New 1,2,3,4-tetrahydroisoquinoline derivatives as modulators of proteolytic cleavage of amyloid precursor proteins

Article abstract of DOI:10.1016/j.bmc.2007.10.101

A type of new 1,2,3,4-tetrahydroisoquinoline derivatives was synthesized via concise procedure from commercially available tetrahydroisoquinoline. These derivatives were delicately designed to possess propargyl-related pharmacophores simulated with a monoamine oxidase inhibitor rasagiline. We investigated the effect of these synthetic tetrahydroisoquinoline derivatives on the regulation of proteolytic processing of amyloid precursor protein (APP) by an ERK-dependent signaling pathway. Additionally, these compounds were also evaluated on the prevention of the proteolytic processing of C99 as γ-secretase inhibitors by using a highly efficient cell-based reporter gene assay for γ-secretase. The results suggested that certain compounds might be explored to possess both sAPPα-releasing stimulation and γ-secretase inhibitory potency, which may reflect the synergetic potential of neuroprotective activities for the treatment of Alzheimer's disease as they possessed both ERK activation and inhibition of amyloidogenic Aβ release.

Full text of DOI:10.1016/j.bmc.2007.10.101

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1957–1965
New 1,2,3,4-tetrahydroisoquinoline derivatives as modulators
of proteolytic cleavage of amyloid precursor proteins
b,
a
b
Ming-Kuan Hu,a,* Yung-Feng Liao, Jung-Fang Chen, Bo-Jeng Wang,
*
Ying-Tsen Tung,b Hui-Ching Linc and Kang-Po Leea
aSchool of Pharmacy, National Defense Medical Center, 161 Minchuan East Road, Section 6, Taipei 114, Taiwan
bLaboratory of Molecular Neurobiology, Institute of Cellular and Organismic Biology, Academia Sinica,
128 Academia Road, Section 2, Taipei 115, Taiwan
cDepartment of Pharmacology, National Defense Medical Center, 161 Minchuan East Road, Section 6, Taipei 114, Taiwan
Received 16 August 2007; revised 31 October 2007; accepted 31 October 2007
Available online 6 November 2007
Abstract—A type of new 1,2,3,4-tetrahydroisoquinoline derivatives was synthesized via concise procedure from commercially avail-
able tetrahydroisoquinoline. These derivatives were delicately designed to possess propargyl-related pharmacophores simulated with
a monoamine oxidase inhibitor rasagiline. We investigated the effect of these synthetic tetrahydroisoquinoline derivatives on the reg-
ulation of proteolytic processing of amyloid precursor protein (APP) by an ERK-dependent signaling pathway. Additionally, these
compounds were also evaluated on the prevention of the proteolytic processing of C99 as c-secretase inhibitors by using a highly
efficient cell-based reporter gene assay for c-secretase. The results suggested that certain compounds might be explored to possess
both sAPPa-releasing stimulation and c-secretase inhibitory potency, which may reflect the synergetic potential of neuroprotective
activities for the treatment of Alzheimer’s disease as they possessed both ERK activation and inhibition of amyloidogenic Ab
release.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
tein kinase (MAPK) pathway involving sequential
activation of mitogen-activated protein kinase (MEK)
and extracellular signal-regulated protein kinase
(ERK).5 Concerning the diverse pathway of APP
processing, another secretase named a-secretase can
mediate alternative processing of APP known as non-
amyloidogenic pathway to produce the soluble form of
amyloid precursor protein a (sAPPa), thus preclude
Ab production. It was therefore interesting to know
how the respective secretase-catalyzed cleavages of
APP can act in response to the control by this signaling
cascade.
One of the pathological hallmarks of Alzheimer’s dis-
ease (AD) is the cerebral accumulation of amyloid pla-
ques,1 and the major constituents of these plaques are
the 40–42 residue amyloid-b peptides (Abs), which are
generated through the proteolysis of amyloid precursor
protein (APP) by sequential actions of a set of mem-
brane-bound proteases termed b- and c-secretases.2
Mounting contributions from cellular and pharmaco-
logical studies, affinity labeling, and biochemical ap-
proaches suggest that c-secretase is an aspartyl
protease and has thus been regarded as a pivotal role
for therapeutic target for AD.3,4 Recently, alternative
investigations have also shown that the proteolytic pro-
cessing of APP was regulated by mitogen-activated pro-
The soluble form of APPa was found to possess potent
neurotropic and neuroprotective activities against oxi-
dative and excitotoxic insults.6,7 In vitro studies have
established the involvement of protein kinase C (PKC)
and PKC-coupled receptors in the non-amyloidogenic
a-secretase pathway of APP cleavage.5 Further investi-
gations indicated that rasagiline (1a, Fig. 1), a MAO-B
inhibitor, and its carbamyl-containing derivative, TV-
3326 (1b), regulate PKC- and MEK-dependent APP
processing in SH-SY5Y neuroblastoma and PC12 cells,8
Keywords: Tetrahydroisoquinolines; Amyloid precursor protein;
c-Secretase; Alzheimer’s disease.
Corresponding authors. Tel.: +886 2 87923100x18896; fax: +886 2
87923169 (M.-K.H.); tel.: +886 2 27899535; fax: +886 2 27858059.
(Y.-F.L.); e-mail addresses: hmk@ndmctsgh.edu.tw; yliao@sinica.
