The discovery of a potent series of carboxamide TRPA1 antagonists

Article abstract of DOI:10.1039/c6md00387g

A series of potent and selective carboxamide TRPA1 antagonists were identified by a high throughput screen. Structure-activity relationship studies around this series are described, resulting in a highly potent example of the series. Pharmacokinetic and skin flux data are presented for this compound. Efficacy was observed in a topical cinnamaldehyde flare study, providing a topical proof of pharmacology for this mechanism. These data suggest TRPA1 antagonism could be a viable mechanism to treat topical conditions such as atopic dermatitis.

Full text of DOI:10.1039/c6md00387g

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RESEARCH ARTICLE
The discovery of a potent series of carboxamide
TRPA1 antagonists†‡
Cite this: DOI: 10.1039/c6md00387g
D. C. Pryde,§ a B. Marron,b C. G. West,b S. Reister,b G. Amato,b K. Yoger,b K. Padilla,b
*
J. Turner,c N. A. Swain,a P. J. Cox,c S. E. Skerratt,a T. Ryckmans,d D. C. Blakemore,a
J. Warmuse and A. C. Gerlachb
A series of potent and selective carboxamide TRPA1 antagonists were identified by a high throughput
screen. Structure–activity relationship studies around this series are described, resulting in a highly potent
example of the series. Pharmacokinetic and skin flux data are presented for this compound. Efficacy was
observed in a topical cinnamaldehyde flare study, providing a topical proof of pharmacology for this mech-
anism. These data suggest TRPA1 antagonism could be a viable mechanism to treat topical conditions such
as atopic dermatitis.
Received 13th July 2016,
Accepted 17th August 2016
DOI: 10.1039/c6md00387g
www.rsc.org/medchemcomm
the pre-synaptic terminals at the level of the spinal cord dor-
sal horn and may more directly modulate synaptic release
Introduction
The transient receptor potential (TRP) family of ion channels
is comprised of 28 distinct channels, many of which have
been implicated as drug targets for a variety of diseases. Sub-
stantial literature supports the involvement of these channels
in the pathophysiology of pain, inflammatory bowel disease,
as well as disorders of the kidney, bladder, skin, respiratory
and cardiovascular system.1,2
TRPA1 is the sole member of the TRPA subfamily, a non-
selective cation channel expressed in nociceptive dorsal root
neurons and visceral tissue that functions as a polymodal
sensory receptor and contributes to inflammatory pain sig-
naling.3 The channel is activated by a diversity of inflamma-
tory mediators such as prostaglandin metabolites, Ca2+, cold
temperatures, 4-hydroxynonenol, allyl isothiocyanate (AITC)
and H2O2 to produce a generator potential for action poten-
tial firing.4 Furthermore, TRPA1 channels are expressed at
and plasticity.5 In humans, a gain of function TRPA1 muta-
tion is linked to familial episodic pain syndrome, a severe de-
bilitating pain condition.6
Supporting evidence for a role of TRPA1 in pain is pro-
vided by selective small molecule TRPA1 inhibitors that are
active in both in vivo electrophysiological and behavioral pain
models.7 These include pre-clinical rodent models of induced
pain, diabetic peripheral neuropathy, visceral pain and pan-
creatitis pain. TRPA1 knockout mice are less sensitive to in-
flammatory pain conditions primarily when measured by me-
chanical endpoints.8
Emerging data also suggests a key role for this channel in
mediating itch, particularly itch signaled via non-
histaminergic pathways that are thought to underpin chronic
itch associated with conditions such as atopic dermatitis.9
Pruritogens, such as chloroquine and BAM8-22,9b which acti-
vate MrgprA3 and MrgprC11 G-protein coupled receptors re-
spectively, have significantly attenuated pruritogenic poten-
tial in TRPA1 knockout mice. TRPA1 is pivotal in thymic
stromal lymphopoietin, IL-31- and endothelin-induced itch,
which are key mediators of itch in atopic dermatitis.10 There-
fore, small molecule TRPA1 inhibitors are hypothesized to
have utility in treating conditions in which chronic pain and/
or itch are significant symptoms.
a Pfizer Worldwide Medicinal Chemistry, Neuroscience and Pain Research Unit,
Portway Building, Granta Park, Great Abington, Cambridgeshire, CB21 6GS, UK
b Icagen, Inc., 4222 Emperor Boulevard, Suite 350, Durham, North Carolina,
NC27703, USA
c Neuroscience and Pain Research Unit, Portway Building, Granta Park, Great
Abington, Cambridgeshire, CB21 6GS, UK
d Pfizer Worldwide Medicinal Chemistry, Ramsgate Road, Sandwich, Kent, CT13
9NJ, UK
e Pfizer Worldwide Medicinal Chemistry, Neuroscience and Pain Research Unit,
Groton, CT, USA
TRPA1 has a tetrameric structure that defines a single
transmembrane pore region and a very long ankyrin repeat
domain in the N-terminal region. The structure of a TRPA1
channel was initially reported at 16 Å resolution using cryo-
EM methods,11 which has been followed by a more recent
higher resolution structure at approximately 4 Å resolution,12
although a detailed understanding of how the channel gates
† All authors are either current or former employees of Pfizer.
‡ Electronic supplementary information (ESI) available: In vitro and in vivo activ-
ity charts, selectivity data and metabolite ID studies for selected compounds.
See DOI: 10.1039/c6md00387g
§ Current address: Curadev UK, Innovation House, Discovery Park, Sandwich,
Kent, CT14 9FF. E-mail: david@curadev.co.uk.
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and functions is still lacking. For example, TRPA1 is cova-
lently activated by many reactive and/or noxious substances,
which have been proposed to react with the many cysteine
residues towards the N-terminus of the protein,13 but it is
not clear how covalent modifications in this region can be
transmitted to gating changes in the pore domain.
