1. Introduction

Figure 1. Structure of model compond 1 and analogues prepared and studied in this work.

Figure 1. Structure of model compond 1 and analogues prepared and studied in this work.

2. Results and Discussion

2.1. 1,4,4-Trisubstituted β–lactam Derivatives

For the first exploratory structure-activity relationships, we decided to prepare and evaluate diastereoisomeric 1,4,4-trisubstituted β–lactam derivatives, which can be more easily prepared than enantiomerically pure analogues. In previous works, we have demonstrated that both 4-S- and 4-R-β-lactams rendered TRPM8 antagonists [37], therefore we expected thet 4-R,S-diastereoisomeric mixtures could be active and allow us to discern among important substituents. Firstly, we proposed the incorporation of different amides in the 4-carboxylate group, including structural diversity with an alkyl, a benzyl, a pyridine group and other related heterocyclic systems, able to slightly increase the TPSA values (Table S1). The six amines selected for the formation of the corresponding amides were isobutylamine, benzylamine, 3,4-dichlorobenzylamine, 2- and 3-picolines (2-, 3-Pic) and 3-aminopyridine (3-Py). In a previous publication on Asp-derived β-lactams, amide derivatives from 4-pyridine and 4-picoline showed lower activity than the corresponding 3-aminopyridyl analogues [35]. Therefore, the 4-picolyl derivative was discarded in this case, while the 2-picoline derivative was prepared, since it had not been explored previously. In addition, pyridyl- and picolyl-derived amides incorporate two protonable nitrogens, which could increase aqueous solubility of final molecules. The 3,4-dichlorobenzylamine was selected because this substitution is present in a kappa opioid agonist with antagonist-like properties in a mouse model related to TRPM8 activation by icilin [39]. Saponification of β–lactam 2a,b (see supporting information for synthetic details), afforded the carboxylic acid intermediate 3ab (a:b = 1:1.8), which was then coupled with the selected amines, in the presence of PyBroP, to allow the preparation of amides 4a,b-9a,b (Scheme 1). These amides were isolated in different diastereoisomeric ratios (Table S2), most probably due to different conversion of the diasteromeric acids 3a and 3b or to the enrichment in a particular diastereoisomer during the purification process.

Scheme 1. Synthetic procedure for the preparation of amides 4a,b-9a,b from β-lactam 2a,b.

Scheme 1. Synthetic procedure for the preparation of amides 4a,b-9a,b from β-lactam 2a,b.

Another way to increase TPSA values is the removal of one of the N-benzyl groups, while the incorporation of different substitutions on the remaining N-benzyl moiety could serve to fine-tune the antagonist activity. To this end, the hydrogenolysis of β-lactam 2a,b, using Pd(OH)₂/C as catalyst in the presence of HCl, gave rise to the primary amine derivative 10a,b in its hydrochloride form (1:2.5, a:b diastereoisomer ratio). The slight enrichement in te major diastereoisomer (b) compared to the starting material is due to the formation of the (3S,6S)-3,6-dibenzyl-1,4-diazabicyclo[4.2.0]octane-5,8-dione 11a (configuration determined through NOE experiments) from minor isomer 10a (Scheme 2). This secondary product was also formed during the reductive amination reaction of compound 10a,b with aldehydes to the corresponding monobenzyl derivatives, complicating the purification steps. To avoid this issue, most reactions in this section were performed with the isobutylamide derivative 12a,b, synthesized from 4a,b. Differently substituted benzaldehydes, containing alkyl or benzyl groups, and electron atrancting or withdrawing groups, and 2-naphtaldehyde were used for the N-monobezyl derivatives preparation (Scheme 2, Table S2). Following some of the activity initial results, detailed later on, most substituents on the benzyl group are at position 3 of the phenyl ring, but some 4-substituted analogues were also prepared to verify preliminary outcomes. Thus, amine derivative 10a,b was treated with the corresponding benzaldehyde, to form the corresponding intermediate imines, which were subsequently reduced with NaBH₃CN to afford β-lactam N-monobenzylated derivatives 13a,b-17a,b (Scheme 2, Table S2). Similarly, the reaction of 12a,b with different aldehydes, containing an aliphatic (Me), two aromatic (Ph, Nf), all halides (F, Cl, Br, I), donor groups (OMe, OPh, OBn) and electron acceptors (CN, NO₂) substituents afforded the corresponding monobenzyl derivatives 18a,b-31a,b (Scheme 2, Table S3).

Scheme 2. Hydrogenolysis of β-lactams 2a,b and 4a,b and reductive amination to N-monobenzylamines.

Scheme 2. Hydrogenolysis of β-lactams 2a,b and 4a,b and reductive amination to N-monobenzylamines.

