Evaluation of the Therapeutic Potential of Sulfonyl Urea Derivatives as Soluble Epoxide Hydrolase (sEH) Inhibitors

The inhibition of soluble epoxide hydrolase (sEH) can reduce the level of dihydroxyeicosatrienoic acids (DHETs) effectively maintaining endogenous epoxyeicosatrienoic acids (EETs) levels, resulting in the amelioration of inflammation and pain. Consequently, the development of sEH inhibitors has been a prominent research area for over two decades. In the present study, we synthesized and evaluated sulfonyl urea derivatives for their potential to inhibit sEH. These compounds underwent extensive in vitro investigation, revealing their potency against human and mouse sEH, with 4f showing the most promising sEH inhibitory potential. When subjected to lipopolysaccharide (LPS)-induced acute lung injury (ALI) in studies in mice, compound 4f manifested promising anti-inflammatory efficacy. We investigated the analgesic efficacy of sEH inhibitor 4f in a murine pain model of tail-flick reflex. These results validate the role of sEH inhibition in inflammatory diseases and pave the way for the rational design and optimization of sEH inhibitors based on a sulfonyl urea template.


Introduction
The pathophysiological process of inflammation is intricate and involves a wide range of mediators and signaling pathways [1,2].Inflammation frequently manifests physiologically as redness, swelling, fever, pain, and even loss of organ function [3].Research conducted over the past few decades has revealed that the arachidonic acid (AA) pathway produces bioactive lipid mediators that control a variety of inflammatory processes [4][5][6].Mammals metabolize polyunsaturated fatty acids like arachidonic acid through the action of cytochrome P450s (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) [4].These are the three principal enzymatic pathways that control the formation of eicosanoids from arachidonic acid.The COX and LOX pathways, which primarily produce pro-inflammatory lipid mediators such as prostaglandins and leukotrienes, have been extensively studied and pharmaceutically targeted [7].On the other hand, the CYP route generates lipid mediators that are both pro-and anti-inflammatory, like the potent anti-inflammatory epoxyeicosatrienoic acids (EETs) and the pro-inflammatory 20-hydroxyeicosatetraenoic acid [8][9][10].The enzyme soluble epoxide hydrolase (sEH) plays a pivotal role in the metabolism process of bioactive lipid signaling molecules [11,12].The substrate-specific hydrolase activity of sEH transforms EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) purported to have inflammatory effects [13,14].
The cytochrome P450 pathway has received a lot of interest lately.EETs are cytochrome P450-derived eicosanoids that can cause vasodilation and inhibit the cytokine-induced inflammatory response in the vasculature, heart, and kidney [15][16][17].EETs are recognized as crucial signaling elements in an organism and have biological effects including blood pressure, vasodilation, and anti-inflammation.It has been demonstrated that sEH inhibition leads to elevated levels of EETs, subsequently manifesting anti-inflammatory, analgesic, and antihypertensive effects [11,12,18].Given the opioid crisis, there is an urgent need for novel medications to address both neuropathic pain and chronic pain without having the same addictive qualities as opioids.Soluble epoxide hydrolase inhibitors stabilize epoxides of polyunsaturated fatty acids (EpFAs), which are endogenous mediators that alleviate inflammation and lessen pain, by acting on the cytochrome P450 branch of the arachidonate cascade.Pharmacological inhibition of sEH in animal models exhibited beneficial effects on the treatment of hypertension, inflammation, and neuropathic pain [19][20][21].For improving the positive effects of EpFAs in a variety of disorders, including neuropathic pain, hypertension, inflammatory bowel disease, and cardiovascular disease, sEH is thought to be an important therapeutic target [22][23][24][25][26][27].
X-ray crystallographic studies demonstrated that sEH has an L-shaped active pocket with the catalytic residues situated at the corner, and the entire pocket is essentially hydrophobic [28,29].Hence, a large number of very potent sEH inhibitors (sEHis) possess lipophilic moieties.Arete Therapeutics developed AR9281 to treat hypertension in diabetic individuals; however, due in significant part to its poor pharmacokinetic features, the drug was not progressed beyond a phase II human clinical trial [30].GlaxoSmithKline's GSK2256294, which was created for chronic obstructive pulmonary disease, is currently undergoing clinical studies for use in obese smokers as well as additional indications such as subarachnoidal hemorrhage and people with diabetes who are insulin resistant (Figure 1A) [31].EicOsis has more recently substituted an aromatic ring for the adamantyl moiety of AR9281 in their drug candidate EC5026, which has completed phase I-a clinical trials in humans for the treatment of neuropathic pain (Figure 1A) [32].Using urea-based motifs with lipophilic units of varying sizes, we recently found that urea derivatives bearing aryl sulfonyl groups manifested subnanomolar inhibition of the human sEH (hsEH) for certain compounds.Therefore, herein, we report a detailed investigation into sulfonyl urea derivatives and their suitability as new hits for sEH inhibition.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 29 substrate-specific hydrolase activity of sEH transforms EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) purported to have inflammatory effects [13,14].The cytochrome P450 pathway has received a lot of interest lately.EETs are cytochrome P450-derived eicosanoids that can cause vasodilation and inhibit the cytokineinduced inflammatory response in the vasculature, heart, and kidney [15][16][17].EETs are recognized as crucial signaling elements in an organism and have biological effects including blood pressure, vasodilation, and anti-inflammation.It has been demonstrated that sEH inhibition leads to elevated levels of EETs, subsequently manifesting anti-inflammatory, analgesic, and antihypertensive effects [11,12,18].Given the opioid crisis, there is an urgent need for novel medications to address both neuropathic pain and chronic pain without having the same addictive qualities as opioids.Soluble epoxide hydrolase inhibitors stabilize epoxides of polyunsaturated fatty acids (EpFAs), which are endogenous mediators that alleviate inflammation and lessen pain, by acting on the cytochrome P450 branch of the arachidonate cascade.Pharmacological inhibition of sEH in animal models exhibited beneficial effects on the treatment of hypertension, inflammation, and neuropathic pain [19][20][21].For improving the positive effects of EpFAs in a variety of disorders, including neuropathic pain, hypertension, inflammatory bowel disease, and cardiovascular disease, sEH is thought to be an important therapeutic target [22][23][24][25][26][27].
X-ray crystallographic studies demonstrated that sEH has an L-shaped active pocket with the catalytic residues situated at the corner, and the entire pocket is essentially hydrophobic [28,29].Hence, a large number of very potent sEH inhibitors (sEHis) possess lipophilic moieties.Arete Therapeutics developed AR9281 to treat hypertension in diabetic individuals; however, due in significant part to its poor pharmacokinetic features, the drug was not progressed beyond a phase II human clinical trial [30].Glax-oSmithKline's GSK2256294, which was created for chronic obstructive pulmonary disease, is currently undergoing clinical studies for use in obese smokers as well as additional indications such as subarachnoidal hemorrhage and people with diabetes who are insulin resistant (Figure 1A) [31].EicOsis has more recently substituted an aromatic ring for the adamantyl moiety of AR9281 in their drug candidate EC5026, which has completed phase I-a clinical trials in humans for the treatment of neuropathic pain (Figure 1A) [32].Using urea-based motifs with lipophilic units of varying sizes, we recently found that urea derivatives bearing aryl sulfonyl groups manifested subnanomolar inhibition of the human sEH (hsEH) for certain compounds.Therefore, herein, we report a detailed investigation into sulfonyl urea derivatives and their suitability as new hits for sEH inhibition.

