Simple chalcones and bis -chalcones ethers as possible pleiotropic agents

Article abstract of DOI:10.3109/14756366.2015.1021253

The synthesis, the antioxidative properties and the lipoxygenase (LOX) and acetylcholinesterase (AChE) inhibition of a number of 4-hydroxy-chalcones diversely substituted as well as of a series of bis-chalcones ether derivatives are reported. The chalcones derivatives were readily produced using a Claisen-Schmidt condensation in a ultra sound bath in good yields. The structures of the synthesized compounds were confirmed by spectral and elemental analysis. Their lipophilicity is experimentally determined by reversed-phase thin-layer chromatography method. Most of them are potent in vitro inhibitors of lipid peroxidation and of LOX. Compounds b2 and b3 were found to be the most potent LOX and AChE inhibitors among the tested derivatives with a significant anti-lipid peroxidation profile. The results led us to propose these enone derivatives as new multifunctional compounds against Alzheimer's disease. The results are discussed in terms of structural and physicochemical characteristics of the compounds. Moreover, the pharmacokinetic profile of these compounds was investigated using computational methods.

Full text of DOI:10.3109/14756366.2015.1021253

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–12
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1021253
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RESEARCH ARTICLE
Simple chalcones and bis-chalcones ethers as possible
pleiotropic agents
Thalia Liargkova1, Dimitra J. Hadjipavlou-Litina1, Caterina Koukoulitsa2, Efstathia Voulgari3, and
Constantinos Avgoustakis3
2
1Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece, Chemistry Department,
University of Athens, Panepistimiopolis-Zografou, Greece, and 3Department of Pharmaceutical Technology and Pharmaceutical Analysis, School of
Pharmacy, University of Patras, Rio Patras, Greece
Abstract
Keywords
The synthesis, the antioxidative properties and the lipoxygenase (LOX) and acetylcholinesterase
(AChE) inhibition of a number of 4-hydroxy-chalcones diversely substituted as well as of a series
of bis-chalcones ether derivatives are reported. The chalcones derivatives were readily
produced using a Claisen–Schmidt condensation in a ultra sound bath in good yields. The
structures of the synthesized compounds were confirmed by spectral and elemental analysis.
Their lipophilicity is experimentally determined by reversed-phase thin-layer chromatography
method. Most of them are potent in vitro inhibitors of lipid peroxidation and of LOX.
Compounds b2 and b3 were found to be the most potent LOX and AChE inhibitors among the
tested derivatives with a significant anti-lipid peroxidation profile. The results led us to propose
these enone derivatives as new multifunctional compounds against Alzheimer’s disease.
The results are discussed in terms of structural and physicochemical characteristics of the
compounds. Moreover, the pharmacokinetic profile of these compounds was investigated
using computational methods.
Acetylcholinesterase inhibitors, bis-chalcones,
chalcones, lipoxygenase inhibitors,
multitarget agents
History
Received 16 December 2014
Accepted 9 February 2015
Published online 23 March 2015
Introduction
the presence of elevated levels of protein oxidation, lipid
peroxidation products and oxidative damage to mitochondria in
AD brain5. Antioxidants protect against the radicals and it is
important to balance an enhanced radical production with a
sufficient supply of antioxidants6. Increasing experimental evi-
dence supports the view that inflammation-generated oxidative
stress contributes to the lesions of AD7. According to the
observed antioxidant activity of the majority of the examined
chalcones, it has been concluded that are convenient templates
when designing useful drugs in treating of Alzheimer’s disease
that involves ROS.
Acetylcholinesterase (AChE) is a carboxyl-esterase which
terminates cholinergic neuro-transmission, by hydrolyzing neuro-
transmitter acetylcholine (ACh) in a synaptic cleft. Reversible
AchE inhibition is implicated in a number of neurological
disorders. It has been noticed that reduced AChE expression is a
common feature of AD patients. However, higher levels of AChE
are observed around b-amyloid plaques8. Thus AChE is another
good target for AD treatment and it is generally accepted that
AChE is associated with b-amyloid plaques. Accordingly, current
approaches to AD treatment aim to an increase of ACh in the
brain by AChE inhibitors9.
Chalcones and their derivatives are natural or synthetic 1,3-diaryl-
2-propenones that may exist in cis and trans isomeric forms, of
which the latter is thermodynamically stable. Due to the enone
system, such molecules present relatively low redox potentials and
have a greater probability of undergoing electron transfer
reactions. They possess an extended number of biological
activities: regulate cholesterol levels, reduce blood pressure,
regulate blood sugar, improve vision and memory, inhibit acetyl-
cholinesterase, inhibit enzymes implicated in inflammation,
reduce joint and muscular pains, enhance liver and kidney
functions, aid sleep, prevent cancer, strengthen the immune
system and beautify the skin and hair1,2
.
Hydroxyl chalcones embrace hydroxyl substitution, a key
group to greatly enhance the antioxidant activity of chalcones, due
to their conversion to phenoxy radicals through the hydrogen
atom transfer mechanism3. However, their effects on the central
nervous system (CNS) are still largely unexplored.
Reactive oxygen species (ROS) have been shown as causative
factors involved in many human degenerative diseases and
antioxidants have been found to have some degree of preventive
and therapeutic effects on these disorders4,5. Evidence for
involvement of free radicals in Alzheimer disease (AD) includes
Lipoxygenases (LOXs) catalyze the first two steps in the
metabolism of arachidonic acid to leukotrienes, important
inflammatory mediators10. Recent findings show that the activa-
tion of brain LOXs is an early event in the pathogenesis of AD11.
Recently showed that AD brains had higher 5-LOX protein levels
than did healthy controls12. 5-Lipoxygenase (5-LOX) acts as a
modulator of Ab peptides formation in vivo. A new study points
Address for correspondence: Dr. Dimitra J. Hadjipavlou-Litina,
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty
of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki
54124, Greece. E-mail: hadjipav@pharm.auth.gr

2
T. Liargkova et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
O
C
c
O
O
Figure 1. Synthetic procedures for the
synthesis of chalcones a1–a4, of diether c
and of bis-chalcones ethers b1–b4.
