An adamantane‐based ligand as a novel chemical tool for thermosensory TRPM8 channel therapeutic modulation

Abstract

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a nonselective thermosensory cation channel expressed in peripheral nociceptor terminals where it transduces cold temperatures and cooling agents such as menthol. TRPM8 dysfunction has been involved in disabling sensory symptoms, such as cold allodynia. In addition, its widespread expression has signaled this channel as a pivotal therapeutic target for a variety of diseases, from peripheral neuropathies to cancer. Thus, the design and therapeutic validation of TRPM8 antagonists is an important endeavor in biomedicine. To address this, we used the multicomponent Passerini and Ugi reactions to design a novel family of TRPM8 modulators using as a scaffold the adamantane ring that exhibits drug‐like qualities. These green chemistry transformations are ideal for the fast synthesis of libraries of medium complexity with minimal or no generation of waste by‐products. We report the identification of a family of TRPM8 agonists and antagonists. Among them, 2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl [1,1′‐biphenyl]‐2‐carboxylate (referred to as compound 23) is a potent and selective antagonist that reduces TRPM8‐induced neuronal firing in primary nociceptor cultures. Compound 23 exhibits 10‐fold higher potency for human TRPM8 (hTRPM8) than for hTRPV1 and hTRPA1 channels. Notably, local administration of compound 23 significantly attenuated oxaliplatin‐induced peripheral cold allodynia by modulating epidermal TRPM8 sensory endings. Thus, α‐acyloxy carboxamide 23 appears as a promising therapeutic candidate to topically intervene on TRPM8‐mediated peripheral neuropathies.

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a cationic thermosensory channel crucial for cold sensation and menthol response. TRPM8 dysfunction leads to sensory symptoms such as cold allodynia. Targeting TRPM8 is important for treating diseases like peripheral neuropathies and cancer. Using green chemistry, we found compound 23, as a potent TRPPM8 antagonist that, locally applied, attenuates oxaliplatin‐induced cold allodynia, showing promise treatment for chemotherapy‐induced neuropathies.

Abbreviations

Ac₂O

acetic anhydride

AITC

allyl isothiocyanate

AMG333

(S)‐6‐(((3‐fluoro‐4‐(trifluoromethoxy)phenyl)(3‐fluoropyridin‐2‐yl)methyl)carbamoyl)nicotinic acid

AMTB

N‐(3‐aminopropyl)‐2‐{[(3‐methylphenyl)methyl]oxy}(20)‐N‐(2‐thienylmethyl)benzamide

AP

action potential

CDCl₃

deuterated chloroform

DMSO‐d₆

deuterated dimethyl sulfoxide

DRG

dorsal root ganglion

EC₅₀

half‐maximal effective concentration

EtOAc

ethyl acetate

HEK293

human embryonic kidney 293 cells

hTRP

human transient receptor potential

IC₅₀

half‐maximal inhibitory concentration

LC‐HRMS

liquid chromatography coupled to high‐resolution mass spectrometry

LC‐UV

liquid chromatography coupled to ultraviolet detector

MEA

multielectrode array

MeOH

methanol

MLM

mouse liver microsome

OIPN

oxaliplatin‐induced peripheral neuropathy

OXP

oxaliplatin

PE

petroleum ether

PF‐05105679

(R)‐3‐[(1‐(4‐fluorophenyl)ethyl)(quinolin‐3‐ylcarbonyl)amino]methylbenzoic acid

POCl₃

phosphorus oxychloride

SEM

standard error of the mean

TEA

triethylamine

TRP

transient receptor potential

TRPA1

transient receptor potential cation channel ankyrin 1

TRPM8

transient receptor potential cation channel melastatin 8

TRPV1

transient receptor potential cation channel vanilloid 1

WS12

(1R,2S,5R)‐N‐(4‐methoxyphenyl)‐5‐methyl‐2‐(propan‐2‐yl)cyclohexane‐1‐carboxamide

Introduction

The transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel expressed in sensory neurons. As a member of the TRP family of ion channels, TRPM8 is involved in thermosensation playing a pivotal role in the perception of environmental cold, and the cooling sensation evoked by menthol [1, 2]. TRPM8 is active in the range of 8–28 °C and participates in the transduction of cold allodynia [3]. Structurally, the channel is a tetrameric protein assembled by four identical subunits arranged around a central aqueous pore. Each subunit contains six transmembrane segments (S1–S6) and intracellular N‐ and C‐terminals [4]. Upon activation, TRPM8 transits to an open conformation that allows the influx of cations, such as calcium (Ca²⁺) and sodium (Na⁺), favoring neuronal depolarization and action potential firing. Electrical impulses are transmitted through the spinal cord towards the sensory processing areas of the brain to elicit the cold sensation [5, 6].

TRPM8 has been involved in inflammation and immune responses in several tissues. For example, TRPM8 influences the release of inflammatory mediators from pulmonary epithelial cells leading to dysregulation of immune responses and chronic inflammation [7, 8]. The suppression of TRPM8 activity alleviated respiratory hypersensitivity in individuals with asthma [9]. Additionally, several studies have explored the role of TRPM8 in cancer cell proliferation, migration, and apoptosis [10, 11] positioning this channel as a potential target for cancer therapy in combination with other treatment modalities [12]. Furthermore, few studies have suggested that TRPM8 is involved in the perception of itch, modulating the transmission of itch‐related neural signals through peripheral pathways [13]. Moreover, the TRPM8 receptor has been also implicated in the etiology of dry eye syndrome, a condition characterized by insufficient tear production or poor tear quality, leading to discomfort and irritation [14, 15]. Notably, positive results of the phase 3 COMET trial of AR‐15512, a novel formulation of a TRPM8 drug candidate for dry eye syndrome, were announced [16].

TRPM8 has been also signaled as pivotal in the pathophysiology of chronic migraine [17, 18] and plays a major role in the pathogenesis of oxaliplatin‐induced cold allodynia (OIPN). Its pharmacological modulation has been explored as a potential strategy for pain alleviation [19, 20]. Thus, the design and validation of TRPM8 modulators are crucial for advancing our understanding on the contribution of TRPM8 to the etiology of neuropathic pain, inflammatory disorders, pruritus, and cancer and to select novel candidates for therapeutic development. Specifically, therapeutic inhibition of TRPM8 may offer antinociception for conditions characterized by cold‐associated pain (i.e., cold allodynia) [21] and provide alternatives to traditional analgesics with unwanted side effects such as opioid drugs [22].

In the past years, the development of TRPM8 modulators has faced challenges due to off‐target effects and species‐specific structural variations [23]. Compounds such as PF‐05105679 and AMG333, both orally active TRPM8 antagonists, progressed into clinical trials with the goal of investigating their potential in cold pain hypersensitivity and alleviating migraine symptoms, respectively [24, 25]. However, these promising compounds did not advance beyond phase I trials due to safety concerns. Thus, the discovery and development of therapeutically useful TRPM8 modulators remain a biomedical challenge.

In the pursuit of identifying new TRPM8 ligands with therapeutic potential, we combined the power of multicomponent reactions to readily achieve molecular diversity with adamantane that exhibits therapeutic properties. Its cyclic aliphatic hydrocarbon inspired us to consider it as a ‘potential surrogate’ for the terpene skeleton of TRPM8 ligands such as menthol, eucalyptol, and camphor. Furthermore, the choice of this caged compound as a potential modulator of the TRPM8 channel was motivated by the fact that the adamantane structure has already been reported capable of inhibiting different ion channels implicated in diseases such as viral infections and neurodegenerative disorders [26, 27]. In addition, the adamantyl group has been considered as a ‘lipophilic bullet’ capable of endowing favorable pharmacokinetic characteristics to molecules, such as a good propensity for crossing biological membranes [27, 28]. Beyond increasing partition coefficients (logP value) by about 3.1 log units, the adamantyl group has been also found to improve drug‐like qualities such as metabolic stability. Of note, the steric bulk of the adamantyl group can impede the access of hydrolytic enzymes (esterases, amidases), thereby increasing the plasma half‐life of lead compounds [27]. These unique properties of the adamantane scaffold make it an ideal candidate for modulating ion channels, showcasing its potential in the development of therapeutics for diseases resulting from TRPM8 channel dysregulation [29].

Among the dozens of multicomponent reactions discovered to date [30, 31, 32, 33] in terms of versatility, efficiency, exploratory power, and experimental simplicity, the Ugi [34, 35, 36, 37] and Passerini reactions [38, 39, 40, 41] remain very attractive. In these two isocyanide‐mediated reactions, four and three components are respectively reacted together to generate α‐aminoacyl amides and α‐acyloxy carboxamides. These multicomponent reactions produce focused libraries and have the advantage of minimizing the formation of waste products, thus fulfilling the criteria to be considered green chemistry [40]. In 2006, we discovered a new variant of the Ugi reaction, named split‐Ugi, whereby replacing a primary amine with a symmetric secondary diamine yielded a new drug‐like molecular scaffold [42].

Here, we report a family of adamantane‐based TRPM8 agonists and antagonists that increase the channel pharmacopeia. Among all ligands tested, the acyloxy carboxamide 23 (referred to as compound 23) is a potent hTRPM8 antagonist showing a > 10‐fold blockade preference over the structurally related thermosensory channels TRPV1 and TRPA1. Furthermore, compound 23 reduced nociceptor excitability and locally attenuated oxaliplatin‐induced in vivo cold allodynia.

Results

Synthesis of compounds using the Passerini, Ugi, and split‐Ugi reactions

To employ the three multicomponent reactions, we utilized 1‐isocyanoadamantane (3) as a lipophilic warhead. This reagent was prepared from 1‐aminoadamantane (1) through the intermediate 1‐adamantylformamide (2), as described previously [43] (Fig. 1). 1‐Isocyanoadamantane (3) was reacted with various commercially available carboxylic acids (4–14) and amines (15–19, 1) (Fig. 1), always employing formaldehyde as the carbonyl component to avoid the introduction of a stereogenic center.

Fig. 1.

Preparation reaction of 1‐isocyanoadamantane 3 and chemical structure of carboxylic acids 4–14 (red color) and amines 15–19, 1 (green color) used in this study.

For the Passerini reaction, we mixed isocyanide 3, formaldehyde, and a carboxylic acid (4–13) in dichloromethane and stirred the three‐component mixture at room temperature. We obtained 10 α‐acyloxy carboxamides (20–29) with excellent yields (Fig. 2A). Employing the Ugi reaction, we obtained 12 α‐aminoacyl amides (30–41) by mixing the 4 components (isocyanide 3, formaldehyde, a carboxylic acid 4–9, 14 and a primary amine 1, 15–18) in methanol and stirring them at 60 °C (Fig. 2B). Finally, we carried out the split‐Ugi reaction [42] to prepare 4 α‐acylpiperazino amides (42–45) using carboxylic acids 4–6 and 9 (Fig. 2B). In this variant of the Ugi reaction, piperazine 19 was added to the other 3 components, instead of a primary amine, maintaining the same experimental conditions. ¹H‐NMR, ¹³C‐NMR spectra, and HRMS spectra of final compounds 20–45 are reported in Figs S1–S78.

Fig. 2.

Passerini and Ugi reactions. (A) Preparation of α‐acyloxy carboxamides 20–29 via the Passerini reaction and compounds obtained. (B) Synthesis of α‐aminoacyl amides 30–41 and α‐acylpiperazino amides 42–45 exploiting the Ugi and split‐Ugi reactions, respectively, and compounds obtained (black color refers to isocyanide component, blue color refers to aldehyde component, red color refers to carboxylic acid component, and green color refers to amine component).

Ca²⁺‐microfluorometry to screen for agonist/antagonist activity of synthesized compounds

We first evaluated the series of compounds 20–29 synthesized using the Passerini‐Adamantane reaction. All compounds were screened for channel agonism and antagonism on recombinant human TRPM8 stably expressed in HEK293 cells (hTRPM8‐HEK293) with Ca²⁺ microfluorimetry. For agonistic activity, we directly assessed compound‐induced Ca²⁺ influx into hTRPM8‐HEK293 cells and compared it with the activity of WS‐12, a well‐known potent hTPRM8 agonist. Compound‐induced TRPM8‐mediated Ca²⁺ influx was confirmed using untransfected HEK293 cells and the channel blocker AMTB. For antagonistic activity, modulation of WS‐12‐induced Ca²⁺ influx was evaluated. The concentration of compounds to reach half of channel activation (EC₅₀) or to block half‐channel activity evoked by WS‐12 (IC₅₀) are reported in Table 1, along with the percentage of maximal activation or blockade (Efficacy).

