Synthesis and evaluation of paeonol derivatives as potential multifunctional agents for the treatment of alzheimer's disease

Article abstract of DOI:10.3390/molecules20011304

(A.H.); Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder characterized by memory loss, language impairment, personality changes and intellectual decline. Taking into account the key pathological features of AD, such as low levels of acetylcholine, beta-amyloid (Aβ) aggregation, oxidative stress and dyshomeostasis of biometals, a new series of paeonol derivatives 5a-5d merging three different functions, i.e., antioxidant, anti-acetylcholinesterase (AChE) activity, metal chelating agents for AD treatment have been synthesized and characterized. Biological assays revealed that compared with paeonol (309.7 μM), 5a-5d had a lower DPPH IC50 value (142.8-191.6 μM). 5a-5d could significantly inhibit hydrogen peroxide-induced neuronal PC12 cell death assessed by MTT assay in the concentration range of 5-40 μM. AChE activity was effectively inhibited by 5a-5d, with IC50 values in the range of 0.61-7.04 μM. 5a-5d also exhibited good metal-chelating ability. All the above results suggested that paeonol derivatives may be promising multifunctional agents for AD treatment.

Full text of DOI:10.3390/molecules20011304

Molecules 2015, 20, 1304-1318; doi:10.3390/molecules20011304
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of Paeonol Derivatives as
Potential Multifunctional Agents for the Treatment of
Alzheimer’s Disease
An Zhou 1,†, Hongfei Wu 2,†, Jian Pan 1,*, Xuncui Wang 2, Jiaming Li 2, Zeyu Wu 1 and
Ailing Hui 1,*
1
Institute of Natural Medicine, Hefei University of Technology, Hefei 230009, Anhui, China;
E-Mails: anzhou0901@163.com (A.Z.); wu38331451@163.com (Z.W.)
2
Anhui Province Key Laboratory of R&D of Chinese Medicine, Anhui University of Chinese
Medicine, Hefei 230038, Anhui, China; E-Mails: wuhongfei2009@126.com (H.W.);
wangxuncui@163.com (X.W.); lijiaming2004@sina.com (J.L.)
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: panjian78@sina.cn (J.P.);
haling@mail.ustc.edu.cn (A.H.); Tel./Fax: +86-551-6290-1862 (J.P. & A.H.).
Academic Editor: Derek J. McPhee
Received: 10 November 2014 / Accepted: 7 January 2015 / Published: 14 January 2015
Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative brain disorder
characterized by memory loss, language impairment, personality changes and intellectual
decline. Taking into account the key pathological features of AD, such as low levels of
acetylcholine, beta-amyloid (Aβ) aggregation, oxidative stress and dyshomeostasis of biometals,
a new series of paeonol derivatives 5a–5d merging three different functions, i.e., antioxidant,
anti-acetylcholinesterase (AChE) activity, metal chelating agents for AD treatment have
been synthesized and characterized. Biological assays revealed that compared with paeonol
(309.7 μM), 5a–5d had a lower DPPH IC50 value (142.8–191.6 μM). 5a–5d could significantly
inhibit hydrogen peroxide-induced neuronal PC12 cell death assessed by MTT assay in the
concentration range of 5–40 μM. AChE activity was effectively inhibited by 5a–5d, with
IC50 values in the range of 0.61–7.04 μM. 5a–5d also exhibited good metal-chelating ability.
All the above results suggested that paeonol derivatives may be promising multifunctional
agents for AD treatment.

