Development of novel azabenzofuran TRPA1 antagonists as in vivo tools

Article abstract of DOI:10.1016/j.bmcl.2014.05.069

The transient receptor potential ankyrin 1 (TRPA1) channel is activated by noxious stimuli including chemical irritants and endogenous inflammatory mediators. Antagonists of this channel are currently being investigated for use as therapeutic agents for t

Full text of DOI:10.1016/j.bmcl.2014.05.069

Accepted Manuscript
Development of Novel Azabenzofuran TRPA1 Antagonists as in vivo Tools
Katrina W. Copeland, Alessandro A. Boezio, Eugene Cheung, Josie Lee, Philip
Olivieri, Laurie B. Schenkel, Qian Wan, Weiya Wang, Mary C. Wells, Beth
Youngblood, Narender R. Gavva, Sonya G. Lehto, Stephanie Geuns-Meyer
PII:
S0960-894X(14)00578-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.069
BMCL 21683
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 April 2014
16 May 2014
20 May 2014
Please cite this article as: Copeland, K.W., Boezio, A.A., Cheung, E., Lee, J., Olivieri, P., Schenkel, L.B., Wan, Q.,
Wang, W., Wells, M.C., Youngblood, B., Gavva, N.R., Lehto, S.G., Geuns-Meyer, S., Development of Novel
Azabenzofuran TRPA1 Antagonists as in vivo Tools, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.05.069
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Development of Novel Azabenzofuran TRPA1 Antagonists as in vivo Tools
Copeland, Katrina W.a,*; Boezio, Alessandro A. a,*; Cheung, Eugeneb; Lee, Josiec; Olivieri, Philipa;
Schenkel, Laurie B. a; Wan, Qiana; Wang, Weiyad; Wells, Mary C.e; Youngblood, Bethd; Gavva,
Narender, R.d; Lehto, Sonya G.d; Geuns-Meyer, Stephaniea
*Corresponding authors. Tel 617-444-5350 and 617-444-5104, email kwoodin@amgen.com and
aboezio@amgen.com
aDepartment of Medicinal Chemistry, Amgen Inc., 360 Binney St., Cambridge, MA 02142 USA
bDepartment of Pharmaceutics, Amgen Inc., 360 Binney St., Cambridge, MA 02142 USA
cDepartment of Neuroscience, Amgen Inc., 360 Binney St., Cambridge, MA 02142 USA
dDepartment of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320 USA
eDepartment of Pharmacokinetics & Drug Metabolism, Amgen Inc., 360 Binney St., Cambridge, MA
02142 USA
Abstract
The transient receptor potential ankyrin 1 (TRPA1) channel is activated by noxious stimuli including
chemical irritants and endogenous inflammatory mediators. Antagonists of this channel are currently
being investigated for use as therapeutic agents for treating pain, airway disorders, and itch. A novel
azabenzofuran series was developed that demonstrated in vitro inhibition of allyl isothiocyanate (AITC)-
induced Ca2+ uptake with nanomolar potencies against both human and rat TRPA1. From this series,
45
compound 10 demonstrated in vivo target coverage in an AITC-induced flinching model in rats while
providing unbound plasma concentrations up to 16-fold higher than the TRPA1 rat IC50.

