Optimization of a novel series of TRPV4 antagonists with in vivo activity in a model of pulmonary edema

Article abstract of DOI:10.1021/ml300449k

High-throughput screening and subsequent hit optimization identified 1-piperidinylbenzimidazoles, exemplified by compound 1, as TRPV4 inhibitors. Lead optimization identified potent TRPV4 blocker 19, which has good target activity and pharmacokinetic properties. Inhibitor 19 was then profiled in an in vivo rat model, demonstrating its ability to inhibit TRPV4-mediated pulmonary edema.

Full text of DOI:10.1021/ml300449k

Letter
pubs.acs.org/acsmedchemlett
Optimization of a Novel Series of TRPV4 Antagonists with In Vivo
Activity in a Model of Pulmonary Edema
Mark A. Hilfiker,*,† Tram H. Hoang,† Johan Cornil,†,⊥ Hilary S. Eidam,† Daniel S. Matasic,‡,#
Theresa J. Roethke,§ Michael Klein,∥ Kevin S. Thorneloe,‡ and Mui Cheung†
†
‡
§
Departments of Chemistry, Biology, and Drug Metabolism and Pharmacokinetics, Metabolic Pathways and Cardiovascular
Therapeutic Area Unit, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States
∥Platform Technology and Science, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville,
Pennsylvania 19426, United States
S
* Supporting Information
ABSTRACT: High-throughput screening and subsequent hit optimization identified 1-piperidinyl-
benzimidazoles, exemplified by compound 1, as TRPV4 inhibitors. Lead optimization identified potent
TRPV4 blocker 19, which has good target activity and pharmacokinetic properties. Inhibitor 19 was
then profiled in an in vivo rat model, demonstrating its ability to inhibit TRPV4-mediated pulmonary
edema.
KEYWORDS: ion channel, calcium channel, vanilloid receptor, transient receptor potential, TRPV4, heart failure, pulmonary edema,
benzimidazole
observed in wild-type mice but notably absent in TRPV4
RPV4 (transient receptor potential vanilloid receptor 4) is
Ta member of the vanilloid family of transient receptor knockout mice.
Similar studies were conducted in mouse models evaluating
potential (TRP) ligand-gated ion channels. TRPV4 channels
are tetrameric with each monomeric unit comprising six trans-
membrane domains. The pore region, located between TM5
and TM6, mediates Ca2+ and Na+ entry across cell membranes
in response to pressure, stretch, temperature, hypotonicity, and
ligand activation.1
TRPV4 is expressed in many organs including lung, kidney,
brain, bladder, liver, choclea, retina, heart, and the vascula-
ture.2,3 Its abundant expression in vascular endothelium and
sensitivity to pressure and stretch have prompted studies to
determine if TRPV4 is implicated in regulating lung
permeability and the formation of pulmonary edema. TRPV4
was linked to elevated pulmonary vascular pressure-mediated
Ca2+ uptake by lung endothelium and subsequent acute lung
injury.4−6 Disruption of endothelial integrity at the alveolar
septal barrier in the lung is a hallmark of acute lung injury in
both respiratory distress syndrome and lung congestion
associated with heart failure. In heart failure patients, elevated
pulmonary venous pressures lead to lung congestion, resulting
in fatigue and shortness of breath (dyspnea).7,8
the effects of heightened pulmonary venous pressure, as occurs
during heart failure.4 Isolated lung preparations from wild-type
mice showed significant increases in lung permeability and
subsequent pulmonary edema in response to elevated
pulmonary venous pressures. This response was substantially
attenuated in TRPV4 knockout mice and wild-type mice
pretreated with the TRPV4 antagonist Ruthenium Red. These
studies make a compelling argument for the discovery and
development of selective TRPV4 antagonists as a treatment for
lung congestion in the heart failure patient.
