Rational design of a novel AMPA receptor modulator through a hybridization approach

Article abstract of DOI:10.1021/ml5004553

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a family of glutamate ion channels of considerable interest in excitatory neurotransmission and associated disease processes. Here, we demonstrate how exploitation of the availa

Full text of DOI:10.1021/ml5004553

Letter
pubs.acs.org/acsmedchemlett
Rational Design of a Novel AMPA Receptor Modulator through a
Hybridization Approach
Nicola Caldwell,†,§ Jonathan E. Harms,‡,§ Kathryn M. Partin,‡ and Craig Jamieson*,†
†Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, U.K.
‡Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1617, United States
S
* Supporting Information
ABSTRACT: The α-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid (AMPA) receptors are a family of glutamate
ion channels of considerable interest in excitatory neuro-
transmission and associated disease processes. Here, we
demonstrate how exploitation of the available X-ray crystal
structure of the receptor ligand binding domain enabled the
development of a new class of AMPA receptor positive allosteric
modulators (7) through hybridization of known ligands (5 and
6), leading to a novel chemotype with promising pharmaco-
logical properties.
KEYWORDS: AMPA receptor, structure-based drug design, hybridization, electrophysiology
Chart 1. Benzamide Derived AMPA Receptor Modulators
he α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic
Tacid (AMPA) receptors are a class of ligand gated ion
channels that are ubiquitously expressed in the central nervous
system (CNS). This subfamily of glutamate receptors is
considered to be responsible for the vast majority of fast
excitatory amino acid neurotransmissions in the CNS.1
A
postulated function of AMPA receptors is in facilitating the
process of synaptic plasticity and long-term potentiation
(LTP), a use dependent potentiation of synaptic efficacy
often considered to be responsible for encoding both learning
and memory. Various classes of AMPA receptor modulators
have demonstrated their potential in enhancing LTP and,
accordingly, are being considered as potential new treatments
for a raft of diseases in the neurosciences area, including
Alzheimer’s disease, schizophrenia, Parkinson’s disease, and
attention deficit hyperactivity disorder.2,3
Four distinct genes (GluA1 to 4) encode subunits of the
AMPA receptor, each consisting of four discernible domains: an
N-terminal region, an extracellular glutamate binding site
(ligand binding domain, LBD), a transmembrane region, and
a C-terminal domain.4 Considerable advances have been made
in solving the three-dimensional structure of the AMPA
receptor. X-ray structures have been reported of protein
complexes representing the LBD5 and more latterly the full-
length receptor.6
Although compounds such as 2 display promising activity in
behavioral paradigms related to cognition,11 relatively high
doses (35 mg/kg) are required, attributable in part to the low in
vitro potency of the compounds (e.g., EC50 = 150 μM in native
tissue preparations for 2).9 In general, the low potency
associated with this compound class can be rationalized
through consideration of the available X-ray crystal structure
of compound 312 (Figure 1).
The binding site is formed through association of two
distinct lobes (S1 and S2) held together by a peptide to give a
C2-symmetrical construct used for X-ray crystallography.5
Examination of the biostructural data indicates that 3 makes
no discernible interactions with the receptor itself and instead
forms a single hydrogen bond with a network of water
molecules. This limited interaction goes some way toward
accounting for the modest potency (37 μM) associated with
the compound and is believed to be conserved in the series. On
the basis of all the above, we reasoned that, by using compound
3 as a starting point, it would be possible to evolve a new lead
On the basis of the strong association with neurological
disorders, significant effort has been invested in the
identification of AMPA receptor positive modulators as
cognition enhancers.7 Pre-eminent among this class of small
molecules are benzamide derivatives such as aniracetam (1),8
the progenitor compound in this series, CX516 (2),9 and
CX614 (3),10 Chart 1.
Received: November 4, 2014
Accepted: February 11, 2015
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
potency compared to the progenitor benzamide series. The
basic design hypothesis is outlined in Chart 2.
Chart 2. Hybridization Strategy Adopted Combining Known
Classes of Modulators, 5 and 6
Figure 1. X-ray crystal structure of 3 in the ligand binding domain of
the GluA2 receptor with key interactions shown.
series by designing more optimal interactions with the receptor
through hybridization with an unrelated class of modulator.
