Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2193–2197

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Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors Vladimir Burmistrov b,c, Christophe Morisseau a, Kin Sing Stephen Lee a, Diyala S. Shihadih a, Todd R. Harris a, Gennady M. Butov b,c, Bruce D. Hammock a,⇑ a b c

Department of Entomology and Nematology, Comprehensive Cancer Center, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA Department of Chemistry and General Chemical Technology, Volzhsky Polytechnic Institute (branch) Volgograd State Technical University, Volzhsky, Russia Volgograd State Technical University, Volgograd, Russia

a r t i c l e

i n f o

Article history: Received 3 January 2014 Revised 6 March 2014 Accepted 7 March 2014 Available online 20 March 2014 Keywords: Soluble epoxide hydrolase Inhibitor Adamantane Isocyanate Diurea

a b s t r a c t A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 lM to 0.4 nM. 1,6(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC50 = 0.5 nM) with a strong binding to sEH (Ki = 3.1 nM) and a moderately long residence time on the enzyme (koff = 1.05  103 s1; t1/2 = 11 min). Ó 2014 Elsevier Ltd. All rights reserved.

In mammals, the soluble epoxide hydrolase (sEH, E.C. 3.3.2.10) is involved in the metabolism of epoxy-fatty acids to vicinal diols through a catalytic addition of a water molecule.1,2 Endogenous substrates for the sEH include cytochrome P450 metabolites of arachidonic acid, such as epoxyeicosatrienoic acids (EETs), and of docosahexaenoic acid, known as EpDPEs.3,4 EETs exert vasodilatory effects through the activation of the Ca2+-activated K+ channels in endothelial cells, which are beneficial in many renal and cardiovascular diseases.5,6 Furthermore, the EETs have some anti-inflammatory and analgesic properties.7 Their conversion to dihydroxyeicosatrienoic acids (DHETs) by sEH produces a molecular that is readily conjugated and removed from the site of action. The inhibition of sEH in vivo by highly selective inhibitors results in an increase of the concentration of EETs and is accompanied by a reduction in angiotensin driven blood pressure in rodent models, but also reduction of inflammatory and painful states, thereby suggesting that sEH is a target for the treatment of hypertension, inflammatory diseases and pain.8–10 Small N,N0 -disubstituted symmetric ureas, such as 1,3-dicyclohexyl urea, were found to be very potent inhibitors of sEH.11–15 However, because of their strong crystalline lattice, these kinds of compounds have poor solubility in many solvents. To improve solubility, asymmetric ureas with a flexible side chain, such as ⇑ Corresponding author. Tel.: +1 530 752 7519; fax: +1 530 751 1537. E-mail address: [email protected] (B.D. Hammock). http://dx.doi.org/10.1016/j.bmcl.2014.03.016 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

AUDA (12-(3-adamantylureido)-dodecanoic acid), were tested and found to be potent sEH inhibitors. While this class of sEH inhibitor shows biological effects when tested in vivo, they are rapidly metabolized, limiting their utility. In contrast, compounds that lack flexible side chain usually have poor physical properties so they show limited biological effects in vivo without careful formulation.16,17 Therefore, to improve the metabolic stability, a third class of conformationally restricted inhibitors, such as AEPU (1-adamantyl-3-(1-acetylpiperidin-4-yl)-urea) or t-AUCB (trans4-((4-(3-adamantylureido)-cyclohexyl)oxy)-benzoic acid), were designed. This latest series includes very potent and more metabolically stable sEH inhibitors that permit in vivo studies. However, these compounds have in general poor solubility, and are quite expensive to synthesize since several steps (3–5) are required. Here, we report the testing of symmetric di-ureas that are simpler to obtain as sEH inhibitors. As shown on Figure 1, a flexible chain was incorporated at the center of the molecules to improve physical properties, while adamantane and urea groups were placed at both ends of the molecules to protect the central flexible chain from metabolism, and to provide the additional possibility of hydrogen bonding to improve potency and solubility. As described on Scheme 1, two simple (one step) and complementary approaches were used to obtain the desired compounds in high yield (>95%). Commercially available 1-isocyanatemethyl adamantane or various adamantyl containing isocyanates18 were

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Figure 1. Compound 3b (blue) docked into the active site of epoxide hydrolase domain of human sEH (cyan). Hydrogen bonds are indicated by dashed lines. However, hydrogen bonds between Tyr466, Tyr383 and the carbonyl of the urea group are not indicated for the sake of clarity.

