Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers

Article abstract of DOI:10.1016/j.bmc.2015.12.024

In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and then tested for inhibitory potency against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the synthesized chalcones to scavenge a stable radical was determined. Structure-activity relationship analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-levels in tissue, we determined the chalcones' effects to rescue neurons subjected to ROS-induced stress created by the addition of β-amyloid peptide. Best protection was provided by chalcones that combined good inhibitory potency with high radical scavenging ability in a single molecule, an observation that points to a potential therapeutic value of this compound class.

Full text of DOI:10.1016/j.bmc.2015.12.024

Accepted Manuscript
Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and
radical scavengers
Emily Hofmann, Jonathan Webster, Thuy Do, Reid Kline, Lindsey Snider,
Quintin Hauser, Grace Higginbottom, Austin Campbell, Lili Ma, Stefan Paula
PII:
S0968-0896(15)30190-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.12.024
BMC 12717
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 October 2015
6 December 2015
14 December 2015
Please cite this article as: Hofmann, E., Webster, J., Do, T., Kline, R., Snider, L., Hauser, Q., Higginbottom, G.,
Campbell, A., Ma, L., Paula, S., Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and
radical scavengers, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.12.024
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Hydroxylated chalcones with dual properties:
xanthine oxidase inhibitors and radical scavengers
Emily Hofmanna, Jonathan Webstera, Thuy Doa, Reid Klinea, Lindsey Snidera, Quintin Hausera,
Grace Higginbottomb, Austin Campbellb, Lili Maa, and Stefan Paulaa,b,c,
*
a Department of Chemistry
Natural Sciences Center
Northern Kentucky University
Highland Heights, KY 41099-1905, U.S.A.
b Department of Chemistry
Purdue University
560 Oval Drive
West Lafayette, Indiana 47907-2084, U.S.A.
c Department of Biochemistry
Purdue University
175 South University Street,
West Lafayette, Indiana 47907-2063, U.S.A.
*corresponding author
Department of Chemistry; Department of Biochemistry
Purdue University
560 Oval Drive
West Lafayette, Indiana 47907-2084, U.S.A.
Tel.: 765-494-4378, Fax: 765-494-0239
email: paulas@purdue.edu
1

Abstract
In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme
xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives
were synthesized by Claisen-Schmidt condensation reactions and then tested for inhibitory potency
against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the
synthesized chalcones to scavenge a stable radical was determined. Structure-activity relationship
analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a
minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two
neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed
previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-
levels in tissue, we determined the chalcones’ effects to rescue neurons subjected to ROS-induced stress
created by the addition of β-amyloid peptide. Best protection was provided by chalcones that combined
good inhibitory potency with high radical scavenging ability in a single molecule, an observation that
points to a potential therapeutic value of this compound class.
keywords: xanthine oxidase, chalcones, enzyme inhibition, radical scavenging, anti-oxidants,
reperfusion injuries, caffeic acid, reactive oxygen species, cell viability
2

Highlights
• synthesized 20 chalcones by Claisen-Schmidt condensation reactions
• determined inhibitory potencies of chalcones against activity of xanthine oxidase and established
structure-activity relationships
• performed molecular docking of chalcones into the crystal structure of xanthine oxidase and
identified critical enzyme/inhibitor interactions
• measured abilities of chalcones to scavenge a stable radical and established structure-activity
relationships
• evaluated the abilities of chalcones to increase viability of neurons exposed to β-amyloid peptide
3

1.
Introduction
Chalcones are natural products with a broad range of bioactivities that are widely found in the plant
kingdom [1, 2]. Structurally, they consist of two aryl groups (A- and B-rings) connected by an α,β-
unsaturated ketone moiety that typically assumes the thermodynamically more stable E configuration.
Whereas the aryl groups can carry a variety of substituents, hydroxyl, methoxy, and alkenyl groups are
by far the most commonly encountered ones in nature. Because of their structural simplicity and the
associated ease of synthesis, chalcones continue to enjoy considerable attention from medicinal chemists
exploring new molecular scaffolds for the design of novel therapeutics [3-5]. Amongst the numerous
bioactivities of chalcones are anticancer and antiviral activities, but they also are known to possess
radical scavenging properties and inhibitory potency against the enzyme xanthine oxidase (XO1). The
latter two properties make chalcones interesting candidates for the development of novel agents for the
treatment of hyperuricemia or the suppression of oxidatively generated stress in tissue.
O
'
2
2
1
'
3
4
3
'
1
A
B
'
4
The chalcone scaffold.
For decades, XO inhibitors have been used for therapy of hyperuricemia, a condition associated
with elevated levels of uric acid in the blood. Hyperuricemia has been linked to cardiovascular and
chronic kidney disease and is also known to cause gout [6, 7], the result of deposition of uric acid
crystals in joints that trigger inflammatory arthritis [8]. Therapeutically used XO inhibitors suppress the
1
Abbreviations: Aβ: β-amyloid peptide; CAPE: caffeic acid phenethylester; DMF: dimethylformamide; DPPH:
2,2-diphenyl-1-picrylhydrazyl; EGTA: ethylene glycol tetraacetic acid; HRMS: high resolution mass
spectrometry; MOMO: methoxymethoxy; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NADPH: Nicotinamide adenine dinucleotide; PBS: phosphate-buffered saline; ROS: reactive oxygen species;
XO: xanthine oxidase;
4

