Benzoxazinones as potent positive allosteric AMPA receptor modulators: Part I

Article abstract of DOI:10.1016/j.bmcl.2011.05.026

AMPA receptors (AMPARs) are an increasingly important therapeutic target in the CNS. Aniracetam, the first identified potentiator of AMPARs, led to the rigid and more potent CX614. This lead molecule was optimized in order to increase affinity towards the

Full text of DOI:10.1016/j.bmcl.2011.05.026

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3923–3926
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzoxazinones as potent positive allosteric AMPA receptor modulators: Part I
⇑
Rudolf Mueller , Yong-Xin Li, Aidan Hampson, Sheng Zhong, Clayton Harris, Christopher Marrs,
Stanislaw Rachwal, Jolanta Ulas, Lena Nielsson, Gary Rogers
Cortex Pharmaceuticals Inc., 15231 Barranca Parkway, Irvine, CA 92618, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
AMPA receptors (AMPARs) are an increasingly important therapeutic target in the CNS. Aniracetam, the
first identified potentiator of AMPARs, led to the rigid and more potent CX614. This lead molecule was
optimized in order to increase affinity towards the AMPA receptor. The substitution of the dioxine with
a benzoxazinone ring system increased the activity and allowed further investigation of the sidechain
SAR.
Received 17 March 2011
Revised 6 May 2011
Accepted 9 May 2011
Available online 14 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
AMPA
Receptor
Modulator
Synthesis
(
L
)-Glutamic acid is the main excitatory neurotransmitter in the
CNS, with the subset of ionotropic glutamatergic postsynaptic
receptors (iGluRs) consisting of N-methyl- -aspartic acid (NMDA),
kainic acid (KA) and -amino-3-hydroxy-5-methyl-4-isoxazole-
The imide structure of aniracetam is hydrolytically labile, which
explains the short in vivo duration of action. With this in mind, the
conformationally rigid benzoxazinone CX614 was synthesized, by
linking the amide oxygen to the benzene ring, in order to transform
the hydrolytically unstable imide into a chemically more stable
molecule (Fig. 1). The methoxy group was also exchanged for the
1,4-dioxine moiety to provide a six-membered ring. Taken to-
D
a
propionic acid (AMPA) receptors. Positive allosteric modulators of
AMPARs (AMPAKINEÒs) have been the subject of a number of stud-
ies and reviews.1–7 Based on neural pathways, these compounds
could be used to treat a range of psychiatric and neurologic disor-
ders8–13 such as Attention Deficit Hyperactivity Disorder (ADHD),
mood disorders, Schizophrenia, Huntington’s, Parkinson’s and Alz-
heimer’s diseases. A subset of AMPAR modulators have been
shown to upregulate the production of brain derived neurotrophic
factor (BDNF).14–16 A structural subset of Ampakines and most
other known potentiators, bind at an extracellular subunit inter-
face, located near the agonist binding site. Most of these modula-
tors increase the binding affinity of glutamate at the agonist site
in a manner analogous to what is seen for the effect of benzodiaze-
pines on the GABA receptor. In addition, various potentiators may
alter the duration of channel opening, desensitisation and/or
receptor rate constants for transmitter binding. It was shown, that
these molecules facilitate the induction of long term potentiation
(LTP), a key element in memory formation and learning, which
could lead to the development of these molecules into cognition
enhancers.17–24
O
O
O
O
O
O
S
S
N
NH
H2N
N
H
O
Cl
Aniracetam
Cyclothiazide
EC2x: 1.6 µM
EC2x > 1000 µM
O
O
Cl
S
NH
O
N
H
O
Idra 21
N
O
O
S
CX 614
EC2x: 2.3 µM
Aniracetam binds at the dimer interface5 of AMPARs, enhancing
the action of glutamate for a brief period of time. However, it is
weak and has a short in vivo half life.
H
N
LY 392098
O
S
EC2X: 0.07 µM
O
⇑
Corresponding author.
E-mail address: rudolfmueller@yahoo.com (R. Mueller).
