Synthesis, receptor binding and activity of iso and azakainoids

Article abstract of DOI:10.1016/j.bmcl.2013.02.046

Two syntheses for the production of an unsubstituted azakainoid are described. The 1,3-dipolar cycloaddition of diazomethane with trans-dibenzyl glutaconate yields a 1-pyrazoline, which may be reduced directly to the pyrazolidine. An unexpected trans-cis

Full text of DOI:10.1016/j.bmcl.2013.02.046

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1949–1952
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Bioorganic & Medicinal Chemistry Letters
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Synthesis, receptor binding and activity of iso and azakainoids
Wentian Wang a, Dragan D. Simovic a, Mingping Di a, Lynne Fieber b, Kathleen S. Rein a,
⇑
a Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Str., Miami, FL 33199, USA
b Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker Cswy, Miami, FL 33149, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two syntheses for the production of an unsubstituted azakainoid are described. The 1,3-dipolar cycload-
dition of diazomethane with trans-dibenzyl glutaconate yields a 1-pyrazoline, which may be reduced
directly to the pyrazolidine. An unexpected trans-cis isomerization is observed during Hg/Al reduction
of the 1-pyrazoline N@N bond. Alternatively, when TMS diazomethane is used as the dipole, the resulting
2-pyrazoline obtained after desilylation may be reduced with NaCNBH3 to provide the trans azakainate
analog exclusively. The synthesis of an unsubstituted isokainoid via Michael addition is also described.
Glutamate receptor binding assays revealed that the azakaniod has a moderate affinity for unspecified
glutamate receptors. Membrane depolarization of Aplysia neurons upon application of the azakainoid
demonstrates that it is an ionotropic glutamate receptor agonist.
Received 4 January 2013
Revised 5 February 2013
Accepted 8 February 2013
Available online 16 February 2013
Keywords:
Kainoids
Synthesis
Glutamate receptor
Receptor binding
Ó 2013 Elsevier Ltd. All rights reserved.
L
-Glutamic acid is a major excitatory neurotransmitter within
the kainite receptor.9 The syntheses, but not the biological activity
of homokainoids was recently described.10 Currently there is no
information on the effect of replacing the C-5 methylene with
nitrogen (azakainoids) or translocation of nitrogen relative to the
ring substituents (isokainoids). Herein we report the first synthe-
ses of unsubstituted aza 3 and isokainoid 4 as well as preliminary
receptor binding and activity studies.
The 1,3-dipolar cycloaddition of diazoalkanes with alkenes
yields 1-pyrazolines (Scheme 1) which may serve as precursors
to azakainoids. Based on preferred Frontier Molecular Orbital
(FMO) interactions11 and the stereospecific suprafacial addition
to the dipolarophile, the pyrrolidine ring of the azakainoids could
be constructed with the correct relative configuration in a single
step. The substituents at the 4 and 5 positions of the pyrazoline
the mammalian central nervous system.1 Two major subgroups
of glutamate receptors are the metabotropic and ionotropic types.
Ionotropic receptors may be further divided into three subgroups
according to their most potent agonist and include the NMDA
(N-methyl-
methylisoxazole-4-propionic acid) and kainate (
receptors.2
-Kainic acid 1 is a naturally occurring non-proteino-
D
-aspartic acid), AMPA ((S)-
a
-amino-3-hydroxy-5-
a-kainic acid)
a
genic amino acid with neuroexcitatory activity towards the mam-
malian central nervous system.3 It acts as a conformationally
restricted analogue of
activates the ionotropic kainate and AMPA receptors with nanomo-
lar and micromolar affinities respectively. As such, -kainic acid
has been used extensively as a probe for neuropharmacological re-
search and kainic acid induced neurotoxicity as an experimental
model for neurodegeneration.4
L-glutamic acid. a-Kainic acid binds to and
a
ring may favor the cis relative configuration if
p interactions are
present in the transition state.12 Thus the 1,3-dipolar cycloaddition
of trans-diethyl glutaconate with diazoalkanes should provide the
appropriate 3,4-trans, 4,5-cis relative configurations in the cycload-
ducts (Scheme 2). The kainoids function as conformationally re-
stricted glutamate receptor agonists. We reasoned that NH could
serve as a bioisostere for the C-5 methylene. If this were the case,
aza analogs could serve as readily accessible functional analogs of
kainic acid. However, 1-pyrazolines tend to be unstable and can
either become oxidized to the pyrazoles or, especially if it results
in conjugation, will isomerize to the 2-pyrazolines (Scheme 1) with
the direction of double bond isomerization dependent on the sub-
stituents. Thus, at least one of the newly formed stereocenters may
be lost.
a
-Kainic acid is the parent of a larger class of synthetic and nat-
urally occurring glutamate receptor agonists known as the kai-
noids. All kainoids possess the pyrrolidine dicarboxylate scaffold
and vary in the substituent at C-4 (Fig. 1) and the configuration
of the three stereocenters. Structure–activity studies of the kai-
noids as well as crystal structures of the binding domain of the kai-
nite receptor in complex with various ligands, have demonstrated
the importance of the absolute configuration of the three stereo-
centers and unsaturation in the C-4 substituent.5–7 The 4-unsubsti-
tuted kainoid, trans-2-carboxy-3-pyrrolidine-3-acetic acid (CPAA)
2 is less potent than 1 and reportedly acts on NMDA receptor sub-
types.8 Substitution at the 5-position eliminates binding affinity for
We described the synthesis of 2-pyrazolines 5–7 by cycloaddi-
tion of trans-diethyl glutaconate with phenyldiazomethane and
diazomethane.13 1-Pyrazolines were not isolated but were
⇑
Corresponding author. Tel.: +1 305 348 6682; fax: +1 305 348 3772.
E-mail address: reink@fiu.edu (K.S. Rein).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.046

