Highly functionalized β-lactams and 2-ketopiperazines as TRPM8 antagonists with antiallodynic activity

The cool sensor transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia, playing a key role in cold hypersensitivity associated to different peripheral neuropathies. Moreover, these channels are aberrantly expressed in different cancers, and seem to participate in tumor progression, survival and invasion. Accordingly, the search for potent and selective TRPM8 modulators attracted great interest in recent years. We describe new heterocyclic TRPM8 antagonist chemotypes derived from N-cloroalkyl phenylalaninol-Phe conjugates. The cyclization of these conjugates afforded highly substituted β-lactams and/or 2-ketopiperazine (KP) derivatives, with regioselectivity depending on the N-chloroalkyl group and the configuration. These derivatives behave as TRPM8 antagonists in the Ca2+ microfluorometry assay, and confirmed electrophysiologically for the best enantiopure β-lactams 24a and 29a (IC50, 1.4 and 0.8 µM). Two putative binding sites by the pore zone, different from those found for typical agonists and antagonists, were identified by in silico studies for both β-lactams and KPs. β-Lactams 24a and 29a display antitumor activity in different human tumor cell lines (micromolar potencies, A549, HT29, PSN1), but correlation with TRPM8 expression could not be established. Additionally, compound 24a significantly reduced cold allodynia in a mice model of oxaliplatin-induced peripheral neuropathy.

Icilin EC 50  www.nature.com/scientificreports/ respectively 33,36 , but both studies were discontinued due to adverse secondary effects, including non-tolerated hot sensations. Therefore, there is still a need for TRPM8 antagonists with improved properties. In this context, we have recently described a series of compounds derived from Phe and Asp/Glu amino acid conjugates and having a monocyclic β-lactam central core, which were able to potently and selectively inhibit the activation of TRPM8 by menthol, cool and voltage 37 . Among this series, the shorter Asp derivative 1 (n = 2, Fig. 1) was more potent than the longer Glu analogue 2 (n = 3, Fig. 1), while all the three benzyl and the Boc hydrophobic moieties are important for activity 37 .
Looking for shorter β-lactam derivatives, bearing four hydrophobic substituents, in this manuscript we describe the preparation of conjugates of Z-phenylalaninol with amino acid derivatives and their cyclization to heterocyclic compounds having a β-lactam or a 2-ketopiperazine central scaffold. Both series of compounds behave as TRPM8 antagonists and, among them, selected β-lactam derivatives display antitumor activity, and antiallodynic properties in a model of chemotherapy-induced cold allodynia.

Results
Design. The preparation of a shorter analogue of compounds 1 and 2 was initially projected starting from Boc-Ser-OBn (n = 1). However, all attempts to condense this Ser derivative with Ns-Phe-OBn, were unsuccessful due to the formation of the corresponding dehydroalanine analogue, as described in related reactions 38 . As the structure of TRPM8 channel was unknown at the moment of conceiving this work, we hypothesized that compounds of general formula I (Fig. 1), which maintain the β-lactam ring, its substitution at position 4, but change the Asp-or Glu-derived N-substituent by phenylalaninol-derived moieties, could constitute a new chemoptype for TRPM8 modulation. Although the changes are not bioisosteric, compounds I preserve two hydrophobic substituents on the N-appendage, which were demonstrated to be important for the activity of 1 and 2.
Chemistry. As previously described for Phe-Asp conjugates 37 , the preparation of chloroacetyl derivatives 10 and 11 started by a Mitsunobu reaction between Ns-L-Phe-OBn (3) and the corresponding Z-phenylalaninol derivative (4 or 5) to give compounds 6 and 7 (Scheme 1). Then, the removal of the nosyl group afforded NH derivatives 8 and 9 that, finally, were reacted with chloroacetyl chloride to give key intermediates 10 and 11 (Scheme 1). The cyclization of these L-Phe-phenylalaninol conjugates in the presence of phosphazene bulky bases (BTPP, BEMP) led almost exclusively to the 2-ketopiperazine (KP) derivatives 13 and 15 (Scheme 1, Table 1), resulting from the cyclization through the NH group of the phenylalaninol-derived moiety. Under these conditions, formation of less than 3% of the expected β-lactams was observed in the HPLC chromatograms of the crude reaction mixtures. The ratio of β-lactams 12 and 14 increased slightly when the smaller Cs 2 CO 3 base was employed in the cyclization, allowing their isolation in 8 and 11% yield, respectively ( Table 1). The chloroacetyl phenylalaninol-D-Phe-OMe-derived conjugate 19, despite bearing a smaller methyl ester that could favor the four-membered ring formation, exclusively led to the KP derivative 20 (Scheme 1, Table 1), independently of the base used. The benzyl ester analogue 21 was prepared by transesterification of 20, in order to compare its activity with those of diastereoisomers 13 and 15.
