Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: A new target for a privileged structure

Article abstract of DOI:10.1016/j.ejmech.2014.09.039

The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α77 nicotinic function. The most potent compound (2,4,2-2,5-2-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

Full text of DOI:10.1016/j.ejmech.2014.09.039

European Journal of Medicinal Chemistry 86 (2014) 724e739
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Chalcones as positive allosteric modulators of
a7 nicotinic
acetylcholine receptors: A new target for a privileged structure
a
b
c
c
ꢀ
ꢀ
Beatriz Balsera , Jose Mulet , Asia Fernandez-Carvajal , Roberto de la Torre-Martínez ,
c
d
d
ꢀ
ꢀ
ꢀ
ꢀ
Antonio Ferrer-Montiel , Jose G. Hernandez-Jimenez , Judith Estevez-Herrera ,
d
e
e
a
ꢀ
ꢀ
Ricardo Borges , Andiara E. Freitas , Manuela G. Lopez , M. Teresa García-Lopez ,
a
a
b
ꢀ
~
ꢀ
ꢀ b
Rosario Gonzalez-Muniz , María Jesús Perez de Vega , Luis M. Valor , Lucie Svobodova ,
Salvador Sala b, Francisco Sala b, Manuel Criado b
,
*
a
ꢀ
Instituto de Química Medica (IQM-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
b Instituto de Neurociencias, Universidad Miguel Hernandez-CSIC, Ramon y Cajal s/n, 03050 Sant Joan d'Alacant, Spain
c Instituto de Biología Molecular y Celular, Universidad Miguel Hernandez, Av. de la Universidad s/n, 03202 Elche, Spain
d Departamento de Farmacología, Universidad de La Laguna, La Cuesta s/n, 38200 La Laguna, Tenerife, Spain
e
ꢀ
ꢀ
Instituto Teofilo Hernando, Departamento de Farmacología, Universidad Autonoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The a7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition dis-
Received 22 July 2014
Received in revised form
9 September 2014
Accepted 11 September 2014
Available online
orders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new
agents that target this receptor has great significance. Positive allosteric modulators might be advan-
tageous, since they facilitate receptor responses without directly interacting with the agonist binding
site. Here we report the search for and further design of new positive allosteric modulators having the
relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chal-
cones substituted with hydroxyl groups at defined locations were identified as optimal and specific
Keywords:
promoters of a
7 nicotinic function. The most potent compound (2,4,20,50-tetrahydroxychalcone, 111) was
a
7 Nicotinic receptor
further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer,
opening the way for future developments around the chalcone structure.
© 2014 Elsevier Masson SAS. All rights reserved.
Positive allosteric modulators
Chalcones
Neuroprotection
Analgesia
1. Introduction
distribution at the central and peripheral nervous systems and their
characteristic features, different of other neuronal nAChRs, such as
Nicotinic acetylcholine receptors (nAChRs) modulate fast syn-
aptic transmission in muscle and nerve cells. They exist as pen-
tamers with a variety of pharmacological and electrophysiological
properties, mainly due to the various combinations of subunits [1].
rapid channel activation and inactivation kinetics, marked sensi-
tivity to certain agonists (choline) and antagonists ( -bungarotoxin,
methyl-lycaconitine) and high Ca2þ permeability [3]. The latter
a
property suggests the involvement of
beyond their channel activity.
a7 nAChRs in processes
In neurons, twelve subunits (a2ea10 and b2eb4) have been iden-
tified and cloned. Functional nAChRs can be observed in heterolo-
Given that a7 nAChRs appear to be engaged in central and pe-
gous expression systems as a result of heteromeric or homomeric
ripheral diseases that involve, among others, cognition disorders
[4,5], schizophrenia [6], pain [7] and inflammation [8], there has
been considerable interest in developing therapeutic agents able to
target this receptor subtype. In this context, ligands that facilitate
agonist-induced responses but avoid direct interaction with the
acetylcholine (ACh) binding site are advantageous, as they should
not interfere with the action of the natural agonist on the different
nAChR subtypes and are less likely to promote desensitization or
downregulation. These positive allosteric modulators (PAMs) se-
assembly of subunits [2]. Within the latter, nAChRs made of only
a7
subunits have received much attention, given their widespread
Abbreviations: ACh, acetylcholine;
a-Bgt, a-bungarotoxin; CFA, complete
Freund's adjuvant; DMEM, Dulbecco's modified eagle's medium; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; nAChR, nicotinic
acetylcholine receptor; PAM, positive allosteric modulator; R/O, rotenone and oli-
gomycin-A.
* Corresponding author.
E-mail address: manuel.criado@umh.es (M. Criado).
lective for
a7 nAChRs have a variety of structures, from calcium ions
[9] and small molecules [10] to proteins [11]. They also have diverse
http://dx.doi.org/10.1016/j.ejmech.2014.09.039
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
725
potency, efficacy and influence on kinetics of agonist-evoked re-
sponses [12,13]. Two large groups of PAMs have been proposed.
Those belonging to group I increase the apparent peak current but
do not largely modify the kinetics of agonistemediated responses.
Galantamine, 5-hydroxyindole, genistein and ivermectin are
representative type I PAMs. PAMs belonging to group II increase the
peak current amplitude and slow the desensitization kinetics.
Representative compounds of this type are PNU-120596, TQS, and
A-867744 (Fig. 1).
compound and 200
meric 7 or heteromeric (
measured and compared with the ones induced only by ACh. For
nAChRs only one pool showed significant current increases (about
3-fold) respect to controls, whereas no current potentiation was
m
M ACh) to oocytes expressing either homo-
a
a4
b
2 and 4) nAChRs. Currents were
a3b
a
7
observed for
pool were individually analyzed as previously and a substance,
isoliquiritigenin, was identified as a specific PAM of 7 nAChRs and
a4b2 and a3b4 nAChRs. Components of the positive
a
named 1 in further analysis (Fig. 2). Compound 1 is a polyhydroxy
substituted chalcone (Fig. 2a)) that enhanced the current evoked by
ACh with little modification of its rising time course, but showing a
slower decay time course (Fig. 2b)). The effect of 1 is dose-
In spite of the advantages of PAMs, caution is needed in using
them as therapeutic agents to treat the disorders mentioned above.
This is mainly due to the high calcium permeability of a7 nAChRs
that in certain cases of excessive and/or sustained receptor acti-
vation could produce unwanted and even deleterious effects.
Therefore, it is important to continue the development of new
compounds that, apparently, might have characteristics similar to
previous ones, but can make available novel structures with ther-
apeutical potential. With this aim we started the screening of a li-
brary of small natural molecules looking for PAMs of different
nAChR subtypes heterologously expressed in Xenopus laevis oo-
cytes. A flavonoid with a chalcone structure, isoliquiritigenin
dependent with an EC50 of 11.6
6.5-fold about the current induced by 200
that is close to the EC50 (Fig. 2c)). The potentiating effect of 1 was
observed throughout a wide range of ACh concentrations (Fig. 2d)),
but not in the absence of agonist, indicating that 1 is not acting as
agonist itself.
m
M and a maximal activation of
mM ACh, a concentration
In our search for
a7 nAChR PAMs, the chalcone scaffold was
further explored with a collection of one hundred differently
substituted chalcones from a commercial source. As in the case of 1,
each compound was individually tested by co-application with ACh
(4,20,40-trihydroxychalcone), showed activity as specific PAM of
a7
nAChRs. This chalcone, present in plants like Glycyrrhiza and Dal-
bergia, has revealed activities against inflammatory [14], oxidative
[15], allergic [16] and carcinogenic [17,18] processes. Also, neuro-
protective effects have been reported [19,20]. Given the relevance
of a7 nAChRs in some of these processes, we decided to perform a
further study of a collection of commercial chalcones differently
to a7 nAChRs (see Table S1, supplementary data). Six compounds
(64, 87, 97, 98, 99, 101) showed activity as PAMs and were further
characterized. As discussed later in more detail, these compounds
have, at least, one OH group in ring A (See Table 1 heading formula),
but preferentially two OHs at positions 20,40 or 20,50. The most
effective was 97 (4,20,50-OH), about five-fold more than 1. Its only
chemical difference with respect to 1, is the presence of a hydroxy
substitution at the 50 position instead of the 40 one.
substituted, which allowed us to refine the molecular requirements
for a7 nAChR activation. Finally, the study of our own synthesized
compounds defined the position and substituents required for
optimal receptor activation. The chalcone with maximal activity
was then tested in different systems, showing its potential as
neuroprotective, analgesic and learning enhancer.
2.2. Neurosecretory effects of compound 97 on adrenal chromaffin
cells
Before continuing the search for compounds with improved
properties, we decided to test 97 in a cellular system where
cholinergic stimulation gives rise to a clearly measurable biological
response. For this purpose, the chromaffin cell of the bovine ad-
renal medulla is very appropriate, since ACh activates nAChRs
triggering catecholamine secretion, which can be amperometrically
detected. In our case, choline was used instead, in order to specif-
2. Results and discussion
2.1. Screening of a library of natural compounds and a collection of
synthetic chalcones
The Greenpharma Natural compound library (Prestwick Chem-
ical, France), composed of 240 natural compounds of small size, was
tested. To speed up the screening, fifty six pools of 4e5 different
ically activate
main cholinergic response in bovine chromaffin cells, which is the
one composed of 3, 4 and probably 5 subunits [21].
a7 nAChRs and not the nAChR responsible of the
compounds each were coapplied with ACh (10
mg/mL of each
a
b
a
Fig. 1. Chemical structures of representative PAMs of a7 nAChRs. Type I and type II PAMs are shown at the upper and lower rows, respectively.

