Design and pharmacological evaluation of PF-4840154, a non-electrophilic reference agonist of the TrpA1 channel

Article abstract of DOI:10.1016/j.bmcl.2011.06.035

TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive, non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identified with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154) is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifensive behaviour in mouse.

Full text of DOI:10.1016/j.bmcl.2011.06.035

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4857–4859
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and pharmacological evaluation of PF-4840154, a non-electrophilic
reference agonist of the TrpA1 channel
Thomas Ryckmans a, , Aisah A. Aubdool b, Jennifer V. Bodkin b, Peter Cox c, Susan D. Brain b,
⇑
Thomas Dupont a, Emma Fairman c, Yoshinobu Hashizume d, Naoko Ishii d, Teruhisa Kato c, Linda Kitching c,
Julie Newman a, Kiyoyuki Omoto a, David Rawson a, Jade Strover c
a Pfizer Worldwide Medicinal Chemistry, Ramsgate Road, Sandwich, Kent CT139NJ, United Kingdom
b Cardiovascular Division, King’s College London, Franklin-Wilkins Building, Waterloo Campus, London SE1 9NH, United Kingdom
c Pfizer Discovery Biology, Ramsgate Road, Sandwich, Kent CT139NJ, United Kingdom
d Pfizer Global Research and Development, Nagoya Laboratories, 5-2 Taketoyo, Aichi 470-2394, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of
chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While
performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive,
non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identi-
fied with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was
optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154)
is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifen-
sive behaviour in mouse.
Received 27 May 2011
Revised 7 June 2011
Accepted 9 June 2011
Available online 21 June 2011
Keywords:
TrPpA1
Agonist
Ó 2011 Elsevier Ltd. All rights reserved.
Pain
Nociception
HTS
TrpA1 knockout
TrpA1 is a member of the Transient Receptor Potential (Trp) ion
channel family and is involved in the perception of nociceptive and
inflammatory pain. This pain can be triggered by mechanical stim-
uli, endogenous mediators such as Bradykinin1 and 4-hydroxynon-
enal (4-HNE 1),2 peroxides and prostaglandins,3 exogenous
chemical substances including pungent natural compounds (Mus-
tard Oil 2) as well as environmental irritants such as formalin,4
acrolein5,6 and lachrymatory agents.7 The mechanism of TrpA1
activation by noxious, electrophilic compounds has been linked
to their covalent binding to the sulfide moiety of specific cysteines
residues situated on the intracellular N-terminus of the TrpA1 pro-
tein.8 The evaluation of TrpA1 as a drug target is complicated by
interspecies differences; for instance, caffeine 3 is reported to be
an agonist of the mouse TRPA1 channel, but an antagonist of the
human channel9 (Fig. 1).
that a large percentage of hits were actually desensitizing agonists,
notably electrophiles.7 Mustard Oil itself desensitizes the TrpA1
channel10 and thus behaves as a functional antagonist, in a fashion
similar to the effect of capsaicin on the TrpV1 receptor.11 As a ref-
erence agonist for in vitro assays, Mustard Oil suffers from serious
drawbacks; it is toxic, volatile, has an unpleasant smell and is a po-
tent lachrymator. Furthermore, in our hands its limited solubility
and aqueous instability was linked to inconsistent screening data.
While alternatives to Mustard Oil such as tear-gas derivatives have
recently been described,12 a non-reactive, non-volatile stable ago-
nist would clearly enable the development of a safer and more ro-
bust screening assay.
Our goal was the identification of a suitable pharmacological
tool, with potent agonist activity at both the rat and the human
As part of an ongoing program to assess the druggability of the
TrpA1 channel, the Pfizer chemical collection was screened using a
FLIPR-based assay at 10 lM concentration in an antagonist format,
OH
O
N
O
N
N
S
using Mustard Oil as the reference agonist. HTS triage indicated
N
C
N
O
⇑
4-HNE 1
Mustard Oil (MO) 2
Corresponding author. Tel.: +44 (0) 1304 643 735; fax: +44 (0) 1304 651 817.
E-mail addresses: thomas.ryckmans@pfizer.com, thomas.ryckmans@gmail.com
Caffeine 3
Figure 1. Structure of HNE 1, Mustard Oil (MO) 2 and caffeine 3.
(T. Ryckmans).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.035

