Identification of orally-bioavailable antagonists of the TRPV4 ion-channel

Article abstract of DOI:10.1016/j.bmcl.2015.06.098

Abstract Antagonists of the TRPV4 receptor were identified using a focused screen, followed by a limited optimization program. The leading compounds obtained from this exercise, RN-1665 23 and RN-9893 26, showed moderate oral bioavailability when dosed to

Full text of DOI:10.1016/j.bmcl.2015.06.098

Accepted Manuscript
Identification of orally-bioavailable antagonists of the TRPV4 ion-channel
Zhi-Liang Wei, Margaret T. Nguyen, Donogh J.R. O’Mahony, Alejandra
Acevedo, Sheila Zipfel, Qingling Zhang, Luna Liu, Michelle Dourado, Candace
Chi, Victor Yip, Jeff DeFalco, Amy Gustafson, Daniel E. Emerling, Michael G.
Kelly, John Kincaid, Fabien Vincent, Matthew A.J. Duncton
PII:
S0960-894X(15)00703-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.098
BMCL 22888
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 June 2015
25 June 2015
29 June 2015
Please cite this article as: Wei, Z-L., Nguyen, M.T., O’Mahony, D.J.R., Acevedo, A., Zipfel, S., Zhang, Q., Liu, L.,
Dourado, M., Chi, C., Yip, V., DeFalco, J., Gustafson, A., Emerling, D.E., Kelly, M.G., Kincaid, J., Vincent, F.,
Duncton, M.A.J., Identification of orally-bioavailable antagonists of the TRPV4 ion-channel, Bioorganic &
Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.098
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Identification of orally-bioavailable
antagonists of the TRPV4 ion-channel
Leave this area blank for abstract info.
Zhi-Liang Wei, Margaret T. Nguyen, Donogh J. R. O’Mahony, Alejandra Acevedo, Sheila Zipfel, Qingling
Zhang, Luna Liu, Michelle Dourado, Candace Chi, Victor Yip, Jeff DeFalco, Amy Gustafson, Daniel E.
Emerling, Michael G. Kelly, John Kincaid, Fabien Vincent* and Matthew A. J. Duncton*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www. elsevier.com
Identification of orally-bioavailable antagonists of the TRPV4 ion-channel
Zhi-Liang Wei, Margaret T. Nguyen, Donogh J. R. O’Mahony, Alejandra Acevedo, Sheila Zipfel, Qingling Zhang,
Luna Liu, Michelle Dourado, Candace Chi, Victor Yip, Jeff DeFalco, Amy Gustafson, Daniel E. Emerling, Michael
G. Kelly, John Kincaid, Fabien Vincent* and Matthew A. J. Duncton*
Renovis, Inc. (a wholly-owned subsidiary of Evotec AG), Two Corporate Drive, South San Francisco, CA 94080, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Antagonists of the TRPV4 receptor were identified using a focused screen, followed by a limited
optimization program. The leading compounds obtained from this exercise, RN-1665 23 and
RN-9893 26, showed moderate oral bioavailability when dosed to rats. The lead molecule, RN-
9893 26, inhibited human, rat and murine variants of TRPV4, and showed excellent selectivity
over related TRP receptors, such as TRPV1, TRPV3 and TRPM8. The overall profile for RN-
9893 may permit its use as a proof-of-concept probe for in vivo applications
Keywords:
