Amino acid and peptide prodrugs of diphenylpropanones positive allosteric modulators of α7 nicotinic receptors with analgesic activity

Article abstract of DOI:10.1016/j.ejmech.2017.10.083

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2′,5′,4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects.

Full text of DOI:10.1016/j.ejmech.2017.10.083

Accepted Manuscript
Amino acid and peptide prodrugs of diphenylpropanones positive allosteric
modulators of α7 nicotinic receptors with analgesic activity
Beatriz Balsera, José Mulet, Salvador Sala, Francisco Sala, Roberto de la Torre, Sara
González-Rodríguez, Adrián Plata, Lieve Naesens, Asia Fernández-Carvajal, Antonio
Ferrer-Montiel, Manuel Criado, María Jesús Pérez de Vega, Rosario González-Muñiz
PII:
S0223-5234(17)30885-1
10.1016/j.ejmech.2017.10.083
EJMECH 9875
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 August 2017
Revised Date: 30 October 2017
Accepted Date: 31 October 2017
Please cite this article as: B. Balsera, José. Mulet, S. Sala, F. Sala, R. de la Torre, S. González-
Rodríguez, Adriá. Plata, L. Naesens, A. Fernández-Carvajal, A. Ferrer-Montiel, M. Criado, Marí.Jesú.
Pérez de Vega, R. González-Muñiz, Amino acid and peptide prodrugs of diphenylpropanones positive
allosteric modulators of α7 nicotinic receptors with analgesic activity, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.083.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Amino acid and peptide prodrugs of diphenylpropanones positive
allosteric modulators of α7 nicotinic receptors with analgesic activity
a
b
b
b
c
c
Beatriz Balsera, José Mulet, Salvador Sala, Francisco Sala, Roberto de la Torre,
c
a
d
Sara González-Rodríguez, Adrián Plata, Lieve Naesens, Asia Fernández-Carvajal,
c
b
a
Antonio Ferrer-Montiel, Manuel Criado, María Jesús Pérez de Vega and Rosario
,a
González-Muñiz*
a
Instituto de Química Médica, IQM-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, 03050 Sant Joan
b
d’Alacant, Spain
c
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avenida de
la Universidad s/n, 03202 Elche (Alicante), Spain
d
Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
Corresponding Author:
Dr. Rosario González-Muñiz
Email: iqmg313@iqm.csic.es
Phone: +34915622900
Fax: +34915644853

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Key words: Diphenylpropanones, Prodrugs, Diketopiperazines, DDP-IV, α7 nicotinic
receptors, Allosteric modulation
Research Highlights
•
Amino acid/peptide prodrugs of 1,3-diphenylpropanone α7 nAChRPAMs are
described
•
•
Val-Val and Val-Pro-Val are suitable promoieties
Val-Pro-Val derivative exhibited long-lasting analgesic effect than the parent
drug
ABSTRACT: α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels
implicated in a number of CNS pathological processes, including pain and psychiatric,
cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive
allosteric modulation of these channels constitutes an interesting approach to achieve
selectivity versus other nicotinic receptors. We have recently described new chalcones
and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs,
which proved to have good analgesic activities but poor pharmacokinetic properties.
Here we report the preparation of amino acid and peptide derivatives as prodrugs of
these modulators with the aim of improving their in vivo biological activity. While the
valine derivative showed very short half life in aqueous solutions to be considered a
prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-
diphenylpropanones, via chemical and enzymatic transformation, respectively.
Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2’,5’,4-trihydroxy-1,3-
diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays.
The best compound, 21, displayed a better profile in the analgesia test than its parent
compound 3, exhibiting about the same potency but long-lasting effects.

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INTRODUCTION
Nicotinic acetyl choline receptors (nAChRs) are ligand-gated ion channels widely
expressed in the nervous system. Among them, α7 receptors (α7 nAChRs) are
implicated in a number of CNS pathological processes, from psychiatric [1-3], and
cognitive disorders [4, 5] to inflammatory processes [6, 7], being associated to different
sorts of pain [8-10], such as inflammatory [8, 11, 12], nociceptive [13, 14], and
neuropathic pain [15-17]. While most of the described modulators of these channels are
agonists, competitive with acetyl choline (Ach), the endogenous ligand, in recent years,
efforts have been focused on the search for positive allosteric modulators (PAMs),
agents that enhanced the positive response evoked by ACh, interacting with the receptor
at allosteric sites. One of the advantages of PAMs is the higher receptor subtype
selectivity comparing with true agonists, given that they bind to sites other than the
well-conserved orthosteric binding domain. This, together with the fact that they show
no effect in the absence of the natural neurotransmitter, made PAMs an interesting
alternative to competitive agonists [18, 19]. There are not many compounds reported as
PAMs of nAChRs, but several of them have been progressed achieving clinical phases
[
20-22].
We have recently described some new chemotypes that proved to be potent PAMs of
the α7 nAChRs with structures of chalcone [23] and 1,3-diphenylpropanone [24]. These
compounds were identified by screening a heterogeneous collection of small natural
molecules, which led to the identification of a polyhydroxy substituted chalcone,
Isoliquiritigenin, as a selective PAM of α7 nAChRs. This finding was the origin of a
research project that permitted the identification of the polyhydroxy derivative 1 (Figure
1
), as a potent and selective PAM of α7 nAChR [23]. To further explore the potential of

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the series, and taking into account the high chemical reactivity that the α,β-unsaturated
carbonyl system confers to the chalcone structure, hydrogenation of the double bond
was performed. This led to the identification of new 1,3-diphenylpropan-1-ones, 2 and 3
(Figure 1), which also behaved as PAMs of the α7 nicotinic receptor with lower but still
significant enhancing activity (see Table 1) [24].
Figure 1. Structure of parent compounds.
The low aqueous solubility and the short times of action found in the in vivo
experiments performed with these compounds [23, 24] prompted us to take a brief in
vitro study of their pharmacokinetic (PK) properties. This study revealed that
compounds 1-3 had in fact a poor PK profile. From the three compounds the best results
were found for compound 3, that showed a bit better aqueous solubility and
permeability than compounds 1 and 2. In particular, the values found for compound 3
-6
are on the range of 160 µM regarding solubility in PBS and 33.5 x 10 cm/s
permeability in the Caco-2 assay. However, the low values obtained for the half-live
time (18-42 min), together with the high intrinsic clearance found in general for the
three studied compounds, 1-3, with values on the range of 165-387 µL/min/mg [24],
was the fact that really encouraged us to prepare some derivatives that could behave as
carrier-prodrugs, especially regarding their in vivo biological behavior, liberating the
parent drug after the in vivo administration in the alive organism.