726
B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
Fig. 2. Effect of compound 1 on
a7 nAChRs. a) Chemical structure of compound 1. b) Ionic currents recorded in a representative oocyte expressing human a7 nAChRs. Currents were
evoked by 600 ms applications of ACh 200
Concentrationeresponse relationship for the potentiating effect of 1 with ACh 200
estimated at 6.5, and an EC50 value of 11.6 M d) Acetylcholine concentrationeresponse relationships in the absence (circles) and in the presence (triangles) of 10
were normalized to the response obtained by ACh 1 mM in control conditions. Continuous lines represent fits to Hill equations, with estimated Imax of 1.3 and 4.3, and EC50 values of
176 M and 52 M, for control and compound 1, respectively.
m
M in the absence (continuous line) and in the presence (dotted line) of 10
M. Continuous line represents the fit to the Hill equation, with a maximal potentiating effect
M of 1. All data
m
M of 1 and recorded at a holding potential of ꢀ80 mV. c)
m
m
m
m
m
Fig. 3 shows the effect of compound 97 on the secretory re-
sponses elicited by choline. As expected, the application of this
conventional ClaiseneSchmidt aldol condensation of the desired
benzaldehyde and acetophenone in EtOH using aqueous NaOH
(40%) as base [22]. The methoxy substituted chalcones were ob-
tained in moderate to excellent yield (41e87%) (Scheme 1). In the
referred condensation conditions, the formation of the E isomer as
a unique product was observed in all cases, as shown by the NMR
data. Demethylation of the methoxy substituted compounds was
accomplished by treatment with BBr3 in DCM under inert atmo-
sphere, which led to the polyhydroxylated analogues after purifi-
cation (Scheme 1) [23]. As described by McOmie et al., the amount
of BBr3 equivalents necessary for completing the deprotection de-
pends on the number of methyl ether groups to be deprotected.
They used at least 1 equiv for each -OMe group, and recommended
one extra equiv for every potentially basic N or O present in the
molecule [23]. In our case, when using the recommended amount
of BBr3, semideprotected products were isolated. For instance,
compound 104 led to formation of the desired tetrahydroxy chal-
cone 111, but the partially deprotected compound 112 was also
isolated. For compound 105 the use of 6 equiv of BBr3 led to the
isolation of partially deprotected compounds 113 and 114, while the
totally deprotected derivative was never isolated, not even using
12 equiv of the reagent. Based on these initial results, the use of
2 equiv of BBr3 for each eOMe, O or N present in the molecule was
decided, in an attempt to obviate this partial deprotection problem.
Using the modified conditions, compounds 106, 107 and 108 led to
the desired 115, 116 and 117, respectively, in moderate yields
(Scheme 1).
a
7
agonist produced modest secretory responses when compared to
the one observed upon Kþ depolarization (Fig. 3a)). Nevertheless,
10 min incubation with 97 largely increased the catecholamine
output elicited by choline. In order to study in detail the effects of
97 in the exocytotic process, we used single cell amperometry.
Fig. 3b) shows a typical recording of an amperometrical trace
resulting of choline application in the presence of 30 mM of 97. A
large increase in the number of secretory spikes was observed and
consequently the secretory response became larger (Fig. 3c)). Thus,
it is evidenced that 97 enhances the function of a7 nAChRs in a
cellular system.
2.3. Synthesis of new chalcone derivatives
Except for the presence of a methyl group at position 20 in 64,
the rest of tested substituents in ring A were inefficient for the
potentiating function of chalcones (Table S1). In fact, most of the
chalcone PAMs contained hydroxy and, to a lesser degree, methoxy
substituents. For this reason, we decided to analyze the effect of the
number and position of hydroxy substituents of chalcones on their
PAM function. With this aim, new compounds were synthesized.
Taking the best compounds from the initial screening as the
starting point, we prepared a small collection of hydroxychalcone
derivatives differently substituted in rings A, B (See Table 1 heading
formula) or both. Additionally, to confirm the importance of the
hydroxy groups of ring B, some chalcones bearing amino or ace-
tylamino groups in position 4 were also synthesized. Finally, the
incorporation of a 5-hydroxyindol moiety replacing ring B was also
contemplated in order to compare with the corresponding phenyl
derivative.
Attempts to prepare the chalcones monohydroxy substituted in
ring A, through the preparation of the corresponding m- and p-
OMe derivatives 109e110 and further deprotection, failed to lead
to the desired OH compounds. In all cases, complex mixtures were
obtained in which the desired compounds were not identified. The
direct condensation of the 2,4-dihydroxybenzaldehyde with the
corresponding monohydroxy substituted acetophenone using
The preparation of the methoxy substituted chalcones (1,3-
diaryl-2-propen-1-ones) 104e110 was achieved by the

B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
727
Table 1
Structures of synthetic chalcones and their potentiation of
a
7 nAChR currents.a
Compdb
2
3
4
5
6
20
30
40
50
60
Imax
EC50 (m
M)c
c
Entry
1
2
3
4
5
6
7
8
22
23
24
25
26
98
99
1
97
OH
H
H
H
H
OH
OH
H
H
H
OH
OH
OH
OH
OH
OH
H
OH
H
OH
OH
OH
H
H
OCH3
OH
OH
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
OCH3
OCH3
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
OH
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
OH
H
OH
H
OH
H
H
OH
H
OH
H
OH
OH
OH
OH
H
H
OH
H
H
H
H
H
H
H
OH
H
OH
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
OH
H
H
H
H
H
H
H
H
H
H
H
e
e
e
e
e
e
e
e
e
e
H
H
2.6 0.18
3.4 0.24
6.5 0.8
33 9.6d
e
4
0.9
1.9 0.4
OH
OH
H
OH
OH
OH
OH
H
11.6 1.4
25 2.8d
e
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
96
125
126
127
111
115
117
101
116
100
123e
122e
138e
64f
19.7 6.1
34.5 2.2
6.1 1.3
38d
H
H
9
0.9
H
8.1d
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
H
OH
H
34 5.5
3.2 0.24d
e
3.3 1.6
8.9d
H
e
OH
OH
OH
OH
OH
OH
OCH3
OH
OCH3
OCH3
OCH3
2.8 0.15
14.5 0.3
e
4
1.3
67 7.1
e
H
e
e
H
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
11.9 2.1
2.3 0.2
2.1 0.32
6.5 0.4
8.6 1.6
7.8 0.52
e
5.4 1.5
10.7 1.6
7.3 1.4
17.2 1.9
3.9 1.4
2.6 1.2
e
OH
CH3
OH
OH
OH
OH
OH
OH
OH
87f
112f
113f
114
133
134
137
OCH3
OH
H
H
H
H
H
H
H
NHAc
NH2
e
e
H
H
e
e
5-OH-indole
3
0.2
1.9 1.3
a
The substituents 2 to 6 and 20 to 60 refer to the positions indicated at the chalcone structure shown above.
Compounds named with numbers 22-101 originated from the first screening of commercial chalcones (see Table S1, Supplementary data), whereas the rest (111-138)
b
were synthesized by us.
c
Values derived from the corresponding dose response curves. Responses were recorded at ꢀ80 mV and normalized with respect to that shown by only ACh (0.2 mM). Data
are the mean SD, with n ꢁ 6.
d
Extrapolated values, as no response saturation could be reached because low solubility at higher concentrations.
Compounds 123, 122 and 138 are Z-isomers of compounds 127, 126 and 111 respectively.
These compounds are the only ones with some substituents different from hydroxyls that showed activity as PAMs.
e
f
Fig. 3. Compound 97 potentiated the secretory responses triggered by choline. a) Thirty-second applications of the
of 97 (30
a7 agonist choline (100 mM, arrows) were used to test the effects
m
M) (see inset for chemical structure) in perifused bovine chromaffin cells recorded by on-line electrochemical detection. A final 30 s pulse of 70 mM high potassium
solution (Kþ) was applied at the end of the experiment to assure the cell responsiveness. b) The amperometrical measurements confirmed that the increase in the total release was
due to an increase in the number of secretory spikes (compare upper and lower parts of this panel, without and with compound 97, respectively) rather than to an increase of
individual net charge. Each spike of oxidation trace came from single exocytotic events. c) Cumulative secretion from experiments as described in b) are summarized with
means SEM. **p < 0.01 Student t test. The number of cells used on each condition is expressed in parentheses.

728
B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
Scheme 1.
different reagents and conditions also failed to lead to the ex-
pected polyhydroxylated compounds. The use of Ba(OH).28H2O
[24] as an alternative to NaOH as base, as well as the acid catalysis
with Lewis acids (Yb(OTf)3, AlCl3) [25] was also tried without
success. Only when F3B.Et2O was used as reagent, the condensa-
tion product was isolated [26]. This procedure worked well for the
m- and p-hydroxy acetophenones, but not for the o-hydroxy
substituted derivative. Analysis of the NMR spectra of the isolated
compounds 122 and 123 indicated that these chalcones corre-
sponded to the Z-isomers (Scheme 2), in contrast with the results
obtained when using NaOH.
Given the lower reactivity of the free hydroxy substituted al-
dehydes in the aldolic condensation, the protection of the 2,4-
hydroxy groups of 121 was necessary. For this purpose the MOM
protecting group was chosen [27]. Condensation of the protected
aldehyde 124 with either o-, m-, or p-hydroxyacetophenone and
further treatment with HCl to remove the MOM group led to the
formation of the expected compounds 125e127 (Scheme 3).
In the case of the o-hydroxy derivative 125, flavanone 128 was
also isolated after treatment with HCl. The formation of the flava-
none isomer in acid medium is in agreement with the study
performed by J. Mai et al. for chalcones o-hydroxy substituted in
ring A [28].
Condensation of the 2,5-dimethoxyacetophenone 129 and the
4-acetylaminobenzaldehyde 130, led to 4-acetylamino-chalcone
131 along with the 4-amino derivative 132, resulting from the
partial deacetylation of 131 during the coupling reaction. Removal
of methoxy groups in 131 gave 133, while treatment of 132 with
BBr3 (4 equiv) allowed the isolation of the fully deprotected com-
pound 134 (Scheme 4). A partially deprotected chalcone was also
detected in this latter reaction (HPLC-MS data).
Finally, substitution of ring B by an indol system was contem-
plated, both to study the effect of the replacement of one of the
phenyl rings by a heterocyclic system, and also because of the ac-
tivity shown by the 5-hydroxyindol as Type I PAM of a7 nAChR
[29,30]. Condensation of 2,5-dimethoxyacetophenone 129 with 5-
methoxyindol-3-carboxaldehyde 135 led to the formation of chal-
cone 136, which upon demethylation gave the expected trihydroxy
derivative 137 (Scheme 5).
All products were purified by flash or medium-pressure
chromatography over silica gel or in reverse phase, as indicated
in each case (Experimental section). Final compounds were
Scheme 2.