production of uric acid, allowing for renal excretion of the uric acid precursors xanthine and
hypoxanthine prior to the formation of uric acid. Allopurinol, a purine analog and prototype XO
inhibitor which has been in use since 1966, is efficacious, but its relatively low potency (IC50: 0.2 – 50
µM; [6]) requires dosages that can cause undesirable side effects, ranging from mild gastrointestinal
upset to more severe hypersensitivity reactions and renal toxicity [9-12]. Even though a number of
promising alternative treatments of hyperuricemia such as recombinant uricase therapy, the use of
interleukin-1 inhibitors, or the targeting of renal urate transporters are currently explored [13-15], XO
inhibitors still remain first-line therapy. As the approval of the high-potency (subnanomolar range; [16]),
non-purine XO inhibitor febuxostat in 2009 shows [17], the development of new XO inhibitors based on
novel structural scaffolds, including that of chalcones, continues to be an active field of current research
[18, 19].
In addition to their traditional use for the treatment of hyperuricemia, XO inhibitors have been
proposed to be useful for the suppression of oxidatively generated stress in tissue, a condition caused by
an imbalance between the production and removal rates of reactive oxygen species (ROS) [20-22]. In
general, the term ROS denotes oxygen radicals, such as the superoxide anion radical, the hydroxyl
radical, the peroxyl radical, or the hydroperoxyl radical, but it also applies to non-radical species that
can convert into radicals, including hydrogen peroxide, hypochlorous acid, or ozone [23]. Elevated ROS
levels inside cells are toxic and cause damage by breaking down biopolymers. Oxidatively generated
stress has been implicated in a number of diseases, among them cancer, neuropathy, inflammatory
diseases, and reperfusion injuries [24-27]. The latter can occur after surgery or ischemic events, such as
heart attacks and strokes, once the blood circulation to oxygen-deprived tissue has been restored. The
causes of reperfusion injuries are multi-facetted and factors like inflammation or the production of ROS
are believed to be involved [28-31]. At present, no approved pharmacological treatment of the damaging
5

effects of ROS is available, but the development of compounds with anti-oxidative properties has been
proposed to overcome this shortcoming [20-22]. In order to rectify the imbalance between ROS
production and removal rates, a suitable compound should prevent the generation of ROS by inhibiting
XO, one of the well-known contributors to ROS production that generates the long-lived ROS species
hydrogen peroxide and the superoxide radical anion. The effectiveness and versatility of such a XO
inhibitor would be substantially enhanced if it were also able to scavenge some of the more stable ROS
(from sources other than XO) before these convert into highly reactive and thus more damaging species,
such as the hydroxyl radical. Although these two properties have been investigated separately for several
compound classes, only a limited number of studies have characterized both properties for the same
molecule.
Certain chalcones are known to inhibit XO and – unlike most other XO inhibitors including
allopurinol and febuxostat – to act as radical scavengers. Even though these two properties have been
appreciated for quite some time, no systematic efforts have been undertaken to date to exploit them for
the development of medicinally useful agents for the reduction of ROS levels in tissue. For instance, no
comprehensive structure-activity relationships (SAR) for chalcone-mediated XO inhibition have been
established since most published studies focused in detail on the in vitro and/or in vivo properties of
single molecules [32-36]. In contrast, more information is available for the radical scavenging ability of
chalcones, but without connecting this information with inhibitory potency, the goal of obtaining
molecules with dual properties remains elusive. As proof-of-principle, such dual property agents have
been described for several non-chalcone molecular scaffolds, among them coumarins or analogs of
caffeic acid [20-22, 37]. Finally, the potential medicinal value of one of these compounds, caffeic acid
phenethyl ester (CAPE), has been demonstrated in animal studies that showed protective effects against
ischemia-induced reperfusion injuries in brain and muscle tissues of rodents [38-40].
6

Chalcones can be synthesized in a relatively straightforward manner, often by acid- or base-
catalyzed Claisen-Schmidt condensation of aryl aldehydes and ketones under homogeneous or
2−
heterogeneous conditions. Typical acid catalysts are AlCl3, RuCl3, silica-H2SO4, and TiO2/SO4 [41,
42] whereas frequently used base catalysts include KOH, NaOH, K2CO3, Ba(OH)2, and MgO [43].
Heterogeneous Claisen–Schmidt condensation reactions are usually assisted by microwave [44] or
ultrasound irradiation [45] to accelerate reaction rates or make the process environmentally more
friendly. Recent reports have identified additional reactions that provide access to the chalcone scaffold,
like the oxidation of alcohols [46], Meyer–Schuster rearrangement of propargylic alcohols [47], and
Palladium-catalyzed carbonylative Heck reactions [48]. However, due to its convenient procedure, broad
substrate scope, and high efficiency, the Claisen-Schmidt condensation method remains the most
attractive choice for the synthesis of chalcones.
As a first step towards designing XO inhibitors with dual properties, we here report on our
efforts to generate basic structure-activity relationship information on XO inhibition and radical
scavenging mediated by hydroxylated chalcones. We first synthesized a selection of 20 chalcones that
varied in number and position of hydroxyl groups at the two phenyl rings (Fig. 1). Next, we measured
their inhibitory potencies against XO activity and their ability to scavenge the stable radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH), a species that is routinely used as a model for long-lived radicals.
Experimental work was complemented by computational docking to elucidate and visualize crucial
interactions between chalcone inhibitors and their target, XO. Lastly, we conducted cell-based viability
assays to determine if the most active chalcones were able to help neurons cope with stress caused by
elevated ROS levels generated artificially by addition of β-amyloid peptide (Aβ). Neurons were chosen
for this assay because this cell-type would potentially suffer from reperfusion injuries after a stroke. The
information obtained in this study can serve as the foundation for future projects aimed at further
7

modifying and refining the properties of chalcones with the ultimate goal of developing this compound
class into medicinally useful agents.
Figure 1.
Chemical structures of chalcones synthesized and evaluated in bioassays.
8

2.
Experimental
2.1. Synthetic chemistry
With the exceptions of 2',5'-dihydroxyacetophenone which was obtained from Thermo Fisher Scientific
(Waltham, MA) and 3,4'-dihydroxyacetophenone which was purchased from Matrix Scientific
(Columbia, SC), all reagents and solvents were received from Sigma/Aldrich (St. Louis, MO) and used
without further purification unless otherwise noted. For thin-layer chromatography, pre-coated Whatman
silica gel F254 plates (GE Healthcare Bio-Sciences, Pittsburgh, PA) were used. Column
chromatography was performed using pre-packed RediSep Rf Silica columns on a CombiFlash Rf flash
chromatography system (Teledyne Isco, Lincoln, NE). NMR spectra were obtained using a Joel 500
MHz spectrometer (Peabody, PA). Chemical shifts were reported in parts per million (ppm) relative to
the tetramethylsilane signal at 0.00 ppm. Coupling constants (J) were reported in Hertz (Hz). The peak
patterns were indicated as follows: s, singlet; d, doublet; t, triplet; dt, doublet of triplet; dd, doublet of
doublet; m, multiplet; q, quartet. High resolution mass spectra (HRMS) were recorded on a Micromass
Q-TOF 2 (Waters, Milford, MA) or a Thermo Scientific LTQ-FT™ mass spectrometer (Waltham, MA)
operating in the electrospray mode.
General procedure for the synthesis of chalcones 1-5.
As outlined in Fig. 2, an aqueous solution of KOH (20% w/v, 2 mL) was added to a stirred solution of
the appropriate acetophenone (1 mmol, 1 equiv.) in ethanol (2 mL). The mixture was stirred at room
temperature for 10 min. After complete dissolution, aryl aldehyde (1 mmol, 1 equiv.) was added slowly
and the reaction mixture was then stirred at room temperature for 24–72 h. After completion, the
mixture was cooled to 0oC on an ice bath and acidified with HCl (10% v/v aqueous solution). In most
cases, the products precipitated out upon acidification with HCl. The crude product was filtered and
9

