The synthesis and structure-activity relationships of 3-amino-4- benzylquinolin-2-ones: Discovery of novel KCNQ2 channel openers

Article abstract of DOI:10.1016/j.bmcl.2004.01.073

3-Amino-4-benzylquinolin-2-ones have been identified as a novel class of KCNQ2 channel openers. Synthesis and SAR is described along with their electrophysiological evaluation as activators of the cloned mKCNQ2 channel expressed in Xenopus laevis oocytes.

Full text of DOI:10.1016/j.bmcl.2004.01.073

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1615–1618
The synthesis and structure–activity relationships of
3-amino-4-benzylquinolin-2-ones:
discovery of novel KCNQ2 channel openers
Piyasena Hewawasam,a,* Nathan Chen,a Min Ding,a Joanne T. Natale,b
Christopher G. Boissard,b Sarita Yeola,b Valentin K. Gribkoff,b John Starretta
and Steven I. Dworetzkyb
aDepartment of Chemistry, The Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
bDepartment of Neuroscience, The Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
Received 5 January 2004; accepted 23 January 2004
Abstract—3-Amino-4-benzylquinolin-2-ones have been identified as a novel class of KCNQ2 channel openers. Synthesis and SAR
is described along with their electrophysiological evaluation as activators of the cloned mKCNQ2 channel expressed in Xenopus
laevis oocytes. The preliminary SAR data suggest the importance of both the trifluoromethylsulfonamido group and electron-
withdrawing substituents on the quinolone nucleus for expression of KCNQ2 channel opening properties.
# 2004 Elsevier Ltd. All rights reserved.
Potassium channels are a structurally diverse and widely
distributed family of transmembrane proteins that are
present in both excitable and nonexcitable cells.1,2 The
KCNQ family of potassium channels which are voltage
dependent is a subgroup of these channels.3,4 KCNQ
channel proteins consist of six transmembrane domains
and a pore-forming P-loop.3,4 Physiological studies
indicate that potassium currents are associated with a
variety of functions including the regulation of the elec-
trical properties of excitable cells.1,2 Although over 60
potassium channel genes have been identified in humans
only a few have been directly linked to human dis-
eases.1À4 Remarkably mutations in four out of five
KCNQ genes underlie diseases including cardiac
arrhythmia,5 epilepsy6 and deafness.7 These disorders
illustrate the physiological relevance of different KCNQ
channels and their potential as drug discovery targets.
including spinal cord and dorsal root ganglion
(DRG).9,10 Originally, Wang et al. reported that co-
assembly of the KCNQ2 and KCNQ3 potassium chan-
nels underlies a native M-current in neurons.10 Subse-
quently, it was shown that several KCNQ isoforms
including KCNQ2 and KCNQ3 can associate to form
heteromeric channels which underlie one type of M-
current, an important regulator of neuronal excitability in
a wide range of neurons.3 Thus, pharmacological modula-
tion of M-currents represents an important and attractive
mechanism for controlling neuronal excitability.
Mutations in genes encoding the KCNQ2 and KCNQ3
channels have been linked to the epileptic condition
benign familial neonatal convulsions (BFNC).6,11,12
Therefore, selective openers of these channels are pre-
dicted to have anticonvulsant properties. Openers of
KCNQ2/3 channels have also been proposed as novel
therapeutic agents for pain. Alleviation of pain is sup-
ported by in vivo data showing that KCNQ2/3 openers
are analgesic in animal models of pain such as formalin
and hot plate tests13 and as well as neuropathic pain.14
Additionally, anxiolytic properties of KCNQ2/3 chan-
nel openers has been demonstrated using the Geller
conflict procedure in rats.13
Unlike KCNQ1, which is predominantly expressed in
human heart and pancreas,5,8 KCNQ2 and KCNQ3
channels are expressed mainly in neuronal tissue
* Corresponding author. Tel.: +1-203-677-7815; fax: +1-203-677-
7702; e-mail: hewawasp@bms.com
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.073

