Novel aromatic-polyamine conjugates as cholinesterase inhibitors with notable selectivity toward butyrylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2014.03.045

Three types of aromatic-polyamine conjugates (6a-6s) were designed, synthesized and evaluated as potential inhibitors for cholinesterases (ChEs). The results showed that anthraquinone-polyamine conjugates (AQPCs) exhibited the most potent acetylcholinesterase (AChE) inhibitory activity with IC ₅₀ values from 1.50 to 11.13 μM. Anthracene-polyamine conjugates (APCs) showed a surprising selectivity (from 76- to 3125-fold) and were most potent at inhibiting butyrylcholinesterase (BChE), with IC₅₀ values from 0.016 to 0.657 μM. A Lineweaver-Burk plot and molecular modeling studies indicated that the representative compounds, 6l and 6k, targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of ChEs. Furthermore, APCs did not affect HepG2 cell viability at the concentration of 100 μM. Consequently, these polyamine conjugates could be thoroughly and systematically studied for the treatment of AD.

Full text of DOI:10.1016/j.bmc.2014.03.045

Bioorganic & Medicinal Chemistry 22 (2014) 3213–3219
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel aromatic–polyamine conjugates as cholinesterase inhibitors
with notable selectivity toward butyrylcholinesterase
,
⇑
⇑
Chen Hong , Wen Luo , Dong Yao, Ya-Bin Su, Xin Zhang, Run-Guo Tian, Chao-Jie Wang
Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng 475004, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three types of aromatic–polyamine conjugates (6a–6s) were designed, synthesized and evaluated as
potential inhibitors for cholinesterases (ChEs). The results showed that anthraquinone–polyamine conju-
gates (AQPCs) exhibited the most potent acetylcholinesterase (AChE) inhibitory activity with IC50 values
Received 24 February 2014
Revised 29 March 2014
Accepted 29 March 2014
Available online 13 April 2014
from 1.50 to 11.13
76- to 3125-fold) and were most potent at inhibiting butyrylcholinesterase (BChE), with IC50 values from
0.016 to 0.657 M. A Lineweaver–Burk plot and molecular modeling studies indicated that the represen-
tative compounds, 6l and 6k, targeted both the catalytic active site (CAS) and the peripheral anionic site
(PAS) of ChEs. Furthermore, APCs did not affect HepG2 cell viability at the concentration of 100 M. Con-
lM. Anthracene–polyamine conjugates (APCs) showed a surprising selectivity (from
l
Keywords:
Polyamine conjugates
Alzheimer’s disease
Acetylcholinesterase
Butyrylcholinesterase
l
sequently, these polyamine conjugates could be thoroughly and systematically studied for the treatment
of AD.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors and the continued use of cholinesterase inhibitors may
lead to improved clinical outcomes.7
Alzheimer’s disease (AD) is characterized by dementia, cogni-
tive impairment, and memory loss and is one of the most common
diseases in elderly people1,2 Current treatment of AD focuses on
acetylcholinesterase (AChE) inhibitors, such as tacrine, donepezil,
rivastigmine and galantamine. However, the potential effective-
ness of such inhibitors in clinical use is often complicated by their
associated side effects. For example, clinical studies have shown
that the AChE inhibitor tacrine causes hepatotoxicity.3 Since AD
is a multi-pathogenic illness, a current drug-discovery strategy is
to develop novel anti-Alzheimer agents with multiple potencies
such as inhibition of both AChE and butyrylcholinesterase (BChE).4
Two major ChEs, AChE and BChE, are involved in the hydrolysis
and regulation of acetylcholine in vertebrates. Various cholinergic
drugs, primarily inhibitors of AChE also function as BChE inhibi-
tors.5 The use of agents with enhanced selectivity for BChE includ-
ing cymserine and MF-8622 indicated potential therapeutic benefit
of inhibiting BChE in AD and related dementias. BChE specific inhi-
bition is unlikely to be associated with adverse events and may
show efficacy without remarkable side effects.6 Therefore BChE
may be considered as an important target for novel drug develop-
ment to treat AD. In the future, the development of specific BChE
Polyamines such as putrescine, spermidine and spermine
(Fig. 1) are aliphatic molecules with amine groups distributed
along their structure and are the most common natural products.
These compounds have always been regarded by medicinal chem-
ists as a universal template8 and many research groups have cho-
sen to focus their investigations on the polyamine metabolic cycle
or the design of polyamine modified drugs.9–12 Our group has cho-
sen to focus on the development of polyamine conjugates as poten-
tial drugs for the treatment of cancer and AD for many years.13–16
Our research has found that quinoline–polyamine conjugates exhi-
bit potent ChEs inhibitory activity and that polyamines occupy the
gorge of AChE.17 Therefore, to further explore the anti-Alzheimer
potential of the polyamine conjugates, we designed and synthe-
sized three types of aromatic–polyamine conjugates and screened
them for their ability to inhibit ChEs.
NH2
Putrecine
H2N
H2N
H2N
H
N
NH2
Spermidine
H
N
NH2
⇑
Corresponding authors. Tel./fax: +86 0371 22864665.
Spermine
N
H
E-mail addresses: luowen83@henu.edu.cn (W. Luo), wcjsxq@henu.edu.cn
(C.-J. Wang).
The first two authors contributed equally to this work.
Figure 1. Chemical structures of putrecine, spermidine and spermine.
http://dx.doi.org/10.1016/j.bmc.2014.03.045
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3214
C. Hong et al. / Bioorg. Med. Chem. 22 (2014) 3213–3219
compound’s ability to inhibit enzyme activity was directly propor-
2. Results and discussion
2.1. Chemistry
tional to the chain length of its polyamine moiety such that four
(6k) and six (6l) methylene groups was the optimal chain length
between two nitrogen atoms associated with the most potent inhi-
bition of BChE. When the distance between these two nitrogen
atoms is too long or too short, the compound is unable to optimally
inhibit BChE.
