Tryptamine-Based Derivatives as Transient Receptor Potential Melastatin Type 8 (TRPM8) Channel Modulators

Article abstract of DOI:10.1021/acs.jmedchem.5b01914

Pharmacological modulation of the transient receptor potential melastatin type 8 (TRPM8) is currently under investigation as a new approach for the treatment of pain and other diseases. In this study, a series of N-substituted tryptamines was prepared to explore the structural requirements determining TRPM8 modulation. Using a fluorescence-based screening assay, we identified two compounds acting as an activator (2-(1H-indol-3-yl)-N-(4-phenoxybenzyl)ethanamine, 21) or an inhibitor (N,N-dibenzyl-2-(1H-indol-3-yl)ethanamine, 12) of calcium influx in HEK293 cells. In patch-clamp recordings, compound 21 displayed a significantly higher potency (EC₅₀ = 40 ± 4 μM) and a similar efficacy when compared to menthol; by contrast, compound 12 produced a concentration-dependent inhibition of menthol-induced TRPM8 currents (IC₅₀ = 367 ± 24 nM). Molecular modeling studies using a homology model of a single rat TRPM8 subunit identified a putative binding site located between the VSD and the TRP box, disclosing differences in the binding modes for the agonist and the antagonist.

Full text of DOI:10.1021/acs.jmedchem.5b01914

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Article
Tryptamine-based derivatives as Transient Receptor
Potential Melastatin type-8 (TRPM8) channel modulators
Alessia Bertamino, Carmine Ostacolo, Paolo Ambrosino, Simona Musella, Veronica
Di Sarno, Tania Ciaglia, Maria Virginia Soldovieri, Nunzio Iraci, Asia Fernandez-
Carvajal, Roberto de la Torre-Martinez, Antonio Ferrer-Montiel, Rosario Gonzalez-Muniz,
Ettore Novellino, Maurizio Taglialatela, Pietro Campiglia, and Isabel Gomez-Monterrey
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01914 • Publication Date (Web): 04 Feb 2016
Downloaded from http://pubs.acs.org on February 5, 2016
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Tryptamine-based derivatives as Transient Receptor Potential Melastatin
type-8 (TRPM8) channel modulators
a,§
b,§
c,§
b
a
Alessia Bertamino , Carmine Ostacolo , Paolo Ambrosino , Simona Musella , Veronica Di Sarno ,
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Tania Ciaglia , Maria Virginia Soldovieri , Nunzio Iraci , Asia Fernandez Carvajal , Roberto de la
TorreꢀMartinez , Antonio FerrerꢀMontiel , Rosario Gonzalez Muniz , Ettore Novellino , Maurizio
Taglialatela , Pietro Campiglia , Isabel GomezꢀMonterrey
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Department of Pharmacy, University of Salerno, Via G. Paolo II 132, 84084, Fisciano (SA), Italy
Department of Pharmacy, University Federico II of Naples, Via D. Montesano 49, 80131, Naples,
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Italy
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Department of Medicine and Health Science, University of Molise, Via de Sanctis, 86100,
Campobasso, Italy
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Institute of Molecular and Cellular Biology, University Miguel Hernández of Elche, 032020, Elche
(Alicante), Spain
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Institute of Medicinal Chemistry, IQMꢀCSIC, c/ Juan de la Cierva 3, 28006, Madrid, Spain
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Abstract
Pharmacological modulation of the transient receptor potential melastatin type 8 (TRPM8) is
currently under investigation as a new approach for the treatment of pain and other diseases. In this
study, a series of Nꢀsubstituted tryptamines was prepared to explore the structural requirements
determining TRPM8 modulation. Using a fluorescenceꢀbased screening assay, we identified two
compounds acting as an activator (2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀNꢀ(4ꢀphenoxybenzyl) ethanamine, 21) or an
inhibitor (N,Nꢀdibenzylꢀ2ꢀ(1Hꢀindolꢀ3ꢀyl) ethanamine, 12) of calcium influx in HEK293 cells. In
patchꢀclamp recordings, compound 21 displayed a significantly higher potency (EC = 40±4 µM) and
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a similar efficacy when compared to menthol; by contrast, compound 12 produced a concentrationꢀ
dependent inhibition of mentholꢀinduced TRPM8 currents (IC =367±24 nM). Molecular modelling
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studies using a homology model of a single rat TRPM8 subunit identified a putative binding site
located between the VSD and the TRP box, disclosing differences in the binding modes for the agonist
and the antagonist.
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Introduction
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Transient receptor potential melastatin typeꢀ8 (TRPM8) is a transmembrane, nonꢀselective
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Ca permeable cation channel, considered as the major sensor for peripheral innocuous cool and its
modulation contributes to a wide range of physiological and pathophysiological processes. One of the
most investigated effects produced by TRPM8 modulation is the analgesia against chronic and
neuropathic pain: in fact, it has been reported that peripheral and central activation of TRPM8 induces
analgesia, specifically reversing the sensitization of the behavioral reflexes elicited by peripheral
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nerve injury. Notably, both TRPM8 agonists and antagonists exert analgesic effects: in particular,
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while TRPM8 agonists produce profound analgesia at very low concentrations, even greater effects
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have been reported for TRPM8 antagonists. This analgesia modulation represents a novel approach
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in a largely unmet therapeutic need. Moreover, many studies have recently revealed novel potential
pharmacological implications for TRPM8 modulators. For example, TRPM8 agonists have been
proposed as useful diagnostic and therapeutic tools for the treatment of prostate cancer and benign
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prostate hyperplasia (BPH).
In fact the activation of TRPM8 receptors enhances apoptosis in
prostate carcinoma cells, highlighting a potential involvement of this channel in androgenꢀresponsive
prostate cancer cell growth. Apart from androgenꢀsensitive prostate cancer, TRPM8 is abnormally
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, 8
overꢀexpressed in other tumor malignancies, like breast cancer,
human pancreatic adenocarcinoma,
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and skin melanoma cells. Concurrently, TRPM8 blockers have been investigated for the treatment
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of overactive and painful bladder syndromes.
In particular, Nꢀ(3ꢀAminopropyl)ꢀ2ꢀ[(3ꢀ
methylphenyl)methoxy]ꢀNꢀ(2ꢀthienylmethyl) benzamide (AMTB), a wellꢀrecognized TRPM8
antagonist, can attenuate the bladder micturition reflex and the nociceptive reflex response in the rat,
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thus decreasing the frequency of volumeꢀinduced bladder contraction. Finally, TRPM8 modulators
could be of interest in treating dry eye syndrome and excessive lacrimation dysfunction, due to
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extensive TRPM8 expression in corneal afferent neurons.
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For these reasons, increasing efforts have been dedicated, both by academia and industry, to
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the design of TRPM8 ligands starting from different structural scaffolds.
Most of the compounds
described in the literature showed high potency on TRPM8 channels, although they also show
agonistic and/or antagonistic properties towards other TRP receptors, such as TRPA1 and TRPV1
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channels.
Recently, some indole alkaloids have been identified, resulting in potent antagonists
with a pronounced selectivity for TRPM8 when compared to TRPA1 or TRPV1 channels. Also 5ꢀ
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substituted tryptamines have been studied as potent TRPM8 blockers. In addition, in a previous
work, we demonstrated that the potency of TRPM8 ligands can be modulated varying substituents on
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the nitrogen group of the aminoisoxazole scaffold.
On the basis of these findings, we chose the tryptamine nucleus as template for the design of a
small library of TRPM8 modulators and synthesized a series of 18 Nꢀsubstituted tryptamine
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derivatives. This series, compared to the above cited tryptamineꢀbased modulators,
are modified
at different positions and have a reduced scaffold complexity than ibogaine alkaloids. These
compounds were tested as TRPM8 modulators by fluorescenceꢀ and electrophysiologyꢀbased (patchꢀ
clamp) assays. Pharmacological activity was rationalized by molecular modelling studies defining
basic structural requirements for TRPM8 agonism and antagonism. In addition, computational studies
allowed the identification of a putative binding site for tryptamineꢀbased TRPM8 modulators.
