Development of multitarget inhibitors for the treatment of pain: Design, synthesis, biological evaluation and molecular modeling studies

Multitarget-directed ligands are a promising class of drugs for discovering innovative new therapies for difficult to treat diseases. In this study, we designed dual inhibitors targeting the human fatty acid amide hydrolase (FAAH) enzyme and human soluble epoxide hydrolase (sEH) enzyme. Targeting both of these enzymes concurrently with single target inhibitors synergistically reduces inflammatory and neuropathic pain; thus, dual FAAH/sEH inhibitors are likely to be powerful analgesics. Here, we identified the piperidinyl-sulfonamide moiety as a common pharmacophore and optimized several inhibitors to have excellent inhibition profiles on both targeted enzymes simultaneously. In addition, several inhibitors show good predicted pharmacokinetic properties. These results suggest that this series of inhibitors has the potential to be further developed as new lead candidates and therapeutics in pain management.


Introduction
Pain results from a wide variety of conditions and illnesses. Managing pain represents a unique challenge to health professionals, which requires multidisciplinary strategies [1,2]. The most effective analgesic drugs currently used to treat moderate-to-severe pain are opioid agonists (e.g., oxycodone) [3]. The endogenous opioid system is a part of the body's natural defense network in the brain, and opioidbased drugs (opioids) interact with opioid receptors, which leads to pain relief [4]. Prolonged use of opioids will eventually lead to tolerance, physical dependence, and addiction [5]. Currently, the most commonly used non-opioid drugs in pain management are nonsteroidal anti-inflammatory drugs (NSAIDs) [6]. The anti-inflammatory action of NSAIDs is a result of their inhibitory effect on the cyclooxygenase (COX) enzymes, that are involved in the metabolism of arachidonic acid into pro-inflammatory prostaglandins [7]. During inflammation, arachidonic acid (AA) is released from the membrane phospholipids by enzyme phospholipase A 2 (PLA2) and converted to different inflammatory mediators (Fig. 1) [8,9]. NSAIDs can treat only mild and moderate pain and, in addition, there are a number of adverse effects associated with their use, but most common are dyspepsia, an increased risk of gastric ulcer, and an increased risk of myocardial infarction [10,11]. Currently there is a great demand for more effective therapeutics to treat acute and chronic pain, and only a few new drugs have been introduced to the market in the past years. One approach toward designing novel analgesics with improved efficacy and reduced adverse effects is the poly-pharmacological approach, i.e., to design a new, single drug that is able to modulate multiple molecular pathways that are involved in pain regulation [12].
Soluble epoxide hydrolase (sEH) is a ubiquitous enzyme widely distributed throughout the body with the most concentrated expression in the liver, kidneys, lungs, and vascular tissues [13]. This enzyme is selective for aliphatic epoxides of fatty acids, such as epoxyeicosatrienoic acids (EETs) [14] (Fig. 1). The EETs are one of the metabolic derivatives of AA [15]. EETs exhibit vasodilatory effects in various arteries and have also been shown to possess analgesic and anti-inflammatory properties [16]. AA is metabolized through three major enzymatic pathways: cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways [14]. The enzyme sEH mediates the addition of water to EETs, leading to the corresponding diols, dihydroxyeicosatrienoic acids (DHETs), which show diminished biological activity [17]. Therefore, inhibition of the enzyme sEH causes an increase in EET concentration, which has beneficial therapeutic effects on pain and inflammation [8]. Thus far, a large body of work has focused on a class of urea-based inhibitors for sEH (e.g. AUDA, TPPU) shown in Fig. 2 [18]. TPPU demonstrated to be active against both acute inflammation and chronic pain conditions in several rodent and primate preclinical models [19][20][21][22].
A recent study by Sasso et al. (2015) showed that the combination of sEH inhibitor TPPU and FAAH inhibitor URB937 causes a significant synergistic reduction in pain behavior in two rodent models of pain [28]. We believe that this synergy may be exploited therapeutically to achieve better pain control at lower drug dosages or to develop dual agents that simultaneously inhibit both FAAH and sEH activities. Previously we have described dual sEH/FAAH inhibitors that are potent on the human form of both enzymes but have poor metabolic stability and water solubility [29,30].
