(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl] -3-(2-fluoro-phenyl)-acrylamide is a potent and efficacious KCNQ2 opener which inhibits induced hyperexcitability of rat hippocampal neurons

Article abstract of DOI:10.1016/j.bmcl.2004.01.069

(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl] -3-(2-fluoro-phenyl)-acrylamide ((S)-2) was identified as a potent and efficacious KCNQ2 opener. This compound demonstrated significant activity in reducing neuronal hyperexcitabil

Full text of DOI:10.1016/j.bmcl.2004.01.069

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1991–1995
(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-
6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide isa potent and
efficaciousKCNQ2 opener which inhibitsinduced hyperexcitability
of rat hippocampal neurons
Yong-Jin Wu,a,* Christopher G. Boissard,b Jie Chen,a William Fitzpatrick,c Qi Gao,d
Valentin K. Gribkoff,b David G. Harden,c Huan He,a Ronald J. Knox,c Joanne Natale,b
Rick L. Pieschl,b John E. Starrett, Jr.,a Li-Qiang Sun,a Mark Thompson,b David Weaver,c
Dedong Wud and Steven I. Dworetzkyb
aDepartment of Neuroscience Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
bDepartment of Neuroscience Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
cDepartment of NewLeads Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
dDepartment of Analytical Sciences, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
Received 4 November 2003; revised 9 January 2004; accepted 14 January 2004
Abstract—(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-2) was
identified as a potent and efficacious KCNQ2 opener. This compound demonstrated significant activity in reducing neuronal
hyperexcitability in rat hippocampal slices, and the inhibition mediated by (S)-2 was reversed by the KCNQ blocker linopirdine.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
rat sympathetic neurons.2,4 This compound produces a
large hyperpolarizing shift in the voltage-dependence of
activation of KCNQ currents, and this particular
mechanism of action probably underlies at least some of
the physiological effects of retigabine. In rat models
KCNQ2 and KCNQ3 are voltage-activated potassium
channels belonging to the 6-transmembrane domain
potassium channel gene family. KCNQ2 and KCNQ3
subunits probably co-assemble in neurons to produce a
native M-current, which is an important regulator of
neuronal excitability because it determines the excit-
ability threshold, firing properties, and responsiveness
of neurons to synaptic inputs. Modulators of the M-
channel have been under clinical investigation: blockers
such as linopirdine for cognition enhancement1 and
openers (e.g., retigabine, Fig. 1) for epilepsy.2 Retiga-
bine has been shown to activate KCNQ channels
expressed in mammalian cells3 and native M-currents in
* Corresponding author. Tel.: +1-203-677-7485; fax: +1-203-677-
7884; e-mail: yong-jin.wu@bms.com
Figure 1.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.069

1992
Y.-J. Wu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1991–1995
of persistent and neuropathic pain, retigabine has been
shown to attenuate nociceptive behaviors.5 Flupirtine
(Fig. 1), a close analogue of retigabine, also shifts the
activation curves towards negative voltages, but it is less
effective than retigabine.6 Both retigabine and flupirtine
demonstrated significant analgesic activity on tactile
allodynia in Chung-lesioned rats.6 Taken together, this
suggests that KCNQ openers may have therapeutic
potential in the treatment of neuropathic pain and
various chronic pain conditions.
zine provided amine 10, which was converted to urea
derivative 11 upon reaction with 3,5-dichlorophenyl
isocyanate (Scheme 2). The X-ray diffraction analysis of
crystalline 11 showed the R configuration (Fig. 2), thus
indicating the R stereochemistry for (R)-2. Since the
amine used for the preparation of (R)-2 was (R)-7,
the amine used for the preparation of (S)-2, 8, and 9
must be (S)-7. Therefore, compounds (S)-2, 8, and 9 are
assigned with S configuration.