B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
729
Scheme 3.
Scheme 4.
crystallized whenever possible. Detailed spectroscopic analysis
2.4. Structureeactivity relationship (SAR) analysis of polyhydroxy-
substituted chalcones
(1H NMR, 13C NMR, MS) permitted the unambiguous character-
ization of the prepared compounds. Identification of the position
of OH and OMe groups in partially deprotected compounds was
accomplished on the basis of bidimensional NMR experiments
(HSQC, HMBC).
Table 1 summarizes the study of compounds in which hydroxy
groups were present at different positions of the chalcone struc-
ture. Information about a few compounds previously analyzed (see
Table S1) has been integrated with the data corresponding to the
new synthesized derivatives to facilitate the SAR discussion.
Considering the whole collection of compounds tested, it seems
that the presence of at least one hydroxy group in each aromatic
ring of the molecule is necessary, but not sufficient for behaving as
While studying the solubility of compound 111 in PBS buffer/
2.5% DMSO, the formation of a certain amount of the Z-isomer was
observed (starting from a 100% E-isomer solution, the formation of
up to 35% of Z-isomer was observed in 5 days, according to HPLC-
data). This transformation was performed at preparative scale to
isolate the Z-isomer (138) and check its behavior in front of the
a
7
a7 nAChR PAM. Thus, compounds totally unsubstituted at ring A, 8
nACh receptor. The photo-isomerization of chalcones has been
previously reported [31], being pH dependent and its feasibility has
also been correlated with the substituents in the aromatic rings
[32].
and 55 (Table S1), as well as non substituted ring B analogues, 25,
26, and 96 (Table 1) were inactive. Compound 24, lacking the 40-OH
group of 1, was ineffective as PAM of
a7 nAChRs. Similarly, other
analogues having only one OH at each ring, like 22 and 23, were

730
B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
Scheme 5.
also inactive. By contrast, trihydroxylated compounds showed
potentiating activity if, at least, one hydroxy group is present in
each of the aromatic rings. As in 1, the combination of mono-
hydroxylated ring B, at either 2 or 4 position, and ring A 20-40- or
20,50-OHs, led to compounds with significant activity (Table 1, en-
tries 6e9). In general, the 4-OH substitution is better than the 2-OH,
and the 20,50-OH combination is preferred over the 20,40 one. In
agreement with this, maximal activity was observed for the pre-
viously studied 97, which has hydroxy groups at positions 4, 20 and
50. The double substitution at ring B (positions 2 and 4) combined
with a single substitution at ring A (positions 20, 30 or 40, com-
pounds 125, 126 and 127, respectively) also produced compounds
able to enhance currents with moderate/high effectiveness,
although in a lesser extent than with 97. For this reason, in the next
step we explored tetrahydroxylated compounds. First, we decided
to combine the substitutions of the best compounds 97 and 125,
namely two OHs at positions 2 and 4 of ring B with the ones present
in ring A of 97 (20 and 50). This yielded compound 111, which had
the highest efficacy, close to 97, but with higher potency. Re-
quirements for hydroxy groups at the mentioned positions were
evident when the tetrahydroxylated compound 117 was tested.
Thus, the change of the OH from position 4 to 6 (ring B), keeping the
rest as in 111, resulted in an inactive compound. Merging one or two
OHs in ring B and three hydroxyl groups in ring A (either at 20,40,50
or 20,40,60) resulted in tetra- and pentahydroxylated compounds
(100, 101, 115 and 116) that reduced drastically or prevented both,
effectiveness and potency. Curiously, the combination of 2,6-OH
OHs at positions 20 and 50 (like 111) whereas methoxy groups are
concentrated at different positions of ring B, suggesting that the
latter can accommodate them without largely affecting PAM ac-
tivity. Nevertheless, the presence of other substituents in this ring,
such as the acetylamino and amino groups in position 4 of com-
pounds 133 and 134, respectively, abolished their PAM activity.
2.5. A comparison between compounds 97 and 111
Fig. 4 features the main similarities and differences between
compounds 97 and 111, the most effective chalcones. When any of
them was applied to oocytes expressing human a7 nAChRs, a large
potentiating effect was observed in ACh-evoked ionic currents
(Fig. 4ced) but, unlike compound 1, the currents decayed very
slowly, so the changes in kinetics were similar to those found with
type II PAMs. A detailed analysis of the kinetics of a7 nAChR cur-
rents induced by these and other chalcones is out of the scope of
the present study. Nevertheless, the diversity of kinetic responses
observed with different chalcones (for instance, 1 and 137 behave
as type I whereas 97 and 111 are type II PAMs) might offer a unique
opportunity to link both aspects,
preparation).
a7 kinetics and PAM structure (in
Both chalcones 97 and 111 showed similar concentration-
dependent effectiveness (>30-fold potentiation at 200 M of ACh,
which is close to its EC50), but 111 was about an order of magnitude
more potent than 97, as the potentiating EC50 were 3.3 M and
25 M for 111 and 97, respectively (Fig. 4eef). Potency of 111 is
comparable to the one observed in similar conditions for other
PAMs, such as PNU-120596 (EC50 3.8e5.8 M) [12,30], TQS (5.5 M)
[30] and A-867744 (1.1 M) [33]. However, maximum efficacy is
m
m
substituents in ring
B
resulted detrimental for activity, as
m
deduced from the lack of potentiation observed with compounds
114 and 117. All these data confirm that the convergence of four OH
groups, preferentially at positions 2, 4, 20 and 50, constitutes the
optimal substitution for activity.
Three Z-isomers, among them the 111 isomer, were also tested.
Compound 123 (isomer of 127) did not show any activity. Com-
pound 122 behaved with moderate activity, like its E-isomer 126.
Finally, 138 was clearly less effective than its isomer 111. Therefore,
no gain is apparent with the Z-isomers.
m
m
m
probably higher for 111 since we observed a 3400% maximal
potentiation over the response with 0.2 mM ACh, whereas
maximum efficacy values were only 541% for 5-hydroxyindol, 418%
for TQS and 455% for PNU-1205964 when using 0.1 mM ACh [30].
The potentiating effect was not much dependent on ACh con-
centration. As shown in Fig. 4geh, when 1 mM of each chalcone was
Although most of the chalcones able to act as PAMs were pol-
yhydroxylated, we detected four compounds with methoxy sub-
stituents having moderate potentiating activity. The three more
effective ones (87, 112 and 113) have in common the presence of
co-applied with ACh the resulting ACh concentrationeresponse
relationships were only slightly shifted to the left, as the ACh EC50
values decreased from 176 mM (control) to 125 mM and 56 mM for
111 and 97, respectively.

B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
731
Fig. 4. Comparison between compounds 97 (left panels) and 111 (right panels). aeb) Chemical structure of chalcones 97 and 111. ced) Ionic currents recorded in representative
oocytes expressing human 7 nAChRs. Currents were evoked by 600 ms applications of ACh 200 M in the absence (continuous line) and in the presence (dotted line) of 30 M of 97
a
m
m
or 3
200
m
M of 111. All currents were recorded at a holding potential of ꢀ80 mV. eef) Concentrationeresponse relationships for the potentiating effect of chalcones co-applied with ACh
m
M. Continuous lines represent the fit to the Hill equation. Maximal potentiating effects were estimated as 33 and 34-fold, for 97 and 111, respectively, with EC50 values of
M for 97 and 111, respectively. Notice that data for 97 are estimates since low solubility beyond 100 M precluded the use of higher concentrations able to saturate
the response. geh) Acetylcholine concentrationeresponse relationships in the absence (circles) and in the presence (triangles) of 1 M of each chalcone. All data were normalized to
the response obtained by ACh 1 mM in control conditions. Continuous lines represent fits to Hill equations, with estimated Imax and EC50 values of 1.3 and 176 M (control), 2.9 and
56 M (with 97), and 8.3 and 125 M (with 111).
25
m
M and 3.3
m
m
m
m
m
m
Consistent with their action as PAMs of
97 and 111 were unable to activate the later in the absence of ACh.
Moreover, when tested for selectivity for 4 and 2 nAChR
a
7 nAChRs, compounds
systems, before continuing the pharmacological exploration, some
preliminary studies on the toxicity of the best compounds of the
series were performed.
a
3
b
a4b
subtypes, they did not show activity as PAMs nor as agonists. Only
a slight decline in the current evoked by ACh was observed
(Table 2).
We first evaluated the cytotoxicity of 97 and 111 by performing
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay on HEK cells. The assay was made using a
range of chalcone concentrations according to their EC50 values.
Compound 97 (Fig. S1a) showed low toxicity even at high con-
2.6. Toxicity studies of selected compounds (97 and 111)
centrations (up to 30
mM) whereas 111 (Fig. S1b) was completely
The conjugated double bond in chalcones is a drug structural
alert, since it can act as Michael acceptor toward nucleophiles.
Moreover, chalcones display different biological activities, and can
be considered within the so called privileged skeletons [34].
Because of this reactivity and promiscuity toward biological
non toxic at concentrations up to 10
m
M. The lack of toxicity for this
concentration was further validated in an alternative cell model, a
primary culture of hippocampal postmitotic neurons (Fig. S2),
delimitating a possible safe range of concentrations for compound
111 in future chronic exposures.