reacted with various amines containing 2, 4, 6 or 8 carbons that are usually used in supramolecular chemistry as guestmonomers.19–21 To vary the X-parameter, several commercially available hydrochlorides of amines were reacted with alkyl di-isocyanates. Compounds containing phenyl and piperidine rings between the urea groups were synthesized as well because those groups commonly confer properties found to be valuable in medicinal chemistry.22–24 Structures of the obtained chemicals were assessed by NMR, while purity was assessed by mass spectrometry and elemental analysis (see Supplemental materials for details). The inhibitor potency of the synthesized compounds was measured using recombinant purified human sEH and CMNPC (cyano(6-methoxynaphthalen-2-yl)methyl ((3-phenyloxiran-2-yl) methyl) carbonate) as a substrate as described.25 For the di-adamantyl urea-based compounds (1a–1f), increasing the length of the flexible chain between the urea groups from 2 to 6 carbons in the compounds 1a–c lead to a >400-fold increase in potency (lower IC50). Further increase of chain length to 8 carbons resulted in a 15fold decrease of inhibition potency for compound 1d, suggesting an optimal length for interaction with the enzyme. 1,4-Diaminobenzene (1e) and piperidine (1f) based disubstituted diureas also showed poor potency, presumably because the significant reduction of flexibility between the urea groups did not permit an optimal positioning of the compounds inside the enzyme active site. In the 2, 3 and 4 series, not only the length and nature of the chain between the urea groups (Z) but also the spacer connecting the urea groups with adamantane (X) were altered as well (Table 2). As found with the first series (Table 1), the presence of an alkyl chain in the middle of the molecule (series 2 and 3) yielded globally more potent inhibitors than the presence of a phenyl group (series 4). While, as observed for series 1, the length of the middle chain influenced potency (globally, series 2 (with 4 carbon)

yielded more potent compounds than series 3 (8 carbon)), the IC50s were markedly influenced by the spacer between the adamantanes and ureas (X), especially in the 3 series. This provides evidence for the orientation of the inhibitor in the active site of human sEH, and raises the possibility that the second urea makes strong polar interactions with the enzyme. Interestingly, changing the bond from the ureas to the adamantane from a 1- (2a and 3a) to a 2- (3a to 3d) position does not alter the potency of the compounds with short central alkyl chains (2a and 2d), but dramatically (500-fold) decreases the potency of a compound with a longer central alkyl chain (3d). For the more potent diadamantyl diureas, we evaluated the inhibitor binding constant (Ki), the inhibitor off rate (koff), and the inhibitor residence time in the enzyme (t1/2) using a Förster resonance energy transfer (FRET) competitive displacement assay developed for the human sEH (Table 3).26 Compounds with the same molecular weight (two adamantane groups, two urea groups and eight carbons comprising various spacers between those groups) were selected for further testing to ensure that any difference arises from specific structural feature and not due to a general effect of the molecules. In general, the trends in potency measured by a kinetic IC50 assay correlated with the equilibrium Ki determination. While the IC50 of compounds 1c, 2b, 3b and 3d was distributed over a 5-fold difference range, the Ki for these four diureas only ranged between 2.4 and 3.4 nM. These values are very similar and are almost equal with the variation of the assay over a narrow range of Kis. Because the urea inhibitors are slow-tight binding ligands of sEH, the IC50s measured after only 5 min incubation, as shown here, probably underestimated the true potency of some compounds, explaining the apparent divergence between IC50 and Ki values.26 The Ki is a ratio of kinetic off and on rates which are independently altered by structure. Ki reflects the strength of the interaction between the inhibitor and the sEH. The inhibitor is only effective in blocking the hydrolysis of endogenous bioactive fatty acid epoxides while it is bound to the target enzyme. Because of this, determination of residence time, the period when the drug is bound to enzyme, is very important for predicting biological activity.26 Interestingly, among the 4 compounds tested, the koff were distributed over a 5-fold difference range and parallel the IC50s. These data indicate that spacers between the urea group and the the adamantane lead to a slower koff and have an optimal length. These results suggest that the molecular flexibility given by the alkyl spacers allows a better fit to the enzyme’s active site, includingformation of a new hydrogen bond by the second urea group. For the diureas without such flexible spacers, formation of new hydrogen bonds is hampered by the rigidity of the inhibitor, which does not allow optimal positioning. To predict bioavailability of disubstituted biureas, the solubility of four the most potent inhibitors were measured in phosphate buffered water and their c log P calculated (Table 4). Diadamantyl diureas with a flexible chain between urea groups are more potent and more soluble that the simple symmetric ureas (DCU and DAU), the first potent sEH inhibitors.11,27 Interestingly, the c log P values suggest that compounds 1c and 2b have drug-like solubility, and thus should have good bioavailability.28 While the four compounds tested have the same mass and atomic composition, 1c is markedly more soluble than the other three. Because the c log P values suggest that the compounds are quite hydrophobic, it is possible that the observed inhibition is due to the aggregation of the chemicals on the enzyme.29 Across the compound tested, increasing the BSA concentration in the buffer by 10-fold resulted in an increase in inhibition potencies (IC50s) by around 2-fold (Table 4), suggesting that the observed inhibition is neither due to non-specific interaction nor to aggregation.29 To further assess the properties of the compounds, we measured their stability in human liver

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a H 2N

H N

NH2

H N

H N

n

H N

n O

O

1a (n = 0) 1b (n = 2) 1c (n = 4) 1d (n = 6)

a H 2N

H N

NH2

H N

H N

O

H N

O

1e

a

H N

NH H2 N

H N

O

b OCN NH2

NCO

O

1f

H N

H N

H N

Z

X

H N

N

X

X

Cl O

O

2a-c (z = (CH2) 4) 3a-c (z = (CH2) 8) 4a-b (z = Ph) NH2

H N

Z

2-4a (x = -) 2-4b (x = -CH(CH3)-) 2-3c (x = -CH(C2H5)-CH2-)

HCl b Z OCN

H N

H N

H N

NCO

H N

Z O

O

2d (z = (CH2) 4) 3d (z = (CH2) 8) Scheme 1. Reagents and conditions: (a) adamant-1-ylmethyl isocyanate (1.9 equiv), DMF, rt, 12 h; (b) triethylamine (2 equiv), DMF, 0–25 °C, 12 h.

