Discovery of potent, soluble and orally active TRPV1 antagonists. Structure-activity relationships of a series of isoxazoles

Article abstract of DOI:10.1016/j.bmcl.2011.01.051

Systematic optimisation of a poorly soluble lead series of isoxazole-3-carboxamides was conducted. Substitution of the 4-position with specific polar functionality afforded the requisite balance of potency, solubility and physicochemical properties. Compo

Full text of DOI:10.1016/j.bmcl.2011.01.051

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4652–4657
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent, soluble and orally active TRPV1 antagonists.
Structure–activity relationships of a series of isoxazoles
Paul Ratcliffe a, , Lynn Abernethy a, Nasrin Ansari b, Ken Cameron a, Tom Clarkson a, Maureen Dempster a,
⇑
David Dunn b, Anna-Marie Easson a, Darren Edwards a, Katy Everett a, Helen Feilden a, Koc-Kan Ho b,
Steve Kultgen b, Peter Littlewood a, John Maclean a, Duncan McArthur a, Deborah McGregor a,
Hazel McLuskey a, Irina Neagu b, Olaf Nimz a, Lesley-Anne Nisbet a, Michael Ohlmeyer b,
Ronnie Palin a, Quynhchi Pham b, Yajing Rong b, Andrew Roughton b, Melanie Sammons a,
Robert Swanson b, Heather Tracey a, Glenn Walker a
a Merck Research Laboratories, MSD, Newhouse, Motherwell, Lanarkshire ML1 5SH, UK
b Pharmacopeia, PO Box 5350, Princeton, NJ 08543, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Systematic optimisation of a poorly soluble lead series of isoxazole-3-carboxamides was conducted. Sub-
stitution of the 4-position with specific polar functionality afforded the requisite balance of potency, sol-
ubility and physicochemical properties. Compound 21a was found to be efficacious in the rat Capsaicin
Hargreaves assay following oral administration.
Received 8 November 2010
Revised 12 January 2011
Accepted 13 January 2011
Available online 26 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
TRPV1 antagonist
Antihyperalgesia
Isoxazoles
The TRPV1 (transient receptor potential vanilloid 1) receptor is
a well-characterised member of the transient receptor potential
(TRP) superfamily of ion channels.1–3 TRPV1 is located both in
the periphery and CNS on C and Ad fibres, the afferents commonly
associated with nociception, and its role in the transmission of pain
signalling 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 includ-
ing 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 signalling.10
This level of target validation prompted a subsequent flurry of
activity within the pharmaceutical industry.11 This effort has led
to the identification of several TRPV1 antagonists that have entered
clinical trials as analgesic agents, and in recent times resulted in
the emergence of clinical data for a number of TRPV1 antago-
nists.12 The outcomes of these trials have been discussed in detail
on a number of occasions already,11,12 however to date no clear
evidence of analgesic efficacy has been demonstrated in man.
Should these new chemical entities relieve symptoms of chronic
pain in man then this class of compounds may offer one of the first
novel mechanisms, in many years, for the treatment of pain. How-
ever, the sensitivity of TRPV1 to heat has suggested a role in main-
tenance of body temperature, and clinical trials of at least one
TRPV1 antagonist have been stopped because of dose-limiting
hyperthermia that occurred at sub-therapeutic doses.11
Indeed, we have previously described the discovery of a novel
isoxazole-3-carboxamide series of TRPV1 antagonists, which was
optimised to a potent, selective and efficacious compound 1,
Figure 1.13 Unfortunately, 1 suffered from poor solubility a physi-
cochemical attribute that appears almost ubiquitous within the
TRPV1 antagonist pharmacophore.
In this communication, we report optimisation of compound 1
and, in particular, our approaches taken to target TRPV1 antago-
nists with improved solubility, and lower plasma protein binding,
⇑
Corresponding author.
E-mail address: Paul.Ratcliffe@grunenthal.com (P. Ratcliffe).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.051

