Pyrazine-Fused Triterpenoids Block the TRPA1 Ion Channel in Vitro and Inhibit TRPA1-Mediated Acute Inflammation in Vivo

Article abstract of DOI:10.1021/acschemneuro.9b00083

TRPA1 is a nonselective cation channel, most famously expressed in nonmyelinated nociceptors. In addition to being an important chemical and mechanical pain sensor, TRPA1 has more recently appeared to have a role also in inflammation. Triterpenoids are natural products with anti-inflammatory and anticancer effects in experimental models. In this paper, 13 novel triterpenoids were created by synthetically modifying betulin, an abundant triterpenoid of the genus Betula L., and their TRPA1-modulating properties were examined. The Fluo 3-AM protocol was used in the initial screening, in which six of the 14 tested triterpenoids inhibited TRPA1 in a statistically significant manner. In subsequent whole-cell patch clamp recordings, the two most effective compounds (pyrazine-fused triterpenoids 8 and 9) displayed a reversible and dose- and voltage-dependent effect to block the TRPA1 ion channel at submicromolar concentrations. Interestingly, the TRPA1 blocking action was also evident in vivo, as compounds 8 and 9 both alleviated TRPA1 agonist-induced acute paw inflammation in mice. The results introduce betulin-derived pyrazine-fused triterpenoids as promising novel antagonists of TRPA1 that are potentially useful in treating diseases with a TRPA1-mediated adverse component.

Full text of DOI:10.1021/acschemneuro.9b00083

Research Article
pubs.acs.org/chemneuro
Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Pyrazine-Fused Triterpenoids Block the TRPA1 Ion Channel in Vitro
and Inhibit TRPA1-Mediated Acute Inflammation in Vivo
†
†
‡,∥
†
‡
‡
̈
̈
̈
̈
Ilari Maki-Opas, Mari Hamalainen, Lauri J. Moilanen, Raisa Haavikko, Tiina J. Ahonen,
Sami Alakurtti,‡,§ Vania M. Moreira,
Katsuhiko Muraki,⊥ Jari Yli-Kauhaluoma,‡
̂
and Eeva Moilanen*,†
†The Immunopharmacology Research Group, Faculty of Medicine and Health Technology, Tampere University and Tampere
University Hospital, 33014 Tampere, Finland
‡Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki,
00014 Helsinki, Finland
§VTT Technical Research Centre of Finland Ltd., 02044 Espoo, Finland
∥Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, U.K.
⊥Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650, Japan
ABSTRACT: TRPA1 is a nonselective cation channel, most
famously expressed in nonmyelinated nociceptors. In addition
to being an important chemical and mechanical pain sensor,
TRPA1 has more recently appeared to have a role also in
inflammation. Triterpenoids are natural products with anti-
inflammatory and anticancer effects in experimental models. In
this paper, 13 novel triterpenoids were created by synthetically
modifying betulin, an abundant triterpenoid of the genus
Betula L., and their TRPA1-modulating properties were
examined. The Fluo 3-AM protocol was used in the initial
screening, in which six of the 14 tested triterpenoids inhibited
TRPA1 in a statistically significant manner. In subsequent whole-cell patch clamp recordings, the two most effective compounds
(pyrazine-fused triterpenoids 8 and 9) displayed a reversible and dose- and voltage-dependent effect to block the TRPA1 ion
channel at submicromolar concentrations. Interestingly, the TRPA1 blocking action was also evident in vivo, as compounds 8
and 9 both alleviated TRPA1 agonist-induced acute paw inflammation in mice. The results introduce betulin-derived pyrazine-
fused triterpenoids as promising novel antagonists of TRPA1 that are potentially useful in treating diseases with a TRPA1-
mediated adverse component.
KEYWORDS: Transient receptor potential channels, TRPA1, inflammation, pain, natural compounds, triterpenoids
due to a direct effect of the disease-inducing exogenous
substance on the ion channel but could be attributed to
endogenous activation of TRPA1. Indeed, endogenous
compounds capable of opening the TRPA1 channel have
recently been characterized, and they include factors released in
inflammatory conditions such as reactive oxygen and nitrogen
species (ROS and RNS, respectively), bradykinin, and some
prostaglandins.5,11 The emerging dualistic role of TRPA1 in
nociception and inflammation has revealed TRPA1 to be a
promising drug target. It has been suggested that blockade of
TRPA1 is likely to provide direct alleviation of pain and
additionally attenuate inflammation. However, although there is
a wide spectrum of compounds known to activate TRPA1, only a
few blockers have been identified.
