Accepted Manuscript

Design, synthesis and evaluation of novel N-phenylbutanamide derivatives as

KCNQ openers for the treatment of epilepsy

Shaoning Yang, Dingqiang Lu, Pingkai Ouyang

PII: S0960-894X(18)30418-9
DOI: https://doi.org/10.1016/j.bmcl.2018.05.019
Reference: BMCL 25836

To appear in: Bioorganic & Medicinal Chemistry Letters

Received Date: 26 April 2018

Revised Date: 7 May 2018
Accepted Date: 8 May 2018

Please cite this article as: Yang, S., Lu, D., Ouyang, P., Design, synthesis and evaluation of novel N-
phenylbutanamide derivatives as KCNQ openers for the treatment of epilepsy, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.019

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Design, synthesis and evaluation of novel N-phenylbutanamide

derivatives as KCNQ openers for the treatment of epilepsy

Shaoning Yang a, b, Dingqiang Lua, *, Pingkai Ouyanga
a
School of Pharmaceutical Sciences, Nanjing University of Technology, Nanjing 211816, P.R.
China
b
Jiangsu Simcere Pharmaceutical Co. Ltd., Nanjing 210042, P.R. China

Abstract
KCNQ (Kv7) has emerged as a validated target for the development of novel
anti-epileptic drugs. In this paper, a series of novel N-phenylbutanamide derivatives
were designed, synthesized and evaluated as KCNQ openers for the treatment of
epilepsy. These compounds were evaluated for their KCNQ opening activity in vitro
and in vivo. Several compounds were found to be potent KCNQ openers. Compound
1 with favorable in-vitro activity was submitted to evaluation in vivo. Results showed
that compound 1 owned significant anti-convulsant activity with no adverse effects. It
was also found to posses favorable pharmacokinetic profiles in rat. This research may
provide novel potent compounds for the discovery of KCNQ openers in treating
epilepsy.

Keywords: N-phenylbutanamide derivatives, KCNQ openers, epilepsy.

*Address correspondence to this author at the School of Pharmaceutical Sciences, Nanjing

University of Technology, Nanjing 211816, P.R. China; E-mail: ludingqiang@njtech.edu.cn.

Epilepsy is a chronic brain disorder with the hallmark of recurrent and

unprovoked seizures. It is the second most common neurological disease with about
65 million patients diagnosed in the world [1]. Although the pathogenesis of epilepsy is
still unclear, it is well recognized that the imbalance between excitation and inhibition
of the central nervous system will lead to seizures, which can be caused by changes in
ion channels, neurotransmitters and glial cells [2-4]. The present anti-epileptic drugs
(AEDs) strategies mainly act on related ion channels and neurotransmitter receptors.
Although more than 20 AEDs have been approved, the presence of drug resistance
and lose control of seizures is still the big issue for current AEDs. Therefore, it is still
necessarily urgent to develop novel AEDs with stronger effect and better safety.
The voltage gated potassium channels (Kv) are widely expressed in the central
and peripheral nervous system and play important roles in many physiological
processes such as the regulation of action potential duration, electrical property of
neurons and neurotransmitter release [5]. Kv7 (KCNQ) belongs to the Kv super-family
and it consists of five subtypes. Among them, Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3)
are mainly expressed in brain, which are mostly associated with epilepsy. The
activation of Kv7 can cause the decrease of neuronal excitability, which is confirmed
to be effective treatment for seizures [6-8 ]. Thus Kv7 has emerged as a potential target
for the development of AEDs [9]. Retigabine (RTG) is the first approved Kv7 opening
agent for the treatment of epilepsy. Besides, other agents in research such as
NS-15370, SF-0034, and PF-05020182 were also reported [10-13]. Therefore, there is
still enthusiastic interest to discover new chemical structures as Kv7 opening agents
for treating epilepsy.

Figure 1. Structures of some compounds targeting Kv7.

In our previous work for the discovery of novel AEDs, we designed a series of
piperidine containing compounds as Kv7 openers based on the structure of RTG and
PF-05020182 using hybridization drug design strategy [14]. As a continuous
optimization, herein we described the design, synthesis and evaluation of a novel
series of N-phenylbutanamide derivatives as Kv7 openers for treatment of epilepsy. In
this series, different fused heterocycles were designed to fix at the N-phenyl tail
(Figure 2). Target compounds were examined for the Kv7 opening activity in vitro.
The potent compounds were further evaluated for anti-convulsant activity and safety
profile in vivo. Meanwhile, the in-vivo pharmacokinetic evaluation was also
performed.

Figure 2. Design of target compounds.

Table 1. Structures of target compounds.

