Identification of pharmacophore model, synthesis and biological evaluation of N-phenyl-1-arylamide and N-phenylbenzenesulfonamide derivatives as BACE 1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.10.059

The pharmacophore model of arylpiperazine amide derivatives was built using Discovery Studio 2.0 software package and the best pharmacophore model (Hypo 1) was validated by Enrichment and ROC method (EF at 2%, 5% and 10% are 30.6, 12.2 and 7.7; AUC of the

Full text of DOI:10.1016/j.bmc.2008.10.059

Bioorganic & Medicinal Chemistry 16 (2008) 10190–10197
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of pharmacophore model, synthesis and biological evaluation
of N-phenyl-1-arylamide and N-phenylbenzenesulfonamide derivatives
as BACE 1 inhibitors
Wenhai Huang a, Haiping Yu b, Rong Sheng 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
a r t i c l e i n f o
a b s t r a c t
Article history:
The pharmacophore model of arylpiperazine amide derivatives was built using Discovery Studio 2.0 soft-
ware package and the best pharmacophore model (Hypo 1) was validated by Enrichment 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). According to the best
pharmacophore model, 11 N-phenyl-1-arylamide, N-phenylbenzenesulfonamide derivatives, compounds
26–28, and 33a–g, were designed to be synthesized and their BACE 1 inhibitory activities were deter-
mined experimentally. Their theoretical results were in good agreement with the experimental values.
Received 6 August 2008
Revised 27 October 2008
Accepted 27 October 2008
Available online 30 October 2008
Keywords:
Compound 33d, which displayed the highest BACE 1 activity (18.33 2.80 lmol/L) among these two ser-
Pharmacophore model
Alzheimer’s disease
BACE 1
ies, was chosen to study the protein binding pattern and the result showed that it was in close contact
with two essential catalytic aspartates (Asp32 and Asp228) of the BACE 1.
Ó 2008 Elsevier Ltd. All rights reserved.
Non-peptidomimetic inhibitor
1. Introduction
ture and high b-secretase inhibitory activity (IC50 values from 0.046
to 20 lmol/L) (Fig. 11a). According to the X-ray crystal structure
Alzheimer’sdisease (AD) is one kind of the most common demen-
tia. It has been characterized by the progressive formation of insolu-
ble amyloid plaques (Ab) and fibrillary tangles.1,2 Plaquesare
extracellular aggregations of a peptide fragment Ab42,3,4 which is
formed by the sequential proteolytic processing of b-amyloid pre-
data for piperidine derivative complexed to renin, the protonated
piperidine nitrogen was positioned between the two catalytic aspar-
tic acid residues Asp32 and Asp228. Kraus and coworkers incorpo-
rated various substituents on the N4-position of the piperazine
ring, replacing the naphthyl ring with various heterocyclic moieties.
These studies confirmed that the substituent on the N4-position of
the piperazine ring is another important feature of the arylpiper-
azine amide derivatives to binding on BACE 1.
cursor protein (APP) by two enzymes, b- and c
-secretase.5–7 There-
fore, the inhibition of the two enzymes is likely to reduce the
production of Ab and thereby delay or halt the progression of AD.
The idea, which
path related to the growth of neural cells, is supported by experi-
mental results, such as the fact that -secretase gene knockout mice
c
-secretase inhibitors could affect the Notch signal
In the present studies, it was to identify the key pharmaco-
phoric features of the arylpiperazine amide derivatives and corre-
sponding pharmacophore models for arylpiperazine amide
derivatives using Discovery Studio 2.0 software package. The vali-
dation of the best pharmacophore model (Hypo 1) was ascertained
by Enrichment and receiver operating curve (ROC) method. The
c
can not survive.8 Taking into account of these observations, develop-
ing b-secretase inhibitors appears to be more encouraging.9,10
Due to low oral bioavailability, metabolic unstability, poor abil-
ity to penetrate central nervous system (CNS) and susceptibility to
P-glycoprotein transport of existing peptidomimetic inhibitors,11–13
researchers are focus on the identification of the non-peptide inhib-
itors in recent years. Several series of non-peptide inhibitors, includ-
ing 1,3,5-trisubstituted aromatic derivatives,14 acylguanidine
derivatives,15 2-amino aromatic heterocyclic derivatives16 and aryl-
piperazine amide derivatives,17–19 have been reported. Among them,
the arylpiperazine amide scaffold has the benefits of its simple struc-
Hypo
1 was used to in silico screen a designed database
(Fig. 11b). As a result, 11 novel N-phenyl-1-arylamide, N-phenyl-
benzenesulfonamide derivatives 26–28, 33a–g were selected and
synthesized considering effects of different skeletons (N-phenyl-
1-arylamide, N-phenylbenzenesulfonamide) and different substit-
uents on the nitrogen of the phenoxyethylamine. BACE 1 inhibitory
activities of these compounds were subsequently evaluated
experimentally. The result showed that the bioactivity data was
consistent with Fit values and cLogP values. In an attempt to
understand the protein binding pattern of these two series, a
molecular docking of compound 33d has also been studied.