Results and Discussion
Chemistry
Derivatives 4-12 were synthesized according the experimental procedure showed in Scheme 1.
A subset of compounds (4-7, scheme 1) was prepared from the Nꢀ1 substituted intermediates 2 and 3.
Reaction of 3ꢀ(2ꢀbromoethyl)ꢀ1Hꢀindole (1) with methyl iodide or 4ꢀphenylbenzyl iodide in
DCM/DMF using NaH as a base, led to derivatives 2 and 3 with 67% and 61% yields, respectively.
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The 4ꢀphenylbenzyl iodide (3a, see supporting information, scheme S1), used in the formation of
intermediate 3, was previously prepared from the corresponding biphenylꢀ4ꢀcarboxylic acid as
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reported elsewhere.
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Scheme 1. Synthesis of the N, 2ꢀsubstituted indolethanamine derivatives 4-12
Nucleophilic displacement of the bromine atom of 1, 2 and 3 by different commercially available
amines was performed in THF/TEA using palladium acetate as a catalyst. Under microwave
conditions the final compounds 4-12 were obtained in 55ꢀ75% yield.
A different strategy was used for the synthesis of compounds 14ꢀ22, which employed
tryptamine 13 as starting material (Scheme 2). Compound 14 was obtained as racemic mixture by
nucleophilic substitution reaction of the tosyl derivative of the (1,2,3,4ꢀtetrahydronaphthalenꢀ4ꢀ
yl)methanol (6a) with tryptamine (13). This intermediate 6a has been synthesized by reducing the
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,2,3,4ꢀtetrahydronaphthaleneꢀ1ꢀcarboxylic acid with lithium aluminium hydride, followed by
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tosylation with 4ꢀmethylbenzeneꢀ1ꢀsulfonyl chloride (see supporting information, scheme S1).
Reductive amination of different commercially available aldehydes with the tryptamine, using
sodium triacetoxyborohydride as reductive agent gave the final compounds 15-21 in 51% ꢀ 85%
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yields (Scheme 2).
Finally, treatment of derivative 21 with methyl iodide using NaH as a base allowed us to
obtained the dimethyl derivative 22 in 37% yield.
Scheme 2. Synthesis of the N, 2ꢀsubstituted indolethanamine derivatives 14-22
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Screening of the activity of the synthesized compounds by Ca -imaging experiments
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To investigate the potential agonistic or antagonistic properties on TRPM8 channels, all
synthesized compounds were tested at three different concentrations (0.5, 5 and 50 ꢀM) by in vitro
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Ca fluorometric assays experiments using Fluo4ꢀNW in HEK293 cells stably expressing mouse
TRPM8 channels. The results obtained are normalized to the effects prompted by 100 ꢀM of the
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canonical agonist menthol or by 10 ꢀM of the TRPM8 antagonist AMTB + 100 ꢀM menthol,
whose effects were reported as + 100% (activation) or ꢀ100% (inhibition), respectively (Figures 1A and
B).
1
As reported in Figure 1A, compounds 4 and 5 showed agonist activity at high concentration (50
ꢀM), being about 2ꢀfold more effective than menthol. At the same concentration, the presence of the
sterically hindered biphenyl ethyl group on the amine moiety (6), led to slight loss of efficacy in
comparison to 4 and 5, maintaining an efficacy similar to menthol. Interestingly, introduction of a
sterically hindered group at Nꢀ1 indole position, such as a biphenyl methyl group, together with
increased flexibility of the substituent on the amine moiety (7 versus 4 or 5), maintained agonistic
efficacy.
The nonꢀsubstituted Nꢀ1 indole derivatives (8-21) were quite productive. A direct linkage of the
amine moiety with different bulky aromatic groups, such as naphthalene and quinolines, gives
compounds 8ꢀ11 unable to act as TRPM8 modulators (Figures 1A and 1B). By contrast, the tertiary
amine obtained by derivatization of tryptamine with a benzyl moiety (12) proved to be an effective and
potent antagonist. Introduction of a methylene spacer between the amine group and the bulky aryl
moiety in 8 led to the potent agonist 15, which showed to be 3ꢀfold more effective than menthol at 50
ꢀ
M. Introduction of substituents at position 2 of the naphthalene ring, such as OMe (16) and OH (17)
induced a shift from agonist to antagonistic activity for these derivatives at high concentration (Fig. 1
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B). A similar effect was observed when the bicyclic system modification involved changes in the
electronic nature of the ring (14) or an increase in ring size (18). In addition, tryptamine benzyl
derivatives 19 and 20 lost the agonistic activity shown by the methylated analogue 4.
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Notably, compound 21 bearing a pꢀmethoxybenzyl group showed significant agonist activity at
0.5 ꢀM, with an efficacy similar to that of menthol. At 50 ꢀM, the efficacy of derivative 21 was nearly
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indolꢀ3ꢀyl)ꢀderivative (22) shifted the agonistic activity to antagonism, with an efficacy comparable to
that of derivative 12, at all tested concentrations (Figure 1B).
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Figure 1: Efficacy of synthesized compounds as TRPM8 agonists (panel A) or antagonists (panel B) at three different
increasing concentrations (0.5 µM, 5 µM, and 50 µM). Values are expressed as mean±SEM of 3 independent
measurements.
To investigate the ability of newlyꢀdescribed TRPM8 modulators to interfere with the activity
of other TRP channels, particularly TRPV1 and TRPA1, two TRPM8 antagonist (12, 22) and one
agonist (21), were also tested in calcium fluorometric experiments in SHꢀSY5Y cells stably expressing
mouse TRPV1 channels and in IMR90 cells natively expressing mouse TRPA1 channels. In these
experiments, the canonical TRPV1 agonist (capsaicin, 10 ꢀM) or antagonist (ruthenium red, 10 ꢀM) as
well as the canonical TRPA1 agonist (AITC, 500 ꢀM) or antagonist (ruthenium red, 10 ꢀM) were used
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as controls. The values obtained for the tested compounds were normalized to the effects exerted by
canonical agonists or agonist + antagonist coꢀexposure.
As reported in Figure 2, compound 12 appeared a rather selective TRPM8 antagonist, showing
weak activity on TRPV1 only at the highest concentration (Figure 2A), and unremarkable activity on
TRPA1 at all concentrations (Figure 2B). Interestingly, the TRPM8 agonist 21 behaved instead as an
antagonist on both TRPV1 and TRPA1 receptors in study, showing in both, full antagonist ability at the
highest concentrations (50 ꢀM; Figs. 2A and 2B). Compound 22 appeared less selective, acting as an
antagonist of both TRPV1 (Fig. 2A) and TRPA1 (Fig. 2B) channels with potency comparable to that on
TRPM8 at all concentrations, and was therefore not further investigated .
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Figure 2: Efficacy of synthesized compounds at three different increasing concentrations (0.5 µM, 5 µM and 50 µM) as
modulators of TRPV1 (vs capsaicin and capsaicin + ruthenium red, panel A) and TRPA1 (vs AITC and AITC + ruthenium
red, panel B). Value are expressed as mean±SEM of 3 indipendent measurements.
Pharmacological properties of derivatives 21 and 12 characterized by patch-clamp
electrophysiology
The previously described multicellular functional assay identified derivatives 21 and 12 as the
most effective and potent agonist and antagonist compounds, respectively, on TRPM8 channels. To
provide direct evidence for this activity, derivatives 21 and 12 were tested in single HEKꢀ293 cells
transiently expressing the rat TRPM8 isoform by patchꢀclamp recordings using a 100ꢀmsec voltage
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ramp ranging from ꢀ100 mV to +100 mV delivered every 4 sec. Perfusion with 500 µM menthol
activated a large, strongly outwardlyꢀrectifying (I+80 mV/Iꢀ80 mV was 90±25; n=39) reversing at a
potential near 0 mV (ꢀ1.3±0.7 mV; n=10) (Figures 3A and 3B). No currents were activated when
menthol was perfused in nonꢀtransfected cells (0 pA/pF at +80 mV; n=4; data not shown). The doseꢀ
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response curve for mentholꢀevoked currents (Figure 3C) at +80 mV showed an EC of 75±4 µM, a
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value close to those reported for rat and mouse isoforms.
