Natural-Product-Derived Transient Receptor Potential Melastatin 8 (TRPM8) Channel Modulators

Article abstract of DOI:10.1021/acs.orglett.6b01222

A library of novel structural hybrids of menthol and cubebol was tested for each derivative's ability to interact with the transient receptor potential subfamily melastatin member 8 (TRPM8) channel. This structure-activity relationship study revealed three potent modulators of the TRPM8 ion channel: a novel agonist (4) with an EC₅₀ value of 11 ± 1 μM, an antagonist (15) with an IC₅₀ value of 2 ± 1 μM, and an allosteric modulator (21) that minimized channel desensitization toward menthol. Each of these novel exocyclic olefin analogues of menthol is readily accessible by synthesis and was tested using Ca²⁺ assays and electrophysiology.

Full text of DOI:10.1021/acs.orglett.6b01222

Letter
pubs.acs.org/OrgLett
Natural-Product-Derived Transient Receptor Potential Melastatin 8
(TRPM8) Channel Modulators
Christina M. LeGay,† Evgueni Gorobets,† Mircea Iftinca,‡ Rithwik Ramachandran,§ Christophe Altier,‡
and Darren J. Derksen*,†
†Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4
‡Department of Physiology & Pharmacology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
§Department of Physiology & Pharmacology, Schulich Medicine & Dentistry, University of Western Ontario, 1151 Richmond Street,
London, Ontario, Canada N6A 3K7
S
* Supporting Information
ABSTRACT: A library of novel structural hybrids of menthol and cubebol was tested for each derivative’s ability to interact with
the transient receptor potential subfamily melastatin member 8 (TRPM8) channel. This structure−activity relationship study
revealed three potent modulators of the TRPM8 ion channel: a novel agonist (4) with an EC50 value of 11 1 μM, an antagonist
(15) with an IC50 value of 2 1 μM, and an allosteric modulator (21) that minimized channel desensitization toward menthol.
Each of these novel exocyclic olefin analogues of menthol is readily accessible by synthesis and was tested using Ca2+ assays and
electrophysiology.
ur group’s interest in anti-inflammatory natural products
Oled us to investigate the structure−activity relationships
(SARs) of the clinically and fundamentally intriguing transient
receptor potential (TRP) channel agonists.1,2 The TRP family
contains more than 30 cation channels, which are divided into
seven main subfamilies, three of which are thermosensitive: the
TRPV (vanilloid) family, the TRPM (melastatin) family, and
the TRPA (ankyrin) family.3,4 All three of these subfamilies
display diverse cation selectivities and biophysical properties,
but their expression within dorsal root and trigeminal ganglia
make them viable targets for the modulation of nociception and
pain.4−6 TRPM8, which is expressed in nearly 15% of all
somatosensory neurons, is of particular interest in terms of
druggability, as it was also found in the epidermis, bladder, and
prostate.6−8 TRPM8 is selective for calcium and is activated by
a range of low temperatures (8−28 °C) and by both natural
and synthetic cooling compounds such as menthol (1), cubebol
(2), and icilin (3) (Figure 1).3−8 In addition to its role as the
primary cold receptor of the somatosensory system, TRPM8
has been suggested as a potential target for cancer therapy
because of the presence of TRPM8 mRNA in prostate tissue
and at elevated levels in tumors of the prostate, breast, colon,
lung, and skin.9,10 TRPM8 has also been identified as a target
Figure 1. Natural and synthetic TRPM8 modulators menthol (1),
cubebol (2), and icilin (3) and initial synthetic intermediates 4 and 5.
for respiratory diseases and for cold hypersensitivity associated
with inflammatory and neuropathic pain.7,9−13 Discovering new
natural product derivatives that are modulators of TRPM8 is a
central goal of this research program because of their potential
therapeutic applications in modulating TRPM8 in chronic
diseases.
