WO2015188060A1 - FLUOROSULFONYL sEH INHIBITORS - Google Patents

Abstract

Fluorosulfonyl-substituted sEH inhibitors are compounds represented by Formula (I): wherein R1 is selected from the group consisting of alkyl, heteroalkyi, C5-C12 cycloalkyi, C5-C12 cycloalkylalkyi, C5-C12 cycloalkylheteroalkyi, arylalkyi, arylheteroalkyi, aryl, and heteroaryl; and R1 can be substituted or unsubstituted; P1 is a primary pharmacophore; P2 is a secondary pharmacophore; P3 is a tertiary pharmacophore; and L1 and L2 are linking groups. The subscript m has a value of 0 or 1; n has a value of 0 or 1; and the compound of Formula (I) includes at least one S02F ("fluorosulfonyl") group covalently bonded thereto.

Description

FLUOROSULFONYL sEH INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit to U.S. Provisional Application No.

62/008,989, filed on June 6, 2014, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

This invention relates to soluble epoxide hydrolase inhibitors. More particularly, this invention relates to soluble epoxide hydrolase inhibitors provided with a

fluorosulfonyl-type group attached to a carbon, oxygen or nitrogen on the inhibitor structure.

BACKGROUND OF INVENTION

Soluble epoxide hydrolase (sEH) is a bifunctional, homodimeric enzyme with hydrolase and phosphatase activity, sEH is highly expressed in the liver, but it is also expressed in tissues such as vascular endothelium, leukocytes, red blood cells, smooth muscle cells, adipocytes, as well as the kidney proximal tubule. sEH metabolizes cis-epoxyeicosatrienoic acids (EETs) as well as other lipid mediators, and as such sEH plays a role in several diseases including hypertension, cardiac hypertrophy,

arteriosclerosis, brain and heart ischemia injury, cancer and pain.

The inhibition of sEH represents a promising therapeutic strategy inasmuch as the inhibition would lead to desirable elevated levels of EETs and attendant beneficial therapeutic effects on blood pressure, inflammation, and cardiac hypertrophy.

sEH inhibitors are known and are described in Hwang et al., J. Med. Chem.

50( 1 6):3825-3840 (2007) as well as in several U.S. patents such as U.S. Patent No,.

7,662,91 0 to Hammock et al., U.S. Patent No. 8,476,043 to Hammock et al. and U.S. Publication No. 2013/0143925 by Hammock et al.

It has now been found that such sEH inhibitors, when derivatized or substituted with a fluorosulfonyl-type group can achieve enhanced solubility and exhibit good or improved sEH inhibitory activity. SUMMARY OF INVENTION

Fluorosulfonyl derivatives of soluble epoxide hydrolase (sEH) inhibitors are useful for treatment of sEH-mediated diseases or conditions.

Fluorosulfonylated sEH compounds are represented by Formula (I):

(I) R'-P'-L'-f P^ L^ P^ wherein R¹ is selected from the group consisting of alkyl, heteroalkyl, C5-C 12 cycloalkyl, C5-C12 cycloalkylalkyl, C5-C12 cycloalkylheteroalkyl, arylalkyl, arylheteroalkyl, aryl, and heteroaryl; and R¹ can be substituted or unsubstituted; P¹ is a primary pharmacophore as further defined hereinbelow; P² is a secondary pharmacophore as further defined hereinbelow; P³ is a tertiary pharmacophore as further defined hereinbelow; L¹ and L² are linking groups, also further defined herein; m has a value of 0 or 1 ; n has a value of 0 or 1 ; and the compound of Formula (I) includes at least one S0₂F ("fluorosulfonyl") group covalently bonded thereto, e.g., by a group such as -S0₂F, -OS0₂F, -N(R )S0₂F,

-N(R³)(CH₂CH₂S0₂F), and -N(CH₂CH₂S0₂F)₂ in which R³ can be substituted or unsubstituted and is selected from the group consisting of hydrogen, C|-C₆ alkyl, C -Q, alkenyl, C₂-C₆ alkynyl, Ci-C₆ haloalkyl (e.g., CF₃, CFI₂CF₃, CC1₃, etc.), aryl, heteroaryl, heterocyclyl, and C -Cg cycloalkyl.

These fluorosulfonyl derivatives of sEH inhibitors possess anti-inflammatory, antiatherosclerotic, antihypertensive, and/or analgesic properties and can be administered to a patient in need of treatment as therapeutic agents orally, parenterally, subcutaneously, intramuscularly, intravenously or topically.

DETAILED DESCRIPTION OF SELECTED EMBODIMENTS

Definitions

"cis-Epoxyeicosatrienoic acids" ("EETs") are biomediators synthesized by cytochrome P450 epoxygenases. "Epoxide hydrolases" ("ΕΗ;" EC 3.3.2.3) are enzymes in the alpha/beta hydrolase fold family that add water to 3 membered cyclic ethers termed epoxides.

"Soluble epoxide hydrolase" ("sEH") is an enzyme which in endothelial, smooth muscle and other cell types converts EETs to dihydroxy derivatives called

dihydroxyeicosatrienoic acids ("DHETs"). The cloning and sequence of the murine sEH is set forth in Grant et al., J. Biol. Chem, 268(23): 17628- 17633 (1993). The cloning, sequence, and accession numbers of the human sEH sequence are set forth in Beetham et al., Arch. Biochem. Biophys. 305(1): 197-201 (1993). The amino acid sequence and the nucleic acid sequence of human sEH is described U.S. Pat. No. 5,445,956. Soluble epoxide hydrolase represents a single highly conserved gene product with over 90% homology between rodent and human (Arand et al., FEBS Lett., 338:251 -256 (1994)).

The terms "treat", "treating" and "treatment" refer to any method of alleviating or abrogating a disease or its attendant symptoms.

The term "therapeutically effective amount" refers to that amount of the compound being administered sufficient to prevent or decrease the development of one or more of the symptoms of the disease, condition or disorder being treated.

The term "modulate" refers to the ability of a compound to increase or decrease the function, or activity, of the associated activity (e.g., soluble epoxide hydrolase).

"Modulation", as used herein in its various forms, is meant to include antagonism and partial antagonism of the activity associated with sEH. Inhibitors of sEH are compounds that, e.g., bind to, partially or totally block the enzyme's activity.

The term "compound" as used herein is intended to encompass not only the specified molecular entity but also its pharmaceutically acceptable, pharmacologically active derivatives, including, but not limited to, salts, prodrug conjugates such as esters and amides, metabolites and the like.

The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The "subject" is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.

As used herein, the term "sEH-mediated disease or condition" and the like refers to a disease or condition characterized by less than or greater than normal, sEH activity. A sEH-mediated disease or condition is one in which modulation of sEH results in some effect on the underlying condition or disease (e.g., a sEH inhibitor or antagonist results in some improvement in patient well-being in at least some patients).

"Parenchyma" refers to the tissue characteristic of an organ, as distinguished from associated connective or supporting tissues.

"Chronic Obstructive Pulmonary Disease" or "COPD" is also sometimes known as "chronic obstructive airway disease", "chronic obstructive lung disease", and "chronic airways disease." COPD is generally defined as a disorder characterized by reduced maximal expiratory flow and slow forced emptying of the lungs. COPD is considered to encompass two related conditions, emphysema and chronic bronchitis. COPD can be diagnosed by the general practitioner using art recognized techniques, such as the patient's forced vital capacity ("FVC"), the maximum volume of air that can be forceably expelled after a maximal inhalation. In the offices of general practitioners, the FVC is typically approximated by a 6 second maximal exhalation through a spirometer. The definition, diagnosis and treatment of COPD, emphysema, and chronic bronchitis are well known in the art and discussed in detail by, for example, Honig and Ingram, in Harrison's Principles of Internal Medicine, (Fauci et al., Eds.), 14th Ed., 1998, McGraw-Hill, New York, pp. 1451 - 1460 (hereafter, "Harrison's Principles of Internal Medicine").

"Emphysema" is a disease of the lungs characterized by permanent destructive enlargement of the airspaces distal to the terminal bronchioles without obvious fibrosis.

"Chronic bronchitis" is a disease of the lungs characterized by chronic bronchial secretions which last for most days of a month, for three months a year, for two years. As the names imply, "obstructive pulmonary disease" and "obstructive lung disease" refer to obstructive diseases, as opposed to restrictive diseases. These diseases particularly include COPD, bronchial asthma and small airway disease.

"Small airway disease." There is a distinct minority of patients whose airflow obstruction is due, solely or predominantly to involvement of the small airways. These are defined as airways less than 2 mm in diameter and correspond to small cartilaginous bronchi, terminal bronchioles and respiratory bronchioles. Small airway disease (SAD) represents luminal obstruction by inflammatory and fibrotic changes that increase airway resistance. The obstruction may be transient or permanent.

The "interstitial lung diseases (ILDs)" are a group of conditions involving the alveolar walls, perialveolar tissues, and contiguous supporting structures. The tissue between the air sacs of the lung is the interstitium, and this is the tissue affected by fibrosis in the disease. Persons with the disease have difficulty breathing in because of the stiffness of the lung tissue but, in contrast to persons with obstructive lung disease, have no difficulty breathing out. The definition, diagnosis and treatment of interstitial lung diseases are well known in the art.

"Idiopathic pulmonary fibrosis," or "IPF," is considered the prototype ILD.

Although it is idiopathic in that the cause is not known, the term refers to a well defined clinical entity.

"Bronchoalveolar lavage," or "BAL," is a test which permits removal and examination of cells from the lower respiratory tract and is used in humans as a diagnostic procedure for pulmonary disorders such as IPF. In human patients, it is usually performed during bronchoscopy.

As used herein, the term "alkyl" refers to a saturated hydrocarbon radical which may be straight-chain or branched-chain (for example, ethyl, isopropyl, t-amyl, or 2,5- dimethylhexyl). This definition applies both when the term is used alone and when it is used as part of a compound term, such as "aralkyl," "alkylamino,"alkylaryl"and similar terms. In some embodiments, alkyl groups are those containing 1 to 24 carbon atoms. All numerical ranges in this specification and claims are intended to be inclusive of their upper and lower limits. Lower alkyl refers to those alkyl groups having 1 to 4 carbon atoms. Additionally, the alkyl and heteroalkyl groups may be attached to other moieties at any position on the alkyl or heteroalkyl radical which would otherwise be occupied by a hydrogen atom (such as, for example, 2-pentyl, 2-methylpent-l -yl and 2-propyloxy).

Divalent alkyl groups may be referred to as "alkylene", and divalent heteroalkyl groups may be referred to as "heteroalkylene" such as those groups used as linkers in the present invention. The alkyl, alkylene, and heteroalkyl moieties may also be optionally substituted with halogen atoms, or other groups such as oxo, cyano, nitro, alkyl, alkylamino, carboxyl, hydroxyl, alkoxy, aryloxy, and the like.

The terms "cycloalkyl" and "cycloalkylene" refer to a saturated hydrocarbon ring and includes bicyclic and polycyclic rings. Similarly, cycloalkyl and cycloalkylene groups having one or more heteroatom (e.g., N, O, or S) in place of a carbon ring atom may be referred to as "heterocycloalkyl" and "heterocycloalkylene," respectively. Examples of cycloalkyl and heteroaryl groups are, for example, cyclohexyl, norbornyl, adamantyl, morpholinyl, thiomorpholinyl, dioxothiomorpholinyl, and the like. The cycloalkyl and heterocycloalkyl moieties may also be optionally substituted with halogen atoms, or other groups such as nitro, alkyl, alkylamino, carboxyl, alkoxy, aryloxy and the like. In some embodiments, cycloalkyl and cycloalkylene moieties are those having 3 to 12 carbon atoms in the ring (e.g., cyclohexyl, cyclooctyl, norbornyl, adamantyl, and the like). In some embodiments, heterocycloalkyl and heterocycloalkylene moieties are those having 1 to 3 hetero atoms (preferably N, O and/or S) substituting for carbon in a 5 or 6-membered ring (e.g., morpholinyl, morpholinylene thiomorpholinyl, thiomorpholinylene,

dioxothiomorpholinyl, dioxothiomorpholinylene piperidinyl, piperidinylene, and the like). Additionally, the term "(cycloalkyl)alkyl" refers to a group having a cycloalkyl moiety attached to an alkyl moiety. Examples are cyclohexylmethyl, cyclohexylethyl, and cyclopentylpropyl.

The term "alkenyl" as used herein refers to an alkyl group as described above which contains one or more sites of unsaturation that is a double bond. Similarly, the term "alkynyl" as used herein refers to an alkyl group as described above which contains one or more sites of unsaturation that is a triple bond. The term "alkoxy" refers to an alkyl radical as described above which also bears an oxygen substituent which is capable of covalent attachment to another hydrocarbon radical (such as, for example, methoxy, ethoxy, aryloxy and t-butoxy).

The term "aryl" refers to an aromatic carbocyclic substituent which may be a single ring or multiple rings which are fused together, linked covalently or linked to a common group such as an ethylene or methylene moiety. Similarly, aryl groups having a heteroatom (e.g. N, O or S) in place of a carbon ring atom are referred to as "heteroaryl". Examples of aryl and heteroaryl groups are, for example, phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-diphenyl- l -ethyl, thienyl, pyridyl and quinoxalyl. The aryl and heteroaryl moieties may also be optionally substituted with halogen atoms, or other groups such as nitro, alkyl, alkylamino, carboxyl, alkoxy, phenoxy and the like. Additionally, the aryl and heteroaryl groups may be attached to other moieties at any position on the aryl or heteroaryl radical which would otherwise be occupied by a hydrogen atom (such as, for example, 2-pyridyl, 3-pyridyl and 4-pyridyl). Divalent aryl groups are "arylene", and divalent heteroaryl groups are referred to as "heteroarylene" such as those groups used as linkers in the present invention.

The terms "arylalkyl", "arylalkenyl" and "aryloxyalkyl" refer to an aryl radical attached directly to an alkyl group, an alkenyl group, or an oxygen which is attached to an alkyl group, respectively. For brevity, aryl as part of a combined term as above, is meant to include heteroaryl as well.

The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C _\-C haloalkyl" is mean to include trifluoromethyl, 2,2,2- trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term "hetero" as used in a "heteroatom-containing alkyl group" (a

"heteroalkyl" group) or a "heteroatom-containing aryl group" (a "heteroaryl" group) refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur or more that none non-carbon atom (e.g., sulfonamide). Similarly, the term "heteroalkyl" refers to an alkyl substituent that is heteroatom- containing, the term "heterocyclic" refers to a cyclic substituent that is heteroatom- containing, the terms "heteroaryl" and heteroaromatic" respectively refer to "aryl" and "aromatic" substituents that are heteroatom-containing, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1 ,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, etc.

The term "hydrophobic radical" or "hydrophobic group" refers to a group which lowers the water solubility of a molecule. In some embodiments, hydrophobic radicals are groups containing at least 3 carbon atoms.

The term "carboxylic acid analog" refers to a variety of groups having an acidic moiety that are capable of mimicking a carboxylic acid residue. Examples of such groups are sulfonic acids, sulfinic acids, phosphoric acids, phosphonic acids, phosphinic acids, sulfonamides, and heterocyclic moieties such as, for example, imidazoles, triazoles and tetrazoles.

The term "substituted" refers to the replacement of an atom or a group of atoms of a compound with another atom or group of atoms. For example, an atom or a group of atoms may be substituted with one or more of the following substituents or groups: halo, cyano, nitro, alkyl, alkylamino, hydroxyalkyl, haloalkyl, carboxyl, hydroxyl, alkoxy,

alkoxyalkoxy, haloalkoxy, thioalkyl, aryl, aryloxy, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl optionally substituted with 1 or more, preferably 1 to 3, substituents selected from halo, halo alkyl and alkyl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, alk(en)(yn)yl groups, halo, cyano, hydroxy, haloalkyl and polyhaloalkyl, preferably halo lower alkyl, especially trifluoromethyl, formyl, alkylcarbonyl, arylcarbonyl that is optionally substituted with 1 or more, preferably 1 to 3, substituents selected from halo, halo alkyl and alkyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino,

arylcarbonylamino, azido, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl and arylaminosulfonyl. When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group.