24h,reflux
K2CO3
CH3
+
CH3
CH3
Br
Br
OH
O
O
O
O
O
b(1-4)
O
25oC
CH3
CH3
Z
Z
Z-CHO
+
O
O
O
O
a(1-4)
ArCOCH3
ZCHO
ZCHO: 4-(4-bromobezyloxy)-benzaldehyde, 3-phenoxybenzaldehyde, 4-dimethyl-amino-cinnamic-
aldehyde, 1-naphthaldehyde
ArCOCH3 : 4-hydroxy-acetophenone
to a new role for 5-LOX in regulating endogenous tau metabol- spectrometer (Bruker Analytische Messtechnik GmbH,
ism in the CNS and supports the hypothesis that its pharmaco- Rheinstetten, Germany) in CDCl3 or DMSO using tetramethylsi-
logic inhibition could be beneficial for AD-related tau lane (TMS) as an internal standard, unless otherwise stated. 13C
neuropathology13.
NMR spectra were obtained at 75.5 MHz on a Bruker AM 300
In the past years, targeted therapies toward a specific spectrometer in CDCl3 or DMSO solutions with TMS as internal
molecular target have been applied. Today, the diligence of reference unless otherwise stated. Mass spectra were determined
inherent redundancy and robustness in many biological networks on a Shimadzu LC-MS 2010 EV under electrospray ionization
and pathways depicts that inhibiting a single target might fall (ESI) conditions [MS (ESI)] (Shimadzu Scientific Instruments,
short of producing the desired therapeutic effect14–16. Multi-target Inc., Columbia, MD). Elemental analyses were obtained in an
drug strategies have emerged as a therapeutic approach to treat acceptable range ( 0.4%) in a Perkin–Elmer 240 B CHN analyzer
diseases that stem from a combination of factors. Multitarget (Perkin–Elmer Corporation Ltd., Lane Beaconsfield, Bucks, UK).
therapeutic strategy can be used to inhibit two or more enzymes, Reactions were monitored by thin-layer chromatography (TLC)
act on an enzyme and a receptor, or affect an ion channel and a by Fluka, on aluminum cards precoated with 0.2 mm of silica gel
transporter17. Using this strategy, a single molecule hits multiple and fluorescent indicator. For reversed-phase TLC (RPTLC)
targets, which participate in pathways implicated to a given silica gel 60 Merck F254 plates were used. Ultra sound bath (US-
disease18. Therefore, it is evident that the treatment of AD could bath) Bandelin Sonorex Typ: RK100H was used. Azobis(2-
benefit from the use of multipotent drugs that present free radical amidinopropane) dihydrochloride (AAPH), 1,1-diphenyl-picryl-
scavenging, anti-inflammatory and AChE inhibitory activity.
hydrazyl (DPPH), nordihydroguaretic acid (NDGA) and Trolox
It is in this context that herein we report the synthesis, the were purchased from the Aldrich Chemical Co. (Milwaukee, WI).
antioxidant as well as the anti-inflammatory and anti-AChE Soybean LOX, linoleic acid sodium salt were obtained from
inhibitory activity of a number of 4-hydroxy-chalcones (Figure 1), Sigma Chemical Co. (St. Louis, MO). For the in vitro tests a
diversely substituted as well as of a series of bis-chalcones UV-1700. PharmSpec Shimadzu UV–Vis double beam spectro-
combined together through an –OCH2CH2CH2O– linkage photometer was used.
(Figure 1, structures b1–b4). The derived b1–b4 molecules
might be used as prodrugs, after an in vivo metabolism cascade,
Synthesis
through which two smaller chalcones will be possible to be taken.
Nowadays in pharmaceutical research new challenges have
been added for which additional considerations must be taken
with respect to toxicological effects, as well as potential
interactions with cytochrome P450, related to avoidance of
Phase 1 metabolism.
Thus, as a contribution to the knowledge about the pharma-
cokinetic properties of the new compounds (simple/double
chalcones) we will investigate their pharmacokinetic profile
using the VolSurf procedure19.
General method for the synthesis of chalcones a (1–4)
A
modified Claisen–Schmidt condensation was performed
between 4-hydroxy acetophenone and the suitable sustituted aryl
aldehyde at a molar ratio 1:1 in absolute ethanol (10 mL). Three
milliliters aqueous KOH (20%) was added. The mixture was
stirred at room temperature in a US-bath the end of the reaction
was monitored by TLC. The mixture was neutralized treated with
aqueous HCl 10%. The precipitate was either filtered and washed
with cold water or extracted with CHCl3 (30 mL ꢀ 3). The
combined organic layers were washed with water and brine and
dried under anhydrous MgSO4. The product was evaporated to
dryness and purified by recrystallization from a proper solvent.
(i) A modification of the above method was performed, in
which the mixture of the reactants was heated in a microwave
oven for 15 min (50 W, 60 ꢁC). Under these experimental
conditions, we have synthesized the known a2 chalcone20.
Experimental
General
All starting materials and solvents were obtained from commer-
cial sources and used without further purification. Melting points
were determined in open glass capillaries using a Mel-TempII
apparatus (Lab. Devices, Holliston, MA). UV–vis spectra were
obtained on a Shimadzu UV-1700 PharmSpec (UVprobe Ver.
2.21) (Kanda-Nishikicho 1-chome, Tokyo, Japan). Infrared spec- 3-(4-((4-Bromobenzyl)oxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-
tra (film as Nujol mulls or KBr) were recorded with a Shimadzu 1-one (a1). 76% yield, yellow solid, m.p. 117–119 ꢁC. Rf: 0.47
FT IR-8101 M (Kanda-Nishikicho 1-chome, Tokyo, Japan). The (2:1, hexane/acetone). Recrystallized from ethanol, IR (KBr):
1H NMR spectra were recorded at 300 MHz on a Bruker AM 300 1710, 1610 cmꢂ1; 1H NMR (CDCl3): d 5.08 (d, 2H), 6.90 (d, 1H)

DOI: 10.3109/14756366.2015.1021253
Simple chalcones and bis-chalcones ethers
3
7.01 (d, 2H), 7.28–7.31 (m, 5H), 7.51–7.72 (m, 3H), 7.83 (m, 2H), 188.7, 188.7; MS (ESI): 891 [M+CH3OH+1]+. Anal. Calcd for
7.97 (d, 1H, J ¼ 9 Hz), 9.89 (s, 1H); 13C NMR (CDCl3): d 40, C47H38Br2O6: C, 65.75; H, 4.46. Found: C, 65.92; H, 4.34.