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In the last decades a lot of work around the development and use of new prodrug
strategies have ended in the improvement of the PK properties of many drugs [25, 26].
Among them, we have considered three different approaches based on the preparation
of amino acid or peptide derivatives since they are easily excreted and non-toxic [27].
On one hand, we planned to prepare amino acid derivatives, in particular Valine-derived
compounds that could act in a similar way as the well-known commercialized
valacyclovir, the valine derivative of the antiherpes agent acyclovir. This approach is
based on the lability of the ester valine bond, which permits the in vivo liberation of the
active substance [28].
On the other hand, we also contemplated the preparation of dipeptide and tripeptide
derivatives following two different strategies found in the literature. The first approach
described by the group of P. Gomes, is based on the preparation of dipeptide derivatives
of the active substance, which will be recovered after the intramolecular cyclization of
the dipeptide fragment, giving rise to the corresponding diketopyperazine (DKP) [29,
3
0]. The second one, described by S. Velazquez and co-workers, is an enzyme-based
prodrug approach centered on the recognition of specific peptide moieties by dipeptidyl
peptidase IV (DPP IV/CD26) [31]. This enzyme is able to cleave oligopeptide moieties
having a Pro residue located in the penultimate position of the N-terminus.
Following the indicated prodrug strategies, in this paper we describe the preparation of
amino acid (Val) and peptide (Val-Val and Val-Pro-Val) derivatives of the selected
compounds 1-3, by ester formation with the OH group in position 4. In the case of Val
derivatives, the new formed ester bond should be sensitive to chemical hydrolysis or to
the action of esterases, which will liberate the parent compounds into the organism. For
Val-Val derivatives, the ability of the dipeptide to form a DKP should control drug
release. Compounds having the tripeptide moiety Val-Pro-Val will suffer first the action

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of CD26, which will liberate Val-Pro dipeptide and the Val-compound, followed by the
chemical or esterase-mediated hydrolysis of the valine residue, which will lead to the
corresponding parent drugs [32]. Moreover, amino acid and peptide prodrugs
presumably would be able to improve drug permeability, because they could be
recognized by specific transporting proteins [33].
RESULTS AND DISCUSSION
Chemistry.
Valine Derivatives. The valine derivative of chalcone 1 was synthesized as depicted in
Scheme 1, by coupling the free 4-hydroxyl group of chalcone 6 with Boc-Val-OH in the
presence of EDC/HOBt as coupling system [34]. Subsequent treatment of the so
obtained product with boron tribromide to deprotect the methoxy groups led to the
expected polyhydroxy chalcone 16, which was isolated as a bromhydrate salt. The
presence of bromhydric acid in the reaction medium also promoted the removal of the
Boc protecting group, leading to the free NH of the valine appendage. The results
2
found after studying the chemical stability of this compound in aqueous solution
revealed its extremely low stability, liberating the parent drug, compound 1, in only 30
min (Figure 2). This result prompted us to desist in the preparation of the valine
derivatives for compounds 2 and 3, going directly to the preparation of dipeptide and
tripeptide analogues.
Dipeptide Derivatives. Compound 17, Val-Val derivative of chalcone 1, was
synthesized from chalcone 6 and the dipeptide Boc-Val-Val-OH, following the same
procedure previously used for the synthesis of analogue 16. Compounds 18 and 19, Val-
Val bioprecursors of propanones 2 and 3, were synthesized following a similar
methodology, from compounds 10 and 11, respectively. These intermediates come from

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the selective hydrogenation of the double bond of the corresponding chalcone analogues
6
and 7, using Pd-C in the presence of Ph S that avoid overreduction of the ketone
2
group (Scheme 1) [35].
The HPLC-MS chromatograms of compound 17 revealed the presence of two peaks
with the same molecular mass. This was indicative of epimerization of the C-terminal
valine residue during the coupling reaction, which led to the formation of two
diastereoisomers. The presence of two diastereoisomers was also detected in the case of
dipeptide analogues 18 and 19. Moreover, in the case of chalcone 17 a certain amount
of the Z-isomer of the linker double bond was also detected. This finding prompted us
to prepare only the tripeptide prodrugs of the saturated propanone analogues, because
the presence of complex, inseparable isomeric mixtures would made rather complicated
to perform the stability studies with these compounds.

