General synthesis of β-alanine-containing spider polyamine toxins and discovery of Nephila polyamine toxins 1 and 8 as highly potent inhibitors of ionotropic glutamate receptors

Article abstract of DOI:10.1021/jm301255m

Certain spiders contain large pools of polyamine toxins, which are putative pharmacological tools awaiting further discovery. Here we present a general synthesis strategy for this class of toxins and prepare five structurally varied polyamine toxins. Elec

Full text of DOI:10.1021/jm301255m

Brief Article
pubs.acs.org/jmc
General Synthesis of β‑Alanine-Containing Spider Polyamine Toxins
and Discovery of Nephila Polyamine Toxins 1 and 8 as Highly Potent
Inhibitors of Ionotropic Glutamate Receptors
Simon Lucas,†,‡ Mette H. Poulsen,† Niels G. Nørager, Anne F. Barslund, Tinna B. Bach,
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: Certain spiders contain large pools of polyamine toxins, which are putative pharmacological tools awaiting further
discovery. Here we present a general synthesis strategy for this class of toxins and prepare five structurally varied polyamine
toxins. Electrophysiological testing at three ionotropic glutamate receptor subtypes reveals that two of these, Nephila polyamine
toxins 1 (NPTX-1) and 8 (NPTX-8), comprise intriguing pharmacological activities by having subnanomolar IC50 values at
kainate receptors.
INTRODUCTION
■
The 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. 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.1−3 In particular, inhibition of iGlu receptors is a
promising strategy for the treatment of a wide range of diseases
such as pain, stroke, and Alzheimer’s disease.1,4,5 Today one
drug targeting iGlu receptors has been approved, memantine,
an open-channel blocker of the N-methyl-D-aspartate (NMDA)
subtype of iGlu receptors, used in the treatment of Alzheimer’s
disease.6,7
Polyamine toxins are a group of small molecule natural
products found in spiders and wasps that like memantine are
open-channel blockers of iGlu receptors.8−10 Polyamine toxins
have found valuable use as pharmacological tools to explore the
neurophysiological role of iGlu receptors based on their high
affinity and selectivity for iGlu receptors8−10 and their ability to
discriminate Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA) and kainate iGlu receptor
subtypes.11 Polyamine toxins from spiders are biosynthesized in
a combinatorial manner, providing a cocktail of structurally
related compounds (Figure 1), most of which remain to be
explored biologically.10,12,13 Thus, spider polyamine toxins have
the general structure as depicted for the prototypical polyamine
toxin argiotoxin-636 (ArgTX-636, 1, Figure 1), composed of an
aromatic headgroup, an optional amino acid linkage, a
polyamine backbone, and an optional amino acid tail.10,12,13
We have recently developed a synthetic methodology for the
synthesis of 1 and analogues using solid-phase synthesis.14 The
procedure, however, does not provide direct access to the large
group of polyamine toxins, such as Joro spider toxin 3 (JSTX-3,
2), which contains a β-alanine moiety in the polyamine region.
JSTX-3 is among the most studied and employed spider toxins,
as it displays potent inhibitory activity particularly at AMPA
Figure 1. General structures of polyamine toxins found in spiders,
illustrated with ArgTX-636 (1) and indicating the four variable
regions. JSTX-3 (2), NSTX-3 (3), clavamine (4), NPTX-8 (5), and
NPTX-1 (6) are structurally related toxins, having a β-alanine moiety
in the polyamine backbone (indicated in JSTX-3) and variations in
three of the four regions of the general structure.
receptors and in contrast to 1 displays only limited activity at
NMDA receptors.10 However, despite these intriguing bio-
logical properties, the challenges of preparing 2 and analogues
have prevented systematic structure−activity relationship
(SAR) studies to improve pharmacological properties. We
therefore decided to explore a revised strategy for the synthesis
Received: August 31, 2012
Published: October 23, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthesis of JSTX-3 (2)
a
Reagents and conditions: (a) Fmoc-Nγ-Trt-L-asparagine, HATU, DIPEA; (b) 20% piperidine in DMF; (c) 2-(2,4-bis(benzyloxy)phenyl)acetic acid,
HATU, DIPEA; (d) TBAF, 55 °C; (e) 3-(2-nitrophenylsulfonamido)propanoic acid, HATU, collidine; (f) 2-(trimethylsilyl)ethyl 4-
hydroxybutylcarbamate, Bu3P, ADDP; (g) 2-nitrobenzenesulfonyl chloride, collidine; (h) 2-(trimethylsilyl)ethyl 3-hydroxypropylcarbamate, Bu3P,
ADDP; (i) DBU, 2-mercaptoethanol; (j) TFA/DCM/TIPS/H2O (75:20:2.5:2.5); (k) H2, Pd(OH)2/C.
of a range of “β-alanine-containing” polyamine spider toxins
(Figure 1).
