Discovery of Nonpungent Transient Receptor Potential Vanilloid 1 (TRPV1) Agonist as Strong Topical Analgesic

Article abstract of DOI:10.1021/acs.jmedchem.9b01046

Paradoxically, some TRPV1 agonists are, at the organismal level, both nonpungent and clinically useful as topical analgesics. Here, we describe the scaled-up synthesis and characterization in mouse models of a novel, nonpungent vanilloid. Potent analgesic activity was observed in models of neuropathic pain, and the compound blocked capsaicin induced allodynia, showing dermal accumulation with little transdermal absorption. Finally, it displayed much weaker systemic toxicity compared to capsaicin and was negative in assays of genotoxicity.

Full text of DOI:10.1021/acs.jmedchem.9b01046

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Brief Article
Discovery of Non-pungent Transient Receptor Potential
Vanilloid 1 (TRPV1) Agonist as Strong Topical Analgesic
Jihyae Ann, Ho Shin Kim, Shivaji A. Thorat, Hee Kim, Heejin Ha, Kwanghyun
Choi, Young Ho Kim, Minseok Kim, Sun Wook Hwang, Larry V. Pearce,
Timothy E. Esch, Noe A. Turcios, Peter M. Blumberg, and Jeewoo Lee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01046 • Publication Date (Web): 08 Nov 2019
Downloaded from pubs.acs.org on November 9, 2019
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Discovery of Non-pungent Transient Receptor Potential Vanilloid 1
(TRPV1) Agonist as Strong Topical Analgesic
Jihyae Ann,† Ho Shin Kim,† Shivaji A. Thorat,† Hee Kim,‡ Hee-Jin Ha,‡ Kwanghyun Choi,‡ Young-Ho
Kim,‡ Minseok Kim,§ Sun Wook Hwang,§ Larry V. Pearce,∥ Timothy E. Esch,∥ Noe A. Turcios,∥ Peter
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M. Blumberg,∥ Jeewoo Lee*,†
† Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826,
Republic of Korea
‡ Medifron DBT, Sandanro 349, Danwon-gu Ansan-si, Gyeonggi-do 15426, Republic of Korea
§
Department of Biomedical Sciences and Department of Physiology, Korea University College of Medicine, Seoul 02841,
Republic of Korea
∥
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD
20892-4255, USA
ABSTRACT: Paradoxically, some TRPV1 agonists are, at the organismal level, both non-pungent and clinically useful as topical
analgesics. Here, we describe the scaled-up synthesis and characterization in mouse models of a novel, non-pungent vanilloid. Potent
analgesic activity was observed in models of neuropathic pain, and the compound blocked capsaicin induced allodynia, showing
dermal accumulation with little transdermal absorption. Finally, it displayed much weaker systemic toxicity compared to capsaicin
and was negative in assays of genotoxicity.
with oral COX-2 inhibitors or NSAIDs to relieve severe pain in
■ INTRODUCTION
adults with osteoarthritis of the knee.8 The advantage of
The Transient Receptor Potential Vanilloid 1 (TRPV1) is a
ligand-gated and nonselective cation channel that is expressed
in the spinal cord and brain and is localized on neurons in
sensory ganglia, with peripheral projection to the skin, muscles,
joints and gut and with central terminals to the spinal dorsal
horn.1 TRPV1 can be activated by multiple factors such as
protons, heat, endogenous ligands and natural vanilloids
including capsaicin and resiniferatoxin. Activation of TRPV1
triggers an influx of calcium and sodium, which initiates a
cascade of events associated with pain transmission, including
membrane depolarization, neuronal firing, and release of pain
transmitters.2
zucapsaicin is the lesser degree of local irritation such as
stinging, burning and erythema in patients.
Resiniferatoxin (RTX, 3) is an extremely potent irritant
tricyclic diterpene. RTX has proven to function
pharmacologically as an ultrapotent agonist for TRPV1,
displaying 103- to 104-fold greater potency than capsaicin.9 The
actions of RTX are mediated by binding, with picomolar
affinity, directly to the capsaicin-binding site on the TRPV1
receptor.10 Whereas capsaicin under normal conditions
produces only short term desensitization of TRPV1 mediated
responses, the apparent desensitization to RTX can be of very
long duration, lasting for weeks and can thus be applicable for
chronic pain relief.11-13
For drug development targeting TRPV1, a recognized and
very important concept is that the physiological response to
agonist engagement is a complex integral of ligand-target
interaction, feedback from cellular signaling mechanisms, and
pharmacokinetics. For example, resiniferatoxin caused partial
separation of acute toxicity versus desensitization/
defunctionalization in rats relative to capsaicin14 and olvanil has
long been known as a non-pungent TRPV1 agonist.15 Thus,
whereas initial evaluation of ligands for receptor engagement is
a valuable first step, in vivo evaluation is essential to further
define the pattern of response.
Capsaicin (CAP, 1) is the prototype of TRPV1 agonists3 and
is clinically used as an analgesic in topical formulations.4,5 The
high concentration of capsaicin (8% capsaicin) as a transdermal
patch (Qutenza) is specifically indicated for the management of
neuropathic pain associated with postherpetic neuralgia.6,7
Topical application of capsaicin causes an initial activation of
TRPV1, causing pungency. However, the persistent activation
of the channel results in high intracellular levels of calcium,
ultimately leading to desensitization/defunctionalization to a
variety of noxious stimuli through functional and
morphological alterations to the peripheral ends of nerve fibers
and consequent pain relief. Topical application of capsaicin
thus combines two actions, a long-term therapeutic effect
following an initial adverse effect of pungency at the
application site as manifested by erythema, pain, pruritus and
papules.6
In our program to develop a non-pungent, highly efficacious
TRPV1 agonist to avoid the adverse effects associated with the
use of capsaicin, we investigated a number of diverse series of
TRPV1 ligands and recently discovered MDR-652 (4) as a
clinical candidate for use in a topical gel. Structurally, MDR-
652 contains the three principal pharmacophores previously
Zucapsaicin (2) is the cis-isomer of capsaicin and was
approved as a topical analgesic in 2010 for use in conjunction
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designated
(hydroxymethyl)phenyl (A-region), urea (B-region) and 2-(t-
butyl)-4-(3-chlorophenyl)thiazole (C-region) groups
corresponding to the 4-hydroxy-3-methoxyphenyl (A-region),
amide (B-region) and 8-methylnonenyl (C-region) groups,
respectively, in capsaicin.
for
capsaicin16 with
the
3-fluoro-4-
reduced to the corresponding alcohol 15 and then converted to
1
2
3
4
the amine of the C-region 16. Finally, the condensation reaction
between carbamate 9 and amine 16 under basic conditions
provided the final product 4. Overall, 4 was obtained in
kilogram scale through a convergent ten-stage synthesis from
readily available 5 and 10.