Previously, our group identified a series of orally active
quinoline carboxamide TRPV4 antagonists capable of attenuat-
ing pulmonary edema in heart failure models.10−12 To further
strengthen this proposed mechanism of action, we sought to
replicate the prior observation of protection against pulmonary
edema via TRPV4 blockade with a novel chemotype. To this
end, 1-(4-piperidinyl)-benzimidazoles were identified from
early hit-to-lead chemistry as having promising TRPV4
antagonist activity (Table 1). In addition to its potency, the
piperidine moiety provided a handle for robust chemical
tractability. A survey of standard amine functionalization
realized that sulfonamides, ureas, and amides had low
micromolar activity (5−7), while the N-phenylpiperidine, 1,
Direct implication of TRPV4 activity in lung injury was
accomplished by studying the effects of TRPV4 agonists on
lung permeability in rats and wild-type and TRPV4 knockout
mice.9 4αPDD and 5,6-EET, both selective TRPV4 agonists,
were found to increase lung permeability in a dose-dependent
manner in isolated rat lungs. This agonist effect was blocked in
rats pretreated with Ruthenium Red, a nonselective TRP
antagonist. Agonist-induced increases in lung permeability were
Received: December 12, 2012
Accepted: January 27, 2013
Published: January 27, 2013
© 2013 American Chemical Society
293
dx.doi.org/10.1021/ml300449k | ACS Med. Chem. Lett. 2013, 4, 293−296

ACS Medicinal Chemistry Letters
Letter
a
Table 1. Lead Identification of Benzimidazole 113
Scheme 1. Synthetic Route
a
Reagents and conditions: (a) Na2CO3, MeCN, 25 °C. (b) Fe,
NH4Cl, EtOH/H2O, 70 °C. (c) Isopropylisothiocyanate, pyridine, 70
°C, then EDC. (d) sec-BuLi, THF, −78 °C, then ClCO2Et. (e) KOH,
THF/EtOH/H2O, 70 °C. (f) Me2NH, T3P, (iPr)2NEt, DCM, 0 °C.
(g) 1 N HCl, DCM, 25 °C. (h) Aryl bromide, Pd(OAc)2, (ortho-
biphenyl)(tBu)2P, Cs2CO3, 1,4-dioxane, 100 °C.
Table 2. Potency and PK Profiles for N-Aryl Piperidine
Analogues18
was identified as being the most potent TRPV4 inhibitor in the
series. Interestingly, analogue 3 was synthesized to evaluate
subtle changes in amine disposition and showed a modest
decrease (∼3-fold) in TRPV4 potency. Given these results,
additional N-arylpiperidines based on lead compound 1 were
evaluated.
Additional structure−activity relationship (SAR) focused on
functionalization of the 2-amino group and the N,N-
dimethylamide. The isopropylamino moiety was found to be
optimal in the 2-position of the benzimidazole with amine
moieties larger than isopropylamine having a substantial loss in
TRPV4 potency. A survey of alternative amide group
substitution also revealed that amides other than N,N-
dimethylamide were not tolerated for TRPV4 activity. As a
result, these residues were conserved with further optimization
focused on surveying SAR at the N-arylpiperidine.
Compounds were synthesized by first preparing 2-nitroani-
line, 8, by SNAr2 addition of 1-tert-butoxycarbonyl (BOC)-4-
aminopiperidine into the requisite 2-fluoronitrobenzene
(Scheme 1). An iron reduction of the nitro group followed
by condensation of the phenylenediamine intermediate with
isopropylisothiocyanate provided benzimidazole 9. The N,N-
dimethylamide group was installed via selective deprotonation
at the C4-position of the benzimidazole followed by alkylation
of the aryl lithium with ethylchloroformate. The resulting ethyl
ester was subject to hydrolysis conditions to afford acid 10.
Subsequent amide coupling with dimethylamine followed by
BOC deprotection yielded the piperidine substrate required for
surveying the N-aryl moiety. This was accomplished through a
palladium-catalyzed coupling of an arylbromide to afford the N-
arylpiperidine generically exemplified by 11.