76.50, 77, 77.40, 110.6, 110.95, 111.70, 117.97, 118.4, 118.85,
1,10-((Propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-(3-
121.74, 123.75, 130.44, 130.67, 132, 136.4, 152.37, 153.89,
190.28. Anal. Calcd for C22H17BrO3: C, 64.56; H, 4.19. Found: C,
64.35; H, 4.07.
phenoxyphenyl)prop-2-en-1-one) (b2). 72% yield, yellow solid,
m.p. 172–174 ꢁC. Rf: 0.68 (2:1, hexane/acetone). Recrystallized
;
from ethanol, IR (KBr): 1680, 1650 cmꢂ1 1H NMR (CDCl3):
1-(4-Hydroxyphenyl)-3-(3-phenoxyphenyl)prop-2-en-1-one
(a2). The modified (i) synthetic method was used. The
analytical data as well as the spectra analysis were in agreement
to the corresponding given in literature20.
d 2.16–2.54 (m, 2H), 4.24–4.28 (m, 4H), 6.93–7.22 (m, 12H),
7.28–7.56 (m, 8H), 7.62–7.69 (m, 4H), 7.75–7.91 (m, 2H), 7.97–
8.07 (m, 4H); 13C NMR (CDCl3): d 29, 54, 54.6, 64.6, 114.4,
118.1, 119, 120.6, 122.7, 123.4, 126, 129.9, 130.2, 130.9, 131.2,
137, 143, 153, 156.9, 157.9, 162.7, 188.5. Anal. Calcd for
C45H36O6: C, 80.34; H, 5.39. Found: C, 80.34; H, 5.43.
5-(4-(Dimethylamino)phenyl)-1-(4-hydroxyphenyl)penta-2,4-dien-
1-one (a3). 79% yield, dark brown solid, m.p. 220–222 ꢁC. Rf:
0.55, 0.55 (2:1, hexane/acetone). Recrystallized from ethanol, IR
(KBr) : 1710, 1620 cmꢂ1; 1H NMR (CDCl3): d 2.61–3.26 (m, 6H),
6.51–7.95 (m, 12H), 12.3 (s, 1H); 13C NMR (CDCl3) d 77.42,
125.43, 126.95, 127.27, 128.56, 128.82, 129.21, 132.64, 141.89,
144.84, 160.82, 163.37, 163.60, 187.20; MS (ESI): 293 [Mꢂ1]+,
293 [M+CH3CH2OH+Na]+. Anal. Calcd for C19H19NO2: C,
77.79; H, 6.53; N, 4.77. Found: C, 77.86; H, 6.77; N, 4.85.
1,10-((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(5-(4-
(dimethylamino) phenyl) penta-2,4-dien-1-one) (b3). 79% yield,
dark brown solid, m.p. 156–158 ꢁC. Rf: 0.55, 0.62 (2:1, hexane/
acetone). Recryctallized from ethanol, IR (KBr): 1710,
1670 cmꢂ1 1H NMR (CDCl3): d 2.29–2.54 (s, 2H), 2.99–3.08
;
(br, 12H), 4.22–4.26 (m, 4H), 6.68–6.94 (m, 10H), 7.31–7.56
(m, 2H), 7.59–7.64 (m, 2H), 7.91–7.98 (m, 10H); 13C NMR
(CDCl3): d 29.4, 64, 100.4, 109, 11.2, 112.4, 114.2, 118.3, 121.5,
124.5, 128.8, 130.5, 130.6, 131.9, 135.6, 141.6, 145.5, 158.3,
186.2. Anal. Calcd for C41H42N2O4: C, 78.57; H, 6,75; N, 4.47.
1-(4-Hydroxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one (a4). see
Ref21.
Synthesis of 1,10-((propane-1,3-diylbis(oxy))bis(4,1-phenylene)) Found: C, 78,4; H, 7.03; N, 4.10.
diethanone (c)
1,10-((Propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-(naphthalen-
1-yl)prop-2-en-1-one) (b4). 74% yield, yellow solid, m.p. 167–
168 ꢁC. Rf: 0.64 (2:1, hexane/acetone). Recrystallized from
petroleum ether:ethylacetate, IR (KBr): 1680, 1630 cmꢂ1 1H
;
NMR (CDCl3): d 2.31–2.54 (m, 2H), 4.2–4.28 (m, 4H), 7.0–7.03
(m, 4H), 7.48–7.64 (m, 8H), 7.86–7.92 (m, 8H), 8.07–8.27
(m, 2H), 8.255 (d, 2H), 8.64 (d, 2H); 13C NMR (CDCl3): d 29.6,
64, 76, 114.4, 117.1, 123.6, 124.7, 124.9, 125.4, 126.3, 126.9,
128.7, 130.6, 130.9, 131.3, 131.8, 132.7, 133.8, 141, 162.7, 18.5;
MS (ESI): 611 [M+Na]+. Anal. Calcd for C41H32O4: C, 83.65;
H, 5.48. Found: C, 83.63; H, 5.87.
4-Hydroxy acetophenone and 1,3-dibromopropane in molar ratio
2:1 was diluted in 50 mL acetone. Anhydrous K2CO3 was added
and the mixture was refluxed for approximately 18 h. The reaction
was completed judged by the negative alcoholic ferric chloride
(3%) test. The mixture was evaporated to dryness. The solid was
treated with water, filtered of and recrystallized from absolute
ethanol.
90% yield, white crystals solid, m.p. 115–117 ꢁC. Rf: 0.53 (2
hexane:1 acetone), IR (KBr): 1750, 1670 cmꢂ1
(CDCl3): d 2.27–2.35 (m, 2H), 2.54 (br, 6H), 4.21–4.25 (m,
4H), 6.87–6.96 (m, 4H), 7.91–7.94 (m, 4H); 13C NMR (CDCl3):
d 26.3, 26.2, 29.1, 64.5, 65, 114.2, 130.6, 130.6, 136.4, 162.7,
196.6, 196.6. Anal. Calcd for C19H20O4: C, 73.06; H, 6.45.