The synthesis of 2 was thus achieved using the revised
procedures, performing the 13-step synthesis on solid-phase
and deprotection of the benzyl groups in solution (Scheme 1).
Purification was easily achieved by preparative HPLC,
providing 2 in >97% purity and a 3% overall yield,
corresponding to 77% per step. We then selected representative
polyamine toxins Nephila spider toxin 3 (NSTX-3, 3),
clavamine (4), Nephila polyamine toxin-8 (NPTX-8, 5), and
Nephila polyamine toxin-1 (NPTX-1, 6), which are all isolated
from the spider Nephila clavata and contain a β-alanine moiety
in the polyamine moiety but represent structurally different
classes, with variations in three of the four regions of the
general polyamine toxin structure (Figure 1). In 3 and 4 the
headgroup and linker amino acid are similar to those in 2,
whereas the polyamine is modified, including addition of amino
acid tails. On the other hand, 5 and 6 contain the same
polyamine moiety as 2 but have different headgroups. Some of
these toxins have been synthesized before by solution phase
methods,15−20 but except for 2, none of them have previously
been biologically evaluated. The synthesis of these four
polyamine toxins was readily achieved by the general synthetic
methodology, using appropriate building blocks, providing the
target compounds 3−6 in high purity and good to excellent
overall yield (see Supporting Information).
CHEMISTRY
■
The methodology developed for the synthesis of 1 was used as
a starting point, i.e., anchoring a monoprotected diamine at a
backbone amide linker (BAL) 7. The loading of monoprotected
diamine was improved using NaBH(OAc)3 in DMF/AcOH
(9:1) instead of NaCNBH3 in DMF/AcOH (99:1). Next the
linker amino acid was coupled, where HATU and DIPEA in
CH2Cl2/DMF (9:1) were used in the coupling of resin-bound
secondary amine to the linker amino acid (Asn) to provide 8,
and subsequently the aromatic acyl group and deprotection of
the primary amine furnished 9. Next, we explored the key step
for introducing the 3-aminopropanoic acid moiety for the
synthesis of 2 using standard coupling reagents, a seemingly
straightforward operation. However, the desired product
underwent fast elimination and the acrylamide side product
was obtained as a major component (see Supporting
Information, Scheme S1). We then systematically evaluated
bases, solvents, and reaction times, which showed that the
combined use of HATU and collidine in DMF for 30 min
provided the desired intermediate 10 in >80% purity and good
yield. In subsequent steps, the polyamine moiety of JSTX-3 was
gradually built up, providing 11 and finally the fully protected
and resin-bound version of JSTX-3 (12). This compound was
cleaved from the resin, with concomitant deprotection of most
protecting groups providing dibenzyl JSTX-3. The final
solution-phase debenzylation step was improved by using
Pd(OH)2/C (Pearlman’s catalyst) and glacial acetic acid
instead of Pd/C and MeOH.14
RESULTS AND DISCUSSION
■
The potency of the five toxins 2−6 to block representative
members of the major iGlu receptor subfamilies from rat was
evaluated using two-electrode voltage-clamp electrophysiology
on Xenopus oocytes expressing the NMDA receptor subtype
GluN1/2A, the AMPA receptor subtype GluA1, and the kainate
receptor subtype GluK1. The concentration−inhibition rela-
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Journal of Medicinal Chemistry
Brief Article
Table 1. Inhibitory Potency of 2−6 at Recombinant iGlu Receptors
a
IC50 (nM)
GluA1
compd
GluN1/2A
GluK1
b
b
c
ArgTX-636 (1)
JSTX-3 (2)
177 [153−205]
135 [121−151]
65 [63−68]
137 [129−145]
NT
19 [19−20]
1710 [1287−2271]
5406 [5275−5540]
>10000
NSTX-3 (3)
clavamine (4)
NPTX-8 (5)
NPTX-1 (6)
164 [157−171]
2575 [2500−2653]
0.82 [0.67−0.99]
1078 [959−1211]
27 [23−32]
150 [144−154]
1390 [1210−1610]
51 [45−57]
0.33 [0.30−0.36]
a
b
Mean IC50 values (95% confidence interval in brackets) determined from 6 to 12 experiments at a membrane potential of −60 mV. Data from
Nelson et al.14 NT: not tested.
