Discovery of a series of aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl) benzamides as TRPA1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.05.013

We describe the discovery and advancement of a novel series of TRPA1 antagonist having an aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamide scaffold. The physical and in vitro DMPK profiles are discussed.

Full text of DOI:10.1016/j.bmcl.2014.05.013

Accepted Manuscript
Discovery of a series of aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)ben-
zamides as TRPA1 antagonists
Sébastien Laliberté, Frédéric Vallée, Pierre-André Fournier, Leanne Bedard,
Jean Labrecque, Jeffrey S. Albert
PII:
S0960-894X(14)00500-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.013
BMCL 21626
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 February 2014
4 May 2014
5 May 2014
Please cite this article as: Laliberté, S., Vallée, F., Fournier, P-A., Bedard, L., Labrecque, J., Albert, J.S., Discovery
of a series of aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamides as TRPA1 antagonists, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.013
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of a series of aryl-N-(3-(alkylamino)-5-
(trifluoromethyl)phenyl)benzamides as TRPA1 antagonists
Sébastien Lalibertéa, Frédéric Vallée a, Pierre-André Fourniera, Leanne Bedardb, Jean Labrecquec and
Jeffrey S. Albertd
hTRPA1
hTRPA1
IC50 0.2.7 M
IC50 0.091 M

1
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of a series of aryl-N-(3-(alkylamino)-5-
(trifluoromethyl)phenyl)benzamides as TRPA1 antagonists
Sébastien Lalibertéa, Frédéric Valléea, Pierre-André Fourniera, Leanne Bedardb, Jean Labrecquec and
Jeffrey S. Albertd*ǂ
aAstraZeneca/OmegaChem, Department of Medicinal Chemistry, Montréal H4S 1Z9, Canada
bAstraZeneca, DMPK, Montréal H4S 1Z9, Canada
cAstraZeneca, Department of Bioscience, Montréal H4S 1Z9, Canada
dAstraZeneca, Department of Chemistry, Montréal H4S 1Z9, Canada
*Corresponding author: Tel.: 514.312.2503, Jeffrey.Albert@IntelliSynRD.com
ǂ Present address: Jeffrey Albert; IntelliSyn R&D; 7171 Frederick-Banting, Montreal, QC, H4S 1Z9, CANADA; Jeffrey.Albert@IntelliSynRD.com
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
We describe the discovery and advancement of a novel series of TRPA1 antagonist having an
aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamide scaffold. The physical and in
vitro DMPK profiles are discussed.
Accepted
Available online
2013Elsevier Ltd. All rights reserved.
Keywords:
TRPA1
antagonist
Ligand gated ion channel
Transient receptor potential ankyrin 1 (TRPA1) is a non-
selective cation channel that is implicated in many aspects of
sensation, including pain. Its restricted expression in sensory
neurons and reported up-regulation following nerve injury are
consistent with a role in pain. Recently, a mutation in the
TRPA1 gene was found to be associated with Familial Episodic
Pain Syndrome.1 The role of TRPA1 in pain is further supported
in various animal models showing reversal of pain behaviors in
High throughput screening for antagonists of TRPA1 led to
the identification of a group of related trifluoromethylaryl-
thiophene-carboxamides as exemplified by 1, 2, and 3 (Table 1,
Figure 1). We initially focused on strategies to increase the
ligand efficiencies12 and reduce the lipophicities (cLogP). We
explored this chemotype by first modifying and truncating
substitution at the 3-position. Changing the secondary amine
functionality to a carbamate (4) was tolerated and served to
improve lipophilicity while tertiary amine analog (5) was inactive
(IC50 >32 M). Further exploration with small secondary alkyl-
substituted anilines led to the identification of 8 and 9 as the
series’ most potent antagonists (IC50 0.39 and 0.29 M,
respectively). Ligand efficiencies were significantly improved
for these smaller compounds; however, lipophilicity still required
improvement.
the rodent by antagonist administration.2-4
Glenmark has
initiated Phase-2 trials with GRC-17536 in two studies, one for
neuropathic pain and another for asthma. Hydra/Cubist has
advanced CB-189625 into Phase-1 for nociceptive pain. Other
therapeutic applications for TRPA1 antagonists that have been
explored include treatments for airway disorders5-6 and bladder
oversensitization.7-8 Various groups have explored different
chemical series as TRPA1 antagonists and this area has been
described in several reviews.9-11 Among the challenges in this
area are that many demonstrated TRPA1 antagonists have one or
more of the following liabilities: high lipophilicity, high
molecular weight, and low solubility. Here we describe a novel
series of TRPA1 antagonists with high in vitro potency and
improved physicochemical properties.
Figure 1.Generic structure of the series identified.

