Design, synthesis, and biological evaluation of a scaffold for iGluR ligands based on the structure of (-)-kaitocephalin

Article abstract of DOI:10.1016/j.bmcl.2008.11.004

The design and synthesis of four pyrrolidine scaffolds that are structurally related to the known ionotropic glutamate receptor antagonist, (-)-kaitocephalin, is described. Additionally, preliminary results of the biological evaluation of these compounds

Full text of DOI:10.1016/j.bmcl.2008.11.004

Bioorganic & Medicinal Chemistry Letters 19 (2009) 132–135
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of a scaffold for iGluR ligands based
on the structure of (ꢀ)-kaitocephalin
Rishi G. Vaswani a, Agenor Limon b, Jorge Mauricio Reyes-Ruiz b, Ricardo Miledi b, A. Richard Chamberlin a,c,
*
a Department of Chemistry, University of California at Irvine, Irvine, CA 92697, USA
b Department of Neurobiology and Behavior, University of California at Irvine, Irvine, CA 92697, USA
c Department of Pharmaceutical Sciences, University of California at Irvine, Irvine, CA 92697, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of four pyrrolidine scaffolds that are structurally related to the known ionotro-
pic glutamate receptor antagonist, (ꢀ)-kaitocephalin, is described. Additionally, preliminary results of the
biological evaluation of these compounds are disclosed.
Received 1 September 2008
Revised 31 October 2008
Accepted 3 November 2008
Available online 6 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Kaitocephalin
iGluR
Receptor antagonist
Excitatory amino acid receptor
AMPA receptor
KA receptor
Library scaffold
Small molecule libraries generated from scaffolds inspired by
natural products have proven to be a rich source of new ligands
for biological targets.1 Our interest in natural product based
libraries was motivated by our efforts to discover new ligands for
the ionotropic glutamate receptors (iGluRs).2 In addition to the
central role iGluRs play in normal neuronal activation, their func-
tion is counterbalanced by a number of pathological processes.3–5
substituted aryl groups are commonly employed for introducing
diversity elements in library synthesis, for example, via palla-
dium-mediated cross-coupling reactions between aryl halides
and boronates, we considered the 5-aryl pyrroline 2 as a possible
scaffold (Fig. 1) for library synthesis.
This structure retains some of the glutamate-like structural ele-
ments known to be required for activity,7 while the phenyl ring
would provide up to five positions for attaching the diversity ele-
ments when appropriately functionalized (halide, triflate, etc.) for
aryl cross-coupling. The fairly substantial structural difference
between 2 and 1 was, however, cause for some concern.
Excessive stimulation of the iGluRs by either L-glutamate (L-Glu)
itself or by any of a large number of iGluR agonists can trigger a
cascade of detrimental cellular events that ultimately lead to neu-
ronal injury or death. This abnormal response to iGluR activation,
known as excitotoxicity, is postulated to play a role in a variety
of neurodegenerative disorders including epilepsy,4,5 Parkinson’s
disease,4 and Alzheimer’s disease.4 It is therefore not surprising
that there is a good deal of interest in discovering new small mol-
ecules that modulate iGluR function.
As a result, before proceeding we decided to study the unsubsti-
tuted core structure 2, beginning with molecular modeling studies
OH
Cl
13
14
As one element of our search for iGluR antagonists, we envi-
sioned the construction of a library based on a scaffold that emu-
lates (ꢀ)-kaitocephalin (1),6 one of the few natural iGluR ligands
with antagonist activity. Stimulated by the current interest in
using specific substructures found within natural products as
library scaffolds, the C1–C7 segment of (ꢀ)-kaitocephalin (1) was
chosen as the template for library synthesis. Since appropriately
Cl
15
16
12
11
10
H3N
H3N
2
CO2
OH
O
CO2
OH
NH
17
HO2C
3
8
9
7
4 CO2
CO2
N
N
H2
H2
18
2
1
Parent Scaffold
* Corresponding author. Tel./fax: +1 949 824 6478.
E-mail address: archambe@uci.edu (A.R. Chamberlin).