TRPV4
2009 Elsevier Ltd. All rights reserved.
Antagonist
RN-9893
RN-1734
Pain
Transient Receptor Potential (TRP) ion channels function as
cellular sensors that can be activated by a variety of stimuli
including pH, osmolarity, temperature changes, mechanical
forces, post-translational modifications and a wide array of small
molecules.1,2 The Transient Receptor Potential Vanilloid (TRPV)
subfamily has been implicated in temperature sensing with some
channels opening in response to noxiously hot (TRPV1 and
TRPV2), or warm temperatures (TRPV3 and TRPV4).3,4 TRPV1
is the prototypical member of the TRPV family. Originally
identified as the molecular target of capsaicin, a pungent
constituent of chili peppers, it has emerged as a compelling
molecular target for the treatment of pain, and a number of
TRPV1 antagonists are currently being assessed in clinical
such as brachyolmia, spondylometaphyseal dysplasia Koslowski
type (SMDK) and metatropic dysplasia, are linked to TRPV4
mutations, most of which appear to increase the basal activity of
the ion-channel.17,18 Additionally, studies from several
laboratories strongly suggest that TRPV4 may play an important
role in neuropathic and inflammatory pain.19-23 In this area, the
finding that TRPV4 may be activated specifically under
pathological conditions could be significant.24,25 Finally, recent
findings indicate that TRPV4 activity modulates bladder
function,26-28 may play a role in metabolic disorders,29 and that
mutations in TRPV4 are responsible for Charcot-Marie-Tooth
disease type 2C.30,31
In addition to GSK1016790A 2, multiple exogenous agonists
of TRPV4 have been characterized. These include semi-synthetic
phorbol esters such as 4-PDD 3,32,33 natural products such as
bisandrographolide 4,34 and synthetic derivatives such as RN-
1747 5 (Figure 1).35 Unfortunately, there are a limited number of
antagonists with which to study the TRPV4 receptor.14,15 Metal-
derived ruthenium red was first characterized as a TRPV4
antagonist, but it has the disadvantage of interacting with at least
fourteen other molecular targets.14,35 More recently, we reported
the discovery of RN-1734 6, a moderately potent small molecule
antagonist suitable for in vitro studies.35 Subsequent to this
report, researchers at Hydra Biosciences and GlaxoSmithKline
described alternative proof-of-concept TRPV4 antagonists, such
as compounds HC-067047 7 and GSK2193874 8 (Figure 1).36,37
In this Letter we detail results of our preliminary investigations to
identify orally-bioavailable antagonists of the TRPV4 receptor,
based on a chemical scaffold differing from those of compounds
7 and 8. Our aim was to discover a small molecule antagonist
trials.5,6 TRPV4,
a close relative of TRPV1, was first
characterized as a molecular osmosensor.7,8 Further studies
revealed that a multiplicity of other stimuli such as warm
temperatures, endogenous ligands (e.g. epoxyeicosanoic acid 5,6-
EET 1), and protein phosphorylation could also lead to channel
activation.9-15 Overall, TRPV4 may function as a cellular signal
integrator of these different modalities in vivo. It is expressed in a
number of tissues including kidneys, lungs, skin, the central
nervous system (CNS), dorsal root ganglia, vascular
endothelium, bladder and cartilage/bone related cells such as
chondrocytes and osteoblasts/osteoclasts.9 Predictably, this
widespread distribution is leading to the discovery of a variety of
roles for TRPV4.13 Most prominently, systemic application of
small molecule TRPV4 agonists, such as GSK1016790A 2,
demonstrated that activation of TRPV4 can lead to a precipitous
drop in blood pressure, vascular leakage and can ultimately be
lethal, suggesting a role for TRPV4 in mediating vasodilation.16
Human genetic studies have also revealed that skeletal dysplasias

which could be used to examine the function of TRPV4 in vivo.
These studies led to the identification of RN-1665 23 and RN-
9893 26,38 whose pharmacological and pharmacokinetic
properties may permit their use as in vivo probes of the TRPV4
receptor.39
Figure 1. Literature agonists and antagonists of TRPV4.
from Table 1, incorporation of hydrophobic substituents at the 4-
position resulted in a favorable increase in potency at TRPV4
(entries 2-6). For instance, addition of a chloropyridyl ring (entry
3), tert-butyl, or a trifluoromethyl group (entries 4 and 5),
resulted in an improvement to the in vitro activity at TRPV4.
When the trifluoromethyl- substituent was moved from the 4-
position to the 3-position of the phenyl ring, a slight reduction in
TRPV4 potency was observed (entry 6). Due to the perceived
liability of tert-butyl groups towards metabolic degradation, the
4-trifluoromethyl analog 13, with an IC50= 0.71 M (entry 5) was
designated as our lead, and was selected for further SAR studies.
