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 prepared and characterized in vitro by Ca2+-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 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 ip), an oxaliplatin-induced cold allodynia (at 10-30 μg sc), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg ip) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.
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 prepared and characterized in vitro by Ca2+-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 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 ip), an oxaliplatin-induced cold allodynia (at 10-30 μg sc), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg ip) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.
The transient receptor
potential melastatin type 8 (TRPM8) is a
member of the thermo-TRP family[1] of polymodal,
nonselective, and Ca2+ permeable ion channel, identified
as the physiological sensor of environmental cold.[2] 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,[5] changes in extracellular osmolarity[6] and phosphatidylinositol 4,5-biphosphate (PIP2).[7]Originally expressed
in a prostate cancer cell line,[8] 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
cornea.[9a,10] TRPM8 is also expressed in visceral tissues
innervated by pelvic or vagal nerves,[11] several tumor cells,[12] macrophages,[13] and different regions in rodents brain.[14] 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 metabolic diseases, among others.[15] 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-treatment with augmented expression
of TRPM8 in sensory neurons,[16] 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 in both 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 findings and the potential activity of TRPM8 antagonists
also in cancer and other pathologies,[21] it is easy to understand the effort of the academic groups and pharmaceutical/biotech
companies to develop potent and selective TRPM8 modulators.[22] To date, two antagonists, the quinoline-3-carboxamido
derivative PF-05105679[18a] and the amino-2-oxoethyl
nicotinic acid derivative AMG-333[20] (Chart ), have been evaluated
for the treatment of cold related pain and migraine, respectively,
although they have not passed phase I studies. In 2017, two undisclosed
structures, named RQ 00434739[23] and Ice
3682,[18d] have reached clinical trials for
the treatment of neuropathic pain in Japan and Israel, respectively.
Chart 1
Structures of Some TRPM8 Antagonists
In the past years, the information obtained through mutagenesis
experiments[24] and molecular modeling studies[25] on the structure–function of TRPM8 channels
has 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 it difficult to rationalize pharmacological results,
particularly in the context of neuropathic pain, where also agonists
of TRPM8 are able to induce analgesia,[27] 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 flycatcherFicedula albicollis (TRPM8FA) using cryoelectron microscopy. The network
of interactions generated from the TRPM8FA/menthol, icilin,
or lipids lays the structural basis for the design and 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 humanTRPM8 using the TRPM8FA structure as template
to rationalize the potent antagonist activity showed by tryptamine[29] and tryptophan-based[18b] TRPM8 modulators (3 and 4, Chart ). In patch-clamp recordings,
these compounds were more potent (IC50 = 367 and 0.2 nM,
respectively) 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 for 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 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-β-carbolines
(THBCs), THBC-based diketopiperazines, and THBC-based hydantoin derivatives,
as well as the results of TRPM8 antagonist activity obtained by assays
of Ca2+fluorescence and patch-clamp measurements. These
data were rationalized by molecular modeling 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 .
Scheme 1
Synthesis of Substituted Tetrahydro-β-carbolines (6a,b and 10–12a,b)
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 acetate as catalyst and microwave
irradiation led to the final trans-(6a) and cis-(6b) THBCs in 62% and 59%
yields, respectively. The relative configuration for 6a and 6b was assigned by ROESY NMR spectra considering
the cross peak between H1 and H3 that is present for 6b (H1, δ 4.96 ppm; H3, δ 3.87 ppm, Figure S8), while it is missing in 6a (H1, δ
5.39 ppm; H3, δ 3.87 ppm, Figure S5). Assuming that 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
(10–12a,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 trans10–12a (35–43% yields) and cis10–12b (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 trans10–12a and cis10–12b as (1R,3S) and (1S,3S), respectively.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 ). Coupling
of these intermediates with NHBoc-β-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, 25a,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, the cross peak between H6 and H12a was investigated
through ROESY NMR. For example, 27b shows a cross peak
between δ 6.16 ppm (H6) and δ 4.30 ppm (H12a, Figure S43) while this correlation is missing
for the diastereoisomer 27a (δ 6.67 ppm; δ
4.18 ppm, Figure S40). The absolute configuration
was attributed considering the retention of the l-tryptophan
chirality.
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)
Final THBC-based hydantoin compounds were obtained through the
synthetic method reported in Scheme . Reaction of the starting THBCs 13, 14a, 14b, 15a, 15b and
the now synthesized 32a and 32b 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, 29a–31a, 33–34a, 29a′–31a′, 33–34a′ in one step and with 35–62% yields. NMR data, αD values, and circular dichroism experiments (see Supporting Information) showed that the reaction
of trans (1R,3S) THBCs14a, 15a, and 32a originated the trans derivatives (5R,11aS) 29a–31a, 33a, and 34a, 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 THBCs15a 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, δ 5.25
ppm; H3, δ 3.99 ppm). After cyclization reaction of 15b to the hydantoine derivative (30a′) the cross
peak between the same hydrogens (H11a δ 4.30 ppm, H5 δ
6.29 ppm) was not detected (Figure S89).
This is in accordance with literature[30] 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 expected, we observed the same NMR chemical shifts and
opposite values for αD (see Experimental
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.
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′)
On the other hand, Scheme reports the synthesis
of the N-aryl hydantoin
derivatives 36a–38a and 36b–38b. In this case, a different chemical approach
is required because of the minor reactivity of anilines. Intermediates
THBC32a and 32b were coupled with 3CF3 or 2F or 4-F-phenyl 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 δ 4.53 ppm and δ 5.86 ppm, respectively, for compound 36b (Figure S76). Absolute configuration
was determined as described above. The formation of the cis intermediates, which 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 the 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 toward trans-conversion, we considered inappropriate the pharmacological testing
of all the cis isomers and we decided to assay only 36b for its pharmacological activity, due to its higher stability
in water environment in comparison with its congeners 37b and 38b, which were almost fully converted to the trans isomers during 60 min 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 min in
39–45% yields.
Scheme 4
Synthesis of THBC-base 2-Arylhydantoin Derivatives
(36–38, Their Enantiomers 36b–38b, and Their Diastereoisomers 36a′–38a′)
Pharmacological Characterization. Screening by Ca2+-Imaging
Assay
TRPM8 blocker activity of all synthesized
compounds was tested by Ca2+ fluorimetric assays using
HEK-293 cells stably expressing the rat isoform of TRPM8 channels,
using menthol and AMTB as prototypical agonist and antagonist, respectively.
All the compounds showed an antagonist activity higher than the canonical
TRPM8 antagonist AMTB, although lower than the lead compound 4 with IC50 values in the 100–0.3 μM
range (Table ).