edu.tw
*
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.101

1958
M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
2. Chemistry
X
N
The synthesis of 7-carbamoyl-1,2,3,4-tetrahydroisoquio-
line derivatives 11a–d and 12a–d is depicted in Scheme 1,
in which 7-hydroxy-tetrahydroisoquinoline 7 was the
critical intermediate for the preparation of these
1,2,3,4-tetrahydroisoquinoline derivatives. First, trifluo-
roacetyl-protection of secondary amine of the starting
material 1,2,3,4-tetrahydroisoquinoline was accom-
plished with trifluoroacetic anhydride and potassium
hydroxide in 72% yields. The following acylation at 7-
position of the N-protected 1,2,3,4-tetraisoquinoline
has been considered a critical step for the synthesis of
the desired products. The acetamide 4 was slowly added
to 1,2-dichloroethane solution with chloroacetyl chlo-
ride and aluminum chloride over 3 h at ambient temper-
ature to provide the desired 7-chloroacetyl derivative 5
with over 90% yields. No 6-chloroacetyl isomer ap-
peared in this reaction. As characterized with 1H
NMR, the H-8 proton of 5 appeared at d = 7.77 as small
doublet splitting, which was shifted downfield as com-
pared to that of compound 4. On the other hand, the
protons H-5 and H-6 were unambiguously identified at
d = 7.81 and d = 7.32 with splitting constants around
7.2 Hz, respectively. For further demonstration of the
regioselective acylation at 7-position of 1,2,3,4-tetra-
hydroisoqunoline ring, an N-propargyl-7-chloroacetyl
analogue 15 was synthesized either through N-propargy-
lation of 3 followed by acylation of the N-propargyl
intermediate 13 or via removal of N-trifluoroacetyl pro-
tection under sodium thiomethoxide in methanol and
sequential N-propargylation. The crystallized form of
15 was obtained to provide an X-ray crystal structure
as depicted in Figure 2. The chemical shifts of H-5, H-
6, and H-8, and their splitting patterns in both 5 and
the N-propargyl analogue 15 are similar to each other,
indicating the right regioselective acylation at 7-position
of tetraisoquinoline ring system under AlCl3-catalytic
conditions.
N
H
1a, X = -H
2
1b, X = -O(CO)-N(Me)Et
Figure 1. Chemical structures of rasagiline (1a), its derivative, TV-
3326 (1b), and selegiline (2).
which resulted in the stimulation of release of the neuro-
protective sAPPa. Nevertheless, selegiline, a well-known
selective MAO-B inhibitor for increasing the efficacy of
levodopa therapy in the treatment of Parkinson’s dis-
ease, also exerted neuroprotective effects in various pre-
clinical models. These results suggested that a crucial
role for PKC-, MEK-dependent pathways may be in-
volved in the enhancement of a-APP release by selegiline
and rasagiline. Taken together, the secretase-mediated
proteolysis of APP can be subject to multiple levels of
regulation by intracellular pathways and the ERK acti-
vation could play a pivotal role in shifting the APP pro-
cessing to the a-secretase-initiated non-amyloidogenic
pathway synergistically blocking c-secretase-dependent
Ab production. Conversely, reduction of formidable
Ab production by interfering c-secretase activity with
enzyme-targeted inhibitors might shift the APP process-
ing to the a-secretase-mediated pathways.10 Thus, con-
trol of Ab production by direct or indirect modulation
of c-secretase activity on APP opens the approaches to-
ward the treatment of Alzheimer’s disease.
Based on the light of these findings, we here manipu-
lated a new type of propargyl-related tetrahydroisoquin-
oline derivatives, simulated as congeners of rasagiline
and selegiline, and made exploitation on the possible
relations between the effects on the c-secretase-mediated
processing and on the modulation on MEK-dependent
signaling of APP.
Cl
Cl
b
NH
e
N
N
O
14
O
O
15
13
d
e
O
Cl
c
b
a
N
O
N
N
O
NH
O
O
Cl
CF3
5
CF3
6
3
4
F3C
O
d
e
f
N
R2
O
R1
6
NH
N
HO
HO
R1
11a-d, 12a-d
7
8, R1 =
9, R1 =
N
N
a, R2 =
c, R2 =
b, R2 =
N
N
d, R2 =
O
Scheme 1. Reagents and conditions: (a) (CF3CO)2O, KOH (b) ClCH2COCl, AlCl3 (c) mCPBA (d) NaSCH3 (e) propargyl bromide or
(bromomethyl)cyclopropane, K2CO3 (f) R2–CO–Cl (10a–d), NaH.

M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
1959
Figure 2. Crystal structure of 7-chloroacetyl-N-propargyl-1,2,3,4-tetrahydroisoquinoline (15) determined by s single-crystal X-ray crystallography.
35
For the introduction of an oxygen function into the 7-
position of 5, Baeyer-Villiger oxidation11 was performed
29.87
30
25
20
15
10
5
27.87
27.76
27.00
by treatment with meta-chloroperbenzoic acid (mCPBA)
in the presence of trifluoroacetic acid at dark conditions
for 3 days to afford the desired 7-chloroacetoxy ana-
logue 6 in 68% yield. The following step for the removal
of the acylated protections was accomplished with so-
dium thiomethoxide in methanol to remove both acetyl
groups at compound 6 simultaneously. Thus, the result-
ing 7-hydroxy-tetrahydroquinoline 7 was obtained as
hydrochloride salt in 54% yields.
24.46
23.76
22.06
19.31
11.49
8.33
1.00
0
D
At penultimate step, N-alkylation of 7 with propargyl or
cyclopropylmethyl bromides was readily carried out in
basic conditions to give the corresponding derivatives
8 and 9, respectively. Following the method previously
reported for the preparation of certain carbamoyl chlo-
rides,12 commercially unavailable N-ethyl-N-methyl-car-
bamoyl chloride (10a) and morpholinecarbonyl chloride
(10d) were synthesized from the corresponding second-
ary amines by using a solution of phosgene in toluene.
Finally, N-ethyl-N-methyl-carbamoyl moiety and its
congeners were successfully incorporated to 8 and 9 in
the presence of 60% sodium hydride to provide a series
of 1,2,3,4-tetrahydroisoquinoline derivatives 11a–d and
12a–d in 50–82% yields.
11a
11c
12a
12c
11b
11d
12b
12d
D + T
Rasagiline
Figure 3. The c-secretase inhibitory activity of 1,2,3,4-tetrahydroiso-
quinoline derivatives 11a–d and 12a–d. T20 cells were treated with
10 lM of each tested compounds (dissolved in DMSO, Abbr. D) in
culture medium containing 1 lg/ml tetracycline (Abbr. T) and incu-
bated at 37 ꢁC for 24 h. The detailed procedures for luciferase reporter
assays for c-secretase inhibition were followed as previously reported.
Each value represents the mean SD of three experiments.
compounds 12a, c, d at 10 lM decreased 35–72% of c-
secretase activity, while rasagiline and most propargyl
analogues were 5-fold less potent as only 6–15% of c-
secretase activity were decreased. The relative potency
of these tetrahydroisoquinoline derivatives suggested
that compounds 12a, c, d could target c-secretase. All
of the tetrahydroisoquinoline derivatives presented no
significant cytotoxicity on the HEK293 cells at this con-
centration as shown in Figure 4. This was the first ever
report for the inhibitory potency of the tetrahydroiso-
quinoline derivatives on the c-secretase activity.