Found: C, 72.98; H, 6.37.
;
1H NMR
Physicochemical studies
Experimental determination of lipophilicity as RM values
RPTLC was performed on silica gel plates impregnated with 55%
(v/v) liquid paraffin in light petroleum ether. The mobile phase
was a methanol/water mixture (70/30, v/v). The plates were
developed in closed chromatography tanks saturated with the
mobile phase at 24 ꢁC. Spots were detected under UV light or by
iodine vapors. RM values were determined from the corresponding
Rf values (from 10 individual measurements) using the equation
RM ¼ log[(1/Rf) ꢂ 1]23.
Synthesis of etherified double chalcones b (1–4)
General synthetic method. A Claisen–Schmidt condensation was
performed between (1,10-((propane-1,3-diylbis(oxy))bis(4,1-phe-
nylene))diethanone) and the appropriate substituted aromatic
aldehyde at a molar ratio 1:2 in absolute ethanol (10 mL)22.
Three milliliters aqueous KOH (20%) was added. The mixture
was stirred at room temperature in a US-bath. The end of the
reaction was monitored by TLC. After the completion of the
reaction the mixture was neutralized treated with aqueous HCl
10%. The precipitate was either filtered and washed with cold
water or extracted with CHCl3 (30 mL ꢀ 3). The combined
organic layers were washed with water and brine and dried
under anhydrous MgSO4. The product was evaporated to dryness
and purified by recrystalization from a proper solvent.
Theoretically calculated values of lipophilicity as clog P
Lipophilicity was theoretically calculated as clog P values in
n-octanol-buffer by CLOGP Programme of Biobyte Corp.24.
Biological experiments
General methods
1,10-((Propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-(4-((4-
bromobenzyl) oxy) phenyl)prop-2-en-1-one) (b1). 89% yield,
light yellow solid, m.p. 197–199 ꢁC. Rf: 0.5(2:1, hexane/acetone).
Each in vitro experiment was performed at least in triplicate
and the standard deviation of absorbance was less than 10% of
the mean.
Recrystallized from acetone, IR (KBr) : 1660, 1630 cmꢂ1 1H
;
NMR (CDCl3): d 2.31–2.35 (m, 2H), 5.06 (s, 4H), 4.24–4.28 (m,
4H), 6.93–7.05 (m, 4H), 7.26–7.31 (m, 8H), 7.38–7.43 (m, 4H),
7.755 (d, 2H), 8.015 (s, 4H), 7.36 (s, 2H), 7.43–7.66 (m, 4H); 13C
NMR (CDCl3): d 45.6, 64.5, 69.4, 66.6, 77, 77.4, 114.3, 115, 120,
122, 128, 129, 130, 130.7, 132, 135, 135.6, 143.7, 151.6, 160.4,
In vitro assays
Determination of the reducing activity of the stable radical
1,1-diphenyl-picrylhydrazyl. To a solution of 1,1-diphenyl-
picrylhydrazyl (DPPH) in absolute ethanol an equal volume of

4
T. Liargkova et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
the compounds dissolved in DMSO was added. A stock solution metabolism is based on the hypothesis that the distance between
(10 mM) of the compounds was used. The concentrations of the reactive center on the protein (oxygen atom attached to the
the final solutions of the compounds were 100 mM. After 20 iron atom) and the interaction points in the protein cavity (GRID-
and 60 min at room temperature the absorbance was recorded MIF) should correlate to the distance between the reactive center
at 517 nm25.
of the molecule (i.e. hydrogen atoms and heteroatoms positions)
and the position of the different atom types in the molecule. This
detailed information can be used by chemists in an early
absorption, distribution, metabolism, and elimination (ADME)
phase to increase the metabolic stability of drug candidates or to
select appropriate chemical modifications leading to a better
pharmacokinetics and metabolic profile.
Inhibition of linoleic acid peroxidation. Production of conju-
gated diene hydroperoxide by oxidation of sodium linoleate in an
aqueous dispersion was monitored at 234 nm26. AAPH is used as a
free radical initiator. Ten microliters of the 16 mM sodium
linoleate solution was added to the UV cuvette containing
0.93 mL of 0.05 M phosphate buffer, pH 7.4 prethermostated at
37 ꢁC. The oxidation reaction was initiated at 37 ꢁC under air by Prediction of BBB permeation and Caco-2 permeability. Blood–
the addition of 50 lL of 40 mM AAPH solution. Oxidation was brain barrier (BBB)33 permeation and Caco-2 cell permeability34
carried out in the presence of aliquots (10 lL) in the assay without of the studied compounds were predicted using VolSurf (ver-
antioxidant. Lipid oxidation was measured in the presence of sion 4, version 4.1.4, Molecular Discovery Ltd., 2005; www.
the same level of DMSO. The rate of oxidation at 37 ꢁC was moldiscovery.com). We used the probes water (OH2), hydropho-
monitored by recording the increase in absorption at 234 nm bic (DRY) and H-bonding carbonyl (O) to generate the 3D
˚
caused by conjugated diene hydroperoxides and compared with interaction energies and a grid space of 0.5 A.
the appropriate standard trolox.
Evaluation of the cytotoxicity
Soybean LOX inhibition study in vitro. In vitro study was
evaluated as reported previously25. The tested compounds
L929 mouse fibroblasts cells were cultured in EMEM supple-
mented with 10% horse serum, 2 mM L-glutamine, 1 mM sodium
dissolved in DMSO were incubated at room temperature with
pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium
sodium linoleate (100 mM) and 0.2 mL of enzyme solution
bicarbonate and 100 lg/mL penicillin–streptomycin at 37 ꢁC in
(1/9 ꢀ 10ꢂ4 w/v in saline). The conversion of sodium linoleate
35
a humidified atmosphere with 5% CO2
.
to 13-hydroperoxylinoleic acid at 234 nm was recorded and
compared with the appropriate standard inhibitor (NDGA).
Several concentrations were used for the determination of
IC50 values.