Table 1
Potency of Synthesized Compounds as
TRPM8 Antagonists
compd
R
R1
R2
configuration
IC50 (μM)a
4
0.09 ± 0.08
6a
OCH3
C6H5
CH2C6H5
1R, 3S
1.3 ± 0.7
6b
OCH3
C6H5
CH2C6H5
1S,3S
1.6 ± 0.9
9
NHCH2(4-F)Ph
H
H
3S
0.9 ± 0.4
10a
NHCH2(4-F)Ph
CH2CH(CH3)2
H
1R,3S
4.6 ± 1.3
10b
NHCH2(4-F)Ph
CH2CH(CH3)2
H
1S,3S
6.2 ± 1.2
11a
NHCH2(4-F)Ph
CH2CH2COOCH3
H
1R,3S
1.1 ± 0.5
11b
NHCH2(4-F)Ph
CH2CH2COOCH3
H
1S,3S
3.0 ± 1.2
12a
NHCH2(4-F)Ph
CH2CH2COOH
H
1R,3S
5.0 ± 1.2
12b
NHCH2(4-F)Ph
CH2CH2COOH
H
1S,3S
22.0 ± 1.4
17
OCH3
H
COCH2CH2NH2
3S
>100
21
H
H
12aS
11.4 ± 1.6
22
H
CH2C6H5
3S,12aS
1.6 ± 0.7
23
H
CH2C6H5
3R,12aS
0.4 ± 0.1
25a
CH2CH(CH3)2
CH2C6H5
3S,6R,12aS
4.1 ± 1.1
25b
CH2CH(CH3)2
CH2C6H5
3S,6S,12aS
1.3 ± 0.6
27a
4-Cl-C6H4
H
6R,12aS
1.5 ± 1.1
27b
4-Cl-C6H4
H
6S,12aS
1.7 ± 0.8
28
H
CH24-(F)-C6H4
11aS
17.8 ± 1.2
29a
CH2CH(CH3)2
CH24-(OCH3)C6H4
5R,11aS
2.8 ± 1.2
29a′
CH2CH(CH3)2
CH24-(OCH3)C6H4
5S,11aR
22.9 ± 1.4
30a
4-(Cl)C6H5
CH24-(CH3)C6H4
5R,11aS
0.8 ± 0.4
30a′
4-(Cl)C6H5
CH24-(CH3)C6H4
5S,11aR
2.3 ± 0.8
31a
4-(Cl)C6H5
CH24-(F)-C6H4
5R,11aS
0.5 ± 0.3
31a′
4-(Cl)C6H5
CH24-(F)C6H4
5S,11aR
>30
33a
4-(F)C6H5
CH2C6H4
5R,11aS
6.4 ± 1.2
33a′
4-(F)C6H5
CH2C6H4
5S,11aR
17.5 ± 1.4
35a
4-(F)C6H5
(CH2)3NH2
5R,11aS
5.1 ± 1.2
35a′
4-(F)C6H5
(CH2)3NH2
5S,11aR
27.2 ± 1.4
36a
4-(F)C6H5
3-(CF3)C6H4
5R,11aS
2.8 ± 1.5
36a′
4-(F)C6H5
3-(CF3)C6H4
5S,11aR
7.8 ± 2.4
36b
4-(F)C6H5
3-(CF3)C6H4
5S,11aS
0.2 ± 0.2
37a
4-(F)C6H5
2-(F)C6H4
5R,11aS
4.4 ± 1.3
37a′
4-(F)C6H5
2-(F)C6H4
5S,11aR
5.1 ± 2.1
38a
4-(F)C6H5
4-(F)C6H4
5R,11aS
6.7 ± 1.2
38a′
4-(F)C6H5
4-(F)-C6H4
5S,11aR
7.2 ± 0.9
AMTB
7.3 ± 1.5
Values are expressed as the mean
± standard deviation of at least three independent measurements.
Values are expressed as the mean
± standard deviation of at least three independent measurements.In the 1,2,3-substituted THBCs
(6, 9–12, and 17) series, the antagonist activity is
conditioned by the relative configuration at 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 (IC50= 1.1 μM
and 3.0 μM for the 11a and 11b, respectively,
and 5 μM and 22 μM 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 IC50 value of 0.9 μM, while the restricted
pseudo dipeptideTrp-β-Ala 17, bearing a polar
3-aminopropanoic chain in position 2, was completely inactive (IC50 > 100 μM).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 μM), but the introduction
of a benzyl substituent at C-3 (22, 23,
and 25) significantly increased activity with IC50 value in the range 4–0.4 μM. 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 (IC50 = 1.6 ± 0.7 μM and 0.4 ± 0.1 μM, 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) (IC50 = 4.1
± 1.1 μM vs 1.3 ± 0.6 μM), while the 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, IC50 = 1.5 ± 1.1 μM and 1.7 ± 0.8 μM,
respectively).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 IC50 in the range 0.5–0.8 μM, feature an aromatic
group at C-5 position and 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 was 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, which was about 3-fold more potent than its enantiomer 36a′ (5S,11aR) (IC50 = 2.8 μM and 7.8 μM, respectively). Compound 36b, the only tested cis derivative of this
series, showed a remarkable higher potency (IC50 = 0.2
μM) than its two trans analogues 36a and 36a′.
Patch-Clamp Electrophysiology
Assay
Functional assay
identified derivatives 6a, 9, 11a, 23, 31a, and 36b to be among
the most effective and potent TRPM8 antagonist compounds with IC50 values in the submicromolar range. To provide direct evidence
for this activity, these derivatives were tested in HEK-293 cells
transiently expressing the humanTRPM8 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 , 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 IC50 of 6.57 ± 1.21
nM and of 4.10 ± 1.52 nM, respectively. The THBC6a 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 μM), antagonized the effect of menthol with an EC50 of 15.41 nM, while its acid free analogue 12a 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 , 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.
Table 2
Full in Vitro Pharmacological Characterization
for Selected Compounds
The activity of compound 36b, which proved
to be a
powerful antagonist of TRPM8 in Ca2+ fluorimetric assay,
was confirmed by patch clamp experiment with an IC50 of
7.67 nM, and an inhibition efficacy of the menthol evoked currents
of 59.4%. In 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).
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
toward TRPV1, TRPA1, and Nav1.7 channels by calcium fluorimetric
experiments. TRPV1 and TRPA1 channels belong to the TRP superfamily
and share a high degree of homology with TRPM8.[1] On the other hand, Nav1.7 channels are reported
to be involved in several neuropathic pain pathways, also modulated
by TRPM8.[31] All the derivatives were unable
to modulate these channels, showing no activity as agonists or antagonists.
Only compounds 6a and 9 showed a negligible
antagonistic activity over Nav1.7 with IC50 >
10 μM (Figure S2).
