Potent and selective inhibitors of the TASK-1 potassium channel through chemical optimization of a bis-amide scaffold

Article abstract of DOI:10.1016/j.bmcl.2014.06.032

TASK-1 is a two-pore domain potassium channel that is important to modulating cell excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for therapeutic benefit, its physiological role needs better characterization; however, designing selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1 selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective hit led to analog 35 which displays a TASK-1 IC ₅₀ = 16 nM with 62-fold selectivity over TASK-3 in an orthogonal electrophysiology assay.

Full text of DOI:10.1016/j.bmcl.2014.06.032

Accepted Manuscript
Potent and selective inhibitors of the TASK-1 potassium channel through chem-
ical optimization of a bis-amide scaffold
Daniel P. Flaherty, Denise S. Simpson, Melissa Miller, Brooks E. Maki, Beiyan
Zou, Jie Shi, Meng Wu, Owen B. McManus, Jeffrey Aubé, Min Li, Jennifer E.
Golden
PII:
S0960-894X(14)00660-X
http://dx.doi.org/10.1016/j.bmcl.2014.06.032
BMCL 21751
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 May 2014
9 June 2014
11 June 2014
Please cite this article as: Flaherty, D.P., Simpson, D.S., Miller, M., Maki, B.E., Zou, B., Shi, J., Wu, M., McManus,
O.B., Aubé, J., Li, M., Golden, J.E., Potent and selective inhibitors of the TASK-1 potassium channel through
chemical optimization of a bis-amide scaffold, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.06.032
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Potent and selective inhibitors of the TASK-1
potassium channel through chemical
optimization of a bis-amide scaffold
Leave this area blank for abstract info.
Daniel P. Flaherty, Denise S. Simpson, Melissa Miller, Brooks E. Maki, Beiyan Zou, Jie Shi, Meng Wu, Owen
B. McManus, Jeffrey Aubé, Min Li, and Jennifer E. Golden

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Potent and selective inhibitors of the TASK-1 potassium channel through
chemical optimization of a bis-amide scaffold
Daniel P. Flaherty a, Denise S. Simpson a, Melissa Miller b,c, Brooks E. Maki a, Beiyan Zou b,c, Jie Shi b,c
Meng Wu c, Owen B. McManus c, Jeffrey Aubé a, Min Li b,c, and Jennifer E. Golden a,
,
a University of Kansas Specialized Chemistry Center, Lawrence, KS 66047, USA
b Johns Hopkins University, School of Medicine, The Solomon H. Snyder Department of Neuroscience, Baltimore, MD 21205, USA
cJohns Hopkins Ion Channel Center, Baltimore, MD 21205, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
TASK-1 is a two-pore domain potassium channel that is important to modulating cell
excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for
therapeutic benefit, its physiological role needs better characterization; however, designing
selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this
study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1
selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective
hit led to analog 35 which displays a TASK-1 IC50 = 16 nM with 62-fold selectivity over TASK-
3 in an orthogonal electrophysiology assay.
Keywords:
TASK1
KCNK3
2009 Elsevier Ltd. All rights reserved.
selective potassium channel inhibitor
bis-amide
chemical probes; however, insight has been achieved with gene
knockout mouse models. For example, genetic deletion of murine
TASK-1 (K2P3.1, KCNK3) channels has been shown to result in
the development of severe hyperaldosteronism.10-12 In separate
studies, the block or knockout of TASK-1was associated with
increased cell death following stroke-related ischemia, suggesting
that these channels provided a neuroprotective effect.9,13-15 These
results were complimented by studies in which TASK-1 null
mice suffered less neurodegeneration in a multiple sclerosis
inflammation/autoimmune model.16,17 The intermediacy of
TASK-1 channels has also been demonstrated in pulmonary
arterial hypertension by means of inhibition by endothelin-1.18-20
Collectively, these outcomes have illuminated and driven efforts
to precisely decipher the contributions of TASK-1 channels to
various pharmacological conundrums. However, the realization
of selective chemical modulators is another approach that could
aid in deconvoluting redundant and complex cellular circuitry
related to TASK conductance.
