Discovery of GSK2193874: An Orally Active, Potent, and Selective Blocker of Transient Receptor Potential Vanilloid 4

Article abstract of DOI:10.1021/acsmedchemlett.7b00094

Transient Receptor Potential Vanilloid 4 (TRPV4) is a member of the Transient Receptor Potential (TRP) superfamily of cation channels. TRPV4 is expressed in the vascular endothelium in the lung and regulates the integrity of the alveolar septal barrier. Increased pulmonary vascular pressure evokes TRPV4-dependent pulmonary edema, and therefore, inhibition of TRPV4 represents a novel approach for the treatment of pulmonary edema associated with conditions such as congestive heart failure. Herein we report the discovery of an orally active, potent, and selective TRPV4 blocker, 3-(1,4′-bipiperidin-1′-ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinolinecarboxamide (GSK2193874, 28) after addressing an unexpected off-target cardiovascular liability observed from in vivo studies. GSK2193874 is a selective tool for elucidating TRPV4 biology both in vitro and in vivo.

Full text of DOI:10.1021/acsmedchemlett.7b00094

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Letter
Discovery of GSK2193874: an Orally Active, Potent and
Selective Blocker of Transient Receptor Potential Vanilloid 4
Mui Cheung, Weike Bao, David J Behm, Carl A Brooks, Michael J. Bury, Sarah E Dowdell,
Hilary S Eidam, Ryan M Fox, Krista Beaver Goodman, Dennis A. Holt, Dennis Lee,
Theresa J Roethke, Robert N Willette, Xiaoping Xu, Guosen Ye, and Kevin S. Thorneloe
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00094 • Publication Date (Web): 20 Mar 2017
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Discovery of GSK2193874: an Orally Active, Potent and Selective
Blocker of Transient Receptor Potential Vanilloid 4
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Mui Cheung,* Weike Bao, David J. Behm, Carl A. Brooks, Michael J. Bury, Sarah E. Dowdell, Hil-
ary S. Eidam, Ryan M. Fox, Krista B. Goodman, Dennis A. Holt, Dennis Lee, Theresa J. Roethke,
Robert N. Willette, Xiaoping Xu, Guosen Ye, Kevin S. Thorneloe
GlaxoSmithKline, Heart Failure Discovery Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic
Area, King of Prussia, Pennsylvania, 19406, United States.
KEYWORDS GSK2193874, TRPV4, TRPV4 antagonist, TRPV4 inhibitor, congestive heart failure, L-type channel inhi-
bition
ABSTRACT: Transient Receptor Potential Vanilloid 4 (TRPV4) is a member of the Transient Receptor Potential (TRP)
superfamily of cation channels. TRPV4 is expressed in the vascular endothelium in the lung and regulates the integrity of
the alveolar septal barrier. Increased pulmonary vascular pressure evokes TRPV4-dependent pulmonary edema, and
therefore, inhibition of TRPV4 represents a novel approach for the treatment of pulmonary edema associated with condi-
tions such as congestive heart failure. Herein we report the discovery of an orally active, potent and selective TRPV4
blocker,
3-(1,4'-bipiperidin-1'-ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-
quinolinecarboxamide (GSK2193874, 28) after addressing an unexpected off-target cardiovascular liability observed from
in vivo studies. GSK2193874 is a selective tool for elucidating TRPV4 biology both in vitro and in vivo.
