Identification of potent, soluble, and orally active TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.01.112

Optimization of a water soluble, moderately potent lead series of isoxazole-3-carboxamides was conducted, affording a compound with the requisite balance of potency, solubility and physicochemical properties for in vivo use. Compound 8e was demonstrated to be efficacious in a rat model of inflammatory pain, following oral administration.

Full text of DOI:10.1016/j.bmcl.2011.01.112

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2559–2563
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent, soluble, and orally active TRPV1 antagonists
Paul Ratcliffe a, , John Maclean a, , Lynn Abernethy b, Tom Clarkson a, Maureen Dempster c,
Anna-Marie Easson a, Darren Edwards a, Katy Everett a, Helen Feilden a, Peter Littlewood b,
Duncan McArthur a, Deborah McGregor c, Hazel McLuskey c, Olaf Nimz a,
⇑
⇑
Lesley-Anne Nisbet a, Ronnie Palin a, Heather Tracey b, Glenn Walker c
a Department of Chemistry, MSD, Newhouse, Lanarkshire ML1 5SH, UK
b Department of Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
c Department of Molecular Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of a water soluble, moderately potent lead series of isoxazole-3-carboxamides was con-
ducted, affording a compound with the requisite balance of potency, solubility and physicochemical
properties for in vivo use. Compound 8e was demonstrated to be efficacious in a rat model of inflamma-
tory pain, following oral administration.
Received 15 December 2010
Revised 24 January 2011
Accepted 25 January 2011
Available online 31 January 2011
Ó 2011 Published by Elsevier Ltd.
Keywords:
TRPV1 antagonist
Antihyperalgesia
Isoxazoles
The TRPV1 (transient receptor potential vanilloid 1) receptor is
a well-characterized member of the transient receptor potential
(TRP) superfamily of ion channels.1–3 TRPV1 is located in both
the periphery and CNS on C and Ad fibres, the afferents commonly
associated with nociception, and its role in the transmission of pain
signaling has been extensively discussed.4,5 In addition to mediat-
ing the effects of exogenous capsaicin (the pungent component of
chilli peppers) primary afferent TRPV1 receptors are thought to
trigger the actions of heat (>43 °C), and protons (pH <6.8) and are
modulated by a variety of endogenous lipid mediators including
anandamide and bradykinin.6 Consequently, TRPV1 is believed to
act as an integrator of nociceptive responses to both chemical
and thermal noxious stimuli.7
TRPV1 knockout mice provided evidence that the channel
played a key role in pain, with a clear attenuation of thermal
hyperalgesia in response to proinflammatory agents.8 TRPV1 also
shows increased expression in pain states in both rat and human.9
In addition to the treatment of inflammatory pain, evidence sug-
gests that a number of other disorders, including urinary urge
incontinence, cough, and irritable bowel syndrome, may be treat-
able through modulation of TRPV1 signaling.10
for the treatment of pain.11 This has lead to several novel com-
pounds entering clinical trials as analgesic agents.12 The outcomes
of these trials have been discussed in detail on a number of occa-
sions,11,12 however to date no clear evidence of analgesic efficacy
has been demonstrated in man. Furthermore, despite being viewed
as potentially the next generation of pain therapeutics, the obser-
vations that TRPV1 plays a role in maintenance of body tempera-
ture, and that blockade of the receptor can cause hyperthermia
both pre-clinically and in humans, has resulted in the termination
of at least one clinical trial because of dose-limiting hyperthermia
that occurred at sub-therapeutic doses (Fig. 1).11
We have previously described the discovery of a novel isoxaz-
ole-3-carboxamide series of TRPV1 antagonists, which was opti-
mized to a
potent, selective and efficacious compound 1.13
Unfortunately, 1 suffered from poor solubility and high plasma
protein binding. This was further optimized to compound 2 which
had greatly improved solubility and lower plasma protein binding,
albeit with reduced in vitro potency.14
In this Letter, we report on the optimization of compound 2,
focusing in particular on our approaches to target TRPV1 antagonists
with increased potency, while maintaining the improved level of
solubility, as well as favorable analgesic and pharmacokinetic
properties.
Over recent years, a substantial effort has been made across all
sectors of the pharmaceutical industry to target TRPV1 antagonists
In an effort to increase potency while retaining solubility, we
set about expanding the structure–activity relationship around
the amine region of the isoxazole (Fig. 2). A general synthesis of
the isoxazole-3-carboxamides has been reported previously,13,14
⇑
Corresponding authors.
E-mail addresses: Paul.Ratcliffe@grunenthal.com (P. Ratcliffe), john.maclean@
merck.com (J. Maclean).
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.01.112