Here, we sought to take alternative approaches to design a new series of dual sEH/FAAH inhibitors that may have a more favorable physio-chemical profile to be used as a possible therapeutic. These inhibitors were designed by integrating a piperidine-sulfonamide-phenyl pharmacophore common to the medicinal chemistry of both selective sEH and FAAH inhibitors.

Design and synthesis
Previously, we identified potent non-urea sEH inhibitors using highthroughput screening (HTS) in combination with structure-activity relationship (SAR) studies. These molecules were derivatives of isonipecotic acid (e.g., inhibitor 1, Fig. 2). Our SAR studies showed that the pharmacophore for the sEH inhibitors should include a central sulfonamide moiety next to the piperidine ring. We also observed that the bulky, hydrophobic groups on the left-hand side of the molecules are positively correlated with inhibitory potency [31,32]. In addition, we were able to successfully co-crystallize one of the non-urea inhibitors with human sEH [33]. Our docking experiments revealed that an amide functional group binds in the proximity of key amino acids needed for catalytic activity, two tyrosines (Y383 and Y466), and one aspartic acid (D335) [34]. The orientation of this amide moiety is similar to the orientation of the urea groups on urea sEH inhibitors (e.g., AUDA, Fig. 2); thus, the amide group likely satisfies the same hydrogen bonding interactions with tyrosine and aspartic acid residues that contribute to highly potent urea inhibitors. Using information obtained from SAR studies in combination with molecular modeling and crystallography data, we were able to determine a particular pharmacophore for this series of sEH inhibitors (e.g., analog 2, shown in red, Fig. 3) required to inhibit the sEH enzyme. Wang et al. (2010) performed an HTS and identified the benzothiazole analog 3 (Fig. 3) as a potent rat FAAH inhibitor having an IC 50 of 18 nM [26]. Their SAR studies indicated that the sulfonamide group, the piperidine ring, and benzothiazole on the left-hand side of the molecule were key components to their activity. The sulfonamide group likely forms hydrogen bonding interactions with the catalytic serine group, similar to the hydrogen-bonded network between the FAAH enzyme and the pyridyl nitrogen and oxazoyl oxygen of the αketo-oxazole FAAH inhibitors [35]. In addition, the modeling study performed by Wang et al. (2010) also indicated that the benzothiazole ring satisfies hydrophobic interactions within the hydrophobic binding pocket of the rat FAAH enzyme that confers extraordinary potency [26]. We also identified several potent 4-phenylthiazole FAAH inhibitors that possess the piperidine moiety connected to the phenyl ring via a sulfonamide bond [36]. These studies have shown that all three components, sulfonamide bond, piperidine ring and benzothiazole/4phenylthiazole moieties, are important for the FAAH inhibition, but more work has to be done in order to access the particular relationship of each of these moieties with the inhibition potencies.
Overall, these extensive SAR studies indicate that modifications to the aromatic ring on the left-and right-hand sides of the pharmacophore (shown in red in Fig. 3) should allow for improved sEH and FAAH inhibition. This data guided our design for the preparation of the dual inhibitors wherein modifications to the right side of the aromatic ring were carried out.
Since inhibition of each enzyme showed analgesic effect individually [37,38], and co-administration of sEH and FAAH inhibitors resulted in a significant synergistic reduction in pain behavior in animal models of pain [28], we hypothesized that dual inhibitors will treat pain at a lower dose, and consequently with fewer side-effects. We decided to employ a Designed Multiple Ligands (DMLs) strategy [12,39] wherein inhibitors used the core pharmacophore (a phenyl ring connected to a piperidine moiety, which is connected to the sulfonamide bond) common to the sEH inhibitor 2 and the FAAH inhibitor 3 with modifications on the aromatic rings on either side. Fig. 3 shows representative structures with the key pharmacophoric regions boxed. Given that the benzothiazole ring contributes to high FAAH inhibitory potency and bulky hydrophobic groups are well-tolerated in that position for sEH inhibitory potency [32,33], we kept this structure constant (general structure 4) while modifying aromatic groups bound to the sulfonamide group.
Following the established synthetic procedure [34] shown in Scheme 1, we started from the readily available 2-(4-aminophenyl) benzothiazole and Boc-isonipecotic acid. EDC coupling yielded the amide 5, which was subjected to Boc-deprotection with trifluoroacetic acid (TFA) which provided the key amine intermediate 6.

