Dual-target-directed 1,3-diphenylurea derivatives: BACE 1 inhibitor and metal chelator against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2010.06.042

Dual-target-directed 1,3-diphenylurea derivatives were designed by hybridizing BACE 1 inhibitor 1 with metal chelator LR-90. A database consisted of 1,3-diphenylurea derivatives was built and screened by the pharmacophore model (Hypo 1) of BACE 1 inhibitor. Based on the predicted results, 11 compounds (6a-d, 9a-g) with favorable Fitvalues were selected, synthesized and evaluated for their BACE 1 inhibitory activities, which showed that the predicted results were in good agreement with the experimental values. Besides, the synthesized compounds also displayed the ability to chelate metal ions. The most effective BACE 1 inhibitor 9f (27.85 ± 2.46 μmol/L) was selected for further receptor-binding studies, the result of which indicated that an essential hydrogen bonds was formed between the urea group of 9f and the catalytic aspartate Asp228.

Full text of DOI:10.1016/j.bmc.2010.06.042

Bioorganic & Medicinal Chemistry 18 (2010) 5610–5615
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dual-target-directed 1,3-diphenylurea derivatives: BACE 1 inhibitor
and metal chelator against Alzheimer’s disease
Wenhai Huang a, Dan Lv a, Haiping Yu b, Rong Sheng a, Sun Chol Kim c, Peng Wu a, Kedi Luo a, Jia Li b,
Yongzhou Hu a,
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang University, Zijingang Campus, Hangzhou 310058, China
b The National Center for Drug Screening, Shanghai 201203, China
c Department of Chemistry, Pyongsong Medical University, Pyongsong, Democratic People’s Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Dual-target-directed 1,3-diphenylurea derivatives were designed by hybridizing BACE 1 inhibitor 1 with
metal chelator LR-90. A database consisted of 1,3-diphenylurea derivatives was built and screened by the
pharmacophore model (Hypo 1) of BACE 1 inhibitor. Based on the predicted results, 11 compounds (6a–d,
9a–g) with favorable Fitvalues were selected, synthesized and evaluated for their BACE 1 inhibitory activ-
ities, which showed that the predicted results were in good agreement with the experimental values.
Besides, the synthesized compounds also displayed the ability to chelate metal ions. The most effective
BACE 1 inhibitor 9f (27.85 2.46 lmol/L) was selected for further receptor-binding studies, the result of
which indicated that an essential hydrogen bonds was formed between the urea group of 9f and the cat-
alytic aspartate Asp228.
Received 7 May 2010
Revised 11 June 2010
Accepted 11 June 2010
Available online 17 June 2010
Keywords:
Alzheimer’s disease
BACE 1 inhibitor
Metal chelators
Ó 2010 Elsevier Ltd. All rights reserved.
Dual-target-directed ligands
1. Introduction
the amyloidogenic pathway, inhibition of BACE 1 is likely to reduce
the production of Ab and thereby delay or halt the progression of
Alzheimer’s disease (AD), the leading cause of dementia in the
elderly, is a complex neurodegenerative disorder. Until now, multi-
ple factors, such as amyloid-b (Ab) aggregation,1,2 metal dysho-
meostasis, oxidative stress, mitochondrial dysfunction and
reduced acetylcholine (ACh) level, have been considered to play
important roles in the pathogenesis of AD.3 The complexity and
multiple etiologies of AD make single-target strategy difficult to
shed desirable therapeutic effect. Thus multi-target-directed
ligand (MTDL) raises as a potentially more effective strategy for
AD treatment.4 MTDL, the goal of which is to enhance efficacy
and improve safety, is rationally designed to hit multiple targets
for a particular disease. In the past two years, many MTDLs have
been reported with improved pharmacological profiles, such as
AChE inhibitors with metal chelate property and AchE-induced
Ab aggregation inhibitory activity,5 BACE1 inhibitors bearing
acetylcholinesterase (AChE) inhibitory activity,6 and cholinesterase
inhibitors with both antioxidant and neuroprotective properties.7
Amyloid-b (Ab), formed by the continuously proteolytic process-
AD.11,12
Recent evidence indicated that dyshomeostasis of biometals (Fe,
Cu, Zn) in the brain may contribute to AD pathology.13 Experiments
also found that the level of metal ions in AD patients is 3–7-folds
higher than that of healthy individuals.14 Therefore, decreasing the
level of metal ions in brain by using metal chelator represents
another rational therapeutic approach for the treating of AD.
Previously, we reported the establishment of pharmacophore
model for BACE 1 inhibitor, Hypo 1, which was validated by Enrich-
ment and ROC method (EF at 2%, 5% and 10% are 30.6, 12.2 and 7.7;
AUC of the ROC curve is 0.93).15 Analysis based on established
pharmacophore model and BACE 1 inhibitory activity revealed that
compound 1 displayed the highest BACE 1 activity among two ser-
ies synthesized N-phenyl-1-arylamide and N-phenylbenzenesul-
fonamide derivatives.
