N-Pyridin-3-yl- and N-quinolin-3-yl-benzamides: Modulators of Human Vanilloid Receptor 1 (TRPV1)

Article abstract of DOI:10.1016/j.bmcl.2008.02.075

High throughput screening of our compound library revealed a series of N-pyridyl-3-benzamides as low micromolar agonists of the human TRPV1 receptor. Synthesis of analogs in this series led to the discovery of a series of N-quinolin-3-yl-benzamides as low

Full text of DOI:10.1016/j.bmcl.2008.02.075

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2730–2734
N-Pyridin-3-yl- and N-quinolin-3-yl-benzamides:
Modulators of Human Vanilloid Receptor 1 (TRPV1)
Michele C. Jetter,a,* James J. McNally,a Mark A. Youngman,a Mark E. McDonnell,a
Adrienne E. Dubin,b Nadia Nasser,b Sui-Po Zhang,a Ellen E. Codd,a Ray W. Colburn,a
Dennis R. Stone,a Michael R. Brandt,a Christopher M. Floresa and Scott L. Daxa
aJohnson & Johnson Pharmaceutical Research and Development, Research and Early Development,
Welsh and McKean Roads, Spring House, PA 19477, USA
bJohnson & Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, San Diego, CA 92121, USA
Received 9 November 2007; revised 27 February 2008; accepted 28 February 2008
Available online 5 March 2008
Abstract—High throughput screening of our compound library revealed a series of N-pyridyl-3-benzamides as low micromolar ago-
nists of the human TRPV1 receptor. Synthesis of analogs in this series led to the discovery of a series of N-quinolin-3-yl-benzamides
as low nanomolar antagonists of human TRPV1.
Ó 2008 Elsevier Ltd. All rights reserved.
Research toward the discovery of new analgesics agents
has focused on identifying ion channels specific to noci-
ceptors and developing agents that modulate the flow of
ions through these channels. Several years ago, the hu-
man vanilloid receptor type 1 (TRPV1) channel was
identified isolated and cloned from afferent nociceptors.1
TRPV1 is activated by capsaicin, the hot component of
chili peppers, as well as other naturally occurring ago-
nists, such as resiniferatoxin (RTX). The channel is also
activated by painful stimuli, such as noxious heat, pro-
tons and certain endogenous lipophilic agonists, such
as anadamide.2 Since the discovery of this channel,
much effort has been put forth in the identification of
TRPV1 modulators as potential therapeutics.3 Our
group has reported several distinct series of novel
TRPV1 antagonists4 (see Fig. 1).
undertook a study to investigate the structure–activity
relationships (SAR) in this series.
3-Aminopyridine was coupled to 4-alkylsubstituted ben-
zoic acids, using standard coupling conditions (HBTU
and diisopropylethylamine in DMF) yielding a series
of pyridylbenzamides that were evaluated in a cell-based
fluorescent TRPV1 functional assay.5 Binding affinity
(Ki) was determined using an assay that measures the
displacement of radiolabeled resiniferatoxin ([3H]-
RTX) from the human TRPV1 receptor6 (see Table 1).
Varying the length of the alkyl chain had a modest effect
on the potency of these compounds in the functional as-
say, the most potent agonist being the n-butyl derivative,
1d. The compound that showed the best binding affinity
in this series was the n-pentyl derivative, 1e. The N-
methylamide 1i was inactive in both receptor binding
and functional assays suggesting that the NH proton
is involved in the interaction with the TRPV1 receptor.
One of the more active leads identified by screening our
internal compound library was 4-pentyl-N-pyridin-3-yl-
benzamide, 1e, a submicromolar agonist of TRPV1.
We observed several structural similarities between cap-
saicin and the initial hit including an alkyl chain, a cen-
tral amide bond, and an aromatic group. Thus we
In most cases, varying the group that linked the pyridine
to the alkylbenzene resulted in a loss of both functional
activity as well as binding affinity (Table 2). Changing
the amide to an aminomethylene (1k) or to the reverse
amide (1l) preserved the 2-atom linkage but resulted in
a loss of binding and functional activity. Insertion of a
1-carbon linking group on either side of amide (1m,1n)
also resulted in analogs with poor activity.
Keywords: TRPV1; Antagonists; Quinolinyl benzamide.
*
Corresponding author. Tel.: +1 610 584 8019; e-mail: michele137@
comcast.net
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.075

