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Inhibition of Microsomal Epoxide Hydrolases by Ureas, Amides, and Amines

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Inhibition of Microsomal Epoxide Hydrolases by Ureas, Amides, and Amines Chem. Res. Toxicol. 2001, 14, 409-415 409 Inhibition of Microsomal Epoxide Hydrolases by Ureas, Amides, and Amines Christophe Morisseau, John W. Newman, Deanna L. Dowdy, Marvin H. Goodrow, and Bruce D. Hammock* Department of Entomology and University of California Cancer Center, University of California, Davis, California 95616 Received August 11, 2000 The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to several diseases, such as emphysema, spontaneous abortion, and several forms of cancer. To provide new tools for studying the function of mEH, inhibition of this enzyme was investigated. Inhibition of recombinant rat and human mEH was achieved using primary ureas, amides, and amines. Several of these compounds are more potent than previously published inhibitors. Elaidamide, the most potent inhibitor that is obtained, has a Ki of 70 nM for recombinant rat mEH. This compound interacts with the enzyme forming a noncovalent complex, and blocks substrate turnover through an apparent mix of competitive and noncompetitive inhibition kinetics. Furthermore, in insect cell cultures expressing rat mEH, elaidamide enhances the toxicity effects of epoxide-containing xenobiotics. These inhibitors could be valuable tools for investigating the physiological and toxicological roles of mEH. Introduction Over the past decade, mEH was also described as mediating the transport of bile acid into hepatocytes (14, Epoxides are highly strained three-membered cyclic 15). The mechanism by which mEH participates in bile ethers that are often electrophilically reactive mutagens, absorption is not known. Obtaining potent mEH inhibi- carcinogens, or cytotoxins (1). Epoxide hydrolases (EH, tors will provide new tools for better understanding the EC 3.3.2.3) catalyze the hydrolysis of epoxides or arene multiple roles of this enzyme. oxides to their corresponding diols (2). Microsomal ep- oxide hydrolase (mEH) is a key hepatic enzyme involved Over the years, several mEH inhibitors have been in the metabolism of numerous xenobiotics, such as described, including 1,1,1-trichloro-2,3-epoxypropane (16), polyaromatic hydrocarbons, which undergo cytochrome- cyclohexene oxide (17), several other cycloalkene oxides dependent epoxidation (3, 4). Additionally, mEH is likely (18), and valpromide (19), and more recently, our labora- involved in the extrahepatic metabolism of these agents, tory described mEH inhibition by metals (20). The former such as naphthalene in the lungs (5). Generally, the compounds, which are inefficient in vivo, are in fact conversion of epoxides to diols results in less mutagenic substrates of mEH and give only transient inhibition of or carcinogenic compounds. However, in some cases, as this enzyme in vitro (2). Valpromide affects the normal for benzo[a]pyrene 4,5-oxide, diol formation can lead to metabolism of carbamazepine by inhibiting the mEH in the stabilization of a secondary epoxide, increasing the vivo (19). However, the anticonvulsant properties of mutagenic and carcinogenic potential of the product (6). valpromide could induce undesirable secondary effects The role of mEH in xenobiotic detoxication is also if it is used as a mEH inhibitor. We recently developed supported by recent polymorphism studies showing a new inhibitors for the soluble epoxide hydrolase (sEH), relationship between this enzyme and susceptibility to an enzyme distantly related to mEH (21). These inhibi- emphysema (7) and several forms of cancer (8). Despite tors are based on urea, carbamate, or amide central the fact that mEH knockout mice do not present an functions. These inhibitors act by mimicking a transition obvious phenotype (9), there are several points suggesting state in the enzyme catalytic mechanism of epoxide an endogenous role for this enzyme. A potential role of hydrolysis (22). Because sEH and mEH have similar mEH in sexual development is supported by the facts mechanisms of catalysis (3, 23), one could expect that that androstene oxide is a very good mEH substrate (10), urea and urea-like compounds would be inhibitors of the and that mEH is an apparent subunit of the anti- mEH also. We describe herein the discovery of new potent estrogen-binding site (11). Such a role could be related and stable inhibitors of mEH and their application in one to the observed relation between mEH polymorphism and in vitro model. spontaneous abortion (12), which has been reported to occur in more than 10% of recognized pregnancies (13). Materials and Methods * To whom correspondence should