5
6
7
8
9
O
O
In vitro Activity. The binding affinity and agonistic potency
of 4 were assessed in vitro by a binding competition assay with
[3H]resiniferatoxin (RTX) and by a functional 45Ca2+ uptake
assay using human/rat TRPV1 heterologously expressed in
Chinese hamster ovary (CHO) cells, as previously described.18
O
O
N
N
H
H
OH
OH
1
2
)
Capsaicin ( )
Zucapsaicin (
10
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S
As shown in Table 1, 4 showed high affinity for human and
rat TRPV1 with Ki = 11.4 and 23.8 nM that was ca. 80-fold
more potent than that of capsaicin in both species. Also, it
H
H
O
N
N
N
F
O
O
H
H
O
O
OH
OH
OCH3
displayed highly potent agonism in hTRPV1 with an EC50
=
Cl
MDR-652 (4
OH
O
O
5.05 nM, which was ca. 5-fold more potent than that of
capsaicin and zucapsaicin. Importantly, in both systems
maximal calcium uptake stimulated by 4 was 50-60% that of
capsaicin, indicating that it functions as a partial agonist
(Figure S1). We have noted elsewhere that there is not full
correspondence between measured TRPV1 ligand affinity and
agonism assayed in cellular systems.19 While both measures are
valuable, we believe that the ligand affinity is a more robust
measure, since the cellular agonism assays will further reflect
the level of exogenous TRPV1 expressed in the cells, rate of
compound uptake, etc. In any case, the most critical measure is
actual in vivo activity. Were TRPV1 receptor affinity by an
agonist to be the only factor determining response, then non-
pungent agonists causing desensitization would be an
unachievable goal.
3
)
Resiniferatoxin (
)
Figure 1. Representative TRPV1 agonists
In this paper, we described the optimized scale-up synthesis
of 4, the agonistic activity of 4 in vitro and in vivo, and its
analgesic activities in acute and neuropathic pain models by
topical and intraperitoneal (i.p.) administration, respectively. In
addition we conducted the pharmacokinetic and toxicological
studies of 4 for its development as a topical agent.
■ RESULTS AND DISCUSSION
Chemistry. The scale-up synthesis of 4 is described in
Scheme 1. For the synthesis of the A-region, commercially
available 2-fluoro-4-nitrobenzoic acid 5 was esterified and then
reduced to the corresponding alcohol 7. The nitro group of 7
was reduced and then reacted with phenylchloroformate to
provide the carbamate intermediate of the A-region 9 employed
for the final coupling reaction. For the synthesis of the C-region,
commercially available 3-chloroacetophenone 10 was alkylated
with dimethylcarbonate to afford the β-ketoester 11, which was
chlorinated with sulfuryl chloride to afford the α-chloro-β-
ketoester 12. The intermediate was condensed with
pivalthioamide 13, prepared from pivalamide by a modified
method17, to provide the thiazole 14. The ester of 14 was
Table 1. In vitro receptor activitya
4
Capsaicin
Zucapsaicin
1080 (±210)
28.2 (±2.3)
hTRPV1 (Ki, nM)
11.4 (±3.9)
1070 (±126)
25.9 (±4.8)
1810 (±270)
44.8 (±3.8)
hTRPV1 (EC50, nM)b 5.05 (±0.88)
rTRPV1 (Ki, nM) 23.8 (±0.87)
rTRPV1 (EC50, nM)c 93 (±17)
a The values are the mean of at least three experiments. The saturating
concentration of capsaicin (3 µMb or 1 µMc) was used to define maximal
response. The EC50 of 4 is expressed relative to the maximal response that
it induces.
Scheme 1. Scale-up synthesis of MDR-652 (4)a
H
F
H2N
F
O2N
F
O2N
PhO
N
F
O2N
F
a
c
d
b
OCH3
OH
OH
O
OH
OH
O
O
6
9
5
7
8
S
H
H
j
N
N
N
F
O
OH
O
O
O
O
S
S
S
Cl
4
N
NH2
O
N
O
O
N
OH
O
NH2
Cl
e
i
g
h
f
+
O
S
13
Cl
Cl
Cl
Cl
Cl
Cl
10
12
11
15
14
16
a
Reagents and conditions: (a) H2SO4, MeOH, reflux, overnight, 95%; (b) NaBH4, MeOH, r.t., 3 h, 78%; (c) Zn, NH4Cl, MeOH-H2O, 50 oC, 4 h, 72%; (d)
phenylchloroformate, pyridine, acetone, r.t., 4 h, 62%; (e) dimethylcarbonate, NaH, THF, 50 oC, overnight, 65%; (f) sulfuryl chloride, CHCl3, reflux, 20 h,
72%; (g) MeOH, reflux, overnight, 71%; (h) LiAlH4, THF, 0 C, 85%; (i) i) SOCl2, CH2Cl2, r.t., ii) NH4OH, CH3CN, 40 C, overnight, 45%; (j) NEt3,
o
o
CH3CN, 15-20 oC, 5 h, 81%.
