Xanthone and Flavone Derivatives as Dual Agents with Acetylcholinesterase Inhibition and Antioxidant Activity as Potential Anti-Alzheimer Agents

Article abstract of DOI:10.1155/2017/8587260

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that is associated with the elderly. The current therapy that is used to treat AD is based mainly on the administration of acetylcholinesterase (AChE) inhibitors. Due to their low eff

Full text of DOI:10.1155/2017/8587260

Hindawi
Journal of Chemistry
Volume 2017, Article ID 8587260, 16 pages
https://doi.org/10.1155/2017/8587260
Research Article
Xanthone and Flavone Derivatives as Dual Agents with
Acetylcholinesterase Inhibition and Antioxidant Activity as
Potential Anti-Alzheimer Agents
Inês Cruz,1 Ploenthip Puthongking,2 Sara Cravo,1,3 Andreia Palmeira,1,3
Honorina Cidade,1,3 Madalena Pinto,1,3 and Emília Sousa1,3
1
´
´
ˆ
ˆ
ˆ
´
´
Laboratorio de Quımica Organica e Farmaceutica, Departamento de Ciencias Quımicas, Faculdade de Farmacia,
Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
2Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, ailand
3
˜
Centro Interdisciplinar de Investigac¸ao Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto,
´
˜
Edifıcio do Terminal de Cruzeiros do Porto de Leixoes, Av. General Norton de Matos s/n, 4050-208 Matosinhos, Portugal
Correspondence should be addressed to Honorina Cidade; hcidade@ff.up.pt and Madalena Pinto; madalena@ff.up.pt
Received 14 November 2016; Revised 9 February 2017; Accepted 13 February 2017; Published 26 March 2017
Academic Editor: Sevgi Kolaylı
Copyright © 2017 Ineˆs Cruz et al. is is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder that is associated with the elderly. e current therapy that
is used to treat AD is based mainly on the administration of acetylcholinesterase (AChE) inhibitors. Due to their low efficacy there is
a considerable need for other therapeutic strategies. Considering that the malfunctions of different, but interconnected, biochemical
complex pathways play an important role in the pathogenesis of this disease, a promising therapy may consist in administration
of drugs that act on more than a target on biochemical scenery of AD. In this work, the synthesis and evaluation of xanthone and
flavone derivatives as antioxidants with AChE inhibitory activity were accomplished. Among the obtained compounds, Mannich
bases 3 and 14 showed capacity to inhibit AChE and antioxidant property, exerting dual activity. Moreover, for the most promising
AChE inhibitors, docking studies on the target have been performed aiming to predict the binding mechanism. e results presented
here may help to identify new xanthone and flavone derivatives as dual anti-Alzheimer agents with AChE inhibitory and antioxidant
activities.
1. Introduction
and galantamine [1]. However, this therapeutic strategy only
is capable of treating the symptoms of AD resulting in
favoring of behavioral and cognitive improvements on AD
patients [2]. Concerning this and that the number of cases of
AD is increasing, it is urgent to find new effective therapeutic
strategies for the treatment of AD [2, 5].
Currently, one of the most interesting approaches for the
treatment of complex diseases, like AD, is based on the
“one molecule, multiple targets” paradigm [6, 7]. Among the
multipotent approaches, the association of AChE inhibition
and antioxidant activity has been considered as an attractive
strategy for the treatment of AD [2, 6] and several multipotent
natural and synthetic compounds have been developed as
potential drug candidates to treat this neurodegenerative
disease [2].
AD is a neurodegenerative disorder that is characterized by
progressive memory loss and cognitive deficits, being the
most common form of dementia observed in the elderly [1, 2].
AD is usually designated as a multifactorial disease, because
its genesis is associated with a diversity of factors, including
genetic, environmental, and endogenous factors. Although
the pathogenesis of AD is not yet fully elucidated, the
formation of senile plaques, the neurofibrillary tangles, loss of
cholinergic neural transmission, and oxidative stress have
been identified as the major characteristic hallmarks of this
disease [1, 3, 4].
e current therapy for AD is based on the administration
of AChE inhibitors, such as tacrine, rivastigmine, donepezil,

2
Journal of Chemistry
Xanthones and flavones frequently found in nature [8–
Ltd., Cheshire, UK). ChromQuest 5.0 (version 3.2.1) sofware
(ermo Fisher Scientific Inc.) managed chromatographic
data. Methanol (HPLC grade) was obtained from Carlo Erba
Reagents (Val de Reuil, Italy), acetic acid (HPLC grade)
was obtained from Romil Pure Chemistry (Cambridge, UK),
and HPLC grade water was obtained from Simplicity5 UV
Ultrapure Water System, Millipore Corporation (USA). Prior
to use, mobile phase solvents were degassed in an ultrasonic
bath for 15 min. All synthesized compounds possessed a
purity of at least 95%.
13] correspond to “privileged structures,” showing a wide
variety of biological activities [9, 11, 14–16]. Among these
activities, the effect on molecular targets of AD have been
widely studied and explored [6, 17–22]. Some xanthones
and flavones have been shown to inhibit AChE and MAO
enzymes [20–27], and the aggregation of Aꢀ peptides [28–
31], as well as to act as antioxidants [21, 32–37].
Herein we describe the synthesis and biological evalua-
tion of xanthone and flavone derivatives with concomitant
AChE inhibitory and antioxidant activities. e planning
focused on the development of aminated derivatives of
xanthones/flavones polyphenols, since FDA-approved acetyl-
cholinesterase inhibitors for AD consist of amine drugs [1].
Moreover, for the most promising AChE inhibitors, docking
studies on the target have been performed aiming to predict
their binding mechanism.
3,6-Dihydroxy-9-oxo-9H-xanthene-9-one (4) and 4-hy-
droxy-3-methoxy-9-oxo-9H-xanthene-1-carbaldehyde (10)
were obtained according to described procedures [38, 39].
2.2. Chemistry
2.2.1. 1,3,8-Trihydroxy-9H-xanthen-9-one (1)
2. Materials and Methods
Method A (Eaton’s Reaction). A mixture of phosphorus pen-
toxide (1.00 g, 3.50 mmol) and methanesulfonic acid (15 mL)
was heated on a steam bath (90∘C) until a clear solution was
obtained (30 min). en, a mixture of phloroglucinol (0.38 g,
3 mmol) and 2,6-dihydroxybenzoic acid (0.46 g, 3 mmol) was
added to the reaction mixture. is mixture was stirred at
reflux (80∘C) for 1 h and the progress of the reaction was
monitored by TLC. en, the resulting mixture was slowly
poured into crushed ice and allowed to stand overnight in
the fridge. e solid was collected by filtration, washed with
water, and dried in oven (60∘C). e crude product was
2.1. Materials. All the reagents and solvents were purchased
from Sigma Aldrich and were used without further purifica-
tion. MW reactions were performed using a glassware setup
for atmospheric-pressure reactions and a 100 mL reactor
(internal reaction temperature measurements with a fiber-
optic probe sensor) and were carried out using an Ethos
MicroSYNTH 1600 Microwave Labstation from Milestone.
All reactions were monitored by TLC carried out on pre-
coated plates (silica gel, 60 F254 Merck) with 0.2 mm of
thickness. e purifications of compounds were performed
by flash column chromatography using Macherey-Nagel
silica gel 60 (0.04–0.063 mm). IR spectra were measured
on a KBr microplate in a FTIR spectrometer Nicolet iS10
from ermo Scientific with Smart OMNI-Transmission
Accessory (Sofware OMNIC 8.3). 1H and 13C NMR spectra
were taken in CDCl (Deutero GmbH) at r.t. or DMSO-d
purified by flash column chromatography (SiO , petroleum
2
ether: EtOAc (9 : 1 v/v)) to afford 1 (yield: 2.07%).
Method B (GSS Reaction). A mixture of zinc chloride
(13.25 g, 97.2 mmol) and phosphorus oxychloride (55 mL) was
submitted to microwave irradiation at 400 W of potency
during 10 min at 60∘C. en, the 2,6-dihydroxybenzoic acid
(5.00 g, 32.40 mmol) was added and the reaction mixture was
subjected to microwave irradiation at 400 W of power for
10 min at 60∘C. Finally, phloroglucinol (4.10 g, 32.40 mmol)
was added and the mixture was submitted to microwave
irradiation at 400 W of power during 60 min at 60∘C. Afer
cooling, the resulting mixture was poured into crushed ice
and extracted successively with CH Cl (9 × 200 mL) and a
3
6
(Deutero GmbH) at r.t., on Bruker Avance 300 instruments
1
(300.13 MHz for H and 75.47 MHz for 13C). ESI-HRMS
experiments were performed at CACTI, University of Vigo
(Spain), on an APEXQe FT-ICR MS (Bruker Daltonics,
USA), equipped with a 7T actively shielded magnet. Ions
were generated using a Combi MALDI-electrospray ioniza-
tion (ESI) source. Ionization was achieved by electrospray,
using a voltage of 4500 V applied to the needle and a
counter voltage of 300 V applied to the capillary. Samples
were prepared by adding a spray solution of 70 : 29.9 : 0.1
2
2
solution of 5% NaHCO (3 × 250 mL). e organic layers were
3
joined and dried over anhydrous Na SO and evaporated
2
4
under reduced pressure. e crude product was purified
(v/v/v) CH OH/water/formic acid or 70 : 29.9 : 0.1 (v/v/v)
CH CN/water/formic acid to a solution of the sample at a
3
by flash column chromatography (SiO , n-hexane: EtOAc
2
3
(9.5 : 0.5 v/v)) to give compound 1 as yellow solid (yield:
v/v ratio of 1 to 5% to give the best signal-to-noise ratio. Data
acquisition was performed using the ApexControl sofware
version 3.0.0, and data processing was performed using the
DataAnalysis sofware, version 4.0, both from Bruker Dal-
tonics. Melting points were obtained in a Ko¨fler microscope
and are uncorrected. e purity of each compound was deter-
mined by analytical HPLC-DAD performed on a SpectraSYS-
TEM (ermo Fisher Scientific, Inc., USA) equipped with
a P4000 pump, an AS3000 autosampler, and a diode array
detector UV8000. e separation was carried out on a 250
× 4.6 mm i.d. FortisBIO C18 (5 ꢁm) (Fortis6 Technologies
2.38%). mp 219-220∘C; IR (KBr, ꢂ (cm−1)): 3500–3400 (OH);
1663 (C=O); 1601, 1565, 1514, 1483 (aromatic C=C); 1293 (C-
1
O); H NMR (300 MHz, DMSO-d ), ꢃ (ppm): 11.89 (1 H, s,
6
OH-1), 11.81 (1 H, s, OH-8), 7.68 (1 H, brt, J = 8.4 Hz, H-6), 7.00
(1 H, dd, J = 8.4, 0.9 Hz, H-5), 6.79 (1 H, dd, J = 8.4, 0.9 Hz,
H-7), 6.38 (1 H, d, J = 2.1 Hz, H-4), 6.21 (1 H, d, J = 2.1 Hz, H-
2); 13C NMR (75.47 MHz, DMSO-d ), ꢃ (ppm): 183.26 (C-9),
6
167.06 (C-3), 162.16 (C-1), 160.27 (C-8), 157.51 (C-4a), 155.49
(C-10a), 137.18 (C-6), 110.60 (C-7), 107.10 (C-5), 106.88 (C-8a),
101.00 (C-9a), 98.70 (C-2), 94.48 (C-4).