TRPV4 is a member of the Transient Receptor Potential
(TRP) superfamily of cation channels and is activated by
heat, hypotonicity and physical stress.1-3 TRPV4 is ex-
pressed in numerous cell types, including endothelial
cells of the lung where it can mediate Ca2+ entry.4 TRPV4
regulates the integrity of the alveolar barrier in the lung
and increases barrier permeability when activated, as
demonstrated in response to the increased vascular
and/or airway pressure.5 Pharmacological activation of
TRPV4 with a small molecule TRPV4 agonist results in
contraction of cultured endothelial cells and pulmonary
edema associated with cardiovascular collapse in vivo.6,7
and mouse in vitro TRPV4 IC50s of 48, 133 and 17 nM, re-
spectively (Figure 1).10 Renovis (Evotec) had reported RN-
1734 as a TRPV4 antagonist with modest potency (human,
rat and mouse TRPV4 IC50s of 2.3, 3.2 and 5.9 µM, respec-
tively) (Figure 1).11 Therefore, discovery of a potent and
selective TRPV4 blocker of utility for in vivo studies was
highly desirable to further understand the complexity of
TRPV4 biology. 12
Herein we report the discovery of an orally active, po-
tent and selective TRPV4 blocker, 3-(1,4'-bipiperidin-1'-
ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-
(trifluoromethyl)phenyl]-4-quinolinecarboxamide
Congestive heart failure patients have a decreased abil-
ity of the left ventricle to pump blood into the peripheral
circulatory system. This results in left ventricular dilation
and a concomitant increase in pulmonary vascular pres-
sure that drives development of pulmonary edema.
Therefore, TRPV4 inhibition represents a potential novel
approach for the treatment of pulmonary edema associat-
ed with congestive heart failure.8 TRPV4 also has been
implicated in many other disease conditions, including
neurogenic pulmonary edema, chronic obstructive pul-
monary disorder (COPD), acute lung injury,9 acute res-
piratory distress syndrome (ARDS), overactive urinary
bladder, pain, genetic motor neuron disorders, cardiovas-
cular disease, and bone related disorders.8
(GSK2193874, 28). GSK2193874 is an excellent tool for
further understanding the biology of TRPV4 both in vitro
and in vivo.8 It is worth noting that after our discovery of
GSK2193874, several orally bioavailable TRPV4 tools have
been reported (Figure 1). These included Pfizer’s azet-
idine-linked
compound,13
GSK’s
piperidinyl-
benzimidazole compound,14 Renovis’ RN-1665 and RN-
9893,15 and Shionogi’s aminothiazole compound.16,17
At the onset of our lead optimization efforts, few small
molecule TRPV4 blockers had been reported, and none
were suitable for oral dosing. Hydra Biosciences had re-
ported a TRPV4 antagonist HC-067047 with human, rat
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2-
appropriately
substituted
benzaldehyde
and
OH
O
CN
CF3
1
2
oxobutanoic acid. Esterification of acid 2 resulted in the
substituted methyl 3-methyl-2-phenyl-4-
O
F
NH
Cl
O
S
O
N
3
4
5
6
7
8
9
quinolinecarboxylate which was then treated with N-
bromosuccinimide and benzoyl peroxide to form an ap-
propriately substituted bromomethyl quinoline. Subse-
quent displacement of the bromomethyl quinoline with
an appropriately substituted piperidine in acetonitrile
afforded the corresponding tertiary amine 3. The methyl
ester was then hydrolyzed to acid by treatment with KOH
in methanol/water. Coupling of the acid with an appro-
priately substituted benzylamine under standard condi-
tions, for example, EDC/HOBT or T3P, provided quino-
line derivatives.
S
N
Cl
N
H
O
Cl
O
Cl
Pfizer TRPV4 antagonist
RN-1734
HC-067047
N
O
O
(Me)2N
O
S
O
N
NH
Cl
Cl
R'
O
N
N
N
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N
H
N
R"
RN-1665
: R' = H, R" = SF3
RN-9893: R' = NO2, R" = CF3
GSK TRPV4 antagonist
CN
CF3
N
H
N
S
N
N
N
N
S
N
N
O
Shionogi TRPV4 antagonist
Figure 1. Structures of TRPV4 antagonists
Quinoline hit 1a was identified as a TRPV4 blocker
from high throughput screening (Figure 2). Compound 1a
has modest TRPV4 potency with human and rat TRPV4
IC50 of 2 and 0.3 µM, respectively, in a FLIPR assay in
HEK293 cells. However, it is a potent neurokinin NK2
and NK3 dual inhibitor with IC50 of 100 and 8 nM, respec-
tively, in FLIPR binding assays. Moreover, 1a has poor
pharmacokinetic properties with high clearance (CL = 52
mL/min/kg) in rats. As our primary goal was to validate
TRPV4 as a pharmacologically relevant target with a po-
tent and selective TRPV4 inhibitor that could be dosed
orally and chronically in rodents, our lead optimization
effort was focused on improving TRPV4 potency, remov-
ing NK2/3 activity, reducing in vivo clearance and improv-
ing oral exposure in rats.