The term "unsubstituted" refers to a native compound that lacks replacement of an atom or a group of atoms.

The terms "sulfonyl fluoride" and "fluorosulfonyl" are used herein generically to refer to substituents comprising -S0₂F attached via the sulfur atom thereof to a carbon, oxygen or nitrogen. Methods of preparing sulfonyl fluoride substituents are well known in the art.

The present invention is based on the discovery that the presence of a

fluorosulfonyl group on the sEH inhibitor (e.g., attached to a carbon, nitrogen, or oxygen atom thereof) provides a sEH inhibitor with improved physical properties.

The present invention provides soluble epoxide hydrolase inhibitors represented by the Formula (I):

and their pharmaceutically acceptable salts; wherein the inhibitor comprises at least one S0₂F group covalently bonded to a carbon, oxygen or nitrogen atom thereof, preferably on an R¹ group or a P¹ group.

In Formula (I), R¹ is selected from the group consisting of alkyl, heteroalkyl, C5-C12 cycloalkyl, C5-C12 cycloalkylalkyl, C₅-C_{) 2} cycloalkylheteroalkyl, arylalkyl, arylheteroalkyl, aryl, and heteroaryl; and R¹ can be substituted or unsubstituted.

P' is a primary pharmacophore selected from the group consisting of -0C(0)O, -OC(0)CH₂-, -CH₂C(0)0-, -OC(O)-, -C(0)0-, -NHC(NH)NH-, -NHC(NH)CH₂-, -CH₂C(NH)NH-,-NHC(NH)-, -C(NH)NH-, -NHC(0)NH-, -OC(0)NH-, -NHC(0)0-, -NHC(S)NH-, -NHC(S)CH₂-, -CH₂C(S)NH-, -SC(0)CH₂-, -CH₂C(0)S-, -SC(NH)CH₂-, - -, -N=C=N-, -CH₂C(0)NH-, -NHC(0)CH₂-, -C(0)NH-, -NHC(O)-,

P 2 is a secondary pharmacophore selected from the group consisting of -NH-, -OC(0)0-, -C(O)-, -CH(OH)-, -0(CH₂CH₂0)_q-, -C(0)0-, -OC(O)-, -NHC(NH)NH-,

-NHC(NH)CH₂-, -CH₂C(NH)NH-, -NHC(0)NH-, -OC(0)NH-, -NHC(0)0-, -C(0)NH-,

NHC(O)-, -NHC(S)NH-, -NHC(S)CH₂-, -CH₂C(S)NH-, -SC(0)CH₂

SC(NH)CH₂-, -CH₂C(NH)S-, -N=C=N-,

C₂-C₆ alkynyl, C, -C₆ haloalkyl, aryl, heteroaryl, heterocyclyl, -0(CH₂CH₂0)_q-R², -OR², -C(0)NHR², -C(0)NHS(0)₂R², -NHS(0)₂R², -OC₂-C₄alkyl-C(0)OR², -C(0)R², -C(0)OR² and carboxylic acid analogs, wherein R² is selected from the group consisting of hydrogen, substituted or unsubstituted C]-C₄ alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl and substituted or unsubstituted aryl C 1 -C4 alkyl.

In Formula (I), the subscripts n and m are each independently 0 or 1 , at least one of n or m is 1 , and the subscript q is 0 to 6.

In Formula (I), L¹ represents a first linking group that is selected from the group consisting of a covalent bond, C i-C₆ alkylene, C3-C6 cycloalkylene, 5-membered ring heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S, 6- membered ring heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S, arylene, and heteroarylene; when m is 1 , L represents a second linking group selected from the group consisting of a covalent bond, Ci -C_{! 2} alkylene, C3-Q, cycloalkylene, 5- membered ring heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S, 6-membered ring heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S (e.g., piperadinylene), arylene, heteroarylene; an amino acid, a dipeptide, a

2 ! dipeptide analog, and a combination of two or more thereof; when m is 0, L is FI. L and L² can be substituted or unsubstituted when they are not covalent bonds. A preferred P¹ group is ureido (-NHC(O)NH-). A preferred R¹ is aryl, particularly phenyl, which can include one or more substituent groups. Non-limiting examples of some preferred P³ groups include substituted or unsubstituted aryl (e.g., phenyl, optionally including one or more substituent), heterocyclyl (e.g., piperidinyl and N-substituted piperidinyl).

In some preferred embodiments, the inhibitor compound of Formula (I) can be represented by Formula (II):

wherein P¹ is as defined for formula (I), but preferably is ureido (-NHC(O)NH-); each x independently is 0, 1 , 2, or 3, provided that at least on x is not 0; and each X¹

independently is selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C(, haloalkyl (e.g., CF₃, CH₂CF₃, CC1₃, etc.), aryl, heteroaryl, heterocyclyl,

-0(CH₂CH₂0)_q-R³, -OR³, -C(0)NHR³, -C(0)NHS0₂R³, -NHS0₂R³, -S0₂R³,

-OC₂-C₄alkyl-C(0)OR³, -C(0)R³, -C(0)OR³ , halogen (e.g., F, CI, Br, I), -S0₂F, -OS0₂F, -N(R )S0₂F, -N(R )(CH₂CH₂S0₂F), and -N(CH₂CH₂S0₂F)₂, wherein R³ can be substituted or unsubstituted and is selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C C₆ haloalkyl (e.g., CF₃, CH₂CF₃, CC1₃, etc.), aryl, heteroaryl, heterocyclyl, and C₃-C₈ cycloalkyl; and wherein at least one X¹ comprises an S0₂F moiety (i.e., a group selected from -S0₂F, -OS0₂F, -N(R )S0₂F,

N(R )(CH₂CH₂S0₂F), and -N(CH₂CH₂S0₂F)₂).

In some preferred embodiments, the inhibitor compound of Formula (I) can be represented by Formula (III):

wherein P¹ is as defined for Formula (I), but preferably is ureido (-NHC(O)NH-); x is 0, 1 , 2, or 3; X¹ is defined as in Formula (II); X² is selected from -C(0)NF1R³,

-SO₂R³, -S0₂R³, C ,-C₆ alkylaryl (e.g., benzyl), C , -Q, alkylaryl-S0₂R³, -C,-C₆ alkylhaloaryl, -C(0)R³, -C(0)OR³ , -S0₂F, -CH₂CH₂S0₂F, and Ci-C₆ alkylaryl-S0₂F (e.g., a -CH₂PhS0₂F group such as 4-fluorosulfonylbenzyl); and wherein at least one of X¹ and X² comprises an S0₂F moiety (i.e., a group selected from - S0₂F, -CH₂CH₂S0₂F, and d-C₆ alkylaryl-S0₂F).

In some preferred embodiments, the inhibitor compound of Formula (I) can be represented by Formula (IV):

wherein P¹ is as defined for Formula (I), but preferably is ureido (-NHC(O)NH-); P² is as defined for Formula (I), but preferably is -0-; x is defined as in Formula (II); and X¹ is defined as in Formula (II).

Treatment of diseases modulated by soluble epoxide hydrolases (sEH) generally involves administering to a subject in need of such treatment an effective amount of a compound of Formula (I), e.g., a compound of Formula (II), (III), or (IV). The dose, frequency and timing of such administering will depend in large part on the selected therapeutic agent, the nature of the condition being treated, the condition of the subject including age, weight and presence of other conditions or disorders, the formulation being administered and the discretion of the attending physician. Preferably, the compositions and compounds of the invention and the pharmaceutically acceptable salts thereof are administered via oral, parenteral, subcutaneous, intramuscular, intravenous or topical routes. Generally, the compounds are administered in dosages ranging from about 2 mg up to about 2,000 mg per day, although variations will necessarily occur depending, as noted above, on the disease target, the patient, and the route of administration. Dosages are administered orally in the range of about 0.05 mg/kg to about 20 mg/kg, more preferably in the range of about 0.05 mg/kg to about 2 mg/kg, most preferably in the range of about 0.05 mg/kg to about 0.2 mg per kg of body weight per day. The dosage employed for the topical administration will, of course, depend on the size of the area being treated.

The sEH inhibitor compounds can be formulated as pharmaceutical compositions for administration as described herein. Such therapeutic compounds can be formulated as a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, vehicle, or diluent, such as an aqueous buffer at a physiologically acceptable pH (e.g., pH 7 to 8.5), a polymer-based nanoparticle vehicle, a liposome, and the like. The

pharmaceutical compositions can be delivered in any suitable dosage form, such as a liquid, gel, solid, cream, or paste dosage form. In one embodiment, the compositions can be adapted to give sustained release of the compound of the sEH inhibitor compounds.

Pharmaceutical compositions comprising therapeutic compounds of Formula (I) can be administered to a subject or patient in a therapeutically effective amount to treat diseases or conditions described herein.

In some embodiments, the pharmaceutical compositions include, but are not limited to, those forms suitable for oral, rectal, nasal, topical, (including buccal and sublingual), transdermal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration. The compositions can, where appropriate, be conveniently provided in discrete dosage units. The pharmaceutical compositions of the invention can be prepared by any of the methods well known in the pharmaceutical arts.

Pharmaceutical formulations suitable for oral administration include capsules, cachets, or tablets, each containing a predetermined amount of one or more of the compounds of Formula (I), as a powder or granules. In another embodiment, the oral composition is a solution, a suspension, or an emulsion. Alternatively, the compounds of Formula (I) can be provided as a bolus, electuary, or paste. Tablets and capsules for oral administration can contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, colorants, flavoring agents, preservatives, or wetting agents. The tablets can be coated according to methods well known in the art, if desired. Oral liquid preparations include, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs. Alternatively, the compositions can be provided as a dry product for constitution with water or another suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, emulsifying agents, nonaqueous vehicles (which may include edible oils), preservatives, and the like. The additives, excipients, and the like typically will be included in the compositions for oral administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The compounds of Formula (I) will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts.

Pharmaceutical compositions for parenteral administration (e.g. by bolus injection or continuous infusion) or injection into amniotic fluid can be provided in unit dose form in ampoules, pre-filled syringes, small volume infusion, or in multi-dose containers, and preferably include an added preservative. The compositions for parenteral administration can be suspensions, solutions, or emulsions, and can contain excipients such as suspending agents, stabilizing agent, and dispersing agents. Alternatively, the compounds of Formula (I) can be provided in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen- free water, before use. The additives, excipients, and the like typically will be included in the compositions for parenteral administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art.

Pharmaceutical compositions for topical administration of the compounds to the epidermis (mucosal or cutaneous surfaces) can be formulated as ointments, creams, lotions, gels, or as a transdermal patch. Such transdermal patches can contain penetration enhancers such as linalool, carvacrol, thymol, citral, menthol, t-anethole, and the like.

Ointments and creams can, for example, include an aqueous or oily base with the addition of suitable thickening agents, gelling agents, colorants, and the like. Lotions and creams can include an aqueous or oily base and typically also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, coloring agents, and the like. Gels preferably include an aqueous carrier base and include a gelling agent such as cross-linked polyacrylic acid polymer, a derivatized polysaccharide (e.g., carboxymethyl cellulose), and the like. The additives, excipients, and the like typically will be included in the compositions for topical administration to the epidermis within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The compounds of Formula (I) will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts.

Pharmaceutical compositions suitable for topical administration in the mouth (e.g., buccal or sublingual administration) include lozenges comprising the compound in a flavored base, such as sucrose, acacia, or tragacanth; pastilles comprising the peptide in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. The pharmaceutical compositions for topical administration in the mouth can include penetration enhancing agents, if desired. The additives, excipients, and the like typically will be included in the compositions of topical oral administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art.

A pharmaceutical composition suitable for rectal administration comprises a compound of the present invention in combination with a solid or semisolid (e.g., cream or paste) carrier or vehicle. For example, such rectal compositions can be provided as unit dose suppositories. Suitable carriers or vehicles include cocoa butter and other materials commonly used in the art. The additives, excipients, and the like typically will be included in the compositions of rectal administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The compounds of Formula (I) will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts.

Pharmaceutical compositions suitable for intra-nasal administration are also encompassed by the present invention. Such intra-nasal compositions comprise a compound of Formula (I) in a vehicle and suitable administration device to deliver a liquid spray, dispersible powder, or drops. Drops may be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents, or suspending agents. Liquid sprays are conveniently delivered from a pressurized pack, an insufflator, a nebulizer, or other convenient means of delivering an aerosol comprising the peptide. Pressurized packs comprise a suitable propellant such as

dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas as is well known in the art. Aerosol dosages can be controlled by providing a valve to deliver a metered amount of the peptide. Alternatively, pharmaceutical compositions for administration by inhalation or insufflation can be provided in the form of a dry powder composition, for example, a powder mix of the compounds of Formula (I) and a suitable powder base such as lactose or starch. Such powder composition can be provided in unit dosage form, for example, in capsules, cartridges, gelatin packs, or blister packs, from which the powder can be administered with the aid of an inhalator or insufflator. The additives, excipients, and the like typically will be included in the compositions of intra-nasal administration within a range of

concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art.

Optionally, the pharmaceutical compositions of the present invention can include one or more other therapeutic agent, such as another sEH inhibitor, e.g., as a combination therapy.

It has previously been shown that inhibitors of sEH can reduce hypertension. Such inhibitors can be useful in controlling the blood pressure of persons with undesirably high blood pressure, including those who suffer from diabetes.

Compounds of Formula (I) can be administered to a subject in need of treatment for hypertension, specifically renal, hepatic, or pulmonary hypertension; inflammation, specifically renal inflammation, vascular inflammation, and lung inflammation; adult respiratory distress syndrome; diabetic complications; end stage renal disease; Raynaud syndrome and arthritis.

Compounds of the invention can also reduce damage to the kidney, and especially damage to kidneys from diabetes, as measured by albuminuria. The compounds of the invention can reduce kidney deterioration (nephropathy) from diabetes even in individuals who do not have high blood pressure. The conditions of therapeutic administration are as described above. cis-Epoxyeicosantrienoic acids ("EETs") can be used in conjunction with the compounds of the invention to further reduce kidney damage. EETs, which are epoxides of arachidonic acid, are known to be effectors of blood pressure, regulators of inflammation, and modulators of vascular permeability. Hydrolysis of the epoxides by sEH diminishes this activity. Inhibition of sEH raises the level of EETs since the rate at which the EETs are hydrolyzed into DHETs is reduced. Without wishing to be bound by theory, it is believed that raising the level of EETs interferes with damage to kidney cells by the

microvasculature changes and other pathologic effects of diabetic hyperglycemia.

Therefore, raising the EET level in the kidney is believed to protect the kidney from progression from microalbuminuria to end stage renal disease.

EETs are well known compounds. EETs useful in the methods of the present invention include 14,15-EET, 8,9-EET and 1 1 ,12-EET, and 5,6 EETs, in that order of preference. Preferably, the EETs are administered as the methyl ester, which is more stable. Persons of skill will recognize that the EETs are regioisomers, such as 8S,9R- and 14R,15S-EET. 8,9-EET, 1 1 ,12-EET, and 14R,15S-EET, are commercially available from, for example, Sigma-Aldrich (catalog nos. E5516, E5641 , and E5766, respectively, Sigma- Aldrich Corp., St. Louis, MO).

