Design, synthesis and in vitro pharmacology of GluK1 and GluK3 antagonists. Studies towards the design of subtype-selective antagonists through 2-carboxyethyl-phenylalanines with substituents interacting with non-conserved residues in the GluK binding sites

Article abstract of DOI:10.1016/j.bmc.2014.07.045

In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal structures of GluK1 in complex with related ligands, the compounds were designed to explore possible interactions with non-conserved residues outside the glutamate ligand binding site and challenge the water binding network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl-substituted urea (compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that this preference may be attributed to contacts between the NH of the urea substituent and non-conserved Ser741 and Ser761 residues.

Full text of DOI:10.1016/j.bmc.2014.07.045

Bioorganic & Medicinal Chemistry 22 (2014) 5368–5377
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and in vitro pharmacology of GluK1 and GluK3
antagonists. Studies towards the design of subtype-selective
antagonists through 2-carboxyethyl-phenylalanines with
substituents interacting with non-conserved residues in the GluK
binding sites
Niklas Sköld, Birgitte Nielsen, Jacob Olsen, Liwei Han, Lars Olsen, Ulf Madsen, Jesper L. Kristensen,
⇑
Darryl S. Pickering, Tommy N. Johansen
University of Copenhagen, Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, Jagtvej 162, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2014
Revised 15 July 2014
Accepted 28 July 2014
Available online 2 August 2014
In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have
designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with
hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal structures of
GluK1 in complex with related ligands, the compounds were designed to explore possible interactions
with non-conserved residues outside the glutamate ligand binding site and challenge the water binding
network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl-substituted urea
(compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that
this preference may be attributed to contacts between the NH of the urea substituent and non-conserved
Ser741 and Ser761 residues.
Keywords:
Glutamate receptors
GluK1 antagonist
GluK3 antagonist
2-Carboxyethylphenylalanine
Structure based design
SAR
Ó 2014 Elsevier Ltd. All rights reserved.
Negishi reaction
1. Introduction
disease states, especially neurodegenerative disorders such as epi-
lepsy and Alzheimer’s disease, whereas much less is known about
Binding of glutamate to ionotropic glutamate receptors (iGluRs)
and subsequent receptor activation are key steps in fast excitatory
synaptic transmission within the human brain, and it is well doc-
umented that normal activity through glutamatergic synapses is
an important element for several essential brain functions includ-
ing cognition, memory and learning processes. However, dysfunc-
tion in glutamatergic transmission has severe consequences on
normal brain function and is implicated in several brain diseases.1
Three distinct receptor classes of iGluRs are known, named after
the selective compounds that activate the individual receptor
the physiological functions and the therapeutic potentials of KA
receptors.1–4 In contrast to AMPA and NMDA receptors, which
are localized postsynaptically, KA receptors are found both post-
and presynaptically, and it is suggested that KA receptors have
modulatory functions in synaptic transmission.2,5 Evidence from
electrophysiological experiments on knockout animals suggests
that GluK3-containing receptors in some brain regions may play
an important role in synaptic plasticity as GluK3 knockout mice
display markedly reduced short- and long-term synaptic potentia-
tion.5,6 Presynaptic KA receptors are believed to function as autore-
ceptors, controlling the release of the neurotransmitters glutamate
and GABA.7,8 The studies of specific KA receptor subtypes have
been greatly hampered by the lack of selective agonists and antag-
onists and especially selective GluK3 receptor antagonists are
needed. Selective agonists, antagonists and modulators have been
obtained only for GluK1-containing receptors and the employment
of these compounds has linked GluK1-containing receptors to
therapeutic targets.4
classes:
(2S,3S,4S)-3-carboxymethyl-4-isopropenylpyrrolidine-2-
carboxylic acid (kainate, KA), (S)-2-amino-3-(3-hydroxy-5-methyl-
4-isoxazolyl)propionic acid (AMPA) and N-methyl-D-aspartic acid
(NMDA) receptors.1 For several years, AMPA and NMDA receptors
have been known to be attractive therapeutic targets in several
⇑
Corresponding author. Tel.: +45 35336412.
E-mail address: tnj@sund.ku.dk (T.N. Johansen).
http://dx.doi.org/10.1016/j.bmc.2014.07.045
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
5369
block11 (7) (Scheme 1). The key intermediate (6, Scheme 1) was
synthesized from chroman-2-one 4 that was selectively nitrated
para to oxygen with fuming nitric acid in a mixture of acetic acid
and acetic anhydride to avoid di-nitration as previously
described.12 Reflux in methanol gave the ring opened methyl ester
5 which was converted into the triflate 6 in a modest 41% overall
yield.
The freshly prepared organozinc compoound 7, generated by
insertion of activated Zn13 into the carbon halogen bond of methyl
(R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate,
was
reacted with 6 using conditions for coupling of amino acid-derived
organozinc reagents with aryl triflates.14 A low and varying yield,
around 20%, in the cross-coupling reaction producing 8 was
observed, and this prompted us to optimize the reaction conditions.
A number of palladium/ligand combinations that have been shown
to be effective in Nigishi cross-coupling reactions with aryl triflates
15
(Pd2(dba)3 in combination with SPhos14 and Pd(dppf)Cl2 and
Pd-PEPPSI-iPr16 with or without SPhos) were evaluated. Pd2(dba)3
in combination with SPhos performed slightly better than
Pd-PEPPSI-iPr in combination with SPhos, whereas Pd(dppf)Cl2
Figure 1. Chemical structure of target 2-carboxyethylphenylalanines and reference
compounds (1–3). R1 = Cl, H. R2 = NH2, NO2, NHCOEt, NHCOPh, NHSO2Et, NHSO2Ph,
NHCONHEt, NHCONHPh, NHCO-o-NO2-Ph, NHCO-m-NO2-Ph, NHCO-p-NO2-Ph.
gave
a
significantly lower conversion. It was furthermore
In this study we attempt to develop compounds differentiating
GluK1 and GluK3 receptor subtypes, and notably GluK3 selective
antagonists through a structure-based approach using 2-carboxy-
ethylphenylalanines. Compounds with this scaffold are known to
block AMPA, GluK1 and GluK3 receptors, but not GluK2 recep-
tors.9,10 Within the current series of 2-carboxyethylphenylalanines
compounds such as 1–3 (Fig. 1) have shown affinity towards GluK3
receptors in the low micromolar range.10 Furthermore, compounds
1 and 2 have been demonstrated to be antagonists in Xenopus laevis
oocytes expressing homomeric GluK1 or GluK3 receptors using
two-electrode voltage clamp techniques.10 An X-ray structure of
compound 1 in complex with the ligand-binding domain (LBD) of
GluK1 has shown that this compound induces a domain opening
similar to what is observed for the partial agonist domoic acid.10
In this X-ray structure the chloro and nitro substituents of com-
pound 1 are placed in a region of the LBD in which four GluK1–
GluK3 non-conserved amino acid residues are present; that is,
S741N, D742E, S761T and Y460F. However Ser741 and Ser761
may be similarly interesting residues as they appear to be adjacent
to the chloro and nitro substituents of 1.10 Asp742 is positioned at
the edge of the binding pocket and Tyr460 is part of a hydrophobic
pocket that has previously been explored at AMPA receptors9 with-
out finding contributions to subtype selectivity.
In the current study we have focused on these non-conserved
amino acid residues and the surrounding water-binding network10
and introduce a series of hydrogen bond donating and accepting
substituents in the 4-position of a series of 2-carboxyethylphenyl-
alanines (Fig. 1), including N-alkyl and N-aryl substituted amides,
sulfonamides and ureas. This series of target compounds were
designed without substituents in the 5-position in order to adjust
the position of the phenylalanine moiety to better fit into the nar-
row pocket containing the non-conserved amino acid residues. For
a few selected compounds, a chloro substituent was maintained as
in 1 in the 5-position in order to systematically extend the struc-
ture-activity relationship of 2-carboxyethylphenylalanines on
AMPA and kainate receptors.
O
OH
O
O
a, b
O
NO2
5
4
c
F
F
F
O
O
S
H
N
O
O
O
O
Boc
7:
O
Zn
I
NO2
6
d
O
O
O
O
Boc
Boc
N
H
O
N
H
O
e
O
O
NO2
NH2
8
9
f
f
O
OH
O
OH
H2N
O
H2N
O
OH
OH
2. Results and discussion
2.1. Chemistry
NO2
NH2
10
11
Scheme 1. (a) HNO3 (fuming) 1.1 equiv, AcOH/Ac2O, 10–20 °C, 1 h; (b) MeOH,
60 °C, 2 h; (c) Tf2O 1.2 equiv, TEA, DCM, 0 °C, 1 h; (d) 7, Pd2(dba)3 0.04 equiv, SPhos
0.08 equiv, LiCl, DMF/THF, rt, overnight; (e) Pd/C 5%, H2, MeOH, rt, 4 h; (f) (i) LiOH
6 equiv, THF/water, rt, overnight, (ii) TFA/DCM overnight.
The target (S)-phenylalanine derivatives were synthesized by
introducing the alanine side chain through a Negishi cross-coupling
reaction using the readily available (S)-serine-based chiral building

5370
N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
demonstrated that the use of Pd-PEPPSI-iPr in the absence of SPhos
resulted in almost no conversion. In order to evaluate the effect of
different qualities of ‘anhydrous’ LiCl we carried out experiments
in which LiCl of different dryness was added and we found that
the yield of the reaction was dependent only to a minor extent on
the conditions used for drying LiCl. LiCl dried with at heat gun for
1 min under vacuum apparently was sufficient. Tuttle et al.17 have
previously reported that increasing the number of equivalents of
the organozinc reagent had a positive effect on yield. This turned
out also to be the case for the preparation of 8, as using the original
conditions14 but with 3 equiv of the organozinc 7 gave a robust
reaction and a markedly improved reaction yield. Compound 8
was then reduced to the aniline derivative 9 in a high yield. Step-
wise deprotection of 8 and 9 with LiOH in THF/water to hydrolyze
the methyl esters and using TFA in DCM to remove the Boc-group
gave 10 and 11, respectively, as zwitterions after preparative HPLC.
