Structure-activity relationship studies of argiotoxins: Selective and potent inhibitors of ionotropic glutamate receptors

Article abstract of DOI:10.1021/jm301602d

Argiotoxin-636 (ArgTX-636), a natural product from the spider Argiope lobata, is a potent but nonselective open-channel blocker of ionotropic glutamate (iGlu) receptors. Here, three series of analogues were designed to exploit selectivity among iGlu receptors, taking advantage of a recently developed solid-phase synthetic methodology for the synthesis of ArgTX-636 and analogues. Initially, the importance of secondary amino groups in the polyamine chain was studied by the synthesis of systematically modified ArgTX-636 analogues, which were evaluated for pharmacological activity at NMDA and AMPA receptors. This led to the identification of two compounds with preference for NMDA and AMPA receptors, respectively. These were further elaborated by systematically changing the aromatic headgroup and linker amino acid leading to compounds with increased potency and selectivity for NMDA and AMPA receptors, respectively. Thus, the first structure-activity relationship study of ArgTX-636 has been carried out and has provided lead compounds for probing the ion channel region of iGlu receptors.

Full text of DOI:10.1021/jm301602d

Article
pubs.acs.org/jmc
Structure−Activity Relationship Studies of Argiotoxins: Selective and
Potent Inhibitors of Ionotropic Glutamate Receptors
Mette H. Poulsen,† Simon Lucas,†,‡ Tinna B. Bach, Anne F. Barslund, Claudius Wenzler,
Christel B. Jensen, Anders S. Kristensen, and Kristian Strømgaard*
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
S
* Supporting Information
ABSTRACT: Argiotoxin-636 (ArgTX-636), a natural product from the spider
Argiope lobata, is a potent but nonselective open-channel blocker of ionotropic
glutamate (iGlu) receptors. Here, three series of analogues were designed to
exploit selectivity among iGlu receptors, taking advantage of a recently
developed solid-phase synthetic methodology for the synthesis of ArgTX-636
and analogues. Initially, the importance of secondary amino groups in the
polyamine chain was studied by the synthesis of systematically modified ArgTX-
636 analogues, which were evaluated for pharmacological activity at NMDA and
AMPA receptors. This led to the identification of two compounds with
preference for NMDA and AMPA receptors, respectively. These were further
elaborated by systematically changing the aromatic headgroup and linker amino
acid leading to compounds with increased potency and selectivity for NMDA
and AMPA receptors, respectively. Thus, the first structure−activity relationship
study of ArgTX-636 has been carried out and has provided lead compounds for probing the ion channel region of iGlu receptors.
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) recep-
tors.
INTRODUCTION
■
The family of ionotropic glutamate (iGlu) receptors are ligand-
gated ion channels that mediate the majority of excitatory
synaptic transmission in the vertebrate brain and are crucial for
normal brain function.1−3 Dysfunction of iGlu receptors is
involved in a range of neurological and psychiatric diseases, and
iGlu receptors are considered important drug targets for brain
diseases. In particular, inhibition of iGlu receptors is a
promising strategy for the treatment of neurodegenerative
diseases such as stroke and Alzheimer’s disease.1 However,
development of selective and clinically effective iGlu receptor
inhibitors has proven to be difficult, and today only a few drugs
directed against iGlu receptors have been approved, one of
which is memantine, an open-channel blocker of the N-methyl-
D-aspartate (NMDA) subtype of iGlu receptors used in the
treatment of Alzheimer’s disease.4−7
Polyamine toxins are a group of small molecules found in
spiders and wasps that like memantine are open-channel
blockers of iGlu receptors.8 Polyamine toxins have found
valuable use as pharmacological tools based on their high
affinity and selectivity for iGlu receptors,8,9 particularly as they
are uniquely selective for iGlu receptors, which are permeable
to Ca2+.10,11 However, polyamine toxins have not yet been
explored as templates for the development of iGlu receptor
drugs, although the selective antagonism of Ca2+-permeable
iGlu receptors has been suggested as a particularly promising
strategy in neuroprotection.12−14 A main concern is that native
polyamine toxins generally distinguish poorly among individual
subtypes of iGlu receptors, such as NMDA and α-amino-3-
Philanthotoxin-433 (PhTX-433, 1, Figure 1) is the
prototypical polyamine toxin15 that has been studied in several
Figure 1. Structures of two prototypical polyamine toxins: philantoxin-
433 (PhTX-433) isolated from the digger wasp Philanthus triangulum
and argiotoxin-636 (ArgTX-636) isolated from the orb weaver spider
Argiope lobata. ArgTX-636 can be divided into four structural regions,
headgroup, linker amino acid, polyamine, and amino acid tail, which
also relates to the biosynthetic origin.
structure−activity relationship (SAR) studies, where partic-
ularly the ease of synthesis by solid-phase synthetic (SPS)
methodologies has been exploited and provided a large number
of PhTX-433 derivatives, some of which particularly show
increased selectivity for AMPA receptors.16−23 Much less
Received: October 30, 2012
Published: January 2, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthesis of Compounds 3−9
a
Reagents and conditions: (a) 2-(3,5-dimethoxy-4-formylphenoxy)ethylpolystyrene, NaBH(OAc)3, DMF/AcOH (9:1); (b) Fmoc-L-Asn(Trt)-OH,
HATU, DIPEA; (c) 20% piperidine in DMF; (d) 2-(2,4-bis(benzyloxy)phenyl)acetic acid, HATU, DIPEA; (e) N-Teoc amino alcohol, Bu3P, ADDP;
(f) TBAF, 55 °C; (g) Fmoc-L-Arg(Pbf)-OH, HATU, DIPEA; (h) DBU, 2-mercaptoethanol; (i) TFA/DCM/TIPS/H2O (75:20:2.5:2.5); (j) H2,
Pd(OH)2/C.
attention has been focused on the wealth of spider polyamine
toxins such as argiotoxin-636 (ArgTX-636, 2, Figure 1)
probably because of their increased structural complexity and
hence more challenging synthetic feasibility.24−26 However,
several spider polyamine toxins are significantly more potent as
iGlu receptor antagonists than PhTX-433, and some even
display a certain degree of selectivity among iGlu receptor
subtypes.24 Thus, spider polyamine toxins are potentially
superior starting points for development of pharmacological
tools such as potent and subtype specific ligands for studies of
iGlu receptors. ArgTX-636 (2) is a polyamine toxin isolated
from the venom of the orb weaver spider Argiope lobata24,25
and is one of the most potent inhibitors of iGlu receptors,
which are blocked in a use- and voltage-dependent manner
presumably by binding to the ion channel region of the
receptors.24 Toxin 2 has, similar to other channel blockers of
iGlu receptors, demonstrated neuroprotective properties27,28
and thus has the potential to be used as a template for
developing therapeutically relevant compounds. However, the
main challenge is that native 2 does not distinguish between
subtypes of iGlu receptors.1,8,9
We have recently developed an efficient SPS methodology
for the generation of 2 and derivatives, and in the same study
we found a notable relationship between modification of the
polyamine moiety of 2 and antagonistic selectivity among
NMDA and AMPA receptors.29 Initially, we found that
modification of the guanidyl moiety was not tolerated, but
changing either one of the secondary amino groups in the
polyamine moiety led to increased selectivity for NMDA and
AMPA receptors, respectively.29 In the same study, molecular
modeling was used to demonstrate that the position of
secondary amines in the polyamine region is important for
key interactions with residues in the iGlu ion channel and that
these interact differentially with NMDA and AMPA receptors,
thus providing an opportunity for design of analogues of 2 with
improved AMPA versus NMDA receptor selectivity. In the
current work, we have explored this rationale by designing a
range of derivatives of 2 and prepared these by the newly
developed SPS methodology, exploiting first the role of
secondary amines in the polyamine region in a systematic
manner and subsequently examining the importance of the
headgroup and linker amino acid region of these derivatives.
RESULTS AND DISCUSSION
■
Design and Synthesis. Initially, we wanted to examine the
importance of the relative position of the secondary amino
group in the polyamine moiety of the two ArgTX-636
derivatives previously synthesized and characterized, namely,
ArgTX-93 and ArgTX-57, which are potent inhibitors and show
about 32- and 11-fold selectivity of NMDA and AMPA
receptors, respectively.29 Thus, using an SPS methodology
similar to what was previously described,29 we systematically
modified the relative position of the secondary amino group in
the polyamine moiety, leading to seven derivatives, ArgTX-39
to ArgTX-93 (3−9, Scheme 1). These were synthesized,
essentially as previously described. Briefly mononosyl protected
diamines with three to nine methylene groups were loaded
onto a backbone amide linker (BAL) by reductive amination
(Scheme 1) followed by successive peptide couplings
generating the headgroup to provide resin-bound intermediates
10−16 (Scheme 1). After liberation of a primary amino group,
N-2-(trimethylsilyl)ethyloxycarbonyl (Teoc) protected amino
alcohols were reacted with 10−16 in Fukuyama−Mitsunobu
reactions, providing the polyamine moiety (17−23). The
terminal protected arginine was then coupled followed by
deprotection of nosyl-protected amines to provide resin-bound,
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a
Scheme 2. Synthesis of Compounds 5a−h and 8a−h
a
Reagents and conditions: (a) Fmoc-L-Asn(Trt)-OH, Fmoc-L-Ala-OH, Fmoc-L-Asp(tBu)-OH, Fmoc-L-His(Trt)-OH, or Fmoc-L-Trp(Boc)-OH,
HATU, DIPEA; (b) 20% piperidine in DMF; (c) 2-(2,4-bis(benzyloxy)phenyl)acetic acid, (2-benzyloxy)phenylacetic acid, (4-benzyloxy)-
phenylacetic acid, phenylacetic acid, or 2-(1H-indol-3-yl)acetic acid, HATU, DIPEA; (d) N-Teoc amino alcohol, Bu3P, ADDP; (e) TBAF, 55 °C; (f)
Fmoc-L-Arg(Pbf)-OH, HATU, DIPEA; (g) DBU, 2-mercaptoethanol; (h) TFA/DCM/TIPS/H2O (75:20:2.5:2.5); (i) H2, Pd(OH)2/C.