further purified by recrystallization from ethanol. In the cases in which no precipitate formed, the
mixture was extracted with ethyl acetate and washed with brine and water. After drying over Na2SO4,
the solvent was removed by rotary evaporation to give the crude product which was further purified by
either recrystallization or automated medium performance liquid chromatography, eluting with an ethyl
acetate/hexanes gradient (0 – 60%).
O
O
O
b
O
b
2
2
2'
A
5'
2'
A
5'
1
1
3'
4'
3
3'
3
1'
6'
1'
6'
CH3
a
H
a
a
OH
4
HO
4'
B
B
HO
+
OH
4
6
6
or
5
5
monohydroxylated chalcones 1-5
b
b
O
O
O
O
b
2
2'
1
3'
HO
4'
3
CH3
c
a
H
MOMO
1'
6'
a
OH
4
MOMO
OMOM
B
OMOM
A
6'
6
5
5'
MOMO- = CH3OCH3O-
di-, tri-, tetra-, penta-, hexahydroxylated chalcones 6 - 21
Figure 2.
Synthesis of chalcones. Reaction conditions: a) 20% KOH, EtOH, room temperature, 24 – 72 h;
b) CH3OCH2Cl, K2CO3, acetone, reflux, 4 h; c) 10% HCl, EtOH, reflux, 15 min.
General procedure for the synthesis of chalcones 6-20.
Methoxymethoxy (MOMO) protection
In a 100 mL oven-dried round bottom flask under argon protection, hydroxylaldehyde or acetophenone
(10 mmol, 1 equiv.) and 60 mL extra dry acetone were combined. After complete dissolution, the
solution was cooled in an ice bath for 10 min and then K2CO3 (100 mmol, 10 equiv.) was added. While
stirring, methoxymethyl chloride (50 mmol, 5 equiv.) was added dropwise. The mixture was first stirred
at 0oC for 30 min and then at under reflux conditions for 4 h. The mixture was cooled to room
temperature and salts were removed by suction filtration. The solvent was removed by rotary
10

evaporation to obtain the crude product which was further purified by automated medium performance
liquid chromatography eluting with an ethyl acetate/hexanes gradient (0 – 20%).
Claisen-Schmidt condensation
To a stirred solution of the appropriate MOMO-protected acetophenone (1mmol, 1 equiv) in ethanol (2
mL), an aqueous solution of KOH (20% w/v, 2 mL) was added. The mixture was stirred at room
temperature for 10 min. After complete dissolution, the appropriate MOMO-protected aryl aldehyde (1
equiv.) was added slowly and the reaction mixture was then stirred at room temperature for 24–72 h.
After completion, the reaction mixture was cooled to 0oC on an ice bath and acidified with HCl (10%
v/v aqueous solution). In most cases, the products precipitated out upon acidification with HCl. The
crude product was filtered and further purified by recrystallization from ethanol. In the cases in which
no precipitate formed, the mixture was extracted with ethyl acetate and washed with brine and water.
After drying over Na2SO4, the solvent was evaporated to give the crude product. The crude product was
further purified by either recrystallization or automated medium performance liquid chromatography
eluting with an ethyl acetate/hexanes gradient (0 – 60%).
MOMO deprotection
MOMO-protected chalcones (1 mmol) were added to ethanol (8 mL), followed by dropwise addition of
HCl (10% aqueous solution, 3.5 mL). The mixture was heated under reflux conditions for 15 min. After
cooling down to room temperature, the mixture was diluted with water (20 mL) and extracted with ethyl
acetate three times (10 mL each). After drying over MgSO4, filtration and evaporation of organic
solvents, the product was obtained in good purity (Fig. 2).
11

(E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one (1) [49]
Synthesized according to the aldol condensation general procedure described above. Rf = 0.70 (10%
1
EtOAc/hex). Yield: 26.7%. H NMR (CDCl3, 500 MHz, ppm): δ 7.93 (1H, d, J = 15.6 Hz), 7.94-7.92
(1H, m), 7.67 (1H, d, J = 16.1 Hz), 7.68-7.66 (2H, m), 7.50 (1H, t, J = 8.7 Hz), 7.45-7.43 (3H, m), 7.03
(1H, d, J = 8.7 Hz), 6.95 (1H, t, J = 7.4 Hz). 13C NMR (CDCl3, 125 MHz, ppm): δ 193.8, 163.7, 145.6,
136.5, 134.7, 131.0, 129.8, 129.1, 128.8, 120.3, 120.1, 118.9, 118.7. HRMS: Calculated for C15H12O2Na
[M+Na] 247.0735, found 247.0728.
(E)-1-(3-hydroxyphenyl)-3-phenylprop-2-en-1-one (2) [50]
Synthesized according to the aldol condensation general procedure described above. Yellow solid. Rf =
1
0.75 (10% EtOAc/hex). Yield: 40.7 %. H NMR (CDCl3, 500 MHz, ppm): δ 7.82 (1H, d, J = 15.6 Hz),
7.68 (1H, s), 7.62-7.61 (2H, m), 7.57 (1H, d, J = 7.8 Hz), 7.51 (1H, d, J = 15.6 Hz), 7.41-7.40 (3H, m),
13
7.37 (1H, t, J = 7.8 Hz), 7.14 (1H, d, J = 7.8 Hz). C NMR (CDCl3, 125 MHz, ppm): δ 190.9, 156.7,
145.7, 139.5, 134.8, 130.9, 130.0, 129.1, 128.7, 122.0, 121.0, 120.7, 115.4. HRMS: Calculated for
C15H12O2Na [M+Na] 247.0735, found 247.0735.
(E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (3) [51]
Synthesized according to the general aldol condensation procedure described above. Yellow solid.
1
Yield: 93.9%. H NMR (acetone-d6, 500 MHz, ppm): δ 9.29 (1H, broad S), 8.09 (2H, d, J = 8.2 Hz),
7.86 (1H, d, J = 16.0 Hz), 7.81 (2H, d, J = 7.8 Hz), 7.74 (1H, d, J = 15.6 Hz), 7.46−7.42 (3H, m), 6.97
(2H, d, J = 8.2 Hz). 13C NMR (acetone-d6, 125 MHz, ppm): δ 187.3, 162.0, 142.9, 135.5, 131.1, 130.4,
130.2, 129.0, 128.5, 122.1, 115.4. HRMS: Calculated for C15H12O2Na [M+Na] 247.0735, found
247.0733.
12

