Discovery of Benzopyridone-Based Transient Receptor Potential Vanilloid 1 Agonists and Antagonists and the Structural Elucidation of Their Activity Shift

Article abstract of DOI:10.1021/acs.jmedchem.0c00982

Among a series of benzopyridone-based scaffolds investigated as human transient receptor potential vanilloid 1 (TRPV1) ligands, two isomeric benzopyridone scaffolds demonstrated a consistent and distinctive functional profile in which 2-oxo-1,2-dihydroquinolin-5-yl analogues (e.g., 2) displayed high affinity and potent antagonism, whereas 1-oxo-1,2-dihydroisoquinolin-5-yl analogues (e.g., 3) showed full agonism with high potency. Our computational models provide insight into the agonist-antagonist boundary of the analogues suggesting that the Arg557 residue in the S4-S5 linker might be important for sensing the agonist binding and transmitting signals. These results provide structural insights into the TRPV1 and the protein-ligand interactions at a molecular level.

Full text of DOI:10.1021/acs.jmedchem.0c00982

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Article
Discovery of Benzopyridone-Based Transient Receptor Potential
Vanilloid 1 Agonists and Antagonists and the Structural Elucidation
of Their Activity Shift
Shivaji A. Thorat,# Yoonji Lee,# Aeran Jung, Jihyae Ann, Songyeon Ahn, Jisoo Baek, Dongxu Zuo,
Nayeon Do, Jin Ju Jeong, Peter M. Blumberg, Timothy E. Esch, Noe A. Turcios, Larry V. Pearce,
Hee-Jin Ha, Young Dong Yoo, Sunhye Hong, Sun Choi,* and Jeewoo Lee*
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ABSTRACT: Among a series of benzopyridone-based scaffolds
investigated as human transient receptor potential vanilloid 1
(TRPV1) ligands, two isomeric benzopyridone scaffolds demon-
strated a consistent and distinctive functional profile in which 2-
oxo-1,2-dihydroquinolin-5-yl analogues (e.g., 2) displayed high
affinity and potent antagonism, whereas 1-oxo-1,2-dihydroisoqui-
nolin-5-yl analogues (e.g., 3) showed full agonism with high
potency. Our computational models provide insight into the
agonist−antagonist boundary of the analogues suggesting that the
Arg557 residue in the S4−S5 linker might be important for sensing the agonist binding and transmitting signals. These results
provide structural insights into the TRPV1 and the protein−ligand interactions at a molecular level.
that both functional activities could have considerable
therapeutic utility.
1. INTRODUCTION
Transient receptor potential vanilloid 1 (TRPV1) is a molecular
integrator of nociceptive stimuli expressed in primary C-fiber
sensory neurons and represents a promising therapeutic target
for the treatment of neuropathic pain and a wide range of other
conditions in which C-fiber sensory neurons are involved.1,2
TRPV1 is nonselective cation channel with high Ca2+
permeability3 and is activated by endogenous factors such as
noxious heat, low pH, and endovanilloids as well as by natural
ligands such as capsaicin and resiniferatoxin (RTX). Also, it is
responsive to the state of the signaling pathways in the cells.3−6
TRPV1 shares many structural features with other ion
channels, such as the voltage-gated potassium channel, being
assembled as a tetramer.3 The recent success of transmission
electron cryomicroscopy (cryo-EM) studies of TRPV1 not only
confirmed that the peripheral domain in each subunit,
encompassing S1−S4 segments, is connected to the pore
domains (S5 and S6) by a short linker, but also showed several
structural states under different conditions: in the absence of
ligand, in the presence of known agonists, that is, capsaicin or
RTX, and in the presence of known antagonist, capsazepine.7,8
The receptor activation leads to an increase in intracellular
Ca2+ that results in excitation of primary sensory neurons and
ultimately in the central perception of harmful stimuli such as
pain, itching, burning, and stinging sensations. The blocking of
this activation could be obtained by antagonism as well as by
desensitization upon persistent activation of agonist, suggesting
The agonist causes the persistent activation of the channel
resulting in high intracellular levels of calcium, ultimately leading
to desensitization/defunctionalization of a variety of noxious
stimuli to the peripheral ends of nerve fibers and consequent
pain relief.9 The antagonists have a therapeutic advantage over
agonists in that they lack the initial excitatory effect preceding
the desensitization, which represents a limiting toxicity for the
systemic application of the agonists. However, their clinical
development has been complicated by side effects such as
hyperthermia and an increased heat perception threshold and by
their nonselective antagonism, which blocks all modes of
TRPV1 activation.10−13 In consequence, both selective
antagonists, which inhibit only the activators related to pain
perception12,13 and nonpungent agonists with minimal initial
excitation14 represent second generation objectives for TRPV1
clinical candidates.
The pharmacophore of TRPV1 ligands can be divided into
three regions, so-called A-, B-, and C-regions, as previously
designated for capsaicin and as described in our prototype
Received: June 9, 2020
Published: January 1, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c00982
© 2021 American Chemical Society
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antagonist 1 (Figure 1).15 The C-region of 1, the 6-
(trifluoromethyl)-2-(4-methylpiperidin-1-yl)pyridine group, in
we established our revised TRPV1 homology model based on
the cryogenic electron microscopy (cryo-EM) structure of rat
TRPV1 (rTRPV1) and performed molecular modeling studies
to gain structural insights into the activities of the ligands by
identifying the key binding interactions critical for their agonism
and antagonism.
2. RESULTS AND DISCUSSION
2.1. Chemistry. The final urea compounds were generally
synthesized by the coupling between the carbamate of the
benzopyridone A-region and the amine of the pyridine/pyrazole
C-region, respectively. The syntheses of the benzopyridone A-
region are represented in Schemes 1−3 and the pyridine and
pyrazole C-region amines were prepared by the previous
reports,16 respectively.
For the syntheses of 2-oxo-1,2-dihydroquinolin-5 (or 8)-yl A-
region analogues (Scheme 1), the nitration of 2-chloroquinoline
afforded a mixture of 5-nitro (5) and 8-nitro (6) isomers in a 1:2
ratio. The isomers were hydrolyzed under acidic conditions into
the corresponding 2-pyridones, respectively, in which 5-nitro
pyridone was further N-methylated. The nitro groups of
benzopyridone intermediates were hydrogenated to the
corresponding amines (7, 8, 12), which were converted to the
corresponding phenyl carbamates (9, 10, 13), respectively.
Finally, the coupling of the carbamates with the pyridine C-
region amine15 provided 2-oxo-1,2-dihydroquinolin-5-yl (2), 1-
methyl-2-oxo-1,2-dihydroquinolin-5-yl (11), and 2-oxo-1,2-
dihydroquinolin-8-yl (14) ureas, respectively.
For the synthesis of 1-oxo-1,2-dihydroisoquinolin-5-yl A-
region analogues (Scheme 2), 1-chloroisoquinoline was hydro-
lyzed to the corresponding 2-pyridone and then nitrated to give
5-nitro isoquinolin-1-one 16. The nitro reduction or N-
methylation followed by reduction of 16 afforded 17 and 18,
respectively, which were converted to the final ureas (3, 19) by
employing the same route as discussed above. For the synthesis
of the 1-oxo-1,2-dihydroisoquinolin-8-yl A-region analogue
(Scheme 2), isoquinoline was brominated and then selectively
nitrated to give 8-nitro isoquinoline 21. The N-oxidation of 21
followed by rearrangement with acetic anhydride17 provided the
corresponding pyridone, which was reduced to amine 22 and
then converted to the final compound (23).
Figure 1. Discovery of benzopyridone-based TRPV1 antagonist and
agonist.
the C-region library has been shown to confer high potency in
our TRPV1 antagonists,16 representing as a prototype of the C-
region for the structure activity relationship (SAR) study of A-
and B-regions. The urea group has been much employed as the
B-region because it provides both high potency and synthetic
accessibility.
In our program to discover novel potent TRPV1 ligands as
drug candidates, we have investigated a series of heterocyclic A-
region analogues in which the B- and C-regions were fixed with a
urea and the prototype pyridine of 1, respectively. Among the
analogues studied, we demonstrated that the two isomeric
benzopyridone scaffolds exhibited distinctive functional profiles
in which 2-oxo-1,2-dihydroquinolin-5-yl (e.g., 2) and 1-oxo-1,2-
dihydroisoquinolin-5-yl (e.g., 3) A-region derivatives provided
potent antagonism and agonism, respectively, with human
TRPV1 (hTRPV1), and their functional activities were
maintained over the range of C-regions investigated.
In this study, we describe the SAR of a series of benzopyridone
A-region analogues to examine their functional activities. With
the selected benzopyridone A-regions, we further investigated
the SAR of the C-region to explore the maintenance of
functional activity as well as to improve the potency. To confirm
the in vitro mechanism of action of the optimized agonist and
antagonist, we evaluated their effects on body temperature in the
animal as an in vivo study. Finally, in order to elucidate the
structural basis for the activity shift of the two isomeric ligands,
For the syntheses of 2-oxo-1,2-dihydroquinolin-6-yl and 1-
oxo-1,2-dihydroisoquinolin-7-yl A-region analogues (Scheme
a
Scheme 1. General Procedures of 2-Oxo-1,2-dihydroquinolin-5 (or 8)-yl A-Region Analogues
a
Reagents and conditions: (a) KNO3, H2SO4 (5 (20%), 6 (40%)); (b) 5 N HCl, THF; (c) 10% Pd/C, EtOH; (d) NaH, MeI; (e) PhOCOCl,
pyridine, THF−DMF (1:1); and (f) X−NH2, NEt3, DMF.
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a
Scheme 2. General Procedures of 1-Oxo-1,2-dihydroisoquinolin A-Region Analogues
a
Reagents and conditions: (a) KNO3, H2SO4; (b) 5 N HCl, THF; (c) NaH, MeI, THF; (d) 10% Pd/C, H2, MeOH; (e) PhOCOCl, pyridine,
ACN−THF (1:1); (f) X−NH2, NEt3, DMF; (g) NBS, H2SO4; (h) H2O2, AcOH; and (i) Ac2O, 10% K2CO3.
a
Scheme 3. General Procedures of 2-Oxo-1,2-dihydroquinolin A-Region Analogues
a
Reagents and conditions: (a)KNO3, H2SO4; (b) DDQ, DCM; (c) mCPBA, DCM; (d) TFAA, DMF; (e) 10% Pd/C, H2, MeOH; (f) PhOCOCl,
pyridine, THF; and (g) X−NH2, NEt3, DMF.
a
Scheme 4. General Procedures of 5-aminoisoquinolinone A-Region Analogue
a
Reagents and conditions: (a) PhOCOCl, pyridine, THF; (b) C-region amine, NEt3, DMF.
