Bioorganic & Medicinal Chemistry Letters 23 (2013) 417–421

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Synthesis and structure–activity relationship of piperidine-derivednon-urea soluble epoxide hydrolase inhibitors

Stevan Pecic a, Svetlana Pakhomova b, Marcia E. Newcomer b, Christophe Morisseau c, Bruce D. Hammock c,Zhengxiang Zhu a, Alison Rinderspacher a, Shi-Xian Deng a,⇑a Department of Medicine, Columbia University, 650 W 168th Street, BB1029, New York, NY 10032, USAb Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USAc Department of Entomology and UCD Cancer Center, University of California, Davis, CA 95616, USA

a r t i c l e i n f o

Article history:Received 27 September 2012Revised 12 November 2012Accepted 20 November 2012Available online 1 December 2012

Keywords:Soluble epoxide hydrolaseNon-urea inhibitorsHypertensionHuman liver microsomes stability

0960-894X/$ - see front matter Published by Elsevierhttp://dx.doi.org/10.1016/j.bmcl.2012.11.084

⇑ Corresponding author. Tel.: +1 212 305 1152; faxE-mail address: sd184@columbia.edu (S.-X. Deng).

a b s t r a c t

A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibi-tion of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thusinhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflam-matory drugs. Structure–activities studies guided optimization of a lead compound, identified throughhigh-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in humanliver microsomal assay suitable for preclinical development.

Published by Elsevier Ltd.

NH

O

NNH

O

N

Soluble epoxide hydrolase (sEH) is a bifunctional homodimericenzyme with hydrolase and phosphatase activity detected in vari-ous species ranging from plants to mammals.1 In humans it ismostly located in liver, kidney, intestinal and vascular tissues.2

The sEH enzyme is selective for aliphatic epoxides of fatty acids,and one of the most important substrates is epoxyeicosatrienoicacid (EET).3 EETs are one of the metabolic derivatives of arachi-donic acid.4 The epoxygenase CYP2 enzymes catalyse the epoxyda-tion of olefin bonds of arachidonic acid generating EETs.5 EETsexhibit vasodilatory effects in various arteries6,7 and possessanti-inflammatory properties.8 sEH mediates the addition of waterto EETs, converting them to the corresponding diols—dihydroxyei-cosatrienoic acids (DHETs), which show abolished or diminishedbiological activity. The inhibition of this enzyme, therefore mayrepresent a promising therapeutic strategy since it would lead toelevated levels of EETs, which could then have beneficial therapeu-tic effects on blood pressure and inflammation.9

The most explored sEH inhibitors to date are urea-based com-pounds and numerous structure–activity relationship (SAR) stud-ies led to discovery of several potent urea-based sEH inhibitorswith IC50s in the lower nanomolar range.10,11 However, the urea-based inhibitors often suffer from poor solubility and stability,which hinders their pharmacological use in vivo.12,13

Ltd.

: +1 212 305 3475.

Amides, carbamates and thioureas have been evaluated as alter-native pharmacophores in an attempt to improve physical proper-ties of urea-based inhibitors.10,11 In this report we disclose ourefforts towards designing novel non-urea, amide-based inhibitorsof sEH.

Previously we identified through high-throughput screening(HTS)14 several hits with a low micromolar to low nanomolar po-tency. A majority of these compounds were urea-based inhibitors.However, several were non-ureas and among them the most po-tent was the derivative of isonipecotic acid, with substantial activ-ity of 20 nM. Our initial SAR studies15 led to discovery ofcompound 1 with an IC50 of 7.9 nM (Fig. 1). Based on its potentinhibition of sEH activity, we continued our evaluation of theright-hand side (2,4,6-trimethylphenyl ring) of 1, and this follow-up SAR study revealed compound 2, with an IC50 of 1.6 nM.16

Herein we report modifications of the left-hand side of non-urea inhibitor 2. In particular, we were focused on the replacement

SO O

SO O

1 IC50 = 7.9 nM 2 IC50 = 1.6 nM

Figure 1. Chemical structures of non-urea inhibitors.

