Design, synthesis and biological evaluation of novel dual inhibitors of acetylcholinesterase and β-secretase

Article abstract of DOI:10.1016/j.bmc.2008.12.067

To explore novel effective drugs for the treatment of Alzheimer's disease (AD), a series of dual inhibitors of acetylcholineterase (AChE) and β-secretase (BACE-1) were designed based on the multi-target-directed ligands strategy. Among them, inhibitor 28

Full text of DOI:10.1016/j.bmc.2008.12.067

Bioorganic & Medicinal Chemistry 17 (2009) 1600–1613
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel dual inhibitors
of acetylcholinesterase and b-secretase
Yiping Zhu a, Kun Xiao a, Lanping Ma a, Bin Xiong a, Yan Fu a, Haiping Yu b, Wei Wang a, Xin Wang a,
b,
Dingyu Hu a, Hongli Peng a, Jingya Li a, Qi Gong a, Qian Chai a, Xican Tang a, Haiyan Zhang a, , Jia Li
,
*
*
Jingkang Shen a,
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR China
b The National Center for Drug Screening, 189 Guoshoujing Road, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To explore novel effective drugs for the treatment of Alzheimer’s disease (AD), a series of dual inhibitors
of acetylcholineterase (AChE) and b-secretase (BACE-1) were designed based on the multi-target-directed
ligands strategy. Among them, inhibitor 28 exhibited good dual potency in enzyme inhibitory potency
Received 13 November 2008
Revised 26 December 2008
Accepted 30 December 2008
Available online 6 January 2009
assay (BACE-1: IC50 = 0.567 lM; AChE: IC50 = 1.83 lM), and also showed excellent inhibitory effects on
Ab production of APP transfected HEK293 cells (IC50 = 98.7 nM) and mild protective effect against hydro-
gen peroxide (H2O2)-induced PC12 cell injury. Encouragingly, intracerebroventricular injection of 28 into
amyloid precursor protein (APP) transgenic mice caused a 29% reduction of Ab1–40 production. Therefore,
28 was demonstrated as a good lead compound for the further study and more importantly, the strategy
of AChE and BACE-1 dual inhibitors might be a promising direction for developing novel drugs for AD
patients.
Keywords:
AD
AChE
BACE-1
Dual inhibitor
HE
Ó 2009 Elsevier Ltd. All rights reserved.
HMC
HEA
1. Introduction
disease.3 b-Secretase (BACE-1), an aspartyl protease, is an attrac-
tive therapeutic target since it plays a key role in the rate-limiting
Alzheimer’s disease (AD) is the most common cause of senile
dementia characterized by a progressive deterioration in cognitive
function, affecting approximately 3% of the population between
the ages of 65–74, and nearly 50% of those 85 years and older. It
has become one of the most costly diseases which brings heavy so-
cial and financial burden to both society and families.1
To date, on the basis of the cholinergic hypothesis, the major
marketed drugs for AD are acetylcholinesterase (AChE) inhibitors,
that is, donepezil, rivastigmine, and galantamine. Unfortunately,
these drugs have been approved for the symptomatic treatment
of AD as they do not address the underlying neuropathology.2 In
2004, memantine was approved by FDA to treat moderate to se-
vere AD patients as a NMDA-receptor antagonist. However, it plays
a limited role in terms of the progression of the disease. Therefore,
extensive research should be carried out to search for more effec-
tive therapy and drugs for the treatment of the disease.
step of Ab generation.4 In addition, BACE-1 gene knockout homozy-
gote mice showed a complete absence of Ab production and no
obvious deficits in basal neurological and physiological functions.5
Since the identification of BACE-1 in 1999, numerous BACE-1
inhibitors have been synthesized and tested in vitro as well in vivo,
showing amyloid lowering properties in animal models (Fig. 1).6
Despite of the success, the actual effects of BACE-1 inhibitors for
AD patients need to be further evaluated. It is still important to ex-
plore novel drug candidate. Considering the complexity of AD, the
classic ‘one molecule, one target’ solution may not be effective en-
ough.7 The novel multi-target-directed strategy has received atten-
tion, since single molecule simultaneously interacts with multiple
targets in the complex neurotoxic cascades would achieve better
efficacy by a complementary manner. Meanwhile, the hybrid
would reduce individual toxicity by special metabolic pathway
compared with the combinational drugs. The recent studies fol-
lowing this strategy have led to the synthesis of several chemically
diverse structures with dual or multiple biological profiles of AD,8
including AChE and monoamine oxidase B (MAO-B) dual inhibitors
(e.g., Ladostigil),8e AChE and serotonin transporter (SER) dual
inhibitors (e.g., 3),8f and AChE, BACE, Ab aggregation inhibiting
and antioxidant multiple functional compounds (e.g., Memoquin)
(Fig. 2).8t Accordingly, considering AChE is the most successful tar-
Recently, the accumulation of the b-amyloid peptide (Ab) in the
brain has been thought to be a key factor in the pathogenesis of the
* Corresponding authors. Tel.: +86 21 50806600 5407; fax: +86 21 50807088
(J.S.).
E-mail addresses: hzhang@mail.shcnc.ac.cn (H. Zhang), jli@mail.shcnc.ac.cn (J.
Li), jkshen@mail.shcnc.ac.cn (J.K. Shen).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.067

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
1601
Figure 1. Representative potent BACE-1 inhibitors.
get for symptomatic treatment of AD and BACE-1 is a crucial factor
of Ab formation for the pathogenesis, our efforts were focused on
designing and synthesizing dual inhibitors that can simultaneously
work on both AChE and BACE-1. Inhibition of AChE activity would
alleviate clinical symptom in short-term therapy, while inhibition
of BACE-1 would have an additive effect by preventing the forma-
tion of Ab and further slowing the progression of the long-term dis-
ease. This therapy may likely result in
a comprehensive
suppression of AD by synergistic manner and obtain higher thera-
peutic effectiveness. More recently, Lorna Piazzi and co-workers
have reported the design and evaluation of the first dual inhibitors
of AChE and BACE-1 (e.g., 4), which further strengthen our hypoth-
esis.8y Herein, we displayed the discovery of novel dual inhibitors
of AChE and BACE-1, which demonstrated not only in vitro en-
zymes inhibitory potency and cellular activity, but also in vivo
functional efficacy.
Scheme 1. Synthesis of designed dual inhibitors 9–14 bearing HE scaffold. Reagents
and conditions: (a) CF3COOH, CH2Cl2, rt; (b) EDCI, HOBt, benzoic acids, DIPEA, DMF,
rt; (c) aq LiOH, THF–H2O; (d) TBDMSCl, imidazole, DMF, rt; (e) EDCI, HOBt, amines,
DIPEA, DMF, rt; (f) nBu4N+Fꢀ, THF, rt.
2. Chemistry
The synthesis of the compounds 9–14 was accomplished as
c
-lactone with lithium hydroxide, followed by protection of the
shown in Scheme 1.9 Removing the N-Boc group of the
c-lactone
c-hydroxyl group with tert-butyldimethylsilyl chloride (TBDMSCl),
6 in the presence of 30% CF3COOH in CH2Cl2, the resulting amine
was subsequently reacted with 3-[(R)-1-(4-fluorophenyl)eth-
ylaminocarbonyl]-5-[methyl(methylsulfonyl)amino]benzoic acid
and 3-[(R)-1-phenylethylaminocarbonyl]-5-[methyl(methylsulfo-
nyl)amino]benzoic acid using EDCI and HOBt as the coupling
agents to yield compounds 7 and 8, respectively.10 Hydrolysis of
then coupling with different amines using EDCI and HOBt to give
the corresponding amides. N-Benzylpiperazine and 4-amino-1-
benzylpiperidine were commercially available. (1-Benzylpiperi-
din-4-yl)methylamine and (1-benzylpiperidin-4-yl)ethylamine
were synthesized according to the literature.11 Removal of the silyl
protecting group by treatment with tetrabutylammoniumfluoride
(nBu4N+Fꢀ) in tetrahydrofuran (THF) afforded the target com-
pounds 9–14.
The general synthesis of various inhibitors containing the
hydroxymethylcarbonyl (HMC) isostere is outlined in Scheme
2.12 The Boc-protected
a-hydroxyl-b-amino acid 15 was coupled
with amines using EDCI, HOBt and DIPEA to provide the amides,
which were removed the Boc protecting group in the presence of
4 N HCl/1,4-dioxane. Then the resulting amine hydrochlorides
were coupled with acids to provide 16–24.
Scheme 2. Synthesis of designed dual inhibitors 16–24 bearing HMC scaffold.
Reagents and conditions: (a) EDCI, HOBt, amines, DIPEA, DMF, rt; (b) 4 N HCl/1,4-
dioxane; (c) EDCI, HOBt, acids, DIPEA, DMF, rt.
Figure 2. Reported hybrids with dual or multiple biological profiles.

