Identification of a Novel TRPM8 Agonist from Nutmeg: A Promising Cooling Compound

Article abstract of DOI:10.1021/acsmedchemlett.7b00104

The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary receptor for innocuous cold stimuli (<28 °C) in humans. TRPM8 agonists such as l-(-)-menthol are widely used as flavors and additives to impart briskness, in addition to medicinal uses for inflammation and pain. Though various natural and synthetic agonists have been explored, only few natural compounds are known. We report herein the identification and characterization of the novel neolignan agonist erythro- and threo-δ^8′-7-ethoxy-4-hydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan (1) with an EC₅₀ of 0.332 μM, which was isolated from a well-known spice, nutmeg (Myristica fragrans Houtt.). Structure activity relationships are also disclosed, showing that the 7-d-menthoxy derivative is the most potent agonist (EC₅₀ = 11 nM). The combination of 1 and l-(-)-menthol has an additive effect, suggesting that neolignan compounds interact with TRPM8 at different sites from those of l-(-)-menthol.

Full text of DOI:10.1021/acsmedchemlett.7b00104

Letter
pubs.acs.org/acsmedchemlett
Identification of a Novel TRPM8 Agonist from Nutmeg: A Promising
Cooling Compound
Tomohiro Shirai,*,† Kentaro Kumihashi,† Mitsuyoshi Sakasai,‡ Hiroshi Kusuoku,‡ Yusuke Shibuya,‡
and Atsushi Ohuchi†
‡
†Kansei Science Research and Biological Science Research, Kao Corporation, 2606 Akabane, Ichikai-Machi, Haga-Gun, Tochigi
321-3497, Japan
S
* Supporting Information
ABSTRACT: The transient receptor potential melastatin 8 (TRPM8) ion
channel is the primary receptor for innocuous cold stimuli (<28 °C) in
humans. TRPM8 agonists such as l-(−)-menthol are widely used as flavors
and additives to impart briskness, in addition to medicinal uses for
inflammation and pain. Though various natural and synthetic agonists have
been explored, only few natural compounds are known. We report herein the
identification and characterization of the novel neolignan agonist erythro- and
threo-Δ8′-7-ethoxy-4-hydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan (1) with
an EC50 of 0.332 μM, which was isolated from a well-known spice, nutmeg
(Myristica fragrans Houtt.). Structure activity relationships are also disclosed,
showing that the 7-d-menthoxy derivative is the most potent agonist (EC50
=
11 nM). The combination of 1 and l-(−)-menthol has an additive effect,
suggesting that neolignan compounds interact with TRPM8 at different sites
from those of l-(−)-menthol.
KEYWORDS: TRPM8, Cooling compound, nutmeg, neolignan, menthol
ransient receptor potential (TRP) channels are one of the
Tlargest groups of ion channels, with at least 28 members in
6 families characterized in humans. Several TRP channels are
expressed in sensory neurons and are receptors for various
chemicals and a wide range of thermal stimuli.1,2 TRPM8, a
member of the melastatin subfamily of TRP channels, is a
primary receptor for innocuous cold stimuli (<28 °C) in
humans.3,4 TRPM8 is also activated by “cooling compounds”
such as menthol, icilin, their analogs, other synthetic
compounds, and a few natural compounds.5−7 Recent findings
have also shown that some pathological conditions, such as
inflammation, pain, prostate cancer, asthma, and dry eye, are
related to different states of TRPM8 expression and activation.
Therefore, drug-like agonists and antagonists have been
developed for clinical applications.5
Synthetic and natural l-(−)-menthol is the TRPM8 agonist
with the most widespread use as an additive in food, drinks,
tobacco, cosmetics, and medicinal products to impart a sense of
cold and briskness or to relieve pain. However, high
concentrations of l-(−)-menthol often create a burning
sensation, unpleasant irritation, and a distinctive smell and
taste, whereas low concentrations do not evoke enough of a
cooling sensation or persist for a sufficient duration.8 Various
natural and synthetic analogs have been explored to overcome
such deficits; for example, synthetic menthancarboxamides are
highly potent TRPM8 agonists, some of which are already in
use.5−7 Other synthetic agonists with completely different
structures and high potencies have also been discovered.9,10
Despite these efforts, few natural agonists have been
developed. Except for menthol and similar compounds, eugenol
and some monoterpenes have been the only known natural
agonists, though their potencies are fairly low.11 Natural
products provide diverse and complex structures that are
often challenging to prepare via synthetic methods.12 Novel
natural agonists might expand the design of TRPM8 agonists
and the field of study by unknown bioactivities.