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Keywords: Alzheimer’s disease; multifunctional agents; paeonol; acetylcholinesterase
inhibitors; metal-chelating; biometal chelators
1. Introduction
Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder characterized by memory loss,
language impairment, personality changes and gradual loss of intellectual ability [1]. Currently, the burden
of AD has affected over 35 million people worldwide, increasing with the aging population. If no efficient
treatment is discovered, the anticipated number of affected population will increase three-fold by
2025 [2]. Although over 100 years have already elapsed since the discovery of AD, effective treatment
methods are still lacking [3]. To date, therapeutic options for symptomatic treatment of AD, which
include three acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine, and galantamine) [4] and
an N-methyl-D-aspartate receptor (NMDAR) antagonist (memantine) [5], could only result in modest
and temporary benefit for memory and cognitive function instead of delaying or stopping the progression
of neurodegeneration. Although the molecular etiology of AD is not completely understood, oxidative
stress, metal ion deregulation, inflammation, beta-amyloid (Aβ) peptide deposits, τ-protein aggregation,
and low levels of acetylcholine (ACh) have been considered to play significant roles in the disease [6,7].
Thus, the development of multitarget agents for simultaneously modulating multiple biological targets
involved in AD has clearly emerged as a successful strategy [8,9]. Among the multiple factors inducing
AD, a deficiency in ACh as neurotransmitter may play an important role in the regulation of learning
and memory processes [10]. ACh is primarily hydrolyzed by AChE, and terminated cholinergic
transmission. Furthermore, a broad range of evidences have shown that AChE could accelerate the
aggregation of Aβ during the early stages of AD [11,12]. As oxidative stress being also an important
player in the pathogenesis and progression of AD, a number of studies has demonstrated that oxidative
stress induced by Aβ deposits is associated with the neurodegeneration of the brain [13], and several
antioxidants have been tested in clinical trials [14]. Recent studies have indicated that biometals (Cu2+,
Fe2+ and Zn2+) also play a very important role in the pathogenesis of AD. High levels of metal ions, like
Cu2+ and Fe2+, have been linked to the production of reactive oxygen species (ROS), which promotes
oxidative stress thus contributing to AD pathogenesis [15]. Thus, the modulation of these biometals in
the brain is also a viable alternative to treat AD patients. In view of multi-pathogenesis of AD, more
efforts are directed to develop multi-target-directed ligands (MTDLs), which has been proven to be a
successful strategy for the design of new drugs for AD [16,17]. Paeonol (2-hydroxy-4-methoxy-
acetophenone), a natural phenolic component isolated from the root bark of Moutan Cortex (Paeonia
suffruticosa), possesses well known antioxidant and anti-inflammatory activities. Recent evidence has
suggested that paeonol could attenuate neurotoxicity and ameliorate cognitive impairment [18]. It is also
proved that paeonol ameliorates neuronal damage via the inhibition of microglia-mediated inflammation
and oxidative stress-induced neuronal damage. In a rat model of AD, treatment with paeonol can protect
against many alterations resulting from administration of Aβ1–42 [19,20]. Our previous research also
showed that paeonol could protect against glutamate-induced PC12 cell death via a ROS mitochondrial
pathway [21]. Considering the cholinergic hypothesis, the importance of biometal ions and oxidative

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stress in the pathological process of AD [22], we are interested in the development of novel paeonol
derivatives as potential multifunctional agents for the treatment of AD. In the present study, a series of
paeonol derivatives were synthesized and then their antioxidant activities, acetylcholinesterase inhibitory,
metal chelating effects and protective effects on H2O2-induced PC12 cell death were investigated.
2. Results and Discussion
2.1. Chemical Synthesis
Donepezil is the first-line drug to prevent AD. Based on the structure of paeonol and donepezil,
a series of new donepezil-like analogs were designed as depicted in Scheme 1. The synthetic pathway
of new paeonol derivatives was shown in Scheme 2. Firstly, various monosubstituted N-alkyl piperazines
(2a–2d) and α-brominated paeonol (4) were prepared according to the slightly modified literatures [23,24],
respectively. Once the intermediate was available, N-alkyl piperazines (2a–2d) reacted with 4 in the
presence of K2CO3/KI to yield the desired 5a–5d.
O
H3CO
N
Donepezil
H3CO
OH
O
N
Designed compounds
R
N
H3CO
OH
O
Paeonol
H3CO
Scheme 1. Design of new donepezil-like analogs based on the structure of paeonol and donepezil.
i
Br
N
+
HN
NH
R
R
NH
2a-2d
1a-1d
OH
O
OH
O
a: R = H
Br
b: R = 4-CH3
c: R = 4-NO2
d: R = 4-Cl
ii
+ CuBr2
H3CO
H3CO
4
3
OH
O
OH
O
N
R
Br
N
N
+
R
iii
NH
H3CO
H3CO
4
5a-5d
2a-2d
(ii) CHCl /EtOAc, Reflux, 6h, 62%; (iii) K2CO3/KI, CHCl3, 6h, RT
Reagents and condition: (i) CHCl3, 0°C,6h;
3
Scheme 2. Synthetic route of the title compounds.