1602
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
Scheme 3. Synthesis of designed dual inhibitors 26–38 bearing HEA scaffold.
Reagents and conditions: (a) amines, isopropyl alcohol, 90 °C, 24 h; (b) 4 N HCl/1,4-
dioxane; (c) EDCI, HOBt, acids, DIPEA, DMF, MW, 70 °C, 10 min.
Compounds 26–38 were obtained from a well-known three-
step procedure (Scheme 3) starting from the requisite erythro
a-
amino epoxide 25, which was prepared according to the literature
procedures.13 Epoxide opening in sealed tube at 90 °C for 24 h
afforded the Boc-protected amino alcohol, which were then depro-
tected under acidic conditions and underwent amide coupling
using facile microwave to provide compounds 26–38.
3. Results and discussion
Figure 3. Structure of designed new hybrid dual inhibitors.
The way we adopted to explore the dual inhibitors of AChE and
BACE-1 was that combining pharmacophores of AChE inhibitors
with those of BACE-1 inhibitors through suitable common scaffold.
As reported, isophthalamide was a widely used pharmacophore in
BACE-1 inhibitors.14,6d–f,10,13d And the small molecular weight
compound 39 without the P01—P02 parts also showed moderate
activity towards BACE-1. It gave us an implication that the S01—S20
pockets of BACE-1 were comparably ‘tolerant’.14e On the other
hand, through studying the crystal structure and related literature
of AChE, we knew that the AChE has a catalytic site at the bottom
of deep and narrow gorge and a peripheral site at the entrance.15
As the drug donepezil of an AChE inhibitor has been widely used
in clinic and studied in academic field, its pharmacophore aroused
our great interest. The N-benzylpiperidine moiety of donepezil,
which forms strong interactions with AChE, locates at the bottom
of the catalytic site of AChE. The other indanone moiety of donepe-
zil, which could be substituted in many other highly potent AChE
inhibitors,16,11 locates at the peripheral site. According to these
analyses, we decided to use HE isostere which has been studied
for a long period to discover BACE-1 inhibitors in our laboratory14d
as the common scaffold, choose isophthalamide moiety and N-ben-
zylpiperidine group at the N-terminus and C-terminus of HE,
respectively to generate dual inhibitor (Fig. 3). In this way, the
introduction of N-benzylpiperidine group to the ligand’s P02 part
may cause little effect to its BACE-1 activity. Meanwhile, the de-
signed hybrid may also display certain AChE activity if effective
interactions occur between the N-benzylpiperidine group and the
catalytic site of AChE and proper location of isophthalamide moi-
ety at the peripheral site. And then we designed and synthesized
compounds 9–14 by using different substitutions of R1 group and
changing the chain length of N-benzylpiperidine amines at the C-
terminus of HE. And we also added the similar N-benzylpiperazine
as the active group of AChE inhibitor for elementary study.
Table 1
Enzyme-inhibiting activities of compounds 9–14
Compd
R1
X
Y
m
BACE-1 IC50
NDa
1.504 0.047
0.616 0.023
1.339 0.145
ND
1.541 0.256
0.462 0.058
ND
(
lM)
AChEb (inhibition%)
9
10
11
12
13
14
39
donepezil
OM99-2
F
F
F
F
H
H
/
/
N
C
C
C
N
C
/
0
0
1
2
0
0
/
0.26
NH
NH
NH
/
NH
/
47.54
45.85
23.79
5.18
48.70
ND
/
/
/
/
/
/
/
/
7.86 0.51 nMc
ND
0.071 0.009
a
’ND’means the IC50 were not determined due to their inhibition activity <50% at
50
l
g/mL.
Inhibition of AChE produced by the tested compounds at 2.5 ꢁ 10ꢀ5 M.
b
c
IC50 of donepezil.
pockets of BACE-1 were comparably ‘tolerant’. Intriguingly, when
N-benzylpiperidine groups were introduced into the ligands’ P20
part, some compounds displayed weak activities against AChE
(10, 11, 12, 14), which encouraged us to further explore dual inhib-
itors that can increase the activity against AChE as well as maintain
or elevate the BACE-1 inhibitory activity.
Then we changed the HE scaffold to HMC, another Asp protease
transition-mimic, for further exploration. As preliminary study, the
hydroxyl of HMC was not stereospecifically synthesized, and the
isobutyl at the P1 position was substituted by the hydrophobic
benzyl group (Fig. 3). As illustrated, our dual inhibitors were con-
ceptually subdivided into three regions for discussion purposes:
an N-terminus (An), a central chain (HMC isostere), and a C-termi-
nus (BPn) (Fig. 4). The An and BPn were individually modified to
probe the optimized activity. Thus, a small 3 ꢁ 3 library was de-
signed and synthesized by choosing different acids (An) and mod-
ifying the chain length of benzylpiperidine amines (BPn). Based on
the above study of HE-based dual inhibitors, it was anticipated that
the introduction of the HMC scaffold to compounds would increase
the activity towards AChE.
These compounds were evaluated the potencies against AChE
and BACE-1 using donepezil and OM99–2 (one of the earliest
inhibitors discovered by Ghosh et al.)17,14d as control, respectively.
As shown in Table 1, through modifying the chain length of ben-
zylpiperidine amines at the C-terminus of HE, except Y = N, all
compounds maintained certain BACE-1 inhibitory activities. It is
likely that the H atom of the C-terminal amide of HE may form
effective hydrogen bond with enzyme that led to great effect on
inhibitory activity against BACE-1. And the comparison among
the active compounds (10, 11, 12, 14 demonstrated the C-terminal
benzylpiperidine groups of HE cause slight effect on BACE-1 activ-
ity, which was consistent with our speculation that the S01—S20

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
1603
Figure 4. A small 3 ꢁ 3 library using HMC transition-state.
bearing HEA, which show similar C-terminal and N-terminal moi-
eties to the HE series, exhibiting good prosperities towards BACE-1
on both enzyme and cell in lots of recent literature.10,6d–f It was
most likely that replacing HMC with HEA may increase the inhib-
itor’s activity towards BACE-1 based on the reported results and
our analysis (Fig. 3). But we were not sure if the deletion of the car-
bonyl group of HMC and choosing the stereospecific hydroxyl of
HEA would cause influence to its AChE activity. Thus, we first syn-
thesized compounds 26–28 to determine whether HEA was an
appropriate common scaffold as we expected.
It was clear that the inhibitory activities of compounds against
BACE-1 increased dramatically compared to the HMC series (Table
3). Interestingly, the activities of compounds towards AChE did not
decrease when the carbonyl group of HMC was deleted and the
requisite stereospecific hydroxyl of HEA was chosen, which dem-
onstrated that HEA was a more suitable common scaffold than
HE and HMC for the dual inhibitors design. Besides, we also ob-
served that inhibitory activities of compounds against AChE in-
creased with the elongation of chain length of N-
benzylpiperidine amines (26 < 27 < 28), which was similar with
the HMC series.
Table 2
Enzyme-inhibiting activities of HMC-based compounds 16–24
Compd
AChEa (inhibition%)
AChE IC50
(l
M)
BACE-1b (inhibition%)
*
16(A1 BP1)
6.38
NDc
ND
1.11 0.12
ND
ND
1.39 0.07
ND
48.99 9.38
27.78 3.43
42.41 4.63
33.30 4.52
34.00 5.05
57.04 9.27
18.56 3.64
19.68 6.48
22.79 4.67
*
17(A1 BP2)
55.32
88.08
5.95
44.93
85.01
3.97
*
18(A1 BP3)
*
19(A2 BP1)
*
20(A2 BP2)
*
21(A2 BP3)
*
22(A3 BP1)
*
23(A3 BP2)
42.41
84.68
ND
1.68 0.27
*
24(A3 BP3)
a
Inhibition of AChE produced by the tested compounds at 2.5 ꢁ 10ꢀ5 M.
b
c
Inhibition of BACE-1 produced by the tested compounds at 50
’ND’ means the IC50 were not determined.
lg/mL.
To further validate N-benzylpiperidine ethyl was the most suit-
able moiety at the C-terminus of HEA, compounds 29–31 were syn-
thesized (Table 3). The results showed a similar trend with the
conclusion discussed above, which was also consistent with the lit-
erature that compounds with two-carbon units distance between
the nitrogen atom and the piperidine ring of benzylpiperidine
showed the highest potencies against AChE.16b,c,e Moreover, we
also found an interesting phenomena that compounds with m = 1
were obviously less potency towards BACE-1 than those with
m = 0, 2 (27 vs 26, 28; 30 vs 29, 31). Since the advantages of N-ben-
zylpiperidine ethyl at C-terminus of HEA on both BACE-1 and AChE
inhibitory activity, no further elaborations were made and N-ben-
zylpiperidine ethyl moiety was adopted as the C-terminus of HEA
in our study to explore further structure-activity relationships
(SAR). The chosen N-terminal residues were a series of isophthala-
mide derivatives with various substituents at 3- and 5-position.
The designed groups at 3- and 5-position were selected according
to the previous studies of BACE-1 inhibitors reported by Merck’s
group10 and ours.14d Methyl(methylsulfonyl)amino, nitro or hydro-
gen was incorporated into 5-position as R4. R5 at the 3-position
was investigated using (R)-1-(4-fluorophenyl)ethyl, (R)-1-phenyl-
ethyl benzyl and N,N-dipropyl.
Encouragingly, compounds bearing HMC scaffold, such as 18, 21
and 24 (IC50 = 1.11, 1.39, 1.68 M), showed greatly increase in po-
l
tency towards AChE compared to the HE-bearing series (Table 2).
And we also found that with elongation of the chain length of N-
benzylpiperidine amines at C-terminus of HMC, the activities of
inhibitors increased obviously (An-HMC-BP3 > An-HMC-BP2 > An-
HMC-BP1), and we have got the best inhibitors when m = 2. But
the influence of groups at the 3- and 5-position of isophthalamides
could not be observed. To our surprise, on the aspect of BACE-1, the
inhibitory activities of compounds decreased dramatically com-
pared to the HE-bearing series. The unexpected results pushed us
to carefully study the reported work about the BACE-1 inhibitors
bearing HMC transition-state mimic.18 Through looking into the
literature, we have got to know when using HMC as the transi-
tion-state mimic, Asn was not a proper amino acid residue at the
P2 position of BACE-1 inhibitors in peptide-scanning experi-
ment.16a Isophthalamide was usually considered as the substitu-
tion for Asn of ligand’s P2 part19,14a and that may explain why we
did not obtain the potent inhibitors when combined HMC and
isophthalamides together. As the weak activities of these com-
pounds towards BACE-1, actual contribution of individual diaste-
reomers has not been determined.
As it could be seen in Table 3, compounds bearing different sub-
stituents at the 3-position of isophthalamides showed closely
enzymatic inhibition against AChE. Among these, compounds con-
taining the N,N-dipropyl groups displayed a little more potency
than those containing the other two groups (32 vs 28, 31; 35 vs
HEA is another important isostere which has been widely used
in the aspartyl protease inhibitors and several HIV protease inhib-
itors adopting it as a scaffold have been approved to be on market
several years ago.20 And there are also multiple BACE-1 inhibitors

1604
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
Table 3
Enzyme-inhibiting activities of compounds 26–38
Compd
m
R4
R5N
AChEa (inhibition%)
11.16
AChE IC50
NDb
(
lM)
BACE-1 IC50 (lM)
O
O
O
O
O
O
O
O
H
N
S
S
S
S
S
S
S
26
0
0.511 0.250
3.999 1.383
0.567 0.159
0.771 0.091
3.233 0.263
0.222 0.047
2.392 0.050
7.021 2.817
2.682 0.017
ND
N
O
H
N
27
28
29
30
31
32
33
34
35
36
37
1
2
0
1
2
2
2
2
2
2
2
2
47.24
84.65
12.22
25.63
88.76
87.64
67.30
87.36
90.02
73.98
62.50
89.04
ND
N
O
H
N
1.83 0.12
N
F
F
F
O
H
N
ND
N
O
H
N
ND
N
O
H
N
1.35 0.06
1.05 0.09
2.53 0.07
2.09 0.18
1.27 0.07
3.06 0.21
N
O
N
N
N
N
H
N
NO2
NO2
NO2
H
F
H
N
H
N
ND
F
H
N
H
11.70 0.06
2.28 0.08
ND
38
H
ND
a
Inhibition of AChE produced by the tested compounds at 2.5 ꢁ 10ꢀ5 M.
b
’ND’ means the IC50 of compounds were not obtained.

