Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction

Article abstract of DOI:10.1016/j.bmcl.2018.09.036

Neuronal voltage-gated potassium channels, K_V7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K_V7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K_V7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K_V7 isoforms was investigated in human detrusor tissue using a panel of K_V7 openers with distinct activity profiles among K_V7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K_V7.4 did not reduce detrusor contraction. This may suggest that the homomeric K_V7.4 channel plays a less significant role in bladder contraction and further investigation is needed.

Full text of DOI:10.1016/j.bmcl.2018.09.036

Accepted Manuscript
Novel KV7 ion channel openers for the treatment of epilepsy and implications
for detrusor tissue contraction
Mark A. Seefeld, Hong Lin, Joerg Holenz, Dave Downie, Brian Donovan,
Tingting Fu, Kishore Pasikanti, Wei Zhen, Matthew Cato, Khuram W.
Chaudhary, Pat Brady, Tania Bakshi, Dwight Morrow, Sridharan Rajagopal,
Swapan Kumar Samanta, Naveena Madhyastha, Bharathi Mohan Kuppusamy,
Robert W. Dougherty, Ravi Bhamidipati, Zainuddin Mohd, Guy A. Higgins,
Mark Chapman, Céline Rouget, Philippe Lluel, Yasuji Matsuoka
PII:
S0960-894X(18)30779-0
https://doi.org/10.1016/j.bmcl.2018.09.036
BMCL 26057
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 August 2018
25 September 2018
27 September 2018
Please cite this article as: Seefeld, M.A., Lin, H., Holenz, J., Downie, D., Donovan, B., Fu, T., Pasikanti, K., Zhen,
W., Cato, M., Chaudhary, K.W., Brady, P., Bakshi, T., Morrow, D., Rajagopal, S., Samanta, S.K., Madhyastha, N.,
Kuppusamy, B.M., Dougherty, R.W., Bhamidipati, R., Mohd, Z., Higgins, G.A., Chapman, M., Rouget, C., Lluel,
P., Matsuoka, Y., Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue
contraction, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.09.036
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H
N
O
O
O
O
Cl
CH3
S
N
H
OH
O
F
F
F
F
14
20
Kv7.2/7.3 EC50 = 0.30 µM (141%) Kv7.2/7.3 EC50 = 0.13 µM (132%)
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Novel KV7 ion channel openers for the treatment of epilepsy and implications for
detrusor tissue contraction
Mark A. Seefelda,*, Hong Lina,b,n, Joerg Holenza, Dave Downiec, Brian Donovand, Tingting Fue, Kishore
Pasikantia, Wei Zhenf, Matthew Catog, Khuram W. Chaudharyg, Pat Bradyd, Tania Bakshih, Dwight
Morrowb,o, Sridharan Rajagopali, Swapan Kumar Samantai, Naveena Madhyasthai, Bharathi Mohan
Kuppusamyi, Robert W. Doughertyj, Ravi Bhamidipatii, Zainuddin Mohdi, Guy A. Higginsk, Mark
Chapmanl, Céline Rougetm, Philippe Lluelm, Yasuji Matsuokaa,*
aNeuroscience Virtual-Proof-of-Concept Discovery Performance Unit, GlaxoSmithKline, Collegeville, PA, United States
bRegenerative Medicine Discovery Performance Unit, GlaxoSmithKline, Collegeville, PA, United States
cScreening Profiling & Mechanistic Biology, Platform Technology Sciences, GlaxoSmithKline, Stevenage, UK
dScreening, Profiling and Mechanistic Biology, Platform Technology Sciences, GlaxoSmithKline, Collegeville, PA, United States
eMechanistic Safety & Disposition, Product Development and Supply, Platform Technology Sciences, GlaxoSmithKline, Shanghai, China
fIntegrated Biological Platform Sciences, Product Development and Supply, Platform Technology Sciences, GlaxoSmithKline, Shanghai, China
gIn vitro / In vivo Translation, Safety Pharmacology, Platform Technology Sciences, GlaxoSmithKline, King of Prussia, PA, United States
hPattern Recognition Receptor Discovery Performance Unit, GlaxoSmithKline, Collegeville, PA, United States
iJubilant Biosys, Bangalore, Karnataka, India
jJubilant Discovery Services, Malvern, PA, United States
kInterVivo Solutions, Toronto, ON, Canada
lIcagen, Durham, NC, United States
mUrosphere SAS, Toulouse, France
nCurrent affiliation: Prelude Therapeutics, Wilmington, DE, United States
oCurrent affiliation: Magenta Therapeutics, Cambridge, MA, Unites States
*Corresponding authors. Email addresses: Mark.A.Seefeld@gsk.com (Mark Seefeld) and Yasuji.Y.Matsuoka@gsk.com (Yasuji Matsuoka)
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Neuronal voltage-gated potassium channels, KV7s, are the molecular mediators of the M current
and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we
report novel small molecule KV7 openers that demonstrate anti-seizure activities in electroshock
and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The
anti-seizure activity was determined to be proportional to the unbound concentration in the