3. Biological results and discussion
First, we investigated the effects of these synthetic tetra-
hydroisoquinolines on the inhibition of c-secretase-med-
iated cleavage of APP. Here, a quantitative cell-based
assay specifically measuring the c-cleavage of APP-C99
was employed. The generation and validation of the
cell-based assay was described previously.13 According
to this method, the fold of activation of luciferase, ex-
pressed in cell nucleus, was measured to determine the
ability of c-secretase on APP processing reversely. Thus,
the effects of a series of propargyl-related tetrahydroiso-
quinoline derivatives are shown in Figure 3. These tetra-
hydroisoquinoline derivatives were structurally related
to rasagiline, a known MAO-B inhibitor. The screening
of these synthetic tetrahydroisoquinolines showed that
Recent evidence was pointing that certain MAO-B
inhibitors including rasagiline, its carbamate derivative,
TV-3326, and selegiline modulated APP processing
through ERK-related signaling pathways to ultimately
reduce the formidable amyloid-b peptide formation.14
The outcome of ERK stimulation was contributed to
indirectly modulate the release of sAPPa. Thus these
contributions possibly hint an alternate approach bene-
fit to the treatment of Alzheimer’s disease. Although the
common propargylamine moiety of these compounds

1960
M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
140%
120%
100%
80%
60%
40%
20%
0%
DMSO
DT
Rasagiline
11a
11b
11c
11d
12a
12b
12c
12d
Compound
Figure 4. Effects of 11a–d and 12a–d on cell viability of HEK293 cells with a dose of 10 lM. Values are means SD, n = 3.
was found to be critical to the activation of ERK signal-
ing, it was found to be independent of their MAO inhib-
itory activity.15 Alternatively, it has been well
established that conformationally restricted modifica-
tions on the biological function-related structures have
been one of the approaches toward new compounds
with improving activities.16 Thus, we chose tetrahydro-
isoquinoline as a rigid skeleton to replace the flexible
alkylamine moiety of selegiline or rasagiline and investi-
gated the possible role in the proteolytic processing of
APP.
the cyclopropylmethyl group of 12a–d to inhibit the
MAO-B might be the ring strain of cyclopropyl group,
which would contribute to the binding and the radical-
mediated ring-opening to the targeted MAO-B at the ac-
tive site.
Taken together, both propargyl- and cyclopropylmeth-
yl-containing tetrahydroisoquinoline derivatives were
shown to keep moderate MAO-B inhibition. The well-
established pharmacophore could be replaced with the
highly strained cyclopropylmethyl moiety as critical
requirement for MAO inhibition, while only cyclopro-
pylmethyl derivatives 12a, c, d displayed moderate
c-secretase inhibition as determined on the cell-based
assays. The a-secretase-mediated processing of APP
was directly or indirectly enhanced by activation of
MEK–ERK pathway by, for instance, rasagiline or
selegiline, as previously described. Therefore, in the next
step, we seek to determine whether these cyclopropyl-
methyl-containing compounds can significantly modu-
late the MEK–ERK signal pathway.
Previous investigations have indicated that the propar-
gylamine moiety of rasagiline or selegiline was shown
to be essential for MAO inhibition.14 Here, all the syn-
thetic propargyl-containing tetrahydroisoquinolines
11a–d showed marginal MAO-B inhibitory activity with
IC50s in the range of 10–20 lM, though they were 10-
fold less potent than rasagiline as shown in Table 1.
On the other hand, the change of the critical propargyl
moiety of 11a–d to cyclopropylmethyl group as in the
analogues 12a–d still maintained the similar activity
against MAO-B. The maintenance of potency for com-
pounds 12a–d was partly attributed to the similar size
and lipophilicity (only an increment of 0.45 of the logP
value) between the cyclopropylmethyl group and prop-
argyl group. Another factor concerning the ability of
The relative levels of ERK activation (p-ERK/ERK, %)
of moderate c-secretase inhibitors 12a, c, d were further
examined in c-30 cells and found that 12c displayed 1.2-
fold stronger ERK activation properties than rasagiline
at concentration of 10 lM, while 12a and 12d showed
comparable potency to rasagiline as shown in Table 2.
Furthermore, the determination of these compounds’
potential to enhance sAPPa secretion using c-30 cells
is summarized in Figure 5. Unlike T20 cells which over-
express APP-C99 as the direct substrate of c-secretase,
c-30 cells constitutively expressed full-length APPs
Table 1. MAO-B inhibitory activity and log P values of tetrahydro-
isoquinoline derivatives 11a–d and 12a–d
O
O
N
N
R2
O
R2
O
11a-d
12a-d
Table 2. The relative levels of ERK activation of certain tetrahydro-
isoquinoline derivatives in treated cells compared with nontreated cells
Compound
R2
IC50 (lM)
log Pa
Rasagiline
11a
11b
2.1 0.6
21.5 1.0
13.9 0.5
15.7 2.2
18.7 1.6
12.8 2.0
14.9 1.1
11.6 0.7
18.5 0.8
2.295
2.252
2.235
2.632
1.567
2.688
2.671
3.088
2.033
Compound
Activation of ERK1/2
(p-ERK/ERK, %)
N-Ethyl-N-methylamino
1-Pyrrolidyl
1-Piperidinyl
11c
11d
D + T
Rasagiline
12a
100
131 14
131 10
1-Morpholinyl
N-Ethyl-N-methylamino
1-Pyrrolidyl
12a
12b
12c
12d
155 28
138 39
*
12c
12d
1-Piperidinyl
1-Morpholinyl
*
Each value represents the mean SD of three experiments. P < 0.05,
a Estimated lipophilicity by CAChe v. 6.1.
significantly different compared with the control.

