Discovery of pyrazole as C-terminus of selective BACE1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.06.027

We recently discovered and reported dual inhibitor 5 of AChE and BACE1 with N-benzylpiperidine ethyl as C-terminus. Compound 5 showed potent inhibitory activities for BACE1, and could reduce endogenous Aβ_1-40 production in APP transgenic mice. In present work, we rapidly identified substituted triazole as the C-terminus of compound 5 by replacing the benzylpiperidine ethyl group with click chemistry and tested these synthesized compounds by in situ screening assay. As revealed by the crystal structures of BACE1 in complex with our triazole compound 12, we found that Pro70 and Thr72 located in the flap region were the critical components for binding with these inhibitors. With the aid of the crystal structure, a new series of five-membered heterocyclic compounds was prepared in order to explore the structure-activity relationship (SAR) of this class of molecules. From these efforts, pyrazole was discovered as a novel C-terminus of BACE1 inhibitors. After further modification of pyrazole with variable substituents, compound 37 exhibited good potency in enzyme inhibition assay (IC₅₀ = 0.025 μM) and compound 33 showed moderate inhibition effects on Aβ production of APP transfected HEK293 cells. Moreover, these pyrazole derivatives demonstrated good selectivity versus cathepsin D. Our results indicated that the vicinity of Pro70 and Thr72 might be utilized as a subsite, and the discovered pyrazole derivatives might provide useful hints for developing novel BACE1 inhibitors as anti-AD drugs.

Full text of DOI:10.1016/j.ejmech.2013.06.027

European Journal of Medicinal Chemistry 68 (2013) 270e283
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of pyrazole as C-terminus of selective BACE1 inhibitors
,
,
Yiquan Zou a 1, Lei Xu b 1, Wuyan Chen a, Yiping Zhu a, Tiantian Chen a, Yan Fu a, Li Li a,
Lanping Ma a, Bing Xiong a, Xin Wang a, Jian Li c, Jianhua He c, Haiyan Zhang a
,
,
*
Yechun Xu a , Jia Li , Jingkang Shen
,
b,
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
c Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 239 Zhang Heng Road, Pudong New District, 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:
We recently discovered and reported dual inhibitor 5 of AChE and BACE1 with N-benzylpiperidine ethyl
as C-terminus. Compound 5 showed potent inhibitory activities for BACE1, and could reduce endogenous
Received 7 November 2012
Received in revised form
26 April 2013
Accepted 6 June 2013
Available online 21 June 2013
Ab1e40 production in APP transgenic mice. In present work, we rapidly identified substituted triazole as
the C-terminus of compound 5 by replacing the benzylpiperidine ethyl group with click chemistry and
tested these synthesized compounds by in situ screening assay. As revealed by the crystal structures of
BACE1 in complex with our triazole compound 12, we found that Pro70 and Thr72 located in the flap
region were the critical components for binding with these inhibitors. With the aid of the crystal
structure, a new series of five-membered heterocyclic compounds was prepared in order to explore the
structureeactivity relationship (SAR) of this class of molecules. From these efforts, pyrazole was
discovered as a novel C-terminus of BACE1 inhibitors. After further modification of pyrazole with variable
Keywords:
Alzheimer’s disease
BACE1
C-terminus
substituents, compound 37 exhibited good potency in enzyme inhibition assay (IC50 ¼ 0.025
mM) and
Click chemistry
In situ screening assay
Pyrazole
compound 33 showed moderate inhibition effects on A production of APP transfected HEK293 cells.
b
Moreover, these pyrazole derivatives demonstrated good selectivity versus cathepsin D. Our results
indicated that the vicinity of Pro70 and Thr72 might be utilized as a subsite, and the discovered pyrazole
derivatives might provide useful hints for developing novel BACE1 inhibitors as anti-AD drugs.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
g
-secretase, respectively [3]. Since
transmembrane proteins, such as Notch, which is involved in cell
differentiation [4] thus chronic high doses of -secretase inhibitors
g-secretase also cleaves other
Alzheimer’s disease (AD), a form of senile dementia, is charac-
terized by a progressive loss of memory and cognitive ability, now
affecting more than 35 million elderly people worldwide with
skyrocketing healthcare costs far exceeding $100 billion annually
[1]. The pathology of this neurodegenerative disorder usually
manifests itself with the presence of extraneuronal aggregation of
g
may disrupt Notch-mediated processes in the gastrointestinal tract,
spleen, and thymus, leading to potential mechanism-based toxicity.
In contrast, Notch inhibition is not expected for b-secretase (b-site
amyloid precursor protein cleaving enzyme 1; BACE1) inhibitors.
Moreover, BACE1 has been identified as the rate-limiting enzyme
plaques composed of
from sequentially proteolytic cleavage of the
protein ( -APP) by two aspartic acid proteases, referred as
b
-amyloid peptides (A
b
b
) [2]. A
-amyloid precursor
- and
b
is derived
for A
b
production [3] and BACE1 knockout homozygote mice show
production and have no reported side ef-
complete absence of A
fects [5e7]. Therefore, BACE1 is considered as a promising target for
developing drugs to treat and/or prevent AD [8].
b
b
b
However, development of agents capable of inhibiting the
BACE1 activity has been proved to be difficult due to the large
binding groove of the enzyme. According to the geometry of the
long binding groove of BACE1 and the original peptide inhibitors
which mimic the sequence pattern of substrate APP [9], BACE1
inhibitors were in general subdivided into three regions: an
N-terminal portion, a central core and a C-terminus. In the last
decade, these sections were individually subjected to modification
Abbreviations: EDCl, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride; HOBt, N-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide; EtOAc, ethyl acetate; DCM, dichloromethane; THF,
tetrahydrofuran.
* Corresponding authors. Tel.: þ86 21 50806600x5407; fax: þ86 21 50807088.
E-mail addresses: hzhang@mail.shcnc.ac.cn (H. Zhang), ycxu@mail.shcnc.ac.cn
(Y. Xu), jli@mail.shcnc.ac.cn (J. Li), jkshen@mail.shcnc.ac.cn (J. Shen).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.027

Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
271
for reducing the peptidic characters. Many efforts have devoted to
find better inhibitors [10], such as via high throughput screening,
fragment-based drug discovery and virtual screening. Among them,
the peptide mimic inhibitors were extensively studies. Although
the N-terminus and central core parts which bound to the hydro-
phobic S1, S3 and polar S2, S4 subpockets had been optimized
extensively (Fig. 1) [11e15], the C-terminus, occupied polar S10, S20,
S30, S40 subpocket, which could hold various sizes and where
different polar groups were rarely explored.
Recently, we have developed a novel series of dual inhibitors of
AChE and BACE1 through introducing N-benzylpiperidine moiety of
donepezil at C-terminus. These inhibitors exhibited good BACE1
inhibitory potency in enzymatic assay, and also showed good
inhibitory effects on Ab production of APP transfected HEK293 cells
and mild protective effect against hydrogen peroxide (H2O2)-
induced PC12 cell injury [15]. This previous study demonstrated
that the C-terminus was also a critical component in BACE1 in-
hibitors and spurred us to explore more novel functional groups
acting as C-terminus of BACE1 inhibitor. To easily track the
improvement from C-terminus of inhibitors, we kept the N-ter-
minus of compound 5 and a facile central core (Fig. 2). By utilizing
click reaction and in situ screening assay, we could quickly assemble
the effective fragments and screen the combined compounds (10e
17) efficiently. Through this technology, we obtained the novel
1,2,3-triazole inhibitors which displayed potent inhibitory effects
toward BACE1 and good selectivity over cathepsin D (Table 1). As
revealed by the crystal structures of BACE1 in complex with com-
pound 12, Pro70 and Thr72 in the flap region were important for
the binding interactions of these inhibitors. To elaborate the
Fig. 2. Click chemistry combined with in situ screening approach for discovering b-
secretase inhibitors.
structureeactivity relationship (SAR) of C-terminus and improve
the cellular activity, we replaced triazole with other five-membered
heterocycles (compound 20e24). Verified by the crystal structure
of BACE1 in complex with compound 20 and 28 and SAR, we found
pyrazole derivatives showed potent activities in BACE1 enzymatic
assay, good inhibitory effects on Ab production of APP transfected
HEK293 cells and good selectivity over cathepsin D. Herein, we
displayed the discovery of pyrazole as novel C-terminus of BACE1
inhibitors which might be beneficial for developing novel selective
BACE1 inhibitors as anti-AD drugs.
2. Results and discussion
2.1. Chemistry
The initial compounds (10e17) were synthesized by click reac-
tion [16]. The Huisgen Cu(I)-catalyzed alkyne-azide cycloaddition
(CuAAC, a paradigm of click chemistry) had become a widely used
strategy for chemical space exploration in drug design [17]. The
synthetic appeal of click reaction relied upon their high yields,
simple reaction conditions, tolerance of oxygen and water, and
simple product isolation [18]. The Cu(I)-catalyzed 1,3-dipolar
cycloaddition reaction between an organic azide and a terminal
alkyne would create 1,2,3-triazole derivatives. The synthesis of in-
termediate azide compound 9 was shown in Scheme 1, and the
Fig. 1. Representative optimized N-terminus of BACE1 inhibitors (1e4).

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Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
Table 1
BACE1, cathepsin D enzyme data and HEK239 cellular-inhibitory activities of compounds 10e17.
Ma
BACE1 IC50
(
mM)
CatD IC50
(
mM)
HEK239-inhibition %c (at 10
9.81
mM)
b
b
Comp.
R
BACE1 in situ-inhibition (%) at 1
m
10
NDd
1.800 ꢁ 0.105
>30
11
12
69.21
88.74
0.099 ꢁ 0.011
0.036 ꢁ 0.005
>30
>30
12.01
13.24
13
14
15
97.86
83.13
90.53
0.264 ꢁ 0.030
0.442 ꢁ 0.027
1.176 ꢁ 0.120
10.56 ꢁ 0.91
13.64 ꢁ 0.77
12.56 ꢁ 0.84
15.81
8.88
10.93
16
17
98.01
1.890 ꢁ 0.154
>30
2.08
14.73
ND
>30
>30
8.36
ND
OM99-2
0.071 ꢁ 0.005
ND
a
The purity of all the in situ compounds were tested by HPLC and ensured the value >75%, and all inhibitory values are means of at least three separate experiments.
All the IC50 values are tested after the compounds were purified and ensured the purity >98%, and IC50 values are means of at least three separate experiments.
All inhibitory values are means of at least three separate experiments.
b
c
d
‘ND’ means the IC50 were not determined.
general synthesis of the combined molecules 10e17 was outlined in
Scheme 2.
To prepare compounds (10e17) for in situ assay, 0.4 mL THF and
0.1 mL H2O were added to each well of a 96-well plate, followed by
adding the azide compound 9 (14.5 mg, 0.025 mmol) and a terminal
alkyne (0.1 mmol) gradually. After shaking a few minutes, sodium
ascorbate (1 mg, 0.005 mmol) and CuI (0.5 mg, 0.0025 mmol) was
added into each well. Then the plate was further been shaking in
shaker for 12 h at 50 ꢀC [19]. TLC/HPLC was used to examine the
accomplishment of reactions, and the identity of the resulting
compounds was confirmed by LC-MS analysis of the crude reaction
mixtures. After identification, the reaction solution was concen-
trated and the residues were used to screen straightly without
further purification.
Scheme 1. Synthesis of compound 9. Reagents and conditions: (a) NaN3, NH4Cl, MeOH, reflux overnight; (b) TFA/DCM ¼ 1/4; (c) EDCI, HOBt, DIPEA, DMF, r.t. overnight.

Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
273
Scheme 2. Synthesis of compounds (10e17). Reagents and conditions: (a) CuI, sodium ascorbate, THF/H2O ¼ 4/1, 50 ꢀC; (b) Ru(PhP3)3Cl2, THF, 50 ꢀC.
All bioactive compounds identified in in situ assay were resyn-
thesized individually for verification with spectra characterization
and further enzymatic studies. Commercially available tert-butyl
(S)-1-((R)-oxiran-2-yl)-2-phenylethyl carbamate (compound 6)
was reacted with sodium azide in methanol to give tert-butyl
(2S,3S)-4-azido-3-hydroxy-1-phenylbutan-2-yl carbamate (com-
pound 7) in good yield. Then removing the N-Boc group of azide
compound 7 was conducted in the presence of 30% TFA in DCM, and
the resulting amine (compound 8) was subsequently reacted with
3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-5-[methyl (methyl-
sulfonyl)amino]benzoic acid using EDCI and HOBt as the coupling
agents to yield compound 9 [15]. The azide compound, a terminal
alkyne, sodium ascorbate, and CuI were stirred for 12 h at 50 ꢀC.
TLC/HPLC was employed to examine the accomplishment of re-
actions. After examination, the reaction was concentrated in vac-
uum to afford the crude 4-substituted 1,2,3-triazole products
(compounds 10e15). The 5-substituted 1,2,3-triazole compounds
(compounds 16e17) were obtained in the presence of Ru(PPh3)2Cl2
with moderate yields.
plate and assayed for their inhibition activity against BACE1.
Compound OM99-2 (one of the earliest inhibitors discovered by
Ghosh et al.) was used as the control [9]. The inhibitory activities of
1,2,3-triazoles against BACE1 were summarized in Table 1.
As shown in Table 1, all compounds showed certain BACE1
inhibitory activities. It was displayed that the triazole scaffold was
suitable for the C-terminal substitution of the BACE1 inhibitor.
Comparison of the active compounds indicated that, the polar S10e
S20 pockets of BACE1 could hold various kinds of substituents, but
the size and the property of substituents on the 1,2,3-triazole were
crucial for the potency. Compound 10, which has not any substit-
uent on the 1,2,3-triazole, displayed moderate inhibitory potency
(IC50 ¼ 1.800
mM). Small hydrophilic groups substituted at the 4-
substituted 1,2,3-triazole (11 and 12) led to more substantial in-
crease in potency than those with large hydrophobic groups such as
phenyl, 4-methoxy phenyl, and cyclohexyl (compounds 13, 14, and
15 respectively). When introduced the hydroxymethyl or phenyl
group into 5-position of triazole, respectively compounds 16 and 17
displayed weak activities against BACE1. Moreover, by comparing
The general synthesis of triazole analogs 20e24 was outlined in
Scheme 3 (the structure of compounds 20e24 was reported in
Table 2).
compound 10 (IC50
¼
1.800
mM) and compound 11
(IC50 ¼ 0.099
mM), it was found that hydroxymethyl group was
important for binding interaction, and the introduced chiral methyl
Epoxide opening in sealed tube at 100 ꢀC for 1 h afforded the
Boc-protected amino alcohol, which were then deprotected under
acidic conditions and underwent amide coupling to provide com-
pounds 20e24 (Table 2).
Similarly, pyrazole derivatives were obtained from epoxide
(compound 6) opening in sealed tube at 100 ꢀC for 1 h afforded the
Boc-protected amino alcohol, which were then deprotected under
acidic conditions and underwent amide coupling to provide com-
pound 28, then hydrolysis under basic conditions and underwent
amide coupling provide compounds 30e37 (Scheme 4, Table 3).
group to compound 11 further increased the activity about 3-fold
(compound 12, IC50 ¼ 0.036
mM). This was suggesting that the
hydroxymethyl might act as a H-bond donor to the residues of
BACE1 and the chiral methyl might have some effects on the
conformation orientation.
It was reported that optimization at C-terminus could
improve the enzymatic selectivity [20]. Since the catalytic
domain of BACE1 was similar to that of other known aspartyl
proteases, we selected cathepsin D as a representative to inves-
tigate the selectivity of these BACE1 inhibitors. Herein, in parallel
all the triazole derivatives were screened for human cathepsin D.
Encouragingly, we found all triazole derivatives inhibited
2.2. In situ screening assay and SAR of triazole derivatives
cathepsin D with IC50 values above 10 mM, suggesting that opti-
mization at C-terminus indeed improves the enzymatic selec-
tivity as expected.
The click reaction product in each well was diluted with 10%
DMSO aqueous solution to 1 mM into another 96-well microtiter
Scheme 3. Synthesis of compound 20e24. Reagents and conditions: (a) K2CO3, DMF 100 ꢀC 1 h; (b) TFA/DCM ¼ 1/4; (c) EDCI, HOBt, DIPEA, DMF, r.t. overnight.

274
Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
ꢀ
Table 2
X-ray crystal structure determined to 1.7 A resolution (Fig. 3). As
expected, The P3-phenyl ring occupied a unique position which
spanned S3 and S4 subsites and caused a significant positional shift
of a protein loop containing residues from 8 to 13 (the 10’s loop)
located in the S3/S4 pocket. The P2-sulfonamide functionality fitted
well into the S2 pocket and made extensive interactions with
BACE1. One of the sulfonamide oxygen atoms formed hydrogen
BACE1 inhibitory activities of triazole isosteres (20e24).
ꢀ
bond via a water molecule with the Ser325 hydroxyl oxygen (3.1 A
and 2.6 A). The same sulfone oxygen also made ionic interactions
ꢀ
with the guanidine side chain of Arg235. Another sulfonamide
oxygen made hydrogen bonds to Thr232 and Asn233 main chain
ꢀ
ꢀ
a
nitrogen atoms (3.2 A and 3.0 A, respectively). While the phenyl
group of compound 12 occupied the S1 region of BACE1, the hy-
droxyl of central core could form effective hydrogen bonds with
Comp.
R
BACE1 IC50 (mM)
ꢀ
ꢀ
10
1.800 ꢁ 0.105
active site aspartic acid residue Asp228 (2.7 A) and Asp32 (2.8 A).
The triazole extended to polar S10 subpocket. And water molecules
played important roles in the hydrogen bond network. It was
shown that there was not only a direct hydrogen bonding inter-
action between the N2 atom of 1,2,3-triazole and the side-chain of
20
21
22
23
24
0.490 ꢁ 0.033
5.900 ꢁ 0.210
0.910 ꢁ 0.027
4.310 ꢁ 0.134
ꢀ
residue Thr72 (3.3 A) but also two indirect hydrogen bonds formed
between the hydroxymethyl group substituted on triazole and the
main-chain of Pro70 via two water molecules. As shown in Table 1,
compound 10 which bound only with Thr72 had moderate binding
affinity toward BACE1. When we introduced the interactions with
Pro70 (compounds 11, 12) it led to great effect on inhibitory activity
against BACE1. It gave us an implication that both Pro70 and
Thr72 located in the flap region were the critical components of
the binding pocket. The interactions between the compound 12
and these two residues made the flap adopt the close conformation,
which strengthened the binding to BACE1 along with other
interactions from the N-terminus and the central core of
compound 12.
2.100 ꢁ 0.078
0.071 ꢁ 0.005
OM99-2
a
IC50 values are means of at least three separate experiments.
2.4. Bioisosterism and pyrazole scaffold
2.3. X-ray crystal structure of triazole derivative 12 bound to BACE1
Although triazole inhibitors exhibited high activity toward
BACE1 in enzymatic assays, their cellular inhibitory potencies were
poor which limited their follow-up studies. Table 1 showed that all
To characterize the binding mode of triazole BACE1 inhibitors,
compound 12 was co-crystallized with human BACE1 providing an
Scheme 4. Synthesis of compound 30e37. Reagents and conditions: (a) K2CO3, DMF 100 ꢀC 1 h; (b) TFA/DCM ¼ 1/4; (c) EDCI, HOBt, DIPEA, DMF, MW, r.t. overnight; (d) NaOH, THF/
EtOH/H2O ¼ 2/2/1, reflux 2 h; (e) amine, EDCI, HOBt, DIPEA, DMF, r.t. overnight.

Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
275
Table 3
BACE1, cathepsin D enzyme data and HEK239 cellular-inhibitory activities of pyrazole derivatives (28e37).
Comp.
R
BACE1 IC50
(
m
M)a
CatD-inhibition (%) at 20
13.23
m
g/mLb
CatD IC50
(
m
M)a
HEK293 inhibition (10
7.59
m
Mb)
HEK293 inhibition (1
6.35
m
Mb)
20
H
0.490 ꢁ 0.033
1.210 ꢁ 0.110
>30
25
ꢂ8.08
>30
ND
ND
28
0.051 ꢁ 0.006
22.17
>30
57.01
24.44
30
31
0.570 ꢁ 0.021
0.256 ꢁ 0.019
ꢂ8.22
ꢂ7.97
>30
>30
56.01
53.37
42.59
40.44
32
33
0.053 ꢁ 0.005
0.230 ꢁ 0.029
ꢂ6.73
>30
>30
54.78
58.69
28.77
46.26
ꢂ15.08
34
35
0.074 ꢁ 0.008
0.076 ꢁ 0.005
ꢂ16.78
ꢂ6.72
>30
>30
48.74
55.66
29.17
25.33
36
37
0.027 ꢁ 0.003
9.60
>30
46.58
36.88
0.025 ꢁ 0.003
0.071 ꢁ 0.005
ꢂ7.42
>30
40.61
ND
32.89
ND
OM99-2
NDc
ND
a
IC50 values are means of at least three separate experiments.
All inhibitory values are means of at least three separate experiments.
‘ND’ means the IC50 were not determined.
b
c
the triazole compounds displayed low inhibitory effects on
endogenous BACE1 activity in HEK293 cells transfected with hu-
tetrazole for preliminary SAR study. We also prepared the imid-
azole analog for examining the importance of this specified nitro-
gen in five-membered heterocycle. The inhibitory activities of these
isosteres against BACE1 were summarized in Table 2.
As shown in Table 2, compounds bearing different triazole iso-
steres at C-terminus showed diverse inhibitory potencies against
man
bAPP695wt (<50% inhibition ratio at 10 mM). In order to
improve cellular inhibitory potency, the other five-membered
heterocyclic rings were employed to substitute the triazole ring.
According to the interaction mode showing in Fig. 3, the N2 atom of
triazole is important to hydrogen bond formation. We selected
some five-membered heterocycles instead of the triazole scaffold,
such as pyrazole, 1,2,4-triazole, the (1H)-tetrazole and the (2H)-
BACE1. As expected, pyrazole 20 (IC50 ¼ 0.490
triazole 22 (IC50 ¼ 0.910 M), exhibited higher potency than the
initial triazole compound 10 (IC50 ¼ 1.800 M). Analyzing the X-ray
mM) and 1,2,4-
m
m