With the key intermediate 9 in hand, three divergent synthetic
routes were followed (Scheme 2) leading to N-substituted amide,
sulfonamide and urea containing target compounds. The amides
12–16 were made by reaction of the corresponding acid chlorides
with 9 using triethylamine as base. For the synthesis of amide 17
the mixed anhydride was used instead of the corresponding acid
chloride. For the synthesis of the sulfonamides 18 and 19 com-
pound 9 was reacted with the appropriate sulfonyl chlorides using
pyridine as base, whereas the urea derivatives 20 and 21 were
obtained in excellent yields by heating 9 together with ethyl- or
phenylisocyanate, respectively. The intermediates 12-21 were
deprotected using a two-step procedure as described above for
the preparation of 10 to give target compounds 22–31.
known. In order to verify an acceptable stereochemical purity of
the target phenylalanine derivatives, experiments were carried out
using a commercially available model compound (methyl (S)-2-
((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propanoate, ee
>99%) as a structurally close related reference compound. To simu-
late conditions used during the two-step deprotection procedure
stereochemically pure (S)-2-((tert-butoxycarbonyl)amino)-3-(4-
nitrophenyl)propanoate was treated in accordance with the
preparation of compound 10 to afford (S)-4-nitrophenylalanine.
The stereochemical purity of the crude (S)-4-nitrophenylalanine
was determined to 98.4% ee by chiral HPLC using a Crownpak
CR(À) column. The identity of the late eluting impurity was con-
firmed by spiking with racemic 4-nitrophenylalanine. Based on
these experiments we conclude that the target compounds isolated
most likely have acceptable high stereochemical purities (>98% ee).
2.2. Pharmacology
All target compounds were evaluated in radioligand binding
studies at recombinant homomeric rat GluK1 and GluK3 receptors
and at native rat AMPA, KA and NMDA receptors (Table 1). None of
the target compounds, nor the reference compounds 1–3, showed
significant affinity at native KA and NMDA receptor sites
(IC50 > 100 lM). In a binding assay of recombinant GluK1 and
GluK3, using [3H]KA as radiolabel, the Ki values for the reference
compounds 1–3 (as racemates) were respectively determined to
be: 3.7, 12 and 21 lM (GluK1) and 11, 5.1 and 14 lM (GluK3),
which are all similar to the Ki values reported previously using
[3H]SYM2081 as radiolabel (Table 1).10
The synthesis of the 5-chloro substituted target compounds 38–
40 is shown in Scheme 3. Regioselective iodination of 32 with sil-
ver sulfate gave 33 in acceptable yield. The aryl iodide then easily
underwent the Negishi cross-coupling reaction with 7, most likely
due to the higher reactivity of iodine compared to triflate.18 The
protected amino acid derivative 34 was then functionalized by
reaction with the appropriate acid chlorides giving amides
35–37. Deprotection in a mixture of 4 M HCl and THF gave the
target amide substituted compounds 38–40.
When comparing the affinities of compounds with a Cl-atom in
the 5-position (1, 3, 38, 39 and 40) with the corresponding com-
pounds without Cl (10, 11, 23, 25 and 26), it is clear that at GluK1
receptors the removal of Cl in the 5-position leads to an approxi-
mately ten-fold drop in affinity (Table 1). At GluK3 receptors, the
presence of a Cl-atom in the 5-position seems to be less important
for compounds with an aromatic substituent, as the GluK3-affinity
of compounds (such as 38, 39 and 40) is only marginally affected
by removal of the Cl substituent (compared to 23, 25 and 26).
Interestingly compounds 38–40 containing aryl substituted
amides at C4 are equipotent with the most potent reference com-
As the target phenylalanine derivatives were prepared as single
enantiomers, the determination of the stereochemical purity could
not be performed directly, as the corresponding R-isomers are not
pound (1) ((S)-1 has a Ki of 1.5 lM at GluK1 and 8.0 l
M at GluK3)10
and show increased GluK1 selectivity. Especially compound 40
shows a significant increase in selectivity towards GluK1 over
GluK3 and AMPA receptors.
For compounds without a Cl-atom at C5, introduction of aro-
matic substituents at C4 improves the binding to GluK1 as well
as GluK3 receptors, as the phenyl substituted amides and ureas
(23 and 31) show an improved affinity compared the correspond-
ing ethyl substituted compounds (22 and 30). The binding affinities
furthermore reveal that introducing a sulfonamide substituent (28
and 29) leads to compounds with no measurable affinity, even for
the aryl substituted sulfonamide 29. Apparently, the sulfonamide
substituent cannot be accommodated by the receptors either due
to unfavorable orientation of hydrogen bond interactions or due
to an unfavorable orientation of the sulfonamide ethyl and phenyl
substituents.
In order to understand the structure-affinity relationships in
more details molecular docking studies of compounds 22 and 23
were carried out using the GluK1 crystal structure 4DLD10
(Fig. 2). These studies indicate that the aromatic substituent of
compound 23 could possibly reach a hydrophobic region formed
by Val765 and Tyr764, thereby gaining affinity. Such a hydropho-
bic region may not be reached by 22 with the aliphatic ethyl sub-
stituent. Furthermore, molecular docking studies reveal the
presence of a hydrophobic region formed by Pro533 and Tyr460
that well accommodates the Cl-atom at C5 of compound 1 (Figs. 2
Scheme 2. (a) RCOCl 1 equiv, TEA 1.5 equiv, DCM, 0 °C, 25 min; (b) Boc-Gly-OH
1 equiv, isobutyl chloroformate 1 equiv, N-ethylmorpholine 1.5 equiv, DCM, rt,
30 min; (c) RSO2Cl 1 equiv, pyridine 1.1 equiv, DCM rt, 48 h; (d) RNCO, THF, 60 °C,
4 h; (e) (i) LiOH 6 equiv, THF/water, rt, overnight. (ii) TFA/DCM overnight

N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
5371
Scheme 3. (a) Ag2SO4 1.1 equiv, I2 1.1 equiv, MeOH, rt, 1 h; (b) Pd2(dba)3 0.04 equiv, SPhos 0.08 equiv, DMF/THF, 60 0C, overnight; (c) RCOCl 1.1 equiv, DIPEA 1.1 equiv, THF,
rt, 1 h; (d) 4 M HCl, THF, reflux, overnight.
Table 1
Binding affinities at recombinant KA receptors and native AMPA receptorsa
Compounds
GluK1 Ki (
lM)
GluK3 Ki (lM)
AMPA-R Ki (lM)
1
2
3
3.0 0.8b
14.4 2.5b
4.7 0.3b
17.0 3.2b
>100c
2.8 [5.56 0.04]
51 [4.29 0.03]
>100
>100
>100
>100
>100
>100
nd
>100
>100
>100
>100
12.3 1.2b
13.3 1.0b
50–100c
>100c
10
11
22
23
24
25
26
27
28
29
30
31
38
39
40
>100c
>100d
>100d
32 9c
50–100c
50–100d
29 4c
50–100d
>100d
50–100d
24 6c
19 2d
>100d
>100d
>100d
>100d
>100d
>100d
>100d
>100
50–100d
2.4 0.3d
6.3 0.6d
1.3 0.3d
20 1d
50–100d
40 4d
34 4d
55 [4.26 0.05]
28 [4.56 0.04]
>100
35 [4.46 0.03]
a
Data are mean values SEM of at least three individual experiments conducted
in triplicate.
b
Data (racemate) reported in Ref. 10.
c
Data obtained using [3H]SYM2081 as radioligand.
d
Data obtained using [3H]KA as radioligand. nd, not determined.
and 3). This hydrophobic region also likely accommodates the Cl-
atoms and may contribute to the increased affinity at least seen
at GluK1 receptor sites for compounds 1, 3, 38, 39 and 40 com-
pared to compounds 10, 11, 23, 25 and 26.
Figure 2. Difference in docking poses between aliphatic and aromatic compounds.
Compound 23 shown in green and 22 shown in yellow. The hydrophobic surface
with an isovalue of À0.5 kcal/mol is shown in yellow.
Even though the affinity at GluK3 is reduced compared to the
reference compounds, the phenyl substituted urea (31), having
no substituent in the 5-position, some preference for GluK3 over
GluK1. The preference can possibly be attributed to the extra NH
in the urea of compound 31, as the docking study predicts the
NH to be close to both non-conserved Ser741 and Ser761 residues.
