Dimethoxybenzohomoadamantane-based soluble epoxide hydrolase inhibitors: in vivo efficacy in a murine model of chemotherapy-induced neuropathic pain

Abstract

The soluble epoxide hydrolase (sEH) has recently emerged as a promising target for the treatment of several pain-related conditions. Herein, we report the design and synthesis of a peripherally restricted sEH inhibitor with high potency and good Drug Metabolism and Pharmacokinetics (DMPK) properties. Molecular dynamics and X-ray crystallography helped reveal the binding of these inhibitors to sEH. The selected compound showed a robust analgesic effect in a dose-dependent manner in a murine model of chemotherapy-induced neuropathic pain (CINP). Moreover, the compound also prevented the development of paclitaxel-induced neuropathic pain. Overall, these results suggest that peripheral inhibition of sEH might constitute a novel therapy to prevent and treat CINP.

Graphical Abstract

Introduction

Soluble epoxide hydrolase (sEH, EPHX2, E.C. 3.3.2.10), an enzyme downstream from the oxidases of the cytochrome P450 pathway of the arachidonic acid cascade¹, has been suggested as a pharmacological target for the treatment of inflammatory and pain-related disorders^2–4. Indeed, sEH inhibitors (sEHIs) are considered now as a promising new class of non-opioid analgesics⁵, with a representative compound, 1 (EC5026, Figure 1), currently in clinical trials for the management of neuropathic pain (NP)⁶, particularly diabetic peripheral neuropathy^7,8, a condition estimated to affect one-third of patients with diabetes mellitus and other conditions involving nerve damage⁹. Interestingly, while this work was ongoing, it was reported that EC5026 also relieved NP induced by three clinically approved chemotherapeutics (oxaliplatin, paclitaxel, and vincristine) in rats¹⁰. The antinociceptive effects of sEHI are thought to be produced (at least partially) at central levels^11,12, and it is unknown whether a peripherally restricted sEHI would be able to induce appreciable antinociception.

Figure 1.

Structures and IC₅₀ values in the hsEH of clinical candidate EC5026, 1, and compounds 2–5. All compounds were evaluated under the same conditions. The blue circle shows the RHS of 2.

Recently, based on our observation that the lipophilic cavity of sEH is flexible enough to accommodate large polycyclic units¹³, we rationally designed a new family of potent urea-based sEHI featuring a benzohomoadamantane scaffold^14–16, a polycyclic unit seldom used in medicinal chemistry^17,18. Several members of this family (e.g. compounds 2–5, Figure 1) were endowed with low nanomolar or even subnanomolar potencies, and a selected candidate, compound 5, presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain¹⁶.

In our previous articles, the structure–activity relationship leading to 5 was thoroughly explored, with particular focus on alternatives to the urea main pharmacophore (e.g. amides)^14,15, different replacements for the nitrogen atom of the right-hand side (RHS) of the urea, and alternative groups at the C-9 position of the benzohomoadamantane core^14–16.

Herein, we report further medicinal chemistry around this family of sEHI¹⁹, studying the effect of introducing electron-withdrawing and electron-donating groups in the benzene ring of the benzohomoadamantane scaffold. Several compounds were synthesised and fully characterised. Interestingly, a combination of molecular dynamics (MD) calculations and X-ray crystallography revealed that this new set of inhibitors adopt a different arrangement in the cavity of the enzyme than the previously observed for the benzene-unsubstituted analogues. After a screening cascade, a peripherally restricted candidate, endowed with high potency at the human, murine and rat sEH and excellent DMPK properties, was subsequently studied in a murine model of chemotherapy-induced neuropathic pain (CINP).

Results and discussion

Design and synthesis of new sEHI

For synthesising the new sEHI, amines 6a–f, previously described by our group, were used as starting materials (Figure 2)^17,18. Taking into account the potency and the DMPK properties of the sEHI derived from these amines (see below), a new amine, 6g, was synthesised from 2,3-dimethoxy-5,6,8,9-tetrahydro-7H-5,9-propanobenzo[7]annulene-7,11-dione, 718, following the route shown in Scheme 1 (see Supplementary Information for further details).

Figure 2.

Benzohomoadamantane amines 6a–g used in this work^17,18.

Scheme 1.

Synthesis of amine 6g. Reagents and conditions: (i) Ph₃PCH₃ I, NaH, anh. DMSO, 90 °C, overnight, 24% yield. (ii) ClCH₂CN, conc. H₂SO₄, AcOH, 0 °C to room temperature, overnight, 39%. (iii) Diethylaminosulfur trifluoride, DCM, −30 °C to room temperature, overnight, 81% yield. (iv) (1) Thiourea, glacial AcOH, abs. Ethanol, reflux, overnight; (2) HCl/Et₂O, 55% overall yield. See the ‘Experimental’ section and Supplementary Information for further details.

The synthesis of the novel urea-based sEHI was straightforward. It involved the reaction of the benzohomoadamantane amines 6a–g with triphosgene to obtain the corresponding isocyanates 11a–g, followed by the addition of the required substituted aminopiperidine of general structure 12 to form the final ureas 13–17 and 19–21. Additionally, catalytic hydrogenation of the nitro group of urea 17 furnished aniline derivative 18 (Scheme 2).

Scheme 2.

Synthesis of the new sEHI. Reagents and conditions: (i) Triphosgene, NaHCO₃, DCM, 30 min; (ii) DCM, overnight; (iii) H₂ 1 atm, PtO₂, ethanol, room temperature, 8 days. See the ‘Experimental’ section and Supplementary Information for further details.

The new compounds were fully characterised through their spectroscopic data and elemental analyses or HPLC/MS. Before starting the synthetic work, we evaluated all the targeted compounds as Pan Assay Interference Compounds (PAINS) using SwissADME and FAFDrugs4 web tools^20,21. None of them gave positive as PAINS.

sEH inhibition and DMPK assays

Some time ago, we reported the synthesis of urea 2, unsubstituted in the benzene ring and featuring a methyl group at C-9 and an acetyl group in the RHS of the molecule (Figure 1)¹⁴. This compound presented potent inhibitory activities against the human and murine enzymes and moderate experimental aqueous solubility (38 µM), but unacceptable lack of stability in human and murine microsomes (Table 1)^14,16. To improve the microsomal stability and the solubility of 2, herein the introduction of a series of electron-donating and electron-withdrawing substituents on the aromatic ring of the benzohomoadamantane moiety was explored. Initially, we synthesised mono-substituted derivatives 13 (with a fluorine atom at C-1), 14 (with a fluorine atom at C-2), 15 (with a methoxy group at C-2), 16 (with an acetyl group at C-2), 17 (with a nitro group at C-2) and 18 (with an amino group at C-2), and the di-substituted derivative 19, featuring methoxy groups at C-2 and C-3. Later, considering the DMPK data obtained for these compounds (see below), two additional ureas, 20 and 21, were synthesised. Gratifyingly, the introduction of this plethora of different substituents in the benzohomoadamantane scaffold was not deleterious for the activity. Indeed, some derivatives were more potent, particularly for the human enzyme, than the unsubstituted urea 2, with several compounds in the low nanomolar or even in the subnanomolar range (Table 1)²². Even the less potent compound, aniline 18, which exhibited IC₅₀ values of 25 and 17.1 nM in the human and murine enzymes, respectively, was an acceptable inhibitor. Taking into account that the sEH presents a hydrophobic pocket, it is reasonable that the addition of the highly polar amino group in the aromatic ring produced a drop in terms of potency (see below the ‘In silico study: molecular basis of benzohomoadamantane/piperidine-based ureas as sEHI’ section for further details). Considering its poor activity and our concerns regarding the potential toxicity of the aniline group, 18 was not further evaluated.

Table 1.

IC₅₀ in human and murine sEH, solubility, permeability (PAMPA-BBB), microsomal stability, inhibition of pooled human cytochromes P450 enzymes and hERG channel and cytotoxicity of novel sEHI.

Compound	sEH IC50 (nM)a		Solubilityb (µM)	Microsomal stabilityc		PAMPA-BBB	Cytochrome inhibitiond						hERG channel inhibitionf
	Human	Murine		Human	Mouse		CYP 1A2	CYP 2C9	CYP 2C19	CYP 2D6	CYP 3A4 (DBF)e	CYP 3A4 (BFC)e
2	4.0	6.0	38	1	0	CNS +	9 ± 1	29 ± 3	19 ± 5	34 ± 1	8 ± 3	19 ± 3	NDg
13	0.9	0.8	45	16	1	CNS +	18 ± 4	6 ± 3	23 ± 1	1 ± 2	9 ± 3	26 ± 1	ND
14	0.9	0.6	28	22	4	CNS +	18 ± 3	12 ± 2	40 ± 1	5 ± 2	13 ± 3	1 ± 2	ND
15	1.2	1.7	89	33	32	CNS ±	13 ± 2	12 ± 3	48 ± 1	8 ± 2	12 ± 4	57 ± 1	ND
16	4.1	6.7	86	92	70	CNS −	11 ± 1	9 ± 3	29 ± 1	7 ± 2	3 ± 2	1 ± 1	ND
17	4.5	4.5	35	100	27	CNS ±	12 ± 3	20 ± 4	36 ± 1	8 ± 3	10 ± 1	2 ± 1	ND
18	25.0	17.1	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
19	1.3	2.2	90	100	70	CNS −	24 ± 1	8 ± 1	25 ± 1	2 ± 2	15 ± 4	18 ± 3	ND
20	0.4	1.9	68	80	96	CNS ±	12 ± 4	22 ± 2	37 ± 3	36 ± 5	43 ± 1	16 ± 5	11 ± 2
21	0.6	2.9	92	100	86	CNS −	19 ± 4	8 ± 1	19 ± 1	15 ± 4	1 ± 1	11 ± 1	9 ± 1

Cytotoxicity was tested by PI staining or by MTT assay after 24h incubation in SH-SY5Y cells; both assays gave CC₅₀ > 100 µM for all the compounds.

^a Reported IC₅₀ values are the average of three replicates. The fluorescent assay as performed here has a standard error between 10% and 20%, suggesting that differences of twofold or greater are significant. Because of the limitations of the assay, it is difficult to distinguish among potencies <0.5 nM²².

^b Solubility measured in a 1% DMSO: 99% Phosphate Buffered Saline buffer solution.

^c Percentage of remaining compound after 60 min of incubation with pooled human and mouse microsomes in the presence of Nicotinamide Adenine Dinucleotide Phosphate Hydrogen at 37 °C.

^d The percentage of cytochrome inhibition was tested at 10 µM.

^e For the study of CYP3A4, two different substrates were used dibenzylfluorescein (DBF) and 7-benzyloxytrifluoromethylcoumarin (BFC).

^f Percentage of inhibition at 10 µM.

^g ND: not determined. See the ‘Experimental’ section for further details.