Maintenance of the negative resting membrane potential of
excitable cells depends significantly on the widely distributed
family of two-pore domain potassium channels (K2P). At the
basal membrane potential, these channels remain constitutively
open, thus enabling potassium ion passage and generating a
background conductance that regulates cell excitability.1-6 The
family shows modulatory susceptibility to various stimuli such as
pH, temperature, neurotransmitters, and pharmaceuticals, but
appears relatively time-independent and nonresponsive to
changes in voltage.7,8 These factors, coupled with variable
expression levels, impart cells with the ability to finely tune ionic
gradients involved with cellular response. The contributions of
voltage-gated (Kv) and inwardly rectifying (Kir) potassium
channels in the establishment and maintenance of resting
membrane potential cannot be ignored; however, the distinctive
biophysical properties of the K2P channels along with their
modulation by physiochemical stimuli favor them in this role.4
Since their identification nearly twenty years ago,1-3 the K2P
subfamily of TASK (TWIK-related-acid-sensitive-K+) channels
has garnered much attention, in part, due to their high density in
tissues impacted by disease and the hypothesis that selective
manipulation of these channels may provide unique opportunities
for therapeutic intervention.9 Efforts to elucidate specific roles
for a particular TASK channel and its relationship to abberant
Selectivity is one of the main obstacles to identifying
compounds that are useful in the physiological examination of
TASK channels.21 For instance, though several TASK family
members are only distantly related,22-25 TASK-1 shares > 50%
amino acid sequence identity with TASK-3 (K2P9.1,
KCNK9),26,27 and has demonstrated similar co-expression
patterns,28,29 sometimes even resulting in heterodimerization.7,30
Nonetheless, promising inhibitory TASK-1 compounds31-34 have
cellular behavior have been complicated by the lack of selective
———
 Corresponding author. Tel.: 785-864-6114; fax: 785-864-8179; e-mail: jengolden@ku.edu

emerged, including the recent reports of biaryl derivative 1 (A-
1899)35 and Merck’s aminopyrimidines, represented by
compound 236 (Fig. 1). The Decher laboratory described A-1899
(1) with a TASK-1IC50 = 7 nM (CHO cells) and 10-fold
selectivity over TASK-3. Compound 2 showed reversed, 10-fold
selectivity towards TASK-3 with a disclosed TASK-1IC50 = 300
nM. Importantly, these moderately selective TASK-1
benchmarks have been embraced as useful pharmacological
tools37-39 though the search continues for high affinity, highly
discriminating TASK-1 inhibitors to aid in the unambiguous
interrogation of TASK-1 related mechanisms. With this in mind,
our team initiated a project aimed at identifying potent and
selective TASK-1 inhibitors.
the exchange of amino benzoic esters in place of a nitroaniline in
the first step, followed by saponification and implementation of
routine amino acid coupling conditions.
Scheme 1. General synthesis for the 1,3-bis-amide scaffold
Reagents: (a) 1,3-nitroaniline, triethylamine, acetonitrile, 70 °C, 3 h, 52 –
92%. (b) Raney nickel, NaBH4, methanol, 0 °C, 2.5 h, 85 – 91%. (c) aryl or
alkyl chloride, triethylamine, acetonitrile, 70 °C, 3 h, 15 – 67%.
Optimization was primarily driven by the cellular TASK-1
and TASK-3 TF assays, and all compounds were profiled at 30
M against the parental TASK-1 cells, as well as KCNQ2,
hERG, and Kir2.1 channels. Selected analogs were subsequently
evaluated
in
TASK-1
and
TASK-3
confirmatory
electrophysiology assays. For the first set of analogs, the parent
scaffold’s unsubstituted phenyl group remained fixed while
changes to the 3-methyl phenyl ring were explored (Table 1).
Figure 1. Reference TASK-1 and TASK-3 inhibitors
Table 1. TASK thallium flux assay data for 1,3-bis-amides
bearing 3-methylphenyl replacements
As part of the NIH Molecular Libraries Probe Production
Centers Network (MLPCN), a high throughput screen of the NIH
Molecular Libraries Small Molecule Repository (MLSMR) was
performed with 339,662 compounds by the Johns Hopkins Ion
Channel Center (JHICC). This campaign, executed to discover
selective TASK-3 inhibitors, included a counterscreen for TASK-
1 selectivity.40 TASK activity was assessed using CHO cells
expressing either TASK-1 or TASK-3 in a fluorescence-based,
thallium flux (TF) assay.41,42 For the most promising hit
compounds, activity for each channel was confirmed in separate
QPatch automated electrophysiology assays directly measuring
whole cell voltage in the same cell line.43 Confirmed hits were
then counterscreened against potassium voltage-gated channels
KCNQ2 (Kv7.2), and KCNH2 (hERG), along with the Kir2.1
channel, resulting in a few validated chemotypes as potential
candidates for further structure-activity relationship (SAR)
optimization. Bis-amide 3 (Fig. 2) was representative of one
TASK-1 selective hit scaffold obtained from this endeavor,
exhibiting TF assay IC50 values of 0.027 M and 0.50 M for
TASK-1 and TASK-3, respectively (19-fold selectivity), and
QPatch assay IC50 values of 0.075 M and 0.69 M for TASK-1
and TASK-3, respectively (9-fold selectivity). No inhibition was
observed for the other channels surveyed (IC50 values > 30 M
for KCNQ2, hERG, and Kir2.1 channels), and the hit rate against
other PubChem assays was less than 0.5%. Given its attractive
TASK-1 activity profile and synthetic feasibility, analogs were
made with the shaded regions of optimization in mind (Fig. 2).
