Oxime derivatives related to AP18: Agonists and antagonists of the TRPA1 receptor

Article abstract of DOI:10.1016/j.bmcl.2009.10.113

AP18 1 was recently disclosed as an antagonist of the TRPA1 receptor by the research group of Patapoutian. However, no detailed structure-activity relationships around 1 have been disclosed. Thus, a small number of oximes related to AP18 were examined in order to characterize the determinants of TRPA1 activity. Congeners of AP18 were found to possess both agonist and antagonist activity, suggesting that AP18 may behave as a covalent antagonist of the TRPA1 ion-channel.

Full text of DOI:10.1016/j.bmcl.2009.10.113

Bioorganic & Medicinal Chemistry Letters 20 (2010) 276–279
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Oxime derivatives related to AP18: Agonists and antagonists
of the TRPA1 receptor
*
*
Jeff DeFalco , Daniel Steiger, Amy Gustafson, Daniel E. Emerling, Michael G. Kelly, Matthew A. J. Duncton
Renovis, Inc. (a wholly-owned subsidiary of Evotec AG), Two Corporate Drive, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
AP18 1 was recently disclosed as an antagonist of the TRPA1 receptor by the research group of Patapou-
tian. However, no detailed structure–activity relationships around 1 have been disclosed. Thus, a small
number of oximes related to AP18 were examined in order to characterize the determinants of TRPA1
activity. Congeners of AP18 were found to possess both agonist and antagonist activity, suggesting that
AP18 may behave as a covalent antagonist of the TRPA1 ion-channel.
Received 8 September 2009
Revised 25 October 2009
Accepted 27 October 2009
Available online 30 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
TRPA1
TRP
Antagonist
Agonist
Transient Receptor Potential
Ankyrin-repeat Transient Receptor Potential 1
Oxime
AP18
The ability to detect and respond to noxious stimuli is critical
for survival and is usually interpreted as a painful experience.
Nociceptors are the sensory neurons that detect noxious stimuli
and transduce them to neural signaling. These cells typically
express one or more Transient Receptor Potential (TRP) receptors:
a functionally-diverse group of six-transmembrane domain ion-
channels capable of responding to a variety of physical and chem-
ical stimuli.1 One such family member, Ankyrin-repeat Transient
Receptor Potential 1 (TRPA1), is expressed in the peptidergic
subset of sensory fibers and has been shown to respond to a variety
of noxious stimuli, including environmental irritants, cold temper-
atures, and pungent plant-metabolites.2,3 Despite differences in
chemical functionality among small molecule irritants that agonize
TRPA1, such as allyl isothiocyanate (mustard oil) 1, iodoacetamide
2, cinnamaldehyde 3 and diallyl disulfide 4, they all activate the
channel via a common mechanism. The agonists act as electro-
philes, covalently modifying conserved cysteine residues present
almost exclusively in the cytoplasmic, amino-terminal domain of
the channel (Fig. 1).2b,4 In the case of cinnamaldehyde (CA), the
Endogenous mediators of inflammation and pain also interact
with TRPA1. Prostaglandins, hydroxynonenal, and reactive oxygen
species have all been shown to act as TRPA1 agonists, consistent
with a role for TRPA1 as a mediator of inflammatory responses.5
Various knock-down or knockout studies in rodents have demon-
strated reduced nociceptive behavior in a number of preclinical
pain models,6 and attenuated airway inflammation in models of
asthma.7 In addition, genetic ablation experiments have shown
that TRPA1 acts downstream of the pronociceptive agent bradyki-
nin,3d further implicating TRPA1 in pain and inflammatory path-
ways. In vivo experiments using TRPA1 inhibitors have yielded
similar results to genetic studies, indicating that inhibition of
TRPA1 can produce analgesic and anti-inflammatory effects, and
underscoring the potential of TRPA1 as a pharmacological target.
At present, four classes of TRPA1 antagonist have been disclosed
(Fig. 2). These are the oxime AP18 5 from the research group of
Patapoutian,8 a series of trichlorosulfide antagonists from Amgen,
for example, AMG7160 6 (related in structure to the TRPA1 agonist,
mustard gas 7),9 a diaminohexane derivative 8 (Patapoutian)8a and
a,b unsaturated bond is attacked in a Michael fashion, and the
resulting covalent addition leads to an opening of the ion-channel,
an influx of calcium into the cell, and ultimately, a perception of a
painful stimulus.
