Polycyclic amides as ube2k modulators for the treatment of cancer

Abstract

The present application provides compounds of Formula (I) and pharmaceutically acceptable salts and compositions thereof for use in the treatment of disorders associated with the modulation of UBE2K.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 956,802, filed January 3, 2020, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Cancer progression is a global concern, with late-stage metastasis being the second leading cause of death worldwide. The ubiquitin-proteasome system (UPS) has recently gained attention as an important target for cancer therapy due to its role in cell proliferation and survival. Ubiquitin (Ub) is a small, highly conserved regulatory protein that covalently binds to proteins as a signal for proteasome degradation. Ubiquitination is a multi-step process that transfers Ub to specific target proteins. Ub conjugation occurs through an enzyme cascade involving Ub-activation (E1), Ub-conjugation (E2), and Ub-linking (E3) enzymes. See, for example, Cancer Biol Ther. 2010 Oct 15; 10(8):737–747. Since the addition and removal of Ub are fundamental processes in all eukaryotic cells, it is not surprising that Ub-metabolizing enzymes are prominently characterized as oncogenes or tumor inhibitors in a variety of cancers and many cancer-related signaling / regulatory pathways. See Cell Cycle 2017; 16(7):634–648.

[0004] Although progress has been made in this field (e.g., in the FDA-approved proteasome inhibitor bortezomib), improved small molecule modulators of UPS are still needed. Summary of the Invention

[0005] This article provides compounds having formula I:

[0006]

[0007] and pharmaceutically acceptable salts and compositions thereof, wherein R 1 R 2 p, Z 1 Z 2 X, ring A, and p are as described herein. The disclosed compounds and compositions regulate (e.g., inhibit) UBE2K and modified forms of UBE2K, namely, but not limited to monoubiquitinated UBE2K, diubiquitinated UBE2K, triubiquitinated UBE2K, and tetraubiquitinated UBE2K, and can be used to treat various cancers. Attached Figure Description

[0008] Figure 1 This demonstrates the UBE2K polyubiquitination activity of certain compounds of the present invention.

[0009] Figure 2 This demonstrates the selective stabilization of monoubiquitinated UBE2K by certain compounds of the present invention.

[0010] Figure 3 This demonstrates the UBE2K-Ub release activity of certain compounds of the present invention.

[0011] Figure 4 This study demonstrates the antitumor efficacy of compound 131 in a xenograft model derived from the MV.4.11 cell line (B-granulocyte monocytic leukemia) in nude mice. Detailed Implementation

[0012] 1. General description of compounds

[0013] This article provides compounds of formula I:

[0014]

[0015] Z 1 and Z 2 Each can be either N or CH independently;

[0016] X is either N or CH;

[0017] Ring A is a phenyl or a 5- to 9-membered heteroaryl group, each optionally surrounded by 1 to 3 groups selected from R. 5 Substitution of groups;

[0018] Y represents CH2, -CHR a -CR a R b 、 or SO;

[0019] R a and R b Each is independently a halogen, (C1-C6)alkyl, or halo(C1-C6)alkyl; or R a and R b Together with the carbon atoms they are bonded to, they form 3 to 6-membered cycloalkyl or 3 to 6-membered heterocyclic groups, each of which is optionally substituted by 1 to 3 groups selected from halogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, (C1-C6)alkylOH, (C1-C6)alkylO, (C1-C6)alkyl and OH;

[0020] R 1 It is a halo(C1-C6)alkyl, halo(C1-C6)alkoxy, or -NR c R dThe two available hydrogen atoms on the halo(C1-C6)alkyl and halo(C1-C6)alkoxy groups may together with the carbon atoms to which they are attached to form a 3- to 6-membered cycloalkyl group, which may optionally be substituted by 1 to 3 groups selected from halogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy and halo(C1-C6)alkoxy groups;

[0021] R c and R d Each of these can be independently hydrogen (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkylO(C1-C6)alkyl, halo(C1-C6)alkylO(C1-C6)alkyl, (C1-C6)alkyl-O-halo(C1-C6)alkyl, halo(C1-C6)alkyl-O-halo(C1-C6)alkyl, or (C1-C6)alkylOH; or R c and R d Together with the nitrogen atoms they are bonded to, they form 4 to 7-membered heterocyclic groups, which are optionally substituted by 1 to 3 groups selected from halogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy and oxo groups.

[0022] R 2 CN, halogen, OH, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, or halo(C1-C6)alkoxy; or R 1 and R 2 When on adjacent carbon atoms, it forms a 5- or 6-membered oxygen-containing heterocyclic group together with the carbon atoms to which they are attached, which is optionally substituted by 1 to 3 groups selected from halogens, (C1-C6)alkyls and halo(C1-C6)alkyls;

[0023] R 3 It is hydrogen, (C1-C6)alkyl, or halo(C1-C6)alkyl;

[0024] R 4 CN, halogen, OH, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, -NH(C1-C6)alkyl, -N[(C1-C6)alkyl]2, or 5 to 6-membered heterocyclic groups; and

[0025] p is 0 or 1.

[0026] 2. Definition

[0027] When used to describe chemical groups that may have multiple connection points, the hyphen (-) indicates the connection point of the group with the defined variable. For example, -NH(C1-C6)alkyl means that the connection point of the group is on the nitrogen atom.

[0028] The terms “halogen” and “halogen” refer to atoms selected from fluorine (fluorinated, -F), chlorine (chlorinated, -Cl), bromine (brominated, -Br), and iodine (iodinated, -I).

[0029] The term "alkyl," when used alone or as part of a larger part, such as "haloalkyl," refers to a saturated straight-chain or branched monovalent hydrocarbon group. Unless otherwise stated, alkyl groups typically have 1-4 carbon atoms, i.e., (C1-C4) alkyl.

[0030] "Alkoxy" refers to an alkyl group linked by an oxygen atom and is represented by -O-alkyl. For example, "(C1-C4)alkoxy" includes methoxy, ethoxy, propoxy, and butoxy.

[0031] The term "haloalkyl" includes mono-, poly-, and perhaloalkyl, wherein the halogen is independently selected from fluorine, chlorine, bromine, and iodine.

[0032] "Haloalkoxy" is a haloalkyl group that is connected to another part by an oxygen atom, such as, but not limited to, -OCHCF2 or -OCF3.

[0033] "Oxygenation" refers to the divalent functional group =O, which is an oxygen atom connected to another atom (usually carbon or sulfur) through a double bond.

[0034] The term "heteroaryl" refers to an aromatic ring of a specific size (e.g., a 5, 6, 7, 8, or 9-membered ring) containing one to four heteroatoms independently selected from N, O, and S. Heteroaryl groups can be monocyclic or bicyclic. Monocyclic heteroaryl groups include, for example, thienyl, furanyl, pyrroleyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridinyl, pyrimidinyl, and the like. Bicyclic heteroaryl groups include groups in which a monocyclic heteroaryl ring is fused to one or more aryl or heteroaryl rings. Non-limiting examples include indolyl, imidazopyridinyl, benzoxazolyl, benzoxadiazolyl, inzolyl, benzimidazolyl, benzothiazolyl, pyrazolyl, thienopyridinyl, thienopyrimidinyl, inzinyl, and the like. When specified, optional substituents on the heteroaryl group may be present at any substituted position.

[0035] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic ring (e.g., 3, 4, 5, 6, or 7-membered ring) of a specific size containing 1 to 4 heteroatoms independently selected from N, O, and S. The heterocyclic ring may be attached to a side group at any heteroatom or carbon atom to form a stable structure. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, oxacyclopropane, thiocyclopropane, azirropropyl, tetrahydrofuranyl, tetrahydrothiophene, tetrahydropyranyl, pyrrolyl, pyridoneyl, pyrrolidoneyl, piperidinyl, oxazolyl, piperazine, dioxane, dioxopentane, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl, oxacyclobutane, azirrobutane, and tetrahydropyrimidinyl. When specified, optional substituents on the heterocyclic group may be present at any substituted position, including, for example, positions where the heterocyclic group is attached.

[0036] The term "cycloalkyl" refers to a monocyclic hydrocarbon of a specific size (e.g., a 3, 4, 5, 6, or 7-membered ring). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, and cyclooctyl. When specified, optional substituents on the cycloalkyl group may be present at any substituted position, including, for example, positions where the cycloalkyl group is linked.

[0037] The disclosed compounds exist in various tautomeric forms and are part of this disclosure. The terms "tautomer" or "tautomeric" refer to two or more interconvertible compounds / substituents resulting from at least one form migration of a hydrogen atom and at least one change in valence. Exemplary tautomers include, for example, the following:

[0038]

[0039] All such isomers of these compounds are explicitly included. Therefore, when a compound is represented by a structural formula or its chemical name herein, all other tautomers that the compound may exist are encompassed within that formula. This includes compounds of formula I in which X is N or C.

[0040] The compounds described herein may exist in pharmaceutically acceptable salt forms. For pharmaceutical use purposes, the salts of the compounds described herein refer to non-toxic "pharmaceutically acceptable salts". Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include, for example, salts of inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid) and organic acids (e.g., acetic acid, benzenesulfonic acid, benzoic acid, methanesulfonic acid, and p-toluenesulfonic acid). The compounds of the present invention having acidic groups such as carboxylic acids can form pharmaceutically acceptable salts with one or more pharmaceutically acceptable bases. Suitable pharmaceutically acceptable basic salts include, for example, ammonium salts, alkali metal salts (e.g., sodium and potassium salts), and alkaline earth metal salts (e.g., magnesium and calcium salts). Compounds having quaternary ammonium groups also contain counter anions, such as chloride, bromide, iodide, acetate, perchlorate, etc. Other examples of this type of salt include hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, benzoate, and salts containing amino acids such as glutamic acid.

[0041] The terms "subject" and "patient" are used interchangeably and refer to mammals in need of treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, a subject is a person in need of treatment.

[0042] As used herein, the term "treatment (treat, treating)" refers to reversing, alleviating, or delaying the onset or progression of a disease or condition or one or more of its symptoms, as described herein. In some respects, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other respects, treatment may be administered in the absence of symptoms. For example, treatment may be administered to susceptible subjects before the onset of symptoms (e.g., based on a history of symptoms and / or based on exposure to a particular organism or other susceptibility factors), i.e., preventative treatment. Treatment may also continue after symptoms have subsided, for example, to delay their recurrence.

[0043] The term "pharmaceutically acceptable w" refers to a non-toxic carrier, adjuvant, or mediator that does not impair the pharmacological activity of the compound formulated with it. Pharmaceutically acceptable carriers, adjuvants, or mediators that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acids in the form of glycerides, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0044] The term “effective amount” or “therapeutic effective amount” refers to the amount of the compound described herein that will elicit a biological or medical response in a subject, for example, a dose between 0.01 and 100 mg / kg body weight / day.

[0045] 3. Compounds

[0046] In the first embodiment, compounds of formula I are provided herein:

[0047]

[0048] Or a pharmaceutically acceptable salt thereof, wherein the variable is as described above.

[0049] In the second embodiment, the compound of formula I has formula II or III:

[0050]

[0051] Or its pharmaceutically acceptable salt, wherein the remaining variables are as described above for Formula I.

[0052] In the third embodiment, the compound of formula I has formula IV:

[0053]

[0054] Or its pharmaceutically acceptable salt, wherein the remaining variables are as described above for Formula I.

[0055] In the fourth embodiment, R in the compound of formula I, II, III or IV or a pharmaceutically acceptable salt thereof 3 The hydrogen is used, and the remaining variables are as described in Formula I or the second embodiment.

[0056] In the fifth embodiment, Y in the compound of formula I, II, III, or IV, or a pharmaceutically acceptable salt thereof, is CH2, SO2, or cyclopropyl, wherein the remaining variables are as described in formula I or the fourth embodiment. Alternatively, as part of the fifth embodiment, Y in the compound of formula I, II, III, or IV, or a pharmaceutically acceptable salt thereof, is CH2, wherein the remaining variables are as described in formula I or the fourth embodiment.

[0057] In the sixth implementation scheme, Z 1 For N and Z 2 CH; Z 1 For CH and Z 2 For N; or Z 1 and Z 2 In compounds of formula I, II, III, or IV, or their pharmaceutically acceptable salts, each is CH, wherein the remaining variables are as described in formula I, or the fourth or fifth embodiment. Alternatively, as part of a sixth embodiment, Z in compounds of formula I, II, III, or IV, or their pharmaceutically acceptable salts. 1 and Z 2 Each is CH, and the remaining variables are as described in Equation I or the fourth or fifth implementation scheme.

[0058] In the seventh embodiment, ring A in the compound of formula I, II, III, or IV, or a pharmaceutically acceptable salt thereof, is phenyl or a 5- to 6-membered heteroaryl group, each optionally surrounded by 1 to 3 groups selected from R 5 The group is substituted, wherein the remaining variables are as described in Formula I or the fourth, fifth or sixth embodiments. Alternatively, as part of the seventh embodiment, ring A in the compound of Formula I, II, III or IV or its pharmaceutically acceptable salt is phenyl, pyridyl, furanyl or pyrazolyl, each optionally replaced by 1 to 3 groups selected from R 5 The group is substituted, wherein the remaining variables are as described in Formula I or the fourth, fifth or sixth embodiments. In another alternative, as part of the seventh embodiment, ring A in the compound of Formula I, II, III or IV or its pharmaceutically acceptable salt is phenyl or furanyl, each optionally replaced by 1 to 3 groups selected from R 5 The group is substituted, wherein the remaining variables are as described in Formula I or the fourth, fifth or sixth embodiments. In another alternative, as part of the seventh embodiment, ring A in the compound of Formula I, II, III or IV or its pharmaceutically acceptable salt is optionally replaced by 1 to 3 groups selected from R 5 The phenyl group is substituted with a group, wherein the remaining variables are as described in Formula I or the fourth, fifth or sixth embodiments.

[0059] In the eighth embodiment, R in the compound of formula I, II, III or IV or a pharmaceutically acceptable salt thereof 1 and R2 A five-membered oxygen-containing heterocyclic group is formed on adjacent carbon atoms and together with the carbon atoms to which they are attached, which is optionally substituted with one or two halogens, wherein the remaining variables are as described in Formula I or embodiments four, five, six or seven. Alternatively, R in compounds of Formula I, II, III or IV or their pharmaceutically acceptable salts. 1 and R 2 A dioxolane group is formed on adjacent carbon atoms and together with the carbon atoms to which they are attached, which is optionally substituted with one or two halogens, wherein the remaining variables are as described in Formula I or the fourth, fifth, sixth or seventh embodiments.

