Structure-activity relationship studies of N-methylated and N-hydroxylated spider polyamine toxins as inhibitors of ionotropic glutamate receptors

Article abstract of DOI:10.1021/jm5004705

Polyamine toxins from spiders and wasps are potent open-channel blockers of ionotropic glutamate (iGlu) receptors. It is well-established that secondary amino groups in the polyamine moiety of these toxins are key to both selectivity and potency at iGlu receptors, still some native spider polyamine toxins comprise both N-methyl and N-hydroxy functionalities. Here, we investigate the effect of both N-methylation and N-hydroxylation of spider polyamine toxins by the synthesis and biological evaluation of the naturally occurring N-methylated argiopinines and pseudoargiopinines I and II, N-hydroxylated Agel-489 and Agel-505, as well as N-methylated analogues of the NMDA and AMPA iGlu receptor subtype selective antagonists ArgTX-93 and ArgTX-48. Efficient synthetic strategies for the synthesis of target compounds were developed, and evaluation of biological activity at AMPA and NMDA receptors identified highly potent and in some cases very selective ligands.

Full text of DOI:10.1021/jm5004705

Article
pubs.acs.org/jmc
Structure−Activity Relationship Studies of N‑Methylated and
N‑Hydroxylated Spider Polyamine Toxins as Inhibitors of Ionotropic
Glutamate Receptors
Niels G. Nørager,† Mette H. Poulsen, Anna G. Jensen, Nanna S. Jeppesen, 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: Polyamine toxins from spiders and wasps are
potent open-channel blockers of ionotropic glutamate (iGlu)
receptors. It is well-established that secondary amino groups in
the polyamine moiety of these toxins are key to both selectivity
and potency at iGlu receptors, still some native spider
polyamine toxins comprise both N-methyl and N-hydroxy
functionalities. Here, we investigate the effect of both N-
methylation and N-hydroxylation of spider polyamine toxins
by the synthesis and biological evaluation of the naturally
occurring N-methylated argiopinines and pseudoargiopinines I and II, N-hydroxylated Agel-489 and Agel-505, as well as N-
methylated analogues of the NMDA and AMPA iGlu receptor subtype selective antagonists ArgTX-93 and ArgTX-48. Efficient
synthetic strategies for the synthesis of target compounds were developed, and evaluation of biological activity at AMPA and
NMDA receptors identified highly potent and in some cases very selective ligands.
INTRODUCTION
■
The ionotropic glutamate (iGlu) receptors are a family of
ligand-gated cation channels that mediate the majority of
excitatory synaptic transmission in the vertebrate brain. The
family comprises three subfamilies, the N-methyl-D-aspartate
(NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA), and kainate receptors.1 These receptors are
considered as promising drug targets, as they play a crucial role
in normal brain function and abnormal activity is believed to
play a critical role in a range of neurological and psychiatric
disorders.1,2
Polyamine toxins are a group of small molecules isolated
Figure 1. Overview of modifications made to argiotoxin-636 from the
orb-weaver spider, Argiope lobata, in recent SAR studies at iGlu
receptors.
from spiders and wasps, which have been shown to be use- and
voltage-dependent open-channel blockers of iGlu receptors.3,4
They have been found to be valuable pharmacological tools for
studying the iGlu receptors due to their high potency and
selectivity.3−5 Most importantly, polyamine toxins are unique
tools to distinguish among AMPA and KA receptor subtypes on
the basis of their Ca2+-permeability, as polyamine toxins only
block Ca2+-permeable iGlu receptors, which is related to the
nature of the amino acid in the so-called Q/R-site.6
A large number of structure−activity relationship (SAR)
studies of polyamine toxins and their iGlu receptor inhibition
have been conducted3,4,7 which have focused on the functional
roles of the four general components that comprise the
polyamine toxin scaffold (Figure 1). Modifications of head-
groups, amino acid linkers, and tails have been performed, while
the polyamine chain has been modified in its length and the
number and positioning of secondary amines. However, only
very limited investigation into the effect of N-functionalization
of the secondary amino groups in the polyamine has been
conducted despite SAR studies having shown the importance of
these groups for both selectivity and potency.8,9
Two classes of N-functionalized naturally occurring poly-
amine toxins have been identified containing N-methylation or
-hydroxylation of the polyamine chain. Notable examples of
these compounds are the family of N-methylated argiopinines
(1 and 2) and pseudoargiopinines (3 and 4) isolated from the
Argiope lobata spider10 and the N-hydroxylated α agatoxins
Received: March 26, 2014
Published: May 13, 2014
© 2014 American Chemical Society
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from the Agelenopsis aperta spider (5 and 6, Figure 2).11,12 To
investigate the influence of the N-functionalization on iGlu
methylated analogues of two polyamine toxin-based NMDA
and AMPA selective iGlu receptor antagonists, ArgTX-93 (7)
and ArgTX-48 (8).9 A recent study has suggested that an
intramolecular hydrogen bond between the polyamine chain
and the headgroup carbonyl is the cause of the selectivity
difference between 7 and 8,8 thus we envisioned that blocking
this hydrogen bond through N-methylation could provide
useful SAR information toward elucidating the active
conformation of this class of compounds.
RESULTS AND DISCUSSION
■
Design and Synthesis. Currently, no synthetic procedures
for preparation of compounds 1−4 have been developed.
These compounds are highly polar and contain several
nucleophilic moieties requiring extensive use of protection
groups, thus we envisioned that a solid-phase organic synthesis
(SPOS) approach would be advantageous. The synthesis was
attempted using a backbone amide linker (BAL),13 which has
previously been extensively used in the SPOS of polyamine
toxins.8,14−16 A mono N-allyloxycarbonyl (N-Alloc) protected
1,5-diaminopentane (9) was loaded onto a polystyrene resin
through reductive amination of a BAL handle (Scheme 1). Boc-
L-Lys(Ns)-OH was subsequently coupled with 2-(1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HATU) and diisopropylethyl amine (DIPEA) to
the resin-bound secondary amine (10), which provided resin-
Figure 2. (A) Structures of natural spider polyamine toxins containing
N-hydroxyl and N-methyl functionalities in the polyamine moiety. (B)
Argiotoxin derivatives, 7 and 8, which have shown to be potent and
selective inhibitors of NMDA and AMPA receptors, respectively.
receptor inhibition, we designed, synthesized, and evaluated
these naturally occurring polyamine toxins as well as their
nonfunctionalized analogues. Furthermore, we synthesized N-
a
Scheme 1. Synthesis of 1−4 and Nonmethylated Analogues 22 and 23
a
Reagents and conditions: (a) BAL polystyrene resin (loading: 0.87 mmol/g), NaBH(OAc)3, DMF/AcOH(9:1), rt; (b) Boc-L-Lys(Ns)-OH, HATU,
DIPEA, DCM/DMF (9:1), rt; (c) N-Teoc 3-amino-1-propanol, Bu3P, ADDP, DCM/THF (1:1), rt; (d) DBU, 2-mercaptoethanol, DMF, rt; (e)
MeI, TEA, DMF, rt (compound 13a) or CH2O, TEA, NaBH(OAc)3, DMF, rt (compound 13b); (f) TBAF, THF, 55 °C; (g) Boc-L-Arg(Pbf)-OH,
HATU, DIPEA, DCM/DMF (9:1), rt; (h) borane dimethylamine, Pd(PPh3)4, DCM, rt; (i) Fmoc-L-Asn(Tr)-OH, HATU, DIPEA, DCM/DMF
(9:1), rt; (j) piperidine, DMF, rt; (k) 2-(4-((2-methoxyethoxy)methoxy)-1H-indol-3-yl)acetic acid (compounds 16a−b) or 2-(1H-indol-3-yl)acetic
acid (compounds 17a−b), HATU, DIPEA, DCM/DMF (9:1), rt; (l) TFA/DCM/EDT/H2O (75:20:2.5:2.5), rt.
