Discovery of dual-acting opioid ligand and TRPV1 antagonists as novel therapeutic agents for pain

Article abstract of DOI:10.1016/j.ejmech.2019.111634

In order to discover a novel type of analgesic, we investigated dual activity ligands with TRPV1 antagonism and mu-opioid receptor affinity with the goal of eliciting synergistic analgesia while avoiding the side effects associated with single targeting.

Full text of DOI:10.1016/j.ejmech.2019.111634

Journal Pre-proof
Discovery of dual-acting opioid ligand and TRPV1 antagonists as novel therapeutic
agents for pain
Hobin Lee, Songyeon Ahn, Jihyae Ann, Heejin Ha, Young Dong Yoo, Young Ho Kim,
Ji-Young Hwang, Kwang-Hyun Hur, Choon-Gon Jang, Larry V. Pearce, Timothy E.
Esch, Nancy E. Lewin, Peter M. Blumberg, Jeewoo Lee
PII:
S0223-5234(19)30768-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.111634
EJMECH 111634
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2019
Revised Date: 19 August 2019
Accepted Date: 19 August 2019
Please cite this article as: H. Lee, S. Ahn, J. Ann, H. Ha, Y.D. Yoo, Y.H. Kim, J.-Y. Hwang, K.-H. Hur,
C.-G. Jang, L.V. Pearce, T.E. Esch, N.E. Lewin, P.M. Blumberg, J. Lee, Discovery of dual-acting opioid
ligand and TRPV1 antagonists as novel therapeutic agents for pain, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111634.
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Graphical Abstract
F3C
TRPV1
H
N
H
binding affinity : Ki = 10.8 nM
antagonism : Ki(ant) = 32 nM
MOR
N
N
N
O
binding affinity : Ki = 29 nM
N
In vivo formalin test
IC50 = 10.5 mg/kg
49

Discovery of Dual-acting Opioid Ligand and TRPV1
Antagonists as Novel Therapeutic Agents for Pain
a
Hobin Lee a, Songyeon Ahn , Jihyae Ann a, Heejin Ha b, Young Dong Yoo b, Young Ho Kim b, Ji-
c
c
c
d
d
Young Hwang , Kwang-Hyun Hur , Choon-Gon Jang , Larry V. Pearce , Timothy E. Esch ,
Nancy E. Lewin d, Peter M. Blumberg d, Jeewoo Lee a,*
a Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul 08826,
Korea
b Medifron DBT, Sandanro 349, Danwon-Gu, Ansan-City, Gyeonggi-Do 15426, Republic of Korea
c Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-
do 16419, South Korea
d
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer
Institute, NIH, Bethesda, MD 20892-4255, USA
* To whom correspondence should be addressed.
Phone: 82-2-880-7846
Fax: 82-2-883-9172
E-mail: jeewoo@snu.ac.kr
Key Words: TRPV1; mu-opioid receptor; analgesic; dual-acting mechanism
1

Abstract
In order to discover a novel type of analgesic, we investigated dual activity ligands with TRPV1
antagonism and mu-opioid receptor affinity with the goal of eliciting synergistic analgesia while
avoiding the side effects associated with single targeting. Based on a combination approach, a series
of 4-benzyl-4-(dimethylamino)piperidinyl analogues were designed, synthesized and evaluated for
their receptor activities. Among them, compound 49 exhibited the most promising dual-acting
activity toward TRPV1 and the mu-opioid receptor in vitro. In vivo, 49 displayed potent, dose-
dependent antinociceptive activity in both the 1st and 2nd phases in the formalin assay. Consistent
with its postulated mechanism, we confirmed that in vivo, as in vitro, compound 49 both
antagonized TRPV1 and functioned as a mu-opioid agonist. This result indicates that dual-acting
TRPV1 antagonist / mu-opioid ligands can be made and represent a new and promising class of
analgesic.
2

1. Introduction
Pain results from a complex interaction between specialized nerves and signalling systems.
Since the transmission of pain signals involves a multitude of parallel processes, a single-target
analgesic may not be as effective as would be suggested by the agent’s high selectivity and potency.
An alternative approach is to seek simultaneous intervention at multiple nodes in these parallel
pathways through a single multi-target drug. There is now abundant validation of this multi-target
drug strategy showing the integration of simultaneous actions on multiple targets.1 A further benefit
of this approach is that the relatively lower affinity of multi-target agents may not only elicit a
synergistic therapeutic effect by the the combination of various mechanistic actions but also show
reduced side effects.
Most strong pain-relieving analgesics currently in use act through the opioid receptor. The
opioid receptor has four subtypes, the mu, delta, kappa and nociceptin receptors.2 Among them, mu-
opioid receptor (MOR) agonists are the most commonly used opioids for pain management. MOR
is an inhibitory G-coupled protein receptor found presynaptically and postsynaptically on various
nerve fibers in both the brainstem and spinal cord. Binding of an agonist to the receptor leads to a
reduction in cAMP, decreasing intracellular Ca2+ levels and inhibiting release of the nerve fiber's
neurotransmitters. The other effect of MOR activation is an increase in potassium efflux out of the
nerve fiber causing hyperpolarization of the nerve.
The transient receptor potential vanilloid 1 (TRPV1) has emerged as a promising
therapeutic target for the treatment of neuropathic pain and a range of other conditions, reflecting
the central role of this nociceptor in the function of C-fiber sensory neurons.3 The receptor
functions as a non-selective cation channel with high Ca2+ permeability. Different classes of
activators, such as low pH, noxious heat, endogenous lipid mediators and natural vanilloids, cause
an increase in intracellular Ca2+, thereby resulting in the excitation of the primary sensory neurons
and the consequent central perception. Therefore, TRPV1 antagonists inhibit this transmission of
3