Compound Structure Compound Structure

1 7

2 8

3 9

4 10
5 11

6 12

As shown in Scheme 1, the key intermediate M-2 was first constructed by

amidation of the starting material M-1 and 3,3-dimethylbutanoyl chloride. Then the
target compounds were obtained by Pd-catalyzed Buchwald coupling of M-2 with the
commercially available or newly prepared secondary amines.

Regents and conditions: i) 3,3-dimethylbutanoyl chloride, triethylamine, DCM, 0 ℃; ii) Pd2(dba)3,

X-Phos, t-BuOK, toluene, 110 ℃.

Scheme 1. The synthetic route of target compounds.

Rb+ flow test is an efficient and reliable method in the high throughput screening
of potasium channel regulators [15, 16]. The target compounds were initially evaluated
by Rb+ flow test, and the results are summarized in Table 2. Selected compounds were
further evaluated by Thallium test and patch-clamp test to verify their activities on
KCNQ2.
It was found that most of the tested compounds showed stronger activity than
RTG in Rb+ flow test. Compound 1 with a thieno[3,2-c]piperidine fragment gave an
EC50 value of 0.02 μM, about 17 fold enhancement compared with that of RTG. When
electron-withdrawing substituents trifluoromethyl or cyano group were introduced to
the ortho-position of sulfur atom, an significant decrease in activity was observed for
compounds 2 and 3 compared with compound 1. Although less potent than compound
1, compounds 2 and 3 were still superior to RTG. A methyl group with electron
donating effect was introduced to the meta-position of sulfur atom to obtain
compound 4 with an EC50 value of 0.3 μM which was equivalent to RTG. But in
comparation with compound 1, compound 4 still afforded a reduced activity.
We then changed the position of sulfur to lead to compounds 5-7, which
containing a thieno[2,3-c]piperidine fragment. Compound 5 showed an equivalent
activity to compound 1. For compound 6, the introduction of electron-withdrawing
fluorine to the ortho-position of sulfur was detrimental to the activity. Compared with
RTG or compound 5, a nearly 10 fold or 150 fold decrease in activity was observed
for compound 6, respectively. While the introduction of electron donating methyl
group in compound 7 slightly influenced the activity compared to compound 5.
Compound 8 was designed as bioisostere of compound 1. As shown in Table 2, a
slightly decrease in activity was observed for compound 8 with an EC50 of 0.034 μM
compared with compound 1 (0.02 μM). To explore the structure activity relationship
of this fragment, the 1-benzazepine which containing a seven membered ring was
introduced in replacement of the piperidine fragment to obtain compounds 9-12.
These compounds were substituted by electron withdrawing halogen or electron
donating methyl group on 7-position of benzazepine ring. As shown in Table 2,
except for the bromo-substituted compound 11, other compounds all exhibited
stronger activity than RTG but weaker than compound 1. The fluoro and chloro
substituted compounds 9 and 10 were apparently superior to bromo-substituted
compound 11. In particular, as observed for compound 12, the replacement of halogen
with an electron donating methyl group significantly elevated the activity.
Table 2. Rb+ flow test results of target compounds.

Rb+ flow test Rb+ flow test

Compound Compound
EC50(μM) EC50(μM)

RTG 0.35 7 0.03

1 0.02 8 0.034

2 0.12 9 0.25

3 0.29 10 0.14

4 0.30 11 1.08

5 0.02 12 0.04

6 2.93

Compounds 1, 5 and 8 with best activity in Rb+ flow test were further evaluated
by Thallium test to confirm the KCNQ2 opening activity [17]. As shown in Table 3,
compound 1 was the most potent one with a two fold activation ratio of RTG. The
isomer compound 5 also presented a 1.5 fold stronger activity than RTG. But it was
unexpected that compound 8 showed very weak activity in this test. These three
compounds were also evaluated by patch-clamp test to verify their channel opening
activity [17]. As shown in Table 4, compounds 1 and 8 were comparable to RTG. And
compound 1 was able to obviously elevate the –⊿V1/2.

Table 3. Thallium test results of target compounds.

