Design, synthesis and evaluation of substituted piperidine based KCNQ openers as novel antiepileptic agents

Article abstract of DOI:10.1016/j.bmcl.2018.04.040

Epilepsy is a kind of disease with complicated pathogenesis. KCNQ (Kv7) is a voltage dependent potassium channel that is mostly associated with epilepsy and thus becomes an important target in the treatment of epilepsy. In this paper, a series of substituted piperidine derivatives targeting KCNQ were designed and synthesized by using scaffold hopping and active substructure hybridization. Compounds were evaluated by fluorescence-based thallium influx assay, Rb⁺ flow assay and electrophysiological patch-clamp assay. Results showed that some compounds possessed more potent potassium channel opening activity than Retigabine. More significantly, compound 11 was found to have good pharmacokinetic profiles in vivo.

Full text of DOI:10.1016/j.bmcl.2018.04.040

Accepted Manuscript
Design, synthesis and evaluation of substituted piperidine based KCNQ openers
as novel antiepileptic agents
Shaoning Yang, Dingqiang Lu, Pingkai Ouyang
PII:
S0960-894X(18)30352-4
https://doi.org/10.1016/j.bmcl.2018.04.040
BMCL 25787
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 February 2018
30 March 2018
14 April 2018
Please cite this article as: Yang, S., Lu, D., Ouyang, P., Design, synthesis and evaluation of substituted piperidine
based KCNQ openers as novel antiepileptic agents, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.04.040
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Design, synthesis and evaluation of substituted piperidine
based KCNQ openers as novel antiepileptic agents
Shaoning Yang a, b, Dingqiang Lua, *, Pingkai Ouyanga
aSchool of Pharmaceutical Sciences, Nanjing University of Technology, Nanjing 211816, P.R.
China
bJiangsu Simcere Pharmaceutical Co. Ltd., Nanjing 210042, P.R. China
Abstract: Epilepsy is a kind of disease with complicated pathogenesis. KCNQ
(Kv7) is a voltage dependent potassium channel that is mostly associated with
epilepsy and thus becomes an important target in the treatment of epilepsy. In this
paper, a series of substituted piperidine derivatives targeting KCNQ were designed
and synthesized by using scaffold hopping and active substructure hybridization.
Compounds were evaluated by fluorescence-based thallium influx assay, Rb+ flow
assay and electrophysiological patch-clamp assay. Results showed that some
compounds possessed more potent potassium channel opening activity than
Retigabine. More significantly, compound 11 was found to have good
pharmacokinetic profiles in vivo.
Keywords: Epilepsy, KCNQ openers, Retigabine, Substituted piperidine.
*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 the world’s oldest recognized disease. It is a functional disorder of
the central nervous system caused by abnormal neuronal discharge in the brain. The
pathogenesis of epilepsy is very complicated and not very clear1-3. Ion channels play a
key role in regulating the excitability of neurons and are considered as effective
targets for treatment of epilepsy4-8. KCNQ (Kv7) is a voltage dependent potassium
channel and is a molecular basis of M channel. KCNQ (Kv7) is mostly responsible for
the regulation of neuronal excitability and frequency of nerve impulses. M channel is
mainly composed of KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) and mutations of these are
commonly found in epilepsy. Activation of KCNQ channels lead to hyperpolarization
of the membrane and decreased neuronal discharge, and is thus considered as a new
therapeutic method for epilepsy9-14. Retigabine (RTG) is the first KCNQ opener agent
and has been approved by EMA and FDA for the treatment of partial-onset seizures.
Therefore, KCNQ openers still attracted much attention for the treatment of epilepsy,
chronic pain and neuropathic pain6-8, 15, 16
.
Figure 1. Design of the target compounds.
Albeit the first approved KCNQ opener, RTG has several disadvantages, such as
instability under light, vulnerability in metabolism, changes in retinal pigment, as well
as other severe side effects17-20. To overcome these drawbacks, several medicinal
chemistry researches have been done based on the core structure of RTG. For example,

PF-05020182, a Kv7 channel opener developed by Pfizer, was designed by replacing
the aniline structure with a 4, 6-dimethoxypyrimidine while introducing an amide
instead of carbamate at the 5-position21, 22. Nevertheless, there is still a need for the
development of new generation KCNQ openers to improve the current weakness of
RTG. Based on the core structure of RTG and PF-05020182, we intended to design a
novel series of substituted piperidine derivatives by introducing the amide group in
PF-05020182 instead of the carbamate group in RTG, and meanwhile two methyl
groups were introduced into the core phenyl scaffold of RTG. Herein, we disclosed
the synthesis and biological evaluation of this series of substituted piperidine
derivatives as novel KCNQ channel openers.
Regents and conditions: i) 3, 3-dimethylbutanoyl chloride, triethylamine, DCM, 0oC; ii) Pd2(dba)3,
Xphos, t-BuOK, toluene, 110 oC; iii) pyridin-2-yl boronic acid, Pd(PPh3)2Cl2, K2CO3, DME-H2O,
80 oC; iv) H2, PtO2, HOAc, MeOH, r.t.
Scheme 1. The synthetic route of compounds 1-4, 6-8 and 16-20.
The target compounds were synthesized through three different routes. As shown
in Scheme 1, the key intermediate M-2 was first constructed by amidation of the
starting material M-1. Then the target compounds 1-3, 6-8, 16-20 were synthesized by
Pd-catalyzed Buchwald coupling reactions of M-2 with the commercially available
substituted piperidines or piperazines, and the target compound 4 was synthesized
through Pd-catalyzed Suzuki coupling reaction to connect with the piperidine 2-yl
group.