(E)-3-(2-hydroxyphenyl)-1-phenylprop-2-en-1-one (4) [52]
Synthesized according to the general aldol condensation procedure described above. Yellow solid.
1
Yield: 26.2%. H NMR (acetone-d6, 500 MHz, ppm): δ 8.18 (1H, d, J = 16.0 Hz), 8.12-8.09 (2H, m),
7.88 (1H, d, J = 16.0 Hz), 7.82 (1H, t, J = 7.3 Hz), 7.65-7.55 (3H, m), 7.28 (1H, d, J = 7.3 Hz), 7.01
13
(1H, t, J = 6.9 Hz), 6.92 (1H, d, J = 6.4 Hz). C NMR (acetone-d6, 125 MHz, ppm): δ 205.4, 157.1,
139.7, 138.7, 132.6, 131.8, 129.0, 128.7, 128.4, 122.2, 121.7, 120.1, 116.3. HRMS: Calculated for
C15H12O2Na [M+Na] 247.0735, found 247.0733.
(E)-3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one (5) [52]
Synthesized according to the aldol condensation general procedure described above. Yellow solid.
1
Yield: 76.2%. H NMR (acetone-d6, 500 MHz, ppm): δ 8.11 (2H, d, J = 8.2 Hz), 7.76-7.52 (7H, m),
13
6.92 (2H, d, J = 8.7 Hz). C NMR (acetone-d6, 125 MHz, ppm): δ 205.4, 160.1, 144.4, 138.7, 132.5,
130.7, 128.6, 128.3, 126.8, 118.9, 116.0. HRMS: Calculated for C15H12O2Na [M+Na] 247.0735, found
247.0737.
(E)-1-(2,4-dihydroxyphenyl)-3-phenylprop-2-en-1-one (6) [53]
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Yellow solid. Rf = 0.61 (30% EtOAc/Hex). Yield: 47.8%. H NMR
(acetone-d6, 500 MHz, ppm): δ 8.12 (1H, d, J = 8.7 Hz), 7.92-7.79 (4H, m), 7.43-7.41 (3H, m), 6.51
(1H, d, J = 8.7 Hz), 6.42 (1H, s). 13C NMR (acetone-d6, 125 MHz, ppm): δ 206.1, 166.5, 164.9, 143.9,
135.1, 132.8, 130.6, 129.0, 128.8, 120.9, 113.7, 108.1, 103.0. HRMS: Calculated for C15H12O3Na
[M+Na] 263.0684, found 263.0695.
13

(E)-3-(3,4-dihydroxyphenyl)-1-phenylprop-2-en-1-one (7)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Yellow solid. Yield: 47.8%. H NMR (acetone-d6, 500 MHz, ppm): δ 8.42
(1H, broad s), 8.10 (2H, d, J = 7.8 Hz), 7.70-7.53 (5H, m), 7.33 (1H, s), 7.20 (1H, d, J = 8.3 Hz), 6.90
13
(1H, d, J = 8.3 Hz), 3.06 (1H, broad s). C NMR (acetone-d6, 125 MHz, ppm): δ 205.5, 148.2, 145.6,
144.8, 138.7, 132.5, 128.6, 128.3, 127.5, 122.4, 119.1, 115.6, 115.0. HRMS: Calculated for C15H12O3Na
[M+Na] 263.0678, found 263.0678.
(E)-3-(3,4-dihydroxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (8)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Dark green solid. Rf = 0.36 (5% CH3OH/CH2Cl2). Yield: 71.4 %. H NMR
(acetone-d6, 500 MHz, ppm): δ 7.65 (1H, d, J = 15.6 Hz), 7.60 (1H, d, J = 7.3 Hz), 7.54 (1H, d, J = 15.6
Hz), 7.52 (1H, s), 7.36 (1H, t, J = 7.8 Hz), 7.32 (1H, s), 7.19 (1H, d, J = 7.8 Hz ), 7.08 (1H, d, J = 7.8
13
Hz), 6.90 (1H, d, J = 8.2 Hz). C NMR (CDCl3, 125 MHz, ppm): δ 189.0, 157.8, 148.2, 145.5, 144.6,
140.3, 129.8, 127.5, 122.4, 119.8, 119.7, 119.2, 115.6, 114.9, 114.8. HRMS: Calculated for C15H12O4Na
[M+Na] 279.0633, found 279.0642.
(E)-3-(3,4-dihydroxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (9)
Synthesized according to the MOMO protection, aldol condensation, and MOMO deprotection general
1
procedure described above. Dark green solid. Rf = 0.48 (30% EtOAc/Hex). Yield: 88.0 %. H NMR
(acetone-d6, 500 MHz, ppm): δ 9.20 (1H, s), 8.56 (1H, s) , 8.16 (1H, s), 8.06 (2H, d, J = 8.2 Hz), 7.62
13
(2H, s), 7.30 (1H, s), 7.17 (1H, d, J = 7.8 Hz), 6.95 (2H, d, J = 8.7 Hz), 6.88 (1H, d, J = 7.8 Hz). C
14