INTRODUCTION
■
Transient receptor potential ankyrin 1 (TRPA1) is a cation
channel expressed predominantly in Aδ- and C-type nociceptive
nerve fibers mediating chemical and mechanical pain. TRPA1 is
a physiological chemoreceptor for potentially harmful exoge-
nous chemicals, and its activation causes immediately
perceivable and often painful sensations.1,2 Indeed, there are
many naturally occurring irritants that activate TRPA1, e.g.,
allicin in garlic (Allium sativum L.), cinnamaldehyde in
cinnamon (Cinnamomum Schaeff.), and allyl isothiocyanate
(AITC) in mustard oil.3−5
A growing body of data supports the idea that the TRPA1 ion
channel also plays a critical role in inflammation.1 Pharmaco-
logical blockade and genetic deletion of TRPA1 have been
shown to alleviate nociception and inflammation in animal
models such as carrageenan-induced paw inflammation,6
monosodium iodoacetate-induced arthritis,7 monosodium
urate crystal-induced gouty arthritis, and formalin-induced
pain.8−10 These adverse effects of TRPA1 activation were not
Received: February 5, 2019
Accepted: April 12, 2019
© XXXX American Chemical Society
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DOI: 10.1021/acschemneuro.9b00083
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Figure 1. Structural formulas of betulin and its triterpenoid derivatives.
Betulin (1) is a widely studied triterpenoid, which can be
found in the birch tree bark (Betula sp. L.). Other triterpenoids
are present in olives and apple peel and are thought to contribute
to some of the health promoting effects of these foodstuffs.
Betulin (1) and some related triterpenoids have been found to
possess anti-inflammatory properties.12−15 There is also
evidence of their therapeutic potential against cancer, HIV,
and protozoal diseases.16−25
this study, we utilized two in vitro protocols, the Fluo 3-AM
intracellular Ca2+ measurement and whole-cell patch clamp
current recording, as well as an in vivo model of acute TRPA1-
mediated inflammation to study the effects of betulin (1) and a
series of its derivatives (2−14) on TRPA1.
RESULTS
■
Chemistry. Thirteen triterpenoids (2−14) were synthesized
as described previously, and their structures are shown in Figure
1, along with that of betulin (1).20−22 In addition, a novel
method for converting betulin to betulonic acid and further to
On the basis of our preliminary screening results, we
postulated that some of the pharmacological features of
triterpenoids might be attributable to blockade of TRPA1. In
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Figure 2. Synthesis of betulonic acid (2) and betulinic acid (3) from betulin (1). Reagents and conditions: (a) Pd(OAc)2, py, toluene, air, 80 °C, 7 h;
(b) NaClO2, NaH2PO4·H2O, 2-methyl-2-butene, tert-butanol, H2O, room temperature, 12 h; (c) NaBH4, NaOH, H2O, 2-propanol, 0 °C, 3 h.
betulinic acid was developed (Figure 2). First, betulin (1) was
oxidized to betulonic aldehyde with air using palladium(II)
acetate as a catalyst. The resulting betulonic aldehyde was
further oxidized to betulonic acid (2) in one pot using sodium
chlorite as an oxidant. Finally, the reduction of betulonic acid
(2) with NaBH4 produced betulinic acid (3) with a good yield.
Six of the 14 Triterpenoids Inhibit TRPA1 in Vitro. The
antagonistic properties of the triterpenoids on TRPA1 were first
Figure 3. Structural formulas of the key triterpenoids 8 and 9.
screened at 10 μM concentrations using the Fluo 3-AM calcium
measurement protocol in human TRPA1 (hTRPA1)-trans-
Further experiments demonstrated that triterpenoids 8 and 9
fected HEK293 cells. The cells were preincubated with the
blocked AITC-induced Ca2+ influx also in HEK293 cells
triterpenoids, and thereafter, TRPA1-mediated Ca2+ influx was
transfected with mTRPA1 (Figure 5). In addition, compounds
induced by adding the known TRPA1 activator AITC.
8 and 9 at concentrations of ≤100 μM did not induce any
Six triterpenoids, namely, compounds 5−9 and 14, inhibited
detectable Ca2+ influx in HEK293 cells transfected with either
AITC-induced Ca2+ influx at a concentration of 10 μM in a
human or mouse TRPA1, ruling out an agonistic effect.
statistically significant manner. The TRPA1 blocking activity
In Whole-Cell Patch Clamp Recordings, Triterpenoids
results of all triterpenoids studied are summarized in Table 1.
8 and 9 Display a Strong and Reversible TRPA1 Blocking
The cytotoxicity of the compounds under the experimental
Activity. To verify the TRPA1 blocking ability of compounds 8
conditions that were used was ruled out with the XTT assay.
and 9, we utilized the whole-cell patch clamp recording
The two most promising compounds, 8 and 9 (Figure 3),
technique. In HEK293 cells transfected with hTRPA1, the
AITC (50 μM)-induced ion currents through TRPA1 were
strongly attenuated at a holding potential of −50 mV when the
cells were treated with compound 8 or 9 (Figure 6A,C).
Interestingly, both compounds exerted a voltage-dependent
blockade of TRPA1 channel currents (Figure 6B,D). The IC50
were examined further in a broader range of concentrations from
0 to 100 μM, and a dose-dependent inhibition was observed
with both compounds (Figure 4). The IC50 values of
compounds 8 and 9 in the Fluo 3-AM Ca2+ influx measurements
were 9.5 and 7.5 μM, respectively.
values of compounds 8 and 9 against HC-030031 sensitive
Table 1. Effects of Triterpenoids (10 μM) on Allyl
TRPA1 channel currents at −100 and −50 mV were each 0.3
μM; at 50 mV, they were 1.3 μM, and at 100 mV, they were 1.8
and 4.4 μM, respectively (Figure 6E,F).
