Exploration of TRPM8 Binding Sites by β-Carboline-Based Antagonists and Their in Vitro Characterization and in Vivo Analgesic Activities

Article abstract of DOI:10.1021/acs.jmedchem.0c00816

Transient receptor potential melastatin 8 (TRPM8) ion channel represents a valuable pharmacological option for several therapeutic areas. Here, a series of conformationally restricted derivatives of the previously described TRPM8 antagonist N,N′-dibenzyl

Full text of DOI:10.1021/acs.jmedchem.0c00816

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Article
Exploration of TRPM8 Binding Sites by #-Carboline-based Antagonists
and Their In Vitro Characterization and in Vivo Analgesic Activities
Alessia Bertamino, Carmine Ostacolo, Alicia Medina, Veronica Di Sarno, Gianluigi Lauro,
Tania Ciaglia, Vincenzo Vestuto, Giacomo Pepe, Manuela Giovanna Basilicata, Simona
Musella, Gerardina Smaldone, Claudia Cristiano, Sara Gonzalez-Rodriguez, Asia Fernández-
Carvajal, Giuseppe Bifulco, Pietro Campiglia, Isabel M. Gomez-Monterrey, and Roberto Russo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00816 • Publication Date (Web): 29 Jul 2020
Downloaded from pubs.acs.org on August 4, 2020
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Exploration of TRPM8 Binding Sites by Carboline-based
Antagonists and Their In Vitro Characterization and in Vivo
Analgesic Activities
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Alessia Bertamino,†,# Carmine Ostacolo,‡,# Alicia Medina, Veronica Di Sarno, Gianluigi Lauro,
∥,#
†
†
Tania Ciaglia, † Vincenzo Vestuto, Giacomo Pepe, Manuela Giovanna Basilicata, Simona
†
†
†
§
†
‡
∥
Musella, Gerardina Smaldone, Claudia Cristiano, Sara Gonzalez-Rodriguez, Asia Fernandez-
Carvajal, Giuseppe Bifulco, Pietro Campiglia,*,†, § Isabel Gomez-Monterrey,*,‡ and Roberto
∥
†
Russo.‡
†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
∥IDiBE, Universitas Miguel Hernández, Avda de la Universidad , 032020, Elche, Spain
§European Biomedical Research Institute (EBRIS), Via S.De Renzi 50, 84125, Salerno, Italy
# These authors contributed equally to this work
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ABSTRACT
Transient receptor potential melastatin 8 (TRPM8) ion channel represents a valuable
pharmacological option for several therapeutic areas. Here, a series of conformationally restricted
derivatives of the previously described TRPM8 antagonist N,N′-dibenzyl tryptophan 4 were
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prepared and characterized in vitro by Ca -imaging and patch-clamp electrophysiology assays.
Molecular modeling studies led to identification of a broad and well-defined interaction network of
these derivatives inside the TRPM8-agonist binding site, underlying their antagonist activity. The
(5R,11aS)-5-(4-chlorophenyl)-2-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1H-
imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione (31a) emerged as a potent (IC50 4.10 ± 1.2
nM), selective, and metabolically stable TRPM8 antagonist. In vivo, 31a showed significant target
coverage in an icilin-induced WDS (at 11.5 mg/kg i.p.), an oxaliplatin-induced cold allodynia (at
10−30 μg s.c.), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg i.p.) mice models. These
results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly
potent modulators of TRPM8-mediated activities.
INTRODUCTION
The transient receptor potential melastatin type 8 (TRPM8) is a member of the thermo-TRP family1
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of polymodal, non-selective, and Ca permeable ion channel, identified as the physiological sensor
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of environmental cold. TRPM8 is activated by a range of innocuous to noxious cold temperatures
10−28°C),2c, 3 natural and synthetic cooling agent,2c, 4 membrane depolarization, changes in
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extracellular osmolarity and phosphatidylinositol-4,5-biphosphate (PIP ).
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Originally expressed in a prostate cancer cell line, TRPM8 was subsequently detected in a subset
of primary afferent neurons in the dorsal root ganglion (DRG) and trigeminal ganglia (TG),2c, 9
which innervate cold highly sensitive tissues, such as skin, oral cavity epithelium, teeth, tongue and
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cornea.9a, 10 TRPM8 is also expressed in visceral tissues innervated by pelvic or vagal nerves,11
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several tumor cells, macrophages , and different regions in rodents brain. Regulation of the
TRPM8-expression and/or -morphological changes in pathological processes involving these tissues
may represent a new opportunity for the therapeutic intervention in pain, cancer, inflammation, and
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metabolic diseases, among others. In particular, there is a large body of evidence that correlates
the hypersensitivity to cold, typical of neuropathic pain models, after nerve injury or oxaliplatin-
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treatment with augmented expression of TRPM8 in sensory neurons, suggesting that blocking the
channel can be a suitable approach to treat these pain conditions. In fact, TRPM8 gene deletion 17 or
pharmacological inhibition of the channel both in animal models and humans is correlated with a
decreased cold hypersensitivity in neuropatic,17-18 chronic visceral pain,19 and also migraine.20
Considering these finding and the potential activity of TRPM8 antagonists also in cancer and other
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pathologies, it is easy to understand the effort of the academic groups and pharmaceutical/biotech
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companies to develop potent and selective TRPM8 modulators. To date two antagonists, the
quinoline-3-carboxamido derivative PF-0510567918a and the amino-2-oxoethyl nicotinic acid
derivative AMG-33320 (Chart 1), have been evaluated for treatment of cold related pain and
migraine, respectively, although they have not passed phase I studies. In 2017, two undisclosed
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18d
structures, named RQ 00434739 and Ice 3682 , have reached clinical trials for the treatment of
neuropathic pain in Japan and Israel, respectively.