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Figure 3. Analgesic activity of 4 in formalin model. Effect of 4 on the an-
In order to check the selectivity of 4 over other TRP family
members, the intracellular calcium influxes mediated by four
other nociceptive TRP channels were examined using a
fluorescence imaging plate reader (FLIPR) assay with
individual TRP-transfected cells. Upon application of 4,
statistically significant calcium influxes occurred only in
TRPV1-transfected cells, but not in cells transfected with
hTRPA1, mTRPA1, mTRPV3 and mTRPM8, whereas these
cells all showed the expected calcium increases in response to
the application of their appropriate TRP family member agonist
(Figure S2), indicating that 4 specifically activates TRPV1.
tinociceptive activity in the formalin test. Animals pretreated with a single
injection of 4 showed significantly less licking compared with vehicle-
treated animals. The data represents the mean±SEM (n=8). *P<0.05,
**P<0.01 compared to vehicle.
1
2
3
4
5
6
7
8
Next, we examined the analgesic activity of 4 in the spinal
nerve ligation (SNL) model. The SNL model is a standard
model of neuropathic pain in which the L5 spinal nerve is tightly
ligated with a surgical suture to cause neuropathic pain. The
ligation was performed 14 days prior to compound testing and
mechanical stimulation was induced with von Frey filaments
(up & down method).24 The i.p. administration of 4 exhibited an
excellent and dose dependent analgesic profile and its activity
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Body Temperature Study. In order to confirm the agonistic
activity of 4 in vivo, we performed the body temperature study
of 4 using a rectal temperature probe in ICR mouse and with
capsaicin as our control.20,21 The administration of 3 mg/kg
capsaicin by i.p. injection decreased the body temperature,
showing the lowest temperature at 30 min. The coadministration
of 3 mg/kg capsaicin with 0.5 mg/kg 4 caused a further decrease
in body temperature compared with that of 3 mg/kg capsaicin
alone (Figure 2A). In addition, 4 displayed a dose-dependent
decrease of body temperature (Figure 2B), supporting that 4
displayed TRPV1 agonism in the intact animal.
(ED50 = 0.5-2 mg/kg) was again superior to gabapentin (ED50
=
> 30 mg/kg). The 5-10 mg/kg dose of 4 blocked the neuropathic
pain completely, indicating 100% maximum possible effect
(MPE) (Figure 4A). In addition, the subcutaneous injection (s.c.)
of 4 also displayed an excellent analgesic outcome with
maximum effect at 30 min after administration, indicating that
the route of administration did not change the strong analgesic
profile (Figure 4B).
A. intraperitoneal
4
4
4
4
10 mg/kg
5 mg/kg
2 mg/kg
1 mg/kg
A.
B.
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Gabapentin 65 mg/kg., i.p.
Saline., i.p.
Vehicle
**
**
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**
**
*
*
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Control
*
Control
CAP 3 mg/kg
CAP 3 mg/kg + 4 0.5 mg/kg
4
4
0.5 mg/kg
5 mg/kg
0
0.5
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hours
0
0
hours
Pre
N1
N7
N14
1
1.5
Pre
N1
N7 N14 0.5
hours
1
1.5
hours
Figure 2. Body temperature study of 4 and capsaicin. Time profile of the
hypothermic response of capsaicin and 4 in ICR mice. Capsaicin or/and 4
was administered intraperitoneally at time point zero, and the body
temperature of mice was measured over 7 h. The data represents the
mean±SEM (n=3).
B. subcutaneous
4
4
4
4 mg/kg
2 mg/kg
1 mg/g
120
100
80
60
40
20
0
4
4
4
4 mg/kg
2 mg/kg
1 mgkg
20
15
10
5
**
Vehicle
**
**
**
**
Vehicle
**
Analgesic Activity. In order to evaluate analgesic activity
of 4, we conducted nociception assays in standard pain models.
The formalin model is a standard model of pain in which 2%
formalin is injected into the dorsal surface of a hindpaw and the
time the animal spends licking the paw is recorded.22,23
Analgesic evaluations both for 1st phase (5 min) and 2nd phase
(20-30 min) were performed (Figure 3). The result revealed an
excellent and dose-dependent analgesic profile after i.p.
administration of 4 with analgesic activity (ED50 = 0.79 mg/kg
for 1st phase and 0.16 mg/kg for 2nd phase) superior to
gabapentin (ED50 = > 40 mg/kg). Of particular note, 1 mg/kg of
4 fully inhibited the 2nd phase pain response, showing ~100% of
the maximum possible effect (MPE).
**
**
*
**
*
*
*
0
0.5
1
1.5
24
Pre N1 N7 N14 0.5
hours
1
1.5 24
hours
Figure 4. Analgesic activity of 4 in neuropathic pain model. Rats treated
with 4 had significantly higher thresholds to pain caused by von Frey fila-
ment post dosing. The data represents the mean±SEM (n=7-8). *p<0.05,
**P<0.01 compared to vehicle.
For the target engagement study of 4, we examined the
analgesic profile of 4 in the capsaicin-induced allodynia
model.25,26 Capsaicin, a full agonist of TRPV1, evokes pain after
injection into the rodent paw. After topical gel application of 4,
the foot withdrawal frequency to mechanical stimulus was
monitored over the 5 min period following capsaicin injection.
As shown in Figure 5, 4 showed an excellent analgesic profile
a dose-dependent manner against capsaicin-induced
allodynia, indicating that the analgesic activity of 4 was
mediated by the desensitization of TRPV1.
1st phase
2nd phase
2nd phase
1st phase
150
100
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200
150
100
50
**
*
**
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**
**
**
in
**
**
** **
1
0
0
Veh. 50 100
Veh. 50 100
Veh. 0.1 0.25 0.5
1
10
Veh. 0.1 0.25 0.5
10
Gabapentin [mg/kg, i.p.]
4 [mg/kg, i.p.]
