Discovery of small molecular (d)-leucinamides as potent, Notch-sparing γ-secretase modulators

Article abstract of DOI:10.1016/j.ejmech.2014.04.006

Structural optimization of the prior lead 3 led to the small molecular (d)-leucinamides with potent modulating activity and Notch-sparing selectivity on the proteolytic processing of amyloid-β precursor proteins. The N-(R)-epoxypropyl analog 10c exhibited

Full text of DOI:10.1016/j.ejmech.2014.04.006

European Journal of Medicinal Chemistry 79 (2014) 143e151
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of small molecular (
D
)-leucinamides as potent,
Notch-sparing -secretase modulators
g
,
Yung-Feng Liao b, Yu-Cheng Tang a, Ming-Yun Chang b, Bo-Jeng Wang b, Ming-Kuan Hu a
*
a School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan
b Laboratory of Molecular Neurobiology, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural optimization of the prior lead 3 led to the small molecular (
D
)-leucinamides with potent
precursor
-secretase modulation compared to DAPT
and showed substantial substrate selection for APP cleavage over Notch cleavage, while N-(2-fluoro)
benzyl analog 10e showed the most potent -secretase inhibition with dull selectivity. The exceptional
suppression of ERK-mediated activation suggested that these potent -secretase modulators may adapt
an alternative pathway to prominently induce the differential inhibition of C99 cleavage by -secretase.
Received 15 August 2013
Received in revised form
22 March 2014
Accepted 4 April 2014
Available online 4 April 2014
modulating activity and Notch-sparing selectivity on the proteolytic processing of amyloid-
proteins. The N-(R)-epoxypropyl analog 10c exhibited potent
b
g
g
g
g
Keywords:
Alzheimer’s disease
Ó 2014 Elsevier Masson SAS. All rights reserved.
Amyloid-b precursor protein
g-Secretase
Notch-sparing selectivity
1. Introduction
data for advanced investigations [8]. Intriguingly, an amino acid
derivative (
hibitor of amyloid synthesis by an A
Generally, these amino acid-derived
three universal components on the basis of structural features: an
aryl group, a sulfonamide moiety, a small alkyl side-chain. There-
fore, attention to the structural optimization on the core fragments
can be worth to pay for developing new modulators for AD
therapies.
D
)-leucinamide 3 was reported to be a nanomolar in-
lowering cellular assay [9].
-secretase inhibitors contain
Alzheimer’s disease (AD) has been characterized as a progres-
sively neurodegenerative disorder with the deposition of amyloid
plaques and intracellular neurofibrillary tangles in the brain [1,2].
Amyloid plaques are predominately composed of 40e42 amino
b
g
acid amyloid-
cleavage of amyloid-
tases [3]. According to the amyloid hypothesis of AD, the generation
and oligomerization of A that results in neuronal insults is the
ultimate cause of the disease [4]. In addition to the APP as the
known substrate, -secretase is also involved in controlling and
b
protein (Ab), which is produced from sequential
b
precursor protein (APP) by
b
- and -secre-
g
b
Contrary to the
age of APP, -secretase was thought to mediate a beneficial cleavage
of APP and generate a neuroprotective, soluble amyloid precursor
protein (sAPP ) in addition to the C-terminal fragment C83, thus
preclude amyloidogenic A production [10]. This -secretase-
b- and g-secretase-mediated proteolytic cleav-
a
g
differentiation in the Notch signaling pathway, a pivotal step that
mediates differentiation in embryonic cells during the develop-
mental stage. Selectivity for APP processing over Notch processing
to avoid certain Notch-related side effects was thus demanded in
a
a
b
a
mediated APP cleavage was thought through downstream activa-
tion from mitogen-activated protein kinases (MAPKs) signaling
the identity of a
treatment of AD [5]. Numerous contributions demonstrated that
certain small molecular arylsulfonamides are potent and selective
g
-secretase modulator targeting the chronic
pathway [11,12]. Accordingly, decreased A
by neuronal protection would be accessed by selective
modulators with ERK-mediated activation, which decrease
secretase-mediated proteolytic processing of APP and alternatively
augment sAPP release [13,14]. From the molecular mechanism
point of view, we planned to develop a type of new modulators of
proteolytic processing of APP as potential agents for Alzheimer’s
therapy [15].
b
formation accompanied
-secretase
-/
g
b
g-
g-secretase inhibitors such as GSI-953 (begacestat, 1) and BMS-
708163 (avagacestat, 2) as shown in Fig. 1 [6,7]. Both compounds
have been in clinical trials in AD patients, while avagacestat was
recently terminated after phase II trials due to lack of supported
a
In our studies to identify new modulators on the proteolytic
processing of APP for AD therapies, we have reported a type of
propargylamine-related small molecules, which exhibited both
* Corresponding author.
E-mail address: hmk@ndmctsgh.edu.tw (M.-K. Hu).
http://dx.doi.org/10.1016/j.ejmech.2014.04.006
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

144
Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
Scheme 2. i) 36% HCl, 37% HCHO, HCl(g), 0 ꢀC.
Fig. 1. Chemical structures of GSI-953 (1), BMS-708163 (2), and a (D)-leucinamide 3.
substitution at the ortho- and para-positions, or difluoro substitu-
tion at the meta-position on the N-benzyl ring gain significant
potency on g-secretase inhibition. They showed 92e103% activity
g
-secretase inhibition activities and ERK-mediated signaling effects
[16]. This stimulated our interest in designing and synthesizing of a
series of small molecular N- arylsulfonyl-( )-leucinamides with
of DAPT and maintained similar potency to the lead compound 3.
These results also indicated that most of the N-(p-chlor-
obenzenesulfonyl) analogs showed slightly more potency than the
D
optimization of the additional N-substitution that led to the dis-
covery of potent modulators with Notch-sparing selectivity on the
proteolytic processing of APP.
corresponding
N-(5-chlorothiophenesulfonyl)
compounds
(i.e.10eeg vs. 11eeg). However, it was noticed that the N-(5-
chlorothiophenesulfonyl) substituted compound 11i is about 3%
more potent than compound 3. The bigger, polar substitution was
clearly disfavored as seen with the N-(8-hydroxy-2-methyl-qui-
nolinyl)methyl analogs 10h and 11h. They were around 5-fold less
potent than the analogs with small, neutral N-benzylic sub-
stitutions, such as 10eeg and 11eeg. Taken together, it is clear that
2. Chemistry
The synthesis of N-substituted N-arylsulfonyl-(D)-leucinamides
was described in Scheme 1. ( )-Leucinamide (4) was coupled to p-
D
chlorobenzenesulfonyl chloride (5) or 5-chlorothiophene-2-
sulfonyl chloride (6) in the presence of Hunig’s base to give the
both N-arylsulfonyl moieties in these substituted (
D
)-leucinamides
are the effective core components for exhibiting
g
-secretase inhi-
corresponding arylsulfonamides
condensed with selected alkyl halides 9aeh in basic conditions to
yield a type of N-substituted ( )-leucinamides 3, 10aeh, and 11aei
in modest yields. For the preparation of the selected alkyl halide, 5-
chloromethyl-2-methyl-8-quinolinol (9h), a solution of 2-methyl-
8-quinolinol (12), concentrated hydrochloride, and 37% formalde-
hyde at an ice bath was treated with gaseous hydrogen chloride for
6 h to afford the desired halide 9h in 48% yield (Scheme 2).
7 and 8, which were each
bition activity and N-fluorobenzyl analogs were generally equi-
potent as seen with 10eeg and 11eeg.
A type of propargylamine-containing small molecules have
been proven to be effective to provide neuroprotective features
through ERK-mediated signaling pathways as previously described
[11,19]. Following the established features, certain N-propargyl-
D
related analogs of (
couple some of the beneficial pharmacological actions. Accordingly,
small molecular ( )-leucinamides 10aed and 11aed, which contain
N-propargyl, -cyclopropyl, and -epoxypropyl moieties were syn-
thesized. As expected, the initial evaluation on modulating the
secretase-mediated APP processing showed that the N-propargyl
analogs 10b and 11b preserved potent -secretase inhibition with
D)-leucinamide were envisioned that would
D
3. Results and discussion
g
-
3.1. Evaluation of selective modulating activity on the
mediated C99 and S3 cleavage
g-secretase-
g
94e99% activity of DAPT, while N-cyclopropyl analogs 10a and 11a
maintained 88e98% potency. For the (R)- and (S)-epoxypropyl an-
alogs, compounds 10c and 10d bearing the N-chlor-
To determinate the modulation of
age of C99 (C99-GL-T20) for the synthesized N-substituted (
leucinamides, the highly efficient and quantitative cell-based
luciferase reporter gene assays as described previously were used
g
-secretase-mediated cleav-
D
)-
obenzenesulfonyl substituent were clearly more favorable for
secretase inhibition than the corresponding chlor-
othiophenesulfonyl analogs 11c and 11d. These results revealed
that N-arylsulfonyl-( )-leucinamides incorporated with N-par-
pargyl-related small moieties can recapitulate activity on -secre-
tase inhibition. All of the synthesized N-substituted )-
g-
[17]. The effects of these synthesized (
either p-chlorobenzenesulfonyl or 5-chlorothiophene-2-sulfonyl
moieties on the -secretase modulation are shown in Table 1.