Chart 1. Structures of some TRPM8 antagonists
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In the last years, the information obtained through mutagenesis experiments24 and molecular
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modeling studies on the structure-function of TRPM8 channels have suggested the existence of
several independent and overlapping pockets in the TRPM8 binding site able to interact with
different antagonist chemotypes.15c, 22, 26 This makes difficult to rationalize pharmacological results,
particularly in the context of neuropathic pain, where also agonist of TRPM8 are able in inducing
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analgesia, as well as to define the molecular basis for TRPM8 antagonism. Recently the group of
Lee 28a, 28b resolved the structure of full-length TRPM8 protein from the collared flycatcher
Ficedula albicollis (TRPM8 ) using cryo-electron microscopy. The network of interactions
FA
generated from the TRPM8 / menthol, icilin, or lipids lays the structural basis for the design and
FA
identification of potent and selective ligands. Importantly, in 2019 two novel structures of TRPM8
complexed with the two antagonists AMTB and TC-I 2014 (PDB codes: 6O6R and 6O72) were
released, thus providing further important structural details for aiding the identification of TRPM8
modulators.28c
In this context, we have also recently generated a homology model of human TRPM8 using the
TRPM8FA structure as template to rationalize the potent antagonist activity showed by a
tryptamine29 and a tryptophan-based18b TRPM8 modulators (3 and 4, Chart 1). In patch-clamp
recordings, these compounds demonstrated to be more potent (IC = 367 and 0.2 nM respectively)
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than the well-known TRPM8 antagonist BCTC. In vivo, compound 4 attenuated icilin-induced
shaking behaviors and reversed oxaliplatin-induced cold allodynia in mice model. Docking studies
disclosed the voltage sensor region (VSLD), in the transmembrane segments portion S1-S4, as a
possible binding site for these derivatives, highlighting the ability of both compounds to affect the
network of interactions established between TM (S1-S4) and the TRP domain at C-terminal of the
channel subunits.
In order to deepen the structural requirements necessary to the TRPM8 antagonist activity of these
indol-based derivatives, we designed and synthesized a new series of conformationally restricted
analogues of 4 pursuing a double aim: a) to increase the metabolic stability of our lead compound
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by decreasing its amino acid character; b) to explore new TRPM8/antagonist interactions leading to
the potential discovery of SAR clues. In this paper, we discuss the design and synthesis of three
different series of tryptophan restricted analogues of the lead compound 4, namely tetrahydro--
carbolines (THBCs), THBC-based diketopiperazines, and THBC-based hydantoin derivatives, as
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well as the results of TRPM8 antagonist activity obtained by assays of Ca fluorescence and patch-
clamp measurements. These data were rationalized by molecular modelling studies defining new
structural requirements for the TRPM8 antagonist activity. Finally, the most potent compound
identified was tested in three different in-vivo pain models.
RESULTS AND DISCUSSION
Chemistry
Tetrahydrobetacarbolines (THBCs) 6a,b, 9 and 10-12a,b, were synthesized as depicted in Scheme
1
.
Scheme 1. Synthesis of substituted Tetrahydro--Carbolines (6a,b and 10-12a,b)
COOCH3
NH
COOCH3
N
COOCH3
NH2
N
Ph
N
H
Ph
H
PhCHO
BzlBr, NaI
5a
6a
TFA, MeOH
Pd(OAc) , THF
N
H
COOCH3
2
COOCH3
W
TEA, W
NH
N
N
H
Ph
N
H
Ph
O
5b
6b
NH
NH
F
R
9
H
O
H
N
O
H
F
N
F
N
R
COOH
NHBoc
H
3 2 2
10a,10b (CH ) CHCH -
NH2
NH2
9
, 10a-12a
NHBoc
F
DCM/TFA
TIS
RCHO
TFA, MeOH
11a,11b CH3OCOCH2CH2-
12a,12b HOCOCH CH -
O
HoBt, HBTU
Dipea, DCM/DMF
NH
N
H
N
N
H
F
2
2
H

W
NH
R
7
8
N
H
10b-12b
Starting from a microwave assisted Pictet-Spengler reaction of L-Trp-OMe with benzaldehyde and
TFA in methanol the THBC intermediates 5a,b were obtained as a diasteroisomeric mixtures (2:1
cis:trans), which were resolved by flash chromatography. N-benzylation reaction of the pure
diastereoisomers with benzyl bromide, sodium iodide, and triethylamine in THF using palladium
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acetate as catalyst and W irradiation, led to the final trans-(6a) and cis-(6b) THBCs in 62 and
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9% yields, respectively. The relative configuration for 6a and 6b was assigned by ROESY NMR
spectra considering the cross peak between H1-H3 that is present for 6b (H1:  4.96 ppm; H3  3.87
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ppm, Figure S8), while it is missing in 6a (H1:  5.39 ppm; H3  3.87 ppm, Figure S5). Assuming
the absolute configuration for the L-tryptophan moiety is maintained in the reaction conditions, the
configuration at C1 was assigned accordingly. The same key correlation was used to assign the
absolute configuration of the other THBCs derivatives (10-12 a,b). Reaction of NBoc-L-Trp-OH
with 4-F-benzylamine, using HoBt and HBTU as coupling agents and DIPEA as base in a mixture
of DCM/DMF, gave the amide intermediate 7, which was then deprotected in DCM/TFA (3:1 v:v).
The free amine 8 was subjected to a Pictet-Spengler reaction with formaldehyde, or
isovaleraldehyde, or methyl-4-oxobutanoate or 4-oxobuthanoic acid in the above described
conditions, leading to the formation of THBC 9 (55% of yield) and the trans-cis mixtures 10-12a,b.
Flash chromatography allowed the separation of the corresponding trans 10-12a (35-43% yields)
and cis 10-12b (33-43% yields) diastereoisomers. On the basis of 2D-NMR correlations and
considering the fixed configuration at C-3 as S, we assigned the configurations to the THBC trans
1
0-12a and cis 10-12b as (1R,3S) and (1S,3S), respectively.
Scheme 2. Synthesis of Tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione derivatives (17,
21-23, 25a, 25b, 27a, and 27b)
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THBC derivatives 13, 14a-b, and 15a-b, were obtained following the procedure described above by
reaction of L-Trp-OMe with formaldehyde, isovaleraldehyde and 4-Cl-benzaldehyde, respectively
(Scheme 2). Coupling of these intermediates with NHBoc--Ala-OH, NHBoc-Gly-OH, NHBoc-L-
Phe-OH, or NHBoc-D-Phe-OH using HoBt, HBTU in DCM/DMF gave the pseudo dipeptide
intermediates 16, 18-20, 24a-b and 26a-b in 25-71% yield, respectively. Boc-deprotection of the
amino group in TFA acid medium of the derivatives 18-20, 24a-b and 26a-b, followed by a
spontaneous intramolecular cyclization provided the final THBC-based diketopiperazines 21-23,
2
5a-b, 27a-b with 73-85% of yields. As expected, the treatment of 16 with TFA gave the
unprotected derivative 17 in quantitative yield. In order to determine the relative configuration for
the tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione derivatives, cross peak
between H6-H12a was investigated trough ROESY NMR. For example, 27b shows a cross peak
between  6.16 ppm (H6) and  4.30 ppm (H12a, Figure S43) while this correlation is missing for
the diastereoisomer 27a ( 6.67 ppm;  4.18 ppm, Figure S40). The absolute configuration was
attributed considering the retention of the L-tryptophan chirality.