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collected after 30 min and weighed (Figure 7B).29 Against the
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80
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0
1
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general idea that all TRPV1 agonists evoke skin irritation and
edema similarly, 4 showed minimal signs of skin irritation and
edema in the animal models whereas capsaicin displayed high
irritation and edema in a concentration-dependent manner. The
result indicated that 4 showed non-pungency and little edema
associated with TRPV1 agonism, in contrast to the behavior of
capsaicin in animal models.
Capsaicin [0.03%/10µl/paw]
**
**
**
**
8
9
Control Veh.
(PBS)
1
10
20
40
A.
B.
4 [mg/ml, topical]
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Capsaicin
4
Capsaicin
4
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Figure 5. Target engagement study of 4. 4 inhibited capsaicin
induced mechanical hyperalgeisa in mice. The data represents
the mean±SEM (n=7-8).
Pharmacokinetic Study. To examine the topical
permeability of 4, we conducted an in vitro transdermal drug
delivery test using diffusion cells, comparing 4 to capsaicin
cream (Figure 6).27,28 Using the Franz cell diffusion system with
porcine skin, 4 in carbopol gel, prepared as a 1% formulation,
was applied to examine dermal accumulation and systemic
absorption. The result indicated that almost all of the 4 applied
remained in the skin, comprised of epidermis and dermis, and
showed much higher dermal accumulation compared to that of
capsaicin. As a result, a negligible amount of 4 was detected in
the acceptor cell, in contrast to the case with capsaicin,
suggesting that there would be little systemic absorption of 4.
The result supported the concept that 4 has promising potential
as a safer dermal agent with minimal potential systemic
absorption.
0
0.05 0.1 0.5
Concentration [mg/ml]
1
5
10 50
0
0.05 0.1 0.5
1
5
10 50
Concentration [mg/ml]
Figure 7. Skin irritation and edema test. (a) ear scratching behavior and (b)
ear swelling elicited by 4 and capsaicin. The data represents the mean±SEM
(n=7-8).
Finally, we conducted standard toxicity studies of 4 compared
to capsaicin (Table 2). In a single-dose toxicity study, the LD50
of 4 was found to be higher than 200 and 2000 mg/kg in i.p. and
p.o. administration, respectively, whereas the LD50 of capsaicin
had low values30,31, indicating that 4 was much safer than
capsaicin. In addition, in a genotoxicity study, 4 proved to be
negative in all assays, including the Ames assay32 or bacterial
reverse mutation study, the in vitro chromosome aberration
assay33 and the in vivo micronucleus assay34 in the rat.
Furthermore, no pathological problems were observed after
daily topical application for 4 weeks in the rat.
Epidermis+Dermis
Capsaicin
Stratum corneum
Capsaicin
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
4
4
Table 2. Toxicity study of 4
4 (mg/kg) CAP (mg/kg)
0.1
2
4
8
0.1
2
4
8
Single-dose tox, LD50 (i.p., mice)
Single-dose tox, LD50 (p.o, mice)
AMES assay
>200
7.5a
118.8b
hours
hours
>2000
Franz cell acceptor
(Transdermal)
negative
negative
negative
0.20
0.15
0.10
0.05
0.00
Capsaicin
4
In vitro chromosome aberration assay
In vivo micronucleus assay, rat (i.p.)
a ref 30, b ref 31
■ CONCLUSION
0.1
2
4
8
hours
In this work, we developed a non-pungent and highly
efficacious TRPV1 agonist as a potential topical analgesic agent
for neuropathic pain. MDR-652 (4) demonstrated high affinity
and potent agonism in human and rat TRPV1 in vitro. The
functional agonism of 4 was confirmed in vivo by showing
hypothermia in body temperature studies in the mouse both in
protocols of coadministration with capsaicin and of single
administration. 4 exhibited an excellent analgesic profile in the
standard pain models - formalin and spinal nerve ligation - and
was superior to capsaicin and gabapentin. In addition, 4
displayed a dose-dependent analgesic profile toward capsaicin-
induced allodynia, indicating it engaged in TRPV1 for its
analgesic activity. An in vitro transdermal delivery test
indicated that most of the 4 remained in the skin and would have
Figure 6. In vitro transdermal delivery test. Retention and permeation
profile of 4 through pig abdominal skin using Franz diffusion cell. The data
represents the mean±SD (n=3)
Toxicology Study. In order to investigate the safety of
agonist 4 as a topical agent, we performed skin irritation and
edema tests comparing it to capsaicin to examine the toxicity
associated with TRPV1 agonism. To evaluate skin irritation, we
conducted the ear scratching test in which a solution of 4 or
capsaicin was applied to the ears of mice and then the number
of scratching movements was recorded during the following 30
min period (Figure 7A).29 For evaluating edema, we performed
the ear swelling test in which a solution of 4 or capsaicin was
applied to the ears of mice and then ear punch biopsies were
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little systematic absorption whereas the majority of the
oil, which was used for the next reaction without further
purification. 1H NMR (400 MHz, CDCl3) δ 8.11 (t, J = 6.14 Hz,
1H), 8.05 (d, J = 6.94 Hz, 1H), 8.00 (d, J = 6.36 Hz, 1H), 3.97
(s, 3H); Mass (FAB) m/z 200 [M + H]+.
1
2
3
4
5
6
7
8
capsaicin was detected in the acceptor cell, indicating that 4 has
promising PK properties as a topical agent. In toxicology studies,
4 showed minimal signs of skin irritation and edema in animal
models and a much higher LD50 (i.e. lower toxicity) compared
to that of capsaicin and 4 was negative in all genotoxicity
studies.
Taken together, these results suggest that 4 is a highly potent
and efficacious TRPV1 ligand with agonist activity having a
promising topical PK profile and no significant toxicity, and it
is currently under clinical development as a topical agent for
neuropathic pain.