Chemistry
In this study, we report in detail the design, synthesis, and biological characterization of sEH inhibitors by utilizing an alkyl amino sulfonyl urea attachment.As seen in Figure 1B, the pharmacophoric features of AUDA and TPPU can be merged to generate sulfonamide-based compounds for their potential to inhibit sEH.Recently, glimepiride, a sulfonyl urea compound, was reported to have sEH inhibitory properties, although previously, sulfonyl ureas were shown to not have sEH inhibition [33,34].Sulfonyl carbamates could be generated from various sulfonamides (1a-l) by reaction with methyl chloroformate, as previously reported [35].Sulfonyl carbamates (3a-l), upon treatment with 1-adamantylamine in refluxing toluene followed by evaporation and trituration, resulted in white solid sulfonylurea products (4a-l).Following our previously published protocol, compound sulfonyl ureas of type 4 could be converted into sulfonyl thioureas in a one-pot manner using N, N-diisopropylethylamine (DIPEA) and phosphoryl chloride (POCl 3 ) followed by treatment with sodium thiosulfate dissolved in a mixture of dioxane and water (8:1) to afford 6a-d as white solids [36].Compound 6d was processed to 7 by the treatment of iodomethane in the same pot (Scheme 1).While maintaining the 4-OCF 3 group, compound 4f was converted to 8a and 8b using 7M ammonia solution in methanol and methyl amine, respectively, by treatment of the imidoyl chloride intermediate obtained from treatment of DIPEA/POCl 3 in toluene.In a parallel set of reactions, the imidoyl chloride intermediate was dissolved in DCM and was treated with various hydrazine moieties.The reaction mixtures were stirred at room temperature for 2 h to afford several hydrazine derivatives, 9a-d (Scheme 1).
mide-based compounds for their potential to inhibit sEH.Recently, glimepiride, a sulfonyl urea compound, was reported to have sEH inhibitory properties, although previously, sulfonyl ureas were shown to not have sEH inhibition [33,34].Sulfonyl carbamates could be generated from various sulfonamides (1a-l) by reaction with methyl chloroformate, as previously reported [35].Sulfonyl carbamates (3a-l), upon treatment with 1-adamantylamine in refluxing toluene followed by evaporation and trituration, resulted in white solid sulfonylurea products (4a-l).Following our previously published protocol, compound sulfonyl ureas of type 4 could be converted into sulfonyl thioureas in a one-pot manner using N, N-diisopropylethylamine (DIPEA) and phosphoryl chloride (POCl3) followed by treatment with sodium thiosulfate dissolved in a mixture of dioxane and water (8:1) to afford 6a-d as white solids [36].Compound 6d was processed to 7 by the treatment of iodomethane in the same pot (Scheme 1).While maintaining the 4-OCF3 group, compound 4f was converted to 8a and 8b using 7M ammonia solution in methanol and methyl amine, respectively, by treatment of the imidoyl chloride intermediate obtained from treatment of DIPEA/POCl3 in toluene.In a parallel set of reactions, the imidoyl chloride intermediate was dissolved in DCM and was treated with various hydrazine moieties.The reaction mixtures were stirred at room temperature for 2 h to afford several hydrazine derivatives, 9a-d (Scheme 1).
In another set of compounds, we modified the earlier route and attached various alkyl amines as depicted in Scheme 2. Sulfonyl carbamates 3b-d were treated with aminoadamantan-1-ol in refluxing toluene to yield sulfonyl ureas 10a-c.Compound 3f was subjected to react with a set of various alkylamines including bicycloamines, adamantan-2-amine, 4-(trifluoromethyl)piperidine, cyclohexylamine, and memantine hydrochloride to form a series of ureas (compounds 11a-d, 12-14, and 15a-b) following a previous protocol (Scheme 2).In another scheme of reactions, compound 3f was refluxed with tertbutyl 4-aminopiperidine-1-carboxylate to form urea 16 (Scheme 3).Deprotection of the Boc group by trifluoroacetic acid (TFA) led to compound 17, which was further reacted with 1-isocyanatoadamantane in the presence of triethylamine in DMF solvent to afford compound 18, containing two urea groups (Scheme 3).In another set of compounds, we modified the earlier route and attached various alkyl amines as depicted in Scheme 2. Sulfonyl carbamates 3b-d were treated with aminoadamantan-1-ol in refluxing toluene to yield sulfonyl ureas 10a-c.Compound 3f was subjected to react with a set of various alkylamines including bicycloamines, adamantan-2-amine, 4-(trifluoromethyl)piperidine, cyclohexylamine, and memantine hydrochloride to form a series of ureas (compounds 11a-d, 12-14, and 15a-b) following a previous protocol (Scheme 2).In another scheme of reactions, compound 3f was refluxed with tert-butyl 4-aminopiperidine-1-carboxylate to form urea 16 (Scheme 3).Deprotection of the Boc group by trifluoroacetic acid (TFA) led to compound 17, which was further reacted with 1-isocyanatoadamantane in the presence of triethylamine in DMF solvent to afford compound 18, containing two urea groups (Scheme 3).

Activity-Guided Design of sEH Inhibitors
The rational development of the sEH inhibitors was guided by their activity profile as measured in in vitro assays through a structure-activity relationship (SAR).The mammalian sEH is a bifunctional enzyme containing an N-terminal phosphatase region and a C-terminal hydrolase domain [37,38].The hydrolysis process of EETs is catalyzed by the C-terminal domain to the corresponding diols.Two tyrosine residues (Tyr381 and Tyr465) make up the hydrolase catalytic pocket of the C-terminal hydrolase.These residues connect with the oxygen atom of the epoxide through two hydrogen bonds.On the other side of Tyr381 and Tyr465, the nucleophilic carboxylic acid Asp333 of the catalytic triad Asp333-Asp495-His523 is orientated and activated by His523 and Asp495 and attacks the epoxide carbon backbone to form an ester bond with the opening epoxide.The corresponding diols are produced because of this ester's subsequent hydrolysis [39,40].The conversion of EETs to their corresponding diols by sEH diminishes the biological activity of epoxides [8,41].
Several findings showed that urea and amide groups fit well in the hydrolase catalytic pocket [14,42].Specifically, the carbonyl oxygen of amide or urea is involved in hydrogen bond formation with tyrosine residues and the NH of ureas or amides serves as a hydrogen bond donor to Asp333.Hence, urea and amide derivatives have been developed as competitive, reversible, and highly potent sEH inhibitors [43,44].Urea derivatives have been created continually due to their high potency, with a focus on improving their solubility and bioavailability to potentially cure EpFA-related disorders.
The first enhancement to the sEH inhibitor design from DCU was the addition of a flexible chain, such as 12-(3-adamantan-1-ylureido)dodecanoic acid (AUDA), on one side of the urea function.While keeping the inhibitory effect, the addition of a flexible side chain greatly increased AUDA's water solubility and decreased the melting point (Figure 1B) [42].The adamantyl group utilized in AUDA produced remarkably high detection sensitivity in an LC-MS analysis as well as high potency in the target sEH in the early stages of sEHi development.
Our study features the synthesis and evaluation of urea derivatives bearing aryl sulfonyl groups as soluble epoxide hydrolase inhibitors.We initiated our design from the urea core, incorporating key structural requisites for manifesting significant sEH inhibition.Strategic substitution at specific positions through SAR development in the lead led to potent inhibitors on human sEH.AUDA was used as the positive control for the fluorescence assay, against which the inhibitory activity of the compounds (evaluated in terms of the IC50 value) was monitored (Table 1).Most of the urea and thiourea compounds were found to have nanomolar to sub micromolar potency against human recombinant sEH.We strategically kept the adamantyl group attached to the urea core as the catalytic site is

