+
258.1. Anal. Calcd for C16H19NO2: C, 74.68; H, 7.44; N, 5.44. Found:
C, 74.65; H, 7.41; N, 5.42.
4.2.4. 2,4-Dichloro-6-((4-fluorobenzylamino)methyl)phenol (4)
Yellow oil. Yield: 74%; 1H NMR (DMSO-d6) d = 1.56 (s, 1H), 3.66
(s, 2H), 3.85 (s, 2H), 7.18–7.29 (m, 6H). MS (ESI) C14H13Cl2FNO
[M+H]+ 300.0. Anal. Calcd for C14H12Cl2FNO: C, 56.02; H, 4.03; Cl,
23.62; F, 6.33; N, 4.67. Found: C, 55.94; H, 4.05; Cl, 23.63; F,
6.31; N, 4.65.
4.2.13. 2-((Butylamino)methyl)-4-chlorophenol (13)
Yellow oil. Yield: 70%; 1H NMR (DMSO-d6) d = 0.91 (t, J = 7.5 Hz,
3H), 1.29–1.41 (m, 2H), 1.52 (s, 1H), 1.57–1.66 (m, 2H), 3.30–3.32
(m, 2H), 3.63 (s, 2H), 6.87 (d, J = 9 Hz, 1H), 7.31–7.35 (m, 1H),
7.54(t, J = 4.5 Hz, 1H), 13.76 (s, 1H). MS (ESI) C11H17ClNO [M+H]+
214.1. Anal. Calcd for C11H16ClNO: C, 61.82; H, 7.55; Cl, 16.59; N,
6.55. Found: C, 61.81; H, 7.52; Cl, 16.56; N, 6.58.
4.2.5. 2,4-Dibromo-6-((4-fluorobenzylamino)methyl)phenol (5)
Yellow oil. Yield: 78%; 1H NMR (DMSO-d6) d = 1.55 (s, 1H), 3.63
(s, 2H), 3.81 (s, 2H), 7.15–7.28 (m, 6H). MS (ESI) C14H13Br2FNO
[M+H]+ 387.9. Anal. Calcd for C14H12Br2FNO: C, 43.22; H, 3.11; Br,
41.08; F, 4.88; N, 3.60. Found: C, 43.26; H, 3.07; Br, 41.03; F,
4.84; N, 3.61.
4.2.14. 4-Bromo-2-((butylamino)methyl)phenol (14)
Yellow oil. Yield: 76%; 1H NMR (DMSO-d6) d = 0.90 (t, J = 7.5 Hz,
3H), 1.29–1.41 (m, 2H), 1.52 (s, 1H), 1.57–1.66 (m, 2H), 3.30–3.33
(m, 2H), 3.63 (s, 2H), 6.85 (d, J = 9 Hz, 1H), 7.29–7.33 (m, 1H), 7.53
(t, J = 4.5 Hz, 1H), 13.71 (s, 1H). MS (ESI) C11H17BrNO [M+H]+ 258.0.
Anal. Calcd for C11H16BrNO: C, 51.18; H, 6.25; Br, 30.95; N, 5.43.
Found: C, 51.13; H, 6.22; Br, 30.91; N, 5.45.
4.2.6. 2-((4-Fluorobenzylamino)methyl)-4,6-dimethylphenol
(6)
Yellow oil. Yield: 83%; 1H NMR (DMSO-d6) d = 1.58 (s, 1H), 2.26
(s, 3H), 2.48 (s, 3H), 3.65 (s, 2H), 3.86 (s, 2H), 7.21–7.34 (m, 6H). MS
(ESI) C16H19FNO [M+H]+ 260.1. Anal. Calcd for C16H18FNO: C, 74.11;
H, 7.00; F, 7.33; N, 5.40. Found: C, 74.09; H, 7.03; F, 7.31; N, 5.46.
4.2.15. 2-((Butylamino)methyl)-4-methylphenol (15)
Yellow oil. Yield: 67%; 1H NMR (DMSO-d6) d = 0.91 (t, J = 7.5 Hz,
3H), 1.28–1.39 (m, 2H), 1.51 (s, 1H), 1.57–1.66 (m, 2H), 2.44 (s, 3H),
3.32–3.35 (s, 2H), 3.63 (s, 2H), 6.90 (d, J = 9 Hz, 1H), 7.33–7.38 (m,
1H), 7.57 (t, J = 4.5 Hz, 1H), 13.77 (s, 1H). MS (ESI) C12H20NO
[M+H]+ 194.2. Anal. Calcd for C12H19NO: C, 74.57; H, 9.91; N,
7.25. Found: C, 74.51; H, 9.92; N, 7.23.
4.2.7. 4-Chloro-2-((4-hydroxybenzylamino)methyl)phenol (7)
Yellow oil. Yield: 90%; 1H NMR (DMSO-d6) d = 1.56 (s, 1H), 3.63
(s, 2H), 3.81 (s, 2H), 6.83 (s, 1H), 7.09–7.22 (m, 4H), 7.27–7.30 (m,
2H), 13.73 (s, 1H). MS (ESI) C14H15ClNO2 [M+H]+ 264.1. Anal. Calcd
for C14H14ClNO2: C, 63.76; H, 5.35; Cl, 13.44; N, 5.31. Found: C,
63.72; H, 5.31; Cl, 13.42; N, 5.33.
