2-(Piperidin-4-yl)acetamides as Potent Inhibitors of Soluble Epoxide Hydrolase with Anti-Inflammatory Activity

The pharmacological inhibition of soluble epoxide hydrolase (sEH) has been suggested as a potential therapy for the treatment of pain and inflammatory diseases through the stabilization of endogenous epoxyeicosatrienoic acids. Numerous potent sEH inhibitors (sEHI) have been developed, however many contain highly lipophilic substituents limiting their availability. Recently, a new series of benzohomoadamantane-based ureas endowed with potent inhibitory activity for the human and murine sEH was reported. However, their very low microsomal stability prevented further development. Herein, a new series of benzohomoadamantane-based amides were synthetized, fully characterized, and evaluated as sEHI. Most of these amides were endowed with excellent inhibitory potencies. A selected compound displayed anti-inflammatory effects with higher effectiveness than the reference sEHI, TPPU.

Taking into account that several adamantane-based and benzene-based ureas are endowed with very potent activity as sEHI [14][15][16][17], that both AR9281 and EC5026 feature an acylpiperidine unit, and that the highly hydrophobic pocket of sEH seems able

Synthesis of the New sEHI
The targeted amides were easily synthesized in low to moderate yields from known benzohomoadamantane amines 2a-e [21][22][23]. The coupling of these amines with carboxylic acids 3 and 7, using either EDC·HCl and HOBt or HATU, provided carbamates 4a-e and amides 6g-h, respectively. Compounds 4a-e were deprotected using HCl in dioxane to give amides 5a-e. Reaction of 5a with acetyl chloride furnished amide 6a. Finally, piperidines 5a-e were reacted with 2-propanesulfonyl chloride to obtain amides 6b-f (Scheme 1). On the other hand, inspired by the structure of amide AS2586114, a very potent sEHI developed by Astellas [24,25], we explored the series of N-aryl derivatives 10a-e (Scheme 2). The removal of the Boc group from the commercially available carboxylic acid 3 using HCl in dioxane, as previously reported [26], provided amino acid 8 [27]. Afterwards, nucleophilic aromatic substitution reactions furnished compounds 9a-d in moderate yields. Reaction of 9d with cyclopropylboronic acid via a Suzuki coupling afforded 9e a good yield. The coupling of amine 2d with carboxylic acids 9a-b yielded final compounds 10a-b. Furthermore, the reaction of 2d with carboxylic acids 9c and 9e followed by hydrolysis of the intermediate carboxylic ester furnished carboxylic acids 10c and 10d, respectively. Similarly, the reaction of amine 2e with 9c followed by hydrolysis led to 10e.