(2-Fluoro-4-nitrophenyl)methanol (7). To a solution of 6 (2.9
kg, 14.56 mol) in MeOH (50 L) at 0 °C was added NaBH4 (2.75
kg, 72.8 mol) portionwise. After stirring for 5 h at room
temperature, the reaction mixture was cooled to 0 oC, water was
carefully added, and the solution concentrated moderately. The
residue was extracted with EtOAc (10 L x 3) and the combined
organic layers were washed with water (10 L) and brine (10 L),
dried over MgSO4 and filtered. The filtrate was concentrated in
vacuo to provide 7 (2.0 kg, 78%) as a pale brown oil, which was
used for the next reaction without further purification. 1H-NMR
(300 MHz, CDCl3) δ 8.08 (dd, J = 2.01, 8.43 Hz, 1H), 7.92 (dd,
J = 2.22, 9.54 Hz, 1H), 7.71 (t, J = 7.32 Hz, 1H), 4.88 (d, J =
4.88 Hz, 2H); Mass (FAB) m/z 172 [M + H]+.
9
10
11
12
13
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■ EXPERIMENTAL SECTION
General. All chemical reagents were commercially
available. Melting points were determined on a melting point
Buchi B-540 apparatus and are uncorrected. Silica gel column
chromatography was performed on silica gel 60, 230–400 mesh,
(4-Amino-2-fluorophenyl)methanol (8). To stirred solution of
7 (2 kg, 11.68 mol) in MeOH at 0 °C was added zinc dust (3 kg,
45.87 mol, 10 μm) followed by ammonium chloride (2.46 kg,
45.98 mol) in water (13 L). The ice bath was removed, and the
resulting mixture was stirred at 50 °C for 4 h. After completion,
the reaction mixture was filtered through a thin Celite pad with
MeOH and the filtrate was concentrated in vacuo. The crude
residue was crystallized with EtOAc/hexanes (1:3) as eluent to
provide 8 (1.16 kg, 72%) as a pale yellow solid. 1H-NMR (300
MHz, CDCl3) δ 7.13 (t, J = 8.04 Hz, 1H), 6.41 (m, 2H), 4.61 (d,
J = 5.88 Hz, 2H), 3.77 (bs, 2H); Mass (FAB) m/z 142 [M + H]+.
Phenyl (3-fluoro-4-(hydroxymethyl)phenyl)carbamate (9).
To a stirred solution of 8 (1.15 kg, 8.15 mol) in acetone (30 L)
was added pyridine (750 mL, 24.44 mol) followed by phenyl
chloroformate (1.075 L, 8.15 mol) at 0°C. After stirring at room
temperature for 5 h, the reaction mixture was concentrated in
vacuo. The residue was extracted with EtOAc (15L x 3) and
washed with water (10 L) and brine (10 L). The combined
organic layers were dried over MgSO4, filtered and
concentrated in vacuo. The resulting residue was dissolved in
EtOAc and recrystallized with EtOAc/hexanes (1:10) to provide
1
Merck. H and 13C NMR spectra were recorded on a JEOL
JNM-LA 300 at 300 MHz, Bruker Analytik DE/AVANCE
Digital 400 at 400 MHz. Chemical shifts are reported in ppm
units with Me4Si as a reference standard. Mass spectra were
recorded on a 6460 Triple Quad LC−MS instrument. The purity
was determined by high-performance liquid chromatography
(HPLC) and was confirmed to be ≥95%. HPLC was performed
on a Water Alliance 2695 separation Module instrument using
a Hydrosphere C18 column (4.6 mm × 250 mm, 5 μm) with a
1.0 mL/min flow rate.
1-((2-(tert-Butyl)-4-(3-chlorophenyl)thiazol-5-yl)methyl)-3-
(3-fluoro-4-(hydroxymethyl) phenyl)urea (4). To a solution
of phenylcarbamate 9 (1.1 kg, 4.21 mol) in acetonitrile was
added amine 16 (963 g, 3.43 mol) followed by triethylamine
(567 mL, 4.06 mol). After stirring at 15-20 °C for 5 h, the
solvent was removed in vacuo and the residue was dissolved in
EtOAc. The organic layer was washed with 1 N HCl, water,
NaHCO3 solution, brine, dried over MgSO4 and concentrated
in vacuo. The residue was purified by silica gel column
chromatography with EtOAc/hexanes (1:2) as eluent to provide
4 (1.25 kg, 81%). For analytical purity, the solid was
recrystallized five times using EtOAc/hexanes (1:10) to yield
the >99% pure form of 4 (1.01 kg) as a white solid. m.p =
132 °C;1H NMR (300 MHz, CDCl3) δ 7.57 (s, 1H, Ar-H), 7.45-
7.42 (m, 1H, Ar-H), 7.35 - 7.31 (m, 2H, Ar-H), 7.26 - 7.24 (m,
2H, Ar-H), 6.91 - 6.87 (dd, J1 = 2.01 Hz, J2 = 2.22 Hz, 1H, Ar-
H), 6.64 (s, 1H, -CH2NH), 5.24 (t, J = 5.85 Hz, 1H, CH2OH),
4.63 (bd, 4H, α-CH2, -CH2OH), 1.42 (s, 9H, -C(CH3)3); 13C
NMR (CDCl3, 125 MHz) δ 177.77, 158.75, 154.79, 147.24,
104.75, 136.65, 133.91, 133.22, 130.38, 129.50, 127.99, 127.60,
126.99, 121.49, 113.18, 104.37, 56.48, 37.26, 36.09, 30.478
(3C); HR-MS (FAB) calcd for C22H23ClFN3O2S [M + H]+
448.1183, found 448.1262. Purity 99.99% (eluent: 0.1 N
phosphoric acid : acetonitrile (45:55, v/v), retention time: 18.64
min)
1
9 (1.32 kg, 62%) as a pale yellow solid. H-NMR (300 MHz,
CDCl3) δ 7.38-7.43 (m, 4H), 7.24 (m, 1H), 7.20 (d, J = 2.22 Hz,
2H), 7.10 (dd, J = 2.01, 8.07 Hz, 1H), 4.71 (d, J = 5.13 Hz, 2H);
Mass (FAB) m/z 262 [M + H]+.