The diastereomeric 1,4,4-trisubstitutes β-lactams were evaluated as modulators of the TRPM8 channel by calcium microfluorography assays. These assays use a HEK293 cell line that stably overexpressed rat TRPM8 channel (rTRPM8). Menthol was used as the agonist during these experiments, whereas AMTB and model compound 1 served as prototype antagonists for comparison.

Within these assays, β-lactam 2a,b, choosen as a pivotal intermediate, displayed submicromolar potency as an antagonists of the TRPM8 channel (Table 1). Among the amide series, the most favorable IC₅₀ values were achieved with compounds 4a,b and 5a,b, sugesting that the methyl ester of 2a,b can be substituted by an aliphatic (4a,b) or benzylic (5a,b) amide, with only marginal loss of activity (Table 1). In contrast, the introduction of two chlorine atoms into the benzylamide group (6a,b) causes a notable reduction in antagonist activity. The amide derived from 2-picoline (8a,b) exhibited micromolar potency, while the 3-aminopyridyl (7a,b) and 3-picolyl (9a,b) derivatives demonstrated lesser potency. When comparing these results to the model antagonist AMTB, it becomes apparent that the 4-CO₂Me derivative 2a,b and its amide counterparts 4a,b and 5a,b exhibit antagonist activity one order of magnitude more potent.

Table 1. Antagonist activity of amides 4a,b-9a,b in rTRPM8 channels (Ca²⁺ microfluorography assay).

Table 1. Antagonist activity of amides 4a,b-9a,b in rTRPM8 channels (Ca²⁺ microfluorography assay).

Compound	R1	a:b	IC50 (μM)	95% confidential intervals
2a,b		1:1.8	0.15 ± 1.23	0.095 to 0.22
4a,b	iBu	1:1.6	0.71 ± 1.20	0.49 to 1.03
5a,b	Bn	1:2.6	0.71 ± 1.27	0.43 to 1.16
6a	(3,4-Cl)Bn		ND	_
6a,b	(3,4-Cl)Bn	1:1.4	ND	_
7a,b	3-Py	1:1.4	11.62 ± 1.30	6.73 to 20.06
8a,b	2-Pic	1:1.8	1.99 ± 1.26	1.24 to 3.17
9a,b	3-Pic	1:1.2	10.69 ± 1.38	5.52 to 20.73
1			1.06 ± 1.21	0.72 to 1.55
AMTB			7.3 ± 1.5	8.82 to 7.85

Within the monobenzyl series, all compounds exibit IC₅₀ values in the micromolar range, displaying slight variation across most of them (1.10 to 12.1 μM), and lower potency in comparison to the dibenzyl model compounds 2a,b and 4a,b. The importance of having at least one benzyl-type group at the N-terminal amino moiety is demonstrated by the substantial decline in antagonist activity observed in 11a (with less than 50% inhibition at 50 μM), although the conformational restriction could also contribute to the lower activity.

Regarding the substituents on the benzyl group, placement at meta (3-) position seems slightly preferred over para (4-) positioning. This observation is drawn from the comparison between compounds 14a,b vs 15a,b, as well as between 20a,b and 21a,b vs 30a,b and 31a,b, respectively. Large ubstituents around position 3, such as Ph (20a,b), Cl (22a,b), Br (16a,b and 23a,b), I (24a,b), and the condensed 2-naphthyl moiety (17a,b and 32a,b), appear to enhance activity. Generally, strong electron-donating groups like OMe (25a,b) and OBn (27a,b), which result in an increased TPSA up to 70 A² (Table S1), exhibit slightly lower potency than their corresponding unsubstituted analogue (13a,b). On the contrary, a distinct behavior is noted between the two analogues bearing strong electron-withdrawing groups (CN and NO₂). While the nitrile-bearing derivative 28a,b ranks as the less effective compound in this series (IC₅₀ = 12.1 µM), the 3-NO₂ analog 29a,b maintains the one-digit micromolar antagonist activity akin to the unsubstituted counterpart. The calculated TPSA values of these latter compounds have increased by more than 25 and 55 units, respectively, when compared to the unsubstituted analogue 13a,b (as shown in Table S1). However, compound 29a,b wasn not pursued further due to the potential interference posed by the NO₂ group, which is signaled as a Pan-Assay Interference Compound (PAIN) alert [40].

All these observations on structure-activity relationships must be approached with the required caution, considering the variations in the ratio of diastereomers among different compounds. In general, the N-monobenzylated derivatives exhibit a reduced activity ranging between one and two orders of magnitude when compared to the corresponding N,N-dibenzylated model compounds. The introduction of different substituents on the phenyl ring does not result in significant impacts on activity, independently of the substituent size and electronis character. Even large substituents (such as Ph or Br) on the N-monobenzyl moiety failed to compensate for the absence of the second benzyl group.

Table 2. Antagonist activity of N-monobenzyl derivatives 13a,b-32a,b in rTRPM8 channels (Ca²⁺ microfluorography assays).

