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  Inhibitors of epoxide hydrolases for the treatment of hypertensionUSPTO Application #: 20060035869Title: Inhibitors of epoxide hydrolases for the treatment of hypertension Abstract: wherein X and Y is each independently nitrogen, oxygen, or sulfur, and X can further be carbon, at least one of R1-R4 is hydrogen, R2 is hydrogen when X is nitrogen but is not present when X is sulfur or oxygen, R4 is hydrogen when Y is nitrogen but is not present when Y is sulfur or oxygen, R1 and R3 are each independently a substituted or unsubstituted alkyl, haloalkyl, cycloalkyl, aryl, acyl, or heterocyclic, or being a metabolite or degradation product thereof.
Biologically stable inhibitors of soluble epoxide hydrolases are provided. The inhibitors can be used, for example, to selectively inhibit epoxide hydrolase in therapeutic applications such as treating inflammation, for use in affinity separations of the epoxide hydrolases, and in agricultural applications. A preferred class of compounds for practicing the invention have the structure shown by Formula 1 (end of abstract)
Agent: Townsend And Townsend And Crew, LLP - San Francisco, CA, US Inventors: Bruce D. Hammock, Christophe H. Morisseau, Jiang Zheng, Marvin H. Goodrow, Tonya Severson, James Sanborn USPTO Applicaton #: 20060035869 - Class: 514114000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Phosphorus Containing Other Than Solely As Part Of An Inorganic Ion In An Addition Salt Doai, Nitrogen, Other Than Nitro Or Nitroso, Bonded Indirectly To Phosphorus The Patent Description & Claims data below is from USPTO Patent Application 20060035869. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10/694,641, filed Oct. 27, 2003, which is a continuation of U.S. application Ser. No. 10/328,495, filed Dec. 23, 2002, now U.S. Pat. No. 6,693,130, which is a continuation of U.S. application Ser. No. 09/721,261, filed Nov. 21, 2000, now U.S. Pat. No. 6,531,506, which is a continuation-in-part of U.S. application Ser. No. 09/252,148, filed Feb. 18, 1999, now U.S. Pat. No. 6,150,415. The contents of each of these applications are incorporated herein by reference. BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention generally relates to methods of treating epoxide hydrolases so as to form complexes therewith, and more particularly relates to compounds, such as ureas, which complex with epoxide hydrolases and can be used to purify, isolate, or inhibit the epoxide hydrolases so complexed. Where compounds such as ureas are immobilized to water insoluble supports, they can be used in affinity separations of epoxide hydrolases. When compounds such as ureas are administered therapeutically, they are useful in treating inflammatory diseases such as adult respiratory distress syndrome or synergizing anti-neoplastic agents. When applied agriculturally, they will selectively inhibit epoxide hydrolase in pest and crop species. [0005] 2. Background of the Invention [0006] Epoxide hydrolases (EH, E.C.3.3.2.3) are enzymes which catalyze the hydrolysis of epoxides including arene oxides to their corresponding diols by the addition of water. EHs play an important role in the metabolism of a variety of compounds including hormones, fatty acid derivatives, chemotherapeutic drugs, carcinogens, environmental pollutants, mycotoxins, and other harmful foreign compounds. [0007] Several members of this ubiquitous enzyme sub-family have been described based on substrate specificity and subcellular localization. Mammalian EHs include cholesterol epoxide hydrolase, leukotriene A4, hydrolase, hepoxilin hydrolase, microsomal epoxide hydrolase (mEH), and soluble epoxide hydrolase (sEH). The latter two enzymes have been extensively studied and found to have broad and complementary substrate selectivity. The microsomal and soluble forms are known to detoxify mutagenic, toxic, and carcinogenic xenobiotic epoxides, are involved in physiological homeostasis, and both are members of the .alpha./.beta.-hydrolase fold family. [0008] U.S. Pat. No. 5,445,956, issued Aug. 29, 1995, inventors Hammock et al., discloses recombinant human and mouse soluble epoxide hydrolase. The mouse enzyme provides a rodent model to evaluate for therapeutic development of human soluble epoxide hydrolase inhibitors. [0009] The search for good sEH inhibitors has been actively pursued for the last about twenty years as reviewed by Hammock et al., Comprehensive Toxicology, (Guengerich, F. P., ed.), Pergamon, Oxford, Vol. 3, Chapter 18, pp. 283-305 (1997). Numerous reagents which selectively modify thiols, imidazoles, and carboxyls irreversibly inhibit sEH. Mullin and Hammock, Arch. Biochem. Biophys., 216, pp. 423-429 (1982) disclosed that chalcone oxides were potent inhibitors of sEH, and Dietze et al., Comp. Biochem. Physiolo., 104B, No. 2, pp. 309-314 (1993) disclosed that