Therefore, we explored novel TRPM8 activators from
botanical and spice extracts by combination with l-(−)-menthol
to find agonists that were not competitors with menthol. The
TRPM8 activities were evaluated through calcium imaging of
human TRPM8-expressing HEK293 cells with fluo-4. This
screening revealed that the ethanol extract of dried nutmeg
(Myristica fragrans Houtt.) seed showed menthol-enhancing
activity and weak TRPM8 activity by itself. Nutmeg and its aril
mace are common and unique spices, widely used in cooking.
Their essential oils, oleoresins, and extracts are also contained
in groceries and commodities as flavors and fragrances. Several
medicinal uses have been studied and are traditionally known,
but no reports have indicated TRPM8 activity or a cooling
effect.13 Though eugenol is contained in the extract, the activity
of the extract could not be explained by eugenol alone.
Received: March 9, 2017
Accepted: May 31, 2017
Published: May 31, 2017
© XXXX American Chemical Society
A
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a
Therefore, we began to identify the TRPM8 agonists in the
nutmeg extract through bioassay-guided purifications with silica
gel column chromatography and semipreparative silica gel
HPLC. Finally, the active fraction produced a single peak in
silica gel HPLC and ODS-HPLC analyses. A wavelength of
maximal absorption at 278 nm by the fraction indicated a
conjugated aromatic moiety. A sodium adduct ion peak in
positive MS mode was observed at m/z 425. However, over 39
carbon peaks were observed in the 13C NMR spectrum
measured in CDCl3, suggesting that there were two isomers of
phenylpropanoid dimers that were not separated by HPLC.
The 1D and 2D NMR spectra indicated that both isomers had
6-methoxyeugenol skeletons because 2 aromatic protons and 6
methoxy protons were obtained by integration, and substitution
of the aromatic rings with allylic groups was observed in the
both isomers. The spectra also revealed that both isomers had
n-propyl skeletons that were substituted with ethoxy groups,
guaiacol moieties, and 4-allyl-2,6-dimethoxyphenyl groups.
From these results, we deduced that the isomers were erythro-
and threo-Δ8′-7-ethoxy-4-hydroxy-3,3′,5′-trimethoxy-8-O-4′-
neolignan (1) (Figure 1).14 Though various 8-O-4′-neolignans
Scheme 1. Diastereoselective Synthesis of 1
a
Reagents and conditions: (a) imidazole, TBSCl, DMF, 92%; (b)
EtMgBr, THF, 0 °C; (c) MnO2, CH2Cl2, 77% (for 2 steps); (d) Br2,
CHCl3; (e) 6 N HCl, MeOH; (f) MOMCl, DIPEA, CH2Cl2, 67% (for
3 steps); (g) 6, K2CO3, acetone, 40 °C, 74%; (h) LiAlH4, THF, 77%
(10% for 8b); (i) NaH, EtI, THF, 0 °C to rt, 82% for 9a, 66% for 9b;
(j) 6 N HCl, THF, 0 °C to rt, 61% for 1a, 72% for 1b; (k) NaBH4, 15-
crown-5, i-PrOH/MeOH, 68% (6% for 8a).
Figure 1. Structure of 1.
have been identified from nutmeg, no 7-ethoxy analogs have
been reported except for a suggestively semisynthetic
compound.14,15 No spectroscopic data were available for 1;
therefore, the structures were elucidated by comparison with
the spectroscopic data of synthetic 1.
1H and 13C NMR chemical shifts of synthetic 1a and 1b and
the purified fraction of nutmeg extract were identical to each
other (see Supporting Information). We determined that the
TRPM8-active compounds in the nutmeg extract were an
approximately 1:1 diastereomixture of 1.
We further investigated the enantiomeric ratio of the isolated
compound 1. Analysis on a Daicel Chiralpak IC column
revealed four peaks, suggesting four stereoisomers in the almost
equal proportions caused by two chiral centers at C-7 and C-8.