[0060] In the ninth embodiment, R in the compound of formula I, II, III or IV or a pharmaceutically acceptable salt thereof 1 It is a halo(C1-C4)alkyl, halo(C1-C4)alkoxy, or -NR c R d And R c It is hydrogen and R d It is a haloalkyl (C1-C4) group; or R c and R d Together they form a 4- to 7-membered heterocyclic group, which is optionally substituted with 1 to 3 groups selected from halogens, (C1-C4) alkyl groups, and oxo groups, wherein the remaining variables are as described in Formula I or the fourth, fifth, sixth, or seventh embodiments. Alternatively, as part of the ninth embodiment, R in the compound of Formula I, II, III, or IV or its pharmaceutically acceptable salt. 1 The heterocycle may be -OCF3, -OCHF2, -OCH2CF3, -CF3, -CH2CF3, -CHF2, piperidinyl, pyrrolidinyl, azircyclic heptyl, morpholinyl, thiomorpholinyl, piperazineyl, or azircyclic butyl, wherein each of the heterocycles is optionally substituted by 1 to 3 groups selected from halogens, (C1-C4) alkyl groups, and oxo groups, wherein the remaining variables are as described in Formula I or the fourth, fifth, sixth, or seventh embodiments.

[0061] In the tenth embodiment, R in the compound of formula I, II, III or IV or a pharmaceutically acceptable salt thereof 2 The R is CN, halogen, (C1-C4)alkyl, halo(C1-C4)alkyl, or (C1-C4)alkoxy, wherein the remaining variables are as described in Formula I or embodiments four, five, six, seven, or nine. Alternatively, as part of embodiment ten, R in compounds of formula I, II, III, or IV or their pharmaceutically acceptable salts. 2 The compound is CN or halogen, wherein the remaining variables are as described in Formula I or embodiments four, five, six, seven, or nine. In another alternative, as part of embodiment ten, R in a compound of Formula I, II, III, or IV, or a pharmaceutically acceptable salt thereof.2 The fluorine is used, wherein the other variables are as described in Formula I or in the fourth, fifth, sixth, seventh or ninth embodiments.

[0062] In the eleventh embodiment, p is 0 in compounds of formula I, II, III or IV or their pharmaceutically acceptable salts, wherein the remaining variables are as described in formula I or the fourth, fifth, sixth, seventh, ninth or tenth embodiments.

[0063] In the twelfth embodiment, R in a compound of formula I, II, III or IV or a pharmaceutically acceptable salt thereof 5 R is a halogen, (C1-C4)alkyl, halo(C1-C4)alkyl, (C1-C4)alkoxy, -N[(C1-C4)alkyl]2, or 6-membered heterocyclic group, wherein the remaining variables are as described in Formula I or the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh embodiments. Alternatively, R is a compound of Formula I, II, III, or IV or a pharmaceutically acceptable salt thereof. 5 The variables are F, Br, Cl, -OCH3, -OCH2CH3, OH, -O(CH2)2CH3, -NMe2, -CH(CH3)2, -C(CH3)3, -OCH(CH3)2, morpholino, -CH3 or -CF3, wherein the remaining variables are as described in Formula I or in the fourth, fifth, sixth, seventh, eighth, ninth, tenth or eleventh embodiments.

[0064] Specific examples of the compounds are provided in the Examples section and are included as part of the thirteenth embodiment herein. Pharmaceutically acceptable salts of these compounds, as well as neutral forms, are also included.

[0065] This document also provides pharmaceutical compositions comprising the compounds described herein; and pharmaceutically acceptable carriers.

[0066] 4. Uses, formulations, and administration

[0067] The compounds and compositions described herein are generally used to modulate the activity of UBE2K. In some respects, the compounds and compositions described herein inhibit the activity of UBE2K.

[0068] In some respects, the compounds and compositions described herein can be used to treat cancer. Therefore, methods of treating cancer are provided herein, comprising administering to a subject in need a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof, or a composition comprising a disclosed compound or a pharmaceutically acceptable salt thereof. Use of the compounds described herein or pharmaceutically acceptable salts thereof, or compositions comprising a disclosed compound or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating cancer is also provided. Compositions comprising the compounds described herein or pharmaceutically acceptable salts thereof, or compositions comprising a disclosed compound or a pharmaceutically acceptable salt thereof, for treating cancer are also provided.

[0069] Cancers that can be treated by the method of the present invention include, but are not limited to, liquid cancers, such as acute myeloid leukemia, acute lymphoblastic leukemia and chronic lymphocytic leukemia; or solid tumors, such as pancreatic cancer, ovarian cancer, breast cancer, colon cancer and gastrointestinal cancer.

[0070] In some respects, the compositions described herein are formulated for administration to subjects requiring such compositions. The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable receptacle. As used herein, the term "parenterally" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally, or intravenously. The sterile injectable form of the compositions described herein may be an aqueous or oily suspension. These suspensions may be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art.

[0071] The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health condition, sex, diet, timing of administration, rate of excretion, combination of drugs, the judgment of the treating physician, and the severity of the specific disease being treated. The amount of the compounds described herein in the composition will also depend on the specific compounds in the composition.

[0072] Implementation

[0073] Representative examples of the disclosed compounds are illustrated in the following non-limiting methods, schemes and examples.

[0074] General synthetic route

[0075] Option 1:

[0076]

[0077] Compound I can be prepared according to general scheme 1 above, wherein, for example, a suitable cyano starting material is reacted with ammonium sulfide (e.g., [NH4]2S) optionally in the presence of a base and at a high temperature to form the corresponding sulfide amine. See step 1. Then, in step 2, the corresponding heteroaryl group is cyclized with a suitable protected amine, wherein PG is an amine protecting group, such as an acid-labile protecting group. The amine is then exposed (e.g., with an acid), and subsequently coupled with a suitable acid using, for example, a diimide-based reagent or the like (see step 3) to form compound I. Variables have the same meaning as described herein.

[0078] Option 2:

[0079]

[0080] Compounds of Formula I can also be prepared according to the general scheme 2 described above, wherein, for example, suitable amino and carboxylic acid starting materials are reacted (e.g., in the presence of a base and optional additives) to form a cyano product in step 1. The cyano group can then be cyclized, for example at high temperature and optionally in the presence of an inorganic base, to form compounds of Formula I in step 2. Variables have the same meaning as described herein.

[0081] Exemplary synthesis

[0082] Synthesis of N-((5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethyl)benzamide (compound 2):

[0083]

[0084] To a solution of 2-(trifluoromethyl)benzoic acid (500 mg, 2.626 mmol) cooled to 0 °C in 2 mL of DMF, add (5-(thiophen-2-yl)-1H-pyrazol-3-yl)methylamine (662 mg, 2.629 mmol, 1 eq), HATU (380 mg, 5.258 mmol), and DIPEA (129 mg, 8.097 mmol). Stir the solution at room temperature for 16 hours. After the reaction is complete, distill off the solvent, add water (10 mL), extract with EtOAc (50 mL × 2 times), and dry with Na₂SO₄. Then concentrate under reduced pressure to obtain the crude product. The crude product was then purified by rapid column chromatography (elution buffer: 20% EtOAc / n-hexane) to give the title compound (650 mg, 70%) as N-((5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethyl)benzamide, a grayish-white solid. 1H NMR (400MHz, DMSO-d6) δ12.77(s,1H),8.94(s,1H),7.77(d,J=8.0Hz,1H),7.40-7.70(m,2H), 7.40(m,2H),7.32(s,1H),7.05(m,1H),6.40-6.48(m,1H),4.40(s,2H).LCMS:M / Z352.1[M+H] +

[0085] The following compounds in Table 1 were prepared using a method similar to that used for compound 1 above, with appropriate starting materials.

[0086] Table 1

[0087]

[0088]

[0089]

[0090]

[0091] Synthesis of 5-fluoro-2-(piperidin-1-yl)-N-((5-(thien-2-yl)-1H-pyrazol-3-yl)methyl)benzamide (compound 80):

[0092]

[0093] Step 1: Methyl 5-fluoro-2-(piperidin-1-yl)benzoate

[0094]

[0095] To a stirred solution of methyl 2,5-difluorobenzoate (500 mg, 2.906 mmol) in DMF (10 mL), piperidine (0.37 mL, 3.488 mmol) was added, followed by the addition of K₂CO₃ (1 g, 7.267 mmol), and the reaction mixture was stirred at 80 °C for 12 hours. After the reaction was complete, the solvent was concentrated under reduced pressure, diluted with water (10 mL), and extracted with EtOAc (2 × 15 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure to give the crude compound. The crude compound was purified by rapid column chromatography (elution: 40% EtOAc in hexane) to give methyl 5-fluoro-2-(piperidin-1-yl)benzoate as a brown solid (350 mg, 50.87%). 1HNMR (400MHz, DMSO-d6): δ7.33-7.36(m,1H),7.26-7.31(m,1H),7.10-7.13(m,1H),3.79(s,3H),2.82-2.87(m,4H),1.58(s,4H),1.48(s,2H).LC-MS m / z(MH):238.0

[0096] Step 2: 5-Fluoro-2-(piperidin-1-yl)benzoic acid

[0097]

[0098] LiOH (200 mg, 4.219 mmol) was added to a stirred solution of methyl 5-fluoro-2-(piperidin-1-yl)benzoate (250 mg, 1.054 mmol) in THF:H₂O (10 mL + 5 mL). The reaction mixture was then stirred at room temperature for 12 hours. After the reaction was complete, the solvent was distilled off, diluted with EtOAc (10 mL), the organic layer was separated, and the aqueous layer was acidified with 1N HCl solution (5 mL) and extracted with EtOAc (2 × 10 mL). The combined organic layers were dried over Na₂SO₄ and concentrated on a rotary evaporator to give 5-fluoro-2-(piperidin-1-yl)benzoic acid (200 mg, 75.47%) as a brown solid.

[0099] 1 ¹H NMR (400MHz, DMSO-d⁶) rotational isomers: δ 18.55 (s, ¹H), 11.97 (s, ¹H), 7.82–7.85 (m, ¹H), 7.69–7.72 (m, ¹H), 7.51–7.56 (m, ¹H), 3.07 (t, J = 5.2 Hz, 4H), 1.89 (bs, 2H), 1.73 (bs, 4H), 1.60 (d, J = 4.8 Hz, 2H); LC-MS m / z (MH): 238.0

[0100] Step 3: 5-Fluoro-2-(piperidin-1-yl)-N-((5-(thien-2-yl)-1H-pyrazol-3-yl)methyl)benzamide

[0101]

[0102] EDC·HCl (385 mg, 2.015 mmol) and HOBt (308 mg, 2.281 mmol) were added to a stirred solution of 5-fluoro-2-(piperidin-1-yl)benzoic acid (300 mg, 1.345 mmol) in DCM (20 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min, and (5-(thiophene-2-yl)-1H-pyrazol-3-yl)methylamine (310 mg, 1.614 mmol) was added. The reaction was stirred at room temperature for 12 h. After the reaction was complete, the solvent was concentrated under reduced pressure, diluted with water (10 mL), and extracted with EtOAc (2 × 15 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure to give the crude compound. The crude compound was purified by rapid column chromatography (elution buffer: 40% EtOAc in hexane) to give 5-fluoro-2-(piperidin-1-yl)-N-((5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)benzamide as a grayish-white solid (11 mg, 21.31%). 1 ¹H NMR (400MHz, DMSO-d6) rotational isomers: δ 12.84 (s, 1H), 10.53 (s, 1H), 7.54–7.64 (m, 1H), 7.32–7.40 (m, 4H), 7.04–7.11 (m, 1H), 6.54 (s, 1H), 4.47–4.56 (m, 2H), 2.78 (s, 4H), 1.47 (s, 4H), 1.39 (s, 2H). LC-MS m / z (MH): 385.0.

[0103] The following compounds in Table 2 were prepared using a method similar to that used for compound 80 above, with appropriate starting materials.

[0104] Table 2

[0105]

[0106] Synthesis of N-((5-(2-methoxyphenyl)-1H-pyrazol-3-yl)methyl)-2-(piperidin-1-yl)benzamide (compound 102):

[0107]

[0108] Step 1: Synthesis of ethyl 5-nitro-1H-pyrazole-3-carboxylate

[0109]

[0110] SOCl2 (8 mL) was added to a stirred solution of 5-nitro-1H-pyrazole-3-carboxylic acid (5 g, 31.84 mmol) in ethanol (50 mL) at 0 °C. The reaction mixture was stirred at 80 °C for 12 hours. After the reaction was complete, the solvent was distilled off, diluted with EtOAc (50 mL), washed once with saturated NaHCO3 solution (50 mL), and then with water (50 mL). The organic layer was separated, dried over Na2SO4, and concentrated on a rotary evaporator to give a crude compound. The crude compound was ground with diethyl ether (25 mL) to give ethyl 5-nitro-1H-pyrazole-3-carboxylic acid as a grayish-white solid (4.5 g, 77.58%). 1 ¹H NMR (400MHz, DMSO-d⁶): rotational isomer δ 15.19 (s, 1H), 7.48 (s, 1H), 4.32–4.37 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H). LC-MS m / z (MH): 186.1

[0111] Step 2: Synthesis of ethyl 5-amino-1H-pyrazole-3-carboxylate

[0112]

[0113] Pd / C (wt / wt, 100 mg) was added to a stirred solution of ethyl 5-nitro-1H-pyrazole-3-carboxylate (10 g, 54.05 mmol) in AcOH:THF (1:1), and the reaction mixture was hydrogenated at 50 psi for 12 h. After the reaction was complete, the mixture was filtered through a diatomaceous earth bed, washed with methanol (2 x 50 m), dried over anhydrous Na2SO4, and concentrated on a rotary evaporator to give ethyl 5-amino-1H-pyrazole-3-carboxylate as a grayish-white solid (8 g, 95.57%). 1 ¹H NMR (400MHz, DMSO-d6) rotational isomers: δ 12.09 (s, 1H), 5.63 (s, 1H), 5.15 (s, 1H), 4.16 (s, 2H), 1.23 (s, 3H); LC-MS m / z (MH): 156.1

[0114] Step 3: Synthesis of ethyl 5-iodo-1H-pyrazole-3-carboxylate

[0115]

[0116] NaNO₂ (658 mg, 9.67 mmol) / H₂O (6 mL) was added to a stirred solution of ethyl 5-amino-1H-pyrazole-3-carboxylate (1.2 g, 7.74 mmol) in HCl (12 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 30 min, and KI (1.6 g, 9.63 mmol) / H₂O (6 mL) was slowly added at the same temperature. The mixture was then warmed to room temperature and stirred for 12 h. After the reaction was complete, the reaction mixture was diluted with cold water (20 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were washed once with a saturated sodium thiosulfate solution (20 mL) and then washed with H₂O (20 mL). The organic layers were dried over Na₂SO₄ and concentrated on a rotary evaporator to give the crude compound. The crude compound was purified by rapid column chromatography (elution buffer: 10% EtOAc in hexane) to give ethyl 5-iodo-1H-pyrazole-3-carboxylate as a grayish-white solid (320 mg, 16%). 1 ¹H NMR (400MHz, DMSO-d⁶) rotational isomers: δ 14.23 (s, ¹H), 6.88 (s, ¹H), 4.27–4.32 (m, ²H), 1.28 (t, J = 7.2 Hz, ³H); LC-MS m / z (MH): 266.92