Table 1.

Screening for agonist/antagonist activity of synthesized compounds 20–45. The experiments were conducted three times independently (N = 3), with each determination performed in triplicate (n = 3). The data are presented as the mean value accompanied by the standard deviation (SD).

Compound	hTRPM8
	R	Activity	EC50 (μm)	IC50 (μm)	Hill coefficient	Efficacy (%)
20		Antagonist		1.8 ± 1.1	0.4 ± 1.2	60
21		Antagonist		1.5 ± 1.1	0.5 ± 2.3	44
22		Agonist	1.1 ± 1.5		0.4 ± 1.3	64
23		Antagonist		0.2 ± 1.3	0.5 ± 0.9	100
24		Agonist	19 ± 2.0		0.6 ± 0.2	19
25		Antagonist		45 ± 2.0	0.8 ± 3.0	57
26		Agonist	1.9 ± 1.6		0.4 ± 6.4	72
27		Agonist	0.6 ± 1.2		0.3 ± 1.6	60
28		Agonist	1.2 ± 1.2		0.1 ± 0.2	87
29		Antagonist		2.3 ± 1.1	0.5 ± 3.0	69
30		Agonist	1.5 ± 1.5		0.7 ± 1.6	92
31		Agonist	0.3 ± 1.9		0.7 ± 2.5	72
32		Agonist	0.4 ± 1.7		0.2 ± 1.5	56
33		Antagonist		1.1 ± 1.2	2.0 ± 7.8	48
34		Agonist	0.2 ± 1.9		0.3 ± 14	54
35		Antagonist		0.6 ± 1.2	2.0 ± 4.3	40
36		Agonist	4.9 ± 1.7		2.3 ± 2.6	47
37		Agonist	0.2 ± 3.0		0.9 ± 9.2	48
38		Agonist	2.0 ± 1.4		0.3 ± 3.6	65
39		Agonist	0.5 ± 1.4		0.4 ± 9.1	64
40		Agonist	1.4 ± 1.4		0.7 ± 2.4	86
41		Agonist	0.9 ± 1.1		2.1 ± 1.0	49
42		Agonist	5.4 ± 1.5		0.4 ± 4.3	73
43		Agonist	9.3 ± 1.5		0.6 ± 1.8	82
44		Agonist	9.6 ± 1.5		0.7 ± 4.0	69
45		Agonist	2.3 ± 1.6		0.7 ± 2.1	54

We found that compounds 20, 21, 23, 25, and 29 behave as hTRPM8 antagonists, blocking WS‐12 responses with potencies ranging from 0.2 to 45 μm (Table 1). The most potent antagonist was compound 23 that exhibited an IC₅₀ value of 0.2 ± 1.3 μm and 100% efficacy. The lowest activity was observed in compound 25, suggesting that a bulky aromatic group at position R was important for potent antagonistic activity. Compounds 22, 24, and 27 exhibited an agonistic activity with potency ranging from 0.6 to 19 μm. Data show that increasing the hydrocarbon side chain (22 and 24) produced a decrease in potency, that can be compensated if the increase in size is accompanied by the addition of nucleophile atoms such as nitrogen and oxygen (27). Interestingly, compounds 26 and 28, containing an aromatic group at the R position, displayed agonist activity contrasting with the antagonistic action of compounds 20 and 29. This suggests that changes in the aromatic ring modulated the interaction of these ligands with molecular determinants of the channel binding site, differentially impacting the allosteric conformational changes needed for channel activation. These results provide a novel family of hTRPM8 agonists and antagonists and signal to α‐acyloxy carboxamide 23 as a potent TRPM8 antagonist with potential therapeutic value.

We next evaluated the α‐aminoacyl amides 30–41 exploiting the Ugi reaction (Table 1). We also found a mixture of agonists and antagonists within this family of compounds, although more enriched in agonists. Hence, compounds 33 and 35 displayed moderate antagonistic activity with IC₅₀ in the low micromolar range. Compound 33 is closely related to compound 23, indicating that the substitution of the ester by an amide did not drastically alter the activity of the compound, substantiating the importance for antagonistic activity of the bulky aromatic rings. Additional support is provided by compound 35. All other α‐aminoacyl amides exhibited agonistic activity with potencies ranging from 0.2 to 5 μm. Again, the substitution of the ester by an amide modestly affected the potency of most compounds (see 22 with 34, 37, 39, and 40). A significant increase in potency was observed for compound 31 (EC₅₀ = 0.3 ± 1.9 μm) as compared with the ester‐containing counterpart 24 (EC₅₀ = 19 ± 2.0 μm). Most notably, at variance with the antagonistic activity of the α‐acyloxy carboxamide 25 (IC₅₀ = 45 ± 2.0 μm), the α‐aminoacyl amide 32 acted as a potent hTRPM8 agonist (EC₅₀ = 0.4 ± 1.7 μm).

Last, functional evaluation of the four α‐acylpiperazino amides (42–45) revealed an agonistic activity on hTRPM8 channels, with lower micromolar potency than the α‐acyloxy carboxamide and α‐aminoacyl amides (Table 1). To highlight specific comparisons: compound 42 can be directly compared to compound 30; compound 44 to compound 36; and compound 45 to compounds 22, 34, 37, 39, and 40. Akin to α‐aminoacyl amide 32, the α‐acylpiperazino amide 43 also behaves as a TRPM8 agonist although with > 10‐fold lower potency.

α‐Acyloxy carboxamide 23 is a potent hTRPM8 competitive WS‐12 antagonist

We selected α‐acyloxy carboxamide 23 as the most potent antagonist (IC₅₀ = 0.2 ± 1.3 μm) for further in vitro and in vivo characterization. We first used patch‐clamp to investigate the potency and blockade mechanism of compound 23 inhibiting WS‐12 evoked ionic currents in hTRPM8‐HEK293 cells. As shown in Fig. 3A, 1 μm of α‐acyloxy carboxamide 23 reduced ≥ 75% WS‐12 evoked inward current. This blockade activity was reversible as virtually all WS‐12‐evoked ionic current could be recovered upon washout (Fig. 3A, bottom panel), thus preventing a use‐dependent side effect. A dose–response curve revealed an IC₅₀ of 0.08 ± 0.07 μm, and a Hill coefficient of 1 (Fig. 3B), suggesting that compound 23 is a potent hTRPM8 antagonist.

Fig. 3.

Compound 23 is a hTRPM8 competitive blocker with nanomolar potency. (A) Representative traces of WS‐12‐evoked ionic currents recorded at a holding potential of −60 mV in HEK293 cells heterologously expressing hTRPM8. Control cells (upper panel) were exposed to two pulses (1 μm) of WS‐12, interspaced by a washing period whereas compound 23‐treated cells (middle panel) were exposed before to 1 min of compound 23 (1 μm) and then to 30s of compound 23 during the second pulse of WS‐12. In the lower panel, the recovery of WS‐12‐evoked currents after compound 23 treatment is shown, indicating a reversible effect of the compound on hTRPM8 activity. (B) Dose–response curve for hTRPM8 current blockade at a holding voltage of −60 mV. The line represents the fit of the experimental data to the Hill equation: $Y=\text{Bottom}+\left(\mathrm{Top}‐\text{Bottom}\right)/\left(1+{10}^{\left(X-{\text{LogIC}}_{50}\right)}\right)$ with a standard slope of 1.0 (Hill coefficient) and a restriction Top (= 100). The fitted value for IC₅₀ was 0.08 ± 0.07 μm. Each point is the mean ± SEM of N = 3, n = 10. (C) WS‐12 dose–response curve in the absence (black) or presence of 0.08 μm compound 23 (blue). The best fitted values for WS‐12 EC₅₀ value were 3.0 μm (95% CI 2.5–3.68 μm) (n = 18) in the absence of 0.1 μm compound 23 and 15 μm (95% CI 12.8–16.8 μm) in its presence (blue curve, n = 16, Top = 100). Hill coefficient in the absence of compound 23 was 1.17 (0.9–1.4) and in its presence was 0.95 (95% CI 0.8–1.0). All data are expressed as mean ± SEM. N = 3. (D) Schild plot derived from the interaction between agonist (WS12) and antagonist (compound 23) at four different concentrations: 0.02, 0.08, 0.1, 0.5 μm. Equation: Y = 1003 × X + 1469; Slope: 1.003; 1/Slope: 0.9973; Std. error: 0.1935. R square: 0.9307; P value: 0.0353. N = 3, n = 10. (E) Representative current density (J)‐V curves elicited by a protocol of voltage steps from −120 to 120 mV in steps of 20 mV in the absence (control) or presence of 0.08 μm compound 23 (n = 10) or 10 μm AMTB (n = 8). In the control condition, an additional pulse of WS‐12 was used to consider channel desensitization. Data were analyzed with two‐way ANOVA, N = 3. Each point is the mean ± SEM. Data were analyzed using a two‐way ANOVA followed by a Sidak post hoc test when appropriate, **P value = 0.0069; ****P value < 0.0001.

To unveil the underlying mechanism of channel blockade (competitive vs noncompetitive) we evaluated the effect of the α‐acyloxy carboxamide 23 on WS‐12 activating potency. For this purpose, we obtained the WS‐12 dose response in the absence and presence of 80 nm compound 23 (corresponding to its IC₅₀ value). As depicted in Fig. 3C, the WS‐12 dose response was right‐shifted to higher concentrations in the presence of compound 23 increasing up to fivefold the WS‐12 EC₅₀ value from 3 to 15 μm, without altering the agonist efficacy, suggesting a competitive blockade mechanism. In support of this tenet, a Schild plot to evaluate the effect of increasing concentrations of compound 23 in the blockade efficacy of WS‐12 revealed a linear relationship (slope of 1.0) consistent with a competitive inhibitory mechanism (Fig. 3D). Thus, these results suggest that α‐acyloxy carboxamide 23 and WS‐12 binding to the same or nearby site in the receptor.

To further substantiate the competitive mechanism of channel blockade, we examined the voltage dependency of compound 23 antagonistic activity (Fig. 3E). For this purpose, we run 100 ms voltage ramps from −120 to 120 mV activated by WS‐12 in the absence and presence of 80 nm α‐acyloxy carboxamide 23. Figure 3E shows the current density (J)‐V relationships for compound 23 and the well‐known TRPM8 competitive antagonist AMTB. Both compounds inhibited WS‐12 in the entire range of depolarizing voltages, indicating channel blockade was not voltage dependent as it would be expected for an uncompetitive antagonist. Thus, these data further support a competitive, voltage‐independent blockade mechanism for compound 23.

Compound 23 marginally blocks TRPV1 and TRPA1 activity

To evaluate receptor selectivity, we investigated the activity of compound 23 on recombinant hTRPV1 and hTRPA1 channels recombinantly expressed in HEK293 cells (Fig. 4). We choose these thermosensory channels because of their structural similarity and expression in peripheral nociceptive terminals. In addition, some ligand cross‐interaction within these channels has been reported [44]. At 1 μm, compound 23 blocked ≈ 50% of hTRPV1‐channel activity (Fig. 4A, top panel), showing an IC₅₀ of 1.1 ± 1.02 μm (Fig. 4A, bottom panel). Similarly, at 5 μm compound 23 blocked ≈ 60% of hTRPA1‐evoked current (Fig. 4B, top panel), exhibiting an IC₅₀ of 3.04 ± 2.01 μm (Fig. 4B, bottom panel). For comparison, 2‐(1,3‐Dimethyl‐2,6‐dioxo‐1,2,3,6‐tetrahydro‐7H‐purin‐7‐yl)‐N‐(4‐isopropylphenyl)acetamide (HC030031), a reference TRPA1 antagonist [45], completely abrogated AITC‐evoked ionic currents at 5 μm (Fig. 4B, top panel). Collectively, these data indicate that α‐acyloxy carboxamide 23 is ≥ 10‐fold more potent blocking hTRPM8 than hTRPV1 and hTRPA1 channels, denoting a preferential selectivity for hTRPM8.

Fig. 4.