The molecular docking studies further indicate that this com-
pound, with no substituent at C5 but a large aromatic 4-substitu-
ent, adopts a pose where the C5 on the phenylalanine ring partly
occupies the hydrophobic pocket that was filled by the Cl-atom
of compound 1 (Fig. 3).
compounds 38–40, showed significant selectivity for GluK1 over
GluK3 and AMPA receptors, whereas one compound, 31, showed
some preference for the GluK3 subtype. The affinity of the target
compounds indicates that strong GluK3-preferring interactions
with any of the non-conserved amino acid residues have not been
obtained. Based on these observations it was decided not to carry
out functional studies. This means that antagonist profiles have
not been confirmed for the four compounds of interest but antago-
nist profiles are assumed based on the functional profiles observed
for structurally closely related reference antagonists 1 and 2.10
However, based on binding affinities and molecular modelling
studies, two hydrophobic regions in the GluK1 receptor site have
been identified. Hydrophobic interaction with these regions seems
to contribute to the observed affinity at GluK1 receptors and to a
smaller extent at GluK3 receptors. The observed activities reveal
that it is possible to accommodate larger substituents into receptor
pocket. However it remains to be exploited if the affinity profile of
31 can be further improved. An enhanced GluK3/GluK1 subtype
selectivity may be obtained through a combination of interactions
2.3. Conclusions
In the present study a series of 2-carboxyethylphenylalanines
with hydrogen bond donating and accepting substituents in the 4-
position have been synthesized and evaluated in vitro at GluK1
and GluK3 receptors. The range of substituents have been selected
aiming at interactions with non-conserved Ser741 and Ser761
residues, directly or through water mediated interactions. Three

5372
N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
0.1% HCOOH and 100% water (by volume) Mobile phase B: 100%
ACN. Isocratic elution A:B. Flow rate: 8 mL/min. NMR spectra were
recorded on 300 MHz Varian or 400, 500, or 600 MHz Bruker instru-
ments. Processing was done with ACDLABS 12.0. Signals are
reported in ppm (d) using the residual solvent peak as internal ref-
erence. Coupling constants (J) are given in Hertz (Hz). Multiplet pat-
terns are designated the following abbreviations, or combinations
of these: br–broad, m–multiplet, s–singlet, d–doublet, t–triplet
and q–quartet. Optical rotations were measured at 22 °C using a
Bellingham + Stanley ADP410 polarimeter. Elemental analysis was
recorded on a Perkin Elmer 2400 CHN Elemental Analyzer and per-
formed by the Department of Physical Chemistry at the University
of Vienna in Austria.
3.1.1. Methyl 3-(2-hydroxy-5-nitrophenyl)propanoate (5)
Fuming nitric acid (12.1 mL, 189 mmol) in acetic acid (38.6 mL)
was added dropwise during 45 min to chroman-2-one (4)
(17.1 mL, 135 mmol) in acetic anhydride (102 mL), keeping the
temperature at 10–20 °C. The reaction was stirred for 1 h at room
temperature and then poured into ice water to give 6-nitrochro-
man-2-one as a light yellow solid (17 g, 88 mmol, 65%) that was
used without further purification in the next step. 1H NMR
(300 MHz, CDCl3): d = 8.13–8.22 (m, 2H), 7.18 (d, J = 8.8 Hz, 1H),
3.08–3.19 (m, 2H), 2.83–2.92 (m, 2H). Crude 6-nitrochroman-2-
one (17 g, 88 mmol) was dissolved in MeOH (150 mL) and refluxed
for 2 h. The reaction was cooled and poured into water (800 mL). A
yellow precipitation was filtered off and dried in a vacuum oven at
60 °C for 24 h to give 5 (13.6 g, 60.4 mmol, 68%) as a light yellow
powder. 1H NMR (300 MHz, CDCl3) d = 8.48–8.65 (m, 1H), 7.98–
8.09 (m, 2H), 6.96 (d, J = 9.7 Hz, 1H), 3.75 (s, 3H), 2.92–3.03 (m,
2H), 2.76–2.87 (m, 2H).
Figure 3. Superimposition of 31 in green on the GluK1 crystal structure in complex
with compound 1 (4DLD) in cyan, illustrating the change in position of the aromatic
ring of 31 compared to the aromatic ring of 1 with a chloride in the 5 position. The
hydrophobic surface with an isovalue of À0.5 kcal/mol is shown in yellow.
3.1.2. Methyl 3-(5-nitro-2-trifluoromethylsulfonyloxyphenyl)-
propanoate (6)
with the identified hydrophobic regions and non-conserved amino
acid residues.
Compound 5 (6.20 g, 27.5 mmol) and TEA (15.4 mL, 110 mmol)
was dissolved in DCM (200 mL) and cooled to À2 °C under N2
before triflic anhydride (5.6 mL, 33.0 mmol) was added dropwise
(reaction temperature À2 to 4 °C).The reaction was stirred for
30 min and poured into water. The organic phase was separated
and washed with 2% NaHCO3 (Â3), brine and dried over Na2SO4.
The organic phase was then filtered through silica twice, and the
silica washed with several portions of DCM. The combined frac-
tions were evaporated to afford 6 as a light yellow oil (9.21 g,
25.5 mmol, 93%). 1H NMR (300 MHz, CDCl3): d = 8.28 (d,
J = 2.6 Hz, 1H), 8.20 (dd, J = 9.1, 2.6 Hz, 1H), 7.49 (d, J = 8.8 Hz,
1H), 3.72 (s, 3H), 3.16 (t, J = 7.3 Hz, 2H), 2.76 (t, J = 7.3 Hz, 2H).
HPLC tR = 1.91 min.
3. Experimental
3.1. Chemistry
Commercially acquired chemicals were used without purifica-
tion. Anhydrous reactions were carried out in oven or flame dried
glassware under nitrogen. Dry solvents (DMF, THF and DCM) were
dried using an SG Water solvent purification system and all other
solvents were dried with 3 Å molecular sieves. Purification by col-
umn chromatography was carried out according to standard proce-
dures using silica gel 60 or by using a CombiFlash Rf System
(Teledyne Isco, Inc.). Merck pre-coated silica gel 60 F254 plates were
used for thin layer chromatography analysis, compounds were
visualized using a combination of UV light (254 nm), anisaldehyde
stain or ninhydrin stain. HPLC was recorded on a Merck Hitachi
LaChrom system using an L-7400 detector using a Chromolith RP-
18e column 50 Â 4.6 mm. Mobile phase A: 0.1% TFA and 100% water
(by volume). Mobile phase B: 0.1% TFA, 90% ACN and 10% water (by
volume). Linear gradient elution 0-5 min 0–90% A. Flow rate: 4 mL/
min. LCMS was recorded on an Agilent 1100 series system using an
3.1.3. (R)-(2-(tert-Butoxycarbonylamino)-3-methoxy-3-
oxopropyl)zinc(II) iodide (7)
To zinc (1.2 g, 18.2 mmol) dried by heating with a heatgun for
1 min under vacuum, DMF (1.5 mL) and 1,2-dibromoethane
(0.08 mL, 0.911 mmol) were added. The reaction was heated to
50 °C and was stirred for 30 min. After returning to rt TMS-Cl
(0.03 mL, 0.2 mmol) was added and the mixture was stirred for
60 min at rt. Methyl (R)-2-(tert-butoxycarbonylamino)-3-iodopro-
panoate (1.00 g, 3.04 mmol) in DMF (1.5 mL) was added dropwise
and an exotermic reaction was observed after stirring for a few sec-
onds. After 10 min the starting material was consumed (judged by
TLC). The concentration of 7 was determined to approximately
0.7 M using a standard titration method.19
Xbridge C18 column (3.5
lm, 100 Â 4.6 mm) with UV detection at
254 nm and 210 nm. Mass detection was done by a Hewlett Packard
Series 1100 MSD mass detection system using atmospheric pres-
sure ionisation electrospray (API-ES). Mobile phase A: 0.1% HCOOH,
5% ACN, 95% water (by volume). Mobile phase B: 0.05% HCOOH, 95%
ACN, 5% water (by volume). Gradient: 0–1 min 100% A, 1–6 min 0–
100% B, 6–10 min 100% B. Flow rate: 1 mL/min. Preparative HPLC
was performed on a Jasco 880-PU system with a TSP Spectra series
3.1.4. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(3-
methoxy-3-oxopropyl)-4-nitrophenyl)propanoate (8)
UV100 detector using an YMC-120A OdDMeSi 5
(250 Â 21 mm) and manual fraction collection. Mobile phase A:
lm column
To 7 (30.0 mL, 21.0 mmol) in DMF, SPhos (0.28 g, 0.67 mmol),
LiCl (1.00 g, 25.2 mmol) (dried for 1 min with heatgun under

N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
5373
vacuum) and Pd2(dba)3 (0.27 g, 0.29 mmol) was added. Compound
6 (3.00 g, 8.40 mmol) in DMF (3 mL) was added and the reaction
slurry was stirred overnight. The reaction was diluted with
150 mL EtOAc and filtered through celite. The organic phase was
washed with sodium pyrosulfite, water (Â2) and brine. Drying over
Na2SO4, filtration and evaporation gives a red–brown oil that was
purified using CombiFlash affording 8 as a yellow oil that partly
crystallizes on standing (2.4 g, 5.67 mmol, 68%). 1H NMR
(300 MHz, CDCl3): d = 8.04 (d, J = 2.35 Hz, 1H), 7.98 (dd, J = 8.4,
2.5 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 5.10 (d, J = 7.9 Hz, 1H), 4.53–
4.66 (m, 1H), 3.73 (s, 4 H), 3.68 (s, 3H), 3.30 (dd, J = 13.9, 6.0 Hz,
1H), 3.11 (d, J = 7.3 Hz, 1H), 3.07 (t, J = 7.3 Hz, 1H), 2.69 (t,
J = 7.3 Hz, 2H), 1.37 (s, 8 H). HPLC tR = 1.8 min. LCMS tR = 6.846 min
[M+H-Boc]+ = 311.
evaporation of the organic phase, purification using CombiFlash
(30–55% EtOAc in heptane) gave the target amide as a solid.
