Synthesis and Optimization of Kv7 (KCNQ) Potassium Channel Agonists: The Role of Fluorines in Potency and Selectivity

Article abstract of DOI:10.1021/acsmedchemlett.9b00097

Based on the potent K_v7 agonist RL-81, we prepared new lead structures with greatly improved selectivity for K_v7.2/K_v7.3 over related potassium channels, i.e., K_v7.3/K_v7.5, K_v7.4, and K_v7.4/7.5. RL-36 and RL-12 maintain an agonist EC_2x of ca. 1 μM on K_v7.2/K_v7.3 in a high-throughput assay on an automated electrophysiology platform in HEK293 cells but lack activity on K_v7.3/K_v7.5, K_v7.4, and K_v7.4/7.5, resulting in a selectivity index SI > 10. RL-56 is remarkably potent, EC_2x 0.11 ± 0.02 μM, and still shows an SI = 2.5. We also identified analogues with significant selectivity for K_v7.4/K_v7.5 over K_v7.2/K_v7.3. The extensive use of fluorine in iterative core structure modifications highlights the versatility of these substituents, including F, CF₃, and SF₅, to span orders of magnitude of potency and selectivity in medicinal chemistry lead optimizations.

Full text of DOI:10.1021/acsmedchemlett.9b00097

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Letter
Synthesis and Optimization of Kv7 (KCNQ) Potassium Channel
Agonists: The Role of Fluorines in Potency and Selectivity
Ruiting Liu, Thanos Tzounopoulos, and Peter Wipf
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00097 • Publication Date (Web): 08 May 2019
Downloaded from http://pubs.acs.org on May 8, 2019
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ACS Medicinal Chemistry Letters
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Synthesis and Optimization of K
The Role of Fluorines in Potency and Selectivity
v
7 (KCNQ) Potassium Channel Agonists:
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†
‡
†,
Ruiting Liu, Thanos Tzounopoulos, Peter Wipf *
†Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260
‡Department of Otolaryngology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15260
KEYWORDS. K 7, KCNQ, potassium channel agonist, retigabine, RL-81
v
ABSTRACT: Based on the potent K
7.2/K 7.3 over related potassium channels, i.e. K
ca. 1 µM on K 7.2/K 7.3 in a high-throughput assay on an automated electrophysiology platform in HEK293 cells, but lack activity
on K 7.3/K 7.5, K 7.4, and K 7.4/7.5, resulting in a selectivity index SI>10. RL-56 is remarkably potent, EC2x 0.11±0.02 µM, and
still shows an SI=2.5. We also identified analogs with significant selectivity for K 7.4/K 7.5 over K 7.2/K 7.3. The extensive use of
, and SF , to span
v
7 agonist RL-81, we have prepared new lead structures with greatly improved selectivity for
K
v
v
v
7.3/K 7.5, K 7.4, and K 7.4/7.5. RL-36 and RL-12 maintain an agonist EC2x of
v
v
v
v
v
v
v
v
v
v
v
v
v
fluorine in iterative core structure modifications highlights the versatility of these substituents, including F, CF
orders of magnitude of potency and selectivity in medicinal chemistry lead optimizations.
3
5
Potassium channels represent the most numerous group
among the ca. 150 cation channels expressed in the human ge-
nome. As such, potassium channels perform a multitude of
physiological responses, such as the frequency and duration of
action potentials in the brain and heart, muscle contraction,
transmitter release, and hormonal secretion. In this realm, the
SMB-1, g-aminobutyric acid (GABA), gabapentine, ICA-27243,
diclofenac, BMS-204352, celecoxib, ML-213, NS-1643, and
compound 14 are representative structures for the chemical
3
,6,7,8
space covered by small molecule K
v
7 activators (Figure 1).
H
N
H
N
H
N
O
O
O
v
five distinct voltage-dependent K 7 channel subunits, encoded
O
O
O
N
H
N
NH
2
N
H
NH
2
N
NH
2
by the KCNQ genes in humans, are of considerable interest to
medicinal chemists. To significant extent, this is due to their
close association with various diseases, as well as the tractabil-
ity of their pharmacological response to small-molecule modi-
F
F
F
Flupirtine
Retigabine
P-Retigabine
O
O
N
N
H
N
1
fiers. Epilepsy, neuropathic pain, tinnitus, migraine, and cardi-
O
O
F
O
H
2
N
2
CO H
O
ovascular, metabolic and psychiatric diseases are frequently as-
N
H
NH
2
N
H
N
2
F
sociated with defective K
v
7 channels. The K
v
7.1 subtype is pri-
F
SF-0034
Acrylamide (S)-1
SMB-1
Gabapentin
marily located in the cell membranes of cardiac tissue, and is
critical for the repolarization of the cardiac action potential; ab-
O
errations can lead to a prolongation of the QT interval. K
v
7.2-
Cl
OH
MeO
MeO
O
Cl
O
H
N
N
O
K
v
7.5 are active in neuronal tissue, and K 7.4 and K 7.5 are also
v
v
F
F
N
N
NH
N
H
N
H
2
N
S
N
N
expressed in skeletal and smooth muscle cells. The neuronal
channels are assembled as homo- or heterotetramers, i.e. they
Cl
OMe
O
CF
3
ICA-27243
Diclofenac
PF-05020182
Celecoxib
v v v v v v
form K 7.2/K 7.3, K 7.3/K 7.4, K 7.4/K 7.5, etc, channel com-
Cl
1
,3
H
N
plexes.