Figure 2. The active site of the hydrolase domain of human sEH complexed with 1.The Fo–Fc electron density map is contoured at 3r. Hydrogen bonds are indicated bydashes. The structure reveals that the 2,4,6-trimethylphenyl group is directed intothe hydrophobic environment, whereas the cycloheptyl moiety is exposed towardthe solvent. Coordinates of the complex deposited as 4HAI with the Protein DataBank. The image was produced using PYMOL.

418 S. Pecic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 417–421

of the cycloheptyl moiety of the 2, following the SAR reported byRose et al.17, and guided by docking model of the previously re-ported protein-inhibitor complex and X-ray cocrystal structure ofhuman sEH and 1.

The X-ray crystallographic structure of human sEH and aninhibitor 4-(3-cyclohexylureido)-carboxylic acid complex (PDBcode: 1ZD3) revealed the catalytic pocket and key structural fea-tures required to inhibit sEH enzyme. Two tyrosine (Tyr383 andTyr466) and one aspartic acid (Asp335) residues, located in thehydrolase catalytic pocket of sEH, are involved in degradation ofEET—tyrosine residues act as hydrogen bond donors to promotethe epoxide ring opening by Asp335.18,19 The urea group of 4-(3-cyclohexylureido)-carboxylic acid fits in the hydrolase catalyticpocket and the carbonyl oxygen of the urea moiety is engaged ina hydrogen bond interaction with Tyr381 and Tyr466 while theN–H acts as a hydrogen bond donor to Asp335 The co-crystal struc-ture of 1 and human sEH was determined (See Supplementarydata) to guide the design of more potent inhibitors. Our closeexamination of the structure revealed that the amide moiety of 1is positioned in the same fashion as the urea group in urea-basedinhibitors, where the amide moiety, instead of urea group, isinvolved in hydrogen bonding with tyrosine and aspartic acidresidues in the catalytic pocket of sEH. We noticed that the 2,4,6-trimethylphenyl ring on the right-hand side of this inhibitor occu-pies the smaller of the two lipophilic pockets in the sEH active site,while the cycloheptyl moiety on the left-hand side is directed

NH

3 4 5

a b

SO2ClO O

NS

O O

O

O

O

Scheme 1. Reagents and condition: (a) Et3N, CH2CL2, rt 24 h; 89% (b) LiOH,

towards the large deep pocket that opens toward solvent, whichallows access for further structural modifications ( Fig. 2). Wetherefore hypothesized that the left-hand segment in 1 could beoptimized to further improve the potency of this group of non-ureasEH inhibitors.

Scheme 1 outlines the synthetic route used to form the left-hand side library of non-urea amide sEH inhibitors. Sulfonamide5 was prepared from two commercially available starting materi-als, methyl isonipecotate 3 and 2,4-dimethylbenzenesulfonylchloride 4. Saponification of this methyl ester with LiOH affordedacid 6. EDC peptide coupling reactions of 6 with various commer-cially available amines provided analogs 7–1 to 7–46.

A sensitive fluorescent based assay was employed to determineIC50 values of these sEH inhibitors. In short, cyano(2-meth-oxynaphthalen-6-yl)methyl trans-(3-phenyloxyran-2-yl) methylcarbonate (CMNPC) was used as the fluorescent substrate. HumansEH (1 nM) was incubated with the inhibitor for 5 min in pH 7.0Bis–Tris/HCl buffer (25 mM) containing 0.1 mg/mL of BSA at30 �C prior to substrate introduction ([S] = 5 lM). Activity wasdetermined by monitoring the appearance of 6-methoxy-2-naph-thaldehyde over 10 min by fluorescence detection with an excita-tion wavelength of 330 nm and an emission wavelength of465 nm. Reported IC50 values are the average of the three repli-cates with at least two datum points above and at least two belowthe IC50.20 In Table 1 are summarized the biological results.