Table 1. Antagonist Effect of Argiotoxin Analogues 2−9 at AMPA and NMDA Receptors
a
IC50 (nM)
b
c
d
compd
m
n
GluA1
GluN1/2A
selectivity
e
e
ArgTX-636 (2)
ArgTX-93 (3)
ArgTX-84 (4)
ArgTX-75 (5)
ArgTX-66 (6)
ArgTX-57 (7)
ArgTX-48 (8)
ArgTX-39 (9)
77 [62−95]
10 [9−11]
8
e
e
8
7
6
5
4
3
2
2
3
4
5
6
7
8
454 [282−428]
83 [73−94]
14 [13−16]
32
10
7
8 [7−10]
118 [100−139]
46 [41−52]
17 [14−21]
21 [19−25]
842 [735−963]
231 [195−274]
>3000
2
e
e
f
78 [67−90]
0.09 (11 )
f
19 [17−22]
171 [29−227]
0.08 (12 )
f
<0.06 (18 )
a
IC50 values determined from nonlinear regression fitting to a logistic equation of composite dose−response data obtained from 5 to 10 oocytes.
b
Numbers in brackets denote the 95% confidence interval for IC50. Inhibition of the current elicited by 300 μM L-glutamate by simultaneous
coapplication of the antagonist in oocytes injected with GluA1i. Inhibition of the current elicited by 100 μM L-glutamate and 100 μM glycine by
simultaneous coapplication of the antagonist in oocytes injected with a 1:1 ratio of GluN1/N2A. Selectivity ratio calculated as IC50(GluA1)/
IC50(GluN1/2A). Data from ref 29. IC50(GluN1/2A)/IC50(GluA1).
c
d
e
f
protected ArgTX derivatives 24−30 (Scheme 1). Subsequent
cleavage from the resin with concomitant cleavage of N-
pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), N-tert-
butoxycarbonyl (Boc), and N-trityl (Trt) protecting groups
provided bis-benzyl ArgTX derivatives. The seven target
compounds 3−9 were obtained in moderate to good yields
after removal of the O-benzyl protecting groups by treatment
with H2 and Pd(OH)2/C.
The initial biological screening of these derivatives led us to
select ArgTX-75 (5) and ArgTX-48 (8) for further structure−
activity relationship (SAR) studies, and derivatives of 5 and 8
were therefore designed and synthesized. Knowing that the
guanidinium moiety is essential for biological activity and
having optimized the polyamine moiety, we next wanted to
modify the headgroup and the linker amino acid, the 2,4-
dihydroxyphenyl acetic acid and asparagine moiety, respec-
tively. These changes were introduced in both 5 and 8 in order
to systematically evaluate their importance for potency and
selectivity at iGlu receptors. A synthetic route similar to that
described above was used where mononosyl protected heptane-
1,7-diamine or butane-1,4-diamine for derivatives of 5 or 8,
respectively, was linked to the resin followed by introduction of
the modifications in the linker amino acid and the aromatic
moiety (Scheme 2). First, the aromatic headgroup was
modified; i.e., the 2,4-dihydroxyphenyl acetic acid moiety was
systematically modified while the asparagine linker was kept
constant. 2-Hydroxyphenylacetic acid, 4-hydroxyphenylacetic
acid, and phenylacetic acid, together with 2-(1H-indol-3-
yl)acetic acid, were introduced instead of 2,4-dihydroxyphenyl-
acetic acid in both 5 and 8 (Scheme 2). For the derivatives with
modifications in the linker amino acid, alanine, aspartate,
histidine, and tryptophan were exchanged with the asparagine
linker, while the 2,4-dihydroxyphenylacetic acid moiety was not
changed. Subsequently, the polyamine chain was elongated by
reaction with N-Teoc protected 5-aminopentan-1-ol or 8-
aminooctan-1-ol for derivatives of 5 or 8, respectively, and by
application of the strategy described above, the 16 derivatives
5a−h and 8a−h were prepared (Scheme 2).
Pharmacology. The inhibitory potency of compounds 3−
9, 5a−h, and 8a−h was evaluated using two-electrode voltage-
clamp (TEVC) electrophysiology with Xenopus laevis oocytes
expressing the flip form of the homomeric AMPA receptor
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Table 2. Antagonist Effect of Argiotoxin Analogues 5a−h at AMPA and NMDA Receptors
a
IC50 values determined from nonlinear regression fitting to a logistic equation of composite dose−response data obtained from 5 to 10 oocytes.
b
Numbers in brackets denote the 95% confidence interval for IC50. Inhibition of the current elicited by 300 μM L-glutamate by simultaneous
coapplication of the antagonist in oocytes injected with GluA1i. Inhibition of the current elicited by 100 μM L-glutamate and 100 μM glycine by
simultaneous coapplication of the antagonist in oocytes injected with a 1:1 ratio of GluN1/N2A. Selectivity ratio calculated as IC50(GluA1)/
IC50(GluN1/2A). Selectivity ratio calculated as IC50(GluN1/2A)/IC50(GluA1).
c
d
e
subtype GluA1 or the heteromeric NMDA receptor subtype
consisting of GluN1 and GluN2A subunits (Figure 2A). IC50
values for the inhibition by compounds 3−9 were determined
at a membrane potential (VH) of −40, −60, and −80 mV
(Tables S1 and S2 and Table 1, respectively), while compounds
5a−h and 8a−h were tested at VH of −60 and −80 mV (Tables
S3 and 2, and Tables S4 and 3, respectively).
Inspired by the conversion of the nonselective iGlu receptor
antagonist ArgTX-636 (2) into 3 and 7 with preference for
NMDA and AMPA receptors, respectively,29 we were
interested in examining the importance of a systematic variation
of the secondary amine of the polyamine tail for their inhibitory
activity at AMPA and NMDA receptors. Thus, the seven
compounds 3−9, where the secondary amino group was
sequentially moved from the N-terminal tail closer to the
almost equally potent at AMPA and NMDA receptors, thus
being nonselective, and generally showed affinities similar to
that of the native toxin 2 (Table 1). On the other hand, when
the secondary amino group was moved closer to the headgroup,
as in compounds 7, 8, and 9, affinities for AMPA receptors
generally increase concomitantly with decreasing affinity for
NMDA receptors, thus showing preference for AMPA
receptors (Table 1). Gratifyingly, we saw that the potency at
AMPA receptors was improved for compound 8 with an IC50 of
19 nM while displaying a 12-fold selectivity relative to NMDA
receptors. Notably, 9 showed even improved selectivity for
AMPA receptors compared to both 7 and 8, which however
was due to lack of inhibitory activity at NMDA rather than
potent activity at AMPA receptors (Table 1). These
observations correlate with previous modeling studies of 2
docked into models of the ion channels of AMPA and NMDA
receptors, respectively.29 These studies showed that the
location of secondary amino groups is important both for
internal hydrogen bonding in 2 and for interaction with key
residues in the channel.29
aromatic headgroup, were tested in the TEVC assay at VH
=
−40 mV (Table S1), −60 mV (Table S2), and −80 mV (Table
1). In general, we observed a distinct correlation between the
location of the secondary amino group and the preference for
either AMPA or NMDA receptors. Compounds 3, 4, and 5
with the secondary amino group closest to the N-terminal tail
showed a preference for NMDA receptors and displayed low
nanomolar affinities for the NMDA receptor. Compound 6 was
Trends similar to those observed at VH = −80 mV (Table 1)
were also observed at VH = −40 mV (Table S1) and −60 mV
(Table S2). Moreover, from these data at the three holding
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Table 3. Antagonist Effect of Argiotoxin Analogues 8a−h at AMPA and NMDA Receptors
a
IC50 values determined from nonlinear regression fitting to a logistic equation of composite dose−response data obtained from 5 to 10 oocytes.
b
Numbers in brackets denote the 95% confidence interval for IC50. Inhibition of the current elicited by 300 μM L-glutamate by simultaneous
coapplication of the antagonist in oocytes injected with GluA1i. Inhibition of the current elicited by 100 μM L-glutamate and 100 μM glycine by
simultaneous coapplication of the antagonist in oocytes injected with a 1:1 ratio of GluN1/N2A. Selectivity ratio calculated as IC50(GluN1/2A)/
c
d
IC50(GluA1).
potentials we could estimate voltage dependency of the
analogues responses, which generally confirmed that all
compounds display voltage-dependent inhibition, as would be
expected from ion channel blockers.8 In conclusion, removal of
a secondary amino group from the polyamine moiety of the
native toxin 2 and systematic manipulation of the remaining
secondary amino group showed a distinct correlation between
location of this amino group and preference for either AMPA
or NMDA receptors, and in some cases improvement in
absolute potency was observed.