Activity-Guided Design of sEH Inhibitors
The rational development of the sEH inhibitors was guided by their activity profile as measured in in vitro assays through a structure-activity relationship (SAR).The mammalian sEH is a bifunctional enzyme containing an N-terminal phosphatase region and a C-terminal hydrolase domain [37,38].The hydrolysis process of EETs is catalyzed by the C-terminal domain to the corresponding diols.Two tyrosine residues (Tyr381 and Tyr465) make up the hydrolase catalytic pocket of the C-terminal hydrolase.These residues connect with the oxygen atom of the epoxide through two hydrogen bonds.On the other side of Tyr381 and Tyr465, the nucleophilic carboxylic acid Asp333 of the catalytic triad Asp333-Asp495-His523 is orientated and activated by His523 and Asp495 and attacks the epoxide carbon backbone to form an ester bond with the opening epoxide.The corresponding diols are produced because of this ester's subsequent hydrolysis [39,40].The conversion of EETs to their corresponding diols by sEH diminishes the biological activity of epoxides [8,41].
Several findings showed that urea and amide groups fit well in the hydrolase catalytic pocket [14,42].Specifically, the carbonyl oxygen of amide or urea is involved in hydrogen bond formation with tyrosine residues and the NH of ureas or amides serves as a hydrogen bond donor to Asp333.Hence, urea and amide derivatives have been developed as competitive, reversible, and highly potent sEH inhibitors [43,44].Urea derivatives have been created continually due to their high potency, with a focus on improving their solubility and bioavailability to potentially cure EpFA-related disorders.
The first enhancement to the sEH inhibitor design from DCU was the addition of a flexible chain, such as 12-(3-adamantan-1-ylureido)dodecanoic acid (AUDA), on one side of the urea function.While keeping the inhibitory effect, the addition of a flexible side chain greatly increased AUDA's water solubility and decreased the melting point (Figure 1B) [42].The adamantyl group utilized in AUDA produced remarkably high detection sensitivity in an LC-MS analysis as well as high potency in the target sEH in the early stages of sEHi development.
Our study features the synthesis and evaluation of urea derivatives bearing aryl sulfonyl groups as soluble epoxide hydrolase inhibitors.We initiated our design from the urea core, incorporating key structural requisites for manifesting significant sEH inhibition.Strategic substitution at specific positions through SAR development in the lead led to potent inhibitors on human sEH.AUDA was used as the positive control for the fluorescence assay, against which the inhibitory activity of the compounds (evaluated in terms of the IC 50 value) was monitored (Table 1).Most of the urea and thiourea compounds were found to have nanomolar to sub micromolar potency against human recombinant sEH.We strategically kept the adamantyl group attached to the urea core as the catalytic site is lipophilic in nature.To increase the polarity and improve the solubility in water, we maintained the substituted aromatic sulfonyl group.To begin with, we changed the aromatic substitution to determine how that plays a role in potency (4a-l).Compound 4a with the phenyl substitution was found to manifest moderate sEH inhibition, with an IC 50 value of 0.10 µM against human recombinant sEH (Table 2).The incorporation of a methyl group at the para position of the phenyl group markedly enhanced potency in compound 4b (IC 50 16.3nM).More, bulkier groups at the para position resulted in a drastic drop in potency (4c-d).Interestingly, when alkoxy groups such as 4-OMe and 4-OCF 3 were present there, it showed very significant sEH inhibitory activity, having IC 50 values of 1.69 and 1.21 nM, respectively (Table 2, Figure S1).But when the position of the -OCF 3 was altered from para to meta (4g) and ortho (4h), sEH inhibition decreased to a significant extent (Table 1).In the next set of compounds, we replaced the para substituents with fluoro and chloro groups in compounds 4j and 4k.Both were promising sEH inhibitors, the latter being more active with IC 50 2 nM.When the substituted phenyl group was replaced with the naphthyl group in compound 4l, it resulted in a marked enhancement in the potency (IC 50 1.21 nM).In our next modifications, we replaced the urea group with a thiourea in compounds 6a-d and methylated thiourea (7).Out of them, 6a and 6d were active, having IC 50 values 0.13 µM and 4.66 nM, respectively.In the next set of compounds (8a-b and 9ad), we incorporated amine and hydrazine substituents, which led to a marked decrease in the potency for most of the compounds.The only exception was compound 9a, with an IC 50 value of 14.2 nM (Table 2).When 1-adamantyl attachment to the urea was substituted with the 3-hydroxy-1-adamantyl group (10a-c) and several bicycloamines (11a-d), the inhibitory activity diminished, advocating for the fact that the presence of 1-adamantylamine is very important.In a subsequent development, we attached an isomer 2-adamantyl group (12) and replaced the 1-adamantyl group with a couple of aliphatic cyclohexyl groups (13-14, 16-17) but none of them showed any activity (Table 1).3,5-Dimethyl-1-adamantyl group replacements in compounds 15a and 15b were found to have significant sEH inhibition, with IC 50 values of 3.87 nM and 0.95 µM, respectively.We synthesized compound 18 with two urea groups in it.It showed sEH inhibition, with an IC 50 value of 38.7 nM (Figure S1).In a nutshell, we can conclude that the strategic structure-activity relationship development led to a set of modestly potent sEH inhibitors with substituents such as adamantyl and substituted phenyl groups in the urea pharmacophore.In the sulfonyl urea-based sEHi, we found the adamantyl group to be indispensable for sEH inhibition in the sulfonyl ureas series.To our satisfaction, we found that most of the urea compounds exhibited nanomolar to sub micromolar sEH inhibition, with compounds 4f and 4l being the most active inhibitors, having IC 50 values of 2.94 and 1.69 nM, respectively, against human recombinant sEH (Table 2).We then investigated their potency against mouse recombinant sEH.Surprisingly, although somewhat precedented, many compounds that were potent in hsEH showed a lower order of potency in mouse recombinant sEH [27,45].lipophilic in nature.To increase the polarity and improve the solubility in water, we maintained the substituted aromatic sulfonyl group.To begin with, we changed the aromatic substitution to determine how that plays a role in potency (4a-l).Compound 4a with the phenyl substitution was found to manifest moderate sEH inhibition, with an IC50 value of 0.10 µM against human recombinant sEH (Table 2).The incorporation of a methyl group at the para position of the phenyl group markedly enhanced potency in compound 4b (IC50 16.3 nM).More, bulkier groups at the para position resulted in a drastic drop in potency (4c-d).Interestingly, when alkoxy groups such as 4-OMe and 4-OCF3 were present there, it showed very significant sEH inhibitory activity, having IC50 values of 1.69 and 1.21 nM, respectively (Table 2, Figure S1).But when the position of the -OCF3 was altered from para to meta (4g) and ortho (4h), sEH inhibition decreased to a significant extent (Table 1).In the next set of compounds, we replaced the para substituents with fluoro and chloro groups in compounds 4j and 4k.Both were promising sEH inhibitors, the latter being more active with IC50 2 nM.When the substituted phenyl group was replaced with the naphthyl group in compound 4l, it resulted in a marked enhancement in the potency (IC50 1.21 nM).In our next modifications, we replaced the urea group with a thiourea in compounds 6a-d and methylated thiourea (7).Out of them, 6a and 6d were active, having IC50 values 0.13 µM and 4.66 nM, respectively.In the next set of compounds (8a-b and 9a-d), we incorporated amine and hydrazine substituents, which led to a marked decrease in the potency for most of the compounds.The only exception was compound 9a, with an IC50 value of 14.2 nM (Table 2).When 1-adamantyl attachment to the urea was substituted with the 3-hydroxy-1-adamantyl group (10a-c) and several bicycloamines (11a-d), the inhibitory activity diminished, advocating for the fact that the presence of 1-adamantylamine is very important.In a subsequent development, we attached an isomer 2-adamantyl group (12) and replaced the 1-adamantyl group with a couple of aliphatic cyclohexyl groups (13-14, 16-17) but none of them showed any activity (Table 1).3,5-Dimethyl-1-adamantyl group replacements in compounds 15a and 15b were found to have significant sEH inhibition, with IC50 values of 3.87 nM and 0.95 µM, respectively.We synthesized compound 18 with two urea groups in it.It showed sEH inhibition, with an IC50 value of 38.7 nM (Figure S1).In a nutshell, we can conclude that the strategic structure-activity relationship development led to a set of modestly potent sEH inhibitors with substituents such as adamantyl and substituted phenyl groups in the urea pharmacophore.In the sulfonyl urea-based sEHi, we found the adamantyl group to be indispensable for sEH inhibition in the sulfonyl ureas series.To our satisfaction, we found that most of the urea compounds exhibited nanomolar to sub micromolar sEH inhibition, with compounds 4f and 4l being the most active inhibitors, having IC50 values of 2.94 and 1.69 nM, respectively, against human recombinant sEH (Table 2).We then investigated their potency against mouse recombinant sEH.Surprisingly, although somewhat precedented, many compounds that were potent in hsEH showed a lower order of potency in mouse recombinant sEH [27,45].lipophilic in nature.To increase the polarity and improve the solubility in water, we maintained the substituted aromatic sulfonyl group.To begin with, we changed the aromatic substitution to determine how that plays a role in potency (4a-l).Compound 4a with the phenyl substitution was found to manifest moderate sEH inhibition, with an IC50 value of 0.10 µM against human recombinant sEH (Table 2).The incorporation of a methyl group at the para position of the phenyl group markedly enhanced potency in compound 4b (IC50 16.3 nM).More, bulkier groups at the para position resulted in a drastic drop in potency (4c-d).Interestingly, when alkoxy groups such as 4-OMe and 4-OCF3 were present there, it showed very significant sEH inhibitory activity, having IC50 values of 1.69 and 1.21 nM, respectively (Table 2, Figure S1).But when the position of the -OCF3 was altered from para to meta (4g) and ortho (4h), sEH inhibition decreased to a significant extent (Table 1).In the next set of compounds, we replaced the para substituents with fluoro and chloro groups in compounds 4j and 4k.Both were promising sEH inhibitors, the latter being more active with IC50 2 nM.When the substituted phenyl group was replaced with the naphthyl group in compound 4l, it resulted in a marked enhancement in the potency (IC50 1.21 nM).In our next modifications, we replaced the urea group with a thiourea in compounds 6a-d and methylated thiourea (7).Out of them, 6a and 6d were active, having IC50 values 0.13 µM and 4.66 nM, respectively.In the next set of compounds (8a-b and 9a-d), we incorporated amine and hydrazine substituents, which led to a marked decrease in the potency for most of the compounds.The only exception was compound 9a, with an IC50 value of 14.2 nM (Table 2).When 1-adamantyl attachment to the urea was substituted with the 3-hydroxy-1-adamantyl group (10a-c) and several bicycloamines (11a-d), the inhibitory activity diminished, advocating for the fact that the presence of 1-adamantylamine is very important.In a subsequent development, we attached an isomer 2-adamantyl group (12) and replaced the 1-adamantyl group with a couple of aliphatic cyclohexyl groups (13-14, 16-17) but none of them showed any activity (Table 1).3,5-Dimethyl-1-adamantyl group replacements in compounds 15a and 15b were found to have significant sEH inhibition, with IC50 values of 3.87 nM and 0.95 µM, respectively.We synthesized compound 18 with two urea groups in it.It showed sEH inhibition, with an IC50 value of 38.7 nM (Figure S1).In a nutshell, we can conclude that the strategic structure-activity relationship development led to a set of modestly potent sEH inhibitors with substituents such as adamantyl and substituted phenyl groups in the urea pharmacophore.In the sulfonyl urea-based sEHi, we found the adamantyl group to be indispensable for sEH inhibition in the sulfonyl ureas series.To our satisfaction, we found that most of the urea compounds exhibited nanomolar to sub micromolar sEH inhibition, with compounds 4f and 4l being the most active inhibitors, having IC50 values of 2.94 and 1.69 nM, respectively, against human recombinant sEH (Table 2).We then investigated their potency against mouse recombinant sEH.Surprisingly, although somewhat precedented, many compounds that were potent in hsEH showed a lower order of potency in mouse recombinant sEH [27,45].