thallium flux (TF)
structure
selectivity
assay dataa
TASK-1
channel
TASK-3
channel
TASK3
IC50/
TASK1
IC50
entry analog
R1
IC50
(M)
IC50
b
%
(M)
1
3
3-CH3-phenyl
97.4 0.027
91.9 0.10
0.50
19
2
3
7
2-CH3-phenyl
4-CH3-phenyl
phenylc
6.5
> 30
23
65
NA
> 1100
6.6
8
56.2
> 3
4
9
100 0.021
94.9 0.22
96.7 0.33
5
10
11
12
13
14
15
16
17
2-OCH3-phenyl
3-OCH3-phenyl
4-OCH3-phenyl
3-F-phenyl
1.5
6
4.3
13
7
97.9
> 3
2.1
0.47
> 670
64
8
78.8 0.045
77.2 0.31
97.3 0.50
99.8 0.052
> 30
20
9
4-F-phenyl
10
11
12
2-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
7.1
14
2.4
46
26.3
> 3
> 30
NA
13
14
15
16
17
18
19
20
21
22
18
19
20
21
22
23
24
25
26
27
2-CF3-phenyl
3-CF3-phenyl
4-CF3-phenyl
4-t-butyl-phenyl
4-N(CH3)2-phenyl
2-furyl
94.7 0.13
94.6 0.16
2.5
>30
> 30
3.2
19
> 190
NA
NA
> 110
160
32
33.7
22.0
> 3
> 3
87.3 0.26
93.2 0.037
72.9 0.80
95.0 0.029
95.6 0.16
94.3 0.086
65.6 1.29
91.3 0.25
94.8 0.079
> 30
5.9
3-furyl
25
2-thiophene
3-thiophene
2-pyridyl
0.97
5.6
33
Figure 2. Hit structure 3 with SAR focus highlighted.
35
The University of Kansas Specialized Chemistry Center (KU
SCC) prepared 117 compounds in the SAR campaign using
standard amide coupling chemistry (Scheme 1). Treatment of
1,3-nitroanilines with substituted benzoyl chlorides 4 afforded
nitro amides which were subsequently reduced with Raney nickel
and sodium borohydride to generate anilines 5. Aniline
intermediates were then coupled with differentially substituted
aryl or alkyl chlorides to generate the desired analogs 6. Reverse
amides were also synthesized for the 1,3-orientation about the
central phenyl ring. Analogous chemistry was utilized except for
2.5
29
23
24
25
28
29
30
3-pyridyl
4-pyridyl
cyclohexyl
>30
>30
5.48
>23
>120
69.4
aData was averaged from n > 2 experiments.
bPercent inhibition of TASK-1 at 3M.
cTASK1 parental cell inhibition was observed (84% at 3M, IC50 = 0.24 M).
NA = not applicable.

Overall, retention of TASK-1 potency pivoted on the steric
requirement of the substituted phenyl ring rather than the
substituent’s electronic nature. For instance, 4-substituted phenyl
analogs showed a > 10-fold loss of TASK activity compared to
the 3-methylphenyl containing parent 3, regardless of the para
substituent’s electron density (compounds 33, 37, 39, 41 and 44-
45). In most cases, 3-phenyl substitution was favored over other
patterns in terms of the greatest gains in combined potency and
TASK-1 selectivity (compounds 18, 21 and 24). The symmetrical
phenyl analog 9 looked promising with comparable potency to
the hit and little TASK-3 liability; however, it also inhibited non-
Interestingly, 4-phenyl substituted analogs in this series
showed some erosion of TASK-1 potency compared to hit 3;
however, TASK-3 inhibition was abolished in each case (> 30
M, Table 2, compounds 34, 37, 44, 48-49 and 52). While
bulkier 2-substituted phenyl groups were not well tolerated
(compounds 33 and 41), smaller and more polar 2- and 3-
substituted derivatives produced a robust boost in TASK-1
potency without a commensurate response in TASK-3 inhibition,
thus generating analogs with low nanomolar TASK-1 potency
and > 100-fold selectivity (Table 2, compounds 35, 43, and 46-
47, and 52). As with previous analogs, no significant inhibition
of KCNQ2, hERG, or Kir2.1 channels was observed with any of
these compounds (> 30 M) unless otherwise noted.
induced parental cells with an IC50 value
= 0.24 M.
Heterocyclic replacements were also examined. The 2-furyl and
2-thiophene derivatives were more potent than their respective 3-
aryl counterparts (23-26). Pyridine variants (27-29), while less
potent, afforded two of three analogs with respectable potency
that were devoid of TASK-3 liability. Several compounds were
identified with notably improved TASK-1 selectivity compared
to hit 3, and no significant inhibition of KCNQ2, hERG, or
Kir2.1 channels was observed (> 30 M).
Given the improvements observed with 2-alkoxyphenyl
analogs 35 and 39, additional compounds featuring this element
were explored (Table 3). Many of these potently inhibited
TASK1 (IC50 < 11 nM) with better selectivity over TASK-3 than
what was observed for the hit compound 3. While inhibition of
TASK-3 was not completely decoupled from enhancement of
TASK-1 potency, in most cases, the margin between the two was
expanded. One of the highlights of this group included the
symmetrical 2-methoxyphenyl bis-amide 53 whose profile
benefitted from a modest improvement in TASK-1 and a more
substantial loss of TASK-3 activity, resulting in selectivity >
190-fold. Attempts were made to capitalize on the finding that
incorporation of a pyridine appeared to abolish TASK-3 liability
(see compounds 28-29, Table 1, and 52 in Table 2); however, the
limitations in TASK-1 potency could not be overcome by the
installation of a 2-methoxy substituent (compound 61, Table 3).