The transient receptor potential ankyrin 1 (TRPA1) channel is a nonselective cation channel that has been
implicated in various physiological and sensory functions including nociception, and thermal, chemical,
and mechanical sensation. It is primarily expressed in the sensory neurons of the dorsal root, trigeminal,
and vagal ganglia, as well as in some non-neuronal tissues like skin and hair cells.1 TRPA1 is activated by
many pungent and reactive compounds including allyl isothiocyanate (AITC, a component of mustard
oil), cinnamaldehyde, allicin (present in garlic), nicotine, and various tear gases.2 These compounds
covalently bind to the cysteine residues in the N-terminus of TRPA1 and allosterically open the
channel.2c,3 This opening increases intracellular Ca2+ concentrations, which initiates multiple downstream
signaling processes, leading to a noxious or pain response.4 Endogenous compounds that are products of
tissue damage related to inflammation and oxidative stress (e.g., hydrogen peroxide,5 reactive oxygen
species (ROS),5,6 4-hydroxy-2-nonenal,7 nitrooleic acid,8 and cycylopentenone prostaglandins9) also
activate TRPA1.10 The level of TRPA1 expression increases in human sensory neurons following an
inflammatory injury and in rodent models of inflammation and neuropathic pain.11 Recently, a family
suffering from Familial Episodic Pain Syndrome (FEPS), which causes episodes of debilitating upper
body pain, was discovered to have a gain-of-function TRPA1 mutation.12 In addition to playing a role in
neurogenic inflammation and pain, TRPA1 functions as a mechanosensor; TRPA1 knock-out mice are
less sensitive to mechanical stimuli as well as chemically-induced AITC flinching.13
The support from human genetics, the known roles for TRPA1 in causing pain and sensitization, and its
localized expression have made TRPA1 antagonists an attractive target for novel analgesics and anti-
inflammatory drugs. Antagonists may have further indications including itch14 and respiratory diseases
such as asthma and COPD.15 Several groups have reported the identification of TRPA1 antagonists16 and
a few compounds entered into clinical trials.17
In previous studies with this target, we and others identified electrophilic TRPA1 antagonists that were
presumed to covalently modify the TRPA1 protein and that exhibited cross-species differences in
antagonist versus agonist behavior.18 Unsatisfied with existing TRPA1 benchmark antagonists, our goal

has been to develop a tool compound that can provide high exposure in vivo (covering multiple fold in
excess of the in vitro IC50) to validate TRPA1 as a potential target for pain.
Figure 1: Profile of Compound 1
A series of tricyclic 3,4-dihydropyrimidine-2-thione TRPA1 antagonists, represented by compound 1
(Figure 1), was recently reported.19 The authors cited poor drug-like properties for this series of
compounds, including low solubility, low metabolic stability, and potential toxicity of the thiourea
moiety.20 In-house profiling of this compound confirmed the low likelihood of achieving robust in vivo
exposure (Figure 1).21 Compound 1 displayed high turnover in human and rat microsomes, which
translated to high in vivo clearance in rat (2.9 L/h/kg) with an associated unbound clearance (Clu) of 290
L/h/kg. Moreover, this clearance coupled with poor solubility led to poor oral bioavailability (%F = 10).
We sought to modify this lead to improve pharmacokinetic properties in order to achieve in vivo target
coverage of TRPA1 toward understanding the therapeutic potential of this mechanism for further
development. Our first strategy was to introduce polarity by including slightly basic heteroatoms to lower
logP, with the expectation that oxidative clearance would be reduced and solubility improved. In
addition, we sought to remove the ketone and thiourea moieties as toxicological liabilities have been
reported for these functional groups22 that may prevent further advancement.
Table 1: Introduction of nitrogen atoms into the side chain

aAntagonisum was measured using a 45Ca2+ fluorimetry assay in both rat and human TRPA1 expressed cells. No agonism was observed for all compounds,
45Ca2+ EC50 > 40 µM. bHCl: 0.01 N hydrogen chloride solution in water; PBS: phosphate buffered saline, pH 7.4; SIF: fasted state simulated intestinal
fluid, pH 6.8 containing 5 mM sodium taurochol, 1.5 mM lecithin, 2.9 mM KH2PO4, 0.22 M KCl. cIn vitro (HLM = human liver microsomes; RLM = rat liver
microsomes). dHLM/RLM data not obtained for the racemate. eNo IC50 value obtained; instead single point 45Ca2+ % Inhibition at 2 µM was 1% in humans
and showed 0% in rat.
Our first tactic toward lowering the logP was to introduce nitrogen atoms into the chiral aromatic ether
side chain of 1 (Table 1).23 First, nitrogen was positioned around the side-chain aryl ring, but these
permutations led to significant losses in potencies (compounds 2-5). Compound 2 showed significant
improvement in solubility and 3 showed a slight improvement in both solubility and human microsomal
stability compared to 1, but poor potency prevented further progression of these compounds.
Replacement of the 3-methoxypropoxy side chain with pyridine rings was better tolerated and compounds
6 & 7 showed TRPA1 antagonism. Both these compounds showed improved solubility, but were unstable
in liver microsomes.