Figure 1. Allosteric AMPAR positive modulators.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.026

3924
R. Mueller et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3923–3926
O
O
gether, these modifications translated into the significantly en-
hanced affinity and stability of CX614.
O
HO
O
HO
O
N
O
i
4
Several pharmacological scaffolds are now known to positively
modulate AMPAR function,25–44 among them are the intensively
studied sulfonamides cyclothiazide, Idra21, LY392098 and their
derivatives. The benzamide type AMPAKINEÒs in our study are
chemically distinct, however, it was shown that the binding sites
of aniracetam, CX614 and cyclothiazide overlap.5
In this Letter, we wish to report the optimization of our lead
CX614 into a series of structurally related derivatives. To study
the role of the 1,4-dioxine ring when CX614 binds to the receptor,
the oxygen atoms of the dioxine ring were moved in 1 and 2, or one
oxygen was replaced with a methylene group to form the pyran
derivative 3 (Fig. 2). Because the geometric shapes of CX614, 1, 2
and 3 are nearly identical, we expexted to be able to determine
the importance of the oxygen atoms in the ring system.
+
O
O
OH
6
7
Scheme 1. Synthesis of intermediate
7 and benzoxazinone 4. Reagents and
conditions: (i) 1.0 equiv 6, 1.6 equiv 4-amino butyraldehyde diethylacetal, 120 °C,
7 min; HCl, 20 °C, 60 min, 64–71% 7 and 10–20% 4.
The reduction of 7 (Scheme 2) with LiBH4 to the colorless alco-
hol 8 required prolonged heating of the reaction mixture. The yel-
low aldehyde 9 was isolated as the main product, when the
reaction temperature was maintained at 20 °C.
Ring closure of 8, using trioxane under acidic conditions yielded
1, which turned out to be slightly more active (EC2x: 1.1 lM) than
CX614. This example shows, that the oxygen can be moved from
position 4 into position 3, without having a significant impact on
activity. Ring closure of 9 with triphenylvinylphosphonium bro-
mide47 yielded the chromene derivative, which was hydrogenated
The effect of these molecules on AMPA induced currents was
evaluated in cultured rat embryonic hippocampal neurons in a
patch clamp electrophysiology assay. The readout was the EC2x
,
the concentration of a compound, which when perfused onto those
neurons, doubled the current flow.45 The comparison to CX614
to 3 (EC2x: 1.1 lM). Replacement of the oxygen with CH2 in the 4-
(EC2x: 2.3
activity of the derivatives in this study. Compound 4 turned out
to be a very potent Ampakine (EC2x: 0.06 M) and was, therefore,
lM) was used as the guideline to optimize the in vitro
position barely affected the activity of the molecule, which demon-
strates, that the oxygen in the 4-position of CX614 is not essential
and can be replaced by other functional groups or substituents,
potentially leading to more potent molecules.
To explore, whether the hetero atom in the 1-position of CX614
is important to receptor binding, we planned to synthesize 2 and 5
(Scheme 3). The Kolbe–Schmitt reaction, using 10 as the starting
material, yielded the bis-acid, which was transformed into the
bis-ester 11 in good overall yield. This material was treated with
4-amino butyraldehyde diethylacetal as before, to yield 5 and 12.
l
chosen to be radiolabeled (3H) and used as a probe for in vitro
receptor binding studies46 since it also binds to the CX614/cyclo-
thiazide binding site. The radioligand binding assay was performed
using a cell membrane preparation from adult rat forebrain.
Besides investigating these modified CX614 derivatives, we
intentionally synthesized the bis-benzoxazinones 4 and 5 in order
to investigate whether symmetrical molecules bind to the receptor
as well.
The bisbenzoxazinone 5 (EC2x: 2.6
clearly less active than its isomer 4 (EC2x: 0.06
12, followed by ring closure gave 2 (EC2x: 22
l
M) is as active as CX614, but
M). Reduction of
M), which is again
The synthesis started with commercially available diethyl 2,5-
dihydroxy-terephthalate 6, which was heated (no solvent) to-
gether with 4-amino butyraldehyde diethyl acetal (Scheme 1).