All of the synthetic anthracene conjugates showed high selec-
tivity for BChE over AChE and the ratio of BChE/AChE selectivity
ranged from 76- to 3125-fold. The selectivity ratios of compounds
were dependent on their inhibitory potential against BChE. The
compounds showed higher inhibitory potential against BChE that
would possess higher BChE/AChE selectivity ratios. Compounds
6k showed the highest inhibitory activity (IC50 = 16 nM) and also
had the highest selectivity ratio (3125-fold). This result indicated
that the anthracene conjugates could favor binding to BChE.
It is report that many aromatic polycyclic such anthrarobin and
anthraquinone have shown diverse activity depending on the func-
tional groups attached18,19 In this paper we chose three kinds of
aromatic polycyclic building blocks (naphthalene, anthracene and
anthraquinone) that are coupled to diverse polyamine motifs
(Fig. 2) to evaluate their inhibition ability to ChEs. The synthesis
of compound 2 was shown in Supplementary data, polyamine skel-
etons 4e–4j were synthesized in our lab16 and the target com-
pounds were synthesized according to Scheme 1. Intermediates
1a, 2a, 3a were obtained by the condensation of chloroacetyl chlo-
ride with 1, 2 and 3 as the starting material. The reaction of 1a–3a
with amines 4a–4j gave the intermediates 5a–5s, and their Boc
groups in the polyamine skeleton were subsequently removed
with 4 M HCl at room temperature to provide hydrochloride salt
target compounds. The structures of the target compounds were
confirmed by 1H NMR, ESI-MS and elemental analysis.
2.3. Kinetic characterization of ChEs inhibition
Graphical analysis of steady state inhibition of the most potent
compounds for AChE and BChE, APCs 6l and 6k, were investigated
to determine the kinetics of ChEs inhibition as shown in Figure 3.21
The Lineweaver–Burk plots showed both increasing slopes and
increasing intercepts for higher inhibitor concentration. The pat-
tern indicated a mixed-type inhibition, hence this kinetic study
suggested that compound 6l bound to both catalytic active site
(CAS) and the peripheral anionic site (PAS) of AChE. A similar inter-
action was found between 6k and BChE.
2.2. In vitro inhibition studies on AChE and BChE
To determine the potential effectiveness of the target com-
pounds 6a–6s for the treatment of AD, their ChEs inhibitory activ-
ity was determined by the method described by Ellman et al,20
using tacrine as a reference compound. The IC50 values for ChEs
inhibition and the selectivity index (SI) are summarized as shown
in Table 1. Due to their poor solubility, compounds 6f and 6g were
not measured. Generally, most of the synthetic compounds tested
(6b–6d, 6h–6o) showed inhibitory selectivity for BChE over AChE.
Anthraquinone–polyamine conjugates (AQPCs) (6p–6s) inhibited
AChE better than naphthalene–polyamine conjugates (NPCs) and
anthracene–polyamine conjugates (APCs), while APCs 6h–6o
exhibited the most potent inhibition of BChE.
2.4. Molecular modeling study
To investigate the interaction mode of compound 6l with
TcAChE (PDB code: 1ACJ), molecular modeling was carried out by
AUTODOCK 4.0 package with PyMOL program as shown in
Figure 4.22,23 The docking result demonstrated that compound 6l
exhibited multiple binding modes with AChE. In the 6l–TcAChE
complex, compound 6l occupied the entire enzymatic CAS, mid-
gorge and PAS. The anthracene moiety was bound to CAS, display-
Compounds 6l, 6q and 6r were the most potent inhibitors of
AChE and had an IC50 value of 2.74, 1.50 and 2.63
while compounds 6k and 6l, the most potent inhibitors of BChE,
had IC50 value of 0.016 and 0.023 M, respectively, which were
lM, respectively,
ing a classic
p–p stacking interaction between Trp84 and Phe330.
l
At the PAS, the end protonated nitrogen atom of the polyamine
much lower than that of tacrine (IC50 = 0.037 lM). Moreover, a
NH2
O
O
NH2
NH2
Ar-NH2=
1
2
3
H2N
H2N
OH
R-NHy=
H2N
4d
H2N
N
NHBoc
n
m
Boc
4a
4b
4h m=1, n=1
N
4i m=2, n=1
H2N
NHBoc
m
4e m=1
4f m=2
4g m=4
H2N
N
N
NHBoc
m
n
m
HN
Boc
Boc
4c
4j m=1, n=2
Figure 2. Building blocks of the designed library.
H
N
H
N
H
N
R
a
b
c
Ar
Cl
x HCl
Ar NH2
Ar
N
H
Ar
R1
O
O
O
1, 2, 3
1a, 2a, 3a
5a-5s
6a-6s
Scheme 1. Reagents and conditions: ClCOCH2Cl, THF, Et3 N, rt, 6 h for 1a and 2a; ClCOCH2Cl, DMF, pyridine, rt, 24 h for 3a; (b) 1.0 equiv of R–NHy, EtOH, Et3N, reflux, 6 h for
5a–5o; 1.0 equiv of R–NH2, CH3CN, K2CO3, KI, rt, 24 h for 5p–5s; (c) 4 M HCl, EtOH, rt, overnight.