P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4652–4657
4653
Cl
of cases this led to an almost complete abolition of TRPV1 antago-
nist activity (data not shown) although one exception was the re-
verse isoxazole-5-carboxamides, which could be synthesized
according to Scheme 2.
O
F3C
N
H
OH
O
N
Benzaldehydes 8 were converted to the oxime 9 under standard
conditions, stirring with NCS in dimethylformamide and in the
presence of HCl in ether gave the chlorooxime 10. Cyclisation to
the isoxazole 11 was achieved by heating the chlorooxime 10,
prop-2-yn-1-ol and triethylamine in toluene. The hydroxyl group
was protected as the acetate by performing the halogenation of
the isoxazole ring with NCS or NBS in acetic acid. Hydrolysis with
LiOH in methanol gave the free alcohol 13 which was oxidised
using TEMPO.15 Amide coupling was performed under standard
conditions to afford the desired compounds 16a–c (Table 2).
Finally, given the limited investigation around the isoxazole 4-
position to date, the influence of further elaboration on solubility,
physicochemical parameters and TRPV1 potency was probed. To
this end, a range of compounds bearing polar substituents were
synthesized according to Schemes 3–5.
1
Figure 1.
C
Cl
D
O
F3C
N
A
H
OH
O
N
B
1
Figure 2.
Bromination of 4a was performed with N-bromosuccinamide in
the presence of benzoyl peroxide, this was then reacted with iso-
propylamine and sodium bicarbonate to yield the amine 20. This
ester was hydrolysed with lithium hydroxide and the resulting acid
coupled with the relevant amine and HATU to afford the desired
amides 21a–d. Hydrolysis of the ester 17 with aqueous TFA gave
a mixture of the hydroxy acid 18a and hydroxy ester 18b which
were readily separated. Compounds 19a–i were synthesised in
one of two ways, where R = H, by simply converting the acid 18a
to the amide using 1-propanephosphonic acid cyclic anhdyride
(T3P) and the appropriate amine or alternatively where R = Et by
reaction of ester 18b with the appropriate amines in acetonitrile
in the presence of triethylamine (Table 3).
4-Bromo-N-cyclopentyl-5-(4-(trifluoromethyl)phenyl)isoxazole-
3-carboxamide 22 has been reported previously.13 This was treated
with n-butyllithium, acetaldehyde or acetone to yield the respec-
tive substituted hydroxylmethyl derivative 23.
Di-ester 24 was synthesized according to the method of Zhao
et al.16 Selective tbutyl ester hydrolysis under acid conditions with
TFA in dichloromethane gave the mono acid 25 in quantitative
yield. Subsequent amide coupling was carried out on the ethyl es-
ter with cyclopentylamine in acetonitrile to furnish 26. This then
allowed for treatment of the remaining carboxylic acid with the
relevant amine in the presence of T3PÒ and triethylamine to yield
the desired compounds 27a–b.
while maintaining favourable analgesic and pharmacokinetic prop-
erties. These efforts culminated in novel ligands with good po-
tency, selectivity and in vivo efficacy following oral administration.
In an effort to introduce solubility, we took three parallel ap-
proaches which involved making structural modifications of the
molecule as outlined in Figure 2. In particular we expanded on
the structure–activity relationships established around the phenyl
ring (region A) as, for example, previous work had demonstrated
that a meta fluoro substituent could modulate solubility. Secondly
variation of the isoxazole ring itself was targeted (region B) and
thirdly introduction of polar substituents on the 3-position of the
isoxazole ring (region C). The generic routes employed to access
these compounds are shown in Schemes 1–3.
The general synthesis of the isoxazole-3-carboxamides has been
reported previously,13 however an alternative approach utilised for
this work is outlined in Scheme 1. For example, synthesis of ethyl
5-bromo-4-methylisoxazole-3-carboxylate 3 according to Adem-
bri14 allowed for a simple Suzuki coupling using the appropriate
boronic acid 2, potassium carbonate and tetrakis(triphenylphos-
phine)palladium(0) to give ethyl 5-phenyl-4-methylisoxazole-
3-carboxylate 4. Hydrolysis with lithium hydroxide in methanol
afforded the acid in near quantitative yield, treatment with oxalyl
chloride followed by coupling with the correct amine give the
desired amides 7a–d (Table 1).
In an effort to probe the impact of the isoxazole moiety, a num-
ber of heterocyclic replacements were synthesized. In the majority
Compounds were evaluated for their ability to modulate influx
of Ca2+ in Chinese Hamster ovary (CHO-k1) cells stably expressing
X
X
X
O
Br
(i)
(ii)
O
O
OH
B
O
OEt
N
OH
O
O
N
OEt
OH
N
2
3
4
5
(iii)
X
O
X
NH
R
O
(iv)
N
O
O
Cl
N
6
7 (a-d)
Scheme 1. Reagents and conditions: (i) K2CO3, tetrakis(triphenylphosphine)palladium(0), aqueous DME, 100 °C; (ii) 1 M lithium hydroxide solution in methanol, aqueous
THF, 60 °C; (iii) oxalyl chloride, DCM, DMF, 25 °C; (iv) RNH2, Et3N, 25 °C.