EETs produced by the endothelium have anti-hypertensive properties and the EETs 1 1 ,12-EET and 14, 15-EET may be endothelium-derived hyperpolarizing factors (EDHFs). Additionally, EETs such as 1 1 ,12-EET have profibrinolytic effects, anti-inflammatory actions and inhibit smooth muscle cell proliferation and migration. In the context of the present invention, these favorable properties are believed to protect the vasculature and organs during renal and cardiovascular disease states.

sEH activity can be inhibited sufficiently to increase the levels of EETs and thus augment the effects of administering sEH inhibitors by themselves. This permits EETs to be used in conjunction with one or more sEH inhibitors to reduce nephropathy in the methods of the invention. It further permits EETs to be used in conjunction with one or more sEH inhibitors to reduce hypertension, or inflammation, or both. Thus, medicaments of EETs can be made which can be administered in conjunction with one or more sEH inhibitors, or a medicament containing one or more sEH inhibitors can optionally contain one or more EETs.

The EETs can be administered concurrently with the sEH inhibitor, or following administration of the sEH inhibitor. It is understood that, like all drugs, inhibitors have half lives defined by the rate at which they are metabolized by or excreted from the body, and that the inhibitor will have a period following administration during which it will be present in amounts sufficient to be effective. If EETs are administered after the inhibitor is administered, therefore, it is desirable that the EETs be administered during the period during which the inhibitor will be present in amounts to be effective to delay hydrolysis of the EETs. Typically, the EET or EETs will be administered within 48 hours of

administering an sEH inhibitor. Preferably, the EET or EETs are administered within 24 hours of the inhibitor, and even more preferably within 12 hours. In increasing order of desirability, the EET or EETs are administered within 10, 8, 6, 4, 2, hours, 1 hour, or one half hour after administration of the inhibitor. Most preferably, the EET or EETs are administered concurrently with the inhibitor.

In some embodiments, the EETs, the compound of the invention, or both, are provided in a material that permits them to be released over time to provide a longer duration of action. Slow release coatings are well known in the pharmaceutical art; the choice of the particular slow release coating is not critical to the practice of the present invention.

EETs are subject to degradation under acidic conditions. Thus, if the EETs are to be administered orally, it is desirable that they are protected from degradation in the stomach. Conveniently, EETs for oral administration may be coated to permit them to passage the acidic environment of the stomach into the basic environment of the intestines. Such coatings are well known in the art. For example, aspirin coated with so-called

"enteric coatings" is widely available commercially. Such enteric coatings may be used to protect EETs during passage through the stomach. An exemplary coating is set forth in the Examples.

While the anti-hypertensive effects of EETs have been recognized, EETs have not been administered to treat hypertension because it was thought endogenous sEH would hydrolyse the EETs too quickly for them to have any useful effect. Surprisingly, it was found during the course of the studies underlying the present invention that exogenously administered inhibitors of sEH succeeded in inhibiting sEH sufficiently that levels of EETs could be further raised by the administration of exogenous EETs. These findings underlie the co-administration of sEH inhibitors and of EETs described above with respect to inhibiting the development and progression of nephropathy. This is an important improvement in augmenting treatment. While levels of endogenous EETs are expected to rise with the inhibition of sEH activity caused by the action of the sEH inhibitor, and therefore to result in at least some improvement in symptoms or pathology, it may not be sufficient in all cases to inhibit progression of kidney damage fully or to the extent intended. This is particularly true where the diseases or other factors have reduced the endogenous concentrations of EETs below those normally present in healthy individuals. Administration of exogenous EETs in conjunction with a sEH inhibitor is therefore expected to be beneficial and to augment the effects of the sEH inhibitor in reducing the progression of diabetic nephropathy.

The present compounds can be used with regard to any and all forms of diabetes to the extent that they are associated with progressive damage to the kidney or kidney function. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. The long-term complications of diabetes include retinopathy with potential loss of vision; nephropathy leading to renal failure; peripheral neuropathy with risk of foot ulcers, amputation, and Charcot joints.

In addition, persons with metabolic syndrome are at high risk of progression to Type 2 diabetes, and therefore at higher risk than average for diabetic nephropathy. It is therefore desirable to monitor such individuals for microalbuminuria, and to administer a sEH inhibitor and, optionally, one or more EETs, as an intervention to reduce the development of nephropathy. The practitioner may wait until microalbuminuria is seen before beginning the intervention. As noted above, a person can be diagnosed with metabolic syndrome without having a blood pressure of 130/85 or higher. Both persons with blood pressure of 1 30/85 or higher and persons with blood pressure below 1 30/85 can benefit from the administration of sEH inhibitors and, optionally, of one or more EETs, to slow the progression of damage to their kidneys. In some embodiments, the person has metabolic syndrome and blood pressure below 130/85.

Dyslipidemia or disorders of lipid metabolism is another risk factor for heart disease. Such disorders include an increased level of LDL cholesterol, a reduced level of HDL cholesterol, and an increased level of triglycerides. An increased level of serum cholesterol, and especially of LDL cholesterol, is associated with an increased risk of heart disease. The kidneys are also damaged by such high levels. It is believed that high levels of triglycerides are associated with kidney damage. In particular, levels of cholesterol over 200 mg/dL, and especially levels over 225 mg/dL, would suggest that sEH inhibitors and, optionally, EETs, should be administered. Similarly, triglyceride levels of more than 215 mg/dL, and especially of 250 mg/dL or higher, would indicate that administration of sEH inhibitors and, optionally, of EETs, would be desirable. The administration of compounds of the present invention with or without the EETs, can reduce the need to administer statin drugs (HMG-CoA reductase inhibitors) to the patients, or reduce the amount of the statins needed. In some embodiments, candidates for the methods, uses and compositions of the invention have triglyceride levels over 215 mg/dL and blood pressure below 130/85. In some embodiments, the candidates have triglyceride levels over 250 mg/dL and blood pressure below 1 30/85. In some embodiments, candidates for the methods, uses and compositions of the invention have cholesterol levels over 200 mg/dL and blood pressure below 130/85. In some embodiments, the candidates have cholesterol levels over 225 mg/dL and blood pressure below 1 30/85.

In other embodiments, compounds of Formula (I) inhibit proliferation of vascular smooth muscle (VSM) cells without significant cell toxicity, (e.g. specific to VSM cells). Because VSM cell proliferation is an integral process in the pathophysiology of atherosclerosis, these compounds are suitable for slowing or inhibition atherosclerosis. These compounds are useful to subjects at risk for atherosclerosis, such as individuals who have had a heart attack or a test result showing decreased blood circulation to the heart. The conditions of therapeutic administration are as described above. The present compounds are particularly useful for patients who have had percutaneous intervention, such as angioplasty to reopen a narrowed artery, to reduce or to slow the narrowing of the reopened passage by restenosis. In some embodiments, the artery is a coronary artery. The compounds can be placed on stents in polymeric coatings to provide a controlled localized release to reduce restenosis. Polymer compositions for implantable medical devices, such as stents, and methods for embedding agents in the polymer for controlled release, are known in the art and taught, for example, in U.S. Pat. Nos. 6,335,029 to Kamath et al.; 6,322,847 to Zhong et al.; 6,299,604 to Raghab et al.; 6,290,722 to Wang et al.; 6,287,285 to Michal et al.; and 5,637,1 13 to Tartaglia et al. In some embodiments, the coating releases the inhibitor over a period of time, preferably over a period of days, weeks, or months. The particular polymer or other coating chosen is not a critical part of the present invention.

The present compounds are useful for slowing or inhibiting the stenosis or restenosis of natural and synthetic vascular grafts. As noted above in connection with stents, desirably, the synthetic vascular graft comprises a material which releases a compound of the invention over time to slow or inhibit VSM proliferation and the consequent stenosis of the graft. Hemodialysis grafts are a particular embodiment.

In addition to these uses, the present compounds can be used to slow or to inhibit stenosis or restenosis of blood vessels of persons who have had a heart attack, or whose test results indicate that they are at risk of a heart attack.

In one aspect, compounds of the invention are administered to reduce proliferation of VSM cells in persons who do not have hypertension. In another group of embodiments, compounds of the invention are used to reduce proliferation of VSM cells in persons who are being treated for hypertension, but with an agent that is not an sEH inhibitor.

The present compounds can be used to interfere with the proliferation of cells which exhibit inappropriate cell cycle regulation. In one important set of embodiments, the cells are cells of a cancer. The proliferation of such cells can be slowed or inhibited by contacting the cells with a compound of the invention. The determination of whether a particular compound of the invention can slow or inhibit the proliferation of cells of any particular type of cancer can be determined using assays routine in the art. In addition to the use of the present compounds, the levels of EETs can be raised by adding EETs. VSM cells contacted with both an EET and a compound of the invention exhibited slower proliferation than cells exposed to either the EET alone or to the a compound of the invention alone. Accordingly, if desired, the slowing or inhibition of VSM cells of a compound of the invention can be enhanced by adding an EET along with a compound of the invention. In the case of stents or vascular grafts, for example, this can conveniently be accomplished by embedding the EET in a coating along with a compound of the invention so that both are released once the stent or graft is in position.

Chronic obstructive pulmonary disease, or COPD, encompasses two conditions, emphysema and chronic bronchitis, which relate to damage caused to the lung by air pollution, chronic exposure to chemicals, and tobacco smoke. Emphysema as a disease relates to damage to the alveoli of the lung, which results in loss of the separation between alveoli and a consequent reduction in the overall surface area available for gas exchange. Chronic bronchitis relates to irritation of the bronchioles, resulting in excess production of mucin, and the consequent blocking by mucin of the airways leading to the alveoli. While persons with emphysema do not necessarily have chronic bronchitis or vice versa, it is common for persons with one of the conditions to also have the other, as well as other lung disorders.

Some of the damage to the lungs due to COPD, emphysema, chronic bronchitis, and other obstructive lung disorders can be inhibited or reversed by administering inhibitors of the enzyme known as soluble epoxide hydrolase, or "sEH". The effects of sEH inhibitors can be increased by also administering EETs. The effect is at least additive over administering the two agents separately, and may indeed be synergistic.

EETs can be used in conjunction with sEH inhibitors to reduce damage to the lungs by tobacco smoke or, by extension, by occupational or environmental irritants. These findings indicate that the co-administration of sEH inhibitors and of EETs can be used to inhibit or slow the development or progression of COPD, emphysema, chronic bronchitis, or other chronic obstructive lung diseases which cause irritation to the lungs.

While levels of endogenous EETs are expected to rise with the inhibition of sEH activity caused by the action of the sEH inhibitor, and therefore to result in at least some improvement in symptoms or pathology, it may not be sufficient in all cases to inhibit progression of COPD or other pulmonary diseases. This is particularly true where the diseases or other factors have reduced the endogenous concentrations of EETs below those normally present in healthy individuals. Administration of exogenous EETs in conjunction with an sEH inhibitor is therefore expected to augment the effects of the sEH inhibitor in inhibiting or reducing the progression of COPD or other pulmonary diseases.

In addition to inhibiting or reducing the progression of chronic obstructive airway conditions, the present compounds also provide new ways of reducing the severity or progression of chronic restrictive airway diseases. While obstructive airway diseases tend to result from the destruction of the lung parenchyma, and especially of the alveoli, restrictive diseases tend to arise from the deposition of excess collagen in the parenchyma. These restrictive diseases are commonly referred to as "interstitial lung diseases", or "ILDs", and include conditions such as idiopathic pulmonary fibrosis. The methods, compositions and uses of the invention are useful for reducing the severity or progression of ILDs, such as idiopathic pulmonary fibrosis. Macrophages play a significant role in stimulating interstitial cells, particularly fibroblasts, to lay down collagen. Without wishing to be bound by theory, it is believed that neutrophils are involved in activating

macrophages, and that the reduction of neutrophil levels found in the studies reported herein demonstrate that the methods and uses of the invention will also be applicable to reducing the severity and progression of ILDs.

In some instances, the 1LD is idiopathic pulmonary fibrosis. In other instances, the ILD is one associated with an occupational or environmental exposure. Exemplars of such ILDs, are asbestosis, silicosis, coal worker's pneumoconiosis, and berylliosis. Further, occupational exposure to any of a number of inorganic dusts and organic dusts is believed to be associated with mucus hypersecretion and respiratory disease, including cement dust, coke oven emissions, mica, rock dusts, cotton dust, and grain dust (for a more complete list of occupational dusts associated with these conditions, see Table 254-1 of Speizer, "Environmental Lung Diseases," Harrison's Principles of Internal Medicine, infra, at pp. 1429-1436). In still other instances, the ILD is sarcoidosis of the lungs. ILDs can also result from radiation in medical treatment, particularly for breast cancer, and from connective tissue or collagen diseases such as rheumatoid arthritis and systemic sclerosis. It is believed that the methods, uses and compositions of the invention can be useful in each of these interstitial lung diseases.

The present compounds can also be used to reduce the severity or progression of asthma. Asthma typically results in mucin hypersecretion, resulting in partial airway obstruction. Additionally, irritation of the airway results in the release of mediators which result in airway obstruction. While the lymphocytes and other immunomodulatory cells recruited to the lungs in asthma may differ from those recruited as a result of COPD or an ILD, it is expected that the invention will reduce the influx of immunomodulatory cells, such as neutrophils and eosinophils, and ameliorate the extent of obstruction. Thus, it is expected that the administration of sEH inhibitors, and the administration of sEH inhibitors in combination with EETs, will be useful in reducing airway obstruction due to asthma.

In each of these diseases and conditions, it is believed that at least some of the damage to the lungs is due to agents released by neutrophils which infiltrate into the lungs. The presence of neutrophils in the airways is thus indicative of continuing damage from the disease or condition, while a reduction in the number of neutrophils is indicative of reduced damage or disease progression. Thus, a reduction in the number of neutrophils in the airways in the presence of an agent is a marker that the agent is reducing damage due to the disease or condition, and is slowing the further development of the disease or condition. The number of neutrophils present in the lungs can be determined by, for example, bronchioalveolar lavage.

Inhibitors of sEH and EETs administered in conjunction with inhibitors of sEH reduce brain damage from strokes. Thus, inhibitors of sEH taken prior to an ischemic stroke will reduce the area of brain damage and are likely to reduce the consequent degree of impairment. The reduced area of damage should also be associated with a faster recovery from the effects of the stroke.

While the pathophysiologies of different subtypes of stroke differ, they all cause brain damage. Hemorrhagic stroke differs from ischemic stroke in that the damage is largely due to compression of tissue as blood builds up in the confined space within the skull after a blood vessel ruptures, whereas in ischemic stroke, the damage is largely due to loss of oxygen supply to tissues downstream of the blockage of a blood vessel by a clot. Ischemic strokes are divided into thrombotic strokes, in which a clot blocks a blood vessel in the brain, and embolic strokes, in which a clot formed elsewhere in the body is carried through the blood stream and blocks a vessel there. But, in both hemorrhagic stroke and ischemic stroke, the damage is due to the death of brain cells. Based on the results observed in our studies, however, we would expect at least some reduction in brain damage in all types of stroke and in all subtypes.

A number of factors are associated with an increased risk of stroke. sEH inhibitors administered to persons with any one or more of the following conditions or risk factors: high blood pressure, tobacco use, diabetes, carotid artery disease, peripheral artery disease, atrial fibrillation, transient ischemic attacks (TIAs), blood disorders such as high red blood cell counts and sickle cell disease, high blood cholesterol, obesity, alcohol use of more than one drink a day for women or two drinks a day for men, use of cocaine, a family history of stroke, a previous stroke or heart attack, or being elderly, will reduce the area of brain damaged due to a stroke. With respect to being elderly, the risk of stroke increases for every 10 years. Thus, as an individual reaches 60, 70, or 80, administration of sEH inhibitors has an increasingly larger potential benefit. As noted below, the administration of EETs in combination with one or more sEH inhibitors can be beneficial in further reducing the brain damage.

The sEH inhibitors and, optionally, EETs, can be administered to persons who use tobacco, have carotid artery disease, have peripheral artery disease, have atrial fibrillation, have had one or more transient ischemic attacks (TIAs), have a blood disorder such as a high red blood cell count or sickle cell disease, have high blood cholesterol, are obese, use alcohol in excess of one drink a day if a woman or two drinks a day if a man, use cocaine, have a family history of stroke, have had a previous stroke or heart attack and do not have high blood pressure or diabetes, or are 60, 70, or 80 years of age or more and do not have hypertension or diabetes.