All these KP derivatives were obtained as mixtures of two diastereoisomers at C1′ in variable proportions ( Table 1). The configuration was indirectly assigned by the preparation of Ala dipeptide derivatives from 13ab (see supplememntary information for details), and applying the known rule of differential HPLC retention times and chemical shifts of the Ala CH 3 group between homochiral and heterochiral dipeptide derivatives 39,40 . β-Lactam derivatives 12 and 14 were also formed as mixtures of two diastereoisomers at C4. Considering that the memory of chirality favors the formation of 4S isomers when starting from L-Phe 39,41,42 , the configuration of the major diastereosiomer was assigned as 4S.
To attempt to obtain β-lactams as single isomers, we prepare enantiopure 2-chloropropanoyl Z-phenylalaninol-Phe-OBn conjugates 22, 23, 27 and 28 (Scheme 2). For this, conjugates 8 and 9 were reacted with 2S-or 2R-chloropropionic acid in the presence of trichloroacetonitrile and triphenylphosphine. We have previously demonstrated that related 2-chloropropanoyl derivatives afforded pure β-lactams, with the stereochemistry at C3, C4 dictated by the configuration of the chloropropionic moiety 43 . In good agreement with our precedents, cyclization of the all S isomer 22 with BTPP led almost exclusively to the formation of the 3S,4S,2´S β-lactam 24a (Table 2), along with less than 11% of the corresponding KP (not isolated). Again, the percentage of conversion to the four-membered ring was higher when Cs 2 CO 3 was used as base (Table 2). Similarly, the basic treatment of the 2′R,2″S-chloropropanoyl derivative 27 with BTPP afforded enatiopure 3S,4S,2′R 2-azetidinone 29a. However, in this case, the indicated β-lactam was obtained along with about 50% of the corresponding KP 30ab (a:b, 81:18). Cyclization of 27 with Cs 2 CO 3 afforded a mixture of β-lactam and KP in the same ratio (48:52), but in this case the 2-azetidinone derivative was obtained as a mixture of two diastereoisomers (29ab, 73:27, see SI for a possible explanation). The KP derivative 26ab was the main reaction product (> 85%) during the treatment of the 2′S,2″R-chloropropanoyl precursor 23, and the minor β-lactam was also obtained as a diastereomeric mixture (25ab, Table 2). Contrastingly, the 2-azetidinone derivative 31ab was the main product (> 70%) after the cyclization of the 2′R,2″R-chloropropanoyl precursor 28. Therefore, the configuration of the linear precursor influenced both the regioselectivity (β-lactam versus KP) and the stereoselectivity in the formation of β-lactam derivatives.
A similar reactivity was observed during the cyclization of Ala derivatives (Supplementary Scheme S1). Accordingly, treatment with BTPP of the chloroacetyl derivative 36 afforded exclusively the 6-membered KP 38ab (a:b, 86:14,  Table 3. Representative recordings of fluorescence obtained in microfluorometry experiments for selected compounds are in Supplementary Fig. S3. No agonist activity was observed for these compounds in the absence of menthol. As shown in Table 3, slightly better antagonist activity was observed for β-lactam with an N-2′R-appendage (compare 2′R-derivatives 14ab, 29a and 31ab to 2′S-analogues 12ab, 24a and 25ab, respectively). However, in the case of distereoisomeric mixtures (ab), the exact contribution to the activity of each individual isomer cannot be assessed. As previously described for the first generation of β-lactam TRPM8 antagonists 37 , the phenyl group at position 4 is important for Ca 2+ entrance inhibition, since the 4-CH 3 derivative 39a was one order of magnitude less active than the 4-Bn analogue 29a.
