6,6-Fused heterocyclic ureas as highly potent TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.12.086

A series of N-[{2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)-pyridin-3-yl}methyl] N′-(6,6-fused heterocyclic) ureas have been investigated as hTRPV1 antagonists. Among them, compound 15 showed highly potent TRPV1 antagonism to capsaicin, with K_i(ant) = 0.2 nM, as well as antagonism to other activators, and it was efficacious in a pain model. A docking study of 15 with our hTRPV1 homology model indicates that there is crucial hydrogen bonding between the ring nitrogen and the receptor, contributing to its potency.

Full text of DOI:10.1016/j.bmcl.2014.12.086

Accepted Manuscript
6,6-Fused heterocyclic ureas as highly potent TRPV1 antagonists
Wei Sun, Hyo-Shin Kim, Sunho Lee, Aeran Jung, Sung-Eun Kim, Jihyae Ann,
Suyoung Yoon, Sun Choi, Jin Hee Lee, Peter M. Blumberg, Robert Frank-
Foltyn, Gregor Bahrenberg, Klaus Schiene, Hannelore Stockhausen, Thomas
Christoph, Sven Frormann, Jeewoo Lee
PII:
S0960-894X(14)01394-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.12.086
BMCL 22331
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 December 2014
23 December 2014
25 December 2014
Please cite this article as: Sun, W., Kim, H-S., Lee, S., Jung, A., Kim, S-E., Ann, J., Yoon, S., Choi, S., Lee, J.H.,
Blumberg, P.M., Frank-Foltyn, R., Bahrenberg, G., Schiene, K., Stockhausen, H., Christoph, T., Frormann, S., Lee,
J., 6,6-Fused heterocyclic ureas as highly potent TRPV1 antagonists, Bioorganic & Medicinal Chemistry Letters
(2015), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.086
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Graphical Abstract
6,6-Fused Heterocyclic Ureas as Highly Potent TRPV1 Leave this area blank for abstract info.
Antagonists
Wei Sun, Hyo-Shin Kim, Sunho Lee, Aeran Jung, Sung-Eun Kim, Jihyae Ann, Suyoung Yoon, Sun Choi, Jin
Hee Lee, Peter M. Blumberg, Robert Frank-Foltyn, Gregor Bahrenberg, Klaus Schiene, Hannelore Stockhausen,
Thomas Christoph, Sven Frormann, Jeewoo Lee*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
6,6-Fused Heterocyclic Ureas as Highly Potent TRPV1 Antagonists
b
Wei Sun a,b, Hyo-Shin Kim b, Sunho Lee b, Aeran Jung b, Sung-Eun Kim b, Jihyae Ann b, Suyoung Yoon ,
Sun Choi c, Jin Hee Lee c, Peter M. Blumberg d, Robert Frank-Foltyn e, Gregor Bahrenberg e, Klaus Schiene
e, Hannelore Stockhausen e, Thomas Christoph e, Sven Frormann e, Jeewoo Lee b,*
a Shenyang Pharmaceutical University, Shenyang 110016, China
b Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
c National Leading Research Laboratory of Molecular Modeling & Drug Design, College of Pharmacy, Graduate School of Pharmaceutical Science and Global
Top 5 Research Program, Ewha Womans University, Seoul 120-750, Korea
d Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA
e Grunenthal Innovation, Grunenthal GmbH, D-52078 Aachen, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of N-[{2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)-pyridin-3-yl}methyl]
N’-(6,6-fused heterocyclic) ureas have been investigated as hTRPV1 antagonists.
Among them, compound 15 showed highly potent TRPV1 antagonism to capsaicin,
with Ki(ant) = 0.2 nM, as well as antagonism to other activators, and it was efficacious
in a pain model. A docking study of 15 with our hTRPV1 homology model indicates
that there is crucial hydrogen bonding between the ring nitrogen and the receptor,
contributing to its potency.