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Scheme 1. Synthesis of valine, dipeptide and tripeptide methoxy derivatives 8, 9, 12-15.
Tripeptide Derivatives. Coupling reaction of compounds 10 and 11 with tripeptide Boc-
Val-Pro-Val-OH [32], followed by treatment with BBr , led to the desired tripeptide
3
derivatives 20 and 21, as mixtures of two diastereoisomers in each case, due again to the

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epimerization of the C-terminal Val residue, as it was mentioned for dipeptide prodrugs
1
8 and 19 (Schemes 1 and 2).
Scheme 2. Synthesis of hydroxyl derivatives 16, 17, 18-21.
Chemical and enzymatic stability of valine, dipeptide and tripeptide derivatives.
Stability in Aqueous Solutions. The chemical stability of all synthesized prodrugs was
determined in PBS (pH = 7.4)/5% DMSO at 25ºC, and monitored by HPLC. As already
commented, this study revealed the extremely low chemical stability in aqueous

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solution of Val derivative 16, releasing the parent compound 1 in a very short time
(100% transformation in 30 min; Figure 2). This result indicated a high lability of the
Val ester bond on this phenol derivative, and therefore the related valine analogues of
compounds 2 and 3 were not prepared.
Figure 2. Chemical stability study of compound 16 in PBS/5% DMSO at time 0 (A)
and 30 (B) min, and compound 19 at time 0 (C) and 150 (D) min.
Dipeptide derivatives 18 and 19 were expected to release the corresponding parent
compounds via the DKP formation (see Figures 2 and 3A). In both cases, after 5 h in
solution around 40% release of the active compound was observed (Figures 2 and S1).
Therefore, they seem appropriate for further activity studies.

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Finally, no significant degradation of tripeptide prodrugs 20 and 21 was observed in
PBS (5% DMSO) solution after 5 hours (Figures 3B and S2). This result revealed that,
as expected, the presence of the enzyme CD26 (DPP IV) seems to be necessary for their
transformation in the corresponding parent drug. As already mentioned, presumably the
enzymatic cleavage that liberates the corresponding valine derivative would take place
first, which in turn will quickly release the desired compound due to its easiness to
suffer hydrolysis in aqueous medium (see Figure 3).
Figure 3. Degradation of dipeptides 18 and 19 (A) and tripeptides 20 and 21 (B)
prodrugs in PBS + 5% DMSO, at 25º C. x: time of injection (min); y: conversion (%); z:
compounds.
Enzymatic Stability of Tripeptide Prodrugs. In the first series of studies, Val-Pro-Val
prodrugs (compounds 20 and 21) were exposed to purified human recombinant CD26

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(Figure S3), with about 70% of parent compounds 2 and 3 released after 15 min, and
total disappearance of the prodrug in approximately 1h. Then, the conversion by native
CD26 in human serum (HS) (Figure 4) or bovine serum (BS) (Figures S4) was
investigated. In human serum, the conversion was slower than with the recombinant
enzyme, as expected, but still very high after 60 min incubation (i.e. 78% for 20 and
8
7% for 21). Prolonged times were needed to recover the drug from the corresponding
prodrug in bovine serum (Figure S4), with total release in about 6 h. The HPLC
chromatograms showed that, when incubated with CD26, HS or BS, both peaks of drug
diastereoisomers disappeared in favor of free compound. Prodrugs 20 and 21 also
showed some spontaneous conversion to the free compound when incubated at 37ºC in
PBS, but this conversion was markedly enhanced by the presence of recombinant or
native CD26.

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Figure 4. Time-dependent conversion of prodrug 20 into its parent drug 2 (A), and
prodrug 21 (B) into its parent drug 3, in human serum containing 20% PBS, at different
times (The red and grey and doted bars correspond to the two diastereoisomers).
Biological Evaluation
Electrophysiological Studies of New Prodrugs. The new di- and tripeptide derivatives
were evaluated for their effect on currents evoked by ACh in Xenopus oocytes
expressing α7 nAChRs. In contrast to parent compounds 1-3 that behave as PAMs of
the α7 nAChR (Table 1, taken from [23, 24] and showing values well over the control
value of 100%, which is the current elicited by 200 µM of ACh alone), all peptide

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derivatives decreased the ACh-evoked currents significantly (Table 1, compounds 17-
2
1), therefore acting as inhibitors of the α7 nAChR, probably as negative allosteric
modulators. It is interesting to note that the methoxylated analogues precursors of the
polyhydroxy parent compounds behave as negative allosteric modulators of the α7
receptors [24]. Taking into account that the amino acid and peptide analogues described
here have the same hydroxyl substituents than their parent compounds, except for the 4-
OH group that it is blocked, these results may be indicative of a key role of this OH for
PAM activity.
Table 1. Percentage Values of Inhibition of α7 nAChR Currents for Prodrugs and
Parent Compounds
%
Current
vs ACh [200 µM]
760±255[23]
89±100[24]
1
a
Compound
R
R
1
2
OH
OH
OH
OH
2
9
3
H
OH
6
66±83[24]
24.5±0.7
26.7±1.2
14.7±5.1
25.3±11.6
24.7±8.1
.
17
18
19
20
21
OH
OH
H
HBr H N-Val-Val-
2
.
HBr H N-Val-Val-
2
.
HBr H N-Val-Val-
2
.
OH
H
HBr H
N-Val-Pro-Val-
N-Val-Pro-Val-
2
.
HBr H
2
a
Compounds were tested at a unique dose of 10 µM, which was co-applied with 200 µM ACh.
Responses were recorded at -80 mV and normalized with respect to that shown by only ACh (200 µM).

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Analgesic Activity. It has been described that PAMs of α7 nAChR show analgesic
effects. In particular PNU-120596, a well-known type II PAM, has been described to
display anti-nociceptive activity in models of persistent and inflammatory pain [13, 36].
In our previous work, compound 3 (10 mg/kg, iv) and PNU-120596 (10 mg/kg, iv),
were checked for the reduction of hyperalgesia resulted from inflammation promoted by
complete Freund’s adjuvant (CFA) in rats. Both compounds showed significant
antinociceptive effects in the mechanical hyperalgesia test, but lacked activity in the
thermal assay [23, 24]. Compound 3 demonstrated a main peak of activity after 2h from
administration, and then a fast decay in the following two hours, just the same behavior
than PNU-120596 [24].
To compare the behavior of the new derivatives with that of the parent compound 3, the
mechanical hyperalgesia test was performed after iv administration of dipeptide 19 and
tripeptide derivative 21 (10 mg/Kg). As depicted in Figure 5, compound 19 (Figure 5A),
the Val-Val derivative of 3, shows an effect quite similar to that of its parent compound
3
, but it exerts its activity in a shorter time, showing its maximal peak after only 30 min
post administration. Then the effect disappears quite fast, decreasing very rapidly, in
only 30 min. This quick and short effect suggests either a fast release and
metabolization of compound 3 in vivo or more probably, a fast liberation just after
injection but a sustained release afterward that do not reach effective concentrations of
the bioactive compound. In contrast, compound 21 (Figure 5B), the Val-Pro-Val
derivative of 3, shows comparable antinociceptive activity to those of model
compounds 3 and PNU-120596, but the effect is statistically significative before, only
3
0 min after administration, reaching its maximal effect at 1 h. Interestingly, this effect
vanishes slowly, lasting for more than 3 h, while PNU derivative and compound 3
decay rapidly after 2 h. This behavior invites to think that our initial hypothesis is valid,