732
B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
Table 2
Activity of 97 and 111 at Human
a
3
b
4 and
a
4
b
2 nAChRs Expressed in Xenopus
Oocytes.a
Receptor
Compound
97
111
a
a
a
3
4
7
b
b
4
2
87 2.6 (5)b
83 4.9 (3)d
93 1.2 (3)c
98 1.8 (3)
1258 95 (7)b
2760 220 (5)b
a
Responses in the presence of 10 mM of the indicated compound and 50 mM ACh
were recorded at ꢀ80 mV and normalized with respect to that shown by only ACh
(100). For comparative purposes, 7 nAChRs were also tested in the same experi-
ment with 200 M ACh and the same concentration of compounds. Data are the
a
m
mean SD, with n indicated in parentheses.
b
p < 0.001.
p < 0.01.
p < 0.05.
c
d
To anticipate possible off-target adverse effects, which can occur
by interaction of our chalcone derivatives with other unintended
targets, we evaluated the effects of the most promising compounds
on isolated organs and arterial blood pressure. Compounds 97 and
111 had no systemic effects on isolated organs of the rat such as
atria (force or beating frequency), vas deferens (spontaneous and
voltage-stimulated contraction), ileum (peristaltic force or fre-
quency) and phrenic-diaphragm muscle preparation, at concen-
Fig. 5. Cytoprotection against cell death induced by 30
oligomycin-A. HT22 cells were preincubated either with 1 or 10
for 24 h, followed by R/O treatment for 24 h. Similar cultures were preincubated with
0.1 -Bgt for 10 min before treatment with compound 111. Each column represents
the mean þ S.E.M. of four different cell batches seeded in triplicate wells. A positive
control was performed by incubating with 10 M of the 7 agonist PNU-282987 in the
same conditions that compound 111. Pretreatment with 0.1 -Bgt was also used in
m
M
rotenone and
1 mM
m
M of compound 111
m
M a
m
a
mM a
this case to prevent the effect of the agonist. Statistical analysis was performed by one-
trations ranging from 0.01 to 100
Compound 111 promoted a dose-dependent reduction in the
heart rate, which was evident after the i.v. infusion of 1 g/kg
(Fig. S3) and a fall in the blood pressure although it was only sig-
nificant at the higher dose tested (100 g/kg). Given the lack of
effect of this compound on isolated atria, its action in vivo probably
reflects a central role or a stimulation of parasympathetic ganglia.
Furthermore, given the antioxidant properties of some hydrox-
ychalcones, including 1 [15], the blood pressure decline induced by
111 could also be related to a decrease in the oxidative stress,
correlated itself to endothelial-dependent relaxation of blood ves-
sels [35].
mM (data not shown).
way ANOVA, followed by NewmaneKeuls test. ***P < 0.001 as compared with the basal
group;
P < 0.001 as compared with R/O group; ꢂ P < 0.05, ꢂꢂꢂ P < 0.001 as
AAA
compared with the corresponding group without preincubation with a-Bgt.
m
m
indicate that 111 deserves further investigations as a neuro-
protective compound.
2.8. Analgesic activity of 111 compared to a typical type II PAM
In general, a low toxicity profile was found for these compounds,
encouraging further pharmacological studies.
Since PAMs of
a7 nAChRs, especially the type II PAM PNU-
120596, are able to produce anti-nociceptive effects [44e46], we
next addressed the question of whether the most potent chalcone,
111, might reduce the thermal or mechanical hyperalgesia that re-
sults from inflammation induced by complete Freund's adjuvant
(CFA). Intraplantar injection of CFA produces a significant inflam-
mation that might last up to ꢁ20 days [47]. Thermal and me-
chanical hyperalgesia in the inflamed paw could be seen 24 h after
CFA injection, as evidenced by the 2-fold reduction in the latency
time response to a mildly noxious thermal or mechanical stimulus
(Fig. 6).
We compared the effects of PNU-120596 and 111 in the CFA test.
Lack of antinociceptive activity in the thermal hyperalgesia test was
observed up to 4 h after the administration of both compounds
(Fig. 6(a and b), respectively). In contrast, they almost abolished
mechanical hyperalgesia at doses of 10 mg/kg (Fig. 6(c and d)). The
analgesic effect was transient and disappeared completely 2 h after
drug inoculation, probably because of pharmacokinetic drug
clearance. Taken together, these results indicate that the level of
analgesic activity exhibited by 111 was comparable to that exerted
by PNU-120596. In spite of this parallelism, we cannot discard the
possibility that 111 is also acting on other analgesic-related path-
ways. For instance, some chalcone derivatives like 20,50-dimethoxy-
3,4-dihydroxychalcone have been reported as potent 5-
lipoxygenase and cyclooxygenase inhibitors [48], whereas 20,3-
dihydroxy-, 20,50-dihydroxy-4-chloro-, and 20,50-dihydroxy-chal-
cones showed remarkable inhibitory effects on hind-paw edema
induced by polymyxin B [49].
2.7. Neuroprotection against cell death induced by rotenone and
oligomycin-A (R/O)
It is well known that treatment with rotenone and oligomycin-A
induces an oxidative impairment through the blockade of mito-
chondrial complexes I and V, respectively [36]. The potential
cytoprotective effect of 111 on this cytotoxicity protocol was veri-
fied in HT22 cells, a hippocampal neuronal cell line having func-
tional cholinergic properties [37] and expressing
a7 nAChR
subunits (Fig. S4). HT22 cells exposed for 24 h to R/O reduced their
viability to 66% compared to control cells (100%), measured as MTT
reduction (Fig. 5). Compound 111 preincubated for 24 h, and
coincubated with 30
additional 24 h-period, significantly increased viability of HT22
cells by 77 and 83% at 1 and 10 M, respectively (Fig. 5). This effect
appears to be mediated by 7 nAChRs since it was prevented by
previous incubation with 0.1 -Bgt, an almost irreversible
antagonist. Moreover, at the same concentration than 111 (10 M),
the specific 7 nAChR agonist PNU-282987 showed a similar neu-
roprotective effect (84% viability), also prevented by -Bgt. These
results are in line with previous ones showing that 7 nAChR ag-
mM rotenone and 1 mM oligomycin-A for an
m
a
mM a
m
a
a
a
onists such as PNU-282987 [38,39], SEN12333 [40] and DMXBA
[41], as well as PAMs such as PNU-120596 [42] and isoliquiritigenin
[19,20,43] display neuroprotective properties. Hence, our data

B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
733
Fig. 6. Effects of PNU-120596 (left panels) and 111 (right panels), in the CFA-induced paw inflammation model. Time course of thermal (a,b) and mechanical (c,d) hyperalgesia in
rats after injection of CFA 0.5 mg/mL (50 L) into the left hind paw with and without administration of compound (10 mg/kg i.v.) The diagram shows the paw withdrawal latencies
m
in response to thermal or mechanical stimulation (n ꢁ 6 rats/group). Data are given as mean SEM with n ¼ 6. 2-way ANOVA with Bonferroni post hoc test. *P < 0.05; **P < 0.01,
***P < 0.001.
2.9. Effect of compound 111 in learning ability
Considering the cognitive effects observed upon
a7 nAChR
stimulation [5], we tested 111 on the water maze Morris' test. We
chose old mice (14 months old) that were tested along 5 successive
days; the results obtained the last day are summarized in Fig. 7.
Each day, two hours before the test, animals received vehicle
(control, C) or 1, 10 or 100
the learning performance was observed, being significant at the
10 g/kg dose. In this case the time required to reach the hidden
mg/kg compound 111. An improvement in
m
platform was reduced to half, suggesting a positive effect of 111 in
cognitive functions as previously observed for other PAM [50].
3. Conclusions
A library of natural compounds was screened in vitro for PAMs of
a
7 nAChRs. A trihydroxy-substituted chalcone, isoliquiritigenin,
showed moderate effectivity as PAM. Further screening of differ-
ently substituted chalcones allowed a detailed SAR analysis that
oriented the synthesis of new compounds with improved efficacy
and potency, especially compound 111, a chalcone tetrahydroxy-
lated at positions 2, 4, 20 and 50. The maximal potentiation was
above 30-fold the current observed with only ACh, with an EC50
Fig. 7. Effect of compound 111 on the spatial memory. Morris water maze was used to
test the effect of 111 on the ability of 14 months old mice to remember the situation of
a hidden platform. The test was repeated daily during the 5 days of treatment with i.p.
injections of vehicle (C) or the indicated doses of 111 administered one hour before the
experiment (n ¼ 5e16 mice). **p < 0.01, Dunnet test.
value of 3.3
also modified in the presence of 111, especially current decay time
was much larger (i.e. 111 inhibited 7 receptor desensitization).
mM. The macroscopic kinetics of a7 nAChR current was
a
related derivatives should be considered useful therapeutic tools, as
our analgesia experiments indicate. Moreover, chalcone 111 pro-
tected against the R/O-induced cell death of hippocampal neuronal
HT22 cells, suggesting its potential as neuroprotective during
increased oxidative stress.
Compound 111 not only showed lack of cytotoxicity on HEK cells
and primary hippocampal neurons but also no systemic effects on
different isolated organs of the rat, except for a slight decline in
blood pressure at high doses. Given the involvement of
a7 nAChRs
in pain and inflammation processes [7,8], compound 111 and future

