7-Hydroxynaphthalen-1-yl-urea and -amide antagonists of human vanilloid receptor 1

Article abstract of DOI:10.1016/j.bmcl.2003.09.090

A series of structurally simple 7-hydroxynaphthalenyl ureas and amides were discovered to be potent ligands of human vanilloid receptor 1 (VR1). 1-(7-Hydroxynaphthalen-1-yl)-3-(4-trifluoromethylbenzyl)urea 5f exhibited nanomolar binding affinity (K_i=1.0 nM) and upon capsaicin challenge, behaved as a potent functional antagonist (IC₅₀=4 nM). The synthesis and structure-activity relationships (SARs) for the series are described.

Full text of DOI:10.1016/j.bmcl.2003.09.090

Bioorganic & Medicinal Chemistry Letters 14 (2004) 531–534
7
-Hydroxynaphthalen-1-yl-urea and -amide antagonists of human
vanilloid receptor 1
a
a
b
b
Mark E. McDonnell, Sui-Po Zhang, Nadia Nasser, Adrienne E. Dubin
a,
and Scott L. Dax *
aJohnson & Johnson Pharmaceutical Research and 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 17 June 2003; accepted 30 September 2003
Abstract—A series of structurally simple 7-hydroxynaphthalenyl ureas and amides were discovered to be potent ligands of human
vanilloid receptor 1 (VR1). 1-(7-Hydroxynaphthalen-1-yl)-3-(4-trifluoromethylbenzyl)urea 5f exhibited nanomolar binding affinity
(K
i
=1.0 nM) and upon capsaicin challenge, behaved as a potent functional antagonist (IC50=4 nM). The synthesis and structure–
activity relationships (SARs) for the series are described.
2003 Elsevier Ltd. All rights reserved.
#
The pungency and antinociceptive properties of Capsi-
cum species have been recognized and used ther-
apeutically for centuries. The active ingredient,
1
capsaicin, is small molecule agonist of vanilloid recep-
tor type 1, a non-selective ligand- and temperature-
gated cation channel. Opening of the vanilloid receptor
Optimization via chemical synthesis, described herein,
rapidly afforded a series of hydroxynaphthenyl ureas
and amides that bind to human VR1 with nanomolar
affinity and behave as functional antagonists of VR1, in
the presence of capsaicin.
1
(VR1) channel leads to influx of cations (particularly
calcium), and subsequent depolarization of subpopula-
tions of mammalian sensory C- and Ad-fibers causing
initially excitation of primary afferents followed by
neuronal desensitization. These pharmacological
actions make capsaicin creams effective in relieving
minor muscle aches, but limited in clinical utility, due to
pungency.
N-Hydroxynaphthenyl ureas were synthesized in
straightforward fashion (Scheme 1). Aminonaphthol 2
was reacted with phenylchloroformate in the presence
of an aqueous base to form the corresponding phenyl-
carbamate 3. Subsequent reaction of hydroxy-
naphthalen-1-yl-carbamic acid phenyl ester 3 with
amines such as substituted anilines, benzylamines, and
phenethylamines (4), in dimethylsulfoxide (DMSO),
afforded the urea final products 5 in good yields. A
trialkylamine base was added to the reaction when the
amine was only available as an acid addition salt. The
choice of dimethylsulfoxide as solvent was important
with respect to obtaining clean and efficient conversion
The cloning of human and rat VR12 has reinvigorated
ꢀ4
research in vanilloids with the promise of developing
5
,6
novel therapeutic agents. In this regard, we chose to
pursue antagonists of VR1, hypothesizing that direct
blockade of the channel could provide a therapeutic
means to treat pain without pungency that is observed
with some agonists such as capsaicin. From a high
throughput functional assay, employing a Fluorometric
TM
7
Imaging Plate Reader (FLIPR ) technology, we iden-
tified urea 1 as a sub-micromolar agonist.
of carbamate 3 to urea 5 under mild conditions. Sepa-
rately, aminonaphthol 2 could be directly reacted with
isocyanates to give 5. Furthermore, amide congeners 6
were prepared simply by coupling aminonaphthol with
benzoic, phenylacetic, or dihydrocinnamic acids, or by
reacting aminonaphthol with the corresponding acid
chloride. O-Protecting groups could be employed to
*
0
Corresponding author. Tel.: +1-215-628-5211; fax: +1-215-628-
297; e-mail: sdax@prdus.jnj.com
3
960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.090

532
M. E. McDonnell et al. / Bioorg. Med. Chem. Lett. 14(200 4) 531–534
9
mask the naphthol (–OH) moiety in cases in which
functionality, other than a phenolic center, was proble-
matic. Along these lines, preliminary studies indicate
that the free phenol is necessary for activity. O-Methyl-
ation abolished VR1 affinity and functional activity.