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
1605
34, 33; 38 vs 37, 36). Similarly, at the 5-position, methyl(methyl-
sulfonyl)amino was a little superior substituent than the nitro
and hydrogen groups (28 vs 33, 36; 31 vs 34, 37; 32 vs 35, 38).
On the contrary, substituents at the 3- and 5-position of isophthal-
amides played a significant role in inhibitory activity towards
BACE-1. The compounds bearing (R)-1-(4-fluorophenyl)ethyl at
the 3-position were about 3-fold and 10-fold more potent than
those bearing (R)-1-phenylethyl benzyl (28 vs 31; 34 vs 33) and
N,N-dipropyl (31 vs 32), respectively. And more clearly, com-
pounds with methyl(methylsulfonyl)amino at the 5-position of
isophthalamides exhibited over 10 times potency than those with
nitro and hydrogen. These results are in agreement with the previ-
ously reported SAR explorations of BACE-1 inhibitors.10,14d
To elucidate the binding modes of dual inhibitor in AChE and
BACE-1, we performed a docking study on inhibitor 31 by utiliz-
ing the advanced AUTODOCK program. Due to many rotatable bonds
contained in ligand, a two-step docking procedure was adopted to
enable the AUTODOCK program to explore such large conformational
space. So the ligand 31 was splitted into two parts. For BACE-1
docking study, we first docked the part containing isophthala-
mide (Fig. 5A, red part in bottom 2D structure diagram) of ligand
31 into BACE-1. Next four lowest conformations were selected
and their torsion angles were fixed in the next docking studies.
Then we connected this part with the remainder part (Fig. 5A,
black part in bottom 2D structure diagram) together, and the
whole ligand 31 was subjected to the docking study again. Finally
the lowest energy conformation was selected for analysis. As
demonstrated in Figure 5A, the isophthalamide moiety of inhibi-
tor 31 adopts a very similar conformation compared with the li-
gand in original crystal structure 2B8L. And the hydroxyl of HEA
scaffold can form effective hydrogen bonds with active site aspar-
tic acid residue Asp228. But the benzylperidine group extends
into the solvent and does not form any specific interactions with
BACE-1. For AChE-ligand docking, a similar two-step procedure
was used but with the part containing benzylpiperidine (Fig. 5B,
red part in bottom 2D structure diagram) to perform docking
first. Then the remainder part (Fig. 5B, black part in bottom 2D
structure diagram) was connected, and after whole molecule
docking study, the final lowest conformation was selected as
the predicted bound conformation. Clearly, the benzylperidine
group of inhibitor 31 properly binds with enzyme in a conforma-
tion similar to donepezil, whereas the isophthalamide moiety
only extends to the outside of binding site and does not make
any specific contact with the residues in AChE. These may shed
light on why the inhibitors of this series only have moderate
binding affinity towards AChE and BACE-1. However, these dock-
ing studies gave the rational explanations that how the dual
inhibitors of BACE-1 and AChE interact with the target proteins,
which may give us some directions to design more potent dual
inhibitors.
According to the results described above, some potent dual or
BACE-1 inhibitors (26, 28, 29, 31, 32, 33 and 34) were selected to
examine the effects on intracellular inhibition of endogenous
BACE-1 activity in HEK293 cells transfected with human
bAPP695wt. Whole cell Ab lowering (sandwich Elisa) was used to
detect inhibitory effect of the compounds on BACE-1 activity.
Among these compounds, 26 (IC50 = 54.6 nM) and 28
(IC50 = 98.7 nM) displayed excellent inhibitory effects in cell-based
assay on Ab production (The IC50 of the other compounds were be-
low 1 lM), which demonstrated N-benzylpiperidine might be a
good group for compounds on cellular activities.
Then the protective effects against free radical of 26 and 28,
which might relate to a self propagating cascade of neurodegener-
ative events,21 were assessed by measuring the ability of the com-
pounds to protect against H2O2 injury according to the reported
protocol with minor modification.22 After 200
lM H2O2 exposure,
cell viability as determined by MTT reduction was markedly de-
creased to 58% (##P < 0.01 vs control). However, compounds 26
and 28 showed mild protective effects against hydrogen perox-
ide-induced PC12 cell injury, which was statistically significant
(Fig. 6).
Due to their favorable overall profiles, we decided to evaluate
the in vivo efficacy. Encouragingly, intracerebroventricular admin-
istration of compounds 26 and 28 to APP transgenic mice, which
contains a transgene encoding the human APP695 with Swedish
double mutation, caused 23% and 29% decrease in endogenous
Ab1–40 production compared to the vehicle-treated control mice,
respectively (Fig. 7). However, further studies will be needed to
verify the pharmacokinetic aspects of these compounds including
the brain penetration.
Figure 5. The docking studies of inhibitor 31. Ligands were depicted as stick model. For BACE-1 docking study (A), the original ligand in crystal structure 2B8L was colored in
cyan and inhibitor 31 was colored in orange. For AChE docking study (B), donepezil was colored in cyan and inhibitor 31 was colored in orange. The 2D skeleton of the
inhibitor 31 at the bottom of the figure is to illustrate how the two-step docking studies were performed. The arrow is indicated the split point and the fragment used in the
first step docking was colored in red. After the first step docking, the black part of molecule was connected and the torsion angle of the red part was fixed in the second step
docking studies.

1606
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
110
90
70
*
*
##
50
30
Control
10
26 ( M)
Hydrogen peroxide (200 μM)
1
10
1
μ
μ
28 ( M)
Figure 6. Protective effects of 26 and 28 on cell injury induced by hydrogen
*
peroxide (200
group P < 0.01 versus H2O2 group.
l
M) in PC12 cells. ##P < 0.01 versus control; P < 0.05 versus H2O2
**
140
120
100
80
Vehicle
Compounds 26 or 28
*
**
60
40
20
0
Vehicle
26
Vehicle
28
Figure 7. Compound 26 or 28 induced Ab1–40 level decreasing in APP transgenic
mouse brain. n = 6–8. Data were MEAN SD expressed as percent of vehicle group.
*
**
P < 0.05, P < 0.01 versus vehicle-treated mice.
Figure 8. The route for discovering AChE and BACE-1 dual inhibitors.
4. Conclusion
which could be considered as a potential multi-target-directed
agent and further studied for therapeutic application in the treat-
ment of AD (Fig. 8). Further studies to evaluate 28 in vivo and to
develop structural refinements are in progress in our laboratory
and will be reported in due course.
To explore novel effective drugs for the treatment of Alzhei-
mer’s disease (AD), three series dual inhibiting molecules of AChE
and BACE-1 were designed and investigated. Applying the frag-
ment-based strategy, HE, HMC and HEA were chosen as the com-
mon scaffolds. These protease transition-mimics were combined
with isophthalamide moiety and N-benzylpiperidine groups at
the N-terminus and C-terminus, respectively. Compared with HE
analogues which displayed strong BACE-1 inhibitory activities
but weak AChE inhibitory activities, HMC derivatives showed
greatly increase of AChE activities at the expense of dramatically
loss of BACE-1 activities. Excitingly, HEA derivatives were obtained
with good inhibiting activities on both AChE and BACE-1, which
demonstrated HEA is a suitable common scaffold for dual inhibi-
tors. Among this series, compounds 26 and 28 showed excellent
activities in the cell-based assay. These two compounds were also
proved to possess anti-oxidative effect against H2O2-induced cyto-
toxicity. More interestingly, our in vivo study further proved that
intracerebroventricular administration of compounds 26 and 28
could markedly decrease the Ab1–40 production in APP transgenic
mice. The present study indicated that hybrid 28 showed AChE
inhibiting activity, as well as Ab lowering and antioxidant function,
5. Experimental
5.1. Chemistry
The 1H NMR (300 MHz or 400 MHz) spectra were recorded on
Varian Mercury-300 or 400 High Performance Digital FT-NMR
using tetramethylsilane as an internal standard and the 13C NMR
(100 MHz) spectra were determined with Varian Mercury-400
High Performance Digital FT-NMR. The LC–MS were carried out
on Thermo Finnigan LCQDECAXP and HRMS were performed with
Finnigan MAT 95, EI: 70 eV, R: 10,000. The purity of the stereospe-
cifical target compounds was recorded on HPLC system (HP 1100
series, Agilent Technologies, Palo Alto, USA) by two different gradi-
ent methods using an YMC ODS column (50 ꢁ 4.6 mm, 5
lm
particle size). In addition, to detect the diastereomers 16–24, an-