3.1.10. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(3-
methoxy-3-oxopropyl)-4-propionamidophenyl)propanoate (12)
Compound 12 (171 mg, 0.39 mmol, 75%) was prepared using
general procedure 2. 1H NMR (400 MHz, DMSO-d6): d = 9.73
(s, 1H), 7.43–7.26 (m, 3H), 7.06 (d, J = 8.0 Hz, 1H), 4.17–4.07
(m, 1H), 3.60 (s, 6 H), 2.99 (dd, J = 14.3, 5.3 Hz, 1H), 2.87–2.76
(m, 3H), 2.56 (t, J = 7.7 Hz, 2H), 2.28 (q, J = 7.5 Hz, 2H), 1.33 (s, 9
H), 1.06 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
d = 172.6, 172.5, 171.7, 155.3, 139.0, 138.0, 130.0, 129.7, 119.1,
116.8, 78.2, 54.4, 51.8, 51.3, 34.4, 32.8, 29.4, 28.1, 26.9, 9.6. LCMS
tR = 6.194 min [M+1-Boc]+ = 337.2.
3.1.5. Methyl (S)-3-(4-amino-2-(3-methoxy-3-
oxopropyl)phenyl)-2-((tert-butoxycarbonyl)amino)-
propanoate (9)
3.1.11. Methyl (S)-3-(4-benzamido-2-(3-methoxy-3-
oxopropyl)phenyl)-2-((tert-butoxycarbonyl)amino)-
propanoate (13)
Compound 8 (2.6 g, 6.4 mmol) and Pd/C (10%, 0.5 g, 0.47 mmol)
was mixed in MeOH (60 mL). The reaction was flask was evapo-
rated and purged with N2 before adding H2 by balloon. The reaction
was stirred at 35 °C for 2 h and then overnight at rt. The reaction
was filtered and evaporated to a light brown oil. Purification using
CombiFlash (30-55% EtOAc in Heptane) gave 9 as a light orange oil
(1.87 g, 4.52 mmol, 96%). 1H NMR (400 MHz, CDCl3): d = 6.87 (d,
J = 7.8 Hz, 1H), 6.44–6.53 (m, 2H), 5.00 (d, J = 7.8 Hz, 1H), 4.40–
4.56 (m, 1H), 3.68 (s, 3H), 3.70 (s, 3H), 3.59 (brs, 2H), 2.99–3.11
(m, 1H), 2.80–2.99 (m, 3H), 2.53–2.63 (m, 2H), 1.40 (s, 9 H). LCMS
t = 5.078 min [M+23] = 403.2 and [M+1-Boc] = 281.2.
Compound 13 (140 mg, 0.289 mmol, 55%) was prepared using
general procedure 2. 1H NMR (400 MHz, CDCl3): d = 10.14 (s, 1H),
7.98–7.88 (m, 2H), 7.65–7.48 (m, 5 H), 7.34 (d, J = 8.3 Hz, 1H),
7.14 (d, J = 8.3 Hz, 1H), 4.21–4.10 (m, 1H), 3.61 (s, 6 H), 3.04 (dd,
J = 14.4, 5.4 Hz, 1H), 2.93–2.79 (m, 3H), 2.65–2.56 (m, 2H), 1.34
(s, 9 H). LCMS tR = 6.798 min [M+1-Boc]+ = 385.2.
3.1.12. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(3-
methoxy-3-oxopropyl)-4-(2-nitrobenzamido)phenyl)-
propanoate (14)
Compound 14 (242 mg, 0.46 mmol, 87%) was prepared using
general procedure 2. 1H NMR (400 MHz, DMSO-d6): d = 10.56
(s, 1H), 8.17–8.10 (m, 1H), 7.89–7.83 (m, 1H), 7.78–7.71 (m, 2H),
7.50–7.41 (m, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H),
4.20–4.11 (m, 1H), 3.66–3.57 (m, 6 H), 3.30 (s, 2H), 3.08–2.98 (m,
1H), 2.87 (t, J = 7.7 Hz, 3H), 2.63–2.55 (m, 2H), 1.34 (s, 9 H). 13C
NMR (101 MHz, DMSO-d6): d = 173.7, 169.9, 163.9, 146.5, 140.1,
137.4, 134.0, 132.7, 131.0, 130.9, 130.5, 129.2, 124.2, 119.9,
117.5, 55.1, 35.7, 33.5, 27.4. HPLC tR = 2.03 min. LCMS tR = 6.516 -
min [M+1-Boc]+ = 430.1.
3.1.6. General procedure 1: Deprotection of methyl esters and
Boc-group
The protected compound 1 equiv was dissolved in THF (0.7 mL)
and LiOH 1 M 6 equiv was added. The reaction was stirred at rt for
overnight. The reaction was evaporated to remove THF and the
reaction was acidified with KHSO4 (1 M). The reaction was
extracted with DCM. TFA was added to the organic phase to a ratio
of DCM:TFA 2:1 and the reaction was stirred overnight. The reac-
tion was evaporated to a solid. The solid was triturated with ether
to remove residual TFA before purification on HPLC.
3.1.13. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(3-
methoxy-3-oxopropyl)-4-(3-
3.1.7. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-nitrophenyl)-
propanoic acid (10)
nitrobenzamido)phenyl)propanoate (15)
Compound 15 (260 mg, 0.49 mmol, 93%) was prepared
using general procedure 2. 1H NMR (400 MHz, DMSO-d6):
d = 10.49 (s, 1H), 8.78 (t, J = 1.9 Hz, 1H), 8.47–8.35 (m, 2H), 7.84
(t, J = 7.9 Hz, 1H), 7.62–7.54 (m, 2H), 7.36 (d, J = 8.0 Hz, 1H), 7.17
(d, J = 8.0 Hz, 1H), 4.22–4.12 (m, 1H), 3.61 (s, 6 H), 3.05 (dd,
J = 14.3, 5.3 Hz, 1H), 2.89 (t, J = 7.7 Hz, 3H), 2.62 (t, J = 7.7 Hz, 2H),
1.34 (s, 9 H). LCMS tR = 6.762 min [M+1-Boc]+ = 430.1.
Compound 10 was prepared using general procedure 1. Recrys-
tallization from water gave 10 (22 mg, 0.18 mmol, 75%) as a white
solid. 1H NMR (300 MHz, 10% D2O in DMSO-d6): d = 7.98 (m, 1H),
7.45 (m, 1H), 7.92 (m, 1H), 3.57 (dd, J = 8.7, 5.30 Hz, 1H), 3.29
(dd, J = 14.8, 5.3 Hz, 1H), 3.01 (dd, J = 14.5, 8.7 Hz, 1H), 2.91
(t, J = 7.3 Hz, 2H), 2.38 (t, J = 7.3 Hz, 2H). HPLC tR = 0.51 min.
3.1.8. (S)-2-Amino-3-(4-amino-2-(2-carboxyethyl)phenyl)-
propanoic acid (11)
3.1.14. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(3-
methoxy-3-oxopropyl)-4-(4-
Compound 11 (23 mg, 0.091 mmol, 27%) was prepared using
general procedure 1. 1H NMR (400 MHz, D2O): d = 8.27 (s, 1H),
7.44 (d, J = 8.3 Hz, 1H), 7.34 (d, J = 2.3 Hz, 1H), 7.32–7.27 (m, 1H),
4.11–4.01 (m, 1H), 3.41 (dd, J = 7.3, 14.6 Hz, 1H), 3.26 (dd,
J = 14.6, 7.8 Hz, 1H), 3.07 (t, J = 7.5 Hz, 2H), 2.78 (t, J = 7.4 Hz, 2H).
LCMS tR = 1.375 min [M+1]+ = 253.1.
nitrobenzamido)phenyl)propanoate (16)
Compound 16 (230 mg, 0.43 mmol, 83%) was prepared using
general procedure 2. 1H NMR (400 MHz, DMSO-d6): d = 10.47 (s,
1H), 8.42–8.32 (m, 2H), 8.21–8.13 (m, 2H), 7.62–7.52 (m, 2H),
7.35 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H), 4.16 (dd, J = 14.4,
8.7 Hz, 1H), 3.61 (s, 6 H), 3.05 (dd, J = 14.6, 5.5 Hz, 1H), 2.94–2.81
(m, 3H), 2.65–2.57 (m, 2H), 1.25 (s, 9 H). LCMS tR = 6.750 min
[M+1-Boc]+ = 430.1.
3.1.9. General procedure 2: Acylation of 9 using acid chlorides
Compound 9 (0.2 g, 0.53 mmol) was dissolved in DCM (3 mL),
TEA (1.5 equiv) was added and the solution was cooled to 0 °C. Acid
chloride (1 equiv) was added and the reaction was stirred for
25 min at rt and followed by LCMS. The reaction was quenched
with NH4Cl (sat) and the organic phase washed with NaHCO3
(sat), water and brine. After drying over Na2SO4, filtration and
3.1.15. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-
((tert-butoxycarbonyl)amino)acetamido)-2-(3-methoxy-3-
oxopropyl)phenyl)propanoate (17)
Boc-Gly-OH (92 mg, 0.526 mmol) was dissolved in DCM (3 mL)
and isobutyl chloroformate (0.069 mL, 0.526 mmol) was added.