c
tionship was determined at these receptor subtypes at a
membrane potential of −60 mV (Table 1) and −80 mV
(Figure 2). Initially, we verified that 2 is a very potent AMPA
range with 0.82 and 0.33 nM for 5 and 6, respectively. We
explored the voltage dependency of inhibitory activity of 5 and
6 by determining IC50 at membrane potential of −80 mV along
with 2 (see Supporting Information, Table S1). A hallmark of
polyamine channel blockers is that they show increased potency
at lower membrane potential, and according to this, we
generally observed increases in potency at all receptor subtypes
when lowering the membrane potential from −60 to −80 mV.
5 and 6 showed IC50 values of 0.28 and 0.19 nM, respectively,
at GluK1 receptors, with similar relative increase in potency at
NMDA and AMPA receptors. To the best of our knowledge
these toxins are thereby the most potent polyamine toxins at a
kainate receptor described to date and among the most potent
iGlu receptor ligands known. In addition, 6 shows a remarkable
selectivity for GluK1 receptors, with >40000-fold preference for
kainate over the GluN1/2A type NMDA receptors and >150-
fold preference over the GluA1 type AMPA receptors. The
GluK1 construct used here is a double cysteine mutant (GluK1-
Y506C-L768C) that prevents the receptor from desensitization
and allows robust measurement of GluK1 activity in oocytes.23
Thus, further studies on native kainate receptors are needed to
fully explore their true pharmacological potential.
Structurally, 5 and 6 are characterized by having a 2-(4-
hydroxy-1H-indol-3-yl)acetic acid or 2-(1H-indol-3-yl)acetic
acid headgroup, whereas 2 has a 2,4-dihydroxyphenylacetic acid
headgroup, while their amino acid linker and polyamine moiety
are identical. Thus, it is tempting to speculate that the indole
headgroup plays a key role for the striking potency at GluK1
receptors. In general, the pharmacology of open ion channel
blockers of kainate receptors is much less explored than for
AMPA and NMDA receptors, where numerous compounds
have been identified and subsequent SAR studies performed.1
In one case, however, derivatives of the wasp polyamine toxin
philanthotoxin-433 (PhTX-433) were demonstrated to be
potent inhibitors of homomeric GluK1.24 Thus, 5 and 6 are
promising starting points for studying this underexplored area
of kainate receptor pharmacology and are also obvious
candidates for future SAR studies.
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−D) Composite concentration−response curves for
clavamine (4) (B), NPTX-1 (6) (C), and NPTX-8 (5) (D) at
GluN1/2A ( ), GluA1 ( ), and GluK1 ( ) receptors at membrane
potentials of −80 mV. Error bars are SEM and are shown when larger
than symbol size.
■
○
▲
receptor antagonist with an IC50 of 65 nM (Table 1).21,22
However, when tested at GluK1 containing receptors, 2 was
even more potent with an IC50 of 19 nM, while potency at
NMDA receptors was significantly less (IC50 = 1.7 μM, Table
1). In contrast, 4 showed drastically reduced affinity for all
tested subtypes (IC50 > 1 μM; Table 1). 3, on the other hand,
showed reasonable potency at AMPA and kainate receptors
with IC50 values of 137 and 164 nM, respectively, but only very
modest activity at NMDA receptors (IC50 = 5.4 μM).
The most remarkable results came from studying the effect of
5 and 6 at AMPA and in particular at kainate receptors. Both
compounds were quite potent inhibitors of AMPA receptors,
with IC50 values of 27 and 51 nM for 5 and 6, respectively, thus
being equally or slightly more potent than 2 (Table 1).
However, at kainate receptors, 5 and 6 demonstrated
unprecedented potency and had IC50 values in the picomolar
CONCLUSION
■
We have developed a general and efficient method for the
synthesis of β-alanine-containing spider polyamine toxins. The
versatility was demonstrated by the synthesis of five
representative toxins, and biological evaluation of these toxins
led to the discovery of 5 and 6 as exceptionally potent kainate
receptor antagonists, and 6 with a noticeable selectivity relative
to NMDA and AMPA receptors. Considering the importance
of the prototypical polyamine toxins as pharmacological tools,
we consider 5 and 6 as highly promising tools for such studies
and as attractive templates for future SAR studies.