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2.2. Biological Evaluation
2.2.1. Free Radical Scavenging Activity in Vitro (DPPH Assay)
Paeonol is well known for its antioxidant property [25]. Based on their interaction with DPPH
(2,2-diphenyl-1-picrylhydrazyl) free radical, antioxidant activities of the new paeonol derivatives were
screened. Concentrations caused 50% loss of the initial DPPH free radical scavenging (IC50) for paeonol
derivatives 5a–5d, which are listed in Table 1. Paeonol is used as the positive control. It could be
observed that all the paeonol derivatives showed greater DPPH radical scavenging activity compared
with paeonol. Their IC50 values were in the range of 142.79–191.64 μM. In the same condition, paeonol
has a value of 309.67 μM. The above results indicated that paeonol derivatives 5a–5d have good radical
scavenging and antioxidant properties. Among the four derivatives, the IC50 values of 5a and 5b are
slightly lower than those of 5c and 5d.
Table 1. Antioxidant and anticholinesterase activities of the Paeonol derivatives 5a–5b.
Compounds Molecular Weight DPPH Scavenging (a IC50, μM) AChE Inhibition (b IC50, μM)
5a
5b
340.1
354.2
385.4
374.1
166.2
379.4
142.79 ± 8.83
157.85 ± 6.88
191.64 ± 8.06
187.32 ± 13.73
309.67 ± 5.41
nt
1.59 ± 0.011
0.61 ± 0.009
7.04 ± 0.032
2.63 ± 0.153
nt
5c
5d
Paeonol
Donepezil
0.053 ± 0.003
a
Notes: : Concentrations caused 50% loss of the initial DPPH free radical scavenging; data are expressed as
the mean ± SD, n = 3; b: AChE from electric eel, n = 3; nt: Not tested.
2.2.2. Inhibitory Effect on PC12 Cell Death Induced by Oxidative Stress
The MTT assay was used to identify the degree of cell death after treatment with H2O2 at various
concentrations. From Figure 1, it could be seen that the PC12 cell viability decreased in a concentration
dependent manner. Compared with control group, the cell viability decreased to 47.1% after being
exposed to 150 μM H2O2 for 5 h and then 150 μM H2O2 was chosen in the following experiments.
The cell viability under the treatment of different concentrations of paeonol and its derivatives are listed
in Figure 2. Paeonol treatment did not cause any apparent effects on PC12 cells when its concentration
was 5 μM. However, 5a–5d showed stronger protection effects at the same concentrations, which is
consistent with the results of DPPH assay. Even if the concentration of paeonol was increased to 40 μM,
its protective effect was only 58.9%. Meanwhile, 5a–5d gave 80.7%, 84.2%, 61.8%, and 64.1% reductions,
respectively. The above results demonstrated that compounds 5a and 5b showed more potent activity at
higher concentrations.

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Figure 1. The MTT assay was used to detect cytotoxicity for various concentrations of H2O2.
Data are shown as mean ± SD (n = 10). (*) compared with control group (p < 0.05
one-way ANOVA).
Figure 2. Cytoprotective effects of paeonol and target compounds 5a–5d in PC12 cells
under H2O2-induced oxidative stress. PC12 cells exposed to 150 µM H2O2 for 5 h after being
pretreated with test compounds for 3 h. Cell viability was determined using the MTT method.
The viability of control cells untreated with test compounds and H2O2 was defined as 100%.
Data are shown as mean ± SD (n = 10). (*) compared with H2O2-treated group (p < 0.05);
(#) compared with control group (p < 0.05 two-way ANOVA).
In comparison to the untreated group (Figure 3A), PC12 cells exhibited a marked decrease in cell
number and morphological alterations such as cell shrinkage and membrane blebbing after being treated
with 150 μM H2O2 for 5 h (Figure 3B). Compared with the H2O2 treated group (Figure 3B), PC12 cells
pretreated with 40 µM paeonol (Figure 3C) and 40 µM 5b (Figure 3D) maintained normal cell
morphology, because of the mitigation of such morphological features. Meanwhile, the results of cell

