Design, synthesis and structure–activity relationships of novel phenylalanine-based amino acids as kainate receptors ligands

Article abstract of DOI:10.1016/j.bmcl.2016.09.075

A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as at cloned homomeric kainate receptors GluK1–GluK3. Among them, six compounds bound to GluK1 receptor subtypes with reasonable affinity (K_ivalues in the range of 4.9–7.5?μM). A structure–activity relationship (SAR) for the obtained series, focused mainly on the pharmacological effect of structural modifications in the 4- and 5-position of the phenylalanine ring, was established. To illustrate the results, molecular docking of the synthesized series to the X-ray structure of GluK1 ligand binding core was performed. The influence of individual substituents at the phenylalanine ring for both the affinity and selectivity at AMPA, GluK1 and GluK3 receptors was analyzed, giving directions for future studies.

Full text of DOI:10.1016/j.bmcl.2016.09.075

Accepted Manuscript
Design, synthesis and structure-activity relationships of novel phenylalanine-
based amino acids as kainate receptors ligands
Ewa Szymańska, Paulina Chałupnik, Katarzyna Szczepańska, Ana Maria
Cuñado Moral, Darryl S. Pickering, Birgitte Nielsen, Tommy N. Johansen,
Katarzyna Kieć-Kononowicz
PII:
S0960-894X(16)31024-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.075
BMCL 24299
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 July 2016
28 September 2016
29 September 2016
Please cite this article as: Szymańska, E., Chałupnik, P., Szczepańska, K., Moral, A.M.C., Pickering, D.S., Nielsen,
B., Johansen, T.N., Kieć-Kononowicz, K., Design, synthesis and structure-activity relationships of novel
phenylalanine-based amino acids as kainate receptors ligands, Bioorganic & Medicinal Chemistry Letters (2016),
doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.075
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Design, synthesis and structure-activity relationships of novel
phenylalanine-based amino acids as kainate receptors ligands
a
a
a
b
Ewa Szymańska, * Paulina Chałupnik, Katarzyna Szczepańska, Ana Maria Cuñado Moral,
b
b
b
a
Darryl S. Pickering, Birgitte Nielsen, Tommy N. Johansen, Katarzyna Kieć-Kononowicz
a
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Medyczna 9, PL 30-688 Kraków, Poland
b
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, DK 2100 Copenhagen Ø, Denmark
Keywords
Ionotropic glutamate receptors, kainate receptors, GluK1, GluK3, phenylalanine
*
Corresponding author: Ewa Szymanska
tel.: +48 012 620 55 80; fax: +48 012 620 55 96. E-mail: ewa.szymanska@uj.edu.pl

Abstract
A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and
pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as
at cloned homomeric kainate receptors GluK1-GluK3. Among them, six compounds bound to
GluK1 receptor subtypes with reasonable affinity (K values in the range of 4.9-7.5 M). A
i
structure-activity relationship (SAR) for the obtained series, focused mainly on the
pharmacological effect of structural modifications in the 4- and 5-position of the
phenylalanine ring, was established. To illustrate the results, molecular docking of the
synthesized series to the X-ray structure of GluK1 ligand binding core was performed. The
influence of individual substituents at the phenylalanine ring for both the affinity and
selectivity at AMPA, GluK1 and GluK3 receptors was analyzed, giving directions for future
studies.
1