[0117] Step 4: Synthesis of ethyl 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxylate

[0118]

[0119] (2-methoxyphenyl)boronic acid (45 mg, 0.296 mmol) and Na₂CO₃ (72 mg, 0.679 mmol) were added to a stirred solution of ethyl 5-iodo-1H-pyrazole-3-carboxylate (100 mg, 0.273 mmol) in 1,4-dioxane-H₂O (8 mL + 2 mL). The reaction mixture was degassed with argon for 10 min, and tetrakis(triphenylphosphine)palladium (31 mg, 0.026 mmol) was added. The reaction was heated at 100 °C for 12 h. After the reaction was complete, the solvent was concentrated under reduced pressure, diluted with water (10 mL), and extracted with EtOAc (2 × 15 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure to give the crude compound. The crude compound was purified by rapid column chromatography (elution buffer: 60% EtOAc in hexane) to give ethyl 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxylate as a grayish-white solid (47 mg, 70.14%). 1¹H NMR (400MHz, DMSO-d⁶) rotational isomers: δ 13.56 (s, ¹H), 7.72 (d, J = 7.6 Hz, ¹H), 7.30–7.39 (m, ¹H), 7.11–7.20 (m, ³H), 7.01–7.05 (m, ¹H), 4.25–4.33 (m, 2H), 3.89 (s, ³H), 1.29 (d, J = 7.2 Hz, ³H); LC-MS m / z (MH): 247

[0120] Step 5: Synthesis of 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxylic acid

[0121]

[0122] LiOH·H2O (260 mg, 6.504 mmol) was added to a stirred solution of ethyl 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxylate (400 mg, 1.62 mmol) in THF:H2O (20 mL + 10 mL) at 0 °C, and the reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was distilled off, diluted with EtOAc (10 mL), the organic layer was separated, and the aqueous layer was acidified with 1N HCl solution (5 mL) and extracted with EtOAc (2 × 25 mL). The combined organic layers were dried over Na2SO4 and concentrated on a rotary evaporator to give 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxylic acid as a brown solid (250 mg, 70.62%). 1 ¹H NMR (400MHz, DMSO-d⁶) rotational isomers: δ 13.21 (s, ¹H), 7.77 (d, J = 6.8 Hz, ¹H), 7.34 (t, J = 8.0 Hz, ¹H), 7.11–7.14 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 3.88 (s, 3H). LC-MS m / z (MH): 219

[0123] Step 6: Synthesis of 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxamide

[0124]

[0125] HATU (650 mg, 1.720 mmol), DIPEA (740 mg, 3.440 mmol), and NH4HCO3 (360 mg, 4.587 mmol) were added to a stirred solution of 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxylic acid (250 mg, 1.146 mmol) in DMF (6 mL) at room temperature and stirred for 12 h. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound was purified by Grace column chromatography, eluting with 80% EtOAc / petroleum ether, to give 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxamide (80 mg, yield: 32.25%) as a grayish-white solid. 1 HNMR (400MHz, DMSO-d6) rotational isomers: δ 13.24 (s, 1H), 7.88–7.94 (m, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.45 (s, 1H), 7.27–7.38 (m, 1H), 6.96–7.19 (m, 4H), 3.89 (s, 3H). LC-MS m / z (MH): 218

[0126] Step 7: Synthesis of (5-(2-methoxyphenyl)-1H-pyrazol-3-yl)methylamine

[0127]

[0128] Lithium aluminum hydride (54 mg, 1.474 mmol) was added to a stirred solution of 5-(2-methoxyphenyl)-1H-pyrazole-3-carboxamide (80 mg, 0.368 mmol) in 10 mL of THF at 0 °C, and the reaction mixture was stirred at 80 °C for 12 h. After the reaction was complete, it was terminated with a slurry of Na₂SO₄, and then EtOAc (20 mL) was added. The mixture was filtered through a diatomaceous earth filter. The filtrate was concentrated under reduced pressure to give (5-(2-methoxyphenyl)-1H-pyrazole-3-yl)methylamine (45 mg, 60%) as a grayish-white solid, which was used in the next step without further purification. LC-MS m / z (MH): 204

[0129] Step 8: N-((5-(2-methoxyphenyl)-1H-pyrazol-3-yl)methyl)-2-(piperidin-1-yl)benzamide

[0130]

[0131] EDC.HCl (63 mg, 0.33 mmol) and HOBt (50 mg, 0.32 mmol) were added to a stirred solution of 2-(piperidin-1-yl)benzoic acid (45 mg, 0.22 mmol) in dichloromethane at 0 °C. The reaction mixture was stirred at 0 °C for 15 min, and (5-(2-methoxyphenyl)-1H-pyrazol-3-yl)methylamine (45 mg, 0.26 mmol) was added to the resulting mixture. The reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was concentrated under reduced pressure, diluted with water (10 mL), and extracted with EtOAc (2 × 15 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give the crude compound. The crude compound was purified by preparative TLC (eluting with 5% MeOH / DCM) to give 5-fluoro-2-(piperidin-1-yl)-N-((5-(thien-2-yl)-1H-pyrazol-3-yl)methyl)benzamide as a grayish-white solid (5.3 mg, 6.16%). 1 ¹H NMR (400MHz, DMSO-d⁶) rotational isomers: δ 12.80 (s, ¹H), 10.22 (s, ¹H), 7.09 (d, J = 7.6 Hz, ¹H), 7.64 (bs, ¹H), 7.40–7.47 (m, ¹H), 7.27–7.29 (m, 2H), 7.18 (t, J = 7.2 Hz, ¹H), 7.09 (d, J = 8.0 Hz, ¹H), 6.99 (bs, ¹H), 6.66 (s, ¹H), 4.51 (s, 2H), 3.85 (s, 3H), 2.80 (s, 4H), 1.49 (s, 4H), 1.38 (s, 2H). LC-MS m / z (MH): 391.0

[0132] The following compounds in Table 3 were prepared using a method similar to that used for compound 102 described above, with appropriate starting materials.

[0133] Table 3

[0134]

[0135]

[0136] Synthesis of 2-(4,4-difluoropiperidin-1-yl)-N-((5-(thiophen-2-yl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 76):

[0137]

[0138] Step 1: Synthesis of methyl 2-bromobenzoate

[0139]

[0140] Concentrated sulfuric acid (8 mL, 150 mmol) was added to a stirred solution of 2-bromobenzoic acid (10 gm, 50 mmol) in methanol (80 mL) at 0 °C, and the reaction mixture was stirred at reflux for 16 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure. The crude product was quenched with cold water and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (50 mL) and saturated sodium bicarbonate solution (50 mL) and dried over sodium sulfate. The organic layers were concentrated to give a pure yellow liquid product, methyl 2-bromobenzoate (9.0 gm, 83% yield). 1 H NMR (400MHz, CDCl3) δ7.79 (dd, J=7.4, 1.9Hz, 1H), 7.65 (dt, J=25.6, 12.6Hz, 1H), 7.42–7.29 (m, 2H), 3.94 (s, 3H).

[0141] Step 2: Synthesis of methyl 2-(4,4-difluoropiperidin-1-yl)benzoate

[0142]

[0143] To a stirred solution of methyl 2-bromobenzoate (250 mg, 1.162 mmol) in 1,4-dioxane (5 mL), 4,4-difluoropiperidine hydrochloride (202 mg, 1.2818 mmol), Xanthphos (335 mg, 0.581 mmol), and Cs₂CO₃ (944 mg, 2.905 mmol) were added. The reaction mixture was degassed five times with argon, and then Pd₂dba₃ (106 mg, 0.1166 mmol) was added. The reaction mixture was stirred at 110 °C for 16 h. After the reaction was complete, the mixture was cooled, quenched with water, and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine and dried over sodium sulfate. The dried organic layers were concentrated under reduced pressure to give a crude product as a yellow liquid. Purification was achieved by rapid column chromatography using ethyl acetate and hexane as eluents. 2-(4,4-difluoropiperidin-1-yl)methyl benzoate was obtained as a pure substance (40 mg, 16%). 1 H NMR (400MHz, CDCl3) δ7.85–7.73 (m, 1H), 7.42 (dd, J = 11.1, 4.4Hz, 1H), 7.10–6. 98(m,2H),3.89(s,3H),3.22–3.07(m,4H),2.16(ddd,J=19.3,13.8,5.6Hz,4H).

[0144] Step 3: Synthesis of 2-(4,4-difluoropiperidin-1-yl)benzoic acid

[0145]

[0146] Water (2 mL) and NaOH (480 mg, 12.078 mmol) were added to a stirred solution of methyl 2-(4,4-difluoropiperidin-1-yl)benzoate (770 mg, 3.019 mmol) in ethanol (8 mL). The reaction mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction mixture was diluted with water, and the aqueous layer was washed with ethyl acetate (2 × 50 mL). The product containing the aqueous layer was acidified with 2N HCl and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried over sodium sulfate. The organic layers were concentrated under reduced pressure to give 2-(4,4-difluoropiperidin-1-yl)benzoic acid (580 mg, 80%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ14.99(s,1H),7.87(d,J=7.5Hz,1H),7.57(t,J=7.5Hz,1H),7.49(d,J= 8.0Hz, 1H), 7.26 (t, J = 7.3Hz, 1H), 3.15 (s, 4H), 2.16 (t, J = 13.9Hz, 4H). LC-MS m / z (M+H): 242.0.

[0147] Step 4: 2-(4,4-difluoropiperidin-1-yl)-N-((5-(thiophen-2-yl)-1H-1,2,4-triazol-3-yl)methyl)benzamide

[0148]

[0149] EDC·HCl (29 mg, 0.156 mmol), HOBt (23 mg, 0.156 mmol), and triethylamine (0.067 mL, 0.468 mmol) were added to a stirred solution of 2-(4,4-difluoropiperidin-1-yl)benzoic acid (25 mg, 0.104 mmol) in dichloromethane (5 mL), followed by the addition of 5-(thiophene-2-yl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (24 mg, 0.114 mmol). The reaction mixture was stirred at room temperature for 16 h. After the reaction was complete, the mixture was quenched with water and extracted with dichloromethane (2 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried over sodium sulfate. The organic layers were concentrated under reduced pressure to give a crude product as a red liquid. The crude product was purified by rapid chromatography using ethyl acetate and hexane as eluents to give 2-(4,4-difluoropiperidin-1-yl)-N-((5-(thiophen-2-yl)-1H-1,2,4-triazol-3-yl)methyl)benzamide as a grayish-white solid (10 mg, 25%).1 H NMR(400MHz,DMSO-d6)δ14.35(s,0.4H),13.97(s,0.6H),9.81(s,0.4H),9.73 (s,0.6H),7.87–7.66(m,2H),7.53(dd,J=11.5,4.2Hz,1H),7.46(t,J=7.7Hz,1 H),7.30(t,J=7.6Hz,1H),7.19(dd,J=13.9,6.4Hz,1H),7.14–7.07(m,1H),4.6 6(d,J=5.6Hz,1.2H),4.57(d,J=5.0Hz,0.8H),3.04(s,4H),2.15(s,4H).LC-MS m / z(M+H):404.1.

[0150] The following compounds in Table 4 were prepared using a method similar to that used for compound 76 above, with appropriate starting materials.

[0151] Table 4

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] Synthesis of 3-(3,3-difluoropyrrolidone-1-yl)-N-((5-(thiophene-2-yl)-1H-1,2,4-triazol-3-yl)methyl)pyrazine-2-carboxamide (compound 87):

[0160]

[0161] Step 1: Synthesis of ethyl 3-(3,3-difluoropyrrolidone-1-yl)pyrazine-2-carboxylate

[0162]

[0163] At 0 °C, CS₂CO₃ (1.2 g, 3.22 mmol) and Et₃N (162.9 mg, 1.61 mmol) were added to a stirred solution of ethyl 3-chloropyridine-2-carboxylate (300 mg, 1.61 mmol) in DMF (10 mL), followed by the addition of 3,3-difluoropyrrolidine·HCl (277.9 mg, 1.93 mmol). The reaction mixture was stirred in a sealed tube at 100 °C for 12 h. After the reaction was complete, the reaction mixture was diluted with water and extracted with EtOAc (3 × 20 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was ground with n-hexane and dried to give ethyl 3-(3,3-difluoropyrrolidine-1-yl)pyridine-2-carboxylate as a yellow slurry (260 mg, 62%). 1 H NMR (400MHz, DMSO-d6) δ8.23 (d, J = 2Hz, 1H), 8.03 (d, J = 2Hz, 1H), 4.50 (q, J = 6.4Hz, 2H), 3.78–3.72 (m, 4H), 2.51–2.40 (m, 2H), 1.46 (t, J = 6.8Hz, 3H).

[0164] Step 2: Synthesis of 3-(3,3-difluoropyrrolidone-1-yl)pyrazin-2-carboxylic acid

[0165]

[0166] LiOH (244.67 mg, 5.83 mmol) was added to a stirred solution of ethyl 3-(3,3-difluoropyrrolidone-1-yl)pyrazin-2-carboxylic acid (250 mg, 0.97 mmol) in THF:H₂O (5 mL: 2 mL) at 0 °C. The reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was acidified with 1 N HCl and extracted with EtOAc (2 × 20 mL). The organic layer was dried over MgSO₄ and concentrated under vacuum to give 3-(3,3-difluoropyrrolidone-1-yl)pyrazin-2-carboxylic acid as a grayish-white solid (200 mg, 90.17%). 1 H NMR (400MHz, DMSO-d6): δ13.50 (s, 1H), 8.28 (d, J = 2.4Hz, 1H), 7.97 (s, 1H), 3.80 (t, J = 12.8Hz, 2H), 3.62 (t, J = 7.2Hz, 2H), 2.56–2.49 (m, 2H).

[0167] Step 3: Synthesis of 3-(3,3-difluoropyrrolidone-1-yl)-N-((5-(thiophene-2-yl)-1H-1,2,4-triazol-3-yl)methyl)pyrazine-2-carboxamide

[0168]

[0169] EDC.HCl (87.57 mg, 0.45 mmol), HOBt (61.8 mg, 0.45 mmol), and Et3N (92.62 mg, 0.91 mmol) were added to a stirred solution of 3-((3,3-difluoropyrrolidone-1-yl)methyl)pyrazin-2-carboxylic acid (70 mg, 0.30 mmol) in CH2Cl2 (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, followed by the addition of (5-(thiophene-2-yl)-1H-1,2,4-triazol-3-yl)methylamine. HCl (72.62 mg, 0.33 mmol) was added, and the reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was washed with water and extracted with CH2Cl2 (2 × 10 mL). The organic layer was dried over MgSO4 and concentrated under vacuum. The crude product was purified by preparative HPLC to obtain 3-(3,3-difluoropyrrolidone-1-yl)-N-((5-(thiophen-2-yl)-1H-1,2,4-triazol-3-yl)methyl)pyrazine-2-carboxamide, which was a grayish-white solid (39 mg, 32.63%). 1 HNMR (400MHz, DMSO-d6): δ13.96(brs,1H),9.26(brs,1H),8.26(s,1H),7.94(s,1H),7.59(d,J=12Hz,2H),7.14(s,1H ),4.55(d,J=5.6Hz,2H),3.80(t,J=13.2Hz,2H),3.66(t,J=11.2Hz,2H),2.49–2.39(m,2H).LC-MS(m / z):391.10(M+H) +

[0170] The following compounds in Table 5 were prepared using a method similar to that used for compound 87 above, with appropriate starting materials.