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
1607
other three gradient methods were set. The optical rotation value
was determined with PerkinElmer-341 (589 nm). All reagents are
of analytical grade pure and used without further purification.
were washed by saturated brine twice (10 mL ꢁ 2). Then the or-
ganic layers were dried over Na2SO4, filtered and evaporated to
give white foamy solid acid, which was used without further puri-
fication (this product turns back slowly to 8 at room temperature).
To a solution of the given acid (0.66 g, 1.14 mmol) in 9 mL DMF
was added imidazole (1.17 g, 25.1 mmol), tert-butyldimethylsilyl
chloride (1.89 g, 12.5 mmol) and stirred at room temperature for
26 h. Then 6 mL CH3OH was added and the mixture stirred for an-
other hour. After that, 30 mL 25% aqueous citric acid was added
and the solution was extracted by EtOAc three times (30 mL ꢁ 3).
The combined organic layers were dried over Na2SO4, filtered
and concentrated to give the residue, which was purified by silica
gel chromatography to afford 0.603 g (2R,4S,5S)-5-{3-[(R)-1-(4-
fluorophenyl)ethylamido]-5-[methyl(methylsulfonyl)amino]benz-
amido}-4-(tert-butyldimethylsilyloxy)-2,7-dimethyloctanoic acid
(white foam solid, yield: 76.3%). 1H NMR (300 MHz, DMSO-d6): d
9.01 (br d, J = 7.6 Hz, 1H), 8.32 (br d, J = 7.9 Hz, 1H), 8.26 (s, 1H),
7.98 (s, 1H), 7.96 (s, 1H), 7.42 (m, 2H), 7.16 (m, 2H), 5.16 (q,
J = 7.2 Hz, 1H), 4.08 (m, 1H), 3.72 (m, 1H), 3.31 (s, 3H), 3.02 (s,
3H), 2.45 (m, 1H), 1.92 (m, 1H), 1.58 (m, 2H), 1.50 (d, J = 7.0 Hz,
3H), 1.22–1.40 (m, 2H), 1.11 (d, J = 7.1 Hz, 3H), 0.93 (d, J = 8.2 Hz,
3H), 0.86 (s, 9H), 0.82 (d, J = 6.3 Hz, 3H), 0.16 (s, 3H), 0.08 (s, 3H).
To a solution of the above acid (0.080 g, 0.115 mmol) in 1 mL
DMF at 0 °C was added N-benzylpiperazine (0.023 mL,
0.132 mmol), HOBt (0.018 g, 0.132 mmol), DIPEA (0.022 mL,
0.126 mmol) and stirred for 10 min. EDCI (0.025 g, 0.132 mmol)
was added and the mixture was allowed to react overnight at room
temperature. Then the resulting mixture was diluted by 30 mL
EtOAc and washed with 1 N HCl aqueous three times
(10 mL ꢁ 3), dilute aqueous NaHCO3 three times (10 mL ꢁ 3) and
saturated brine once (10 mL), sequentially. The organic layers were
dried over Na2SO4, filtered and evaporated to give the crude prod-
uct amide, which was used directly in the next step. To a solution
of the given amide in 1 mL THF was added 0.3 mL 1.0 M tetrabuty-
lammoniumfluoride in THF. The mixture was allowed to react for
24 h and concentrated in vacuum. The residue was purified by pre-
parative thin layer chromatography or silica gel chromatography to
afford the target compound 9, yield: 64.2%. 1H NMR (300 MHz,
CD3OD): d 8.22 (s, 1H), 8.01 (s, 2H), 7.40 (m, 2H), 7.28 (m, 5H),
7.04 (m, 2H), 5.23 (q, J = 7.0 Hz, 1H), 4.20 (m, 1H), 3.58 (m, 5H),
3.52 (s, 2H), 3.38 (s, 3H), 3.18 (m, 1H), 2.99 (s, 3H), 2.42 (m, 4H),
1.98 (m, 1H), 1.68 (m, 2H), 1.58 (d, J = 7.0 Hz, 3H), 1.42 (m, 2H),
1.12 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 6.6 Hz, 6H); 13C NMR
(100 MHz, CDCl3): 175.6, 165.9, 164.9, 163.0, 160.6 (d,
J = 244 Hz), 142.0, 138.9, 137.1, 135.8, 135.6, 129.0, 128.2, 128.1,
127.9, 127.8, 127.3, 124.1, 115.4, 115.2, 70.4, 62.6, 53.1, 52.9,
52.5, 48.9, 45.7, 41.8, 40.7, 37.9, 37.2, 35.4, 32.2, 24.8, 23.2, 21.9,
21.6, 17.2; LC-MS: m/z 738.7 [M+H]+; HRMS: calcd for
5.2. N-[(R)-1-(4-Fluorophenyl)ethyl]-N0-{(S)-3-methyl-1-
[(2S,4R)-4-methyl-5-oxo-tetrahydrofuran-2-yl]butyl}-5-
[methyl(methylsulfonyl)amino]isophthalamide (7)
To a solution of CF3COOH (4 mL) in CH2Cl2 (10 mL) was added 6
(1.40 g, 4.91 mmol). The mixture was at room temperature for 0.5 h,
and then it was concentrated under reduced pressure to afford the
crude product used without further purification. To a solution of
the product (0.23 g, 1.26 mmol) in DMF (8 mL) at 0 °C was added
3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-5-[methyl(methylsu-
lfonyl)amino]benzoic acid (0.496 g, 8, 1.26 mmol), HOBt (0.179 g,
1.32 mmol), DIPEA (0.22 ml, 1.32 mmol) and stirred for 10 min. EDCI
(0.254 g, 1.32 mmol) was added and the mixture was allowed to re-
act overnight at room temperature. Then the resulting mixture was
diluted by 100 mL EtOAc and washed with 1 N HCl aqueous solution
three times (30 mL ꢁ 3), dilute aqueous NaHCO3 three times
(30 mL ꢁ 3) and saturated brine once (30 mL), sequentially. The or-
ganic layers were dried over Na2SO4, filtered and evaporated to give
the residue, which was purified by silica gel chromatography to af-
ford 0.66 g 7 (white foam solid, yield: 93%). 1H NMR (300 MHz,
CDCl3): d 8.12 (s, 1H), 7.96 (m, 2H), 7.32 (m, 2H), 7.01 (m, 2H),
6.83 (br, 1H), 6.81 (br, 1H), 5.21 (m, 1H), 4.62 (m, 1H), 4.48 (m,
1H), 3.38 (s, 3H), 2.84 (s, 3H), 2.62 (m, 1H), 2.40 (m, 1H), 1.99 (m,
1H), 1.68 (m, 2H), 1.53 (d, J = 7.0 Hz, 3H), 1.44 (m, 1H), 1.25 (d,
J = 7.2 Hz, 3H), 0.94 (d, J = 6.4 Hz, 6H); 13C NMR (100 MHz, CDCl3):
180.8, 166.0, 164.7, 163.1 (d, J = 244 Hz), 160.6, 142.2, 138.7, 135.6,
135.2, 128.1, 128.0, 127.8, 127.7, 124.1, 115.5, 115.3, 80.5, 50.8,
49.1, 40.9, 37.9, 35.6, 34.3, 32.5, 24.7, 23.1, 21.8, 21.7, 16.4; LC-MS:
m/z 1123.1 [2M+H]+; HRMS: calcd for C28H36N3O6NaSF [M+Na]+
584.2207, found 584.2208.
5.3. N-{(S)-3-methyl-1-[(2S,4R)-4-methyl-5-oxo-tetrahydro-
furan-2-yl]butyl}-5-[methyl(methylsulfonyl)amino]-N0-[(R)-1-
phenylethyl]isophthalamide (8)
Compound 8 was obtained from 6 and 5-[methyl(methylsul-
fonyl)amino]-3-[(R)-1-phenylethylaminocarbonyl] benzoic acid
according to the similar procedure used to prepare 7, yield: 90%.
1H NMR (300 MHz, CDCl3): d 8.15 (s, 1H), 7.98 (m, 2H), 7.22–7.34
(m, 5H), 6.93 (br, 1H), 6.85 (br, 1H), 5.23 (m, 1H), 4.62 (m, 1H),
4.48 (m, 1H), 3.33 (s, 3H), 2.87 (s, 3H), 2.60 (m, 1H), 2.39 (m,
1H), 2.00 (m, 1H), 1.68 (m, 2H), 1.56 (d, J = 6.9 Hz, 3H), 1.44 (m,
1H), 1.25 (d, J = 7.4 Hz, 3H), 0.94 (d, J = 6.5 Hz, 6H); 13C NMR
(100 MHz, CDCl3): 180.8, 166.1, 164.8, 142.9, 142.1, 135.7, 135.1,
128.6, 128.2, 127.3, 126.0, 124.2, 80.5, 50.8, 49.7, 40.8, 37.8, 35.5,
34.3, 32.4, 24.7, 23.1, 21.8, 21.7, 16.4; LC–MS: m/z 1087.1
[2M+H]+; HRMS: calcd for C28H37N3O6NaS [M+Na]+ 566.2301,
found 566.2277.
C39H53N5O6FS [M+H]+ 738.3701, found 738.3733; ½a 2D2
= ꢀ36.0 (c
ꢂ
1.11, acetone).
5.4.2. N-{(4S,5S,7R)-2-[7-(1-Benzylpiperidin-4-yl)]carbamoyl-5-
hydroxy-2-methyloct-4-yl}-N0-[(R)-1-(4-fluorophenyl)ethyl]-5-
[methyl(methylsulfonyl)amino]isophthalamide (10)
5.4. General method for preparation of compounds 9–14
(exemplified by 9)
Compound 10 was obtained from 8 and 4-amino-1-benzylpi-
peridine, yield: 51%. 1H NMR (300 MHz, CD3OD): d 8.25 (s, 1H),
8.02 (s, 2H), 7.45–7.32 (m, 7H), 7.06 (m, 2H), 5.22 (q, J = 6.9 Hz,
1H), 4.19 (m, 1H), 3.80 (s, 2H), 3.62 (m, 2H), 3.38 (s, 3H), 3.29
(m, 2H), 3.10 (m, 2H), 2.96 (s, 3H), 2.65 (m, 1H), 2.38 (m, 2H),
1.72–1.40 (m, 7H), 1.12 (d, J = 7.2 Hz, 3H), 1.01 (d, J = 7.3 Hz, 3H),
0.99 (d, J = 7.3 Hz, 3H), 0.94 (d, J = 6.2 Hz, 3H); 13C NMR
(100 MHz, CD3OD): 178.9, 169.1, 168.0, 164.9, 162.5 (d,
J = 242 Hz), 144.2, 141.7, 138.0, 137.5, 132.0, 130.1, 129.9, 129.8,
129.7, 129.6, 126.3, 116.6, 116.4, 72.8, 63.2, 59.9, 54.5, 53.0, 50.8,
40.5, 39.5, 39.2, 38.8, 36.484, 26.6, 25.2, 24.3, 22.7, 22.6, 21.2,
19.6, 14.4; LC–MS: m/z 752.7 [M+H]+; HRMS: calcd for
5.4.1. N-[(4S,5S,7R)-7-(4-Benzylpiperazin-1-yl)carbamoyl-5-
hydroxy-2-methyloct-4-yl]-N0-[(R)-1-(4-fluorophenyl)ethyl]-5-
[methyl(methylsulfonyl)amino]isophthalamide (9)
Compound 8 (0.64 g, 1.14 mmol) was dissolved in 5 mL THF at
0 °C and 6 mL aqueous 1 N LiOH was added. The mixture was al-
lowed to stir overnight at room temperature. Then the organic sol-
vent was evaporated and 25% aqueous citric acid solution was
added to adjust the pH to 3–4. The solution was extracted with
EtOAc three times (20 mL ꢁ 3) and the combined organic layers