All KP derivatives were assayed as mixtures of two diastereoisomers, therefore the structure-activity relationships should be considered as tendencies, not as absolute statements. As for the β-lactam derivatives, the configuration of the stereocenter coming from the phenylalaninol moiety seems to dictate the antagonist activity, with 5R-KPs more potent than 5S-isomers (compare 15ab to 13ab) ( Table 3). The 1′-configuration appears also to play a role for the inhibition of menthol-induced TRPM8 activation, with a preference for the 1′R-(21ab, 10:90) over the 1′S-isomer (13ab, 80:20). The OBn group in 21ab could participate in the direct interaction with the TRPM8 channel, as the corresponding OMe analogue 20ab shows an important drop in activity. In the 3-methyl derivatives, a 3R,5R configuration (in 30ab) is preferred over the 3S,5S combination (in 26ab), while the 3S,5R-configured diastereoiomers (32ab) showed the lowest activity in this series. In this case, a 4-CH 3 group led to slightly less active derivatives (38ab, 40ab) compared to the corresponding 4-Bn analogues (15ab and 30ab, respectively), although the fall in activity due to this modification is less acute than in the β-lactam series.
β-Lactam and 2-ketopiperazine derivatives were also assayed for their activity in cell expressing hTRPV1 channels. No significant antagonist activity was measured for any derivatives within both chemotypes (Supplementary Tables S1, S2, and Fig. S3), indicating their selectivity for TRPM8 channels.
The TRPM8 antagonist activity of the enantiopure β-lactams 24a and 29a was further confirmed electrophysiologically by Patch-clamp experiments, using the whole cell configuration in HEK293 cells expressing TRPM8 channels.
As shown in Fig. 2, perfusion with 100 µM menthol gives rise to a strongly outward rectifying ionic current characterized by the presence of negligible current at negative potential and the presence of a linear current increase (ohmic) at positive voltages ≥ 40 mV (I-50 mV/I + 120 mV = 0.07). When 10 µM of 24a was applied ( Fig. 2A, blue), an important reduction on the menthol-mediated TRPM8 activity at depolarizing voltages (+ 120 mV) was observed. A similar behaviour was detected for diastereomeric β-lactam 29a (Fig. 2C, blue). The dose-response curve for both compounds was obtained at a holding potential of -60 mV ( Supplementary  Fig. S4). The IC 50 values were 1.4 ± 1.1 µM for 24a (Fig. 2B) and 0.8 ± 1.1 µM for 29a (Fig. 2D).
Docking studies. In order to investigate possible binding pockets within the TRPM8 channel for these families of KP and β-lactam TRPM8 antagonists, we performed computational studies with compounds 13a, 24a, and 29a. A model of the rat TRPM8 channel, created from the cryo-electron microscopy structure of the Ficedula albicollis (PDB code 6BPQ) 24 , was used, and docking simulations were performed with the software implemented in Yasara [44][45][46] .
These docking studies predicted that the three compounds most likely (> 80% solutions) interact with the TRPM8 by the pore zone, with two major solutions having the best binding energies ( Supplementary Fig. S5, Table S3). Site 1 was identified in the middle of the transmembrane region, mainly involving TM5 (S5) and www.nature.com/scientificreports/ TM6 (S6) of one monomer and segments of an adjacent subunit (S5 or S6 and/or the S5-S6 segment forming the pore). The second binding compartment, Site 2, correspond to the cytosolic mouth of the pore, involving the loops connecting TM6 and TRP domains of the 4 protein subunits forming the channel. As for the compounds, mainly hydrophobic interactions can be distinguished at both binding pockets, with some π-π stacking and a few H-bonds identified. Ketopiperazine 13a binds TRPM8 channel at Site 1 through two π-π stacking connections, a face-to-face stacked interaction between the phenyl group of the 1′-Bzl moiety and Y963 at subunit 1 (S6), and secondly a T-shaped (edge-to-face) contact encompassing the phenyl group of the α-CO 2 Bzl moiety and F874 residue of TRPM8 subunit 3 (S5) (Supplementary Fig. S6). Among hydrophobic interactions, the Ph group of the Cbz moiety occupies a hydrophobic pocket delineated by residues located at subunit 3 (F870, L873, F874, I962, L965, and I969) and subunit 2 (I844). In addition, the 1′-Bn moiety is also involved in hydrophobic interactions through L871 and F874 (Subunit 3), while the phenyl group of the 5-Bn is in touch with I962 and Y963, also at subunit 3. At Site 2, a face-to-face π-π stacking involves the Ph group of the Cbz and the aromatic ring of the Y981 residue (subunit 1). Moreover, the hydrophobic interactions comprise the four Ph rings of 13a and residues from the four channel subunits (see Supplementary Fig. S7).