Table 2: Modification of the core
aAntagonisum was measured using a 45Ca2+ fluorimetry assay in both rat and human TRPA1 expressed cells. No agonism was observed for all compounds,
45Ca2+ EC50 > 40 µM. bHCl: 0.01 N hydrogen chloride solution in water; PBS: phosphate buffered saline, pH 7.4; SIF: fasted state simulated intestinal
fluid, pH 6.8 containing 5 mM sodium taurochol, 1.5 mM lecithin, 2.9 mM KH2PO4, 0.22 M KCl. cIn vitro (HLM = human liver microsomes; RLM = rat liver
microsomes. dHLM/RLM data not obtained for the racemate.
Modifying the core instead led to more dramatic changes in the physical-chemical properties of this series
(Table 2).24 Replacing the carbonyl of indenone 1 by incorporating the oxygen into the ring resulting in a
benzofuran (compound 8), led to a significant loss in potency on rat and human TRPA1, and no marked
change in solubility or microsomal stability. Addition of a fluorine atom adjacent to the oxygen
(compound 9) led to an increase in both potency and microsomal stability. Finally, the replacement of the
fluorine in 9 with a nitrogen (compound 10) created a new azabenzofuran core with significantly restored
potency, especially against rat TRPA1. As anticipated, the installation of an ionizable nitrogen reduced
the clogP, which led to an improvement in both solubility and microsomal stability of the compound.

When the 3-(pyridin-2-ylmethoxy)phenyl side chain of 7 was combined with the new azabenzofuran core,
the solubility was further increased in acidic media but a loss of potency was also observed (compound
11).
Figure 2: Inactive replacements for the thiourea moiety
a
Compounds had either TRPA1 45Ca2+ IC50 > 40 µM or inhibition <20% at 2 µM. Compounds 16-19 did not show agonism,
45Ca2+ EC50 > 40 µM; Compounds 12-15 were not tested. b TRPA1 45Ca2+ IC50 rat was 6.3 µM for this compound; no agonsim
observed.
In addition to improving the physical-chemical properties, we also sought to replace the thiourea in either
the indenone or azabenzofuran core, depending on synthetic ease (Figure 2). Methylating the thiourea
(compound 12) led to loss of activity on TRPA1. Replacing the cyclic thiourea with dihydropyrimidine
13, methyl dihydropyrimidine 14, and amino dihydropyrimidine 15 also led to loss of activity. In the
azabenzofuran core, the substitution of the thiourea for a urea to produce 16 led to a dramatic loss in
potency on TRPA1.25 Converting the urea to a lactam to form compound 17 also did not show any
antagonism. The thiourea bioisosteres26 sulfamide 18 and 2-cyanoguanidine 19 were also showed no
antagonism of TRPA1. Thus our preliminary screening results showed that the thiourea as essential for
maintaining potency on TRPA1. The importance of the thiourea may perhaps be attributed to the
potential for covalent binding with cysteine residues present in the protein.27
Scheme 1: Synthesis of 10 via the Biginelli reaction

The azabenzofuran thioureas, as exemplified by 10, were synthesized using a modification of the three-
component Biginelli reaction reported by Gijsen et al. (Scheme 1).28 Replacing the hydrochloric acid as
previously reported with sodium bisulfate in the coupling increased the yields from ~10% to above 50%.
The most potent racemic products were then separated using supercritical fluid chromatography (SFC,
Chiralpak AD-H column) to provide each enantiomer with >99% ee.
Table 3. In Vitro and In Vivo ADME Properties of Compound 1 and 10
1
230
10
90
RLM (µL / min mg)
Rat plasma protein binding (fu)a
Ratb iv CL / CLu (L / h / kg)
Vss (L / kg) / T1/2 (h)
0.010
2.9 / 290
4.7 / 2.1
0.005 / 10
--
0.085
3.0 / 36
4.0 / 1.2
--
Unbound rat Cmax (µM)c,d / % F at 10 mg/kg
at 30 mg/kg
0.082 / 13
0.612 / 79
at 100 mg/kg
--
a fu: unbound fraction; separation method = equilibrium dialysis. b In vivo experiments with male Sprague-Dawley rats (n = 3); iv, 0.5 mg/kg (DMSO). c All
dose levels were run po in 2% HPMC, 1% Tween 80 in H2O, pH 2.2 with MSA. dThe 10 and 30 mg/kg groups of animals were fasted during PK studies, the
100 mg/kg were fed.
The rat in vivo clearance and steady state volume of distribution were very similiar for 1 and 10 (Table 3).
The unbound clearance (CLu) of azabenzofuran 10 was approximately 8-fold better than 1 as a result of its
higher free fraction and comparable clearance. The improved solubility of 10 relative to 1 allowed
compound dosing at 100 mg/kg, which likely saturated the primary elimination mechanisms based on the
nonlinear increase in bioavailability between the 30 and 100 mg/kg doses (13% compared to 79%). The
incorporation of polarity in the azabenzofuran replacement of the indenone core resulted in a slight loss of
potency, which was well made up for by the reduction in unbound clearance and increase in solubility.