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P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2559–2563
NH
Cl
O
O
F3C
F3C
N
N
H
OH
O
N
H
OH
O
N
1
2
Figure 1. Structures of progenitor examples 1 and 2.
(Table 1, Fig. 3). Introduction of methyl 8a or ethyl 8b units mod-
erately improved TRPV1 potency (pIC50’s of 7.6) without effecting
solubility, albeit at the expense of a log unit increase in lipophilic-
ity (e Log D).16 Retaining a secondary amine, we began to probe the
steric constraints around the amine nitrogen, the effect of varia-
tions in lipophilicity/polarity and of course changes which would
influence amine basicity. As is illustrated by 8c, 8d, 8i and 8j, con-
straining the isopropyl group in the form of a cycloalkyl or hetero-
alkyl group was tolerated, and indeed for 8c lead to improvement
in TRPV1 potency, showing a pIC50 of 8.5. Significantly, the e Log D
of these compounds was increasing with potency. An extreme
example was provided by the introduction of the 1,1,1-trifluoro-
propan-2-amine moiety 8h, which increased potency dramatically
to furnish our most active compound to date (pIC50 of 9.8) but lost
solubility, probably as a consequence of both reducing the amine
basicity and increasing lipophilicity. Noting that polarity in the
form of ether functionality was tolerated (8i–j with pIC50’s of 7.9
and 7.1, respectively), the potential to balance potency and lipo-
philicity was further probed by synthesizing compounds 8k–s.
While the sulfone moiety present in 8q and the amide moiety in
8s furnished compounds with reduced TRPV1 antagonist activity,
there appeared to be tolerance for a wide range of functionalities,
especially for hydroxyl groups, as well as cyclic and acyclic ethers,
leading to compounds that combined good levels of potency and
solubility, for example, 8l and 8n with pIC50’s of 8.6 and 8.5 and
solubilities of 79 and 89 mg/L, respectively.17 Finally, as can be
seen from 8e to 8g, a happy compromise of our desired properties
could also be achieved through subtle modification of the alkyl
group.
1
2
R R N
O
F3C
N
H
O
N
OH
Figure 2. Generic scaffold of examples 8a–s.
however for the purposes of probing this polar region more exten-
sively an alternative route was established (Scheme 1). Starting
from compound 3,14 simple bromination of the pendant methyl
substituent was achieved in near quantitative yield using N-bro-
mosuccinimide in the presence of benzoyl peroxide to give com-
pound 4. Treatment with trifluoroacetic acid in the presence of
water furnished the hydroxy acid, compound 5, which could be re-
acted with thionyl chloride in toluene and DMF to simultaneously
furnish the acid chloride and transform the hydroxyl moiety into
the desired alkyl halide 6. Amide formation was achieved using
the appropriate amine in the presence of triethylamine and DCM
which furnished the important precursor, compound 7, for probing
tolerance to amine containing compounds 8a–s.
Compounds were evaluated for their ability to modulate influx
of Ca2+ in cells (CHO-K1) stably expressing human TRPV1 (VR1)
using a Molecular Devices FLIPR. TRPV1 agonists stimulated intra-
cellular fluorescence when applied to the channel, while antago-
nists inhibited the fluorescent response when co-applied with an
agonist. Thus the same TRPV1 transfected cell line was used in
both agonist and antagonist assays. Results are reported as pIC50
and are an average of at least two independent experiments in
duplicate.15
As illustrated in Table 2, a number of compounds were selected
for in vitro ADME and hERG profiling. Apart from 8f and 8h, they
demonstrated good stability in human hepatic microsomes in vi-
tro. In some cases there did appear to be a disconnect between
Initial investigations centered on subtle modifications to com-
pound 2, probing tolerance to the presence of a tertiary amine
Br
OH
O
O
O
OEt
OEt
OH
(a)
(b)
N
N
N
98%
86%
O
O
O
F3C
F3C
F3C
3
4
5
O
S
(c)
Cl
Cl
100%
Cl
O
Cl
O
O
1
2
Cl
R R N
(e)
(d)
N
H
OH
N
H
OH
N
61%
N
O
F3C
N
O
F3C
O
F3C
8a-s
7
6
Scheme 1. Reagents and conditions: (a) N-bromosuccinimide, benzoyl peroxide, CCl4, 88 °C, 4 h, (98%); (b) TFA, water, microwave, 15 min, 150 °C, (86%); (c) thionyl chloride,
toluene, DMF, 90 °C, 4 h, (100%); (d) (1R, 3S)-3-aminocyclohexanol, Et3N, DCM; rt, 2 h, (61%); (e) NHR1, DIPEA, acetonitrile, microwave, 15 min, 165 °C, (7–68%).

