Synthesis of aminoalkyl-substituted coumarin derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.01.010

Alzheimer's disease, one of the most common forms of dementia, is a progressive neurodegenerative disorder symptomatically characterized by declines in memory and cognitive abilities. To date, the successful therapeutic strategy to treat AD is maintaining levels of acetylcholine by inhibiting acetylcholinesterase (AChE). In the present study, coumarin derivatives were designed and synthesized as AChE inhibitors based on the lead structure of scopoletin. Of those synthesized, pyrrolidine-substituted coumarins 3b and 3f showed ca. 160-fold higher AChE inhibitory activities than scopoletin. These compounds also ameliorated scopolamine-induced memory deficit in mice when administered orally at the dose of 1 and 2 mg/kg.

Full text of DOI:10.1016/j.bmc.2014.01.010

Bioorganic & Medicinal Chemistry 22 (2014) 1262–1267
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of aminoalkyl-substituted coumarin derivatives
as acetylcholinesterase inhibitors
Seung Ok Nam a, , Dong Hyun Park a, , Young Hun Lee a, Jong Hoon Ryu a,b, Yong Sup Lee a,c,
⇑
a Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
b Department of Oriental Pharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Republic of Korea
c Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease, one of the most common forms of dementia, is a progressive neurodegenerative
disorder symptomatically characterized by declines in memory and cognitive abilities. To date, the
successful therapeutic strategy to treat AD is maintaining levels of acetylcholine by inhibiting acetylcho-
linesterase (AChE). In the present study, coumarin derivatives were designed and synthesized as AChE
inhibitors based on the lead structure of scopoletin. Of those synthesized, pyrrolidine-substituted couma-
rins 3b and 3f showed ca. 160-fold higher AChE inhibitory activities than scopoletin. These compounds
also ameliorated scopolamine-induced memory deficit in mice when administered orally at the dose of
1 and 2 mg/kg.
Received 30 November 2013
Revised 4 January 2014
Accepted 6 January 2014
Available online 11 January 2014
Keywords:
Acetylcholinesterase inhibitor
Alzheimer’s disease
Coumarin
Ó 2014 Elsevier Ltd. All rights reserved.
Scopoletin
Galantamine
Memory
1. Introduction
Recently, pharmaceutical agents developed based on the b-amy-
loid hypotheses such as semagacestat (c-secretase inhibitor), bap-
Alzheimer’s disease (AD), one of the most common forms of
dementia, is a progressive neurodegenerative disorder symptomat-
ically characterized by declines in memory and cognitive abilities.1
AD is the fourth leading cause of death in developed countries, and
because most AD patients are over 65 years old, numbers are
expected to increase in parallel with population aging.2 The esti-
mated number of AD patients worldwide in 2006 was 26.6 million
and is predicted to increase to 106.8 million in 2050.3 The major
pathological features of AD are b-amyloid plaque deposition and
the presence of neurofibrillary tangles in the brain.4 However,
the precise causes of AD at the molecular level remain unclear de-
spite many suggested hypotheses. Further effective therapy that
can delay the neurodegeneration is not available yet.5
A deficit in cholinergic neurotransmission, due to the degener-
ation of cholinergic neurons, in the brain is believed to be one of
the major causes of the memory impairments associated with
AD.6 The maintenance of acetylcholine (ACh) levels in the synaptic
cleft by inhibiting acetylcholinesterase (AChE), which is responsi-
ble for the degradation of ACh, is the only known means of treating
AD. The four AChE inhibitors used in clinical practice for treatment
of AD are tacrine, donepezil, galantamine, and rivastigmine.7
ineuzumab and solanezumab (anti-Ab antibody) were failed in
phase III clinical trials.8,9 Therefore, novel AChE inhibitors possess-
ing less adverse effects and more cognitive enhancing effects could
contribute to extend the choices to AD patients and clinical
practitioners.
(ꢀ)-Galantamine (1) is an alkaloid present in the Caucasian
snow-drop (Galanthus woronowii) and in bulbs of the Amaryllida-
ceae family, and was recently approved by the US FDA and Europe
for the treatment of AD.10 (ꢀ)-Galantamine (1) is a selective,
reversible, competitive AChE inhibitor and allosteric modulator of
neural nicotinic acetylcholine receptors.11 The clinical experiences
of galantamine in AD patients are promising, but it is less potent
than other approved AChE inhibitors like tacrine and often causes
peripheral side effects.12 Furthermore, the production of galanta-
mine requires a lengthy series of reactions because of its complex
structure.
Pharmacophore modeling provides a useful tool of identifying
hit compounds of pharmacologic interest.13 Recently, scopoletin
(2) (a widely distributed coumarin derivative found in the Solana-
ceae family) was identified as a potential AChE inhibitor by virtual
screening of a 3D database of natural products based on the com-
plex structure of galantamine-AChE.14 Furthermore, although
scopoletin has much less AChE inhibitory activity (IC50 = 168.6
than galantamine (1, IC50 = 3.2 M), it was found to increase extra-
cellular ACh concentrations in rat brain to the same level as 1.
lM)
⇑
Corresponding author. Tel.: +82 2 961 0370; fax: +82 2 966 3885.