Previous studies on urea-based inhibitors containing piperidinemoiety have shown that the hydrophobic cycloalkyl groups on theleft-hand side of the molecules are positively correlated withinhibitory potency.17 Among this set of analogs we identified sev-eral inhibitors possessing improved or similar potency comparingto the lead compound 2, specifically compound 7–10 showed anIC50 of 0.4 nM, the most potent amide non-urea sEH inhibitor re-ported to date. Replacement of cycloalkyl ring with a more com-pact phenyl ring (compound 7–12), resulted in 15-fold drop inpotency against the human sEH. However, introduction of the phe-nyl ring allowed us access to electronically and sterically diversestructures, and as well attachment of various polar groups, whichcould in turn improve physical and pharmacokinetic properties.Placement of fluorine or bromine in the ortho position did not sig-nificantly change the potency of the non-urea inhibitors (7–13 and7–15), while chlorine and methyl group decreased the potency for10 and 30-fold, respectively (7–14 and 7–16).

Polar hydroxyl group in ortho position showed clear negative ef-fect on potency in non-urea based compounds (7–17). Althoughthe para substitution is generally tolerated, placement of polar sub-stituents resulted in less potent inhibitors.

Placement of methoxy group in para position (compound 7-18)did not significantly changed the potency comparing to compound7-12, while introduction of hydroxyl group in the same position(compound 7-19 can be observed as a metabolite of 7-18) led totwo fold decreased potency presumably because of unfavorableelectron character and increased polarity. Similar results, but withmore drastically change in potency can be observed based onresults for methyl ester compound 7-21 and its corresponding car-boxylic acid compound 7-22. We synthesized the 4-trifluorometh-oxyphenyl analog 7-23, since it has been reported previously that

6 7-1 to 7-46

cNS

O O

OH

NS

O O

HN

O

R

THF/H2O, rt, 24 h; 91% (c) R-NH2, EDC, DMAP, CH2CL, rt, 24 h; 65–92%.

Table 1The biological results for sulfonamide analogs 7–1 to 7–46

NS

O O

HN

O

R

Compound R IC50a,b (nM) Compound R IC50 (nM)

7-1 18 7-24 4.6

7-2 6900 7-25 29

7-3 5.2 7-26 102

7-4N

Boc

263 7-27 41

7-5 5.8 7-28 2.8

7-6 1.7 7-29 6.9

7-7

F1.1 7-30

Cl8.7

7-8

Br0.6 7-31 20

7-9

OH1.2 7-32

O25

7-10HO

0.4 7-33

O

20

7-11 O

O

8.5 7-34HOOC

17

7-12F3CO

23 7-35O2N

12

7-13 N 20 7-36N

H2N

O

43

7-14 NO

250 7-37 6.7

7-15F3C

30 7-38 SF3C

O

O

1.6

7-16F3C

640 7-39F

290

7-17Cl

2200 7-40

Br45

(continued on next page)

S. Pecic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 417–421 419

Table 1 (continued)

Compound R IC50a,b (nM) Compound R IC50 (nM)

7-18

Cl

Cl

25 7-41

F3C

Cl78

7-19

Cl

Cl55 7-42 8.3

7-20N

6.0 7-43

N2.3

7–21

N13 7-44

N23

7-22 O

O

110 7-45HOOC

0.6

7-23

O

O5.2 7-46

O

O O30,000

a Reported IC50 values are the average of three replicates. The fluorescent assay as performed here has a standard error between 10% and 20% suggesting that differences oftwo fold or greater are significant.

b t-AUCB that has an IC50 between 1 and 2 nM was used as positive control.