brain. KV7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of
these channels may cause localized undesired effects. Therefore, the impact of individual KV7
isoforms was investigated in human detrusor tissue using a panel of KV7 openers with distinct
activity profiles among KV7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in
detrusor tissue, yet a compound that has significantly reduced activity on homomeric KV7.4 did
not reduce detrusor contraction. This may suggest that the homomeric KV7.4 channel plays a
less significant role in bladder contraction and further investigation is needed.
Keywords:
KCNQ
Opener
Epilepsy
Bladder
2009 Elsevier Ltd. All rights reserved.
Neuronal voltage-gated potassium channels composed of the following heterodimeric KV7 isoforms, KV7.2, 7.3, 7.4, and 7.5, are
coded by KCNQ2-5, respectively. KV7 ion channels are the molecular mediators of the M current and regulate membrane excitability in
the central and peripheral neuronal systems.1 Genetic mutations in KCNQ3 and KCNQ4, are associated with excitability disorders
including multiple forms of epilepsy and deafness.2, 3 Therefore, KV7 channel opening is a potential therapeutic approach for aberrant
neuroexcitation conditions.4
Previously reported small molecule KV7-openers suppress neuronal activity at sub-micromolar concentrations in vitro. Suppression
of neuronal activities with KV7-openers translate to functional endpoints in a wide range of pre-clinical models.5 In human clinical studies,
Retigabine (1) and Flupirtine (2) (Figure 1) that act on pan-KV7.2-7.5 channels demonstrated clinical efficacy in partial epilepsy6 and
multiple forms of pain,7 respectively. However, these molecules have undesired effects including of discolouration to the skin, nails, lips
and ocular tissues (Retigabine)8 and hepatotoxicity (Flupirtine)9, 10 that are suspected as KV7-unrelated actions. Furthermore, the frequency
of urinary adverse events such as urinary retention was increased in patients receiving Retigabine compared with placebo in clinical
studies.11 Urinary adverse events were suspected as target-related because some KCNQ isoforms are expressed in human urinary bladder
smooth muscle12 and pan-KV7 openers affected bladder contractibility ex vivo in human12 and guinea pig.13
While KV7 channel openers have demonstrated clinical efficacy, improved therapeutic agents are desired. Discovery efforts leading
to chemically distinct KV7.2/7.3 channel opener series (1-5, Figure 1) have been described in the literature5, 14-16 and, except for the ICA-
27243 (5) series of compounds, all have been reported to activate human pan-KV7-mediated currents. In this study, we aimed to discover
novel Kv7 isoform-selective openers for the treatment of epilepsy and to investigate the implications of KV7 isoform activities in human
urinary bladder contraction.
A high throughput screen of the GSK compound collection using the heterodimeric KV7.2/7.3 channel (See Supplemental Information
for methods of all biological experiments) revealed two similar, but structurally distinct lead series, Benzamide series (Table 1) and
Cyclohexamide series (Table 2).
F
H
N
O
O
X
N
O
N
H
H
NH2
1, Retigabine, X=CH
2, Flupirtine, X=N
3, ML-213
OCH3
O
F
F
HN
N
N
H3CO
O
N
N
NH
OCH3
Cl
4, PF05020182
5, ICA-27243
Figure 1. Representative KV7.2/7.3 openers
The synthesis of the benzamide series is depicted in scheme 1. Ortho-bromo benzoic acids 6 were coupled with phenolic reagents
via an SnAr reaction to provide biaryl ethers 7. Subsequent amide coupling with chiral benzyl amines gave benzamide products (Table
1).

