N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor

Article abstract of DOI:10.1016/j.bmcl.2012.07.032

A series of potent antagonists of the ion channel transient receptor potential A1 (TRPA1) was developed by modifying lead structure 16 that was discovered by high-throughput screening. Based on lead compound 16, a SAR was established, showing a narrow region at the nitro-aromatic R¹ moiety and at the warhead, while the R² side had a much wider scope including ureas and carbamates. Compound 16 inhibits Ca²⁺-activated TRPA1 currents reversibly in whole cell patch clamp experiments, indicating that under in vivo conditions, it does not react covalently, despite its potentially electrophilic ketone.

Full text of DOI:10.1016/j.bmcl.2012.07.032

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5485–5492
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor
Karl S. A. Vallin a, , Karin J. Sterky a, Eva Nyman b, Jenny Bernström b,c, Rebecka From a, Christian Linde a,
⇑
Alexander B. E. Minidis a, Andreas Nolting b,c, Katja Närhi a, Ellen M. Santangelo a,
Fernando W. Sehgelmeble a, Daniel Sohn a, Jennie Strindlund d, Dirk Weigelt a,
⇑
a Medicinal Chemistry, CNSP iMed, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
b Discovery Sciences, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
c Discovery Sciences, AstraZeneca R&D, Innovative Medicines, SE-43183 Mölndal, Sweden
d Neuroscience, CNSP iMed, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of potent antagonists of the ion channel transient receptor potential A1 (TRPA1) was developed
by modifying lead structure 16 that was discovered by high-throughput screening. Based on lead com-
pound 16, a SAR was established, showing a narrow region at the nitro-aromatic R1 moiety and at the
warhead, while the R2 side had a much wider scope including ureas and carbamates. Compound 16 inhib-
its Ca2+-activated TRPA1 currents reversibly in whole cell patch clamp experiments, indicating that under
in vivo conditions, it does not react covalently, despite its potentially electrophilic ketone.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 June 2012
Revised 4 July 2012
Accepted 6 July 2012
Available online 14 July 2012
Keywords:
Ion channel
TRPA1
Antagonist
TRPA1 is a nonselective cation channel,1 which has gained high
interest as a target for future analgesics during recent years.2
O
S
O
S
A
large number of agonists for TRPA1 have been reported,3 many of
them being of electrophilic nature such as acrolein (1),4 allyl isothi-
ocyanate (2),5 allicin (3),6 cinnamaldehyde (4),4 ozone7 or the
highly potent CR (5) and other tear gas ingredients (Fig. 1).8 It
has been speculated that TRPA1 is a part of a warning system
against noxious chemicals.9
O
N
S
H
1
2
3
4
H
N
HO
H
N
O
O
O
O
O
But also reversible TRPA1 agonists have been found, such as
H
NO2
O
9-tetrahydrocannabinol (6),10 nifedipine (7),11 the TRPM8 agonist
5
6
7
D
menthol (8),12 the TRPV1 agonist BCTC (9),13 acetic acid (10),14 or
carbon dioxide.15 Even simple ions like intracellular Ca2+ and
Zn2+ have been reported to open the TRPA1 channel.16
O
O
N
H
N
Cl
OH
OH
N
In contrast, far fewer antagonists have been reported, many of
them with a very limited scope (Fig 2). Most appear in patent
applications such as de-aza caffeine-derived compound 11
(Hydra)17 and similar compounds 12 (Glenmark)18 tricyclic pyrim-
idones and –thiones (13, Janssen)19 decalines (14, Merck)20 and
trichloro(sulfanyl)ethyl benzamides (15, Amgen).21
N
8
9
10
Figure 1. TRPA1 agonists.
campaign and led to the finding of the potent singleton 16
(Fig. 3). This compound features two groups, a nitro, and an aro-
matic (electrophilic) ketone, which we sought to replace. The nitro
group since it is known to impact several drug properties (e.g. sol-
ubility) and is known to carry toxicity risks, the latter also associ-
ated with ketone groups.24
Our aim was to indentify novel TRPA1 antagonists and in order
to avoid covalently binding activators we developed a fluorescent
assay where Ca2+ was used as stimulating agonist of TRPA1. Ca2+
has been suggested to bind to the EF-hand motif in the intracellular
N-terminus of the channel.22,23 This assay was used in a HTS
During the course of the project we switched the agonist in the
assay from Ca2+ to Zn2+ for reasons of assay stability and a possible
in vitro/in vivo translation. The Ca2+ assay initially had stability
⇑
Corresponding authors.
E-mail address: karl_sa.vallin@bredband.net (K.S.A. Vallin).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.032