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suggesting that compound 21 is continuously metabolized through the action of CD26
enzyme in plasma. These results indicate that the presence of compound 3 in the
organism at effective concentration is longer when administered in the form of prodrug
2
1. Moreover, the fact that compounds 19 and 21, show analgesic effects in the in vivo
assay is an evidence of their capacity to really act as carrier prodrugs. In fact, in the in
vitro electrophysiology tests these compounds display an opposite behavior to that of
parent compound 3, blocking instead of activating the receptor.
Figure 5. Effect of compounds 19 and 21 in the CFA-induced paw inflammation model.
Time course of mechanical hyperalgesia in rats after injection of complete Freund’s
adjuvant (CFA) 0.5 mg/mL (50 µL) into the right hind paw with and without
administration of compound (10 mg/kg i.v.). The diagram shows the paw withdrawal
latencies in response to mechanical stimulation (n ≥ 6 rats/group). Data are given as
mean ± SEM n = 6. Two-way ANOVA with Bonferroni post-hoc test. *P <0,05; **P
<0,01, ***P <0,001. A) and B) correspond to the model compounds.
Despite an apparent better aqueous solubility of prodrugs 19 and 21, the in vivo test was
performed again by iv administration to be able to compare with the results obtained for
2
4
the parent compound 3. As by nature prodrugs should be chemically or enzymatically
unstable molecules, the measurement of PK properties was not attempted. It is expected
that the permeability of both prodrugs could be better, because it could take place
through active transport, involving amino acid/peptide transporters, while the polar
parent compound is supposed to permeate only through passive diffusion. On the other

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hand, the long-lasting analgesic activity points to a sustained release of the active
principle in vivo .
CONCLUSIONS
Previous studies that started with the in vitro screening of a library of natural
compounds, allowed the identification of some chalcones and 1,3-propanones as PAMs
of the α7 nACh receptors [23, 24]. Pharmacological studies of these compounds
revealed the promising biological properties of some of them, including significant
antinociceptive effects in the mechanical hyperalgesia assay performed in rats, and
cognitive enhancement in the Morris test. However, their poor pharmacokinetic profile
prompted us to prepare some derivatives able to behave as prodrugs, thus releasing the
parent compounds in vivo. We report here the preparation of the amino acid, di- and
tripeptide derivatives of the best compounds of the series, 1-3, which were designed as
carrier bioprecursors of the parent compounds. With the exception of the Val derivative
that hydrolyzes very quickly in aqueous solution, Val-Val and Val-Pro-Val derivatives
showed chemical and enzymatic stabilities compatible with their use as prodrugs. The
biological evaluation of these compounds showed that they behave as inhibitors of α7
nAChR currents in electrophysiological assays in vitro, in contrast to parent compounds
1
-3 that are PAMs. However, the in vivo evaluation of selected compounds 19 and 21
revealed that both display analgesic activity, thus indicating the release of the parent
compound 3 from the peptide precursors. Compound 21, the Val-Pro-Val tripeptide
derivative, which should release 3 through the action of CD26 and a spontaneous
hydrolysis of the Val residue, exhibited comparable analgesic activity than 3 and
PNU120596 but with an interesting long-lasting effect, a result good enough to justify
further work on this series of compounds.

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ACKNOWLEDGEMENTS
Funding: This work was supported by the Spanish Ministerio de Economía y
Competitividad (MINECO) SAF2011-22802 and SAF2015-66275-C2-R, and
the Generalitat Valenciana, PROMETEO/2014/011. BBP thanks the CSIC for a
predoctoral fellowship (JAE-Predoc from Junta para la Ampliación de Estudios, co-
financed by FSE).
EXPERIMENTAL SECTION
Chemistry. 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 (230-400 mesh) was used for flash
chromatography. Some compounds were purified by flash chromatography using SNAP
2g KP-C18-HS cartridges on an ISOLERA ONE (Biotage) apparatus. Mixtures of
CH CN (solvent A), H O + 0.05% TFA (solvent B) were used as mobile phase. Unless
1
3
2
indicated, all compounds were isolated in a purity ≥ 95% (HPLC data). Analytical
HPLC-MS was performed on a Waters equipment coupled to a single quadrupole ESI-
TM
MS (Waters Micromass ZQ 2000) using a reverse-phase SunFire C18 4.6 x 50 mm
column (3.5 µm) at a flow rate of 1 mL/min and by using a diodo array UV detector.
Mixtures of CH CN + 0.08% formic acid (solvent A) and H O + 0.1% formic acid
3
2
(solvent B) were used as mobile phase (gradient of 15 to 95% of A in B in 5 or 10 min,
as indicated in each case). HRMS (EI+) 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
1
System 500 spectrometer, operating at 300, 400 or 500 MHz for H and at 75, 100, and