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W. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1949–1952
Figure 1. Representative kainoids: a-kainic acid 1, the unsubstituted kainoid CPAA
2, and corresponding azakainoid 3 and isokainoid 4.
Scheme 3. 2-Pyrazolines proved resistant to reduction (see text).
Scheme 4. Reagents and conditions: (a) CH2N2, Et2O, rt, 2 h; (b) Al/Hg, MeOH/ H2O,
60 °C; (c) CbzCl, CH2Cl2, NaHCO3 (aq), rt, overnight, 95% (three steps).
Scheme 1. 1, 3-Diploar cycloadditions with diazoalkanes produce 1-pyrazolines
which readily isomerize to 2-pyrazolines.
immediately isomerized to the 2-pyrazolines and protected as the
Cbz derivatives to prevent oxidation to the pyrazoles (Scheme 3).
The 2-pyrazolines proved to be extremely resistant to reduction
using hydride reducing agents (NaCNBH3, LiBH4, LiAlH4, BH3,
LiEt3BH, Et3SiH, and Bu3SiH), catalytic reduction (H2, Pd/C) or
SmI2. Only limited success was achieved with NaBH4 in refluxing
methanol. We therefore sought to reduce the 1-pyrazolines
in situ prior to isomerization which, we anticipated, would have
the added benefit of preserving the relative stereochemistry of
the 1-pyrazoline. Again, we found the 1-pyrazolines to be resistant
to a variety of reducing agents. However, the use of a mercury/alu-
minum almagam14 proved to be effective and provided the pro-
tected pyrazolidine 8 in 95% yields after quenching the reaction
with Cbz chloride (Scheme 4).
We were surprised to find that pyrazolidine 8 was isolated as a
50:50 mixture of cis and trans isomers. Our initial thought was that
double bond isomerization to the conjugated 2-pyrazoline had oc-
curred prior to the reduction. However, hydrogenolysis of the pro-
tected 2-pyrazoline 5, followed by treatment with the Hg/Al
almagam failed to produce any pyrazolidine (Scheme 5). The Hg/
Scheme 5. Reagents and conditions: (a) H2, Pd/C, EtOH (100%); (b) Al/Hg, MeOH/
H2O, 60 °C.
Al amalgam reduction is performed under slightly basic conditions
which apparently causes the isomerization of the C-3 stereocenter.
cis-8 and trans-8 were readily separated chromatographically.
The production of both cis and trans isomers of 8 prompted us to
seek an alternate route to the unsubstituted aza-kainoid precursor,
trans-8. Carreira and co-workers described 1,3-dipolar cycloaddi-
tions with TMS diazomethane as a key step in the synthesis of
aza-proline analogs.15–17 The initially formed 1-pyrazolines from
TMS diazomethane may yield three different types of products,
Scheme 6. 1,3-Dipolar cycloadditon with TMS diazomethane may yield three
Scheme 2. Proposed synthesis of aza-kainoids by reduction of 1-pyrazolines.
products.

W. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1949–1952
1951
arising from double bond isomerization in either direction or
desilylation (Scheme 6).18 Carreira reported that products arising
from desilylation could be formed exclusively under certain condi-
tions.19 Similarly, the 1,3-dipolar cycloaddition of TMS diazometh-
ane with trans-dibenzyl glutaconate, followed by desilylation could
provide trans-8 after reduction of the C@N double bond and Cbz
protection. While we previously had difficulties in reducing 2-pyr-
azolines (Scheme 3), we reasoned that the unconjugated C@N dou-
ble bond would be more readily reduced.
TMS diazomethane reacts with dibenzyl glutaconate which,
upon quenching with Cbz-Cl, yields a mixture of two 2-pyrazolines
9 and 11 (Scheme 7). Under the conditions employed, 10 was not
observed. After flash chromatography, the desilylated 2-pyrazoline
9 was obtained in 28% yield for the two steps. 2-Pyrazoline 9 was
reduced using NaCNBH4 followed by an additional Cbz protection
step to yield trans-8 in 58% yield for the two steps. A one step-4-
group-deprotection by hydrogenolysis in the presence of 5% palla-
dium on carbon in methanol provided a quantitative yield of 3.
We reasoned that the unsubstituted isokainoid 4, could serve as
a conformationally restricted glutamate agonist with selectivity
distinct from the kainoids. It was prepared in five steps as outlined
in Scheme 8. The reductive alkylation of b-alanine ethyl ester with
benzaldehyde provided N-benzyl-b-alanine ethyl ester. Alkylation
with ethyl-4-bromocrotonate provided the precursor 12, which
was cyclized via a Michael addition of the enolate affected by treat-
ment with LiHMDS to provide pyrrolidine 13. Acid catalyzed
hydrolysis of the ethyl ester followed by hydrogenolysis provided
the isokainoid, trans-4-(carboxymethyl)pyrrolidine-3-carboxylic
acid 4. NOESY correlations confirmed the anticipated trans relative
configuration of 4 (Fig. 2).
Scheme 8. Reagents and conditions: (a) BrCH2CH@CHCO2Et, K2CO3, CH3CN, rt,
overnight (61%); (b) LiHMDS, THF, À78 °C to rt 25 min (79%); (c) (i) 1 N HCl, reflux
overnight; (ii) H2 (50 psi), Pd/C, 26 h; (75%, two steps).
Figure 2. NOESY correlations for determination of relative configuration of
isokainoid 4.
Finally, the unsubstituted kainoid, trans-CPAA 2, was prepared
in racemic form (Scheme 9). The cyclization precursor 14 was pre-
pared as previously described20 and cyclized according to the pro-
cedure described by Karoyan et al.21 followed by hydrolysis of the
ester and nitrile groups, and hydrogenolysis provided cis-CPAA.
Heating cis-CPAA in water at 190 °C for 12 h provided a separable
80:20 mixture of cis and trans CPAA (2).21
Azakainoid 3, isokainoid 4 and trans-CPAA 2 were tested in
receptor binding assays in rat brain against [3H]NMDA, [3H]AMPA,
[3H]kainic acid and [3H]glutamic acid. With the single exception of
azakainoid 3, none of the compounds demonstrated affinity for
glutamate receptors. Azakainoid 3 competitively inhibited uniden-
tified [3H]
L-Glu binding in rat brain with an EC50 of 12 lM. (Fig. 3).
Agonist activity of 3 was tested in neurons of the marine neural
model organism, Aplysia californica. Figure 4 shows cell responses
in patch clamp recordings from buccal S cluster neurons dissoci-
Scheme 9. Reagents and conditions: (a) (i) LDA, THF, À20 °C to À100 °C; (ii) ZnBr2,
À100 °C to rt 2 h; (iii) CuCN/2LiCl, 0 °C, TsCN, rt overnight (33%); (b) (i) HCl 6 N,
reflux; (ii) H2/Pd then Dowex 50-X8 (45% yield); (c) H2O, 190 °C, 12 h; (79% after
purification as the Cbz derivative)
Scheme 7. Reagents and conditions: (a) TMSCH2N2, benzene:hexane, reflux, 8 h;
(b) CbzCl, CH2Cl2, NaHCO3 (aq), rt, overnight (36% 9 and 11, two steps); (c) NaCNBH3
(6 equiv), HOAc, rt, 5 h; (d) CbzCl, CH2Cl2, NaHCO3 (aq), rt, overnight, 58% (two
steps); (e) H2, Pd/C, MeOH (100%).
Figure 3. Competitive displacement receptor binding assay for 3. Error bars
represent the range of values from two trials.

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W. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1949–1952
receptor desensitization to agonist actions of 3 on this time scale
did not occur.
Azakainate and isokainate analogs 3 and 4 were synthesized in
order to test the hypothesis that the pyrazolidine and pyrrolidine
analogs of kainic acid could serve as glutamate receptor agonists.
Compound 3 exhibited an affinity for unspecified glutamate recep-
tors in rat brain and an excitatory effect on Aplysia neurons that
may result from its action on
the agonist actions of this kainate derivative are non-desensitizing,
much like the actions of
-glutamate on these cells.25 By analogy
L-glutamate receptors. Interestingly,
L
with the kainoids, it is highly likely that azakainoids which are
appropriately substituted at the C-4 position will be significantly
more potent glutamate agonists than 3. Alternate syntheses of
such compounds are currently being explored.
Acknowledgments
D.D.S. and M.D. are grateful for Dissertation Year Fellowships
from the University Graduate School. The authors acknowledge
the National Resource for Aplysia, NIH P40 OD010952-17 for ani-
mals and Stephen Carlson for assistance with electrophysiology.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.046.
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may be unique receptors.24 In Figure 4A, 3 applied repetitively at
2 s intervals evoked 8–10 mV depolarizations of the cell membrane
from its resting membrane potential of ꢀÀ37 mV. Figure 4B dem-
onstrates that an excitatory inward current underlies the depolar-
ization. The current showed no decrease in amplitude when it was
repetitively elicited by multiple applications of 3. Trains of P6 re-
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amplitude in seven experiments (data not shown), suggesting that
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