M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
1961
sAPP-alpha secretion
5
4
3
2
1
0
DMSO
Rasagiline
12a
12c
12d
Figure 5. The secretion of sAPPa was determined by analysis of the immunoblots. The units obtained are relative to the signal from cells treated with
solvent (DMSO). Each value indicates the mean SD of three experiments.
along with human PS1, Aph-1a2, and Pen-2, and were
an ideal cell model for the detection of a-cleavage of
APP.18 The production of sAPPa in the conditioned
media of c-30 cells would be defined as the a-secretase
activity. Substantial effects of these compounds, 12a,
12c, and 12d, on a-secretase activity at 10 lM were ob-
served. The effect on sAPPa secretion correlates nicely
with the ERK activation and also showed modest
improvement as compared to rasagiline, suggesting that
the inhibition of c-secretase and the activation of sAPPa
secretion could be simultaneously mediated by ERK
activation in c-30 and T20 cells. Recently, Kim and
coworkers found that the inhibition of ERK1/2 activity
dramatically increased c-secretase activity in several
different cell types, while raising ERK1/2 activity by
adding purified active ERK1/2 significantly reduced
c-secretase activity,17 suggesting that ERK1/2 is an
endogenous negative regulator of the c-secretase acti-
vity. Consistent with this notion, we also found that
c-secretase activity in T20 cells treated with the MEK
inhibitor PD98059 was dramatically increased, concomi-
tant with the increased levels of secreted Ab40 in the
conditioned media (Liao, Y. F. et al. unpublished data).
(d) downfield from tetramethylsilane. The deuterated
NMR solvents contained 99.8–99.9% deuterium in the
indicated position and were obtained from CIL, Inc.
1
(Massachusetts, USA). H NMR coupling constants (J
values) are listed in hertz (Hz) and spin multiplicities
are reported as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (b). Elemental analyses
were determined using a Perkin-Elmer 240 EA analyzer.
Fast atom bombardment mass spectra (FABMS) were
acquired on a Finnigan Mat 95S mass spectrometer.
Specific rotations were measured with an Optical Activ-
ity AA-5 Polarimeter. Chromatography refers to flash
chromatography on silica gel (silica gel 60, 230–400
mesh ASTM, E. Merck). Melting points were recorded
on a Thomas Hoover capillary melting point apparatus
in open capillary tubes and are uncorrected.
5.1.1. N-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline
(4). To an ice-cooled solution of trifluoroacetic anhy-
dride (9 ml) in CH2Cl2 (32.4 ml) was added dropwise a
solution of 1,2,3,4-tetrahydroisoquinoline (8 ml,
60 mmol) in CH2Cl2 and stirred under ice-cooling for
3.5 h. A solution of KOH (4.74 g, 85 mmol) in water
(72 ml) was then added, and the reaction mixture was
stirred for further 2 h at room temperature. The result-
ing mixture was extracted with CH2Cl2 and the organic
layer was dried over MgSO4. The residue was taken on a
silica gel column (n-hexane/EtOAc = 3:1 as eluents) to
give a yellow oil (9.92 g, 72%) of the title compound;
TLC Rf = 0.58 (n-hexane/EtOAc = 1:1). 1H NMR
(300 MHz, CDCl3): d 7.29–7.19 (m, 4H, Aryl Hs); 4.80
(s, 2H, Ph-CH2–N); 3.91–3.86 (m, 2H, Ph-CH2–CH2–
N); 3.00 (br s, 2H, Ph-CH2–CH2–N). FABMS m/z:
230 (M + 1) (C11H10 NOF3).
4. Conclusion
In conclusion, we have developed a type of new 1,2,3,4-
tetrahydroisoquinoline derivatives in order to evaluate
its modulation on the proteolytic processing of amyloid
precursor protein. The results presented above indicate
that these N-cyclopropylmethyl derivatives possess sAP-
Pa-releasing stimulation, which correlates with their ef-
fects on ERK activation, and also display c-secretase
inhibitory potency. These results may reflect the syner-
getic potential of neuroprotective activities for the treat-
ment of Alzheimer’s disease as they possessed both
sAPPa secretion through ERK activation and simulta-
neous inhibition of amyloidogenic Ab release.
5.1.2. 7-Chloroacetyl-N-trifluoroacetyl-1,2,3,4-tetrahy-
droisoquinoline (5). To a suspension of AlCl3 (24.16 g)
in CH2Cl2 (162 ml) was added chloroacetyl chloride
(15 ml) dropwise at 0–5 ꢁC under argon for 20 min
and left to warm to room temperature. To this mixture
was added 4 (9.29 g, 40 mmol) over a period of 30 min at
room temperature. The resulting mixture was then stir-
red for an additional 3 h and poured onto a mixture
of ice-cold water (407 ml). The mixture was stirred for
5 min, and the layers were separated. The aqueous layer
was extracted with CH2Cl2 (2 · 200 ml). The combined
organic layers were washed with water (2 · 240 ml)
and 5% aqueous NaHCO3 (3 · 240 ml). The organic
layer was dried and the solvent evaporated to give a
white solid (10.47 g, 88%). TLC Rf = 0.4 (n-hexane/
5. Experimental
5.1. Chemistry
All reagents were commercial materials and were used
directly unless otherwise noted. DMF was dehydrated
˚
over 4 A molecular sieves. NMR spectra were recorded
1
on a Varian Gemini at 300 MHz for H and at 75 MHz
for 13C. Chemical shifts are reported in parts per million

1962
M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
EtOAc = 3:1). mp: 120–122 ꢁC; 1H NMR (300 MHz,
CDCl3): d 7.78 (q, J = 4 Hz, 1H, Aryl H), 7.34–7.26
(m, 2H, Aryl Hs), 4.87 (s, 2H, Ph-CH2–N), 4.67 (s,
2H, –CH2–C@O), 3.95–3.87 (m, 2H, –CH2–N), 3.06–
3.01 (m, 2H, Ph-CH2ꢀ). FABMS m/z: 306 (M + 1)
(C13H11NO2ClF3).
(2.0 g, 12 mmol) in CH3CN (18 ml) was added a solu-
tion of 96% (bromomethyl)cyclopro- pane (0.6 ml,
6.3 mmol). The reaction mixture was stirred at room
temperature under nitrogen for 25 h and filtered. The fil-
trate was washed with water and the aqueous layer was
extracted with ethyl acetate (20 ml x 2). The organic
layer was dried over MgSO4. The residue was taken
on a silica gel column (DCM/methanol = 9:1 as eluents)
to give a yellow solid (0.37 g, 57%) of the title com-
pound; TLC Rf = 0.3 (DCM/MeOH = 9:1). mp: 118–
119 ꢁC, 1H NMR (300 MHz, CDCl3 ): d 6.87 (d,
J = 8.1 Hz, 1H, Aryl H), 6.58 (dd, J = 8.1 Hz, 2.1 Hz,
1H, Aryl H); 6.24 (d, J = 2.1 Hz, 1H, Aryl H), 3.58 (s,
2H, Ph-CH2–N), 2.87 (dd, J = 5 Hz, 4H, Ph-CH2–
CH2–N), 2.48 (d, J = 7 Hz, 2H, N–CH2); 1.13–1.10 (m,
1H); 0.61–0.57 (m, 2H), 0.21 (q, J = 4.8 Hz, 2H). FAB-
MS m/z: 204 (M + 1) (C13H17NO).