At Site 1, β-Lactams 24a and 29a occupy similar areas of the transmembrane region, involving two neighboring channel subunits, but their poses are clearly different, with the β-lactam ring pointing to the upper and   4 and Supplementary Fig. S8). Additionally, three π-π interactions contribute to the complex stabilization, two T-shaped contacts comprising the Ph group of the Cbz moiety and W877 and F881 side-chains (both at S5), and a face-to-face sandwich between the aromatic groups of 2′-Bn and Y963 residue (S6), all at channel monomer 3. This monomer contributes also to the complex with nine hydrophobic interactions among a series of residue side-chains of the channel and the four Ph groups of the antagonist, while subunit 2 (S5 and S5-S6 loop) add three additional hydrophobic contacts (specified in Supplementary Fig. S8). The diastereoisomeric 3S,4S,2′R compounds 29a is fixed to the channel through a H-bond (NH → CO of G913, S5-S6 segment, subunit 1) and a parallel displaced π-π connection (OBn-Y963 of S6, subunit 1, Supplementary Fig. S10). Also, a number of residues at subunit 1 provide hydrophobic interactions with the four Ph groups of 29a. As previously, the contiguous monomer (subunit 4) also supplies additional stabilizing interactions, in this case involving three residues at the pore-forming S5-S6 segment ( Supplementary Fig. S10). Main residues involved in the hydrophobic interactions of 24a and 29a at Site 2 are Y981 (from three out of four subunits), T982 of monomer 4, and I985 of two subunits. In addition, a H-bond between the CO group of Y981 (subunit 1, S6) and the NH group of 24a, and a π-π displaced stacking interaction involving the phenyl groups of the 2′-Bn moiety in 29a and Y981 side-chain (Subunit 1, S6), contribute to the respective stabilization of the complexes at Site 2 (Fig. 4, and Supplementary Figs. S9 and S11). Interestingly, most residues of rTRPM8 suggested as important for the interaction with β-lactams and KPs at both sites 1 and 2 are highly conserved in hTRPM8 ( Supplementary Fig. S12).

Growth inhibitory activity in tumor cells.
A number of recent experimental evidences position TRPM channels as important players in cancer growth and progression 47 . Among these channels, the aberrant expression of the TRPM8 subtype has been described in different human malignant tumors, including those of prostate, pancreas, breast, colon, and skin, among others 3 . More importantly, sometimes the TRPM8 overexpression was associated to poor prognosis of cancer patients. In good agreement, several, structurally different TRPM8 antagonists demonstrated good antitumor activity in prostate 32,48,49 , and others human tumor cell lines 50 . A few  Tables 4  and S4, and compared to those of the well-known chemotherapeutic agent doxorubicin. As shown in Table 4, compound 24a displays in vitro cytotoxic activity in the micromolar range in three out of four assayed tumor cell lines, with no activity against the MDA-MB-231 breast cell line (at 10 µM/mL). Compared to 24a, slightly lower potencies were measured for β-lactam 29a in lung, colon and pancreas tumor cell lines, but contrastingly it displays better, although moderate, in vitro cytotoxic activity in the breast cell line. Hence, no significant influence neither of the configuration of the β-lactam derivative nor of the TRPM8 antagonist potency was observed on the antiproliferative activity of these compounds. In general, these activities were one order of magnitude less potent than the control doxorubicin.
Three steroisomeric 2-KP derivatives, having different TRPM8 antagonist capacity, were also assayed for their antitumoral activity (Table 4). In this case, compound 30ab, showing submicromolar TRPM8 antagonist activity, is only moderately active in A549 and PNS1 cell lines, with no significant cytotoxicity in the colon and breast cell lines. Compound 15ab, with micromolar potency as TRPM8 antagonist, is only cytotoxic in the pancreas cell line, while the less potent analogue 13ab did not show any significant antiproliferative activity. It seems that for the 2-KP series the in vitro cytotoxicity follows the same order than TRPM8 antagonist potency. The lower antitumor potential, compared to β-lactams, could be due either to the evaluation of diasteroisomeric mixtures in 2-KPs versus enantiopure β-lactams, or to the fact that the cytotoxicity of β-lactam derivatives is independent of the TRPM8 activity or both.  54 . Because this lack of information and the controversial results, we cannot assure that the antitumor activity displayed by our compounds is partially due to a high expression of TRPM8 in the indicated cell lines or if it could be an effect totally independent of these channels.