Since this change led to a dramatic improvement in unbound plasma concentration (Cu) for 10, 14-fold
over the rat IC50 at the 100 mg/kg dose, this compound was advanced into an AITC-induced flinching
model.
Figure 3: AITC (0.1%)-Induced Flinching in Rats
Compound 10,29 as well as its inactive enantiomer ent-10,30 which served as a negative control, were then
evaluated in a target-coverage model using AITC-induced flinching in rats (Figure 3).31 AITC is an
agonist of TRPA1 that covalently modifies key cysteine and lysine residues in the N-terminus of the
channel, and activates the C-mechano-heat nociceptors to release neuropeptides leading to inflammatory
edema, hyperalgesia and pain.32 Male Harlan Sprague-Dawley rats33 were dosed orally with either vehicle
(2% HPMC/1% Tween-80), the TRPA1 antagonist 10, or ent-10. After 1 hour, each animal’s left ventral
hind paw was injected with the agonist AITC (0.1%). The nocifensive behaviors (including flinching and
licking) were then observed and recorded during the first minute post injection. The unbound plasma
concentrations (Cu) for the 10, 30, and 100 mg/kg doses were 0.048 0.010 (n=8), 0.22 0.04 (n=6), and
0.74 0.14 (n=8) µM, respectively, covering the in vitro rat TRPA1 IC50 at approximately 1X, 5X, and
16X, respectively. Significant reversal of AITC-induced flinches was observed at 30 and 100 mg/kg with

compound 10.34 As expected, the inactive enantiomer ent-10 showed no blockade of AITC-induced
flinches at 100 mg/kg with an exposure (Cu = 0.37 0.12 µM) in a range where 10 was efficacious,
supporting the likely on-mechanism effect of 10.
In conclusion, we have described a novel azabenzofuran thiourea core that displays good in vitro
antagonism of the TRPA1 channel. The genesis of the series came from modification of Compound 1 in
an attempt to improve solubility and clearance by lowering the logP and introducing ionizable atoms.
Although we were unable to replace the thiourea, we identified a compound (10) that provides an
unbound plasma concentration 16-fold in excess of the in vitro IC50 and demonstrates significant effect in
an on-target biochemical challenge study (AITC-induced flinching). Further investigations with this tool
compound in various pain models will be disclosed in due course.
Acknowledgements
The authors would like to thank Loren Berry for measuring plasma protein binding, Heather Eastwood for
collecting solubility data, Xingwen Li and Virginia Berry for bioanalysis of the PK and PD studies, and
Min-Hwa Jasmine Lin and Yohannes Teffera for input to pharmacokinetic and drug metabolism
investigations. We’d also like to thank Dawn Zhu for assistance with the AITC-flinch model and
Margaret Chu-Moyer for editing input.
1
aStory, G. M.; Peier, A. M.; Reeve, A. J.; Eid, S. R.; Mosbacher, J.; Hricik, T. R.; Earley, T. J.; Hergardern, A.C.;
b
Andersson, D. A.; Hwang, S. W.; McIntyre, P.; Jegla, T.; Bevan, S.; Patapoutian, A. Cell, 2003, 112, 819. Nagata,
c
K.; Duggan, A.; Kumar, G.; Gracía-Añoveros, J. J. Neurosci., 2005, 25, 4052. Kobayashi, K.; Fukuoka, T.; Obata,
K.; Yamanaka, H.; Dai, Y.; Tokunaga, A.; Noguchi, K. J. Comp. Neurol, 2005, 493, 596. dAndre, E,; Campi, B.;
Materazzi, S.; Trevisani, M.; Amadesi, S.; Massi, D.; Creminon, C.; Vaksman, N.; Nassini, R.; Civelli, M.; Baraldi,
e
P. G.; Poole, D. P.; Bunnett, N. W.; Geppetti, P.; Patacchinni, R. J. J. Clin. Invest., 2008, 118, 2574. Corey, D. P.;
Gracía-Añoveros, J.; Holt, J. R.; Kwan, K. Y.; Lin, S. Y.; Vollrath, M. A.; Amalfitano, A.; Cheung, E. L.; Derfler,
B. H.; Duggan, A.; Geleoc, G. S.; Gray, P. A.; Hoffman, M. P.; Rehm, H. L.; Tamasauskas, D.; Zhang, D. S.
f
Nature, 2004, 432, 723. Nassenstein, C.; Kwong, K.; Taylor-Clark, T.; Kollarik, M.; MacGlashan, D. M.; Braun,
A.; Undem, B. J. J. Physiol., 2008, 586, 1595.
2
aJordt, S. E.; Bautista, D. M.; Chuang, H. H. Nature, 2004, 427, 260. bMacpherson, L. J.; Geierstanger, B. H.;
Viswanath, V.; Bandell, M.; Eid, S. R.; Hwang, S. W.; Patapoutian, A. Curr. Biol., 2005, 15, 929. cHinman, A.;