Figure 1. (ꢀ)-Kaitocephalin (1) and targeted parent aryl-appended scaffold 2.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.004

R. G. Vaswani et al. / Bioorg. Med. Chem. Lett. 19 (2009) 132–135
133
to assess whether the proposed simplified scaffold might be a rea-
H3N
H3N
CO2
OH
CO2
OH
sonable ligand for the iGluRs. Although one might argue that dock-
ing the natural product should come first, we chose to begin with
the much simpler 2, more or less as a model for 1, which has many
more degrees of rotational freedom and is a more difficult model-
ing problem. A further complication is that it is not at all obvious—
in contrast to most glutamate-like iGluR ligands—how the highly
functionalized kaitocephalin core might be oriented in the binding
site. There clearly are two ‘embedded glutamates’ within this sub-
structure that might control the ligand orientation, that is, one in
N
N
CO2
CO2
H2
H2
2
3
Parent Scaffold
o
-Me-Scaffold
H3N
H3N
CO2
OH
CO2
OH
N
N
CO2
CO2
H2
which the C-2 nitrogen mimics the glutamate
a-amino group,
H2
and a very different arrangement in which the pyrrolidine ring
nitrogen does so (similar to kainate; see below). If that issue could
be resolved, molecular modeling of 2 might provide information
concerning which positions of the aryl ring might best tolerate
the presence of substituents, which would in turn be instructive
for our later library design. The parent scaffold 2 was therefore
docked into the glutamate binding cleft of Gouaux’s X-ray struc-
ture of the iGluR2-kainate construct (Fig. 2)8 in these two starting
orientations, and others. The resultant minimized structures indi-
cated that the complex in which the scaffold pyrrolidine ring is ori-
ented similarly to that of kainate produced the lowest energy
structures. In these, the phenyl ring was readily accommodated
in the binding site, with one of the ortho-positions directed to-
wards the mouth of the binding cleft and therefore likely to be
amenable to substitution (yellow arrow in Fig. 2). The other aryl
positions appear to be either more sterically encumbered or
buried.
The model shown in Figure 2 provided some reassurance that
structures such as 2 might act as acceptable surrogates for (ꢀ)-kai-
tocephalin (1) and, with suitable aryl substituents, as library scaf-
folds; further, it also suggested that the ortho-position of the aryl
ring would best tolerate substituents in such libraries. We should
emphasis that although this is a simplistic analysis it does provide
a testable hypothesis regarding where the diversity elements
should eventually be attached to the phenyl ring, or more directly
not be attached because of steric constraints; further modeling will
be required to identify potentially favorable binding interactions
between the substituents and residues in the binding site. As an
initial experimental assessment of this model, particularly the ste-
ric tolerances at the different positions of the aryl ring, three addi-
tional steric probes were envisioned in addition to the parent
scaffold (Fig. 3): the ortho-, meta-, and para-substituted tolyl deriv-
atives 3, 4, and 5, respectively, with the ortho-tolyl analog 3
predicted to be a better ligand than the meta- and para-counter-
parts, according to the model. To investigate these computational
4
5
m
-Me-Scaffold
p
-Me-Scaffold
Figure 3. Targeted analogs based on modeling predictions.
predictions, analogs 2, 3, 4, and 5 were synthesized and submitted
to biological evaluation.
The ultimate success of this library approach depends on the
development of an expedient, efficient, and scaleable synthetic
strategy to access the various aryl substituted scaffolds. The spe-
cific route chosen for preparing the parent library scaffold 2
(Scheme 1) evolved from the principles established in our recent
synthesis of (ꢀ)-kaitocephalin,9 in both cases utilizing primarily
substrate-controlled manipulations for the iterative installation
of the requisite stereocenters. We envisioned the construction of
an enantiomerically pure 2,5-disubstituted pyrrolidine core to
which the remaining carbon framework would be appended. A
key element of the plan was to exploit the inherent functionality
present in the synthetic intermediate to differentiate the multiple
functional groups, thereby avoiding extensive protecting group
manipulations.