3), quinolin-2-one and isoquinolin-2-one were selectively
nitrated, respectively, and then converted to the final ureas
(26, 29) by following the same routes as discussed above. For
the synthesis of the 2-oxo-1,2-dihydroquinolin-7-yl A-region
analogue, 7-nitroquinoline 31 was prepared from 1,2,3,4-
tetrahydroquinoline by selective nitration followed by ring
oxidation. The N-oxidation of 31 followed by rearrangement
provided the corresponding pyridone 32, which was converted
to the final compound (33).
For the syntheses of the C-region analogues of 2 and 3
(Scheme 4), 5-aminoquinolin-2-one 7 and 5-aminoisoquinolin-
1-one 17 were converted to the corresponding carbamates,
respectively, which were reacted with various pyridine and
pyrazole C-region amines previously reported16 to provide the
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a
final 2-oxo-1,2-dihydroquinolin-5-yl (34−54) and 1-oxo-1,2-
dihydroisoquinolin-5-yl (55−67) derivatives.
2.2. Structure Activity Relationship. The binding
affinities and functional potencies of TRPV1 ligands with the
benzopyridone A-region in Table 1 were assessed in vitro by a
Table 2. SAR of the Pyridine C-Region in 2 on hTRPV1 a
a
Table 1. SAR of Benzopyridone A-Region on hTRPV1
a
The values are the mean of at least three experiments.
a
Table 3. SAR of the Pyrazole C-Region in 2 on hTRPV1
a
NE: no effect, WE: weak effect; the values are the mean of at least
b
three experiments. NE, results of at least two experiments. Only
fractional calcium uptake compared to 1 μM capsaicin. Only
c
fractional antagonism to 30 nM capsaicin.
binding competition assay with [3H]RTX and by a functional
45Ca2+ uptake assay using hTRPV1 heterologously expressed in
Chinese hamster ovary (CHO) cells, as previously described.18
For the agonism assay, a saturating concentration of capsaicin (1
μM) was used to define maximal response. For the antagonism
assay, the dose-dependent inhibition of the capsaicin (30 nM)-
stimulated calcium uptake was measured. The Ki values for
antagonism take into account the competition between
capsaicin and the antagonist.19
a
The values are the mean of at least three experiments.
heterologously expressed in recombinant human embryonic
kidney (HEK) cells.15 The antagonistic activity was measured by
inhibition of TRPV1 activation by capsaicin (10 nM) and was
To evaluate the antagonistic potency of the compounds in
Tables 2 and 3 for hTRPV1, assays were conducted using a
fluorometric imaging plate reader (FLIPR) with hTRPV1
compared to that of the prototype antagonist, BCTC (Ki(CAP) =
2.5 nM). To measure the agonistic potency of the compounds in
Table 4 for hTRPV1, a functional 45Ca2+ uptake assay as
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described in Table 1 was used and the activities were compared
to that of the prototype agonist, capsaicin (EC50 = 44.8 nM).
group of the pyridone in 3 was critical for interacting with the
receptor for agonism. Next, we explored the positional isomers
of 2 and 3. 2-Oxo-1,2-dihydroquinolin-8-yl (14) and 1-oxo-1,2-
dihydroisoquinolin-8-yl (23) analogues showed very weak
binding affinity and antagonism, indicating that their ring
amides directed to the urea probably caused unfavorable
interaction with the receptor. Other positional analogues, 26,
29, and 33, were also explored as positional isomers. Whereas
the 2-oxo-1,2-dihydroquinolin-6-yl (26) and 1-oxo-1,2-dihy-
droisoquinolin-7-yl (29) analogues were a partial agonist and a
weak antagonist, respectively, the 2-oxo-1,2-dihydroquinolin-7-
yl analogue (33) was found to be very potent antagonist with a
a
Table 4. SAR of the C-Region in 3 on hTRPV1
K
i(ant) = 0.83 nM.
The findings of potent antagonist 2 and agonist 3 prompted us
to explore the SAR of their C-region for further optimization.
First, the 2-position in the pyridine C-region of 2 was modified
with representative substituents selected from our previous
study.15 Their antagonistic activities were measured by the
FLIPR assay and their values are represented in Table 2. In this
assay, 2 showed excellent antagonism with an IC50 = 0.27 nM,
which was ca. 10-fold more potent than BCTC. The pyridine C-
region analogues, including 2-alkylamino (34−37), 2-alkyloxy
(38−44), 2-alkylthio (45), and alkyl/aryl (46−48) groups, all
exhibited potent antagonism within the range of IC50 = 0.75−
5.21 nM. In addition, the selected pyrazole C-region16 analogues
of 2 were also examined as shown in Table 3. They also displayed
potent antagonism with a range of IC50 = 0.64−6.17 nM, similar
to that found with the pyridine C-region. The result indicated
that the 2-oxo-1,2-dihydroquinolin-5-yl A-region proved a
robust antagonistic scaffold providing potent antagonism
tolerant of significant variation in the C-region.
Next, we explored the C-region analogues of agonist 3. In the
calcium influx assay, 3 was found to be ca. 4.5-fold more potent
than capsaicin. As conducted in the SAR of antagonist 2, a series
of 2-substituted pyridine and pyrazole C-region analogues of 3
(55−67) were investigated and all were found to be agonists.
Among them, compounds 56, 58, and 65 displayed more potent
agonism than 3 with values of IC50 = 5.81, 2.79, and 3.07 nM,
which were 8−16 fold more potent than that of capsaicin. The
results confirmed that 1-oxo-1,2-dihydroisoquinolin-5-yl A-
region proved to be a promising agonistic scaffold showing
potent agonism regardless of the C-region.
a
b
The values are the mean of at least three experiments. All showed
no antagonistic activity in the assay.
First, we investigated the structural and positional benzopyr-
idone isomers in the A-region analogues with the B- and C-
regions fixed with a urea and prototype pyridine. The 2-oxo-1,2-
dihydroquinolin-5-yl A-region analogue (2) showed high
affinity with a Ki = 0.72 nM and potent antagonism with a
K
i(ant) = 3.94 nM, which was 4-fold higher in binding affinity and
3-fold weaker in antagonistic potency compared to reference 1,
respectively. Its N-methyl analogue (11) exhibited similar
binding affinity and antagonism compared to 2, suggesting that
N−H of pyridone was not involved in hydrogen bonding with
the receptor for activity. On the contrary, the 1-oxo-1,2-
dihydroisoquinolin-5-yl A-region analogue (3), a reverse amide
surrogate of 2, proved to be a full and potent agonist with an
EC50 = 9.6 nM. However, its N-methylation, providing 19, led to
much reduction in binding affinity and caused the shift of
functional activity toward antagonism, revealing that the N−H
2.3. In vivo Study. It should be noted that TRPV1
modulates not only pain perception, but also other sensory
modalities such as thermal control and heat perception.1 In
Figure 2. Body temperature study of 2 and 3.
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Figure 3. Homology model of hTRPV1. (A) Monomeric model. Six transmembrane helices (S1−S6) including pre-S1 helix, S4−S5 linkers, pore-helix,
and TRP domain are marked. The bound ligand, CAPZ, is depicted in sticks. (B) Tetrameric model. The four chains are marked with A−D. Note that
the ligand-binding site is composed of S3, S4, and the S4−S5 linkers of one monomer, and the S5 and S6 of the adjacent monomer (black box). (C)
Generation of the MD-based multiple receptor conformations. Input system for MD simulations was represented. The graph below is for the root
mean squared deviation (rmsd) plot of the 100 ns production run. From the final 30 ns MD-simulated trajectories, 300 conformations were sampled
and clustered based on their rmsd values. Among the 11 clusters, highly populated clusters were chosen, and the protein conformation with lowest total
energy in each cluster is selected.
particular, the regulation of body temperature is a predominant
function of TRPV1 in which agonism causes a decrease in body
temperature (hypothermia) and antagonism elicits an increase
in body temperature (hyperthermia).
constructed. The model was further refined by molecular
dynamics (MD) simulations to obtain a more reliable structure.
The ensemble docking study of TRPV1 ligands was carried out
with the multiple conformation structures, representing the
flexibility of the protein. Using our homology models and
docking method, which were confirmed to be reliable by
screening the known actives from decoy compounds, the
binding modes of the representative benzopyridone-based
agonists and antagonists were investigated.
2.5. Homology Modeling. In order to investigate the
structural basis of the pharmacological activity shift of the
analogues, we conducted computational studies. Although
several cryo-EM structures of rTRPV1 were available,8 the
side chains that are essential for ligand binding interactions are
not fully resolved, so that these structures may not be directly
utilized for docking studies. Therefore, homology models of
hTRPV1 were constructed by using these cryo-EM structures
bound to an agonist, RTX (PDB id: 5IRX), and an antagonist,
CAPZ (PDB id: 5IS0) as templates (Figure 3A). The quality of
the initial monomer model was evaluated with a Ramachandran
plot20 and ERRAT scores21 (Figure S1A), indicating that the
backbone conformations and nonbonded atomic interactions
are well modeled within the acceptable range. Then, the
tetramer model was built by aligning each monomer model to
In order to confirm the antagonistic activities of 2 in vivo, we
performed a capsaicin-induced hypothermia test in mice (Figure
2A).14 Consistent with its in vitro mechanism of action,
antagonist 2 after oral administration (3 mg/kg) blocked ca.
50% of the hypothermia induced by intraperitoneal (ip)
injection of capsaicin (3 mg/kg). In addition, we measured
the effect on body temperature of agonist 3 in mice (Figure 2B).
The administration of 3 by ip injection (3 mg/kg) caused a drop
in body temperature over −2 °C for 30−60 min, indicating that
3 proved to be a full agonist consistent with its in vitro
mechanism.
2.4. Molecular Modeling Studies. Recently, the single-
particle cryo-EM structures of rTRPV1 were released.7,8
Although the resolution is not quite high enough for drug
discovery purposes and some important side chains are not fully
resolved, the structures showed homotetrameric functional
states with the typical ligands bound to the intracellular site, the
so-called vanilloid pocket, which is formed by two adjacent
chains.8 Using these structures as templates, the tetrameric
homology model of the hTRPV1 transmembrane domain was
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Figure 4. Binding modes of (A) the agonist 3 and (B) the antagonist 2 represented in ball-and-sticks with the carbon atoms in magenta and green-blue,
respectively. The interacting residues are depicted in sticks with their carbon atoms in gray, and the hydrogen bonds are marked in black-dashed lines.
The secondary structure of the hTRPV1 model is displayed in ribbon. The nonpolar hydrogen atoms are undisplayed for visual convenience.
the tetrameric template structure (Figure 3B), and further
refined by energy-minimization and MD simulations. Because
the sequence identity between hTRPV1 and rTRPV1 is almost
∼93%, their overall backbone geometries can be considered to
be remarkably similar. Therefore, we applied harmonic restraints
to the backbone atoms to maintain the overall conformations
from the experimentally determined structures. Note that the
purpose of this homology modeling process was to accurately
model the side chains and also to consider the dynamic aspects
of the receptor’s binding sites.