D)-leucinamides containing
D
g
g
(D
First, the screening showed evidence that the lead compound 3 and
its thiophenesulfonyl analog 11i bearing N-piperonyl substituent
leucinamides, with only the exception of 11i, presented no signif-
icant cellular toxicity as shown in Fig. 2.
exhibited potent inhibition of
g-secretase, which showed almost
We next examined whether these N-arylsulfonyl-(
mides can circumvent the -secretase-mediated S3 cleavage of
Notch, which would reflect the selectivity for the -secretase-
D)-leucina-
equal potency to the prominent DAPT in modulating
mediated C99 cleavage [18]. Intriguingly, all the synthesized N-
fluorobenzyl compounds 10eeg and 11eeg proved effective in
g-secretase-
g
g
mediated proteolytic processing of APP over Notch. Accordingly,
their influence on the processing of S3 cleavage of Notch was
measured by using a stable cell line (NG) constitutively expressing
establishing that the (D)-leucinamides with either monofluoro
N
D
E as previously reported [17]. The relative potency of
tase-mediated S3 cleavage of Notch referred as fold of activation for
luciferase gene expression by the synthesized N-arylsulfonyl ( )-
leucinamides was described in Fig. 3. The results indicated that all
of the effective -secretase inhibitors with the exception of 10c
were shown to significantly block -secretase-mediated S3 cleav-
age of N E in NG cells, suggesting that these compounds were
consequentially lack of selectivity for -mediated processing of APP
over Notch. We found that 62% activity of the -secretase were
g-secre-
D
g
g
D
g
Scheme 1. i) diisopropylethylamine, 0 ꢀC e r.t. ii) alkyl halides (9aei), K2CO3, r.t.
g

Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
145
Table 1
Inhibition of
g
-secretase-mediated C99 cleavage by the synthesized N-arylsulfonyl-(
D
)-leucinamides using a luciferase reporter gene assay. The production of T20 cells has
g/mL hygromycin, 5 g/mL blasticidin, and 250 g/mL zeocin (DMEM-
been reported previously [20]. T20 cells were maintained in DMEM supplemented with 10% FBS, 200
m
m
m
HZB). The stably transfected T20 cells were detached from culture dishes by trypsinization, washed with PBS, and resuspended in DMEM-HZB, followed by plating onto 96-well
microplates (2 ꢁ 104 cells/50
m mM in the presence of
L/well) and incubating at 37 ꢀC for 24 h. Tested compounds diluted in DMEMHZB were added to a final concentration of 10
tetracycline (1 m
g/mL). Treatments were terminated after incubation at 37 ꢀC for 24 h by directly adding an equivalent volume of the Steady-Glo luciferase assay reagent
(Promega, Madison, WI, USA), and luciferase signals from each well were performed immediately with the luminescence plate reader available in our Institute. Triplicates of
each compound treatment were assayed. Luciferase signals from the stable line without tetracycline induction and tested compound treatment was referred to as one-fold of
activation. Parallel testing with a cell line constitutively expressing only the luciferase reporter gene (e.g., under control of a CMV promoter) was performed as a control panel.
Compound 10
R
Reduction of C99 cleavage (%)
Compound 11
R
Reduction of C99 cleavage (%)
a
88.5 ꢂ 1.5
a
98.9 ꢂ 1.2
b
c
99.2 ꢂ 1.0
89.7 ꢂ 1.2
63.4 ꢂ 1.6
b
c
94.3 ꢂ 1.6
39.2 ꢂ 3.0
22.9 ꢂ 4.2
d
d
e
f
103.1 ꢂ 1.2
98.9 ꢂ 1.0
e
f
99.6 ꢂ 3.0
93.1 ꢂ 2.5
g
97.3 ꢂ 1.0
23.7 ꢂ 1.4
g
92.1 ꢂ 1.0
h
h
i
20.2 ꢂ 1.3
3
98.9 ꢂ 2.0
101.9 ꢂ 1.0
DAPT
100.0 ꢂ 1.0
maintained under the treatment of 10
analog 10c to mediate the processing of S3 cleavage, while DAPT
was shown to completely block the -secretase-mediated proteo-
lytic process. The (S)-isomer 10d exhibited a little better selectivity
than 10c, but it was less active for the inhibition of the -secretase-
mediated C99 cleavage. Similarly, other N-epoxypropyl analogs 11c
and 11d and compounds 10h and 11h did not influence the
secretase-mediated processing of S3; they were also lack of inhi-
bition activity on -secretase-mediated C99 processing as
described above. Apparently, the N-(R)-epoxypropyl analog 10c
was the best potent and Notch-sparing -secretase modulator
among the synthesized N-substituted ( )-leucinamides.
m
M of N-(R)-epoxypropyl
propargylamine. The way to incorporate the N-propargyl-related
moieties to the core ( )-leucinamides did not provide additive
pharmacologic effects as we first proposed to. The results suggested
that these -secretase modulators were lack of beneficial effects on
stimulating -secretase activity; on the contrary, they might
slightly boost the overall -secretase activity (based on our previ-
ous data). The exceptional actions on ERK-mediated signaling
possibly posed an alternative pathway for these modulators to
induce the differential inhibition of C99 cleavage as they all
D
g
g
g
a
g
g-
g
exhibited strong g-secretase inhibition activity. Nevertheless, only
g
the N-(R)-epoxypropyl analog 10c showed substantial Notch-
sparing selectivity as described above, the N-(R)-epoxypropyl
moiety in the compound 10c may play crucial roles in the differ-
D
ential modulation and substrate selectivity on the
mediated processing of C99 cleavage.
g-secretase-
3.2. Modulation of ERK-mediated signaling
In the following step, we sought to determine whether these
compounds can significantly modulate the MEK-ERK signal
4. Conclusion
pathway because the
a-secretase-mediated processing of APP
would be slightly enhanced directly or indirectly through the
activation of MEK-pathway by certain N-propargyl-related com-
pounds as previously described. Thus, the relative ERK activation
In this study, we designed and synthesized a type of small
molecular ( )-leucinamides and investigated their selective inhi-
bition activities on the -secretase-mediated proteolytic processing
D
g
(p-ERK/ERK, %) of the selected
10e were further examined in
[16,19]. The results were shown in Fig. 4 and indicated that all the
selected -secretase inhibitors unexpectedly suppressed ERK acti-
vation compared to the prevailing ERK-activated rasagiline and
g
-secretase inhibitors 3,10b,10c, and
between APP and Notch, and their modulation on the ERK-signaling
pathway. The results demonstrated that the small molecular N-(R)-
g
-30 cells as previously described
epoxypropyl-(
hibition with promising selectivity on the
APP cleavage over Notch cleavage, while other potent analogs were
D
)-leucinamide 10c exhibited potent
g-secretase in-
g
g-secretase-mediated

146
Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
Fig. 2. Viability of host cells in the presence of the synthesized N-arylsulfonyl-(D)-
leucinamides. T20 cells (5 ꢁ 104/100
m
L/well) were seeded onto the wells of 96-well
microplates in culture medium containing 10
m
M of respective compounds and incu-
bated at 37 ꢀC for 24 h. Viable cells were determined using the CellTiter 96Ò Aqueous
Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) as specified in
the manufacturer’s instructions. Briefly, following the addition of the combined MTS/
PMS solution (20 m
L/well) microplates were incubated for 3 h at 37 ꢀC. The conversion
Fig. 4. The modulation of ERK-mediated signaling assay for the selected (D)-leucina-
mides and the western blot images from 3 independent assays. The stimulation of the
ERKs was measured by using anti-phospho-ERK1/2 and anti-ERK1/2 antibodies (Cell
of MTS into formazan in viable cells was quantitated by the absorbance at 490 nm
using a Synergy HT ELISA plate reader (BioTek, Winooski, VT, USA). The number of
living cells in culture was directly proportional to the absorbance at 490 nm. Viable
cells in culture medium containing vehicle alone (1% DMSO, Control) were referred to
as 100% viability. The background absorbance shown at devoid of cells was subtracted
from these data.
Signaling Technology, Danvers, MA, USA) as previously described [16]. Briefly,
g-
30 cells were grown in six-well plates at 5 ꢁ 105 cells/well and incubated with culture
medium at 37 ꢀC for 18 h, followed by an additional incubation with culture medium
containing 1
the medium with DMEM containing 0.5% FBS and treated it with the tested com-
pounds (10 M each). After treatment, reactions were stopped by placing cells on ice
m
g/mL tetracycline at 37 ꢀC for 18 h. Before the experiments, we replaced
m
and aspirating the medium. Cells were harvested and lysed in 50 mM TriseHCl (pH
8.0), 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 1% Triton X-100, and
protease- and phosphatase-inhibitor cocktails. Protein concentration was determined
by the BCA assay (Pierce, Rockford, IL, USA). Each cell lysate, which contained 50 mg
proteins, was separated on 12% SDS-polyacrylamide electrophoresis gels, immuno-
blotted, and identified using anti-phospho-ERK1/2 or anti-ERK1/2 antibody.
obviously devoid of substrate selection. The dual actions on
exhibiting potent
ERK activation suggest that these potent
may adapt an alternative pathway to induce the differential inhi-
bition of C99 cleavage by -secretase. It is worth to pursue the
g-secretase inhibition and inversely suppressing
g-secretase modulators
g
relative epoxypropyl-containing compounds further to see if it is a
prerequisite entity for the selective modulation of C99 cleavage.