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a
Scheme 2. Synthesis of N-Hydroxylated Polyamine Toxins 5, 6, and 31 and Nonhydroxylated Analogues 33a−c
a
Reagents and conditions: (a) BAL polystyrene resin (loading: 0.87 mmol/g), NaBH(OAc)3, DMF/AcOH(9:1), rt; (b) 2-(1H-indol-3-yl)acetic acid
(compound 26a), 2-(4-((2-methoxyethoxy)methoxy)-1H-indol-3-yl)acetic acid (compound 26b), or 2-(2,4-bis((2-methoxyethoxy)methoxy)-
phenyl)acetic acid (compound 26c), HATU, DIPEA, DCM/DMF (9:1), rt; (c) O-TBDPS 1,3-propanediol, Me3P, ADDP, DCM/THF (1:1), rt; (d)
DBU, 2-mercaptoethanol, DMF, rt; (e) allyl chloroformate, collidine, DCM, rt; (f) TBAF, THF, 55 °C; (g) 36, Bu3P, ADDP, DCM/THF (1:1), rt;
(h) borane dimethylamine, Pd(PPh3)4, DCM, rt; (i) Davis’ oxaziridine, DCM, rt; (j) DBU, 2-mercaptoethanol, DMF, rt; (k) TFA/DCM/EDT/H2O
(75:20:2.5:2.5), rt; (l) ethyl trifluoroacetate, MeOH, −78 °C → 0 °C, then (Boc)2O, MeOH 0 °C → rt; then concd aq NH3 to pH > 11 (55%); (m)
4-nitrobenzenesulfonyl chloride, TEA, DCM, 0 °C → rt (80%).
bound intermediate 11 that was further elongated through a
Fukuyama−Mitsunobu reaction with N-2-(trimethylsilyl)-
ethyloxycarbonyl (N-Teoc) protected 1-amino-3-propanol and
using 1,1-(azodicarbonyl)dipiperidine (ADDP) and Bu3P as
redox pair.17 This yielded the key intermediate 12 (Scheme 1),
where the polyamine chain had been assembled with
orthogonal protection groups that allows independent chain
elongation in three directions. The synthesis proceeded with
deprotection of the N-Ns group followed by either mono-
methylation in a one-pot reductive amination procedure with
methanol, triethylamine (TEA), and sodium triacetoxyborohy-
dride or dimethylations with iodomethane and TEA to provide
intermediates 13a−b (Scheme 1). The N-Teoc protection
group of these intermediates was removed with tetra-n-
butylammonium fluoride (TBAF), and Boc-L-Arg(Pbf)-OH
was coupled to the resulting primary amine. Palladium
catalyzed deprotection of the N-Alloc group, followed by
coupling with Fmoc-L-Asn(Tr)-OH, yielded resin-bound 15a−
b, which were subsequently coupled to either O-2-methox-
yethoxymethyl (O-MEM) protected 4-hydroxyindolyl (16a−b)
or indolyl (17a−b) acetic acids (Scheme 1). Final cleavage of
the compounds from the resin and concomitant removal of the
acid labile protection groups provided the desired compounds
1−4 (Scheme 1, 3−13% yield, 12 steps, 75−84% average yields
per step). The yields of compounds 1 and 2 were lower, as side
product formation was observed during the resin cleavage and
deprotection step, likely due to alkylations by electrophiles
formed during the removal of the O-MEM group. In an attempt
to limit these side reactions addition of four different
scavengers during cleavage from the resin were examined:
triisopropylsilane (TIPS), 1,2-ethanedithiol (EDT), thioanisole,
and thiophenol, with EDT resulting in formation of less side
products. The corresponding nonalkylated analogues (22 and
23) were synthesized following a similar approach but omitting
the above-described N-methylation step (Scheme 1, 3−9%
yield, 11 steps, 71−80% average yields per step).
The naturally occurring N-hydroxylated polyamine toxins 5
and 6 have previously been synthesized in solution.18,19
However, to circumvent the tedious purification steps and
enable parallel synthesis of the target compounds and their
analogues, we developed a solid-phase procedure (Scheme 2).
The synthesis commenced with loading of mono N-Ns
protected 1,3-diaminopropane (24) onto a polystyrene resin
through reductive amination of a BAL handle (Scheme 2A).
Three phenyl and indolyl acetyl headgroups were subsequently
coupled to the resin-bound secondary amine (25), providing
intermediates 26a−c, which were reacted with mono O-tert-
butyldiphenylsilyl (O-TBPDS) protected 1,3-propanediol in a
Fukuyama−Mitsunobu reaction (Scheme 2). Use of the less
hindered PMe3 phosphine source was found to be advanta-
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a
Scheme 3. Synthesis of N-Methylated Analogues of 7 and 8
a
Reagents and conditions: (a) BAL polystyrene resin (loading: 0.87 mmol/g), NaBH(OAc)3, DMF/AcOH(9:1), rt; (b) Boc-L-Arg(Pbf)-OH,
HATU, DIPEA, DCM/DMF (9:1), rt; (c) N-Teoc 9-amino-1-nonanol or N-Teoc 4-amino-1-butanol, Bu3P, ADDP, DCM/THF (1:1), rt; (d) DBU,
2-mercaptoethanol, DMF, rt; (e) CH2O, TEA, NaBH(OAc)3, DMF, rt (compound 41a) or MeI, TEA, DMF, rt (compound 41b); (f) TBAF, THF,
55 °C; (g) Fmoc-L-Asn(Tr)-OH, HATU, DIPEA, DCM/DMF (9:1), rt; (h) piperidine, DMF, rt; (i) 2-(2,4-bis(benzyloxy)phenyl)acetic acid,
HATU, DIPEA, DCM/DMF (9:1), rt; (j) TFA/CH2Cl2/TIPS/H2O (75:20:2.5:2.5), rt; (k) H2, Pd(OH)2/C, AcOH, rt.
geous compared to PBu3, which is generally used in the
Tsunoda protocol, as previously reported when employing diols
in the Fukuyama−Mitsunobu reaction.20 Protection group
substitution of 27a−c, through sequential N-Ns removal and N-
Alloc protection yielding intermediates 28a−c, was conducted,
as it was envisioned that the N-Alloc protection group could be
orthogonally removed prior to an N-hydroxylation step, thus
ensuring regioselectivity at this secondary amino group. The
remaining part of the polyamine chain was synthesized through
deprotection of the O-TBDPS group with TBAF, followed by
an “inverse” Fukuyama−Mitsunobu reaction with an appropri-
ately protected spermine analogue (36),20 which was readily
synthesized from commercially available spermine (Scheme
2B).21 In the subsequent key steps, N-Alloc deprotection of
resin-bound 29a−c was followed by selective oxidation of the
free secondary amine to provide intermediates 30a−c (Scheme
2A). The oxidants m-chloroperoxybenzoic acid (mCPBA),
hydrogen peroxide, and Davis’ oxaziridine were examined for
N-hydroxylation, and it was found that the latter reagent
provided the best result in terms of yield and purity.22
Repetitive treatments with Davis’ oxaziridine were required to
drive the reaction to completion, and only minor side product
formation, such as overoxidation to the nitrone as previously
reported when oxidizing secondary amines with this reagent,
was observed.18,23 Furthermore, NMR analysis showed no sign
of indole oxidation. Finally, N-Ns deprotection, followed by
cleavage from the resin and concomitant removal of the
remaining acid labile protection groups, provided the desired
N-hydroxylated compounds 5, 6, and 31 (Scheme 2A, 3−8%,
11 steps, 74−78% average yields per step). In comparison to
previously published synthetic procedures,18,19 lower absolute
yields were obtained. However, the present procedure required
fewer synthetic steps and circumvented cumbersome purifica-
tions, thus resulting in an efficient procedure suitable for SAR
studies of this class of compounds. The corresponding
nonhydroxylated analogues were synthesized using the same
procedure but omitting the protection group substitution and
oxidation steps, yielding 33a−b (Scheme 2A, 9−10%, 7 steps,
71−72% average yields per step).
Finally, mono- and di-N-methylated analogues of 7 and 8,
respectively, were synthesized following a revised version of a
previously reported solid-phase synthesis procedure (Scheme
3).14 The initial loading of a N-Ns protected diamine onto the
resin, coupling of Boc-L-Arg(Pbf)-OH to the primary amine,
and Fukuyama−Mitsunobu elongation with an N-Teoc
protected amino alcohol, yielding intermediate 40, were
performed as described previously.14 At this point, the N-Ns
protecting group was selectively removed and subsequent N-
methylation of the resulting secondary amine provided
intermediates 41a−b (Scheme 3). Subsequent N-Teoc
deprotection and coupling with Fmoc-L-Asn(Tr)-OH were
performed following standard procedures, and the desired
products 44a−b and 45a−b were isolated after resin cleavage
and concomitant protection group removal (Scheme 3, 5−11%
yield, 11 steps, 76−82% average yields per step).