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1
3
1
25 MHz for C recording. To confirm the NMR peak assignments, COSY and HSQC
experiments were performed when necessary (For H and C numbering see Table 1
heading formula).
General procedure for the synthesis of chalcones. 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 0ºC, and an aqueous solution of
NaOH 40% (1.87 mL) was slowly added. After 15h 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 H O and
2
brine. After drying over MgSO , the solvent was removed to dryness and the resulting
4
residue crystallized from MeOH. Characterization data for all new methoxy-substituted
chalcones can be found in the SI.
General procedure for the preparation of 1,3-diphenyl-1-propanones. To a solution
of the corresponding chalcone (0,30 mmol) in MeOH (30 mL) and diphenylsulfide
(0.003 mmol), Pd/C 10% (10% weight) was added. After hydrogenation, at room
temperature and 20 psi during 24h, the catalyst was filtered off, the solvent evaporated
under vacuum, and the residue purified by flash chromatography (AcOEt/Hexane).
Typical procedure for the Coupling Reaction of Amino Acids and Peptides. To a
solution of the corresponding 4-hydroxy chalcone or 1,3-diphenyl-1-propanone (0.25
mmol) in Cl CH /THF (4/1 mL), Boc-Val-OH, or Boc-Val-Val-OH or Boc-Val-Pro-
2
2
Val-OH (0.50 mmol), EDC chlorhydrate (0.50 mmol) and DMAP (0.05 mmol). The
reaction was stirred at room temperature for 15 h. The organic phase was successively
washed with citric acid 5%, NaHCO 10%, and brine. After drying over anhydrous
3
MgSO , the solvent was removed and the residue chromatographed to give the desired
4

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compound. Characterization data for all the methoxy-substituted derivatives can be
found in the SI.
General procedure for the deprotection of methoxy substituted compounds. To a
previously cooled solution (0ºC) of the corresponding methoxy substituted compound
(
0.60 mmol) in dried DCM (15 mL), a 1M solution of BBr in DCM (2 equiv for each
3
MeO- group plus 2 more equiv for every group containing a potentially basic N or O)
was slowly added under Ar atmosphere. After stirring 24-48 h at room temperature
under Ar, H O was added to the reaction mixture. Then DCM was removed under
2
reduced pressure and the residue extracted with EtOAc. The organic extracts were
washed with H O and brine, dried over Na SO and then evaporated. The crude product
2
2
4
was purified as indicated in each case.
(E)-1-(2’,5’-Dihydroxyphenyl)-3-[2-hydroxy-4-(L-Val)oxy]phenyl-2-propenone
(
16). Obtained using 10 equiv of BBr . Reddish solid (39% yield), mp: 194-197ºC.
3
Purification by reverse phase flash chromatography, (eluent: from 10 to 30% of ACN in
H O + 0.05% TFA). HPLC (Sunfire): t = 5.11 min (10 min gradient from 15 to 95% of
2
R
1
A in B). H RMN (500 MHz, DMSO-d ) δ: 1.08, 1.11 (d, 3H, J = 6.9 Hz, γ-Val), 2.33
6
(m, 1H, β-Val), 4.24 (d, 1H, J = 4.5 Hz, α-Val), 6.72 (dd, 1H, J = 8.5, 2.2 Hz, 5-H),
6
.79 (d, 1H, J = 2.4 Hz, 3-H), 6.84 (d, 1H, J = 8.9 Hz, 3’-H), 7.03 (dd, 1H, J = 8.9, 3.0
Hz, 4’-H), 7.43 (d, 1H, J = 3.0 Hz, 6`-H), 7.89 (d, 1H, J = 15.7 Hz, H ), 7.97 (d, 1H, J =
α
+
8
1
.5 Hz, 6-H), 8.03 (d, 1H, J = 15.7 Hz, H ), 8.53 (bs, 3H, NH ), 9.23 (s, 1H, OH),
β
3
1
3
0.94 (s, 1H, OH), 11.23 (s, 1H, OH) ppm. C NMR (125 MHz, DMSO-d ) δ: 17.7 (γ-
6
Val), 18.3 (γ-Val), 29.6 (β-Val), 57.4 (α-Val), 109.1 (C-3), 112.60 (C-5), 114.8 (C-6’),
18.4 (C-4’), 120.0 (C-3’), 121.0 (C-1), 121.4 (C ), 124.3 (C-1’), 130.5 (C-6), 138.87
1
α
(
C ), 149.5 (C), 152.0 (C), 154.6 (C), 158.3 (C), 167.47 (CONH), 193.3 (CO) ppm. MS
β