The crude amide cyclized under acidic workup conditions to yield
the very versatile intermediate 7 and 4 (which consists of three
isomers). The products of this reaction were separated easily using
column chromatography, which enabled us to quickly isolate 50 g
batches of 7 for further use. The original synthesis of 4, using an ex-
cess of amine, resulted in an almost quantitative yield of 4. The
conditions in Scheme 1 were optimized in order to maximize the
yield of 7.
l
l
clearly less active than its isomers CX614 and 1.
Based on these observations, it is clear, that the oxygen in posi-
tion 1 of CX614 is important for in vitro binding activity. We
wanted to modify the remaining ring-system further, and the ben-
zoxazinone ring appeared to be an ideal starting point, since in-
creased activity was observed for 4 and this structure presented
an interesting scaffold, to which side chains can be attached via
the nitrogen atom. However, we had tested only six-membered
derivatives and needed to investigate five- and seven-membered
O
O
7
O
N
O
N
i
O
O
O
O
O
O
O
O
O
HO
HO
HO
1
EC2x: 1.1 µM
2
EC2x: 22 µM
N
N
O
O
N
O
N
1
O
8
9
2
3
iii, iv
ii
4
O
O
O
O
O
CX 614
3
EC2x: 1.1 µM
EC2x: 2.3 µM
O
O
N
N
O
O
O
O
O
O
O
O
1
3
N
N
O
N
N
O
O
Scheme 2. Synthesis of 1,3-dioxine
1 and dihydrochromene 3. Reagents and
conditions: (i) 1.0 equiv 7, 2 equiv LiBH4, 20 °C, 2 h, 20–48% 8 and 27–42% 9; (ii)
dioxane, excess trioxane, CuSO4, H2SO4, 20 °C, 18 h, 76% 1; (iii) toluene/DMF
1.2 equiv NaH, 2 equiv CH2CHPPh3Br, 2 h, 110 °C, 54%; (iv) Pd/C/H2, AcOEt, 2.5 h,
86% 3.
EC2x: 0.06 µM
5
4
EC2x: 2.6 µM
Figure 2. Modification of the lead CX614.

R. Mueller et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3923–3926
3925
M)
Table 1
O
O
Influence of alkyl-substitution on AMPAR activity
O
O
ii
M) Slice ampl.b % (
l
M) Kic
(l
i
a
Compound Substituent
EC2x
(
l
OH
HO
OH
HO
15a
15b
15c
15d
15e
15f
15g
15h
15i
H–
Methyl–
Ethyl–
nPropyl–
iPropyl–
Cyclopropyl–
Propargyl–
Allyl–
5.0
0.1
0.2
2.5
0.9
0.8
0.25
0.5
5 [30]
19 [10]
17 [10]
0 [1]
3 [10]
10 [10]
6 [3]
—
—
—
9 [10]
0 [1]
225
18
20
70
100
26
7
18
—
163
33
330
10
11
iii
O
O
O
O
O
O
N
N
EtO
N
2-Methylpropyl– 41
O
15k
15l
15m
Cyclopentyl–
FCH2CH2–
CF3CH2–
7.0
0.5
8.8
HO
5
12
iv
EC2x values45 are defined as the concentration of compound required to double
a
v
the steady-state current induced by glutamate.
O
O
In vitro slice assay:49 percentage increases in the amplitude of fEPSP, recorded
b
at indicated concentration (— = no data available).
Displacement of tritiated AMPAKINE radioligand 4 in cell membrane prepara-
tions from adult rat forebrain.