Pharmacology. To address the biological activity at iGlu
receptors of the compounds prepared, their inhibitory
potencies were evaluated at NMDA (GluN1/2A) and AMPA
(GluA1i) receptors recombinantly expressed in Xenopus laevis
oocytes, using two-electrode voltage-clamp electrophysiology.
Compound potencies were assessed by measuring their degree
of inhibition of agonist-evoked currents at a concentration of
100 nM (Figure 3), and for selected compounds full
concentration−inhibition relationships were determined at
these receptor subtypes at a membrane potential of −80 mV
(Table 1).
Compounds 1−4 were previously investigated at native iGlu
receptors from rat pyramidal neurons, which suggested that
these compounds are inhibitors of AMPA and kainate
receptors.24 Here we observed that all compounds 1−4 showed
>40% inhibition of the AMPA receptor at a concentration of
100 nM (Figure 3A) and a propensity toward the mono-N-
methylated analogues (2 and 4) being more potent than their
di-N-methylated counterparts (1 and 3). The nonmethylated
analogues 22 and 23 showed inhibition of AMPA receptors in
the same range (84% and 81% inhibition, respectively). All six
compounds showed significantly lower inhibition at the NMDA
receptor (<20%), and as observed for the AMPA receptor, N-
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while 1 and 2 show similar selectivity but lower receptor
inhibition (Table 1).
Similarly, the four N-methylated analogues of 7 and 8 were
examined for their inhibitory potency at AMPA and NMDA
receptors (Figure 3B). Initially, the native compounds 7 and 8
were confirmed as potent and selective inhibitors of the NMDA
and AMPA receptors, respectively, as demonstrated recently.9
In a previous study combing synthesis, biological testing, and
molecular modeling, we suggested such differences in AMPA
versus NMDA receptor activity is caused by different active
compound conformations due to intramolecular hydrogen
bonds between the secondary amines in the polyamine chain
and the carbonyl groups in the headgroups.8 Examination of the
inhibitory potencies of N-methylated analogues 44a−b and
45a−b furthermore support this hypothesis for selectivity.
Specifically, di-N-methylation of the secondary amine in the
NMDA receptor selective 7, compound 44b, which removes
hydrogen bond donor capacity, led to remarkable reduction in
inhibition at the NMDA receptor, while mono-N-methylation,
as in 44a, preserves hydrogen bond donor capacity, although
adding steric bulk around the secondary amine, did not change
the inhibition significantly compared to 7 (Figure 3B). A
similar, but less pronounced, tendency was observed for mono-
and di-N-methylation of the AMPA receptor selective 8, as di-
N-methylation (45b) led to a relatively larger reduction in
inhibition of the AMPA receptor compared to the correspond-
ing mono-N-methylated analogue (45a) (Figure 3B). Collec-
tively, this indicates that hydrogen bond donation by the
secondary amines is central for determining activity of 7 and 8
at AMPA and NMDA receptors. The observed reduced degree
of inhibition upon N-methylation could in principle also be
caused by unfavorable steric clash from the introduced methyl
groups, however, this was not observed for the mono-N-
methylated analogues.
Figure 3. Screening of % inhibition of agonist-evoked currents
(L‑glutamate (100 μM) and glycine (100 μM) for GluN1/2A and
L‑glutamate (300 μM) for GluA1i) by 100 nM N-functionalized
polyamine toxins at NMDA (black bars) and AMPA (gray bars)
receptors expressed in oocytes and held at a membrane potential of
−80 mV.
Table 1. IC50 Values of Selected Compounds
Finally, the N-hydroxylated compounds 5−6 and 31 and
their nonhydroxylated analogues (33a−c) were tested for
activity at AMPA and NMDA receptors (Figure 3C). The
naturally occurring N-hydroxylated polyamine toxins 5 and 6
were highly potent inhibitors of the NMDA receptors with
>90% inhibition of agonist-induced currents from a 100 nM
concentration, as previously suggested.25−27 Interestingly, the
indole headgroups seemed to be critical for these effects, as
substitution of the native headgroup with a phenyl acetyl group
(31), as found for example in argiotoxin-636, reduced
inhibition dramatically. In contrast, replacement of the N-
hydroxylate group by a secondary amine did not affect NMDA
receptor inhibition, and both N-hydroxylated compounds 5 and
6 as well as their nonhydroxylated analogues 33a−b showed
inhibition >90% (Figure 3C). Finally, we observed that removal
of the N-hydroxyl group in 31, yielding 33c, led to a significant
increase in NMDA receptor inhibition. For all six compounds,
the degree of inhibition at AMPA receptors was reduced
compared to their NMDA receptor activity and no distinct
correlation between the presence of the N-hydroxylation and
the degree of inhibition was observed (Figure 3C). Finally, IC50
values were obtained from full concentration−inhibition curves
for 5 at AMPA and NMDA receptors and verified that 5 is
indeed a very potent, but also nonselective, inhibitor of iGlu
receptors, with IC50 values of 12 and 11 nM at AMPA and
NMDA receptors, respectively (Table 1).
a
IC50 (nM)
b
c
compd
GluN1/2A
GluA1i
1
3852 [3120−4755]
1634 [1276−2094]
387 [225−665]
12 [7−20]
123 [103−146]
52 [43−63]
15 [11−19]
11 [7−16]
2
22
5
a
Mean IC50 values (95% confidence interval in brackets) determined
from 4−18 different oocytes at a membrane potential of −80 mV.
Inhibition of the current elicited by 100 μM L-glutamate and 100 μM
glycine by simultaneous coapplication of the antagonist at oocytes
injected with a 1:10 ratio of GluN1/N2A. Inhibition of the current
elicited by 300 μM L-glutamate by simultaneous coapplication of the
b
c
antagonist at oocytes injected with GluA1i.
methylation led to a lower degree of inhibition. We then
performed full concentration−inhibition correlations for
compounds 1, 2, and 22 at AMPA and NMDA receptors
(Figure 4), which allow a precise measure of the effect of
different degrees of N-methylation on inhibitory potency. In
conformity with the single concentration determination, we
observed that increasing N-methylation led to decreased
potency at both AMPA and NMDA receptors (Table 1),
with a strong preference for AMPA receptors. In fact,
compound 22 is a selective (26-fold vs NMDA receptors)
and very potent (IC50 = 15 nM) inhibitor of AMPA receptors,
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Figure 4. Determination of compound inhibitory potency (IC50) at GluA1 and GluN1/2A receptors. Composite concentration−inhibition curves
for inhibition by 1 (left), 2 (middle), and 22 (right) at AMPA (gray circles) and NMDA (black squares) receptors at a membrane potential of −80
mV. Data points represent the means SEM of experiments with >4 oocytes per curve.
spectrometer (at 400 or 100 MHz, respectively) or an Avance 600
spectrometer (at 600 or 150 MHz, respectively), using CDCl3,
CD3OD, or DMSO-d6 as solvent. Chemical shifts are reported in ppm
CONCLUSION
■
In summary, we have developed solid-phase synthetic method-
(δ) using residual solvent as an internal standard; CDCl3, 7.26 (1H),
77.16 (13C) ppm; CD3OD. 3.31, 49.00 ppm; DMSO-d6, 2.50, 39.52
ppm. Coupling constants (J) are given in Hz. The purity of the
compounds was determined by LC-MS. LC-MS analysis was
performed on an Agilent 6410 triple quadrupole mass spectrometer
instrument using electron spray coupled to an Agilent 1200 HPLC
system (ESI-LC/MS) with autosampler and diode array detector using
a gradient (0%−100% B over 3 min, then 100% B for 3 min) of the
binary solvent system of water/acetonitrile/TFA (A, 95/5/0.1%, and
B, 5/95/0.1%) 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 ELS detector (ELSD), which was used
for estimation of the purity of the final products. Analyses at
intermediate stages of the synthesis were performed by cleaving
samples from the resin as follows: a 1 mL screwcap vial was charged
with a 1 mg sample of the resin and 0.4 mL TFA/DCM/EDT
(75:20:5). The vial was agitated periodically over 1 h and filtered using
a syringe filter and disposable syringe. The crude product was analyzed
by LC-MS. High resolution mass spectra (HRMS) were obtained
using a Micromass Q-Tof II instrument.
Mono-N-methylation: General Procedure. The resin was
swelled in DMF (4 mL) for 30 min. The solvent was drained, 37%
formaldehyde in water (0.12 mL, 1.5 mmol), triethylamine (0.11 mL,
0.75 mmol), and sodium triacetoxyborohydride (0.16 g, 0.75 mmol)
were added, and the mixture was agitated for 2 h at rt. The solvent was
drained, and the resin was washed with DMF, DCM, MeOH, and
DCM (3 × 4 mL each) and dried in vacuo.