Preliminary SAR analysis revealed that, despite the near
symmetrical nature of the scaffold, the dissimilar terminal aryl
moieties dramatically influenced potency and selectivity. As a
result, analogs were generated in which the 3-methylphenyl
group of hit 3 was held constant while replacements for the
unsubstituted phenyl ring were surveyed (Table 2).
Table 2. TASK thallium flux assay data for 1,3-bis-amides
bearing phenyl group replacements
thallium flux (TF)
structure
selectivity
Table 3. TASK thallium flux assay and selectivity data for 1,3-
bis-amides bearing dual terminal phenyl group replacements
assay dataa,b
TASK-3
IC50/
TASK-1
TASK-1
channel
TASK-3
channel
entry analog
thallium flux (TF)
structure
selectivity
assay dataa,b
R2
IC50
(M)
IC50
(M)
c
IC50
%
TASK-1 TASK-3
TASK-3
IC50/
entry analog
channel
channel
1
2
3
4
5
6
7
8
9
31
32
33
34
35
36
37
38
39
2-CH3-phenyl
3-CH3-phenylc
2-i-propyl-phenyl
4-t-butyl-phenyl
2-OCH3-phenyl
3-OCH3-phenyl
4-OCH3-phenyl
95.5 0.091
95.6 0.030
94.5 0.33
96.6 0.19
96.6 0.004
92.0 0.031
0.96
0.15
1.7
11
5.0
5.2
>160
100
69
TASK-1
IC50
R1
R2
IC50
(M)
IC50
(M)
c
%
2-OCH3-
phenyl
2-OCH3-
phenyl
>30
0.39
2.2
1
2
3
4
5
7
53
54
55
56
57
58
96.6 0.010
1.88
0.41
190
100
33
2-OCH3-
phenyl
2-OCF3-
phenyl
92.1 0.004
69.3
1.3
>30
2.3
>23
15
2-OCH3-
phenyl
2-OEt-
phenyl
92.2 0.002 0.066
2,5-di-OCH3-phenyl 93.2 0.15
2-OCH2CH3-phenyld 100 0.009
0.41
46
2-OCH3-
phenyl
2-CH3-
phenyl
100 0.073
100 0.007
100 0.011
2.2
1.0
31
10
11
12
40
41
42
2-OCF3-phenyl
93.4 0.029
0.75
2.7
26
13
58
2-isopropoxyphenyl 100 0.21
2-OCH3-
phenyl
2-i-propyl
phenyl
140
66
2-Cl-phenyl
92.0 0.023
1.34
2-OCF3-
phenyl
2-i-propyl
phenyl
13
14
15
16
17
18
19
20
21
22
43
44
45
46
47
48
49
50
51
52
3-Cl-phenyl
4-Cl-phenyl
3-F-phenyl
92.2 0.003
77.9 0.15
93.7 0.025
91.9 0.014
79.3 0.010
0.48
>30
0.60
1.4
160
>200
24
0.73
2-isopro-
poxy
phenyl
2-OCF3-
phenyl
8
59
93.4 0.001
0.11
110
2-CF3-phenyl
3-CF3-phenyl
4-CF3-phenyl
4-N(CH3)2-phenyl
2-furyl
100
550
NA
>200
19
5.5
2-OEt-
phenyl
2-CF3-
phenyl
9
60
61
92.8 0.002
90.0 0.18
0.30
2.43
150
20.2
>3
>30
>30
2.7
2-OCH3-
4-pyridyl
3-CH3-
phenyl
100 0.15
92.8 0.14
94.1 0.005
96.0 0.095
10
13.5
aData was averaged from n > 2 experiments.
bPercent inhibition of TASK-1 at 3M.
2-thiophene
4-pyridyl
0.33
> 30
66
> 310
aData was averaged from n > 2 experiments.
bPercent inhibition of TASK-1 at 3M.
chERG IC50 = 12.4 M.
Attention was shifted to the connectivity of the amide groups
to the central phenyl ring, though none were found to be superior
to those augmentations already discussed. The corresponding
NA = not applicable.

1,2-bis-amide featuring the same substituted phenyl moieties as
hit 3 showed a 10-fold loss in TASK-1 activity (IC50 = 0.26 M;
TASK-3 IC50 = 17.8 M). Reversing the amide on either side of
the parent structure 3 led to a > 40-fold loss in TASK-1 activity
(data not shown). While replacing the 3-tolyl amide of 3 with 3-
tolylsulfonamide eliminated TASK-3 activity (IC50 > 30 M),
TASK-1 potency was significantly diminished (IC50 = 1.5 M).