Regents and conditions: i) Pd(PPh3)2Cl2, K2CO3, DME-H2O, 80 oC; ii) H2, 10% Pd-C, MeOH; iii)
TFA, DCM, r.t.; iv) 4-fluoro-2,6-dimethyl-nitrobenzene, K2CO3, DMF, 100oC; v) sodium
hyposulfite, MeOH-H2O, sealed, 100oC; vi) 3,3-dimethylbutanoyl chloride, triethylamine, DCM,
0oC.
Scheme 2. The synthetic route of compounds 9-15.
As illustrated in Scheme 2, the substituted 4-phenylpiperidine intermediate M-6
was initially obtained by three-step reactions including Suzuki coupling-reduction,
catalytic hydrogenation and de-protection of Boc group from the commercially
available M-3, and then reacted with 4-fluoro-2, 6-dimethylnitrobenzene to afford
intermediate M-7. Subsequently, M-7 was reduced to generate the
piperidine-substituted 2, 6-dimethylaniline M-8, which then underwent amidation as
described above to give the target compounds 9-15.
Regents and conditions: i) n-BuLi, DMF, THF, -78 oC; ii) NaBH4, MeOH, 0oC; iii) MsCl, CH3CN,
0oC to r.t.; iv) piperidine/tetrahydroquinoline, K2CO3, CH3CN, 50oC; v) tetrahydroisoquinoline,

NaBH(OAc)3, 1,2-dichloroethane, 0oC.
Scheme 3. The synthetic route of compounds 5, 21 and 22.
As shown in Scheme 3, for introducing methylene group between the side chain
and the benzene ring, the key aldehyde intermediate M-9 was prepared by using
n-BuLi-DMF complex. The piperidine/quinolin-1-substituted target compounds 5 and
22 were obtained through the reduction of methanesulfonyl-nucleophilic substitution,
and the target compound 21 was then obtained through condensation-reduction of
M-9 with isoquinolines.
The activity of the newly synthesized compounds was initially evaluated on
KCNQ2 at 10μM by fluorescence-based thallium influx assay23. The results were
summarized as activation ratio in Table 1. When R was an un-substituted piperidine,
then the KCNQ2 activation ratio was 69.83%, which was better than that of RTG
(39.22%), while the piperidines ring was substituted with 4-dimethyl (2) or
4-carbonyl group (3), then the activation ratio of KCNQ2 was significantly increased
(100.00% and 110.34%). When the connecting position of the piperidine group was
shifted from position 1 to 2 (4) or when methylene group was inserted between the
piperidine group and the core (5), then the KCNQ2 activation of the compounds were
substantially lost (0.83% and 10.34%).
Various substituents on the piperidine ring were investigated to explore the
structure-activity relationship. Approximately complete loss of activity was found for
compounds 6 and 7, which contained an aliphatic alkaline group piperidine and
morpholine, respectively on C-4 position of piperidine. Compound 8 with a benzene
group in this position also failed to show obvious activation on KCNQ2. Introduction
of a fluorine atom at position 2 of the benzene ring (9) showed that the activation ratio
(43.96%) was retained same as that of RTG. The introduction of
2-trifluoromethylphenyl (10) pulled down the activation activity on KCNQ2, which
may be due to the greater steric hindrance of trifluoromethyl group. When the
substituents on the phenyl group were shifted from position 2- to 3, compounds 11

(3-F), 12 (3-CF3) and 13 (3-Cl) displayed significantly better activity than RTG
(3.7-fold to 4.2-fold).
Table 1. Activation activity of target compounds on KCNQ2
KCNQ2
KCNQ2
Compd
R
activation ratio Compd
(%, at 10μM)
R
activation ratio
(%, at 10μM)
1
2
69.83
100.00
110.34
0.83
12
13
14
15
16
17
18
19
20
21
22
149.12
168.20
41.20
88.34
29.74
42.64
52.06
88.34
0.28
3
4
5
10.34
0.02
6
7
1.13
8
0.63
9
43.96
-47.16
151.94
10
11
5.11
138.84