Table 1 IC50 values for diadamantyl urea-based sEH inhibitors 1a–f

H N

H N

H N

H N

Z O

a

O

Z

n

#

IC50a (nM)

–(CH2)n–

2 4 6 8

1a 1b 1c 1d

179.2 26.3 0.4 7.3

1e

779.6

1f

39.7

As determined via a kinetic fluorescent assay.25

microsomes (Table 4). While the compounds were stable in the absence of NADPH, they were metabolized in its presence, probably by P450s. Compounds 2b and 3d were the most stable, suggesting that secondary adamantyl are more stable than tertiary. Compared to 1b, in presence of NADPH, 2b and 3b are more stable suggesting that a methyl on the carbon linker between the urea and adamantyl is preferable. To understand the increased potency of the diureas, compound 3b was manually docked into the active site of the human sEH using the ‘bioMedCAChe 5.0’ software (Fujitsu computer Systems Corporation). For this, the published X-ray crystal structure of the human sEH complexed with a urea-based ligand (PDB accession number 1ZD3) was used. After docking, the ligand and the amino acid residues within 8.0 Å from the ligand were minimized on MM geometry (MM3) as described.17 As expected, one of the urea groups of 3b forms strong hydrogen bonds with the catalytic residues (Asp335, Tyr383 and Tyr466) of the sEH. Surprisingly, the other urea group of 3b forms a hydrogen bond with Ser374 side chain. This is a novel hydrogen bond for this family of sEH ligands, and probably explains the unexpected high potency of compound

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Table 2 IC50 values for diadamantyl-based sEH inhibitors 2a–d, 3a–d and 4a–b

H N

H N

H N

X

X O

O

#

2a

X

Z H2 C

—

C H2

H2 C C H2

H2 C

H C

2b

H2 C

CH3 H2 C

Mp (°C)

7.2

251.1–253.4

1.2

257.1–258.9

2.3

210.9–213.7

997

256.3–257.8

0.5

191.8–192.9

1440

130.1–131.4

19.5

362.4–364.1

161

285.2–286.1

10.4

274.2–276.0

2.6

254.6–255.4

H2 C C H2

C H2

IC50a (nM)

H2 C C H2

C H2

CH 3

2c

H N

Z

CH C H2

3a

H2 C

—

C H2

H2 C

H C

3b

C H2

CH 3 CH3

H2 C

H2 C C H2

H2 C

H2 C C H2

C H2

C H2

H2 C

3c

H2 C C H2

H2 C

H2 C C H2

C H2 H2 C

C H2

H2 C

H2 C C H2

C H2

CH C H2

4a

—

H C

4b CH 3

H N

H N

H N

X

X O

O

a

2d

—

3d

—

H2 C

H2 C C H2

C H2 H2 C C H2

H N

Z

H2 C C H2

H2 C C H2

H2 C C H2

As determined via a kinetic fluorescent assay.25

3b compared to 3a and 3c. For these later compounds, the steric bulky groups (adamantane for 3a and 1-(2-methylbutyl)adamantane for 3c) next to the urea function should impede the formation of this extra bond with the enzyme. We described the synthesis and structure–activity relationships of a series of di-adamantyl diureas containing various spacers between the two urea groups. The data show that ureas with small flexible linkers between the adamantane and the urea group show excellent inhibition potency along with good binding parameters. Diureas with phenyl spacers between urea

groups showed poor activity due to the small size of molecule and the lack of flexibility. We showed that compounds with flexible adamantanes have significantly better binding to the enzyme than those without, as described by the Ki and koff values. This can be explained by the fact that the flexible chain allows second urea group to form additional hydrogen bonds with Ser374. This is the first time that this residue is shown to be involved in sEH ligand binding. Lower mp of the flexible diureas could have a positive indirect effect on their ease of formulation as well.