Considering the crucial roles of BACE 1 and metal ions in AD
pathology, we focused on dual-target-directed ligands that can
simultaneously impact on BACE 1 and metal ions in this study.
Based on the previous work, a series of novel 1,3-diphenylurea
derivatives were designed by hybridizing metal chelator LR-90
with BACE 1 inhibitor 1 (Fig. 1). A database consisted of 1,3-
diphenylurea derivatives was built and screened by Hypo 1. Based
on Fitvalues, 11 1,3-diphenylurea derivatives were picked out, syn-
thesized and subsequently evaluated for their BACE 1 inhibitory
activity, as well as the ability to chelate metal ions. Docking
ing of b-amyloid precursor protein (APP) by b and c
-secretase,8–10 is
thought to play a central role in the pathogenesis of AD. Since
b-secretase (BACE 1) mediated cleavage of APP is the first step of
* Corresponding author. Tel./fax: +86 571 88208460.
E-mail addresses: huyz@zju.edu.cn, huyz@zjuem.zju.edu.cn (Y. Hu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.042

W. Huang et al. / Bioorg. Med. Chem. 18 (2010) 5610–5615
5611
Figure 1. Structure of parent compound (1), metal chelator LR-90 and 1,3-diphenylurea derivatives (2).
analysis of 9f with corresponding receptor protein was carried out
2.2. Chemistry
to study its binding pattern.
The synthetic route for designed 1,3-diphenylurea derivatives
6a–d and 9a–g are illustrated in Scheme 1. Reaction of 4-bromo-
2-nitrophenol with 1,2-dibromoethane afforded bromide 3, which
was reacted with morpholine in refluxing acetonitrile to yield com-
pound 4. Reduction of 4 with stannous chloride afforded 5 accord-
ing to the known methods.15 Target compounds 6a–d were
obtained in good yields by the reaction compound 5 with substi-
tuted isocyanatobenzenes.
Bromide 3 was reduced with stannous chloride, and then re-
acted with substituted isocyanatobenzenes to yield 8a–g in good
yields. Followed reaction of 8a–g with various amines obtained
target compounds 9a–g.
2. Results and discussion
2.1. Build designed database and virtual screening
The designed database consisted of 1,3-diphenylurea deriva-
tives was built using the Build 3D database protocol within the
Pipeline Pilot software. Enumerate stereoisomers protocol was
used to expend the database, which contains 206,259 compounds.
Followed Lipinski filtration (AlogP was changed as ꢀ3, with the
remaining parameters were set as default) led to a reduced set of
99,494 derivatives. All compounds were then optimized by the Dis-
covery Studio 2.1 software package.
The pharmacophore model of BACE 1 inhibitors (Hypo 1) was
built in previous work.15 Discovery Studio 2.1/Ligand Pharmaco-
phore Mapping protocol was used to screen the designed database
using Hypo 1 as the template. Compounds 6a–d, 9a–g with high
Fitvalues (3.25–4.25, the maximum of the Fitvalue is 5.00) were
picked out and synthesized.
2.3. BACE 1 inhibitory activity
All synthesized compounds were evaluated for their BACE 1
inhibitory activities using a fluorescence resonance energy transfer
(FRET) assay, with potent peptidomimetic inhibitor OM99-2 as
the positive control. Compounds with BACE 1 inhibitory rates at
Scheme 1. Reagents and conditions: (a) BrCH2CH2Br, 40%NaOH, reflux; (b) morpholine, CH2Cl2, rt; (c) SnCl2Á2H2O, EtOH/THF, rt; (d) isocyanatobenzene, CH2Cl2, reflux.

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W. Huang et al. / Bioorg. Med. Chem. 18 (2010) 5610–5615
20
lg/mL not less than 50% were tested for IC50 values. The results
On the other hand, the BACE 1 inhibitory activities of tested
are summarized in Table 1.
compounds were highly dependent on the structures of 2-amino-
ethoxy side chains on ring A. Compounds, whose R1 were secondary
amines, exhibited more potent inhibition (e.g., 9b–g). The result im-
plied that introduction of 2-tertiary amines side chains to ring A
failed to enhance BACE 1 inhibitory activity. Comparing the IC50 val-
ues of compounds 9b and 9e revealed that a small cyclic amine R1
(9b) was beneficial for BACE 1 inhibitory potency, while compound
with a long chain amine on ring A (9f) showed more effective BACE
1 activity than compound with a small chain amine (9g).
As shown in Table 1, compounds 9a–g showed more potent
activity than compounds 6a–d. Compounds 9c and 9f displayed
equivalent or more potent BACE 1 inhibitory activities in compar-
ison with compound 1. Compound 9f, R1 is 1,6-diaminohexane,
exhibited the most potent BACE 1 inhibitory activity with an IC50
value of 27.85 2.46 lmol/L.
In series 6, compound 6d with a chlorine atom at the 2-position
of ring B, showed potent activity with an inhibitory rate of 36.8%.