To determine the absolute configurations of each peak, we
prepared the four stereoisomers from optically pure inter-
mediates as follows (Scheme 2). (S)- and (R)-MTPA
esterification of the synthetic erythro intermediate 8a and
Diastereoselective synthesis of 1 was achieved by following
the reported diastereoselective reduction of the ketone at C-7
in similar compounds (Scheme 1).16 The phenolic group of
vanillin (2) was protected by a TBS group to give the TBS
ether 3. A Grignard reaction with ethyl magnesium bromide
and oxidation of the secondary alcohol with manganese dioxide
afforded the ketone 4, and the α-position was brominated. In
this bromination, the TBS group was partly eliminated, and the
crude product was subsequently treated with hydrochloric acid
to afford a completely deprotected α-bromoketone. Reprotec-
tion of the phenolic group by MOMCl gave the MOM ether 5,
which was condensed with 4-allyl-2,6-dimethoxyphenol (6) to
afford the ketone 7. To synthesize the erythro isomer, 7 was
reduced by lithium aluminum hydride to give the erythro-
secondary alcohol 8a preferentially (77% yield compared with
10% yield of the threo-isomer 8b). The relative configurations
were deduced by the coupling constant between the C-7 and C-
8 protons. 8a had a smaller coupling constant (2.6 Hz) than 8b
(8.6 Hz), similar to other 8-O-4′-neolignans. Furthermore, the
MOM group of 8a was deprotected here to give the reported
erythro compound to determine the relative configurations (see
Supporting Information).15 Then, 8a was alkylated with ethyl
iodide to afford the erythro-ethyl ether 9a, and subsequent
deprotection of the MOM group with hydrochloric acid in
THF gave the erythro isomer 1a. Next, 7 was reduced by
sodium borohydride with crown ether to afford 8b preferen-
tially (68% yield compared with 6% yield of 8a). 8b was
alkylated with ethyl iodide to afford the threo-ethyl ether 9b,
which afforded the threo isomer 1b following deprotection. The
a
Scheme 2. Preparation of the Four Stereoisomers of 1
a
Reagents and conditions: (a) (S)-(+)-MTPACl or (R)-(−)-MTPACl,
Et3N, DMAP, CH2Cl2, 78−79% as a diastereomixture; (b) 50 w/v%
NaOH aq, MeOH, 93−100%.
B
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threo intermediate 8b enabled the separation of the
enantiomers and determination of the absolute configurations
by a modified Mosher method (see Supporting Information).17
Hydrolysis of the (S)-MTPA ester 10aa-s, which had a (7R,8S)
configuration, gave the (7R,8S)-secondary alcohol 8aa. The
(7S,8R) isomer 8ab was synthesized in the same way, and the
optically pure threo isomers 8ba, which has a (7R,8R)
configuration, and 8bb, which has a (7S,8S) configuration,
were also obtained using the same method as for synthetic 8b.
Each isomer was alkylated with ethyl iodide and deprotected as
indicated before to afford four stereoisomers of 1. By
comparing the retention times in the chiral column of 1 from
the nutmeg extract and the synthetic standards, the absolute
configurations of all peaks were determined (see Supporting
Information).
From this result, it was suspected that the identified 1 was an
artifact because most identified 8-O-4′-neolignans in nutmeg
had erythro configurations and ethanol was used as an
extraction solvent.13 We identified the erythro-7-hydroxy analog
named myrislignan 11a and the (7S,8R)-7-acetoxy analog 12ab
and observed that the amount of 1 in the nutmeg extract was
increased in ethanol, suggesting that 11a and/or 12ab were
gradually converted to 1. Thus, the majority of identified 1
should be an artifact. On the other hand, LC/MS/MS analysis
of nutmeg extract purified without using ethanol contained a
trace amount of 1 (see Supporting Information). Further
experiments including biosynthetic studies are required to
determine the actual naturality of 1; however, the existence of
natural 1 is suggested.
Table 1. EC50 Values of 1 and Its Enantiomers
a
a
Compounds
Configuration
EC50(M8)(μM)
EC50(A1)(μM)
1
4 isomers
0.332 0.046
10.1 1.0
19.1 0.7
96.0 5.1
N.D.
/-Menthol
1aa
7R,8S
7S,8R
7R,8R
7S,8S
1.13 0.55
1.15 0.44
0.583 0.276
0.087 0.017
1ab
N.D.
1ba
N.D.