L929 cells were plated into 24-well plates at a density 5 ꢀ 104
cells/well and allowed to attach and grow for 24 h. The
supernatant in each well then replaced with medium containing
various concentrations of a1, a2, a3, a4, c, b1, b2, b3 and b4: 1,
10, 20, 50 and 100 lM. After 24 h incubation, the supernatant was
removed and the cells were washed with PBS. The cells were
detached with 0.25% trypsin, transferred to FACS tubes and then
centrifuged (1600 rpm for 5 min) and the pellet washed with PBS.
After washing, the cells in the pellet were incubated with 5 lL
propidium iodide (PI) solution (1 mg/mL) for 1 min.
Glutathione conjugation. Stock solutions of the compounds
were prepared in water using phosphate buffer solution (PBS) pH
7.4 and in order to achieve dissolution the solvent contained
approximately 10% DMSO. The concentrations of the solutions
were chosen so that the absorption maxima were between 0.5
and 1. The test compounds are incubated for 24 h at 37 ꢁC and
their UV spectra were recorded. All determinations were carried
out in duplicate. The error limits of the e values were approxi-
mately 2%. The experiment was repeated in the presence of GSH
using thiol/test compound, 2/1 and incubation at 37 ꢁC for 24 h
The PI fluorescence (cell death) was determined with flow
cytometry, FACS Calibur, Coulter Epics XL-MCL. The analysis
of flow cytometry data was performed with WinMDI analysis
program.
and their UV spectra were recorded25,27
.
Results and discussion
Inhibition of acetylocholinesterase in vitro28. A modified
Ellman procedure was followed. The inhibitory activity was
measured by the change in absorbance at 412 nm. The assay uses
the thiol ester acetylthioline (0.01 M), which is hydrolyzed by
AChE (3.5 U/mL) to produce thiocholine and acetate. The
thiocholine reduces DTNB (0.01 M in phosphate 0.1 M pH 7)
liberating nitrobenzoate, which absorbs at 412 nm. A 10-mM
stock solution of the compounds in DMSO was used, from which
several dilutions were made for the determination of IC50 values.
The experiment was performed in phosphate buffer pH 8 (0.1 M).
As a standard inhibitor physostigmine was used.
Chemistry
The chalcone analogs a (1–4) and b (1–4) were successfully
synthesized via Claisen–Schmidt condensation, using KOH as a
catalyst in a US-bath. In our study, we found that the use of
the US-bath greatly affected the reactivity as well as the yields.
The synthesis of the chalcones a (1–4), of bis-chalcones b (1–4)
and of the diether c, is shown in Figure 2. Chalcones a2 and a4
have been synthesized earlier by other investigators20,21. Herein
we used modified synthetic procedures with better yields and less
reaction time. In all cases, the use of US-bath diminished the
reaction time and enhanced yields. Their structural characteristics
and physicochemical properties were in agreement with the
literature.
The structures of the new derivatives (a1, a3, c, b1–b4) have
been analyzed and (Figure 2) confirmed spectroscopically [IR, 1H
NMR, 13C NMR, MS (ESI)] and by their elemental analysis. All
the chalcones (a and b) present the characteristic absorption in the
IR (nujol) (cmꢂ1 3200 (O–H), 1720 (C¼O), 1625 (C¼C)). H
Computational methods
The structures a1–a4 and b1–b4 were generated using SYBYL29
Sybyl molecular modeling package running on a Linux operating
system, and their energy were minimized using the Powell method
with a convergent criterion provided by the Tripos force field30.
1
Prediction of the site of metabolism. MetaSite31,32 is a compu- NMR spectroscopy did not succeed to delineate the (E)/(Z)
tational procedure specifically designed to predict the site of structure. Previous findings22 supported the (E) conformation.
metabolism of compounds, which metabolized by several cyto- The products were obtained in good yields (over 70%). The
chromes (CYP1A1, CYP1A2, CYP2C9, CYP2B6, CYP2C19, physical data of the synthesized compounds are given in details in
CYP2D6, CYP2E1 and CYP3A4). The prediction of the site of the experimental session.

DOI: 10.3109/14756366.2015.1021253
Simple chalcones and bis-chalcones ethers
5
HO
(A) HO
a2
a1
O
O
O
Br
O
HO
HO
a4
a3
O
O
N
(B)
Br
Br
H3C
CH3
H3C
CH3
N
N
CH2
H2C
b3
O
O
O
O
b1
O
O
O
O
O
O
b4
O
O
b2
O
O
O
O
O
O
O
O
Figure 2. (A) 4-Hydroxy-substituted chalcones a (1–4). (B) Bis-chalcones etherified derivatives b (1–4).
Table 1. Lipophilicity values: (i) theoretically calculated as clog P and
(ii) experimentally determined as RM.
Physicochemical studies
Lipophilicity is a significant physicochemical property determin-
ing distribution, bioavailability, metabolic activity and elimin-
ation. Thus, we tried to determine experimentally their
lipophilicity from RPTLC method as RM values and to compare
them with the corresponding theoretically calculated clog P
values24 in n-octanol-buffer. This is considered to be a reliable,
fast and convenient method for expressing lipophilicity23. Apart
from the important role of lipophilicity for the kinetics of
biologically active compounds, antioxidants of hydrophilic or
lipophilic character are both needed to act as radical scavengers in
the aqueous phase or as chain-breaking antioxidants in biological
membranes.
Compounds
clog P
RM ( SD)
a1
a2
a3
a4
c
b1
b2
b3
b4
6.05
5.60
3.66
4.67
3.68
12.16
11.07
9.00
9.23
0.34 (0.003)
0.36 (0.001)
ꢂ0.69 (0.005)
ꢂ0.26 (0.002)
ꢂ0.23 (0.002)
0.23 (0.02)
ꢂ0.68 (0.005)
0.89 (0.005)
0.77 (0.007)
From our results (Table 1), it can be concluded that RM values
could be used as a successful relative measure of the overall
lipophilic/hydrophilic balance of these molecules.
RM values were significantly correlated to the theoretical clog P
values. Two derivatives have been omitted from this correlation
(b1 and b2). Their clog P values were high (unrealistic range of
lipophilicity in nature). This conclusion was supported by the
statistical data given with Equation (1) (e.g. correlation coeffi-
cients, F values).