Recently, several groups have studied potential binding
sites of common TRPA1 ligands14 many of which are pro-
posed to bind to the menthol binding site based on site-
directed mutagenesis data. A number of different small mole-
cule chemotypes with activity at the TRPA1 channel have
been reported in the literature15 and several of these have
progressed to human clinical trials.16 A selection of these
chemotypes 1–7 are shown in Fig. 1. We describe here our
own efforts to identify and develop a potent and selective se-
ries of TRPA1 antagonists starting from a high throughput
screening hit.
Fig. 2 File screening hit, identified using a FLIPR assay and confirmed
in PatchXpress electrophysiology.
about the high lipophilicity of the hit (clog P 5.0), and how
dependent on this high lipophilicity potency in the series
might be.
However, when further profiled in a broad panel of 132 ion
channels, transporters, receptors and enzymes, 8 showed excel-
lent selectivity. When profiled against the other TRP family
members implicated in pain, it also showed complete selectivity
over the TRPV1 and TRPM8 channels. The synthesis of this
compound was short and efficient, and we were able to develop
a very straightforward synthesis of compounds from this series
as described in the Experimental section below.
Results
Screening hit
Our screening was initially based on a high throughput
FLIPR assay, which identified actives and were then con-
firmed using a stable medium throughput electrophysiology
assay on the PatchXpress platform. The data presented below
in the tables are all confirmed PatchXpress data except where
stated. For selected compounds we also tested passive mem-
brane permeability as RRCK permeability17 and human he-
patic microsome stability18 on selected compounds as a gen-
eral indication of compound quality.
A high throughput FLIPR screen of the corporate com-
pound collection identified several hit series, the most prom-
ising of which was based on a piperidine amide scaffold
8 shown in Fig. 2. This compound showed good potency in
the initial FLIPR assay, and when confirmed in the
PatchXpress assay showed significantly higher potency with
an IC50 of 17 nM. At this stage, we did have some concerns
Core changes
We screened some close analogues of the initial hit 8 from our
compound collection to build early SAR as shown in Table 1.
Piperazine analogues were not active, either in a more po-
lar template 13, or a more lipophilic substituted piperazine
scaffold 9. Truncating the left hand side phenyl ring down to
the smaller isopropyl group 10 also lost activity, as did cap-
ping the amide grouping with a methyl group (14) or replac-
ing the benzyl group with more polar heterocyclic systems 12
and 11. At this stage we also confirmed that 8 had very poor
metabolic stability and low permeability in the RRCK assay
which was, for the most part, replicated across the analogues
depicted in Table 1.
Piperidinol unit and stereoselectivity
These initial SAR findings suggested that large improvements
in the metabolic stability or physicochemical properties of
the initial lead would be challenging, with significant struc-
tural changes around the piperidine ring poorly tolerated.
However, improving metabolic stability in the series now be-
came a major area of focus for the project to indicate the fu-
ture potential of the chemotype to provide an orally available
compound. To further explore these observations, several
more analogues were designed around a downsized right
hand side template which featured a cyclopropyl ring in the
benzylic position as a less lipophilic core structure (Table 2).
The downsized system 15 was somewhat weaker than its
cyclopentyl counterpart 8, but was 1 log unit less lipophilic.
Contracting the benzyl substituent to a phenyl substituent 16
further reduced lipophilicity, but ablated activity. Separating
the individual enantiomers of the benzylic analogue revealed
that most of the activity resided in the R enantiomer 18, with
the S isomer 17 being some 10 fold less active. The more
Fig. 1 Examples of chemotypes with reported activity at TRPA1.
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Table 1 Early SAR of the primary screening hit
Comp
R1
R2
H
MWt. (clog P)
TRPA1 IC50a (nM)
17
HLM (μL min−1 mg−1
>320
)
RRCK (× 10−6 cm s−1
)
8
509 (5.0)
1
9
H
H
503 (6.3)
461 (4.5)
>1000
>1000
312
<1
<1
10
>320
11
12
H
H
530.1 (4.2)
517.0 (3.7)
26 700
1900
42
1
3
>320
13
14
H
440 (4.4)
523 (5.5)
>3000
>1000
>320
>320
3
Me
ND
a
PX electrophysiology IC50. All compounds were racemic.
active R enantiomer had TRPA1 potency of around IC50 50
nM, although its metabolic stability was still very poor.
It was of concern that all of the compounds in Table 2, de-
spite reductions in global lipophilicity, all continued to show
rapid metabolic turnover in human hepatic microsomes. Me-
tabolite identification studies19 with 8 showed that com-
pounds within this series were being oxidised in several
places around the molecule. The major metabolites resulted
from dehydration and aromatisation of the piperidine ring or
oxidations around the amide group and its substituents.
These data indicated a need to further overall reduce the
global lipophilicity of the series to improve the probability of
achieving metabolic stability and to seek more metabolically
robust replacements of the piperidine unit.
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Table 2 Piperidinol and benzyl variations of the hit compound
Comp
R1
Stereo-centrea
MWt. (clog P)
TRPA1 IC50a (nM)
73
HLM (μL min−1 mg−1
>320
)
RRCK (× 10−6 cm s−1
)
15
rac
481 (3.9)
2
16
rac
467 (3.6)
>3000
>320
4
17
18
S
481 (3.9)
481 (3.9)
474
53
>320
>320
ND
ND
R
a
PX electrophysiology IC50. rac indicates compounds were racemic.
Amide substituent
group more fully with various right hand side spiro-fusions
and phenyl substitutions in place (Table 4). Simple deletion
of the hydroxyl group from the piperidine 29 lost activity
compared to the alcohol equivalent 8, as did the ring
contracted pyrrolidine 30 and the tropane system 37. Delet-
ing the hydroxyl from the pyrrolidine core then gave a further
5 fold loss in potency 31.
Interestingly, all other changes examined led to a complete
loss in activity, including a spiro-fused oxetane 32, spiro-fused
cyclopropane 33, bridged piperidine 34 and spiro-fused lactam
36. It was also interesting that deletion of the trifluoromethyl
group from the pyrrolidine core in 35 led to a complete loss of
activity. Each of these changes had increased the basic pKa of
the central N atom from around pKa ∼6 with up to almost pKa
∼8 with some of these latter changes20 and it is possible that
more basic groups which are more ionised at neutral pH's are
poorly tolerated at the TRPA1 channel.