nociceptive signaling from the periphery to the CNS, providing therapeutic potential for novel
analgesic and antiinflammatory agents.4
In order to discover novel analgesics with a multi-target mechanism, we investigated new
dual-target compounds acting both as a MOR ligand and a TRPV1 antagonist. This type of
compound should not only block pain transmission in both the peripheral and central nervous
system but may also reduce the side effects associated with single targeting, such as reinforcement
disorders, nausea, drowsiness and respiratory depression from mu-receptor agonism and marked
hyperthermia and impaired noxious heat sensation from TRPV1 antagonism.
Figure 1. Design of novel dual-acting scaffold
In order to design a dual-acting scaffold, we combined the key pharmacophores of the
potent TRPV1 antagonist 1 (GRT12360)5 and the piperidine MOR agonist 2 (Meperidine) to
generate a hydrid 3 (Figure 1). In this design, the 4-methylpiperidinyl moiety in 1 was replaced
with the structurally similar 4-benzyl-4-(dimethylamino)piperidinyl (BDP) group that was known
as a novel synthetic opioid (NSO)6. In addition, the pyridine and propanamide groups of 1 were
switched with the synthetically more accessible phenyl and urea surrogates, respectively. In this
new scaffold 3, a variety of substituted phenyl and heterocycles present in potent TRPV1
antagonists previously reported were incorporated into the A-region. The synthesized compounds
4

were assayed in vitro for MOR binding affinity and TRPV1 receptor activity. Then, the most
promising selected dual-acting compound in the series was tested in the formalin model to evaluate
its in vivo antinociceptive activity. Finally, it was confirmed that this compound indeed targeted
the mu-opioid receptor and TRPV1 in vivo, as it did in vitro.
2. Results and discussion
2.1. Chemistry
The C-region amine 8 was synthesized from commercially available 1-(tert-butoxy
carbonyl)-4-piperidone 4 in 5 steps (Scheme 1). The 4-keto group of 4 was converted to the 4-
cyano-4-dimethylamino moiety by the Strecker reaction in which the cyano group was substituted
with a benzyl group by Grignard reagent to afford 6. The piperidine 6 was reacted with 2-fluoro-4-
(trifluoromethyl)benzonitrile under basic conditions to give N-aryl piperidine 7. The nitrile of 7 was
reduced to the corresponding amine 8 which was converted to phenyl carbamate 9. Either amine 8
or carbamate 9 was used for the coupling reaction with the corresponding A-region carbamate or
amine, respectively, to provide the final compounds.
Scheme 1. Synthesis of the C-region.
5

Reagent and Conditions: (a) dimethylamine, KCN, MeOH, H2O, 0 oC to r.t, 24 h, 99%; (b) BnMgCl,
THF, reflux, 3 h, 55%; (c) CF3CO2H, CH2Cl2, r.t, 1 h, 95%; (d) 2-fluoro-4-
(trifluoromethyl)benzonitrile, K2CO3, 18-crown-6-ether, MeCN, reflux, overnight, 37%; (e)
.
BH2 SMe2, THF, reflux, overnight, 11%; (f) phenyl chloroformate, THF, MeCN, pyridine, 0 oC to r.t,
1 h, 99%.
For the synthesis of 4-methylsulfonamidophenyl A-region analogues (Scheme 2), the
propionic acid 105 was coupled with the C-region amine 8 under EDC conditions to give the
propanamide 11. The phenyl carbamates, 12 and 14,7 were condensed with amine 8 to provide the
ureas 13 and 15 as one-carbon elongated A-region analogues. The benzylamine 167 was reacted
with carbamate 9 to give the urea 17 as a one-carbon elongated B-region analogue.
Scheme 2. Synthesis of 3-fluoro-4-methylsulfonamidophenyl A-region analogues
Reagent and conditions: (a) amine 8, HOBt, EDC, TEA, 1,4-dioxane, r.t, overnight, 42%; (b) amine
8, DMAP, MeCN, 50 oC, overnight, 41-45%; (c) carbamate 9, DBU, DMF, 100 oC, overnight, 34%.
6

As the A-region bioisosteres of 15, its sulfamoylamino and hydroxyl surrogates were
synthesized (Scheme 3). Starting from 2-fluoro-4-nitrobenzyl amine 18, the amine was converted to
the N-Boc sulfamoylamino group using chlorosulfonyl isocyanate and t-butanol and then the 4-nitro
group was transformed to the phenylcarbamate 19. For the synthesis of the hydroxyl analogue, 2-
fluoro-4-nitrobenzoic acid 21 was reduced to the corresponding alcohol and then the 4-nitro group
was converted to the phenylcarbamate 22. The condensation of 19 and 22 with amine 8 provided
the final sulfamoylamino and hydroxyl analogues, 20 and 23, respectively.
Scheme 3. Synthesis of (4-sulfamoylamino)methyl and 4-hydroxymethyl A-region analogues
o
Reagent and conditions: (a) ClSO2-N=C=O , t-BuOH, CH2Cl2, TEA, 0 C to r.t., 75%; (b) 10%
Pd/C, H2, THF-EtOH (1:1), r.t ., 3 h, 95%; (c) phenyl chloroformate, pyridine, THF, MeCN, r.t., 1 h,
90%; (d) amine 8, DMAP, MeCN, 50 oC, overnight, 55-63%; (e) CF3CO2H, CH2Cl2, r.t, 1 h, 64%;
(f) LiAlH4, Et2O, reflux, 30 min, 55%.
For the synthesis of 7-hydroxynaphthalen A-region analogues (Scheme 4), commercially
available 8-aminonaphthalen-2-ol 24 was converted to the corresponding phenylcarbamate 25 or
reduced and then carbamoylated to the tetrahydronaphthalene 27. The condensation of 25 and 27
with amine 8 provided the final products, 26 and 28, respectively.
7

Scheme 4. Synthesis of 7-hydroxynaphthalen A-region analogues
Reagent and conditions: (a) pyridine, phenyl chloroformate, acetone, 0 oC to r.t, 30 min, 70-85%; (b)
2N HCl in 1,4-dioxane, PtO2, H2, ethanol, 45 oC, overnight, 65%; (c) amine 8, DMAP, MeCN, 50
oC, overnight, 59-60%.
For the synthesis of dihydrobenzofuran A-region analogue (Scheme 5), 2,3-
dihydrobenzofuran 29 was converted to the desired 6-nitro-2,3-dihydrobenzofuran 31 in 6 steps.
The nitro group of 31 was tranformed to the corresponding phenyl carbamate 32 which was
condensed with amine 8 to afford the final compound 33.
Scheme 5. Synthesis of the 2,3-dihydrobenzofuran A-region analogue.
Reagent and conditions: (a) acetyl chloride, AgNO3, MeCN, 0 oC, 1 h, 35%; (b) 10 % Pd/C, H2,
MeOH, THF, r.t., overnight, 82%; (c) acetic anhydride, 1,4-dioxane, r.t, overnight, 86%; (d) HNO3,
acetic acid, r.t, overnight, 28%; (e) c.HCl, ethanol, reflux, 2 h, 65%; (f) isoamylnitrite, THF, reflux,
41%; (g) 10 % Pd/C, H2, MeOH, r.t., overnight, 75%; (h) phenyl chloroformate, THF, MeCN,
pyridine, 0 oC to r.t., 1 h, 87%; (i) amine 8, DMAP, MeCN, 50 oC, overnight, 30%.
8