Because the ligand binding to a target protein is closely related
to the dynamic motions of the protein, considering a protein’s
local flexibility or multiple conformations is crucial to increase
the accuracy in modeling studies.22 By using the MD
simulations, we obtained a diverse subset of the receptor
conformation structures to cover various binding site geo-
metries. The resulting MD-derived multiple conformations were
clustered based on their backbone’s rmsd values, and the
representative conformations (i.e., highly populated and low-
energy state ones) were selected for ensemble docking studies
(Figure 3C). The reliability of the models was evaluated by
prescreening known active compounds (142 known TRPV1
antagonists with Ki < 1 nM in the ChEMBL database23) from
decoy compounds (3517 drug-like molecules, which have
molecular weights of 250−600 in the NCI drug database24).
The model performed quite well for screening the known actives
with the area under the receiver operating characteristic curve of
0.87 (Figure S2B).
Phe591, Ala666, and Leu670). The most significant differences
between the binding modes of the agonists and antagonists were
observed in the polar interaction at the bottom pocket. In the
case of 3 with agonistic activity, the pyridone ring’s carbonyl and
NH groups showed hydrogen bonds with Arg557 and Ser512,
respectively (Figure 4A). However, the reverse amide surrogate
2 cannot maintain the hydrogen bond with the residues at the
bottom pocket, leading to loss of the agonistic activity (Figure
4B).
This tendency is also confirmed by other analogues with the
same scaffold. For example, 56 and 58 (agonists, Figure 5A)
with the A-region of 3 showed H-bonding interactions with
Arg557, while 35 and 37 (antagonists, Figure 5B) with the A-
2.6. Binding Mode Comparison of Agonists and
Antagonists. For the selected representative analogues,
ensemble docking studies were performed by utilizing the
selected conformational ensembles of the hTRPV1 models. As
shown in Figure 4, the agonist and antagonist showed similar
binding modes with (i) the A-region, which occupies the deep
bottom of the vanilloid pocket, forming hydrophobic
interactions with Tyr511, Leu553, Ala566, Ile569, and Ile573,
(ii) the B-region with the urea linker maintaining the key
hydrogen bonds to Tyr511 and Thr550, and (iii) the C-region
aromatic group, which forms additional hydrophobic inter-
actions with the upper part residues (Phe543, Ala546, Leu547,
Figure 5. Overlaid binding modes of (A) the agonists 3 (magenta), 56
(light-pink), and 61 (purple) and (B) the antagonists 2 (green-blue),
35 (teal), and 37 (cyan). The interacting residues are depicted in sticks
with their carbon atoms in gray, and the hydrogen bonds are marked in
black-dashed lines. The secondary structure of the hTRPV1 model is
displayed in ribbon. The nonpolar hydrogen atoms are undisplayed for
visual convenience.
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Figure 6. Binding modes of the analogues represented in ball-and-sticks with the carbon atoms in (A) sky-blue (19), (B) light-teal (11), (C) pale-cyan
(33), (D) light-blue (29), (E) purple (14), (F) slate (23), and (G) green (26). The interacting residues are depicted in sticks with their carbon atoms
in gray, and the hydrogen bonds are marked in black-dashed lines. The secondary structure of the hTRPV1 model is displayed in ribbon. The nonpolar
hydrogen atoms are undisplayed for visual convenience.
region of 2 did not. In this scaffold, the R groups, the southern
part of the C-region, can definitely contribute the hydrophobic
interactions but face toward the solvent-exposed site so that the
wide range of substituted groups did not alter the scaffold’s
pharmacological profiles (Tables 2 vs 4).
bonding with Arg557 with different binding geometry (Figure
6G).
3. CONCLUSIONS
We investigated a series of benzopyridone-based scaffolds as
hTRPV1 ligands. Of those, the two isomeric benzopyridones, 2
and 3, demonstrated distinctive functional profiles in which,
whereas the N-(2-oxo-1-dihydroquinoline-5-yl) scaffold (2)
displayed high affinity and potent antagonism, the N-(1-oxo-1,2-
dihydroisoquinolin-5-yl) scaffold (3) showed full agonism with
high potency. The SAR of the two scaffolds indicated that (1)
the functional activities of the two scaffolds have been
maintained regardless of the C-region and (2) the hydrogen
bonding between pyridone of 3 and the receptor was critical for
agonism. The body temperature study in mice indicated that
antagonist 2 inhibited capsaicin-induced hypothermia and
agonist 3 showed hypothermia in mice, consistent with the in
vitro mechanism of action. Computational studies showed that
the H-bonding interactions of our agonists with Arg557 in the
S4−S5 linker, along with the induced-fit effect, appear to be
important for sensing the agonist binding and transmitting
signals. These results provide a more precise understanding of
In addition, the compound 19, which is the N-methyl
analogue of 3, cannot maintain the hydrogen bond with the
residues at the bottom pocket (Figure 6A), leading to loss of
agonism as well as binding affinity. It seems that, because of the
spatial limitation, the additional methyl group slightly alters the
binding geometry resulting in the loss of key H-bonding to the
bottom residues. In the case of other antagonists 11, 14, 23, 29,
and 33, the best binding modes also did not maintain H-bonding
interaction with Arg557 although they can sometimes make H-
bonding with Ser512 or Ala566 at the bottom (Figure 6B−F).
The strong polar interaction with Arg557 appears to be crucial
for the analogues’ agonistic activities, and the proper positioning
of the pyridone ring’s carbonyl group makes this key interaction
possible. Gao et al. also reported that Arg557 is essential for
agonist binding because the agonist RTX binds to TRPV1 and
coordinates the interaction between Arg557 and Glu570,
consequently pulling the S4−S5 linkers away from the central
axis to facilitate the opening of the lower gate.8 Interestingly, 26,
which showed partial agonistic activity, also maintains the H-
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washed with brine. The organic phase was dried over MgSO4, filtered,
and concentrated in vacuo to provide bromoisoquinoline N-oxide,
which was used in the next step without further purification. A
suspension of bromoisoquinoline N-oxide (1.0 mmol) in Dimethyl-
formamide (DMF) (1 mL/mmol) was treated with trifluoroacetic
anhydride (10 mmol) at 0 °C. The clear solution was stirred at room
temperature overnight then evaporated in vacuo. The residue was
diluted with aqueous NaHCO3 solution and extracted with EtOAc
several times. The combined organic extracts were washed with brine,
dried over MgSO4, and evaporated. The residue was purified by column
chromatography on silica gel to afford the desired product.
4.6. General Procedure for N-Methylation (Procedure E). To a
solution of 5-nitroisoquinolin-1(2H)-one (1.0 mmol) in DMF, sodium
hydride (60% suspension oil, 1.5 mmol) was added at 0 °C and stirred
for 30 min. Methyl iodide (1.3 mmol) was added and the mixture was
stirred for 5 h. The reaction mixture was quenched by dropwise
addition of water at 0 °C and extracted with EtOAc, washed with brine
several times, dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography to afford the
desired compound.
4.7. General Procedure for Catalytic Hydrogenation (Proce-
dure F). Palladium on charcoal (10% w/w) was added to a solution of
appropriate nitroquinolinone (1.0 mmol) in EtOAc or MeOH and
stirred under a balloon of hydrogen for 18 h. The reaction mixture was
filtered through Celite pad washing with EtOAc. The filtrate was
concentrated under reduced pressure and purified by column
chromatography to afford the desired compound.
4.8. General Procedure for Carbamation (Procedure G). To a
solution of the appropriate amine (1.0 mmol) in THF was added
pyridine (1.1 mmol) and phenyl chloroformate (1.0 mmol) at 0 °C and
stirred at room temperature for 2 h. The reaction mixture was quenched
with water and extracted with EtOAc several times. The combined
organic layer was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography to afford the desired product.
the compounds’ actions at the atomic level and the rationale for
designing compounds with specific pharmacological profiles.
4. EXPERIMENTAL SECTION
4.1. General. All chemical reagents and solvents were commercially
available. Melting points were determined on a melting point Buchi B-
540 apparatus and are uncorrected. Silica gel column chromatography
1
was performed on a silica gel 60, 230−400 mesh, Merck. H and 13
C
NMR spectra were recorded on a JEOL JNM-LA 300 at 300 MHz and
JEOL JNM-ECZ 400S at 400 MHz. Chemical shifts are reported in
ppm units with Me4Si as a reference standard. Mass spectra were
recorded on an Agilent, Q-TOF 6530 LC−MS instrument. All final
compounds were purified up to more 95% purity, as determined by
high-performance liquid chromatography (HPLC). HPLC was
performed on an Agilent 1120 Compact LC (G4288A) instrument
using an Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm).
4.2. General Procedure for Nitration (Procedure A). To a
mixture of quinoline or qunolinone (1.0 mmol) in H2SO4 was added
potassium nitrate (0.99 mmol) at 0 °C. The reaction mixture was
allowed to warm to room temperature and stirred for 3 h. After
completion, the reaction mixture was poured onto ice, neutralized with
NaHCO3, and extracted with EtOAc several times. The combined
organic layer was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography to afford the desired product.
4.3. General Procedure for Bromination (Procedure B). To a
solution of isoquinoline (1.0 mmol) in concentrated H2SO4 (1.29 mL/
mmol) was added N-bromosuccinimide (1.2 mmol) by portions at −10
°C. The reaction mixture was allowed to warm to room temperature
and stirred for 24 h. Then, the reaction mixture was poured onto ice
water and neutralized with NaOH. The water was extracted with
EtOAc, dried over MgSO4, and filtered and concentrated in vacuo. The
residue was purified by silica gel column chromatography to afford the
desired product.
4.9. General Procedure for Urea Coupling (Procedure H). To a
solution of the appropriate carbamate (1.0 mmol) in DMF was added
amine (2.0 mmol) and triethylamine (1.0 mmol) and stirred at 50 °C
for 15 h. After completion, the reaction mixture was cooled to room
temperature, diluted with water, and extracted with EtOAc several
times. The combined organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
silica gel column chromatography to afford the desired product.
4.9.1. 2-Chloro-5-nitroquinoline (5). From 4, procedure A, yield
30%, yellow solid; 1H NMR (300 MHz, CDCl3): δ 8.99 (d, J = 9.18 Hz,
1H), 8.40 (d, J = 7.68 Hz, 1H), 8.34 (d, J = 8.61 Hz, 1H), 7.84 (t, J =
8.13 Hz, 1H), 7.63 (d, J = 8.97 Hz, 1H).
4.9.2. 2-Chloro-8-nitroquinoline (6). From 4, procedure A, yield
60%, yellow solid; 1H NMR (400 MHz, CDCl3): δ 8.19 (d, J = 8.6 Hz,
1H), 8.08 (dd, J = 7.3, 1.2 Hz, 1H), 8.02−8.04 (m, 1H), 7.62−7.66 (m,
1H), 7.52−7.54 (m, 1H).