[0171] Table 5

[0172]

[0173]

[0174] Synthesis of N-((5-(4-methylthiophen-3-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (compound 16):

[0175]

[0176] Step 1: N-((1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0177]

[0178] HATU (46.1 g, 0.121 mmol) was added to a stirred solution of 2-(trifluoromethoxy)benzoic acid (25 g, 0.121 mmol) in DMF (250 mL) at 0 °C, followed by (2H-pyrazol-3-yl)methylamine (11.7 g, 0.1213 mmol) and DIPEA (39.1 g, 0.303 mmol). The reaction mixture was then stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was diluted with water (2.5 L) and extracted with EtOAc (2 x 500 mL). The combined organic layers were washed once with H2O (250 mL), saturated NaHCO3 solution (250 mL), and finally with brine (250 mL). The organic layers were dried over Na2SO4 and concentrated to give the crude compound. The crude compound was purified by rapid column chromatography (elution buffer: 70% EtOAc / petroleum ether) to give N-((1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (18.3 g, 53.0% yield) as a grayish-white solid. 1 H NMR(400MHz,DMSO)δ12.64(d,J=47.1Hz,1H),8.87(d,J=42.1Hz,1H),7.76–7.50 (m,3H),7.49–7.27(m,2H),6.15(d,J=12.1Hz,1H),4.42(t,J=11.1Hz,2H).LC-MS m / z(M+H):286.1.

[0179] Step 2: N-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0180]

[0181] 3,4-Dihydro-2H-pyran (5.39 g, 64.1 mmol) was added to a stirred solution of N-((1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (18.3 g, 64.15 mmol) in toluene (400 mL) at room temperature. The reaction was then heated at 80 °C for 4 h. After the reaction was complete, the solvent toluene was distilled off, and the residue was diluted with EtOAc (250 mL) and washed once with saturated NaHCO3 solution (100 mL) and H2O (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the crude compound. The crude product was then ground with petroleum ether (200 mL) and stirred for 12 h. The solid was filtered and dried under vacuum to obtain N-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (12.57 g, 51.5%), which was a grayish-white solid. 1 H NMR (400MHz, DMSO) δ8.87 (s, 1H), 7.80 (d, J = 2.2Hz, 1H), 7.57 (t, J = 7.0Hz, 2 H),7.46–7.38(m,2H),6.20(d,J=2.2Hz,1H),5.32(d,J=10.3Hz,1H),4.38( d,J=5.9Hz,2H),3.90(d,J=11.0Hz,1H),3.67–3.52(m,1H),2.07(dd,J=24. 7,11.0Hz,1H),1.98–1.80(m,2H),1.65(s,1H),1.51(d,J=3.5Hz,2H).LC-MS m / z(M+H):370.1.

[0182] Step 3: N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0183]

[0184] 1.6 M n-butyllithium / hexane (6.31 g, 98.61 mmol) was added to a stirred solution of N-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (18.2 g, 49.30 mmol) in 200 mL of dry THF at -78 °C for 10 min. The reaction mixture was then stirred at the same temperature for 1 h. Iodine (13.76 g, 54.2 mmol) / 200 mL of dry THF was added to the resulting mixture over 15 min. After the addition of iodine was complete, the reaction mixture was slowly heated to -20 °C and stirred for 45 min. After the reaction was complete, it was carefully quenched with 200 mL of saturated NaHSO3 solution and extracted with EtOAc (2 × 150 mL). The combined organic layers were dried over Na2SO4 and concentrated to give the crude compound. The crude compound was purified by rapid column chromatography (elution buffer: 20% EtOAc in petroleum ether) to give N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (12.57 g, 51.5%), as a grayish-white solid. 1 H NMR(400MHz,DMSO)δ8.91(t,J=5.6Hz,1H),7.63–7.53(m,2H),7.44(dd,J=14.6 ,7.6Hz,2H),6.43(s,1H),5.33(d,J=9.8Hz,1H),4.36(d,J=5.6Hz,2H),3.90(d ,J=10.9Hz,1H),3.59(dd,J=17.3,7.5Hz,1H),2.27(dd,J=22.8,9.5Hz,1H),1. 97(d,J=12.3Hz,1H),1.83(d,J=12.1Hz,1H),1.67(s,1H),1.50(s,2H).,LC-MS m / z(M+H):396.0.

[0185] Step 4: N-((5-(4-methylthiophene-3-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide

[0186]

[0187] Na₂CO₃ (34.71 mg, 0.327 mmol) was added to a stirred solution of N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (60 mg, 0.131 mmol) and 4-methylthiophene-3-boronic acid (20.66 mg, 0.157 mmol) in 1,4-dioxane:water (5 mL:1 mL). The reaction mixture was then degassed with argon for 10 min, followed by the addition of tetrakis(triphenylphosphine)palladium (15.13 mg, 0.0130 mmol). The resulting mixture was heated at 100 °C for 12 h. After the reaction was complete, the mixture was diluted with H₂O (5 mL) and extracted with EtOAc (2 × 10 mL). The combined organic layers were dried over Na₂SO₄ and concentrated to give the crude compound. It was further purified by preparative TLC to obtain N-((5-(4-methylthiophen-3-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (34 mg, 57.74%), which was a grayish-white solid. 1 H NMR (400MHz, DMSO) δ8.93(s,1H),7.57(dd,J=15.8,5.4Hz,3H),7.44(dd,J=15.6,7.6Hz,2H),7.34(s,1H),6.26(s,1H),5.02(d,J=9.5Hz,1H), 4.43(s,2H),3.91(d,J=11.1Hz,1H),3.43(s,1H),2.33(d,J=14.2Hz,1H),2.09(d,J=16.3Hz,3H),1.90(s,1H),1.47(s,3H),1.22(s,2H).LC-MS m / z(M+H):466.2.

[0188] Using a method similar to that used for compound 16 above, the following intermediate compounds in Table 6 were prepared using appropriate starting materials.

[0189] Table 6

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] Step 5: N-((5-(4-methylthiophen-3-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0199]

[0200] 1,4-Dioxane-HCl (4.0 M, 3 mL) was added to a stirred solution of N-((5-(4-methylthiophen-3-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (34 mg, 0.073 mmol) in dichloromethane (3 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 4 h. After the reaction was complete, the mixture was concentrated and further co-distilled with dichloromethane (2 × 10 mL) to give a crude substance. Further treatment was performed by adding H₂O (10 mL), followed by alkalization with saturated NaHCO₃ solution and extraction with EtOAc (2 × 5 mL). The combined organic layers were dried over Na₂SO₄ and concentrated to give the crude compound. It was then purified by preparative TLC (elution buffer: 30% EtOAc + hexane) to give N-((5-(4-methylthiophen-3-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (19 mg, 68.24%), as a grayish-white solid. 1 H NMR (400MHz, DMSO) δ12.77(d,J=41.1Hz,1H),8.92(d,J=39.6Hz,1H),7.66(s,1H),7.58(d,J=7.0Hz,2H),7.43(s,2H),7. 24(d,J=43.8Hz,1H),6.38(d,J=20.0Hz,1H),4.55–4.36(m,2H),2.37(s,1H),2.30(d,J=14.2Hz,2H),1.22(s,1H).LC-MS m / z(MH):382.1.

[0201] The following compounds in Table 7 were prepared using a method similar to that used for compound 16 above, with appropriate starting materials.

[0202] Table 7

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] Synthesis of N-((5-(2,5-dihydroxyphenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (compound 40):

[0213]

[0214] BBr3 / dichloromethane (1.0 M, 4 mL) was added to a stirred solution of N-((5-(2,5-dimethoxyphenyl)-1H-pyrazole-3-yl)methyl)-2-(trifluoromethoxy)benzamide (90 mg, 0.365 mmol) in dichloromethane (5 mL) at 0 °C. The resulting mixture was stirred at room temperature for 12 h. After the reaction was complete, it was quenched with an aqueous solution of NaHCO3 (5 mL) and extracted with 10% MeOH / DCM (2 x 10 mL). The combined organic layers were dried over Na2SO4 and concentrated to give a crude compound. It was purified by preparative TLC (3% MeOH in DCM) to give N-((5-(2,5-dihydroxyphenyl)-1H-pyrazole-3-yl)methyl)-2-(trifluoromethoxy)benzamide as a grayish-white solid (24 mg, 34.28%). 1 H NMR(400MHz,DMSO)δ12.80(d,1H),9.85(d,1H),8.93(d,2H),7.62(d,2H),7.4 5(s,2H),6.99(s,1H),6.71(s,1H),6.59(s,2H),4.47(d,2H)LC-MS(ESI):m / z 393.9(M+H) +

[0215] The following compounds in Table 8 were prepared using a method similar to that used for compound 40 above, with appropriate starting materials.

[0216] Table 8

[0217]

[0218]

[0219]

[0220]

[0221] Synthesis of 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 126):

[0222]

[0223] Step 1: Synthesis of 2-methoxybenzothioamide

[0224]

[0225] An ammonium sulfide solution (650 mL, 5 vol) was added to a stirred solution of 2-methoxybenzonitrile (130 g, 977 mmol) in pyridine (1200 mL) at 0 °C, followed by the addition of triethylamine (150 mL, 1075 mmol). The reaction mixture was then stirred at 55 °C for 12 h. The reaction was monitored by TLC (30% EtOAc / hexane). After the reaction was complete, the mixture was diluted with cold water (4.0 L), filtered, and dried under reduced pressure to give 2-methoxybenzothioamide (145 g, yield: 89%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ9.94br(s,1H),9.30(brs,1H),7.68(d,J=7.6Hz,1H),7 .36(t,J=8.0Hz,1H),7.04(d,J=8.4Hz,1H),6.94(t,J=7.2Hz,1H),3.79(s,3H).

[0226] Step 2: Synthesis of tert-butyl ((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)carbamate

[0227]

[0228] A stirred solution of 2-methoxybenzothioamide (61 g, 365 mmol) and (2-hydrazino-2-oxoethyl) tert-butyl carbamate (207 g, 1095 mmol) / pyridine (300 mL) was heated at 120 °C for 48 h. After the reaction was completed by TLC monitoring, the solution was diluted with water (500 mL) and extracted with EtOAc (2 x 600 mL). The organic layer was separated, washed with saturated NH4Cl solution (500 mL) and brine solution (500 mL), dried over Na2SO4, filtered, and evaporated to give the crude compound. The crude product was washed with diethyl ether to give ((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)tert-butyl carbamate (27 g, yield: 24%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ13.45(s,1H),8.03(d,J=7.2Hz,1H),7.44(t,J=7.6Hz,1H),7.22- 7.16(m,2H),7.06(t,J=7.2Hz,1H),4.18(d,J=5.6Hz,2H),3.94(s,3H),1.38(s,9H).LC-MS m / z(M+H):305.0.

[0229] Step 3: Synthesis of (5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride:

[0230]

[0231] 4M HCl / 1,4-dioxane-HCl (54 mL, 2 vol) was added to a stirred solution of ((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)carbamate (27 g, 89 mmol) in DCM (150 mL) for 10 minutes at 0 °C. The reaction mixture was stirred at room temperature for 4 h. TLC showed the completion of the starting material and the formation of the polar point (5% MeOH / DCM). After the reaction was complete, the mixture was diluted with petroleum ether (200 mL), filtered to form a free solid, washed with diethyl ether (200 mL), and dried under vacuum to give (5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (25 g, yield: 99%) as a grayish-white solid. 1H NMR(400MHz, DMSO-d6)δ8.66(brs,3H),8.05(d,J=8.0Hz,1H),7.51-7.47(m,1H),7.22(d,J= 8.4Hz,1H),7.10(t,J=7.6Hz,1H),6.01(brs,3H),4.13(d,J=5.6Hz,2H),3.95(s,3H).LC-MS m / z(M+H):205.1.

[0232] Step 4: Synthesis of 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide:

[0233]

[0234] At 0 °C, EDC·HCl (30 g, 156 mmol), HOBt (21 g, 156 mmol), and (5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (25 g, 104 mmol) were added to a stirred solution of 2-(difluoromethoxy)benzoic acid (20 g, 104 mmol) in DCM (500 mL), followed by the addition of triethylamine (44 mL, 312 mmol). The reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the solid was filtered through a diatomaceous earth bed, and the filtrate was washed once with saturated NaHCO3 solution (500 mL), saturated NH4Cl solution (1 Lit), and brine solution. The organic layer was separated, dried over Na2SO4, and concentrated under reduced pressure to give the crude product. The crude product was ground with acetonitrile (500 mL), stirred for 1 h, filtered, washed with diethyl ether (100 mL), and dried under vacuum to give 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (19 g, yield: 56%), which was a grayish-white solid. 1 H NMR (400MHz, DMSO-d6): δ13.55(s,1H),8.76(bs,1H),8.07(d,J=7.6Hz,1H),7.61(d,J=7.6Hz,1H),7.5 2(t,J=7.6Hz,1H),7.45(t,J=8.0Hz,1H),7.36-6.99(m,5H),4.53(d,J=5.6Hz,2H),3.94(s,3H).LC-MS m / z(M+H):374.9.