For the aqueous stability experiment, 100% of ML365 remained
after 48 hours. When exposed to a 5-fold concentration of DTT
for 8 hours, 100% of ML365 (35) was found without any
detectable adduct formation or dimerization.49
Table 5. Solubility data for select bis-amide TASK-1 inhibitors
pION buffer
pH 0/6.2/7.4
PBS buffer
pH 7.4
With a number of analogs demonstrating promising TASK-1
selectivity and potency in the TF assay, data confirmation was
sought in the electrophysiology assay for a select subset of
compounds (Table 4). With the exception of compounds 47 and
52, TASK-1 data between the TF and QPatch assays was
reasonably congruent (IC50 < 5-fold difference). A stronger
correlation between the two assays was observed for TASK-3,
differing by < 2.6-fold except for compound 39. Though the
QPatch analysis revealed reduced TASK-1 selectivity compared
to the TF assay, several bis-amides were found to have improved
selectivity compared to reported compound A-1899 (1).
Compound 35 was noteworthy in that it retained low nanomolar
activity (IC50 = 16 nM) in the TASK-1 QPatch assay with 62-fold
selectivity over TASK-3. Considering the potency and selectivity
profiles in both assays, compound 35 was chosen as a flagship
probe (ML365) which was worthy of more elaborate
characterization.
entry analog
g/mL
(M)
g/mL
(M)
R1
R2
3-CH3-
phenyl
<0.01 for all
(0.030 for all)
0.27
(0.82)
1
2
3
phenyl
2-OCH3-
phenyl
3-CH3-
phenyl
<0.01/0.06/0.07
(0.028/0.17/0.19)
0.010
(0.028)
35
3-CH3-
phenyl
30.9/31.4/31.4
(93.2/94.8/94.8)
8.2
(24.7)
3
4
52
60
4-pyridyl
2-OEt-
phenyl
2-CF3-
phenyl
0.07/0.05/0
(0.16/0.12/0)
0.38
(0.89)
2-OCH3-
4-pyridyl
3-CH3-
phenyl
43.4/46.7/44.3
((120.1/129.2/122.6)
0.36
(1.0)
5
61
In summary, a TASK-3 directed screening campaign revealed
a promising TASK-1-selective bis-amide scaffold as part of a
counterscreening effort. Guided by two differentiated cellular
assays, optimization led to several potent TASK-1 inhibitors with
the benefit of substantially improved selectivity over the related
TASK-3 channel and no liability on KCNQ2, hERG, and Kir2
channels. Compared to reported inhibitors which have maximally
shown 10-fold TASK-1 selectivity, compound 35 (ML365)
demonstrated > 60-fold selectivity for TASK-1 over the related
TASK-3 channel when assessed by an orthogonal
electrophysiological assay. Ultimately, the discovery of 35 and
its analogs represents an important milestone in the development
of useful agents with sufficient specificity to better define the
pharmacological role of the TASK-1 channel. The preliminary
SAR supports that future endeavors focused on additional bis-
amide derivatives featuring key functionality will likely lead to
compounds with improved solubility and superior TASK-1
selectivity.
Table 4. Correlation of TASK TF and QPatch assay data
thallium flux
TF assay
selectivity
QPatch
QPatch
selectivity
(TF)
assay dataa
assay dataa
entry analog
TASK-1 TASK-3
channel channel
TASK-1 TASK-3
channel channel
TASK-3
IC50/
TASK-3
IC50/
TASK-1
IC50
TASK-1
IC50
IC50
(M)
IC50
(M)
IC50
(M)
IC50
(M)
1
2
3
4
5
6
3
0.027
0.004
0.009
0.029
0.010
0.095
0.50
0.39
0.41
0.75
5.5
19
100
46
0.075
0.016
0.011
0.060
0.37
0.69
0.99
0.038
0.99
3.94
12.5
9
35
39
40
47
52
62
34
16
11
6
26
550
> 310
> 30
2.14
Acknowledgments
7
8
53
60
0.010
0.002
1.88
0.30
190
150
0.030
0.010
0.71
0.25
24
25
The authors gratefully acknowledge funding from the
National Institutes of Health, grant numbers R03 MH090849-01,
R03 MH090837-01 and R03 DA031670-01 to M. Wu and partial
funding in support of chemistry performed at the University of
Kansas Specialized Chemistry Center (NIH U54HG005031) and
screening performed at the Johns Hopkins Ion Channel Center
(NIH U54 MH084691). The authors also express their sincere
appreciation to Dr. Douglas A. Bayliss, University of Virginia
Health System, for the donation of stably KCNK3-expressing
CHO cells.
aData was averaged from n > 2 experiments.
The bis-amide series was constructed to refine the overall
activity profile; however, some architecture was assembled with
physiochemical properties in mind. The initial hit compound (3)
was poorly soluble in aqueous phosphate buffer saline (PBS) and
at all three pH levels of pION buffer.44,45 Incorporation of alkyl
ethers and pyridine derivatives improved aqueous solubility
compared to the hit compound 3 (Table 5). Solubility of
compound 35 (probe ML365) was also assessed in assay media.
For the TF assay medium46 compound 35 was determined to have
a kinetic solubility reading of 0.47 g/mL, or 1.30 M. Solubility
of 35 in QPatch assay medium47 was found to be 0.17 g/mL, or
0.47 M. Though these are modest values, the solubility for 35
was 325- and 29-fold over the compound’s IC50 values for the TF
and QPatch assays, respectively.