* Corresponding author. Tel.: +86 571 88208460.
E-mail addresses: huyz@zju.edu.cn (Y. Hu), huyz@zjuem.zju.edu.cn (Y. Hu).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.059

W. Huang et al. / Bioorg. Med. Chem. 16 (2008) 10190–10197
10191
Figure 1. The structures of arylpiperazine amide derivatives 1a and N-phenyl-1-arylamide and N-phenylbenzenesulfonamide derivatives 1b.
2.2. Common feature-based pharmacophore model
2. Materials and methods
2.1. Data preparation
We employed the Discovery Studio 2.0/HipHop approach to
evaluate the common features required for binding. A training set
consisting of three arylpiperazine amide derivatives was submitted
for pharmacophore model generation based on common chemical
features. All the parameters were kept at the default setting except
the Best method for the Conformation Generation and Fitting.
Twenty-one positives for the pharmacophore model study were
taken from the literatures (Fig. 2),17,18 and one thousand two hun-
dred negatives were retrieved from Advanced Chemical Directory
(ACD) database using Random Percent Filter protocol by Pipeline Pi-
lot software. The training set was consisted of three positives and
the test set was consisted of 18 positives and 1200 negatives. All
compounds were optimized by Discovery Studio 2.0 software
package.
2.3. Model validation
The validation of any pharmacophore model should be ascer-
tained and there are a number of approaches to quantitate models.
Figure 2. The structure of three positives used in training set (2–4) and 18 positives used in test set (5–22) for the pharmacophore model study.

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W. Huang et al. / Bioorg. Med. Chem. 16 (2008) 10190–10197
The simplest and most often used method is Enrichment (EF) at a
given percentage. EF is defined as:
Every model should be ascertained, and the most often used
and simplest is enrichment (EF) at a given percentage of the data-
base. The EF of test set consisting of one thousand two hundred
and eighteen compounds screened by Hypo 1 at 2%, 5% and 10%
are 30.6, 12.2 and 7.7, respectively. In addition, the ROC curve of
the test set screening by Hypo 1 shows in Figure 4 and AUC is
0.93 which means that a selected randomly active compound has
a higher score than a randomly selected inactive compound 9.3
times out of 10.
TP
TP þ FP n
N
EF ¼
where TP is the number of true positives, FP is the number of false
positives, N is the number of the total compounds and n is the
number of the total positive compounds.20
In addition, the receiver operating curve (ROC) metric, allowed
quick calculation of sensitivity and specificity from a comparison
between in vitro and in silico, had also been applied to evaluate
the validation of the model.21
The results of the EF and ROC metric suggested that the Hypo 1
would be valuable and reliable in identifying the compounds for
BACE 1 inhibitory activity.
3.2. Chemistry
(a) Sensitivity (Se): the likelihood that an event will be detected
if that event is present. Se is defined as:
Discovery Studio 2.0/Ligand Pharmacophore Mapping protocol
was used with Hypo 1 as pharmacophore model to screen the de-
signed database. Compounds 26–28a, 33a–f with high Fit values
(2.80–4.06, the maximum of the Fit value is 5.00) were picked
out and synthesized. In order to investigate the predictive accuracy
of the Hypo 1 for inactive compounds, 28b and 33g with low Fit
values (1.70 and 1.89) were also prepared.
TP
Se ¼
TP þ FN
where FN is the number of false negatives.
(b) Specificity (Sp): the likelihood that the absence of an event
will be detected. Sp is defined as:
N-Phenyl-1-arylamide and N-phenylbenzenesulfonamide deri-
vatives 26–28 were prepared according to the procedure depicted
in Scheme 1. Reaction of 4-bromo-2-nitrophenol with 1,2-dibro-
moethane afforded compound 23, which was refluxed with
morpholine in acetonitrile to yield 24, followed by reduction of ni-
tro group with stannous chloride to get 25, a key intermediate
which was subsequently reacted with substituted aryl chloride to
yield target compounds 26–28 in good yields.
TN
Sp ¼
TN þ FP
where TN is the number of true negatives.
(c) The ROC curve is a function of (1 ꢀ Sp) versus the Se, and the
area under the ROC curve (AUC) is the important way of measuring
the performance of the test.
Compounds 33a–g were synthesized according to the proce-
dure depicted in Scheme 2. The hydroxyl of 4-bromo-2-nitrophe-
nol was protected with methoxy methyl (MOM) group to get
compound 29, which was reduced to 30a with stannous chloride,
while catalytic hydrogenation with Pd/C led to the loss of the bro-
mine atom to get 30b. The aniline derivatives 30a,b were subse-
quently acylated with 2-naphthyl acetyl chloride, followed by
treatment with diluted hydrochloric acid to afford intermediates
31a–b. The resulting compounds were reacted with 1,2-dibromo-
ethane to get compounds 32a–b, which were subsequently reacted
with various amine derivatives to obtain target compounds 33a–g.