Clot dissolving agents, such as tissue plasminogen activator (tPA), reduce the extent of damage from ischemic strokes if administered in the hours shortly after a stroke. tPA, for example, is approved by the FDA for use in the first three hours after a stroke. Thus, at least some of the brain damage from a stoke is not instantaneous, but occurs over a period of time or after a period of time has elapsed after the stroke. It is therefore believed that administration of sEH inhibitors, optionally with EETs, can also reduce brain damage if administered within 6 hours after a stroke has occurred, more preferably within 5, 4, 3, or 2 hours after a stroke has occurred, with each successive shorter interval being more preferable. Even more preferably, the inhibitor or inhibitors are administered 2 hours or less or even 1 hour or less after the stroke, to maximize the reduction in brain damage. Persons of skill are well aware of how to make a diagnosis of whether or not a patient has had a stroke. Such determinations are typically made in hospital emergency rooms, following standard differential diagnosis protocols and imaging procedures.

The sEH inhibitors and, optionally, EETs, are administered to persons who have had a stroke within the last 6 hours who: use tobacco, have carotid artery disease, have peripheral artery disease, have atrial fibrillation, have had one or more transient ischemic attacks (TIAs), have a blood disorder such as a high red blood cell count or sickle cell disease, have high blood cholesterol, are obese, use alcohol in excess of one drink a day if a woman or two drinks a day if a man, use cocaine, have a family history of stroke, have had a previous stroke or heart attack and do not have high blood pressure or diabetes, or are 60, 70, or 80 years of age or more and do not have hypertension or diabetes.

The conditions of therapeutic administration for all of these indications are as described above.

The compounds of the present invention can be prepared by a variety of methods as outlined generally in the schemes below.

Scheme 1 - Introduction of a Secondary Pharmacophore (Ketone)

Scheme 1 illustrates general methods that can be used for preparation of compounds of the invention having a secondary pharmacophore that is a ketone functional group. While the scheme is provided for the synthesis of l -(4-hydroxyphenyl)-3-(4- oxodecyl)urea, one of skill in the art will understand that a number of commercially available isocyanates could be used in place of 3-chlorophenyl isocyanate, and that shorter or longer analogs of ethyl 4-aminobutyric acid or hexylbromide could also be employed.

In Scheme 1 :

(a) Benzopheonone imine, CH₂C1 , rt;

(b) DIBAL, THF, -78°C;

(c) Mg/I₂, hexylbromide, THF, rt;

(d) acetic anhydride, DMSO, rt;

(e) IN HCl/dioxane, rt;

(f) benzyloxyphenyl isocyanate, TEA, DMF, ambient room temperature (rt);

(g) hydrogenolysis with 10% palladium; and

(h) Borax buffer/dichloromethane, S0₂F₂ (e.g., VIKANE brand sulfuryl fluoride), 6 hours.

As shown in Scheme 1 , ethyl 4-aminobutyrate hydrochloride (available from Aldrich Chemical Co., Milwaukee, WI, USA) is combined with benzophenone imine at room temperature to provide intermediate (i). DIBAL (diisobutylaluminum hydride) reduction of the ester group provides an aldehyde moiety that is then reacted, without isolation, with a Grignard reagent (prepared in situ) to provide intermediate alcohol (ii). Oxidation of the alcohol moiety to a ketone provides (iii) which can then be deprotected to form the amino-ketone (iv). Reaction of (iv) with a benzyloxy isocyanate provides the phenoxy compound (v), which is then reacted with sulfuryl fluoride (S0₂F₂).

Preparation of illustrative fluorosulfonyl derivatives of sEH inhibitors is further illustrated om the non-limiting Examples provided below.

EXAMPLE 1 : Synthesis of (^-(S-O-^-methylbutanoy piperidin^- yl)ureido)benzenesulfonyl fluoride

CH₂CI₂, 0 °C to rt, 2h

4-Aminobenzenesulfonyl fluoride (80 mg, 457 μιηοΐ, 1 .0 eq) and triethyl amine (50 mg, 495 μηιοΐ, 1 .1 eq) were dissolved in CH₂C1₂ (5 mL) with stirring at -78 °C. Triphosgene (50 mg, 183 μιηοΐ, 0.37 eq) dissolved in CH₂C1₂ (5 mL) was added dropwise at -78 °C. The reactants were then warmed to 0 °C and stirred for 30 minutes (min.).

Thereafter the reactants and reaction products were cooled to 0 °C. (S)- \ -(4- aminopiperidin-l -yl)-2-methylbutan-l -one (92 mg, 548 μιηοΐ, 1 .2 eq) and triethyl amine (50 mg, 495 μιηοΐ, 1.1 eq) dissolved in CH₂C1₂ (5 mL) were added slowly and the resulting reaction mixture was further stirred at room temperature for 12 hours (h). The reaction was then quenched with the addition of HC1 solution (1 M, 15 mL). An organic layer was collected from the reaction mixture and the remaining aqueous layer was further extracted with ethyl acetate (EtOAc) three times. The obtained organic layers were combined and washed with saturated NaCl solution. The washed organic layer was dried over anhydrous magnesium sulfate and was concentrated under vacuum. The obtained product (35mg; 20%) was eluted by flash chromatography with (EtOAc: Hexane/ 7:3). The product was further purified by crystallization (MeOH with water). Yield 20 %. Ή NMR (d₆-DMSO, 300 Mhz): d 0.70-0.90 (m, 3H), 0.97 (t, J= 6 Hz, 3H), 1.2- 1 .4 (m, 3H), 1.4-1.6 (m, 1 H), 1.7-1.9 (m, 2H), 2.7-2.9 (m, 2H), 3.17 (t, J = 12 Hz, 1 H), 3.7-3.8 (m, 1 H), 3.89 (d, J = 10.2 Hz, 1H), 4.22 (br, 1 H), 6.56 (t, J = 7.5 Hz, 1 H), 7.73 (d, J = 9 Hz, 2H), 7.96 (d, J = 9 Hz, 2H), 9.21 (d, J = 7.5 Hz, 1 H); Melting point (^°C): 263.3-265.6 (264.5).

EXAMPLE 2: Synthesis of (S 4-(3-(l-(2-methylbutanoyl)piperidin-4- yl)ureido)phenyl sulfurofluoridate

CH₂CI₂, 0 °C to rt, 2h

4-Aminophenyl sulfurofluoridate (also known as aniline-4-fluoi Sul fate; 80 mg, 419 μιτιοΐ, 1 .0 eq) and triethyl amine (Et₃N; 46.5 mg, 461 μι^ηοΐ, 1 .1 eq) were dissolved in CH₂C1₂ (5 mL) with stirring at -78 °C. Triphosgene (46 mg, 1 55 μιηοΐ, 0.37 eq) dissolved in CH₂C1₂ (5 mL) was added dropwise at -78 °C. The reactants were then warmed to 0 °C and stirred for 30 min. Thereafter the reactants and reaction products were cooled to 0 °C. (¾)-l-(4-aminopiperidin-l-yl)-2-methylbutan-l-one (84 mg, 461 μηιοΐ, 1.2 eq) and Et₃N (46.5 mg, 461 μηιοΐ, 1.1 eq) dissolved in CH₂CI₂ (DCM; 5 mL) were added slowly and the resulting reaction mixture was further stirred at room temperature for 12 h. The reaction was then quenched with the addition of HC1 solution (1M, 15 mL). An organic layer was collected from the reaction mixture and the remaining aqueous layer was further extracted with EtOAc three times. The obtained organic layers were combined and washed with saturated NaCl solution. The washed organic layer was dried over anhydrous magnesium sulfate and was concentrated under vacuum. The obtained product (85 mg; 50.6%) was eluted by flash chromatography with (EtOAc: Hexane/ 7:3). The product was further purified by crystallization (MeOH with water). Yield 50.6 %. Ή NMR (d₆-DMSO, 300 Mhz): d 0.80-0.90 (m, 3H), 0.97 (t,J=5Hz, 3H), 1.2-1.4 (m, 3H), 1.4-1.6 (m, 1H), 1.7- 1.9 (m, 2H), 2.7-2.9 (m, 2H), 3.16 (t, J = 12 Hz, 1H), 3.6-3.8 (m, 1H), 3.88 (d,J= 12.6 Hz, 1H), 4.21 (br, 1H), 6.32 (t, J= 7.5 Hz, 1H), 7.43 (d, J= 9 Hz, 2H), 7.54 (d, J= 9 Hz, 2H), 8.68 (d,J=8 Hz, 1H); Melting point (^°C): 186.5-188.0 (187.3).

EXAMPLE 3: Synthesis of4-(3-(l-propionylpiperidin-4yl)ureido)

benzenesulfonyl fluoride

3 (A). 26 mg ( 0.1 mmol scale) tert-butyl (l-propionylpiperidin-4-yl)carbamate dissolved in 0.5 mL DCM, 0.5 mL trifluoroacetic acid (TFA) added into the solution at room temperature. Reaction mixture was stirred at room temperature for 6 hours. The solvent and excess TFA was removed by rotary evaporation and dried under high vacuum. The crude TFA salt was dissolved in DCM again and directly submitted for next step.

3 (B). 1.1 eq Et₃N was added to free the TFA/amine salt; using DCM as solvent. The crude product was purified by column chromatography (100% EtOAc, Rf: 0.10) to give the product as a white solid (0.1 mmol scale, 18 mg, 51% yield for two steps). Analytically pure sample was obtained by prepare-HPLC purification. Ή NMR (400 MHz, Acetone-i/₆) δ 9.14 (s, 1H), 7.92 - 7.82 (m, 4H), 6.63 (s, 1H), 4.34 (d, J= 13.4 Hz, 1H), 3.93 - 3.75 (m, 2H), 3.16 (dd, J= 14.0, 10.7 Hz, 1H), 2.32 (q, J= 7.4 Hz, 3H), 1.97 - 1.77 (m, 4H), 1.53- 1.20 (m, 5H), 1.01 (td,J=7.4, 1.9 Hz, 3H); ¹⁹F NMR (376 MHz, Acetone-</₆) 567.00; ESI-MS (m/z): 358 [MH]'.

EXAMPLE 4: Synthesis of 4-(3-(l-propionylpiperidin-4-yl)ureido)phenyl

sulfurofluoridate

Exact Mass: 253. 2

Molecular Weight: 253.25

4 (A). 26 mg ( 0.1 mmol scale) tert-butyl (l-propionylpiperidin-4-yl)carbamate dissolved in 0.5 mL DCM, 0.5 mL TFA added into the solution at room temperature. Reaction mixture was stirred at room temperature for 6 hours. The solvent and excess TFA was removed by rotary evaporation and dried under vacuum. The crude TFA salt was dissolved in DCM again and directly submitted for next step.

4 (B). 1.1 eq Et₃N was added to free the TFA/amine salt using DCM as solvent. The crude product was purified by column chromatography (100% EtOAc, Rf: 0.14) to give the product as a white solid (0.1 mmol scale, 24 mg, 65% yield for two steps ). Ή NMR (400 MHz, Methanol-.^) δ 7.55 - 7.45 (m, 2H), 7.34 - 7.26 (m, 2H), 4.37 (dtd, J = 13.6,4.0, 1.8 Hz, 1H), 3.90 (dtd,J= 14.1,4.0, 1.8 Hz, 1H), 3.82 (tt,J= 10.5,4.1 Hz, 1H), 3.22 (ddd,J= 14.2, 11.5, 2.9 Hz, 1H), 2.87 (ddd, J= 14.0, 11.6,3.1 Hz, 1H), 2.41 (q,J = 7.5 Hz, 2H), 2.08- 1.87 (m, 2H), 1.47- 1.27 (m, 2H), 1.10 (t, J =7.5 Hz, 3H);

l⁹F NMR (376 MHz, Methanol-J₄) δ 35.02; ESI-MS (m/z): 374 [MH]. EXAMPLE 5: Synthesis of 4-(((lr,4r)-4-(3-(4-(fluorosuIfonyl)phenyl)

cyclohexyl)oxy)benzoic acid

S0₂F HOOC 60 °C/24hours

24 mg (0.1 mmol) 4-(((lr,4r)-4-aminocyclohexyl)oxy)benzoic acid was dissolved in 0.5 mL dimethylformamide (DMF), 15μΙ, Et N was added to the solution. The reaction mixture was stirred at room temperature for 5 min, and 0.1 mL 4- isocyanatobenzenesulfonyl fluoride/DCM solution (0.1 mmol, 1 eq) was added by syringe, followed by 1 mg of l ,4-diazabicyclo[2.2.2]octane (DABCO). Reaction was heated at 60 °C by an oil bath for 24 hours and was monitored by LCMS. After removal of the solvent by rotary evaporation, crude mixture was directly loaded to a column; purification by column chromatography (5% methanol/DCM, product is very poor solubility in

DCM/methanol, this step requires much excess elution solvent, and significant loss of product) to give the product as a white solid (0.1 mmol scale, 15 mg, 34% yield for two steps). Analytically pure sample was obtained by prepare-HPLC purification afterward. 1H NMR (400 MHz, Acetone-^) δ 9.03 (s, 1 H), 8.02 - 7.83 (m, 6H), 7.09 - 6.95 (m, 2H), 6.53 (d, J = 7.6 Hz, 1 H), 4.54 - 4.43 (m, 1 H), 3.73 (d, J = 6.7 Hz, l H), 2.24 - 2.14 (m, 2H), 1 .69 - 1.44 (m, 4H), ¹⁹F NMR (376 MHz, Acetone- ) δ 67.00; ESI-MS (m/z): 437 [MH]⁺.

EXAMPLE 6: Synthesis of 4-(((ls,4s)-4-(3-(4-((fluorosulfonyI)oxy)phenyl)

ureido) cyclohexyl)oxy)benzoic acid

24 mg (0. 1 mmol) 4-(((lr,4r)-4-aminocyclohexyl)oxy)benzoic acid was dissolved in 0.5 niL DMF, 15μΙ, Et₃N was added to the solution. Reaction mixture stirred at room temperature for 5 min. O. lmL 4-isocyanatophenyl sulfurofluoridate/DCM solution

(O. l mmol, l eq) was added by syringe, followed by 1 mg DABCO. Reaction was heated at 60 °C by an oil bath for 24 hours and was monitored by LCMS. Solvent was removed by rotary evaporation. Crude mixture was directly loaded to a column; purification by column chromatography (Rf = 0.41 , 100% EtOAc ) to give the product as a white solid ( 0.1 mmol scale, 25 mg, 53% yield for two steps). 1H NMR (400 MHz, Methanol-d₄) δ 7.99 - 7.90 (m, 2H), 7.56 - 7.47 (m, 2H), 7.36 - 7.26 (m, 2H), 7.03 - 6.92 (m, 2H), 4.43 (ddd, J =

10.0, 6.0, 4.0 Hz, 1 H), 3.73 - 3.57 (m, 1 H), 2.22 - 2.13 (m, 2H), 2.08 (dt, J = 13.6, 3.8 Hz, 2H), 1.60 (tdd, J = 12.8, 9.9, 3.2 Hz, 2H), 1 .45 (tdd, J = 13.0, 10.5, 3.2 Hz, 2H); ¹⁹F NMR (376 MHz, Methanol-d₄) δ 34.97; ESI-MS (m/z): 453 [MH] ¹ .

The physical properties of soluble epoxide hydrolase (sEH) inhibitors embodying the present invention were evaluated by the procedures set forth below.