1616
P. Hewawasam et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1615–1618
Retigabine (1) has been shown to activate human
KCNQ2 and KCNQ3 channels expressed either alone
or as heteromultimer in Xenopus oocytes.15 A variety of
pyridyl benzamide analogues such as 2 has been claimed
as KCNQ channel openers following evaluation in a
86Rb+ efflux assay in KCNQ2-transfected CHO cells.16
In pursuit of identifying novel activators of neuronal
KCNQ2 channels that would be useful in modulation
of neuronal hyperexitability, we synthesized a series of
3-amino-4-benzylquinolin-2-one derivatives by modifi-
cation of 3-amino-4-arylquinolin-2-one template that
has been identified previously as a maxi-K channel
activator pharmacophore.17 (Chart 1).
Chart 1.
As illustrated in Scheme 1, preparation of 3-amino-4-
benzylquinolin-2-ones required the (2-aminophenyl)-
benzyl ketones (8) as precursors. Reaction of ortho-
lithiated N-Boc-aminobenzotrifluoride derivatives (3)
with DMF gave the corresponding N-Boc-2-amino-
benzaldehyde derivatives (4).18 Addition of Grignard
reagent 5 to a mixture of 4 and NaH in ether provided
the desired benzyl alcohol derivatives 6. Oxidation of
the alcohols 6 with PCC followed by deprotection of the
boc group afforded the desired (2-aminophenyl)benzyl
ketones 8. Chloroacetylation of 8 with chloroacetyl
chloride in the presence of pyridine gave the corres-
ponding N-chloroacetyl derivatives (9). Upon heating a
solution of 9 in anhydrous pyridine at reflux for 15–30
min, the initially formed a-pyridinium salt undergoes
cyclodehydration to afford the pyridinium chloride (10).
Hydrazinolysis of 10 with hydrazine hydrate in ethanol
at reflux for 1–2 h provided the desired 3-amino-4-ben-
zylquinolin-2-one derivatives 11.
Scheme 1. (a) (1) BuLi (2.2 equiv), THF, À40 ꢀC (2) DMF; 58–86%
t
(b) NaH, ether, 0 ꢀC then add 5; 25–37% (c) PCC, anhydrous NaOAc,
DCM; 90–93% (d) 3N HCl, EtOH, reflux; 90–95% (e) ClCH2COCl,
pyridine, DCM, 0–23 ꢀC; 88–92% (f) anhydrous pyridine, reflux; 74–
.
89% (g) NH2NH2 H2O, EtOH, reflux; 70–83% (h) BBr3, DCM, 0–
23 ꢀC; 90–95% (i) (1) (CF3SO2)2O, pyridine, 23 ꢀC (2) 3N NaOH,
THF–MeOH, 23 ꢀC; 40–68%.
The 3-amino group of 11 was derivatized as (trifluoro-
methyl)sulfonamides. In order to monosulfonylate the
amino group, 11 was persulfonylated with excess tri-
fluoromethanesulfonic anhydride and then treated with
NaOH in MeOH–THF to afford the desired mono-
sulfonylated derivatives 13. Demethylation of 11 and 13
with BBr3 was carried out where a methoxyl is present
to afford the phenols 12 and 14, respectively. The 3-
amino-4-benzylquinolin-2-one derivatives prepared by
these methods are compiled in Table 1.
Whole-cell membrane current recordings were achieved
using standard two-electrode voltage clamp techni-
ques.19 Voltage-clamp protocols typically comprised a
series of voltage steps of approximately 5 seconds in
duration, in +10 mV steps from a holding potential of
À90 mV to a maximal potential of +40 mV. Records
were digitized at 5 kHz and stored on a computer using
pClamp data acquisition and analysis software (Axon
Instruments). All compounds were tested in at least 3
different oocytes to evaluate the effect of a single drug
concentration (1, 5 or 20 mM) and expressed as the
average percentage change in mKCNQ2 current relative
to drug-free control (100%). The results obtained are
listed in Table 1 along with data for Retigabine, which
allows the efficacy comparison of the 3-amino-4-benzyl-
quinolin-2-one derivatives to a prototypical KCNQ2
channel opener.
The ability of the target compounds to open KCNQ2
channel was assessed under two-electrode voltage-clamp
conditions by determining their ability to increase
cloned mouse KCNQ2 (mKCNQ2) mediated outward
currents expressed in Xenopus laevis oocytes. Oocytes
were prepared and injected using standard techniques.
Each oocyte was injected with approximately 50 mL of
mKCNQ2 and then impaled with electrodes (1–2 Mꢀ).