N
X
AUC ¼
SeðxÞ½ð1 ꢀ SpÞðxÞ ꢀ ð1 ꢀ SpÞðx ꢀ 1Þꢁ
x¼2
Here, Se(x) is the percent of the true positives versus the total
positives at rank position x, (1 ꢀ Sp)(x) is the percent of the false
positives versus the total negatives at rank position x.
3. Results and discussion
3.1. Pharmacophore model and validation
For the HipHop pharmacophore analysis, three highly active
arylpiperazine amide derivatives were chosen as a training set to
generate the pharmacophore model based on common chemical
features. Ten pharmacophore models were obtained and the best
pharmacophore model (Hypo 1) with the highest score 38.78 con-
tained five chemical features: one pos-ionizable (PI), two ring aro-
matic (RA1 and RA2), one hydrophobic (HP), and one hydrogen
bond acceptor (HBA) (Fig. 3, 3a).
3.3. BACE 1 activity study
Eleven compounds were evaluated for their BACE 1 activities
using a fluorescence resonance energy transfer (FRET) assay, with
potent peptidomimetic inhibitor OM99-2 as positive control. As
shown in Table 1, at 20
compounds, and their IC50 values were 18.33 2.80
47.09 3.37 mol/L, respectively. Unfortunately, the activity of
lg/mL, 33d and 33e were the most active
lmol/L and
l
Figure 3. (a) The best pharmacophore model (Hypo 1); (b) Hypo 1 aligned to compound 28b; (c) Hypo 1 aligned to compound 33e.

W. Huang et al. / Bioorg. Med. Chem. 16 (2008) 10190–10197
10193
stituents on the nitrogen of the phenoxyethylamine revealed that
compounds with 1-naphthalenyl acetyl group as aromatic ring moi-
ety (26, cLogP = 4.53), hydrophobic group and secondary amine as
substitutes on the nitrogen of the phenoxyethylamine (33d,
cLogP = 4.68 and 33e, cLogP = 5.95) demonstrated more potent
inhibitory activity against BACE 1. Compared with the structure
and the activity of the compounds 33a and 33g, it was worth noting
that the cLogP value of the more potent compound 33a
(cLogP = 5.22) was greater than compound 33g (cLogP = 4.19)
which only differed in structure by the presence of a bromine atom.
These bioactivity data were also consistent with cLogP values.
3.4. Molecular docking study
In an attempt to understand the molecular interaction between
33d and BACE 1, a molecular docking study was performed using
the AUTODOCK 3.0 with the crystal structure of OM99-2/BACE 1
complex (PDB ID: 1W51) as model. The docking and subsequent
scoring were performed using default parameters. The result dis-
closed that the binding pattern of 33d into the BACE 1 was similar
to the crystal structure of Vertex in the patent application
WO02088101 (Fig. 5).19 Hydrogen bonding interactions were ob-
served between acylamide groups and Thr72, Gln73, Thr231.
Hydrophobic interactions between S1 pocket which lined with aro-
matic side chain (Try71) and naphthyl group provided a compo-
nent of the affinity of 33d. The nitrogen of the
phenoxyethylamine formed hydrogen bonds with catalytic aspar-
Figure 4. The red curve is the ROC curve of the test set; The green curve is the ROC
curve of the random classification of the compounds.
the synthesized compounds did not increase compared to the lead
compound. However, we can still find some SAR of the compounds.
A correlation between the Fit values comparing the best phar-
macophore model (Hypo 1) and the inhibitory activities has been
observed. Nine compounds 26–28b, 33a–f (such as compound
33e in Fig. 3, 3c), which were predicted with high Fit values by
the Hypo 1, showed medium to good activities, while 7b and 12g
with low Fit values (such as compound 28b in Fig. 3, 3b) showed
weak BACE 1 inhibitory activities.
Insight into the observed effects of different skeletons (N-phe-
nyl-1-arylamide, N-phenylbenzenesulfonamide) and different sub-
0
0
tates, Asp32 and Asp228, with distance of 2.73 ÅA and 2.66 ÅA,
Scheme 1. Syntheses of compounds 23–28. Reagents and condition: (a) BrCH2CH2Br, 40%NaOH, reflux; (b) morpholine, CH2Cl2, rt; (c) SnCl2ꢂ2H2O, EtOH/THF, rt; (d) acyl
chloride, CH2Cl2, rt; (e) substituted phenyl sulfonyl chloride, CH2Cl2, rt.
Scheme 2. Syntheses of compounds 33a–g. Reagents and condition: (a) MOMCl, K2CO3, acetone, rt; (b) SnCl2ꢂ2H2O, EtOH/THF, rt; or Pd-C, EtOH, rt; (c) naphthalenyl acetyl
chloride, CH2Cl2, rt; (d) BrCH2CH2Br, 40%NaOH, reflux; (e) amine, CH2Cl2, rt.

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W. Huang et al. / Bioorg. Med. Chem. 16 (2008) 10190–10197
Table 1
The BACE 1 activity of N-phenyl-1-arylamide and N-phenylbenzenesulfonamide derivatives.