Isothiocyanate (AITC, 50 μM)-Induced TRPA1 Activation
Measured by Ca2+ Influx in HEK293 Cells Transfected with a
a
Plasmid Encoding hTRPA1
In additional experiments, we examined the reversibility of the
inhibitory effects of compounds 8 and 9 on TRPA1. Because
high concentrations of AITC may desensitize TRPA1 during the
recordings, we exploited 10 μM AITC to activate TRPA1 in
these experiments. As shown in Figure 7A−H, both pyrazine-
fused triterpenoids (compounds 8 and 9) exhibited inhibitory
effects on 10 μM AITC-induced currents at a holding potential
of −50 mV comparable to those seen with the 50 μM AITC-
induced currents (see also panels E and F of Figure 6). This
suggests that the concentration of the TRPA1 agonist had little
or no influence on the inhibitory effect of compounds 8 and 9.
After the current amplitude had stabilized during the application
of compounds 8 and 9, they were removed from the bathing
solution while maintaining 10 μM AITC. The recovery of AITC-
induced TRPA1 activation was complete after the withdrawal of
concentrations of compounds 8 and 9 of 0.3 and 3 μM, while the
recovery was partial, but still obvious, after concentrations of
compounds 8 and 9 of 30 μM (Figure 7C,F−H). This
demonstrates that compounds 8 and 9 are reversible blockers
of hTRPA1 at concentrations of ≤3 μM, but at 30 μM, the
blockade is partially irreversible.
compound
control
HC-030031 (100 μM)
1 (10 μM)
2 (10 μM)
3 (10 μM)
4 (10 μM)
5 (10 μM)
6 (10 μM)
7 (10 μM)
% of control
100
22.24 2.2***
98.89 4.0
83.32 8.9
83.65 3.9
83.42 5.0
85.52 3.2*
79.08 3.2**
79.55 4.2**
61.32 3.1**
64.09 4.0**
85.65 15.1
86.48 6.6
96.48 6.3
96.90 6.8
75.50 6.0**
8 (10 μM)
9 (10 μM)
10 (10 μM)
11 (10 μM)
12 (10 μM)
13 (10 μM)
14 (10 μM)
a
TRPA1 inhibition by triterpenoids is presented as a percentage of
the area under the curve (AUC) in relation to the untreated AITC
control. Results are expressed as means the standard error of the
mean. *p < 0.05. **p < 0.01. ***p < 0.001.
Triterpenoids 8 and 9 Attenuate TRPA1-Mediated
Acute Inflammation in Vivo. As compounds 8 and 9 were
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Figure 4. Compounds 8 and 9 inhibited allyl isothiocyanate (AITC, 50 μM)-induced Ca2+ influx in HEK293 cells transfected with a plasmid encoding
hTRPA1 in a dose-dependent manner. Fluo 3-AM-loaded cells were preincubated for 30 min with compound 8 (1−100 μM), compound 9 (1−100
μM), or the known TRPA1 antagonist HC-030031 (100 or 200 μM) before intracellular Ca2+ measurement. Panels A and C show representative
curves displaying relative fluorescence units after treatment with compounds 8 and 9, respectively. Panels B and D show the areas under the curve
(AUC; from 15 to 45 s) of the response to AITC with or without treatment with 8 and 9, respectively. The background fluorescence was recorded for
15 s before the addition of the TRPA1 agonist AITC. The increase in fluorescence in response to AITC was measured for 30 s, after which the control
ionophore, ionomycin (1 μM), was added and fluorescence was measured for an additional 30 s. The results are normalized to the background and
expressed as means + the standard error of the mean (SEM) (n = 6−8). *p < 0.05. **p < 0.01. ***p < 0.001.
highly promising TRPA1 blockers in vitro, we determined
whether they could inhibit TRPA1-mediated inflammation in
vivo. Hence, the effects of compound 8 and 9 on AITC-induced
acute inflammation were investigated in the mouse paw edema
model. The mice were injected intraplantarly with the TRPA1
agonist AITC, and the increase in the volume of the paw caused
by the inflammatory edema was measured in mice pretreated
with the compounds and compared with the increase in those
receiving vehicle only. Systemically administered compounds 8
(10 mg/kg, intraperitoneally (ip)) and 9 (10 mg/kg, ip) both
attenuated the AITC-induced inflammatory edema with an
effect similar to that of the known TRPA1 antagonist HC-
030031 [300 mg/kg, intragastrically (ig)], used as a positive
control. The results are summarized in Figure 8.
likely to be generalized to humans, as well. Furthermore, in the
whole-cell patch clamp recordings, the blocking effect of the
compounds was completely reversible at concentrations of ≤3
μM.