Molecular Docking Study with 4f and 4l
To evaluate the binding mode of sulfonyl urea-based sEHi, we conducted molecular docking of lead compounds (Figure 2A-D).The structure of sEHi complexed to LK864 (1-[(4~{S})-9-propan-2-ylsulfonyl-1-oxa-9-azaspiro[5.5]undecan-4-yl]-3-[[4-(trifluoromethyloxy)phenyl]methyl]urea (PDB:6FR2) was chosen for modeling because it produced the best prediction of the binding pose of the "native" ligand compared to re-docking for other structures of sEH-urea complexes in PDB.RMSD for the prediction was 0.975 Å. Sulfonyl ureas indeed dock similarly to ureas: nitrogens of the urea group form hydrogen bonds with Asp335 carboxyl, while carbonyl forms a hydrogen bond with the hydroxyl of Tyr383 (Figure 2).Although the sulfonyl group is not involved in hydrogen bond formation, its computed contribution to binding energy per atom in 4f (4.3%) exceeds that of the urea (3.75%) and (trifluoromethyloxy)phenyl groups (2.2%).Only the adamantyl group contributes more, 5% per atom.Remarkably, adamantyl groups of AUDA, 4f, and 4l (Figure 2A-C) overlap completely and fill the corresponding part of the pocket cavity completely, thus explaining the preference for the group in the most active compounds.It is possible that another bulky group may work as well or better during the optimization of inhibitors if it would allow for additional interactions with nearby Q384.Meanwhile, a subpocket occupied by the (trifluoromethyloxy)phenyl group of 4f or the naphthyl moiety of 4l is not filled completely.Thus, the affinity and solubility of the derivatives can be further improved by hydrogen bonding to the Ser415 sidechain of the pocket and interactions with the main chain of the 415-419 loop.The carboxyl group of AUDA forms an additional hydrogen bond with His420 (Figure 2A), with ionic interaction also possible.However, the high flexibility of AUDA's long hydrocarbon chain, leading to an entropic penalty, is a likely reason for the observed higher Kd that is improved in more rigid derivatives.Notably, the compounds in this work had a higher potency for human sEH but showed a lower order of potency for mouse sEH.Docking results, however, were unable to pinpoint the species-difference inhibitory potency seen for this set of compounds.

Molecular Docking Study with 4f and 4l
To evaluate the binding mode of sulfonyl urea-based sEHi, we conducted molecular docking of lead compounds (Figure 2A-D).The structure of sEHi complexed to LK864 (1-[(4~{S})-9-propan-2-ylsulfonyl-1-oxa-9-azaspiro[5.5]undecan-4-yl]-3-[[4-(trifluoromethyloxy)phenyl]methyl]urea (PDB:6FR2) was chosen for modeling because it produced the best prediction of the binding pose of the "native" ligand compared to re-docking for other structures of sEH-urea complexes in PDB.RMSD for the prediction was 0.975 Å. Sulfonyl ureas indeed dock similarly to ureas: nitrogens of the urea group form hydrogen bonds with Asp335 carboxyl, while carbonyl forms a hydrogen bond with the hydroxyl of Tyr383 (Figure 2).Although the sulfonyl group is not involved in hydrogen bond formation, its computed contribution to binding energy per atom in 4f (4.3%) exceeds that of the urea (3.75%) and (trifluoromethyloxy)phenyl groups (2.2%).Only the adamantyl group contributes more, 5% per atom.Remarkably, adamantyl groups of AUDA, 4f, and 4l (Figure 2A-C) overlap completely and fill the corresponding part of the pocket cavity completely, thus explaining the preference for the group in the most active compounds.It is possible that another bulky group may work as well or better during the optimization of inhibitors if it would allow for additional interactions with nearby Q384.Meanwhile, a subpocket occupied by the (trifluoromethyloxy)phenyl group of 4f or the naphthyl moiety of 4l is not filled completely.Thus, the affinity and solubility of the derivatives can be further improved by hydrogen bonding to the Ser415 sidechain of the pocket and interactions with the main chain of the 415-419 loop.The carboxyl group of AUDA forms an additional hydrogen bond with His420 (Figure 2A), with ionic interaction also possible.However, the high flexibility of AUDA's long hydrocarbon chain, leading to an entropic penalty, is a likely reason for the observed higher Kd that is improved in more rigid derivatives.Notably, the compounds in this work had a higher potency for human sEH but showed a lower order of potency for mouse sEH.Docking results, however, were unable to pinpoint the species-difference inhibitory potency seen for this set of compounds.

Molecular Docking Study with 4f and 4l
To evaluate the binding mode of sulfonyl urea-based sEHi, we conducted molecular docking of lead compounds (Figure 2A-D).The structure of sEHi complexed to LK864 (1-[(4~{S})-9-propan-2-ylsulfonyl-1-oxa-9-azaspiro[5.5]undecan-4-yl]-3-[[4-(trifluoromethyloxy)phenyl]methyl]urea (PDB:6FR2) was chosen for modeling because it produced the best prediction of the binding pose of the "native" ligand compared to re-docking for other structures of sEH-urea complexes in PDB.RMSD for the prediction was 0.975 Å. Sulfonyl ureas indeed dock similarly to ureas: nitrogens of the urea group form hydrogen bonds with Asp335 carboxyl, while carbonyl forms a hydrogen bond with the hydroxyl of Tyr383 (Figure 2).Although the sulfonyl group is not involved in hydrogen bond formation, its computed contribution to binding energy per atom in 4f (4.3%) exceeds that of the urea (3.75%) and (trifluoromethyloxy)phenyl groups (2.2%).Only the adamantyl group contributes more, 5% per atom.Remarkably, adamantyl groups of AUDA, 4f, and 4l (Figure 2A-C) overlap completely and fill the corresponding part of the pocket cavity completely, thus explaining the preference for the group in the most active compounds.It is possible that another bulky group may work as well or better during the optimization of inhibitors if it would allow for additional interactions with nearby Q384.Meanwhile, a subpocket occupied by the (trifluoromethyloxy)phenyl group of 4f or the naphthyl moiety of 4l is not filled completely.Thus, the affinity and solubility of the derivatives can be further improved by hydrogen bonding to the Ser415 sidechain of the pocket and interactions with the main chain of the 415-419 loop.The carboxyl group of AUDA forms an additional hydrogen bond with His420 (Figure 2A), with ionic interaction also possible.However, the high flexibility of AUDA's long hydrocarbon chain, leading to an entropic penalty, is a likely reason for the observed higher Kd that is improved in more rigid derivatives.Notably, the compounds in this work had a higher potency for human sEH but showed a lower order of potency for mouse sEH.Docking results, however, were unable to pinpoint the species-difference inhibitory potency seen for this set of compounds.

Molecular Docking Study with 4f and 4l
To evaluate the binding mode of sulfonyl urea-based sEHi, we conducted molecular docking of lead compounds (Figure 2A-D).The structure of sEHi complexed to LK864 (1-[(4~{S})-9-propan-2-ylsulfonyl-1-oxa-9-azaspiro [5.5]undecan-4-yl]-3-[[4-(trifluoromethyloxy)phenyl]methyl]urea (PDB:6FR2) was chosen for modeling because it produced the best prediction of the binding pose of the "native" ligand compared to re-docking for other structures of sEH-urea complexes in PDB.RMSD for the prediction was 0.975 Å. Sulfonyl ureas indeed dock similarly to ureas: nitrogens of the urea group form hydrogen bonds with Asp335 carboxyl, while carbonyl forms a hydrogen bond with the hydroxyl of Tyr383 (Figure 2).Although the sulfonyl group is not involved in hydrogen bond formation, its computed contribution to binding energy per atom in 4f (4.3%) exceeds that of the urea (3.75%) and (trifluoromethyloxy)phenyl groups (2.2%).Only the adamantyl group contributes more, 5% per atom.Remarkably, adamantyl groups of AUDA, 4f, and 4l (Figure 2A-C) overlap completely and fill the corresponding part of the pocket cavity completely, thus explaining the preference for the group in the most active compounds.It is possible that another bulky group may work as well or better during the optimization of inhibitors if it would allow for additional interactions with nearby Q384.Meanwhile, a subpocket occupied by the (trifluoromethyloxy)phenyl group of 4f or the naphthyl moiety of 4l is not filled completely.Thus, the affinity and solubility of the derivatives can be further improved by hydrogen bonding to the Ser415 sidechain of the pocket and interactions with the main chain of the 415-419 loop.The carboxyl group of AUDA forms an additional hydrogen bond with His420 (Figure 2A), with ionic interaction also possible.However, the high flexibility of AUDA's long hydrocarbon chain, leading to an entropic penalty, is a likely reason for the observed higher Kd that is improved in more rigid derivatives.Notably, the compounds in this work had a higher potency for human sEH but showed a lower order of potency for mouse sEH.Docking results, however, were unable to pinpoint the species-difference inhibitory potency seen for this set of compounds.