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V. Burmistrov et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2193–2197 Table 3 IC50, Ki and koff for a selection of the best compounds

a b

#

IC50a (nM)

Kib (nM)

koffb (103 s1)

t1/2b (min)

1c 2b 3b 3d

0.4 1.2 0.5 2.6

3.4 2.7 3.1 2.4

1.16 1.42 1.05 4.61

10 8 11 2.5

As determined via a kinetic fluorescent assay.25 As determined via FRET-based displacement assay.26

Table 4 IC50 and water solubility of the selected compounds

BSA 0.1 (mg/mL)

1c 2b 3b 3d DCUc DAUc

Solubilityb (lM)

IC50a (nM)

#

0.4 1.2 0.5 2.6 3.0 1.2

c Log Pd

Microsomal stabilitye (%)

BSA 1.0 (mg/mL)

0.7 2.5 0.8 4.1 5.1 1.3

NADPH

50 < S < 75 10 < S < 25 10 < S < 25 5 < S < 10 5 < S < 10 5 < S < 10

4.54 4.34 6.01 4.96 2.28 2.84



+

102 92 100 100 100 93

3 24 11 36 22 19

a

As determined via a kinetic fluorescent assay.25 Solubilities were measured in sodium phosphate buffer (pH 7.4, 0.1 M) containing 1% of DMSO. c DCU: 1,3-dicyclohexyl urea; DAU: 1,3-diadamantyl urea. d Calculated using ChemBioDraw Uultra v12.0 (PerkinElmer, Waltham, MA). e Percent of compound (1 lM) remaining after 30 min incubation with human liver microsomes (1 mg/mL) at 37 °C with or without NADPH generating system. Results are triplicate average. 5–10% errors were obtained. b

Acknowledgments The reported study was partially funded by National Institute of Environmental Health Sciences (NIEHS) grant R01 ES002710, NIEHS Superfund Research Program grant P42 ES004699 and Russian Foundation for Basic Research (RFBR) grant No. 12-03-33044. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 03.016. References and notes 1. Arand, M.; Grant, D. F.; Beetham, J. K.; Friedberg, T.; Oesch, F.; Hammock, B. D. FEBS Lett. 1994, 338, 251. 2. Oesch, F. Xenobiotica 1973, 3, 305. 3. Zeldin, D. C.; Kobayashi, J.; Falck, J. R.; Winder, B. S.; Hammock, B. D.; Snapper, J. R.; Capdevilla, J. H. J. Biol. Chem. 1993, 268, 6402. 4. Spector, A. A.; Fang, X.; Snyder, G. D.; Weintraub, N. L. Prog. Lipid Res. 2004, 43, 55. 5. Fleming, I.; Rueben, A.; Popp, R.; Fisslthaler, B.; Schrodt, S.; Sander, A.; Haendeler, J.; Falck, J. R.; Morisseau, C.; Hammock, B. D.; Busse, R. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 2612. 6. Imig, J. D. Expert Opin. Drug Metab. Toxicol. 2008, 4, 165. 7. 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. 8. Spiecker, M.; Liao, J. K. Arch. Biochem. Biophys. 2005, 433, 420. 9. Imig, J. D.; Zhao, X.; Capdevilla, J. H.; Morisseau, C.; Hammock, B. D. Hypertension 2002, 39, 690.

10. Imig, J. D.; Zhao, X.; Zaharis, C. Z.; Olearczyk, J. J.; Pollock, D. M.; Newman, J. W.; Kim, I. H.; Watanabe, T.; Hammock, B. D. Hypertension 2005, 46, 975. 11. Morisseau, C.; Goodrow, M. H.; Dowdy, D.; Zheng, J.; Greene, J. F.; Sanborn, J. R.; Hammock, B. D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8849. 12. McElroy, N. R.; Jurs, P. C.; Morisseau, C.; Hammock, B. D. J. Med. Chem. 2003, 46, 1066. 13. Kim, I. H.; Morisseau, C.; Watanabe, T.; Hammock, B. D. J. Med. Chem. 2004, 47, 2110. 14. Kim, I. H.; Heirtzler, F. R.; Morisseau, C.; Nishi, K.; Tsai, H. J.; Hammock, B. D. J. Med. Chem. 2005, 48, 3621. 15. Schmelzer, K. R.; Kubala, L.; Newman, J. W.; Kim, I. H.; Eiserich, J. P.; Hammock, B. D. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9772. 16. Hwang, S. H.; Wecksler, A. T.; Zhang, G.; Morisseau, C.; Nguyen, L. V.; Fu, S. H.; Hammock, B. D. Bioorg. Med. Chem. Lett. 2013, 23, 3732. 17. Hwang, S. H.; Tsai, H. J.; Liu, J. Y.; Morisseau, C.; Hammock, B. D. J. Med. Chem. 2007, 50, 3825. 18. Butov, G. M.; Pershin, V. V.; Burmistrov, V. V. Russ. J. Org. Chem. 2011, 47, 606. 19. Butov, G. M.; Burmistrov, V. V.; Saad, K. R. J. Chem. Chem. Eng. 2012, 6, 774. 20. Raymo, F. M.; Stoddart, J. F. Chem. Rev. 1999, 99, 1643. 21. Zubovich, E. A.; Burmistrov, V. V.; Butov, G. M. Butlerov Comm. 2013, 33, 65. 22. Kowalski, J. A.; Swinamer, A. D.; Muegge, I.; Eldrup, A. B.; Kukulka, A.; Cywin, C. L.; De Lombaert, S. Bioorg. Med. Chem. Lett. 2010, 20, 3703. 23. Kato, Y.; Fuchi, N.; Saburi, H.; Nishimura, Y.; Watanabe, A.; Yagi, M.; Nakadera, Y.; Higashi, E.; Yamada, M.; Aoki, T. Bioorg. Med. Chem. Lett. 2013, 23, 5975. 24. Pecic, S.; Pakhomova, S.; Newcomer, M. E.; Morisseau, C.; Hammock, B. D.; Zhu, Z.; Deng, S. X. Bioorg. Med. Chem. Lett. 2013, 23, 417. 25. Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock, B.; Hammock, B. D. Anal. Biochem. 2005, 343, 66. 26. Lee, K. S. S.; Morisseau, C.; Yang, J.; Wang, P.; Hwang, S. H.; Hammock, B. D. Anal. Biochem. 2013, 434, 259. 27. Morisseau, C.; Goodrow, M. H.; Newman, J. W.; Wheelock, C. E.; Dowdy, D. L.; Hammock, B. D. Biochem. Pharmacol. 2002, 63, 1599. 28. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev. 2001, 46, 3. 29. McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. J. Med. Chem. 2002, 45, 1712.