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K. S. A. Vallin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5485–5492
a halogen at the para position (compounds 18–20), increased bind-
S
Ar
HN
ing activity was found compared to the unsubstituted phenyl 17,
especially with chlorine (19a). The phenyl substitution SAR
seemed rather narrow and by moving the chlorine to the meta po-
sition all activity was lost (21). Dichloride substitution though was
accepted (22). Further efforts to replace the nitro group in the
highly potent compound 16 resulted in decreased activity, for
example, nitrile in 23, or no activity at all when changing to sulfone
group (24) or amino moieties as in 25–26. By separating racemic
19a we observed that only one isomer was active, 19b versus
19c. Early attempts to crystallise a single enantiomer failed, as well
as efforts to synthesise compounds starting from enantiomerically
pure valine, which led to inconclusive outcomes, most probably
due to racemisation issues. On the other hand, the absolute config-
uration of a similar compound sharing the same warhead has been
determined to be (R) via vibrational circular dicroism (VCD)
experiments.31
HN
F
O
N
O
N
O
O
N
N
N
N
Ar
O
12
N
11
O
F
O
Br
O
H
N
S
HN
OH
NH
O
HN
Cl
Cl
Cl
Cl
15
S
H
13
14
Figure 2. TRPA1 antagonists.
NO2
We next turned our attention to the impact on in vivo potency
of the R2 substituents. As summarized in Table 2, SAR at this side of
the warhead seem to be broader compared to the R1 region. Benzo-
nitrile (23) was used as the default R1 analogue due to its good sol-
O
N
H
16
O
ubility of 150 l
M.32 Both electron withdrawing and donating
Figure 3. Lead compound.
groups were tolerated on the phenyl ring. Chlorine in the para po-
sition (27) showed a significant increase in activity compared to
the unsubstituted phenyl ring (23) and the electron-rich methoxy
group (28). Disubstituted phenyl rings with di-chloro (29), or Cl/F
(30, 31) showed activities similar to the plain phenyl (23). Interest-
ingly, introduction of CF3 in the 5-position in combination with Cl
in the 2-position showed high activity (32). Removing the chloro
substituent at the 2-position furnished a 10-fold activity drop
(33). Introducing heteroaromatic groups at the R2 position toler-
ated as well, especially with a bulky alkyl group as the ortho-sub-
stituent (34–37). Even a long, non-functionalized alkyl group as the
R2 moiety was found active (38). Results from the introduction of
an amino or ether linker at the R2 position, which generated ureas
and carbamates, are disclosed in Table 3. Secondary ureas showed
no or low activity, whereas N-alkylation increased potency signif-
icantly (39–44). The corresponding carbamate analogues showed
similar activities (45 and 46). We also discovered that by introduc-
ing a fluoro atom at the meta position in the R1 region the activity
was increased (47). Making the R1 region less lipophilic by use of a
pyridine (48) led to loss of activity. However, no general correla-
tion between lipophilicity and potency in the Zn2+ assay was ob-
served, when the 31 compounds active in the zinc assay were
examined.33
As shown in Table 4, the linker region had a very tight SAR.
Lipophilic bulky iso-propyl-(19a) and tert-butyl groups (49), as
the alkyl part were tolerated. The cyclopropyl group as in 50 was
acceptable, whereas, surprisingly, the unbranched n-propyl (51)
had no in vitro activity at all. The attempt to remove potential
racemisation issues of the chiral sp3 carbon by introducing an
additional methyl group resulted in reduced potency (52).34 Rigid-
ification of the tert-butyl group to bicyclic 53 was unfavourable as
was making the symmetric cyclobutyl compound 54. Removing
the central alkyl chain resulted in inactive 55. Combining high po-
tency fragments from R1, R2 and the alkyl chain generated active
issues, which later were recognized to depend on the sporadic addi-
tion of probenicid, an inhibitor for the anion-exchange protein in
Chinese hamster ovar (CHO) cells. The root cause was (at that time
the unknown fact) that probenicid is a TRPA1 agonist.25,26 Once the
issues due to probenicid in the Ca2+ assay had been identified and
the results from the effected experiments had been discarded, the
Zn2+ had been developed as well. Zn2+ is a TRPA1 agonist that has
been suggested to interact with an intracellular C-terminal site in
the channel.27–29 Zn2+ has been shown to induce nocifensive
behaviour via activation of TRPA127 and using an in vitro assay with
Zn2+as agonist may be of translational value as the relationship
between in vitro IC50 values and effect of in vivo models would be
clearer and easier to interpret. In the series described here no major
differences in antagonistic potency (IC50) were found when the
agonist was changed. When comparing the assays the compound
IC50 values correlated well and the ranking order of potency stayed
the same (Supplementary data) and we therefore decided to treat
the data as if it came from one assay regardless of the agonist used.
This explains why some of the compounds in this study only have
IC50 values in either the Zn2+ or the Ca2+ assay. However, for a
structurally different series there was a clear difference between
the assays and those observations will be published later on.30 To
be certain that the observed inhibition was not due to auto fluores-
cence caused by potential inherent spectrophotometric properties
of test compounds, the activity on all compounds with pIC50 >5
were confirmed in
a automated electrophysiology assay on
IonWorks (Molecular Devices, data not shown).
Herein we report our SAR study effort divided into three major
parts: Substitution modifications at positions R1, R2 and of the
warhead (Fig. 4, Tables 1–3).
We initially prepared a series of substituted phenyl moieties in
the R1 region and the SAR is summarized in Table 1. Introduction of
O
alkyl
R1
R2
N
H
O
Warhead
Figure 4. Warhead.