C. Hong et al. / Bioorg. Med. Chem. 22 (2014) 3213–3219
3215
Table 1
Inhibition of ChEs activity and selectivity index of the synthesized compounds
H
N
Ar
R1
x HCl
O
6a-6e, 6f-6o, 6p-6s
a
b
Compds
Ar
R1
x
1
IC50 for AChE (
l
M)
IC50 for BChE (
lM)
Selectivity indexc
6a
>50
>50
>50
—
HN
OH
6b
6c
2
4.39 0.17
>11.4
N
HN
HN
NH2
2
2
3
>50
10.28 0.04
>4.9
HN
HN
N
H
NH2
6d
6e
>50
>50
—
47.55 2.02
>1.1
—
2
N
H
N
H
NH2
4
1
>50
—
2
6fd
—
N
6gd
6h
1
1
—
—
—
HN
HN
>50
0.502 0.041
>99.6
OH
6i
6j
2
2
>50
>50
0.161 0.004
0.280 0.007
>310.6
>178.6
N
HN
HN
NH2
NH2
HN
2
4
6k
2
>50
0.016 0.001
>3125
HN
HN
NH2
6l
2
3
3
2.74 0.12
>50
0.023 0.002
0.305 0.007
0.164 0.002
119.1
>163.9
153.1
N
H
NH2
NH2
6m
6n
HN
HN
N
H
25.11 2.24
2
N
H
N
H
NH2
6o
6p
4
1
>50
0.657 0.015
>50
>76.1
<0.20
2
11.13 0.36
HN
HN
OH
O
O
NH2
2
2
6q
6r
2
3
1.50 0.24
2.63 0.15
>50
<0.03
HN
N
H
NH2
32.96 0.71
<0.08
HN
N
H
N
H
NH2
6s
4
7.22 0.24
>50
<0.14
5.8
2
Tacrine
—
—
0.215 0.001
0.037 0.004
a
b
c
AChE from electric eel; IC50, 50% inhibitory concentration (means SEM of three experiments).
BChE from equine serum; IC50, 50% inhibitory concentration (means SEM of three experiments).
Selectivity index = IC50 (AChE)/IC50 (BChE).
d
Due to the poor solubility, compounds 6f and 6g were not measured.
establishes a cation–
p
interaction with Trp279. The result showed
with our target compounds. As shown in Table 2, the results indi-
that compound 6l was able to bind both CAS and PAS of AChE
which was in agreement with the result of kinetic study. Because
the crystal structure of BChE from equine serum has not been
reported, so we did not perform the docking study for compound
6k with BChE.
cated that NPCs (6a–6e) and APCs (6h–6o), showed no obvious
effect on cell viability at concentrations of 100 lM, compared with
tacrine, they had lower toxicity on cell viability. In the case of
AQPCs (6p–6s), they showed more toxicity than NPCs and APCs
with IC50 value from 11.52 to 24.25
mitoxantrone.
lM, but were less toxic than
2.5. Methyl thiazolyl tetrazolium (MTT) assay of HepG2 cell
viability
3. Conclusion
To determine the potential cytotoxic effect of our synthesized
compounds, the human hepatoma cell line HepG2 was treated
In summary, three types of novel aromatic–polyamine conjugates
6a–6s were synthesized and subjected to biological evaluation.

3216
C. Hong et al. / Bioorg. Med. Chem. 22 (2014) 3213–3219
50
40
30
20
10
0
30
non inhibitor
non inhibitor
[6k]= 5 nM
[6k]=10 nM
[6k]=20 nM
[6l]=1.0uM
[6l]=2.0uM
[6l]=4.0uM
25
20
15
10
5
0
-10
-5
0
5
10
-1
15
20
-10
-5
0
5
10
-1
15
20
-1
-1
[S] mM
[S] mM
Figure 3. Lineweaver–Burk plot for compound 6l (left) and 6k (right) with AChE and BChE, respectively.
Table 2
MTT assay of HepG2 cell viability
a
Compds
IC50 (lmol/L)
6a
6b
6c
6d
>100
>100
>100
>100
>100
—
6e
6fb
6gb
—
6h
6i
6j
6k
6l
6m
6n
6o
>100
>100
>100
>100
>100
>100
>100
>100
6p
6q
6r
6s
22.96 3.59
24.25 2.04
15.64 1.41
11.52 0.82
98.73 3.42
11.05 1.60
Tacrine
Mitoxantrone
a
IC50, 50% inhibitory concentration for HepG2 of target compounds, (mean-
SEM of three experiments).
Due to the poor solubility, compounds 6f and 6g were not measured.
s
b
analyses were performed on a Gmbe VarioEL Elemental Instru-
ment. Flash column chromatography was performed with silica
gel (200–300 mesh) purchased from Qingdao Haiyang Chemical
Co. Ltd.
Figure 4. Docking model for compound 6l with AChE.
The results have shown that AQPCs were moderately potent AChE
inhibitors with IC50 values at the micromolar range, APCs were
selective and the most potent inhibitors of BChE, the range of
BChE/AChE selectivity was from 76- to 3125-fold, and conjugates
4.2. General procedures for the preparation of intermediate 1a
and 2a
6k and 6l had an IC50 value of 0.016 and 0.023 lM, respectively,
which were much lower than that of tacrine. A Lineweaver–Burk
plot and molecular modeling study showed that 6k and 6l targeted
both the CAS and PAS of ChEs. In addition, the APCs did not show
A mixture of compound 1 (4.30 g, 30 mmol), triethylamine
(8.35 mL, 60 mmol) and THF (50 mL) were stirred at ice bath until
completely dissolved, and then chloroacetyl chloride (6 mL,
75 mmol) was added dropwise. The suspension was stirred for
6 h (monitored by TLC). And then the solvent was removed in vac-
uum, the residue was poured into water and filtered, and the filter
cake was washed by water, dried to obtain brown solid, which was
purified by flash chromatography to give white solid 1a. Yield 80%,
1H NMR (400 MHz, DMSO-d6) dH: 10.29 (br s, 1H), 8.05 (d,
J = 7.5 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H),
7.68 (d, J = 7.2 Hz, 1H),7.55–7.60 (m, 2H), 7.52 (t, J = 7.8 Hz, 1H),
4.45 (s, 2H). ESI-MS m/z: 220.7 (M+H)+.
obvious toxicity on HepG2 cell viability at 100 lM in vitro. These
novel polyamine conjugates, especially APCs should be further
evaluated as potent, low heptotoxic ChEs inhibitors for the
treatment of AD.
