Potent soluble epoxide hydrolase inhibitor

Abstract

The present invention relates to potent soluble epoxide hydrolase inhibitors. The present invention discloses a series of novel soluble epoxide hydrolase (sEH) inhibitors with improved physical properties that enhance the drug-like properties of the soluble epoxide hydrolase inhibitors for treating sEH-related diseases such as chronic diabetic neuropathic pain.

Description

[0001] This application is a divisional application. The application date of the original application is March 27, 2015, the application number is 201580015513.2, and the name of the invention is “Effective soluble epoxide hydrolase inhibitors”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 USC §119(e) to U.S. Serial No. 61 / 971,410, filed on March 27, 2014, the entire contents of which are incorporated herein by reference in their entirety.

[0004] Government Rights Statement

[0005] This invention was made in part with government support under Grant No. R43ES025598 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0006] The present invention relates generally to compounds and, more particularly, to compounds that inhibit soluble epoxide hydrolase (sEH) and methods of making and using the same. Background Art

[0007] Soluble epoxide hydrolase (sEH, EC 3.3.2.10) is a bifunctional enzyme encoded by the Ephx2 gene in mammals. sEH is found in the cytosol and peroxisomal parts of the cytosol and hydrolyzes specific epoxides (including epoxy fatty acids) to the corresponding 1,2-diols, while different regions of the protein also have lipid phosphate phosphatase activity.

[0008] Epoxy fatty acids (EpFA), metabolites from the cytochrome P450 (CYP450) pathway in the arachidonic acid cascade, are important lipid mediators. Epoxyeicosatrienoic acid (EET), epoxyeicosatetraenoic acid, and epoxydocosapentaenoic acid, which are epoxide metabolites of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, respectively, from the CYP450 pathway, have been shown to have anti-inflammatory, vasoregulatory, analgesic, and angiogenic effects.

[0009] EpFAs are significantly metabolized by sEH to their corresponding 1,2-diols, which reduce their activity. Therefore, stabilizing EETs in vivo by inhibiting sEH can be anti-inflammatory, antihypertensive, and analgesic by stabilizing EETs in vivo. Therefore, sEH is an important drug target.

[0010] Most diabetic patients will eventually develop renal failure, hypertension and / or stroke. In addition, about 65% of diabetic patients will develop peripheral neuropathy. In addition to many other complications, people with diabetic neuropathic pain experience spontaneous pain, hyperalgesia and allodynia, which greatly affects the patient's quality of life. It is believed that high blood sugar is the initial cause of the degeneration of peripheral nerve fibers that causes pain. However, active blood sugar control can only control the progression of neuronal degeneration, but cannot reverse neuropathy. Although semi-effective treatments for diabetic neuropathy are available, including tricyclic antidepressants and selective serotonin reuptake inhibitors, they have side effects that limit their use. Therefore, a kind of alternative therapy with no or greatly reduced side effects is needed.

[0011] Recent studies have shown that soluble epoxide hydrolase (sEH) inhibitors are analgesics in a model of diabetic neuropathic pain. The results suggest that sEH inhibitors are more effective, useful, and have fewer side effects than the clinically approved drug gabapentin.

[0012] Over the years, several groups have reported the synthesis and evaluation of sEH inhibitors with different central pharmacophores (potency ranging from micromolar to nanomolar range), including but not limited to amides, carbamates and ureas. Disubstituted ureas are one of the effective central pharmacophores for inhibiting sEH because the urea nitrogen forms a tight hydrogen bond with the active residue Asp337 and the chemistry is easily accessible.

[0013] Many of the most effective compounds generally have poor physical properties. Efforts to improve physical properties (including water solubility, hydrophilicity), reduce clogP and lower melting point generally result in reduced potency and less desirable pharmacokinetics. This may also lead to poor absorption, poor pharmacokinetic properties, and may require heroic formulation. Therefore, it is necessary to further optimize the structure and improve its physical properties, which can simplify the drug formulation process and improve the oral bioavailability of sEH inhibitors with disubstituted ureas as central pharmacophores.

[0014] Evidence strongly suggests that the retention time (t1 / 2) of the inhibitor on the target enzyme is one of the most important parameters affecting the efficacy in vivo. Inhibitors with long retention times have a long action time on the target enzyme and a long effect of conversion in vivo. However, most of the sEH inhibitors reported so far do not have an optimal retention time on the enzyme. The sEH inhibitors with a methyl group at R7 of the following formula (II) have a short t1 / 2, which can explain their lack of efficacy in humans. Therefore, sEH inhibitors with longer retention times (t1 / 2) are necessary for committing to this pharmaceutical target. Summary of the invention

[0015] In one aspect, the present invention provides a compound of structural formula (I), or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof.

[0016]

[0017] in:

[0018] R1 is selected from the group consisting of: trifluoromethyl- or trifluoromethoxy-;

[0019] R2 is selected from the group consisting of: H- or F-;

[0020] R3 is selected from the group consisting of: H-, trifluoromethyl- or -CH2CO2H;

[0021] X is selected from carbon, nitrogen or oxygen;

[0022] Y is selected from carbon, nitrogen or oxygen;

[0023] subscript m is 0, 1, or 2; and

[0024] Subscript n is 1 or 2.

[0025] In another aspect, the present invention provides a compound of structural formula (II), or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof.

[0026]

[0027] in:

[0028] X is selected from carbon or nitrogen;

[0029] Y is selected from carbon, nitrogen or oxygen;

[0030] R5 is selected from the group consisting of: trifluoromethyl- or trifluoromethoxy-;

[0031] R6 is selected from the group consisting of: H- or F-; and

[0032] R7 is selected from the group consisting of 1H-pyrrole, furan, pyran, tetrahydropyran, 4,5-dihydrofuran, 2-methylfuran, 3-methylfuran, ethyl-, isopropyl-, cyclopropyl-, 2-methylbutyl- and (S)-2-methylbutyl-.

[0033] The compounds include all pharmaceutically acceptable derivatives thereof, such as salts, prodrugs, soft drugs, solvates and hydrates.

[0034] Certain embodiments also provide pharmaceutical compositions comprising one or more compounds disclosed herein, together with a pharmaceutically acceptable carrier, and methods of making and using the compounds and compositions.

[0035] In another aspect, the present invention provides a method for inhibiting a soluble epoxide hydrolase, comprising contacting the soluble epoxide hydrolase with an inhibitory amount of a compound having the above formula (I) or (II).

[0036] In a related aspect, the invention provides a method for treating a disease regulated by a soluble epoxide hydrolase, the method comprising administering to a subject in need of such treatment an effective amount of a compound having a formula selected from formula (I) or (II) above. In one aspect, the effective amount is a therapeutically effective amount.

[0037] In another aspect, the present invention provides a method for increasing the water solubility of a compound of formula (I) by incorporating a heteroatom at X.

[0038] In a related aspect, the invention provides a method for increasing the water solubility of a compound of formula (II) by incorporating a heterocyclic ring at R7.

[0039] In another aspect, the present invention provides a method for lowering the melting point of a compound of formula (I) by incorporating fluorine at R2.

[0040] In a related aspect, the present invention provides a method for lowering the melting point of a compound of formula (II) by incorporating fluorine at R6.

[0041] In another aspect, the invention provides a method of increasing the aqueous solubility of a compound of formula (I) in the pH range of 1-7 by incorporating oxygen at X and subscript m is 0, 1 or 2 and n is 1 or 2.

[0042] In a related aspect, the present invention provides a method for increasing the aqueous solubility of a compound of formula (II) in the pH range of 1-7 by incorporating a heterocyclic ring at R7.

[0043] In another aspect, the present invention provides a method for increasing the retention time (t1 / 2) of a compound of formula (I) on a soluble epoxide hydrolase by incorporating a trifluoromethoxy- group at R1.

[0044] In a related aspect, the present invention provides a method for increasing the retention time (t1 / 2) of a compound of formula (II) on a soluble epoxide hydrolase by incorporating a trifluoromethoxy group at R5 and an isopropyl group, a cyclopropyl group, a 2-methylbutyl group or a (S)-2-methylbutyl group at R7.

[0045] In another aspect, the invention provides a method for monitoring the activity of a soluble epoxide hydrolase (sEH). The method comprises contacting a soluble epoxide hydrolase with a compound of formula (I) or (II) in an amount sufficient to produce a detectable change in the optical fluorescence of the soluble epoxide hydrolase, the detectable change being produced by interacting with one or more tryptophan residues present in the catalytic site of the sEH.

[0046] In another aspect, the present invention provides a method for treating a disease or condition in a subject. The method comprises administering to a subject in need of such treatment an effective amount of a compound having a formula selected from formula (I) or (II) above. In an embodiment, the disease or condition is pain, such as diabetic neuropathic pain, inflammatory pain, or postoperative pain. In some embodiments, neuropathic pain may be the result of nerve damage caused by diabetes (whether congenital or otherwise) or other types of neurological disorders, such damage caused by external forces (such as accidents, surgery, or disease states, including infections) or drugs used to treat another ailment. In a related embodiment, the disease is an epileptic disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a graph showing the relationship of the dipole moments of the different heterocycles attached at R7 of formula (II) and it does not correlate with the enhanced solubility obtained as shown in Table II.

[0048] Figure 2 is a graph showing the experimental logP relationship for different heterocycles attached at R7 of formula (II) and it does not correlate to the enhanced solubility obtained as shown in Table II.

[0049] Figure 3 is a graph showing the solubility of selected inhibitors at pH 7.4.

[0050] Figure 4 is a graph showing the solubility of selected inhibitors at pH 3.0.

[0051] Figure 5 The solubility of selected compounds at pH 3.0 is plotted along with their potency (K i ) changes with the curve.

[0052] Figure 6 is a graph showing the anti-nociceptive response effects of inhibitors with improved drug-like properties, and these inhibitors have much better in vivo efficacy than the clinically approved drug gabapentin at 100x the dose.

[0053] Figure 7( is a graph showing the anticonvulsant efficacy of sEH inhibitors (0.3 mpk, sc) relative to sc PTZ). Among the five 1,3-disubstituted urea based sEH inhibitors, compound 29 delayed the tonic phase of seizures in wild type Swiss mice (*=p<0.05).

[0054] Figure 8 is a graph showing the CNS protective effects of sEH inhibitors. Several sEH inhibitors prevented mice from dying from PTZ-induced seizures.

[0055] Fig. 9 is a series of graphs showing that the solubility of compounds 1 and 29 increases with increasing pH. DETAILED DESCRIPTION

[0056] Abbreviations and Definitions

[0057] "Epoxide hydrolases" ("EH"; EC 3.3.2.3) are enzymes in the alpha / beta-fold hydrolase family that add water to 3-membered cyclic ethers called epoxides.

[0058] "Soluble epoxide hydrolase" ("sEH") is an enzyme located in endothelial, smooth muscle and other cell types that converts EETs to dihydroxy derivatives called dihydroxyeicosatrienoic acids ("DHETs"). The cloning and sequence of murine sEH are described in Grant et al., J. Biol. Chem. 268(23): 17628-17633 (1993). The cloning, sequence and accession number of the human sEH sequence are described in Beetham et al., Arch. Biochem. Biophys. 305(1): 197-201 (1993). The amino acid sequence of human sEH is also described in SEQ ID NO: 2 of U.S. Pat. No. 5,445,956; the nucleic acid sequence encoding human sEH is described in nucleotides 42-1703 of SEQ ID NO: 1 of that patent. The evolution and nomenclature of the gene is discussed in Beetham et al., DNA Cell Biol. 14(1):61-71 (1995). Soluble epoxide hydrolases represent a single highly conserved gene product with greater than 90% homology between rodents and humans (Arand et al., FEBS Lett., 338:251-256 (1994)).

[0059] The terms "treat", "treating" or "treatment" refer to any method of alleviating or eliminating a disease or its attendant symptoms.

[0060] The term "therapeutically effective amount" refers to the amount of the compound being administered sufficient to prevent or reduce the development of one or more symptoms of the disease, condition or disorder being treated.

[0061] The term "modulate" refers to the ability of a compound to increase or decrease the function or activity of an associated activity (e.g., soluble epoxide hydrolase). "Modulation" as used herein in various forms is intended to include antagonism and partial antagonism of an activity associated with sEH. An inhibitor of sEH is, for example, a compound that binds, partially or completely blocks the activity of the enzyme.

[0062] The term "compound" as used herein is intended to include not only the specified molecular entity, but also pharmaceutically acceptable, pharmacologically active derivatives thereof, including but not limited to salts, prodrug conjugates such as esters and amides, metabolites, and the like.

[0063] As used herein, the term "composition" is intended to include a product comprising the specified ingredients in the specified amounts, as well as any product resulting, directly or indirectly, from combination of the specified ingredients in the specified amounts. "Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0064] "Subject" as defined herein includes animals, such as mammals, including but not limited to primates (eg, humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In some embodiments, the subject is a human.

[0065] As used herein, the term "sEH-mediated disorder or disease" and the like refers to a disease or condition characterized by less than or greater than normal sEH activity. A she-mediated disorder or disease is one in which modulation of sEH results in some effect on the condition or disease (e.g., a sEH inhibitor or antagonist results in some improvement in patient health in at least some patients). These conditions and diseases may include seizures such as epilepsy, renal disease, cardiomyopathy, hypertension, pain, inflammation, inflammatory pain, postoperative pain, neuropathic pain, diabetic neuropathic pain, tissue trauma or pain resulting therefrom, acute inflammation, inflammation from sepsis, pancreatitis, various traumas such as brain injury, and tissue damage such as lacerations of the musculature, brain surgery, hemorrhagic shock, and immune-mediated organ damage, adult respiratory distress syndrome, emphysema, chronic bronchitis, obstructive lung disease, chronic obstructive pulmonary disease (COPC), small airway disease, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, burns or pain in skin diseases such as dermatitis, chemical burns, thermal burns, reddening of the skin, and chemically induced lesions, neuralgia, pain due to trauma or irritation of peripheral nerves close to the surface of the skin.

[0066] As used herein, the term "alkyl" refers to a saturated hydrocarbon group (e.g., ethyl, isopropyl, tert-amyl, or 2,5-dimethylhexyl) that may be straight or branched. This definition applies when the term is used alone and when it is used as part of a compound term, such as "aralkyl," "alkylamino," 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 include their upper and lower limits. A low alkyl group refers to an alkyl group having 1-4 carbon atoms. In addition, the alkyl and heteroalkyl groups may be connected to other moieties (such as, for example, 2-pentyl, 2-methylpentyl-1-yl, and 2-propyloxy) at any position on the alkyl or heteroalkyl group that is otherwise occupied by a hydrogen atom. A divalent alkyl group may be referred to as an "alkylene group," and a divalent heteroalkyl group may be referred to as a "heteroalkylene group," 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.

[0067] The terms "cycloalkyl" and "cycloalkenyl" refer to saturated hydrocarbon rings and include bicyclic and polycyclic rings. Similarly, cycloalkyl and cycloalkenyl groups with heteroatoms (e.g., N, O, or S) replacing carbon ring atoms may be referred to as "heterocycloalkyl" and "heterocycloalkylene," respectively. Examples of cycloalkyl and heteroaryl groups are, for example, cyclohexyl, norbornyl, adamantyl, morpholinyl, thiomorpholinyl, dioxythiomorpholinyl, and the like. 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 cycloalkenyl 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 heteroatoms in the ring (e.g., morpholinyl, thiomorpholinyl, dioxythiomorpholinyl, piperidinyl, and the like). Furthermore, the term "(cycloalkyl)alkyl" refers to a group having a cycloalkyl portion attached to an alkyl portion. Examples are cyclohexylmethyl, cyclohexylethyl and cyclopentylpropyl.