Br
O
O
O
OH
OH
a
CF3
CF3
6
7
SO2CH3
H2N
O
O
SO2CH3
N
H
b
8
CF3
Scheme 1
. Synthesis of benzamide derivatives.
Reagents and conditions: (a) Phenol, CuI, K2CO3, DMF, 130 °C,
16h; (b) HOBt, EDC, DIPEA, DMF, 60 °C, 2h
Cyclohexamide compounds (Scheme 2) were synthesized starting from 4,4-difluoro cyclohexanone (9) and dimethyl carbonate
to afford ketoester 10. Converting 10 to the corresponding enol triflate 11 and subsequent cross-coupling with phenylboronic acid gave
the unsaturated aryl ester 12. Hydrogenation followed by saponification under basic conditions provided the racemic trans-cyclohexyl
carboxylic acid 13. Amide coupling with appropriately substituted chiral benzyl amines yielded KV7.2/7.3 openers which were separated
into pure diastereomers using chiral HPLC (Table 2). Only one enantiomer in the cyclohexamide series was determined to be consistently
active in the KV7.2/7.3 primary assay and this absolute stereochemistry was confirmed by an X-ray crystal structure of compound 14
(Supplemental Figure 1, CCDC code: 1855751).
O
O
O
O
OTf
O
O
O
O
O
a
b
F
F
F
F
F
F
11
10
9
OH
B
O
OH
O
OH
O
d, e
c
F
F
F
F
12
(+) - 13
(R)
H2N
H
Cl
O
N
Cl
OH
OH
F
f,g
F
14
Scheme 2
. Synthesis of aryl amide analogs.
Reagents and conditions: (a) NaH, DMF, 0 °C - RT, 16h;
(b) Commin's reagent, NaH, THF, 0°C - RT,16h; (c) Pd(dppf)Cl2·CH2Cl2,
Na2CO3, Toluene, H2O, EtOH, 90 °C, 16h; (d) H2, Pd/C, MeOH, RT 16h;
(e) NaOEt, EtOH, 90 °C; (f) HATU, DIPEA, THF, RT, 16h;
(g) Chiral prep HPLC.
In the benzamide series, thioether screening lead 15 (Table 1) was metabolically unstable in pooled human liver microsomes
(HLM, >3 mL/min/g liver) as were phenyl ether analogs lacking electron withdrawing groups (EWGs) at R1 (not shown). Alkyl
substituents at R were tolerated, however, unsubstituted phenyl ethers provided the highest level of KV7.2/7.3 activation. Functionality at
R2 was limited to methyl or hydroxymethyl with the specific stereochemistry shown. The methyl sulfone at the 3-position of the benzylic
ring remained unchanged as it provided the best combination of solubility and KV7.2/7.3 activity.
R
R2
O
O
CH3
2
1
S
N
O
H
R1
Table 1. Benzamide series potencies (µM)
Cmpd R R1 R2 EC50a Sol.(µM)b
15 SPh 5-CF3 H 0.20 (175) 34
16 OPh 5-CF3 H 0.16 (245) 23
8 OPh 5-CF3 CH3 0.04 (134) 57
17 OPh 5-CF3 CH2OH 0.08 (77) 11