A focused screen of our corporate collection, using RN-1747
5 and RN-1734 6 as templates, identified benzamide 9 as a
moderately active antagonist of TRPV4, blocking the entry of
calcium into cells expressing human and rat TRPV4 receptors
stimulated with the TRPV4 agonist, 4α-PDD 3, applied at an
EC80 concentration (Table 1, entry 1).11,35 Therefore, we initiated
a campaign to improve upon the potency of compound 9.
Table 1. Preliminary SAR with a TRPV4 hit.
The importance of the amide and sulfonamide groups to
TRPV4 activity was next examined. For instance, replacement of
the amide in compound 13, with a sulfonamide to give compound
15, resulted in a greater than 10-fold reduction in TRPV4
potency (IC50 > 10M; Table 2, entry 2 cf. entry 1). Likewise, the
importance of the sulfonamide moiety in compound 13 was
clearly illustrated by replacing it with an amide group, resulting
in compound 16, which also had an IC50 >10 M (Table 2, entry
3 cf. entry 1).
IC50 hTRPV4a
8.53 (n =1)
4.93 (n =1)
0.93 (n = 6)
0.39 (n = 2)
0.71 (n = 7)
2.17 (n = 1)
IC50 rTRPV4a
4.3 (n = 1)
Entry
Compound
R
1
2
3
4
5
6
4-H
9
Table 2. Replacement of amide and sulfonamide.
6.49 (n = 1)
3.9 (n = 2)
10
11
12
13
14
4-
4-
N
Cl
N
4-t Bu
4-CF3
3-CF3
1.05 (n = 2)
0.94 (n = 4)
1.94 (n = 1)
Entry
Compound
X
Y
IC50 hTRPV4a
0.71 (n = 7)
>10 (n = 3)
>10 (n = 3)
IC50 rTRPV4a
0.94 (n = 4)
>10 (n = 2)
>10 (n = 5)
SO2
SO2
C=O
1
2
3
13
15
16
C=O
SO2
C=O
a. Agonist = 4a -PDD at an EC80 concentration; average of n=1-7 experiments.
a . Agonist = 4a -PDD at an EC80 concentration; average of n=2-7 experiments.
Structure-activity relationships of substituents on the benzoate
core of compound 9 were initially examined. As can be seen

Changes to the isopropylpiperazine in compound 13 were also
examined (Table 3). Switching the isopropylpiperazine in 13
(IC50 = 0.71 M) to a dimethyl group resulted in a reduction of
TRPV4 activity to 4.22 M (17; entry 2). Enlarging the
piperazine to a 7-membered homopiperazine ring, resulted in
compound 18, which exhibited two-fold lower activity than the
parent (IC50 = 1.32 M; entry 3). It was also possible to exchange
the piperazine in compound 13 for a piperidine with an exocyclic
basic nitrogen to give compounds 19 and 20, with IC50 values of
2.11 M and 0.91 M respectively (entries 4 and 5). Exchanging
the isopropyl group in 13 with other alkyl, or cyclo-alkyl
structures was also permissible (entries 6 and 7). For instance, N-
cyclopentyl congener 22 had similar TRPV4 activity to parent
compound 13 (entry 7).
Table 4. Further development of SAR around benzenoid ring.
IC50 hTRPV4a
0.71 (n = 7)
0.26 (n = 4)
0.38 (n = 5)
0.54 (n = 2)
0.42 (n = 13)
0.93 (n = 4)
IC50 rTRPV4a
0.94 (n = 4)
0.39 (n = 2)
0.37 (n = 5)
0.49 (n = 5)
0.66 (n = 9)
2.92 (n = 3)
Entry
Compound
R
4-CF3
1
2
3
4
5
6
13
23
24
25
26
27
4-SF5
Table 3. SAR of piperazine ring.