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viability also clearly indicated that 5a–5d could attenuate the cytotoxic effects on PC12 cells induced
by H2O2 (Figure 2).
Figure 3. Identification of morphological cell changes with phase-contrast microscopy.
(A) PC12 control cells; (B) PC12 cells exposed to 150 μM H2O2 for 5 h. There is a significant
decrease in the cell number; (C,D) PC12 cells preincubated with (C) paeonol (40 µM) or
(D) 5b (40 μM) for 3 h and then exposed to 150 μM H2O2 for 5 h. All the photos were taken
5 h after exposure to H2O2 (visualized under a light microscope at ×100 magnification).
2.2.3. Anticholinesterase Activity
All the compounds 5a–5d were evaluated against AChE (from electric eel) using the method of
Ellman et al. [26], with donepezil as the reference compound. As the IC50 values shown in Table 1, all
the compounds demonstrated moderate inhibition activities with IC50 values in the range of micromolar
to sub-micromolar. Among the tested derivatives, 5b with para methyl substituent on phenyl ring was
the most potent one in this series (IC50 = 0.61 μM). The other three compounds presented poor inhibitory
activity, which had no substituent (5a) or electron-withdrawing groups (5c and 5d) on phenyl ring.
2.2.4. Molecular Docking
To explore a possible interacting mode of the compound 5b with TcAChE (PDB code: 1EVE),
a molecular modeling study was performed with the MOE software program. As shown in Figure 4A,B,
compound 5b moiety occupied the peripheral anionic site (PAS) of enzyme and possessed potential
hydrophobic interaction with residues Tyr334, Phe331, Phe331, Phe290 and Tyr279. Moreover, the
linker was long enough for the paeonol substituted group arriving in the catalytic active site (CAS) of
AChE, it adopted an appropriate orientation for its binding to CAS via a π-π interaction with Trp84 and
potentially induced a hydrogen bond interaction with residues His440 and Gly118. The simultaneous
binding of 5b with the CAS and PAS of TcAChE provided an explanation for its demonstrated moderate
inhibitory activity for AChE and thus revealed a mixed-type inhibition for this compound.

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Figure 4. Representations of the molecular model of the complex formed between
compound 5b and TcAChE (PDB code: 1EVE). (A) 3D representation of the ligand-enzyme
binding interactions (hydrogen bonds as green dashed lines); (B) 2D schematic
representation of the hydrogen bonding and hydrophobic interactions.
2.2.5. Metal-Chelating Study
The chelating ability of the most potent compound 5b for the biologically relevant metal ions such as
Cu2+, Fe2+ and Zn2+ in methanol was studied by UV-vis spectrophotometer [27,28]. Figure 5A showed
that UV absorption of compound 5b in methanol changed with addition of Cu2+ or Fe2+, while little
change occurring after titrating with Zn2+. The electronic spectra of 5b exhibited a blue shift (the peak
at 275 nm shifted to 271 nm) after addition of CuCl2 and the absorbance dropped dramatically, indicating

Molecules 2015, 20
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that 5b could interact with Cu2+ effectively. The absorption intensity change could also be observed
after adding FeSO4. The stoichiometry of complex 5b-Cu (II) was determined using the molar ratio
method [29,30], by titrating solutions of compound 5b with ascending amounts of CuCl2. The UV
spectra were used to obtain the absorbance of 5b complex and different concentrations of CuCl2
at 375 nm. As shown in Figure 5B, absorbance firstly increased and then reached the saturation point.
The point for the two straight lines intersected at the mole fraction of 0.98, indicating a 1:1 stoichiometry
for the 5b-Cu(II) complex.
Figure 5. (A) The UV spectrum of compound 5b (40 μM) alone or in the presence of FeSO4,
CuCl2 or ZnCl2 (40 μM for all the metals in methanol); (B) Determination of the stoichiometry
of complex 5b-Cu (II) by using molar ratio method through titrating methanol solution of
compound 5b with ascending amounts of CuCl2. The final concentration of test compound 5b
was 40 μM, and the final concentration of Cu2+ ranged from 2–80 μM.
3. Experimental Section
3.1. Chemistry
Paeonol (purity > 99%) was purchased from Nanjing Ze-Lang Medical Technology Co., Ltd
(Nanjing, China). In the synthesis experiments, all the reagents and solvents were reagent grade and used