1608
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
C40H55N5O6SF [M+H]+ 752.3857, found 752.3898; ½a 2D2
ꢂ
= ꢀ31.3 (c
NMR (100 MHz, CD3OD): 178.9, 169.2, 168.0, 145.5, 144.2, 138.0,
137.6, 131.9, 130.2, 130.0, 129.7, 128.6, 127.8, 126.4, 72.8, 63.2,
59.9, 54.3, 51.4, 50.1, 40.1, 39.2, 38.8, 36.4, 31.4, 26.6, 25.2, 24.3,
22.7, 22.6, 21.2, 19.4, 14.4; LC-MS: m/z 734.6 [M+H]+; HRMS: calcd
1.64, acetone).
5.4.3. N-{(4S,5S,7R)-2-[7-(1-Benzylpiperidin-4-yl)]methyl-
carbamoyl-5-hydroxy-2-methyloct-4-yl}-N’-[(R)-1-(4-fluoro-
phenyl)ethyl]-5-[methyl(methylsulfonyl)amino]isophthal-
amide (11)
for C40H56N5O6S [M+H]+ 734.3951, found 734.3958; ½a 2D2
= ꢀ39.0 (c
ꢂ
0.86, acetone).
Compound 11 was obtained from 8 and (1-benzyl-4-piperidi-
nyl)methylamine, yield: 43%. 1H NMR (300 MHz, CD3OD): d 8.31
(s, 1H), 8.02 (s, 2H), 7.58–7.35 (m, 7H), 7.02 (m, 2H), 5.22 (q,
J = 7.1 Hz, 1H), 4.18 (m, 1H), 4.01 (m, 2H), 3.60 (m, 1H), 3.38 (s,
3H), 3.25 (m, 5H), 2.99 (s, 3H), 2.82 (m, 1H), 2.72–2.63 (m, 3H),
1.92 (m, 1H), 1.70–1.48 (m, 7H), 1.12 (d, J = 6.6 Hz, 3H), 1.02 (d,
J = 7.2 Hz, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.4 Hz, 3H); 13C
NMR (100 MHz, CD3OD): 179.7, 169.1, 168.0, 164.9, 162.5 (d,
J = 242 Hz), 144.2, 141.8, 138.1, 137.5, 132.6, 130.5, 130.3, 129.9,
129.8, 126.3, 116.6, 116.4, 72.7, 62.6, 59.9, 54.5, 54.1, 50.9, 45.2,
40.6, 39.8, 39.4, 38.9, 36.6, 26.6, 25.2, 24.4, 22.8, 22.7, 21.1, 19.7,
14.4; LC-MS: m/z 766.8 [M+H]+; HRMS: calcd for C41H57N5O6SF
5.5. General method for preparation of compounds 16–24
(exemplified by 16)
5.5.1. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]carbamoyl-
3-hydroxy-1-phenylbut-2-yl}-5-[methyl(methylsulfo-
nyl)amino]-N0-[(R)-1-phenylethyl]isophthalamide (16)
To a solution of 15 (0.030 g, 0.102 mmol) in 10 mL DMF at 0 °C
was added 4-amino-1-benzylpiperidine (0.020 g, 0.104 mmol),
HOBt (0.014 g, 0.104 mmol), DIPEA (0.018 mL, 0.104 mmol) and
stirred for 10 min. EDCI (0.020 g, 0.104 mmol) was added and the
mixture was allowed to react overnight at room temperature. Then
the resulting mixture was diluted by 30 mL EtOAc and washed
with 1 N HCl aqueous three times (10 mL ꢁ 3), dilute aqueous
NaHCO3 three times (10 mL ꢁ 3) and saturated brine once
(20 mL), sequentially. The organic layers were dried over Na2SO4,
filtered and concentrated. The crude product was dissolved in
1 mL 4 N HCl/1,4-dioxane and stirred for 2 h. The organic solvent
was evaporated to give the amine hydrochloride. To a solution of
the resulting product in 2 mL DMF was added 3-[(R)-1-phenyleth-
ylaminocarbonyl]-5-[methyl(methylsulfonyl)amino]benzoic acid
(0.035 g, 0.093 mmol), HOBt (0.014 g, 0.104 mmol), DIPEA
(0.018 mL, 0.104 mmol) and stirred for 10 min. EDCI (0.020 g,
0.104 mmol) was added and the mixture was allowed to react
overnight at room temperature. Then the resulting mixture was di-
luted by 30 mL EtOAc and washed with 1 N HCl aqueous three
times (10 mL ꢁ 3), dilute aqueous NaHCO3 three times
(10 mL ꢁ 3) and saturated brine once (20 mL), sequentially. The or-
ganic layers were dried over Na2SO4, filtered and concentrated. The
residue was purified by silica gel chromatography to give 16 as yel-
low foam solid, yield: 49%. 1H NMR (300 MHz, CDCl3): d 7.55–8.13
(m, 5H), 7.09–7.34 (m, 13H), 5.22 (m, 1H), 4.56 (m, 1H), 4.36 (m,
0.5H), 4.12 (m, 0.5H), 3.72 (m, 1H), 3.58 (m, 2H), 2.68–3.22 (m,
10H), 2.20 (m, 2H), 1.79 (m, 1H), 1.58 (m, 4H), 1.25 (m, 2H); LC-
MS: m/z 726.5 [M+H]+; HRMS: calcd for C40H48N5O6S [M+H]+
726.3325, found 726.3318.
[M+H]+ 766.4014, found 766.3995; ½a 2D2
= ꢀ29.9 (c 1.86, acetone).
ꢂ
5.4.4. N-{(4S,5S,7R)-2-[7-(1-Benzylpiperidin-4-yl)]ethylc
arbamoyl-5-hydroxy-2-methyloctan-4-yl}-N’-[(R)-1-(4-
fluorophenyl)ethyl]-5-[methyl(methylsulfonyl)amino]isoph-
thalamide (12)
Compound 12 was obtained from 8 and 2-(1-benzylpiperidin-4-
yl)ethylamine, yield: 53%. 1H NMR (300 MHz, CD3OD): d 8.25 (s,
1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.43 (m, 2H), 7.32 (m, 5H), 7.02
(m, 2H), 5.23 (q, J = 7.1 Hz, 1H), 4.21 (m, 1H), 3.63 (s, 2H), 3.58
(m, 1H), 3.36 (s, 3H), 3.16 (m, 2H), 2.98 (s, 3H), 2.92 (m, 2H),
2.61 (m, 1H), 2.18 (m, 2H), 1.90 (m, 2H), 1.62 (m, 4H), 1.58 (d,
J = 7.0 Hz, 3H), 1.45–1.23 (m, 6H), 1.13 (d, J = 7.3 Hz, 3H), 0.95 (d,
J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3): 176.9, 166.0, 164.7,
163.0, 160.6 (d, J = 244 Hz), 142.1, 139.2, 136.0, 135.8, 135.6,
129.6, 128.3, 128.0, 127.9, 127.5, 123.9, 115.3, 115.1, 70.9, 62.7,
53.2, 52.4, 49.1, 40.6, 38.4, 37.6, 37.0, 35.6, 35.5, 33.0, 31.1, 24.8,
23.2, 22.0, 21.8, 17.7; LC-MS: m/z 780.7 [M+H]+; HRMS: calcd for
C42H59N5O6SF [M+H]+ 780.4170, found 780.4177; ½a 2D2
= ꢀ33.8 (c
ꢂ
0.62, acetone).
5.4.5. N-{(4S,5S,7R)-2-[7-(4-Benzylpiperazin-1-yl)]carbamoyl-5-
hydroxy-2-methyloct-4-yl}-5-[methyl(methylsulfonyl)amino]-
N0-[(R)-1-phenylethyl]isophthalamide (13)
Compound 13 was obtained from 7 and N-benzylpiperazine,
yield: 47%. 1H NMR (300 MHz, CD3OD): d 8.23 (s, 1H), 8.01 (s,
2H), 7.19–7.41 (m, 10H), 5.25 (q, J = 7.0 Hz, 1H), 4.22 (m, 1H),
3.58 (m, 5H), 3.52 (s, 2H), 3.38 (s, 3H), 3.20 (m, 1H), 2.99 (s, 3H),
2.40 (m, 4H), 1.98 (m, 1H), 1.67 (m, 2H), 1.59 (d, J = 7.0 Hz, 3H),
1.42 (m, 2H), 1.11 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.6 Hz, 6H); 13C
NMR (100 MHz, CDCl3): 175.5, 165.8, 164.7, 142.9, 142.0, 137.3,
135.8, 135.7, 129.0, 128.5, 128.2, 127.9, 127.3, 127.2, 126.2,
123.9, 70.3, 62.6, 53.1, 52.9, 52.5, 49.6, 45.7, 41.8, 40.9, 37.9,
37.3, 35.4, 32.3, 24.8, 23.2, 22.0. 21.6, 17.2; LC-MS: m/z 720.6
[M+H]+; HRMS: calcd for C39H53N5O6SNa [M+Na]+ 742.3614, found
5.5.2. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]methyl-
carbamoyl-3-hydroxy-1-phenylbut-2-yl}-5-[methyl-
(methylsulfonyl)amino]-N0-[(R)-1-phenylethyl]isophthalamide
(17)
Compound 17 was obtained from 15, (1-benzyl-4-piperidi-
nyl)methylamine and 3-[(R)-1-phenylethylaminocarbonyl]-5-
[methyl(methylsulfonyl)amino]benzoic acid, yield: 39%. 1H NMR
(300 MHz, CDCl3): d 8.24 (m, 1H), 7.78–8.02 (m, 3H), 7.07–7.46
(m, 14H), 5.26 (m, 1H), 4.66 (m, 1H), 4.36 (m, 0.5H), 4.11 (m,
0.5H), 3.62 (m, 2H), 2.69–3.29 (m, 12H), 1.88–2.14 (m, 2H), 1.16–
1.60 (m, 8H); LC-MS: m/z 741.0 [M+H]+; HRMS: calcd for
C41H50N5O6S [M+H]+ 740.3482, found 740.3448.
742.3659; ½a 2D2
= ꢀ38.3 (c 0.36, acetone).
ꢂ
5.4.6. N-{(4S,5S,7R)-2-[7-(1-Benzylpiperidin-4-yl)]carbamoyl-5-
hydroxy-2-methyloct-4-yl}-5-[methyl(methylsulfonyl)amino]-
N0-[(R)-1-phenyl ethyl]isophthalamide (14)
Compound 14 was obtained from 7 and 4-amino-1-benzylpi-
peridine, yield: 41%. 1H NMR (300 MHz, CD3OD): d 8.26 (s, 1H),
8.03 (s, 1H), 7.99 (s, 1H), 7.21–7.42 (m, 10H), 5.27 (q, J = 7.1 Hz,
1H), 4.20 (m, 1H), 3.79 (s, 2H), 3.58 (m, 2H), 3.38 (s, 3H), 3.26
(m, 2H), 3.09 (m, 2H), 2.98 (s, 3H), 2.62 (m, 1H), 2.44 (m, 2H),
1.90 (m, 2H), 1.45–1.75 (m, 5H), 1.13 (d, J = 7.1 Hz, 3H), 1.01 (d,
J = 7.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H); 13C
5.5.3. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]ethyl- carba-
moyl-3-hydroxy-1-phenylbut-2-yl}-5-[methyl- (meth ylsulfo-
nyl)amino]-N0-[(R)-1-phenylethyl]isophthalamide (18)
Compound 18 was obtained from 15, (1-benzyl-4-piperidi-
nyl)ethylamine
and
3-[(R)-1-phenylethylaminocarbonyl]-5-
[methyl(methylsulfonyl)amino]benzoic acid, yield: 43%. 1H NMR
(300 MHz, CDCl3): d 7.46–8.04 (m, 4H), 6.89–7.03 (m, 14H), 5.20
(m, 1H), 4.44 (m, 1H), 4.36 (m, 0.6H), 4.09 (m, 0.4H), 3.46 (m,
2H), 2.70–3.29 (m, 12H), 1.89–2.20 (m, 3H), 1.09–1.61 (m, 9H);