[0068] The term "alkenyl" as used herein refers to an alkyl group as described above containing one or more unsaturated sites that are double bonds. Similarly, the term "alkynyl" as used herein refers to an alkyl group as described above containing one or more unsaturated sites that are triple bonds.

[0069] The term "alkoxy" refers to an alkyl group as described above further bearing an oxygen substituent capable of being covalently linked to another hydrocarbon group (such as, for example, methoxy, ethoxy, aryloxy and tert-butoxy).

[0070] The term "aryl" refers to an aromatic carbocyclic substituent that can be a monocyclic or polycyclic fused together, covalently connected or connected to a common group, such as an ethylene or methylene moiety. Similarly, an aryl with a heteroatom (e.g., N, O, or S) that replaces a carbon ring atom is referred to as a "heteroaryl". Examples of aryl and heteroaryl are, for example, phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-diphenyl-1-ethyl, thienyl, pyridyl, and quinoxalyl. Aryl and heteroaryl moieties may also be optionally substituted by halogen atoms or other groups, such as nitro, alkyl, alkylamino, carboxyl, alkoxy, phenoxy, and the like. In addition, aryl and heteroaryl may be connected to other moieties (such as, for example, 2-pyridyl, 3-pyridyl, and 4-pyridyl) at any position on an aryl or heteroaryl that is otherwise occupied by a hydrogen atom. A divalent aryl is an "arylidene", and a divalent heteroaryl is referred to as a "heteroarylidene", such as those groups used as linkers in the present invention.

[0071] The terms "arylalkyl", "arylalkenyl" and "aryloxyalkyl" refer to aryl groups attached directly to an alkyl, alkenyl, or to an oxygen on an alkyl, respectively. For simplicity, as part of the above combined terms, aryl is also intended to include heteroaryl.

[0072] Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "C1-C6 haloalkyl" is intended to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0073] The term "hetero" as used in "heteroatom-containing alkyl" ("heteroalkyl") or "heteroatom-containing aryl" ("heteroaryl") refers to a molecule, bond or substituent in which one or more carbon atoms are replaced with non-carbon atoms, such as nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur, or those that are not non-carbon atoms at all (e.g., sulfonamide). Similarly, the term "heteroalkyl" refers to an alkyl substituent containing a heteroatom, the term "heterocycle" refers to a cyclic substituent containing a heteroatom, the terms "heteroaryl" and "heteroaromatic" refer to "aryl" and "aromatic" substituents containing heteroatoms, respectively, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated aminoalkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, and the like, and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, and the like.

[0074] The term "hydrophobic group" or "hydrophobic group" refers to a group that reduces the water solubility of a molecule. In some embodiments, the hydrophobic group is a group containing at least 3 carbon atoms.

[0075] The term "hetero" as used in "heteroatom" refers to any atom other than carbon or hydrogen, for example nitrogen, oxygen, sulfur, phosphorus or silicon.

[0076] The term "carboxylic acid analogue" refers to various groups having an acidic portion that can simulate a carboxylic acid residue. Examples of these groups are sulfonic acid, sulfinic acid, phosphoric acid, phosphonic acid, phosphinic acid, sulfonamide, and heterocyclic moieties such as, for example, imidazole, triazole, and tetrazole.

[0077] The term "substituted" means that one atom or an atom group of a compound is replaced by another atom or an atom group. For example, an atom or an atom group can be substituted by one or more of the following substituents or groups: halo, cyano, nitro, alkyl, alkylamino, hydroxyalkyl, haloalkyl, carboxyl, hydroxy, alkoxy, alkoxyalkoxy, haloalkoxy, sulfanyl, aryl, aryloxy, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl (which is optionally substituted by 1 or more, preferably 1 to 3 substituents selected from halo, haloalkyl and alkyl), aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, alk(ene / ynyl), halo, cyano, hydroxy, haloalkyl and polyhaloalkyl (preferably halogenated lower alkyl, especially trifluoromethyl), formyl, alkylcarbonyl, arylcarbonyl (which is optionally substituted by 1 or more, preferably 1 to 3 substituents selected from halo The term "substituted" may be substituted with alkyl, alkylsulfonyl ...

[0078] The term "unsubstituted" refers to a naturally occurring compound having no replacement atoms or groups of atoms.

[0079] Overview

[0080] In recent decades, 1,3-disubstituted ureas have emerged as one of the potent central pharmacophores of soluble epoxide hydrolases (sEHs). However, sEH inhibitors with 1,3-disubstituted ureas always suffer from poor solubility and high melting points, which make them difficult to formulate and have less druglikeness.

[0081] Solubility and melting point are important parameters that affect drug-like properties, and the ease of formulation is greatly affected by these two parameters. However, these two parameters are difficult to predict and change. In addition, in the past, modifications to these two parameters often had side effects on the potency of the inhibitor.

[0082] Inhibitors with flexible substituents on one end of 1,3-disubstituted ureas have lower melting points, which facilitates the drug formulation process. However, these inhibitors are generally metabolically unstable and have poor oral bioavailability. Therefore, inhibitors with restricted or rigid substituents have been synthesized, but they have much higher melting points (>180°C), which makes them poor drug candidates, and there are no reports of any specific modifications to reduce the melting point of the sEH inhibitors.

[0083] The binding pocket of sEH is very hydrophobic. Therefore, sEH inhibitors with good potency at the nM level always carry hydrophobic substituents at both ends of the 1,3-disubstituted urea. These substituents always lead to very poor water solubility of the inhibitors over a wide pH range. These properties lead to difficulties and many problems in drug formulation and always lead to poor bioavailability.

[0084] In order to improve the solubility of sEH inhibitors, polar substituents have been incorporated into sEH inhibitors. However, such incorporation often results in reduced potency against sEH.

[0085] It has been shown that residence time (t1 / 2) plays an important role in in vivo efficacy, but there are no reports on increasing the residence time of sEH inhibitors. sEH inhibitors with short alkyl chains have been reported to have short t1 / 2, which may explain their lack of in vivo efficacy in human clinical trials.

[0086] Inhibitors that can penetrate the blood-brain barrier are important for the treatment of neurological diseases. However, it is difficult to identify compounds that can cross the blood-brain barrier and compounds that are peripherally restricted because they cannot cross the blood-brain barrier.

[0087] The present invention is derived from the discovery that certain sEH inhibitors can be further functionalized to provide more potent sEH inhibitors with improved physical properties.

[0088] As described herein, the introduction of heterocycles into the inhibitors increased the aqueous solubility and oral availability of the sEH inhibitors. Further modifications were determined to lower the melting point and significantly increase the potency on the target.

[0089] Compound

[0090] In one aspect, the present invention provides a compound of structural formula (I), or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof.

[0091]

[0092] in:

[0093] R1 is selected from the group consisting of: trifluoromethyl- or trifluoromethoxy-;

[0094] R2 is selected from the group consisting of: H- or F-;

[0095] R3 is selected from the group consisting of: H-, trifluoromethyl- or -CH2CO2H;

[0096] X is selected from carbon, nitrogen or oxygen;

[0097] Y is selected from carbon, nitrogen or oxygen;

[0098] subscript m is 0, 1, or 2; and

[0099] Subscript n is 1 or 2.

[0100] In another aspect, the present invention provides a compound of structural formula (II), or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof.

[0101]

[0102] in:

[0103] X is selected from carbon or nitrogen;

[0104] Y is selected from carbon, nitrogen or oxygen;

[0105] R5 is selected from the group consisting of: trifluoromethyl- or trifluoromethoxy-;

[0106] R6 is selected from the group consisting of: H- or F-; and

[0107] R7 is selected from the group consisting of 1H-pyrrole, furan, pyran, tetrahydropyran, 4,5-dihydrofuran, 2-methylfuran, 3-methylfuran, ethyl, isopropyl, cyclopropyl, 2-methylbutyl and (S)-2-methylbutyl-.

[0108] The compounds include all pharmaceutically acceptable derivatives thereof, such as salts, prodrugs, soft drugs, solvates and hydrates.

[0109] Representative compounds of the invention are presented in Table I below.

[0110] Table I: sEH inhibitors (physical properties and potency relative to human sEH)

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] Abbreviations: Mol.Weight: molecular weight; Sol: solubility; K i : inhibition constant of affinity-purified recombinant human sEH; elogP: experimental log P; clogP: calculated log P; -: not determined.

[0120] a The elogP was determined by HPLC and calibrated with the eLogP of 4 selected inhibitors determined by the shake flask method.

[0121] b clogP was calculated by CLogPDriver of Cambridge Soft CLogP from ChemBioOffice 2012.

[0122] c K i is measured by a previously published FRET shift assay (Lee et al., Anal. Chem. 2013, see Example 53)

[0123] d The enzyme t 1 / 2 It was defined as the time required for the fluorescence signal of the drug separated from recombinant affinity purified human sEH to be reduced to half.

[0124] In exemplary embodiments, the present invention provides the following compounds from Table I: 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-isobutyrylpiperidin-4-yl)urea (Compound 19); 1-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (Compound 1); 1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (Compound 1). )-3-(3-fluoro-4-(trifluoromethoxy)phenyl)urea (Compound 24); (S)-1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(2-methylbutanoyl)piperidin-4-yl)urea (Compound 26); and 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea (Compound 29).

[0125] Certain embodiments of the present invention also provide pharmaceutical compositions comprising one or more compounds disclosed herein, together with a pharmaceutically acceptable carrier, and methods of making and using the compounds and compositions.

[0126] Compound preparation

[0127] The compounds of the present invention can be prepared by a variety of methods as summarized in the general synthetic schemes shown below, which outline three synthetic routes, each of which is discussed in further detail in Example 1.

[0128] General synthetic scheme

[0129] Synthesis route 1

[0130]

[0131] Synthesis Path 2

[0132]

[0133] Synthesis Path 3

[0134]

[0135] To improve solubility, heterocyclic rings were incorporated into the inhibitors at R7 (Formula II). However, interestingly, most heterocyclic rings were able to improve inhibitor solubility at pH 7.4 compared to the sEH inhibitors of Formula (II) having a hydrophobic alkyl group at R7 as shown in Formula (II) (Table II and Figure 3 ).

[0136] Table II: Compound Solubility (at pH 7 and pH 3)

[0137]

[0138]

[0139]

[0140] Abbreviations: Sol: solubility; K i : Inhibition constant; ND: Not determined

[0141] a The solubility of the drug was measured in phosphate buffer at pH 7.4.

[0142] b The solubility of the drug was measured in acetate buffer at pH 3.

[0143] Unlike all other heterocycles, incorporation of tetrahydropyran into R7 of sEH inhibitor (Formula (II)) can significantly improve the solubility of sEH inhibitor compared to sEH inhibitor with hydrophobic alkyl at R7 of Formula (II) (Table II and Figure 3 ; Compounds 1, 8, 27 and 29).

[0144] The enhanced solubility due to tetrahydropyran at R7 is surprising since it is not related to the dipole moment of the heterocycle or the experimental logP of the heterocycle ( Figure 1 and 2 ).

[0145] Surprisingly, compared to sEH inhibitors of formula (II) with alkyl substituents at R7 (ethyl, cyclopropyl, 2-sec-butyl), sEH inhibitors with heterocyclic rings showed at least a 3-fold improvement in solubility at pH 3 (the pH of gastric acid) compared to solubility at pH 7, while maintaining inhibitory potency in the single digit nanomolar level (Tables 1 and 2 and Figure 4 and 5 ).

[0146] In addition to other heterocycles, sEH inhibitors with tetrahydropyran at R7 (Formula (II)) showed unexpectedly enhanced solubility at pH 3. The solubility of the inhibitors with tetrahydropyran at R7 at acidic pH 3 was at least 5 times higher than any other sEH inhibitors with alkyl at R7 of Formula (II), and several of these inhibitors (Compounds 1, 8, and 27) were much better at pH 3 than the most soluble 1,3-disubstituted urea sEH inhibitor (Compound APAU) (Table II and Table II). Figure 4 ). This improves the drug-likeness of this series of sEH inhibitors. Because the stomach, one of the main organs for drug absorption, has a highly acidic environment. The exceptionally improved solubility of inhibitors with tetrahydropyran at pH 3 improves their absorption and oral bioavailability, meeting an important need in this field.

[0147] Thus, the present invention provides a method for increasing the water solubility of compounds of formula (I) by incorporating a heteroatom at X.

[0148] In a related aspect, the invention provides a method for increasing the water solubility of a compound of formula (II) by incorporating a heterocyclic ring at R7.

[0149] Modification of the inhibitor structure usually has a small and significant stochastic effect on the melting point of the compound. However, unexpectedly and surprisingly, the addition of fluorine atoms at R2 of Formula I and at R6 of Formula II lowered the melting point of this series of sEH inhibitors by an average of about 20°C, while improving the potency (Tables I and III). Melting point reduction not only eases the formulation process of the drug but also generally improves the oral bioavailability of the drug and reduces the problem of stabilizing polymorphs. Therefore, improvements that reduce high melting points have been explored in the industry.

[0150] Table III: Melting points of compounds (Incorporation of fluorine at the R2 position of formula (I) or at the R6 position of formula (II) reduces the melting point of sEH inhibitors (°C))

[0151]

[0152]

[0153] Abbreviations: Sol: solubility

[0154] a Melting point change = melting point of the inhibitor with meta-F substitution on the aromatic urea - melting point of the original inhibitor.

[0155] b Potency enhancement = K of original inhibitor i / m-F substituted inhibitor K i .

[0156] Fluorine substitution at R6 of formula (II) is unique in that substitutions including halogens other than fluorine at R6 of formula (II) all increase the melting point of the inhibitor, and most of those substitutions hinder the potency of the inhibitor (Tables I and Figure 6 ).

[0157] Furthermore, in this case, the addition of fluorine at R2 of formula (I) or at R6 of formula (II) increased the potency of the sEH inhibitor by about 2-fold (Table I). Thus, by increasing potency on the target, this modification reduces crystal stability and increases ease of formulation (as indicated by a decrease in melting point).

[0158] Thus, the present invention provides a method for lowering the melting point of compounds of formula (I) by incorporating fluorine at R2.

[0159] In a related aspect, the present invention provides a method for lowering the melting point of a compound of formula (II) by incorporating fluorine at R6.

[0160] The combination of these different approaches to improve the inhibitor, i.e., incorporation of a fluorine atom at R6 of formula (II) and tetrahydropyran at R7, resulted in surprisingly strong, sub-nanomolar potency and remarkably high aqueous solubility (>100 μg / mL) (compound 27, Table 2 and Figure 3 and 4 ) and a lower melting point (Table III). These modifications significantly improved the drug-likeness of the inhibitors compared to all previous sEH inhibitors.

[0161] pH titration of compounds 1 and 29 showed a significant increase in solubility when the pH was below 4 ( Fig. 9 ).