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
These authors equally contributed to this study.
l
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.010

S. O. Nam et al. / Bioorg. Med. Chem. 22 (2014) 1262–1267
1263
Accordingly, the aim of this study was to develop new coumarin
2. Chemistry
derivatives starting from scopoletin with enhanced AChE inhibi-
tory activities (Fig. 1).
The syntheses of aminoethyl-substituted coumarin derivatives
3a–3d were accomplished using the procedures shown in
Scheme 1. C-7 MOM-protected esculetin (4), which was obtained
from esculetin, as previously described,16 was used as a starting
material. Compound 4 was reacted with four commercially avail-
able aminoethyl chlorides in the presence of 1.5 equivalents of
K2CO3 and 1 equiv of Cs2CO3, to provide 5a–5d in 20–51% yields.17
The MOM group in 5a–5d was removed using 3 N HCl in methanol
to afford 3a–3d as hydrochloride salts in 85–95% yields.
Since many aminopropyl chlorides are not available commer-
cially, aminopropyl-substituted coumarin derivatives 3e–3j were
prepared by synthesizing 3-halopropyloxy coumarin 6, as shown
in Scheme 2. Compound 4 was reacted with 1-bromo-3-chloropro-
pane to give 3-chloropropyl-substituted coumarin. 3-Bromopro-
pyl-substituted coumarin was also obtained as a minor product
and the combined yield of the 3-halopropyl-substituted coumarin
6 was ca. 80%. Because both chloropropyloxy- and bromopropyl-
oxy coumarins give the same products in next step, the mixture
of 3-halopropyl-substituted coumarin 6 was reacted with various
amines in the presence of K2CO3 and Cs2CO3 to afford 7. Subse-
quent removal of the MOM group of 7 using 3 N HCl in methanol
afforded 3e–3j as hydrochloride salts in 16–38% overall yields for
two steps from 6.
A pharmacophore model generated from the three-dimensional
structure of the galantamine-AChE complex and virtual screening
based on this information indicated that the C-7 hydroxyl, lactone
oxygen, and C-6 methoxy methyl in scopoletin are important fea-
tures for AChE inhibitory activity, because they probably interact
with the catalytic site of AChE as a H-bond donor, a H-bond accep-
tor, and a hydrophobic group, respectively.14,15 Accordingly, based
on these previous findings, we modified the C-6 methoxy methyl in
2 by incorporating aminoalkyl substituents. Additionally, amino
groups were introduced into alkyl chain to increase water solubil-
ity, and thus, enhance oral bioavailability, and the effect of the
length of the alkyl chain between C-6 oxygen and amino moieties
was explored. Resultantly, a series of coumarin derivatives 3a–3j
were synthesized via a simple three-step sequence of reactions
from scopoletin, and screened for their AChE inhibitory activities.
In addition, synthesized compounds were also assessed for their
memory ameliorating abilities in a mouse model of scopolamine-
induced memory impairment using the passive avoidance task.
OCH3
H
•
HCl
H3CO
HO
O
R1R2N
OH
n
3. Results and discussion
O
O
HO
O
O
N
CH3
3.1. The AChE inhibitory effects of coumarin derivatives
1, Galantamine
2
3
,Scopoletin
To determine inhibitory activities of compounds 3a–3j on AChE,
in vitro AChE inhibition assays were conducted according to the
Figure 1. Structures of galantamine (1), scopoletin (2) and target compounds (3).
R1R2(CH2)2Cl
• HCl
R1R2N
O
R1R2N
O
HO
K2CO3, Cs2CO3
3N-HCl
MeOH
MOMO
O
O
DMF, rt.
HO
O
3a~3d
O
MOMO
O
O
4
5a~5d
NR1R2
:
NR1R2
:
45%
3a
3b
3c
3d
N
N
5a
5b
5c
5d
96%
condition:
see ref. 14
N
N
51%
21%
20%
84%
85%
88%
HO
HO
N
N
O
O
N
N
esculetin
O
O
Scheme 1.
R1R2NH
K2CO3, Cs2CO3
Cl
Br
Cl
(Br)
O
K2CO3, Cs2CO3
4
MOMO
O
O
DMF, rt.
DMF, rt
80%
6
• HCl
R1R2N
O
R1R2N
O
3N-HCl
MeOH
MOMO
O
O
HO
3e~3j
O
O
7
NR1R2
:
3e
3f
3h
3i
N
N
16%
19%
16%
38%
O
N
N
N
N
CH3
CH3
CH3
3g
17% 3j
30%
N
H
Scheme 2.