For the synthesis of quinolinone and isoquinolinone analogues (Scheme 6), the 5-
aminoquinolin-2-one 34 and 5-aminoisoquinolin-1-one 36 previously reported8 were converted the
corresponding phenyl carbamates and then coupled with the amine 8 to provide the quinolin-2-one
35 and isoquinolin-1-one 37, respectively.
Scheme 6. Synthesis of the quinolinone A-region analogues.
Reagent and conditions: (a) phenyl chloroformate, THF, MeCN, Pyridine, 0 oC to r.t., 1 h, 80-86%;
(b) amine 8, DMAP, MeCN, 50 oC, overnight, 30-40%.
Finally, for the synthesis of quinoline and isoquinoline analogues (Scheme 7),
aminoquinolines and aminoisoquinolines (38-43), which were commercially available or previously
reported9, were converted to the corresponding phenyl carbamate or trichloroacetamide, which were
reacted with the amine 8 to provide the final compounds (44-49), respectively.
9

Scheme 7. Synthesis of the quinoline A-region analogues.
Reagent and conditions: (a) [Method A] phenyl chloroformate, pyridine, THF, MeCN, 0 oC to r.t, 2
h for 44, 45, 47, 48, 49, 60-75% [Method B] Trichloroacetyl chloride, TEA, CH2Cl2 for 46, 63%; (b)
[Method A] amine 8, DMAP, MeCN, 50 oC, overnight for 44, 45, 47, 48, 49, F [Method B] amine 8,
DBU, MeCN, reflux, overnight for 46, 25%.
2.2. In vitro activity
The binding affinities and potencies as TRPV1 agonists/antagonists of dual-acting ligands
were assessed in vitro by a binding competition assay with [3H]resiniferatoxin (RTX) and by a
45
functional Ca2+ uptake assay using human TRPV1 heterologously expressed in Chinese hamster
ovary (CHO) cells, as previously described.10 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. The
binding affinities as mu opioid ligands were measured by a binding competition assay with [3H]-D-
Ala2-MePhe4-Glyol5-enkephalin (DAMGO).11,12 The Ki values was calculated from the measured
IC50 values using the Cheng-Prusoff equation.13 The results are shown in Table 1 together with
those for morphine (Ki = 6.08 nM) and 1 (Ki(ant) = 2.22 nM) as references.
First, we investigated 4-methylsulfonamidophenyl A-region analogues. The direct
substitution of the 4-methylpiperidinyl group in 1 with the BDP group, providing 11, showed
10

similar activity in the binding affinity and antagonism toward TRPV1 compared to 1. However, its
binding affinity to MOR decreased significantly compared to that of morphine. The urea B-region
analogues of 11 (13, 15, 17) were next examined. Compound 13, the urea surrogate of 11, displayed
weak activity on both TRPV1 and MOR. On the other hand, whereas compound 15, the one-carbon
elongated analogue in the A-region, exhibited improved TRPV1 antagonism, compound 17, the
one-carbon elongated analogue in the B-region, displayed enhanced MOR activity. However,
neither compound showed the desired dual-acting activity. The sulfamide analogue 20 showed
improved MOR activity comparable to 17, but reduced TRPV1 antagonism compared to 15. The 4-
hydroxymethyl analogue 23 did not displayed any substantial improvement in either receptor
activity compared to 15.
Next, we investigated the bicyclic A-region analogues. Previously, the 7-
hydroxynaphthalen-1-yl14 and 7-hydroxytetrahydronaphthalen-1-yl15 urea analogues showed potent
TRPV1 antagonism. Based on that fact, we examined their BDP C-region analogues, 26 and 28.
Both showed high affinity and potent TRPV1 antagonism; however, their MOR activity was modest.
In addition, the 2,3-dihydrobenofuran-6-yl urea analogue 33 was examined, but the receptor
activities were also moderate. Recently, we discovered two distinct A-regions for TRPV1 functional
activity, quinolin-2-one-5-yl (34) and isoquinolin-1-one-5-yl (35), where derivatives of 34
displayed potent antagonism and derivatives of 36 displayed potent agonism, regardless of the C-
region.8 Similarly, their BDP C-region analogues (35, 37) displayed the same functional activities,
respectively, however the potencies in TRPV1 and MOR were moderate.
Finally, we explored quinoline and isoquinoline A-region analogues. Previously, we
reported that 6,6-fused heterocyclic ureas displayed highly potent TRPV1 antagonism.9 Based on
that, we investigated their BDP C-region analogues including quinolin-7-yl (44), isoquinolin-7-yl
(45), isoquinolin-6-yl (46), isoquinolin-8-yl (47), isoquinolin-5-yl (48) and quinolin-5-yl (49)
derivatives.
11

Whereas the β-substituted ring analogues (44-46) exhibited moderate TRPV1 antagonism, the α-
substituted ring analogues (47-49) displayed potent TRPV1 antagonism. Among them, compounds
46 and 49 showed reasonable MOR activities with Ki = 17 and 29 nM, respectively, that were just
3- and 5-fold less potent than that of morphine. Considering the two receptor activity, compound 49
was selected as most promising candidate with a dual-acting mechanism.
Table 1. In vitro activity of 4-benzyl-4-(dimethylamino)piperidinyl derivatives on hTRPV1 and
MOR
TRPV1a
Agonism
EC50 (nM)
MOR
Binding Affinity
Ki (nM)
R
Binding Affinity
Antagonism
Ki(ant) (nM)
Ki (nM)
Morphine
1 (rac)
6.08
7.9 (±1.6)
NE
NE
2.22 (±0.47)
8.2 (±1.7)
11
13
15
17
20
3.79 (±0.19)
428
368
1159 (±35)
16.4 (±0.90)
266 (±69)
NE
NE
NE
NE
WE
25.0 (±1.4)
85.9 (±7.0)
120 (±19)
322
76.2
72.4
15.3 (±3.6)
12