[0162] Thus, the present invention provides a method for increasing the aqueous solubility of a compound of formula (I) in the pH range of 1-7 by incorporating oxygen at X and the subscript m is 2 and n is 2.

[0163] In a related aspect, the present invention provides a method for increasing the aqueous solubility of a compound of formula (II) in the pH range of 1-7 by incorporating a heterocyclic ring at R7.

[0164] In embodiments, the pH ranges from about 1-7, 2-5, 2-4, 3-4, or about 1, 2, 3, 4, 5, 6, or 7.

[0165] The incorporation of a bulky alkyl group at R7 of Formula II and a trifluoromethoxy group at R5 (e.g., compound 26) resulted in an inhibitor with a longer retention time (t1 / 2 -20 min) than APAU (t1 / 2 -6 min), which demonstrated better in vivo efficacy, the values ​​of the validation method being disclosed herein (Table I).

[0166] Thus, the present invention provides a method for increasing the retention time (t1 / 2) of compounds of formula (I) to soluble epoxide hydrolases by incorporating a trifluoromethoxy-group at R1.

[0167] Table IV: Melting points and K values ​​of compounds i (Incorporation of a substituent other than fluorine at the R2 position of formula (I) or at the R6 position of formula (II) increases the melting point (°C) of the sEH inhibitor)

[0168]

[0169]

[0170] aMelting point change = melting point of the inhibitor when there is substitution on the aromatic urea - melting point of the original inhibitor.

[0171] The compounds of the present disclosure were determined to protect mice from convulsions and associated lethality as determined by a standard chemically induced seizure assay, indicating that the compounds of the present invention are capable of crossing the blood-brain barrier of the subject (Examples 52 and Figure 7 ).

[0172] As discussed in Example 54 below, in a pain response assay using type I diabetic rats, the compounds of the present disclosure outperformed the registered standard drug gabapentin for neuropathic pain and the standard sEH inhibitor (compound APAU). The compounds of the present disclosure reached high efficacy levels more quickly. In addition, the compounds of the present disclosure showed higher potency and higher efficacy compared to the standards in the art ( Figure 6 ).

[0173] Furthermore, compounds of the present disclosure exhibit significant efficacy in epileptic seizure assays due to the compounds' ability to cross the blood-brain barrier, thereby readily penetrating the CNS and protecting subjects from epileptic seizures (Example 52).

[0174] Methods and therapeutic uses

[0175] Administration of sEH inhibitors has been found to have many beneficial applications, as described in U.S. Pat. Nos. 8,815,951, 8,513,302, 8,399,425, 8,501,783, 7,662,910, and U.S. Patent Publication Nos. 2015 / 0065540 and 2015 / 0017267, which are incorporated herein by reference in their entirety.

[0176] Therefore, the present invention provides a method for inhibiting a soluble epoxide hydrolase, comprising contacting the soluble epoxide hydrolase with an inhibitory amount of a compound having the above formula (I) or (II).

[0177] Preferably, the inhibitor inhibits sEH without significantly inhibiting microsomal epoxide hydrolase ("mEH"). Preferably, at a concentration of 500 μM, the inhibitor inhibits sEH activity by at least 50% without inhibiting mEH activity by more than 10%. Preferred compounds have an IC of 50 (inhibitory potency or, by definition, the concentration of inhibitor that reduces enzyme activity by 50%) is less than about 500 μM. Preferably, IC 50 Less than 500 μM of the inhibitor, along with the IC 50 Lower the preferred IC 50 Less than 200 μM, more preferably 100 μM, and more preferably IC 5050 μM, 40 μM, 30 μM, 25 μM, 20 μM, 15 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM or even less. Assays for determining sEH activity are known in the art and described elsewhere herein.

[0178] The compounds disclosed herein may exist as therapeutically acceptable salts. The present invention includes compounds listed above in the form of salts, including acid addition salts. Suitable salts include those formed with organic and inorganic acids. These acid addition salts are generally pharmaceutically acceptable. However, salts that are not pharmaceutically acceptable salts may be utilized in the preparation and purification of the compounds in question. Base addition salts may also be formed and are pharmaceutically acceptable. For a more complete discussion of the preparation and selection of salts, reference is made to Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P. Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

[0179] Thus, the present invention provides a method of treating a sEH-mediated disease or condition as defined herein. The method comprises administering to a subject in need of such treatment an effective amount of a compound having a formula selected from formula (I) or (II). In one aspect, the effective amount is a therapeutically effective amount.

[0180] As used herein, the term "therapeutically acceptable salt" means a salt or zwitterionic form of a compound disclosed herein, which is water or oil soluble or dispersible and is acceptable for treatment as defined herein. Salts can be prepared in the final separation and purification of the compound or prepared separately by reacting the appropriate compound in the form of a free base with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate (benzenesulfonate / besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate, hemisulfate, enanthate, caproate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (hydroxyethyl sulfonate) , lactate, maleate, malonate, racemic mandelate, mesitylene sulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphonate, picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate, tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate (para-toluenesulf onate / p-tosylate), and undecanoate. Meanwhile, the basic groups in the compounds disclosed herein can be methyl, ethyl, propyl and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfate; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides quaternized. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. Salts can also be formed by coordination with alkali metal or alkaline earth metal ions. Thus, the present invention encompasses sodium, potassium, magnesium and calcium salts of the compounds disclosed herein, and the like.

[0181] Base addition salts can be prepared by reacting a carboxyl group with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a metal cation, or with ammonia or an organic primary, secondary, or tertiary amine in the final separation and purification of the compound. The cation of the therapeutically acceptable salt includes lithium, sodium, potassium, calcium, magnesium, and aluminum, and nontoxic quaternary ammonium cations, such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N, N- dimethylaniline, N- methylpiperidine, N- methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N, N- dibenzylphenethylamine, 1- ephedrine (1-ephenamine) and N, N'- dibenzylethylenediamine. Other representative organic amines that can be used to form base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine, etc.

[0182] Although the compounds of the present invention may be administered as raw chemicals, they may also be provided as pharmaceutical preparations. Therefore, pharmaceutical preparations are provided herein, which include one or more certain compounds disclosed herein, or one or more pharmaceutically acceptable salts, esters, prodrugs, amides, or solvates thereof, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the preparation, and not harmful to its recipient. Suitable formulations depend on the selected route of administration. Any well-known technology, carrier, and excipient can be used appropriately and are understood in the art, such as Remington's Pharmaceutical Sciences. The pharmaceutical compositions disclosed herein can be manufactured in any manner known in the art, for example, by means of conventional mixing, dissolving, granulating, making dragees, grinding, emulsifying, encapsulating, encapsulating or compressing methods.

[0183] The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including skin, buccal, sublingual and intraocular) administration, although the most suitable route may depend, for example, on the condition and disorder of the recipient. The formulations can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. In general, these methods include the steps of combining a compound of the invention or a pharmaceutically acceptable salt, ester, amide, prodrug or solvate thereof ("active ingredient") with a carrier that constitutes one or more additional ingredients. In general, the formulations are prepared by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier or a carrier of both, and then, if necessary, shaping the resulting product into the desired formulation.

[0184] Formulations of the compounds disclosed herein suitable for oral administration can be provided in the form of discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be provided as a bolus, electuary or paste.

[0185] Pharmaceutical preparations that can be used orally include tablets, push fit capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Tablets can be prepared by compression or molding, optionally with one or more additional ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, an inert diluent or a lubricant, a surfactant or a dispersant. Molded tablets can be formed by molding a mixture of a powdered compound moistened with an inert liquid diluent using a suitable machine. The tablets may optionally be coated or scored and may be formulated to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should have a dosage suitable for this mode of administration. The push fit capsules may contain the active ingredient in admixture with a filler such as lactose; a binder such as starch; and / or a lubricant such as talc or magnesium stearate; and optionally a stabilizer. In soft capsules, the active compound can be dissolved in or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycol. In addition, a stabilizing agent can be added. The core of the dragee has a suitable coating. For this purpose, a concentrated sugar solution can be used, which can optionally contain gum arabic, talcum, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture. Dye or pigment can be added to tablet or dragee coating to identify or characterize the different combinations of active compound dosage.

[0186] The compound can be formulated into a parenteral administration form for injection, such as bolus injection or continuous infusion. The preparation for injection can be a unit dose form, such as in an ampoule or a multi-dose container, and a preservative is added. The composition can be in the following form, such as a suspension, solution or emulsion dissolved in an oily or aqueous vehicle, and can contain a formulation agent such as a suspending agent, a stabilizer and / or a dispersant. The preparation can be provided in a unit dose or multi-dose container, such as a sealed ampoule and a vial, and can be stored in a powder form or under lyophilized conditions, and only a sterile liquid carrier, such as saline or sterile pyrogen-free water, needs to be added before use. Ready-to-use injection solutions and suspensions can be prepared from previously described sterile powders, granules and tablets, etc.

[0187] Preparations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compound, which may contain antioxidants, buffers, bacteriostats, and solutes that can make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of high concentration solutions.

[0188] In addition to the aforementioned preparations, the compound can also be formulated as a depot preparation. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Therefore, for example, the compound can be formulated with a suitable polymer or hydrophobic substance (e.g., an emulsion in an acceptable oil) or an ion exchange resin, or a slightly soluble derivative (e.g., a slightly soluble salt).

[0189] For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, pastilles, or gels formulated in conventional manner. These compositions may contain the active ingredient on a flavored basis such as sucrose and acacia or tragacanth.

[0190] The compounds may also be formulated in rectal compositions such as suppositories or sustained release enemas, eg, containing conventional suppository bases such as cocoa butter, polyethylene glycol, or other glycerides.

[0191] Certain compounds disclosed herein may be administered topically, i.e., by non-systemic administration. This includes external application of compounds disclosed herein to the epidermis or buccal cavity, as well as instillation of such compounds into the ears, eyes, and noses, so that the compounds do not enter the bloodstream significantly. In contrast, systemic administration refers to oral, intravenous, intraperitoneal, and intramuscular administration.

[0192] Preparations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration into the skin to the inflamed site (such as gels, liniments, lotions, creams, ointments or pastes) and drops suitable for eye, ear or nose administration. The active ingredient for topical administration may comprise, for example, 0.001% to 10% w / w (by weight) of the preparation. In certain embodiments, the active ingredient may comprise up to 10% w / w. In other embodiments, it may comprise less than 5% w / w. In certain embodiments, the active ingredient may comprise 2% w / w to 5% w / w. In other embodiments, it may comprise 0.1% to 1% w / w of the preparation.

[0193] In addition to the active ingredient, the topical eye, ear and nose preparations of the present invention may also include excipients. Excipients commonly used in these preparations include, but are not limited to, tonicity agents, preservatives, chelating agents, buffers and surfactants. Other excipients include solubilizers, stabilizers, comfort enhancers, polymers, softeners, pH-adjusters and / or lubricants. Any of the various excipients that can be used in the preparations of the present invention include water, water and water-miscible solvents, such as mixtures of C1-C7 alkanols, vegetable oils or mineral oils, containing 0.5% to 5% non-toxic water-soluble polymers, natural products such as alginates, pectins, tragacanth gum, karaya gum, guar gum, xanthan gum, carrageenan, agar and gum arabic, starch derivatives such as starch acetate and hydroxypropyl starch, and other synthetic products such as polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid and mixtures of these products. The concentration of the excipient is generally 1 to 100,000 times the concentration of the active ingredient. In preferred embodiments, the excipients included in the formulation are generally selected based on their inertness toward the active ingredient components of the formulation.

[0194] For ocular, otic and nasal preparations, suitable tonicity adjusting agents include, but are not limited to, mannitol, sodium chloride, glycerol, sorbitol and the like. Suitable buffers include, but are not limited to, phosphates, borates, acetates and the like. Suitable surfactants include, but are not limited to, ionic and nonionic surfactants (however, nonionic surfactants are preferred), RLM 100, POE 20 cetylstearyl ether, such as CS20 and poloxamer, such as F68.

[0195] The formulations described herein may include one or more preservatives. Examples of such preservatives include parabens, sodium perborate, sodium chlorite, alcohols such as chlorobutanol, benzyl alcohol or phenylethyl alcohol, guanidine derivatives such as polyhexamethylene biguanide, sodium perborate, polyquaternium-1, amino alcohols such as AMP-95, or sorbic acid. In certain embodiments, the formulations may be self-preserving, so no preservative is required.

[0196] For administration to the eye, ear or nose, the formulation may be a solution, suspension or gel. In a preferred aspect, the formulation is topically applied to the eye, nose or ear in the form of an aqueous solution in the form of drops. The term "aqueous" generally refers to an aqueous formulation wherein the formulation is greater than 50%, more preferably greater than 75%, and in particular greater than 90% by weight of water. These drops may be delivered in single-dose ampoules, which may preferably be sterile and therefore do not require the provision of antibacterial components of the formulation. Alternatively, the drops may be delivered from a multi-dose bottle, which may preferably include a device that extracts any preservative from the delivered formulation, such as a device well known in the art.

[0197] For ocular conditions, the components of the invention may be delivered to the eye as a concentrated gel or similar vehicle or as a dissolvable insert placed under the eyelid.

[0198] The preparations of the present invention suitable for topical administration to the eye are preferably isotonic or slightly hypotonic to counteract any hyperosmotic properties of the tears due to evaporation and / or disease. This may require a tonicity agent to bring the osmotic pressure concentration of the preparation to a level of or approaching 210-320 milliosmoles / kilogram (mOsm / kg). The preparations of the present invention generally have an osmotic pressure concentration in the range of 220-320 mOsm / kg, and preferably have an osmotic pressure concentration in the range of 235-300 mOsm / kg. The ocular preparations are generally formulated as sterile aqueous solutions.

[0199] In certain ocular embodiments, the compositions of the present invention are formulated with one or more tear substitutes. Various tear substitutes are known in the art and include, but are not limited to: monomeric polyols such as glycerol, propylene glycol, and ethylene glycol; polymeric polyols such as polyethylene glycol; cellulose esters such as hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl cellulose; dextran such as dextran 70; vinyl polymers such as polyvinyl alcohol; and carbomers such as carbomer 934P, carbomer 941, carbomer 940, and carbomer 974P. Certain formulations of the present invention may be used with contact lenses or other ophthalmic products.

[0200] Preferably the formulation is prepared using a buffer system that maintains the formulation at about pH 4.5 to about pH 8. Most preferably the formulation pH is from 6 to 8.

[0201] In a specific embodiment, the preparation of the present invention is applied once a day. However, the preparation may also be prepared for use at any frequency of application, including once a week, once every 5 days, once every 3 days, once every 2 days, twice a day, three times a day, four times a day, five times a day, six times a day, eight times a day, once an hour, or any higher frequency. This frequency of application may also be maintained different durations according to the treatment regimen. The specific treatment regimen duration may be changed between a single application to a plan that continues for several months or years. The preparation is applied in different doses, but a typical dosage is one to two drops in each application, or a considerable amount of gel or other preparations. Those of ordinary skill in the art will be skilled in determining the treatment regimen for specific indications.