4.9.3. 5-Aminoquinolin-2(1H)-one (7). From 5, procedures D
(method A) and F, yield 45% (in 2 steps), white solid; 1H NMR (300
MHz, DMSO-d6): δ 11.39 (s, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.10 (t, J =
8.0 Hz, 1H), 6.44 (d, J = 7.8 Hz, 1H), 6.33 (d, J = 7.8 Hz, 1H), 6.26 (d, J
= 9.6 Hz, 1H), 5.86 (s, 2H).
4.4. General Procedure for Aromatization (Procedure C). To a
solution of 30 (1.0 mmol) in CH2Cl2 (1.2 mL/mmol) was added 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (2.0 mmol) by portions at 0
°C. The reaction mixture was allowed to warm to room temperature
and stirred for 4 h. The precipitate was filtered off and washed with
CH2Cl2. The combined filtrate was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel to afford the desired product.
4.5. General Procedure for Quinolinone Synthesis (Proce-
dure D). 4.5.1. Method A. To a solution of the appropriate
chloroquinoline (1.0 mmol) in tetrahydrofuran (THF) was added 5
N HCl and refluxed for 15 h. After completion, the reaction mixture was
cooled to room temperature and diluted with water and extracted with
EtOAc several times. The combined organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
silica gel column chromatography to afford the desired product.
4.5.2. Method B. To a stirred solution of appropriate bromoisoqui-
noline (1.0 mmol) in acetic acid was slowly added 30% hydrogen
peroxide (10 mmol) and refluxed for 3 h. The mixture was cooled to
room temperature and the reaction mixture was extracted with EtOAc
and water. The organic phase was dried over MgSO4, filtered, and
concentrated in vacuo to provide bromoisoquinoline N-oxide, which
was used in the next step without further purification. The crude
bromoisoquinoline N-oxide (1.0 mmol) was suspended in acetic
anhydride (2 mL/mmol) and refluxed for 3 h. After cooling, the solvent
was removed under reduced pressure, the residue was dissolved in 10%
potassium carbonate solution (3 mL/mmol), and stirred at room
temperature for 1 h. The cold suspension was adjusted to pH = 6 using
5% citric acid solution and the crystals were filtered and concentrated.
The obtained residue was triturated with ether to afford the desired
product.
4.9.4. 5-Amino-1-methylquinolin-2(1H)-one (8). From 5, proce-
1
dure D (method A), E and F, yield 62% (in 3 steps), white solid; H
NMR (300 MHz, DMSO-d6): δ 8.09 (d, J = 9.7 Hz, 1H), 7.23 (t, J = 8.2
Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 8.0 Hz, 1H), 6.37 (d, J =
9.7 Hz, 1H), 5.94 (s, 2H), 3.51 (s, 3H).
4.9.5. Phenyl (2-Oxo-1,2-dihydroquinolin-5-yl)carbamate (9).
1
From 7, procedure G, yield 91%, pale yellow solid; H NMR (300
MHz, DMSO-d6): δ 11.37 (s, 1H), 9.31 (s, 1H), 8.06 (d, J = 9.9 Hz,
1H), 7.16 (m, 3H), 6.73 (d, J = 8.0 Hz, 3H), 6.42 (d, J = 7.8 Hz, 1H),
6.32 (d, J = 7.8 Hz, 1H), 6.25 (d, J = 9.6 Hz, 1H).
4.5.3. Method C. To a solution of appropriate bromoisoquinoline
(1.0 mmol) in CH2Cl2 (9 mL/mmol) was added 3-chloroperox-
ybenzoic acid (1.4 mmol) at 0 °C. The mixture was stirred at room
temperature for 18 h. The reaction was diluted with CH2Cl2 and
4.9.6. Phenyl (1-Methyl-2-oxo-1,2-dihydroquinolin-5-yl)-
carbamate (10). From 8, procedure G, yield 93%, pale yellow solid;
1H NMR (300 MHz, DMSO-d6): δ 9.32 (s, 1H), 8.46 (d, J = 9.87 Hz,
1H), 7.81 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 9.5 Hz, 1H), 7.53 (d, J = 7.6
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Hz, 1H), 7.15 (t, J = 8.0 Hz, 2H), 6.76 (m, 3H), 6.42 (d, J = 7.8 Hz, 1H),
3.66 (s, 3H).
4.9.7. 8-Aminoquinolin-2(1H)-one (12). From 6, procedures D
(method A) and F, yield 61% (in 2 steps), white solid; 1H NMR (400
MHz, DMSO-d6): δ 10.80 (s, 1H), 7.76 (d, J = 9.8 Hz, 1H), 6.82−6.91
(m, 2H), 6.76 (dd, J = 7.7, 1.5 Hz, 1H), 6.40 (d, J = 9.8 Hz, 1H), 5.47 (s,
2H).
4.9.8. Phenyl (2-Oxo-1,2-dihydroquinolin-8-yl)carbamate (13).
From 12, procedure G, yield 93%, white solid;1H NMR (400 MHz,
DMSO-d6): δ 9.27 (s, 1H), 7.98 (d, J = 9.8 Hz, 1H), 7.38 (d, J = 7.3 Hz,
1H), 7.27 (t, J = 7.6 Hz, 1H), 7.09−7.17 (m, 3H), 6.71 (t, J = 7.9 Hz,
3H), 6.56 (d, J = 9.8 Hz, 1H).
4.9.9. 5-Nitroisoquinolin-1(2H)-one (16). From 15, procedures A
and D (method A), yield 63% (in 2 steps), white solid; 1H NMR (400
MHz, DMSO-d6): δ 11.79 (s, 1H), 8.57 (d, J = 7.8 Hz, 1H), 8.46 (d, J =
6.0 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 6.97 (d, J
= 7.6 Hz, 1H).
4.9.10. 5-Aminoisoquinoline-1(2H)-one (17). From 16, procedure
F, yield 91%, white solid; 1H NMR (400 MHz, DMSO-d6): δ 11.03 (s,
1H), 7.38 (d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 5.6
Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.66 (d, J = 7.5 Hz, 1H), 5.60 (s, 2H).
4.9.11. 5-Amino-2-methylisoquinolin-1(2H)-one (18). From 16,
procedures E and F, yield 65% (in 2 steps), pale yellow solid; 1H NMR
(400 MHz, DMSO-d6): δ 7.37 (d, J = 7.6 Hz, 1H), 7.28 (d, J = 7.2 Hz,
1H), 7.11 (t, J = 8.0 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.68 (d, J = 7.6
Hz, 1H), 5.59 (s, 2H), 3.42 (s, 3H).
(m, 2H), 0.97 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ
161.6, 160.6, 155.5, 137.4, 134.4, 131.0, 129.6, 128.4, 126.7, 126.0,
123.9, 121.0, 113.8, 98.9, 50.0, 33.8, 30.2, 21.8; HRMS (FAB): calcd for
C24H25F3N5O2 [M + H]+, 460.1882; found, 460.1967.
4.9.20. 1-(1-Methyl-2-oxo-1,2-dihydroquinolin-5-yl)-3-((2-(4-
methylpiperidin-1-yl)-6-(trifluoromethyl)-pyridin-3-yl)methyl)urea
(11). From 10, procedure H, yield 75%, white solid, mp = 214.9−215.7
°C; 1H NMR (300 MHz, DMSO-d6): δ 8.85 (s, 1H), 8.05 (d, J = 10.0
Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.60−7.40
(m, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.09 (t, J = 5.6 Hz, 1H), 6.64 (d, J =
9.9 Hz, 1H), 4.36 (m, 2H), 3.61 (s, 3H), 3.39 (m, 2H), 2.79 (m, 2H),
1.72 (m, 2H), 1.65−1.50(br s, 1H), 1.33 (m, 2H), 0.96 (d, J = 6.3 Hz,
3H); 13C NMR (100 MHz, DMSO-d6): δ 160.7, 160.6, 155.5, 140.5,
137.4, 136.8, 133.5, 130.9, 119.6, 115.0, 113.8, 112.1, 109.2, 50.0, 33.8,
30.2, 29.3, 21.8; HRMS (FAB): calcd for C24H27F3N5O2 [M + H]+,
474.2039; found, 474.2115.
4.9.21. 1-((2-(4-Methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-8-yl)urea (14). From 13,
procedure H, yield 68%, white solid, mp = 246.8−247.4 °C; 1H NMR
(300 MHz, DMSO-d6): δ 10.90 (s, 1H), 8.47 (s, 1H), 7.94 (t, J = 9.9
Hz, 2H), 7.68 (br, s, 1H), 7.48 (dd, J = 7.5 Hz, 2H), 7.16 (t, J = 7.5 Hz,
1H), 6.82 (br s, 1H), 6.55 (d, J = 8.7 Hz, 1H), 4.35 (s, 2H), 3.41 (m,
2H), 2.82 (t, J = 11.7 Hz, 2H), 1.75 (d, J = 12 Hz, 2H), 1.56 (br s, 1H),
1.32 (d, J = 10.8 Hz, 2H), 0.97 (d, J = 6.0 Hz, 3H); 13C NMR (100
MHz, DMSO-d6): δ 161.7, 160.5, 155.7, 140.9, 137.4, 131.1, 123.1,
121.6, 120.0, 113.8, 50.0, 33.8, 30.2; HRMS (FAB): calcd for
C23H25F3N5O2 [M + H]+, 460.1882; found, 460.1967.
4.9.12. 5-Bromo-8-nitroisoquinoline (21). From 20, procedures B
4.9.22. 1-(2-Methyl-1-oxo-1,2-dihydroisoquinolin-5-yl)-3-((2-(4-
methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)methyl)urea
(19). From 18, procedures G and F, yield 81% (in 2 steps), white solid,
1
and A, yield 32% (in 2 steps), yellow powder; H NMR (300 MHz,
CDCl3): δ 10.01 (s, 1H), 8.84 (d, J = 5.8 Hz, 1H), 8.17 (m, 3H).
4.9.13. 8-Aminoisoquinoline-1(2H)-one (22). From 21, procedures
1
mp = 245.9−247.6 °C; H NMR (300 MHz, DMSO-d6): δ 8.58 (s,
1
D (method B) and F, yield 45% (in 2 steps); H NMR (300 MHz,
1H), 8.09 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.45 (m, 3H),
7.05 (m, 1H), 6.70 (d, J = 7.7 Hz, 1H), 4.36 (d, J = 5.7 Hz, 2H), 3.50 (s,
3H), 2.78 (m, 2H), 1.72 (m, 2H), 1.29 (m, 2H), 0.95 (d, J = 6.4 Hz,
3H); 13C NMR (100 MHz, DMSO-d6): δ 161.1, 160.6, 155.5, 137.4,
134.4, 133.1, 130.9, 128.6, 126.3, 126.0, 123.3, 120.9, 113.8, 99.1, 50.0,
36.3, 33.8, 30.2, 21.8; HRMS (FAB): calcd for C23H27F3N5O2 [M +
H]+, 474.2039; found, 474.2115.
CDCl3): δ 11.92 (s, 1H), 7.71 (d, J = 9.1 Hz, 1H), 7.31 (t, J = 8.8 Hz,
1H), 6.86 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 7.8 Hz, 1H), 6.44 (s, 2H),
6.18 (d, J = 9.1 Hz, 1H).