Table 2. Antagonist activity of N-monobenzyl derivatives 13a,b-32a,b in rTRPM8 channels (Ca²⁺ microfluorography assays).

Compound	XR1	R2	a:b	IC50 (μM)	95% confidential intervals
2a,b	OMe	-	1:1.8	0.15 ± 1.23	0.095 to 0.22
4a,b	NHiBu	-	1:1.6	0.71 ± 1.20	0.49 to 1.03
11a	-	-	-	ND	ND
13a,b	OMe	H	1:3.6	3.95 ± 1.20	2.66 to 5.85
14a,b	OMe	3-Me	1:3.6	3.58 ± 1.41	1.74 to 7.35
15a,b	OMe	4-Me	1:5.6	3.90 ± 1.47	1.74 to 8.74
16a,b	OMe	3-Br	1:2	1.10 ± 1.38	0.56 to 2.16
17a,b	OMe	(2-Nph)CH2	1:4	2.60 ± 1.23	1.69 to 3.98
18a,b	OMe	4-OPr	1:9.5	8.90 ± 1.40	4.37 to 18.10
19a,b	NHiBu	3-Me	1:1.5	3.97 ± 1.31	2.28 to 6.93
20a,b	NHiBu	3-Ph	1:1.1	2.03 ± 1.32	1.15 to 3.60
21a,b	NHiBu	3-F	1:2.6	5.15 ± 1.46	2.37 to 11.22
22a,b	NHiBu	3-Cl	1:2.4	2.57 ± 1.32	1.45 to 4.53
23a,b	NHiBu	3-Br	1:2.9	3.30 ± 1.36	1.73 to 6.29
24a,b	NHiBu	3-I	1:2.7	3.83 ± 1.28	2.33 to 6.29
25a,b	NHiBu	3-OMe	1:2.5	4.48 ± 1.25	2.83 to 7.09
26a,b	NHiBu	3-OPh	1:5.7	8.22 ± 1.30	4.79 to 14.08
27a,b	NHiBu	3-OBn	1:1.5	3.27 ± 1.37	1.72 to 6.20
28a,b	NHiBu	3-CN	1:1.1	12.10 ± 1.31	6.99 to 20.94
29a,b	NHiBu	3-NO2	1.2:1	2.12 ± 1.28	1.28 to 3.50
30a,b	NHiBu	4-Ph	1:1.9	4.57 ± 1.22	3.07 to 6.82
31a,b	NHiBu	4-F	1.1:1	8.32 ± 1.30	4.87 to 14.22
32a,b	NHiBu	3,4-Ph (2-Nph)	1:1.1	1.81 ± 1.21	1.22 to 2.68
1				1.06 ± 1.21	0.72 to 1.55
AMTB				7.30 ± 1.50	8.82 to 7.85

2.2. 1,3,4,4-Tetrasubstituted, Enantiopure β–lactam Derivatives

The challenges in making precise comparisons among compounds of the previous 1,4,4-trisubstituted series, due to the variability in the diastereoisomeric ratio and the requirement to study pure isomers for further pharmaceutical development, prompted us to transition to a new series of diastereo- and enantiopure β-lactam derivatives. In this newly designed series, the most promising substituents identified earlier were incorporated onto a 1,3,4,4-tetrasubstituted β–lactam scaffold. These enantiopure β–lactams can be synthesized using chiral 2-chloropropanoic acid derivatives, as previously reported [38,41].

Staring from compound 1, the tert-butyl ester underwent acidic hydrolysis to yield the free carboxylic acid intermediate 33. This intermediate was subsequently coupled to isobutyl, bencyl and 2-picolyl amines to generate the corresponding amides, 34-36, with very good yield (Scheme 3). Hydrogenolysis of compound 1, using Pd(OH)₂ as catalyst, resulted in a mixture of the monobenzyl derivative 37 and the fully deprotected amino analogue 38, which were separated using column chromatography. A similar reaction, starting from compound 34, rendered the free amino derivative 39 in almost quantitative yield. Further trestments of compounds 38 and 39 with differently substituted benzaldehydes, followed by reduction of the resulting imine intermediates with NaBH₃CN, led to the synthesis of the expected N-monobenzyl derivatives 40-45 (Scheme 3, Table S3).

Scheme 3. Preparation of enantiopure β–lactam derivatives.

Scheme 3. Preparation of enantiopure β–lactam derivatives.