ACCEPTED MANUSCRIPT
+
+
+
(
ESI ): m/z 372.4 [M + H] . HRMS (ESI+) m/z ([M + H] calcd for C H NO
2
0
21
6
3
72.14416; found 372.14320
(E)-1-(2’,5’-Dihydoxyphenyl)-3-[2-hydroxy-4-(L-Val-L,D-Val)oxy]phenyl-2-
propenone (17). Obtained using 20 equiv of BBr . Red solid (72% yield), mp. = 189-
3
1
91ºC. Purification by reverse phase flash chromatography, eluent: from 2 to 30%
gradient of ACN in H O + 0.05% TFA. HPLC (Sunfire): Mixture of diastereoisomers
2
1
(
47:53) t = 5.92 and 6.12 min (10 min gradient from 15 to 95% of A in B). H RMN
R
(
500 MHz, DMSO-d ) δ: 0.95, 0.96 (d, 3H, J = 7.0 Hz, γ-Val), 0.98, 1.00 (d, 3H, J =
6
6
.9 Hz, γ-Val), 1.02, 1.03 (d, 3H, J = 6.8 Hz, γ-Val), 1.06, 1.07 (d, 3H, J = 6.8 Hz, γ-
Val), 2.14 (m, 1H, β-Val), 2.27 (m, 1H, β-Val), 3.75, 3.78 (m, 1H, α-Val), 4.41, 4.50 (
dd, 1H, J = 7.9, 6.1 Hz, α-Val), 6.62, 6.66 (dd, 1H, J = 8.6, 2.3 Hz, 5-H), 6.70, 6.72 (d,
1
3
7
H, J = 2.3 Hz, 3-H), 6.80, 6.84 (d, 1H, J = 8.9 Hz, 3’-H), 7.02, 7.04 (dd, 1H, J = 8.9,
.0 Hz, 4’-H), 7.41, 7.42 (d, 1H, J = 3.0 Hz, 6’-H), 7.86, 7.87 (d, 1H, J = 15.7 Hz, H ),
α
.92, 7.93 (d, 1H, J = 8.6 Hz, 6-H), 8.02, 8.03 (d, 1H, J = 15.7 Hz, H ), 8.14 (bs, 3H,
β
+
NH ), 8.81 (d, 1H, J = 6.8 Hz, CONH), 8.93 (d, 1H, J = 7.9 Hz, CONH), 9.23 (s, 1H,
3
13
OH), 10.18 (s, 1H, OH), 10.83, 10.85 (s, 1H, OH), 11.82, 11.83 (s, 1H, OH), ppm. C
NMR (125 MHz, DMSO-d ) δ: 17.3, 17.5 (γ-Val), 18.1, 18.25 (γ-Val), 18.2, 18.44 (γ-
6
Val), 18.7, 18.9 (γ-Val), 29. 7, 30.0 (β-Val), 30.0, 31.0 (β-Val), 57.1, 57.2 (α-Val), 57.7,
5
1
1
1
8.1 (α-Val), 109.1, 109.2 (C-3), 112.7, 112.8 (C-5), 114.7 (C-6’), 118.3, 118.4 (C-4’),
19.5, 119.6 (C-1), 120.9, 120.9 (C-3’), 121.1, 121.12 (Cα), 124.0, 124.3 (C-1’), 130.4,
30.4 (C-6), 139.1 (Cβ), 149.3, 149.5 (C), 152.7, 152.8 (C), 154.6, 154.6 (C), 158.3 (C),
68.6, 168.7 (CONH), 169.3, 169.6 (OCO), 193.3 (CO) ppm. MS (ESI+): m/z 471.2 [M
+
+
H] .

ACCEPTED MANUSCRIPT
1
-(2’,5’-Dihydoxyphenyl)-3-[2-hydroxy-4-(L-Val-L,D-Val)oxy]phenyl-1-propanone
(
18). Obtained using 20 equiv of BBr . Colorless solid (69% Yield), mp = 162-165ºC).
3
Purification by reverse phase flash chromatography, eluent: 2 to 30% gradient of ACN
in H O + 0.05% TFA. HPLC (Sunfire): Mixture of diastereoisomers (56:44) t = 6.16
2
R
1
and 6.30 min (10 min gradient from 15 to 95% of A in B). ). H RMN (500 MHz,
DMSO-d ) δ: 0.94, 0.95 (d, 3H, J = 6.9 Hz, γ-Val), 0.97, 0.98 (d, 3H, J = 6.9 Hz, γ-
6
Val), 1.01, (d, 3H, J = 6.8 Hz, γ-Val), 1.04, 1.05 (d, 3H, J = 6.8 Hz, γ-Val), 2.11 (m, 1H,
β-Val), 2.24 (m, 1H, β-Val), 2.85 ( m, 2H, Hz, H ), 3.25, 3.26 (t, 2H, J = 7.5 Hz H ),
β
α
3
6
8
7
.74, 3.77 (d, 1H, J = 5.3 Hz, α-Val), 4.38, 4.48 (dd, 1H, J = 8.1, 6.5 Hz, α-Val) , 6.43,
.47 (dd, 1H, J = 8.3, 2.4 Hz, 5-H), 6.53, 6.55 (d, 1H, J = 2.4 Hz, 3-H), 6.81 (d, 1H, J =
.9 Hz, 3’-H), 6.98 (dd, 1H, J = 8.9, 3.0 Hz, 4’-H), 7.14, 7.15 (d, 1H, J = 8.3 Hz, 6-H),
+
.21 (d, 1H, J = 3.0 Hz, 6’-H), 8.10 (bs, 3H, NH ), 8.76, 8.89 (d, 1H, J = 8.1 Hz,
3
1
3
CONH), , 9.16, 9.17 (s, 1H, OH), 9.82, 9.83 (s, 1H, OH), 11.23 (s, 1H, OH) ppm. C
NMR (125 MHz, DMSO-d ) δ: 17.2, 17.5 (γ-Val), 18.1, 18.2 (γ-Val), 18.3, 18.5 (γ-Val),
6
1
8.8, 19.0 (γ-Val), 24.2, 24.3 (C ), 29.7, 30.0 (β-Val), 30.0, 30.2 (β-Val), 38.8, 38.8
α
(
C ), 57.1, 57.2 (α-Val), 57.6, 58.1 (α-Val), 108.1, 108.1 (C-3), 111.4 (C-4’), 111.6 (C-
β
3
’), 114.7 (C-5), 118.4 (C-6’), 120.1, 120.2 (C-1), 124.1 (C-1’), 125.1 (C), 130.2, 130.2
(C-6), 149.4 (C), 153.5 (C), 155.8 (C), 168.6, 168.6 (CONH), 169.7, 169.9 (OCO),
+
+
2
04.9 (CO) ppm. MS (ESI+): m/z 473.3 [M + H] . HRMS (ESI+) m/z ([M + H] calcd
for C H N O 473.22823; found 473.22990.
2
5
32
2
7
1
-(2’,5’-Dihydroxyphenyl)-3-[4-(L-Val-L,D-Val)oxy]phenyl-1-propanone
(19).
Obtained using 20 equiv of BBr , Colorless solid (69% yield), mp = 129-131ºC.
3
Purification by reverse phase flash chromatography, eluent: 2 to 30% gradient of ACN
in H O + 0.05% TFA. HPLC (Sunfire): Mixture of diastereoisomers (53:47) t = 6.00
2
R
1
and 6.21 min (10 min gradient from 15 to 95% of A in B). H RMN (500 MHz, DMSO-