Kainate (KA) receptors are cation-selective ligand-gated ion channels that belong to the
family of ionotropic glutamate receptors together with -amino-3-(3-hydroxy-5-methyl-4-
isoxazolyl)propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. They are
widely expressed in the mammalian central nervous system, particularly in the hippocampus,
where they act principally as the modulators of synaptic transmission and neuronal
1
excitability. Functional KA receptors are tetrameric structures formed by the combination of
subunits GluK1–5, which are assembled into two dimers of two homo- or two heteromeric
subunits. It has been shown that GluK1-3 may form functional homomeric or heteromeric
receptors, whereas GluK4 and GluK5 only form functional receptors in combination with
2
,3
subunits GluK1, GluK2 or GluK3.
In recent years, dynamic growth has been reported in the understanding of the biophysical
properties and function of KA receptors in the brain. A particular problem in the studies on
this type of receptors is the relative lack of specific pharmacological tools. Most known KA
receptors agonists, including AMPA and kainate themselves, act on both AMPA and KA
4
receptors, similar to the early non-NMDA receptor antagonists (such as the isoxazol-based
3
-6
(
S)-ATPO, Fig. 1A). Despite an extensive effort, only a few antagonists (such as UBP302
and LY466195, Fig. 1A) with preference for GluK1 receptors compared to GluK3 receptors
3
,4,7-10
and recombinant AMPA receptors have been reported.
GluK1 antagonists have been
1
1,12
11
shown to be highly effective in animal models of epilepsy,
neurodegeneration,
3
,12,13
14
neuropathic pain
and migraine.
Recently, we have reported two series of competitive non-NMDA receptor antagonists built
6
,15-18
on the phenylalanine scaffold.
2-Carboxyethylphenylalanines 1 (Fig. 1B) were found to
1
5,16
block AMPA, GluK1 and GluK3 receptors, but not GluK2 receptors.
The analysis of
1
2
structure-activity relationship (SAR) within the series showed that both R and R positions
are structural factors influencing AMPA/GluK1 and GluK3 selectivity. Introduction of a
2

4
-nitro-5-chloro substitution pattern into the phenylalanine ring was beneficial for both
AMPA and homomeric kainate receptors affinity, whereas a hydrogen atom or a polar amino
group in the 4-position introduced several-fold selectivity for GluK1 and GluK3 receptors.
Within the series of biaryl phenylalanine analogues based on the scaffold 2 (Fig. 1B) it was
shown that only compounds containing a 3’-carboxylate, such as 2a and 2b, bound selectively
6
to GluK1 receptors. In this series, only the 4,5-dichloro substitution pattern of the
phenylalanine ring has been studied.
Figure 1. (A) Structures of selected AMPA and kainate receptor antagonists. (B) Design of the new
phenylalanine-based ligands of AMPA and kainate receptors.
Here, we report the synthesis and biological evaluation of a new series of biaryl-based
phenylalanine analogues, with a carboxyl-benzene (or a carboxyl-thiophene ring) as a distal
aromatic function (Fig. 1B). To find the substitution pattern optimal for kainate receptors
affinity, the structural modifications were focused on positions 5- and 4- of the phenylalanine
ring. Various combinations of small-sized groups, both polar and lipophilic, hydrogen bond
3

donors and acceptors, were investigated. All the target amino acids were pharmacologically
characterized by radioligand binding at native AMPA, KA and NMDA receptors in rat brain
membranes. All the compounds were further evaluated in radioligand binding assays at
homomeric recombinant rat GluK1-3 receptors, expressed in Sf9 insect cell membranes.
The general procedure for the synthesis of the target amino acids is outlined in Schemes 1-3.
6
,19-23
Modified literature methods
bromophenyl intermediates 7-14 and 16, starting from commercially available substituted
-methylanilines (Scheme 1) or 2-chloro-4-methylphenol (Scheme 2). Oxidative bromination
with NaClO and HBr followed by Sandmayer reaction using CuCl afforded 7 and 8. The
were applied to obtain the suitable substitution pattern of
4
3
2
removal of an amine group performed through diazotization yielded compounds 9 and 10.
Oxidation of amine 4 to 2-bromo-4-methylnitrobenzene 11 was performed by means of
3
-chloroperbenzoic acid (mCPBA). BOC-protection of the amino group, carried out in the
mixture of dioxane and water in the presence of Na CO was successful in the case of amine
, but these conditions were not sufficient for 5 and 6. To obtain 13 and 14, a catalytic amount
2
3
4
of 4-dimethylaminopyridine (DMAP) was used in addition to tert-butoxycarbonyl anhydride.
The reaction was carried out in tetrahydrofuran (THF) for 72 hours at room temperature, in
the presence of triethylamine (TEA) as the base. To yield 16, bromination of 2-chloro-4-
methylphenol followed by alkylation of the hydroxy group was applied.
The treatment of bromotoluene precursors 7-14 and 16 with N-bromosuccinimide (NBS)
under free radical conditions and the following substitution with the sodium salt of diethyl
acetamidomalonate were performed in one step, without the isolation of benzyl bromides
(
Scheme 3). The target amino acids 3a-i were prepared as a result of a number of Suzuki
coupling reactions of intermediates 17-25 with 3-cyanophenylboronic acid, followed by
deprotection in acidic environment. In most cases, the product of the cross-coupling reaction
was refluxed in the mixture of concentrated hydrochloric acid, acetic acid and water for 24
4