Di-N-methylation: General Procedure. The resin was swelled in
DMF (4 mL) for 30 min. The solvent was drained, methyl iodide
(0.19 mL, 3.0 mmol) and triethylamine (0.22 mL, 1.50 mmol) were
added, and the mixture was agitated for 2 h at rt. The solvent was
drained, and the resin was washed with DMF, DCM, MeOH, and
DCM (3 × 4 mL each) and dried in vacuo.
N-Hydroxylation: General Procedure. The resin was swelled in
dry DCM (4 mL) for 30 min. The resin was drained, and a solution of
(+)-(8,8-dichlorocamphorylsulfonyl)oxaziridine (0.22 g, 0.75 mmol)
in dry DCM (4 mL) was added. The mixture was agitated for 3 h at rt,
drained, washed with DMF, DCM, MeOH, and DCM (3 × 4 mL
each), and the resin dried in vacuo. The procedure was repeated three
times.
Preparation of 1−4. The BAL resin (0.18 g, 0.15 mmol) was
swelled in DMF (4 mL) for 1 h. The resin was drained, and a solution
of allyl (5-aminopentyl)carbamate (9, 0.28 g, 1.50 mmol) in dry
DMF/glacial acetic acid (9:1, 4 mL) was added. After agitation for 15
min, sodium triacetoxyborohydride (0.32 g, 1.5 mmol) was added. The
reaction mixture was agitated overnight at rt. The solvent was drained,
and the resin was washed with DMF, DIPEA (10% in DMF), DMF,
DCM, MeOH, and DCM (3 × 4 mL each) and dried in vacuo. The
resin (10) was swelled in DCM (4 mL) for 30 min and then drained.
A solution of Boc-L-Lys(Ns)-OH (0.32 g, 0.75 mmol) and HATU
(0.28 g, 0.75 mmol) in DCM/DMF (9:1, 4 mL) was added, followed
ologies for selective N-methylation and N-hydroxylation of
secondary amines in the polyamine moiety of polyamine toxins.
We have then exploited these methodologies for the synthesis
of three groups of polyamine toxins: the naturally occurring N-
methylated 1−4, N-hydroxylated 5 and 6, N-methylated
analogues of 7 and 8, as well as their corresponding
nonfunctionalized analogues. These compounds were used to
investigate the influence of N-methylation and N-hydroxyla-
tions in the polyamine part of polyamine toxins for inhibition of
iGlu receptor subtypes.
Examination of the 1−4 resulted in the discovery of a new
group of potent and selective AMPA receptor antagonists. We
found that N-methylations of polyamine toxins generally did
not affect selectivity between AMPA and NMDA receptors
while the inhibitory potency was gradually reduced upon N-
methylation. In particular, di-N-methylation of 7 and 8 resulted
in loss of inhibitory potency, indicating that the hydrogen bond
donating property of these secondary amines is important for
iGlu receptor inhibition of this class of compounds. Finally, N-
hydroxylated α agatoxins 5 and 6 were found to be highly
potent NMDA receptor antagonists, with 5 also having high
potency for AMPA receptor antagonism. Furthermore, their
nonhydroxylated analogues (33a−b) showed comparable
inhibition of NMDA receptors, thus indicating that the N-
hydroxylation is not crucial to iGlu receptor inhibition, which
would simplify future SAR studies of this class of polyamine
toxins.
EXPERIMENTAL SECTION
■
Chemistry: General Procedures. Unless otherwise stated,
starting materials were obtained from commercial suppliers and were
used without further purification. The BAL-resin used was 2-(3,5-
dimethoxy-4-formylphenoxy)ethyl polystyrene, with a loading of 0.87
mmol/g, which was purchased from Novabiochem. 2-(Trimethylsilyl)-
ethyl 3-hydroxypropylcarbamate,28 2-(trimethylsilyl)ethyl (4-
hydroxybutyl)carbamate,29 2-(trimethylsilyl)ethyl (9-hydroxynonyl)-
carbamate,14 N-(3-aminopropyl)-2-nitrobenzenesulfonamide,30 N-(8-
aminooctyl)-2-nitrobenzenesulfonamide,14 3-((tert-butyldiphenylsilyl)-
oxy)propan-1-ol,31 2-(2,4-bis(benzyloxy)phenyl)acetic acid,32 and allyl
(5-aminopentyl)carbamate33 were prepared according to literature
procedures. THF, DMF, and DCM were dried, degassed, and
scrubbed using a Glass Contour solvent purification system
immediately before use. Preparative HPLC was performed on 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 water/acetonitrile/formic acid (A, 95/5/0.1%; and B, 5/95/
0.1%) with a flow rate of 20 mL/min and UV detection at 215 and 254
nm. 1H NMR and 13C NMR spectra were recorded on an Avance 400
4945
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Journal of Medicinal Chemistry
Article
by DIPEA (0.27 mL, 1.5 mmol). The reaction mixture was agitated
overnight at rt. The resin was drained, washed with DMF, DCM,
MeOH, and DCM (3 × 4 mL each), and dried in vacuo. The resin
(11) was swelled in dry THF/DCM (1:1, 2 mL) for 30 min. The
mixture was flushed with nitrogen and kept under an atmosphere of
nitrogen throughout the reaction. A solution of 2-(trimethylsilyl)ethyl
3-hydroxypropylcarbamate (0.17 g, 0.75 mmol) in THF/DCM (1:1, 1
mL) was added, followed by tributylphosphine (0.19 mL, 0.75 mmol).
After agitation for 15 min, a solution of ADDP (0.19 g, 0.75 mmol) in
THF/DCM (1:1, 1 mL) was added. The mixture was agitated for 3 h,
then drained, washed with DMF, DCM, MeOH, and DCM, and dried
in vacuo. The procedure was repeated twice. The resin (12) was
swelled in DMF (4 mL) for 30 min. After draining, solutions of DBU
(0.09 mL, 0.60 mmol) in DMF (2 mL) and 2-mercaptoethanol (0.04
mL) in DMF (2 mL) were added. The mixture was agitated for 30
min, drained, and washed with DMF (3 × 4 mL). The procedure was
repeated until the solution was colorless (three times). The resin was
washed with DMF, DCM, MeOH, and DCM (3 × 4 mL each) and
dried in vacuo. The resin was subsequently methylated using the two
general procedures. The resin (13) was swelled in dry THF (4 mL) for
30 min and heated to 55 °C. A 1.0 M solution of TBAF in THF (0.75
mL, 0.75 mmol) was added slowly, and the mixture was agitated for 30
min at 55 °C. The mixture was cooled to rt, drained, and washed with
THF (3 × 4 mL), and the procedure was repeated once. The resin was
washed with DMF, DCM, MeOH, and DCM (3 × 4 mL each) and
dried in vacuo. Boc-L-Arg(Pbf)-OH (0.40 g, 0.75 mmol) was coupled
to the resin using the same procedure as for 10. The resin (14) was
swelled in dry DCM (2 mL). The mixture was flushed with nitrogen
and kept under an atmosphere of nitrogen throughout the reaction. A
solution of borane dimethylamine complex (0.35 g, 6.0 mmol) in dry
DCM (1 mL) was added, and after 5 min of agitation a solution of
tetrakis(triphenylphosphine)palladium(0) (0.017 g, 0.015 mmol) in
dry DCM (1 mL) was added. The mixture was agitated at rt for 2 h,
drained, washed with sodium diethyldithiocarbamate trihydrate (0.02
M in DMF), DMF, DCM, MeOH, and DCM (3 × 4 mL each), and
the resin dried in vacuo. Fmoc-L-Asn(Tr)-OH (0.45 g, 0.75 mmol)
was coupled to the resin using the same procedure as for 10. The resin
(15) was then swelled in DMF (4 mL) for 30 min, drained, and a
solution of piperidine (20% in DMF, 4 mL) was added. After agitation
for 20 min, the resin was drained and washed with DMF (3 × 4 mL).
The treatment was repeated once. The resin was washed with DMF,
DCM, MeOH, and DCM (3 × 4 mL each) and dried in vacuo. The
resin was swelled in DCM (4 mL) for 30 min and then drained. 2-
(1H-Indol-3-yl)acetic acid (0.13 g, 0.75 mmol) or 2-(4-((2-
methoxyethoxy)methoxy)-1H-indol-3-yl)acetic acid (54, 0.21 g, 0.75
mmol) were coupled to the resin using the same procedure as for 10.
