Discovery of small molecule antagonists of TRPV1

Article abstract of DOI:10.1016/j.bmcl.2004.05.028

Small molecule antagonists of the vanilloid receptor 1 (TRPV1, also known as VR1) are disclosed. Ureas such as 5 (SB-452533) were used to explore the structure activity relationship with several potent analogues identified. Pharmacological studies using electrophysiological and FLIPR Ca²⁺ based assays showed compound 5 was an antagonist versus capsaicin, noxious heat and acid mediated activation of TRPV1. Study of a quaternary salt of 5 supports a mode of action in which compounds from this series cause inhibition via an extracellularly accessible binding site on the TRPV1 receptor.

Full text of DOI:10.1016/j.bmcl.2004.05.028

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3631–3634
Discovery of small molecule antagonists of TRPV1
Harshad K. Rami,a,* Mervyn Thompson,a Paul Wyman,b Jeffrey C. Jerman,c
Julie Egerton,a Stephen Brough,c Alexander J. Stevens,a Andrew D. Randall,a
Darren Smart,a Martin J. Gunthorpea and John B. Davisa
aNeurology and GI CEDD, New Frontiers Science Park, GlaxoSmithKline, Third Avenue, Harlow, Essex CM19 5AW, UK
bPsychiatry CEDD, New Frontiers Science Park, GlaxoSmithKline, Third Avenue, Harlow, Essex CM19 5AW, UK
cDiscovery Research, New Frontiers Science Park, GlaxoSmithKline, Third Avenue, Harlow, Essex CM19 5AW, UK
Received 17 March 2004; revised 12 May 2004; accepted 14 May 2004
Available online
Abstract—Small molecule antagonists of the vanilloid receptor 1 (TRPV1, also known as VR1) are disclosed. Ureas such as 5 (SB-
452533) were used to explore the structure activity relationship with several potent analogues identified. Pharmacological studies
using electrophysiological and FLIPR Ca2þ based assays showed compound 5 was an antagonist versus capsaicin, noxious heat and
acid mediated activation of TRPV1. Study of a quaternary salt of 5 supports a mode of action in which compounds from this series
cause inhibition via an extracellularly accessible binding site on the TRPV1 receptor.
Ó 2004 Elsevier Ltd. All rights reserved.
The pungent component of hot chili pepper, capsaicin
and its analogues, are activators of the ion channel
originally called vanilloid receptor 1 (VR1) but now
referred to as TRPV1 according to an updated nomen-
clature.1 Activation of TRPV1 on sensory neurons by
capsaicin, heat or low pH is thought to lead to an influx
of calcium and sodium ions through the integral channel
causing a depolarisation of the cell and eventually
leading to the sensation of pain. In humans, activation
of TRPV1 by topical application of capsaicin initially
induces pain that is followed by densensitisation leading
to an analgesic effect. Several potent TRPV1 agonists
have been identified2 but the side effects associated with
the development of these agents have hindered rapid
progress. A TRPV1 antagonist, however, offers a rapid
onset of analgesic action by simply blocking TRPV1
mediated signalling potentially avoiding the side effects
associated with an agonist induced desensitisation
strategy. Furthermore, because of its different mode of
action, a TRPV1 antagonist may offer an improved
safety profile in comparison with established analgesics
such as NSAIDs3 and hence may have therapeutic
potential for the treatment of chronic pain arising from
both tissue or nerve injury.
In 1992, capsazepine4 was reported as the first compet-
itive TRPV1 antagonist. This compound, however, has
nonspecific actions, at both voltage-gated calcium
channels5 and nicotinic acetylcholine receptors,6 at
concentrations commonly used to block TRPV1 medi-
ated responses. We recently reported7 that SB-366791
O
HO
N
H
HO
Capsaicin
Cl
S
H
N
HO
HO
N
Capsazepine
O
OMe
N
H
Keywords: TRPV1; Vanilloid antagonist; VR1; SB-452533; SB-366791.
* Corresponding author. Tel.: +44-(0)-1279-627818; fax: +44-(0)-1279-
622122; e-mail: harshad.rami@gsk.com
Cl
SB-366791
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.028