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
1609
LC-MS: m/z 754.6 [M+H]+; HRMS: calcd for C42H52N5O6S [M+H]+
754.3638, found 754.3611.
3H), 0.74 (m, 3H); LC-MS: m/z 658.7 [M+H]+; HRMS: calcd for
C37H47N5O6Na [M+Na]+ 680.3424, found 680.3443.
5.5.4. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]carbamoyl-
3-hydroxy-1-phenylbut-2-yl}-N0-[(R)-1-(4-fluorophenyl)ethyl]-
5-[methyl(methylsulfonyl)amino]isophthalamide (19)
Compound 19 was obtained from 15, 4-amino-1-benzyl-
piperidine and 3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-5-
[methyl(methylsulfonyl)amino]benzoic acid, yield: 26%. 1H NMR
(300 MHz, CDCl3): d 8.10 (m, 1H), 7.54–7.96 (m, 3H), 7.10–7.38
(m, 11H), 6.92 (m, 2H), 5.21 (m, 1H), 4.54 (br, 1H), 4.34 (m,
0.5H), 4.14 (m, 0.5H), 3.70 (m, 4H), 2.61–3.24 (m, 9H), 2.24 (m,
2H), 1.81 (m, 1H), 1.53 (m, 4H), 1.25 (m, 2H); LC-MS: m/z 744.8
[M+H]+; HRMS: calcd for C40H46N5O6SFNa [M+Na]+ 766.3051,
found 766.3029.
5.5.9. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]ethylcar-
bamoyl-3-hydroxy-1-phenylbut-2-yl}-N0,N0-dipropyl-5-
nitroisophthalamide (24)
Compound 24 was obtained from 15, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-(dipropylcarbamoyl)-5-nitrobenzoic acid,
yield: 45%. 1H NMR (300 MHz, CDCl3): d 8.48 (s, 0.5H), 8.41 (s,
0.5H), 8.25 (s, 0.5H), 8.18 (m, 1H), 8.02 (s, 0.5H), 7.96 (m, 1H),
7.10–7.29 (m, 9H), 5.28 (m, 1H), 4.57 (m, 1H), 4.33 (m, 0.5H),
4.19 (m, 0.5H), 3.44 (m, 4H), 3.27 (m,2H), 3.10 (m, 3H), 2.88 (m,
3H), 2.16 (m, 1H), 1.86 (m, 2H), 1.27–1.78 (m, 6H), 1.20 (m, 4H),
0.95 (m, 3H), 0.73 (m, 3H); LC-MS: m/z 672.8 [M+H]+; HRMS: calcd
for C38H50N5O6 [M+H]+ 672.3761; found 672.3748.
5.5.5. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]methyl-
carbamoyl-3-hydroxy-1-phenylbut-2-yl}-N0-[(R)-1-(4-fluoroph-
enyl)ethyl]-5-[methyl(methylsulfonyl)amino]isophthalamide
(20)
Compound 20 was obtained from 15, (1-benzyl-4-piperidi-
nyl)methylamine and 3-[(R)-1-(4-fluorophenyl)ethylaminocarbon-
yl]-5-[methyl(methylsulfonyl)amino]benzoic acid, yield: 53%. 1H
NMR (300 MHz, CDCl3): d 8.25 (m, 1H), 7.76–7.95 (m, 3H), 6.86–
7.40 (m, 11H), 6.86 (m, 2H), 5.23 (m, 1H), 4.62 (m, 1H), 4.35 (m,
0.5H), 4.12 (m, 0.5H), 4.00 (m, 2H), 3.61 (m, 2H), 2.74–3.23 (m,
10H), 1.88–2.14 (m, 2H), 1.18–1.57 (m, 8H); LC-MS: m/z 758.8
[M+H]+; HRMS: calcd for C41H49N5O6SF [M+H]+ 758.3388, found
758.3400.
5.6. General method for preparation of compounds 26–38
(exemplified by 26)
5.6.1. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[(1-benzylpiperidin-
4-yl)amino]-propyl}-5-[methyl(methylsulfonyl)amino]-N0-[(R)-
1-phenylethyl]isophthalamide (26)
To a solution of 25 (0.026 g, 0.100 mmol) in 1 mL isopropylalco-
hol was added 4-amino-1-benzylpiperidine (0.060 g, 0.312 mmol)
and stirred at 90 °C for 24 h. Then the resulting mixture was di-
rectly isolated by simply preparative thin layer chromatography
to offer the pure product. The product was dissolved in 1 mL 4 N
HCl/1,4-dioxane and stirred for 2 h. To a solution of the resulting
product in 1 mL DMF was added 3-[(R)-1-phenylethylaminocar-
bonyl]-5-[methyl(methylsulfonyl)amino]benzoic acid (0.010 g,
0.050 mmol), HOBt (0.007 g, 0.052 mmol), DIPEA (0.009 mL,
0.104 mmol) and stirred for 10 min. EDCI (0.010 g, 0.052 mmol)
was added and the mixture was allowed to react overnight at room
temperature. Then the resulting mixture was diluted by 30 mL
EtOAc and washed with 1 N HCl aqueous three times
(10 mL ꢁ 3), dilute aqueous NaHCO3 three times (10 mL ꢁ 3) and
saturated brine once (20 mL), sequentially. The organic layers were
dried over Na2SO4, filtered and concentrated. The residue was puri-
fied by preparative thin layer chromatography to give 26 as yellow
foam solid, yield: 33%. 1H NMR (400 MHz, CD3OD): d 8.00 (m, 1H),
7.96 (m, 1H), 7.78 (m, 1H), 7.08–7.40 (m, 15H), 5.22 (q, J = 7.0 Hz,
1H), 4.27 (m, 1H), 3.79 (m, 1H), 3.53 (s, 2H), 3.32 (s, 3H), 3.22
(m, 1H), 2.78–2.98 (m, 8H), 2..65 (m, 1H), 2..07 (m, 2H), 1.91 (m,
2H), 1.56 (d, J = 7.0 Hz, 3H), 1.48 (m, 2H); 13C NMR (100 MHz,
CDCl3): 166.3, 164.7, 143.3, 141.9, 137.8, 137.7, 135.7, 135.4,
129.0, 128.4, 128.4, 128.1, 127.6, 127.1, 127.0, 126.4, 126.2,
123.9, 70.0, 62.7, 55.3, 51.8, 49.7, 48.0, 37.6, 36.3, 35.2, 31.3,
21.8; LC-MS: m/z 712.6 [M+H]+; HRMS: calcd for C40H50N5O5S
5.5.6. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]ethylc-
arbamoyl-3-hydroxy-1-phenylbut-2-yl}-N0-[(R)-1-(4-fluoro-
phenyl)ethyl]-5-[methyl(methylsulfonyl)amino]iso phthal
amide (21)
Compound 21 was obtained from 15, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-
5-[methyl(methylsulfonyl)amino]benzoic acid, yield: 38%. 1H NMR
(300 MHz, CDCl3): d 7.74–8.22 (m, 4H), 6.89–7.30 (m, 13H), 5.16
(m, 1H), 4.62 (m, 1H), 4.09 (m, 1H), 3.59 (m, 2H), 2.70–3.21 (m,
12H), 2.03 (m, 3H), 1.14–1.60 (m, 9H); LC-MS: m/z 772.8 [M+H]+;
HRMS: calcd for C42H51N5O6SF [M+H]+ 772.3544, found 772.3553.
5.5.7. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]carbamoyl-
3-hydroxy-1-phenylbut-2-yl}-N0,N0-dipropyl-5-
nitroisophthalamide (22)
Compound 22 was obtained from 15, 4-amino-1-benzylpiperi-
dine and 3-(dipropylcarbamoyl)-5-nitrobenzoic acid, yield: 36%.
1H NMR (300 MHz MHz, CDCl3): d 8.54 (m, 1H), 8.25 (m, 1H),
8.03 (m, 1H), 7.88 (m, 1H), 7.08–7.39 (m, 9H), 5.30 (m, 1H), 4.52
(m, 1H), 3.76 (m, 1H), 3.56 (s, 2H), 3.44 (t, J = 7.5 Hz, 2H), 2.86–
3.16 (m, 6H), 2.31 (m, 2H), 1.45–1.94 (m, 8H), 0.66–0.99 (m, 6H);
LC-MS: m/z 644.7 [M+H]+; HRMS: calcd for C36H45N5O6 Na
[M+Na]+ 666.3268, found 666.3272.
712.3533, found 712.3576; ½a D20
= ꢀ56.5 (c 1.10, CH2Cl2).
ꢂ
5.6.2. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[(1-benzylpiperidin-
4-yl)methylamino]-propyl}-5-[methyl(methylsulfonyl)amino]-
N0-[(R)-1-phenylethyl]isophthalamide (27)
Compound 27 was obtained from 25, 2-(1-benzylpiperidin-4-
5.5.8. N-{[2S,3(R,S)]-2-[4-(1-Benzylpiperidin-4-yl)]meth-
ylcarbamoyl-3-hydroxy-1-phenylbut-2-yl}-N0,N0-dipropyl-5-
nitroisophthalamide (23)
yl)methylamine
and
3-[(R)-1-phenylethylaminocarbonyl]-5-
[methyl(methylsulfonyl)amino]benzoic acid, yield: 54%. 1H NMR
(400 MHz, CD3OD): d 8.03 (t, J = 1.3 Hz, 1H), 7.96 (t, J = 1.3 Hz,
1H), 7.78 (t, J = 2.0 Hz, 1H), 7.18–7.40 (m, 14H), 7.11 (m, 1H),
5.48 (s, 1H), 5.22 (q, J = 7.0 Hz, 1H), 4.27 (m, 1H), 3.81 (m, 1H),
3.52 (s, 2H), 3.30 (s, 3H), 3.23 (m, 1H), 2.75–2.95 (m, 8H), 2.58
(m, 2H), 2.02 (m, 2H), 1.75 (m, 2H), 1.57 (d, J = 7.0 Hz, 4H), 1.27–
1.55 (m, 2H); 13C NMR (100 MHz, CDCl3): 166.4, 164.7, 143.4,
142.0, 137.7, 137.6, 135.8, 135.0, 129.2, 129.1, 128.5, 128.5,
128.2, 127.8, 127.2, 127.2, 126.5, 126.3, 124.1, 69.9, 63.0, 54.8,
54.8, 52.8, 51.3, 49.9, 37.7, 36.1, 35.6, 34.2, 29.7, 22.0; LC-MS:
Compound 23 was obtained from 15, (1-benzyl-4-piperidi-
nyl)methylamine and 3-(dipropylcarbamoyl)-5-nitrobenzoic acid,
yield: 61%. 1H NMR (300 MHz, CDCl3): d 8.58 (t, J = 1.7 Hz, 0.5H),
8.50 (t, J = 1.7 Hz, 0.5H), 8.32 (d, J = 9.0 Hz, 0.5H), 8.20 (m, 1H),
8.16 (m, 0.5H), 8.03 (m, 0.5H), 7.97 (d, J = 8.0 Hz, 0.5H), 7.56 (br,
0.5H),7.09–7.46 (m, 8.5H), 4.61–4.85 (m, 2H), 4.37 (m, 0.5H),
4.21 (m, 0.5H), 3.75–3.92 (m, 1H), 3.70 (s, 1H), 3.42 (t, J = 6.9 Hz,
2H), 2.77–3.50 (m, 8H), 2.17 (m, 2H), 1.43–1.73 (m, 9H), 0.96 (m,