Scheme 1. General synthesis of quinoline derivatives. a)
KOH, H2O/EtOH; b) 2-oxobutanoic acid, EtOH; c)
(COCl)2, DMF, CH2Cl2, then MeOH; d) benzoyl peroxide,
NBS, CCl4; e) R2-substituted piperidine, CH3CN; f) KOH,
MeOH/H2O; g) R3, R3a-substituted benzylamine, T3P, i-
Pr2NEt, CH2Cl2.
Table 1. Biochemical activities of substituted phenyl ana-
logs in human and rat TRPV4 FLIPR assay
hTRPV4
IC50 (µM)a
2
rTRPV4
IC50 (µM)a
0.32
Figure 2. Quinoline hit 1a and chemistry strategies focused
Compound
R1
on R1-R4 optimization.
1a
4
H
We divided quinoline 1a into four regions for structure
activity relationship (SAR) exploration (Figure 2): 2-aryl
substitution (R1), amine modification (R2), benzyl amide
modification (R3, R3a) and quinoline substitution (R4).
Quinoline derivatives were synthesized using standard
conditions as described in Scheme 1.18,19 3-Methyl-2-
phenyl-4-quinolinecarboxylic acid 2 was prepared by
treatment of the appropriately substituted 1H-indole-2,3-
dione and ethyl aryl ketone with KOH in refluxing etha-
nol. Alternatively, compound 2 was prepared by treat-
ment of the appropriately substituted aniline with the
4-Cl
0.32
0.016
0.016
0.1
5
4-CF3
4-Me
4-OMe
3-Cl
0.4
6
1.58
7
5.01
0.32
8
0.2
0.008
0.0025
0.013
0.63
9a
10
11
3-CF3
3-Me
3-OMe
0.08
0.5
3.98
aIC50 values are the mean of at least two independent
experiments.
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First we focused on the aryl substitution on the 2-
Interestingly when R3 or R3a is CF3, the potency difference
for these two analogs (20 and 21) was not as significant as
the methyl analogs (9a and 9b). Non chiral benzyl am-
ides were synthesized to understand stereochemistry
preference (17, 22, and 23). The unsubstituted benzyl am-
ide (17) and dimethyl benzyl amide (22) maintained mod-
est TRPV4 potency. Interestingly the cyclopropyl benzyl
amide (23) possessed superior TRPV4 potency as 9a and
20 analogs.
1
2
3
4
5
6
7
8
position of the quinoline, as this position was known to
be sensitive to NK2 and NK3 activity. Substitution of
electron-donating groups such as Me and OMe at 4-
position (6, 7) or OMe at 3-position (11) of the phenyl ring
maintained TRPV4 potency (Table 1). Methyl at 3-
position (10) of the phenyl ring improved human and rat
TRPV4 potency over compound 1a by 4x and 24x, respec-
tively. Interestingly substitution of electron-withdrawing
groups such as Cl and CF3 in both positions (4, 5, 8, 9a)
improved human and rat TRPV4 potency by up to 25 and
128 fold, respectively, over unsubstituted quinoline hit 1a.
3-CF3 compound (9a) is the most potent analog with rat
TRPV4 IC50 of 2.5 nM. Compound 9a was evaluated for
NK2 and NK3 selectivity, and as expected, 9a has reduced
NK2 and NK3 activity with IC50s of 1.3 and 2.5 µM, respec-
tively, representing ≥520 fold selectivity over rat TRPV4
potency.