EXAMPLE 7: Synthesis of 4-isocyanatobenzenesulfonyl fluoride

4-isocyanatobenzenesulfonyl fluoride

4-isocyanatobenzenesulfonyl fluoride

7 (A) General procedure for synthesis of aromatic sulfonyl fluorides by saturated KHF/H₂O/CH₃CN procedure: KHF₂ (71 g; 2.3 eq) was dissolved in H₂0 (200 mL) to make a saturated solution (endothermic reaction), which was treated with a solution of 4- bromobenzene- 1 -sulfonyl chloride ( 100 g, 1 eq) in acetonitrile (400 mL). The reaction mixture was stirred at room temperature for 3 h, monitored by GC-MS. After that time, the reaction mixture was transferred to a separatory funnel and the organic layer was collected. The aqueous phase was extracted with EtOAc (300 mL), and the combined organic extracts were washed with 10% aqueous NaCl (2x), saturated sodium chloride ( l x), dried over sodium sulfate, and concentrated by rotary evaporation to give the desired product as a white solid (91.5 g, 98% yield), mp 63-64 °C. Ή NMR: (400MHz, CDC1₃) δ 7.88 (dd, J = 9.2Hz, 2Hz, 2H), 7.78 (dd, J = 9.2Hz, 2Hz, 2H); ¹³C NMR: (101 MHz, CDC1₃)5 133.1, 131.9 (d,J = 25.5Hz), 131.3, 129.8; ¹⁹F NMR (376 MHz, CDC1₃) δ +65.9; EI (m/z): 240/238 [M]\

The following compounds were prepared by general method 7(A):

(a) 4-Nitrobenzene-l-sulfonyl fluoride: white solid (22.6 g, 97% yield), mp 75- 76 °C. Ή NMR (400 MHz, CDC1₃) δ 8.49 (d, J= 8Hz, 2H), 8.25 (d, J= 8Hz, 2H); ¹³C NMR (101 MHz, CDCI3) δ 151.9, 138.4 (d, J =27.3Hz), 130.1, 125.0; ¹⁹FNMR(376 MHz, CDCI₃) δ +66.0; EI-MS (m/z): 205 [M]⁺.

(b) 3-Nitrobenzene-l-sulfonyl fluoride: white solid (22.3 g, 97%o yield), mp 42- 43 °C. Ή NMR (400 MHz, CDCI₃) δ 8.86 (t, J= 2.4Hz, 1H), 8.64 (d, J= 8Hz, 1H), 8.35 (d,J=8Hz, 1H), 7.93 (t,J=8Hz, 1H); ^l3CNMR(101 MHz, CDCI₃) δ 148.4, 134.9 (d,J =28.3Hz), 133.8, 131.4, 130.0, 123.9; ¹⁹F NMR (376 MHz, CDC1₃) δ +65.9; El-MS (m/z): 205 [M] ' .

7(B).4-Aminobenzenesulfonyl fluoride: In a 500ml flask, 4.05 g (19.8 mmol) of 4-Nitrobenzene-l-sulfonyl fluoride was dissolved into 200 mL EtOH/H₂0 (8:1) solution at room temperature.1.1 g (1 eq) NH₄CI was added into the solution. The solution was stirred for 10 min, 5.5 gram (5 eq) iron powder was added in one portion. The reaction mixture was stirred at room temperature and open to air. TLC and GCMS were used to monitor reaction, which was directly filtered when complete by Biichner funnel. The filter cake was carefully washed with 100 mL EtOAc three times. The filtrate was concentrated by rotary evaporation, extracted and washed with 200 mL DCM three times. The DCM solution was dried over sodium sulfate and concentrated. The pure product was obtained as yellow oil. (2.6 gram, 78% yield). Ή NMR (400 MHz, Chloroform-*/) δ 7.71 (d, J= 8.7 Hz, 2H), 6.86 -6.41 (m, 2H),4.53 (s, 2H); ^1:,CNMR(101 MHz, CDCI₃) δ 153.43, 130.89, 119.18(d,J = 23 Hz), 114.03; ¹⁹F NMR (376 MHz, CDC1₃) δ 67.51; EI-MS (m/z): 175 [M]'.

7(C). 4-Isocyanatobenz.enesulfonyl fluoride. In a 10 mL flask, 128 mg (0.43 eq) of triphosgene (Caution: triphosgene is toxic, a well functional fume hood is necessary for this procedure) was dissolved in 1 mL DCM. Solution temperature was kept at about 0 °C by an ice bath and protected by an Ar balloon. 175 mg ( l mmol) 4-aminobenzenesulfonyl fluoride was dissolved into 1 mL DCM and this solution was added into the flask slowly by syringe over 10 min, followed by 0.14 mL ( 1 eq) Et₃N. After the addition, the ice bath was removed and the reaction mixture was stirred at room temperature for 2 hours.

Reaction can be monitored by quenching the solution with methanol, then submitting the sample to LCMS. The solution was concentrated by rotary evaporation, and 4-isocyanato benzenesulfonyl fluoride was obtained as a yellow oil (mixed with Et^N/HCl salt). The isocyanate product can be used as it is, or dissolved in DCM then directly use for next steps. This intermediate is not stable at room temperature as a crude product. A rapid wash with 1M HC1, followed by saturated sodium chloride, and drying over sodium sulfate afforded a material that could be stored for up to one week;. 4-isocyanatobenzenesulfonyl fluoride was obtained as yellow solid.

EXAMPLE 8: Synthesis of 4-(3-(4-ethynylphenyl)ureido)benzenesulfonyI fluoride

1 mmol 4-isocyanatobenzenesulfonyl fluoride (crude oil) was dissolved into 4 mL diethylether at room temperature. 1 17mg ( 1 eq) 4-ethynylaniline was added into the solution. Reaction mixture was stirred for 5 min at room temperature. 2.5 mg DABCO ( l ,4-diazabicyclo[2.2.2]octane, 2%,) was added. The reaction mixture was stirred at room temperature overnight. After removal of solvent by rotary evaporation, the crude product was purified by column chromatography (75 :25 hexane:EtOAc Rf: 0.25) to give the product as a white solid (285 mg, 90% yield for two steps); mp 1 93- 1 95 °C. When the same reaction was repeated at 2.5 gram scale, a slightly higher yield was obtained.

Ή NMR (400 MHz, Methanol-^) δ 7.90 - 7.83 (m, 2H), 7.76 - 7.66 (m, 2H), 7.44 - 7.37 (m, 2H), 7.37 - 7.30 (m, 2H), 3.34 (s, 1 H); ¹ C NMR ( 101 M Hz, Methanol-J₄) 1 53.63, 147.93, 140.43, 133.68, 133.68, 130.89, 125.80(d, J= 23 Hz), 119.82, 119.37, 118.01, 84.29, 77.76; ¹⁹F NMR (376 MHz, Methanol-^) δ 65.82; ESI-MS (m/z): 319 [MH]⁺ .

EXAMPLE 9: Synthesis of 4-(3-(4-cyclopropylphenyl)ureido)

benzenesulfon l fluoride

4-(3-(4-cyclopropylphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a yellow solid according to the general procedure in Example 2. The crude product was purified by column chromatography (25:75; hexane:EtOAc, Rf: 0.13) to give the product (0.25 mmol scale, 63 mg, 75% yield for two steps); mp 188-190 °C

(decompose). Ή NMR (400 MHz, Acetone-J₆) 58.86 (s, 1H), 8.31 (s, 1H), 8.06 - 7.84 (m, 4H), 7.42 (d, J= 8.1 Hz, 2H), 7.03 (d, J= 8.1 Hz, 2H), , 1.87 (td, J= 8.5, 4.4 Hz, 1H), 0.98 - 0.81 (m, 2H), 0.69 - 0.56 (m, 2H); ESI-MS (m/z): 335 [MH]' .

EXAMPLE 10: Synthesis of 4-(3-(4-vinylphenyl)ureido)benzenesulfonyl fluoride

4-(3-(4-viiiylphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a yellow solid according to the general procedure in Example 2. The crude product was purified by column chromatography (hexane:EtOAc 50/50, Rf: 0.67) to give the product (0.25 mmol scale, 72 mg, 92% yield for two steps); mp 98-100 °C; Ή NMR (400 MHz, Methanol-^) δ 7.97 - 7.87 (m, 2H), 7.80 - 7.70 (m, 2H), 7.49 - 7.30 (m, 4H), 6.66 (dd, ./ = 17.6, 10.9 Hz, 1H), 5.67 (dd, J = 17.6, 1.0 Hz, 1H), 5.12 (dd, J = 10.9, 1.0 Hz, 1H); ^!9F NMR (376 MHz, Methanol-J₄) δ 65.65; ESI-MS (m/z): 321 [MH]'. EXAMPLE 11 : Synthesis of 4-(3-(4-isopropylphenyI)

benzenesulfon l fluoride

4-(3-(4-isopropylphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (hexane:EtOAc 50/50, Rf: 0.78) to give the product (0.25 mmol scale, 71 mg, 84% yield for two steps); mp 172-175 °C; Ή NMR (400 MHz, Methanol-^) δ 7.90 (d, J= 9.0 Hz, 2H), 7.78 - 7.71 (m, 2H), 7.37 - 7.27 (m, 2H), 7.19 - 7.10 (m, 2H), 2.84 (p, J= 6.9 Hz, 1H), 1.21 (s, 3H), 1.20 (s, 3H); ¹⁹F NMR (376 MHz, Methanol-^) δ 65.67; ESI-MS (m/z): 337 [MH] ' .

EXAMPLE 12: Synthesis of 4-(3-(4-ethylphenyl)ureido)benzenesulfonyl fluoride

4-(3-(4-ethylphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white crystal according to the general procedure in Example 2. The crude product was purified by column chromatography (hexane:EtOAc 25/75, Rf: 0.25) to give the product (0.5 mmol scale, 151 mg, 94% yield for two steps); mp 198-201 °C; Ή NMR (400 MHz, Methanol-^) δ 8.00 - 7.87 (m, 2H), 7.83 - 7.72 (m, 2H), 7.41 - 7.27 (m, 2H), 7.21 - 7.07 (m, 2H), 2.60 (q, J = 7.6 Hz, 2H), 1.21 (t, J= 7.6 Hz, 3H); ^{l 9}F NMR (376 MHz, Methanol- c/₄) δ 65.57; ESI-MS (m/z): 323 [MH] ' . EXAMPLE 13: Synthesis of 4-(3-(3-ethynylphenyf)ureido)benzenesuIfonyl fluoride

4-(3-(3-ethynylphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a yellow solid according to the general procedure in Example 2. The crude product was purified by column chromatography (25 :75 hexane:EtOAc, Rf: 0.25) to give the product as a white solid (1 mmol scale, 270 mg, 85% yield for two steps); mp 190- 192 °C; Ή NMR (400 MHz, Acetone-t/₆) 6 8.90 (s, 1 H), 8.45 (s, 1 H), 7.98 - 7.91 (m, 2H), 7.90 - 7.83 (m, 2H), 7.74 (t, J = 1.9 Hz, 1 H), 7.49 (ddd, J = 8.2, 2.3, 1.1 Hz, 1 H), 7.27 (t, J = 7.9 Hz, 1 H), 7.14 (dt, J = 7.7, 1 .3 Hz, 1 H), 3.60 (s, 1 H); ¹³C NMR (101 MHz, Acetone) 8 152.59, 147.81 , 139.96, 130.60, 129.79, 127.16, 124.85(d, J = 25Hz), 1 23.50, 122.91 , 120.36, 1 19.14, 1 19.06, 83.98, 78.98; ¹⁹F NMR (376 MHz, Acetone) 5 65.82; ESI-MS (m/z): 319 [MH]⁺.

EXAMPLE 14: Synthesis of 3-(3-(4-ethynylphenyI)ureido)benzenesulfonyI fluoride

3-(3-(4-ethynylphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a yellow solid according to the general procedure in Example 2. The crude product was purified by column chromatography (75 :25 hexane:EtOAc Rf: 0.33) to give the product as a yellow solid (0.5 mmol scale, 121 mg, 76% yield for two steps); mp 1 66- 1 68 °C; Ή NMR (400 MHz, Acetone-r/ ) δ 8.77 (s, l H), 8.56 - 8.48 (m, 2H ), 7.88 - 7.80 (m, ] H), 7.71 - 7.61 (m, 2H), 7.60 - 7.53 (m, 2H), 7.45 - 7.36 (m, 2H), 3.53 (s, 1 H); ESI-MS (m/z): 319 [MH] ' . EXAMPLE 15: Synthesis of 4-(3-(3-(trifluoromethyl)phenyl)ureido) benzenesulfonyl fluoride

4-(3-(3-(triJluoromethyl)phenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (75 :25 hexane:EtOAc, Rf: 0.38) to give the product as a white solid (0.5 mmol scale, 145 mg, 80% yield for two steps); mp 202-

204 °C; Ή NMR (400 MHz, Acetone-^) δ 8.96 (s, 1 H), 8.68 (s, l H), 8.05 (t, J = 2.1 Hz, 1 H), 8.01 - 7.95 (m, 2H), 7.94 - 7.86 (m, 2H), 7.74 - 7.66 (m, 1 H), 7.51 (td, J= 8.0, 1.0 Hz, 1 H), 7.35 (ddt, J= 7.9, 1.8, 0.9 Hz, 1 H); ¹³C NMR (101 MHz, Acetone) 5152.48, 147.59, 140.59, 131.19(q, J= 32 Hz), 130.52, 130.36, 125.00(d, J = 24 Hz), 124.90(q, J = 272 Hz), 122.97, 1 19.80(q, J = 4 Hz), 1 19.14, 1 15.92(q, J= 4 Hz); ESI-MS (m/z): 363

[MH] .

EXAMPLE 16: Synthesis of 4-(3-(4-chlorophenyl)ureido)benzenesulfonyl fluoride

4-(3-(4-chlorophenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (75 :25 hexane:EtOAc, Rf: 0.24) to give the product as a white solid (0.5 mmol scale, 151 mg, 92% yield for two steps); mp 224-226 °C; Ή NMR (400 MHz, Acetone- ) δ 8.88 (s, 1 H), 8.48 (s, 1 H), 8.08 - 7.78 (m, 4H), 7.56 (t, J = 6. 1

Hz, 2H), 7.42 - 7. 12 (m, 2H); ^L C NMR ( 101 MHz, Acetone) δ 1 52.45, 147.81 , 1 38.67, 1 30.58, 129.36, 129.27, 127.93, 124.83(d, J = 24 Hz), 121 .16, 121 .06, 120.95, 1 19.07; ESI-MS (m/z): 329 [MH]⁺. EXAMPLE 17 : Synthesis of teri-b tyl4-((lS,2R)-2-(3-(4-( luorosulfonyl)

phenyl)ureido -l,2-diphenylethyl)piperazine-l-carboxylate

tert-butyl4-((lS,2R)-2-(3-(4-(fluorosulfonyl)phenyl)ureido)-l,2- diphenylethyl)piperazine-l-carboxylate was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.26) to give the product (0.25 mmol scale, 120 mg, 81 % yield for two steps); mp 148-151 °C; Ή NMR (400 MHz, Methanol- d₄) 8 7.81 - 7.72 (m, 2H), 7.58 - 7.51 (m, 2H), 7.30 - 7.03 (m, 10H), 5.52 (d, J= 7.9 Hz, 1 H), 3.69 (d, J = 7.9 Hz, 1 H), 3.22 (d, J = 5.7 Hz, 4H), 2.43 (dt, J - 10.7, 5.0 Hz, 2H), 2.35 - 2.20 (m, 2H), 1.37 (s, 9H); ¹³C NMR (101 MHz, Methanol-^) δ 156.14, 155.73, 148.29, 142.83, 136.74, 130.84, 130.10, 129.00, 128.93, 128.66, 128.09, 127.94, 125.00(d, J = 25Hz), 1 18.79, 80.97, 75.03, 54.57, 51.17, 28.64; ¹⁹F NMR (376 MHz, Methanol-^) δ 66.26; ESI-MS (m/z): 583 [MH] ' .