2
We then evaluated changes to the carboxamide substituent
(Table 2). The thiophene could be replaced by a phenyl group
(10), resulting in approximately 2-fold increase in potency
relative to 8. Additionally, 3-pyridyl 12 and 4-pyridyl 13 were
tolerated, albeit with a reduced potency, whereas 2-pyridyl 11
was not tolerated (IC50 >32 M). It was encouraging to find that
potency could be retained for some of the pyridyl analogs while
driving lipophilicity in the right direction. Next, a number of
mono and bis-substituted phenyl analogs were prepared.
Table 2.
trifluoroaryl-N-propyl-carboxamides.
Antagonist potency for various 1-substituted
Derivatives 14 and 15 had reduced activity in comparison to
10; however, the 2,6-bis-fluorinated derivative, 16 (IC50 0.11
M), was the most active compound yet seen.
F
F
F
5
N
R
3
1
H
Table 1. Antagonist potency for 3-substituted trifluoroaryl-
thiophene-carboxamides.
IC50
Compd
R
LE
cLogP
4.8
(M)a
F
F
F
1
O
5
N
H
10
0.20
>32
0.94
2.6
0.40
O
S
R
N
3
O
O
O
H
N
N
H
11
12
13
14
4.3
3.9
3.9
5.0
IC50
AZ
R
LE
cLogP
4.5
(M)a
N
H
N
N
0.36
0.33
0.33
S
N
H
1
2.7
0.30
N
H
N
H
2
2.9
0.29
4.9
O
N
1.4
3
4
5
3.9
2.3
0.27
0.32
--
5.5
3.8
6.1
H
N
F
H
O
O
F
F
N
H
O
N
H
15
1.2
0.32
0.38
5.1
O
F
N
>32
N
16
17
0.11
>32
4.3
2.5
H
F
6
1.3
0.33
5.2
N
H
-NH2
a Results (human receptor) are the mean of at least 2 experiments.
Experimental errors are within 20% of value based on replicate
measurements.
N
H
7
8
9
0.79
0.39
0.29
0.36
0.40
0.40
4.9
4.5
4.3
N
H
Table 3. Antagonist potency for R1 and R2 modifications.
N
H
R2
a Results (human receptor) are the mean of at least 2 experiments.
Experimental errors are within 20% of value based on replicate
measurements.
O
F
F
R1
N
H
Based on the results for compound 16, we maintained the 2,6-
bis-fluoaryl amide group and surveyed other N3-substitued
anilines.
The carboxamide group appears essential as
evidenced by the inactivity of 17. Analogs with small alkyl
groups (e.g. 18 and 19) retained similar, or possibly stronger
potency as 16 (Table 3). N-alkylation of the aniline (affording
20) and preparation of the oxo derivatives (21) both led to
complete loss of detectable activity, underlying the importance of
the NH group of that aniline. Replacement of the CF3 group by
a CH3 group led to a 17-fold reduction in potency (22).
IC50
Cmpd
R1
R2
LE cLogP
(M)a
16
18
19
20
21
22
PrN(H)-
EtN(H)-
iPrN(H)-
PrN(Me)-
PrO-
CF3
CF3
CF3
CF3
CF3
CH3
0.11
0.38
0.4
4.3
3.8
4.1
4.9
4.6
3.4
0.074
0.091 0.38
>32
>32
PrN(H)-
1.9
0.35