M. C. Jetter et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2730–2734
2731
Figure 1. Effect of compound 6i on CFA-induced hyperalgesia in the rat.
Table 1. Human TRPV1 binding affinities and functional activity of 4-
alkyl-N-pyridin-3-yl-benzamides
Table 3. Human TRPV1 binding affinities and functional activity of
benzamide compounds
Compound Ar
Binding affinity Functional
Ki (nM)
Compound
n
R
Binding affinity Functional activity
Ki (nM)
activity
IC50 (lM)
EC50 (lM)
1a
1b
1c
1d
1e
1f
0
1
2
3
5
6
7
9
4
H
H
H
H
H
H
H
H
NT
NT
NT
626
421
1740
546
607
>10.0
2.7
0.7
1d
2
3-Pyr
Ph
626
6272
29
EC50 = 0.3
>30
3
7-OH-1 Naphthyl
5-Isoquinoline
3-Quinoline
0.042
2.4
0.23
0.3
1.7
4
72
53
5
3.7
2.9
1g
1h
1i
>30
>30
series, the explanation for the shift in functional activity
is not clear.
CH3 15,000
NT, not tested.
While the naphthol compound 3 showed excellent
antagonist activity, the series suffered from metabolic
liability due to the phenolic group and was not further
developed. Modification of the terminal alkyl group in
the amidoquinoline series, 5, had a profound effect on
the binding affinity and the potency in the calcium flux
assay (Table 4). Increasing the chain length of the alkyl
group from propyl, 5a, to heptyl, 5d, resulted in de-
creased functional potency and binding affinity. Further
increases in chain length resulted in significant reduction
in binding and functional activity. The bulky tert-butyl
group imparted good binding affinity as well as good
Table 2. Human TRPV1 binding affinities and functional activity of
derivatives with various linking groups
Compound
L
Binding affinity Functional activity
Ki (nM)
EC50 (lM)
1e
1k
1l
–NHCO–
–NHCH2–
–CONH–
421
>3000
>3000
1.7
>5.0
>5.0
4.2
1m
1n
–CH2NHCO– >3000
–NHCOCH2– 7260
>5.0
Table 4. Human TRPV1 binding affinities and functional activity of 3-
quinolinyl benzamides
Replacement of the pyridine of compound 1d with other
aromatic groups had a profound effect on both binding
and function (Table 3). A simple benzene replacement
resulted in a compound, 2, with little binding or func-
tional activity. The naphthol, 3, isoquinoline, 4 and
quinoline, 5, amide analogs showed improved binding
potency 8- to 20-fold over 1d. Significantly, these ana-
logs were the first to exhibit antagonist activity. This
attenuation of functional activity prompted our group
to further investigate these series. Appendino et al. re-
ported that insertion of an iodine atom in the 60 position
in the vanillyl moiety of TRPV1 agonists caused a sim-
ilar shift in functional activity from agonist to antago-
nist.7 The authors suggest the change in functional
activity may be due to a difference in the way the 60-iodo
derivatives bind to the TRPV1 receptor. Within our own
Compound
R
X
Binding affinity
Ki (nM)
Functional
activity
IC50 (lM)
5a
5b
5c
5d
5e
5f
H
n-Propyl
n-Hexyl
n-Heptyl
n-Octyl
60
190
3520
2080
980
24
0.13
0.15
>30
>30
>30
0.041
>30
H
H
H
H
n-Nonyl
tert-Butyl
n-Propyl
H
Me
5g
NT
NT, not tested.