sEH Inhibition and Microsomal Stability
The potency of all the new compounds as inhibitors of the human and murine sEH was tested using a previously reported sensitive fluorescent-based assay using baculovirus expressed recombinant human and mouse sEH [28]. As shown in Table 1, known urea 1 is a low nanomolar inhibitor of the human (IC 50 = 3.1 nM) and murine sEH (IC 50 = 6.0 nM) but endowed with very poor microsomal stability. Previous studies with other families of sEHI have shown that amides are suitable pharmacophores for sEHI. For these families of compounds, the amide function significantly improves the physical properties compared to the corresponding ureas, such as increasing solubility and reducing melting points [17,20].
For this reason, we first explored the effect of replacing the urea group of 1 by an amide, leading to compound 6a. Typically, previous structure-activity relationship (SAR) studies indicated that the inhibition potencies of amides do not affect mouse sEH compared to the corresponding ureas, whereas this change leads to reduced inhibition potency for human sEH [20]. Indeed, amide 6a was a low nanomolar inhibitor of murine sEH (IC 50 = 0.4 nM) but was less potent against the human enzyme (IC 50 = 34.5 nM). Furthermore, as expected, the melting point of 6a (mp 85-86 • C) was lower than that of the corresponding urea 1 (mp 206-207 • C). Unfortunately, microsomal stability of 6a was still very low (Table 1). Next, we briefly explored the right-hand side (RHS) of the molecule. Thus, the acyl group of 6a was replaced by either an isopropylsulfonyl group or a benzyl group to obtain compounds 6b and 6g, respectively. Both compounds showed potencies in the same order than 6a, but again presented unacceptable microsomal stabilities.
Separately and noteworthy, we have recently found in a related series of sEHI that the substitution of the methyl group at C-9 of the polycyclic structure in 11a by halogen atoms, as in 11b and 11c, led to a significative increase in the microsomal stability, particularly in murine microsomes (Table 2) [19].
Thus, we synthesized and evaluated halogenated compounds 6c and 6d with the aim of improving the microsomal stability of 6b. Interestingly, this modification resulted in an improvement in the potency on the human enzyme, but only a very moderate enhancement in the microsomal stability (Table 1). Two further derivatives were synthesized, 6e and 6f, bearing a hydrogen and a deuterium atom at C-9, respectively. Once again, both compounds were shown to be excellent sEHI in the human and murine enzymes, but the microsomal activity did not improve. Considering that within this series of amides, 6c was the compound with the highest human and murine microsomal stability, we selected the fluorinated amine 2d as the starting material for further SAR studies, keeping constant the left-hand side of the molecule and modifying the RHS. First, we replaced the isopropylsulfonyl group of 6c by a benzyl unit, leading to 6h. Unfortunately, this change led to a reduction in the potency and, once again, no improvement over the stabilities of the C-methyl analog, 6g, was observed.
Next, we explored the series of N-aryl derivatives 10a-d (Scheme 2). All these compounds were endowed with sub-nanomolar potency against human and murine sEH, and, interestingly, benzoic acid 10c emerged as a better compound, with improved microsomal stabilities at human and mice. Finally, we synthesized the chlorinated analog 10e, another potent sEH inhibitor, but with lower microsomal stabilities than 10c (Table 1).

Cytotoxicity and Anti-Inflammatory Properties of 10c
Taking into account the high potency and improved microsomal stability of 10c, we evaluated its cytotoxicity in SH-SY5Y neuroblastoma cells by propidium iodide (PI) staining after 24 h of incubation. Neither 10c nor the well-known sEHI TPPU [29] showed cytotoxicity at the highest concentration tested (100 µM). Namely, the calculated percentages of cell death were similar to that of the control treatment (vehicle: DMSO 0.1%) for both tested compounds, 10c and TPPU (n = 10-12).
It is well known that the pro-inflammatory agent lipopolysaccharide (LPS), a component of the bacterial wall, induces a phenotypic change in the macrophages and in the brain microglia, the main players of the innate immune system. These cells become reactive to fight the infection and show increased phagocytic activity, release of oxygen species and release of inflammatory mediators, including nitric oxide, pro-inflammatory cytokines, and eicosanoids. However, dysregulation in the immune response with age and infections may result in age-related ailments and progressive neurodegeneration [30]. Of note, nitric oxide is a key signaling molecule, which increased generation by the inducible nitric oxide synthase (iNOS) enzyme may initiate deleterious inflammatory processes [31].
In order to evaluate the ability of the selected 10c in inhibiting the nitric oxide generation by activated glial cells [32] we used microglial BV2 cells activated with LPS. As shown in Figure 3, treatment with LPS led to an increased release of nitric oxide as indicated by the analysis of nitrite levels in the conditioned media. Gratifyingly, co-incubation with 10c completely inhibited the pro-inflammatory effects of LPS. Statistical comparison of means showed that 10c at 50 µM or 100 µM reduced nitric oxide generation to levels indistinguishable from control levels despite the presence of LPS. In contrast, the reference compound TPPU was less effective and only partially inhibited LPS effects. TPPU at 100 µM reduced the nitric oxide release induced by LPS to approximately 50%, which was significantly higher than control wells. Notably, the statistical comparison of nitrite levels between both sEHI compounds showed significantly higher protection by 10c than TPPU against inflammatory cell injury by 1 µg/mL of LPS. Figure 3. Anti-inflammatory effects of 10c in activated BV2 microglial cells. Nitrite levels in the culture media, that indicate nitric oxide generation induced by LPS, were decreased to cell resting levels by co-incubation with 10c. However, TPPU was less effective and lead to a partial decrease. Values are mean ±SEM of n = 10-15. Statistics: * p < 0.001 compared to the corresponding control group without LPS; # p < 0.001 compared to the corresponding LPS group without anti-inflammatory agents; $ p < 0.001 compared to the corresponding LPS concentration treated with 10c.