O
O
MeO
OH HO
O
Cl
F
Ph
K
v
7.2/K 7.3 heterotetramers are slow-activating, voltage-de-
v
NH
OH
O
F
3
C
N
N
H
CF
3
F
pendent, non-inactivating, potassium channels that are open at
hyperpolarizing (subthreshold) potentials. Accordingly, they
control the subthreshold membrane potential and serve as pow-
erful brakes on neuronal firing activity. Genetic or acquired re-
F
3
C
N
H
H
F
BMS-204352
ML-213
NS-1643
Compound 14
v
Figure 1. Structures of K 7 channel activators.
ductions in the activity of K
native K 7 currents in neurons, are linked with brain disorders
that are characterized by neuronal hyperexcitability, including
v v
7.2/K 7.3 heterodimers, underlying
4
Pharmacological activation of K 7 channels by retigabine, an
v
v
FDA approved anti-epileptic drug that serves as a pan-agonist
5
v v
at K 7.2-K 7.5 channels, prevents seizures, neuropathic pain
anxiety, mania, tinnitus and ADHD. Consequently, K
7.5 channel activators are capable to compensate for a con-
stitutive decline in K 7 channel activity, and thus decrease neu-
ronal excitability and block neurotransmitter release.
Flupirtine, retigabine, P-retigabine, SF-0034, acrylamide (S)-1,
v
7.2-
9
,10
and the development of tinnitus. In the CNS of vertebrates,
7.2/K 7.3 (KCNQ2/3) propagate the M-current, a musca-
rinic-inhibited trigger of neuronal excitability. Moreover, natu-
ral, non-pharmacologically driven recovery in K 7.2/K 7.3
K
v
K
v
v
v
3
v
v
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channel activity is linked to resilience to noise-induced tinni-
Page 2 of 9
NHCO
2
Et
NHCO Et
2
CF
3
1
1
zone 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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tus. However, the possibility for severe side effects with re-
tigabine, including urinary retention, blue skin and retinal dis-
coloration resulted in a “black box” warning by the FDA for
this drug, at first limiting its use to patients who had not re-
sponded to alternative treatments, until the drug was discontin-
R'
F
5
S
N
H
NH2
R3
N
H
NH2
NHCO
2
R
N
H
NH
2
NR-45
NR-04
zone 2
NHCO
2
3
-i-Pr
NHCO
2
Et
zone 1
N
H
NH
2
N
H
NH
2
1
2
ued in 2017. We hypothesized that these undesirable side ef-
fects were due in part to the poor selectivity of retigabine among
F
F
F
F
C
RL-86
RL-81
K
v
7.2-K
v
7.5 channels, as well as the low solubility of its meta-
v
Figure 2. Structures of K 7 channel activators.
bolic degradation products. Interestingly, exposure of retiga-
bine to a hypervalent iodine reagent as a model for the oxidative
metabolism of xenobiotic substances generated the colored,
With RL-81 as a new lead structure, we focused our attention
on zone 2 modifications, in particular the position and the num-
ber of fluorine groups on the three unsubstituted carbons of the
triaminobenzene moiety, in part motivated by the goal to find
potent analogs where the dimerization site in 2 was blocked. We
have also re-optimized structural features of zone 1 and zone 3,
and developed a computational SAR model.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
2
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7
8
9
0
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2
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8
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0
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poorly soluble dimer 2, whose structure was confirmed by x-ray
analysis (Scheme 1). Dimer 2 has also been identified as an im-
1
4
purity in the retigabine API.
Scheme 1. Oxidation of retigabine and x-ray structure of sym-
metrical dimer 2.
The syntheses of RL-81 and 19 new analogs are summarized
in Schemes 2-5. S Ar of benzylamines 3a-3c with fluoroarenes
N
F
NHCO
2
Et
4a and 4b in the presence of catalytic iodine generated anilines
5a-d in 63-84% yield (Scheme 2). Reduction of the nitro group
and treatment with ethyl chloroformate led to RL-81, RL-73,
RL-02, and RL-72 in 47%, 45%, 37%, and 24% yield, respec-
tively. RL-81 was treated with Togni’s trifluoromethylating re-
PhI(O
2
CCH
3
)
2
H
N
NH
HN
2
N
H
NH
2
2
CH Cl
2
; 27%
F
EtO
2
CHN
F
Retigabine (1)
NH
NHCO Et
2
2
2
1
8
5
agent 6 in acetonitrile to obtain the R trifluoromethyl deriva-
tive RL-073, albeit in a low yield of 17%. For the preparation
of the zone 3 modified iso-propyl and cyclo-propyl carbamates
RL-32 and RL-56, nitroaniline 5a was reduced with Pd/C under
an atmosphere of hydrogen gas in ethanol, and the resulting di-
amine was selectively acylated with iso-propyl chloroformate
and cyclo-propyl chloroformate in 63% and 40% yield, respec-
tively.