1bb
N.D.
a
N = 3−5 (mean SEM). N.D. = not determined.
did not activate the TRPV1, TRPV3, or TRPV4 channels (data
not shown).
The TRPM8 activity of other 8-O-4′-neolignans from
nutmeg extract and their analogs were also investigated. In
addition to 11a and 12ab, the (7S,8R)-4-methoxy-7-acetoxy
analog 13ab and (7S,8R)-4-methoxy analog 14ab were
obtained by purification and derivatization. Interestingly,
natural 12ab and 13ab were optically active, whereas 11a was
racemic (see Supporting Information). The substitution of
various alkoxy groups at C-7 was carried out using acids and
alcohols from natural 11a, which is the main nonvolatile
component in nutmeg, through a quinone methide inter-
mediate (Scheme 3).18 This biomimetic alternation was
Scheme 3. Acid-Induced Conversion of the Substituents at
C-7 with a Configurational Randomization through a
Quinone Methide Intermediate
The activities of TRPM8 and other TRP channels were
measured for 1 and its stereoisomers (Figure 2). A mixture of
possible with primary and secondary alcohols using hydro-
chloric acid, whereas it produced a low yield with tert-butanol
because of the bulkiness. This is a fairly convenient method to
prepare various analogs, though the configuration at C-7 is
epimerized. The same reaction was applied for 12ab. We
prepared 10 analogs 15−24 from 11a using the following 10
alcohols: methanol, n-hexanol, n-octanol, n-decanol, 2-butox-
yethanol, iso-propanol, sec-butanol, cyclohexanol, l-menthol,
and d-menthol. The TRPM8 activities of these analogs were
measured, and their EC50 values were calculated (Table 2). The
4-methoxy analogs 13ab and 14ab completely lost the activity,
suggesting that the 4-hydroxy group is essential for the activity.
The 7-hydroxy analog 11a and 7-acetoxy analog 12ab also
showed low activity, suggesting that the alkoxy groups at C7 are
also important for the activity. Longer alkoxy chains resulted in
smaller EC50 values (18 < 17 < 16 ≤ 1 < 15), and bulkier
alkoxy groups resulted in smaller EC50 values as well (24 < 23 <
22 < 20 < 21 < 1). The introduction of an ether bond did not
affect the activity significantly (19 versus 16 and 17). The d-
menthoxy analog 24 had the lowest EC50 of 11 nM, which was
smaller than that of the synthetic menthanecarboxamide WS-12
(EC50 = 66 nM), a representative cooling compound. Though
Figure 2. Dose−response curve of 1 and l-menthol: (a) for TRPM8;
(b) for TRPA1. N = 6 (mean SEM).
the four stereoisomers of 1 had an EC50 of 0.332 μM, which is
the lowest of the natural products with known TRPM8 activity.
Additionally, 1bb, which has a (7S,8S) configuration, produced
the lowest EC50 value (0.087 μM) compared with the other
isomers, which had EC50 values of 0.583−1.15 μM (Table 1).
1 also exhibited weak TRPA1 activity (EC50 = 19.1 μM) like
l-menthol (EC50(M8) = 10.1 μM, EC50(A1) = 96.0 μM).
TRPA1 activity often causes irritation, and higher selectivity for
TRPM8 is preferable for use as a cooling additive. The
submicromolar TRPM8 EC50 value of 1 presumably enables
display of the cooling effect in vivo with relatively lower TRPA1
activity. Therefore, 1 is a promising novel cooling compound. 1
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Table 2. TRPM8 Potency SAR of 8-O-4′-Neolignan
Derivatives
Figure 3. (a) Effect of a 745Y mutation in TRPM8. N = 3 (mean
SEM). (b) Additive effect of TRPM8 activity with 1 and l-menthol. N
= 4 (mean SEM).
a
b
dr was calculated from 1H NMR data. N = 3 (mean
SEM).
c
Mixture of four stereoisomers. dr = 12:26:26:36 for 23 and 24.
1
Calculated from H NMR data.
it was challenging to separate the stereoisomers of 24, the
(7S,8S) isomer 24bb is expected to be a more potent agonist.
