Discovery and SAR of a novel series of GIRK1/2 and GIRK1/4 activators

Article abstract of DOI:10.1016/j.bmcl.2013.07.002

This Letter describes a novel series of GIRK activators identified through an HTS campaign. The HTS lead was a potent and efficacious dual GIRK1/2 and GIRK1/4 activator. Further chemical optimization through both iterative parallel synthesis and fragment library efforts identified dual GIRK1/2 and GIRK1/4 activators as well as the first examples of selective GIRK1/4 activators. Importantly, these compounds were inactive on GIRK2 and other non-GIRK1 containing GIRK channels, and SAR proved shallow.

Full text of DOI:10.1016/j.bmcl.2013.07.002

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5195–5198
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of a novel series of GIRK1/2 and GIRK1/4 activators
Susan J. Ramos-Hunter d,e, Darren W. Engers a,b,c, Kristian Kaufmann b, Yu Du b, Craig W. Lindsley a,b,c,d,e
,
C. David Weaver a,e, Gary A. Sulikowski d,e,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
d Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
e Vanderbilt Institute of Chemical Biology, Vanderbilt University, Vanderbilt University Medical Center, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes a novel series of GIRK activators identified through an HTS campaign. The HTS lead
was a potent and efficacious dual GIRK1/2 and GIRK1/4 activator. Further chemical optimization through
both iterative parallel synthesis and fragment library efforts identified dual GIRK1/2 and GIRK1/4 activa-
tors as well as the first examples of selective GIRK1/4 activators. Importantly, these compounds were
inactive on GIRK2 and other non-GIRK1 containing GIRK channels, and SAR proved shallow.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 14 May 2013
Revised 27 June 2013
Accepted 1 July 2013
Available online 18 July 2013
Keywords:
GIRK
Kir3.x
Activators
Thallium flux
The G protein regulated Inwardly Rectifying Potassium (K+)
(GIRK) channels are a family of inward-rectifying potassium chan-
nels also known as the Kir3 family, whose physiological role is to
modulate the excitability of the various cell types in which they
are expressed.1–4 Four GIRK channel subunits are expressed, as
either homo- or heterodimers, in mammals: GIRK1 (Kir3.1), GIRK2
(Kir3.2), GIRK3 (Kir3.3) and GIRK4 (Kir3.4). GIRK1–GIRK3 are the
predominant subunits in the CNS, with GIRK4 expressed at low
levels.1–7 Multiple lines of evidence support important roles for
GIRK in a variety of physiological processes including the control
of heart rate and electrical excitability in a variety of neuronal pop-
ulations, leading to postulates that GIRK channel modulation as
potential target for a variety of therapeutic indications including
pain, epilepsy, and reward/addiction.1–7 However, a complete lack
of selective and effective GIRK activators has prevented further tar-
get validation for the many indications where GIRK activation is
speculated to be of potential benefit.1–7 Indeed, the only com-
pounds that are known to activate GIRK are alcohols (e.g., ethanol)
at concentrations of hundreds of millimolar and, recently, the com-
pound naringin has been identified as a GIRK activator; however,
MLSCN10 mGlu8 screening project, where mGlu8 activity was mea-
sured via a thallium flux-based readout of GIRK1/GIRK2.11 Once
hits for mGlu8 (mGlu8/Gqi9 calcium flux assay) were triaged, multi-
ple hits remained that were putative GIRK activators with poten-
cies ranging from ꢀ1
lM to 10 lM. We recently reported on the
one series (Fig. 1) from this effort, represented by HTS hit 1
(VU0032230), that was subsequently optimized to afford the first
selective (ꢀ10-fold vs GIRK1/4, inactive on GIRK2/3 and inactive
F
O
O
N
N
N
F
N
H
N
H
N
N
H
N
H
1
2
, VU0032230
,VU0456810 (ML297)
GIRK1/2 EC50 = 0.16 µM, 194%
GIRK1/4 EC50 1.8 µM, 93%
inactive on GIRK2/3
GIRK1/2 EC50
= 1.4 µM, 97%
GIRK1/4 EC50 = 0.69 µM, 93%
=
inactive on GIRK2/3
the reported EC50 is in excess of 100 l
M.7–9
O
Based on this limitation in the field, we elected to develop a
suite of small molecule GIRK probes. Towards this goal, we identi-
fied GIRK activator leads by mining HTS hits obtained from an
N
S
O
N
H
O
Cl
3
, VU0259369
GIRK1/2 EC50 = 1.1 µM, 100%
GIRK1/4 EC50 = 1.1 µM, 104%
⇑
Corresponding author.