Scheme 3. Synthesis of Tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione derivatives (28, 29a-31a,
33a, 35a and their enantiomers 29a’-31a’, 33a’, 35a’)
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O
N
F
N
Triphosgene, TEA
-F-BzlNH2, THF
O
1
3
4
N
H
28
O
O
N
N
OCH3
or
OCH3
N
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O
O
Triphosgene, TEA
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4-CH3O-BzlNH2, THF
N
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H
H
29a
29a'
O
O
N
N
R
R
R
Triphosgene, TEA
4-R-BzlNH2, THF
N
N
O
CH3 30, 30'
or
1
5a, 15b
F
31, 31'
N
N
H
H
Cl
Cl
3
0a, 31a
30a', 31a'
O
O
N
N
N
N
O
O
BzlNH2
COOCH3
or
N
N
H
H
NH
3
3a
33a'
Triphosgene
THF, TEA
F
F
N
H
O
O
O
O
NHBoc
NHBoc
NH2
NH2
N
N
N
N
F
N
N
N
N
32a or 32b
O
O
DCM/TFA/TIS
O
O
or
or
BocNH(CH2)3NH2
N
H
N
H
N
H
N
H
34a
34a'
35a
35a'
F
F
F
F
Final THBC-based hydantoin compounds were obtained through the synthetic method reported in
scheme 3. Reaction of the starting THBCs 13, 14a, 14b, 15a, 15b, and the now synthesized 32a and
3
2b with triphosgene and different amines, such as 4-F, 4-OMe, 4-Me-benzylamine, benzylamine,
and NHBoc propylendiamine in THF using TEA as base, led to the final hydantoin derivatives 28,
9a-31a, 33-34a, 29a’-31a’, 33-34a’, in one step and with 35-62% yields. NMR data,  values,
2
D
and circular dichroism experiments (see Supporting Information) showed that the reaction of trans
(1R, 3S) THBCs 14a, 15a, and 32a originated the trans derivatives (5R,11aS) 29a-31a, 33a and
3
4a, while the cyclization reaction from the cis analogues (1S, 3S) 14b, 15b, and 32b led to the
formation of the trans enantiomers (5S,11aR) 29a’-31a’, 33a’, and 34a’. For instance, the
configuration of THBCs 15a and 15b was assessed by ROESY NMR, investigating the correlation
between H3 and H1, assuming retention of configuration for the L-tryptophan moiety. As shown in
figure S89, only compound 15b showed the investigated correlation (H1:  5.25 ppm; H3  3.99
ppm). After cyclization reaction of 15b to the hydantoine derivative (30a’) the cross peak between
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the same hydrogens (H11a  4.30 ppm, H5  6.29 ppm) was not detected (Figure S89). This is in
3
0
accordance with literature, that describes the epimerization at C-3 position of the (1S, 3S) THBCs
during the cyclization process, resulting in the formation of the most stable trans (5S,11aR) THBC-
based hydantoin derivatives. Moreover, for all the THBC-based hydantoin enantiomeric couples, as
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
expected, we observed the same NMR chemical shifts and opposite values for  (see experimental
D
section), and specular circular dichroism spectra (Figure S89). Removal of the Boc protecting group
from 34a and 34a’ using TFA and triisopopylsilane (TIS) in dichloromethane led to the final
products 35a and 35a’, respectively.
On the other hand, Scheme 4 reports the synthesis of the N-aryl hydantoin derivatives 36a-38a and
3
6b-38b. In this case, a different chemical approach is required because of the minor reactivity of
anilines. Intermediates THBC 32a and 32b were coupled with 3CF , or 2F, or 4-F-phenyl
3
isocyanate in basic medium of TEA. In these conditions, we obtained the corresponding (5R, 11aS)
trans- and (5S, 11aS) cis-hydantoins (36a-38a and 36b-38b, respectively), which were isolated and
characterized by 2D-NMR spectroscopy. In particular, the cis configuration was evidenced by the
correlation between H11a and H5, corresponding to  4.53 ppm and  5.86 ppm, respectively, for
compound 36b (Figure S76). Absolute configuration was determined as described above. The
formation of the cis intermediates, that was not observed with the N-benzyl or N-alkyl analogs, can
be explained by the increased stability of the kinetic control species due to the higher rigidity of this
structure. However, C11a epimerization was not suppressed and we noticed that the cis conformers
converted to their thermodynamically more stable trans congeners (5S, 11aR) 36a’-38a’, with a
conversion kinetic depending on experimental conditions. High temperatures and alcoholic solvents
such as methanol and ethanol, favored the conversion to trans-derivative while in aqueous media at
room temperature the cis conformers were more stable (Figure S2). Therefore, given the
spontaneous trend of cis-hydantoins towards trans-conversion, we considered inappropriate the
pharmacological testing of all the cis conformers and we decided to assay only 36b for its
pharmacological activity, due to its higher stability in water environment in comparison with its
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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
congeners 37b and 38b, that were almost fully converted to the trans-conformers during 60 minutes
regardless of the solvent used (Figure S1). In addition, the corresponding C-11a epimers 36a’- 38a’
could be obtained directly, by reaction of 32b with the corresponding isocyanates and TEA at 60°C
for 30 minutes in 39-45% yields.
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
Scheme 4. Synthesis of THBC-base 2-arylhydantoin derivatives (36-38, their enantiomers 36b-38b and their
diastereoisomers 36a’-38a’)
O
O
COOCH3
COOCH3
R
O
R
O
N
N
NH
or
NH
N
N
RNCO
or
N
N
H
TEA, THF, RT
N
H
N
H
H
32a
F
32b
F
36a-38a
36b-38b
F
F
R
O
3
2
4
-CF -Ph 36a-b, 36a'
R
3
N
-F-Ph
-F-Ph
37a-b, 37a'
38a-b, 38a'
N
O
RNCO
EtOH
TEA, THF, 60°C
N
over time or 
H
36a'-38a'
F
Pharmacological characterization
2+
Screening by Ca -Imaging Assay
2
+
TRPM8 blocker activity of all synthesized compounds was tested by Ca fluorimetric assays using
HEK-293 cells stably expressing the rat isoform of TRPM8 channels, using menthol and AMTB as
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prototypical agonist and antagonist, respectively. All the compounds showed an antagonist activity
higher than the canonical TRPM8 antagonist AMTB, despite lower than the lead compound 4 with
IC50s in the 100-0.3 M range (Table 1).