4.9.14. 6-Nitroquinoline-2(1H)-one (25). From 24, procedure A,
yield 41%, yellow powder;1H NMR (300 MHz, DMSO-d6): δ 8.41 (s,
1H), 8.05 (d, J = 9.3 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.18 (d, J = 9.1
Hz, 1H), 6.44 (d, J = 9.3 Hz, 1H).
4.9.15. 7-Nitroisoquinolin-1(2H)-one (28). From 27, procedure A,
yield 31%, yellow powder; 1H NMR (400 MHz, CDCl3): δ 9.78 (br s,
1H), 9.26 (d, J = 2.5 Hz, 1H), 8.45 (dd, J = 9.2, 2.5 Hz, 1H), 7.68 (d, J =
8.6 Hz, 1H), 6.61 (d, J = 7.4 Hz, 1H).
4.9.16. 7-Nitroquinoline (31). From 30, procedures A and C, yield
42% (in 2 steps), pale brown solid; 1H NMR (300 MHz, DMSO-d6): δ
9.13 (d, J = 4.2 Hz, 1H), 8.82 (s, 1H), 8.60 (d, J = 7.6 Hz, 1H), 8.38 (d, J
= 8.9 Hz, 1H), 8.30 (d, J = 8.9 Hz, 1H), 7.79 (d, J = 4.2 Hz, 1H).
4.9.17. 7-Nitroquinoline-2(1H)-one (32). From 31, procedure D
(method C), yield 57%, yellow solid; 1H NMR (300 MHz, DMSO-d6):
δ 8.15 (s, 1H), 8.05 (d, J = 9.6 Hz, 1H), 7.93 (m, 2H), 6.70 (d, J = 9.5
Hz, 1H), 3.36 (br s, 1H).
4.9.18. 1-((2-(4-Methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(2-oxo-1,2-dihydro-quinolin-5-yl)urea (2). From 9,
procedure H, yield 82%, white solid, mp = 253.6−254.9 °C; 1H NMR
(300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.74 (s, 1H), 8.03 (d, J = 9.9
Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.44 (d, J =
7.7 Hz, 1H), 7.36 (t, J = 8.1 Hz, 1H), 7.02 (t, J = 5.7 Hz, 1H), 6.96 (d, J
= 8.1 Hz, 1H), 6.50 (d, J = 9.9 Hz, 1H), 4.35 (d, J = 5.3 Hz, 2H), 2.75
(m, 2H), 1.70 (m, 2H), 1.54 (br s, 1H), 1.29 (m, 1H), 0.94 (d, J = 6.4
Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 161.5, 160.6, 155.4,
139.7, 137.3, 136.3, 134.7, 130.9, 130.5, 123.0, 120.5, 120.3, 113.9,
113.8, 111.0, 109.6, 50.0, 33.8, 30.2, 21.8; HRMS (FAB): calcd for
C23H25F3N5O2 [M + H]+, 460.1882; found, 460.1972.
4.9.19. 1-((2-(4-Methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(1-oxo-1,2-dihydro-isoquinolin-5-yl)urea (3). From
17, procedures G and H, yield 72% (in 2 steps), white solid, mp =
240.3−242.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.32 (br s, 1H),
8.57 (s, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.89 (m, 2H), 7.47 (d, J = 7.8 Hz,
1H), 7.41 (t, J = 8.1 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.09 (t, J = 5.7 Hz,
1H), 6.68 (d, J = 7.5 Hz, 1H), 4.38 (d, J = 5.4 Hz, 2H), 3.34 (m, 2H),
2.83 (t, J = 11.7 Hz, 2H), 1.73 (d, J = 12.6 Hz, 2H), 1.55 (br s, 1H), 1.36
4.9.23. 1-((2-(4-Methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(1-oxo-1,2-dihydroisoquinolin-8-yl)urea (23). From
22, procedures G and H, yield 76% (in 2 steps), white solid, mp =
1
246.8−247.4 °C; H NMR (300 MHz, DMSO-d6): δ 11.92 (s, 1H),
8.99 (s, 1H), 7.73 (m, 3H), 7.45 (m, 3H), 7.24−6.89 (m, 3H), 6.42 (d, J
= 9.5 Hz, 1H), 4.30 (d, J = 5.0 Hz, 2H), 2.77 (t, J = 12.3 Hz, 2H), 1.72
(d, J = 12.3 Hz, 2H), 1.66 (s, 1H), 1.29 (q, J = 12.5 Hz, 2H), 0.96 (d, J =
6.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 164.5, 160.6, 154.9,
142.9, 139.2, 137.5, 133.0, 131.0, 128.2, 123.0, 120.3, 117.8, 114.1,
113.7, 111.7, 106.4, 50.0, 33.8, 30.2, 21.8; HRMS (FAB): calcd for
C23H25F3N5O2 [M + H]+, 460.1882; found, 460.1959.
4.9.24. 1-((2-(4-Methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-6-yl)urea (26). From 25,
procedures F, G, and H, yield 42% (in 3 steps), white solid, mp =
1
248.6−249.3 °C; H NMR (300 MHz, DMSO-d6): δ 11.58 (s, 1H),
9.25 (s, 1H), 7.77 (m, 3H), 7.44 (m, 2H), 7.27−7.15 (m, 2H), 6.42 (d, J
= 9.5 Hz, 1H), 4.30 (d, J = 5.0 Hz, 2H), 2.77 (t, J = 12.3 Hz, 2H), 1.72
(d, J = 12.3 Hz, 2H), 1.66 (s, 1H), 1.29 (q, J = 12.5 Hz, 2H), 0.96 (d, J =
6.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 161.5, 160.5, 155.6,
140.0, 137.2, 134.8, 133.7, 131.4, 123.0, 122.2, 120.3, 119.2, 115.4,
115.3, 113.8, 50.0, 33.8, 30.2, 21.8; HRMS (FAB): calcd for
C23H25F3N5O2 [M + H]+, 460.1882; found, 460.1975.
4.9.25. 1-((2-(4-Methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(1-oxo-1,2-dihydroisoquinolin-7-yl)urea (29). From
28, procedures F, G, and H, yield 57% (in 3 steps), white solid, mp =
247.6−248.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.44 (dd, J = 8.6,
1.8 Hz, 1H), 7.70 (m, 2H), 7.48 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 7.7 Hz,
1H), 6.90 (d, J = 7.1 Hz, 1H), 6.51 (d, J = 7.1 Hz, 1H), 4.42 (s, 2H),
3.37 (d, J = 12.8 Hz, 2H), 2.82 (t, J = 11.2 Hz, 2H), 1.69 (d, J = 11.6 Hz,
2H); 13C NMR (100 MHz, DMSO-d6): δ 161.5, 160.6, 155.2, 138.8,
137.3, 132.0, 131.1, 126.7, 126.4, 123.4, 113.7, 105.4, 50.0, 33.8, 30.2,
21.8; HRMS (FAB): calcd for C23H25F3N5O2 [M + H]+, 460.1882;
found, 460.1967.
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4.9.26. 1-((2-(4-Methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-7-yl)urea (33). From 32,
procedures F, G, and H, yield 63% (in 3 steps), pale yellow solid, mp =
248.1−249.3 °C; H NMR (300 MHz, DMSO-d6): δ 11.56 (s, 1H),
NMR (300 MHz, DMSO-d6): δ 11.71 (s, 1H), 8.75 (s, 1H), 8.04 (d, J =
9.8 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.46 (d, J
= 7.5 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 6.95−6.93 (m, 2H), 6.51 (d, J =
9.8 Hz, 1H), 4.35−4.30 (m, 4H), 1.74 (m, 2H), 1.41 (m, 2H), 1.30−
1.25 (m, 4H), 0.83 (t, J = 6.7 Hz, 3H); HRMS (FAB): calcd for
C23H26F3N4O3 [M + H]+, 463.1879; found, 463.1957.
1
9.19 (s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 9.3 Hz, 1H), 7.55 (s,
1H), 7.47 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 9.9 Hz, 1H), 7.14 (dd, J = 8.5
Hz, 1.8 Hz, 1H), 6.90 (s, 1H), 6.27 (d, J = 9.5 Hz, 1H), 4.34 (d, J = 5.4
Hz, 1H), 3.44 (d, J = 12.4 Hz, 2H), 2.79 (t, J = 11.7 Hz, 2H), 1.73 (d, J =
10.8 Hz, 2H), 1.56 (m, 1H), 1.34−1.26 (m, 2H), 0.97 (d, J = 6.3 Hz,
3H); 13C NMR (100 MHz, DMSO-d6): δ 162.3, 160.6, 154.9, 142.3,
139.9, 139.8, 137.2, 131.0, 128.2, 123.0, 120.3, 118.5, 113.8, 112.6,
102.2, 50.0, 33.8, 30.2, 21.8; HRMS (FAB): calcd for C23H25F3N5O2
[M + H]+, 460.1882; found, 460.1964.
4.9.27. 1-(2-Oxo-1,2-dihydroquinolin-5-yl)-3-((2-(piperidin-1-yl)-
6-(trifluoromethyl)pyridin-3-yl)methyl)urea (34). From 7, procedures
G and H, yield 61% (in 2 steps), white solid, mp = 265.1−267.0 °C; 1H
NMR (300 MHz, DMSO-d6): δ 11.65 (s, 1H), 10.50 (s, 1H), 9.20 (m,
1H), 8.27 (d, J = 9.9 Hz, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.49 (d, J = 7.8
Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 6.89 (d, J =
7.8 Hz, 1H), 6.39 (d, J = 10.0 Hz, 1H), 4.29 (d, J = 5.4 Hz, 2H), 1.62 (m,
10H); HRMS (FAB): calcd for C22H23F3N5O2 [M + H]+, 446.1726;
found, 446.1815.
4.9.28. 1-((2-(4-Benzylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (35). From 7,
procedures G and H, yield 54% (in 2 steps), white solid, mp = 273.6−
274.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.77 (s,
1H), 8.04 (d, J = 9.8 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 7.5
Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.37 (t, J = 8.2 Hz, 1H), 7.31−7.26
(m, 2H), 7.21−7.18 (m, 3H), 7.06 (m, 1H), 6.96 (d, J = 8.2 Hz, 1H),
6.51 (d, J = 10.9 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.45 (d, J = 12.9 Hz,
2H), 2.74 (t, J = 10.9 Hz, 2H), 2.57 (d, J = 6.6 Hz, 2H), 1.68 (d, J = 11.9
Hz, 2H), 1.70 (m, 1H), 1.37 (m, 2H); HRMS (FAB): calcd for
C29H29F3N5O2 [M + H]+, 536.2195; found, 536.2272.