E-mail address: gary.a.sulikowski@vanderbilt.edu (G.A. Sulikowski).
inactive on GIRK2/3
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.002
Figure 1. Structures and GIRK activities of HTS leads 1 and 3, and the optimized
GIRK1/2 activator 2.

5196
S. J. Ramos-Hunter et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5195–5198
fragment libraries
on othe K+ channels) GIRK1/2 activator 2 (VU0456810, also known
alternate
as ML297).12 Importantly, 2 was centrally penetrant and estab-
lished anti-epileptic properties in mice via GIRK1/2 acitvation.
The HTS also identified another attractive, and structurally distinct,
dual GIRK1/2 and GIRK1/4 activator 3 (VU0259369), and in this let-
ter, we detail the synthetic strategy and SAR for this lead.
heteroaryls
substituents
O
N
S
N
H
O
O
Cl
alternate
substituents
alternate
sulfonamides/polar moieties
linker
3, VU0259369
The optimization strategy for 3 is shown in Figure 2. Here, we
elected to pursue divergent approaches in parallel, that consisted
of iterative parallel library synthesis and a fragment library ap-
proach, to enable both the optimization as well as an attempt to
identify a minimum pharmacophore.13,14 In short order, over 100
analogs were synthesized (via standard acylation chemistry) and
assayed in the GIRK1/2 and GIRK1/4 thallium flux assays, and
SAR proved to be shallow for this series, with >50% of the analogs
displaying no GIRK activity. The itertaive parallel synthesis effort,
surveying modifications to the parent 3, afforded few actives
(Fig. 3). Of the actives, bromine effectively replaced the chlorine
atom, as in 3a, in contrast deletion of the methyl group, providing
3b, maintained potency and efficacy at GIRK1/4, but diminished
potency and lost efficacy at GIRK1/2. Moving the halogen of 3 from
the 2- to the 4-position, as in 3c and 3d, also led to a diminution in
potency at both GIRK1/2 and GIRK1/4. Figure 4 highlights the
GIRK1/2 and GIRK1/4 concentration response curves for 3 and 3a.
All attempts to replace or modify the N,N-dimethyl sulfonamide
group, incorporation of heterocycles or modification of the linker
moiety led to a complete loss of GIRK activity. Fortunately, the
fragment libraries proved more productive.
Figure 2. Chemical optimization plan for 3, consisting of iterative parallel synthesis
and fragment libraries.
O
O
N
N
S
N
H
S
N
H
O
O
Br
O O
Cl
3b
3a
, VU0474530
, VU0474527
GIRK1/2 EC50 = 3.3 µM, 55%
GIRK1/4 EC50 = 1.6 µM, 123%
GIRK1/2 EC50 = 1.4 µM, 108%
GIRK1/4 EC50 = 1.6 µM, 114%
inactive on GIRK2/3
inactive on GIRK2/3
Cl
F
O
O
N
N
S
N
H
S
N
H
O
O
O
O
3d
3c
, VU0474532
, VU0474523
GIRK1/2 EC50 = 3.6 µM, 107%
GIRK1/4 EC50 = 5.2 µM, 108%
GIRK1/2 EC50
GIRK1/4 EC50
=
=
2.3 µM, 71%
2.2 µM, 64%
inactive on GIRK2/3
inactive on GIRK2/3
For the fragment libraries (Fig. 2), we held the 5-(N,N-dimethyl-
sulfonamide)-4-methylphenyl moeity 4 constant, and prepared li-
braries of amide 5 and urea congeners 6 (Scheme 1). In parallel, we
Figure 3. Representative active GIRK activators from iterative parallel synthesis
effort around 3.