Next, solubility, permeability through the blood-brain barrier (BBB), cytotoxicity, cytochrome inhibition, and microsomal stability were experimentally measured for compounds 13–17 (Table 1). While compounds 14 and 17 exhibited limited solubility, with values lower than 40 µM, the other compounds displayed good to excellent solubility values. Additionally, the compounds were further tested for predicted brain permeation in the widely used in vitro parallel artificial membrane permeability assay-blood-brain barrier (PAMPA-BBB) model (see ‘Experimental’ section and Table S7 for details)²³. As parent compound 2, new ureas 13 and 14 showed CNS +, proving their potential capacity to reach CNS, whereas 15 and 17 presented uncertain BBB permeation (CNS ±). More polar 16 and 19 showed predicted low BBB permeation. Interestingly, non-BBB penetrant sEHI candidates are highly attractive for the treatment of peripheral pain to avoid unwanted central side effects.

Using the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) assays, the novel sEHI showed to be non-cytotoxic in SH-SY5Y cells at the highest concentration tested (100 µM). Moreover, inhibition of five cytochrome P450 enzymes (1A2, 2C9, 2C19, 2D6, and 3A4) was measured, as some isoforms, such as CYPs 2C19 and 2C9, are main producers of epoxyeicosatrienoic acids (EETs), the substrates of the sEH²⁴. Gratifyingly, at 10 µM, none of the compounds significantly inhibit these subfamilies of cytochromes (Table 1).

Finally, the microsomal stability of 13–17 and 19 in human and murine microsomes was explored. All the compounds showed better results than 2, although the values of 13, 14, and 15 were still unacceptable. Three compounds, 16, 17, and 19, were endowed with very high human microsomal stabilities, although only 16 and 19 also presented high values in the murine microsomes (Table 1).

Of relevance, it is worth mentioning that while 2 and 19 are non-chiral, urea 16 and the other mono-substituted analogues 13–18 are chiral molecules, that were synthesised and evaluated as racemic mixtures. For this reason, we selected achiral dimethoxy urea 19 over chiral 16 for further work.

Previously, the introduction of a halogen atom at the C-9 position of the unsubstituted benzohomoadamantane scaffold was found to increase the microsomal stability^14,16. Also, it is known that the acyl chain of piperidine-based sEHI is a suitable position for metabolism²⁵. Therefore, we decided to replace the methyl group of C-9 and the acetyl group of 19 by a fluorine atom and a tetrahydro-2H-pyran-4-carbonyl unit, respectively. Thus, we synthesised compound 21 and, for comparison, 20 (Scheme 2). Gratifyingly, both compounds were endowed with excellent potencies and very good DMPK properties (see Table 1). Additionally, hERG inhibition assays were also performed to characterise them further. None of them significantly inhibit hERG at 10 µM.

Overall, after performing the above-mentioned screening cascade, compound 21 emerged as a very promising candidate. It exhibited excellent inhibitory activities against the human (IC₅₀ = 0.6 nM) and murine (IC₅₀ = 2.9 nM) enzymes, high metabolic stability, and excellent solubility, and it was not cytotoxic. Furthermore, it neither significantly inhibited the cytochromes tested nor hERG.

Highly lipophilic scaffolds in small molecules such as adamantane or fluorene could emulate the characteristics of misfolded proteins, thereby leading to the protein degradation mediated by chaperones or directly recruited by the proteasome²⁶. Considering the hydrophobicity of the benzohomoadamantane scaffold and to ensure the inhibition mechanism of action, the protein levels of human soluble epoxide hydrolase (hsEH) were monitored after 4 h treatment with compound 21 at different concentrations in the HEK293 cell line. No significant degradation was observed in a 10-fold titration (10 nM to 10 µM) of compound 21 (Figure S5), demonstrating that this molecule likely has a single mechanism of action.

In silico study: molecular basis of benzohomoadamantane/piperidine-based ureas as sEHI

Incorporating electron-withdrawing and -donating groups at the C-2 and C-3 positions of the benzohomoadamantane scaffold can impact the binding mode of sEHI compared to previously reported unsubstituted adamantane or benzohomoadamantane urea-based inhibitors. The L-shaped active site of sEH consists of the left-hand side (LHS) and the RHS hydrophobic pockets, which are bridged by a polar narrow channel (Figure 3(A)). The central channel is defined by the side chains of residues D335, Y383, and Y466, responsible for reacting to epoxide substrates and recognising the urea moiety of sEHI. Available inhibitor-bound sEH X-ray structures show that bulky hydrophobic groups directly connected to urea can occupy either the RHS (e.g. PDB 4Y2X or 5ALZ) or the LHS (e.g. PDB 3WKE). Likewise, long chains with polar groups at their ends can also extend through the length of both sites with the polar groups located near the entrances of the RHS and LHS pockets (e.g. PDB 5AM3 or 6AUM). MD simulations showed that the sEH pocket presents large fluctuations in volume (70–700 ų) in the apo state and high adaptability and restricted flexibility when the inhibitors are bound, indicating that there is enough space to fit bulky substituents such as benzohomoadamantane within both LHS and RHS (Figure 3(A))¹⁴. Considering the plasticity of the sEH active site to specifically accommodate each inhibitor, rigid molecular docking protocols typically struggle to predict the correct binding mode of new sEHI^27,28. Unravelling the preferred binding mode of benzohomoadamantane-based sEHI is key for further development.

Figure 3.

(A) hsEH active site regions and volume. (B) BenzoH-RHS-1 binding pose. Overlay of most populated conformations of 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. The benzohomoadamantane moiety occupies the RHS pocket, while the piperidine group is placed in the LHS pocket. The central urea unit establishes hydrogen bonds with Asp335, Tyr466, and Tyr383. Water molecules are depicted as spheres. Relative binding affinity calculated with respect of BenzoH-RHS-1. (C) BenzoH-LHS-1 binding pose. Overlay of most populated conformations of 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. The benzohomoadamantane moiety occupies the LHS pocket while the piperidine group is placed in the RHS pocket. The central urea unit establishes hydrogen bonds with Asp335, Tyr466, and Tyr383. Water molecules are depicted as spheres. (D) BenzoH-RHS-2 binding pose. Overlay of most populated conformations of compound 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. The benzohomoadamantane moiety occupies the RHS pocket, while the piperidine group is placed in the LHS pocket. The central urea unit establishes hydrogen bonds with Asp335, Tyr466, and Tyr383. (E) Rotation and orientation of the benzohomoadamantane moiety represented by the dihedral angle: C (urea moiety), N (urea moiety), C (benzohomoadamantane), C (benzohomoadamantane). Histogram plot of the dihedral angle that describes the rotation of the benzohomoadamantane moiety along the MD simulations of BenzoH-RHS-1 (blue), BenzoH-LHS-1 (grey), and BenzoH-RHS-2 (purple). (F) Water occupation in the sEH active site. Representation of the normalised kernel density plot of the water distribution in the vicinity of methoxy groups (<4 Å) along the MD simulations of BenzoH-RHS-1 (blue), BenzoH-LHS-1 (grey), and BenzoH-RHS-2 (purple). PDB 6I5E was used as starting point for molecular modelling.

Here, we studied the mechanism of binding of 17, 18, 20, and 21 through extensive MD simulations and MM/GBSA calculations. To simplify the discussion, we will focus on the results obtained for 21, the most promising compound, while the complete results for 17, 18, and 20 can be found in the Supplementary Information (see Table S3 and Figures S6–S8). To explore the possible binding modes of selected benzohomoadamantane urea-based sEHI, the structure of sEH C-terminal domain (CTD) corresponding to apo sEH (PDB 6I5E) was used²⁹. Using this structure as a reference to place the compounds in the binding pocket ensures that the active site of sEH is not preorganised towards any specific inhibitor. Since the interactions of the urea moiety with sEH active site residues are well-known, two main orientations of compound 21 in the sEH pocket were explored while keeping the symmetric urea moiety bound to D335, Y383, and Y466 using t-AUCB (PDB 5AM3) as a reference. One orientation presents the benzohomoadamantane scaffold in the LHS and the piperidine moiety in the RHS pocket, while the other corresponds to the opposite orientation with the benzohomoadamantane in the RHS and piperidine in the LHS. For each orientation, a series of conformations were generated by setting the dihedral angle that connects the urea moiety to the benzohomoadamantane ring to different values (varying the angle in 60° spans). From these 12 conformations (6 for each orientation), we submitted 3 replicas of 500 ns of MD simulations. Due to the active site constraints, the 12 sets of MD simulations evolved to 3 stable binding poses (see Figure 3(B–D)). Two with the benzohomoadamantane in the RHS (BenzoH-RHS-1 and BenzoH-RHS-2 pose) and one in the LHS (BenzoH-LHS-1 pose), with average dihedral angles of 179.8°, 57.4°, and 65.5° respectively (Figure 3(E)). From these three binding modes, we ran five replicas of 1000 ns of MD simulations that were collectively analysed to retrieve the binding affinity and flexibility of the inhibitor, as well as active site rearrangements and water interactions.

The binding affinity of each binding pose was estimated with MM/GBSA calculations. BenzoH-RHS-1 is predicted as the most stable binding pose followed by BenzoH-LHS-1 (ΔΔG = 1.7 kcal/mol) and BenzoH-RHS-2 (ΔΔG = 6.7 kcal/mol). In all binding poses, the benzohomoadamantane remains quite rigid, while the piperidine-pyran moiety is significantly more flexible, exploring multiple conformations in the binding pocket. This is translated with some fluctuations in the active site volume when 21 is bound, indicating the plasticity of the sEH active site (see Figure S9). In the BenzoH-RHS-1 pose, the benzene of the benzohomoadamantane scaffold is oriented towards the entrance of the RHS pocket (Figure 3(B)). Indeed, the methoxy groups in C-2 and C-3 positions are near the solvent-exposed RHS entrance defined by S415 and F497 residues, while the fluorine in C-9 is oriented towards the inner hydrophobic RHS pocket. In BenzoH-LHS-1, the methoxy groups of the benzohomoadamantane scaffold are oriented towards the LHS entrance (Figure 3(C)), while in the less stable BenzoH-RHS-1 pose, the methoxy groups are located in the inner hydrophobic cavity of the RHS (Figure 3(D)). By monitoring the presence of water molecules around the methoxy groups, we obtained that the average number of water molecules is higher in BenzoH-RHS-1 followed by BenzoH-LHS-1 and BenzoH-RHS-2, indicating that in the most plausible binding pose the methoxy groups are surrounded and stabilised by the water molecules located in the RHS pocket entrance (Figure 3(B,F)).

The preferred orientation for 21 observed in MD simulations contrasts with previous results obtained for unsubstituted benzenes (e.g. 2 and 5) where the hydrophobic benzohomoadamantane moiety was predicted to occupy the LHS while the long chains with polar groups at their ends extended through the RHS¹⁴. These observations are in line with the higher hydrophobic character of unsubstituted benzohomoadamantane with respect to bulkier and polar methoxy-based compound 21. In terms of the preferred orientation for 17, 18, and 20 compounds, a similar trend is observed for the highly active compound 20 (ΔΔG = 2.0 kcal/mol for BenzoH_LHS-1), while both orientations present nearly equal binding affinity in mono-substituted 17 (ΔΔG = 0.4 kcal/mol for BenzoH_LHS-1) and 18 (ΔΔG = −0.7 kcal/mol for BenzoH_LHS-1) compounds. The presence of water molecules near the amino group of compound 18 is significantly enhanced in comparison to other compounds 17, 20, and 21 (see Figures S6–S8). The access of water molecules in the hydrophobic pocket may weaken the interaction with active site residues, which is in line with the significantly higher IC₅₀ of 18.