2-F,4-CF3
2,4-(CF3)2
2-NO2,4-CF3
2-NH2,4-CF3
IC50 hTRPV4a
0.71 (n =7)
4.22 (n =1)
1.32 (n = 1)
2.11 (n = 1)
0.91 (n = 1)
1.28 (n = 1)
0.8 (n = 1)
IC50 rTRPV4a
Entry
Compound
R
a . Agonist = 4-PDD at an EC80 concentration; average of n=2-13 experiments.
1
2
3
4
5
6
7
0.94 (n = 4)
13
17
18
19
20
21
22
N
N
Next, in vivo pharmacokinetic profiles for a very limited
selection of compounds identified from our SAR studies were
investigated. For example, RN-1665 23 and RN-9893 26 were
evaluated further. As can be seen, both 23 and 26 showed
reasonable clearance, volume of distribution and half-life values
in rats. Both compounds also showed moderate oral
bioavailability (%F), with RN-9893 26 approaching towards an
oral bioavailability of 50% when dosed at ca. 30 mg/kg (Table
5). RN-9893 26 was also bioavailable when dosed via the intra-
peritoneal route of administration (see Supplemental Material).
Additional relevant information for compounds 23 and 26
includes solubility (0.15 μM for RN-1665 23; 45.5 μM for RN-
9893 26 at pH 7.4), and plasma protein binding (rPPB=99.9%
and hPPB=99.9% for RN-1665 23; rPPB=99.0% and
hPPB=99.0% for RN-9893 26).
N
5.74 (n = 1)
nd
nd
nd
nd
nd
N
N
N
N
N
N
N
N
N
N
a . Agonist = 4-PDD at an EC80 concentration; average of n=1-7 experiments; nd: not determined.
Table 5. Pharmacokinetic data for RN-1665 23 and RN-9893 26
when dosed to Wistar rats.
After
investigating
structure-activity
around
the
Compound
RN-1665 23
RN-9893 26
isopropylpiperazine moiety, we returned to exploring the effect
of further substituents on the benzoate core of compound 13
(Table 4). In recent years the pentafluorosulfanyl group has been
shown to be a useful bioisostere for trifluoromethyl substituents
in many biological applications.40,41 Accordingly, we applied
i.v.
dose (mg/kg)
Cl (L/h/kg)
1.05
1.40
1.35 ±0.04
46767 ±1450
2.01 ±0.01
1.03 ±0.03
1.42 ±0.30
60632 ±11942
1.44 ±0.20
0.71 ±0.07
AUCinf (min*ng/ml)
Vss (L/kg)
such
a strategy to our lead-molecule 13. Gratifyingly,
pentafluorosulfanyl congener 23 was also active at TRPV4, with
an IC50 of 0.26 M (entry 2). Separately, it was found that
compound 13 tolerated the presence of additional substituents at
the 2-position of the aromatic ring. For instance, the presence of a
2-fluoro, 2-trifluoromethyl, or 2-nitro group resulted in potent
TRPV4 antagonists (entries 3-5). Reduction of the nitro-group in
compound 26 (entry 5), to provide aniline 27 (entry 6), also gave
a moderately-active TRPV4 antagonist.
Half-life (h)
p.o.
dose (mg/kg)
Cmax (ng/ml)
AUCinf (min*ng/ml)
F (%)
1.80
29.73
3823 ±1556
604089 ±200185
46.8 ±15.5
143 ±75
18153 ±4735
22.7 ±5.9
0.15 M
Solubility at pH7.4
45.5 M

family members, and a wide range of other biological targets in
vitro. Importantly, RN-9893 inhibits the TRPV4 receptor when it
is activated by exogenous agonists such as the phorbol ester, 4α-
PDD, and by more physiologically relevant agonists, such as
hypotonicity. The compounds identified in this report may be of
use to those examining the function of the TRPV4 receptor in
vivo, and, given their alternative chemical scaffold, will
complement the other small molecule antagonist probes of
TRPV4 that have recently been disclosed in the primary
literature.