Perfusion of derivative 21 (100 µM) in rTRPM8ꢀtransfected HEKꢀ293 cells also triggered the
appearance of large outwardly rectifying currents, reversing around 0 mV (ꢀ1.1±0.3 mV; n=10; Figures
3
D and 3E). By contrast, perfusion of derivative 12 (100 µM) failed to activate TRPM8 current (data
not shown). When compared to menthol, derivative 21 displayed a significantly higher potency (EC50
was 40±4 µM; n =3ꢀ10) and a similar efficacy (current density at +80 mV was 134±13 and 140±7 for
derivative 21 and menthol, respectively; p > 0.05) (Figure 3F). Notably, the presence of the 1ꢀmethylꢀ
4
ꢀphenoxybenzene group in this derivative (Scheme 2) appears to be crucial for TRPM8 activation, as
the derivative 11 which carries an isoquinoline group showed no agonist activity (0 pA/pF at +80 mV;
n=4; data not shown), as also suggested by the fluorescence assay (Figure 1A).
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Figure 3: Mentholꢀ and derivative 21ꢀinduced currents in HEK293 cells expressing TRPM8. A, D) Representative traces of
currents evoked by a 100ꢀms voltage ramp ranging from ꢀ100 mV to +100 mV applied every 4 sec, in control solution
(
(
CTL; black trace; A,D), during application of menthol (green trace; A) or derivative 21 (gray trace; D) or after washout
W; red trace; A,D). B, E) Timeꢀcourse of currents recorded at +80 mV (blue symbols) or ꢀ80 mV (red symbols) in single
HEK293 cells expressing TRPM8 upon exposure to menthol (500 µM; B) or derivative 21 (100 µM; E). The duration of
compounds exposure is indicated by the bar on top of the traces. C, F) Concentration–response curves for TRPM8 current
activation by menthol (C) or derivative 21 (F). Peak current data were expressed as pA/pF (to facilitate comparison among
cells of different sizes), and expressed as a function of agonist concentrations. The solid lines represent fits of the
n
experimental data to the following binding isotherm: y = max/(1 + x/EC ) , where x is the drug concentration and n the Hill
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coefficient. The fitted values for n were 0.97 ± 0.05 or 3.0 ± 0.6 for menthol or derivative 21, respectively. Each point is the
mean ± SEM of 4–13 (for menthol) or 3ꢀ10 (for derivative 21) determinations, each obtained in different cells.
To investigate possible antagonistic effects exerted by the newlyꢀsynthesized compounds, we
investigated their ability to inhibit mentholꢀevoked responses. As previously reported, the canonical
TRPM8 antagonist BCTC (3 µM) produced a fast and complete inhibition of mentholꢀgated TRPM8
33
currents, which largely recovered after drug washout (Figures 4A and 4B). Similarly, perfusion with
the same concentration (3 µM) of derivative 12, produced a complete inhibition of mentholꢀinduced
TRPM8 currents at +80 mV (Figures. 4C and 4D), with slower kinetics when compared to those of
BCTC. TRPM8 block by derivative 12 appeared concentrationꢀdependent, with an IC of 367±24 nM
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(
n=3ꢀ9;), a value slightly lower than that reported for BCTC (475 nM) . These effects were largely
reversible upon drug washout (Figure 3C), although with much slower kinetics when compared to
those of BCTC.
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Figure 4: BCTC and derivative 12 block TRPM8ꢀmediated responses evoked by menthol in TRPM8ꢀexpressing HEK293
cells. A, C) Timeꢀcourse of currents recorded at +80 mV or ꢀ80 mV in HEK293 cells expressing TRPM8 upon application
of 500 µM menthol (A,C), 500 µM menthol + 3 µM BCTC (A) or 500 µM menthol + 3 µM derivative 12 (C). B, D) I–V
curves obtained in HEK293 cells expressing TRPM8 and exposed to control solution (CTL; black trace; B,D), 500 µM
menthol (green trace; B,D), 500 µM menthol + 3 µM BCTC (red trace; B) or to 500 µM menthol + 3 µM derivative 12 (red
trace; D). Traces shown in B, D correspond to the currents measured at the time points shown in colors (green or red circles)
in A, C.
Molecular modeling and structural rationale
In a recent paper, Taberner et al. reported the central role of S6ꢀTRP box linker (amino acids
3
4
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80–992 in rat TRPM8) in the gating mechanism of TRPM8 channel. As shown by their homology
models based on the experimentally solved structures of TRPV1 (PDB ID: 3J5P), the conformational
changes in the voltage sensors by activating stimuli could be associated to a widening of the S6 Cꢀend,
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which in turn might disrupts the intersubunit interactions in the region of Tyr981, thus favoring channel
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opening.
To investigate the binding characteristics of newlyꢀsynthesized TRPM8 modulators with
particularly attention to the difference between agonist and antagonist binding which might be
indicative of the residues involved in TRPM8 gating, we ran a series of molecular dynamic simulations
using a homology model of a rat TRPM8 subunit. Notably, human, rat, and mouse TRPM8 amino acid
sequences are highly similar within the modeled regions; the percent of overall identity/similarity is:
93.8%/97.4% (human versus mouse), 93.8%/97.3% (human versus rat), and 98.6%/99.5% (mouse
versus rat). More importantly, some of the critical residues involved in drug binding (namely Leu697,
Ile710, Tyr754, Leu843, Glu1004, and Arg1008; see below) are strictly conserved among the three
species. Overall, these data strongly suggest a high level of structural similarity in the binding sites for
the described modulators among TRPM8 receptors belonging to humans, rats, and mice. We ran
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SiteMap calculations on the whole monomer to detect potential binding sites. The best site identified
(BP1 – Figure S1), has a volume of 791 Å and a site score of 1.093 (the cutoff value generally used to
distinguish between drugꢀbinding and nonꢀdrugꢀbinding sites is 0.80), and is located between S1, S2,
S3, and S4 helices, while at the bottom is delimited by residues 990ꢀ1010. The proximity of the binding
site to the 980ꢀ992 region suggests that the binding of a molecule to BP1 might influence the
conformation of this linker region in the TRP domain pivotal for coupling stimuli sensing to pore
opening. We ran automated molecular docking simulations to predict the binding mode into BP1 of the
most potent antagonist (12) and agonist (21) in our series. The bestꢀdocked models were then submitted
to 12 ns long molecular dynamics simulations to analyze proteinꢀligand contacts and protein
conformational rearrangements at the level of BP1 and of the 980ꢀ992 region.
BP1ꢀ 12 and BP1ꢀ 21 complexes are stabilized by similar interactions, such as hydrophobic
contacts with Leu843, Ile701, Leu697, πꢀπ stacking with Tyr754 and electrostatic interactions with the
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Glu1004 side chain. A part from these similar interactions, the antagonist and the agonist also show
different interactions that could be responsible for different conformational rearrangements of the 980ꢀ
992 region. In particular, 21 makes a hydrogen bond with the phenoxyl group of Tyr754 by its aminoꢀ
linker and a second hydrogen bond, with the backbone of Arg1008, provided by the indole NH (see
figure S2A). The different interaction of 12 or 21 to TRPM8 possibly brings to differences in the
conformation of BP1, regarding in particular Arg1008. In the closed model of TRPM8 (APO),
Arg1008 is projected into BP1, stabilized by hydrogen bonds with Glu1004 and Tyr754 (Figure 5A).
