Design, synthesis, and acetylcholinesterase inhibitory activity of novel coumarin analogues

Article abstract of DOI:10.1016/j.bmc.2008.07.068

Three series (series A-C) of coumarin analogues with phenylpiperazine functions as substitution were designed and synthesized for studying their potential for treating Alzheimer's (AD) disease. Their anticholinesterase activities were assayed according to Ellmann's method against freshly prepared acetylcholinesterase (AChE) from Electrophorus electricus using donepezil as the reference compound. Pharmacological study and preliminary structure-activity relationships showed that coumarins with substitution on positions 3 and/or 4 have parallel anti-AchE activities compared with the reference compound.

Full text of DOI:10.1016/j.bmc.2008.07.068

Bioorganic & Medicinal Chemistry 16 (2008) 8011–8021
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and acetylcholinesterase inhibitory activity of novel
coumarin analogues
Xiang Zhou a, Xiao-Bing Wang a, Tao Wang b, Ling-Yi Kong a,
*
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
b Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series (series A–C) of coumarin analogues with phenylpiperazine functions as substitution were
designed and synthesized for studying their potential for treating Alzheimer’s (AD) disease. Their anti-
cholinesterase activities were assayed according to Ellmann’s method against freshly prepared acetylcho-
linesterase (AChE) from Electrophorus electricus using donepezil as the reference compound.
Pharmacological study and preliminary structure–activity relationships showed that coumarins with
substitution on positions 3 and/or 4 have parallel anti-AchE activities compared with the reference
compound.
Received 14 May 2008
Revised 22 July 2008
Accepted 23 July 2008
Available online 29 July 2008
Keywords:
Alzheimer’s disease
Acetylcholinesterase inhibitor
Design
Ó 2008 Elsevier Ltd. All rights reserved.
Synthesis
Coumarin
1. Introduction
the neurodegenerative cascade of the disease, but only mitigate
some of the symptoms temporarily.
Alzheimer’s disease (AD), a progressive and degenerative disor-
der of the brain, is believed to be the most common cause of
dementia among the elderly. AD is associated with a loss of the
presynaptic markers of the cholinergic system in the brain areas
related to memory and learning, and is characterized by the pres-
ence of amyloid deposits and neurofibrillary tangles in the brain of
afflicted individuals.1,2 Up to now, medicines to cure Alzheimer’s
disease have received significant attention for many years. The
molecular causes of this condition remain unknown in spite of
the existence of several theories regarding the pathogenesis of
AD.3–6 As a result, most of the existing medicines can only delay
the development of the state of AD, but cannot cure the disease
in nature. Now, the most widely accepted biochemical theory of
the disease, known as the cholinergic hypothesis,7–10 is that the
decline in cognitive and mental functions associated with AD is
related to the loss of cortical cholinergic neurotransmission. One
rational way to enhance cholinergic neurotransmission is to inhibit
an enzyme11–13 responsible for the metabolic breakdown of acetyl-
choline (ACh). So up to now most of the drugs approved for AD
treatment are acetylcholinesterase (AChE) inhibitors (AChEIs)
which can enhance cholinergic neurotransmission by increasing
acetylcholine (ACh) availability in the synaptic cleft. Considering
the mechanism of AChEIs, they are not expected to interfere with
Recently, studies have shown that AChEIs can be effective over a
longer period14 for that AChE plays an important role in Ab depo-
sition.15–17 It seems likely that AChE interacts with Ab and pro-
motes the formation of amyloid fibril through a pool of amino
acids located in proximity of the peripheral anionic binding site
(PAS) of the enzyme.15 Moreover, it has been shown that molecules
interact either exclusively with PAS or with both catalytic and
peripheral binding sites, which can also prevent the pro-aggrega-
tion of AChE toward Ab.16 Compounds showing dual bindings with
AChE are intriguing, and they represent a new type of therapeutic
agents through the prevention of Ab aggregation. Recent research
revealed that several AChEIs not only facilitate cholinergic trans-
mission, but also interfere with the synthesis, deposition and
aggregation of toxic amyloid-b-peptides (Ab).18,19 On this basis,
AChEIs have become the leading strategy for the development of
anti-AD agents. The current interest in these drugs has received
considerable attention too.20,21 Some anti-AChE agents, such as ta-
crine, donepezil, rivastigmine, and ensaculin (Fig. 1), show a mod-
est induce of the improvement in memory and cognitive
functions,22 and have been used to treat AD clinically for a long
time. But only ensaculin, a coumarin derivative, has appeared to
prevent or slow down the progressive neurodegeneration in these
compounds.22
At the same time, the three-dimensional (3-D) X-ray structure
of AChE from Torpedo California electric organ has been reported
for further study of the enzyme and corresponding inhibitors.23
* Corresponding author. Tel.: +86 25 8539 1289; fax: +86 25 8530 1528.
E-mail address: lykong@jlonline.com (L.-Y. Kong).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.068

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X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
coumarin analogues with phenylpiperazine substituted at position
NH2
O
6 of coumarin as series A, at position 3 as series B and at position 4
as series C were designed and synthesized. In the designed com-
pounds, the following hypothesis was expected: (1) the coumarin
ring, 2H-chromen-2-one heterocycle, a heterocyclic moiety com-
prising in ensaculin with cognitive functions, demonstrated to be
compatible with a high anti-AChE potency,33,34 acted as the periph-
eral anonic site, which can interact with the peripheral binding
site;24 (2) the nitrogen atom from the phenylpiperazine groups
acted as the positive charge center presented in many potent AChE
inhibitors,35 which can interact with the catalytic center of AChE
demonstrated by the X-ray crystallographic studies of the AChE/
donepezil and AChE/galantamine complexes;36,37 and (3) the phe-
nyl ring connecting with the piperazine ring acted as the choline
binding site as shown in Figure 3. Furthermore, a linking chain
bearing different amounts of carbon atoms might have the chance
to line the wall of the AChE gorge.23
N
H3CO
H3CO
N
tacrine
donepezil
N
O
N
O
N
O
N
O
H3CO
O
O
rivastigmine
ensaculin
Figure 1. Structures of the acetyl cholinesterase inhibitors as FDA approved
Alzheimer’s disease therapeutics.
The X-ray structure of a transition state analogue complex of AChE
has also been reported.24 The active sites of AChE were shown in
Figure 2. The figure suggested that the active sites of AChE consist
at least the following binding sites: (1) an anionic substrate (AS)
binding site, such as Trp84, Glu199, and Phe330, which contains
a small number of negative charges where the quaternary ammo-
nium part of ACh and various active sites bind through a preferen-
tial interaction of quaternary nitrogens with the p electrons of
aromatic groups; (2) an ecstatic site (ES), which contains the cata-
lytic triad Ser200-His440-Glu32718; (3) an acyl binding site (ABS),
Phe288, and Phe299, which binds to the acetyl group of ACh.19 Be-
sides these, AChE also has a peripheral anionic site (PAS), such as
Trp279, Tyr70, Tyr121, Asp72, Glu199, and Phe290, which may
bind to 9-aminoacridine and 9-amino-1,2,3,4-tetrahydroacridine
(tacrine).25–28
As mentioned above, ensaculin composed of a benzopyran with
a piperazine substituted moiety (Fig. 1) has been used clinically as
its HCl salt with trade name of KA-672 HCl for treating AD as AChEI
for a long time.29 So ensaculin, a coumarin analogue, was chosen to
be the parent compound in this study, and the analogues designed
were expected to have anti-AChE activity. Coumarin, naturally
occurring phytochemicals with fragrance found in many plant spe-
cies with a wide range of biological activities, such as anti-inflam-
matory, anti-tumour, anti-allergic and anti-HIV-1 activities, has
received significant attention for many years.30–32 So considering
the active sites of AChE and ensaculin structure, three series of
2. Results and discussion
2.1. Chemistry
Series A, B, and C were designed and synthesized as shown in
Schemes 1–3, respectively. The key immediate 1 was prepared by
p-cresol and malonic acid using ZnCl2 as catalyst and POCl3 as sol-
vent. Then, 1 reacted with (CH3)2SO4 to give methylated product 2
or reacted with POCl3 to give chlorinated product 4. 2 or 4 was
bromided with NBS to give 3 or 5. 3 or 5 reacted with correspond-
ing substituents and the target compounds 6–20 were obtained
(Scheme 1).