RM ¼ 0:246ð0:133Þ clog P ꢂ 1:200ð0:782Þ;
ð1Þ
N ¼ 7; R ¼ 0:906; R2 ¼ 0:820; S ¼ 0:270;
F1;5 ¼ 22:78; ꢀ ¼ 0:01:

6
T. Liargkova et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 2. Interaction–RA% with DPPH (RA%); % inhibition of lipid peroxidation (AAPH%); in vitro inhibition of soybean
LOX (IC50 or %); in vitro inhibition of AChE (IC50 or %).
RA% 100 lM
20 min
RA% 100 lM
60 min
AAPH%
at 100 lM
IC50 or %LOX inh.
at 100 lM
IC50 or % AChE
at 100 lM
Compounds
a1
a2
a3
a4
c
b1
b2
b3
1.0
3.0
18.0
7.0
No
36.0
No
6.0
4.0
17.0
3.0
No
55.0
8.0
94.0
94.0
40.0
96.0
20.0
51.0
78.0
100.0
100.0
63.0
14.0%
10.0%
100 lM
44.0%
Mo
40.0%
55.0 lM
56.0 lM
55.0 lM
7.0%
26.0%
100.0 lM
25.0%
13.0%
23.0%
49.0 lM
52.0 lM
100.0 lM
17.0
19.0
14.0
50.0
b4
Trolox
Physostigmin
NDGA
0.05 lM*
81.0
83.0
28 mM
No, no activity under the reported experimental conditions; confidence limits less than 10%. *76% at 100 lM.
Calculation methods
DPPH, the simple 4-hydroxy chalcones a (1–4) did not reduce
DPPH. The decrease/absence of the RA seems to be correlated to
steric hindrance. The bis-chalcones b1 and b4 showed interesting
reducing ability (50–55%), compared to the reference compound
NDGA (83%). The highest RA (55%) is observed by b1 after
60 min. Although in most cases the interaction remained constant
after 60 min, an increase was observed for bis-chalcones b1 and
b4. Saking into consideration the fact that a (1–4), present very
low, if any, RA on DPPH, it is apparent that bis-chalcones b1 and
b4 do lead to compounds with improved antioxidant activity
compared to the simpler molecules (a1 and a4). The ether linkage
does not seem to offer to the interaction with DPPH because the
Some physicochemical properties such as the energy values of
the lowest and highest unoccupied molecular orbital (ELUMO and
EHOMO) of the chalcones, the electrostatic potential and so on
were calculated by the program Spartan v. 5.1.3 (Wavefunction
Inc.) on energy minimized structures. These results were
subjected to a multivariate analysis in order to describe better
the activity of the compounds in terms of quantitative structure
activity relationships.
Biological evaluation
The obtained products were evaluated as potential antioxidants/ simple intermediate c does not show any interaction with DPPH at
anti-inflammatory agents and inhibitors of AChE. The anti- 100 lM. Taking under consideration the calculated physicochem-
inflammatory activity of the synthesised compounds was tested as ical properties of the molecules, such as the energy values of the
in vitro inhibition of LOX activity. Their antioxidant potential was lowest and highest unoccupied molecular orbital (ELUMO and
evaluated by the offered inhibition of lipid peroxidation, and by EHOMO), the electrostatic potential, and so on (Table 3), it seems
their interaction with 1,1-diphenyl-2-picrylhydrazyl stable radical. that their reducing ability RA is mainly correlated with EHOMO
.
The antioxidant ability of a compound must be evaluated in a Thus, b1 and b4 with the lowest EHOMO values (ꢂ0.2286 and
variety of milieus. In this way, factors such as solubility or steric ꢂ0.214549) are the most potent.
hindrance which may be of overriding importance in one
The low interaction values observed with b2 and b3 bis-
environment in another can be varied. Thus, we have used two chalcones can be attributed to the absence of easily oxidizable
different types of assays to measure in vitro antioxidant activity: functionalities like the ones (two catechol subunits) present in
(a) the interaction with the stable free radical DPPH and (b) the NDGA as well as to their EHOMO values. Lipophilicity does not
interaction with the water-soluble azo compound AAPH.
Both require a spectrophotometric measurement and a certain
seem to play an important role.
In the AAPH assay, the highly reactive alkylperoxyl radicals
reaction time in order to obtain reproducible results36. The DPPH are intercepted mainly by a hydrogen atom transfer from the
method is described as a simple, rapid and convenient method antioxidant. Therefore, particularly effective hydrogen atom
independent of sample polarity for screening many samples for transfer agents are compounds with high hydrogen atom donating
radical scavenging activity37. The use of the free radical reactions ability, that is compounds with low heteroatom-H bond dissoci-
initiator AAPH is recommended as more appropriate for ation energies and/or compounds from which hydrogen abstrac-
measuring radical-scavenging activity in vitro, because the tion leads to sterically hindered radicals as well as compounds
activity of the peroxyl radicals produced by the action of AAPH from which abstraction of hydrogen leads to C-centered radicals
shows a greater similarity to cellular activities such as lipid stabilized by resonance.
peroxidation26.
The interaction/reducing activity (RA) of the examined (AAPH%) in Table
Perusal of the percentage anti-lipid peroxidation activity
showed high inhibition of lipid
2
compounds with the stable free radical DPPH is shown in peroxidation, higher than trolox (63%), with the exception of a3
Table 2. This interaction, which indicates their radical scavenging (40%) and b1 (51%). Judging separately the anti-lipid peroxida-
ability in an iron-free system, was measured at 100 lM (20/ tion activity of the two chalcones groups, it seems that within the
60 min). For the sake of comparison, already established RA% simple chalcones (a1–a4), low lipophilicity (as clog P value), led
values for the intermediate molecule c were included in the table. to low inhibitory ability (a3 ¼ 40%, clog P ¼ 3.66). The bis-
In the DPPH assay, the dominant chemical reaction involved is chalcones b1 and b2 were found less active compared to the
the reduction of the DPPH radical by an electron transfer from corresponding simple chalcones a1 and a2 due to their stereo-
the antioxidant38. Particularly effective such antioxidants are the chemistry and bulk. Among the bis-chalcones (b1–b4) the
phenoxide anions from phenolic compounds like catechol and antioxidant activity was found to be higher for b3/b4 chalcones,
derivatives, such as NDGA. Regarding their ability to reduce which presented the lower clog P values (9.0–9.23) within the

DOI: 10.3109/14756366.2015.1021253
Simple chalcones and bis-chalcones ethers
7
Table 3. Physicochemical properties calculated by the program Spartan v. 5.1.3.