NMR (acetone-d6, 125 MHz, ppm): δ 187.2, 161.7, 147.9, 145.5, 143.6, 130.9, 127.7, 122.1, 119.0,
115.6, 115.3, 114.9. HRMS: Calculated for C15H12O4Na [M+Na] 279.0633, found 279.0641.
(E)-1-(2,4-dihydroxyphenyl)-3-(3-hydroxyphenyl)prop-2-en-1-one (10) [54]
Synthesized according to the MOMO protection, aldol condensation, and MOMO deprotection general
1
procedure described above. Orange solid. Rf = 0.50 (30% EtOAc/Hex). Yield: 59.5 %. H NMR
(acetone-d6, 500 MHz, ppm): δ 8.14 (1H, d, J = 8.7 Hz), 7.88-7.77 (2H, m), 7.31-7.26 (3H, m), 6.94
13
(1H, d, J = 7.8 Hz), 6.47 (1H, d, J = 8.7 Hz), 6.37 (1H, s). C NMR (acetone-d6, 125 MHz, ppm): δ
192.0, 166.9, 165.1, 157.9, 144.2, 136.5, 132.8, 130.1, 120.8, 120.3, 117.8, 115.3, 113.7, 108.1, 103.0.
HRMS: Calculated for C15H12O4Na [M+Na] 279.0633, found 279.0630.
(E)-1-(2,6-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one (11)
Synthesized according to the MOMO protection, aldol condensation, and MOMO deprotection general
procedure described above. Orange solid. Yield: 88.6 %. 1H NMR (acetone-d6, 500 MHz, ppm): δ 8.64
(1H, s), 8.30 (1H, s), 8.05 (1H, d, J = 15.6 Hz), 7.76 (1H, d, J = 15.6 Hz), 7.26 (1H, d, J = 8.5 Hz), 7.23
(1H, s), 7.11 (1H, d, J = 8.3 Hz), 6.90 (1H, d, J = 8.2 Hz), 6.45 (2H, d, J = 8.3 Hz). 13C NMR (acetone-
d6, 125 MHz, ppm): δ 194.5, 162.3, 148.4, 145.6, 144.1, 135.9, 127.7, 124.5, 122.6, 115.7, 114.8, 110.9,
107.8. HRMS: Calculated for C15H12O5Na [M+Na] 295.0582, found 295.0591.
(E)-1-(2,5-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one (12)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Brown solid. Yield: 80.5 %. H NMR (acetone-d6, 500 MHz, ppm): δ 7.80
(1H, d, J = 15.1 Hz), 7.67 (1H, d, J = 15.6 Hz), 7.58 (1H, s), 7.38 (1H, s), 7.24 (1H, d, J = 8.2 Hz), 7.11
(1H, d, J = 9.2 Hz), 6.92 (1H, d, J = 8.2 Hz), 6.83 (1H, d, J = 9.2 Hz). 13C NMR (acetone-d6, 125 MHz,
15

ppm): δ 156.9, 149.4, 148.8, 146.0, 145.7, 127.2, 124.7, 123.0, 120.0, 118.6, 117.4, 115.7, 115.3, 114.7.
HRMS: Calculated for C15H12O5Na [M+Na] 295.0582, found 295.0582.
(E)-1,3-bis(3,4-dihydroxyphenyl)prop-2-en-1-one (13) [53]
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Dark red solid. Yield: 34.0 %. H NMR (acetone-d6, 500 MHz, ppm): δ
7.64-7.61 (3H, m), 7.58 (1H, s), 7.30 (1H, s), 7.16 (1H, d, J = 8.2 Hz), 6.94 (1H, d, J = 8.7 Hz), 6.88
(1H, d, J = 8.3 Hz). 13C NMR (acetone-d6, 125 MHz, ppm): δ 150.1, 148.0, 145.5, 145.2, 143.6, 131.2,
127.7, 122.1, 122.1, 119.0, 115.6, 115.4, 114.9, 114.8. HRMS: Calculated for C15H12O5Na [M+Na]
295.0582, found 295.0576.
(E)-3-(3,4-dihydroxyphenyl)-1-(3,5-dihydroxyphenyl)prop-2-en-1-one (14)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Black solid. Yield: 84.0 %. H NMR (acetone-d6, 500 MHz, ppm): δ 7.62
(1H, d, J = 15.6 Hz), 7.43 (1H, d, J = 15.6 Hz), 7.30 (1H, s), 7.16 (1H, d, J = 7.8 Hz), 7.04 (2H, s), 6.98
(1H, d, J = 8.2 Hz), 6.59 (1H, s). 13C NMR (acetone-d6, 125 MHz, ppm): δ 189.1, 171.5, 170.3, 158.9,
148.3, 145.6, 144.6, 140.9, 127.4, 122.3, 119.3, 115.6, 114.8, 106.8, 106.8. HRMS: Calculated for
C15H12O5Na [M+Na] 295.0582, found 295.0578.
(E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one (15) [54]
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Red solid. Rf = 0.19 (5% CH3OH/CH2Cl2). Yield: 78.7 %. H NMR
(acetone-d6, 500 MHz, ppm): δ 8.11 (1H, d, J = 8.7 Hz), 7.76 (1H, d, J = 15.2 Hz), 7.69 (1H, d, J = 15.2
Hz), 7.35 (1H, s), 7.22 (1H, d, J = 8.3 Hz), 6.90 (1H, d, J = 8.2 Hz), 6.46 (1H, d, J = 8.8 Hz), 6.36 (1H,
16