Journal of Chemistry
3
2.2.2. 1,8-Dihydroxy-3-methoxy-9H-xanthen-9-one (2). A mix-
ture of 1,3,8-trihydroxyxanthone (1) (0.30 g, 1.23 mmol),
methyl iodide (0.803 mL, 12.30 mmol), and anhydrous
K CO (0.21 g, 1.24 mmol) in anhydrous acetone (40 mL)
again for 30 min and cooled down to room temperature.
en, crushed ice was added to the reaction mixture and
stirred for 15 min. e solution was kept in refrigerator
for overnight. e solid thus obtained was collected and
washed with ice-water to give a brown solid (9.280 g) that
2
3
was stirred at reflux (60∘C) for 3 h. en, the solid was filtered,
the solvent was removed under reduced pressure, and the
crude product was purified by crystallization with acetone
was further purified by open column chromatography (SiO ,
2
chloroform : acetone, several proportions). e fractions
eluted with chloroform : acetone (9 : 1) were gathered and the
solvent evaporated to furnish a white solid corresponding to
3,6-dihydroxy-9-oxo-9H-xanthene-4,5-dicarbaldehyde (5,
and by flash column chromatography (SiO , n-hexane)
2
to yield compound 2 as yellow needle solid (yield: 74%).
mp 170–172∘C; IR (KBr, ꢂ (cm−1)): 3500–3400 (OH); 1662
(C=O); 1603, 1568, 1505, 1470 (aromatic C=C); 2959, 2925,
2852 (aliphatic C-H); 1294 (C-O); 1H NMR (300 MHz,
yield: 4.0%). mp >220∘C; 1H NMR (300 MHz, DMSO-d ), ꢃ
6
(ppm): 10.65 (2 H, s, 4-CHO, 5-CHO), 8.25 (2 H, d, J = 8.8 Hz,
H-1, H-8), 7.07 (2 H, d, J = 8.8 Hz, H-2, H-7); 13C NMR
(75.47 MHz, DMSO-d6), ꢃ (ppm):191.12 (4-CHO, 5-CHO),
172.69 (C-9) 166.67 (C-3, C-6), 156.76 (C-10a, C-4a), 133.89
(C-1, C-8), 115.28 (C-2, C-7), 113.89 (C-8a, C-9a), 109.86 (C-4,
C-5). ESI-HRMS (+) m/z: Anal. Calcd for C H O (M+H)+:
CDCl ), ꢃ (ppm): 11.99 (1 H, s, OH-1), 11.92 (1 H, s, OH-8),
3
7. 5 6 (1 H, brt, J = 8.4 Hz, H-6), 6.87 (1 H, dd, J = 8.4, 0.8 Hz,
H-5), 6.78 (1 H, dd, J = 8.4, 0.8 Hz, H-7), 6.42 (1 H, d, J
= 2.3 Hz, H-4), 6.35 (1 H, d, J =2.3 Hz, H-2), 3.89 (3 H, s,
3-OCH ); 13C NMR (75.47 MHz, CDCl ), ꢃ (ppm): 184.63
3
3
15
9
6
(C-9), 167.35 (C-3), 162.97 (C-1), 161.31 (C-8), 157.78 (C-4a),
156.14 (C-10a), 136.80 (C-6), 110.89 (C-7), 106.96 (C-5),
106.96 (C-8a), 102.70 (C-9a), 97.41 (C-2), 93.10 (C-4), 55.93
(3- OCH ). ESI-HRMS (+) m/z: Anal. Calcd for C H O
285.03936; found: 285.03902.
2.2.5. General Procedure for the Reductive Amination. e
carbaldehyde xanthone (5 or 10, 0.1 mmol) was dissolved
in methanol (5 mL) and 2.4 equimolar of the appropriate
3
14 11
5
(M+H)+: 259.06010; found: 259.06014.
amine (0.24 mmol) was added to the solution under N gas.
2
2.2.3. 2-((Dimethylamino)methyl)-1,8-dihydroxy-3-methoxy-9H-
xanthen-9-one (3). Dimethylamine (0.420 mL, 0.8395 mmol,
4.35 equiv.) was cooled in the ice bath for about 5 min and
37% formaldehyde solution (0.104 mL, 1.28 mmol) and acetic
acid (0.965 mL, 16.85 mmol) were added dropwise. e
reaction mixture was stirred at room temperature for 1 h and
then 2 was added (0.050 g, 0.193 mmol). e mixture was
stirred at room temperature for 6 days and then treated with
water (5 mL) and stirring was continued for about 12 h. e
resulting solid was filtered and the filtrate was treated with
10% NaOH until the pH was 9∼10. Finally, the precipitate
was also filtered, washed with water, dried, and then
A 4.0 equimolar quantity of NaBH(OAc) (0.4 mmol) was
3
added to the mixture solution. e reaction mixture was
stirred at room temperature for 30 min. en, 2.0 equimolar
quantity of AcOH was added to the reaction mixture. e
reaction mixture was stirred at room temperature under
N
gas for overnight. Afer the solvent was evaporated,
2
the crude product was further purified by flash column
chromatography (SiO , chloroform : methanol : ammonium
2
hydroxide (90 : 10 : 2 v/v/v)) to afford compounds 6–9, 11, and
12 in 32–51% yield.
(1) 4,5-Bis(((2-(diethylamino)ethyl)amino)methyl)-3,6-dihy-
droxy-9H-xanthen-9-one (6). Pale yellow solid (yield: 51%).
purified by flash column chromatography (SiO , petroleum
2
ether : chloroform : ammonium hydroxide (95 : 5 : 1 v/v/v)) to
get 3 as yellow solid (yield: 6.29%). mp 118–120∘C; IR (KBr, ꢂ
(cm−1)): 3600–3400 (OH); 1653 (C=O); 1605, 1558, 1539, 1490
(aromatic C=C); 2923, 2853 (aliphatic C-H); 1296 (C-O);
mp >220∘C; IR (KBr, ꢂ (cm−1)): 3446 (OH, broad spectrum);
1602 (C=O); 1431 (aromatic C=C); 1283 (C-O); 1201 (C-N);
1083 (C-O-C). 1H NMR (300 MHz, DMSO-d ), ꢃ (ppm): 7.86
6
(2 H, d, J = 8.8 Hz, H-1, H-8), 6.73 (2 H, d, J = 8.8 Hz, H-2, H-
7), 4.25 (4 H, s, 4-CH , 5-CH ), 2.85 (4 H, t, J = 6.2 Hz, CH )
1230 (C-N); 1H NMR (300 MHz, CDCl ), ꢃ (ppm): 12.09 (1
2
2
2
3
2.67 (4 H, t, J = 6.8 Hz, CH ), 2.57 (8 H, m, CH ), 0.98 (12 H, t,
H, s, OH-1), 12.09 (1 H, s, OH-8), 7.55 (1 H, brt, J = 8.3 Hz,
H-6), 6.86 (1 H, brd, J = 8.3 Hz, H-5), 6.78 (1 H, brd, J = 8.3,
2
2
J = 6.8 Hz, CH ); 13C NMR (75.47 MHz, DMSO-d ), ꢃ (ppm):
3
6
173.70 (C-9) 166.70 (C-3, C-6), 155.10 (C-4a, C-10a), 126.40
(C-1, C-8), 117.60 (C-2, C-7), 111.70 (C-8a, C-9a), 107.80 (C-4,
C-5), 50.02 (C-CH ), 46.64 (C-CH ), 44.80 (C-CH ), 42.00
Hz, H-7), 6.44 (1 H, s, H-4), 3.94 (3 H, s, 3-OCH ), 3.64 (2
3
H, s, CH N), 2.37 (6 H, s, N(CH ) ); 13C NMR (75.47 MHz,
2
3 2
CDCl ), ꢃ (ppm): 184.33 (C-9), 165.79 (C-3), 161.39 (C-1),
2
2
2
3
(4-CH , 5-CH ), 11.42 (C-CH ). ESI-HRMS (+) m/z: Anal.
161.39 (C-8), 157.69 (C-4a), 155.96 (C-10a), 136.58 (C-6),
110.90 (C-7), 107.85 (C-5), 106.79 (C-8a), 102.70 (C-9a), 103.5
(C-2), 90.05 (C-4), 56.34 (3-OCH ), 49.97(CH N), 44.96
2
2
3
Calcd for C H N O (M+H)+: 485.31223; found: 485.31119.
27 41
4
4
3
2
(2) 3,6-Dihydroxy-4,5-bis(piperidin-1-ylmethyl)-9H-xanthen-
9-one (7). Pale yellow solid (yield: 37%). mp >220∘C; IR (KBr,
ꢂ (cm−1)): 3424 (OH, broad spectrum); 2955 (aliphatic C-H);
1616 (C=O); 1431, 1438 (aromatic C=C); 1289 (C-O); 1200 (C-
(N(CH ) ). ESI-HRMS (+) m/z: Anal. Calcd for C H O
3 2
21 25
6
(M+H)+: 316.11795; found: 316.11740.
2.2.4. 3,6-Dihydroxy-9-oxo-9H-xanthene-4,5-dicarbaldehyde
(5). A mixture of 3,6-dihydroxy-9H-xanthen-9-one (4,
5.545 g, 0.024 mol) and hexamethylenetetramine (HMTA,
17.1 g, 0.12 mol) was dissolved in AcOH (60 mL). e reaction
mixture was refluxed for 3–6 h. Afer the reaction mixture
was cooled down to room temperature, 80 mL of 15%
HCl was added to the reaction. e mixture was refluxed
N); 1087 (C-O-C). 1H NMR (300 MHz, DMSO-d ), ꢃ (ppm):
6
7. 91 (2 H, dd, J = 8.8, 1.4 Hz, H-1, H-8), 6.91 (1 H, d, J = 8.7 Hz,
H-2/H-7), 6.78 (1 H, d, J = 8.7 Hz, H-7/H-2), 4.78 (4 H, s, 4-
CH , 5-CH ), 2.61 (8 H, m, CH N), 1.52 (12 H, m, CH ); 13
C
2
2
2
2
NMR (75.47 MHz, DMSO-d ), ꢃ (ppm): 174.18 (C-9) 164.56
6
(C-3, C-6), 154.81 (C-4a, C-10a), 126.09 (C-1, C-8), 113.86