Retaining the benzylic left hand side group, we next explored
SAR around the right hand side amide substituent with a view
to reducing lipophilicity. It was immediately apparent (Table 3)
that there was more toleration of structural changes in this re-
gion, with TRPA1 activity apparent in structures that featured
heteroaromatic (20 and 28), carbocyclic (19, 21, 23) and ali-
phatic (25, 26, 27) substituents, many with clogP below 4.
We confirmed once more in the hexyl pair of substituents 25
and 26 that most of the activity resided in the R enantiomeric
series. It was also apparent that bulky and lipophilic substitu-
ents such as that in 24 did not guarantee activity and in fact
could be detrimental to potency as was also the case with the
di-substituted oxazole 22. We tested a selection of compounds
from the heterocyclic and alkyl chemotypes for metabolic stabil-
ity and found them all uniformly to possess poor stability, de-
spite in many cases much reduced lipophilicity.
In the next set of targets we sought to address the meta-
bolic liability of the piperidine unit.
Tool compound identification
The SAR we had developed to this point had showed how
sensitive the series was to significant structural changes and
lipophilicity reduction. Furthermore, in seeking a suitable
tool compound for further probing of the pharmacology of
Piperidine analogues
At this point, we incorporated our structure–activity data to
date into a set of targets that sought to explore the piperidine
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Table 3 Amide analogues of the lead series
Comp
R1
Stereo-centrea
MWt (clog P)
TRPA1 IC50a (nM)
101
HLM (μL min−1 mg−1
>320
)
RRCK (× 10−6 cm s−1
)
19
rac
468.3 (5.1)
ND
20
rac
455.2 (3.8)
159
>320
ND
21
22
rac
rac
438.2 (2.1)
479.2 (1.8)
1000
149
146
ND
ND
>10 000b
23
24
rac
rac
426.2 (3.8)
476.3 (4.8)
851
>320
>320
ND
ND
>10 000b
25
26
27
S
414.5 (3.5)
414.5 (3.5)
440.2 (2.6)
1700b
71b
ND
ND
284
ND
ND
31
R
rac
91
28
rac
493.2 (3.6)
83
285
8
a
PX electrophysiology IC50. rac indicates compounds were racemic. b FLIPR IC50
.
TRPA1 we had not identified a metabolically stable com-
pound suitable for oral dosing. However, we had identified a
number of potent and selective TRPA1 antagonists, and
returned to the original early screening hit 8 to work this up
into a tool compound for further evaluation of the target.
The enantiomers were separated and again showed that the
major activity resided in the R enantiomer 39 (Fig. 3).
When tested in single rig electrophysiology, 39 showed
excellent potency of IC50
< 1 nM, and demonstrated
potent activity across several species of TRPA1, albeit
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Table 4 Piperidine variations
Comp
R1
H
R2
R3/R4
MWt (clog P)
TRPA1 IC50a (nM)
673
HLM (μL min−1 mg−1
)
RRCK (× 10−6 cm s−1
)
29
492.2 (6.5)
238
6
30
31
H
F
464.2 (4.6)
480.2 (5.8)
309
ND
ND
ND
ND
1451
32
F
468.3 (4.6)
>10 000b
>320
ND
33
34
35
F
F
F
474.2 (5.2)
427.0 (5.3)
428.5 (4.2)
>10 000b
>10 000b
>10 000b
>320
>320
170
0.3
ND
ND
36
37
F
F
484.0 (3.2)
>10 000b
168
ND
ND
536.6 (4.9)
825
>320
b
All compounds were racemic.a PX electrophysiology IC50
.
FLIPR IC50.
somewhat less potent at the pig channel (Fig. 4). It
showed high turnover in hepatic microsomes, low perme-
ability, low aqueous solubility and high plasma stability
indicating there to be no amide hydrolysis contributing to
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the degradation of the compound during the metabolism
assays.
It showed excellent selectivity in a broad panel of 112
screens and no genetic toxicity flags. 39 showed no activity at
either the TRPM8 or the TRPV1 channels. Overall, this com-
pound represented an excellent tool compound to further ex-
plore TRPA1 pharmacology.
Binding site elucidation
As mentioned above, there has been an increase in literature
activity recently in trying to elucidate the binding sites of
small molecule ligands of the TRPA1 channel. Most publica-
tions have centred on a region around S5 that has been
shown to affect the binding of menthol, and related terpene
molecules, centred around the S873 and T874 residues.14
This suggests that the channel can be modulated through
binding either to the N-terminal region which is rich in cyste-
ine residues, or to this S5 region.
In order to generate some hypotheses of where our tool
compound series was binding to the TRPA1 channel, we gen-
erated several ortholog chimeras based on hTRPA1 and the
opossum version of the channel (oTRPA1) due to its diversity
of sequence in the pore region. Chimeras were made in
which the S5 region of the two ortholog species were
swapped, as shown in Fig. 5.
Fig. 4 Further profile data for compound 39.
for environmental irritants. In these conditions, effective
agents would be efficacious at the local site of administration
and not require a long systemic pharmacokinetic half-life.
The rat pharmacokinetics of 39 were measured and
showed moderate to high clearance and low oral bioavailabil-
ity (clearance 42 ml min−1 kg−1, Vdss 3.99 L kg−1, T1/2 4.4 h, F
5%). The systemic pharmacokinetics of 39 following a topi-
cally applied dose of compound were also examined in the
rat. A dose of 0.68 mg per animal was given using a 27 mg
mL−1 solution of 39 in 70% ethanol/30% polyethyleneglycol
applied to a 5 cm2 area of skin and systemic levels of 39 mon-
itored for 24 hours. At all time points the levels of compound
observed were low, and well below IC50 concentrations. In-
deed at most of the time points compound levels were below
the limit of quantitation in the bioanalysis assay. We exam-
ined the skin flux properties of the compound which showed
low skin permeability (0.1 × 10−5 cm h−1) but high concentra-
tions in both the epidermal layer (3128 μg g−1) and in the der-
mal layer (640 μg g−1) following topical administration,
confirming the compound had good skin retention properties.