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without further purification unless otherwise indicated. All the reactions were monitored using TLC.
Column chromatography was performed using silica gel (200–300 mesh). All yields of the products refer
to isolated yield. IR spectra were obtained on Thermo Scientific Nicolet 8700 system. NMR spectra
were obtained on Agilent VNMRS (600 MHz) with TMS as internal standard, and the chemical shifts
were recorded in ppm along with coupling constants (J values) in Hz. Multiplicities were designated as
singlet (s), broad singlet (br), doublet (d), triplet (t) and multiplet (m). Mass spectra were recorded by
electrospray ionization (ESI) on a Thermo LCQ Advantage MAX spectrometry with direct insertion probe.
Elemental analysis (EA) was obtained by a PE 2400 (Perkin Elmer, Waltham, MA, USA) analyzer.
3.2. Synthesis
General procedure for the synthesis of 2a–2d: Anhydrous piperazine (18.0 g, 0.21 mol) was dissolved
in chloroform (60 mL). Benzyl bromide 1a–1d (0.05 mol) in chloroform (30 mL) was added dropwise
to the solution at 0 °C. The reaction mixture was stirred for about 6 h at room temperature. Then, the
mixture was washed with 5% K2CO3 solution (80 mL × 2) and water (80 mL × 4). The organic layer
was dried over anhydrous Na2SO4. The filtrate was evaporated in vacuo and the residue was
recrystallized from n-hexane to give the desired products 2a–2d.
1-Benzylpiperazine (2a): Yield 77%, 1H-NMR (CDCl3, 600 MHz): δ 7.28–7.31 (m, 4H), 7.22–7.24 (m,
1H), 3.47 (s, 2H), 2.85–2.89 (m, 4H), 2.38–2.43 (m, 4H), 1.82 (br, 1H) ppm.
1
1-(4-Methylbenzyl)piperazine (2b): Yield 77%, H-NMR (CDCl3, 600 MHz): δ 7.16–7.22 (m, 2H),
7.06–7.11 (m, 2H), 3.45 (s, 2H), 2.82–2.87 (m, 4H), 2.37–2.42 (m, 4H), 2.31 (s, 3H), 1.74 (br, 1H) ppm.
1-(4-Nitrobenzyl) piperazine (2c): Yield 22%,1H-NMR (CDCl3, 600 MHz): δ 8.21 (d, J = 8.9 Hz, 2H),
7.60 (d, J = 9.0 Hz, 2H), 3.58 (s, 2H), 3.02–3.15 (m, 4H), 2.58–2.74 (m, 4H) ppm.
1-(4-Chlorobenzyl) piperazine (2d): Yield 53%, 1H-NMR (CDCl3, 600 MHz): δ 7.25–7.29 (m, 4H), 3.45
(s, 2H), 2.89–2.94 (m, 4H), 2.39–2.45 (m, 4H), 1.73 (br, 1H) ppm.
Synthesis of 2-bromo-1-(2-hydroxy-4-methoxy-phenyl)-ethanone 4: Powdered cupric bromide (13.4 g,
60 mmol) was added into a solution of paeonol (5.0 g, 30 mmol) in chloroform (100 mL) and ethyl
acetate (100 mL). The mixture was refluxed at 90 °C for 6 h until the reation was complete (monitored
by TLC). After cooling, the mixture was filtered. The filtrate was washed with H2O (150 mL × 4),
dried over anhydrous Na2SO4. The filtrate was evaporated in vacuo and the residue was purified by
chromatography on silica gel column with ethyl acetate/petroleum ether as the elution. The resulting
bromidated panonol 4 (4.5 g) was obtained as white powder. Yield 62%, 1H-NMR (CDCl3, 600 MHz):
δ 6.75–8.15 (s, 3H), 3.95 (s, 3H), 2.59 (s, 2H) ppm.
General procedure for the synthesis of 5a–5d: 2-Hydroxy-4-methoxy-1-(2-bromo)-acetophenon 4 (2.1 g,
5 mmol) and N-substituted benzyl piperazine 2a–2d (10 mmol) were dissolved in chloroform (60 mL).
Anhydrous K2CO3 (3.46 g, 25 mmol) and catalytic KI (0.17 g, 0.5 mmol) were added to the solution.
The reaction mixture was stirred for about 6 h at room temperature. Then, the solution was washed with
H2O (60 mL × 3). The organic layer was dried, filtered and evaporated. The resulting crude product was