Biological evaluation and Structure-activity relationship studies
All synthesized analogs 4-1 to 4-30 were tested in vitro in both human sEH and human FAAH inhibition assays. The inhibition potencies of analyzed analogs against both enzymes are summarized in Table 1. Our initial SAR investigation started with the synthesis of the thiophene-2-yl analog, 4-1. This analog showed inhibition potency in the low nanomolar range for human FAAH enzyme (IC 50 = 16 nM), but only moderate inhibition potency at the human sEH enzyme (IC 50 = 420 nM). The introduction of bromine and chlorine atoms on the thiophene ring (analog 4-2) led to significantly diminished inhibition potencies at both enzymes. We decided to replace the thiophene ring with a phenyl ring, which allowed us to access many sterically and electronically diverse chemical groups, which could in turn improve inhibition profiles at both enzymes. The phenyl analog 4-3, showed excellent inhibition potency with the human FAAH enzyme, having an IC 50 of 8.6 nM, but only low micromolar inhibition for human sEH enzyme (IC 50 = 1100 nM). Fluoro-, chloro-, bromo-and methyl-groups placed at the ortho position (4-4, 4-5, 4-6, and 4-7, respectively) were all well tolerated in the human FAAH binding pocket and led to low nanomolar inhibition potency on human FAAH enzyme. The bulkier, electron-donating methoxy-group (4-8) was less potent for human FAAH (IC 50 = 80 nM). Placement of chloro-, bromo-, or methyl-groups in the ortho position improved potency at the sEH enzyme relative to the unsubstituted inhibitor . This led to the best dual sEH/FAAH inhibitor of the series (4-5) with equally high potency for sEH (IC 50 = 9.6 nM) and FAAH (IC 50 = 7 nM). Next, we introduced the same replacements into the meta-position on the phenyl ring. We noticed that adding fluoro-, chloro-, bromo-, methyl-or methoxygroups at the meta position (4-9, 4-10, 4-11, 4-12 and 4-13, respectively) had comparable potencies relative to the unsubstituted inhibitor (4-3). These inhibitors retain low nanomolar inhibition potencies for human FAAH enzyme with low potency at the human sEH enzyme (IC 50 s ranged from 620 to 2400 nM). Modification of the para position with fluoro-, chloro, bromo-, methyl-and methoxy-substitutions (4-14, 4-15, 4-16, 4-17 and 4-18, respectively) led to a loss of potency towards FAAH relative to the unsubstituted inhibitor . Surprisingly, the introduction of the 2,4-disubstitutions for fluoro-, chloro-, bromoand methoxy-groups (4-19, 4-20, 4-21, 4-22 and 4-23, respectively) led to inhibitors with potency at both enzymes comparable to the same single ortho-substitutions and improved potency relative to para-substitutions. Thus, the benefit from ortho-substitutions is greater than the loss from para-substitutions. The fluoro-and methyl-3,5-disubstitutions (4-24 and 4-25) had low potency at both enzymes (IC 50 s > 10,000 nM on FAAH and 1,000 nM on sEH). Additionally, the fluoro-, chloro-, methyl-and isopropyl-tri-substitutions (4-26, 4-27, 4-28 and 4-29, respectively) and the pentafluoro-substitution (4-30) had lower potency than the 2,4-disubstituted molecules. This suggests compounds that have high bulk are unfavored in the active sites of FAAH and sEH.