R2
R1
O
R3
Compound
OM99-2
R1
R2
R3
Inhibition at 20
102.09 2.87a
lg/mL (%)
Fit value
cLogP
26
Br
Br
Br
Br
1-Naphthalenylacetamide
Phenylpropanamide
39.35 6.64
13.84 7.30
14.27 5.96
ND
3.94
3.25
2.80
1.70
4.53
3.92
4.77
3.76
N
O
27
N
N
N
O
O
O
28a
28b
4-Bromobenzenesulfonamide
Benzenesulfonamide
N
OE t
O
33a
Br
1-Naphthalenylacetamide
24.74 3.15
3.92
5.22
N
H
OH
33b
33c
Br
Br
1-Naphthalenylacetamide
1-Naphthalenylacetamide
15.08 9.02
34.77 7.93
3.86
4.06
3.75
5.00
O
N
OEt
N
H
NH2
33d
33e
Br
Br
1-Naphthalenylacetamide
1-Naphthalenylacetamide
83.70 1.30
64.46 3.47
3.96
4.00
4.68
5.95
4
H
N
33f
Br
H
1-Naphthalenylacetamide
1-Naphthalenylacetamide
41.81 9.91
ND
3.68
1.89
6.37
4.16
N
N
OEt
O
33g
a
OM99-2 was tested at 2 lg/mL.
respectively, consistent with the known binding mode of arylpip-
erazine amide derivatives.18 Moreover, amino group at the end of
the alkyl chain had hydrogen bonding interactions with Lys-107,
Phe-108 as designed, which may explain the high activity of the
compound.
ROC curve is 0.93) to predict the activity of designed database con-
sisting of N-phenyl-1-arylamide and N-phenylbenzenesulfonamide
derivatives. Nine compounds (26–28a, 33a–f) with high Fit values
comparing Hypo 1 and 2 compounds (28b, 33g) with low Fit values
were selected and synthesized. The theoretical results of them
were in good agreement with the experimental values. Compound
33d (IC50 = 18.33 2.80 lmol/L) was the most effective inhibitor
4. Conclusions
against BACE 1 and as a typical example of refined structure. Dock-
ing study of 33d indicated that substituents on the nitrogen of the
phenoxyethylamine could make some additional hydrogen bonds
with certain residues. These observations may direct our design
in the further.
In this work, we built pharmacophore model with good predic-
tive ability (EF at 2%, 5% and 10% are 30.6, 12.2 and 7.7; AUC of the
5. Experimental
5.1. Chemistry
All solvents used were of analytical grade. Melting points were
recorded on a Buchi apparatus and are uncorrected, IR spectra
were recorded on a Bruker VECTOR 22 FTIR spectrophotometer.
1H NMR spectra were recorded on a Bruker AM 400 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.
5.1.1. 4-Bromo-1-(2-bromoethoxy)-2-nitrobenzene (23)
To a solution of 4-bromo-2-nitrophenol (1.09 g, 5 mmol) in 1,2-
dibromoethane (5 mL), 40% NaOH solution (5 mL) and tetrabutyl
ammonium bromide (0.05 g) were added, the mixture was re-
fluxed for 4 h, cooled to room temperature, the organic phase
Figure 5. The docking result of inhibitor 33d in complex with BACE 1.

W. Huang et al. / Bioorg. Med. Chem. 16 (2008) 10190–10197
10195
was separated, and the aqueous phase was extracted with CH2Cl2
5.1.7. 4-Bromo-N-[5-bromo-2-(2-morpholinoethoxy)phenyl]-
benzenesulfonamide (28a)
(15 mL). The combined organic extract was washed with brine,
dried over anhydrous Na2SO4, evaporated under reduced pressure,
the crude solid was purified by silica-gel column chromatography
(PE/EtOAc 10:1) to get a yellow solid (1.3 g, 80% yield), mp 76–
78ꢀC. IR (KBr) cmꢀ1: 2932, 1604, 1521. 1H NMR (CDCl3) d: 3.65
(2H, t, J = 6.4 Hz, –CH2Br), 4.38 (2H, t, J = 6.4 Hz, –OCH2), 6.98
(1H, d, J = 9.2 Hz, ArH), 7.62 (1H, dd, J1 = 9.2 Hz, J2 = 2.4Hz, ArH),
7.97 (1H, d, J = 2.4 Hz, ArH).
Compound 28a was obtained (0.013 g, 10% yield) as a yellow oil,
mp 127–130ꢀC. IR (KBr) cmꢀ1: 2935, 2845, 1599, 843. 1H NMR
(CDCl3) d: 2.52–2.60 (6H, m, –CH2N, morpholine), 3.95 (4H, m,
morpholine), 4.05 (2H, t, J = 5.6 Hz, –ArOCH2), 6.78 (1H, d,
J = 9.2 Hz, ArH), 7.21 (1H, dd, J1 = 8 Hz, J2 = 2.4 Hz, ArH), 7.56–7.62
(4H, m, ArH), 7.77 (1H, m, ArH). ESI-MS m/z: 471 (M+).