Compound 23 modestly inhibits currents of hTRPV1 and hTRPA1 expressed in HEK293 cells. (A) Representative capsaicin (0.5 μm)‐evoked hTRPV1 inward currents recorded at a holding potential of −60 mV, for control cells (untreated) and cells treated with 1 μm compound 23 or 10 μm of Capsazepine. Red lines represent the duration of the capsaicin pulse (top panel). Dose–response curve for hTRPV1 current blockade at a holding voltage of −60 mV. The line represents the fitted curve of the experimental data to the Hill equation. The fitted value for IC₅₀ was 1 μm ± 1.02. Each point is the mean ± SEM of n = 6 (bottom panel). (B) Representative AITC (60 μm) evoked hTRPA1 inward current recorded at a holding potential of −60 mV, for control cells (untreated) and cells treated with 5 μm compound 23 or 5 μm of HC030031. Orange lines represent the duration of the AITC pulse (top panel). Dose–response curve for hTRPA1 current blockade at a holding voltage of −60 mV. The line represents fits of the experimental data to the Hill equation. The fitted value for IC₅₀ was 3.04 μm ± 2.01. Each point is the mean ± SEM of n = 8 (bottom panel).

Modeling of α‐acyloxy carboxamide 23 docking in the menthol WS‐12 site of the hTRPM8 channel

To further support the blocking mechanism and selectivity, we performed molecular docking of compound 23 on the hTRPM8 channel (Fig. 5). Notably, compound 23 readily accommodates into the menthol binding pocket located between the S1, S2, S3, and S4 transmembrane domains in the closed conformation (Fig. 5A,B). The biphenyl group of compound 23 interacts with residues R842, H845, and I846 in S4, and W798 in S3 of hTRPM8. Similarly, the adamantane group contacts V849 and L853 in S4, F738 in S1, and L1001 and Y1005 in the TRP helix. Additional interactions are a salt bridge with H845 and two π‐cation involving R842 and H845 in S4 (Fig. 5C).

Fig. 5.

Molecular docking of compound 23 in TRPM8 channels. (A) Side and top view of human TRPM8 structure (PDB ID: 8BDC) used for docking studies. Subunits are colored differently. The tetrameric nature of the channel and the pore are clearly seen. The red square roughly indicates the simulation box built around the menthol binding site to accommodate compound 23. (B) Detail of the human TRPM8 menthol binding pocket with bound compound 23 (orange color). Residues mainly involved in ligand interactions were F738 (S1), W798 (S3), R842, H845, I846, V849, L853 (S4), and L1001, Y1005 (TRP helix). Interactions are mainly hydrophobic, although a salt bridge and π‐cation interactions are also observed. (C) Detail of compound 23 bound to the mouse TRPM8 binding pocket (equivalent view to panel B). Residues interacting with compound 23 (orange) were Q785 (S2), D796, W798, N799, D802 (S3), H845 (S4), and E1004 (TRP helix). (D) Compound 23 (orange) and WS‐12 (cyan) or AMTB (green) superimposed in the human TRPM8 menthol binding site for comparison. (E) Compound 23 (orange) and WS‐12 (cyan) or AMTB (green) superimposed in the mouse TRPM8 menthol binding site for comparison. Figures were constructed using open‐source pymol v3.0 (https://pymol.org/).

The superimposition of compound 23, WS‐12, and AMTB bound to the receptor menthol binding pocket (Fig. 5D) shows that compound 23 partially occupies the site of WS‐12, consistent with the observed competitive mechanism of channel blockade. The estimated binding energies to the hTRPM8 receptor reveal that the largest value corresponds to compound 23 (−10.41 kcal·mol⁻¹ vs. −9.93 kcal·mol⁻¹ for AMTB and −7.72 kcal·mol⁻¹ for WS‐12). The higher binding energy of compound 23 as compared to WS‐12 may be due to the polar and hydrophobic contacts with residues located mainly in S4 and the TRP helix that involve both the biphenyl and the adamantane groups of compound 23.

For comparison, we also modeled the interaction of compound 23 with the menthol binding site of mouse TRPM8 (mTRPM8, Fig. 5E). Docking of compound 23 into this binding site appears to involve slightly different interactions than those observed in the human ortholog. At variance with hTRPM8, where compound 23 accommodates into a faintly polar environment with the biphenyl and the adamantane groups closer to S2 and S3 transmembrane domains, in the murine TRPM8 compound 23 interacts with Q785 (S2), D796, W798, N799, D802 in S3, H845 in S4, and E1004 in the TRP helix. The reduced hydrophobic interactions noticed in the mTRPM8 binding site may underlie the lower binding energy estimated (−9.96 kcal·mol⁻¹) as compared to the human ortholog. This lower binding energy suggests a lower blocking potency of compound 23 blocking mTRPM8.

Compound 23 blocks neuronal TRPM8 and modulates WS‐12‐induced neuronal firing

To examine the in vivo activity of compound 23, we first investigated its inhibitory potency on neuronal TRPM8 channels expressed in murine primary nociceptor cultures by patch‐clamp. As seen in Fig. 6A, 5 μm of compound 23 blocked 60% of WS‐12‐evoked ionic currents in primary cultures of dorsal root ganglion neurons. A dose–response curve revealed an IC₅₀ of 0.43 ± 0.75 μm (Fig. 6B). This result indicates a lower potency of compound 23 inhibiting the murine TRPM8 as compared with the human ortholog consistent with the predicted lower binding energy to mTRPM8. Alternatively, it could also be due to distinct gating mechanisms of both orthologs or the influence of the neuronal environment.

Fig. 6.

Compound 23 blocks WS‐12‐induced TRPM8 activation in murine nociceptors. (A) Representative WS‐12‐evoked currents in DRG neurons of neonatal rats recorded at a holding potential of −60 mV. For control conditions (untreated) cells were exposed to two pulses (1 μm) of WS‐12, interspaced by a washing period, whereas treated cells were exposed before to 1 min of compound 23 (1 μm) and then to 30 s of compound 23 during the second pulse of WS‐12. (B) Dose–response curve for mTRPM8 current blockade at a holding voltage of −60 mV. The line represents fits of the experimental data to the Hill equation. The fitted value for IC₅₀ was 0.43 μm ± 0.75. Each point is the mean ± SEM of N = 3, n = 8.

Complementarily, we examined the effect of compound 23 on WS‐12‐evoked action potentials in primary cultures of murine DRG sensory neurons. For this task, we recorded neuronal excitability using multielectrode arrays (MEAs). The IC₅₀ value corresponding to 0.43 μm was used to examine compound 23 induced inhibition of neuronal excitability. A protocol of two sequential pulses of WS‐12 interspersed with a washing pulse was applied to correct for channel desensitization (Fig. 7A, upper recording). Compounds 23 and AMTB (0.43 and 10 μm, respectively) were applied 1 min before and during the second WS‐12 pulse (Fig. 7A, second, third, and fourth recordings). As depicted in Fig. 7B, compound 23 virtually abolished action potential firings induced by WS‐12 application in murine primary afferent neurons (≥ 75%). Thus, compound 23 appears to be a potent inhibitor of WS12‐evoked firing frequency, even surpassing the efficacy of AMTB at equimolar concentrations (Fig. 7B).

Fig. 7.

Compound 23 reduces WS‐12‐induced excitability of murine nociceptors. (A) Multielectrode array (MEA) recordings, with representative traces showing WS‐12‐evoked action potential (AP) firing under different conditions: control, 0.43 μm compound 23, 0.43 μm AMTB, and 10 μm AMTB. WS‐12 (1 μm) was applied in two consecutive pulses, with compounds added 1 min before and during the second pulse. The protocol concluded with a 15 s pulse of 40 mm KCl to ensure neuronal viability. (B) Normalized WS12‐induced firing (P2/P1) in the absence (vehicle) and presence of antagonists (0.43 μm compound 23, 0.43 μm AMTB and 10 μm AMTB) were compared. Data were analyzed with one‐way ANOVA followed by Dunnett's test; P values for statistical differences are indicated. The number of independent experiments (N) was 3, with 94 electrodes per condition. The data points are plotted with error bars representing the standard deviation (SD). (C) Multielectrode array (MEA) recordings, with representative traces showing Cap‐evoked action potential (AP) firing under different conditions: control and 1 μm compound 23. Cap (0.5 μm) was applied in two consecutive pulses, with compounds added 1 min before and during the second pulse. The protocol concluded with a 15 s pulse of 40 mm KCl to ensure neuronal culture viability. (D) Normalized capsaicin‐induced firing (P2/P1) in the absence (vehicle) and presence of antagonist (1 μm compound 23) were compared. The data points are plotted with error bars representing the standard deviation (SD). The number of independent experiments (N) was 3, with 63 electrodes per condition. Data were analyzed with Mann–Whitney test; P values for statistical differences are indicated. (E) Representative traces showing AITC‐evoked action potential (AP) firing under different conditions: control and 5 μm compound 23. AITC (100 μm) was applied in two consecutive pulses, with compounds added 1 min before and during the second pulse. The protocol concluded with a 15 s pulse of 40 mm KCl to ensure neuronal culture viability. (F) Normalized AITC‐induced firing (P2/P1) in the absence (vehicle) and presence of antagonist (5 μm compound 23) were compared. Data were analyzed with Mann–Whitney test; P values for statistical differences are indicated. The data points are plotted with error bars representing the standard deviation (SD). The number of independent experiments (N) was 3, with 67 electrodes per condition.

Additionally, we investigated the effect of compound 23 on capsaicin and AITC‐induced excitability in murine nociceptors. Employing an IC₅₀ value of 1 μm for TRPV1 and 5 μm for TRPA1 of firing inhibition by compound 23 (Fig. 4), we interrogated its ability to modulate neuronal excitability. As for TRPM8, our experimental design involved a protocol of dual agonist pulses interspersed with a washing pulse to minimize agonist‐induced channel desensitization. Compound 23 was administered 1 min prior to and during the second agonist pulse. Figure 7C–F show that compound 23 attenuated capsaicin and AITC responses by 30% and 40%, respectively, consistent with a more potent blockade of TRPM8 channels and a mild cross‐reactivity with TRPV1 and TRPA1 akin to other modulators of this channel family [46]. Collectively, these results indicate that α‐acyloxy carboxamide 23 is a novel TRPM8 antagonist that potently and selectively blocks TRPM8 channel activity and reduces neuronal excitability induced by receptor agonists.

Local application of compound 23 reduces oxaliplatin‐induced peripheral cold allodynia

Given the inhibitory activity of compound 23 and the antinociceptive effects of TRPM8 antagonists described in models of oxaliplatin (OXP)‐induced neuropathy [47, 48, 49], we conducted a behavioral experiment in mice to assess the antinociceptive effects of compound 23 after repeated oxaliplatin administration. Oxaliplatin was intraperitoneally (i.p.) administered every other day for 5 days at 6 mg·kg⁻¹, reaching a total accumulative dose of 18 mg·kg⁻¹. The acetone test, based on the duration of the licking response after the application of acetone drops to the hind paws (Fig. 8A–C, Before OXP vs. After OXP), revealed a significant enhancement in the nociceptive response to cold after OXP treatment (Fig. 8A–C, P < 0.01 Before OXP vs. After OXP). The OXP‐induced sensitization to cold stimuli was also evident with the dry ice test, as evidenced by the significant reduction in the latency to cold stimulation through the application of a dry ice pellet against the paw on a glass surface (Fig. 8B, P < 0.05 Before OXP vs. After OXP).

Fig. 8.

Local application of 23 alleviates oxaliplatin‐induced sensitivity to cold. (A) Repeated administration of oxaliplatin increased the duration of responses to cold induced by acetone application (A, ^# P < 0.05 Before vs After OXP, two‐way ANOVA). However, no significant effect of systemic 23 was found 30 or 90 min after its intraperitoneal (i.p.) administration. (B) Oxaliplatin induced a significant decrease in the withdrawal latency to cold induced by dry ice application (B, ^# P < 0.05 Before vs After OXP, two‐way ANOVA). Vehicle‐treated mice retained significant cold sensitization after i.p. treatment (B, ^$,# P < 0.05, Before OXP vs 30 min after Vehicle, two‐way ANOVA), whereas this difference was not evident in 23‐treated mice. However, 23 did not exhibit cold antinociceptive effects following systemic treatment. (C) Oxaliplatin increased the duration of the responses to acetone‐induced cold (C, ^## P < 0.01 Before vs After OXP, two‐way ANOVA) and 23 alleviated oxaliplatin‐induced cold sensitivity 30 and 90 min after its local subcutaneous administration in the paw (i.pl., *P < 0.05 After OXP vs. 30 min after 23, **P < 0.01 After OXP vs. 90 min after 23). Two‐way ANOVA followed by Tukey post hoc test when appropriate. Bars represent average values and error bars represent SEM. Dots are the individual values of each mouse (N = 6–7).