Overall, MD simulations suggest that bulkier and polar methoxy groups show preference towards the entrance of the RHS pocket. Since the predicted binding affinities between the two most stable binding poses are in close proximity, X-ray crystallography experiments were undertaken to confirm the exact orientation of the highly active methoxy-substituted benzohomoadamantane compounds and validate the computational protocol used. This harbours important information for further computer-aided rational development of substituted benzohomoadamantane scaffolds.

X-ray crystallography

Considering that the abovementioned MD simulations did not show significant changes in the binding mode of 20 and 21 and taking into account the limited supply of both compounds, 21 was used for the in vivo studies (see below), whilst the crystal structure of 20 bound to hsEH CTD was solved (Table S4, PDB ID: 9F1A). The complex crystallised in space group I2, with two hsEH CTD molecules in the asymmetric unit forming a crystallographic homodimer, analogous to the previously solved crystal structure of the apo hsEH CTD (Figure 4(A))²⁹. Superposition of the bound structure with the apo hsEH CTD (PDB id: 6I5E) gave an overall backbone RMSD of 0.78 Å, indicating similarity of the apo and bound structures. A local backbone RMSD (for residues within 5 Å of the catalytic pocket) of 0.92 Å suggests minor changes in and around the catalytic site, particularly affecting the Ca atoms of functional residues (epoxide positioners Y383 and Y466, and catalytic triad D335, D496, H524), as well as the side chains of M339 and M419. These observations are consistent with the presence of 20 within the active site of the enzyme.

Figure 4.

X-ray crystal structures of 20 in complex with hsEH CTD (9F1A). (A) Crystallographic symmetry. The crystallographic homodimeric structure (comprising chain A and chain B) is shown as cartoon with the catalytic pocket being presented as surface (cyan). The 20 ligand is shown as sticks (orange). (B–D) Interactions of 20 with hsEH CTD in conformation 1 (B), 2 (C), and 3 (D) on chain A. Residues involved in hydrogen bonding in all conformations are shown in cyan, while residues participating in hydrophobic or van der Waals contacts in all conformations are coloured in purple. Residues stabilising specific conformations (1, 2, or 3) are represented with green sticks. Hydrogen bonds are displayed as yellow dotted lines. (E,F) Superpositions of different conformations of ligand 20 in chain A (E) and chain B (F). The two epoxide positioners Y383 and Y466 and the catalytic triad residue D335 are in white. Ligand conformations 1, 2, and 3 are coloured in pink, magenta, and brown, respectively.

The compound 20 was found to adopt a conformation in the bound state which recapitulates the binding mode of other dialkylurea inhibitors^30,31 sharing a similar array of hydrogen bond interactions: the oxygen of the 20 urea group accepts hydrogen bonds from the side chains of the two epoxide positioners Y383 and Y466, whilst the urea NH groups donate H-bonds to the catalytic triad residue D335 (Figure 4(B–D)). Clear electron density defines the location and conformation of the bulky benzohomoadamantane scaffold of 20 occupying the F267 pocket (RHS). This moiety establishes van der Waals contacts with protein residues of a hydrophobic surface delineated by F267, F387, L408, M419 and V498. The dimethoxybenzene groups are involved in van der Waals with H524 and W525.

The substituted acylpiperidine end of 20 is accommodated within the W336 niche (LHS) (Figure 4(B–E)). Interestingly, and in excellent agreement with the abovementioned MD calculations that predict significant flexibility for the piperidine-pyran unit, the crystal structure captured three different conformations of this moiety, specifically Conformation 1, 2, and 3 in chain A of the crystallographic homodimer and Conformation 1 and 2 in chain B (Figure 4(E,F)). This suggests a certain degree of flexibility of the bound compound at this end. In all these conformations hydrophobic and van der Waals contacts with W336, F381 and M339 side chains are present (Figure 4(B–D), Table S5), although distinct interacting surfaces participate in the positioning of the tetrahydropyran ring in the alternative orientations, specifically: T360, P361, L499, M503, F381 and M339 in conformation 1, F381 in conformation 2 and W336, M339, and Y466 in conformation 3 (Figure 4(B–D), Table S5). A possible H-bond between the oxygen of the tetrahydropyran with Q384 could also be observed in conformation 2 (Figure 4(C), Table S5). This pattern of protein-ligand contacts agrees with the highest observed ligand occupancy for conformation 1 compared to conformation 2 and 3 (Table S5), possibly suggesting that it could be the most populated binding mode for 20. Interestingly, the carboxyl moiety of acylpiperidine fails to engage in interactions with the protein, probably contributing to the observed fluctuations of this part of the compound in the catalytic pocket. These multiple orientations suggest that a crystal structure may not be able to capture all the possible binding modes of 20, endorsing the need for a computational complementary approach (as for above). Interestingly, and in agreement with the aforementioned MD calculations, 20 appears to bind to the protein in a different orientation compared to another urea-based inhibitor, t-AUCB (PDB id: 3WKE) whose adamantane moiety is lodged in the W336 niche (LHS). In the crystals analysed, no evidence of the opposite binding conformation for 20 could be observed.

Pharmacokinetic study of compound 21

Compound 21, endowed with good inhibitory activity on sEH, very good solubility, and excellent safety profile, was selected for in vivo studies. This compound, predicted to be peripherally restricted according to the in vitro PAMPA-BBB assay, might potentially allow us to determine the effect of selective peripheral sEH inhibition in a pain model for the first time. Firstly, a study was conducted to determine the pharmacokinetic profile in the plasma and brain of compound 21 when administered by a subcutaneous route at a single dose of 5 mg/kg. The compound was analysed in the brain and plasma (time 0.08–18 h after dosing). As shown in Table 2, plasma absorption of 21 is good, reaching C_max (1.6 µg/mL) at 2 h after dosing. The compound disappeared from the plasma progressively, and the half-life in the elimination phase (HL_β) was calculated to be around 1.0 h. The narrow differences in AUC₀^last and AUC₀^inf showed complete exposure and good bioavailability. Interestingly, the compound was undetectable in all brain samples collected at multiple time points, making it impossible to calculate the brain-to-plasma ratio. As 21 demonstrated significant absorption after subcutaneous administration, it was subsequently evaluated in in vivo efficacy studies.

Table 2.

Plasma pharmacokinetic parameters in male CD1 mice for compound 21 after 5 mg/kg single dose subcutaneous administration.^a

Compound	21
Dose	5 mg/kg
HLβ (h)	1.0
Tmax (h)	2.0
Cmax (µg/mL)	1.6
AUClast (µg * h/mL)	5.5
AUCinf (µg * h/mL)	5.6

^a See ‘Experimental’ section and Table S6 and Figure S10 in the Supplementary Information.

In vivo efficacy studies

CINP is a severe side effect of several anticancer agents, such as oxaliplatin, cisplatin, carboplatin, and paclitaxel^32,33. The prevalence of CIPN is high, affecting up to 50% of patients at standard doses and nearly all patients at high doses, it is the primary dose-limiting factor of several chemotherapy treatments and can last even after stopping the treatment, compromising the probability of survival of cancer patients. Although several analgesics are prescribed to treat painful CIPN, they are either ineffective (Non-Steroidal Anti-Inflammatory Drugs, NSAIDs) or endowed with severe side effects (narcotics, gabapentinoids). Therefore, there are no current therapies that offer adequate relief for CINP^34–36.

The sensitivity to mechanical stimulation in the paw of mice was tested 10 days after treatment with paclitaxel or its vehicle. Mice treated with the vehicle of the antineoplastic did not alter their threshold force for paw withdrawal. However, animals treated with paclitaxel showed a statistically significant reduction in their mechanical threshold, denoting the presence of tactile allodynia (Figure 5(A)). The subcutaneous administration (s.c.) of 21 (2.5 and 5 mg/kg), a peripherally restricted sEHI, dose-dependently and fully reversed paclitaxel-induced tactile allodynia, as mechanical threshold increased up to values equivalent to those obtained in mice treated with the vehicle of paclitaxel (Figure 5(A)). In contrast, animals treated with the solvent of 21, 5% dimethyl sulfoxide (DMSO) in saline (s.c.), did not significantly modify paclitaxel-induced mechanical allodynia during the 150 min test period (Figure 5(A)). We also tested the effects of the oral (p.o.) administration of 21 (5 and 10 mg/kg), and found that it also dose-dependently and fully reversed paclitaxel-induced tactile allodynia, increasing mechanical threshold up to values indistinguishable to those from mice treated with the vehicle of the antineoplastic, whereas neuropathic animals treated p.o. with the solvent of 21 did not show alterations in mechanical threshold, which remained low during the test period (Figure S11). Therefore, our results suggest that 21 is fully compatible with oral administration to achieve full antineuropathic effects.

Figure 5.

Effect of the subcutaneous (s.c.) compound 21 in paclitaxel (PTX)-induced neuropathic pain. Threshold force for paw withdrawal (g) was tested as a measure of PTX-induced sensory hypersensitivity. Baseline sensitivity to mechanical stimulation was evaluated (PRE), and then animals were treated once daily for 5 days with PTX (2 mg/kg, intraperitoneal) or its vehicle (Vh). (A) Time-course of the effect of a single s.c. administration of 21 (2.5 and 5 mg/kg) or its solvent (5% DMSO), 10 days after the first PTX administration (n = 8 animals in all groups except in the group treated with PTX and 5% DMSO, which had n = 10). (B) Effect of the administration of 21 (5 mg/kg, s.c.) or gabapentin (GBP) associated with MSPPOH (20 mg/kg, s.c.) or their solvent (5% DMSO) at day 10 after PTX (n = 8, 10, 8, 8, 10, 8, 8 from left to right in the bars represented in the figure). (C) Time-course of the effect of PTX associated with 21 (5 mg/kg, s.c.) or its solvent 30 min before each PTX administration (n = 8 animals in each group). (A–C) Each point or bar and vertical line represents the mean ± SEM of the values obtained in the mice tested. Statistically significant differences between values from animals treated with PTX vs Vh: *p < 0.05; and 21 vs 5% DMSO: ^#p < 0.05. (B) 21 associated with MS-PPOH or its solvent: ^†p < 0.05. (A,C) Two-way repeated measures ANOVA. (B) One-way ANOVA. Tukey post hoc test was used in all cases.