Molecular
Modeling and Structural Rationale
The TRPM8
three-dimensional structures 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 both of 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 first re-evaluated
by considering the TC-I 2014-bound TRPM8 protein structure (PDB code 6O72), chosen as reference
system since it featured a better resolution if compared with that
originally complexed with AMTB (PDB code 6O6R). In particular, the obtained docking
poses of the lead compound 4 revealed a binding mode
different from what was reported in the original paper,[43] in which an homology modeling structure of the
protein was accounted. Indeed, in the TC-I 2014-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 ). Also, H-bonds were detected for compound 4 with Asn732 and Gln776 (Figure ).
Figure 1
Compound 4 (colored by atom type; C orange,
N blue,
O red, polar H light gray) in docking with TRPM8 (represented in green
ribbons; residues colored by atom type; C gray, N blue, O red, polar
H light gray) 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 code 6O72).
Compound 4 (colored by atom type; C orange,
N blue,
O red, polar H light gray) in docking with TRPM8 (represented in green
ribbons; residues colored by atom type; C gray, N blue, O red, polar
H light gray) 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 code 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
activity 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-β-carboline
(6a, 9, 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) 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 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 and 3). First, the tetrahydro-β-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 A). On the other hand, the careful analysis of the superimposed
poses of 4 and 6a highlighted a similar
total shape (Figure B), 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 A).
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 gray, N blue, O red, polar H light
gray) 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 code 6O72).
Figure 3
Predicted binding modes of (A) compound 9 (colored
by atom type, C light violet), (B) 11a (colored by atom
type, C gray), (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 pale 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 gray, N blue, O red, polar H light gray) 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 code 6O72).
(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 gray, N blue, O red, polar H light
gray) and (B) superposition between the predicted binding modes of 6a and 4 into the TRPM8TC-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 code 6O72).Predicted binding modes of (A) compound 9 (colored
by atom type, C light violet), (B) 11a (colored by atom
type, C gray), (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 pale 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 gray, N blue, O red, polar H light gray) 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 code 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-fluorobenzyl function, whereas π–π
stacking contacts were detected with Phe729 and Tyr995 through the
indole moiety (Figure A). The introduction of a substituent at C-1, as in compounds 11a, 11b, 12a, 12b,
determined a similar accommodation in the TRPM8 binding site (Figure B–E). Specifically,
in the cases of compounds 11a and 12a the
tetrahydro-β-carboline moiety was oriented in front of Phe729
and Tyr995 residues, while the 4-fluorobenzyl substituent interacted
again with Arg998 through a π–cation and with Tyr736
through a π–π stacking (Figure B and Figure D). Also, the acid moiety in 12a allowed
a further H-bond interaction with Arg998 (Figure D). On the other hand, the different stereochemical
arrangements of the related analogs 11b and 12b (featuring 1S,3S configuration,
instead of 1R,3S as for compounds 11a and 12a) determined a slightly different
binding mode. Specifically, for compound 11b, the 4-fluorobenzyl
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 C). 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-fluorobenzyl substituent showed
in this case a π–π interaction with Tyr736 (Figure E)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 a different placement in 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 F). Concerning
compound 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 G). As expected,
a similar binding mode was detected for compound 36a,
featuring the same absolute configurational pattern of 31a, but the presence of a 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 I). 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 (Figure H and Figure J). 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 Phe736 through the terminal 3-(CF3)-aryl moiety (also
able to interact with Arg998 through a π–cation) as well
as the π–π interaction with Phe729 and Tyr995 with
the indole moiety (Figure K). 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
on 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.
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 , almost
all the compounds proved to be stable in the absence of metabolic
cofactors (NADPH or UDP-GlcUA/NADPH) except for 11a,
showing unspecific metabolic liability (black bars). In fact, after
60 min in contact with liver microsomes, in the absence of any metabolic
cofactors, 11a turnover was 66.5 ± 3.8%. When the
phase I metabolism conditions were mimicked (see protocol I, material
and methods section), compound 4 was massively metabolized
with a turnover percentage of 98.3 ± 3.1% (Figure , 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 cofactors. As shown in Figure (white bar), 31a proved to
have a slow metabolic turnover (46.0 ± 2.3%)[32] in the experimental conditions used and was then selected
for the in vivo pharmacological assays.
Figure 4
In vitro metabolic stability
assays for the selected compounds:
(a) compounds’ unspecific metabolism in absence of cofactors
calculated as ([ ]t60/[ ]t0) × 100, black
bars; (b) compounds’ metabolic stability under phase I metabolism
(gray bars, protocol I; see materials and methods section); (c) compound 31a metabolic stability under phase I + phase II metabolism
(white bar, protocol II; see materials and methods section).
In vitro metabolic stability
assays for the selected compounds:
(a) compounds’ unspecific metabolism in absence of cofactors
calculated as ([ ]t60/[ ]t0) × 100, black
bars; (b) compounds’ metabolic stability under phase I metabolism
(gray bars, protocol I; see materials and methods section); (c) compound 31a metabolic stability under phase I + phase II metabolism
(white bar, protocol II; see materials and methods 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 min before the challenge with icilin (1 mg/kg ip) and WDS was recorded
for 30 min. In the vehicle-treated group, a mean of about 128 shakes
were counted (Figure , white column). As expected, from the metabolic stability experiments,
the pretreatment with 4 (10 mg/kg ip) significantly decreased
the number of icilin-induced WDS 0.5 h after the injection (Figure ; **p < 0.01 vs vehicle treated mice); no effect was observed at 2
h. On the contrary, 31a (11.5 mg/kg ip) showed a significant
effect at both 0.5 and 2 h after the injection (Figure ; *p < 0.05 and **p < 0.01 vs vehicle treated mice).
Figure 5
Comparative effect of 4 and 31a on icilin-induced
WDS in Swiss CD1 mice. After ip injection of icilin (10 mg/kg), the
number of wet-dog shakes (WDS) were counted over 30 min. Data are
given as the mean ± SEM n = 6, two-way ANOVA
with Bonferroni post hoc test: *p < 0.05; **p < 0.01.
Comparative effect of 4 and 31a on icilin-induced
WDS in Swiss CD1mice. After ip injection of icilin (10 mg/kg), the
number of wet-dog shakes (WDS) were counted over 30 min. Data are
given as the mean ± SEM n = 6, two-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
has 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 , 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 inhibitory effect, which
was maximum after 15 min. This effect was still evident 30 min after
administration of a 30 μg dose (Figure ). These data suggest that 31a may be a viable therapeutic scaffold for the treatment of CIPN.
Figure 6
Dose-dependent
inhibition of nocifensive paw licking given by compound 31a (1, 10, and 30 μg, sc) in oxaliplatin-induced cold
allodynia in C57/BL6 mice. Data are given as the 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.