As shown in Scheme 2, series B were prepared through the reac-
tion of salicylic aldehyde and diethyl malonate to give the interme-
diate 21. Then, 21 was hydrolyzed with NaOH and 22 was
obtained. 22 was chlorided with SOCl2 to give the key intermediate
23. At last, 23 reacted with corresponding substituents, and the
target compounds 24–37were obtained.
Series C were prepared from the key intermediate 1 (Scheme 3).
Then 1 was chlorided with POCl3 to give 4. Lastly, 4 reacted with
O
H3CO
choline binding site
Phe290
H3CO
Substrate
N
Trp279
peripheral anonic site
positive charge centor
Tyr70
PAS
Asp72
Phe
330
Tyr121
X
4
+
14
19
20
13
5
11
10
9
6
Me3N
3
21
N
N
18
Phe288
Phe299
ABS
OH
R
23 22
17
16
7
AS
2
CH3
O
O
1
Trp84
8
A
O
O
C
O
17
16
23
19
22
Gly118
Gly119
Ala201
4
5
n
p
O
10
Ar1
m
N
Y
Ar2
6
11
N
N
21
3
ACCS
18
X
R
7
13
14
20
9
2
O
O
8
1
B
OH
His440
16
14
17
Glu327
23
22
21
11
5
4
N
3
13
N
H3C
10
18
6
R
19
Ser200
20
7
9
2
O
O
8
ES
1
C
Figure 2. The active site and the PAS of AchE. Numbers refer to residue positions in
Torpedo California AchE.16
Figure 3. Design strategy of the target compounds.

X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
8013
OCH3
H3C
b
OH
O
O
H3C
H3C
2
a
+
COOHCH2COOH
O
O
OH
Cl
O
1
H3C
c
O
OCH3
4
BrH2C
BrH2C
X
O
O
O
d
RH2C
e
3
O
Cl
6-20
O
O
5
Compound
R
X
Compound
R
X
HN
N
HN
N
6
OCH3
OCH3
OCH3
OCH3
14
Cl
Cl
Cl
Cl
H3C
HN
N
CH3
HN
N
CH3
7
8
9
15
16
17
HN
N
HN
N
OCH3
H3C
HN
N
HN
N
H3CO
HN
N
Cl
HN
HN
N
OCH3
10
OCH3
18
Cl
HN
N
N
OC2H5
11
12
13
OCH3
OCH3
OCH3
19
20
Cl
H3CO
O
HN
N
HN
N
HN
N
OCH3
H3C
H3C
O
HN
N
Scheme 1. The preparation of series A. Reagents and conditions: (a) ZnCl2, POCl3, 60 °C; (b) (CH3)2SO4, K2CO3, reflux; (c) POCl3, reflux; (d) NBS, BPO, reflux; (e) RH, NaHCO3,
reflux.
the corresponding substituents to give the target compounds 38–
50.
and/or 4 of coumarin ring are better than that of the 6-substituted
coumarins. Most of the 6-substituted coumarins did not show anti-
AChE activities. On comparison of the target 6-substituted couma-
rins and ensaculin, we found that there is one atom in the linking
chain between coumarin backbone and phenylpiperazine in the 6-
substituted coumarins, while there are four atoms in ensuculin
(Fig. 3). So, we can conclude that the amounts of the atoms in
the linking chain are important for anti-AChE activities of 6-phe-
nylpiperazine coumarin analogues for that one atom cannot reach
the requirement for the gorge.19
The structures of the target compounds were characterized by
1H NMR, IR, and ESI-MS. One compound of each series was charac-
terized by 13C NMR, too. The purity of the target compounds was
analyzed by HPLC. Besides, the structure of compound 16 was fur-
ther confirmed by single crystal X-ray diffraction.
2.2. Inhibition of AChE
To determine the therapeutic potency of the new coumarin
derivatives for treating AD, their anticholinesterase activities
(compounds 6–20, 24–50) were assayed according to Ellmann’s
method38 against freshly prepared AChE from electrophorus elec-
tricus using donepezil as reference compound. Inhibition of AChE
activities of the synthesized compounds is shown in Table 1.
The data listed in Table 1 clearly show that most of the designed
compounds exhibited moderate inhibitory activities toward the
cholinesterase. In general, the AChE inhibitory activities of com-
pounds with phenylpiperazine substitutions on the positions 3
The 4-phenylpiperazine substituted coumarin compounds 45 6-
methyl-4-(4-phenylpiperazin-1-yl)2H-chromen-2-one and 47 6-
methyl-4-(4-(4-methyl-benzoyl)piperazin-1-yl)2H-chromen-2-one
showed significant activities with IC50 4.5 lmol/L and 5.3 lmol/L,
respectively. There are two carbones between the carbonyl-carbon
atom (C2) and piperazine ring in the 4-substituted coumarins (Fig.
3), which is in agreement with donepezil, the positive compound.
It suggests that the distance between carbonyl-carbon atom and
nitrogen atom of piperazine ring is important for the anti-AChE
activities.

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X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
O
O
O
OH
a
OC2H5
b
OH
+
CH2(COOC2H5)2
O
CHO
O
O
O
21
22
O
O
c
Cl
d
R
O
O
O
24-37
23
Compound
R
Compound
R
HN
N
HN
N
CH3
24
31
Cl
HN
CH3
HN
N
25
32
Cl
O
HN
O
26
27
28
33
34
35
HN
N
CH3
O
HN
N
HN
N
H3
C
O
HN
N
OCH3
HN
N
Cl
O
HN
N
HN
N
29
30
36
37
H3CO
Cl
O
HN
N
Cl
HN
N
NO2
Scheme 2. The preparation of series B. Reagents and conditions: (a) piperidine, CH3COOH, reflux; (b) NaOH, ethol, reflux; (c) SOCl2, reflux; (d) RH, K2CO3, reflux.
All compounds with benzoylpiperazine groups as substitutions,
33–37, 47–50, showed some anti-AChE activities (Table 1). It sug-
gests that benzoylpiperazine groups are helpful for AChE inhibitory
activities.
precoated Merck silica gel Kiesegel 60 F254 plates, and the spots
were detected under UV light (254 nm). The flash chromatography
was conducted using silica gel 230–400 mesh. IR spectra were
measured on a Jasco FT/IR-430 spectrophotometer. 1H and 13C
NMR spectra were recorded at 500 MHz on a Bruker ARX 300 spec-
trometer. The chemical shifts are reported downfield in ppm (d)
relative to internal TMS, and coupling constants are reported in
Hertz (Hz). Splitting patterns describe apparent multiplicities,
and are designated as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), or br s (broad singlet). Mass spectra analysis was
performed on a Quattro microMS Micromass UK mass spectrome-
ter, and was recorded on an electrospray ionization mass spec-
trometer as the value m/z. The HPLC spectra were recorded on a
Agilent 1100 HPLC system equipped with an analytical ODS col-
umn (406 ꢀ 150 mm).
3. Conclusion
Three series of conformationally restricted coumarin deriva-
tives by linking a substituted phenylpiperazine moiety to the cou-
marin backbone as potential inhibitors of AChE for curing PAD
were designed and synthesized. Preliminary structure–activity
relationships revealed that the substitutions on the positions 3
and/or 4 of coumarin ring are more helpful than that on the posi-
tion 6 for inhibiting AChE. The stronger inhibitory activity of the
target compound having smaller substitution on the phenylpiper-
azine proves that the AChE’s active sites can apparently be reached
only through a deep and narrow catalytic gorge.11 So we can con-
clude that coumarins having phenylpiperazine substitution on the
positions 3 and/or 4 with a suitable linking chain show significant
anti-AChE activities. They can be considered as interesting inhibi-
tors in the search of new therapies for curing AD. The study of their
anti-AChE activities led to a novel family of potent anti-AChE li-
gands, which are coumarin analogues having substitution on posi-
tions 3 and/or 4.
4.1.1. 4-Hydroxy-6-methylcoumarin (1)
The compound 1 was prepared by the classic synthetic way of
4-hydroxy coumarin. 0.07 mol p-cresol, 0.07 mol malonic acid and
30 g ZnCl2 were dissolved in 20 ml POCl3. The mixture was kept
for 24 h at 60 °C. Then, the reaction mixture was poured into
250 ml ice water and deposited overnight. The precipitation was
filtered and recrystallized in 5% ethanol to give 1, 5.91 g, yield
48%.
4.1.2. 4-Methoxy-6-methyl coumarin (2)
4. Experimental
4.1. Chemistry
The compound
2 was prepared with 0.02 mol 1 and
0.04 mol (CH3)2SO4 dissolved in 200 ml acetone with 0.06 mol
K2CO3. The mixture was refluxed for 24 h, and then the solvent
was removed on vacuum. The residue was treated with flash
silica gel (cyclohexane/acetone = 7:3) to give 2, 3.30 g, yield
86.8%.