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
In the 1, 2, 3-substituted THBCs (6, 9-12, and 17) series, the antagonist activity is conditioned by
the relative configuration at the position 1 when long and linear aliphatic chains are used. Thus, the
trans derivatives (1R,3S) 11a and 12a are around 4 times more active than the corresponding cis
diastereoisomers (1S,3S) 11b and 12b (IC = 1.1 M and 3.0 M for the 11a and 11b, respectively
5
0
and 5M and 22 M for 12a and 12b, respectively). The influence of the configurational pattern
was not observed for other diastereoisomer couples bearing bulkier alkyl or planar aryl substituents
in C-1 (6a/6b, 10a/10b). Indeed, the unsubstituted derivative at position C-1 (9) had an interesting
antagonistic activity with an IC value of 0.9 M, while the restricted pseudo dipeptide Trp--Ala
50
1
7, bearing a polar 3-aminopropanoic chain in position 2, was completely inactive (IC > 100 M).
5
0
Further expansion of the structure from TBHCs to diketopiperazines (21-23, 25a-b, 27a-b) retained
the antagonist activity. The unsubstituted TBHC-based diketopiperazine 21, for instance,
maintained good potency (11.4 M), but the introduction of a benzyl substituent at C-3 (22, 23, and
2
5) significantly increased activity with IC50 value in the range 4-0.4 M. In this case, the
configuration at position 3 did not significantly influence compound potencies that were
comparable for the (3R,12aS) diastereoisomer 23 and its 3-epimer (3S,12aS) 22 (IC s = 1.6±0.7
5
0
M and 0.4±0.1 M, respectively).
The trans derivative 25a (3S,6R,12aS) bearing the isobutyl moiety at C-6 was significantly less
potent than its cis diastereoisomer 25b (3S,6S,12aS) (IC = 4.1±1.1M vs 1.3±0.6 M), while the
5
0
introduction of the 4-Cl-phenyl moiety at position C-6 (27) was well tolerated and no differences in
potency were evidenced between the 6R,12aS and the 6S,12aS isomers (27a and 27b, IC = 1.5± 1.1
5
0
M and 1.7 ± 0.8 M respectively).
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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
Finally, the diketopiperazines ring was simplified to the five-membered hydantoin system. For this
series (28, 29a-31a, 33a, 35a, 36a-38a, 36b and their enantiomers 29a’-31a’, 33a’, 35a’-38a’) the
most active compounds 30a and 31a, with IC in the range 0.5-0.8 M, feature an aromatic group
5
0
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
at C-5 position, a benzyl group at N-2 and retain the trans configuration, 5R,11aS. The
unsubstituted compound 28 and the C-6 alkyl derivative 29a showed reduced potency. On the other
hand, the N-benzyl trans isomers (5S,11aR) 29a’-31a’, 33a’, 35a’, were less active than their
corresponding trans 5R,11aS enantiomers. This difference is not statistically significant for the N-
aryl derivatives (37a/37a’ and 38a/38a’), except for the compound 36a (5R,11aS), containing a 3-
trifluoromethyphenyl substituent at N2, that was about 3-fold more potent than its enantiomer
3
6a’(5S,11aR) (IC50 = 2.8 M and 7.8M, respectively). Compound 36b, the only tested cis
derivative of this series, showed a remarkable higher potency (IC = 0.2 M) than its two trans
5
0
analogues 36a and 36a’.
Table 1. Potency of Synthesized Compounds as TRPM8 Antagonists
Comp.
R
R
1
R
2
Configuration IC (M)
5
0
4
0.09 ± 0.08
1.3±0.7
1.6±0.9
0.9±0.4
4.6± 1.3
6.2± 1.2
1.1 ±0.5
3.0 ±1.2
5.0 ±1.2
22.0 ±1.4
>100
6
6
9
1
1
1
1
a
OCH
OCH
3
3
C
C
6
6
H
H
5
5
CH
CH
2
2
C
C
6
6
H
H
5
5
1R, 3S
1S,3S
3S
b
NHCH
NHCH
NHCH
NHCH
NHCH
NHCH
NHCH
2
2
2
2
2
2
2
(4-F)Ph
(4-F)Ph
(4-F)Ph
H
H
H
H
H
H
H
H
0a
0b
1a
1b
CH
CH
2
2
CH(CH
CH(CH
3
3
)
)
2
1R,3S
1S,3S
1R,3S
1S,3S
1R,3S
1S,3S
3S
2
(4-F)Ph CH
(4-F)Ph CH
2
2
CH
CH
2
2
COOCH
COOCH
3
3
12a
(4-F)Ph
(4-F)Ph
CH
CH
2
2
CH
CH
2
2
COOH
COOH
1
1
2
2b
7
OCH
3
H
H
COCH
2
CH
H
2
NH
2
1
12aS
11.4±1.6
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2
2
2
2
2
H
CH
CH
CH
CH
2
2
2
2
C
C
C
C
6
6
6
6
H
H
H
H
5
5
5
5
3S,12aS
3R,12aS
3S,6R,12aS
3S,6S,12aS
6R,12aS
6S,12aS
11aS
1.6 ±0.7
0.4 ±0.1
4.1±1.1
3
H
5a
5b
CH
CH
2
2
CH(CH
CH(CH
3
3
)
)
2
2
1.3±0.6
27a
4-Cl-C
4-Cl-C
H
6
H
H
4
H
H
1.5± 1.1
1.7 ± 0.8
17.8 ± 1.2
2.8 ±1.2
22.9 ± 1.4
0.8 ±0.4
2.3 ± 0.8
0.5 ±0.3
>30
2
2
2
2
7b
8
6
4
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
aValues are
2 6 4
CH 4-(F)-C H
9a
9a’
CH
CH
2
2
CH(CH
CH(CH
3
3
5
5
5
5
)
)
2
CH
CH
2
2
4-(OCH
4-(OCH
3
3
) C
) C
6
H
H
4
4
5R,11aS
5S,11aR
5R,11aS
5S,11aR
5R,11aS
5S,11aR
5R,11aS
5S,11aR
5R,11aS
5S,11aR
5R,11aS
5S,11aR
5S,11aS
5R,11aS
5S,11aR
5R,11aS
5S,11aR
expressed as
the mean ±
standard
2
6
30a
4-(Cl)C
4-(Cl)C
6
6
6
6
H
H
H
H
CH
CH
2
2
4-(CH
4-(CH
3
3
)C
)C
6
6
H
H
4
4
30a’
3
3
3
3
1a
4
-(Cl)C
CH
CH
2
4-(F)-C
6 4
4-(F)C H
6
H
4
deviation of
at least three
independent
measuremen
ts.