The use of 2-fluoro-4-trifluoromethylbenzylamine 3d intro-
duced an additional fluorine group into the zone 1 moiety, and
in an analogous series of S Ar, zinc reduction, and car-
N
v v
To generate potent K 7.2/K 7.3-selective channel activators,
bamoylation transformations, ethyl carbamate RL-18 and cy-
clo-propyl carbamates RL-35, RL-36, RL-46, and RL-50 were
obtained in moderate to good yields (Scheme 3). In this series
of analogs, we also introduced up to two additional fluorine sub-
we previously partitioned retigabine’s chemical structure into
15
three distinct zones (Figure 2). In these preliminary structure-
activity relationship (SAR) studies, we mainly modified zones
1 and 3, and subsequently combined the beneficial modifica-
tions found for zones 1 and 3 with a previously known benefi-
cial modification on zone 2, which had also been used in the
5
6
stituents at zone 2 R and R positions.
10
development of the retigabine analog SF-0034. Our guiding
principle for these preliminary SAR studies was to modulate
both steric and, in particular, electronic features in zone 1 by
Scheme 2. Preparation of RL-81 and 6 structurally closely re-
lated analogs from mono-substituted benzylamines 3a-c.
R5
NO
2
R5
R2
NO
2
3 2
Et N, I (cat.)
introducing fluoride (F), trifluoromethyl (CF
3
) and pen-
NH
2
+
R2
N
H
NH
2
DMSO, 120 °C
63-84%
tafluorosulfanyl (SF
methyl” substituent.
selective benzylamines NR-45, NR-04, and RL-86 (Fig. 2).
Specifically, by introducing a CF -group at the 4-position of the
5
), which is considered a “super-trifluoro-
R1
F
NH
2
R4
16,17
R4
R1
This strategy led us to the potent and
1
5
3a (R
3
1
=CF , R
2
=H)
4a (R
4
=F, R
5
=H)
5a (R
1
1
=CF , R
2
,R
2
5
=H, R =F)
4
3
3
1
2
4
5
5
4
b (R =H, R =CF
3
)
4b (R =H, R =F)
5b (R ,R =H, R =CF
3
, R =F)
1
2
1
5
2
4
3
3c (R =H, R =SF
5
)
5
5c (R ,R =H, R =SF , R =F)
1
2
4
5
5
3
d (R =CF , R ,R =H, R =F)
benzylamine moiety, combined with a fluorine atom at the 3-
position of the aniline ring, we generated RL-81, a new
R5
NHCO
2
R7
1
2
4 2
. Zn, NH Cl, MeOH, H O, rt
or Pd/C, H , EtOH, rt
2
K
v
7.2/K
v
7.3-specific activator (EC50 = 190 nM) that was ca. 3
R2
R1
N
H
NH
2
times more potent than the most active retigabine analog, SF-
7
. ClCO
2
R , DIPEA, CH
2
Cl
2
R4
O
0
K
034 (EC50 = 0.60 µM), at shifting the voltage dependence of
0 °C to rt; 24-63%
1
5
1
2
5
4
7
RL-81 (R =CF
3
, R ,R =H, R =F, R =Et)
v
7.2/K
v
7.3 channels to more hyperpolarized potentials.
O
1
5
2
4
7
RL-73 (R ,R =H, R =CF
3
, R =F, R =Et)
I
Hence, RL-81 became a promising lead structure for the devel-
opment of clinical candidates for treating or preventing neuro-
logical disorders associated with neuronal hyperexcitability, in-
cluding tinnitus and epilepsy.
RL-02 (R
1
,R
5
=H, R
2
5
=SF , R
4
4
=F, R =Et)
7
6
1
2
5
7
CF
3
RL-72 (R =CF
3
, R ,R =H, R =F, R =Et)
1
5
2
4
7
MeCN, 85 °C; 17%
RL-073 (R ,R =CF
3
2
, R =H, R =F, R =Et)
5
1
4
7
RL-32 (R =CF
3
3
, R ,R =H, R =F, R =i-Pr)
1
2
5
4
7
RL-56 (R =CF
, R ,R =H, R =F, R =c-Pr)
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Scheme 3. Preparation of 5 analogs containing the 2-fluoro-4-
Scheme 5. Preparation of 3 analogs containing the 3-pyridyl-
methylamine and the 3-cyano-2-pyridylmethylamine moieties
in zone 1.
1
2
3
4
5
6
7
8
9
trifluoromethylbenzylamine moiety in zone 1.
R6
R6
R5
NO2
NH2
NO2
R5
NO2
NH2
NO2
Et
3
N, I
2
(cat.)
Et N, I2 (cat.)