Compounds
MW
Volume As
Surface area
Dipole (Debye)
Sm2 total energy (kcal/m)
c
626.79
858.62
672.77
626.79
464.56
409.27
316.35
293.36
274.31
750.99
860.24
769.3
750.26
544.35
406.73
362.75
353.57
316.89
760.47
855.76
757.36
756.21
546.24
408.65
317.24
372.09
327.46
1.667435
3.324089
2.256212
1.60489
0.238953
3.59584
2.273257
3.841782
1.974221
b1
b2
b3
b4
a1
a2
a3
a4
ꢂ24.44
ꢂ8.32
31.65
ꢂ38.39
ꢂ26.11
ꢂ20.39
ꢂ7.89
ꢂ5.45
EHOMO
ELUMO
E (bandgap)
Elpotmin
Elpotmax
c
ꢂ0.265419
ꢂ0.22806
0.058602
0.19129
0.324021
0.41935
ꢂ51.9398
ꢂ41.5485
ꢂ38.5631
ꢂ57.3943
ꢂ38.253
36.8835
27.0606
21.6093
28.5348
18.1817
53.9305
76.9638
73.2086
75.1948
b1
b2
b3
b4
a1
a2
a3
a4
ꢂ0.244199
ꢂ0.268259
ꢂ0.214549
ꢂ0.305302
ꢂ0.309998
ꢂ0.268314
ꢂ0.277466
0.180312
0.062454
0.187093
0.0878556
0.081237
0.06715
0.424511
0.330714
0.401642
0.393158
0.391235
0.335464
0.34597
ꢂ55.138
ꢂ47.1648
ꢂ50.8953
ꢂ47.4675
0.068504
subgroup. The intermediate c presents low anti-lipid peroxidation
ability.
In an attempt to get deeper into the implicated physicochem-
ical properties, we decided to analyze the % inhibition values
Regression analyses including all the values of anti-lipid induced by the compounds at 100 mM (LOX %, Table 2) instead
peroxidation (AAPH %) revealed that lipophilicity, as RM values of the IC50 values, due to the small number of data. We found that
(experimentally determined lipophilicity), was the main physico- EHOMO, the energy value of the highest unoccupied molecular
chemical parameter influencing their inhibitory activity and orbital, was the main physicochemical parameter influencing their
this was supported by the statistical data presented within inhibitory activity (Equation 3). Again bis-chalcones b1 and b2
Equation (2).
were rejected from correlation (3). Their EHOMO (Table 3) values
did not present great differences.
log AAPH% ¼ 0:383ð0:351ÞRM þ 1:701ð0:197Þ;
ð2Þ
log % LOX ¼ 10:442ð6:269ÞEHOMO þ 4:435ð1:687Þ; ð3Þ
N ¼ 7; R ¼ 0:782; R2 ¼ 0:612; S ¼ 0:184;
F1;5 ¼ 7:89; ꢀ ¼ 0:01:
Two bis-chalcones b1 and b2 did not follow this model of
activity. Their acivity was not correlated with lipophilicity. Both
present unrealistic high clog P values.
In an attempt to investigate the mechanism of the anti-
inflammatory activity of chalcones and bis-chalcones, their
in vitro effect on soybean LOX was examined. Soybean LOXs
convert linoleic acid to 13-hydroperoxylinoleic acid, producing a
conjugated diene that absorbs at 234 nm. It has been shown that
inhibition of soybean LOX activity by nonsteroidal anti-inflam-
matory agents is qualitatively similar to their inhibition of the rat
mast cell LOX and may be used as a simple qualitative screen for
such activity39.
N ¼ 7; R ¼ 0:886; R2 ¼ 0:786; S ¼ 0:206;
F1;5 ¼ 18:37; ꢀ ¼ 0:01:
There is no universally accepted approach to evaluate the
relative potency of different substances to cause LOX inhibition.
However, most of the LOX inhibitors are antioxidants or free
radical scavengers40. Herein, the results from the anti-lipid
peroxidation activity studies support the anti-LOX activity for
the bis-halcones b1–b4.
We investigated in vitro the inhibitory activity of a and b on
AChE activity, using acetylthiocholine as substrate28. The applied
method is based on the colorimetric determination of thiocholine
produced through the catalytic activity of AChE, by its reaction
with dithio-bisnitrobenzoic acid. The potential of these com-
pounds to act as AChE inhibitors can be considered beneficial for
their prospective nootropic action and may contribute to the
mechanisms of action of reported structurally related hydroxyl
chalcones41.
Bis-chalcone derivatives b2 and b3 were found to present
significant and close IC50 values (49/52 lM) followed by b4 with
the exception of b1. The simple a chalcones were found less
active with a % inhibition range from 7% to 26% (Table 2).
Only a3 presented interesting inhibition. Again it was observed
that the transformation to a bis-chalcone with an ether linkage led
to more potent analogues with more potent representatives the
a2–b3/b2–b3.
NDGA, a known inhibitor of soybean LOX, has been used as a
reference compound (IC50 28 mM/93% at 100 lM).
Perusal of the %/IC50’s inhibition values (Table 2) showed that
the most potent, and almost equipotent, inhibitors were bis-
chalcones b2, b3 and b4 with the exception of b1. The
intermediate ether c was found inactive whereas simple 4-hydroxy
chalcones a1, a2 and a4 exhibited activities in the range 10–44%
at 100 lM. It is important to be noticed that the transformation of
a3 to the bis-chalcone ether b3 led to a more potent derivative
(IC50 ¼ 56 lM). Lipophilicity was referred as an important
physicochemical property for LOX inhibition40. This physico-
chemical property could explain the increase of anti-LOX activity
in b3 (clog P values for a3 ¼ 3.66, whereas b3 ¼ 9.00). For the
same reason b1, 2 and 4 exhibited higher activities (in terms of %
or IC50 values) than the simple chalcones.