Dose-dependent
inhibition of nocifensive paw licking given by compound 31a (1, 10, and 30 μg, sc) in oxaliplatin-induced cold
allodynia in C57/BL6 mice. Data are given as the 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 end point is withdrawal
to a noxious stimulus, a fact that has been questioned during the
past years for their unsatisfactory translation.[34] In particular, this test integrates information on temperature
perception distinguishing exploratory behavior from thermal preference
behavior.[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 A), and no statistical differences
were evidenced at the different time points (Figure B). No effects on temperature preferences
were observed after 31a administration in sham-mice (data
not shown). This value slightly differs from the previously reported
by Touska et al.[35a] but 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 25.88 °C ± 1.08 °C, for
CCImice, p = 1.452 vs sham mice, Figure A and Figure B). 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 A), which was most
prominent during the first 45 min of exposure as shown in Figure B (**p < 0.01 vs sham mice) and extending to 60 min. This is in accordance
with the cold-seeking behaviors reported during inflammatory states.[35d] Considering that thermosensation is mediated
by the primary afferent Aδ and C fibers,[35e] where TRPM8 is particularly represented,[2] its role in the cold-seeking behaviors of CCI animal seems
evident. In fact, intraperitoneal administration of the TRPM8 antagonist 31a (11.5 mg/kg) significantly reverted this behavior to 33.30
°C ± 1.44 °C (Figure A; °p < 0.05 vs CCI 14 days).
Similar enhanced thermal tolerance has been recently reported when
the antihyperalgesic drug clonidine was administered in a CCImouse
model.[34] Moreover, the mice behavior is
also in accordance with 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 B; °°°p < 0.001 and °°°°p < 0.0001 vs CCI-mice) also showing a preference for an even warmer
temperature than sham animals during the first 15 min (Figure B; #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 to decrease the body temperature. This effect could probably
account for the thermal preference expressed by animals treated with 31a at 15 min.
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 the mean ± SEM, n =
6, two-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 the mean ± SEM n = 6, two-way ANOVA
with Bonferroni post hoc test: *p < 0,05 and **p < 0.01 vs vehicle treated; °°°p < 0.001 and °°°°p < 0.0001 vs CCI-mice; #p < 0.05
vs sham mice.
(A) Thermal preference behavior of Swiss CD1mice 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 the mean ± SEM, n =
6, two-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
CD1mice (white dots), CD1mice 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 the mean ± SEM n = 6, two-way ANOVA
with Bonferroni post hoc test: *p < 0,05 and **p < 0.01 vs vehicle treated; °°°p < 0.001 and °°°°p < 0.0001 vs CCI-mice; #p < 0.05
vs sham mice.The efficacy and the rapid onset
of action further confirm the
efficacy of compound 31a as TRPM8 antagonists.
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 improving 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 an efficient and potent TRPM8 antagonist activity in the nanomolar
range. Using a new TRPM8 three-dimensional protein structure, we rationalized
the SAR of this series of compounds by identifying the structural
and stereochemical requirements that determine their competitive antagonist
activity. One of the synthesized compounds, 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, 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. These last data are 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−27c] Our results confirm the validity of the 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.
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 precoated 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 reported in δ values (ppm) relative to internal
Me4Si for 1H and 13C NMR and to CFCl3 for 19F NMR. J values were reported
in hertz (Hz). 1H 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, two LC-30AD pumps, a DGU-20 A5R degasser,
an SPD-M20A photodiode array detector, a CTO-20AC column oven, a SIL-30AC
autosampler, and a Kinetex C18 150 mm × 2.1 mm × 2.6 μm
(100 Å) column (Phenomenex, Bologna, Italy). The optimal mobile
phase consisted of 0.1% HCOOH/H2O v/v (A) and 0.1% HCOOH/ACN
v/v (B). Analysis was performed in gradient elution as follows: 0–13.00
min, 5–65% B; 13–14.00 min, 65–95% B; 14–15.00
min, isocratic to 95% B; then 3 min for column re-equilibration. Flow
rate was 0.5 mL min–1. Column oven temperature was
set to 40 °C. Injection volume was 5 μL of sample. The
following PDA parameters were applied: sampling rate, 12.5 Hz; detector
time constant, 0.160 s; cell temperature, 40 °C. Data acquisition
was set in the range 190–800 nm, and chromatograms were monitored
at 230 nm. Analytical RP-HPLC confirmed that all final compounds had
a purity of >95%. For quantitative analysis, the calibration curve
was obtained in a concentration range of 2.5–40 μM with
five concentration levels and triplicate injections of each level
were run. Peak areas were plotted against corresponding concentrations,
and the linear regression was used to generate a calibration curve
with R2 values of ≥0.999 (Table S1).Stability analysis for 36b was performed using the same chromatographic conditions
reported above but with the following elution gradient: 0–13.00
min, 15–65% B; 13–14.00 min, 65–95% B; 14–15.00
min, isocratic to 95% B; then 3 min for column re-equilibration.All circular dichroism spectra were recorded using a JASCO J810
spectropolarimeter at 25°C in the range λ = 260–190
nm (1 mm path length, 1 nm bandwidth, four accumulations, and a scanning
speed of 10 nm min–1). Compounds were dissolved
in methanol at a concentration of 0.100 mM. Spectra were corrected
for the solvent contribution.
General Procedure A: Pictet–Spengler
Reaction
1 mmol of l-tryptophan methyl ester or
(S)-2-amino-N-(4-fluorobenzyl)-3-(1H-indol-3-yl)propanamide (8) was dissolved
in methanol
and added with the proper aldehyde (1.5 equiv) and trifluoroacetic
acid (1.5 equiv). The mixture was subjected to a microwave assisted
closed vessel reaction for 45 min at 110 °C.[36] The mixture was then evaporated in vacuo, and the residue was dissolved in dichloromethane and was washed
three times with water. The organic phase was extracted, dried over
Na2SO4, filtered, and concentrated under vacuum.
The crude products were purified by flash chromatography using mixtures
of n-hexane/ethyl acetate as mobile phase.
General
Procedure B: Coupling Reactions
1 mmol of the
proper carboxylic acid was dissolved in dichloromethane/DMF (4:1 v:v)
and added with HoBt (1.2 equiv), HBTU (1.2 equiv), DIPEA (2.4 equiv),
and the corresponding amine (1.2 equiv) and stirred at room temperature
overnight. Then, the solvent was evaporated in vacuum, and the residue
was dissolved in dichloromethane and washed with water (3 times),
a saturated solution of NaHCO3 (3 times), and a solution
of citric acid (10% w:w). The organic phase was extracted, dried over
Na2SO4, filtered, and concentrated under vacuum.
The crude products were purified by flash chromatography using mixtures
of n-hexane/ethyl acetate as mobile phase.
General
Procedure C: Boc Removal
The N-Boc protected
intermediate (0.2 mmol) was dissolved in a mixture
of TFA/DCM (1/3, v/v), and triisopropylsilane (TIS, 0.25 equiv) was
added. Reaction was stirred at room temperature for 2 h. Then, a solution
of NaOH (2 N) was added dropwise until pH 7. The mixture was diluted
with water and dichloromethane, and the organic phase was extracted,
dried over Na2SO4, filtered, and concentrated
under vacuum. The crude products were purified by flash chromatography
using mixtures of n-hexane/ethyl acetate as mobile
phase.