O
N
c
O
2
Scheme 3. Synthesis of reversed benzoxazinone 5 and 1,3-dioxine 2. Reagents and
conditions: (i) 1.0 equiv 10, 3 equiv KHCO3, 800 psi CO2, 230 °C, 6 h, 97%; (ii) EtOH,
SOCl2, 90 °C, 8 h, 69% 11; (iii) 2.3 equiv 4-amino butyraldehyde dimethylacetal,
120 °C, 15 min, then HCl concd 10% 5 and 73% 12; (iv) 3.8 equiv LiBH4, 70 °C, 2.5 h,
65%; (v) dioxane, excess trioxane, cat. H2SO4, 18 h, 20 °C, 33% 2.
ring, the phenol of 7 was first alkylated with 1,2-dibromoethane
to form 16, then the halogen was substituted with ethylamine
and the seven-membered ring closed to form 17.
The six-membered ring derivative 15c is clearly superior to the
5- and 7-ring analogs 14 and 17 by comparison of the EC2x val-
ues.45 Consequently we focused our attention on a series of deriv-
atives of 15c (as shown in Table 1).
rings in comparison. The synthetic route (Scheme 4) to these mol-
ecules started in each case with 7, which was treated with hydrox-
ylamine and thionylchloride to form the isoxazole derivative 13.
Alkylation occurred only on the nitrogen to form the five-mem-
bered ring in 14. The benzoxazinones 15a–m were synthesized48
by hydrolyzing ester 7, followed by amide formation using stan-
dard conditions. Ring closure with trioxane and an acid catalyst
formed compound 15c (R = Et). In order to synthesize the larger
The derivatives 15a–m were synthesized following the general
procedure in Scheme 4. A small substituent on the nitrogen pro-
vides optimum affinity as demonstrated by 15b, 15c and 15g,
while the unsubstituted derivative 15a loses activity both in the
binding assay and the patch clamp electrophysiology assay. The
larger substituents in 15i and 15k are clearly detrimental.
The fluorine in 15l has only a minimal effect, however, the three
fluorines in 15k reduce the activity significantly, in addition to
having a negative effect on solubility.
O
O
The slice data49, reflecting activity in a neuronal network, sup-
port the results based on binding data and EC2x (single cell record-
ings) that smaller alkyl substituents are preferable in comparison
to larger ones (compare 15b, 15c and 15e).
O
O
N
ii
N
N
N
O
O
O
HO
14 EC2X: 13 µM
13
In summary, the oxygen in position 1 of CX614 proved to be
important for in vitro AMPAR activity, whereas the oxygen in posi-
tion 4 can be replaced or moved into position 3 without activity
change. A six-membered ring attached to the central benzene ring
is favorable over five- or seven-membered rings. A second benzox-
azinone system increases AMPAR activity up to 23-fold in combi-
nation with a small substituent and 38-fold when a second
pyrrolo benzoxazinone system is attached. Radioligand binding
data, EC2x and slice data correlate for the most part when the alkyl
derivatives (Table 1) are compared. This approach permits attach-
ment of a wide variety of nitrogen bound substituents, which will
be investigated further.
i
O
O
N
HO
O
O
iii
N
N
O
EtOOC
R
O
v
iv
15a-m
7
15c:
EC2X: 0.2 µM
Br
vi
O
O
O
vii
O
N
N
References and notes
N
O
O
EtOOC
O
1. Morrow, J. A.; Maclean, J. K. F.; Jamieson, C. Curr. Opin. Drug Discovery Dev.
2006, 9, 571.
16
17 EC2X: 12 µM
2. Jin, R.; Clark, S.; Weeks, A. M.; Dudman, J. T.; Gouaux, E.; Partin, K. M. J.
Neuroscience 2005, 25, 9027.
3. Arai, A. C.; Kessler, M. Curr. Drug Targets 2007, 8, 583.
4. Harpsøe, K.; Liljefors, T.; Balle, T. J. Mol. Graphics Modell. 2008, 26, 874.
5. Harpsøe, K.; Varming, T.; Gouliaev, A. H.; Peters, D.; Liljefors, T. J. Mol. Graphics
Modell. 2007, 26, 213.
6. Sun, Y.; Olson, R.; Horning, M.; Armstrong, N.; Mayer, M.; Gouaux, E. Nature
2002, 417, 245.
7. Arai, A. C.; Xia, Y.-F.; Rogers, G.; Lynch, G.; Kessler, M. J. Pharmacol. Exp. Ther.
2002, 303, 1075.
