Identification of a Potent Tryptophan-Based TRPM8 Antagonist with in Vivo Analgesic Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b00545

TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of N,N′-dibenzyl tryptamine 1, a potent TRPM8 antagonist, was prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably transfected HEK293 cells. The tryptophan derivative 14 was identified as a potent (IC₅₀ 0.2 ± 0.2 nM) and selective TRPM8 antagonist. In vivo, 14 showed significant target coverage in both an icilin-induced WDS (at 1-30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1-1 μg s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1-4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain.

Full text of DOI:10.1021/acs.jmedchem.8b00545

Subscriber access provided by NAGOYA UNIV
Article
Identification of a Potent Tryptophan-based
TRPM8 Antagonist With in vivo Analgesic Activity
Alessia Bertamino, Nunzio Iraci, Carmine Ostacolo, Paolo Ambrosino, Simona Musella,
Veronica Di Sarno, Tania Ciaglia, Giacomo Pepe, Marina Sala, Maria Virginia Soldovieri,
Ilaria Mosca, Sara Gonzalez-Rodriguez, Asia Fernández-Carvajal, Antonio Ferrer-Montiel,
Ettore Novellino, Maurizio Taglialatela, Pietro Campiglia, and Isabel M. Gomez-Monterrey
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00545 • Publication Date (Web): 25 Jun 2018
Downloaded from http://pubs.acs.org on June 26, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Identification of a Potent Tryptophan-based TRPM8 Antagonist
With in vivo Analgesic Activity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†
#
† #
‡ #
§
‡
Alessia Bertamino, , Nunzio Iraci, , Carmine Ostacolo, , Paolo Ambrosino, , Simona Musella,
†
†
†
†
§
Veronica Di Sarno, Tania Ciaglia, Giacomo Pepe, Marina Sala , Maria Virginia Soldovieri,
§
∥
∥
∥
Ilaria Mosca, Sara GonzalezꢀRodriguez, Asia Fernandez Carvajal, Antonio FerrerꢀMontiel,
‡
‡
* †
* ‡
Ettore Novellino, Maurizio Taglialatela, Pietro Campiglia, , and Isabel GomezꢀMonterrey ,
†Department of Pharmacy, University of Salerno, Via G. Paolo II 132, 84084, Fisciano, Salerno,
Italy
‡
Department of Pharmacy, University Federico II of Naples, Via D. Montesano 49, 80131, Naples,
Italy
§
8
Department of Medicine and Health Science V. Tiberio, University of Molise, Via F. de Sanctis,
6100, Campobasso, Italy
‡Department of Neuroscience, Reproductive Sciences and Dentistry, University Federico II of
Naples, Via Pansini, 5, 80131, Naples, Italy
∥
Institute of Molecular and Cellular Biology, Universitas Miguel Hernández, Avda de la
Universidad , 032020, Elche, Spain
#
These authors contributed equally to this work
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target
for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of N,N’ꢀ
dibenzyl tryptamine 1, a potent TRPM8 antagonist, were prepared and screened using a
fluorescenceꢀbased in vitro assay based on mentholꢀevoked calcium influx in TRPM8 stablyꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
transfected HEK293 cells. The tryptophan derivative 14 was identified as a potent (IC 0.2±0.2
50
nM) and selective TRPM8 antagonist. In vivo, 14 showed significant target coverage in both an
icilinꢀinduced WDS (at 1ꢀ30 mg/kg s.c.) and oxaliplatinꢀinduced cold allodynia (at 0.1ꢀ1 ꢀg s.c.)
mice models. Molecular modeling studies identified the putative binding mode of these antagonists,
suggesting that they could influence an interaction network between the S1ꢀ4 transmembrane
segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new
pharmacophoric scaffold for the design of highly potent modulators of TRPM8ꢀmediated pain.
ACS Paragon Plus Environment

Page 3 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Transient receptor potential melastatin type 8 (TRPM8) channel is a transmembrane Ca ꢀ
2
+
1
2,3
permeable nonselective channel triggered by cold temperatures (10−28 °C), as well by some
2,4
specific compounds, such as menthol and icilin, also known as cooling compounds. Moreover,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
TRPM8 is sensitive to transmembrane potential, phosphatidylinositolꢀ4,5ꢀbiphosphate (PIP2),
7
ꢀ9
and different synthetic molecules. TRPM8 channels are widely expressed in different tissues such
10
11
12
13
as skin and mucosae, prostate and bladder, lung epithelium and artery myocytes. However,
the biological significance of TRPM8 in these tissues remains largely undisclosed.
In the last years, a correlation between TRPM8 overexpression and cold allodynia or hyperalgesia
14, 15
has been disclosed in animal models of nerve injury or inflammation.
Moreover, the typical
hyperalgesic response to some types of pain is strongly reduced when TRPM8 antagonists are
1
6, 17
administered or in TRPM8 knockout mice.
evidenced during the evolution of androgenꢀsensitive prostate cancer and other malignancies.
These findings underscore the high therapeutic potential of these channels in both cancer and
Upregulation of TRPM8 levels has been also
18, 19
8,20ꢀ23
pain.
Efforts to identify TRPM8 have strongly increased in the last decade and a wide variety
24,25
of chemotypes have been recently disclosed as agonists or antagonists.
Among antagonists,
26
BCTC, a piperazine carboxamide derivative, was shown to inhibit prostate tumor cells growth,
while the benzamideꢀbased blocker, AMTB, proved remarkable therapeutic efficacy in animal
27
models of overactive and painful bladder syndromes. A benzimidazoleꢀbased antagonist,
identified by a Janssen’s research group, demonstrated an excellent inhibitory activity when
2
8
challenged in animal models of neuropathic pain. Furthermore, researchers from Pfizer identified
29
the isoquinoline derivative PFꢀ05105679, endowed with clinical efficacy in human cold allodynia.
Our research group has recently identified an indoleꢀbased derivative [N,Nꢀdibenzylꢀ2ꢀ(1Hꢀindolꢀ3ꢀ
yl)ethanamine, 1, Figure 1] inhibiting mentholꢀinduced currents in HEK293 cells stably expressing
30
TRPM8. In patchꢀclamp recordings, compound 1 showed concentrationꢀdependent TRPM8
inhibition with higher potency (IC = 367 ± 24 nM) and selectivity than the reference antagonist
50
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
BCTC.
Starting from these results, a new small library of indole derivatives was designed, synthesized and
characterized, obtaining new SAR clues and new potent antagonists of TRPM8 channel with
significantly improved potency over the parent compound 1 and potent in vivo antiꢀnociceptive
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31
activity. Taking advantage of the recently reported cryoꢀelectron microscopy structure of TRPM8,
molecular modeling studies provided some clues about the binding mode of the new TRPM8
antagonists being herein described.
ACS Paragon Plus Environment