Molecular modeling studies
Our design and evaluation of synthesized analogs were complemented with in silico experiments. Since the crystal structure of the human FAAH enzyme has not been reported, we built and evaluated a homology model for the human FAAH enzyme [36]. We docked all synthesized analogs, 4-1 to 4-30, in both the human FAAH enzyme homology model and the human sEH enzyme crystal structure derived from PDB: 4HAI. Docking scores obtained in these experiments are shown in Table 1 and all non-covalent interactions are shown in Table 2A and Table 2B for FAAH and sEH enzymes, respectively. The ICM docking score represents unitless approximations of the binding free energy between the inhibitor and the enzyme where a lower docking score suggests a higher chance the inhibitor is bound to the enzyme [40]. For the human FAAH enzyme, the low values for the potential energy (docking scores) were obtained for all the analogs that also show in vitro low nanomolar inhibition potencies (e.g., 4-7, 4-11, 4-12, 4-21, 4-23, etc.). Analysis of these values show that most of the obtained docking energies are correlated with inhibitory potency (R 2 = 0.3255, p = 0.0023) with the exception of compounds with very poor potency on FAAH (IC 50 > 10,000) (Fig. 4A). This suggests that the potency of these inhibitors is primarily based on Van der Waals interactions between the enzyme active site and inhibitors. After visual inspection of the top binding modes of the most active inhibitors, we noticed that all docked compounds are located in the proximity of S241 and S217, both residues of the catalytic triad, S241-S217-K142, which is responsible for the hydrolytic cleavage of the amide bond of the substrate anandamide by the FAAH enzyme [25]. We were able to define the most important residues within the binding pocket of the FAAH enzyme and tried to explain the increased in vitro inhibition potencies by observing and analyzing the type of contacts of the most active FAAH inhibitor identified in this study, 4-4, with the inhibitory potency of 1.3 nM, and other analogs with low nanomolar inhibition potencies, e.g., 4-5, 4-6, 4-10 and 4-22. The aromatic part of the inhibitor 4-4 is found to be embedded between several hydrophobic amino acid residues (F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433, M436, T488, V491, I530, W531) and forms other important non-covalent interactions (M191, G216, S217, L380, L401, G485, M495), which we believe all contribute to high inhibitory potency of this analog (Table 2A). The best dual inhibitor identified herein, 4-5 has a very similar defined binding pocket, showing the importance of selected hydrophobic and other non-covalent interactions for the low nanomolar inhibition potency (Fig. 5A, Fig. 5B, Table 2A). The poor correlation of docking energies with in vitro experiments were observed with only three analogs, 4-24, 4-25 and 4-30, but visual inspection of these compounds within the binding pocket of the human FAAH homology model reveals the absence of several important contacts with residues, S241 and S217, that are present with the biologically active analogs, which could explain the lower inhibition potencies of these three analogs (Table 2A).
The docking experiments of all synthesized inhibitors in the human sEH enzyme model revealed that most of the analogs are located in the proximity of key amino acids within the catalytic pocket that are involved in the hydrolysis of EETs. Compared with docking scores for the FAAH enzyme, the docking scores of sEH poorly correlated with sEH potency (Fig. 4B). All potent sEH inhibitors (e.g. (Table 1), however, the analogs that were inactive in the in vitro inhibition assay, also have good docking scores in our docking experiments. This possibly indicates that sEH potency is primarily determined based on hydrogen bonding interactions with the catalytic residues rather than hydrophobic interactions with the binding pocket which probably is responsible for the good docking scores in this series of analogs. Our previous molecular modeling studies and X-ray crystallographic structure showed that two tyrosine residues (Y383 and Y466) and one aspartic acid residue (D335), located in the hydrolase catalytic pocket of sEH, are involved in hydrogen bonding with the inhibitors. One of the most potent sEH inhibitors identified in this study, and the best dual inhibitor, 4-5, is in the close proximity of these three amino acid residues (Fig. 6A, Fig. 6B, Table 2B). This inhibitor forms a hydrogen bond with D335 via amide bond, and has several important hydrophobic interactions (F267, W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428, M469, N472, V498, L499, H524), that, combined with other noncovalent interactions (P268, Q384, Y466, W525), contribute to the high inhibition potency of this compound. We also noticed that all low   (continued on next page) nanomolar sEH inhibitors identified in this study are docked in the proximity of the key amino acid residues, Y383, Y466, and D335, located in the catalytic site of the sEH enzyme (Table 2B), while inactive compounds lack this interaction. Finally, we looked at the binding poses of the potent dual inhibitors, 4-5, 4-6, 4-20, 4-21, and 4-22, within both, human FAAH and sEH enzyme binding pockets. We observed that the benzothiazole moiety of all aforementioned ligands is located in the proximity of the two hydrophobic residues, V422 and L433 in the human FAAH binding pockets, while this group probably interacts with one of the methionine residues (M419 or M469) within human sEH binding pocket.

In silico ADMET studies
Based on the encouraging biological results described above, we performed some selected absorption, distribution, metabolism, excretion, and toxicology (ADMET) predictions to assess their drug-like properties. Unfavorable ADMET properties have been identified as a major cause of drug candidate failure in the pharmaceutical industry [41,42]. In silico ADMET prediction represents the use of computer modeling software to understand structure-property relationships and predicts the in vivo behavior of potential drug candidates in the human body [43].