We recently reported a class of thiophene-amide derived
compounds (4 and 5),13,14 which more fully occupy the
receptor binding site, establishing additional interactions with
the allosteric domain (Figure 2). The X-ray crystal structure of
The pyrazole fragment from amide 5 was chosen for
inclusion in the new template based on its highly conserved
binding mode as determined through previous biostructural
work,13,15 thus providing confidence of how it is likely to
interact with the receptor as part of a hybridized series.
From consideration of the benzamide portion, we elected to
base our hybrid compound on the tricyclic benzamide-derived
scaffold represented by 6 (Chart 2). This progenitor
compound, which is formally related to both 2 and 3, has
previously been reported as a modulator of the AMPA
receptor16 and was selected as the basis of this approach as it
offered greater synthetic tractability.
Docking of the proposed hybrid compound 7 using the
GOLD algorithm17 into the GluA2 LBD gave a favorable pose
(Figure 3), with similar hydrophobic interactions with the
Figure 2. X-ray structure of compounds 4 (green) and 5 (blue) in the
GluA2 LBD. Both compounds span the entire allosteric site, making
interactions with distal hydrophobic pockets.
these compounds shows hydrophobic interactions of the
pyrazole and benzothiophene rings with Pro and Leu residues,
respectively. In addition, a hydrogen bonding interaction can be
observed between the primary amide and a proximal Ser
residue. This results in this class of compound having
significantly improved potency with EC50 values in the low
micromolar range (e.g., EC50 for 5 = 0.40 μM), as well as
profound effects on desensitization and deactivation (vide
infra). In a related study,15 we have also demonstrated how the
pyrazole motif could be used in a hybridization approach,
combining features of unrelated classes of AMPA receptor
modulators to furnish a new class of ligand.
In this letter, we illustrate how this approach can be extended
to generate a novel series of AMPA receptor modulators
though combination of the benzamide and thiophene amide
derived systems. From consideration of the binding modes of
both the benzamide series and the more recent thiophene-
derived systems, we concluded that hybridizing elements of
both series could lead to a novel chemotype with enhanced
Figure 3. Docked pose of proposed benzamide hybrid compound 7 in
the GluA2 LBD.
allosteric domain being observed as for progenitor compounds,
4 and 5. An additional hydrophobic interaction was observed
with the trifluoromethyl group, providing confidence that
synthesis of related analogues would furnish new templates
capable of interacting with the receptor.
Given the high level of structural information available at the
outset of our synthesis campaign, we envisaged a focused
chemistry effort, targeting specific compounds in order to
ascertain the feasibility of our hybridization approach.
Accordingly, in addition to compound 7 we selected the
analogues shown in Chart 3 for preparation.
On the basis of the modeling data shown in Figure 3, a
methylene linker unit was considered to be essential in order to
achieve optimal interaction with the allosteric site.
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cholorination and alkylation to furnish the requisite pyrazole
derivative 7. The corresponding sulfonamide system 9 was
accessed in an analogous fashion, starting from the aryl sulfonyl
chloride building block 17.
Chart 3. Additional Hybrid Compounds Targeted for
Synthesis
Each of the analogues were then examined in an electro-
physiological assay in order to assess their efficacy as positive
allosteric modulators of the AMPA receptor. In this study, two
separate splice variants of the GluA2 receptor, termed flip and
flop were employed. These variations can lead to differences in
the kinetic and pharmacological properties of the channel and
particularly manifest in the allosteric site where an N754S
mutation occurs.7 On this basis we sought to explore the
pharmacology of our nascent compounds against both splice
variants.
AMPA receptor potentiation by an exogenous ligand is a
product of its impact on two processes: deactivation and
desensitization. Deactivation involves the channel being at rest
following dissociation of the endogenous ligand (glutamate),
whereas desensitization involves the closure of the channel with
glutamate still bound. For the AMPA receptors, these processes
occur on a very rapid time scale (1−2 and 10 ms, respectively)
and are pivotal in shaping the duration and amplitude of
response to glutamate at a synapse. Inhibition of either or both
of these responses will lead to potentiation of the receptor.
Accordingly, we sought to determine the effects of our novel
compounds against both desensitization and deactivation.