NH2
+
NH2
3
+
N
H
N
H
NH2
F
DMSO, 120 °C
51-81%
F
NH2
DMSO, 120 °C
85%
F3C
F
F
F3C
N
F
F
F3C
F
F
F3C
N
5
6
5
6
3d
4a (R ,R =H)
5e (R ,R =H)
3g
4a
5m
5
6
5
6
4
4
4
c (R =F, R =H)
5f (R =F, R =H)
5
6
5
6
d (R =H, R =F)
5g (R =H, R =F)
NHCO
2
R7
5
6
5
6
e (R ,R =F)
5h (R ,R =F)
1. Zn, NH Cl, MeOH, H O, rt
4
2
2
or Pd/C, H , EtOH, rt
R6
N
NH
2
1
. Zn, NH
MeOH, H
4
Cl
5
6
7
7
H
RL-18 (R ,R =H, R =Et)
2
. ClCO
2
R , DIPEA, CH
2
Cl
2
F
R5
NHCO2R7
NH2
O, rt
5
6
7
F
3
C
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
RL-35 (R ,R =H, R =c-Pr)
0 °C to rt; 28-60%
5
6
7
RL-24 (R
RL-67 (R =c-Pr)
7
7
=Et)
RL-36 (R =F, R =H, R =c-Pr)
7
2. ClCO
2
R , DIPEA
, 0 °C to rt
20-55%
N
H
5
6
7
RL-46 (R =H, R =F, R =c-Pr)
RL-50 (R ,R =F, R =c-Pr)
CH
2
Cl
2
F
5
6
7
F3C
F
NO
2
CHO
NO
2
1. TsOH, xylene, 140 °C
+
A further diversification of the RL-81 lead structure was ac-
complished by the use of (5-(trifluoromethyl)pyridin-2-
yl)methanamine 3e and (3-fluoro-5-(trifluoromethyl)pyridin-2-
yl)methanamine 3f (Scheme 4). Reduction of the nitro group in
N
N
H
NH
2
F
3
C
H
2
N
NH
2
4
2. NaBH , MeOH, 0 °C
N
F
31%
3
F C
CN
F
CN
3h
4f
5n
NHCO Et
2
5
i, isolated in 75% yield from the coupling of 3e with 4a, with
Pd/C and acylation with ethyl chloroformate provided RL-31 in
3% yield. A zinc reduction and cyclo-propyl chloroformate
4 2
1. Zn, NH Cl, MeOH, H O, rt
N
H
NH
2
2
. ClCO
2
Et, DIPEA; 43%
N
F
3
F C
4
CN
RL-23
were used for the preparation of RL-68 in 23% yield from 5i.
The penta- and hexa-fluorinated intermediates 5j, 5k, and 5l
were obtained from the coupling of 3f with 4a, 4c and 4d in
45%, 58%, and 37% yield, respectively. Zinc reduction and
treatment with cyclo-propyl chloroformate then yielded the cor-
responding carbamates RL-96, RL-01, and RL-12 in 33%,
The biological evaluation of the new analogs, including RL-
1 as a positive standard, was performed in the Lilly OIDD pro-
8
2
0,21
gram.
Ion channel agonistic potency was determined in a
high-throughput assay using the automated electrophysiology
IonWorks Barracuda (IWB) platform in HEK293 cells (Table
37%, and 41%, respectively.
1). The resulting 10-point concentration-response curves pro-
Finally, we also synthesized analogs with a pyridine in zone
by coupling of amine 3g with nitrobenzene 4a, which gave
vide a measure of compound efficacy by determining the ago-
nist concentration that doubles the conductance at the voltage
1
S
N
Ar product 5m in 85% yield (Scheme 5). Reduction and ac-
ylation of 5m provided the carbamates RL-24 and RL-67 in
0% and 28% yield, respectively. The 2-pyridyl analog RL-23,
2
2
leading to 15% channel activation (EC2x). The lower the EC2x
value, the more potent the agonist.
6
Compared to the literature standards, flupirtine (FP) and re-
tigabine (RG), our previous lead structure RL-81 demonstrated
ca. 8-50 times higher (vs FP) or slightly higher (vs RG) efficacy
which also contained a cyano group next to the nitrogen of the
heterocycle as a chemical replacement mimicking a pyridazine
1
9
ring, was generated in an overall yield of 13% by a reductive
amination of pyridine 3h with aniline 4f, followed by zinc re-
duction and acylation.
at all tested K
mainly interested in optimizing central (K
ripheral (K 7.4/K 7.5) tissue activities, we also calculated a se-
lectivity index (SI), defined as the inverse ratio of the EC2x val-
v
7 channels (Table 1) in this assay. Since we were
v
7.2/K 7.3) over pe-
v
v
v
Scheme 4. Preparation of 5 analogs containing the 2-pyridyl-
methylamine moiety in zone 1.
ues
EC2x(K
for
these
two
channel
7.2/K 7.3); the higher this ratio, the
7.2/K 7.3 channels).
Interestingly, RL-81 (SI=0.3) proved to be considerably more
potent at K 7.4/K 7.5 than FP (SI=1.8), suggesting a potentially
types
(i.e.