Upon 21 binding this arginine side chain appears projected outside of BP1 (Figure 5B), with the ligand
indole ring claiming its space and hydrogen binding to Arg1008 backbone. Compound 21 makes
further hydrogen bonds with the side chain carboxyl of Glu1004, which is shifted away from the
position it has in the APO closed model, and with the hydroxyl group of Tyr754. Therefore, the binding
of 21 caused an increase in the Arg1008ꢀGlu1004 distance when compared to that measured in the
APO TRPM8 configuration (Figure 5D). This conformational rearrangement at the bottom of BP1
prompts an evident shift of the TRP domain helix, that makes the conformation of the 980ꢀ992 region
looser and prone to rearrange as described below. In the case of 12 binding, Arg1008 is instead
pointing again toward the BP1 cavity, keeping its interaction with Glu1004 as in the APO form
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(Figures 5CꢀD).
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Figure 5: Arg1008 hydrogen bond network (in yellow dashes) in Apo TRPM8 (A) and its changes upon ligand binding: (B)
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1 bound conformation of TRPM8 is depicted in cyan, ligand in yellow sticks and apo TRPM8 is depicted in green as
reference. (C) 12 bound conformation of TRPM8 is depicted in green, 12 in yellow sticks and 21 bound TRPM8 is depicted
in cyan as reference. Arg1008ꢀGlu1004 distances (D) and 980ꢀ992 region RMSDs (E) recorded during 12ns long MD
trajectories
.
Moreover, compound 12 establishes a wider hydrogenꢀbonding network, interacting even with
Gln1003 and Glu662, possibly stabilizing the TRP domain helix which might be responsible for its
antagonistic nature. Indeed, looking at the RMSD of the 980ꢀ992 region as a function of time and of the
bound ligand (Figure 5E), it is evident that the conformation of TRPM8/12 is much closer to the APO
TRPM8 than TRPM8/21. This behavior indicates that the binding of 21 makes the 980ꢀ992 region
prone to conformational rearrangement events.
We used the predicted bioactive conformations of 12 and 21 to summarize the most stable interactions
involved in the binding of these ligands as representative antagonists and agonists of this tryptamineꢀ
based series (Figure 6). The pharmacophore model for tryptamineꢀbased agonists accounts for the
interaction with Ile837, Leu806, Ile701 (H1), Leu697, Leu843, Ile844 (H2), Glu1004 (D2/P1),
Arg1008 (D3), Tyr754 (D1/R1), and Leu757 and Met758 (R1) (Figure 6A).
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Figure 6. Pharmacophore models for tryptamineꢀbased TRPM8 agonists (A) and antagonists (B). Chemical features are
depicted as spheres and colorꢀcoded as follows: yellow, H = hydrophobic, blue, P = positive charge, green, D = HB donor,
orange, R = aromatic ring. 21 and 12 are represented as references in panels A and B, respectively.
On the other hand, the antagonist model accounts for the interaction with Ile701, Leu751, Leu697,
Leu843 (R1), Glu1004 (P1/D1), Val800 (H1), Arg1008 (R2) and Tyr754 (R1/R2) (Figure 6B). The two
pharmacophore models might help to understand which chemical features are required to endow a
ligand with agonist or antagonist capabilities. Despite binding to the same site, the two models present
in fact substantial differences that may cause the observed differences induced in TRPM8
conformation. All the agonists reported in this paper fit, at least in part, the agonist model. All of them
in fact place correctly D1/P1, D2 (compounds 4, 5, 6, 7, 15, and 21) and H1 features (21). R1 location
is always occupied by a large substituent claiming the space of Arg1008 side chain and in same cases
this moiety engages in HB with the Arg1008 backbone. By contrast R2 antagonist feature has to be,
when present, a small substituent that does not occupy the Arg1008 room. H1 is probably the feature
that boosts the activity of 21, since it is the only one that perfectly fits it. Regarding the antagonists
model, H1, P1 and D1 seem to be the mandatory feature for exerting antagonism (compounds 12 and
both conformers of 22), while R1 could boost activity by πꢀπ interaction with Tyr754, and a
relatively small R2 substituent could be engaged, by its π cloud, in interaction with Tyr754 and the
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positively charged Arg1008 side chain, improving its antagonist capabilities. Looking at the fittings of
the tryptamineꢀbased agonists and antagonists sets, it seems that the mandatory features to exert
agonism are H2, P1, D1 and D2, while on the other side H1, P1 and D1 are necessary for antagonism
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(see Table S1).
Conclusions
Here we report the synthesis and pharmacological evaluation of a small library based on the tryptamine
scaffold designed as TRPM8 ion channel modulators. Despite the limited number of compounds
investigated, two of the synthesized derivatives, compounds 12 and 21, showed to be efficient and
potent TRPM8 antagonist and agonist, respectively. In fact, 12 produced a complete inhibition of
mentholꢀinduced TRPM8 currents with a potency higher than that reported for the wellꢀknown TRPM8
antagonist BCTC. Meanwhile the agonist 21 showed a potency and efficacy significantly higher than
menthol. In addition, compound 12 showed unremarkable activity on TRPV1 and TRPA1 at different
concentrations while the agonist 21 behaved as an antagonist on both TRPV1 and TRPA1 receptors at
high concentration. Computational studies allowed us to hypothesize a binding site and two
pharmacophore prototypes for this series of tryptamineꢀbased TRPM8 modulators. The proposed
structural model identifies the same binding site for agonist and antagonist, but two binding modes
differing in the “interaction network”. Since structural models have to be used with caution, further
investigations are needed to confirm the structural hypothesis herein proposed. The results of this study
provide valuable information for the future design of new and specific TRPM8 modulators that could
be useful for the treatment of different diseases involving a deregulation of this ion channel.
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Experimental section
General. Reagents, starting materials, and solvents were purchased from SigmaꢀAldrich (Milan, Italy)
and used as received. Reactions were carried out with magnetic stirring in roundꢀbottomed flasks
unless otherwise noted. Moistureꢀsensitive reactions were conducted in ovenꢀdried glassware under a
positive pressure of dry nitrogen, using preꢀdried, freshly distilled solvents. Microwave assisted
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reactions were performed in a Biotage Initiator reactor. Analytical thin layer chromatography (TLC)
was performed on preꢀcoated glass silica gel plates 60 (F254, 0.25 mm, VWR International).
Purifications were performed by flash column chromatography on silica gel (230ꢀ400 mesh, Merck
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Millipore). NMR spectra were recorded on Varian Mercuryꢀ400 apparatus. HNMR and C NMR
spectra were recorded with a Varianꢀ400 spectrometer, operating at 400 and 100 MHz, respectively.
Chemical shifts are reported in δ values (ppm) relative to internal Me Si, and J values are reported in
4
hertz (Hz). The following abbreviations are used to describe peaks: s (singlet), d (doublet), dd (double
double), t (triplet), q (quadruplet) and m (multiplet). ESIꢀMS experiments were performed on an
Applied Biosystem API 2000 tripleꢀquadrupole spectrometer. Combustion microanalyses were
performed on a Carlo Erba CNH 1106 analyzer, and were within 0.4% of calculated values and
confirmed >95% purity for the final products. Analytical RPꢀHPLC was performed on a Phenomenex
Synergi Fusion RPꢀ80A (75 x 4.6 mm, 4 ꢀM), with a flow rate of 1 ml/min, using a tunable UV
detector at 254 nm. Mixtures of CH CN and 0.05% TFA in H O were used as mobile phase.
3
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General procedure for the synthesis of derivatives 2 and 3.