K. S. A. Vallin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5485–5492
5487
Table 1
SAR at the R1 region
O
R1
N
H
O
Compounda
R1
pIC50 Zn2+,b
pIC50, Ca2+,b
NO2
16
6.6
6.5
NV
5.3
17
18
<4.5c
F
5.1
Cl
Br
19a, 5.9, racemate
19b, 6.4, isomer 1
19c, NV, isomer 2
5.6
6.0
NV
19
20
21
5.9
5.1
NV
<4.5c
Cl
Cl
22
23
NR
NR
5.4
5.4
Cl
CN
O
S
24
25
NV
NV
NV
NV
O
NH2
N
26
<4.5c
NV
a
All compounds were racemates except 19b–c.
NV = no value (no observed effect), NR = not run.
pIC50 <4.5 = 30–50% inhibition in highest concentration.
b
c
substance 56 with a good solubility profile (80
l
M).32 The nitro
that the mechanism of TRPA1 inhibition by 16 does not involve
the irreversible formation of covalent bonds with the ion channel
protein.
bioisostere benzooxodiazole 57 was active. Analogue 58 with the
original R1 moiety was highly active, although with an unfavour-
able solubility of less than 1
l
M.32
With regards to the general in vivo safety of ketones in drugs,
there seems to be a lack of general studies and no generic standard
assays are available. Generally, a ketone moiety could be expected
to react with amines (lysine or N-termini), thiols (cysteine) or alco-
hols (serine). Although covalent bonds could be formed, the hemi-
acetal-type products from serine or cysteine would be instable. For
example, a hemiacetal from a ketone and Porcine Pancreatic Elas-
tase has been reported,36 but could only be observed in the enzyme
itself. The reaction of a ketone with a lysine moiety would result in
an imine, which also can be a reversible reaction. Mutation studies
suggest that the described compounds most likely bind in the pore
region of TRPA1.37 As there are no cysteine or lysine residues in
that particular region, covalent binding to TRPA1 can be considered
to be unlikely. This would be furthermore in accordance to the re-
sults of the patch clamp experiments (vide supra).
Introduction of a methylene spacer in the linker region had a
negative impact in compounds tested (Fig 5 and 59–61). As pointed
out initially, we wanted to replace the ketone functionality, due to
potential toxicity liabilities. Generating isosteres by removing the
carbonyl (62), reducing the ketone (63), ring formation to the R1 re-
gion to benzotriazole 64, or sulfonamide 65 all led to inactive com-
pounds. Methylation of the nitrogen showed that an N–H as donor
is crucial for the activity (66). Efforts to move the H-donor function
to the aromatic R2 moiety (67), or to change the amide to sulfon-
amide 68, as well as reversing the amide as in 69, also generated
only compounds which lacked activity.
Whole cell patch clamp experiments were performed to con-
firm whether the ketone containing original HTS hit 16 acted on
TRPA1 in a reversible manner. Application of 10 lM compound
16 resulted in a rapid and complete inhibition of Ca2+ activated
TRPA1 currents. Washout of 16 led to full recovery of the TRPA1
currents within seconds.35 The reversibility of inhibition suggests
In addition, the general reactivity of 15 compounds was deter-
mined in a human microsome gluthathione (GSH) trapping as-
say.38 Of those tested, nine compounds did not show any adducts