Keywords:
Vanilloid Receptor 1
TRPV1 Antagonist
Analgesic
2009 Elsevier Ltd. All rights reserved.
Molecular Modeling
The transient receptor potential V1 (TRPV1) receptor1 is a
(Figure 1).5 The pharmacophoric analysis of these leads indicates
that the 5-isoquinolinyl urea as the A/B-region and 4-substituted
benzyl group as the C-region represent principal pharmacophores,
respectively.
molecular integrator of nociceptive stimuli, including protons,
heat, inflammatory endogenous mediators and natural vanilloids
such as capsaicin and resiniferatoxin. Since the receptor
activation leads to an increase in intracellular Ca2+ that results in
excitation of primary sensory neurons and ultimately the central
perception of pain, TRPV1 antagonists have been actively
developed as novel analgesic and antiinflammatory agents,
particularly for chronic pain and inflammatory hyperalgesia.2
Although hyperthermia associated with TRPV1 antagonism is in
some instances problematic, TRPV1 is still one of most attractive
drug targets for the treatment of neuropathic pain. The clinical
development of TRPV1 antagonists in the pharmaceutical
industry has been extensively reviewed.3
As part of our continuing program for the discovery of
novel TRPV1 antagonists for the treatment of neuropathic pain,
we have investigated a set of novel antagonists with a N-6,6-
fused heteroaryl urea moiety as the A/B-region and a library of
substituted heteroaryls as the C-region, which was designed
based on the our pharmacophore model with the above leads.
Previously, the Abbott group reported a series of ureas with
a
variety of 6,6-fused nitrogen heterocycles as TRPV1
antagonists, among which the 5-isoquinoline-containing
compound 1 exhibited good oral bioavailability and in vivo
activity in animal models (Figure 1).4 Similarly, the Johnson &
Johnson group had described N-isoquinoline-5-yl-N’-aralkyl
ureas and amide antagonists, among which compound
2
exhibited excellent potency in receptor binding and antagonism
Figure 1. Discovery of potent TRPV1 antagonists

The extensive and systematic structure-activity relationship
investigation of the series indicated that the 5-isoquinolinyl urea
moiety as the A/B-region and a series of {2-substituted-6-
(trifluoromethyl)-pyridin-3-yl}methyl moieties as the C-region6
contributed high potency in antagonism. Among them, we
identified compound 15 to be highly potent for antagonism of
capsaicin, with an IC50 = 0.2 nM, as well as a potent antagonist
for stimulation by heat or N-arachidonoyl dopamine (NADA),
and it is thus about 90 fold more potent than the parent
compound 1 under the same conditions.
aminoquinazoline for 11 and 12 were synthesized from the
anilines, 1,3-diaminobenzene and 1,4-diaminobenzene,
respectively, by Chilin’s method8 (Scheme 4). 8-Amino
isoquinoline for 14 was prepared by catalytic hydrogenation from
commercially available 5-bromo-8-nitro isoquinoline (Scheme
5).4 4-Amino quinoline for 17 was prepared from commercially
available 4-nitroquinoline N-oxide using Fe/AcOH reduction
(Scheme 6). 4-Amino isoquinoline for 18 was prepared from
commercially available 4-bromoisoquinoline in two steps
(Scheme 7). 5-Amino quinoxaline for 20 was prepared from 3-
nitro-1,2-diaminobenzene according to the literature procedure
(Scheme 8). 10
In this paper, we report the structure activity relationships
of the A-region with 6,6-fused heterocycles in the series while
the B/C-region was fixed as the N-[{2-(4-methylpiperidin-1-yl)-
6-(trifluoromethyl)-pyridin-3-yl}methyl]urea group. Further
biological characterization and molecular modeling of compound
15 will also be described.
The target compounds (3-21) were synthesized in general
by the carbamylation of the aryl amine A-region with phenyl or
trichloromethyl chloroformates, followed by coupling with the
pyridylmethyl amine of the C-region, as represented in Scheme
1.
Scheme 3. Reagents and conditions: (a) NaBH4, BF3 etherate, 1,2-
dimethoxyethane, >99%; (b) oxalyl chloride, DMSO, NEt3, CH2Cl2, 50%; (c)
NH2NH2·H2O, CH2Cl2, EtOH, 51%; (d) H2, 10% Pd/C, MeOH, 95%
Scheme 4. Reagents and conditions: (a) ClCO2Et, NEt3, THF, 90-97%; (b)
(1) HMTA, TFA and (2) KOH, K3Fe(CN)6, EtOH, H2O, 32-47%; (c)
CCl3COCl, NEt3, CH2Cl2, 75-78%; (d) R-NH2, DBU, MeCN, 32-39%
Scheme 1. Reagents and conditions: (a) Method A: PhOCOCl, pyridine,
CH3CN-THF or Method B: CCl3COCl, TEA, CH2Cl2; (b) Method A: R-NH2,
TEA, DMSO or Method B: R-NH2, DBU, CH3CN
The C-region amine,
2-(4-methylpiperidin-1-yl)-6-
(trifluoromethyl)-pyridin-3-methylamine, was synthesized by the
nucleophilic addition of 4-methylpiperidine to 2-chloro-6-
trifluoromethyl-3-pyridinecarbonitrile which was prepared in
three steps starting from ethyl vinyl ether as shown in Scheme 2.