hours, except for the methoxy-substituted compound 31, for which aqueous hydrobromic acid
was used. The racemic mixtures of final products were purified by evaporation of the acid(s),
neutralization of pH followed by reverse-phase liquid chromatography. All final amino acids
were obtained in their zwitterionic form.
Scheme 1. Synthesis of compounds 5-14. Reagents and conditions: (i) 48% HBr, NaClO
ii) 1. CuCl , tert-BuNO , acetonitrile, 65 °C, 2. 20% HCl; (iii) 50% H PO , NaNO , acetic acid/water/HCl,
°C→ r.t.; (iv) 77% mCPBA, toluene, reflux; (v) for R = H: BOC anhydride, Na CO , dioxane/water, r.t.; for
3
, acetic acid, 5→45 °C;
(
2
2
3
2
2
1
0
2
3
1
2
R = Cl, NO : BOC anhydride, DMAP, TEA, tetrahyfrofuran, r.t.
Scheme 2. Synthesis of compounds 15 and 16. Reagents and conditions: (i) HBr, NaClO
3
, acetic acid, 5→30 °C;
(
ii) CH J, K CO , dimethylformamide, r.t.
3
2
3
5

Scheme 3. Synthesis of compounds 17-34 and 3a-i. Reagents and conditions: (i) NBS, azobisisobutyronitrile,
carbon tetrachloride, reflux; (ii) NaH, diethyl acetamidomalonate, dimethylformamide, r.t.; (iii) 3-
2 3 2
cyanophenylboronic acid, PdCl (PPh ) , triethylamine, DME/water, 50 °C; (iv) HCl or HBr, reflux.
All final amino acids 3a-i were characterized pharmacologically in the radioligand binding
assays at native ionotropic glutamate receptors (rat membrane preparations) as well as cloned
2
4-30
rat homomeric subtypes, GluK1-3.
The binding data are shown in Table 1, together with
6
those previously reported for 2a and 2b, cited here as reference compounds. In addition to
the previous binding data, the present work also examined analogues 2a and 2b for their
affinity at the GluK2 and GluK3 receptors.
All compounds were found to be inactive at native NMDA receptors as well as homomeric
GluK2 receptors (K
only analogue that demonstrated micromolar affinity for native kainate receptors (K
Table 1). Weak AMPA binding was observed for some of phenylalanines for which K
i
> 100 M, data not shown). Within the series investigated, 3h was the
= 32 M,
values
i
i
varied in the range of 38-65 M. This effect seems to be associated with the presence of a
chloro atom or nitro group in the 5-position of the phenylalanine ring with concomitant
unsubstituted 4-position (3d, 3g), while a polar hydroxyl or amine group in position 4
markedly reduced AMPA affinity.
At KA receptors, a structural modification in the 4- and 5-position of the phenylalanine ring
affected both affinity and selectivity at homomeric GluK1 and GluK3 receptors. The
6