Page 5 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
RESULT AND DISCUSSION
Design and Chemistry
For the design of the new TRPM8 antagonist library we opted to retain the tryptamine scaffold,
which allows efficient and extensive structural modifications.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Structure of newly proposed antagonists
The indole moiety was substituted at Nꢀ1, Cꢀ2 and Cꢀ5 (I, Figure 1), while the benzyl rings on the
amine group were decorated or substituted (II). The conformational architecture of the parent
compound 1 was also altered by the introduction of constraints on the indole moiety (III), on the diꢀ
benzyl group (II) or replacing the tryptamine template with tryptophan (IV).
New tryptamine derivatives 2-4 were synthesized as depicted in scheme 1. Compounds 1 and 2
were obtained by a double nucleophilic substitution of tryptamine or 5ꢀbenzyloxytryptamine with a
30
large excess of benzyl bromide in DCM, using DIPEA as base. Nꢀ1 methylation of 1 with CH
3
I or
addition of 2,4ꢀdinitrobenzenesulphenyl chloride at Cꢀ2 in acid media yielded derivatives 3 and 4,
respectively.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 1. Synthesis of Dibenzyl Tryptamine Derivatives 1-4.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reductive amination of benzaldehyde with tryptamine using sodium borohydride in dry methanol
yielded linear compound 5, while the same reaction in acidic media led to racemic tetrahydroꢀbetaꢀ
carboline 6 (Scheme 2). Final compounds 7, 8 and 9-11 were synthesized via palladiumꢀcatalyzed
alkylation of the intermediates 5 or 6, respectively, with the corresponding alkyl, phenylalkyl, or
phenacyl bromides. Compounds 9-11 were tested as racemic mixtures since all attempts to resolve
these mixtures, or the starting beta carboline mixture 6, by chiral semi preparative HPLC, failed.
ACS Paragon Plus Environment

Page 7 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 2. Synthesis of N,NꢀDisubstituted Tryptamine Derivatives (7, 8) and 2ꢀsubstitutedꢀ1ꢀphenylꢀ2,3,4,9ꢀtetrahydroꢀ
1Hꢀpyrido[3,4ꢀb]indole (9-11).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Nucleophilic displacement of the 3ꢀ(2ꢀbromoethyl)indole by the commercially available 2,3ꢀ
dihydroꢀ1Hꢀbenzo[d,e]isoquinoline (12) led to the restricted analogue 13 (Scheme 3). The synthesis
of compounds 14-16 was performed starting from L or D tryptophan methyl ester, using the same
procedure described for derivative 1. Finally, alkaline hydrolysis of compound 14 gave the acid
derivative 15.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 3. Synthesis of 1ꢀ(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)ꢀ2,3ꢀdihydroꢀ1Hꢀbenzo[d,e]quinoline (13) and L and Dꢀtryptophan
derivatives (14-16).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
Pharmacological screening by Ca -imaging experiments
The activity of the synthesized compounds as TRPM8 blockers was tested in vitro by evaluating
2+
2+
their ability to counteract mentholꢀinduced increase in intracellular Ca levels in Ca fluorimetric
assays in HEK293 cells stably expressing the rat isoform of TRPM8 channels. As reported in Table
1
, eight of the twelve compounds synthesized proved to be more potent than the specific antagonist
Nꢀ(3ꢀaminopropyl)ꢀ2ꢀ[(3ꢀmethylpheny)methoxy]ꢀNꢀ(2ꢀthienylmethyl) benzamide hydrochloride
AMTB), used as reference compound.
(
ACS Paragon Plus Environment

Page 9 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound
IC50 (ꢀM)
1
2
3
4
7
8
9
3.2±0.8
1.9±0.9
3.3±1.3
2.2±0.7
0.6±0.5
3.00±0.9
3.3±1.3
> 100
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10
11
13
1.6±0.9
11.8±2.2
0.04±0.09
8.2±2.1
6.5±1.4
14
15
AMTB
Table 1: Potency of synthesized compounds as TRPM8 antagonists. Values are expressed as the mean ± standard
deviation of three independent measurements.
The functionalization at the positions 5 and 2 of the indole ring is well tolerated and the compounds
2
and 4 weakly improve the potency over compound 1.
The methylation at the indole Nꢀ1 of compound 3, slightly decreased the activity respect to
analogues 2 and 4, although maintaining the potency in comparison to 1. Compound 7, containing a
methyl cyclohexylꢀ in place of a benzylꢀ group, proved to be fiveꢀfold more effective and potent
than 1 (IC50 0.6±0.5 ꢀM versus 3.2±0.8ꢀM), while the introduction of a strong electronꢀ
2
withdrawing group, such as NO , at position 4 in one of the aryl moieties (8), maintained the
antagonist potency in the micromolar range. Interestingly, the tetrahydroꢀβꢀcarboline 9 as restricted
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
analog of 8, still show micromolar activity, while the replacement of the NO group with a COOH
2
one at the same position (derivative 10) caused the loss of the antagonist activity. The substitution
at Nꢀ2 of the benzyl moiety by the more hydrobophic acetophenone group (compound 11), allowed
to recover and even increase the antagonistic potency. On the other hand, the conformational
restriction introduced using the bulky 2,3ꢀdihydroꢀ1Hꢀbenzoisoquinoline substituent, proved to be
deleterious for the activity. Thus, compound 13 resulted 4 times less active than 1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, the replacement of tryptamine with the tryptophan scaffold led to the most interesting
derivatives. Dibenzyl tryptophan 14, showing nanomolar potency, is indeed the most potent
TRPM8 blocker reported so far. It is interesting to note that the acid derivative 15 resulted 200
times less potent than 14. We wondered whether this behaviour was mainly a matter of
pharmacodynamic or pharmacokinetic properties, thus we challenged the membrane crossing
properties of the two compounds by using the Parallel Artificial Membrane Permeability Assay
−
6
(
PAMPA, SI Figure S1). We found that the ester derivative 14 (Papp = 0.97±0.21 × 10 cm/s,
−6
QPlogPO/W = 5.4) was less prone to cross membranes than 15 (Papp = 3.17 ±0.81 × 10 cm/s,
QPlogPO/W = 2.9, Figure S1). Thus, it is plausible that the dramatic decrease in activity is mainly
due to the pharmacodynamic properties of the investigated molecules. The selectivity of the most
potent TRPM8 modulators (7 and 14) over TRPV1, was also investigated by calcium fluorimetric
experiments in SHꢀSY5Y cells stably expressing mouse TRPV1 channels, using the prototypical
TRPV1 agonist (capsaicin, 10 ꢁM) or antagonist (ruthenium red, 10 ꢁM) as controls. Both
compounds displayed high selectivity for TRPM8 channels, showing no activity on TRPV1. (SI,
Figure S2)
Pharmacological properties of compounds 7, 14, and 16 by patch-clamp electrophysiology
As described, 7 and 14 derivatives were identified as the most potent TRPM8 antagonists by a
2+
multiꢀcellular Ca fluorimetric assay. To further characterize such pharmacological activity in a
single cellꢀbased electrophysiological assay, we tested the ability of these compounds to counteract
ACS Paragon Plus Environment