Figure 4. GIRK activator concentration response curves (CRC) measuring thallium flux. (A) GIRK1/2 (EC50 = 1.1
hit 3; (B) GIRK1/2 (EC50 = 1.4 M, 108%) and GIRK1/4 (EC50 = 1.6
M, 114%) CRC for related analog 3a.11
lM, 110%) and GIRK1/4 (EC50 = 1.1 lM, 104%) CRCs for the HTS
l
l

S. J. Ramos-Hunter et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5195–5198
5197
held the 2-(2-chlorophenyl)acetamide portion constant, and trea-
ted the acid chloride 7 with a variety of anilines to provide analogs
8. Yields for these libraries ranged from 22% to 95% based on the
coupling partners employed.15
The amide congeners 5 proved to be uniformly inactive, with
the exception of a few C4- to C8-aliphatic amide congeners; how-
engendered a slight preference for GIRK1/2 (Table 1). This effort
provided dual GIRK1/2 and GIRK1/4 activators such as 6b, 6f and
6j, with a range of full to partial efficacies. This is an important
finding, as it is not yet clear if a full or partial GIRK activator will
be more ideal in terms of in vivo efficacy, safety and tolerability,
and having a range of chemical tools will enable these issues to
be addressed. Analogs such as 6h, with a 4-CF3 moiety, proved to
ever, the GIRK1/2 EC50s were in the 4–10 lM range with modest
efficacies (23–91%). In contrast, the urea congeners 6 possessed ro-
be selective for GIRK1/2 (EC50 = 1.5
lM) over GIRK1/4 (EC50
bust SAR, low micromolar potencies, high efficacy (71–120%) and
>10 M), but with low efficacy (26%). Electron rich congeners, such
l
as 6i, were uniformly inactive at both GIRK1/2 and GIRK1/4.
In analogs 8, where the eastern 2-(2-chlorophenyl)acetamide
portion was held constant and diverse anilines were surveyed,
interesting SAR emerged (Table 2). SAR was very shallow, and un-
like analogs 6, all analogs 8 were devoid of activity at GIRK1/2,
affording GIRK1/4 preferring activators. A 4-Cl,3-CF3phenyl amide
derivative 8e, was a very potent GIRK1/4 activator (EC50 = 0.24 -
O
O
a
b
N
Ar
N
N
S
N
H
N
H
S
N
H
R
S
NH2
O
O
O
O
O
O
5
4
6
l
M), but displayed weak, partial activation (17%) of the channel,
O
O
c
and was inactive on GIRK1/2. Other electron withdrawing substit-
uents in the 3-position, such as Cl (8g) or CF3 (8h), were selective,
partial GIRK1/4 activators; interestingly, electron donating substi-
uents in the 3-position, such as 8i, were inactive at both GIRK1/2
and GIRK1/4. None of the analogs 6 or 8 were active at non-GIRK1
containing GIRK channels, further enabling them as valuable tool
Cl
ArHN
Cl
Cl
7
8
Scheme 1. Reagents and conditions: (a) RCOCl, TEA, DMF, rt, 16 h, 45–95%; (b)
RNCO, DMF, rt, 22–88%; (c) ArNH2, TEA, DMF, rt, 37–92%.
Table 1
Structures and GIRK activities of analogs 6
O
N
S
Ar
N
N
H
H
O
O
6
Compd
Ar
GIRK1/2
GIRK1/4
Efficacya (%)
EC50 (lM)
Efficacya (%)
a
a
EC50
(lM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
Ph
4-ClPh
3-Cl,4-MePh
4-OMePh
2-FPh
3-FPh
2-ClPh
4-CF3Ph
2,5-diOMePh
3-CNPh
1.4
3.3
2.9
1.7
3.9
1.1
2.7
1.5
>10
3.9
94
92
91
101
111
89
94
26
ND
83
3.9
2.8
6.2
5.6
7.8
1.8
6.8
>10
>10
3.3
100
72
115
104
72
55
106
ND
ND
29
6j
a
Potency values were obtained from triplicate determinations and the reported efficacy values shown are standardized to the efficacy of 2, arbitrarily designated to 100%.
ND = not determined.
Table 2
Structures and GIRK activities of analogs 8
O
Ar
N
H
Cl
8
Compd
Ar
GIRK1/2
GIRK1/4
Efficacya (%)
EC50 (lM)
Efficacya (%)
a
a
EC50
(lM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
4-F,3-SO2MePh
3-t-BuPh
3-OCHF2Ph
3-Cl,5-CF3Ph
4-Cl,3-CF3Ph
4-ClPh
3-ClPh
4-CF3Ph
3-MePh
>10
5.9
ND
44
>10
2.7
7.8
>10
0.24
>10
1.8
ND
76
119
ND
17
ND
22
20
>10
>10
>10
>10
>10
>10
>10
ND
ND
ND
ND
ND
ND
ND
2.1
>10
ND
a
Potency values were obtained from triplicate determinations and the reported efficacy values shown are standardized to the efficacy of 2, arbitrarily designated to 100%.