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M. C. Jetter et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2730–2734
Scheme 1. Reagents and conditions: (a) HBTU, DIEA, BOC-NHPhCO2H, CH3CN, heat; (b) HNR1R2, DMSO, 100 °C; (c) TFA, DCM; (d)
aldehyde, ketone, or paraformaldehyde, Me4NBH(OAc)3, DCE, heat.
antagonist potency (5f). As was seen previously in the
pyridyl series, N-methylation of the amide, 5g, resulted
in the loss of both functional and binding activity.
in the simpler alkyl series (see Table 4). However, the 14-
carbon chain derivative, 6h, showed a lack of binding or
functional activity. The N-methyl-N-cyclohexyl analog
6i exhibited excellent binding and antagonist potency
at the human TRPV1 receptor. Replacement of the N-
methyl with NH (6j) reduced both binding and func-
tional activity, as did the replacement of the N-methyl
substituent with the larger N-propyl group (6k). Inser-
tion of a methylene between the N and the cyclohexyl
group gave a compound (6l) with a similar binding affin-
ity but with somewhat reduced antagonist potency. Dur-
ing the course of this SAR study, discrepancies were
sometimes seen between the functional and binding
activities of the compounds. These slight differences
could be attributed to the nature of the cellular-based
functional assay as compared to the radioligand binding
assay.5,6
In the course of this study, we determined that tertiary
amines were suitable replacements for the terminal alkyl
group. The quinoline p-benzamides were readily pre-
pared8 by coupling 3-aminoquinoline with Boc-pro-
tected p-amino benzoic acid, using HBTU coupling
conditions, followed by deprotection to yield the free
amine. The N-alkyl groups were then installed sequen-
tially by reductive amination with a suitable carbonyl
component. Alternatively, 3-aminoquinoline was cou-
pled to p-fluorobenzoic acid followed by the reaction
with an appropriate amine at high temperature in
DMSO (Scheme 1).
As shown in Table 5, there was a significant effect of the
chain length of R1 on the binding affinity. The n-propyl
compound 6a, exhibited only modest binding affinity for
the human TRPV1 receptor, while the longer n-octyl
analog 6f exhibited a 10-fold increase in binding affinity.
Interestingly, this trend seems to be opposite to that seen
Compound 6i was further examined for its in vitro prop-
erties and pharmacokinetic profile. Against other activa-
tors of the human TRPV1 receptor such as low pH,
anandamide, and the phorbol ester PMA, 6i was a func-
tional antagonist with IC50 values of 0.16 lM, 0.033 lM
and 0.035 lM, respectively. Selectivity of compound 6i
was demonstrated when assayed at 10 lM against a pa-
nel of >50 different receptors, ion channels, and trans-
porters (Cerep; Paris, France), exhibiting <50%
inhibition at all targets tested. In the presence of rat or
human liver microsomes, 6i showed modest in vitro
rat and human metabolic stability (45% and 25%
remaining after 15 min, respectively). The pharmacoki-
netic profile of compound 6i was studied following
intravenous (iv) and oral (po) dosing in male Sprague–
Dawley rats. The in vivo clearance of 6i was moderate
(20 mL/min/kg), the elimination half-life (t1/2) was equal
to 100 min and the compound had good oral bioavail-
ability (F = 70%). Subsequently, compound 6i was eval-
uated for in vivo efficacy in an established rodent model
of hyperalgesia.9 In the rat, at an oral dose of 30 mg/kg,
compound 6i produced a significant (>80%) reversal of
thermal hyperalgesia induced by complete Freund’s
adjuvant (CFA) (Fig. 2).
Table 5. Human TRPV1 binding affinities and functional activity of 3-
quinolinyl p-amino benzamides
Compound R1
R2
Binding Functional
affinity activity
Ki (nM) IC50 (lM)
6a
6b
6c
6d
6e
6f
n-Propyl
n-Butyl
Me
Me
267
83
0.39
0.066
0.054
0.086
0.12
0.19
0.15
>30
0.022
10
n-Pentyl
n-Hexyl
n-Heptyl
n-Octyl
Me
Me
38
70
Me
Me
24
15
6g
6h
6i
n-Nonyl
(CH2)13Me
c-Hexyl
c-Hexyl
c-Hexyl
Me
Me
23
In addition, 6i was orally efficacious in a rodent model
of experimental colitis.10 These initial in vivo efficacy
data gathered for compound 6i support the continued
study of the 3-quinolinyl p-aminobenzamide series of
TRPV1 antagonists.
1471
8
Me
H
p-n-Propyl
6j
156
85
6k
6l
10
0.17
CH2-c-hexyl Me
16