1a’
3a
4-(Cl)C
2
4-(F)C
4-(F)C
4-(F)C
4-(F)C
4-(F)C
4-(F)C
4-(F)C
4-(F)C
4-(F)C
4-(F)C
6
H
5
CH
CH
2
2
C
C
6
6
H
H
4
4
6.4 ±1.2
17.5±1.4
5.1 ±1.2
27.2 ± 1.4
2.8 ±1.5
7.8 ± 2.4
0.2 ± 0.2
4.4±1.3
3a’
6
6
6
6
6
6
6
6
6
6
H
H
H
H
H
H
H
H
H
H
5
5
5
5
5
5
5
5
5
5
35a
(CH
(CH
2
2
)
)
3
NH
NH
2
2
3
3
3
3
5a’
6a
3
3-(CF
3-(CF
3-(CF
3
3
3
)C
)C
)C
6
6
6
H
H
H
4
Patch-
6a’
6b
4
4
Clamp
37a
2-(F)C
2-(F)C
4-(F)C
6
6
6
H
H
H
4
4
4
37a’
5.1 ±2.1
6.7 ± 1.2
7.2 ± 0.9
7.3 ± 1.5
Electroph
ysiology
Assay.
3
3
8a
8a’
4
-(F)C
6 4
4-(F)-C H
AMTB
Functional assay identified derivatives 6a, 9, 11a, 23, 31a and 36b among the most effective and
potent TRPM8 antagonist compounds with IC values in the submicromolar range. To provide
5
0
direct evidence for this activity, these derivatives were tested in HEK-293 cells transiently
expressing the human TRPM8 isoform by whole-cell voltage clamp experiments. Moreover, we
decided to perform whole-cell voltage clamp experiments also for compounds 12a and 31a’, in
order to further highlight the pharmacophoric properties of the ester group in 11a and of the
stereocenters of 31a. As shown in Table 2, the well-known TRPM8 antagonist BCTC (300 nM),
used as reference, produced a complete inhibition of menthol-gated TRPM8 currents, with an IC50
of 501 nM. THBC-based diketopiperazine 23 and the hydantoin derivatives 31a have concentration-
dependent antagonistic activity, showing IC50s of 6.57±1.21 nM and of 4.10±1.52 nM respectively.
The THBC 6a and 9 showed decreased potency. The propanoic ester derivative 11a, identified as a
potent inhibitor of menthol-induced increase of intracellular Ca2+ levels (IC50 = 0.8 M),
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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
antagonized the effect of menthol with an EC of 15.41 nM, while its acid free analogue 12a
5
0
inhibited only 34% of the menthol-induced current at the maximum concentration of 300 nM. To
determine the role of the relative configuration at the stereocenters in the hTRPM8- blocking
activity of compound 31a, the pharmacological effect of its 5S,11aR enantiomer, namely 31a’, was
also investigated. As shown in Table 2, 300 nM of 31a’ weakly inhibited menthol-induced currents
showing very weak efficacy (11% inhibition) when compared to the 5R,11aS enantiomer, therefore
confirming the crucial role of the configurations in the pharmacological properties of this series of
compounds.
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
The activity of compound 36b, that proved to be a powerful antagonist of TRPM8 in Ca2+
fluorimetric assay, was confirmed by patch clamp experiment with an IC of 7.67 nM and an
5
0
inhibition efficacy of the menthol evoked currents of 59.4%. In the light of the reported
spontaneous epimerization of the cis isomer 36b to its trans congener (36a’) we hypothesized that
the cis-isomer contributed mainly to this pharmacological activity. Thus, 36b was assayed in a time
course stability test, and results confirmed that the percentage of epimerization was negligible
during patch-clamp electrophysiology assays (Figure S1).
Table 2. Full in vitro pharmacological characterization for selected compounds
%
Inhibition 300 M
menthol-evoked
IC50 Ca2+
50
IC (nM)
Comp.
Structure
(M)
currents at 300 nM
compounds
COOMe
N
6a
0.9±1.9
0.9±0.4
62.5 ±2.3
59.4±6.6
12.30±4.21
36.85±8.04
N
H
F
O
NH
NH
9
N
H
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7
8
9
F
O
NH
NH
1
1
1a
2a
1.1 ±0.5
5.0 ±1.2
0.4 ±0.1
62.1±4.2
34.1±9.6
61.1±6.3
61.7±8.7
11.6±3.6
15.46±5.63
N
H
COOMe
F
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
O
NH
NH
n.a.
N
H
COOH
O
N
NH
O
2
3
6.57±1.21
4.10±1.52
n.a
N
H
O
N
N
F
3
1a
O
0
.6 ±1.2
N
H
Cl
O
N
N
F
O
3
1a’
>30
N
H
Cl
CF3
O
N
N
36b
O
0.2 ±0.2
59.4±9.7
7.67±1.74
N
H
F
Selectivity studies
The most potent compounds identified by patch clamp studies (6a, 9, 11a, 23, 31a and 36b) were
subjected to further in vitro characterization by assessment of their selectivity towards TRPV1,
TRPA1 and Nav1.7 channels by calcium fluorimetric experiments. TRPV1 and TRPA1 channels
1
belong to the TRP superfamily and share a high degree of homology with TRPM8. On the other
hand, Nav1.7 channels are reported to be involved in several neuropathic pain pathways, also
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modulated by TRPM8. All the derivatives were unable to modulate these channels, showing no
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activity both as agonists or antagonists. Only compounds 6a and 9 showed a negligible antagonistic
activity over Nav1.7 with IC50s > 10 M (Figure S2).
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Molecular Modelling and Structural Rationale.
The TRPM8 three-dimensional structures co-complexed with the two antagonists AMTB and TC-I
2014 (PDB codes: 6O6R and 6O72) released by Diver et al. in 2019 revealed new important details
for developing potential modulators of this protein.28c The preliminary analysis and superposition
of the both the TRPM8 structures revealed a very similar protein architecture when bound with the
two different antagonists. Starting from these premises, the binding mode of the lead compound 4
was firstly re-evaluated considering the TC-I 2014-bound TRPM8 protein structure (PDB code:
6
O72), chosen as reference system since it featured a better resolution if compared with that
originally co-complexed with AMTB (PDB code: 6O6R). In particular, the obtained docking poses
of the lead compound 4 revealed a binding mode different from what reported in the original
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paper, in which an homology modeling structure of the protein was accounted. Indeed, in the TC-I
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014-bound protein structure (PDB code: 6O72), compound 4 adopted a particular shape in which
one aromatic function was in front of another one establishing an intramolecular π-π stacking
interaction. Specifically, the aromatic functions of 4 were π-π stacked with several residues
stabilizing the ligand/protein complex and allowing a large set of additional interactions, such as H-
bond contacts. Indeed, the indole function of 4 was involved in both π-π stacking (with Tyr736) and
π-cation (with Arg998) interactions, whereas one benzyl function also established an edge-to-face
π-π stacking with Phe729 (Figure 1). Also, H-bonds were detected for compound 4 with Asn732
and Gln776 (Figure 1).
