N-Isoquinolin-5-yl-N′-aralkyl-urea and -amide antagonists of human vanilloid receptor 1

Article abstract of DOI:10.1016/j.bmcl.2004.04.038

Starting from a low micromolar agonist lead identified by high-throughput screening, series of N-isoquinolin-5-yl-N′-aralkyl ureas and analogous amides were developed as potent antagonists of human vanilloid receptor 1 (VR1). The synthesis and structure-activity relationships (SAR) of the series are described.

Full text of DOI:10.1016/j.bmcl.2004.04.038

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3053–3056
0
N-Isoquinolin-5-yl-N -aralkyl-urea and -amide antagonists of
human vanilloid receptor 1
a
Michele C. Jetter, Mark A. Youngman, James J. McNally, Sui-Po Zhang,
a
a
a
b
b
Adrienne E. Dubin, Nadia Nasser and Scott L. Dax
a,
*
a
Johnson & Johnson Pharmaceutical Research and Development, Welsh and McKean Roads, Spring House, PA 19477, USA
b
Johnson & Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, San Diego, CA 92121, USA
Received 10 February 2004; revised 12 April 2004; accepted 13 April 2004
0
Abstract—Starting from a low micromolar agonist lead identified by high-throughput screening, series of N-isoquinolin-5-yl-N -
aralkyl ureas and analogous amides were developed as potent antagonists of human vanilloid receptor 1 (VR1). The synthesis and
structure–activity relationships (SAR) of the series are described.
ꢀ
2004 Elsevier Ltd. All rights reserved.
The discovery and cloning of vanilloid receptors has
ushered in a new era of pharmaceutically based research
lead molecule, specifically the polar heterocyclic ‘head’
portion, apparently responsible for conferring func-
tional activity toward the channel, a urea or amide
linking group, and lastly, a lipophilic ‘tail’. In the first
regard, we observed that replacement of the 3-pyridyl
group with an isoquinoline moiety resulted in a dramatic
switch in functional activity, namely conversion of VR1
agonists to antagonists. Agonists stimulate calcium flux
1–4
aimed at delivering novel analgesics. Our labs
and
5;6
others have reported on various series of small mole-
cules that modulate vanilloid receptor type 1 (VR1).
Moreover, some congeners are reported to be effective in
rodent models of pain. For example, a series of N-phenyl-
4-pyridin-2-yl-piperazine carboxamides, exemplified by
the potent VR1 antagonist BCTC (N-(4-tert-butyl-
phenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1-
(effective concentrations (EC50
)
values reported)
whereas antagonists block capsaicin-induced flux (re-
ported as inhibitory concentrations (IC ) values). With
N-isoquinolin-5-yl-4-pentyl-benzamide (2) and N-iso-
(
behavioral effects of acute, inflammatory, and neuro-
2H)-carboxamide), have been shown to reverse
50
7
0
pathic pain in rats.
quinolin-5-yl-N -4-butylphenyl-urea (3) serving as pro-
totypes, we synthesized and evaluated urea and amide
analogs in attempt to enhance in vitro potency.
From a high throughput functional assay, employing
TM
FLIPR
(Fluorometric Imaging Plate Reader) tech-
2
nology and HEK cells transfected with hVR1, we
identified 4-pentyl-N-pyridin-3-yl-benzamide (1) as a
sub-micromolar agonist. Our decision to pursue this
N
H
N
O
‘
hit’ arose from recognition that this molecule possessed
H
N
2: hVR1: Ki = 71 nM;
hVR1: IC50 = 2.4 µM
antagonist
structural features similar to the naturally occurring
VR1 agonist capsaicin, from which the first VR1
antagonist, capsazepine, was elaborated. Thus, in
keeping with the approach Sandoz researchers under-
hit-to-lead
O
N
N
1
:
hVR1: EC50 = 0.9 µM
H
N
H
N
agonist
8
–10
took in developing capsaicin SAR, our efforts
focused upon exploration of three distinct regions of our
O
3
:
hVR1: Ki = 330 nM;
hVR1: IC50 = 490 nM
antagonist
Keywords: Vanilloid; VR1 antagonists; Pain.