Scheme 5. Reagents and conditions: (a) H2, 10% Pd/C, MeOH, >99%
Scheme 6. Reagents and conditions: (a) Fe, AcOH, >99%
Scheme 7. Reagents and conditions: (a) benzophenoneimine, Pd(OAc)2,
NEt3, Cs2CO3, DPPF, BINAP, toluene, 58%; (b) 2M HCl, THF, EtOH, 97%
Scheme 2. Reagents and conditions: (a) (CF3CO)2O, pyridine, CHCl3, 50%;
(b) NCCH2CONH2, K2CO3, toluene, 35%; (c) POCl3, 90%; (d) 4-
methylpiperidine, K2CO3, 18-C-6, CH3CN, 90%; (e) 2M BH3-SMe2 in THF,
70%
The A-region 6,6-fused aryl amines were either obtained
from commercial sources or prepared by the modification of
known methods as illustrated in Schemes 3-8. The A-region
amines for the syntheses of 3-9, 13, 15, 16, 19, and 21 were
commercially available. 6-Amino phthalazine for 10 was
prepared from 4-nitro phthalic acid in four steps according to the
literature procedure (Scheme 3).7 7-Aminoquinazoline and 6-
Scheme 8. Reagents and conditions: (a) glyoxal, EtOH, 90%; (b) Fe, AcOH,
76%

Table 1. In vitro hTRPV1 antagonism to capsaicin
The synthesized compounds were evaluated for their ability
to inhibit the effect of the given concentration of capsaicin (100
nM) on hTRPV1 as determined using a fluorescence imaging
plate reader (FLIPR) and inhibitory values were expressed as (f)
6a
Ki(ant)
.
The results are presented in Table 1, together with the
potencies of the parent antagonists 1 and 2.
The A-region of the synthesized compounds has one of the
two geometries, –fused (compounds 3-12) or –fused
(compounds 13-21), depending on the relative position of the A-
region to the B-region urea. Among the –fused analogues,
compounds 3-9 include 7 different mono-aza heterocycles and
their antagonism appears to be sensitive to the position of the
nitrogen. Generally, whereas the distal aza-compounds 4-6
showed good antagonism with Ki(ant) values ranging from 7-9 nM,
the proximal aza-compounds 3 and 9 were weak or inactive
although compounds 7 and 8 were exceptional. The diaza
analogues phthalazine 10 and quinazoline 11 showed similar
potency to the distal aza surrogates, whereas quinazoline 12 was
found to be an agonist.
R
(f) Ki(ant)
(nM)
R
(f) Ki(ant)
(nM)
1
18.3
2
13.8
-Fused
-Fused
3
4
NE
7.1
8.4
7.2
WE
9.5
WE
8.9
7.8
AG
13
14
15
16
17
18
19
20
21
91.6
0.4
Among the –fused A-region analogues, compounds 13-19
also represent the 7 different mono-aza analogues as above. As
found for the –fused derivatives, the distal aza-analogues 14-16
also exhibited excellent antagonism in the subnanomolar range.
In particular, isoquinolin-5-yl urea 15 displayed highly potent
antagonism with Ki = 0.2 nM, which was about 90 fold more
potent than compound 1 under the same conditions. The
structural comparision of the C-regions between compounds 1
and 15 indicated that the 4-methylpiperidinyl moiety in
compound 15 contributes high potency in antagonism probably
due to enhanced specific binding interactions with the receptor.
On the other hand, the proximal aza-analogues 17-19 showed
quite different functional profiles. Whereas compound 18 showed
potent antagonism, compounds 17 and 19 exhibited agonism and
loss of activity, respectively. The diaza analogues quinoxaline 20
and naphthyridine 21 displayed reduced potency.