Similarly, simple naphthalenyl congeners, lacking the
expressing human VR1 cDNA (hVR1/HEK293) were
3
incubated with [ H]-RTX and the test compounds.
3
After incubation and centrifugation, bound [ H]-RTX
was quantified and from this, K values were deter-
i
mined. Ureas 5 and amides 6 were also assayed for
ability to antagonize capsaicin-induced calcium flux
2
+
TM
8
-OH moiety, were poorly active (data not shown).
using a Ca -sensitive fluorescent dye and FLIPR
1
0
technology (Molecular Devices, Inc.). hVR1/HEK
4
Compounds 5–6 were assayed for binding affinity at
hVR1 using a modified version of the method pre-
Cells were first challenged by exposure to compound (5
and 6) to determine if they possess inherent agonist
activity. Antagonist potency was subsequently determined
using a similar protocol except that after incubation
with compound, cells were challenged with capsaicin at
a concentration eliciting ꢁ80% maximal response.
8
viously reported by Szallasi and Blumberg (Table 1).
Membranes harvested from a HEK293 cell line stably
Our starting point was 1-benzyl-3-(7-hydroxynaphtha-
len-1-yl)-urea 5a which had good binding affinity
(
antagonism of VR1 (IC =45 nM). N-Alkylated con-
K =94 nM) and exhibited significant functional
i
5
0
geners were poorly active in terms of binding affinity
(
N-Me 5l >N-Me 5b ꢁN-Et 5c >N-benzyl 5d) sugges-
tive of a requisite free NH group. The carbamate inter-
mediate 4 was also inactive (IC =6300 nM). However,
5
0
in the case of the ureas, addition of lipophilic inert
electron-withdrawing groups onto the benzylic portion
of the urea enhanced both binding affinity and func-
tional antagonist potency. The preferred site of sub-
stitution was the distal end of the molecule; specifically
para-substitution was slightly favored over meta-analo-
gues and ortho-analogues (not shown). Thus, 3-tri-
fluoromethylated urea 5e represented a significant
Scheme 1. (a) PhOC(O)Cl/aq NaHCO
3
/CH
2
Cl
2
(92%); (b) DMSO
ꢂ
(
(
45–97%); (c) Ar-L-N¼C¼O/MeCN, microwave, 100 C 5 min (45%);
d) Ar-l-CO
/CH
2
H, PyBROP, DIPEA, DMF; or Ar-l-C(O)Cl/aq
Cl (13–39%).
NaHCO
3
2
2
Table 1. Human VR1 binding affinities and functional activity of ureas 5 and amides 6a
X
L
R
Binding affinity
K
Functional activity
IC50 (nM)
i
(nM)
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
O
Nil
(H)
(H)
(H)
(H)
Ia
94
Ia
1110
3280
6300
45
Ia
Ia
Ia
26
4
ndb
27
nd
nd
15
nd
nd
90
17
21
770
320
100
a
b
c
d
e
f
g
h
i
NH
NMe
NEt
NCH Ph
NH
NH
NH
NH
NH
NH
NH
NMe
NH
NH
–CH
–CH
–CH
–CH
–CH
–CH
2
–
–
–
–
–
–
a
2
2
2
2
2
2
(H)
3-CF
4-CF
4-CF
3
3
3
2.1
1.0
4.0
1.0
7.0
2.8
2.0
581
53
1.0
–CH(CH
3
)–
–CH
–CH
–CH
–CH
–CH
2
2
2
2
2
–
4-OCF
3
–
–
–
–
3,4-diF
2,4-diCl
3,4-diCl
3,4-diCl
(H)
3,4-diCl
3,4-diCl
4-Cl
j
k
l
m
n
k
o
p
–(CH
–(CH
–CH
–CH
2
)
2
–
–
2
)
2
–CH
–CH
–CH
Nil
—
2
2
2
–
–
–
2
–
–
4.6
9.8
2
Nil
Nil
—
3,4-diCl
4-Cl
—
1830
28% inh @10 mM
120
c
q
CPZ
a
Ia, inactive (>10 uM).
nd, not done.