The in vivo experiments demonstrated that compounds 8 and
9 attenuated TRPA1-mediated inflammation induced by AITC,
indicating that these two triterpenoids when administered
systemically have appropriate pharmacokinetics to ease paw
inflammation, most likely by inhibiting TRPA1 activation. It is
noteworthy that the two triterpenoids were active at doses that
were smaller than that of the widely used TRPA1 antagonist
HC-030031. Mice exhibited no adverse effects due to treatment
with the triterpenoids during the short duration of the
experiment.
The detailed molecular mechanism of action of pyrazine-
fused triterpenoids (compounds 8 and 9) on the TRPA1
channel cannot be determined on the basis of the results of this
study, though both triterpenoids inhibited the channel currents
in a voltage-dependent manner. Previous studies of TRPA1
blockers have revealed versatile binding sites and mechanisms of
action. A very effective TRPA1 antagonist A-967079 seems to
attach to the TRPA1 channel pore vestibule where it interacts
with the phenyl moiety of F944;26 the site of HC-030031 action
is the N855 residue of the channel, and interaction with the C-
DISCUSSION
■
These results clearly indicate that the two pyrazine-fused
triterpenoids, compounds 8 and 9 (Figure 3), effectively inhibit
TRPA1 activation in a dose-dependent manner in vitro and
significantly attenuate acute inflammation mediated by TRPA1
in vivo. The observed blockade of both the human and the
mouse TRPA1 channel in vitro suggests that the compounds’
inhibitory effects on TRPA1-mediated conditions in mice are
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In this study, we show that the effects of compounds 8 and 9
were reversible at least up to a concentration of 3 μM, while their
action was only partly reversible at 30 μM. Moreover, the
recovery after the removal was relatively slow even at 0.3 μM,
suggesting that these triterpenoids bind tightly to the TRPA1
channel. Because both compounds 8 and 9, but not the other
triterpenoids that were investigated, have a pyrazine ring in their
structure (Figure 1), this moiety appears to be critical for their
blocking efficacy, reversibility, and/or recovery rate after their
removal. The conversion of the carboxylic acid in compound 8
to the respective amide in compound 9 did not seem to affect the
TRPA1 blocking properties of the compound. Even though
pyridine derivative compound 10 did not exert a blockade
similar to that of compounds 8 and 9, the effects of other six-
membered rings on the 2,3-position should be determined in the
future. Further extensive studies will be required to clarify the
detailed molecular mechanisms of TRPA1 blocking by pyrazine-
fused triterpenoids.
The IC50 values of compounds 8 and 9, around 0.3 μM each at
−50 mV in whole-cell patch clamp recordings against 50 μM
AITC, were lower than that of the most widely used TRPA1
blocker HC-030031 (∼1 μM against 5 μM AITC-induced
human TRPA1 currents).10 However, the IC50 values calculated
from intracellular Ca2+ influx measurements were higher: 9.5
and 7.5 μM for compounds 8 and 9, respectively. Similarly, the
IC50 value of HC-030031 was higher in the Ca2+ measurement
assay (6.2 μM) than in the patch clamp experiments.10 It is also
notable that IC50 values of compounds 8 and 9 were increased at
positive membrane potentials in whole-cell patch clamp
experiments. This depolarization-induced reduction in the
blockade of TRPA1 by both compounds 8 and 9 may in part
explain the difference in IC50 values between Fluo 3-AM and
whole-cell patch clamp experiments. The activation of TRPA1
induces depolarization of TRPA1-expressing HEK293 cells in
the Fluo 3-AM experiments and thus may attenuate the blocking
efficacy of compounds 8 and 9 against TRPA1. For comparison
to the identified IC50 value of 0.3 μM for betulin-derived
compounds 8 and 9, the naturally occurring stilbenoids
pinosylvin and resveratrol have been shown to block TRPA1
activity with IC50 values of 16.7 and 12.9 μM in whole-cell patch
clamp recordings, respectively,29 and gallic acid with an IC50 of
11 μM.30 Recently described synthetically modified quinazoli-
none-based purinone 27 and N-isopropylglycine sulfonamide-
based compound 20 have increased potencies, while the most
widely used traditional TRPA1 blockers are less potent than
compounds 8 and 9 identified in this study.31,32
Laavola et al. have recently assessed the anti-inflammatory
properties of compounds 8 and 9.15 A significant decrease in the
level of inducible nitric oxide synthase (iNOS) expression was
observed in J774 macrophages treated with both compounds,
which is likely to result in the impaired production of nitric oxide
(NO) under inflammatory conditions. As NO is a known
TRPA1 activator,11 compounds 8 and 9 may downregulate
TRPA1-mediated responses in inflammatory conditions both
directly by blocking the channel (as shown in this study) and
indirectly by reducing the level of synthesis of endogenous
TRPA1 activators such as NO. Moreover, the antagonistic
effects of compounds 8 and 9 on TRPA1 channels may at least
partially explain the previously reported attenuating effects of
these compounds on inflammatory gene expression,15 because
TRPA1 activation has been shown to upregulate the expression
of pro-inflammatory factors, like cyclo-oxygenase-2, and
interleukins 1, 6, and 8 in inflammatory conditions.6,7,33−35
Figure 5. Triterpenoids 8 and 9 inhibited allyl isothiocyanate (AITC,
50 μM)-induced Ca2+ influx in HEK293 cells transfected with a plasmid
encoding mTRPA1. Fluo 3-AM-loaded cells were preincubated for 30
min with compound 8 (100 μM), compound 9 (100 μM), or the known
TRPA1 antagonist HC-030031 (100 μM). In panels A and B, the areas
under the curve (AUC) of the response of the fluorescence to AITC
with or without treatment with 8 and 9, respectively, are presented. The
background fluorescence was recorded for 15 s before addition of
AITC. The increase in fluorescence in response to AITC was measured
for 30 s. The results are normalized to background and expressed as
means + SEM (n = 5−8). *p < 0.05. **p < 0.01. ***p < 0.001.