Using the ICM-Chemist-Pro tool, we started our investigations (see Table 3) by calculating simple physicochemical descriptors, such as cLogP and aqueous solubility (cLogS). It has been shown previously that these parameters are good predictors for drug candidate permeability [44,45]. High logP values usually mean poor absorption/barrier penetration and is also an integral part of the well-known Lipinski Rule of 5 prediction [46]. All synthesized analogs in this study, 4-1 to 4-30, are in the agreement with Lipinski Rule of 5 in terms of cLogP, the number of hydrogen bond acceptors and the number of hydrogen bond donors. Several inhibitors have molecular weights that exceed 500 g/mol, but these numbers are very close to 500 Da and as the prediction rule states, potential orally active drugs should not violate more than one of the four criteria. Therefore, we believe that these inhibitors represent excellent candidates for future follow-up SAR studies and drug development. In addition, another important rule that predicts oral bioavailability, Veber's Rule, states that drug candidates will have good oral bioavailability if the number of rotatable bonds present in a molecule is less than 10 [47]. All compounds synthesized in this study has 6 to 9 rotatable bonds. The logS for aqueous solubility of a molecule, expressed in mol/L, helps to predict oral absorption. The acceptable range for logS is between −6.5 and −0.5 mol/liter. In this study, all synthesized analogs showed values close to, but below, −6.5 mol/L, suggesting a moderate aqueous solubility. Improving solubility of these potential drug candidates, if needed, can be addressed in the future guided design of follow-up inhibitors or in the drug formulation process. The next important parameter to consider for the drug design and development is permeability, and we assessed it using the Caco-2 prediction tool [48]. The ICM-Chemist-Pro Caco-2 prediction scores higher than −5 suggest a highly permeable drug candidate, while scores of below −6 represent a poorly permeable compound. As shown in Table 3, all compounds synthesized in this study have predicted scores for Caco-2 between −5 and −6, suggesting moderately permeable drug candidates. We also wanted to predict the plasma half-life of synthesized dual inhibitors. Our analysis showed that inhibitors 4-26, 4-27 and 4-28 have the longest predicted half-life in hours (10.10 h) whereas the inactive compound 4-2 had the shortest predicted half-life of 1.2 h. The most potent inhibitors identified in this study, 4-5, 4-6, 4-20, 4-21, and 4-22 showed a moderate predicted half-life of 3.2 to 5.7 h. As part of our Tox studies, we analyzed several important toxicology descriptors, LD50, hERG inhibition, and the Tox score. Prediction of the hERG inhibition is performed because pharmacological blockade of the hERG channel results in a severe life-threatening cardiac side effect possibly causing sudden death, leading to the withdrawal of many drugs from the development process [49,50]. The ICM-Chemist-Pro tool predicts that scores above or equal to 0.5 will probably exhibit some hERG inhibition at 100 µM or less, while compounds with predicted values below 0.5 will likely not be hERG inhibitors. No compounds synthesized in this study exceeded a value of 0.5. As part of the toxicity study predictions, we calculated the Tox score for each compound synthesized. This value represents the identification of potentially toxic parts/bi-products (during metabolism) of the molecule. The only compound that showed a toxicity score was analog 4-30. Finally, we calculated a "drug-like" properties for each synthesized compound. This is a purely empirical value and is based on several factors calculated above. The scores are on a scale of −1 to 1 and should not exceed 1. Only two compounds, 4-15 and 4-29, slightly exceed a value of 1. In addition, these two analogs show moderate and low inhibition profiles against both enzymes, respectively, and will not be considered for future follow up studies.

Conclusion
We successfully designed, synthesized, and identified several potent dual FAAH/sEH inhibitors. Inhibitors 4-5, 4-6, 4-20, 4-21, and 4-22 were identified as the most potent compounds in the benzothiazole series against both enzymes. Molecular modeling studies of these compounds revealed important residues within the catalytic sites of both enzymes that are responsible for the low nanomolar potencies of these inhibitors. This knowledge will be used in future design and follow-up SAR studies. In addition, these selected dual inhibitors showed acceptable predicted ADMET properties, and further study is currently underway to test their stability in liver microsome assays. In addition, the inhibitors of both enzymes have been individually investigated as potential therapeutics in many diseases, i.e., cardiovascular [51], pulmonary [52,53], and Parkinson's disease [54,55]. In this study, we observed several novel compounds that have inhibitor activity for one of these two enzymes. Compounds that were found to inhibit only one particular enzyme, sEH (e.g., 4-18) or FAAH (e.g., 4-4), will be very useful to further develop as novel therapeutics for the aforementioned diseases and other disorders beyond pain.