3
a Results (human receptor) are the mean of at least 2 experiments.
Experimental errors are within 20% of value based on replicate
measurements.
microsomes supplemented with 5 mM GSH, as detected by MS/MS
using precursor ion scan. Result shows the molecular weight added
h
to the parent-glutathione adduct of the primary metabolite.
available.
Not
The most potent antagonists in the series were further profiled
and the results are shown in Table 4. The compounds profiled
demonstrate significantly improved potencies while retaining
high ligand efficiency and acceptable lipophilicity as compared
to 1. However, characterization of selected antagonists for their
in vitro DMPK properties revealed a number of key liabilities.
Clearance in human liver microsomes is much higher than
desired and human plasma protein binding is high (< 1% free).
Moreover, every tested compound showed relatively poor
solubility in aqueous media as well as low to moderate cell
permeability as exemplified by the low Caco-2 permeability. In
terms of drug-drug interaction potential, both compounds tested
proved to be potent competitive inhibitors of CYP1A2, and
analogue 10 demonstrated significant time-dependent inhibition
(TDI) of CYP 2D6. Also, compound 10 showed a potential for
metabolic activation as GSH adducts were detected upon
incubation in human liver microsomes. Interestingly, the
fluorinated analogue, 16, did not show these issues as no CYP
TDI activity was observed and no GSH adducts were observed
upon incubation, suggesting the unsubstituted phenyl group was
related to both problems. Further reductions in lipophilicity may
serve to address the physiochemical properties issues like the
poor solubility, the high hCLint and high plasma protein binding.
Overall, this novel high affinity series of TRPA1 antagonists may
serve as a starting point for lead optimization.
Supplementary Material
Supplementary information (representative experimental
conditions for the synthesis of compounds (10, 16, 18 and 19)
and characterization data, as well as testing assay protocols can
be found in the online version at ???
References
(1)
Kremeyer, B.; Lopera, F.; Cox, J. J.; Momin, A.;
Rugiero, F. et al. A gain-of-function mutation in
TRPA1 causes familial episodic pain syndrome. Neuron
2010, 66, 671.
(2)
(3)
Cai, X. A new tr(i)p to sense pain: TRPA1 channel as a
target for novel analgesics. Expert Rev. Neurother.
2008, 8, 1675.
Bang, S.; Hwang, S. W. Polymodal ligand sensitivity of
TRPA1 and its modes of interactions. J. Gen. Physiol.
2009, 133, 257.
(4)
(5)
Jordt, S.-E. Trigeminal TRPs and the scent of pain.
Pain 2011, 152, 4.
Bessac, B. F.; Jordt, S.-E. Breathtaking TRP channels:
TRPA1 and TRPV1 in airway chemosensation and
reflex control. Physiology 2008, 23, 360.
Table 4. Physical and DMPK properties of selected compounds.
(6)
Caceres, A. I.; Brackmann, M.; Elia, M. D.; Bessac, B.
F.; del Camino, D. et al. A sensory neuronal ion channel
essential for airway inflammation and hyperreactivity in
asthma. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 9099.
Everaerts, W.; Gevaert, T.; Nilius, B.; De Ridder, D. On
the origin of bladder sensing: Tr(i)ps in urology.
Neurourol. Urodyn. 2008, 27, 264.
Skryma, R.; Prevarskaya, N.; Gkika, D.; Shuba, Y.
From urgency to frequency: facts and controversies of
TRPs in the lower urinary tract. Nat. Rev. Urol. 2011, 8,
617.
Compd
hTRPA1 IC50 (M)
LE
10
0.20
0.4
4.8
8
154
<1
6.2
16
0.11
0.38
4.3
7
96
<1
2.7
18
0.074
0.4
3.8
5
19
0.091
0.38
4.1
1
73
<1
6.9
cLogP
(7)
(8)
Solubilitya (M)
hCLintb (μL/min/mg)
Human PPBc (%free )
Caco-2d
115
<1
nah
(1E-6 cm/s)
CYP Competitive
Inhibitione IC50 (μM)
CYP TDIf, %shift
0.14
(1A2)
35
(2D6)
+18
1.5
(1A2)
None
--
--
--
--
--
--
(9)
Strassmaier, T.; Bakthavatchalam, R. Transient receptor
potential A1 modulators. Curr. Top. Med. Chem. 2011,
11, 2227.
GSH adduct
-2
formation in HLMg
a
(10)
Rech, J. C.; Eckert, W. A., III; Maher, M. P.; Banke, T.;
Bhattacharya, A. et al. Recent advances in the biology
and medicinal chemistry of TRPA1. Future Med. Chem.
2010, 2, 843.
Thermodynamic solubility measured in a pH 7.4 buffer using
b
HPLC with UV detection for quantification. Metabolic stability
performed in human liver microsomes (1 mg/mL) in presence of
c
NADPH using test compound at 1 μM. Protein binding of test
compound at 10 μM in human plasma, as determined by equilibrium
(11)
(12)
Baraldi, P. G.; Preti, D.; Materazzi, S.; Geppetti, P.
Transient Receptor Potential Ankyrin 1 (TRPA1)
Channel as Emerging Target for Novel Analgesics and
Anti-Inflammatory Agents. J. Med. Chem. 2010, 53,
5085.
d
dialysis.
Transwell assay. Apparent permeability (apical to
basolateral) across a Caco-2 cell monolayer measured at pH 7.4
e
using test compound at 10 μM applied on the apical side.
Competitive CYP450 (1A2, 2C9, 2C19, 2D6, 3A4) inhibition
screening assay in human liver microsomes, using standard probe
substrates and detected by LC-MS/MS. f CYP450 (1A2, 2C9, 2D6,
3A4) time-dependent inhibition IC50 shift screening assay in human
liver microsomes at 10 μM test compound. TDI risk is considered
LE = Ligand efficiency. Here we define LE = -
RT(ln(IC50))/(heavy atom count).
significant if % shift is >20%; data for compounds that meet this
g
criterion are shown.
GSH adduct formation in human liver