Scheme 4. Synthesis of five-, six- and seven- membered rings. Reagents and
conditions: (i) 1.0 equiv 7, 1.6 equiv H2NOH, 3 equiv NaOH, 2 h, 20 °C; 2.3 equiv
SOCl2, 15 min, 3 equiv NEt3, 60 min, 20 °C, 73% 13; (ii) 3.6 equiv K2CO3, 2.5 equiv
EtI, 58 °C, 18 h, 50% 14; (iii) 15 equiv KOH, MeOH/water, 1.5 h, 50 °C; (iv) 1.0 equiv
DMAP, 1.0 equiv HOBT, 2.5 equiv NEt3, 2.0 equiv EDCI, 1.3 equiv amine, DMF, 20 °C,
4 h; (v) CHCl3, excess trioxane, cat. H2SO4, 2 h, 70 °C, 20–90% (three steps) 15a–m.
(vi) 2.5 equiv K2CO3, excess BrCH2CH2Br, 110 °C, four days, 76%; (vii) EtNH2, 4 equiv
KOH, 1 equiv HOBT, 1 equiv DMAP, 1 equiv NEt3, 3 equiv EDCI, three days, 20 °C,
59% 17.

3926
R. Mueller et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3923–3926
8. Marenco, S.; Weinberger, D. R. CNS Drugs 2006, 20, 173.
9. Alt, A.; Nisenbaum, E. S.; Bleakman, D.; Witkin, J. M. Biochem. Pharmacol. 2006,
77, 1273.
10. Kanju, P. M.; Parameshwaran, K.; Sims, C.; Bahr, B. A.; Shonesy, B. C.;
Suppiramaniam, V. Exp. Neurol. 2008, 214, 55.
Mosley, J.; Neve, M.; Oliosi, B.; Profeta, R.; Smith, K. J.; Smith, P. W.; Spada, S.;
Thewlis, K. M.; Yusaf, S. P. J. Med. Chem. 2010, 53, 5801.
39. Ward, S. E.; Bax, B. D.; Harries, M. Br. J. Pharmacol. 2010, 160, 181.
40. Grove, S. J. A.; Jamieson, C.; Maclean, J. K. F.; Morrow, J. A.; Rancovic, Z. J. Med.
Chem. 2010, 53, 7271.
11. O’Neill, M. J.; Bleakman, D.; Zimmerman, D. M.; Nisenbaum, E. S. Curr. Drug
Targets: CNS Neurol. Disord. 2004, 3, 181.
12. Sanacora, G.; Zarate, C. A.; Krystal, J. H.; Manji, H. K. Nature Rev. 2008, 7, 426.
13. Hashimoto, K. Brain Res. Rev. 2009, 61, 105.
41. 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. Bioorg. Med. Chem. Lett. 2011,
21, 805.
14. Lauterborn, J. C.; Lynch, G.; Vanderklish, P.; Arai, A.; Gall, C. M. J. Neurosci. 2000, 8.
15. Lauterborn, J. C.; Pineda, E.; Chen, L. Y.; Ramirez, E. A.; Lynch, G.; Gall, C. M.
Neuroscience 2009, 159, 283.
16. Lauterborn, J. C.; Troung, G.; Baudry, M.; Bi, X.; Lynch, G.; Gall, C. J. Pharmacol.
Exp. Ther. 2003, 307, 297.
17. Lynch, G. Curr. Opin. Pharmacol. 2004, 4, 4.
18. Lynch, G.; Gall, C. M. Trends Neurosci. 2006, 29, 554.
19. O’Neill, M. J.; Dix, S. IDrugs 2007, 10, 185.
42. Jamieson, C.; Campbell, R. A.; Cumming, I. A.; Gillen, K. J.; Gillespie, J.;
Kazemier, B.; Kiczun, M.; Lamont, Y.; Lyons, A. J.; Maclean, J. K. F.; Martin, F.;
Moir, E. M.; Morrow, J. A.; Pantling, J.; Rancovic, Z.; Smith, L. Bioorg. Med. Chem.