ꢀ(2ꢀBromoethyl)indole (1, 1.0 eq) was dissolved in a mixture of anhydrous DCM/DMF (2/1 v/v)
3
under magnetic stirring and the temperature was set to 0°C. To this solution, 1.5 equivalents of NaH
were added portionwise and the mixture was allowed to react for 30 minutes. Then, 1.5 equivalents of
alkyl iodide [methyl iodide or 4ꢀ[phenyl]iodomethylbenzene (3a)] in DCM were added dropwise and
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the reaction was warmed to room temperature and maintained under stirring for further 12h. Then,
reaction was quenched by 10% aqueous solution of citric acid and washed with brine. Organic layer
was separated, dried over anhydrous Na SO , filtered and evaporated in vacuo. Crude products were
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purified by column chromatography using nꢀhexane/ethyl acetate (4:1 v:v) as mobile phase.
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-(2-Bromoethyl)-1-methyl-1H-indole (2).
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Yield 67%. H NMR (400 MHz,CDCl ) δ 3.39ꢀ3.46 (m, 4H, CH ); 3.79 (s, 3H, CH ); 6.97 (s, 1H);
3
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.20 (t, 1H, J = 7.6 Hz); 7.28 (d, 1H, J = 8.0 Hz); 7.34 (t, 1H, J = 8.0 Hz); 7.62 (d, 1H, J = 8.0 Hz).
+
ESIMS m/z calcd for C H BrN, 237.02; found 238.11 (M+H )
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Synthesis of 1-(Iodomethyl)-4-phenylbenzene intermediate (3a).
To one equivalent of biphenylꢀ4ꢀcarboxylic acid (1a) dissolved in dry THF was added portionwise
LiAlH (3.0 eq), under stirring at room temperature (Scheme 2). After 2 hours the solution was washed
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three times with HCl 2N and then water and the organic layer was dried over anhydrous Na SO ,
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filtered and concentrated. The resultant 4ꢀbiphenylmethanol (2a) was purified by flash chromatography
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using ethyl acetate as solvent and was obtained as oil in 89% yield. H NMR (400 MHz,CDCl ) δ 2.98
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bs, 1H, OH); 4.70 (s, 2H, CH ); 7.33ꢀ7.43 (m, 5H, aryl); 7.59ꢀ7.61 (m, 4H, Aryl).
2
Then, treatment of 2a with triphenylphosphine and I , in dry DCM, according to the procedure
2
2
7
1
previously described, gave the title compound in 65% yield. H NMR (400 MHz,CDCl ) δ 4.53 (s,
3
2
H, CH ); 7.15 (d, 2H, J = 8.0 Hz); 7.24 (d, 2H, J = 8.0 Hz); 7.43ꢀ7.55 (m, 5H).
2
1-(Biphenyl-4-ylmethyl)-3-(2-bromoethyl)-1H-indole (3).
Prepared from intermediate 3a and 3ꢀ(2ꢀbromoethyl)indole as described above. Yield 61%. H NMR
400 MHz,CD OD) δ 3.27 (t, 1H, J = 7.4 Hz, CH ); 3.38ꢀ3.46 (m, 2H, CH ); 3.81 (t, 1H, J = 7.6 Hz,
1
(
3
2
2
CH ); 5.35 (s, 2H, CH ); 7.07 (s, 1H); 7.14ꢀ7.61 (m, 13H). ESIMS m/z calcd for C H BrN, 391.08;
2
2
23 20
+
found 392.13 (M+H ).
General procedure for the synthesis of derivatives 4-12.
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One equivalent of 3ꢀ(2ꢀbromoethyl)indole (1) or intermediate 2 or 3 was dissolved in THF and 1.5 eq
of the proper amine, 1.5 eq of TEA, 1.5 eq of NaI and 0.3 eq of (CH COO) Pd were added to this
3
2
solution (Scheme I). The reaction was conducted under ꢀW, at 100°C, for 20 minutes. The resulting
mixture was filtered through Celite, dried in vacuo and reconstituted in DCM. The organic phase was
washed with water (3 x 50 mL), dried over anhydrous Na SO , filtered, concentrated and purified by
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column chromatography using DCM/MeOH as mobile phase.
N-(4-Methoxybenzyl)-2-(1-methyl-1H-indol-3-yl)ethanamine (4)
1
Synthesized starting from 3 and 4ꢀmethoxybenzylamine, yield 71%. H NMR (400 MHz,CDCl ) δ 2.92
3
(
t, 2H, J = 7.6 Hz, CH ); 3.19 (t, 2H, J = 7.6 Hz, CH ); 3.49 (s, 6H, 2CH ); 3.72 (s, 2H, CH ); 6.81 (s,
2 2 3 2
13
1
H); 7.07ꢀ7.15 (m, 3H); 7.20 (t, 1H, J = 6.8 Hz); 7.24ꢀ7.30 (m, 3H); 7.52 (d, 1H, J = 8.0 Hz). C NMR
(
100 MHz, CDCl ) δ 22.7, 41.6, 50.8, 57.1, 109.1, 110.0, 112.4, 118.6, 118.8, 121.4, 121.9, 124.2,
3
+
1
26.4, 128.0, 128.3, 135.3, 199.8. ESIMS m/z calcd for C H N O, 294.17; found 295.20 (M+H ).
1
9
22
2
N-(4-Chlorobenzyl)-2-(1-methyl-1H-indol-3-yl)ethanamine (5)
1
Synthesized starting from 3 and 4ꢀchlorobenzylamine, yield 69%. H NMR (400 MHz,CDCl ) δ 2.93ꢀ
3
3
.00 (m, 4H, 2CH ); 3.74 (s, 3H, CH ); 3.77 (s, 2H, CH ); 6.87 (s, 1H); 7.10 (t, 1H, J = 8.0 Hz); 7.20ꢀ
2 3 2
1
3
7.30 (m, 6H); 7.58 (d, 1H, J = 8.0 Hz). C NMR (100 MHz, CDCl ) δ 25.57, 32.56, 49.40, 53.06,
3
1
09.2, 110.0, 112.3, 118.7, 118.9, 121.6, 126.7, 127.8, 128.4, 129.4, 132.5, 137.1, 138.7. ESIMS m/z
+
calcd for C H ClN , 298.12; found 299.17 (M+H ).
1
8
19
2
2-([1,1'-Biphenyl]-4-yl)-N-(2-(1-methyl-1H-indol-3-yl)ethyl)ethanamine (6).
1
Synthesized starting from 3 and 2ꢀ(4ꢀbiphenyl)ethylamine, yield 65%. H NMR (400 MHz,CD OD) δ
3
3
7
.03 (t, 2H, J = 8.8 Hz, CH ); 3.17 (t, 2H, J = 8.6 Hz, CH ); 3.32ꢀ3.37 (m, 4H, CH ); 3.76 (s, 3H, CH );
2 2 2 3
13
.06ꢀ7.11 (m, 2H); 7.20 (t, 1H, J = 7.2 Hz); 7.31ꢀ7.44 (m, 6H); 7.56ꢀ7.60 (m, 5H). C NMR (100
MHz, CD OD) δ 22.0, 31.7, 48.4, 109.4, 118.0, 119.0, 121.8, 126.7, 127.3, 127.5, 128.7, 129.1, 135.5.
3
+
ESIMS m/z calcd for C H N , 354.21; found 355.26 (M+H ).
2
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-(1-([1,1'-Biphenyl]-4-ylmethyl)-1H-indol-3-yl)-N-(3,4-dimethoxyphenethyl)ethanamine
(7).
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1
Synthesized starting from 3 and 3,4ꢀdimethoxyphenthylamine, yield 75%. H NMR (400
MHz,CD OD) δ 2.91 (t, 2H, J = 7.2 Hz, CH ); 3.17ꢀ3.26 (m, 4H, CH ); 3.34 (t, 2H, J = 7.4 Hz, CH );
3
2
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0
3
7
.77 (s, 3H, OCH ); 3.79 (s, 3H, OCH ); 5.39 (s, 2H, CH ); 6.83ꢀ6.87 (m, 2H); 7.09 (t, 1H, J = 8.0 Hz);
3 3 2
1
3
.11ꢀ7.24 (m, 5H); 7.37ꢀ7.41 (m, 4H); 7.50ꢀ7.55 (m, 4H); 7.62 (d, 1H, J = 8.0 Hz). C NMR (100
MHz, CD OD) δ 22.1, 31.7, 48.8, 49.2, 55.2, 109.2, 110.1, 112.1, 112.3, 118.3, 119.3, 120.9, 112.0,
3
1
26.7, 126.9, 127.0, 127.2, 127.4, 127.7, 128.7, 129.0, 137.3, 140.7, 148.6 149.6. ESIMS m/z calcd for
+
C H N O , 490.26; found 491.31 (M+H ).