trans-3-phenylglycidols were potent chiral inhibitors of sEH. [0010] Copending U.S. Ser. No. 08/909,523, filed Aug. 12, 1997, Hammock et al., suggests the treatment of pulmonary diseases with epoxide hydrolase inhibitors such as chalcone oxides, and describes assays for epoxide hydrolase inhibitors. Among the epoxide hydrolase inhibitors taught are alternative enzyme substrates, such as the epoxide of methyl oleate and other fatty acids and esters or methyl epoxyoctadecenoate and phenyl glycidiols, as well as chalcone oxides. [0011] EH enzymes have been reported to be present in a wide variety of species including bacteria, yeast, fungi, plants, nematodes, insects, birds, fish, and mammals. Indeed, they appear to be present in most, if not all, organisms, and have multiple roles. Plant epoxide hydrolases are also known. For example, fatty acid epoxide hydrolases from apple fruit skin, soybean seedlings, and rice plants have been described. The cDNAs encoding epoxide hydrolase from potato, cress, and tobacco have been isolated and cloned. Stapleton et al., Plant J., 6, pp. 251-258 (1994); Kiyosue et al., Plant J., 6, pp. 259-269 (1994); Guo et al., Plant J., 15, pp. 647-656 (1998). These plant epoxide hydrolases show a high homology with mammalian soluble epoxide hydrolase, but they are 30% shorter on the N-terminus. [0012] Epoxide hydrolases in insects and other arthropods function in the metabolism of endogenous chemical mediators like juvenile hormone and degradation of plant allelochemicals which defend the plant against insects. These enzymes in plants catalyze the hydration of epoxystearic acid to the corresponding .alpha.,.beta.-diols which are important intermediates in the cutin biosynthesis and which have some anti-fungal activity. [0013] Epoxides and diols are key synthetic intermediates in the production of both bulk and speciality organic chemicals. Thus, biosynthetic mechanisms to convert epoxides to diols under gentle, regio and stereospecific conditions are very important. The ability to test if a biosynthetic pathway involves an epoxide by the use of a selective inhibitor can be important in the search for new biosynthetic enzymes and the use of high affinity binding agents in the rapid affinity purification of epoxide hydrolases has proven very important in the study of the mammalian soluble epoxide hydrolases. [0014] The presently known high affinity binding agents for affinity purification of the mammalian soluble epoxide hydrolases include thiols such as benzylthiol, alkyl or terpenoid thiols reacted with epoxy activated SEPHAROSE separation media (SEPHAROSE is available from Pharmacia). These affinity chromatography columns bind a variety of proteins, many of which have a lipophilic catalytic site. The soluble epoxide hydrolase can be selectively eluted from these columns with chalcone oxides, generally as described by Prestwich, Proc. Natl. Acad. Sci. USA, 82, pp. 1663-1667 (1985) and Wixtrom et al., Analyt. Biochem, 169, pp. 71-80 (1988). However, to date there are no affinity purification systems for other epoxide hydrolases which could be used for the initial isolation and cloning of the enzymes, as well as for the isolation of epoxide hydrolases for industrial purposes. New high affinity binding agents for various epoxide hydrolases, particularly for mammalian soluble epoxide hydrolases, remain a useful goal. Also, eluting agents which are competitive, rather then irreversible inhibitors, could be valuable. SUMMARY OF THE INVENTION [0015] In one aspect of the present invention, a method of treating an epoxide hydrolase is provided that is useful to purify, isolate, or inhibit the target epoxide hydrolase by complexing with a free form or immobilized compound so that the activity of the complexed epoxide hydrolase is modified with respect to enzymatically active, uncomplexed epoxide hydrolase. Compounds useful for forming complexes with epoxide hydrolases in practicing this invention include epoxide hydrolase transition state mimics. For example, ureas, amides, and carbamates can mimic the enzyme transition state or other transient intermediates along the reaction coordinate when these compounds stably interact with the enzyme catalytic site. [0016] A preferred class of compounds with this complexing ability for practice in accordance with the invention has the structure shown by Formula 1. wherein X and Y is each independently nitrogen, oxygen, or sulfur, and X can further be carbon, at least one of R.sub.1-R.sub.4 is hydrogen, R.sub.2 is hydrogen when X is nitrogen but is not present when X is sulfur or oxygen, R.sub.4 is hydrogen when Y is nitrogen but is not present when Y is sulfur or oxygen, R.sub.1 and R.sub.3 are each independently a substituted or unsubstituted alkyl, haloalkyl, cycloalkyl, aryl, acyl, or heterocyclic. [0017] Where the modified activity of the complexed epoxide hydrolase is enzyme inhibition, then particularly preferred compound embodiments have an IC.sub.50 (inhibition potency or, by definition, the concentration of inhibitor which reduces enzyme activity by 50%) of less than about 500 .mu.M. For example, we have discovered that the five compounds listed in Table 1 have an IC.sub.50 for mouse soluble epoxide hydrolase of less than about 0.1 .mu.M and less than about 0.8 .mu.M for human soluble epoxide hydrolase. TABLE-US-00001 TABLE 1 Mouse sEH Human sEH Inhibitor Structure No. IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) 2 0.09 .