Replacement the 2-chlorine atom of ring B with either a 3-nitro
group or a 4-methoxy group (6b, 6c) decreased activity.
2.4. Metal chelating effect
All compounds were tested for their metal chelating effect using
difference UV–vis spectra recorded in methanol at 298 K with
wavelength ranging from 210 to 400 nm. Numerical subtraction
of the spectra of the copper/iron ion alone and the compound alone
from the spectra of the mixture obtained the difference UV–vis
spectra due to complex formation.5 Results showed that all 1,3-
diphenylurea derivatives were potent metal chelators. The UV–
vis spectra (Fig. 2a) and the difference UV–vis spectra between 9f
and copper ion (Fig. 2b) were shown as an example. In difference
Table 1
The BACE 1 activity of 1,3-diphenylurea derivatives
R1
O
H
N
H
N
A
B
R2
O
UV–vis spectra, the absorption of 9f (25
with the increased concentration of copper ion from 1.25 to
50 mol/L. The result using iron ion was similar with that of copper
lmol/L) decreased along
Br
l
d
Compd
R1
R2
Inhibition at
20
g/mLa(%)
IC50
mol/L)
Fitvalue
ion. These observations indicated that there was an interaction be-
tween 9f and copper/iron ion. On the other hand, compound 1 pro-
duced no significant spectral change when mixed with up to 20-
fold FeSO4 or CuCl2 (Fig. 2c), indicating that the observed metal
ion binding was attributed to specific interactions between metal
ions and the urea moiety of 1,3-diphenylurea derivatives.
l
(l
OM99-2
1
6a
6b
6c
6d
102.1 2.9b
83.7 1.3
24.5 1.6
20.3 9.7
0.25 0.03
—
—
—
—
—
3.96
3.92
3.95
3.81
4.20
Morpholine
Morpholine
Morpholine
Morpholine
H
m-NO2
p-OCH3 20.9 1.4
o-Cl
36.8 10.8
In order to investigate the ratio of ligand/metal ion in the com-
plex, a fixed amount of compound 9f (25
lmol/L) was mixed with
9a
9b
9c
o-Cl
61.9 3.3
>44.25
>42.82
3.99
3.93
N
growing amounts of copper ion (2.5–50 mol/L) and tested the dif-
l
H
N
ference UV–vis spectra. It was observed that the maximum inten-
sity of difference spectra reached at 2:1 ratio, demonstrating that
the ratio of ligand/metal ion (Cu2+) in the complex was 2:1.
o-Cl
o-Cl
87.7 0.5
79.4 4.0
N
3
29.40 6.92 3.53
H
2.5. Molecular docking study
N
H
OH
3
9d
9e
9f
o-Cl
o-Cl
o-Cl
67.5 4.1
77.1 2.7
NAc
>42.46
4.13
In an attempt to understand the molecular interaction be-
tween 9f and BACE 1, a molecular docking study was performed
using the Discovery Studio 2.1/CDOCKER protocol. The crystal
structure of OM99-2/BACE 1 complex (PDB ID: 2ZHR) was used
as the template. Docking and subsequent scoring studies were
performed using default parameters. The result disclosed that
compound 9f made several important interactions along the ac-
tive-site of BACE 1 with different mode in comparison with com-
pound 1.15 As shown in Figure 3a, the bromo substituted benzene
ring of 9f made hydrophobic contacts with the S1 pocket, and
H
37.63 8.92 3.25
27.85 2.46 4.25
N
N
H
NH2
NH2
4
N
H
9g
o-Cl
77.9 0.5
>45.25
4.12
a
b
c
Data are means standard deviation of three independent experiments.
OM99-2 was tested at 2 g/mL.
There is an interference of compound with fluorescence resonance energy
l
transfer (FRET) assay.
d
IC50 value only determined for compounds attaining over 50% BACE 1 inhibition
formed classical
p–p stacking with the benzene ring of Tyr71.
at 20 lg/mL.
Hydrogen bonding interactions were observed between urea
Figure 2. (a) The UV–vis spectra of 9f. (The purple curve is the UV–vis spectra of compound alone. Curves with other colors are the UV–vis spectra of compound mixed with
different concentrations of copper ion.); (b) The difference UV–vis spectra between 9f and copper ion. (Curves with other colors are the difference UV–vis spectra between
compound 9f mixed with different concentrations of copper ion.); (c) The difference UV–vis spectra between compound 1 and copper ion.

W. Huang et al. / Bioorg. Med. Chem. 18 (2010) 5610–5615
5613
Figure 3. (a) The binding pattern of 9f into the BACE 1; (b) The binding pattern of compound 1 into the BACE 1.
group and Thr72 with a distance of 2.27 Å. The urea group formed
two hydrogen bonds with catalytic aspartate Asp228, with
distances of 2.33 and 2.15 Å, respectively. Besides, hydrophobic
interactions with the S3 pocket and hexanamine group further in-
creased the affinity of 9f. The different binding modes of 9f and 1
may explain the fact that compound 9f showed more potent
activity than compound 1.