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Journal of Medicinal Chemistry
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5.8 Hz, 1H), 7.04 (s, 1H), 6.96−6.87 (m, 2H), 6.41 (dd, J = 1.6, 6.7
Hz, 1H), 4.66 (t, J = 6.3 Hz, 1H), 3.28−2.96 (m, 14H), 2.88−2.62 (m,
4H), 2.56−2.48 (t, J = 5.5 Hz, 2H), 2.12−2.01 (m, 2H), 1.83−1.70
(m, 4H), 1.55−1.24 (m, 6H), 1.10 (q, J = 7.8 Hz, 2H). 13C NMR (75
MHz, CD3OD) δ 176.8, 173.9, 173.6, 172.0, 170.8, 150.0, 143.9, 124.0,
121.6, 120.1, 110.2, 105.9, 102.1, 51.0, 44.6, 44.0, 39.2, 39.0, 37.4, 36.9,
32.7, 30.4, 28.6, 28.5, 24.3, 23.8, 23.3, 23.0. HRMS (EI) exact mass
calcd for C29H49N8O5 [MH+] 589.3826; found 589.3837. Purity
(ELSD): 99%.
EXPERIMENTAL SECTION
■
General Synthesis Procedure. See Supporting Information.
(S)-N1-(5-(3-(4-(3-Aminopropylamino)butylamino)-
propanamido)pentyl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate) (JSTX-3, 2).18 Yield: 3.7
mg (2.6%). 1H NMR (300 MHz, CD3OD): δ 6.95 (d, J = 8.4 Hz, 1H),
6.34 (d, J = 2.4 Hz, 1H), 6.28 (dd, J = 8.1, 2.4 Hz, 1H), 4.66 (t, J = 6.4
Hz, 1H), 3.53 (d, J = 15.0 Hz, 1H), 3.41 (d, J = 14.7 Hz, 1H), 3.29−
3.17 (m, 4H), 3.17 −2.95 (m, 10H), 2.69 (d, J = 6.3 Hz, 2H), 2.66−
2.50 (m, 2H), 2.17−1.99 (m, 2H), 1.88−1.67 (m, 4H), 1.58−1.34 (m,
4H), 1.34−1.16 (m, 2H). 13C NMR (75 MHz, CD3OD) δ 175.2,
174.9, 173.2, 171.9, 159.1, 157.3, 132.9, 114.3, 108.1, 103.9, 52.1, 48.4,
48.0, 46.0, 45.1, 40.3, 40.1, 38.8, 38.0, 37.7, 31.8, 29.9, 25.6, 24.9, 24.5,
24.3. HRMS (EI) exact mass calcd for C27H48N7O6 [MH+] 566.3666;
found 566.3651. Purity (ELSD): 97%.
ASSOCIATED CONTENT
* Supporting Information
■
S
Information on chemistry and synthesis of 2-(4-(benzyloxy)-
1H-indol-3-yl)acetic acid, experimental procedures for biology,
Scheme S1, and a table. This material is available free of charge
via the Internet at http://pubs.acs.org.
(S)-N1-((R)-1,5-Diamino-1-imino-6,15-dioxo-2,7,12,16-tetraa-
zahenicosan-21-yl)-2-(2-(2,4-dihydroxyphenyl)acetamido)-
succinamide Tris(2,2,2-trifluoroacetate) (NSTX-3, 3).16 3 was
prepared in similar manner to JSTX-3. Instead of steps g and h,
acylation with Boc-L-Arg(Pbf)-OH is performed, similar to step b.
Yield: 9.1 mg (5.4%). 1H 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), 4.64
(t, J = 6.3 Hz, 1H), 3.86 (t, J = 6.3 Hz, 1H), 3.51 (d, J = 15.1 Hz, 1H),
3.41 (d, J = 15.1 Hz, 1H), 3.26−3.00 (m, 12H), 2.68 (d, J = 6.1 Hz,
2H), 2.61 (t, J = 6.3 Hz, 2H), 1.92−1.85 (m, 2H), 1.75−1.59 (m, 6H),
1.49−1.40 (m, 4H), 1.34−1.20 (m, 2H). 13C NMR (75 MHz,
CD3OD) δ 175.2, 175.0, 173.1, 171.9, 170.0, 159.1, 158.7, 157.3,
132.9, 114.3, 108.1, 103.9, 54.3, 52.1, 45.0, 41.9, 40.28, 40.25, 39.9,
38.9, 37.6, 31.8, 29.95, 29.88, 29.83, 27.4, 25.7, 24.9, 24.5. HRMS (EI)
exact mass calcd for C30H53N10O7 [MH+] 665.4099; found 665.4100.