P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2559–2563
2561
Table 1
SAR of amine modifications; effect on potency and solubility
Table 1 (continued)
Compd Structure
TRPV1 pIC50
5.9
Solkin (mg LÀ1
)
e Log D
Compd Structure
TRPV1 pIC50
Solkin (mg LÀ1
)
e Log D
O
S
8q
26
3.6
3.6
2
7.2
82
3.0
NH
O
N
N
O
8r
8.3
5.5
31
30
8a
7.6
7.6
8.5
80
85
21
4.0
4.1
4.5
N
O
H2N
8b
8s
3.6
N
N
*
ND—not determined.
8c
NH
8d
7.8
9.0
26
78
4.3
4.3
NH
O
N
F3C
H
N
OH
O
NH
8e
Figure 3.
NH
8f
8.1
8.9
77
3.5
4.4
the in vitro stabilities in human and rat microsomes with com-
pounds 8e, 8g and 8n having significantly reduced stability in rat
compared to human, but compounds 8c, 8f and 8h having compa-
rable stability across species. Apart from compound 8g, the
remaining compounds in Table 2 displayed similar or lower affinity
for the hERG receptor, and all demonstrated moderate to good lev-
els of unbound compound as illustrated by the rat and human plas-
ma protein binding assays.
Based on the combination of TRPV1 antagonist potency, inher-
ent solubility and favourable overall physicochemical characteris-
tics, compounds 8e and 8l were selected for further ADME
profiling. As demonstrated in Table 3, compound 8l has low to
moderate in vivo clearance in rat, achieves a moderate plasma
half-life of 4.3 h, but suffers from poor oral bioavailability (4%).
Pleasingly, compound 8e demonstrated improved oral bioavail-
ability (33%) as compared to compound 2. Furthermore, the rat
in vitro and in vivo clearance (Tables 2 and 3) appear to correlate
well, with compound 8e having a relatively high rate of metabolic
clearance, as anticipated due to its low in vitro microsomal stabil-
ity and because its lower plasma protein binding, as compared to
8l, leads to a larger free fraction in rat. Perhaps as a consequence
of the extensive volume of distribution a moderate half-life of
4 h was also observed.
NH
8g
ND*
F3C
O
8h
8i
9.8
7.9
3.5
6.6
5.6
4.5
NH
N
O
8j
7.1
8.0
82
87
3.7
4.7
N
HO
CF3
8k
NH
OH
8l
8.6
8.0
79
81
4.2
4.1
NH
OH
Overall, compound 8e demonstrated an acceptable pharmacoki-
netic profile sufficient to proceed to in vivo efficacy testing in a rat
model of inflammatory pain. The efficacy of compound 8e was
evaluated in the capsaicin Hargreaves assay, a functional in vivo
assay of TRPV1 induced thermal hyperalgesia in rats.18 In the cap-
8m
NH
saicin Hargreaves model, 8e (3, 10, 30
ment) significantly reversed the thermal hyperalgesia induced by
capsaicin, with an MED of 30 mol/kg (see Fig. 4).
lmol/kg; po; 2 h pre-treat-
HO
8n
8o
8p
8.5
6.9
8.1
89
80
83
3.5
3.7
4.0
NH
l
In summary, a series of isoxazole derivatives was optimized
through a systematic SAR study of the tolerance to varying degrees
of polarity at the isoxazole 4-position. This led to the identification
of compounds with a better balance of solubility, physicochemical
properties and in vitro potency. In particular compounds 8e and 8l
were identified as the most promising examples from this series
and both were progressed into animal studies. The improved oral
bioavailability of compound 8e lead to its advancement into effi-
cacy testing where it showed statistically significant attenuation
O
NH
O
N