5.1.3. 7-Chloroacetoxy-N-trifluoroacetyl-1,2,3,4-tetrahy-
droisoquinoline (6). Compound 5 (8.4 g, 27 mmol) was
dissolved in anhydrous CH2Cl2 (58 ml) and 3-chloroper-
oxybenzoic acid (12.3 g) was added in one portion. The
suspension was cooled to 0 ꢁC, and trifluoroacetic acid
(2.0 ml) was added dropwise over 5–10 min. The reac-
tion flask was protected from light, and the mixture
was stirred for 3–5 days at room temperature, poured
onto water (100 ml), and neutralized with ammonium
hydroxide solution. The layers were separated, and the
aqueous layer was extracted with CH2Cl2 (60 ml). After
purification on
EtOAc = 2:1) the title compound was obtained as a yel-
a
silica gel column (n-hexane/
5.1.7. General procedure for the preparation of 11a–d and
12a–d. To a stirred and ice-cooled solution of 8 or 9
(1.0 equiv) in CH3CN was added each of N,N-dialkyl-
carbamyl chloride (10a–d), followed by dropwise addi-
tion of NaH (60% in mineral oil, 1.3 equiv). The
reaction mixture was stirred for 2 h at room temperature
under argon. After evaporation of the solvent in vacuo,
water was added and extracted with EtOAc (3·). The
organic layers were combined, dried over MgSO4, and
evaporated to dryness in vacuo. The residue was purified
by column chromatography to afford title compound.
low oil (4.24 g, 48%); TLC Rf = 0.5 (n-hexane/
1
EtOAc = 3:1). H NMR (300 MHz, CDCl3): d 7.20 (t,
J = 7 Hz, 1H, Aryl H), 7.01–6.93 (m, 2H, 2H, Aryl
Hs), 4.75 (s, 2H, Ph-CH2–N), 4.33 (s, 2H, CO–CH2–),
3.90–3.82 ( m, 2H, N–CH2–), 2.97–2.92 (m, 2H, Ph-
CH2–). FABMS m/z: 322 (M + 1) (C13H11NO3ClF3).
5.1.4. 7-Hydroxy-1,2,3,4-tetrahydroisoquinoline hydro-
chloride salt (7). To a solution of 6 (3.6 g, 11 mmol) in
MeOH (100 ml) was added 15% of aqueous NaSMe
(10 ml, 107 mmol) at 20 ꢁC. After 2 h, the mixture was
acidified with 3 N HCl to pH 1. The resulting precipitate
was collected as a white solid: Rf = 0.2 (DCM/
5.1.7.1. 7-(N-Methyl-N-ethylcarbamoyloxy)-N-prop-
argyl-1,2,3,4-tetrahydroisoquinoline (11a). Purified on a
silica gel column (EtOAc/n-hexane = 2:1); Yield 50%;
1H NMR (300 MHz, CDCl3): d 7.08 (d, J = 8 Hz, 1H,
Aryl H); 6.88 (d, J = 7 Hz, 1H, Aryl H), 6.80 (s, 1H,
Aryl H), 3.76 (s, 2H, Ph-CH2–N), 3.50 (d, J = 2 Hz,
2H, N–CH2–CCH), 3.42(q, J = 7 Hz, 2H, N–CH2–
CH3), 3.04–2.81 (m, 7H, N–CH3, Ph-CH2–CH2–N),
2.28–2.26 (m, 1H, C„CH), 1.25–1.15 (m, 3H, N–
CH2–CH3); IR (KBr): m cmꢀ1 = 3300, 2923, 1715. FAB-
MS m/z: 273 (M + 1) (C16H20N2O2).
1
MeOH = 5:1), H NMR (300 MHz, D2O): d 7.15–7.00
(m, 3H, 2H, Aryl Hs), 4.22 (d, J = 6 Hz, 2H, Ph-CH2–
N), 3.80–3.58 (m, 2H, Ph-CH2–CH2–N), 2.65 (t,
J = 5.4 Hz, 2H, Ph-CH2–CH2–N); FABMS m/z: 186
(M + 1) (C9H11NOÆHCl).
5.1.5. 7-Hydroxy-N-propargyl-1,2,3,4-tetrahydroisoquin-
oline (8). To a stirred mixture of hydrochloride salt 7
(1.7 g, 11 mmol) and K2CO3 (3 g, 22 mmol) in CH3CN
(33 ml) was added a solution of 80% propargyl bromide
(1.3 ml, 11 mmol). The reaction mixture was continued
to stir at room temperature under nitrogen for 25 h and
filtered. The filtrate was washed with water and the aque-
ous layer was extracted with EtOAc (2 · 40 ml). The or-
ganic layer was dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. The resulting residue was ta-
ken on a silica gel column (DCM/MeOH = 9:1 as eluents)
to give a brown solid (110 mg, 50%) of the title com-
pound: Rf = 0.4 (DCM/MeOH = 9:1); FABMS: m/z 188
5.1.7.2. 7-(1-Pyrrolidinecarbamoyloxy)-N-propargyl-
1,2,3,4-tetrahydroisoquinoline (11b). Purified on a silica
gel column (EtOAc/n-hexane = 2:1); Yield 53%; 1H
NMR (300 MHz, CDCl3): d 7.08 (d, J = 8 Hz, 1H, Aryl
H), d 6.91 (dd, J = 8 Hz, 2 Hz, 1H, Aryl H), 6.83 (d,
J = 2 Hz, 1H, Aryl H), 3.81 (s, 2H, Ph-CH2), 3.55–
3.44 (m, 6H, 2 N–CH2–CCH), 2.94–2.90 (m, 4H, N–
CH2–CH2), 2.30 (t, J = 3 Hz, 1H, C„CH), 1.98–1.89
(m, 4H); IR (KBr): m cmꢀ1 3375, 2951, 1715. FABMS
m/z: 285 (M + 1) (C17H20N2O2).