Percent inhibition @ 10 uM; CPZ, capsazepine.
b
c

M. E. McDonnell et al. / Bioorg. Med. Chem. Lett. 14(200 4) 531–534
533
breakthrough in being nearly two orders of magnitude
more potent in terms of binding affinity (K =2.1 nM)
5. Lee, J.; Lee, J.; Kim, J.; Kim, S. Y.; Chun, M. W.; Cho,
H.; Hwang, S. W.; Oh, U.; Park, Y. H.; Marquez, V. E.;
Beheshti, M.; Szabo, T.; Blumberg, P. M. Bioorg. Med.
Chem. 2001, 9, 19.
i
and 2-fold more potent as a functional antagonist
(
IC =26 nM) compared to the unsubstituted analogue
50
6
. Dax, S.; Dubin, A.; Jetter, M.; Nasser, N.; Shah, C.;
Swanson, D.; Carruthers, N. I. Vanilloid Receptor
Antagonists: Structure Activity Relationships via Parallel
and Targeted Synthesis. In 17th International Symposium
on Medicinal Chemistry, Barcelona, Spain, Sept 1–5, 2002.
5
benzyl)-urea 5f was even more potent with a K value of
a. 1-(7-Hydroxynaphthalen-1-yl)-3-(4-trifluoromethyl-
i
1 nM and an IC50 value of 4 nM. Methylation at the
benzylic carbon center retained strong VR1 affinity (5g:
K =4 nM). The trifluoromethylether congener 5h also
i
exhibited exquisite binding affinity (K =1 nM) but was
i
7. Thavonekham, B. Synthesis 1997, 1189.
8. Szallasi, A.; Blumberg, P. M. Naunyn-Schmiedeberg’s
Arch. Pharmacol. 1993, 347, 84.
less active in the functional assay (IC =27 nM) relative
5
0
3
to the directly trifluoromethylated 5f. The incorporation
of additional halogen substituents had relatively minor
impact on binding and functional activity, evident by
9. [ H]-RTX binding assay using hVR1/HEK293 cell mem-
branes. Cloning and generation of stable cell lines expressing
human VR1. Human VR1 was cloned and stably expres-
sed in HEK293 cells (hVR1/HEK293) as described by
Grant et al. in J. Pharm. Exp. Ther. 2002, 300, 9. Pre-
paration of membranes. hVR1/HEK293 were homo-
genized with a Polytron twice and centrifuged at 3000 rpm
for 10 min in HEPES buffer containing 20 mM HEPES,
1
1
highly active 5i (3,4-difluoro), 5j (2,4-dichloro) and 5k
3,4-dichloro) which all displayed low nanomolar affi-
nity for VR1.
(
Conversion of the urea moiety into an amide linkage
retained VR1 activity. For example, 3-(3,4-dichloro-
phenyl)-N-(7-hydroxynaphthalen-1-yl)-propionamide
2
pH 7.4, NaCl 5.8 mM, sucrose 320 mM, MgCl 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. Incu-
1
2
6
IC =17 nM) as the urea analogue 5k. The mono-
k
possessed similar affinity and potency (K =4.6 nM;
i
5
0
ꢂ
bation procedure. Incubations for 60 min at 37 C were
performed in a total volume of 0.5 mL that contained
chloro analogue 6o was also potent (K =9.8 nM;
i
IC =21 nM). However, moving the aryl moiety closer
5
0
3
1
(
20 mg/mL membrane protein and 0.3–0.6 nM [ H]-RTX
NEN, Boston, MA, USA) in the HEPES buffer. After
to the amide center significantly reduced activity, evi-
dent by both 2-(3,4-dichlorophenyl)-N-(7-hydroxy-
incubation, the samples were cooled on ice and 100 mg of
a-acid glycoprotein were added followed by centrifuga-
tion at 13,000 rpm for 15 min. The supernatant was aspi-
rated and the tips of tubes were cut off into 6-mL vials.