O
O
I
S
4
N C S
NH2
Ph
H
S
1
2
3
* Corresponding author. Tel.: +1 917 345 3183.
E-mail addresses: jeff.defalco@gmail.com (J. DeFalco), mattduncton@yahoo.com
(M.A.J. Duncton).
Figure 1. Representative covalent agonists of TRPA1 (site of reaction with
cytoplasmic N-terminal cysteine residues denoted by arrow).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.113

J. DeFalco et al. / Bioorg. Med. Chem. Lett. 20 (2010) 276–279
277
Br
Cl
NH2OH.HCl
NaOH, H2O,
EtOH, reflux
H
S
R3
R3
OH
Cl
S
N
N
OH
O
Cl
N
R1
R1
O
Cl
Cl Cl
Cl
AMG7160 6
R2
R2
16, 17, 21, 22, 23 & 26
AP18 5
OH
mustard gas 7
O
O
N
Scheme 1. Preparation of oximes from aldehyde or ketone precursors.
N
H
N
H
H
N
N
N
N
O
OH
8
HC-030031 9
the para-chloro group of AP18 was replaced with a meta-methyl
substituent; the resulting compound (14) had comparable agonist
activity to CA (entry 7). Deleting one, or more methyl groups in
AP18 also had a marked effect upon TRPA1 agonist activity. For
example, deletion of the C3 methyl group in AP18 resulted in the
emergence of TRPA1 agonist behavior (15, entry 8). Deletion of
both methyl groups in AP18 gave rise to a particularly potent
agonist, which had similar relative efficacy to CA, but was substan-
tially more potent in terms of its EC50 (compound 16; entry 9).
Significantly, compound 17, a geometrical isomer of 16, was less
potent in its agonist behavior at TRPA1 (compare entries 9 and 10).
It is also interesting to note the SAR for compound 11 and its
demethylated congeners. For instance, selective deletion of a
methyl group in compound 11 gave rise to 18 and 19, which both
showed weak agonist activity at TRPA1 (entries 11 and 12). In con-
trast to the behavior observed with congeners of AP18 above, dele-
tion of both methyl groups in 11 resulted in a compound (20) with
no agonist activity (entry 13; compare entries 9 and 13).
Figure 2. Literature antagonists of TRPA1.
a series of purine acetamides, exemplified by HC-030031 9, from
Hydra Biosciences.10 While the exact mechanism of action for
these antagonists is not known, it is possible that compounds such
as 5 and 6 are capable of covalently modifying TRPA1, but are
unable to trigger a conformational change that would result in
channel activation. In this Letter we explore the structure–activity
relationship of simple analogues related to AP18 5, in an effort to
characterize the determinants of agonist and antagonist activities
against human TRPA1. Our results may suggest that AP18 behaves
as a covalent modifier of TRPA1. Additionally, we detail 3-methyl-
4-phenylbut-3-en-2-one oxime 11, a congener of AP18, as a new
antagonist of the TRPA1 receptor.11
In order to undertake a preliminary examination of the struc-
ture–activity relationships (SAR) around AP18, a number of related
oximes were purchased from commercial sources. Additionally,
further oximes were prepared using the chemistry detailed in
Scheme 1. Thus, an appropriate cinnamaldehyde or benzylidene-
acetone starting material was reacted with hydroxylamine hydro-
chloride, in a mixture of aqueous NaOH and EtOH, to afford the
desired oxime product.12 For some reactions, both oxime geomet-
rical isomers could be isolated; the (E)-isomer was presumed to
predominate for such reactions.12b,c In total, 19 oximes were pur-
chased, or synthesized, for evaluation as modulators of TRPA1
(Fig. 3).13
Compounds were tested for an ability to agonize the TRPA1 ion-
channel by assessing the influx of 45Ca into TRPA1-expressing cells