4.2.1. General procedure for the preparation of compounds 6a–d
To a solution of 5 (0.22 mmol) in CH2Cl2 (5 mL), isocyanatoben-
zene (0.2 mmol) was added, and the mixture was refluxed for 1 h.
After removal of the solvent, the residue was purified by silica gel
column chromatography (PE/EtOAc/TEA = 1:1:0.1) to afford 6.
4.2.1.1. 1-(5-Bromo-2-(2-morpholinoethoxy)phenyl)-3-phenyl-
urea 6a. White solid (79%), mp 170–173 °C. IR (KBr): 2810, 1602,
1496, 1384 cmÀ1 1H NMR (400 MHz, CDCl3): d 2.51–2.53 (t, 4H,
;
3. Conclusions
J = 4.4 Hz, morpholine), 2.74–2.77 (t, 2H, J = 5.2 Hz, –CH2N), 3.74–
3.76 (t, 4H, J = 4.4 Hz, morpholine), 4.06–4.09 (t, 2H, J = 5.2 Hz, –Ar-
OCH2), 6.66–6.68 (d, 1H, J = 8.4 Hz, ArH), 7.00–7.03 (dd, 1H,
J1 = 8.8 Hz, J2 = 2.0 Hz, ArH), 7.06–7.09 (t, 1H, J = 7.6 Hz, ArH),
7.29–7.33 (t, 2H, J = 8.0 Hz, ArH), 8.40–8.42 (d, 1H, J = 7.6 Hz,
ArH), 7.48 (s, 1H, –CONH), 8.19 (s, 1H, –CONH), 8.47–8.48 (d, 1H,
J = 2.0 Hz, ArH); 13C NMR (100 MHz, CDCl3): d 53.2, 55.1, 65.3,
66.6, 112.8, 114.4, 121.5, 122.3, 122.5, 123.1, 124.3, 124.6, 127.7,
129.1, 136.1, 146.2, 152.4; MS (ESI): m/z = 420 [M+H]+.
In this work, the strategy of hybridizing metal chelator LR-90
with BACE 1 inhibitor 1 was utilized to design 1,3-diphenylurea
derivatives as Dual-Target-Directed ligands. The designed database
of 1,3-diphenylurea derivatives was built and screened by a previ-
ous built and validated BACE 1 inhibitor pharmacophore model.
Eleven compounds (6a–d, 9a–g) with high Fitvalues were selected,
synthesized and evaluated for their biological activities, which re-
vealed that the theoretical results were in good agreement with
the experimental values. Compound 9f (27.85 2.46 lmol/L) was
tested as the most effective BACE 1 inhibitor. Docking study of 9f
indicated that the urea group made central hydrogen bonds with
the catalytic aspartate Asp228. All synthesized compounds also
showed the ability to chelate metal ions. The MTDL design strategy
used in this study may thus shed instructive light on the treatment
of AD in the near future.
4.2.1.2. 1-(5-Bromo-2-(2-morpholinoethoxy)phenyl)-3-(3-nitr-
ophenyl)urea 6b. Yellow solid (84%), mp 211–213 °C. IR (KBr):
2807, 1647, 1599, 1346 cmÀ1 1H NMR (400 MHz, CDCl3): d 2.55–
;
2.57 (m, 4H, morpholine), 2.81–2.84 (t, 2H, J = 5.2 Hz, –CH2N),
3.77–3.80 (t, 4H, J = 4.4 Hz, morpholine), 4.07–4.10 (t, 2H,
J = 5.2 Hz, –ArOCH2), 6.65–6.67 (d, 1H, J = 8.4 Hz, ArH), 7.00–7.03
(dd, 1H, J1 = 8.4 Hz, J2 = 2.0 Hz, ArH), 7.37–7.41 (t, 1H, J = 8.4 Hz,
ArH), 7.79–7.81 (dd, 1H, J1 = 8.0 Hz, J2 = 1.6 Hz, ArH), 7.89–7.91
(dd, 1H, J1 = 8.0 Hz, J2 = 1.6 Hz, ArH), 8.24–8.25 (m, 1H, ArH),
8.46–8.47 (d, 1H, J = 3.2 Hz, ArH), 8.68 (s, 1H, –CONH), 8.83 (s,
1H, –CONH); 13C NMR (100 MHz, CDCl3): d 53.2, 55.7, 65.7, 66.8,
112.6, 114.8, 121.5, 122.5, 123.1, 123.6, 124.3, 129.2, 131.6,
136.1, 145.8, 147.4, 152.8; MS (ESI): m/z = 465 [M+H]+.