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors (Supplemental data) Vladimir Burmistrovb,c, Christophe Morisseaua, Kin Sing Stephen Leea, Diyala S. Shihadiha, Todd. R. Harrisa, Gennady M. Butovb,c and Bruce D. Hammocka*

a Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA, 95616, USA b Department of Chemistry and General Chemical Technology, Volzhsky Polytechnic Institute (branch) Volgograd State Technical University, Volzhsky, Russia c Volgograd State Technical University, Volgograd, Russia

Volgograd State Technical University, Volgograd, Russia

1   

Synthetic procedures General. All reagents and solvents were obtained from commercial suppliers and were used without further purification. All reactions, unless otherwise described, were performed under an inert atmosphere of dry nitrogen. Melting points were determined on an OptiMelt melting point apparatus. 1H NMR spectra were recorded at 500 MHz “Bruker DRX500”. Mass spectra were measured with Hewlett Packard GC 5890 Series II/MSD 5972 Series. Elemental analyses were determined at Volgograd State Technical University, Russia. General Procedure for the synthesis diadamantyl biureas 1a-f from adamantyl isocyanate and diamines. To a solution of 1-isocyanatemethyl adamantane (1 mmol) in DMF (10 mL) was added corresponding diamine (0.45 equiv) at 0°C. The reaction mixtures were allowed to slowly warm to room temperature overnight. The reaction mixtures were poured into water, and the resulting precipitates were collected and washed with 1 N HCl solution followed by water. The crude product was purified by silica gel chromatography. 1,2-(Ethylene)bis{[(adamant-1-yl)methyl]urea} (1a). The general method above was used with 1,2-diaminoethane to afford a white solid (0.21 g, 99% yield). Mp 211.0-212.3 оС. MS (ESI) m/z: 443 (0.9%, [M+1]+). Anal. (C26H42N4O2) С 70.53%, Н 9.57%, N 12.67%. 1,4-(Tetramethylene)bis{(adamant-1-yl)methyl]urea} (1b). The general method above was used with 1,4-diaminobutane to afford a white solid (0.20 g, 99% yield). Mp 176.7-179.2 оС. 1H NMR (DMSO-d6): δ 7.66-7.63 (t, J = 13 Hz, 4H), 3.01-2.98 (d, 8H), 1.95-1.33 (m, 34H). MS (ESI) m/z: 470 (3.1%, [М]+). Anal. (C28H46N4O2) С 71.45%, Н 9.87%, N 11.87%. 1,6-(Hexamethylene)bis{[(adamant-1-yl)methyl]urea} (1c). The general method above was used with 1,6-diaminohexane to afford a white solid (0.23 g, 96% yield). Mp 218,4-219,4 оС. MS (ESI) m/z: 499 (3.5%, [М+1]+). Anal. (C30H50N4O2) С 72.26%, Н 10.06%, N 11.18%. 1,8-(Oktamethylene)bis{[(adamant-1-yl)methyl]urea} (1d). The general method above was used with 1,8-diaminooctane to afford a white solid (0.25 g, 98% yield). Mp 163.9-164.2 оС. 1H NMR (DMSO-d6): δ 5.68-5.65 (t, J = 3.5 Hz, 4H), 2.97-2.68 (m, 16H), 1.94-1.24 (m, 34H). MS (ESI) m/z: 527 (3.0%, [М+1]+). Anal. (C32H54N4O2) С 73.02%, Н 10.22%, N 10.54%. 1,4-(Phenylene)bis{[(adamant-1-yl)methyl]urea} (1e). The general method above was used with 1,4-diaminobenzene to afford a white solid (0.24 g, 97% yield). Mp 238.7-240.0 оC. 1H NMR (DMSO-d6): δ 11.84-11.83 (dd, J = 6 Hz, 4H arom), 7.35-7.32 (t, J = 2.4 Hz, 2H), 7.217.20 (s, J = 2.7 Hz, 2H), 1.94-1.44 (m, 34H). MS (ESI) m/z: 491 (0.5%, [М+1]+). Anal. (C30H42N4O2) С 73.48%, Н 8.51%, N 11.42%. 1-{[1-((Adamant-1-yl)methylcarbamoyl)piperidin-4-yl]methyl}-3-[(adamant-1yl)methyl]urea (1f). The general method above was used with 4-aminomethylpiperidine to afford a white solid (0.24 g, 98% yield). Mp 127.2-128.1 оС. MS (ESI) m/z: 497 (45.0%, [М+1]+). Anal. (C30H48N4O2) С 72.59%, Н 9.54%, N 11.27%. General Procedure for the synthesis diadamantil biureas 2a-d, 3a-d, 4a-b from adamantylamine hydrochlorides and diisocyanates. To a solution of 1,42   