5. Experimental section
5.1. Chemistry
All chemicals and solvents were commercial materials and were
ꢀ
used directly unless otherwise noted. DMF was dehydrated over 4 A
molecular sieves. Reactions were monitored by thin layer chro-
matography using Echo silica gel F254 plates visualized under UV
irradiation along with staining with phosphomolybdic acid/heat, or
iodine. Melting points were determined with a Thomas Hoover
capillary melting point apparatus in open capillary tubes and were
uncorrected. A Varian Gemini-300 spectrometer was employed for
1H and 13C NMR spectra with tetramethylsilane or chloroform as an
internal reference. Fast atom bombardment mass spectra (FABMS)
were acquired on a Finnigan Mat 95S mass spectrometer. Chro-
matography refers to flash chromatography on silica gel (silica gel
60, 230e400 mesh ASTM, E. Merck, Darmstadt, Germany).
Fig. 3. Inhibition of
(D)-leucinamides using a luciferase reporter gene assay. A HEK293-derived stable cell
line (N7) that was constitutively expressing N E was used to examine the inhibition of
-secretase-mediated S3 cleavage of Notch by the tested compounds as previously
g-secretase-mediated S3 cleavage of Notch by the N-arylsulfonyl-
D
g
described [20]. N7 cells were plated onto12-well microplates in 1 mL/well DMEM
supplemented with 10% FBS at 5 ꢁ 105 cells/well. After incubation at 37 ꢀC overnight,
cells were treated with compounds at 10 m C
M as described above and incubated at 37 ꢀ
for 24 h. Treated cells were harvested using PBS containing 20 mM EDTA and dissolved
in 50
m
L of 1ꢁ PLB, followed by centrifugation at 13,200g for 5 min to remove cell
debris. The protein concentrations of clarified supernatants were determined using a
BCA protein assay reagent kit, and cell extracts containing equivalent amounts of
proteins were resolved by SDS-PAGE and analyzed by Western blotting using an anti-
Notch(Val1744) polyclonal antibody.

Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
147
5.1.1. N-(4-Chlorobenzenesulfonyl)-(
To mixture of diisopropylethylamine (DIPEA, 0.53 mL,
3.0 mmol) and ( )-leucinamide hydrochloride (4,170 mg, 1.0 mmol)
D
)-leucinamide (7)
H2), 0.79 (3H, d, J ¼ 6.6 Hz, CH3), 0.81 (3H, d, J ¼ 6.6 Hz, CH3), 0.98e
1.08 (1H, m, cyclopropane-H), 1.17e1.26 (1H, m, CHH), 1.38e1.45
(1H, m, CHH), 1.87e1.97 (1H, m, CH3CHCH3), 3.15 (2H, dd, J ¼ 6.9,
1.5 Hz, NCH2), 4.28 (1H, t, J ¼ 7.2 Hz, COCH), 5.91 (1H, br, NH), 6.51
(1H, br, NH), 6.91 (2H, d, J ¼ 3.9 Hz, thiophene-H), 7.39 (2H, d,
a
D
in CH2Cl2 (10 mL) was added slowly 4-chlorobenzenesulfonyl
chloride (5, 250 mg, 1.2 mmol). The resulting mixture was stirred
at room temperature overnight, then diluted with CH2Cl2, washed
with H2O, and dried over MgSO4. The resulting residue was purified
by silica gel chromatography to give 7 (258 mg, 85%) as a white
powder; tlc Rf ¼ 0.55 (EtOAc/n-hexane ¼ 1 : 1); UV lmax (MeOH) nm
J ¼ 3.9 Hz, thiophene-H); 13C NMR (CDCl3)
d 4.6, 5.6, 11.2, 22.3, 22.4,
24.8, 29.6, 37.3, 49.9, 57.5, 126.5, 131.8, 137.3, 138.8, 172.9; FABMS:
365 [M þ H]þ; HRFABMS: Calcd for C14H22N2O3S2Cl [M þ H]þ
365.0763; found 365.0764.
(log ε): 256 (0.244); mp 228e229 ꢀC; [
a
]
D
þ3.7ꢀ (c 0.003, MeOH);
cLogP ¼ 2.56; 1H NMR (DMSO-d6)
d
0.70 (3H, d, J ¼ 6.6 Hz, CH3),
0.80 (3H, d, J ¼ 6.6 Hz, CH3), 1.27e1.38 (2H, m, CHCH2), 1.55e1.50
(1H, m, CH3CHCH3), 3.64 (1H, t, J ¼ 7.2 Hz, COCH), 6.88 (1H, br, NH),
7.27 (1H, br, NH), 7.61 (2H, d J ¼ 8.7 Hz, Ar-H), 7.76 (2H, d, J ¼ 8.7 Hz,
5.1.5. N-(4-Chlorobenzenesulfonyl)-N-propargyl-(D)-leucinamide
(10b)
Compound 10b was prepared from 7 (0.15 g, 0.5 mmol) and
propargyl bromide (9b, 0.09 mL, 1.0 mmol) following the same
protocol as for 10a. Purification by silica gel column chromatog-
raphy gave 10b (99 mg, 58%) as a yellow oil; tlc Rf ¼ 0.46 (EtOAc/n-
hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 285 (0.445);
Ar-H), 7.95 (1H, br, NH); 13C NMR (DMSO-d6)
d 21.7, 23.2, 24.3, 42.2,
55.0, 128.9, 129.4, 1378.4, 140.5, 173.3; FABMS: 305 [M þ H]þ.
5.1.2. N-(5-Chlorothiophene-2-sulfonyl)-( )-leucinamide (8)
D
To a mixture of diisopropylethylamine (0.53 mL, 3. 0 mmol) and
[
a
]
D þ33.3ꢀ (c 0.002, MeOH); cLogP ¼ 3.76; 1H NMR (CDCl3)
d 0.75
(D
)-leucinamide$HCl (4, 0.17 g, 1.0 mmol) in CH2Cl2 (10 mL) was
(3H, d, J ¼ 6.3 Hz, CH3), 0.78 (3H, d, J ¼ 6.6 Hz, CH3), 1.31e1.50 (2H,
m, CH2), 1.77e1.86 (1H, m, CH3CHCH3), 2.22 (1H, t, J ¼ 2.4 Hz, CCH),
4.04 (1H, dd, J ¼ 18.6, 2.4 Hz, NCHH), 4.30 (1H, t, J ¼ 7.5 Hz, COCH),
4.38 (1H, dd, J ¼ 18.6, 2.4 Hz, NCHH), 5.44 (1H, br, NH), 6.33 (1H, br,
added slowly 5-chlorothiophene-2-sulfonyl chloride (6, 0.26 g,
1.20 mmol). The resulting mixture was stirred at room temperature
overnight, and then diluted with CH2Cl2, washed with H2O, and
dried over MgSO4. The crude residue was purified by silica gel
chromatography to give 8 (267 mg, 86%) as a white powder; tlc
Rf ¼ 0.50 (EtOAc/n-hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 288
NH), 7.50 (2H, d, J ¼ 8.7 Hz, Ar-H), 7.88 (2H, d, J ¼ 8.7 Hz, Ar-H); 13
C
NMR (CDCl3)
d 21.9, 22.3, 24.5, 33.6, 37.7, 58.0, 73.1, 78.8, 129.0,
129.2, 138.0, 139.6, 172.6; FABMS: 343 [M þ H]þ; HRFABMS: Calcd
(1.583); mp 228e229 ꢀC;
[a
]
þ10.0ꢀ (c 0.002, MeOH);
0.70 (3H, d, J ¼ 6.6 Hz, CH3),
for C15H20N2O3SCl [M þ H]þ 343.0885; found 343.0884.
D
cLogP ¼ 2.34; 1H NMR (DMSO-d6)
d
0.80 (3H, d, J ¼ 6.6 Hz, CH3), 1.27e1.38 (2H, m, CHCH2), 1.50e1.55
(1H, m, CH3CHCH3), 3.64 (1H, t, J ¼ 7.2 Hz, COCH), 6.88 (1H, br, NH),
7.27 (1H, s, NH), 7.61 (2H, d J ¼ 8.7 Hz, thiophene-H), 7.76 (2H, d,
J ¼ 8.7 Hz, thiophene-H), 7.95 (1H, br, NH); 13C NMR (DMSO-d6)
5.1.6. N-(5-Chlorothiophene-2-sulfonyl)-N-propargyl-(D)-
leucinamide (11b)
Compound 11b was prepared form 8 (0.16 g, 0.5 mmol) and
propargyl bromide (9b, 0.09 mL, 1.0 mmol) using the procedure for
the synthesis of 10a. Purification by silica gel column chromatog-
raphy gave 11b (26 mg, 15%) as a yellow oil; tlc Rf ¼ 0.36 (EtOAc/n-
hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 288 (1.587);
d
21.7, 23.2, 24.4, 42.1, 55.2, 128.0, 131.8, 134.6, 141.0, 173.2; FABMS:
311 [M þ H]þ.