5.1.8. N-[5-Bromo-2-(2-morpholinoethoxy)phenyl]benzene
sulfonamide (28b)
5.1.2. 4-[2-(4-Bromo-2-nitrophenoxy)ethyl]morpholine (24)
A solution of 23 (0.975 g, 3 mmol) and morpholine (0.518 mL,
6 mmol) in CH2Cl2 (5 mL) was stirred at room temperature for
3 h, the mixture was evaporated under reduced pressure to afford
yellow oil, which was then purified by silica-gel column chroma-
tography (PE/EtOAc/TEA 10:1:0.1) to give a yellow oil (0.914 g,
92% yield). IR (KBr) cmꢀ1: 2925, 2863, 1599, 1511. 1H NMR (CDCl3)
d: 2.57 (t, 4H, J = 4.4 Hz, morpholine), 2.82 (2H, t, J = 5.6 Hz,
–CH2N), 3.69 (4H, t, J = 4.4 Hz, morpholine), 4.20 (2H, t, J = 5.6 Hz,
–OCH2), 6.97 (1H, d, J = 9.2 Hz, ArH),7.59 (1H, dd, J1 = 9.2 Hz,
J2 = 3.2 Hz, ArH), 7.95 (2H, d, J = 3.2 Hz, ArH).
Compound 28b was obtained (0.094 g, 85% yield) as a white so-
lid, mp 103–105ꢀC. IR (KBr) cmꢀ1: 2929, 1595, 1497, 761, 689. 1H
NMR (CDCl3) d: 2.50–2.58 (6H, m, –CH2N, morpholine), 3.92 (4H,
t, J = 4 Hz, morpholine), 4.01 (2H, t, J = 4.8 Hz, –ArOCH2), 6.74
(1H, d, J = 8.4 Hz, ArH), 7.16 (1H, dd, J1 = 8 Hz, J2 = 2 Hz, ArH),
7.40–7.44 (2H, m, ArH), 7.50–7.52 (1H, m, ArH), 7.74–7.76 (3H,
m, ArH). ESI-MS m/z: 441 (M+).
5.1.9. N-(5-Bromo-2-hydroxyphenyl)-1-naphthalenylacetamide (31a)
To a solution of 4-bromo-2-nitrophenol (1.09 g, 5 mmol) in ace-
tone (25 mL), K2CO3 (2.76 g, 20 mol) and MOMCl (0.49 mL,
6.5 mmol) was added, the mixture was stirred at room tempera-
ture for 2 h. The organic phase was separated and the solvent
was removed under reduced pressure to get yellow oil (29), to
which ethanol (5 mL) and THF (5 mL) were added to get a homog-
enous solution. The reaction mixture was added SnCl2ꢂ2H2O
(3.39 g, 15 mmol) in a portion and then stirred at room tempera-
ture for 4 h, evaporated under reduced pressure to afford a white
solid, to which 15% NaOH (5 mL) was added, stirred for 0.5 h, ex-
tracted with ether and dried by Na2SO4. Then removed the ether
under reduced pressure to afford an oil (30a), which was mixed
with 1-naphthalenylacetyl chloride (0.74 g, 3.6 mmol) and 15 mL
CH2Cl2, stirred at room temperature for 1 h. The solvent was re-
moved and 3 N HCl (3 mL), isopropanol (3 mL) and THF (3 mL)
was added, stirred for 36 h. Volatiles were removed and remained
solid was purified by a silica-gel column chromatography (PE/
EtOAc 1:1) to afford a white solid (0.57 g, 32% yield), mp 207–
209ꢀC. IR (KBr) cmꢀ1: 3365, 1668, 1588, 1532, 735, 674. 1H NMR
(DMSO-d6) d: 4.26 (2H, s, –COCH2), 6.79 (1H, d, J = 8 Hz, ArH),
7.04–7.07 (1H, m, ArH), 7.48–7.56 (4H, m, ArH), 7.84 (1H, d,
J = 8 Hz, ArH), 7.92–7.95 (1H, m, ArH), 8.10–8.13 (2H, m, ArH),
9.43 (1H, bs, –NHCO), 10.24 (1H, s, –OH).
5.1.3. 5-Bromo-2-(2-morpholinoethoxy)benzenamine (25)
To a solution of 24 (0.993 g, 3 mmol) in the mixture of ethanol
(5 mL) and 5 mL THF (5 mL), SnCl2ꢂ2H2O (3.39 g, 15 mmol) was
added in a portion, the mixture was stirred at room temperature
for 4 h. Volatiles were removed and remained solid was added
the 15% NaOH (5 mL), stirred for 0.5 h in ice bath. The reaction
mixture was extracted with ether, separated the organic phase
which was then removed to get crude solid. The solid was purified
by silica-gel column chromatography (PE/EtOAc/TEA 10:1:0.1) to
give a yellow oil (0.452 g, 50% yield), mp 66–67ꢀC. IR (KBr) cmꢀ1
:
3380, 3311, 3196, 2923, 2877, 1591, 1503, 1458. 1H NMR (CDCl3)
d: 2.56–2.58 (4H, m, morpholine), 2.77 (2H, t, J = 5.2 Hz, –CH2N),
3.72 (4H, t, J = 4.8 Hz, morpholine), 3.99–4.01 (2H, m, –NH2), 4.08
(2H, t, J = 5.2 Hz, –OCH2), 6.65 (1H, d, J = 8 Hz, ArH), 6.77 (1H, dd,
J1 = 8 Hz, J2 = 2.4 Hz, ArH), 6.83 (2H, d, J = 2.4 Hz, ArH).