EXAMPLE 18 : Synthesis of tert-butyl4-((lS,2R)-2-(3-(3-(fluorosulfonyI)

phenyl) ureido)-! ^' ,2-diphenylethyl)piperazine- 1 ^' -carboxylate

tert-butyl4-((lS,2R)-2-(3-(3-(fluorosulfonyl)phenyl)ureido)-l,2- diphenylethyl)piperazine-l-carboxylate was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.26) to give the product as a white solid (0.25 mmol scale, 124 mg, 83% yield for two steps); mp 133-135 °C; ^l3C NMR (101 MHz, Methanol-J₄) δ 156.34, 156.24, 142.87(d, J= 27 Hz), 136.84, 134.59, 134.35, 131.37, 130.21, 129.03, 128.98, 128.69, 128.16, 127.96, 125.88, 122.13, 118.07, 81.05, 75.15, 54.60, 51.23, 28.63; ¹⁹FNMR(376 MHz, Methanol-^) δ 64.24; ESI-MS (m/z): 583 [MH]'.

EXAMPLE 19: Synthesis of 4-(3-(3,5-bis(trifluoromethyl)phenyI)ureido)

benzenesulfonyl fluoride

4-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (75:25 hexane:EtOAc, Rf: 0.38) to give the product (0.5 mmol scale, 131 mg, 61%> yield for two steps); mp 255-256 °C; Ή NMR (400 MHz, Acetone-</₆) δ 9.12 (s, 1H), 9.00 (s, l H), 8.23 - 8.15 (m, 2H), 8.04 - 7.96 (m, 2H), 7.95 - 7.86 (m, 2H), 7.68 - 7.60 (m, lH); ¹ C NMR (101 MHz,

Acetone) 5152.52, 152.45, 147.31, 141.92, 132.35(q, J= 33 Hz), 130.59, 125.64, 125.39(d, J=25Hz), 124.12(q,J=272 Hz), 119.46, 119.37, 116.21(m, J= 24 Hz); ESI-MS (m/z): 431 [MH]'. EXAMPLE 20: Synthesis of 4-(3-(2,4-difluorophenyl)urcido)

benzenesulfonyl fluoride

4-(3-(2,4-difluorophenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (75:25 hexane:EtOAc, Rf: 0.37) to give the product (0.5 mmol scale, 124 mg, 75% yield for two steps); mp 208-210 °C; Ή NMR (400 MHz, Methanol-^) δ 8.01 (td, J= 9.1, 5.9 Hz, 1H), 7.94 - 7.84 (m, 2H), 7.80 - 7.68 (m, 2H), 7.05-6.83 (m, 2H); ¹³CNMR(101 MHz, Methanol-^) δ 161.02(d, J= 11 Hz), 158.58(d,J= 12 Hz), 155.87(d,J= 12 Hz), 153.86, 153.43(d,J= 12 Hz), 147.98, 130.98, 126.00(d,J=24 Hz), 124.34(d, J= 6 Hz), 124.25(d, J= 6 Hz), 124.19, 119.33, 112.04(d,J = 4 Hz), 111.82(d,J=4Hz), 104.62(d, J= 26 Hz) 104.39(d, J= 26 Hz); ¹⁹F NMR (376 MHz, Methanol-i4) δ 65.65, -117.79, -126.41. ESI-MS (m/z): 331 [MH]¹. EXAMPLE 21: Synthesis of 3-(3-(4-(fluorosulfonyl)phenyl)ureido)benzenesulfonyl fluoride

3-(3-(4-(fluorosulfonyl)phenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.22) to give the product (0.5 mmol scale, 96 mg, 51% yield for two steps); mp 215-218 °C; Ή NMR (400 MHz, Methanol-i4) 68.35 (t, J= 2.0 Hz, 1H), 8.00 - 7.82 (m, 3H), 7.80 - 7.69 (m, 4H), 7.67-7.51 (m, 2H);¹ C NMR (101 MHz, Methanol-^) δ 153.48, 147.62, 141.84, 134.57(d,J=24 Hz), 131.48, 130.88, 126.60, 126.10(d, J= 24 Hz), 123.11, 119.63, 119.60, 119.54, 118.74; ^l9F NMR (376 MHz, Methanol-^) δ 65.81, 64.30; ESI-MS (m/z): 377 [MH] ' .

EXAMPLE 22: Synthesis of 3,3'-(carbonylbis(azanediyl))dibenzenesulfonyl fluoride

F0₂S

3, 3 '-(carbonylbis(azanediyl))dibenzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.23) to give the product (0.5 mmol scale, 123 mg, 65% yield for two steps); mp 195- 197 °C; Ή NMR (400 MHz, Methanol-^) δ 8.32 (t, J = 2.0 Hz, 1 H), 7.84 - 7.75 (m, 1 H), 7.70 - 7.54 (m, 2H); ¹³C NMR (101 MHz, Methanol-^) δ 154.07, 142.13, 134.64(d, J = 25 Hz), 131.53, 126.69, 123.01 , 1 18.86; ^{, 9}F NMR (376 MHz, Methanol-i/4) δ 64.15; ESI-MS (m/z): 377 [MH]⁺.

EXAMPLE 23: Synthesis of 3-(3-(2-hydroxyphenyl)ureido)benzenesulfonyl fluoride

3-(3-(2-hydroxyphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a light pink solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc; Rf: 0.22) to give the product (0.5 mmol scale, 93 mg, 60% yield for two steps); mp 77-80 °C; Ή NMR (400 MHz, Methanol-J₄) 6 8.37 (t, J = 2.0 Hz, 1 H), 7.91 (dd, J = 7.9, 1 .6 Hz, 1 H), 7.76 - 7.69 (m, 1 H), 7.66 - 7.51 (m, 2H), 6.90 - 6.75 (m, 5H); ESI-MS (m/z): 31 1 [MH] ' .

EXAMPLE 24: Synthesis of 4,4'-(carbonylbis(azancdiyl))dibenzenesulfonyI fluoride

4,4 '-(carbonylbis(azanediyl))dibenzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0. 16) to give the product (0.5 mmol scale, 140 mg, 74%, yield for two steps); mp 237-238 °C; Ή NMR (400 MHz, Methanol-i/4) δ 8.09 - 7.90 (m, 4H), 7.87 - 7.74 (m, 4H); ^{, 3}C NMR ( 101 MHz, Methanol- ck) δ 147.70, 131.04, 129.54(d, J = 24 Hz), 1 19.79; ¹⁹F NMR (376 MHz, Methanol-^) δ 65.48; ESI-MS (m/z): 377 [MH]⁺.

EXAMPLE 25: Synthesis of4-(3-(2-hydroxyphenyl)ureido)benzenesulfonyl fluoride

4-(3-(2-hydroxyphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.25) to give the product (0.5 mmol scale, 103 mg, 68% yield for two steps); mp 95-99 °C; Ή NMR (400 MHz, Methanol-^) δ 7.89 (dd, J = 8.0, 1.6 Hz, 1 H), 7.86 - 7.79 (m, 2H), 7.74 - 7.64 (m, 2H), 6.88 - 6.68 (m, 3H), 6.71 - 6.50 (m, 1 H); ^{I 3}C NMR (101 MHz, Methanol-^) δ 154.21 , 148.30, 147.77, 130.87, 127.80, 125.37(d, J= 24 Hz), 124.44, 121.00, 120.60, 120.54, 1 19.09, 1 17.69, 1 15.54; ¹⁹F NMR (376 MHz, Methanol-</₄) δ 65.88; ESI-MS (m/z): 31 1 [MH] ¹.

EXAMPLE 26: Synthesis of4-(3-(4-chloro-3-(trifluoromethyl)

phenyl)ureido)benzenesuIfonyl fluoride

4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benzenesulfo was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.25) to give the product (0.5 mmol scale, 173 mg, 87% yield for two steps); mp 222-224 °C; Ή NMR (400 MHz, Methanol-^) δ 7.97 (d, J = 2.6 Hz, 1 H), 7.95 - 7.87 (m, 2H), 7.81 - 7.72 (m, 2H), 7.61 (ddd, J = 8.7, 2.7, 0.7 Hz, 1 H), 7.46 (dd, J = 8.8, 0.8 Hz, 1 H);

¹³C NMR ( 101 MHz, Methanol-^) δ 1 53.71 , 147.88, 139.64, 1 33.04, 1 30.98, 129.37(d, J = 31 Hz), 126.33, 125.57, 124.40, 122.87, 119.60, 118.91, 118.86; ¹⁹FNMR(376 MHz, Methanol-^) δ 65.58, -63.63; ESI-MS (m/z): 397 [MH]' .

EXAMPLE 27: Synthesis of 3-(3-(4-chloro-3-(trifluoromethyl)phenyl)

ureido)benzenesulfonyl fluoride

3-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benz,enesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.38) to give the product as a white solid (0.5 mmol scale, 153 mg, 77% yield for two steps); mp 171-174 °C 1H NMR (400 MHz, Methanol-^) δ 8.30 (t, J= 2.0 Hz, 1H), 7.90 (d, J =2.6 Hz, 1H), 7.73 - 7.65 (m, 1H), 7.62-7.47 (m, 4H), 7.38 (d, J = 8.8 Hz, 1H); ¹ C NMR (101 MHz, Methanol-^) 5154.03, 142.10, 139.61, 134.54(d, J= 24 Hz), 132.90, 131.40, 130.86, 129.41, 129.10, 126.48, 125.77, 125.50, 124.21, 122.85, 122.79, 118.77, 118.72, 118.68; ¹⁹F NMR (376 MHz, Methanol-i/4) δ 64.24, -63.50; ESI-MS (m/z): 397 [MH]'.

EXAMPLE 28: Synthesis of 3-(3-(5-chIoro-2-hydroxyphenyl)ureido)

benzenesulfonyl fluoride

3-(3-(5-chloro-2-hydroxyphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a brown solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.26) to give the product (0.5 mmol scale, 121 mg, 70% yield for two steps); mp 81-82 °C Ή NMR (400 MHz, Methanol-^) δ 8.28 (t, J= 2.0 Hz, 1 H), 8.02 (d, J= 2.5 Hz, 1 H), 7.69 (dt, J 8.0, 1.7 Hz, 1H), 7.58 - 7.45 (m, 2H), 6.79 - 6.66 (m, 3H); ^l C NMR (101 MHz,

Methanol-^) δ 154.21 , 145.99, 142.45, 134.53(d, J = 24 Hz), 131.38, 129.38, 126.13, 125.18, 123.17, 122.49, 1 19.97, 1 18.23, 1 15.97; ¹⁹F NMR (376 MHz, Methanol-^) δ 64.22. ESI-MS (m/z): 345 [MH .

EXAMPLE 29 : Synthesis of 4-(3-(5-chloro-2- hydroxyphenyl)ureido)benzenesulfonyl fluoric!

4-(3-(5-chloro-2-hydroxyphenyl)ureido)benzenesulfonyl fluoride was prepared and isolated as a yellow solid according to the general procedure in Example 2. The crude product was purified by column chromatography (66:33 hexane:EtOAc, Rf: 0.2) to give the product (0.5 mmol scale, 108 mg, 62% yield for two steps); mp 207-208 °C; Ή NMR (400 MHz, Methanol-^) δ 8.08 (d, J= 2.5 Hz, 1H), 7.90 - 7.84 (m, 2H), 7.78 - 7.67 (m, 2H), 6.78 (dd, J = 8.5, 2.5 Hz, 1H), 6.72 (d, J= 8.5 Hz, 1H); ¹³C NMR (101 MHz, Methanol-^) δ 153.74, 148.17, 146.01 , 130.91 , 129.35, 125.64(d, J = 24 Hz), 125.20, 123.28, 1 19.93, 1 19.15, 1 15.96; ¹⁹F NMR (376 MHz, Methanol-^) δ 65.77; ESI-MS (m/z): 345 [MH]⁺.

EXAMPLE 30: Synthesis of 4-(3-(l-(methylsulfonyl)piperidin

yl)ureido)benzenesulfonyl fluoride

4-(3-(l-(methylsulfonyl)piperidin-4-yl)ureido)beiizenesulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 2. The crude product was purified by column chromatography (hexane: EtOAc 50/50, Rf: 0.1 1) to give the product (0.1 mmol scale, 26 mg, 70% yield for two steps); mp 272-274 °C; Ή NMR (400 MHz, DMSO-t/₆) 5 9.27 (s, 1 H), 8.05 - 7.85 (m, 2H), 7.80 - 7.61 (m, 2H), 6.63 (d, J = 7.6 Hz, 1 H), 2.92 - 2.77 (m, 5H), 1.91 (dd, J = 13.0, 3.9 Hz, 2H), 1.54™ 1.36 (m, 2H); ¹⁹F NMR (376 MHz, DMSO) δ 68.26; ESI-MS (m/z): 380 [MH]⁺.

EXAMPLE 31 : Synthesis ofl-(4-ethynylphenyl)-3-(l-(methylsulfonyl)

piperidin-4-yl)urea

l-(4-ethynylphenyl)-3-(l-(methylsulfonyl)piperidin-4-yl)urea was prepared and isolated as a yellow solid according to the general procedure in Example 2 from 1 -ethynyl- 4-isocyanatobenzene. The crude product was purified by column chromatography (100% EtOAc, hexane:EtOAc 50/50, Rf: 0.10) to give the product (0.1 mmol scale, 27 mg, 85% yield for two steps); mp 238-240 °C; Ή NMR (400 MHz, Methanol-^) δ 7.33 (s, 4H), 3.75 - 3.59 (m, 4H), 3.34 (s, 2H), 2.97 - 2.86 (m, 2H), 2.83 (s, 3H), 2.08 - 1.96 (m, 2H), 1.61 - 1.47 (m, 3H); ESI-MS (m/z): 322 [MH]⁺.

EXAMPLE 32: Synthesis of tert-butyl 4-(3-(4-((fIuorosulfonyl)oxy)

phenyl)ureido)piperidine-l-carboxylate

₂F

-isocyanatophenyl sulfurofluoridate

32 (A). 4-Aminophenyl fluorosulfonate was readily prepared and isolated as abrown solid (mp 41 -42 °C) in 91 % yield (8.0 g) by reaction of 4-aminophenol with sulfuryl fluoride for 6 hours in DCM using 3 eq of Et₃N. 'HNMR (400 MHz, CDC1₃) δ

7.08 (d, J = 8.5 Hz, 1 H), 6.65 (d, J = 8.7 Hz, 1 H), 3.87 (br s, 1 H); ^{l 3}C MR ( 101 MHz, CDC1₃) 6 146.9, 142.1 , 121 .8, 1 15.6, 1 15.5; ¹⁹F NMR (376 MHz, CDC1₃) δ +35.5; EI- MS (m/z): 191 [M]⁺.

32 (B). 4-isocyanatophenyl sulfurofluoridate was prepared and isolated as a yellow oil according to the general procedure in Example 7(C).

32 (C). tert-butyl 4-(3-(4-((fluorosulfonyl)oxy)phenyl)ureido)piperiditie-l- carboxylate was prepared and isolated as a white solid according to the general procedure in Example 8. The crude product was purified by column chromatography (50:50 hexane:EtOAc, Rf: 0.15) to give the product as a white solid (0.25 mmol scale, 93 mg, 88% yield for two steps); Ή NMR (400 MHz, Methanol-^) δ 7.57 - 7.47 (m, 2H), 7.39 - 7.23 (m, 2H), 3.99 (dt, J = 14.1 , 3.7 Hz, 2H), 3.75 (tt, J = 10.5, 4.1 Hz, 1 H), 2.96 (s, 2H), 2.01 - 1.78 (m, 2H), 1.46 (s, 9H), 1.44 - 1.27 (m, 2H); ¹⁹F NMR (376 MHz, Methanol-^) δ 35.02; ESI-MS (m/z): 318 [MH] ' -100.