We have earlier shown that modifications of both polyamine
moiety and headgroup of the prototypical polyamine toxin
PhTX-433 (1) influence the inhibitory potency and selectiv-
ity,17,18,20,22 and we have also seen for spider polyamine toxins
that the headgroup in particular is highly important for selective
inhibition of iGlu receptors.26 Thus, having exploited system-
atic modifications of the polyamine moiety of 2 and knowing
that changes in the tail amino acid, particularly modifying the
guanidinium group of arginine, lead to substantial loss in
inhibitory activity at both AMPA and NMDA receptors,29 we
focused on the importance of changes in the headgroup (2,4-
dihydroxyphenylacetic acid) and linker amino acid (asparagine)
region (Figure 1). We selected the two derivatives, 5 and 8, that
in our initial screening showed most promise and designed a set
of derivatives where the same changes in the headgroup and
linker amino acid were introduced into 5 and 8, respectively
(Scheme 2). Since we had no prior knowledge on the
importance of changes in this region, we introduced subtle
changes in the 2,4-dihydroxyphenylacetic acid moiety, as well as
more drastic modifications of the asparagine amino acid linker,
and a total of 16 analogues of 5 and 8 were prepared.
First, we looked into the importance of the number and
position of headgroup hydroxyl groups by substituting the 2,4-
dihydroxyphenylacetic acid moiety with 2-hydroxyphenylacetic
acid, 4-hydroxyphenylacetic acid, and phenylacetic acid. Finally,
we also substituted the 2,4-dihydroxyphenylacetic acid moiety
with a 2-(1H-indol-3-yl)acetic acid because a range of
structurally similar spider toxins, such as Nephila polyamine
toxin-8 (NPTX-8),24,26 contain this indole moiety as the
headgroup. Next, we tested the consequence of substituting the
amino acid linker asparagine, choosing four different amino
acids (alanine, aspartic acid, histidine, and tryptophan) with
alkyl, carboxylic acid, or aromatic side chains. These
modifications were conveniently introduced using the same
SPS methodology as described above (Scheme 2).
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Figure 2. Inhibitory potency of toxins at recombinant iGlu receptor subtypes. (A) Representative two-electrode voltage-clamp current recording
illustrating the standard testing protocol. Oocytes expressing iGlu receptor were exposed to maximally effective concentrations of agonist, followed
by increasing concentrations of the test toxin plus agonist (B, C). Composite concentration−response curves for 5d, in which the headgroup of 5 has
been replaced with 2-(1H-indol-3-yl)acetic acid (B), and for 8a, in which the headgroup of 8 has been replaced with 2-hydroxyphenylacetic acid (C)
■
▲
at GluN1/2A ( ) and GluA1 ( ) receptors at membrane potentials of −80 mV. Error bars are SEM and are shown when larger than symbol size.
Modification of the position and number of headgroup
hydroxyl groups of 5, with preference for the NMDA receptor,
generally reduced potency at NMDA receptors as observed for
compounds 5a−c, whereas potency at AMPA receptors was
largely unaffected. The selectivity profile of 5a−c was thereby
changed to largely nonselective inhibitors (Table 2). In
contrast, when an indole headgroup is introduced, as in 5d,
potency at NMDA receptors was improved with an IC50 of 5
nM whereas potency at AMPA receptors was improved only
about 2-fold, thereby slightly improving the selectivity relative
to 5 (Figure 2B). The most pronounced effect observed at
NMDA receptors was obtained by substituting the amino acid
linker asparagine with aspartic acid (5f), which resulted in a
131-fold loss of potency, while potency at AMPA receptors was
largely unaffected, thereby shifting the preference to AMPA
receptors. Substitution with alanine (5e) resulted in a 5-fold
loss of potency at NMDA receptors, whereas AMPA receptor
potency was not affected. Replacing the asparagine linker with
histidine (5g) improved potency at AMPA receptors slightly,
while the reverse was true for NMDA receptor potency.
Notably, introducing a tryptophan (5h), i.e., introducing an
indole group similar to 5d, resulted in significant improvement
of potency at both NMDA and AMPA receptors with IC50
values of 5 and 17 nM, respectively (Table 2). Thus, changes in
the headgroup and linker amino acid region of 5 led to
identification of 5d and 5h both having IC50 of 5 nM at NMDA
receptors, and 5d showed improved selectivity compared to 5.
Interestingly, both 5d and 5h contain an indole moiety in either
the headgroup or linker amino acid, suggesting that such a
moiety is particularly useful in improving NMDA receptor
potency. A similar observation that an indole headgroup
improved potency at iGlu receptors was recently found for
structurally related compounds, the Nephila polyamine toxins.26
We also evaluated the biological consequences of introducing
the same changes into the headgroup and linker amino acid
region of the AMPA receptor, preferring compound 8 (Table
3). Modifications of the hydroxylation pattern of the 2,4-
dihydroxyphenylacetic acid moiety of 8, compounds 8a−c, as
well as substitution with an indole (8d), had only minor effect
on potency at AMPA receptors with IC50 values from 37 to 94
nM (Table 3). In contrast, the effect on NMDA receptor
potency was much more drastic, as significant reductions were
observed for all analogues, and hence, the selectivity for AMPA
receptors was drastically improved, and 8a (Figure 2C) and 8c
showed 77-fold and >32-fold selectivity, respectively, for AMPA
vs NMDA receptors. Replacing the asparagine linker showed
similar tendencies; AMPA receptor potency was not much
affected except when substituting with aspartic acid (8f), while
potency at NMDA receptors was dramatically reduced except
for introduction of tryptophan (8h) (Table 3).
In order to get an estimate of the voltage dependency, which
is a hallmark of this class of inhibitors, we also tested
compounds 5a−h and 8a−h at a holding potential (VH) of −60
mV (Tables S3 and S4). Generally, we observed that all
analogues were less potent at VH = −60 mV compared to IC50
measured at VH = −80 mV, as would be expected from voltage-
dependent inhibitors. This suggests that all the investigated
compounds still act as voltage-dependent channel blockers of
AMPA and NMDA receptors.
CONCLUSION
■
We have used ArgTX-636 (2) as a template for the
development of potent and selective iGlu receptor ligands
and have performed the first SAR study of spider polyamine
toxins in general and 2 in particular. A recently developed solid-
phase synthesis methodology was exploited for the synthesis of
two classes of analogues: first, there is the importance of
secondary amines of the polyamine moiety, and second, the
headgroup and linker amino acid were systematically examined
and evaluated for activity at AMPA and NMDA receptors. For
the first class of analogues, compounds 3−9, we observed a
distinct correlation between the location of the secondary
amino group in the polyamine moiety and inhibition of AMPA
and NMDA receptors: if the amino group was closer to the
headgroup, compounds showed preference for NMDA
receptors, whereas if the amino group was closer to the
amino acid tail, preference for AMPA receptors was observed.
Two analogues, 5 and 8, were selected for further derivatization
in the headgroup and linker amino acid region. The two regions
had not been previously investigated for argiotoxin analogues.
Changes in these two regions generally had more pronounced
biological effect than those in the polyamine region. For
analogues of 5 it was observed that highly potent and
reasonable NMDA receptor selective ligands could be
developed by introducing indole moieties, exemplified with
compound 5d. On the other hand, introducing the same
modifications in 8 led to fairly potent and highly selective
AMPA receptors ligands such as 8a. Thus, exploiting SAR
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CH2Cl2 (1:1, 6 mL) under nitrogen. A solution of a N-Teoc amino
alcohol (2.25 mmol) in dry THF/CH2Cl2 (1:1, 3 mL), tributylphos-
phine (2.25 mmol), and a solution of 1,1′-(azadicarbonyl)dipiperidine
(ADDP, 2.25 mmol) in dry THF/CH2Cl2 (1:1, 3 mL) were added
successively. The mixture was stirred at room temperature under
nitrogen for 3 h. The resin was drained and washed with DMF,
CH2Cl2, MeOH, and CH2Cl2 (3 × 16 mL each) and dried in a
vacuum. The procedure was repeated two more times, and the
resulting resin was dried in a vacuum. The above resin was suspended
in dry THF (16 mL) under nitrogen at 50 °C. A solution of TBAF (1
M in THF, 1.80 mmol) was added slowly, and the mixture was stirred
at 50 °C for 30 min. The resin was drained and washed with DMF,
CH2Cl2, MeOH, and CH2Cl2 (3 × 16 mL each) and dried in a
vacuum. The resin was then treated with a solution of Boc-L-Arg(Pbf)-
OH (2.25 mmol) and HATU (2.25 mmol) in CH2Cl2/DMF (9:1, 16
mL), followed by DIPEA (4.50 mmol), and agitated for 2 h at room
temperature and subsequently drained and washed with DMF,
CH2Cl2, MeOH, and CH2Cl2 (3 × 16 mL each). The resin was
treated with DBU (1.80 mmol) in DMF (6 mL) and mercaptoethanol
(1.80 mmol) in DMF (6 mL) for 30 min. The resin was drained and
washed with DMF (5 × 12 mL). The procedure was repeated three
more times. The resin was washed with DMF, CH2Cl2, MeOH, and
CH2Cl2 (3 × 16 mL each) and then treated with a solution of TFA/
CH2Cl2/triisopropylsilane/H2O (75:20:2.5:2.5 v/v/v/v, 12 mL) for 2
h. The resin was drained and washed with MeOH (6 mL) and CH2Cl2
(6 mL). The solutions of the cleaved product and the washings were
combined and evaporated in a vacuum and purified by preparative
HPLC. A screw cap vial was charged with a magnetic stir bar and the
bis-benzyl protected argiotoxin derivative followed by glacial acetic
acid (4 mL). Pd(OH)2/C (10% w/w) was added and the vial sealed
with a rubber septum. Hydrogen was bubbled through the solution for
about 30 min. The mixture was filtered with a 0.2 μm syringe filter and
washed several times with MeOH. The combined washings were
concentrated in vacuo to give an off-white solid which was purified by
preparative HPLC and lyophilized to give the argiotoxin derivative as a
fluffy white solid. Compounds 2, 3, and 7 were prepared previously.20
(S)-N1-(8-(4-((S)-2-Amino-5-guanidinopentanamido)-
butylamino)octyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate (ArgTX-84, 4). Yield:
64.5 mg (14.7%). 1H NMR (300 MHz, CD3OD): δ = 6.93 (d, J = 8.3
Hz, 1H), 6.34 (d, J = 2.5 Hz, 1H), 6.28 (dd, J = 8.3, 2.5 Hz, 1H), 4.66
(t, J = 6.3 Hz, 1H), 3.87 (t, J = 6.6 Hz, 1H), 3.50 (d, J = 14.9 Hz, 1H),
3.41 (d, J = 14.9 Hz, 1H), 3.21−3.29 (m, 4H), 2.90−3.14 (m, 6H),
2.69 (d, J = 6.3 Hz, 2H), 1.90 (m, 2H), 1.59−1.74 (m, 8H), 1.46 (m,
2H), 1.25−1.35 (m, 8H). 13C NMR (75 MHz, CD3OD): δ = 175.1,
174.0, 173.0, 169.9, 159.1, 158.7, 157.3, 132.8, 114.2, 108.1, 103.9,
54.3, 52.0, 41.9, 40.5, 40.0, 38.9, 37.6, 30.2, 30.0, 29.8, 29.8, 27.5, 27.3,
27.2, 27.2, 25.7, 24.8. HPLC-ELS: 100%. m/z = 636.5 [MH+]. HRMS
(EI) exact mass calculated for C30H53N9O6 [MH+] 636.4197. Found:
636.4194.