General Procedure D: Hydantoin Synthesis
Diastereoisomerically
pure tetrahydro-β-carbolines (0.2 mmol) were dissolved in THF,
and 0.4 equiv of triphosgene was added. The pH was adjusted to 8 by
addition of TEA, and the mixture was stirred at room temperature for
10 min. Then, the proper amine (1.2 equiv) was added and the resulting
mixture was refluxed for 1 h. After cooling to room temperature, the
solvent was evaporated, the residue reconstituted in dichloromethane
and washed with water (3 times). The organic phase was extracted,
dried over Na2SO4, filtered, and concentrated
under vacuum. The crude products were purified by flash chromatography
using mixtures of n-hexane/ethyl acetate as mobile
phase.
General Procedure E: Hydantoin Synthesis
Tetrahydro-β-carboline 32a or 32b (0.2 mmol) was dissolved in THF, and
1.2 equiv of trimethylamine and 1.2 equiv of the proper isocyanate
were added. The mixture was stirred at room temperature for 30 min.
The solvent was evaporated, the residue reconstituted in dichloromethane
and washed with water (3 times). The organic phase was extracted,
dried over Na2SO4, filtered, and concentrated
under vacuum. The crude products were purified by flash chromatography
using mixtures of n-hexane/ethyl acetate as mobile
phase.
General Procedure F: Hydantoin Synthesis
Tetrahydro-β-carboline 32b (0.2 mmol) was dissolved in THF, and 1.2 equiv of trimethylamine
and 1.2 equiv of the proper isocyanate were added. The mixture was
stirred at room temperature for 30 min and then refluxed for further
30 min. After cooling to room temperature, the solvent was evaporated,
the residue reconstituted in dichloromethane and washed with water
(3 times). The organic phase was extracted, dried over Na2SO4, filtered, and concentrated under vacuum. The crude
products were purified by flash chromatography using mixtures of n-hexane/ethyl acetate as mobile phase.
Compound 7a was obtained using
general procedure A in 33% yield, using benzaldehyde as starting material.
Spectral data were in accordance with literature.[37] FC in n-hexane/ethyl acetate 1/1, R = 0.37.
Compound 7b was obtained using
general procedure A in 41% yield, using benzaldehyde as starting material.
Spectral data were in accordance with literature.[37] FC in n-hexane/ethyl acetate 1/1, R = 0.44.
Compound 11a was dissolved
in a mixture of NaOH 6 N/methanol (9/1 v/v) and stirred for 90 min
at room temperature. The mixture was then buffered to pH 7 using HCl
(6N) and extracted three times with ethyl acetate. The organic phases
were collected, dried over Na2SO4, filtered,
and concentrated in vacuo. The final product was
purified by the use of reverse phase preparative HPLC using a Synergi
fusion column (4 μm, 80A, 150 mm × 21.2 mm, Phenomenex
Torrence, CA, USA) as stationary phase and a gradient elution with
acetonitrile 0.1% TFA (A) and water 0.1% TFA (B) (from 5% to 90% of
A in 22 min). Flow rate was set at 20 mL/min. Retention time was 9.10
min. White powder (42% yield). 1H NMR (400 MHz, CD3OD): δ: 2.01–2.08 (m, 1H, CH); 2.11–2.19 (m, 1H, CH); 2.47 (t, 2H, CH2, J = 7.0 Hz); 2.88 (dd, 1H,
CH, J′=9.2, J″= 15.2 Hz); 3.10 (dd, 1H,
CH, J′ = 4.6, J″= 15.3 Hz); 3.90 (dd, 1H,
CH, J′= 4.7, J″= 9.3 Hz); 4.20 (dd, 1H, CH, J′ = 3.6, J″= 9.0 Hz); 4.45 (s, 2H,
CH2); 6.96–7.09 (m, 4H, aryl);
7.29–7.35 (m, 3H, aryl); 7.42 (d, aryl, 1H, J = 7.7 Hz). 13C NMR (100 MHz, CD3OD) δ:
24.4, 31.0, 34.8, 41.8, 51.2, 52.5, 106.1, 110.4, 114.6, 114.8, 117.1,
118.2, 120.7, 126.9, 128.9, 129.0, 134.7, 135.6, 136.5, 163.2, 174.1.
HR-MS m/z calcd for C22H22FN3O3 [(M + H)]+:
396.1718; found 396.1725.
Intermediate 13 was synthesized
in 89% yield according to the general procedure A starting from l-tryptophan methyl ester and formaldehyde. The product was
isolated by filtration from the reaction mixture. Spectral data were
in accordance with literature.[37]
Synthesized in 36% yield according to
the general procedure A starting from tryptophan methyl ester and
isovaleraldehyde. FC in hexane/ethyl acetate 1/1, R = 0.40. Spectral data were in accordance with literature.[38]
Synthesized in 40% yield according to
the general procedure A starting from tryptophan methyl ester and
isovaleraldehyde. FC in hexane/ethyl acetate 1/1, R = 0.44. Spectral data were in accordance with literature.[38]
Synthesized in 33% yield according to
the general procedure A starting from tryptophan methyl ester and
4-chlorobenzaldehyde. FC in hexane/ethyl acetate 1/1, R = 0.45. Spectral data were in accordance with literature.[39]
Synthesized in 46% yield according to
the general procedure A starting from tryptophan methyl ester and
4-chlorobenzaldehyde. FC in hexane/ethyl acetate 1/1, R = 0.51. Spectral data were in accordance with literature.[39]
Synthesized in 71% yield according to the
general procedure B starting from intermediate 13 and N-Boc-β-Ala-OH. FC ethyl acetate/n-hexane 1/2. R = 0.65. Spectral data
were in accordance with literature.[36]
Compound 18 was synthesized
in 71% yield starting from intermediate 13 and N-Boc-Gly-OH following the general procedure B. FC in ethyl
acetate/n-hexane 1/1, R = 0.25. Spectral data were in accordance with literature.[36]
Compound 19 was synthesized
in 68% yield starting from intermediate 13 and N-Boc-L-Phe-OH following the general procedure
B. FC in ethyl acetate/n-hexane 2/3, R = 0.30. Spectral data were in accordance with literature.[36]
Compound 20 was synthesized
in 65% yield starting from intermediate 13 and N-Boc-D-Phe-OH following the general procedure
B. FC in ethyl acetate/n-hexane 2/3, R = 0.30. Spectral data were in accordance with literature.[36]
Compound 24a was synthesized
in 25% yield starting from intermediate 14a and N-Boc-L-Phe-OH following the general procedure
B. FC in ethyl acetate/n-hexane 1/2, R = 0.60. Spectral data were in accordance with literature.[36]
Compound 24b was synthesized
in 29% yield starting from intermediate 14b and N-Boc-L-Phe-OH following the general procedure
B. FC in ethyl acetate/n-hexane 1/2, R = 0.65. Spectral data were in accordance with literature.[36]
Compound 26a was synthesized
in 62% yield starting from intermediate 15a and N-Boc-Gly-OH following the general procedure B. FC in ethyl
acetate/n-hexane 1/2, R = 0.35. Spectral data were in accordance with literature.[36]
Compound 26b was synthesized
in 52% yield starting from intermediate 15b and N-Boc-Gly-OH following the general procedure B. FC in ethyl
acetate/n-hexane 1/2, R = 0.45. Spectral data were in accordance with literature.[36]
Synthesized in 35% yield from l-tryptophan methyl ester and 4-fluorobenzaldehyde following the general
procedure A, as previously described.[39] FC in ethyl acetate/n-hexane 1/2, R = 0.36. Spectral data were in accordance with literature.