No apparent, significant cytotoxic effects were observed for the β-lactam derivatives in HEK293 cells (up to 500 µM concentration, MTT assay).

Antiallodynic effects in vivo.
Cold allodynia (painful sensation at cold temperatures that do not usually cause pain) and cold hyperalgesia (increased sensitivity to distressing cold temperatures) are associated to different peripheral neuropathies 55 . Several chemotherapeutic agents in first clinical line induce peripheral neuropathies (known as CIPN), affecting million patients worldwide and limiting the dose administered to them, as well as the quality of life of many survivors 56 . In oxaliplatin CIPN, the increased sensitivity to cold has been correlated to an augmented expression of TRPM8 channels, among others [4][5][6]57 . In good correlation, there are recent experimental evidences describing that TRPM8 antagonists are able to decrease oxaliplatin-induced allodynia and cold hypersensitivity 34,58 . According to these discoveries, we decided to explore the effects of β-lactam 24a in an in vivo model of oxaliplatin CIPN, using acetone assay for monitoring cold allodynia. In male mice, the injection of oxaliplatin on days 1, 3 and 5 at a 6 mg/kg dose produces peripheral cold allodynia. As shown in Fig. 4, the intraplantar (i.pl.) administration of β-lactam derivative 24a (1 μg), attenuates the cold-induced paw licking in a significant manner 15 min after administration, showing the maximum activity from 30 to 60 min. At a 3 µg dose, the antagonist activity is clearly increased at 15 min, and firmly maintained up to 60 min.

Discussion
To search for new TRPM8 antagonist chemotypes, we explore the base-assisted cyclization of linear phenylalaninol-Phe conjugates, which afforded chiral β-lactam and/or 2-ketopiperazine (KP) heterocyclic derivatives. The regioselectivity (β-lactam versus KP) was dependent on the chloroalkyl substituent and the configuration of the linear precursor. While 2-chloroacetyl derivatives gave almost exclusively to the KP six-membered ring heterocycle, the cyclization of 2-chloropropanoyl analogues is governed by the configuration of both the  Data are given ± SME (n = 5). **P < 0.05; ***P < 0.001; ****P < 0.0001.
Scientific RepoRtS | (2020) 10:14154 | https://doi.org/10.1038/s41598-020-70691-x www.nature.com/scientificreports/ phenylalaninol-derived (2′) and the 2-chloropropanoyl (2″) stereocenters. In short, 2′S,2″S and 2′R,2″R isomers provides β-lactams as the very major component of the reaction, while the KP heterocycle predominates after the cyclization of the 2′S,2″R diastereoisomer, and the 2′S,2″R linear precursor provides almost the same amount of the four-and six-membered heterocyclic systems. The epimerization at the C-3 stereocenter in 3-methyl-β-lactam derivatives, not previously observed for related 2-azetidinones 43 , was low for 3S,4S-configured compounds and more important for 3R,4R-analogues. Both, the phenylalaninol-Phe-derived β-lactams and KPs behave as new TRPM8 antagonist chemotypes, blocking the channel activation by menthol (Ca 2+ entry assay) with micromolar or submicromolar potencies, and did not show activity at hTRPV1. Single isomer β-lactams 24a and 29a display IC 50 values of 2.4 and 0.4 µM, respectively, indicating that a 2′R-configuration of the phenylalaninol-derived substituent is preferred for TRPM8 antagonist activity. These antagonist activities were further confirmed using electrophysiology experiments, with Patch-clamp measurements sustaining that the 2′R diastereoisomer is slightly more potent than the corresponding 2′S isomer. In general, these phenylalaninol-Phe-derived β-lactams maintain significant TRPM8 blockade activity, although they showed somewhat decreased potency compared to the longer Asp-Phe analogues 37 . For KPs, a 1′R-and a 5R-configuration seem to favor the inhibition of TRPM8 channel activation.