9
Table 3. Biochemical activities of substituted piperidine
analogs in human and rat TRPV4 FLIPR assays
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Next we evaluated whether there was a stereochemistry
preference for binding to TRPV4. Both R- and S- enanti-
omers for compounds 1 and 9 were synthesized using en-
antiomerically pure benzyl amines. As shown in Table 2,
there was no significant stereo preference when R1 is H
(1a vs. 1b). Surprisingly, when R1 is 3-CF3, the (S)-
enantiomer (9a) was 25 and 160 fold more potent than the
corresponding (R)-enantiomer (9b) in human and rat
TRPV4 respectively, demonstrating a stereo preference
for (S)-enantiomer.
hTRPV4
IC50 (µM)a
0.08
rTRPV4
IC50 (µM)a
0.0025
>25
Compound
R2
9a
12
13
14
15
16
piperidine
H
>25
NMe2
2.51
0.32
morpholine
piperazine
cyclohexane
0.05
0.004
0.79
3.16
>25
>25
aIC50 values are the mean of at least two independent
experiments.
Table 2. Stereoselectivity of quinoline analogs
Table 4. Biochemical activities of substituted amine ana-
logs in human and rat TRPV4 FLIPR assays
hTRPV4
IC50 (µM)a
rTRPV4
IC50 (µM)a
0.32
Compound
R1
Stereochemistry
1a
1b
9a
9b
H
S
R
S
2
2
H
0.63
3-CF3
3-CF3
0.08
2
0.0025
0.4
hTRPV4
IC50 (µM)a
0.08
rTRPV4
IC50 (µM)a
0.0025
0.05
Compound
R3
R3a
R
aIC50 values are the mean of at least two independent
experiments.
9a
17
18
19
20
21
22
23
Me
H
H
H
0.5
Et
H
0.16
0.006
iPr
CF3
H
H
0.13
0.025
We then evaluated the SAR around piperidine substitu-
tion while keeping R1 as 3-CF3, and R3 as S-Me. Piperi-
dine-piperidine analog (9a) and piperidine-morpholine
derivative (14) had optimal TRPV4 potency (Table 3). The
terminal piperidine or morpholine was important for
TRPV4 activity as compounds without those moieties
have reduced TRPV4 activities (12, 13, 15 and 16).
H
0.04
0.0025
0.008
CF3
Me
0.1
Me
1
0.04
cyclopropyl
0.05
0.0025
aIC50 values are the mean of at least two independent
experiments.
Since chirality at the alpha position of benzyl amide
(R3) played an important role in TRPV4 potency (Table 2),
we explored additional SAR at that region. As shown in
Table 4, increasing size of the R3 group from methyl to
ethyl and isopropyl decreased TRPV4 potency (9a, 18, 19).
Quinoline substitution (R4) was then examined. A vari-
ety of substitutions were generally tolerated. Exemplar
data for substitutions at the 6- or 7- position are shown in
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mg/kg with Cmax of ~20 µM (free fraction of 0.94 µM).
No effect was observed at 1 mg/kg with Cmax of ~7 µM
(free fraction of ~0.3 µM).
Table 5. In general, compounds with 7-ethoxy substitu-
1
2
3
4
5
6
7
8
tion (27 and 30) are more potent in the human TRPV4
assay than the corresponding 7-methoxy analogs (26 and
29). Electron-withdrawing groups such as bromo are tol-
erated at both 6- and 7- positions (24, 25, 28). Interest-
ingly all substitutions maintained similar rat TRPV4 po-
tency.
To understand if the depressor effect of 9a observed in
dog was a TRPV4 mechanism-based or an off-target ef-
fect, 9a was administered to TRPV4 KO and WT mice
while monitoring BP and HR. Compound 9a was dosed
at 30 mg/kg via an iv infusion reaching a terminal plasma
concentration of 10-11 µM. As observed in dog, BP and HR
were reduced with 9a administration. The responses were
similar in KO and WT mice, suggesting 9a possesses off-
target activity that contributes to the reduction in BP and
HR.
Table 5. Biochemical activities of substituted quinolines
in human and rat TRPV4 FLIPR assays
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To investigate what off-target activity might contribut-
ed to the reduction in BP and HR, we evaluated 9a in a
number of assays including rabbit ventricular wedge,
HCN4 (pace maker channel), CaV1.2 (L-type channel),
CaV2.2 (N-type channel), CaV3.2 (T-type channel), and in
vitro contractility in mouse isolated aorta and right atria
assays. Based on the overall results, we concluded that
the off-target effect observed with 9a was likely due to
frequency-dependent L-type channel inhibition with an
IC30 of 1.7 µM at 0.1 Hz, and an IC30 of 0.12 µM at 2 Hz.