734
B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
Cognitive impairment is observed upon deficits in cholinergic
neurotransmission driven, at least in part, by 7 nAChRs [4,5].
Presumably as a consequence of its potentiation of 7 nAChRs,
compound 111 showed an improvement in cognitive performance
when used in aged mice during the Morris' test. These results
suggest the potential therapeutic utility of 111 in aging-related
neurodegenerative conditions like Alzheimer's disease.
4.1.3. General procedure for the synthesis of polyhydroxylated
chalcones
a
a
To a previously cooled solution (0 ꢄC) of the corresponding
methoxy substituted chalcone (0.609 mmol) in dried DCM (15 mL),
a 1 M solution of BBr3 in DCM (2 equiv for each MeO- group plus 2
more equiv for every group containing a potentially basic N or O)
was slowly added under Ar atmosphere. After stirring 24e48 h at
room temperature under Ar, H2O was added to the reaction
mixture. If a solid precipitate was formed, it was separated by
filtration and washed with H2O and DCM. When no precipitate was
observed, the product was extracted with EtOAc. The organic ex-
tracts were washed with H2O and brine, dried over Na2SO4 and
then evaporated. The crude product was purified as indicated in
each case.
Because of the high efficacy in potentiating a7 nAChRs and the
consequent promising effects as neuroprotective, analgesic and
cognitive enhancer, we conclude that 111 and other chalcone de-
rivatives represent a novel class of modulators with relatively
simple structure to be used as departure point in future
developments.
4. Experimental section
4.1.3.1. (E)-1-(20,50-Dihydroxyphenyl)-3-(2,4-dihydroxyphenyl)-2-
propen-1-one (111). Prepared from 104 using 10 equiv of Br3B,
which led also to the formation of 112. Red solid (0.048 g, 29%) mp
180e182 ꢄC. Purified twice by reverse-phase MPLC; gradient: 2 to
25 of CH3CN in H20 þ 0.05% TFA. HPLC-MS: tR ¼ 3.89 min (5 min
4.1. Chemistry
4.1.1. General
All reagents were of commercial quality. Solvents were dried
and purified by standard methods. Analytical TLC was performed
on aluminum sheets coated with a 0.2 mm layer of silica gel 60
F254. Silica gel 60 (230e400 mesh) was used for flash chroma-
tography. Some compounds were purified by MPLC using SNAP 12g
KP-C18-HS cartridges on an ISOLERA ONE (Biotage) apparatus.
Mixtures of CH3CN (solvent A), H2O þ 0.05% TFA (solvent B) were
used as mobile phase. Unless indicated, all compounds were iso-
lated in a purity ꢁ95% (HPLC data). Analytical HPLC-MS was per-
formed on a Waters equipment coupled to a single quadrupole ESI-
MS (Waters Micromass ZQ 2000) using a reverse-phase SunFire™
gradient). 1H NMR (400 MHz, DMSO-d6)
d: 6.33 (dd, 1H, J ¼ 8.6,
2.3 Hz, 5-H), 6.40 (d, 1H, J ¼ 2.3 Hz, 3-H), 6.81 (d, 1H, J ¼ 8.8 Hz, 30-
H), 7.00 (dd, 1H, J ¼ 8.8, 2.9 Hz, 40-H), 7.41 (d, 1H, J ¼ 2.9 Hz, 60-H),
7.67 (d, 1H, J ¼ 15.5 Hz, Ha), 7.67 (d, 1H, J ¼ 8.6 Hz, 6-H), 8.04 (d, 1H,
J ¼ 15.5 Hz, Hb), 9.14, 10.07, 10.33 and 12.24 (s, 1H, OH). 13C NMR
(100 MHz, DMSO-d6)
114.44 (C), 116.26 (C
(C-6), 141.37 (C
d
:102.48 (C-3), 108.20 (C-5), 113.25 (C-60),
a
),118.28 (C-30),120.58 (C),123.96 (C-40), 131.29
b
), 149.28 (C), 154.96 (C), 159.62 (C), 161.94 (C),
193.31 (CO) ppm. EM (ESIþ): m/z 273.5 (MþH)þ. HRMS (ESIþ) m/z
calcd for C15H12O5 [Mþ1]þ 273.0757, found 273.0763.
C18 4.6 ꢃ 50 mm column (3.5
mm) at a flow rate of 1 mL/min and by
4.1.3.2. (E)-1-(20,50-Dihydroxyphenyl)-3-(2,4-dimethoxyphenyl)-2-
propen-1-one (112). Prepared from 104 using 5 equiv of Br3B which
led also to the formation of 111. Red oil (0.45 g, 13%). Purified by
column chromatography, eluent: CH2Cl2: MeOH, 30: 1. HPLC-MS:
tR ¼ 9.23 min (10 min gradient). 1H NMR (400 MHz, DMSO-d6):
using a diodo array UV detector. Mixtures of CH3CN þ 0.08% formic
acid (solvent A) and H2O þ 0.1% formic acid (solvent B) were used as
mobile phase (gradient of 15e95% of A in B in 5 or 10 min, as
indicated in each case). HRMS (EIþ) was carried out in an Agilent
6520 Accurate-Mass Q-TOF LC/MS equipment. NMR spectra were
recorded on a Varian-INOVA 300, a Bruker-AVANCE 300, a Varian-
MERCURY 400, a Varian-INOVA 400 or a Varian System 500 spec-
trometer operating at 300, 400 or 500 MHz for 1H and at 75, 100,
and 125 MHz for 13C recording. To confirm the NMR peak assign-
ments, COSY and HSQC experiments were performed when
necessary (For H and C numbering see Table 1 heading formula).
Microwave assisted reactions were carried out in a Biotage Initiator
Classic synthesizer. Melting points were determined on a Mettler
MP70 apparatus and are uncorrected.
d
3.85 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.646.64 (m, 2H, 3-H and 5-
H), 6.82 (d, 1H, J ¼ 8.9 Hz, 30-H), 7.01 (dd, 1H, J ¼ 8.9, 2.8 Hz, 40-H),
7.44 (d, 1H, J ¼ 2.8 Hz, 60-H), 7.76 (d, 1H, J ¼ 15.6 Hz, Ha), 7.91 (d, 1H,
J ¼ 8.4 Hz, 6-H), 8.04 (d, 1H, J ¼ 15.6 Hz, Hb), 9.16 and 12.08 (s, 1H,
OH). 13C NMR (100 MHz, DMSO-d6)
d: 46.62 (OCH3), 46.92 (OCH3),
89.34 (C-3), 97.53 (C-5), 105.68 (C-60), 106.75 (C-30), 109,11 (C),
109.58 (Ca), 111.69 (C), 115.18 (C-40), 121.85 (C-6), 130.66 (Cb), 140.39
(C), 145.88 (C), 151.28 (C), 154.48 (C), 184.22 (CO) ppm. EM (ESIþ):
m/z 301.3 (MþH)þ. HRMS (ESIþ) m/z calcd for C17H16O5 [MþH]þ
301.1071, found 301.1081.
Compounds 1e101 were commercially available and purchased
to Indofine, New Jersey, USA.
Structure of compounds 104,105,109, 110, 127,131 and 132 were
confirmed by comparison of their physical data with those reported
in the literature.
4.1.3.3. (E)-1-(20,50-Dihydroxyphenyl)-3-(2-hydroxy-4,6-
dimetoxyphenyl)-2-propen-1-one (113). Prepared from 105 using
6 equiv of Br3B, which led also to the formation of 114. Red oil
(0.019 g, 9%, 91% purity). Purification by MPLC in reverse-phase,
eluent: gradient from 10 to 40% of A in B. HPLC-MS: tR ¼ 4.41 min
4.1.2. General procedure for the synthesis of methoxy-substituted
chalcones
(5 min gradient). 1H NMR (400 MHz, DMSO-d6)
d: 3.79 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 6.17 (s, 1H, 3-H), 6.18 (s, 1H, 5-H), 6.81 (d,
1H, J ¼ 8.9 Hz, 30-H), 6.99 (dd, 1H, J ¼ 8.9, 2.9 Hz, 40-H), 7.23 (d, 1H,
J ¼ 2.9 Hz, 60-H), 7.93 (d, 1H, J ¼ 15.6 Hz, Ha), 8.22 (d, 1H, J ¼ 15.6 Hz,
Hb), 9.24, 9.35 and 12.34 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6)
To a solution of the conveniently substituted benzaldehyde
(1.91 mmol) in EtOH (15 mL), the desired acetophenone
(1.66 mmol) was added. The solution was cooled at 0 ꢄC, and an
aqueous solution of NaOH 40% (1.87 mL) was slowly added. After
15 h of stirring at room temperature water was added and the
organic solvent was removed at reduced pressure. The organic
phase was extracted with EtAcO, and the organic extracts were
washed with H2O and brine. After drying with MgSO4, the solvent
was removed to dryness and the resulting residue crystallized from
MeOH. Characterization data for all new methoxy-substituted
chalcones can be found in the SI.
d: 55.35 (OCH3), 56.03 (OCH3), 90.43 (C-3), 93.77 (C-5), 104.47 (C),
113.65 (C-60), 118.19 (Ca), 118.57 (C-30), 120.43 (C), 124.04 (C-40),
137.08 (Cb), 149.27 (C), 155.11 (C), 160.87 (C), 161.74 (C), 163.47 (C),
193.93 (CO) ppm. EM (ESIþ): m/z 317.4 (M þ H)þ. HRMS (ESIþ) m/z
calcd for C17H16O6 [MþH]þ 317.1020, found 317.1005.
4.1.3.4. (E)-1-(20,50-Dihydroxyphenyl)-3-(2,6-dihydroxy-4-
methoxyphenyl)-2-propen-1-one (114). Prepared from 105 using