Docking studies, using a homology model of rat TRPM8 channel, built on the cryo-electron microscopy structure of the TRPM8 from Ficedula albicollis 24 , propose two putative binding sites, by the pore zone, for the phenylalaninol-Phe-derived heterocyclic compounds described here. The first site involves transmembrane S5 and S6 of one channel subunit and the S5 or S6 and/or the S5-S6 segment forming the pore of one adjacent monomer, suggesting an allosteric modulation of the channel. The second most probable binding point is located at the bottom part of the pore, involving mainly hydrophobic interaction among the phenyl rings of the molecules and hydrophobic and aromatic residues of the four channel subunits, with the compound acting as a channel blocker. The sites predicted by these models of interaction differs from those proposed for tryptophan-derived antagonists 33 , and that from AMTB and TC-I/TRPM8 complexes solved by cryo-electron microscopy 26 , which adopt different poses within the channel, but all around the menthol-binding pocket (delineated by the lower half of the TM4-TM5 helices and the TRP domain) 25 . The larger volume of our molecules could be behind this different behaviour.
TRPM8 channels are overexpressed in a number of tumors, like prostate, melanoma, lung and colon adenocarcinomes, and some TRPM8 antagonists demonstrated good antitumor activity 32,48,49 . Interestingly, enantiopure β-lactams 24a and 29a exhibited non-selective antitumor activity in different tumor cell lines, showing micromolar potency in four of them, while 2-KP regioisomeric compounds displayed lower antifloliferative activity. No direct correlation between TRPM8 antagonist and antitumor activity could be established. Abnormalities in TRPM8 expression was also found in models of chemotherapy-induced peripheral neuropathy 57 . To evaluate the usefulness of this family of compounds in relieving persistent pain associated to antineoplastic agents, we evaluated β-lactam 24a in a mice model of oxaliplatin-induced peripheral neuropathy. Compound 24a demonstrated significant antiallodynic effects in vivo, with maximum activity from 30 to 60 min. Compared to literature reported results, the antiallodynic activity of compound 24a seems superior or comparable to that of other described TRPM8 antagonists. Thus, its potency is higher than that of a described spirochromene derivative 59 , and seems to span longer than that of a Trp-OMe derivative, which showed more potent antagonist activity at the Ca 2+ assay than 24a 34 . At 1 µg i.pl., β-lactam derivative 24a showed slightly lower potency and similar duration of action than a biphenyl amide TRPM8 antagonist recently reported 60 .
In conclusion, the two phenylalaninol-Phe-derived heterocyclic systems, highly functionalized β-lactam and/or 2-ketopiperazine, allow the identification of new hits for TRPM8 modulation. Therefore, these two new chemotypes could constitute the starting point for further modifications on the road to improved compounds for future therapeutic applications in both pain and cancer.    46 , implemented in Yasara, in which a total of 800 flexible docking runs were set and clustered around the putative binding sites. The program then performed a simulated annealing optimization of the complexes, which moved the structure to a nearby stable energy minimum, by using the implemented Assisted Model Building with Energy Refinement (AMBER03) force field 63 . The Yasara pH command was set to 7.0, to ensure that molecules preserved their pH dependency of bond orders and protonation patterns. The best binding energy complex in each cluster was stored, analyzed, and used to select the best orientation of the interacting partners.
Cytotoxicity Assay. Triplicate cultures were incubated for 72 h in the presence or absence of test compounds in dose-response curves (10 concentrations, typically ranging from 10 to 0.0026 µg/mL). A colorimetric assay using sulforhodamine B (SRB) was adapted for quantitative measurement of cell growth and cytotoxicity 27 . A more detailed information on this assay is provides in the supplementary information. GI 50 is the compound concentration that produces 50% inhibition on cell growth as compared to control cells.
In vivo anti-allodynic effects. Male C57-mice (≈30 g) (Harlam, Holland) were used for the 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.
As previously described 34 , oxaliplatin (Tocris) was dissolved in water with gentle warming and was subcutaneously (s.c.) 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. The compound 24a stock was prepared in DMSO (Sigma-Aldrich) and diluted in saline for injections. Compound at different doses (1 to 3 μg) was injected into the plantar surface (25 μL) of the right hind paw of mice. Cold chemical thermal sensitivity was assessed using acetone drop method. 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 (10μL) was applied gently on to 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.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.