Two Hz (i.e. 120 beats per minute) is considered to be
more physiological relevant than the 0.1 Hz.20 Minimal or
no activities were observed with 9a in all other assays
evaluated.
hTRPV4 rTRPV4
Compound
R3
R3a
R4
IC50
IC50
(µM)a
(µM)a
9a
20
24
25
26
27
23
28
29
30
Me
CF3
CF3
CF3
CF3
CF3
H
H
H
H
H
H
H
H
0.08
0.04
0.06
0.06
0.05
0.01
0.05
0.04
0.25
0.08
0.0025
0.0025
0.0025
0.0032
0.004
6-Br
7-Br
7-OMe
7-OEt
H
0.0032
0.0025
0.002
Table 6. L-type channel inhibition activity for TRPV4
analogs
cyclopropyl
cyclopropyl
7-Br
cyclopropyl 7-OMe
cyclopropyl 7-OEt
0.0025
0.0016
aIC50 values are the mean of at least two independent
experiments.
Among all the analogs that were synthesized and evalu-
ated, 9a represented an exemplar tool compound with
excellent rodent TRPV4 activity. Compound 9a was eval-
uated in a PK study in rat and found to have reduced
clearance [CL = 25 mL/min/kg) and significantly im-
proved oral bioavailability (~100% F) and exposure
(DNAUC = 0.8 µg*h/mL/(mg/kg)] in Sprague-Dawley
(SD) rats. Next we assessed selectivity of 9a against a
panel of cardiac ion channels. Compound 9a was evalu-
ated against hERG (PatchXpress), CaV1.2 (PatchXpress)
and NaV1.5 (IonWorks) and found to have IC50s of 1.2, 4.5
and 40 µM, respectively, representing ≥480 fold selectivi-
ty over rat TRPV4 potency. Based on the potency, selec-
tivity and PK, 9a was progressed as the first TRPV4 inhib-
itor for in vivo evaluation.
hTRPV4 rTRPV4
CaV1.2
IC30 (µM)
Compound
R3
R3a
R4
H
IC50
IC50
(µM)a
(µM)a
0.1 (2) Hz
1.7 (0.12)
9a
27
28
Me
H
H
0.08
0.01
0.04
0.0025
0.0032
0.002
7-
OEt
CF3
10 (12)
cyclopropyl
7-Br
10.5 (10.5)
aIC50 values are the mean of at least two independent
experiments.
In order to further understand the SAR relating to fre-
quency dependent L-type channel inhibition, a set of
structurally diverse quinolines with potent TRPV4 poten-
cy were evaluated for L-type channel inhibition using
electrophysiology patch assay. Interestingly substitution
at the quinoline improves the L-type channel selectivity.
A shown in Table 6, 27 and 28 showed significant im-
provement in L-type selectivity over 9a and a lack of fre-
quency dependence. Upon further selectivity profiling,
Compound 9a was evaluated in an anesthetized dog CV
study monitoring blood pressure (BP), heart rate (HR),
cardiac output and left ventricular (LV) function. Dogs
were pretreated with 1 and 3 mg/kg of 9a dosed intrave-
nously (n=4).
QTc prolongation was observed with
compound treatment, consistent with hERG inhibition
measured in vitro (IC50 = 1.2 µM). However, 9a reduced
BP, HR, cardiac output and LV function unexpectedly at 3
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All animal studies were performed in compliance with the
we identified 27 as a PXR activator with EC50 of 1.3 µM
1
2
3
4
5
6
7
8
Guide for the Care and Use of Laboratory Animals as pub-
lished by the US National Institutes of Health and were ap-
proved by the Institutional Animal Care and Use Committee
of GlaxoSmithKline.
while 28 is not.