Using these reagents, the activity of 39 was investigated as
shown in Fig. 6. 39 was most potent against the hTRPA1
channel, but lost almost all activity when the S5 region was
swapped to that of the opossum sequence. 39 had almost no
activity against oTRPA1, but to a large extent its activity was
rescued by insertion of the human S5 sequence.
Together, these data indicated the importance of the S5
region of the channel for the bioactivity of the series.
Topical flare study
At this point in the project we had identified a potent and se-
lective series and demonstrated using computational and
mutation studies that the series bound in the menthol bind-
ing site region of S5 but we had been unable to increase met-
abolic stability to the extent that it could be considered a use-
ful oral series. We were aware of the extensive literature that
had been published on the role of TRPA1 in topical condi-
tions including itch21 and chronic cough22 as a chemosensor
Fig. 3 Separated enantiomers of 8.
Fig. 5 Design of the hTRPA1 and oTRPA1 ortholog chimeras.
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skin. At each timepoint, the AUC figures for 50 mg ml−1 and
100 mg ml−1 doses were very similar and equally effective at re-
ducing cinnamaldehyde-induced flare (P < 0.01). Three pre-
dose timepoints were examined in this study. Only the 30 min
data are shown in Fig. 7. Theoretically, a longer pre-dose time
allows the compound time to transition through the outer skin
layers and engage with TRPA1 receptors within the skin,
but all pre-dose times significantly (P
< 0.01) reduced
cinnamaldehyde-induced flare. However 30 min appeared to be
sufficient time for the compound to traverse relatively thin rat
skin. Increasing the pre-dose time potentially also allows more
time to be lost to metabolic processes in the skin, non-specific
protein binding or passing through the skin and being systemi-
cally metabolised. Confirming these theories were outwith the
scope of these studies.
Fig. 6 Carboxamide 39 potency is dependent on the TRPA1 S5 helix.
TRPA1 species ortholog chimeras were generated to identify the
critical region of interaction, stably expressed in hEK293 cells and
evaluated using whole-cell voltage clamp electrophysiology. Opossum
TRPA1 (oTRPA1) was selected as the human (hTRPA1) chimeric partner
because of divergence in the channel pore region (68% identity in S5–
S6) hTRPA1/oTRPA1 chimeras were generated by swapping the 27
amino acid comprising S5. hS5 designates chimeras in which hTRPA1
amino acids 867–893 were swapped into oTRPA1. oS5 designates chi-
meras in which oTRPA1 amino acids 870–896 were swapped into
hTRPA1. The mean IC50 (95% confidence interval, n) was (●) hTRPA1 =
0.72 nM (95% CI = 0.60–0.85 nM, n = 6), ( ) hS5 = 20 nM (95% CI =
17–25 nM, n = 5), ( ) oS5 = 1578 nM (95% CI = 1426–1745 nM, n = 4)
and ( ) oTRPA1 ≥ 3000 nM (n = 4).
Comparing these data to those obtained with a 50 mg mL−1
dose of 4 (Fig. 8), the two datasets look very similar, despite the
diversity of skin flux and retention data displayed by the two
compounds. The 60 min pre-dose data is shown in Fig. 8.
Further study is required to identify an optimal skin flux
and skin retention compound profile, but these data clearly
show that a topically-administered TRPA1 antagonist can ab-
late an inflammatory flare response.
Conclusions
We designed a topical flare study in the rat, in which test
compound was formulated in vehicle, applied to the skin and
left for a defined period of time. After this time, a 20% v/v
formulation of cinnamaldehyde in the study vehicle was then
applied to the same location and blood flow measured in the
dosed region by laser Doppler sonography. The test com-
pound chosen for this initial study was the racemate 8. Re-
sponses were compared directly to an example of a literature
TRPA1 antagonist, for which we chose compound 4 as a
small molecule analogue of similar lipophilicity and in vitro
potency to 8 (compound 4 showed human TRPA1 PX IC50 10
nM and rat TRPA1 PX IC50 8 nM in our hands). This com-
pound also offered a useful comparator as our data suggested
it to have a higher skin flux of 8 × 10−5 cm h−1, but lower con-
centrations retained in both the dermis (232 μg g−1) and epider-
mis (86 μg g−1) compared to 8 for an equivalent administered
dose. Typically, the literature23 would suggest that for effective
transdermal delivery of compounds, they should be low molec-
ular weight (<400) and moderately polar (logP < 4), which was
consistent with the properties of 4 and our skin flux data find-
ings, and provided an excellent contrasting profile to 8.
Fig. 7 shows the cinnamaldehyde-induced flare data for
8 at two different concentrations. Systemic pharmacokinetic
analysis showed no measurable levels of any compound in
the systemic circulation. Skin samples were not analysed due
to difficulties extracting meaningful values.
Topically applied 8 significantly reduced cinnamaldehyde-
mediated flare in rat skin. Comparing the two dose groups, in-
creasing the concentration of topically applied 8 made no differ-
ence to the effect on cinnamaldehyde-induced erythema in rat
We have identified potent selective inhibitors of the TRPA1 ion
channel based on a carboxamide template. Efforts to enhance
the in vitro ADME properties of this series with a goal to improve
the oral bioavailability of this series, proved futile. However, an
exemplar compound from this series accumulated to high levels
in skin and engaged with the target, in the absence of signifi-
cant systemic exposure, when administered topically in ro-
dent CA flare studies. The skin is a major neurosensory organ
Fig.