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S. O. Nam et al. / Bioorg. Med. Chem. 22 (2014) 1262–1267
modification of Ellman’s method18,19 using mouse brain homoge-
nates. Scopoletin and galantamine were used as standards, and re-
sults are summarized in Table 1. Initially, preliminary screening for
tested for their effects on scopolamine-induced learning and
memory impairments using the passive avoidance task. The
reversal of scopolamine-induced memory deficit in the passive
avoidance task has been widely used to screen for agents that
ameliorate cognition dysfunction. During the acquisition trial,
no differences in latencies were observed among tested groups,
which indicated that 3b and 3f do not affect the general behavior
of mice. The activity profiles of 3b and 3f in this model are shown
in Figure 2
AChE inhibitory activities were performed at 100
lM. Selected
compounds exhibiting more than 50% inhibition, were re-screened
at half-log scale concentrations to elucidate their IC50 values. Al-
most all synthesized compounds were found to be more potent
than scopoletin. Furthermore, potency was found to be influenced
substantially by amine substituents, but not by alkyl chain length.
The pyrrolidine-substituted coumarins 3b and 3f were found to
have the most potent inhibitory activities with IC50 values of
For retention trials, the mean step-through latency of scopol-
amine-treated (1 mg/kg, ip) mice was significantly shorter than
that of saline-treated controls (P <0.05). Galantamine (1 mg/kg,
p.o.) as a positive control significantly increased step-through ver-
sus scopolamine treated mice. Compounds 3b and 3f also antag-
onized scopolamine-induced memory deficit in mice when orally
administered at 0.25, 0.5 and 5 mg/kg. These compounds reversed
clearly the shortened step-through latency induced by scopol-
amine. Inverted U-shaped dose–response relationships were
found with significantly (P <0.05) longer latencies for doses of
2 mg/kg (3b) and 1 mg/kg (3f) as compared with the scopolamine
treated animals. Such inverted U-shaped dose–response relation-
ship which was observed in compound 3b or 3f has been well
recognized in the research area of learning and memory. Cala-
brese reviewed a general biological model to attempt to explain
this dose–response, namely, the hormetic dose–response model.20
Under the same conditions, the relative effects of 3b (2 mg/kg)
and 3f (1 mg/kg) to increase step-through latency were similar
to that of galantamine (1 mg/kg, p.o.) (Fig. 2). Finally, coumarin
derivatives 3b and 3f exhibited memory enhancing effects even
higher than galantamine by increasing the latency on passive
avoidance response in scopolamine-induced memory deficit
model.
6.85 and 2.87
than scopoletin (IC50 = 476.37
antamine (IC50 = 2.50 M). The diethylamine, that is, the open ana-
lM, and the potency of 3f was ca. 160-fold higher
l
M) and nearly equal to that of gal-
l
log of pyrrolidine ring, and piperidine, and isopropylamine-
substituted coumarins also showed potent AChE inhibitory activi-
ties. However, replacement of the pyrrolidine ring in 3b or 3f with
a morpholine or a piperazine ring, both of which have additional
hetero atoms, significantly reduced AChE inhibition (i.e., 3d, 3h–
3j) implicating the requirement of hydrophobic interactions in this
binding region of the enzymes for strong inhibition.
3.2. The amelioration of scopolamine-induced memory deficit
in mice
Many efforts have been made, based on the cholinergic
hypothesis, to develop AChE inhibitors that improve cognition
functions in AD patients.6 Thus, as the synthesized coumarin
derivatives showed potent AChE inhibitory effects in vitro, we
sought to determine whether they ameliorate impaired learning
and memory functions in vivo. Accordingly, the compounds 3b
and 3f, which had the greatest AChE inhibitory activities, were
Table 1
The acetylcholinesterase (AChE) inhibitory activities of 3a–3j, scopoletin, and galantamine
• xHCl
R1R2N
O
n
HO
O
O
3a-3j
Compounds
n
R1R2
% AChE inhibition at 100 (
l
M)
AChE inhibition IC50 (l
M)a
N
3a
3b
3c
3d
3e
3f
1
1
1
1
2
2
2
69.42
93.95
75.48
37.68
70.4
18.65 2.37
6.85 0.38
12.16 2.22
—
N
N
N
O
N
30.53 4.28
2.87 0.17
19.09 1.53
N
N
95.14
76.29
3g
N
N
3h
3i
2
2
2.44
—
—
O
N
40.44
CH3
CH3
CH3
3j
2
80.79
26.98 2.95
N
H
Scopoletin
Galantamine
2.49
91.94
476.37 79.54
2.50 0.14
a
50% Inhibitory concentration (means SEMs of three experiments).

S. O. Nam et al. / Bioorg. Med. Chem. 22 (2014) 1262–1267
1265
250
200
150
100
50
Aquisition trial
Retention trial
#
#
#
#
#
#
*
0
(mg/kg, p.o)
Control
0
1
0.5
Scopolamine (i.p., 1 mg/kg)
Galantamine 3b
1
2
5
0.5
1
2
1
3f
Figure 2. Effects of 3b, 3f, and galantamine on the passive avoidance task in scopolamine-induced memory deficit model. ⁄P <0.05 versus vehicle-treated controls. #P <0.05
versus scopolamine-treated group. Data are expressed as mean SEM.