4.9.34. 1-((2-Isobutoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-3-
(2-oxo-1,2-dihydroquinolin-5-yl)urea (41). From 7, procedures G and
H, yield 67% (in 2 steps), white solid, mp = 278.8−280.5 °C; 1H NMR
(300 MHz, DMSO-d6): δ 11.71 (s, 1H), 8.75 (s, 1H), 8.04 (d, J = 9.5
Hz, 1H), 7.79 (m, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 6.6 Hz,
1H), 7.35 (m, 1H), 6.95 (m, 2H), 6.50 (d, J = 10.2 Hz, 1H), 4.34 (m,
2H), 4.11 (d, J = 6.0 Hz, 2H), 0.99 (d, J = 5.8 Hz, 6H); HRMS (FAB):
calcd for C21H22F3N4O3 [M + H]+, 435.1566; found, 435.1634.
4.9.35. 1-((2-(Cyclobutylmethoxy)-6-(trifluoromethyl)pyridin-3-
yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (42). From 7,
procedures G and H, yield 71% (in 2 steps), white solid, mp =
1
271.2−272.1 °C; H NMR (300 MHz, DMSO-d6): δ 11.71 (s, 1H),
8.76 (s, 1H), 8.04 (d, J = 9.8 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.56 (d, J
= 8.0 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 6.95−
6.92 (m, 2H), 6.50 (d, J = 9.8 Hz, 1H), 4.32−4.30 (m, 4H), 2.75 (m,
1H), 2.04 (m, 2H), 1.87 (m, 4H); HRMS (FAB): calcd for
C22H22F3N4O3 [M + H]+, 447.1566; found, 447.1640.
4.9.36. 1-((2-(Cyclopentylmethoxy)-6-(trifluoromethyl)pyridin-3-
yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (43). From 7,
procedures G and H, yield 68% (in 2 steps), white solid, mp =
1
273.6−275.1 °C; H NMR (300 MHz, DMSO-d6): δ 11.71 (s, 1H),
8.76 (s, 1H), 8.04 (d, J = 9.8 Hz, 1H), 7.80 (d, J = 7.3 Hz, 1H), 7.56 (d, J
= 8.0 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 6.96−
6.93 (m, 2H), 6.50 (d, J = 9.8 Hz, 1H), 4.32 (d, J = 5.8 Hz, 2H), 4.22 (d,
J = 6.9 Hz, 2H), 2.36 (m, 1H), 1.75 (m, 2H), 1.56 (m, 4H), 1.36 (m,
2H); HRMS (FAB): calcd for C23H24F3N4O3 [M + H]+, 461.1722;
found, 461.1795.
4.9.29. 1-(2-Oxo-1,2-dihydroquinolin-5-yl)-3-((2-(pyrrolidin-1-yl)-
6-(trifluoromethyl)pyridin-3-yl)methyl)urea (36). From 7, procedures
G and H, yield 61% (in 2 steps), white solid, mp = 291.6−292.2 °C; 1H
NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.69 (s, 1H), 8.04 (d, J =
9.8 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.58 (m, 1H), 7.37 (m, 1H), 7.11
(d, J = 7.9 Hz, 1H), 6.96 (d, J = 7.4 Hz, 1H), 6.52 (d, J = 7.4 Hz, 1H),
4.43 (d, J = 5.4 Hz, 2H), 3.54 (m, 4H), 1.89 (m, 4H); HRMS (FAB)
calcd for C21H21F3N5O2 [M + H]+, 432.1569; found, 432.1689.
4.9.30. 1-((2-(Dipropylamino)-6-(trifluoromethyl)pyridin-3-yl)-
methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (37). From 7, pro-
cedures G and H, yield 67% (in 2 steps), white solid, mp = 292.3−293.8
4.9.37. 1-((2-(Benzyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-
3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (44). From 7, procedures G
and H, yield 61% (in 2 steps), white solid, mp = 275.1−276.2 °C; 1H
NMR (300 MHz, DMSO-d6): δ 11.73 (s, 1H), 8.77 (s, 1H), 8.04 (d, J =
10.2 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.52−7.59 (m, 3H), 7.32−7.39
(m, 5H), 7.00 (m, 1H), 6.51 (d, J = 10.4 Hz, 1H), 5.45 (s, 1H), 4.37 (m,
2H); HRMS (FAB): calcd for C24H20F3N4O3 [M + H]+, 469.1409;
found, 469.1497.
4.9.38. 1-((2-(Cyclohexylthio)-6-(trifluoromethyl)pyridin-3-yl)-
methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (45). From 7, pro-
cedures G and H, yield 77% (in 2 steps), white solid, mp = 276.2−279.4
1
1
°C; H NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.81 (s, 1H),
°C; H NMR (400 MHz, DMSO-d6): δ 11.70 (s, 1H), 8.79 (s, 1H),
8.06 (d, J = 9.9 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 3.5 Hz,
1H), 7.38−7.34 (m, 2H), 7.14 (m, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.49
(d, J = 9.9 Hz, 1H), 4.32 (d, J = 5.6 Hz, 2H), 3.17 (t, J = 6.2 Hz, 4H),
1.50 (m, 4H), 0.81 (t, J = 7.3 Hz, 6H); HRMS (FAB): calcd for
C23H27F3N5O2 [M + H]+, 462.2039; found, 462.2111.
8.06 (d, J = 9.8 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.8 Hz,
1H), 7.56 (d, J = 7.9 Hz, 1H), 7.36 (t, J = 8.1 Hz, 1H), 7.07 (t, J = 5.6
Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H), 6.51 (d, J = 9.8 Hz, 1H), 4.27 (d, J =
5.4 Hz, 2H), 3.91 (m, 1H), 2.06 (m, 2H), 1.74 (m, 2H), 1.61−1.29 (m,
6H); HRMS (FAB): calcd for C23H24F3N4O2S [M + H]+, 477.1494;
found, 477.1521.
4.9.31. 1-((2-Butoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-3-(2-
oxo-1,2-dihydroquinolin-5-yl)urea (38). From 7, procedures G and H,
4.9.39. 1-((2-(4-Methylcyclohexyl)-6-(trifluoromethyl)pyridin-3-
yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (46). From 7,
procedures G and H, yield 62% (in 2 steps), white solid, mp =
1
yield 64% (in 2 steps), white solid, mp = 294.2−295.1 °C; H NMR
(300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.76 (s, 1H), 8.05 (d, J = 9.87
Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.48 (d, J =
7.5 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 6.97−6.94 (m, 2H), 6.52 (d, J =
9.8 Hz, 1H), 4.38−4.32 (m, 4H), 1.73 (m, 2H), 1.45 (m, 2H), 0.93 (t, J
= 7.5 Hz, 3H); HRMS (FAB) calcd for C21H22F3N4O3 [M + H]+,
435.1566; found, 435.1640.
1
280.3−282.5 °C; H NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H),
8.70 (s, 1H), 8.01 (d, J = 9.7 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.70 (d, J
= 7.8 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.01
(m, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.51 (d, J = 9.6 Hz, 1H), 4.49 (d, J =
5.4 Hz, 2H), 3.06 (m, 1H), 1.85−1.54 (m, 9H), 1.02−0.90 (m, 3H);
HRMS (FAB): calcd for C24H26F3N4O2 [M + H]+, 459.1930; found,
459.2003.
4.9.32. 1-(2-Oxo-1,2-dihydroquinolin-5-yl)-3-((2-(pentyloxy)-6-
(trifluoromethyl)pyridin-3-yl)methyl)urea (39). From 7, procedures
G and H, yield 59% (in 2 steps), white solid, mp = 276.3−277.8 °C; 1H
NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.76 (s, 1H), 8.05 (d, J =
9.8 Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.48 (d, J
= 7.5 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 6.97−6.94 (m, 2H), 6.52 (d, J =
9.87 Hz, 1H), 4.37−4.32 (m, 4H), 1.77 (m, 2H), 1.46−1.28 (m, 4H),
0.88 (t, J = 7.14 Hz, 3H); HRMS (ESI): calcd for C22H23F3N4O3 [M +
H]+, 448.1722; found, 449.1799.
4.9.40. 1-((2-(3-Isopropylphenyl)-6-(trifluoromethyl)pyridin-3-yl)-
methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (47). From 7, pro-
cedures G and H, yield 58% (in 2 steps), white solid, mp = 281.3−282.7
1
°C; H NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.72 (s, 1H),
8.12 (d, J = 8.1 Hz, 1H), 7.98 (m, 2H), 7.42 (m, 6H), 7.12 (s, 1H), 6.96
(d, J = 8.1 Hz, 1H), 6.51 (d, J = 9.9 Hz, 1H), 4.42 (d, J = 5.4 Hz, 2H),
2.98 (m, 1H), 1.24 (d, J = 6.9 Hz, 6H); HRMS (FAB): calcd for
C26H24F3N4O2 [M + H]+, 481.1773; found, 481.1790.
4.9.33. 1-((2-(Hexyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-
3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (40). From 7, procedures G
and H, yield 62% (in 2 steps), white solid, mp = 285.4−286.2 °C; 1H
4.9.41. 1-((2-(4-Fluorophenyl)-6-(trifluoromethyl)pyridin-3-yl)-
methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (48). From 7, pro-
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cedures G and H, yield 61% (in 2 steps), white solid, mp = 268.5−269.2
7.18 (bt, 1H), 6.67 (d, J = 7.1 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 3.45 (d,
J = 12.4 Hz, 2H), 2.74 (d, J = 12.0 Hz, 2H), 2.7 (m, 1H), 1.70−1.66 (m,
3H), 1.35 (m, 2H); HRMS (FAB): calcd for C29H29F3N5O2 [M + H]+,
536.2195, found, 536.2264.
1
°C; H NMR (400 MHz, DMSO-d6): δ 11.69 (s, 1H), 8.73 (s, 1H),
8.13 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 9.8 Hz, 1H), 7.94 (d, J = 7.9 Hz,
1H), 7.68−7.64 (m, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.39−7.33 (m, 3H),
7.09 (t, J = 5.5 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 9.8 Hz,
1H), 4.43 (d, J = 5.4 Hz, 2H); HRMS (FAB): calcd for C23H17F4N4O2
[M + H]+, 457.1209; found, 457.1286.
4.9.42. 1-((1-(3-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-
yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (49). From 7,
procedures G and H, yield 60% (in 2 steps), white solid, mp =
269.5−270.3 °C; 1H NMR (300 MHz, CD3OD): δ 8.66 (d, J = 8.1 Hz,
1H), 8.44 (d, J = 7.5 Hz, 1H), 7.66 (t, J = 8.1 Hz, 1H), 7.39 (d, J = 7.5
Hz, 1H), 7.21 (d, J = 7.0 Hz, 1H); HRMS (FAB): calcd for
C21H16ClF3N5O2 [M + H]+, 462.0866; found, 462.0940.