A survey of N-arylpiperidine groups indicated that a wide
variety of substitution was tolerated for TRPV4 activity (Table
2). In addition, favorable pharmacokinetic properties could be
realized as exemplified by early analogue 12. It became evident
that incorporating large hydrophobic groups in the aryl group’s
para-position achieved the highest levels of TRPV4 potency,
a
Clearance measurements derived from iv leg in units of mL/min/kg;
b
T1/2 derived from oral leg measured in hours. Not quatifiable: target
compounds were not observed at detectable levels in isolated rat
plasma samples.
but this often resulted in decreased oral exposure in the rat as
exemplified by analogue 15. Interestingly, ester 17 was devised
in an effort to maintain the potency of tert-butyl analogue 14
and incorporate a synthetic handle for further functionalization
and was found to have excellent potency. Unfortunately, this
compound proved to be highly unstable in vivo in the rat, with
no quantifiable concentrations observed following either iv or
oral administration. Analysis of plasma samples did reveal the
294
dx.doi.org/10.1021/ml300449k | ACS Med. Chem. Lett. 2013, 4, 293−296

ACS Medicinal Chemistry Letters
Letter
Figure 1. Effect of TRPV4 agonists and antagonists on rat MAP and lung wet weights.18
presence of the corresponding carboxylic acid, 18. Believing
ester hydrolysis to be the principle clearance mechanism,
carboxylic acid 18 was surveyed and found to have no activity
against TRPV4. We hypothesized that the improved potency of
ester 17 was due to the incorporation of a hydrogen bond
acceptor and reasoned that the combination of the gem-
dimethyl group and a metabolically stable H-bond acceptor
would lead to an optimized inhibitor. To this end, nitriles 19
and 20 were surveyed and found to have excellent in vitro
potency with acceptable rat PK profiles for evaluating TRPV4
activity in vivo.
In an effort to establish a connection between TRPV4
activity and pulmonary edema, a rat in vivo model was
developed, whereby lung wet weights and mean arterial
pressure are measured with iv administration of a selective
TRPV4 agonist, GSK1016790A.14,15 Thirty minutes prior to
challenge with the agonist, animals were pretreated with
vehicle, 3 or 10 mg/kg iv of TRPV4 inhibitor 19.
GSK1016790A in the vehicle-treated group demonstrated a
dramatic dose-dependent decrease in mean arterial pressure
(MAP) and a significant increase in pulmonary edema (as
assessed by wet lung weight/body weight ratio) (Figure 1).
Nitrile 19 was evaluated in this study as it represents a good
tool compound; typical to this chemical series, its antagonist
activity in the rat (IC50 = 32 nM) is identical to its measured
human activity and does not have any TRPV4 agonist activity
in either species. In addition, ion channel selectivity for 19 is
excellent with no observed activity at concentrations greater
than 10 μM for the human Ca v1.2 (L-type), K v1.5, or Na v1.5
ion channels.16
In conclusion, a novel series of small molecules have been
identified with potent TRPV4 antagonist activity and good ion
channel selectivity. In addition, many compounds in this series
demonstrate good DMPK properties, making this series useful
in studying the pharmacology of TRPV4 activity in vivo. To
this end, compound 19 demonstrated protection against
TRPV4-mediated pulmonary edema in the rat. These
preliminary results showing in vivo activity with a second
chemical class of TRPV4 antagonists compliment our previous
studies.10−12 Together, this body of data adds weight to the
evidence that lung permeability is closely linked to TRPV4
activity and that future TRPV4 antagonists have the potential to
alleviate respiratory distress in patients suffering from heart
failure.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures, assay details, and procedures for
pharmacokinetic and pharmacodynamic studies. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 610-270-4472. Fax: 610-270-6609. E-mail: mark.a.
hilfiker@gsk.com.
Present Addresses
⊥Chimie ParisTech, 11, rue Pierre et Marie Curie, 75231 Paris,
France.
#Department of Biology, The Pennsylvania State University,
University Park, Pennsylvania 16802, United States.