We also tested the effects of gabapentin, a first-line treatment for neuropathic pain (PMID: 27345098), as a control antineuropathic drug in mice treated with paclitaxel. Administration of gabapentin (10–20 mg/kg, s.c.) also induced a dose-dependent and full reversion of tactile allodynia, reaching the maximum effect from 45 to 90 min after gabapentin administration (as shown in Figure S12). It is worth noting that the dose of gabapentin needed to achieve a maximum antiallodynic effect (20 mg/kg) was higher than the doses used of 21 in the experiments commented above.

We then studied the effect of N-methanesulfonyl-6–(2-proparyloxyphenyl)hexanamide (MS-PPOH), an inhibitor of microsomal CYP450s which is responsible for the production of EETs³⁷. Treatment with MS-PPOH (20 mg/kg, s.c.) did not alter paclitaxel-induced tactile allodynia (Figure 5(B)), suggesting that production of EETs in neuropathic animals is not enough to alter the mechanical threshold. We then studied the effects of the association of 21 with MS-PPOH. Experiments were performed at the peak effect of 21 (90 min) and at the dose able to fully reverse neuropathic hypersensitivity (5 mg/kg). Treatment with MS-PPOH fully abolished the effect of compound 21 (Figure 5(B)), indicating that the antiallodynic effect of 21 was mediated by sEH inhibition leading to accumulation of EETs. Importantly, MS-PPOH was completely unable to modify the antiallodynic effect induced by gabapentin (20 mg/kg, s.c.) (Figure 5(B)), which is known to act through the α₂δ subunit of voltage-gated Ca²⁺ channels and not through sEH inhibition (PMID: 27345098). These findings support the selectivity of the pharmacological strategies used. Altogether, our results suggest that peripheral inhibition of sEH by 21 is able to fully reverse paclitaxel-induced neuropathic pain once it has been developed.

The effect of peripheral sEH inhibition on the development of paclitaxel-induced neuropathic pain was also studied. The animals received a s.c. injection of 21 (or 5% DMSO in saline for the control group) 30 min before each dose of paclitaxel, and tested allodynia on several posttreatment days. Animals treated with paclitaxel and 5% DMSO showed a marked tactile allodynia which lasted for 14 days, and mechanical threshold came back to normal values (i.e. threshold values in mice treated with the vehicle of paclitaxel) 21 days after the first administration of paclitaxel (Figure 5(C)). Mice treated with compound 21 showed no signs of sensory hypersensitivity at any time point tested (Figure 5(C)). This is important, since although the onset of nerve insult for CINP is known (i.e. the administration of the antineoplastic), no agents can be currently recommended for the prevention of CINP³⁸. According to our results, peripheral inhibition of sEH might constitute a novel therapy to prevent CINP. Paclitaxel is known to induce peripheral neuroinflammation, a process which participates in neuropathic pain development³⁹. Considering the known participation of sEH in the inflammatory response (as previously commented in the ‘Introduction’ section), it could be hypothesised that a decrease in paclitaxel-induced peripheral neuroinflammation could account for the antineuropathic effects induced by peripheral sEH inhibition seen here.

Conclusions

Several studies have identified sEH as a promising target for several diseases, particularly inflammatory and pain-related disorders. Herein we report that the introduction of two methoxy groups in the benzohomoadamantane scaffold led to very potent sEHI with improved DMPK properties. Further in vitro profiling of these new compounds allowed to select a promising candidate for in vivo efficacy studies. MD calculations and X-ray crystallography agree that the benzohomoadamantane unit of these inhibitors is oriented towards the entrance of the RHS pocket, whilst significant flexibility is exhibited by the piperidine-pyran unit in the LHS W336 niche. Compound 21, which is a peripherally restricted sEHI, was tested in paclitaxel-induced neuropathic pain in mice, as a model of CINP, revealing its robust analgesic effect^10,40. The results suggest that targeting peripheral sEH might be useful for the treatment or prevention of CINP. The clinical utility of peripherally restricted sEHI may depend on the pathophysiological context and the target tissues involved. Thus, our compound may not be suitable for all indications under investigation for sEH inhibition. However, in the case of CINP, our results show that peripheral inhibition is sufficient to produce a therapeutic benefit. Of note, sedation or abuse potential limit the use of some of the current centrally-penetrant analgesics (e.g. opioid drugs, gabapentinoids). Considering that peripherally restricted analgesics are expected to lack potentially relevant centrally induced side effects, we consider that our finding with the sEHI herein disclosed are important and could pave the way for novel efficacious and safer analgesic compounds.

Experimental

Chemistry: general methods¹⁹

General chemistry methods were previously reported^16,19. See also supplementary information for further details.

1-(1-Acetylpiperidin-4-yl)-3–(1-fluoro-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (13)

To a solution of 1-fluoro-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride, 6a (150 mg, 0.53 mmol), in dichloromethane (DCM) (3 mL) saturated aqueous NaHCO₃ solution (3 mL) and triphosgene (58 mg, 0.20 mmol) were added. The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (3 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added 1-(4-aminopiperidin-1-yl)ethan-1-one, 12a, (91 mg, 0.64 mmol). The reaction mixture was stirred at room temperature overnight and the solvent was evaporated under vacuum to obtain a yellow oil (320 mg). Column chromatography (SiO₂, DCM/methanol mixtures) gave 1-(1-acetylpiperidin-4-yl)-3–(1-fluoro-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea, 13 (160 mg, 73% yield), as a white solid, mp 122–123 °C. IR (NaCl disk): 3351, 2944, 2918, 2861, 1642, 1618, 1555, 1462, 1362, 1321, 1238, 1137, 1066, 976, 885, 798, 749. cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 0.90 (s, 3 H, C9–CH₃), 1.18 [dq, J = 12.0 Hz, J′ = 4.0 Hz, 2 H, 3′(5′)-H_ax], 1.42–1.54 (complex signal, 2 H, 10-H_ax, 13-H_ax), 1.59–1.67 (complex signal, 10-H_eq, 13-H_eq), 1.71 (d, J = 11.6 Hz, 1 H, 8-H_a), 1.77–1.89 (complex signal, 2 H, 3′-H_eq or 5′-H_eq, 8-H_b), 1.89–2.05 (complex signal, 4 H, 3′-H_eq or 5′-H_eq, 6-H_ax, 12-H_ax, 6-H_eq or 12-H_eq), 2.06 (s, 3 H, COCH₃), 2.14 (m, 1 H, 12-H_eq or 6-H_eq), 2.72 (m, 1 H, 2′-H_ax or 6′-H_ax), 3.05–3.16 (complex signal, 2 H, 6′-H_ax or 2′-H_ax, 11-H, 3.64–3.78 (complex signal, 3 H, 4′-H, 6′-H_eq or 2′-H_eq, 5-H), 4.41 (dm, J = 12.8 Hz, 1 H, 2′-H_eq or 6′-H_eq), 4.66–4.71 (complex signal, 2 H, C4′–NH, C7–NH), 6.79–6.86 (complex signal, 2 H, 2-H, 4-H), 6.98 (td, J = 8.0 Hz, J′ = 5.6 Hz, 1 H, 3-H). ¹³C-NMR (100.5 MHz, CDCl₃) δ: 21.4 (CH₃, COCH₃), 28.5 (C, C9), 28.6 (CH, C5), 32.2 (CH₃, C9–CH₃), 32.4 (CH₂, C5′ or C3′), 33.6 (CH₂, C3′ or C5′), 39.3 (CH₂, C6 or C12), 39.5 (CH₂, C12 or C6), 40.6 (CH₂, C10 or C13), 40.7 (CH₂, C2′ or C6′), 40.9 (CH₂, C13 or C10), 41.0 (d, ³J_C-F = 2.21 Hz, CH, C11), 45.4 (CH₂, C6′ or C2′), 46.7 (CH, C4′), 47.9 (CH₃, C8), 53.3 (C, C7), 113.1 (²J_C-F = 24.85 Hz, CH, C2), 123.3 (⁴J_C-F = 3.02 Hz, CH, C4), 126.8 (³J_C-F = 9.35 Hz, CH, C3), 128.7 (²J_C-F = 28.84 Hz, C, C11a), 132.6 (³J_C-F = 13.36 Hz, C, C4a), 154.4 (C, NHCONH), 159.1 (¹J_C-F = 242.75 Hz, C, C1-F), 169. 1 (C, COCH₃). HRMS: Calcd for [C₂₄H₃₂FN₃O₂ + H]⁺: 414.2551; found: 414.2554.

1-(1-Acetylpiperidin-4-yl)-3–(2-fluoro-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (14)

To a solution of 2-fluoro-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride, 6b (150 mg, 0.53 mmol), in DCM (3 mL) saturated aqueous NaHCO₃ solution (3 mL) and triphosgene (59 mg, 0.20 mmol) were added. The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (3 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added 1-(4-aminopiperidin-1-yl)ethan-1-one, 12a, (91 mg, 0.64 mmol). The reaction mixture was stirred at room temperature overnight and the solvent was evaporated under vacuum to obtain a yellowish oil (165 mg). Column chromatography (SiO₂, DCM/methanol mixtures) gave 1-(1-acetylpiperidin-4-yl)-3–(2-fluoro-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea, 14 (103 mg, 47% yield), as a white solid, mp 269–270 °C. IR (NaCl disk): 3357, 2919, 2856, 1644, 1620, 1555, 1499, 1453, 1361, 1342, 1320, 1228, 1153, 1138, 1064, 967, 863, 818 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 0.90 (s, 3 H, C9–CH₃), 1.17 [dq, J = 11.6 Hz, J′ = 4.0 Hz, 2 H, 3′(5′)-H_ax], 1.49 (m, 2 H, 10-H_ax, 13-H_ax), 1.57–1.64 (complex signal, 2 H, 10-H_eq, 13-H_eq), 1.76 (s, 2 H, 8-H), 1.85 (m, 1 H, 5′-H_eq or 3′-H_eq), 1.91–2.13 (complex signal, 8 H, COCH₃, 6-H, 12-H, 2′-H_eq or 6′-H_eq), 2.70 (m, 1 H, 2′-H_ax or 6′-H_ax), 2.98 (t, J = 6.0 Hz, 1 H, 11-H or 5-H), 3.04 (t, J = 6.0 Hz, 1 H, 5-H or 11-H), 3.11 (m, 1 H, 6′-H_ax or 2′-H_ax), 3.69–3.78 (complex signal, 2 H, 4′-H, 6′-H_eq or 2′-H_eq),4.42 (d, J = 13.6 Hz, 1 H, 2′-H_eq or 6′-H_eq), 4.58–4.65 (complex signal, 2 H, C7–NH, C4′–NH), 6.69–6.75 (complex signal, 2 H, 3-H, 1-H), 6.97 (dd, J = 8.0 Hz, J′ = 5.6 Hz, 1 H, 4-H). ¹³C-NMR (100.5 MHz, CDCl₃) δ: 21.4 (CH₃, COCH₃), 32.2 (CH₃, C9–CH₃), 32.4 (CH₂, C5′ or C3′), 33.6 (C, C9), 33.7 (CH₂, C3′ or C5′), 39.6 (CH₂, C6 or C12), 39.9 (CH₂, C12 or C6), 40.3 (CH, C5 or C11), 40.7 (CH₂, C2′ or C6′), 40.9 (CH, C11 or C5), 41.2 (CH₂, C10, C13), 45.4 (CH₂, C6′ or C2′), 46.7 (CH, C4′), 47.9 (CH₂, C8), 53.2 (C, C7), 112.1 (d, ²J_C-F = 20.32 Hz, CH, C1), 114.6 (d, ²J_C-F = 20.72 Hz, CH, C3), 129.3 (d, ³J_CF = 7.94 Hz, CH, C4), 142.5 (d, ⁴J_CF = 3.11 Hz, C, C4a), 148.4 (d, ³J_CF = 6.63 Hz, C, C11a), 156.4 (C, NHCONH), 161.1 (d, ¹J_CF = 243.45, C, C2-F), 169.1 (C, COCH₃). HRMS: Calcd for [C₂₄H₃₂FN₃O₂ + H]⁺: 414.2551; found: 414.2553.