studies of spider polyamine toxins clearly provides a useful and
efficient way to develop notable tools for studies of iGlu
receptors.
EXPERIMENTAL SECTION
■
Chemistry. General Procedures. Unless otherwise stated,
starting materials were obtained from commercial suppliers and were
used without further purification. 2-(3,5-Dimethoxy-4-
formylphenoxy)ethylpolystyrene with a loading of 0.92 mmol/g was
purchased from Novabiochem. N-(Aminoalkyl)-2-nitrobenzenesulfo-
namide, N-(trimethylsilyl)ethoxycarbonylamino alcohols, 2-(2,4-bis-
(benzyloxy)phenyl)acetic acid, (2-benzyloxy)phenylacetic acid, and
(4-benzyloxy)phenylacetic acid were prepared as previously de-
scribed.20 Tetrahydrofuran (THF) was distilled under nitrogen from
sodium/benzophenone immediately before use. N,N-Dimethylforma-
mide (DMF) and dichloromethane were dried using AldraAmine
trapping packets. Proton (1H) NMR spectra were recorded on a
Varian spectrometer, Mercury Plus (300 MHz), and carbon (13C)
NMR spectra were recorded on a Varian spectrometer, Gemini 2000
(75 MHz). Chemical shifts (δ) are reported in parts per million (ppm)
with reference to tetramethylsilane (TMS) as internal standard. The
following abbreviations are used for the proton spectra multiplicities: s,
singlet; bs, broad singlet; d, doublet; dd, double doublet, t, triplet; q,
quartet; m, multiplet. Coupling constants (J) are reported in hertz
(Hz). Preparative HPLC was performed on a Agilent 1100 system
using a C18 reverse phase column (Zorbax 300 SB-C18, 21.2 mm ×
250 mm) with a linear gradient of the binary solvent system of H2O/
ACN/TFA (A, 95/5/0.1; B, 5/95/0.1) with a flow rate of 20 mL/min.
Analytical HPLC was performed on an Agilent 1100 system with a
C18 reverse phase column (Zorbax 300 SB-C18 column, 4.6 mm ×
150 mm), flow rate of 1 mL/min, and a linear gradient of the binary
solvent system of H2O/ACN/TFA (A, 95/5/0.1; B, 5/95/0.1). Mass
spectra were obtained with an Agilent 6410 triple quadrupole mass
spectrometer instrument using electron spray coupled to an Agilent
1200 HPLC system (ESI-LC/MS) with a C18 reverse phase column
(Zorbax Eclipse XBD-C18, 4.6 mm × 50 mm), autosampler, and diode
array detector using a linear gradient of the binary solvent system of
H2O/ACN/formic acid (A, 95/5/0.1; B, 5/95/0.086) with a flow rate
of 1 mL/min. During ESI-LC/MS analysis evaporative light scattering
(ELS) traces were obtained with a Sedere Sedex 85 light scattering
detector. Compound identity of all tested compounds was confirmed
by ESI-LC/MS, which also provided purity data (all >95%, UV and
ELSD). High-resolution mass spectra were obtained using a
Micromass Q-Tof II instrument, and results were all within 5 ppm
of theoretical values.
General Synthetic Procedure A. Compounds 2−9. 2-(3,5-
Dimethoxy-4-formylphenoxy)ethylpolystyrene resin (0.45 mmol) was
treated with a solution of N-(aminoalkyl)-2-nitrobenzenesulfonamide
(4.50 mmol) in DMF/acetic acid (9:1, 16 mL). The reaction mixture
was agitated for 2 min, and NaBH(OAc)3 (4.50 mmol) was added in
one portion. The mixture was agitated for 12 h at room temperature.
The solvents were drained, and the resin was washed with DMF,
DIPEA (10% in DMF), DMF, CH2Cl2, MeOH, CH2Cl2 (3 × 16 mL
each) and dried in a vacuum. A solution of Fmoc-L-Asn(Trt)-OH
(2.25 mmol) and HATU (2.25 mmol) in CH2Cl2/DMF (9:1, 16 mL)
was added to the above resin followed by DIPEA (4.50 mmol). The
mixture was agitated for 12 h, and the resin was subsequently washed
with DMF, CH2Cl2, MeOH, CH2Cl2 (3 × 16 mL each) and dried
under reduced pressure. The product was treated with 20% piperidine
in DMF (v/v, 16 mL) and the mixture agitated for 2 min at room
temperature. The resulting resin was washed with DMF (3 × 16 mL),
treated again with 20% piperidine in DMF (v/v, 16 mL) for 20 min,
and then washed with DMF, CH2Cl2, MeOH, and CH2Cl2 (3 × 16
mL each). This resin was treated with a solution of 2-(2,4-
bis(benzyloxy)phenyl)acetic acid (2.25 mmol) and HATU (2.25
mmol) in CH2Cl2/DMF (9:1, 16 mL), followed by DIPEA (4.50
mmol). The mixture was agitated for 2 h at room temperature and
subsequently drained and washed with DMF, DCM, MeOH, and
DCM (3 × 16 mL each). The above resin was suspended in dry THF/
(S)-N1-(7-(5-((S)-2-Amino-5-guanidinopentanamido)-
pentylamino)heptyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate (ArgTX-75, 5). Yield:
1
24.3 mg (5.5%). H NMR (300 MHz, CD3OD): δ = 6.93 (d, J = 8.3
Hz, 1H), 6.33 (d, J = 2.2 Hz, 1H), 6.26 (dd, J = 8.3, 2.5 Hz, 1H), 4.64
(t, J = 6.3 Hz, 1H), 3.84 (t, J = 6.6 Hz, 1H), 3.51 (d, J = 15.1 Hz, 1H),
3.40 (d, J = 15.1 Hz, 1H), 3.19−3.26 (m, 4H), 3.09 (m, 2H), 2.84−
2.96 (m, 4H), 2.69 (d, J = 6.6 Hz, 2H), 1.88 (m, 2H), 1.53−1.69 (m,
8H), 1.38−1.47 (m, 4H), 1.24−1.35 (m, 6H). 13C NMR (75 MHz,
CD3OD): δ = 175.2, 175.0, 173.1, 169.8, 159.1, 158.7, 157.3, 132.9,
114.3, 108.1, 103.9, 54.2, 52.0, 41.9, 40.4, 40.3, 38.9, 37.4, 30.0, 30.0,
30.0, 29.2, 27.0, 27.0, 26.9, 26.7, 25.7, 25.0. HPLC-ELS: 100%. m/z =
636.4 [MH+]. HRMS (EI) exact mass calculated for C30H53N9O6
[MH+] 636.4197. Found: 636.4193.