5.1.3. N-(4-Chlorobenzenesulfonyl)-N-cyclopropylmethyl-(D)-
leucinamide (10a)
[
a
]
D þ33.3ꢀ (c 0.002, MeOH); cLogP ¼ 3.12; 1H NMR (CDCl3)
d 0.86
To a mixture of 7 (0.30 g, 1 mmol) in DMF (6 mL) was added
potassium carbonate (0.83 g, 6.0 mmol), potassium iodide (0.01 g,
0.06 mmol), and (chloromethyl)cyclopropane (9a, 0.28 mL,
3.0 mmol) at room temperature. The mixture was stirred for 48 h,
poured into brine, then extracted with CH2Cl2. The organic layer
was separated, dried over MgSO4, and concentrated. The crude
residue was purified by silica gel column chromatography to give
10a (100 mg, 28%) as a yellow oil. tlc Rf ¼ 0.46 (EtOAc/n-hexane ¼ 1
(6H, d, J ¼ 6.3 Hz, CH3), 1.50e1.60 (2H, m, CH2), 1.80e1.89 (1H, m,
CH3CHCH3), 2.27 (1H, t, J ¼ 2.4 Hz, CCH), 4.11 (1H, dd, J ¼ 18.9,
2.7 Hz, NCHH), 4.21 (1H, dd, J ¼ 18.9, 2.7 Hz, NCHH), 4.35 (1H, t,
J ¼ 7.8 Hz, COCH), 5.49 (1H, br, NH), 6.20 (1H, br, NH), 6.94 (1H, d,
J ¼ 3.9 Hz, thiophene-H), 7.49 (1H, d, J ¼ 4.2 Hz, thiophene-H); 13
C
NMR (CDCl3) d 22.1, 22.3, 24.7, 33.9, 37.7, 48.1, 58.1, 73.3, 78.4, 126.7,
132.6, 137.9, 138.0, 172.2; FABMS: 349 [M þ H]þ; HRFABMS: Calcd
for C13H18N2O3S2Cl [M þ H]þ 349.0449; found 349.0448.
: 1); UV lmax (MeOH) nm (log ε): 288 (1.057); [
a
]
D þ24.1ꢀ (c 0.003,
MeOH); cLogP ¼ 3.76; 1H NMR (CDCl3)
d 0.26e0.36 (2H, m, cyclo-
propane-H2), 0.46e0.59 (2H, m, cyclopropane-H2), 0.72 (6H, d,
J ¼ 6.6 Hz, CH3), 0.98e1.15 (2H, m, cyclopropane-H, CHH), 1.25e1.35
(1H, m, CHH), 1.86e2.03 (1H, m, CH3CHCH3), 3.16 (2H, dd, J ¼ 6.6,
5.4 Hz, NCH2), 4.23 (1H, t, J ¼ 6.9 Hz, COCH), 5.57 (1H, br, NH), 6.67
(1H, br, NH), 7.49 (2H, d, J ¼ 8.7 Hz, Ar-H), 7.78 (2H, d, J ¼ 8.7 Hz, Ar-
5.1.7. N-(4-Chlorobenzenesulfonyl)-N-((R)-2,3-epoxypropyl)-(D)-
leucinamide (10c)
Compound 10c was prepared from 7 (0.61 g, 2.0 mmol) and (R)-
epichlorhydrin (9c, 0.47 mL, 6. 0 mmol) using the procedure for the
synthesis of 10a. Purification by silica gel column chromatography
gave 10c (216 mg, 30%) as a yellow oil; tlc Rf ¼ 0.37 (EtOAc/n-
H); 13C NMR (CDCl3)
d 4.5, 5.7, 11.2, 22.3, 24.6, 37.2, 49.8, 57.1, 128.6,
129.3, 138.7, 139.3, 173.1; FABMS: 359 [M þ H]þ; HRFABMS: Calcd
hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 284 (0.218); [
a
]
D ꢃ4.5ꢀ
for C16H24N2O3SCl [M þ H]þ 359.1198; found 359.1196.
(c 0.002, MeOH); cLogP ¼ 2.69; 1H NMR (CDCl3)
d 0.77 (3H, d,
J ¼ 6.6 Hz, CH3), 0.79 (3H, d, J ¼ 6.6 Hz, CH3), 1.10e1.18 (1H, m, CHH),
1.33e1.42 (1H, m, CHH), 1.82e1.91 (1H, m, CH3CHCH3), 2.60 (1H, dd,
J ¼ 4.5, 2.4 Hz, CHOCHH), 2.81 (1H, t, J ¼ 4.5 Hz, CHOCHH), 3.10e
3.16 (1H, m, CHOCHH), 3.31 (1H, dd, J ¼ 15.6, 6.3 Hz, NCHH), 3.54
(1H, dd, J ¼ 15.6, 4.2 Hz, NCHH), 4.32 (1H, t, J ¼ 7.5 Hz, COCH), 5.64
(1H, br, NH), 6.50 (1H, br, NH), 7.50 (2H, d, J ¼ 8.7 Hz, Ar-H), 7.83
5.1.4. N-(5-Chlorothiophene-2-sulfonyl)-N-cyclopropylmethyl-(D)-
leucinamide (11a)
Compound 11a was prepared from 8 (0.31 g, 1.0 mmol) and
(chloromethyl)cyclopropane (9a, 0.28 mL, 3.0 mmol) following the
procedure for the synthesis of 10a. Purification by silica gel column
chromatography gave 11a (29 mg, 8%) as a yellow oil; tlc Rf ¼ 0.30
(EtOAc/n-hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 288 (1.622);
(2H, d, J ¼ 8.7 Hz, Ar-H); 13C NMR (CDCl3)
d 22.2, 22.3, 24.6, 37.7,
46.8, 47.3, 50.7, 57.8, 128.8, 129.3, 138.1, 139.7, 172.2; FABMS: 361
[M þ H]þ; HRFABMS: Calcd for C15H22N2O4SCl [M þ H]þ 361.0991;
found 361.0990.
[
a
]
D þ15.1ꢀ (c 0.003, MeOH); cLogP ¼ 3.49; 1H NMR (CDCl3)
d 0.30e
0.34 (2H, m, cyclopropane-H2), 0.50e0.56 (2H, m, cyclopropane-

148
Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
5.1.8. N-(4-Chlorobenzenesulfonyl)-N-((S)-2,3-epoxypropyl)-(
leucinamide (10d)
D
)-
NMR (CDCl3)
d
0.71 (3H, d, J ¼ 6.6 Hz, CH3), 0.79 (3H, d, J ¼ 6.6 Hz,
CH3), 1.17e1.26 (1H, m, CHH), 1.28e1.37 (1H, m, CHH), 1.80e1.89
(1H, m, CH3CHCH3), 4.33 (1H, t, J ¼ 7.2 Hz, COCH), 4.47 (1H, d,
J ¼ 15.9 Hz, NCHH), 4.63 (1H, d, J ¼ 16.5 Hz, NCHH),5.18 (1H, br, NH),
6.28 (1H, br, NH), 6.96 (1H, t, J ¼ 9.6 Hz, Ar-H), 7.09 (1H, t, J ¼ 7.5 Hz,
Ar-H), 7.46 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.49 (2H, t, J ¼ 7.8 Hz, Ar-H), 7.70
Compound 10d was prepared from 7 (0.61 g, 2.0 mmol) and (S)-
epichlorhydrin (9d, 0.47 mL, 6.0 mmol) following the same protocol
as for 10a. Purification by silica gel column chromatography gave
10d (180 mg, 25%) as a yellow oil; tlc Rf ¼ 0.46 (EtOAc/n-hexane ¼ 1
: 1); UV lmax (MeOH) nm (log ε): 286 (0.328); [
a
]
þ4.2ꢀ (c 0.002,
(2H, d, J ¼ 8.7 Hz, Ar-H); 13C NMR (CDCl3)
d 22.1, 22.3, 24.7, 37.4,
D
MeOH); cLogP ¼ 2.69; 1H NMR (CDCl3)
d
0.80 (3H, d, J ¼ 6.3 Hz,
42.0, 58.0, 115.4 (2JC-F ¼ 22.4 Hz, C-3), 123.3 (2JC-F ¼ 13.1 Hz, C-1),
124.2 (3JC-F ¼ 3.5 Hz, C-4), 128.8, 129.4, 129.9 (4JC-F ¼ 8.0 Hz, C-5),
131.5 (3JC-F ¼ 3.5 Hz, C-6), 138.6, 139.7, 161.0 (1JC-F ¼ 246.1 Hz, C-2),
171.6; FABMS: 414 [M þ H]þ; HRFABMS: Calcd for C19H24FN2O3SCl
[M þ H]þ 414.1182; found 414.1184.
CH3), 0.81 (3H, d, J ¼ 6.6 Hz, CH3), 1.27e1.36 (1H, m, CHH), 1.43e
1.49 (1H, m, CHH), 1.70e1.79 (1H, m, CH3CHCH3), 2.58 (1H, dd,
J ¼ 4.5, 2.4 Hz, CHOCHH), 2.81 (1H, t, J ¼ 4.5 Hz, CHOCHH), 3.15e
3.19 (1H, m, CHOCHH), 3.30 (1H, dd, J ¼ 15.9, 6.3 Hz, NCHH), 3.41
(1H, dd, J ¼ 15.9, 4.2 Hz, NCHH), 4.30 (1H, t, J ¼ 7.5 Hz, COCH), 5.45
(1H, br, NH), 6.31 (1H, br, NH), 7.51 (2H, d, J ¼ 8.7 Hz, Ar-H), 7.80 (2H,
5.1.12. N-(5-Chlorothiophene-2-sulfonyl)-N-(2-fluorobenzyl)-(
leucinamide (11e)
D)-
d, J ¼ 8.7 Hz, Ar-H); 13C NMR (CDCl3)
d 22.1, 22.4, 24.6, 38.0, 46.9,
47.6, 50.5, 57.5, 128.8, 129.5, 137.9, 139.8, 172.3; FABMS: 361
[M þ H]þ; HRFABMS: Calcd for C15H22N2O4SCl [M þ H]þ 361.0991;
found 361.0993.