1-(1-Acetylpiperidin-4-yl)-3–(2-methoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (15)

To a solution of 2-methoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride, 6e (150 mg, 0.51 mmol), in DCM (3 mL) saturated aqueous NaHCO₃ solution (3 mL) and triphosgene (56 mg, 0.19 mmol) were added. The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (3 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added 1-(4-aminopiperidin-1-yl)ethan-1-one, 12a (87 mg, 0.61 mmol). The reaction mixture was stirred at room temperature overnight and the solvent was evaporated under vacuum to obtain a brown oil (256 mg). Column chromatography (SiO₂, DCM/methanol mixtures) gave 1–(1-acetylpiperidin-4-yl)-3–(2-methoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea, 15 (121 mg, 56% yield), as a white solid, mp 116–117 °C. IR (NaCl disk): 3359, 2905, 2861, 1644, 1619, 1551, 1501, 1452, 1360, 1343, 1319, 1267, 1227, 1153, 1136, 1042, 973, 807, 736 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (s, 3 H, C9–CH₃), 1.16 [m, 2 H, 5′(3′)-H_ax], 1.44–1.55 (complex signal, 2 H, 10-H_ax, 13-H_ax), 1.57–1.65 (complex signal, 2 H, 10-H_eq, 13-H_eq), 1.75–1.93 (complex signal, 5 H, 8-H, 6-H_ax, 12-H_ax, 5′-H_eq or 3′-H_eq), 2.01 (dm, J = 12.0 Hz, 1 H, 3′-H_eq or 5′-H_eq), 2.06 (s, 3 H, COCH₃), 2.12 (m, 2 H, 6-H_eq, 12-H_eq), 2.70 (m, 1 H, 2′-H_ax or 6′-H_ax), 2.95–3.04 (complex signal, 2 H, 5-H, 11-H), 3.10 (m, 1 H, 6′-H_ax or 2′-H_ax), 3.67–3.77 (complex signal, 5 H, C2–OCH₃, 4′-H, 6′-H_eq or 2′-H_eq), 4.42 (dm, J = 13.6 Hz, 1 H, 2′-H_eq or 6′-Heq), 4.54–4.63 (complex signal, 2 H, C7–NH, C4′–NH), 6.56–6.63 (complex signal, 2 H, 1-H, 3-H), 6.95 (d, J = 8.0 Hz, 4-H). ¹³C-NMR (100.5 MHz, CDCl₃) δ: 21.4 (CH₃, COCH₃), 32.3 (CH₃, C9–CH₃), 32.4 (CH₂, C5′ or C3′), 33.5 (C, C9), 33.6 (CH₂, C3′ or C5′), 39.8 (CH₂, C12 or C6), 40.2 (CH, C5 or C11), 40.3 (CH₂, C6 or C12), 40.7 (CH₂, C2′ or C6′), 41.3 (CH, C11 or C5), 41.6 (CH₂, C10 or C13), 45.4 (CH₂, C6′ or C2′), 46.7 (CH, C4′), 47.9 (CH₂, C8), 53.4 (C, C7), 55.2 (CH₃, C2–OCH₃), 110.3 (CH, C1), 114.0 (CH, C3), 128.9 (CH, C4), 138.8 (CH, C4a), 147.7 (C, C11a), 156.4 (C, NHCONH), 157.8 (C, C2), 169.0 (C, COCH₃). HRMS: Calcd for [C₂₅H₃₅N₃O₃ + H]⁺: 426.2571; found: 426.2760.

1-(2-Acetyl-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)-3–(1-acetylpiperidin-4-yl)urea (16)

To a solution of 1-(7-acetyl-9-methyl-6,7,8,9,10,11-hexahydro-5H-5,9:7,11-dimethanobenzo[9]annulen-2-yl)ethan-1-one hydrochloride, 6d (300 mg, 0.98 mmol), in DCM (5 mL) and saturated aqueous NaHCO₃ solution (3.52 mL) was added triphosgene (145 mg, 0.49 mmol). The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (5 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added 1-(4-aminopiperidin-1-yl)ethan-1-one, 12a, (167 mg, 1.17 mmol). The reaction mixture was stirred at room temperature overnight and the solvent was evaporated under vacuum to obtain a yellow gum (483 mg). Column chromatography (SiO₂, DCM/methanol mixtures) gave 1-(2-acetyl-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)-3–(1-acetylpiperidin-4-yl)urea, 16 (324 mg, 76% yield), mp 144–145 °C. IR (NaCl disk): 3363, 3005, 2918, 2861, 2239, 1679, 1619, 1552, 1453, 1426, 1361, 1320, 1272, 1229, 1203, 1137, 1106, 1057, 973, 950, 917, 830, 731, 645 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 0.91 (s, 3 H, C9–CH₃), 1.09–1.20 [complex signal, 2 H, 5′(3′)-H_ax], 1.49 (d, J = 13.6 Hz, 2 H, 10-H_ax, 13-H_ax), 1.65 (dd, J = 13.2 Hz, J′ = 6.0 Hz, 2 H, 10-H_eq, 13-H_eq), 1.75 (dm, J = 11.6 Hz, 1 H, 8-H_a), 1.79–1.97 (complex signal, 4 H, 8-H_b, 5′-H_eq or 3′-H_eq, 6-H_ax, 12-H_ax), 2.01 (m, 1 H, 2′-H_eq or 6′-H_eq), 2.05 (s, 3 H, NCOCH₃), 2.09 (m, 1 H, 6-H_eq or 12-H_eq), 2.21 (m, 1 H, 12-H_eq or 6-H_eq), 2.55 (s, 3 H, C2–COCH₃), 2.65–2.75 (complex signal, 1 H, 2′-H_ax or 6′-H_ax), 3.07–3.17 (complex signal, 3 H, 5-H, 11-H, 6′-H_ax or 2′-H_ax), 3.66–3.77 (complex signal, 2 H, 4′-H, 6′-H_eq or 2′-H_eq), 4.42 (d, J = 13.6 Hz, 1H, 2′-H_eq or 6′-H_eq), 4.71 (d, J = 6.8 Hz, 1 H, C4′–NH), 4.76 (s, 1 H, C7–NH), 7.12 (d, J = 7.6 Hz, 1 H, 1-H), 7.62–7.67 (complex signal, 2 H, 3-H, 4-H). ¹³C-NMR (100.5 MHz, CDCl₃) δ: 21.4 (CH₃, NCOCH₃), 26.6 (CH₃, C2–COCH₃), 31.5 (C, C9), 32.2 (CH₃, C9–CH₃), 32.4 (CH₂, C5′ or C3′), 33.6 (CH₂, C3′ or C5′), 39.3 (CH₂, C12 or C6), 39.8 (CH₂, C6 or C12), 40.7 (CH₂, C2′ or C6′), 41.0 (CH₂, C10, C13), 41.07 (CH, C5 or C11), 41.1 (CH, C11 or C5), 45.4 (CH₂, C6′ or C2′), 46.7 (CH, C4′), 47.7 (CH₂, C8), 53.2 (C, C7), 126.8 (CH, C3), 127.6 (CH, C4), 128.3 (CH, C1), 135.3 (C, C2), 146.7 (C, C11a), 152.4 (C, C4a), 156.4 (C, NHCONH), 169.0 (C, NCOCH₃), 198.3 (C, C2–COCH₃). HRMS: Calcd for [C₂₆H₃₅N₃O₃ + H]⁺: 438.2751; found: 438.2746.

1-(1-Acetylpiperidin-4-yl)-3–(9-methyl-2-nitro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (17)

To a solution of 9-methyl-2-nitro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride, 6c (600 mg, 1.94 mmol), in DCM (10 mL) saturated aqueous NaHCO₃ solution (10 mL) and triphosgene (213 mg, 0.72 mmol) were added. The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (5 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added 1-(4-aminopiperidin-1-yl)ethan-1-one, 12a, (331 mg, 2.33 mmol). The reaction mixture was stirred at room temperature overnight and the solvent was evaporated under vacuum to obtain a brown solid (840 mg). Column chromatography (SiO₂, DCM/methanol mixtures) gave 1-(1-acetylpiperidin-4-yl)-3–(9-methyl-2-nitro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea, 17 (640 mg, 75% yield), as a yellowish solid, mp 155–156 °C. IR (NaCl disk): 3360, 2918, 2237, 1619, 1552, 1522, 1454, 1345, 1322, 1266, 1230, 1164, 1137, 1081, 974, 949, 911, 865, 838, 798, 761, 731, 644 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 0.93 (s, 3 H, C9–CH₃), 1.17 [dq, J = 11.2 Hz, J′ = 3.6 Hz, 2 H, 3′(5′)-H_ax], 1.51 (complex signal, 2 H, 10-H_ax, 13-H_ax), 1.65–1.88 (complex signal, 5 H, 10-H_eq, 13-H_eq, 8-H, 5′-H_eq or 3′-H_eq), 2.02–2.11 (complex signal, 8 H, COCH₃, 6-H, 12-H, 3′-H_eq or 5′-H_eq), 2.70 (m, 1 H, 2′-H_ax or 6′-H_ax), 3.12 (m, 1 H, 6′-H_ax or 2′-H_ax), 3.18 (complex signal, 2 H, 5-H, 11-H), 3.73 (complex signal, 2 H, 4′-H, 6′-H_eq or 2′-H_eq), 4.55 (d, J = 7.6 Hz, 1 H, C4′–NH), 4.62 (s, 1 H, C7–NH), 7.18 (m, 1 H, 1-H). ¹³C-NMR (100.5 MHz, CDCl₃) δ: 21.4 (CH₃, COCH₃), 32.1 (CH₃, C9–CH₃), 32.4 (CH₂, C5′ or C3′), 33.6 (C, C9), 33.7 (CH₂, C3′ or C5′), 39.0 (CH₂, C6 or C12), 39.4 (CH₂, C12 or C6), 40.5 (CH₂, C10 or C13), 40.7 (CH₂, C2′ or C6′), 41.0 (CH, C5, C11), 45.4 (CH₂, C6′ or C2′), 46.8 (CH, C4′), 47.7 (CH₂, C8), 121.6 (CH C1), 122.8 (CH, C3), 128.9 (CH, C4), 146.2 (C, C4a or C11a), 147.8 (C, C11a or C4a), 154.16 (C, C2), 156.3 (C, NHCONH), 169.1 (C, COCH₃). HRMS: Calcd for [C₂₄H₃₂N₄O₄ + H]⁺: 441.2496; found: 441.2495.