The resin (16 and 17) was swelled in DCM (4 mL) for 30 min and
then drained. A solution of TFA/DCM/EDT/H2O (75:20:2.5:2.5, 4
mL) was added. The mixture was agitated for 3 h, drained, and the
solution collected. The resin was washed with DCM (2 mL) and
MeOH (2 mL), and the washings were collected. The solutions were
combined and evaporated by a stream of nitrogen. The residue was
purified by preparative HPLC, yielding compounds 1−4.
tetracosan-24-yl)succinamide Tetrakis(2,2,2-trifluoroacetate) (2).
Prepared with 54 and monomethylation. Yield: 5.8 mg, 3.2% (85%
per step over 12 steps). 1H NMR (600 MHz, CD3OD) δ 7.07 (s, 1H),
6.97−6.92 (m, 2H), 6.45 (d, J = 7.0 Hz, 1H), 4.68 (t, J = 6.4 Hz, 1H),
3.92−3.79 (m, 3H), 3.74 (t, J = 6.6 Hz, 1H), 3.46−3.41 (m, 1H),
3.28−2.99 (m, 11H), 2.91−2.83 (m, 2H), 2.82 (s, 3H), 2.75−2.67 (m,
2H), 2.00−1.65 (m, 10H), 1.57−1.29 (m, 6H), 1.20−1.12 (m, 2H).
13C NMR (150 MHz, CD3OD) δ 174.8, 173.6, 171.8, 168.3, 161.8,
161.6, 157.3, 150.5, 139.2, 122.8, 122.5, 116.7, 107.3, 103.8, 53.8, 52.8,
50.6, 40.3, 38.7, 36.0, 34.0, 30.6, 28.3, 28.0, 24.1, 24.0, 23.4, 23.2, 21.6.
HRMS (EI) exact mass calculated for C36H60N12O6 [MH+] 745.4837;
found 745.4811. Purity (ELSD): 99%.
(S)-6-((5-((S)-2-(2-(1H-Indol-3-yl)acetamido)-4-amino-4-
oxobutanamido)pentyl)amino)-5-amino-N-(3-((S)-2-amino-5-
guanidinopentanamido)propyl)-N,N-dimethyl-6-oxohexan-1-ami-
nium Tetrakis(2,2,2-trifluoroacetate) (3). Prepared with 2-(1H-indol-
3-yl)acetic acid and dimethylation. Yield: 17.8 mg, 10.0% (83% per
step over 12 steps). 1H NMR (400 MHz, CD3OD) δ 7.56 (dt, J = 8.0,
1.3 Hz, 1H), 7.38 (dt, J = 8.0, 0.8 Hz, 1H), 7.22 (s, 1H), 7.13 (ddd, J =
8.0, 7.0, 1.0 Hz, 1H), 7.04 (ddd, J = 8.0, 7.0, 1.3 Hz, 1H), 4.68 (dd, J =
7.3, 6.0 Hz, 1H), 3.90 (t, J = 6.8 Hz, 1H), 3.79 (t, J = 6.5 Hz, 1H),
3.75−3.73 (m, 2H), 3.45−3.38 (m, 1H), 3.29−3.11 (m, 10H), 3.09−
3.03 (m, 1H), 2.99 (s, 3H), 2.98 (s, 3H), 2.72−2.59 (m, 2H), 1.98−
1.82 (m, 6H), 1.81−1.62 (m, 4H), 1.51−1.34 (m, 6H), 1.26−1.18 (m,
2H). 13C NMR (100 MHz, CD3OD) δ 174.9, 173.2, 170.4, 169.9,
163.2, 162.9, 158.8, 138.2, 128.5, 125.2, 122.8, 120.2, 119.5, 112.6,
109.4, 65.2, 63.5, 54.2, 52.0, 51.1, 41.7, 40.3, 40.1, 37.8, 37.6, 33.9,
32.1, 29.7, 29.6, 29.5, 25.5, 24.7, 23.8, 23.2, 22.9. HRMS (EI) exact
+
mass calculated for C36H63N12O5 [M+] 743.5044; found 743.5029.
Purity (ELSD): 99%.
(S)-2-(2-(1H-Indol-3-yl)acetamido)-N1-((6S,17S)-1,6,17-triamino-
1-imino-12-methyl-7,18-dioxo-2,8,12,19-tetraazatetracosan-24-yl)-
succinamide Tetrakis(2,2,2-trifluoroacetate) (4). Prepared with 2-
(1H-indol-3-yl)acetic acid and monomethylation. Yield: 23.2 mg,
13.0% (84% per step over 12 steps). 1H NMR (400 MHz, CD3OD) δ
7.55 (dt, J = 7.5, 1.0 Hz, 1H), 7.38 (dt, J = 8.0, 1.0 Hz, 1H), 7.22 (s,
1H), 7.13 (ddd, J = 8.0, 7.0, 1.3 Hz, 1H), 7.04 (ddd, J = 8.0, 7.0, 1.0
Hz, 1H), 4.70−4.67 (m, 1H), 3.88 (t, J = 6.8 Hz, 1H), 3.79−3.73 (m,
3H), 3.44−3.35 (m, 1H), 3.25−2.98 (m, 11H), 2.76 (s, 3H), 2.73−
2.59 (m, 2H), 1.98−1.81 (m, 6H), 1.80−1.62 (m, 4H), 1.50−1.33 (m,
6H), 1.26−1.18 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 174.9,
173.2, 170.5, 169.9, 163.2, 162.9, 158.8, 138.2, 128.5, 125.2, 122.8,
120.2, 119.5, 116.7, 112.6, 109.4, 55.1, 54.2, 52.0, 41.7, 40.3, 40.1, 37.7,
33.9, 32.1, 29.7, 29.6, 25.5, 24.7, 23.0. HRMS (EI) exact mass
calculated for C35H61N12O5 [MH+] 729.4888; found 729.4871. Purity
(ELSD): 99%.
Preparation of 22 and 23. Resin 12 was Teoc-deprotected and
coupled to Boc-L-Arg(Pbf)-OH (0.40 g, 0.75 mmol) using the same
procedure as 13, and the formed intermediate (18) was Alloc-
deprotected and coupled to Fmoc-L-Asn(Tr)-OH (0.45 g, 0.75 mmol)
in the same manner as 14. The resin (19) was Fmoc-deprotected and
coupled to 2-(1H-indol-3-yl)acetic acid (0.13 g, 0.75 mmol) or 2-(4-
((2-methoxyethoxy)methoxy)-1H-indol-3-yl)acetic acid (54, 0.21 g,
0.75 mmol) using the same procedure as for 15, and the intermediates
20−21 were Ns-deprotected using the same procedure as for 12,
cleaved from the resin, and purified by preparative HPLC, yielding
compounds 22 and 23.
(S)-5-Amino-6-((5-((S)-4-amino-2-(2-(4-hydroxy-1H-indol-3-yl)-
acetamido)-4-oxobutanamido)pentyl)amino)-N-(3-((S)-2-amino-5-
guanidinopentanamido)propyl)-N,N-dimethyl-6-oxohexan-1-ami-
nium Tetrakis(2,2,2-trifluoroacetate) (1). Prepared with 54 and
1
(S)-2-(2-(4-Hydroxy-1H-indol-3-yl)acetamido)-N1-((6S,17S)-
1,6,17-triamino-1-imino-7,18-dioxo-2,8,12,19-tetraazatetracosan-
24-yl)succinamide Tetrakis(2,2,2-trifluoroacetate) (22). Yield: 4.4
mg, 2.5% (71% per step over 11 steps). 1H NMR (600 MHz, CD3OD)
δ 7.07 (s, 1H), 6.97−6.92 (m, 2H), 6.45 (d, J = 7.0 Hz, 1H), 4.68 (t, J
= 6.2 Hz, 1H), 3.92−3.78 (m, 3H), 3.73 (t, J = 6.6 Hz, 1H), 3.47−3.42
(m, 1H), 3.29−3.20 (m, 4H), 3.14−2.96 (m, 7H), 2.75−2.67 (m, 2H),
1.98−1.80 (m, 6H), 1.78−1.66 (m, 4H), 1.49−1.27 (m, 6H), 1.20−
1.11 (m, 2H). 13C NMR (150 MHz, CD3OD) δ 174.8, 173.6, 171.8,
169.3, 168.3, 157.3, 150.5, 139.2, 122.8, 122.5, 116.6, 107.3, 103.8,
52.7, 50.6, 45.1, 40.3, 38.8, 36.1, 35.9, 34.0, 30.6, 28.3, 28.2, 28.1, 26.0,
25.5, 24.1, 23.2, 21.5. HRMS (EI) exact mass calculated for
C34H59N12O6 [MH+] 731.4681; found 731.4663. Purity (ELSD): 99%.