Protein binding (rat)
99.91 ±0.01
99.92 ±0.01
99.0 ±0.1
99.0 ±0.1
Protein binding (human)
aData is the mean value ±standard deviation
As mentioned previously, the TRPV4 receptor can be
activated by multiple stimuli in vivo, such as hypotonicity,
endogenous ligands, warm temperature and protein
phosphorylation. As such, it can be regarded as a multimodal
receptor, similar to its cousin, the TRPV1 receptor (which is
activated by high temperature, acidic pH and endogenous small
molecules). In order to determine if RN-989326 could block both
the effects of exogenous ligands, and those of more
physiologically-relevant conditions, we determined the IC50
against rTRPV4 when the receptor was activated by either 4α-
PDD, or 20% hypotonicity (Figure 2). RN-9893 26 inhibited
rTRPV4 with an IC50 of 0.57 μM when 4α-PDD was used as an
agonist. Similarly, RN-9893 26 inhibited rTRPV4 with an IC50 of
2.1 μM when hypotonicity was used as the activating stimulus,
indicating that RN-9893 is capable of antagonizing both
activation modalities.42 These results were similar to those
observed with the related TRPV4 antagonist, RN-1734 6.35
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Figure 2. Dose response curves obtained for RN-9893 26 against
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Finally, we assessed the selectivity of RN-9893 26 against a
small panel of human TRP receptors, both those agonized by heat
(TRPV1 and TRPV3), and by cold (TRPM8). RN-9893 26
showed no inhibition of TRPV1 at a concentration of 10 μM, an
IC50 >30 μM against TRPV3 and an IC50 of approximately 30 μM
against TRPM8. The results from this small selectivity panel
indicate that RN-9893 is a relatively selective TRPV4 antagonist
when evaluated against related family members. Additionally,
profiling of this compound in a panel of 54 binding assays
against common biological targets revealed good selectivity
against other proteins, as significant inhibition at 10 M was only
observed against the M1 muscarinic receptor (see Supplemental
Material). Overall, the in vitro pharmacology profile and in vivo
pharmacokinetic characteristics for RN-9893 26, suggest that this
molecule may be suitable as a proof-of-concept probe of TRPV4
function (Table 6).
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bioavailable inhibitors of the TRPV4 receptor, such as the pen-
tafluorosulfamyl derivative RN-1665 23, and the tri-substituted
benzamide RN-9893 26. The most promising compound from
our studies, RN-9893 26, is a potent antagonist at human, rat and
mouse TRPV4 receptors, and is selective against related TRP

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42. While we noticed slightly different potencies of RN-9893 for
blockade of TRPV4 when stimulated with 4PDD, or
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hypotonic stimulus.
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Table 6. Pharmacology profile of RN-9893 26.
IC50
Kb
IC50
Kb
IC50
Kb
IC50
IC50
IC50
Rat PK,
F (%)
hTRPV4 hTRPV4 rTRPV4 rTRPV4 mTRPV4 mTRPV4 hTRPV1 hTRPV3 hTRPM
(M)a
Entry
Compound
(M)b
(M)a
(M)b
(M)a
(M)b
(M)c
(M)d 8 (M)e
> 30 30
46.8 ±
15.5
1
RN -9893 26
0.42
0.1
0.66
0.12
0.32
0.057
> 10
a. 4-PDD at an EC80 used as agonist. b. 4-PDD used as agonist. c. Capsaicin EC50 used as agonist. d. camphor EC50 used as agonist. e. Menthol EC50 used as
agonist
the selectivity profile of RN-9893 26 in a binding assay panel of
54 biological targets (conducted by Cerep).
Supplementary Material
Corresponding Author
Supplemental material includes pharmacokinetic curves for
dosing of RN-9893 26 to Wistar rats (po, iv, ip), experimental
details for key compounds RN-1665 23 and RN-9893 26, the
45Ca uptake assay protocol for assessment of TRPV4 activity, and
*E.mail: fabien_vincent_us@yahoo.com; Present address: Pfizer Inc,
445 Eastern Point Road, Groton, CT 06340, USA.
*E-mail: mattduncton@yahoo.com