We next investigated the antinociceptive effect of systemic compound 23 (5 mg·kg⁻¹ i.p.) as compared to its vehicle in both the acetone and the dry ice tests. Measurements were conducted 30 and 90 min after compound 23 or vehicle i.p. administration. As shown in Fig. 8A,B, instillation of compound 23 did not significantly affect the licking response to acetone drops nor the withdrawal latencies to dry ice application as compared to vehicle. This unexpected lack of antinociceptive activity may be because of poor pharmacokinetic due to the metabolic instability of the ester bond that is susceptible to esterase hydrolysis.

To circumvent the potential systemic metabolic instability, we investigated if a local application in the animal paw produced antinociceptive activity. As illustrated in Fig. 8C, local subcutaneous application of compound 23 produced a significant antinociceptive effect in the acetone test, evident at 30 and 90 min after compound administration (P < 0.05 at 30 min, P < 0.01 at 90 min, After OXP vs. 30 and 90 min after compound 23). Collectively, these results suggest that α‐acyloxy carboxamide 23 exhibits a significant antinociceptive effect following local application to the peripheral endings, substantiating that systemic treatment is limited by a poor pharmacokinetics of the antagonist.

Compound 23 exhibits mild metabolic stability

Because of the lack of systemic activity of compound 23, we evaluated the in vitro plasmatic and hepatic metabolic stability. To this aim, compound 23 was exposed to mouse plasma and mouse liver microsome (MLM) fractions, and the residual substrate was measured after 1 h. Under these conditions, the residual substrate was about 97% and 90% in plasma and microsomes, respectively, denoting that this molecule suffers from mild hydrolysis of the ester group. However, when the microsomal monooxygenase system was activated by the NADPH regenerating system, the residual substrate dropped about 42% suggesting a susceptibility towards oxidative metabolism.

Metabolic biotransformation of compound 23 was investigated by tandem high‐resolution mass spectrometry using the software compound discoverer 3.2™. Alongside enzymatic hydrolysis giving M1a and M1b metabolites, mono and multi‐aliphatic oxidation was the predominant biotransformation occurring on the adamantane ring M2–M9 (Table 2; Fig. 9) and (Tables S1 and S2, Figs S79 and S80 for full spectral data) also demonstrate by the relative abundancy of some of them estimated based on the total ion current (TIC) measurement. Notably, the susceptibility of the adamantane ring to undergo microsomal oxidation is consistent with previous published findings [50, 51]. Indeed, some synthetic cannabinoid mono‐, di‐, and even tri‐hydroxylate derivatives of the adamantane group were revealed after hepatic phase I biotransformation.

Table 2.

Phase I metabolites of the compound 23 incubated in mouse liver microsomes and detected by LC‐HRMS analysis.

	Retention time (min)	Theoretical [M + H]+	Measured [M + H]+	Δ Da p.p.m.	Metabolite relative abundancy %
23	10.71	390.20637	390.20584	1.36	–
M1a	4.87	210.14886	210.14871	0.71	18.7
M1b	6.07	199.07536	199.07563	1.36	n.d. a
M2	6.94	406.20129	406.20118	0.27	11.5
M3	4.42	422.19620	422.19601	0.45	14.2
M4	4.82	422.19620	422.19602	0.43	1.5
M5	5.34	422.19620	422.19598	0.52	6.2
M6	5.67	422.19620	422.19577	1.02	12.8
M7	3.36	438.19111	438.19092	0.43	4.5
M8	4.20	438.19111	438.19077	0.78	29.0
M9	7.02	420.18055	420.18018	0.88	1.5

^a Not determined due to poor ionization in positive ion modality.

Fig. 9.

Proposed in vitro phase I metabolic scheme for the compound 23.

Discussion

Cumulative evidence supports a role of TRPM8 in transducing both innocuous and harmful cold thermal signals under physiological and pathological conditions [52, 53, 54]. Accordingly, TRPM8 is considered a pivotal clinical target, and the development of therapeutically useful modulators is a significant pharmaceutical and medical unmet goal. Akin to TRPV1 antagonists, most of systemic TRPM8 modulators tested have failed clinical development due to safety concerns. However, topical formulations of TRPM8 modulators directed to attenuate receptor pathological overactivity in conditions such as dry eye syndrome [14] or pruritus [23] have reported promising results. In this context, the salient contribution of this study is the design of hTRPM8 ligands with potential therapeutic properties based on multicomponent reactions such as the Ugi and Passerini reactions that capitalize on the drug‐like properties of the adamantane group. These reactions enable the rapid assembly of focused chemical libraries using green chemistry criteria [40]. Although medicinal chemists have extensively utilized the Ugi reaction in drug discovery [32], the Passerini reaction has been less popular. This reluctance stems from the apprehension of medicinal chemists towards esters, which are considered enzymatically unstable and unsuitable for systemic drug administration. In contrast to this belief, we have demonstrated that depending on the nature of the carboxylic acid utilized, Passerini adducts can be obtained that are resistant to plasma and hepatic esterases [55]. Nonetheless, these ester‐containing soft compounds have been shown useful in improving the safety of topically acting vanilloid‐based soft antagonists [56].

Among all compounds tested, α‐acyloxy carboxamide 23 stems as a potent TRPM8 competitive antagonist, substantiated by Schild plot analysis. Furthermore, compound 23 exhibited high blockade potency (IC₅₀ of 80 nm) and efficacy (100% response block). Structurally, α‐acyloxy carboxamide 23 displays a bulky moiety that readily fits into the menthol binding site of the receptor stabilizing the channel in the closed state, most likely by restricting the movement of the S4 and TRP domains necessary to drive the allosteric conformation that leads to the opening of the inner channel gate [57]. Computational docking studies show the potential binding interactions of compound 23 to hTRPM8 and mTRPM8 orthologs and provide evidence for the lower blockade potency on the murine channel. These differences appear to be mediated by a slightly different orientation of the compound within the receptor binding site that impacts the binding energy and consequently the blockade potency. Interestingly, the capacity of compound 23 to effectively bind to the channel closed state is compatible with a competitive blockade mechanism, as this site is readily accessible for agonists to bind and trigger the allosteric conformation to the open state. It is also tempting to hypothesize that occupancy of a binding site of the four available in the native tetrameric channel may suffice to prevent channel opening by the agonist, as suggested by the estimated hill coefficient from the dose–response curves. In this regard, it has been reported that efficient opening of TRPM8 channels requires occupancy of the four binding sites [58] thus interference with a single binding site by antagonists may suffice to lock the channel in the closed state. Nonetheless, additional experiments are needed to unveil the structural determinants of compound 23 binding, including site‐specific mutagenesis, along with biophysical analysis of its blockade activity.

Fast unblocking kinetics of α‐acyloxy carboxamide 23 unblock, that is, fast k _off, ensures reversible activity, which is important to reduce a use‐dependent channel block. This property is associated with high‐affinity antagonists exhibiting a slow unblockage kinetics, which limits their therapeutic utility because of undesirable side effects, as exemplified by the high affinity of TRPV1 blockers [59] or the NMDA blocker MK‐801 [60]. In this regard, compounds endowed of fast on/off kinetics appear to be safer for therapeutic applications. As TRPM8 is a physiologically relevant channel transducing environmental temperature detection for proper body temperature homeostasis and prevention of tissue damage, preservation of this physiological activity is crucial to prevent alterations in body temperature unwanted effects described with previous antagonists including hot sensation, gut motility deficits, and cold burns [61, 62]. Absence of use‐dependent block minimizes the manifestation of unwanted side effects, increasing the safety profile of drug candidates.

A complementary property of α‐acyloxy carboxamide 23 is represented by its channel selectivity, as evidenced by its preferential action on TRPM8 channels, and lower interaction with structurally related thermosensory channels such as TRPV1 and TRPA1, which are also highly expressed in nociceptive neurons and contribute to the etiology of peripheral neuropathies. Interestingly, cross‐reactivity of agonists and antagonists of these thermosensory channels has been reported [46]. For instance, an interaction of capsaicin with TRPM8 and menthol with TRPV1 and TRPA1 channels has been described [46]. This cross‐recognition is due to conserved similarities between the receptor membrane domain structuring the ligand binding sites, which have evolved to preferentially recognize one ligand but conserve the essential features to be mildly recognized by others.

Drug‐off target effects may raise safety concerns in contrast to highly selective drugs. However, it should be noted that although drug selectivity has been considered a pivotal goal for therapeutic leads and long pursued by medicinal chemistry programs, it is being realized that some cross‐reactivity of drugs with structurally similar receptors may enhance their therapeutic value as peripheral neuropathies involve the participation of various thermosensory receptors.

Absence of use‐dependency also in off‐target interactions minimizes the appearance of side effects that could compromise the therapeutic index. Furthermore, a multitarget cross‐activity may be beneficial in cancer patients treated with chemotherapy that develop a distal peripheral neuropathy, where a contribution of TRPM8, TRPV1, and TRPA1 channels has been reported [63]. The possibility of differentially targeting all three channels with a single compound could be a promising therapeutic strategy to target this disease. In support of this tenet, local application of α‐acyloxy carboxamide 23 exhibited significant antinociceptive activity attenuating OIPN‐induced cold allodynia.

A systemic administration of compound 23, however, failed to reduce the cold allodynia that accompanies OIPN. This lack of therapeutic effect is most likely due to a low in vivo metabolic stability of compound 23 that resulted in poor pharmacokinetics. In this regard, although the bulky aromatic groups present in the molecule slowed the hydrolysis when exposed to plasma and microsomal fractions, it did not fully prevent the breakdown of the ester under oxidative metabolism when the microsomal monooxygenase system was activated. Though this metabolic instability may be seen as a therapeutic limitation, metabolically sensitive soft drugs are ideal candidates for local applications whereby the therapeutic activity needs to be peripherally restricted to avoid potential interference with a pharmacological treatment, such as in cancer patients suffering OIPN. In addition, this lack of systemic distribution helps to reduce any potential off‐target effect on physiologically acting thermosensory channels in other tissues, which would raise safety concerns. Thus, drugs that act epidermally and are enzymatically hydrolyzed at the dermis appear relevant for therapeutic interventions of peripheral neuropathies. In this regard, the anti‐allodynic local activity of compound 23 paves the way to develop this compound for the topical treatment of this disabling sensory condition suffered by cancer patients.

In conclusion, our findings indicate that α‐acyloxy carboxamide 23 is a potential therapeutic TRPM8 antagonist that attenuates pathological cold allodynia arising from a common peripheral neuropathy in cancer patients. The in vitro pharmacological properties of compound 23, that is, its potency and efficacy, reversible inhibition, receptor selectivity, lack of use‐dependent block, and relative peripheral stability, make it a potential drug candidate for local therapeutic intervention of peripheral neuropathies mediated by dysfunctional TRPM8 channels.

Materials and methods

Chemistry

General experimental methods

Commercially available reagents and solvents were purchased from Sigma‐Aldrich (Merck Life Science, Milan, Italy) or Alfa Aesar (Thermo Fisher Scientific, Monza, Italy) and were used without further purification. Melting points were determined in open glass capillary with a Stuart scientific SMP3 apparatus and are uncorrected. Infrared spectra were acquired with a FT‐IR Thermo‐Nicolet Avatar. ¹H‐NMR and ¹³C‐NMR spectra were recorded on a JEOL ECP 300 MHz spectrometer (JEOL, Basiglio, Italy). Chemical shifts (δ) are reported in parts per million (p.p.m.) referenced to the residual solvent peak. The multiplicity of each signal is designated using the following abbreviations: s (singlet), d (doublet), t (triplet), q (quadruplet), quint (quintuplet), m (multiplet), br s (broad singlet), dd (doublet of doublets). Coupling constants (J) are reported in Hertz (Hz). High‐resolution ESI‐MS spectra were acquired on a Thermo Scientific Q‐Exactive™ Plus Hybrid Quadrupole‐Orbitrap™ mass spectrometer. The spectra were recorded by infusion into the ESI source using methanol as the solvent. Flash column chromatography was performed on silica gel (Merck Kieselgel 60, 230–400 mesh ASTM, Milan, Italy). Thin layer chromatography (TLC) was carried out on plates with a layer thickness of 0.25 mm (Merck Silica gel 60 F₂₅₄); when necessary, they were developed with KMnO₄ reagent or Dragendorff reagent. 1‐Isocyanoadamantane 3 was synthesized as described previously [43]. Compounds 1, 4–14, and 15–19 were commercially available. The purity of final compounds was ≥ 95% and was determined by high‐performance liquid chromatography coupled with an ultraviolet–visible detector using the instrumentation and methods reported in Figs S81–S92.