[0235] Alternatives to compound 126 for the preparation of 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide:

[0236]

[0237] Step 1: Synthesis of N-(cyanomethyl)-2-(difluoromethoxy)benzamide

[0238]

[0239] HATU (15.1 g, 39.8 mmol), DIPEA (10.3 g, 79 mmol), and 2-aminoacetonitrile HCl (2.4 g, 26 mmol) were added to a stirred solution of 2-(difluoromethoxy)benzoic acid (5 g, 26 mmol) in DMF (20 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 16 h. After the reaction was confirmed to be complete by TLC, the reaction mixture was diluted with ice-cold water (50 mL) and extracted with EtOAc (2 x 50 mL). The organic layer was separated, washed with ice-cold water (3 x 100 mL), then washed with brine solution (2 x 100 mL), and finally dried over Na2SO4. The solution was concentrated under reduced pressure to give a crude compound. The crude product was dissolved in diethyl ether (50 mL), then ground with pentane (2 x 50 mL), the precipitate was filtered, and dried under vacuum to give N-(cyanomethyl)-2-(difluoromethoxy)benzamide (3.5 g, yield ~58%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6): 8.98 (t, J=5.2Hz, 10.4Hz, 1H), 7.56 (t, J=8Hz, 16.4Hz, 2H), 7.33 (t, J=8Hz,15.6Hz,1H),7.26(d,J=8Hz,1H)6.99(d,J=73.6Hz,1H),4.29(d,J=5.6Hz,2H).LC-MS m / z(MH):227.1

[0240] Stage 2: Synthesis of 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide

[0241]

[0242] Potassium carbonate (0.95 g, 6.89 mmol) was added to a stirred solution of N-(cyanomethyl)-2-(difluoromethoxy)benzamide (3.5 g, 13.78 mmol) and 2-methoxybenzoylhydrazine (3.44 g, 20.6 mmol) in n-BuOH (20 mL). The resulting reaction mixture was heated at 110 °C for 16 h. After confirming the completion of the reaction by TLC, the reaction mixture was evaporated under vacuum and diluted with water (50 mL), followed by extraction with EtOAc (2 × 50 mL). The organic layer was separated, washed with brine (20 mL) and water (50 mL), and finally dried over Na₂SO₄ and concentrated to give a brown crude compound. The crude product was diluted with acetonitrile (5 mL), stirred for 15 minutes, and the precipitated white solid was filtered off. The solid was washed with diethyl ether (2 x 20 mL) to give 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (BRG-0399) (1.9 g, yield ~37%), which was a grayish-white solid. 1 H NMR (400MHz, DMSO-d6): δ13.5(s,1H),8.76(s,1H),8.06(d,J=7.6Hz,1H),7.60(d,J=7.2Hz,1H),7.52(t,J=7. 6Hz,15.2Hz,1H),7.45(t,J=7.6Hz,15.2Hz,1H),7.36-6.99(m,5H),4.52(d,J=5.2Hz,2H),3.94(s,3H).LC-MS m / z(MH):375.1.

[0243] The following compounds in Table 9 were prepared using a method similar to that used for compound 126 described above, with appropriate starting materials.

[0244] Table 9

[0245]

[0246]

[0247]

[0248] Synthesis of N-((5-(3-methoxypyridin-2-yl)-1H-1,2,4-triazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (compound 34):

[0249]

[0250] Step 1: Methyl 3-methoxypyridinecarboxylate

[0251]

[0252] K₂CO₃ (2.48 g, 17.97 mmol) was added to a stirred solution of 3-methoxypyridinecarboxylic acid (1 g, 7.19 mmol) in acetone (10 mL) at 0 °C, followed by the addition of methyl iodine (2.23 g, 15.75 mmol). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was concentrated and diluted with H₂O (25 mL) and extracted with EtOAc (2 x 15 mL). The combined organic layers were dried over Na₂SO₄ and concentrated on a rotary evaporator to give the crude compound. The crude product was purified by rapid column chromatography to give methyl 3-methoxypyridinecarboxylate as a yellow slurry (570 mg, 47.50%). 1 H NMR (400MHz, DMSO): δ8.16 (dd, J=4.5, 1.1Hz, 1H), 7.64 (d, J=8.6Hz, 1H), 7.54 (dd, J=8.6, 4.5Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H). LC-MS (ESI): m / z 168.1(M+H) +

[0253] Step 2: 3-Methoxypyridinecarboxylhydrazine

[0254]

[0255] Hydrazine hydrate (213 mg, 6.66 mmol) was added to a stirred solution of methyl 3-methoxypyridinecarboxylate (560 mg, 3.33 mmol) in ethanol (10 mL) at room temperature, and the mixture was stirred at 70 °C for 12 h. After the reaction was complete, the mixture was concentrated and the resulting crude compound was ground with petroleum ether to give 3-methoxypyridinecarboxyhydrazide as a brown slurry (530 mg, 94.6%). 1 H NMR (400MHz, DMSO): δ9.40 (s, 1H), 8.10 (d, J = 4.5Hz, 1H), 7.53 (d, J = 8.4Hz, 1H), 7.43 (dd, J = 8.5, 4.6Hz, 1H), 4.43 (s, 2H), 3.79 (s, 3H). LC-MS (ESI): m / z 168.2(M+H) +

[0256] Step 3: N-(cyanomethyl)-2-(trifluoromethoxy)benzamide

[0257]

[0258] HATU (5.5 g, 14.56 mmol) and DIPEA (3.76 g, 29.12 mmol) were added to a stirred solution of 2-(trifluoromethoxy)benzoic acid (2 g, 9.7 mmol) in DMF (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, then 2-aminoacetonitrile 2 (897 mg, 9.70 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was diluted with ice-cold water (100 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over Na2SO4 and concentrated under vacuum. The crude product was purified by combi-flash column chromatography (elution: 40% EtOAc in hexane) to give N-(cyanomethyl)-2-(trifluoromethoxy)benzamide as a white solid (1.8 g, 82.19%). 1 H NMR (400MHz, DMSO): δ9.18 (s, 1H), 7.65-7.60 (m, 2H), 7.48 (dd, J=14, 3.5Hz, 2H), 4.30 (d, J=5.6Hz 2H). LC-MS (ESI): m / z 245.1 (M+H) +

[0259] Step 4: N-((5-(3-methoxypyridin-2-yl)-1H-1,2,4-triazol-3-yl)methyl)-2-(trifluoromethyl)benzamide

[0260]

[0261] 3-Methoxypyridinecarboxylhydrazine (44.34 mg, 0.26 mmol) and K₂CO₃ (15.20 mg, 0.11 mmol) were added to a stirred solution of N-(cyanomethyl)-2-(trifluoromethoxy)benzamide / n-butanol (2 mL), and the mixture was stirred in a microwave oven at 165 °C for 30 min. After the reaction was complete, the reaction mixture was diluted with cold water and extracted with EtOAc (2 x 10 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under vacuum to give a crude compound. The crude compound was purified by preparative TLC to give N-((5-(3-methoxypyridin-2-yl)-1H-1,2,4-triazol-3-yl)methyl)-2-(trifluoromethyl)benzamide as a grayish-white solid (30 mg, 34.5%). 1H NMR (400MHz, DMSO): δ14.01(s,1H),8.95(s,1H),8.27(s,1H),7.69–7.56(m,3H),7.52–7.39(m,3H),4.54(s,2H),3.92(s,3H).LC-MS(ESI):m / z 394.1(M+H) +

[0262] Synthesis of 2-(difluoromethoxy)-N-(1-(5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)cyclopropyl)benzamide (compound 118):

[0263]

[0264] Step 1: N-(1-Cyanocyclopropyl)-2-(Difluoromethoxy)benzamide

[0265]

[0266] TEA (3.0 mL, 21.28 mmol), EDC·HCl (1.52 mg, 7.98 mmol), and HOBt (1.07 mg, 7.98 mmol) were added to a stirred solution of 2-(difluoromethoxy)benzoic acid (1 g, 5.32 mmol) and 1-aminocyclopropane-1-carboxynitrile hydrochloride (747 mg, 6.40 mmol) in DMF (20 mL) at 0 °C. The resulting reaction mixture was then stirred at room temperature for 12 h. After the reaction was complete, the mixture was diluted with ice-cold water and extracted with EtOAc. The combined organic layers were washed with ice-cold water (3 × 100 mL), brine (2 × 100 mL), and dried over Na₂SO₄. The crude product was concentrated and then purified by rapid column chromatography to give N-(1-cyanocyclopropyl)-2-(difluoromethoxy)benzamide (560 mg, yield: 42%) as a grayish-white solid. 1 HNMR (400MHz, DMSO): δ9.20 (s, 1H), 7.51-7.56 (m, 2H), 6.94-7.34 (m, 3H), 1.53-1.57 (m, 2H), 1.18-1.22 (m, 2H). LC-MS (ESI): m / z 400.1 (M+H) +

[0267] Step 2: 2-(difluoromethoxy)-N-(1-(5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)cyclopropyl)benzamide

[0268]

[0269] K₂CO₃ (100 mg, 0.72 mmol) was added to a stirred solution of N-(1-cyanocyclopropyl)-2-(difluoromethoxy)benzamide (200 mg, 0.79 mmol) and 2-methoxybenzoylhydrazine (200 mg, 1.2 mmol) in n-BuOH at room temperature. The resulting reaction mixture was heated at 120 °C for 16 h. After the reaction was complete, the mixture was concentrated under vacuum, diluted with water, and extracted with EtOAc. The organic layer was washed with brine and water, and finally dried over Na₂SO₄. The solution was concentrated on a rotary evaporator and then purified by rapid column chromatography to give 2-(difluoromethoxy)-N-(1-(5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)cyclopropyl)benzamide (50 mg, yield: 16%) as a grayish-white solid. 1 H NMR (400MHz, DMSO): δ13.37(s,1H),9.04(s,1H),7.99(d,J=7.6Hz,1H),7.58(d,J=7.6Hz,1H),7.52(t,J=8.0H z,1H),7.44(t,J=7.2Hz,1H),6.94-7.37(m,6H),3.93(s,3H),1.41(s,2H),1.20-.132(m,4H).LC-MS(ESI):m / z 400.1(M+H) +

[0270] Synthesis of 2-(difluoromethoxy)-5-fluoro-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 138):

[0271]

[0272] Step 1: 2-(difluoromethoxy)-5-fluorobenzaldehyde:

[0273]

[0274] 4-Fluoro-2-hydroxybenzaldehyde (1 g, 7.14 mmol) was added to a stirred solution of KOH (8.09 g, 142.8 mmol) in acetonitrile (20 mL) and water (20 mL) at -20 °C, followed by dropwise addition of diethyl (bromodifluoromethyl)phosphonate (3.80 g, 14.28 mmol) over 30 min. After the reaction was complete, the mixture was diluted with EtOAc (20 mL), the organic layer was separated, and dried over Na2SO4. The organic layer was concentrated, and the crude compound was then purified by rapid column chromatography (elution: 10% EtOAc in hexane) to give 2-(difluoromethoxy)-5-fluorobenzaldehyde as a yellow slurry (700 mg, 51.8%). 1H NMR (400MHz, DMSO): δ10.20 (d, J=2.8Hz, 1H), 7.65 (ddd, J=9.0, 8.0, 3.3Hz, 1H), 7.59 (dd, J=8.3, 3.2Hz, 1H), 7.46 (dd, J=9.0, 4.2Hz, 1H), 7.32 (t, J=73.6Hz, 1H).

[0275] Step 2: 2-(difluoromethoxy)-5-fluorobenzoic acid:

[0276]

[0277] Sodium dihydrogen phosphate (1.02 g, 6.56 mmol) was added to a vigorously stirred solution of 2-(difluoromethoxy)-5-fluorobenzaldehyde (500 mg, 2.62 mmol) in THF:tert-butanol:H₂O (10 mL) at 0 °C, followed by the sequential addition of 2-methyl-2-butene (473.4 mL, 2.29 mmol) and NaClO₂. The mixture was warmed to room temperature and stirred for 1 h. After the reaction was complete, the reaction mixture was acidified with 1N HCl (5 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were dried over Na₂SO₄ and concentrated to give 2-(difluoromethoxy)-5-fluorobenzoic acid as a white solid (400 mg, 73.93%). 1 H NMR (400MHz, DMSO): δ13.49(s,1H),7.61(dd,J=8.7,3.2Hz,1H),7.49(ddd,J=8.9,8 .0,3.3Hz,1H),7.34(dd,J=9.0,4.5Hz,1H),7.10(t,J=74.3Hz,1H).GC-MS(ESI):m / z 206(M) +

[0278] Step 3: 2-Difluoromethoxy-5-fluoro-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide:

[0279]

[0280] EDC·HCl (113.02 mg, 0.72 mmol), HOBt (98.37 mg, 0.72 mmol), and TEA (147.07 mg, 1.45 mmol) were added to a stirred solution of 2-(difluoromethoxy)-5-fluorobenzoic acid (100 mg, 0.48 mmol) in dichloromethane (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, and then (5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (139.8 mg, 0.58 mmol) was added. The mixture was warmed to room temperature and stirred for 12 h. After completion, the reaction mixture was washed once each with saturated NH4Cl (20 mL), saturated NaHCO3 solution (20 mL), and brine (20 mL). The combined organic layers were dried over Na2SO4 and concentrated. The crude product was purified by rapid column chromatography (elution: 40% EtOAc in hexane) to give compound 2-(difluoromethoxy)-5-fluoro-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide as a grayish-white solid (60 mg, 31.5%). 1 H NMR (400MHz, DMSO): δ13.56(s,1H),8.89(s,1H),8.06(d,J=7.6Hz,1H),7.49–7.36(m,4H),7.31(t,J=4.3Hz,1H),7.19(d, J=8.4Hz,1H),7.12(t,J=68Hz,1H)7.07(t,J=7.5Hz,1H),6.94(s,2H),4.53(d,J=5.6Hz,2H),3.95(s,3H).LC-MS(ESI):m / z 393.3(M+H) +

[0281] Synthesis of 2-(difluoromethoxy)-5-fluoro-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide

[0282]

[0283] EDC.HCl (113.02 mg, 0.72 mmol), HOBt (98.37 mg, 0.72 mmol), and TEA (147.07 mg, 1.45 mmol) were added to a stirred solution of 2-(difluoromethoxy)-5-fluorobenzoic acid (100 mg, 0.48 mmol) in dichloromethane (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, and then (5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methoxyamine hydrochloride (129.36 mg, 0.58 mmol) was added. The mixture was warmed to room temperature and stirred for 12 h. After the reaction was complete, the reaction mixture was washed once each with saturated NH4Cl (20 mL), saturated NaHCO3 solution (20 mL), and brine (20 mL). The combined organic layers were dried over Na2SO4 and concentrated. The crude product obtained therefrom was purified by rapid column chromatography (elution buffer: 40% EtOAc in hexane) to give 2-(difluoromethoxy)-5-fluoro-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide, as a grayish-white solid (40 mg, 20.10%). 1 H NMR (400MHz, DMSO): δ13.70(s,1H),8.90(s,1H),7.77(s,1H),7.46–7.37(m,2H),7.31(t,J=4.4Hz,2H) ,7.21(dd,J=8.8,4.3Hz,1H),7.13(t,J=64Hz,1H),4.53(d,J=5.5Hz,2H),3.94(s,3H).LC-MS(ESI):m / z 411.1(M+H) + .

