α-Aryl pyrrolidine sulfonamides as TRPA1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2015.11.088

A series of α-aryl pyrrolidine sulfonamide TRPA1 antagonists were advanced from an HTS hit to compounds that were stable in liver microsomes with retention of TRPA1 potency. Metabolite identification studies and physicochemical properties were utilized as a strategy for compound design. These compounds serve as starting points for further compound optimization.

Full text of DOI:10.1016/j.bmcl.2015.11.088

Bioorganic & Medicinal Chemistry Letters 26 (2016) 495–498
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
a
-Aryl pyrrolidine sulfonamides as TRPA1 antagonists
⇑
Vishal A. Verma , Daniel G. M. Shore, Huifen Chen, Jun Chen, Steven Do, David H. Hackos,
⇑
Aleks Kolesnikov, Joseph P. Lyssikatos, Suzanne Tay, Lan Wang, Anthony A. Estrada
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of
a-aryl pyrrolidine sulfonamide TRPA1 antagonists were advanced from an HTS hit to com-
Received 21 September 2015
Revised 20 November 2015
Accepted 24 November 2015
Available online 25 November 2015
pounds that were stable in liver microsomes with retention of TRPA1 potency. Metabolite identification
studies and physicochemical properties were utilized as a strategy for compound design. These com-
pounds serve as starting points for further compound optimization.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
TRPA1
Metabolic stability
Ion channel
The non-selective cation channel transient receptor potential
ankyrin 1 (TRPA1) has been proposed as a polymodal irritant
sensor of noxious exogenous [e.g., cinnamaldehyde, allyl-isothio-
cyanate (AITC), acrolein] and endogenous [4-hydoxynonenal
(4-HNE), methylglyoxal, oxidized lipids] chemicals.1 It is expressed
predominantly in primary afferent nociceptive neurons in the
dorsal root ganglia (DRG), trigeminal ganglia (TG), and nodose
ganglia (NG).2 The cryo EM single particle structure of TRPA1
was recently solved (ꢀ4 Å), and revealed a homotetrameric config-
uration with at least 16 ankyrin repeats in the N-terminal domain.3
Bridging the ankyrin repeats and the transmembrane domains
(S1–S6) is a linker domain that possesses lysine and cysteine resi-
dues which play a critical role in channel activation through reac-
tion with electrophilic ligands.1 Multiple genetic studies have been
reported that suggest TRPA1-dependent roles in neuropathic
pain,4,5 asthma,6–8 and non-histaminergic itch.9,10 As a result,
many academic and industrial groups have reported progress in
the development of TRPA1 antagonists for the potential treatment
of a variety of clinical indications including compounds that have
progressed into the clinic.11–16
liability of this series, as the predicted hepatic clearance from both
human and rat liver microsomes was high. Metabolite identifica-
tion studies indicated that primary sites of metabolism were the
methylenes of the pyrrolidine ring as well as the aryl sulfonamide.
Sequential modifications were made in order to first probe the tol-
erance of these positions to change with respect to TRPA1 potency.
As such, a series of fluorinated pyrrolidine derivatives were
synthesized in order to mitigate oxidative metabolism of the
pyrrolidine methylenes as well as to decrease the logD of the
compounds. Fluorination at the 3 position of the pyrrolidine ring
led to an improvement in potency but no significant increase in
metabolic stability (2, Table 1). While the measured logD of the
4-fluoro compound 3 indicated a decreased lipophilicity that trans-
lated into moderately improved stability, it was accompanied by a
loss in potency. The 4-gem-difluoro target 4 was equipotent to 3-F
compound 2 but led to a loss of the moderate stability gained in 3.
The 5 position of the pyrrolidine was probed in order to investigate
a location for the installation of more polar groups to improve sta-
bility and solubility. Incorporation of a hydroxymethyl group at the
5 position (5) led to an increase in polarity as well as a small
increase in potency but did not exhibit any improvement in stabil-
ity. Nonetheless, these compounds indicated that mono-fluorina-
tion of the 3 position and di-fluorination of the 4 position on
various scaffolds have the potential to lead to potency increases
and so were envisioned to be combined with further modifications
to impart an overall increase in metabolic stability while
improving or maintaining potency.
As part of our own program targeting TRPA1 antagonists, com-
pound 1 was identified through a high-throughput screening
effort. In vitro evaluation of this compound indicated a moderate
potency of 0.11 lM against the human TRPA1 channel with a high
logD17 (4.4) resulting in a low lipophilic ligand efficiency (LLE) of
2.6. This series exhibited selectivity for TRPA1, as no activity was
measured against the TRPV1, TRPC6 and TRPM8 channels. Initial
efforts were directed towards improving the in vitro metabolic
The two fold increase in potency of the hydroxymethyl
compound 5 prompted the synthesis of various derivatives at this
position targeting compounds with improved physicochemical
⇑
Corresponding authors.
http://dx.doi.org/10.1016/j.bmcl.2015.11.088
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