18 OPh 5-SF5 H 0.08 (185) 7
19 OPh 5-SF5 CH2OH 0.02 (101) 121
20 OPh 4-F,5-F CH3 0.13 (132) 145
ameasured using IWQ (% of Maximal response to ML213)
bkinetic aqueous solubility measured using CAD or CLND
Initially, poor aqueous solubility was observed in the cyclohexamide series. This issue was resolved by the addition of a
hydroxymethyl group at position R. Again, the stereospecificity at this position was limited to that shown in Table 2. Stabilities under
HLMs were compromised without EWGs on the cyclohexyl ring. Gem-difluoro substitution on the cyclohexyl ring provided stability and
avoided the introduction of an additional chiral center. The phenyl group adjacent to the carboxamide was ideally left unsubstituted much
like the phenyl group in the benzamide series. Lipophilic substituents at the meta-position of the benzyl ring improved target activity
without sacrificing significant solubility. Methylsulfone substitution was not well-tolerated on the benzyl ring in this series.
H
O
N
R2
R
R1
R1
Table 2. Cyclohexamide series potencies (µM)
Cmpd R R1 R2 EC50a Sol.(µM)b
21 CH3 H H 0.25(115) 57
22 CH2OH H H 0.79(165) 455
23 CH2OH F H 1.3(108) 332
24 CH3 F H 0.20(140) 25
14 CH2OH F 3-Cl 0.30(141) 318
25 CH2OH F 3-CF3 0.08(96) 119
ameasured using IWQ (% of Maximal response to ML213)
bkinetic aqueous solubility measured using CAD or CLND
The selection criteria for progressing compounds to in vivo studies were minimally: aqueous kinetic solubilities of >100 µM,
KV7.2/7.3 channel opening EC50 ≤0.30 µM (IonWorks™ Quattro), and HLM clearance values under 3 mL/min/g liver. All compounds
had P-glycoprotein (P-gp) ratios of less than 2. Some compounds elicited complex curves wherein the currents measured were actually
depressed as compared with vehicle controls at more depolarized potentials (i.e., generally higher than -10 mV). These compounds were
not advanced to in vivo studies. Examples of IV curves of compounds in QPatch (see supplemental section) that met with our progression
criteria are indicated in Figure 2.
compound 14
1.00
control
0.048 M; V1/2 = 1.8 mV
0.24 M; V1/2 = 10.2 mV
1.2 M; V1/2 = 21.0 mV
0.75
6 M; V1/2 = 29.5 mV
30 M; V1/2 = 27.8 mV
control 2
compound 3 (ML-213)
3 M; V1/2 = 24.7 mV
0.50
0.25
0.00
-100
-80
-60
-40
-20
0
20
voltage (mV)
compound 20
control
0.016 µM; V1/2 = 0.4 mV
0.08 µM; V1/2 = 5.1 mV
0.4 µM; V1/2 = 14.3 mV
2 µM; V1/2 = 23.1 mV
10 µM; V1/2 = 26.8 mV
control 2
compound 3 (ML-213)
3 µM; V1/2 = 28.4 mV
1.00
0.75
0.50
0.25
0.00
-100
-80
-60
-40
-20
0
20
voltage (mV)
Figure 2. Examples of IV curve
Prior to in vivo testing in rodents, activities of representative compounds from both chemical series 14 and 20 were
measured in primary cultured rat cortical neurons using a multiple-electrode array system (see supplemental section). Both

compounds inhibited spontaneous neuronal firing activities at similar concentrations to those observed with cell lines over-
expressing KV7 isoforms. IC50 values of 0.28 and 0.11 µM were determined for compounds 14 and 20, respectively.
Compounds 14 and 20 were also found to be sufficiently brain penetrant (Table 3) to progress to maximum electroshock-
threshold (MES-T, Figure 3A) and Rotarod (Figure 3B) studies that measure anti-seizure effects and CNS tolerability, respectively. Dose-
dependent increases in seizure threshold with no meaningful effects in the Rotarod model were similar to the results observed with
the experimental control ICA-27243 (Compound 5 in Figure 3A and B). Similar results were obtained using the pentylenetetrazol
(PTZ)-induced seizure model (Supplemental Figure 2). In addition, pharmacodynamic (PD) effects observed in both the PTZ and
MES-T mouse models correlated well with the predicted target engagement across compounds at all dose levels tested
(Supplemental Figure 3).
Table 3. Pharmacokinetic parameters for selected
KV7.2/7.3 channel openers following intravenous
dosing in mice
Cmpd Clb DNAUC0~t Br/Bl (mL/min/kg) ((µg.h/mL) ratio
/mg/kg))
14 48 ± 2.1 0.51 ± 0.5 0.8
20 31 ± 2.6 0.33 ± 0.5 1.3
Figure 3. MES-T seizure and Rotarod studies
After determining that compounds from these two series could achieve efficacy in seizure models, we sought to understand if
the compounds demonstrated isoform selectivity amongst the KV7.2-7.5 channels. Compounds meeting the previously described selection
criteria for activity and solubility were cross-screened against the heterodimeric KV7.3/7.5 channel using the IonWorks™ Barracuda
platform (Molecular Devices). All compounds demonstrated similar levels of activities in both KV7.2/7.3 and KV7.3/7.5 assays (Table 4).
To further scrutinize the binding site interactions, select compounds were tested in a cell line that has point mutations (KV7.2W236L and
KV7.3W265L) in the pore domain (Table 5). No compounds from these two series showed measurable activity up to 10 µM in this assay,
suggesting that these compounds bind in the KV7.2/7.3 pore domain. The activity of Icagen compound ICA-27243 (5) was unaffected in
this mutant KV7.2/7.3 assay, supporting reports of compound binding in the voltage sensor domain.17
Table 4. Activities in human KV7.2-7.5 channels (µM)a
Cmpd KV7.2/7.3 KV7.3/7.5 KV7.4
14 0.10 0.36 0.10
19 0.06 0.08 >10
20 0.20 0.20 0.16
24 0.40 0.40 4.0
25 0.05 0.25 0.02
ML-213 0.04 0.50 1.6
ICA-27243 0.46 >10 >10
aactivities determined by SyncroPatch platform.
Table 5. Activities in human wildtype and mutant KV7.2/7.3 channels (µM)a
Cmpd WT KV7.2/7.3 KV7.2W236L/
KV7.3W265L
15 0.02 >30
16 0.05 >30
21 0.10 >30
ML-213 0.04 >11
ICA-27243 0.46 0.43-1.81