General procedure A for the synthesis of α‐acyloxy carboxamides 20–29

To a solution of the corresponding carboxylic acid (0.62 mmol, 1 equiv) in dichloromethane (2 mL), 37% aqueous formaldehyde solution (2.48 mmol, 4 equiv) and 1‐isocyanoadamantane (3) (0.62 mmol, 1 equiv) were added. The reaction mixture was stirred at 25 °C for 18 h. After completion of the reaction, water was added, and the product was extracted with dichloromethane (x2). The combined organic layers were washed with aqueous saturated Na₂CO₃ solution (x1), brine (x1) and dried over sodium sulfate. After evaporation of the solvent under reduced pressure, the crude material was purified by flash column chromatography.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl benzoate (20)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution, and benzoic acid (4) according to general procedure A. After extraction, the crude was purified by flash column chromatography using PE/EtOAc 9 : 1 as eluent to give a white solid; yield 83%; mp 155–157 °C; IR (KBr) $\tilde{\nu }$ 3275, 3090, 2916, 2853, 1727, 1657, 1271, 1129, 709 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 8.05 (d, J = 8.3 Hz, 2H), 7.62 (t, J = 7.3 Hz, 1H), 7.49 (d, J = 7.5 Hz, 2H), 5.79 (br s, 1H), 4.70 (s, 2H), 2.09 (br s, 3H), 2.04 (br s, 6H), 1.68 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 165.9, 165.2, 133.7, 129.8, 129.2, 128.8, 63.7, 52.2, 41.7, 36.3, 29.5 p.p.m.; HRMS (ESI⁺): m/z = 314.17464 [M + H]⁺; calcd. for C₁₉H₂₃O₃N + H⁺: 314.17507.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl 2‐phenylacetate (21)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and phenylacetic acid (5) according to general procedure A. After extraction, the crude was purified by flash column chromatography using PE/EtOAc 8 : 2 as eluent to give a white solid; yield 84%; mp 78–80 °C; IR (KBr) $\tilde{\nu }$ 3292, 3065, 2911, 2851, 1750, 1671, 1544, 1234, 1134 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.33–7.25 (m, 5H), 5.47 (br s, 1H), 4.39 (s, 2H), 3.66 (s, 2H), 1.99 (br s, 3H), 1.81 (br s, 6H), 1.60 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 169.6, 165.6, 133.4, 129.1, 128.8, 127.4, 63.1, 51.8, 41.3, 41.2, 36.1, 29.3 p.p.m.; HRMS (ESI⁺): m/z = 328.19034 [M + H]⁺; calcd. for C₂₀H₂₅O₃N + H⁺: 328.19072.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl acetate (22)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and acetic acid (6) according to general procedure A. After extraction, the product is obtained as a white solid; yield 91%; mp 102–104 °C; IR (KBr) $\tilde{\nu }$ 3292, 3077, 2909, 2849, 1745, 1662, 1558, 1225, 1095, 999, 605 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 5.83 (br s, 1H), 4.31 (s, 2H), 2.03 (br s, 3H), 1.96 (br s, 3H), 1.90 (br s, 6H), 1.56 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 169.3, 165.6, 63.0, 51.9, 41.3, 36.1, 29.2, 20.6 p.p.m.; HRMS (ESI⁺): m/z = 252.15916 [M + H]⁺; calcd. for C₁₄H₂₁O₃N + H⁺: 252.15942.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl [1,1′‐biphenyl]‐2‐carboxylate (23)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and [1,1′‐biphenyl]‐2‐carboxylic acid (7) according to general procedure A. After extraction, the product is obtained as a colorless oil which taken up with methanol precipitated as a white solid, yield 89%; mp 53–55 °C; IR (KBr) $\tilde{\nu }$ 3059, 2908, 2850, 1734, 1685, 1533, 1273, 1132, 747 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.83 (d, J = 7.7 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.42–7.27 (m, 7H), 5.27 (br s, 1H), 4.32 (s, 2H), 2.00 (br s, 3H), 1.81 (br s, 6H), 1.60 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 167.3, 165.0, 141.9, 140.9, 131.7, 130.6, 130.1, 129.8, 128.4, 128.0, 127.5, 127.3, 63.6, 51.6, 41.0, 36.1, 29.2 p.p.m.; HRMS (ESI⁺): m/z = 390.20607 [M + H]⁺; calcd. for C₂₅H₂₇O₃N + H⁺: 390.20637.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl butyrate (24)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and butanoic acid (8) according to general procedure A. After extraction, the product is obtained as a colorless oil; yield 95%; IR (KBr) $\tilde{\nu }$ 3297, 3073, 2908, 2849, 1749, 1664, 1551, 1421, 1170, 1109 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 5.78 (br s, 1H), 4.32 (s, 2H), 2.27 (t, J = 7.3 Hz, 2H), 1.96 (br s, 3H), 1.90 (br s, 6H), 1.57 (br s, 8H), 0.86 (t, J = 7.5 Hz, 3H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 171.9, 165.8, 62.8, 51.8, 41.3, 36.1, 35.7, 29.2, 18.2, 13.5 p.p.m.; HRMS (ESI⁺): m/z = 280.19042 [M + H]⁺; calcd. for C₁₆H₂₅O₃N + H⁺: 280.19072.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl cyclopentane carboxylate (25)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and cyclopentanecarboxylic acid (9) according to general procedure A. After extraction, the crude was purified by flash column chromatography using PE/EtOAc 9 : 1 as eluent to give a white solid; yield 83%; mp 104–106 °C; IR (KBr) $\tilde{\nu }$ 3280, 2910, 2851, 1743, 1673, 1558, 1153, 1096, 997, 814 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 5.76 (br s, 1H), 4.34 (s, 2H), 2.73 (quint, J = 7.6 Hz, 1H), 1.98 (br s, 3H), 1.91 (br s, 6H), 1.88–1.49 (m, 14H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 175.0, 165.9, 62.8, 51.8, 43.4, 41.4, 36.1, 29.9, 29.3, 25.7 p.p.m.; HRMS (ESI⁺): m/z = 306.20594 [M + H]⁺; calcd. for C₁₈H₂₇O₃N + H⁺: 306.20637.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl nicotinate (26)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and nicotinic acid (10) according to general procedure A. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 5 : 5 as eluent to give a white solid; yield 82%; mp 155–157 °C; IR (KBr) $\tilde{\nu }$ 3273, 3094, 2913, 2852, 1732, 1658, 1568, 1271, 1134, 741 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 9.13 (s, 1H), 8.70 (d, J = 3.4 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.33 (dd, J = 8.0/4.9 Hz, 1H), 5.95 (br s, 1H), 4.63 (s, 2H) 1.97 (br s, 3H), 1.92 (d, J = 2.1 Hz, 6H), 1.56 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 165.2, 164.0, 153.8, 150.7, 137.1, 125.2, 123.4, 63.6, 52.1, 41.4, 36.1, 29.2 p.p.m.; HRMS (ESI⁺): m/z = 315.16979 [M + H]⁺; calcd. for C₁₈H₂₂O₃N₂ + H⁺: 315.17032.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl 4‐acetamidobutanoate (27)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and 4‐acetamidobutanoic acid (11) according to general procedure A. After extraction, the product is obtained as a white solid; yield 94%; mp 102–104 °C; IR (KBr) $\tilde{\nu }$ 3560, 3415, 3074, 2908, 2850, 1742, 1697, 1547, 1361, 1175 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.03 (br s, 1H), 6.25 (br s, 1H), 4.32 (s, 2H), 3.14 (q, J = 6.1 Hz, 2H), 2.31 (t, J = 6.9 Hz, 2H), 1.93 (br s, 3H), 1.88 (br s, 6H), 1.83 (s, 3H), 1.73 (quint, J = 6.7 Hz, 2H), 1.53 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 171.9, 170.7, 166.0, 62.7, 52.0, 41.2, 38.4, 36.0, 31.3, 29.2, 24.6, 22.7 p.p.m.; HRMS (ESI⁺): m/z = 337.21169 [M + H]⁺; calcd. for C₁₈H₂₈O₄N₂ + H⁺: 337.21218.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl 4‐chlorobenzoate (28)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and 4‐chlorobenzoic acid (12) according to general procedure A. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 as eluent to give a white solid; yield 91%; mp 165–167 °C; IR (KBr) $\tilde{\nu }$ 3307, 3074, 2906, 2849, 1720, 1658, 1552, 1117, 855, 764, 686 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.94 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.6 Hz, 2H), 5.81 (br s, 1H), 4.64 (s, 2H), 2.03 (br s, 3H), 1.98 (br s, 6H), 1.63 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 165.6, 164.4, 140.1, 131.1, 129.0, 127.6, 63.7, 52.2, 41.6, 36.2, 29.4 p.p.m.; HRMS (ESI⁺): m/z = 348.13573 [M + H]⁺; calcd. for C₁₉H₂₂O₃ClN + H⁺: 348.13610.

2‐((3S,5S,7S)‐adamantan‐1‐ylamino)‐2‐oxoethyl 3‐nitrobenzoate (29)

The title compound was prepared from 1‐isocyanoadamantane (3), 37% aqueous formaldehyde solution and 3‐nitrobenzoic acid (13) according to general procedure A. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 9 : 1 and PE/EtOAc 7 : 3 as eluents to give a white solid; yield 76%; mp 122–124 °C; IR (KBr) $\tilde{\nu }$ 3282, 3083, 2905, 2848, 1729, 1533, 1352, 1263, 1143, 715 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 8.78 (s, 1H), 8.39–8.32 (m, 2H), 7.64 (t, J = 8.0 Hz, 1H), 5.89 (br s, 1H), 4.70 (s, 2H), 2.01 (br s, 3H), 1.98 (br s, 6H), 1.61 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 165.1, 163.4, 148.2, 135.4, 131.0, 129.9, 127.8, 124.6, 64.0, 52.3, 41.5, 36.2, 29.3 p.p.m.; HRMS (ESI⁺): m/z = 359.15987 [M + H]⁺; calcd. for C₁₉H₂₂O₅N₂ + H⁺: 359.16015.

General procedure B for the synthesis of α‐aminoacyl amides 30–41

To a solution of 1‐isocyanoadamantane (3) (0.070 g, 0.40 mmol, 1 equiv) in methanol (3 mL), paraformaldehyde (0.026 g, 0.60 mmol, 1.5 equiv), the corresponding amine (0.40 mmol, 1 equiv) and the corresponding carboxylic acid (0.40 mmol, 1 equiv) were added. The reaction mixture was stirred at 60 °C for 4 h. After completion of the reaction, water was added and the product was extracted with dichloromethane (x2). The combined organic layers were washed with water (x1), NaOH 2 m (x1), brine (x1) and dried over sodium sulfate. After evaporation of the solvent under reduced pressure, the crude material was purified by flash column chromatography.

N‐(2‐(((3S,5S,7S)‐adamantan‐1‐yl)amino)‐2‐oxoethyl)‐N‐cyclopropylbenzamide (30)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclopropylamine (15) and benzoic acid (4) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 8 : 2 as eluent to give a white solid; yield 73%; mp 71–72 °C; IR (KBr) $\tilde{\nu }$ 3445, 3316, 3066, 2906, 2849, 1645, 1626, 1548, 1426, 728, 702 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.55–7.34 (m, 5H), 6.05 (br s, 1H), 4.06 (s, 2H), 2.96–2.88 (m, 1H), 2.05 (br s, 3H), 1.98 (br s, 6H), 1.65 (br s, 6H), 0.60–0.50 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 173.4, 168.5, 136.7, 130.0, 128.2, 127.4, 53.5, 52.0, 41.6, 36.4, 33.6, 29.5, 9.8 p.p.m.; HRMS (ESI⁺): m/z = 353.22214 [M + H]⁺; calcd. for C₂₂H₂₈O₂N₂ + H⁺: 353.22235.