[0202] Topical or transdermal gels may generally include a mixture of volatile solvents, non-volatile solvents and water. In certain embodiments, the volatile solvent component of the buffered solvent system may include lower (C1-C6) alkyl alcohols, lower alkyl glycols and lower glycol polymers. In further embodiments, the volatile solvent is ethanol. The volatile solvent component is believed to be used as a penetration enhancer, while also producing a cooling effect on the skin when it evaporates. The non-volatile solvent portion of the buffered solvent system is selected from lower alkylene glycols and lower glycol polymers. In certain embodiments, propylene glycol is used. The non-volatile solvent slows down the evaporation of the volatile solvent and reduces the vapor pressure of the buffered solvent system. The amount of this non-volatile solvent component, like the volatile solvent, is determined by the pharmaceutical compound or drug used. When there is too little non-volatile solvent in the system, the pharmaceutical compound may crystallize due to the evaporation of the volatile solvent, while an excess may result in a lack of bioavailability due to poor drug release in the solvent mixture. The buffer component of the buffered solvent system may be selected from any buffer commonly used in the art; in certain embodiments, water is used. Typical ratios of ingredients are about 20% non-volatile solvent, about 40% volatile solvent, and about 40% water. There are several optional ingredients that can be added to the topical composition. These include, but are not limited to, chelating agents and gelling agents. Suitable gelling agents can include, but are not limited to, semisynthetic cellulose derivatives (such as hydroxypropyl methylcellulose), synthetic polymers, galactomannan polymers (such as guar gum and its derivatives), and cosmetic agents.

[0203] Lotions include those suitable for application to the skin or eyes. Eye lotions may include a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those used to prepare drops. Lotions or liniments applied to the skin may also include an agent that accelerates drying and cools the skin, such as ethanol or acetone and / or a moisturizer, such as glycerol or an oil, such as castor oil or peanut oil.

[0204] Creams, ointments or pastes are semisolid preparations of active ingredients for external use. They can be prepared by mixing the active ingredients in finely divided or powdered form, either alone or in a solution or suspension in an aqueous or non-aqueous fluid, using an oleaginous or non-oleaginous base with the aid of a suitable machine. The base may include: hydrocarbons, such as hard, soft or liquid paraffin, glycerine, beeswax, metallic soaps; mucilages; oils of natural origin, such as almond oil, corn oil, peanut oil, castor oil or olive oil; lanolin or its derivatives or fatty acids, such as stearic acid or oleic acid with alcohols, such as propylene glycol or macrogranules. The preparation may incorporate any suitable surfactant, such as anionic, cationic or nonionic surfactants, such as sorbitan esters or their polyethylene oxide derivatives. Suspending agents, such as natural gums, cellulose derivatives or inorganic materials, such as silicate silicas and other ingredients, such as lanolin, may also be included.

[0205] Drops can contain sterile water or oily solutions or suspensions and can be prepared by dissolving the active ingredient in a bactericide and / or fungicide and / or any other suitable preservative, and in a suitable aqueous solution including a surfactant in certain embodiments. Then, the resulting solution can be clarified by filtration, transferred to a suitable container, which is subsequently sealed and sterilized by autoclaving or maintained at 98-100°C for half an hour. Alternatively, the solution can be sterilized by filtration and transferred to a container by aseptic technique. Examples of bactericides and fungicides suitable for inclusion in drops are phenylmercuric nitrate or phenylmercuric acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for preparing oily solutions include glycerol, dilute alcohols and propylene glycol.

[0206] Formulations for topical administration in the mouth, e.g., buccal or sublingually, include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.

[0207] For administration by inhalation, the compound can be conveniently delivered by an insufflator, atomizer pressurized package or other convenient means of delivering an aerosol spray. The pressurized package may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the compound according to the present invention can be prepared in the form of a dry powder composition, for example a powder mixture of the compound and a suitable powder base (such as lactose or starch). The powder composition can be provided in the form of a unit dose, for example, in a capsule, cartridge, gelatin or blister package, from which the powder can be administered by means of an insufflator or insufflator.

[0208] Preferred unit dosage formulations are those containing an effective dose, as herein below recited, or an appropriate fraction thereof, of the active ingredient.

[0209] It should be understood that in addition to the ingredients particularly mentioned above the formulations described above may include other agents conventional in the art having the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0210] The compound can be administered orally or by injection at a dosage of 0.1 to 500 mg / kg per day. The dosage range for adults is generally 5 mg to 2 g / day. Tablets or other forms of presentation provided in discrete units can conveniently contain a certain amount of one or more compounds, which are effective at such dosages or as multiple identical dosages, for example, units containing 5 mg to 500 mg, usually about 10 mg to 200 mg.

[0211] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

[0212] The compound can be administered in various modes, such as orally, topically, or by injection. The exact amount of the compound administered to the patient will be the responsibility of the attending physician. The specific dosage level for any particular patient will depend on various factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, the exact condition being treated, and the severity of the indication or condition being treated. The route of administration may also vary according to the condition and its severity.

[0213] In some cases, it may be appropriate to administer at least one of the compounds described herein (or a pharmaceutically acceptable salt, ester, or prodrug thereof) in combination with another therapeutic agent. By way of example only, if one of the side effects experienced by a patient receiving one of the compounds herein is hypertension, then an antihypertensive agent may be appropriately administered in combination with the initial therapeutic agent. Or, by way of example only, the therapeutic effect of one of the compounds described herein may be enhanced by administering an adjuvant (i.e., an adjuvant that has only a small therapeutic benefit on its own, but when combined with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Or, by way of example only, the benefit experienced by the patient may be increased by administering one of the compounds described herein with another therapeutic agent (which also includes a treatment regimen) that also has a therapeutic benefit. By way of example only, in the treatment of diabetic neuropathic pain that includes administering one of the compounds described herein, an increased therapeutic benefit may be obtained by also providing the patient with another diabetes therapeutic agent. In any case, regardless of the disease, disorder, or condition being treated, the overall benefit experienced by the patient may be the simple addition of the two therapeutic agents or the patient may experience a synergistic benefit.

[0214] In any case, multiple therapeutic agents (at least one of which is a compound disclosed herein) can be administered in any order or even simultaneously. If administered simultaneously, the multiple therapeutic agents can be provided in a single, unified form, or in multiple forms (by way of example only, as a single pill or as two separate pills). One of the therapeutic agents can be provided in multiple doses, or both compounds can be provided in multiple doses. If not simultaneously, the time between multiple doses can be of any duration, ranging from a few minutes to four weeks.

[0215] The following examples are provided to further illustrate embodiments of the present invention, but are not intended to limit the scope of the present invention. Although they are typical examples that can be used, other procedures, methods or techniques known to those skilled in the art can be substituted.

[0216] Example

[0217] Example 1-51

[0218] All reagents and solvents were purchased from commercial suppliers and used directly without further purification. Unless otherwise stated, all syntheses were performed in a dry nitrogen atmosphere. Reactions were determined by thin layer chromatography (TLC, Merck F 254 On silica gel 60 aluminum plates, the spots are visible under lamplight or UV light (254 nm) or monitored by staining with an oxidizing solution (KMnO4 staining). The purity is tested using the same TLC system, and all final products appear as a single spot on the TLC. Column chromatography is performed with silica gel.

[0219] 1 H-NMR spectra were recorded on a Varian QE-300 spectrometer using deuterated chloroform (CDCl3; δ = 7.24 ppm) or deuterated dimethyl sulfoxide (DMSO-d6) containing TMS as an internal standard. 13 C-NMR spectra were performed on a Varian QE-300 TM The spectrometer was recorded at 75 MHz.

[0220] The purity of the inhibitors reported herein was determined by the following methods: 1) HPLC-UV using an Agilent 1200 TM Series HPLC Series, equipped with Phenomenex TMLuna2C18 reverse phase column (C18, 4.6mm×150mm, 5μm), coupled to AgilentG1314 UV-vis detector (detection at 230nm), 90 minutes under isocratic flow of methanol: water (2:1 by volume); or 2) H-NMR. The lowest purity obtained is reported. The inhibitor was dissolved in EtOH at 100μM and 10μL was injected on HPLC. Purity is based on the percentage of total peak area at 230nm using HPLC-UV. The purity estimate is compared with the value from H-NMR. The aniline present in the final product is estimated from H-NMR. The lowest purity obtained is reported. Purity is further supported by the following techniques as described in the supplementary materials: LC / MS monitoring total ion flow, TLC in several systems, clear melting points and other occasional techniques. Elemental analysis was performed by LCC of MIDWESTMICRO Laboratory.

[0221] The syntheses of tert-butyl 4-(3-(4-(trifluoromethyl)phenyl)ureido)piperidine-1-carboxylate, 1-(piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea, 1-(piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, and tert-butyl 4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidine-1-carboxylate have been previously reported.

[0222] Example 1

[0223] General synthetic scheme for sEH inhibitors

[0224] The compounds of the present invention are prepared by a variety of methods as outlined in the general synthetic schemes below.

[0225] General synthetic scheme

[0226] Synthesis route 1

[0227]

[0228] Synthesis Path 2

[0229]

[0230] Synthesis Path 3

[0231]

[0232] Synthesis route 1

[0233] Step 1

[0234] The corresponding isocyanate (1 equivalent) and 4-amino-1-Boc-piperidine (1.1 equivalents) are dissolved in CH2Cl2 (50mM, corresponding to isocyanate) and stirred at room temperature for 12 hours. The reaction is quenched by adding water. The organic layer is separated and the water layer is extracted 4 times with EtOAc (EtOAc: water layer / 1: 1 (v: v)). The organic layer merged is dried over anhydrous magnesium sulfate and concentrated under vacuum, and further purified by flash chromatography to obtain the corresponding Boc-protected urea.

[0235] Step 2

[0236] The Boc protected urea from step 1 is dissolved in HCl solution (2M, MeOH) to make a reaction mixture (186mM, Boc protected urea). The resulting solution is refluxed for 2 hours. Remove solvent under vacuum and adjust the crude reaction product to pH 12 with NaOH. Filter the precipitation and dry under high vacuum. The final unprotected urea is used as the skeleton for the next step synthesis.

[0237] Step 3

[0238] Unless otherwise specified, unprotected urea (1 eq.), EDCI (1.5 eq.), DMAP (1.5 eq.) and the corresponding carboxylic acid (1.5 eq.) from step 2 were dissolved in CH2Cl2 (8.3 mM, unprotected urea) and stirred at room temperature overnight (12 hours). The reaction was quenched by adding HCl solution (1 M). The organic layer was collected and the aqueous layer was extracted 4 times with EtOAc (EtOAc: aqueous layer / 1: 1 (v: v)). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated under vacuum and further purified by flash chromatography.

[0239] Synthesis Path 2

[0240] Step 1

[0241] The corresponding amine (1 equivalent) and triethylamine (1.5 equivalents) were dissolved in CH2Cl2 (54mM corresponding to amine) and stirred at -78°C. Triphosgene (0.45 equivalent) dissolved in CH2Cl2 (20mM, corresponding triphosgene) was added dropwise at -78°C. The reaction was then warmed to room temperature and stirred for 30 minutes. The reaction was cooled to 0°C. The corresponding piperidine (1.5 equivalents) and triethylamine (1.5 equivalents) dissolved in CH2Cl2 (54mM, corresponding piperidine) were slowly added and the reaction was further stirred at room temperature for 12 hours. The reaction was quenched by adding HCl solution (2M). The organic layer was collected and the aqueous layer was further extracted 3 times with EtOAc (EtOAc: aqueous layer / 1:1). The combined organic layer was washed with saturated NaCl solution. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The product can be used without further purification.

[0242] Step 2

[0243] The Boc protected urea from step 1 is dissolved in HCl solution (2M, MeOH) to make a reaction mixture (186mM, Boc protected urea). The resulting solution is refluxed for 2 hours. Remove solvent under vacuum and adjust the crude reaction product to pH 12 with NaOH. Filter the precipitation and dry under high vacuum. The final unprotected urea is used as the skeleton for the next step synthesis.

[0244] Step 3

[0245] Unless otherwise specified, unprotected urea (1 eq.), EDCI (1.5 eq.), DMAP (1.5 eq.) and the corresponding carboxylic acid (1.5 eq.) from step 2 were dissolved in CH2Cl2 (8.3 mM, unprotected urea) and stirred at room temperature overnight (12 hours). The reaction was quenched by adding HCl solution (1 M). The organic layer was collected and the aqueous layer was extracted 4 times with EtOAc (EtOAc: aqueous layer / 1: 1 (v: v)). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated under vacuum and further purified by flash chromatography.

[0246] Synthesis Path 3

[0247] The corresponding amine (1 equivalent) and triethylamine (1.5 equivalents) are dissolved in CH2Cl2 (54mM corresponding to amine) and stirred at -78°C. Triphosgene (0.45 equivalent) dissolved in CH2Cl2 (20mM, corresponding triphosgene) is added dropwise at -78°C. The reaction is then warmed to room temperature and stirred for 30 minutes. The reaction is cooled to 0°C. The corresponding piperidine (1.5 equivalents) and triethylamine (1.5 equivalents) dissolved in CH2Cl2 (54mM, corresponding piperidine) are slowly added and the reaction is further stirred at room temperature for 12 hours. The reaction is quenched by adding HCl solution (2M). The organic layer is collected and the water layer is further extracted 3 times with EtOAc (EtOAc: water layer / 1:1). The combined organic layer is washed with saturated NaCl solution. The organic layer is dried over anhydrous magnesium sulfate and concentrated in vacuo. The product is purified by flash chromatography.

[0248] Example 2

[0249] Synthesis of tert-butyl 4-(3-(3-fluoro-4-(trifluoromethoxy)phenyl)ureido)piperidine-1-carboxylate (A)

[0250] plan

[0251]

[0252] Meta-fluoro-4-(trifluoromethoxy)aniline (500mg, 2.56mmol) and triethylamine (388mg, 3.84mmol) are dissolved in CH2Cl2 (4mL) and triphosgene (341mg, 1.15mmol) solution dissolved in CH2Cl2 (5mL) is added dropwise at -78°C. The reaction mixture is stirred at 0°C for 1 hour and then cooled to -78°C. 4-amino-1-Boc-piperidine (769mg, 3.84mmol) and triethylamine (388mg, 3.84mmol) are dissolved in CH2Cl2 (4mL) and the suspension is added dropwise at -78°C to the reaction mixture. The reaction mixture is stirred at room temperature for 2 hours. The reaction is quenched by adding water. The organic layer is separated and the organic layer is further washed 4 times by HCl solution (1M). The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to give the final crude product (1.05 g, purity 86%, 2.13 mmol, yield 83.4%). Impurity 38 was purified by column chromatography using EtOAc:Hex (1:1).

[0253] 1 H NMR (d6-DMSO, 300Mhz): A: (s,1H),7.66(dd,J=13.5,2.4Hz,1H),7.38(t,J=8.1Hz,1H),7.10(d,J=9Hz,1H),6.33(d,J=7.5Hz,1H),3.81(d,J =12.9Hz,2H),3.6-3.8(m,1H),2.8-3.0(m,2H),1.78(dd,J=12.3Hz,3.3Hz,2H),1.40(s,9H),1.2-1.4(m,2H); 38: (s,1H),7.69(dd,J=14.9,2.4Hz,1H),7.46(t,J=9Hz,1H),7.2-7.3(m,1H)

[0254] Example 3

[0255] Synthesis of 1-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0256] plan

[0257]

[0258] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) was reacted with tetrahydro-2H-pyran-4-carboxylic acid (51.5 mg, 396 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The product was further purified by recrystallization from methanol and water. White crystals were collected by filtration.