Page 11 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mentholꢀevoked responses in HEK293cells transiently expressing rTRPM8 by patchꢀclamp
recordings. Application of menthol (500 ꢁM) resulted a large, strongly outwardly rectifying current
(I+80mV/I−80mV = 94±19; n = 66) that reverted near to 0 mV (ꢀ1.1±0.4 mV; n=66), consistent with the
1
wellꢀknown poor selectivity of rTRPM8 among monovalent cations (Fig. 3A). As previously
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
30
reported, menthol EC50 was 75±4 ꢁM, a value consistent with that shown for rTRPM8 when
1
expressed in Xenopus oocytes (67 ꢁM). The nonꢀtransfected cells were insensitive to menthol
(data not shown), confirming that mentholꢀactivated currents were strictly dependent on the
expression of rTRPM8. As also shown in the timeꢀcourse experiments of Fig. 2A and 2B (left
panels), 500 ꢁM mentholꢀevoked rTRPM8 currents were rather stable for at least 1 min; during
menthol exposure, addition of 500 nM 7 (Fig. 2A) or 100 nM 14 (Fig. 2B), almost fully blocked
mentholꢀgated rTRPM8 currents, thus showing potent antagonistic properties. The inhibitory effects
of both 7 and 14 were fully reversible, as mentholꢀevoked currents were largely recovered upon
washout of the drugs. The inhibitory effects of both 7 and 14 compounds on mentholꢀevoked
rTRPM8 currents were concentrationꢀdependent (Fig. 2C, left panel), showing IC50s of 3.5±0.6 nM
(n=28) and 0.2±0.2 nM (n=16), respectively (Fig. 2C, right panel); these values are well below
3
1
those reported for the canonical TRPM8 antagonist BCTC (IC =475 nM) as well as for the
5
0
30
reference compound 1 (IC =367 nM). Noteworthy, comparison of the absolute IC50 values
50
2+
calculated from intracellular Ca and electrophysiological assays for both compounds 7 and 14
confirmed the rank order of potency for these compounds, with 14 being more potent than 7.
To determine the role of the chiral center in the rTRPM8ꢀblocking activity of compound 14, the
pharmacological effects of its Dꢀenantiomer 16 was also investigated. As shown in Figure 2C, 16
showed a similar efficacy but a >4000ꢀtimes reduced potency (IC =865±26 nM; n=20) when
50
compared to the Lꢀenantiomer 14, therefore suggesting a crucial role played by the chiral center in
the pharmacological properties shown by compound 14.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Compounds 7 and 14 block mentholꢀevoked TRPM8 currents in HEK293 cells. (A,B left) Timeꢀcourse of
currents recorded at +80 mV or −80 mV in HEK293 cells transiently expressing TRPM8 upon application of 500 ꢁM
menthol + derivative 7 (500 nM, A) or 500 ꢁM menthol + derivative 14 (100 nM, B). (A, B right) Representative
current traces from a single TRPM8ꢀtransfected HEK293 cell subsequently exposed to control solution (CTL, black
trace), 500 ꢁM menthol (green traces), 500 ꢁM menthol + 500 nM derivative 7 (red trace; A), or 500 ꢁM menthol + 100
nM derivative 14 (red trace; B). Traces shown are those indicated in each respective color (black, green, red) in the
timeꢀcourse shown in the left panels. (C) Doseꢀresponse curve for 7-, 14-, and 16–induced inhibition of TRPM8
currents (left panel), and resulting IC50 for each compound (right panel).
ACS Paragon Plus Environment

Page 13 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Molecular modeling and structural rationale.
In order to rationalize the SAR of 1 and other analogues, we previously built a model of TRPM8
31
32
bound to compound 1, based on the homology model by Taberner et al., which highlighted the
pivotal role of the linker between S6 and TRP box (amino acids 980−992 in rat TRPM8) for the
channel gating. In particular, we used a simplified version of the model and proposed that our
antagonists docked at a putative binding site in the VSLD cavity. This putative binding region is
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
33
34
supposed to bind diverse chemotypes, such as βꢀlactam derivatives, naphtyl derivatives,
3
5
tetrahydroisoquinolines and 2,5ꢀdiketopiperazines. In this work, we created a new model of
human TRPM8 using the recently published ficedula albicollis TRPM8 (TRPM8FA) structure as
template. The TRPM8FA experimental model shows significant differences with respect to TRPV1,
TRPV2 and previously reported TRPM8 models, and redefines the location of several residues
31
involved in modulators binding.
Focusing on the region formed by TM helices S1ꢀ6 and TRP box (residues 723ꢀ1013), we modeled
36
a human TRPM8 monomer by homology with TRPM8FA, using the Prime software.
It is worth noting that TRPM8FA shows a high similarity with human TRPM8, an this similarity gets
higher when focusing on the binding region delimited by TM helices S1ꢀ6 and TRP box. (Table S1)
In the resulting model, the VSLD region appears almost divided into two layers, with a very
hydrophobic top one and a bottom one populated by charged residues interacting mutually. In
particular, R1008 interacts with E1004, D781 and E782, while R842 interacts with D802 and E782.
R784 points instead outside of the VSLD cavity (Figure 3A).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Ionic interactions (represented as magenta dashed lines) between charged residues of the VSLD cavity in the
apo protein (A) and upon binding of compound 1 (B).
3
7,38
We performed an Induced Fit Docking (IFD)
simulation of compound 1 on the homology model
and submitted the best scoring 1/TRPM8 complex to a 48ns long molecular dynamics simulation.
The simulations showed the indole NH interacting with Asp781, and the nitrogen of the linker with
Glu782. One of the two phenyl rings can engage cationꢀπ interactions with arginines 842 and 1008.
Residues Y1005, F839 and F744 can make πꢀπ interactions with the three rings of the antagonist.
Further interactions, mainly hydrophobic, are observed with residues F738, Y745, L778, I846, and
L1009 (Figure 4).
ACS Paragon Plus Environment