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P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2559–2563
Table 2
In vitro PK, hERG and plasma protein binding data for selected compounds
Compd
HLM CLint
l minÀ1 mgÀ1
RLM CLint
l minÀ1 mgÀ1
Rat Hep. CLint
l minÀ1106 cellsÀ1
hERG pKi
(% inhibition at 100 lM)
PPB%Bound
(Human)
PPB% Bound
(Rat Wistar)
(
l
)
(
l
)
(
l
)
2
<12
17
12
48
21
66
12
12
<12
<12
107
55
144
49
<6
<6
12
5.2
75
72.9
93.6
75.8
ND*
ND*
96.3
88.4
83.2
8c
8e
8f
8g
8h
8l
(75)
(86)
5.3
88.2
82.3
ND*
ND*
95.2
74.4
63.9
ND*
ND*
ND*
ND*
10
5.9
(59)
(83)
(69)
41
93
8n
*
ND—not determined.
Table 3
In vivo PK, for selected compounds. All compounds were dosed at 10 mg/kg (po) and 2 mg/kg (iv)
Compd
CL (ml minÀ1 kgÀ1
)
Vss (L kgÀ1
)
T1/2 (h)
po
po
%F
Cmax
(lM)
Tmax (h)
2
8e
8l
12.5
69
13.5
5.9
7.6
3.5
7.9
4.0
4.3
0.72
0.42
0.23
2
2
1.5
20
33
4
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Figure 4. The effects of po administration of 8e (3, 10, 30 lmol/kg) on paw
withdrawal latency (PWL) in the capsaicin Hargreaves assay.
of the acute inflammatory thermal response in the rat capsaicin
Hargreaves assay (p <0.01). We believe 8e will be a valuable tool
in exploring the potential role of TRPV1 antagonists in pain, and
in potentially elucidating and interpreting the relationship be-
tween TRPV1 antagonism and hyperthermia.
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K.-K.; Kultgen, S.; Littlewood, P.; McArthur, D.; McGregor, D.; McLuskey, H.;
Neagu, I.; Nimz, O.; Nisbet, L.-A.; Ohlmeyer, M.; Pham, Q.; Ratcliffe, P. D.; Rong,
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14. Ratcliffe, P. D.; Abernethy, L.; Ansari, N.; Cameron, K.; Clarkson, T.; Dempster,
M.; Dunn, D.; Easson, A-M.; Edwards, D.; Everett, K.; Feilden, H.; Ho, K.-K.;
Kultgen, S.; Littlewood, P.; McArthur, D.; McGregor, D.; MacLean, J.; McLuskey,
H.; Neagu, I.; Nimz, O.; Nisbet, L-A.; Ohlmeyer, M.; Palin, R.; Pham, Q.; Rong, Y.;
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Acknowledgments
We would like to thank our colleagues in the Analytical section
for structure and purity determination of all compounds, through-
out the duration of the work described in this article (2007–2008).
References and notes
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and 0.5 mM Probenecid), at room temperature for 1 h (25
this, 12.5 of test compound, prepared in assay buffer containing 4%
dimethylsulfoxide, was added by the FLIPR (Molecular Devices, Corp) to each
ll/well). Following
l
l

P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2559–2563
2563
well and the subsequent change in the fluorescence of the cells measured to
assess agonist activity. Ten minutes after test compound addition, 12.5 l of 30
mixture was then vortex mixed (1500 rpm) for 24 0.5 h at 21 2 °C. After
mixing, the resultant solution/suspension was filtered under vacuum using a
l
nM capsaicin, prepared in assay buffer, was added to each well and the change
in the fluorescence of the cells measured again. In this way, the same assay was
used to assess both the agonist activity and antagonist activity of test
compounds.
filter plate (Millipore Multiscreen HTS, 0.4 lM). The concentration of the
compound in the filtrate was determined by High Performance Liquid
Chromatography (HPLC) running a generic acid gradient method with UV
detection at 230 nm. Peak areas from analysis of the diluted filtrates were
quantified by comparison to a calibration line prepared by injecting onto the
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HPLC three different volumes of a 50 lM solution of the test compound in
17. Aqueous solubilities were determined using a medium-throughput adaptation
of a shake-flask methodology, known as the ‘SolKin’ method. A 10 mM solution
of the test compound in DMSO was added to 0.05 M phosphate buffered saline
pH 7.4 such that the final concentration of DMSO was 2%. The resultant
DMSO. Solubilities were determined in duplicate for each test compound and
average values reported.
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