[0259] Yield: 56 mg, 135 μmol, yield 51%. Purity (H-NMR): ≥95%

[0260] 1 H NMR (d6-DMSO, 600Mhz): (s,1H),7.46-7.48(m,2H),7.21(d,J=9Hz,2H),6.24(d,J=7.8Hz,1H),4.19 (d,J=13.2Hz,1H),3.88(d,J=13.8Hz,1H),3.83(m,2H),3.70-3.72(m,1H),3 .36-3.40(m,2H),3.16(t,J=12Hz,1H),2.85-2.90(m,1H),2.76-2.80(m,1H) ,1.79-1.89(m,2H),1.55-1.62(m,2H),1.48-1.52(m,2H),1.20-1.32(m,2H)

[0261] Example 4

[0262] Synthesis of 1-(1-(1H-pyrrole-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0263] plan

[0264]

[0265] The reaction was carried out according to Synthesis Path 1, Step 3. Piperidine B (80 mg, 264 μmol) was reacted with tetrahydro-2H-pyran-4-carboxylic acid (51.5 mg, 396 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0266] Yield: 61 mg, 154 μmol, yield 58.3%. Purity (H-NMR): ≥95%

[0267] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),8.57(s,1H),7.47(d,J=9.6Hz,2H),7.21(d,J=9Hz,2H),7.08(d,J=1.2Hz,1H),6.75-6.77(m,1H),6.22- 6.26(m,2H),4.19(d,J=13.5Hz,2H),3.72-3.74(m,1H),3.08-3.12(m,2H),1.83-1.86(m,2H),1.26-1.37(m,2H)

[0268] Example 5

[0269] Synthesis of 1-(1-(furan-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0270] plan

[0271]

[0272] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) was reacted with tetrahydro-2H-pyran-4-carboxylic acid (51.5 mg, 396 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water.

[0273] Yield: 64 mg, 161 μmol, yield 61%. Purity (H-NMR): ≥95%

[0274] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),8.02(s,1H),7.74(s,1H),7.47(d,J=9.6Hz,2H),7.21(d,J=9Hz,2H),6.65(s,1H),6.26(d,J=6. 9Hz,2H),3.8-4.4(br,2H),3.74-3.65(m,1H),2.9-3.2(br,2H),1.87(d,J=9.9Hz,2H),1.29-1.40(m,2H)

[0275] Example 6

[0276] Synthesis of (R)-1-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0277] plan

[0278]

[0279] The reaction is carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) is reacted with (R)-tetrahydrofuran-3-carboxylic acid (46.2 mg, 396 μmol). The product is purified by flash chromatography and eluted with ethyl acetate. The collected fractions are dried in vacuo to obtain a white solid. The product is further purified by recrystallization using methanol and water.

[0280] Yield: 58 mg, 145 μmol, yield 55%. Purity (H-NMR): ≥95%

[0281] 1 H NMR (d6-DMSO, 300Mhz): (d,J=5.4Hz,1H),7.47(d,J=9.6Hz,2H),7.21(d,J=9Hz,2H),6.23-6.26(m,1H),4.65(t,J=6.3Hz,1H),4.05-4.10(m,1H),3.89(d ,J=14.7Hz,1H),3.6-3.8(m,3H),3.15(q,J=14.7Hz,1H),2.81(q,J=15.3Hz,1H),1.9-2.1(m,2H),1.7-1.9(m,4H),1.2-1.4(m,2H)

[0282] Example 7

[0283] Synthesis of 1-(1-(4,5-dihydrofuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0284] plan

[0285]

[0286] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) was reacted with tetrahydro-2H-pyran-4-carboxylic acid (45.4 mg, 396 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (8:2). The collected fractions were dried in vacuo to give a white solid.

[0287] Yield: 68 mg, 170 μmol, yield 65%. Purity (H-NMR): ≥95%

[0288] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.47(d,J=8.7Hz,2H),7.21(d,J=8.7Hz,2H),6.99(s,1H),6.23(d,J=7.8Hz,1H),4.37(t,J=9.6Hz,2H),4.04(d ,J=13.5Hz,2H),3.6-3.8(m,1H),3.04(t,J=11.4Hz,2H),2.79(t,J=10.5Hz,2H),1.84(d,J=9.6Hz,2H),1.2-1.4(m,2H)

[0289] Example 8

[0290] Synthesis of 1-(1-(2-methylfuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0291] plan

[0292]

[0293] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) was reacted with 2-methylfuran-3-carboxylic acid (50 mg, 396 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (8: 2). The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water.

[0294] Yield: 59 mg, 144 μmol, yield 54%. Purity (H-NMR): ≥95%

[0295] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.56(d,J=1.8Hz,1H),7.47(d,J=8.7Hz,2H),7.21(d,J=8.7Hz,2H),6.49(d,J=1.8Hz,1H),6.25(d,J= 7.5Hz,1H),4.0-4.4(br,1H),3.6-3.8(m,2H),3.0-3.2(m,2H),2.30(s,3H),1.83-1.88(m,2H),1.2-1.4(m,2H)

[0296] Example 9

[0297] Synthesis of 1-(1-(3-methylfuran-2-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0298] plan

[0299]

[0300] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) was reacted with 3-methylfuran-2-carboxylic acid (50 mg, 396 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (8: 2). The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water.

[0301] Yield: 53 mg, 129 μmol, yield 49%. Purity (H-NMR): ≥95%

[0302] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.67(s,1H),7.48(d,J=9.6Hz,2H),7.21(d,J=8.7Hz,2H),6.48(s,1H),6.27(d,J=7.8Hz,1H), 4.0-4.2(br,2H),3.7-3.9(m,1H),3.0-3.2(br,2H),2.14(s,3H),1.88(d,J=9.3Hz,2H),1.3-1.4(m,2H)

[0303] Example 10

[0304] Synthesis of 1-(1-(tetrahydro-2H-pyran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0305] plan

[0306]

[0307] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) was reacted with tetrahydro-2H-pyran-3-carboxylic acid (50 mg, 396 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water.

[0308] Yield: 43 mg, 104 μmol, yield 39%. Purity (H-NMR): ≥95%

[0309] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.47(d,J=9.3Hz,2H),7.21(d,J=8.7Hz,2H),6.23(d,J=7.8Hz,1H),4.1-4.2(m,1H),3. 6-3.9(m,4H),3.1-3.4(m,3H),2.7-2.9(m,2H),1.7-1.9(m,3H),1.5-1.7(m,3H),1.1-1.4(m,2H)

[0310] Embodiment 11

[0311] Synthesis of 1-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0312] plan

[0313]

[0314] The reaction was carried out according to the synthetic route 1, step 3. Piperidine D (47 mg, 164 μmol) was reacted with tetrahydro-2H-pyran-4-carboxylic acid (32 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The product was further purified by recrystallization from methanol and water. White crystals were collected by filtration.

[0315] Yield: 50 mg, 125 μmol, yield 76.8%. Purity (H-NMR): ≥95%

[0316] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.57(s,4H),6.34(d,J=7.5Hz,1H),4.19(d,J=12.3Hz,1H),3.8-3.9(m,3H),3.7-3.8(m,1H),3.38 (t,J=11.4Hz,3H),3.18(t,J=9.9Hz,1H),2.7-2.9(m,2H),1.7-1.9(m,2H),1.4-1.7(m,2H),1.1-1.4(m,2H)

[0317] Example 12

[0318] Scheme for the synthesis of 1-(1-(furan-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0319]

[0320] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (47 mg, 164 μmol) was reacted with furan-3-carboxylic acid (32 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (8:2). The product was further purified by recrystallization from methanol and water. White crystals were collected by filtration.

[0321] Yield: 43 mg, 113 μmol, yield 69%. Purity (H-NMR): ≥95%

[0322] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),8.03(s,1H),7.74(s,1H),7.57(s,4H),6.65(s,1H),6.36(d,J=7.8Hz,1H),3.8 -4.4(br,2H),3.7-3.9(m,1H),3.0-3.4(br,2H),1.87(d,J=11.7Hz,2H),1.3-1.5(m,2H)

[0323] Example 13

[0324] Synthesis of (R)-1-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0325] plan

[0326]

[0327] The reaction was carried out according to Synthesis Route 1, Step 3. Piperidine D (47 mg, 164 μmol) was reacted with (R)-tetrahydrofuran-3-carboxylic acid (28.7 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0328] Yield: 42 mg, 109 μmol, yield 66.9%. Purity (H-NMR): ≥95%

[0329] 1 H NMR (d6-DMSO, 300Mhz): (d,J=4.8Hz,1H),7.57(s,4H),6.36(d,J=4.2Hz,1H),4.66(t,J=6.3Hz,2H),4.14(t,J=13.2Hz,1H),3.96(d,J=15.9Hz ,1H),3.6-3.8(m,3H),3.15(q,J=14.4Hz,1H),2.82(d,J=12.3Hz,1H),1.9-2.1(m,2H),1.7-1.9(m,4H),1.2-1.5(m,2H)

[0330] Embodiment 14

[0331] Synthesis of 1-(1-(4,5-dihydrofuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0332] plan

[0333]

[0334] The reaction was carried out according to Synthesis Route 1, Step 3. Piperidine D (47 mg, 164 μmol) was reacted with 4,5-dihydrofuran-3-carboxylic acid (28.4 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0335] Yield: 45 mg, 117 μmol, yield 72.1%. Purity (H-NMR): ≥95%

[0336] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.57(s,4H),6.99(s,1H),6.34(d,J=7.5Hz,1H),4.37(t,J=9.6Hz,2H),4.05(d,J=13.5Hz,2H ),3.6-3.8(m,1H),3.04(t,J=11.1Hz,2H),2.79(t,J=9.6Hz,2H),1.85(d,J=10.5,2H),1.2-1.4(m,2H)

[0337] Embodiment 15

[0338] Synthesis of 1-(1-(2-methylfuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0339] plan

[0340]

[0341] The reaction was carried out according to the synthetic route 1, step 3. Piperidine D (47 mg, 164 μmol) was reacted with 2-methylfuran-3-carboxylic acid (30 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (8:2). The product was further purified by recrystallization from methanol and water. White crystals were collected by filtration.

[0342] Yield: 48 mg, 121 μmol, yield 74.5%. Purity (H-NMR): ≥95%

[0343] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.57(s,4H),6.48(d,J=1.8Hz,1H),6.35(d,J=7.8Hz,1H),4.0-4.3(br,2H), 3.6-3.9(m,1H),3.0-3.2(br,2H),2.30(s,3H),1.85(d,J=10.5,2H),1.2-1.4(m,2H)

[0344] Example 16

[0345] Synthesis of 1-(1-(3-methylfuran-2-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0346] plan

[0347]

[0348] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (47 mg, 164 μmol) was reacted with 3-methylfuran-2-carboxylic acid (30 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (8:2). The product was further purified by recrystallization from methanol and water. White crystals were collected by filtration.

[0349] Yield: 45 mg, 114 μmol, yield 69.9%. Purity (H-NMR): ≥95%

[0350] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.67(s,1H),7.57(s,4H),6.48(s,1H),6.38(d,J=7.5Hz,1H),3.9-4.2(br,2H ),3.7-3.9(m,1H),3.0-3.2(br,2H),2.14(s,3H),1.89(d,J=9.9,2H),1.3-1.5(m,2H)

[0351] Embodiment 17

[0352] Synthesis of 1-(1-(tetrahydro-2H-pyran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0353] plan

[0354]

[0355] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (47 mg, 164 μmol) was reacted with tetrahydro-2H-pyran-3-carboxylic acid (32.4 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The product was further purified by recrystallization from methanol and water. White crystals were collected by filtration.

[0356] Yield: 42 mg, 105 μmol, yield 64.6%. Purity (H-NMR): ≥95%

[0357] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.57(s,4H),6.38(d,J=7.5Hz,1H),4.16(d,J=12.6Hz,1H),3.6-3.9(m,4H) ,3.1-3.3(m,3H),2.7-2.9(m,2H),1.7-1.9(m,3H),1.5-1.7(m,3H),1.2-1.4(m,2H)

[0358] Embodiment 18

[0359] Synthesis of 1-(1-(1H-pyrrole-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0360] plan

[0361]

[0362] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (47 mg, 164 μmol) was reacted with 1H-pyrrole-3-carboxylic acid (27.2 mg, 245 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0363] Yield: 52 mg, 137 μmol, yield 83.9%. Purity (H-NMR): ≥95%

[0364] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.57(s,4H),7.08(s,1H),6.76(d,J=1.8Hz,m),6.34(d,J=7.8Hz,1H),6.22(s,1H),4.18 (d,J=13.5Hz,1H),3.7-3.9(m,1H),3.09(t,J=10.2Hz,2H),1.86(d,J=9.9Hz,2H),1.2-1.4(m,2H)

[0365] Embodiment 19

[0366] Synthesis of 1-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0367] plan

[0368]

[0369] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (80 mg, 264 μmol) was reacted with 2-methylfuran-3-carboxylic acid (61 mg, 528 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to obtain a white solid. The product was further purified by recrystallization using methanol and water.

[0370] Yield: 59 mg, 144 μmol, yield 54%. Purity (H-NMR): ≥95%

[0371] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.47(d,J=9.3Hz,2H),7.21(d,J=8.7Hz,2H),6.23(d,J=7.2Hz,1H),4.18(d,J=12.9Hz,1H),3.8-3.9(m,2H),3.6- 3.8(m,3H),3.3-3.4(m,1H),3.1-3.3(m,1H),2.82(t,J=12Hz,2H),1.9-2.1(m,2H),1.84(t,J=12.9Hz,2H),1.2-1.4(m,2H)

[0372] Embodiment 20

[0373] Synthesis of 1-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea

[0374] plan

[0375]

[0376] The reaction is carried out according to synthetic route 1, step 3. Piperidine D (80 mg, 278 μmol) is reacted with 2-methylfuran-3-carboxylic acid (65 mg, 556 μmol). The product is purified by flash chromatography and eluted with ethyl acetate. The collected fractions are dried in vacuo to obtain a white solid. The product is further purified by recrystallization using methanol and water.

[0377] Yield: 64 mg, 166 μmol, yield 60%. Purity (H-NMR): ≥95%

[0378] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.57(s,4H),6.33(d,J=6.9Hz,1H),4.19(d,J=13.5Hz,1H),3.8-3.9(m,2H),3.6-3.8(m,4H),3.3- 3.4(m,1H),3.1-3.3(m,1H),2.83(t,J=12Hz,1H),1.9-2.1(m,2H),1.84(t,J=13.8Hz,2H),1.2-1.4(m,2H)

[0379] Embodiment 21

[0380] Synthesis of 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-isobutyrylpiperidin-4-yl)urea

[0381] plan

[0382]

[0383] The reaction was carried out according to synthetic route 2. 3-Fluoro-4-(trifluoromethoxy)aniline (109 mg, 559 μmol) was reacted with 1-(4-aminopiperidin-1-yl)-2-methylpropan-1-one (143 mg, 838 μmol). The product was purified by flash chromatography using ethyl acetate: hexane (7: 3). The product was further purified by recrystallization using methanol and water.