4.2.16. 2-((Butylamino)methyl)-4,6-dichlorophenol (16)
Yellow oil. Yield: 72%; 1H NMR (DMSO-d6) d = 0.91 (t, J = 7.5 Hz,
3H), 1.29–1.41 (m, 2H), 1.52 (s, 1H), 1.57–1.66 (m, 2H), 3.30–3.34
(m, 2H), 3.63 (s, 2H), 7.32 (s, 1H), 7.52 (t, J = 4.5 Hz, 1H), 13.76 (s,
1H). MS (ESI) C11H16Cl2NO [M+H]+ 248.1. Anal. Calcd for
C11H15Cl2NO: C, 53.24; H, 6.09; Cl, 28.57; N, 5.64. Found: C,
53.20; H, 6.05; Cl, 28.56; N, 5.65.
4.2.8. 4-Bromo-2-((4-hydroxybenzylamino)methyl)phenol (8)
Yellow oil. Yield: 71%; 1H NMR (DMSO-d6) d = 1.54 (s, 1H), 3.63
(s, 2H), 3.81 (s, 2H), 6.81 (s, 1H), 7.07–7.21 (m, 4H), 7.25–7.28 (m,
2H), 13.66 (s, 1H). MS (ESI) C14H15BrNO2 [M+H]+ 308.0. Anal. Calcd
for C14H14BrNO2: C, 54.56; H, 4.58; Br, 25.93; N, 4.55. Found: C,
54.52; H, 4.51; Br, 25.92; N, 4.56.
4.2.17. 2,4-Dibromo-6-((butylamino)methyl)phenol (17)
Yellow oil. Yield: 60%; 1H NMR (DMSO-d6) d = 0.90 (t, J = 7.5 Hz,
3H), 1.29–1.41 (m, 2H), 1.52 (s, 1H), 1.57–1.66 (m, 2H), 3.28–3.31
(m, 2H), 3.63 (s, 2H), 7.30 (s, 1H), 7.53 (t, J = 4.5 Hz, 1H), 13.71 (s,
1H). MS (ESI) C11H16Br2NO [M+H]+ 336.0. Anal. Calcd for
C11H15Br2NO: C, 39.20; H, 4.49; Br, 47.41; N, 4.16. Found: C,
39.17; H, 4.45; Br, 47.37; N, 4.13.
4.2.9. 2-((4-Hydroxybenzylamino)methyl)-4-methylphenol (9)
Yellow oil. Yield: 82%; 1H NMR (DMSO-d6) d = 1.56 (s, 1H), 2.48
(s, 3H), 3.65 (s, 2H), 3.81 (s, 2H), 6.85 (s, 1H), 7.10–7.23 (m, 4H),
7.29–7.32 (m, 2H), 13.71 (s, 1H). MS (ESI) C15H18NO2 [M+H]+
244.1. Anal. Calcd for C15H17NO2: C, 74.05; H, 7.04; N, 5.76. Found:
C, 74.01; H, 7.03; N, 5.72.
4.2.18. 2-((Butylamino)methyl)-4,6-dimethylphenol (18)
Yellow oil. Yield: 71%; 1H NMR (DMSO-d6) d = 0.91 (t, J = 7.5 Hz,
3H), 1.28–1.39 (m, 2H), 1.51 (s, 1H), 1.57–1.66 (m, 2H), 2.24 (s, 3H),
2.44 (s, 3H), 3.28–3.31 (m, 2H), 3.63 (s, 2H), 7.37 (s, 1H), 7.57 (t,
J = 4.5 Hz, 1H), 13.77 (s, 1H). MS (ESI) C13H22NO [M+H]+ 208.2.
Anal. Calcd for C13H21NO: C, 75.32; H, 10.21; N, 6.76. Found: C,
75.26; H, 10.27; N, 6.71.
4.2.10. 2,4-Dichloro-6-((4-hydroxybenzylamino)methyl)phenol
(10)
Yellow oil. Yield: 78%; 1H NMR (DMSO-d6) d = 1.55 (s, 1H), 3.63
(s, 2H), 3.81 (s, 2H), 7.09–7.22 (m, 4H), 7.27–7.30 (m, 2H), 13.73 (s,
1H). MS (ESI) C14H14Cl2NO2 [M+H]+ 298.0. Anal. Calcd for
C14H13Cl2NO2: C, 56.39; H, 4.39; Cl, 23.78; N, 4.70. Found: C,
56.33; H, 4.35; Cl, 23.76; N, 4.67.
4.3. General procedure for ureas (1a–18a) and thioureas (1b–
18b) preparations
4.2.11. 2,4-Dibromo-6-((4-hydroxybenzylamino)methyl)phenol
(11)
To chloroform solution of amines (1–18) (1.5 mmol), phenyliso-
cyanate or phenylisothiocyanate (1.5 mmol) was slowly added with
stirring. The mixture was refluxed for 2–4 h. After this time, the
product was filtered and purified by recrystallization from ethanol.