Compound 11e was prepared from 8 (0.16 g, 0.52 mmol) and 2-
fluorobenzyl bromide (9e, 0.13 mL, 1.06 mmol) following the same
protocol as for 10a. Purification by silica gel column chromatog-
raphy gave 11e (53 mg, 24%) as a white solid; tlc Rf ¼ 0.56 (EtOAc/n-
hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 289 (1.333); mp 135e
5.1.9. N-(5-Chlorothiophene-2-sulfonyl)-N-((R)-2,3-epoxypropyl)-
(D)-leucinamide (11c)
136 ꢀC; [
a]
D þ8.7ꢀ (c 0.002, MeOH); cLogP ¼ 4.37; 1H NMR (CDCl3)
Compound 11c was prepared from 8 (0.62 g, 2.0 mmol) and (R)-
d
(ppm) 0.74 (3H, d, J ¼ 6.6 Hz, CH3), 0.83 (3H, d, J ¼ 6.6 Hz, CH3),
epichlorhydrin (9c, 0.47 mL, 6.00 mmol) following the same pro-
tocol as for 10a. Purification by silica gel column chromatography
gave 11c (132 mg, 18%) as a pale yellow oil; tlc Rf ¼ 0.36 (EtOAc/n-
1.23e1.32 (1H, m, CHH), 1.38e1.47 (1H, m, CHH), 1.82e1.91 (1H, m,
CH3CHCH3), 4.35 (1H, t, J ¼ 7.2 Hz, COCH), 4.50 (1H, d, J ¼ 15.9 Hz,
NCHH), 4.66 (1H, d, J ¼ 15.9 Hz, NCHH), 5.24 (1H, br, NH), 6.14 (1H,
br, NH), 6.91 (1H, d, J ¼ 3.9 Hz, thiophene-H), 7.00 (1H, t, J ¼ 9.3 Hz,
Ar-H), 7.12 (1H, t, J ¼ 7.5 Hz, Ar-H), 7.28 (1H, t, J ¼ 6.6 Hz, Ar-H), 7.15
(1H, d, J ¼ 4.2 Hz, thiophene-H) 7.53 (1H, t, J ¼ 7.8 Hz, Ar-H); 13C
hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 288 (1.305); [
a
]
D ꢃ6.7ꢀ
(c 0.002, MeOH); cLogP ¼ 2.43; 1H NMR (CDCl3)
d 0.85 (3H, d,
J ¼ 7.2 Hz, CH3), 0.89 (3H, d, J ¼ 6.6 Hz, CH3),1.18e1.30 (1H, m, CHH),
1.44e1.56 (1H, m, CHH),1.86e1.95 (1H, m, CH3CHCH3), 2.62 (1H, dd,
J ¼ 4.5, 2.4 Hz, CHOCHH), 2.83 (1H, t, J ¼ 4.5 Hz, CHOCHH), 3.16e
3.21 (1H, m, CHOCHH), 3.32 (1H, dd, J ¼ 15.9, 6.0 Hz, NCHH), 3.53
(1H, d, J ¼ 15.9, 4.8 Hz, NCHH), 4.35 (1H, dd, J ¼ 7.8, 6.6 Hz, COCH),
5.48 (1H, br, NH), 6.35 (1H, br, NH), 6.95 (2H, d, J ¼ 4.2 Hz, thio-
phene-H), 7.46 (2H, d, J ¼ 3.9 Hz, thiophene-H); 13C NMR (CDCl3)
NMR (CDCl3)
d
22.2, 22.4, 24.9, 37.4, 41.9, 42.0, 58.2, 115.3 (2JC-
¼ 16.2 Hz, C-3), 123.2 (2JC-F ¼ 9.9 Hz, C-1), 124.2 (3JC-F ¼ 2.6 Hz, C-
F
4), 126.6, 129.8 (4JC-F ¼ 6.2 Hz, C-5), 131.2 (3JC-F ¼ 2.6 Hz, C-6), 132.1,
137.8, 138.1, 161.0 (1JC-F ¼ 184.6, C-2), 171.4; FABMS: 420 [M þ H]þ;
HRFABMS: Calcd for C17H22FN2O3S2Cl [M þ H]þ 420.0747; found
420.0745.
d
22.3, 22.5, 24.8, 37.7, 46.9, 47.4, 50.6, 58.1, 126.8, 132.3, 137.7, 138.2,
171.7; FABMS: 367 [M þ H]þ; HRFABMS: Calcd for C13H20N2O4S2Cl
5.1.13. N-(4-Chlorobenzenesulfonyl)-N-(4-fluorobenzyl)-(
leucinamide (10f)
D)-
[M þ H]þ 367.0555; found 367.0556.
Compound 10f was prepared form 7 (0.11 g, 0.37 mmol) and 4-
fluorobenzylbromide (9f, 0.19 mL, 0.75 mmol) following the same
protocol as for 10a. Purification by silica gel column chromatog-
raphy gave 10f (75 mg, 49%) as a white solid; tlc Rf ¼ 0.61 (EtOAc/n-
hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 284 (0.376); mp 146e
5.1.10. N-(5-Chlorothiophene-2-sulfonyl)-N-((S)-2,3-epoxypropyl)-
(D)-leucinamide (11d)
Compound 11d was prepared from 8 (0.62 g, 2.0 mmol) and (S)-
epichlorhydrin (9d, 0.47 mL, 6.00 mmol) using the procedure for
the synthesis of 10a. Purification by silica gel column chromatog-
raphy gave 11d (125 mg, 17%) as a yellow oil; tlc Rf ¼ 0.42 (EtOAc/n-
147 ꢀC; [
a
]
D þ26.7ꢀ (c 0.003, MeOH); cLogP ¼ 4.63; 1H NMR (CDCl3)
d
0.67 (3H, d, J ¼ 6.6 Hz, CH3), 0.76 (3H, d, J ¼ 6.6 Hz, CH3), 1.09e1.18
hexane); UV lmax (MeOH) nm (log ε): 288 (1.430); [
a
]
þ6.2ꢀ (c
(1H, m, CHH), 1.26e1.37 (1H, m, CHH), 1.79e1.88 (1H, m,
CH3CHCH3), 4.29 (1H, t, J ¼ 7.2 Hz, COCH), 4.36 (1H, d, J ¼ 15.9 Hz,
NCHH), 4.57 (1H, d, J ¼ 15.6 Hz, NCHH),5.18 (1H, br, NH), 6.21 (1H,
br, NH), 6.98 (2H, t, J ¼ 8.7 Hz, Ar-H), 7.33 (2H, dd, J ¼ 8.4, 5.4 Hz, Ar-
H), 7.46 (2H, d, J ¼ 8.7 Hz, Ar-H), 7.66 (2H, t, J ¼ 8.4 Hz, Ar-H); 13C
D
0.002, MeOH); cLogP ¼ 2.43; 1H NMR (CDCl3)
d 0.89 (6H, d,
J ¼ 6.3 Hz, CH3), 1.31e1.48 (1H, m, CHH), 1.52e1.58 (1H, m, CHH),
1.72e1.81 (1H, m, CH3CHCH3), 2.60 (1H, dd, J ¼ 4.5, 2.4 Hz,
CHOCHH), 2.83 (1H, t, J ¼ 4.5 Hz, CHOCHH), 3.17e3.22 (1H, m,
CHOCHH), 3.34 (1H, dd, J ¼ 15.9, 6.0 Hz, NCHH), 3.54 (1H, dd,
J ¼ 15.9, 4.2 Hz, NCHH), 4.35 (1H, t, J ¼ 7.5 Hz, COCH), 5.39 (1H, br,
NH), 6.14 (1H, br, NH), 6.94 (2H, d, J ¼ 3.9 Hz, thiophene-H), 7.43
NMR (CDCl3)
d
22.0, 22.4, 24.7, 37.9, 47.9, 57.7, 115.2 (2JC-F ¼ 16.0 Hz,
C-3 & C-5), 128.6, 129.4, 130.7 (3JC-F ¼ 6.1 Hz, C-2 & C-6), 132.0 (4JC-
¼ 2.4 Hz, C-1), 138.5, 139.5, 162.4 (1JC-F ¼ 184.3 Hz, C-4), 171.6;
F
(2H, d, J ¼ 3.9 Hz, thiophene-H); 13C NMR (CDCl3)
d
22.3, 22.6, 24.9,
FABMS: 414 [M þ H]þ; HRFABMS: Calcd for C19H24FN2O3SCl
38.4, 47.0, 48.0, 50.7, 58.0, 126.9, 132.3, 137.9, 138.1, 172.1; FABMS:
367 [M þ H]þ; HRFABMS: Calcd for C13H20N2O4S2Cl [M þ H]þ
367.0555; found 367.0557.
[M þ H]þ 414.1182; found 414.1183.