ND = not determined.

5198
S. J. Ramos-Hunter et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5195–5198
Figure 5. GIRK activator concentration response curves (CRC) measuring thallium flux. (A) GIRK1/4 (EC50 = 0.24
for 8g.11
lM, 17%) CRC for 8e; (B) GIRK1/4 (EC50 = 1.8 lM, 22%) CRCs
dispensed to daughter plates using a Labcyte Echo555 and diluted to twofold
over their target assay concentration with 20 mM HEPES-buffered (pH 7.4)
Hanks Balanced Salt Solution (HBSS), hereafter referred to as Assay Buffer,
using a Thermo Fisher Combi. Twenty-thousand HEK-293 cells/well stably
transfected with the ion channel subunits of interest (e.g., GIRK1/GIRK2,
GIRK1/GIRK4) were plated into 384-well, black-walled, clear-bottom, amine-
coated coated plates in 20 lL/well alpha-minimal essential medium (MEM)
supplemented with 10% (v/v) fetal bovine serum and incubated overnight in a
5% CO2 incubator at 37 °C. Cell culture medium was removedfrom cell plates
and replaced with 20 lL/well Assay Buffer. Twenty microliters/well of 0.5 lM
compounds to better understand selective GIRK channel activation
(see Fig. 5).
In summary, we have described the synthesis and one of the
first accounts of SAR for a novel series of rarely described GIRK
activators, indicating that GIRK activators can be identified from
HTS campaigns and optimized. SAR proved shallow, and an itera-
tive parallel synthesis approach provided little improvement in
terms of potency or efficacy at GIRK1/2 or GIRK1/4. Instead a frag-
ment library approach afforded a diverse array of GIRK activators
that included dual GIRK1/2 and GIRK1/4 activators, GIRK1/2 pre-
ferring activators and GIRK1/4 selective activators possessing a
wide range of efficacy (from weak partial to full activation), and
devoid of activity at non-GIRK1-containing GIRK channels. Addi-
tional characterization and refinements are in progress and will
be reported in due course.
of the thallium-sensitive dye, Thallos-AM (TEFlabs, Austin TX) in Assay Buffer
was added to cell plates. Cell plates were incubated for ꢀ60 min at room
temperature and then dye-loading solution was removed from cell plates and
replaced with 20
transferred to a Hamamatsu FDSS 6000 and a double-addition protocol was
initiated. After 10s, 20 L/well of 20 M test compound in 0.2% DMSO and
Assay Buffer was added. After 4 min 10
L/well of a 5Â sodium bicarbonate-
lL/well Assay Buffer. Dye loaded and washed cell plates were
l
l
l
based thallium stimulus buffer (20 mM HEPES pH 7.4, 135 mM NaHCO3, 2 mM
CaSO4, 1 mM MgSO4,5 mM glucose, 12 mM Tl2SO4) was added and 2 more
minutes of data collection followed. Fluorescence data were collected at 1 Hz.
Data analysis: Waveform signals (fluorescence intensity vs time normalized by
dividing each fluorescence value (F) by the initial fluorescence value for each
trace (F0)) were reduced to single values by subtracting the average normalized
waveform from vehicle control wells from each wave on the plate followed by
obtaining the slope of the change in fluorescence immediately after the
Acknowledgments
Vanderbilt is a member of the MLPCN and houses the Vander-
bilt Specialized Chemistry Center for Accelerated Probe Develop-
ment. This work was supported by the NIH/MLPCN grant U54
MH084659 (C.W.L.), the Vanderbilt Department of Pharmacology
and William K. Warren, Jr. who funded the William K. Warren, Jr.
Chair in Medicine (to C.W.L.). Funding for the NMR instrumenta-
tion was provided in part by a grant from NIH (S10 RR019022).
addition of the thallium stimulus. Slope values were normalized as
a
percentage of the slope obtained by the addition of a maximally effective
concentration of the GIRK activator, ML297 (e.g., an efficacy value of 100%
means that the compound’s maximum apparent efficacy equals that of
ML297). Curve fits for normalized slope values were obtained using a four-
parameter logistic equation in the Excel add-in, XLfit.