23
26
27.9 (±7.6)
NE
NE
81 (±19)
240
137
0.46 (±0.07)
8.9 (±1.2)
28
33
35
37
44
45
46
47
48
49
0.95 (±0.20)
41.4 (±8.1)
1.67 (±0.50)
111 (±15)
NE
NE
10.1 (±0.78)
31.3 (±9.5)
30.6 (±3.8)
NE
261
225
240
105
166
177
17.0
205
507
29
NE
43.7 (±6.7)
NE
14.1 (±2.9)
7.33 (±0.26)
62 (±10)
42 (±14)
NE
56.1 (±7.2)
156 (±50)
18.68 (±1.2)
8.8 (±4.1)
32 (±14)
NE
12.3 (±4.3)
0.67 (±0.13)
10.8 (±1.6)
NE
NE
NE
a
NE: no effect, WE: weak effect; values for TRPV1 are the mean of at least three experiments;
values for MOR represent single experiments.
2.3. In vivo activity
13

In order to evaluate antinociceptive activity of compound 49, we conducted the formalin
assay in ICR mice.16,17 In this assay, the compound was administered by intraperitoneal (i.p.)
injection 30 min before subcutaneous (s.c.) injection of 2% formalin solution into the hindpaw and
then the pain response of licking and biting of the injected paw was evaluated over the next 30 min.
The evaluation was performed over two discrete phases (1st phase, 0-5 min; 2nd phase, 20-30 min)
of the response upon formalin injection. The 1st Phase (0-5 min) models acute pain, whereas the 2nd
phase (20-30 min) models chronic pain. As shown in Figure 2, compound 49 exhibited potent
antinociceptive activity in a dose-response fashion with an ED50 of 23.9 mg/kg in the 1st phase and
10.5 mg/kg in the 2nd phase, respectively. In addition, at a dose of 40 mg/kg compound 49 was able
to inhibit the pain behavior completely in phase 2.
Figure 2. Antinociceptive activity of compound 49 in formalin assay
2.4. Target engagement study
To determine whether the antinociceptive effect of compound 49 was derived from in vivo
MOR activation and TRPV1 antagonism, we confirmed the target engagement of 49 employing the
capsaicin-induced paw licking model18 and a MOR antagonist pain model19.
The involvement of TRPV1 in the antinociceptive activity of 49 was investigated using the
capsaicin-induced paw licking model as a model of pain induced by capsaicin. As shown in Figure
14

3A, compound 49 administered intraperitoneally showed a dose-dependent reduction in the
capsaicin-induced nociceptive response, producing 64.8 and 89.6% inhibition against capsaicin-
induced pain compared to vehicle at doses of 20 and 40 mg/kg, respectively. These results confirm
that capsaicin-induced pain, which is mediated through TRPV1, was antagonized in vivo by 49.
The involvement of MOR in the antinociceptive activity of 49 was investigated using
naloxone, a MOR antagonist, in the formalin model. As shown in Figure 3B, intraperitoneal
administration of compound 49 decreased the paw licking time by 52.5 and 90.2 % in the 1st and 2nd
phases at a dose of 30 mg/kg, respectively. However, following pretreatment with naloxone (10
mg/kg, i.p.), the licking time in response to compound 49 was reduced to 5.7 and 47.9 % in 1st and
2nd phases, respectively, indicating that the analgesic effect of 49 was significantly reversed by
naloxone in both phases of the formalin-induced nociception.
A.
B.
Figure 3. (A) Compound 49 blocks capsaicin-induced licking behavior in mice. Results are
expressed as mean ± SEM (n = 3-4). **p<0.01 compared to vehicle-treated group. (B) Pretreatment
of naloxone significantly inhibits the analgesic effect of compound 49 in the formalin test. Results
are expressed as mean ± SEM (n=3-4). *p<0.05; **p<0.01 compared with vehicle-treated group:
**p<0.01 compared with 49 group.
15

3. Conclusion
In order to discover a novel analgesic with a multi-target mechanism, we investigated dual-
acting compounds with MOR ligand and TRPV1 antagonist activities. This type of compound could
potentially not only block the pain transmission in both the peripheral and central nervous system
but also minimize the side effects associated with single targeting. For the design of dual-acting
compounds, we combined the 4-benzyl-4-(dimethylamino)piperidinyl group known as NSO with a
potent TRPV1 antagonist template to generate a new scaffold in which the A-region was variably
modified with substituted aryl groups, including 3-fluoro-4-methylsulfonamidophenyl analogues,
(tetrahydro)naphthalene, and 6,6-fused heterocycles used as the A-region in previous potent TRPV1
antagonists. Among them, compound 49 displayed the most promising dual-acting activity with
Ki(ant) = 32 nM for TRPV1 and Ki = 29 nM for MOR. An in vivo study of compound 49 in the
formalin test indicated that 49 showed potent and dose-dependent antinociceptive activity with ED50
= 23.9 and 10.5 mg/kg in 1st and 2nd phases, respectively. The target engagement study indicated
that capsaicin-induced nociception was significantly reduced by 49 and the antinociceptive effect of
49 in the formalin model was blocked by the pretreatment of naloxone, suggesting that the
antinociceptive effect of 49 was derived from both TRPV1 antagonism and MOR activation. The
discovery of a new dual-acting compound with activity as a TRPV1 antagonist and as a MOR
ligand presents a new direction for the development of novel analgesics to overcome the problems
associated with single targeting.
4. Experimental
4.1. Chemistry
All chemical reagents were commercially available. Silica gel column chromatography was
performed on silica gel 60, 230-400 mesh, Merck. Nuclear magnetic resonance (1H NMR) spectra
were recorded on a JEOL JNM-LA 300 and a Bruker Avance 400 MHz FTNMR spectrometer.
16