4. Experimental
4.1. General
All solvents used were of analytical grade. Melting points were
recorded on a Buchi apparatus and uncorrected, IR spectra were re-
corded on a Bruker VECTOR 22 FTIR spectrophotometer. 1H NMR
spectra were recorded on a Bruker Avance III 500 M instrument
(chemical shifts are expressed as d values relative to TMS as inter-
nal standard). Mass spectra (MS) were recorded on an Esquire-LC-
00075 spectrometer. The designed database of 1,3-diphenylurea
derivatives was built by Pipeline Pilot software. The pharmaco-
phore model (Hypo 1) of BACE 1 inhibitors was built in previous
work.15 Molecular model studies were performed using the Dis-
covery Studio 2.1.
4.2.1.3. 1-(5-Bromo-2-(2-morpholinoethoxy)phenyl)-3-(4-meth
oxyphenyl)urea 6c. White solid (70%), mp 178–182 °C. IR
(KBr):2965, 2936, 2804, 1628, 1597, 1351 cmÀ1 1H NMR (400
;
MHz, CDCl3): d 2.50–2.55 (m, 4H, morpholine), 2.70–2.73 (m, 2H,
–CH2N), 3.74–3.76 (m, 4H, morpholine), 3.81 (s, 3H, –OCH3),
4.04–4.07 (t, 2H, J = 5.6 Hz, –ArOCH2), 6.65–6.67 (d, 1H, J = 8.0 Hz,
ArH), 6.88–6.90 (d, 2H, J = 8.8 Hz, ArH), 7.01–7.04 (dd, 1H,
J1 = 8.8 Hz, J2 = 2.0 Hz, ArH), 7.08 (s, 1H, –CONH), 7.30–7.32 (d, 2H,
J = 8.8 Hz, ArH), 7.88 (s, 1H, –CONH), 8.47–8.48 (d, 1H, J = 2.0 Hz,
ArH); 13C NMR (100 MHz, CDCl3): d 53.1, 55.1, 55.4, 65.5, 66.2,
113.1, 115.2, 121.4, 123.0, 123.5, 124.4, 127.6, 129.0, 131.8, 135.8,
146.2, 152.1, 159.1; MS (ESI): m/z = 450 [M+H]+.
4.2. Chemistry
Compound 5 was synthesized using 4-bromo-2-nitrophenol as
the starting material according to the known methods.15

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W. Huang et al. / Bioorg. Med. Chem. 18 (2010) 5610–5615
4.2.1.4. 1-(5-Bromo-2-(2-morpholinoethoxy)phenyl)-3-(2-chlo-
7.45–7.47 (d, 1H, J = 8.0 Hz, ArH), 8.00–8.02 (d, 1H, J = 8.0 Hz,
ArH), 8.27–8.28 (d, 1H, J = 2.4 Hz, ArH), 8.95 (s, 1H, –CONH), 9.02
(s, 1H, –CONH); 13C NMR (100 MHz, CDCl3): d 24.1, 27.8, 35.1,
55.4, 58.1, 64.3, 112.1, 114.2, 121.3, 121.5, 122.3, 123.1, 124.2,
127.6, 129.2, 130.5, 135.9, 146.2, 153.2; MS (ESI): m/z = 466
[M+H]+.
rophenyl)urea 6d. White solid (76%), mp 94–100 °C. IR (KBr):
2801, 1633, 1599, 1350 cmÀ1 1H NMR (400 MHz, CDCl3): d 2.64–
;
2.66 (t, 4H, J = 4.4 Hz, morpholine), 2.89–2.92 (t, 2H, J = 5.2 Hz, –
CH2N), 3.82–3.84 (t, 4H, J = 4.4 Hz, morpholine), 4.09–4.12 (t, 2H,
J = 5.2 Hz, –ArOCH2), 6.66–6.68 (d, 1H, J = 8.0 Hz, ArH), 6.99–7.04
(m, 2H, ArH), 7.18–7.22 (t, 1H, J = 8.0 Hz, ArH), 7.28–7.31 (m, 1H,
ArH), 7.58–7.59 (t, 1H, J = 2.0 Hz, –ArH), 8.22 (s, 1H, –CONH),
8.48 (s, 1H, –CONH), 8.50–8.51 (d, 1H, J = 1.2 Hz, ArH); 13C NMR
(100 MHz, CDCl3): d 53.2, 55.1, 65.3, 66.1, 112.3, 114.1, 121.5,
122.6, 122.9, 123.2, 124.5, 127.6, 129.1, 130.8, 136.3, 146.1,
153.5; MS (ESI): m/z = 454 [M+H]+.