Recently, we reported (S)-N-[1-(3-morpholin-4-yl-
phenyl)-ethyl]-3-phenyl-acrylamide [(S)-1, Fig. 1] as a
novel KCNQ2 opener with excellent oral bioavailability
in dogs and rats.7 This compound demonstrated sig-
nificant oral activity in the cortical spreading depression
model of migraine, suggesting that KCNQ2 openers
may also have potential for the treatment of some types
of migraine headache.
Despite the recent advances in the KCNQ area, there is
still a pressing need for the development of KCNQ
channel openers with increased potency compared with
retigabine and acrylamide (S)-1. In an effort to increase
the potency of (S)-1, we retained the morpholine nitro-
gen of (S)-1 in the same relative orientation, but fused
the morpholine onto the phenyl ring. Through this
approach we hoped to retain a KCNQ recognition ele-
ment (the basic nitrogen) while decreasing the entropy
of the resulting molecule. The concept of restricting
rotatable bonds to improve potency has been employed
extensively in medicinal chemistry optimization pro-
grams.8 This investigation led to the identification of (S)
-N-[1-(4-cyclopropylmethyl -3,4-dihydro-2H-benzo-
[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide
((S)-2, Fig. 1) as a potent and efficacious KCNQ2 opener.
This report describes the synthesis and abbreviated
SAR of acrylamide (S)-2, its effects on KCNQ-mediated
currents, and its activity on neuronal hyperexcitability
in rat hippocampal slices.
.
Scheme 1. Reagents and conditions: (a) NH2OH HCl, NaOH (10 N),
.
THF, reflux, 87%; (b) Raney Ni, H2 (50 psi), NH3 H2O, MeOH, 94%;
(c) LiAlH4, THF, À78 ꢀC, 95%; (d) 3-(2-fluoro-phenyl)-acrylic acid,
.
EDAC HCl, DMAP, Et3N, CH2Cl2, 91%; (e) chiral HPLC (AD
2. Chemistry
column, 75% hexanes/25% EtOH); (f) K2CO3, acetone, cyclopro-
pylmethyl bromide for 2 (78%), EtI for 8 (86%), and MeI for 9 (77%);
(g) K2CO3, acetone, cyclopropylmethyl bromide, 74%.
The synthesis of (S)-2 and its close analogues started
with commercially available 6-acetyl-4H-benzo[1,4]-
oxazin-3-one (3) (Scheme 1). This ketone was converted
to oxime 4 through reaction with hydroxylamine hydro-
chloride. Raney nickel reduction of this oxime furnished
amine (Æ)-5, which was further reduced with lithium
aluminum hydride to provide diamine (Æ)-6. Direct
conversion of 4 to (Æ)-6 with lithium aluminum hydride
proved to be of much lower yield than the two-step
procedure via (Æ)-5. Diamine (Æ)-6 was coupled with
3-(2-fluoro-phenyl)-acrylic acid to afford acrylamide
(Æ)-7. Chiral HPLC separation provided two enantio-
mers: (S)-7 and (R)-7. Compound (S)-7 underwent
alkylation with appropriate alkyl halides to afford (S)-
2,9 8 and 9. Compound (R)-2 was prepared from (R)-7
in the same fashion as (S)-2 (Scheme 1).
Scheme 2. Reagents and conditions: (a) NH2NH2, EtOH, 100 ꢀC,
63%; (b) 3,5-dichlorophenyl isocyanate, i-Pr2NEt, CH2Cl2, 41%.
The absolute configuration of (S)-2, (R)-2, 8, and 9 was
established as follows. Treatment of (R)-2 with hydra-

Y.-J. Wu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1991–1995
1993
Figure 2. Thermal ellipsoid plot (35% ellipsoids) of crystalline 11.