Nonspecific binding was measured in the presence of
200 nM unlabeled RTX in 4 mL scintillation liquid using
a Packard scintillation counter. Data analysis. Percent
1
3
naphthalen-1-yl)-acetamide 6p and 3,4-dichloro-N-(7-
1
4
hydroxynaphthalen-1-yl)-benzamide 6q.
In conclusion, ureas and amides derived from 8-amino-
naphthalen-2-ol and containing an appropriately posi-
tioned halosubstituted aryl moiety, display low nano-
molar affinity for human VR1 and behave as functional
antagonists of the channel upon capsaicin challenge.
These structurally simple molecules may provide a
starting point for the development of VR1 antagonists
as a potentially new family of analgesic agents. Indeed,
(
%)) inhibition=(total) bindingꢀtotal binding in pres-
cence of compound */100 (total) binding ꢀ non specific
binding. K values were obtained from Prism (GraphPad,
i
San Diego, CA, USA) calculated using equation of
Cheng–Prusoff ðKi ¼ IC50=ð1 þ ½LIGANDŠÞ=KdÞ.
4
1
0. hVR1/HEK cells were seeded on poly-d-lysine coated 96-
1
5
N-[4-(methylsulfonylamino)benzyl]-thioureas recently
have been disclosed as VR1 antagonists, thus demon-
strating that simple benzylic ureas, if appropriately
functionalized, can effectively modulate VR1.
well, black-walled plates (BD 354640) and 2 days later
loaded with Fluo-3/AM for 1 h and subsequently tested for
2
+
agonist-induced increases in intracellular Ca levels using
TM
FLIPR technology. Cells were challenged with single
concentrations of compound (3, 4, and 5) and intracellular
+
+
Ca
CAP to all wells to achieve a final CAP concentration of
5 nM to fully activate VR1. Antagonist potency was
was measured for 3 min prior to the addition of
Acknowledgements
1
determined using the protocol described by McDonnell et
al. (Bioorg. Med. Chem. 2002, 12, 1189). Data were ana-
lyzed using Prism software to calculate IC50 values.
We thank Michele Jetter, Jim McNally, Mark Young-
man, Andrea Works, Devin Swanson, Chandra Shah,
Nick Carruthers and Bill Jones for their input and
assistance.
1
1. 1-(3,4-Dichlorobenzyl)-3-(7-hydroxynaphthalen-1-yl)-urea
(5k). 3,4-Dichlorobenzylamine (53 uL, 0.39 mmol) and (7-
hydroxynaphthalen-1-yl)-carbamic acid phenyl ester
(
125 mg, 0.35 mmol) were combined and stirred at ambi-
ent temperature in DMSO (3 mL) overnight. The product
was purified by directly injecting the crude reaction onto a
reverse phase prep-HPLC (gradient 10–90% MeCN,
0.1% TFA). The appropriate fractions were lyophilized to
yield 1-(7-hydroxynaphthalen-1-yl)-3-(3,4-dichloro-phe-
References and notes
1
2
. Jancso, J. Bull. Millard Fillmore Hosp. (Buffalo) 1960, 7,
3.
. Caterina, M. J.; Schumacher, M. A.; Tominaga, M.;
5
+
nylmethyl)-urea (yield=63 mg, 49%). MS (MH ) 362.4;
analytical reverse-phase HPLC (gradient 10–90% MeCN,
Rosen, T. A.; Levine, J. D.; Julius, D. Nature (London)
1
1
997, 389, 816.
. Julius, D. J.; Caterina, M.; Brake, A. US Patent
,335,180, Jan 1, 2002.
. McIntyre, P.; James, I. F. US Patent 6,406,908, June 18,
002.
0.1% TFA) t =4.23 min, 99%. H NMR (CD OD) d
R
3
3
4
4.35 (s, 2H), 7.09 (dd, 1H, J=8.8, 2.4 Hz), 7.21–7.26 (m,
3H), 7.44–7.49 (m, 3H), 7.62 (d, 1H, J=8.2 Hz), 7.73 (d,
6
1H, J=8.9 Hz); HRMS: calcd for
360.0433; found 360.0432.