introduction of a 4-nitro group to the unsubstituted 2b induced affinity at GluK3 receptors and
did not affect GluK1 binding (3b), while 4-chloro or 4-amino groups (3a and 3c, respectively)
led to a noticeable reduction in binding affinity at GluK1 receptor. A different effect was
observed in the case of substituents in 5-position. The 5-chloro analogue (3d) was found to be
equipotent at GluK1 receptors compared to 2b and showed micromolar affinity at GluK3
receptors, whereas the 5-nitro group (3g) induced 4-fold reduction in GluK1 affinity. The
addition of the second substituent to 5-chloro or 5-nitro structures resulted in the active
GluK1 ligands 3e, 3f, 3h and 3i with micromolar range of affinity (K
interesting effect was observed in the case of compounds possessing a polar group in the
-position of the phenylalanine ring. The 4-amino group seemed to substantially diminish the
i
= 4.9-7.5 M). An
4
affinity at GluK3 receptor (3c, 3e, 3i), while 4-hydroxy group together with the 5-chloro
substituent of 3f was found to be a substitution pattern beneficial for GluK3 receptors. Thus,
the 4-amino-5-chloro-substituted biphenylalanine 3e, the most potent compound at GluK1
within the present series, presented over 20-fold selectivity vs. GluK3 (and native AMPA)
receptors, while its close (and equipotent at GluK1 receptors) analogue 3f showed only 2-fold
difference between GluK1 and GluK3 subtypes affinities.
Table 1. Binding pharmacology at native AMPA and KA receptors (rat brain membranes) as well as homomeric
a
recombinant GluK1 and GluK3 receptors.
cloned homomeric receptors
GluK1 GluK3
(M) (M)
native receptors
3
3
 HAMPA
 HKA
1
2
Compound
R
R
K
i
K
i
K (M)
i
K (M)
i
7

b
d
c
c
(
S)-ATPO
2.9
0.6
nd.
16
> 100
nd.
e
UBP302
4.0
nd.
nd.
f
f
LY466195
0.05
8.9
nd.
b
g
g
g
g
2
2
3
a
b
a
Cl
H
Cl
H
2.8
> 100
> 100
> 100
> 100
> 100
> 100
> 100
b
7.9
H
Cl
35
> 100
> 100
> 100
[
[
[
[
[
[
[
4.46 ± 0.02]
3
b
H
NO
2
7.2
23
65
5.15 ± 0.03]
[4.64 ± 0.02]
4.19 ± 0.04
3
3
3
3
3
c
d
e
f
H
NH
2
26
> 100
> 100
> 100
> 100
> 100
> 100
> 100
4.58 ± 0.01]
Cl
Cl
Cl
NO
H
5.7
25
51
5.26 ± 0.09]
[4.60 ± 0.02]
4.31 ± 0.09
NH
OH
H
2
4.9
> 100
 100
5.33 ± 0.08]
5.5
11
> 100
5.31 ± 0.13]
[4.95 ± 0.04]
g
2
2
2
32
> 100
> 100
> 100
38
4.50 ± 0.06]
4.43 ±0.03
3
3
h
i
NO
NO
Cl
6.1
62
32
[
5.23 ± 0.07]
4.21 ± 0.05
4.50 ± 0.02
NH
2
7.5
> 100
> 100
[5.15 ± 0.08]
a
i
Data are given as mean of at least three separate experiments conducted in triplicate. Mean pK ± SEM (M) are
b
6
c
5
d
8
indicated in brackets. Data from ref. . IC50 values, data from ref. . Data from ref. : K
i
value for inhibition of
e
9
glutamate-evoked currents in HEK293 cells expressing homomeric KA receptors. Data from ref. : IC50 values
f
10
g
6
in an Xenopus oocyte assay. Data from ref. . IC50 values, data from ref. . nd. – not determined.
8

To analyze the structure-activity relationship of the new target compounds in more details,
molecular docking studies to the GluK1 ligand binding core (LBD) were undertaken. Due to a
size of designed ligands, an X-ray structure of GluK1 LBD with bound antagonist: (S)-1-(2'-
amino-2'-carboxyethyl)-3-[(2-carboxythien-3-yl)methyl]thieno[3,4-d]pyrimidin-2,4-dione
31
(
PDB code: 3S2V) has been chosen as a template. The results of docking showed, that
inside the binding pocket biphenylalanines adopt the binding mode similar to that observed
for most of α-amino acid-based competitive antagonists co-crystallized with the GluK1 and
GluA2 LBDs (Fig. 2). Detailed analysis of high scored docking poses of (S)-3e and (S)-3f
1
2
revealed the presence of two pockets surrounding the substituents in R and R positions of
the phenylalanine ring. The one formed by Glu456, Tyr459, Pro531, Thr755, Ser756 and
Tyr779 (full-length numbering including the signal peptide of isoform GluR5-1, UniProtKB
identifier P22756-1) seems to accommodate equally well chlorine atom and a nitro group,
however, lack of a substituent in this position is connected with a significant decrease of a
docking score. Despite a high hydrogen bonding potential in this region of the receptor, no
2
such an interaction is observed for docked ligands. The substituent in R position is
surrounded by residues Ser736, Ser756 and Met752. Among them Ser756 seems to exert the
strong influence on GluK1 affinity of the compounds, probably interacting through a
hydrogen bond with a hydroxy or amino group of ligands (Fig. 2). Furthermore, both residues
Ser736 and Ser756 of GluK1 receptor are non-conserved. In GluK3 receptor in these
positions there are asparagine and threonine, and in the AMPA subunit GluA2: threonine and
methionine, respectively. Those differences may contribute to the observed
AMPA/GluK1/GluK3 selectivity of the target biphenylalanines.
9