276
Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
likeness and potencies of these five isosteres, we chose pyrazole
as a functional group at the entrance of the C-terminus of the in-
hibitor for further SAR study.
2.5. Biological data and SAR of pyrazole derivatives
Structural analysis revealed that there were still two water
molecules in the place of two water molecules in triazole’s
conformation in the polar S10eS20 subpocket (Fig. 4A). As we all
know that water molecules could act as not only hydrogen donor
but also hydrogen acceptor. In the view of the above analysis, the
ester and amide aroused our great interest to be considered as
substituent to the pyrazole scaffold. Their carboxyl oxygen atoms
might act as hydrogen acceptor through water molecule binding
with Pro70, which was expected to strengthen the inhibitory po-
tency. Subsequently 4-ester and 4-amide substituted pyrazole de-
rivatives were designed and investigated the BACE1 and cathepsin
D inhibitory activities. The structureeactivity relationship (SAR) of
4-substitued pyrazoles was summarized in Table 3.
As we expected, the 4-substituted carbethoxyl compound 28
(IC50 ¼ 0.051
mM) had nearly ten-fold potency than that of the
compound 20, suggesting that the carbethoxyl might act as a
hydrogen acceptor to form direct or indirect hydrogen bonding
interactions with Pro70 in the active site of BACE1. Fortunately an
X-ray crystal structure of BACE1 in complex with compound 28 was
obtained and the interaction mode was illustrated in Fig. 4B. The
carboxyl oxygen of compound 28 formed one water-mediated
Fig. 3. The crystal structures of BACE1 in complex with compound 12 (PDB ID code:
3UQX).
crystal structure of BACE1 in complex with compound 12 and
compound 20, we found pyrazole and triazole could overlap
perfectly, and pyrazole fitted at the entrance of C-terminus. There
was a direct hydrogen binding interaction between the N2 atom of
ꢀ
ꢀ
hydrogen bond with the residue Pro70 (3.4 A and 2.9 A) in addi-
tion to the direct hydrogen bond that was formed between the N2
atom of pyrazole ring and the residue Thr72 (3.3 A). These results
ꢀ
ꢀ
pyrazole and Thr72 (3.1 A) as well. The other three isosteres
were in agreement with our previous speculation, and this might
be the reason that the inhibitory potencies were greatly strength-
ened. It was most likely that the amide analogs bound with enzyme
in a conformation similar to compound 28. Clearly, cycle amides
(compounds 34e37) were more potent than chain amides (31, 33)
(compounds 21, 23, 24) displayed weak potencies against BACE1.
Compound 21 bearing imidazole group could not form a hydrogen
bond with Thr72, which led to a dramatically decrease of inhibitory
activity, and the IC50 value was dropped to 5.9
mM. This result
confirmed the importance of the hydrogen bond between the N2
atom of pyrazole and Thr72. The inhibitory potencies of two tet-
razole analogs (23, 24) decreased. This might be due to some
changes in their physicochemical property. Given to the drug-
(Table 3). The suberyl amide compound 37 (IC50 ¼ 0.025
m
M) had
nearly 3-fold more potent than that of OM99-2 (IC50 ¼ 0.071
mM).
Meanwhile, all the pyrazole derivatives showed low inhibitory ac-
tivities toward cathepsin D and the inhibition ratios were all below
Fig. 4. A. Superposition of the crystal structures of BACE1 in complex with compound 12 (green) and 20 (magenta) (PDB ID code: 3UQX, 3UQU). The position of the two compounds
was overlapped well. B. The crystal structures of BACE1 in complex with compound 28 (PDB ID code: 3UQW). The compounds were shown in sticks and two residues, Thr72 and
Pro70, were displayed with line. The dash lines represented the hydrogen bonds. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)

Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
277
50% at 20
(IC50 ¼ 1.210
decreased dramatically comparing with its triazole analog 11
M). Therefore, we didn’t further optimize pyrazole
analogs with hydroxy group, and paid our attention to pyrazole
analogs with carbonyl group.
m
g/mL. To our surprise, the potency of compound 25
1100series, Agilent Technologies, Palo Alto, USA) by two different
mM) with hydroxymethyl substituted at 4-position,
gradient methods using an YMC ODS column (50 ꢃ 4.6 mm, 5
mm
particle size). The optical rotation value was determined with
PerkinElmer-341 (589 nm). All reagents are of analytical grade pure
and used without further purification.
(IC50 ¼ 0.099
m
According to intriguing findings described above, all the pyr-
azole analogs were selected to examine the cellular inhibition of
endogenous BACE1 activity in HEK293 cells transfected with hu-
4.1.1. tert-Butyl (2S,3S)-4-azido-3-hydroxy-1-phenylbutan-2-
ylcarbamate (7)
To a solution of compound 6 (770 mg, 2.93 mmol) in methanol
(20 mL) was added NH4Cl (282 mg, 5.27 mmol), NaN3 (457 mg,
7.03 mmol). The mixture was heated to reflux overnight. and then
the solvent was concentrated under reduced pressure. Water
(30 mL) was added into the reaction. The solution was extracted
with EtOAc twice (30 mL ꢃ 2) and the combined organic layers
were washed by diluted HCl, saturated NaHCO3, saturated brine
twice (15 mL ꢃ 2) by turns. Then the organic layers were dried over
Na2SO4, filtered and evaporated to give the residue, which was
purified by silica gel chromatography to afford 7 (810 mg, 90.3%). 1H
man
bAPP695wt. The percentage of whole cell Ab reduction
(sandwich Elisa) was used to monitor inhibitory effects of the
compounds. The results showed that all compounds displayed
moderate inhibitory effects on the cell-based assay at 1 mM, which
demonstrated that pyrazole acting as C-terminus scaffold were
better than triazole analogs according to the cellular activities
(Table 3). Among these, compounds 30 and 33 exhibited good in-
hibition effects on Ab production in the cell-based assay. Although
that cycle amides were more potent than chain amides toward
BACE1 in enzymatic assay, while, chain amides were better than
cycle amides in cellular assay. This might be due to the better
permeability of the chain amides than the cycle amides. In recent
years, there have been reported many BACE1 inhibitors with better
drug-like properties [10], such as lower molecular weight, higher
bioavailability, the better BBB penetration. We are aware that our
compounds have undesirable drug-like properties. But in the pre-
sent work, the aim is to explore the novel scaffold binding to S10
subpocket. The pyrazole was found that is a good functional group
as C-terminus scaffold, which can contribute to the design of new
inhibitors by incorporating the pyrazole ring. However, further
studies will be needed to test the adaptability of this scaffold.
NMR (300 MHz, CDCl3):
d
7.29e7.27 (m, 5H), 4.60 (d, J ¼ 8.4 Hz, 1H),
3.82e3.78 (m, 2H), 3.42e3.37 (m, 3H), 2.90e2.93 (m, 2H), 1.37 (s,
9H); 13C NMR (100 MHz, CDCl3):
d 156.1, 137.8, 129.4, 128.5, 126.3,
79.8, 72.1, 55.1, 53.8, 38.2, 28.2; LCMS (ESI): 306.7 [M þ H]þ; HRMS:
calcd for C15H22N4O3Na 329.1590, found C15H22N4O3Na 329.1602;
19
[a
]
¼ ꢂ51.5ꢀ (c 1.000, MeOH).
D
4.1.2. N1-((2S,3S)-4-Azido-3-hydroxy-1-phenylbutan-2-yl)-N3-
((R)-1-(4-fluorophenyl)ethyl)-5-(N-methylmethylsulfonamido)
isophthalamide (9)
To a solution of CF3COOH (1 mL) in CH2Cl2 (4 mL) was added 7
(92 mg, 0.3 mmol). The mixture was at room temperature for 3 h,
and then it was concentrated under reduced pressure to afford the
crude product 8 used without further purification. To a solution of
the product (92 mg, 0.3 mmol) in DMF (8 mL) was added 3-[(R)-1-
(4-fluorophenyl)ethylaminocarbonyl]-5-[methyl(methylsulfonyl)
amino]benzoic acid (106 mg, 0.27 mmol), EDCI (52 mg, 0.27 mmol),
3. Conclusion
Toward the goal to obtain novel C-terminus groups for BACE1
inhibitors, we utilized the click chemistry and in situ screening
approach to design and investigate triazole derivatives on the basis
of the previous study of our group. Among these triazole de-
HOBt (37 mg, 0.27 mmol), DIPEA (99 mL, 0.6 mmol) was added and
the mixture was allowed to react overnight at room temperature.
Then the resulting mixture was diluted by 50 mL EtOAc and washed
with dilute HCl aqueous solution twice (30 mL ꢃ 2), saturated
aqueous NaHCO3 twice (30 mL ꢃ 2) and saturated brine once
(30 mL) sequentially. The organic layers were dried over Na2SO4,
filtered and evaporated to give the residue, which was purified by
silica gel chromatography to afford 9 (170 mg, 97.4%). 1H NMR
rivatives, compound 12 (IC50 ¼ 0.036
inhibiting potency, comparable to that of OM99-2 (IC50
0.071 M). With the aid of the crystal structure of BACE1 in complex
mM) exhibited better enzyme-
¼
m
with compound 12, we found that Pro70 and Thr72 which located
in the flap region were the critical components for interaction with
substituted triazole group. On the basis of structural analysis and
bioisosterism, pyrazole derivatives were prepared to extend the
SAR of this class of molecules and improve the cellular activities. As
a result, pyrazole derivatives, which maintained potent BACE1 ac-
tivities in enzymatic assay and good selectivity over cathepsin D,
showed improvement of cellular activities, especially compound 30
and 33. From the present work, the pyrazole scaffold could be
considered as better C-terminus of BACE1 inhibitors comparing
that of triazole. And Pro70 and Thr72 might be taken as promising
subsite for developing selective BACE1 inhibitors as anti-AD drugs.
(300 MHz, CD3OD):
d
8.08 (s, 1H), 7.98 (t, J ¼ 1.5 Hz, 1H), 7.86 (t,
J ¼ 1.2 Hz, 1H), 7.42 (m, 2H), 7.27 (m, 4H), 7.16 (m, 1H), 7.06 (m, 2H),
5.22 (m, 1H), 4.40 (m, 1H), 3.85 (m, 1H), 3.32 (m, 5H), 2.96e3.06 (m,
2H), 2.95 (s, 3H), 1.57e1.52 (d, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz,
CDCl3):
d
165.9, 164.8, 161.9 (d, J ¼ 245 Hz, 1C), 142.1, 138.5, 137.3,
135.6, 135.4, 129.2, 128.7, 127.9, 127.8, 127.7, 127.6, 126.8, 124.1, 70.4,
54.8, 53.6, 49.2, 37.9, 37.8, 35.5, 21.6; LCMS (ESI): 583.0 [M þ H]þ;
HRMS: calcd for C28H31N6O5FSNa 605.1958, found C28H31N6O5FSNa
20
605.1964; [
a
]
¼ ꢂ79.2ꢀ (c 0.5, MeOH).
D
4. Experimental section
4.2. General method for preparation of compounds 10e17
(exemplified by 10)
4.1. General methods
4.2.1. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(1H-1,2,3-triazol-1-yl)butan-2-yl)-5-(N-methylmethylsu-
lfonamido)isophthalamide (10)
The 1H NMR (300 MHz or 400 MHz) spectra were recorded on
Varian Mercury-300 or 400 High Performance Digital FT-NMR us-
ing tetramethylsilane as an internal standard and the 13C NMR
(100 MHz) spectra were determined with Varian Mercury-400 High
Performance Digital FT-NMR. The LCeMS 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 stereo-
specifical target compounds was recorded on HPLC system (HP
To a solution of compound 9 (29 mg, 0.05 mmol) in THF (0.8 mL)
and H2O (0.2 mL), was added ethynyltrimethylsilane (28
mL,
0.2 mmol), followed by addition of sodium ascorbate (2 mg,
0.01 mmol) and CuI (1 mg, 0.005 mmol). This reaction was heated
at 50 ꢀC for 12 h. The reaction mixture was concentrated under
reduced pressure to afford the crude product used without further