Natural products including terpenes and alkaloids, such as
resiniferatoxin and voacangine, respectively, have been a major
source of ligands for the TRP channels.10,14−16 The small-
molecule natural products 1 and 2 are strong activators of the
TRPM8 channel.7,17 Given the structural similarities of 1 and 2,
Received: April 26, 2016
© XXXX American Chemical Society
A
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Figure 2. Evaluation of TRPM8 modulators by calcium measurements. Menthol (1), cubebol (2), icilin (3), and ethanol (Z) were used as controls.
(A) Calcium mobilization in HEK cells transfected with TRPM8 after injection of analogues 1, 2, 4, and 5 at 100 μM and 3 at 90 μM. * indicates
nontransfected HEK cells used as a control. (B) Calcium mobilization in HEK cells transfected with TRPM8 after injection of analogues at 500 μM.
(C) Calcium mobilization evoked by 100 μM menthol in HEK cells transfected with TRPM8 after stimulation with analogues at 500 μM.
we recognized the value of a systematic study of synthetic
analogues of these compounds with the goals of developing a
better understanding of how these natural products are
involved in regulating the cooling sensation mediated by the
TRPM8 channel and of discovering selective modulators.
Although high-resolution crystal structures of closely related
TRP channels (TRPV1 and TRPA1) exist, no crystal structure
of the TRPM8 channel has been reported to date.18−20 A SAR
study of natural product analogues and their interactions with
TRPM8 is crucial to gain information on the therapeutically
significant TRPM8 binding site.20
Scheme 1. (R)-(−)-Carvone-Based Synthesis of Allyl
Alcohols
To efficiently prepare a new generation of small-molecule
TRPM8 modulators, developing a synthetic route that would
allow for the production of a variety of analogues was essential.
On the basis of the brevity and reproducibility of the synthesis,
the work of Hodgson et al.21 was followed for the preparation
of initial quantities of cubebol. Two intermediates of this route,
allylic alcohol 4 and tricyclic ketone 5 (Figure 1), were
evaluated for their impact on TRPM8. As an initial qualitative
screen, fluorescent calcium imaging in TRPM8-transfected
HEK cells was used to evaluate 2, 4, and 5 at high
concentration (Figure 2A). Notably, 4 appeared to be an
agonist of TRPM8 with a calcium mobilization greater than that
of 2, while 5 was not significantly active. Given that 4 clearly
interacts with the TRPM8 receptor, we turned our attention to
the development of a revised, low-cost synthesis of 4 from
readily available (R)-(−)-carvone (6) instead of menthol as
used in the Hodgson method.21 This alternative route avoids
the use of high-cost reagents to access the multigram quantities
of 4 required for generating analogues.
Our synthetic route began with 6, which was transformed
into (R)-dihydrocarvone (7) via stereoselective reduction with
sodium dithionite under phase-transfer conditions (Scheme
1).22,23 These conditions were chosen to selectively reduce the
conjugated olefin, and the minor diastereomer that is formed
can be easily separated from the product using silica gel column
chromatography. Hydrogenation over PtO2 in ethanol was
employed to prepare (R,R)-tetrahydrocarvone (8) from 7. PtO2
was found to be more selective than Pd/C, which resulted in
the formation of a substantial amount (ca. 20%) of another
diastereomer resulting from double-bond migration. The same
sequence of four steps (silyl enol ether formation, Rubottom
oxidation, Wittig olefination, and silyl removal) was used to
transform 6, 7, and 8 into the corresponding allylic alcohols 10,
12, and 4 (Scheme 1).
effect of ester substituents on the binding of allylic alcohol 4
with TRPM8, the syntheses of the acetate (14) and the
benzoate (15) were carried out (Scheme 2). In order to
elucidate the effect of the exocyclic double bond, 16, 17, 18,
and 19 were synthesized as analogues of Frescolat ML,24
Coolact 5,25 (−)-menthyl succinate,26 and N,N-dimethyl
(−)-menthyl succinamide,27 respectively (Scheme 2).