be addressed: Department of Chemicals. General laboratory reagents were purchased Entomology, University of California, Davis, CA 95616. Phone: (530) from either Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO), 752-7519. Fax: (530) 752-1537. E-mail: [email protected]. and used without further purification. Compounds 15-29, 40, 10.1021/tx0001732 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/10/2001 410 Chem. Res. Toxicol., Vol. 14, No. 4, 2001 Morisseau et al. Table 1. Inhibition of Rat mEH c-SO Hydrolysis by Table 2. Inhibition of RmEH by Several Pharmacophores N-Octylureas a Melting points (mp) are uncorrected. b The enzyme and the inhibitor are incubated together for 5 min at 30 °C before the a Melting points (mp) are uncorrected. “_” indicated that the addition of substrate (c-SO, 50 µM). Results are means ( SD of melting points of the commercial compound were not measured. three separate experiments. b The enzyme and the inhibitor are incubated together for 5 min at 30 °C before the addition of substrate (c-SO, 50 µM). Results ( and 41 were obtained from Aldrich, while compound 37 was are means SD of three separate experiments. purchased from Sigma. Compounds 1-10, 13, and 39 were Table 3. Inhibition of Rat mEH by Amines and Amides synthesized by the condensation of the appropriate isocyanate with Diverse Alkyl Chains and amine, whereas compounds 11 and 12 were synthesized by the condensation of the appropriate isocyanate and alcohol as previously described (21). Compounds 14, 30-36, and 38 were synthesized by the condensation of the appropriate acyl chloride and ammonia as described by Roe et al. (24). Reaction products were purified by recrystallization. Purities of >95% were obtained for all of the compounds that were studied. Assignment of purities was carried out using NMR and mass spectroscopy data. In addition to sharp melting points and single spots on silica gel TLC, the obtained products were characterized using 1H and/or 13C NMR (General Electric QE-300 instrument), infrared, and mass spectroscopy [for GC/MS, Hewlett-Packard 6890 gas chromatograph (San Jose, CA) equipped with a 30 m × 0.25 mm × 0.25 µm film DB-5ms column (J&W Scientific, Folsom, CA) and a 5973 mass spectra detector; for electrospray ionization (ESI-MS), positive mode Fisons Quattro BQ ap- paratus (Altrancham, England), 5 µL/min flow of a 1:1:0.05 (v:v:v) acetonitrile/water/formic acid mixture, with a cone voltage of 50 V; fast atom bombardment mass spectra were generated on a Kratos MS-50 mass spectrometer (Kratos Analytical, Manchester, U.K.)]. Melting point and reaction yields are listed in Tables 1-3. Acquired IR, NMR, and MS data used for structural confirmation can be found as Supporting Information. The detailed syntheses of two ureas and an amide representing these reaction pathways are included below. Compound 1 (N-Octylurea). Octyl isocyanate (0.88 mL, 0.77 g, 5.0 mmol) and 6 mL of 1 M ammonium hydroxide were vigorously shaken for 3 h. The precipitate was collected, washed with water followed by an acetone wash, and vacuum-oven dried to obtain 0.77 g (90%) of 1. The isolated solid had an mp of 96.0- 97.5 °C, which was unchanged by recrystallizations from either a methanol/water mixture (5:2, v:v) or a hexanes/ethyl acetate mixture (4:1, v:v): IR (KBr) 3393 (s, NH), 1657 (s, CdO), 1603 -1 1 (vs), 1537 cm (s, amide II); H NMR (CDCl3) δ 4.5 (m, 1 H, NH), 4.3 (m, 2 H, NH2), 3.15 (dd, J ) 7.0, 6.0 Hz, 2 H, CH2-1), 1.5 (m, 2 H, CH2-2), 1.3 (m, 10 H, CH2-3-7), 0.88 (t, J ) 6.6 Hz, 13 3H,CH3); C NMR (DMSO-d6) δ 158.7 (CdO), 39.3 (C-1), 31.2 (C-2), 30.0 (C-4), 28.8 (C-5 or -6), 28.7 (C-5 or -6), 26.4 (C-3), Compound 7 (N-Cyclopropyl-N′-octylurea). To 0.115 g 22.1 (C-7), 13.8 (C-8); fast atom bombardment MS m/z (relative (2.00 mmol) of cylcopropylamine in 10 mL of hexane were added intensity) 345 (2M + H+, 10%), 173 (M + H+, 100%). 0.310 g (2.00 mmol) of octyl isocyanate and 1 drop of 1,8- Microsomal Epoxide Hydrolase Inhibition Chem. Res. Toxicol., Vol. 14, No. 4, 2001 411 -1 -1 diazabicyclo[5.4.0]undec-7-ene. After 1 day at ambient temper- nmol min mL for the substrate c-SO. Substrate (1.5 < [S]final ature, compound 7 was obtained (0.405 g, 95%) as white < 50 µM) was then added. Velocity was measured as described crystals: mp 57.5-59.0 °C; IR (KBr) 3339 (m, NH), 1626 (vs, under IC50 Assay Conditions. For each inhibitor concentration, -1 1 CdO), 1573 cm (s, amide II); H NMR (CDCl3) δ 5.17 (br, 2 linear relations are produced between the inverse of the velocity H, 2 NH), 3.20 (dt, J ) 6.6, 6.3 Hz, 2 H, CH2-1), 2.4 (m, 1 H, and the inverse substrate concentration (Lineweaver-Burk CH), 1.5 (m, 2 H, CH2-2), 1.3 (m, 10 H, CH2-3-7), 0.88 (t, 3 H, representation).
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