Page 15 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Bound conformation of compound 1, depicted as yellow sticks. TRPM8 is depicted as blue cartoons and
sticks. Dashed lines represent πꢀ π, πꢀcation, salt bridge and Hꢀbond interactions between TRPM8 and compound 1, and
are colorꢀcoded in green, red, magenta and yellow, respectively.
It is interesting to note how the architecture of the ionic interactions described above for the
unbound protein changes upon binding of 1 (Figure 3B). Indeed, D781, which previously interacted
with R1008, now moves away from the cavity and interacts with R784. R1008 continues to interact
with E782 but establishes two new interactions with F802 and with the phenyl ring of the ligand.
R842 no longer interacts with E782 but continues to interact with D802 and establishes a new
interaction with one of the phenyl rings of the ligand. It seems that the addition of the positive
charge of the ligand and the displacement of D781 tend to neutralize the negative charge of the
pocket and this could stabilize an inactive conformation of the channel.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We used the predicted 1/TRPM8 complex, after MD equilibration (see materials and methods), as
target for constrained molecular docking simulations of compounds 2-4, 7-11 and 13-16 (Figure
39
S2aꢀi), using the Glide program.
Docking of compound 3 (SI, Figure S3a) shows a deviation of the indole moiety caused by the
methylation of NH, and loss of the interaction with D781. Since the compound still shows a good
activity we suggest that despite this interaction could increase the activity, its lack does not
substantially compromise it.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Modifications at position 5 and 2 of the indole ring are well tolerated. The accommodation of the
benzyl ether group in 2 (SI, Figure S3b), near to the residues L778, L774, L806, F839, and F748,
slightly improves the activity of the product with respect to 1, whereas the additional phenyl sulfane
of 4 (SI, Figure S3c) points toward the membrane and does not influence the activity
The cyclohexyl group of compound 7 (SI, Figure S3d), which is docked in the hydrophobic region
of the site surrounded by residues L778, L806, F748, L774, F839 and Y745, leads to a 5ꢀfold
activity improvement suggesting that in this binding region, a hydrophobic and aliphatic moiety
may interact stronger than an aromatic one.
Compounds 8 and its more conformational restricted analogue 9 address the nitro group in different
directions (SI, Figures S3e,f), showing that it could probably be tolerated in two different areas of
the pocket. The restrained strengthening of the 9 scaffold prevents NH indole from interacting with
D781 showing, once again, that this interaction is not necessary for the activity.
Substitution of the nitro group of 9 with a carboxyl one, as in compound 10 (SI, Figure S3g), leads
to a dramatic loss of activity; bound conformations of 9 and 10 show the nitro and carboxyl groups
docked in the same area. Although these compounds have been tested as racemic mixtures, our
modeling study indicates that the
as confirmed even by the most potent compound 11 (SI, Figure S3h), whose 4ꢀsubstituted benzyl is
replaced by a –CH ꢀbenzoyl group.
R enantiomer is the most active in this tetrahydroꢀcarboline series
2
ACS Paragon Plus Environment

Page 17 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Docking of compound 13 (SI, Figure S3i), highlights the lack of any potential cationꢀπ interaction
at the level of R1008, revealing a potential mechanism for its decreased potency when compared to
compound 14 (see below). It is however important to note that the binding mode of this compound
suggests that the hydrophobic "roof" area could accommodate, besides phenyl groups or aliphatic
substituents as in compound 7, even larger substituents.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Dibenzyl tryptophan derivative 14, indeed the most potent TRPM8 inhibitor reported so far, with an
IC50 in the subꢀnanomolar range (0.2 nM, see Fig. 2C), shares most of its interactions with the
parent compound 1. The 14/TRPM8 complex, during a 48ns long molecular dynamics simulation,
reveals an interaction with Y745, which is reported as a critical residue for the binding of menthol
40
and SKF963635. The interaction with E781 is weaker than 1, and this confirms once again that
this hydrogen bond is not fundamental for the activity of the antagonists (Figure 5). The additional
carboxymethyl group docks in a hydrophobic area surrounded by residues V742, I846, L1009,
N741 and Y745.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Bound conformation of compound 14, depicted as yellow sticks. TRPM8 is depicted as blue cartoons and
sticks. Dashed lines represent πꢀ π, πꢀcation, and salt bridge interactions between TRPM8 and compound 14, and are
colorꢀcoded in green, red and magenta, respectively.
This feature, together with the negative net charge of the site, could explain the dramatic loss of
activity showed by its acid derivative 15. The inversion of chiral configuration as in compound 16,
projects the carboxymethyl group to an area surrounded by R1008, E782, L778 and D781 (Figure
6), and the proximity of charged residues could contribute to the loss of activity shown by the 16
enantiomer.
Our molecular modeling studies highlight the plausible importance of several VSLD cavity residues
in the binding of TRPM8 modulators. Indeed, charged residues like D781, E782, R784, D802,
R842, E1004 and R1008 could be likely involved in the equilibrium between functional states of
the channel, and our docking simulations show the interaction of these residues with our indole
ACS Paragon Plus Environment

Page 19 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
derivatives. In particular, E782, R842, R1008 are always predicted to be involved in ionic and πꢀ π
stacking interactions with our inhibitors. If, on one side, the above mentioned charged residues may
look like the most important VSLD residues in the gating mechanism, several others may be
responsible for an efficient inhibitor binding. The interaction with hydrophobic residues V742,
L778, L806, I846, F748, L774, F839, Y745, V742 and L1009 appears indeed desirable for an
efficient antagonist activity. Overall, using a simplified channel model, our molecular studies gave
some clues about the gating and modulation mechanisms of TRPM8, that need to be challenged
through further inꢀdepth experimental and computational studies.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Bound conformation of compound 16, depicted as magenta sticks. TRPM8 is depicted as blue cartoons and
sticks. Compound 14 is represented as yellow sticks for reference.
In vivo pharmacological characterization of compound 14
Wet-dog shakes assay
To determine whether the observed in vitro TRPM8 inhibitory activity of compound 14 also
translates in TRPM8ꢀdependent antinociceptive activity in vivo, a “wetꢀdog shakes” (WDS) assay
4
1
was performed. In this assay, the potent TRPM8 agonist icilin (10mg/kg intraperitoneally i.p.),
was injected in mice treated with 14 or vehicle. Mice TRPM8 is known to share high homology
30
with rat TRPM8, that has been used for in vitro assays. Icilin injections in mice produce vivid and
28,42
quantifiable TRPM8ꢀmediated shaking behaviors.
The compound 14 (1, 10 and 30 mg/kg) or its
vehicle were administered subcutaneously (s.c.) 30 minutes before icilin injection . As shown in
ACS Paragon Plus Environment