To boost the yield of the sequential silyl enol ether formation
and Rubottom oxidation steps and to prepare a number of
derivatives without the terminal alkene, triethylchlorosilane
(TESCl) was used instead of trimethylsilyl chloride (TMSCl)
in the first step (20, Scheme 2). This change of the hydroxyl
protecting group resulted in a noticeable increase in yield for
both the silyl enol ether preparation step (from 69 to 78%) and
the Rubottom oxidation step (from 62 to 66%). These
improved results can be attributed to the stability of the TES
protecting group, which is 64 times more stable toward acid
and 10−100 times more stable toward base than the TMS
moiety.28 TES-protected hydroxy ketone 20 was then used for
the preparation of various derivatives, including diol 21 via
reduction and deprotection, amino alcohol 22 via reductive
amination and deprotection, and TES-protected allylic alcohol
23 via Wittig olefination (Scheme 2).
The qualitative evaluation of each derivative’s ability to
activate TRPM8 was again conducted using a fluorescence-
based calcium assay in TRPM8-transfected HEK cells.29 On the
basis of the high concentration of compounds used for the
From allylic alcohol 4, a variety of analogues were prepared
via alcohol functionalization. These compounds were synthe-
sized using established synthetic methods. To examine the
B
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suspected to have antagonist activity on menthol-induced
TRPM8 currents. Indeed, pretreatment with 15 (50 μM)
completely blocked the TRPM8 current induced by menthol
(100 μM, n = 6). At +100 mV under control conditions, the
peak current density was 13 2 pA/pF compared with 140
20 pA/pF after the application of menthol and 17 3 pA/pF
after the application of 15 followed by menthol (see the SI for
more details). Benzoate derivative 15 exhibited dose-dependent
inhibition of the TRPM8 currents with a half-maximal
inhibitory concentration (IC50) of 2 1 μM.
Scheme 2. Syntheses of Unsaturated Menthol Analogues
14−19, Diol 21, and Amino Alcohol 22
The desensitization of the TRPM8 channels that occurs after
long-term or repeated stimulation by cold or menthol is a well-
known phenomenon and is dependent on the presence of
extracellular Ca2+.17 The peak of the menthol-evoked currents
at +100 mV was reduced (56 3% decrease, n = 4) between
consecutive applications of menthol. When the TRPM8-
transfected HEK cells were pretreated with diol 21 (100 μM)
between applications of menthol, the current density of the
second stimulation was reduced by only 27
2% (n = 6).
These results indicate that 21 is an allosteric modulator of
TRPM8 that significantly prevents menthol-induced channel
desensitization.
The growing amount of data on TRPM8 accumulated since
its discovery provides evidence that this channel has roles more
diverse than simply being a cold sensor. TRPM8 has been
shown to be involved in the cold hypersensitivity and
hyperalgesia that appear in neuropathic conditions (e.g.,
complex regional pain syndrome, trigeminal neuralgia, periph-
eral nerve injury, or chemotherapy-induced neuropathy) and
are often accompanied by cold hypoesthesia.5,6,13,30 Further-
more, TRPM8 has been implicated in certain types of cancer as
well as chronic inflammatory diseases including inflammatory
bowel disease.9,10,31,32 Novel, potent modulators of TRPM8
could provide pharmacological tools in the treatment of various
pathological conditions, as indicated by the recent finding that
TRPM8 antagonists are viable agents for the treatment of both
cold hypersensitivity and hyperalgesia.5,13 The current SAR
study of several menthol derivatives and their interactions with
the TRPM8 channel has revealed three potent modulators. The
in vitro assays conducted demonstrate the agonistic activity of
allylic alcohol 4, the antagonistic activity of benzoate derivative
15, and allosteric modulation of the TRPM8 channel by diol
21. To further investigate the implications of modulating this
channel, a second library of menthol derivatives is being
assembled on the basis of these results. With a second
generation of menthol derivatives in hand, in vivo evaluations
of the best candidates from each library (including derivatives 4,
15, and 21) will be conducted to develop a better under-
standing of the implications of modulating the TRPM8 channel
using small-molecule natural product analogues.