tetramethylenediisocyanate (for compounds 2a-d), 1,8-octamethylenediisocyanate (for compounds 3a-d) or 1,4-diisocyanatobenzene (for compounds 4a-b) 1 mmol in DMF (15 mL) was added corresponding amine hydrochloride (2.2 equiv) and triethylamine (2.2 equiv) at 0°C. The reaction mixture were allowed to slowly warm to room temperature overnight. The reaction mixture were poured into water, and the resulting precipitates were collected and washed with 1 N HCl solution followed by water. The crude product was purified by silica gel chromatography. 1,4-(Tetramethylene)bis[(adamant-1-yl)urea] (2a). The general method above was used with 1-aminoadmantane hydrochloride to afford a white solid (0.43 g, 98% yield). Mp 251.1-253.4 о С. 1H NMR (DMSO-d6): δ 5.62-5.59 (t, J = 7.6 Hz, 2H), 5.42-5.41 (s, J = 13.4 Hz, 2H), 2.912.89 (dd, J = 33 Hz, 8H), 1.97-1.28 (m, 30H). MS (ESI) m/z: 443 (0.7%, [М+1]+). Anal. (C26H42N4O2) С 70.45%, Н 9.56%, N 12.61%. 1,4-(Tetramethylene)bis{[(adamant-1-yl)ethyl-1]urea} (2b). The general method above was used with 1-(adamant-1-yl)ethanamine hydrochloride to afford a white solid (0.47 g, 96% yield). Mp 257.1-258.9 оС. 1H NMR (DMSO-d6): δ 5.69-5.66 (t, J = 17 Hz, 2H), 5.50-5.47 (d, J = 11 Hz, 2H), 2.98-2.94 (dd, J = 17 Hz, 8H), 1.93-1.33 (m, 32H), 0.88-0.86 (d, J = 98 Hz, 6H). MS (ESI) m/z: 499 (2.4%, [М+1]+). Anal. (C30H50N4O2) С 72.83%, Н 10.15%, N 11.14%. 1,4-(Tetramethylene)bis{[(adamant-1-yl)sec-butyl-1]urea} (2c). The general method above was used with 1-(adamant-1-yl)butan-2-amine hydrochloride to afford a white solid (0.53 g, 96% yield). Mp 210.9-213.7 оС. 1H NMR (DMSO-d6): δ 5.56-5.54 (d, J = 13 Hz, 2H), 5.40-5.38 (t, J = 10 Hz, 2H), 3.60-3.56 (t, 6H), 2.96-2.93 (d, 4H), 1.88-1.06 (m, 44H). MS (ESI) m/z: 555 (1.0%, [М+1]+). Anal. (C34H58N4O2) С 73.53%, Н 10.41%, N 10.20%. 1,4-(Tetramthylene)bis[(adamant-2-yl)urea] (2d). The general method above was used with 2aminoadmantane hydrochloride to afford a white solid (0.43 g, 99% yield). Mp 274.2-276.0 оС. 1 H NMR (DMSO-d6): δ 6.02-5.99 (d, J = 35 Hz, 2H), 5.83-5.89 (t, J = 35Hz, 2H), 2.99-2.95 (dd, J = 60 Hz, 8H), 1.83-1.33 (m, 30H). MS (ESI) m/z: 443 (7.3%, [М+1]+). Anal. (C26H42N4O2) С 70.53%, Н 9.54% N 12.67%. 1,8-(Octamethylene)bis[(adamant-1-yl)urea] (3a). The general method above was used with 1-aminoadmantane hydrochloride to afford a white solid (0.47 g, 96% yield). Mp 256.3-257.8 о С. 1H NMR (DMSO-d6): δ 5.57-5.54 (t, J = 16 Hz, 2H), 5.38-5.36 (d, J = 20 Hz, 2H), 2.92-2.72 (m, 16H), 1.98-0.96 (m, 30H). MS (ESI) m/z: 499 (0.4%, [М+1]+). Anal. (C30H50N4O2) С 72.30%, Н 10.09%, N 11.31%. 1,8-(Octamethylene)bis{[(adamant-1-yl)ethyl-1]urea} (3b). The general method above was used with 1-(adamant-1-yl)ethanamine hydrochloride to afford a white solid (0.54 g, 99% yield). Mp 191.8-192.9 оС. 1H NMR (DMSO-d6): δ 5.65-5.61 (t, J = 17 Hz, 2H), 5.48-5.46 (d, J = 8 Hz, 2H), 2.98-2.93 (m, 18H), 1.93-1.24 (m, 30H), 0.89-0.86 (d, 6H). Найдено, %). MS (ESI) m/z: 555 (2.1%, [М+1]+). Anal. (C34H58N4O2) С 73.55%, Н 10.34%, N 10.09%. 1,8-(Octamethylene)bis{[(adamant-1-yl)sec-buthyl-1]urea} (3c) The general method above was used with 1-(adamant-1-yl)butan-2-amine hydrochloride to afford a white solid (0.59 g, 98% yield). Mp 130.1-131.4 оС. 1H NMR (DMSO-d6): δ 5.57-5.54 (t, J = 16 Hz, 2H), 5.44-5.41 (d, J 3   