33
34
2
2
N-[2-(1H-Indol-3-yl)ethyl]naphthalen-1-amine (8).
Synthesized starting from 1 and 1ꢀnaphtalenlamine, yield 55%. H NMR (400 MHz, CDCl ) δ 3.26 (t,
1
3
2
H, J = 8.0 Hz, CH ); 3.64 (t, 2H, J = 8.2 Hz, CH ); 6.70 (d, 1H, J=7.8 Hz); 7.06 (s, 1H); 7.20 (t, 1H,
2 2
J=7.2 Hz); 7.25ꢀ7.27 (m, 2H); 7.37ꢀ7.43 (m, 4H); 7.66 (d, 1H, J= 7.6 Hz); 7.72 (d, 1H, J=8.0 Hz); 7.80
1
3
(d, 1H, J=8.2 Hz); 7.98 (s, 1H, NH). C NMR (100 MHz, CDCl ) δ 25.1; 44.3; 104.7, 111.5; 113.6;
3
1
17.5, 119.0, 119.8, 120.1, 122.3; 122.5; 123.7; 124.8, 125.9, 126.9; 127.6; 128.9; 133.4; 136.7, 143.7.
+
ESIꢀMS m/z calcd for C H N , 286.15; found 287.20 (M+H ).
2
0
18
2
N-[2-(1H-Indol-3-yl)ethyl]naphthalen-2-amine (9).
1
Synthesized starting from 1 and 2ꢀnaphtalenlamine, yield 58%. H NMR (400 MHz, CDCl ) δ 3.16 (t,
3
2H, J = 7.8 Hz CH ); 3.58 (t, 2H, J = 8.0 Hz, CH ); 6.81ꢀ6.85 (m, 2H); 7.08 (s, 1H); 7.12ꢀ7.24 (m, 4H);
2
2
1
3
7
.33ꢀ7.40 (m, 3H); 7.59 (d, 1H, J=8.4 Hz); 7.64 (d, 1H, J=8.2 Hz); 8.07 (s, 1H, NH). C NMR (100
MHz, CDCl ) δ 25.1; 44.1; 104.8, 111.4, 118.4, 119.0, 119.7 122.1, 122.3; 122.4; 123.0; 126.1; 126.5;
3
126.8; 127.4; 127.8; 129.1; 133.7; 136.5; 146.0. ESIꢀMS m/z calcd for C H N , 286.15; found 287.17
2
0
18
2
+
(
M+H ).
2
-(1H-Indol-3-yl)-N-(quinolin-5-yl-methyl)ethanamine (10).
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Synthesized starting from 1 and 5ꢀaminoquinoline, yield 63%. H NMR (400 MHz, CDCl ) δ 3.26 (t,
3
2
1
H, J = 8.0 Hz, CH ); 3.62 (t, 2H, J = 8.2 Hz, CH ); 6.82 (d, 1H, J=7.6 Hz); 7.12ꢀ7.48 (m, 7H); 7.69 (d,
2 2
1
3
H, J=7.8 Hz); 8.15 (s, 1H, NH); 8.37 (d, 1H, J=4.0 Hz); 9.13 (d, 1H, J=4.0 Hz). C NMR (100 MHz,
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CDCl ) δ 24.9; 44.1; 107.9; 111.6; 113.0; 113.6, 116.2; 118.9; 119.4; 119.8, 122.3; 122.6; 127.4; 128.4;
3
+
136.5; 138.7; 141.9; 146.6, 153.0. ESIꢀMS m/z calcd for C H N , 287.14; found 288.26 (M+H ).
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9
17
3
2
-(1H-Indol-3-yl)-N-(isoquinolin-5-ylmethyl)ethanamine (11).
1
Synthesized starting from 1 and 5ꢀaminoisoquinoline, yield 67%. H NMR (400 MHz, CDCl ) δ 3.23
3
(
t, 2H, J = 6.4 Hz, CH ); 3.61 (t, 2H, J = 6.2 Hz, CH ); 7.82 (d, 1H, J=8.0 Hz); 7.15ꢀ7.54 (m, 7H); 7.71
2 2
13
(
d, 1H, J=8.0 Hz); 8.11 (s, 1H, NH); 8.39 (d, 1H, J=7.2 Hz); 9.17 (s,1H). C NMR (100 MHz, CDCl )
3
δ 25.9; 49.9; 111.4; 114.9; 116.9, 119.0, 119.5, 122.3, 122.4, 127.0; 127.3; 127.5; 127.7; 130.1; 134.7;
+
1
34.9; 135.4; 136.6; 143.3; 153.2. ESIMS m/z calcd for C H N 287.14; found 288.24 (M+H ).
1
9
17
3
N,N-Dibenzyl-2-(1H-indol-3-yl)ethanamine (12).
1
Synthesized starting from 1 and dibenzylamine, yield 73%. H NMR (400 MHz, CDCl ) δ 2.84 (t, 2H,
3
J = 5.6 Hz, CH ); 3.01 (t, 2H, J = 6.0 Hz, CH ); 3.73 (s, 4H, 2CH ); 6.90 (s, 1H); 7.06 (t, 1H, J = 8.2
2
2
2
Hz, aryl); 7.18 (t, 1H, J = 8.0 Hz); 7.26 (t, 2H, J = 8.4 Hz); 7.32ꢀ7.35 (m, 5H); 7.40ꢀ7.43 (m, 5H); 7.85
1
3
(
s, 1H, NH). C NMR (100 MHz, CDCl ) δ 23.3; 54.1; 58.5; 111.2; 114.8; 119.1, 119.3, 121.6, 122.0
3
1
27.0; 127.8; 128.4; 129.0; 136.4; 140.1. ESIꢀMS m/z calcd for C H N , 340.19 found 341.27
24 24 2
+
(M+H ).
Synthesis of N-[(1,2,3,4,Tetrahydronaphthalen-1-yl)methyl]-2-(1H-indol-3-yl)ethanamine (14).
1,2,3,4-Tetrahydronaphthalen-1-yl)methyl 4-methylbenzenesulfonate (6a)
(
(1,2,3,4ꢀTetrahydronaphthalenꢀ1ꢀyl)methanol (5a) was synthesized by reduction of 1,2,3,4ꢀ
tetrahydronaphthalen carboxylic acid (4a) following the procedure described for intermediate 2a. Yield
1
9
5% H NMR (400 MHz,CDCl ) δ 1.80ꢀ1.89 (m, 2H, CH ); 2.02ꢀ2.11 (m, 2H, CH ); 2.79ꢀ2.90 (m, 3H,
3
2
2
CH and CH); 3.90ꢀ3.96 (m, 2H, CH O); 7.12ꢀ7.23 (m, 4H).
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Then, to a solution of 5a in DCM was added ClꢀTos (2.0 eq), TEA (2.0 eq), and DMAP (0.7 eq). After
hour at room temperature, the solution was washed with brine. The organic layer was dried over
anhydrous Na SO , filtered, concentrated and purified with flash chromatography in nꢀExane/Ethyl
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1
acetate 30:20. The title compound 6a was obtained in 61% yield. H NMR (400 MHz, CDCl ) δ 1.67ꢀ
3
1
8
.73 (m, 2H, CH ); 1.85ꢀ1.91 (m, 2H, CH ); 2.48 (s, 3H, CH ); 2.68ꢀ2.74 (m, 2H, CH ); 3.18 (t, 1H, J =
2 2 3 2
.0 Hz, CH); 4.07 (t, 1H, J = 7.8 Hz, CH O); 4.21ꢀ4.26 (m, 1H, CH O); 7.05ꢀ7.13 (m, 4H); 7.36 (d, 2H,
2
2
J = 8.0 Hz, tosyl); 7.86 (d, 2H, J = 8.0 Hz, tosyl).