SI=
v
7.4/K
v
7.5)/EC2x(K
v
v
R6
R3
R6
R5
better the selectivity of the agent for the K
NO
2
v
v
R5
R
3
NO
2
3 2
Et N, I (cat.)
NH
2
+
N
H
NH
2
N
3
DMSO
0-120 °C
37-75%
F
NH
2
v
v
F
3
C
N
F
6
F
3
F C
adverse property profile, and setting the stage for further me-
dicinal chemistry optimizations. One of our first modifications
5
6
3
5
6
3
e (R =H)
4a (R ,R =H)
5i (R ,R ,R =H)
3
5
6
3
5
6
3f (R =F)
4c (R =F, R =H)
5j (R =F, R ,R =H)
5
6
3
5
6
4
d (R =H, R =F)
5k (R ,R =F, R =H)
was to move the CF
to the meta-position. To our delight, the resulting analog, RL-
3, was twice as potent at K 7.2/K 7.3 than RL-81, while sub-
stantially decreasing activity at K 7.4/K 7.5, resulting in a su-
perior SI=4.7. These findings were further substantiated by the
SF -containing analog RL-02, which showed an equivalent
3
group in zone 1 in RL-81 from the para-
3
6
5
5
l (R ,R =F, R =H)
R6
7
v
v
1. Zn, NH
or Pd/C, H , EtOH, rt
2
4
Cl, MeOH, H
2
O, rt
R5
NHCO
2
R7
R3
v
v
N
H
NH
2
7
2
. ClCO
0
2
R , DIPEA, CH
2
Cl
2
N
F
°C to rt; 23-43%
F
3
C
5
3
5
6
7
channel activity profile to RL-73, and also had a more favorable
SI=1.8. Since structurally the only difference between RL-73
and RL-02 is the switch from a meta-CF - to a meta-SF -
3 5
RL-31 (R ,R ,R =H, R =Et)
3
5
6
7
RL-68 (R ,R ,R =H, R =c-Pr)
3
5
6
7
RL-96 (R =F, R ,R =H, R =c-Pr)
3
5
6
7
RL-01 (R ,R =F, R =H, R =c-Pr)
3
6
5
7
RL-12 (R ,R =F, R =H, R =c-Pr)
substituent in zone 1, these assay data further validate the po-
tential for biological mimicry between the trifluoromethyl and
1
6,17
the pentafluorosulfanyl groups.
7.2/K 7.3 vs K 7.4/K 7.5 selectivity is high, both RL-73 and
RL-02 have low EC2x values for K 7.3/K 7.5 and K 7.4 in the
.2-0.6 µM range, comparable to RL-81’s EC2x of 0.29 and 0.10
Interestingly, while their
K
v
v
v
v
v
v
v
0
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µM. Therefore, we continued our structure-activity studies and
prepared several zone 2 and zone 3 modifications of RL-81.
and the ortho-aniline nitrogen. We briefly also explored the pos-
sibility that a hindered rotation at the zone 1 benzylamine moi-
ety could be responsible for the lack of activity of the o,o’-di-
substituted aniline RL-50. However, a potential energy analysis
(HF-6-31G*) suggested a barrier of only 1.5-2.5 kcal/mol for
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
5
Moving the fluorine atom from R to R in RL-72 had a det-
rimental effect on the EC2x for K 7.2/K 7.3, reducing it 5-fold
vs RL-81 to 1.26 µM. However, the selectivity was now superb,
with EC2x’s of >10, 4.65, and >10 µM for K 7.3/K 7.5, K 7.4,
and K 7.4/7.5, respectively, accounting for an SI>7. In contrast,
installing a CF
v
v
2
the C-NH-CH -C dihedral angle for both RL-50 and the o-
v
v
v
monosubstituted reference compound RL-46, and geometri-
v
21
5
cally similar energy minima. Accordingly, we hypothesize
that K 7.2-5 channel potency is largely due to the electro-
3
-group at the R position in RL-073 completely
abrogated activity at all channel types. This position appears to
be very sensitive to steric bulk.
Surprisingly, changing the ethyl carbamate in zone 3 to an
iso-propyl carbamate slightly increased the potency of the re-
v
static/H-bonding interactions at the carbamate oxygen and the
ortho-aniline, with decreased electron density being detri-
mental. In contrast, steric and conformational effects at the car-
bamate and the benzylamine moieties appear to influence chan-
nel selectivity. This rationalization is also supported by litera-
ture evidence. Based on mutation and modeling studies, the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
v v
sulting RL-32 at K 7.2/K 7.3, but vastly decreased the
K
v
7.4/7.5 EC2x to 4.98 µM, leading to an SI=31 – 100x better
than the SI=0.3 of RL-81. The SI=2.5 of the corresponding cy-
clo-propyl carbamate analog, RL-56, fits this trend, since the
steric dimensions of a cyclo-propane are roughly in between
those of the iso-propane and the ethane groups. RL-56 proved
tryptophan residue W236 in K
tively, W265, W242, W235 in K
v
7.2 (or, analogously and respec-
7.3, K 7.4, K 7.5) is thought
v
v
v
to be critical for binding, and participates in hydrogen bonding
interactions with carbamate or amide groups of small molecule
to be the most active analog on K
with an EC2x of 0.11±0.02 µM. However, both RL-32 and RL-
6 also still showed <0.4 µM EC2x potencies at K 7.3/K 7.5 and
7.4 channels.