s). 13C NMR (acetone-d6, 125 MHz, ppm): δ 192.0, 166.8, 164.8, 148.4, 145.6, 144.8, 132.5, 127.4,
122.7, 117.6, 115.6, 115.2, 113.7, 107.9, 103.0. HRMS: Calculated for C15H12O5Na [M+Na] 295.0582,
found 295.0587.
(E)-3-(2,3-dihydroxyphenyl)-1-(3,4-dihydroxyphenyl)prop-2-en-1-one (16)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Brown solid. Yield: 57.0 %. H NMR (acetone-d6, 500 MHz, ppm): δ 8.09
(1H, d, J = 16.1 Hz), 7.81 (1H, d, J = 15.6 Hz), 7.62 (1H, s), 7.61 (1H, d, J = 6.4 Hz), 7.28 (1H, d, J =
7.8 Hz), 6.95 (1H, d, J = 7.8 Hz), 6.92 (1H, d, J = 7.8 Hz), 6.74 (1H, t, J = 8.0 Hz). 13C NMR (acetone-
d6, 125 MHz, ppm): δ 187.6, 151.5, 149.9, 145.6, 145.1, 138.4, 131.3, 122.5, 122.1, 121.9, 119.7, 119.6,
116.4, 115.3, 114.8. HRMS: Calculated for C15H11O5 [M-H] 271.0601, found 271.0608.
(E)-3-(2,5-dihydroxyphenyl)-1-(3,4-dihydroxyphenyl)prop-2-en-1-one (17)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Black solid. Yield: 88.4 %. H NMR (acetone-d6, 500 MHz, ppm): δ 8.04
(1H, d, J = 15.6 Hz), 7.74 (1H, d, J = 16.1 Hz), 7.62 (1H, s) 7.60 (1H, d, J = 8.3 Hz), 7.21 (1H, s), 6.95
(1H, d, J = 8.2 Hz), 6.84 (1H, d, J = 8.7 Hz), 6.78 (1H, d, J = 8.7 Hz). 13C NMR (acetone-d6, 125 MHz,
ppm): δ 187.8, 150.5, 150.3, 150.0, 145.1, 138.6, 131.2, 122.7, 122.0, 121.5, 118.9, 117.0, 115.4, 115.0,
113.9. HRMS: Calculated for C15H11O5 [M-H] 271.0601, found 271.0614.
(E)-3-(3,4-dihydroxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one (18)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Red solid. Yield: 97.1 %. H NMR (acetone-d6, 500 MHz, ppm): δ 7.76
17

(1H, d, J = 15.2 Hz), 7.70 (1H, d, J = 8.7 Hz), 7.69 (1H, d, J = 15.6 Hz), 7.35 (1H, s), 7.23 (1H, d, J =
13
8.2 Hz), 6.90 (1H, d, J = 8.3 Hz), 6.50 (1H, d, J = 8.7 Hz). C NMR (acetone-d6, 125 MHz, ppm): δ
192.5, 153.0, 151.6, 148.3, 145.4, 144.7, 132.3, 127.4, 122.7, 122.4, 117.7, 115.6, 115.2, 114.0, 107.4.
HRMS: Calculated for C15H11O6 [M-H] 287.0550, found 287.0557.
(E)-1-(3,4-dihydroxyphenyl)-3-(2,3,4-trihydroxyphenyl)prop-2-en-1-one (19)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
procedure described above. Brown solid. Yield: 44.0 %. 1H NMR (D2O, 500 MHz, ppm): δ 8.46 (1H, d,
J = 8.2 Hz), 7.58 (1H, d, J = 8.7 Hz), 7.28-7.24 (m, 2H), 7.05 (1H, d, J = 8.7 Hz), 6.97 (1H, s), 6.46 (1H,
d, J = 8.2 Hz). 13C NMR (D2O, 125 MHz, ppm): δ 191.2, 169.0, 168.5, 159.2, 158.3, 143.6, 137.7,
125.3, 122.5, 121.0, 117.4, 115.4, 113.8, 111.8, 110.0. HRMS: Calculated for C15H11O6 [M-H]
287.0550, found 287.0558.
(E)-1,3-bis(2,3,4-trihydroxyphenyl)prop-2-en-1-one (20)
Synthesized according to the MOMO protection, aldol condensation, and general MOMO deprotection
1
procedure described above. Brown solid. Yield: 95.7 %. H NMR (acetone-d6, 500 MHz, ppm): δ 8.19
(1H, d, J = 15.6 Hz), 7.83 (1H, d, J = 15.6 Hz), 7.60 (1H, d, J = 9.2 Hz), 7.24 (1H, d, J = 8.7 Hz), 6.50
(2H, d, J = 9.2 Hz). 13C NMR (acetone-d6, 125 MHz, ppm): δ 192.9, 153.0, 151.3, 148.4, 147.3, 140.6,
132.5, 132.3, 122.0, 121.0, 117.4, 115.0, 114.1, 107.9, 107.3. HRMS: Calculated for C15H11O7 [M-H]
303.0499, found 303.0509.
18

2.2. Activity assays
Materials for bioassays
Bovine XO, xanthine, potassium phosphate, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were received from Sigma/Aldrich.
Dimethylformamide (DMF), and phosphate-buffered saline (PBS) were purchased from Fisher
Scientific. Murine Neuro-2A cells were received from Eton Bioscience (San Diego, CA) whereas
growth medium, serum, trypsin, and antibiotics were obtained from Atlanta Biologicals (Atlanta, GA).
Aβ 25-35 was from Abbiotec (San Diego, CA).
Determination of inhibitory potency against XO activity
The rate of XO-catalyzed conversion of xanthine to uric acid was determined spectroscopically by
measuring the concomitant absorbance change at 295 nm for five min [7]. XO (0.1 units/mg) was
suspended in buffer (20 mM phosphate, pH 7.5) and the reaction was triggered by adding the enzyme
buffer (final XO concentration: 39 µg/mL) to xanthine dissolved in the same buffer (final concentration:
46 µM; total volume: 200 µL), using 96-well plastic plates capable of transmitting UV light that were
placed in a microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA). Assays were
conducted in the absence and presence of potential inhibitors at 11 different concentrations. The exact
inhibitor concentrations used in the assay depended on the potency of the test compound and were
chosen so that the IC50 value was located approximately in the center of the concentration range.
Reaction rates were obtained by linear regression of the absorbance versus time traces and then fit to a
three-parameter logistic equation. Inhibitory potencies were expressed as IC50 values [55], the inhibitory
concentrations that reduced XO activity by 50%.
19