Chemical shifts are reported in ppm units with Me4Si as a reference standard. Mass spectra were
recorded on a VG Trio-2 GC-MS and a 6460 Triple Quad LC/MS.
4.1.1. Synthesis of the C-region
4.1.1.1. tert-Butyl 4-cyano-4-(dimethylamino)piperidine-1-carboxylate (5)
To a solution of 4 (1.0 mmol) in MeOH was added dimethylamine solution (7.0 mmol) and
dimethylamine hydrochloride (3.0 mmol). Conc. HCl (0.5 mmol) was then added to the reaction
o
mixture dropwise at 0 C over 10 min and the mixture stirred for 1 h at room temperature.
Potassium cyanide (3.0 mmol) was added in portions to the reaction mixture and stirred 24 h at
room temperature. After completion, the reaction mixture was 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
1
to afford 5 as a white solid in 99% yield. H NMR (CDCl3, 300 MHz) δ 3.97 (d, J = 13.2 Hz, 2H),
3.14 (bt, J = 11.7 Hz, 2H), 2.35 (s, 6H), 2.08 (bd, J = 13.5 Hz, 2H), 1.69-1.59 (m, 2H), 1.46 (s, 9H);
FAB-MS m/z 254 [M+H]+.
4.1.1.2. 4-Benzyl-N,N-dimethylpiperidin-4-amine (6)
To a solution of 5 (1.0 mmol) in anhydrous THF was added benzylmagnesium chloride (2.0 M in
THF, 2.24 mmol). After the reaction mixture was refluxed for 3 h, the reaction mixture was cooled
to room temperature, diluted with aq. NH4Cl 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.
By following the general procedure described in 4.1.2.1, the Boc group was deprotected to give 6 as
1
a white solid in 55% yield. H NMR (CDCl3, 400 MHz) δ 7.03 - 7.37 (m, 5H), 3.19 (t, J = 12.2 Hz,
2H), 2.95 (d, J = 12.1 Hz, 2H), 2.66 (s, 2H), 2.25 (s, 6H), 1.88 (d, J = 14.0 Hz, 2H) 1.61 (dd, J = 3.4
Hz, 16.5 Hz, 6H); FAB-MS m/z 219 [M+H]+.
17

4.1.1.3. 2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzonitrile (7)
To a solution of 6 (1.0 mmol) in CH3CN, potassium carbonate (2.2 mmol) was added 18-crown-6
ether (0.1 mmol) and 2-fluoro-4-(trifluoromethyl)benzonitrile (0.9 mmol). The reaction mixture was
refluxed for 15 h and cooled to room temperature. The mixture was 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
1
to afford 7 as a white solid in 37% yield. H NMR (CDCl3, 400 MHz) δ 7.55 (d, J = 8.0 Hz, 1H),
7.35 (d, J = 4.6 Hz, 2H), 7.26 (d, J = 7.2 Hz, 1H), 7.07 - 7.21 (m, 4H), 3.21 - 3.33 (m, 4H), 2.71 (s,
2H), 2.33 (s, 6H), 1.93 (dd, J = 2.1, 14.4 Hz, 2H), 1.61 (dd, J = 4.2, 13.5 Hz, 2H); FAB-MS m/z 388
[M+H]+.
4.1.1.4. 1-(2-(Aminomethyl)-5-(trifluoromethyl)phenyl)-4-benzyl-N,N-dimethylpiperidin-4-amine
(8)
To a solution of 7 (1.0 mmol) in THF, borane dimethylsulfide complex (2 M in THF, 5.0 mmol)
was added dropwise and refluxed for 15 h. The mixture was cooled to 0 oC and quenched by water
until the remaining borane was inactivated. The reacton mixture was extracted with CH2Cl2 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 colunm chromatography to afford 8 as
1
a pale yellow solid in 11% yield. H NMR (CDCl3, 300 MHz) δ 7.45 (s, 1H), 7.29 - 7.34 (m, 3H),
7.16 - 7.23 (m, 4H), 4.73 (bs, 2H), 3.92 (t, J = 7.3 Hz, 2H), 3.16 (t, J = 11.2 Hz, 2H), 2.71 - 2.75 (m,
4H), 2.36 (s, 6H), 1.98 (d, J = 12.8 Hz, 2H); FAB-MS m/z 392 [M+H]+.
4.1.1.5. Phenyl (2-(4-benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)carb-
amate (9)
The title compound was obtained by following the general procedure of 4.1.2.9 as a white solid in
99% yield. 1H NMR (CDCl3, 300 MHz) δ 7.07 - 7.69 (m, 13H), 4.44 (d, J = 5.9 Hz, 2H), 3.31 (t, J
18

= 11.3 Hz, 2H), 2.73 (m, 4H), 2.36 (s, 6H), 1.92 (d, J = 12.5 Hz, 2H), 1.58 (m, 3H); FAB-MS m/z
512 [M+H]+.
4.1.2. General procedures
4.1.2.1. Boc deprotection.
To a solution of N-Boc protected compound (1.0 mmol) in CH2Cl2 was added dropwise
trifluoroacetic acid (20 mmol) at 0 °C and stirred for 1 h at room temperature. Then, the reaction
mixture was quenched with saturated NaHCO3 solution 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 flash column chromatography on silica gel to afford the desired
product.
4.1.2.2. N-Boc sulfamide.
Chlorosulfonyl isocyanate (CSI, 1.1 mmol) was dissolved in CH2Cl2 and cooled to 0 oC. A solution
of tert-butyl alcohol (1.1 mmol) in CH2Cl2 was added dropwise and stirring continued for 30 min at
room temperature. The resulting solution was slowly added into a solution of 18 (1.0 mmol) in
CH2Cl2 and triethylamine (1.1 mmol) at 0 °C. The resulting solution was allowed to warm to room
temperature. The reaction mixture was diluted with CH2Cl2 and washed with 0.1 N HCl and water.
The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was
puried by column chromatography on silica gel to afford the desired product.
4.1.2.3. Carboxylic acid reduction.
To a stirred suspension of LiAlH4 (3.0 mmol) in ether was added dropwise a solution of 21 in ether
at 0 oC over 15 min. The mixture was refluxed for 30 min, then the reaction cooled to 0 oC and
19

quenched with 1N HCl. The precipitate was filtered off and washed with ether. The combined
filtrate was dried over MgSO4, filtered and concentrated in vacuo. The residue was puried by
column chromatography on silica gel to afford the desired product.
4.1.2.4. Catalytic Hydrogenation
Method A. To a solution of nitro compound (1.0 mmol) in MeOH / THF (1:1) was added 10% Pd/C
and charged with hydrogen gas. The mixture was stirred for 2 h at room temperature. After
completion, the mixture was filtered through celite and the filtrate was concentrated in vacuo. The
residue was puried by column chromatography on silica gel to afford the desired product.
Method B. To a solution of 24 in EtOH, 2N HCl in 1,4-dioxane and PtO2 was added and charged
with hydrogen gas. The reaction mixture was stirred at 45 oC for 15 h. After completion, the mixture
was filtered through celite and the filtrate was concentrated in vacuo. The compound was used
without further purification.
4.1.2.5. Nitration
Method A. A solution of 29 (1.0 mmol) in CH3CN was cooled to 0 oC and was added with silver
nitrate (1.0 mmol) and acetyl chloride (1.0 mmol). The reaction mixture was stirred at 0 oC for 1 h.
After completion, the reaction mixture was diluted with water and extracted with EtOAc several
times. The combined organic layer was washed with brine, dried over MgSO4 and concentrated in
vacuo. The residue was purified by silica gel column chromatography to afford the desire product.
Method B. To a mixture of acetic acid and nitric acid (1.4 mmol) was added dropwise a solution of
o
30 (1.0 mmol) in acetic acid at 0 C and stirred at room temperture for 15 h. After completion, ice
was added to the reaction mixture and the participate was filtered and washed with water several
times. The filtrate was dried over MgSO4, concentrated in vacuo and the combined solid was used
for the next step without further purification.
20