Synthesized in 44% yield from l-tryptophan methyl ester and 4-fluorobenzaldehyde following the general
procedure A, as previously described.[39] FC in ethyl acetate/n-hexane 1/2, R = 0.41. Spectral data were in accordance with literature.
For measurement
of the potency of the compounds, fluorimetric experiments were performed
using HEK-293 cells (CRL-1573TM, American Type Culture Collection,
LGC Promochem, Molsheim, France) that stably express ratTRPM8. The
cells were seeded in 96-well plates (Corning Incorporated, Corning,
NY) at a cell density of 40 000 cells 2 days before treatment.
On the day of treatment, the medium was replaced with 100 μL
of the dye loading solution Fluo-4 NW supplemented with probenecid
2.5 mM.For the assessment of selectivity of target compounds,
fluorimetric experiments were performed using HEK-293 cells lines
stably transfected with either hTRPA1 or hNav1.7 and CHO-K1 stably
transfected with hTRPV1. HEK-293 cells were cultured in EMEM (MEM
Eagle Earl’s salts balanced salt solution, Lonza, Walkersville,
USA), 5 mL of 200 mM Ultraglutamine1 (Lonza), 5 mL of 100× penicillin/streptomycin
(Lonza), 50 mL of fetal bovine serum (Euroclone, Milan, Italy), 2
mL of 100 mg/mL G418 (InvivoGen, San Diego, USA). CHO-K1 cells were
grown in DMEM F-12 (1:1) mixture (Lonza), 5 mL of 100 mM sodium pyruvate
(Lonza), 25 mL of 7.5% sodium bicarbonate (Lonza), 6.5 mL of 1 M HEPES
(Lonza), 5 mL of 100× penicillin/streptomycin (Lonza), 50 mL
of fetal bovine serum (Euroclone), 0.25 mL of 10 mg/mL puromycin (InvivoGen),
and 0.5 mL of 100 mg/mL zeocin (InvivoGen).For patch-clamp
experiments, HEK-293/TRPM8 exon1 K3 cells were
cultured in minimum essential medium with Earle’s salts, without l-glutamine (Euroclone) supplemented with 5 mL of 200 mM Ultraglutamine
1 in 0.85% NaCl solution (Lonza), 5 mL of 100× penicillin/streptomycin
(Lonza), 0.2 mL of 10 mg/mL puromycin (InvivoGen; final concentration
0.4 μg/mL), and 50 mL of fetal bovine serum (Sigma-Aldrich,
Milan, Italy).
Fluorimetric Assays
The tested molecules
dissolved
in DMSO were added at the desired concentrations, and the plates were
incubated in darkness at 37 °C in a humidified atmosphere of
5% CO2 for 60 min. The fluorescence was measured using
instrument settings appropriate for excitation at 485 nm and emission
at 535 nm (POLARstar Omega BMG LABtech). A baseline recording of four
cycles was recorded prior to stimulation with the agonist (100 μM
menthol for TRPM8). The TRPM8 antagonist, 10 μM AMTB, was added
to the medium containing the corresponding agonist to induce channel
blockade. The changes in fluorescence intensity were recorded during
15 cycles more. The higher concentration of DMSO used in the experiment
was added to the control wells. The cells’ fluorescence was
measured before and after the addition of various concentrations of
test compounds. The fluorescence values obtained are normalized to
that prompted by the corresponding agonist (for channel activating
compounds) or upon agonist and antagonist coexposure (for channel
blocker compounds).
Selectivity Assays
The analysis
was performed in 384-well
clear bottom black walled polystyrene plates, (Thermo Scientific,
Waltham, USA) for CHO-K1 cells and in 384-well clear bottom black
polystyrene walled poly-d-Lys coated plates (TwinHelix, Rho,
Italy) for HEK-293 cells. Compound dilution was performed in 96-well
U bottom plates (Thermo Scientific), and then compounds were transferred
into 384-well V bottom polypropylene barcoded plates (Thermo Scientific).
To assess the activity of the selected compound over TRPA1 and TRPV1,
cells were seeded in 384 MTP in complete medium (25 μL/well)
at 10 000 cells/well concentration. 24 h after seeding, the
culture medium was removed and cells were loaded with 20 μL/well
of 0.5× calcium sensitive dye (Fluo-8 NW, AAT Bioquest, Sunnyvale,
USA) in assay buffer. To assess the activity of the selected compound
over Nav1.7, cells were seeded at 15 000 cells/well in 384
MTP in complete medium (25 μL/well). 24 h after seeding, the
culture medium was removed and cells were loaded with 20 μL/well
of 0.5× membrane potential dye (FLIPR membrane potential assay
kits Blue, Molecular Devices LLC, San Jose, USA) in assay buffer.
Plates were incubated for 1 h at room temperature in the dark. Then,
10 μL/well of test compounds and controls were injected at 3×
concentration, and the signal of the emitted fluorescence was recorded
using FLIPRTETRA apparatus (FortèBio, Fremont, USA). Then,
a second injection of 15 μL/well of 3× reference activator
(at ∼EC80) was performed analyzing the signal of
the emitted fluorescence. Allyl isothiocyanate (AITC, Sigma-Aldrich),
capsaicine (Sigma-Aldrich), and veratridine (Sigma-Aldrich) were used
as reference agonists, while HC-030031 (Sigma-Aldrich), capasazepine
(Sigma-Aldrich), and tetrodotoxine (Tocris bioscience, Bristol, U.K.)
were used as reference antagonists for TRPA1, TRPV1, and Nav1.7 assaying,
respectively.