Yellow oil. Yield: 79%; 1H NMR (DMSO-d6) d = 1.56 (s, 1H), 3.63
(s, 2H), 3.81 (s, 2H), 7.07–7.21 (m, 4H), 7.25–7.28(m, 2H), 13.66 (s,
1H). MS (ESI) C14H14Br2NO2 [M+H]+ 385.9. Anal. Calcd for

310
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4.3.1. 1-(5-Chloro-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylurea (1a)
4.3.9. 1-(2-Hydroxy-5-methylbenzyl)-1-(4-hydroxybenzyl)-3-
phenylurea (9a)
Mp 172–173 °C; yield: 85%; 1H NMR (DMSO-d6) d = 4.39 (s, 2H),
4.72 (s, 2H), 6.81 (d, J = 8.4 Hz, 1H), 7.03 (s, 1H), 7.08–7.18 (m, 4H),
7.24–7.34 (m, 6H), 9.61 (s, 1H), 10.11 (s, 1H). MS (ESI)
C21H19ClFN2O2 [M+H]+ 385.1. Anal. Calcd for C21H18ClFN2O2: C,
65.54; H, 4.71; Cl, 9.21; F, 4.94; N, 7.28. Found: C, 65.48; H, 4.67;
Cl, 9.17; F, 4.92; N, 7.29.
Mp 148–149 °C; yield: 77%; 1H NMR (DMSO-d6) d = 2.49 (s, 3H),
4.43 (s, 2H), 4.76 (s, 2H), 6.83 (d, J = 8.4 Hz, 1H), 7.01 (s, 1H), 7.05–
7.13 (m, 3H), 7.24–7.38 (m, 7H), 9.43 (s, 1H), 10.07 (s, 1H). MS (ESI)
C22H23N2O3 [M+H]+ 363.2. Anal. Calcd for C22H22N2O3: C, 72.91; H,
6.12; N, 7.73. Found: C, 72.89; H, 6.09; N, 7.71.
4.3.10. 1-(3,5-Dichloro-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-
3-phenylurea (10a)
4.3.2. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylurea (2a)
Mp 141–142 °C; yield: 72%; 1H NMR (DMSO-d6) d = 4.27 (s, 2H),
4.68 (s, 2H), 6.88–7.21 (m, 10H), 7.55 (s, 1H), 10.41 (s, 1H). MS (ESI)
C21H19Cl2N2O3 [M+H]+ 417.1. Anal. Calcd for C21H18Cl2N2O3: C,
60.44; H, 4.35; Cl, 16.99; N, 6.71. Found: C, 60.42; H, 4.31; Cl,
16.95; N, 6.67.
Mp 158–159 °C; yield: 92%; 1H NMR (DMSO-d6) d = 4.42 (s, 2H),
4.73 (s, 2H), 6.80 (d, J = 8.4 Hz, 1H), 7.07–7.43 (m, 11H), 9.44 (s,
1H), 10.12 (s, 1H). MS (ESI) C21H19BrFN2O2 [M+H]+ 429.1. Anal.
Calcd for C21H18BrFN2O2: C, 58.75; H, 4.23; Br, 18.61; F, 4.43; N,
6.53. Found: C, 58.70; H, 4.22; Br, 18.56; F, 4.39; N, 6.52.
4.3.11. 1-(3,5-Dibromo-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-
3-phenylurea (11a)
4.3.3. 1-(4-Fluorobenzyl)-1-(2-hydroxy-5-methylbenzyl)-3-
phenylurea (3a)
Mp 130–131 °C; yield: 79%; 1H NMR (DMSO-d6) d = 4.39 (s, 2H),
4.77 (s, 2H), 6.92–7.24 (m, 10H), 7.57 (s, 1H), 10.34 (s, 1H). MS (ESI)
C21H19Br2N2O3 [M+H]+ 505.0. Anal. Calcd for C21H18Br2N2O3: C,
49.83; H, 3.58; Br, 31.57; N, 5.53. Found: C, 49.85; H, 3.53; Br,
31.54; N, 5.51.
Mp 172–173 °C; yield: 86%; 1H NMR (DMSO-d6) d = 2.46 (s, 3H),
4.43 (s, 2H), 4.70 (s, 2H), 6.83 (d, J = 8.4 Hz, 1H), 7.01 (s, 1H), 6.98–
7.12 (m, 2H), 7.23–7.38 (m, 8H), 9.42 (s, 1H), 10.08 (s, 1H). MS
(ESI) C22H22FN2O2 [M+H]+ 365.2. Anal. Calcd for C22H21FN2O2: C,
72.51; H, 5.81; F, 5.21; N, 7.69. Found: C, 72.48; H, 5.83; F, 5.16;
N, 7.63.
4.3.12. 1-(2-Hydroxy-3,5-dimethylbenzyl)-1-(4-
hydroxybenzyl)-3-phenylurea (12a)
Mp 127–128 °C; yield: 81%; 1H NMR (DMSO-d6) d = 2.32 (s, 3H),
2.50 (s, 3H), 4.38 (s, 2H), 4.73 (s, 2H), 7.00–7.32 (m, 10H), 7.57 (s,
1H), 10.37 (s, 1H). MS (ESI) C23H25N2O3 [M+H]+ 377.1. Anal. Calcd
for C23H24N2O3: C, 73.38; H, 6.43; N, 7.44. Found: C, 73.51; H,
6.25; N, 7.53.
4.3.4. 1-(3,5-Dichloro-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylurea (4a)
Mp 138–139 °C; yield: 95%; 1H NMR (DMSO-d6) d = 4.29 (s, 2H),
4.76 (s, 2H), 6.90–7.23 (m, 11H), 7.55 (s, 1H). MS (ESI)
C21H18Cl2FN2O2 [M+H]+ 419.1. Anal. Calcd for C21H17Cl2FN2O2: C,
60.16; H, 4.09; Cl, 16.91; F, 4.53; N, 6.68. Found: C, 60.12; H,
4.05; Cl, 16.89; F, 4.51; N, 6.65.