Page 21 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 7, compound 14 produced a marked, doseꢀdependent antinociceptive behavior; at the highest
dose of 30 mg/kg, 14 inhibited WDS like cold hypersensitivity in mice by 63%, thus showing an
efficacy comparable to that observed with a standard analgesic dose of gabapentin (25 mg/kg, s.c.)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Effect of 14 on icilinꢀinduced WDS in C57BL/6J mice. Compound 14 or vehicle was administered 30
minutes before icilin injection. Gabapentin was administered 1h before icilin injection. After i.p. injection of icilin (10
mg/kg), the number of wetꢀdog shakes (WDS) were counted over 30 min. Data are given as mean ± SEM (n = 6). 2ꢀ
way ANOVA with Bonferroni post hoc test. *P <0,05; **P <0,01, ***P <0,001,**** P <0,001.
Cold allodynia
As indicated in the introduction, increasing evidence suggest that TRPM8 plays a critical role in
mouse models of chemotherapyꢀinduced neuropathic pain evoked by oxaliplatin (OXP), a condition
mimicking cold hypersensitivity provoked by chemotherapyꢀinduced peripheral neuropathy
4
3
(
CIPN). In fact, acute and chronic OXPꢀinduced cold hypersensitivity have been reproduced in
44, 45
rats and mice and correlated with TRPM8 expression and function.
The TRPM8 agonist
4
6
menthol has shown antiꢀallodynic activity in a neuropathic rat model
and its topical
4
7
administration showed analgesic efficacy in human chemotherapyꢀinduced neuropatic pain.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
However, it has been reported that, in a rodent model, acute cold allodynia after OXP injection was
alleviated by the TRPM8 blockers Nꢀ(2ꢀaminoethyl)ꢀNꢀ[4ꢀ(benzyloxy)ꢀ3ꢀmethoxybenzyl]ꢀN’ꢀ(1S)ꢀ
48
1
ꢀ(phenyl) ethyl] urea and TCꢀI 2014. According to these findings we investigated the effect of
our antagonist 14 in an OXPꢀinduced cold allodynia model, using acetone for cooling stimulation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Considering that cold pain threshold is increased from ≈12 °C to ≈ 26 °C in OXPꢀtreated patients,
48
acetone stimulation is considered to evoke pain in OXPꢀtreated mice.
Figure 8. Oxaliplatin (OXP)ꢀinduced cold allodynia and effects of TRPM8 blocker (14). Mice were given three i.p.
injection of OXP (6 mg/kg) or the vehicle (VH) on alternate days. Cold allodynia was evaluated by the acetone test.
Timeꢀcourse of cold allodynia after OXP injection. Effects of the TRPM8 blocker at 0,1 (A) or 1.0 ꢀg (B). Data are
given as mean ± SEM (n = 6). 2ꢀway ANOVA with Bonferroni post hoc test. *P <0,05; **P <0,01, ***P <0,001.
The activity of compound 14 was evaluated at day 7 after intraperitoneal administration of three
OXP doses (6 mg/kg) in C57/BL6 mice, when cold allodynia had developed. After single
subcutaneous administration of 14 (0.1 and 1 ꢀg), attenuated cold allodynia was evident at 15 min
and reaches the maximum inhibition 30 min after administration (Figure 8A and 8B) in a dose
dependent manner.
Although highly potent, compound 14 showed a short lasting effect in both the animal models used.
We argued that this effect could be due to the presence of the labile ester moiety, readily prone to
generate the less active acid derivative 15. This hypothesis was challenged by enzymatic stability
assay in mice serum and chemical stability assay in phosphate buffer. The compound resulted stable
ACS Paragon Plus Environment

Page 23 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
to chemical hydrolysis when dissolved in phosphate buffer at pH 7.4 for 1 hour (Figure S4).
Contrariwise, 14 is almost completely metabolized during a 1 hour time course experiment in mice
serum, generating 15 as the major metabolite (Figure S4 and S5). It must be considered that, the
high potency of derivative 14 could also be justified on the basis of a multitargeted activity. Many
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
43,44
different ionotropic and metabotropic receptors
have been described to mediate oxaliplatinꢀ
induced neuropathy. Among these, the TRPA1 receptors, located also on primary afferent neurons,
49
50
is involved in mechanical and cold allodynia. Recently, Pan et al. described that TRPA1 and
TRPM8 promote a local vasodilatation that aggravate the oxaliplatinꢀinduced peripheral neuropathy
and the that blockade of TRPA1 and/or TRPM8 receptors attenuated this process. According to
these results a “double block" of TRPM8 and TRPA1 could represent a potential pharmacological
advantage. To address this hypothesis, studies on TRPA1 activity with different tryptophan
derivatives are ongoing.
CONCLUSIONS
In this work the synthesis and the pharmacological characterization of a small library of TRPM8
antagonists has been reported. One of the synthesized derivatives, compound 14, proved to be the
most potent in vitro TRPM8 antagonist identified so far, producing a selective and complete
TRPM8 inhibition with potency in the subnanomolar range. In vivo studies showed that compound
14 suppresses TRPM8ꢀmediated cold hypersensitivity and significantly reduces cold allodynia in
vivo. According to these findings, 14 represents a new lead compound and a new pharmacological
tool to investigate TRPM8ꢀmediated signaling. Moreover, a new TRPM8 homology model has been
built, allowing to hypothesize a binding mode for this series of TRPM8 modulators, to rationalize
their pharmacological activity and to provide useful findings for the rational development of new
TRPM8 blockers.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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 predried, freshly distilled solvents. Microwave
assisted reactions were performed in a Biotage Initiator+ reactor. Analytical thin layer
chromatography (TLC) was performed on precoated glass silica gel plates 60 (F254, 0.25 mm,
VWR International). Purifications were performed by flash column chromatography on silica gel
(230−400 mesh, Merck Millipore). 1D and 2D NMR spectra were recorded with Bruker Avance
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(400 MHz) spectrometer, at room temperature. Spectra were referenced to residual chloroform
1
13
1
13
(
7.24 ppm, H; 77.23 ppm, C) or methanol (3.31 ppm, H; 49.15 ppm, C). Chemical shifts are
reported in δ values (ppm) relative to internal Me Si, and J values are reported in hertz (Hz). The
4
following abbreviations are used to describe peaks: s (singlet), d (doublet), dd (double doublet), t
(triplet), q (quartet), and m (multiplet). HRꢀMS experiments were performed using an LTQꢀ
OrbitrapꢀXLꢀETD mass spectrometer (Thermo Scientific, Bremen, Germany), using electrospray
ionization. Optical rotations were measured on an Atago Polax 2ꢀL polarimeter, at the concentration
of 0,1g/100ml. Analytical RPꢀHPLC was performed on a Phenomenex Synergi Fusion RPꢀ80A (75
mm × 4.6 mm, 4 ꢁm), with a flow rate of 1 mL/min, using a tunable UV detector at 254 nm.
Mixtures of CH
purity ≥ 95%.
3 2
CN and 0.05% TFA in H O were used as mobile phase. All compounds showed a
N,N-dibenzyl-2-(1H-indol-3-yl)ethanamine (1) and N,N-dibenzyl-2-(5-(benzyloxy)-1H-indol-
30
3-yl)ethanamine (2) were obtained according to the synthetic procedure previously described.
N,N-dibenzyl-2-(5-(benzyloxy)-1H-indol-3-yl)ethanamine (2)
ACS Paragon Plus Environment