1610
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
m/z 726.6 [M+H]+; HRMS: calcd for C41H51N5O5SNa [M+Na]+,
1.57(d, J = 7.0 Hz, 5H), 1.20–1.50 (m, 3H); 13C NMR (100 MHz,
CDCl3): 166.3, 164.6, 162.9, 160.5 (d, J = 244 Hz), 141.9, 139.2,
138.1, 137.8, 135.6, 135.3, 129.1, 129.0, 128.4, 128.0, 128.0,
127.9, 127.5, 126.9, 126.4, 124.0, 115.3, 115.1, 70.1, 63.2, 55.3,
53.3, 51.2, 49.1, 47.2, 37.7, 36.1, 35.3, 35.2, 33.5, 31.9, 21.9; LC-
MS: m/z 758.6 [M+H]+; HRMS: calcd for C42H52N5O5FS 758.3751,
748.3509, found 748.3543; ½a D20
= ꢀ42.4 (c 0.90, CH2Cl2).
ꢂ
5.6.3. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-propyl}-5-[methyl(methylsulfo-
nyl)amino]-N0-[(R)-1-phenylethyl]isophthalamide (28)
Compound 28 was obtained from 25, 2-(1-benzylpiperi-
din-4-yl)ethylamine and 3-[(R)-1-phenylethylaminocarbonyl]-5-
[methyl(methylsulfonyl)amino]benzoic acid, yield: 50%. 1H NMR
(400 MHz, CD3OD): d 8.02 (m, 1H), 7.97 (m, 1H), 7.78(m, 1H),
7.15–7.40 (m, 15H), 5.48 (m, 1H), 5.22 (q, J = 7.0 Hz, 1H), 4.26 (m,
1H), 3.79 (m, 1H), 3.50 (s, 2H), 3.31 (s, 3 H), 3.18 (m, 1 H), 2.75–
2.95 (m, 10H), 2.00 (m, 2H), 1.77 (m, 2H), 1.57 (d, J = 7.0 Hz, 5H),
1.20–1.50 (m, 3H); 13C NMR (100 MHz, CDCl3): 166.4, 164.8,
143.3, 141.9, 137.9, 137.8, 135.7, 135.2, 129.2, 129.0, 128.5,
128.4, 128.1, 127.6, 127.2, 127.0, 126.4, 126.3, 124.1, 70.1, 63.2,
55.1, 53.4, 51.1, 49.8, 47.1, 37.7, 36.0, 35.3, 34.8 33.4, 31.8, 21.9;
LC-MS: m/z 740.5 [2M+H]+; HRMS: calcd for C42H54N5O5S
found 758.3774; ½a D22
= ꢀ53.1 (c 0.55, CH2Cl2).
ꢂ
5.6.7. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-propyl}-N0,N0-dipropyl-5-
[methyl(methylsulfonyl)amino]isophthalamide (32)
Compound 32 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-(dipropylcarbamoyl)-5-[methyl(methylsulfo-
nyl)amino]benzoic acid, yield: 39%. 1H NMR (400 MHz, CDCl3): d
7.69 (m, 1H), 7.56 (m, 1H), 7.49 (m, 1H), 7.23–7.31 (m, 10H),
5.30 (m, 1H), 4.35 (m, 1H), 3.69 (m, 1H), 3.47 (m, 4H), 3.30 (s,
3H), 3.13 (m, 2H), 3.01 (m, 2H), 2.79–2.88 (m, 7H), 2.68 (m,
2H), 1.93 (m, 2H), 1.64 (m, 4H), 1.48 (m, 3H), 1.28 (m, 4 H),
0.97 (s, 3H), 0.73 (s, 3H); 13C NMR (100 MHz, CDCl3): 169.8,
166.1, 141.6, 138.2, 138.1, 137.9, 135.9, 129.3, 129.2, 128.4,
128.1, 127.7, 126.9, 126.4, 124.7, 123.9, 70.2, 63.3, 54.4, 53.6,
51.1, 50.8, 47.2, 46.7, 37.8, 36.1, 35.5, 35.0, 33.5, 32.0, 21.8,
20.6, 11.4, 10.9; LC-MS: m/z 720.3 [M+H]+; HRMS: calcd for
[M+H]+, 740.3846, found 740.3862;½a 2D2
= ꢀ34 (c 0.30, CH2Cl2).
ꢂ
5.6.4. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[(1-benzylpiperidin-
4-yl)amino]-propyl}-N0-[(R)-1-(4-fluorophenyl)ethyl]-5-
[methyl(methylsulfonyl)amino]isophthalamide (29)
Compound 29 was obtained from 25, 4-amino-1-benzylpi-
peridine and 3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-5-
[methyl(methylsulfonyl)amino]benzoic acid, yield: 33%. 1H NMR
(400 MHz, CD3OD): d 8.05 (m, 1H), 7.96 (m, 1H), 7.80 (m, 1H),
7.41 (m, 2H), 7.18–7.35 (m, 12H), 5.48 (s, 1H), 5.22 (q, J = 7.0 Hz,
1H), 4.27 (m, 1H), 3.81 (m, 1H), 3.51 (s, 2H), 3.31 (s, 3H), 3.23
(m, 1H), 2.79–2.99 (m, 8H), 2.67 (m, 1H), 2.09 (m, 2H), 1.92 (m,
2H), 1.54 (d, J = 7.0 Hz, 3H), 1.48 (m, 2H); 13C NMR (100 MHz MHz,
CD3OD): 159.1, 158.1, 155.0, 152.5 (d, J = 242 Hz), 134.1, 131.6,
130.3, 128.7, 127.8, 127.5, 121.2, 120.8, 119.9, 119.8, 119.7,
119.6, 119.3, 119.0, 117.8, 116.4, 62.8, 54.1, 47.0, 46.5, 45.3, 43.4,
40.8, 40.4, 28.7, 27.9, 26.3, 22.2, 21.9, 12.6; LC-MS: m/z 730.8
[M+H]+; HRMS: calcd for C40H49N5O5FS [M+H]+, 730.3438, found
C40H58N5O5S [M+H]+, 720.4159, found 720.4142; ½a 2D2
= ꢀ15.6 (c
ꢂ
0.45, CH2Cl2).
5.6.8. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-propyl}-5-nitro-N0-[(R)-1-phenyl-
ethyl]isophthalamide (33)
Compound 33 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-[(R)-1-phenylethyl]carbamoyl-5-[methyl-
(methylsulfonyl)amino]benzoic acid, yield: 41%. 1H NMR
(400 MHz, CDCl3): d 8.65 (m, 1H), 8.49 (m, 1H), 8.43 (m, 1H),
8.34 (d, J = 8.3 Hz, 1H), 7.25–7.40 (m, 13H), 7.03 (d, J = 7.5 Hz,
1H), 5.29 (m, 1H), 4.42 (m, 1H), 3.76 (m, 1H), 3.46 (s, 2H), 3.02
(m, 1H), 2.78–2.95 (m, 5H), 2.64 (m, 2H), 1.88 (m, 2H), 1.59 (m,
5H), 1.41 (m, 2H), 1.26 (m, 3H); 13C NMR (100 MHz, CDCl3):
164.5, 163.3, 148.1, 142.4, 138.2, 137.5, 136.5, 136.3, 131.1,
129.2, 129.1, 128.7, 128.6, 128.1, 127.6, 126.9, 126.7, 126.2,
124.7, 123.9, 69.4, 63.4, 55.8, 53.6, 51.1, 49.8, 47.5, 36.7, 36.3,
33.7, 32.2, 21.5; LC-MS: m/z 678.3 [M+H]+; HRMS: calcd for
730.3458; ½a 2D2
= ꢀ56.3 (c 0.95, CH2Cl2).
ꢂ
5.6.5. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[(1-benzylpiperidin-
4-yl)methylamino]-propyl}-N0-[(R)-1-(4-fluorophenyl)ethyl]-5-
[methyl(methylsulfonyl)amino]isophthalamide (30)
Compound 30 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)methylamine and 3-[(R)-1-(4-fluorophenyl)ethylaminocarbon-
yl]-5-[methyl(methylsulfonyl)amino]benzoic acid, yield: 66%. 1H
NMR (400 MHz, CD3OD): d 8.01 (m, 1H), 7.95 (m, 1H), 7.78 (m,
1H), 7.41 (m, 2H), 7.00–7.35 (m, 12H), 5.48 (s, 0.5H), 5.22 (q,
J = 7.0 Hz, 1H), 4.27 (m, 1H), 3.77 (m, 1H), 3.49 (s, 2H), 3.31 (s,
3H), 3.24 (m, 1H), 2.65–2.95 (m, 8H), 2..49 (m, 2H), 1.99 (m, 2H),
1.71 (m, 2H), 1.57 (d, J = 7.0 Hz, 4H), 1.20–1.35 (m, 2H); 13C NMR
(100 MHz, CDCl3): 165.8, 164.5, 163.1, 160.7 (d, J = 244 Hz),
142.0, 138.6, 138.3, 137.6, 135.7, 135.6, 129.1, 128.5, 128.1,
127.9, 127.8, 127.4, 127.0, 126.8, 126.5, 123.8, 69.9, 63.4, 55.9,
55.0, 53.4, 51.3, 48.9, 37.7, 36.4, 35.7, 35.2, 30.3, 21.6; LC-MS: m/
C40H48N5O5 [M+H]+, 678.3655, found 678.3639; ½a 2D2
= ꢀ45.6 (c
ꢂ
0.55, CH3OH).
5.6.9. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-
benzylpiperidin-4-yl)ethylamino]-propyl}-N’-[(R)-1-(4-
fluorophenyl)ethyl]-5-nitroisophthalamide (34)
Compound 34 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)ethanamine and 3-[(R)-1-(4-fluorophenyl)ethyl]carbamoyl-5-
[methyl(methylsulfonyl)amino]benzoic acid, yield: 52%. 1H NMR
(400 MHz, CDCl3): d 8.62 (s, 1H), 8.53 (s, 1H), 8.47 (s, 1H), 7.54
(m, 1H), 7.10–7.40 (m, 11H), 6.95 (m, 2H), 5.30 (m, 1H), 4.39 (br,
1H), 3.90 (br, 1H), 3.46 (s, 2H), 2.60–3.20 (m, 8H), 1.89 (m, 2H),
1.47–1.59 (m, 7H), 1.25 (m, 3H); 13C NMR (100 MHz, CDCl3):
164.8, 163.4, 163.1, 160.7 (d, J = 245 Hz), 148.1, 138.5, 137.8,
137.3, 136.3, 135.8, 131.0, 139.2, 129.0, 128.6, 128.1, 128.0,
127.9, 127.0, 126.8, 125.1, 124.3, 115.5, 115.3, 69.5, 63.2, 55.4,
53.0, 50.8, 49.3, 47.2, 36.2, 34.9, 33.5, 31.9, 21.7; LC-MS: m/z
696.7 [M+H]+; HRMS: calcd for C40H47N5O5F [M+H]+, 696.3561,
z
744.3 [M+H]+; HRMS: calcd for C41H51N5O5FS [M+H]+,
744.3595, found 744.3660; ½a D20
= ꢀ27.8 (c 0.40, CH2Cl2).
ꢂ
5.6.6. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpiperi-
din-4-yl)ethylamino]-propyl}-5-[methyl(methylsulfonyl)-
amino]-N0-[(R)-1-(4-fluorophenyl)ethyl]isophthalamide (31)
Compound 31 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-
5-[methyl(methylsulfonyl)amino]benzoic acid, yield: 53%. 1H
NMR (400 MHz, CD3OD): d 8.05 (m, 1H), 7.97 (m, 1H), 7.80 (m,
1H), 7.42 (m, 2H), 7.00–7.35 (m, 12H), 5.48 (s, 1H), 5.22 (q,
J = 7.0 Hz, 1H), 4.26 (m, 1H), 3.87 (m, 1H), 3.52 (s, 2H), 3.31 (s,
3H), 3.30 (m, 1H), 2.80–3.02 (m, 10H), 2.03 (m, 2H), 1.71 (m, 2H),
found 696.3572; ½a D22
= ꢀ40.2 (c 0.45, CH3OH).
ꢂ
5.6.10. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-propyl}-N0,N0-dipropyl-5-
nitroisophthalamide (35)
Compound 35 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-(dipropylcarbamoyl)-5-[methyl(methylsulfo-