4
Journal of Chemistry
(C-2, C-7), 113.03 (C-8a, C-9a), 108.16 (C-4, C-5), 53.41 (C-
CH N), 48.65 (4-CH , 5-CH ), 30.76 (C-CH ). ESI-HRMS
7. 60 (1 H, d, J = 7.8 Hz, H-5), 7.41 (1 H, dt, J = 1.0, 8.0 Hz,
H-6), 7.33 (1 H, s, H-2), 4.15 (2 H, s, 1-CH ), 3.02 (2 H, s,
2
2
2
2
2
(+) m/z: Anal. Calcd for C H N O (M+H)+: 423.22783;
CH ), 2.93 (2 H, m, CH ), 2.41–2.69 (8 H, m, CH ); 13C
25 31
2
4
2
2
2
found: 423.22580.
NMR (75.47 MHz, DMSO-d ), ꢃ (ppm): 177.14 (C-9), 1172.15
6
(C-10a), 154.67 (C-3), 151.31 (C-4a), 146.70 (C-4), 134.80 (C-
6), 132.74 (C-8), 126.11 (C-1), 123.94 (C-8a), 121.72 (C-7),
(3) 3,6-Dihydroxy-4,5-bis((4-(2-hydroxyethyl)piperazin-1-
yl)methyl)-9H-xanthen-9-one (8). Pale yellow solid (yield:
32%). mp >220∘C; (KBr, ꢂ (cm−1)): 3454 (OH, broad spec-
trum); 2925 (aliphatic C-H); 1644 (C=O); 1604, 1525, 1469,
1416 (aromatic C=C); 1323 (C-O); 1221 (C-N); 1075 (C-O-C).
117.60 (C-9a), 113.68 (C-5), 108.70 (C-2), 58.33 (C-CH OH),
2
56.03 (3-OCH ), 52.77 (C-1CH ), 48.65 (C-CH N), 45.65 (C-
3
2
2
CH N), 43.22 (C-CH N). ESI-HRMS (+) m/z: Anal. Calcd
2
2
for C H N O5 (M+H)+: 385.17580; found: 385.17425.
1H NMR (300 MHz, CDCl ), ꢃ (ppm): 8.14 (2 H, d, J = 8.8 Hz,
21 25
2
3
H-1, H-8), 6.83 (2 H, d, J = 8.8 Hz, H-2, H-7), 4.06 (4 H, s,
4-CH , 5-CH ), 3.67 (4 H, t, J = 5.3 Hz, CH OH), 2.25 (20
2.2.6. 8-((Dimethylamino)methyl)-5,6,7-trihydroxy-2-phenyl-
4H-chromen-4-one (14). To a solution of baicalein (13)
(0.2 g, 0.74 mmol) in methanol (100 mL) 37% (v/v) formalde-
hyde solution (0.018 mL, 0.98 mmol) and dimethylamine
(1.00 mL, 2.00 mmol) were added dropwise. e mixture was
stirred at room temperature for 1 h 30 min. e precipi-
tate was collected, filtered, and washed several times with
methanol. en, the precipitate was purified by flash column
2
2
2
H, m, CH ); 13C NMR (75.47 MHz, CDCl ), ꢃ (ppm): 170.79
2
3
(C-9) 159.30 (C-3, C-6), 149.4 (C-4a, C-10a), 122.64 (C-1, C-
8), 109.65 (C-8a, C-9a), 109.37 (C-2, C-7), 101.95 (C-4, C-5),
54.41 (C-CH OH), 53.20 (4-CH , 5-CH ), 49.26 (C-CH N),
2
2
2
2
48.15 (C-CH N), 47.80 (C-CH N).
2
2
(4) 1,1ꢀ-(((3,6-Dihydroxy-9-oxo-9H-xanthene-4,5-diyl)bis(meth-
ylene))bis(piperazine-4,1-diyl))bis(ethan-1-one) (9). Pale yel-
low solid (yield: 42%). mp >220∘C; IR (KBr, ꢂ (cm−1)): 3448
(OH, broad spectrum); 2921, 2851 (aliphatic C-H); 1624, 1602
(C=O); 1474, 1430, 1400 (aromatic C=C); 1265 (C-O); 1210 (C-
chromatography (SiO , chloroform : methanol : ammonium
2
hydroxide (90 : 10 : 2 v/v/v)) to afford compound 14 as orange
solid (yield: 10.50%). mp 286–288∘C; IR (KBr, ꢂ (cm−1)):
3500–3250 (OH); 1667 (C=O); 1608, 1572, 1536, 1468 (aro-
matic C=C); 2955, 2919 (aliphatic C-H); 1287 (C-O); 1247
N); 1073 (C-O-C). 1H NMR (300 MHz, DMSO-d ), ꢃ (ppm):
6
(C-N); 1H NMR (300 MHz, CDCl ), ꢃ (ppm): 12.52 (1 H, s,
7. 96 (2 H, d, J = 8.7 Hz, H-1, H-8), 6.91 (2 H, d, J = 8.8 Hz,
H-2, H-7), 4.05 (4 H, brs, 4-CH , 5-CH ), 2.50 (16H, m,
3
OH-5), 7.84–7.82 (2 H, m, Ar-2ꢀ,6ꢀ-H), 7.54–7.52 (3 H, m, Ar-
2
2
3ꢀ,4ꢀ,5ꢀ-H), 6.63 (1 H, s, H-3), 4.01 (2 H, s, CH N), 2.45 (6 H,
CH ), 1.81 (6 H, s, CH ); 13C NMR (75.47 MHz, DMSO-d ),
2
2
3
6
s, N (CH ) ); 13C NMR (75.47 MHz, CDCl ), ꢃ (ppm): 182.53
ꢃ (ppm): 168.30 (C-9, COCH ) 162.80 (C-3, C-6), 155.40 (C-
3 2
3
3
(C-4), 162.60 (C-2), 153.65 (C-7), 147.17 (C-8a), 145.43 (C-5),
132.05 (C-4ꢀ), 131.43 (C-1ꢀ), 129.10 (C-3ꢀ,5ꢀ), 126.02 (C-2ꢀ,6ꢀ),
119.78 (C-6), 105.18 (C-4a), 104.31 (C-3), 98.54 (C-8), 55.39
(CH N), 44.47 (N(CH ) ).
4a, C-10a), 126.40 (C-1, C-8), 113.60 (C-2, C-7), 113.40 (C-8a,
C-9a), 109.40 (C-4, C-5), 52.70 (4-CH , 5-CH ), 45.50 (C-
2
2
CH N), 21.20 (C-CH ). ESI-HRMS (+) m/z: Anal. Calcd for
2
3
C H N O (M+H)+: 509.23946; found: 509.23910.
2
3 2
27 33
4
6
(5) 1-(((2-(Diethylamino)ethyl)amino)methyl)-4-hydroxy-3-
methoxy-9H-xanthen-9-one (11). Pale yellow solid (yield:
35%). mp >220∘C; IR (KBr, ꢂ (cm−1)): 3448 (OH, broad
spectrum); 2921, 2851 (aliphatic C-H); 1624 (C=O); 1607, 1586,
1467 (aromatic C=C); 1320 (C-O); 1203 (C-N); 1071 (C-O-C).
2.3. Biological Activity
2.3.1. DPPH Radical Scavenging Assay. Scavenging activity
for stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical
was evaluated according to a method previously described
[40–42] with some modifications. A solution of DPPH radical
in methanol was prepared daily and protected from light.
1H NMR (300 MHz, DMSO-d ), ꢃ (ppm): 8.00 (1 H, d, J =
6
8.5 Hz, H-8), 7.58 (1 H, d, J = 8.0 Hz, H-6), 7.34 (1 H, dt, J =
1.7, 8.5 Hz, H-7), 7.05 (1 H, s, H-2), 6.73 (1 H, d, J = 8.8 Hz, H-
Reaction mixtures containing 100 ꢁL of methanol or DMSO
solutions of tested compounds (100–1.56 ꢁM) and 100 ꢁL of
DPPH (0.03 mg/mL) methanolic solution were shaken in a
96-well microtiter plate and incubated for 30 min in darkness
at room temperature (until stable absorbance values were
5), 4.20 (2 H, s, 1-CH ), 3.86 (3 H, s, 3-OCH ), 2.71 (2 H, t, J
2
3
= 6.09 Hz, CH ), 2.50 (6 H, m, CH ), 0.94 (6 H, t, J = 7.1 Hz,
2
2
CH ); 13C NMR (75.47 MHz, DMSO-d ), ꢃ (ppm): 177.26 (C-
3
6
9), 161.11 (C-8b), 154.60 (C-3), 151.50 (C-10a), 147.16 (C-4),
134.82 (C-6), 125.83 (C-8), 123.71 (C-7), 121.67 (C-1), 121.28
obtained). Controls containing 100 ꢁL of methanol instead
(C-8a), 117.58 (C-9a), 113.55 (C-5), 110.92 (C-2), 55.82 (C-
of compounds, blanks containing 200 ꢁL of methanol, and
sample’s blanks containing 100 ꢁL of methanol instead of
DPPH methanolic solution were also made. e absorbances
were measured at 520 nm in a microplate reader (BioTek
Instruments, Vermont, US). e percentage of inhibition
activity was calculated according to the following formula:
CH ), 52.14 (C-CH ), 51.75 (1-CH ), 46.20 (3-OCH ), 45.51
2
2
2
3
(C-CH ), 11.70 (C-CH ). ESI-HRMS (+) m/z: Anal. Calcd for
2
3
C H N O (M+H)+: 371.19653; found: 371.19573.
21 27
2
4
(6) 4-Hydroxy-1-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-
3-methoxy-9H-xanthen-9-one (12). Pale yellow solid (yield:
39%). mp >220∘C; IR (KBr, ꢂ (cm-1)): 3422 (OH, broad
spectrum); 2950, 2834 (aliphatic C-H); 1603 (C=O); 1460,
1412 (aromatic C=C); 1274 (C-O); 1235 (C-N); 1079 (C-O-
DPPH radical scavenging effect (%) = [1 − (Abssample
−
Abssample’s blank/Abscontrol − Absblank)] × 100. Ascorbic acid
was used as positive control. All the tests were performed in
triplicate. e scavenging activities of the tested compounds
towards DPPH radical were expressed as the effective concen-
C). 1H NMR (300 MHz, DMSO-d ), ꢃ (ppm): 8.10 (1 H, dd,
6
J = 1.5, 8.0 Hz, H-7), 7.80 (1 H, dt, J = 1.7, 8.6 Hz, H-8),
tration at which DPPH radical was scavenged by 50% (IC ).
50