3. Results and discussion
Figure 3A and 3B show the effects of (S)-2 on the same
HEK 293 cell at a test potential of À30 mVand À110
mVrespectively. The very large enhancement of
KCNQ2 current measured at À30 mVin the presence
of 10 mM (S)-2 is particularly striking because of the
change in the kinetic properties of the KCNQ2 current
produced by (S)-2. The control current has the expected
sigmoid activation kinetics characteristic of KCNQ
currents (lower trace), whereas in the presence of (S)-2,
the KCNQ2 current evoked by stepping from À80 to
À30 mVis largely instantaneous with an additional
slowly activating current component on top of the
instantaneous current fraction. This dramatic change
from sigmoid to instantaneous current activation is
reflected in the shape of the conductance-voltage G/V
relationship of KCNQ2 in the presence of (S)-2 shown
in Figure 3C. The current traces in Figure 3B show the
large inward KCNQ2 currents produced following a
membrane potential step from À80 mVto À110 mV.
The KCNQ2 current flows into the cell because the
+
command potential of À110 mVis below the K equi-
librium potential, EK+ (ꢁÀ90 mV). This result indi-
cates that (S)-2 produces a very large and profound
hyperpolarizing shift in the voltage-dependence of acti-
Figure 3. Effect of (S)-2 (10 mM) on mKCNQ2 currents stably
expressed in HEK 293 cells measured by the whole-cell patch clamp
method. A and B show representative data from a single cell of the
dramatic increase in KCNQ2 current measured at test potentials of
À30 mVand À110 mV, respectively, following a step command from
À80 mVin the absence and presence of 10 mM (S)-2. C shows the
effect of 10 mM (S)-2 on the conductance–voltage relationship for
mKCNQ2. Data shown is the mean ÆSEM (n=6).
vation of KCNQ2. The voltage for half-activation (V0.5
was À15.9 mVin control conditions and À52.7 mVin
the presence of 10 mM (S)-2, a 36.8 mVshift in the
)
hyperpolarization direction. Note that Figure 3C only
shows the normalized outward conductance changes
produced by (S)-2, which is the conventional method
for displaying G/Vdata. The numerical shift in V
is
0.5
actually an underestimate of the total change in voltage-
dependence of the conductance.
Figure 4 shows the concentration–response relationship
for (S)-2 mediated increase of KCNQ2 current from
single HEK 293 cells voltage-clamped at À40 mV. The
threshold concentration is ꢁ10 nM to 30 nM, and the
onset of action is a few seconds (time course data not
shown) and steady-state activity is reached in approxi-
mately 30 s. Following removal of (S)-2, there is rapid
and complete reversal of the enhancement of KCNQ2
current. The EC50 for (S)-2 was estimated from the
fit to data to be 0.063Æ0.01 mM with a Hill slope (HS)
of 1.8. The HS >1 suggests the mechanism of action
of (S)-2 may involve positive cooperative binding, a
relatively common observation in drug receptor/channel
interactions.10,11
Figure 4. Concentration dependent augmentation effect of mouse
KCNQ2 currents by (S)-2. Data shown is the mean ÆSEM for 5 cells.

1994
Y.-J. Wu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1991–1995
The mechanism of action of (S)-2 is in large part a hyper-
polarizing shift in the voltage dependence of activation of
the KCNQ2 conductance. In addition, the instanta-
neous kinetics of activation observed in the presence of
(S)-2 are strikingly different from the activation kinetics
of non-treated cells, which implies that (S)-2 converts
KCNQ2 channels from being strongly voltage-depen-
dent to a conformational state essentially devoid of
voltage-dependence in the physiological membrane
potential range. In any given neuron where natively
expressed KCNQ2 channels contribute significantly to
setting and maintaining the resting membrane potential,
one might expect (S)-2 to hyperpolarize and therefore
essentially clamp the membrane potential close to E+K .
The net effect of this would be that any such neurons
would become electrically ‘silent’. Such a mechanism of
action of (S)-2 would be entirely consistent with its
ability to strongly inhibit the firing frequency of hippo-
campal neurons described in this report. Interestingly,
the mode switch in activation kinetics produced by (S)-2
is similar to that observed with (S)-1,7 but importantly
(S)-2, unlike (S)-1, does not significantly inhibit
KCNQ2 conductance at positive membrane potentials,
which could simplify its physiological effects in the
CNS. Direct electrophysiological recordings from neu-
rons expressing KCNQ currents should be able to dis-
tinguish the functional significance of not inhibiting
KCNQ conductance at positive potentials.