4. Experimental section
4.1. Chemistry
1H NMR spectra were recorded with a Bruker AV-400 spectrom-
eter at 400 MHz. MS spectra were recorded on a Shimadzu LCMS-
2010A instrument with an ESI mass selective detector. Elemental
9-Aminoanthracene 2 was treated with chloroacetyl chloride
according to general procedure to give the intermediate 2a as yel-
low solid. Yield 89%, 1H NMR (400 MHz, DMSO-d6) dH: 10.60 (brs,

C. Hong et al. / Bioorg. Med. Chem. 22 (2014) 3213–3219
3217
1H), 8.63 (s, 1H), 8.09–8.15 (m, 4H), 7.53–7.60 (m, 4H), 4.60 (s, 2H).
2H), 3.33 (t, J = 7.2 Hz, 2H), 3.13 (t, J = 7.6 Hz, 2H), 1.82–1.98
(m, 4H). ESI-MS m/z: 272.2 (M+Hꢁ2HCl)+. Anal. calcd for C16H23-
Cl2N3Oꢂ0.9 H2O: C, 53.31; H, 6.93; N, 11.66; found: C, 53.45; H,
6.98; N, 11.68.
ESI-MS m/z: 270.1 (M+H)+.
4.3. Procedures for the preparation of intermediate 3a
A mixture of 1-aminoanthraquinone 3 (1.8 g, 8 mmol), pyridine
(1 mL) and DMF (70 mL) were stirred at ice bath until completely dis-
solved, then chloroacetyl chloride (2.8 g, 24 mmol) was added drop-
wise, the suspension was stirred for 24 h at room temperature. After
thereaction finished(monitoredbyTLC),the reactionwaspouredinto
ice water and filtered, the filter cake was washed by ether and recrys-
tallized from EtOH to give pale yellow solid 3a. Yield 66%, 1H NMR
(400 MHz, DMSO-d6) dH: 12.72 (br s, 1H), 8.98 (d, J = 8.3 Hz, 1H),
8.25 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.01 (d, J = 7.6 Hz,
1H), 7.92–7.96 (m, 3 H), 4.59 (s, 2H). ESI-MS m/z: 300.7 (M+H)+.
4.10. 2-(4-(3-Aminopropylamino)butylamino)-N-(naphthalen-
1-yl)acetamide hydrochloride (6d)
White solid, yield 71.8%, 1H NMR (400 MHz, D2O) dH: 8.07–8.10
(m, 1H), 8.02 (t, J = 6.8 Hz, 2H), 7.62–7.72 (m, 4H), 4.38 (s, 2H), 3.33
(t, J = 6.8 Hz, 2H), 3.15–3.25 (m, 6H), 2.12–2.20 (m, 2H), 1.89–1.95
(m, 4H). ESI-MS m/z: 329.3 (M+Hꢁ3HCl)+. Anal. calcd for C19H31Cl3-
N4Oꢂ0.6ꢂH2O: C, 50.87; H, 7.23; N, 12.49; found: C, 50.99; H, 7.40;
N, 12.44.
4.11. 2-(3-(4-(3-Aminopropylamino)butylamino)propylamino)-
N-(naphthalen-1-yl)acetamide hydrochloride (6e)
4.4. Procedures for the preparation of material 4h, 4i and 4j
The Boc protected polyamines 4h, 4i and 4j were prepared in
White solid, yield 75.2%, 1H NMR (400 MHz, D2O) dH: 8.07–
8.10 (m, 1H), 8.02 (t, J = 6.8 Hz, 2H), 7.62–7.72 (m, 4H), 4.38 (s,
2H), 3.40 (t, J = 7.9 Hz, 2H), 3.28 (t, J = 7.9 Hz, 2H), 3.12–3.22
(m, 8 H), 2.26–2.34 (m, 2H), 2.09–2.14 (m, 2H), 1.82–1.86 (m,
4H). ESI-MS m/z: 386.4 (M+Hꢁ4HCl)+. Anal. calcd for C22H39Cl4-
N5Oꢂ0.2ꢂH2O: C, 49.39; H, 7.42; N, 13.09; found: C, 49.11; H,
7.60; N, 12.96.
our previous work.16
4.5. General procedures for the preparation of intermediates
5a–5o
A mixture of 1a or 2a (3 mmol), Et3N (6 mL), 4a–4j (4.5 mmol),
and 20 mL EtOH were stirred for 6 h under reflux (monitored by
TLC). After cooling to the room temperature, the solvent was
removed, the residue was diluted with CH2Cl2 (35 mL) and washed
with 10% NaCO3 (30 mL ꢀ 3). The organic layer was dried over Na2-
SO4, filtered, concentrated in vacuo, and purified by flash column
chromatography with chloroform/methanol elution.
4.12. N-(Anthracen-9-yl)-2-(diethylamino)acetamide
hydrochloride (6f)
Pale yellow solid, yield 66.8%, 1H NMR (400 MHz, D2O) dH:
8.60 (s, 1H), 8.10 (d, J = 7.8 Hz, 2H), 7.97 (d, J = 8.6 Hz, 2H),
7.52–7.61 (m, 4H), 4.60 (s, 2H), 3.40 (q, J = 7.4 Hz, 4H), 1.38 (t,
J = 7.3 Hz, 6H). ESI-MS m/z: 307.2 (M+HꢁHCl)+. Anal. calcd for
4.6. General procedures for the preparation of intermediates
6a–6o
C
20H23ClN2Oꢂ0.4 H2O: C, 68.62; H, 6.85; N, 8.00; found: C,
68.47; H, 6.92; N, 7.96.
The respective N-Boc protected intermediates 5a–5o were dis-
solved in EtOH (10 mL) and stirred at 0 °C for 10 min. Then 4 M
HCl was added dropwise at 0 °C. The reaction mixture was stirred
at room temperature overnight and a white precipitate was gener-
ated. The precipitate was concentrated and washed several times
with absolute ethanol and ether, and dried under vacuum to give
the pure target compounds 6a–6o.