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K. S. A. Vallin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5485–5492
Table 2
SAR at the R2 region
CN
O
R2
N
H
O
Compound
R2
pIC50 Zn2+
NR
pIC50 Ca2+
5.4
23
27
28
29
30
31
NR
5.2
5.6
5.4
5.7
6.4
4.9
5.3
5.5
NV
Cl
O
Cl
Cl
Cl
Cl
F
F
Cl
F
F
32
6.5
6.4
F
F
F
33
34
5.3
6.3
5.5
6.0
F
N
O
35
36
5.4
5.7
5.1
5.8
N
N
O
O
37
38
5.8
6.0
5.4
5.7
N
7
(among them compound 16), 7 showed GSH-adducts on levels
between 17% and 40% compared to the standard clozapine and
only 1 compound formed GSH-adducts in high rate: 41 was found
6 times more reactive as clozapine. However, there was no corre-
lation between TRPA1 inhibition and adduct formation. Although
this assay typically is used to determine the risk of reactive metab-
olite formation, it can be used as an indicator for reactive group
containing compounds, as would be the case to see whether or
not the ketone moiety would react with either a free amino or free
thiol group (in glutathione) and thus useful as an indicator for
potential disadvantageous covalent binding to proteins in e.g. the
liver or other organs in vivo. The present warhead does not seem
to show this type of reactive behaviour.
The majority of the compounds have been prepared by the
route described in Scheme 1, where protected amino acid 49–0
was converted to Weinreb-amide 49–1, which was reacted with
a Grignard-reagent to form the ketone functionality in 49–2. The
amine was then de-protected (49–3) and reacted with an acid
chloride, alternatively with an acid and an appropriate coupling
reagent, to form the desired product 49. The alternative route
described in Scheme 2 made use of an intermediate 4-isopropyl-
2-phenyloxazol-5(4H)-one (19a–1), which was treated with an
acid chloride and subsequently hydrolyzed to form the desired
product 19a.39
In summary, a series of potent TRPA1 inhibitors were developed
by modifying lead structure 16. Based on the lead compound, SAR

K. S. A. Vallin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5485–5492
5489
Table 3
Ureas and carbamates
X
A
O
R2
N
H
Y
O
Compound
R2
X/Y/A
pIC50 Zn2+
pIC50 Ca2+
F
39
CN/H/C
NV
NV
N
H
N
40
41
CN/H/C
CN/H/C
NV
5.3
NV
5.3
H
N
42
43
44
CN/H/C
Cl/H/C
Cl/H/C
NR
6.2
5.6
5.4
6.6
5.7
N
N
N
H
45
46
47
48
Cl/H/C
CN/H/C
CN/F/C
Cl/H/N
6.1
5.6
5.7
4.8
6.0
5.2
5.9
5.3
O
O
O
O
Table 4
SAR at the warhead
Compound
Structure
pIC50 Zn2+a
pIC50 Ca2+a
Cl
Cl
Cl
O
19a
5.9
6.4
5.7
5.6
N
H
O
O
O
49
50
6.0
5.7
N
H
O
O
N
H
Cl
Cl
O
O
51
52
NV
NV
NV
N
H
O
O
5.5
N
H
(continued on next page)