Compounds were tested at a single concentration of 30 μM,
with the highly potent analogue 5 (which was previously shown
The site contains a “saddle” region in the center of the C2-
symmetrical domain; therefore, a flexible linker was included in
the design of compound 7. The corresponding fused analogue
8 was targeted as a control, with this more rigid structure not
anticipated to be accommodated in the allosteric site. The
sulfonamide hybrid 9 was selected in order to probe spatial
requirements in the region of the hydrophobic pocket.
Additionally, previous work on benzamide related derivatives
had indicated that sulfonamide derivatives could be tolerated in
this region of the molecule,18 as well as other sulfonamide
derived compounds, which target hydrogen bond interactions
with Pro105.19,20
Target compounds 7−9 were prepared as shown in Scheme
1. Base-mediated amidation of the benzoate ester 1121 followed
by SNAr cyclization furnished tricylic intermediate 14. This
could be converted to target compound 8 in two steps (copper-
mediated arylation and subsequent reduction). Alternatively, 14
could be subjected to a carbonylation procedure with
Herrmann’s catalyst,22 followed by a sequence of reduction,
a
Scheme 1. Preparation of Target Compounds
a
Reagents and conditions: (a) (1) AcCl, MeOH, 0 °C; (2) 100 °C, 87%; (b) 10 mol % BEMP, MeCN, 25 °C, 92%; (c) NaH, DMF, 120 °C, 70%;
(d) trans-bis(acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II), [HP(tBu)3]BF4, DBU, Mo(CO)6, MeCN/MeOH, 120 °C (μW), 62%; (e)
LiAlH4, THF, 25 °C, 54%; (f) (1) SOCl2, CH2Cl2, 25 °C; (2) (3-(trifluoromethyl)-1H-pyrazol-4-yl)methanol, K2CO3, DMF, 60 °C, 26% (over 2
steps); (g) (1) 3-(trifluoromethyl)-1H-pyrazole-4-carbaldehyde, CuI, trans-1,2-diaminocyclohexane, K2CO3, 1,4-dioxane, 180 °C (μW); (2) NaBH4,
MeOH, 25 °C, 11% (over 2 steps); (h) Et3N, CH2Cl2, 25 °C, 98%; (i) as (c), 72%; (j) trans-bis(acetato)bis[o-(di-o-tolylphosphino)benzyl]-
dipalladium(II), [HP(tBu)3]BF4, DBU, Mo(CO)6, MeCN/MeOH, 110 °C, 87%; (k) as (e), 70%; (l) as (f), 22% (over 2 steps).
C
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to be effective against both processes and tested here at a
concentration of 100 μM14) used for comparison. Earlier efforts
from our laboratories23 have shown how comparing electro-
physiological profiles within a series can be used to determine
relative efficacy and affinity of individual exemplars using a
single test concentration.
even at the lower concentrations tested, but no longer showed
an effect on flip GluA2 (p = 0.64, Figure 4A). This flop isoform
specific effect of 7 was also observed for desensitization kinetics,
displaying a slowed onset of desensitization for flop receptors
(p < 0.001) not observed on flip isoforms (p = 0.77) or with
compound 5 (Figure 4B). Despite this isoform specificity
relating to onset of desensitization, compound 7 showed an
enhanced steady-state current on both flip (p < 0.001) and flop
(p < 0.001) receptors (Figure 4C). Our previous study23
suggests that slowing of desensitization together with robust
block of steady-state current is indicative of a compound with
high efficacy and high affinity. In this regard, compound 7
represents a significant advance on compound 5 (vide supra).
These apparently detached patterns of receptor kinetics were
further observed with the hybridized compound 9. This
compound showed no observable effect on deactivation kinetics
(flip, p = 0.64; flop. p = 0.46; Figure 4A) or steady-state current
(flip, p = 0.24; flop, p = 0.25; Figure 4C), but demonstrated a
most pronounced effect on desensitization kinetics specific to
the flop isoform (p < 0.001, Figure 4B), with a slight effect on
flip receptors (p = 0.05). Finally, there was no observed
modulation by compound 8 on either isoform for any tested
measure (deactivation, flip, p = 0.12, flop, p = 0.96;
desensitization, flip, p = 0.54, flop, p = 0.87; SS/Peak, flip, p
= 0.20, flop, p = 0.55).