General Methods
Commercially available reagents and solvents were used without further purification unless stated otherwise. Preparative normal phase chromatography was performed on a CombiFlash Rf 150 (Teledyne Isco) with pre-packed RediSep Rf silica gel cartridges. Thin-layer chromatography was performed with aluminum-backed sheets with silica gel 60 F254 (Merck, ref 1.05554), and spots were visualized with UV light and 1% aqueous solution of KMnO 4 . Melting points were determined in open capillary tubes with an MFB 595010M Gallenkamp. Next, 400 MHz 1H and 100.6 MHz 13C NMR spectra were recorded on a Varian Mercury 400 or on a Bruker 400 Avance III spectrometers. The chemical shifts are reported in ppm (δ scale) relative to internal tetramethylsilane, and coupling constants are reported in Hertz (Hz). Assignments given for the NMR spectra of selected new compounds have been carried out on the basis of DEPT, COSY 1 H/ 1 H (standard procedures), and COSY 1 H/ 13 C (gHSQC and gHMBC sequences) experiments. IR spectra were run on Perkin-Elmer Spectrum RX I, Perkin-Elmer Spectrum TWO or Nicolet Avatar 320 FT-IR spectrophotometers. Absorption values are expressed as wavenumbers (cm −1 ); only significant absorption bands are given. High-resolution mass spectrometry (HRMS) analyses were performed with an LC/MSD TOF Agilent Technologies spectrometer. The elemental analyses were carried out in a Flash 1112 series Thermofinnigan elemental microanalyzer (A5) to determine C, H and N. The structure of all new compounds was confirmed by elemental analysis and/or accurate mass measurement, IR, 1 H NMR, and 13 C NMR (check them in Supplementary Materials). The analytical samples of all the new compounds, which were subjected to pharmacological evaluation, possessed purity ≥95% as evidenced by their elemental analyses.

Conclusions
Starting from a previous series of ureas with high potency as sEHI but poor microsomal stability, several amides were synthesized and fully characterized. Amide 10c, endowed with excellent potency, tolerable microsomal stability, and no cytotoxicity, emerged as a promising compound. 10c was highly effective in the inhibition of nitric oxide generated in microglial BV2 cells activated by LPS. Furthermore, it showed higher effectiveness than TPPU, the reference sEHI, that was reported to have anti-inflammatory properties in the brain of mouse models of Alzheimer's disease [37,38]. The results indicate 10c as an excellent candidate for further in vitro characterization as an anti-inflammatory and neuroprotective agent although its limited microsomal stability may prevent in vivo development. Overall, the results emphasize the significance of sEH as a druggable target in therapies involving inflammatory processes.

Patents
A PCT patent application has been filed. See PCT WO2019/243414A1 (priority data 20 June 2018).

Data Availability Statement: Data is contained within the article and Supplementary Materials.
Conflicts of Interest: S.C. and S.V. are inventors of the Universitat de Barcelona patent application on sEHI WO2019/243414. C.M. and B.D.H. are inventors of the University of California patents on sEHI licensed to EicOsis. None of the other authors has any disclosures to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.