Molecular Docking Study with 4f and 4l
To evaluate the binding mode of sulfonyl urea-based sEHi, we conducted molecular docking of lead compounds (Figure 2A-D).The structure of sEHi complexed to LK864 (1-[(4~{S})-9-propan-2-ylsulfonyl-1-oxa-9-azaspiro [5.5]undecan-4-yl]-3-[[4-(trifluoromethyloxy)phenyl]methyl]urea (PDB:6FR2) was chosen for modeling because it produced the best prediction of the binding pose of the "native" ligand compared to re-docking for other structures of sEH-urea complexes in PDB.RMSD for the prediction was 0.975 Å. Sulfonyl ureas indeed dock similarly to ureas: nitrogens of the urea group form hydrogen bonds with Asp335 carboxyl, while carbonyl forms a hydrogen bond with the hydroxyl of Tyr383 (Figure 2).Although the sulfonyl group is not involved in hydrogen bond formation, its computed contribution to binding energy per atom in 4f (4.3%) exceeds that of the urea (3.75%) and (trifluoromethyloxy)phenyl groups (2.2%).Only the adamantyl group contributes more, 5% per atom.Remarkably, adamantyl groups of AUDA, 4f, and 4l (Figure 2A-C) overlap completely and fill the corresponding part of the pocket cavity completely, thus explaining the preference for the group in the most active compounds.It is possible that another bulky group may work as well or better during the optimization of inhibitors if it would allow for additional interactions with nearby Q384.Meanwhile, a subpocket occupied by the (trifluoromethyloxy)phenyl group of 4f or the naphthyl moiety of 4l is not filled completely.Thus, the affinity and solubility of the derivatives can be further improved by hydrogen bonding to the Ser415 sidechain of the pocket and interactions with the main chain of the 415-419 loop.The carboxyl group of AUDA forms an additional hydrogen bond with His420 (Figure 2A), with ionic interaction also possible.However, the high flexibility of AUDA's long hydrocarbon chain, leading to an entropic penalty, is a likely reason for the observed higher Kd that is improved in more rigid derivatives.Notably, the compounds in this work had a higher potency for human sEH but showed a lower order of potency for mouse sEH.Docking results, however, were unable to pinpoint the species-difference inhibitory potency seen for this set of compounds.

Molecular Docking Study with 4f and 4l
To evaluate the binding mode of sulfonyl urea-based sEHi, we conducted molecular docking of lead compounds (Figure 2A-D).The structure of sEHi complexed to LK864 (1-[(4~{S})-9-propan-2-ylsulfonyl-1-oxa-9-azaspiro [5.5]undecan-4-yl]-3-[[4-(trifluoromethyloxy)phenyl]methyl]urea (PDB:6FR2) was chosen for modeling because it produced the best prediction of the binding pose of the "native" ligand compared to re-docking for other structures of sEH-urea complexes in PDB.RMSD for the prediction was 0.975 Å. Sulfonyl ureas indeed dock similarly to ureas: nitrogens of the urea group form hydrogen bonds with Asp335 carboxyl, while carbonyl forms a hydrogen bond with the hydroxyl of Tyr383 (Figure 2).Although the sulfonyl group is not involved in hydrogen bond formation, its computed contribution to binding energy per atom in 4f (4.3%) exceeds that of the urea (3.75%) and (trifluoromethyloxy)phenyl groups (2.2%).Only the adamantyl group contributes more, 5% per atom.Remarkably, adamantyl groups of AUDA, 4f, and 4l (Figure 2A-C) overlap completely and fill the corresponding part of the pocket cavity completely, thus explaining the preference for the group in the most active compounds.It is possible that another bulky group may work as well or better during the optimization of inhibitors if it would allow for additional interactions with nearby Q384.Meanwhile, a subpocket occupied by the (trifluoromethyloxy)phenyl group of 4f or the naphthyl moiety of 4l is not filled completely.Thus, the affinity and solubility of the derivatives can be further improved by hydrogen bonding to the Ser415 sidechain of the pocket and interactions with the main chain of the 415-419 loop.The carboxyl group of AUDA forms an additional hydrogen bond with His420 (Figure 2A), with ionic interaction also possible.However, the high flexibility of AUDA's long hydrocarbon chain, leading to an entropic penalty, is a likely reason for the observed higher Kd that is improved in more rigid derivatives.Notably, the compounds in this work had a higher potency for human sEH but showed a lower order of potency for mouse sEH.Docking results, however, were unable to pinpoint the species-difference inhibitory potency seen for this set of compounds.

Molecular Docking Study with 4f and 4l
To evaluate the binding mode of sulfonyl urea-based sEHi, we conducted molecular docking of lead compounds (Figure 2A-D).The structure of sEHi complexed to LK864 (1-[(4~{S})-9-propan-2-ylsulfonyl-1-oxa-9-azaspiro [5.5]undecan-4-yl]-3-[ [4-(trifluoromethyloxy) phenyl]methyl]urea (PDB:6FR2) was chosen for modeling because it produced the best prediction of the binding pose of the "native" ligand compared to re-docking for other structures of sEH-urea complexes in PDB.RMSD for the prediction was 0.975 Å. Sulfonyl ureas indeed dock similarly to ureas: nitrogens of the urea group form hydrogen bonds with Asp335 carboxyl, while carbonyl forms a hydrogen bond with the hydroxyl of Tyr383 (Figure 2).Although the sulfonyl group is not involved in hydrogen bond formation, its computed contribution to binding energy per atom in 4f (4.3%) exceeds that of the urea (3.75%) and (trifluoromethyloxy)phenyl groups (2.2%).Only the adamantyl group contributes more, 5% per atom.Remarkably, adamantyl groups of AUDA, 4f, and 4l (Figure 2A-C) overlap completely and fill the corresponding part of the pocket cavity completely, thus explaining the preference for the group in the most active compounds.It is possible that another bulky group may work as well or better during the optimization of inhibitors if it would allow for additional interactions with nearby Q384.Meanwhile, a subpocket occupied by the (trifluoromethyloxy)phenyl group of 4f or the naphthyl moiety of 4l is not filled completely.Thus, the affinity and solubility of the derivatives can be further improved by hydrogen bonding to the Ser415 sidechain of the pocket and interactions with the main chain of the 415-419 loop.The carboxyl group of AUDA forms an additional hydrogen bond with His420 (Figure 2A), with ionic interaction also possible.However, the high flexibility of AUDA's long hydrocarbon chain, leading to an entropic penalty, is a likely reason for the observed higher Kd that is improved in more rigid derivatives.
Notably, the compounds in this work had a higher potency for human sEH but showed a lower order of potency for mouse sEH.Docking results, however, were unable to pinpoint the species-difference inhibitory potency seen for this set of compounds.[46,47].Residues involved in contact with the compounds are numbered.

In Vitro ADME and In Vivo Pharmacokinetic Study
Based on the sEH inhibition, we shortlisted compounds 4f and 4l, with similar structural features except for their nature in terms of aromatic substitution, to evaluate further.4f with 4-trifluoromethoxyphenyl and 4l with the naphthyl group were evaluated in a panel of in vitro ADME assays [48,49].They were assessed through a series of established assays, providing us insights into how in vitro pharmacokinetic properties are affected by structural modifications in the scaffold.We investigated aqueous solubility at pH 7.4, Caco-2 permeability, and plasma protein binding, and performed a microsomal stability.assay, through a series of established in vitro experiments (Table 3).Compound 4f was found to have moderate aqueous solubility as compared to 4l.This is due to the presence of a more polar -OCF3 group in 4f in comparison to the highly hydrophobic naphthyl group in 4l.It is noteworthy that both 4f and 4l demonstrated good human plasma stability even after 2 h.Caco-2 permeability for these compounds showed that these compounds are brain penetrable, and their efflux ratios (<2) indicated that 4f and 4l are not Pgp substrates.4f and other sulfonyl urea-based sEHi were docked into PDB:6FR2 using ICM-Pro software version 3.9-3b (Molsoft, San Diego, CA, USA) [46,47].Residues involved in contact with the compounds are numbered.

In Vitro ADME and In Vivo Pharmacokinetic Study
Based on the sEH inhibition, we shortlisted compounds 4f and 4l, with similar structural features except for their nature in terms of aromatic substitution, to evaluate further.4f with 4-trifluoromethoxyphenyl and 4l with the naphthyl group were evaluated in a panel of in vitro ADME assays [48,49].They were assessed through a series of established assays, providing us insights into how in vitro pharmacokinetic properties are affected by structural modifications in the scaffold.We investigated aqueous solubility at pH 7.4, Caco-2 permeability, and plasma protein binding, and performed a microsomal stability.
Assay, through a series of established in vitro experiments (Table 3).Compound 4f was found to have moderate aqueous solubility as compared to 4l.This is due to the presence of a more polar -OCF 3 group in 4f in comparison to the highly hydrophobic naphthyl group in 4l.It is noteworthy that both 4f and 4l demonstrated good human plasma stability even after 2 h.Caco-2 permeability for these compounds showed that these compounds are brain penetrable, and their efflux ratios (<2) indicated that 4f and 4l are not Pgp substrates.
Next, we proceeded with in vivo investigations using compound 4f after taking into account all of the previously mentioned parameters as all of the in vitro pharmacokinetic parameters of this compound are in the ideal range.We have explored the in vivo pharmacokinetic profile of compounds 4f, 4l, and 15a.Both the compounds 4f and 15a are structurally very similar, only differing in the aliphatic amine attachment.We found that the plasma half-life was short for compounds 4f and 15a (<2 h) after both oral and intraperitoneal administration (10 mg/kg) of the compounds in an in vivo pharmacokinetics study (Figure 3), although the C max value of 4f was found to be better than that of 15a.Compound 4l also exhibited a promising C max value after oral administration (10 mg/kg), though the half-life value is similar to compound 4f.Their intraperitoneal PK profiles were also evaluated (Supplementary Materials; Figure S2).Next, we proceeded with in vivo investigations using compound 4f after taking into account all of the previously mentioned parameters as all of the in vitro pharmacokinetic parameters of this compound are in the ideal range.We have explored the in vivo pharmacokinetic profile of compounds 4f, 4l, and 15a.Both the compounds 4f and 15a are structurally very similar, only differing in the aliphatic amine attachment.We found that the plasma half-life was short for compounds 4f and 15a (<2 h) after both oral and intraperitoneal administration (10 mg/kg) of the compounds in an in vivo pharmacokinetics study (Figure 3), although the Cmax value of 4f was found to be better than that of 15a.Compound 4l also exhibited a promising Cmax value after oral administration (10 mg/kg), though the half-life value is similar to compound 4f.Their intraperitoneal PK profiles were also evaluated (Supplementary Materials; Figure S2).