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purified by using flash column chromatography on silica gel with ethyl acetate/petroleum ether as elution
solvent to give yellow oil 5a–5d.
2-(4-Benzylpiperazin-1-yl)-1-(2-hydroxy-4-methoxyphenyl) ethanone (5a): Yield 78%, yellow oily
product. IR (film): ν 3026.0, 2938.1, 2813.0, 1715.2, 1616.1, 1507.7, 1441.8, 838.9, 747.0 cm−1.
1H-NMR (CDCl3, 600 MHz): δ 9.96 (s, 1H), 7.79 (d, J = 6.0 Hz, 1H), 7.28 (d, J = 6.0 Hz, 4H), 7.22 (d,
J = 6.0 Hz, 1H), 6.40 (d, J = 6.0 Hz, 1H), 6.38 (t, J = 6.0 Hz, 2H), 3.77 (s, 3H), 3.62 (s, 2H), 3.49 (s,
2H), 2.62(m, 4H), 2.52 (m, 4H) ppm. 13C-NMR (CDCl3, 150 MHz): δ 168.71, 140.35, 134.49, 131.83,
130.90, 129.81, 110.32, 104.00, 65.48, 58.46, 55.73, 55.15 ppm. ESI-MS: m/z 341.2 [M+H]+. Anal.
Calcd. for C20H24N2O3: C, 70.56; H, 7.11; N 8.23; O 14.10. Found: C, 70.32; H, 7.18; N, 8.36.
1-(2-Hydroxy-4-methoxyphenyl)-2-(4-(4-methylbenzyl) piperazin-1-yl) ethanone (5b): Yield 57%, pale
yellow solid. IR (KBr): ν 2939.0, 2812.8, 1701.3, 1611.9, 1506.7, 1441.1, 829.9 cm−1. 1H-NMR (CDCl3,
600 MHz): δ 9.96 (s, 1H), 7.84 (d, J = 12.0 Hz, 1H), 7.20 (d, J = 12.0 Hz, 2H), 7.13 (d, J = 12.0 Hz,
2H), 6.44 (d, J = 6.0 Hz,1H), 6.41 (t, J = 6.0 Hz, 1H), 3.83 (s, 3H), 3.66 (s, 2H), 3.49 (s, 2H), 2.65 (m,
4H), 2.51 (m, 4H), 2.33 (s, 3H) ppm. 13C-NMR (CDCl3, 150 MHz): δ 169.79, 164.30, 161.29, 132.00,
129.90, 127.11, 124.19, 120.87, 106.51, 102.98, 96.60, 95.44, 91.13, 58.65, 57.85, 50.76, 48.36, 47.72,
16.37 ppm. ESI-MS: m/z 355.3 [M+H]+. Anal. Calcd. for C21H26N2O3: C, 71.16; H, 7.39; N, 7.90. Found:
C, 70.24; H, 7.40; N, 7.98.
1-(2-Hydroxy-4-methoxyphenyl)-2-(4-(4-nitrobenzyl) piperazin-1-yl) ethanone (5c): Yield 31%, yellow
solid. IR (KBr): ν 2942.1, 2809.6, 1632.6, 1615.4, 1515.6, 1350.8, 858.4, 829.5 cm−1. 1H-NMR (CDCl3,
600 MHz): δ 8.25 (d, J = 12.0 Hz, 2H), 7.88 (d, J = 12.0 Hz, 1H), 7.58 (d, J = 6.0 Hz, 2H), 6.51 (t,
J = 6.0 Hz, 1H), 6.47 (s, 1H), 3.90 (s, 3H), 3.77 (s, 2H), 3.68 (s, 2H), 2.74 (m, 4H), 2.64 (m, 4H) ppm.
13C-NMR (CDCl3, 150 MHz): δ 161.36, 160.30, 142.40, 141.22, 126.87, 124.75, 118.78, 103.01, 96.46,
57.17, 50.80, 48.37, 47.94 ppm. ESI-MS: m/z 444.10 [M+H]+. Anal. Calcd. for C20H23N3O5: C, 62.33;
H, 6.01; N, 10.90. Found: C, 62.41; H, 5.98; N, 10.97.
2-(4-(4-Chlorobenzyl) piperazin-1-yl)-1-(2-hydroxy-4-methoxypheny) ethanone (5d): Yield 44%, pale
1
yellow solid. IR (KBr): ν 2936.6, 2811.8, 1637.9.1, 1510.5, 1489.6, 1455.1, 815.3 cm−1. H-NMR
(CDCl3, 600 MHz): δ 7.82 (d, J = 12.0 Hz, 1H), 7.23–7.26 (m, 4H), 6.42 (t, J = 6.0 Hz, 1H), 6.40 (t,
13
J = 6.0 Hz, 1H), 3.82 (s, 3H), 3.66 (s, 2H), 3.48 (s, 2H), 2.64 (m, 4H), 2.53 (m, 4H) ppm. C-NMR
(CDCl3, 150 MHz): δ 168.75, 138.98, 135.53, 134.43, 133.05, 131.06, 110.37, 103.96, 64.66, 58.16,
55.74, 55.15 ppm. ESI-MS: m/z 375.2 [M+H]+. Anal. Calcd. for C20H23ClN2O3: C, 64.08; H, 6.18; N,
7.47. Found: C, 64.14; H, 6.02; N, 7.42.
3.3. DPPH Radical Scavenging Activity
The free radical scavenging capacities of paeonol and target compounds 5a–5d were evaluated by the
recognized method. The stable radical DPPH was used as a reagent in this spectrophotometric assay.
Two milliliters of various concentrations of the test compounds (0.08–0.8 mM) in methanol were added
into 2 mL of 0.3 mM DPPH methanol solution; therefore, the appropriate amount of methanolic solution
of each the test compounds was added to obtain cuvette concentrations ranging from 0.04–0.4 mM. After