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Figure 1. Compound 4 (colored by atom type: C orange, N blue, O red, polar H light grey) in docking with TRPM8
(represented in green ribbons; residues colored by atom type: C grey, N blue, O red, polar H light grey) in the TC-I
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014 ligand binding site. H bonds are represented with green dotted lines, π-cation interactions with yellow dotted lines,
and π- π interactions with light blue dotted lines. (PDB ID: 6O72)
In order to shed light about the possible mechanism of action of the reported β-carboline-based
TRPM8 antagonists, molecular docking calculations (Glide software) were performed. With the aim
of rationalizing the molecular basis behind the different antagonistic power of the tested molecules,
we specifically investigated the predicted protein-ligand complexes related to most representative
compounds 6a, 9, 11a, 11b, 12a, 12b, 23, 31a, 31a’, 36a, 36a’, 36b. In this way, we investigated
both the influence of the molecular architecture, namely accounting the tetrahydro--carboline (6a,
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, 11a, 11b, 12a, 12b), tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione (23),
tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione (31a, 31a’, 36a, 36a’, 36b)
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scaffolds, while also considering the effects of the different substituents as well as the impact of the
specific stereoarrangements for the three chemotypes on the observed biological activity.
The subsequent analysis of the ligand docking poses on this specific protein structure highlighted
further details for clarifying the action of the investigated compounds at a molecular level (Figures
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and 3). Firstly, the tetrahydro--carboline-based compound 6a, more conformationally restricted
if compared with its parent compound 4, showed a slightly different binding mode due to the flip of
the indole moiety (Figure 2a). On the other hand, the careful analysis of the superimposed poses of
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and 6a highlighted a similar total shape (Figure 2b), and this was further confirmed by detecting a
similar set of key interactions for both the compounds, such as the π-π stacking with Phe729 and the
polar contacts with Gln776 and Asn790 . Also, an additional π-π was detected with Tyr995,
whereas the terminal benzyl moiety established a partial π-π contact with Tyr736 (Figure 2a).
Figure 2. a) Predicted binding modes of 6a (colored by atom type: C pink) in docking with TRPM8 (represented in
green ribbons; residues colored by atom type: C grey, N blue, O red, polar H light grey) and b) superposition between
the predicted binding modes of 6a and 4 into the TRPM8 TC-I 2014 ligand binding site. H bonds are represented with
green dotted lines, π-cation interactions with yellow dotted lines, and π- π interactions with light blue dotted lines. (PDB
ID: 6O72)
Compound 9 occupied the TRPM8 binding site showing π-cation interactions with Arg832 and
Arg998 and further π-π interactions with Tyr736 (as in the starting compound 4, vide supra) and
Phe1003 through the 4-fluoro-benzyl function, whereas π-π stacking contacts were detected with
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Phe729 and Tyr995 through the indole moiety (Figure 3A). The introduction of a substituent at C-1,
as in compounds 11a, 11b, 12a, 12b, determined a similar accommodation in the TRPM8 binding
site (Figures 3B-E). Specifically, in the cases of compounds 11a/12a the tetrahydro--carboline
moiety was oriented in front of Phe729 and Tyr995 residues, while the 4-fluoro-benzyl substituent
interacted again with Arg998 through a π-cation and with Tyr736 through a π-π stacking (Figures
3B and 3D). Also, the acid moiety in 12a allowed a further H-bond interaction with Arg998 (Figure
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D). On the other hand, the different stereochemical arrangements of the related analogs 11b/12b
(featuring 1S,3S configuration, instead of 1R,3S as for compounds 11a/12a) determined a slightly
different binding mode. Specifically, for compound 11b, the 4-fluoro-benzyl substituent was
inserted in a deep cavity in front of Phe1003, while the π-π stacking interactions with Phe729 and
Tyr995 were again detected as well as further H-bonds with Asn732 and Arg998 (Figure 3C). A
quite similar binding mode was observed for compound 12b, in which the terminal carboxylate
function was involved in H-bond interactions with Asn732 and Arg998, whereas the 4-fluoro-
benzyl substituent showed in this case a π-π interaction with Tyr736 (Figure 3E)
The introduction of a conformational restriction in compound 23, featuring four fused rings
(tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione scaffold), determined
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different occupation of the binding site, namely with the indole moiety establishing a π-cation
interaction with Arg998 and Arg832, whereas the terminal benzyl moiety made further π-π contacts
with Phe729 and Tyr995 (Figure 3F). Concerning compounds 31a, again featuring four fused rings
(tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione scaffold), the presence of a
substituent at C-5 determined a flip of the indole moiety, able to interact with Phe729 and Tyr995
through π-π stacking contacts, as previously observed for 11a, 11b, 12a, 12b that, interestingly, also
featured an additional substituent at C-1, corresponding to C-5 in 31a/31a’. Also, the 4-Cl-phenyl
substituent at C-5 determined further π-π interaction with Tyr736, whereas an H-bond contact was
established with Arg998 (Figure 3G). As expected, a similar binding mode was detected for
compound 36a, featuring the same absolute configurational pattern of 31a, but the presence of a
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phenyl substituent at N-2 instead of a benzyl determined a slightly different accommodation of the
tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione core and the consequent lack of
the π-π stacking between the aromatic substituent at C-5 and Tyr736 (as observed for 31a), replaced
by an additional π-cation with Arg998 (Figure 3I). On the other hand, the corresponding
enantiomeric species of 31a and 36a, namely compounds 31a’ and 36a’, respectively, showed a
different occupation of the TRPM8 binding site due to the different stereoarrangements, especially
for what concerns the position of the terminal substituted benzyl and aryl moieties, not in line with
all the above reported structure-activity observations, suggesting the poor consistency of this mode
of binding that could explain the detected related decreases of antagonistic activity against TRPM8
(Figures 3H and 3J). Interestingly, compound 36b, the only one of the series featuring the 5S,11aS
absolute configuration, showed a three-dimensional arrangement onto the TRPM8 compatible with
the establishment of the key interactions with the receptor counterpart, namely the π-π stacking with
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Phe736 through the terminal 3-(CF )-aryl moiety (also able to interact with Arg998 through a π-
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cation) as well as the π-π interaction with Phe729 and Tyr995 with the indole moiety (Figure 3K).
In summary, the comparison of the predicted binding modes related to the reference compound 4
and of the new identified TRPM8 inhibitors disclosed a similar accommodation in the ligand
binding site, with the subsequent respect of a network of specific interactions with key residues in
the receptor counterpart (e.g. Phe729, Tyr736, Tyr995, Arg998). These in silico results shed light
about the rationalization of the observed antagonistic activity of the new identified compounds,
providing structural insights for the development of new agents able to interfere with the activity of
this target. Starting from these encouraging data at a molecular level, we then moved to the
investigation of specific molecular properties of the identified compounds (e.g. in vitro metabolism,
vide infra) for selecting the most promising items and for further deepening their antagonistic
pharmacological profile against TRPM8.