terminal region exerts synergistic effects on the inhibition.27
Because compounds 8 and 9 exerted voltage-dependent
blockade against TRPA1, it is possible that these triterpenoids
interact with certain amino acid residues in the channel that are
located close to the electrical fields within the plasma membrane.
In addition, HC-030031 has been reported to be a rapidly acting
and reversible blocker.10 Menthol is a terpene-related
compound, which has been shown to have an effect on
TRPA1. Its binding site in mouse TRPA1 has been reported to
be transmembrane domain 5, which is essential for the channel’s
ability to sense menthol. Nine other pore residues were also
identified to account for the species-specific alteration of
menthol’s action as either an agonist or an antagonist of
TRPA1.28
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Figure 6. Triterpenoids 8 and 9 inhibited allyl isothiocyanate (AITC)-induced membrane currents measured with whole-cell patch clamp recording in
HEK293 cells transfected with hTRPA1. In panels A and C, the current trace displays the change of inward currents at −50 mV that was evoked by
compounds 8 (0.3, 3, and 30 μM) and 9 (0.3, 3, and 30 μM), respectively, in the presence of 50 μM AITC. To determine the zero current level of
TRPA1 components, 30 μM HC-030031 (HC) was applied at the end of each experiment. In panels B and D, a current−voltage (I−V) relationship
was constructed to use a ramp waveform pulse from −150 to 100 mV for 100 ms. Six I−V relationships were illustrated before (cont) and after
application of AITC (50 μM AITC) and during simultaneous application of 0.3, 3, or 30 μM compound 8 (+30 μM 8) and 50 μM AITC in panel B.
The experimental conditions for panel D were identical to those utilized for panel B except for application of compound 9. In panels E and F, each
relative amplitude (relative) was calculated as a current size at −100, −50, 50, and 100 mV between before and after application of 8 and 9, respectively,
and summarized as a dose−response relationship. Maximum and minimum amplitudes of the current were obtained in the presence of AITC (1.0) and
in the presence of both AITC and HC-030031 (0). The results in panels E and F are expressed as means SEM. *p < 0.05 by two-way analysis of
variance. In panels E and F, the numbers of replicates are given in parentheses next to the markers.
These results have laid the foundation for a wider assessment
of the effects of pyrazine-fused triterpenoids in different animal
models of diseases involving TRPA1 activation. If one considers
the positive effects of other TRPA1 blockers, treatment of
neuropathic pain has traveled the longest path; phase II clinical
trials of compound GRC 17536 have been conducted, with
favorable results.36 Some novel directions in TRPA1 research
have emerged. TRPA1 has a notable role in the pathogenesis of
allergic contact dermatitis, and therefore, TRPA1 blockers might
be useful in its treatment.37 Our recent studies have shown that
in primary human articular chondrocytes TRPA1 is expressed
and TRPA1 blockers and/or genetic deletion of TRPA1 can
alleviate pain and inflammation in experimental models of
osteoarthritis and gout.7,8,33 Other inflammatory conditions
such as colitis and asthma are also potential targets of TRPA1
blockers.38,39 The most widely used TRPA1 blocker in animal
experiments, HC-030031, was unfortunately discovered to
possess poor pharmacokinetic properties.40 Therefore, the
pharmacokinetic profile as well as the safety profile of
compounds 8 and 9 should be characterized in the future,
although our preliminary data did not reveal any obvious
concerns.
mediated inflammation in vivo. They could be useful lead
compounds when developing new drugs for alleviating TRPA1-
mediated adverse conditions.
METHODS
■
Triterpenoid Synthesis. General Experimental Procedures.
Commercially available reagents were used without further purification.
Crude betulin (UPM, Lappeenranta, Finland) was recrystallized from
2-propanol/H2O azeotrope to yield 99% pure betulin as a white solid.
All solvents were of high-performance liquid chromatography grade.