All purchased starting materials and reagents were used with-
out further purification unless noted. Reaction progress was mon-
itored using analytical thin layer chromatography (TLC) on

X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
8015
4.2.1. 4-Methoxy-6-((4-(2-methylphenyl)piperazin-1-yl)meth-
yl)coumarin (6)
OH
O
H3C
a
+
COOHCH2COOH
H3C
OH
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 6, yield 19%, colorless powder, mp
O
1
131–133 °C; MS (ESI) m/z 365.2 (M+H)+; IR (KBr)
c
cmꢁ1: 2949,
R
Cl
H3C
H3C
1747, 1456, 1376, 1121, 770. 1H NMR (CDCl3, 500 MHz) d 7.78 (s,
1H, H-5), 7.57 (m, 1H, H-7), 7.29 (m, 1H, H-8), 7.14–7.17 (m, 2H,
Ph), 6.95–7.03 (m, 2H, Ph), 5.69 (s, 1H, H-3), 4.00 (s, 3H, OCH3),
3.62 (m, 2H, N1(CH2)), 2.94 (m, 4H, N4(CH2)2), 2.62 (m, 4H,
N1(CH2)2), 2.29 (s, 3H, CH3). 13C NMR (CDCl3, 125 MHz) d 162.9 (C-
2), 90.1 (C-3), 166.4 (C-4), 126.5 (C-5), 133.4 (C-6), 131.0 (C-7),
123.1 (C-8), 152.5 (C-9), 115.3 (C-10), 56.3 (OCH3), 62.4 (C-11),
53.6 (C-13), 51.7 (C-14), 53.6 (C-16), 51.7 (C-17), 151.4 (C-18),
118.9 (C-19), 126.5 (C-20), 116.7 (C-21), 123.2 (C-22), 123.1 (C-
23), 17.8 (CH3). HPLC analysis 96.4% (V(Methol): V(H2O) = 65:35,
tR = 6.71 min).
c
b
O
O
O
O
4
38-50
Compound
R
Compound
R
HN
N
HN
HN
N
38
45
H
3
CO
Cl
HN
N
OCH
N
3
39
46
Cl
O
HN
N
CH
3
40
41
42
47
48
49
HN
N
CH3
4.2.2. 4-Methoxy-6-((4-p-tolylpiperazin-1-yl)methyl)coumarin (7)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 7, yield 16%, colorless powder, mp
O
HN
N
HN
N
H
C
3
123–125 °C; MS (ESI) m/z 365.1 (M+H)+; IR (KBr)
c
cmꢁ1 2959,
O
HN
N
Cl
1756, 1610, 1513, 1456, 1260, 802. 1H NMR (CDCl3, 500 MHz) d
7.78 (s, 1H, H-5), 7.57 (d, 1H, H-7, J = 8.10 Hz), 7.27 (d, 1H, H-7,
J = 8.46 Hz), 7.05–7.07 (m, 2H, Ph), 6.82–6.86 (m, 2H, Ph), 5.69 (s,
1H, H-3), 3.99 (s, 3H, OCH3), 3.61 (m, 2H, N1(CH2)), 3.16 (m, 4H,
N4(CH2)2), 2.62 (m, 4H, N1(CH2)2), 2.26 (s, 3H, CH3). HPLC analysis
97.0% (V(Methol): V(H2O) = 65:35, tR = 9.67 min).
HN
N
Cl
O
HN
N
HN
N
CH
O
3
43
44
50
Cl
HN
4.2.3. 4-Methoxy-6-((4-(4-methoxyphenyl)piperazin-1-yl)meth-
yl)coumarin (8)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 8, yield 20%, colorless powder, mp
Scheme 3. The preparation of series C. Reagents and conditions: (a) ZnCl2, POCl3,
60 °C; (b) POCl3, reflux; (c) RH, K2CO3, reflux.
4.1.3. 6-Bromomethyl-4-methoxycoumarin (3)
126–128 °C; MS (ESI) m/z 381.1 (M+H)+; IR (KBr)
c
cmꢁ1 2959,
0.003 mol 2 was added to 34 ml anhydrous CCl4 with the reac-
tion system protected by N2. When the mixture was refluxed for
0.5 h, 0.0028 mol NBS (1-bromopyrrolidine-2,5-dione) and
100 mg BPO (methyl benzoperoxoate) were added to the reaction
mixture. After the reaction mixture was refluxed for 5 h, the addi-
tional 100 mg BPO was added again, and the reaction continued for
3 h. The reaction mixture was cooled down to room temperature
and filtered. The filtrate was evaporated under reduced pressure.
The flash chromatography (cyclohexane/acetone = 7:3) was per-
formed to give 3, 0.32 g, yield 39.6%.
1756, 1610, 1513, 1456, 1103, 802. 1H NMR (CDCl3, 500 MHz)d
7.78 (s, 1H, H-5), 7.57 (d, 1H, H-7, J = 8.10 Hz), 7.27 (d, 1H, H-8,
J = 8.46 Hz), 7.05–7.07 (m, 2H, Ph), 6.82–6.86 (m, 2H, Ph), 5.69 (s,
1H, H-3), 3.99 (s, 3H, OCH3), 3.61 (m, 2H, N1(CH2)), 3.16 (m, 4H,
N4(CH2)2), 2.62 (m, 4H, N1(CH2)2), 2.26 (s, 3H, CH3). HPLC analysis
99.0% (V(Methol): V(H2O) = 65:35, tR = 4.35 min).
4.2.4. 4-Methoxy-6-((4-phenylpiperazin-1-yl)methyl)coumarin (9)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 9, yield 20%, colorless powder, mp
136–137 °C; MS (ESI) m/z 351.2 (M+H)+; IR (KBr)
c
cmꢁ1 2938,
4.1.4. 4-Chloro-6-methylcoumarin (4)
1716, 1629, 1458, 1373, 934. 1H NMR (CDCl3, 500 MHz)d 7.78 (s,
1H, H-5), 7.57 (d, 1H, H-7, J = 8.1 Hz), 7.24–7.31 (m, 3H, Ph, H-8),
6.85–6.93 (m, 3H, Ph), 5.69 (s, 1H, H-3), 3.99 (s, 3H, OCH3), 3.61
(m, 2H, N1(CH2)), 3.22 (m, 4H, N4(CH2)2), 2.62 (m, 4H, N1(CH2)2).
HPLC analysis 97.6% (V(Methol): V(H2O) = 65:35, tR = 7.92 min).
0.005 mol 1 and 0.005 mol triethylamine were added to 5 ml
POCl3. The mixture was refluxed for 30 min, and the resultant was
poured into water. Then the solution was extracted with methylene
chloride. The methylene chloride layer was dried with anhydrous
Na2SO4 and evaporated under reduced pressure. The flash chroma-
tography (cyclohexane/acetone = 10:1) was performed to give 4,
0.8 g, yield 82.3%.
4.2.5. 4-Methoxy-6-((4-(4-chlorophenyl)piperazin-1-yl)methyl)-
coumarin (10)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 10, yield 14%, colorless powder, mp
4.1.5. 6-Bromomethyl-4-chlorocoumarin (5)
The compound 5 was prepared from 0.003 mol 4 and 0.003 mol
NBS by the same way of synthesis of 3. The target compound 5,
0.22 g was obtained, yield 26.3%.
130–132 °C; MS (ESI) m/z 384.7 (M+H)+; IR (KBr)
c
cmꢁ1 2823,
1709, 1603, 1379, 1248, 1217, 821. 1H NMR (CDCl3, 500 MHz) d
7.77 (s, 1H, H-5), 7.57 (m, 1H, H-7), 7.28 (m, 1H, H-8), 7.17–7.20
(m, 2H, Ph), 6.80–6.84 (m, 2H, Ph), 5.69 (s, 1H, H-3), 3.99 (s, 3H,
OCH3), 3.60 (m, 2H, N1(CH2)), 3.17 (m, 4H, N4(CH2)2), 2.62 (m,
4H, N1(CH2)2), 2.29 (s, 3H, CH3). HPLC analysis 95.6% (V(Methol):
V(H2O) = 65:35, tR = 12.45 min).