Lett. 2010, 20, 6072.
43. Jamieson, C.; Basten, S.; Campbell, R. A.; Cumming, I. A.; Gillen, K. J.; Gillespie,
J.; Kazemier, B.; Kiczun, M.; Lamont, Y.; Lyons, A. J.; Maclean, J. K. F.; Moir, E.
M.; Morrow, J. A.; Papakosta, M.; Rancovic, Z. Bioorg. Med. Chem. Lett. 2010, 20,
5753.
20. Arai, A.; Kessler, M.; Rogers, G.; Lynch, G. J. Pharmacol. Exp. Ther. 1996, 278, 627.
21. Staubli, U.; Rogers, G.; Lynch, G. Proc. Natl. Acad. Sci. 1994, 91, 777.
22. Raymond, C. R. Trends Neurosci. 2007, 30, 167.
44. Cannazza, G.; Jozowiak, K.; Parenti, C.; Braghiroli, D.; Carrozzo, M. M.; Puia, G.;
Losi, G.; Baraldi, M.; Lindnaer, W.; Wainer, I. W. Bioorg. Med. Chem. Lett. 2009,
19, 1254.
23. Lamprecht, R.; LeDoux, J. Nature Rev., Neurosci. 2004, 5, 45.
24. Lynch, M. A. Physiol. Rev. 2004, 84, 87.
45. Patch clamp recording and EC2x estimate: On cultured hippocampal neurons
(Sprague–Dawley rat cells removed at embryonic day 18, 4–7 days in culture),
25. Bloss, E. B.; Hunter, R. G.; Waters, E. M.; Munoz, C.; Bernard, K.; McEwen, B. S.
Exp. Neurol. 2008, 210, 109.
26. Francotte, P.; De Tullio, P.; Goffin, E.; Dintilhac, G.; Graindorge, E.; Fraikin, P.;
Lestage, P.; Danober, L.; Thomas, J. Y.; Caignard, D. H.; Pirotte, B. J. Med. Chem.
2007, 50, 3153.
500 lM Glutamate-induced currents with or without AMPA receptor
modulators were measured under whole-cell recording with patch-clamp
amplifiers (Axopatch 200B; MDS, Inc., Toronto, Canada) at room temperature.
All compounds or saline were applied by DAD-12 or DAD-VC superfusion
system (ALA Scientific Instruments Inc., New York). The 13–1 or 17–1 tip was
27. Zivcovic, I.; Thompson, D. M.; Bertolino, M.; Uzunov, D.; Dibella, M.; Costa, E.;
Guidotti, A. Pharmacol. Exp. Ther. 2009, 272, 300.
28. Murray, T. K.; Whalley, K.; Robinson, C. S.; Ward, M. A.; Hicks, C. A.; Lodge, D.;
Vandergriff, J. L.; Baumbarger, P.; Siuda, E.; Gates, M.; Ogden, A. M.; Skolnick,
P.; Zimmerman, D. M.; Nisenbaum, E. S.; Bleakman, D.; O’Neill, M. J. Pharmacol.
Exp. Ther. 2003, 306, 752.
placed approximately 100–150 lM from the cell. Cells (voltage-clamped at
À80 mV) were pre-treated for 20 s with the saline/modulators before the
application of glutamate/glutamate plus the modulator for 1 s. The mean value
of plateau current between 600 to 900 ms after application of glutamate was
measured and used as the parameter to evaluate the compound’s effect. EC2x
value is defined as the concentration (in
lM) of the compound required to
29. Francotte, P.; Tullio, P.; Podona, T.; Diouf, O.; Fraikin, P.; Lestage, P.; Danober,
L.; Thomas, J.-Y.; Caigard, D.-H.; Pirotte, B. Bioorg. Med. Chem. 2008, 16, 9948.
30. Zarrinmayeh, H.; Bleakman, D.; Gates, M. R.; Yu, H.; Zimmerman, D. M.;
Ornstein, P. L.; McKennon, T.; Arnold, M. B.; Wheeler, W. J.; Skolnick, P. J. Med.