upon exposure to test compounds. Cinnamaldehyde (CA) was used
as a positive control in each experiment. The results from our
investigations are shown in Table 1.13 Some notable features in
the structure–activity relationships (SAR) were apparent. As
expected AP18 5, displays no agonist activity at the TRPA1 recep-
tor. Likewise, when the para-chloro group of AP18 was moved to
the ortho-position (compound 10), or was replaced with a hydro-
gen atom (compound 11), or nitro group (compound 12), no ago-
nist activity was observed (entries 2–5). However, when the
chloro group of AP18 was replaced with a methoxy substituent,
to give compound 13, weak agonist activity at TRPA1 was noted
(entry 6). This agonist activity was even more pronounced when
Table 1
TRPA1 agonist activity of AP18 and related oximes
a
Entry
Compound
Agonist
Relative efficacy to CAa
EC50
(lM)
1
2
CA 3
5
Yes
No
1.0
0
51.4 ( 5.8)
nd
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
No
No
No
0
0
0
nd
nd
nd
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
0.23 (n = 1)
0.84 ( 0.03)
0.66 ( 0.07)
0.85 ( 0.12)
0.46 (n = 1)
0.18 ( 0.08)
0.27 ( 0.02)
0
0.85 ( 0.01)
0.21 ( 0.15)
0.13 ( 0.03)
0.87 ( 0.07)
0.54 ( 0.16)
0.15 (n = 1)
<0.1
46.1 (n = 1)
14.4 ( 3.3)
27.6 ( 1.2)
9.4 ( 0.6)
nd
69.4 ( 10.1)
96.6 ( 3.2)
nd
63.1 ( 19.4)
nd
nd
15.9 ( 9.6)
11.5 ( 2.0)
nd
nd
a
Values are means of two or three experiments (standard error is given in
brackets). nd = not determined.
OH
N
OH
OH
OH
OH
OH
N
N
N
N
N
N
N
Cl
10
MeO
Cl
11
17
12
18
13
19
14
20
15
21
NO2
OH
Cl
OH
OH
OH
OH
N
OH
N
N
N
N
Cl
16
22
Cl
OH F3C
NH2
OH
O
O
OH
OH
OH
N
tBu
N
N
N
N
OH
F3C
Me2N
Cl
CF3
27
23
24
25
26
Figure 3. Compounds tested for activity at TRPA1.

278
J. DeFalco et al. / Bioorg. Med. Chem. Lett. 20 (2010) 276–279
Table 2
with cysteines). Oximes 15–17, which are all demethylated ana-
TRPA1 antagonist activity of oximes related to AP18
logues of AP18, are agonists of the TRPA1 receptor. Given the struc-
tural homology between AP18 and 15–17, it is conceivable that
AP18 could be acting as a covalent modifier of the TRPA1 receptor.
Under this hypothesis, the presence of the additional methyl
group(s) in AP18 prevent the receptor from adopting an active con-
formation (open ion-channel) despite binding and cysteine modifi-
cation.16 In order to gain experimental support for such a model, it
would be necessary to characterize fragments of the N-terminal
domain of TRPA1 upon exposure to AP18 and 15–17. The presence
of covalently modified cysteine residues can be ascertained from
mass-spectrometry measurements. Similar approaches have been
used previously to demonstrate that noxious chemicals bind in a
covalent manner,4 and could illuminate the covalent or non-cova-
lent mechanism by which AP18 and the oxime agonists and antag-
onists described in this Letter modulate TRPA1 activity.
In conclusion, this Letter describes the agonist and antagonist
behavior of a set of oximes related to the literature TRPA1 antago-
nist, AP18. The results indicated that demethylated versions of
AP18 behaved as agonists at TRPA1, and may suggest that AP18
is a covalent modifier of the TRPA1 receptor. In addition, our stud-
ies revealed that a related AP18 derivative, 3-methyl-4-phenylbut-
3-en-2-one oxime 11, also acted as a TRPA1 antagonist.
a
Entry
Compound
Antagonist
IC50 (lM)
1
2
3
4
5
6
7
8
5
Yes
Yes
No
No
No
No
No
No
2.8 ( 0.3)
2.7 ( 0.4)
11
15
18
20
25
26
27
nd
nd
nd
nd
nd
nd
a
Values are means of two experiments (standard error is given in brackets).
nd = not determined.