The initial biological evaluation of this series of 1,3,4,4-tetrasubstituted β-lactams was performed by calcium microfluorography assays using the indicated HEK293 cell line overexpressing rTRPM8 channels. As outlined in Table 3, submicromolar IC₅₀ values were achieved for the three prepared amide derivatives, with their antagonist activities ranking as follows: 34 (R¹ = ⁱBu) > 35 (R¹ = Bn) > 36 (R¹ = CH₂(2-Pic)). These levels of potency surpass that of the O^tBu model compound 1 and are one order of magnitude higher than the reference antagonist AMTB. In line with previous findings, most of the N-monobenzylic compounds displayed reduced potency compared to their dibenzyl analogues. Furthermore, substitution at the 3-position of the benzyl aromatic ring appears to be slightly favored over the 4-position (comparison of 40 vs 41). Compounds with 3-Ph (42), 3-Br (44) and 2-Nf (45) exhibited similar micromolar activities. Unfortunately, our efforts to increase the TPSA values by the incorporation of additional heteroatoms in amides and N-monobenzyl derivatives resulted mostly in lower potencies compared to model compound 1. Interestingly, amides 34 and 35 improved the TRPM8 antagonist activity, although the increases in TPSA values are tiny for these compounds (Table S1).

Table 3. Antagonist activity of enantiopure β–lactam derivatives at rTRPM8 channels (Ca²⁺ microfluorography assays).

Table 3. Antagonist activity of enantiopure β–lactam derivatives at rTRPM8 channels (Ca²⁺ microfluorography assays).

Compound	XR1	R2	R3	IC50 (µM)	95% Confidence Intervals
1	OtBu		Bn	1.06 ± 1.21	0.72 to 1.55
34	NHiBu		Bn	0.30 ± 1.26	0.19 to 0.50
35	NHBn		Bn	0.49 ± 1.27	0.30 to 0.79
36	NHCH2(2-Pic)		Bn	0.84 ± 1.32	0.48 to 1.49
37	OtBu		H	3.06 ± 1.25	2.67 to 3.51
40	OtBu		H	6.36 ± 1.41	3.08 to 13.17
41	OtBu		H	9.26 ± 1.41	4.44 to 19.35
42	NHiBu		H	2.19 ± 1.36	1.15 to 4.17
43	NHiBu		H	4.03 ± 1.35	2.14 to 7.60
44	NHiBu		H	3.27 ± 1.31	1.86 to 5.75
45	NHiBu		H	3.56 ± 1.40	1.74 to 7.31
AMTB				7.3 ± 1.50	6.82 to 7.85

2.3. Activity in hTRPM8 Channels and Electrophysiology Assays

To further characterize this family of compounds, calcium microfluorography assays were conducted using the HEK293 cell line, which expresses the human isoform of the TRPM8 channel. Amide derivatives 34 and 35 were selected, and their outcomes were compared with those of the model compound 1 (Table 4). These new β–lactams exhibit a somewhat diminished antagonist activity, as evidenced by higher IC₅₀ values, when evaluated in hTRPM8 as compared to rTRPM8. This behaviour is similar to those observed for other TRPM8 antagonists [42].

Table 4. Antagonist activity of β-lactams 34 and 35 in hTRPM8 (Ca²⁺ microfluorommetry) and electrophysiology in rTRPM8 channels (Patch-clamp assays).

Table 4. Antagonist activity of β-lactams 34 and 35 in hTRPM8 (Ca²⁺ microfluorommetry) and electrophysiology in rTRPM8 channels (Patch-clamp assays).

Compound	Ca2+ Microfluorography Assays				Patch-Clamp Assay
	rTRPM8 IC50 (µM)	95% Confidence Intervals	hTRPM8 IC50 (µM)	95% Confidence Intervals	rTRPM8 IC50 (µM)	95% Confidence Intervals
1	1.06 ± 1.21	0.72 to 1.55	1.74 ± 1.19	1.23 to 2.45	0.60 ± 1.66	0.20 to 1.76
34	0.30 ± 1.26	0.19 to 0.50	2.58 ± 1.24	1.65 to 4.02	0.16 ± 0.82	0.10 to 0.271
35	0.49 ± 1.27	0.30 to 0.79	3.33 ± 1.28	2.03 to 5.48	0.48 ± 1.33	0.18 to 1.23

To confirm the antagonistic activity of the most potent compounds, electrophysiology studies were conducted using the Patch-Clamp technique on single HEK293 cells expressing the rat TRPM8 channel (Table 4, Figure 2). As depicted in Figure 2A, perfusion with 100 µM menthol (shown in blue) resulted in a strongly outward rectifying ion current. This is evident by a negligible current at negative potentials and a linear intensification of the ohmic current at positive voltages. When applying compound 34 at a concentration of 5 µM, a noticeable reduction in menthol-induced activity at depolarizing voltages was observed (indicated by the red line), compared to menthol alone. Dose-response relationships for both tested compounds were established through triplicate experiments at various concentrations, applying a holding voltage of −60 mV (as shown in Figure 2B). These compounds effectively blocked the TRPM8-mediated and menthol-induced responses. In these assays, both derivatives displayed higher potency than the model β–lactam 1, being the the isopropylamide derivative 34 the most potent, with a efficacy in the low nanomolar range (Table 4).