4.13. N-(anthracen-9-yl)-2-(cyclopropylamino)acetamide
hydrochloride (6g)
Pale yellow solid, yield 67.2%, 1H NMR (400 MHz, D2O) dH:
8.67 (s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 8.07 (d, J = 8.6 Hz, 2H),
7.57–7.65 (m, 4H), 4.55 (s, 2H), 2.89–2.95 (m, 1H), 0.99 (d,
J = 5.6 Hz, 4H). ESI-MS m/z: 291.1 (M+HꢁHCl)+. Anal. calcd for
4.7. 2-(3-Hydroxypropylamino)-N-(naphthalen-1-yl)acetamide
hydrochloride (6a)
C
19H19ClN2Oꢂ0.1ꢂH2O: C, 69.44; H, 5.89; N, 8.52; found: C,
69.39; H, 5.90; N, 8.43.
White solid, yield 67.1%, 1H NMR (400 MHz, D2O) dH: 8.08–8.10
(m, 1H), 8.02 (t, J = 7.2 Hz, 2H), 7.63–7.73 (m, 4H), 4.38 (s, 2H), 3.83
(t, J = 11.9 Hz, 2H), 3.40 (t, J = 14.8 Hz, 2H), 2.06–2.12 (m, 2H). ESI-
MS m/z: 259.1 (M+HꢁHCl)+. Anal. calcd for C15H19ClN2O2: C, 61.12;
H, 6.50; N, 9.50; found: C, 61.11; H, 6.56; N, 9.42.
4.14. N-(Anthracen-9-yl)-2-(3-hydroxypropylamino)acetamide
hydrochloride (6h)
Pale yellow solid, yield 65.9%, 1H NMR (400 MHz, D2O) dH: 8.50
(s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.6 Hz, 2H), 7.46–7.55 (m,
4H), 4.44 (s, 2H), 3.69 (t, J = 6.0 Hz, 2H), 3.28 (t, J = 7.7 Hz, 2H),
1.92–1.99 (m, 2H). ESI-MS m/z: 309.1 (M+H-HCl)+. Anal. calcd for
4.8. 2-(2-(Dimethylamino)ethylamino)-N-(naphthalen-1-
yl)acetamide hydrochloride (6b)
C
19H21ClN2O2ꢂ0.2 H2O: C, 65.49; H, 6.19; N, 8.04; found: C, 65.43;
H, 6.02; N, 7.96.
White solid, yield 68.4%, 1H NMR (400 MHz, D2O) dH: 8.09–8.12
(m, 1H), 8.04 (t, J = 6.7 Hz, 2H), 7.64–7.72 (m, 4H), 4.48 (s, 2H),
3.71-3.79 (m, 4H), 3.09 (s, 6H). ESI-MS m/z: 272.1 (M+Hꢁ2.3HCl)+.
Anal. calcd for C16H23Cl2N3Oꢂ0.3 HClꢂ0.4 H2O: C, 53.02; H, 6.70; N,
11.59; found: C, 52.93; H, 6.99; N, 11.64.
4.15. N-(Anthracen-9-yl)-2-(2-
(dimethylamino)ethylamino)acetamide hydrochloride (6i)
Pale yellow solid, yield 64.8%, 1H NMR (400 MHz, D2O) dH: 8.59
(s, 1H), 8.09 (d, J = 8.2 Hz, 2H), 8.01 (d, J = 8.7 Hz, 2H), 7.51–7.61 (m,
4H), 4.59 (s, 2H), 3.70 (t, J = 7.9 Hz, 2H), 3.62 (t, J = 7.5 Hz, 2H), 2.96
(s, 6H). ESI-MS m/z: 322.2 (M+Hꢁ2.3HCl)+. Anal. calcd for C20H25-
Cl2N3Oꢂ0.3ꢂHCl: C, 59.27; H, 6.29; N, 10.37; found: C, 59.25; H,
6.47; N, 10.16.
4.9. 2-(4-Aminobutylamino)-N-(naphthalen-1-yl)acetamide
hydrochloride (6c)
White solid, yield 72.3%, 1H NMR (400 MHz, D2O) dH: 8.08–
8.10 (m, 1H), 8.02 (t, J = 7.8 Hz, 2H), 7.61–7.72 (m, 4H), 4.38 (s,

3218
C. Hong et al. / Bioorg. Med. Chem. 22 (2014) 3213–3219
4.16. 2-(3-Aminopropylamino)-N-(anthracen-9-yl)acetamide
hydrochloride (6j)
stirred at room temperature for 30 min, then intermediate 3a
(0.9 g, 3 mmol) was added and stirred at room temperature for
24 h (monitored by TLC). The solvent was removed in vacuum,
the residue was diluted with CH2Cl2 (35 mL) and then washed with
10% NaCO3 (30 mL ꢀ 3). The organic layer was dried over NaSO4,
filtered, concentrated in vacuo, and purified by flash column chro-
matography with chloroform/methanol elution to give 5p–5s.
Pale yellow solid, yield 69.2%, 1H NMR (400 MHz, D2O) dH: 8.59
(s, 1H), 8.08 (d, J = 30.2 Hz, 4H), 7.57–7.63 (m, 4H), 4.57 (s, 2H),
3.37 (t, J = 7.7 Hz, 2H), 3.15 (t, J = 7.7 Hz, 2H), 2.16–2.24 (m, 2H).
ESI-MS m/z: 308.2 (M+Hꢁ2HCl)+. Anal. calcd for C19H23Cl2N2Oꢂ0.6
H2O: C, 58.35; H, 6.24; N, 10.74; found: C, 58.42; H, 6.09; N, 10.59.