Another set of oximes (21–27) were also tested for agonist
activity at TRPA1. All of these compounds exhibited agonist behav-
ior, although the magnitude of the effect was variable (entries 14–
20). Interestingly, as was seen in the case of compound 16 and its
isomer 17 mentioned above, the stereochemistry of the oxime
group seemed to play a role in the magnitude of agonist response.
Thus, the agonist activity of compound 21 differed significantly to
that of its geometrical isomer 22 (compare entries 14 and 15; note
the similarities to entries 9 and 10).14
A select number of non-agonist oxime analogues, or those with
a low relative efficacy, were tested for an ability to antagonise the
effect of CA to TRPA1-expressing cells. The results are shown in Ta-
ble 2.13 As can be seen, only AP18 5 and compound 11 were found
to act as antagonists. Thus, the results in Tables 1 and 2 indicate
that a number of compounds appeared to be neither agonists of
TRPA1 nor antagonists of its activation by CA (e.g., compounds
15, 18, 20, 25–27).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.113.
References and notes
1. Forareviewsee:Patapoutian,A.;Tate, S.;Woolf, C.J. Nat.Rev. DrugDisc.2009, 8, 55.
2. For reviews of TRPA1 see: (a) Cai, X. Expert Rev. Neurother. 2008, 8, 1675; (b)
Cebi, M.; Koert, U. ChemBioChem 2007, 8, 979.
Finally, the pA2 values for the antagonists AP18 5 and 11 were
determined using CA as an agonist (Fig. 4). The results indicated
that AP18 has a pA2 value of 1.26 0.15
lM, while compound 11
3. (a) Story, G. M.; Peier, A. M.; Reeve, A. J.; Eid, S. R.; Mosbacher, J.; Hricik, T. R.;
Earley, T. J.; Hergarden, A. C.; Andersson, D. A.; Hwang, S.; McIntyre, P.; Jegla, T.;
Bevan, S.; Patapoutian, A. Cell 2003, 112, 819; (b) Jordt, S.-E.; Bautista, D. M.;
Chuang, H.-H.; McKemy, D. D.; Zygmunt, P. M.; Hogestatt, E. D.; Meng, I. D.;
Julius, D. Nature 2004, 427, 260; (c) Bandell, M.; Story, G. M.; Hwang, S.;
Viswanath, V.; Eid, S. R.; Petrus, M. J.; Earley, T. J.; Patapoutian, A. Neuron 2004,
41, 849; (d) Bautista, D. M.; Jordt, S.-E.; Nikai, T.; Tsuruda, P. R.; Read, A. J.;
Poblete, J.; Yamoah, E. N.; Basbaum, A. I.; Julius, D. Cell 2006, 124, 1269; (e)
Macpherson, L. J.; Xiao, B.; Kwan, K. Y.; Petrus, M. J.; Dubin, A. E.; Hwang, S.;
Cravatt, B.; Corey, D. P.; Patapoutian, A. J. Neurosci. 2007, 27, 11412.
4. (a) Hinman, A.; Chuang, H.; Bautista, D. M.; Julius, D. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 19564; (b) Macpherson, L. J.; Dubin, A. E.; Evans, M. J.; Marr, F.;
Schultz, P. G.; Cravatt, B. F.; Patapoutian, A. Nature 2007, 445, 541.
5. (a) Taylor-Clark, T. E.; Undem, B. J.; MacGlashan, D. W.; Ghatta, S.; Carr, M. J.;
McAlexander, M. A. Mol. Pharmacol. 2008, 73, 274; (b) 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. U.S.A. 2007, 104, 13519.
6. McNamara, C. R.; Mandel-Brehm, J.; Bautista, D. M.; Siemens, J.; Deranian, K. L.;
Zhao, M.; Hayward, N. J.; Chong, J. A.; Julius, D.; Moran, M. M.; Fanger, C. M.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 13525.