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Figure 3. Predicted binding modes of: a) compound 9 (colored by atom type: C light violet), b) 11a (colored by atom
type: C grey), c) 11b (colored by atom type: C purple), d) 12a (colored by atom type: C yellow), e) 12b (colored by
atom type: C orange), f) 23 (colored by atom type: C pale blue), g) 31a (colored by atom type: C paded red), h) 31a’
(colored by atom type: C red-orange); i) 36a (colored by atom type: C violet); j) 36a’ (colored by atom type: C light
purple); k) 36b (colored by atom type: C light green) in docking with TRPM8 (represented in green ribbons; residues
colored by atom type: C grey, N blue, O red, polar H light grey) in the TC-I 2014 ligand binding site. H bonds are
represented with green dotted lines, π-cation interactions with yellow dotted lines, and π- π interactions with light blue
dotted lines. (PDB ID: 6O72)
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In vitro metabolism
The most potent compounds analyzed by patch clamp electrophysiological assays were further
characterized for their metabolic stability using human liver microsomes as in vitro model.
Compound 4 was used as reference, considering that its main pitfall was represented by metabolic
instability that the newly synthesized compounds were aimed in overcoming. As shown in figure 4,
almost all the compounds proved to be stable in absence of metabolic cofactors (NADPH or UDP-
GlcUA/NADPH) except for 11a, showing unspecific metabolic liability (black bars). In fact, after
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0 minutes in contact with liver microsomes, in the absence of any metabolic co-factors, 11a
turnover was 66.5±3.8%. When the phase I metabolism condition were mimicked (please see
protocol I, material and methods section), compound 4 was massively metabolized with a turnover
percentage of 98.3 ± 3.1% (Figure 4, gray bars), in accordance with our previously reported data.18b
Indeed, the newly synthesized analogues showed improved metabolic stability with a metabolic
turnover in the range 1.1-72.0% under phase I metabolism conditions. In particular, compound 31a
with a phase I metabolic turnover of 26.5±3.9% was the most stable compound. For these reasons,
stability of derivative 31a was further challenged using a different protocol that involved both phase
I and phase II metabolic co-factors. As shown in figure 4 (white bar), 31a proved a slow metabolic
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turnover (46.0±2.3%) in the experimental conditions used and was then selected for the in vivo
pharmacological assays.
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Figure 4: In vitro metabolic stability assays for the selected compounds. a) compounds unspecific metabolism in
absence of co-factors calculated as ([ ]t60/[ ]t )×100, black bars; b) compounds metabolic stability under phase I
metabolism (gray bars, protocol I, please see material and method section); c) compound 31a metabolic stability under
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phase I + phase II metabolism (white bar, protocol II, please see material and method section).
In vivo experiments
Effect of 4 and 31a on icilin-induced WDS
Initially, we have evaluated the capability of TRPM8 antagonist 31a in blocking the spontaneous
wet-dog shake (WDS) induced by icilin in comparison with its precursor derivative 4 at equimolar
doses. Due to the difference in metabolic stability, a prolonged pharmacological effect of 31a was
expected. For this purpose, 4 and 31 were administrated 30 minutes before the challenge with icilin
(1 mg/kg i.p.) and WDS were recorded for 30 min. In the vehicle-treated group, a mean of about
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28 shakes were counted (Figure 5, white column). As expected, from the metabolic stability
experiments, the pre-treatment with 4 (10 mg/kg i.p) significantly decreased the number of icilin-
induced WDS 0.5 hours after the injection (Figure 5; **p< 0.01 vs. vehicle treated mice); no effect
was observed at 2 hours. On the contrary, 31a (11.5 mg/kg i.p) showed a significant effect both at
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.5 and 2 hours after the injection (Figure 5; *p<0.05 and **p < 0.01 vs. vehicle treated mice).
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Figure 5: Comparative effect of 4 and 31a on icilin-induced WDS in Swiss CD1 mice. After i.p. injection of icilin (10
mg/kg), the number of wet-dog shakes (WDS) were counted over 30 min. Data are given as mean ± SEM n = 6. 2-way
ANOVA with Bonferroni post hoc test. *P <0,05; **P <0,01.
Effect of 31a in neuropathic pain models
TRPM8 plays a critical role in mouse models of chemotherapy-induced neuropathic pain evoked by
oxaliplatin (OXP), a condition mimicking cold hypersensitivity provoked by chemotherapy-induced
peripheral neuropathy (CIPN). Both acute and chronic OXP-induced cold hypersensitivity have
been reproduced in rats and correlated with TRPM8 expression and function. Mizoguchi et al.33
reported that, in a rodent model, acute cold allodynia after OXP injection was alleviated by the
TRPM8 blockers N-(2-aminoethyl)-N-[4-(benzyloxy)-3- methoxybenzyl]-N’-(1S)-1-(phenyl) ethyl]
urea and TC-I 2014. According to these findings, we investigated the effect of our antagonist 31a in
an OXP-induced cold allodynia model, using acetone for cooling stimulation. Considering that the
cold pain threshold is increased from ≈12 °C to ≈ 26 °C in OXP-treated patients, acetone
stimulation is considered to evoke pain in OXP-treated mice.
The activity of compound 31a was evaluated 7 days after three intraperitoneal injections of OXP (6
mg/kg) in C57/BL6 mice, when cold allodynia had developed. As shown in figure 6, a single
subcutaneous administration of 1 μg of 31a was not effective in inhibiting the (OXP)-induced
cold allodynia, whereas injections of 10 and 30 μg of our compound showed a remarkable
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inhibitory effect, which was maximum after 15 minutes. This effect is still evident 30 minutes
after administration of 30 μg dose (Figure 6). This data suggests that 31a may be a viable
therapeutic scaffold for the treatment of CIPN.
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Figure 6: Dose-dependent inhibition of nocifensive paw licking given by compound 31a (1, 10 and 30 µg, s.c.) in
oxaliplatin-induced cold allodynia in C57/BL6 mice. Data are given as mean ± SEM n = 6. Statistical analysis was two-
way ANOVA followed by post hoc Bonferroni test by multiple comparison. ***p<0.001, ****p<0.0001
Further we investigated the efficacy of 31a in a chronic constriction injury (CCI) model of
neuropathic pain, using a thermal gradient ring assay. This assay deeply differs from the canonical
reflexive measures of nociception, in which the endpoint is withdrawal to a noxious stimulus; a fact
34
that have been questioned during the last years for their unsatisfactory translation. In particular,
this test integrates information on temperature perception distinguishing exploratory behaviour from
thermal preference behaviour.35a Thus, we measured the thermal preference location of sham, CCI-
mice, and CCI-mice treated intraperitoneally with 31a in a thermal gradient assay equilibrated
between 15 and 40°C.