J = 9.6 Hz), 5.21 (2H, s), 4.27 (2H, t, J = 5.6 Hz), 3.42 (3H, s), 3.05
(2H, t, J = 5.6 Hz), 2.82 (4H, q, J = 7.2 Hz), 0.98 (6H, t, J = 7.2 Hz).
4. Conclusions
New coumarin derivatives were designed and synthesized using
the structure of scopoletin as a starting point. Almost all synthe-
sized compounds potently inhibited AChE than scopoletin. Of the
synthesized, pyrrolidine-substituted coumarins, 3b and 3f had
the greatest inhibitory effects, and the potency of 3f was ca. 160-
fold higher than scopoletin, and nearly equal to that of galanta-
mine. Oral administrations of 3b and 3f also ameliorated scopol-
amine-induced memory deficit in mice, and the effects of 3b
(2 mg/kg) and 3f (1 mg/kg) on step-through latency were greater
than galantamine (1 mg/kg). These results suggest that 3b and 3f
have the ability to maintain ACh levels in the synaptic cleft, and
thus, to improve learning and memory by inhibiting AChE. Further-
more, the syntheses of 3b and 3f are relatively straightforward
because these compounds do not have stereogenic centers as gal-
antamine in the structures.
5.2.1.1. 7-(Methoxymethoxy)-6-(2-(pyrrolidin-1-yl)ethoxy)-2H-
chromen-2-one (5b).
The compound 5b (73 mg) was pre-
pared from 4 (100 mg, 0.45 mmol) and 1-(2-chloroethyl)pyrroli-
dine (116 mg, 0.68 mmol) using the procedure described for 5a.
Yield 51%. 1H NMR (400 MHz, CD3OD) d 7.79 (1H, d, J = 9.6 Hz),
7.13 (1H, s), 7.03 (1H, s), 6.20 (1H, d, J = 9.6 Hz), 5.21 (2H, s), 4.10
(2H, t, J = 5.6 Hz), 3.40 (3H, s), 2.89 (2H, t, J = 5.6 Hz), 2.62–2.65
(4H, m), 1.73–1.79 (4H, m).
5.2.1.2. 7-(Methoxylmethoxy)-6-(2-(piperidin-1-yl)ethoxy)-2H-
chromen-2-one (5c).
The compound 5c (16 mg) was pre-
pared from 5 (50 mg, 0.23 mmol) and 1-(2-chloroethyl)piperidine
(54 mg, 0.29 mmol) using the procedure described for 5a. Yield
21%. 1H NMR (400 MHz, CDCl3) d 7.60 (1H, d, J = 9.6 Hz), 7.17
(1H, s), 7.00 (1H, s), 6.24 (1H, d, J = 9.6 Hz), 5.27 (2H, s), 4.37 (2H,
br s), 3.49 (3H, s), 3.07 (2H, br s), 2.82 (2H, br s), 2.66 (2H, br s),
1.55 (4H, br s), 1.25 (2H, br s).
5. Experimental section
5.1. Instrumentation and chemicals
5.2.1.3.
chromen-2-one (5d).
7-(Methoxylmethoxy)-6-(2-(morpholinoethoxy)-2H-
The compound 5d (30 mg) was pre-
NMR spectra were recorded on a Brucker Avance 400 spectrom-
eter operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR
spectra. Analytical thin-layer chromatography (TLC) was per-
formed on TLC Silica gel (thickness 0.20 mm). Column chromatog-
raphy was performed with silica gel Merck Silica gel 60 (230–
400 mesh). All solvents, chemicals and reagents were purchased
from Aldrich, Acros or TCI.
pared from 5 (100 mg, 0.45 mmol) and 4-(2-chloroethyl)morpho-
line (126 mg, 0.68 mmol) using the procedure described for 5a.
Yield 20%. 1H NMR (400 MHz, CDCl3) d 7.60 (1H, d, J = 9.6 Hz),
7.14 (1H, s), 6.98 (1H, s), 6.31 (1H, d, J = 9.6 Hz), 5.30 (2H, s), 4.31
(2H, br s), 3.86 (4H, br s), 3.54 (3H, s), 2.66–3.02 (6H, m).