[0284] Synthesis of 2-(difluoromethoxy)-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)nicotinamide (compound 143) and 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)nicotinamide (compound 144)

[0285] Step 1: 2-Hydroxynicotinic acid methyl ester:

[0286]

[0287] Thionyl chloride (5 mL) was added to a stirred solution of 2-hydroxynicotinic acid (5 g, 27.8 mmol) in methanol (75 mL) at 0 °C, and the mixture was stirred at 75 °C for 12 h. After the reaction was complete, the reaction mixture was concentrated under vacuum to give a crude product. The mixture was alkalized with saturated NaHCO3 solution (20 mL) and extracted with 10% methanol / dichloromethane (2 × 25 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give methyl 2-hydroxynicotinic acid as a grayish-white solid (2.3 g, 41.8%). 1 H NMR (400MHz, DMSO): δ12.08 (s, 1H), 8.04 (dd, J = 7.1, 2.2Hz, 1H), 7.65 (dd, J = 6.3, 2.2Hz, 1H), 6.25 (t, J = 6.7Hz, 1H), 3.71 (s, 3H). LC-MS (ESI): m / z 154.1(M+H)

[0288] Step 2: 2-(difluoromethoxy)nicotinic acid methyl ester:

[0289]

[0290] Sodium hydride (357 mg, 14.9 mmol) was added in portions to a stirred solution of methyl 2-hydroxynicotinic acid (1.9 g, 12.4 mmol) in DMF at 0 °C and stirred for 10 min. Then, 2-chloro-2,2-difluoroacetic acid (1.94 g, 14.9 mmol) was added and the mixture was stirred at 125 °C for 2 h. After the reaction was complete, the mixture was diluted with ice-cold water (25 mL) and extracted with 10% methanol / dichloromethane (2 × 10 mL). The combined organic layers were concentrated under reduced pressure to give the crude compound. This crude compound was purified by rapid column chromatography to give methyl 2-(difluoromethoxy)nicotinic acid as a light brown solid. 1 H NMR (400MHz, DMSO): δ8.48 (dd, J=4.9, 1.9Hz, 1H), 8.33 (dd, J=7.6, 1.9Hz, 1H), 7 .79(t,J=72.2Hz,2H),7.42(dd,J=7.6,4.9Hz,1H),3.87(s,3H).LC-MS(ESI):m / z 154.1(M+H)

[0291] Step 3: 2-(difluoromethoxy)nicotinic acid:

[0292]

[0293] LiOH (122.8 mg, 5.1 mmol) was added to a stirred solution of methyl 2-(difluoromethoxy)nicotinic acid (260 mg, 1.27 mmol) in THF:H₂O (10 mL: 5 mL) at 0 °C, and the reaction mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction mixture was concentrated, diluted with H₂O (10 mL), and extracted with EtOAc (2 × 10 mL). The aqueous layer was separated and acidified with 1 N HCl solution (5 mL), followed by extraction with 10% methanol / dichloromethane (2 × 15 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under vacuum to give 2-(difluoromethoxy)nicotinic acid as a grayish-white solid (60 mg, 24.7%). 1 H NMR (400MHz, DMSO): δ13.44 (s, 1H), 8.43 (dd, J = 4.9, 1.9 Hz, 1H), 8.30 (dd, J = 7.6, 1.9 Hz, 1H), 7.78 (t, J = 72.4 Hz, 1H), 7.39 (dd, J = 7.6, 4.9 Hz, 1H).

[0294] Step 4: 2-(difluoromethoxy)-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)nicotinamide (compound 143):

[0295]

[0296] EDC.HCl (106.4 mg, 0.55 mmol), HOBt (75.01 mg, 0.55 mmol), and triethylamine (112 mg, 1.11 mmol) were added to a stirred solution of 2-(difluoromethoxy)nicotinic acid (70 mg, 0.37 mmol) in CH2Cl2 (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, and (5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (95.74 mg, 0.37 mmol) was added, followed by stirring at room temperature for 12 h. After the reaction was complete, the mixture was washed successively with saturated NH4Cl (20 mL), saturated NaHCO3 solution (20 mL), and brine (20 mL). The combined organic layers were dried over Na2SO4 and concentrated to give the crude compound. The crude product was then purified by rapid column chromatography (elution buffer: 50% EtOAc in hexane) to obtain 2-(difluoromethoxy)-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)nicotinamide, which was a grayish-white solid (60 mg, 41.23%). 1H NMR (400MHz, DMSO): δ13.73(s,1H),8.83(s,1H),8.35(dd,J=4.9,1.9Hz,1H),8.11(dd,J=7.5,1.8Hz,1H),7.74(t,J=72.1Hz,1H) ,7.81(dd,J=9.4,3.2Hz,1H),7.42–7.29(m,2H),7.22(dd,J=9.1,4.5Hz,1H),4.56(d,J=5.5Hz,2H),3.95(s,3H).LC-MS(ESI):m / z 394.2(M+H)

[0297] Step 5: 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)nicotinamide (compound 144):

[0298]

[0299] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (76.02 mg, 0.39 mmol), hydroxybenzotriazole (53.58 mg, 0.39 mmol), and triethylamine (80.10 mg, 0.79 mmol) were added to a stirred solution of 2-(difluoromethoxy)nicotinic acid (50 mg, 0.26 mmol) in CH2Cl2 (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, and then (5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (63.6 mg, 0.26 mmol) was added and stirred at room temperature for 12 h. After the reaction was complete, the mixture was diluted with aqueous NH4Cl and extracted with dichloromethane. The organic layer was washed successively with 1N HCl, aqueous NH4CO3, and brine. The organic layer was dried with MgSO4, concentrated under vacuum, and the crude compound obtained was purified by rapid column chromatography to give the product as a white solid (60 mg, 76%). 1 H NMR (400MHz, DMSO): δ13.58(s,1H),8.82(s,1H),8.35(dd,J=4.9,1.8Hz,1H),8.09(t,J=8.1Hz,2H),7.73(t,J=72.1Hz,1H),7.46(t,J=7.5Hz,1 H),7.37(dd,J=7.5,4.9Hz,1H),7.19(d,J=8.4Hz,1H),7.07(t,J=7.5Hz,1H),4.56(d,J=5.4Hz,2H),3.95(s,3H).LC-MS(ESI):m / z376.1(M+H))

[0300] The following compounds in Table 10 were prepared using methods similar to those described above, with appropriate starting materials.

[0301] Table 10

[0302]

[0303]

[0304]

[0305] Synthesis of 2-(difluoromethoxy)-3-fluoro-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 135) and 2-(difluoromethoxy)-3-fluoro-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 136):

[0306]

[0307] Step 1: Methyl 2-(difluoromethoxy)-3-fluorobenzoate

[0308]

[0309] Acetonitrile (30 mL) was added to a cold-stirred solution of KOH (6.58 g, 117.5 mmol) in water (30 mL), and the solution was further cooled to -20 °C. Methyl 2-fluoro-6-hydroxybenzoate (1 g, 5.87 mmol) was added to the resulting mixture, followed by dropwise addition of diethyl (bromodifluoromethyl)phosphonate (3.13 g, 11.7 mmol), and the reaction mixture was stirred at -20 °C for 30 min. After the reaction was complete, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated under vacuum. The crude compound was purified by rapid column chromatography to give methyl 2-(difluoromethoxy)-3-fluorobenzoate as a yellow slurry (520 mg, 40.31%). 1 H NMR (400MHz, DMSO): δ7.69-7.64 (m, 2H), 7.47-7.43 (m, 1H), 7.12 (t, J = 73.6Hz, 1H), 3.98 (s, 3H).

[0310] Step 2: 2-(difluoromethoxy)-3-fluorobenzoic acid

[0311]

[0312] LiOH (954.5 mg, 22.7 mmol) was added to a stirred solution of methyl 2-(difluoromethoxy)-6-fluorobenzoate (500 mg, 2.27 mmol) in THF:H₂O (2:1) at 0 °C, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was acidified with 1N HCl, then extracted with 10% methanol / CH₂Cl₂ solution. The organic layer was dried with MgSO₄ and concentrated under vacuum to give 2-(difluoromethoxy)-3-fluorobenzoic acid as a grayish-white solid. 1 H NMR (400MHz, DMSO): δ13.53 (s, 1H), 7.70–7.57 (m, 2H), 7.44 (dt, J = 13.2, 6.6Hz, 1H), 7.10 (t, J = 73.8Hz, 1H).

[0313] Step 3: 2-(difluoromethoxy)-3-fluoro-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 135):

[0314]

[0315] EDC.HCl (139.07 mg, 0.72 mmol), HOBt (111.50 mg, 0.72 mmol), and triethylamine (147 mg, 1.45 mmol) were added to a stirred solution of 2-(difluoromethoxy)-3-fluorobenzoic acid (100 mg, 0.48 mmol) in CH2Cl2 (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, and (5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (125 mg, 0.48 mmol) was added, followed by stirring at room temperature for 12 h. After the reaction was complete, the mixture was diluted with aqueous NH4Cl and extracted with CH2Cl2. The organic layer was then washed successively with 1N HCl, aqueous NH4CO3, and brine. The organic layer was then dried over MgSO4 and concentrated under vacuum to obtain the crude product. The product was then purified by rapid column chromatography to obtain 2-(difluoromethoxy)-3-fluoro-N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide, a grayish-white solid (70 mg, 35.17 g / L). 1H NMR (400MHz, DMSO): δ13.71(s,1H),8.98(d,J=5.2Hz,1H),8.04-8.01(m,1H),7.89(dd,J=9.2,3.2Hz,1H),7.43( d,J=8.4Hz,1H),7.35-7.29(m,2H),7.23-7.20(m,1H),7.37(t,J=73.2Hz,1H),4.53(d,J=5.2Hz,2H),3.94(s,3H)

[0316] Step 4: 2-(difluoromethoxy)-6-fluoro-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 36):

[0317] EDC.HCl (139.07 mg, 0.72 mmol), HOBt (111.50 mg, 0.72 mmol), and triethylamine (147 mg, 1.45 mmol) were added to a stirred solution of 2-(difluoromethoxy)-3-fluorobenzoic acid (100 mg, 0.48 mmol) in dichloromethane (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, and ((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (116.53 mg, 0.48 mmol) was added, followed by stirring at room temperature for 12 h. After the reaction was complete, the mixture was diluted with aqueous NH4Cl solution and extracted with CH2Cl2. The organic layer was then washed successively with 1N HCl, aqueous NH4CO3 solution, and brine. The organic layer was then dried over MgSO4 and concentrated to give the crude product. The crude compound was purified by rapid column chromatography to give 2-(difluoromethoxy)-6-fluoro-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide as a white solid (70 mg, 36.75%). 1 H NMR (400MHz, DMSO): δ13.55(s,1H),8.97(s,1H),8.06(d,J=7.7Hz,1H),7.56–7.38(m,4H), 7.12(ddd,J=67.7,53.3,45.4Hz,3H),4.52(d,J=5.6Hz,2H),3.95(s,3H).LC-MS(ESI):m / z 393.29(M+H)

[0318] Examples of N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (compound 127):

[0319]

[0320] EDC.HCl (348.93 mg, 1.82 mmol), HOBt (278.72 mg, 1.82 mmol), and triethylamine (367.68 mg, 3.63 mmol) were added to a stirred solution of 2-(trifluoromethoxy)benzoic acid (250 mg, 1.21 mmol) in CH2Cl2 (10 mL) at 0 °C. The resulting mixture was stirred for 10 min, and (5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methylamine hydrochloride (313.12 mg, 1.21 mmol) was added, followed by stirring at room temperature for another 12 h. After the reaction was complete, the mixture was diluted with aqueous NH4Cl and extracted with CH2Cl2. The organic layer was then washed successively with 1N HCl, aqueous NH4CO3, and brine. The crude compound was then dried over MgSO4 and concentrated under vacuum to obtain a crude compound, which was purified by rapid column chromatography to obtain the product N-((5-(5-fluoro-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide, which was a grayish-white solid (300 mg, 60%). 1 H NMR (400MHz, DMSO): δ13.68(s,1H),8.92(s,1H),7.79(d,J=7.8Hz,1H),7.60(dd,J=16.6,7.6Hz,2H),7.4 5(dd,J=21.1,7.7Hz,2H),7.32(s,1H),7.22(s,1H),4.52(d,J=5.3Hz,2H),3.94(s,3H).LC-MS(ESI):m / z 411.24(M+H)

[0321] Synthesis of 2-(difluoromethoxy)-N-((5-(2-hydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 60):

[0322]

[0323] Step 1: Methyl 2-hydroxybenzoate

[0324]

[0325] H₂SO₄ (0.8 mL) was added to a stirred solution of 2-hydroxybenzoic acid (1 g, 6.57 mmol) in methanol at 0 °C. The reaction mixture was then heated to 75 °C and stirred for 8 h. After the reaction was complete, the mixture was concentrated under vacuum, diluted with water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to give methyl 2-hydroxybenzoate (1.8 g, yield: 81%) as a colorless liquid. 1 H NMR (400MHz, DMSO-d6): δ10.49 (s, 1H), 7.83 (dd, J = 8.0Hz, 1.6Hz, 1H), 7.54–7.50 (m, 1H), 6.90-6.91 (m, 2H), 3.88 (s, 3H).

[0326] Step 2: 2-Hydroxybenzoylhydrazine

[0327]

[0328] Hydrazine hydrate (1 mL) was added to a stirred solution of methyl 2-hydroxybenzoate (1 g, 6.57 mmol) in ethanol at room temperature. The reaction mixture was heated under reflux for 3 h. After the reaction was complete, the mixture was concentrated under vacuum, diluted with water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to give 2-hydroxybenzoyl hydrazine (480 mg, yield: 48%) as a white solid. 1 H NMR (400MHz, DMSO-d6): δ12.50(brs,1H),10.05(brs,1H),15.60(dd,J=8.0Hz,1.2Hz ,1H),7.38–7.34(m,1H),6.89–8.82(m,2H),4.64(brs,2H).LC-MS(m / z):153.1(M+H)+

[0329] Step 3: 2-(difluoromethoxy)-N-((5-(2-hydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 60):

[0330]

[0331] K₂CO₃ (61 mg, 0.44 mmol) was added to a stirred solution of N-(cyanomethyl)-2-(difluoromethoxy)benzamide (200 mg, 0.88 mmol) and 2-hydroxybenzoylhydrazine (202 mg, 1.32 mmol) in n-BuOH at room temperature. The resulting reaction mixture was irradiated with microwave at 165 °C for 50 min. After the reaction was complete, the mixture was concentrated under vacuum to obtain a crude product, which was purified by rapid column chromatography to give 2-(difluoromethoxy)-N-((5-(2-hydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (110 mg, yield: 35%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6): δ14.18(brs,1H),11.42(brs,1H),8.85(brs,1H),7.92(brs,1H) ,7.64–7.53(m,2H),7.33–6.96(m,6H),4.61(d,J=2.8Hz,2H).LC-MS(m / z):361.1(M+H)+

[0332] Synthesis of 2-(difluoromethoxy)-N-((5-(2,3-dihydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 61):

[0333]

[0334] Step 1: Methyl 3-(benzyloxy)-2-hydroxybenzoate

[0335]