The mean temperature to which the sham animal located during the observation time was
27.9°C±0.35°C (Figure 7A) and no statistical differences were evidenced at the different time
points (Figure 7B). No effects on temperature preferences were observed after 31a administration in sham-
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mice (data not shown). This value slightly differs from the previously reported by Touska et al.,35a but
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is consistent with gender, age and strain differences within animals used. The same temperature
preference was observed in CCI-mice 7 days after ligation (mean preferred temperature
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5.88°C±1.08°C, for CCI mice, p=1.452 vs sham mice, Figure 7A and 7B). However, 14 days after
ligation, when the neuropathic pain and the related nociceptive disorders are well-known to
occur,35b, 35c the CCI animals displayed a marked preference for colder areas (mean temperature=
22.80°C±0.61°C, *p<0.05 vs sham mice, Figure 7A), which is most prominent during the first 45
min of exposure as shown in Figure 7B (**p < 0.01 vs. sham mice) and extending to 60 min. This is
in accordance with the cold-seeking behaviours reported during inflammatory states.35d Considering
that thermosensation is mediated by the primary afferent Aδ and C fibers,35e where TRPM8 is
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particularly represented, its role in the cold-seeking behaviours of CCI animal seems evident. In
fact, intraperitoneal administration of the TRPM8 antagonist 31a (11.5 mg/kg) significantly
reverted this behaviour to 33.30°C±1.44°C (Figure 7A; °p<0.05 vs CCI 14 days). Similar enhanced
thermal tolerance has been recently reported when the antihyperalgesic drug clonidine was
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administered in a CCI mouse model. Moreover, the mice behaviour is also in accordance with
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previous data that describe TRPM8 deficient mice (TRPM8 ) as rather preferring warmer than
colder areas.35a It should be noted that mice treated with 31a immediately recognized warmer zones
as preferable to colder areas compared to vehicle CCI-mice (Figure 7B; °°°p < 0.001 and
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°°°p<0.0001 vs. CCI-mice) also showing a preference for an even warmer temperature than sham
#
animals during the first 15 minutes (Figure 7B; p<0.05 vs sham mice). It is questionable why this
transient effect was recorded, but it must be considered that TRPM8 antagonists are able in
decreasing the body temperature. This effect could probably account for the thermal preference
expressed by animals treated with 31a at 15 minutes.
The efficacy and the rapid onset of action further confirm the efficacy of compound 31a as TRPM8
antagonists.
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Figure 7: A) Thermal preference behavior of Swiss CD1 mice 7 days (light blue bar) and 14 days (green bar) after CCI.
The blue bar represents the effect of compound 31a administration at 14 days of CCI. Data are given as mean ± SEM n
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6. 2-way ANOVA with Bonferroni post hoc test. *P <0,05 vs sham mice; ° P <0,05 vs 14 days CCI .B) Time course
thermal preference behavior of sham Swiss CD1 mice (white dots), CD1 mice 7 days (light blue dots) and 14 days
(green dots) after CCI. The blue dots represent the time course effect of compound 31a administration at 14 days of
CCI. Data are given as mean ± SEM n = 6. 2-way ANOVA with Bonferroni post hoc test. *P <0,05 and **p < 0.01 vs.
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vehicletreated. °°°p < 0.001 and °°°°p<0.0001 vs. CCI-mice. p<0.05 vs sham mice.
CONCLUSIONS
Following our interest in the TRPM8 modulation and taking into account the in vivo promising
results obtained with a tryptophan-based TRPM8 antagonist (4), in this work we describe the
synthesis and the pharmacological characterization of different conformationally restricted
analogues of this hit compound, designed with the dual objective of exploring the structural
requirements for antagonizing TRPM8 at molecular level and improve the metabolic stability of our
hit compound. Some of the synthesized compounds featuring tetrahydrocarboline,
tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione, and tetrahydro-1H-imidazo
[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione chemical structures, showed a efficient and potent
TRPM8 antagonist activity in the nanomolar range. Using a new TRPM8 homology model, we
rationalized the SAR of this series of compounds identifying the structural and stereochemical
requirements that determinate their competitive antagonist activity. One of the synthesized
compounds,
the
(5R,11aS)-5-(4-chlorophenyl)-2-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1H-
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imidazo[1',5':1,6] pyrido[3,4-b]indole-1,3(2H)-dione, 31a, has a slow metabolic turnover and both
overcomes TRPM8- mediated cold hypersensitivity over time, as measured in the WDS assay, and
displays acute antinociceptive response 15 min after its application in an oxaliplatin-induced cold
allodynia model. In addition, 31a also shows remarkable analgesic activity in an animal model of
CCI-induced hyperalgesia. This last data is in agreement with the results obtained with 4 in other
models of neuropathic pain,27d but differ with those obtained by other authors who demonstrate the
efficacy of the TRPM8 agonists in animal models of injury-induced neuropathic pain.27a-c Our
results confirm the validity of indole nucleus in the design of potent TRPM8 modulators, adding
one more piece to the puzzle that composes the TRPM8’s complex biology in the transmission and
modulation of pain.
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Experimental section
General. All reagents and solvents used were purchased from Sigma-Aldrich (Milan, Italy), unless
otherwise stated. Reactions were performed under magnetic stirring in round-bottomed flasks unless
otherwise noted. Moisture-sensitive reactions were conducted in oven-dried glassware under
nitrogen stream, using freshly distilled solvents. TLC analysis of reaction mixtures was performed
on pre-coated glass silica gel plates (F254, 0.25 mm, VWR International), while crude products
were purified by the Isolera Spektra One automated flash chromatography system (Biotage,
Uppsala, Sweden), using commercial silica gel cartridges (SNAP KP-Sil, Biotage). NMR spectra
were recorded on a Bruker Avance 400 MHz apparatus, at room temperature. Chemical shifts were
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reported in δ values (ppm) relative to internal Me Si, for H and C NMR, and CFCl for F NMR.
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J values were reported in hertz (Hz). H-NMR and 19F NMR peaks were described using the
following abbreviations: s (singlet), d (doublet), t (triplet) and m (multiplet). HR-MS spectra were
recorded by LTQ-Orbitrap-XL-ETD mass spectrometer (Thermo Scientific, Bremen, Germany),
equipped with an ESI source. Analytical RP-HPLC analysis of final products was performed
through a Nexera UHPLC system (Shimadzu, Kyoto, Japan) consisting of a CBM-20A controller,
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