For the next SAR iteration, we introduced a 2-fluoro-4-tri-
v v
7.2/K 7.3 prepared to date,
2
3,24
agonists.
v
In contrast, K 7.1, which lacks a corresponding W
residue, is not activated by retigabine-type molecules.
5
v
v
K
v
fluoromethylbenzylamine moiety in zone 1, and systematically
varied fluorinations in zone 2. The parent compound in this se-
ries, RL-18, was both potent, EC2x=0.18±0.11 µM, and moder-
ately selective, SI=1.7 (Table 1). Changing the ethyl to the cy-
clo-propyl carbamate decreased potency and SI (i.e. RL-35),
5
6
but, as previously found, adding fluorine at R or R , recovered
a high selectivity, generating an SI>10 and >7, respectively, for
NHCO
2
Et
RL-81
EC2x 0.26 µM
RL-36 and RL-46. In agreement with the data found for RL-
N
H
NH
2
5
F
7
2, the R -fluorinated RL-36 also lacked agonist activity at
3
F C
K
v
7.3/K
v
7.5, K
v
7.4, and K
v
7.4/7.5, and, due to its remaining
7.2/K 7.3, RL-36 therefore
sub-micromolar EC2x=0.93 at K
v
v
represents an overall significant improvement over RL-81. In
contrast, the hepta-fluorinated RL-50 lost all activity at these
channels.
In a final round of SAR investigations, we replaced the tri-
fluorobenzene in zone 1 with trifluoromethylated pyridines. In-
F
F
NHCO
2
c-Pr
RL-50
EC2x >10 µM
itially, we focused on the 2-pyridyl analogs RL-31, RL-68, RL-
N
H
NH
2
3
9
6, RL-01, RL-12 and RL-23. With the exception of the R -
F
F
3
C
F
fluorinated RL-96 and RL-12, these analogs demonstrated ei-
3
4
6
ther low selectivity or low potency. The R ,R ,R -trifluorinated
RL-12, in particular, maintained a respectable 1.00 µM EC2x at
K 7.2/K 7.3, with an SI>10 and no detectable agonist activity
v v
at other channels. Compared to their 2-pyridyl isomers RL-31
and RL-68, the 3-pyridyl analogs RL-24 and RL-67 showed
slightly increased selectivity, but essentially equivalent potency,
demonstrating that among the studied chemotypes, the fluorina-
tion pattern was the most significant determinant of selectivity
and activity.
F
NHCO
2
c-Pr
RL-46
EC2x 1.47 µM
N
H
NH
2
F
F
3
C
F
Since substitution with fluorine has profound effects on the
electron distribution in conjugated p-systems, we examined the
electrostatically encoded electron-density surfaces of RL-81 vs
RL-50, which has additional fluorinations at the benzylamine
Figure 3. Maps of electron-density surface encoded with elec-
trostatic potential for RL-81 (top) and RL-50 (middle), and
RL-46 (bottom). Colors reflects a property range of +170
2
5
5
6
kJ/mol (blue) to -310 kJ/mol (red).
moiety and, especially, at R and R of the triaminobenzene,
rendering it inactive at all K 7 channel types (Figure 3). Most
v
significantly, the electron density at the carbamate oxygen and
the ortho-aniline nitrogen is significantly decreased as a result
of the two additional fluorinations at the triaminobenzene moi-
ety in RL-50. In comparison, RL-46, which only has one addi-
An alternative, but possibly complementary, interpretation of
the differential selectivities observed for fluorinated and heter-
ocyclic analogs of retigabine and RL-81 could be their prefer-
ence for different binding sites on the channels, and hence dif-
ferent mechanisms of action. While retigabine binds to the pore
6
tional fluorine atom at R vs RL-81 and is still quite active,
shows an intermediate electron density at the carbamate oxygen
v v
domain on K 7.2-K 7.5, other compounds have been suggested
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2
6
to target the voltage-sensing domain. In the future, we plan to
perform experimental and computational studies to further in-
vestigate this hypothesis on our lead compounds.