[0384] Yield: 150 mg, 383 μmol, yield 68.6%. Purity (H-NMR): ≥95%

[0385] 1 H NMR (d6-DMSO, 600Mhz): (s,1H),7.67(dd,J=13.89Hz,1.2Hz,1H),7.38(t,J=9.6Hz,1H),7.12(d,J=9.6H z,1H),6.35(d,J=13.8Hz,1H),4.20(d,J=13.2Hz,1H),3.84(d,J=13.8Hz,1H),3. 7-3.8(m,1H),3.15(t,J=12Hz,1H),2.8-2.9(m,1H),2.77(t,J=11.4Hz,1H),1.86 (d,J=10.8Hz,1H),1.79(d,J=11.4Hz,1H),1.2-1.4(m,2H),0.98(t,J=7.8Hz,6H)

[0386] Embodiment 22

[0387] Synthesis of 1-(3-fluoro-4-(trifluoromethyl)phenyl)-3-(1-isobutyrylpiperidin-4-yl)urea

[0388] plan

[0389]

[0390] The reaction was carried out according to synthetic route 2. 3-Fluoro-4-(trifluoromethoxy)aniline (100 mg, 558 μmol) was reacted with 1-(4-aminopiperidin-1-yl)-2-methylpropan-1-one (142 mg, 838 μmol). The product was purified by flash chromatography using ethyl acetate: hexane (7:3). The product was further purified by recrystallization using methanol and water.

[0391] Yield: 120 mg, 320 μmol, yield 68.6%. Purity (H-NMR): ≥95%

[0392] 1H NMR (d6-DMSO, 300Mhz): (s,1H),7.66(d,J=14.7Hz,1H),7.58(t,J=8.7Hz,1H),7.20(d,J=8.7Hz,1H),6.46(d,J=7.5Hz,1H),4.20(d,J=12.9Hz,1H),3.85(d,J=13.5Hz,1 H),3.6-3.8(m,1H),3.16(t,J=12Hz,1H),2.8-2.95(m,1H),2.77(t,J=11 .4Hz,1H),1.83(t,J=14.1Hz,2H),1.2-1.4(m,2H),0.98(d,J=5.7Hz,6H)

[0393] Embodiment 23

[0394] Synthesis of N-(3-fluoro-4-(trifluoromethoxy)phenyl)-2-(1-isobutyrylpiperidin-4-yl)acetamide

[0395] plan

[0396]

[0397] The reaction was carried out according to Synthesis Route 1, Step 3. Piperidine H (38 mg, 118 μmol) was reacted with isobutyric acid (12.3 mg, 142.9 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0398] Yield: 120 mg, 320 μmol, yield 68.6%. Purity (H-NMR): ≥95%

[0399] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.85(dd,J=12.9Hz,1.8Hz,1H),7.49(t,J=9Hz,1H),7.36(d,J=9.6Hz,1H),4.36(d,J=12.6Hz,1H),3.91(d,J=12.9Hz,1H),3.00(t, J=11.7Hz,1H),2.7-2.9(m,1H),2.5-2.6(m,1H),2.27(d,J=6.9Hz,2H),1.9-2.1(m,1H),1.70(t,J=14.4Hz,2H),1.1-1.2(m,2H),0.98(s,6H)

[0400] Embodiment 24

[0401] Synthesis of 2-(1-(2-oxo-2-(4-(3-(4-(trifluoromethyl)phenyl)ureido)piperidin-1-yl)ethyl)cyclohexyl)acetic acid

[0402] plan

[0403]

[0404] 2,2'-(cyclohexane-1,1-diyl)diacetic acid (35mg, 175μmol), EDCI (30mg, 157μmol) and DMAP (21.2mg, 157μmol) were dissolved in THF (20mL), and piperidine D (75mg, 261μmol) suspended in THF (4mL) was added to the reaction mixture at room temperature. The reaction mixture was stirred at room temperature for 12 hours and quenched by adding HCl solution (1M). The organic layer was collected and the water layer was further extracted 3 times by ethyl acetate. The combined organic layer was dried in vacuo and purified by flash chromatography using 1% methanol in ethyl acetate and monitored by TLC. The collected fractions were combined and dried in vacuo to obtain the final white precipitate.

[0405] Yield: 15 mg, 31.9 μmol, yield 18.3%. Purity (H-NMR): ≥95%

[0406] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.56(s,4H),6.35(d,J=7.8Hz,1H),4.2-4.3(m,1H),3.92(d,J=12.9Hz,1H),3.6-3.8(m,1H),3 .14(t,J=11.4Hz,1H),2.7-2.9(m,1H),2.55(s,2H),1.7-1.9(m,2H),1.5-1.7(br,2H),1.2-1.5(m,6H)

[0407] Melting point (℃): 171.8-176.7

[0408] Embodiment 25

[0409] Synthesis of 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-propionylpiperidin-4-yl)urea

[0410] plan

[0411]

[0412] To the suspension of piperidine B (30mg, 93.5μmol) and triethylamine (141mg, 141.7μmol) propionic anhydride (18.2mg, 141.7μmol) was added dropwise. The reaction was stirred at room temperature for 12 hours and quenched by adding silica gel (50mg). The slurries were dried in a vacuum and flash chromatography was applied. The product was eluted with ethyl acetate: hexane (9:1) and monitored by TLC. The collected fractions were dried in a vacuum and recrystallized by methanol and water. The final white crystals were collected by filtration.

[0413] Yield: 33 mg, 87.5 μmol, yield 93%. Purity (H-NMR): ≥95%

[0414] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.66(dd,J=13.5Hz,2.4Hz,1H),7.39(t,J=9Hz,1H),7.10(d,J=9Hz,1H),6.34(d,J=7.5Hz,1H),4.18(d,J=12.9Hz,1H),3.6-3.8( m,2H),3.12(t,J=11.7Hz,1H),2.77(t,J=11.7Hz,1H),2.31(q,J=7.5,2H),1.82(d,J=12.3Hz,1H),1.2-1.4(m,2H),0.98(t,J=7.5Hz,6H)

[0415] Embodiment 26

[0416] Synthesis of 1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

[0417] plan

[0418]

[0419] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (30 mg, 93.4 μmol) was reacted with cyclopropanecarboxylic acid (12 mg, 140 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (9: 1). The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water.

[0420] Yield: 31 mg, 93.4 μmol, yield 85%. Purity (H-NMR): ≥95%

[0421] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.66(d,J=13.5Hz,1H),7.39(t,J=9Hz,1H),7.11(d,J=8.7Hz,1H),6.36(d,J=7.8Hz,1H),4.16(b r,2H),3.6-3.8(m,1H),3.2-3.3(m,1H),2.7-2.9(m,1H),1.7-2.0(m,3H),1.1-1.4(m,2H),0.6-0.8(m,4H)

[0422] Embodiment 27

[0423] Synthesis of (R)-1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)

[0424] Urea

[0425] plan

[0426]

[0427] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (30 mg, 93.4 μmol) was reacted with (R)-tetrahydrofuran-3-carboxylic acid (16 mg, 140 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water (32 mg, 76.3 μmol, 81.7% yield).

[0428] Yield: 32 mg, 76.3 μmol, yield 81.7%. Purity (H-NMR): ≥95%

[0429] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.66(dd,J=13.8Hz,2.4Hz,1H),7.39(t,J=8.7Hz,1H),7.11(d,J=9Hz,1H),6.35(d,J=7.8Hz,1H),4.18(d,J=12.9Hz,1H),3.8 -3.9(m,2H),3.6-3.8(m,4H),3.3-3.4(m,1H),3.1-3.2(m,1H),2.81(t,J=12Hz,1H),1.9-2.1(m,2H),1.7-1.9(m,2H),1.1-1.4(m,2H)

[0430] Embodiment 28

[0431] Synthesis of (S)-1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(2-methylbutyryl)piperidin-4-yl)urea

[0432] plan

[0433]

[0434] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (30 mg, 93.4 μmol) was reacted with (S)-2-methylbutyric acid (14 mg, 140 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water (30 mg, 83.9 μmol, 79.2% yield).

[0435] Yield: 30 mg, 83.9 μmol, yield 79.2%. Purity (H-NMR): ≥95%

[0436] 1 H NMR (d6-DMSO, 600Mhz): (d,J=17.4Hz,1H),7.66(dd,J=13.8Hz,2.4Hz,1H),7.39(t,J=9Hz,1H),7.11(d d,J=9Hz,1.2Hz,1H),6.3-6.4(m,1H),4.23(t,J=13.2Hz,1H),3.88(d,J=10.8H z,1H),3.6-3.8(m,1H),3.1-3.2(m,1H),2.6-2.8(m,2H),1.8-1.9(br,1H),1.7 -1.8(m,1H),1.5-1.6(m,1H),1.1-1.4(m,3H),0.9-1.0(m,3H),0.7-0.9(m,3H)

[0437] Embodiment 29

[0438] Synthesis of (R)-1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)urea

[0439] plan

[0440]

[0441] The reaction was carried out according to synthetic route 1, step 3. Piperidine B (30 mg, 93.4 μmol) was reacted with tetrahydro-2H-pyran-3-carboxylic acid (18 mg, 140 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid. The product was further purified by recrystallization using methanol and water.

[0442] Yield: 34 mg, 78.4 μmol, yield 84%. Purity (H-NMR): ≥95%

[0443] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.66(dd,J=13.5Hz,2.4Hz,1H),7.39(t,J=8.7Hz,1H),7.11(d,J=9Hz,1H),6.34(d,J=7.5Hz,1H),4.16(d, J=11.4Hz,1H),3.6-4.0(m,4H),3.1-3.3(m,2H),2.7-2.9(m,2H),1.7-1.9(m,3H),1.5-1.6(m,3H),1.1-1.4(m,3H)

[0444] Embodiment 30

[0445] Synthesis of 1-(4-(trifluoromethoxy)phenyl)-3-(1-(1-(trifluoromethyl)cyclopropane-1-carbonyl)piperidin-4-yl)urea

[0446] plan

[0447]

[0448] The reaction is carried out according to synthetic route 1, step 3. Piperidine E (50 mg, 165.0 μmol) is reacted with 1-(trifluoromethyl)cyclopropane-1-carboxylic acid (30.5 mg, 198 μmol). The product is purified by flash chromatography and eluted with ethyl acetate: hexane (4: 1). The collected fractions are dried in vacuo to obtain a white solid. The product is further purified by recrystallization using methanol and water.

[0449] Yield: 60 mg, 142 μmol, yield 71.6%. Purity (H-NMR): ≥95%

[0450] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.57(s,4H),6.36(d,J=7.8Hz,1H),4.10(d,J=12.9Hz,2H),3.6-3.8(m ,1H),2.9-3.2(br,2H),1.87(d,J=9.3Hz,2H),1.2-1.4(m,4H),1.1-1.2(s,2H)

[0451] Embodiment 31

[0452] Synthesis of 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea

[0453] plan

[0454]

[0455] The reaction is carried out according to synthetic route 1, step 3. Piperidine B (60 mg, 186.9 μmol) is reacted with tetrahydrofuran-2H-pyran-4-carboxylic acid (48.6 mg, 373.8 μmol). The product is purified by flash chromatography and eluted with ethyl acetate. The collected fractions are dried in vacuo to obtain a white solid. The product is further purified by recrystallization using methanol and water.

[0456] Yield: 58 mg, 134 μmol, yield 71.6%. Purity (H-NMR): ≥95%

[0457] 1 H NMR (d6-DMSO, 600Mhz): (s,1H),7.67(dd,J=13.2Hz,2.4Hz,1H),7.37(t,J=9Hz,1H),7.11(d,J=9.6 Hz,1H),6.35(d,J=7.2Hz,1H),4.20(d,J=13.2Hz,1H),3.8-3.9(m,3H),3.7 -3.8(m,1H),3.3-3.4(m,2H),3.16(t,J=12Hz,1H),2.8-2.9(m,1H),2.7-2. 9(m,1H),1.7-1.9(m,2H),1.5-1.7(m,2H),1.4-1.5(m,2H),1.2-1.4(m,2H)

[0458] Embodiment 32

[0459] Synthesis of 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea

[0460] plan

[0461]

[0462] The reaction is carried out according to synthetic route 1, step 3. Piperidine B (60 mg, 186.9 μmol) is reacted with 1-(trifluoromethyl)cyclopropane-1-carboxylic acid (57.6 mg, 373.8 μmol). The product is purified by flash chromatography and eluted with ethyl acetate. The collected fractions are dried in vacuo to obtain a white solid. The product is further purified by recrystallization using methanol and water.

[0463] Yield: 60 mg, 131 μmol, yield 70.2%. Purity (H-NMR): ≥95%

[0464] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.67(dd,J=13.2Hz,2.1Hz,1H),7.39(t,J=9Hz,1H),7.11(d,J=8.7Hz,1H),6.37(d,J=7.5Hz,1H),4.1 0(d,J=13.2Hz,1H),3.6-3.8(m,1H),3.0-3.2(br,1H),1.86(d,J=11.7Hz,2H),1.2-1.4(m,4H),1.1-1.2(s,2H)

[0465] Embodiment 33

[0466] Synthesis of 1-(3-fluoro-4-(trifluoromethyl)phenyl)-3-(1-propionylpiperidin-4-yl)urea

[0467] plan

[0468]

[0469] The reaction was carried out according to the synthesis of inhibitor 23. Briefly, piperidine D (40 mg, 131.1 μmol) was reacted with propionic anhydride (25 mg, 192 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0470] Yield: 42 mg, 116 μmol, yield 88.7%. Purity (H-NMR): ≥95%

[0471] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.65(d,J=14.1Hz,1H),7.56(t,J=9Hz,1H),7.19(d,J=8.7Hz,1H),6.85(d,J=7.2Hz,1H),4.14(d,J=12.6Hz,1H),3.6-3.8( m,2H),3.11(t,J=11.1,1H),2.79(t,J=12.1Hz,1H),2.2-2.4(m,2H),1.79(t,J=12.6Hz,2H),1.1-1.4(m,2H),0.96(t,J=7.5Hz,3H)

[0472] Embodiment 34

[0473] Synthesis of 1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(3-fluoro-4-(trifluoromethyl)phenyl)urea

[0474] plan

[0475]

[0476] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (40 mg, 131.1 μmol) was reacted with cyclopropanecarboxylic acid (17 mg, 197.4 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (9: 1). The collected fractions were dried in vacuo to give a white solid.