Methyl 3-(3-chlorophenyl)-3-oxopropanoate (11). To a 100 L
reactor charged with THF (50 L) was carefully added sodium
hydride (3.83 kg, 159.64 mol) surrounded by an ice bath. 3-
Chloroacetophenone (10, 5 kg, 32.34 mol) in THF (5 L) was
added dropwise to the mixture over 1 h at 0 °C, followed by
dropwise addition of dimethyl carbonate (5.45 L, 64.68 mol) in
THF (6 L) at 0 °C. The reaction mixture was stirred at 0 °C for
30 min and then heated to 50 °C for 4 h. The mixture was cooled
to 0 °C and quenched by the addition of aqueous NH4Cl
solution. The resulting mixture was extracted with EtOAc (20 L
x 3), washed with water, dried over MgSO4 and concentrated in
vacuo. The residue was purified by silica gel column
chromatography with EtOAc/hexanes (1:10) as eluent to
provide 11 (4.5 kg, 65%) as a brown oil. 1H NMR (400 MHz,
CDCl3) δ 7.91 (d, J = 1.6 Hz, 1H), 7.79-7.81 (m, 1H), 7.75 (m,
0.3 H, enol form), 7.62-7.64 (m, 0.3 H, enol form), 7.56 (dd, J
= 8.2, 3.2 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.34 (m, 0.3 H, enol
form), 3.97 (s, 2H), 3.80 (s, 1H, enol form), 3.75 (s, 3H); Mass
(FAB) m/z 213 [M + H]+.
Methyl 2-fluoro-4-nitrobenzoate (6). To a solution of 2-
fluoro-4-nitrobenzoic acid (5, 3 kg, 15.06 mol) in MeOH (40 L)
was added H2SO4 (180 mL) and heated to reflux for 15 h. After
completion of the reaction by TLC, MeOH was removed under
reduced pressure and the residue was partitioned between
EtOAc and water. The aqueous layer was extracted with EtOAc
(15 L x 3) and the combined organic layers were washed with
brine, dried over MgSO4 and filtered. The filtrate was
concentrated in vacuo to provide 6 (2.98 kg, 95%) as a brown
Methyl 2-chloro-3-(3-chlorophenyl)-3-oxopropanoate (12).
To a solution of 11 (4.5 kg, 21.16 mol) in chloroform (40 L) was
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Journal of Medicinal Chemistry
Page 6 of 8
added dropwise sulfuryl chloride (1.7 L, 21.16 mol) at 0 °C and
yellow solid. 1H-NMR (400 MHz, CDCl3) δ 7.63 (t, J = 1.8 Hz,
1 H), 7.48 (dt, J = 7.4, 1.6 Hz, 1 H), 7.28-7.36 (m, 2 H), 4.14 (s,
2 H), 1.44 (s, 10 H); Mass (FAB) m/z 281 [M + H]+.
1
2
3
4
5
6
7
8
the solution was then heated to reflux for 20 h. The reaction was
allowed to cool to room temperature and diluted with water. The
organic layer was washed with water (20 L x 3), dried over
MgSO4, filtered and concentrated in vacuo. The residue was
purified by silica gel column chromatography with
EtOAc/hexanes (1:5) as eluent to provide 12 (3.76 kg, 72%) as
a pale brown oil. 1H-NMR (400 MHz, CDCl3) δ 7.96 (t, J = 1.8
Hz, 1H), 7.86 (dt, J = 7.8, 1.4 Hz, 1H), 7.60 (dq, J = 7.9, 1.1 Hz,
1H), 7.45 (t, J = 8.0 Hz, 1H), 5.59 (s, 1H), 3.88 (s, 3H); Mass
(FAB) m/z 246 [M + H]+.
■
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at http://pubs.acs.org.
Molecular formula strings (CSV); Agonism data of 4; Selectiv-
ity study of 4; Biological and pharmacokinetic methods.
9
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11
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2,2-Dimethylpropanethioamide (13). To
a solution of
■ AUTHOR INFORMATION
pivalamide (2.6 kg, 25.70 mol) in diethyl ether (40 L) and
toluene (20 L) was added phosphorus pentasulfide (4 kg, 17.99
mol) and the mixture was stirred at 45 °C for 5 h to provide a
tacky yellow solid and a supernatant layer. The supernatant was
filtered and the yellow solid was extracted with diethyl ether (5
L x 3). The combined organic layers were evaporated in vacuo
and the residue was purified by silica gel column
chromatography with EtOAc/hexanes (1:1) as eluent to provide
Corresponding Author
* Phone, 82-2-880-7846; E-mail, jeewoo@snu.ac.kr.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENT
1
13 (2.87 kg, 63 %) as a pale brown oil. H-NMR (400 MHz,
This work was supported by the Midcareer Researcher Program
(NRF-2019R1A2C2006837) funded by the National Research
Foundation of Korea (NRF) and by the Intramural Research
Program of the National Institutes of Health, Center for Cancer
Research, National Cancer Institute (Project Z1A BC 005270).
CDCl3) δ 7.62 (s, 1H), 6.96 (s, 1H), 1.36 (s, 9H) ; Mass (FAB)
m/z 118 [M + H]+.
Methyl
2-(tert-butyl)-4-(3-chlorophenyl)thiazole-5-
carboxylate (14). To a solution of 12 (3.7 kg, 15.04 mol) in
MeOH (30 L) was added thioamide 13 (1.85 kg, 15.78 mol) and
the solution was then refluxed for 15 h. The reaction mixture
was concentrated in vacuo and the residue was dissolved in
EtOAc (20 L). The organic layer was washed with water, brine,
dried over MgSO4, and concentrated in vacuo. The residue was
recrystallized with isopropyl alcohol/hexanes to provide 14
■ ABBREVIATIONS
TRPV1, Transient Receptor Potential Vanilloid 1; CAP,
capsaicin; RTX, resiniferaxin; CHO, chinese hamster ovary;
SNL, spinal nerve ligation; MPE, maximum possible effect
1
(3.28 kg, 71%) as a pale yellow solid. H-NMR (400 MHz,
CDCl3) δ 7.77 (d, J = 1.4 Hz, 1H), 7.67 (td, J = 4.4, 2.3 Hz, 1H),
7.40-7.31 (2H), 3.80 (s, 3H), 1.47 (s, 9H); Mass (FAB) m/z 310
[M + H]+.