Anhydrous solvents were purchased from Sigma-Aldrich (St. Louis,
MO). All reactions in anhydrous solvents were performed in oven-dried
glassware under an inert atmosphere of anhydrous argon or nitrogen.
Thin-layer chromatography (TLC) was performed on E. Merck
(Darmstadt, Germany) silica gel 60 backed plates, with visualization
by ultraviolet illumination and staining with 5% H2SO4 in MeOH.
Melting points were obtained with a Sanyo Gallenkamp (Moriguchi,
Osaka, Japan) apparatus without correction. The Fourier transform
infrared (FTIR) spectra were recorded on a Nicolet (Waltham, MA)
iS50 FTIR instrument using a built-in diamond ATR. The 1H and 13
nuclear magnetic resonance (NMR) spectra were measured on a Bruker
(Billerica, MA) Avance III 500 MHz NMR spectrometer. 1H and 13
C
C
NMR spectra were recorded in solution, in CDCl3. HRMS spectra were
measured to determine the purity of all tested compounds on a Waters
Acquity UPLC system (Waters, Milford, MA) equipped with a Synapt
G2 HDMS mass spectrometer (Waters).
In conclusion, this study discovered two pyrazine-fused
triterpenoids (compounds 8 and 9) which were found to block
the TRPA1 ion channel reversibly in vitro and alleviate TRPA1-
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Figure 7. Recovery of allyl isothiocyanate (AITC)-induced membrane currents after removal of compounds 8 and 9. In panels A and D, the trace
displays the change in inward currents at −50 mV that was evoked by application and removal of compounds 8 (0.3 μM) and 9 (0.3 μM), respectively,
in the presence of 10 μM AITC. To determine the zero-current level of TRPA1 components, the known TRPA1 antagonist A-967079 (A96, 5 μM) was
applied at the end of each experiment. The experimental conditions for panels B and E and panels C and F were identical to those utilized for panels A
and D except for the concentrations of compounds 8 and 9 of 3 and 30 μM, respectively. In panels G and H, the relative amplitude was calculated as a
current size at −50 mV between the zero current and the current amplitude before and after application of each compound and after their removal but
in the presence of AITC. The results in panels G and H are expressed as means + SEM. *p < 0.05 against each first column, and #p < 0.05 against each
second column, tested by Tukey’s multiple-comparison test. In panels G and H, the numbers of replicates are given in parentheses above the columns.
Figure 8. Triterpenoids 8 and 9 inhibited TRPA1-mediated acute paw inflammation in mice. Mice were pretreated with compound 8 (10 mg/kg, ip),
compound 9 (10 mg/kg, ip), the known TRPA1 blocker HC-030031 (300 mg/kg, ig), or the vehicle 1 h (8, 9, and vehicle) or 2 h (HC-030031) prior
to the subcutaneous administration of the TRPA1 agonist allyl isothiocyanate (AITC) in the paw. The paw volume was measured with a
plethysmometer before and 3 and 6 h after AITC paw injection. The results are normalized to the control paw injected with the vehicle (means + SEM;
n = 6). *p < 0.05. **p < 0.01. ***p < 0.001.
Triterpenoids. A novel method was developed to synthesize
betulonic and betulinic acid from betulin, as detailed below. The
other 11 betulin derivatives were prepared as described previously.20−22
Betulonic Acid (2). Palladium(II) acetate (1.2 g, 6 wt %) and
pyridine (5 mL) were added to toluene (500 mL). The mixture was
heated to 80 °C, and betulin (1) (20.0 g, 45.2 mmol) was added in small
portions under vigorous stirring and air bubbling (flow rate of 1000
mL/min). After 7 h, the reaction was quenched (monitored by GC) by
adding N-acetyl-L-cysteine (8.0 g, 66.0 mmol) to scavenge Pd(II). The
reaction mixture was cooled to room temperature, and the residue was
filtered and washed with toluene (3 × 20 mL). The resulting filtrate
containing betulonic aldehyde was used in the next reaction step in one
pot without further purification. tert-Butanol (170 mL) and 2-methyl-2-
butene (30 mL) were added to the filtrate (containing 20 g of betulonic
aldehyde, GC purity of 88%) under a N2 flow. NaClO2 (80%, 17.0 g,
150 mmol) and NaH2PO4·H2O (25.2 g, 183 mmol) in water (200 mL)
G
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were added over 30 min, and stirring was continued at room
temperature under a N2 atmosphere for 15 h. The organic and aqueous
phases were separated, and a 1.7 M solution of NaOH in H2O (40 mL)
was added to the organic phase and stirred for 0.5 h. Half of the solvent
was evaporated off in vacuo, and the formed precipitate was filtered,
washed with toluene (4 × 20 mL), and dried overnight in vacuo to yield
sodium betulonate (16.6 g, 36.0 mmol). Sodium betulonate was treated
with a 3% solution of HCl in H2O (200 mL), and the resulting mixture
was stirred for 2.5 h. The formed precipitate was filtered, washed with
water (5 × 30 mL), and dried in vacuo at 60 °C overnight to give
betulonic acid (14.9 g, 32.8 mmol), in 73% yield from betulin. Its
spectral data were identical to those reported in the literature.21
Betulinic Acid (3). Betulonic acid (2) (13.0 g, 28.6 mmol) was added
to a mixture of water (130 mL), a 5% solution of NaOH in H2O (26
mL), and 2-propanol (120 mL). NaBH4 (1.20 g, 31.7 mmol) was
added, and the reaction mixture was stirred for 3 h in an ice bath. The
alkaline solution was acidified by adding a 10% solution of HCl in H2O
(130 mL). The precipitated betulinic acid was filtered, washed with
water (4 × 50 mL), and dried in vacuo to yield crude betulinic acid (12.2
g, 94%). A batch of crude betulinic acid (5.0 g) was dissolved in
refluxing ethanol (50 mL) and allowed to crystallize at 4 °C overnight.