4.3.13. 1-Butyl-1-(5-chloro-2-hydroxybenzyl)-3-phenylurea
(13a)
Mp 123–124 °C; yield: 67%; 1H NMR (DMSO-d6) d = 0.87 (t,
J = 7.5 Hz, 3H), 1.12–1.18 (m, 2H), 1.52–1.62 (m, 2H), 3.04–3.09
(m, 1H), 3.28 (t, J = 7.5 Hz, 2H), 4.97 (s, 2H), 6.84 (d, J = 9 Hz, 1H),
7.08–7.14 (m, 3H), 7.23–7.30 (m, 4H), 9.17 (s, 1H), 10.06 (s, 1H).
MS (ESI) C18H22ClN2O2 [M+H]+ 333.1. Anal. Calcd for C18H21ClN2O2:
C, 64.96; H, 6.36; Cl, 10.65; N, 8.42. Found: C, 64.93; H, 6.32; Cl,
10.61; N, 8.40.
4.3.5. 1-(3,5-Dibromo-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylurea (5a)
Mp 133–134 °C; yield: 80%; 1H NMR (DMSO-d6) d = 4.26 (s, 2H),
4.80 (s, 2H), 6.93–7.24 (m, 11H), 7.55 (s, 1H). MS (ESI)
C21H18Br2FN2O2 [M+H]+ 507.0. Anal. Calcd for C21H17Br2FN2O2: C,
49.63; H, 3.37; Br, 31.45; F, 3.74; N, 5.51. Found: C, 49.59; H,
3.35; Br, 31.43; F, 3.71; N, 5.53.
4.3.14. 1-(5-Bromo-2-hydroxybenzyl)-1-butyl-3-phenylurea
(14a)
4.3.6. 1-(4-Fluorobenzyl)-1-(2-hydroxy-3,5-dimethylbenzyl)-3-
phenylthiourea (6a)
Mp 121–122 °C; yield: 71%; 1H NMR (DMSO-d6) d = 0.89 (t,
J = 7.5 Hz, 3H), 1.04–1.12 (m, 2H), 1.54–1.64 (m, 2H), 3.12–3.16
(m, 1H), 3.23 (t, J = 7.5 Hz, 2H), 4.97 (s, 2H), 6.84 (d, J = 9 Hz, 1H),
7.08–7.14 (m, 3H), 7.21–7.29 (m, 4H), 9.18 (s, 1H), 10.04 (s, 1H).
MS (ESI) C18H22BrN2O2 [M+H]+ 377.1. Anal. Calcd for
C18H21BrN2O2: C, 57.30; H, 5.61; Br, 21.18; N, 7.43. Found: C,
57.28; H, 5.57; Br, 21.17; N, 7.41.
Mp 128–129 °C; yield: 77%; 1H NMR (DMSO-d6) d = 2.23 (s, 3H),
2.45 (s, 3H), 4.28 (s, 2H), 4.72 (s, 2H), 6.96–7.29 (m, 11H), 7.55 (s,
1H). MS (ESI) C23H24FN2O2 [M+H]+ 379.2. Anal. Calcd for
C23H23FN2O2: C, 73.00; H, 6.13; F, 5.02; N, 7.40. Found: C, 72.95;
H, 6.11; F, 5.03; N, 7.36.
4.3.7. 1-(5-Chloro-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-3-
phenylurea (7a)
4.3.15. 1-Butyl-1-(2-hydroxy-5-methylbenzyl)-3-phenylurea
(15a)
Mp 164–165 °C; yield: 87%; 1H NMR (DMSO-d6) d = 4.42 (s, 2H),
4.68 (s, 2H), 6.81 (d, J = 8.4 Hz, 1H), 6.98 (s, 1H), 7.08–7.31 (m,
10H), 9.48 (s, 1H), 10.01 (s, 1H). MS (ESI) C21H20ClN2O3 [M+H]+
383.1. Anal. Calcd for C21H19ClN2O3: C, 65.88; H, 5.00; Cl, 9.26; N,
7.32. Found: C, 65.83; H, 4.95; Cl, 9.22; N, 7.29.
Mp 127–128 °C; yield: 68%; 1H NMR (DMSO-d6) d = 0.90 (t,
J = 7.5 Hz, 3H), 1.05–1.11 (m, 2H), 1.53–1.62 (m, 2H), 2.45 (s, 3H),
3.03–3.08 (m, 1H), 3.27 (t, J = 7.5 Hz, 2H), 4.96 (s, 2H), 6.83 (d,
J = 9 Hz, 1H), 7.10–7.17 (m, 3H), 7.23–7.34 (m, 4H), 9.21 (s, 1H),
10.07 (s, 1H). MS (ESI) C19H25N2O2 [M+H]+ 313.2. Anal. Calcd for
C19H24N2O2: C, 73.05; H, 7.74; N, 8.97. Found: C, 73.08; H, 7.67;
N, 8.93.
4.3.8. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-3-
phenylurea (8a)
Mp 155–156 °C; yield: 85%; 1H NMR (DMSO-d6) d = 4.46 (s, 2H),
4.85 (s, 2H), 6.80 (d, J = 8.4 Hz, 1H), 7.05–7.40 (m, 11H), 9.48 (s,
1H), 10.11 (s, 1H). MS (ESI) C21H20BrN2O3 [M+H]+ 427.1. Anal. Calcd
for C21H19BrN2O3: C, 59.03; H, 4.48; Br, 18.70; N, 6.56. Found: C,
59.01; H, 4.42; Br, 18.66; N, 6.53.