5374
N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
The reaction was stirred for 5 min before N-ethylmorpholine
(0.1 mL, 0.789 mmol) was added and stirring was continued for
30 min before 9 (200 mg, 0.526 mmol) was added. The reaction
was stirred overnight and quenched with NH4Cl (sat). After separa-
tion of phases, the organic phase was washed with NaHCO3 (sat),
water and brine, dried over Na2SO4 and evaporated to an orange
oil that was purified on CombiFlash using 30–55% EtOAc in Hep-
3.1.20. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(3-
methoxy-3-oxopropyl)-4-(3-phenylureido)phenyl)propanoate
(21)
Compound 21 (190 mg, 0.38 mmol, 96%) was prepared using
general procedure 3. 1H NMR (400 MHz, CDCl3): d = 7.41–7.31
(m, 4 H), 7.22 (brs, 1H), 7.13 (tt, J = 1.9, 6.7 Hz, 2H), 7.09–7.03
(m, 1H), 6.57 (brs, 1H), 6.51 (br. s, 1H), 5.06 (d, J = 8.0 Hz, 1H),
4.58–4.50 (m, 1H), 3.72 (s, 3H), 3.66 (s, 3H), 3.15 (dd, J = 14.3,
6.0 Hz, 1H), 2.96 (dt, J = 7.8, 3.3 Hz, 3H), 2.62 (t, J = 7.7 Hz, 2H),
1.40 (s, 9H). LCMS tR = 6.697 min [M+H-Boc]+ = 400.1.
tane as eluent to give 17 as
a light-brown solid (256 mg,
0.476 mmol) 91%.1H NMR (400 MHz, DMSO-d6): d = 7.40–7.28 (m,
2H), 7.08 (d, J = 8.3 Hz, 1H), 6.99 (t, J = 6.3 Hz, 1H), 4.18–4.08 (m,
1H), 3.68 (d, J = 6.0 Hz, 1H), 3.64–3.54 (m, 6 H), 3.00 (dd, J = 14.3,
5.5 Hz, 1H), 2.84 (t, J = 7.5 Hz, 3H), 2.75–2.67 (m, 1H), 2.60–2.53
3.1.21. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-
(m, 2H), 1.39 (s,
9
H), 1.33 (s,
9
H). LCMS tR = 6.460 min
propionamidophenyl)propanoic acid (22)
[M+1-Boc]+ = 438.3.
Compound 12 (85 mg, 0.195 mmol) was dissolved in THF and
LiOH (1.2 mL, 1.2 mmol) was added and stirred at rt for 5 h. The
reaction was evaporated to remove THF and the reaction was acid-
ified with KHSO4 (2 M). A yellow oil was formed and the mixture
was extracted with DCM. TFA was added to a ratio of DCM:TFA
2:1 and the reaction was stirred for 30 min. LCMS shows complete
conversion. The reaction was evaporated to an oil that was
triturated to a solid with diethyl ether. The solvent was washed
with diethyl ether, filtered and dried in a vacuum oven for 2 h
3.1.16. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-
(ethylsulfonamido)-2-(3-methoxy-3-
oxopropyl)phenyl)propanoate (18)
Compound 9 (200 mg, 0.526 mmol) was dissolved in DCM
(3 mL). Pyridine (0.047 mL, 0.578 mmol) was added followed by
ethanesulfonyl chloride (0.050 mL, 0.526 mmol). The reaction
was stirred at rt for 48 h and quenched with NH4Cl (satd). The
organic phase was washed with NaHCO3 (satd), water and brine,
dried over Na2SO4 and evaporated to an oil. The crude oil was puri-
fied via CombiFlash (30–55% EtOAc in heptane) to give 18 (170 mg,
0.36 mmol, 68%).
giving 22 as
a
solid (57 mg, 0.185 mmol, 95%). 1H NMR
(600 MHz, D2O): d = 7.35 (s, 1H), 7.31–7.23 (m, 2H), 4.07–4.01
(m, 1H), 3.39 (dd, J = 14.9, 6.1 Hz, 1H), 3.11 (dd, J = 14.7, 8.8 Hz,
1H), 3.01–2.95 (m, 2H), 2.72 (t, J = 7.3 Hz, 2H), 2.42 (q, J = 7.7 Hz,
2H), 1.18 (t, J = 7.7 Hz, 3H). 13C NMR (151 MHz, D2O): d = 177.5,
177.0, 173.0, 140.2, 136.6, 131.1, 130.0, 122.5, 120.4, 54.9, 34.5,
32.9, 29.8, 26.7, 9.3. HPLC tR = 1.04 min. LCMS tR = 1.124 min
[M+H]+ = 309.1.
1H NMR (400 MHz, CDCl3): d = 7.11–7.06 (m, 1H), 7.03 (s, 1H),
7.02–6.97 (m, 1H), 6.26 (brs, 1H), 5.04 (d, J = 8.3 Hz, 1H), 4.60–
4.49 (m, 1H), 3.73 (s, 3H), 3.69 (s, 3H), 3.22–3.07 (m, 3H), 3.04–
2.90 (m, 4 H), 2.66–2.57 (m, 2H), 1.40 (s, 9H). HPLC tR = 1.78 min.
3.1.17. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(3-
methoxy-3-oxopropyl)-4-
3.1.22. (S)-2-Amino-3-(4-benzamido-2-(2-
carboxyethyl)phenyl)propanoic acid (23)
(phenylsulfonamido)phenyl)propanoate (19)
Compound 13 (100 mg, 0.206 mmol) was dissolved in THF
(0.7 mL) and LiOH (1.2 mL, 1.2 mmol) was added. The reaction
was stirred at rt for 90 min. The reaction was evaporated, more
water was added and the reaction was acidified with KHSO4
(1 M) to pH 2. A light yellow precipitation was formed and filtered
of. The solid was dissolved in DCM:TFA (2:1) and stirred for 30 min.
The reaction was evaporated to a yellow oil. The crude was dis-
solved in water and pH was adjusted by TEA/TFA to approximately
pH 4 when a precipitation was formed. The solid was separated
and dried (16.8 mg, 0.047 mmol, 23%). 1H NMR (400 MHz, D2O):
d = 7.82 (d, J = 7.3 Hz, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.49 (t,
J = 7.4 Hz, 2H), 7.26–7.15 (m, 3H), 3.42 (dd, J = 7.9, 6.4 Hz, 1H),
3.00 (dd, J = 13.9, 6.4 Hz, 1H), 2.94–2.85 (m, 2H), 2.78 (dd,
J = 13.8, 8.0 Hz, 1H), 2.48–2.37 (m, 2H). 13C NMR (101 MHz, D2O):
d = 182.5, 182.4, 169.8, 141.2, 137.9, 135.0, 133.1, 131.8, 130.9,
128.7, 127.4, 123.2, 120.7, 57.5, 38.7, 37.5, 28.8. LCMS tR = 1.82
Compound 9 (200 mg, 0.526 mmol) was dissolved in DCM
(3 mL). Pyridine (0.047 mL, 0.578 mmol) was added followed by
benzenesulfonyl chloride (0.067 mL, 0.526 mmol). The reaction
was stirred at rt for 48 h and quenched with NH4Cl (satd). The
organic phase washed with NaHCO3 (satd), water and brine,
dried over Na2SO4 and evaporated to an oil. The crude oil was
purified via CombiFlash (30–55% EtOAc in heptane) to give
19 (227 mg, 0.436 mmol, 83%). 1H NMR (400 MHz, CDCl3):
d = 7.74 (d, J = 7.5 Hz, 2H), 7.59–7.50 (m, 1H), 7.49–7.39 (m, 2H),
6.98 (d, J = 8.3 Hz, 1H), 6.93–6.79 (m, 2H), 6.35 (brs, 1H), 5.00
(d, J = 8.8 Hz, 1H), 4.56–4.43 (m, 1H), 3.67 (s,
(dd, J = 6.4, 14.2 Hz, 1H), 3.01–2.80 (m, 3H), 2.56–2.44 (m, 2H),
1.39 (s, 9 H). HPLC tR = 1.99 min.
6 H), 3.09
3.1.18. General procedure 3: preparation of urea derivatives 20
and 21
Compound 9 (150 mg, 0.394 mmol) was dissolved in dry THF
(2 mL) and isocyanate 1 equiv was added. The reaction was stirred
for 15 min and then heated to 60 °C for 4 h. Evaporation gave a
solid that was used without further purifications.
min, [M+H]+ = 357.1. Anal. Calcd. for C19H20N2O5Á1.3 H2O:
C
60.09; H 6.00; N 7.38; Found: C 59.80 H 5.69 N 7.29.
3.1.23. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-(2-
nitrobenzamido)phenyl)propanoic acid (24)
Compound 24 (80 mg, 0.2 mmol, 44%) was prepared using gen-
eral procedure 1. 1H NMR (400 MHz, DMSO-d6): d = 8.16–8.10 (m,
1H), 7.90–7.82 (m, 1H), 7.78–7.70 (m, 2H), 7.52–7.43 (m, 2H),
7.22 (d, J = 8.3 Hz, 1H), 3.42 (dd, J = 8.3, 5.0 Hz, 1H), 3.20 (dd,
J = 14.7, 4.9, Hz, 1H), 2.95–2.79 (m, 3H), 2.53–2.44 (m, 2H). 13C
NMR (101 MHz, DMSO-d6): d = 174.2, 170.4, 164.4, 147.0, 140.6,
137.9, 134.5, 133.2, 131.5, 131.4, 131.0, 129.7, 124.7, 120.5,
118.0, 55.6, 36.2, 34.0, 27.9. LCMS tR = 1.60 min [M+H]+ = 402.