GluA2 flip or flop receptor cDNA was transiently transfected
and expressed in HEK 293 cells. Outside-out membrane
patches containing homomeric GluA2 were pulled and
positioned within the control stream of a two-barrel flowpipe.
This stream was then rapidly switched to the adjacent
compound-containing stream for 1 or 500 ms to record
receptor deactivation or desensitization, respectively.
As demonstrated previously,14 compound 5 was effective at
modulating deactivation of both flip (p < 0.05) and flop (p <
0.05) GluA2 homomers (Figure 4A), one of a limited number
The electrophysiological data presented above indicates that
for meaningful activity at the GluA2 receptor an appropriate
spacer must be incorporated into the hybridized compounds
(cf. compounds 7 and 8). This observation is consistent with
our earlier design hypothesis informed through consideration of
the X-ray structure of the allosteric site. The new hybrid
compound 7 is able to robustly block the process of
desensitization against both flip and flop receptor isoforms,
showing encouraging levels of efficacy as an AMPA receptor
modulator and compares favorably with progenitor compounds
such as 2, 3, and 5.
The subtype preference observed with compound 7 for
slowing of on-set of desensitization and deactivation is not
readily explained through use of the available biostructural data
and attempts to generate biostructural data have so far not
proven to be fruitful. It can be remarked, however, that this
subtype preference has been observed for progenitor
compounds (e.g., 3) and so is likely to be a function of the
benzamide chemotype.10
Lastly, the sulfonamide analogue 9, despite slowing on-set of
desensitization, does not exhibit any measurable degree of
efficacy as a desensitization blocker as evidenced by its
negligible effect on the steady-state to peak ratio. This indicates
a strong preference for the benzamide moiety as a preferred
chemotype.
Figure 4. Electrophysiology data for tested compounds in both flip
and flop receptors showing (A) deactivation, (B) desensitization, and
(C) steady-state/peak ratios.
In summary, hybridization of two distinct lead series with
contrasting levels of affinity and efficacy has furnished a new
chemotype with a high amplitude of modulation of the AMPA
receptor. This new chemotype represented by compound 7
compares very favorably with highly potent modulators (e.g., 5)
identified previously in terms of tonic potency, affinity, and
efficacy. The use of biostructural information has greatly
enabled the design process and led to the expedient generation
of new tools with which to further interrogate the biology of
the AMPA receptor.
of compounds that demonstrates a modulatory effect on flip
deactivation. Similarly, though not affecting desensitization
kinetics (Figure 4B; flip, p = 0.82; flop, p = 0.85), compound 5
increased the steady-state current observed with receptor
desensitization on both isoforms (Figure 4C; flip and flop, p
< 0.001). On the basis of our recent analysis of decay current
produced by the onset of desensitization,23 behavior of this type
is indicative of a compound with low affinity and low efficacy.
By comparison, compound 7 was significantly more effective
than 5 at modulating deactivation of flop isoforms (p < 0.001),
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(12) Jin, R.; Clark, S.; Weeks, A. M.; Dudman, J. T.; Gouaux, E.;
Partin, K. M. Mechanism of positive allosteric modulators action on
AMPA receptors. J. Neurosci. 2005, 25, 9027−9036.
(13) Jamieson, C.; Campbell, R. A.; Martin, F.; Moir, E. M.; Morrow,
J. A.; Pantling, J.; Rankovic, Z.; Smith, L.; Cumming, I. A.; Gillen, K. J.;
Gillespie, J.; Kazemier, B.; Kiczun, M.; Lamont, Y.; Lyons, A. J.;
Maclean, J. K. F. A novel series of positive modulators of the AMPA
receptor: structure-based lead optimization. Bioorg. Med. Chem. Lett.
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(14) Harms, J. E.; Benveniste, M.; Maclean, J. K. F.; Partin, K. M.;
Jamieson, C. Functional analysis of a novel positive allosteric
modulator of AMPA receptors derived from a structure-based drug
design strategy. Neuropharmacology 2013, 64, 45−52.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures (synthesis and pharmacology) and
spectral data for all compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +44-141-548-4830. E-mail craig.jamieson@strath.ac.uk.