= 22 Hz, 2H), 3.61-3.56 (dd, 2H), 3.03-2.87 (m, 16H), 1.91-0.74 (m, 44H). MS (ESI) m/z: 611 (30.5%, [М+1]+). Anal. (C38H66N4O2) С 74.64%, Н 10.88%, N 9.29%. 1,8-(Octamethylene)bis[(adamant-2-yl)urea] (3d). The general method above was used with 2-aminoadmantane hydrochloride to afford a white solid (0.49 g, 99% yield). Mp 254.6-255.4 о С. 1H NMR (DMSO-d6) 5.99-5.96 (d, J = 18 Hz, 2H), 5.78-5.75 (t, J = 24 Hz, 2H), 3.66-3.64 (t, 2H), 2.99-2.93 (m, 16H), 1.84-1.20 (m, 30H). MS (ESI) m/z: 499 (0.5%, [М+1]+). Anal. (C30H50N4O2) С 72.31%, Н 9.92%, N 11.35%. 1,4-(Phenylene)bis[(adamant-2-yl)urea] (4a). The general method above was used with 2aminoadmantane hydrochloride to afford a white solid (0.45 g, 98% yield). Mp 362.4-364.1 оС. 1 H NMR (DMSO-d6): 8.50-8.20 (dd, J = 4 Hz, 4H arom), 7.22-7.21 (s, J = 20 Hz, 2H), 6.38-6.34 (d, J = 16 Hz, 2H), 1.94-1.54 (m, 30H). MS (ESI) m/z: 463 (0.6%, [М+1]+). Anal. (C28H38N4O2) С 72.73%, Н 8.34%, N 12.08%. 1,4-(Phenylene)bis{[(adamant-1-yl)ethyl-1]urea} (4b). The general method above was used with 1-(adamant-1-yl)ethanamine hydrochloride to afford a white solid (0.45 g, 98% yield). Mp 285.2-286.1 оС. 1H NMR (DMSO-d6): 8.50-8.20 (dd, J = 4 Hz, 4H arom), 7.27-7.26 (s, 2H), 5.85-5.83 (d, J = 12 Hz, 2H), 1.98-0.94 (m, 38H). MS (ESI) m/z: 519 (0.8%, [М+1]+). Anal. (C32H46N4O2) С 74.16%, Н 8.83%, N 10.93%. Solubility determination. Each inhibitor (1mg) was added into PB (0.1 M Sodium Phosphate, pH 7.4, 300 µL). The suspension was incubated at 30 ˚C for 24h and was cooled to rt for 1h. The precipitates were centrifuged at 10,000 rpm for 10 min at rt by Centrifuge 5415D (Eppendorf, Hauppauge, NY). The supernatant was collected into a new 1.5 mL eppendorf tube and was further diluted by methanol for 5 times. The solution was kept at rt for 15 min to precipitate the salt from the solution. The solution was then centrifuged at 10,000 rpm for 10 min at rt by AccuspinTM MicroR (thermo Fisher, Freemont, CA). The supernatant was transferred to vial and was kept at -20˚C before LC/MS-MS analysis. FRET-Displacement assay procedure Measurement in 96-well plates. All the measurement for FRET- based displacement assay was run in TECAN Infinite® M1000 Pro. Pre-treatment of 96-well plate: In order to prevent nonspecific binding of sEH or its inhibitors on the surface of 96-well plate, the 96 well plates were pre-incubated with PB with 0.1% gelatin overnight at rt. The gelatin coats the plate and prevents non-specific binding to the plate by sEH and inhibitors. The buffer was discarded and the plate was dried before use. Assay procedure: ACPU (one equivalent to sEH, 10 mM, Ethanol) was added to the sEH solution and was incubated for 2h at rt. The sEH-ACPU mixture (20 nM, 100 mM sodium phosphate, 0.1 % gelatin, pH 7.4, 150 uL) was added to each well. The baseline fluorescence (F0) (λexcitation at 280 nm, λemission at 450 nm) of the samples was measured. Because DMSO has been known to quench fluorescence. DMSO was served as a control (FDMSO). The desired concentration of inhibitors which is the concentration that quenched 100% of the fluorescence signal, was added at the first well and was further diluted by 2-fold across the rest of the wells. Based on our study, 12 data points which correspond to 12 different concentrations of the inhibitor, should be enough to calculate an accurate Ki. The samples were incubated at 30 4   