Patch-Clamp Experiments
HEK-293/TRPM8exon
1 cells are
seeded 72 or 96 h before experiment at a concentration of 4 and 2.5
million cells, respectively, onto a T225 flask. Just before the experiments,
cells are washed twice with D-PBS without Ca2+/Mg2+ (Euroclone, Milan, Italy) and detached from the flask with trypsin–EDTA
(Sigma-Aldrich, Milan, Italy; diluted 1/10). Cells are then resuspended
in the suspension solution, 25 mL of EX-CELL ACF CHO medium (Sigma-Aldrich,
Milan, Italy); 0.625 mL of HEPES (Lonza, Walkersville, USA); 0.25
mL of 100× penicillin/streptomycin (Lonza, Walkersville, USA),
0.1 mL of soybean trypsin inhibitor 10 mg/mL (Sigma-Aldrich, Milan,
Italy), and placed on an automated patch-clamp platform (QPatch 16X,
Sophion Bioscience, Ballerup, Denmark).Menthol was used as
reference agonist, and a stock solution (1 M, 100% DMSO) was prepared
the day of the experiment from the powder; an intermediate stock of
300 mM was prepared from the 1 M stock in 100% DMSO, and the final
dilution was performed in the extracellular solution to obtain a working
concentration of 300 μM (1:1000, 0.1% final DMSO concentration).
Stock solutions of the testing compounds (10 mM; 100% DMSO; stored
at −20 °C) were prepared the day of the experiment; an
intermediate stock for each compound (300 μM) was prepared from
the 10 mM stock in 100% DMSO, and the working dilutions were performed
just before the experiments in the extracellular solution containing
300 μM menthol. The highest concentration tested was 300 nM,
with serial dilutions (1:10) in the extracellular solution. DMSO was
balanced to keep it constant throughout all the solutions in the same
experiment (0.2% final DMSO concentration). Standard whole-cell voltage
clamp experiments are performed at room temperature using the multihole
technology. For the voltage clamp experiments on humanTRPM8, data
are sampled at 2 kHz. After establishment of the seal and the passage
in the whole cell configuration, the cells are challenged by a voltage
ramp (20 ms step at −60 mV; 100 ms ramp −60/+100 mV;
20 ms step at +100 mV; return to −60 mV) every 4 s. The potential
antagonistic effect on humanTRPM8 current of target compounds was
evaluated after application of the agonist (menthol, 300 μM)
alone and in the presence of the compound under investigation at increasing
concentrations. Output: outward current evoked by the voltage ramp,
measured in the step at +100 mV. The intracellular solution contained
(mM) 135 CsCl, 10 BAPTA, 10 HEPES, 4 Na2ATP (pH 7.2 with
CsOH). The extracellular solution contained (mM) 145 NaCl, 4 KCl,
1 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose (pH 7.4
with NaOH).
Computational Details
3D structures
of TRPM8 in complex
with TC-I 2014 antagonist (PDB code: 6O72)[28c] were prepared
using the Schrödinger Protein Preparation Wizard workflow.[40] Specifically, water molecules were deleted,
cap termini were included, all hydrogen atoms were added, and bond
orders were assigned. Finally, the .pdb files were converted to the
.mae file.The grids for the subsequent molecular docking calculations
were generated accounting the related position of TC-I 2014 on the
receptor binding sites. In this way, the cocrystallized ligands were
also automatically removed from the original binding sites.The library of investigated compounds (see Results
and Discussion) was prepared using LigPrep software (Schrodinger
Suite).[41] Specifically, all the possible
tautomers and protonation states at pH = 7.4 ± 1.0 were generated
for each compound, and finally the structures were minimized using
the OPLS 2005 force field.Molecular docking experiments were
performed using Glide software
(Schrödinger Suite),[42] setting the
Extra Precision [XP] mode. For this step, 20 000 poses were
kept in the starting phase of docking, and 1200 poses for energy minimization
were selected. The scoring window for keeping the initial poses was
set to 400.0, and a scaling factor of 0.8 related to van der Waals
radii with a partial charge cutoff of 0.15, based on a 0.5 kcal/mol
rejection cutoff for the obtained minimized poses, was considered.
In the output file, 10 poses for each compound were saved.
In Vitro
Metabolic Stability Using Liver Microsomes
Protocol I
Each
sample (2.5 mM) was incubated with
100 mM phosphate buffer (pH 7.4) and 20 mg/mL of liver microsomes
(Thermo Fisher Scientific, Bremen, Germany). After preincubation in
water bath for 5 min, the mixture was incubated with 20 mM NADPH (protocol
I) at 37 °C for 60 min in a Thermomixer comfort (Eppendorf, Hamburg,
Germany).
Protocol II
For the measurement
of UGT activity the
microsomes were preincubated with alamethicin, which forms pores in
microsomal membranes, promoting access of substrate and cofactor to
UGT enzymes. Subsequently, each sample was incubated with 100 mM phosphate
buffer, 500 mM magnesium chloride, 10 mM NADPH, and 20 mM UDP-GlcUA
at 37 °C for 60 min.Finally, the reactions from both protocols
(protocols I and II) were stopped by the addition of 200 μL
of ice-coldmethanol, and then samples were centrifuged at 10 000
rpm at 25 °C for 5 min (Eppendorf microcentrifuge 5424, Hamburg,
Germany). The supernatants were collected and injected in UHPLC-PDA.The control at 0 min was obtained by addition of the organic solvent
immediately after incubation with microsomes. As the positive control,
testosterone was used, while the negative controls were prepared by
incubation up to 60 min without NADPH and UDP-GlcUA/NADPH for protocols
I and II, respectively. The negative control is essential to detect
problems such as nonspecific protein binding or heat instability.
The extent of metabolism is expressed as a percentage of the parent
compound turnover using the following equation, as previously described:[43]
Animals
C57-mice (males, 5 week old, ∼30 g)
(Harlan, The Netherlands) were used for the oxaliplatin-induced neuropathic
pain study. All experiments were approved by the Institutional Animal
and Ethical Committee of the Universidad Miguel Hernandez where experiments
were conducted, and they were in accordance with the guidelines of
the Economic European Community and the Committee for Research and
Ethical Issues of the International Association for the Study of Pain.
All parts of the study concerning animal care were performed under
the control of veterinarians.The WDS was performed in Wistar
male rats (300–350 g), and the thermal ring experiment was
performed on male Swiss CD1mice (30–35 g) purchased from Charles
Rivers (Calco-Lecco-Italy) and then housed in the animal care facility
of the Department Experimental of Pharmacology, University of Naples.
The animals were acclimated to their environment for 1 week, and food
and water were available ad libitum. All behavioral tests were performed
between 9:00 am and 1:00 pm, and animals were used only once. Procedures
involving animals and their care were conducted in conformity with
international and national law and policies (EU Directive 2010/63/EU
for animal experiments, ARRIVE guidelines, and the Basel declaration
including the 3R concept). All procedures reported here were approved
by the Institutional Committee on the Ethics of Animal Experiments
(CVS) of the University of Naples Federico II and by “Ministero
della Salute” under Protocol No. 851/2016. All efforts were
made to minimize animal suffering, and at the end of all experiments,
the animals were euthanized by CO2overdose.