496
V. A. Verma et al. / Bioorg. Med. Chem. Lett. 26 (2016) 495–498
Table 1
Table 2
TRPA1 antagonist logDs, potencies and liver microsome-predicted hepatic stability
TRPA1 antagonist logDs, potencies and liver microsome-predicted hepatic stability
data for pyrrolidine modifications
data for hydroxymethyl modifications
Compd Structure
logDa IC50
M)
HLM Clb
(mL/min/kg) (mL/min/kg)
RLM Clb
Compd Structure
logDa IC50 HLM Clb
RLM Clb
(
l
(l
M) (mL/min/kg) (mL/min/kg)
1
4.4 0.110
19
18
46
36
6
3.9
2.7
0.270 19
45
53
7
8
>20 12
2
3
4
3.9 0.020
3.7
4.3
>18 18
42
37
2.6 0.380
13
21
9
>20 18
4.2 0.023
19
19
45
40
a
pH 7.4.
b
Human and rat predicted hepatic clearance using liver microsomes using
the formula based on the well-stirred model: predicted hepatic Cl = (QH Â
f
ubLM Â Clint)/(QH + fubLM Â Clint) where QH is species liver blood flow, fubLM is
fraction unbound in liver microsomes and in the absence of measured microsomal
binding fubLM is assumed to equal 1.
5
3.3 0.063
a
Table 3
pH 7.4.
b
TRPA1 antagonist logDs, potencies and liver microsome-predicted hepatic stability
Human and rat predicted hepatic clearance using liver microsomes using the
data for sulfonamide modifications
formula based on the well-stirred model: predicted hepatic Cl = (QH Â
f
ubLM Â Clint)/(QH + fubLM Â Clint) where QH is species liver blood flow, fubLM is
Compd Structure
logDa IC50 HLM Clb
RLM Clb
fraction unbound in liver microsomes and in the absence of measured microsomal
binding fubLM is assumed to equal 1.
(
lM) (mL/min/kg) (mL/min/kg)
properties. Capping the hydroxyl group as the methyl ether (6) led
to a loss in potency (Table 2). Incorporation of basic amines as
replacements for the hydroxyl group were not tolerated, as the
N-methyl and N,N-dimethyl containing compounds 7 and 8 led
to complete loss of activity. The less basic trifluoroethylamine 9
also was not tolerated. Additionally, the increased polarity of some
of these compounds did not translate into improvements in meta-
bolic stability.
Attention was next turned to the substituted phenylsulfon-
amide, as metabolite ID studies indicated this was a site of
extensive metabolism. Targeting improved physicochemical
characteristics, replacement of the phenyl group with a pyridyl
functionality (10 and 11) led to loss of activity as well as no
improvement in stability (Table 3). Conversion of the sulfonamide
moiety to the amide 12 as well as the benzyl amine 13 also was not
tolerated.
10
1.1
2.0
>20
>20
20
18
53
43
11
12
3.1
4.0
>20
6.7
nm
nm
nm
nm
13
Derivatives of the substituents on the phenyl sulfonamide were
next explored. Removal of the methyl groups as well as the fluorine
by replacement with an unsubstituted phenyl group in 14 did lead
to a more polar compound as expected (logD of 2.2 compared to
3.3) but also led to complete loss of activity (Table 4). Removal
of only the 4-F substituent (15) led to a drop in potency while
excision of a single methyl group (16) led to only a two-fold drop
a
pH 7.4.
b
Human and rat predicted hepatic clearance using liver microsomes using the
formula based on the well-stirred model: predicted hepatic Cl = (QH Â
f
ubLM Â Clint)/(QH + fubLM Â Clint) where QH is species liver blood flow, fubLM is
fraction unbound in liver microsomes and in the absence of measured microsomal
binding fubLM is assumed to equal 1.