+-. 0.01 0.16 .+-. 0.01 4 0.06 .+-. 0.01 0.13 .+-. 0.02 15 0.09 .+-. 0.01 0.72 .+-. 0.01 18 0.07 .+-. 0.01 0.15 .+-. 0.01 187 0.05 .+-. 0.01 0.42 .+-. 0.03 [0018] In Table 1, the compound 187 is an amide and illustrates that the pharmacophore can be more general than ureas or carbamates. [0019] The enzymes of interest for this invention typically are able to distinguish enantiomers. Thus, in choosing an inhibitor for use for an application in accordance with the invention it is preferred to screen different optical isomers of the inhibitor with the selected enzyme by routine assays so as to choose a better optical isomer, if appropriate, for the particular application. The pharmacophores described here can be used to deliver a reactive functionality to the catalytic site. These could include alkylating agents such as halogens or epoxides or Michael acceptors which will react with thiols and amines. These reactive functionalities also can be used to deliver fluorescent or affinity labels to the enzyme active site for enzyme detection. [0020] Inhibition of soluble epoxide hydrolase can be therapeutically effective to treat an inflammatory disease, such as adult respiratory distress syndrome. This is because suitable epoxide hydrolase inhibitors retard or prevent an inflammatory response in a patient via an inhibition of formation of one or more polyunsaturated lipid metabolites, such as to inhibit the formation of dihydroxy-oxy-eicosadienoates, or DiHOxyEDEs in the arachidonic acid series of oxylipins. Arachidonic acid epoxides (EETs) and in some cases the corresponding diols (DHETs) are widely known to be biologically active as well. They are thought to be involved in the onset and severity of fever (Kozak, 1998), they inhibit prostaglandin E2 production in vascular smooth muscle (Fang, 1998), mediate bradykinin induced vasodilation of heart (Fulton, 1998), induce vasodilation (Oltman, 1998; Imaoka, 1998), and have other physiological effects. The soluble epoxide hydrolase appears to bioactivate an inflammatory-derived mediator, which suggests the need for effective and site-specific inhibitors of epoxide hydrolase, such as are provided as one aspect of the present invention. The inhibitor of epoxide hydrolase can also be used therapeutically to treat fever, inflammatory disease, and hypertension. Thus, inhibitors of the invention act to reduce the conversion of lipid epoxides to the corresponding diols, as in conversion of EEP's to DHET's. [0021] In another aspect of this invention, formation of an epoxide hydrolase complex is useful to purify or to isolate the targeted epoxide hydrolase. For example, the Formula 1 compound may be derivatized so as to be immobilized to a water insoluble support. When one contacts the support with enzymatically active epoxide hydrolase, such as to elute an aqueous solution having epoxide hydrolase through the support, then the complex formation causes a selective separation of the epoxide hydrolase. [0022] Inhibitors, such as certain analogues and active derivatives of the Formula 1 compound, can also be used to elute epoxide hydrolases from these and other supports. As earlier noted, useful compounds to complex with epoxide hydrolases in practicing this invention include epoxide hydrolase transition state dicyclohexyl mimics, such as ureas and carbamates which can mimic the enzyme transition state when they stably interact with the enzyme catalytic site. We have found that analogs of the Formula 1 compound, which can also function as selective inhibitors of soluble epoxide hydrolase, include compounds with the Formula 2 structure (where X, Y, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 may be as described for Formula 1) but wherein Z is oxygen or sulfur and W is carbon, phosphorous, or sulfur. An illustrative several such inhibitor compounds of the Formula 2 structure are shown in Table 2. Compound 12 may be converted to the corresponding carbodiimide (compound 1) and then to the corresponding urea (compound 2). TABLE-US-00002 TABLE 2 Mouse sEH Human sEH Inhibitor Structure No. IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) 1 0.25 .+-. 0.02 0.47 .+-. 0.01 11 3.8 .+-. .01 7.5 .+-. 0.4 12 99 .+-. 5 20 .+-. 1 Continue reading... 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