In Vivo Therapeutic Efficacy Study
3.4.1.Analgesic Effects of 4f and 4l in Pain Models sEH inhibitors have been implicated to have analgesic properties.Therefore, we investigated first the analgesic effects of 4f and 4l in tail-flick and hot plate pain models.We found that both 4f and 4l demonstrate similar analgesic activity to AUDA after a single intraperitoneal injection at 10 mg/kg in a tail-flick model (Figure 4).However, none of the sEH inhibitors, including AUDA, showed any analgesic effect in the hot plate assay, suggesting mechanisms other than CNS-mediated.We found that both 4f and 4l demonstrate similar analgesic activity to AUDA after a single intraperitoneal injection at 10 mg/kg in a tail-flick model (Figure 4).However, none of the sEH inhibitors, including AUDA, showed any analgesic effect in the hot plate assay, suggesting mechanisms other than CNS-mediated.

Effects of 4f and 4l on Lipopolysaccharide (LPS)-Induced Acute Lung Inflammation
The increased activity of soluble epoxide hydrolase promotes inflammatory conditions.Acute lung injury (ALI) is a state of acute inflammation that results in the disruption of the lung endothelium and epithelial cells [50].Characteristic features of ALI include the loss of alveolar-capillary membrane integrity, vascular permeability, excessive neutrophil transmigration, and the release of pro-inflammatory mediators and cytotoxic components.LPS causes acute inflammation resulting in an increased number of immune cell infiltrations to the lungs and the expression of cytokines and other inflammatory mediators, eventually leading to mortality in mice.In the present study, an LPS-induced acute lung injury model in mice was used to evaluate the sEH inhibitors for their role in the attenuation of inflammation.LPS increased cell numbers in bronchoalveolar lavage (BAL), indicating an increased infiltration of immune cells (Figure 5A); treatment with either prototypic sEH inhibitor AUDA (10 mg/kg, i.p.) or 4f (10 mg/kg, i.p.) similarly attenuated the number of cells in BAL (Figure 5A).On the other hand, neither AUDA nor novel sEH inhibitors' (4f and 4l) effect on LPS induced increases in albumin (Figure 5B) and total protein (Figure 5C) in bronchoalveolar lavage fluid (BALF).Increased albumin levels and total protein levels are indicators of vascular permeability and alveolar-capillary membrane integrity, respectively.Since sEH inhibition by AUDA and 4f similarly reduced cell infiltration (Figure 5A), this endorsed sEH's role in the inflammatory process.Therefore, we investigated the gene expression of inflammatory markers in lungs (Figure 5D).Interleukin 6, TNF-alpha, and interleukin 1B are the major inflammatory cytokines.Both AUDA and 4f treatments attenuated LPS-induced expressions of inflammatory cytokines such as interleukin 6 (IL-6) and interleukin 1B (IL-1B) (Figure 5D).However, 4l had no effect on reducing these.Additionally, both AUDA and 4f similarly attenuated the expression of CXCL1 (Figure 5D).Notably, the effect of 4f was more pronounced in reducing IL-6 and CCL-2 expressions (Figure 5D).These results suggest that sEH inhibition has no direct effect on preventing vascular permeability (Figure 5B) and alveolar-capillary membrane integrity (Figure 5C) in LPS-induced acute lung injury.Importantly, these results demonstrate that 4f achieved comparable and potent in vivo sEH inhibition to that of AUDA in the lungs.

Effects of 4f and 4l on Lipopolysaccharide (LPS)-Induced Acute Lung Inflammation
The increased activity of soluble epoxide hydrolase promotes inflammatory c tions.Acute lung injury (ALI) is a state of acute inflammation that results in the disru of the lung endothelium and epithelial cells [50].Characteristic features of ALI includ loss of alveolar-capillary membrane integrity, vascular permeability, excessive neutr transmigration, and the release of pro-inflammatory mediators and cytotoxic co nents.LPS causes acute inflammation resulting in an increased number of immun infiltrations to the lungs and the expression of cytokines and other inflammatory m tors, eventually leading to mortality in mice.In the present study, an LPS-induced lung injury model in mice was used to evaluate the sEH inhibitors for their role i sion of CXCL1 (Figure 5D).Notably, the effect of 4f was more pronounced in reducing IL-6 and CCL-2 expressions (Figure 5D).These results suggest that sEH inhibition has no direct effect on preventing vascular permeability (Figure 5B) and alveolar-capillary membrane integrity (Figure 5C) in LPS-induced acute lung injury.Importantly, these results demonstrate that 4f achieved comparable and potent in vivo sEH inhibition to that of AUDA in the lungs.

Experimental Section 4.1. Chemical Synthesis and General Methods
All starting materials, reagents, and solvents were purchased from commercial suppliers and used without further purification.Air-sensitive reactions were carried out under a dry nitrogen or argon atmosphere.RediSepRf silica gel columns were used for column chromatographic purification on the Teledyne ISCO CombiFlash system. 1 H NMR spectra were recorded at 400 MHz (Jeol) and 800 MHz (Bruker) frequencies, and 13 C NMR spectra were recorded at 100 MHz (Jeol) and 200 MHz (Bruker) frequencies in CDCl 3 or DMSO-d 6 solvent using tetramethylsilane (TMS) as the internal standard.Mass spectra (HRMS) were recorded on a VG 7070E spectrometer or a JEOL SX102a mass spectrometer.Melting points were determined on an MPA100 Stanford Research System apparatus.Thin-layer chromatography (TLC) analyses were carried out on Analtech/Miles Scientific silica gel GHLF 0.25 mm plates using various gradients of EtOAc/n-hexanes or CHCl 3 /MeOH.TLC plates were visualized under UV light at 254 nm wavelengths.All compounds tested had ≥95% purity, which was determined by a combination of TLC, 1 H NMR, 13 C NMR, liquid chromatography−mass spectrometry (LC−MS), and high-resolution mass spectrometry.Detection in LC−MS was carried out on an Agilent 1200 system using an Agilent EC-C18, Poroshell, 2.7 µM (3 mm × 50 mm) where the mobile phase was 50-98% acetonitrile (0.1% formic acid).The LC−MS chromatogram showed the expected molecular (MH + ) ions as well as a single peak at UV (254 nm).
General procedure A for the synthesis of sulfonyl carbamates (3a-l) [35,51]: synthesis of methyl tosylcarbamate (3a): 10 g benzenesulfonamide (1a) was dissolved in dry ACN (60 mL).Triethylamine (22 mL, 2.5 equiv.) was added to the solution.Methyl chloroformate 2 (7.4 mL, 1.5 equiv.) was added dropwise to the reaction mixture in an ice-cold condition.The reaction mixture was stirred at room temperature for 12 h.Completion of the reaction was determined by thin-layer chromatography.ACN was removed under a vacuum.The residue was then dissolved in ethyl acetate and the organic layer was extracted with a saturated sodium bicarbonate solution.The aqueous part was acidified with conc.HCl to obtain a precipitate, which was filtered, dried, and concentrated to give compound 3a as a white solid (9.20 g, 67%). 1 H NMR (400 MHz; CDCl 3 ): δ 7.85 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 3.72 (s, 3H), 1.87 (s, 3H).
General procedure B for the synthesis of adamantyl ureas (4a-l): 5.0 g suitable sulfonyl carbamate (3) was dissolved in 50 mL toluene.To the solution, adamantyl amine (0.8 equiv.) was added and the reaction mixture was refluxed for 4 h.Toluene was evaporated, and to it, isopropanol was added and triturated to produce a white solid product (4a-l).
Synthesis of N-((adamantan-1-yl)amino)(amino)methylene)-4-(trifluoromethoxy) benzenesulfonamide (8a): Compound 4f (0.5 g, 1.19 mmol) was reacted with POCl 3 (0.22 mL, 2.38 mmol) in dry toluene in the presence of N, N-diisopropylethylamine (0.52 mL, 3.0 mmol) as a base.Solvent was evaporated and the intermediate 5d formed was reacted with 1 mL ammonia in a methanol solution (7M) according to general procedure D to form compound 8a (0.31 g, 62%).Mp 221-223 • C; 1 H NMR (400 MHz; CDCl 3 ): δ 7.93 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 5.99 (s, 2H), 2.10 (s, 3H), 1.92 (s, 6H), 1.66 (t, J = 12.0 Hz, 6H). 13   3  Aliquots of 5 µL of 20.00 mM, 10.00 mM, 5.00 mM, 2.50 mM, 1.25 mM, 0.63 mM, 0.31 mM, 0.16 mM DMSO stock solutions of the control (highly soluble 2 ′ -deoxy-5-fluorouridine and insoluble raloxifen hydrochloride) and the test compounds were loaded in 96-well microplates with clear flat bottoms in duplicates.Then, 250 µL of PBS was dispensed into each well using a Multidrop Combi dispenser to reach the concentrations of 400.0 µM, 200.0 µM, 100.0 µM, 50.0 µM, 25.0 µM, 12.5 µM, 6.25 µM, 3.125 µM for each compound with 2% final DMSO.Then, the resulting solutions were incubated for 15 min at room temperature.The effective range of this assay is approximately 5-200 µM.The solubility of each compound was calculated using the dedicated fitting algorithm.The physical base of nephelometric titration fitting is based on the quick increase in turbidity when a concentration of the tested compound reaches its solubility level.An intersection of the regression line of the high-level turbidity points with the calculated baseline was plotted for each compound.The baseline is a linear regression line plotted for fully soluble control compound DOFU.Each concentration point signal of the test article was compared with the corresponding signal of DOFU.Concentration points with a statistically higher signal of the test article compared to DOFU were assigned as concentrations with some compound sedimentation and then were used for linear fit plotting.The abscissa of the intersection of a test article and DOFU linear fits is, therefore, the kinetic solubility of the compound.