EXAMPLE 33 : S nthesis of 4-(3-(l-(methyIsuIfonyl)piperidin-4-yl)

ureido)phenyl sulfurofluoridate

4-(3-(l-(methylsulfonyl)piperidin-4-yl)ureido)phenyl sulfurofluoridate was prepared according to the general procedure in Example 2; mp 227-229; Ή NMR (400 MHz, Methanol-^) δ 7.56 - 7.48 (m, 2H), 7.36 - 7.27 (m, 2H), 3.76 - 3.60 (m, 3H), 2.96 - 2.86 (m, 2H), 2.84 (s, 3H), 2.09 - 1 .94 (m, 2H), 1 .54 (dtd, J = 12.8, 1 1 .0, 4.1 Hz, 2H); ^{l 9}F NMR (376 MHz, Methanol-^) δ 35.00; ESI-MS (m/z): 396 [MH] ' . EXAMPLE 34: Synthesis of 4-(3-(l-(fluorosulfonyl)piperidin-4-yl)ureido)phenyl sulfurofluoridate

34 (A). General Procedure for Synthesis of sulfamoyl fluorides. Secondary amine (1 eq), Ν,Ν-dimethylaminopyridine (DMAP; 0.5-1 eq) and triethylamine (2 eq) are mixed in CH₂C1₂ (0.33 M) in a round-bottom flask filled to one-third capacity. The flask is sealed with septa and evacuated. S0₂F₂ gas is introduced from a balloon. The reaction mixture is stirred vigorously at room temperature for about 3 to 18 h, and reaction progress is monitored by GC or LC-MS. Upon completion, the mixture is concentrated, dissolved in EtOAc, washed with 1 N HC1 and brine, dried over MgS0₄, and concentrated to provide the desired compound, usually in quite pure form. In some cases, additional purification is performed by passage through a short silica gel column.

34 (B). 4-((3-Methylbutylidene)amino)piperidine-l-sulfamoyi fluoride was prepared according the general procedure 34(A), above, with 0.5 eq DMAP and 2 eq Et₃N. Upon completion, the reaction mixture was washed with water, dried, and concentrated. The product was obtained as a yellow oil yield (1.8 g, 70% yield, accounting for DMAP contamination in Ή NMR). For characterization, the imine group was hydrolyzed

(treatment with isopropanol/water mixture at 50 °C for 1.5 hours) followed by treatment with methanesulfonic acid (MsOH) to make the shelf stable salt. EI-MS (m/z): 183 [MH] ' .

34(C). 4-(3-(l-(fluorosulfonyl)piperidin-4-yl)ureido)phenyl sulfurofluoridate

4-(3-(l-(fluorosulfonyl)piperidin-4-yl)ureido)phenyl sulf urofluoridate was prepared and isolated as a white solid according to the general procedure in Example 8. The crude product was purified by column chromatography (50:50 hexane:EtOAc, Rf: 0.57) to give the product as a white solid; mp 168-170 °C; Ή NMR (400 MHz, Methanol- d₄) δ 7.54 - 7.48 (m, 2H), 7.33 - 7.27 (m, 2H), 3.88 - 3.80 (m, 2H), 3.77 (td, J = 6.4, 3.2 Hz, 1 H), 3.24 (ddt, J = 13.1 , 1 1.5, 3.0 Hz, 2H), 2.1 1 - 1 .98 (m, 2H), 1 .59 (dtd, J = 13.7, 1 1 .0, 4.2 Hz, 2H); ¹⁹F NMR (376 MHz, Methanol-^) δ 35.06; ESI-MS (m/z): 400 [MH]⁺.

EXAMPLE 35: Synthesis of 4-(3-(4-ethynylphenyl)ureido)piperidine-l-sulfonyl fluoride

4-(3-(4-ethynylphenyl)ureido)piperidine-l-sulfonyl fluoride was prepared and isolated as a white solid according to the general procedure in Example 8 from 1 -ethynyl- 4-isocyanatobenzene. The crude product was purified by column chromatography (50:50 hexane:EtOAc, Rf: 0.70) to give the product as a white solid; mp 227-229 °C; Ή NMR (400 MHz, Methanol-^) δ 7.33 (s, 4H), 3.89 - 3.80 (m, 2H), 3.80 - 3.70 (m, 1 H), 3.34 (s, l H), 3.27 - 3.19 (m, 2H), 2.1 1 - 1.97 (m, 2H), 1 .68 - 1.52 (m, 2H); ¹⁹F NMR (376 MHz, Methanol-i/4) δ 39.06; ESI-MS (m/z): 326 [MH]⁺.

EXAMPLE 36: Synthesis of 4-(3-(4-(fluorosulfonyI)phenyl)ureido)piperidine-l- sulfonyl fluoride

4-(3-(4-(fluorosulfonyI)phenyl)ureido)piperidine-l-sulfonyl fluoride was isolated as a white solid according to the general procedure in Example 8 from 4- isocyanatobenzenesulfonyl. The crude product was purified by column chromatography (50:50 hexane:EtOAc; Rf: 0.56) to give the product as a yellow gel; Ή NMR (400 MHz, Methanol-d₄) δ 7.93 - 7.86 (m, 2H), 7.72 - 7.65 (m, 2H), 3.92 - 3.73 (m, 3H), 3.29 - 3.21 (m, 2H), 2.06 (dt, J = 13.2, 3.8 Hz, 2H), 1.71 - 1.52 (m, 2H); ^{l 9}F NMR (376 MHz, Methanol-d₄) 5 65.58, 39.15; ESI-MS (m/z): 384 [MH] ' .

EXAMPLE 37: Synthesis of 2-(4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidin- l-yl)ethane-l-suIfonyl fluoride

quantitative

37 (A). General procedure for the reaction of primary and secondary amines with ESF (adapted from Krutak, J.J.; Burpitt, R.D.; Moore, W.H.; Hyatt, J.A. J. Org. Chem. 1979, 44, 3847-3858). The starting amine (1 equiv) is dissolved in organic solvent (usually CH₂C1₂ or THF, 0.1 -0.5 M in substrate) and treated with ESF (1 -2.5 equiv). The reaction mixture is stirred at room temperature for several minutes to several hours, monitoring conversion by LC-MS. Upon completion, the solvent and excess of ESF are removed by rotary evaporation and dried under vacuum, usually providing clean product. When purification by column chromatography is mentioned, it was done to remove trace impurities.

37 (B). 2-(4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidin-l-yl)etha sulfonyl fluoride was prepared and isolated as a yellow solid (quantitative yield. 22 rag) according to the general ESF procedure 37(A), above. The piperidine compound was mixed with 1.1 eq ESF (ethenesulfonyl fluoride) in DCM/CH₃CN at room temperature. Reaction finished in 10 minutes, mp 180-182 °C; Ή NMR (400 MHz, Methanol-^) δ 7.54 - 7.39 (m, 2H), 7.23 - 7.01 (m, 2H), 4.37 (d, J = 13.1 Hz, 2H), 3.85 (s, 1 H), 3.77 - 3.48 (m, 4H), 3.14 (s, 2H), 2.21 - 2.07 (m, 2H), 1.92 (d, J = 12.8 Hz, 2H); ^{l 9}F NMR (376 MHz, Methanol-^) 6 54.78, -59.37; ESI-MS (m/z): 414 [MH] ' . EXAMPLE 38: Synthesis of 4-((4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidin- l-yl)methyl)benzenesulfonyl fluoride

Crude 4-(bromomethyl)benzene-l -sulfonyl fluoride (13 mg; 0.05 mmol) and the amine (1 eq, 15 mg; 0.05 mmol) were mixed in CH3CN (0.5 mL) at room temperature. Potassium carbonate (7.5 mg; 1 .1 eq) was added and the reaction mixture was stirred at room temperature overnight, monitored by LC-MS. After completion, the reaction mixture was concentrated by rotary evaporation and the residue purified by column

chromatography (50:50 hexane:EtOAc Rf: 0.12) giving the desired product as a white solid (20 mg, 85% yield); Ή NMR (400 MHz, Methanol-^) δ 8.06 - 7.98 (m, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.46 - 7.37 (m, 2H), 7.18 - 7.09 (m, 2H), 3.70 (s, 2H), 3.62 (dt, J = 10.7, 6.1 Hz, 1 H), 2.94 - 2.76 (m, 2H), 2.27 (t, J = 1 1.1 Hz, 2H), 1.95 (dd, J = 13.1 , 4.0 Hz, 2H), 1 .63 - 1.46 (m, 2H); ¹⁹F NMR (376 MHz, Methanol-^) δ 64.63, -59.39; ESI-MS (m/z): 476[MH]⁺.

EXAMPLE 39: Synthesis of 3-(4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidine- l-carbonyI)benzenesulfonyl fluoride

F0₂S ^_¾¾i^C0₂H (COCI)₂, D F (cat.) F0₂S _{^ ^}COCI

CH₂CI₂, 6h

quant.

39(A). 3-(Fhwrosulfonyl)bem.oyl chloride. 3-(Fluorosulfonyl)benzoic acid (2.1 g; 10 mmol) was dissolved in CH2CI2 (20 mL) at room temperature and oxalyl chloride (40 mL of 1 solution in CH2CI2, 2 eq) was added at 0 °C, followed by slow addition of DMF (0.2 mL). The reaction was stirred at room temperature for 6-8 hours. After that time, solvent and volatiles were removed by rotary evaporation to give 3- (fluorosulfonyl)benzoyl chloride as a yellow oil, which was used directly in the next step.

39(B). 3-(4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidine-l- carbonyl)benzenesulfonyl fluoride. Amine (15mg, 0.05 mmol) was dissolved in 1 mL DCM at room temperature.1 eq Et₃N was added (5mg, dilution in 0.1 mL DCM) by syringe.3-(Fluorosulfonyl)benzoyl chloride in DCM solution (leq in 0.1 mL DCM solution) was added last in one portion. Reaction was monitored by LCMS. The reaction was stirred at room temperature for 2 hours. After that time, solvent and volatiles were removed by rotary evaporation and the residue purified by column chromatography (50:50 hexane:EtOAc, Rf: 0.11) giving the desired product as a colorless oil (21 mg, 81% yield); Ή NMR (400 MHz, Methanol-*^) δ 8.19 (dt,J= 8.0, 1.5 Hz, 1H), 8.14 (t,J= 1.7 Hz, 1H), 7.94 (dt,J=7.8, 1.4 Hz, 1H), 7.85 (t, 7=7.8 Hz, 1H), 7.48 - 7.40 (m, 2H), 7.20-7.11 (m, 2H), 4.64-4.38 (m, 1H), 3.89 (tt, 7= 10.5,4.1 Hz, 1H), 3.63 (d,7= 13.6 Hz, 1H), 3.15 (d, 7 = 13.4 Hz, 1H), 2.15- 1.84 (m, 2H), 1.67- 1.40 (m, 2H); ¹⁹F NMR (376 MHz,

Methanol-^) δ 64.50, -59.38; ESI-MS (m/z): 490 [MH]⁺.

EXAMPLE 40: Synthesis of 4-(3-(l-(pent-4-yn-l-ylsulfonyl)piperidin

yl)ureido)phenyl sulfurofluoridate

4-(3-(l-(pent-4-yn-l-ylsulfonyl)piperidin-4-yl)ureido)phenyl sulfurofluoridate.

4-(3-(Piperidin-4-yl)ureido)phenyl sulfofluoridate-TFA salt was prepared by treatment of tert-b\xiy\ 4-(3-(4-((fluorosulfonyl)oxy)phenyl)ureido)piperidine-l -carboxylate with TFA in DCM at room temperature for 4 hrs; ESI-MS: 318 [MH] ' . The reaction mixture was then concentrated, co-evaporated with toluene several times, and dried. Crude material (0.16 mmol), above, was dissolved in acetonitrile/water (4: 1 ratio, 0.1 M in substrate) and treated with solution of pent-4-yne- l -sulfonyl fluoride (2.6 eq) in acetonitrile and potassium carbonate (about 4.8 equiv.). Reaction mixture was stirred at rt overnight, monitored by LCMS; ESI-MS: 448 [MH]⁺. Product was purified by preparative TLC (hexane/EtOAc - 1/1 ; Rf: 0.38). 70 mg (quant.) of product was isolated as a white solid; mp 153-155 °C; Ή NMR (400 MHz, Methanol-^) δ 7.56 - 7.48 (m, 2H), 7.36 - 7.27 (m, 2H), 3.81 - 3.59 (m, 3H), 3.18 - 3.09 (m, 2H), 3.01 (ddd, J = 12.5, 1 1.3, 2.7 Hz, 2H), 2.40 - 2.31 (m, 3H), 1.99 (dddd, J = 16.6, 14.4, 8.4, 4.0 Hz, 4H), 1.60 - 1 .46 (m, 2H); ¹⁹F NMR (376 MHz, Methanol-^) 5 35.04; ESI-MS (m/z): 448 [MH] ' .

EXAMPLE 41: 4-(3-(4-(trifluoromethoxy)phenyl)ureido)

piperidine-l-sulfonyl fluoride

4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidine-l-sulfonyl fluoride was prepared and isolated as a white solid according to the procedure of Example 2. The crude product was purified by column chromatography (50:50 hexane:EtOAc Rf: 0.75) to give the product as a white solid; mp 197- 198 °C; Ή NMR (400 MHz, Methanol-^) δ 7.48 - 7.38 (m, 2H), 7.15 (dq, J = 8.0, 0.9 Hz, 2H), 3.90 - 3.71 (m, 3H), 3.28 - 3.16 (m, 2H), 2.1 1 - 1 .95 (m, 2H), 1 .67 - 1 .52 (m, 2H); ¹⁹F NMR (376 MHz, Methanol-^) δ 39.08, -59.40; ESI-MS (m/z): 386 [MH] ¹ .

EXAMPLE 42: Evaluation of sEH Inhibitors

42 (A). LogP Determination, Shake-flask method. The inhibitor ( 10 111M, 20 \xL of EtOH) was added to a mixture of water (saturated with octanol, 1 mL) and octanol (saturated with water, l mL). The mixture was shaken (220 rpm) at rt for overnight in a sealed vial (4 mL). The sample ( 100 μί) from each layer was collected and diluted in a series of dilution (x l O, x l OO, l OOO) in MeOH and kept at -20 °C before it was analyzed by LC/MS-MS.

42 (B). Preparation of solubility sample. Each inhibitor ( 1 mg) was added into the phosphate buffer (PB; 0.1 M sodium phosphate, pH 7.4, 300 μί) to create a suspension. The suspension was then incubated and was shaken (220 rpm) at 30 °C for 24 h. The suspension was cooled down to rt for 1 h. The precipitates were centrifuged (10,000 rpm, 10 min, rt). The supernatant was further diluted 10 times by methanol. The solution was kept on ice for 15 min to precipitate all the salts. The solution was centrifuged (10,000 rpm, 10 min, 4 °C) and the supernatant was transferred to vial and was kept at -20 °C before LC/MS-MS analysis.

42 (C). Enzyme preparation. Expression and purification of recombinant sEH followed the published procedure in Morisseau et al., Arch. Biochem. Biophys. 378:321 - 332 (2000). Briefly, sEH from human and rat were expressed in high yield in a baclovirous expression system. The sEH in the supernatant from insect cell culture was purified by affinity chromatography to yield high specific activity and apparent homogeneity on SDS-PAGE. The enzyme was frozen by liquid nitrogen in small aliquots and thawed once at 0 ^°C immediately before use.

42 (D). ICs determination for human sEH Inhibitors. ICso values for the sEH inhibitors were determined by fluorescent according to published procedures. IC₅o, the concentration of the inhibitor that blocks 50% of the enzyme activity, was determined based on regression of at least five datum points with a minimum of two points in the linear region of the curve on either side of the ICso.

42 (E). FRET-Displacement assay procedure. The Forster Resonance Energy Transfer (FRET) assay was carried out as described in Lee et al., Analytical Biochemistry 434(2):259-268 (201 3). In order to prevent leaching of fluorescence impurities from the plastic tube and non-specific binding to sEH inhibitors, the inhibitor stock solution ( 1 0 mM, DMSO) was stored in glass vials. In addition, sEH was diluted to desired

concentration (20 nM) with sodium phosphate buffer (PB; 100 mM sodium phosphate, pH 7.4, 0.01 % gelatin) to avoid loss of protein from non-specific binding to the cuvette surface. All buffer used in this assay was filtered by sterilized filtration unit (Millipore© Durapore© PVDF Membrane, pore size: 0.22 μητ).