1-(1-Acetylpiperidin-4-yl)-3–(2-amino-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (18)

To a solution of 1-(1-acetylpiperidin-4-yl)-3–(9-methyl-2-nitro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea, 17 (260 mg, 0.59 mmol), in EtOH (17 mL) was added PtO₂ (20 mg). The mixture was hydrogenated at room temperature and atmospheric pressure for 8 days. The resulting suspension was filtered and the filtrate was evaporated under vacuum to obtain a dark brown solid (223 mg), which was dissolved in DCM (10 mL) and washed with Et₂O obtaining a white solid (140 mg). Column chromatography (SiO₂, DCM/methanol mixtures) gave 18 as a white solid (82 mg, 34% yield), mp 150–151 °C. IR (NaCl disk): 3344, 3006, 2905, 2853, 1614, 1556, 1505, 1454, 1360, 1344, 1320, 1303, 1266, 1229, 1194, 1162, 1136, 1060, 974, 868, 820, 734 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (s, 3 H, C9–CH₃), 1.15 [m, 2 H, 5′(3′)-H_ax], 1.43–1.53 (complex signal, 2 H, 10-H_ax, 13-H_ax), 1.56–1.62 (complex signal, 2 H, 10-H_eq, 13-H_eq), 1.75–1.86 (complex signal, 5 H, 8-H, 5′-H_eq or 3′-H_eq, 6-H_ax, 12-H_ax), 1.99 (dm, J = 12.0 Hz, 1 H, 3′-H_eq or 5′-H_eq), 2.04–2.10 (complex signal, 4 H, COCH₃, 6-H_eq or 12-H_eq), 2.15 (m, 1 H, 12-H_eq or 6-H_eq), 2.69 (m, 1 H, 2′-H_ax or 6′-H_ax), 2.85 (t, J = 6.0 Hz, 1 H, 11-H or 5-H), 2.94 (t, J = 6.0 Hz, 1 H, 5-H or 11-H), 3.09 (m, 1 H, 6′-H_ax or 2′-H_ax), 3.67–3.76 (complex signal, 2 H, 4′-H, 6′-H_eq or 2′-H_eq), 4.41 (dm, J = 13.6 Hz, 1 H, 2′-H_eq or 6′-H_eq), 4.62 (d, J = 8.0 Hz, 1 H, C4′–NH), 4.66 (s, 1 H, C7–NH), 6.37–6.41 (complex signal, 2 H, 1-H, 3-H), 6.82 (d, J = 7.6 Hz, 1 H, 4-H). ¹³C-NMR (100.5 MHz, CDCl₃) δ: 21.4 (CH₃, COCH₃), 32.29 (CH₃, C9–CH₃), 32.36 (CH₂, C5′ or C3′), 33.5 (C, C9), 33.6 (CH₂, C3′ or C5′), 39.8 (CH₂, C12 or C6), 40.2 (CH, C5 or C11), 40.6 (CH₂, C6 or C12), 40.7 (CH₂, C2′ or C6′), 41.2 (CH, C11 or C5), 41.7 (CH₂, C10, C13), 45.4 (CH₂, C6′ or C2′), 46.7 (CH₂, C8), 53.4 (C, C7), 112.4 (CH, C1), 115.2 (CH, C3), 128.9 (CH, C4), 136.9 (C, C2), 144.3 (C, C4a), 147.4 (C, C11a), 156.5 (C, NHCONH), 169.0 (C, COCH₃). HRMS: Calcd for [C₂₄H₃₅N₄O₂ + H]⁺: 411.2755; found: 411.2756.

1-(1-Acetylpiperidin-4-yl)-3–(2,3-dimethoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (19)

To a solution of 2,3-dimethoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride, 6f (150 mg, 0.46 mmol), in DCM (3 mL) saturated aqueous NaHCO₃ solution (3 mL) and triphosgene (51 mg, 0.17 mmol) were added. The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (3 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added -1-(4-aminopiperidin-1-yl)ethan-1-one, 12a (79 mg, 0.55 mmol). The reaction mixture was stirred at room temperature overnight and the solvent was evaporated under vacuum to obtain a yellow oil (334 mg). Column chromatography (SiO₂, DCM/methanol mixtures) gave 1-(1-acetylpiperidin-4-yl)-3–(2,3-dimethoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea, 19 (168 mg, 80% yield), as a white solid, mp 127–128 °C. IR (NaCl disk): 3365, 3052, 2913, 2862, 2834, 1643, 1616, 1553, 1516, 1452, 1360, 1343, 1320, 1293, 1252, 1232, 1168, 1137, 1092, 1019, 974, 863, 801, 734 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 0.90 (s, 3 H, C9–CH₃), 1.16 [m, 2 H, 3′(5′)-H_ax], 1.51 [d, J = 13.2 Hz, 2 H, 10(13)-H_ax], 1.61 [dd, J = 13.6 Hz, J′ = 6.4 Hz, 2 H, 10(13)-H_eq], 1.80 (s, 2 H, 8-H), 1.82–1.93 [complex signal, 3 H, 6(12)-H_ax, 3′-H_eq or 5′-H_eq], 1.99–2.05 (dm, J = 12.4 Hz, 1 H, 5′-H_eq or 3′-H_eq), 2.06 (s, 3 H, COCH₃), 2.12 [dd, J = 12.8, J′ = 6.4 Hz, 2 H, 6(12)-H_eq], 2.69 (m, 1 H, 2′-H_ax or 6′-H_ax), 2.96 [t, J = 6.0 Hz, 2 H, 5(11)-H], 3.10 (m, 1 H, 6′-H_ax or 2′-H_ax), 3.67–3.77 (complex signal, 2 H, 4′-H, 6′-H_eq or 2′-H_eq), 3.82 [s, 6 H, C2(3)-OCH₃], 4.38 (d, J = 7.6 Hz, 1 H, 4′-NH), 4.44 (dm, J = 13.2 Hz, 1 H, 2′-H_eq or 6′-H_eq), 4.50 (s, 1 H, 7-NH), 6.59 [s, 2 H, 1(4)-H]. ¹³C-NMR (100.5 MHz, CDCl₃) δ: 21.4 (CH₃, COCH₃), 32.3 (CH₃, C9–CH₃), 32.4 (CH₂, C3′ or C5′), 33.6 (C, C9), 33.6 (CH₂, C5′ or C3′), 40.1 [CH₂, C6(12)], 40.7 [CH, C5(11), CH₂, C2′ or C6′], 41.4 [CH₂, C10(13)], 45.4 (CH₂, C6′ or C2′), 46.9 (CH, C4′), 47.9 (CH₂, C8), 53.5 (C, C7), 56.0 [CH₃, C2(3)–OCH₃], 112.2 [CH, C1(4)], 138.7 [C, C4a(11a)], 146.7 [C, C2(3)], 156.3 (C, NHCONH), 169.0 (C, COCH₃). HRMS: Calcd for [C₂₆H₃₇N₃O₄ + H]⁺: 456.2857; found: 456.2859.

1-(2,3-Dimethoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)-3–(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea (20)

To a solution of 2,3-dimethoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride, 6f (170 mg, 0.52 mmol) in DCM (4.5 mL) saturated aqueous NaHCO₃ solution (4 mL) and triphosgene (58 mg, 0.19 mmol) were added. The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (4 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added (4-aminopiperidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone, 12b (111 mg, 0.52 mmol). The mixture was stirred overnight at room temperature and the solvent was then evaporated. Column chromatography (SiO_2, DCM/methanol mixtures) provided 1-(2,3-dimethoxy-9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)-3–(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea, 20, as a white solid (73 mg, 27% yield), mp 142–146 °C. IR (ATR): 3359, 2908, 2843, 1634, 1613, 1548, 1512, 1443, 1292, 1253, 1167, 1122, 1088, 1016, 983, 950, 869, 799 cm⁻¹. ¹H (400 MHz, CDCl₃): δ 0.90 (s, 3H, C9–CH₃), 1.15 [m, 2 H, 3′(5′)-H_ax], 1.48–1.65 [complex signal, 6 H, 3″(5″)-H_ax, 10(13)-H₂], 1.80 (s, 2 H, 8-H₂), 1.82–1.85 [complex signal, 3 H, 3″(5″)-H_eq, 3′-H_eq or 5′-H_eq], 1.89 [d, J = 12.8 Hz, 6(12)-H_ax], 2.04–2.15 [complex signal, 3 H, 5′-H_eq or 3′-H_eq, 6(12)-H_eq], 2.65–2.75 (complex signal, 2 H, 4″-H, 2′-H_ax or 6′-H_ax), 2.95 [t, J = 5.6 Hz, 2 H, 5(11)-H], 3.10 (t, J = 12.8 Hz, 1 H, 6′-H_ax or 2′-H_ax), 3.43 [t, J = 11.2 Hz, 2 H, 2″(6″)-H_ax], 3.75 (m, 1 H, 4′-H), 3.81 (m, 1 H, 2′-H_eq or 6′-H_eq), 3.82 (s, 6 H, 2 OCH₃), 3.98 [m, 2 H, 2″(6″)-H_eq], 4.37 (d, J = 7.6 Hz, 1 H, HNCONH), 4.44 (m, 1 H, 6′ or 2′-H_eq), 4.48 (s, 1 H, HNCONH), 6.59 [s, 2 H, 1(4)-H]. ¹³C (100.6 MHz, CDCl₃): δ 29.2 (CH₂, C3″ or 5″), 29.3 (CH₂, C5″ or 3″), 32.4 (CH₃, C9–CH₃), 32.5 (CH₂, C3′ or 5′), 33.7 (C, C9), 34.2 (CH₂, C5′ or 3′), 37.7 (CH, C4″), 40.3 [CH₂, C6(12)], 40.8 [CH₂, C10(13)], 41.2 (CH₂, C2′ or 6′), 41.5 [CH, C5(11)], 44.4 (CH₂, C6′ or 2′), 47.2 (CH, C4′), 48.1 (CH₂, C8), 53.6 (C, C7), 56.1 (CH₃, 2 OCH₃), 67.3 [CH₂, C2″(6″)], 112.3 [CH, C1(4)], 138.8 [C, C4a(11a)], 146.7 [C, C2(3)], 156.4 (C, HNCONH), 172.9 (C, COC4″). HRMS: Calcd for [C₃₀H₄₃N₃O₅ + H]⁺: 526.3275, found: 526.3286.