N‐(2‐(((3S,5S,7S)‐adamantan‐1‐yl)amino)‐2‐oxoethyl)‐N‐cyclopropylbutyramide (31)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclopropylamine (15) and butanoic acid (8) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 and PE/EtOAc 5 : 5 as eluents to give an off‐white solid; yield 72%; mp 126–127 °C; IR (KBr) $\tilde{\nu }$ 3312, 3073, 2906, 2849, 1679, 16 345, 1554, 1428, 1362, 1290, 1275, 744 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 5.87 (br s, 1H), 3.85 (s, 2H), 2.81–2.73 (m, 1H), 2.52 (t, J = 7.3 Hz, 2H), 2.01 (br s, 3H), 1.91 (br s, 6H), 1.73–1.61 (m, 8H), 0.94 (t, J = 7.3 Hz, 3H), 0.91–0.81 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 176.8, 169.0, 53.2, 51.8, 41.6, 36.4, 35.9, 31.7, 29.5, 18.5, 14.1, 9.3 p.p.m.; HRMS (ESI⁺): m/z = 319.23778 [M + H]⁺; calcd. for C₁₉H₃₀O₂N₂ + H⁺: 319.23800.

N‐(2‐(((3S,5S,7S)‐adamantan‐1‐yl)amino)‐2‐oxoethyl)‐N‐cyclopropylcyclopentanecarboxamide (32)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclopropylamine (15) and cyclopentanecarboxylic acid (9) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 9 : 1 and PE/EtOAc 8 : 2 as eluents to give a white solid; yield 52%; mp 152–153 °C; IR (KBr) $\tilde{\nu }$ 3407, 3321, 2906, 2849, 1681, 1630, 1546, 1429, 1361, 1273 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 5.91 (br s, 1H), 3.85 (s, 2H), 3.42–3.32 (m, 1H), 2.83–2.76 (m, 1H), 1.99–1.54 (m, 23H), 0.86–0.80 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 180.5, 169.0, 53.3, 51.6, 41.5, 41.4, 36.3, 31.5, 30.6, 29.4, 26.3, 9.1 p.p.m. HRMS (ESI⁺): m/z = 345.25329 [M + H]⁺; calcd. for C₂₁H₃₂O₂N₂ + H⁺: 345.25365.

N‐(2‐(((3S,5S,7S)‐adamantan‐1‐yl)amino)‐2‐oxoethyl)‐N‐cyclopropyl‐[1,1′‐biphenyl]‐2‐carboxamide (33)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclopropylamine (15) and [1,1′‐biphenyl]‐2‐carboxylic acid (7) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 8 : 2 and PE/EtOAc 7 : 3 as eluents to give a white solid; yield 49%; mp 83–84 °C; IR (KBr) $\tilde{\nu }$ 3313, 3060, 2906, 2850, 1683, 1629, 1542, 1453, 745, 701 cm⁻¹; ¹H‐NMR (300 MHz, DMSO‐d ₆ 75 °C) δ 7.48–7.35 (m, 9H), 6.94 (br s, 1H), 3.76 (br s, 2H), 3.30 (br s, 1H), 2.01–1.84 (m, 9H), 1.64 (br s, 6H), 0.56–0.24 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, DMSO‐d ₆ 75 °C) δ 172.0, 167.0, 139.7, 137.6, 136.5, 129.0, 128.6, 128.1, 128.0, 127.2, 127.1, 126.7, 51.9, 50.3, 40.9, 35.8, 31.4, 28.6, 7.5 p.p.m.; HRMS (ESI⁺): m/z = 429.25359 [M + H]⁺; calcd. for C₂₈H₃₂O₂N₂ + H⁺: 429.25365.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(N‐cyclopropylacetamido)acetamide (34)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclopropylamine (15) and acetic acid (6) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 and PE/EtOAc 5 : 5 as eluents to give a white solid; yield 70%; mp 73–74 °C; IR (KBr) $\tilde{\nu }$ 3330, 3053, 2906, 2849, 1660, 1544, 1422, 1360, 1291, 1030 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 5.86 (br s, 1H), 3.82 (s, 2H), 2.81–2.74 (m, 1H), 2.19 (s, 3H), 1.97 (br s, 3H), 1.90 (br s, 6H), 1.59 (br s, 6H), 0.83–0.76 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 174.3, 168.6, 52.7, 51.8, 41.4, 36.3, 32.3, 29.4, 22.4, 9.1 p.p.m.; HRMS (ESI⁺): m/z = 291.20628 [M + H]⁺; calcd. for C₁₇H₂₆O₂N₂ + H⁺: 291.20670.

N‐(2‐(((3S,5S,7S)‐adamantan‐1‐yl)amino)‐2‐oxoethyl)‐N‐cyclopropylbenzo[b]thiophene‐2‐carboxamide (35)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclopropylamine (15) and benzo[b]thiophene‐2‐carboxylic acid (14) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 and PE/EtOAc 5 : 5 as eluents to give a white solid; yield 23%; mp 193–194 °C; IR (KBr) $\tilde{\nu }$ 3411, 3309, 3142, 2907, 2851, 1666, 1600, 1515, 1390, 1288, 961, 760 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) signals are referred to the main rotamer, δ 7.89–7.79 (m, 3H), 7.44–7.33 (m, 2H), 6.04 (br s, 1H), 4.10 (s, 2H), 3.22–3.14 (m, 1H), 2.14–1.97 (m, 9H), 1,71–1.63 (m, 6H), 0.89–0.82 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) signals are referred to the main rotamer, δ 168.3, 166.5, 141.0, 138.8, 137.7, 127.9, 126.3, 125.2, 124.8, 122.4, 54.6, 52.1, 41.6, 36.4, 33.4, 29.5, 10.7 p.p.m.; HRMS (ESI⁺): m/z = 409.19433 [M + H]⁺; calcd. for C₂₄H₂₈O₂N₂ + H⁺: 409.19443.

N‐(2‐(((3S,5S,7S)‐adamantan‐1‐yl)amino)‐2‐oxoethyl)‐N‐cyclopropyl‐2‐phenylacetamide (36)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclopropylamine (15) and phenylacetic acid (5) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 as eluent to give an off‐white solid; yield 53%; mp 119–120 °C; IR (KBr) $\tilde{\nu }$ 3329, 3025, 2905, 2851, 1723, 1689, 1639, 1454, 1300, 1042, 742, 696 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.29–7.20 (m, 5H), 5.84 (br s, 1H), 3.92 (s, 2H), 3.88 (s, 2H), 2.77–2.69 (m, 1H), 2.00 (br s, 3H), 1.85 (br s, 6H), 1.61 (br s, 6H), 0.89–0.81 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 174.6, 168.5, 135.0, 129.1, 128.7, 126.9, 53.1, 51.7, 41.4, 41.1, 36.3, 32.0, 29.4, 9.5 p.p.m.; HRMS (ESI⁺): m/z = 367.23767 [M + H]⁺; calcd. for C₂₃H₃₀O₂N₂ + H⁺: 367.23800.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(N‐cyclohexylacetamido)acetamide (37)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde; cyclohexylamine (16) and acetic acid (6) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 and PE/EtOAc 5 : 5 as eluents to give a white solid; yield 78%; mp 77–78 °C; IR (KBr) $\tilde{\nu }$ 3309, 2907, 2851, 1669, 1627, 1538, 1453, 1416, 1359, 1241, 976 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) signals are referred to the main rotamer, δ 6.36 (br s, 1H), 3.74 (s, 2H), 3.50–3.41 (m, 1H), 2.10 (s, 3H), 1.99–1.87 (m, 9H), 1.80–1.46 (m, 12H), 1.33–1.16 (m, 4H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) signals are referred to the main rotamer, δ 171.3, 169.6, 58.7, 51.4, 48.4, 47.4, 41.4, 36.3, 31.1, 30.4, 29.3, 25.8, 25.0, 21.7 p.p.m.; HRMS (ESI⁺): m/z = 333.25320 [M + H]⁺; calcd. for C₂₀H₃₂O₂N₂ + H⁺: 333.25365.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(N‐((3S,5S,7S)‐adamantan‐1‐yl)acetamido)acetamide (38)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, (3S,5S,7S)‐adamantan‐1‐amine (1), and acetic acid (6) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 and PE/EtOAc 5 : 5 as eluents to give a white solid; yield 87%; mp 239–240 °C; IR (KBr) $\tilde{\nu }$ 3302, 3064, 2908, 2849, 1691, 1623, 1549, 1414, 1359, 988, 902 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 5.80 (br s, 1H), 3.78 (s, 2H), 2.11 (br s, 6H), 2.02–1.92 (m, 15H), 1.61 (br s, 12H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 172.1, 168.8, 59.2, 52.1, 49.6, 41.6, 40.0, 36.2, 36.1, 30.1, 29.3, 25.8 p.p.m.; HRMS (ESI⁺): m/z = 385.28461 [M + H]⁺; calcd. for C₂₄H₃₆O₂N₂ + H⁺: 385.28495.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(N‐butylacetamido)acetamide (39)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, butan‐1‐amine (17), and acetic acid (6) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 and PE/EtOAc 5 : 5 as eluents to give a white solid; yield 70%; mp 124–125 °C; IR (KBr) $\tilde{\nu }$ 3296, 2907, 2853, 1644, 1537, 1414, 1305, 1245, 985, 708 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) signals are referred to the main rotamer, δ 6.20 (br s, 1H), 3.75 (s, 2H), 3.26 (t, J = 7.4 Hz, 2H), 2.05–1.87 (m, 12H), 1.60–1.46 (m, 8H), 1.24–1.20 (m, 2H), 0.85 (t, J = 7.0 Hz, 3H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) signals are referred to the main rotamer, δ 171.2, 168.5, 52.0, 50.4, 47.1, 41.4, 36.2, 30.6, 29.3, 21.1, 19.9, 13.7 p.p.m.; HRMS (ESI⁺): m/z = 307.23759 [M + H]⁺; calcd. for C₁₈H₃₀O₂N₂ + H⁺: 307.23800.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(N‐benzylacetamido)acetamide (40)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, benzylamine (18), and acetic acid (6) according to general procedure B. After extraction, the product was obtained as a white solid; yield 78%; mp 195–196 °C; IR (KBr) $\tilde{\nu }$ 3312, 3030, 2920, 2854, 1642, 1533, 1409, 1344, 1004, 722, 693 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) signals are referred to the main rotamer, δ 7.35–7.26 (m, 5H), 5.90 (br s, 1H), 4.62 (s, 2H), 3.83 (s, 2H), 2.16–1.61 (m, 18H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) signals are referred to the main rotamer, δ 171.7, 167.9, 136.2, 129.1, 127.9, 126.8, 53.5, 52.0, 51.3, 41.7, 36.5, 29.6, 21.5 p.p.m.; HRMS (ESI⁺): m/z = 341.22196 [M + H]⁺; calcd. for C₂₁H₂₈O₂N₂ + H⁺: 341.22235.

N‐(2‐(((3S,5S,7S)‐adamantan‐1‐yl)amino)‐2‐oxoethyl)‐N‐benzyl‐2‐phenylacetamide (41)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, benzylamine (18), and phenylacetic acid (5) according to general procedure B. After extraction, the crude was purified by flash column chromatography, using PE/EtOAc 7 : 3 as eluent to give a white solid; yield 63%; mp 60–61 °C; IR (KBr) $\tilde{\nu }$ 3300, 3063, 3029, 2906, 2849, 1681, 1632, 1545, 1453, 1359, 724, 697 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) signals are referred to the main rotamer, δ 7.31–7.26 (m, 10H), 5.98 (br s, 1H), 4.60 (s, 2H), 3.84 (s, 2H), 3.77 (s, 2H), 2.02–1.60 (m, 15H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) signals are referred to the main rotamer, δ 172.2, 167.6, 135.8, 134.6, 129.0, 128.9, 128.6, 127.9, 127.1, 126.7, 52.5, 51.8, 51.2, 41.4, 40.7, 36.3, 29.4 p.p.m.; HRMS (ESI⁺): m/z = 417.25337 [M + H]⁺; calcd. for C₂₇H₃₂O₂N₂ + H⁺: 417.25365.