(S)-N1-(6-(6-((S)-2-Amino-5-guanidinopentanamido)-
hexylamino)hexyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate (ArgTX-66, 6). Yield:
1
26.3 mg (6.0%). H NMR (300 MHz, CD3OD): δ = 6.93 (d, J = 8.0
Hz, 1H), 6.33 (d, J = 2.5 Hz, 1H), 6.26 (dd, J = 8.0, 2.5 Hz, 1H), 4.62
(t, J = 6.6 Hz, 1H), 3.84 (t, J = 6.6 Hz, 1H), 3.50 (d, J = 15.1 Hz, 1H),
1177
dx.doi.org/10.1021/jm301602d | J. Med. Chem. 2013, 56, 1171−1181

Journal of Medicinal Chemistry
Article
3.40 (d, J = 15.1 Hz, 1H), 3.09−3.26 (m, 6H), 2.92 (t, J = 7.7 Hz, 2H),
2.86 (t, J = 7.9 Hz, 2H), 2.69 (m, 2H), 1.88 (m, 2H), 1.61−1.71 (m,
4H), 1.53−1.60 (m, 4H), 1.46 (m, 2H), 1.37−1.42 (m, 4H), 1.26−
1.33 (m, 4H). 13C NMR (75 MHz, CD3OD): δ = 175.1, 175.0, 173.1,
169.7, 159.1, 158.6, 157.3, 132.9, 114.3, 108.1, 103.9, 54.2, 52.0, 49.0,
48.8, 41.9, 40.7, 40.3, 39.0, 37.4, 30.2, 30.0, 30.0, 27.6, 27.3, 27.1, 27.1,
27.0, 27.0, 25.7. HPLC-ELS: 100%. m/z = 636.4 [MH+]. HRMS (EI)
exact mass calculated for C30H53N9O6 [MH+] 636.4197. Found:
636.4176.
(EI) exact mass calculated for C30H53N9O5 [MH+] 620.4248. Found:
620.4226.
(S)-N1-(7-((5-((S)-2-Amino-5-guanidinopentanamido)pentyl)-
amino)heptyl)-2-(2-phenylacetamido)succinamide Tris(2,2,2-
1
trifluoroacetate (5c). Yield: 13.4 mg (4.7%). H NMR (300 MHz,
CD3OD): δ = 7.17−7.31 (m, 5H), 4.65 (t, J = 6.8 Hz, 1H), 3.84 (t, J =
6.4 Hz, 1H), 3.56 (s, 2H), 3.13−3.26 (m, 6H), 2.91−2.99 (m, 4H),
2.58−2.76 (m, 2H), 1.80−1.97 (m, 2H), 1.57−1.73 (m, 8H), 1.40−
1.53 (m, 4H), 1.29−1.39 (m, 6H). 13C NMR (75 MHz, CD3OD): δ =
174.7, 174.0, 173.0, 169.8, 158.7, 136.7, 130.3, 130.2, 129.7, 129.6,
128.0, 54.2, 52.1, 43.7, 41.9, 40.4, 38.0, 30.1, 29.9, 29.6, 29.5, 27.4,
27.3, 27.1, 27.0, 25.7, 25.0. HPLC-ELS: 98%. m/z = 604.4 [MH+].
HRMS (EI) exact mass calculated for C30H53N9O4 [MH+] 604.4299.
Found: 604.4277.
(S)-N1-(4-(8-((S)-2-Amino-5-guanidinopentanamido)-
nonylamino)propyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate (ArgTX-48, 8). Yield:
76.8 mg (17.5%). 1H NMR (300 MHz, CD3OD): δ = 6.94 (d, J = 8.3
Hz, 1H), 6.35 (d, J = 2.2 Hz, 1H), 6.28 (dd, J = 8.3, 2.2 Hz, 1H), 4.61
(t, J = 6.3 Hz, 1H), 3.86 (t, J = 6.6 Hz, 1H), 3.51 (d, J = 15.1 Hz, 1H),
3.42 (d, J = 15.1 Hz, 1H), 3.20−3.26 (m, 6H), 2.87−2.92 (m, 4H),
2.71 (m, 2H), 1.89 (m, 2H), 1.55−1.72 (m, 10H), 1.34−1.38 (m, 8H).
13C NMR (75 MHz, CD3OD): δ = 175.2, 175.0, 173.3, 169.7, 159.0,
2-(2-(1H-Indol-3-yl)acetamido)-N1-(7-(5-(2-amino-5-
guanidinopentanamido)pentylamino)heptyl)succinamide
Tetrakis(2,2,2-trifluoroacetate (5d). Yield: 14.1 mg (4.3%). 1H
NMR (300 MHz, CD3OD): δ = 7.53 (d, J = 8.0 Hz, 1 H), 7.35 (d, J =
8.3 Hz, 1 H), 7.19 (s, 1 H), 7.10 (t, J = 7.5 Hz, 1 H), 7.01 (t, J = 7.4
Hz, 1 H), 4.67 (t, J = 7.6 Hz, 1 H), 3.84 (t, J = 7.4 Hz, 1 H), 3.73 (s, 2
H), 3.21 (t, J = 7.2 Hz, 4 H), 3.13−3.02 (m, 2 H), 2.92−2.70 (m, 4
H), 2.65 (d, J = 5.8 Hz, 2 H), 1.98−1.77 (m, 2 H), 1.72−1.47 (m, 9
H), 1.43−1.14 (m, 16 H). 13C NMR (75 MHz, CD3OD): δ = 173.8,
173.7, 171.9, 168.7, 137.1, 127.4, 124.1, 121.7, 119.1, 118.3, 111.5,
108.2, 53.2, 51.0, 40.8, 39.4, 39.2, 36.7, 33.0, 28.9, 28.9, 28.2, 28.2,
25.9, 25.9, 25.8, 25.7, 24.6, 23.9, 21.4. HPLC-ELS: 99%. m/z = 643.5
[MH+]. HRMS (EI) exact mass calculated for C32H54N10O4 [MH+]
643.4408. Found: 643.4401.
158.7, 157.2, 132.9, 114.2, 108.1, 103.9, 54.2, 52.1, 49.0, 41.8, 40.8,
39.4, 38.9, 37.4, 30.4, 30.2, 29.9, 28.0, 27.6, 27.4, 27.3, 25.6, 24.2.
HPLC-ELS: 100%. m/z = 636.5 [MH+]. HRMS (EI) exact mass
calculated for C30H53N9O6 [MH+] 636.4197. Found: 636.4190.
(S)-N1-(3-(9-((S)-2-Amino-5-guanidinopentanamido)-
nonylamino)propyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate (ArgTX-39, 9). Yield:
1
22.8 mg (5.2%). H NMR (300 MHz, CD3OD): δ = 6.93 (d, J = 8.3
Hz, 1H), 6.32 (d, J = 2.5 Hz, 1H), 6.25 (dd, J = 8.3, 2.5 Hz, 1H), 4.57
(t, J = 5.8 Hz, 1H), 3.83 (t, J = 6.6 Hz, 1H), 3.45 (s, 2H), 3.32 (m,
2H), 3.24−3.18 (m, 4H), 2.90 (t, J = 7.2 Hz, 2H), 2.86−2.65 (m, 4H),
1.92−1.77 (m, 4H), 1.70−1.58 (m, 4H), 1.53 (t, J = 6.3 Hz, 2H),
1.39−1.33 (m, 10H). 13C NMR (75 MHz, CD3OD): δ = 175.4, 174.9,
174.6, 169.7, 159.1, 158.7, 157.2, 132.9, 114.2, 108.1, 103.8, 54.2, 52.0,
49.3, 46.2, 41.9, 40.9, 38.9, 37.2, 36.9, 30.6, 30.5, 30.5, 30.4, 30.0, 28.2,
27.7, 27.7, 27.6, 25.7. HPLC-ELS: 100%. m/z = 636.5 [MH+]. HRMS
(EI) exact mass calculated for C30H53N9O6 [MH+] 636.4197. Found:
636.4183.
(S)-2-Amino-N-(5-((7-((S)-2-(2-(2,4-Dihydroxyphenyl)-
acetamido)propanamido)heptyl)amino)pentyl)-5-guanidino-
pentanamide Tris(2,2,2-trifluoroacetate (5e). Yield: 49.6 mg
(17.7%). 1H NMR (300 MHz, CD3OD): δ = 6.88 (d, J = 7.3 Hz, 1H),
6.29 (s, 1H), 6.23 (d, J = 8.0 Hz, 1H), 4.21 (q, J = 7.0 Hz, 1H), 3.82 (t,
J = 6.4 Hz, 1H), 3.47 (d, J = 14.7 Hz, 1H), 3.35 (d, J = 15.7 Hz, 1H),
2.98−3.24 (m, 6H), 2.77−2.95 (m, 4H), 1.75−1.94 (m, 2H), 1.59−
1.71 (m, 4H), 1,47−1.58 (m, 4H), 1.33−1.46 (m, 4H), 1.20−1.33 (m,
9H). 13C NMR (75 MHz, CD3OD): δ = 175.1, 175.0, 169.8, 158.9,
158.7, 157.4, 132.8, 114.5, 108.0, 103.9, 54.2, 51.0, 49.0, 41.8, 40.4,
40.3, 38.7, 30.1, 29.9, 29.9, 29.4, 27.1, 27.0, 26.8, 25.7, 25.0, 18.1.
HPLC-ELS: 100%. m/z = 593.4 [MH+]. HRMS (EI) exact mass
calculated for C29H52N8O5 [MH+] 593.4139. Found: 593.4137.