[0336] K₂CO₃ (3.3 g, 23.8 mmol) and benzyl bromide (0.85 mL, 7.14 mmol) were added to a stirred solution of methyl 2,3-dihydroxybenzoate (1 g, 5.95 mmol) in a mixture of chloroform and methanol. The reaction mixture was stirred at 60 °C for 8 h. After the reaction was complete, the mixture was concentrated under vacuum, diluted with water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to give a crude product, which was purified by rapid column chromatography to give methyl 5-(benzyloxy)-2-hydroxybenzoate (600 mg, yield: 38%) as a colorless liquid. 1H NMR (400MHz, DMSO-d6): δ10.09(s,1H),7.43(d,J=7.2Hz,2H),7.38(t,J=7.2Hz,2H),7.33–7.29(m, 2H),7.24–7.21(m,1H),6.93(d,J=8.8Hz,1H),5.05(s,2H),3.88(s,3H).LC-MS(m / z):259.07(M+H) +

[0337] Step 2: 3-(benzyloxy)-2-hydroxybenzoylhydrazine

[0338]

[0339] Hydrazine hydrate (1 mL) was added to a stirred solution of methyl 5-(benzyloxy)-2-hydroxybenzoate (600 mg, 2.32 mmol) in ethanol at room temperature. The reaction mixture was then refluxed for 3 h. After the reaction was complete, the mixture was concentrated under vacuum and the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to give 3-(benzyloxy)-2-hydroxybenzoyl hydrazine (430 mg, yield: 43%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6): δ9.21(s,1H),7.43(d,J=7.2Hz,2H),7.37(t,J=6.8Hz,2H),7.32–7.30(m,2H),7.09(dd, J=9.2Hz,3.2Hz,1H),7.08(d,J=8.8Hz,1H),5.07(s,2H),4.51(brs,2H),3.79(s,1H).LC-MS(m / z):273.17(M+H) +

[0340] Step 3: N-((5-(3-(benzyloxy)-2-hydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide

[0341]

[0342] K₂CO₃ (61 mg, 0.44 mmol) was added to a stirred solution of N-(cyanomethyl)-2-(difluoromethoxy)benzamide (200 mg, 0.88 mmol) and 3-(benzyloxy)-2-hydroxybenzoylhydrazine (300 mg, 1.16 mmol) in n-BuOH at room temperature. The reaction mixture was irradiated with microwave at 165 °C for 50 min. After the reaction was complete, the mixture was concentrated under vacuum to give a crude product. This crude product was then purified by rapid column chromatography to give N-((5-(3-(benzyloxy)-2-hydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide (120 mg, yield: 29%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6): δ14.37(s,1H),11.53(brs,1H),11.25(brs,1H),8.90(brs,1H),7.53–7.52(m,2H),7.47(d,J=7.6Hz,2 H),7.40–7.30(m,5H),7.25(d,J=7.6Hz,1H),7.14(s,1H),6.83(brs,1H),5.15(S,2H),4.65(brs,2H).LC-MS(m / z):467.2(M+H) +

[0343] Step 4: 2-(difluoromethoxy)-N-((5-(2,3-dihydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 61):

[0344]

[0345] Pd / C (100 mg) was added to a stirred solution of N-((5-(3-(benzyloxy)-2-hydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide (120 mg, 0.25 mmol) in ethanol at room temperature. The reaction mixture was stirred at room temperature under a hydrogen atmosphere of 50 Psi for 12 h. After the reaction was complete, the reaction mixture was filtered through a diatomaceous earth bed and concentrated under reduced pressure to obtain a crude product, which was purified by preparative HPLC to give 2-(difluoromethoxy)-N-((5-(2,3-dihydroxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (30 mg, yield: 18%) as a white solid. 1H NMR (400MHz, DMSO-d6): δ11.27(brs,1H),9.20(brs,1H),9.17(s,1H),7.60(d,J=7.2Hz,1H),7.55(t ,J=8.0Hz,1H),7.37–7.32(m,2H),7.26(d,J=8.4Hz,1H),7.15(s,1H),6.75(t,J=8Hz,1H),4.60(brs 2H),3.15(s,1H).LC-MS(m / z):377.10(M+H) +

[0346] Synthesis of 2-(difluoromethoxy)-N-((5-(5-hydroxy-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 63):

[0347]

[0348] Step 1: Methyl 5-(benzyloxy)-2-hydroxybenzoate

[0349]

[0350] K₂CO₃ (3.3 g, 23.8 mmol) and benzyl bromide (0.85 mL, 7.14 mmol) were added to a stirred solution of methyl 2,5-dihydroxybenzoate (1 g, 5.95 mmol) in chloroform and methanol. The reaction mixture was then stirred at 60 °C for 8 h. After the reaction was complete, the mixture was concentrated under vacuum, diluted with water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product obtained was purified by rapid column chromatography to give methyl 5-(benzyloxy)-2-hydroxybenzoate (470 mg, yield: 30%) as a colorless liquid. 1 H NMR (400MHz, DMSO-d6): δ10.09(s,1H),7.43(d,J=7.2Hz,2H),7.38(t,J=7.2Hz,2H),7.33–7.29(m, 2H),7.24–7.21(m,1H),6.93(d,J=8.8Hz,1H),5.05(s,2H),3.88(s,3H).LC-MS(m / z):259.07(M+H) +

[0351] Step 2: Methyl 5-(benzyloxy)-2-methoxybenzoate

[0352]

[0353] K₂CO₃ (1.29 g, 9.3 mmol) and methyl iodoforme (0.44 mL, 6.9 mmol) were added to a stirred solution of methyl 5-(benzyloxy)-2-hydroxybenzoate (1.2 g, 4.65 mmol) in DMF. The resulting reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to give methyl 5-(benzyloxy)-2-methoxybenzoate (1.0 g), a colorless liquid. 1 H NMR (400MHz, DMSO-d6): δ7.42(d,J=6.8Hz,2H),7.38(t,J=7.2Hz,2H),7.32(t,J=4.8Hz,1H),7.25(d,J=3.2Hz,1H),7 .15(dd,J=8.8Hz,3.2Hz,1H),7.07(d,J=8.0Hz,1H),5.07(s,2H),3.76(s,3H),3.74(s,3H).LC-MS(m / z):273.10(M+H) +

[0354] Step 3: 5-(benzyloxy)-2-methoxybenzoylhydrazine

[0355]

[0356] Hydrazine hydrate (1 mL) was added to a stirred solution of methyl 5-(benzyloxy)-2-methoxybenzoate (1 g, 3.6 mmol) in ethanol at room temperature. The resulting reaction mixture was refluxed for 3 h. After the reaction was complete, the mixture was concentrated under vacuum, diluted with water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to give 5-(benzyloxy)-2-methoxybenzoyl hydrazine (430 mg, yield: 43%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6): δ9.21(s,1H),7.43(d,J=7.2Hz,2H),7.37(t,J=6.8Hz,2H),7.32–7.30(m,2H),7.09(dd, J=9.2Hz,3.2Hz,1H),7.08(d,J=8.8Hz,1H),5.07(s,2H),4.51(brs,2H),3.79(s,1H).LC-MS(m / z):273.17(M+H) +

[0357] Step 4: N-((5-(5-(benzyloxy)-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide

[0358]

[0359] K₂CO₃ (61 mg, 0.44 mmol) was added to a stirred solution of N-(cyanomethyl)-2-(difluoromethoxy)benzamide (200 mg, 0.88 mmol) and 5-(benzyloxy)-2-methoxybenzoylhydrazine (360 mg, 1.32 mmol) in n-BuOH at room temperature. The reaction mixture was then microwave-irradiated at 165 °C for 50 min. The resulting reaction mixture was then concentrated under vacuum to give a crude product (200 mg), which was used in the next step without further purification.

[0360] Step 5: 2-(difluoromethoxy)-N-((5-(5-hydroxy-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 63):

[0361]

[0362] Pd / C (160 mg) was added to a stirred solution of N-((5-(5-(benzyloxy)-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide (200 mg, 0.42 mmol) in ethanol at room temperature. The reaction mixture was stirred at room temperature under a hydrogen atmosphere (50 Psi) for 12 h. The resulting reaction mixture was filtered through a diatomaceous earth bed and concentrated under reduced pressure to give a crude product. It was further purified by preparative HPLC to give 2-(difluoromethoxy)-N-((5-(5-hydroxy-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (30 mg, yield: 18%) as a white solid. 1 H NMR (400MHz, DMSO-d6): δ13.45(s,1H),9.20(s,1H),8.75(brs,1H),7.60(d,J=7.6Hz,1H),7.54–7.50(m,2H),7.35–7.33(m,1H),7.24 (d,J=8.4Hz,1H),7.16(s,1H),7.01–6.98(m,1H),6.82(d,J=3.2Hz,1H),4.52(d,J=5.6Hz,2H),3.85(s,3H).LC-MS(m / z):391.10(M+H) +

[0363] Synthesis of 2-(difluoromethoxy)-N-((5-(3-hydroxy-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 62):

[0364]

[0365] Step 1: Synthesis of methyl 3-(benzyloxy)-2-hydroxybenzoate

[0366]

[0367] K₂CO₃ (410 mg, 2.973 mmol) was added to a stirred solution of methyl 2,3-dihydroxybenzoate (1) (500 mg, 2.973 mmol) in acetone (10 mL) at 0 °C, followed by the addition of (bromomethyl)benzene (423.8 mg, 3.568 mmol) and tetrabutylammonium bromide (191.7 mg, 0.594 mmol). The reaction mixture was stirred at room temperature for 12 h. The resulting reaction mixture was concentrated under reduced pressure, and the crude product obtained was diluted with cold water and extracted with ethyl acetate (2 × 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product obtained was purified by column chromatography (using 2% EtOAc in n-hexane) to give methyl 3-(benzyloxy)-2-hydroxybenzoate (100 mg, 13%) as a white solid. 1 H NMR (400MHz, DMSO-d6): δ10.60(s,1H),7.45(d,J=7.2Hz,2H),7.40–7.29(m,4H),7.27( d,J=8Hz,1H),6.84(t,J=8Hz,1H),5.14(s,2H),3.89(s,3H).LC-MS(m / z):259.30(M+H) +

[0368] Step 2: Synthesis of methyl 3-(benzyloxy)-2-methoxybenzoate

[0369]

[0370] K₂CO₃ (410 mg, 2.973 mmol) was added to a stirred solution of methyl 3-(benzyloxy)-2-hydroxybenzoate (500 mg, 1.937 mmol) in DMF (20 mL) at 0 °C, followed by the addition of methyl iodoforme (412.4 mg, 2.905 mmol). The reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was quenched with ice water and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The resulting solid was ground with diethyl ether and dried under vacuum to give methyl 3-(benzyloxy)-2-methoxybenzoate (450 mg, 85.3%) as a grayish-white solid. 1H NMR (400MHz, DMSO-d6): δ17.45(d,J=7.2Hz,2H),7.39(t,J=7.2Hz,2H),7.35–7.31(m,2H),7 .09(dd,J=8.4Hz,1.6Hz,1H),7.04(t,J=7.6Hz,1H),5.14(s,2H),3.95(s,3H),3.91(s,3H).

[0371] Step 3: Synthesis of 3-(benzyloxy)-2-methoxybenzoylhydrazine

[0372]

[0373] Hydrazine hydrate (0.5 mL) was added to a stirred solution of methyl 3-(benzyloxy)-2-methoxybenzoate (450 mg, 1.65 mmol) in ethanol (20 mL) at 0 °C. The resulting reaction mixture was stirred at 90 °C for 12 h and concentrated. The residue obtained was diluted with ice-cold water and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The resulting solid was ground with diethyl ether and dried under reduced pressure to give 3-(benzyloxy)-2-methoxybenzoyl hydrazine (250 mg, 55.5%) as a grayish-white solid.

[0374] 1 H NMR (400MHz, DMSO-d6): δ9.27(s,1H),7.47(d,J=7.6Hz,2H),7.40(t,J=7.2Hz,2H),7.33(t,J=7.2H z,1H),7.21(dd,J=8.0Hz,1.6Hz,1H),7.09–7.01(m,2H),5.15(s,2H),4.47(brs,2H),2.93(s,3H).

[0375] Step 4: Synthesis of N-((5-(3-(benzyloxy)-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide

[0376]

[0377] K₂CO₃ (76.2 mg, 0.552 mmol) was added to a stirred solution of N-(cyanomethyl)-2-(difluoromethoxy)benzamide (250 mg, 1.105 mmol) in n-BuOH (10 mL) at 0 °C, followed by the addition of 3-(benzyloxy)-2-methoxybenzoylhydrazine (331 mg, 1.215 mmol). The resulting reaction mixture was microwave-stirred at 165 °C for 50 min. After the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product obtained therefrom was purified by column chromatography (using 60% EtOAc in n-hexane) to give N-((5-(3-(benzyloxy)-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide (170 mg, 32%) as a brown solid. 1 H NMR (400MHz, DMSO-d6): δ13.63(s,1H),8.54(t,J=5.6Hz,1H),7.60(t,J=8.4Hz,2H),7.54–7.49(m,3H),7.42(t,J=7.2Hz,2H),7.36– 7.30(m,2H),7.26(t,J=8.8Hz,2H),7.17(t,J=7.6Hz,2H),5.19(s,2H),4.53(d,J=5.6Hz,2H),3.84(s,3H).LC-MS(m / z):481.20(M+H) +

[0378] Step 5: Synthesis of 2-(difluoromethoxy)-N-((5-(3-hydroxy-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (compound 62):

[0379]

[0380] Pd / C (170 mg) was added to a stirred solution of N-((5-(3-(benzyloxy)-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)-2-(difluoromethoxy)benzamide (170 mg, 0.354 mmol) in methanol (20 mL). The reaction mixture was stirred under a hydrogen balloon at room temperature for 12 h. After the reaction was complete, the mixture was filtered through a short diatomaceous earth bed, washed with methanol, dried over anhydrous Na2SO4, and concentrated. The crude product thus obtained was purified by column chromatography (using 50% EtOAc in n-hexane) to give 2-(difluoromethoxy)-N-((5-(3-hydroxy-2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)methyl)benzamide (45 mg, 32.6%) as a brown solid. 1 H NMR (400MHz, DMSO-d6): δ9.71(brs,1H),8.79(t,J=5.2Hz,1H),7.62(dd,J=8.0Hz,1.6Hz,1H),7.55–7.50(m,1H),7.39–7.31 (m,2H),7.24(d,J=8.4Hz,1H),7.17(s,1H),7.03–6.95(m,2H),4.54(d,J=7.6Hz,2H),3.78(s,3H).LC-MS(m / z):391.1(M+H) +

[0381] Synthesis of N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)sulfonyl)-2-(trifluoromethoxy)benzamide (compound 123) and 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)sulfonyl)benzamide (compound 124):

[0382]

[0383] Step 1: 5-(2-methoxyphenyl)-4H-1,2,4-triazol-3-thiol:

[0384]