To test the feasibility of this approach we constructed the par-
ent scaffold 2 commencing with the formation of the requisite phe-
nyl incorporated trans-2,5-disubstituted pyrrolidine ring. Of the
various established methods for the construction of 2,5-disubsti-
tuted pyrrolidine rings,10 we chose the addition of organocopper
reagents to the chiral N-acyliminium ion (derived from (R)-pyro-
glutamic acid). To implement this approach required the formation
of aminal 6, which was chemically derived in three steps from (R)-
pyroglutamic acid.9 Once aminal 6 was in hand, transmetalation of
phenyl Grignard with a suspension of CuBrꢁDMS in Et2O, formed
the desired organocopper species. Subsequent addition of BF3ꢁOEt2
and the N-acyliminium precursor 6 produced 7 as a 20:1 mixture
of diastereomers (trans:cis) in 95% yield.11
Further functionalization to give the desired pyrrolidine substi-
tution pattern next required the stereoselective introduction of the
tetrasubstituted C4, followed by formation the C3 secondary alco-
hol. The formation of the tetrasubstituted center was established
with excellent diastereoselectivity via a Claisen condensation of
the lithium enolate derived from 7 and N-acylimidazole 8,9 pro-
ducing the b-keto ester 9 in 48% yield as a single detectable diaste-
reomer. After an exhaustive investigation of reducing agents the
diastereoselective reduction of the ketone 9 was achieved by
employing DIBALH in THF to afford alcohol 10 as a single diastereo-
mer in 86% yield, again with no evidence of the undesired
diastereomer.
Hydrolysis of the N,O-acetonide was accomplished next with
80% aqueous AcOH and subsequent chemoselective TEMPO-cata-
lyzed oxidation of the resultant primary alcohol furnished carbox-
ylic acid 11 in 97% yield.12 Deprotection of the methyl ester was
effected with LiI13 in hot EtOAc and treatment with TFA in CH2Cl2
removed the Boc carbamates; reverse phase HPLC purification of
the crude reaction mixture furnished 2 as the bis-trifluoroacetate
ammonium salt in 53% yield.14 The o-tolyl, m-tolyl, and p-tolyl
scaffolds, 3,15 4,16 and 5,17 respectively, were synthesized in an
Figure 2. Docking of parent scaffold 2 into Gouaux’s iGluR2 construct. See text for
details.

134
R. G. Vaswani et al. / Bioorg. Med. Chem. Lett. 19 (2009) 132–135
Boc
N
O
O
O
N
N
i. Ph–MgBr, THF
N
Boc
N
ii. CuBr·DMS, Et2O
O
8
–45 ºC
DiBAL-H
CO2Me
CO2Me
MeO
Ph
Ph
N
iii. BF3·OEt2, Et2O
–78 ºC to 0 ºC
N
LDA (1.3 equiv)
THF, –78 ºC
CO2Me
THF, –78 ºC
Boc
Boc
Boc
86%
95%
48% isolated
81% brsm
6
7
9
d.r. (trans:cis) = 20:1
Single Diastereomer
O
H
N
N
H3N
CO2H
Boc
CO2
OH
Boc
i. 80% AcOH (aq)
i. LiI, EtOAc
OH
CO2Me
OH
Ph
Ph
Ph
ii. TEMPO (cat), NaOCl (cat)
NaClO2 (2.5 equiv)
CH3CN/pH 6.0 buffer, 60 ºC
N
N
Boc
N
ii. TFA, CH2Cl2
CO2
CO2Me
H2
Boc
53%
11
2
10
97%
Parent Scaffold
Single Diastereomer
(20% overall yield)
H3N
H3N
H3N
CO2
OH
CO2
OH
CO2
OH
N
N
N
CO2
CO2
CO2
H2
H2
H2
3
4
5
o
-Me-Scaffold
m
-Me-Scaffold
p
-Me-Scaffold
(10% overall yield)
(9% overall yield)
(20% overall yield)
Scheme 1. Synthesis of parent scaffold 2 and the ortho-, meta-, and para-tolyl analogs 3, 4, and 5, respectively.
analogous sequence by substituting the appropriate tolyl-Grignard
for the organocuprate reaction in the initial arylation of the
N-acyliminium precursor 6. This sequence should provide an expe-
dient and efficient method for the eventual preparation of various
substituted aryl scaffolds for library synthesis, and in the current
application provided sufficient quantities of each of the four ana-
logs for biological evaluation.