Supplementary Material
The synthesis50 of compound 35 is generally representative of
most analogs discussed in this manuscript; however,
supplementary material associated with this article, including
general synthetic methods and biological assay protocols that are
not available in cited references, can be found in the online
version, at: X
Probe ML365 (35) was also evaluated for aqueous stability in
PBS, for susceptibility to nucleophilic addition and formation of
conjugates when treated with thiol-bearing dithiothreitol (DTT).48

References and notes
(23) Decher, N.; Maier, M.; Dittrich, W.; Gassenhuber, J.;
Bruggemann, A.; Busch, A. E.; Steinmeyer, K. FEBS Lett.
2001, 492, 84.
(24) Girard, C.; Duprat, F.; Terrenoire, C.; Tinel, N.; Fosset,
M.; Romey, G.; Lazdunski, M.; Lesage, F. Biochem.
Biophys. Res. Comm. 2001, 282, 249.
(25) Kim, D.; Gnatenco, C. Biophys. Res. Comm. 2001, 284,
923.
(26) Kim, Y.; Bang, H.; Kim, D. J. Biol. Chem. 2000, 275,
9340.
(27) Meadows, H. J.; Randall, A. D. Neuropharmacol. 2001,
40, 551.
(28) Karschin, C.; Wischmeyer, E.; Preisig-Muller, R.; Rajan,
S.; Derst, C.; Grzeschik, K. H.; Daut, J.; Karschin, A. Mol.
Cell. Neurosci. 2001, 18, 632.
(29) Talley, E. M.; Solórzano, G.; Lei, Q.; Kim, D.; Bayliss, D.
A. J. Neurosci. 2001, 21, 7491.
(30) Czirják, G.; Enyedi, P. J. Biol. Chem. 2002, 277, 5426.
(31) Mathie, A.; Veale, E. L. Curr. Opin. Invest. Drugs 2007, 8,
555.
(1) Goldstein, S. A. N.; Bockenhauer, D.; O'Kelly, I.;
Zilberberg, N. Nat. Rev. Neurosci. 2001, 2, 175.
(2) Talley, E. M.; Sirois, J. E.; Lei, Q.; Bayliss, D. A. The
Neuroscientist 2003, 9, 46.
(3) Lesage, F.; Lazdunski, M. Am. J. Physiol. Renal Physiol.
2000, 279, F793.
(4) Bayliss, D. A.; Barrett, P. Q. Trends Pharmacol. Sci. 2008,
29, 566.
(5) Bayliss, D. A.; Talley, E. M.; Sirois, J. E.; Lei, Q.
Respiration Physiol. 2001, 129, 159.
(6) Kim, D. Curr. Pharm. Des. 2005, 11, 2717.
(7) Talley, E. M.; Bayliss, D. A. J. Biol. Chem. 2002, 277,
17733.
(8) Patel, A. J.; Honore, E. Trends Neurosci. 2001, 24, 339.
(9) Es-Salah-Lamoureux, Z.; Steele, D. F.; Fedida, D. Trends
Pharmacol. Sci. 2010, 31, 587.
(10) Heitzmann, D.; Derand, R.; Jungbauer, S.; Bandulik, S.;
Sterner, C.; Schweda, F.; El Wakil, A.; Lalli, E.; Guy, N.;
Mengual, R.; Reichold, M.; Tegtmeier, I.; Bendahhou, S.;
Gomez-Sanchez, C. E.; Aller, M. I.; Wisden, W.; Weber,
A.; Lesage, F.; Warth, R.; Barhanin, J. EMBO J. 2008, 27,
179.
(11) Davies, L. A.; Hu, C.; Guagliardo, N. A.; Sen, N.; Chen,
X.; Talley, E. M.; Carey, R. M.; Bayliss, D. A.; Barrett, P.
Q. Proc. Natl. Acad. Sci. 2008, 105, 2203.
(12) Guagliardo, N. A.; Yao, J.; Bayliss, D. A.; Barrett, P. Q.
Mol. and Cell. Endocrinol. 2011, 336, 47.
(13) Muhammad, S.; Aller, M. I.; Maser-Gluth, C.;
Schwaninger, M.; Wisden, W. Neurosci. 2010, 167, 758.
(14) Bittner, S.; Budde, T.; Wiendl, H.; Meuth, S. G. Brain
Pathol. (Zurich, Switzerland) 2010, 20, 999.
(15) Meuth, S. G.; Kleinschnitz, C.; Broicher, T.; Austinat, M.;
Braeuninger, S.; Bittner, S.; Fischer, S.; Bayliss, D. A.;
Budde, T.; Stoll, G.; Wiendl, H. Neurobiol. Dis. 2009, 33,
1.
(16) Meuth, S. G.; Bittner, S.; Meuth, P.; Simon, O. J.; Budde,
T.; Wiendl, H. J. Biol. Chem. 2008, 283, 14559.
(17) Bittner, S.; Meuth, S. G.; Gobel, K.; Melzer, N.;
Herrmann, A. M.; Simon, O. J.; Weishaupt, A.; Budde, T.;
Bayliss, D. A.; Bendszus, M.; Wiendl, H. Brain 2009, 132,
2501.