Journal of Chemistry
5
e IC value was obtained by interpolation from linear
in a microplate reader (BioTek Instruments, Vermont, US).
50
regression analysis.
Controls containing 20 ꢁL of compound vehicle (methanol or
DMSO) instead of compounds and blanks containing 20 ꢁL
of buffer (0.1% (w/v) bovine serum albumin in 50 mM Tris-
HCl) instead of enzyme and 20 ꢁL of compound vehicle
(methanol or DMSO) instead of compounds were made. In
this assay, sample’s blanks containing 20 ꢁL of buffer (0.1%
(w/v) bovine serum albumin in 50 mM Tris-HCl) instead
of AChE were also performed. Percentage of enzymatic
inhibition was calculated as percentage of inhibition = 100 −
[((Abssample − Abssample’s blank)/(Abscontrol − Absblank)) × 100].
Every experiment was done in triplicate and galantamine
was used as positive control. e inhibitory activities of the
purified compounds towards AChE were expressed as the
effective concentration at which AChE was inhibited by 50%
(IC ). e IC value was obtained by interpolation from
2.3.2. Ferrous Ion Chelating Assay. e chelation of ferrous
ions (Fe2+) was assessed by the methodology described by
Dinis et al. with modifications [43]. is assay was per-
formed in a 96-well plate and the absorbance was measured
at 562 nm. Briefly, 10 ꢁL of FeCl (50 ꢁM) was added to
2
50 ꢁL of methanolic solution of compounds in different
concentrations (100–50 ꢁM) and 120 ꢁL of methanol. e
mixture was allowed to stand 5 min and the reaction was
initiated by the addition of 20 ꢁL of 3-(2-pyridyl)-5,6-bis(4-
phenylsulphonic acid)-1,2,4-triazine (ferrozine, 100 ꢁM) and
the mixture was shaken at room temperature for 10 min.
en, the absorbance of the solution was measured in
a microplate reader (BioTek Instruments, Vermont, US).
50
50
linear regression analysis.
Controls containing 50 ꢁL of solvent instead of compounds
and blanks containing 50 ꢁL of solvent instead of compounds
and 20 ꢁL milliQ6 purified water instead of ferrozine were
made. In this assay, sample’s blanks containing 20ꢁL milliQ
purified water instead of ferrozine were also performed.
All tests and analyses were carried out in triplicate and
EDTA was used as positive control. e percentage of
inhibition of Fe2+–ferrozine complex formation was given
2.4. Statistical Analyses. Data were reported as means
standard error of the mean (SEM) of at least three inde-
pendent experiments. Statistical analysis of the results was
performed with GraphPad Prism (GraphPad Sofware, San
Diego, CA). Unpaired ꢄ-test was carried out to test for any
significant differences between the means. Differences at the
5% confidence level were considered significant.
below: % inhibition = [((Abscontrol − Absblank) − (Abssample
−
Abssample’s blank))/(Abscontrol − Absblank)] × 100. e chelat-
ing activities of the purified compounds towards ferrous
ions were expressed as the effective concentration at which
Fe2+–ferrozine complex formation was inhibited in 50%
(IC ). e IC value was obtained by interpolation from
2.5. Docking Studies. Crystal structure of AChE (PDB code:
4EY7) [47], downloaded from the protein databank (PDB)
[48], was used for the study. Structure files of 14 known AChE
inhibitors and compounds 2, 3, 6–9, and 14 were created
and minimized using the chemical structure drawing tool
Hyperchem 7.5 (Hypercube, FL, USA) [49]. Structure-based
docking was carried out using AutoDock Vina (Molecular
Graphics Lab, CA, USA) [50]. e active site was defined
50
50
linear regression analysis.
2.3.3. Copper Chelating Activity. e chelation of copper ions
was assessed by the methodology described by Brown et al.
[44] and adapted to a 96-well plate [45]. Stock solutions
of each compound (1 mM) were prepared in methanol or
DMSO. en 50 ꢁM of these solutions was prepared with PBS
(10 mM) pH 7.4 and 50 ꢁL of each was taken to 96-well UV
plate (well A-D). Aferward, 100 ꢁL of ultrapure water (well
˚
as the region of AChE that comes within 12 A from the
crystallographic ligand. Default settings for small molecule-
protein docking were used throughout the simulations. Top
9 poses were collected for each molecule and the lowest
docking score value was associated with the more favorable
binding conformation. PyMol1.3 (Schro¨dinger, NY, USA) [51]
and MOE (Chemical Computing Group, Montreal, Canada)
[52] were used for visual inspection of results and graphical
representations.
A), or 50 ꢁL of ultrapure water and 50 ꢁL CuSO (200 ꢁM)
4
(well B), or 50 ꢁL of ultrapure water and 50 ꢁL of CuSO
4
(100 ꢁM) (well C), or 50 ꢁL of EDTA (500 ꢁM) and 50 ꢁL
CuSO (200 ꢁM) (well D) were added. e absorption spectra
4
were recorded between 200 and 800 nm in a microplate
reader (BioTek Instruments, Vermont, US), and the scans in
the presence of 1 : 1 (green spectrum) and 2 : 1 (blue spectrum)
of copper-to-compound ratios were compared with the scan
with compound alone (red spectrum). All tests and analyses
were carried out in triplicate.
3. Results and Discussion
3.1. Chemistry. e synthesis of xanthones 1, 2, and 3 was
achieved according to the general reaction pathway outlined
in Scheme 1. 1,3,8-Trihydroxyxanthone (1) was obtained by
one-step synthesis using two different approaches: Eaton’s
reaction and Grover Shah and Shah (GSS) reaction. In
Eaton’s reaction, 1 was obtained by the condensation of 2,6-
dihydroxybenzoic acid with phloroglucinol in the presence
of a mixture of phosphorus pentoxide-methanesulfonic acid
(Eaton’s reagent) as condensing agent (Scheme 1) [53]. In the
GSS reaction xanthone 1 was synthesized by the condensation
of the same building blocks in the presence of zinc chloride in
phosphorus oxychloride, instead of Eaton’s reagent [54]
(Scheme 1). Although this condensation agent has been
2.3.4. AChE Inhibitory Assay. AChE inhibitory activity was
measured according to the method of Ellman et al. [46] with
some modifications using a 96-well plate reader. Briefly, in the
performed assay 20 ꢁL of 0.22 U/mL AChE from Electropho-
rus electricus was added to the wells containing 20 ꢁL of tested
compounds (100 and 75 ꢁM in methanol or DMSO), 100 ꢁL
of 3 mM 5,5ꢀ-dithiobis(2-nitrobenzoic acid) (DTNB), and
20 ꢁL of 15 mM acetylthiocholine iodide. Absorbance of the
colored end product was measured at 412 nm for 10 minutes