4.23. General procedures for the preparation of the target
compounds 6p–6s
4.17. 2-(4-Aminobutylamino)-N-(anthracen-9-yl)acetamide
hydrochloride (6k)
The respective N-Boc protected intermediates 5p–5s were dis-
solved in EtOH (10 mL) and stirred at 0 °C for 10 min. Then 4 M
HCl was added dropwise at 0 °C. The reaction mixture was stirred
at room temperature overnight. The solution typically gave a pale
yellow solid as a precipitate. The solid was concentrated and
washed several times with absolute ethanol and ether, and dried
under vacuum to give the pure target compounds 6a–6s.
Pale yellow solid, yield 74.2%, 1H NMR (400 MHz, D2O) dH: 8.61
(s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.7 Hz, 2H), 7.52–7.61 (m,
4H), 4.49 (s, 2H), 3.26 (t, J = 7.4 Hz, 2H), 3.01 (t, J = 7.7 Hz, 2H),
1.73–1.87 (m, 4H). ESI-MS m/z: 322.2 (M+Hꢁ2HCl)+. Anal. calcd
for C20H25Cl2N3Oꢂ0.4 H2O: C, 59.82; H, 6.48 ; N, 10.46; found: C,
59.90; H, 6.43; N, 10.40.
4.18. 2-(6-Aminohexylamino)-N-(anthracen-9-yl)acetamide
4.24. N-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-2-(3-
hydrochloride (6l)
hydroxypropylamino)acetamide hydrochloride (6p)
White solid, yield 71.4%, 1H NMR (400 MHz, D2O) dH: 8.69 (s,
1H), 8.20 (d, J = 7.6 Hz, 2H), 8.11 (d, J = 8.7 Hz, 2H), 7.63–7.72 (m,
4H), 4.58 (s, 2H), 3.33 (t, J = 7.7 Hz, 2H), 3.06 (t, J = 7.5 Hz, 2H),
1.84–1.92 (m, 2H), 1.72–1.79 (m, 2H), 1.51–1.54 (m, 4H). ESI-MS
m/z: 349.3 (M+Hꢁ2HCl)+. Anal. calcd for C22H31Cl2N3O2: C, 60.00;
H, 7.09; N, 9.54; found: C, 59.93; H, 7.06; N, 9.44.
Orange solid, yield 68.8%, 1H NMR (400 MHz, D2O) dH: 8.36 (d,
J = 9.4 Hz, 1H), 7.67–7.76 (m, 4H), 7.52–7.55 (m, 2H), 4.29 (s, 2H),
3.91 (t, J = 5.7 Hz, 2H), 3.48 (t, J = 7.4 Hz, 2H), 2.13–2.20 (m, 2H).
ESI-MS m/z: 339.2 (M+HꢁHCl)+. Anal. calcd for C19H19ClN2O4ꢂ0.3
H2O: C, 60.02; H, 5.20; N, 7.37; found: C, 59.93; H, 7.31; N, 7.31.
4.25. 2-(4-Aminobutylamino)-N-(9,10-dioxo-9,10-
4.19. 2-(3-(3-Aminopropylamino)propylamino)-N-(anthracen-
dihydroanthracen-1-yl)acetamide hydrochloride (6q)
9-yl)acetamide hydrochloride (6m)
Bright yellow solid, yield 76.2%, 1H NMR (400 MHz, D2O) dH:
8.46 (d, J = 6.8 Hz, 1H), 7.84–7.88 (m, 2H), 7.62–7.76 (m, 4H),
4.33 (s, 2H), 3.38 (t, J = 7.3 Hz, 2H), 3.18 (t, J = 7.7 Hz, 2H), 1.88–
2.04 (m, 4H). ESI-MS m/z: 352.2 (M+Hꢁ2HCl)+. Anal. calcd for C20-
H23Cl2N3O3ꢂ0.2 H2O: C, 56.13; H, 5.51; N, 9.82; found: C, 56.06; H,
5.18; N, 9.63.
Yellow solid, yield 69.6%, 1H NMR (400 MHz, D2O) dH: 8.72 (s,
1H), 8.23 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.7 Hz, 2H), 7.65–7.74 (m,
4H), 4.66 (s, 2H), 3.47 (t, J = 7.5 Hz, 2H), 3.25–3.35 (m, 4H), 3.18
(t, J = 7.8 Hz, 2H), 2.29–2.37 (m, 2H), 2.14–2.22 (m, 2H). ESI-MS
m/z: 365.3 (M+Hꢁ3HCl)+. Anal. calcd for C22H33Cl3N4O2ꢂ0.1 H2O:
C, 53.52; H, 6.78; N, 11.25; found: C, 53.78; H, 6.81; N, 10.95.
4.26. 2-(4-(3-Aminopropylamino)butylamino)-N-(9,10-dioxo-
4.20. 2-(4-(3-Aminopropylamino)butylamino)-N-(anthracen-9-
9,10-dihydroanthracen-1-yl)acetamide hydrochloride (6r)
yl)acetamide hydrochloride (6n)
Bright yellow solid, yield 80.8%, 1H NMR (400 MHz, D2O) dH: 8.48
(d, J = 8.3 Hz, 1H), 7.86–7.90 (m, 2H), 7.73–7.78 (m, 4H),7.66 (t,
J = 8.1 Hz, 1H), 4.34 (s, 2H), 3.38 (t, J = 7.0 Hz, 2H), 3.24-3.28 (m,
4H), 3.19 (t, J = 7.8 Hz, 2H), 2.14–2.22 (m, 2H), 1.91–2.04 (m, 4H).
ESI-MS m/z: 309.3 (M+Hꢁ3HCl)+. Anal. calcd for C23H31Cl3N4O3ꢂ0.1
H2O: C, 53.16; H, 6.05; N, 10.78; found: C, 53.07; H, 5.74; N, 10.68.