(18) Olschewski, A.; Li, Y.; Tang, B.; Hanze, J.; Eul, B.; Bohle,
R. M.; Wilhelm, J.; Morty, R. E.; Brau, M. E.; Weir, E. K.;
Kwapiszewska, G.; Klepetko, W.; Seeger, W.; Olschewski,
H. Circ. Res. 2006, 98, 1072.
(19) Tang, B.; Li, Y.; Nagaraj, C.; Morty, R. E.; Gabor, S.;
Stacher, E.; Voswinckel, R.; Weissmann, N.; Leithner, K.;
Olschewski, H.; Olschewski, A. J. Resp. Cell Mol. Biol.
2009, 41, 476.
(20) Seyler, C.; Duthil-Straub, E.; Zitron, E.; Gierten, J.;
Scholz, E. P.; Fink, R. H. A.; Karle, C. A.; Becker, R.;
Katus, H. A.; Thomas, D. British J. Pharmacol. 2012, 165,
1467.
(21) Beltran, L. R.; Dawid, C.; Beltran, M.; Gisselmann, G.;
Degenhardt, K.; Mathie, K.; Hofmann, T.; Hatt, H. Front.
Pharmacol. 2013, 4, 141.
(32) Maingret, F.; Patel, A. J.; Lazdunski, M.; Honore, E.
EMBO J. 2001, 20, 47.
(33) Brendel, J.; Goegelein, H.; Wirth, K.; Kamm, W.; Gmbh,
S.-A. D., Ed. Germany, 2007; WO 2007124849.
(34) Brendel, J.; Goegelein, H.; Wirth, K.; Kamm, W.; [DE]),
S.-A. D. G. F., Ed. United States, 2009; US 2009149496.
(35) Streit, A. K.; Netter, M. F.; Kempf, F.; Walecki, M.;
Rinné, S.; Bollepalli, M. K.; Preisig-Müller, R.; Renigunta,
V.; Daut, J.; Baukrowitz, T.; Sansom, M. S. P.; Stansfeld,
P. J.; Decher, N. J. Biol. Chem. 2011, 286, 13977.
(36) Coburn, C. A.; Luo, Y.; Cui, M.; Wang, J.; Soll, R.; Dong,
J.; Hu, B.; Lyon, M. A.; Santarelli, V. P.; Kraus, R. L.;
Gregan, Y.; Wang, Y.; Fox, S. V.; Binns, J.; Doran, S. M.;
Reiss, D. R.; Tannenbaum, P. L.; Gotter, A. L.; Meinke, P.
T.; Renger, J. J. ChemMedChem 2012, 7, 123.
(37) Schiekel, J.; Lindner, M.; Hetzel, A.; Wemhoner, K.;
Renigunta, V.; Schlichthorl, G.; Decher, N.; Oliver, D.;
Daut, J. Cardiovas. Res. 2013, 97, 97.
(38) Cotten, J. F. Anesth. Analg. 2013, 116, 810.
(39) Bittner, S.; Bauer, M. A.; Ehling, P.; Bobak, N.; Breuer, J.;
Herrmann, A. M.; Golfels, M.; Wiendl, H.; Budde, T.;
Meuth, S. G. Exp. Neurol. 2012, 238, 149.
(40) A manuscript describing the screening and development
efforts leading to a TASK-3 selective probe (ML308) is in
preparation.
(41) Weaver, C. D.; Harden, D.; Dworetzky, S. I.; Robertson,
B.; Knox, R. J. J. Biomol.Screen. 2004, 9, 671.
(42) Center, J. H. I. C. In PubChem; March 28, 2013 ed.;
National Center for Biotechnology Information:
https://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=652
212, 2013, AID 652212.
(43) Kutchinsky, J.; Friis, S.; Asmild, M.; Taboryski, R.;
Pedersen, S.; Vestergaard, R. K.; Jacobsen, R. B.;
Krzywkowski, K.; Schroder, R. L.; Ljungstrom, T.; Helix,
N.; Sorensen, C. B.; Bech, M.; Willumsen, N. J. Assay
Drug Dev. Technol. 2003, 1, 685.
(44) Solubility data was obtained from the Sanford Burnham
Medical Institute Exploratory Pharmacology Core under
the direction of Dr. Layton Smith. Assays were performed
by A. Mangravita-Novo.
(22) Reyes, R.; Duprat, F.; Lesage, F.; Fink, M.; Salinas, M.;
Farman, N.; Lazdunski, M. J. Biol. Chem. 1998, 273,
30863.

was capped and heated at 80 °C for 5 h. After cooling to
room temperature, the solution was concentrated, and the
residue was dissolved in CH2Cl2 (10 mL). The organic
extract was washed with saturated aqueous NaHCO3 (2x 8
(45) Kinetic solubility assessment was performed in Pion
Prisma HT Universal buffer (pION) and in phosphate
buffer saline (PBS) at room temperature (23 °C). PBS is
137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate
dibasic, and 2 mM potassium phosphate monobasic.