Figure 2. Compound 34 blocks TRPM8-mediated responses evoked by menthol in rTRPM8-expressing HEK293 cells. (A) IV curves obtained after exposure to vehicle solution (green trace), 100 µM menthol (blue trace) and 100 µM menthol + 10 µM 34 (red trace). Peak current data were expressed as pA/pF (to allow comparison among different size cells). Each point is the mean ± SEM of n = 15. (B) Concentration/response curves for rTRPM8 current blockade by 34, at a holding voltage of −60 mV. The solid line represents fits of the experimental data to the following equation: Y = Bottom + (Top-Bottom)/(1 + 10^((X-LogIC50))) with a standard slope of −1.0 (Hill coefficient) and a restriction of the minimum (Top = 100). The fitted value for IC₅₀ was 0.16 ± 0.82. Each point is the mean ± SEM of n = 15.

Figure 2. Compound 34 blocks TRPM8-mediated responses evoked by menthol in rTRPM8-expressing HEK293 cells. (A) IV curves obtained after exposure to vehicle solution (green trace), 100 µM menthol (blue trace) and 100 µM menthol + 10 µM 34 (red trace). Peak current data were expressed as pA/pF (to allow comparison among different size cells). Each point is the mean ± SEM of n = 15. (B) Concentration/response curves for rTRPM8 current blockade by 34, at a holding voltage of −60 mV. The solid line represents fits of the experimental data to the following equation: Y = Bottom + (Top-Bottom)/(1 + 10^((X-LogIC50))) with a standard slope of −1.0 (Hill coefficient) and a restriction of the minimum (Top = 100). The fitted value for IC₅₀ was 0.16 ± 0.82. Each point is the mean ± SEM of n = 15.

We also investigated the effect of β-lactams derivatives on the resting membrane potential (RMP) of DRG sensory neurons. Under current clamp conditions in patch-clamp assays, small sensory neurons displayed a RMP of ~-50 mV (Figure 3A, upper left trace). Exposure of the neurons to 10 µM of compound 34 or 35 did not alter the RMP of these neurons (Figure 3A, lower right traces and Figure 3B). As control to ensure that neurons were able to exhibit action potential firing they were exposed to 100 µM menthol (Figure 3A, right trace, and Figure 3B).

Figure 3. β-lactams derivatives 34 and 35 do not affect the membrane resting potential of sensory neurons. (A) Representatives recordings of resting membrane potential measured under current-clamp configuration. The traces show the action potentials (APs) firings from DRG neuron elicited by 100 μM Menthol (left trace) or in the presence of vehicle (0.5% DMSO) or the β-lactams derivatives (left traces). (B) Changes in the Vm in the absence and in the presence of 10 μM of of the β-lactams derivatives. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons (ns = no significance, **** = 0.0001) and given as mean ± SEM; n ≥ 40 cells from 4 cultures.

Figure 3. β-lactams derivatives 34 and 35 do not affect the membrane resting potential of sensory neurons. (A) Representatives recordings of resting membrane potential measured under current-clamp configuration. The traces show the action potentials (APs) firings from DRG neuron elicited by 100 μM Menthol (left trace) or in the presence of vehicle (0.5% DMSO) or the β-lactams derivatives (left traces). (B) Changes in the Vm in the absence and in the presence of 10 μM of of the β-lactams derivatives. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons (ns = no significance, **** = 0.0001) and given as mean ± SEM; n ≥ 40 cells from 4 cultures.

2.4. Activity in Other TRP Channels and Pain-Related Peripheral Receptors

To asess the selectivity for the TRPM8 channel, the effect of compound 34 was examined on different ion channels and receptors implcated in pain-related processes. First, some TRP channels that play a role in temperature integration and nociception were selected [31,43], including hTRPV1, hTRPV3, hTRPA1 and TRPM3. Additionally, the evaluation encompassed the hASIC3 channel, with widespread distribution within the peripheral nervous system and contributions to excitability of primary sensory neurons [44]. In each instance, the compound was subjected to evaluation at a consistent concentration of 10 µM. As outlined in Table 5, it is evident that compound 34 did not exhibit noteworthy modulatory effects on any of the assessed TRP and ASIC3 channels. Importantly, amide derivative 34 did not show activation on the TRPV1 channel, which was about 30% in the model compound 1 [38]. Compound 35 underwent evaluation as a potential antagonist for the TRPM3 channel, revealing a non-significant degree of inhibition (4.1 ± 10.8) in the Ca²⁺ influx induced by pregnenolone sulfate.

Table 5. Functional activity of the selected β-lactam 34 (10 μM) in Ca²⁺ microfluorography assays of TRPV1, TRPV3, TRPA1, TRPM3 and ASIC3 channels and percentages of inhibition of specific binding at CGRPR, CB2 and M3 receptors.