General procedure for the preparation of benzothiazole-phenyl analogs
The mixture of N-Boc-4-piperidinecarboxylic acid (250 mg, 1.09 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC (252 mg, 1.31 mmol) and a catalytic amount of 4-dimethylaminopyridin, DMAP were dissolved in anhydrous tetrahydrofuran, THF (40 mL). The reaction mixture was stirred at room temperature for 1.5 h under an argon atmosphere. Then, 2-(4-aminophenyl) benzothiazole (197 mg, 0.87 mmol) was added to the stirring solution. The reaction mixture was stirred at room temperature for 48 h under an argon atmosphere. After removal of the solvent under reduced pressure, the residue was dissolved in ethyl acetate (40 mL). The mixture was transferred to a separatory funnel and the organic layer was washed with an aqueous solution of 1 M hydrochloric acid, HCl (3x25 mL), followed by an aqueous solution of saturated sodium bicarbonate, NaHCO 3 (25 mL), dried over anhydrous sodium sulfate, Na 2 SO 4 , filtered and concentrated. The crude product 5 was purified by flash chromatography The amide 5 (250 mg, 0.57 mmol) was dissolved in anhydrous dichloromethane, DCM (10 mL), and stirred in an ice bath at 0 °C. Trifluoroacetic acid, TFA (1 mL, 11.4 mmol) was added dropwise into the solution and the reaction mixture was stirred at room temperature for 24 h under an argon atmosphere. Following concentration in vacuo, the crude product was triturated with diethyl ether and filtered. The product 6 was obtained as a TFA salt and used for next step without further purification. A small amount was free-based and used for NMR mixture was warmed to room temperature. Corresponding benzenesulfonyl chloride (0.33 mmol) was added to the reaction mixture and the reaction mixture was stirred for 48 h at room temperature under an argon atmosphere. Next, the mixture was transferred to a separatory funnel where the organic layer was washed with an aqueous solution of saturated NaHCO 3 (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by flash chromatography (1:4 ethyl acetate/hexane solvent system) and recrystallized from diethyl ether.

sEH and FAAH IC 50 assay conditions
Measurement of sEH potency was performed using cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-phenyloxyran-2-yl) methyl carbonate (CMNPC) as the fluorescent substrate [56]. Human sEH (1 nM) was incubated with the inhibitor for 5 min in pH 7.0 Bis-Tris/HCl buffer (25 mM) containing 0.1 mg/mL of bovine serum albumin (BSA) at 30 °C prior to substrate introduction ([S] = 5 μM). Activity was determined by monitoring the appearance of 6-methoxy-2-naphthaldehyde over 10 min by fluorescence detection with an excitation wavelength of 330 nm and an emission wavelength of 465 nm. Reported IC 50 values are the average of the three replicates with at least two data points above and at least two below the IC 50 . Measurement of FAAH potency was performed using the substrate N-(6-methoxypyridin-3-yl) octanamide (OMP) ([S]final = 50 μM) in sodium phosphate buffer (0.1 M, pH = 8, 0.1 mg/mL BSA). Progress of the reaction was measured by fluorescence detection of 6-methoxypyridin-3-amine at an excitation wavelength of 303 nm and an emission wavelength of 394 nm at 37 °C by the use of microplate reader (Molecular Devices., CA, USA). All experiments were run in triplicate, and values reported as average +/-SD. The substrate OMP was synthesized following a previously reported synthetic procedure and reaction conditions [36].

Molecular modeling
For the docking studies of the dual sEH/FAAH inhibitors, a crystal structure of human soluble epoxide hydrolase complexed with N-cycloheptyl-1-(mesitylsulfonyl)piperidine-4-carboxamide (PDBfile: 4HAI) [33] and a homology model of human FAAH enzyme [36] were used. PDB file 4HAI was first converted to an ICM file and the inhibitor Ncycloheptyl-1-(mesitylsulfonyl) piperidine-4-carboxamide was removed. Docking experiments were performed following the program guidelines. ICM scores were obtained after this procedure. ADMET properties for all synthesized target analogs were calculated using the ICM Chemist program.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.