4.3.16. 1-Butyl-1-(3,5-dichloro-2-hydroxybenzyl)-3-
phenylturea (16a)
Mp 136–137 °C; yield: 61%; 1H NMR (DMSO-d6) d = 0.88 (t,
J = 7.5 Hz, 3H), 1.02–1.10 (m, 2H), 1.51–1.61 (m, 2H), 3.01–3.05

H.-Q. Li et al. / Bioorg. Med. Chem. 18 (2010) 305–313
311
(m, 1H), 3.20 (t, J = 7.5 Hz, 2H), 4.97 (s, 2H), 7.04–7.11 (m, 3H),
4.3.24. 1-(4-Fluorobenzyl)-1-(2-hydroxy-3,5-dimethylbenzyl)-
7.23–7.30 (m, 4H), 7.55 (s, 1H). MS (ESI) C18H21Cl2N2O2 [M+H]+
367.1. Anal. Calcd for C18H20Cl2N2O2: C, 58.86; H, 5.49; Cl, 19.31;
N, 7.63. Found: C, 58.86; H, 5.46; Cl, 19.29; N, 7.61.
3-phenylthiourea (6b)
Mp 136–137 °C; yield: 72%; 1H NMR (DMSO-d6) d = 2.24 (s, 3H),
2.47 (s, 3H), 4.77 (s, 2H), 5.13 (s, 2H), 7.04–7.35 (m, 11H), 7.57 (s,
1H). MS (ESI) C23H24FN2OS [M+H]+ 395.2. Anal. Calcd for
C23H23FN2OS: C, 70.02; H, 5.88; F, 4.82; N, 7.10; S, 8.13. Found: C,
70.05; H, 5.81; F, 4.86; N, 7.11; S, 8.15.
4.3.17. 1-Butyl-1-(3,5-dibromo-2-hydroxybenzyl)-3-phenylurea
(17a)
Mp 129–130 °C; yield: 55%; 1H NMR (DMSO-d6) d = 0.90 (t,
J = 7.5 Hz, 3H), 1.04–1.12 (m, 2H), 1.53–1.59 (m, 2H), 3.03–3.06
(m, 1H), 3.24 (t, J = 7.5 Hz, 2H), 4.96 (s, 2H), 7.08–7.14 (m, 3H),
7.22–7.33(m, 4H), 7.55 (s, 1H). MS (ESI) C18H21Br2N2O2 [M+H]+
455.0. Anal. Calcd for C18H20Br2N2O2: C, 47.39; H, 4.42; Br, 35.03;
N, 6.14. Found: C, 47.36; H, 4.41; Br, 35.99; N, 6.10.
4.3.25. 1-(5-Chloro-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-3-
phenylthiourea (7b)
Mp 168–169 °C; yield: 81%; 1H NMR (DMSO-d6) d = 4.88 (s, 2H),
5.11 (s, 2H), 6.81 (d, J = 8.4 Hz, 1H), 7.04 (s, 1H), 7.12–7.37 (m,
10H), 9.48 (s, 1H), 10.01 (s, 1H). MS (ESI) C21H20ClN2O2S [M+H]+
399.1. Anal. Calcd for C21H19ClN2O2S: C, 63.23; H, 4.80; Cl, 8.89;
N, 7.02; S, 8.04. Found: C, 63.21; H, 4.83; Cl, 8.82; N, 6.98; S, 8.03.
4.3.18. 1-Butyl-1-(2-hydroxy-3,5-dimethylbenzyl)-3-
phenylurea (18a)
Mp 133–134 °C; yield: 59%; 1H NMR (DMSO-d6) d = 0.90 (t,
J = 7.5 Hz, 3H), 1.02–1.09 (m, 2H), 1.50–1.58 (m, 2H), 2.32 (s, 3H),
2.50 (s, 3H), 3.04–3.08 (m, 1H), 3.23 (t, J = 7.5 Hz, 2H), 4.97 (s,
2H), 7.10–7.15 (m, 3H), 7.24–7.35 (m, 4H), 7.55 (s, 1H). MS (ESI)
C20H27N2O2 [M+H]+ 327.2. Anal. Calcd for C20H26N2O2: C, 73.59;
H, 8.03; N, 8.58. Found: C, 73.58; H, 8.00; N, 8.61.
4.3.26. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-3-
phenylthiourea (8b)
Mp 161–162 °C; yield: 78%; 1H NMR (DMSO-d6) d = 4.88 (s, 2H),
5.19 (s, 2H), 6.80 (d, J = 8.4 Hz, 1H), 7.11–7.46 (m, 11H), 9.48 (s,
1H), 10.11 (s, 1H). MS (ESI) C21H20BrN2O2S [M+H]+ 443.1. Anal.
Calcd for C21H19BrN2O2S: C, 56.89; H, 4.32; Br, 18.02; N, 6.32; S,
7.23. Found: C, 56.87; H, 4.26; Br, 18.04; N, 6.30; S, 7.18.
4.3.19. 1-(5-Chloro-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylthiourea (1b)
4.3.27. 1-(2-Hydroxy-5-methylbenzyl)-1-(4-hydroxybenzyl)-3-
phenylthiourea (9b)
Mp 185–186 °C; yield: 82%; 1H NMR (DMSO-d6) d = 4.88 (s, 2H),
5.11 (s, 2H), 6.81 (d, J = 8.4 Hz, 1H), 7.04 (s, 1H), 7.12–7.21 (m, 4H),
7.29–7.37 (m, 6H), 9.63 (s, 1H), 10.14 (s, 1H). MS (ESI)
C21H19ClFN2OS [M+H]+ 401.1. Anal. Calcd for C21H18ClFN2OS: C,
62.92; H, 4.53; Cl, 8.84; F, 4.74; N, 6.99; S, 8.00. Found: C, 62.91;
H, 4.55; Cl, 8.78; F, 4.71; N, 6.96; S, 8.02.