General procedure C for the synthesis of α‐acylpiperazino amides 42–45

To a solution of piperazine (19) (0.037 g, 0.40 mmol, 1 equiv) in methanol (3 mL) were added paraformaldehyde (0.031 g, 0.60 mmol, 1.5 equiv), the corresponding carboxylic acid (0.40 mmol, 1 equiv) and 1‐isocyanoadamantane (3) (0.070 g, 0.40 mmol, 1 equiv) sequentially at room temperature. The reaction mixture was heated at 60 °C for 3 h, and the solvent was evaporated. The crude material was purified by flash column chromatography.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(4‐benzoylpiperazin‐1‐yl)acetamide (42)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, piperazine (19), and benzoic acid (4) according to general procedure C. The crude was purified by flash column chromatography using EtOAc as eluent to give a white solid; yield 62%; mp 141–142 °C; IR (KBr) $\tilde{\nu }$ 3346, 3019, 2909, 2848, 1668, 1638, 1530, 1458, 1428, 1138, 1017, 715 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.35 (br s, 5H), 6.75 (br s, 1H), 3.72–3.44 (m, 4H), 2.88 (s, 2H), 2.50 (br s, 4H), 2.03–1.94 (m, 9H), 1.64 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 170.4, 168.3, 135.6, 129.8, 128.6, 127.0, 62.2, 53.3 (4C), 51.2, 41.7, 36.3, 29.4 p.p.m.; HRMS (ESI⁺): m/z = 382.24859 [M + H]⁺; calcd. for C₂₃H₃₁O₂N₃ + H⁺: 382.24890.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(4‐(cyclopentanecarbonyl)piperazin‐1‐yl)acetamide (43)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, piperazine (19), and cyclopentanecarboxylic acid (9) according to general procedure C. The crude was purified by flash column chromatography using EtOAc as eluent to give a white solid; yield 75%; mp 118–119 °C; IR (KBr) $\tilde{\nu }$ 3270, 3062, 2907, 2852, 2811, 1672, 1644, 1552, 1514, 1452, 1239, 814, 715 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 6.80 (br s, 1H), 3.56 (br s, 2H), 3.47 (br s, 2H), 2.90–2.74 (m, 3H), 2.46–2.40 (m, 4H), 2.01–1.91 (m, 9H), 1.76–1.48 (m, 14H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 174.6, 168.5, 62.0, 53.4, 53.0, 51.2, 45.4, 41.8, 41.6, 40.9, 36.2, 30.0, 29.3, 25.9 p.p.m.; HRMS (ESI⁺): m/z = 374.27984 [M + H]⁺; calcd. for C₂₂H₃₅O₂N₃ + H⁺: 374.28020.

N‐((3S,5S,7S)‐adamantan‐1‐yl)‐2‐(4‐(2‐phenylacetyl)piperazin‐1‐yl)acetamide (44)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, piperazine (19), and phenylacetic acid (5) according to general procedure C. The crude was purified by flash column chromatography, using EtOAc as eluent to give a white solid; yield 83%; mp 145–146 °C; IR (KBr) $\tilde{\nu }$ 3321, 3019, 2908, 2848, 1664, 1630, 1511, 1456, 1241, 1143, 736, 723 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 7.28–7.15 (m, 5H), 6.70 (br s, 1H), 3.67 (s, 2H), 3.59 (br s, 2H), 3.40–3.37 (m, 2H), 2.78 (s, 2H), 2.41–2.38 (m, 2H), 2.26–2.23 (m, 2H), 2.02–1.91 (m, 9H), 1.61 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 169.4, 168.2, 134.8, 128.7, 128.4, 126.8, 62.0, 53.0, 52.8, 51.1, 46.0, 41.7, 41.6, 40.9, 36.2, 29.3 p.p.m.; HRMS (ESI⁺): m/z = 396.26419 [M + H]⁺; calcd. for C₂₄H₃₃O₂N₃ + H⁺: 396.26455.

2‐(4‐acetylpiperazin‐1‐yl)‐N‐((3S,5S,7S)‐adamantan‐1‐yl)acetamide (45)

The title compound was prepared from 1‐isocyanoadamantane (3), paraformaldehyde, piperazine (19), and acetic acid (6) according to general procedure C. The crude was purified by flash column chromatography, using EtOAc/MeOH 95 : 5 as eluent to give a white solid; yield 76%; mp 135–136 °C; IR (KBr) $\tilde{\nu }$ 3322, 3008, 2915, 2848, 2901, 1667, 1654, 1512, 1427, 1273, 1245, 1006, 990 cm⁻¹; ¹H‐NMR (300 MHz, CDCl₃) δ 6.73 (br s, 1H), 3.55 (br s, 2H), 3.41 (br s, 2H), 2.84 (s, 2H), 2.47–2.39 (m, 4H), 2.02–1.92 (m, 12H), 1.61 (br s, 6H) p.p.m.; ¹³C‐NMR (75 MHz, CDCl₃) δ 168.9, 168.2, 62.1, 53.2, 52.9, 51.2, 46.2, 41.6, 41.4, 36.2, 29.3, 21.3 p.p.m.; HRMS (ESI⁺): m/z = 320.23295 [M + H]⁺; calcd. for C₁₈H₂₉O₂N₃ + H⁺: 320.23325.

Biology

Animals

C57BL/6JRccHSd female mice (15‐23‐week‐old, 24–35 g; Harlan, the Netherlands) bred at the animal facility at Universidad Miguel Hernández de Elche (UMH, Elche, Spain) were used to assess the antinociceptive effects of compound 23 on the model of chemotherapy‐induced neuropathic pain. Housing conditions were maintained at 21 ± 1 °C and 55 ± 20% relative humidity in a controlled light/dark cycle (light on between 8:00 a.m. and 8:00 p.m). Care was taken to minimize the number of animals used and the pain and stress they experienced. Animal experimentation procedures were conducted under the approval of the Institutional Animal and Ethical Committee at UMH, following the guidelines of the European Community (2010/63/EU), and the Committee for Research and Ethical Issues of the International Association for the Study of Pain [64]. The study protocol received approval from the Ethical Committee of Universidad Miguel Hernández de Elche (UMH, Elche, Spain) and the regional government (approval code: 2022 VSC PEA 0078‐2). For the in vivo behavioral experiments evaluating the effects of the TRPM8 antagonist on mice with chemotherapy‐induced neuropathy, male C57BL/6JRccHSd mice (15–23 weeks old; Harlan, the Netherlands) were utilized. The animals were bred and housed at the UMH Animal Facility (Servicio de Experimentación Animal, UMH, Elche, Spain). The experimental protocol was reviewed and approved by the Institutional Animal and Ethical Committee of UMH.

Primary cultures of dorsal root ganglia neurons

Primary cultures of neonatal dorsal root ganglia were used for multielectrode array (MEA) and patch‐clamp experiments following established methodologies previously described [65]. Neonatal dorsal root ganglia (DRGs) were obtained from Wistar rats (3–5 days old) and were isolated and digested with 0.25% (w/v) collagenase (type IA) in DMEM GlutaMax with 1% (v/v) penicillin/streptomycin (P/S) solution for 1 h at 37 °C in a 5% CO₂ Thermo Scientific incubator. Following digestion, DRGs were mechanically dissociated and passed through a 100 μm cell strainer to obtain single‐cell suspensions. Suspensions were washed with DMEM GlutaMax with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (P/S). Cells were then seeded in microelectrode array chambers coated with poly‐l‐lysine (8.3 μg·mL⁻¹) and laminin (5 μg·mL⁻¹). After 1 h, the medium was replaced with DMEM GlutaMax, 10% (v/v) FBS, and 1% (v/v) P/S, supplemented with mouse 2.5S NGF 50 ng·mL⁻¹ and 1.25 μg·mL⁻¹ cytosine arabinoside. Isolated mouse cells were incubated with 0.67% (w/v) collagenase type XI and 3% (w/v) dispase (Gibco) in INC mix medium (in mm): 155 NaCl, 1.5 K₂HPO₄, 5.6 HEPES, 4.8 NaHEPES, and 5 glucose for 1 h (37 °C, 5% 3 CO₂). Primary cultures of mouse DRGs were used for patch‐clamp experiments. Mouse DRG were mechanically dissociated using a glass Pasteur pipette. Single‐cell suspensions were passed through a 100 μm cell strainer and washed with DMEM GlutaMax plus 10% FBS (Invitrogen (Thermo Fisher Scientific), Barcelona, Spain) and 1% P/S. For each experiment, cells were seeded at the required density on 12 mm coverglass slides in a 24‐well plate or microelectrode array chambers previously coated with poly‐l‐lysine (8.33 μg·mL⁻¹) and laminin (5 μg·mL⁻¹). After 2 h, the medium was replaced with DMEM GlutaMax, 10% FBS, and 1% P/S, supplemented with mouse 2.5 s NGF 50 ng·mL⁻¹ (Promega). All experiments in patch‐clamp were conducted 48 h after cell seeding.

Calcium microfluorimetric assay

Assays were conducted to assess the channel activity of the recombinant hTRPM8 stably expressed in HEK‐293 cells. HEK‐293‐hTRPM8 cells were cultured in a monolayer using a DMEM GlutaMax with 10% FBS and 1% P/S and maintained at 37 °C in 5% CO₂. For experiments, cells were prepared and seeded at the indicated densities. To evaluate compound effectiveness against hTRPM8 activity, microfluorometry‐based calcium flux assays were performed using Fluo‐4 NW Ca²⁺ dye and fluorescence detection. HEK cell lines expressing hTRPM8 were seeded in 96‐well plates and incubated with the dye‐loading solution. Ion channel activity was measured using a plate reader, and fluorescence intensity changes were recorded following the addition of vehicle, compound at varying concentrations, and antagonist (10 μm AMTB). Data analysis included calculating the Z‐factor for each assay and normalizing compound effects to capsaicin‐induced fluorescence. Data were analyzed to determine the concentration exerting half‐maximal inhibition (IC₅₀) or activation (EC₅₀) of agonist‐induced calcium elevation, using graphpad prism8® software (Boston, MA, USA). All experiments were conducted in triplicate, and results are presented as mean ± standard deviation.

Patch‐clamp recordings from recombinant cells

Recording through patch‐clamp techniques was performed on HEK293 cells cultured in DMEM GlutaMax with 10% FBS and 1% P/S. The cell lines were authenticated following the standardization of STR Profiling guidelines (ANSI/ATCC ASN‐0002‐2022) using the CLA Identifier Plus PCR Amplification Kit (Thermo Fisher‐A44660) to analyze 16 highly variant human STRs. The cells were confirmed to be mycoplasma‐free. These cells were transiently transfected with plasmids encoding hTRPV1, hTRPM8, or hTRPA1 using Lipofectamine 3000. Transfected cells were seeded on 12 mm Ø glass coverslips treated with poly‐l‐lysine solution and recorded 2 days post‐transfection. The intracellular pipette solution included (in mm) 150 NaCl, 3 MgCl₂, 5 EGTA, and 10 HEPES, pH 7.2 with CsOH, while the extracellular solution contained (in mm) 150 NaCl, 6 CsCl, 1 MgCl₂, 1.5 CaCl₂, 10 glucose, and 10 HEPES, pH 7.4 with NaOH. An EPC‐10 amplifier with Patchmaster software was used in whole‐cell experiments. Patch pipettes, created from thin‐wall borosilicate capillary glass tubing, were pulled with a Micropipette puller to a final resistance of 2–8 MΩ when filled with the internal solution. Recordings were acquired at 10 kHz and low‐pass filtered at 3 kHz, discarding recordings with leak currents > 200pA or series resistance > 20 MΩ. In voltage‐clamp recordings, cells were held at a constant potential, and the application of modulators was carried out using a gravity‐driven perfusion system. Total currents were normalized to the first current peak evoked by the activating stimuli. To study the effect of compound 23 on TRPM8 voltage dependence, a voltage step protocol from −120 to 120 mV was used with 100 ms steps of 20 mV from a holding potential at 0 mV. Leak currents were not subtracted, and conductance was calculated using the equation: G = I/(V − V _r), where I is the measured ionic current, V is the applied voltage, and V _r is the reversal potential that for the ionic conditions used was set to 0 mV.