5.1.14. N-(5-Chlorothiophene-2-sulfonyl)-N-(4-fluorobenzyl)-(
D)-
leucinamide (11f)
5.1.11. N-(4-Chlorobenzenesulfonyl)-N-(2-fluorobenzyl)-(
leucinamide (10e)
D
)-
Compound 11f was prepared from 8 (0.11 g, 0.37 mmol) and 4-
fluorobenzylbromide (9f, 0.19 mL, 0.75 mmol) following the same
protocol as for 10a. Purification by silica gel column chromatog-
raphy gave 11f (105 mg, 68%) as a white solid; tlc Rf ¼ 0.53 (EtOAc/
n-hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 289 (2.192); mp
Compound 10e was prepared from 7 (0.16 g, 0.53 mmol) and 2-
fluorobenzyl bromide (9e, 0.13 mL, 1.06 mmol) using the procedure
for the synthesis of 10a. Purification by silica gel column chroma-
tography gave 10e (169 mg, 77%) as a white solid; tlc Rf ¼ 0.58
(EtOAc/n-hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 286 (0.238);
143e144 ꢀC; [
(CDCl3)
a
]
D
þ13.5ꢀ (c 0.004, MeOH); cLogP ¼ 4.37; 1H NMR
d
0.72 (3H, d, J ¼ 6.6 Hz, CH3), 0.83 (3H, d, J ¼ 6.6 Hz, CH3),
mp 172e173 ꢀC; [
a
]
D
þ25.0ꢀ (c 0.003, MeOH); cLogP ¼ 4.63; 1H
1.21e1.27 (1H, m, CHH), 1.36e1.43 (1H, m, CHH), 1.80e1.97 (1H, m,

Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
149
CH3CHCH3), 4.33 (1H, t, J ¼ 7.5 Hz, COCH), 4.40 (1H, d, J ¼ 15.6 Hz,
5.1.18. N-(4-Chlorobenzenesulfonyl)-N-( 8-hydroxy-2-methyl-
quinolin-5-yl)methyl-( )-leucinamide (10h)
NCHH), 4.56 (1H, d, J ¼ 15.6 Hz, NCHH), 5.18 (1H, br, NH), 6.38 (1H,
br, NH), 6.91 (1H, d, J ¼ 4.2 Hz, thiophene-H), 7.00 (2H, t, J ¼ 8.7 Hz,
Ar-H), 7.29 (1H, d, J ¼ 3.9 Hz, thiophene-H) 7.37 (2H, dd, J ¼ 8.7,
D
To a mixture of 7 (0.12 g, 0.38 mmol) in CHCl3 (6 mL) was added
DIPEA (0.13 mL, 0.75 mmol), potassium iodide (0.01 g), and 9h
(0.16 mg, 0.75 mmol) at room temperature. The mixture was stirred
overnight, poured into brine, then extracted with CHCl3. The
organic layer was separated, washed with 5% NaHCO3, brine, and
then dried over MgSO4, and concentrated. The resulting residue
was purified by silica gel column chromatography to give 10h
(62 mg, 34%) as a yellow solid; tlc Rf ¼ 0.65 (EtOAc/n-hexane ¼ 1 :
1); UV lmax (MeOH) nm (log ε): 313 (0.489); mp 166e167 ꢀC;
5.7 Hz, Ar-H); 13C NMR (CDCl3)
d 22.1, 22.4, 24.7, 37.9, 48.1, 58.0,
115.4 (2JC-F ¼ 21.2 Hz, C-3 & C-5),126.7,130.8 (3JC-F ¼ 8.0 Hz, C-2 & C-
6), 132.0 (4JC-F ¼ 2.0 Hz, C-1), 132.1, 137.9, 138.7, 162.6 (1JC-
¼ 245.0 Hz, C-4), 171.5; FABMS: 420 [M þ H]þ; HRFABMS: Calcd
F
for C17H22FN2O3S2Cl [M þ H]þ 420.0747; found 420.0746.
5.1.15. N-(3,5-Difluorobenzyl)-N-(4-chlorobenzenesulfonyl)-(
D)-
leucinamide (10g)
[a
]
þ9.7ꢀ (c 0.003, MeOH); cLogP ¼ 4.92; 1H NMR (CDCl3)
d 0.58
D
Compound 10g was prepared from 7 (0.30 g, 1 mmol) and 3,5-
difluorobenzyl bromide (9g, 0.26 mL, 2.0 mmol) using the proce-
dure for the synthesis of 10a. Purification by silica gel column
chromatography gave 10g (207 mg, 48%) as a white solid; tlc
Rf ¼ 0.63 (EtOAc/n-hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 286
(3H, d, J ¼ 6.6 Hz, CH3), 0.76 (3H, d, J ¼ 6.6 Hz, CH3), 0.93e1.01 (1H,
m, CHH), 1.33e1.44 (1H, m, CHH), 1.79e1.88 (1H, m, CH3CHCH3),
2.80 (3H, s, Ar-CH3), 4.31 (1H, t, J ¼ 8.7 Hz, COCH), 4.69 (1H, d,
J ¼ 14.7 Hz, NCHH), 5.03 (1H, d, J ¼ 14.7 Hz, NCHH), 5.29 (1H, br,
NH), 5.97 (1H, br, NH), 7.03 (1H, d, J ¼ 8.1 Hz, Ar-H), 7.33 (2H, d,
J ¼ 7.8 Hz, Ar-H), 7.41 (1H, d, J ¼ 8.4 Hz, Ar-H), 7.41 (2H, d, J ¼ 8.7 Hz,
(0.183); mp 176e177 ꢀC; [
1H NMR (CDCl3)
a
]
D þ29.0ꢀ (c 0.003, MeOH); cLogP ¼ 4.77;
d
0.70 (3H, d, J ¼ 6.6 Hz, CH3), 0.79 (3H, d,
Ar-H), 7.69 (2H, d, J ¼ 8.7 Hz, Ar-H), 8.69 (1H, d, J ¼ 8.7 Hz, Ar-H); 13
C
J ¼ 6.6 Hz, CH3), 1.04e1.13 (1H, m, CHH), 1.31e1.40 (1H, m, CHH),
1.74e1.84 (1H, m, CH3CHCH3), 4.33 (1H, dd, J ¼ 8.1, 6.9 Hz, COCH),
4.34 (1H, d, J ¼ 15.9 Hz, NCHH), 4.57 (1H, d, J ¼ 16.5 Hz, NCHH), 5.27
(1H, br, NH), 6.18 (1H, br, NH), 6.71 (1H, tt, J ¼ 8.7, 2.4 Hz, Ar-H), 6.88
(2H, d, J ¼ 6.0 Hz, Ar-H), 7.49 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.70 (2H, d,
NMR (CDCl3) d 21.1, 21.9, 22.9, 25.0, 29.8, 37.3, 46.8, 57.3, 109.5,
120.6, 123.2, 125.6, 128.8, 129.2, 130.1, 137.8, 139.5, 151.9, 156.9,
171.7; FABMS: 476 [M þ H]þ; HRFABMS: Calcd for C23H27N3O4SCl
[M þ H]þ 476.1413; found 476.1412.
J ¼ 8.7 Hz, Ar-H); 13C NMR (CDCl3)
d 22.0, 22.4, 24.7, 37.9, 47.9, 57.7,
103.2 (2JC-F ¼ 18.8 Hz, C-4), 111.3 (2,4JC-F ¼ 14.0, 5.3 Hz, C-2 & C-6),
128.6, 129.5, 138.1, 139.8, 140.7 (3JC-F ¼ 6.6 Hz, C-1), 162.8 (1,3JC-
5.1.19. N-(5-Chlorothiophene-2-sulfonyl)-N-(8-hydroxy-2-methyl-
quinolin-5-yl)methyl-( )-leucinamide (11h)
D
¼ 186, 9.5 Hz, C-3 & C-5), 171.2; FABMS: 431 [M þ H]þ; HRFABMS:
Compound 11h was prepared from 8 (0.12 g, 0.38 mmol) and 9h
(0.16 mg, 0.75 mmol) using the procedure for the synthesis of 10h.
Purification by silica gel column chromatography gave 11h (33 mg,
18%) as a yellow solid; tlc Rf ¼ 0.61 (EtOAc/n-hexane ¼ 1 : 1); UV
F
Calcd for C19H22F2N2O3SCl [M þ H]þ 431.1010; found 431.1012.