Caco-2 Permeability (Bienta Ltd.)
Caco-2 cells were cultured in 75 cm 2 flasks to 80-90% confluence according to the ATCC and Millipore recommendations in a humidified atmosphere at 37 • C and 5% CO 2 .Cells were detached with a trypsin/EDTA solution, resuspended in the complete medium containing DMEM high glucose (4.5 mg/L) with L-glutamine (4 mM) supplemented with 10% heat-inactivated Fetal Bovine Serum, 1% non-essential amino acids, and 730 nM puromycin, and seeded at a density of 5 × 105 cells in a 75 cm 2 flask.After 5 days, cells were trypsinized and resuspended in the complete medium to a final concentration of 600 × 10 3 cells/mL.Of the cell suspension, 400 µL was added to each well of an HTS 24-Multiwell Insert System and 25 mL of prewarmed complete medium was added to the feeder tray.Caco-2 cells were incubated in the Multiwell Insert System for 6-10 days before the transport experiments.The medium in the filter plate and feeder tray was refreshed every other day.Prior to the transport experiment, the integrity of the monolayer was verified by measuring the transepithelial electrical resistance (TEER) for every well using a Millicell-ERS system ohm meter.The final TEER values were within the range of 150-600 Ω × cm 2 , as required for the assay conditions.The 24-well insert plate was removed from its feeder plate and placed in a new sterile 24-well transport analysis plate.The inserts were washed with PBS after medium aspiration.Ketoprofen, atenolol, digoxin, and quinidine were used as reference compounds.
To determine the rate of the compounds' transport in the apical (A)-to-basolateral (B) direction, 300 µL of the test compound dissolved in transport buffer (Hanks' BSS (9.5 g/L) and NaHCO 3 (0.35 g/L) with MgSO 4 to final concentration 0.81 mM, CaCl 2 to final concentration 1.26 mM, HEPES to final concentration 25 mM, pH adjusted to 7.4) was added into the filter wells; 1000 µL of transport buffer was added to the transport analysis plate wells.To determine transport rates in the basolateral (B)-to-apical (A) direction, 1000 µL of the test compound solutions was added into the wells of the transport analysis plate, and the wells in the filter plate were filled with 300 µL of buffer (apical compartment).
The effect of the inhibitor on the Pgp-mediated transport of the tested compounds was assessed by determining the bidirectional transport in the presence or absence of verapamil.The Caco-2 cells were preincubated for 30 min at 37 • C with 100 µM of verapamil in both apical and basolateral compartments.After removal of the preincubation medium, the test compounds with verapamil (100 µM) in transport buffer were added in donor wells, while the receiver wells were filled with the appropriate volume of transport buffer with 100 µM of verapamil.The final amounts of test and reference compounds were 10 µM each.The plates were incubated for 90 min at 37 • C under continuous shaking at 100 rpm.Aliquots of 75 µL were taken from the donor and receiver compartments for LC-MS/MS analysis.All samples were mixed with 2 volumes of acetonitrile followed by protein sedimentation by centrifuging at 10,000 rpm for 10 min.Supernatants were analyzed using the HPLC system coupled with a tandem mass spectrometer.

Plasma Protein Binding (Bienta Ltd.)
The assay was performed in a multiple-use 96-well dialysis unit (HTD96b dialyzer).Each individual well unit consisted of 2 chambers separated by a vertically aligned dialysis membrane of predetermined pore size (MWCO 12-14 kDa).An amount of 120 µL of non-diluted plasma spiked with the compound (1 µM, final DMSO concentration 1%) was added to one chamber, and the same volume of PBS buffer with pH 7.4 was added to the other chamber.The HTD96b dialyzer was covered with adhesive sealing film and incubated at 37 • C and shaken at 100 rpm for 5 h.For sample preparation, an aliquot of the content of each chamber was mixed with the same volume of the blank opposite matrix.To define the non-specific loss of the compound during this assay, a standard solution was created by mixing an aliquot of spiked plasma with a blank buffer without dialysis.Two aliquots of the standard solution were incubated at 37 • C and shaken at 100 rpm for 5 h (recovery samples).The other two aliquots were immediately diluted with acetonitrile and stored at 4 • C until LC-MS/MS analysis (stability samples).All samples were diluted 5-fold with 100% methanol with subsequent plasma protein sedimentation by centrifuging at 6000 rpm for 5 min.Supernatants were analyzed using the HPLC system coupled with a tandem mass spectrometer.

Microsomal Stability (Bienta Ltd.)
Human microsomal incubations were carried out in 96-well plates in 5 aliquots of 30 µL each (one for each time point).Human liver microsomal incubation medium comprised of phosphate buffer (100 mM, pH 7.4), MgCl 2 (3.3 mM), NADPH (3 mM), glucose-6phosphate (5.3 mM), glucose-6-phosphate dehydrogenase (0.67 units/mL) with 0.42 mg of liver microsomal protein per ml.In the control reactions, the NADPH-cofactor system was substituted with phosphate buffer.Test compounds (2 µM, final solvent concentration 1.6%) were incubated with microsomes at 37 • C and shaken at 100 rpm.Each reaction was performed in duplicates.Five time points over 40 min were analyzed.The reactions were stopped by adding 5 volumes of methanol containing the internal standard to the incubation aliquots, followed by protein sedimentation by centrifuging at 5500 rpm for 4 min.Supernatants were analyzed using the HPLC system coupled with a tandem mass spectrometer.

Molecular Docking Study
AUDA, 4f, 4l, and 15a compounds were docked into the active site of human sEH.4f and other sulfonyl urea-based sEHis were docked into PDB:6FR2 using ICM-Pro software version 3.9-3b (Molsoft, San Diego, CA, USA) [46,47].Residues involved in contacts with the compounds are numbered.The PDB files for the docking study are included in the Supplementary Materials.

In Vitro sEH Activity Assay
A fluorescence-based sEH activity assay provided a convenient method for screening human epoxide hydrolase inhibitors.The assay utilized (3-Phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME) as a substrate.When the epoxide moiety of PHOME was hydrolyzed by epoxide hydrolase, an intramolecular cyclization occurred, which resulted in the release of a cyanohydrin under basic conditions.The cyanohydrin quickly decomposed into cyanide ions and the highly fluorescent 6-methoxy-2-naphthaldehyde, which was analyzed using an excitation wavelength of 330 nm and emission wavelength of 465 nm [52].A fluorescence-based sEH activity assay kit was obtained from Cayman Chemical (item number: 10011671).The inhibitory function of the compounds on sEH activity was tested as detailed in the product manual from Cayman Chemical.Briefly, 3 mL sEH assay buffer (10×) was diluted with 27 mL HPLC-grade water.This final assay buffer, 25 mM bis-tris (pH 7.0), was used in the assay and for the dilution of the soluble epoxide hydrolase (sEH) enzyme.Amounts of 12 µL each of human and mouse recombinant sEH were thawed on ice.Prior to assaying, 588 µL cold assay buffer was added directly to the enzyme vial.An amount of 190 µL assay buffer and 5 µL solvent (40% DMSO in assay buffer) were added to the background wells.In the 100% initial activity wells, 185 µL assay buffer, 5 µL sEH, and 5 µL solvent were added.In inhibitor wells, 185 µL assay buffer, 5 µL solution of the synthesized compounds, and 5 µL of recombinant enzyme solution were added.To start the reaction, 5 µL of PHOME was added to each well at a final concentration of 0.25 mM.The microwell plate was shaken carefully for 10 s and covered with a plate cover.It was incubated for 15 min at 25 • C. The cover plate was removed, and fluorescence was read using an excitation wavelength of 330 nm and emission wavelength of 465 nm.
Average fluorescence (AF) of the background wells and 100% initial activity in the wells and of each of the inhibitors were determined.The background AF was subtracted from the 100% initial activity and inhibitor AFs.The % sEH activity is calculated as (inhbitited AF/% 100 initial activity) × 100.
To calculate the half maximal inhibitory concentration (IC 50 ) values of the potent inhibitors, a dose-dependent fluorescence assay was performed.Multiple concentrations of the inhibitors were tested followed by plotting of the percent activity as a function of inhibitor concentration to determine the IC 50 values.