Figure 2. The highest ranked docking poses of (S)-3e (purple) and (S)-3f (cyan) at the GluK1 LBD (3S2V
template). Depending on the rotameric position of the side chain, Ser756 may interact with either amino of 3e or
hydroxy group of 3f. The shape of the GluK1 binding site (in mesh) was calculated for 3S2V.
In summary, the studies described in the present work have clarified the structure-activity
relationship of the new series of phenylalanine derivatives based on the [1,1'-biphenyl]-3-
carboxylic acid scaffold and acting as ligands of non-NMDA receptors. The influence of
substituents in the 4- and 5-position of the phenylalanine on binding affinity to native AMPA
and cloned homomeric GluK1 and GluK3 receptors was the main object of the study. The
results clearly point at the significance of the nitro or hydroxyl group in the 4-position and the
chloro atom in the 5-position for affinity at both GluK1 and GluK3 receptor subtypes.
Furthermore, the analysis of the present results and the SAR established for the
phenylalanines previously described based on the 2-carboxyethylphenylalanine scaffold
1
5,17
(
1)
shows 2-6 fold higher GluK1 receptor affinity of biphenyl analogues 3 compared to
phenylalanines with structure 1 with the same substitution pattern in the phenylalanine ring.
As the biphenylalanine core seems to be optimal for GluK1 ligands, a 5-chloro-4-nitro
biphenyl-based analogue is expected to possess high GluK1 and GluK3 affinity and this
compound will be included in our future studies. On the other hand, as the amino group in the
1
0

4
-position of 3 reduces binding at both native AMPA and homomeric GluK3 receptors, 4-
amino substitution should be taken into account in the design of selective GluK1 ligands
based on the biphenylalanine core.
Acknowledgements
The financial support of this research by the National Science Centre Poland
(
2014/15/B/NZ7/00908) is gratefully acknowledged.
Supplementary data
Supplementary data (full experimental details, compound characterization data, H-NMR and
1
1
3
C-NMR spectra as well as pharmacology and molecular modeling protocols) associated with
this article can be found in the online version.
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3

List of Figures, Schemes and Tables – color online only (black-and-white in print)
Figure 1. (A) Structures of selected AMPA and kainate receptor antagonists. (B) Design of
the new phenylalanine-based ligands of AMPA and kainate receptors.
Figure 2. The highest ranked docking poses of (S)-3e (purple) and (S)-3f (cyan) at the GluK1
LBD (3S2V template). Depending on the rotameric position of the side chain, Ser756 may
interact with either amino of 3e or hydroxy group of 3f. The shape of the GluK1 binding site
(
in mesh) was calculated for 3S2V.
Scheme 1. Synthesis of compounds 5-14. Reagents and conditions: (i) 48% HBr, NaClO
3
,
acetic acid, 5→45 °C; (ii) 1. CuCl , tert-BuNO , acetonitrile, 65 °C, 2. 20% HCl; (iii) 50%
2
2
H
3
PO
2
2
, NaNO , acetic acid/water/HCl, 0 °C→ r.t.; (iv) 77% mCPBA, toluene, reflux; (v) for
1
1
R = H: BOC anhydride, Na
2
CO
3
, dioxane/water, r.t.; for R = Cl, NO
2
: BOC anhydride,
DMAP, TEA, tetrahyfrofuran, r.t.
Scheme 2. Synthesis of compounds 15 and 16. Reagents and conditions: (i) HBr, NaClO
acetic acid, 5→30 °C; (ii) CH J, K CO , dimethylformamide, r.t.
3
,
3
2
3
Scheme 3. Synthesis of compounds 17-34 and 3a-i. Reagents and conditions: (i) NBS,
azobisisobutyronitrile, carbon tetrachloride, reflux; (ii) NaH, diethyl acetamidomalonate,
dimethylformamide, r.t.; (iii) substituted phenyl- or thienylboronic acid, PdCl
2
(PPh
3
2
) ,
triethylamine, DME/water, 50 °C; (iv) HCl or HBr, reflux.
Table 1. Binding pharmacology at native AMPA and KA receptors (rat brain membranes) as
a
well as homomeric recombinant GluK1 and GluK3 receptors.
Footnote:
1
4