278
Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
purification. The remaining solid was dissolved in THF (1 mL).
Tetrabutylammonium fluoride (52 mg, 0.2 mmol) was added and
the mixture was allowed to react overnight at room temperature.
The mixture was filtered and evaporated to give the residue, which
was purified by silica gel chromatography to afford 10 (21 mg,
(m, 1H), 8.14e8.12 (m, 1H), 7.98e7.97 (m, 1H), 7.89e7.88 (m, 1H),
7.77e7.74 (m, 2H), 7.44e7.37 (m, 4H), 7.33e7.21 (m, 5H), 7.15e7.12
(m, 1H), 7.09e7.04 (m, 2H), 5.26e5.19 (q, J ¼ 6.7 Hz, 1H), 4.63e4.57
(m, 1H), 4.53e4.39 (m, 2H), 4.21e4.19 (m, 1H), 3.34 (s, 3H), 3.12e
2.99 (m, 2H), 2.94 (s, 3H), 1.57e1.54 (d, J ¼ 6.7 Hz, 3H); 13C NMR
70.0%). 1H NMR (300 MHz, CD3OD):
d
8.14 (m, 1H), 8.00 (s, 2H), 7.89
(100 MHz, CDCl3):
d
166.2, 164.8, 161.9 (d, J ¼ 245 Hz, 1C), 147.3,
(s, 1H), 7.70 (s, 1H), 7.42e7.40 (m, 2H), 7.30e7.21 (m, 4H), 7.17e7.14
(m, 1H), 7.08e7.05 (t, J ¼ 8.7 Hz, 2H), 5.25e5.19 (q, J ¼ 7.0 Hz, 1H),
4.63e4.57 (m, 1H), 4.50e4.37 (m, 2H), 4.19e4.16 (m, 1H), 3.34 (s,
3H), 3.13e2.99 (m, 2H), 2.95 (s, 3H),1.57e1.52 (d, J ¼ 7.0 Hz, 3H); 13C
142.1, 138.7, 137.4, 135.8, 135.3, 129.7, 129.3, 128.8, 128.7, 128.3,
128.0, 127.9, 126.8, 125.4, 124.0, 121.4, 115.6, 115.4, 70.4, 54.7, 54.1,
49.2, 38.0, 37.9, 35.6, 21.7; LCMS (ESI): 685.2 [M þ H]þ; HRMS: calcd
for C36H37N6O5FSNa 707.2428, found C36H37N6O5FSNa 707.2393;
17
NMR (100 MHz, CDCl3):
d
166.03, 164.82, 162.80 (d, J ¼ 245 Hz, 1C),
[a
]
¼ ꢂ60.7ꢀ (c 0.95, MeOH).
D
141.88, 139.64, 137.74, 137.58, 135.46, 135.11, 133.14, 129.22, 128.14,
127.91, 126.36, 125.33, 124.08, 115.13, 115.07, 70.67, 58.58, 54.31,
53.95, 49.27, 37.74, 35.33, 23.63; LCMS (ESI): 609.1 [M þ H]þ;
4.2.5. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-1-phenylbutan-2-yl)-
5-(N-methylmethylsulfonamido)isophthalamide (14)
HRMS: calcd for C30H33FN6O5SNa 631.2115, found C30H33FN6O5SNa
17
631.2114; [
a
]
¼ ꢂ34.0ꢀ (c 0.35, MeOH).
Compound 14 was obtained from
9 and 1-ethynyl-4-
D
methoxybenzene (26 L, 0.2 mmol) according to the similar pro-
m
4.2.2. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
(4-(hydroxylmethyl)-1H-1,2,3-triazol-1-yl)-1-phenylbutan-2-yl)-5-
(N-methylmethylsulfonamido)isophthalamide (11)
cedure used to prepare 11. The crude product was purified by silica
gel chromatography to afford 14 (30 mg, 82.1%). 1H NMR (300 MHz,
CD3OD): d 8.19 (s, 1H), 8.12 (s, 1H), 7.98e7.97 (m, 1H), 7.89e7.87 (m,
To a solution of compound 9 (29 mg, 0.05 mmol) in THF (0.8 mL)
and H2O (0.2 mL), was added prop-2-yn-1-ol (12 mL, 0.2 mmol),
1H), 7.68e7.65 (m, 2H), 7.46e7.40 (m, 2H), 7.31e7.21 (m, 4H), 7.18e
7.12 (m, 1H), 7.08e7.03 (m, 2H), 6.97e6.94 (m, 2H), 5.26e5.20 (q,
J ¼ 7.0 Hz, 1H), 4.63e4.57 (m, 1H), 4.52e4.45 (m, 1H), 4.42e4.39 (m,
1H), 4.24e4.21 (m, 1H), 3.81 (s, 3H), 3.33 (s, 3H), 2.97e3.15 (m, 2H),
2.95 (s, 3H), 1.58e1.54 (d, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz,
followed by addition of sodium ascorbate (2 mg, 0.01 mmol) and
CuI (1 mg, 0.005 mmol). This reaction was heated at 50 ꢀC for 12 h.
The reaction mixture was concentrated under reduced pressure to
afford the crude product, which was purified by silica gel chro-
matography to afford 11 (23 mg, 70.1%). 1H NMR (300 MHz,
CDCl3):
d
166.1, 164.6, 163.1 (d, J ¼ 245 Hz, 1C), 160.6, 159.5, 147.0,
142.1, 138.4, 137.1, 135.7, 135.0, 129.5, 128.4, 127.7, 127.6, 126.7, 123.9,
122.4120.6, 115.6, 115.1, 114.1, 70.5, 55.1, 54.5, 53.8, 49.1, 38.0, 37.8,
CD3OD): d 8.12 (s, 1H), 7.99e7.98 (m, 1H), 7.94 (s, 1H), 7.89e7.88 (m,
1H), 7.43e7.40 (m, 2H), 7.30e7.21 (m, 4H), 7.17e7.14 (m, 1H), 7.09e
7.03 (t, J ¼ 9.0 Hz, 2H), 5.27e5.20 (q, J ¼ 7.0 Hz, 1H), 4.66 (s, 2H),
4.59e4.53 (m, 1H), 4.49e4.45 (m, 1H), 4.41e4.33 (m, 1H), 4.17e4.15
(m, 1H), 3.35 (s, 3H), 3.12e2.99 (m, 2H), 2.96 (s, 3H), 1.58e1.54 (d,
35.4, 21.6; LCMS (ESI): 715.1 [M
þ
H]þ; HRMS: calcd for
C37H39N6O6FSNa 737.2534, found C37H39N6O6FSNa 737.2514;
18
[a
]
¼ ꢂ52.6ꢀ (c 1.10, MeOH).
D
J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d
166.6, 164.9, 160.5 (d,
4.2.6. N1-((2S,3S)-4-(4-Cyclohexyl-1H-1,2,3-triazol-1-yl)-3-
hydroxy-1-phenylbutan-2-yl)-N3-((R)-1-(4-fluorophenyl)ethyl)-5-
(N-methylmethylsulfonamido)isophthalamide (15)
J ¼ 245 Hz, 1C), 141.7, 139.0, 137.7, 137.4, 135.7, 135.0, 133.1, 129.1,
128.4, 127.6, 126.5, 124.7, 123.9, 115.3, 115.0, 70.9, 55.4, 54.2, 53.6,
48.8, 37.6, 37.3, 35.5, 21.6; LCMS (ESI): 639.1 [M þ H]þ; HRMS: calcd
Compound 15 was obtained from 9 and ethynylcyclohexane
for C31H35N6O5FSNa 661.2221, found C31H35N6O5FSNa 661.2231;
(26
pare 11. The crude product was purified by silica gel chromatog-
raphy to afford 15 (29 mg, 82.1%). 1H NMR (300 MHz, CD3OD):
8.12
mL, 0.2 mmol) according to the similar procedure used to pre-
15
[
a]
¼ ꢂ107ꢀ (c 0.15, MeOH).
D
d
4.2.3. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
(4-((R)-1-hydroxy ethyl)-1H-1,2,3-triazol-1-yl)-1-phenylbutan-2-
yl)-5-(N-methylmethylsulfonamido) isophthalamide (12)
(s, 1H), 8.00 (s, 1H), 7.89 (s, 1H), 7.72 (s, 1H), 7.45e7.40 (m, 2H),
7.29e7.21 (m, 4H), 7.17e7.14 (m, 1H), 7.08e7.03 (t, J ¼ 8.7 Hz, 2H),
5.26e5.22 (q, J ¼ 7.0 Hz, 1H), 4.54e4.40 (m, 2H), 4.37e4.30 (m, 1H),
4.17e4.14 (m,1H), 3.35 (s, 3H), 3.11e2.98 (m, 2H), 2.96 (s, 3H), 2.69e
2.65 (m, 1H), 1.99e1.96 (m, 2H), 1.80e1.70 (m, 3H), 1.59e1.57 (d,
J ¼ 7.0 Hz, 3H), 1.45e1.25 (m, 5H); 13C NMR (100 MHz, CDCl3):
Compound 12 was obtained from 9 and (R)-but-3-yn-2-ol
(16
pare 11. The crude product was purified by silica gel chromatog-
raphy to afford 12 (21 mg, 62.7%). 1H NMR (300 MHz, CD3OD):
8.12
mL, 0.2 mmol) according to the similar procedure used to pre-
d
d
166.0, 164.7, 163.2 (d, J ¼ 245 Hz, 1C), 153.2, 142.2, 138.6, 138.1,
(s, 1H), 7.99 (s, 1H), 7.89 (s, 2H), 7.44e7.40 (m, 2H), 7.27e7.23 (m,
4H), 7.17e7.16 (m, 1H), 7.12e7.04 (m, 2H), 5.27e5.20 (q, J ¼ 7.0 Hz,
1H), 4.96e4.94 (m, 1H), 4.57e4.51 (m, 1H), 4.49e4.42 (m, 1H),
4.40e4.32 (m, 1H), 4.17e4.15 (m, 1H), 3.35 (s, 3H), 3.12e2.97 (m,
2H), 2.96 (s, 3H), 1.58e1.56 (d, J ¼ 7.0 Hz, 3H), 1.51e1.49 (d,
137.3, 135.8, 135.2, 129.1, 128.2, 127.7, 126.7, 123.8, 121.1, 115.5, 115.1,
70.1, 58.5, 54.3, 53.9, 49.0, 37.9, 37.6, 35.4, 34.8, 32.6, 25.8, 21.7;
LCMS (ESI): 691.2 [M þ H]þ; HRMS: calcd for C36H43N6O5FSNa
17
713.2897, found C36H43N6O5FSNa 713.2898; [
MeOH).
a
]
¼ ꢂ86ꢀ (c 0.30,
D
J ¼ 6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 166.6, 165.1, 161.8 (d,
J ¼ 245 Hz, 1C), 141.9, 138.9, 137.5, 137.4, 135.9, 135.1, 129.2, 128.7,
128.0, 127.9, 126.8, 124.2, 122.7, 115.4, 115.2, 72.1, 62.4, 54.6, 53.2,
49.1, 37.8, 35.8, 35.7, 22.8, 21.6; LCMS (ESI): 653.1 [M þ H]þ; HRMS:
4.2.7. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-1-phenylbutan-2-yl)-5-
(N-methylmethylsulfonamido)isophthalamide (16)
To a solution of compound 9 (29 mg, 0.05 mmol) in THF
(1.0 mL) prop-2-yn-1-ol (12 mL, 0.2 mmol) was added, followed by
calcd for C32H37FN6O6SNa 675.2377, found C32H37FN6O6SNa
23
675.2390; [
a
]
¼ ꢂ36ꢀ (c 0.20, MeOH).
D
addition of Cp*Ru (PPh3)3Cl2 (4 mg, 0.005 mmol). This reaction
was heated to reflux under argon for 8 h. The resulting mixture
was concentrated under reduced pressure to afford the crude
product, which was purified by silica gel chromatography to afford
4.2.4. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(4-phenyl-1H-1,2,3-triazol-1-yl)butan-2-yl)-5-(N-meth-
ylmethylsulfonamido)isophthalamide (13)
Compound 13 was obtained from 9 and ethynylbenzene (22
mL,
16 (17 mg, 51.1%). 1H NMR (300 MHz, CD3OD):
d 8.12 (s, 1H), 7.99
0.2 mmol) according to the similar procedure used to prepare 11.
The crude product was purified by silica gel chromatography to
(s, 1H), 7.88 (s, 1H), 7.63 (s, 1H), 7.45e7.40 (m, 2H), 7.32e7.22 (m,
4H), 7.17e7.14 (m, 1H), 7.09e7.03 (t, J ¼ 8.7 Hz, 2H), 5.27e5.20 (q,
J ¼ 7.1 Hz, 1H), 4.73e4.62 (m, 2H), 4.64e4.57 (m, 1H), 4.53e4.42
afford 13 (31 mg, 91.5%). 1H NMR (300 MHz, CD3OD):
d 8.30e8.29

Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
279
(m, 2H), 4.27e4.23 (m, 1H), 3.36 (s, 3H), 3.14e2.99 (m, 2H), 2.96 (s,
4.2.10. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
(1H-imidazol-1-yl)-1-phenylbutan-2-yl)-5-(N-methylmethylsul-
fonamido)isophthalamide (21)
3H), 1.59e1.56 (d, J ¼ 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d
166.26, 164.59, 161.06 (d, J ¼ 245 Hz, 1C), 142.08, 138.71, 137.41,
135.79, 135.37, 129.32, 128.59, 127.80, 127.69, 126.64, 123.89,
115.78, 115.04, 70.20, 69.47, 65.9, 53.72, 51.10, 49.23, 37.97, 35.50,
29.63, 29.00, 21.81; LCMS (ESI): 639.1 [M þ H]þ; HRMS: calcd for
Compound 21 was obtained from 6 and imidazole (51 mg,
0.75 mmol) according to the similar procedure used to prepare 20.
The crude product was purified by silica gel chromatography to
C31H35N6O6FSNa 661.2221, found C31H35N6O6FSNa 661.2213;
afford 21 (141 mg, 88.7%). 1H NMR (300 MHz, CD3OD):
d 8.10 (s, 1H),
17
[
a
]
¼ ꢂ58.0ꢀ (c 0.20, MeOH).
7.99 (s, 1H), 7.89 (s, 1H), 7.44e7.39 (m, 2H), 7.28e7.20 (m, 5H), 7.17e
7.14 (m, 2H), 7.08e7.03 (m, 3H), 5.24e5.22 (q, J ¼ 6.6 Hz, 1H), 4.48e
4.44 (m, 1H), 4.21e4.18 (m, 1H), 4.00e3.97 (m, 2H), 3.34 (s, 3H),
3.03e2.99 (m, 2H), 2.94 (s, 3H), 1.58e1.55 (d, J ¼ 6.6 Hz, 3H); 13C
D
4.2.8. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(5-phenyl-1H-1,2,3-triazol-1-yl)butan-2-yl)-5-(N-methy-
lmethylsulfonamido)isophthalamide (17)
NMR (100 MHz, CDCl3)
d
166.05, 164.78, 162.91 (d, J ¼ 245 Hz, 1C),
Compound 17 was obtained from 9 and ethynylbenzene (22
mL,
142.35, 139.91, 138.95, 137.55, 135.67, 135.19, 131.22, 129.36, 128.76,
128.24, 128.11, 126.87, 123.87, 115.54, 115.24, 112.40, 70.07, 54.12,
49.28, 38.18, 37.84, 35.86, 31.83, 22.54; LCMS (ESI): 608.3 [M þ H]þ;
0.2 mmol) according to the similar procedure used to prepare 16.
The crude product was purified by silica gel chromatography to
afford 17 (21 mg, 58.8%). 1H NMR (300 MHz, CD3OD):
d
8.00e7.98
HRMS: calcd for C31H35N5O5FS 608.2343, found C31H35N5O5FS
15
(m, 2H), 7.78 (s, 1H), 7.74 (s, 1H), 7.48e7.41 (m, 4H), 7.31e7.21 (m,
7H), 7.17e7.12 (m, 1H), 7.09e7.03 (t, J ¼ 8.7 Hz, 2H), 5.28e5.21 (q,
J ¼ 7.1 Hz, 1H), 4.49e4.44 (m, 1H), 4.42e4.26 (m, 3H), 3.34 (s, 3H),
3.07e2.96 (m, 2H), 2.95 (s, 3H), 1.58e1.56 (d, J ¼ 7.1 Hz, 3H); 13C
608.2315; [
a
]
¼ ꢂ32.5ꢀ (c 0.04, CHCl3).
D
4.2.11. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(1H-1,2,4-triazol-1-yl)butan-2-yl)-5-(N-methylmethy-
lsulfonamido)isophthalamide (22)
NMR (100 MHz, CDCl3):
d
166.1, 164.7, 162.9 (d, J ¼ 245 Hz, 1C),
141.6, 139.2, 138.7, 137.5, 135.8, 135.2, 132.5, 129.6, 129.2, 128.8,
128.3, 128.2, 127.8, 126.7, 126.0, 124.2, 115.7, 115.1, 69.9, 53.8, 51.7,
48.8, 38.1, 37.4, 35.4, 29.6, 21.5; LCMS (ESI): 685.2 [M þ H]þ; HRMS:
Compound 22 was obtained from 6 and 1,2,4-triazole (52 mg,
0.75 mmol) according to the similar procedure used to prepare 20.
The crude product was purified by silica gel chromatography to
calcd for C36H37N6O5FSNa 707.2428, found C36H37N6O5FSNa
afford 22 (137 mg, 86.2%). 1H NMR (300 MHz, CD3OD):
d 8.21 (s,1H),
15
707.2435; [
a
]
¼ ꢂ90.3ꢀ (c 0.35, MeOH).
8.12 (s, 1H), 7.99 (s, 1H), 7.95 (s, 1H), 7.89 (s, 1H), 7.44e7.40 (m, 2H),
7.28e7.21 (m, 4H), 7.17e7.12 (m, 1H), 7.08e7.03 (t, J ¼ 8.7 Hz, 2H),
5.24e5.22 (q, J ¼ 7.2 Hz, 1H), 4.45e4.40 (m, 1H), 4.37e4.31 (m, 1H),
4.25e4.11 (m, 2H), 3.34 (s, 3H), 3.10e2.95 (m, 2H), 2.97 (s, 3H),
D
4.2.9. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(1H-pyrazol-1-yl)butan-2-yl)-5-(N-methylmethylsulfo-
namido)isophthalamide (20)
1.58e1.56 (d, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 166.08,
To a solution of compound 6 (131 mg, 0.50 mmol) in DMF
(2 mL) was added pyrazole (51 mg, 0.75 mmol), K2CO3 (138 mg,
1.00 mmol). The mixture was heated at 100 ꢀC for 1 h. Reaction
mixture was cooled, ice cold water (30 mL) was added and then
extracted with EtOAc twice (30 mL ꢃ 2) and the combined organic
layers were washed by saturated brine (30 mL). Then the organic
layers were dried over Na2SO4, filtered and evaporated to afford
the crude product used without further purification, To a solution
of CF3COOH (1 mL) in CH2Cl2 (4 mL) was added the remaining
solid (92 mg, 0.30 mmol). The mixture was at room temperature
for 3 h, and then it was concentrated under reduced pressure to
afford the crude product 19 used without further purification. To a
solution of the product (99 mg, 0.30 mmol) in DMF (8 mL) was
added 3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-5-[methyl
(methylsulfonyl)amino]benzoic acid (106 mg, 0.27 mmol), EDCI
164.85, 163.03 (d, J ¼ 245 Hz, 1C), 151.15, 143.97, 141.94, 138.69,
137.35, 135.71, 135.32, 129.15, 128.59, 127.88, 127.80, 126.73, 124.34,
115.43, 115.22, 69.78, 53.80, 53.54, 49.04, 37.86, 37.79, 35.49, 21.56;
LCMS (ESI): 609.3 [M þ H]þ; HRMS: calcd for C30H34N6O5FS
15
609.2295, found C30H34N6O5FS 609.2307; [
CHCl3).
a]
¼ ꢂ251.9ꢀ (c 0.21,
D
4.2.12. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(1H-tetrazol-1-yl)butan-2-yl)-5-(N-methylmethylsul-
fonamido)isophthalamide (23) and N1-((R)-1-(4-fluorophenyl)
ethyl)-N3-((2S,3S)-3-hydroxy-1-phenyl-4-(2H-tetrazol-2-yl)butan-
2-yl)-5-(N-methylmethylsulfonamido)isophthalamide (24)
To a solution of compound 6 (131 mg, 0.50 mmol) in DMF (2 mL)
was added tetrazole (53 mg, 0.75 mmol), K2CO3 (138 mg,
1.00 mmol). The mixture was heated at 100 ꢀC for 1 h. Reaction
mixture was cooled, ice cold water (30 mL) was added and then
extracted with EtOAc twice (30 mL ꢃ 2) and the combined organic
layers were washed by saturated brine (30 mL). Then the organic
layers were dried over Na2SO4, filtered and evaporated to afford the
crude product used without further purification, To a solution of
CF3COOH (1 mL) in CH2Cl2 (4 mL) was added the remaining solid
(92 mg, 0.30 mmol). The mixture was stirred at room temperature
for 3 h, and then it was concentrated under reduced pressure to
afford the crude product used without further purification. To a
solution of the product (100 mg, 0.30 mmol) in DMF (8 mL) was
(52 mg, 0.27 mmol), HOBt (37 mg, 0.27 mmol), DIPEA (99
mL,
0.60 mmol) was added and the mixture was allowed to react
overnight at room temperature. Then the resulting mixture was
diluted by 50 mL EtOAc and washed with dilute HCl aqueous
solution (30 mL), saturated aqueous NaHCO3 (30 mL) and satu-
rated brine (30 mL) sequentially. The organic layers were dried
over Na2SO4 and evaporated to give the residue, which was pu-
rified by silica gel chromatography to afford 20 (149 mg, 91.3%). 1H
NMR (300 MHz, CD3OD):
d 8.12 (s, 1H), 7.99 (s, 1H), 7.89 (s, 1H),
7.63 (s, 1H), 7.46e7.39 (m, 3H), 7.26e7.19 (m, 4H), 7.16e7.13 (m,
1H), 7.08e7.02 (t, J ¼ 8.4 Hz, 2H), 6.24 (s, 1H), 5.24e5.22 (q,
J ¼ 6.9 Hz, 1H), 4.41e4.36 (m, 1H), 4.29e4.23 (m, 1H), 4.15e4.10
(m, 2H), 3.34 (s, 3H), 3.08e2.93 (m, 2H), 2.94 (s, 3H), 1.58e1.55 (d,
added
(methylsulfonyl)amino]benzoic acid (106 mg, 0.27 mmol), EDCI
(52 mg, 0.27 mmol), HOBt (37 mg, 0.27 mmol), DIPEA (99 L,
3-[(R)-1-(4-fluorophenyl)ethylaminocarbonyl]-5-[methyl
m
J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d
165.60, 164.47, 163.21
0.60 mmol) was added and the mixture was allowed to react
overnight at room temperature. Then the resulting mixture was
diluted by 50 mL EtOAc and washed with dilute HCl aqueous so-
lution (30 mL), saturated aqueous NaHCO3 (30 mL) and saturated
brine (30 mL) sequentially. The organic layers were dried over
Na2SO4, filtered and evaporated to give the residue, which was
purified by silica gel chromatography to afford 23 (68 mg, 41.3%)
(d, J ¼ 245 Hz, 1C), 142.28, 139.82, 138.44, 137.19, 135.83, 135.45,
130.34, 129.35, 128.65, 127.89, 127.27, 126.48, 123.55, 115.62,
115.19, 105.70, 70.40, 53.46, 49.01, 38.19, 37.51, 35.36, 29.48, 21.51;
LCMS (ESI): 608.1 [M þ H]þ; HRMS: calcd for C31H35N5O5FS
15
608.2343, found C31H35N5O5FS 608.2322; [
MeOH).
a
]
¼ ꢂ42.2ꢀ (c 0.09,
D