6
Journal of Chemistry
OH
O
9
OH
1
OH
O
OH
OH
OH
8
a
8a
9a
COOH
OH
2
3
7
6
c
+
O 4a
10
1
OH
O
10a
b
OCH3
HO
OH
5
4
2
d
OH
O
OH
CH3
N
CH3
OCH3
O
3
Scheme 1: Reagents and conditions for the synthesized xanthones 1, 2, and 3. (a) Eaton’s reaction: P O /CH SO H, 90∘C, 30 min; 2,6-
2
5
3
3
dihydroxybenzoic acid/phloroglucinol, 80∘C, 1 h; (b) GSS reaction: (i) ZnCl , POCl , MW (400 W), 60∘C, 10 min; (ii) 2,6-dihydroxybenzoic
2
3
acid, MW (400 W), 60∘C, 10 min; (iii) phloroglucinol, MW (400 W), 60∘C, 60 min; (c) CH I, K CO , 60∘C, 3 h; (d) Mannich reaction: (i)
3
2
3
dimethylamine/HCHO/CH COOH, room temperature, 1 h; (ii) addition of xanthone 2, room temperature, 6 days. e numbering used
3
concerns the NMR assignments.
shown to be efficient for the synthesis of hydroxylated
xanthones, this synthetic methodology is associated with low
yields [9, 55, 56]. Taking these into account, some experimen-
tal modifications to the classic GSS reaction were performed
including the stepwise addition of reactants as described
before and the use of microwave assisted organic synthesis
(MAOS), instead of conventional thermal heating (classical
synthesis). Particularly, in the original GSS methodology,
a mixture of a hydroxybenzoic acid, a phenol, fused zinc
chloride, and phosphorus oxychloride was heated together
on a water bath. Instead, in the GSS reaction here reported,
firstly, a mixture of fused zinc chloride and phosphorus
oxychloride was irradiated by MW at 400 W for 10 min
at 60∘C; then the hydroxybenzoic acid was added and the
mixture was subjected to MW irradiation at 400 W for 10 min
at 60∘C and afer the appropriated phenol was added and
the mixture was once more submitted to MW at 400 W for
60 min at 60∘C to afford xanthone 1 (Scheme 1). In gen-
eral, both approaches showed short reaction times (Eaton’s
reaction: 1 h 30 min; modified GSS reaction by MW: 1 h
20 min) and low yields. Although purification of 1 in both
reactions was time consuming, this process was more com-
plex in the GSS reaction. In fact, in this methodology, three
different procedures had been performed: extraction with
CH Cl in order to remove a brown slimy material; extraction
Scheme 1, this methylated derivative 2 reacted with formalde-
hyde and dimethylamine through Mannich reaction to afford
the respective Mannich base derivative 3.
e aminated xanthones 6–9, 11, and 12 were obtained by
reductive amination from the corresponding carbaldehydes
5 and 10 (Scheme 2). Both precursors were obtained from
oxygenated xanthones by a Duff formylation according to the
described procedures [38, 39].
8-((Dimethylamino)methyl)-5,6,7-trihydroxy-2-phenyl-
4H-chromen-4-one (14) was obtained according to the
method previously described by Zhang et al. [57].
Concerning this synthetic approach, aminoflavone 14 was
achieved from the reaction of baicalein (13) with formal-
dehyde and dimethylamine by Mannich reaction (Scheme 3).
3.2. Biological Activity
3.2.1. DPPH Radical Scavenging Activity. e DPPH assay
has been widely used to assess the antioxidant potential of
compounds. Using this assay, the capacity of compounds 1–3,
6–9, and 11–14 to transfer labile H-atoms to this free radical
was measured (Table 1).
While no promising DPPH radical scavenging activity
was observed for xanthones 1–3 and 6–9 at 100 ꢁM, the
aminated derivatives 11 and 12 exhibited some scavenging
activity, suggesting that the introduction of amino side chains
at C-1, instead of C-4,5, is favorable for this activity. Among
the compounds tested the baicalein derivative14 was the most
2
2
with 5% NaHCO to eliminate traces of benzoic acid; and a
3
flash column chromatography to get 1. In contrast, in Eaton’s
reaction, xanthone 1 was obtained using merely flash chro-
matography as purification method. In conclusion, according
to these results, Eaton’s reaction seemed to be more suitable
than GSS for the synthesis of 1, because it was a cleaner
approach, requiring less purification procedures.
active, showing an IC value of 30.40 1.28 ꢁM, very similar
50
to the precursor baicalein (13) (IC = 35.09 4.13 ꢁM),
50
suggesting that the presence of 8-(dimethylamino)methyl
group on the flavone skeleton did not significantly alter its
ability to scavenge free radicals.
e hydroxylated xanthone 1 was then methylated with
methyl iodide under basic conditions to afford compound
2 (Scheme 1). Following the synthetic route as shown in
3.2.2. Ferrous Ion Chelating Activity. Among the transition
metals, iron is well known as playing a crucial role in