ACCEPTED MANUSCRIPT
d6) δ: 0.93, 0.94 (d, 3H, J = 7.0 Hz, γ-Val), 0.96, 0.98 (d, 3H, J = 7.0 Hz, γ-Val), 1.02
(d, 3H, J = 6.8 Hz, γ-Val), 1.05, 1.06 (d, 3H, J = 6.8 Hz, γ-Val), 2.12 (m, 1H, β-Val),
2
.26 (m, 1H, β-Val), 2.94 ( m, 2H, H ), 3.24 (overlapped with H O signal; t, 2H, J = 7.5
β
2
Hz, H ), 3.74, 3.77 (d, 1H, J = 5.0 Hz, α-Val), 4.39, 4.48 ( dd, 1H, J = 7.9, 6.2 Hz, α-
α
Val), 6.81 (d, 1H, J = 8.9 Hz, 3’-H), 6.97, 6.99 ( dd, 2H, J = 8.9, 2.9 Hz, 3-H and 5-H),
7
.02 (dd, 1H, J = 8.7, 2.0 Hz, 4’-H), 7.21 (d, 1H, J = 3.0 Hz, 6’-H), 7.33, 7.35 (d, 2H,
+
J = 8.5 Hz, 2-H and 6-H), 8.10 (bs, 3H, NH ), 8.78, 8.90 (d, 1H, J = 6.9 Hz, CONH), ,
3
1
3
9
.18, 9.19 (s, 1H, OH), 11.17, 11.18 (s, 1H, OH) ppm. C NMR (125 MHz, DMSO-d )
6
δ: 17.3, 17.5 (γ-Val), 18.2, 18.2 (γ-Val), 18.3, 18.4 (γ-Val), 18.9, 19.1 (γ-Val), 28.7,
2
5
5
1
8.7 (C ), 29.6, 29.7 (β-Val), 30.0, 30.0 (β-Val), 40.5, 40.5 (C ), 57.1, 57.2 (α-Val),
α β
7.8, 58.1 (α-Val), 114.7 (C-6’), 118.4 (C-3’), 120.3, 120.3 (C-1), 121.2, 121.3 (C-3, C-
), 124.1 (C-4’), 129.5, 129.6 (C-2, C-6), 139.1 (C), 148.3 (C), 149.4 (C), 153.4 (C),
68.62, 168.7 (CONH), 169.8, 170.1 (OCO), 204.2 (CO) ppm. MS (ESI+): m/z 457.3
+
+
[
M + H] . HRMS (ESI+) m/z [M + H] calcd for C H N O 457.23331; found
25 32 2 6
4
1
57.23240.
-(2’,5’-Dihydroxyphenyl)-3-[-2-hydroxy-4-(L-Val-L-Pro-L,D-Val)oxy]phenyl-1-
propanone (20). Obtained using 20 equiv of BBr . Colorless solid (48% yield), mp =
3
1
70-173ºC. Purification by reverse phase flash chromatography, eluent: 2 to 30%
gradient of ACN in H O + 0.05% TFA. HPLC (Sunfire): Mixture of diastereoisomers
2
1
(
38:62) t = 5.85 and 5.99 min (10 min gradient from 15 to 95% of A in B). H RMN
R
(
500 MHz, DMSO-d ) δ: 0.94-1.03 ( m, 12H, , γ-Val), 1.87 (m, 2H, γ-Pro), 1.97 (m,
6
1
H, β-Val), 2.13 (m, 2H, β-Pro), 2.20 (m, 1H, β-Val), 2.84 (t, 2H, J = 7.5 Hz, H ), 3.26
β
(
t, 2H, J = 7.5 Hz, H ), 3.50 (m, 1H, δ-Pro), 3.71 (m, 1H, δ-Pro), 3.99 (d, 1H, J = 5.6
α
Hz, α-Val), 4.32, 4.36 ( dd, 1H, J = 8.0, 6.2 Hz, α-Val), 4.56 (m, 1H, α-Pro), 6.42, 6.44
dd, 1H, J = 8.1, 2.4 Hz, 5-H), 6.53, 6.55 (d, 1H, J = 2.4 Hz, 3-H), 6.80 (d, 1H, J = 8.9
(