Molecules 2015, 20
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a 60 min incubation period at room temperature in the dark, the absorbance was read at 517 nm against
the corresponding blank. Inhibition of free radical DPPH in percent (I%) was calculated using the
following Equation (1):
Ablank − A
I% = (
sample )×100
(1)
Ablank
Where Ablank is the absorbance of DPPH solution (control solution) against the blank, and Asample is the
absorbance of the tested compound against the blank. Concentrations caused 50% loss of the initial
DPPH free radical scavenging.
3.4. Protection of PC12 Cells against Hydrogen Peroxide-Induced Damage
3.4.1. Cell Culture
PC12 (rat pheochromocytoma) cells were obtained from the Chinese Academia Sinica (Shanghai,
China). Cells were cultured at 37 °C under a humidified 5% CO2 atmosphere in RPMI1640 medium
supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin G and 100 μg/mL streptomycin.
Before treatment, the differentiated PC12 cells were plated at appropriate density on 96-well culture
plates and cultured for 24 h. In all experiments, cells were preincubated with different concentrations of
paeonol and its derivatives dissolved in DMSO and diluted with DMEM with final concentration of
DMSO less than 0.1% for 3 h, and later, H2O2 was added to the medium. Not all of the compounds were
removed after the addition of H2O2. The sample was prepared as stock solution in DMSO and diluted
with DMEM with the final concentration of DMSO less than 0.1%, 0.1% (v/v) DMSO having no
protective or toxic effect by itself.
3.4.2. Cell Viability Assay
Cell viability following exposure to H2O2 was measured by the MTT reduction assay according to
our previous study [21]. Before the treatment, differentiated PC12 cells were seeded on 96-well culture
plates at a density of 1 × 105 cells/well and cultured for 24 h. Then, the medium was changed and
cells were treated with paeonol and its derivatives (5 µM, 10 µM, 20 µM, 40 µM) for 3 h, respectively.
After this time, the cells were exposed to 150 µM H2O2 for 5 h. Then, 20 µL of MTT solution (5 mg/mL)
was added to 200 µL culture medium of each well. Four hours later, the medium was removed, and the
resultant formazan product was dissolved by addition of 150 µL of DMSO. The absorbance of MTT was
measured at 570 nm with a microplate reader (ELX 800; Bio-TEK instruments, Inc., Winooski, VT,
USA). The results were expressed as percentage of cell viability (%), assuming that of control cells
is 100%.
3.5. In Vitro AChE Inhibition Assay
Acetylcholinesterase (AChE, E.C.3.1.1.7, from the electric eel), 5,5'-dithiobis-(2-nitrobenzoic acid)
(DTNB), acetylthiocholine iodide (ATCI) and donepezil hydrochloride were purchased from Sigma
Aldrich. The inhibitory activities against AChE of paeonol and target compounds 5a–5d were performed
according to the method developed by Ellman et al. [26], using donepezil as the reference compound.