7 Normalised flare response to topical trans-cinnamaldehyde
(20% solution in ethanol) in animals dosed topically with vehicle or the
selective TRPA1 blocker 8, n = 8 per group. Each data point represents
mean blood flow in a 5 minute laser Doppler scan over a 2.5 cm2 scan
area. Baseline measurements were taken for 10 minutes before
treatment was delivered topically. Laser Doppler scans continued
during the pre-treatment then for a further 5 minutes to establish a
post-dose baseline. 50 μl of 20% trans-cinnamaldehyde solution was
administered topically to the centre of the scan area using a 12 mm
polypropylene coated aluminium Finn chamber for a 10 minute dura-
tion. Doppler scans were not recorded during this period. The Finn
chamber was then removed and laser Doppler flowmetry scans were
then recorded for 30 minutes.
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was added 4-(trifluoromethyl)piperidin-4-ol (2.72 g, 9.61
mmol) and potassium phosphate (8.16 g, 38.4 mmol) and the
reaction was stirred at room temperature for 18 hours. The
reaction was diluted with EtOAc, washed with water, dried
over magnesium sulphate and concentrated in vacuo. The res-
idue was purified using silica gel column chromatography
eluting with 0–25% EtOAc in heptanes to afford the title com-
pound (1.93 g, 60%). HPLC analysis: column: XBridge C18
150 × 4.6 mm, 5 micron; mobile phase A: water with 0.1%
TFA, mobile phase B: MeCN with 0.1% TFA. Gradient: 5% B for
0–1.5 min, 5–100% B for 1.5–10 min, 100% B for 10–11 min
and 100–5% B for 11–12 min. Flow rate 1.5 mL min−1. Rt = 5.96
minutes. 1H NMR (400 MHz, CDCl3): δ 1.50–2.00 (m, 4H), 3.05–
3.20 (m, 2H), 3.20–3.38 (m, 3H), 3.75 (s, 3H), 3.75–3.85 (m, 1H),
4.40–4.52 (m, 1H), 7.20–7.42 (m, 5H) ppm.
Fig. 8 Normalised flare response to topical trans-cinnamaldehyde
(20% solution in ethanol) in animals dosed topically with vehicle or the
selective TRPA1 blocker 4,
conducted as described for 8.
n = 8 per group. Experiments were
where the symptoms of pain and itch in chronic diseases are
most often first perceived, therefore we hypothesize that carb-
oxamide based inhibitors of TRPA1 could offer potential safe
and effective topical treatments for pain or itch.
(S)-2-(4-Hydroxy-4-(trifluoromethyl)piperidin-1-yl)-3-
phenylpropanoic acid
Experimental
An example of the synthetic routes followed to prepare the com-
pounds contained herein is shown below for compound 39.
(R)-Methyl 3-phenyl-2-
(((trifluoromethyl)sulfonyl)oxy)propanoate
A
solution of (S)-methyl 2-(4-hydroxy-4-(trifluoromethyl)-
piperidin-1-yl)-3-phenylpropanoate (1.3 g, 3.924 mmol) in 6 N
HCl (aq) (30 mL) was heated to 90 °C for 18 hours. The reac-
tion was cooled to −10 °C with a brine/ice mixture and
treated with 6 N NaOH (aq) until pH = 3–4. The aqueous mix-
ture was extracted into EtOAc four times. The organic layers
were combined, washed with brine, dried over sodium sul-
phate and concentrated in vacuo to afford the title compound
that was used directly in the next step (1.2 g, 97%). MS m/z
To a cooled solution (−10 °C) of (R)-methyl 2-hydroxy-3-
phenylpropanoate (0.99 g, 5.49 mmol) in DCM (14 mL) was
added 2,6-lutidine (0.77 mL, 6.6 mmol) followed by
trifluoromethanesulfonic anhydride (1.11 mL, 6.59 mmol)
dropwise. The reaction was stirred at this temperature for 1
hour. The reaction was quenched by the addition of water and
stirred for 2 minutes. The solution was extracted into DCM and
the organic layer was collected, dried over magnesium sulphate
and concentrated in vacuo. The residue was purified using silica
gel column chromatography eluting with 0–25% EtOAc in hep-
1
318 [M + H]+. H NMR (400 MHz, MeOH-d4): δ 2.00–2.30 (m,
4H), 3.10–3.30 (m, 2H), 3.40–3.65 (m, 3H), 3.75–3.85 (m, 1H),
4.30–4.50 (m, 1H), 7.25–7.45 (m, 5H) ppm.
(S)-2-(4-Hydroxy-4-(trifluoromethyl)piperidin-1-yl)-3-
phenylpropanamide
1
tanes to afford the title compound as an oil (1.435 g, 84%). H
NMR (400 MHz, CDCl3): δ 3.15–3.25 (m, 1H), 3.30–3.40 (m, 1H),
3.85 (s, 3H), 5.10–5.15 (m, 1H), 7.10–7.40 (m, 5H) ppm.
(S)-Methyl-2-(4-hydroxy-4-(trifluoromethyl)piperidin-1-yl)-3-
phenylpropanoate
A solution of (S)-2-(4-hydroxy-4-(trifluoromethyl)piperidin-1-yl)-
3-phenylpropanoic acid (1211 mg, 3.816 mmol) and
carbonyldiimidazole (1180 mg, 7.25 mmol) in THF (38 mL)
was stirred at room temperature for 1 hour before cooling to
0 °C. Ammonium hydroxide (2.97 mL) dropwise was added
and the reaction was stirred at 0 °C for 30 minutes. The reac-
tion was diluted with water and extracted into EtOAc. The or-
ganic layer was collected, dried over magnesium sulphate
and concentrated in vacuo. The residue was purified using
To a solution of (R)-methyl 3-phenyl-2-(((trifluoromethyl)-
sulfonyl)oxy)propanoate (3 g, 9.6 mmol) in EtOAc (24.6 mL)
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silica gel column chromatography eluting with 0–75% EtOAc
in heptanes to afford the title compound (1 g, 83%) as a yel-
low oil which was used immediately in the next step.
the title compound as a colourless oil (600 mg, 74%). MS m/z
303 [M + H]+ 1H NMR (400 MHz, MeOH-d4): δ 1.75–1.80 (m,
3H), 1.83–1.90 (m, 1H), 2.35–2.50 (m, 2H), 2.55–2.65 (m, 3H),
2.75–2.80 (m, 2H), 2.90–3.03 (m, 2H), 7.15–7.30 (m, 5H) ppm.