ACCEPTED MANUSCRIPT
Hz, 3’-H), 6.98 (dd, 1H, J = 8.9, 3.0 Hz, 4’-H), 7.12, 7.13 (d, 1H, J = 8.1 Hz, 6-H),
+
7
.21 (d, 1H, J = 3.0 Hz, 6’-H), 8.0 (bs, 3H, NH ), 8.45, 8.47 (d, 1H, J = 8.0 Hz,
3
1
3
CONH), 9.17, 9.18 (s, 1H, OH), 9.79, 9.81 (s, 1H, OH), 11.24 (s, 1H, OH) ppm. C
NMR (125 MHz, DMSO-d ) δ: 17.1, 17.2 (γ-Val), 18.1, 18.3 (γ-Val), 18.4, 18.5 (γ-Val),
6
1
3
5
9.0, 19.0 (γ-Val), 24.2 (C ), 24.6, 24.7 (γ-Pro), 29.2, 29.4 (β-Pro), 29.4, 29.6 (β-Val),
α
0.0, 30.3 (β-Val), 38.8 (C ), 47.4, 47.5 (δ-Pro), 55.7, 55.7 (α-Val), 57.4, 57.6 (α-Val),
β
9.2, 59.4 (α-Pro), 108.1, 108.2 (C-3), 111.6 (C-5), 114.7 (C-6’), 118.4 (C-4’), 120.1
(C-3’), 124.1 (C), 124.9, 124.9 (C), 130.1, 130.2 (C-6), 149.1 (C), 149.4 (C), 153.5 (C),
1
55.8 (C), 166.7, 167.0 (CONH), 170.1, 170.2 (CONH), 171.4, 171.7 (OCO), 204.9
+
+
(
CO) ppm. MS (ESI+): m/z 570.4 [M + H] , 552.4 [M - H O] . HRMS (ESI+) m/z [M +
2
+
H] calcd for C H N O 570.28099; found 570.28226.
3
0
39
3
8
1
-(2’,5’-Dihydroxyphenyl)-3-[-2-hydroxy-4-(L-Val-L-Pro-L,D-Val)oxy]phenyl-1-
propanone (21). Obtained using 20 equiv of BBr . Colorless solid (41% yield), mp =
3
1
30-133ºC). Purification by reverse phase flash chromatography, eluent: 2 to 30%
gradient of ACN in H O + 0.05% TFA. HPLC (Sunfire): Mixture of diastereoisomers
2
1
(
49:51) t = 5.81 and 6.03 min (10 min gradient from 15 a 95% of A in B). H RMN
R
(
500 MHz, DMSO-d ) δ: 0.94-1.03 ( m, 12H, , γ-Val), 1.81 (m, 2H, γ-Pro), 1.95 (m,
6
1
H, β-Val), 2.12 (m, 2H, β-Pro), 2.21 (m, 1H, β-Val), 2.93 (t, 2H, J = 7.5 Hz, H ), 3.
β
(
overlapped with H O signal; t, 2H, J = 7.5 Hz, H ), 3.49 (m, 1H, δ-Pro), 3.70 (m, 1H,
2
α
δ-Pro), 3.98 ( s, 1H, α-Val), , 4.36 (m, 1H, α-Val), 4.55 (m, 1H, α-Pro), 6.80 (d, 1H, J
8.9 Hz, 3’-H), 6.96, 6.98 (d, 1H, J = 6.6 Hz, 3-H and 5-H) 6.97 (d, 1H, J = 8.6 Hz, 4’-
=
H), 7.21 (d, 1H, J = 3.00 Hz, 6’-H), 7.31, 7.32 (d, 2H, J = 8.5 Hz, 2-H and 6-H), 8.03
+
(
sa, 3H, NH ), 8.47, 8.48 (d, 1H, J = 7.7 Hz, CONH), 9.21 (s, 1H, OH), 11.18 (s, 1H,
3
1
3
OH) ppm. C NMR (125 MHz, DMSO-d ) δ: 17.6, 17.6 (γ-Val), 18.6, 18.6 (γ-Val),
6
1
8.8, 18.8 (γ-Val), 19.5, 19.5 (γ-Val), 25.1, 25.2 (γ-Pro), 29.1 (C ), 29.6, 29.8 (β-Pro),
α

ACCEPTED MANUSCRIPT
2
5
1
1
9.9, 30.1 (β-Val), 30.4, 30.6 (β-Val), 40.9 (C ), 47.8, 47.9 (δ-Pro), 56.2 (α-Val), 58.0,
β
8.1 (α-Val), 59.6, 59.7 (α-Pro), 115.1 (C-6’), 118.8 (C-3’), 120.7, 120.7 (C), 121.7,
21.8 (C-3, C-5), 124.5 (C-4’), 129.8, 129.9 (C-2, C-6), 139.4 (C), 148.8 (C), 149.9 (C),
53.8 (C), 167.2, 167.4 (CONH), 170.7, 170.8 (CONH), 171.9, 172.1 (OCO), 204.7
+
+
(
CO) ppm. MS (ESI+): m/z 554.3 [M + H] . HRMS (ESI+) m/z [M + H] calcd for
C H N O 554.28608; found 554.28563.
30
39
3
7
Biological procedures.
Oocyte expression & electrophysiological studies. The human α7 nAChR cDNA was
cloned in a derivative of the pSP64T vector containing part of the pBluescript olylinker.
Capped mRNA was synthesized in vitro using SP6 RNA polymerase, the
mMESSAGEmMACHINE kit from Ambion (Thermo Fisher Scientific, Madrid, Spain)
and the pSP64T derivative mentioned above. Defoliculated Xenopus laevis oocytes
were injected with 5 ng cRNA in 50 nl of sterile water. All experiments were performed
within 2–3 days after cRNA injection. Unless otherwise specified, compounds were
preapplied 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 α7 nAChR 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°C. Current records were measured with
Clampfit 10.0 (MDS Analytical Technologies, Sunnyvale, CA, USA). Normalized peak
currents were obtained by dividing the maximum value of the current obtained in the
presence of 10 µM compound by the maximum value of the current obtained in control
conditions.
Analgesic assay. Male Wistar rats (250–300 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

ACCEPTED MANUSCRIPT
accordance with the guidelines of the Economic European Community and the
Committee for Research (Directive 2010/63/EU) 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 plantar surface (50 μl) of the left hind paw of rats [37].
Compounds were dissolved in DMSO, dilute in physiologic saline (0.9% sodium
chloride) and injected IV at a total volume of 0,5 ml. The final DMSO concentration
was 2,5%. Compounds were administered at 10 mg/kg i.v. 24 h after CFA injection.
The mechanical allodynia was monitored 24 h after CFA injection and up to 4 h after
administering the compounds. Paw withdrawal 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 30–40 min to allow
accommodation. The paw withdrawal latency was obtained as the mean of three
consecutive assessments at each time point (at least 10 s between repeated
measurements of the same paw).
Conflicts of interest: none.
Authors contribution: Idea: RGM, MJPV; Synthesis and chemical stability: BB, AP;
Electrophysiology: JM, SS, FS; In vivo experiments: SGR,RT; Enzymatic stability: LN;
Supervision: AF, AFM, MC, MJPV, RGM; This article has been written through
contribution of all authors, which have approved the final version.

ACCEPTED MANUSCRIPT
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