420 S. Pecic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 417–421

this moiety is more metabolically stable replacement for cycloalkylrings.12 We observed fourfold increase in potency for this com-pound compared with compound 7-12, thus it was selected for fur-ther pharmacokinetic studies. Interestingly, the analog 7-24,showed five fold increased activity comparing to phenyl compound7-12, despite the presence of the high polarity nitro functionality.The metabolic stability for this inhibitor was evaluated as well.We also introduced a basic nitrogen (piperidine and morpholinerings in para position; analogs 7-25, 7-26 and 7-27) in order to al-low formulation of the inhibitor as a salt. These modifications didnot improve the potency, similar to other polar substituents in thisposition. On the other hand, the inhibition potencies increasedwhen small non-polar para or meta substituents were added (7-28, 7-29, 7-30 and 7-31). Since it has been known that halogenscan enhance polarity and decrease the rate of metabolism degrada-tion due to their electron withdrawing effect on the aromatic ring,we decided to prepare a set of analogs containing various halogensin different position on the left-hand side phenyl moiety. Thefluorinated, chlorinated and brominated para-phenyl compounds(7-32, 7-33 and 7-34, respectively) did not show significantimprovement in activity compared to compound 7-12. Placementof two chlorine atoms in meta, and meta and para positions showeda twofold and threefold lower IC50 against human sEH enzyme,7-35 and 7-37, respectively.

Future rational optimization of these meta, para disubstitutedhalogens might yield additional more potent compounds. Inclusionof 2-naphthalene on the left side of the molecule 7-38 resulted inhigh potency against the human enzyme, which is already shownin recent literature,17 thus, we decided to test in vitro metabolicprofile for this compound. We tried to introduce a nitrogen in thismoiety in order to improve physical properties and the ease offormulation. Various aminoquinolines were attached via differentposition to central non-urea moiety. 5-Aminoquinoline derivative7-39 led to five fold lower potency against sEH, 3-aminoquinolinederivative 7-40 showed 30-fold diminished potency, while 6- and8-aminoquinoline analogs 7-41 and 7-42, led to even moredrastically decreased potency, 50-fold and 180-fold, respectively.

The poor performance of aminoquinolines returned our attentionto 2-naphthalene moiety, where we tried to introduce polar groupin position 6. Methylester analog 7-43 showed slight decrease inactivity, while corresponding carboxylic acid 7-44 had 15-foldslower inhibition then the 2-naphthalene analog. Interestingly,3,4-methylenedioxybenzene analog 7-45 led us to discovery ofsubnano molar potent inhibitor of human sEH enzyme.

The selected non-urea sEH inhibitors were profiled in humanliver microsomal assay22 as a predictor of in vivo oxidative metab-olism (Table 2). Our results from this assay indicated that althoughcompounds with hydrophobic cycloalkyl substituents on theright-side of the non-urea piperidine scaffold such as cyclohexyl,methylcyclohexyl, cycloheptyl, cyclooctyl or adamantyl were morepotent inhibitors of sEH, compared to compounds with aromaticmoiety, these compounds showed poor metabolic profile in humanliver microsomal assay. Evaluation of the in vitro metabolic stabil-ity for aromatic compounds revealed intermediate metabolicprofiles for compounds with para-substitution (compounds 7-23and 7-24), with the exception of the carboxylic acid derivative7-44, which demonstrated excellent in vitro metabolic stabilityin human liver microsomes.

In summary, our structure–activity relationship of the lead 2 re-veals some particular structural requirements to the left-hand sidepart of the piperidine amide-based sEH inhibitors. A varying degreeof bulky, nonpolar cycloalkyl rings are well tolerated in this regionby target enzyme. In contrast, proper substitution on the phenylring is crucial for attaining good potency, emphasizing the impor-tance of the small nonpolar groups and halogens in para position asa recognition element for sEH, suggesting that left-hand side phe-nyl is in a relatively close proximity to a several hydrophobic res-idues located in the large, non-polar pocket of sEH that openstowards solvent, and probably participating in a p-stacking inter-action with them. The above observations are corroborated bythe costructure of 1 with sEH.