in Table 1, the potency appears to increase as the alkyl
substituent attached to the oxazine nitrogen becomes
bulkier. Interestingly, the size of the alkyl group has lit-
tle impact to the efficacy of these analogues with the
exception of the unsubstituted analogue (S)-7. Of spe-
cial note is that the (R)-2 exhibited no KCNQ2 opener
activity when tested at 10 mM, whereas (S)-2 is a highly
potent and efficacious KCNQ2 opener, suggesting that
the S configuration may play an important role in
opening KCNQ2 channels for this series of acrylamides.
Preliminary studies using thallium(I) influx assay7
showed that compounds (S)-2, (S)-7, 8, and 9, like (S)-
1,7 also activated other members of the KCNQ family.
Further electrophysiological characterization is required
in order to understand the activation of other KCNQ
channels.
Compound (S)-2 was examined for its ability to reduce
spontaneous neuronal discharges in rat hippocampal
slices. In this assay, the induction of spontaneous neu-
ronal bursting was achieved by bathing slices to an
artificial cerebrospinal fluid (CSF) containing zero
magnesium (MgSO4) and low calcium (CaCl2 1.5 mM).
The resulting multiple-unit extracellular electrical activ-
ity was recorded by advancing an electrode 50–150 mm
into the CA1 region of the hippocampus. This electrical
activity was then amplified using a differential amplifier,
and the number of events per min was collected and
analyzed. The analyzed data was expressed in Hz and
reported as percent inhibition of compound free con-
trol. Figure 5A depicts the multi-unit response of a sin-
gle hippocampal slice to the application of (R)-2
(inactive enantiomer), (S)-2 (active enantiomer) and (S)-
2 in the presence of linopirdine, a KCNQ blocker.
Application of (R)-2 (2.5 mM) did not provide any
reduction on the neuronal bursting, whereas addition of
(S)-2 (2.5 mM) resulted in a significant inhibition on
neuronal hyperexcitability. After maximal inhibition by
(S)-2 was achieved, the KCNQ blocker linopirdine (10
mM) was applied concurrently with (S)-2 (2.5 mM).
Complete reversal of the (S)-2 mediated inhibition was
produced by co-application in this example.
Whole-cell patch-clamp evaluation on recombinant
mouse KCNQ2 channels expressed in HEK 293 cells
demonstrates that (S)-2 is a potent and efficacious
opener of KCNQ2 channels. As shown in Table 1, (S)-2
has an improved potency over (S)-1 and retigabine by a
factor of ꢁ55-fold and 22-fold, respectively. The effi-
cacies of test compounds were normalized to the efficacy
of a specific reference compound,12 to yield an E/Eref
ratio. The ratio is calculated by dividing the maximum
current amplitude produced by the test compound by
the maximum current amplitude produced by the refer-
ence compound. The benefit of using an E/Eref value is
that it allows ranking of compound efficacies indepen-
dent of the amount of basal KCNQ2 current expression.
The E/Eref’s of (S)-2, (S)-1 and retigabine are 1.83,
1.40, and 1.60, respectively (Table 1). Thus, (S)-2 is 31%
and 14% more efficacious in opening KCNQ2 channels
than (S)-1 and retigabine, respectively.
In order to establish a concentration–response curve for
(S)-2 (Fig. 5B), slices were exposed to a range of con-
centrations (100 nM–2.5 mM). A minimum of 12 slices
were used for each compound concentration. The EC50
calculated for (S)-2 was 0.70 mM. It should be noted
that (R)-2 had no effect on neuronal discharge when
tested at the concentration producing maximal inhibi-
tion by (S)-2 (2.5 mM) (Fig. 5B), which is consistent with
the inability of this concentration of (R)-2 to activate
KCNQ2 expressed in HEK 293 cells (data not shown).
Concurrent application of (S)-2 (2.5 mM) and lino-
pirdine (10 mM) significantly reduced the inhibition
relative to (S)-2 (2.5 mM) alone (Fig. 5B).