In Vivo Pharmacokinetic Experiment
Twenty 8-9-week-old male C57BL/6J mice were taken for the in vivo pharmacokinetic experiment.They were divided into four groups, each containing five mice.Each group was used for either oral or intraperitoneal administration of compound 4f or 15a.Both the compounds were reconstituted in 5% DMSO, 5% TWEEN 80 in saline water (pH = 7.4).The mice were orally and intraperitoneally administered compounds at 10 mg/kg dose.Blood samples were collected at 0, 0.5, 1, 2, 4, 6, and 24 h after administration of the compound, and serum was isolated with the compound.For the pharmacokinetic study, 10 µL of serum samples from mice were extracted with a 2:1 mixture of LC/MS-grade chloroform and methanol, followed by intermittent vortexing, and then centrifuged at 4000 rpm for 10 min at 4 • C. Thereafter, the supernatant was collected and analyzed by LC−MS/MS to measure the concentrations of compounds 4f and 15a in the serum [53].

Bronchoalveolar Lavage Collection from Mice (BALF)
BALF was collected from anesthetized mice by lavaging lungs two times with 0.8 mL Hanks' balanced salt solution without calcium and magnesium (HBSS) (Sigma-Aldrich, St. Louis, MI, USA).Supernatants were collected as BALF for further analyses.

Total Cell Count in Bronchoalveolar Lavage
The number of cells in the BALF was estimated using an automated cell counter, the Countess II FL cell counter (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Level of Albumin in BALF
Albumin levels were measured by the Mouse Albumin Elisa Assay kit (Cat # E99-134), which was obtained from Bethyl Laboratories.BALF samples from the LPS group were diluted at a 1:2000 ratio.

Level of Total Protein in BALF
Total protein in BALF samples was measured using the Pierce BCA protein assay kit (Cat # 23225) as detailed in the product manual (Thermo Fisher Scientific).

RNA Isolation and qPCR
The right middle lobe was immediately placed in an RNA later solution and stored at −80 • C until the RNA extraction procedure.RNA extraction was performed using RNeasy Mini Kits from Qiagen (Valencia, CA, USA).One microgram of total RNA was reverse transcribed to cDNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA).Expression of the target genes was quantified using gene-specific primers and PowerSYBRGreen master mix (Thermo Fisher Scientific) in a QuantStudio 3 Real-Time PCR instrument (Thermo Fisher Scientific).Predesigned mouse IL-6 (QT00098875), TNF-α (QT00104006), IL-1β (QT01048355), CXCL1 (QT00115647), CCL2 (QT00167832), CXCL3 (QT00151599) and CXCL9 (QT00097062) primers were purchased from Qiagen (Valencia, CA, USA).Fold changes in gene expression relative to the housekeeping gene TATA-Box Binding Protein (Tbp, QT00198443) were determined.Gene expression values were calculated based on the 2 ∆∆Ct method.

Tail-Flick Reflex Model
A tail-flick reflex test was performed as previously described [55,56].The parameters of the tail-flick instrument (7360, Ugo Basile Instruments, Varese, Italy) were set up to keep the baseline at 2.5-4.0 s.Mice were fixed and the part about 1.5-2 cm away from the tail of the mouse was aligned with the infrared emission hole of the instrument.The time the mouse spends on the infrared emission hole surface is automatically recorded on the instrument when the mouse shows tail flick.Latencies of mice of less than 2 s or more than 5 s were excluded.After the detection of the baseline (0 min), AUDA (10 mg/kg) and compounds 4f and 4l (10 mg/kg) or the vehicle (1:1:18 ratio DMSO/TWEEN 80/saline) were administrated by intraperitoneal (i.p.) injection, and the latencies were measured at 15, 30, 60, 90, and 120 min after the drugs' injection.The cut-off time in the tail-flick reflex test was set at 8 s to avoid sensitization and damage to the skin and to achieve the maximum possible effect (MPE%) = (measured value − basic value) × 100/(cut-off − basic value).C57BL/6J male mice obtained from The Jackson Laboratory (Bar Harbor, ME, USA) weighing 28 g were used for the tail-flick test.Animal procedures were conducted following the rules and regulations of the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) under the protocols of LPS-GK1.

Hot Plate Model
Thermal nociception in the hot plate test was assessed with a commercially available apparatus (Hotplate Analgesia Meter, Columbus Instruments) consisting of a metal plate to a constant temperature of 55.0 ± 0.1 • C. Mice were put into the hot plate testing instrument to adapt for at least 15 min.The device was connected to a manually operated timer that recorded the amount of time the mouse spent on the heated surface before showing signs of nociception (e.g., licked its paws or jumped), the cut-off time was 60 s.The baseline (basic pain threshold, the response was more than 25 s, and the jumping mice were excluded.)was calculated as the mean of three readings recorded before testing at intervals of 15 min.The time course of latency was then determined at 15, 30, 45, 60, 90, and 120 min after compound treatment.Data were analyzed as time-course curves of the percentage of maximum effect MPE% = (post drug latency − baseline latency) × 100/(cut-off time − baseline latency).C57BL/6J male mice obtained from The Jackson Laboratory (Bar Harbor, ME, USA) weighing 28 g were used for the hot plate test.Animal procedures were conducted following the rules and regulations of the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) under the protocols of LPS-GK1.AUDA (10 mg/kg), compounds 4f and 4l (10 mg/kg), or the vehicle (1:1:18 ratio DMSO/TWEEN 80/saline) were injected intraperitoneally (i.p.) [57].

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA).One-way ANOVA followed by Bonferroni's multiple comparisons testing was performed.p < 0.05 was considered significant.
Statistical analyses for tail-flick and hot plate experiments were performed using GraphPad Prism 9 (GraphPad Software Inc.).Two-way ANOVA was followed by Tukey's multiple comparisons testing.

Conclusions
The availability of high-quality EpFA and sEH inhibitors to stabilize them contributed to the discovery of a multitude of biological effects mediated by the CYP450 branch of the arachidonate cascade by several academic investigators worldwide.Substantial studies have shown that EpFAs play essential roles in the pathology of inflammation and neuropathic pain.Our study further demonstrates the underlying molecular mechanisms of EpFAs/sEHI actions as an analgesic strategy for inflammation and pain management.Recently, several clinical trials of sEH inhibitors targeting different diseases have emphasized the importance of sEH as a promising therapeutic target [21,32,45,58].Strategic substitution at specific positions of sulfonyl ureas through structure-activity relationship development led to a set of potent sEH inhibitors in this study.Preliminary in vitro fluorescence assays and pharmacokinetic experiments identified compound 4f as the most suitable candidate for in vivo studies.We performed tail-flick reflex and hot plate pain models to determine the analgesic efficacy of our lead.To our satisfaction, compound 4f manifested significant efficacy in the tail-flick reflex pain model.Further, lipopolysaccharide (LPS)-induced acute lung injury (ALI) studies in mice demonstrated that our lead is more efficacious than known sEH inhibitor AUDA in reducing the gene expressions of several inflammatory cytokines in vivo.The results from this study show that sulfonyl urea derivatives show inhibition of sEH and suggest that the solubility and potency of the compounds can be optimized further.Collectively, these results pave the way for further optimization of sulfonyl urea derivatives as sEH inhibitors for the treatment of inflammation and pain.

Supplementary Materials:
The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/molecules29133036/s1.This material is available free of charge via the Internet: Representative NMR spectra, HRMS and LCMS chromatograms and additional screening data.

Figure 1 .
Figure 1.(A) Chemical structures of sEH inhibitors in clinical trials.(B) Our rational design of sulfonyl urea scaffold containing requisite features exhibiting sEH inhibition.

Figure 1 .
Figure 1.(A) Chemical structures of sEH inhibitors in clinical trials.(B) Our rational design of sulfonyl urea scaffold containing requisite features exhibiting sEH inhibition.

3. 4 .
In Vivo Therapeutic Efficacy Study 3.4.1.Analgesic Effects of 4f and 4l in Pain Models sEH inhibitors have been implicated to have analgesic properties.Therefore, we investigated first the analgesic effects of 4f and 4l in tail-flick and hot plate pain models.

Table 1 .
Analysis of human sEH inhibition through structure-activity relationship (SAR) and their physicochemical properties.a tPSA (theoretical polar surface area) and ClogP obtained from Chem-Draw 22.2.

Table 1 .
Analysis of human sEH inhibition through structure-activity relationship (SAR) and their physicochemical properties.a tPSA (theoretical polar surface area) and ClogP obtained from ChemDraw 22.2.

Table 1 .
Analysis of human sEH inhibition through structure-activity relationship (SAR) and their physicochemical properties.a tPSA (theoretical polar surface area) and ClogP obtained from ChemDraw 22.2.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 2 .
Dose-dependent assay results for potent sEH inhibitors.

Table 3 .
In vitro pharmacokinetic profiles of potent compounds.

Table 3 .
In vitro pharmacokinetic profiles of potent compounds.