*
Corresponding author. Tel.: +1-215-628-5211; fax: +1-215-628-3297;
e-mail: sdax@prdus.jnj.com
As reported herein, these efforts led to optimized
isoquinolin-5-yl-ureas and amides that possess
0
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.038

3054
M. C. Jetter et al. / Bioorg. Med. Chem. Lett. 14(200 4) 3053–3056
sub-nanomolar binding affinity (K
i
values given) for
series shifted the capsaicin concentration dependence to
the right in a parallel fashion in whole cell patch clamp
experiments on endogenously expressed rat VR1 (Dubin
et al., unpublished observations). Collectively, these
data allow us to speculate that isoquinolin-5-yl-ureas
and -amides act upon a common binding site also shared
by capsaicin and resiniferatoxin. Furthermore, these
small molecule hVR1 antagonists did not exhibit any
overt cytotoxic effects during the course of conducting
in vitro evaluations.
human VR1 in a transfected cell line and behave as
functional antagonists of VR1 at nanomolar concen-
trations, upon capsaicin challenge.
0
N-Isoquinolin-5-yl-N -aralkyl ureas 7 were prepared in a
straightforward manner, from the reaction of phenyl
carbamates of 5-aminoisoquinolines with anilines, benz-
yl amines, or phenethylamines (6), using dimethylsulf-
oxide as solvent. The intermediate isoquinolin-5-yl-
carbamic acid phenyl ester 5 was obtained via reaction
of 5-aminoisoquinoline with phenylchloroformate.
Separately, 5-aminoisoquinoline 4 could be directly
reacted with phenethyl-, benzyl- or, aryl-isocyanates to
yield ureas 7. N-Isoquinolin-5-yl-aralkylamides 8 were
generated from the coupling of aminoisoquinolines to
benzoic, phenylacetic, or phenylpropionic acids using
standard protocols. Urea products 7 could be directly
Optimized compounds were found to be one to two
orders of magnitude more potent than capsazepine.
Carbamate precursor 5 was inactive and N-methylation
of the urea and amide functionality abolished activity at
hVR1 (data not shown). In addition to an unmasked
(free N–H) urea or amide linking group, a requirement
for good in vitro potency is the proper positioning of an
isoquinoline group and an aryl moiety that contains a
highly lipophilic substituent at the distal end. The pre-
ferred spacing groups (L) are short alkyl moieties with
13–15
isolated from the reaction by precipitation;
adducts 8 were obtained via extraction and purification
amide
16
by chromatography (Scheme 1).
benzylic versions (L ¼ –CH –) being optimal as illus-
2
Isoquinolin-5-yl-derived ureas and amides 7–8 were
assayed for binding affinity at hVR1 by measuring dis-
placement of radiolabeled resiniferatoxin (RTX),
employing a modified version of the method previously
trated by benzylic ureas 7j and 7i compared to their
phenyl/phenethyl homologs 7c/7r, and 7b/7q, respec-
tively. This general trend carries over to highly potent
propionamide congeners. Interestingly, this feature was
noted in a series of 7-hydroxynaphthalen-1-yl-urea and
11
reported by Szallasi and Blumberg. The compounds
were also assayed for their ability to antagonize capsa-
2
-amide VR1 antagonists previously disclosed.
2þ
icin-induced calcium flux using a Ca -sensitive fluo-
TM
rescent dye and FLIPR
technology (Molecular
Devices, Inc.) under conditions previously reported by
With respect to aryl substituents, lipophilicity appears to
be an overriding factor rather than electronics since
both alkylated and halogenated congeners are excep-
tionally potent. Thus although the t-butyl benzyl and
phenethyl ureas 7l and 7s are exquisitely potent, the
halogenated analog 7k is comparable. However, com-
pounds with less lipophilic aryl tails, such as mono-
substituted variations, are significantly less active (e.g.,
compare 7b vs 7d and 7g vs 7k). Moreover, the place-
ment of nonlipophilic electron-donating aryl substitu-
ents is detrimental as evidenced by the methoxylated
compounds 7e and 7n. However, in contrast, the triflu-
oromethoxylated congeners (7f,o) exhibit low nano-
12
1–3
our group.