4.2. General procedure for the preparation of compounds 6–13
0.001 mol 3, 0.001 mol substituted phenylpiperazine deriva-
tives, and 0.002 mol potassium carbonate were added to a mixed
solvent containing 15 ml acetone and 15 ml alcohol. The mixture
was refluxed for 48 h, then the resultant was evaporated. The flash
chromatography (EtOAc/hexane) was performed to give the target
compounds, respectively.
4.2.6. 4-Methoxy-6-((4-(2-methoxyphenyl)piperazin-1-yl)meth-
yl)coumarin (11)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 11, yield 20%, colorless powder, mp

8016
X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
Table 1
Table 1 (continued)
Inhibition of AchE activities of the synthesized compounds
Compound Series
R
AchE
Compound Series
R
AchE
inhibition
inhibition
(IC50 SD)a
(IC50 SD)a
(lmol/L)
(lmol/L)
26
B
101 0.1
HN
HN
O
N
HN
HN
N
6
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
19 0.1
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
9.3 0.05
97 0.2
H3C
, OCH3
27
28
B
B
6.7 0.02
7.5 0.03
H3C
7
N
CH
3 , OCH3
HN
HN
N
OCH3
8
HN
HN
N
OCH
3 , OCH3
N
29
B
11 0.05
H3CO
N
9
, OCH3
30
31
B
B
98 0.1
HN
HN
N
N
Cl
10
11
12
13
14
15
16
17
18
19
20
24
25
HN
HN
N
N
Cl
, OCH3
7.6 0.04
CH3
Cl
H3CO
32
B
14 0.04
HN
N
, OCH3
O
N
Cl
O
HN
OCH3
, OCH3
33
34
35
36
B
B
B
B
18 0.02
15 0.03
21 0.1
26 0.2
O
N
HN
HN
N
CH3
HN
HN
O
N
, OCH3
N
, Cl
O
HN
HN
N
O
Cl
HN
HN
N
CH3
, Cl
N
N
Cl
, Cl
H3C
N
O
N
37
B
34 0.3
HN
HN
HN
HN
NO2
H3CO
, Cl
N
38
39
C
C
249 0.2
18 0.2
N
N
OCH
3 , Cl
H3CO
HN
N
OCH3
HN
HN
OC2H5
HN
, Cl
40
C
61 0.03
N
HN
HN
N
CH3
N
H3C
H3C
,
N
41
42
C
C
7.9 0.02
HN
HN
N
H3C
N
808 0.1
HN
Cl
CH3

X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
8017
7.73 (s, 1H, H-5), 7.53 (d, 1H, H-7, J = 9.00 Hz), 7.26 (m, 1H, H-8),
7.38–7.40 (m, 5H, Ph), 5.69 (s, 1H, H-3), 3.99 (s, 3H, OCH3), 3.57
(m, 2H, N1(CH2)), 3.79 (m, 2H, N4(CH2)), 3.44 (m, 2H, N4(CH2)),
2.53 (m, 4H, N1(CH2)2). HPLC analysis 97.6% (V(Methol):
V(H2O) = 65:35, tR = 6.48 min).
Table 1 (continued)
Compound Series
R
AchE
inhibition
(IC50 SD)a
(lmol/L)
43
44
C
C
901 0.1
875 0.3
HN
HN
N
O
CH3
4.3. General procedure for the preparation of compounds 14–20
0.001 mol 5, 0.001 mol substituted phenylpiperazine deriva-
tives, and 0.002 mol potassium carbonate were added to a mixed
solvent containing 15 ml acetone and 15 ml alcohol. The mixture
was refluxed for 48 h, and then the resultant was evaporated.
The flash chromatography (EtOAc/hexane) was performed to give
the target compounds, respectively.
45
46
C
C
4.5 0.03
HN
HN
N
N
Cl
827 0.1
4.3.1. 4-Chloro-6-((4-phenylpiperazin-1-yl)methyl)coumarin (14)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 14, yield 10%, colorless powder, mp
Cl
113–115 °C; MS (ESI) m/z 355.1 (M+H)+; IR (KBr)
c
cmꢁ1 3440,
O
1726, 1500, 1454, 1241, 921, 753. 1H NMR (CDCl3, 500 MHz) d
7.84 (s, 1H, H-5), 7.64 (m, 1H, H-7), 7.34 (m, 1H, H-8), 7.24–7.27
(m, 2H, Ph), 6.86–6.93 (m, 3H, Ph), 6.61 (s, 1H, H-3), 3.65 (m, 2H,
N1(CH2)), 3.22 (m, 4H, N4(CH2)2), 2.64 (m, 4H, N1(CH2)2). 13C
NMR (CDCl3, 125 MHz) d 159.0 (C-2), 115.4 (C-3), 152.2 (C-4),
125.5 (C-5), 134.0 (C-6), 129.1 (C-7), 119.8 (C-8), 151.2 (C-9),
129.1 (C-10), 61.9 (C-11), 53.1 (C-13), 49.1 (C-14), 53.1 (C-16),
49.1 (C-17), 149.6 (C-18), 117.8 (C-19), 129.1 (C-20), 117.0 (C-
21), 129.1 (C-22), 116.1 (C-23). HPLC analysis 97.0% (V(Methol):
V(H2O) = 65:35, tR = 6.78 min).
47
48
49
C
C
C
5.3 0.02
7.4 0.04
16 0.2
HN
HN
HN
N
N
N
CH3
O
O
Cl
O
4.3.2.4-Chloro-6-((4-p-tolylpiperazin-1-yl)methyl)coumarin (15)
Purified by flash silica gel column chromatography (EtOAc/
hexane = 1:7) to afford compound 15, yield 10%, colorless powder,
HN
N
50
C
21 0.1
Cl
mp 127–129 °C; MS (ESI) m/z 369.2 (M+H)+; IR (KBr) cmꢁ1 3087,
c
1778, 1614, 1571, 1290, 1240, 1223, 812. 1H NMR (CDCl3,
500 MHz) d7.84 (s, 1H, H-5), 7.65 (d, 1H, H-7, J = 8.10 Hz), 7.34
(d, 1H, H-8, J = 8.45 Hz), 7.06–7.08 (m, 2H, Ph), 6.82–6.85 (m,
2H, Ph), 6.61 (s, 1H, H-3), 3.65 (m, 2H, N1(CH2)), 3.17 (m, 4H,
N4(CH2)2), 2.64 (m, 4H, N1(CH2)2), 2.26 (s, 3H, CH3). HPLC analysis
96.7% (V(Methol): V(H2O) = 65:35, tR = 13.78 min).
Donepezil
0.11 0.01
The bold font stands for that the corresponding compounds exhibited inhibitory
some activities toward the cholinesterase.
a
Data are means standard deviation of duplicate independent experiments.
128–130 °C; MS (ESI) m/z 381.2 (M+H)+; IR (KBr)
c
cmꢁ1 3075,
1707, 1629, 1604, 1575, 1456, 1378, 986. 1H NMR (CDCl3,
500 MHz)d 7.78 (s, 1H, H-5), 7.57 (d, 1H, H-7, J = 8.10 Hz), 7.28
(m, 1H, H-8), 6.85–6.99 (m, 4H, Ph), 5.69 (s, 1H, H-3), 3.99 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 3.63 (m, 2H, N1(CH2)), 3.11 (m, 4H,
N4(CH2)2), 2.67 (m, 4H, N1(CH2)2), 2.26 (s, 3H, CH3). HPLC analysis
96.1% (V(Methol): V(H2O) = 65:35, tR = 5.65 min).