4.2.2.3. 1-(5-Bromo-2-(2-(hexylamino)ethoxy)phenyl)-3-(2-chlo
rophenyl)urea 9c. Red oil (45%), IR (KBr): 3304, 3261, 2955, 2851,
1692, 1594, 1542, 1441, 1403 cmÀ1 1H NMR (400 MHz, CDCl3): d
;
1.22–1.35 (m, 9H, –NHCH2CH2CH2CH2CH2CH3), 1.80–1.82 (m, 2H,
–NHCH2CH2CH2–), 2.93–2.97 (t, 1H, J = 10.0 Hz, –NHCH2CH2CH2–),
3.35–3.37 (t, 2H, J = 5.2 Hz, –NHCH2CH2O), 4.28–4.30 (t, 2H,
J = 5.2 Hz, –NHCH2CH2O), 6.59–6.61 (d, 1H, J = 11.0 Hz, ArH),
6.96–7.02 (m, 2H, ArH), 7.21–7.25 (t, 1H, J = 10.0 Hz, ArH), 7.31–
7.33 (d, 2H, J = 10.0 Hz, ArH), 8.04–8.06 (d, 1H, J = 10.0 Hz, ArH),
8.47 (d, 1H, J = 2.4 Hz, ArH), 8.93 (s, 1H, –NHCO), 9.25 (s, 1H, –
NHCO); 13C NMR (100 MHz, CDCl3): d 14.1, 21.3, 25.9, 29.9, 30.6,
52.3, 53.5, 65.2, 111.2, 114.2, 121.5, 121.6, 122.1, 122.9, 124.4,
127.4, 129.1, 130.4, 135.9, 146.1, 153.1; MS (ESI): m/z = 468 [M+H]+.
4.2.1.5. 1-(5-Bromo-2-(2-bromoethoxy)phenyl)-3-(2-chlorophe-
nyl)urea 8. To a solution of 3 (0.975 g, 3 mmol) in ethanol (3 mL)
and THF (3 mL), SnCl2Á2H2O (2.03 g, 9 mmol) was added in a por-
tion and then stirred at room temperature for 4 h. The solvent
was evaporated under reduced pressure to afford a white solid,
to which 15% NaOH (3 mL) was added, stirred at room temperature
for 0.5 h. The reaction mixture was extracted with ether and
washed with water and brine. The organic layer was dried over
Na2SO4, filtered and concentrated under vacuum to afford com-
pound 7 as a red oil, which was then mixed with 1-chloro-2-isocy-
anatobenzene (0.51 g, 3.3 mmol) and 10 mL CH2Cl2, and refluxed
for 1 h. The solvent was removed, and the remained solid was puri-
fied by silica gel column chromatography (PE/EtOAc = 2:1) to af-
4.2.2.4. 1-(5-Bromo-2-(2-(5-hydroxypentylamino)ethoxy)phe-
nyl)-3-(2-chlorophenyl)urea 9d. Yellow oil (49%), IR (KBr): 3304,
3261, 2934, 2859, 1701, 1646, 1593, 1477, 1410 cmÀ1 1H NMR
;
(400 MHz, CDCl3):
d 1.52–1.69 (m, 6H, –NHCH2CH2CH2CH2),
2.82–2.85 (m, 2H, –NHCH2 CH2CH2), 3.15–3.18 (t, 2H, J = 6.0 Hz, –
OCH2CH2NH), 3.53 (m, 2H, –OH), 3.63–3.65 (m, 1H, –CH2NHCH2),
3.66–3.69 (m, 2H, –CH2OH), 4.10–4.12 (t, 2H, J = 6.0 Hz, –ArOCH2),
6.65–6.67 (d, 1H, J = 10.0 Hz, ArH), 6.94–6.98 (t, 1H, J = 9.2 Hz,
ArH), 7.01–7.04 (dd, 1H, J1 = 10.0 Hz, J1 = 3.2 Hz, ArH), 7.22–7.24
(d, 1H, J = 10.0 Hz, ArH), 7.31–7.33 (d, 1H, J = 10.0 Hz, ArH), 8.20–
8.22 (d, 1H, J = 9.2 Hz, ArH), 8.41 (s, 1H, –NHCO), 8.51–8.52 (d,
1H, J = 3.2 Hz, ArH), 9.21 (s, 1H, –NHCO); 13C NMR (100 MHz,
CDCl3): d 22.4, 28.9, 29.1, 51.3, 53.6, 65.2, 65.8, 110.9, 113.9,
121.3, 121.8, 122.3, 123.0, 124.7, 128.0, 129.3, 131.0, 136.2,
145.9, 152.8; MS (ESI): m/z = 470 [M+H]+.
ford 8 as yellow oil (29%), IR (KBr): 2807, 1647, 1599, 1346 cmÀ1
;
1H NMR (400 MHz, CDCl3): d 3.66–3.69 (t, 2H, J = 6.4 Hz, –CH2Br),
4.31–4.34 (t, 2H, J = 6.4 Hz, –CH2O), 6.74–6.77 (d, 1H, J = 11.2 Hz,
ArH), 7.02–7.12 (m, 3H, ArH), 7.27–7.31 (t, 1H, J = 10.0 Hz, ArH),
7.37–7.39 (dd, 1H, J1 = 10.0 Hz, J2 = 1.2 Hz, ArH), 7.48 (s, 1H, –
CONH), 8.10–8.15 (t, 1H, –ArH), 8.42–8.43 (d, 1H, J = 2.0 Hz, ArH);
13C NMR (100 MHz, CDCl3): d 33.2, 70.1, 115.1, 116.3, 121.3,
121.4, 122.2, 123.0, 124.4, 127.4, 129.3, 130.7, 136.2, 146.2,
153.2; MS (ESI): m/z = 449 [M+H]+.