The activities of the parent compound 2 and its methyl analogs
(3, 4, and 5) were screened in oocytes injected with mRNA from rat
cerebral cortex.18 Potential AMPA/KA receptor antagonist activity
was assayed based on the membrane current inhibition at a con-
100
90
80
70
60
50
40
30
20
10
0
centration of 100 lM for each analog against 100 lM of kainate
as the control. Experiments were quite reproducible in multiple
oocyte preparations (N = 7), generally with a standard error of a
few percent.
2
3
4
5
Analogues (100 μm)
In the antagonist activity assay (Fig. 4), the parent phenyl scaf-
fold 2 proved to be a reasonably potent antagonist that reduces
the kainate control current by approximately 28% (to 72% of con-
Figure 4. Antagonist activity assay of compounds 2, 3, 4, and 5, respectively. See
text for conditions.
trol) at a concentration of 100
methyl substituted phenyl scaffolds, 3, 4, and 5, respectively, also
showed activity in antagonizing 100 M kainate currents. Signifi-
lM. The ortho-, meta-, and para-
It was gratifying that initial screening of the analogs 2, 3, 4, and
5 revealed a reasonable level of antagonist activity (Fig. 4) for each,
with the most active of the group, 3, exhibiting a potency compa-
rable to kainate itself (albeit as an antagonist, in contrast to the
agonist activity of kainate). In addition, the relative potencies of
the four compounds are consistent with the modeling predictions
suggesting that the ortho-position of the aryl ring should tolerate
additional steric bulk well and thus may be the preferred point
of attachment of diversity elements in future libraries, while ana-
logs with substituents in the meta- and the para-positions may
be less potent because of greater respective steric constraints in
the binding site. Although the results obtained from the initial
l
cantly, from the perspective of our binding model (Fig. 2), the
ortho-methyl substituted phenyl scaffold 3 was more potent than
either of the other methyl substituted analogs: the meta- and
para-methyl substituted phenyl scaffolds, 4 and 5 reduced kainate
control current by approximately 25% (75 5% of control) and
17% (83 6% of control), respectively, while the ortho-methyl
substituted phenyl scaffold 3 reduced kainate current by half
(49 6% of control). None of the analogs showed agonist activity
on AMPA/KA receptors when tested alone at concentrations of
100 lM (N = 5).

R. G. Vaswani et al. / Bioorg. Med. Chem. Lett. 19 (2009) 132–135
135
Discover _3 module on Insight II version 2002. Dynamics were carried out at
300 K. Only the residues of the S1S2 construct within 7.0 Å of the docked ligand
were allowed to relax during the minimization and dynamics simulation.
9. Vaswani, R. G.; Chamberlin, A. R. J. Org. Chem. 2008, 73, 1661.
screen are far from comprehensive, they provide considerable
additional motivation for the development of these kaitocepha-
lin-inspired scaffolds as novel iGluR antagonists.
10. Pichon, M.; Figadere, B. Tetrahedron: Asymmetry 1996, 7, 927.
11. (a) Wistrand, L. G.; Skrinjar, M. Tetrahedron 1991, 47, 573; (b) Collado, I.;
Ezquerra, J.; Pedregal, C. J. Org. Chem. 1995, 60, 5011; (c) Thaning, M.; Wistrand,
L. G. Acta Chem. Scand. 1992, 46, 194; (d) Ludwig, C.; Wistrand, L. G. Acta Chem.
Scand. 1994, 48, 367; (e) Ludwig, C.; Wistrand, L. G. Acta Chem. Scand. 1990, 44,
707; (f) Skrinjar, M.; Wistrand, L. G. Tetrahedron Lett. 1990, 31, 1775.
12. Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tschaen, D. M.; Grabowski, E. J. J.; Reider, P. J.
J. Org. Chem. 1999, 64, 2564.
Acknowledgments
We are grateful for support from the National Institute of Neu-
rological Disorders and Stroke (NS-27600 to A.R.C.) and from the
American Health Assistance Foundation (A2006-054 to R.M.).