Dose–response relationships for WS‐12 channel activation were normalized to the response of the channel to a saturating concentration of the agonist. Similarly, dose–response curves for blockade activity were normalized with respect to the response in the absence of the blocker. The experimental data were fitted to the Hill equation: $Y=\text{Bottom}+\left(\mathrm{Top}‐\text{Bottom}\right)/(1+{10}^{\left(\left[\text{ligand}\right]-{\text{LogIC}}_{50}\left(\text{or}\mathrm{Log}\left({\mathrm{EC}}_{50}\right)\right)\right)})$ using prism 9 software. Top was fixed to 100, as the unblocked response or the maximal response for WS‐12.

Patch‐clamp recordings from DRG nociceptors

Two days after being seeded on 12 mm Ø glass coverslips treated with poly‐l‐lysine solution and Laminin (Sigma‐Aldrich), whole‐cell patch‐clamp recordings were conducted on sensory DRG neurons from adult mice. The intracellular pipette solution consisted of (in mm): 4 NaCl, 110 K gluconate, 1 CaCl₂, 30 KCl, 2 MgCl₂, 10 HEPES, 4 ATP, 0.4 GTP, and 10 EGTA, with a pH of 7.2 adjusted with KOH. The extracellular solution comprised (in mm): 140 NaCl, 4 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, 5 glucose, and 20 mannitol, with a pH of 7.4 adjusted with NaOH.

Microelectrode array (MEA)

Microelectrode array measurements were conducted using 60‐electrode thin MEA chips, featuring 30 μm diameter electrodes and 200 μm inter‐electrode spacing, including an integrated reference electrode (Multi Channel Systems GmbH, Reutlingen, Germany). The MEA1060 System (Multi Channel Systems GmbH) and mc rack software version 4.3.0 were employed to record the electrical activity of primary sensory neurons. Short 30 s applications (termed P1 and P2) of WS‐12 were applied using a continuous perfusion system (2 mL·min⁻¹). Between each stimulus, cells underwent 4 min and 30 s washes with external solution. Treated cells were perfused with compound 23, 1 min before and together with P2. The protocol concluded with the application of 40 mm KCl to confirm neuronal excitability and viability. All measurements were conducted at approximately 34.5 °C using the Multichannel Systems Temperature Controller. For microelectrode array analysis, data were processed using MC_RACK spike sorter with a sample rate of 25 kHz, applying a Butterworth high‐pass 2nd order filter with a 200 Hz cutoff. For AITC analysis, a cutoff of 500 Hz was applied. An evoked spike was defined when the amplitude of neuronal electrical activity reached a threshold established by automatic estimation at −4.7 μV Std. For AITC analysis, an evoked spike was defined when the amplitude of neuronal electrical activity reached a threshold established by automatic estimation at −5 μV Std. Spiking activity was measured for 60 s immediately following the instillation of activating stimuli. Electrodes lacking electrical activity in the first agonist pulse were excluded. The recorded signals were then analyzed to extract the mean spike frequency for each pulse (P1–P2). The ratio P2/P1 of mean spike frequency was calculated and normalized to the vehicle for comparison across different conditions.

Model of chemotherapy‐induced neuropathic pain

Chemotherapy‐induced sensitization was induced by repeated administration of oxaliplatin (ref#2623; Tocris, Bristol, UK). Oxaliplatin was freshly prepared every day by dissolution in 5% dextrose in warmed distilled water at 37 °C. The chemotherapeutic was administered intraperitoneally every other day for 5 days at a 6 mg·kg⁻¹ and in a volume of 10 mL·kg⁻¹, reaching an accumulated dose of 18 mg·kg⁻¹ after the three injections.

Compound 23 treatment

The systemic effect of compound 23 was assessed after intraperitoneal administration at 5 mg·kg⁻¹, dissolved in a solution of 2% Cremophor and 5% DMSO in saline, with a volume of 10 mL·kg⁻¹. This dose was chosen based on the relative potency of the compound in the in vitro studies when compared to the canonical antagonist AMTB. Nociceptive sensitivity was evaluated for 30‐ and 90‐min postinjection using the acetone drop test (7 days after the first oxaliplatin dose) and the dry ice test (9 days after the last oxaliplatin dose).

The local effect of compound 23 or its vehicle was assessed 11 days after beginning the oxaliplatin treatment through subcutaneous intraplantar injection of 1 μg compound 23 or vehicle (2% Cremophor and 5% DMSO in saline) in the ventral side of the right hind paw, administered in a volume of 10 μL [66]. This dose was chosen based on previous works [48] and considering the relative potency of the compound in the in vitro studies. Nociceptive sensitivity was evaluated 30 and 90 min after the intraplantar injection with the acetone test [66].

Behavioral assessment of cold sensitivity

Prior to conducting behavioral experiments, mice underwent a 2‐day acclimatization period to the experimental conditions, during which they were handled and habituated to the male experimenter for a minimum of 2 min per day and mouse [66]. Additionally, the animals were familiarized with each testing environment 2 h per day, placed individually in Plexiglas® chambers (10 × 10 × 14 cm). Every day of evaluation, the mice spent an additional hour of habituation in the testing environment before the measurement of nociceptive sensitivity.

Acetone drop test

Mice were placed over a metal grid and allowed to habituate for approximately 1 h. Acetone (179124; Sigma‐Aldrich) was applied in 20 μL drops onto the mid‐plantar surface of the right hind paw by using a 200 μL pipette with a plastic tip manually curved [66]. Responses were recorded using an iPhone SE camera (Apple, Cupertino, CA, USA), and quantification of paw‐licking responses was conducted afterwards by a blinded observer. The responses were measured for 1 min after acetone application, with a digital stopwatch (Xnote Stopwatch Version 1.63 2011 Dmitry Nikitin) and were averaged for both hindpaws after the systemic treatment with compound 23 or conducted only in the right hind paw after the intraplantar treatment. For each measurement, the paws were sampled three times, and the mean was calculated. The interval between each application of acetone was at least 3 min [66].

Dry ice test

The dry ice test was conducted as previously described [67] with some modifications. Mice underwent a 1‐h habituation period on a 6 mm thick glass surface (Flores Valles, Madrid, Spain). Subsequently, a hand‐made probe was prepared by cutting the top of a 3 mL syringe (DicoNEX; ZARYS International Group, Zabrze, Poland) and drilling the syringe with a 25 G needle (BD Microlance 3; Beckton Dickinson & Co Ltd, Louth, Ireland) to prevent the accumulation of CO₂ gas. Powdered dry ice was used to fill the probe. The ice was compacted by pressing the plunger against the bench until a dense pellet at least 1 cm long was obtained. The flattened pellet was then applied to the glass surface using the probe, targeting the right and left hindpaws. A minimum interval of 5 min was maintained between applications. To determine the threshold value for each mouse, the latency to paw withdrawal was recorded for each hindpaw and subsequently averaged. The glass surface was always kept dry.

Statistical analysis

For the analysis of in vitro studies, all data are expressed as mean ± SEM. The number of replicates is indicated in the figure legends. In vitro data were statistically analyzed using one‐way ANOVA followed by the Bonferroni post hoc test of multiple comparisons as indicated, or unpaired, two‐tailed Student t‐test for some experiments are also indicated. For one‐way ANOVA, we report the F (DFn, DFd) and the P value, along with the P values derived from the Bonferroni post hoc test. The P value for all the analyses was set at 0.05, and the value obtained is reported.

For the analysis of behavioral experiments, graphpad prism 9 was used (GraphPad Software Inc., San Diego, CA, USA). An ANOVA with two factors (Within Factor ‘OXP treatment’, between factor ‘23 vs Vehicle group’) and their interaction was first used to assess the effect of the oxaliplatin treatment and to assess possible baseline differences between treatment groups. A subsequent two‐way ANOVA was used to study the effects of compound 23 treatment (Within Factor ‘Time Point’, between factor ‘23 vs Vehicle group’ and their interaction). Post hoc Tukey's multiple comparisons tests were run whenever the interaction was significant and differences were considered statistically significant when P value was below 0.05.

Metabolic stability

Incubation in mouse plasma

The standard incubation mixture (100 μL final volume) was carried out by dissolving the tested substrate (100 μm) in DMSO (5% final volume) in preincubated plasma at 37 °C. The mixture was shaken for 60 min at 37 °C. Control incubations were carried out without substrate. Each incubation was stopped by addition of 200 μL of ice‐cold acetonitrile, vortexed, and centrifuged at 13 000 r.p.m. for 10 min. The supernatants were analyzed by LC‐UV [68].

Incubation in mouse liver microsomes

The standard incubation mixture (250 μL final volume) was carried out in a 50 mm Tris (tris[hydroxymethyl]aminomethane) buffer (pH 7.4) containing 150 mm KCl, 1.5 mm, 3.3 mm MgCl₂, 1.3 mm NADPNa₂, 3.3 mm glucose 6‐phosphate, 0.4 units·mL⁻¹ glucose 6‐phosphate dehydrogenase, acetonitrile as cosolvent (1% of total volume), and the substrate (50 μm). After pre‐equilibration of the mixture, an appropriate volume of MLM suspension was added to give a final protein concentration of 1.0 mg·mL⁻¹. The mixture was shaken for 60 min at 37 °C using a horizontal shaking thermostatic bath while protecting the samples from light. Control incubations were carried out without the presence of substrate, or NADPH regenerating system, or microsomes. Each incubation was stopped by the addition of 250 μL of ice‐cold acetonitrile, vortexed, and centrifuged at 13 000 r.p.m. for 5 min [68]. The supernatants were analyzed by LC‐UV and LC‐HRMS equipment (see Tables S1 and S2, Fig. S79 for full parameters setting in the Supporting Information).

Data processing

The metabolic stability of the compound 23 was determined in vitro by measuring the residual peak area after incubation by LC‐UV analysis. Samples were further processed by LC‐HRMS equipment for metabolite profiling. Raw data files were processed using both xcalibur® and compound discoverer 3.2® software (Thermo Scientific) using a customized workflow for the detection and identification of the expected and unknown metabolites (Fig. S80).

Molecular modeling and docking

Human and mouse TRPM8 structures (PDB ID 8BDC, 8E4N) [4, 69] were used to explore the binding of compound 23. The docking procedure was performed with the AutoDock4 algorithm [70] implemented in Yasara [71, 72]. Briefly, a local docking procedure was accomplished using either human or mouse TRPM8 structures, and compound 23. The search space was limited to a simulation box built around the well‐known agonist or antagonist ligands (WS‐12, AMTB). A total of 500 flexible docking runs were set and clustered around the selected binding sites. The program performs a simulated annealing minimization of the complexes, which moves the structure to a stable energy minimum, by using the implemented AMBER 99 (Assisted Model Building with Energy Refinement) force field [73]. 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. The theoretical affinities of ligands at its binding site were determined by calculating the binding energy of the ligand‐receptor complex. The binding energy was obtained by measuring the energy at infinite distance (the unbound state) and subtracting from that value the energy of the complex at the bound state. Figures were drawn using the open‐source pymol v2.6 (The PyMOL Molecular Graphics System, at http://www.pymol.org/). Interactions were determined in PLIP (https://plip‐tool.biotec.tu‐dresden.de/plip‐web/plip/index), a fully automated web server [74] to identify noncovalent interactions between macromolecules and ligands. The same molecular modeling methodology was used by Lamberti et al. [75].

Conflict of interest

The authors declare no conflict of interest.

Author contributions

UG, AF‐M, GCT, and AF‐C wrote and revised the manuscript with input from all the authors, drafting the work or reviewing it critically for important intellectual content. UG and FT synthesized the compounds. SA performed metabolic stability, HRMS analysis, and purity of final compounds. AL performed the biological evaluation, patch‐clamp, and MEA experiments and wrote the manuscript. DC performed in vivo experiments and wrote and revised the manuscript. LB performed MEA experiments and revised the manuscript. GFB performed docking simulations. All the authors have approved the final version of the manuscript.

Peer review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1111/febs.70065.

Supporting information

Figs S1–S78. ¹H‐NMR, ¹³C‐NMR and HRMS spectra of final compounds 20–45.

Figs S79–S80. LC‐UV methods for purity and metabolic stability evaluation and LC‐HRMS metabolic profile of compound 23 in mouse liver microsomes.

Figs S81–S92. Purity of selected active compounds.

Tables S1–S2. LC‐UV methods for purity and metabolic stability evaluation and LC‐HRMS metabolic profile of compound 23 in mouse liver microsomes.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the main text and the Supporting Information. The data that support the findings of this study are available from the corresponding authors upon reasonable request.