3.1.19. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(3-
ethylureido)-2-(3-methoxy-3-oxopropyl)phenyl)propanoate
(20)
Compound 20 (176 mg, 0.390 mmol, 99%) was prepared using
general procedure 3. 1H NMR (400 MHz, CDCl3): d = 7.15 (brs,
1H), 7.04 (s, 2H), 6.21 (brs, 1H), 5.04 (d, J = 9.0 Hz, 1H), 4.71 (brs,
1H), 4.59–4.48 (m, 1H), 3.72 (s, 3H), 3.67 (s, 3H), 3.35–3.26 (m,
2H), 3.14 (dd, J = 14.3, 6.0 Hz, 1H), 2.95 (dt, J = 7.7, 3.0 Hz, 3H),
2.64–2.57 (m, 2H), 1.40 (s, 9 H), 1.17 (t, J = 7.2 Hz, 3H). LCMS
tR = 6.054 min [M+H-Boc]+ = 352.1.
[
a
]D: +12 (c = 0.5 in 0.1 M HCl). Anal. Calcd. for C19H19N3O7Á0.8
H2O 0.1 TFA: C 53.98; H 4.88, N 9.84; Found: C 53.77, H 4.66, N
9.82.

N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
5375
3.1.24. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-(3-
nitrobenzamido)phenyl)propanoic acid (25)
1H), 6.90 (d, J = 2.3 Hz, 1H), 6.83 (dd, J = 8.3, 2.3 Hz, 1H), 3.34 (dd,
J = 7.9, 5.1 Hz, 1 H), 3.08 (dd, J = 14.8, 5.0 Hz, 1H), 2.83–2.69 (m,
3H), 2.44–2.26 (m, 2H) 13C NMR (101 MHz, DMSO-d6): d = 173.6,
140.5, 139.8, 136.1, 132.7, 131.1, 129.2, 126.6, 120.3, 117.7, 99.5,
54.9, 35.6, 33.3, 27.3 LCMS tR = 1.94 min [M+H]+ = 393. Anal. Calcd.
for C18H20N2O6SÁ0.9H2OÁ0.3 HCOOH: C 52.03, H 5.34, N 6.63;
Found: C 51.68, H 4.96, N 6.60.
Compound 25 (15 mg, 0.037 mmol, 20%) was prepared using
general procedure 1. 1H NMR (400 MHz, DMSO-d6, D2O, NaOD):
d = 8.80–8.75 (m, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.13–8.06 (m, 1H),
7.54 (t, J = 7.9 Hz, 1H), 7.11–7.01 (m, 2H), 6.89 (d, J = 8.0 Hz, 1H),
3.16 (dd, J = 9.7, 3.9 Hz, 1H), 3.01 (dd, J = 13.6, 3.5 Hz, 1H), 2.81–
2.64 (m, 2H), 2.35 (dd, J = 13.6, 10.3 Hz, 1H), 2.18 (t, J = 8.0 Hz,
2H) 13C NMR (101 MHz, DMSO-d6): d = 180.0, 179.2, 164.6, 151.0,
147.9, 145.5, 140.6, 135.0, 130.0, 129.1, 128.8, 125.2, 123.1,
123.0, 122.1, 57.8, 40.5, 40.0, 30.1HPLC tR = 1.25 min Anal. Calcd.
for C19H19N3O7Á0.95 H2OÁ0.4 TFA: C 51.24, H 4.63, N 9.05; Found:
C 51.16, H 4.49, N 9.10.
3.1.29. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-(3-
ethylureido)phenyl)propanoic acid (30)
Compound 30 (96 mg, 0.297 mmol, 76%) was prepared using
general procedure 1. 1H NMR (400 MHz, D2O): d = 7.26–7.12 (m,
3H), 4.15 (brs, 1H), 3.92 (dd, J = 9.0, 6.0 Hz, 1H), 3.69 (brs, 1H),
3.35 (dd, J = 14.7, 5.9 Hz, 1H), 3.20 (q, J = 7.2 Hz, 2H), 3.11–2.87
(m, 3H), 2.75–2.64 (m, 2H), 1.12 (t, J = 7.3 Hz, 3H) 13C NMR
(151 MHz, D2O): d = 178.1, 173.8, 158.3, 140.3, 137.8, 131.1,
128.7, 121.9, 119.8, 55.6, 35.0, 34.8, 33.0, 26.9, 14.4. LCMS
3.1.25. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-(4-
nitrobenzamido)phenyl)propanoic acid (26)
Compound 16 (100 mg, 0.189 mmol) was dissolved in THF
(0.7 mL) and LiOH 1 M (1.14 mL, 1.14 mmol) was added. The reac-
tion was stirred at rt for 90 min. The reaction was evaporated to
remove THF and the reaction was acidified with KHSO4 (1 M). A
yellow oil was formed and the reaction was extracted with DCM.
TFA was added to a ratio of DCM:TFA 2:1 and the reaction was stir-
red for 30 min. The reaction was evaporated to a yellow oil. The
crude was dissolved in water and pH was adjusted by TEA/TFA to
approximately pH 4 affording 26 (28.4 mg, 0.071 mmol, 38%) as a
solid. 1H NMR (400 MHz, D2O): d = 8.30–8.23 (m, J = 9.0 Hz, 2H),
8.01–7.94 (m, J = 8.8 Hz, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.01–6.93
(m, 2H), 3.43 (t, J = 7.2 Hz, 1H), 2.99 (dd, J = 13.9, 6.1 Hz, 1H),
2.93–2.84 (m, 2H), 2.77 (dd, J = 13.9, 8.2 Hz, 1H), 2.46–2.37 (m,
2H) 13C NMR (101 MHz, D2O): d = 182.7, 182.6, 168.4, 148.4,
144.8, 140.9, 131.2, 130.8, 129.6, 128.5, 123.7, 123.6, 123.5,
121.2, 116.8, 57.6, 38.9, 37.4, 28.9 HPLC tR = 1.27 min Anal. Calcd.
for C19H19N3O7Á1.8 H2O: C 52.61, H 5.25, N 9.69; Found: C 52.36,
H 5.00, N 9.49.
tR = 1.299 min [M+H]+ = 324.1. [
a]D: +8 (c = 0.5 in 0.1 M HCl). Anal.
Calcd. for C15H21N3O5Á1.4 HCOOH: C 50.80, H 6.19, N 10.84; Found:
C 50.22, H 5.90, N 11.45.
3.1.30. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-(3-
phenylureido)phenyl)propanoic acid (31)
Compound 31 (78 mg, 0.21 mmol, 55%) was prepared using
general procedure 1. 1H NMR (600 MHz, DMSO-d6): d = 10.23
(brs, 1H), 9.92 (brs, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.42 (s, 1H), 7.23
(t, J = 8.1 Hz, 2H), 7.05 (s, 2H), 6.94–6.86 (m, 1H), 3.54 (t,
J = 6.1 Hz, 1H), 3.10–2.96 (m, 2H), 2.83 (t, J = 7.2 Hz, 2H), 2.44 (dt,
J = 8.0, 2.8 Hz, 2H) 13C NMR (151 MHz, DMSO-d6): d = 174.3,
172.1, 153.6, 141.2, 140.3, 139.6, 130.9, 129.0, 127.6, 121.6,
118.6, 118.5, 118.4, 116.7, 55.4, 36.0, 33.3, 28.0. LCMS tR = 4.645 -
min [M+H]+ = 372.1. [
a]D: +12 (c = 0.5 in 0.1 M HCl). Anal. Calcd
for C19H21N3O5Á0.85 H2O: C 59.01, H 5.92, N 10.87; Found: C
58.39, H 5.29, N 10.64.
3.1.26. (S)-2-Amino-3-(4-(2-aminoacetamido)-2-(2-
carboxyethyl)phenyl)propanoic acid (27)
3.1.31. Methyl 3-(5-amino-4-chloro-2-iodophenyl)propanoate
(33)
Compound 27 (52 mg, 0.168 mmol, 90%) was prepared using
general procedure 1. 1H NMR (600 MHz, DMSO-d6): d = 10.40 (s,
1H), 7.43 (dd, J = 8.3, 2.0 Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 7.18 (d,
J = 8.4 Hz, 1H), 4.01 (t, J = 7.3 Hz, 1H), 3.76 (s, 2H), 3.12 (dd,
J = 14.5, 7.5 Hz, 1H), 3.04 (dd, J = 14.7, 7.3 Hz, 1H), 2.83 (t,
J = 7.7 Hz, 2H), 2.52–2.47 (m, 2H) 13C NMR (151 MHz, DMSO-d6):
d = 173.5, 170.5, 164.6, 140.3, 137.3, 130.8, 128.9, 119.4, 117.2,
To a solution of commercially available methyl 3-(3-amino-4-
chlorophenyl)propanoate 32 (3.39 g, 15.9 mmol) dissolved in
50 mL MeOH, Ag2SO4 (5.46 g, 17.5 mmol) was added. I2 (4.44 g,
17.5 mmol) was dissolved in 75 mL MeOH and added by addition
funnel over 10 min and stirred for 1 h at rt. The reaction mixture
was filtered through celite and concentrated in vacuo. Redissolved
in 100 mL EtOAc and made basic with 2 M NaOH before washing
with Na2S2O3, brine, dried over Na2SO4, filtered and concentrated
in vacuo. The crude product was purified by flash chromatography
(1:4 EtOAc:Hept). Evaporation to an orange oil (3.66 g, 10.8 mmol)
68%. 1H NMR (400 MHz, CDCl3): d = 7.63 (s, 1H), 6.71 (s, 1H), 3.69
(s, 3H), 2.93 (t, J = 7.8 Hz, 2H), 2.59 (t, J = 7.8 Hz, 2H) 13C NMR
(101 MHz, CDCl3): d = 172.8, 142.9, 142.3, 138.7, 118.3, 116.4,
84.2, 51.7, 35.2, 34.1 GCMS tR = 12.7 min, [M]+ = 339.