Thallium test Thallium test

Compound KCNQ2 activation ratio Compound KCNQ2 activation ratio

(%, at 10 μM) (%, at 10 μM)

RTG 40.21 5 64.80

1 84.24 8 4.87
 Table 4. Patch-clamp test results of target compounds.

patch-clamp test patch-clamp test

Compound –⊿V1/2 Compound –⊿V1/2

I/I0 I/I0
(mV) (mV)
RTG 2.12 + 0.32 40.00 + 2.80 5 0.67 + 0.07 53.31 + 2.08

1 1.63 + 0.11 66.21 + 5.55 8 1.65 + 0.09 46.10 + 4.01

Based on the above results in vitro, compound 1 was advanced to evaluation in

vivo. The anti-convulsant evaluation were conducted in maximal electroshock (MES)
test in mouse [19]. Mice presented hind legs rigidity after stimulation were regarded as
epilepsy positive. Rotating-rod test was performed to evaluate the neurotoxicity.
Compound 1 was dosed at 0.3 mg/kg, 1 mg/kg, 3 mg/kg and 5 mg/kg while RTG was
dosed at 25 mg/kg. As shown in Table 5, compound 1 gave a significant
anti-convulsant activity with a calculated EC50 of 1.83 mg/kg in MES test while no
adverse effects were observed during rotating-rod test and the pharmacokinetic
experiment in rats. The anti-convulsant activity of compound 1 at a dosage of 5 mg/kg
was close to that of RTG at 25 mg/kg.

Table 5. Anti-convulsant and neurotoxic results of compound 1.

Anti-convulsant activity Neurotoxic activity

No. of No. of No. of No. of

Compound Dose (i.p.) % %
mice mice with mice mice

tested EP tested falling

RTG 25 mg/kg 20 3 15 20 0 0

1 5 mg/kg 10 2 20 10 0 0
1 3 mg/kg 10 2 20 10 0 0

1 1 mg/kg 10 9 90 10 0 0

1 0.3 mg/kg 10 7 70 10 0 0

To evaluate brain permeability of compound 1, drug concentrations in brain and

plasma at 5 min, 30 min and 2 h after administration (i.v. or i.g.) were determined in
mice. The concentration ratio of brain and plasma were calculated. As shown in Table
6 and Table 7, compound 1 was able to quickly enter brain and keep a stable
distribution between brain and plasma within 2 h.
Table 6. Concentrations in brain and plasma of compound 1 by i.v.(1.5 mg/kg).

Time 2h 30 min 5 min

Mice No. 1 2 3 1 2 3 1 2 3

Brain (ng/g) 1.28 0.97 1.55 15.79 16.35 24.89 145.82 112.99 160.72

Mean 1.27 19.01 139.84

Plasma (ng/mL) 8.65 5.91 8.99 116.70 129.68 163.90 877.79 625.84 807.94

Mean 7.85 136.76 770.52

Mean Ratio 0.16 + 0.01 0.14 + 0.01 0.18 + 0.02

Table 7. Concentrations in brain and plasma of compound 1 by i.g.(15 mg/kg).

Time 2h 30 min 5 min

Mice No. 1a 2 3 1 2 3 1 2 3

Brain (ng/g) 5.41 4.52 22.55 26.37 41.19 25.93 21.46 27.70

Mean 4.96 30.04 25.03

Plasma (ng/mL) 61.74 44.09 173.77 227.55 329.07 226.10 172.31 203.43

Mean 52.91 243.46 200.61

Mean Ratio 0.09 + 0.01 0.12 + 0.01 0.12 + 0.01

a: Mouse died after administration.

Table 8. Rat PK profile of compound 1 by i.v..

Rat

(1 mg/kg, i.v., 5% EtOH/10% Hydroxypropyl-beta-cyclodextrin )

compound
T1/2 Tmax Cmax AUClast Vz_obs CL_obs MRTlast

(h) (h) (ng/mL) (h*ng/mL) (mL/kg) (mL/h/kg) (h)

1 5.49 0.03 1057.80 687.21 11249.55 1416.50 2.17

Pharmacokinetic properties of compound 1 was also evaluated in rat by

intravenous injection (i.v.). As shown in Table 8, at a dosage of 1 mg/kg, compound 1
gave a favorable half life (T1/2 = 5.49 h) and moderate plasma exposure (AUClast =
687.21 h*ng/mL) and clearance (CL_obs = 1416.50 mL/h/kg). The above in vitro and
in vivo evaluation results indicated that compound 1 could serve as a potential lead for
development of AEDs.

In this research, we designed, synthesized and evaluated a novel series of

N-phenylbutanamide derivatives as KCNQ openers for treatment of epilepsy. These
compounds were firstly evaluated for their potassium ion channel opening activity in
vitro, some compounds were found to be potent KCNQ openers. Compound 1 with
favorable in-vitro activity was submitted to evaluation in vivo. Results showed that
compound 1 owned significant anti-convulsant activity with no adverse effects. It was
also found to posses favorable pharmacokinetic profiles in rat. This research may
provide novel potent compounds for the discovery of KCNQ openers in treating
epilepsy.

Abbreviations
KCNQ (Kv7), voltage gated potassium channel 7; RTG, Retigabine; FDA, Food and
Drug Administration; AEDs, anti-epileptic drugs; PK, pharmacokinetic; MES,
maximal electroshock; EP, epilepsy.
Supplementary Information.