5.1.16. N-(3,5-Difluorobenzyl)-N-(5-chlorothiophene-2-sulfonyl)-
(D
)-leucinamide (11g)
lmax (MeOH) nm (log ε): 361 (1.712); mp 164e165 ꢀC; [
a
]
D þ8.6ꢀ (c
Compound 11g was prepared from 8 (0.31 g, 1 mmol) and 3,5-
0.004, MeOH); cLogP ¼ 4.66; 1H NMR (CDCl3)
d 0.74 (3H, d,
difluorobenzyl bromide (9g, 0.26 mL, 2.0 mmol) using the proce-
dure for the synthesis of 10a. Purification by silica gel column
chromatography gave 11g (209 mg, 48%) as a white solid; tlc
Rf ¼ 0.58 (EtOAc/n-hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 289
J ¼ 6.6 Hz, CH3), 0.83 (3H, d, J ¼ 6.6 Hz, CH3), 1.23e1.32 (1H, m,
CHH), 1.38e1.47 (1H, m, CHH), 1.82e1.91 (1H, m, CH3CHCH3), 4.35
(1H, t, J ¼ 7.2 Hz, COCH), 4.50 (1H, d, J ¼ 15.9 Hz, NCHH), 4.66 (1H, d,
J ¼ 15.9 Hz, NCHH), 5.24 (1H, br, NH), 6.14 (1H, br, NH), 6.91 (1H, d,
J ¼ 3.9 Hz, thiophene-H), 7.00 (1H, t, J ¼ 9.3 Hz, Ar-H), 7.12 (1H, t,
J ¼ 7.5 Hz, Ar-H), 7.28 (1H, t, J ¼ 6.6 Hz, Ar-H), 7.15 (1H, d, J ¼ 4.2 Hz,
(1.347); mp 162e163 ꢀC; [
a
]
D þ13.0ꢀ (c 0.002, MeOH); cLogP ¼ 4.51;
1H NMR (CDCl3)
d
0.74 (3H, d, J ¼ 6.6 Hz, CH3), 0.85 (3H, d, J ¼ 6.6 Hz,
CH3), 1.14e1.23 (1H, m, CHH), 1.38e1.47 (1H, m, CHH), 1.75e1.84
(1H, m, CH3CHCH3), 4.36 (1H, t, J ¼ 8.1 Hz, COCH), 4.43 (1H, d,
J ¼ 16.5 Hz, NCHH), 4.58 (1H, d, J ¼ 16.5 Hz, NCHH), 5.33 (1H, br,
NH), 6.06 (1H, br, NH), 6.72 (1H, tt, J ¼ 8.7, 2.4 Hz, Ar-H), 6.93 (2H, d,
J ¼ 7.5 Hz, Ar-H), 6.93 (2H, d, J ¼ 4.2 Hz, thiophene-H), 7.35 (2H, d,
thiophene-H) 7.53 (1H, t, J ¼ 7.8 Hz, Ar-H); 13C NMR (CDCl3)
d 22.0,
22.5, 24.8, 25.0, 37.7, 46.9, 57.8, 108.9, 120.6, 122.5, 123.1, 125.4,
126.6, 129.6, 132.3, 132.7, 137.8, 139.1, 152.3, 158.9, 171.4; FABMS:
482 [M þ H]þ; HRFABMS: Calcd for C21H25N3O4S2Cl [M þ H]þ
482.0978; found 482.0977.
J ¼ 4.2 Hz, thiophene-H); 13C NMR (CDCl3)
d 22.1, 22.4, 24.8, 38.1,
48.0, 58.0,103.2 (2JC-F ¼ 19.0 Hz, C-4),111.2 (2,4JC-F ¼ 14.0, 5.3 Hz, C-2
& C-6), 126.7, 132.2, 137.8, 138.1, 140.8 (3JC-F ¼ 6.5 Hz, C-1), 162.9
5.1.20. N-(4-Chlorobenzenesulfonyl)-N-piperonyl-(D)-leucinamide
(3)
1,3
(
J
¼ 186, 9.5 Hz, C-3 & C-5), 171.2; FABMS: 437 [M þ H]þ;
C-F
HRFABMS: Calcd for C17H20F2N2O3S2Cl [M þ H]þ 437.0574; found
Compound 3 was prepared from 7 (0.12 g, 0.38 mmol) and
piperonyl chloride (9i, 0.13 mg, 0.76 mmol) using the procedure
for the synthesis of 10a. Purification by silica gel column chro-
matography gave 3 (114 mg, 69%) as a white solid; tlc Rf ¼ 0.40
(EtOAc/n-hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 298
437.0576.
5.1.17. 5-Chloromethyl-2-methyl-8-quinolinol (9h)
To a mixture of 2-methyl-8-quinolinol (1.46 g, 10 mmol), 1.6 mL
of 36% HCl, and 1.6 mL of 37% formaldehyde was treated with
hydrogen chloride gas at an ice bath for 6 h. The solution was then
allowed to stand at room temperature for 2 h without stirring. The
yellow solid formed was collected on a filter, washed with 90%
alcohol, dried over MgSO4, and concentrated to give 9h (0.94 g,
48%) as a yellow solid; tlc Rf ¼ 0.25 (EtOAc/n-hexane ¼ 1 : 1); UV
(2.010); mp 126e127 ꢀC;
cLogP ¼ 4.45; 1H NMR (CDCl3)
[
d
a
]
þ20.0ꢀ (c 0.003, MeOH);
D
0.71 (3H, d, J ¼ 6.6 Hz, CH3), 0.78
(3H, d, J ¼ 6.6 Hz, CH3), 1.17e1.24 (1H, m, CHH), 1.31e1.37 (1H, m,
CHH), 1.80e1.90 (1H, m, CH3CHCH3), 4.29 (1H, d, J ¼ 15.3 Hz,
NCHH), 4.31 (1H, t, J ¼ 7.2 Hz, COCH), 4.48 (1H, d, J ¼ 15.6 Hz,
NCHH), 5.26 (1H, br, NH), 5.94 (2H, s, CH2), 6.25 (1H, br, NH), 6.70
(1H, t, J ¼ 7.8 Hz, Ar-H), 7.33 (2H, dd, J ¼ 8.4, 5.4 Hz, Ar-H), 7.46
(2H, d, J ¼ 8.7 Hz, Ar-H), 7.66 (2H, t, J ¼ 8.4 Hz, Ar-H); 13C NMR
lmax (MeOH) nm (log ε): 323 (0.783); mp 241e242 ꢀC; [
a
]
D ꢃ10.0ꢀ
(c 0.002, MeOH); cLogP ¼ 3.14; 1H NMR (DMSO-d6)
d 2.97 (3H, s,
CH3), 5.29 (2H, s, CH2),7.53 (1H, d, J ¼ 7.8 Hz, Ar-H), 7.80 (1H, d,
(CDCl3) d 22.2, 22.3, 24.6, 37.9, 48.6, 57.7, 101.1, 107.9, 109.3, 122.6,
J ¼ 8.4 Hz, Ar-H), 8.01 (1H, d, J ¼ 9.0 Hz, Ar-H), 9.10 (1H, d, J ¼ 9.0 Hz,
128.7, 129.2, 129.8, 138.6, 139.3, 147.3, 147.7, 172.2; FABMS: 439
[M
H]þ; HRFABMS: Calcd for C20H24N2O5SCl [M H]þ
439.1096; found 439.1098.
Ar-H); 13C NMR (DMSO-d6)
d
20.8, 60.5, 115.9, 124.4, 126.5, 128.8,
þ
þ
129.3, 129.8, 143.1, 147.6, 157.5, 228.5; FABMS: 208 [M þ H]þ.

150
Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
5.1.21. N-(5-Chlorothiophene-2-sulfonyl)-N-piperonyl-(
leucinamide (11i)
D
)-
5 min to remove cell debris. The protein concentrations of clarified
supernatants were determined using a BCA protein assay reagent
kit, and cell extracts containing equivalent amounts of proteins
were resolved by SDS-PAGE and analyzed by Western blotting us-
ing an anti-Notch(Val1744) polyclonal antibody.
Compound 11i was prepared from 8 (0.16 g, 0.37 mmol) and
piperonyl chloride (9i, 0.13 mg, 0.76 mmol) following the same
protocol as for 10a. Purification by silica gel column chromatog-
raphy gave 11i (115 mg, 70%) as a white solid; tlc Rf ¼ 0.30 (EtOAc/n-
hexane ¼ 1 : 1); UV lmax (MeOH) nm (log ε): 289 (2.241); mp 111e
112 ꢀC; [
a]
D þ11.8ꢀ (c 0.002, MeOH); cLogP ¼ 4.19; 1H NMR (CDCl3)
5.2.3. Cell viability assay
d
0.73 (3H, d, J ¼ 6.6 Hz, CH3), 0.82 (3H, d, J ¼ 6.6 Hz, CH3), 1.24e1.33
T20 cells (5 ꢁ104/100
m
L/well) were seeded onto the wells of 96-
(1H, m, CHH), 1.37e1.45 (1H, m, CHH), 1.77e1.86 (1H, m,
CH3CHCH3), 4.32 (1H, t, J ¼ 7.2 Hz, COCH), 4.47 (1H, d, J ¼ 15.3 Hz,
NCHH), 4.34 (1H, d, J ¼ 15.3 Hz, NCHH), 5.64 (1H, br, NH), 5.94 (2H,
s, CH2), 6.15 (1H, br, NH), 6.71 (1H, d, J ¼ 8.1 Hz, Ar-H), 6.81 (1H, d,
J ¼ 8.1 Hz, Ar-H), 6.88 (1H, s, Ar-H), 6.90 (1H, d, J ¼ 3.9 Hz, thio-
phene-H), 7.30 (1H, d, J ¼ 3.9 Hz, thiophene-H); 13C NMR (CDCl3)
well microplates in culture medium containing 10 mM of respective
compounds and incubated at 37 ꢀC for 24 h. Viable cells were
determined using the CellTiter 96Ò Aqueous Non-Radioactive Cell
Proliferation Assay (Promega, Madison, WI, USA) as specified in the
manufacturer’s instructions. Briefly, following the addition of the
combined MTS/PMS solution (20 mL/well) microplates were incu-
d
22.2, 22.4, 24.8, 37.8, 48.8, 59.0, 101.1, 108.2, 109.2, 122.6, 126.5,
bated for 3 h at 37 ꢀC. The conversion of MTS into formazan in
viable cells was quantitated by the absorbance at 490 nm using a
Synergy HT ELISA plate reader (BioTek, Winooski, VT, USA). The
number of living cells in culture was directly proportional to the
absorbance at 490 nm. Viable cells in culture medium containing
vehicle alone (1% DMSO, Control) were referred to as 100% viability.