Chem. 2001, 44, 302.
double the steady-state current induced by glutamate.
46. In a test run with 4, we were able to achieve 95% H/D exchange, using D2 gas
and Crabtree’s catalyst. The active, tritiated enantiomer was synthesized
accordingly, isolated by HPLC (Chiralpak IA) and used in binding studies.
47. Schweizer, E. E.; Liehr, J.; Monaco, D. J. J. Org. Chem. 1968, 33, 2416.
48. Rogers, G. A.; Allan, M.; Harris, C.; Huang, J.; Marrs, C. M.; Mueller, R.; Rachwal,
S. U.S. 7799913, 2010.
31. Magnus, N. A.; Stazak, M. A.; Udodong, U. E.; Wepsiec, J. P. Org. Process Res. Dev.
2006, 10, 899.
32. Phillips, D.; Sonnenberg, J.; Arai, A. C.; Vaswani, R.; Krutzik, P. O.; Kleisli, T.;
Kessler, M.; Granger, R.; Lynch, G.; Chamberlin, A. R. Bioorg. Med. Chem. 2002,
10, 1229.
49. In vitro slice assay: Transverse 300–400 lM hippocampal slices were taken
from adult male Sprague–Dawley rats (4–6 week old) and incubated on nylon
net in a rapid-flow linear interface recording chamber. The chamber was
perfused with oxygenated artificial cerebral spinal fluid (ACSF). Field EPSP
(excitatory postsynaptic potential) recordings were made with glass electrodes
filled with 2 M NaCl (1–5 megaohm), placed in the stratum radiatum or
stratum oriens of CA1. Electrical stimuli were produced with twisted bipolar
nichrome wire electrodes in the Schaffer-commissural fiber afferents. EPSP was
induced by activating Schaffer-commissural fiber using pair pulse stimulation
with a pulse interval at 200 ms delivered at 0.05 Hz. The amplitude, half-width
and area were measured for each response and plotted against time. After a
stable baseline was established (20–30 min), test compound was infused for
30 min to observe the effect. Then the solution was switched to control ACSF
and continued to record until drug effect was washed back to the baseline.
Most of the compounds were made into stock solution by dissolving in DMSO
then adding into ACSF. If DMSO concentration has to be >0.05%, DMSO was also
added into the control ACSF. Both Control ACSF and test compound were
infused to the recording chamber bath by gravity infusion system.
33. Zarrinmayeh, H.; Tromiczak, E.; Zimmerman, D. M.; Rankl, N.; Ho, K. H.;
Dominguez, E.; Castano, A.; Escribano, A.; Fernandez, C.; Jimenez, A.; Hornback,
W. J.; Nisenbaum, E. S. Bioorg. Med. Chem. Lett. 2006, 20, 5203.
34. Fernandez, M.-C.; Castano, A.; Dominguez, E.; Escribano, A.; Jiang, D.; Jimenez, A.;
Hong, E.; Hornback, W. J.; Nisenbaum, E. S.; Rankl, N.; Tromiczak, E.; Vaught, G.;
Zarrinmayeh, H.; Zimmerman, D. M. Bioorg. Med. Chem. Lett. 2006, 20, 5057.
35. Ptak, C. P.; Ahmaed, A. H.; Oswald, R. E. Biochemistry 2009, 8594.
36. Thewlis, K. M.; Aldegheri, L.; Harries, M. H.; Mookherjee, C.; Oliosi, B.; Ward, S.
E. Bioorg. Med. Chem. Lett. 2010, 16, 7116.
37. 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.; Woolard, P. M.; Yusaf, S. P. J. Med. Chem. 2011, 54, 78.
38. Ward, S. E.; Harries, M.; Aldegheri, L.; Andreotti, D.; Ballantine, S.; Bax, B. D.;
Harris, A. J.; Harker, A. J.; Lund, J.; Melarange, R.; Mingardi, A.; Mookherjee, C.;