The enantiomers were then separated using a Chiralpak AD-H.
Mobile phase: 30% IPA in heptanes modified with 0.1% DEA in
IPA. Flow rate: 18 mL min−1 (preparative); 1 mL min−1 (analyti-
(S)-1-(1-Amino-3-phenylpropan-2-yl)-4-
(trifluoromethyl)piperidin-4-ol
cal).
(S)-1-(1-Amino-3-phenylpropan-2-yl)-4-(trifluoromethyl)-
3.95 minutes and (R)-1-(1-amino-3-
piperidin-4-ol, Rt
=
phenylpropan-2-yl)-4-(trifluoromethyl)piperidin-4-ol, Rt = 4.95
minutes.
rac-1-(4-Chlorophenyl)-N-{2-[4-hydroxy-4-
(trifluoromethyl)piperidin-1-yl]-3-
phenylpropyl}cyclopentanecarboxamide
To a solution of (S)-2-(4-hydroxy-4-(trifluoromethyl)piperidin-1-
yl)-3-phenylpropanamide (919 mg, 2.90 mmol) in THF (14.5
mL) was added a 0.5 M solution of LiAlH4 in DME (14.5 mL)
and the reaction was heated to 65 °C for 6 hours followed by
heating at 50 °C for 18 hours. The reaction was cooled to −7
°C and quenched by the addition of water (0.3 mL), followed
by 15% (aq) NaOH solution (0.3 mL) followed by water (0.9
mL) and stirred for 1 hour. The reaction was diluted with
diethyl ether and filtered through a pad of sodium sulphate.
The filtrate was collected and concentrated in vacuo to afford
the title compound as a white solid (778 mg, 87%). MS m/z
303 [M + H]+. 1H NMR (400 MHz, CDCl3): δ 1.60–1.76 (m,
3H), 1.82–1.92 (m, 1H), 2.21–2.30 (t, 2H), 2.46–2.72 (m, 5H),
2.85–2.99 (m, 2H), 7.08–7.27 (m, 5H) ppm.
To
a
solution of rac-1-(1-amino-3-phenylpropan-2-yl)-4-
(trifluoromethyl)piperidin-4-ol (50 mg, 0.214 mmol) in DMF (1
mL) was added 1-(4-chlorophenyl)cyclopentanecarboxylic acid
(37 mg, 0.165 mmol), DIPEA (0.035 mL, 0.198 mmol) and
EDCI (38 mg, 0.198 mmol), followed by HOBt (30 mg, 0.198
mmol) and the reaction was stirred at room temperature for
18 hours. Water was added and the reaction stirred for a fur-
ther 2 hours. DCM was added with further stirring for 1 hour
followed by elution through a phase separation cartridge.
The organic filtrate was concentrated in vacuo. The residue
was dissolved in MeOH and treated with ethereal HCl with
standing for 18 hours. The resulting suspension was filtered
and triturated with EtOAc, heptanes and TBME to afford the
title compound as the hydrochloride salt (69 mg, 82%). 1H
NMR (400 MHz, DMSO-d6): δ ppm 1.50–1.60 (m, 4H), 1.70–
1.90 (m, 4H), 2.15–2.25 (m, 2H), 2.40–2.48 (m, 2H), 2.70–2.80
(m, 1H), 3.05–3.25 (m, 6H), 3.47–3.62 (m, 2H), 6.38 (br s, 1H),
7.20–7.40 (m, 9H), 7.80 (br m, 1H). MS m/z 509 [M + H]+.
A shorter racemic synthesis of this amine was achieved in
fewer steps as below.
rac-1-(1-Amino-3-phenylpropan-2-yl)-4-
(trifluoromethyl)piperidin-4-ol
A solution of phenylacetaldehyde (355 mg, 2.96 mmol) and
trimethylsilylcyanide (293 mg, 2.96 mmol) in diethyl ether
(1.5 mL) was treated with zinc iodide (5 mg) and stirred at
room temperature for 15 minutes. 4-(Trifluoromethyl)-
piperidin-4-ol (500 mg, 2.96 mmol) in MeOH (5 mL) was
added and the reaction heated to reflux for 18 hours. The re-
action was cooled and concentrated in vacuo, azeotroping
with toluene. The residue was dissolved in THF (5 mL),
treated with LiAlH4 (2 M solution in THF, 2.2 mL, 4.43 mmol)
and stirred at room temperature for 48 hours. The reaction
was diluted with TBME (10 mL) and treated with water (0.17
mL), 15% (aq) NaOH solution (0.17 mL) followed by another
aliquot of water (0.51 mL). Sodium sulphate was added and
the reaction was filtered and concentrated in vacuo. The resi-
due was purified using silica gel column chromatography
eluting with DCM : MeOH : NH3, 90 : 9 : 1 to 80 : 18 : 2 to afford
(S) and (R)-1-(4-chlorophenyl)-N-{2-[4-hydroxy-4-
(trifluoromethyl)piperidin-1-yl]-3-phenylpropyl}
cyclopentanecarboxamide
To
a suspension of (S)-1-(1-amino-3-phenylpropan-2-yl)-4-
(trifluoromethyl)piperidin-4-ol (70 mg, 0.232 mmol) and
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1-(4-chlorophenyl)cyclopentanecarboxylic acid (57.3 mg, 0.255
mmol) in acetonitrile (0.8 mL) was added triethylamine
(0.133 mL, 0.928 mmol) followed by propylphosphonic anhy-
dride (50% wt solution in EtOAc, 0.21 mL, 0.35 mmol). The
reaction was stirred at room temperature for 1.5 hours after
which the solution was purified directly by silica gel column
chromatography eluting with 0–30% EtOAc in heptanes to af-
ford the title compound (75 mg, 64%). [α]2D0 = +9.6 in DCM
[20 mg mL−1]. ee determination column: ChiralTech AD-H,
250 × 4.6 mm, 5 micron. Mobile phase A: CO2; mobile phase
B: MeOH with 0.2% ammonium hydroxide. Gradient: 5% B
at 0.00 min, 60% B at 9.00 min; hold to 9.5 min and return
to 5% B at 10 min. Flow rate 3 mL min−1. Rt = 5.05 minutes,
ee = 95%.