Purity (ELSD): >99%.
AUTHOR INFORMATION
Corresponding Author
*Phone: +45 3533 6114. E-mail: kristian.stromgaard@sund.ku.
■
dk.
Present Address
‡Global Drug Discovery, Grunenthal GmbH, D-52078 Aachen,
̈
Germany.
Author Contributions
†These authors contributed equally.
Notes
(S)-N1-((6S,12S)-1,6-Diamino-1-imino-12-methyl-7,10,13,22-
tetraoxo-2,8,11,14,19,23-hexaazaoctacosan-28-yl)-2-(2-(2,4-
dihydroxyphenyl)acetamido)succinamide Tris(2,2,2-trifluoroa-
cetate) (Clavamine, 4).15 4 was prepared in similar manner to JSTX-
3. Instead of steps g and h, the following was performed first, acylation
with Fmoc-L-Ala-OH, like step b, then step c, and acylation with
Fmoc-L-Gly-OH like step b, then step c, and finally acylation with Boc-
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the Alfred Benzon Foundation for an
Investigator Fellowship to S.L. and to GluTarget for a Ph.D.
fellowship to M.H.P. Dr. Jared K. Nelson is thanked for initial
contributions to the project.
1
L-Arg(Pbf)-OH like step b. Yield: 21.6 mg (11.8%). H NMR (300
MHz, CD3OD) δ 6.92 (d, J = 8.3 Hz, 1H), 6.32 (d, J = 2.5 Hz, 1H),
6.25 (dd, J = 8.2, 2.1 Hz, 1H), 4.63 (t, J = 6.2 Hz, 1H), 4.29−4.19 (m,
1H), 4.04−3.88 (m, 3H), 3.49 (d, J = 15.0 Hz, 1H), 3.40 (d, J = 15.0
Hz, 1H), 3.26−3.16 (m, 7H), 3.14−2.95 (m, 5H), 2.67 (d, J = 6.1 Hz,
2H), 2.60 (t, J = 5.7 Hz, 2H), 2.00−1.85 (m, 2 H), 1.75−1.38 (m,
11H), 1.34 (d, J = 7.3 Hz, 3H), 1.25 (q, J = 7.5 Hz, 2H). 13C NMR (75
MHz, CD3OD) δ 175.2 (2C), 175.0, 173.1, 171.9, 170.9, 170.6, 159.0,
158.7, 157.3, 132.9, 114.3, 108.1, 103.9, 54.2, 52.1, 51.1, 45.1, 43.3,
41.9, 40.3 (2C), 39.5, 38.8, 37.6, 32.0, 29.9, 29.9, 29.8, 29.7, 27.5, 25.3,
24.9, 24.4, 18.3. HRMS (EI) exact mass calcd for C36H61N12O9 [MH+]
793.4685; found 793.4682. Purity (ELSD): >99%.
ABBREVIATIONS USED
■
ADDP, 1,1-(azodicarbonyl)dipiperidine; AMPA, α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DIPEA, diisopropylethyl-
amine; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetrame-
thyluronium hexafluorophosphate; JSTX, Joro spider toxin;
NMDA, N-methyl-D-aspartate; NPTX, Nephila polyamine
toxin; Ns, 2-nitrobenzenesulfonamide; NSTX, Nephila spider
toxin; Pbf, pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl;
TBAF, tetra-n-butylammonium fluoride; Teoc, 2-
(trimethylsilyl)ethyloxycarbonyl
(S)-2-(2-(1H-Indol-3-yl)acetamido)-N1 -(5-(3-(4-(3-
aminopropylamino)butylamino)propanamido)pentyl)-
succinamide Tetrakis(2,2,2-trifluoroacetate) (NPTX-8, 5).17
5
was prepared in similar manner to JSTX-3 using 2-(1H-indol-3-
yl)acetic acid in step c and without the final debenzylation step. Yield:
5.7 mg (3.3%). H NMR (300 MHz, CD3OD) δ 7.54 (d, J = 7.7 Hz,
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