3632
H. K. Rami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3631–3634
was a novel, potent and selective competitive inhibitor
of human and rat TRPV1 that represented an improved
tool compound for the study of TRPV1 function.
Synthesis of the urea analogues (1–20) was readily
accomplished from commercially available isocya-
nates and N-(2-aminoethyl)-N-ethyl-m-toluidine. Other
required substituted amines were conveniently prepared
from the appropriate substituted N-ethyl anilines and
2-aminoethyl bromide hydrochloride in toluene at reflux
(2:1 stoichiometry) (Fig. 1).9
More recently, other workers have sought to exploit this
exciting area and several different structural classes of
TRPV1 antagonists have been published.8
As part of our continuing effort to identify novel TRPV1
templates, a high throughput screen (HTS) was per-
formed using a FLIPR (fluorescence imaging plate
reader)-based Ca2þ assay versus our in-house compound
collection. This Fluo-3 based assay allowed the identi-
fication of compounds, which were capable of anta-
gonising the capsaicin-mediated activation of human
TRPV1 stably expressed in 1321N1 cells, as previously
described.7 Compound 5 was identified as the most
potent urea TRPV1 antagonist (pKb 7.8) from the HTS
campaign. We therefore initiated an investigation of the
SAR of this urea template using array chemistry and a
detailed characterisation of the antagonist properties of
this compound versus the acid- and heat-mediated
activation of TRPV1 using FLIPR and electrophysio-
logical techniques (see Figs. 2 and 3).
Compounds were screened in the FLIPR7;10 assay using
capsaicin as the agonist and the data are presented in
Table 1.
Scrutiny of the substitution pattern on the LHS Ar
group revealed that good antagonist activity was re-
tained with 2-Cl 4 but surprisingly the corresponding
fluoro 3 was 10-fold less potent. The unsubstituted
compound 1 also showed an acceptable level of potency.
Interestingly, for bromo substitution the order of po-
tency was 2-Br > 3-Br > 4-Br. The dichloro analogues (9,
10) showed comparable potency to 5 but the 1-naphthyl
compound 8 was less active. Further SAR work estab-
lished N-ethyl group (R1) as being optimum as
replacement by NH (11), N-methyl (12), N-benzyl (13)
or N-acetyl (14) did not enhance activity.
H2NCH2CH2Br.HCl
Toluene, reflux, 24h
R
R
H2N
H
N
N
Et
Et
Figure 1. Preparation of N-(2-aminoethyl)-N-ethyl-phenyl derivatives.
Figure 2. Inhibition of human TRPV1 receptors by 5. (A and B) Whole cell currents were recorded from HEK293. TRPV1 cells using the patch
clamp technique. TRPV1-mediated responses to 1 lM capsaicin (CAP) or acid (pH 5.3) were reversibly inhibited by co-application of 1 lM 5 (n ¼ 3).
Similarly, in (C), 1 lM 5 was demonstrated to inhibit the native capsaicin receptor present in rat dorsal root ganglia neurons (n ¼ 4). (D) Con-
centration–response curves for capsaicin-evoked Ca2þ-entry in HEK293. TRPV1 cells recorded using a fluorescence imaging plate reader (FLIPR)10
under control conditions, or in the presence of increasing concentrations of 5 (10–300 nM), as indicated. (E) Schild analysis of the data shown in (D)
exemplified a competitive mechanism of antagonism of TRPV1 by 5, with a pA2 ¼ 8.01.

H. K. Rami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3631–3634
3633
Figure 3. Inhibition of the heat-mediated activation of human TRPV1 receptors by 5. Whole cell currents evoked by noxious heat (50 °C, applied for
the duration indicated by the bar) were recorded from HEK293. TRPV1 cells using the patch clamp technique.11 Heat-activated currents were
reversibly inhibited by 1 lM 5 (93 2% inhibition relative to control, n ¼ 3).
Table 1. Antagonist activity at TRPV1
Having established key SAR we then turned our atten-
tion towards investigation of the binding site of ureas
such as 5. Interestingly, the capsaicin binding site on
R2
H
N
H
N
TRPV1 has been reported to be accessible from the
intracellular side of the receptor.11 However, recent data
now suggests that intracellular application of vanilloids
is insufficient for activation of TRPV1.12 The true
location of the capsaicin binding site on TRPV1 there-
fore remains unclear. As 5 is a competitive inhibitor of
the capsaicin binding site we thought it would be
interesting to determine its site of action. Quaternary
salts are unlikely to permeate through cell membranes
and therefore the methiodide salt of 5 was prepared by
stirring in acetone with methyl iodide at ambient tem-
perature for 7 days.
N
Ar
R1
O
Compound
Ar
Ph
R1
R2
pKb
1
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
H
7.2
6.9
6.8
7.7
7.8
7.4
7.2
7.1
7.7
7.6
<6.8
7.4
<6.7
<6.6
7.2
6.9
8.1
7.6
7.8
8.2
7.7
7.5
2
2-Me–Ph
2-F–Ph
3
4
2-Cl–Ph
2-Br–Ph
3-Br–Ph
4-Br–Ph
5
6
7
8
1-Naphthyl
2,3-diCl–Ph
2,5-diCl–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
2-Br–Ph
9
10
11
12
Br
I-
Me
CH2Ph
COMe
Et
Me
H
N
H
N
+
13
14
Me
N
Et
O
15
16
21
Et
Et
2-Me
4-Me
3-F
17
18
Et
Et
19
20
3,4-diF
3-F-4-Me
This quaternary salt 21 showed moderate antagonist
activity in the FLIPR assay (pKb 7.0) in comparison
with 5. Electrophysiological assays suggested that its
binding site and, therefore, the binding site for this
template is likely to be accessible only from the extra-
cellular surface as 21 was inactive when applied intra-
cellularly at up to 100 lM concentration and yet was
clearly active when applied extracellularly (Fig. 4).
Et
SB-366791
Capsazepine
Substitution (R2) on RHS phenyl ring was investigated
next and unsubstituted phenyl (15) and 2-Me (16)
showed lower activity but in contrast the 4-Me analogue
(17) was more potent. Replacement of the 3-Me by 3-F
substituent (18) maintained activity, as did the 3,4-diF
pattern (19). Encouragingly, a combination of 3-F-4-Me
substitution (20) gave the most potent compound.
Compound 5 was also potent as an antagonist at the
cloned recombinant rat TRPV1 receptor in HEK293
cells with a pKb of 7.7 versus capsaicin and pIC50 of 7.0
versus pH. This level of activity was confirmed in native
receptor: 5 showed a pKb of 7.4 versus capsaicin-evoked
responses in rat dorsal root ganglian neurones. Fur-
thermore, good potency was also observed for 5 in
HEK293 cells at the cloned guinea pig TRPV1 receptor
(pKb of 7.5) and the human TRPV1 receptor (pKb 7.7)
versus capsaicin in the same cell line.
Compound 5 was investigated in electrophysiology
experiments using the whole cell patch clamp method
and its mode of action was studied in detail in further
FLIPR experiments. The data show that 5 is a potent
and reversible inhibitor of capsaicin, low pH and heat-
mediated activation of human TRPV1 (see Figs. 2 and
3) and acts as a competitive inhibitor at the capsaicin
binding site on the receptor (Fig. 2D and E).
In conclusion, we have identified a potent series of
TRPV1 antagonists, as exemplified by 5, which show