Page 25 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
Colourless oil (71% yield) H NMR (400 MHz, CDCl
= 8.4 Hz); 3.73 (s, 4H, C ); 4.97 (s, 2H, C
.29ꢀ7.38 (m, 5H, aryl); 7.42ꢀ7.44 (m, 6H, aryl); 7.51 (d, 2H, aryl,
NMR (100 MHz, CDCl ) δ 23.2; 53.8; 58.4; 71.0; 102.2; 111.7; 112.9; 114.4; 122.3; 126.8; 127.7;
31 2
27.8; 127.9; 128.2; 128.5; 128.8; 131.6; 137.7; 140.0; 153.1. HRꢀMS m/z calcd for C31H N O
3
) δ 2.83 (t, 2H, C
2
H J = 8.4 Hz); 2.98 (t, 2H,
C
H
2
J
H
2
H ); 6.92 (s, 3H, aryl); 7.23ꢀ7.25 (m, 3H, aryl);
2
13
7
J = 7.6 Hz); 7.80 (s, 1NH); C
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
+
[
(M+H) ]: 447.2431; found 447.2439
Synthesis of N,N-dibenzyl-2-(1-methyl-1H-indol-3-yl)ethanamine (3)
N,Nꢀdibenzylꢀ2ꢀ(1Hꢀindolꢀ3ꢀyl)ethanamine (1, 1.0 equiv) 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,
1
1
.2 equiv of NaH were added portionwise and the mixture was allowed to react for 30 min. Then,
.2 equiv of methyl iodide in DCM were added dropwise and the reaction was warmed to room
temperature and maintained under stirring for further 12 h. The reaction was quenched by 10%
aqueous solution of citric acid and washed with brine. Organic layer was separated, dried over
2 4
anhydrous Na SO , filtered, and evaporated in vacuo. Crude products were purified by column
chromatography using nꢀhexane/ethyl acetate (4:1 v:v) as mobile phase.
1
Colourless oil (65% yield) H NMR (400 MHz, CDCl
3
) δ 2.83 (t, 2H, C
); 6.76 (s, 1H, aryl); 7.05 (t, 1H, aryl,
= 7.6 Hz); 7.34 (t, 3H, aryl, = 7.2 Hz); 7.39ꢀ7.43 (m,
H
2
,
J
= 8.0 Hz); 3.00 (t, 2H,
C
H
2
,
J
= 8.0 Hz); 3.72 (s, 7H, C
H
2
and C
H
3
J
= 7.6 Hz); 7.21
(
t, 1H, aryl, = 7.2 Hz); 7.27 (d, 3H, aryl,
J
J
J
13
6H, aryl); C NMR (100 MHz, CDCl
3
) δ 23.0; 32.5; 54.2; 58.4; 109.0; 113.0; 118.5; 119.0; 121.4;
+
1
3
26.3; 126.8; 128.2; 128.8; 139.9. HRꢀMS m/z calcd for C H N [(M+H) ]: 355.2169; found
25
27
2
55.2177.
Synthesis of N,N-dibenzyl-2-(2-((2,4-dinitrophenyl)thio)-1H-indol-3-yl)ethanamine (4)
30
N,Nꢀdibenzylꢀ2ꢀ(1Hꢀindolꢀ3ꢀyl)ethanamine
(1, 1.0 equiv) was dissolved in a mixture of
CH COOH/DMF (4/1 v/v) under magnetic stirring at room temperature. Then, 2,4ꢀdinitro sulphenyl
3
chloride (2.0 equiv) was added and the solution was stirred for 2h. Then, the reaction was quenched
by 10% aqueous solution of sodium carbonate, extracted with DCM (3 x 50 mL) and the organic
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
layer was dried over anhydrous Na SO , filtered, and evaporated in vacuo. Final compound 4 was
2
4
obtained after flash chromatography using a mixture of ethyl acetate/nꢀhexane (4.8/0.2 v/v) as
eluent.
1
Yellow oil (52% yield) H NMR (400 MHz, CDCl ) δ 2.66 (t, 2H, CH J = 8.0 Hz); 3.07 (t, 2H,
3
2,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
H
2,
J
= 8.0 Hz); 3.71 (s, 4H, C
H
2
); 6.71 (d, 1H, aryl,
J
= 12.0 Hz); 7.13ꢀ7.25 (m, 7H, aryl); 7.31ꢀ
1
3
7
.42 (m, 7H, aryl); 7.93 (d, 1H, aryl, J = 8.2 Hz); 8.08 (s, 1NH); 9.07 (s, 1H, aryl). C NMR (100
MHz, CDCl
3
) δ 22.5; 29.7; 53.3; 58.4; 111.4; 117.6; 120.1; 120.5; 121.5; 124.9; 126.9; 127.0;
+
127.5; 128.1; 128.6; 137.7; 139.4; 143.8; 144.8. HRꢀMS m/z calcd for C30
39.1748; found 539.1760.
Synthesis of Derivatives N-benzyl-2-(1H-indol-3-yl)ethanamine (5)
H
27
N
4
O
4
S [(M+H) ]:
5
Tryptamine (1.0 equiv) was dissolved in dry methanol at room temperature under nitrogen
atmosphere. To this solution 1.0 equiv of benzaldehyde was added and the mixture was reacted for
12h. Then, 2.0 equiv of sodium borohydride were added portionwise and the mixture was reacted
for further 3h. Methanol was then evaporated in vacuo and the mixture was reconstituted in DCM
2 4
and washed with brine (3 x 50 mL). The organic phase was separated, dried over Na SO , filtered,
and concentrated in vacuo. The crude product was purified by column chromatography using a
mixture of DCM/MeOH (4.5/0.5 v/v) as eluent, obtaining intermediate 5 as a colourless oil in 78%
yield.
1
H NMR (400 MHz, CDCl
3
) δ 2.87 (t, 2H, C
H, CH2); 6.97ꢀ7.02 (m, 2H, aryl); 7.09 (t, 1H, aryl,
H, aryl, = 8.0 Hz); 7.51 (d, 1H, aryl). HRꢀMS m/z calcd for C H N [(M+H) ]: 251.1543;
H
2
,
J
= 8.2 Hz); 2.96 (t, 2H, C
2
H , J = 8.2 Hz); 4.91 (s,
2
1
J
= 8.0 Hz); 7.20ꢀ7.26 (m, 5H, aryl); 7.35 (d,
+
J
1
7
19
2
found 251.1549
Synthesis of (R,S)-1-phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (6)
Tryptamine (1.0 equiv) was dissolved in a solution of DCM/CH COOH (4:1 v/v) at room
3
temperature and benzaldehyde (1.0 equivalent) was added. The mixture was stirred for 3h and then
was quenched by 1N aqueous NaOH. The organic phase was extracted one more time. Then it was
ACS Paragon Plus Environment