4.9.50. 1-(1-Oxo-1,2-dihydroisoquinolin-5-yl)-3-((2-(pyrrolidin-1-
yl)-6-(trifluoromethyl)pyridin-3-yl)methyl)urea (57). From 17, pro-
cedures G and H, yield 60% (in 2 steps), white solid, mp = 230.8−231.2
°C; 1H NMR (300 MHz, DMSO-d6): δ 11.31 (d, J = 5.3 Hz, 1H), 8.83
(s, 1H), 8.16 (dd, J = 7.9, 1.2 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.72 (d,
J = 7.7 Hz, 1H), 7.45 (t, J = 5.6 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.19
(m, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.85 (d, J = 7.5 Hz, 1H), 4.44 (d, J =
5.4 Hz, 2H), 3.55 (t, J = 6.6 Hz, 4H), 1.90 (m, 4H); HRMS (FAB):
calcd for C21H21F3N5O2 [M + H]+, 432.1569; found, 432.1589.
4.9.51. 1-((2-(Dipropylamino)-6-(trifluoromethyl)pyridin-3-yl)-
methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (58). From 17,
procedures G and H, yield 75% (in 2 steps), white solid, mp = 214.4−
216.7 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.34 (d, J = 5.3 Hz, 1H),
8.56 (s, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.81 (d, J
= 7.7 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 7.25 (t, J = 7.3 Hz, 1H), 7.07 (t, J
= 5.7 Hz, 1H), 6.67 (d, J = 7.5 Hz, 1H), 4.35 (d, J = 5.7 Hz, 2H), 3.19 (t,
J = 7.0 Hz, 4H), 1.45−1.57 (m, 4H), 0.83 (t, J = 7.3 Hz, 6H); HRMS
(FAB): calcd for C23H27F3N5O2 [M + H]+, 462.2039; found, 462.2115.
4.9.52. 1-((2-Butoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-3-(1-
oxo-1,2-dihydroisoquinolin-5-yl)urea (59). From 17, procedures G
and H, yield 59% (in 2 two steps), white solid, mp = 212.9−213.2 °C;
1H NMR (300 MHz, DMSO-d6): δ 11.29 (s, 1H), 8.56 (s, 1H), 8.11 (d,
4.9.43. 1-((1-(3-Fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-
yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (50). From 7,
procedures G and H, yield 71% (in 2 steps), white solid, mp =
1
294.9−295.7 °C; H NMR (300 MHz, DMSO-d6): δ 11.74 (s, 1H),
8.63 (s, 1H), 7.98 (d, J = 9.9 Hz, 2H), 7.65 (m, 2H), 7.45 (m, 4H), 6.99
(m, 2H), 6.88 (s, 1H), 6.52 (d, J = 9.6 Hz, 1H), 4.48 (d, J = 5.7 Hz, 2H);
HRMS (FAB): calcd for C21H16F4N5O2 [M + H]+, 446.1162; found,
446.1238.
4.9.44. 1-(2-Oxo-1,2-dihydroquinolin-5-yl)-3-((1-(m-tolyl)-3-(tri-
fluoromethyl)-1H-pyrazol-5-yl)methyl)urea (51). From 7, procedures
G and H, yield 63% (in 2 steps), white solid, mp = 294.2−295.6 °C; 1H
NMR (300 MHz, DMSO-d6): δ 11.7 (s, 1H), 8.65 (s, 1H), 7.98 (d, J =
9.9 Hz, 1H), 7.45 (m, 6H), 7.03 (t, J = 5.7 Hz, 1H), 6.97 (d, J = 8.1 Hz,
1H), 6.82 (s, 1H), 6.50 (d, J = 9.9 Hz, 1H), 4.41 (d, J = 5.7 Hz, 2H),
2.48 (s, 3H); HRMS (FAB): calcd for C22H19F3N5O2 [M + H]+,
442.1413; found, 442.1479.
J = 7.0 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.69
(m, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.23 (t, J =
7.0 Hz, 1H), 7.00 (m, 1H), 6.68 (d, J = 7.1 Hz, 1H), 4.35 (m, 4H),
1.70−1.79 (m, 2H), 1.42−1.49 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H);
HRMS (FAB): calcd for C21H22F3N4O3 [M + H]+, 435.1566; found,
435.1578.
4.9.45. 1-((1-(3-Isopropylphenyl)-3-(trifluoromethyl)-1H-pyrazol-
5-yl)methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (52). From 7,
procedures G and H, yield 56% (in 2 steps), white solid, mp = 253.6−
254.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.65 (s,
1H), 7.98 (d, J = 9.9 Hz, 1H), 7.49 (m, 6H), 7.03 (t, J = 5.1 Hz, 1H),
6.96 (d, J = 1.2 Hz, 1H), 6.85 (s, 1H), 6.50 (d, J = 9.9 Hz, 1H), 4.41 (d, J
= 5.4 Hz, 2H), 3.00 (m, 1H), 1.22 (d, J = 6.2 Hz, 6H); HRMS (FAB):
calcd for C24H23F3N5O2 [M + H]+, 470.1726; found, 470.1799.
4.9.46. 1-((3-(tert-Butyl)-1-(3-chlorophenyl)-1H-pyrazol-5-yl)-
methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (53). From 7, pro-
cedures G and H, yield 54% (in 2 steps), white solid, mp = 257.1−258.3
4.9.53. 1-((2-Isobutoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-3-
(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (60). From 17, procedures G
and H, yield 63% (in 2 steps), white solid, mp = 213.8−214.3 °C; 1H
NMR (300 MHz, DMSO-d6): δ 11.33 (d, J = 5.1 Hz, 1H), 8.63 (s, 1H),
8.12 (dd, J = 7.8, 1.1 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.83 (d, J = 7.5
Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.49 (d, J =
7.3 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.23 (t, J = 5.8 Hz, 1H), 7.07 (t, J =
5.8 Hz, 1H), 6.07 (d, J = 7.3 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 4.13 (d, J
= 6.3 Hz, 2H), 2.09 (p, J = 6.6 Hz, 1H), 1.01 (d, J = 6.6 Hz, 6H); HRMS
(FAB): calcd for C21H22F3N4O3 [M + H]+, 435.1566; found, 435.1578.
4.9.54. 1-((2-(Cyclobutylmethoxy)-6-(trifluoromethyl)pyridin-3-
yl)methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (61). From
17, procedures G and H, yield 74% (in 2 steps), white solid, mp =
213.8−214.2 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.33 (d, J = 5.4
Hz, 1H), 8.59 (s, 1H), 8.12 (d, J = 6.7 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H),
7.82 (d, J = 7.5 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.38 (t, J = 8.0 Hz,
1H), 7.24 (d, J = 5.8 Hz, 1H), 7.00 (t, J = 5.6 Hz, 1H), 4.33 (d, J = 6.6
Hz, 4H), 2.77 (m, 1H), 2.10−1.99 (m, 6H); HRMS (FAB): calcd for
C22H22F3N4O3 [M + H]+, 447.1566; found, 447.1579.
4.9.55. 1-((2-(Cyclopentylmethoxy)-6-(trifluoromethyl)pyridin-3-
yl)methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (62). From 17,
procedures G and H, yield 76% (in 2 steps), white solid, mp = 232.3−
234.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.33 (s, 1H), 8.60 (s,
1H), 8.10 (d, J = 6.9 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 7.5
Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.23 (t, J =
5.6 Hz, 1H), 7.02 (t, J = 6.0 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H), 4.32 (d, J
= 5.4 Hz, 2H), 4.22 (d, J = 6.9 Hz, 2H), 2.34 (p, J = 7.3 Hz, 1H), 1.78−
1.76 (m, 2H), 1.60−1.48 (m, 4H), 1.38−1.32 (m, 2H); HRMS (FAB):
calcd for C23H24F3N4O3 [M + H]+, 461.1722; found, 461.1787.
4.9.56. 1-((2-(Benzyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-
3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (63). From 17, procedures
G and H, yield 81% (in 2 steps), white solid, mp = 231.6−232.6 °C; 1H
NMR (300 MHz, DMSO-d6): δ 11.30 (s, 1H), 8.59 (s, 1H), 8.11 (d, J =
7.7 Hz, 1H), 7.87 (d, J = 8.1 Hz, 2H), 7.54 (m, 4H), 7.32−7.40 (m,
4H), 7.23 (t, J = 7.3 Hz, 1H), 7.06 (m, 1H), 6.68 (d, J = 7.3 Hz, 1H),
5.46 (s, 2H), 4.38 (d, J = 5.7 Hz, 2H); HRMS (FAB): calcd for
C24H20F3N4O3 [M + H]+, 469.1409; found, 469.1501.
1
°C; H NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.56 (s, 1H),
7.98 (d, J = 9.9 Hz, 1H), 7.52 (m, 5H), 7.30 (t, J = 7.8 Hz, 1H), 6.98 (d,
J = 8.1 Hz, 1H), 6.92 (t, J = 5.4 Hz, 1H), 6.51 (d, J = 9.9 Hz, 1H), 6.37
(s, 1H), 4.45 (d, J = 5.4 Hz, 2H), 1.29 (s, 9H); HRMS (FAB): calcd for
C24H25ClN5O2 [M + H]+, 450.1619; found, 450.1630.
4.9.47. 1-((3-(tert-Butyl)-1-(3-fluorophenyl)-1H-pyrazol-5-yl)-
methyl)-3-(2-oxo-1,2-dihydroquinolin-5-yl)urea (54). From 7, pro-
cedures G and H, yield 63% (in 2 steps), white solid, mp = 250.9−251.2
1
°C; H NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.74 (s, 1H),
8.03 (d, J = 9.9 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.55 (d, J = 7.7 Hz,
1H), 7.44 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 8.1 Hz, 1H), 7.02 (t, J = 5.7
Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 9.9 Hz, 1H), 4.35 (d, J =
5.3 Hz, 2H), 2.75 (m, 2H), 1.70 (m, 2H), 1.54 (br s, 1H), 1.29 (m, 1H),
0.94 (d, J = 6.4 Hz, 3H); HRMS (FAB): calcd for C24H25FN5O2 [M +
H]+, 434.1914; found, 434.1989.
4.9.48. 1-(1-Oxo-1,2-dihydroisoquinolin-5-yl)-3-((2-(piperidin-1-
yl)-6-(trifluoromethyl)pyridin-3-yl)methyl)urea (55). From 17, pro-
cedures G and H, yield 61% (in 2 steps), white solid, mp = 231.2−232.3
°C; 1H NMR (300 MHz, DMSO-d6): δ 11.32 (d, J = 5.3 Hz, 1H), 8.58
(s, 1H), 8.10 (d, J = 6.7 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.84 (d, J =
8.2 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.24 (t, J
= 7.7 Hz, 1H), 7.09 (t, J = 7.7 Hz, 1H), 6.67 (d, J = 7.5 Hz, 1H), 4.37 (d,
J = 5.9 Hz, 2H), 3.11 (m, 4H), 1.67 (m, 6H); HRMS (FAB): calcd for
C22H22F3N5O2 [M + H]+, 446.1726; found, 446.1790.