Chuang, H. H.; Bautista, D. M.; Julius, D. Proc. Natl. Acad. Sci. USA. 2006, 103, 19564. Bandell, M.; Story, G.
M.; Hwant, S. W.; Viswanath, V.; Eid, S. R.; Petrus, M. J.; Earley, T. J.; Patapoutian, A. Neuron, 2004, 41, 849.
Talavera, K.; Gees, M.; Karashima, Y.; Meseguer, V. M.; Vanoirbeek, J. A.; Damann, N.; Everaerts, W.; Wouter,
B.; Benoit, M.; Janssens, A.; Vennekens, R.; Viana, F.; Benoit, N.; Nilius, B.; Voets, T. Nat. Neurosci., 2009, 12,
1293.
3
Macpherson, L. J.; Dubin, A. E.; Evans, M. J.; Marr, F.; Schultz, P. G.; Cravatt, B. F.; Patapoutian, A. Nature,
2007, 445, 541.
4
Chen, J.; McGaraughty, S.; Kym, P. R. TRP Channels in Drug Discovery, 2012, 1, 43.
5 Sawada, Y.; Hosokawa, H.; Matsumura, K.; Kobayashi, S. Eur. J. Neurosci., 2008, 27, 1131.
6 Bessac, B. F.; Sivula, M.; von Hehn, C. A.; Escalera, J.; Cohn, L.; Jordt, S. E. J. Clin. Invest., 2008, 118, 1899.
7
Trevisani, M.; Siemens, J.; Materazzi, S.; Bautista, D. M.; Nassini, R.; Campi, B.; Imamachi, N.; Andre, E.;
Patacchini, R.; Cottrell, G. S.; Gatti, R.; Basbaum, A. I.; Bunnett, N. W.; Julius, D.; Geppetti, P. Proc. Natl. Acad.
Sci. USA, 2007, 104, 13519.
8 Taylor-Clark, T. E.; Ghatta, S.; Bettner, W.; Undem, B. J. Mol. Pharmacol., 2009, 75, 820.
9
Materazzi, S.; Nassini, R.; Andre, E.; Campi, B.; Amadesi, S.; Trevisani, M.; Bunnet, N. W.; Patacchini, R.;
Geppetti, P. Proc. Natl. Acad. Sci. USA, 2008, 105, 12045.
10 aTakahashi, N.; Mizuno, Y.; Kozai, D.; Yamamoto, S.; Kiyonaka, S.; Shibata, T.; Uchida, K.; Mori, Y. Channels,
b
2008, 27, 1131. Andersson, D. A.; Gentry, C.; Moss, S.; Bevan, S. J. Neurosci., 2008, 28, 2485.
11
aObata, K.; Katsura, H.; Mizushima, T.; Yamanaka, H.; Kobayashi, K.; Dai, Y.; Fukuoka, T.; Tokunaga, A.;
b
Tominaga, M.; Noguchi, K. J. Clin. Invest.; 2005, 115, 2393. Anand, U.; Otto, W. R. Neurosci. Lett., 2008, 438,
c
221. da Costa, D. S.; Meotti, F. C.; Andrade, E. L.; Leal, P. C.; Motta, E. M.; Calixto, J. B. Pain, 2010, 148, 431.
12 Kremeyer, B.; Lopera, F.; Cox, J. J.; Momin, A.; Rugiero, F.; Marsh, S.; Woods, C. G.; Jones, N. G.; Paterson, K.
J.; Fricker, F. R.; Villegas, A.; Acosta, N.; Tineda-Trujilo, N. G.; Ramirez, J. D.; Zea, J.; Burley, M.-W.; Bedoya,
G.; Bennett, D. L. H.; Wood, J. N.; Ruiz-Linares, A. Neuron, 2010, 66, 671.
13
aKwan, K. Y.; Allchorne, A. J.; Vollrath, M. A.; Christensen, A. P.; Zhang, D.-S.; Woolf, C. J.; Corey, D. P.
b
Neuron, 2006, 50, 277. Bautista, D. M.; Movahed, P.; Hinman, A.; Axelsson, H. E.; Sterner, O.; Hogestatt, E. D.;
Julius, D.; Jordt, S.-E.; Zygmunt, P. E. Proc. Natl. Acad. Sci. USA, 2005, 102, 12248.
b
14 aXiao, B.; Patapoutian, A. Nat. Neurosci., 2011, 14, 540. Wilson, S. R.; Gerhold, K. A.; Bifolck-Fisher, A.; Liu,
Q.; Patel, K. N.; Bautista, D. M. Nat. Neurosci., 2011, 14, 595.