Retigabine 0.32 >30
aactivities determined by 86Rb efflux assay
It has been reported that KV7.4 is implicated in pain and urinary bladder contraction. Therefore, we measured the effects of
compound 19, which has significantly reduced activity on KV7.4, in a bladder contraction model.
Urinary adverse events were reported more frequently in patients receiving the pan-Kv7 activator Retigabine (1), as compared
with placebo, although most patients were able to continue with treatment.11 KCNQ isoforms are expressed in human detrusor and KV7
openers were shown to influence bladder contraction.12, 18 In this study, we have examined expression of KCNQ isoforms (Supplemental
Figure 4) and effects on human bladder contraction ex vivo (Figure 4). We tested compounds that have diverse KCNQ isoform activity
profiles to determine how different isoform profiles influence bladder contraction. While the inhibition of bladder contraction was clearly
associated with KV7 activation, it wasn’t clear how individual KV7.2-7.5 isoforms may contribute. Compound 19, which has significantly
reduced homomeric KV7.4 activity, did not reduce impact on bladder contraction, and this may suggest that the homomeric KV7.4 channel
plays a less critical role in bladder contraction. Further studies are needed to conclude a role of KV7 isoforms in bladder contraction.
Figure 4. Effects of KV7 openers with differential activities on KV7.2/7.3, 7.4 and 7.3/7.5 isoforms on carbachol-induced
contraction in human bladder.
In the present study, we have identified novel pan-KV7 openers, that are free from structural alerts. Representatives of novel
KV7 openers demonstrated anti-seizure effects in a dose-dependent manner without meaningful changes in the Rotarod CNS tolerability
model. We found that effects on bladder contraction depend on pan-KV7 activity, but perhaps not on homomeric KV7.4 activity. Further
investigation is required to conclude the roles of individual KV7 isoforms in bladder contraction.
Acknowledgments
We acknowledge Drs. Gareth Young, Lishuang Cao, Alex Gutteridge, Wayne Rycroft, GlaxoSmithKline, for their technical input in
electrophysiology, bioinformatics, and seizure models; Drs. James Cooper, Carol Wilson, Neil Brickel, GlaxoSmithKline, for critical
review of the manuscript; Dr. Ravichandran Sankaranarayanan, Jubilant Biosys, for his program management. We also thank Professor
W. Clegg and Dr. M. R. Probert for the GSK-funded crystal structure of compound 14.
References and notes
1. Wang HS, Pan Z, Shi W, et al. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science.
1998;282(5395): 1890-1893.
2. Biervert C, Schroeder BC, Kubisch C, et al. A potassium channel mutation in neonatal human epilepsy. Science. 1998;279(5349): 403-406.
3. Kubisch C, Schroeder BC, Friedrich T, et al. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant
deafness. Cell. 1999;96(3): 437-446.
4. Jentsch TJ, Schroeder BC, Kubisch C, Friedrich T, Stein V. Pathophysiology of KCNQ channels: neonatal epilepsy and progressive deafness.
Epilepsia. 2000;41(8): 1068-1069.
5. Miceli F, Soldovieri MV, Martire M, Taglialatela M. Molecular pharmacology and therapeutic potential of neuronal Kv7-modulating drugs.
Curr Opin Pharmacol. 2008;8(1): 65-74.
6. Porter RJ, Burdette DE, Gil-Nagel A, et al. Retigabine as adjunctive therapy in adults with partial-onset seizures: integrated analysis of three
pivotal controlled trials. Epilepsy Res. 2012;101(1-2): 103-112.
7. Devulder J. Flupirtine in pain management: pharmacological properties and clinical use. CNS Drugs. 2010;24(10): 867-881.
8. Garin Shkolnik T, Feuerman H, Didkovsky E, et al. Blue-gray mucocutaneous discoloration: a new adverse effect of ezogabine. JAMA
Dermatol. 2014;150(9): 984-989.
9. Douros A, Bronder E, Andersohn F, et al. Flupirtine-induced liver injury--seven cases from the Berlin Case-control Surveillance Study and
review of the German spontaneous adverse drug reaction reporting database. Eur J Clin Pharmacol. 2014;70(4): 453-459.
10. Michel MC, Radziszewski P, Falconer C, Marschall-Kehrel D, Blot K. Unexpected frequent hepatotoxicity of a prescription drug, flupirtine,
marketed for about 30 years. Br J Clin Pharmacol. 2012;73(5): 821-825.
11. Brickel N, Gandhi P, VanLandingham K, Hammond J, DeRossett S. The urinary safety profile and secondary renal effects of retigabine
(ezogabine): a first-in-class antiepileptic drug that targets KCNQ (K(v)7) potassium channels. Epilepsia. 2012;53(4): 606-612.