The title compounds may also be prepared from rac-1-(4-
chlorophenyl)-N-{2-[4-hydroxy-4-(trifluoromethyl)piperidin-1-yl]-
3-phenylpropyl}cyclopentanecarboxamide prepared as above.
The racemate was separated into two enantiomers using pre-
parative chiral chromatography as described below: Chiralpak
IA, 4.6 × 250 mm, 5 micron. Mobile phase: hexane : DCM :
EtOH : DEA 90: 8 : 2 : 0.1. Flow rate: 1 mL min−1, Rt = 8.351 mi-
nutes and Rt = 10.068 minutes. The first eluting isomer is (S)-1-
(4-chlorophenyl)-N-{2-[4-hydroxy-4-(trifluoromethyl)piperidin-1-
yl]-3-phenylpropyl} cyclopentane-carboxamide, ee = 100%. The
second eluting isomer is (R)-1-(4-chlorophenyl)-N-{2-[4-hydroxy-
4-(trifluoromethyl)piperidin-1-yl]-3-phenylpropyl} cyclopentane-
carboxamide, ee = 99.62%.
Group IX: 100 mg ml−1 8 (5 μl cm−2, topical); 90 minute
pre-dose.
Formulation and dosing. 8 was formulated into a solution
of 70 : 30 ethanol : propylene glycol and dosed at 6 μl cm−2.
To a weighing of the compound (equivalent to a final
concentration of 50 or 100 mg mL−1), ethanol was added to
70% of the final volume. This was sonicated until dissolved,
prior to addition of 30% of final volume of propylene glycol.
This was mixed well to give a final clear solution for
dosing. trans-Cinnamaldehyde (99%, Sigma-Aldrich, Munich,
Germany) was dissolved in 100% ethanol at 20% v/v and
dosed topically at 50 μl dose volume.
Laser Doppler imaging protocol. Male Sprague Dawley rats
were weighed and randomised to one of 2 treatment groups,
placed in an anaesthetic chamber and anaesthetized with a
5% isoflurane/O2 mix. Animals were then moved to a
homoeothermic heat mat and anaesthesia maintained at
approximately 2% isoflurane/O2 during the laser Doppler
scanning via nose cone for the duration of the study. The
abdomen was then shaved to allow laser Doppler flowmetry
scans to be taken over an approximate area of 2.5 cm2.
Baseline scans were recorded for 10 min, subsequently 15 μl
of compound or vehicle (70 : 30 ethanol : propylene glycol)
was then applied to the centre of the scan area. The
treatment was left to penetrate the skin for 30–90 minutes
while baseline blood flow was measured. After the treatment
period, a further 5 post-dose Doppler scans were completed
to establish a post-dose baseline before 50 μl of 20% trans-
cinnamaldehyde (99%, Sigma-Aldrich, Munich, Germany)
dissolved in 100% ethanol was administered topically to the
centre of the scan area using a 12 mm polypropylene coated
aluminium Finn chamber on Scanpor tape (Biodiagnostics
Ltd, Worcestershire) applied for a 10 minute duration. The
Finn chamber was removed and laser Doppler flowmetry
scans were then recorded for 30 minutes. In all cases, the op-
erator was blinded to treatment identity.
The compounds prepared from the chiral separation
method are identical by α-rotation and retention time to the
compounds prepared as the single enantiomer described
above.
MS m/z 509 [M + H]+. 1H NMR (400 MHz, DMSO-d6): δ
1.30–1.80 (m, 10H), 2.20–2.30 (m, 1H), 2.35–2.60 (m, 6H),
2.65–2.85 (m, 4H), 3.00–3.15 (m, 1H), 5.50 (br s, 1H), 6.95–
7.00 (m, 1H), 7.05–7.15 (m, 2H), 7.20–7.35 (m, 6H) ppm.
Sample collection schedule. Terminal PK samples were
taken from all animals via cardiac puncture at study
endpoint (70–150 min post topical dose depending on pre-
dose time). Blood samples were centrifuged and resultant
plasma samples were frozen at −20 °C prior to bioanalysis.
Equipment and supplies list. MoorLDI2 laser Doppler
imager (Moor Instruments Ltd, Axminster, UK).
Data analysis. All data design, randomization and analysis
were handled in accordance with previous discussions with
the Non-Clinical Statistics group. In brief, area under the
curve (AUC) analysis was carried out for normalized blood
flow following cinnamaldehyde challenge. AUC values were
then compared across groups using one-way ANOVA.
4 topical study
In vivo efficacy studies
All experiments were performed in compliance with the laws
and institutional guidelines of the UK Home Office.
8 topical study
Treatment groups. Male Sprague Dawley rats were weighed
and randomised to one of the following treatment groups:
Group I: vehicle (5 μl cm−2, topical); 30 minute pre-dose.
Group II: 50 mg ml−1 8 (5 μl cm−2, topical); 30 minute pre-
dose.
Group III: 100 mg ml−1 8 (5 μl cm−2, topical); 30 minute
pre-dose.
Group IV: vehicle (5 μl cm−2, topical); 60 minute pre-dose.
Group V: 50 mg ml−1 8 (5 μl cm−2, topical); 60 minute pre-
dose.
Treatment groups. Male Sprague Dawley rats were weighed
and randomised to one of the following treatment groups, N
= 8 per group:
Group VI: 100 mg ml−1 8 (5 μl cm−2, topical); 60 minute
pre-dose.