5.2.2. 6-(2-(Diethylamino)ethoxy)-7-hydroxy-2H-chromen-2-
one hydrochloride (3a)
5.2. Syntheses
A solution of 5a (50 mg, 0.16 mmol) in 3 N HCl (0.5 ml) and
MeOH (2 ml) was stirred at 85 oC for 3 h. The solvent was removed,
and the residue was recrystallized from ethyl acetate and n-hexane
to afford 3a (47 mg, 96%) 1H NMR (400 MHz, DMSO-d6) d 7.94 (1H,
d, J = 9.6 Hz), 7.34 (1H, s), 6.92 (1H, s), 6.25 (1H, d, J = 9.6 Hz), 4.38
(2H, br s), 3.53 (2H, br s), 3.21 (4H, q, J = 7.2 Hz), 1.27 (6H, t,
J = 7.2 Hz); 13C NMR (100 MHz, CD3OD) d 162.3, 151.5, 150.6,
144.5, 143.6, 111.8, 111.7, 111.3, 103.0, 63.4, 50.8, 48.4, 7.8.
5.2.1. 6-(2-(Diethylamino)ethoxy)-7-(methoxymethoxy)-2H-
chromen-2-one (5a)
To
a
solution of 415 (83 mg, 0.37 mmol) and 2-(diethyl-
amino)ethyl chloride (96 mg, 0.56 mmol) in DMF (5 ml) was added
K2CO3 (77 mg, 0.56 mmol) and Cs2CO3 (120 mg, 0.37 mmol). The
reaction mixture was stirred at rt for 3 d. The mixture was concen-
trated in vacuo and partitioned between H2O and CH2Cl2. The sep-
arated organic layer was washed with 10% NaOH and brine, dried
over MgSO4, and filtered. The solvent was removed under reduced
pressure and the residue was solidified from ethyl acetate and n-
hexane to give 5a (54 mg, 45%). 1H NMR (400 MHz, CDCl3) d 7.55
(1H, d, J = 9.6 Hz), 7.06 (1H, s), 6.97 (1H, s), 6.24 (1H, d,
5.2.2.1. 7-Hydroxy-6-(2-(pyrrolidin-1-yl)ethoxy)-2H-chromen-
2-one hydrochloride (3b).
The compound 3b (60 mg) was
prepared from 5b (73 mg, 0.23 mmol) using the procedure de-
scribed for 3a. Yield 84%. 1H NMR (400 MHz, CD3OD) d 7.87 (1H,

1266
S. O. Nam et al. / Bioorg. Med. Chem. 22 (2014) 1262–1267
d, J = 9.5 Hz), 7.27 (1H, s), 6.85 (1H, s), 6.26 (1H, d, J = 9.5 Hz), 4.40
(2H, t, J = 5.6 Hz), 3.72 (2H, t, J = 5.6 Hz), 3.37–3.38 (2H, m), 3.30–
3.34 (2H, m), 2.17–2.21 (4H, m); 13C NMR (100 MHz, CD3OD) d
163.6, 152.8, 152.1, 145.8, 145.0, 113.3, 113.2, 112.8, 104.5,
65.8,55.5, 55.0, 49.7, 49.3, 49.2, 23.9.
NMR (400 MHz, CD3OD) d 7.87 (1H, d, J = 9.4 Hz), 7.20 (1H, s),
6.81 (1H, s), 4.23 (2H, t, J = 5.0 Hz), 3.30–3.42 (6H, m), 2.30–2.32
(2H, m), 1.93 (4H, br s); 13C NMR (100 MHz, CD3OD) d 163.7,
151.5, 146.1, 145.9, 145.8, 113.0, 112.7, 112.6, 104.1, 68.5, 56.8,
54.7, 24.8, 24.3.
5.2.2.2. 7-Hydroxy-6-(2-piperidin-1-yl)ethoxy)-2H-chromen-2-
5.2.4.2. 7-Hydroxy-6-(3-(piperidin-1-yl)propoxy)-2H-chromen-
one hydrochlroride (3c).
The compound 3c (10 mg) was pre-
2-one hydrochloride (3g).
The compound 3g (15 mg) was
pared from 5c (12 mg, 0.04 mmol) using the procedure described
for 3a. Yield 85%. 1H NMR (400 MHz, DMSO-d6) d 7.76 (1H, d,
J = 9.6 Hz), 7.16 (1H, s), 6.73 (1H, s), 6.14 (1H, d, J = 9.6 Hz), 4.33
(2H, br s), 3.52–3.59 (2H, m), 3.38–3.42 (2H, m), 2.91 (2H, br s),
1.71–2.05 (6H, m); 13C NMR (100 MHz, CD3OD) d 163.6, 152.9,
152.2, 145.7, 145.0, 113.4, 113.3, 112.8, 104.5, 64.5, 54.7, 24.2,
22.7.
prepared from 6 (100 mg, 0.33 mmol) and piperidine (22 mg,
0.26 mmol) using the procedure described for 3e. Yield 17%. 1H
NMR (400 MHz, CD3OD) d 7.87 (1H, d, J = 9.4 Hz), 7.20 (1H, s),
6.83 (1H, s), 6.25 (1H, d, J = 9.4 Hz), 4.24 (2H, t, J = 5.4 Hz), 3.32–
3.42 (6H, m), 2.17–2.33 (4H, m), 1.80–2.00 (4H, m); 13C NMR
(100 MHz, CD3OD) d 162.2, 149.9, 144.5, 144.2, 111.3, 111.1,
110.1, 102.5, 66.7, 55.0, 52.9, 23.3, 22.8, 21.2.