1
[M + H]+; mp: 82–83 ꢁC; H NMR (300 MHz, CDCl3):
d 6.91 (d, J = 8.1 Hz, 1H, Aryl H), 6.60 (d, J = 8.1 Hz,
1H, Aryl H), 6.38 (s, 1H, Aryl H), 3.69 (d,
J = 23 Hz,2H, Ph-CH2–N), 3.51 (d, J = 1.8 Hz, 2H, N–
CH2–C„C–), 2.84 (t, J = 4 Hz, 4H, Ph-CH2–CH2–N),
2.31 (t, J = 2 Hz, 1H, C„CH). FABMS m/z: 188
(M + 1) (C12H13NO).
5.1.7.3. 7-(1-Piperidinecarbamoyloxy)-N-propargyl-
1,2,3,4-tetrahydroisoquinoline (11c). Purified on a silica
gel column (EtOAc/n-hexane = 2:1); Yield 50%; mp:
72–73 ꢁC; 1H NMR (300 MHz, CDCl3): d 7.08 (d,
J = 8 Hz, 1H, Aryl H), 6.87 (dd, J = 8 Hz, 2 Hz, 1H,
Aryl H), 6.80 (d, J = 2 Hz, 1H, Aryl H), 3.77 (s, 2H,
Ph-CH2–N), 3.52 (t, J = 15 Hz, 4H, N–CH2ꢀ), 2.94–
2.85 (m, 4H, Ph-CH2–CH2–N); 2.28 (t, J = 2 Hz, 1H,
C„CH), 1.63 (s, 6H,); IR (KBr): m cmꢀ1 = 3255, 2926,
1717. FABMS m/z: 299 (M + 1) (C18H22N2O2).
5.1.6. 7-Hydroxy-N-cyclopropylmethyl-1,2,3,4-tetrahy-
droisoquinoline (9). To a stirred mixture of hydrochlo-
ride salt 7 (0.9 g, 6 mmol) and potassium carbonate

M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
1963
5.1.7.4. 7-(1-Morpholinecarbamoyloxy)-N-propargyl-
1,2,3,4-tetrahydroisoquinoline (11d). Purified on a silica
gel column (EtOAc/n-hexane = 1:2); Yield 82%); 1H
NMR (300 MHz, CDCl3): d 7.10 (d, J = 8.4 Hz, 1H,
Aryl H), 6.90 (dd, J = 8.4 Hz, 2.4 Hz, 1H, Aryl H),
6.81 (d, J = 2.4 Hz, 1H, Aryl H), 3.83–3.56 (m, 12H,
2–OCH2CH2N–, Ph-CH2–N, N–CH2–C„CH), 2.96–
2.92 (m, 4H, Ph-CH2–CH2–N), 2.32 (t, J = 2.4 Hz, 1H,
–C„CH); IR (KBr): m cmꢀ1 = 3232), 2951, 1706. FAB-
MS m/z: 301 (M + 1) (C17H20N2O3).
5.1.8. N-Propargyl-1,2,3,4-tetrahydroisoquinoline (13).
To a stirred mixture of 3 (2 ml, 15 mmol) and K2CO3
(2.07 g, 15 mmol) in CH3CN (30 ml) was added a solu-
tion of 80% propargyl bromide (1.7 ml, 15 mmol). The
reaction mixture was continued to stir at room temper-
ature under nitrogen for 25 h and filtered. The filtrate
was washed with water and the aqueous layer was ex-
tracted with EtOAc (2 · 40 ml). The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The resulting residue was taken on a silica gel
column (DCM/MeOH = 9:1) to give a brown solid
(1.28 g, 75%) of the title compound: Rf = 0.3 (DCM/
MeOH = 9:1); mp: 102–104 ꢁC; 1H NMR (300 MHz,
CDCl3): d 7.38–7.16 (m, 4H, Aryl Hs), 3.92 (s, 2H,
Ar-CH2–N), 3.65 (d, J = 2.4 Hz, 2H, N–CH2–C„C–),
3.08 (t, J = 5.4 Hz, 4H, Ph-CH2–CH2–N), 2.97 (t,
J = 5.2 Hz, 4H, Ph-CH2–CH2–N), 2.41 (t, J = 2.4 Hz,
1H, C„CH). FABMS m/z: 172 (M + 1) (C12H13N).
5.1.7.5. 7-(N-Methyl-N-ethylcarbamoyloxy)-N-cyclo-
propylmethyl-1,2,3,4-tetrahydroisoquinoline (12a). Puri-
fied on a silica gel column (DCM/MeOH = 10:1);
1
Yield 50%; H NMR ( 300 MHz, CDCl3 ): d 7.06 (d,
J = 8 Hz, 1H, Aryl H); 6.84 (s, 1H, Aryl H); 6.81 (s,
1H, Aryl H); 3.71 (s, 2H, Ph-CH2–N); d 3.44, 3.39
(dd, J = 7 Hz, 2H, CH3–N–CH2CH3); 3.03–2.75 (m,
7H, CH3–N–CH2CH3, Ph-CH2–CH2–N); 2.42 (d,
J = 6 Hz, 2H, N–CH2); 1.24–1.17 (m, 3H, -CH3); 1.14–
0.94 (q, J = 7 Hz, 1H); 0.58–0.52 (m, 2H); 0.19–0.14
(m, 2H); IR (KBr): m cmꢀ1 = 2924, 1715. FABMS m/z:
289 (M + 1) (C17H24N2O2).
5.1.9. 7-Chloroacetyl-1,2,3,4-tetrahydroisoquinoline (14).
To a solution of 5 (4.4 g, 14 mmol) in MeOH (50 ml)
was added 15% of aqueous NaSMe (5 ml, 53 mmol) at
20 ꢁC. After 2 h, the reaction mixture was concentrated
in vacuo, diluted with EtOAc (50 ml), and washed with
10% aqueous sodium thiosulfate (2 · 20 ml) and brine.