The above EC50 measurements were also carried out on
several analogues structurally related to (S)-2. As shown
Table 1. Whole cell patch-clamping data (À40 mV)
Compd
EC50 (mM)a
E/Erefa,b
(S)-1
(S)-2
8
9
(S)-7
Retigabine
3.28Æ0.05
0.06Æ0.01
0.20Æ0.02
0.94Æ0.14
2.0Æ0.1
1.40Æ0.06
1.83Æ0.02
2.1Æ0.3
1.8Æ0.2
1.2Æ0.1
We have hypothesized that a KCNQ opener would be
efficacious in reducing the induced neuronal hyperexcit-
ability in rat hippocampal slices, by virtue of increasing
K+ efflux from the cells, stabilizing the cell membrane,
and thus making it harder for depolarizing stimuli to
1.30Æ0.04
1.60Æ0.05
a These values are the meanÆSEM (n=2–5).
b The efficacy of the test compound relative to the reference com-
pound.12

Y.-J. Wu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1991–1995
1995
and most probably other KCNQ channels. In vitro
testing of (S)-2 in hyperexcited hippocampal slices sug-
gests that KCNQ2 openers may have potential for the
treatment of CNS disorders characterized by neuronal
hyperexcitability, such as migraine, epilepsy, bipolar
disorder and neuropathic pain.
Referencesand notes
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9. Data for (S)-2: 1H NMR (CDCl3, 400 MHz) d 7.68 (d,
J=16 Hz, 1H), 7.48–7.43 (m, 1H), 7.34–7.25 (m, 1H),
7.15–7.04 (m, 2H), 6.75 (m, 2H), 6.66–6.59 (m, 1H), 6.50
(d, J=16 Hz, 1H), 5.76 (d, J=7.6 Hz, 1H), 5.17–5.07 (m,
1H), 4.23 (t, J=4.4 Hz, 2H), 3.41 (t, J=4.4 Hz, 2H), 3.14
(d, J=6.4 Hz, 2H), 1.54 (d, J=6.8 Hz, 3H), 0.99–1.04 (m,
1H), 0.52–0.57 (m, 2H), 0.20–0.24 (m, 2H). 13C NMR
(CDCl3, 100 MHz) d 3.6 (d, J=10 Hz), 8.1, 21.4, 46.6,
49.2, 55.2, 64.7, 111.1, 114.7, 116.2 (d, J=22 Hz), 116.4,
122.0 (d, J=11 Hz), 123.0 (d, J=8 Hz), 124.4 (d, J=4
Hz), 129.8 (d, J=3 Hz), 130.8 (d, J=8 Hz), 134.1, 135.6,
136.0, 143.5, 161.4 (d, J=252 Hz), 164.7. [a]2D0 +5.7 (c
3.08, CH2Cl2).
Figure 5. A. Example of the multi-unit response of a single hippo-
campal slice to the application of (R)-2 (2.5 mM), followed by pro-
longed application of (S)-2 (2.5 mM). After maximal inhibition by (S)-
2 was achieved, linopirdine (Lin., 10 mM) was applied concurrently
with (S)-2 (2.5 mM). B. Group concentration–response relationship
generated for (S)-2 in hippocampal slices (n=12–36).
spread. Consistent with this hypothesis, the active
enantiomer (S)-2 demonstrated a significant inhibition
of the neuronal discharge in a concentration-related
fashion, while no reduction was observed with the inac-
tive enantiomer (R)-2. Furthermore, the effect of (S)-2
was mediated specifically via modulation of KCNQ
channel function as shown by the reversal of (S)-2
mediated inhibition by the KCNQ blocker linopirdine
in this hippocampal slice model.
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12. A manuscript describing the structure and activity of the
reference compound will be submitted to Bioorg. Med.
Chem. Lett.
4. Conclusion
Compound (S)-2 was identified as a potent and effica-
cious KCNQ2 opener and is expected to be a valuable
tool in further studying the pharmacology of KCNQ2