(6S,24S)-1,6-Diamino-24-(2-(2,4-dihydroxyphenyl)-
acetamido)-1-imino-7,23dioxo-2,8,14,22-tetraazahexacosan-
26-oic Acid Tris(2,2,2-trifluoroacetate (5f). Yield: 36.6 mg
General Synthetic Procedure B. Compounds 5a−h and 8a−
h. Compounds were prepared according to the general synthetic
procedure A with the following exceptions. For compounds 5a−d and
8a−d the use of 2-(2,4-bis(benzyloxy)phenyl)acetic acid was replaced
with (2-benzyloxy)phenylacetic acid (a), (4-benzyloxy)phenylacetic
acid (b), phenylacetic acid (c), or 2-(1H-indol-3-yl)acetic acid (d), and
when the last two compounds were employed the debenzylation step
was avoided. For compounds 5e−h and 8e−h, the use of Fmoc-L-
Asn(Trt)-OH was replaced with the use of Fmoc-L-Ala-OH (e),
Fmoc-L-Asp(tBu)-OH (f), Fmoc-L-His(Trt)-OH (g), and Fmoc-L-
Trp(Boc)-OH (h).
1
(12.5%). H NMR (300 MHz, CD3OD): δ = 6.90 (d, J = 8.0 Hz,
1H), 6.31 (d, J = 1.9 Hz, 1H), 6.24 (dd, J = 1.9, 8.3 Hz, 1H), 4.62 (t, J
= 6.3 Hz, 1H), 3.83 (t, J = 6.3 Hz, 1H), 3.48 (d, J = 14.9 Hz, 1H), 3.37
(d, J = 14.9 Hz, 1H), 3.01−3.25 (m, 6H), 2.79−2.95 (m, 4H), 2.70−
2.78 (m, 2H), 1.77−1.93 (m, 2H), 1,48−1.72 (m, 8H), 1.33−1.47 (m,
4H), 1.20−1.32 (m, 6H). 13C NMR (75 MHz, CD3OD): δ = 175.3,
174.1, 172.9, 169.8, 159.0, 158.7, 157.3, 132.8, 114.3, 108.1, 103.9,
54.2, 51.7, 49.0, 41.9, 40.4, 38.9, 36.6, 30.1, 30.0, 29.9, 29.3, 27.1, 27.0,
26.8, 25.7, 25.0. HPLC-ELS: 100%. m/z = 637.4 [MH+]. HRMS (EI)
exact mass calculated for C30H52N8O7 [MH+] 637.4037. Found:
637.4026.
(S)-N1-(7-((5-((S)-2-Amino-5-guanidinopentanamido)pentyl)-
amino)heptyl)-2-(2-(2-hydroxyphenyl)acetamido)succinamide
1
Tris(2,2,2-trifluoroacetate (5a). Yield: 42.1 mg (14.6%). H NMR
(300 MHz, CD3OD): δ = 7.03−7.15 (m, 2H), 6.72−6.84 (m, 2H),
4.64 (t, J = 6.0 Hz, 1H), 3.84 (t, J = 6.4 Hz, 1H), 3.60 (d, J = 14.0 Hz,
1H), 3.49 (d, J = 15.0 Hz, 1H), 3.16−3.25 (m, 4H), 3.00−3.15 (m,
2H), 2.76−2.97 (m, 4H), 2.68 (d, J = 6.0, 2H), 1.75−1.96 (m, 2H),
1.59−1.72 (m, 4H), 1.49−1.59 (m, 4H), 1.36−1.48 (m, 4H), 1.22−
1.32 (m, 6H). 13C NMR (75 MHz, CD3OD): δ = 174.9, 174.6, 173.0,
169.8, 158.7, 156.5, 132.4, 129.7, 123.3, 121.1, 116.4, 54.2, 52.1, 41.8,
40.5, 40.4, 39.5, 37.6, 30.1, 29.9, 29.3, 27.0, 26.8, 25.6, 25.0. HPLC-
ELS: 100%. m/z = 620.4 [MH+]. HRMS (EI) exact mass calculated for
C30H53N9O5 [MH+] 620.4248. Found: 620.4240.
(S)-2-Amino-N-(5-((7-((S)-2-(2-(2,4-dihydroxyphenyl)-
acetamido)-3-(1H-imidazol-5-yl)propanamido)heptyl)amino)-
pentyl-5-guanidinopentanamide Tetrakis(2,2,2-trifluoroace-
1
tate (5g). Yield: 31.6 mg (9.4%). H NMR (300 MHz, CD3OD): δ
= 8.69 (s, 1H), 7.12 (s, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.29 (d, J = 2.2
Hz, 1H), 6.23 (dd, J = 2.2, 8.3 Hz, 1H), 4.61 (q, J = 5.0 Hz, 1H), 3.83
(t, J = 6.6 Hz, 1H), 3.38 (s, 2H), 3.14−3.23 (m, 6H), 2.85−3.06 (m,
6H), 1.79−1.95 (m, 2H), 1.52−1.69 (m, 8H), 1.36−1.49 (m, 4H),
1.25−1.35 (m, 6H). 13C NMR (75 MHz, CD3OD): δ = 175.3, 171.9,
169.8, 159.0, 158.7, 157.2, 134.8, 132.7, 131.2, 118.3, 114.2, 108.0,
103.9, 54.2, 53.7, 41.9, 40.6, 40.4, 38.7, 30.2, 30.0, 29.8, 28.2, 27.5,
27.4, 27.2, 27.0, 25.7, 25.0. HPLC-ELS: 100%. m/z = 659.4 [MH+].
HRMS (EI) exact mass calculated for C32H54N10O5 [MH+] 659.4357.
Found: 659.4355.
(S)-N1-(7-((5-((S)-2-Amino-5-guanidinopentanamido)pentyl)-
amino)heptyl)-2-(2-(4-hydroxyphenyl)acetamido)succinamide
1
Tris(2,2,2-trifluoroacetate (5b). Yield: 16.5 mg (5.7%). H NMR
(300 MHz, CD3OD): δ = 7.10 (d, J = 8.3 Hz, 2H), 6.72 (d, J = 8.3 Hz,
2H), 4.65 (t, J = 5.8 Hz, 1H), 3.86 (t, J = 6.3 Hz, 1H), 3.47 (s, 2H),
3.11−3.28 (m, 6H), 2.90−3.00 (m, 4H), 2.56−2.75 (m, 2H), 1.52−
1.74 (m, 8H), 1.40−1.51 (m, 4H), 1.24−1.40 (m, 6H). 13C NMR (75
MHz, CD3OD): δ = 174.7, 174.6, 173.0, 169.8, 158.7, 157.5, 131.3,
127.3, 116.4, 54.2, 52.1, 42.9, 42.0, 41.9, 40.4, 37.9, 30.1, 29.9, 29.5,
27.2, 27.0, 25.7, 25.0. HPLC-ELS: 97%. m/z = 620.4 [MH+]. HRMS
(S)-2-Amino-N-(5-(7-((S)-2-(2-(2,4-dihydroxyphenyl)-
acetamido)-3-(1H-indol-2-yl)propanamido)heptylamino)-
1178
dx.doi.org/10.1021/jm301602d | J. Med. Chem. 2013, 56, 1171−1181

Journal of Medicinal Chemistry
Article
pentyl)-5-guanidinopentanamide Tetrakis(2,2,2-trifluoroace-
175.4, 175.3, 169.7, 158.9, 158.7, 157.3, 132.9, 114.5, 107.9, 103.8,
54.2, 51.4, 50.0, 41.8, 40.8, 39.2, 38.5, 30.4, 30.3, 29.9, 28.0, 27.6, 27.4,
27.4, 25.6, 24.3, 17.9. HPLC-ELS: 100%. m/z = 593.4 [MH+]. HRMS
(EI) exact mass calculated for C29H52N8O5 [MH+] 593.4139. Found:
593.4138.
(6S,24S)-1,6-Diamino-24-(2-(2,4-dihydroxyphenyl)-
acetamido)-1-imino-7,23-dioxo-2,8,17,22-tetraazahexacosan-
26-oic Acid Tris(2,2,2-trifluoroacetate (8f). Yield: 49.3 mg (17%).
1H NMR (300 MHz, CD3OD): δ = 6.91 (d, J = 8.2 Hz, 1 H), 6.31 (d,
1
tate (5h). Yield: 10.8 mg (3.1%). H NMR (300 MHz, CD3OD): δ
= 7.63−7.52 (m, 1 H), 7.50 (d, J = 7.9 Hz, 1 H), 7.30 (d, J = 7.9 Hz, 1
H), 7.11−6.93 (m, 3 H), 6.82 (d, J = 8.0 Hz, 1 H), 6.28 (d, J = 2.5 Hz,
1 H), 6.24 (dd, J = 2.5, 8.2 Hz, 1 H), 4.52 (t, J = 6.7 Hz, 1 H), 3.85 (t,
J = 6.6 Hz, 1 H), 3.39 (d, J = 3.7 Hz, 2 H), 3.27−3.07 (m, 8 H), 3.02−
2.80 (m, 6 H), 1.99−1.81 (m, 2 H), 1.74−1.08 (m, 24 H). 13C NMR
(75 MHz, CD3OD): δ = 175.0, 173.8, 159.0, 157.3, 183.0, 132.7,
128.7, 124.5, 122.5, 119.9, 119.4, 114.1, 112.3, 110.6, 108.1, 103.9,
56.0, 54.3, 41.9, 40.4, 40.3, 38.8, 30.0, 29.9, 29.4, 28.8, 27.2, 27.1, 27.0,
29.6, 25.7, 25.0. HPLC-ELS: 100%. m/z = 708.5 [MH+]. HRMS (EI)
exact mass calculated for C32H57N9O5 [MH+] 708.4561. Found:
708.4545.