M. C. Jetter et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2730–2734
2733
20
PreCFA
15
B.; Gonzales, L.; Hoey, K.; Rizzolio, M.; Bogenstaetter,
M.; Codd, E.; Lee, D.; Zhang, S.-P.; Chaplan, S.;
Carruthers, N. J. Med. Chem. 2005, 48, 1857; (e) Jetter,
M. C.; Youngman, M. A.; McNally, J. J.; McDonnell, M.
E.; Dax, S. L.; Dubin, A. E.; Nasser, N.; Codd, E. E.;
Zhang, S.-P.; Flores, C. M. Bioorg. Med. Chem. Lett.
2007, 17, 6160.
Vehicle
*
Compound 6i
30 mg/kg po
* P<0.05
10
5
5. hTRPV1/HEK cells were seeded on poly-D-lysine coated
96-well, black-walled plates (BD 354640) and 2 days later
loaded with Fluo-3/AM for 1 h and subsequently tested
for agonist-induced increases in intracellular Ca2+ levels
using FLIPRTM technology. Cells were challenged with
single concentrations of compound and intracellular Ca++
was measured for 3 min prior to the addition of CAP to all
wells to achieve a final CAP concentration of 15 nM to
fully activate TRPV1. Antagonist potency was determined
using the protocol described by McDonnell et al. Bioorg.
Med. Chem. 2002, 12, 1189 (data were analyzed using
Prism software to calculate IC50 values).
0
0
100
Time (min)
Figure 2.
In summary, a series of 3-pyridyl benzamide agonists of
human TRPV1 receptor was identified through broad
screening of our internal compound library. Modifica-
tion of the initial HTS hit, compound 1e, led to the dis-
covery of novel 3-quinolinyl p-aminobenzamides that
act as functional antagonists at the TRPV1 receptor.
In particular, compound 6i was extensively profiled in
several in vitro and in vivo assays. Compound 6i exhib-
ited relatively high functional antagonist activity at the
human TRPV1 receptor whether activated by capsaicin,
low pH or phorbol ester PMA. Oral in vivo efficacy was
demonstrated by 6i in rodent models of inflammatory
pain and colitis. These results suggest that further opti-
mization of the 3-quinolinyl p-amino benzamide core to
improve solubility, metabolic stability, and pharmacoki-
netic properties may lead to TRPV1 antagonists that
have potential therapeutic utility for the treatment of
inflammatory pain and gastrointestinal disorders.
6. [3H]-RTX binding assay using hVR1/HEK293 cell mem-
branes. Cloning and generation of stable cell lines
expressing human TRPV1. Human TRPV1 was cloned
and stably expressed in HEK293 cells (hVR1/HEK293) as
described by Grant et al. in J. Pharm. Exptl Ther. 2002,
300, 9. Preparation of membranes. Human TRPV1/
HEK293 were homogenized with a Polytron twice and
centrifuged at 3000 rpm for 10 min in Hepes buffer
containing 20 mM Hepes, pH 7.4, NaCl 5.8 mM, sucrose
320 mM, MgCl2 2 mM, CaCl2 0.75 mM, and KCl 5 mM.
The supernatant was centrifuged at 18,000 rpm for 20 min.
The pellet was saved in a tube and 10 mL assay buffer was
added into the tube. The pellet and buffer were mixed with
a Polytron. Incubation procedure. Incubations for 60 min
at 37 °C were performed in a total volume of 0.5 mL that
contained 120 lg/mL membrane protein and 0.3–0.6 nM
[3H]-RTX (NEN, Boston) in the Hepes buffer. After
incubation, the samples were cooled on ice and 100 lg of
a-acid glycoprotein was added followed by centrifugation
at 13,000 rpm for 15 min. The supernatant was aspirated
and the tips of tubes were cut off into 6 mL vials. Non-
specific binding was measured in the presence of 200 nM
unlabeled RTX in 4 mL scintillation liquid using a
Packard scintillation counter. Data analysis. Percent (%)
inhibition = (total binding ꢀ total binding in presence of
References and notes
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*
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8. 4-(Cyclohexyl-methyl-amino)-N-quinolin-3-yl-benzamide.
Tetramethylammonium triacetoxyborohydride (2.64 g,
0.010 mol) was added to a mixture of 4-cyclohexylamino-
N-quinolin-3-yl-benzamide (1.58 g, 0.0046 mol) and para-
formaldehyde (0.928 g, 0.032 mol) in 1,2-dichloroethane
(50 mL). The resultant mixture was heated at reflux for 6 h.
The solvent was evaporated in vacuo. The crude product
was purified by flash chromatography eluting with a
gradient of methanol (3–5%) in dichloromethane. The
product was triturated with diethyl ether to give a colorless
1
solid (1.21 g). MS (MH+): 360; H NMR (DMSO-d6): d
1.07–1.22 (m, 1H), 1.36–1.72 (m, 7H), 1.76–1.85 (m, 2H),
2.83 (s, 3H), 3.70–3.81 (m, 1H), 6.86 (d, J = 9 Hz, 2H), 7.55–
7.69 (m, 2H), 7.91–7.98 (m, 4H), 8.81 (d, J = 2.2 Hz, 1H),
9.15 (d, J = 2.4 Hz, 1H) and 10.29 (s, 1H).
9. Complete Fruend’s adjuvant (CFA; 100 lL emulsion of
saline and heat-killed Mycobacterium tuberculosis in min-
eral oil) was injected into a single hind paw of male

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M. C. Jetter et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2730–2734
Sprague–Dawley rats. Each rat was placed in a test
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10. Kimball, E. S.; Wallace, N. H.; Schneider, C. R.;
D’Andrea, M. R.; Hornby, P. J. Neurogastroenterol.
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chamber on a warm glass surface and allowed to acclimate
for approximately 10 min. Response latencies to the
radiant thermal stimulus were recorded for each animal
prior to and 24 h following the injection of CFA.
Immediately following the post-CFA latency assessment,
test compound or vehicle was administered orally to the