Mp 147–148 °C; yield: 83%; 1H NMR (DMSO-d6) d = 2.46 (s, 3H),
4.90 (s, 2H), 5.10 (s, 2H), 6.85 (d, J = 8.4 Hz, 1H), 7.04 (s, 1H), 7.08–
7.16 (m, 3H), 7.29–7.43 (m, 7H), 9.44 (s, 1H), 10.07 (s, 1H). MS (ESI)
C22H23N2O2S [M+H]+ 379.1. Anal. Calcd for C22H22N2O2S: C, 69.81;
H, 5.86; N, 7.40; S, 8.47. Found: C, 69.80; H, 5.82; N, 7.42; S, 8.45.
4.3.20. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylthiourea (2b)
4.3.28. 1-(3,5-Dichloro-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-
3-phenylthiourea (10b)
Mp 170–171 °C; yield: 85%; 1H NMR (DMSO-d6) d = 4.88 (s, 2H),
5.19 (s, 2H), 6.80 (d, J = 8.4 Hz, 1H), 7.11–7.46 (m, 11H), 9.46 (s,
1H), 10.13 (s, 1H). MS (ESI) C21H19BrFN2OS [M+H]+ 445.0. Anal.
Calcd for C21H18BrFN2OS: C, 56.64; H, 4.07; Br, 17.94; F, 4.27; N,
6.29; S, 7.20. Found: C, 56.60; H, 4.09; Br, 17.97; F, 4.24; N, 6.23;
S, 7.16.
Mp 152–153 °C; yield: 68%; 1H NMR (DMSO-d6) d = 4.75 (s, 2H),
5.14 (s, 2H), 6.92–7.25 (m, 10H), 7.56 (s, 1H), 10.43 (s, 1H). MS (ESI)
C21H19Cl2N2O2S [M+H]+ 433.1. Anal. Calcd for C21H18Cl2N2O2S: C,
58.20; H, 4.19; Cl, 16.36; N, 6.46; S, 7.40. Found: C, 58.22; H,
4.17; Cl, 16.33; N, 6.43; S, 7.38.
4.3.29. 1-(3,5-Dibromo-2-hydroxybenzyl)-1-(4-hydroxybenzyl)-
3-phenylthiourea (11b)
4.3.21. 1-(4-Fluorobenzyl)-1-(2-hydroxy-5-methylbenzyl)-3-
phenylthiourea (3b)
Mp 158–159 °C; yield: 65%; 1H NMR (DMSO-d6) d = 4.78 (s, 2H),
5.16 (s, 2H), 6.97–7.28 (m, 10H), 7.57 (s, 1H), 10.41 (s, 1H). MS (ESI)
C21H19Br2N2O2S [M+H]+ 521.0. Anal. Calcd for C21H18Br2N2O2S: C,
48.30; H, 3.47; Br, 30.60; N, 5.36; S, 6.14. Found: C, 48.28; H,
3.43; Br, 30.57; N, 5.34; S, 6.12.
Mp 176–177 °C; yield: 88%; 1H NMR (DMSO-d6) d = 2.46 (s, 3H),
4.90 (s, 2H), 5.10 (s, 2H), 6.85 (d, J = 8.4 Hz, 1H), 7.04 (s, 1H), 7.08–
7.16 (m, 2H), 7.29–7.43 (m, 8H), 9.47 (s, 1H), 10.08 (s, 1H). MS (ESI)
C22H22FN2OS [M+H]+ 381.1. Anal. Calcd for C22H21FN2OS: C, 69.45;
H, 5.56; F, 4.99; N, 7.36; S, 8.43. Found: C, 69.48; H, 5.52; F, 4.95; N,
7.33; S, 8.40.
4.3.30. 1-(2-Hydroxy-3,5-dimethylbenzyl)-1-(4-
hydroxybenzyl)-3-phenylthiourea (12b)
4.3.22. 1-(3,5-Dichloro-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylthiourea (4b)
Mp 145–146 °C; yield: 62%; 1H NMR (DMSO-d6) d = 2.31 (s, 3H),
2.48 (s, 3H), 4.81 (s, 2H), 5.18 (s, 2H), 7.04–7.35 (m, 10H), 7.57 (s,
1H), 10.41 (s, 1H). MS (ESI) C23H25N2O2S [M+H]+ 393.2. Anal. Calcd
for C23H24N2O2S: C, 70.38; H, 6.16; N, 7.14; S, 8.17. Found: C, 70.34;
H, 6.15; N, 7.12; S, 8.14.
Mp 157–159 °C; yield: 78%; 1H NMR (DMSO-d6) d = 4.78 (s, 2H),
5.17 (s, 2H), 6.92–7.25 (m, 11H), 7.56 (s, 1H). MS (ESI)
C21H18Cl2FN2OS [M+H]+ 435.0. Anal. Calcd for C21H17Cl2FN2OS: C,
57.94; H, 3.94; Cl, 16.29; F, 4.36; N, 6.43; S, 7.37. Found: C,
57.96; H, 3.91; Cl, 16.27; F, 4.34; N, 6.41; S, 7.35.