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K. S. A. Vallin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5485–5492
Table 4 (continued)
Compound
Structure
pIC50 Zn2+a
pIC50 Ca2+a
Cl
O
53
NV
NR
N
H
O
N
O
Cl
O
54
55
NV
NV
NV
NV
N
H
O
Cl
O
N
H
O
CN
O
56
57
6.7
6.4
8.0
NR
NR
NR
N
F
O
N
H
O
N
O
N
N
N
N
O
O
CF3
NO2
O
Cl
58
N
H
O
CF3
a
NV = no value (no observed effect), NR = not run.
O
N
N
Cl
N
F
O
H
N
60
H
O
N
H
O
59
61
O
O
Cl
O
Cl
O
HN
O
N
N
N
N
H
62
H
N
OH
63
64
Cl
Cl
Cl
O
HO
O
O
N
O
N
H
N
O
N
O
O
O
S
65
68
66
67
O
O
Cl
N
H
N
O
S
O
F
N
H
O
O
69
Figure 5. Warhead changes, leading to inactive compounds.
studies have been focused on modifications at the R1- and R2-posi-
tion, as well as the warhead. Despite these efforts, nitro-containing
compounds remained among the most potent, with low solubility
severely limiting in vivo testing potential. Two different control
studies could conclude that the presence of an aromatic ketone
in the inhibitor has no negative influence: (1) Patch clamp studies
showed that 16 does not form unwanted covalent bonds to the
channel protein, and (2) GSH-trapping experiments showed little
or no reactivity in the assay for many compounds, among them
the original hit 16.

K. S. A. Vallin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5485–5492
5491
O
O
H
N
O
H
N
O
H
N
O
O
a
b
O
N
OH
O
O
Cl
O
49-2
49-1
49-0
O
O
d
c
H2N
H
N
Cl
O
49
Cl
49-3
Scheme 1. Reagents and conditions: (a) N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride, N,O-dimethylhydroxylamine hydrochloride, N-
ethyl-N-isopropylpropan-2-amine, DCM, rt, 48 h; (b) (4-chlorophenyl)magnesium bromide, THF, rt, 16 h; (c) 5–6 N HCl in 2-propanol, MeOH, rt, 16 h; (d) benzoyl chloride,
pyridine, 0–25 °C, 16 h.
O
O
H
N
H
N
O
a
b, c
O
OH
N
O
O
Cl
19a
19a-0
19a-1
Scheme 2. Reagents and conditions: (a) ethyl chlorocarbonate, triethylamine, THF, 0–25 °C 1 h; (b) 4-chlorobenzoyl chloride, MgCl2, triethylamine, THF, 0–25 °C, 30 min; (c)
6 M HCl, 0–50 °C, 30 min.
Nature Neurosci. 2007, 10, 277; (c) Hu, H.; Bandell, M.; Petrus, M. J.; Zhu, M. X.;
Patapoutian, A. Nature Chem. Biol 2009, 5, 183.
Acknowledgments
17. Ng, H.; Hang, J. U.S. Patent 2010/0249154, 2010.
The authors would like to thank Erwan Arzel, Robert Pendrill,
Jonas Johansson, Anna Laurell, Sandra Lindberg, Jens Nolsöe and
the chemists at Naeja for the synthesis of several of the analogues.
18. Muthuppalniappan, M.; Thomas, A.; Sukeerthi, K.; Margal, S.; Khairatkar-Joshi,
N.; Mukhopadyay, I.; Gullapalli, S. International Patent WO 2010/004390,
2010. Chaudhari, S.S.; Kumar, S.; Thomas, A.; Patil, N.P.; Kadam, A.B.;
Deshmukh, V.G.; Dhone, S.V.; Chikhale, R.P.; Khairatkar-Joshi, N.;
Mukhopadyay, I. International Patent WO 2010/109287, 2010. Kumar, S.;
Thomas, A.; Margal, S.; Khairatkar-Joshi, N.; Mukhopadyay, I. International
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.07.
032.
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data.

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