Nitrile 19 dosed at 3 and 10 mg/kg providing plasma
concentrations of 1.26 (42 nM free) and 3.39 μM (112 nM
free), respectively.17 A dose-dependent decrease in agonist-
induced MAP decrease was observed with complete blockade
of the agonist affect with 10 mg/kg of compound 19. No effect
on MAP was observed in the pretreatment phase prior to
agonist challenge. While no effect on the lung/body weight
ratio was observed at 3 mg/kg, a significant decrease in wet
lung weight was observed at the 10 mg/kg dose as compared to
vehicle-treated rats. These results indicate that compound 19 is
active in vivo and able to inhibit TRPV4 activity, consistent
with prior observations.10−12
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
TRPV4, transient receptor potential vanilloid receptor 4; SAR,
structure−activity relationship; BOC, tert-butoxycarbonyl; T3P,
1-propanephosphonic acid cyclic anhydride; MAP, mean
arterial pressure; hERG, human ether-a-go-go related gene
potassium channel; LW/BW ratio, wet lung weight/body
weight ratio
295
dx.doi.org/10.1021/ml300449k | ACS Med. Chem. Lett. 2013, 4, 293−296

ACS Medicinal Chemistry Letters
Letter
(15) Mendoza, S. A.; Fang, J.; Gutterman, D. D.; Wilcox, D. A.;
Bubolz, A. H.; Li, R.; Suzuki, M.; Zhang, D. X. TRPV4-mediated
endothelial Ca2+ influx and vasodilation in response to shear stress.
Am. J. Physiol. Heart Circ. Physiol. 2010, 298, H466.
(16) Details for the Ca v1.2 (L-type), K v1.5, Na v1.5, and hERG ion
channel selectivity assays can be found in the Supporting Information.
(17) Plasma protein binding was measured for compound 19 and
found to be 4.4 and 3.3% unbound in human and rat plasmas,
respectively.
(18) All animal studies were conducted in accordance with the GSK
Policy on the Care, Welfare, and Treatment of Laboratory Animals
and were reviewed by the Institutional Animal Care and Use
Committee at GSK.
REFERENCES
■
(1) Everaerts, W.; Nilius, B.; Owsianik, G. The vanilloid transient
receptor potential channel TRPV4: From structure to disease. Prog.
Biophys. Mol. Biol. 2010, 103, 2−17.
(2) Vincent, F.; Duncton, A. J. TRPV4 Agonists and Antagonists.
Curr. Top. Med. Chem. 2011, 11 (17), 2216−2226.
(3) Bakthavatchalam, R.; Kimball, S. D. Modulators of Transient
Receptor Potential Ion Channels. Annu. Rep. Med. Chem. 2010, 45,
37−53 and references therein.
(4) Jian, M.-Y.; King, J. A.; Al-Mehdi, A.-B.; Liedtke, W.; Townsley,
M. I. High Vascular Pressure-Induced Lung Injury Requires P450
Epoxygenase-Dependent Activation of TRPV4. Am. J. Respir. Cell Mol.
Biol. 2008, 38, 386−392.
(5) Yin, J.; Hoffmann, J.; Kaestle, S. M.; Neye, N.; Wang, L.; Baeurle,
J.; Liedtke, W.; Wu, S.; Kuppe, H.; Pries, A. R.; Kuebler, W. M.
Negative-feedback loop attenuates hydrostatic lung edema via a
cGMP-dependent regulation of transient receptor potential Vanilloid
4. Circ. Res. 2008, 103, 966−974.
(6) Kuebler, W. M.; Ying, X.; Bhattacharya, J. Pressure-induced
endothelial Ca2+ oscillations in lung capillaries. Am. J. Physiol. Lung Cell
Mol. Physiol. 2002, 282, L917−L923.
(7) Bernard, G. R.; Artigas, A.; Brigham, K. L.; Carlet, J.; Falke, K.;
Hudson, L.; Lamy, M.; LeGall, J. R.; Morris, A.; Spragg, R. The
American European Consensus Conference on ARDS. Definitions,
mechanisms, relevant outcomes, and clinical trial coordination. Am. J.
Respir. Crit. Care Med. 1994, 149, 818−824.