N-[(1,2,3,4,Tetrahydronaphthalen-1-yl)methyl]-2-(1H-indol-3-yl)ethanamine (14).
A 10 mL sealable flask, equipped with a stir bar, was charged with 1 eq of tosylate 6a, and 2.6 eq of
tryptamine (13, Scheme 3), dissolved in THF. The flask was sealed, heated to 60–70°C, and allowed to
stir at that temperature overnight. The resulting slurry was cooled to room temperature, added with
DCM and washed with brine. The organic layer solvents was dried over Na SO , filtered and
2
4
evaporated under vacuum. The resulting mixture was separated by silica gel chromatography using
1
DCM/MeOH 9/1 as eluent. Yield 69%. H NMR (400 MHz, CDCl ) δ 1.68ꢀ1.71 (m, 2H, CH ); 1.82ꢀ
3
2
1
.85 (m, 2H, CH ); 2.65ꢀ2.68 (m, 2H, CH ); 3.02ꢀ3.05 (m, 2H, CH ); 3.25ꢀ3.29 (m, 5H, CH and 2CH );
2 2 2 2
6.96 (d, 1H, J=8.0 Hz); 7.00ꢀ7.14 (m, 5H); 7.21 (t, 1H, J = 8.4 Hz); 7.36 (d, 1H, J=8.2 Hz); 7.62 (d,
13
1
5
H, J=8.4 Hz); 8.08 (s, 1H, NH); C NMR (100 MHz, CDCl ) δ 19.2; 22.8; 26.1; 29.3; 36.0; 49.1;
3
4.1; 111.6; 113.0; 118.8, 120.0, 122.9, 123.1, 126.2, 126.8, 127.5, 128.7, 129.8; 136.0; 136.9, 143.7.
+
ESIMS m/z calcd for C H N , 304.19; found 305.43 (M+H )
2
1
24
2
General procedure for the synthesis of derivatives (15-22).
Tryptamine (1.0 eq) was dissolved in a solution of DCM:CH COOH (5:1 v/v) at room temperature. To
3
this solution 2.0 equivalents of the proper aldheyde were added and the mixture was warmed to reflux
for 1.5h. Then, 1.8 equivalents of sodium triacetoxyborohydride were added portionwise and the
mixture was allowed to reflux for further 3ꢀ5h. After cooling to room temperature, NaOH 1N was
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added. The organic phase was separated and extracted one more time with the alkaline solution. Then it
was dried over Na SO , filtered and concentrated in vacuo. The crude products were purified by
2
4
column chromatography using mixtures of DCM/MeOH as eluent.
-(1H-Indol-3-yl)-N-(naphthalen-1-ylmethyl)ethanamine (15).
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1
Synthesized starting from 13 and 1ꢀnaphthaldehyde, yield 75%. H NMR (400 MHz,CDCl ) δ 3.05 (t,
3
2
H, J = 6.8 Hz, CH ); 3.13 (t, 2H, J = 6.8 Hz, CH ); 4.27 (s, 2H, CH ); 6.97 (s, 1H); 7.20 (t, 1H, J = 7.2
2 2 2
Hz); 7.33ꢀ7.48 (m, 4H); 7.64 (d, 1H, J=7.8 Hz); 7.74 (d, 1H, J=8.2 Hz); 7.84 (d, 1H, J=7.8 Hz); 8.00
1
3
(d, 2H, J=4.0 Hz). C NMR (100 MHz, CDCl ) δ 25.7, 49.8, 51.4, 111.1, 113.9, 118.9, 119.3, 121.9,
3
1
22.0, 123.5, 125.4, 125.5, 125.9, 126.0, 127.4, 127.7, 128.7, 131.7, 133.8, 135.7. ESIMS m/z calcd for
+
C H N , 300.16; found 301.18 (M+H ).
21
20
2
2-(1H-Indol-3-yl)-N-[(7-methoxynaphthalen-1-yl)methyl]ethanamine (16).
Synthesized starting from 13 and 2ꢀmethoxyꢀ1ꢀnaphthaldehyde, yield 63%. H NMR (400 MHz,
CDCl ) δ 3.05ꢀ3.09 (m, 4H, CH ); 3.61 (s, 3H, CH ); 4.35 (s, 2H, CH ); 7.01ꢀ7.19 (m, 3H); 7.28ꢀ7.44
1
3
2
3
2
1
3
(
m, 6H); 7.76 (d, 1H, J=7.8 Hz); 7.90 (d, 1H, J=7.0 Hz); 8.14 (s, 1H, NH). C NMR (100 MHz,
CDCl ) δ 25.1; 42.6; 48.1; 56.0; 94.6, 111.4; 112.7; 119.0, 119.6, 122.3, 122.7, 122.9; 123.6; 127.3;
3
128.8; 129.9; 130.7; 131.7; 136.5, 142.8. ESIMS m/z calcd for C H N O 330.17; found 331.21
2
1
20
2
+
(
M+H ).
1
-[(2-(1H-Indol-3-yl)ethyl)amino]methyl]naphthalen-2-ol (17).
1
Synthesized starting from 13 and 2ꢀhydroxyꢀ1ꢀnaphthaldehyde, yield 51%. H NMR (400 MHz,
CDCl ) δ 3.05ꢀ3.09 (m, 4H, 2CH ); 4.41 (s, 2H, CH ); 7.07ꢀ7.14 (m, 3H); 7.20 (t, 1H, J = 7.0 Hz);
3
2
2
7.26ꢀ7.42 (m, 3H); 7.60 (d, 1H, J=8.0 Hz); 7.65 (d, 1H, J=8.0 Hz); 7.73 (d, 2H, J=8.0 Hz); 8.08 (s, 1H,
1
3
NH) C NMR (100 MHz, CDCl ) δ 25.2; 47.4; 48.7; 111.4; 111.5; 112.8; 113.0; 118.9, 119.6, 119.7,
3
1
21.1, 122.5; 122.6; 126.6; 127.5; 128.6; 129.0; 129.3; 131.9; 137.4, 142.7. ESIMS m/z calcd for
+
C H N O, 316.16; found 317.40 (M+H ).
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-(1H-Indol-3-yl)-N-[(10-methylanthracen-9-yl)methyl]ethanamine (18).
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Synthesized starting from 13 and 10ꢀmethylꢀ9ꢀanthracenecarbaldehyde, yield 58%. H NMR (400
MHz, CDCl ) δ 3.04−3.07 (m, 4H, 2CH ); 3.26 (t, 2H, J = 6.4 Hz, CH ); 3.48 (s, 3H, CH ); 6.92 (s,
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0
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H); 7.10 (t, 1H, J = 8.0 Hz); 7.20 (t, 1H, J = 8.0 Hz); 7.32 (d, 1H, J=8.0 Hz); 7.39 (t, 2H, J = 7.8 Hz);
7.46 (t, 2H, J = 8.0 Hz, aryl); 7.56 (d,1H, J=8.0 Hz); 7.95 (s, 1H, NH); 8.19 (d, 2H, J=8.0 Hz); 8.29 (d,
1
3
2
1
H, J=8.0 Hz). C NMR (100 MHz, CDCl ) δ 17.1; 25.5; 45.6; 49.7; 111.4; 111.5;112.2; 113.0; 119.7,
3
22.2, 122.4, 124.6; 125.0; 125.7; 125.9; 136.5; 142.7. ESIMS m/z calcd for C H N , 364.19 found
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6
24
2
+
365.48 (M+H ).
5
[(2-(1H-Indol-3-yl)ethylamino)methyl]-2-methoxyphenol (19).