P. Hewawasam et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1615–1618
1617
Table 1. Structure of 3-amino-4-benzylquinolin-2-one derivatives and effect on KCNQ2-mediated outward current in Xenopus laevis oocytes
expressing the cloned mKCNQ2
Compda
R1
R2
R3
R4
R5
R6
R
% Increase in mKCNQ2 current
@ 20 mM (@À40 mV)b
11a
12a
12b
H
H
H
H
H
CF3
H
CF3
H
H
H
H
H
OH
OH
H
Cl
Cl
H
H
H
103Æ4 (n=3)
122Æ11 (n=5)
89Æ1 @ 1 mM (n=5)
Insol. @ 5 or 20 mM
131Æ3 (n=5)
13a
13b
13c
13d
13e
14a
14b
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
H
H
H
H
Cl
Cl
H
SO2CF3
SO2CF3
SO2CF3
SO2CF3
SO2CF3
SO2CF3
SO2CF3
CF3
CF3
H
CF3
CF3
CF3
223Æ12 (n=5)
254Æ17 (n=5)
231Æ24 (n=7)
220Æ22 (n=6)
OH
OH
277Æ15 (n=5)
H
Cl
271Æ21 (n=6)
190Æ10 @ 1 mM (n=5)
284Æ14 (n=7)
14c
H
H
CF3
H
OH
Cl
SO2CF3
217Æ6 @ 1 mM (n=5)
143Æ6 @ 100 nM (n=5)
269Æ10 (n=3)
1 (Retigabine)
1
a All new compounds were characterized by H NMR, LRMS, and elemental analysis.
b All recordings were measured at À40 mV.
The structure–activity relationship data presented in
Table 1 provide a rudimentary understanding of the
KCNQ2 channel opening pharmacophore of 3-amino-
4-benzylquinolin-2-ones. In this preliminary study, the
optimal substitution pattern required for KCNQ2
channel opening activity was probed. Unsubstituted
3-amino-4-benzylquinolin-2-one (11a) was inactive.
Incorporation of CF3 group and 5-chloro-2-hydroxy-
benzyl moiety (12a) resulted in a slight increase in
channel opening activity. Similarly, derivatization of the
3-amino group as (trifluoromethyl)sulfonamide (13a)
also had modest enhancement of channel opening
activity. However, the most dramatic improvement in
KCNQ2 channel opening activity was observed upon
incorporation of these three elements together onto the
3-amino-4-benzylquinolin-2-one pharmacophore. It is
also apparent that (trifluoromethyl)sulfonamido group
and CF3 moiety work in tandem to exert the biggest
enhancement in channel opening activity. This observa-
tion indicates an important relationship between
increasing acidity of the 3-amino moiety and enhanced
channel opening activity. Additionally, both methoxy
and hydroxy moieties also enhance the channel opening
activity. Removal of the chlorine atom as shown in 14a
did not affect the opening activity. A majority of the
sulfonamide derivatives (13b–e, 14a–c) were shown to
be efficacious KCNQ2 channels openers with greater
than 200% increase in outward currents at 20 mM. In
comparison, these novel KCNQ2 openers are approach-
ing or, in some cases, equaling the efficacy of Retigabine
(1). The most potent KCNQ2 opener (14c) identified
from this series showed robust opening of the channel
even at 100 nM. The EC50 value of 14c was determined
to be 663 nM.
both an electron-withdrawing substituent on the quino-