42 (F). Measurement in Quartz cuvette. The sEH (10 nM, 3 mL, 100 mM sodium phosphate, pH 7.4, 0.01 % gelatin) was stirred and pre-incubated with \ -((3s, 5s, 7 s)- adamantan-1 -yl)-3-(l -(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl)piperidin-4-yl)urea (ACPU) reporting ligand (1 equivalent, 20 nM, 100 mM sodium phosphate, pH 7.4) at 30 °C for 1 h in a quartz cuvette. The fluorescence (455 nm, emission slit width = 12 nm, excited at 280 nm, excitation slit width = 1.0 nm) was measured. The enzyme-ligand complex was titrated with different sEH inhibitors at varying concentration until no more fluorescence quenching was observed. The relative fluorescence intensity was plotted against the concentration of inhibitor.

42 (G). Pre-treatment of96-well plates. All the measurement for FRET- based displacement assay in 96-well plate format was done in TEC AN Infinite*' Ml 000 Pro. As mentioned, in order to prevent non-specific binding of sEH or inhibitor on the 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 of sEH and sEH inhibitors to the plate. The buffer was discarded and the plate was dried before use.

42 (H). Assay procedure. The sEH stock was diluted to the desired concentration (20 nM) by PB (100 mM sodium phosphate, 0.1 % gelatin, pH 7.4). ACPU (one equivalent to sEH, 10 mM, Ethanol) was added to the sEH solution and was incubated for 2 h 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) (λ_εχ0ίΐ;ιΐίοη at 280 nm, Emission at 450 nm) of the samples was measured after the z-position and gain were optimized automatically by the fluorometers. The z and gain value was noted and will be used for the later fluorescent measurement. Because DMSO has been known to quench fluorescence. 1 % DMSO in PB was served as a control (F_DMSO)- The desired concentration of inhibitors which is the concentration that 100% of sEH was bound to inhibitor, 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 have enough data to calculate the accurate K for the inhibitors. The samples were incubated at 30 °C for 1.5 hours. Then, the fluorescence ( _excitation at 280 nm, _emi_ssion at 450 nm) of the samples was measured using the z-position and gain values that previously obtained. The obtained fluorescence signals were transformed as below and were used to calculated the K_\ of the inhibitors according to "Curve fitting" section below.

Initiated fluorescence = F_DMso (well x) / F₀ <_Weii x)

Saturated fluorescence = F_al the saturated concentration (well X) / Fo (at well X)

Observed fluorescence = F(_wen x) / F₀ (_Weii x)

42 (I). Curve fitting. The data manipulation and calculation were based on the original paper by Wang et al , FEBS Lett. 360: 1 1 1 -1 14 (1995), with modifications suggested by Roehrl et al , Proc. Natl. Acad. Sci. USA, 101:7554-7559 (2004).

The displacement assay is based on a three-state equilibrium binding model. This is modeled as described below (Eq 1 ):

[Rl] + L => R + l + L » [RL] + (Eq l ) with [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 ) consists of the sEH -inhibitor complex, sEH and sEH-reporting ligand complex. In this study, the relative fluorescence intensity (F_?) was plotted against the concentration of sEH inhibitor and the resulting curve was fitted into equation (Eq 2) derived by Wang et al. for three-state equilibrium.

. 1/2

2 (a²-3b)^1/2 cos(0/3) -a] / {3 K_dl + [2 (a²-3b)^{1/ 2} cos (0/3)-a] (Eq 2)

with a = K_dl + K_d2 + L + I-R;

b = K_d2 (L-R) + K_dl (l-R) + K_dl K_d2 ; c = -K_dlK_d2R; and

Θ = arccos j (-2a³ + 9ab-27c)/ 2

where Relative Fluorescence = (observed fluorescence - fluorescence at saturation)/(initiated fluorescence - fluorescence at saturation)

1 = the concentration of added unlabeled competing ligand;

R = the total concentration of sEH;

L— The total concentration of reporting ligand;

Ken = The dissociation constant of reporting ligand (found by fluorescent binding assay), and ;

Kd2 ⁼ The inhibition constant of inhibitors

42 (J). k₀ff measurement procedure. The sEH (8 μΜ) was pre-incubated with the selected inhibitor (8.8 μΜ, 100 mM PB buffer, pH 7.4) for 1.5 h at rt. The sEH-inhibitor complex was then diluted 40 times with ACPU (20 μΜ, 100 mM sodium phosphate buffer, pH 7.4). The fluorescence ^excitation at 280 nm, _emission at 450 nm) was monitored immediately for every 30s up to 5100s. The fluorescence Emission at 450 nm) data was plotted against time (s). The resulting curve was fitted to single exponential growth and the relative k_0/j was obtained.

The obtained results are compiled in Table 1.

TABLE 1

Physical properties of soluble epoxide hydrolase (sEH)

inhibitors and potency against human sEH ic₅₀ ^b i^c tl/2^d Solubility"

g/mL _eLogpf

Structure M.W.^a (hsEH) (hsEH) (

(hsEH)

(μΜ))

nM) (nM) (min-1)

In Table 1 :

a M.W. - molecular weight.

b IC50 values were determined by a fluorescent substrate assay using

cyano(2-methoxynapthalen-6-yl)methyl (3-phenyloxiran-2-yl)methyl carbonate) (5 μΜ) with human sEH ( 1 nM). ^c Ki was determined by FRET-based displacement assay described by Lee et al,

Analytical Biochemistry 434(2) 256-26% (2013). The stated results are average of duplicates with ± S.D.

d k_0jj was determined by FRET-based displacement assay described by Lee et al. A pre- incubated human sEH-inhibitor complex (8 μΜ) was diluted by 40 times by fluorescent reporter-APCU (2 μΜ, 0.1 M Sodium Phosphate, pH 7.4). The fluorescent enhancement O it = 280 nra, λβ,ηύ = 450) was measured over time (5100s). The results are the average of triplicates with ± S.D. tl/2 = In (2)1 k_off , which describes the half-life of enzyme- inhibitor complex.

e Solubility was measured with sodium phosphate buffer (0.1 M, pH 7.4) according to the method described by Tsai et al, European J. Pharm. Sci. 40:222 (2010).

f eLogP stands for experimental log P, determined by shake-flask method.

The activity of a library of sulfonyl fluoride-based sEH inhibitors against human sEH was evaluated according to the same procedures and the results are shown in Table 2.

TABLE 2

Sulfonyl fluoride library inhibitors and potency against human sEH

The IC50 values in Table 2 were determined by a fluorescent substrate assay using cyano(2-methoxynapthalen-6-yl)methyl (3-phenyloxiran-2-yl)methyl carbonate) (5 μΜ) with human sEH ( 1 nM), as for the data in Table 1 .

The IC50 data in Tables 1 and 2 clearly demonstrate that the flurosulfonyl sEH inhibitor compounds described herein exhibit significant inhibitor activity. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. The terms "consisting of and "consists of are to be construed as closed terms, which limit any compositions or methods to the specified components or steps, respectively, that are listed in a given claim or portion of the specification. In addition, and because of its open nature, the term "comprising" broadly encompasses compositions and methods that "consist essentially of or "consist of specified components or steps, in addition to compositions and methods that include other components or steps beyond those listed in the given claim or portion of the specification. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

All numerical values obtained by measurement (e.g., weight, concentration, physical dimensions, removal rates, flow rates, and the like) are not to be construed as absolutely precise numbers, and should be considered to encompass values within the known limits of the measurement techniques commonly used in the art, regardless of whether or not the term "about" is explicitly stated.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate certain aspects of the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WE CLAIM:

1. A compound represented by Formula (I):

(I) _Rl_pl— _Ll ^ p2 ^_ _L2 ^ p_3)m

wherein:

R¹ is selected from the group consisting of alkyl, heteroalkyl, C5-C₁₂ cycloalkyl, C5-Ci₂ cycloalkylalkyl, C₅-C i₂ cycloalkyiheteroalkyl, arylalkyl, arylheteroalkyl, aryl, and heteroaryl; and R¹ can be substituted or unsubstituted;

P¹ is a primary pharmacophore selected from the group consisting of -OC(0)0-, -OC(0)CH₂-, -CH₂C(0)0-, -OC(O)-, -C(0)0-, -NHC(NH)NH-, -NHC(NH)CH₂-, -CH₂C(NH)NH-,-NHC(NH)-, -C(NH)NH-, -NHC(0)NH-, -OC(0)NH-, -NHC(0)0-, -NHC(S)NH-, -NHC(S)CH₂-, -CH₂C(S)NH-, -SC(0)CH₂-, -CH₂C(0)S-, -SC(NH)CH₂-, - -, -N=C=N-, -CH₂C(0)NH-, -NHC(0)CH₂-, -C(0)NH-, -NHC(O)-,

₂

P is a secondary pharmacophore selected from the group consisting of -NH-, -OC(0)0-, -C(O)-, -CH(OH)-, -0(CH₂CH₂0)_q-, -C(0)0-, -OC(O)-, -NHC(NH)NH-, -NHC(NH)CH₂-, -CH₂C(NH)NH-, -NHC(0)NH-, -OC(0)NH-, -NHC(0)0-, -C(0)NH-, -NHC(O)-, -NHC(S)NH-, -NHC(S)CH₂-, -CH₂C(S)NH-, -SC(0)CH₂-, -CH₂C(0)S-, - -, -CH₂C(NH)S-, -N=C=N-,

P^' is a tertiary pharmacophore selected from the group consisting of C₂-C_f, alkenyl, C₂-C₆ alkynyl, C _rC₆ haloalkyl, aryl, heteroaryl, heterocyclyl, -0(CH₂CH₂0)_q-R², -OR², -C(0)NHR², -C(0)NHS(0)₂R², -NHS(0)₂R², -OC₂-C₄alkyl-C(0)OR², -C(0)R², -C(0)OR² and carboxylic acid analogs, wherein R² is selected from the group consisting of hydrogen, substituted or unsubstituted C j -C₄ alkyl, substituted or unsubstituted Ci-Cx cycloalkyl, substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl and substituted or unsubstituted aryl C i -C₄ alkyl;

n and m are each independently 0 or 1 , and at least one of n or m is 1 ;

q is 0, 1 , 2, 3, 4, 5, or 6; L¹ represents a first linking group that is selected from the group consisting of a covalent bond, C)-C₆ alkylene, C₃-C₆ cycloalkylene, 5-membered ring heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S, 6-membered ring

heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S, arylene, and heteroarylene;

when m is 1 , L² represents a second linking group selected from the group consisting of a covalent bond, Cj-Ci₂ alkylene, C₃-C₆ cycloalkylene, 5-membered ring heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S, 6- membered ring heterocycloalkylene comprising 1 to 3 heteroatoms selected from N, O, and S, arylene, heteroarylene; an amino acid, a dipeptide, a dipeptide analog, and a

combination of two or more thereof; when m is 0, U is H;

L¹ and L² can be substituted or unsubstituted when they are not covalent bonds; and the compound of Formula (I) includes at least one S0₂F ("fluorosulfonyl") group covalently bonded thereto.

The compound of claim 1 wherein the compound is represented by Formul

wherein:

each x independently is 0, 1 , 2, or 3, provided that at least on x is not 0; and each X¹ independently is selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, C,-C₆ haloalkyl, aryl, heteroaryl, heterocyclyl, -0(CH₂CH₂0)_q-R³, -OR³, - C(0)NHR³, -C(0)NHS0₂R³, -NHS0₂R³, -S0₂R³, -OC₂-C₄alkyl-C(0)OR³, -C(0)R³, - C(0)OR³ , halogen, -S0₂F, -OS0₂F, -N(R³)S0₂F, -N(R )(CH₂CH₂S0₂F), and

-N(CH₂CH₂S0₂F)₂;

R³ can be substituted or unsubstituted and is selected from the group consisting of hydrogen, C|-C₆ alkyl, C₂-C₆ alkenyl, C₂-C alkynyl, Ci-C₆ haloalkyl, aryl, heteroaryl, heterocyclyl, and C₃-C₈ cycloalkyl; and wherein at least one X¹ comprises an S0₂F moiety.

3. The compound of claim 2 wherein P¹ is -NHC(0)NH-.

4. The compound of claim 1 wherein the compound is represented by Formula

wherein:

x is 0, 1 , 2, or 3;

X¹ is selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ haloalkyl, aryl, heteroaryl, heterocyclyl, -0(CH₂CH₂0)_q-R³, -OR³, -C(0)NHR³,

-C(0)NHS0₂R³, -NHS0₂R³, -S0₂R³, -OC₂-C₄alkyl-C(0)OR³, -C(0)R³, -C(0)OR³, halogen, -S0₂F, -OS0₂F, -N(R³)S0₂F, -N(R )(CH₂CH₂S0₂F), and -N(CH₂CH₂S0₂F)₂;

X² is selected from -C(0)NHR³, -C(0)NHS0₂R³, -S0₂R³, -S0₂R³, C,-C₆ alkylaryl (e.g., benzyl), Q-Q alkylaryl-S0₂R³, -C,-C₆ alkylhaloaryl, -C(0)R³, -C(0)OR³ , -S0₂F, -CH₂CH₂S0₂F, and C,-C₆ alkylaryl-S0₂F;

and wherein at least one of X¹ and X² comprises an S0 F moiety.

5. The compound of claim 4 wherein P¹ is -NHC(0)NH-.

The compound of claim 1 wherein the compound is represented by Formula

wherein:

each x independently is 0, 1 , 2, or 3, provided that at least on x is not 0; and each X¹ independently is selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, C C₆ haloalkyl, aryl, heteroaryl, heterocyclyl, -0(CH₂CH₂0)_q-R³, -OR³, -C(0)NHR³, -C(0)NHS0₂R³, -NHS0₂R³, -S0₂R³, -OC₂-C₄alkyl-C(0)OR³, -C(0)R³, -C(0)OR³ , halogen, -S0₂F, -OS0₂F, -N(R )S0₂F, -N(R³)(CH₂CH₂S0₂F), and

-N(CH₂CH₂S0₂F)₂;

R³ can be substituted or unsubstituted and is selected from the group consisting of hydrogen, Cj-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C]-C₆ haloalkyl, aryl, heteroaryl, heterocyclyl, and C3-C8 cycloalkyl;

and wherein at least one X¹ comprises an S0₂F moiety. 7. The compound of claim 6 wherein P¹ is -NHC(0)NH-.

8. The compound of claim 6 wherein P is -0-.

9. The compound of claim 6 wherein P¹ is -NHC(0)NH- and P² is -0-.

10. The compound of claim 1 and having the structural formula:

The com tural formula

The compound of claim 1 and having the structural formul,

The compound of claim 1 and having the structural formula:

O

H H

The compound of claim 1 and having the structural formula:

compound of claim 1 and having the structural formula:

16. A pharmaceutical composition comprising the compound of any one of claims 1 to 15 in combination with a pharmaceutically acceptable carrier, vehicle, or diluent.

17. Use of the compound of any one of claims 1 to 15 for treating a disease associated with soluble epoxide hydrolase.

1 8. Use of the compound of any one of claims 1 to 15 for the preparation of a medicament for treating a disease associated with soluble epoxide hydrolase.

19. The use of claim 17 or claim 18 wherein the disease is hypertension, cardiac hypertrophy, arteriosclerosis, brain and heart ischemia injury, cancer, pain or a

combination of two or more thereof.

20. A method of treating a disease associated with soluble epoxide hydrolase comprising administering the compound of any one of claims 1 to 15 to a patient with the disease.

21. The method of claim 20 wherein the disease is hypertension, cardiac hypertrophy, arteriosclerosis, brain and heart ischemia injury, cancer, pain or a

combination of two or more thereof.