J = 2.5 Hz, 1 H), 6.25 (dd, J = 2.5, 8.1 Hz, 1 H), 4.56 (t, J = 6.2 Hz, 1
H), 3.82 (t, J = 6.5 Hz, 1 H), 3.48 (d, J = 14.9 Hz, 1 H), 3.39 (d, J =
15.1 Hz, 1 H), 3.11−3.26 (m, 6 H), 2.86 (t, J = 7.8 Hz, 4 H), 2.73−
2.80 (m, 2 H), 1.74−1.95 (m, 2 H), 1.43−1.72 (m, 10 H), 1.34 (s, 8
H). 13C NMR (75 MHz, CD3OD): δ = 175.4, 174.1, 173.2, 169.7,
159.0, 157.3, 133.0, 114.3, 108.0, 103.8, 54.2, 51.9, 49.0, 41.8, 40.8,
39.4, 38.8, 36.5, 30.4, 30.3, 30.0, 28.0, 27.7, 27.4, 27.3, 25.6, 24.3.
HPLC-ELS: 100%. m/z = 637.4 [MH+]. HRMS (EI) exact mass
calculated for C30H52N8O7 [MH+] 637.4037. Found: 637.4036.
(S)-2-Amino-N-(8-((4-((S)-2-(2-(2,4-dihydroxyphenyl)-
acetamido)-3-(1H-imidazol-5-yl)propanamido)butyl)amino)-
octyl)-5-guanidinopentanamide Tetrakis(2,2,2-trifluoroace-
(S)-N1-(4-((8-((S)-2-Amino-5-guanidinopentanamido)octyl)-
amino)butyl)-2-(2-(2-hydroxyphenyl)acetamido)succinamide
1
Tris(2,2,2-trifluoroacetate (8a). Yield: 66.8 mg (23%). H NMR
(300 MHz, CD3OD): δ = 7.04−7.19 (m, 2 H), 6.75−6.88 (m, 2 H),
4.56−4.69 (m, 1 H), 3.86 (t, J = 6.2 Hz, 1 H), 3.61 (d, J = 15.1 Hz, 1
H), 3.53 (d, J = 14.9 Hz, 1 H), 3.22 (t, J = 6.2, 6 H), 2.88 (t, J = 7.4, 4
H), 2.63−2.79 (m, 2 H), 1.78−1.97 (m, 2 H), 1.43−1.78 (m, 10 H),
1.36 (s, 8 H). 13C NMR (75 MHz, CD3OD): δ = 173.9, 173.6, 172.3,
168.6, 157.6, 155.4, 131.4, 128.7, 122.2, 120.0, 115.3, 53.2, 51.2, 47.9,
47.5, 40.8, 39.8, 38.4, 36.4, 29.3, 29.2, 28.9, 27.0, 26.6, 26.4, 26.3, 24.6,
23.2. HPLC-ELS: 100%. m/z = 620.4 [MH+]. HRMS (EI) exact mass
calculated for C30H53N9O5 [MH+] 620.4248. Found: 620.4256.
(S)-N1-(4-((8-((S)-2-Amino-5-guanidinopentanamido)octyl)-
amino)butyl)-2-(2-(4-hydroxyphenyl)acetamido)succinamide
1
tate (8g). Yield: 69.9 mg (21%). H NMR (300 MHz, CD3OD): δ
= 8.68 (s, 1 H), 7.12 (s, 1 H), 6.83 (d, J = 8.0 Hz, 1 H), 6.28 (d, J = 2.5
Hz, 1 H), 6.22 (dd, J = 2.5, 8.0 Hz, 1 H), 4.54 (q, J = 5.3 Hz, 1 H),
3.81 (t, J = 6.4 Hz, 1 H), 3.37 (s, 2 H), 3.14−3.25 (m, 6 H), 2.96−3.13
(m, 2 H), 2.88 (t, J = 7.7 Hz, 4 H), 1.75−1.95 (m, 2 H), 1.44−1.69
(m, 10 H), 1.32 (s, 8 H). 13C NMR (75 MHz, CD3OD): δ = 175.4,
172.2, 169.7, 159.0, 157.2, 134.8, 132.7, 131.2, 118.3, 114.2, 108.0,
103.8, 54.2, 54.1, 49.1, 41.8, 40.8, 39.7, 38.6, 30.4, 30.3, 30.0, 28.1,
27.9, 27.7, 27.4, 27.4, 25.6, 24.5. HPLC-ELS: 99%. m/z = 659.4
[MH+]. HRMS (EI) exact mass calculated for C32H54N10O5 [MH+]
659.4357. Found: 659.4355.
(S)-2-Amino-N-(8-(4-((S)-2-(2-(2,4-dihydroxyphenyl)-
acetamido)-3-(1H-indol-2-yl)propanamido)butylamino)octyl)-
5-guanidinopentanamide Tetrakis(2,2,2-trifluoroacetate (8h).
Yield: 29.4 mg (8.4%). 1H NMR (300 MHz, CD3OD): δ = 7.49 (d, J =
7.96 Hz, 1 H), 7.30 (d, J = 8.3 Hz, 1 H), 7.06 (t, J = 7.3, 1 H), 7.01−
6.91 (m, 2 H), 6.82 (d, J = 8.3 Hz, 1 H), 6.28 (d, J = 2.4 Hz, 1 H), 6.24
(dd, J = 2.5, 8.03 Hz, 1 H), 4.45 (t, J = 6.7 Hz, 1 H), 3.83 (t, J = 6.6
Hz, 1 H), 3.39 (s, 2 H), 3.28−3.09 (m, 8 H), 3.08−2.94 (m, 1 H),
2.92−2.71 (m, 4 H), 1.96−1.74 (m, 2 H), 1.72−1.29 (m, 20 H). 13C
NMR (75 MHz, CD3OD): δ = 172.6, 171.6, 167.0, 156.4, 154.6, 135.4,
130.1, 126.0, 122.0, 119.9, 117.2, 116.7, 111.5, 109.7, 107.9, 105.4,
101.2, 53.7, 51.6, 39.2, 38.2, 36.7, 36.0, 27.8, 27.7, 27.3, 26.0, 25.4,
25.1, 24.8, 24.6, 23.0, 21.6. HPLC-ELS: 100%. m/z = 708.5 [MH+].
HRMS (EI) exact mass calculated for C32H57N9O5 [MH+] 708.4561.
Found: 708.4559.
Electrophysiology. In Vitro cRNA Transcription. The cDNA
encoding rat GluA1i or GluN1/2A subunits were inserted into the
vectors pGEM-HE or pCIneo, respectively, for preparation of high-
expression cRNA transcripts. Plasmids were grown in Top10 E. coli
bacteria (Invitrogen, Carlsbad, CA) and isolated by using column
purification (Qiagen, La Jolla, CA). The cRNA was synthesized from
the above cDNAs by in vitro transcription using the mMESSAGE
mMACHINE T7 mRNA-capping kit (Ambion, Austin, TX) according
to the protocol supplied by the manufacturer.
Oocyte Electrophysiology. Mature female Xenopus laevis (Nasco,
Modesto, CA) were anesthetized using 0.1% ethyl 3-aminobenzoate,
and their ovaries were surgically removed. The ovarian tissue was
dissected and treated with collagenase in Ca2+-free Barth’s medium (2
mg/mL) for 1−2 h at room temperature. On the second day, oocytes
were injected with 25 nL of cRNA (1 ng/nL for GluA1i or 0.05 ng/nL
for GluN1/2A) and incubated at 17 °C in Barth’s medium (in mM: 88
NaCl, 1 KCI, 0.33 Ca(NO3)2, 0.41 CaC12, 0.82 MgSO4, 2.4 NaHCO3,
10 HEPES; pH 7.4) with gentamicin (0.10 mg/mL). Oocytes were
used for recordings after 3−6 days postinjection and were voltage-
clamped with the use of a two-electrode voltage clamp (Dagan
Corporation, Minneapolis, MN) with both microelectrodes filled with
3 M KCl. Recordings were made while the oocytes were continuously
superfused with frog Ringer’s solution (in mM: 115 NaCl, 2 KCI, 1.8
1
Tris(2,2,2-trifluoroacetate (8b). Yield: 38.4 mg (13%). H NMR
(300 MHz, CD3OD): δ = 7.09 (d, J = 8.3 Hz, 2 H), 6.72 (d, J = 8.3
Hz, 2 H), 4.60 (t, J = 6.5 Hz, 1 H), 3.85 (t, J = 6.2 Hz, 1 H), 3.47 (s, 2
H), 3.13−3.28 (m, 6 H), 2.92 (t, J = 7.57 Hz, 4 H), 2.59−2.76 (m, 2
H), 1.77−1.98 (m, 2 H), 1.44−1.75 (m, 10 H), 1.37 (s, 8 H). 13C
NMR (75 MHz, CD3OD): δ = 174.7, 174.7, 173.3, 169.7, 158.7, 157.5,
131.3, 127.3, 116.4, 54.2, 52.3, 49.0, 48.5, 42.8, 41.9, 40.8, 39.4, 37.8,
30.4, 30.3, 30.0, 28.1, 27.7, 27.4, 27.4, 25.6, 24.3. HPLC-ELS: 98%. m/
z = 620.4 [MH+]. HRMS (EI) exact mass calculated for C30H53N9O5
[MH+] 620.4248. Found: 620.4234.