5.2.2.3. 7-Hydroxy-6-(2-morpholinoethoxy)-2H-chromen-2-one
5.2.4.3. 7-Hydroxy-6-(3-morpholinopropoxy)-2H-chromen-2-
hydrochloride (3d).
The compound 3d (25 mg) was prepared
one hydrochloride (3h).
The compound 3h (14 mg) was pre-
from 5d (29 mg, 0.09 mmol) using the procedure described for 3a.
Yield 88%. 1H NMR (400 MHz, CDCl3) d 7.92 (1H, d, J = 9.6 Hz), 7.33
(1H, s), 6.86 (1H, s), 6.26 (1H, d, J = 9.6 Hz), 4.39 (2H, m), 3.87–4.05
(4H, m), 3.47–3.59 (4H, m), 3.20 (2H, m); 13C NMR (100 MHz, CD3-
OD) d 163.5, 152.8, 152.1, 145.7, 144.9, 113.5, 113.4, 112.8, 104.4,
65.0, 64.2, 57.4, 53.5.
pared from (100 mg, 0.33 mmol) and morpholine (23 mg,
6
0.26 mmol) using the procedure described for 3e. Yield 16%. 1H
NMR (400 MHz, CD3OD) d 7.93 (1H, d, J = 9.5 Hz), 7.25 (1H, s),
6.84 (1H, s), 6.25 (1H, d, J = 9.5 Hz), 4.26 (2H, t, J = 5.4 Hz), 3.97–
4.07 (4H, m), 3.48–3.54 (4H, m), 3.32–3.33 (2H, m), 2.36–2.42
(2H, m); 13C NMR (100 MHz, CD3OD) d 163.9, 152.8, 151.6, 146.2,
145.8, 112.9, 112.7, 111.7, 104.1, 68.1, 65.1, 56.8, 53.4, 24.6.
5.2.3. 6-(3-Chloropropoxy)-7-(methoxymethoxy)-2H-chromen-
2-one (6)
5.2.4.4. 7-Hydroxy-6-(3-(4-methylpiperazin-1-yl)propoxy)-2H-
To a mixture of 4 (79 mg, 0.35 mmol) and 1-bromo-3-chloro-
propane (165 mg, 1.05 mmol) in DMF (5 ml) was added K2CO3
(75 mg, 0.54 mmol) and Cs2CO3 (114 mg, 0.35 mmol). The reaction
mixture was stirred at rt for 1 d. The mixture was concentrated in
vacuo and partitioned between CH2Cl2 and H2O. The organic layer
was separated and was with 10% NaOH and brine, dried over
MgSO4, and filtered. The solvent was removed under reduced pres-
sure and the residue was purified by column chromatography
(ethyl acetate/n-hexane = 2:1) to give 6 (92 mg, 86%, calculated
based on 3-chloropropoxy compound). For major peaks; 1H NMR
(400 MHz, CDCl3) d 7.54 & 7.50 (1H, two d, J = 9.5 Hz), 7.06 &
6.99 (1H, two s), 6.88 & 6.86 (1H, two s), 6.23 & 6.22 (1H, two d,
J = 9.5 Hz), 5.20 (2H, s), 4.12–4.16 (2H, m), 3.58–3.72 (2H, m),
3.44 (3H, s), 2.18–2.28 (2H, m).
chromen-2-one dihydrochloride (3i).
The compound 3i
(34 mg) was prepared from 6 (53 mg, 0.18 mmol) and 1-methylpi-
perazine (23 mg, 0.22 mmol) using the procedure described for 3e.
Yield 38%. 1H NMR (400 MHz, CD3OD) d 7.87 (1H, d, J = 9.5 Hz), 7.20
(1H, s), 6.83 (1H, s), 6.25 (1H, d, J = 9.5 Hz), 4.26 (2H, t, J = 5.5 Hz),
3.55–3.60 (4H, m), 3.32–3.34 (4H, m), 3.03 (3H, s), 2.36–2.43 (2H,
m); 13C NMR (100 MHz, CD3OD) d 163.9, 151.6, 146.1, 145.9, 145.8,
113.1, 112.8, 112.7, 104.1, 67.9, 54.1, 51.2, 35.4, 24.9.
5.2.4.5.