1-(9-Fluoro-2,3-dimethoxy-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)-3–(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea (21)

To a solution of 9-fluoro-2,3-dimethoxy-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride, 6g (100 mg, 0.31 mmol), in DCM (3 mL) saturated aqueous NaHCO₃ solution (2.5 mL) and triphosgene (33.5 mg, 0.11 mmol) were added. The biphasic mixture was stirred at room temperature for 30 min and then the two phases were separated and the organic layer was washed with brine (3 mL), dried over anh. Na₂SO₄, filtered and evaporated under vacuum to obtain 1–2 mL of a solution of the isocyanate in DCM. To this solution was added (4-aminopiperidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone, 12b (64.8 mg, 0.31 mmol). The mixture was stirred overnight at room temperature and the solvent was then evaporated. Column chromatography (SiO_2, DCM/methanol mixtures) provided 1–(9-fluoro-2,3-dimethoxy-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)-3–(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea, 21, as a yellowish solid (35 mg, 22% yield), mp 230–233 °C. IR (ATR): 3351, 2938, 2853, 1679, 1596, 1546, 1516, 1468, 1445, 1264, 1214, 1161, 1126, 1091, 1020, 1010, 988, 874, 801, 585 cm⁻¹. ¹H (400 MHz, CDCl₃): δ 1.18 [m, 2 H, 3′(5′)-H_ax], 1.52–1.67 [complex signal, 4 H, 3″(5″)-H₂], 1.82–1.98 [complex signal, 5 H, 10(13)-H_ax, 6(12)-H_ax, 3′-H_eq or 5′-H_eq], 2.03–2.22 [complex signal, 5 H, 10(13)-H_eq, 6(12)-H_eq, 5′-H_eq or 3′-H_eq], 2.22 (m, 2 H, 8-H₂), 2.65–2.78 (complex signal, 2 H, 4″-H, 2′-H_ax or 6′-H_ax), 3.12 [m, 2 H, 5(11)-H], 3.46 [t, J = 11.6 Hz, 2 H, 2″(6″)-H_ax], 3.49 (s, 1 H, 6′-H_ax or 2′-H_ax], 3.75 (m, 1 H, 4′-H), 3.83 (m, 1 H, 6′-H_eq or 2′-H_eq), 3.84 (s, 6 H, 2 OCH₃), 4.01 [m, 2 H, 2″(6″)-H_eq], 4.24 (d, J = 7.6 Hz, 1 H, HNCONH), 4.41 (s, 1 H, HNCONH), 4.50 (d, J = 11.2 Hz, 1 H, 2′-H_eq or 6′-H_eq), 6.61 [s, 2 H, 1(4)-H]. ¹³C (100.6 MHz, CDCl₃): δ 29.2 (CH₂, C3″ or C5″), 29.3 (CH₂, C5″ or C3″), 32.5 (CH₂, C3′ or C5′), 34.2 (CH₂, C5′ or C3′), 37.8 (CH, C4″), 39.3 [CH, d, ³J_C-F = 14.1 Hz, C5(11)], 39.7 [CH₂, C6(12)], 40.4 [CH₂, d, ²J_C-F = 20.1 Hz, C10(13)], 41.2 (CH₂, C2′ or C6′), 44.4 (CH₂, C6′ or C2′), 46.8 (CH₂, d, ²J_C-F = 18.1 Hz, C8), 47.3 (CH, C4′), 56.2 (CH₃, 2 OCH₃), 57.1 (d, ³J_C-F = 11.1 Hz, C, C7), 67.3 (CH₂, C2″ or C6″), 67.4 (CH₂, C6″ or C2″), 94.5 (C, d, ¹J_C-F = 177.1 Hz, C9), 112.3 [CH, C1(4)], 137.2 [C, C4a(11a)], 147.1 [C, C2(3)], 156.0 (C, HNCONH), 173.0 (C, NCOC4″). HRMS: Calcd for [C₂₉H₄₀FN₃O₅ + H]⁺: 530.3025; found: 530.3017.

In vitro biological methods

Several in vitro assays (inhibitory activities towards human and mouse sEH²², PAMPA-BBB permeability²³, aqueous solubility, cytotoxicity in SH-SY5Y cells, microsomal stability, cytochrome P450 inhibition, and hERG inhibition) were carried out following described methodologies previously used in our group (see the Supplementary Information for complete details)^14–16.

sEH immunoblotting

HEK293 cells (SIGMA) were cultured with DMEM supplemented with 10% Foetal Bovine Serum, 2 mM L-Glutamine, 100 U μg⁻¹ penicillin–streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% CO₂. Once the cells reached about 80–90% confluency, they were seeded in 6-well plates (1.2 × 106 cells/mL in each well) to evaluate the effect of the treatment with compound 21. After 4 h of compound treatment, cells were disrupted with radioimmunoprecipitation assay buffer (RIPA) lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) to isolate the detergent-soluble proteins. The total protein fraction was quantified using BCA Protein Quantification Kit (Thermo Fisher Scientific) and normalised amounts of protein were loaded into TGX 4–20% SDS-Electrophoresis Gel (BioRad) and resolved at 180 V. The resolved proteins were electrotransferred onto PVDF membranes with the iBlot2 system (Invitrogen). Then, the membranes were blocked with tris-buffered saline with 0.1% Tween® 20 detergent (TBST) + 5% non-fat dry milk 1 h at room temperature. After blocking, membranes were probed with specific primary antibodies (Abcam) at appropriate dilutions and incubated at 4 °C overnight. After several washes with TBST, membranes were probed with the corresponding secondary antibody (Jackson ImmunoResearch). Final washes were performed before chemiluminescent detection. Enhanced Chemiluminescence (ECL) western blotting substrate (Thermo Fisher Scientific) was used to detect the HRP conjugates on the membrane with Amersham Imager 680 System (Cytiva). The images obtained were analysed using Image J software. See Table S2 for the list of antibodies used.

In silico studies. MD simulation details

The force-field parameters for compounds 17, 18, 20, and 21 were generated using the ANTECHAMBER module of AMBER 2241 using the general AMBER force field (GAFF)⁴², with partial charges set to fit the electrostatic potential generated at the HF/6-31G(d) level by the RESP model⁴³. The charges were calculated according to the Merz − Singh − Kollman scheme^44,45 using Gaussian 0946_.

MD simulations of sEH were carried out using PDB 6I5E (C-terminal crystallised in the apo state) as a starting point to manually place the compounds. The coordinates of the sEHI t-AUCB in PDB 5AM3 and piperidine-based compound in PDB 5ALZ were a reference for placing compounds 17, 18, 20, and 21 in the two possible orientations: a. benzohomoadamantane group in the LHS while piperidine group in the RHS and b. benzohomoadamantane group in the RHS while piperidine in the LHS. From these two sets of orientations, we generated six conformations of the benzohomoadamantane in each pocket (changing the dihedral angle that connects the urea moiety and the adamantane in 60° spans). Then, conventional MD simulations were used to explore the conformational plasticity of sEH in the presence of 17, 18, 20, and 21 bound in the active site. All simulations were performed using the AMBER ff14SB force field⁴⁷. The protonation states of protein residues were predicted using the H++ server (http://biophysics.cs.vt.edu/H++). The MD simulations were performed using the following protonation of histidine residues: HIE146, HIE239, HIP251, HID265, HIP334, HIE420, HIE506, HIE513, HIE518, and HIP524.

After the system preparation, each system was immersed in a pre-equilibrated truncated octahedral box of water molecules with an internal offset distance of 10 Å. All systems were neutralised with explicit counterions (Na⁺ or Cl⁻) if required. Then, we performed a two-stage geometry optimisation. Firstly, a short minimisation of the positions of water molecules with positional restraints on the solute by a harmonic potential with a force constant of 500 kcal mol⁻¹Å⁻² was done. The second stage corresponds to an unrestrained minimisation of all the atoms in the simulation cell. Then, the systems were progressively heated in six 50 ps steps, increasing the temperature by 50 K each step (0 − 300 K) under constant-volume, periodic-boundary conditions, and the particle-mesh Ewald approach⁴⁸ to introduce long-range electrostatic effects. For these steps, a 10 Å cut-off was applied to Lennard-Jones and electrostatic interactions. Bonds involving hydrogen were constrained with the SHAKE algorithm⁴⁹. Harmonic restraints of 10 kcal mol⁻¹ were applied to the solute, and the Langevin equilibration scheme was used to control and equalise the temperature⁵⁰. The time step was kept at 2 fs during the heating stages, allowing potential inhomogeneities to self-adjust. Each system was then equilibrated for 2 ns with a 2 fs timestep at a constant pressure of 1 atm (NPT ensemble). Finally, conventional MD trajectories at a constant volume and temperature (300 K) were collected. In total, we carried out five replicas of 1000 ns MD simulations for sEH in the presence of 17, 18, 20, and 21 gathering a total of 20 μs of MD simulation time. Each MD simulation was clusterised based on active site residues, and the structures corresponding to the most populated clusters were used for the noncovalent interactions analysis. We monitored the presence of water molecules using the watershell function of the cpptraj MD analysis program⁵¹. Binding affinities (kcal/mol) of compounds 17, 18, 20, and 21 were computed using the MM/GBSA method as implemented in AMBER 22 using the frames corresponding to the last 750 ns of each simulation.

Protein expression and purification

The hsEH CTD (amino acids 230–555) was cloned as described⁵². The recombinant protein was expressed in Escherichia coli BL21-AI cells (Invitrogen) in ZYM5052 auto-induction medium⁵³. A single colony of transformed cells was inoculated in 200 mL of ZYM5052 medium and cultured at 30 °C and 200 rpm overnight. The culture was diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 in six 1 L Erlenmeyer flasks with fresh ZYM5052 and cultured at 37 °C and 200 rpm until their OD₆₀₀ reached 0.6. The cells were then incubated at 18 °C and 200 rpm overnight. Cells were harvested by centrifugation at 5000 g for 30 min at 4 °C. Pellets were kept at −20 °C. The purification of hsEH CTD was performed as described⁵². Briefly, the protein was purified in three steps: nickel-immobilised metal affinity chromatography, benzylthio-sepharose column, and a size exclusion step. Purified protein was dialysed in the crystallisation buffer, 300 mM NaCl, 500 mM Tris-HCl pH 7.4, 3 mM DL-dithiothreitol at 4 °C overnight. After dialysis, the protein was concentrated to 10 mg/mL and stored at −80 °C.