4654
P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4652–4657
(i)
(ii)
(iii)
Cl
R1
OH
R1
R1
R1
O
N
O
NOH
NOH
8
9
11
10
(iv)
O
X
X
X
N
(vi)
(v)
O
OH
OH
R1
R1
R1
O
O
O
O
N
N
14
13
12
(viii)
(vii)
O
O
X
NH
R2
R1
O
R1
O
O
N
N
15
16 (a-c)
X = Br or Cl
Scheme 2. Reagents and conditions: (i) NH2OHÁHCl, aqueous EtOH, KOH, 25 °C; (ii) NCS, DMF, 25 °C; (iii) prop-2-yn-1-ol, MePh, Et3N, 25 °C; (iv) AcOH, concd H2SO4, NBS or
NCS, 120 °C; (v) 1 M lithium hydroxide in methanol, aqueous THF, 60 °C; (vi) TEMPO, NaH2PO4, NaClO2, bleach, MeCN, H2O, 35 °C, then Na2SO3, 25 °C; (vii) 1 M lithium
hydroxide in methanol, aqueous THF, 60 °C; (viii) HOBt, EDCI, R2NH2, Et3N, DCM, 25 °C.
O
O
O
O
O
O
O
O
NH
Br
NH
NHR
O
O
O
(iii)
(iv)
(i)
(ii)
N
N
N
N
F3C
F3C
F3C
F3C
4a
17
20
21 (a-d)
(v)
R1
O
O
OH
OH
N
OR
R2
(vi) or (vii)
N
N
O
O
F3C
F3C
18 (a R = H)
(b R = Et)
19 (a-i)
Scheme 3. Reagents and conditions: (i) NBS, benzoyl peroxide, CCl4; (ii) isopropylamine, sodium bicarbonate, THF (iii) lithium hydroxide, THF; (iv) NH2R, HATU, NEt3, DCM;
(v) TFA, H2O (vi) R1NH2, T3PÒ, NEt3, DCM; (vii) R2NH2, MeCN, NEt3.
human TRPV1 (VR1) using a Molecular Devices FLIPR. TRPV1 ago-
nists stimulated intracellular fluorescence when applied to the
channel, while antagonists 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.
Initial investigations centered on incorporation of the 3-fluoro
moiety into analogue 1a to give compound 1b, however in this in-
stance it led to no beneficial effect on solubility. A similar outcome
was achieved for compound 1d, although in this case a 10-fold in-
crease in potency was observed compared to the des-Fluoro ana-
logue 1c. Our next attempt to improve intrinsic solubility was to
affect a twist around the phenyl-isoxazole bond by the introduc-
tion of an ortho substituent. Given the scale-able preparation of
the 4-methyl isoxazole core, and ability to functionalize the 3-
and 5-positions with the requisite groups, a small array of com-
pounds were swiftly prepared according to Scheme 1.Introduction
of the ortho ethoxy group did indeed have a positive effect on the
intrinsic solubility with compounds 7b and 7c achieving aqueous
solubilities of 27 and 28 mg LÀ1 respectively.17 However, it re-
sulted in a lowering of potency and the presence of the ortho sub-
stituent alone was not sufficient to maintain this improvement
instead; a combination of the ortho substituent and a pendant hy-
droxyl group in region D was also required for solubility as demon-
strated by compound 7d.
Reversing the substitution on the central isoxazole did result in
more soluble compounds as exemplified by 16a–c. However, in
general they were less potent at the TRPV1 receptor, with the best
compromise coming in the form of 16b.