4.1.2.6. N-Acetylation
To a solution of the appropriate amine (1.0 mmol) in 1,4-dioxane was added acetic anhydride (2.1
mmol) and the mixture was stirred at room temperature for 15 h. After completion, the solution was
diluted with water and extrated 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.
4.1.2.7. Deacetylation
A suspension of N-acetyl compound (2.07 mmol) and conc. HCl (6.5 mL) in EtOH was refluxed for
2 h. The resulting solution was concentrated under a reduced pressure and carefully neutralized with
NH4OH solution to pH 8. The reaction mixture was extracted with EtOAc, dried over MgSO4,
filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography
to afford the desired product.
4.1.2.8. Deamination
To a solution of the appropriate aryl amine (1.0 mmol) in THF was added isoamylnitrile (3.0 mmol)
and the mixture was refluxed for 3 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.
4.1.2.9. Carbamation
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 oC and stirred at room temperature for 1 h. The reaction
mixture was quenched with water and extracted with EtOAc several times. The combined organic
layer was washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The residue
21

was purified by silica gel colunm chormatography to afford the desired product.
4.1.2.10. Trichloroacetamide
To a solution of the appropriate amine (1.0 mmol) in CH2Cl2 was added triethylamine (1.0 mmol)
and trichloroacetyl chloride (1.1 mmol) at 0 oC and stirred at room temperature for 15 h. After
completion, the reaction mixture was 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.
4.1.2.11. Urea coupling
Method A. To a solution of the appropriate carbamate (1.0 mmol) in CH3CN was added amine (2.0
mmol) and 4-dimethylaminopyridine (1.0 mmol) and stirred at 50 oC 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 wash with brine, dried with MgSO4, filtered and
concentrated in vacuo. The residue was purified by silica gel column chromatography
(CH2Cl2:MeOH=20:1) to afford the desired product in 60-80 % yield.
Method B. To a solution of trichloroacetamide or carbamate (1.0 mmol) in DMF was added the
appropriate amine (1.1 mmol) and 1,8-diazabicyclo(5.4.0)undec-7-ene (2.5 mmol). After stirring at
o
100 C for 15 h, the reaction mixture was cooled to room temperature, diluted with water and
extracted with EtOAc several times. The combined organic layer was dried with MgSO4, filtered
and concentrated in vacuo. The residue was purified by silica gel column chromatography
(CH2Cl2:MeOH=20:1) to afford the desired product in 70-90% yield.
4.1.2.12. EDC coupling
To a solution of 10 (1.0 mmol) in 1,4-dioxane, 8 (1.05 mmol) was added 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (1.5 mmol), hydroxybenzotriazole (1.5 mmol) and
22

triethylamine (2.5 mmol). After stirring at room temperature for 15 h, the reaction mixture was
diluted with H2O 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 (CH2Cl2:MeOH=20:1) to afford the desired product in 85% yield.
4.1.3. Chemical Spectra
4.1.3.1.
N-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (11)
42 % yield, white solid, mp = 80-86 oC; 1H NMR (300 MHz, CD3OD) δ 6.99-7.35 (m, 11H), 4.25 (s,
2H), 3.54 (m, 1H), 2.96 (d, J = 11.7 Hz, 2H), 2.88 (s, 3H), 2.76 (s, 2H), 2.62 (m, 2H), 2.36 (s, 6H),
1.94 (d, J = 11.0 Hz, 2H), 1.58 (m, 2H), 1.33 (d, J = 7.0 Hz, 3H); HRMS (FAB) calcd for
C32H38F4N4O3S [M + H]+ 635.2679, found: 635.2668.
4.1.3.2.
N-(4-(3-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-
ureido)-2-fluorophenyl)methanesulfonamide (13)
41 % yield, white solid, mp = 115-120 oC; 1H NMR (300 MHz, DMSO) δ 8.44 (bs, 1H), 7.16 - 7.33
(m, 11H), 7.10 (d, J = 8.04 Hz, 2H), 4.15 (d, J = 5.5 Hz, 2H), 2.96 (m, 5H), 2.65 (m, 4H), 2.28 (s,
6H), 1.87 (m, 2H), 1.33 (m, 2H); HRMS (FAB) calcd for C30H35F4N5O3S [M + H]+ 622.2475,
found: 622.2476.
4.1.3.3.
N-(4-(3-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-
ureido)-2-fluorobenzyl)methanesulfonamide (15)
45 % yield, white solid, mp = 204-207 oC; 1H NMR (300 MHz, DMSO) δ 8.89 (s, 1H), 7.19 - 7.46
(m, 11H), 7.01 (dd, J = 1.8, 8.4 Hz, 1H), 6.66 (t, 1H), 4.21 (d, J = 5.4 Hz, 2H), 4.08 (d, J = 6.0 Hz,
2H), 2.99 (t, J = 11.0 Hz, 2H), 2.84 (s, 3H), 2.70 (m, 4H), 2.30 (s, 6H), 1.92 (d, J = 12.0 Hz, 2H),
1.43 (t, 2H); HRMS (FAB) calcd for C31H37F4N5O3S [M + H]+ 636.2632, found: 636.2625.
23