References and Notes

1. Ngugi, A.K.; Bottomley, C.; Kleinschmidt, I.; Sander, J.W.; Newton, C.R. Epilepsia, 2010, 51,
883.
2. Fisher, R.S.; Acevedo, C.; Arzimanoglou, A.; Bogacz, A.; Cross, J.H.; Elger, C. E.; Engel Jr, J.;
Forsgren, L.; French, J. A.; Glynn, M.; Hesdorffer, D.C.; Lee, B.I.; Mathern, G.W.; Moshe,
S.L.; Perucca, E.; Scheffer, I.E.; Tomson, T., Watanabe, M.; Wiebe, S. Epilepsia, 2014,
55,475.
3. Scharfman, H.E. Curr. Neurol. Neurosci. Rep. 2007, 7, 348.
4. Emilien, G.; Maloteaux, J. M. Int. J. Pharmacol. Ther. 1998, 36, 181.
5. Jan, L.Y.; Jan, Y.N. J. Physiol. 2012, 590, 2591.
6. Robbins, J. Pharmacol. Ther. 2001, 90, 1.
7. Brown, D.A.; Passmore, G.M. Br. J. Pharmacol. 2009, 156, 1185.
8. Schroder, B.C.; Kubisch, C.; Stein, V.; Jentsch, T.J. Nature 1998, 396, 687.
9. Grunnet, M.; Strobak, D.; Houqaard, C.; Christophersen, P. Eur. J. Pharmacol. 2014, 726,
133.
10. Eskioqlou, E.; Perrenoud, M.P.; Ryvlin, P.; Novy, J. Curr. Pharm. Des. 2017, 23, 6389.
11. Dacoren, J.E.; Claffey, M.M.; Snow, S.L.; Reese, M.R.; Arora, G.; Butler, C.R.; Boscoe, B.P.;
Chenard, L.; DeNinno, S.L.; Drozda, S.E.; Duplantier, A.J.; Moine, L.; Rogers, B.N.; Rong,
S.B.; Schuyten, K.; Wright, A.S.; Zhang, L.; Serpa, K.A.; Weber, M.L.; Stolyar, P.; Whisman,
T.L.; Baker, K.; Tse, K.; Clark, A.J.; Rong, H.J.; Mather, R.J.; Lowe III, J.A. Bioorg.
Med.Chem. Lett. 2015, 25, 4941.
12. Dalby-Brown, W.; Jessen, C.; Hougaard, C.; Jensen, M.L.; Jacobsen, T.A.; Nielsen, K.S.;
Erichsen, H.K.; Grunnet, M.; Ahring, P.K.; Christophersen, P.; Strobak, D.; Jorgensen, S. Eur.
J. Pharmacol. 2013, 709, 52.
13. Kalappa, B.I.; Soh, H.; Duiqnan, K.M.; Furuya, T.; Edwards, S.; Tzinqounis, A.V.;
Tzounopoulos, T. J. Neurosci. 2015, 35, 8829.
14. Yang, S.N.; Lu D.Q.; Ouyang, P.K. Bioorg. Med.Chem. Lett. 2018, 28, 1731.
15. Wang, K.; McIlvain, B.; Tseng, E.; Kowal, D.; Jow, F.; Shen, R.; Zhang, H.; Shan, Q.J.; He, L.;
Chen, D.; Lu, Q.; Dunlop, J. Assay Drug Dev. Technol. 2004, 2, 525.
16. Qi, J.; Zhang, F.; Mi, Y.; Fu, Y.; Xu, W.; Zhang, D.; Wu, Y.; Du, X.; Jia, Q.; Wang, K.; Zhang,
H. Eur. J. Med. Chem. 2011, 46, 934.
17. Beacham, D.W.; Blackmer, T.; O' Grady, M.; Hanson, G.T. J. Biomol. Screen. 2010, 15, 441.
18. Jow, F.; Tseng, E.; Maddox, T.; Shen, R.; Kowal, D.; Dunlop, J.; Mekonnen, B.; Wang, K.
Assay Drug Dev. Technol. 2006, 4, 443.
19. Castel-Branco, M.M.; Alves, G.L.; Fiqueiredo, I.V.; Falcao, A.C.; Caramona, M.M. Methods
Find. Exp. Clin. Pharmacol. 2009, 31, 101.
Graphical abstract
Hightlights

A series of novel N-phenylbutanamide derivatives as KCNQ openers for treatment of

epilepsy were investigated.

KCNQ opening activity was evaluated in vitro and in vivo.

Compound 1 was found to possess significant anti-convulsant activity with good

safety profile and pharmacokinetic property.