4.3.3. 4-Chloro-6-((4-o-tolylpiperazin-1-yl)methyl)coumarin (16)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 16, yield 13%, colorless powder, mp
103–105 °C; MS (ESI) m/z 369.1 (M+H)+; IR (KBr)
c
cmꢁ1 3054,
2818, 1724, 1615, 1454, 1354, 1011, 843. 1H NMR (CDCl3,
500 MHz) d7.85 (s, 1H, H-5), 7.68 (m, 1H, H-7), 7.34 (m, 1H, H-8),
7.15–7.26 (m, 2H, Ph), 6.62–7.04 (m, 2H, Ph), 6.61 (s, 1H, H-3), 3.67
(m, 2H, N1(CH2)), 3.19 (m, 4H, N4(CH2)2), 2.65 (m, 4H, N1(CH2)2),
2.29 (s, 3H, CH3). Crystal data: empirical formula, C21H21Cl1N2O2;
molecular weight, 368.85; crystal dimensions, 0.50 ꢀ 0.48 ꢀ 0.27
4.2.7. 4-Methoxy-6-((4-(4-methoxybenzoyl)piperazin-1-yl)meth-
yl)coumarin (12)
Purified by flash silica gel column chromatography (EtOAc:hex-
ane = 1:5) to afford compound 12, yield 21%, colorless powder, mp
124–126 °C; MS (ESI) m/z 409.1 (M+H)+; IR (KBr)
c
cmꢁ1 3075,
ꢀ
mm; triclinic; P1; a = 6.010(2) Å, b = 11.126(3) Å, c = 13.829(3) Å;
c
= 85.155(2)°; V = 918.7(5) Å3;
1707, 1629, 1604, 1575, 1456, 1378. 1H NMR (CDCl3, 500 MHz) d
7.99 (s, 1H, H-5), 7.51 (br d, 1H, H-7), 7.23 (m, 1H, H-8), 7.11–
7.23 (m, 4H, Ph), 5.63 (s, 1H, H-3), 3.98 (s, 3H, OCH3), 2.37 (s, 3H,
CH3), 3.63 (m, 2H, N1(CH2)), 3.54 (m, 6H, N4(CH2)2, CH2), 2.56 (m,
4H, N1(CH2)2). HPLC analysis 96.2% (V(Methol): V(H2O) = 65:35,
tR = 6.06 min).
a
= 86.026(3)°, b = 87.900(3)°,
Z = 2; Dx = 1.333 mg mꢁ3; R1 = 0.0462;
MoK radiation cell parameters from 3199 reflections; h = 1.48–
25.01°;
= 0.226 mmꢁ1; T = 298(2) K. HPLC analysis 97.3% (V(Meth-
x
R2 = 0.1104; GOOF = 1.019;
a
l
ol): V(H2O) = 65:35, tR = 6.31 min).
4.3.4. 4-Chloro-6-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-
coumarin (17)
4.2.8. 4-Methoxy-6-((4-benzoylpiperazin-1-yl)methyl)coumarin
(13)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 17, yield 12%, colorless powder, mp
Purified by flash silica gel column chromatography (EtOAc:hex-
ane = 1:5) to afford compound 13, yield 16%, colorless powder, mp
116–118 °C; MS (ESI) m/z 385.2 (M+H)+; IR (KBr)
c
cmꢁ1 3440,
1727, 1614, 1570, 1121, 1012, 921. 1H NMR (CDCl3, 500 MHz) d
131–134 °C; MS (ESI) m/z 379.2 (M+H)+; IR (KBr)
c
cmꢁ1 3075,
1707, 1629, 1604, 1378, 1249, 986. 1H NMR (CDCl3, 500 MHz) d
7.84 (s, 1H, H-5), 7.68 (m, 1H, H-7), 7.34 (m, 1H, H-8), 6.85–6.98

8018
X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
(m, 4H, Ph), 6.61 (s, 1H, H-3), 3.86 (s, 3H, OCH3), 3.67 (m, 2H,
N1(CH2)), 3.11 (m, 4H, N4(CH2)2), 2.68 (m, 4H, N1(CH2)2). HPLC
analysis 95.3% (V(Methol): V(H2O) = 65:35, tR = 8.04 min).
chromatography (EtOAc/hexane) was performed to give the target
compounds, respectively.
4.4.1. 3-(4-Phenylpiperazin-1-carbonyl)coumarin (24)
Purified by flash silica gel column chromatography (EtOAc/
hexane = 1:5) to afford compound 24, yield 22%, colorless pow-
4.3.5. 4-Chloro-6-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)-
coumarin (18)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 18, yield 8%, colorless powder, mp
der, mp 232–234 °C; MS (ESI) m/z 335.1 (M+H)+; IR (KBr) cmꢁ1
c
2831, 1718, 1469, 1234, 754. 1H NMR (CDCl3, 500 MHz) d 8.00
(s, 1H, H-4), 7.64 (m, 1H, H-7), 7.58 (m, 1H, H-5), 7.41 (m, 1H,
H-8), 7.32 (m, 1H, H-6), 3.99 (m, 2H, N4(CH2)), 3.60 (m, 2H,
N4(CH2)), 3.33 (m, 2H, N1(CH2)), 3.26 (m, 2H, N1(CH2)). 13C
NMR (CDCl3, 125 MHz) d 157.9 (C-2), 116.8 (C-3), 132.9 (C-4),
125.2 (C-5), 124.9 (C-6), 128.6 (C-7), 118.4 (C-8), 154.2 (C-9),
120.7 (C-10), 163.5 (C-11), 47.1 (C-13), 49.5 (C-14), 49.8 (C-
16), 42.1 (C-17), 143.4 (C-18), 116.8 (C-19), 129.3 (C-20), 116.9
(C-21), 129.3 (C-22), 116.9 (C-23). HPLC analysis 97.3% (V(Meth-
ol): V(H2O) = 65:35, tR = 3.36 min).
121–124 °C; MS (ESI) m/z 385.1 (M+H)+; IR (KBr)
c
cmꢁ1 3090,
2950, 1779, 1512, 1180, 1142, 831. 1H NMR (CDCl3, 500 MHz) d
7.84 (s, 1H, H-5), 7.65 (d, 1H, H-7, J = 8.17 Hz), 7.34 (d, 1H, H-8,
J = 8.46 Hz), 6.82–6.91 (m, 4H, Ph), 6.61 (s, 1H, H-3), 3.76 (s, 3H,
OCH3), 3.65 (m, 2H, N1(CH2)), 3.11 (m, 4H, N4(CH2)2), 2.64 (m, 4H,
N1(CH2)2). HPLC analysis 96.5% (V(Methol): V(H2O) = 65:35,
tR = 8.18 min).
4.3.6. 4-Chloro-6-((4-(4-ethoxyphenyl)piperazin-1-yl)methyl)-
coumarin (19)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 19, yield 19%, colorless powder, mp
4.4.2. 3-(4-Methylpiperazin-1-carbonyl)coumarin (25)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 25, yield 28%, colorless powder, mp
121–123 °C; MS (ESI) m/z 399.1 (M+H)+; IR (KBr)
c
cmꢁ1 2939,
1717, 1563, 1342, 1323, 1300, 1198. 1H NMR (CDCl3, 500 MHz) d
7.65 (s, 1H, H-5), 7.54 (m, 1H, H-7), 7.49 (m, 1H, H-8), 7.45 (m,
1H, Ph), 7.18–7.20 (m, 2H, Ph), 6.81–6.84 (m, 2H, Ph, H-3), 4.45
(q, 2H, OCH2, J = 7.13 Hz), 3.65 (m, 2H, N1(CH2)), 3.17 (m, 4H,
N4(CH2)2), 2.64 (m, 4H, N1(CH2)2), 1.43 (t, 3H, CH3, J = 7.13 Hz).
HPLC analysis 95.9% (V(Methol): V(H2O) = 65:35, tR = 9.67 min).
220–222 °C; MS (ESI) m/z 273.1 (M+H)+; IR (KBr) cmꢁ1 3423, 1726,
c
1622, 1476, 1285, 1127, 762. 1H NMR (CDCl3, 500 MHz) d 7.90 (s,
1H, H-4), 7.58 (ddd, 1H, H-7, J = 1.59 Hz, 7.37, 1.18 Hz), 7.53 (dd,
1H, H-5, J = 1.53, 7.63 Hz), 7.35 (m, 1H, H-8), 7.31 (ddd, 1H, H-6,
J = 1.49, 7.53, 1.85 Hz), 3.79 (m, 2H, N4(CH2)), 3.41 (m, 2H, N4(CH2)),
2.51 (m, 2H, N1(CH2)), 2.45 (m, 2H, N1(CH2)), 2.33 (s, 3H, CH3). HPLC
analysis 99.3% (V(Methol): V(H2O) = 65:35, tR = 2.42 min).
4.3.7. 4-(4-o-Tolylpiperazin-1-yl)-6-((4-o-tolylpiperazin-1-yl)-
methyl)coumarin (20)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:3) to afford compound 20, yield 8%, colorless powder, mp
4.4.3. 3-(Morpholino-4-carbonyl)coumarin (26)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 26, yield 29%, colorless powder, mp
169–171 °C; MS (ESI) m/z 509.3 (M+H)+; IR (KBr)
c
cmꢁ1 2952,
180–182 °C; MS (ESI) m/z 260.0 (M+H)+; IR (KBr)
c
cmꢁ1 3424,
1715, 1617, 1433, 1371, 1028, 765. 1H NMR (CDCl3, 500 MHz)d
7.69 (s, 1H, H-5), 7.51 (m, 1H, H-7), 7.32 (m, 1H, H-8), 6.96–7.23
(m, 8H, Ph), 5.79 (s, 1H, 3-H), 3.65 (m, 2H, N1(CH2)), 3.18 (m, 4H,
1721, 1646, 1270, 1238, 1109, 1065, 995, 773. 1H NMR (CDCl3,
500 MHz) d 7.95 (s, 1H, H-4), 7.58 (m, 1H, H-7), 7.54 (m, 1H, H-
5), 7.33–7.37 (m, 2H, H-6, H-8), 3.79 (m, 2H, OCH2), 3.72 (m, 2H,
N1(CH2)), 3.41 (m, 2H, N1(CH2)). HPLC analysis 98.1% (V(Methol):
V(H2O) = 65:35, tR = 3.63 min).