5.1.4. General procedure for the preparation of compounds 26–
28
To a solution of 25 (0.25 mmol) in CH2Cl2 (5 mL), acyl chloride
or the substituted phenyl sulfonyl chloride (0.3 mmol) was added,
and the mixture was stirred at room temperature for 1 h. Volatiles
were removed and the residue was purified by a silica-gel column
chromatography (PE/EtOAc/TEA 3:1:0.1 or 2:1:0.1).
5.1.10. N-(2-Hydroxyphenyl)-1-naphthalenylacetamide (31b)
Using the same method as 4-bromo-2-nitrophenol (1.09 g,
5 mmol) to get 29, which was then dissolved in the ethanol
(5 mL), 10% Pd–C (0.24 g) was added. The mixture was stirred at
room temperature for 4 h under hydrogen balloon, filtered to re-
move Pd/C. The filtrate was evaporated under reduced pressure
to get an oil (30b). 0.776 g of 31b was prepared as described for
31a but starting from 30b (56% yield). IR (KBr) cmꢀ1: 3359, 2924,
2851, 1653, 1591, 1509, 741, 698. 1H NMR (CDCl3) d: 4.26 (2H, s,
–NHCOCH2–), 5.32 (1H, s, –OH), 6.54 (1H, d, J = 8Hz, ArH), 6.70–
6.74 (1H, m, ArH), 6.97–7.09 (2H, m, ArH), 7.53–7.63 (4H, m,
ArH), 7.92–7.99 (3H, m, ArH), 8.69 (1H, bs, –NHCO–).
5.1.5. N-[5-Bromo-2-(2-morpholinoethoxy)phenyl]-1-
naphthalenylacetamide (26)
Compound 26 was obtained (0.094 g, 80% yield) as a white so-
lid, mp 77–79ꢀC. IR (KBr) cmꢀ1: 2930, 2851, 1681, 1593, 1525,
788. 1H NMR (CDCl3) d: 2.11–2.23 (6H, m, –CH2N, morpholine),
3.60–3.64 (4H, m, morpholine), 3.73–3.76 (2H, m, –CH2CO),
4.21(2H, s, –CH2O), 6.52 (1H, d, J = 7.6Hz, ArH), 7.03–7.05 (1H, m,
ArH), 7.51–7.54 (4H, m, ArH), 7.75 (1H, bs, –NHCO), 7.90 (2H, s,
ArH), 8.01 (1H, s, ArH), 8.58 (1H, s, ArH). ESI-MS m/z: 469 (M+).
5.1.6. N-[5-Bromo-2-(2-morpholinoethoxy)phenyl]-3-
phenylpropanamide (27)
5.1.11. N-[5-Bromo-2-(2-bromoethoxy)phenyl]-1-naphthalenyl
acetamide (32a)
Compound 27 was obtained (0.074 g, 68% yield) as a yellow oil.
IR (KBr) cmꢀ1: 2925, 2855, 1687, 1593, 1519, 753, 699. 1H NMR
(CDCl3) d: 2.51–2.53 (4H, m, morpholine), 2.70–2.74 (4H, m,
–COCH2CH2, –OCH2CH2), 3.04 (2H, t, J = 8 Hz, –COCH2), 3.69 (4H,
t, J = 4.8 Hz, morpholine), 4.09 (2H, t, J = 5.6 Hz, –OCH2), 6.73 (1H,
d, J = 8.8 Hz, ArH), 7.10–7.32 (6H, m, ArH), 8.20 (1H, bs, –NHCO),
8.59 (1H, s, ArH). ESI-MS m/z: 433 (M+).
Prepared as described for 23 but starting from 31a to get the
yellow oil (25% yield). IR (KBr) cmꢀ1: 3381, 2926, 1682, 1592,
1520, 1463, 711, 674. 1H NMR (CDCl3) d: 3.02 (2H, t, J = 5.6 Hz,
–CH2Br), 3.91 (2H, t, J = 5.6 Hz, –OCH2), 4.23 (1H, s, –COCH2),
6.51 (1H, d, J = 8 Hz, ArH), 7.04–7.07 (1H, m, ArH), 7.55–7.60 (4H,
m, ArH), 7.78 (1H, bs, –NHCO), 7.89–7.94 (2H, m, ArH), 8.03 (1H,
d, J = 7.6 Hz, ArH), 8.59–8.60 (1H, d, J = 2 Hz, ArH).