B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
735
6 equiv of Br3B, which led also to the formation of 113. Red oil
(0.049.g, 26%, 90% purity). Purified by reverse-phase MPLC.
Gradient from 10 to 40% of CH3CN in H20 þ 0.05% TFA. HPLC-MS:
mp > 92 ꢄC(dec). Purified by reverse-phase MPLC. Gradient from 2 to
50 of CH3CN in H2O þ 0.05% TFA; gradient: 2e50% of CH3CN in
H2O þ 0.05% TFA. HPLC-MS: tR ¼ 8.47 min (10 min gradient). 1H
tR ¼ 3.83 min (5 min gradient) 1H NMR (400 MHz, DMSO-d6)
d:
NMR (400 MHz, DMSO-d6)
d
: 6.30 (dd, 1H, J ¼ 8.3, 2.3 Hz, 5-H), 6.36
3.71 (s, 3H, OCH3), 6.03 (s, 2H, 3-H and 5-H) (These two protons are
equivalent) 6.81 (d, 1H, J ¼ 8.9 Hz, 30-H), 6.98 (dd,1H, J ¼ 8.9, 2.8 Hz,
40-H), 7.21 (d, 1H, J ¼ 2.8 Hz, 60-H), 7.95 (d, 1H, J ¼ 15.5 Hz, Ha), 8.24
(d, 1H, J ¼ 2.3 Hz, 3-H), 6.97 (m, 2H, 40-H and 60-H), 7.50 (td, 1H,
J ¼ 8.3, 7.8, 1.6 Hz, 50-H), 7.73 (d, 1H, J ¼ 8.3 Hz, 6-H), 7.77 (d, 1H,
J ¼ 15.4 Hz, Ha), 8.13 (d, 1H, J ¼ 15.4 Hz, Hb), 8.14 (dd, 1H, J ¼ 7.3,
1.2 Hz, 30-H), 10.07 (s, 1H, OH), 10.31 (s, 1H, OH), 12.98 (s, 1H, OH)
(d, 1H, J ¼ 15.5 Hz, Hb), 12.43, 13.30, 13.42 and 13.69 (s, 1H, OH). 13
C
NMR (100 MHz, DMSO-d6)
d
: 55.05 (OCH3), 93.01 (C-3, C-5), 104.08
ppm. 13C NMR (100 MHz, DMSO-d6)
d: 102.44 (C-3), 108.28 (C-5),
(C), 113.61 (C-60), 117.63 (Ca), 118.53 (C-30), 120.41 (C), 123.98 (C-40),
138.11 (Cb), 149.23 (C), 155.16 (C), 160.78 (C), 163.13 (C), 194.01 (CO).
EM (ESIþ): m/z 303.4 (MþH)þ. HRMS (ESIþ) m/z calcd for C16H14O6
[MþH]þ 303.0863, found 303.0868.
113.23 (C-30), 115.69 (C-60), 117.77 (C-40), 119.03 (C-50), 120.56 (Ca),
130.29 (C-6), 130.81 (C), 135.89 (Cb), 141.39 (C), 159.71 (C), 162.20
(C), 162.20 (C), 193.66 (CO) ppm. EM (ESIþ): m/z 257.3 (MþH)þ,
239.3 (MþH e H2O). HRMS (ESIþ) m/z calcd for C15H12O4 [MþH e
H2O]þ 239.0703, found 239.0705.
4.1.3.5. (E)-3-(2-Hydroxyphenyl)-1-(20,40,50-trihydroxyphenyl)-2-
propen-1-one (115). Prepared from 106 using 10 equiv of Br3B.
Amorphous red solid (0.038 g, 23%) mp > 212 ꢄC(dec). Purified by
reverse-phase MPLC. Gradient: 10e30% CH3CN in H2O þ 0.05% TFA.
HPLC-MS: tR ¼ 5.36 min (10 min gradient). 1H NMR (400 MHz,
4.1.3.9. (E)-3-(2,4-Dihydroxyphenyl)-1-(30-hydroxyphenyl)-2-
propen-1-one (126). Amorphous red solid (0.065 g, 18%),
mp > 75 ꢄC(dec). Purified by reverse-phase MPLC. Gradient from 2 to
50 of CH3CN in H20 þ 0.05% TFA; gradient: 2e50% of CH3CN in
H20 þ 0.05% TFA. HPLC-MS: tR ¼ 6.62 min (10 min gradient). 1H
DMSO-d6)
d
: 6.32 (s, 1H, 60-H), 6.87 (t, 1H, J ¼ 7.4 Hz, 5-H), 6.94 (d,
1H, J ¼ 8.2 Hz, 3-H), 7.27 (ddd, 1H, J ¼ 8.2, 7.4, 1.5 Hz, 4-H), 7.45 (s,
NMR (400 MHz, DMSO-d6)
d: 6.31 (dd, 1H, J ¼ 8.5, 2.1 Hz, 5-H), 6.38
1H, 30-H), 7.80 (d, 1H, J ¼ 15.7 Hz, Ha), 7.80 (dd, 1H, J ¼ 1.5 Hz, 6-H),
(d, 1H, J ¼ 2.1 Hz, 3-H), 6.97 (dd, 1H, J ¼ 7.8, 1.2 Hz, 40-H), 7.30 (t, 1H,
J ¼ 7.8 Hz, 50-H), 7.34 (d, 1H, J ¼ 1.2 Hz, 20-H), 7.45 (dd, 1H, J ¼ 7.8,
1.2 Hz, 60-H), 7.56 (d, 1H, J ¼ 15.6 Hz, Ha), 7.64 (d, 1H, J ¼ 8.5 Hz, 6-
H), 7.92 (d, 1H, J ¼ 15.6 Hz, Hb), 9.77, 9.99 and 10.23 (s, 1H, OH) ppm.
8.06 (d, 1H, J ¼ 15.6 Hz, Hb), 10.31 and 13.13 (s, 1H, OH) ppm. 13
C
NMR (100 MHz, DMSO-d6) d
: 103.11 (C-30), 111.72 (C), 115.07 (C-60),
116.26 (C-3), 119.48 (C-5), 120.36 (C), 121.41 (Ca), 129.30 (C-4),
132.00 (C-6), 138.40 (Cb), 139.02 (C), 154.99 (C), 157.30 (C), 159.34
(C), 191.36 (CO) ppm. EM (ESIþ): m/z 273.4 (MþH)þ. HRMS (ESIþ) m/
z calcd for C15H12O5 [MþH]þ 273.0757, found 273.0752.
13C NMR (100 MHz, DMSO-d6)
d: 102.50 (C-3), 108.03 (C-5), 113.34
(C), 114.41 (C-20), 117.37 (Ca), 119.11 (C-60), 119.64 (C-40), 120.94 (C-
50),129.76 (C-6), 130.61 (C), 140.21 (Cb), 157.65 (C), 159.20 (C),161.44
(C), 189.23 (CO) ppm. EM (ESIþ): m/z 257.3 (MþH)þ, 239.3 (MþH e
H2O). HRMS (ESIþ) m/z calcd for C15H12O4 [MþH]þ 257.0808, found
257.0801.
4.1.3.6. (E)-3-(2,4-Dihydroxyphenyl)-1-(20,40,50-trihydroxyphenyl)-2-
propen-1-one (116). Prepared from 107 using 10 equiv of Br3B.
Amorphous red solid (0.063 g, 36%) mp > 250 ꢄC(dec). Purified by
reverse-phase MPLC. Gradient: from 10 to 30 of CH3CN in
H2O þ 0.05% TFA. HPLC-MS: tR ¼ 4.42 min (10 min gradient). 1H
4.1.3.10. (E)-3-(2,4-Dihydroxyphenyl)-1-(40-hydroxyphenyl)-2-
propen-1-one (127). Amorphous red solid (0.025 g, 20%),
mp > 185 ꢄC(dec) (lit.48 187e188 ꢄC, 90% purity). Purified by reverse-
phase MPLC. Gradient from 2 to 50 of CH3CN in H20 þ 0.05% TFA;
NMR (400 MHz, DMSO-d6)
d
: 6.29 (s, 1H, 60-H), 6.32 (dd, 1H, J ¼ 8.7,
2.3 Hz, 5-H), 6.39 (d, 1H, J ¼ 2.3 Hz, 3-H), 7.42 (s, 1H, 30-H), 7.58 (d,
1H, J ¼ 15.5 Hz, Ha), 7.63 (d, 1H, J ¼ 8.7 Hz, 6-H), 7.97 (d, 1H,
J ¼ 15.5 Hz, Hb), 8.71, 10.00, 10.26, 10.41 and 13.35 (s, 1H, OH) ppm.
gradient: 2e50% of CH3CN in H20
þ
0.05% TFA. HPLC-MS:
tR ¼ 1.00 min (10 min gradient). 1H NMR (400 MHz, DMSO-d6)
d:
13C NMR (100 MHz, DMSO-d6)
d
: 102.51 (C-30), 103.1 (C-3), 108.06
6.30 (dd, 1H, J ¼ 8.7, 2.3 Hz, 5-H), 6.37 (d, 1H, J ¼ 2.3 Hz, 3-H), 6.87
(d, 2H, J ¼ 8.8 Hz, 20-H, 60-H), 7.61 (d,1H, J ¼ 15.9 Hz, Ha), 7.91 (d,1H,
J ¼ 15.9 Hz, Hb), 7.95 (d, 1H, J ¼ 8.7 Hz, 6-H), 7.96 (d, 2H, J ¼ 8.8 Hz,
30-H, 50-H), 9.93, 10.15 and 10.33 (s, 1H, OH) ppm. 13C NMR
(C-5), 111.69 (C), 113.39 (C-60), 114.86 (C), 116.16 (Ca), 131.08 (C-6),
138.19 (Cb), 139.89 (C), 154.48 (C), 159.16 (C), 159.27 (C), 161.48 (C),
191.36 (CO) ppm. EM (ESþ): 289.5 (MþH)þ. HRMS (ESIþ) m/z calcd
þ
for C15H12O6 [MþH] 289.0712, found 289.0704. EM (ESIþ): m/þz
(100 MHz, DMSO-d6) d: 102.52 (C-3), 107.94 (C-5), 113.53 (C), 115.32
289.5 (MþH)þ. HRMS (ESIþ) m/z calcd for C15H12O6 [MþH]
(C-50, C-30), 117.12 (Ca), 129.84 (C-6), 130.18 (C), 130.76 (C-20, C-60),
138.93 (Cb), 158.93 (C), 161.15 (C), 161.69 (C), 187.39 (CO) ppm. EM
(ESIþ): m/z 239.3 (MþH e H2O). HRMS (ESIþ) m/z calcd for C15H12O4
[MþH e H2O]þ 239.0703, found 239.0701.
289.0707, found 289.0704.
4.1.3.7. (E)-1-(20,50-Dihydroxyphenyl)-3-(2,6-dihydroxyphenyl)-2-
propen-1-one (117). Prepared from 108 using 10 equiv of Br3B.
Amorphous red solid (0.062 g, 38%) mp > 250 ꢄC(dec). Purified by
reverse-phase MPLC. Gradient from 10 to 30 of CH3CN in H2O þ
0.05% TFA; gradient: 10e30% of CH3CN in H2O þ 0.05% TFA. HPLC-
MS: tR ¼ 5.04 min (10 min gradient). 1H NMR (400 MHz, DMSO-d6)
4.1.3.11. (E)-3-(4-Acetylaminophenyl)-1-(20,50-dihydroxyphenyl)-2-
propen-1-one (133). Prepared from 131 using 8 equiv of Br3B.
Amorphous red solid (0.069 g, 38%, 92% purity) mp > 237 ꢄC(dec)
.
(HPLC-MS: tR ¼ 4.18 min (5 min gradient). 1H NMR (400 MHz,
d
: 6.41 (d, 2H, J ¼ 8.2 Hz, 3-H and 5-H), 6.83 (d, 1H, J ¼ 8.9 Hz, 30-H),
DMSO- d6)
d
: 2.08 (s, 3H, OCH3), 6.84 (d, 1H, J ¼ 8.8 Hz, 30-H), 7.04
7.00 (dd, 1H, J ¼ 8.9, 2.9 Hz, 40-H), 7.06 (t, 1H, J ¼ 8.2 Hz, 4-H), 7.22
(d, 1H, J ¼ 2.9 Hz, 60-H), 8.10 (d, 1H, J ¼ 15.6 Hz, Ha), 8.26 (d, 1H,
(dd, 1H, J ¼ 8.8, 2.9 Hz, 40-H), 7.50 (d, 1H, J ¼ 2.9 Hz, 60-H), 7.68 (d,
2H, J ¼ 8.6 Hz, 2-H and 6-H), 7.74 (d, 1H, J ¼ 15.6 Hz, Ha), 7.80 (d, 1H,
J ¼ 15.6 Hz, Hb), 7.83 (d, 2H, J ¼ 8.6 Hz, 3-H and 5-H), 10.20 (s, 1H,
J ¼ 15.6 Hz, Hb), 9.26, 10.36, 10.36 and 12.14 (s, 1H, OH) ppm. 13
C
NMR (100 MHz, DMSO-d6)
d: 106.65 (C-3, C-5), 109.73 (C), 113.80
NH), 11.87 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6)
d: 24.16
(C-60), 118.63 (C-30), 120.60 (C-40), 120.99 (Ca), 124.23 (C), 132.52 (C-
4), 137.58 (Cb), 149.36 (C), 154.99 (C), 159.37 (C), 159.37 (C), 194.25
(CO) ppm. EM (ESIþ): m/z 273.3 (M þ H)þ. HRMS (ESIþ) m/z calcd for
(CeCH3), 115.01 (C-60), 118.27 (C-30), 118.83 (C-2 and C-6), 120.07
(Ca), 120.89 (C), 124.27 (C-40), 129.08 (C), 130.10 (C-3 and C-5),
141.84 (C), 144.35 (Cb), 149.45 (C), 154.66 (C), 168.70 (CeCOeNH),
193.11 (CeCOeC) ppm. EM (ESIþ): m/z 298.5 (MþH)þ. HRMS (ESIþ)
m/z calcd for C17H15NO4 [MþH]þ 298.1074, found 298.1074.
C
15H12O5 [MþH]þ 273.0757, found 273.0759.
4.1.3.8. (E)-3-(2,4-Dihydroxyphenyl)-1-(20-hydroxyphenyl)-2-
propen-1-one (125). Compound 128 was also isolated from the
same reaction medium. Amorphous red solid (0.045 g, 12%),
4.1.3.12. (E)-3-(4-Aminophenyl)-1-(20,50-dihydroxyphenyl)-2-
propen-1-one (134). Obtained from 132 (0.106 mmol) using 8 equiv