1
Synthesized starting from 13 and vanillin, yield 58%. H NMR (400 MHz, CD OD) δ 2.84 (t, 2H,
3
CH , J=7.4 Hz ); 2.92 (t, 2H, CH , J=7.4 Hz); 3.59 (s, 2H, CH ); 3.67 (s, 3H, CH ); 6.61 (d, 1H, J=8.0
2
2
2
3
Hz ); 6.71 (s, 1H); 6.73 (d, 1H, J=8.0 Hz); 6.94ꢀ6.98 (m, 2H); 7.05 (t, 1H, J=8.0 Hz); 7.31 (d, 1H,
1
3
J=8.0 Hz); 7.48 (d, 1H, J=8.0 Hz). C NMR (100 MHz, CD OD) δ 24.7, 48.6, 52.8, 55.1, 111.2,
3
1
11.8, 112.1, 115.0, 118.2, 121.2, 121.3, 122.4, 122.5, 127.4, 130.1, 137.0, 145.9, 147.9. ESIMS m/z
+
calcd for C H N O , 296.15; found 297.16 (M+H ).
18 20
2
2
N-(3,4-Dimethoxybenzyl)-2-(1H-indol-3-yl)ethanamine (20).
1
Synthesized starting from 13 and 3,4ꢀdimethoxybenzaldehyde, yield 72%. H NMR (400 MHz, CDCl )
3
δ 3.01ꢀ3.15 (m, 4H, 2CH ); 3.80 (s, 2H, CH ); 3.82 (s, 6H , 2CH ); 6.75 (d, 1H, J=8.0 Hz); 6.81 (d, 1H,
2
2
3
J=8.0 Hz); 6.88 (s, 1H); 7.03 (s, 1H); 7.10 (t, 1H, J=4.0 Hz); 7.18 (t, 1H, J=4.0 Hz); 7.35 (d, 1H, J=8.0
1
3
Hz); 7.58 (d, 1H, J=8.0 Hz); 8.10 (s, 1H, NH). C NMR (100 MHz, CDCl ) δ 25.4; 49.0; 53.5; 56.0;
3
111.0, 111.4; 111.5; 112.7; 119.0, 119.5, 120.8, 122.3; 122.4; 127.4; 130.1; 137.0; 145.9, 147.9.
+
ESIMS m/z calcd for C H N O , 310.17; found 311. 25 (M+H ).
1
9
22
2
2
2-(1H-Indol-3-yl)-N-(4-phenoxybenzyl)ethanamine (21)
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Journal of Medicinal Chemistry
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Synthesized starting from 13 and 4ꢀphenoxybenzaldehyde, yield 83%. H NMR (400 MHz, CD OD) δ
3
3
7
5
.12 (t, 2H, CH J=8.8 Hz); 3.24 (t, 2H, CH J=8.4 Hz); 4.08 (s, 2H , CH ); 6.95ꢀ7.05 (m, 5H); 7.10ꢀ
2, 2, 2
1
3
.18 (m, 3H); 7.31ꢀ7.42 (m, 5H); 7.53 (d, 1H, J=8.0 Hz). C NMR (100 MHz, CD OD) δ 24.5; 50.7;
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0
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0
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2.7; 111.4; 113.4; 119.8; 120.6; 120.9, 121.3, 123.6, 124.9; 125.9; 128.8; 129.0; 131.9; 133.4; 139.2;
+
158.8, 160.8. ESIMS m/z calcd for C H N O, 342.17; found 343. 25 (M+H )
2
3
22
2
N-Methyl-2-(1-methyl-1H-indol-3-yl)-N-(4-phenoxybenzyl)ethanamine (22)
Compound 21 (1 eq) was dissolved in a mixture of anhydrous DCM/DMF (2/1 v/v) under magnetic
stirring and the temperature was set to 0°C. To this solution, 3 equivalents of NaH were added
portionwise and the mixture was allowed to react for 30 minutes. Then, 3 equivalents of methyl iodide
in dry DCM were added dropwise and the reaction was warmed to room temperature and maintained
under stirring for further 12h. Then, reaction was quenched by 10% aqueous solution of citric acid and
washed with brine. Organic layer was separated, dried over anhydrous Na SO , filtered and evaporated
2
4
in vacuo. Crude product was purified by column chromatography using DCM/MeOH (9:1 v:v) as
1
mobile phase. H NMR (400 MHz ,CDCl ) δ 3.14ꢀ3.18 (m, 5H, CH and CH ); 3.78 (s, 3H, CH ); 3.84
3
3
2
3
(
t, 2H, J = 8.0 Hz, CH ); 4.99 (s, 2H, CH ); 6.95 (d, 2H, J=8.0 Hz); 7.05 (d, 2H, J=8.0 Hz); 7.17 (t, 1H,
2 2
13
J=7.6 Hz); 7.18ꢀ7.24 (m, 4H); 7.35 (t, 2H, J = 8.0 Hz); 7.59 (d, 3H, J = 8.0 Hz). C NMR (100 MHz,
CDCl ) δ 19.3, 32.8, 49.7, 63.7, 67.0, 109.5, 118.2, 118.4, 119.3, 119.9, 120.7, 121.9, 124.5, 126.9,
3
1
28.2, 130.0, 134.8, 136.8, 155.4, 159.9. ESIMS m/z calcd for C H N O, 370.49; found 371.55
25 26 2
+
(M+H ).
Pharmacology
Cell culture and transfections
For fluorescence assays, cells stably expressing TRP channels (SHꢀSY5Y for TRPV1, HEK for
TRPM8 and IMR90 for TRPA1) were cultured in a monolayer at 37ºC in a humidified atmosphere of
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Page 29 of 51
Journal of Medicinal Chemistry
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% CO in Earle’s minimum essential medium with Earle’s salts supplemented with 10% fetal calf
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serum,1% nonessential amino acids, 2 mM Lꢀglutamine, 100 µg streptomycin/ml, 100 U penicillin/ml,
and the correspondent antibiotic for each stable cell line (0.4 µg/ml puromycin for ShꢀSY5Y and 400
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ꢀg/ml G418 for HEKꢀCR1).
For electrophysiological experiments, HEK293 cells were grown in 100ꢀmm plastic Petri dishes
in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, penicillin (100 U/ml), and
streptomycin (100 U/ml) in a humidified atmosphere at 37°C with 5% CO . The cells were seeded on
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glass coverslips (Carolina Biological Supply Company, Burlington, NC) and transfected the next day
with 3.6 µg rat TRPM8 receptor cDNA (a gift from Dr. Felix Viana, Alicante Institute of
Neuroscience, Elche, Spain) using Lipofectamine 2000 (Invitrogen, Milan, Italy). A plasmid encoding
for the Enhanced Green Fluorescent Protein (Clontech, Palo Alto, CA) was used as a transfection
marker. Total cDNA in the transfection mixture was kept constant at 4 ꢁg.
Fluorescence assays.
For fluorescence assays, the cells were seeded in 96ꢀwell plates (Corning Incorporated,
Corning, NY) at a cell density of 40,000 cells 2 days before treatment. The day of treatment the
medium was replaced with 100 ꢀL of the dye loading solution Fluoꢀ4 NW supplemented with
probenecid 2.5 mM. Then the tested molecules dissolved in DMSO were added at the desired
concentrations and the plate(s) were incubated in darkness at 37°C in a humidified atmosphere of 5%
CO for 60 minutes. The fluorescence was measure using instrument settings appropriate for excitation
2
at 485 nm and emission at 535 nm. (POLARstar Omega BMG LAB tech). A baseline recording of 4
cycles was recorded prior to stimulation with the agonist (10 ꢀM capsaicin for TRPV1, 100 ꢀM
menthol for TRPM8, and 100 ꢀM AITC for TRPA1). Each antagonist (10 ꢀM Ruthenium Red for
TRPV1 and TRPA1, 10 ꢀM AMTB for TRPM8) was added to the medium containing the
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