Perusal of Clog P and IC50 or %AChE values in Tables 1 and 2
revealed that the role of lipophilicity on the inhibition of AChE
was not well defined in this series of compounds.

8
T. Liargkova et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 3. ?Inhibition % of AChE induced
by100 lM of compounds a3, b (2–4) in
relation to the time (0–30 min).
Table 4. Stability studies and incubation with GSH.
a/a
knm (max)
emax
a1
a1 + 2GSH
a2
a2 + 2GSH
a3
a3 + 2GSH
a4
a4 + 2GSH
c
c + 2GSH
b1
b1 + 2GSH
b2
b2 + 2GSH
b3
b3 + 2GSH
b4
b4 + 2GSH
340
340
256
256
326
326
326
326
245
245
246
246
341
341
320
320
443
443
2200
1800
14 200
8600
2700
5000
11 500
15 500
0
600
1500
1500
12 700
3700
9900
0
12 460
0
Analyzing this data set as a wholesome, lipophilicity did not
seem to affect absolutely the AChE inhibition. Within the a1–b4
subgroup lower clog P values (a3 ¼ 3.66) were correlated
to higher activity. This concept was not followed by bis-
chalcones.
To further investigate the inhibition mechanism, time depend-
ence of AChE inhibition by compounds a3, b2, b3 and b4 was
subsequently probed. For the time 0–30 min the data showed a
continued increase (Figure 3). The inhibition seems to be time
dependent.
Figure 4. Cytotoxicity of chalcones (a1–a4) and Bis-chalcones–ethers
(b1–b4) on L929 cells (24-h incubation) expressed as PI % values.
Glutathione (GSH) conjugation constitutes an in vivo antioxi-
dant protective mechanism, by which reactive electrophilic
compounds are detoxified by virtue of its nucleophilic sulphydryl
group. The nucleophilic addition of GSH to electron-deficient
carbon double bonds occurs mainly in compounds with a,b-unsat-
urated double bonds like chalcones demonstrating preferential
reactivity toward thiols in contrast to amino and hydroxyl
groups27. In our case, we have studied the possibility of alkylation
of our mono/bis-chalcones with a cellular thiol such as GSH,
leading to the adducts A. In most instances the double bond was
rendered electron deficient by resonance or conjugation with the
carbonyl group.
whereas bis-b3 and b4 were highly conjugated with GSH
(Table 4). The others, e.g. a3 and b1 did not exhibit any
interaction. It seems that the stereochemistry of substituent Z
affects the alkylation.
H
C
H
H
H
GSH
H
Z
C
Z
C
C
C
O
X
C
O
X
SG
It should be emphasized that chalcones a1, a2 and b2 were
conjugated with 2 GSH, a4 presented slight alkylation rate
Adducts A

DOI: 10.3109/14756366.2015.1021253
Simple chalcones and bis-chalcones ethers
9
Figure 5. Histogram (left) and graphical visualization (right) of predicted (A) CYP1A1, (B) CYP1A2, (C) CYP2D6 metabolic sites for compound b2.
Four different shades of color are used; from dark (lowest probability) to white (highest). The major metabolic site is highlighted (blue color).
Cytotoxicity
Molecular docking studies
The cytotoxicity of the synthesized derivatives was determined
Prediction of the site of metabolism by CYP1A1, CYP1A2 and
using the PI fluorescence method35,42 in the presence of different
CYP2D6
concentrations (1–100 lM) of these compounds.
The results are presented in Figure 4 in the form a bar-graph of
the % cell survival values as PI % values for the examined
compounds. All tested chalcones a (1–4) showed low cytotoxi-
cities in the whole area of concentrations examined (from 1 to
100 lM), with the noticeable exception of a2 and a4. Considering
the bis-chalcone ethers, b3 was found to present high toxicity
started at the concentration of 1 lM (81%) reaching the highest
values at 20 lM (100%). The rest bis-derivatives follow, with
the exception of b2 whose toxicity started increasing significantly
at the concentration of 20 lM reaching the highest values at
100 lM (55%).
CYP family is one of the important steroid-metabolizing enzymes.
In general, metabolic transformations tend to reduce the bioavail-
ability of a compound and, in turn, the pharmacological profile.
Therefore, we have computed the prediction of the site of
metabolism using the MetaSite software (version 5.0.3, Molecular
Discovery Ltd., 2014; www.moldiscovery.com). MetaSite can be
used to evaluate the metabolic stability from the 3D structure of a
drug candidate prior to experimental measurements. Three CYP
enzyme isoform CYP1A1, CYP1A2 and CYP2D6 were selected for
the prediction of the site of metabolism for compound b2. Figure 5
reports the histogram with the PSM values (Site of Metabolism

10 T. Liargkova et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 6. In silico: (A) Caco-2 permeability and (B) BBB permeation.
Probability), for each of the substrate atoms considered in the
computation by the three enzymes. It is observed that the most
probable sites of metabolism for the enzymes CYP1A1 and
CYP1A2 are the hydrogens of the –OCH2CH2CH2O– linkage,
while for theenzyme CYP2D6the most probable site ofmetabolism
are the para-hydrogens of the two O-benzyl groups.
In silico Caco-2 permeability and BBB permeation
The compounds were projected on the precalculated models of
VolSurf: Caco-2 cell absorption and BBB passage. Regarding to
the Caco-2 cell permeability, the 2D partial least squares (PLS)
score model can discriminate between permeable and less

DOI: 10.3109/14756366.2015.1021253
Simple chalcones and bis-chalcones ethers
11
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permeability. The PLS score space of the BBB model is divided
into, (i) a region (left) in which BBB ranges from negative values
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Acknowledgements
We would like to thank Dr A. Leo and Biobyte Corp., 201 West 4th Str.,
Suite 204, Claremont, CA 91711, USA for free access to the C-QSAR
program. We are also grateful to Prof. Gabriele Cruciani (Laboratory of
Chemoinformatics and Molecular Modeling, School of Chemistry,
University of Perugia, Italy) for kindly providing us the MetaSite and
Volsurf programs.
Declaration of interest
The authors report no conflicts of interest.
´
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