15 aBessac, B. F.; Jordt, S. E.; Physiology, 2008, 23, 360. bSimon, S. A.; Liedtke, W. J. Clin. Invest., 2008, 118,
2383. cCaceras, A. I.; Brackmann, M.; Elia, M. D.; Bessac, B. F.; del Camino, D.; D’Amours, M.; Witek, J. S.;
Fanger, C. M.; Chong, J. A.; Hayward, N. J.; Homer, R. J.; Cohn, L.; Huang, X.; Moran, M. M.; Jordt, S. E. Proc.
Natl. Acad. Sci. USA, 2009, 106, 9099. dGeppetti, P.; Patacchini, R.; Nassini, R.; Materazzi, S. Lung, 2010, 188,
e
S63. Grace, M. S.; Belvisi, M. G. Pulm. Pharmacol. Ther., 2011, 24, 286.
16
For recent reviews on TRPA1 as a target for therapuetic agents see these references and the compounds cited
a
therein: See reference 4. bBaraldi, P. G.; Preti, D.; Materazzi, S.; Geppetti, P. J. Med. Chem., 2010, 53, 5085.
cRadresa, O.; Dahllof, H.; Nyman, E.; Nolting, A.; Albert, J. S.; Raboisson, P. The Open Pain Journal, 2013, 6,
d
137. Khot, S. S.; Shaikh, H. Y.; Patil, R. S. Int. J. Pharm. Sci. Res., 2013, 6, 2035. eBrederson, J.-D.; Kym, P. R.;
f
g
Szallasi, A. Eur. J. of Pharm., 2013, 716, 61. Premkumar, L.; Abooj, M. Life Sciences, 2013, 92, 415. Salat, K.;
Moniczewski, A.; Librowski, T. Cur. Med. Chem., 2013, 20, 1409. hWaskielewicz, A. M.; Gunia, A.; Szkaradek,
i
Other references: De
N.; Sloczynska, K.; Krupinska, S.; Marona, H. Cur. Med. Chem., 2013, 20, 1241.
j
Petroclellis, L.; Moriello, A. S.; Expert. Opin. Ther. Patents, 2013, 23, 119. Baraldi, P. G.; Romagnoli, R.;
Saponaro, G.; Tabrizi, M. A.; Baraldi, S.; Pedretti, P.; Fusi, C.; Nassini, R.; Materazzi, S.; Geppetti, P.; Preti, D.
Biorg. Med. Chem., 2012, 20, 1690. kVallin, K. S. A.; Sterky, K. J.; Nyman, E.; Bernstrom, J.; From, R.; Linde, C.;
Minidis, A. B. E.; Nolting, A.; Narhi, K.; Santangelo, E. M.; Sehgelmeble, F. W.; Sohn, D.; Strindlund, J.; Weigelt,
D. Biorg. Med. Chem., 2012, 22, 5485. lOrtar, G.; Moriello, A. S.; Morera, E.; Nalli, M.; Di Marzo, V.; De
Petrocellis, L. Biorg. Med. Chem. Lett., 2013, 23, 5614. mMorera, E.; De Petrocellis, L.; Morera, L.; Moriello, A.
S.; Nalli, M.; Di Marzo, V.; Ortar, G. Biorg. Med. Chem. Lett., 2012, 22, 1674. nMaione, S.; Costa, B.; Piscitelli,
F.; Morera, E.; De Chiaro, M.; Comelli, F.; Boccella, S.; Guida, F.; Verde, R.; Ortar, G.; Di Marzo, V. Pharm. Res.,
o
2013, 76, 98. Wu, X.; Kimball, S. D.; Chen, P.; Zhou, D.; Peng, S. US Patent 8530487 B1, 2013.
17aCubist Pharmaceuticals’/Hydra Biosciences’ CB-625: Clinical trials were halted due to solubility concerns; see
b
Cubist Pharmaceuticals quarterly report (Form 10-Q) filed on 05/07/2013 for further details . Glenmark’s GRC
17536 is currently in Phase II clinical trials: Muthuppalniappan, M. T. A.; Kumar, S.; Margal, S.; Khairatkar-Joshi,
N.; Mukhopadhyay, I.; Gullapalli, S. US Patent 20090325987 A1 20091231, 2009.