Measurement of DPPH scavenging activities
The abilities of compounds to scavenge the stable radical DPPH were conducted by mixing 50 µL of a
freshly prepared solution of DPPH in ethanol (0.2 mM) with 150 µL of the test compound in ethanol at
several concentrations ranging from 5 to 50 µM [56]. Samples were placed in a 96-well plate and
incubated for 30 min at room temperature in the dark. The disappearance of the DPPH absorbance at a
wavelength of 517 nm, an indicator of radical scavenging, was measured with a plate reader.
Assessment of cell viability in the presence of Aβ
The ability of chalcones to improve the viability of cells exposed to cytotoxic Aβ was assessed
according to an established protocol using Neuro-2A neuroblastoma cells [20, 21]. According to
previous work, exposure to self-aggregating β-amyloid peptide induces elevated levels of ROS via
activation of NADPH oxidase [57]. Cells were grown according to the supplier’s guidelines in
Dulbecco’s minimum essential medium complemented with 10% of fetal bovine serum and 100
units/mL penicillin-streptomycin at 37ºC in an atmosphere of 95% air and 5% carbon dioxide. For
seeding, cells were washed with PBS, treated with trypsin for no longer than one minute, and then
placed in 100 µL aliquots in a 96-well plate at a density of 2,000-4,000 cells/mL. After 6 hrs of
incubation, a 10 µL aliquot of 250 µM test compound, 15 µL of 833 µM Aβ, and 25 µL of MTT buffer
(5 mg/mL in PBS) were added to each well [58]. After six hrs of incubation, 100 µL of extraction buffer
(prepared by addition of 2.5% v/v of a mixture composed of four parts of acetic acid and one part of
20% w/v SDS prepared in a 50:50 mix of DMF and water, pH 4.7) was added. After six hrs on an
incubating shaker (25 rpm) in the dark at 37ºC, the absorbances of the samples were measured at 570 nm
with a microplate reader.
20

2.3. Computation of inhibitor binding poses by computational ligand docking
Inhibitor structures of 8, 9, 15, 16, and 20 were modeled in MOE (version 2013.08; Chemical
Computing Group, Montreal, Canada) and energy-minimized using the MMFF94s force field. All
minimization parameters were kept at their default settings, except for the dielectric constant, which was
set to a value of 4 to account for the relatively hydrophobic character of the protein interior. Docking of
chalcones was performed with the program GOLD (version 5.2; Cambridge Crystallographic Data
Centre, UK) [59, 60] using the X-ray crystal structure of the XO/xanthine complex (Protein Data Bank
entry 3EUB) [61]. The protein structure was prepared in GOLD for docking by adding hydrogen atoms
and deleting xanthine. All other non-protein entities such as prosthetic groups remained unchanged. The
scoring function selected for docking was ChemScore [62, 63] and the genetic algorithm of GOLD was
executed at the default settings, performing 30 independent and identical repeats. The docking site was
defined as the area occupied by the deleted xanthine ligand plus a 6 Å wide zone in its immediate
proximity, yielding a docking area that was large enough to accommodate each of the docked chalcones.
21

3.
Results and Discussion
3.1. Synthesis of chalcones
Simple mono-substituted chalcones were successfully prepared through the Claisen-Schmidt
condensation of the corresponding acetophenones and benzaldehydes (Compounds 1-5). For the
synthesis of di-, tri-, tetra-, penta-, and hexasubstituted chalcones, a three-step route was utilized (Figure
2). More specifically, the hydroxyl groups in aldehydes or acetophenones were protected by MOMO
groups under basic reflux conditions [64]. Claisen-Schmidt condensation of these protected aldehydes
and acetophenones provided enones [65, 66]. In the case of mono-, di-, or tri- substituted chalcones, the
corresponding enone products mostly precipitated after acidic workup. These enones were then filtered
and further purified by recrystallization. In the cases of tetra-, penta-, and hexasubstituted chalcones, the
enone products did not precipitate and an acid/base extraction was utilized instead to obtain crude
products. Enone compounds were treated with 10% HCl to remove the MOMO protecting groups [67]
and yield the chalcone compounds (Scheme 1, Compounds 6-20). The chalcones with four or more
hydroxyl groups showed poor solubility in organic solvents. Their solubility in water was pH-dependent,
1
with poor solubility at low pH and high solubility at high pH. H-NMR spectra revealed that the
coupling constants of the two olefinic protons were around 16 Hz, indicating the E configuration of the
1
olefinic bond in these chalcones. All synthesized compounds were fully characterized by H NMR and
13C NMR spectroscopy and by HRMS.
3.2. Inhibition of XO activity by chalcones
Among the 20 tested chalcones (see Fig. 3 for a representative result), 15 displayed measurable
inhibitory potencies (Table 1 and Fig. 1), with IC50 values ranging from 1.2 (18) to 290 µM (5).
Inspection of the chemical structures of the active chalcones showed a general requirement for three or
22

more hydroxyl groups for good potency, with compounds 18 and 15 being the most potent inhibitors in
the test pool (IC50 values of 1.2 and 1.3 µM, respectively). As the comparison of the potencies of 7 and 9
and of 8 and 13 revealed, the presence of hydroxyl groups in position 4′ of the A-ring increased a
compound’s potency. A similar trend was observed for position 3′, where an additional hydroxyl group
rendered chalcones more active (9 versus 13 and 7 versus 8). Findings pertaining to position 2′ were
more ambiguous, as evident from a potency increase seen for 9 versus 15 and for 13 versus 18, but a
decrease observed for 8 versus 12.
Figure 3.
Representative XO activity inhibition assay for compounds 12(ꢀ) and 15(ꢁ). The lines represent
fits of a three-parameter logistic curve to the data points.
Structural variations in the B-ring were more limited, but an increase in potency was noted by
the introduction of a hydroxyl group in positions 3 (6 versus 10) and 4 (16 versus 19). In contrast, an
additional hydroxyl group in position 2 was detrimental to potency (13 versus 19 and 18 versus 20).
23

3.3. Computational prediction of chalcone binding to XO
Despite the availability of a relatively large number of high resolution X-ray crystal structures of bovine
XO in complex with various small molecules [16, 61, 68-70], no structural information exists for the
binding of chalcones, leaving the molecular details of interactions between this inhibitor class and XO
ambiguous. In the absence of crystallographic information, computational tools like ligand docking can
provide valuable insights into critical enzyme/inhibitor interactions [71]. Among numerous commercial
and academic docking routines, the program GOLD is known for its reliability and accuracy, which was
the main reason for employing it for the investigation of chalcone binding to XO [60, 72].
Docking was limited to a representative subset of high potency inhibitors (8, 9, 15, 16, and 20)
since their interactions with the enzyme were presumably strong and therefore most likely predicted
accurately by GOLD. For simplicity, initial docking focused on compounds 8 and 9 since their three
hydroxyl groups represented the minimum structural requirement for high potency inhibition. Docking
yielded consensus orientations, implying that the majority of independent repeats of the docking
protocol generated identical solutions (21/30 for 8 and 17/30 for 9). Control docking runs for 8 with a
larger binding site (8Å instead of 6Å) resulted in virtually the same results but required longer
computation times, which is why the smaller site was used for all subsequent runs. As shown in Fig. 4
(middle panel), the two hydroxyl groups on the B ring of 8 formed hydrogen bonds with the side chain
carboxyl groups of Glu802 and Glu1261 whereas the carbonyl oxygen was engaged with the hydroxyl
groups of Ser876. The docking results for 15, 16, and 20 were more diverse, but all contained predicted
poses that overlapped with those seen for 8 and 9, which were used for further analysis (Fig. 4, upper
panel). The XO side chains involved in binding of 15 and 16 were the same as for 8 and 9, whereas 20,
the only compound with six hydroxyl groups, was predicted to form additional, albeit weaker hydrogen
bonds to Thr1010 and Arg880. A second major driving force to inhibitor binding came from
24