Both isoquinolin-5-yl-ureas and -amides 7–8 exhibited
excellent binding affinity to VR1 and behaved as func-
tional antagonists of the channel (Table 1). Overall,
i
binding affinity (K values) correlated with functional
inhibitory concentrations in terms of potency, within an
order of magnitude, despite the use of different agonists
in each assay, namely resiniferatoxin (RTX) in the case
of binding and capsaicin (CAP) as challenge for func-
tional antagonism. Furthermore, antagonists from this
molar potency.
A
compound lacking any aryl
substitution 7m was poorly active further supporting the
necessity of added lipophilicity to the distal end of the
aralkyl urea in order to maximize in vitro potency.
Propionamides 8a–d and cinnamides 8e–f followed
similar trends in that the more lipophilic aryl substitu-
ents were preferred (compare 8c vs 8a). Collectively, the
results of our studies suggest that a preliminary working
pharmacophore for VR1 binding and antagonism con-
sists simply of an isoquinolinyl-derived urea or amide
coupled to a lipophilic tail. However, it is clear from our
N
N
H
N
R'
HN
a
R
NH
2
O
+
L
O
6
4
5
b
c
d
R'
N
H
N
R
N
L
O
2;4
previous research, as well as work from other labo-
5;6
N
H
7
L
ratories, that heterocycles and functional groups other
than an isoquinoline moiety can confer antagonist
properties to similarly designed small molecules.
N
R
O
8
L = -(CH
2 n
) - where n = 0-2,
-
CH=CH-
In summary, we report upon a series of isoquinolin-5-yl-
ureas and -amides that bind to human VR1 with
nanomolar affinity and behave as functional antagonists
of VR1, upon capsaicin challenge, with similar potency.
These structurally simple molecules, recently disclosed
Scheme 1. (a) PhOC(O)Cl/aq NaHCO
45–97%); (c) Ar–L–N@C@O/CH Cl
HBTU, or HATU, DIEA/DMF; or Ar–L–C(O)Cl, DIEA/CH
20–65%).
3
/CH
(50–80%); (d) Ar–L–CO
CN
2
Cl
2
(92%); (b) DMSO
(
2
2
2
H,
3
(

M. C. Jetter et al. / Bioorg. Med. Chem. Lett. 14(200 4) 3053–3056
3055
0
a
Table 1. Human VR1 binding affinities and functional activity of N-aralkyl-N -isoquinolin-5-yl-derived ureas 7 and amides 8
3
R
4
N
H
N
X
L
O
X
L
R
Binding affinity
(nM)
Functional activity
IC50 (nM)
K
i
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
a
b
c
d
e
f
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
Nil
Nil
3-CF
4-CF
3
19
32
225
170
32
45
3
3
160
17
Nil
Nil
3,4-Di-Cl
3,5-Di-CF
4-CH O–
O–
3
1.4
193
1.3
–CH
–CH
–CH
–CH
–CH
–CH
–CH
–CH
2
2
2
2
2
2
2
2
–
–
–
–
–
–
–
–
3
4-CF
4-Cl
3
g
h
i
31
23
5
6
3-CF
4-CF
3
11
3
3
j
3,4-Di-Cl
9
4
k
l
4-Cl, 3-CF
4-t-Bu
(H)
3
0.8
0.3
1440
6
3
m
n
o
p
q
r
–(CH
–(CH
–(CH
–(CH
–(CH
–(CH
–(CH
2
2
2
2
2
2
2
)
2
2
2
2
2
2
2
–
1000
680
11
19
19
10
3
)
)
)
)
)
)
–
–
–
–
–
–
4-CH
3
O–
O–
1290
6
4-CF
3-CF
4-CF
3
3
3
41
22
17
3,4-Di-Cl
4-t-Bu
4-Cl
s
1.3
a
b
c
d
e
f
–(CH
–(CH
–(CH
–(CH
2
2
2
2
)
)
)
)
2
2
2
2
–
–
–
–
98
60
4
74
38
13
410
1500
5
4-CF
3
4-t-Amyl
4-t-Bu
(H)
11
400
–CH@CH–
–CH@CH–
4-t-Bu
1.2
120
Capsazepine
100
1
7;18
in patent applications,
might serve as a template for
vanilloid VR1 receptor: McDonnell, M. E.; Zhang, S.-P.;
Dubin, A. E.; Dax, S. L. Bioorg. Med. Chem. Lett. 2002,
the design of future VR1 antagonists with the goal of
providing novel therapeutics for the treatment of pain in
humans.