12. Svalo J, Sheykhzade M, Nordling J, Matras C, Bouchelouche P. Functional and molecular evidence for Kv7 channel subtypes in human
detrusor from patients with and without bladder outflow obstruction. PLoS One. 2015;10(2): e0117350.
13. Afeli SA, Malysz J, Petkov GV. Molecular expression and pharmacological evidence for a functional role of kv7 channel subtypes in Guinea
pig urinary bladder smooth muscle. PLoS One. 2013;8(9): e75875.
14. Brueggemann LI, Haick JM, Cribbs LL, Byron KL. Differential activation of vascular smooth muscle Kv7.4, Kv7.5, and Kv7.4/7.5 channels
by ML213 and ICA-069673. Mol Pharmacol. 2014;86(3): 330-341.
15. Davoren JE, Claffey MM, Snow SL, et al. Discovery of a novel Kv7 channel opener as a treatment for epilepsy. Bioorg Med Chem Lett.
2015;25(21): 4941-4944.
16. Wu YJ, Boissard CG, Greco C, et al. (S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]- 3-phenylacrylamide: an orally bioavailable KCNQ2 opener
with significant activity in a cortical spreading depression model of migraine. J Med Chem. 2003;46(15): 3197-3200.
17. Wang AW, Yang R, Kurata HT. Sequence determinants of subtype-specific actions of KCNQ channel openers. J Physiol. 2017;595(3): 663-
676.
18. Bientinesi R, Mancuso C, Martire M, Bassi PF, Sacco E, Curro D. KV7 channels in the human detrusor: channel modulator effects and gene
and protein expression. Naunyn Schmiedebergs Arch Pharmacol. 2017;390(2): 127-137.
Supplementary Materials
X-Ray crystal structure of compound 24 (Supplemental Figure 1), synthetic procedures including yields, seizure protection in PTZ-
induced seizure model (Supplemental Figure 2), methods and some data for electrophysiological and biological experiments, PK/PD
analysis in MES-T and PTZ models (Supplemental Figure 3), and quantification of KCNQ isoforms mRNA expression in the bladder
(Supplemental Figure 4), were included in Supplementary Section.



Novel pan-Kv7 openers for the treatment of epilepsy are discussed
Compounds from two series of Kv7 openers show efficacy in PTZ and MES-T seizure models
Bladder contraction is associated with pan-KV7 activity, but perhaps not with homomeric Kv7.4 activity as
reported
Graphical Abstract
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H
N
O
O
O
O
Cl
CH3
S
N
H
OH
O
F
F
F
F
14
20
Kv7.2/7.3 EC50 = 0.30 µM (141%) Kv7.2/7.3 EC50 = 0.13 µM (132%)