13. Fisher, J. W.; Trinkle, K. L. Tetrahedron Lett. 1994, 35, 2505.
14. Characterization for 2: 1H NMR (500 MHz, D2O/CH3CN (10:1), 315 K) d 7.79–
7.72 (m, 5H), 5.01 (dd, J = 12.1, 5.6, 1H), 4.85 (br s, 1H), 4.68 (br s, 1H), 2.85
(dd, J = 12.1. 5.7, 1H), 2.69–2.54 (m, 1H and dd, J = 13.2, 5.9), 2.45 (qd, J = 12.2,
6.2); 13C NMR (100 MHz, D2O, uncalibrated) d 173.7, 170.6, 134.4, 130.0,
129.6, 128.0, 76.7, 70.5, 64.5, 55.7, 33.0, 30.3; HRMS (ESI/methanol) m/z Calcd
References and notes
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8. Molecular modeling was performed in collaboration with Dr. Xiaohong Shou.
Molecular dynamics and minimizations of the ligand/iGluR2S1S2 construct
complex were carried out on a Silicon Graphics Octane 2 computer using the
for C14H18N2O5 (M+Na)+ 317.1113. Found 317.1104;
H2O).
½ ꢂ ꢀ18.5° (c 0.11,
a 2D5
15. Characterization for 3: 1H NMR (500 MHz, D2O) d 7.58–7.54 (m, 1H), 7.37–7.31
(m, 3H), 5.03 (dd, J = 12.2, 5.5, 1H), 4.62 (s, 1H), 4.48 (s, 1H), 2.60 (dd, J = 12.4,
5.9, 1H), 2.42 (s, 3H), 2.38–2.31 (m, 2H), 2.28–2.19 (m, 1H); 13C NMR (125 MHz,
D2O, uncalibrated) d 173.8, 170.6, 137.8, 132.4, 131.0, 129.6, 127.0, 126.3, 76.7,
70.4, 59.9, 55.5, 32.8, 29.4, 18.6; HRMS (ESI/methanol) m/z Calcd for
C15H20N2O5 (M+Na)+ 331.1270. Found 331.1265; ½a 2D5
ꢀ42.3° (c 0.11, H2O).
ꢂ
16. Characterization for 4: 1H NMR (500 MHz, D2O) d 7.39–7.35 (m, 2H), 7.31–7.29
(m, 2H), 4.71 (dd, J = 12.5, 5.3, 1H), 4.64 (s, 1H), 4.47 (s, 1H), 2.62–2.58 (m, 1H),
2.40–2.32 (m, 2H and s, 3H), 2.21–2.14 (m, 1H); 13C NMR (125 MHz, D2O,
uncalibrated) d 173.4, 170.3, 139.7, 134.1, 130.5, 129.33, 128.3, 124.7, 76.3,
70.2, 64.3, 55.4, 32.8, 30.1, 20.4; HRMS (ESI/methanol) m/z Calcd for
C15H20N2O5 (M+H)+ 309.1451. Found 309.1450; ½a 2D5
ꢀ24.2° (c 0.10, H2O).
ꢂ
17. Characterization for 5: 1H NMR (500 MHz, D2O) d 7.42 (app d, J = 8.2, 2H), 7.32
(app d, J = 7.9, 2H), 4.73 (dd, J = 12.1, 5.4, 1H), 4.60 (d, J = 0.85, 1H), 4.51 (s, 1H),
2.60–2.56 (m, 1H), 2.40–2.30 (m, 2H and s, 3H), 2.21–2.13 (m, 1H); 13C NMR
(125 MHz, D2O, uncalibrated) d 173.7, 170.5, 140.3, 131.2, 129.9, 127.7, 76.4,
70.4, 64.1, 55.5, 32.8, 30.1, 20.3; HRMS (ESI/methanol) m/z calcd for
C15H20N2O5 (M+H)+ 309.1451. Found 309.1445; ½a 2D5
ꢀ23.8° (c 0.12, H2O).
ꢂ
18. Gundersen, C. B.; Miledi, R.; Parker, I. Proc. R. Soc. B., Biol. Sci. 1984, 221, 127.