53.2, 40.9, 34.6, 32.6, 26.9 Anal. Calcd. for
C
14H19N3O5Á0.25
H2OÁ2.05 TFA: C 39.70, H 3.97, N 7.67; Found: C 39.76, H 4.02, N
7.72.
3.1.27. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-
(ethylsulfonamido)phenyl)propanoic acid (28)
Compound 28 (85 mg, 0.25 mmol, 67%) was prepared using
general procedure 1. 1H NMR (400 MHz, DMSO-d6): d = 7.19 (d,
J = 8.3 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H), 6.98 (dd, J = 8.3, 2.3, Hz,
1H), 3.44 (dd, J = 8.3, 5.3 Hz, 1H), 3.16 (dd, J = 14.7, 5.1 Hz, 1H),
3.04 (q, J = 7.4 Hz, 2H), 2.89–2.75 (m, 3 H), 2.48–2.41 (m, 2H),
1.18 (t, J = 7.4 Hz, 3H) 13C NMR (101 MHz, DMSO-d6): d = 173.7,
140.6, 136.8, 130.9, 130.7, 120.0, 117.6, 54.9, 44.8, 35.5, 33.3,
3.1.32. Methyl (S)-3-(4-amino-5-chloro-2-(3-methoxy-3-
oxopropyl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate
(34)
To 33 (3.54 g, 8.55 mmol), Pd2(dba)3 (20 mg, 0.022 mmol) and
dry DMF. SPhos (18.3 mg, 0.044 mmol) dissolved in dry THF
(0.75 mL) was added followed by 7 (6.7 g, 17.1 mmol) and stirred
at 60 °C overnight. The mixture was diluted with EtOAc, filtered
through celite, washed with Na2S2O3, water, brine and dried over
Na2SO4. Filtration and evaporation gives a crude that was purified
by flash chromatography (1:3 to 1:1 EtOAc:Hept), 34 was isolated
in two fractions which afforded a yield of 2.08 g (48%) and 1.75 g
(40%). The first fraction contained traces of 33 (10%) and the second
fraction had some unknown impurity, no further purification was
done. 1H NMR (400 MHz, CDCl3): d = 6.97 (s, 1H), 6.61 (s, 1H),
27.4, 8.0 LCMS tR = 1.20 min [M+H]+ = 345. [
a]D: +16 (c = 0.5 in
0.1 M HCl). Anal. Calcd. for C14H20N2O6SÁ1.0 HCOOH: C 46.15, H
5.68, N 7.18; Found: C 45.99, H 5.59, N 7.31.
3.1.28. (S)-2-Amino-3-(2-(2-carboxyethyl)-4-
(phenylsulfonamido)phenyl)propanoic acid (29)
Compound 29 (86 mg, 0.22 mmol, 50%) was prepared using
general procedure 1. 1H NMR (400 MHz, DMSO-d6): d = 7.80–7.73
(m, 2H), 7.64–7.57 (m, 1H), 7.57–7.50 (m, 2H), 7.08 (d, J = 8.3 Hz,

5376
N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
5.03 (d, J = 8.0 Hz, 1H), 4.48 (m, 1H), 3.72 (s, 3H), 3.67 (s, 3H), 3.06
(dd, J = 14.2, 5.9 Hz, 1H), 2.80–2.91 (m, 3H), 2.55–2.59 (m, 2H), 1.41
(s, 9H) 13C NMR (101 MHz, CDCl3): d = 173.0, 172.5, 155.1, 141.5,
138.9, 130.8, 125.1, 117.4, 116.5, 80.0, 54.2, 52.3, 51.7, 35.1, 34.4,
28.2, 26.9.
3.1.36. General procedure 3: Preparation of target compounds
38–40
The protected compound was dissolved in 2.5 mL THF and
added 3.5 mL 4 M HCl (aq). The solution was refluxed for 8–15 h.
Upon completion, THF was evaporated and 2.5 mL H2O was added
before isolating the deprotected compound by preparative HPLC.
3.1.33. Methyl (S)-3-(4-benzamido-5-chloro-2-(3-methoxy-3-
oxopropyl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate
(35)
3.1.37. (S)-2-Amino-3-(4-benzamido-2-(2-carboxyethyl)-5-
chlorophenyl)propanoic acid (38)
To a solution of 34 (0.3 g, 0.72 mmol) in 2 mL dry THF, DIPEA
(0.15 mL, 0.87 mmol) and benzoyl chloride (0.10 ml, 0.87 mmol)
were added and stirred 1 h at rt. A second addition of benzoyl chlo-
ride (0.025 mL, 0.216 mmol) was added and stirred for 2 h. The
reaction mixture was diluted with 20 mL EtOAc, washed with
NaHCO3(Sat), brine, dried over Na2SO4 filtered and concentrated
in vacuo. The crude product was purified by flash chromatography
(1:1 EtOAc:Hept) and concentrated in vacuo. Redissolved in EtOAc
and washed with K2CO3(Satd) (Â2) then concentrated in vacuo to
give 35 (0.275 g, 0.53 mmol, 74%) as a colorless solid. 1H NMR
(400 MHz, CDCl3): d = 8.47 (s, 1H), 8.38 (s, 1H), 7.90–7.92 (m,
2H), 7.51–7.62 (m, 3H), 7.18 (s, 1H), 5.09 (d, J = 8.0 Hz, 1H), 4.53–
4.58 (m, 1H), 3.74 (s, 3H), 3.70 (s, 3H), 3.16–3.21 (m, 1H, 2.93–
3.05 (m, 3H), 2.66–2.70 (m, 2H), 1.42 (s, 9H) 13C NMR (101 MHz,
CDCl3): d = 172.9, 172.3, 165.1, 155.0, 139.3, 134.5, 133.6, 132.1,
131.2, 130.2, 128.9, 127.0, 121.5, 120.7, 80.0, 54.0, 52.3, 51.7,
35.0, 34.6, 28.2, 27.0.
Compound 38 (0.028 g, 72 lmol, 38%) was prepared using gen-
eral procedure 3. 1H NMR (400 MHz, D2O): d = 7.78–7.86 (m, 2H),
7.58–7.64 (m, 1H), 7.45–7.53 (m, 3H), 7.39 (s, 1H), 4.26 (dd, J = 8.4,
6.9 Hz, 1H), 3.39 (dd, J = 14.8, 6.8 Hz, 1H), 3.16 (dd, J = 14.8, 8.5 Hz,
1H), 2.86–2.95 (m, 2H), 2.62–2.71 (m, 2H) 13C NMR (101 MHz,
DMSO-d6): d = 173.5, 169.9, 165.3, 163.1, 139.1, 134.8, 134.0,
133.4, 131.8, 130.7, 128.4, 127.7, 126.8, 54.3, 35.1, 33.2, 26.6. LCMS
tR = 4.48 min [M+H]+ = 391. Anal. Calcd. for C19H19ClN2O5Á1.1 H2O: C
55.57, H 5.20, N 6.82; Found: C 55.26, H 4.77, N 6.68.
3.1.38. (S)-2-Amino-3-(2-(2-carboxyethyl)-5-chloro-4-(3-
nitrobenzamido)phenyl)propanoic acid (39)
Compound 39 (0.03 g, 69 lmol, 39%) was prepared using gen-
eral procedure 3 and isolated as a colorless solid 1H NMR
(400 MHz, D2O): d = 8.66 (s, 1H), 8.42 (ddd, J = 8.3, 2.3, 0.8 Hz,
1H), 8.22 (d, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.48 (s, 1H),
7.42 (s, 1H), 4.28 (dd, J = 8.3, 6.8 Hz, 1H), 3.42 (dd, J = 14.8,
6.8 Hz, 1H), 3.20 (dd, J = 14.8, 8.3 Hz, 1H), 2.93 (t, J = 7.4 Hz, 2H),
2.69 (t, J = 7.4 Hz, 2H) 13C NMR (101 MHz, DMSO-d6): d = 173.6,
169.6, 163.4, 147.8, 139.3, 135.8, 135.4, 134.1, 132.8, 130.8,
130.3, 128.7, 127.1, 126.4, 122.5, 54.6, 35.2, 33.4, 26.7. LCMS
tR = 4.70 min [M+H]+ = 436. Anal. Calcd. for C19H18ClN3O7Á0.1
H2O: C 52.15, H 4.19, N 9.60; Found: C 52.00, H 3.98, N 9.43.
3.1.34. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(5-chloro-
2-(3-methoxy-3-oxopropyl)-4-(3-
nitrobenzamido)phenyl)propanoate (36)
To a solution of 34 (0.3 g, 0.723 mmol) in 5 mL dry THF, DIPEA
(0.14 mL, 0.795 mmol) and 3-nitrobenzoyl chloride (0.173 g,
0.932 mmol) were added and stirred for 30 min at rt. The reaction
mixture was diluted with 15 mL EtOAc, washed with NaHCO3
(satd), brine, dried over Na2SO4, filtered and concentrated in vacuo.
The crude product was purified by flash chromatography (3:7
EtOAc:Hept) to give 36 (331 mg, 0.586 mmol, 81%) as a colorless
solid.1H NMR (400 MHz, CDCl3): d = 8.76 (d, J = 1.9 Hz, 1H), 8.45
(ddd, J = 8.3, 2.3, 1.0 Hz, 1H), 8.39 (s, 1H), 8.37 (s, 1H), 8.22–8.25
(m, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.22 (s, 1H), 5.11 (d, J = 7.5 Hz,
1H), 4.57 (q, J = 6.8 Hz, 1H), 3.75 (s, 3H), 3.71 (s, 3H), 3.20 (dd,
J = 14.2, 5.9 Hz, 1H), 2.93–3.09 (m, 3H), 2.63–2.74 (m, 2H), 1.42
(s, 9H) 13C NMR (101 MHz, CDCl3): d = 172.8, 172.2, 162.7, 155.0,
148.5, 139.5, 136.2, 132.7, 132.2, 130.4, 130.2, 126.6, 122.2,
121.9, 121.1, 80.1, 54.0, 52.4, 51.8, 34.9, 34.7, 28.2, 27.0 LC MS
tR = 6.92 min, [M+H-Boc]+ = 464.