a
Data are given as mean of at least three separate experiments conducted in triplicate. Mean
b
6
c
5
pK
i
± SEM (M) are indicated in brackets. Data from ref . IC50 values, data from ref .
d
8
Data from ref : K
i
value for inhibition of glutamate-evoked currents in HEK293 cells
e
9
expressing homomeric KA receptors. Data from ref : IC50 values in an Xenopus oocyte
f
10
g
6
assay. Data from ref . IC50 values, data from ref . nd. – not determined.
1
5

Graphical abstract
Graphical abstract
Design, synthesis and structure-activity relationships of novel
phenylalanine-based amino acids as kainate receptors ligands
a
a
a
b
Ewa Szymańska, * Paulina Chałupnik, Katarzyna Szczepańska, Ana Maria Cuñado Moral,
b
b
b
a
Darryl S. Pickering, Birgitte Nielsen, Tommy N. Johansen, Katarzyna Kieć-Kononowicz
1
6

Table 1. Binding pharmacology at native AMPA and KA receptors (rat brain membranes) as
a
well as homomeric recombinant GluK1 and GluK3 receptors.
cloned homomeric
receptors
GluK1 GluK3
native receptors
3
3
 HAMPA
 HKA
1
2
Compound
R
R
K
i
(M)
K
i
(M)
i
K (M)
i
K (M)
b
c
c
(
S)-
2.9
nd.
16
> 100
ATPO
d
e
UBP302
0.6
4.0
nd.
nd.
nd.
nd.
f
f
LY46619
5
0.05
8.9
b
b
g
g
2
2
a
Cl
H
Cl
H
2.8
7.9
> 100
> 100
> 100
> 100
> 100
g
g
b
> 100
> 100
3
a
H
Cl
35
> 100
> 100
> 100
[
[
4.46 ± 0.02]
3
b
H
H
NO
2
2
7.2
23
65
5.15 ± 0.03] [4.64 ± 0.02] 4.19 ± 0.04
3
c
NH
26
> 100
> 100
> 100
[
4.58 ± 0.01]
1
7

3
d
Cl
H
5.7
25
51
> 100
> 100
> 100
> 100
32
[
5.26 ± 0.09] [4.60 ± 0.02] 4.31 ± 0.09
3
3
3
e
f
Cl
NH
OH
H
2
4.9
> 100
11
 100
[
[
[
5.33 ± 0.08]
Cl
5.5
> 100
5.31 ± 0.13] [4.95 ± 0.04]
g
NO
NO
NO
2
2
2
32
> 100
> 100
> 100
38
4.50 ± 0.06]
4.43 ±0.03
3
3
h
i
Cl
6.1
62
[
[
5.23 ± 0.07]
4.21 ± 0.05 4.50 ± 0.02
> 100 > 100
NH
2
7.5
5.15 ± 0.08]
a
Data are given as mean of at least three separate experiments conducted in triplicate. Mean
b
6
c
5
pK
i
± SEM (M) are indicated in brackets. Data from ref. . IC50 values, data from ref. .
d
8
Data from ref. : K
i
value for inhibition of glutamate-evoked currents in HEK293 cells
e
9
expressing homomeric KA receptors. Data from ref. : IC50 values in an Xenopus oocyte
f
10
g
6
assay. Data from ref. . IC50 values, data from ref. . nd. – not determined.
1
8