1
2
3
4
5
6
7
8
9
Table 1. Efficacy of RL-81 Analogs in Human K
v
7 Channel Conductance Studies
R6
R5
NHCO R7
R3
Y
2
R2
N
H
NH2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R4
R7
X
R1
cmpd
X
Y
R1
R2
R3
R4
R5
R6
K
v
7.2/3
EC2x±SD EC2x±SD EC2x±SD
µM] [µM] [µM]
K
v
7.3/5
K
v
7.4
K
v
7.4/5
SI
EC2x±SD
[µM]
[
FP
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
N*
H
F
F
F
H
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
F
Et
Et
Et
Et
Et
Et
Et
2.00±0.69 1.58±0.69 5.62±1.86
0.33±0.08 0.34±0.04 ND
3.50±2.68
ND
1.8
ND
0.3
4.7
1.8
>7
RG
RL-81 CH
RL-73 CH
RL-02 CH
RL-72 CH
RL-073 CH
RL-32 CH
RL-56 CH
RL-18 CF
RL-35 CF
RL-36 CF
RL-46 CF
RL-50 CF
CF
H
3
0.26±0.13 0.29±0.06 0.10±0.04
0.14±0.05 0.20±0.08 0.40±0.13
0.36±0.25 0.21±0.11 0.65±0.39
0.09±0.04
0.66±0.01
0.65±0.28
>10
CF
3
H
SF
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1.26±0.31
>10
>10
>10
4.65±0.95
>10
CF
H
H
H
H
F
3
>10
ND
31
i-Pr 0.16±0.11 0.16±0.10 0.19±0.10
c-Pr 0.11±0.02 0.23±0.10 0.38±0.30
4.98±1.12
0.28±0.03
0.30±0.26
0.35±0.17
>10
2.5
1.7
0.6
>10
>7
Et
0.18±0.11 0.34±0.01 0.11±0.04
c-Pr 0.59±0.08 0.47±0.01 0.21±0.19
c-Pr 0.93±0.21
c-Pr 1.47±0.22
>10
>10
>10
>10
0.55±0.14
>10
H
F
>10
F
c-Pr
>10
>10
ND
0.9
1.2
>7
RL-31
RL-68
RL-96
RL-01
RL-12
RL-23
N
N
N
N
N
N
H
H
H
F
H
H
H
H
F
Et
0.66±0.25 0.79±0.26 0.85±0.14
0.59±0.34
0.96±0.05
>10
c-Pr 0.79±0.03 0.72±0.05 0.90±0.02
c-Pr 1.39±0.20
c-Pr >10
c-Pr 1.00±0.50
>10
>10
>10
>10
>10
>10
F
>10
ND
>10
1.5
2.1
1.9
F
H
H
H
H
>10
CCN CF
H
H
H
H
H
H
Et
Et
1.60±0.19 1.30±0.16 2.17±0.37
0.61±0.34 0.69±0.35 0.95±0.13
2.33±0.89
1.27±0.05
1.36±0.70
RL-24 CH
RL-67 CH
N
N
CF
CF
c-Pr 0.71±0.23 0.69±0.19 1.13±0.72
4
N*: FP has a pyridine ring nitrogen at the R -substituted carbon position; EC2x data were obtained from 10-point concentration curves and 2-5
individual assay determinations, with the exception of FP, for which 6-431 repeats were determined; SD=standard deviation; SI=selectivity index=
v v
EC2x(K 7.4/5)/EC2x(K 7.2/3); ND=not determined.
In conclusion, we have prepared and characterized analogs of
the potent K 7 agonist RL-81, and obtained several new lead
structures with greatly improved selectivity for K 7.2/K 7.3
over the other tested potassium channels, i.e. K 7.3/K 7.5,
7.4, and K 7.4/7.5. Specifically, RL-36 and RL-12 maintain
an agonist EC2x of ca. 1 µM on K 7.2/K 7.3 in a high-through-
put assay on the automated IWB platform in HEK293 cells, but
lack activity on K 7.3/K 7.5, K 7.4, and K 7.4/7.5, resulting in
v v v v
K 7.2/K 7.3 over K 7.4/K 7.5, i.e. it is almost an order of mag-
v
nitude more selective than RL-81 (SI=0.3). Accordingly, RL-
56, RL-36, and RL-12 represent promising new lead structures
for the therapeutic use of selective potassium channel agonists
in epilepsy, neuropathic pain, anxiety, mania, ADHD, depres-
sion, migraines, and tinnitus. It is also worth mentioning that
the R substitution on RL-36 should prevent the oxidative di-
merization observed for RG; further studies to test this hypoth-
esis, including in vivo metabolite identification, are planned. In
a preliminary experiment, subjecting RL-36 and the R ,R -
difluorinated RL-50 to the oxidative conditions from Scheme 1
v
v
v
v
K
v
v
v
v
5
v
v
v
v
a selectivity index SI>10. We have also identified an analog of
RL-81, i.e. RL-56, with a ca. 3 times more potent EC2x of
0.11±0.02 µM. Furthermore, RL-56 also shows an SI=2.5 for
5
6
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did not produce a corresponding dimer product by LCMS anal-
Supporting Information
Page 6 of 9
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ysis, and starting materials were recovered in 43% and 51%
yield, respectively, based on NMR integration of the crude re-
action mixture in the presence of an internal standard. A meta-
bolic stability analysis of RG, RL-81, and eight analogs in
pooled human (HLM) and male mouse (MLM) liver micro-
somes suggested that the pyridine-containing RL-24 (HLM
Experimental details and H and 13C NMR spectra for new syn-
1
thetic intermediates and products. Assay information from the Eli
Lilly OIDD program, and details of dihedral angle conformational
analysis. The Supporting Information is available free of charge on
the ACS Publications website.
t
t
1/2=406 min) and the iso-propyl carbamate RL-32 (HLM
1/2=451 min) have longer half-lives than RL-81 (HLM t1/2=344
AUTHOR INFORMATION
min). Only the cyclo-propyl carbamate RL-36 (HLM t1/2=76
min) has a substantially shorter half-live than RL-81, but the
microsomal degradation of all tested analogs is significantly
faster than RG (HLM t1/2=970 min), which we hypothesize will
prove advantageous in overcoming RG’s undesired toxicities
(see Tables 1 and 2 in the Supporting Information for compre-
hensive HLM and MLM data).