[0477] Yield: 45 mg, 121 μmol, yield 91.9%. Purity (H-NMR): ≥95%

[0478] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.65(d,J=14.1Hz,1H),7.56(t,J=9Hz,1H),7.19(d,J=8.7Hz,1H),6.47(d,J=6.9Hz,1H),4.16(b r,2H),3.6-3.8(m,1H),3.2-3.3(m,1H),2.7-2.9(m,1H),1.7-2.0(m,3H),1.1-1.4(m,2H),0.6-0.8(m,4H)

[0479] Embodiment 35

[0480] Synthesis of (R)-1-(3-fluoro-4-(trifluoromethyl)phenyl)-3-(1-(tetrahydrofuran-3-carbonyl)piperidin-4-yl)urea

[0481] plan

[0482]

[0483] The reaction was carried out according to Synthesis Route 1, Step 3. Piperidine D (40 mg, 131.1 μmol) was reacted with (R)-tetrahydrofuran-3-carboxylic acid (23 mg, 197.4 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0484] Yield: 41 mg, 102 μmol, yield 77.5%. Purity (H-NMR): ≥95%

[0485] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.65(d,J=14.7Hz,1H),7.58(t,J=9Hz,1H),7.19(d,J=8.7Hz,1H),6.45(d,J=6.9Hz,1H),4.20(d,J=13.2Hz,1H),3.8-3.9(m,2H),3. 6-3.8(m,4H),3.3-3.4(m,1H),3.10(t,J=14.7Hz,1H),2.81(t,J=12.1Hz,1H),2.00(q,J=7.5Hz,2H),1.84(t,J=13.5Hz,2H),1.1-1.4(m,2H)

[0486] Embodiment 36

[0487] Synthesis of (S)-1-(3-fluoro-4-(trifluoromethyl)phenyl)-3-(1-(2-methylbutyryl)piperidin-4-yl)urea

[0488] plan

[0489]

[0490] The reaction was carried out according to Synthesis Path 1, Step 3. Piperidine D (40 mg, 131.1 μmol) was reacted with (S)-2-methylbutyric acid (20 mg, 197.4 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (4: 1). The collected fractions were dried in vacuo to give a white solid.

[0491] Yield: 36 mg, 92.4 μmol, yield 70.5%. Purity (H-NMR): ≥95%

[0492] 1 H NMR (d6-DMSO, 300Mhz): (d,J=4.5Hz,1H),7.66(d,J=14.1Hz,1H),7.58(t,J=9Hz,1H),7.19(d,J=8.7Hz,1H),6.4-6.5(m,1H),4.2-4.3(br,1H),3.87(d,J= 12.9Hz,1H),3.6-3.8(m,1H),3.16(t,J=12Hz,1H),2.6-2.9(m,2H),1.8-1.9(m,2H),1.4-1.6(m,3H),0.97(s,3H),0.8-0.9(m,3H)

[0493] Embodiment 37

[0494] Synthesis of 1-(3-fluoro-4-(trifluoromethyl)phenyl)-3-(1-(tetrahydro-2H-pyran-3-carbonyl)piperidin-4-yl)urea

[0495] plan

[0496]

[0497] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (40 mg, 131.1 μmol) was reacted with tetrahydro-2H-pyran-3-carboxylic acid (22.8 mg, 197.4 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0498] Yield: 41 mg, 98.2 μmol, yield 74.9%. Purity (H-NMR): ≥95%

[0499] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.65(d,J=14.7Hz,1H),7.58(t,J=8.4Hz,1H),7.18(d,J=9Hz,1H),6.45(d,J=6.9Hz,1H),4.16(d,J=1 3.5Hz,1H),3.6-4.0(m,4H),3.1-3.4(m,3H),2.7-3.0(m,2H),1.8-2.0(m,3H),1.5-1.8(s,3H),1.1-1.4(m,2H)

[0500] Embodiment 38

[0501] Synthesis of 1-(3-fluoro-4-(trifluoromethyl)phenyl)-3-(1-(1-(trifluoromethyl)cyclopropane-1-carbonyl)piperidin-4-yl)urea

[0502] plan

[0503]

[0504] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (40 mg, 131.1 μmol) was reacted with 1-(trifluoromethyl)cyclopropane-1-carboxylic acid (30 mg, 197.4 μmol). The product was purified by flash chromatography and eluted with ethyl acetate: hexane (9: 1). The collected fractions were dried in vacuo to give a white solid.

[0505] Yield: 46 mg, 104 μmol, yield 79.5%. Purity (H-NMR): ≥95%

[0506] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.65(d,J=14.1Hz,1H),7.58(t,J=9Hz,1H),7.19(d,J=8.7Hz,1H),6.48(d,J=7.2Hz,1H) ,4.10(d,J=12.3Hz,2H),3.6-3.8(m,1H),3.0-3.2(br,2H),1.87(d,J=9.9Hz,2H),1.1-1.5(m,6H)

[0507] Embodiment 39

[0508] Synthesis of 1-(3-fluoro-4-(trifluoromethyl)phenyl)-3-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea

[0509] plan

[0510]

[0511] The reaction was carried out according to synthetic route 1, step 3. Piperidine D (40 mg, 131.1 μmol) was reacted with tetrahydro-2H-pyran-4-carboxylic acid (22.8 mg, 197.4 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0512] Yield: 46 mg, 108 μmol, yield 82.2%. Purity (H-NMR): ≥95%

[0513] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.66(d,J=14.1Hz,1H),7.58(t,J=8.4Hz,1H),7.20(d,J=8.7Hz,1 H),6.45(d,J=7.5Hz,1H),4.19(d,J=12.3Hz,1H),3.8-4.0(m,3H),3.6-3.8 (m,1H),3.38(t,J=11.4Hz,2H),3.16(t,J=12Hz,1H),2.8-3.0(m,1H),2.78 (t,J=11.7Hz,1H),1.84(t,J=15.3Hz,2H),1.4-1.7(m,4H),1.2-1.4(m,2H)

[0514] Embodiment 40

[0515] Synthesis of 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-isobutyrylpiperidin-4-yl)urea

[0516] plan

[0517]

[0518] The reaction was carried out according to synthetic route 2. 5-(Trifluoromethyl)pyridine-2-aniline (100 mg, 617 μmol) was reacted with (S)-1-(4-aminopiperidin-1-yl)-2-methylbutan-1-one (174 mg, 944 μmol). The product was purified by flash chromatography using ethyl acetate. The product was further purified by recrystallization using methanol and water.

[0519] Yield: 153 mg, 411 μmol, yield 73.6%. Purity (H-NMR): ≥95%

[0520] 1 H NMR (d6-DMSO, 300Mhz): (d,J=7.5Hz,1H),8.66(s,1H),8.12(d,J=8.7Hz,1H),7.75(d,J=11.7Hz,1H),6.5-6.6(m,1H),4.2-4.4(br,1H),3.88(d,J=13.5Hz,1H), 3.7-3.8(m,1H),3.16(t,J=10.8Hz,1H),2.6-2.9(m,2H),1.7-2.0(m,2H),1.4-1.6(m,1H),1.1-1.4(m,3H),0.97(s,3H),0.8-0.9(m,3H)

[0521] Embodiment 41

[0522] Synthesis of 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(morpholine-4-carbonyl)piperidin-4-yl)urea

[0523] plan

[0524]

[0525] The reaction was carried out according to synthetic route 2. Piperidine B (40 mg, 124 μmol) was reacted with morpholine (12 mg, 138 μmol). The product was purified by flash chromatography using 1% methanol in ethyl acetate. The product was further purified by recrystallization using methanol and water.

[0526] Yield: 46 mg, 106 μmol, yield 85.4%. Purity (H-NMR): ≥95%

[0527] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.66(dd,J=13.5,2.4Hz,1H),7.39(t,J=8.4Hz,1H),7.10(d,J=8.7Hz,1H),6.33(d,J=7.5Hz,1H),3.6-3 .7(m,1H),3.4-3.6(m,5H),3.11(t,J=4.2Hz,3H),2.87(t,J=11.1Hz,1H),1.79(d,J=10.2Hz,2H),1.2-1.4(m,2H)

[0528] Embodiment 42

[0529] Synthesis of 2-(1-(cyclopropanecarbonyl)piperidin-4-yl)-N-(3-fluoro-4-(trifluoromethoxy)phenyl)acetamide

[0530] plan

[0531]

[0532] The reaction was carried out according to Synthesis Route 1, Step 3. Piperidine H (38 mg, 118 μmol) was reacted with cyclopropanecarboxylic acid (12.3 mg, 142.9 μmol). The product was purified by flash chromatography and eluted with ethyl acetate. The collected fractions were dried in vacuo to give a white solid.

[0533] Yield: 32 mg, 82.4 μmol, yield 69.8%. Purity (H-NMR): ≥95%

[0534] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.85(dd,J=12.9,2.1Hz,1H),7.50(t,J=9Hz,1H),7.35(d,J=8.7Hz,1H),4.2-4.4(m,2H),3.0-3.2(m, 1H),2.57(t,J=12Hz,1H),2.28(d,J=6.9Hz,2H),1.9-2.1(m,2H),1.6-1.8(m,2H),1.0-1.2(m,2H),0.69(m,4H)

[0535] Embodiment 43

[0536] Synthesis of ((S)-N-(3-fluoro-4-(trifluoromethoxy)phenyl)-2-(1-(2-methylbutyryl)piperidin-4-yl)acetamide

[0537] plan

[0538]

[0539] The reaction was carried out according to Synthesis Path 1, Step 3. Piperidine H (38 mg, 118 μmol) was reacted with (S)-2-methylbutyric acid (14 mg, 142.9 μmol). The product was purified by flash chromatography and eluted with ethyl acetate). The collected fractions were dried in vacuo to give a white solid (27 mg, 66.8 μmol, 56.6% yield).

[0540] Yield: 27 mg, 66.8 μmol, yield 56.6%. Purity (H-NMR): ≥95%

[0541] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.84(dd,J=12.6,2.4Hz,1H),7.50(t,J=9Hz,1H),7.36(d,J=9Hz,1H ),4.39(d,J=12.3Hz,1H),3.95(d,J=12.6Hz,1H),3.00(t,J=12.3Hz,1H),2.6 -2.8(m,1H),2.27(d,J=6.9Hz,2H),1.9-2.1(m,1H),1.6-1.8(m,2H),1.5-1.6 (m,1H),1.2-1.4(m,1H),1.0-1.2(m,2H),0.96(t,J=6Hz,3H),0.7-0.9(m,3H)

[0542] Embodiment 44

[0543] Synthesis of N-(3-fluoro-4-(trifluoromethoxy)phenyl)-2-(1-isobutyrylpiperidin-4-yl)acetamide

[0544] plan

[0545]

[0546] The reaction was carried out according to Synthesis Route 1, Step 3. Piperidine H (38 mg, 118 μmol) was reacted with isobutyric acid (12.6 mg, 142.9 μmol). The product was purified by flash chromatography and eluted with ethyl acetate). The collected fractions were dried in vacuo to give a white solid.

[0547] Yield: 30 mg, 76.8 μmol, yield 65.1%. Purity (H-NMR): ≥95%

[0548] 1 H NMR (d6-DMSO, 300Mhz): (s,1H),7.84(dd,J=12.9,1.8Hz,1H),7.49(t,J=9Hz,1H),7.36(d,J=9.6Hz,1H),4.36(d,J=12.6Hz,1H),3.91(d,J=12.9Hz,1H),3.0 0(t,J=11.7Hz,1H),2.8-2.9(m,1H),2.5-2.6(m,1H),2.27(d,J=6.9Hz,2H),1.9-2.1(m,1H),1.70(t,J=13.5Hz,2H),0.9-1.2(m,8H)

[0549] Embodiment 45

[0550] Synthesis of (S)-1-(3-chloro-4-(trifluoromethoxy)phenyl)-3-(1-(2-methylbutyryl)piperidin-4-yl)urea

[0551] plan

[0552]

[0553] The reaction was carried out according to synthetic route 2. 3-Chloro-4-(trifluoromethoxy)aniline (50 mg, 236 μmol) was reacted with (S)-1-(4-aminopiperidin-1-yl)-2-methylbutan-1-one (65 mg, 353 μmol). The product was purified by flash chromatography using ethyl acetate. The product was further purified by recrystallization using methanol and water.

[0554] Yield: 78 mg, 185 μmol, yield 78.4%. Purity (H-NMR): ≥95%

[0555] 1 H NMR (d6-DMSO, 300Mhz): (d,J=4.5Hz,1H),8.65(s,1H),8.13(d,J=8.7Hz,1H),6.4-6.6(m,1H),4.2-4.3(br,1H),3.88(d,J=13.5Hz,1H),3.6-3.8(m ,1H),3.13(t,J=12Hz,1H),2.6-2.9(m,2H),1.8-1.9(m,2H),1.4-1.6(m,1H),1.2-1.4(m,3H),0.97(s,3H),0.8-0.9(m,3H)

[0556] Embodiment 46

[0557] Synthesis of (S)-1-(3-chloro-4-(trifluoromethoxy)phenyl)-3-(1-(2-methylbutyryl)piperidin-4-yl)urea

[0558] plan

[0559]

[0560] The reaction was carried out according to synthetic route 2. 3-bromo-4-(trifluoromethoxy)aniline (55 mg, 234 μmol) was reacted with (S)-1-(4-aminopiperidin-1-yl)-2-methylbutan-1-one (65 mg, 353 μmol). The product was purified by flash chromatography using ethyl acetate. The collected fractions were combined and dried in vacuo.

[0561] Yield: 81 mg, 174 μmol, yield 74.4%. Purity (H-NMR): ≥95%

[0562] 1 H NMR (d6-DMSO, 300Mhz): (d,J=4.5Hz,1H),7.97(d,J=1.8Hz,1H),7.3-7.4(m,2H),6.4-6.6(m,1H),4.2-4.3(br,1H),3.88(d,J=11.7Hz,1H),3.6-3.8( m,1H),3.15(t,J=12.9Hz,1H),2.6-2.9(m,2H),1.7-1.9(m,2H),1.4-1.6(m,1H),1.2-1.4(m,3H),0.97(s,3H),0.8-0.9(m,3H)

[0563] Embodiment 47

[0564] Synthesis of (S)-1-(1-(2-methylbutyryl)piperidin-4-yl)-3-(4-(trifluoromethoxy)-3-(trifluoromethyl)phenyl)urea

[0565] plan

[0566]

[0567] The reaction was carried out according to synthetic route 2. 4-(trifluoromethoxy)-3-(trifluoromethyl)aniline (55 mg, 224 μmol) was reacted with (S)-1-(4-aminopiperidin-1-yl)-2-methylbutan-1-one (64 mg, 347 μmol). The product was purified by flash chromatography using ethyl acetate. The product was further purified by recrystallization using methanol and water.

[0568] Yield: 73 mg, 160 μmol, yield 71.6%. Purity (H-NMR): ≥95%

[0569] 1 H NMR (d6-DMSO, 300Mhz): (d,J=8.4Hz,1H),8.06(d,J=2.4Hz,1H),7.63(dd,J=9.6,2.4Hz,1H),7.51 (d,J=11.7Hz,1H),6.42(t,J=7.2Hz,1H),4.2-4.3(br,1H),3.88(d,J=13. 5Hz,1H),3.6-3.8(m,1H),3.15(t,J=12.9Hz,1H),2.6-2.8(m,2H),1.7-1. 9(m,2H),1.4-1.6(m,1H),1.2-1.4(m,3H),0.9-1.1(m,3H),0.8-0.9(m,3H)

[0570] Embodiment 48

[0571] Synthesis of (S)-1-(1-(2-methylbutyryl)piperidin-4-yl)-3-(4-(trifluoromethoxy)-3-(trifluoromethyl)phenyl)urea

[0572] plan

[0573]

[0574] The reaction was carried out according to synthetic route 2. 3,4-bis(trifluoromethoxy)aniline (60 mg, 230 μmol) was reacted with (S)-1-(4-aminopiperidin-1-yl)-2-methylbutan-1-one (64 mg, 347 μmol). The product was purified by flash chromatography using ethyl acetate. The product was further purified by recrystallization using methanol and water (68 mg, 144 μmol, yield 62.7%).