18 aKlionsky, L. Tamir, R.; Goa, B.; Wang, W.; Immke, D. C.; Nishimura, N.; Gavva, N. R. Mol. Pain, 2007, 3, 39.
bChen, J.; Zhang, X. F. J. Neurosci., 2008, 28, 5063.
19 Gijsen, H. J. M.; Berthelot, D.; De Cleyn, M. A. J.; Geuens, I.; Brone, B.; Mercken, M. Bioorg. Med. Chem. Lett.
2012, 22, 797.
20aBlagg, Julian; Burger’s Medicinal Chemistry, Drug Discovery, and Development, Seventh Ed. 2010, 318.
bEdwards, P. J.; Sturino, C. Curr. Med. Chem., 2011, 18, 3116 and references cited therein
21 Data reported in Figure 1 obtained internally.
22 aKetone toxicity: Evans, D. C.; Watt, A. P.; Nicoll-Griffith, D.; Baillie, T. A. Chem. Res. Toxicol., 2004, 17, 3.
bThiourea toxicity: see reference 20.
23 Compounds were screened for antagonism using a 45Ca2+ fluorimetry assay in both rat and human TRPA1
expressed cells. See reference 18 for experimental details on the fluorimetry assay used for this program.
24 Racemic compounds that showed potency were further separated using chiral chromatography and were further
tested as individual enantiomers. Assignment of stereochemistry was made in analogy to Compound 1 , see
reference 19. As was observed by Gijsen et al., one enantiomer in these new compounds was potent in the 45Ca2+
assay, and the other enantiomer showed minimal TRPA1 antagonism.
25 This trend was similarly observed in the indenone core as reported by Gijsen et al. in reference 19.
a
26 For reviews on bioisosteres, including those for the thiourea see: Patani, G. A.; LaVoie, E. J. Chem. Rev., 1996,
b
96, 3147. Meanwell, N. A. J. Med. Chem., 2011, 54, 2529. cLima, L. M.; Barreiro, E. J. Cur. Med. Chem., 2005,
12, 23.
27 Chuang, H.-H.; Lin, S. Proc. Natl. Acad. Sci. USA. 2009, 106, 20097.
28
Zeynizadeh, B.; Dilmaghani, K. A.; Yari, M. Phosphorous, Sulfur and Silicon and the related Elements, 2009,
184, 2465.
29 Assignment of stereochemistry was made in analogy to Compound 1, see reference 24.
30 Data for ent-10: 45Ca2+ human IC50: >40 mM; 45Ca2+ rat IC50: undefined (failed to achieve proper hill fit to
generate IC50 curve).
31For experimental details on the AITC mechanical challenge model used for this program see: de Oliveira, C.;
Garami, A.; Lehto, S.; Pakai, E.; Tekus, V.; Pohoczky, K.; Youngblood, B.; Wang, W.; Kort, M.; Kym, P.; Pinter,
E.; Gavva, N.; Romanovsky, A.. Journal of Neuroscience, 2014, 34 , 4445.
32