dimethylation. Yield: 8.0 mg, 4.4% (77% per step over 12 steps). H
NMR (600 MHz, CD3OD) δ 7.07 (s, 1H), 6.98−6.93 (m, 2H), 6.45
(d, J = 7.0 Hz, 1H), 4.67 (t, J = 6.2 Hz, 1H), 3.94−3.80 (m, 3H), 3.76
(t, J = 6.6 Hz, 1H), 3.49−3.43 (m, 1H), 3.32−3.21 (m, 7H), 3.16−
3.07 (m, 4H), 3.04 (m, 6H), 2.75−2.69 (m, 2H), 2.02−1.78 (m, 8H),
1.74−1.67 (m, 2H), 1.48−1.30 (m, 6H), 1.20−1.10 (m, 2H). 13C
NMR (150 MHz, CD3OD) δ 174.8, 173.6, 171.8, 169.0, 168.3, 157.3,
150.5, 139.2, 122.8, 122.5, 116.7, 107.3, 103.8, 52.8, 50.6, 49.7, 40.3,
38.7, 36.2, 36.0, 34.0, 30.7, 28.3, 28.2, 28.0, 24.1, 23.2, 22.4, 21.8, 21.5.
+
HRMS (EI) exact mass calculated for C36H63N12O6 [M+] 759.4994;
found 759.4999. Purity (ELSD): 98%.
(S)-2-(2-(4-Hydroxy-1H-indol-3-yl)acetamido)-N1-((6S,17S)-
1,6,17-triamino-1-imino-12-methyl-7,18-dioxo-2,8,12,19-tetraaza-
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(S)-2-(2-(1H-Indol-3-yl)acetamido)-N1-((6S,17S)-1,6,17-triamino-
1-imino-7,18-dioxo-2,8,12,19-tetraazatetracosan-24-yl)-
succinamide Tetrakis(2,2,2-trifluoroacetate) (23). Yield: 15.3 mg,
Preparation of 33a−c. The resin (27) was coupled to tert-butyl
(4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)-
amino)butyl)(3-(2-nitrophenylsulfonamido)propyl)carbamate (36,
0.31 g, 0.45 mmol) using the same procedure as for 11. The resin
(32) was Ns-deprotected using the standard procedure shown for 12,
cleaved from the resin, and purified by preparative HPLC, yielding
compounds 33a−c.
1
8.7% (80% per step over 11 steps). H NMR (400 MHz, CD3OD) δ
7.55 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.22 (s, 1H), 7.13
(dt, J = 7.6, 1.0 Hz, 1H), 7.04 (dt, J = 7.6, 1.0 Hz, 1H), 4.69 (t, J = 6.7
Hz, 1H), 3.88 (t, J = 6.8 Hz, 1H), 3.77−3.73 (m, 3H), 3.44−3.35 (m,
1H), 3.26−3.10 (m, 6H), 3.08−2.92 (m, 5H), 2.72−2.60 (m, 2H),
1.95−1.79 (m, 6H), 1.77−1.62 (m, 4H), 1.50−1.32 (m, 6H), 1.25−
1.17 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 175.0, 173.2, 170.7,
169.9, 163.3, 162.9, 158.8, 138.2, 128.5, 125.2, 122.8, 120.2, 119.4,
116.7, 112.6, 109.3, 54.2, 52.0, 46.5, 41.7, 40.3, 40.2, 37.7, 37.4, 33.9,
32.0, 29.7, 29.6, 27.4, 26.8, 25.5, 24.8, 22.9, 20.8. HRMS (EI) exact
mass calculated for C34H59N12O5 [MH+] 715.4731; found 715.4710.
Purity (ELSD): 99%.
Preparation of 5, 6, and 31. N-(3-Aminopropyl)-2-nitro-
benzenesulfonamide (24, 0.39 g, 1.50 mmol) was loaded onto a
BAL resin using the standard procedure as shown for 9. The resin (25)
was coupled to 2-(1H-indol-3-yl)acetic acid (0.13 g, 0.75 mmol), 2-(4-
((2-methoxyethoxy)methoxy)-1H-indol-3-yl)acetic acid (54, 0.21 g,
0.75 mmol), or 2-(2,4-bis((2-methoxyethoxy)methoxy)phenyl)acetic
acid (48, 0.18 g, 0.75 mmol) in a similar manner to 10. The resin (26)
was coupled to 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol (0.24 g,
0.75 mmol) using the same procedure as for 11, using 1 M
trimethylphosphine in THF (0.75 mL, 0.75 mmol) as the phosphine
source. The resin (27) was Ns-deprotected as described for 12, and
the resin was then swelled in dry DCM (2 mL) for 30 min. Collidine
(0.12 mL, 0.90 mmol) was added, followed by a solution of allyl
chloroformate (0.15 mL, 1.35 mmol) in DCM (2 mL). The mixture
was agitated for 1 h at rt, drained, washed with DMF, DCM, MeOH,
and DCM, and the resin dried in vacuo. The resin (28) was TBDPS-
deprotected using the same protocol as 13 and coupled to tert-butyl
(4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)-
amino)butyl)(3-(2-nitrophenylsulfonamido)propyl)carbamate (36,
0.31 g, 0.45 mmol) as described for 11. The resin (29) was Alloc-
deprotected using the same procedure as for 14, followed by
hydroxylation using the general procedure. The resin (30) was Ns-
deprotected following the protocol used for 12, cleaved, and purified
by preparative HPLC, yielding compounds 5, 6, and 31.
N-(20-Amino-4,8,12,17-tetraazaicosyl)-2-(indolin-3-yl)acetamide
Pentakis(2,2,2-trifluoroacetate) (33a). Prepared using 2-(1H-indol-3-
yl)acetic acid. Yield: 9.8 mg, 6.9% (78% per step over 11 steps). Yield:
1
14.2 mg, 9.1% (71% per step over 7 steps). H NMR (400 MHz,
CD3OD) δ 7.55 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.21 (s,
1H), 7.12 (dt, J = 7.2, 1.1 Hz, 1H), 7.03 (dt, J = 7.7, 1.1 Hz, 1H), 3.70
(s, 2H), 3.15−2.99 (m, 14H), 2.92−2.83 (m, 4H), 2.16−1.96 (m, 6H),
1.86−1.76 (m, 6H). 13C NMR (100 MHz, CD3OD) δ 175.2, 162.1,
161.6, 136.9, 127.2, 123.9, 121.4, 118.8, 118.1, 114.9, 111.3, 108.1,
44.7, 36.7, 35.5, 32.7, 26.5, 24.3, 23.1. HRMS (EI) exact mass
calculated for C26H48N7O [MH+] 474.3920; found 474.3913. Purity
(ELSD): 99%.
N-(20-Amino-4,8,12,17-tetraazaicosyl)-2-(4-hydroxyindolin-3-yl)-
acetamide Pentakis(2,2,2-trifluoroacetate) (33b). Prepared using 54.
1
Yield: 15.2 mg, 9.6% (72% per step over 7 steps). H NMR (400
MHz, CD3OD) δ 7.03 (s, 1H), 6.95−6.88 (m, 2H), 6.40 (dd, J = 7.0,
1.5 Hz, 1H), 3.81 (s, 2H), 3.37−3.32 (m, 2H), 3.15−2.91 (m, 18H),
2.14−1.97 (m, 6H), 1.85−1.77 (m, 6H). 13C NMR (100 MHz,
CD3OD) δ 178.2, 152.2, 140.7, 124.1, 123.9, 118.2, 108.7, 105.0, 45.9,
37.8, 36.4, 35.1, 30.7, 27.7, 25.4, 24.2, 24.0. HRMS (EI) exact mass
calculated for C26H48N7O2 [MH+] 490.3869; found 490.3861. Purity
(ELSD): 99%.
N-(20-Amino-4,8,12,17-tetraazaicosyl)-2-(2,4-dihydroxyphenyl)-
acetamide Pentakis(2,2,2-trifluoroacetate) (33c). Prepared using 48.