Journal of Chemistry
7
O
O
O
a
HO
HO
OH
O
OH
4
CHO
CHO
5
b
O
R
R
OH
N
N
N
6 =
HN
8 =
N
N
O
HO
OH
CH3
CH2
H2C
7 =
R
R
N
9 =
O
6–9
R
CHO
O
O
H2C
b
O
OCH3
O
OCH3
OH
OH
10
11-12
R
NH
N
N
11 =
12 =
OH
N
Scheme 2: Reagents and conditions for the synthesis of carbaldehyde xanthone5 and aminated xanthones6–9 and 11-12. (a) (1) HMTA/AcOH,
reflux, 3 h, (2) 15% HCl, reflux, 30 min; (b) amine (2.4 eq), NaBH (OAc) (4 eq), AcOH (2 eq), MeOH, room temperature, overnight.
3
CH3
5ꢀ
6ꢀ
1ꢀ
4ꢀ
3ꢀ
N
H3C
HO
8
8a
4a
O
O
HO
HO
2
7
2ꢀ
O
a
6
3
4
5
OH
HO
OH
O
13
14
Scheme 3: Reagents and conditions for the synthesized flavone 14. (a) Dimethylamine/HCHO/MeOH, room temperature, 1 h 30 min. e
numbering used concerns the NMR assignments.

8
Journal of Chemistry
Table 1: DPPH radical scavenging activity of tested compounds.
also the presence of a 2-(dimethylamino)methyl group on the
xanthonic scaffold can favor the chelating activity.
% of scavenging DPPH
Compounds
IC (ꢁM)
Considering flavonoids, compounds 13 and 14 showed
interesting chelating activity at 100 ꢁM, suggesting that the
catechol moiety in the structure of these flavones was associ-
ated with their ferrous ion chelating capacity. In fact, it was
reported that the presence of ortho-OH groups offers appro-
priate geometric and electronic environments to bind metal
ions [6, 21]. Moreover, considering the chelating activity of
13 (% of chelation at 100 ꢁM = 18.24 3.46) and 14 (% of
chelation at 100 ꢁm = 49.90 0.23), it is possible to infer
that the presence of 8-(dimethylamino)methyl group in the
baicalein derivative 14 was associated with an improvement
of the ferrous ion chelating activity.
50
radical at 100 ꢁM
1
2
3
6
7
8
9
11
12
13
14
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.a.
n.a.
n.a.
∗∗
5.92 0.84
∗
2.32 0.25
∗
5.74 0.83
n.a.
∗∗∗
∗∗∗
61.62 0.72
69.26 2.11
∗∗∗
∗∗∗
108.99 0.87
47.09 1.35
∗∗∗
∗∗∗
35.09 4.13
30.40 1.28
96.03 0.42
3.2.3. Copper Chelating Activity. As iron, copper (II) ions also
promote oxidative damage in AD by the generation of ROS
via the Fenton reaction. In addition, this transition metal still
has the ability to interfere with protein aggregation processes
implicated in the pathogenesis of this disease [1, 59, 60]. e
copper chelating activity of compounds 1–3, 6–9, and 11–14
was assessed by UV-Vis spectroscopy, mainly through the
detection of bathochromic shifs in the spectra of the tested
compounds in the presence of Cu2+ ions at pH 7.4. For this,
the UV-Vis spectra of each tested compound in PBS at pH 7.4
alone (red spectra, Figure 1) and in the presence of a solution
of 50 ꢁM CuSO (blue spectra, Figure 1) or 25 ꢁM CuSO
∗∗∗
< 0.01;
∗∗∗
93.93 0.20
Results are given as mean
SEM of three independent experiments
performed in triplicate; n.a.: not active; n.d.: not determined. Ascorbic acid
∗
∗
∗∗
ꢂ
ꢂ
(positive control): IC : 20.40 0.000213 ꢁM .
< 0.05;
50
∗∗∗
ꢂ
< 0.001.
Table 2: Iron chelating activity of tested compounds.
Compounds
% of chelation at 100 ꢁM
IC (ꢁM)
50
∗
1
2
3
6
7
8
9
11
12
13
14
12.19 2.55
n.d.
n.a.
n.d.
65.94 1.57
n.d.
∗
∗
4
4
88.40 0.56
(green spectra, Figure 1) were recorded. In addition, the UV-
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
Vis spectrum of each compound in the presence of 25 ꢁM
n.d.
CuSO afer addition of 25 ꢁM EDTA (pink spectra, Figure 1)
4
n.d.
was also registered.
n.d.
n.d.
n.d.
n.d.
e changes observed in the spectrum of xanthone
derivative 1 afer the addition of Cu2+ (blue and green
spectra, Figure 1(a)) in comparison with the spectrum in the
absence of this metal ion (red spectrum, Figure 1(a)) and the
regeneration of the original spectrum (pink spectrum, Fig-
ure 1(a)) afer the addition of EDTA suggest that 1 has
Cu2+ chelating effect. Nevertheless, no chelating activity was
observed for the 1 methylated derivative (2) since all spectra
are overlapped (Figure 1(b)).
∗
18.24 3.46
∗∗
∗∗
49.90 0.23
100 0.21
Results are given as mean
SEM of three independent experiments
performed in triplicate; n.a.: not active; n.d.: not determined. EDTA (positive
∗∗
∗
∗∗
ꢂ
ꢂ
control): IC : 30.66 0.17 ꢁM
.
< 0.05;
< 0.001.
50
For xanthone 2 Mannich base derivative (3) some chelat-
ing activity was observed. In fact, interactions of 3 with
Cu2+ ions at 25 ꢁM (green spectrum, Figure 1(c)) and 50 ꢁM
(blue spectrum, Figure 1(c)) produced bathochromic shif
in bands at 250 nm and 370 nm. e comparison of results
obtained for 2 and 3 indicates that the aminomethylation at
C-2 of xanthone nucleus potentiates its chelating activity. For
derivatives 6–9, 11, and 12 no chelating activity was detected
since the spectrum of each compound in the presence of
copper was overlapped with the spectrum of the compound
alone (data not shown).
production of reactive oxygen species (ROS) by Fenton
reaction, which contribute to the oxidative stress commonly
found in AD [58]. e ability of compounds 1–3, 6–9, and
11–14 to chelate ferrous ions (Fe2+) was evaluated by the
ferrozine-based colorimetric assay. Only compounds 1, 3, 13,
and 14 had ferrous ion chelating activity at 100 ꢁM (Table 2).
Among the tested xanthones, compound 3 was the most
potent, showing an IC value of 65.94 1.57 ꢁM. e com-
50
parison of the ferrous ion chelating effects of tested xanthones
suggests that not only the presence but also the position
of the amino side chains in the xanthone scaffold may
influence this effect. In fact, although the presence of amino
groups at position C-2 in derivative 3 is associated with a
chelating effect, the presence of this group at C-1 (derivatives
11 and 12) or C-4,5 (derivatives 6–9) is not associated with
any effect. Moreover, when comparing the results obtained
for xanthones 1–3 it is verified that not only the presence of
a hydroxyl group instead of a methoxy group at C-3 but
e chelation of Cu2+ by baicalein (13) and its Mannich
base derivative 14 is also evidenced by the chances in the UV-
Vis spectra. For baicalein (13) a hypochromic shif in band at
266 nm (blue and green spectra, Figure 1(d)) and a slight
bathochromic shif in band at 359 nm afer the addition of
Cu2+ were detected. Moreover, the addition of EDTA (pink
spectrum, Figure 1(d)) regenerated the original spectrum. For
baicalein Mannich base derivative 14 the chelation of this ion