While low nanomolar and sub-nanomolar potencies wereachieved, the majority of tested inhibitors in this non-urea basedseries were still not as stable in human liver microsome assay as

Table 2Stability in human liver microsomes (t1/2) for selected compounds

Compound hLMa t1/2 (min) CLint, appb (mL/min/kg) Compound hLMa t1/2 (min) CLint, app

b (mL/min/kg)

2 5.5 220 7-24 180 7.07-3 14 90 7-37 25 507-6 14 90 7-38 36 357-9 2.4 520 7-42 8.7 1407-11 3.7 340 7-44 220 5.67-20 11 120 7-45 36 357-23 46 28

a Data represents averages of duplicate determination. hLMt1/2 is the half life in human liver microsomes.b CLint, app is apparent intrinsic clearance

S. Pecic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 417–421 421

some other non-urea sEH inhibitors previously reported.21 How-ever, compound 7-4423 showed good stability and it will be furtherexplored in various in vivo experiments. Our future work will focusas well on possible combinations of these data obtained from thisSAR study and combinations of related fragments that would beused for future design of highly potent inhibitors with improvedpharmacokinetic profiles.

Acknowledgments

This work was supported in part by NIEHS R01 ES002710 andNIH R01 HL 107887 (MEN). Preliminary structural work was per-formed at the Center for Advanced Microstructures and Devices(Baton Rouge), funded in part by the Louisiana Governors’ Biotech-nology Initiative. The work includes research conducted at theAdvanced Photon Source on the Northeastern Collaborative AccessTeam beamlines, which are supported by grants from the NationalCenter for Research Resources (5P41RR015301-10) and the Na-tional Institute of General Medical Sciences (8 P41 GM103403-10) from the National Institutes of Health. Use of the AdvancedPhoton Source, an Office of Science User Facility operated for theU.S. Department of Energy (DOE) Office of Science by Argonne Na-tional Laboratory, was supported by the U.S. DOE under ContractNo. DE-AC02-06CH11357. We thank Dr. David Neau for assistancewith data collection. BDH is a George and Judy Senior fellow of theAmerican Asthma Foundation.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.084.

References and notes

1. Newman, J. W.; Morisseau, C.; Hammock, B. D. Prog. Lipid Res. 2005, 44, 1.2. Hammock, B. D.; Grant, D.; Storms, D. In Comprehensive Toxicology; Sipes, I.,

McQueen, C., Gandolfi, A., Eds.; Pergamon: Oxford, 1997; p 283.3. Imig, J. D.; Hammock, B. D. Nat. Rev. Drug Disc. 2009, 8, 794.4. Chacos, N.; Falck, J. R.; Wixtrom, C.; Capdevila, J. Biochem. Biophys. Res. Commun.

1982, 104, 916.5. Spector, A. A.; Fang, X.; Snyder, G. D.; Weintraub, N. L. Prog. Lipid Res. 2004, 43,

55.6. Larsen, B. T.; Gutterman, D. D.; Hatoum, O. A. Eur. J. Clin. Invest. 2006, 36, 293.

7. Capdevila, J. H.; Falck, J. R.; Harris, R. C. J. Lipid Res. 2000, 41, 163.8. Node, K.; Huo, Y.; Ruan, X.; Yang, B.; Spiecker, M.; Ley, K.; Zeldin, D. C.; Liao, J. K.

Science 1999, 285, 1276.9. Yu, Z.; Xu, F.; Huse, L. M.; Morisseau, C.; Draper, A. J.; Newman, J. W.; Parker, C.;

Graham, L.; Engler, M. M.; Hammock, B. D.; Zeldin, D. C.; Kroetz, D. L. Circ. Res.2000, 87, 992.

10. Marino, J. P., Jr. Curr. Top. Med. Chem. 2009, 9, 452.11. Shen, H. C. Expert Opin. Ther. Pat. 2010, 20, 941.12. Hwang, S. H.; Tsai, H. J.; Liu, J. Y.; Morisseau, C.; Hammock, B. D. J. Med. Chem.