Table 5. Functional activity of the selected β-lactam 34 (10 μM) in Ca²⁺ microfluorography assays of TRPV1, TRPV3, TRPA1, TRPM3 and ASIC3 channels and percentages of inhibition of specific binding at CGRPR, CB2 and M3 receptors.

Compound	% Channel Inhibition at 10 μM					% Binding at 10 μM
	hTRPV1	hTRPV3	hTRPA1	TRPM3	ASIC3	hCGRPR	hCB2	hM3
1	19.4 ± 1.7	−6.3 ± 5.9	4.2 ± 1.1	ND	6.0 ± 0.9	−1.0 ± 4.3	7.3 ± 0.3	−4.1 ± 3.1
34	8.9 ± 2.9	17.3 ± 5.7	2.0 ± 3.4	6.9 ± 10.7	2.2 ± 9.6	−11.9 ± 5.2	7.0 ± 2.1	5.2 ± 3.2

hTRPV1: transient receptor potential vanilloid, type 1. hTRPV3: transient receptor potential vanilloid, type 3. hTRPA1: transient receptor potential ankirin, type 1. ASIC3: acid sensing ion cannel, subunit 3. hCGRPR: human calcitonin gen-related peptide receptor. hCB2: human cannabinoid receptor, subtype 2. hM3: human muscarin receptor, subtype 3. In all cases, data is from two experiments in duplicate. Ago.: assay for agonist activity. Antago.: assay for antagonist activity (see methods). Agonists used for activation: TRPV1 (Capsaicin, 30 nM), TRPV3 (2-Aminoethoxydiphenyl borate, 2-APB. 30 µM), TRPA1 (Allylisothiocyanate, 10 µM), TRPM3 (pregnenolone sulfate, 50 µM), ASIC3 (Buffer, pH 5.5). Reference antagonists: TRPV1 (Capsazepin, IC₅₀ 1.3·10⁻⁷), TRPV3 (Ruthenium red, IC₅₀ 2.5·10⁻⁷ M), TRPA1 (Ruthenium red, 10 µM), TRPM3 (isosakuranetin, 10 µM; IC₅₀ 50 nM), ASIC3 (Amiloride. 1 mM). Radioligand for hCGRPR: [125]hGCRPα, agonist hGCRPα (1 µM). Radioligand for hCB2: [3H]WIN 55212-2, agonist WIN 55212-2 (5 µM). Radioligand for hM3: [3H]4-DMAP, agonist 4-DMAP (1 µM).

Furthermore, the investigation extended to measuring the binding affinity of compound 34 to calcitonin gene-related peptide hCGRPR, cannabinoid hCB2 and muscarinic hM3 receptors, all relevant to different pain conditions [45,46,47,48,49]. As shown in Table 5, this compound show inability to displace the radioligands for hCB2, hM3, and hCGRPR receptors.

2.5. Antinociceptive Activity in a Mouse Model of Chemotherapy-Induced Cold Allodynia

It is established that treatments with the chemotherapeutic drug oxaliplatin (OXA) induce painful peripheral neuropathy, known as CIPN, which becomes more pronounced in cold conditions [50]. The TRPM8 channel has been implicated in mouse models of CIPN pain induced by OXA [20,51]. In fact, the OXA-induced peripheral neuropathy mouse model is being used for the pharmacological characterizion of TRPM8 antagonists. For this peripheral neuropathy assay, mice were subcutaneously (s.c.) injected with OXA was subcutaneously (s.c.) injected at a dose of 6 mg/kg dose on days 1, 3 and 5 to male mice, to induce incresed sensitivity to cold, which can be monitored through the acetone drop test.

Consecuently, compounds 34 and 35 were assessed for their ability to alleviate OXA-induced cold hypersensitivity after intraplantar administration (i.pl., 3, 1 and 0.1 μg). As depicted in Figure 4A,B, both compounds, 34 and 35 exhibited a dose-dependent and significant reduction the cold-induced paw licking. This effect was observed 15 min post-administration and sustained up to 60 min. These findings parallel those reported for a biphenylamide N-spiro[4.5]decan-8-yl derivative which demonstrated antiallodynic activity lasting up to 60 minutes at a 1µg dose, despite possessing higher nanomolar potency on rat TRPM8.[52] Notably, compounds 34 and 35 displayed greater potency and a longer lasting action compared to another group of TRPM8 antagonists with an imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione central scaffold.[53] The superior in vivo activity of this β–lactam family might be attributed to their distinct mode of interaction, as suggested by modeling studies.