Corresponding Author
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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1
2
3
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5
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7
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0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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6
7
8
9
0
*pwipf@pitt.edu.
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script.
Our SAR analysis demonstrates the ability of fluorine sub-
stituents to substantially alter the potency and selectivity of
pharmaceutical candidates. While the utility of fluorinated drug
candidates has been highlighted in particular for their increases
in metabolic stability, conformational effects, pKa modulation,
and for applications as bioisosteric replacements, fewer studies
Funding Sources
This work was supported in part by DOD Award
W81XWH1810623.
Notes
27
have focused on their effects on specific target affinity. The
CCDC 1899172 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_re-
quest@ccdc.cam.ac.uk, or by contacting The Cambridge Crystal-
lographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: +44 1223 336033
extensive use of fluorine as the guiding principle in iterative
core structure modifications in this work further supports the
versatility of fluorine-containing substituents, including F, CF
3
,
and SF , to span orders of magnitude of potency and selectivity
5
in lead optimization.
While our primary goal was the preparation of a K
with a high selectivity for K 7.2/K 7.3 over K 7.4/K
v v v v
v
7 agonist
7.5, we
note that this work also identified several analogs with signifi-
cant selectivity for K 7.4/K 7.5 over K 7.2/K 7.3, i.e. com-
pounds with an SI <1, such as RL-81 and RL-35. K 7.4 is the
primary potassium channel in the smooth muscle of the bladder,
ACKNOWLEDGMENT
v
v
v
v
OIDD screening data supplied courtesy of Eli Lilly and
Company—used with Lilly's permission, and gratefully
acknowledged. The authors thank T. S. Maskrey (University of
Pittsburgh) for project management and technical assistance, S. J.
Geib (University of Pittsburgh) for the x-ray analysis of 2, and J.
C. Burnett and R. Cao (University of Pittsburgh) for preliminary
v
2
8
v
where it serves to regulate contractility. Activation of K 7.4
leads to membrane hyperpolarization and a resultant loss of
contractile function, a possible etiology for the urinary retention
side effect of RG. While currently there is no clinical prece-
v
K 7 channel modeling studies.
v v
dence for selective agonists of K 7.4 and K 7.5, other medicinal
2
9
chemistry efforts toward this goal have been reported. There-
fore, RL-81 and RL-35 could form a foundation for the devel-
opment of much more selective K 7.4/K 7.5 activators, with po-
v v
tential applications for the treatment of visceral smooth muscle-
related diseases.
ABBREVIATIONS
KCNQ, potassium channel, voltage-gated, KQT-like subfamily;
ADHD, attention deficit hyperactivity disorder; HEK293, human
embryonic kidney 293; SAR, structure-activity relationship.
ASSOCIATED CONTENT
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SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”).
1
2
3
4
5
6
7
8
9
F
NHCO
2
Et
NHCO
2
-c-Pr
2
NHCO -i-Pr
F
N
H
NH
2
N
H
NH
2
N
H
NH
2
F
F
N
F
F
3
C
F
3
C
3
F C
RL-81
RL-56
RL-12
EC2x 1.0±0.50 µM; SI>10
EC2x 0.26±0.13 µM; SI=0.3
EC2x 0.11±0.02 µM; SI=2.5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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For information on the, as of 2/1/2019 discontinued, OIDD program, please see: https://openinnovation.lilly.com/dd/login.jsp.
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Physicochemical properties of agonists were calculated with Instant JChem 18.13.0 (ChemAxon, Cambridge, MA). A HF-6-31G* dihe-
dral angle/conformational energy analysis was performed with Spartan 18 v. 1.3.0 (Wavefunction, Inc, Irvine, CA).
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For a related, but antagonist assay set up, see: Priest, B. T.; Cerne, R.; Krambis, M. J.; Schmalhofer, W. A.; Wakulchik, M.; Wilenkin,
B.; Burris, K. D. Automated Electrophysiology Assays, In Assay Guidance Manual. Sittampalam, S.; Coussens, N. P.; Brimacombe, K.;
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Advancing Translational Sciences Bethesda (MD) 2018; https://www.ncbi.nlm.nih.gov/books/NBK53196/pdf/Bookshelf_NBK53196.pdf;
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Calculations performed with Spartan’10 (Wavefunction, Inc., Irvine CA), using the PM6 semiempirical parametrization set.
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Synthesis and Optimization of Kv7 (KCNQ) Potassium Channel Agonists: The Role of Fluorines in Potency
and Selectivity
Ruiting Liu,† Thanos Tzounopoulos,‡ Peter Wipf†,*
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