Drug Treatment
For the oxaliplatin-induced neuropathic
pain assay, oxaliplatin (Tocris) was dissolved in water with gentle
warming and was subcutaneously (sc) injected on days 1, 3, and 5 at
a 6 mg/kg dose. The day 7 after administration, experiments were performed.
Together with oxaliplatin injection, saline and a 5% mannitol solution
were intraperitoneally injected to prevent kidney damage and dehydration. 31a stock was prepared in DMSO (Sigma-Aldrich) and diluted
in saline for injections. Compound 31a at different doses
(1 to 30 μg) was injected into the plantar surface (25 μL)
of the right hind paw of mice.For the other in vivo assays,
compound 4 and 31a were dissolved in PEG
400 10% v/v, Tween 80 5% v/v, and sterile saline 85% v/v and injected
once intraperitoneally at the equimolar doses of 10 mg/kg for 4 and 6.7 mg/kg for 31a. Control group was only
treated with vehicle.
Icilin-Induced “Wet-Dog” Shaking
in Rats
Icilin, a TRPM8 agonist, was used to induce shaking
in mice.[44] Animals were first habituated
to the testing
room for 30 min. After that they were randomized into treatment groups
and treated with vehicle or TRPM8 antagonists. Icilin was administered
intraperitoneally (ip) at 1 mg/kg dissolved in 1% Tween 80/H2O 30 or 120 min after drugs. The number of intermittent but rhythmic
“wet-dog-like” shakes (WDS) of neck, head, and trunk
in each animal was counted for a period of 30 min following icilin
administration.
Oxaliplatin-Induced Neuropatic Pain Model
Cold chemical
thermal sensitivity was assessed using acetone drop method.[18b] Mice were placed in a metal mesh cage and allowed
to habituate for approximately 30 min in order to acclimatize them.
Freshly dispensed acetone drop (10 μL) was applied gently onto
the mid-plantar surface of the hind paw. Cold chemical sensitive reaction
with respect to paw licking was recorded as a positive response (nociceptive
pain response). The responses were measured for 20 s with a digital
stopwatch. For each measurement, the paw was sampled twice and the
mean was calculated. The interval between each application of acetone
was approximately 5 min.
Chronic Constriction Injury (CCI) Model of
Neuropathic Pain
Neuropathic pain behavior was induced by
ligation of the sciatic
nerve as described previously.[27d] Briefly,
mice were first anesthetized with xylazine (10 mg/kg ip) and ketamine
(100 mg/kg ip), and the left thigh was shaved and scrubbed with betadine,
and then a small incision in the middle left thigh (2 cm in length)
was performed to expose the sciatic nerve. The nerve was loosely ligated
at two distinct sites (spaced at a 2 mm interval) around the entire
diameter of the nerve using silk sutures (7–0). The surgical
area was closed and finally scrubbed with betadine. In sham-operated
animals, the nerve was exposed but not ligated. Drug effects were
evaluated 7 and 14 days after ligation.
Thermal Gradient Ring
We utilized the thermal gradient
ring from Ugo-Basile previously using a modified protocol from Touska
et al., 2016.[35] The apparatus consists
of a circular running track where each side of the ring is divided
into 12 zones, in which the temperature is proportionally distributed
from 15 to 40 °C, and each sector represents an increment of
2.27 °C. Before the experiment, on day 1, all mice were habituated
to the apparatus for 30 min with the aluminum floor acclimatized to
room temperature (22–24 °C). On day 2, mice were injected
and 30 min after were placed in the apparatus and measured for 60
min using 15–40 °C. Data on preference temperature in
time course were collected from the video-tracking software Any-Maze
connected to the apparatus.
Data Analysis
Data are reported
as the mean ±
standard error of the mean (sem) values of at least three independent
experiments each in triplicate. Statistical analysis was performed
by analysis of variance test, and multiple comparisons were made by
Bonferroni’s test by using Prism 5 (GraphPad Software, San
Diego, CA, USA). p-values smaller than 0.05 were
considered significant.
Authors: Luciano De Petrocellis; Francisco J Arroyo; Pierangelo Orlando; Aniello Schiano Moriello; Rosa Maria Vitale; Pietro Amodeo; Aránzazu Sánchez; Cesáreo Roncero; Giulia Bianchini; M Antonia Martín; Pilar López-Alvarado; J Carlos Menéndez Journal: J Med Chem Date: 2016-06-14 Impact factor: 7.446
Authors: Purificación Ordás; Pablo Hernández-Ortego; Hugo Vara; Carlos Fernández-Peña; Alfonso Reimúndez; Cruz Morenilla-Palao; Ana Guadaño-Ferraz; Ana Gomis; Mark Hoon; Félix Viana; Rosa Señarís Journal: J Comp Neurol Date: 2019-06-02 Impact factor: 3.215
Authors: Carmen De Caro; Claudia Cristiano; Carmen Avagliano; Alessia Bertamino; Carmine Ostacolo; Pietro Campiglia; Isabel Gomez-Monterrey; Giovanna La Rana; Oreste Gualillo; Antonio Calignano; Roberto Russo Journal: Int J Mol Sci Date: 2019-11-07 Impact factor: 5.923
Authors: Ryan Patel; Leonor Gonçalves; Mathew Leveridge; Stephen R Mack; Alan Hendrick; Nicola L Brice; Anthony H Dickenson Journal: Pain Date: 2014-07-30 Impact factor: 6.961
Authors: María Ángeles Bonache; Pedro Juan Llabrés; Cristina Martín-Escura; Roberto De la Torre-Martínez; Alicia Medina-Peris; Laura Butrón; Isabel Gómez-Monterrey; Ana María Roa; Gregorio Fernández-Ballester; Antonio Ferrer-Montiel; Asia Fernández-Carvajal; Rosario González-Muñiz Journal: Int J Mol Sci Date: 2021-02-27 Impact factor: 5.923
Authors: Nunzio Iraci; Carmine Ostacolo; Alicia Medina-Peris; Tania Ciaglia; Anton M Novoselov; Andrea Altieri; David Cabañero; Asia Fernandez-Carvajal; Pietro Campiglia; Isabel Gomez-Monterrey; Alessia Bertamino; Alexander V Kurkin Journal: Int J Mol Sci Date: 2022-02-13 Impact factor: 5.923
Authors: Cristina Martín-Escura; Alicia Medina-Peris; Luke A Spear; Roberto de la Torre Martínez; Luis A Olivos-Oré; María Victoria Barahona; Sara González-Rodríguez; Gregorio Fernández-Ballester; Asia Fernández-Carvajal; Antonio R Artalejo; Antonio Ferrer-Montiel; Rosario González-Muñiz Journal: Int J Mol Sci Date: 2022-02-28 Impact factor: 5.923
Chart 1
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7