736
B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
of Br3B. Red solid (0.091 g, 58%, 93% purity) mp > 252 ꢄC(dec). Pu-
rified by preparative centrifugally accelerated radial thin-layer
chromatography (chromatotron), eluent: MeOH: CH2Cl2 (1:60)
HPLC-MS: tR ¼ 4.22 min (5 min gradient). 1H NMR (400 MHz,
(with 2.5% DMSO). After five days the formation of a 35% of Z isomer
was detected by HPLC-MS (an explanation has been included in the
discussion section). The product was lyophilized and the inorganic
salts were filtered off. The crude product was purified by reverse-
phase MPLC (three times). Amorphous red solid (5 mg, 20%, 85%
purity, no isomer E was detected at the sample). Gradient from 2 to
25 of CH3CN in H2O þ 0.05% TFA; gradient: 2e25% of CH3CN in
H2O þ 0.05% TFA. HPLC-MS: tR ¼ 3.13 min (5 min gradient). 1H NMR
DMSO-d6)
d: 6.03 (bs, 2H, NH2), 6.60 (d, 2H, J ¼ 8.3 Hz, 2-H and 6-
H), 6.79 (d, 1H, J ¼ 8.9 Hz, 30-H), 7.00 (dd, 1H, J ¼ 8.9, 2.7 Hz, 40-H),
7.50 (d, 1H, J ¼ 2.7 Hz, 60-H), 7.56e7.60 (m, 3H, 3-H, 5-H and Ha),
7.72 (d, 1H, J ¼ 14.8 Hz, Hb), 9.11 and 12.37 (s, 1H, OH) ppm. EM
(ESIþ): m/z 256.4 (MþH)þ. HRMS (ESIþ) m/z calcd for C15H13NO3
[MþH]þ 256.0968, found 256.0964.
(400 MHz, DMSO-d6)
d: 6.26 (dd, 1H, J ¼ 8.4, 2.4 Hz, 5-H), 6.33 (d,
1H, J ¼ 2.4 Hz, 3-H), 6.80 (d, 1H, J ¼ 8.8 Hz, Ha), 6.90 (d, 1H,
J ¼ 8.8 Hz, Hb), 6.99 (dd, 1H, J ¼ 8.4, 3.2 Hz, 40-H), 7.11 (d, 1H,
J ¼ 8.9 Hz, 30-H), 7.18 (d, 1H, J ¼ 3.1 Hz, 60-H), 7.22 (d, 1H, J ¼ 8.4 Hz,
6-H), 9.23, 9.41, 9.63 and 11.33 (brs, 1H, OH). EM (ESIþ): m/z 273.5
(MþH)þ. HRMS (ESIþ) m/z calcd for C15H12O5 þ H]þ 273.0757, found
273.0759.
4.1.3.13. (E)-1-(20,50-dihydroxyphenyl)-3-(5-hydroxy-1H-indol-3-yl)-
2-propen-1-one (137). Prepared from 136 using 10 equiv of Br3B
following the general procedure described above for polyhydroxy
chalcones. Amorphous red solid (0.180 g, 80%). mp > 240 ꢄC(dec)
.
(HPLC-MS: tR ¼ 5.45 min (10 min gradient). 1H NMR (400 MHz,
DMSO-d6)
d
: 6.77 (dd,1H, J ¼ 8.7, 2.5 Hz, 6-H), 6.82 (d,1H, J ¼ 8.7 Hz,
7-H), 7.00 (dd, 1H, J ¼ 8.7, 2.9 Hz, 40-H), 7.31 (d, 1H, J ¼ 8.7 Hz, 30-H),
7.31, (d,1H, J ¼ 2.5 Hz, 40-H), 7.41 (d,1H, J ¼ 2.9 Hz, 60-H), 7.45 (d,1H,
J ¼ 15.2 Hz, Ha), 8.06 (d, 1H, J ¼ 3.0 Hz, 2-H), 8.10 (d, 1H, J ¼ 15.2 Hz,
Hb), 11.35 (d,1H, J ¼ 3.0 Hz, NH) ppm. 13C NMR (100 MHz, DMSO-d6)
4.2. Pharmacology
4.2.1. Oocyte expression and electrophysiological studies
All human nAChR cDNAs were cloned in derivatives of the
pSP64T vector containing part of the pBluescript polylinker. Capped
mRNA was synthesized in vitro using SP6 RNA polymerase, the
mMESSAGE-mMACHINE kit from Ambion (Thermo Fisher Scienti-
fic, Madrid, Spain) and the pSP64T derivatives mentioned above.
Defoliculated X. laevis oocytes were injected with 5 ng of each
subunit cRNA in 50 nL of sterile water. All experiments were per-
formed within 2e3 days after cRNA injection [51].
Unless otherwise specified, chalcones were pre-applied in the
bath for 2 min and then co-applied with ACh through a pipette held
very close to the oocyte for fast application.
Functional expression of each receptor was estimated as the
peak ionic current evoked by 0.6 s application of 0.2 mM ACh
at ꢀ80 mV. All experiments were performed at 22 ꢄC. Current re-
cords were measured with Clampfit 10.0 (MDS Analytical Tech-
nologies, Sunnyvale, CA, USA).
d
: 104.74 (C-4), 112.45 (C), 112.73 (C-30), 112.84 (C-60), 113.18 (C-6),
113.97 (Ca), 118.38 (C-7), 120.55 (C), 123.66 (C-40), 126.06 (C), 131.80
(C), 134.85 (C-2), 140.70 (Cb), 149.26 (C), 152.88 (C), 154.98 (C),
192.48 (CO) ppm. EM (ESIþ): m/z 296.4 (MþH)þ. HRMS (ESIþ) m/z
calcd for C17H13NO4 [MþH]þ 296.0917, found 296.0927.
4.1.4. Preparation of polyhydroxy Z-chalcones 122 and 123
To a solution of 2,4-dihydroxybenzaldehyde (0.4 g, 2.93 mmol)
and the corresponding hydroxyacetophenone (0.2 g, 1.468 mmol)
in 1,4-dioxane (12 mL), BF3.Et2O (0.91 mL, 7.34 mmol) was added.
The reaction mixture was stirred at room temperature for 24 h.
After that, the precipitated solid was filtered and washed with
methylene chloride leading to 122, or 123 according to the
hydroxyacetophenone used as starting material.
4.1.4.1. (Z)-3-(2,4-Dihydroxyphenyl)-1-(30-hydroxyphenyl)-2-
propen-1-one (122). Amorphous red solid (0.3 g, 80%, 92% purity),
mp > 167 ꢄC(dec). (HPLC-MS: tR ¼ 2.09 min (5 min gradient). 1H NMR
Normalized peak currents were obtained by dividing the
maximum value of the current obtained in the presence of chalcone
by the maximum value of the current obtained in control condi-
tions. Doseeresponse curves for the peak current obtained with
(500 MHz, DMSO-d6)
d
: 7.26 (ddd, 1H, J ¼ 8.1, 2.2, 0.9 Hz, 40-H), 7.49
(dd, 1H, J ¼ 9.0, 2.2 Hz, 5-H), 7.52e7.59 (m, 2H, 3-H, 60-H), 7.82 (t,
1H, J ¼ 2.2 Hz, 20-H), 7.96 (dd, 1H, J ¼ 8.1, 0.9 Hz, 50-H), 8.29 (d, 1H,
J ¼ 9.0 Hz, 6-H), 8.57 (d, 1H, J ¼ 8.5 Hz, Ha), 9.37 (d, 1H, J ¼ 8.5 Hz,
ACh were fitted to the Hill equation: Normalized current ¼ Imax
/
(1 þ (EC50/[ACh])nH). Data are expressed as mean SEM.
Hb), 10.28 (brs, 2H, OH) ppm. 13C NMR (125 MHz, DMSO-d6)
d:
102.60 (C-3), 112.70 (C), 113.32 (Ca), 115.00 (C-20), 119.06 (C), 120.16
(C-5), 120.33 (C-50), 122.70 (C-40), 123.01 (C-60), 133.30 (C-6), 154.90
(Cb), 158.49 (C), 159.49 (C), 161.49 (C), 189.20 (CO) ppm. EM (ESIþ):
m/z 239.3 (MþH e H2O). HRMS (ESIþ) m/z calcd for C15H12O4 [MþH
e H2O]þ 239.0703, found 239.0698.
4.2.2. Bovine chromaffin cells amperometry
Bovine chromaffin cells were isolated by adrenal medulla
digestion with collagenase IA and further Urografin® centrifuga-
tion, as described [52]. Cells were suspended in a 1:1 mixture of
Dulbecco's modified Eagle's (DMEM) and HAM's-F12 media sup-
plemented with 5% fetal calf serum, 50 IU/mL penicillin and 50 mg/
4.1.4.2. (Z)-3-(2,4-Dihydroxyphenyl)-1-(40-hydroxyphenyl)-2-
propen-1-one (123). Amorphous red solid (0.188 g, 50%),
mp > 188 ꢄC(dec). (HPLC-MS: tR ¼ 1.20 min (5 min gradient). 1H NMR
mL gentamicin and incubated at 37 ꢄC/5% CO2 and plated on 12-mm
diameter glass coverslips at 5 ꢃ 104 cells density. Cells were used at
room temperature between 1 and 3 days of culture. Carbon fiber
(400 MHz, DMSO-d6)
d
: 7.11 (d, 2H, J ¼ 9.0 Hz, 20-H and 60-H), 7.40
microelectrodes of 5 mm radius (Thornel P-55; Amoco Corp.,
Greenville SC), were prepared as described [53]. Electrodes were
calibrated in a flow cell and accepted for cell studies when the
application of noradrenaline (50 mM) resulted in an oxidation cur-
rent of 300e400 pA, which is reduced by 80e100 pA under stop-
flow conditions. Amperometric measurements were performed
with the carbon fiber microelectrode gently touching the cell
membrane. Signals were low-pass filtered at 1 kHz and collected at
4 kHz using locally written software (LabView for Macintosh, Na-
tional Instruments, Austin, TX, USA). Data analysis was conducted
using locally written macros for IGOR (Wavemetrics, Lake Oswego,
OR) [54].
(dd, 1H, J ¼ 8.9, 2.2 Hz, 5-H), 7.51 (d, 1H, J ¼ 2.2 Hz, 3-H), 8.18 (d, 1H,
J ¼ 8.9 Hz, 6-H), 8.41 (d,1H, J ¼ 8.7 Hz, Ha), 8.45 (d, 2H, J ¼ 9.0 Hz, 30-
H, 50-H), 9.19 (d, 1H, J ¼ 8.7 Hz, Hb) ppm. 13CNMR (100 MHz, DMSO-
d6)
d
: 102.52 (C-3), 112.36 (C), 113.51 (Ca), 117.28 (C-30, C-50), 119.59
(C), 120.96 (C-5), 130.42 (C-6), 132.60 (C-20 and C-60), 153.18 (Cb),
158.68 (C), 161.43 (C), 165.96 (C), 187.41 (CO) ppm. EM (ESIþ): m/z
239.3 (MþH
e
H2O). HRMS (ESIþ) m/z calcd for
C
15H12O4 þ HeH2O]þ 239.0703, found 239.0705.
4.1.4.3. (Z)-1-(20,50-Dihydroxyphenyl)-3-(2,4-dihydroxyphenyl)-2-
propen-1-one (138). Prepared from 111 (20 mg) using 40 mL of PBS