Intriguingly, 24 had a smaller EC50 than the l-menthoxy
analog 23 (EC50 = 28 nM), though l-menthol is more potent
than d-menthol. It was speculated that the interaction sites of
these neolignans are different from those of menthols because
of the entirely novel skeleton compared to the reported
TRPM8 agonists. Previous reports have noted that one of the
key residues in the interaction between l-menthol and TRPM8
is Tyr745, located in the middle of putative transmembrane
segment 2.19 Therefore, we prepared Y745H-TRPM8 through
a point mutation at this residue, and the activity of 1 was
measured (Figure 3). Though the activity of l-menthol
significantly disappeared with the mutated channel, the activity
of 1 was not affected at all. Additionally, 1 did not evoke a
competitive effect with l-menthol but an additive effect,
suggesting that the interaction sites of 1 are different from
those of l-menthol (Figure 3). Future studies exploring the
actual interaction sites will be directed toward designing fresh
agonists and understanding the molecular dynamics of TRPM8.
Lastly, the cooling effect of 1 was evaluated by a simple
mouth washing test. 1 or l-menthol (0.02%) was dissolved in
water with a surfactant (EMANON CH-40, 0.6%), and the
cooling effect was scored for 30 min after washing (score: 0−5,
in increments of 0.5) (Figure 4). l-Menthol evoked a cooling
sensation soon after washing, whereas 1 evoked this sensation a
few minutes later. This difference was partly due to the
difference in absorption properties because of the high
molecular weight of 1 (402 Da) and the low molecular weight
of l-menthol (156 Da). However, further investigation of the
kinetic differences in TRPM8 activity is required to discuss the
Figure 4. Cooling effect of 1 and l-menthol by a simple mouth
washing test. N = 6 (mean SEM).
precise kinetic differences. This absorption discrepancy
probably caused a difference in the duration of the cooling
sensation as well; thus, 1 exhibited a long-lasting cooling effect,
which is unique and industrially valuable. Therefore, 1 is a
promising cooling compound and clearly exhibits TRPM8
activity in vivo.
In summary, we identified a novel TRPM8 agonist 1 that is
the most potent agonist discovered from natural sources to
date. 8-O-4′-Neolignans are major nonvolatile components in
nutmeg, and the safety of 1 might be ensured experientially. 1
did not compete with l-menthol but showed an additive effect.
The high molecular weight of 1 resulted in a cooling effect with
a long duration time. These features make 1 an attractive
cooling compound. Additionally, we demonstrated the easy
alternation of the alkoxy group at C-7 under acidic conditions,
and more potent derivatives were discovered. This reaction can
be utilized in industrial processes, and various structural
modifications would be useful for the preparation of labeled
derivatives as chemical tools for studying the TRPM8 channel
at the molecular level.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00104.
■
S
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Purification procedures, synthetic procedures, compound
characterization, and experimental protocols for the
assays (PDF)
AUTHOR INFORMATION
■
(13) Abourashed, E. A.; El-Alfy, A. T. Chemical Diversity and
Pharmacological Significance of the Secondary Metabolites of Nutmeg
(Myristica fragrans Houtt.). Phytochem. Rev. 2016, 15, 1035−1056.
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271−273.
Corresponding Author
*E-mail: shirai.tomohiro@kao.co.jp.
ORCID
Tomohiro Shirai: 0000-0001-6278-4021
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(15) Kasahara, H.; Miyazawa, M.; Kameoka, H. Absolute
Configuration of 8-O-4′-Neolignans from Myristica fragrans. Phyto-
chemistry 1995, 40, 1515−1517.
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Neolignans. Phytomedicine 2001, 8, 454−459.
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Funding
Kao Corporation is the funder of the research presented in this
study.
Notes
The authors declare the following competing financial
interest(s): All authors are employees of Kao Corporation.
(18) Kawabe, Y.; Ishikawa, R.; Akao, Y.; Yoshida, A.; Inai, M.;
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ACKNOWLEDGMENTS
■
We thank H. Hashimoto for fruitful discussion and providing
nutmeg samples; J. Nakamura, N. Saito, Y. Suzuki, and M.
Matsuura for kind support; and T. Wakimoto and T. Kuranaga
(Hokkaido University) for discussion about the biosynthesis of
neolignans. We also thank the DAICEL Corporation for the
chiral column screening.
ABBREVIATIONS
■
HEK, human embryonic kidney; ODS, octadecyl silica; TBS,
tert-butyldimethylsilyl; MOM, methoxymethyl; MTPA, α-
methoxy-α-(trifluoromethyl)phenylacetic
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