lone nucleus and the presence of a phenolic hydroxyl for
effective activation of KCNQ2 channels.
References and notes
1. Shieh, C. C.; Coghlan, M.; Sullivan, J. P.; Gopa-
lakrishnan, M. Pharmacol. Rev. 2000, 52, 557.
2. Coghlan, M. J.; Carroll, W. A.; Gopalakrishnan, M. J.
Med. Chem. 2001, 44, 1627.
3. Jentsch, T. J. Nat. Rev. Neurosci. 2000, 1, 21.
4. Robbins, J. Pharmacol. Ther. 2001, 90, 1.
5. Wang, Q.; Curran, M. E.; Splawski, I.; Burn, T. C.; Mill-
holland, J. M.; VanRaay, T. J.; Shen, J.; Timothy, K. W.;
Vincent, G. M.; de Jager, T.; Schwartz, P. J.; Toubin,
J. A.; Moss, A. J.; Atkinson, D. L.; Landes, G. M.; Con-
nors, T. D.; Keating, M. T. Nat. Genet. 1996, 12, 17.
6. Biervert, C.; Schroeder, B. C.; Kubisch, C.; Berkovic,
S. F.; Propping, P.; Jentsch, T. J.; Steinlein, O. K. Science
1998, 279, 403.
7. Kubisch, C.; Schroeder, B. C.; Friedrich, T.; Lutjohann,
B.; El-Amraoui, A.; Marlin, S.; Petit, C.; Jentsch, T. J.
Cell 1999, 96, 437.
8. Yang, W. P.; Levesque, P. C.; Little, W. A.; Conder,
M. L.; Shalaby, F. Y.; Blanar, M. A. Proc. Natl. Acad.
Sci. 1997, 94, 4017.
9. Tinel, N.; Lauritzen, I.; Chouabe, C.; Lazdunski, M.;
Borsotto, M. FEBS Lett. 1998, 438, 171.
10. Wang, H. S.; Pan, Z.; Shi, W.; Brown, B. S.; Wymore,
R. S.; Cohen, I. S.; Dixon, J. E.; McKinnon, D. Science
1998, 282, 1890.
11. Charlier, C.; Singh, N. A.; Ryan, S. G.; Lewis, T. B.; Reus,
B. E.; Leach, R. J.; Leppert, M. Nat. Genet. 1998, 18, 53.
12. Singh, N. A.; Charlier, C.; Stauffer, D.; DuPont, B. R.;
Leach, R. J.; Melis, R.; Ronen, G. M.; Bjerre, I.; Quat-
tlebaum, T.; Murphy, J. V.; McHarg, M. L.; Gagnon, D.;
Rosales, T. O.; Peiffer, A.; Anderson, V. E.; Leppert, M.
Nat. Genet. 1998, 18, 25.
13. Wickenden, A. D.; Rigdon, G. C.; McNaughton-Smith,
G. A.; Gross, M. F. WO Patent 0,110,381 2001.
14. Blackburn-Munro, G.; Jensen, B. S. Eur. J. Pharmacol.
2003, 460, 109.
In summary, we have identified a novel class of KCNQ2
channel openers and demonstrated that channel opening
activity is enhanced by the presence of a (trifluoro-
methyl)sulfonamido group. The preliminary structure–
activity data for this series suggests the importance of

1618
P. Hewawasam et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1615–1618
15. Main, M. J.; Cryan, J. E.; Dupere, J. R.; Cox, B.;
Clare, J. J.; Burbidge, S. A. Mol. Pharmacol. 2000, 58,
253.
16. McNaughton-Smith, G. A.; Gross, M. F.; Wickenden, A.
D. US Patent 6,372,767 2002.
17. Hewawasam, P.; Fan, W.; Knipe, J.; Moon, S. L.; Bois-
sard, C. G.; Gribkoff, V. K.; Starrett, J. E., Jr. Bioorg.
Med. Chem. Lett. 2002, 12, 1779.
18. (a) Muchowski, J. M.; Venuti, M. C. J. Org. Chem. 1980,
45, 4798. (b) Takagishi, S.; Katsoulos, G.; Schlosser, M.
Synlett 1991, 360.
19. Stuhmer, W. Methods Enzymol. 1992, 207, 319.