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All the in vitro assays were carried out in 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) using a Shimadzu
UV-2550 UV-Vis Spectrophotometer. Paeonol and 5a–5d were dissolved in DMSO and then diluted by
phosphate buffer (pH 8.0) to seven different concentrations in the range of 10−3–10−9 M. AChE solutions
were prepared in double-distilled water to 2.0 units/mL in 2 mL aliquots. The assay medium contained
phosphate buffer, pH 8.0 (1 mL), 50 µL of 0.01 M DTNB, 10 µL of enzyme and 50 µL of acetylthiocholine
iodide. Test compounds were added to assay solution and preincubated at 37 °C with enzyme for 15 min,
followed by the addition of substrate. Activity was determined by measuring the increase in absorbance
at 412 nm at 1 min intervals at 37 ± 0.2 °C. For determining the blank value, 50 µL buffer replaced the
AChE solution. Each concentration was assayed three times and 50% inhibitory concentration (IC50)
was calculated graphically from inhibition curves.
3.6. Molecular Docking
To further investigate the interaction mode for AChE, molecular docking study was carried out by
Molecular Operating Environment (MOE 2008.10) software package. The crystal complex (PDB
code: 1EVE) was downloaded from the protein data bank [31]. The protein was added to hydrogens and
calculated with partial charges (Gasteiger methodology). Energy minimization (MMFF94x, gradient:
0.05) was performed. The structure was energy minimized in AMBER force filed to an RMSD of
0.05 Å where the implicit solvated environment was specified and the stabilized conformation was
saved. The 3D structure of the strongest AChE inhibitor 5b was built using the builder interface of MOE
program, and docked into the active site of the protein after energy minimized. All parameters were left
as default values. Finally, the conformation with the lowest docking score was selected for analyzing the
interactions between the AChE and 5d.
3.7. Metal-Chelating Studies
The metal-chelating studies were carried out in methanol using a UV-vis spectrophotometer
(SHIMADZC UV-2550PC). FeSO4, CuCl2 or ZnCl2 solution was prepared to 200 μM using methanol.
The test compounds 5a–5d were also dissolved in methanol to 400 μM. To investigate the metal binding
ability of compounds, a mixture of 100 μL test compound solution was added with 200 μL FeSO4
solution (CuCl2 or ZnCl2) and 700 μL methanol in a 1 cm quartz cuvette. The solution was incubated
for 30 min at room temperature and then the absorption spectra were recorded with wavelength ranging
from 200–500 nm. The stoichiometry of complex 5b-Cu (II) was determined using the molar ratio
method [29,30], by titrating solutions of compound 5b with ascending amounts of CuCl2. The final
concentration of tested compound 5b was 40 μM, and the final concentration of Cu2+ ranged from
4–80 μM. The recorded UV spectra data were treated by numerical subtraction of CuCl2 and 5b at
corresponding concentrations, and then plotted versus the mole fraction of 5b.
3.8. Statistical Analysis
Data were represented as mean ± SD. Comparisons between several groups were performed by
analysis of variance (ANOVA). A value of p < 0.05 was considered to be statistically significant.

Molecules 2015, 20
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4. Conclusions
In summary, a series of novel paeonol derivatives 5a–5d were designed, synthesized and characterized
as multifunctional agents for the treatment of AD. Their structures were identified by means of spectral
data and elemental analysis. Their antioxidant activities were evaluated by DPPH radical-scavenging activity
assays and their protective effects on H2O2-induced cytotoxicity in PC12 cells by the MTT assay method.
Compared with paeonol, 5a–5d possessed stronger antioxidant activity and protective effects on
H2O2-induced PC12 cell death. In addition, these compounds also had moderate AChE inhibition
properties, with IC50 values ranging from micromolar to sub-micromolar. Among them, compound 5b,
with para methyl substituent on phenyl ring was found to bind to Cu2+ and assemble into a metal
complex. Further investigations of AD candidates based on these results are in progress.
Acknowledgments
The authors gratefully acknowledge the financial support from the National Natural Science Foundation
of China (No. 81303245 and 21302037) and the Key Project of the National Science Fund of Anhui
Province (No. KJ2013A171).
Author Contributions
An Zhou performed whole experiments and wrote the paper. Hongfei Wu and Xuncui Wang
contributed to finishing bioassays and analyzing the data of biological activity. Jiaming Li, Zeyu Wu
and Ailing Hui supported the Zhou’s experiments and shared the work of structural investigations.
Jian Pan and Ailing Hui conceived and designed the experiments. All authors have read and approved
the final version of the article.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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