The formed crystals were filtered and dried in vacuo to produce very
pure betulinic acid (4.1 g, 9.0 mmol) in 82% yield. Its spectral data were
identical to those reported in the literature.21
(adjusted to pH 7.2 with CsOH)]. Membrane currents and voltage
signals were converted into a digital form using an analog−digital
converter (PCI6229; National Instruments Japan Corp., Tokyo,
Japan). Data acquisition and current imaging of whole-cell currents
were performed using WinEDR version 3.38 developed by J. Dempster
(University of Strathclyde). A ramp voltage protocol from −150 to 100
mV over 100 ms was applied every 5 s from a holding potential of −50
mV. A HEPES-buffered bathing solution [10 mM CsCl, 5.9 mM KCl,
137 mM NaCl, 14 mM glucose, 1.2 mM MgCl2, and 10 mM HEPES
(adjusted to pH 7.4 with NaOH)] was used. All experiments were
performed at 25 1 °C, and the studied drugs, AITC (10 and 50 μM),
compound 8 (0.3−100 μM), compound 9 (0.3−100 μM), HC-030031
(30 μM), and A-967079 (5 μM), were dissolved in dimethyl sulfoxide
(final concentration of ≤0.13%).
In Vivo Model of TRPA1-Mediated Inflammation. Male C57BL/
6N mice were obtained from Scanbur Research A/S (Karlslunde,
Denmark). The mice were housed at the Tampere University animal
facility (12 h:12 h light:dark cycle, temperature of 22 1 °C, food and
water provided ad libitum). Animal experiments were carried out in
accordance with the institutional, national, and European (Directive
2010/63/EU) legislation for the protection of animals used for
scientific purposes and approved by the National Animal Experiment
Board. Anesthesia was performed with ketamine (75 mg/kg, ip, Ketalar;
Pfizer Oy Animal Health, Helsinki, Finland) and medetomidine (0.375
mg/kg, ip, Domitor; Orion Oyj, Espoo, Finland).
The specific TRPA1 agonist AITC was used to induce inflammatory
paw edema by injecting 50 μL of sterile endotoxin-free PBS containing
15 mM AITC into the hind paw of anesthetized mice. The contralateral
paw was injected with the corresponding volume of the vehicle. Prior to
the injection of AITC, the mice were dosed with either compound 8 or
9 (both 10 mg/kg in PBS and 10% DMSO given intraperitoneally 1 h
before AITC) or with the vehicle or with the known TRPA1 antagonist
HC-030031 [300 mg/kg in 50% polyethylene glycol, 40% 1,2-
propanediol, and 10% Glucosteril (50 mg/mL) (Baxter Oy, Vantaa,
Finland) given intragastrically 2 h before AITC]. The paw volumes
were measured before and 3 and 6 h after the AITC injections with a
plethysmometer (Ugo Basile, Comerio, Italy). The volume change in
the vehicle-injected control paw was subtracted from the volume
change in the AITC-injected paw, and the results are given in
microliters.
Statistical Analysis. The statistical significance of the results was
analyzed with GraphPad InStat 3.10 for Windows (GraphPad Software,
Inc., San Diego, CA) and with Origin9.1J (OriginLab, Northampton,
MA) using two-way or one-way analysis of variance with Dunnett’s,
Bonferroni’s, or Tukey’s multiple-comparison test. The results are
expressed as means SEM (standard error of the mean) with *p < 0.05,
**p < 0.01, and ***p < 0.001.
Effects of Triterpenoids on TRPA1-Mediated Responses.
Reagents. Reagents (unless otherwise indicated) were provided by
Sigma Chemical Co. (St. Louis, MO).
Cell Culture. HEK293 human embryonic kidney cells (American
Type Culture Collection, Manassas, VA) were cultured at 37 °C in 5%
CO2 in Eagle’s minimum essential medium (EMEM) supplemented
with fetal bovine serum (10%), sodium pyruvate (1 mM), sodium
bicarbonate (1.5%), non-essential amino acids (1 mM each, all from
Lonza, Verviers, Belgium), streptomycin (100 mg/mL), penicillin (100
units/mL), and amphotericin B (all from Invitrogen, Paisley, U.K.).