Figure 4. Effects of compounds 34 (A) and 35 (B) on the oxaliplatin (OXP)-induced cold hypersensitivity (drop acetone test). Peripheral neuropathy was induced in male mice by injecting OXA (6 mg/kg, s.c.) on days 1, 3 and 5. Control animals were treated with vehicle. Compounds 34 (A) and 35 (B) (3 µg/i.pl., 1 µg/i.pl. and 0.1 µg/i.pl.) were administered to the OXP-treated animals, and the time-course of cold hypersensitivity was measured. Data are given ± SEM (n = 5). Statistical analysis, comparing with oxaliplatin-treated mice, was performed by two-way ANOVA followed by post hoc Bonferroni test by multiple comparisons: **** p < 0.0001.

Figure 4. Effects of compounds 34 (A) and 35 (B) on the oxaliplatin (OXP)-induced cold hypersensitivity (drop acetone test). Peripheral neuropathy was induced in male mice by injecting OXA (6 mg/kg, s.c.) on days 1, 3 and 5. Control animals were treated with vehicle. Compounds 34 (A) and 35 (B) (3 µg/i.pl., 1 µg/i.pl. and 0.1 µg/i.pl.) were administered to the OXP-treated animals, and the time-course of cold hypersensitivity was measured. Data are given ± SEM (n = 5). Statistical analysis, comparing with oxaliplatin-treated mice, was performed by two-way ANOVA followed by post hoc Bonferroni test by multiple comparisons: **** p < 0.0001.

2.6. Insights into the Mode of Interaction of Selected β–lactams with the TRPM8 Channel

To shed light on the mechanism by which β–lactam amides and monobenzyls interact with the TRPM8 channel, we select compounds 34, 35, and 37, which represent the most potent TRPM8 antagonists within their respective series. For our docking studies, we employed software tools in Yassara suite of programs, and the interactions were analyzed with Yasara and Protein-Ligand Interaction Profiler (PLIP). Docking simulations were performed on a model of rat TRPM8 channel generated from the cryoelectron microscopy (cryo-EM) structure of Ficedula albicollis TRPM8 (PDB code 6BPQ) [54].

Initially, we used the TRPM8 structure encompassing transmembrane helices, as well as the internal and extracellular loops of the indicated tetrameric channel. As outlined in Table 6, the most prominent binding solutions for the three compounds consistently aligned them within or close to the pore channel. Curiously, these calculations did not provided any solution in which compounds 34, 35, or 37 bind to the menthol binding pocket [55].

Table 6. Percentage of main docking solutions (and binding energy estimation in kcal/mol) obtained in molecular docking studies using Yasara. rTRPM8 with extracellular loops.

Table 6. Percentage of main docking solutions (and binding energy estimation in kcal/mol) obtained in molecular docking studies using Yasara. rTRPM8 with extracellular loops.

Subsite	Channel Location	34	35	37
1	Pore, external tower	20.0 (9.09)	14.4 (7.82)	28.2 (9.85)
2	Pore, high S3-S4, S6	13.8 (8.51)	16.4 (10.11)	7.4 (7.40)
3	Inner pore, S5S6, S5 loops	29.3 (9.81)	24.6 (9.40)	31.8 (7.62)
4	Pore, internal mouth	6 .0 (11.21)	7.6 (10.68)	13.9 (8.49)

Upon analyzing the most populated solutions for these compounds, we identified four primary binding subsites within the TRPM8 tetrameric protein. Progressing from the top (extracellular) to the bottom (intracellular) regions, we observed that 14 to 28% of the solutions were clustered around the tower formed by the external loops (Subsite 1), being the second subsite most populated and that of the better biding energy in the case of monobenzyl derivative 37. A second subsite (Subsite 2), involving transmembrane helices S3 and S4 of a protein subunit and S6 of an adjacent monomer, was found to be more populated for the dibenzyl amide derivatives 34 and 35 in comparison to the monobenzyl analogue 37. Subsite 3, bounded by interactions with transmembrane helices S5 and S6 from a single monomer, along with certain loops near the pore helix on the same monomer and a neighboring protein subunit, emerged as the most populated binding site for all three compounds. Subsite 4, located within the cytoplasmic pore loops, accumulated a greater number of solutions for the smaller monobenzyl derivative 37 when compared to the dibenzyl analogues 34 and 35. However, for the latter compounds, subsite 4 corresponded to the binding pocket with the better binding energy.

To investigate whether the extracellular loops in the previous model play a role in guiding the distribution of β–lactam derivatives into cavities along the pore zone, as previously hypothesized for related analogs [36,38], an additional structure of the channel was incorporated into the modeling studies (compounds 34 and 37). This alternate structure retained the transmembrane helices and inner pore loops but excluded the extracellular loops. Within this framework, many different solutions emerged, but subsites 2, 3 and 4 remained the most frequently populated solutions, with an increased preference for subsite 4 in the case of the smaller compound 37 (see SM for details). Once more, no interactions with the menthol binding site were observed.