1
2, 1189.
4
5
. Codd, E.; Dax, S. L.; Jetter, M.; McDonnell, M.;
McNally, J. J.; Youngman, M. WO 2003097586A1.
. TRPV1 Agonists and Antagonists: Appendino, G.;
Munoz, E.; Fiebich, B. L. Expert Opin. Ther. Pat. 2003,
1
3, 1825, and references cited therein.
Acknowledgements
6. Small Molecules Targeting the Vanilloid Receptor Com-
plex as Drugs for Inflammatory Pain: Planells-Cases, R.;
Garcia-Martinez, C.; Royo, M.; Perez-Paya, E.; Carreno,
C.; Messeguer, F. A. A.; Ferrer-Montiel, A. Drugs Future
We thank Mark McDonnell, Andrea Works, Neela-
kandha Mani, Chandra Shah, Devin Swanson, and
Nick Carruthers for their valuable input into this pro-
ject.
2
. Pomonis, J. D.; Harrison, J. E.; Mark, L.; Bristol, D. R.;
003, 28, 787, and references cited therein.
7
8
9
Valenzano, K. J.; Walker, K. J. Pharm. Exp. Ther. 2003,
3
06, 387.
. Walpole, C. S.; Wrigglesworth, R.; Bevan, S.; Campbell,
E. A.; Dray, A.; James, I. F.; Perkins, M. N.; Reid, D. J.;
Winter, J. J. Med. Chem. 1993, 36, 2362.
. Walpole, C. S.; Wrigglesworth, R.; Bevan, S.; Campbell,
E. A.; Dray, A.; James, I. F.; Masdin, K. J.; Perkins,
M. N.; Winter, J. J. Med. Chem. 1993, 36, 2373.
References and notes
1
. 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, September 1–
10. Walpole, C. S.; Wrigglesworth, R.; Bevan, S.; Campbell,
E. A.; Dray, A.; James, I. F.; Masdin, K. J.; Perkins, M.
N.; Winter, J. J. Med. Chem. 1993, 36, 2381.
3
5
, 2002.
11. [ H]-RTX binding assay using hVR1/HEK293 cell mem-
2
3
. 7-Hydroxy-naphthalen-1-yl-urea and -amide antagonists
of human vanilloid receptor 1: McDonnell, M. E.; Zhang,
S.-P.; Nasser, N.; Dubin, A. E.; Dax, S. L. Bioorg. Med.
Chem. Lett. 2004, 14, 531.