N4(CH2)2), 2.64 (m, 4H, N1(CH2)2), 3.44 (m, 4H, N0 (CH2)2), 3.18
1
(m, 4H, N0 (CH2)2), 2.36 (s, 3H, CH3), 2.29 (s, 3H, CH3). HPLC analysis
4
97.4% (V(Methol): V(H2O) = 65:35, tR = 2.16 min).
4.4.4. 3-(4-o-Tolylpiperazine-1-carbonyl)coumarin (27)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 27, yield 25%, colorless powder, mp
4.3.8. Ethyl-2-oxo-2H-chromene-3-carboxylate (21)
0.005 mol salicylaldehyde, 0.005 mol malonic acid, 0.05 ml
piperidine, and 0.005 ml glacial acetic acid were added to 20 ml
anhydrous alcohol. The mixture was refluxed for 3 h, and then
the resultant was poured into 50 ml hot water. The water was
cooled down to room temperature, and the residue was filtered
and recrytallized with 95% alcohol to give 21, 0.7 g, yield 64.3%.
164–166 °C; MS (ESI) m/z 349.2 (M+H)+; IR (KBr) cmꢁ1 2821, 1726,
c
1631, 1369, 1243, 994, 724. 1H NMR (CDCl3, 500 MHz) d 7.95 (s, 1H,
H-4), 7.55–7.59 (m, 2H, H-7, H-5), 7.33–7.37 (m, 2H, H-6, H-8),
7.00–7.18 (m, 4H, Ph), 3.93 (m, 2H, N4(CH2)), 3.55 (m, 2H, N4(CH2)),
2.99 (m, 2H, N1(CH2)), 2.94 (m, 2H, N1(CH2)), 2.32 (s, 3H, CH3). HPLC
analysis 96.3% (V(Methol): V(H2O) = 65:35, tR = 2.62 min).
4.3.9. 2-Oxo-2H-chromene-3-carboxylic acid (22)
0.02 mol 21 and 250 ml of 0.5% NaOH solution were added to
50 ml alcohol. The mixture was refluxed for 2 h, and then it was
acidified with HCl to pH 2. The mixture was cooled down to 0 °C
and crystallized with ethanol to give 22, 3.29 g, yield 86.7%.
4.4.5. 3-(4-(4-Methoxyphenyl)piperazine-1-carbonyl)coumarin (28)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 28, yield 23%, colorless powder, mp
140–142 °C; MS (ESI) m/z 365.2 (M+H)+; IR (KBr)
c
cmꢁ1 2918,
1716, 1636, 1446, 1243, 1036, 763. 1H NMR (CDCl3, 500 MHz)
d7.95 (s, 1H, H-4), 7.60 (m, 1H, H-7), 7.55 (m, 1H, H-5), 7.33–7.38
(m, 2H, H-6, H-8), 6.91 (m, 2H, Ph), 6.84 (m, 2H, Ph), 3.93 (m, 2H,
N4(CH2)), 3.55 (m, 2H, N4(CH2)), 3.16 (m, 2H, N1(CH2)), 3.09 (m,
2H, N1(CH2)), 3.77 (s, 3H, OCH3). HPLC analysis 95.3% (V(Methol):
V(H2O) = 65:35, tR = 2.63 min).
4.3.10. 2-Oxo-2H-chromene-3-carbonyl chloride (23)
0.002 mol 22 was added to 5 ml sulfurous oxychloride. The mix-
ture was refluxed for 3 h, and then the resultant was removed with
simple distillation to give simple 23, 3.95 g. The simple compound
can be used directly without purification.
4.4. General procedure for the preparation of compounds 24–37
4.4.6. 3-(4-(2-Methoxyphenyl)piperazine-1-carbonyl)coumarin (29)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 29, yield 29%, colorless powder, mp
0.001 mol 23, 0.001 mol substituted phenylpiperazine deriva-
tives, and 0.002 mol potassium carbonate were added to a mixed
solvent containing 15 ml acetone and 15 ml alcohol. The mixture
was refluxed for 6 h, then the resultant was evaporated. The flash
146–147 °C; MS (ESI) m/z 365.1 (M+H)+; IR (KBr)
c
cmꢁ1 2944,
2904, 1738, 1633, 1237, 930, 753. 1H NMR (CDCl3, 500 MHz) d

X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
8019
7.95 (s, 1H, H-4), 7.54–7.59 (m, 2H, H-7, H-5), 7.33–7.37 (m, 2H, H-
6, H-8), 6.88–6.93 (m, 4H, Ph), 3.97 (m, 2H, N4(CH2)), 3.59 (m, 2H,
N4(CH2)), 3.16 (m, 2H, N1(CH2)), 3.11 (m, 2H, N1(CH2)), 3.87 (s, 3H,
OCH3). HPLC analysis 96.3% (V(Methol): V(H2O) = 65:35,
tR = 2.63 min).
1H, H-4), 7.60 (m, 1H, H-7), 7.55 (m, 1H, H-5), 7.26–7.41 (m, 6H,
H-6, H-8, Ph), 3.42–3.78 (m, 8H, N1(CH2)2, N4(CH2)2). HPLC analysis
97.5% (V(Methol): V(H2O) = 65:35, tR = 3.16 min).
4.4.13. 3-(4-(2-Chlorobenzoyl)piperazine-1-carbonyl)coumarin
(36)
4.4.7. 3-(4-(4-Chlorophenyl)piperazine-1-carbonyl)coumarin (30)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 30, yield 23%, colorless powder, mp
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 36, yield 16%, colorless powder, mp
200–203 °C; MS (ESI) m/z 397.0 (M+H)+; IR (KBr)
c
cmꢁ1 3041,
128–130 °C; MS (ESI) m/z 369.2 (M+H)+; IR (KBr)
c
cmꢁ1 3425, 1718,
1705, 1641, 1434, 1290, 1241, 1175. 1H NMR (CDCl3, 500 MHz) d
7.97 (s, 1H, H-4), 7.56 (m, 1H, H-7), 7.40 (m, 1H, H-5), 7.26–7.38
(m, 6H, H-6, H-8, Ph), 3.32–4.07 (m, 8H, N1(CH2)2, N4(CH2)2). HPLC
analysis 98.1% (V(Methol): V(H2O) = 65:35, tR = 2.76 min).
1635, 1443, 1284, 997, 692. 1H NMR (CDCl3, 500 MHz) d 7.96 (s, 1H,
H-4), 7.54–7.60 (m, 2H, H-7, H-5), 7.33–7.38 (m, 2H, H-6, H-8),
7.26–7.30 (m, 2H, Ph), 6.91–6.95 (m, 2H, Ph), 3.94 (m, 2H, N4(CH2)),
3.56 (m, 2H, N4(CH2)), 3.29 (m, 2H, N1(CH2)), 3.22 (m, 2H, N1(CH2)).
HPLC analysis 96.4% (V(Methol): V(H2O) = 65:35, tR = 2.92 min).
4.4.14. 3-(4-(4-Nitrobenzoyl)piperazine-1-carbonyl)coumarin (37)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 37, yield 25%, colorless powder, mp
4.4.8. 3-(4-(4-Methylphenyl)piperazine-1-carbonyl)coumarin (31)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 31, yield 22%, colorless powder, mp
224–226 °C; MS (ESI) m/z 408.0 (M+H)+; IR (KBr)
c
cmꢁ1 3433,
1710, 1633, 1348, 1286, 1241, 1175, 1093, 840. 1H NMR (CDCl3,
500 MHz) d 8.30 (m, 2H, Ph), 7.99 (s, 1H, H-4), 7.56–7.64 (m, 4H,
H-5, H-7, Ph), 7.34–7.38 (m, 2H, H-6, H-8), 3.36–3.91 (m, 8H,
156–157 °C; MS (ESI) m/z 349.1 (M+H)+; IR (KBr)
c
cmꢁ1 3030,
2825, 1726, 1630, 1476, 1151, 996, 970, 754. 1H NMR (CDCl3,
500 MHz) d7.95 (s, 1H, H-4), 7.54–7.59 (m, 2H, H-7, H-5), 7.32–
7.37 (m, 2H, H-6, H-8), 6.88–7.32 (m, 4H, Ph), 3.94 (m, 2H,
N4(CH2)), 3.56 (m, 2H, N4(CH2)), 3.23 (m, 2H, N1(CH2)), 3.17 (m,
2H, N1(CH2)), 2.28 (s, 3H, CH3). HPLC analysis 98.3% (V(Methol):
V(H2O) = 65:35, tR = 2.63 min).