hydrophobic interactions, which exceeded the energetic contributions of hydrogen bonding by a factor
of about three for all compounds docked. In comparison to the binding site entrance, the walls and the
bottom of the cavity are more hydrophobic (Fig. 4, lower panel), allowing for favorable hydrophobic
interactions of an inhibitor’s B-ring and the carbon-carbon double bond with nonpolar side chains
(Phe941, Phe1009, Phe1013, Leu648, Leu873, Val1011, Ala910, Ala1078, Ala1079, and Pro1076).
Contributions to the docking score arising from factors other than hydrogen bonding and hydrophobic
interactions, such as unfavorable energy terms due to steric clashes and the loss of conformational
freedom upon ligand binding, were minor and could therefore be safely omitted.
25

Figure 4.
Docking-predicted poses of chalcones in the XO binding site. Upper panel: Consensus binding
poses of chalcones 8, 9, 15, 16, and 20. Middle panel: Predicted hydrogen bond interactions (thin grey lines)
between 8 and the XO binding site. Lower panel: Front and side views of 8 in the binding site with a hydrophobic
index map superimposed on the binding site of XO (colors in decreasing hydrophobicity: brown – green – blue).
26

3.4. DPPH radical scavenging by chalcones
Among the twenty tested chalcones, thirteen were capable of scavenging the DPPH radical to a
noticeable degree. The ability to reduce the stable DPPH radical is a frequently considered measure for a
compound’s potential to scavenge long-lived radicals. In a typical experiment, DPPH was incubated
with the test compound at several concentrations up to 100 µM for 30 min (Fig. 5). The DPPH
absorbance was then and converted into radical scavenging activity according to:
ꢈꢉꢊꢋꢉ
ꢈꢌꢍꢎꢉꢏꢍꢐ
ꢀꢁꢂꢃꢄꢃꢂꢅꢆ =ꢆꢆꢇ1 −ꢆ
ꢑ ꢆ100ꢆ%
The control sample was devoid of a scavenger and reported activities were obtained at a chalcone
concentration of 20 µM (Table 1).
Inspection of the DPPH radical scavenging abilities revealed that the chalcones could be clearly
divided into two groups with unique behavior. They either had clearly detectable activities varying from
39 to 92% (7-9 and 11-20) or were essentially inactive (all other compounds). Some chalcones exceeded
the scavenging ability of the known antioxidant ascorbic acid noticeably (Fig. 5). The obvious structural
feature that all active compounds shared was the presence of two hydroxyl groups located at
neighboring carbon atoms at one or both phenyl rings of the chalcone scaffold. This observation was in
agreement with the results of a study on phenolic compounds that showed that two hydroxyl groups in
1,2 or 1,4 positions were optimal for radical scavenging activity. The observed behavior was accounted
for using resonance structures showing increased stabilization of a formed radical by a second, electron-
donating hydroxyl group in the indicated positions [73]. Moreover, a comparison of the standard
reduction potentials of catechol (530 mV), resorcinol (720 mV), and hydroquinone (459 mV) shows the
higher likelihood of 1,2- and 1,4-dihydroxybenzenes to become oxidized [74].
27

Figure 5.
Representative DPPH radical scavenging assay. Bar heights indicate the amount of DPPH radical
remaining in a sample after incubation with a potential scavenger. The first bar (R) represents a reference
conducted in the absence of test compounds whereas the next three bars depict samples in the presence of the
known scavenger ascorbic acid (AA), compound 8, and compound 10 (all at 20 µM). The arrows indicate the
percentage of DPPH scavenged by the test compound, calculated as described under Materials and Methods.
3.5. Ability of chalcones to protect cells from Aβ-induced cytotoxicity
To be of use for therapeutic purposes, compounds need to be able to suppress ROS-related stress in
living cells. As most of the chalcones in this study were capable of inhibiting XO and/or scavenging the
DDPH radical, we evaluated their ability to rescue cells with elevated ROS-levels. As a simple model
for the evaluation of chalcones under in situ conditions, they were added to cultured Neuro-2A cells that
had been exposed to Aβ, an agent known to activate NADPH oxidase and thereby generate ROS [57].
Neurons were chosen as a model since they are a potential target of stroke-induced reperfusion injuries.
A MTT-based cell viability assay permitted the study of the effect of the added chalcones on cell
survival rates. Cell viability percentages (Table 1) were obtained by dividing the MTT absorbance at
570 nm by the absorbance of a control sample not subjected to ROS-induced stress (Fig. 6).
28

Figure 6.
Representative results of a cell viability assay under Aβ-induced cytotoxicity for compounds 10
and 12. Bar heights correspond directly to the absorbance of the dye MTT at 570 nm, a direct measure for
viability of Neuro 2A cells. The first two bars represent control measurements in the absence of chalcones in the
absence and presence of Aβ, respectively. The third and fourth bars represent measurements for compound 12,
which exhibited good rescue properties. Bars five and six were obtained for compound 10, a poor rescue
compound.
The results summarized in Table 1 showed that compounds offering the best level of protection
were those with dual activities (15, 17, 18, and 20), i.e. good DPPH scavengers (>80% scavenging
activity) with high inhibitory potencies against XO activity (IC50 < 10 µM). The only exception to this
trend was 8, which offered only modest protection against ROS-induced stress. Interestingly, 8 was the
only compound with dual activities that possessed three hydroxyl groups, whereas 15, 17, 18, and 20
had between four and six hydroxyl groups and were therefore more polar. It is conceivable that the
difference in polarity and hydrophobicity caused the protective ability of 8 to differ from those of the
29