Light white soldi, yield 64.6%, 1H NMR (400 MHz, D2O) dH: 8.42
(s, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.41–7.52
(m, 4H), 4.44 (s, 2H), 3.20 (t, J = 7.5 Hz, 2H), 2.96–3.06 (m, 6H),
1.94–2.02 (m, 2H), 1.71–1.81 (m, 4H). ESI-MS m/z: 379.3
(M+Hꢁ3HCl)+. Anal. calcd for C23H33Cl3N4Oꢂ0.7 H2O: C, 55.19; H,
6.93; N, 11.19; found: C, 55.36; H, 7.06; N, 10.90.
4.27. 2-(3-(4-(3-Aminopropylamino)butylamino)propylamino)-
N-(9,10-dioxo-9,10-dihydroanthracen-1-yl)acetamide
hydrochloride (6s)
4.21. 2-(3-(4-(3-Aminopropylamino)butylamino)propylamino)-
N-(anthracen-9-yl)acetamide hydrochloride (6o)
Pale yellow solid, yield 62.9%, 1H NMR (400 MHz, D2O) dH: 8.56 (s,
1H), 8.06 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 8.8 Hz, 2H), 7.49–7.58 (m,
4H), 4.52 (s, 2H), 3.31 (t, J = 7.9 Hz, 2H), 3.15 (t, J = 8.0 Hz, 2H),
3.00-3.09 (m, 8 H), 2.13–2.21 (m, 2H), 1.97–2.05 (m, 2H), 1.69–
1.73 (m, 4H). ESI-MS m/z: 436.4 (M+Hꢁ4HCl)+. Anal. calcd for C26-
H43Cl4N5O2ꢂ0.4 H2O: C, 51.47; H, 7.28; N, 11.54; found: C, 51.48; H,
7.50; N, 11.30.
Bright yellow solid, yield 64.7%, 1H NMR (400 MHz, D2O) dH: 8.52
(d, J = 8.4 Hz, 1H), 7.95-8.00 (m, 2H), 7.69–7.83 (m, 3 H), 4.36 (s, 2H),
3.40 (t, J = 7.9 Hz, 2H), 3.28 (t, J = 7.9 Hz, 2H), 3.12–3.22 (m, 8 H),
2.26–2.34 (m, 2H), 2.09–2.14 (m, 2H), 1.82–1.86 (m, 4H). ESI-MS
m/z: 465.5 (M+Hꢁ4HCl)+. Anal. calcd for C26H39Cl4N5O3ꢂ0.1 H2O: C,
50.92; H, 6.44; N, 11.42; found: C, 51.04; H, 6.15; N, 11.11.
5. Biological activity
4.22. General procedures for the preparation of intermediates
5p–5s
5.1. In vitro inhibition studies on AChE and BChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyr-
ylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum), 5,50-dithi-
A mixture of polyamine (4a, 4f, 4i or 4j) (3 mmol), K2CO3
(0.83 g,6.0 mmol), KI (catalytic amount) in CH3CN (40 mL) was

C. Hong et al. / Bioorg. Med. Chem. 22 (2014) 3213–3219
3219
obis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocho-
line chloride (ATC), butylthiocholine chloride (BTC), and tarcine
were purchased from Sigma Aldrich. Tacrine and synthesized
derivatives were dissolved in H2O and then diluted in 0.1 M KH2-
PO4/K2HPO4 buffer (pH 8.0) to provide a final concentration range.
All the assays were under 0.1 M KH2PO4/K2HPO4 buffer, pH 8.0,
using a Shimadzu UV-2450 Spectrophotometer. Enzyme solutions
were prepared to give 2.0 units/mL in 2 mL aliquots. The assay
cells by the conversion of MTT to a purple formazan precipitate
as previously described.24 Cells were seeded into 96-well plates
at 5 ꢀ 103 cells/well. After 12 h, 0.1, 1.0, 10, 30, 100
lM of com-
pounds were subsequently added and incubated for 48 h. The cell
viability was determined by using MTT colorimetry, measuring the
absorption at 590 nm. Controls were taken as having 100% viabil-
ity. Each concentration was tested in triplicate, and IC50 values
were calculated graphically from log concentration–inhibition
curve (Origin 7.5 software).
medium contained phosphate buffer, pH 8.0 (1 mL), 50
lL of
0.01 M DTNB, 10 L of enzyme, and 50 L of 0.01 M substrate
l
l
(ATC). The substrate was added to the assay medium containing
enzyme, buffer, and DTNB with inhibitor after 15 min of incubation
time. The activity was determined by measuring the increase in
absorbance at 412 nm at 1 min intervals at 37 °C. Calculations
were performed according to the method of the equation in Ellman
et al. In vitro BChE assay use the similar method described above.
Each concentration was assayed in triplicate.
Acknowledgments
We thank the Natural Science Foundation of China (Nos.
21172053, 21302041), the Postdoctoral Science Foundation of
China (No. 2012M521391), and the Postdoctoral Science Founda-
tion of Henan Province (No. 2011015) for financial support of this
study.
5.2. Kinetic characterization of AChE inhibition
Supplementary data
Six different concentrations of substrate were mixed in the 1 mL
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.045.
0.1 M KH2PO4/K2HPO4 buffer (pH 8.0), containing 50
lL of DTNB,
10 L AChE, and 50 L substrate. Test compound was added into
l
l
the assay solution and pre-incubated with the enzyme at 37 °C
for 15 min, followed by the addition of substrate. Kinetic character-
ization of the hydrolysis of ATC catalyzed by AChE was done spec-
trometrically at 412 nm. A parallel control with no inhibitor in the
mixture, allowed adjusting activities to be measured at various
times. BChE assay use the similar method described above.
References and notes
1. Kung, H. F.; Lee, C. W.; Zhuang, Z. P.; Kung, M. P.; Hou, C.; Plössl, K. J. Am. Chem.
Soc. 2001, 123, 12740.