C H N Cl O :
18 14 2 2 2
2

534
M. E. McDonnell et al. / Bioorg. Med. Chem. Lett. 14(200 4) 531–534
1
2. 3-(4-Chlorophenyl)-N-(7-hydroxynaphthalen-1-yl)-propion-
amide (6o). Into a round-bottom flask equipped with a stir
bar was added 5 mL DMF, 3-(4-chlorophenyl)propanoic
347.8; analytical reverse-phase HPLC (gradient 10-90%
1
MeCN, 0.1% TFA) t =4.3 min, ꢁ99%. H NMR
R
3
(CD OD) d 3.83 (s, 2H), 7.09 (dd, 1H, J=8.8, 2.4 Hz),
acid (0.27 g, 1.2 mmol), iPr
aminonapth-7-ol (0.2 g, 1.2 mmol) and lastly PyBroP
0.585 g, 1.25 mmol). The reaction was stirred overnight at
2
NEt (0.22 mL, 1.26 mmol), 1-
7.17 (d, 1H, J=2.3 Hz), 7.23 (t, 1H, J=8.0), 7.35–7.42 (m,
2H), 7.51 (d, 1H, J=8.2 Hz), 7.61 (d, 1H, J=2.0 Hz), 7.66
(d, 1H, J=8.3 Hz), 7.74 (d, 1H, J=8.9 Hz); HRMS: calcd
(
room temperature. The product was purified by directly
injecting the crude reaction onto a reverse phase prep-
HPLC (gradient 10–90% MeCN, 0.1% TFA). The
appropriate fractions were lyophilized to yield 3-(4-chloro-
2 2
for C18H13NCl O : 345.0318; found 345.0323.
14. 3,4-Dichloro-N-(7-hydroxynaphthalen-1-yl)-benzamide (6q).
Synthesized in the same manner as 6o, substituting 3,4-
dichlorobenzoic acid for-(4-chlorophenyl)propanoic acid.
+
phenyl) - N - (7 - hydroxynaphthalen - 1 - yl) - propionamide
+
(yield=6.2 mg, 3%). MS (MH ) 333.3; analytical reverse-
phase HPLC (gradient 10–90% MeCN, 0.1% TFA)
(yield=150 mg, 13%). MS (MH ) 325.8, Analytical
reverse-phase HPLC (gradient 10–90% MeCN, 0.1%
1
t =4.3 min, ꢁ99%. H NMR (CD Oꢀ) d 7.10 (dd, 1H,
R
3
1
TFA) t
(
R
=3.5 min, ꢁ96%. H NMR (DMSO-d
6
) d 2.74
m, 2H), 3.0 (m, 2H), 7.09 (dd, 1H, J=8.8, 2.3 Hz), 7.19–
J=9.0 Hz), 7.21 (s, 1H), 7.31 (t, 1H, J=7.6), 7.35–7.42
(m, 2H), 7.46 (d, 1H, J=7.3 Hz), 7.72–7.8 (m, 3H), 7.95
(d, 1H, J=8.9 Hz), 8.24 (s, 1H); HRMS: calcd for
C H NCl O : 331.0170; found 331.01668.
7.39 (m, 7H), 7.45 (d, 1H, J=7.2 Hz), 7.62 (d, 1H,
J=7.9 Hz), 7.76 (d, 1H, J=8.9 Hz), 9.7 (s, 1H); HRMS:
calcd for C19 16NClO : 325.0864; found 325.0869.
1
7
11
2
2
H
2
15. Lee, J.; Lee, J.; Kang, M.; Shin, M.; Kim, J.-M.; Kang,
S.-U.; Lim, J.-O.; Choi, H.-K.; Suh, Y.-G.; Park, H.-G.;
Oh, U.; Kim, H.-D.; Park, Y.-H.; Ha, H.-J.; Kim, Y.-H.;
Toth, A.; Wang, Y.; Tran, R.; Pearce, L. V.; Lundberg,
D. J.; Blumberg, P. M. J. Med. Chem. 2003, 46, 3116.
1
3. 2-(3,4-Dichlorophenyl)-N-(7-hydroxynaphthalen-1-yl)-acet-
amide (6p). Synthesized in the same manner as 6o, sub-
stituting 3,4-dichlorophenylacetic acid for 3-(4-chloro-
+
phenyl)propanoic acid. (yield=84 mg, 39%). MS (MH )