4.9.49. 1-((2-(4-Benzylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-
3-yl)methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (56). From
17, procedures G and H, yield 60% (in 2 steps), white solid, mp =
231.8−232.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.34 (d, J = 4.9
Hz, 1H), 8.58 (s, 1H), 8.09 (d, J = 7.1 Hz, 1H), 7.89−7.82 (m, 2H),
7.46 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.31−7.28 (m, 4H),
4.9.57. 1-((2-(Cyclohexylthio)-6-(trifluoromethyl)pyridin-3-yl)-
methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (64). From 17,
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procedures G and H, yield 54% (in 2 steps), white solid, mp = 264.8−
265.4 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.31 (br s, 1H), 8.63 (s,
1H), 8.10 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.6
Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.24 (t, J =
6.5 Hz, 1H), 7.12 (bt, 1H), 6.69 (d, J = 7.5 Hz, 1H), 4.28 (d, J = 5.4 Hz,
2H), 3.92 (m, 1H), 2.05−1.94 (m, 4H), 1.58−1.23 (m, 6H); HRMS
(FAB): calcd for C23H24F3N4O2S [M + H]+, 477.1494; found,
477.1513.
4.9.58. 1-((2-(3-Isopropylphenyl)-6-(trifluoromethyl)pyridin-3-yl)-
methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (65). From 17,
procedures G and H, yield 68% (in 2 steps), white solid, mp = 287.2−
288.4 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.30 (s, 1H), 8.54 (s,
1H), 8.12 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 7.9, 1.2 Hz, 1H), 7.92 (d, J =
8.0 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.46−7.32 (m, 5H), 7.21 (t, J =
7.3 Hz, 1H), 7.10 (bt, 1H), 6.53 (d, J = 7.3 Hz, 2H), 4.42 (d, J = 5.4 Hz,
2H), 2.97 (p, J = 6.7 Hz, 1H), 1.23 (d, J = 6.9 Hz, 6H); HRMS (FAB):
calcd for C26H24F3N4O2 [M + H]+, 481.1773; found, 481.1842.
4.9.59. 1-((1-(3-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-
yl)methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (66). From
17, procedures G and H, yield 71% (in 2 steps), white solid, mp =
229.0−231.2 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.31 (br s, 1H),
8.45 (s, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.76 (s,
1H), 7.36 (s, 3H), 7.30 (t, J = 8.1 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.03
(t, J = 5.7 Hz, 1H), 6.88 (s, 1H), 6.61 (d, J = 7.5 Hz, 1H), 4.48 (d, J = 5.4
Hz, 2H); HRMS (FAB): calcd for C21H16ClF3N5O2 [M + H]+,
462.0866; found, 462.0939.
4.9.60. 1-((3-(tert-Butyl)-1-(3-chlorophenyl)-1H-pyrazol-5-yl)-
methyl)-3-(1-oxo-1,2-dihydroisoquinolin-5-yl)urea (67). From 17,
procedures G and H, yield 81% (in 2 steps), white solid, mp = 224.8−
225.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.32 (br s, 1H), 8.39 (s,
1H), 8.10 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.57 (s, 1H),
7.51 (m, 3H), 7.38 (t, J = 8.1 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H), 7.18 (t, J
= 8.7 Hz, 1H), 6.97 (t, J = 5.7 Hz, 1H), 6.78 (m, 1H), 6.62 (d, J = 7.5
Hz, 1H), 6.38 (s, 1H), 4.45 (d, J = 5.1 Hz, 2H), 1.29 (s, 9H); HRMS
(FAB): calcd for C24H25ClN5O2 [M + H]+, 450.1619; found, 450.1690.
4.10. Computational Studies. 4.10.1. Homology Modeling. The
primary sequence of hTRPV1 was retrieved from UniProtKB
(accession: Q8NER1), and the transmembrane region (residues
417−712) was used in the homology modeling. As template structures,
the cryo-EM structures of rTRPV1 bound to RTX (PDB id: 5IRX)8 and
CAPZ (PDB id: 5IS0)8 were used for the agonist-bound and
antagonist-bound models, respectively. The corresponding residues
from Asn604 to Ser627 of hTRPV1 were not solved in the template
structure and identified as being located far from the ligand-binding
site; these 24 residues were truncated. Based on the sequence alignment
using the Clustal program,25 the homology model of the hTRPV1
monomer was constructed using the “Build Homology Models”
protocol of MODELER 9v1526 implemented in Discovery Studio.
The bound ligands were copied during the model building. The 10
models were derived, and the model with the lowest probability density
function total energy was chosen for further optimization. The models
were further refined by using the “Loop refinement (MODELER)” and
“Side-chain refinement” protocols. After building the tetrameric model
by aligning the monomer models to the tetrameric template structure,
the transmembrane region was predicted by performing the “Add
Membrane and Orient Molecule” protocol with the Generalized Born
with simple SWitching (GBSW)27 as an implicit solvent model. The
generated tetramer model with the implicit membrane was energy
minimized using the CHARMM force field with partial charges of CFF.
The GBSW implicit solvent model was used with the dielectric constant
set as 2 for the membrane nonpolar region and an implicit solvent
dielectric constant set as 80 for the water solvent. Some constraints such
as harmonic restraints to the backbone atoms with a force constant of 5
and the SHAKE constraint to the bonds including hydrogen atoms were
applied.
solvated with explicit water molecules and ionized with 0.15 M NaCl.
After 10 ns equilibration with the NVT ensemble, the production runs
with the NPT ensemble were carried out for 100 ns. From the final 30 ns
simulated trajectories, 300 protein conformations were sampled and
clustered using the clustering tool in the VMD plugin to calculate and
visualize the clusters of conformations. The clustering was carried out
with the residues within 6 Å of the complexed ligand. The number of
target clusters was set to 3 and a distance function was set to a rmsd with
a cutoff of 1.0 Å as the maximal distance value between two similar
frames. After clustering the protein conformations into 11 clusters, the
conformations with the lowest total energy were selected from each
cluster.
4.10.3. Ensemble Docking. Each of the protein conformation was
prepared using the “Protein Preparation Wizard” implemented in
Maestro v. 11.4 (Schrodinger, LLC, NY). 3D structures of the ligands
were prepared using the “LigPrep” module in Maestro. The generation
of ionization states significantly populated in pH 7.4 was performed
with Ionizer. The geometries of the generated structures were energy-
minimized using optimized potential for liquid simulation (OPLS) 3
force field,31 while keeping the chirality from the input files throughout
the calculations. The ensemble docking studies were performed using
the “Virtual screening workflow” protocol, which includes the Glide
docking. The grids of all protein conformations were added into the
receptor table with the default parameters. The Glide SP docking of the
ligand was performed first with a van der Waals radii scaling factor of 0.8
for ligand nonpolar atoms and a partial charge cutoff of 0.15. The 30
poses were generated for each ligand and for each protein
conformation, keeping all the good scoring states. The ensemble
docking results were then merged to generate a maximum of 30 poses
based on the glide Emodel scores. The top five poses of the resulting
docked complexes were then refined by energy minimization with the
OPLS3 force field.
All of the molecular graphic figures were generated using PyMOL
software (http://www.pymol.org). All computational studies were
undertaken on an Intel Xeon Octa-Core 2.67 GHz workstation with
Linux CentOS release 6.7.
4.10.4. Body Temperature Measurements. The rectal measurement
procedure was executed by lifting the tail and inserting a rectal probe
thermometer (RET-2, Physitemp Instruments, Clifton, NJ, USA), with
minimal restraint achieved by holding the base of the tail. The rectal
temperature measurement procedure was executed over 1 min. In this
study, we followed the national guidelines for conducting animal
experiments. All procedures for animal tests were approved by the
Medifron Animal Care and Use Committee (approval number,
Medifron 2017-1, IACUC).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00982.
■
sı
HPLC purities of the final compounds and the figures of
molecular modeling (PDF)
Molecular formula strings and biological data (PDB)
3D representation of Figure 3 (PDB)
Molecular formula strings of the prepared compounds
(CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Jeewoo Lee − Laboratory of Medicinal Chemistry, College of
Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea; orcid.org/0000-0002-7832-6719; Phone: 82-2-
880-7846; Email: jeewoo@snu.ac.kr; Fax: 82-2-888-0649
Sun Choi − Laboratory of Molecular Modeling & Drug Design,
College of Pharmacy and Graduate School of Pharmaceutical
Sciences, Ewha Womans University, Seoul 03760, Republic of
Korea; orcid.org/0000-0002-7669-7954; Phone: 82-2-
4.10.2. MD Simulations. The final models were subjected to MD
simulations using GROMACS v.5.1.428 with CHARMM36 force
fields.29 The input system with the 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphatidylcholine lipid membrane was constructed by using the
“Membrane Builder” module in CHARM-GUI.30 The system was
382
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3277-4503; Email: sunchoi@ewha.ac.kr; Fax: 82-2-3277-
2851
ACKNOWLEDGMENTS
■
This work was supported by the Midcareer Researcher Program
(NRF-2019R1A2C2006837) funded by the National Research
Foundation of Korea (NRF), the Bio & Medical Technology
Development Program (NRF-2019M3E5D4065251) and the
Medical Research Center (MRC) grant (no.
2018R1A5A2025286) through the National Research Founda-
tion of Korea (NRF). We thank the Korea Institute of Science
and Technology Information (KISTI) National Supercomput-
ing Center for providing computing resources (KSC-2019-CRE-
0076). This work was also supported in part by the Intramural
Program of the Center for Cancer Research, National Cancer
Institute, NIH (project Z1A BC 005270).
Authors
Shivaji A. Thorat − Laboratory of Medicinal Chemistry, College
of Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea
Yoonji Lee − Laboratory of Molecular Modeling & Drug Design,
College of Pharmacy and Graduate School of Pharmaceutical
Sciences, Ewha Womans University, Seoul 03760, Republic of
Korea; College of Pharmacy, Chung-Ang University, Seoul
06974, Korea
Aeran Jung − Laboratory of Medicinal Chemistry, College of
Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea
Jihyae Ann − Laboratory of Medicinal Chemistry, College of
Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea
Songyeon Ahn − Laboratory of Medicinal Chemistry, College of
Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea
Jisoo Baek − Laboratory of Medicinal Chemistry, College of
Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea
Dongxu Zuo − Laboratory of Medicinal Chemistry, College of
Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea
Nayeon Do − Laboratory of Medicinal Chemistry, College of
Pharmacy, Seoul National University, Seoul 08826, Republic of
Korea
ABBREVIATIONS
■
TRPV1, transient receptor potential vanilloid 1; cryo-EM,
transmission electron cryomicroscopy; hTRPV1, human
TRPV1; rTRPV1, rat TRPV1; SAR, structure activity relation-
ship; RTX, resiniferatoxin; CHO, Chinese hamster ovary;
FLIPR, fluorometric imaging plater reader; HEK, human
embryonic kidney; BCTC, 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-
dimethylethyl)phenyl]-1-piperazinecarboxamide; ip, intraperi-
toneal; MD, molecular dynamics; rmsd, root mean squared
deviation
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