N1(CH2)2,
N4(CH2)2).
HPLC
analysis
97.7%
(V(Methol):
V(H2O) = 65:35, tR = 3.45 min).
4.5. General procedure for the preparation of compounds 38–50
0.001 mol 4, 0.001 mol substituted phenylpiperazine deriva-
tives, and 0.002 mol potassium carbonate were added to a mixed
solvent of 15 ml acetone and 15 ml alcohol. The mixture was re-
fluxed for 10 h, and then the resultant was evaporated. The flash
chromatography (EtOAc/hexane) was performed to give the target
compounds, respectively.
4.4.9. 3-(4-(2,5-Dichlorophenyl)piperazine-1-carbonyl)coumarin
(32)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 32, yield 18%, colorless powder, mp
104–106 °C; MS (ESI) m/z 403.0 (M+H)+; IR (KBr)
c
cmꢁ1 3421,
1722, 1639, 1363, 1231, 995, 751. 1H NMR (CDCl3, 500 MHz) d
7.97 (s, 1H, H-4), 7.61 (m, 1H, H-7), 7.56 (m, 1H, H-5), 7.37 (m,
1H, H-8), 7.34 (m, 1H, H-6), 7.29 (m, 1H, Ph), 7.05 (m, 1H, Ph),
6.99 (m, 1H, Ph), 3.98 (m, 2H, N4(CH2)), 3.59 (m, 2H, N4(CH2)),
3.16 (m, 2H, N1(CH2)), 3.13 (m, 2H, N1(CH2)). HPLC analysis 98.2%
(V(Methol): V(H2O) = 65:35, tR = 3.36 min).
4.5.1.6-Methyl-4-(4-(2-methoxyphenyl)piperazin-1-yl)coumarin
(38)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 38, yield 19%, colorless powder, mp
131–133 °C; MS (ESI) m/z 351.1 (M+H)+; IR (KBr)
c
cmꢁ1 3441,
2832, 1713, 1596, 1376, 1242, 1029, 936. 1H NMR (CDCl3,
500 MHz)d 7.46 (s, 1H, H-5), 7.33 (m, 1H, H-7), 7.28 (m, 1H, 8-H),
6.94–7.09 (m, 4H, Ph), 5.80 (s, 1H, H-3), 3.49 (m, 4H, N4(CH2)2),
3.36 (m, 4H, N1(CH2)2), 3.93 (s, 3H, OCH3), 2.45 (s, 3H, CH3). 13C
NMR (CDCl3, 125 MHz) d 162.6 (C-2), 98.2 (C-3), 161.2 (C-4),
124.5 (C-5), 132.5 (C-6), 123.7 (C-7), 121.2 (C-8), 152.4 (C-9),
116.1 (C-10), 21.1 (CH3), 51.3 (C-13), 50.3 (C-14), 51.3 (C-16),
50.3 (C-17), 152.4 (C-18), 132.9 (C-19), 111.7 (C-20), 117.6 (C-
21), 118.6 (C-22), 55.9 (C-23), 55.6 (OCH3). HPLC analysis 98.3%
(V(Methol): V(H2O) = 70:30 tR = 8.52 min).
4.4.10. 3-(4-(4-Methylbenzoyl)piperazine-1-carbonyl)coumarin
(33)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 33, yield 13%, colorless powder, mp
92–94 °C; MS (ESI) m/z 377.1 (M+H)+; IR (KBr) cmꢁ1 3436, 1727,
c
1627, 1286, 1711, 1050, 751. 1H NMR (CDCl3, 500 MHz) d 7.96 (s,
1H, H-4), 7.59 (m, 1H, H-7), 7.55 (m, 1H, H-5), 7.33–7.37 (m, 2H,
H-6, H-8), 7.32–7.37 (m, 2H, Ph), 7.21–7.30 (m, 2H, Ph), 3.41–
3.77 (m, 8H, N1(CH2)2, N4(CH2)2), 2.37 (s, 3H, CH3). HPLC analysis
96.3% (V(Methol): V(H2O) = 65:35, tR = 4.54 min).
4.5.2. 6-Methyl-4-(4-(4-methoxyphenyl)piperazin-1-yl)coumarin
(39)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 39, yield 17%, colorless powder, mp
4.4.11. 3-(4-Benzoylpiperazine-1-carbonyl)coumarin (34)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 34, yield 19%, colorless powder, mp
110–112 °C; MS (ESI) m/z 363.0 (M+H)+; IR (KBr)
c
cmꢁ1 3427,
146–148 °C; MS (ESI) m/z 351.2 (M+H)+; IR (KBr)
c
cmꢁ1 3441,
1720, 1627, 1429, 1245, 1172, 1009, 563. 1H NMR (CDCl3,
500 MHz) d 7.96 (s, 1H, H-4), 7.60 (m, 1H, H-7), 7.54 (m, 1H, H-
5), 7.31–7.41 (m, 7H, H-6, H-8, Ph), 3.42–3.78 (m, 8H, N1(CH2)2,
N4(CH2)2). HPLC analysis 97.9% (V(Methol): V(H2O) = 65:35,
tR = 6.34 min).
2829, 1696, 1561, 1374, 1236, 1030, 938, 823. 1H NMR (CDCl3,
500 MHz) d 7.43 (s, 1H, H-5), 7.32 (m, 1H, H-7), 7.25 (m, 1H, 8-
H), 6.92–6.99 (m, 4H, Ph), 5.78 (s, 1H, H-3), 3.48 (m, 4H,
N4(CH2)2), 3.34 (m, 4H, N1(CH2)2), 3.91 (s, 3H, OCH3), 2.42 (s, 3H,
CH3). HPLC analysis 99.1% (V(Methol): V(H2O) = 70:30,
tR = 4.81 min).
4.4.12. 3-(4-(4-Chlorobenzoyl)piperazine-1-carbonyl)coumarin
(35)
4.5.3. 6-Methyl-4-(4-(4-methylphenyl)piperazin-1-yl)coumarin (40)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 40, yield 15%, colorless powder, mp
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:5) to afford compound 35, yield 12%, colorless powder, mp
128–130 °C; MS (ESI) m/z 397.0 (M+H)+; IR (KBr)
c
cmꢁ1 3034,
126–128 °C; MS (ESI) m/z 335.1 (M+H)+; IR (KBr)
c
cmꢁ1 3448,
1719, 1629, 1246, 1172, 748. 1H NMR (CDCl3, 500 MHz) d 7.97 (s,

8020
X. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 8011–8021
1688, 1557, 1516, 1425, 1384, 1238, 1204, 937, 840. 1H NMR (CDCl3,
500 MHz) d 7.45 (s, 1H, H-5), 7.36 (m, 1H, H-7), 7.29 (m, 1H, H-8),
7.16–7.17 (m, 2H, Ph), 6.96–6.97 (m, 2H, Ph), 5.81 (s, 1H, H-3),
3.41–3.44 (m, 8H, N1(CH2)2, N4(CH2)2), 2.46 (s, 3H, CH3), 2.34 (s,
3H, CH3). HPLC analysis 96.3% (V(Methol): V(H2O) = 70:30,
tR = 4.71 min).
3.46 (m, 4H, N4(CH2)2), 3.32 (m, 4H, N1(CH2)2), 2.45 (s, 3H, CH3).
HPLC analysis 97.7% (V(Methol): V(H2O) = 70:30, tR = 2.92 min).