. Synthesis and in vitro evaluation of a novel iodinated
resiniferatoxin derivative that is an agonist at the human
branes. Cloning and generation of stable cell lines express-
ing human VR1. Human VR1 was cloned and stably
expressed in HEK293 cells (hVR1/HEK293) as described
by Grant et al. J. Pharm. Exp. Ther. 2002, 300. Prepara-
tion of membranes. hVR1/HEK293 were homogenized
with a Polytron twice and centrifuged at 3000 rpm for

3056
M. C. Jetter et al. / Bioorg. Med. Chem. Lett. 14(200 4) 3053–3056
1
0 min in HEPES buffer containing 20 mM HEPES,
pH 7.4, NaCl 5.8 mM, sucrose 320 mM, MgCl 2 mM,
CaCl 0.75 mM, and KCl 5 mM. The supernatant was
14. 1-Isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea (7i):
Synthesized in the same manner as (7b) substituting 4-
2
2
trifluoromethyl-benzyl isocyanate for 4-trifluoromethyl-
1
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
phenyl isocyanate (2.1 g, 60%; mp 216–218 ꢁC). H NMR
(MeOH-d ): d 4.4 (s, 2H), 7.4–7.6 (m, 5H), 7.7 (d, 1H), 7.8
4
13
(d, 1H), 8.1 (d, 1H), 8.3 (d, 1H), 9.1 (s, 1H); C NMR
(CDCl , CD OD): d 44 (CH ), 115 (2C), 123 (2C), 126
(2C), 128 (2C), 130 (3C), 134, 142 (2C), 144, 153, 157
(C@O); IR (KBr, thin film, cm ): 3276, 1636 (C@O),
1568, 1327, 1111, 1068, 828; MS (MH ): 346.1; HRMS
3
3
2
were performed in a total volume of 0.5 mL that contained
3
ꢁ1
120 lg/mL membrane protein and 0.3–0.6 nM [ H]-RTX
þ
(
NEN, Boston) in the HEPES buffer. After incubation, the
þ
samples were cooled on ice, and 100 lg of a-acid glyco-
protein were added followed by centrifugation at
(m=z): MH calcd for C18
346.117.
H
15
F
3
N
3
O, 346.117; found,
1
3,000 rpm for 15 min. The supernatant was aspirated
15. 1-Isoquinolin-5-yl-3-[2-(4-trifluoromethyl-phenyl)-ethyl]-
urea (7q): Synthesized in the same manner as (7b)
substituting 4-trifluoromethyl phenethyl isocyanate for 4-
and the tips of tubes were cut off into 6 mL vials.
Nonspecific binding was measured in the presence of
2
00 nM unlabeled RTX in 4 mL scintillation liquid using a
trifluoromethyl-phenyl isocyanate (0.56 g, 78%; mp 214–
1
Packard scintillation counter. Data analysis. Percent (%)
inhibition ¼ (total binding)total binding in presence of
216 ꢁC). H NMR (MeOH-d
4
): d 3.0 (t, 2H), 3.6 (t, 2H),
7.5–7.6 (m, 4H), 7.65 (t, 1H), 7.75 (d, 1H), 7.85 (d, 1H), 8.2
1
3
compound)*100/(total binding)nonspecific binding). K
i
(d, 1H), 8.5 (d, 1H), 9.2 (s, 1H); C NMR (CDCl
CD OD): d 36 (CH ), 41 (CH ), 115, 123 (2C), 124, 126
(3C), 128, 129 (2C), 130, 134, 142, 144, 153, 157 (C@O);
3
,
values were obtained from Prism (GraphPad, San Diego,
CA) calculated using equation of Cheng–Prusoff
3
2
2
ꢁ1
(K
i
¼ IC50=ð1 þ ½LIGANDꢀ=K
d
).
2. hVR1/HEK cells were seeded on poly-
IR (KBr, thin film, cm ): 3303, 1633 (C@O), 1578, 1332,
1127, 1071, 827; MS (MH ): 360.5; HRMS (m=z): MH
calcd for C19 O, 360.132; found, 360.120.
3
þ
þ
1
D
-lysine coated 96-
well, black-walled plates (BD 354640) and 1–2 days later
loaded with Fluo-3/AM for 1 h and subsequently tested
for agonist-induced increases in intracellular Ca levels
TM
using FLIPR technology. Cells were challenged on line
16 3 3
H F N
16. N-Isoquinolin-5-yl-3-(4-trifluoromethyl-phenyl)-propion-
amide (8b): 4-(Trifluoro-methyl)hydrocinnamic acid
2
þ
2 2
(0.0018 mol, 0.39 g) was dissolved in 25 mL CH Cl ,
with compounds (7 and 8) at final concentrations ranging
from 0.3 nM to 30 lM in half-log increments. Intracellular
Ca was measured for 3 min prior to the addition of CAP
followed by the addition of diisopropylethyl amine
0
(0.0018 mol, 0.23 g). O-(7-Aza-benzotriazol-1-yl)-N,N,N ,
0
2
þ
N -tetramethyluronium hexa-fluorophosphate (HATU)
to all wells to achieve a final CAP concentration of 15 nM
(
protocol described by McDonnell et al. (Bioorg. Med.