aSee references 2a, 2c, 3 and 9 and Andrade, E. L.; Luiz, A. P.; Ferreira, J.; Calixto, J. B. Neuroscience, 2008,
152, 511.
33 All experimental procedures were approved by the Institutional Animal Care and Use Committee at an AAALAC-
accredited facility, and adhere to the National Research Council’s Guide for the Care and Use of Laboratory
Animals: Eighth Ed., Washington, DC: The National Academies Press (US); 2011.
34 No significant effects on open-field activity were observed with this compound.
Supporting Information:
A mixture of 3-(3-methoxypropoxy)benzaldehyde (7.19 g, 37.0 mmol), sodium hydrogensulfate
hydrate (0.102 g, 0.740 mmol), furo[2,3-b]pyridin-3(2H)-one (5.0 g, 37.0 mmol) and thiourea
(5.63 g, 74.0 mmol) in 100.0 mL of THF was heated at 80°C for 48 h until complete by LCMS.
The reaction mixture was then cooled to rt and diluted with water (300 mL) and extracted with
EtOAc (2 × 200 mL). The organic extracts were washed with brine (1 × 150 mL) and dried over
Na SO . The solution was filtered and concentrated in vacuo to give a yellow solid. The crude
2
4
material was absorbed onto a plug of silica gel and purified via column chromatography
(Biotage) using a 350 g column eluting with 0 % to 30% DCM :EtOAc to provide 4-(3-(3-
methoxypropoxy)phenyl)-3,4-dihydropyrido[3',2':4,5]furo[3,2-d]pyrimidine-2(1H)-thione (7.95
g, 21.52 mmol, 58% yield) as yellow solid. The compound was then separated into its
enantiomers via prep-scale supercritical fluid chromatography using a Chiralpak AD-H 2 x 15
cm, 5 micron column with an isocratic 45% carbon dioxide / 55% methanaol with 0.2% diethyl
amine solvent system at 60 mL/min, to yield R-4-(3-(3-methoxypropoxy)phenyl)-3,4-
dihydropyrido[3',2':4,5]furo[3,2-d]pyrimidine-2(1H)-thione (10, 3.8 g, 48% yield). MS ESI m/z

= 370.0 (M+H). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.93 (quin, J=6.33 Hz, 2 H) 3.23 (s, 3
H) 3.45 (t, J=6.26 Hz, 2 H) 4.00 (t, J=6.36 Hz, 2 H) 5.97 (s, 1 H) 6.79 - 6.98 (m, 3 H) 7.31 (t,
J=8.02 Hz, 1 H) 7.35 - 7.45 (m, 1 H) 8.13 - 8.39 (m, 2 H) 9.32 (s, 1 H) 10.88 - 11.21 (m, 1 H).
-1.38° (c 1.0, EtOH)

O
N
O
Compound 10 provides an unbound plasma concentration 16-fold in
excess of the in vitro TRPA1 IC50 and demonstrates significant
effect in an on-target biochemical challenge study (allyl
isothiocyanate-induced flinching).
O
NH
HN
S
10
TRPA1 45Ca2+ IC50 rat ( M): 0.045
TRPA1 45Ca2+ IC50 human ( M): 0.17