The organic layer was evaporated and taken on a silica
gel column (DCM/MeOH = 9:1) to give a white solid
(1.98, 68%): Rf = 0.4 (DCM/MeOH = 8:1), 1H NMR
(300 MHz, CDCl3): d 7.76 (d, J = 4.2 Hz, 1H, Aryl H),
7.24–7.18 (m, 2H, Aryl Hs), 4.66 (s, 2H, Ph-CH2–N),
4.44 (s, 2H, –CH2–C@O), 3.76–4.65 (m, 2H, –CH2–N),
2.98–2.86 (m, 2H, Ph-CH2–). FABMS m/z: 210
(M + 1) (C11H12NOCl).
5.1.7.6. 7-(1-Pyrrolidinecarbamoyloxy)-N-cyclopro-
pylmethyl-1,2,3,4-tetra-hydroisoquinoline (12b). Purified
on a silica gel column (DCM/MeOH = 20:1); Yield
50%; 1H NMR (300 MHz, CDCl3):
d 7.08 (d,
J = 8.1 Hz, 1H, Aryl H), 6.90 (d, J = 8.1 Hz, 1H, Aryl
H); d 6.84 (s, 1H, Aryl H), 3.80 (s, 2H, Ph-CH2–N),
3.54 (t, J = 5.1 Hz, 2H, N–CH2–), 3.46 (t, J = 5.1 Hz,
2H, N–CH2–), 2.92 (br s, 4H, Ph-CH2–CH2–N); d 2.51
(d, J = 4.8 Hz, 2H, N–CH2–C„C–); 1.92 (br s, 4H,
–CH2–CH2–); d 1.03 (br s, 1H); 0.58 (dd, J = 6.3 Hz,
1.5 Hz, 2H); 0.20 (d, J = 1.5 Hz, 2H); IR (KBr):
5.1.10. 7-Chloroacetyl-N-propargyl-1,2,3,4-tetrahydro-
isoquinoline (15). Via acetylation of 13. According to
the reaction procedure for preparing 5, 13 (1.28 g,
7.5 mmol) was acetylated to give 15 as a white solid
(0.85 g, 46%). Via propargylation of 14: A mixture of
14 (0.8 g, 3.8 mmol) and K2CO3in CH3CN was treated
with a solution of 80% propargyl bromide (0.45 ml,
3.9 mmol) to give 15 as a white solid (0.38 g, 40%);
m cmꢀ1 = ,2876,
(M + 1)(C18H24N2O2).
1715.
FABMS
m/z:
301
5.1.7.7. 7-(1-Piperidinecarbamoyloxy)-N-cyclopro-
pylmethyl-1,2,3,4-tetrahydroisoquinoline (12c). Purified
on a silica gel column (DCM/MeOH = 12:1); Yield
50%; 1H NMR ( 300 MHz, CDCl3): d 7.06 (d,
J = 8.4 Hz, 1H, Aryl H); 6.86 (dd, J = 8.4 Hz, 2.4 Hz,
1H, Aryl H), 6.80 (d, J = 2.4 Hz, 1H, Aryl H), 3.71 (s,
2H, Ph-CH2–N), 3.57 (br s, 2H, N–CH2–), 3.50 (br s,
2H, N–CH2–), 2.92–2.81 (m, 4H, Ph-CH2–CH2–N),
2.44 (d, J = 6.9 Hz, 2H, N–CH2), 1.62 (br s, 6H);
1.00–0.95 (m, 1H); 0.59–0.53 (m, 2H); 0.20–0.15 (m,
2H); IR (KBr): m cmꢀ1 = 2924, 1717. FABMS m/z: 315
(M + 1)(C19H26N2O2).
1
Rf = 0.35 (n-hexane/EtOAc = 4:1); mp: 108–110 ꢁC; H
NMR (300 MHz, CDCl3): d 7.75 (q, J = 5.2 Hz, 1H,
Aryl H), 7.70 (s, 1H, Aryl H), 7.28 (d, J = 5.0 Hz, 1H,
Aryl H ), 4.67 (s, 2H, –CH2–C@O), 4.06 (s, 2H, Ph-
CH2–N), 3.73 (br s, 2H, N–CH2ꢁCH), 3.16–3.10 (m,
2H, Ph-CH2–), 2.45 (br s, 1H, C„CH),. FABMS m/z:
248 (M + 1) (C14H14ClNO2).
5.2. X-ray crystal structure determination of 7-chloro-
acetyl-N-propargyl-1,2,3,4-tetrahydroisoquinoline (15)
5.1.7.8. 7-(1-Morpholinecarbamoyloxy)-N-cyclopro-
pylmethyl-1,2,3,4-tetrahydroisoquinoline (12d). Purified
on a silica gel column (DCM/MeOH = 10:1); Yield
A
selected single crystal (dimensions 0.30 mm ·
51%; 1H NMR (300 MHz, CDCl3):
d
7.08 (d,
0.25 mm · 0.20 mm) of 15 was mounted onto a glass fi-
ber and set up on a Nonius Kappa-CCD diffractometer.
Diffraction data were collected at room-temperature Mo
J = 8.2 Hz, 1H, Aryl H), 6.86 (d, J = 8.2 Hz, 1H,
Aryl H), 6.81 (s, 1H, Aryl H), 3.75–3.57 (m, 10H,
O-(CH2–CH2)2–N-, Ph-CH2–N), 2.90 (t, J = 5.7 Hz,
4H, –CH2–N ), 2.80 (t, J = 5.7 Hz, 4H, Ph-CH2–),
2.42 (d, J = 6.6 Hz, N–CH2–), 0.98–0.92 (m, 1H),
0.59–0.53 (m, 2H), 0.19–0.15 (m, 2H); IR (KBr):
m cmꢀ1 = 2924, 1717. FABMS m/z: 317 (M + 1)
(C18H24N2O3).
˚
Ka radiation (k = 0.71073 A). Unit cell parameter deter-
mination, data collection strategy, and integration were
carried out with the Nonius EVAL-14 suite of pro-
grams. The data were corrected from absorption by a
multi-scan method. The structure was solved by direct
methods with SHELXS-86, refined by full matrix

1964
M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
least-squares on F2. Graphics were carried out with
DIAMOND. All non-H atoms were refined with aniso-
tropic displacement parameters and H atoms were sim-
ply introduced at calculated positions (riding model).
Crystallographic data: C14H14ClNO, MW, 247.70;
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 0 cells/well was subtracted from
these data.