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
1611
nyl)amino]benzoic acid, yield: 49%. 1H NMR (400 MHz, CD3OD): d
5.7. Biology
8.61 (m, 1H), 8.29 (m, 1H), 8.14 (m, 1H), 7.10–7.28 (m, 10H),
4.38 (m, 1H), 3.83 (m, 1H), 3.46 (m, 4H), 3.20 (m, 3 H), 2.78 (m,
7H), 1.90 (m, 2H), 1.22–1.80 (m, 11H), 0.95 (m, 3H), 0.72 (m,
3H); 13C NMR (100 MHz, CDCl3): 168.1, 164.7, 147.8, 138.7,
137.8, 136.2, 131.4, 129.5, 129.2, 128.5, 128.3, 127.5, 126.5,
123.7, 123.0, 69.6, 62.8, 54.8, 53.2, 51.2, 50.9, 47.0, 46.8, 35.9,
33.0, 31.2, 29.6, 21.8, 20.6, 11.4, 11.0; LC-MS: m/z 658.6 [M+H]+;
HRMS: calcd for C38H52N5O5 [M+H]+, 658.3968, found 696.3951;
This section describes the various assays used to assess the bio-
logical profiles of the compounds reported in this paper.
5.8. Enzyme-based assay of AChE
AChE is from rat cortex homogenate. The AChE activity was
evaluated by studying the hydrolysis of acetylthiocholine (ATCh)
following the method of Ellman as reported in detail elsewhere.
Five different concentrations of each compound were used in order
to obtain inhibition of AChE activity comprised between 20% and
80%. The assay solution consisted of a 0.1 M phosphate buffer pH
½
a 2D2
ꢂ
= ꢀ27.1 (c 0.35, CH2Cl2).
5.6.11. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-propyl}-N0-[(R)-1-phenylethyl]isoph-
thalamide (36)
8.0, with the addition of 340
acid), 0.035 unit/mL of human recombinant AChE derived from hu-
man serum (Sigma Chemical), and 550 M of substrate (acetylthi-
l
M 5,50-dithio-bis(2-nitrobenzoic
Compound 36 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-{[(R)-1-phenylethyl]carbamoyl}benzoic acid,
yield: 44%. 1H NMR (400 MHz, CDCl3): d 8.10 (s, 1H), 7.86 (d,
J = 7.8 Hz, 1H), 7.70 (d, J = 10.8 Hz, 1H), 7.47 (d, J = 4.6 Hz, 1H),
7.16–7.40 (m, 14H), 6.85 (d, J = 7.3Hz, 1H), 5.30 (m, 1H), 4.39
(m, 1H), 3.78 (m, 1H), 3.46 (s, 2H), 2.98 (d, J = 6.9 Hz, 2H),
2.81 (m, 4H), 2.64 (m, 2H), 1.87 (m, 2H), 1.58 (d, J = 7.0 Hz,
5H), 1.44 (m, 2H), 1.25 (m, 3H); 13C NMR (100 MHz, CDCl3):
167.2, 165.6, 143.2, 138.2, 138.0, 137.5, 134.6, 133.9, 130.7,
129.9, 130.7, 129.9, 129.2, 129.1, 128.8, 128.6, 128.5, 128.1,
127.3, 127.0, 126.6, 126.3, 125.3, 69.9, 63.2, 54.4, 53.5, 51.0,
49.4, 47.0, 36.1, 33.4, 31.9, 29.6, 21.8; LC-MS: m/z 651.3
[M+H]+; HRMS: calcd for C40H48N4O3F [M+H]+, 651.3710, found
651.3701; LC-MS: m/z 633.3 [M+H]+; HRMS: calcd for
l
ocholine iodide). Test compounds were added to the assay solution
and preincubated at 37 °C with the enzyme for 20 min followed by
the addition of substrate. Assays were done with a blank contain-
ing all components except AChE in order to account for nonenzy-
matic reaction. The reaction rates were compared, and the
percent inhibition due to the presence of test compounds was cal-
culated. Each concentration was analyzed in triplicate, and IC50
values were determined graphically from log concentration–inhi-
bition curves.
5.9. Enzyme-based assay of BACE-1
C40H49N4O3 [M+H]+, 633.3805, found 633.3818; ½a 2D2
ꢂ
= ꢀ34.0 (c
Recombinant human b-secretase ectodomain (amino acid resi-
dues 1–460) was expressed as a secreted protein with a C-terminal
His tag in insect cells using baculovirus infection. The BACE activity
was determined at room temperature by monitoring the hydrolysis
of FRET substrate DABCYL-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-
0.75, CH2Cl2).
5.6.12. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpiperi-
din-4-yl)ethylamino]-propyl}-N0-[(R)-1-(4-fluorophenyl)ethyl]-
isophthalamide (37)
EDANS (SynPep Corp, USA). In a typical 100
lL assay mixture con-
Compound 37 was obtained from 25, 2-(1-benzylpiperidin-4-
yl)ethylamine and 3-{[(R)-1-(4-fluorophenyl)ethyl]carbamoyl}-
benzoic acid, yield: 46%. 1H NMR (400 MHz, CDCl3): d 8.05 (s,
1H), 7.80 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.55 (d, J =
8.9 Hz, 1H), 7.10–7.42 (m, 11H), 6.91 (m, 3H), 5.23(m, 1H), 4.31
(m, 1H), 3.72 (m, 1H), 3.39 (s, 2H), 2.89 (d, J = 6.9 Hz, 2H), 2.78
(m, 4H), 2.58 (m, 2H), 1.81 (m, 2H), 1.50 (d, J = 7.0 Hz, 5H), 1.36
(m, 2H), 1.17 (m, 3H); 13C NMR (100 MHz, CDCl3): 167.5, 166.4,
162.9, 160.5 (d, J = 245 Hz), 139.4, 137.6, 137.4, 137.2, 134.6,
134.3, 133.6, 131.1, 130.3, 129.4, 129.3, 129.1, 128.8, 128.5,
128.2, 128.1, 128.0, 127.9, 127.8, 127.2, 127.0, 126.6, 125.4,
115.3, 115.1, 69.9, 63.1, 54.4, 53.2, 50.9, 48.9, 46.8, 35.6, 33.6,
33.2, 32.1, 22.0; LC-MS: m/z 651.3 [M+H]+; HRMS: calcd for
taining 100 mM ammonium acetate, pH 4.0, 20
l
M substrate, and
50 nM purified recombinant human BACE-1/Fc, the enzyme activ-
ity was continuously monitored with excitation 355 nm/emission
460 nm filter set for 20 min and the initial rate of the hydrolysis
was determined using the early linear region of the enzymatic
reaction kinetic curve.
5.10. Cellular Ab lowering assay in APP transfected HEK293
cells
Human embryonic kidney 293 cell transfected with APP695
cDNA containing the Swedish double mutation (HEK293sw), a line
known for its tendency to generate high level of aggregating Ab,
was used to examine the effects of the compounds on BACE-1
activity. HEK293sw cells were seeded into 6-well plates with a to-
tal volume of 1.5 mL of the cell suspension in Dulbecco’s modified
Eagle medium (DMEM, Gibco), supplemented with 10% (v/v) heat-
inactivated Fetal bovine serum. In the meantime, different concen-
trations of the compounds were added to the cultures and incu-
bated for 24 h at 37 °C and 5% CO2. The conditioned medium was
removed from the culture wells and Ab1–40 peptide levels in the
media were analyzed by Human b Amyloid 1–40 Colormetric Elisa
kit (BioSource International, Inc. California, USA) after 24 h.
C40H48N4O3F [M+H]+, 651.3710, found 651.3701; ½a 2D2
= ꢀ52.9 (c
ꢂ
0.85, CH2Cl2).
5.6.13. (1S,2R)-N-{1-Benzyl-2-hydroxy-3-(S)-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-propyl}-N0,N0-dipropylisophthal-
amide (38)
The title compound 38 was obtained from 25, 2-(1-ben-
zylpiperidin-4-yl)ethylamine and 3-(dipropylcarbamoyl)benzoic
acid, yield: 46%. 1H NMR (400 MHz, CD3OD): d 7.70 (m, 1H),
7.56 (m, 1H), 7.49 (m, 2H), 7.10–7.45 (m, 10H), 4.26 (m, 1H),
3.84 (m, 1H), 3.51 (s, 2H), 3.46 (m, 2H), 3.27 (m, 1H), 3.13
(m, 2H), 2.86 (m, 7H), 2.02 (m, 2H), 1.72 (m, 4H), 1.51 (m,
4H), 1.28 (m, 3H), 0.99 (t, J = 7.7 Hz, 3H), 0.69 (t, J = 7.7 Hz,
3H); 13C NMR (100 MHz, CDCl3): 170.9, 167.2, 138.2, 137.8,
137.4, 134.4, 129.3, 129.2, 128.6, 128.4, 128.1, 127.9, 126.9,
126.4, 125.2, 70.1, 63.3, 54.0, 53.5, 51.2, 50.7, 47.0, 46.4,
36.2, 35.0, 33.3, 31.9, 21.8, 20.6, 11.4, 11.0; LC-MS: m/z 613.6
[M+H]+; HRMS: calcd for C38H53N4O3 [M+H]+, 613.4188, found
613.4133.
5.11. Cell protective test in PC12 cells
The antioxidative activity against H2O2-induced cytotoxicity
was tested according to the reported protocol.22 Briefly, PC12 cells
were seeded into 96-well plates at a density of 1 ꢁ 105 cells per mL
in DMEM medium (Gibco), supplemented with 10% heat-inacti-
vated bovine calf serum. Experiments were carried out 24 h after
cells were seeded. The compounds were dissolved with 2% DMSO

1612
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
first, and then diluted with phosphate-buffered saline (PBS). After
pretreatment with the compounds for 2 h, H2O2 was added to
PC12 cell cultures for 30 min, and then replaced with fresh DMEM
medium (without phenol red). Assays for cell viability were per-
formed 24 h after cultured in fresh medium. Cell survival was eval-
uated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT, Sigma) reduction. The amount of MTT formazan
was quantified by determining the absorbance at 570 and
630 nm using Universal Microplate Reader (Bio-Tek). Data were
expressed as means SD and evaluated for statistical significance
with one-way ANOVA followed by Duncan’s multiple range tests.
5.13.3. AChE
The 3-dimensional crystal structure of AChE was retrieved from
the PDB database (the access PDB code: 1EVE). Similar to docking
studies for BACE-1, a two-step procedure was utilized for docking
inhibitors 31 into the AChE binding site. The ligand was separated
into two parts (see Fig. 5B), namely benzylpiperidine containing
part (red colour) and the remainder part (black colour). In AChE
docking studies, we first docked the benzylpiperidine containing
part into the binding site. Four conformations were selected and
the torsion angles were fixed in the second docking step. The
remainder part was then connected with the pre-docked part to
make complete ligands, which were subjected to the second dock-
ing study. Finally the lowest conformations for three inhibitors
were selected to investigate the interaction mechanism.
5.12. Animal-based experiment
Experiments were carried out under principles of Regulations of
Experimental Animal Administration issued by the State Commit-
tee of Science and Technology of the People’s Republic of China
on November 14, 1988 and followed the guidelines of the Animal
Care and Use Committee of Shanghai Institute of Materia Medica.
APP transgenic mice, 3–4 months old were generously provided
by Prof. Stephen Brimijoin (Mayo Clinic, Rochester), and used for
both vehicle-treated and compound-treated groups (n = 8 per
group). Compound 26 (10 nmol/mice) and compound 28 (8 nmol/
mice) was stereotaxically administered into the cerebral ventricle
(Bregma ꢀ0.2 mm, 1.0 mm lateral, 2.5 mm depth). For control, dis-
tilled H2O was injected. After 4 h, the hemispheres were isolated
quickly on ice, and the samples were processed and analyzed for
the Ab1–40 level following the instruction of Human b Amyloid 1–
40 Colormetric Elisa kit (BioSource International, Inc. California,
USA). Data were expressed as means SD and evaluated for statis-
tical significance Independent T-test.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (Grant 30230400, 30672538,
30572169), the Ministry of Science and Technology of China (No.
2004CB518907), ‘863’ Hi-Tech Program of China (Grant 2004 AA2
Z3781), and the State Key Program of Basic Research of China
(2004GB518907). We are also most grateful to Prof. Stephen Brimi-
join (Mayo Clinic, Rochester) and Prof. Zhou Zhang (Shanghai Insti-
tute of Materia Medica, Chinese Academy of Sciences) for providing
and breeding the transgenic mice.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.12.067.
5.13. Docking
References and notes
5.13.1. General AUTODOCK parameter setting
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The AUTODOCK Tool software was utilized to prepare the proteins
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5.13.2. BACE-1
The 3-dimensional crystal structure of BACE-1 was retrieved
from the PDB database (the access PDB code: 2B8L). A two-step
procedure was adopted to enable AUTODOCK program to explore large
conformation space in inhibitors. For BACE-1 docking studies, the
ligand was separated into two parts (see Fig. 5A), namely isoph-
thalamide containing part (red colour) and the remainder part
(black colour). We first docked the isophthalamide containing part
into the BACE-1 binding site, and four lowest energy conformations
were selected and the torsion angles in this part will be fixed in the
second docking step. Then we connected this part with the remain-
der part to make the complete ligand, which were docked into the
BACE-1 binding site again. Finally the lowest energy conformations
were selected for analyzing the interactions between the BACE-1
and inhibitor.

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 1600–1613
1613
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