(8) Matthay, M. A.; Zimmerman, G. A.; Esmon, C.; Bhattacharya, J.;
Coller, B.; Doerschuk, C. M.; Floros, J.; Gimbrone, M. A., Jr.;
Hoffman, E.; Hubmayr, R. D.; Leppert, M.; Matalon, S.; Munford, R.;
Parsons, P.; Slutsky, A. S.; Tracey, K. J.; Ward, P.; Gail, D. B.; Harabin,
A. L. Future research directions in acute lung injury: Summary of
National Heart, Lung, and Blood Institute working group. Am. J.
Respir. Crit. Care Med. 2003, 167, 1027−1035.
(9) Alvarez, D. F.; King, J. A.; Weber, D.; Addison, E.; Liedtke, W.;
Townsley, M. I. Transient Receptor Potential Vanilloid 4-Mediated
Disruption of the Alveolar Septal Barrier: A Novel Mechanism of
Acute Lung Injury. Circ. Res. 2006, 99, 988−995.
(10) Thorneloe, K. S.; Cheung, M.; Bao, W.; Alsaid, H.; Lenhard, S.;
Jian, M.-Y.; Costell, M.; Maniscalco-Hauk, K.; Krawiec, J. A.; Olzinski,
A.; Gordon, E.; Lozinskaya, I.; Elefante, L.; Qin, P.; Matasic, D. S.;
James, C.; Tunstead, J.; Donovan, B.; Kallal, L.; Waszkiewicz, A.;
Vaidya, K.; Davenport, E. A.; Larkin, J.; Burgert, M.; Casillas, L. N.;
Marquis, R. W.; Ye, G.; Eidam, H. S.; Goodman, K. B.; Toomey, J. R.;
Roethke, T. J.; Jucker, B. M.; Schnackenberg, C. G.; Townsley, M. I.;
Lepore, J. J.; Willette, R. N. An Orally Active TRPV4 Channel Blocker
Prevents and Resolves Pulmonary Edema Induced by Heart Failure.
Sci. Transl. Med. 2012, 4, 159ra148.
(11) Brooks, C. A.; Cheung, M.; Eidam, H. S.; Fox, R. M.; Hilfiker,
M. A.; Manas, E. S.; Ye, G. 4-Quinolinecarboxamide derivatives as
TRPV4 antagonists and their preparation and use for the treatment of
diseases. WO 2011/119704 A1, September 29, 2011.
(12) Brooks, C. A.; Cheung, M.; Eidam, H. S.; Fox, R. M.; Hilfiker,
M. A.; Manas, E. S.; Ye, G. 4-Quinolinecarboxamide derivatives as
TRPV4 antagonists and their preparation and use for the treatment of
diseases. WO 2011/119701 A1, September 29, 2011.
(13) TRPV4 potency was determined by measuring the blockade of
agonist GSK634775-induced calcium immobilization through human,
recombinant TRPV4 channels in HEK cells on a fluorescent light
imaging plate reader (FLIPR). The reported potencies are an average
of at least two measurements with a SEM 0.1 for the pIC50. Assay
details can be found in the Supporting Information.
(14) Willette, R. N.; Bao, W.; Nerurkar, S.; Yue, T-l.; Doe, C. P.;
Stankus, G.; Turner, G. H.; Ju, H.; Thomas, H.; Fishman, C. E.;
Sulpizio, A.; Behm, D. J.; Hoffman, S.; Lin, Z.; Lozinskaya, I.; Cassillas,
L. N.; Lin, M.; Trout, R. E. L.; Bartholomew, J. V.; Thorneloe, K.;
Lashinger, E. S. R.; Figueroa, D. J.; Marquis, R.; Xu, X. Systematic
Activation of the Trasient Receptor Potential Vanilloid Subtype 4
Channel Causes Endothelial Failure and Circulatory Collapse: Part 2.
J. Pharmacol. Exp. Ther. 2008, 326, 443.
296
dx.doi.org/10.1021/ml300449k | ACS Med. Chem. Lett. 2013, 4, 293−296