ꢀ
˚
˚
monoclinic, P1 (No. 2); a, 13.275(2) A; b, 14.472(4) A;
3
˚
˚
c, 6.529(2) A; V, 1250.54(9) A ; Z, 4; Dcalc, 1.310 mg/
m3; F(000), 516; l, 0.288 mmꢀ1; 2864 observed data,
with I > 2r (I).
5.3.4. Cell culture and cell lines. Human embryonic kid-
ney cells (HEK293) were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS) and 0.1 mg/ml penicillin
and streptomycin. T-REx293 cells were purchased from
Invitrogen and cultured in DMEM supplemented with
10% FBS and 5 mg/ml blasticidin. The generation of sta-
bly transfected cell lines, T20 and c-30, has been de-
5.3. Pharmacological evaluation
5.3.1. Reagents. Anti-ERK1/2 MAP kinase antibody
and anti-MEK antibody were purchased from Cell Sig-
naling Technology, Inc. Horseradish peroxidase-conju-
gated anti-rabbit IgG was from Santa Cruz
Biotechnology, Inc. Horseradish peroxidase-conjugated
anti-mouse IgG and ECL Western Blotting detection re-
agents were from Amersham Biosciences. Dulbecco’s
modified Eagle’s medium (DMEM) and fetal bovine ser-
um (FBS) were from Invitrogen and Biological Indus-
tries Ltd (Kibbtuz Beit Haemek, Israel), respectively.
All other reagents were of at least reagent grade and ob-
tained from standard suppliers.
scribed previously.13 Cells were incubated in
a
humidified incubator at 37 ꢁC in 5% CO2.
5.3.5. MAO-B assays.
5.3.5.1. Fluorometric assay of rat brain protein
content. MAO-B enzyme preparations were prepared
from brain cortex of decapitalized male F344/N rats.
The brain cortex in PBS solution was frozen with liquid
nitrogen and kept at ꢀ80 ꢁC for 3 days. The homoge-
nate was then obtained by centrifugation (14,000 rpm,
10 min) of the frontal cortex in 0.1 M potassium phos-
phate buffer (pH 7.4) at 4 ꢁC and the supernatant was
taken as the enzyme source for MAO. A standard solu-
tion containing 0.1 M potassium phosphate buffer and
albumin were prepared in different concentration
grades. Ten microliters of each solution was taken and
added with 200 ll of BCA kit. Following the similar
method, the above supernatant from brain cortex was
diluted and 10 ll of stock solution taken and added with
200 ll of BCA kit. Both standard and sample solutions
were incubated at 37 for 30 min. Each of 200 ll of the
solutions was taken to 96-well microplate and the pro-
tein contents were measured with a fluorescence micro-
plate reader by determination of optical density at
595 nm.
5.3.2. Cell-based c-secretase assays. The generation of
T20 cells has been reported previously.9 T20 cells were
routinely maintained in DMEM supplemented with
10% FBS, 200 lg/ml hygromycin, 5 lg/ml blasticidin,
and 250 lg/ml zeocin (DMEM-HZB). The stably trans-
fected T20 cells were detached from culture dishes by
trypsinization, washed with PBS, and resuspended in
DMEM-HZB, followed by plating onto 96-well micro-
plates (2 · 104 cells/50 ll/well) and incubating at 37 ꢁC
for 24 h. Preliminarily, compounds diluted in DMEM-
HZB were added to a final concentration of 10 lM in
the presence of tetracycline (1 lg/ml). Treatments were
terminated after incubation at 37 ꢁC for 24 h by directly
adding an equivalent volume of the Steady-Glo lucifer-
ase assay reagent (Promega), 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
signal from the stable line without tetracycline induction
and 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.
5.3.5.2. MAO-B inhibitory assays. The supernatant
(protein content, 80 lg/ml) in 96-well microplate was
added with 1 U/ml HRP, 1 mM benzylamine, and
50 lM Amplex red in 0.1 M potassium phosphate buffer
followed by the tested sample, which was prepared in
200 lg/ml in DMSO as stock solution. These mixtures
were incubated at room temperature for 60 min and
optical density determined by a fluorometric reader.
Inhibition ð%Þ ¼ 1 ꢀ ½ðOD595 of sample=protein
ꢀ OD595 of sampleÞ=OD595 of blankꢂ
ꢃ 100%
5.3.3. Cell viability assay. T20 cells (5 · 104/100 ll/well)
were seeded onto the wells of 96-well microplates in cul-
ture medium containing 10 lM of respective compounds
and incubated at 37 ꢁC for 24 h. Viable cells were deter-
mined using the CellTiter 96ꢂ Aqueous Non-Radioac-
tive Cell Proliferation Assay (Promega) as specified in
the manufacturer’s instructions. Briefly, following the
addition of the combined MTS/PMS solution (20 ll/
well) microplates were incubated 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 Syn-
ergy HT ELISA plate reader (BioTek). The number of
living cells in culture was directly proportional to the
5.3.6. Assay for extracellular signal-regulated kinase
(ERK) activity. The activation of the ERKs was mea-
sured by using anti-phospho-ERK1/2 and anti-ERK1/
2 antibodies (Cell Signaling Technology). In brief, T20
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 med-
ium containing 1 lg/ml tetracycline at 37 ꢁC for 18 h.
Before the experiments, we replaced the medium with

M.-K. Hu et al. / Bioorg. Med. Chem. 16 (2008) 1957–1965
1965
DMEM containing 0.5% FBS and treated it with the
Cheng-Kung
acknowledged.
University
are
also
gratefully
tested compounds (10 lM each). After treatment, reac-
tions were stopped by placing cells on ice and aspirating
the medium. Cells were harvested and lysed in 50 mM
Tris-HCl (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). Each cell lysate, which contained 50 lg pro-
teins, was separated on 12% SDS–polyacrylamide elec-
trophoresis gels, immunoblotted, and identified using
anti-phospho-ERK1/2 or anti-ERK1/2 antibody.
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Acknowledgments
This work was supported by the National Science
Council, Taiwan, of the Republic of China (NSC
93-2320-B-016-046 to M.H. and NSC 95-3112-B-001-
006 to Y.-F.L.) and Academia Sinica (to Y.-F.L.).
High-resolution mass spectra analyses performed by
the National Science Council Regional Instruments
Center at National Taiwan University and National