Page 27 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
dried over Na SO , filtered, and evaporated. The crude product was purified by column
2
4
chromatography using a mixture of DCM/MeOH (4.5/0.5 v/v) as eluent, obtaining intermediate 6 as
a Colourless oil in 82% yield
1
H NMR (400 MHz, CDCl ) δ 2.72ꢀ2.79 (m, 1H, CH2); 2.83ꢀ2.91 (m, 1H, CH2); 3.04ꢀ3.11 (m, 1H,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
H
2
); 3.29ꢀ3.34 (m, 1H, C
H
2
); 5.11 (s, 1H, C
H
); 7.02ꢀ7.09 (m, 2H, aryl); 7.15 (d, 1H, aryl,
J
= 8.0
Hz); 7.24ꢀ7.29 (m, 5H, aryl); 7.43 (s, 1NH); 7.47 (d, 1H, aryl,
J = 8.0 Hz). HRꢀMS m/z calcd for
+
C
17
H
17
N
2
[(M+H) ]: 249.1386; found 249.1395
General Procedure for the Synthesis of Derivatives 7, 8.
Compound 5 (1.0 equiv) was dissolved in acetone and NaI (0.8 equiv), Cs
2
3
CO (2.0 equiv),
Pd(OAc) (0.01 equiv) were added together with the proper alkyl or aryl bromide (0.8 equiv). The
2
reaction was refluxed for 12h. The resulting mixture was filtered through Celite, dried in vacuo, and
reconstituted in DCM. The organic phase was washed with water (3 × 50 mL), dried over
2 4
anhydrous Na SO , filtered, concentrated, and purified by column chromatography using ethyl
acetate/nꢀhexane as mobile phase.
N-benzyl-N-(cyclohexylmethyl)-2-(1H-indol-3-yl)ethanamine (7)
1
Colourless oil (39% yield) H NMR (400 MHz, CDCl ) δ 0.69ꢀ0.78 (m, 2H, CH2); 1.03ꢀ1.13 (m,
3
3H, C
H2
.87 (s, 1H, aryl); 6.99 (t, 1H, aryl,
.0 Hz); 7.22 (t, 3H, aryl, = 8.0 Hz); 7.28 (t, 2H, aryl,
.79 (s, 1NH); C NMR (100 MHz, CDCl ) δ 23.1; 26.2; 26.9; 31.9; 36.2; 54.8; 59.4; 61.4; 111.0;
H
2
); 1.40ꢀ1.47 (m, 1H, C
H
); 1.58ꢀ1.62 (m, 3H, C
H
2
); 1.73ꢀ1.77 (m, 2H, C
H
2
); 2.24 (d, 2H,
= 8.2 Hz); 3.58 (s, 2H, CH2);
= 8.0 Hz); 7.15 (t, 1H, aryl,
= 8.0 Hz);
C
,
J
= 8.0 Hz); 2.67 (t, 2H, CH2
,
J
= 8.2 Hz); 2.84 (t, 2H, CH2
,
J
6
8
7
1
J
= 8.0 Hz); 7.08 (t, 1H, aryl,
J
J =
J
J
= 8.0 Hz); 7.40 (d, 1H, aryl,
J
1
3
3
15.0; 118.9; 119.1; 121.4; 121.8; 126.6; 127.6; 128.0; 128.8; 136.2; 140.5. HRꢀMS m/z calcd for
+
C H N [(M+H) ]: 347.2482; found 347.2490
24
31
2
N-benzyl-2-(1H-indol-3-yl)-N-(4-nitrobenzyl)ethanamine (8)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Yellow oil (53% yield) H NMR (400 MHz, CDCl
= 7.2 Hz); 3.74 (s, 2H, C ); 3.76 (s, 2H, C
= 8.0 Hz); 7.30ꢀ7.47 (m, 9H, aryl); 7.94 (s, 1NH); 8.08 (d, 2H, aryl,
3
) δ 2.85 (t, 2H, C
2
H , J = 7.2 Hz); 3.01 (t, 2H,
C
H
2
,
J
H
2
H
2
); 6.94 (s, 1H, aryl); 7.04 (t, 1H, aryl,
J
J
= 8.0
= 8.8
Hz); 7.19 (t, 1H, aryl,
J
13
Hz); C NMR (100 MHz, CDCl
3
) δ 23.3; 54.3; 57.8; 58.6; 111.1; 114.3; 118.7; 119.2; 121.5;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
22.0; 123.3; 127.1; 127.4; 128.4; 128.8; 129.1; 136.2; 139.2; 146.9; 148.1. HRꢀMS m/z calcd for
+
C H N O [(M+H) ]: 386.1863; found 386.1869
24 24
3
2
General Procedure for the Synthesis of Derivatives 9-11.
Final compounds 9-11 were obtained by reaction of intermediate 6 with the proper alkyl or aryl
bromide in the same conditions described for compounds 7 and 8.
(
R,S)-2-(4-nitrobenzyl)-1-phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (9)
1
Yellow oil (48% yield) H NMR (400 MHz, CDCl
3
) δ 2.73 (dd, 1H, Hꢀ4’, J’ = 6.0, J” = 1.8 Hz);
2.78 (d, 1H, Hꢀ4”,
J
= 7.8 Hz); 2.86ꢀ3.02 (m, 1H, Hꢀ3’); 3.16 (dd, 1H, Hꢀ3”, J’ = 1.8, J” = 16.0
benzyl, = 16.0 Hz); 3.98 (d, 1H, C benzyl, = 16.0 Hz); 4.71 (s, 1H, Hꢀ
Hz); 3.51 (d, 1H, C
H
2
J
H
2
J
1
); 7.11ꢀ7.17 (m, 2H, aryl); 7.22 (d, 1H, aryl,
J
= 8.0 Hz); 7.29 (s, 1NH); 7.35ꢀ7.43 (m, 3H, aryl);
13
7
.47 (d, 2H, aryl, = 8.4 Hz); 7.55 (d, 3H, aryl,
J
J
= 8.0 Hz); 8.20 (d, 2H, aryl,
J
= 8.0 Hz); C
NMR (100 MHz, CDCl ) δ 21.3; 48.9; 57.8; 65.1; 108.8; 110.8; 118.3; 119.5; 121.7; 123.5; 127.1;
3
+
1
28.4; 134.4; 136.3; 140.9; 147.1; 147.7. HRꢀMS m/z calcd for C24
H
22
N
3
O
2
[(M+H) ]: 384.1707;
found 384.1715
(R,S)-4-((1-phenyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)methyl)benzoic acid (10)
1
White solid (45% yield) H NMR (400 MHz, DMSO) δ 2.63ꢀ2.71 (m, 1H, Hꢀ4’); 2.75ꢀ2.82 (m,
2
J
1
H, Hꢀ3); 2.98ꢀ3.03 (m, 1H, Hꢀ4”); 3.61 (d, 1H, C
= 12.6 Hz); 4.77 (s, 1H, Hꢀ1); 6.96 (t, 1H, aryl,
H, aryl, = 7.8 Hz); 7.29ꢀ7.37 (m, 5H, aryl); 7.43 (d, 1H, aryl,
H
2
benzyl,
= 7.8 Hz); 7.01 (t, 1H, aryl,
= 8.0 Hz); 7.47 (d, 2H, aryl,
= 8.0 Hz); 10.35 (s, 1NH). C NMR (100 MHz, DMSO) δ 20.6; 46.9;
J
= 12.0 Hz); 3.78 (d, 1H, C
2
H benzyl,
J
J
= 7.8 Hz); 7.23 (d,
J
J
J =
13
7.8 Hz); 7.92 (d, 2H, aryl,
J
4
1
9.1; 57.4; 63.1; 107.5; 111.6; 118.1; 118.8; 121.1; 126.9; 128.0; 128.7; 129.3; 129.8; 130.0; 134.9;
+
23 2 2
36.8; 142.1; 145.3; 167.7. HRꢀMS m/z calcd for C25H N O [(M+H) ]: 383.1754; found 383.1761
ACS Paragon Plus Environment