has a pA2 of 1.91 0.09
l
M.15
The results presented above are significant and may yield in-
sights into the action of AP18 5 at TRPA1. It is assumed that the
activities observed for all oxime agonists presented in Table 1 re-
sult from a similar mechanism of action to that observed with
CA. That is, the oxime agonists are assumed to act as Michael
acceptors, reacting with cysteine residues located in the N-termi-
nal domain of the TRPA1 receptor. This covalent modification re-
sults in a conformational change, allowing opening of the ion-
channel and an influx of calcium into the cell. For those compounds
with reduced efficacy compared to CA, it is not apparent whether
this property is due to an inability to react with every cysteine that
is modified by CA, a less efficacious transduction of the reaction
with cysteines to channel activation, or a difference in ‘on–off’ rate
(i.e., a difference compared to CA in the reversibility of the reaction
Compound 11
Compound 5
2500
2500
63uM
63uM
20uM
6.3uM
2uM
0.63uM
0
20uM
6.3uM
2uM
0.63uM
0
2000
1500
1000
500
0
2000
1500
1000
500
0
-7
-6
-5
-4
-3
-7
-6
-5
-4
-3
Log[CA], M
Log[CA], M
Figure 4. pA2 determination of AP18 5 and related oxime antagonist 11.

J. DeFalco et al. / Bioorg. Med. Chem. Lett. 20 (2010) 276–279
279
7. Caceres, 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. U.S.A. 2009,
106, 9099.
13. See Supplementary data for experimental procedures and a representative
copy of the agonist and antagonist dose–response graphs for each compound
described in this Letter.
14.
A
representative
selection
of
substituted
cinnamaldehyde
and
8. (a) Patapoutian, A.; Jegla, T. J. WO2007098252; Chem. Abstr. 2007, 147, 292253;
(b) Petrus, P.; Peier, A. M.; Bandell, M.; Hwang, S. W.; Huynh, Y.; Olney, N.;
Jegla, T.; Patapoutian, A. Mol. Pain 2007, 3, :40.
benzylideneacetone derivatives related to the oximes described in Table 1
were also tested for their ability to act as TRPA1 ligands. In each case, we found
that the compounds functioned as TRPA1 agonists.
9. Klionsky, L.; Tamir, R.; Gao, B.; Wang, W.; Immke, D. C.; Nishimura, N.; Gavva,
N. R. Mol. Pain 2007, 3, 39.
15. See Ref. 8b for the ability of AP18 5 to act as an antagonist of TRPA1 when
cinnamaldehyde, iodoacetamide, mustard oil (allyl isothiocyanate) and
noxious cold were used as agonists. Confirmation of the antagonist
properties of AP18 using electrophysiology experiments are also described in
Ref. 8b.
16. A reviewer of this manuscript proposed that compounds 5 and 11 could also
act as antagonists by ‘binding’ in the same pocket as cinnamaldehyde.
However, the presence of additional methyl substitutions prevented reaction
with the cysteine residues of TRPA1. We think this hypothesis to be unlikely,
since literature suggests that chemical modulation of TRPA1 is dictated by the
reactivity of a molecule, rather than its shape (see Refs. 1 and 4 for further
information).
10. (a) Moran, M. M.; Fanger, C.; Chong, J. A.; Mcnamara, C.; Zhen, X.; Mandel-
Brehm, J. WO2007073505; Chem. Abstr. 2007, 147, 118074; (b) Ng, H.; Weigele,
M.; Moran, M.; Chong, J.; Fanger, C.; Larsen, G. R.; Del Camino, D.; Hayward, N.;
Adams, S.; Ripka, A. WO2009002933; Chem. Abstr. 2009, 150, 98047; (c) Eid, S.
R.; Crown, E. D.; Moore, E. L.; Liang, H. A.; Choong, K.-C.; Dima, S.; Henze, D. A.;
Kane, S. A.; Urban, M. O. Mol. Pain 2008, 4, 48.
11. After the submission of this Letter a series of related oxime derivatives from
Abbott Laboratories were also disclosed as antagonists of the TRPA1 receptor.
See Perner, R. J.; Kort, M. E.; Didomenico, S.; Chen, J.; Vasudevan, A. WO
2009089083; Chem. Abstr. 2009, 151, 148153.
12. (a) Zielinski, W. Pol. J. Chem. 1978, 52, 2233; (b) Unterhalt, B.; Eljabour, S. Archiv
der Pharmazie 1986, 319, 666; (c) Jeong, H. J.; Park, H.-Y.; Jeong, Y.; Jeong, T.-S.;
Lee, W. S. Bioorg. Med. Chem. Lett. 2006, 16, 5576.