4.2.2. General procedure for the preparation of compounds 9a–g
To a solution of 8 (0.045 g, 0.1 mmol) in 3 mL anhydrous aceto-
nitrile, substituted amine (0.12 mmol) was added and the mixture
was refluxed for 3 h. The mixture was evaporated under reduced
pressure to dryness and the residue was purified by silica gel col-
umn chromatography (PE/EtOAc/TEA = 3:1:0.1) to give 9.
4.2.2.5. 1-(5-Bromo-2-(2-(cyclopropylamino)ethoxy)phenyl)-3-
(2-chlorophenyl)urea 9e. Yellow oil (71%). IR (KBr): 3442, 3319,
2926, 2856, 1701, 1653, 1529, 1475, 1446, 1409 cmÀ1 1H NMR
;
(400 MHz, CDCl3): d 0.54–0.57 (m, 2H, –NHCHCH2CH2), 0.82–0.87
(m, 2H, –NHCHCH2CH2), 2.23–2.24 (m, 1H, –NHCH), 3.14–3.16 (t,
2H, J = 6.0 Hz, –NHCH2), 4.13–4.16 (t, 2H, J = 6.0 Hz, –ArOCH2),
6.73–6.75 (d, 1H, J = 10.0 Hz, ArH), 6.97–7.00 (t, 1H, J = 9.2 Hz,
ArH), 7.28–7.30 (t, 1H, J = 9.2 Hz, ArH), 7.32–7.35 (d, 2H,
J = 9.2 Hz, ArH), 7.79 (s, 1H, –NHCO), 8.18–8.20 (d, 1H,
J = 10.0 Hz, ArH), 8.47 (d, 1H, J = 2.4 Hz, ArH), 8.67 (s, 1H, –NHCO);
13C NMR (100 MHz, CDCl3): d 10.4, 29.1, 52.9, 66.1, 111.2, 114.2,
121.4, 121.9, 123.1, 123.3, 124.8, 128.7, 129.2, 131.1, 137.1,
146.1, 153.0; MS (ESI): m/z = 424 [M+H]+.
4.2.2.1. 1-(5-Bromo-2-(2-(piperidin-1-yl)ethoxy)phenyl)-3-(2-
chlorophenyl)urea 9a. White solid (82%), mp 140–142 °C. IR
(KBr): 2936, 2800, 1694, 1594, 1538, 1442, 1409 cmÀ1 1H NMR
;
(400 MHz, CDCl3): d 1.51 (m, 2H, piperidine), 1.66–1.72 (m, 4H,
piperidine), 2.43–2.61 (m, 4H, piperidine), 2.85–2.88 (t, 2H,
J = 5.2 Hz, –CH2N), 4.05–4.08 (2H, t, J = 5.2 Hz, –ArOCH2), 6.64–
6.66 (d, 1H, J = 8.8 Hz, ArH), 6.92–6.97 (m, 1H, ArH), 6.99–7.03
(m, 1H, ArH), 7.22–7.26 (t, 1H, J = 8.8 Hz, ArH), 7.31–7.34 (dd, 1H,
J1 = 8.0 Hz, J2 = 1.6 Hz, ArH), 8.29–8.31 (dd, 1H, J1 = 8.0 Hz,
J2 = 1.6 Hz, –ArH), 8.44 (s, 1H, –CONH), 8.55–8.56 (d, 1H,
J = 2.0 Hz, ArH), 9.32 (s, 1H, –CONH); 13C NMR (100 MHz, CDCl3):
d 23.9, 25.1, 53.7, 57.0, 63.8, 112.2, 114.2, 121.5, 121.9, 122.5,
123.1, 124.3, 127.5, 129.1, 130.8, 136.0, 146.1, 152.6; MS (ESI):
m/z = 452 [M+H]+.
4.2.2.6. 1-(2-(2-(6-Aminohexylamino)ethoxy)-5-bromophenyl)-
3-(2-chlorophenyl)urea 9f. Red oil (52%). IR (KBr): 3318, 2938,
1697, 1591, 1531, 1476, 1409 cmÀ1 1H NMR (400 MHz, CDCl3): d
;
1.31–1.32 (m, 4H, –NH2CH2CH2CH2CH2), 1.41–1.45 (m, 4H, –
NH2CH2CH2 CH2CH2CH2), 2.76–2.79 (m, 3H, –NHCH2, –NH2),
3.13–3.15 (t, 2H, J = 5.6 Hz, –CH2NH), 3.18–3.29 (m, 4H, –NHCH2,
NH2CH2–), 4.13–4.15 (t, 2H, J = 5.6 Hz, –ArOCH2), 6.67–6.69 (d,
1H, J = 11.2 Hz, ArH), 6.92–6.96 (t, 1H, J = 10.0 Hz, ArH), 6.99–7.02
(dd, 1H, J1 = 11.2 Hz, J1 = 3.0 Hz, ArH), 7.19–7.24 (t, 1H,
J = 10.0 Hz, ArH), 7.29–7.31 (d, 1H, J = 10.0 Hz, ArH), 8.16–8.18 (d,
1H, J = 10.0 Hz, ArH), 8.48–8.49 (d, 1H, J = 3.2 Hz, ArH), 8.52 (s,
1H, –NHCO), 9.23 (s, 1H, –NHCO); 13C NMR (100 MHz, CDCl3): d
23.7, 23.9, 33.1, 33.4, 47.1, 54.3, 55.6, 65.3, 66.6, 111.8, 115.0,
121.6, 121.9, 123.3, 123.8, 124.9, 128.8, 130.1, 131.4, 137.6,
145.8, 153.4; MS (ESI): m/z = 483 [M+H]+.