3.1.39. (S)-2-Amino-3-(2-(2-carboxyethyl)-5-chloro-4-(4-
nitrobenzamido)phenyl)propanoic acid (40)
Compound 40 (0.031 g, 73 lmol, 41%) was prepared using gen-
eral procedure 3 and isolated as a colorless solid 1H NMR
(400 MHz, D2O): d = 8.27 (d, J = 8.8 Hz, 2H), 7.97 (d, J = 8.8 Hz,
2H), 7.43 (s, 1H), 7.38 (s, 1H), 4.23 (dd, J = 8.3, 7.0 Hz, 1H), 3.37
(dd, J = 14.8, 6.8 Hz, 1H), 3.15 (dd, J = 14.8, 8.5 Hz, 1H), 2.88
(t, J = 7.3 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H) 13C NMR (101 MHz,
DMSO-d6): d = 173.5, 169.6, 163.9, 149.3, 139.7, 139.3, 135.8,
132.8, 130.8, 129.2, 128.6, 127.0, 123.6, 54.6, 35.2, 33.4, 26.7 Anal.
Calcd. for C19H18ClN3O7Á1 H2O: C 50.28, H 4.44, N 9.26; Found: C
50.11, H 4.18, N 9.04.
3.2. Receptor binding studies
3.1.35. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(5-chloro-
2-(3-methoxy-3-oxopropyl)-4-(4-
nitrobenzamido)phenyl)propanoate (37)
Radioligand displacement studies were performed as previously
described at native rat AMPA, KA and NMDA receptors20 and at
recombinant rat homomeric GluK1(Q)1b and GluK3a receptors.20
For recombinant receptors assays, either [3H]SYM2081 or [3H]KA
was used as the radioligand.
To a solution of 34 (0.3 g, 0.72 mmol) in 2 mL dry THF, DIPEA
(0.15 mL, 0.86 mmol) and 4-nitrobenzoyl chloride (0.147 g,
0.79 mmol) was added and stirred for 35 min at rt. The reaction
mixture was diluted with 20 mL EtOAc, washed with K2CO3(satd),
brine, dried over Na2SO4 filtered and concentrated in vacuo. The
crude product was purified by flash chromatography (1:4 to 7:13
to 9:11 to 1:1 EtOAc:Hept) to afford 37 (0.317 g, 0.561 mmol,
78%) as a yellow solid. 1H NMR (400 MHz, CDCl3): d = 8.34–8.43
(m, 4H), 8.04–8.10 (m, 2H), 7.21 (s, 1H), 5.11 (d, J = 8.0 Hz, 1H),
4.50–4.62 (m, 1H), 3.75 (s, 3H), 3.70 (s, 3H), 3.20 (dd, J = 14.3,
6.0 Hz, 1H), 2.92–3.09 (m, 3H), 2.63–2.73 (m, 2H), 1.42 (s, 9H)
13C NMR (101 MHz, CDCl3): d = 172.9, 172.2, 163.1, 155.0, 150.0,
140.0, 139.6, 133.0, 132.3, 130.4, 128.3, 124.2, 121.8, 121.0, 80.2,
54.0, 52.5, 51.8, 35.0, 34.8, 28.2, 27.1.
3.3. Molecular docking
3.3.1. Protein preparation
The receptor modelwas prepared for dockingas follows:The pro-
tein (PDB 4DLD) was prepared by adding hydrogens and removal of
remote water molecules by the Protein Preparation Wizard software
in Maestro.21 SiteMap22–24 was used to identify favorable regions for
water molecules. Four of the crystal water molecules bind in regions
where the hydrogen bonding acceptor surface has an energy of
À15 kcal/mol or below. These water molecules were included in

N. Sköld et al. / Bioorg. Med. Chem. 22 (2014) 5368–5377
5377
2. Contractor, A.; Mulle, C.; Swanson, G. T. Trends Neurosci. 2011, 34, 154.
3. Lerma, J.; Marques, Joana M. Neuron 2013, 80, 292.
4. Jane, D. E.; Lodge, D.; Collingridge, G. L. Neuropharmacology 2009, 56, 90.
5. Perrais, D.; Veran, J.; Mulle, C. Trends Pharmacol. Sci. 2010, 31, 516.
the subsequent docking (see below). The kept water molecules are
numbered in 4DLD as 401, 407, 491 and 538.
6. Pinheiro, P. S.; Perrais, D.; Coussen, F.; Barhanin, J.; Bettler, B.; Mann, J. R.;
Malva, J. O.; Heinemann, S. F.; Mulle, C. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
12181.
7. Schmitz, D.; Mellor, J.; Nicoll, R. A. Science 1972, 2001, 291.
8. Lauri, S. E.; Bortolotto, Z. A.; Bleakman, D.; Ornstein, P. L.; Lodge, D.; Isaac, J. T.;
Collingridge, G. L. Neuron 2001, 32, 697.
9. Szymanska, E.; Frydenvang, K.; Contreras-Sanz, A.; Pickering, D. S.; Frola, E.;
Serafimoska, Z.; Nielsen, B.; Kastrup, J. S.; Johansen, T. N. J. Med. Chem. 2011, 54,
7289.
3.3.2. Ligand preparation
The structures were generated by ChemDraw and minimized
using OPLS-2005 force field using default settings. Epik25–27 in
Maestro was then used to determine protonation and the com-
pounds with the lowest energy state penalty were selected.
3.3.3. Docking calculations
10. Venskutonyte, R.; Frydenvang, K.; Valades, E. A.; Szymanska, E.; Johansen, T. N.;
Kastrup, J. S.; Pickering, D. S. ChemMedChem 2012, 7, 1793.
11. Rilatt, I.; Caggiano, L.; Jackson, R. F. W. Synlett 2005, 2701.
12. Katritzky, A. R.; Ji, F. B.; Fan, W. Q.; Beretta, P.; Bertoldi, M. J. Heterocycl. Chem.
1992, 29, 1519.
13. Knochel, P.; Yeh, M. C. P.; Berk, S. C.; Talbert, J. J. Org. Chem. 1988, 53, 2390.
14. Dexter, C. S.; Jackson, R. F. W.; Elliott, J. Tetrahedron 2000, 56, 4539.
15. Rottlander, M.; Palmer, N.; Knochel, P. Synlett 1996, 573.
16. Nasielski, J.; Hadei, N.; Achonduh, G.; Kantchev, E. A. B.; O’Brien, C. J.; Lough, A.;
Organ, M. G. Chem. Eur. J. 2010, 16, 10844.
The selected compounds were docked into the GluK1 receptor
using Glide XP and standard settings. For compounds giving no
poses or poses that significantly deviated from that of the crystal-
lized ligand the setting ‘restrict docking to reference position’
was used for the (S)-2-carboxyethyl-phenylalanine part of the
compound.
The grid was generated by selecting the ligand and using a 20 Å
box. The original ligand was re-docked into the receptor with an
RMSD of 0.4 Å compared to the experimental binding mode.
17. Tuttle, J. B.; Azzarelli, J. M.; Bechle, B. M.; Dounay, A. B.; Evrard, E.; Gan, X.;
Ghosh, S.; Henderson, J.; Kim, J.-Y.; Parikh, V. D.; Verhoest, P. R. Tetrahedron
Lett. 2011, 52, 5211.
18. Tsuji, J. Palladium Reagents and Catalysts—New Perspectives for the 21st Century;
John Wiley & Sons Ltd, 2004.
Acknowledgments
19. Krasovskiy, A.; Knochel, P. Synthesis-Stuttgart 2006, 890.
20. Assaf, Z.; Larsen, A. P.; Venskutonyte, R.; Han, L.; Abrahamsen, B.; Nielsen, B.;
Gajhede, M.; Kastrup, J. S.; Jensen, A. A.; Pickering, D. S.; Frydenvang, K.;
Gefflaut, T.; Bunch, L. J. Med. Chem. 2013, 56, 1614.
This study was supported by the GluTarget Program and the
Hørslev Foundation.
21. Maestro, Schrödinger, LLC: New York, NY, 2012.
22. Halgren, T. Chem. Biol. Drug Des. 2007, 69, 146.
23. Halgren, T. A. J. Chem. Inf. Model. 2009, 49, 377.
Supplementary data
24. SiteMap, Schrödinger, LLC: New York, NY, 2012.
25. Shelley, J. C.; Cholleti, A.; Frye, L. L.; Greenwood, J. R.; Timlin, M. R.; Uchimaya,
M. J. Comput. Aided Mol. Des. 2007, 21, 681.
26. Greenwood, J. R.; Calkins, D.; Sullivan, A. P.; Shelley, J. C. J. Comput. Aided Mol.
Des. 2010, 24, 591.
Supplementary data (XP-docking scores) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2014.07.045.
27. Epik, Schrödinger, LLC: New York, NY, 2012.
References and notes
1. Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F. S.; Vance, K. M.;
Ogden, K. K.; Hansen, K. B.; Yuan, H.; Myers, S. J.; Dingledine, R. Pharmacol. Rev.
2010, 62, 405.