12. Kauffman, K.; Days, E.; Romaine, I.; Du, Y.; Sliwoski, G.; Morrison, R.; Denton, J.;
Niswender, C. M.; Daniels, J. S.; Sulikowski, G. A.; Xie, S.; Lindsley, C. W.;
Weaver, C. D. ACS Chem. Neurosci. 2013, in press. http://dx.doi.org/10.1021/
cn400062a.
13. Kennedy, J. P.; Williams, L.; Bridges, T. M.; Daniels, R. N.; Weaver, D.; Lindsley,
C. W. J. Comb. Chem. 2008, 10, 345.
14. Lindsley, C. W.; Wisnoski, D. D.; Leister, W. H.; O’Brien, J. A.; Lemiare, W.;
Williams, D. L., Jr.; Burno, M.; Sur, C.; Kinney, G. G.; Pettibone, D. J.; Tiller, P. R.;
Smith, S.; Duggan, M. E.; Hartman, G. D.; Conn, P. J.; Huff, J. R. J. Med. Chem.
2004, 47, 5825.
References and notes
1. Kubo, Y.; Reuveny, E.; Slesinger, P. A.; Jan, Y. N.; Jan, L. Y. Nature 1993, 364, 802.
2. Lesage, F.; Duprat, F.; Fink, M.; Guillemare, E.; Coppola, T.; Lazdunski, M.;
Hugnot, J. P. FEBS Lett. 1994, 353, 37.
3. Kobayashi, T.; Ikeda, K.; Ichikawa, T.; Abe, S.; Togashi, S.; Kumanishi, T.
Biochem. Biophys. Res. Commun. 1995, 208, 1166.
4. Karschin, C.; Dissmann, E.; Stuhmer, W.; Karschin, A. J. Neurosci. 1996, 16, 3559.
5. Luscher, C.; Slesinger, P. Nat. Rev. Neurosci. 2010, 11, 301.
6. Krapivinsky, G.; Gordon, E. A.; Wickman, K.; Velimirovic´, B.; Krapivinsky, L.;
Clapham, D. E. Nature 1995, 374, 135.
7. Kobayashi, T.; Ikeda, K.; Kojima, H.; Niki, H.; Yano, R.; Yoshioka, T.; Kumanishi,
T. Nat. Neurosci. 1999, 2, 1091.
8. Yow, T. T.; Pera, E.; Absalom, N.; Heblinski, M.; Johnston, G. A.; Hanrahan, J. R.;
Chebib, M. Br. J. Pharmacol. 2011, 163, 1017.
9. Aryal, P.; Dvir, H.; Choe, S.; Slesinger, P. A. Nat. Neurosci. 2009, 12, 988.
10. The MLSCN evolved into the MLPCN in 2008. For more information on the
MLPCN and further details on the HTS effort, see: www.mli.nih.gov/mli/mlpcn.
11. Thallium flux assay protocol: Compounds were dissolved in DMSO, transferred
to 384-well polypropylene plates, and serially diluted in DMSO using and
Agilent Bravo (11-points, threefold dilutions). Serially diluted plates were
15. General
urea
library:
To
a
solution
of
5-amino-N,N,2-
trimethylbenzenesulfonamide (4) (15 mg, 0.07 mmol, 1.0 equiv) in DMF
(1.0 mL) was added an isocyanate (0.07 mmol, 1.0 equiv) at rt. After 12 h, the
reaction was purified by reverse phase HPLC to afford the desired urea. General
Amide Library: To a solution of 5-amino-N,N,2-trimethylbenzenesulfonamide
(4) (15 mg, 0.07 mmol, 1.0 equiv) in DMF and TEA (4:1, 1.0 mL) was added the
acid chloride (0.084 mmol, 1.1 equiv) at rt. Once the reaction was complete by
LCMS, the reaction was filtered and purified by reverse phase HPLC to afford
the desired amide.
General
urea
library:
To
a
solution
of
5-amino-N,N,2-
trimethylbenzenesulfonamide (4) (15 mg, 0.07 mmol, 1.0 equiv) in DMF and
TEA (4:1, 1.0 mL) was added the acid chloride (0.084 mmol, 1.1 equiv) at rt.
Once the reaction was complete by LCMS, the reaction was filtered and purified
by reverse phase HPLC to afford the desired amide.