P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4652–4657
4655
Table 1
Structure–activity relationships (SAR) of phenyl and isoxazole rings
Table 2
Effects of reversing isoxazole ring on potency and solubility
Compound Structure
TRPV1 Solkin
pIC50
Compound Structure
TRPV1 Solkin
pIC50
17
17
(mg LÀ1
)
(mg LÀ1
)
O
O
O
O
Cl
Cl
1a
9.3
<1
1a
9.3
<1
OH
OH
OH
OH
N
H
N
H
F3C
F3C
F3C
Cl
N
N
N
O
O
O
Cl
Cl
16a
8.4
9.2
5
2
N
H
N
H
F3C
1b
1c
1d
9.2
8.3
9.2
<1
3
N
O
F
O
Cl
O
O
Cl
OH
N
1d
H
OH
N
Cl
Cl
N
O
H
N
F
F
O
O
Cl
Cl
OH
OH
OH
N
H
N
H
Cl
16b
8.5
11
2
O
N
N
N
N
O
O
O
F
O
O
O
Cl
1e
7.3
6.1
38
47
OH
N
N
H
F3C
Cl
F
7a
7b
8.7
7.9
4
H
N
O
O
F
O
Cl
16c
OH
N
H
F
OH
OH
N
H
N
27
O
Finally we explored the SAR around the 4-position of the isox-
azole. Introduction of a methyl hydroxyl substituent, in most cases,
led to a significant increase in solubility but again a substantial
reduction in potency as exemplified by compounds 19a–f. Further-
more, substitution on the methylene adjacent to the isoxazole
diminished activity at the TRPV1 receptor (23a–b). Despite this,
compound 19a showed a good level of in vitro stability, low hERG
activity and pleasingly lower plasma protein binding in both hu-
man and rat. Encouraged by the tolerance for a polar substituent
at the isoxazole 4-position, we embarked upon further exploration
of extended polar functionality including acids, amides and
amines. This led to compounds 21a–b, 26 and 27a–b. Amides
and acids were only weakly tolerated, however introduction of
O
O
N
H
Cl
Cl
7c
6.7
7.9
28
<1
N
N
O
O
O
O
O
N
H
7d
R1 R2
HO
O
O
O
H
N
H
N
Br
(i)
N
N
O
(ii)
22
23 (a) R1=Me; R2=H
(b) R1=Me; R2=Me
F3C
F3C
Scheme 4. Reagents and conditions: (i) nBuLi, acetaldehyde, THF; (ii) nBuLi, acetone, THF.
O
O
O
O
O
O
H
N
H
N
O
O
O
O
R1
O
O
(i)
(ii)
(iii)
O
HO
HO
N
R2
N
N
N
N
O
O
F3C
F3C
F3C
F3C
24
25
26
27 (a-b)
Scheme 5. Reagents and conditions: (i) TFA/DCM; (ii) cyclopentylamine, MeCN; (iii) R1R2NH, T3PÒ, NEt3, DCM.