2007, 50, 3825.13. Watanabe, T.; Schulz, D.; Morisseau, C.; Hammock, B. D. Anal. Chim. Acta 2006,

559, 37.14. AID:1026; Pubchem 2008.15. Xie, Y.; Liu, Y.; Gong, G.; Smith, D. H.; Yan, F.; Rinderspacher, A.; Feng, Y.; Zhu,

Z.; Li, X.; Deng, S. X.; Branden, L.; Vidovic, D.; Chung, C.; Schurer, S.; Morisseau,C.; Hammock, B. D.; Landry, D. W. Bioorg. Med. Chem. Lett. 2009, 19, 2354.

16. Pecic, S.; Deng, S. X.; Morisseau, C.; Hammock, B. D.; Landry, D. W. Bioorg. Med.Chem. Lett. 2012, 22, 601.

17. Rose, T. E.; Morisseau, C.; Liu, J. Y.; Inceoglu, B.; Jones, P. D.; Sanborn, J. R.;Hammock, B. D. J. Med. Chem. 2010, 53, 7067.

18. Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D. W. Biochemistry2004, 43, 4716.

19. Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D. W. Protein Sci.2006, 15, 58.

20. Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock, B.; Hammock, B. D.Anal. Biochem. 2005, 343, 66.

21. Taylor, S. J.; Soleymanzadeh, F.; Eldrup, A. B.; Farrow, N. A.; Muegge, I.;Kukulka, A.; Kabcenell, A. K.; De Lombaert, S. Bioorg. Med. Chem. Lett. 2009, 19,5864.

22. In vitro human liver microsomal metabolic stability: Microsomal stability wasassessed in pooled human liver microsomes (Celsis, Edison, NJ). All reactionswere carried out for 90 min at 37 �C in an NADPH-generating system consistingof glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and NADP+

(Sigma, St., Louis, MO). Positive control incubations proceeded with 7-ethoxycoumarin as the substrate. Reactions were terminated by addingmethanol. The mixtures were centrifuged and the supernatants wereevaporated. The residues were reconstituted in mobile phase (85% ACN; 15%H2O) and subjected to LC/MS analysis.

23. Analytical data for the representative compounds. Compound 7–10: 1H NMR(400 MHz, CDCl3): d 7.78–7.76 (d, J = 8 Hz, 1H) 7.09 (s, 1H), 7.07 (s, 1H), 3.96–3.89 (m, 1H), 3.71–3.66 (d, J = 9.6 Hz, 2H), 2.69 (t, J = 12 Hz, 2H), 2.55 (s, 3H),2.35 (s, 3H), 2.10–2.03 (m, 1H), 1.86–1.82 (d, J = 10.8 Hz, 2H), 1.79–1.68 (m,4H), 1.66–1.42 (m, 14H); 13C NMR (100 MHz, CDCl3) d 172.5, 143.7, 138.1,133.7, 133.1, 130.6, 126.8, 49.6, 44.9, 43.0, 32.8, 28.8, 27.5, 25.9, 24.1, 21.6,20.8; ESI-MS (M++H): 407; Compound 7–44: 1H NMR (400 MHz, DMSO-d6): d10.27 (s, 1H), 8.45 (s, 1H), 8.34 (s, 1H), 7.99–7.97 (d, J = 9.2 Hz, 1H), 7.88–7.85(d, J = 10 Hz, 1H), 7.82–7.80 (d, J = 8.8, 1H), 7.68–7.66 (d, J = 8.4 Hz, 1H), 7.61–7.59 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H) 7.20–7.18 (d, J = 7.6 Hz, 1H), 3.65–3.62 (d,J = 12.4 Hz, 2H), 2.67 (t, J = 11.6 Hz, 2H), 2.53 (s, 3H), 2.35 (s, 3H), 1.92–1.90 (d,J = 11.2 Hz, 2H), 1.69–1.59 (dd, J = 11.6 and 9.2 Hz, 2H); 13C NMR (100 MHz,DMSO-d6) d 173.8, 168.0, 143.9, 139.5, 137.7, 136.3, 134.2, 134.0, 133.4, 131.0,130.7, 130.5, 129.3, 127.4, 127.2, 121.2, 115.5, 115.2, 45.1, 28.7, 21.6, 21.5,20.9; ESI-MS (M�+H): 465.