■ REFERENCES
(1) Szallasi, A.; Blumberg, P. M. Vanilloid (capsaicin) receptors
and mechanisms. Pharmacol. Rev. 1999, 51, 159-211.
(2) Tabrizi, M. A.; Baraldi, P. G.; Baraldi, S.; Gessi, S.; Merighi,
S.; Borea, P. A. Medicinal chemistry, pharmacology, and
clinical implications of TRPV1 receptor antagonists. Med. Res.
Rev. 2017, 37, 936-983.
(3) Caterina, M. J.; Schumacher, M. A.; Tominaga, M.; Rosen,
T. A.; Levine, J. D.; Julius, D. The capsaicin receptor: a heat-
activated ion channel in the pain pathway. Nature 1997, 389,
816–824.
(4) Fattori, V.; Hohmann, M. S.; Rossaneis, A. C.; Pinho
Ribeiro, F. A.; Verri, W. A. Capsaicin: Current understanding
of its mechanisms and therapy of pain and other pre-clinical and
clinical uses". Molecules 2016, 21, E844.
(5) Frias, B.; Merighi, A. Capsaicin, nociception and pain.
Molecules 2016, 21, E797.
(6) Anand, P.; Bley, K. Topical capsaicin for pain management:
therapeutic potential and mechanism of action of the new high-
concentration capsaicin 8% patch. British J. Anaesthesia 2011,
107, 490-502.
(7) Derry, S.; Rice, A. S.; Cole, P.; Tan, T.; Moore, R. A.
Topical capsaicin (high concentration) for chronic neuropathic
pain in adults. Cochrane Database of Systematic Reviews.
2017, 1, CD007393.
(8) Sałat, K.; Jakubowska, A.; Kulig K. Zucapsaicin for the
treatment of neuropathic pain. Expert Opin. Investig. Drugs.
2014, 23, 1433-1440.
(9) Szallasi, A.; Blumberg, P. M. Resiniferatoxin, a phorbol-
related diterpene, acts as an ultrapotent analog of capsaicin the
(2-(tert-Butyl)-4-(3-chlorophenyl)thiazol-5-yl)methanol
(15). To a solution of 14 (3.2 kg, 10.35 mol) in THF (60 L) was
added lithium aluminium hydride (420 g, 11.06 mol) at 0 °C and
stirring was continued at room temperature for 4 h. The reaction
mixture was cooled to 0 °C, water was added dropwise, and the
mixture was then concentrated in vacuo. The residue was
dissolved in EtOAc and the organic layer was washed with
water, brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to provide 15 (2.47 kg, 85%) as a brown
oil, which was used for the next reaction without further
purification. 1H-NMR (400 MHz, CDCl3) δ 7.67 (d, J = 1.8 Hz,
1 H), 7.51 (td, J = 4.4, 2.3 Hz, 1 H), 7.29-7.35 (m, 2 H), 4.79 (s,
2 H), 2.44 (s, 1 H), 1.44 (s, 10 H); Mass (FAB) m/z 282 [M +
H]+.
(2-(tert-Butyl)-4-(3-chlorophenyl)thiazol-5-yl)methanamine
(16). To a solution of 15 (2.45 kg, 8.71 mol) in CH2Cl2 (30 L)
was added thionyl chloride (548 mL, 7.56 mol) at 0 °C. After
stirring at room temperature for 2 h, the reaction mixture was
concentrated in vacuo. The residue was dissolved in acetonitrile
(30 L) and then tetrabutyl ammonium bromide (100 g) was
added followed by dropwise addition of ammonium hydroxide
solution (20 L) at 0 °C. After stirring at 40 °C for 15 h, the
reaction mixture was concentrated in vacuo, and the residue was
diluted with water. The aqueous layer was extracted with EtOAc
(10L x 3) and the combined organic layers were washed with
water, brine, dried over MgSO4 and concentrated in vacuo. The
residue was purified by silica gel column chromatography with
EtOAc/hexanes (1:2) as eluent to provide 16 (1.1 kg, 45%) as a
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Journal of Medicinal Chemistry
irritant constituent in red pepper. Neuroscience 1989, 30, 515-
520.
(24) Kim, S. H.; Chung, J. M. An experimental model for
peripheral neuropathy produced by segmental spinal nerve
ligation in the rat. Pain. 1992, 50, 355-363.
(25) Barrett, A. C.; Smith, E. S.; Picker, M. J. Capsaicin-
induced hyperalgesia and mu-opioid-induced antihyperalgesia
in male and female Fischer 344 rats. J. Pharmacol. Exp. Ther.
2003, 307, 237-245.
(26) Gavva, N. R.; Bannon, A. W.; Hovland, D.N. Jr.; Lehto, S.
G.; Klionsky, L.; Surapaneni, S.; Immke, D. C.; Henley, C.;
Arik, L.; Bak, A.; Davis, J.; Ernst, N.; Hever, G.; Kuang, R.; Shi,
L.; Tamir, R.; Wang, J.; Wang, W.; Zajic, G.; Zhu, D.; Norman,
M. H.; Louis, J. C.; Magal, E.; Treanor, J. J. Repeated
administration of vanilloid receptor TRPV1 antagonists
attenuates hyperthermia elicited by TRPV1 blockade. J.
Pharmacol. Exp. Ther. 2007, 323, 128-137.
(27) Fang, J-Y.; Wu, P-C.; Huang, Y-B.; Tsai, Y-H. In vitro
permeation study of capsaicin and its synthetic derivatives from
ointment bases using various skin types. Int. J. Pharm. 1995,
126, 119-128.
(28) Wang, Y-Y.; Hong, C-T.; Chiu, W-T.; Fang, J-Y. In vitro
and in vivo evaluations of topically applied capsaicin and
nonivamide from hydrogels Int. J. Pharm. 2001, 224, 89-104.