X-ray crystallography

hsEH CTD was crystallised using hanging drops in pre-greased 24-well plates (Greiner Bio-One). Each reservoir contained 300 µL of precipitant buffer (20% PEG 3350, 0.2 M malic acid, 0.05 M HEPES pH 7.4, 10% ethylene glycol). The hanging drop was prepared by mixing 3 µL of 10 mg/mL hsEH CTD protein with 1 µL of precipitant buffer. After 5 days at 4 °C, cubic crystals were obtained. To obtain the complex of 20 bound to hsEH CTD, hsEH CTD protein crystals were soaked in precipitant buffer containing 2 mM 20, incubated for 3 h, and flash frozen in liquid nitrogen. The diffraction data were collected at the Diamond Light Source (Didcot, UK) on I24 beamline. The crystals analysed were selected based on best diffraction data such as resolution and CC_1/2, as well as their space group (I₁₂₁) compatible with a molecular replacement approach using the deposited PDB file 6I5E of apo hsEH CTD²⁹. The unit cell parameters were a = 93.0188, b = 81.7558, and c = 108.112 Å. The data were first integrated by XIA2 dials⁵⁴ and then reduced and scaled using AIMLESS in the CCP4 i2 software⁵⁵. PHENIX⁵⁶ was used to perform molecular replacement using PDB 6I5E²⁹, as well as a further refinement of the structure. The density map was explored and manually built in COOT⁵⁷. The ligand 20’s coordinate file was fitted within structure based on electron density during manual refinement. Occupancy of alternative conformations of the ligand was analysed by PHENIX⁵⁶. The final structure was visualised using PyMOL⁵⁸. Analysis of the contacts between hsEH CTD and ligand were performed in LigPlot+⁵⁹, PLIP⁶⁰, and PyMOL⁵⁸.

Pharmacokinetic study

Animals

For pharmacokinetic studies, 24 male CD1 mice (weight, 40–50 g; age, 8 weeks), obtained from (Envigo, Spain), were randomised to different groups and housed 4 mice per cage (IVC GM500, Tecniplast SpA), equipped with DVC boards underneath the cage and fixed to the rack. Mice were kept in an environmentally controlled room: air replacement every 10 min, constant temperature (21 ± 3 °C) and humidity, and on a 12/12 h day/night cycle. Procedures for the brain dissection, and extractions followed the ARRIVE standard ethical guidelines (European Communities Council Directive 2010/63 and Guidelines for the Care and Use of Mammals in Neuroscience and Behavioural Research, National Research Council 2003) and were approved by the Institutional Animal Care and Generalitat de Catalunya (approval number: #10291, 1/28/2018).

Drug delivery, sample collection, and sample preparation

Formulations were prepared the day of the study. The vehicle was 10% of 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich, Ref. 332607-25G). Mice were administered with 21 at a dose of 5 mg/kg by subcutaneous route. The volume of administration was 10 mL/kg. Animals were weighed before each administration to adjust the required volume. Blood samples (n = 3) were collected after the euthanasia of mice (by cervical dislocation) at different times (0.08, 0.25, 0.5, 1, 2, 3, 6 and 18 h). Plasma was separated by centrifugation for 10 min and stored at −80 °C until analysis by HPLC. Frozen plasma samples were thawed at room temperature, and 25 µL of acetonitrile were added to a 100 µL of plasma sample. The sample was vortexed for 30 s and centrifuged (14 000 rpm/min) for 5 min. The supernatant was transferred to an injection bottle, and 25 µL was injected into the chromatographic system. Mouse’s brain was carefully removed from the skull and stored at −80 °C up to use for molecular determinations. Frozen brain samples were weighed after thawing. Then, 1 mL of acetonitrile containing 0.1% formic acid was added to each brain sample. The mixture was treated in a Tissuelyser (Qiagen, Germany) at 50 Hz for 5 min, followed by centrifugation for 15 min at 15 000 rpm. Then, 200 μL of each sample were transferred into HPLC vials, and 2.8 μL of the sample were injected.

Instruments and analysis conditions

The HPLC system was a Perkin Elmer LC (Perkin Elmer INC, Massachusetts, U.S.) consisting of a Flexar LC pump, a chromatography interface (NCI 900 network), a Flexar LC autosampler PE, and a Waters 2487 dual λ absorbance detector. The chromatographic column was a Synergy™ Hydro-RP, 4 µm, 80 A (4.6 × 250 mm-Phenomenex, USA). Flow was 0.8 mL/min and the mobile phase was 0.05 M oxalic acid (30%): acetonitrile (70%) in isocratic conditions. The elution time of 21 was 4.1 min. The compound was detected at 280 nm. The assay had a range of 0.025–10 µg/mL. The calibration curves were constructed by plotting the peak area ratio of the analysed peak against known concentrations.

Pharmacokinetic analysis

The plasma concentrations of 21 versus time were analysed as previously described (see supporting information for details) but using the trapezoidal rule in the interval 0–18 h¹⁶.

Experimental animals

Experiments were performed in 94 female WT-CD1 (Charles River, Barcelona, Spain) mice weighing 25–30 g. Mice were acclimated in our animal facilities for at least 1 week before testing and were housed in a room under controlled environmental conditions: 12/12 h day/night cycle, constant temperature (22 ± 2 °C), air replacement every 20 min, and they were fed a standard laboratory diet (Harlan Teklad Research Diet, Madison, WI, USA) and tap water ad libitum until the beginning of the experiments. Behavioural test was conducted during the light phase (from 9.00 to 15.00 h), and randomly throughout the oestrous cycle. The mice were handled in accordance with international standards (European Communities Council directive 2010/63), and the experimental protocols were approved by institutional (Research Ethics Committee of the University of Granada, Spain) and regional (Junta de Andalucía, Spain) authorities (approval number: 24/10/2023/093). Mice were euthanised by cervical dislocation.

Paclitaxel-induced neuropathic pain

The antineoplastic paclitaxel (Tocris Cookson Ltd., Bristol, UK) was dissolved in a solution of 50% Cremophor EL and 50% absolute ethanol to obtain a concentration of 6 mg/mL. This paclitaxel solution was diluted in sterile physiological saline to a final concentration of 2 mg/10 mL just before its administration. For control treatment, the vehicle of paclitaxel solution was also diluted just before its administration in saline at the same proportion as the paclitaxel solution. Paclitaxel (2 mg/kg) was administered intraperitoneally (i.p.) in a volume of 10 mL/kg once per day for 5 consecutive days (cumulative dose of 10 mg/kg); a schedule of paclitaxel treatment that we previously reported that produces a painful neuropathy in mice⁶¹. The control group was administered with the vehicle for paclitaxel according to the same schedule.

Procedure to measure mechanical allodynia

The measurement of mechanical allodynia was performed as previously published⁶¹. See supplementary information for further details.

Drugs and drug administration

To test for the effect of compound 21 and gabapentin (Tocris Cookson Ltd.) once neuropathic pain was fully developed, the compounds were administered on day 10 after the first paclitaxel injection, and mechanical sensitivity was assessed 45, 90 and 150 min after injection. Compound 21 and gabapentin were dissolved in 5% DMSO (Merck KGaA, Darmstadt, Germany) in physiological sterile saline (0.9% NaCl). Drug solutions were prepared immediately before the start of the experiments and injected s.c. in a volume of 5 mL/kg into the interscapular area. To test for the effects of MS-PPOH (Cayman Chemical Company, Ann Arbour, MI, USA), a selective inhibitor of microsomal CYP450 oxidase³⁷, on the effects induced by 21 or gabapentin, it was dissolved in DMSO 5% and cyclodextrin 40% in saline, and administered 5 min before injection of the other compound. The effects of MS-PPOH were measured 90 min after injection of 21 or gabapentin, since it was the peak of their antiallodynic effects (see Results for details). Control groups were administered with the vehicle of the compounds at the same volume. Administrations of 21, MS-PPOH or their solvents were made in different areas of the interscapular zone to avoid mixture of the drug solutions and any physicochemical interaction between them.

To test for the influence of 21 in the development of paclitaxel-induced neuropathic allodynia, we s.c. administered the sEHI 30 min before each paclitaxel injection, and then evaluated mechanical sensitivity at 10, 14, and 21 days after the first injection of paclitaxel (or its vehicle).

Funding Statement

This work was funded by the Spanish Ministerio de Ciencia, Innovación y Universidades, MICIU/AEI/10.13039/501100011033: Grants PID2020-118127RB-I00 and PID2023-147004OB-I00 (to S.V.), PID2019-108691RB-I00 (to E.J.C.), PDC2021-121096 (to M.P. and C.S.), PID2021-127693OB-I00 (to C.G.), PID2022-141676NB-I00 (to F.F.); PID2021-129034NB-I00 and PDC2022-133950-I00 (to S.O.); the Generalitat de Catalunya (2021 SGR 00357 and 2021 SGR 00487); the European Research Council (ERC-2015-StG-679001-NetMoDEzyme and ERC-2022-CoG-101088032 to S.O.); the Xunta de Galicia (ED431C 2022/20); and the European Regional Development Fund (ERDF); Ministry of Science and Innovation, Spain, with funds from the European Union NextGenerationEU (PRTRC17.I1) and the Autonomous Community of Galicia within the framework of the 2023 Biotechnology Plan Applied to Health (to M.I.L. and J.B.). Partial support was provided by NIH-NIEHS River Award R35 ES030443 and, NIH-NIEHS Superfund Program P42 ES004699 to B.D.H. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. M.R.C. was granted access to I24 beamline by the Diamond Light Source for (proposal number: MX32787). S.C. and A.L.T. received PhD fellowships from the Universitat de Barcelona (APIF grant) and the Spanish Ministerio de Educación, Cultura y Deporte (FPU15/00889 grant), respectively. A.B.-M., C.E., and M.S.-C. were supported by the Research Personnel Training Program (FPI grants PRE2019-087468, PRE2022-104952, and PRE2020-096203, respectively) of the Spanish Ministerio de Ciencia, Innovación y Universidades (MICIU) and Agencia Estatal de Investigación (AEI). Q.Q. is funded by the King’s-China Scholarship Council PhD Scholarship programme. F.F. received Spanish MICINN for a Ramón y Cajal fellowship (RYC2020-029552-I). C.C.-T. received Science Postdoctoral Fellowship from a Schmidt Futures program.

Author contributions

S.V. conceived the idea. S.C., B.J., C.E., and A.L.T. synthesised and chemically characterised the compounds. E.J.C. designed the in vivo experiments. M.S.-C. carried out the in vivo experiments. Q.Q., F.P., and M.R.C. carried out protein expression and purification and the X-ray crystallography and analysed the structural data. F.F., C.C.-T., and S.O. performed MD calculations. C.M. and B.D.H. performed the determination of the IC50 in human and murine sEH. C.V., M.I.L., and J.M.B. carried out DMPK studies. C.B., R.C., and C.S. performed cytotoxicity studies. C.G.-F., M.P., and B.P. performed the pharmacokinetics. A.B.-M. performed the immunoblot experiments. S.C., B.J., C.G., S.O., M.R.C., F.F., E.J.C., and S.V. analysed the data. S.C., C.C.-T., M.R.C., F.F., E.J.C., and S.V. wrote, edited, and reviewed the manuscript with feedback from all the authors. All authors have given approval to the final version of the manuscript.

Disclosure statement

S.V. is a member of the Editorial Board of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this article. S.C., A.L.T., C.G., M.P., C.G.-F. and S.V. are inventors of the Universitat de Barcelona patent application on sEHIs WO2019/243414 and WO2022/200105. C.M. and B.D.H. are inventors of the University of California patents on sEHIs licenced to EicOsis. None of the other authors has any disclosures to declare.

Data availability statement

The data that support the findings of this study are available from the corresponding author, S. V., upon reasonable request.