4858
T. Ryckmans et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4857–4859
O
O
O
O
O
O
O
N
OH
NH
R1
N
N
H
c, d
N
N
H
a, b
N
O
N
Cl
N
Cl
N
N
e
R3
N
N
NH2
N
R4
N
NH
R2
N
Cl
N
*
NH
Cl
N
Cl
Cl
N
Cl
7
Ph
8
5
4
5
6
O
9a: R1=
9b: R1=
R1
Figure 2. Structure of non-covalent hit 3, versatile template
compounds 6.
5
and target
9c: R1=
9d: R1=
*
*
N
N
H
N
N
N
NH
N
Ph
channel, appropriate solubility and some measure of selectivity
against other drug targets to enable further assessment of the
TrpA1 channel pharmacology.
O
*
9
N
Preliminary triage of non-electrophilic ligands of the TrpA1
channel identified compound 4 as a weak (6 lM) agonist of the
human TrpA1 channel. The central core of 4 is a parallel chemis-
try-enabled template, as the commercially available 2,4-dichloro-
Scheme 1. Synthetic methodology for the variation of R1. Reagents and conditions:
(a) methanol (1.2 equiv), Et3N (1.2 equiv), DCM, 0 °C, 2 h, 57%; (b) isobutylamine
(1.05 equiv), Et3N (1.05 equiv), DCM, 0 °C, 16 h, 82%; (c) 4-benzylpiperazine
(1.1 equiv), Et3N (1.1 equiv), acetonitrile, rt 1 h, 97%; (d) NaOH (20 equiv), MeOH–
water (50:50), reflux, 1 h, 99%; (e) amine R1-NH2 (1 equiv), Et3N (1 equiv), HATU
(1 equiv), DMF, 6 h, rt.
pyrimidine-5-carbonyl chloride
5 is amenable to sequential
functionalisation13 to 2,4-diamino-pyrimidine-5-carboxylic acid
amide derivatives 6 (Fig. 2).
In order to limit the very large number of potential targets, sub-
stituents were optimized sequentially. Preliminary data obtained
by screening nearest neighbours of 4 showed that small alkyl
groups such as cyclopropyl and isobutyl at R2 were beneficial to
agonist potency. Replacing the 4-benzyl piperidine with the more
polar and synthetically enabled 4-benzylpiperazine designated 8
as a key intermediate for optimization of R1 (Scheme 1).
Pleasingly, 9a (R1 = benzyl) was readily identified as a moder-
ately potent agonist of both the rat and human TrpA1 channels
(Table 1). The 3-picolyl group (9b) was tolerated, but the 2-picolyl
(9c) and methoxyethyl (9d) derivatives significantly lost potency.
Keeping R1 as benzyl and R2 as either cyclopropyl or isobutyl, a
range of piperazines was prepared from intermediates 12 and 13
(Scheme 2).
Table 1
Agonist activitya at the rat and human TrpA1 channels15
Compound
rTrpA1 EC50, nM
hTrpA1 EC50, nM
2 (MO)
9a
9b
9c
9d
14a
14b
15a
15b
15c
16a
5813 246 (n = 330)
1120 165 (n = 10)
79
49
na
na
na
27
na
175
215
2250
581
5600
186
468
30% (10
848
lM)
na
109
97 4.7 (n = 136)
23 0.64 (n = 773)
Amides 14a and 14b showed very different potencies, raising
the possibility that the affinity of 14b is due to the reaction be-
tween a sulfur atom from a cysteine residue of the protein and
the fluoroaromatic ring, a reaction known to occur at room tem-
perature.14 Reductive aminations on derivative 15 showed that
the cyclopropyl derivative was markedly less potent than its isobu-
tyl analogue (15a vs 9a). While n-butyl derivative 15b displayed
low activity, the cyclic derivative 15c showed some potency at
both the rat and human receptor (Table 1).
a
Values are geometric means of a minimum of two experiments, unless stated
otherwise.
Having established the selectivity of PF-4840154 in vitro, we
needed to establish its functional activity at the TrpA1 channel
in vivo.
The mouse Formalin test is a well established pain model in
which intraplantar (i.pl.) injection of the TrpA1 activator formalin
elicits paw licking behaviour.4 In a similar experiment, we exam-
ined the effects of intraplantar (i.pl.) injections of PF-4840154 on
licking behaviour in TrpA1 WT and KO mice. The i.pl. injections
of PF-4840154 caused a significant increase in the time spent lick-
ing of the treated paws as compared to vehicle (n = 4). PF-
4840154-evoked nocifensive behaviours were significantly less in
TrpA1 KO mice. Furthermore, pre-treatment with the selective
TrpA1 antagonist HC-0300314 significantly reduced the time spent
licking the injected paws in PF-4840154-treated animals.15 Thus
both genetic and pharmacological disruption of the TrpA1 channel
blocked the effects of PF-4840154 in this model, demonstrating the
functional agonism of PF-4840154 at the TrpA1 channel (Fig. 3).
In conclusion, having conducted an HTS we identified a novel
class of non-covalent TrpA1 agonists that was successfully utilized
as a replacement for the well established yet problematic reference
agonist Mustard Oil. PF-4840184 proved to be a potent and selec-
tive agonist of the human and rat TrpA1 channel in vitro, and a po-
tent functional agonist of the mouse TrpA1 channel in vivo. This
compound is adequately soluble at pH 7.2, stable in aqueous solu-
tion, non-volatile, and proved to be vastly superior to Mustard Oil
as a new reference agonist for the HTS of compound collections.
No attempts were made to separate the enantiomers of 16a.
Further profiling of PF-4840154 was initiated to establish its suit-
ability as a reference agonist of the TrpA1 channel.
The compound was stable in aqueous solution, with a solubil-
ity (pH 7.2) of 3
lg/mL (6.4
lmol/L). This solubility, while lim-
ited, proved to be well above the top concentration (1.5
lmol/
L) needed to use PF-4840154 as reference agonist in a screening
assay.15
Before ascertaining that the in vitro activity of PF-4840154 at
the TrpA1 channel was indeed translated into in vivo functional
efficacy, the compound was screened on a range of targets to assess
its selectivity. Significant blockade of the hERG channel16 was ob-
served (IC50 580 nM). When tested on a broad range of over 100
proteins (Cerep Panel), micromolar activity on less than 10% of
the target set was observed with weak inhibition of reference com-
pound binding at the Sigma (1.1
tors, the chloride channel (1.5
and Noradrenaline (2.8 M) transporters. In our in-house screens,
lM) and the D3 (1.9
l
M) recep-
lM) and the Dopamine (1.6
lM)
l
PF-4840154 had no effect on established pain targets such as the
hTrpV1, hTrpV4 and hTrpM8 channels, in either agonist or antago-
nist modes. This selectivity suggests that the pharmacology of the
compound is likely to be TrpA1 mediated.