[0575] Yield: 68 mg, 144 μmol, yield 62.7%. Purity (H-NMR): ≥95%

[0576] 1 H NMR (d6-DMSO, 300Mhz): (d,J=7.5Hz,1H),7.91(s,1H),7.47(d,J=11.7Hz,1H),7.28(dd,J=8.7,2.4Hz,1H),6.39(t,J=7.8Hz,1H),4.2-4.3(br,1H),3.88(d,J=12.3Hz ,1H),3.6-3.8(m,1H),3.15(t,J=11.4Hz,1H),2.6-2.8(m,2H),1.7-1. 9(m,2H),1.4-1.6(m,1H),1.2-1.4(m,3H),0.97(s,3H),0.8-0.9(m,3H)

[0577] Embodiment 49

[0578] Synthesis of ethyl 4-(3-(3-fluoro-4-(trifluoromethoxy)phenyl)ureido)piperidine-1-carboxylate

[0579] plan

[0580]

[0581] To the suspension of piperidine B (30mg, 93.5μmol) and triethylamine (11.6mg, 115μmol), ethyl chloroformate (29.6mg, 274μmol) was added dropwise. The reaction was stirred at room temperature for 12 hours and quenched by adding silica gel (50mg). The slurries were dried in a vacuum and flash chromatography was applied. The product was eluted with ethyl acetate: hexane (4:1) and monitored by TLC. The collected fractions were dried in a vacuum to obtain a final white powder.

[0582] Yield: 87.1% (32 mg, 81.4 μmol) Purity: ≥95% (H-NMR)

[0583] 1 H NMR (d6-DMSO, 600Mhz): (s,1H),7.66(dd,J=14.1,2.4Hz,1H),7.38(t,J=9Hz,1H),7.09(d,J=12Hz,1H),6.34(d,J=9Hz,1H),4.02(q,J=6.9Hz,2H ),3.85(d,J=13.5Hz,2H),3.6-3.8(m,1H),2.8-3.0(m,2H),1.79(t,J=10.2Hz,2H),1.2-1.4(m,2H),1.18(t,J=7.2Hz,3H)

[0584] Embodiment 50

[0585] Synthesis of ethyl 4-(3-(3-fluoro-4-(trifluoromethyl)phenyl)ureido)piperidine-1-carboxylate

[0586] plan

[0587]

[0588] To the suspension of piperidine D (30mg, 97μmol) and triethylamine (13.1mg, 129μmol), ethyl chloroformate (31.9mg, 294μmol) was added dropwise. The reaction was stirred at room temperature for 12 hours and quenched by adding silica gel (50mg). The slurries were dried in a vacuum and flash chromatography was applied. The product was eluted with ethyl acetate: hexane (4:1) and monitored by TLC. The collected fractions were dried in a vacuum to obtain a final white powder.

[0589] Yield: 90.2% (33 mg, 87.5 μmol) Purity: ≥95% (H-NMR)

[0590] 1 H NMR (d6-DMSO, 600Mhz): (s,1H),7.65(d,J=14.1Hz,1H),7.58(t,J=8.7Hz,1H),7.19(d,J=9Hz,1H),6.45(d,J=7.5Hz,1H),4.02(q,J=6.9Hz,2H),3. 85(d,J=13.5Hz,2H),3.6-3.8(m,1H),2.8-3.0(m,2H),1.80(dd,J=12.6,3.3Hz,2H),1.2-1.4(m,2H),1.18(t,J=7.2Hz,3H)

[0591] Embodiment 51

[0592] Synthesis of 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(2-methylbutyryl)piperidin-4-yl)urea

[0593] plan

[0594]

[0595] The reaction is carried out according to synthetic route 1, step 3. Piperidine B (52 mg, 161.8 μmol) is reacted with (S)-2-methylbutyric acid (24 mg, 243 μmol). The product is purified by flash chromatography and eluted with ethyl acetate. The collected fractions are dried in vacuo to obtain a white solid. The product is further purified by recrystallization using ethyl acetate and hexane.

[0596] Yield: 52 mg, 145 μmol, yield 89.9%. Purity (H-NMR): ≥95%

[0597] 1 H NMR (d6-DMSO, 300Mhz): (d,J=6.9Hz,1H),7.67(dd,J=13.5Hz,2.4Hz,1H),7.39(t,J=9.3Hz,1H),7.11(d,J=8.7Hz,1H),6.3-6.4(m,1H),4.23(m,1H),3.88(d,J=10.8 Hz,1H),3.6-3.8(m,1H),3.1-3.2(m,1H),2.6-2.8(m,2H),1.7-1.9(m, 2H),1.5-1.6(m,1H),1.1-1.4(m,3H),0.9-1.0(m,3H),0.7-0.9(m,3H)

[0598] Embodiment 52

[0599] Seizure determination

[0600] Pentylenetetrazol (PTZ)-induced epileptic seizure model

[0601] The central nervous system penetration efficacy of sEH inhibitors was studied using a standard acute test involving the administration of a convulsive prodrug, pentylenetetrazol (PTZ). In the subcutaneous pentylenetetrazol (PTZ) test, 80 mg / kg of PTZ was administered by subcutaneous route, and the time of onset of the first clonic seizure (clonic), tonic hindlimb extension, and lethality (tonic spasm) was monitored for 30 minutes. PEG400 was used as a vehicle to completely dissolve the inhibitor of sEH to obtain a clear solution. One hour before the administration of the convulsive prodrug with a single differentiated dose according to earlier data, the inhibitor or vehicle was administered by intraperitoneal route. This dose of PTZ produced 100% lethality within 30 minutes after administration.

[0602] Compound 29 was found to protect mice from convulsions and associated lethality, indicating that the compounds claimed herein can cross the blood-brain barrier ( Figure 7 ). PTZ is a chemical that induces epileptic seizures. This assay is believed to be highly translatable from mice to humans. In this epileptic seizure assay, which depends entirely on the ability of a compound to cross the blood-brain barrier, compound 29 exhibited significant efficacy, indicating that this compound readily penetrates the CNS and protects mice from epileptic seizures ( Figure 7 and 8 ).

[0603] Embodiment 53

[0604] FRET-shift assay procedure

[0605] The FRET assay for the compounds of Table I was performed as previously described to determine K i (Lee et al., Analytical Biochemistry 434 (2013) 259-268). To prevent fluorescent impurities from leaching out of the plastic tube and nonspecifically binding to the sEH inhibitor, the inhibitor stock solution (10 mM, DMSO) was stored in a glass vial. In addition, sEH was diluted to the desired concentration (20 nM) with sodium phosphate buffer (PB) (100 mM sodium phosphate, pH 7.4, 0.01% gelatin) to avoid nonspecific binding to the cuvette surface and protein loss. All buffers used in this assay were sterile filtered ( Durapore PVDF membrane, pore size: 0.22um) filtration

[0606] Measurement in 96-well plates

[0607] All measurements in 96-well plate format based on FRET shift assays were performed on TECAN The assay was performed in a M1000Pro 96-well fluorescence plate reader.

[0608] Pretreatment of 96-well plates:

[0609] To prevent non-specific binding of sEH or inhibitors to the 96-well plate, the 96-well plate was pre-incubated with PB and 0.1% gelatin at room temperature overnight. The gelatin coated the plate and prevented non-specific binding of sEH and sEH inhibitors to the plate. The buffer was discarded and the plate was dried before use.

[0610] Determination procedure

[0611] sEH stock solution was diluted to the desired concentration (20 nM) with PB (100 mM sodium phosphate, 0.1% gelatin, pH 7.4). ACPU (1 equivalent to sEH, 10 mM, ethanol) was added to the sEH solution and incubated for 2 hours at room temperature. sEH-ACPU mixture (20 nM, 100 mM sodium phosphate, 0.1% gelatin, pH 7.4, 150 uL) was added to each well.

[0612] The baseline fluorescence (F0) of the sample is measured after the z position (λ 激发 is 280nm, λ 发射 The z and gain values ​​were recorded and used for subsequent fluorescence measurements. Since DMSO is known to quench fluorescence, 1% DMSO in PB was used as a control (F DMSO ). The desired inhibitor concentration is the concentration at which sEH binds 100% to the inhibitor, which is added to the first well and further diluted 2-fold in all remaining wells. Based on our studies, the 12 reference points correspond to 12 different inhibitor concentrations, providing important data to calculate the exact K of the inhibitor. i The samples were incubated at 30 °C for 1.5 h. Subsequently, the fluorescence (λ 激发 is 280nm, λ 发射 The obtained fluorescence signal was converted as follows and used to calculate the K of the inhibitor according to the following "Curve Fitting" section. i .

[0613] Initial fluorescence = F DMSO(孔X) / F 0(孔X)

[0614] Saturated fluorescence = F 在饱和浓度下(孔X) / F 0(孔X)

[0615] Observed fluorescence = F (孔X) / F 0(孔X)

[0616] Curve Fitting

[0617] Ki The curve fitting of the assay was reported previously (Lee et al.). Data Processing and K i Calculations were based on the original work of Wang (Febs Letters 360 (1995) 111-114) with modifications proposed by Roehrl et al. (PNAS 101 (2004) 7554-7559).

[0618] The displacement assay is based on a three-state equilibrium binding model. This is modeled as follows (Equation 1)

[0619]

[0620] where [RI] represents receptor or enzyme inhibitor complex;

[0621] L represents the reporter ligand;

[0622] I represents inhibitor;

[0623] [RL] represents receptor or enzyme-reporter ligand complex.

[0624] The three-state equilibrium (Equation 1) consists of the sEH-inhibitor complex, sEH, and the sEH-reporter ligand complex. In this study, the relative fluorescence intensity (F3) was plotted against the concentration of the sEH inhibitor, and the resulting curve was fitted to the equation derived by Wang for the three-state equilibrium (Equation 2).

[0625] F3=[2(a 2 -3b) 1 / 2 cos(θ / 3)-a] / [3K d1 +[2(a 2 -3b) 1 / 2 cos(θ / 3)-a] (Equation 2)

[0626] Where a = K d1 +K d2 +L+IR;

[0627] b=K d2 (LR)+K d1 (I·R)+K d1 K d2 ;

[0628] c=-K d1 K d2 R; and

[0629] θ=arccos{(-2a 3 +9ab-27c) / [2(a 2-3b ) 3 / 2 ]}.

[0630] Where F3 = relative fluorescence = (observed fluorescence - saturation fluorescence) / (initial fluorescence - saturation fluorescence)

[0631] I = concentration of added unlabeled competing ligand;

[0632] R = total concentration of sEH;

[0633] L = total concentration of reporter ligand;

[0634] K d1 = the dissociation constant of the reporter ligand (found by fluorescence binding assay), and;

[0635] K d2 = inhibition constant of the inhibitor

[0636] k off Measurement procedure

[0637] k off Measurements were performed as described previously.

[10] sEH (8 μM) was pre-incubated with the selected inhibitor (8.8 μM, 100 mM PB buffer, pH 7.4) at room temperature for 1.5 h. The sEH-inhibitor complex was then diluted 40-fold with ACPU (20 μM, 100 mM sodium phosphate buffer, pH 7.4). Fluorescence (λ) was immediately monitored every 30 s until 5100 s. 激发 is 280nm, λ 发射 The fluorescence (λ) is plotted against time (seconds). 发射 The resulting curve was fitted with a single exponential growth and the related k off .

[0638] Embodiment 54

[0639] Measurement of pain response

[0640] Hierarchical model for measuring pain response in diabetic rats

[0641] Male Sprague-Dawley rats weighing 250-300 g were used. Animals were individually housed under standard conditions with free access to food and water and maintained for at least 1 week before the experiment. Pain was quantified using the von Frey mechanical allodynia test. All experiments were performed at the same time of day during the daylight photoperiod.

[0642] First, rats were trained to the laboratory during two separate time periods. On the day of induced neuropathy, the basic response of animals to the test was measured, and then streptozotocin (55mg / kg) was administered intravenously to induce a diabetic state. The development of neuropathic pain in animals was followed over time, and the pain occurred in 7-10 days. The test compound was then administered subcutaneously about one week after the onset of pain symptoms. As shown in the illustration, pain response was measured over a period of several hours. The compound was prepared by dissolving it in a vehicle to obtain the amount shown in the illustration. At least six animals were used in each group, but twelve rats were used in some experiments. The von Frey test was carried out by stimulating the plantar surface of the rat hind paw with a calibrated filament connected to a force sensor (IITC Inc, WoodlandHills CA). With the application of this filament, the amount of force applied to the paw increased, and when the force reached its threshold, the animal withdrew its paw. The force at this moment was electronically displayed by a display connected to the force sensor.

[0643] In a pain response assay using type I diabetic rats, the compounds of the present disclosure outperformed the registered standard drug for neuropathic pain, gabapentin, and the standard sEH inhibitor APAU. The compounds of the present disclosure reached high efficacy levels more quickly. In addition, the compounds of the present disclosure showed higher potency and higher efficacy compared to the standards in the field ( Figure 6 ).

[0644] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and variations are encompassed within the spirit and scope of the present invention. Accordingly, the present invention is limited only by the following claims.

Claims

1. A compound of formula (I): or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R1 is trifluoromethoxy-; R2 is F-; R3 is selected from the group consisting of: H-, trifluoromethyl- or -CH2CO2H; X is selected from carbon, nitrogen or oxygen; Y is nitrogen; subscript m is 1 or 2; and Subscript n is 1 or 2.

2. A compound of formula (II) or an optically pure stereoisomer or a pharmaceutically acceptable salt thereof, wherein: X is carbon; Y is nitrogen; R5 is selected from the group consisting of: trifluoromethyl- or trifluoromethoxy-; R6 is selected from the group consisting of: H- or F-; and R7 is tetrahydropyran.

3. The compound of claim 2, wherein the compound is selected from the group consisting of: 1-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (Compound 1); and 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea (Compound 29).

4. The compound according to claim 3, wherein the compound is 1-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (Compound 1).

5. The compound of claim 3, wherein the compound is 1-(3-fluoro-4-(trifluoromethoxy)phenyl)-3-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)urea (Compound 29). The compound according to claim 1 , wherein X is selected from nitrogen or oxygen.

7. The compound according to claim 2, wherein R6 is fluoro.

8. The compound of claim 1, wherein X is oxygen, subscript m is 2, and subscript n is 2.

9. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for inhibiting the activity of soluble epoxide hydrolase (sEH).

10. Use of the compound according to any one of claims 1 to 5 in the preparation of a reagent for monitoring the activity of soluble epoxide hydrolase (sEH).

11. Use of a compound according to any one of claims 1 to 5 in the preparation of a medicament for treating or ameliorating a soluble epoxide hydrolase (sEH)-mediated condition or disease in a subject.

12. The use according to claim 11, wherein the sEH-mediated condition or disease is selected from the group consisting of pain, epilepsy, renal disease, cardiomyopathy and inflammation.

13. The use according to claim 12, wherein the pain is neuropathic pain.

14. The use according to claim 13, wherein the neuropathic pain is caused by nerve damage.

15. The use according to claim 14, wherein the nerve damage is caused by diabetes.

16. The use according to claim 12, wherein the pain is caused by trauma.

17. The use according to claim 12, wherein the subject suffers from type 1 or type 2 diabetes.

18. The use according to claim 12, wherein the pain is inflammatory pain.

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