Cells were cultivated on a 96-well plate and transfected transiently with
hTRPA1 (0.2 μg/well, pCMV6-XL4; Origene, Rockville, MD) or
mTRPA1 plasmid (0.2 μg/well, Mm17807; Gene Copoeia Inc.). The
cells were transfected 20 h before the experiments were started with
Lipofectamine 2000 (Invitrogen, 0.5 μL/well).
Intracellular Ca2+ Measurements. For intracellular Ca2+ measure-
ments, transiently transfected HEK293 cells were loaded with Fluo 3
acetoxymethyl (AM) ester [2.5 μM Fluo 3-AM in Hanks’ Basic Salt
Solution (Lonza) (pH 7.45) with 25 mM HEPES, 1 mg/mL bovine
serum albumin, 2.5 mM probenecid, and 0.08% Pluronic F-127] for 40
min at room temperature. Thereafter, the cells were washed; a buffer
solution containing the studied compounds was added to the wells, and
the cells were incubated for 30 min at 37 °C. Free intracellular Ca2+
concentrations were measured with a Victor3 1420 multilabel counter
(PerkinElmer, Waltham, MA) at wavelengths of 485 nm (emission)
and 535 nm (excitation). The basal level of fluorescence was measured
for 15 s before adding the TRPA1 agonist allyl isothiocyanate (AITC,
50 μM), and thereafter, the measurements were continued for 30 s.
Finally, ionomycin (1 μM), was applied to induce a robust Ca2+ influx.
The TRPA1 opening properties of the compounds were measured
using the same protocol with the exception of adding the compounds of
interest instead of AITC. In these measurements, AITC (50 μM) was
used as a positive control, and the TRPA1 antagonist HC-030031
(100−200 μM) was utilized to estimate the TRPA1 mediation of Ca2+
influx.
AUTHOR INFORMATION
Corresponding Author
■
*Faculty of Medicine and Health Technology, Tampere
University, 33014 Tampere, Finland. E-mail: eeva.moilanen@
tuni.fi.
ORCID
̈
Ilari Maki-Opas: 0000-0003-0918-040X
̂
Vania M. Moreira: 0000-0001-6169-5035
Jari Yli-Kauhaluoma: 0000-0003-0370-7653
The cytotoxic effects of all of the triterpenoids studied were
evaluated with the modified XTT test (Cell Proliferation Kit II, Roche
Diagnostics, Mannheim, Germany).
Author Contributions
R.H., T.J.A., S.A., V.M.M., and J.Y.-K. designed and synthesized
the triterpenoids, and the chemistry part was conceived,
supervised, and funded by J.Y.-K. I.M.-O., M.H., L.J.M., K.M.,
and E.M. designed and carried out the in vitro and in vivo
bioactivity experiments and data analysis, and the bioactivity
part was conceived, supervised, and funded by K.M. (patch
clamp experiments) and by E.M. (other in vitro and in vivo
experiments). I.M.-O. drafted the manuscript and was supported
by M.H., L.J.M., K.M., J.Y.-K., and E.M. All authors participated
Whole-Cell Patch Clamp Recordings. Whole-cell patch clamp
recordings were performed on HEK293 cells transiently transfected
with a plasmid encoding hTRPA1 (pIRES2-AcGFP1; Takara, Tokyo,
Japan). The cells successfully expressing TRPA1 were identified using
green fluorescent protein. The resistance of electrodes was 3−7 MΩ
when they were filled with a solution mimicking intracellular conditions
[30 mM CsCl, 110 mM cesium aspartate, 1 mM MgCl2, 10 mM EGTA,
10 mM HEPES, 6.25 mM CaCl2, and 2 mM ATP disodium salt
H
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ACS Chemical Neuroscience
Research Article
of TRPA1 channel activation by cysteine-reactive inflammatory
mediators. Channels 2, 287−298.
in the interpretation of the data and revised the manuscript
critically for important intellectual content.
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of triterpenoid skeletal diversity. Phytochemistry 65, 261−291.
Funding
The authors acknowledge Tor, Joe and Pentti Borg Foundation,
The Paulo Foundation, The Academy of Finland (Project
317507), and the Medical Research Fund of Tampere
University Hospital for financially supporting the bioactivity
studies and the European Commission (Contracts FP7-KBBE-
227329-ForestSpeCs and FP7-HEALTH-602919-GLORIA),
The Academy of Finland (Project 252308), and the Marjatta
and Eino Kolli Foundation for supporting the chemistry part of
this study.
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Notes
The authors declare no competing financial interest.
(16) Dehelean, C. A., Soica, C., Ledeti, I., Aluas, M., Zupko, I.,
ACKNOWLEDGMENTS
̌
Galusç an, A., Cinta-Pinzaru, S., and Munteanu, M. (2012) Study of the
■
betulin enriched birch bark extracts effects on human carcinoma cells
and ear inflammation. Chem. Cent. J. 6, 137.
Ms. Terhi Salonen is warmly thanked for skillful technical
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