2. Förstl, H.; Kurz, A. Eur. Arch. Psychiatry Clin. Neurosci. 1999, 249, 288.
3. Cummings, J. L.; Doody, R.; Clark, C. Neurology 2007, 69, 1622.
4. Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini,
M.; Melchiorre, C. J. Med. Chem. 2008, 51, 347.
6. Molecular modeling
5. Meng, F. C.; Mao, F.; Shan, W. J.; Qin, F.; Huang, L.; Li, X. S. Bioorg. Med. Chem.
Lett. 2012, 22, 4462.
6. Greig, N. H.; Utsuki, T.; Ingram, D. K.; Wang, Y.; Pepeu, G.; Scali, C.; Yu, Q. S.;
Mamczarz, J.; Holloway, H. W.; Giordano, T.; Chen, D.; Furukawa, K.;
Sambamurti, K.; Brossi, A.; Lahiri, D. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
17213.
7. Woo, Y. J.; Lee, B. H.; Yeun, G. H.; Kim, H. J.; Won, M. H.; Kim, S. H.; Lee, B. L.;
Park, J. H. Bull. Korean Chem. Soc. 2011, 32, 2593.
8. Tumiatti, V.; Milelli, A.; Minarini, A.; Rosini, M.; Bolognesi, M. L.; Micco, M.;
Andrisano, V.; Bartolini, M.; Mancini, F.; Recanatini, M.; Cavalli, A.; Melchiorre,
C. J. Med. Chem. 2008, 51, 7308.
9. Bolognesi, M. L.; Banzi, R.; Bartolini, M.; Cavalli, A.; Tarozzi, A.; Andrisano, V.;
Minarini, A.; Rosini, M.; Tumiatti, V.; Bergamini, C.; Fato, R.; Lenaz, G.; Hrelia,
P.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. J. Med. Chem. 2007, 50, 4882.
10. Simoni, E.; Bergamini, C.; Fato, R.; Tarozzi, A.; Bains, S.; Motterlini, R.; Cavalli,
A.; Bolognesi, M. L.; Minarini, A.; Hrelia, P.; Lenaz, G.; Rosini, M.; Melchiorre, C.
J. Med. Chem. 2010, 53, 7264.
11. Cen, J.; Liu, L.; He, L.; Liu, M.; Wang, C. J.; Ji, B. S. Int. J. Dev. Neurosci. 2012, 30,
584.
12. Elsinghorst, P. W.; Hartig, W.; Gundisch, D.; Mohr, K.; Trankle, C.; Gutschow, M.
Curr. Top. Med. Chem. 2011, 11, 2731.
13. Tian, Z. Y.; Xie, S. Q.; Mei, Z. H.; Zhao, J.; Gao, W. Y.; Wang, C. J. Org. Biomol.
Chem. 2009, 7, 4651.
14. Xie, S. Q.; Wang, J. H.; Ma, H. X.; Cheng, P. F.; Zhao, J.; Wang, C. J. Toxicology
2009, 263, 127.
15. Wang, J. H.; Chen, Z. Y.; Xie, S. Q.; Zhao, J.; Wang, C. J. Bioorg. Med. Chem. Lett.
2010, 20, 6421.
16. Wang, Y. X.; Zhang, X. B.; Zhao, J.; Xie, S. Q.; Wang, C. J. J. Med. Chem. 2012, 55,
3502.
17. Luo, W.; Huang, K.; Zhang, Z.; Hong, C.; Wang, Y. Q. Acta Pharmacol. Sin. 2013,
48, 269.
18. Sheu, S. Y.; Chiang, H. C. Anticancer Res. 1997, 17, 3293.
19. Shi, D. H.; Huang, W.; Li, C.; Wang, L. T.; Wang, S. F. Bioorg. Med. Chem. 2013, 21,
1064.
20. Ellman, G. L.; Courtney, K. D., Jr.; Andres, B.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
21. Tang, H.; Ning, F. X.; Wei, Y. B.; Huang, S. L.; Huang, Z. S.; Chan, A. S.; Gu, L. Q.
Bioorg. Med. Chem. Lett. 2007, 17, 3765.
The crystal structure of the torpedo AChE complexed with
tacrine (code ID: 1ACJ) was obtained in the Protein Data Bank after
eliminating the inhibitor and water molecules. The 3D Structure of
6l was built and performed geometry optimization by molecular
mechanics. Further preparation of substrates included addition of
Gasteiger charges, removal of hydrogen atoms and addition of their
atomic charges to skeleton atoms, and finally, assignment of
proper atomic types. Autotors was then used to define the rotat-
able bonds in the ligand.
Docking studies were carried out using the AUTODOCK 4.0 pro-
gram. Using ADT, Polar hydrogen atoms were added to amino acid
residues and Gasteiger charges were assigned to all atoms of the
enzyme. The resulting enzyme structure was used as an input for
the AUTOGRID program. AUTOGRID performed a precalculated
atomic affinity grid maps for each atom type in the ligand plus
an electrostatics map and a separate desolvation map present in
the substrate molecule. All maps were calculated with 0.375 Å
spacing between grid points. The centre of the grid box was placed
at the bottom of the active site gorge (AChE [2.781 64.383 67.971]).
The dimensions of the active site box were set at 50 ꢀ 46 ꢀ 46 Å.
Flexible ligand docking was performed for the compounds.
Docking calculations were carried out using the Lamarckian
genetic algorithm (LGA) and all parameters were the same for each
docking. To ensure the reliability of the results, the docking proce-
dures were repeated 10 independent times for the compound and
the obtained orientations were analyzed.
22. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
7. MTT assay of HepG2 cell viability
23. DeLano, W.L. The PyMOL Molecular Graphics System. DeLano Scientific. San
Carlos, CA, 2002
24. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
Cells were cultured at 37 °C under a 5% CO2 atmosphere. The
antiproliferative ability of compounds was evaluated in HepG2