5
0.2
6
0.7
7
AG
1.8
8
The SAR analysis indicates that a hydrogen bonding
interaction between the ring nitrogen of the A-region and the
receptor is critical for antagonism, as indicated by the positional
sensitivity of the ring nitrogen for activity, and further indicates
that the ring nitrogen needs to be present at the distal area of the
ring. Additional hydrogen bonding at the proximal area may also
be important because compounds 8 and 18, containing a proximal
nitrogen at the same position, also showed good potency.
9
NE
48.1
NE
10
11
12
Since compound 15 was identified as the most potent
antagonist to capsaicin in this series, its antagonism to other
activators including pH, N-acetyl dopamine (NADA), and heat
was also evaluated as shown in Table 2. The potency of
compound 15 for antagonism to capsaicin was ca. 70-90 fold
higher than reference compounds 1 and 2 under the same
conditions. In addition, compound 15 also antagonized N-
acetyldopamine (NADA) and heat with high potency. However,
it appeared not to antagonize pH activation under our system.
AG: agonism, WE: weakly effective (7: 40% inhibition at 1 uM, 9: 10%
inhibition at 1 uM), NE: not effective
a
The analgesic activity of compound 15 was examined in the
formalin animal model. When administered orally in mice,
compound 15 was found to be efficacious at 0.3 and 10 mg/kg,
respectively, where it showed 18% and 73% maximal possible
effect (% MPE). The detailed in vivo evaluation will be presented
elsewhere.
Table 2. in vitro antagonism of compound 15 for various
activators of hTRPV1
CAP
pH
NADA
HEAT(45oC)
(f) Ki (nM)
IC50 (nM)
(f) Ki (nM)
IC50 (nM)
0.2
NE
0.02
0.55

(A)
(B)
(C)
LP
0.14
0.12
0.09
0.07
0.05
0.03
0.01
Leu547
Thr550
-0.02
-0.04
-0.06
Leu515
-0.08
-0.11
-0.13
Tyr511
-0.15
-0.17
-0.19
Ser512
Figure 2. Flexible docking result of 15 in the hTRPV1 model
(A) Binding mode of 15. The key interacting residues are marked and displayed as a capped-stick with carbon atoms in white. The helices are colored in gray and
the helices of the neighboring monomer are displayed as a line ribbon. The ligand is depicted as a ball-and-stick with carbon atoms in magenta and its van der
Waals surface is presented with lipophilic potential property (LP) which ranges from brown (highest lipophilic area) to blue (highest hydrophilic area). Hydrogen
bonds are drawn in black dashed lines and non-polar hydrogens are undisplayed for clarity. (B) Surface of hTRPV1 and 15. The Fast Connolly surface of
hTRPV1 was generated by MOLCAD and colored by the lipophilic potential. For clarity, the surface of hTRPV1 is Z-clipped and that of the ligand is in its
carbon color. (C) Lipophilic potential property of 15.
In order to investigate the key interactions of compound 15
with the receptor, we carried out the docking study of compound
15 with the human TRPV1 homology model, which was built on
our rat TRPV1 homology model as reported recently11 and
modified to include the sequence differences between rat and
human TRPV1 in the vicinity of the binding site. As shown in
Figure 2, compound 15 fitted well to the binding site of hTRPV1
which has a deep bottom hole, surrounded by Tyr511 and
Ser512, and an upper hydrophobic region containing Leu547.
The isoquinoline group of 15 in the A-region occupied the deep
bottom hole and formed a hydrogen bonding with Ser512. In
addition, its urea group as the B-region interacted with Tyr511
by hydrogen bonding. The C-region of 15 oriented toward the
upper region of the binding site and the hydrophobic CF3 group
fitted very well, making the hydrophobic interaction with
Leu547. This docking result identified binding interactions
between the three regions of 15 and hTRPV1 and the high
potency of 15 can be understood in terms of criticial hydrogen
bonding between the isoquinoline nitrogen and Ser512.
Acknowledgments
This research was supported by Grants from Grunenthal, the
Korea Health Technology R&D Project (HI13D2358), National
Research Foundation of Korea (R11-2007-107-02001-0), the
National Leading Research Lab program (2011-0028885), and in
part by the Intramural Research Program of NIH, Center for
Cancer Research, NCI (Project Z1A BC 005270).
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In summary, we have investigated a series of N-[{2-(4-
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N’-(6,6-fused heterocylic) ureas as hTRPV1 antagonists. Among
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