4656
P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4652–4657
Table 3
Table 3 (continued)
Effects of water solubilising groups on potency and solubility
Compound Structure
TRPV1 Solkin
pIC50
17
(mg LÀ1
)
Compound Structure
TRPV1 Solkin
pIC50
17
(mg LÀ1
)
OH
N
O
O
O
O
O
Cl
26
5.8
72
N
H
F3C
1
9.3
<1
OH
N
H
F3C
F3C
O
N
O
OH
NH
O
19a
7.1
73
OH
OH
21a
7.2
7.2
6.5
82
N
H
OH
N
F3C
N
O
H
N
O
O
O
OH
19b
19c
19d
19e
19f
5.8
5.8
5.5
6.9
6.8
6
70
66
35
1.5
1
N
H
F3C
F3C
F3C
F3C
F3C
F3C
F3C
F3C
NH
O
O
O
N
21b
4
O
N
H
F3C
OH
OH
O
N
N
H
N
O
NH
OH
21c
13
O
O
O
O
O
O
O
N
H
F3C
N
N
H
N
O
OH
NH
21d
6.1
12
N
H
N
H
F3C
N
O
O
O
O
O
N
O
OH
N
H
N
an isopropyl amine in this region achieved compounds such as 21a,
with potency and solubility equivalent to 19a.
OH
23a
23b
<1
<1
The in vitro profile of compounds 19a and 21a are summarised
below. As demonstrated in Table 4, both compounds are stable to
rat and human hepatic microsomes in vitro. They have lower to
moderate affinity for the hERG receptor and moderate to high plas-
ma protein binding.
N
H
N
OH
5.3
N
H
Compound 21a was selected for further profiling, and was
found to have good stability in human and rat hepatocytes with
N
OH
an intrinsic clearance of <6 l
l/min/106 cells in both, moderate
affinity for hERG with a pKi of 5.2 and moderate plasma protein
binding in both rat and human. As demonstrated in Table 5, com-
pound 21a has low to moderate clearance in rat, and achieves a rel-
atively long plasma half-life of 7.9 h, perhaps as a consequence of
the extensive volume of distribution.
N
H
19g
19h
19i
7.7
7.4
6.8
1.4
N
F
F
OH
O
N
Overall, compound 21a demonstrated an acceptable pharmaco-
kinetic profile to proceed to in vivo testing. The efficacy of com-
pound 21a was evaluated in the Capsaicin Hargreaves assay, a
functional in vivo assay of TRPV1 induced thermal hyperalgesia
73
F3C
F3C
H
N
O
OH
OH
O
in rats.19 In the Capsaicin Hargreaves model, 21a (3, 10, 30
l
mol/
N
H
31
kg; po; 2 h pre-treatment) significantly reversed the thermal
hyperalgesia induced by capsaicin, with an MED of 30
(see Fig. 3).
N
O
lmol/kg
F
In summary, a series of isoxazole derivatives was optimised
through a systematic SAR study of the aryl portion, central isoxaz-
ole ring and 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 19a and
21a were identified as the most promising examples from this ser-
ies and compound 21a was progressed into animal studies. Despite
the lower in vitro potency as compared to compound 1a, com-
pound 21a showed clear attenuation of the acute inflammatory
thermal response in the rat Capsaicin Hargreaves assay.
NH
N
O
O
O
27a
27b
5.3
5.7
1.6
N
F3C
F3C
H
O
N
O
<1
N
H
N
O

P. Ratcliffe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4652–4657
4657
Table 4
In vitro pharmacokinetic data for selected compounds
Compound Solkin
(mg LÀ1
HLM CLint
l minÀ1 mgÀ1
RLM CLint
l minÀ1 mgÀ1
hERG pKi (% inhibition at
100 M)
PPB %bound
(Human)
PPB %bound (Rat
Wistar)
17
)
(
l
)
(
l
)
l
1
19a
21a
<1
73
82
<12/<12
<12/27
<12/<12
<12/<12
<12/27
<12/<12
Inactivea
(71%)
5.2 (81%)
99.0
91.9
75.0
99.3
90.9
72.9
Abbreviations: HLM CLint: intrinsic clearance in human liver microsomes; RLM CLint: intrinsic clearance in rat liver microsomes; hERG pKi: inhibition constant in a hERG
channel binding assay (dofetilide); PPB %bound: percentage of compound bound to plasma protein.18
a
Apparent inactivity may have been a result of low solubility under the assay conditions.
Table 5
In vivo pharmacokinetic data for compound 21a
Compound
CL (ml minÀ1 kgÀ1
)
Vss (L kgÀ1
)
T1/2 (h)
po
Tmax (h)
%F
Cmax
(lM)
AUClast (h
2728
l )
g LÀ1
21a
12.5
5.9
7.9
0.72
2
20
Abbreviations: CL: plasma clearance; Vss: steady state volume of distribution; T1/2: biological half-life; Cmax: peak plasma concentration after oral administration; AUClast: area
under the curve at final tested concentration; %F: oral bioavailability.
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Vehicle
30µmol/kg
Figure 3. Reversal of induced hyperalgesia by 21a in the Capsaicin Hargreaves
model. Paw withdrawal latency (PWL) is shown on the y-axis.
Acknowledgements
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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