(46) TF assay medium conditions: 1x HBSS, pH 7.4
(47) QPatch assay medium conditions:14 mM NaCl, 140 mM
KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 10 mM HEPES,
pH 7.4
(48) Compound 35 was dissolved at 10 micromolar in 1:1
PBS/acetonitrile at pH 7.4 (1% DMSO) and incubated at
room temperature with no nucleophile present or 50
micromolar dithiotreitol.
mL), separated, and dried (MgSO4). Filtration and
concentration generated a residue that was purified by
reverse-phase MPLC (10 - 100% acetonitrile:water) to
afford
2-methoxy-N-(3-(3-
methylbenzamido)phenyl)benzamide (0.16 g, 0.43 mmol,
33 % yield). 1H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H),
8.25 (dd, J1 = 7.8 Hz, J2 = 1.9 Hz, 1H), 8.14 (br. s, 1H),
8.12 (br. t, J = 2.0 Hz, 1H), 7.67 (br. s, 1H), 7.66 – 7.59
(m, 2H), 7.51 – 7.46 (m, 1H), 7.39 – 7.30 (m, 4H), 7.14 –
7.09 (m, 1H), 7.03 (br. d, J = 8.4 Hz, 1H), 4.05 (s, 3H),
2.59 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.00,
163.37, 157.19, 138.89, 138.74, 138.56, 134.86, 133.31,
132.50, 132.42, 129.50, 128.56, 127.70, 124.06, 121.59,
121.56, 116.16, 115.76, 111.89, 111.49, 56.23, 21.32.
LCMS retention time: 3.154 min. LCMS purity at 214 nm:
100%. HRMS: m/z calcd for C22H20N2O3 (M + H+)
361.1474, found 361.1557.
(49) Assays were performed by Patrick Porubsky at the
University of Kansas Specialized Chemistry Center.
Experimental details can be found in the supplemental
material.
(50) Synthesis of compound 35: Step 1, preparation of 3-
methyl-N-(3-nitrophenyl)benzamide: To a vial containing
acetonitrile (10 mL) was added 3-nitroaniline (0.50 g, 3.62
mmol), NEt3 (0.56 mL, 3.98 mmol), and 3-methylbenzoyl
chloride (0.50 mL, 3.80 mmol). The vial was capped, and
the resulting solution was heated at 75 °C for 3 h. After
cooling to room temperature, the solution was
concentrated, the residue was dissolved in CH2Cl2 (10 mL)
and then it was washed with saturated aqueous NaHCO3 (2
x 5 mL). The separated organic extract was dried (MgSO ,
)
4
filtered and adsorbed onto silica. Purification by MPLC (0
-
30% EtOAc:hexanes) afforded 3-methyl-N-(3-
nitrophenyl)benzamide (0.86 g, 3.36 mmol, 93% yield) as
an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ
10.67 (s, 1H), 8.81 (t, J = 2.2 Hz, 1H), 8.21 (ddd, J = 8.2,
2.2, 1.0 Hz, 1H), 7.97 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.86 -
7.75 (m, 2H), 7.67 (t, J = 8.2 Hz, 1H), 7.52 - 7.41 (m, 2H),
2.42 (d, J = 0.8 Hz, 3H). Step 2, preparation of N-(3-
aminophenyl)-3-methylbenzamide: To a 500 mL round-
bottom
flask
was
added
3-methyl-N-(3-
nitrophenyl)benzamide (0.86 g, 3.36 mmol) in MeOH and
CH2Cl2 (1.5:1, 16.5 mL total). The reaction was cooled to
0 °C and Raney Nickel (0.020 g, 0.34 mmol) was added to
the flask.
After portionwise addition of sodium
borohydride (0.32 g, 8.39 mmol), the reaction mixture was
allowed to warm to room temperature and then stirred for 3
h. The solution was filtered through Celite to remove any
residual Raney Nickel, and the collected filtrate was
diluted with CH2Cl2 (15 mL). After washing with water
(2x 15 mL), the CH2Cl2 layer separated, dried (MgSO ,
)
4
filtered and adsorbed onto silica. Purification by MPLC (0
- 10% MeOH:CH2Cl2) afforded N-(3-aminophenyl)-3-
methylbenzamide (0.69 g, 3.03 mmol, 90% yield). 1H
NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 7.77 – 7.65
(m, 2H), 7.44 – 7.30 (m, 2H), 7.11 (t, J = 2.1 Hz, 1H), 6.96
(t, J = 7.9 Hz, 1H), 6.85 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H),
6.31 (ddd, J = 7.9, 2.2, 1.1 Hz, 1H), 2.40 (s, 3H). Step 3,
preparation
methylbenzamido)phenyl)benzamide (35).
of
2-methoxy-N-(3-(3-
To vial
a
containing acetonitrile (10 mL) was added N-(3-
aminophenyl)-3-methylbenzamide (0.30 g, 1.32 mmol),
NEt3 (0.28 mL, 1.98 mmol) and 2-methoxybenzoyl
chloride (0.22 mL, 1.45 mmol), and the resulting solution