The pharmacokinetic (PK) properties for compound 28
were evaluated in both rat and dog and found to have
half-lives and oral exposure suitable for oral dosing in
chronic animal models (Rat PK: iv CL = 7.3 mL/min/kg,
po t1/2 = 10 h, % F = 31; Dog PK: iv CL = 6.9 mL/min/kg,
po t1/2 = 31 h, %F = 53). Compound 28 was profiled
against TRP channels and was selective against TRPV1,
TRPA1, TRPC3, TRPC6, TRPM8 (IC50 >25 µM). In addi-
tion, 28 showed no blood pressure or heart rate effect in
rats when dose up to 30 mg/kg.8 GSK2193874 (28) is the
first-in-class orally bioavailable TRPV4 inhibitor which
demonstrated ability to improve pulmonary functions in a
number of heart failure models.8
ACKNOWLEDGMENT
We thank all members of the TRPV4 Program Team at Glax-
oSmithKline for their contribution. We thank Ronggang Liu
for synthesizing compounds 4, 9a, 20 and 21, and Deyou Sha
for synthesizing compound 23. We thank J.P Jarworski, Mi-
chael Klein and Dwight Morrow for TRPV4 FLIPR data; Brian
Donovan for hERG, CaV1.2 and NaV1.5 data; Barry Brown and
Khuram Chaudhary for rabbit ventricular wedge data; Clare
Townsend for HCN4 data; Joe Lin and Irina Lozinskaya for L-
type and T-type channel data; Erin Davis, Melanie Nord and
Yaming Su for PK data; and Chris P. Doe and Jerry Stankus
for dog hemodynamic data.
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In summary, starting from the quinoline screening hit
1a, a lead optimization effort identified 9a as a potential
in vivo tool compound. Unfortunately 9a exhibited unex-
pected hemodynamic effect in dogs. By evaluating 9a in
various in vitro cardiac channel assays and in vitro con-
tractility assays in mouse isolated aorta and right atria, we
concluded that the off-target effect observed with 9a was
likely due to frequency-dependent L-type channel inhibi-
tion. Further lead optimization resulted in the discovery
of GSK2193874 (28), a highly potent, selective and orally
active inhibitor of the TRPV4 channel. GSK2193874 is an
excellent in vitro and in vivo tool for target validation
studies probing the biology of TRPV4. Additional phar-
macology data of GSK2193874 including TRPV4 potency
across species, potency against different TRPV4 stimuli,
selectivity data on CEREP panel and activities in pulmo-
nary edema and heart failure models had been disclosed
by Thorneloe et al.8 Further SAR optimization leading to
the identification of the pre-clinical candidates will be
reported in due course.
ABBREVIATIONS
BP, blood pressure; compd, compound; CL, clearance; CV,
cardiovascular; DNAUC, dose normalized area under the
curve; FLIPR, fluorometric imaging plate reader; HR, heart
rate; iv, intravenous; PK, pharmacokinetic; PXR, pregnane X
receptor; SAR, structure-activity relationship; TRPV, transi-
ent receptor potential vanilloid; KO, knockout; WT, wild
type.
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ASSOCIATED CONTENT
Supporting Information. Material and methods for TRPV4
FLIPR assays, ion channel assays (hERG, Cav1.2, Nav1.5) and
hemodynamic measurement in anesthetized mice and dogs.
Compound synthesis and spectroscopic characterization of
9a and 28. This material is available free of charge via the
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AUTHOR INFORMATION
Corresponding Author
* E-mail: mui.h.cheung@gsk.com
Present Addresses
N/A
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding Sources
The authors declare the following competing financial inter-
ests: All authors are current or past employees of Glax-
oSmithKline and/or stockholders of GlaxoSmithKline.
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Discovery of GSK2193874: an Orally Active, Potent and Selective
Blocker of Transient Receptor Potential Vanilloid 4
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Mui Cheung,* Weike Bao, David J. Behm, Carl A. Brooks, Michael J. Bury, Sarah E. Dowdell, Hil-
ary S. Eidam, Ryan M. Fox, Krista B. Goodman, Dennis A. Holt, Dennis Lee, Theresa J. Roethke,
Robert N. Willette, Xiaoping Xu, Guosen Ye, Kevin S. Thorneloe
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