(29) Son, M. H.; Bae, E. J.; Kim, H. K.; Shin, M. S.; Kim, S. H.;
Kim, W. B.; Yang, J. Studies on the local irritation of DA-5018,
a new capsaicin derivative. J. Appl. Pharmacol. 1997, 5, 150-
157.
(30) Nagabhushan, M.; Bhide, S. V. Mutagenicity of chili
extract and capsaicin in short-term tests.
Environ. Mutagen. 1985, 7, 881-888.
1
2
3
4
5
6
7
8
(10) Biro, T.; Acs, G.; Acs, P.; Modarres, S.; Blumberg, P. M.
Recent advances in understanding of vanilloid receptors: a
therapeutic target for treatment of pain and inflammation in skin,
J. Investig. Dermatol. Symp. Proc. 1997, 2, 56-60.
(11) Liu, L.; Simon, S. A. The influence of removing
extracellular Ca2+ in the desensitization responses to capsaicin,
zingerone and olvanil in rat trigeminal ganglion neurons. Brain
Res. 1998, 809, 246-252.
(12) Szallasi, A.; Joo, F.; Blumberg, P. M. Duration of
desensitization and ultrastructural changes in dorsal root ganglia
in rats treated with resiniferatoxin, an ultrapotent capsaicin
analog, Brain Res. 1989, 503, 68-72.
(13) Brown, D. C. Resiniferatoxin: The evolution of the
“molecular scalpel” for chronic pain relief. Pharmaceuticals
2016, 9(3), E47.
(14) Szallasi, A.; Blumberg, P.M. Resiniferatoxin, a phorbol-
related diterpene, acts as an ultrapotent analog of capsaicin, the
irritant constituent of red pepper. Neuroscience 1989, 30, 515-
520.
(15) Brand, L.; Berman, E.; Schwen, R.; Loomans, M.; Janusz,
J.; Bohne, R.; Maddin, C.; Gardner, J., Lahann,T.; Farmer, R.
NE-19550: a novel, orally active anti-inflammatory analgesic.
Drugs Exp. Clin. Res. 1987, 13, 259-265.
(16) Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.; Campbell,
E. A.; Dray, A.; James, I. F.; Perkins, M. N.; Reid, D. J.; Winter,
J. Analogues of capsaicin with agonist activity as novel
analgesic agents; Structure-activity studies. 1. The aromatic “A-
region. J. Med. Chem. 1993, 36, 2362-2372.
(17) Orr, D.; Tolfrey, A.; Percy, J. M.; Frieman, J.; Harrison, Z.
A.; Campbell-Crawford, M.; Patel, V. K. Single-step
microwave-mediated synthesis of oxazoles and thiazoles from
3-oxetanone: A synthetic and computational study. Chem. Eur.
J. 2013, 19, 9655-9662.
(18) Pearce, L. V.; Ann, J.; Jung, A.; Thorat, S. A.; Herold, B.
K. A.; Habtemichael, A. D.; Blumberg, P. M.; Lee, J. Novel
radiolabeled vanilloid with enhanced specificity for human
transient receptor potential vanilloid 1 (TRPV1). J. Med.
Chem. 2017, 60, 8246-8252.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(31) Saito, A.; Yamamoto M. Acute oral toxicity of capsaicin in
mice and rats. J. Toxicol. Sci. 1996, 21, 195-200.
(32)
Mortelmans,
K.;
Zeiger,
E.
The
Ames
Salmonella/microsome mutagenicity assay. Mutat. Res. 2000,
455, 29-60.
(33) Ishidate, M. Jr.; Miura, K. F.; Sofuni, T. Chromosome
abrerration assays in genetic toxicology testing in vitro. Mutat.
Res. 1998, 404, 167-172.
(34) Hayashi, M. The micronucleus test-most widely used in
vivo genotoxicity test. Genes and Environment 2016, 38, 18.
(19) Szallasi, A.; Blumberg, P.M.;Annicelli, L.L.; Krause, J.E.;
Cortright, D.N.; The cloned vanilloid receptor VR1 mediates
both R-type biding and C-type calcium response in dorsal root
ganglion neurons. Mol. Pharmacol. 1999, 56, 581-587.
(20) Shimizu, I.; Iida, T.; Horiuchi, N.; Caterina, M. J. 5-
Iodoresiniferatoxin evokes hypothermia in mice and is a partial
transient receptor potential vanilloid 1 agonist in vitro. J.
Pharmacol. Exp. Ther. 2005, 314, 1378-1385.
(21) Fosgerau, K.; Weber, U. J.; Gotfredsen, J. W.; Jayatissa,
M.; Buus, C.; Kristensen, N. B.; Vestergaard, M.; Teschendorf,
P.; Schneider, A.; Hansen, P.; Raunsø, J.; Køber, L.; Torp-
Pedersen, C.; Videbaek, C.; Drug-induced mild therapeutic
hypothermia obtained by administration of a transient receptor
potential vanilloid type 1 agonist. BMC Cardiovasc Disord.
2010,
10, 51.
(22) Dubuisson, D.; Dennis, S. G. The formalin test: a
quantitative study of the analgesic effects of morphine,
meperidine, and brain stem stimulation in rats and cats. Pain.
1997, 4, 161-174.
(23) Coderre, T.J.; Vaccarino, A.L.; Melzack, R. Central
nervous system plasticity in the tonic pain response to
subcutaneous formalin injection. Brain Res. 1990, 535, 155-158.
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Table of Contents (TOC) graphic
1
2
TRPV1 Agonist
3
4
MDR-652 (4)
Capsaicin (1)
5
6
7
8
O
S
H
H
O
N
N
N
F
N
H
O
OH
OH
9
Cl
high affinity
10
11
12
13
14
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17
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moderate affinity
strong analgesic effect
moderate analgesic effect
pungent
non-pungent
low systemic toxicity
systemic toxicity
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