4.5.10.6-Methyl-4-(4-(4-methylbenzoyl)piperazin-1-yl)coumarin
(47)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 47, yield 18%, colorless powder, mp
4.5.4. 6-Methyl-4-(4-o-tolylpiperazin-1-yl)coumarin (41)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 41, yield 16%, colorless powder, mp
123–125 °C; MS (ESI) m/z 363.1 (M+H)+; IR (KBr) cmꢁ1 3425,
c
2832, 1718, 1635, 1443, 1284, 1234. 1H NMR (CDCl3, 500 MHz) d
7.38 (m, 3H, H-5, Ph), 7.26–7.35 (m, 4H, H-7, H-8, Ph), 5.75 (s,
1H, H-3), 3.90 (m, 4H, N4(CH2)2), 3.27 (m, 4H, N1(CH2)2), 2.42–
2.45 (m, 6H, 2ꢀ CH3). HPLC analysis 95.6% (V(Methol):
V(H2O) = 70:30, tR = 3.91 min).
132–134 °C; MS (ESI) m/z 335.1 (M+H)+; IR (KBr)
c
cmꢁ1 3431,
2831, 1713, 1596, 1375, 1242, 1212, 936, 816. 1H NMR (CDCl3,
500 MHz) d 7.48 (s, 1H, H-5), 7.35 (m, 1H, H-7), 7.29 (m, 1H, 8-
H), 7.24–7.27 (m, 2H, Ph), 7.17 (m, 1H, Ph), 7.09 (m, 1H, Ph), 5.81
(s, 1H, H-3), 3.47 (m, 4H, N4(CH2)2), 3.21 (m, 4H, N1(CH2)2), 2.46
(s, 3H, CH3), 2.41 (s, 3H, CH3). HPLC analysis 97.1% (V(Methol):
V(H2O) = 70:30, tR = 2.15 min).
4.5.11. 6-Methyl-4-(4-benzoylpiperazin-1-yl)coumarin (48)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 48, yield 19%, colorless powder, mp
136–138 °C; MS (ESI) m/z 349.1 (M+H)+; IR (KBr)
c
cmꢁ1 3441,
4.5.5. 6-Methyl-4-(4-(4-chlorophenyl)piperazin-1-yl)coumarin (42)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 42, yield 19%, colorless powder, mp
1694, 1629, 1444, 1424, 1372, 1187, 707. 1H NMR (CDCl3,
500 MHz) d 7.45 (m, 5H, Ph), 7.35 (s, 1H, H-5), 7.32 (m, 1H, H-7),
7.24 (m, 1H, H-8), 5.73 (s, 1H, H-3), 3.25–3.98 (m, 8H, N1(CH2)2,
N4(CH2)2), 2.41 (s, 3H, CH3). HPLC analysis 97.1% (V(Methol):
V(H2O) = 70:30, tR = 3.35 min).
133–135 °C; MS (ESI) m/z 355.1 (M+H)+; IR (KBr)
c
cmꢁ1 3443,
2835, 1709, 1596, 1426, 1379, 1023, 938. 1H NMR (CDCl3,
500 MHz) d 7.44 (s, 1H, H-5), 7.36 (m, 1H, H-7), 7.28–7.31 (m,
3H, H-8, Ph), 6.95–6.97 (m, 2H, Ph), 5.81 (s, 1H, H-3), 3.42–3.45
(m, 8H, N1(CH2)2, N4(CH2)2), 2.46 (s, 3H, CH3). HPLC analysis
96.5% (V(Methol): V(H2O) = 70:30, tR = 2.43 min).
4.5.12.6-Methyl-4-(4-(4-chlorobenzoyl)piperazin-1-yl)coumarin
(49)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 49, yield 22%, colorless powder, mp
4.5.6. 6-Methyl-4-(4-methylpiperazin-1-yl)coumarin (43)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 43, yield 17%, colorless powder, mp
126–128 °C; MS (ESI) m/z 383.0 (M+H)+; IR (KBr) cmꢁ1 3434,
c
1720, 1644, 1626, 1430, 1261, 1204, 1011. 1H NMR (CDCl3,
500 MHz) d 7.43 (m, 4H, Ph), 7.34 (s, 1H, H-5), 7.32 (m, 1H, H-7),
7.24 (m, 1H, H-8), 5.72 (s, 1H, H-3), 3.25–3.89 (m, 8H, N1(CH2)2,
N4(CH2)2), 2.41 (s, 3H, CH3). HPLC analysis 96.2% (V(Methol):
V(H2O) = 70:30, tR = 4.71 min).
126-129 °C; MS (ESI) m/z 259.1 (M+H)+; IR (KBr)
c
cmꢁ1 3084,
2930, 1695, 1613, 1371, 1240, 1101, 1006, 934, 833. 1H NMR
(CDCl3, 500 MHz) d 7.36 (s, 1H, H-5), 7.21–7.30 (m, 2H, H-7, H-
8), 5.71 (s, 1H, H-3), 3.29 (m, 4H, N1(CH2)2), 2.66 (m, 4H,
N4(CH2)2), 2.41 (s, 6H, 2 ꢀ CH3). HPLC analysis 96.2% (V(Methol):
V(H2O) = 70:30, tR = 5.12 min).
4.5.13.6-Methyl-4-(4-(2-chlorobenzoyl)piperazin-1-yl)coumarin
(50)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 50, yield 26%, colorless powder, mp
4.5.7. 6-Methyl-4-morpholino-coumarin (44)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 44, yield 23%, colorless powder, mp
166–168 °C; MS (ESI) m/z 383.0 (M+H)+; IR (KBr) cmꢁ1 3434,
c
1720, 1644, 1626, 1568, 1430, 1011. 1H NMR (CDCl3, 500 MHz) d
7.43 (m, 1H, H-5), 7.32–7.89 (m, 4H, Ph), 7.31 (m, 1H, H-7), 7.24
(m, 1H, H-8), 5.73 (s, 1H, H-3), 3.25–3.89 (m, 8H, N1(CH2)2,
N4(CH2)2), 2.41 (s, 3H, CH3). HPLC analysis 97.2% (V(Methol):
V(H2O) = 70:30, tR = 4.52 min).
106–108 °C; MS (ESI) m/z 246.1 (M+H)+; IR (KBr)
c
cmꢁ1 3441,
2964, 1693, 1562, 1448, 1424, 1202, 948. 1H NMR (CDCl3,
500 MHz) d 7.36 (s, 1H, H-5), 7.31 (m, 1H, H-7), 7.24 (m, 1H, H-8),
5.71 (s, 1H, H-3), 3.94 (m, 4H, N1(CH2)2), 3.24 (m, 4H, N4(CH2)2),
2.42 (s, 3H, CH3). HPLC analysis 97.6% (V(Methol): V(H2O) = 70:30,
tR = 3.52 min).
4.6. In vitro AChE inhibition assay
4.5.8. 6-Methyl-4-(4-phenylpiperazin-1-yl)coumarin (45)
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 45, yield 15%, colorless powder, mp
AChE activity was measured repeated twice by the spectropho-
tometric method reported by Ellman et al.25 with some modifica-
tions. Brain homogenate was used as the enzyme source. The
whole brain except for the cerebellum was homogenized in 9 vol-
umes of 100 mM sodium phosphate buffer (pH 7.0). The test com-
pounds were dissolved in dimethyl sulfoxide (DMSO). The AChE
activity was expressed as a change in OD at 412 nm.
125–127 °C; MS (ESI) m/z 321.1 (M+H)+; IR (KBr)
c
cmꢁ1 3432,
1710, 1596, 1560, 1203, 1024, 939. 1H NMR (CDCl3, 500 MHz) d
7.41 (s, 1H, H-5), 7.32–7.37 (m, 2H, H-7, Ph), 7.25 (m, 1H, 8-H),
7.01–7.26 (m, 3H, Ph), 5.79 (s, 1H, H-3), 3.48 (m, 8H, N1(CH2)2,
N4(CH2)2), 2.43 (s, 3H, CH3). HPLC analysis 98.6% (V(Methol):
V(H2O) = 70:30, tR = 4.70 min).
Acknowledgment
4.5.9.6-Methyl-4-(4-(2,5-dichlorophenyl)piperazin-1-yl)coumarin
(46)
This research work was supported by the Teaching and Re-
search Award Program for Outstanding Young Teachers in Higher
Education Institutions of MOE, P.R. China (for L.-Y. Kong).
Purified by flash silica gel column chromatography (EtOAc/hex-
ane = 1:7) to afford compound 46, yield 22%, colorless powder, mp
141–143 °C; MS (ESI) m/z 389.1 (M+H)+; IR (KBr)
c
cmꢁ1 3441,
References and notes
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(m, 1H, Ph), 7.12 (m, 1H, Ph), 7.03 (m, 1H, Ph), 5.80 (s, 1H, H-3),
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