4.2.2.2. 1-(5-Bromo-2-(2-(cyclohexylamino)ethoxy)phenyl)-3-
(2-chlorophenyl)urea 9b. White solid (62%), mp 108–109 °C. IR
(KBr): 3306, 3265, 2927, 2851, 1691, 1593, 1541, 1444,
1408 cmÀ1 1H NMR (400 MHz, DMSO): d 0.96–1.23 (m, 6H, –
;
NCHCH2CH2CH2CH2CH2), 1.51–1.54 (m, 1H, –NCH), 1.62–1.83 (m,
4H, –NCHCH2CH2CH2CH2CH2), 2.39–2.44 (m, 1H, –NH), 2.92–2.95
(t, 2H, J = 5.6 Hz, –CH2NH), 4.11–4.14 (2H, t, J = 5.6 Hz, –ArOCH2),
7.01–7.13 (m, 3H, ArH), 7.28–7.32 (t, 1H, J = 8.4 Hz, ArH),

W. Huang et al. / Bioorg. Med. Chem. 18 (2010) 5610–5615
5615
4.2.2.7. 1-(2-(2-(3-Aminopropylamino)ethoxy)-5-bromophenyl)
-3-(2-chlorophenyl)urea 9g. Red oil (44%). IR (KBr): 3323, 2938,
1698, 1592, 1534, 1479, 1411 cmÀ1 1H NMR (400 MHz, CDCl3): d
formation. A fixed amount of 9f (25
ing amounts of copper ion (1.25–50
ence UV–vis spectra to investigate the ratio of ligand/metal in the
l
l
mol/L) was mixed with grow-
mol/L) and tested the differ-
;
1.72–1.75 (m, 2H, –NHCH2CH2CH2NH2), 2.81–2.89 (m, 4H, –NHCH2
CH2CH2NH2), 3.09–3.11 (t, 2H, J = 5.6 Hz, –OCH2CH2NH), 4.07–4.09
(t, 2H, J = 5.6 Hz, –ArOCH2), 6.68–6.70 (d, 1H, J = 10.4 Hz, ArH),
6.95–6.99 (t, 1H, J = 8.4 Hz, ArH), 7.03–7.06 (dd, 1H, J1 = 10.4 Hz,
J1 = 3.0 Hz, ArH), 7.25–7.26 (d, 1H, J = 8.4 Hz, ArH), 7.33–7.35 (d,
1H, J = 8.4 Hz, ArH), 8.28–8.30 (d, 1H, J = 10.4 Hz, ArH), 8.48 (s,
1H, –NHCO), 8.56–8.57 (d, 1H, J = 3.2 Hz, ArH), 9.03 (s, 1H, –NHCO);
13C NMR (100 MHz, CDCl3): d 33.1, 41.1, 47.1, 50.3, 67.8, 112.5,
115.9, 120.6, 121.4, 123.7, 124.8, 125.0, 128.6, 130.6, 131.3,
136.9, 146.9, 152.3; MS (ESI): m/z = 441 [M+H]+.
complex.
Acknowledgments
The authors thank the National Key Tech Project for Major Cre-
ation of New drugs (NO. 2009ZX09501-003) and the 2008 Zhejiang
Innovation Program for Graduates for financial support.
References and notes
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All compounds were tested as BACE 1 inhibitors, using a fluo-
rescence resonance energy transfer (FRET) assay, which used puri-
fied insect-expressed BACE 1 and a specific substrate. An excitation
wavelength of 355 nm and an emission wavelength of 460 nm
were used to monitor the hydrolysis of substrate. Compounds with
inhibitory rates at 20 lg/mL upon 50% were tested for IC50 values.
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4.4. Spectrophotometric measurement of complex with Cu and
Fe5
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10190.
All compounds were tested as metal chelators, using difference
UV–vis spectra recorded in methanol at 298 K with wavelength
ranging from 210 to 400 nm. Numerical subtraction of the spectra
of the metal alone and the compound alone from the spectra of the
mixture obtained the difference UV–vis spectra due to complex