(S)-N1-(4-((8-((S)-2-Amino-5-guanidinopentanamido)octyl)-
amino)butyl)-2-(2-phenylacetamido)succinamide Tris(2,2,2-
htrifluoroacetate (8c). Yield: 41.9 mg (15%). 1H NMR (300
MHz, CD3OD): δ = 7.10−7.30 (m, 5 H), 4.58 (t, J = 6.5 Hz, 1 H),
3.82 (t, J = 6.3, 1 H), 3.55 (s, 2 H), 3.08−3.24 (m, 6 H), 2.79−2.97
(m, 4 H), 2.55−2.75 (m, 2 H), 1.71−1.94 (m, 2 H), 1.41−1.70 (m, 12
H), 1.32 (s, 10 H). 13C NMR (75 MHz, CD3OD): δ = 174.6, 174.0,
173.3, 169.7, 136.7, 130.2 (2 C), 129.6 (2 C), 128.0, 54.2, 52.3, 49.0,
48.5, 43.6, 41.8, 40.8, 39.4, 37.9, 37.9, 30.4, 30.3, 29.9, 28.0, 27.7, 27.4,
27.4, 27.4, 25.6, 24.3. HPLC-ELS: 99%. m/z = 604.4 [MH+]. HRMS
(EI) exact mass calculated for C30H53N9O4 [MH+] 604.4299. Found:
604.4290.
2-(2-(1H-Indol-3-yl)acetamido)-N1-(4-(8-(2-amino-5-
guanidinopentanamido)octylamino)butyl)succinamide
Tetrakis(2,2,2-trifluoroacetate (8d). Yield: 17.1 mg (5.2%). 1H
NMR (300 MHz, CD3OD): δ = 7.54 (d, J = 7.6 Hz, 1 H), 7.35 (d, J =
8.0 Hz, 1 H), 7.20 (s, 1 H), 7.11 (t, J = 7.6 Hz, 1 H), 7.02 (t, J = 7.3
Hz, 1 H), 4.61 (t, J = 6.5 Hz, 1 H), 3.84 (t, J = 6.7 Hz, 1 H), 3.73 (s, 2
H), 3.28−3.05 (m, 8 H), 2.88 (s, 1 H), 2.85−2.71 (m, 4 H), 2.68 (d, J
= 6.4 Hz, 2 H), 1.9 −1.79 (m, 2 H), 1.73−1.29 (m, 22 H), 1.25 (d, J =
6.1 Hz, 2 H). 13C NMR (75 MHz, CD3OD): δ = 174.9, 174.8, 173.3,
169.6, 138.2, 128.5, 125.2, 122.7, 120.2, 119.4, 112.6, 109.3, 54.2, 52.2,
48.5, 41.9, 40.8, 39.3, 37.5, 34.0, 30.4, 30.3, 30.0, 28.1, 27.7, 27.4, 27.2,
25.7, 24.2, 22.5. HPLC-ELS: 99%. m/z = 643.5 [MH+]. HRMS (EI)
exact mass calculated for C32H54N10O4 [MH+] 643.4408. Found:
643.4405.
(S)-2-Amino-N-(8-((4-((S)-2-(2-(2,4-dihydroxyphenyl)-
acetamido)propanamido)butyl)amino)octyl)-5-guanidinopen-
tanamide Tris(2,2,2-trifluoroacetate (8e). Yield: 76.4 mg (27%).
1H NMR (300 MHz, CD3OD): δ = 6.93 (d, J = 8.0 Hz, 1 H), 6.33 (d,
J = 1.7 Hz, 1 H), 6.27 (dd, J = 1.7, 7.8 Hz, 1 H), 4.19 (q, J = 7.0 Hz, 1
H), 3.85 (t, J = 6.2 Hz, 1 H), 3.51 (d, J = 14.8 Hz, 1 H), 3.39 (s, 1 H),
3.04−3.29 (m, 6 H), 2.70−2.95 (m, 4 H), 1.75−1.97 (m, 1 H), 1.42−
1.75 (m, 10 H), 1.36 (s, 13 H). 13C NMR (75 MHz, CD3OD): δ =
1179
dx.doi.org/10.1021/jm301602d | J. Med. Chem. 2013, 56, 1171−1181

Journal of Medicinal Chemistry
Article
BaC12, 5 HEPES; pH 7.6). The test compounds were dissolved in frog
Ringer’s solution and added by bath application. Recordings were
made at room temperature at holding potentials in the range from −80
to −40 mV. Antagonist concentration−response curves were
constructed by measuring the maximal current induced by the
saturating concentration of agonist (300 μM glutamate for GluA1i;
100 μM glutamate and 100 μM glycine for GluN1/N2A) and then
applying increasing concentrations of antagonist in the presence of the
appropriate agonist. Data from individual oocytes were fitted to the
equation I = Imin + [(Imax − Imin)/(1 + ([antagonist]/IC50)nH)], where I
is the agonist-evoked current at a given antagonist concentration, Imax
is the agonist-evoked current in the absence of antagonist, Imin is the
agonist-evoked current in the presence of the highest antagonist
concentration, [antagonist] is the concentration of antagonist, and nH
is the Hill slope. The IC50 (concentration of antagonist producing 50%
of Imax) values were determined by an iterative least-squares fitting
routine. Concentration−response curves were determined in triplicate
for three to eight individual oocytes.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Tables S1, S2, S3, and S4 listing IC50 values. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
(12) Pellegrini-Giampietro, D. E.; Gorter, J. A.; Bennett, M. V.;
Zukin, R. S. The GluR2 (GluR-B) hypothesis: Ca2+-permeable AMPA
receptors in neurological disorders. Trends Neurosci. 1997, 20, 464−
470.
■
Corresponding Author
*Phone: +45 3533 6114. E-mail: kristian.stromgaard@sund.ku.
dk.
(13) Liu, S. J.; Savtchouk, I. Ca2+ permeable AMPA receptors switch
allegiances: mechanisms and consequences. J. Physiol. 2012, 590, 13−
20.
Present Address
‡Department of Medicinal Chemistry, Boehringer Ingelheim
RCV GmbH & Co. KG, A-1121 Vienna, Austria.
(14) Beattie, M. S.; Ferguson, A. R.; Bresnahan, J. C. AMPA-receptor
trafficking and injury-induced cell death. Eur. J. Neurosci. 2010, 32,
290−297.
(15) Eldefrawi, A. T.; Eldefrawi, M. E.; Konno, K.; Mansour, N. A.;
Nakanishi, K.; Oltz, E.; Usherwood, P. N. Structure and synthesis of a
potent glutamate receptor antagonist in wasp venom. Proc. Natl. Acad.
Sci. U.S.A. 1988, 85, 4910−4913.
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
(16) Strømgaard, K.; Brierley, M. J.; Andersen, K.; Sløk, F. A; Mellor,
I. R.; Usherwood, P. N. R.; Krogsgaard-Larsen, P.; Jaroszewski, J. W.
Analogues of neuroactive polyamine wasp toxins that lack inner basic
sites exhibit enhanced antagonism toward a muscle-type mammalian
nicotinic acetylcholine receptor. J. Med. Chem. 1999, 42, 5224−5234.
(17) Strømgaard, K.; Brier, T. J.; Andersen, K.; Mellor, I. R.; Saghyan,
A.; Tikhonov, D.; Usherwood, P. N. R.; Krogsgaard-Larsen, P.;
Jaroszewski, J. W. Solid-phase synthesis and biological evaluation of a
combinatorial library of philanthotoxin analogues. J. Med. Chem. 2000,
43, 4526−4533.
(18) Kromann, H.; Krikstolaityte, S.; Andersen, A. J.; Andersen, K.;
Krogsgaard-Larsen, P.; Jaroszewski, J. W.; Egebjerg, J.; Strømgaard, K.
Solid-phase synthesis of polyamine toxin analogues: potent and
selective antagonists of Ca2+-permeable AMPA receptors. J. Med.
Chem. 2002, 45, 5745−5754.
(19) Jørgensen, M. R.; Olsen, C. A.; Mellor, I. R.; Usherwood, P. N.
R.; Witt, M.; Jaroszewski, J. W.; Franzyk, H. The effects of
conformational constraints and steric bulk in the amino acid moiety
of philanthotoxins on AMPAR antagonism. J. Med. Chem. 2005, 48,
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(20) Jensen, L. S.; Bølcho, U.; Egebjerg, J.; Strømgaard, K. Design,
synthesis and pharmacological characterization of polyamine toxin
derivatives: novel, potent ligands for the pore-forming region of
AMPA receptors. ChemMedChem 2006, 1, 419−428.
ACKNOWLEDGMENTS
■
We are grateful for support from Alfred Benzon Foundation
(S.L.), GluTarget (M.H.P.), and the Lundbeck Foundation
(K.S.).
ABBREVIATIONS USED
■
ADDP, 1,1-(azodicarbonyl)dipiperidine; AMPA, α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid; ArgTX, argiotox-
in; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DIPEA, diisopro-
pylethylamine; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; iGlu, ionotropic
glutamate; NMDA, N-methyl-D-aspartate; Ns, 2-nitrobenzene-
sulfonamide; Pbf, pentamethyl-2,3-dihydrobenzofuran-5-sulfon-
yl; TBAF, tetra-n-butylammonium fluoride; Teoc, 2-
(trimethylsilyl)ethyloxycarbonyl; TEVC, two-electrode voltage
clamp
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