The stabilizing interactions among the external loops of the channel (Subsite 1) and amide derivatives 34 and 35 are characterized by hydrophobic contacts with L889, V903, V919, T922, T923, F926, K937, and L939, among others, from a single protein monomer (Figure 4A). Using PLIP program, both compounds establishes a π–cation interconnection, between R890 and the Ph ring of the 4-Bn group in 34, and between K937 and one of the aromatic rings of the N-Bn₂ moiety in 35. Calculations conducted with Yasara identified some π–π interactions involving F900 and/or F926. Furthermore, both software programs indicate that the CO group of the β–lactam ring in 35 engages in a hydrogen-bond with NH/NH₂ protons of the R890 guanidino group.

Within subsite 2 (Figure 4B), amides 34 and 35 exhibit two π–π interactions involving F815 and W954 of the protein and different phenyl groups of the small-molecule. Additionally, different hydrophobic contacts are observed, mainly delimited by Y808 (TM S3), and F815, I832 and I831 (TM S4) of a monomer, and by P949 and F951 of TM S6 of an adjacent protein unit (Figure 4B).

Deeper within the pore channel, subsite 3 corresponds with the most populated docking solutions for both compounds 34 and 35. This subsite entails two transmembrane helices from a protein subunit, namely S5 and S6, and two loops positioned at the exit of the pore helix, one from the same monomer, and the other from a contiguous protein in the tetramer and its S5. At this subsite, both software programs identify an H-bond involving the α–CO of G913 residue, acting as the acceptor, while the 4-CONH amide donates its proton to the interaction (identified in both compounds, 34 and 35). In addition, several hydrophobic contacts occur among different parts of the indicated small-molecules (NBn₂, 2’-Bn, 3-Me, and ⁱBu or Bn amide group) and different protein residues. These include interactions with L909 and L915 of the S5-S6 connector, at the end of pore-helix (two subunits), and L959, V960, I962 and Y963 of transmembrane S6 (Figure 4C). Residues W877 or F874 from S5 are also implicated in the complex stabilizing interactions.

At the pore internal mouth (Subsite 4), amide compounds 34 and 35 interconnect with residues spanning all four protein units. The hydrophobic interactions are primarily enabled by Y981 (4 subunits), I985 (3), T982 (1) and M978 (1) residues. However, both compounds adopt different poses within the channel and establish a singular H-bond, between the α–CO of V983 and the 4-CONH proton in 34 (Figure 4D), while the 4-CO of the β–lactam connects with the α–NH proton of residue I985 in 35. Additionally, the aromatic side-chains of Tyr981 residues are able of engaging π–π stacking and T-edge interactions with the aromatic rings of either 2´-Bn and N-Bn₂ in 34, or with that of 4-Bn in 35.

The monobenzyl derivative 37 has binding interactions with the same four subsites as its dibenzyl analogue 34. However, the population of solutions is increased at subsites 1, 3 and 4. Residues of the protein implicated in the hydrophobic interconnections with 37 are similar to those above described for the dibenzyl compound at pockets 2-4. However, a different pose was identified at subsite 1, where Y924 residues from all four protein subunits enclose the molecules with the assistance of D920 from a single monomer. Moreover, a π–π stacking interaction is formed between one of these Y924 residues and the phenyl ring at 2´-position of 37. This compound also engages in two H-bonds, involving the 2’-NH and the CO of T982, and the CO of the β–lactam ring and the α–NH of one of the Y981 residues.

The recently resolved cryo-EM structures of TRPM8 complexed with known antagonists, like AMTB and TC-I2014, situated these compounds within a well-defined pocket, formed by the lower part of the TM S1-S4 helices and the TRP domain [56,57]. It encompasses a quite big box that includes a combination of hydrophobic and charged residues. As indicated above, our docking studies to identify potential interaction points of our β–lactams within the TRPM8 tetramer did not lead to any results directed to this precise pocket, thus suggesting a different mode of action for this series of compounds.

Figure 5. Main subsites for the interaction of compound 34 with TRPM8 channel including extracellular loops. Top and side views of the TRPM8 channels indicate the approximate localization of the docking subsites. Subsite 1 (A); Subsite 2 (B); Subsite 3 (C); Subsite 4 (D). Interactions are drawn with dashed lines and colored gray for hydrophobic, green for π-stacking, and orange for cation-pi interactions. Hydrogen bonds are drawn with continuous blue lines.

Figure 5. Main subsites for the interaction of compound 34 with TRPM8 channel including extracellular loops. Top and side views of the TRPM8 channels indicate the approximate localization of the docking subsites. Subsite 1 (A); Subsite 2 (B); Subsite 3 (C); Subsite 4 (D). Interactions are drawn with dashed lines and colored gray for hydrophobic, green for π-stacking, and orange for cation-pi interactions. Hydrogen bonds are drawn with continuous blue lines.

3. Materials and Methods

4. Conclusions

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