Author Contributions
§These authors contributed equally to this work. All authors
have given approval to the final version of the manuscript.
(15) Jamieson, C.; Maclean, J. K. F.; Brown, C. I.; Campbell, R. A.;
Gillen, K. J.; Gillespie, J.; Kazemier, B.; Kiczun, M.; Lamont, Y.; Lyons,
A. J.; Moir, E. M.; Morrow, J. A.; Pantling, J.; Rankovic, Z.; Smith, L.
Structure based evolution of a novel series of positive modulators of
the AMPA receptor. Bioorg. Med. Chem. Lett. 2011, 21, 805−811.
(16) Bursi, R.; Erdemli, G.; Campbell, R.; Hutmacher, M. M.;
Kerbusch, T.; Spanswick, D.; Jeggo, R.; Nations, K. R.; Dogterom, P.;
Schipper, J.; Shahid, M. Maximum Tolerated Dose Evaluation of the
AMPA Modulator Org 26576 in Healthy Volunteers and Depressed
Patients. Psychopharmacology 2011, 218, 713−724.
Funding
We thank the University of Strathclyde and Tenovus Scotland
for financial support.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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(17) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible docking.
J. Mol. Biol. 1997, 267, 727−748.
Mass spectrometry data was acquired at the EPSRC UK
National Mass Spectrometry Facility at Swansea University.
(18) Ward, S. E.; Harries, M.; Aldegheri, L.; Austin, N. E.; Ballantine,
S.; Ballini, E.; Bradley, D. M.; Bax, B. D.; Clarke, B. P.; Harris, A. J.;
Harrison, S. A.; Melarange, R. A.; Mookherjee, C.; Mosley, J.; Dal
Negro, G.; Oliosi, B.; Smith, K. J.; Thewlis, K. M.; Woollard, P. M.;
Yusaf, S. P. Integration of Lead Optimization with Crystallography for
a Membrane-Bound Ion Channel Target: Discovery of a New Class of
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ABBREVIATIONS
■
AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
CNS, central nervous system; GluA, ionotropic glutamate
receptor; LBD, ligand binding domain; LTP, long-term
potentiation
(19) Ornstein, P. L.; Zimmerman, D. M.; Arnold, B. M.; Bleisch, T.
J.; Cantrell, B.; Simon, R.; Zarrinmayeh, H.; Baker, R. S.; Gates, M.;
Tizzano, J. P.; Bleakman, D.; Mandelzys, A.; Jarvie, K. R.; Ho, K.;
Deverill, M.; Kamboj, R. K. Biarylpropylsulfonamides as Novel, Potent
Potentiators of 2-Amino-3- (5-methyl-3-hydroxyisoxazol-4-yl)- prop-
anoic Acid (AMPA) Receptors. J. Med. Chem. 2000, 43, 4354−4358.
(20) Patel, N. C.; Schwarz, J.; Hou, X. J.; Hoover, D. J.; Xie, L.; Fliri,
A. J.; Gallaschun, R. J.; Lazzaro, J. T.; Bryce, D. K.; Hoffman, W. E.;
Hanks, A. N.; McGinnis, D.; Marr, E. S.; Gazzard, J. L.; Hajos, M.;
Scialis, R. J.; Hurst, R. S.; Schaffer, C. L.; Pandit, J.; O’Donnell, C. J.
Discovery and Characterization of a Novel Dihydroisoxazole Class of
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Re-
ceptor Potentiators. J. Med. Chem. 2013, 56, 9180−9191.
(21) Caldwell, N.; Jamieson, C.; Simpson, I.; Tuttle, T. Organobase-
catalyzed amidation of esters with amino alcohols. Org. Lett. 2013, 15,
2506−2509.
(22) Herrmann, W. A.; Brossmer, C.; Ofele, K.; Reisinger, C. P.;
Priermeier, T.; Beller, M.; Fischer, H. Palladacycles as Structurally
Defined Catalysts for the Heck Olefination of Chloro- and
Bromoarenes. Angew. Chem., Int. Ed. 1995, 34, 1844−1848.
(23) Weeks, A. M.; Harms, J. E.; Partin, K. M.; Beneveniste, M.
Functional insight into development of positive allosteric modulators
of AMPA receptors. Neuropharmacology 2014, 85, 57−66.
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