1
Yield: 14.8 mg, 9.5% (71% per step over 7 steps). H NMR (400
MHz, CD3OD) δ 6.94 (d, J = 8.3 Hz, 1H), 6.34 (d, J = 2.5 Hz, 1H),
6.28 (dd, J = 8.3, 2.5 Hz, 1H), 3.44 (s, 2H), 3.36−3.31 (m, 2H), 3.16−
2.97 (m, 18H), 2.16−2.04 (m, 6H), 1.90−1.77 (m, 6H). 13C NMR
(100 MHz, CD3OD) δ 177.2, 159.1, 157.6, 132.9, 119.6, 116.7, 114.3,
107.9, 103.8, 45.9, 38.4, 37.8, 36.4, 27.7, 25.4, 24.2. HRMS (EI) exact
mass calculated for C24H47N6O3 [MH+] 467.3709; found 467.3703.
Purity (ELSD): 99%.
Preparation of 44a−b and 45a−b. N-(3-Aminopropyl)-2-
nitrobenzenesulfonamide (0.19 g, 0.75 mmol) or N-(8-aminooctyl)-
2-nitrobenzenesulfonamide (0.25 g, 0.75 mmol) was loaded onto a
BAL resin using the standard procedure as shown for 9. The resin (38)
was coupled to Boc-L-Arg(Pbf)-OH (0.40 g, 0.75 mmol) using the
same procedure as for 10, and 2-(trimethylsilyl)ethyl 4-hydroxybu-
tylcarbamate (0.18 g, 0.75 mmol) or 2-(trimethylsilyl)ethyl 9-
hydroxynonylcarbamate (0.23 g, 0.75 mmol) was coupled onto the
resin as described for 11. The resin (40) was Ns-deprotected using the
same procedure as for 12, followed by methylation using the two
general procedures. The resin (41) was Teoc-deprotected and coupled
to Fmoc-L-Asn(Tr)-OH (0.45 g, 0.75 mmol) using the same
procedure as for 13, and the intermediate 42 was Fmoc-deprotected
and coupled to 2-(2,4-bis(benzyloxy)phenyl)acetic acid (0.26 g, 0.75
mmol) using the same procedure as for 15. 43 was cleaved from the
resin following the standard protocol as described for 16, using TIPS
instead of EDT as scavenger, and the residue was purified by
preparative HPLC. The benzyl-protected intermediate was dissolved
in glacial acetic acid (8 mL) under an atmosphere of nitrogen.
Pd(OH)2/C (10% w/w) was added, and hydrogen was bubbled
through the solution for 5 min. The mixture was then stirred for 3 h
under a static atmosphere of hydrogen. The mixture was filtered and
the solid washed with MeOH (4 mL). The organic phases were
collected, water (10 mL) was added, and the solvent was removed by
lyophilization. The crude product was purified by preparative HPLC,
yielding compounds 44a−b and 45a−b.
N-(20-Amino-4-hydroxy-4,8,12,17-tetraazaicosyl)-2-(indolin-3-
yl)acetamide Tetrakis(2,2,2-trifluoroacetate) (5). Prepared using 2-
(1H-indol-3-yl)acetic acid. Yield: 9.8 mg, 6.9% (78% per step over 11
steps). 1H NMR (400 MHz, CD3OD) δ 7.57 (dt, J = 8.0, 1.3 Hz, 1H),
7.38 (dt, J = 8.0, 1.0 Hz, 1H), 7.22 (s, 1H), 7.15−7.11 (m, 1H), 7.07−
7.02 (m, 1H), 3.67 (s, 2H), 3.14−2.93 (m, 14H), 2.76−2.67 (m, 4H),
2.12−2.03 (m, 4H), 1.96−1.86 (m, 2H), 1.84−1.72 (m, 6H). 13C
NMR (100 MHz, CD3OD) δ 175.6, 138.2, 128.6, 125.0, 122.8, 120.1,
119.6, 112.6, 110.0, 57.9, 45.8, 37.8, 37.1, 34.0, 25.4, 24.2. HRMS (EI)
exact mass calculated for C26H48N7O2 [MH+] 490.3869; found
490.3850. Purity (ELSD): 99%.
N-(20-Amino-4-hydroxy-4,8,12,17-tetraazaicosyl)-2-(4-hydrox-
yindolin-3-yl)acetamide Tetrakis(2,2,2-trifluoroacetate) (6). Pre-
pared using 54. Yield: 4.9 mg, 3.4% (74% per step over 11 steps).
1H NMR (400 MHz, CD3OD) δ 7.04 (s, 1H), 6.96−6.90 (m, 2H),
6.41 (dd, J = 7.0, 1.3 Hz, 2H), 3.82 (s, 2H), 3.37−3.32 (m, 2H), 3.14−
2.90 (m, 18H), 2.12−1.99 (m, 6H), 1.83−1.76 (m, 6H). 13C NMR
(100 MHz, CD3OD) δ 177.9, 151.9, 140.9, 124.3, 118.5, 108.5, 105.5,
45.8, 37.6, 36.4, 35.0, 30.6, 27.9, 25.0, 24.0. HRMS (EI) exact mass
calculated for C26H48N7O3 [MH+] 506.3819; found 506.3822. Purity
(ELSD): 98%.
N-(20-Amino-4-hydroxy-4,8,12,17-tetraazaicosyl)-2-(2,4-
dihydroxyphenyl)acetamide Tetrakis(2,2,2-trifluoroacetate) (31).
Prepared using 48. Yield: 5.1 mg, 3.5% (74% per step over 11
steps). 1H NMR (400 MHz, CD3OD) δ 6.95 (d, J = 8.2 Hz, 1H), 6.34
(d, J = 2.3 Hz, 1H), 6.29 (dd, J = 8.0, 2.3 Hz, 1H), 3.45 (s, 2H), 3.36−
3.31 (m, 2H), 3.18−2.96 (m, 18H), 2.19−2.06 (m, 6H), 1.92−1.79
(m, 6H). 13C NMR (100 MHz, CD3OD) δ 177.6, 159.5, 157.7, 133.0,
119.8, 114.2, 107.8, 103.6, 45.8, 38.2, 37.8, 36.5, 27.7, 25.4, 24.1.
HRMS (EI) exact mass calculated for C24H47N6O4 [MH+] 483.3659;
found 483.3648. Purity (ELSD): 98%.
(S)-N1-(9-((3-((S)-2-Amino-5-guanidinopentanamido)propyl)-
(methyl)amino)nonyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate) (44a). Prepared using N-(3-
aminopropyl)-2-nitrobenzenesulfonamide, 2-(trimethylsilyl)ethyl 9-
hydroxynonylcarbamate, and monomethylation. Yield: 9.0 mg, 6.0%
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1
(77% per step over 11 steps). H NMR (600 MHz, CD3OD) δ 6.96
Oocyte Electrophysiology. Mature female Xenopus laevis (Nasco,
Modesto, CA) were anesthetized using 0.1% ethyl 3-aminobenzoate
and their ovaries surgically removed using approved animal protocols.
Ovarian tissue was dissected and treated with collagenase (1 mg/mL)
in Ca2+-free Barth’s medium (in mM: 82.5 NaCl, 2 KCl, 5 HEPES,
0.82 MgCl2) for 1−2 h at rt. For expression of recombinant AMPA
and NMDA receptors, ocytes were injected with 25 nL of cRNA (1
ng/nL for GluA1i or 0.5 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 2−4
days postinjection and were voltage clamped with the use of a OC-
725C two-electrode voltage clamp amplifier (Warner Instruments,
Hamden, CT) 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 BaCl2, 5 HEPES; pH 7.6). Test compounds were dissolved in frog
Ringer’s solution and added by bath application. Recordings were
made at rt at a holding potential of −80 mV. Antagonist
concentration−inhibition 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 GluN1/2A) and then applying increasing concentrations of
antagonist in the presence of the appropriate agonist. Composite
concentration−inhibition data from experiments at 3−8 individual
oocytes were plotted using GraphPad Prism software (GraphPad
Software, Inc., San Diego, CA) and fitted by an iterative least-squares
routine 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 maximum current, Imin is the minimum
current, [antagonist] is the concentration of antagonist, and nH is the
Hill slope. The IC50 is the concentration of antagonist producing 50%
(d, J = 8.1 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 6.30 (dd, J = 8.1, 2.2 Hz,
1H), 4.69 (t, J = 6.2 Hz, 1H), 3.91 (t, J = 6.8 Hz, 1H), 3.52−3.41 (m,
3H), 3.31−3.05 (9H), 2.86 (s, 3H), 2.73−2.68 (m, 2H), 2.03−1.86
(m, 4H), 1.76−1.66 (m, 4H), 1.51−1.45 (m, 2H), 1.41−1.25 (m,
10H). 13C NMR (150 MHz, CD3OD) δ 173.7, 171.7, 169.1, 161.8,
161.6, 157.7, 157.3, 155.9, 131.4, 117.8, 115.8, 112.8, 106.7, 102.5,
53.5, 52.7, 50.3, 40.3, 39.0, 37.4, 36.1, 28.7, 28.6, 28.5, 28.3, 26.0, 25.9,
24.1, 23.6. HRMS (EI) exact mass calculated for C31H56N9O6 [MH+]
650.4354; found 650.4337. Purity (ELSD): 99%.