SUPPORTING INFORMATION

Protein Crystallization. Crystals of the enzyme were obtained using the hanging drop vapor-

diffusion method by mixing equal volumes of protein (8-12 mg/mL concentration in 100 mM

sodium phosphate pH 7.4, 3 mM DTT ) and the reservoir solution (30% PEG 3350, 0-10%

sucrose) at 4º C. The crystals grew in approximately one week and belonged to the hexagonal

space group P6522. The sEH·1 complex was obtained by soaking sEH crystals in modified

mother liquor (35% PEG 3350, 50 mM sodium phosphate pH 7.4) supplemented with 1 mM

solution of 1 for 1-7 days at room temperature.

Data Collection. Prior to the data collection, a suitable crystal was dipped for 30 seconds in a

modified mother liquor solution (35% PEG 3350) with the addition of 10% sucrose as a

cryoprotectant. Diffraction data were collected at 100 K at the NE-CAT beamline 24-ID-C at the

Advance Photon Source equipped with the Pilatus 6M detector. The images were processed and

scaled using the Xia2 [1]. Data collection and data processing statistics are given in Supplementary

Table 1.

Crystal Structure Determination. The molecular replacement procedure was applied to locate a

solution using the program MOLREP [2]. A monomer of human sEH (PDB accession code 1S8O)

was used as a search model. The positioned MR model was refined using the maximum likelihood

refinement in REFMAC [2] with the TLS parameters generated by the TLSMD server [3]. Coot [4]

was used for model building throughout the refinement. The final model consists of protein residues

1-548, one phosphate anion, one magnesium cation, compound 1 and 27 water molecules.

Supplementary Table 1. Data Collection and Refinement Statistics.

Wavelength (Å) 0.97920 Resolution (Å) 2.55 Temperature (K) 100 Space group P6522 Cell dimensions a (Å) 92.31 c (Å) 243.98 Number of molecules per asymmetric unit 1 No. of unique reflections 20 842 Rsym

1,2 (%) 7.0 (70.0)

Completeness (%) 99.2 (95.9) Redundancies 6.5 (5.2) I/(I) 18.0 (1.8)

Refinement statistics

Resolution range (Å) 79.95 – 2.55 No. of reflections used in refinement 19 762 cutoff used in refinement none R /Rfree

3 (%) 18.01/23.57 Number of refined atoms Protein 4328 Heterogen atoms 34 Water 27 Average B-factors (Å2) Protein 50.9 Water 46.0 Phosphate ion 51.0 Inhibitor 108.4 Magnesium 43.0 R.m.s. deviations Bonds (Å) 0.009 Angles () 1.397

a Ramachandran plot (%)

Favored

89.1

Allowed 9.8 Generous 0.8 Disallowed 0.2

1 Values in parentheses are for the highest-resolution shell. 2 Rsym = ∑|Ii - <Ii> |/ ∑Ii, where Ii is the intensity of the ith observation and <Ii> is the mean intensity of the reflection 3 R = ∑|| Fo | - | Fc ||/∑|Fo|, where Fo and Fc are the observed and calculated structure factors amplitudes. Rfree is calculated using 5.1% of reflections omitted from the refinement.

References 1. Winter, G., J. Appl. Cryst., 2010. 43: p. 186-190. 2. Bailey, S., The Ccp4 Suite - Programs for Protein Crystallography. Acta Crystallogr D-

Biological Crystallography, 1994. 50: p. 760-763. 3. Painter, J. and E.A. Merritt, TLSMD web server for the generation of multi-group TLS

models. Journal of Applied Crystallography, 2006. 39: p. 109-111. 4. Emsley, P., Lohkamp, B., Scott, W., Cowtan, K., Acta Crystallographica Section D -

Biological Crystallography, 2010. 66: p. 486-501.