280
Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
and 24 (74 mg, 45.4%). 1H NMR (300 MHz, CD3OD):
d
8.68 (s, 1H),
4.2.16. N1-((2S,3S)-4-(4-Carbamoyl-1H-pyrazol-1-yl)-3-hydroxy-
1-phenylbutan-2-yl)-N3-((R)-1-(4-fluorophenyl)ethyl)-5-(N-meth-
ylmethylsulfonamido)isophthalamide (30)
8.13 (s, 1H), 7.98 (s, 1H), 7.89 (s, 1H), 7.44e7.39 (m, 2H), 7.30e7.21
(m, 4H), 7.17e7.13 (m, 1H), 7.08e7.02 (t, J ¼ 8.7 Hz, 2H), 5.24e5.21
(q, J ¼ 7.5 Hz, 1H), 4.76e4.73 (m, 2H), 4.54e4.49 (m, 1H), 4.41e4.38
(m, 1H), 3.34 (s, 3H), 3.10e2.95 (m, 2H), 2.97 (s, 3H), 1.58e1.55 (d,
Two equivalent NaOH (80 mg, 2 mmol) was dissolved in the
solvent of THF/EtOH/H2O ¼ 2/2/1, then compound 28 (670 mg,
1 mmol) was added into the solution. The reaction was heated to
reflux for 2 h. Then the PH was adjusted to 4 by diluted HCl, and
extracted by EtOAc (30 mL ꢃ 2) and washed with saturated brine
once (50 mL) sequentially. The organic layers were dried over
Na2SO4, filtered and evaporated to give the crude product acid 29,
which was used directly in the next step. To a solution of the above
acid (65 mg, 0.10 mmol) in 2 mL DMF was added ammonium
chloride (6.0 mg, 0.11 mmol), HOBt (18 mg, 0.132 mmol), DIPEA
J ¼ 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 166.55, 165.16, 163.02
(d, J ¼ 245 Hz, 1C), 144.07, 138.88, 137.31, 135.68, 135.22, 129.14,
128.61, 128.18, 127.94, 127.86, 126.78, 124.21, 115.42, 115.21, 70.36,
53.86, 52.30, 49.26, 37.76, 37.55, 35.73, 21.66; LCMS (ESI): 610.1
[M þ H]þ; HRMS: calcd for C29H32N7O5FSNa 632.2067, found
15
C29H32N7O5FSNa 632.2097; [
a
]
¼ ꢂ301.8ꢀ (c 0.22, CHCl3).
D
4.2.13. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(2H-tetrazol-2-yl)butan-2-yl)-5-(N-methylmethylsulfo-
namido)isophthalamide (24)
(45
mL, 0.30 mmol) and stirred for 10 min. EDCI (25 mg,
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 diluted aqueous HCl
aqueous (30 mL), dilute aqueous NaHCO3 (30 mL) and saturated
brine (30 mL) sequentially. The organic layers were dried over
Na2SO4, filtered and evaporated to give the crude product amide,
which was purified by silica gel chromatography to afford the target
1H NMR (300 MHz, CD3OD):
d 9.07 (s, 1H), 8.09 (s, 1H), 7.99 (s,
1H), 7.87 (s, 1H), 7.44e7.40 (m, 2H), 7.31e7.22 (m, 4H), 7.18e7.16 (m,
1H), 7.08e7.02 (t, J ¼ 8.4 Hz, 2H), 5.24e5.22 (q, J ¼ 7.2 Hz,1H), 4.68e
4.61 (m, 1H), 4.51e4.42 (m, 2H), 4.18e4.13 (m, 1H), 3.35 (s, 3H),
3.10e2.95 (m, 2H), 2.97 (s, 3H), 1.58e1.56 (d, J ¼ 7.2 Hz, 3H); 13C
NMR (100 MHz, CDCl3):
d
166.18, 164.95, 163.09 (d, J ¼ 245 Hz, 1C),
142.00, 138.62, 137.19, 135.58, 135.22, 129.18, 128.64, 128.02, 127.90,
127.75, 126.80, 124.25, 115.48, 115.27, 69.94, 56.99, 53.85, 49.12,
37.84, 37.69, 35.48, 21.58; LCMS (ESI): 610.2 [M þ H]þ; HRMS: calcd
compound 30 (55 mg, 84.2%). 1H NMR (300 MHz, CD3OD):
d 8.13 (s,
1H), 8.10 (s, 1H), 7.99 (s, 1H), 7.89 (s, 2H), 7.44e7.40 (m, 2H), 7.28e
7.20 (m, 4H), 7.16e7.14 (m, 1H), 7.07e7.02 (t, J ¼ 6.3 Hz, 2H), 5.24e
5.21 (q, J ¼ 6.9 Hz, 1H), 4.44e4.39 (m, 1H), 4.32e4.26 (m, 1H), 4.14e
4.11 (m, 2H), 3.34 (s, 3H), 3.09e2.98 (m, 2H), 2.95 (s, 3H), 1.58e1.56
for C29H32N7O5FSNa 632.2067, found C29H32N7O5FSNa 632.2065;
15
[
a]
¼ ꢂ188.1ꢀ (c 0.26, CHCl3).
D
(d, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 166.12, 165.11,
4.2.14. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
(4-(hydroxyl methyl)-1H-pyrazol-1-yl)-1-phenylbutan-2-yl)-5-(N-
methylmethylsulfonamido)isophthalamide (25)
Compound 25 was obtained from 6 and (1H-pyrazol-4-yl)
methanol (74 mg, 0.75 mmol) according to the similar procedure
used to prepare 20. The crude product was purified by silica gel
chromatography to afford 25 (118 mg, 74.2%). 1H NMR (300 MHz,
163.17, 160.75 (d, J ¼ 245 Hz, 1C), 141.84, 139.16, 138.73, 137.52,
135.86, 135.37, 132.92, 129.14, 128.52, 127.74, 126.64, 124.52, 123.94,
117.05, 115.61, 115.14, 70.65, 56.12, 53.94, 49.12, 37.89, 35.46, 29.65,
21.52; LCMS (ESI): 651.1 [M þ H]þ; HRMS: calcd for C32H35N6O6F-
15
NaS 673.2221, found C32H35N6O6FNaS 673.2234; [
0.08, CHCl3).
a
]
¼ ꢂ135.0ꢀ (c
D
CDCl3):
d
8.01 (s, 1H), 7.94 (s, 1H), 7.82 (s, 1H), 7.43 (s, 1H), 7.36e7.32
4.2.17. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
(4-(methyl carbamoyl)-1H-pyrazol-1-yl)-1-phenylbutan-2-yl)-5-
(N-methylmethylsulfonamido)isophthalamide (31)
Compound 31 was obtained from 29 and methylamine hydro-
chloride (7.5 mg, 0.11 mmol) according to the similar procedure
used to prepare 30. The crude product was purified by silica gel
chromatography to afford 31 (53 mg, 80.1%). 1H NMR (300 MHz,
(m, 3H), 7.28e7.21 (m, 2H), 7.20e7.19 (m, 1H), 7.08e6.96 (m, 4H),
5.29e5.27 (q, J ¼ 7.2 Hz, 1H), 4.48 (s, 2H), 4.39e4.36 (m, 1H), 4.13e
4.02 (m, 3H), 3.33 (s, 3H), 3.05e3.02 (m, 2H), 2.86 (s, 3H), 1.59e1.56
(d, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 165.70, 164.66,
163.18 (d, J ¼ 245 Hz, 1C), 142.14, 139.03, 138.56, 137.38, 135.91,
135.45, 129.69, 129.21, 128.51, 127.88, 127.45, 126.74, 123.98, 121.52,
115.63, 115.42, 71.42, 56.00, 55.10, 53.78, 48.90, 38.10, 37.91, 35.38,
CD3OD):
d 8.14 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.83 (s,
21.75; LCMS (ESI): 637.2 [M þ H]þ; HRMS: calcd for C32H36N5O6F-
1H), 7.45e7.40 (m, 2H), 7.28e7.20 (m, 4H), 7.16e7.14 (m, 1H), 7.08e
7.02 (t, J ¼ 8.7 Hz, 2H), 5.24e5.22 (q, J ¼ 7.2 Hz, 1H), 4.40e4.38 (m,
1H), 4.31e4.25 (m, 1H), 4.16e4.11 (m, 2H), 3.34 (s, 3H), 3.09e3.01
(m, 2H), 2.82 (s, 3H), 2.98 (s, 3H), 1.58e1.56 (d, J ¼ 7.2 Hz, 3H); 13C
15
NaS 660.2268, found C32H36N5O6FNaS 660.2257; [
0.20, CHCl3).
a
]
¼ ꢂ34.0ꢀ (c
D
4.2.15. Ethyl-1-((2S,3S)-3-(3-((R)-1-(4-fluorophenyl)ethylcarbam-
oyl)-5-(N-methylmethylsulfonamido)benzamido)-2-hydroxy-4-
phenylbutyl)-1H-pyrazole-4-carboxylate (28)
Compound 28 was obtained from 6 and ethyl 1H-pyrazole-4-
carboxylate (105 mg, 0.75 mmol) according to the similar proce-
dure used to prepare 20. The crude product was purified by silica
gel chromatography to afford 28 (122 mg, 76.6%). 1H NMR
NMR (100 MHz, CDCl3): d 166.15, 165.08, 163.74, 162.92 (d,
J ¼ 245 Hz, 1C), 141.93, 139.43, 138.80, 137.81, 135.63, 135.22, 132.06,
129.28, 128.43, 128.27, 127.99, 126.46, 124.31, 117.89, 115.26, 115.05,
70.58, 54.13, 51.15, 49.23, 42.32, 37.86, 35.63, 26.10, 21.99; LCMS
(ESI): 665.2 [M þ H]þ; HRMS: calcd for C33H37N6O6FNaS 687.2377,
15
found C33H37N6O6FNaS 687.2390; [
a]
¼ ꢂ68.8ꢀ (c 0.26, CHCl3).
D
(300 MHz, CD3OD):
d
8.14 (s, 1H), 8.11 (s, 1H), 7.98 (s, 1H), 7.88 (s,
4.2.18. N1-((2S,3S)-4-(4-(Dimethylcarbamoyl)-1H-pyrazol-1-yl)-3-
hydroxy-1-phenylbutan-2-yl)-N3-((R)-1-(4-fluorophenyl)ethyl)-5-
(N-methylmethylsulfonamido)isophthalamide (32)
Compound 32 was obtained from 29 and dimethylamine hy-
drochloride (9.0 mg, 0.11 mmol) according to the similar procedure
used to prepare 30. The crude product was purified by silica gel
chromatography to afford 32 (53 mg, 78.4%). 1H NMR (300 MHz,
1H), 7.84 (s,1H), 7.45e7.39 (m, 2H), 7.28e7.20 (m, 4H), 7.16e7.14 (m,
1H), 7.08e7.02 (t, J ¼ 9.0 Hz, 2H), 5.24e5.22 (q, J ¼ 6.6 Hz, 1H),
4.42e4.40 (m, 1H), 4.28e4.21 (m, 1H), 4.26e4.23 (q, J ¼ 6.9 Hz, 2H),
4.15e4.12 (m, 2H), 3.35 (s, 3H), 3.05e2.98 (m, 2H), 2.95 (s, 3H),
1.58e1.55 (d, J ¼ 6.6 Hz, 3H), 1.33e1.28 (t, J ¼ 6.9 Hz, 3H); 13C NMR
(100 MHz, CDCl3):
d
165.90, 164.67, 163.07 (d, J ¼ 245 Hz, 1C),
160.64, 142.09, 141.24, 138.62, 137.30, 135.72, 135.30, 133.82, 129.21,
128.59, 127.88, 127.54, 126.61, 123.99, 115.55, 115.22, 114.85, 70.41,
60.34, 56.00, 55.76, 53.70, 49.03, 37.82, 35.41, 21.43, 14.15; LCMS
CD3OD): d 8.11 (s, 1H), 8.03 (s, 1H), 7.98 (s, 1H), 7.89 (s, 1H), 7.77 (s,
1H), 7.45e7.40 (m, 2H), 7.28e7.20 (m, 4H), 7.16e7.14 (m, 1H), 7.08e
7.03 (t, J ¼ 8.7 Hz, 2H), 5.24e5.22 (q, J ¼ 6.9 Hz, 1H), 4.43e4.37 (m,
1H), 4.31e4.25 (m, 1H), 4.15e4.12 (m, 2H), 3.35 (s, 3H), 3.22 (s, 3H),
3.03 (s, 3H), 3.05e2.97 (m, 2H), 2.95 (s, 3H), 1.58e1.56 (d, J ¼ 6.9 Hz,
(ESI): 680.2 [M þ H]þ; HRMS: calcd for C34H39N5O7FS 680.2554,
15
found C34H39N5O7FS 680.2546; [
a
]
¼ ꢂ72.7ꢀ (c 0.30, CHCl3).
D

Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
281
3H); 13C NMR (100 MHz, CDCl3):
d
166.08, 164.83, 164.74, 163.03 (d,
4.2.22. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-4-
J ¼ 245 Hz, 1C), 142.00, 139.46, 138.91, 138.88, 137.57, 135.76, 135.28,
129.20, 128.56, 127.92, 127.84, 126.62, 124.14, 116.76, 115.38, 115.17,
70.53, 56.02, 53.83, 48.97, 39.10, 37.84, 35.97, 35.52, 21.60; LCMS
(4-(morpholine-4-carbonyl)-1H-pyrazol-1-yl)-1-phenylbutan-2-
yl)-5-(N-methylmethylsulfonamido)isophthalamide (36)
Compound 36 was obtained from 29 and morpholine
(10.0 mg, 0.11 mmol) according to the similar procedure used to
prepare 30. The crude product was purified by silica gel chro-
matography to afford 36 (51 mg, yield: 71.3%). 1H NMR
(ESI): 679.2 [M þ H]þ; HRMS: calcd for C34H39N6O6FNaS 701.2534,
15
found C34H39N6O6FNaS 701.2547; [
a
]
¼ ꢂ43.0ꢀ (c 0.30, CHCl3).
D
4.2.19. N1-((2S,3S)-4-(4-(Diethylcarbamoyl)-1H-pyrazol-1-yl)-3-
hydroxy-1-phenylbutan-2-yl)-N3-((R)-1-(4-fluorophenyl)ethyl)-5-
(N-methylmethylsulfonamido)isophthalamide (33)
(300 MHz, CD3OD): d 8.11e8.10 (m, 1H), 7.99e7.98 (m, 2H),
7.90e7.89 (m, 1H), 7.72 (s, 1H), 7.45e7.40 (m, 2H), 7.28e7.20 (m,
4H), 7.17e7.14 (m, 1H), 7.08e7.03 (t, J ¼ 6.9 Hz, 2H), 5.24e5.22
(q, J ¼ 6.9 Hz, 1H), 4.39e4.35 (m, 1H), 4.30e4.24 (m, 1H), 4.15e
4.12 (m. 2H), 3.71e3.65 (m, 8H), 3.35 (s, 3H), 3.04e2.98 (m, 2H),
2.95 (s, 3H), 1.58e1.56 (d, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz,
Compound 33 was obtained from 29 and diethylamine (8.0 mg,
0.11 mmol) according to the similar procedure used to prepare 30.
The crude product was purified by silica gel chromatography to
afford 33 (57 mg, 81.0%). 1H NMR (300 MHz, CD3OD):
d
8.11 (s, 1H),
CDCl3)
d
165.98, 164.62, 163.50, 160.67 (d, J ¼ 245 Hz, 1C), 142.12,
7.98 (s, 2H), 7.90 (s, 1H), 7.72 (s, 1H), 7.45e7.40 (m, 2H), 7.27e7.19
(m, 4H), 7.16e7.08 (m, 1H), 7.04e7.03 (t, J ¼ 8.7 Hz, 2H), 5.24e5.22
(q, J ¼ 6.9 Hz, 1H), 4.42e4.37 (m, 1H), 4.31e4.25 (m, 1H), 4.18e4.13
(m, 2H), 3.50 (s, 4H), 3.35 (s, 3H), 3.05e2.98 (m, 2H), 2.97 (s, 3H),
1.58e1.56 (d, J ¼ 6.9 Hz, 3H), 1.20 (s, 6H); 13C NMR (100 MHz,
139.20, 138.67, 137.38, 135.71, 135.27, 132.86, 129.24, 128.67,
127.95, 127.61, 126.77, 123.98, 116.26, 115.56, 115.34, 70.49,
66.67, 55.92, 55.87, 53.92, 49.05, 45.74, 37.88, 35.38, 21.69;
LCMS (ESI): 721.3 [M þ H]þ; HRMS: calcd for C36H41N6O7FSNa
15
743.2639, found C36H41N6O7FSNa 743.2684; [
0.21, CHCl3).
a
]
¼ ꢂ100.5ꢀ (c
D
CDCl3):
d
166.16, 164.85, 163.95, 163.08 (d, J ¼ 245 Hz, 1C), 142.05,
138.89, 138.70, 137.58, 135.87, 135.27, 133.03, 129.24, 128.58, 127.93,
127.85, 126.65, 124.07, 117.25, 115.43, 115.22, 70.48, 55.92, 54.03,
50.60, 49.01, 37.87, 37.72, 35.53, 21.65, 12.99; LCMS (ESI): 707.2
4.2.23. N1-((2S,3S)-4-(4-(Azepane-1-carbonyl)-1H-pyrazol-1-yl)-
3-hydroxy-1-phenylbutan-2-yl)-N3-((R)-1-(4-fluorophenyl)ethyl)-
5-(N-methylmethylsulfonamido)isophthalamide (37)
[M þ H]þ; HRMS: calcd for C36H43N6O6FNaS 729.2847, found
15
C36H43N6O6FNaS 729.2834; [
a
]
¼ ꢂ26.3ꢀ (c 0.40, CHCl3).
Compound 37 was obtained from 29 and azepane (11.0 mg,
0.11 mmol) according to the similar procedure used to prepare 30.
The crude product was purified by silica gel chromatography to
D
4.2.20. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(4-(pyrrolidine-1-carbonyl)-1H-pyrazol-1-yl)butan-2-
yl)-5-(N-methylmethylsulfonamido)isophthalamide (34)
afford 37 (59 mg, yield: 81.2%). 1H NMR (300 MHz, CD3OD):
d 8.12
(s, 1H), 7.98 (s, 2H), 7.90 (s, 1H), 7.73 (s, 1H), 7.45e7.40 (m, 2H),
7.25e7.20 (m, 4H), 7.16e7.14 (m, 1H), 7.08e7.03 (t, J ¼ 8.4 Hz, 2H),
5.24e5.22 (q, J ¼ 6.9 Hz, 1H), 4.40e4.35 (m, 1H), 4.30e4.25 (m,
1H), 4.16e4.12 (m, 2H), 3.70e3.66 (t, J ¼ 6.0 Hz, 2H), 3.62e3.58 (t,
J ¼ 6.0 Hz, 2H), 3.35 (s, 3H), 3.04e2.98 (m, 2H), 2.95 (s, 3H), 1.77
(s, 4H), 1.58 (s, 4H), 1.58e1.56 (d, J ¼ 6.9 Hz, 3H); 13C NMR
Compound 34 was obtained from 29 and pyrrolidine (8.0 mg,
0.11 mmol) according to the similar procedure used to prepare 30.
The crude product was purified by silica gel chromatography to
afford 34 (52 mg, 74.1%). 1H NMR (300 MHz, CD3OD):
d 8.12e8.10
(m, 2H), 7.99 (s, 1H), 7.89 (s, 1H), 7.85 (s, 1H), 7.45e7.40 (m, 2H),
7.25e7.20 (m, 4H), 7.16e7.14 (m, 1H), 7.08e7.03 (t, J ¼ 8.7 Hz, 2H),
5.24e5.22 (q, J ¼ 6.9 Hz, 1H), 4.40e4.38 (m, 1H), 4.30e4.27 (m, 1H),
4.16e4.13 (m, 2H), 3.69e3.68 (m, 2H), 3.56e3.51 (t, J ¼ 6.9 Hz, 2H),
3.35 (s, 3H), 3.04e2.98 (m, 2H), 2.95 (s, 3H), 1.99e1.91 (m, 4H),
(100 MHz, CDCl3):
d
166.10, 164.82, 164.56, 163.09 (d, J ¼ 245 Hz,
1C), 142.06, 139.09, 138.90, 137.56, 135.86, 135.28, 133.23, 129.25,
128.60, 127.94, 127.72, 126.66, 124.05, 117.35, 115.44, 115.23, 70.46,
55.98, 53.99, 49.06, 46.43, 37.87, 35.53, 29.63, 27.24, 26.71, 21.67;
1.58e1.56 (d, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d
165.98,
LCMS (ESI): 733.4 [M þ H]þ; HRMS: calcd for C38H46N6O6FS
15
164.80, 163.06, 162.50 (d, J ¼ 245 Hz, 1C), 142.03, 139.48, 138.92,
137.57, 135.79, 135.29, 133.67, 129.24, 128.58, 127.86, 127.73, 126.64,
124.15,117.85, 115.41, 115.20, 70.47, 56.16, 53.88, 49.00, 48.32, 46.92,
37.87, 35.53, 23.95, 21.65; LCMS (ESI): 705.4 [M þ H]þ; HRMS: calcd
733.3184, found C38H46N6O6FS 733.3148; [
CHCl3).
a
]
¼ ꢂ46.0ꢀ (c 0.20,
D
4.3. Enzyme-based assay of BACE1 and cathepsin D
for C36H42N6O6FS 705.2871, found C36H42N6O6FS 705.2884;
15
[
a]
¼ ꢂ50.4ꢀ (c 0.25, CHCl3).
Recombinant human -secretase ectodomain (amino acid resi-
b
D
dues 1e460) 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-
4.2.21. N1-((R)-1-(4-Fluorophenyl)ethyl)-N3-((2S,3S)-3-hydroxy-1-
phenyl-4-(4-(piperidine-1-carbonyl)-1H-pyrazol-1-yl)butan-2-yl)-
5-(N-methylmethylsulfonamido)isophthalamide (35)
Compound 35 was obtained from 29 and piperidine (9.0 mg,
0.11 mmol) according to the similar procedure used to prepare 30.
The crude product was purified by silica gel chromatography to
EDANS (SynPep Corp, USA). In a typical 100
containing 100 mM ammonium acetate, pH 4.0, 20
m
L assay mixture
mM substrate,
and 50 nM purified recombinant human BACE1/Fc, the enzyme
activity 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.
afford 35 (56 mg, 78.2%). 1H NMR (300 MHz, CD3OD):
d 8.12 (s, 1H),
7.98 (s, 1H), 7.95 (s, 1H), 7.89 (s, 1H), 7.68 (s, 1H), 7.45e7.40 (m, 2H),
7.27e7.20 (m, 4H), 7.16e7.14 (m, 1H), 7.08e7.03 (t, J ¼ 8.1 Hz, 2H),
5.25e5.22 (q, J ¼ 7.5 Hz, 1H), 4.40e4.39 (m, 1H), 4.30e4.24 (m, 1H),
4.15e4.12 (m, 2H), 3.66e3.62 (m, 4H), 3.35 (s, 3H), 3.06e2.97 (m,
2H), 2.95 (s, 3H),1.70e1.60 (m, 6H),1.58e1.56 (d, J ¼ 7.5 Hz, 3H); 13C
Cathepsin D activity was measured in 100 mM formate buffer,
pH 3.1, using Mca-GKPILFFRLK(DNP)-D-R-NH2 (2
mM). Test sub-
NMR (100 MHz, CDCl3):
d
166.11, 164.77, 163.43, 163.13 (d,
stances were serially diluted from 10 M to 10 nM, inhibition assays
m
J ¼ 245 Hz, 1C), 142.09, 138.93, 137.57, 135.84, 135.30, 132.81, 129.25,
128.61, 127.95, 127.88, 127.79, 126.68, 124.04, 116.87, 115.48, 115.27,
70.50, 57.62, 55.92, 54.01, 49.01, 37.91, 37.77, 35.57, 26.35, 24.43,
were done in black 96-wellplates (Costar) by adding the respective
enzyme in assay buffer. Plates were incubated at room temperature
for 1 h, and residual enzymatic activity was measured after addition
of substrate in a Spectramax Gemini fluorescence plate reader
(Molecular Devices, Sunnyvale, CA). IC50 values were calculated
using the Microsoft Excel extension XL-fit.
21.68; LCMS (ESI): 719.3 [M þ H]þ; HRMS: calcd for C37H44N6O6FS
15
719.3027, found C37H44N6O6FS 719.3014; [
a
]
¼ ꢂ44.1ꢀ (c 0.27,
D
CHCl3).