7-Hydroxy-6-(3-(isopropylamino)propoxy)-2H-chro-
The compound 3j (24 mg)
men-2-one hydrochloride (3j).
was prepared from 6 (100 mg, 0.33 mmol) and isopropylamine
(15 mg, 0.26 mmol) using the procedure described for 3e. Yield
30%. 1H NMR (400 MHz, CD3OD) d 7.86 (1H, d, J = 9.4 Hz), 7.19
(1H, s), 6.80 (1H, s), 6.22 (1H, d, J = 9.4 Hz), 4.25 (2H, t,
J = 5.6 Hz), 3.46 (1H, m), 3.32–3.36 (2H, m), 2.22–2.28 (2H, m),
1.40–1.41 (6H, m); 13C NMR (100 MHz, CD3OD) d 163.8, 152.9,
151.7, 145.9, 113.0, 112.7, 111.9, 104.0, 68.7, 52.1, 44.7, 27.1, 19.3.
5.2.4. 6-(3-(Diethylamino)propoxy)-7-hydroxy-2H-chromen-2-
one hydrochloride (3e)
To a solution of 6 (90 mg, 0.30 mmol) and diethylamine (17 mg,
0.20 mmol) in DMF (4 ml) was added K2CO3 (48 mg, 0.40 mmol)
and Cs2CO3 (75 mg, 0.20 mmol). The reaction mixture was stirred
at rt for 3 d. The mixture was concentrated in vacuo and parti-
tioned between H2O and CH2Cl2. The separated organic layer was
washed with 10% NaOH and brine, dried over MgSO4, and filtered.
After evaporation of solvent, the residue was treated with 3 N HCl
(1 ml) in MeOH (2 ml) and stirred at 85 °C for 3 h. The reaction
mixture was concentrated and partitioned between H2O and CH2-
Cl2, and the separated aqueous layer was washed with CH2Cl2.
The aqueous layer was concentrated and was recrystallized from
dichloromethane and ethyl acetate to afford 3e (12 mg, 16%). 1H
NMR (400 MHz, CD3OD) d 7.75 (1H, d, J = 9.4 Hz), 7.09 (1H, s),
6.71 (1H, s), 6.12 (1H, d, J = 9.4 Hz), 4.13 (2H, t, J=5.5 Hz), 3.36–
3.33 (4H, m), 3.22–3.20 (2H, m), 2.12–2.16 (2H, m), 1.26 (6H, q,
J = 7.3 Hz); 13C NMR (100 MHz, CD3OD) d 163.8, 151.8, 145.9,
145.9, 112.9, 112.6, 112.2, 104.2, 68.2, 51.3, 49.3, 24.9, 9.2.
5.3. Acetylcholinesterase inhibition assay
Acetylcholinesterase activity assays were carried out using
acetylthiocholine iodide as synthetic substrate based on colorimet-
ric method, as described elsewhere.18,19 Whole brains of male ICR
mice (25–30 g) were homogenized in a glass Teflon homogenizer
(Eyela, Japan) containing 50 volumes of phosphate buffer (pH 8.0,
0.1 M), and then centrifuged at 14,000 rpm for 20 min at 4 °C.
The supernatant obtained was used as a source of enzyme for the
assay. Each drug was initially dissolved in dimethyl sulfoxide
(DMSO) and diluted to various concentrations immediately before
use. An aliquot of diluted drug solution was then mixed with
640
man’s reagent (10 mM 5,50-dithio-bis[2-nitrobenzoic acid] and
15 mM sodium bicarbonate), and the enzyme source (100 l) and
pre-incubated at room temperature for 10 min. Then 5 l of acetyl-
ll of phosphate buffer (0.1 M, pH 8.0), 25 ll of buffered Ell-
l
l
thiocholine iodide solution (75 mM) was added to this mixture and
5.2.4.1. 7-Hydroxy-6-(3-(pyrrolidin-1-yl)propoxy)-2H-chromen-
mixed. Absorbance was measured at 410 nm after 10 min for the
2-one hydrochloride (3f).
The compound 3f (15 mg) was
reaction adding 5 ll of acetylthiocholine iodide solution (75 mM)
prepared from 6 (92 mg, 0.31 mmol) and pyrrolidine (17.0 mg,
and to the reaction mixtures (OPTIZEN 2120UV, Mecasys Co. Ltd,
Korea). The concentration of each drug required to inhibit
0.24 mmol) using the procedure described for 3e. Yield 19%. 1H

S. O. Nam et al. / Bioorg. Med. Chem. 22 (2014) 1262–1267
1267
acetylcholinesterase activity by 50% (IC50) was calculated using an
5.4.3. Statistical analysis
enzyme inhibition dose response curve. Galantamine was used as a
positive control.
Data from the passive avoidance task was analyzed by Kruskal–
Wallis non-parametric test fol lowed by Dunn’s post hoc test. All
statistical analysis was processed using Sigmastat software (Systat
Software, IL 60606). Statistical significance was accepted for P val-
ues of <0.05.