Page 29 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
R,S)-1-phenyl-2-(1-phenyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)ethanone (11)
1
Colourless oil (60% yield) H NMR (400 MHz, CDCl
3
) δ 2.87ꢀ2.92 (m, 1H, Hꢀ4’); 2.98ꢀ3.03 (m,
benzyl, = 16.4
= 16.4 Hz); 5.00 (s, 1H, Hꢀ1); 7.12ꢀ7.17 (m, 2H, aryl); 7.22 (d, 1H,
= 7.2 Hz); 7.29 (s, 1NH); 7.36ꢀ7.43 (m, 7H, aryl); 7.54 (t, 2H, aryl, = 8.4 Hz); 7.87 (d, 2H,
= 8.4 Hz); C NMR (100 MHz, CDCl ) δ 20.9; 49.0; 60.3; 63.5; 109.2; 110.8; 118.3; 119.4;
1
H, Hꢀ4”); 3.06ꢀ3.11 (m, 1H, Hꢀ3’); 3.27ꢀ3.31 (m, 1H, Hꢀ3”); 3.83 (d, 1H, C
H
2
J
Hz); 4.15 (d, 1H, C benzyl,
H
2
J
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aryl,
aryl,
J
J
J
13
3
121.7; 127.1; 128.2; 128.4; 128.7; 129.5; 130.0; 133.1; 134.0; 136.1; 136.3; 140.4; 198.1. HRꢀMS
+
m/z calcd for C25
H
23
N
2
O [(M+H) ]: 367.1805; found 367.1812
Synthesis of 2-(2-(1H-indol-3-yl)ethyl)-2,3-dihydro-1H-benzo[de]isoquinoline (13)
One equivalent of 3ꢀ(2ꢀbromoethyl)indole was dissolved in acetone and 1.5 equiv of 2,3ꢀdihydroꢀ
1
Hꢀbenzo[de]isoquinoline (12), 2.0 equiv of Cs CO , 1.0 equiv of NaI, and 0.01 equiv of
2 3
(
CH
mixture was filtered through Celite, dried in vacuo, and reconstituted in DCM. The organic phase
was washed with water (3 × 50 mL), dried over anhydrous Na SO , filtered, concentrated, and
3 2
COO) Pd were added to this solution. The reaction was refluxed for 12h. The resulting
2
4
purified by column chromatography using DCM/MeOH as mobile phase.
1
Colourless oil (69% yield) H NMR (400 MHz, CDCl
3
) δ 2.93 (t, 2H, C
); 6.97 (s, 1H, aryl); 7.03ꢀ7.17 (m, 4H, aryl); 7.26 (d, 1H, aryl,
= 7.2 Hz); 7.57 (d, 1H, aryl, = 7.6 Hz); 7.63 (d, 2H, aryl, = 8.0
) δ 23.5; 56.9; 58.2; 111.1; 114.3; 119.0; 119.4;
21.7; 122.4; 125.4; 126.5; 127.5; 128.2; 133.2; 133.4; 136.3; 156.6. HRꢀMS m/z calcd for
2
H , J = 7.6 Hz); 3.10 (t, 2H,
C
=
H
2
,
J
= 7.2 Hz); 4.03 (s, 4H, C
H
2
J
8.0 Hz); 7.33 (t, 2H, aryl,
J
J
J
1
3
Hz); 7.92 (s, 1NH); C NMR (100 MHz, CDCl
3
1
+
C H N [(M+H) ]: 313.1699; found 313.1708
22
21
2
General Procedure for the Synthesis of Derivatives 14-16.
N,Nꢀdibenzyl derivatives of or ꢀTryptophanꢀOMe (14, 16) were obtained by nucleophilic
L
D
substitution using an excess of (bromomethyl)benzene in the same conditions described for the
synthesis of compound 1 and 2. Starting from 14, an alkaline hydrolysis of ester group using NaOH
in H O/MeOH (4/1 v/v) led to acid compound 15.
2
ACS Paragon Plus Environment