3634
H. K. Rami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3631–3634
Figure 4. Inhibition of human TRPV1 receptors by the quaternary salt 21. (A) The antagonist activity of the quaternary salt 21 was confirmed by
demonstrating inhibition of TRPV1-mediated whole-cell responses to 1 lM capsaicin (n ¼ 3). (B) Application of a high concentration (100 lM) of 21
to the intracellular side of the receptor did not result in inhibition of capsaicin-mediated responses (n ¼ 5). In these experiments 21 was included in the
intracellular solution and a pre-application period of >4 min was employed after whole cell mode was achieved to ensure that the compound had
sufficient time to diffuse into the cell. (C) In the same test cells as those studied in (B) subsequent extracellular addition of compound 21 to responses
recorded at both negative ()70 mV) and positive (+70 mV) membrane potentials resulted in a full blockade of TRPV1 (n ¼ 5).
5. Docherty, R. J.; Yeats, J. C.; Piper, A. S. Br. J. Pharmacol.
1997, 121, 1461–1467.
activity against capsaicin, noxious heat and low pH
mediated activation of TRPV1. In addition 5 is equi-
potent across species (human, rat and guinea pig) with a
good level of antagonistic activity. High intrinsic clear-
ance determined in vitro in both rat and human liver
microsomes13 means that 5 is unsuitable for in vivo
studies. However, it does provide an excellent tool
compound for the study of the pharmacology of TRPV1
in vitro or ex vivo. Further work that addresses the
pharmacokinetics properties of 5 (SB-452533)14 is
ongoing and will be reported in a future publication.
6. Liu, L.; Simon, S. A. Neurosci. Lett. 1997, 228, 29–32.
7. Gunthorpe, M. J.; Rami, H. K.; Jerman, J. C.; Smart, D.;
Gill, C. H.; Soffin, E. M.; Luis Hannan, S.; Lappin, S. C.;
Egerton, J.; Smith, G. D.; Worby, A.; Howett, L.; Owen,
D.; Nasir, S.; Davies, C. H.; Thompson, M.; Wyman, P.
A.; Randall, A. D.; Davis, J. B. Neuropharmacology 2004,
46, 133–149.
8. Bakthavatchatam, R.; Blum, C. A.; Brielmann, H. L.;
Caldwell, T. M.; De Lombaert, S. WO Patent 02/08221;
Suh, H.; Oh, U. T.; Kim, H. D.; Lee, J. W.; Park, H. G.;
Park, Y. H. WO Patent 02/16317, WO Patent 02/16318,
WO Patent 02/16319; McIntyre, P.; James, I. F. U.S.
Patent 6,406,908; Bo,Y. Y.; Chakrabarti, P. P.; Chen, N.;
Doherty, E. M.; Fotsch, C. H. WO Patent 03/049702.
9. Rami, H. K.; Thompson, M.; Wyman, P. WO Patent 02/
072536.
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B.; Smart, D. Br. J. Anaesth. 2002, 89, 882–887.
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M.; Bonnet, F.; Ebrahim, S.; Escolar, G.; Jage, J.; Pocock,
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1992, 107, 544–552.
13. Stevens, A. J. unpublished results. Intrinsic clearance rates
in liver microsomes were rat >50 and human 41 mL/min/g
liver.
14. Characterisation data for
5
(SB-452533): 1H NMR
(250 MHz, CDCl3) d (ppm): 8.00 (d, 1H), 7.50 (d, 1H),
7.26 (m, 1H), 7.10 (m, 1H), 6.92 (m, 1H), 6.55 (m, 4H),
4.95 (br, 1H), 3.47 (m, 4H), 3.37 (q, 2H), 2.30 (s, 3H), and
1.14 (t, 3H). MHþ ¼ 376, 378.