5.4. In vivo assay
5.4.1. Animals
Male ICR mice (25–30 g) were purchased from the Orient Co.,
Ltd, a branch of Charles River Laboratories (Seoul, Republic of Kor-
ea). Animals were housed 5 per cage with food and water available
Acknowledgements
This work was supported by the Basic Science Research Pro-
gram of the Korean National Research Foundation (NRF) funded
by the Ministry of Education, Science and Technology of Korean
Government (#2012-006431).
ad libitum and maintained under
a constant temperature
(23 1 °C) and humidity (60 10%) under a 12 h light/dark cycle
(light on 07:30–19:30 h). All behavioral tasks were conducted be-
tween 10:00 h and 16:00 h. Animal treatment and maintenance
were conducted in accordance with the Principle of Laboratory
Animal Care (NIH publication No. 85-23, revised 1985) and the
Animal Care and the Use Guidelines of Kyung Hee University,
Republic of Korea.
References and notes
1. Pakaski, M.; Kalman, J. Neurochem. Int. 2008, 53, 103.
2. Farlow, M. R. Alzheimer Dis. Assoc. Disord. 1994, 8, S58.
3. Brookmeyer, R. E.; Ohnson, J. E.; Ziegler-Graham, K.; Arrighi, H. M. Alzheimer’s
Dement. 2007, 3, 186.
4. Perl, D. P. Mt Sinai J. Med. 2010, 77, 32.
5.4.2. Passive avoidance task
5. Van Marum, R. J. Fundam. Clin. Pharmacol. 2008, 22, 265.
6. Francis, P. T.; Palmer, A. M.; Snape, M.; Wilcock, G. K. J. Neurol. Neurosurg.
Psychiatry 1999, 66, 137.
7. Francotte, P.; Graindorge, E.; Boverie, S.; de Tullio, P.; Pirotte, B. Curr. Med.
Chem. 2004, 11, 1757.
8. Tayeb, H. O.; Murray, E. D.; Price, B. H.; Tarazi, F. I. Expert Opin. Biol. Ther. 2013,
13, 1075.
9. Blennow, K.; Zetterberg, H.; Haass, C.; Finucane, T. Nat. Med. 2013, 19, 1214.
10. Bores, G. M.; Kosley, R. W. Drugs Future 1996, 21, 621.
11. Lilienfeld, S. CNS Drug Rev. 2002, 8, 159.
12. Wilcock, G. K.; Lilienfeld, S.; Gaens, E. BMJ 2000, 321, 1445.
13. Langer, T.; Krovat, E. M. Curr. Opin. Drug Disc. Dev. 2003, 6, 370.
14. Rollinger, J. M.; Hornick, A.; Langer, T. J. Med. Chem. 2004, 47, 6248.
15. Nam, S. O.; Yun, Y. D.; Park, D. H.; Ryu, J. H.; Lee, Y. S. Yakhak Hoeji 2011, 55,
473.
16. Nemoto, T.; Ohshima, T.; Shibasaki, M. Tetrahedron 2003, 59, 6889.
17. Grice, C. A.; Tays, K. L.; Savall, B. M.; Wei, J.; Butler, C. R.; Axe, F. U.; Bembenek,
S. D.; Fouri, A. M.; Dunford, P. J.; Lundeen, K.; Coles, F.; Xue, X.; Riley, J. P.;
Williams, K. N.; Karlsson, L.; Edwards, J. P. J. Med. Chem. 2008, 51, 4150.
18. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
Passive avoidance tasks were carried out in identical illumi-
nated and non-illuminated boxes (20 ꢁ 20 ꢁ 20 cm), separated by
a guillotine door (5 ꢁ 5 cm) as described elsewhere.19 The illumi-
nated compartment contained a 50 W bulb, and the floor of the
non-illuminated compartment (20 ꢁ 20 ꢁ 20 cm) was composed
of 2 mm stainless steel rods spaced 1 cm apart. For an acquisition
trial, mice were initially placed in the illuminated compartment;
the door between the two compartments was opened 10 s later.
When the mice entered the dark compartment, the door automat-
ically closed and an electrical foot shock (0.5 mA) for 3 s was deliv-
ered through the stainless steel rods. One hour before the
acquisition trial, mice were orally administered 0.9% vehicle saline
or various doses of test compounds dissolved in 0.9% saline (0.5, 1,
2, or 5 mg/kg for 3b, 0.25, 0.5, 1, 2 mg/kg for 3f). The galantamine
(1 mg/kg) was orally administered as a positive control. Scopol-
amine (1 mg/kg) was intraperitoneally administered 30 min before
the acquisition trial. Twenty-four hours after the acquisition trial,
retention trials were conducted by re-placing mice in the illumi-
nated compartment. The time taken for a mouse to enter the dark
compartment after opening the door was defined as latency for
both acquisition and retention trials. Latency to enter the dark
compartment was recorded up to 300 s to avoid ceiling effects.
19. Kim, D. H.; Hung, T. M.; Bae, K. H.; Jung, J. W.; Lee, S.; Yoon, B. H.; Cheong, J. H.;
Ko, K. H.; Ryu, J. H. Eur. J. Pharmacol. 2006, 542, 129.
20. Calabrese, E. J. Crit. Rev. Toxicol. 2008, 38, 419.