10196
W. Huang et al. / Bioorg. Med. Chem. 16 (2008) 10190–10197
5.1.12. N-[2-(2-Bromoethoxy)phenyl]-1-naphthalenyl
acetamide (32b)
5.1.18. N-{5-Bromo-2-[2-(cyclohexylamino)ethoxy]phenyl}-1-
naphthalenylacetamide (33e)
Prepared as described for 32a but starting from 31b to get the
yellow oil (80% yield). IR (KBr) cmꢀ1: 3382, 2925, 2867, 1668,
1593, 1510, 1456, 762, 699. 1H NMR (CDCl3) d: 2.99 (2H, t,
J = 5.6 Hz, –CH2Br), 3.90 (2H, t, J = 5.6 Hz, -OCH2), 4.21 (1H, s,
–COCH2), 6.63 (1H, d, J = 8 Hz, ArH), 6.92–6.94 (2H, m, ArH),
7.50–7.54 (4H, m, ArH), 7.78 (1H, bs, –NHCO), 7.87–7.92 (2H, m,
ArH), 8.03–8.05 (1H, m, ArH), 8.35–8.37 (1H, m, ArH).
Compound 33e was obtained (0.078 g, 65% yield) as a yellow oil.
IR (KBr) cmꢀ1: 2956, 2925, 2854, 1630, 1459, 762, 689. 1H NMR
(CDCl3) d: 1.53–1.81 (10H, m, –NCHCH2CH2CH2CH2CH2), 2.06 (1H,
m, –NCH), 2.53 (2H, t, J = 5.2 Hz, –ArOCH2CH2), 3.73 (2H, t,
J = 5.2 Hz, –ArOCH2), 4.23 (2H, s, –COCH2), 6.56 (1H, d, J = 8.8 Hz,
ArH), 7.04 (1H, dd, J1 = 8 Hz, J2 = 2 Hz, ArH), 7.50–7.57 (4H, m,
ArH), 7.87–8.05 (4H, m, 3ArH, –NHCO), 8.55 (1H, d, J = 2 Hz, ArH).
ESI-MS m/z: 481 (M+).
5.1.13. General procedure for the preparation of compounds
33a–g
5.1.19. N-{2-[2-(Benzyl-methylamino)ethoxy]-5-bromopheny}-
1-naphthalenylacetamide (33f)
To a solution of 32a (0.115 g, 0.25 mmol) or 32b (0.096 g,
0.25 mmol) in 3 mL anhydrous acetonitrile, substituted amine
(0.3 mmol) was added and the mixture was stirred at room tem-
perature for one day. The mixture was evaporated under reduced
pressure to dryness and the residue was purified by a silica-gel col-
umn chromatography (PE/EtOAc/TEA 3:1:0.1 or PE/EtOAc/MeOH
3:1:0.1).
Compound 33f was obtained (0.07 g, 56% yield) as a white solid,
mp 113–115ꢀC. IR (KBr) cmꢀ1: 2925, 1643, 1594, 1526, 1483, 735,
696. 1H NMR (CDCl3) d: 2.12 (3H, s, –NCH3), 2.25–2.28 (2H, m, –Ar-
OCH2CH2), 3.44 (2H, s, –NCH2Ar), 3.76 (2H, t, J = 6 Hz, –ArOCH2), 4.18
(2H, s, –COCH2), 6.54 (1H, d, J = 9.2 Hz, ArH), 7.04 (1H, dd, J1 = 8.4Hz,
J2 = 2.4Hz, ArH), 7.27–7.37 (9H, m, ArH, –NHCO), 7.83–8.02 (4H, m,
ArH), 8.59 (1H, d, J = 2.4Hz, ArH). ESI-MS m/z: 503 (M+).
5.1.14. Ethyl 2-{2-[4-bromo-2-(2-naphthalen-1-yl-
acetamido)phenoxy]ethyl-methyl-amino}acetate (33a)
Compound 33a was obtained (0.097 g, 78% yield) as a white so-
lid, mp 68–70ꢀC. IR (KBr) cmꢀ1: 3389, 2976, 2925, 1739, 1683,
1598, 1527, 1450, 751. 1H NMR (CDCl3) d: 1.18–1.21 (3H, m,
–CH2CH3), 2.27 (3H, s, –NCH3), 2.49 (2H, t, –ArOCH2CH2), 3.10
(2H, s, NCH2CO-), 3.75 (2H, t, J = 6.4 Hz, –ArOCH2), 4.05 (2H, q,
J = 7.2 Hz, –COOCH2), 4.20 (2H, s, –NHCOCH2), 6.54 (1H, d,
J = 8.8 Hz, ArH), 6.99–7.02 (1H, m, J1 = 8.8Hz, J2 = 2.4Hz, ArH),
7.45–7.50 (4H, m, ArH), 7.80–7.86 (2H, m, ArH), 7.99 (1H, d,
J = 8.0Hz, ArH), 8.10 (1H, bs, –NHCO-), 8.54 (1H, d, J = 2.0Hz,
ArH). ESI-MS m/z: 499 (M+).
5.1.20. Ethyl 2-{methyl 2-[2-(2-naphthalen-1-yl-
acetamido)phenoxy]ethyl-amino}acetate (33g)
Compound 33g was obtained (0.089 g, 85% yield) as a yellow oil.