4.3.31. 1-Butyl-1-(5-chloro-2-hydroxybenzyl)-3-phenylthiourea
(13b)
4.3.23. 1-(3,5-Dibromo-2-hydroxybenzyl)-1-(4-fluorobenzyl)-3-
phenylthiourea (5b)
Mp 149–150 °C; yield: 56%; 1H NMR (DMSO-d6) d = 0.88 (t,
J = 7.5 Hz, 3H), 1.22–1.34 (m, 2H), 1.54–1.64 (m, 2H), 3.42–3.46
(m, 1H), 3.69 (t, J = 7.5 Hz, 2H), 4.96 (s, 2H), 6.84 (d, J = 9 Hz, 1H),
7.10–7.16 (m, 3H), 7.27–7.34 (m, 4H), 9.20 (s, 1H), 10.06 (s, 1H).
MS (ESI) C18H22ClN2OS [M+H]+ 349.1. Anal. Calcd for
C18H21ClN2OS: C, 61.97; H, 6.07; Cl, 10.16; N, 8.03; S, 9.19. Found:
C, 61.96; H, 6.03; Cl, 10.14; N, 8.01; S, 9.15.
Mp 144–145 °C; yield: 74%; 1H NMR (DMSO-d6) d = 4.75 (s, 2H),
5.19 (s, 2H), 6.97–7.28 (m, 11H), 7.57 (s, 1H). MS (ESI)
C21H18Br2FN2OS [M+H]+ 523.9. Anal. Calcd for C21H17Br2FN2OS: C,
48.11; H, 3.27; Br, 30.48; F, 3.62; N, 5.34; S, 6.12. Found: C,
48.14; H, 3.28; Br, 30.44; F, 3.60; N, 5.32; S, 6.11.

312
H.-Q. Li et al. / Bioorg. Med. Chem. 18 (2010) 305–313
4.3.32. 1-(5-Bromo-2-hydroxybenzyl)-1-butyl-3-
phenylthiourea (14b)
proteins were eluted with 250 and 500 mM imidazole and dialyzed
against 50 mM NaCl, 20 mM HEPES, 10% glycerol, and 1 g/mL
l
Mp 145–146 °C; yield: 55%; 1H NMR (DMSO-d6) d = 0.89 (t,
J = 7.5 Hz, 3H), 1.22–1.34 (m, 2H), 1.54–1.64 (m, 2H), 3.42–3.46
(m, 1H), 3.69 (t, J = 7.5 Hz, 2H), 4.97 (s, 2H), 6.84 (d, J = 9 Hz, 1H),
7.08–7.14 (m, 3H), 7.25–7.33 (m, 4H), 9.18 (s, 1H), 10.04 (s, 1H).
MS (ESI) C18H22BrN2OS [M+H]+ 393.1. Anal. Calcd for
C18H21BrN2OS: C, 54.96; H, 5.38; Br, 20.31; N, 7.12; S, 8.15. Found:
C, 54.94; H, 5.35; Br, 20.28; N, 7.10; S, 8.13.
each of aprotinin, leupeptin, and pepstatin for 2 h. The entire puri-
fication procedure was performed at 4 °C or on ice.24
Both EGFR and HER-2 kinase assays were set up to assess the le-
vel of autophosphorylation based on DELFIA/Time-Resolved Fluo-
rometry. Compounds 1a–18a and 1b–18b were dissolved in 100%
DMSO and diluted to the appropriate concentrations with 25 mM
HEPES at pH 7.4. In each well, 10 íL of compound was incubated
with 10
zyme (1:80 dilution in 100 mM HEPES) for 10 min at room temper-
ature. Then, 10 L of 5_buffer (containing 20 mM HEPES, 2 mM
MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM
ATP-50 mM MgCl2 was added for 1 h. Positive and negative con-
trols were included in each plate by incubation of enzyme with
or without ATP-MgCl2. At the end of incubation, liquid was aspi-
rated, and plates were washed three times with wash buffer. A
lL (12.5 ng for HER-2 or 5 ng for EGFR) of recombinant en-
4.3.33. 1-Butyl-1-(2-hydroxy-5-methylbenzyl)-3-
phenylthiourea (15b)
l
Mp 142–142 °C; yield: 61%; 1H NMR (DMSO-d6) d = 0.90 (t,
J = 7.5 Hz, 3H), 1.23–1.36 (m, 2H), 1.55–1.64 (m, 2H), 2.49 (s, 3H),
3.42–3.47 (m, 1H), 3.71 (t, J = 7.5 Hz, 2H), 4.97 (s, 2H), 6.86 (d,
J = 9 Hz, 1H), 7.12–7.19 (m, 3H), 7.28–7.39 (m, 4H), 9.21 (s, 1H),
10.07 (s, 1H). MS (ESI) C19H25N2OS [M+H]+ 329.2. Anal. Calcd for
C19H24N2OS: C, 69.47; H, 7.36; N, 8.53; S, 9.76. Found: C, 69.41;
H, 7.32; N, 8.56; S, 9.75.
l
l
75 lL (400 ng) sample of europiumlabeled anti-phosphotyrosine
antibody was added to each well for another 1 h of incubation.