˚C for 1.5h. Then, the fluorescence (λexcitation at 280 nm, λemission at 450 nm) of the samples were measured using the z and gain value previously obtained. The obtained fluorescence signal was transformed as below and was used to calculated the Ki of the inhibitors according to “Curve fitting” section below. Initiated fluorescence = FDMSO (well X) / F0 (well X) Saturated fluorescence = Fat the saturated concentration (well X) / F0 (at well X) Observed fluorescence = F(well X) / F0 (well X) Curve fitting. The curve fitting for Ki determination was reported before. 1 The data manipulation and Ki calculation were based on the original paper by Wang et al. with some modifications suggested by Roehrl et al. 2,3 The displacement assay is based on a three-state equilibrium binding model. This is modeled as described below (Eq. 1) (Eq. 1) [RI] stands for receptor or enzyme-inhibitor complex; L stands for reporting ligand; I stands for inhibitors; [RL] stands for receptor or enzyme-reporting ligand complex. The three-state equilibrium (Eq. 1) is consisted of the sEH-inhibitor complex, sEH and sEHreporting ligand complex. In this study, the relative fluorescence intensity (F3) was plotted against the concentration of sEH inhibitor and the curve was fitted into equation (Eq. 2) derived by Wang et al. for three-state equilibrium. 3 with

F

2 a ‐3b

with

where saturation)/

⁄

cos θ⁄3 ‐a / 3K a b c

K K L K L‐R K ‐K K R; and

θ

arccos

‐2a

2 a ‐3b I‐R; I‐R

⁄

cos θ⁄3 ‐a

(Eq. 2)

K K ;

9ab‐27c / 2 a

‐

⁄

.

F3 = Relative Fluorescence = (observed fluorescence – fluorescence at (initiated fluorescence – fluorescence at saturation) I = the concentration of added unlabeled competing ligand; R = the total concentration of sEH; L = The total concentration of reporting ligand; Kd1 = The dissociation constant of reporting ligand (found by fluorescent binding assay), and ; Kd2 = The inhibition constant of inhibitors.

koff measurement procedure. The koff measurement was conducted as described before. 1 The sEH (8 µM) was incubated with inhibitor (8.8 µM, 100 mM PB buffer, pH 7.4) for 1.5 h at rt. The sEH-inhibitor complex was diluted 40 times with ACPU (20 µM, 100 mM Sodium phosphate buffer, pH 7.4). The fluorescence (λexcitation at 280 nm, λemission at 450 nm) was monitored immediately every 30s up to 5100s. The fluorescence (λemission at 450 nm) was plotted against time (s). The resulting curve was fitted to single exponential growth and the relative koff 5   

was obtained. This procedure was then applied to other inhibitors using the same procedure as described above for ACPU. Microsomal stability. The stability of the sEH inhibitors was determined using human liver microsomes as described.4 the inhibitors ([I]final = 1 M) to a suspension of human liver microsomes ([E]final= 1 mg/mL) in potassium phosphate buffer (0.1M pH7.4) containing 3mM MgCl2 and 1 mM EDTA. After 5 minutes incubation at 37 °C, the reaction was started by the addition of NADPH generating system (buffer was added in the control tubes). After 30 minutes at 37 °C, the reaction was stopped by the addition of one volume of methanol containing CUDA (200 nM) as surrogate. The amount of remaining inhibitors were determine by LC/MS/MS. The stability is reported as a percentage of compound remaining after 30 minutes in these conditions. Results are triplicate average. Manual docking. Compound 3b was manually docked into the active site of the human sEH using the “bioMedCAChe 5.0” software (Fujitsu computer Systems Corporation). For this, the published X-ray crystal structure of the human sEH complexed with a urea-based ligand (PDB accession number 1ZD3) was used. After docking, the ligand and the amino acid residues within 8.0 Å from the ligand were minimized on MM geometry (MM3) as described.5 References 1. Lee, K. S. S.; Morisseau, C.; Yang, J.; Wang, P.; Hwang, S. H.; Hammock, B. D. Anal Biochem. 2013, 434, 259. 2. Roehrl, M. H. A.; Kang, S. H.; Aramburu, J.; Wagner, G.; Rao, A.; Hogan, P. G. Proc Acad Natl Sci USA. 2004, 101, 7554. 3. Wang, Z. X. FEBS Letters 1995, 360, 111. 4. Kim, I.H.; Heirtzler, F.R.; Morisseau, C.; Nishi, K.; Tsai, H.J.; Hammock, B.D. J. Med. Chem. 2005, 48, 3621. 5. Hwang, S.H.; Tsai, H.J.; Liu, J.Y.; Morisseau, C.; Hammock, B.D. J. Med. Chem. 2007, 50, 3825.

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