Journal of Chemistry
9
0,400
0,350
0,300
0,250
0,200
0,150
0,100
0,050
3,000
2,500
2,000
1,500
1,000
0,500
0,000
0,000
200
300
400
500
600
700
800
200
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
(25ꢁM)
(25ꢁM)
(25ꢁM) + CuSO4 50ꢁM
(25ꢁM) + CuSO4 25ꢁM
(25ꢁM) + CuSO4 50ꢁM
(25ꢁM) + CuSO4 25ꢁM
(25ꢁM) + CuSO4 25ꢁM + EDTA 125 ꢁM
(25ꢁM) + CuSO4 25ꢁM + EDTA 125 ꢁM
(a)
(b)
0,700
0,600
0,500
0,400
0,300
0,200
0,100
0,000
0,450
0,400
0,350
0,300
0,250
0,200
0,150
0,100
0,050
0,000
200
300
400
500
600
700
800
200
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
(25ꢁM)
(25ꢁM)
(25ꢁM) + CuSO4 50ꢁM
(25ꢁM) + CuSO4 25ꢁM
(25ꢁM) + CuSO4 50ꢁM
(25ꢁM) + CuSO4 25ꢁM
(25ꢁM) + CuSO4 25ꢁM + EDTA 125 ꢁM
(25ꢁM) + CuSO4 25ꢁM + EDTA 125 ꢁM
(c)
(d)
0,600
0,500
0,400
0,300
0,200
0,100
0,000
200
300
400
500
600
700
800
Wavelength (nm)
(25ꢁM)
(25ꢁM) + CuSO4 50ꢁM
(25ꢁM) + CuSO4 25ꢁM
(25ꢁM) + CuSO4 25ꢁM + EDTA 125 ꢁM
(e)
Figure 1: UV-Vis spectra of 25 ꢁM of compounds 1 (a), 2 (b), 3 (c), 13 (d), and 14 (e) in PBS at pH 7.4 (red spectra), afer the addition of 25 ꢁM
CuSO (green spectra), 50 ꢁM CuSO (blue spectra), and 25 ꢁM of CuSO plus 125 ꢁM of EDTA (pink spectra).
4
4
4

10
Journal of Chemistry
Table 3: AChE inhibitory activity of tested compounds.
Table 4: Docking scores of known AChE inhibitors and compounds
2, 3, 6–9, and 14.
Compounds
% of inhibition at 100 ꢁM
IC (ꢁM)
50
Compound
Docking scores (kcal⋅mol−1
)
1
2
3
6
7
8
9
11
12
13
14
n.a.
n.d.
n.d.
∗
Trichlorfon
Echothiophate
Isoflurophate
Pyridostigmine
Neostigmine
Parathion
Rivastigmine
Tacrine
Ladostigil
Physostigmine
Huperzine A
Ungeremine
Galantamine
Donepezil
2
3
6
7
8
9
−5.7
−5.9
−5.9
−6.3
−7. 5
−7. 6
−7. 9
−8.8
−9.1
−9.1
−9.7
−10
34.04 3.12
∗∗
∗∗
49.08 2.78
100.00 2.76
∗∗
29.28 5.79
n.d.
∗
17.34 2.45
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
∗
21.50 4.02
∗∗
29.74 3.70
n.a.
n.a.
n.a.
∗∗
∗∗
69.34 0.72
81.99 1.16
Results are given as mean
performed in triplicate; n.a.: not active; n.d.: not determined. Galantamine
SEM of three independent experiments
∗∗
∗
∗∗
ꢂ
ꢂ
(positive control): IC : 6.59 0.15 ꢁM
.
< 0.05;
< 0.01.
50
−10.2
−12.1
−9.1
−9.4
−7. 8
−5.7
induced a bathochromic shif in band at 268 nm for both Cu2+
concentrations (50 ꢁM and 25 ꢁM, blue and green spectra,
Figure 1(e)) and the original spectrum was recovered afer the
addition of EDTA (pink spectrum, Figure 1(e)).
−7. 6
−7. 9
−10
3.2.4. AChE Inhibitory Activity. e AChE inhibitory activity
of compounds 1–3, 6–9, and 11–14 was screened by the
Ellman spectrophotometric assay with some modifications
(Table 3) [46].
14
with compounds already described in the literature as
AChE inhibitors [61], being these used as positive controls.
For controls, docking scores values ranging from −5.7 to
−12.1 kcal⋅mol−1 were obtained (Table 4). e most stable
complex (14: AChE) originated a free binding energy within
that range (−10 kcal⋅mol−1) in accordance with in vitro studies
(Table 3). Compounds 2 and 3 are also predicted as forming
stable complexes with AChE, with docking scores of −9.1 and
−9.4 kcal⋅mol−1, respectively. Amines 6, 7, and 9 presented
docking scores of −7. 8, −7. 6, and −7. 9 kcal⋅mol−1, respectively,
whereas less active amine 8 presented a docking score of
−5.7 kcal⋅mol−1 (Table 4).
Although no effect was observed for 1,3,8-trihydroxy-
xanthone (1) and xanthones 11 and 12 with an amino group
at C-1, xanthone derivatives possessing amino groups at C-
4,5 (compounds 6–9) or C-2 (compound 3) revealed AChE
inhibitory activity at 100 ꢁM, being derivative 3 the most
potent. Overall, these results indicate that the introduction
of an amino side chain at C-2 or 4,5 seems to favor the AChE
inhibitory activity relatively to position 1.
Regarding the tested flavones, in opposition to baicalein
(13) which did not possess AChE inhibitory activity, its
derivative 14 was revealed to be a moderate AChE inhibitor,
with an IC value of 81.99 1.16 ꢁM. As for xanthone deriva-
50
tives 1–3, the introduction of 8-(dimethylamino)methyl
group on the flavone skeleton is also associated with the
increase of AChE inhibition.
In order to further understand the binding mode of the
most potent inhibitors (2, 3, and 14) to AChE, a careful
inspection of the most stable docking pose of these small
molecules was performed (Figure 2). AChE is a serine
hydrolase possessing a narrow, long, and hydrophobic cavity
composed by two subsites: the esteratic and anionic subsites
[61]. e esteratic subsite, where AChE is hydrolyzed to
acetate and choline, contains the catalytic triad of three amino
acids: Ser203, His447, and Glu334, the lining of which con-
tains mostly aromatic residues that form a narrow entrance
to the catalytic Ser203 [62]. e activation of Ser203 allows
the acylation between hydroxyl group of that residue and
AChE oxygen. In the anionic site, the indole side chain of the
conserved residue Trp86 makes a cation-ꢅ interaction with
the quaternary amino group of AChE. A peripheral site is
composed of several aromatic residues, lining a hydrophobic
region that traps AChE and transfers it to the deep catalytic
site [63].
In summary, concerning the results obtained for antiox-
idant activity (DPPH radical scavenging and ferrous and
copper ions chelating activities) and the AChE inhibitory
property for all studied xanthones and flavones, it can be
seen that some of them showed both activities. Particularly,
Mannich base analogues 3 and 14 exhibited interesting AChE
inhibitory effect and antioxidant activity through different
ways. While aminoxanthone 3 exerted its antioxidant activity
by chelating ferrous and copper ions, aminoflavone 14 was an
antioxidant agent through radical scavenging and ferrous and
copper ions chelating effects.
3.3. Docking Studies. In this work, compounds 2, 3, 6–9,
and 14 have been discovered as AChE inhibitors. ere-
fore, docking studies were performed for this series along