4.1.3.4.
N-(4-((3-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-
ureido)-methyl)-2-fluorophenyl)methanesulfonamide (17)
34% yield, white solid, mp = 85-90 oC; 1H NMR (300 MHz, DMSO) δ 9.5 (bs, 1H), 7.86 (bs, 1H),
7.02 - 7.33 (m, 10H), 6.58 (t, 1H), 6.49 (t, 1H), 4.14 (m, 4H), 2.98 (s, 3H), 2.69 (m, 4 H), 2.29 (s, 6
H), 1.90 (d, J = 14.0 Hz, 2H), 1.10 - 1.50 (m, 4H), 0.81 (m, 2 H); HRMS (FAB) calcd for
C31H37F4N5O3S [M + H]+ 636.2632, found: 636.2624.
4.1.3.5.
N-(4-(3-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-
ureido)-2-fluorobenzyl)aminosulfonamide (20)
64% yield, white solid, mp = 98-101 oC; 1H NMR (300 MHz, DMSO) δ 8.90 (s, 1H), 7.19 - 7.41 (m,
11H), 6.93 - 7.00 (m, 2H), 6.63 (s, 2H), 4.20 (d, J = 5.7 Hz, 2H), 3.99 (d, J = 6.2 Hz, 2H), 2.97 (d, J
= 9.9 Hz, 2H), 2.69 (m, 4H), 2.29 (s, 6H), 1.93 (m, J = 10.1 Hz, 2H), 1.43 (bs, 2H); HRMS (FAB)
calcd for C30H36F4N6O3S [M + H]+ 637.2584, found: 637.2580.
4.1.3.6.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-(3-
fluoro-4-(hydroxymethyl)phenyl)urea (23)
55% yield, white solid, mp = 89-94 oC; 1H NMR (300 MHz, CD3OD) δ 7.32 - 7.41 (m, 2H), 7.09 -
7.25 (m, 8H), 6.90 (dd, J = 1.8, 8.2 Hz, 1H), 4.48 (s, 2H), 4.29 (s, 2H), 3.02 (t, J = 11.3 Hz, 2H),
2.69 - 2.77 (m, 4H), 2.37 (s, 6H), 1.92 (d, J = 6.2 Hz, 2H), 1.68 (t, J = 10.4 Hz, 2H); HRMS (FAB)
calcd for C30H34F4N4O2 [M + H]+ 559.2696, found: 559.2699.
4.1.3.7.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-(7-
hydroxynaphthalen-1-yl)urea (26)
o
59% yield, light pink solid, mp = 123-127 C; 1H NMR (300 MHz, CD3OD) δ 7.74 (d, J = 8.4 Hz,
1H), 7.63 (d, J = 8.0 Hz, 1H), 7.07 - 7.48 (m, 12H), 4.37 (s, 2H), 3.03 (t, J = 11.3 Hz, 2H), 2.75 (s,
24

2H), 2.67 (d, J = 11.3 Hz, 2H), 2.36 (s, 6H), 1.88 (d, J = 13.4 Hz, 2H), 1.53 (t, J = 7.1 Hz, 2H);
HRMS (FAB) calcd for C33H35F3N4O2 [M + H]+ 557.2790, found: 557.2788.
4.1.3.8.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-(7-
hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)urea (28)
60% yield, white solid, mp = 176-179 oC; 1H NMR (300 MHz, CDCl3) δ 7.39 - 7.29 (m, 3H), 6.99 -
7.21 (m, 8H), 6.00 (bs, 1H), 5.30 (bs, 1H), 4.38 (d, J = 3.8 Hz, 2H), 4.11 (m, 1H), 3.05 (t, J = 11.2
Hz, 2H), 2.91 - 2.81 (m, 4H), 2.70 (s, 2H), 2.62 (m, 2H), 2.33 (s, 6H), 1.87 (d, J = 12.3 Hz, 2H),
1.45 (t, J = 11.9 Hz, 2H); HRMS (FAB) calcd for C33H39F3N4O2 [M + H]+ 581.3103, found:
581.3102.
4.1.3.9.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-(2,3-
dihydrobenzofuran-6-yl)urea (33)
o
1
30% yield, white solid, mp = 87-93 C; H NMR (400 MHz, CD3OD) δ 7.45 (d, J = 8.0 Hz, 1H,),
7.40 (s, 1 H), 7.18 - 7.33 (m, 6 H), 7.04 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 1.7 Hz, 1H), 6.68 (dd, J =
1.8, 7.8 Hz, 1H), 4.51 (t, J = 8.6 Hz, 2H), 4.37 (s, 2H), 3.08 - 3.13 (m, 4H) 2.88 (s, 2H), 2.83 (m,
2H) 2.48 (s, 6H), 2.01 (m, 2H), 1.75 (m, 2H); HRMS (FAB) calcd for C31H35F3N4O2 [M + H]+
553.2790, found: 553.2787.
4.1.3.10.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-(2-
oxo-1,2-dihydroquinolin-5-yl)urea (35)
40% yield, white solid, mp = 125-130 oC; 1H NMR (300 MHz, CD3OD) δ 7.99 (d, J = 8.4 Hz, 1H),
7.12 - 7.51 (m, 11H), 6.57 (d, J = 9.9 Hz, 1H), 4.41 (s, 2H), 3.11 (t, J = 10.8 Hz, 2H), 2.79 (m, 4H),
2.41 (s, 6H), 1.97 (t, J = 14.1 Hz, 2H), 1.65 (t, J = 10.6 Hz, 2H); HRMS (FAB) calcd for
C32H34F3N5O2 [M + H]+ 578.2743, found: 578.2747.
25