preliminary screen, all of the synthetic analogues of 4 were
essentially inactive as agonists (Figure 2B). Suspecting that the
analogues could potentially interact with TRPM8 but were
unable to induce calcium mobilization, we evaluated the
analogues as antagonists by pretreating cells with a test
compound followed by addition of menthol (Figure 2C).
Benzoate derivative 15 gave a reduced fluorescence response
consistent with an antagonistic modulator. Unexpectedly, diol
21 gave a pronounced enhancement in fluorescence. With these
results in hand, compounds 4, 15, and 21 were evaluated for
TRPM8-mediated calcium mobilization via electrophysiological
measurements using the conventional whole-cell patch clamp
method (see the Supporting Information (SI) for more details).
When applied at a concentration of 100 μM at room
temperature, compound 4 and menthol (1) both evoked a
TRPM8 current in transfected HEK cells. Upon exposure to 4
and 1, the TRPM8 current density elicited by depolarization
steps from a holding potential of 0 mV to +100 mV showed
similar increases of 3.6 times (from 48 13 to 170 18 pA/
pF, n = 5) and 4.1 times (from 34 9 to 140 16 pA/pF, n =
5) from the basal current, respectively (see the SI for more
details). It was found that 4 and 1 both evoke a TRPM8 current
in a dose-dependent manner with half-maximal effective
concentrations (EC50) of 11 1 and 33 3 μM respectively,
demonstrating the potency of allylic alcohol 4 to activate the
TRPM8 channel.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b01222.
Experimental procedure, physical characterization of the
products, calcium assay methods, and electrophysiology
methods and figures (PDF)
Through the preliminary screen of the derivatives by
fluorescent calcium imaging, benzoate derivative 15 was
C
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Letter
113−8. (c) Liao, M.; Cao, E.; Julius, D.; Cheng, Y. Curr. Opin. Struct.
Biol. 2014, 27, 1−7.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: dderksen@ucalgary.ca.
Author Contributions
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(24) Tejima, H. Japan Patent 2 000 239 142, 2000.
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All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
D.J.D. holds the Alberta Children’s Hospital Foundation Junior
Chair in Medicinal Chemistry and is supported by funding from
the Alberta Children’s Hospital Research Institute, the Alberta
Children’s Hospital Foundation, the Charbonneau Cancer
Institute, and the University of Calgary. C.M.L. was funded by
the Natural Sciences and Engineering Research Council of
Canada (NSERC) Canada Graduate Scholarships-Master’s
Program (CGS-M). C.A. holds a Canada Research Chair in
Inflammatory Pain (Tier 2) and is supported by funding from
the Canadian Institutes of Health Research (CIHR) and
NSERC. Funding was also received by R.R. and C.A. from the
Prostate Cancer Canada Movember Discovery Grant D2014-8.
(30) Dai, Y. Semin. Immunopathol. 2016, 38, 277−291.
(31) Liu, Z.; Wu, H.; Wei, Z.; Wang, X.; Shen, P.; Wang, S.; Wang,
A.; Chen, W.; Lu, Y. J. Cancer Res. Clin. Oncol. 2016, DOI: 10.1007/
s00432-015-2112-1.
(32) Ramachandran, R.; Hyun, E.; Zhao, L.; Lapointe, T. K.;
Chapman, K.; Hirota, C. L.; Ghosh, S.; McKemy, D. D.; Vergnolle, N.;
Beck, P. L.; Altier, C.; Hollenberg, M. D. Proc. Natl. Acad. Sci. U. S. A.
2013, 110, 7476−7481.
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