V. A. Verma et al. / Bioorg. Med. Chem. Lett. 26 (2016) 495–498
497
Table 4
in activity. However, removal of both methyl groups (in the C5
unsubstituted series) led to abrogation of potency (17). A key isos-
teric replacement of a methyl group with a chlorine in 18 indicated
no loss in activity and thus compound 19 was next synthesized,
replacing both methyl groups with chlorines. Compound 19 was
equipotent to 1 but critically exhibited a significant improvement
in metabolic stability in both human and rat liver microsomes. This
result was also notable in that it appeared to obviate the need for
fluoro substitution on the pyrrolidine ring for metabolic stability.
This 3,5-dichloro-4-fluoro phenyl sulfonamide moiety was also
placed on the initial pyrrolidine system lacking a C5 hydroxymethyl
(20) but led to a three fold loss in activity. Importantly, however,
compound 20 also displayed an increase in stability in the human
and rat microsome assays relative to compound 1, supporting the
trend that the chlorine for methyl substitutions are able to amelio-
rate the metabolic liabilities with this series. It should be noted that
it cannot be ruled out that potential differences in microsomal
binding could account for this stability increase.
TRPA1 antagonist logDs, potencies and liver microsome-predicted hepatic stability
data for aryl sulfonamide substituent modifications
Compd Structure
logDa IC50 HLM Clb
RLM Clb
(lM) (mL/min/kg) (mL/min/kg)
14
2.2
3.2
>19
20
53
28
15
0.290 19
0.190 18
16
17
2.8
3.2
37
The synthesis of compound 19 began from the ethyl ester of
N-Boc pyroglutamate (21, Scheme 1). Aryl Grignard addition gave
ketone 22, which was followed by Boc deprotection and
subsequent cyclization. Reduction to amino alcohol 23 was
accomplished with sodium borohydride. The diastereomers were
separated and formation of the sulfonamide then resulted in the
final compound 19.
6.6
nm
nm
Additional evaluation of compound 19 indicated selectivity for
the human TRPA1 channel over the rat channel as was typical for
this series (Table 5). Moderate improvement of the logD from
the initial hit had been achieved, resulting in a modest increase
in the LLE. Hepatic clearance calculated from human and rat liver
microsomes indicated relative stability in this in vitro assay. Poten-
tially as a result of its moderate logD, compound 20 was mem-
brane permeable, highly plasma protein bound and exhibited low
kinetic aqueous solubility.
The progress described herein represents advancement of an
HTS hit guided by physicochemical properties and utilizing both
TRPA1 potency as well as in vitro evaluation of metabolic stability
in liver microsomes to arrive at starting points for further com-
pound optimization. Steep structure activity relationships in terms
of both potency and metabolic stability have been established for
many positions of these compounds. Areas for further refinement
include increasing potency through increased LLE as well as
increasing solubility by driving down logD.
18
19
3.6
3.8
4.6
0.130 19
40
13
12
0.110 7.3
20
0.350 6
a
pH 7.4.
b
Human and rat predicted hepatic clearance using liver microsomes using the
formula based on the well-stirred model: predicted hepatic Cl = (QH Â
f
ubLM Â Clint)/(QH + fubLM Â Clint) where QH is species liver blood flow, fubLM is
Table 5
fraction unbound in liver microsomes and in the absence of measured microsomal
binding fubLM is assumed to equal 1.
In vitro and physicochemical properties of compound 19
Human TRPA1 IC50
(
l
M)
0.11
7.3
3.9
3.1
7.3
13
5.7
99.4
<1
Rat TRPA1 IC50
logD (pH 7.4)
LLE
(lM)
HLM Cla (mL/min/kg)
RLM Cla (mL/min/kg)
MDCK (10À6 cm/s)
Human PPB (%)
Kinetic aqueous solubility (lM)
a
Human and rat predicted hepatic clearance using liver microsomes using the
formula based on the well-stirred model: predicted hepatic Cl = (QH Â
Scheme 1. Synthesis of target compound 19. Reagents and conditions: (a) 4-F-
PhMgBr (1.2 equiv), 2-MeTHF, 0 °C, 30 min (91%); (b) TFA (4.0 equiv), CH2Cl2, 40 °C,
3 h; (c) NaBH4 (6.0 equiv), MeOH (70% over two steps); (d) separation; (e) ArSO2Cl
(1.5 equiv), Et3N (2.0 equiv), CH2Cl2, 16 h (39%).
f
ubLM Â Clint)/(QH + fubLM Â Clint) where QH is species liver blood flow, fubLM is
fraction unbound in liver microsomes and in the absence of measured microsomal
binding fubLM is assumed to equal 1.

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V. A. Verma et al. / Bioorg. Med. Chem. Lett. 26 (2016) 495–498
8. Nassini, R.; Materazzi, S.; Andre, E.; Sartiani, L.; Aldini, G.; Trevisani, M.;
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