282
Y. Zou et al. / European Journal of Medicinal Chemistry 68 (2013) 270e283
4.4. Cellular A
b
lowering assay in APP transfected HEK293
20 mM TriseHCl, pH 7.5. In order to obtain crystals with different
protein packing patterns, two mutations, K75A and E77A, were
introduced into BACE1 together.
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,
The active BACE1 monomer in the elution buffer was concen-
trated to 8e10 mg/mL. Cocrystallization of compound 12, 20 and 28
with mutated BACE1 was performed at room temperature using the
hanging drop vapor-diffusion method, by mixing the solution of the
protein-compound complex with an equal volume of a precipitant
solution, 1.7 M Li2SO4/100 mM HEPES, pH 7.5. The protein-
compound complex was prepared by adding the compound to
the protein solution to reach a final concentration of 0.5 mM ligand.
The perfluoropolyether, PFO-X175/08 (Hampton Research), was
used as cryoprotectant for all the crystals.
was used to examine the effects of the compounds on BACE1 ac-
tivity. HEK293sw cells were seeded into 6-well plates with a total
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 con-
centrations of the compounds were added to the cultures and
incubated for 24 h at 37 ꢀC and 5% CO2. The conditioned medium
was removed from the culture wells and Ab1e40 peptide levels in
the media were analyzed by Human
b Amyloid 1e40 Colorimetric
Elisa kit (BioSource International, Inc. California, USA) after 24 h.
4.6. Structure determination and refinement
Data were collected at 100 K on beamline BL17U (at wavelength
0.9793 A) at the Shanghai Synchrotron Radiation Facility (SSRF)
4.5. Protein purification and crystallization
ꢀ
(Shanghai, China) for the co-crystallized structures. The data were
processed with the HKL2000 [22], software packages, and the
structures were then solved by molecular replacement, using the
CCP4 program MOLREP [23]. The search model used for the crystals
was the apo wild type BACE1 structure (PDB ID code: 3TPL). The
structures were refined using the CCP4 program REFMAC5 [23]
combined with the simulated-annealing protocol implemented in
the program PHENIX [24]. With the aid of the program Coot [25],
compound, water molecules, and others were fitted into to the
initial FoeFc maps. The complete statistics, as well as the quality of
the solved structures, are shown in Table 4.
A detailed description of production of recombinant human
BACE1 has been done in our previous publication [21]. Briefly,
BACE1 was expressed in Escherichia coli as inclusion bodies that
were then denatured and refolded into the active monomer. A
cDNA fragment encoding BACE1 residues 43e454 was cloned into
pET28a with a TEV protease cleavage site following a six-residue
His-Tag added at the N-terminus. After refolding, nickel beads (Ni
SepharoseÔ High Performance, Amersham Biosciences, Uppsala,
Sweden) were used to concentrate the protein. The eluted BACE1
was then injected into a 124 mL Hiload Superdex75 column from
which it was eluted with 1 mM DTT/0.5 M urea/150 mM NaCl/
Acknowledgments
Table 4
Data collection and refinement statistics.
This work was financially supported by the National S&T Major
Projects “Key New Drug Creation and Manufacturing Program” of
China (Grant Nos. 2009ZX09301-001, and 2009ZX09501-010), the
“100 Talents Project” of CAS (to Y. X.), the Shanghai Pujiang Program
(Grant No. 10PJ1412000), and the National Natural Science Foun-
dation of China (Grant No. 21172233).
Compound 12
Compound 20
Compound 28
Pdb code
3UQX
C2221
3UQU
C2221
3UQW
C2221
Space group
Cell dimensions
ꢀ
a (A)
104.55
128.28
76.39
104.47
128.24
76.18
107.32
131.54
78.54
ꢀ
b (A)
ꢀ
c (A)
Appendix A. Supplementary data
ꢀ
Wavelength (A)
0.9792
0.9791
1.0089
Reflectionsa
56,267 [53,400] 56,630 [53,726] 28,642 [27,042]
1.70e81.04
1.70e1.73
ꢀ
Resolution range (A)
Highest-resolution
1.70e80.99
1.70e1.73
2.20e83.15
2.20e2.24
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.027.
ꢀ
shell (A)
Redundancyb
13.5 (7.2)
11.2 (8.1)
10.6 (9.8)
I/
s
(I)b
27.7 (3.67)
99.0 (89.2)
15.89/18.62
40.99 (7.54)
99.7 (97.6)
16.32/19.50
11.53 (4.92)
100.0 (100.0)
17.51/20.69
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