RTG
40.21
Furthermore, the activation activity on KCNQ2 was reduced (1.9-fold to
3.6-fold), but was still comparable to RTG when the substituent on the phenyl group
shifted from position 3- to 4, such as 4-CF3 (14) and 4-Cl (15). When the phenyl
group at position 4 of piperidine was replaced with pyridin-4-yl (16) or pyridin-3-yl
(17), the activity was less potent compared to 11-13, but was equivalent to RTG. The
structure-activity relationship of compounds 8-17 suggested that the 4-position of
piperidine was substituted by electron-deficient aryl group, showing more favorable
for activity.
Replacement of piperidine with piperazine produced compounds 18, 19 and 20.
Compared with compound 12, although a big loss in activity was observed for
compound 18, the activation ratio was still higher than that of RTG. In contrast, a
2-fold increase in activity was observed for compound 19 compared with 14. For the
pyridine substituted compound 20, no significant activity was obtained. An attempt
was made to introduce the fused benzene rings at 3, 4-position and 2, 3-position of the
piperidine ring to afford compound 21 and 22, respectively. Interestingly, 21 showed
no significant activity, while 22 exerted a 3.5-fold higher activity than RTG
(138.84%).
Table 2 Results of Rb+ flow by atomic absorption spectrophotometry and
electrophysiological patch-clamp test
Rb+ flow
patch-clamp test
Compd
EC50
Efficacy
–⊿V1/2
I/I0
(μM)
(%)
(mV)
RTG
0.35
1.58
210
210
2.12 + 0.32
1.53 + 0.23
40.0 + 2.8
25 + 3.0
2
0.13
0.22
0.33
148
180
160
NTa
NT
NT
3
NT
11
2.76 + 0.35 13.33 + 0.57
22
a NT means “Not Tested”.

Atomic absorption spectrophotometry, a faster and more reliable method, of the
Rb+ flow was conducted to further verify the activating activity on KCNQ224, 25
.
Compounds 2, 3, 11 and 22 were selected for evaluation. As shown in Table 2, all
these compounds presented significant activating activity on KCNQ2. The potency of
compound 2 was the same as that of RTG, showing an EC50 value of 1.58μM which
was about 5-fold that of RTG. For compounds 3, 11 and 22, the efficacy and EC50
values were both slightly lower than RTG.
Electrophysical patch clamp technology is the "gold standard" of ion channel
research and is an important technique for studying the ion channel function25, 26
.
Compounds 2 and 22 were further evaluated for their regulation on the ion channel by
patch-clamp technique. The results showed that compounds 2 and 22 led to
conductivity (I/I0) enhancement, and the activity of compound 22 was slightly better
than that of RTG. At the same time, compounds 2 and 22 also led to half-maximum
activation voltage left shift (-ΔV1/2) increase, which was similar to RTG.
Table 3 Rat PK profile of compound 11 by i.v.a
T1/2 Tmax
Cmax
AUClast
Vz_obs
CL_obs
MRTlast
(hr)
Compd
(hr) (hr) (μg/mL)
(hr*μg/mL)
(L/kg)
(L/hr/kg)
1.38 0.03
9.11
8.36
0.24
0.12
2.61
11
a 1 mg/kg, 5% EtOH/10% Hydroxypropyl-beta-cyclodextrin.
To investigate the drug-likeability of the active compounds, compound 11 was
further evaluated for the pharmacokinetic (PK) properties in rat. As shown in Table 3,
compound 11 displayed high exposure (8.36 hr×μg/mL) and peak concentration (9.11
μg/mL) at a dose of 1mg/kg i.v., while possessing moderate clearance (0.12 L/hr/kg)
and good half-life (1.38 hr). These results indicated that compound 11 showed
satisfactory metabolic properties, and hence deserved further research and
development as highly active lead compound.
In summary, a series of novel substituted piperidine derivatives as KCNQ
openers were investigated. Based on the core structure of RTG and the side chain of
PF-05020182, target compounds were designed by using scaffold hopping and active

substructure hybridization. The target compounds were synthesized through three
different routes according to the structural characteristics. Biological evaluation
showed some compounds with solid activating activities on KCNQ2 through thallium
influx assay, Rb+ flow assay and electrophysiological patch-clamp assay. Compound
11 possessed favorable PK profiles in rat. These results indicated that the substituted
piperidine derivatives were a series of potent KCNQ openers.
Abbreviations
KCNQ (Kv7), voltage gated potassium channel 7; KCNQ2 (Kv7.2), voltage gated
potassium channel 7.2; KCNQ3 (Kv7.3), voltage gated potassium channel 7.3; RTG,
Retigabine; EMA, European Medicines Agency; FDA, Food and Drug Administration;
PK, pharmacokinetic.
Supplementary Information.
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Graphical Abstract
Design, synthesis and evaluation of substituted piperidine based KCNQ openers
as novel antiepileptic agents
Shaoning Yang a, b, Dingqiang Lua, *, Pingkai Ouyanga
aSchool of Pharmaceutical Sciences, Nanjing University of Technology, Nanjing 211816, P.R.
China
bJiangsu Simcere Pharmaceutical Co. Ltd., Nanjing 210042, P.R. China
*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

Hightlights
A series of novel substituted piperidine derivatives as KCNQ openers for treatment of
epilepsy were investigated.
Some compounds were found to be potent KCNQ2 openers.
Compound 11 also possessed favorable PK profiles in rat.