Journal of Chemistry
11
His
447
Glu-202
Glu
202
Phe
338
Tyr
337
Phe-338
O
Tyr
133
HO
HO
Gly
120
O
HO
Tyr-133
Gly
126
Gly
121
Trp
N+
86
Asp
74
Ser-125
Tyr
341
Ser
125
Tyr
124
Asn
87
(a)
(b)
(c)
Figure 2: (a) AChE (surface) and docked 14 (blue sticks). (b) AChE active site (surface) bound to 14 (blue sticks). Residues involved in
hydrogen interactions (yellow broken line) and ꢅ stacking interactions (yellow double arrow) are displayed using a stick model. AChE
carbon, oxygen, nitrogen, and hydrogen are represented in green, red, blue, and grey, respectively. (c) 2D depiction of the 14 docked into
the binding site of AChE highlighting the protein residues that form the main interactions with the different structural units of the inhibitor.
Hydrogen-bonding interactions are represented with yellow broken lines. ꢅ stacking interactions are represented with a yellow double arrow.
Receptor residues that are close to 14, but whose interactions with the ligand are weak or diffuse, such as collective hydrophobic or electrostatic
interactions, are also represented (all the ones that have no indication for hydrogen-bonding or ꢅ stacking). Hydrophobic residues are colored
with a green interior, polar residues are colored in light purple, basic residues are annotated by a blue ring, and acidic residues are annotated
with a red ring. Solvent accessible surface area of 14 is plotted directly onto the atoms in the form of a blue smudge.
e interactions between inhibitors such as donepezil,
galantamine, huperzine A, and the enzyme, as observed from
their crystallographic structures (Figure 3), are characterized
by hydrogen interactions and ꢅ-ꢅ stacking and cation-ꢅ
interactions involving aromatic residues of AChE [47]. Most
ligands are located at the bottom of the binding groove that
forms a hydrophobic pocket base, although larger ligands as
donepezil extend to the periphery of the pocket.
docking algorithm used, it has steric complementarity with
a small lateral groove, establishing several van der Waals,
hydrophobic, and permanent dipole-induced dipole interac-
tions with residues such as Asp74, Trp86, Asn87, Tyr124, and
Ser125.
Compound 3 establishes one polar interaction with
residue Tyr124 and one cation-ꢅ interaction with Trp86,
whereas compound 2 establishes polar interactions with
Tyr-337 and His-447 and stacking interactions with Trp86
(Figure 4).
e narrower gorge in AChE results in a conformation
where the phenyl ring of 14 packs against the aromatic
hydrophobic portions of the side chain of Phe338. Packing
in this region is quite tight, and only the smallest substituents
might be accommodated. In contrast, the chromenone ring
system of the flavone molecule faces a larger, negatively
charged, and hydrophilic cavity, which includes hydroxyl
and carbonyl groups of Gly120, Ser125, Tyr133, and Glu202.
Upon analysis of the interaction of 14 in AChE binding
site domains, it is observed that the residues Gly120, Ser125,
Tyr133, and Glu202 participate in H-interactions. ese
residues are present in the buried region of the groove.
ꢅ stacking interactions are established between the phenyl
ring of the 14 and Phe338. Although 14 has a protonable
amine group, and cation-ꢅ interactions between protonated
nitrogen and AChE aromatic residues are described as being
important for the activity of the highly potent inhibitors,
such as donepezil (Figures 3(a) and 3(b)), this interaction is
not predicted to occur between 14 and AChE binding site
(Figures 2(b) and 2(c)). e binding affinity of 14 allows
not only the establishment of several polar interactions, but
also stacking interactions with Phe338 favored by the torsion
angle between the chromenone and the benzene ring (39.41∘).
Although 8-(dimethylamino)methyl group of compound 14
is not predicted to be evolved in polar interactions by the
In conclusion, molecular modifications on the flavone
scaffold placing the amine group on different positions
on A ring will be further explored in order to facilitate
a cation-ꢅ interaction. Moreover, molecular modifications
that would allow the establishment of hydrogen interactions
with the catalytic residue Ser203 (described for galantamine;
Figures 3(c) and 3(d)) and elongation of the molecule that
would favor the establishment of additional ꢅ-ꢅ or cation-ꢅ
interactions with residues such as Trp86, Trp286, or Tyr337
(described for donepezil and huperzine A; Figures 3(a), 3(b),
3(e), and 3(f)) are foreseen as being important for the AChE
inhibitory ability of flavones.
4. Conclusions
AD is the most prevalent form of dementia and it is known
that the malfunctions of different, but interconnected, bio-
chemical complex pathways are related to this pathogenesis.
Although inhibition of AChE is one of the most accepted
therapy strategies for AD, the AChE inhibitors that have
been approved for commercial use show lack of selectivity
for AChE and AD patients suffer from side-effects. erefore,

12
Journal of Chemistry
Tyr
72
Trp
286
His
447
Trp
86
Tyr
124
O
Gly
448
+
N
O
Glu
202
O
Ser
293
Tyr
341
Tyr
337
Phe
295
Phe
338
(a)
(b)
Asp
74
Gly
122
Tyr
337
TYr
124
His
447
N+
Trp
86
Phe
338
Phe
297
O
O
O
Phe
295
Gly
120
Ser
203
Glu
202
Gly
121
(c)
(d)
Trp
86
His
447
Tyr
337
Gly
126
Tyr
119
Glu
202
N+
Tyr
133
Ser
203
O
N
Ser
125
Gly
120
Gly
121
Tyr
124
Gly
122
(e)
(f)
Figure 3: AChE active site (surface) bound to crystallographic donepezil (pdb ID: 4EY7) (a), galantamine (pdb ID: 4EY6) (c), and huperzine
A (pdb ID: 4EY5) (e) (blue sticks). Residues involved in hydrogen interactions (yellow broken line), ꢅ stacking interactions (yellow double
arrow), and ꢅ-cation interactions (orange double arrow) are displayed using a stick model. AChE carbon, oxygen, nitrogen, and hydrogen are
represented in green, red, blue, and grey, respectively. 2D depiction view of crystallographic donepezil (b), galantamine (d), and huperzine A
(f) in AChE active site. Hydrogen and ꢅ stacking are represented with the same color scheme. Receptor residues that are close to the ligand,
but whose interactions with the ligand are weak or diffuse, such as collective hydrophobic or electrostatic interactions, are also represented
(all the ones that have no indication for hydrogen-bonding). Solvent accessible surface area of the ligand is plotted directly onto the atoms in
the form of a blue smudge. Solvent accessible surface area for the receptor residues is plotted as a blue halo.
this multifactorial disease requires new therapeutic strategies.
Considering that concept, the present work focused on
obtaining new AChE inhibitors with antioxidant activity
based on the xanthonic and flavonic scaffolds. From this
study, the Mannich base derivatives 3 and 14 emerged as dual
agents with AChE inhibition and antioxidant activity. e
synthesis of salt form of these amines to obtain bioactive
water-soluble compounds will allow further investigating
their potential as dual AChE inhibitors and antioxidants [64].
Conflicts of Interest
e authors declare that there are no conflicts of interest
regarding the publication of this paper.
Authors’ Contributions
Ineˆs Cruz and Ploenthip Puthongking performed the syn-
thesis, purification, and the evaluation of biological activity

Journal of Chemistry
13
Ser
125
Tyr
337
Tyr
124
HIS-447
TYR-337
Gly
121
Asp
74
O
O
OH
TRP-86
Tyr
133
Phe
338
OH
OH
Gly
120
Trp
86
Asp
74
Tyr
124
Tyr
341
(a)
(b)
His
447
Asp
74
Phe
295
Val
294
Tyr
337
O
O
O
Phe
338
N+
OH
OH
Trp
286
Tyr
341
Tyr
124
Trp
86
(c)
Figure 4: AChE active site (surface) bound to 2 (pink sticks) and 3 (purple sticks) (pdb ID: 4EY7) (a); residues involved in hydrogen
interactions (yellow broken line), ꢅ stacking interactions (yellow double arrow), and ꢅ-cation interactions (orange double arrow) are displayed
using a stick model. AChE carbon, oxygen, nitrogen, and hydrogen are represented in green, red, blue, and grey, respectively. 2D depiction
view of 2 (b) and 3 (c) docked into AChE active site. Hydrogen, ꢅ stacking, and ꢅ-cation are represented with the same color scheme. Receptor
residues that are close to the ligand, but whose interactions with the ligand are weak or diffuse, such as collective hydrophobic or electrostatic
interactions, are also represented (all the ones that have no indication for hydrogen-bonding). Solvent accessible surface area of the ligand is
plotted directly onto the atoms in the form of a blue smudge. Solvent accessible surface area for the receptor residues is plotted as a blue halo.
(both contributed equally to this work); Sara Cravo con-
tributed to the structure elucidation of compounds; Andreia
Palmeira performed the docking studies; Honorina Cidade
and Madalena Pinto conceived and designed the work; Hon-
orina Cidade, Madalena Pinto, and Em´ılia Sousa contributed
to acquisition, analysis, and interpretation of data and wrote
the paper. All authors read, critically revised, and approved
the final manuscript.
Operacional Factores de Competitividade (POFC) pro-
gramme, under the Strategic Funding UID/Multi/04423/
2013, the project PTDC/MAR-BIO/4694/2014 (Reference
POCI-01-0145-FEDER-016790; Project 3599, Promover a P
roduc¸a˜o Cient´ıfica e Desenvolvimento Tecnolo´gico e a
Constituic¸a˜o de Redes Tema´ticas (3599-PPCDT)), PTDC/
DTPFTO/1981/2014 (POCl-01-0145-FEDER-016581), and
PTDC/AAGTEC/0739/2014 (POCI-01-0145-FEDER-016793)
in the framework of the programme PT2020 as well as by the
project INNOVMAR-Innovation and Sustainability in the
Management and Exploitation of Marine Resources (Ref-
erence NORTE-01-0145-FEDER-000035, within Research
Line NOVELMAR), supported by North Portugal Regional
Operational Programme (NORTE 2020), under the PORTU-
GAL 2020 Partnership Agreement, through the European
Regional Development Fund (ERDF). e authors also thank
Acknowledgments
is work was partially supported through national funds
provided by FCT/MCTES-Foundation for Science and Tech-
nology from the Minister of Science, Technology and Higher
Education (PIDDAC) and European Regional Develop-
ment Fund (ERDF) through the COMPETE-Programa

14
Journal of Chemistry
for the grant of Erasmus Mundus Action 2, Lotus Unlimited
project of P. Puthongking. is paper is partially based on the
Master thesis of Ineˆs Cruz.
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