N-(9-((S)-4-Amino-2-(2-(2,4-dihydroxyphenyl)acetamido)-4-oxo-
butanamido)-N-(3-((S)-2-amino-5-guanidinopentanamido)propyl)-
N,N-dimethylnonan-1-aminium Tris(2,2,2-trifluoroacetate) (44b).
Prepared using N-(3-aminopropyl)-2-nitrobenzenesulfonamide, 2-
(trimethylsilyl)ethyl 9-hydroxynonylcarbamate, and dimethylation.
1
Yield: 7.4 mg, 4.9% (76% per step over 11 steps). H NMR (600
MHz, CD3OD) δ 6.96 (d, J = 8.1 Hz, 1H), 6.36 (d, J = 2.6 Hz, 1H),
6.30 (dd, J = 8.1, 2.6 Hz, 1H), 4.68 (t, J = 6.2 Hz, 1H), 3.93 (t, J = 6.6
Hz, 1H), 3.53−3.42 (m, 3H), 3.38−3.34 (m, 2H), 3.31−3.24 (m, 6H),
3.14−3.10 (m, 1H), 3.09 (s, 6H), 2.70 (d, J = 6.6 Hz, 2H), 2.05−1.86
(m, 4H), 1.79−1.67 (m, 4H), 1.51−1.45 (m, 2H), 1.39−1.25 (m,
10H). 13C NMR (150 MHz, CD3OD) δ 173.7, 171.7, 169.0, 161.6,
157.7, 157.3, 155.9, 131.4, 117.8, 115.8, 112.9, 106.7, 102.5, 64.3, 61.6,
52.7, 50.4, 49.9, 40.4, 39.0, 37.4, 36.2, 36.1, 28.7, 28.4, 28.3, 26.0, 25.7,
+
24.1, 22.5, 22.0. HRMS (EI) exact mass calculated for C32H58N9O6
[M+] 664.4510; found 664.4498. Purity (ELSD): 99%.
(S)-N1-(4-((8-((S)-2-Amino-5-guanidinopentanamido)octyl)-
(methyl)amino)butyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate) (45a). Prepared using N-(8-
aminooctyl)-2-nitrobenzenesulfonamide, 2-(trimethylsilyl)ethyl 4-hy-
droxybutylcarbamate, and monomethylation. Yield: 8.8 mg, 5.9% (77%
per step over 11 steps). 1H NMR (600 MHz, CD3OD) δ 6.98 (d, J =
8.1 Hz, 1H), 6.37 (d, J = 2.2 Hz, 1H), 6.30 (dd, J = 8.3, 2.1 Hz, 1H),
4.60 (t, J = 6.1 Hz, 1H), 3.86 (t, J = 6.6 Hz, 1H), 3.53 (d, J = 15.0 Hz,
1H), 3.41 (d, J = 15.0 Hz, 1H), 3.28−3.22 (m, 5H), 3.12−2.91 (m,
4H), 2.79−2.69 (m, 5H), 1.95−1.85 (m, 2H), 1.73−1.51 (m, 10H),
1.44−1.36 (m, 8H). 13C NMR (150 MHz, CD3OD) δ 173.8, 173.6,
172.2, 168.3, 161.6, 161.4, 157.7, 157.3, 155.9, 131.7, 113.0, 106.7,
102.4, 55.4, 52.7, 50.7, 40.4, 39.3, 38.9, 37.3, 35.6, 28.8, 28.7, 28.4,
26.5, 26.1, 24.1. HRMS (EI) exact mass calculated for C31H56N9O6
[MH+] 650.4354; found 650.4338. Purity (ELSD): 99%.
of Imax
.
ASSOCIATED CONTENT
■
S
* Supporting Information
Preparation of building blocks 36, 48, and 54, as well as 1H and
13C NMR spectra of final compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
N-(4-((S)-4-Amino-2-(2-(2,4-dihydroxyphenyl)acetamido)-4-
oxobutanamido)butyl)-8-((S)-2-amino-5-guanidinopentanamido)-
N,N-dimethyloctan-1-aminium tris(2,2,2-trifluoroacetate) (45b).
Prepared using N-(8-aminooctyl)-2-nitrobenzenesulfonamide, 2-
(trimethylsilyl)ethyl 4-hydroxybutylcarbamate, and dimethylation.
■
Corresponding Author
*Phone: +45 3533 6114. Fax: +45 3533 6040. E-mail: kristian.
stromgaard@sund.ku.dk.
1
Yield: 16.8 mg, 11.1% (82% per step over 11 steps). H NMR (600
Present Address
†For N.G.N.: Nuevolution A/S, Rønnegade 8, DK-2100
Copenhagen, Denmark.
MHz, CD3OD) δ 6.98 (d, J = 8.4 Hz, 1H), 6.37 (d, J = 2.6 Hz, 1H),
6.31 (dd, J = 8.4, 2.6 Hz, 1H), 4.60 (t, J = 6.1 Hz, 1H), 3.87 (t, J = 6.6
Hz, 1H), 3.53 (d, J = 15.0 Hz, 1H), 3.41 (d, J = 15.0 Hz, 1H), 3.40−
3.35 (m, 1H), 3.28−3.23 (m, 5H), 3.19−3.14 (m, 4H), 2.93 (m, 6H),
2.77 (dd, J = 15.8, 7.0 Hz, 1H), 2.71 (dd, J = 15.8, 5.1 Hz, 1H), 1.96−
1.85 (m, 2H), 1.74−1.49 (m, 10H), 1.45−1.36 (m, 8H). 13C NMR
(150 MHz, CD3OD) δ 173.5, 172.3, 168.3, 157.7, 157.4, 131.8, 113.3,
106.7, 102.4, 64.2, 63.6, 52.7, 50.8, 49.7, 40.3, 39.3, 37.6, 35.6, 28.8,
28.7, 28.4, 26.5, 25.9, 25.6, 24.1, 22.1, 18.9. HRMS (EI) exact mass
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for support from the GluTarget (University of
Copenhagen) for a Ph.D. fellowship to N.G.N. and M.H.P.
+
calculated for C32H58N9O6 [M+] 664.4510; found 664.4515. Purity
(ELSD): 99%.
ABBREVIATIONS USED
Electrophysiology: In Vitro cRNA Transcription. cDNA
encoding rat GluA1i or GluN1/2A subunits inserted into the vectors
pGEM-HE or pCIneo, respectively, were used for preparation of
cRNA transcripts. Plasmid DNA were grown in Top10 Escherichia coli
bacteria (Invitrogen, Carlsbad, CA) and purified using the NucleoSpin
■
ADDP, 1,1-(azodicarbonyl)dipiperidine; Alloc, allyloxycarbon-
yl; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid; ArgTX, argiotoxin; DIPEA, diisopropylamine; EDT, 1,2-
ethanedithiol; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; mCPBA, m-chloro-
peroxybenzoic acid; MEM, 2-methoxyethoxymethyl; NMDA,
N-methyl-D-aspartate; Ns, 2-nitrobenzenesulfonamide; Pbf,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; TBAF,
tetra-n-butylammonium fluoride; TBDPS, tert-butyldiphenylsil-
Midi purification kit (Macherey-Nagel GmbH, Duren, Germany)
̈
according to the protocol supplied by the manufacturer and linearized
by restriction enzyme digestion. cRNA was synthesized from linearized
cDNAs by in vitro transcription using the mMESSAGE mMACHINE
T7 mRNA-capping kit (Ambion, Austin, TX) according to the
protocol supplied by the manufacturer.
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yl; TEA, triethylamine; Teoc, 2-(trimethylsilyl)-
ethyloxycarbonyl; TIPS, triisopropylsilane
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