4.1.3.11.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-(1-
oxo-1,2-dihydroisoquinolin-5-yl)urea (37)
30% yield, white solid, mp = 104-109 oC; 1H NMR (400 MHz, CD3OD) δ 8.14 (d, J = 8.1 Hz, 1H),
7.48 (m, 2H), 7.39 (s, 1 H, Ar), 7.33 (d, J = 7.8 Hz, 1H), 7.13 - 7.26 (m, 8H), 4.40 (s, 2 H), 3.10 (t, J
= 10.9 Hz, 2H), 2.78 (s, 2H), 2.73 (d, J = 11.4 Hz, 2H), 2.40 (s, 6H), 1.96 (d, J = 13.5 Hz, 2H), 1.62
(t, J = 11.2 Hz, 2H); HRMS (FAB) calcd for C32H34F3N5O2 [M + H]+ 578.2743, found: 578.2747.
4.1.3.12.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-
(quinolin-7-yl)urea (44)
46% yield, white solid, mp= 103-106 oC; 1H NMR (300 MHz, DMSO) δ 9.08 (s, 1H), 8.75 (dd, J =
1.8, 4.2 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.13 (s, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.55 (dd, J = 2.0,
8.8 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.19 - 7.33 (m, 7H), 6.75 (bs, 1 H),
4.26 (d, J = 5.5 Hz, 2H), 3.01 (t, J = 11.2 Hz, 2H), 2.70 - 2.74 (m, 4H), 2.31 (s, 6H), 1.93 (d, J =
18.0 Hz, 2H), 1.45 (bs, 2H); HRMS (FAB) calcd for C32H34F3N5O [M + H]+ 562.2794, found:
562.2791.
4.1.3.13.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-
(isoquinolin-7-yl)urea (45)
51% yield, white solid, mp = 123-127 oC; 1H NMR (300 MHz, CD3OD) δ 9.06 (s, 1H), 8.27 (d, J =
5.9 Hz, 1H), 8.18 (s, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.70 (m, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.42 (s,
1H), 7.33 (d, J = 7.7 Hz, 1H), 7.16 - 7.29 (m, 5H), 4.44 (s, 2H), 3.12 (t, J = 10.8 Hz, 2H), 2.85 (m, 4
H), 2.45 (s, 6H), 2.02 (d, J = 12.6 Hz, 2H), 1.74 (bs, 2H); HRMS (FAB) calcd for C32H34F3N5O [M
+ H]+ 562.2794, found: 562.2799.
4.1.3.14.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-
(isoquinolin-6-yl)urea (46)
26

o
1
25% yield, white solid, mp = 99-102 C; H NMR (400 MHz, CD3Cl3) δ 9.06 (s, 1H), 8.38 (d, J =
5.8 Hz, 1H), 7.92 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 5.8 Hz, 1H), 7.26 - 7.42 (m, 5H),
7.06-7.14 (m, 4H), 4.45 (s, 2H), 3.16 (t, J = 9.4 Hz, 2H), 2.79 (m, 4H), 2.42 (s, 6H), 2.01 (m, 2H),
1.75 (bs, 2H); HRMS (FAB) calcd for C32H34F3N5O [M + H]+ 562.2794, found: 562.2800.
4.1.3.15.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-
(isoquinolin-8-yl)urea (47)
42% yield, white solid, mp = 144-147 oC; 1H NMR (300 MHz, CDCl3) δ 9.32 (bs, 1H), 8.48 (d, J =
5.8 Hz, 1H), 7.60 (m, 4H), 7.34 (d, J = 7.9 Hz, 1H) 7.31 (s, 1H), 7.02 - 7.22 (m, 7H), 5.84 (bs, 1H),
4.40 (d, J = 5.00 Hz, 2H), 3.05 (t, J = 10.8 Hz, 2H), 2.65 (m, 4H,), 2.32 (s, 6H), 1.88 (m, 2H), 1.44
(m, 2H); HRMS (FAB) calcd for C32H34F3N5O [M + H]+ 562.2794, found: 562.2800.
4.1.3.16.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-
(isoquinolin-5-yl)urea (48)
45 % yield, white solid, mp = 106-113 oC; 1H NMR (300 MHz, CD3OD) δ 9.12 (s, 1H), 8.32 (d, J =
6.03 Hz, 1H), 7.80 (m, 2H), 7.56 (m, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 7.25 (d, J = 7.5 Hz,
1H), 7.04 - 7.14 (m, 5H), 4.45 (s, 2H), 3.01 (t, J = 12.5 Hz, 2H), 2.71 (m, 4 H), 2.32 (s, 6H), 1.89 (d,
J = 14.1 Hz, 2H), 1.57 (bs, 2H); HRMS (FAB) calcd for C32H34F3N5O [M + H]+ 562.2794, found:
562.2784.
4.1.3.17.
1-(2-(4-Benzyl-4-(dimethylamino)piperidin-1-yl)-4-(trifluoromethyl)benzyl)-3-
(quinolin-5-yl)urea (49)
53 % yield, white solid, mp = 183-191 oC; 1H NMR (300 MHz, DMSO) δ 8.87 (d, J = 4.0 Hz, 1H),
8.83 (s, 1 H), 8.48 (d, J = 8.6 Hz, 1H), 7.96 (d, J = 6.2 Hz, 1H), 7.65 (m, 2H), 7.52 - 7.56 (dd, J =
4.02, 8.61 Hz, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.24 - 7.29 (m, 3H), 7.18 (m,
3H), 6.97 (m, 1H), 4.27 (d, J = 5.3 Hz, 2H), 3.00 (t, J = 13.6 Hz, 2H), 2.71 (m, 4H), 2.29 (s, 6H),
27

1.92 (m, 2H), 1.43 (t, J = 11.2 Hz, 2H); HRMS (FAB) calcd for C32H34F3N5O [M + H]+ 562.2794,
found: 562.2784.
4.2. Biology
4.2.1. TRPV1 assay
4.2.1.1. Cell culture
As previously reported,10 the selected stable CHO cell line expressing human TRPV1 (Tet-Off
system) was cultured in maintaining medium (Ham's F-12 supplemented with 10% fetal bovine
serum, 0.025M HEPES pH 7.5, 250 µg/mL Geneticin, and 1mg/L tetracycline). For [3H]RTX
competition binding experiments, cells were seeded in 75 cm2 culture flasks in maintaining media
and grown to approximately 90% confluency after 48 h. The flasks were then washed with DPBS
(without Ca2+ or Mg2+) and the medium was replaced with inducing medium (Ham's F12
supplemented with 10% fetal bovine serum and 0.025 M HEPES pH 7.3) for an additional 48 h.
The cells were harvested in Trypsin-EDTA and DPBS (no Ca2+ or Mg2+) and pelleted by
centrifugation at 1000 rpm for 5 min. The pellets were stored at −20 °C until use.
4.2.1.2. Radioligand binding assay
As previously reported,10 competition binding studies were performed in the presence of a fixed
concentration of [3H]RTX and various concentrations of competing ligands. The binding assay
mixtures were handled in borosilicate tubes and contained DPBS (with Ca2+ and Mg2+), bovine
serum albumin (0.25 mg/mL), [3H]RTX (100 pM), and various concentrations of the ligands for a
total volume of 350 µL. Non-specific binding was determined in the presence of non-radioactive
RTX (100 nM). The tubes were incubated for 60 min in a 37 °C water bath. The mixtures were then
cooled on ice for approximately 10 min, after which bovine glycoprotein fraction VI (100 µL; 2
mg/mL) was added to reduce nonspecific binding. The ice-cold contents were then transferred to
28