Chem. 2002, 12, 1189). Data were analyzed using Prism
software to calculate IC50 values.
(0.0021 mol, 0.80 g), was added and the reaction mixture
was stirred at room temperature for 15 min.
5-Aminoisoquinoline (0.0015 mol, 0.22 g) was added and
the reaction mixture was stirred at room temperature for
16 h. The reaction mixture was washed with 25 mL of
saturated sodium bicarbonate, with 25 mL of brine, dried
over magnesium sulfate, and evaporated in vacuo. The
residue was chromatographed on silica gel eluting with 3%
EC80). Antagonist potency was determined using the
1
3. 1-Isoquinolin-5-yl-3-(4-trifluoromethyl-phenyl)-urea (7b):
The 5-aminoisoquinoline (0.002 mol, 0.29 g) was dissolved
in 10 mL of methylene chloride. The 4-trifluoromethyl-
phenyl isocyanate (0.0022 mol, 0.41 g) was slowly added
via syringe to the stirred solution. The reaction mixture
was stirred at room temperature for 4 h. The resultant
precipitate was collected by vacuum filtration. The
collected solid was triturated with hexane (2 · 5 mL). This
material was recrystallized from ethyl acetate to yield
MeOH/CH
brown solid (0.25 g, 48%; mp 155–157 ꢁC). H NMR
(MeOH-d ): d 2.9 (t, 2H), 3.2 (t, 3H), 7.5–7.65 (m, 3H),
7.7–7.8 (m, 3H), 7.9–8.0 (m, 2H), 8.5 (d, 1H), 9.2 (s, 1H);
2 2
Cl . The product was obtained as an orange-
1
4
1
3
C NMR (CDCl
126 (3C), 127, 128, 129 (4C), 131, 132, 142, 145, 152, 173
3
, CD
3
2 2
OD): d 38 (CH ), 43 (CH ), 116,
1
ꢁ1
product (0.48 g, 72%; mp 188–190 ꢁC). H NMR (MeOH-
(C@O); IR (KBr, thin film, cm ): 3303, 1633 (C@O),
1578, 1332, 1127, 1071, 827; MS (MH ): 345.121; HRMS
(m=z): MH calcd for C19
345.122.
þ
d
1
4
): d 7.2 (d, H), 7.4 (t, 1H), 7.5 (d, 1H), 7.6 (t, 1H), 7.7 (d,
H), 7.8 (s, 1H), 7.9 (d, 1H), 8.35 (d, 1H), 8.6 (d, 1H), 9.2
þ
H
16
F
3
N
2
O, 344.3; found,
1
3
(
s, 1H); C NMR (CDCl
3
, CD
25, 126, 127 (2C), 129, 130, 134, 143 (2C), 153, 154
3
OD): d 119, 123, 124 (2C),
1
17. Brown, R. E.; Doughty, V. A.; Hollingworth, G. J.; Jones,
A. B.; Lindon, M. J.; Moyes, C. R.; Rogers, L. WO
2003080578A1, 2003.
18. Codd, E.; Dax, S. L.; Jetter, M.; McDonnell, M.;
McNally, J. J.; Youngman, M. WO 2004007459A2, 2004.
ꢁ
1
(
1
C@O); IR (KBr, thin film, cm ): 3253, 1605 (C@O),
þ
557, 1317, 1113, 1066, 840; MS (MH ): 332.4; HRMS
þ
(m=z): MH calcd for C17
H
13
F
3
N
3
O, 332.101; found,
332.101.