Group VII: vehicle (5 μl cm−2, topical); 90 minute pre-dose.
Group VIII: 50 mg ml−1 8 (5 μl cm−2, topical); 90 minute
pre-dose.
Group I: vehicle (6 μl cm−2, topical); 60 minute pre-dose.
Group II: 4 (50 mg ml−1, 6 μl cm−2, topical); 60 minute
pre-dose.
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The above treatment groups were dosed prior to trans-
cinnamaldehyde application.
Formulation and dosing. 4 was formulated into a solution
of 70 : 30 ethanol : propylene glycol and dosed at 6 μl cm−2
oTRPA1 amino acids 870–896 replaced hTRPA1 amino acids
867–983. All gene synthesis and seamless cloning was
performed by GenScript USA.
Stable clonal cell lines for each chimeric construct were
generated by transfecting chimera plasmid DNA into 293-T-
REx cell line (Invitrogen) and selecting with 150 μg ml−1
hygromycin and 5 μg ml−1 blasticidin (at 37 °C with 10%
CO2). 1 μg ml−1 tetracycline was used to induce hTRPA1 ex-
pression for functional assays.
trans-cinnamaldehyde
(99%,
Sigma-Aldrich,
Munich,
Germany) was dissolved in 100% ethanol at 20% v/v and
dosed topically at 50 μl dose volume.
Laser Doppler imaging protocol. Male Sprague Dawley rats
were weighed and randomised to one of 2 treatment groups,
placed in an anaesthetic chamber and anaesthetized with a
5% isoflurane/O2 mix. Animals were then moved to a
homoeothermic heat mat and anaesthesia maintained at
approximately 2% isoflurane/O2 during the laser Doppler
scanning via nose cone for the duration of the study. The
abdomen was then shaved to allow laser Doppler flowmetry
scans to be taken over an approximate area of 2.5 cm2.
Baseline scans were recorded for 10 min, subsequently 15 μl
of compound (50 mg ml−1 4) or vehicle (70 : 30 ethanol :
propylene glycol) will then be applied to the centre of the
scan area. The treatment was left to penetrate the skin for 60
minutes while baseline blood flow was measured. After the
treatment period, a further 5 post-dose Doppler scans were
completed to establish a post-dose baseline before 50 μl of
20% trans-cinnamaldehyde (99%, Sigma-Aldrich, Munich,
Germany) dissolved in 100% ethanol was administered topi-
cally to the centre of the scan area using a 12 mm polypropyl-
ene coated aluminium Finn chamber on Scanpor tape (Bio-
diagnostics Ltd, Worcestershire) applied for a 10 minute
duration. The Finn chamber was removed and laser Doppler
flowmetry scans were then recorded for 30 minutes.
Whole-cell patch clamp electrophysiology. Whole-cell patch
clamp recordings were obtained using patch pipettes with
resistances of 1.5 to 3.0 MΩ when filled with the internal
solution, consisting of (in mM) 90 CsCl, 32 CsF, 10 HEPES,
10 EGTA, 10 Cs4 BAPTA, 1 MgCl2, 5 Mg ATP, and 0.1 Na GTP,
pH adjusted to 7.3 with CsOH. The external solution
consisted of (in mM) 132 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgCl2,
10 HEPES, and 5 Glucose, pH adjusted to 7.4 with NaOH. To
ensure complete dialysis with pipette solution, recordings
began 1 min after establishment of whole-cell configuration.
Subsequently, 150 μM cinnamaldehyde was perfused with the
external solution to activate TRPA1 channels. Currents were
recorded at room temperature with an Axopatch 200B ampli-
fier and filtered at 1 kHz with a low-pass Bessel filter. Voltage
protocols were elicited using Clampex 10.2 software and
consisted of 150 ms. Depolarizing ramps from −40 mV
followed by 100 ms steps to +40 mV and then to −40 mV re-
peated every 5 seconds from a holding potential of 15 mV.
Currents were digitized using a Digidata 1440A data acquisi-
tion interface and analyzed using Clampfit 10.2 software. For
these experiments the average steady state current at −40 mV
was analyzed for each test condition. For potency experi-
ments, cumulative concentration–responses were generated.
Individual concentration–response relationships were fitted
to a logistic equation with floating potency, slope and Emax.
Individual concentration-relationships were then normalized
to their fitted Emax to visualize all data on a single graph.
Sample collection schedule. Terminal PK samples were
taken from all animals via cardiac puncture at study
endpoint (180 min post oral dose). Blood samples were
centrifuged and resultant plasma samples were frozen at −20
°C prior to bioanalysis.
Equipment and supplies list. MoorLDI2 laser Doppler
imager (Moor Instruments Ltd, Axminster, UK).
Data analysis. All data design, randomization and analysis
were handled in accordance with previous discussions with
Non-Clinical Statistics group. In brief, area under the curve
(AUC) analysis was carried out for normalized blood flow fol-
lowing cinnamaldehyde challenge. AUC values were then
compared across groups using one-way ANOVA.
Acknowledgements
The authors would like to thank Neil Flanagan and Thean
Yeoh for the skin flux and skin retention data.
Notes and references
Plasmids and cell lines. Full-length human TRPA1
(hTRPA1)cDNA (Genbank accession Y10601) was cloned into
inducible vector pcDNA5/TO (Invitrogen, Carlsbad, CA). DNA
representing full-length opossum TRPA1 (oTRPA1, Mono-
delphis domestica XP001378427) was codon-optimized, syn-
thesized and cloned into pcDNA5/TO. The hS5 chimera was
generated by gene synthesis of human S5 DNA which was
cloned (using seamless cloning technology) into opossum
TRPA1 to replace oS5. The oS5 chimera was generated by syn-
thesis of opossum S5 DNA which was cloned into human
TRPA1 to replace hS5. This resulted in a 27 amino acid swap
such that for the hS5 chimera, hTRPA1 amino acids 867–893
replaced oTRPA1 amino acids 870–896. For the oS5 chimera,
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