IR (KBr) cmꢀ1: 2926, 1733, 1678, 1593, 1453, 733, 698. 1H NMR
(CDCl3) d: 1.18 (3H, t, J = 7.2 Hz, –CH2CH3), 2.25 (3H, s, –NCH3),
2.37 (2H, t, J = 5.6 Hz, –ArOCH2CH2), 3.07 (2H, s, –NCH2CO), 3.75
(2H, t, J = 5.6 Hz, –ArOCH2), 4.05 (2H, q, J = 6.4 Hz, –OCH2CH3),
4.19 (2H, s, –NHCOCH2), 6.60 (1H, d, J = 9.2 Hz, ArH), 6.87–6.90
(2H, m, ArH), 7.43–7.54 (4H, m, ArH), 7.80–7.86 (2H, m, ArH),
8.00–8.03 (2H, m, 1ArH, –NHCO), 8.32–8.34 (1H, dd, J1 = 6.8 Hz,
J2 = 2.4 Hz, ArH). ESI-MS m/z: 420 (M+).
5.1.15. N-{5-Bromo-2-[2-(3-hydroxypropylamino)ethoxy]
phenyl}-2-(naphthalen-1-yl) acetamide (33b)
5.2. In vitro BACE 1 inhibit activity screening
Compound 33b was obtained (0.097 g, 85% yield) as a yellow
oil. IR (KBr) cmꢀ1: 3380, 2923, 2856, 1683, 1593, 1511,762, 689.
1H NMR (CDCl3) d: 1.66–1.69 (2H, m, –CH2CH2OH), 2.66–2.84
(4H, m, –CH2NHCH2), 3.76–3.81 (4H, m, –ArOCH2,CH2OH), 4.26
(2H, s, –COCH2), 6.52 (1H, d, J = 8.8 Hz, ArH), 7.03 (1H, dd,
J1 = 8.4 Hz, J2 = 1.6 Hz, ArH), 7.48–7.55 (4H, m, ArH), 7.85–7.91
(2H, m, 2ArH), 8.05 (1H, d, J = 7.6 Hz, ArH), 8.22 (1H, s, –NHCO),
8.52 (1 H, d, J = 1.6 Hz, ArH). ESI-MS m/z: 457(M+).
All the compounds were assayed as BACE 1 inhibitors, using a
fluorescence resonance energy transfer (FRET) assay, which used
purified insect-expressed BACE 1 and a specific substrate. An exci-
tation wavelength of 355 nm and an emission wavelength of 460
nm were used to monitor the hydrolysis of substrate. The com-
pound of which the inhibitory activity at 20 lg/mL was upon
50% had been tested the IC50
.
Acknowledgement
5.1.16. Ethyl-1-{2-[4-bromo-2-2-(naphthalen-1-yl)acetamid-
ophenoxy]ethyl}piperidine-4-carboxylate (33c)
The authors thank the National Natural Science Foundation of
China (30572239) for financial support.
Compound 33c was obtained (0.114 g, 85% yield) as a white so-
lid, mp 94–96ꢀC. IR (KBr) cmꢀ1: 2926, 2854, 1730, 1629, 1595,
1523, 1484, 764, 689. 1H NMR (CDCl3) d: 1.66–1.69 (3H, m,
–CH2CH3), 1.81–2.17 (9H, m, piperidine CH2), 2.64–2.67 (2H, m,
–CH2N), 3.73 (2H, t, J = 6.4Hz, -OCH2), 4.12 (2H, q, J = 7.6Hz,
–CH2CH3), 4.21 (2H, s, –COCH2), 6.53 (1H, d, J = 9.2 Hz, ArH), 7.02
(1H, dd, J1 = 8.8 Hz, J2 = 2.4 Hz, ArH), 7.50–7.56 (4H, m, ArH),
7.86–7.92 (3H, m, 2ArH, –NHCO), 8.00 (1H, d, J = 6.8 Hz, ArH),
8.57 (1H, d, J = 2 Hz, ArH). ESI-MS m/z: 539 (M+).
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phenyl}-1-naphthalenylacetamide (33d)
Compound 33d was obtained (0.083 g, 67% yield) as a yellow oil.
IR (KBr) cmꢀ1: 3382, 2929, 2854, 1649, 1595, 1522, 1481, 762, 699.
1H NMR (CDCl3) d: 1.29–1.43 (8H, m, cyclohexane CH2, H-2, H-3, H-4,
H-5), 2.43–2.46 (4H, m, cyclohexane CH2, H-1, H-6), 2.64 (2H, t,
J = 6.8 Hz, –ArOCH2CH2), 3.66 (2H, t, J = 6.8 Hz, –ArOCH2), 4.16 (2H,
s, –COCH2), 6.51 (1H, d, J = 8.8 Hz, ArH), 6.98 (1H, dd, J1 = 8.8 Hz,
J2 = 2.4 Hz, ArH), 7.46–7.52 (4H, m, ArH), 7.83–7.98 (4H, m, 3ArH,
1-NHCO), 8.52 (1H, d, J = 2.4Hz, ArH). ESI-MS m/z: 498 (M+).

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