After washing, enhancement solution was added and the signal
was detected by Victor (Wallac Inc.) with excitation at 340 nm
and emission at 615 nm. The percentage of autophosphorylation
inhibition by the compounds was calculated using the following
equation: 100% ꢁ [(negative control)/(positive control–negative
control)]. The IC50 was obtained from curves of percentage inhibi-
tion with eight concentrations of compound. As the contaminants
in the enzyme preparation are fairly low, the majority of the signal
detected by the anti-phosphotyrosine antibody is from EGFR or
HER-2.
4.3.34. 1-Butyl-1-(3,5-dichloro-2-hydroxybenzyl)-3-
phenylthiourea (16b)
Mp 155–156 °C; yield: 51%; 1H NMR (DMSO-d6) d = 0.88 (t,
J = 7.5 Hz, 3H), 1.22–1.34 (m, 2H), 1.54–1.64 (m, 2H), 3.42–3.46
(m, 1H), 3.69 (t, J = 7.5 Hz, 2H), 4.96 (s, 2H), 7.10–7.16 (m, 3H),
7.27–7.34 (m, 4H), 7.55 (s, 1H). MS (ESI) C18H21Cl2N2OS [M+H]+
383.1. Anal. Calcd for C18H20Cl2N2OS: C, 56.40; H, 5.26; Cl, 18.50;
N, 7.31; S, 8.36. Found: C, 56.43; H, 5.23; Cl, 18.45; N, 7.28; S,
8.33%.
4.3.35. 1-Butyl-1-(3,5-dibromo-2-hydroxybenzyl)-3-
phenylthiourea (17b)
4.5. Cell proliferation assay
Mp 148–149 °C;. yield: 47%; 1H NMR (DMSO-d6) d = 0.90 (t,
J = 7.5 Hz, 3H), 1.23–1.36 (m, 2H), 1.55–1.64 (m, 2H), 3.42–3.47
(m, 1H), 3.71 (t, J = 7.5 Hz, 2H), 4.97 (s, 2H), 7.11–7.17 (m, 3H),
7.25–7.36(m, 4H), 7.55 (s, 1H). MS (ESI) C18H21Br2N2OS [M+H]+
471.0. Anal. Calcd for C18H20Br2N2OS: C, 45.78; H, 4.27; Br, 33.84;
N, 5.93; S, 6.79. Found: C, 45.74; H, 4.25; Br, 33.82; N, 5.91; S, 6.76.
The antiproliferative activity was determined using a standard
(MTT)-based colorimetric assay (Sigma), using 5-fluorouracil and
genistein 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 indi-
cated compounds at final concentrations ranging from 0.1 to
100
of an MTT solution (10
100 L 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 mea-
sured at 570 nm on an LX300 Epson Diagnostic microplate reader.
Survival ratios are expressed in percentages with respect to un-
treated cells. IC50 values were determined from replicates of 6
wells from at least two independent experiments.
l
g/mL. After 48 h, cell survival was determined by the addition
4.3.36. 1-Butyl-1-(2-hydroxy-3,5-dimethylbenzyl)-3-
phenylthiourea (18b)
lL of 5 mg/mL MTT in PBS). After 4 h,
l
Mp 136–137 °C; yield: 52%; 1H NMR (DMSO-d6) d = 0.90 (t,
J = 7.5 Hz, 3H), 1.23–1.36 (m, 2H), 1.55–1.64 (m, 2H), 2.32 (s, 3H),
2.49 (s, 3H), 3.42–3.47 (m, 1H), 3.71 (t, J = 7.5 Hz, 2H), 4.96 (s,
2H), 7.12–7.17 (m, 3H), 7.28–7.39 (m, 4H), 7.55 (s, 1H). MS (ESI)
C20H27N2OS [M+H]+ 343.2. Anal. Calcd for C20H26N2OS: C, 70.14;
H, 7.65; N, 8.18; S, 9.36. Found: C, 70.11; H, 7.62; N, 8.15; S, 9.33.
4.6. Molecular docking modeling
4.4. Preparation, and purification of HER-2 and EGFR and
inhibitory assay
Molecular docking of compound 7b into the three-dimensional
EGFR complex structure (1M17.pdb, downloaded from the PDB)
was carried out using the AUTODOCK software package (version 4.0)
as implemented through the graphical user interface AUTODOCKTOOLS
(ADT 1.4.6).
A 1.7 kb cDNA encoded for human HER-2 cytoplasmic domain
(HER-2-CD, amino acids 676–1245) and 1.6 kb cDNA encoded for
the EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186)
were cloned into baculoviral expression vectors pBlueBacHis2B
and pFASTBacHTc, separately. A sequence that encodes (His)6
was located at the 50 upstream to the HER-2 and EGFR sequences.
Sf-9 cells were infected for 3 days for protein expression. Sf-9 cell
pellets were solubilized at 0 °C in a buffer at pH 7.4 containing
50 mM HEPES, 10 mM NaCl, 1% Triton, 10 íM ammonium molyb-
Acknowledgments
This work was supported by National Basic Research Programs
(973) of China (No. 2008CB418004 & No. 2008CB418201) and the
Major Projects on Control and Rectification of Water Body Pollu-
tion (Water Special Project) (No. 2008ZX07526-001).
date, 100
lM sodium vanadate, 10
lg/mL aprotinin, 10
lg/mL
leupeptin, 10
lg/mL pepstatin, and 16
lg/ mL benzamidine HCl
for 20 min followed by 20 min centrifugation. Crude extract super-
natant was passed through an equilibrated Ni-NTA superflow
packed column and washed with 10 mM and then 100 mM imidaz-
ole to remove nonspecifically bound material. Histidinetagged
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