B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
737
4.2.3. Cytotoxicity assay
table. A mobile radiant heat source was located under the table and
focused on the hind paw. Paw withdrawal latencies were defined as
the time taken by the rat to remove its hind paw from the heat
source. A cutoff point of 25 s was set to prevent tissue damage.
The mechanical allodynia was monitored 24 h after CFA injec-
tion and up to 4 h after administering the compounds. Paw with-
drawal latency to mechanical stimulation was assessed with an
automated testing device consisting of a steel rod that is pushed
against the plantar surface of the paw with increasing force until
the paw is withdrawn (Dynamic Plantar Aesthesiometer; Ugo
Basile). The maximum force was set at 50 g to prevent tissue
damage and the ramp speed was 2.5 g/s. Rats were placed in test
cages with a metal grid bottom. They were kept in the test cages for
30e40 min to allow accommodation. The paw withdrawal latency
was obtained as the mean of 3 consecutive assessments at each
time point (at least 10 s between repeated measurements of the
same paw).
Cell viability was assessed by the detection of mitochondrial
activity in living cells using a modified colorimetric analysis of Blue
Tetrazolium Bromide Thiazolyl (MTT) [55,56]. Briefly, HEK293 cells
(2 ꢃ 104 cells/well) were subcultured in 96-well plates, grown until
80e90% confluence, and incubated with increasing concentrations
of chalcones for 24 h. Following treatment, 10
(5 mg/mL in phosphate buffered saline) was added to each well and
further incubated for 4 h at 37 ꢄC. Subsequently, 100
L of DMSO
was added to each well to dissolve any deposited formazan
resulting from cleavage and reduction of MTT by active mito-
chondrial dehydrogenases [55]. The optical density of each well
was measured at 540 nm with a microplate reader (Polastar BMG
LABTECH, Offenberg, Germany).
Primary hippocampal neurons were seeded in 24-well plates at
a density of 13 ꢃ 104 cells/well (see supporting information for
detailed culture procedures). The day of plating was considered day
in vitro 1 (1DIV). Neurons were incubated with compound 111 at
9DIV and measurements for cell viability and death were con-
ducted 24 h later (10DIV) in three independent cultures. Cell
viability was estimated using the MTT assay previously described
for HEK293 cells whereas cell death was estimated using the lactate
dehydrogenase assay (Cytotoxicity Detection kit PLUS (LDH), Roche
Applied Science), according to manufacturer's instructions. As
positive controls (100% of cytotoxicity), 2e3 wells per culture were
treated with 1% Triton X-100, whose percentage of LDH activity in
the medium related to basal (DMSO) was 387.4 29.4%.
mL of MTT solution
m
4.2.6. Morris water maze test [58]
A plastic circular pool of 90 cm diameter was filled with
reconstituted powdered milk to keep the surface opaque. For
testing purposes, the pool was divided in eight equidistant entry
points (N, NE, E, SE, S, SW, W, NW) along the whole circumference
and a 10 cm diameter transparent circular disc placed one centi-
meter below the surface close to the SW point. The milky water was
kept at 27 1 ꢄC and changed every other day. Several visual cues
were positioned around the pool to facilitate the orientation of
mice. Every animal performed 4 consecutive trials for 5 consecutive
days, each trial as follows: with the experimenter standing
immobile and silent on the same spot, the mouse was deposited in
the pool face to the pool wall and left to swim until it reached the
hidden escape disc or last swimming for 60 s (after which the
mouse was placed on the disc), whatever happened first. The
subject was allowed to rest on the disc for 30 s before being
immersed again. After a 4 trials session, each one starting at a
different place of the pool, the animal was cleaned and dried with a
paper towel and a soft infrared light and returned to its cage. Each
trial was recorded and analysis was performed using our dedicated
software [59]. For analytical purposes, a mouse is considered as
having reached the disc if it climbed completely or partially onto it
and stood there for at least 2 s. Any mouse which was unable to
swim or sunk frequently was removed from the experiment and its
previous results discarded.
4.2.4. Culture and viability assay of hippocampal neuronal HT22
cells
HT22 cells were cultured in DMEM, supplemented with 10%
heat-inactivated fetal bovine serum, 100 units/mL penicillin, and
100
mg/mL streptomycin. Cultures were seeded into flasks con-
taining supplemented medium and kept at 37 ꢄC in a humidified
atmosphere of 5% CO2 and 95% air. For assays, HT22 cells were sub-
cultured in 48-well plates at a seeding density of 1 ꢃ 105 cells per
well. Cells were treated with compound 111 before confluence in
DMEM with 10% fetal bovine serum. Cells were used at a passage
below 13.
Cell viability of HT22 cells was assessed by the MTT assay
[55,56]. Briefly, 50 mL of the MTT labeling reagent, at a final con-
centration of 0.5 mg/mL, was added to the DMEM of each well at
the end of the treatments period and the plate was placed in a
humidified incubator at 37 ꢄC with 5% CO2 and 95% air (v/v) for an
additional 2 h period. The resulting formazan was spectrophoto-
metrically assayed as above. Results were compared to control
samples, treated with vehicle (DMEM), to which 100% viability was
attributed.
Transparency declarations
The authors declare no conflict of interest.
4.2.5. Analgesic activity
Male Wistar rats (250e300 g) were obtained from Janvier,
France. All experiments were approved by the Institutional Animal
and Ethical Committee of the Universidad Miguel Hernandez
where experiments were conducted and they were in accordance
with the guidelines of the Economic European Community and the
Committee for Research and Ethical Issues of the International
Association for the Study of Pain. All parts of the study concerning
animal care were performed under the control of veterinarians.
CFA emulsion (1:1 oil/saline, 0.5 mg/mL) was injected into the
Acknowledgments
ꢀ
We thank Sara Gonzalez-Reyes for her assistance in the syn-
thesis of two starting compounds and Susana Gerber for technical
assistance. This work was supported by grants from Spanish Min-
istry of Science and Innovation (currently Ministry of Economy and
Competitivity): The Spanish Ion Channel Initiative-CONSOLIDER
INGENIO 2010 (CSD2008-00005), SAF2009-09323, BFU2008-
02160, SAF2011-22506, SAF2011-22802, SAF2012-23332, BFU2010-
15822 and BFU2012-39092-C02-02. B.B. thanks the CSIC for a
predoctoral fellowship (JAE-Predoc from Junta para la Ampliacion
de Estudios, co-financed by FSE). The Instituto de Neurociencias is a
“Centre of Excellence Severo Ochoa”.
plantar surface (50 mL) of the left hind paw of rats [57]. Compounds
were administered at 10 mg/kg i.v. 24 h after CFA injection. Thermal
hyperalgesia was monitored 24 h after CFA injection and up to 4 h
after administering the compounds with an Ugo Basile Plantar Test
(Hargreaves Apparatus). In brief, rats were habituated to an appa-
ratus consisting of individual Perspex boxes on an elevated glass
ꢀ

738
B. Balsera et al. / European Journal of Medicinal Chemistry 86 (2014) 724e739
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dx.doi.org/10.1016/j.ejmech.2014.09.039.
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