The background absorbance shown at devoid of cells was sub-
tracted from these data.
129.7, 132.0, 137.7, 138.5, 147.4, 147.8, 171.6; FABMS: 445 [M þ H]þ;
HRFABMS: Calcd for C18H22N2O5S2Cl [M þ H]þ 445.0661; found
445.0663.
5.2. Pharmacological evaluation
5.2.1. Reagents
Anti-MEK antibody and anti-ERK1/2 MAP kinase antibody were
purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Horseradish peroxidase-conjugated anti-rabbit IgG was obtained
from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish
peroxidase-conjugated anti-mouse IgG and ECL Western Blotting
detection reagents were obtained from Amersham Biosciences
(Piscataway, NJ, USA). Dulbecco’s modified Eagle’s medium
(DMEM) and fetal bovine serum (FBS) were from Invitrogen and
Biological Industries Ltd (Kibbtuz Beit Haemek, Israel), respectively.
All other reagents were reagent grade and obtained from standard
suppliers abroad.
5.2.4. Cell culture and cell lines
Human embryonic kidney cells (HEK293) were incubated in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS) and 0.1 mg/mL penicillin and strep-
tomycin. T-REx293 cells were purchased from Invitrogen and
cultured in DMEM supplemented with 10% FBS and 5 mg/mL
blasticidin. The generation of stably transfected cell lines, T20 and
g
-30, has been described previously [21]. Cells were incubated in a
humidified incubator at 37 ꢀC in 5% CO2.
5.2.2. Cell-based g-secretase assays
The production of T20 cells has been reported previously [20].
T20 cells were maintained in DMEM supplemented with 10% FBS,
5.2.5. Assay for extracellular signal-regulated kinase (ERK)
activation
200
m
g/mL hygromycin, 5
m
g/mL blasticidin, and 250
m
g/mL zeocin
The stimulation of the ERKs was measured by using anti-
phospho-ERK1/2 and anti-ERK1/2 antibodies (Cell Signaling Tech-
(DMEM-HZB). The stably transfected T20 cells were detached from
culture dishes by trypsinization, washed with PBS, and resus-
pended in DMEM-HZB, followed by plating onto 96-well micro-
nology, Danvers, MA, USA) as previously described [16]. Briefly, g-
30 cells were grown in six-well plates at 5 ꢁ 105 cells/well and
plates (2 ꢁ 104 cells/50
m
L/well) and incubating at 37 ꢀC for 24 h.
incubated with culture medium at 37 ꢀC for 18 h, followed by an
Tested compounds diluted in DMEMHZB were added to a final
additional incubation with culture medium containing 1 mg/mL
concentration of 10
mM in the presence of tetracycline (1
mg/mL).
tetracycline at 37 ꢀC for 18 h. Before the experiments, we replaced
the medium with DMEM containing 0.5% FBS and treated it with
Treatments were terminated after incubation at 37 ꢀC for 24 h by
directly adding an equivalent volume of the Steady-Glo luciferase
assay reagent (Promega, Madison, WI, USA), and luciferase signals
from each well were performed immediately with the lumines-
cence plate reader available in our Institute. Triplicates of each
compound treatment were assayed. Luciferase signals from the
stable line without tetracycline induction and tested compound
treatment was referred to as one-fold of activation. Parallel testing
with a cell line constitutively expressing only the luciferase re-
porter gene (e.g., under control of a CMV promoter) was performed
as a control panel.
the tested compounds (10
mM each). After treatment, reactions
were stopped by placing cells on ice and aspirating the medium.
Cells were harvested and lysed in 50 mM TriseHCl (pH 8.0),
150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 1% Triton
X-100, and protease- and phosphatase-inhibitor cocktails. Protein
concentration was determined by the BCA assay (Pierce, Rockford,
IL, USA). Each cell lysate, which contained 50
mg proteins, was
separated on 12% SDS-polyacrylamide electrophoresis gels,
immunoblotted, and identified using anti-phospho-ERK1/2 or anti-
ERK1/2 antibody.
To examine compound inhibition of
cleavage of Notch, we generated a HEK293-derived stable line (N7)
that was constitutively expressing N E as previously described
g-secretase-dependent S3
D
5.2.6. Data analysis
[20]. N7 cells were plated onto12-well microplates in 1 mL/well
DMEM supplemented with 10% FBS at 5 ꢁ 105 cells/well. After in-
cubation at 37 ꢀC overnight, cells were treated with compounds at
The data presented were means ꢂ SD and triplicates of each
compound treatment were measured in the experiments. The ef-
fects of the tested compounds on the biological outcome were
statistically examined using a one-way analysis of variance. Dun-
nets’s test was applied to compare individual compounds. In all
cases, P < 0.05 was accepted to denote significance.
10
cells were harvested using PBS containing 20 mM EDTA and dis-
solved in 50
L of 1ꢁ PLB, followed by centrifugation at 13,200g for
m
M as described above and incubated at 37 ꢀC for 24 h. Treated
m

Y.-F. Liao et al. / European Journal of Medicinal Chemistry 79 (2014) 143e151
151
evaluation of BMS-708163, a potent, selective and orally bioavailable
retase inhibitor, Medicinal Chemistry Letters 1 (2010) 120e124.
g-sec-
Author contributions
[8] Bristol-Myers Squibb Company. Available online: http://www.bms.com/news/
features/2012/Pages/AvagacestatDevelopmentStatus.aspx.
[9] E.A. Kitas, G. Galley, R. Jakob-Roetne, A. Flohr, W. Wostl, H. Mauser, A.M. Alker,
C. Czech, L. Ozmen, P. David-Pierson, D. Reinhardtc, H. Jacobsen, Substituted 2-
oxo-azepane derivatives are potent, orally active g-secretase inhibitors, Bio-
organic & Medicinal Chemistry Letters 18 (2008) 304e308.
[10] H. Meizane, J.C. Dodart, C. Mathis, S. Little, J. Clemens, S.M. Paul, A. Ungerer,
Y.-C.T. carried out the synthesis of the designed compounds. M.-
Y.C. and B.-J.W. performed the pharmacological experiments. Y.-F.L.
conceived and designed the pharmacological experiments. M.-K.H.
conceived and designed the targeted compounds. Y.-F.L. and M.-
K.H. analyzed the data.
Memory-enhancing effects of secreted forms of the
b-amyloid precursor
protein in normal and amnestic mice, Proceedings of the National Academy of
Sciences of the United States of America 95 (1998) 12683e12688.
Conflicts of interest
[11] M. Yogev-Falach, T. Amit, O. Bar-Am, Involvement of MAP kinase in the
regulation of amyloid precursor protein processing by novel cholinesterase
inhibitors derived from rasagiline, FASEB Journal 16 (2002) 1674e1676.
[12] O. Bar-Am, M. Yogev-Falach, T. Amit, Regulation of protein kinase C by the
anti-Parkinson drug, MAO-B inhibitor, rasagiline and its derivatives, Journal of
Neurochemistry 89 (2004) 1119e1125.
The authors declare no conflict of interest.
Acknowledgments
[13] T.A. Comery, R.L. Martone, S. Aschmies, K.P. Atchison, G. Diamantidis, X. Gong,
H. Zhou, A.F. Kreft, M.N. Pangalos, J. Sonnenberg-Reines, J.S. Jacobsen,
This work was financially supported by the National Science
Council Taiwan (NSC 98-2320-B-016-004-MY3 to M.-K. Hu and NSC
98-2320-B-001-014-MY2 to Y.-F. Liao). High-resolution mass
spectra analyses performed by the National Science Council
Regional Instruments Center at National Chiao Tung University are
also acknowledged.
K.L. Marquis, Acute
g-secretase inhibition improves contextual fear condi-
tioning in the Tg2576 mouse model of Alzheimer’s disease, Journal of
Neuroscience 25 (2005) 8898e8902.
[14] A.Y. Hung, C. Haass, R.M. Nitsch, W.Q. Qiu, M. Citron, R.J. Wurtman,
J.H. Growdon, D.J. Selkoe, Activation of protein kinase C inhibits cellular
production of the amyloid
b-protein, Journal of Biological Chemistry 268
(1993) 22959e22962.
[15] J. Desdouits-Magnen, F. Desdouits, S. Takeda, Regulation of secretion of Alz-
heimer amyloid precursor protein by the mitogen-activated protein kinase
cascade, Journal of Neurochemistry 70 (1998) 524e530.
Appendix A. Supplementary data
[16] M.K. Hu, Y.F. Liao, J.F. Chen, B.J. Wang, Y.T. Tung, H.C. Lin, K.P. Lee, New 1,2,3,4-
tetrahydroisoquinoline derivatives as modulators of proteolytic cleavage of
amyloid precursor proteins, Bioorganic & Medicinal Chemistry 16 (2008)
1957e1965.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.006.
[17] Y.F. Liao, B.J. Wang, W.M. Hsu, H. Lee, C.Y. Liao, S.Y. Wu, H.T. Cheng, M.K. Hu,
Unnatural amino acid-substituted (hydroxyethyl)urea peptidomimetics
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