T. Ryckmans et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4857–4859
4859
O
O
O
N
N
H
Ph
N
N
H
Ph
a, b
N
Cl
c
N
N
NH
R2
Cl
N
NH
R2
Cl
N
Cl
HN
12 R2=cPr
13 R2=iBu
5
10 R2=cPr
11 R2=iBu
e
d
O
O
14a R5=
14b R5=
N
N
Ph
N
O
N
Ph
*
H
H
N
N
NH
R2
N
N
NH
F
*
R6
N
R5
N
O
N
15 R2=cPr
16 R2=iBu
14
F
O
15a R6=
15b R6=
15c R6=
N
N
H
Ph
*
*
*
O
N
NH
N
16a
Scheme 2. Reagents and conditions: (a) benzylamine (1.05 equiv), Et3N (1.05 equiv), DCM, À10 °C, 2 h 86%; (b) cyclopropylamine or isobutylamine (1.05 equiv), Et3N
(1.05 equiv), DCM, 0 °C, 16 h, 75–82%; (c) piperazine (10 equiv), acetonitrile, rt, 1 h, 93–96%, amine (4-benzylpiperazine) (1.1 equiv), Et3N (1.1 equiv), acetonitrile, rt 1 h, 97%;
(d) acid (1 equiv), Et3N (1 equiv), HATU (1 equiv), DMF, 6 h, rt; (e) aldehyde (1.5 equiv), NaBH(OAc)3 (2 equiv), DCM, AcOH 1%, 0 °C ? rt, 16 h, 78–89% with tetrahydro-pyran-
3-carbaldehyde.
Figure 3. The in vivo effect of PF-4840184 is mediated by TrpA1. (A) Total time (s) spent licking the TRPA1 agonist (16a, PF-4840154, 30 nmol) or vehicle injected paws of
female TRPA1+/+ or TRPA1À/À mice (n = 4) over 1hr period. In TrpA1+/+ mice, PF-4840154 elicits robust licking behaviour upon i.pl. administration, but has no significant effect
on TrpA1À/À animals. (B) Total time (s) spent licking the TRPA1 agonist (16a, PF-4840154, 30 nmol) or vehicle injected paws of female CD1 mice pretreated with TrpA1
antagonist HC030031 (100 mg/kg) or vehicle (10% DMSO in saline) over 1 h period (n = 4). Pre-treatment with the selective TrpA1 antagonist HC-030031 abolishes the effect
of PF-4840154 on licking behaviour. All data are represented as mean SEM and analysed by One-Way ANOVA followed by Bonferroni’s test (A) ⁄⁄⁄p <0.001 versus
vehicle-treated paws and ##p <0.01 versus TRPA1 agonist-treated paws, (B) ⁄⁄⁄⁄p <0.001 versus vehicle-treated paws and ####p <0.0001 TRPA1-agonist treated paws of mice
pre-treated with HC030031.
6. Bandell, M.; Story, G. M.; Hwang, S. W.; Viswanath, V.; Eid, S. R.; Petrus, M. J.;
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