[0385] Trimethyl isothiocyanate (1.97 g, 15.04 mmol) was added to a stirred solution of 2-methoxybenzoyl hydrazine (2.5 g, 15.04 mmol) in ethanol (25 mL) at 0 °C, and the reaction mixture was heated at 90 °C for 4 h. Then, 4 M NaOH solution (25 mL) was added to the resulting reaction mixture, and the mixture was stirred at 90 °C for another 4 h. The reaction mixture was then concentrated, diluted with H₂O (10 mL), and acidified with HCl (40 mL, 4 M). The resulting precipitate was filtered and dried under vacuum to give 5-(2-methoxyphenyl)-4H-1,2,4-triazol-3-thiol (2.1 g mg, 67.37%) as a grayish-white solid. 1 H NMR (400MHz, DMSO) δ13.61(s,1H),13.11(s,1H),7.63(d,J=6.4Hz,1H),7.49(t,J=7. 2Hz,1H),7.15(d,J=8.4Hz,1H),7.04(t,J=7.2Hz,1H),3.83(s,3H).LC-MS(ESI):m / z 208.1(M+H)

[0386] Step 2: 5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-sulfonamide:

[0387]

[0388] 4M HCl (10 mL) and 4% NaOCl (10 mL) were added dropwise to a stirred solution of 5-(2-methoxyphenyl)-4H-1,2,4-triazol-3-thiol (500 mg, 15.04 mmol) in dichloromethane (10 mL) at 0 °C while maintaining the temperature below 5 °C. The mixture was then stirred for another 15 min at the same temperature. The organic layer was separated from the resulting mixture, and an aqueous solution of NH4OH was added and stirred for 12 h. After the reaction was complete, the organic layer was collected and concentrated under reduced pressure. Water was removed by co-evaporation with toluene to give 5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-sulfonamide as a grayish-white solid (2.1 g, 67.37%). LC-MS (ESI): m / z 255.1 (M+H)

[0389] Step 3: N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)sulfonyl)-2-(trifluoromethoxy)benzamide (compound 123):

[0390]

[0391] EDC·HCl (113.09 mg, 0.589 mmol), HOBt (79.58 mg, 0.588 mmol), DMAP (5 mg), and triethylamine (98.98 mg, 0.98 mmol) were added to a stirred solution of 5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-sulfonamide (100 mg, 0.393 mmol) and 2-(trifluoromethoxy)benzoic acid (81.06 mg, 0.393 mmol) in dichloromethane (20 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 12 h and concentrated. The resulting residue was diluted with H₂O (10 mL) and extracted with 10% methanol / dichloromethane (3 × 15 mL). The combined organic layers were dried over Na₂SO₄ and concentrated to give the crude compound. The crude compound was purified by preparative HPLC to obtain N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)sulfonyl)-2-(trifluoromethoxy)benzamide, which was a grayish-white solid (14 mg, 8%). 1 H NMR (400MHz, DMSO) δ13.75(s,1H),8.08(d,J=6.8Hz,1H),7.33(d,J=8.0Hz,1H),7.46(t,J=7.2Hz,1H),7.40(t, J=8.4Hz,1H),7.32(t,J=7.6Hz,1H),7.20(t,J=9.6Hz,2H),7.09(t,J=8.0Hz,1H),3.95(s,3H).LC-MS(ESI):m / z 443.1(M+H)

[0392] Step 4: 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)sulfonyl)benzamide (compound 124):

[0393]

[0394] Pyridine (168.48 mg, 2.130 mmol) and freshly prepared 2-(difluoromethoxy)benzoyl chloride (220 mg, 1.065 mmol) were added to a stirred solution of 5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-sulfonamide (261.20 mg, 1.065 mmol) in dichloromethane (20 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was diluted with H2O (20 mL) and extracted with 10% methanol / dichloromethane (3 × 15 mL). The combined organic layers were dried over Na2SO4 and concentrated to give a crude compound. This crude compound was purified by preparative HPLC to give 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl)sulfonyl)benzamide (22 mg, 4.86%) as a grayish-white solid. 1 H NMR (400MHz, DMSO) δ7.99 (d, J = 8.0Hz, 1H), 7.67 (m, 1H), 7.46 (t, J = 8.4Hz, 1H), 7.35 (t, J = 7.2Hz, 1H), 6.88-7.21 (m, 6H), 3.94 (s, 3H). LC-MS (ESI): m / z 425.1(M+H)

[0395] Synthesis of 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-pyrazol-3-yl)sulfonyl)benzamide (compound 121):

[0396]

[0397] Step 1: 5-(2-methoxyphenyl)-1H-pyrazole-3-amine

[0398]

[0399] Hydrazine hydrate (10 mL) was added to a stirred solution of methyl 3-(2-methoxyphenyl)-3-oxopropionitrile (2.5 g, 14.27 mmol) in ethanol (10 mL), followed by a catalytic amount of acetic acid. The resulting mixture was stirred at 80 °C for 24 h and concentrated. The residue was washed twice with toluene (10 mL) and dried under vacuum to give 5-(2-methoxyphenyl)-1H-pyrazole-3-amine (2 g, 74%) as a yellow viscous liquid. 1H NMR (400MHz, DMSO) δ11.56(bs,1H),7.62(d,J=7.6Hz,1H),7.26(dd,J=1.6,8.8Hz, 1H),7.07(d,J=8.4Hz,1H),6.95(t,J=7.2Hz,1H),4.58(bs,2H),3.84(s,3H).LC-MS m / z(M+H):190.1.

[0400] Step 2: 5-(2-methoxyphenyl)-1H-pyrazole-3-sulfonamide

[0401]

[0402] Thionyl chloride (44.85 mL, 0.618 mmol) was added dropwise to a suspension of CuCl (0.204 g, 2.1 mmol) in water (265 mL) under vigorous stirring at 0 °C. The resulting solution was stirred overnight at room temperature to give a pale yellow solution. Separately, a solution of NaNO2 (0.33 g, 4.8 mmol) / water (4 mL) was added dropwise to a solution of 5-(2-methoxyphenyl)-1H-pyrazole-3-amine (0.62 g, 4.1 mmol) in concentrated HCl (4 mL) at -10 °C. The resulting deep orange solution was stirred at -10 °C for 30 min and then added to a copper(I) solution (10.6 mL) from the first step at -5 °C for 5 min. The resulting reaction mixture was stirred at -5 °C for 1 h and extracted with ethyl acetate (10 mL x 3). The combined organic layers were concentrated under vacuum to give a yellow solid. The solid was dissolved in THF (20 mL) and cooled to 0 °C, followed by the dropwise addition of ammonia (10 mL, 28 wt%). The resulting reaction mixture was stirred at 0 °C for 2 hours and then concentrated under vacuum. The crude product was purified by rapid chromatography (using dichloromethane and methanol as eluents) to give 5-(2-methoxyphenyl)-1H-pyrazole-3-sulfonamide as a grayish-white solid (85 mg, 10%). 1 H NMR (400MHz, DMSO) δ13.53 (s, 1H), 7.71 (dd, J = 1.2, 7.6Hz, 1H), 7.40-7.37 (m, 3H), 7. 18–7.16(d,J=8.4Hz,1H),7.05(t,J=7.6Hz,1H),6.97(d,J=2.0Hz,1H),3.89(s,3H).

[0403] Step 3: 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-pyrazol-3-yl)sulfonyl)benzamide (compound 121):

[0404]

[0405] HATU (72.13 mg, 0.1897 mmol), 5-(2-methoxyphenyl)-1H-pyrazole-3-sulfonamide (40 mg, 0.1581 mmol), and DIPEA were added to a stirred solution of 2-(difluoromethoxy)benzoic acid (44 mg, 0.2371 mmol) in DMF (1 mL) at 0 °C, and the resulting mixture was stirred at room temperature for 16 h. The mixture was then quenched with ice-cold water and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with a brine solution (50 mL), dried over sodium sulfate, and concentrated to give a crude product. The crude product was further purified by preparative HPLC to give 2-(difluoromethoxy)-N-((5-(2-methoxyphenyl)-1H-pyrazole-3-yl)sulfonyl)benzamide (28 mg, 41.87%) as a grayish-white solid. 1 H NMR (400MHz, DMSO) δ13.84(s,1H),12.61(s,1H),7.76(d,J=7.2Hz,1H),7.58(t,J=6.8Hz,1H),7.52( d,J=7.2Hz,1H),7.41(t,J=7.2Hz,1H),7.31(t,J=6.8Hz,1H),7.24–6.95(m,5H),3.91(s,3H).LC-MS m / z(MH):423.39.

[0406] N-[[5-(2-methoxyphenyl)-1H-pyrazol-3-yl]sulfonyl]-2-(trifluoromethoxy)benzamide (compound 122):

[0407]

[0408] Compound 122 was synthesized using the same method described for compound 121. Yield: 25%. LC-MS m / z (MH): 442.1.

[0409] Biochemical and cell experiments

[0410] Biochemical Regulation of UBE2K Polyubiquitination Activity by Small Molecule Regulators .

[0411] In vitro polyubiquitination activity was determined using 3 μM UBE2K, 300 nM UBE1, and 200 μM Ub in 50 mM Tris pH 8.0, 1 mM TCEP buffer containing 0.05% Tween 20, 4 mM ATP, and 10 mM MgCl2. Reactions were performed at various compound concentrations, incubated at 37 °C for 3 h, and quenched with a non-reducing sample-loaded dye. 4–20% Criterion was used for assay. TMTGX Unstained TM Samples were analyzed using protein gel and 4–20% Criterion staining gels, and images of unstained and post-Coomassie staining were performed using BioRadImager. Compound titrations were performed in 1X PBS-P+ (GE) buffer containing 3% DMSO. Run buffer was prepared immediately prior to experiments using freshly opened DMSO. The compound stock solution (in DMSO, provided by Berg) was first diluted to DMSO-free 1X PBS-P+ to match to 3% DMSO. The final concentration of this 3% DMSO matching solution was determined by the concentration of the original stock solution (i.e., 3 mM for a 100 mM DMSO stock solution). The matching stock solution was then diluted to 100 μM with 1X PBS-P+ (GE) buffer containing 3% DMSO, and serial dilutions were performed.

[0412] In polyubiquitination assays in unstained gels, the compound stabilized monoubiquitinated UBE2K, leading to a reduction in multiubiquitinated products. The opposite was observed in stained gels, where the polyubiquitination polymerization following compound treatment utilized less ubiquitin. Both effects were dose-dependent. Figure 1 The analysis was performed by five independent experiments (N=5). Unstained gels utilize in-gel compounds that enhance the fluorescence of tryptophan amino acids upon exposure to UV light. Natural ubiquitin lacks tryptophan residues, while UBE2K and UBE1 contain tryptophan. Therefore, single-Ub UBE2K bands are more readily detected on unstained gels.

[0413] UBE2K Selective Trial

[0414] The selectivity of the compounds of this invention for UbE2K relative to other E2s was tested in an in vitro polyubiquitination assay as described above. E2-ubiquitin conjugates from the E2 enzyme family, namely UBE2D4, UBE2E1, UBE2Q2, UBE2S, and UBE2W, were selected. The ability of the compounds of this invention to stabilize monoubiquitinated E2 and multi-Ub products was observed at 500 μM. Although the compounds of this invention stabilized mono-Ub UBE2K and reduced multi-Ub chains, the same results were not observed for other representatives of E2 in the assay. E2 has a highly conserved active site, and the observation that the compounds of this invention do not affect or modulate other E2s confirms the allosteric sites involved in these molecules. Results are as follows: Figure 2 As shown.

[0415] Praja 1 trial

[0416] This assay used UBE2K thioester-linked ubiquitin and small molecule regulators to influence the ability of ubiquitin release into the Praja1 RING domain and the ability to polyubiquitinate PRAJA1. The level of polyubiquitination was measured using an ELISA and a combination of a primary antibody against Ub A5 (AF594) and a secondary antibody conjugated to alkaline phosphatase and the Attophos AP fluorescent substrate system (goat polyclonal anti-mouse AP). Fluorescence was read using a Teacan Spark 10M plate reader at excitation wavelengths of 435 nm and emission wavelengths of 555 nm.

[0417] The compounds of this invention were observed to regulate the release of ubiquitin from UBE2K and the polyubiquitination of Praja1RING protein in a concentration-dependent manner. A decrease in ELISA signaling indicates a reduction in the release of polyubiquitin-producing Praja1RING. The compounds of this invention were observed to reduce polyubiquitination of Praja1. See also... Figure 3 .

[0418] Cell viability assay

[0419] Using Cell Titer Fluor TM Cell viability was determined using a Promega G6080 assay. MIA PaCa-2 cells were grown in DMEM medium containing 10% FBS and 1% Pen / Strep / Amphotericin B. Cells were trypsinized and counted using a Nexcelom cytometer. 50,000 cells / 100 μl were seeded per well in Greiner black / clear 96-well plates. Cells used in the workflow should be within 10 passages of the stock vial. These cells were cultured in parallel for multiple passages in three different lineages, and five complete plates were seeded once for each lineage. The small molecule compound was provided as a 100 mM stock solution / DMSO. Diluted series plates were prepared using a 1:3 dilution to achieve a 7-point concentration on a half-logarithmic scale. After adding the compound, cells were incubated at 37°C and 5% CO2 for 72 h.

[0420] For each test compound condition, three replicates were used. For each reference compound, two replicates were used. At the end of the 72-hour incubation, the used culture medium was discarded. Then, 100 μl of GF-AFC diluted in DMEM (serum-free and phenol red-free) was added at a concentration of 1:2000 (5 μl / 10 ml). Cells were incubated with reaction buffer at 37°C for 1 h. Fluorescence was then read on a plate reader using an excitation wavelength of 390 nm and an emission wavelength of 505 nm. All raw data were analyzed in Microsoft Excel 2010 and normalized relative to the DMSO medium control. Relative results were copied to GraphPad Prism for nonlinear regression analysis and IC50 and other dose-response parameters (minimum, maximum, Hill slope, etc.) were determined using the log(inhibitor) vs. reaction equation. The results are shown in Table 11. The values ​​are as follows: A represents IC50 <1.0mM, B represents IC50 from 1mM to 10mM, and C represents IC50 >10mM.

[0421] Table 11

[0422]

[0423]

[0424] Materials and Methods for In Vivo PoC Study in a Tumor Growth Inhibition-Mouse Xenograft Model

[0425]

[0426]

[0427] Cell lines and tumor models:

[0428] The K-562 cancer cell line was derived from the American Type Culture Collection (ATCC), USA. Cells were grown in IMDM medium (Sigma, Cat#30-2005) containing 10% FBS (Invitrogen, Cat#10438-026) and 1% penicillin-streptomycin (Invitrogen, Cat#15140-122). To establish xenografts, when cells reached approximately 70-80% confluence, they were collected by trypsin digestion, and 5 million K-562 cells were suspended in 200 μL of serum-free medium and mixed with matrix gel at a 1:1 ratio. The cells were then subcutaneously implanted into the right flank of SCIDBg mice using a 1 mL BD syringe attached to a 24-gauge needle.

[0429] Randomization

[0430] Ten days after cell seeding, once the K-562 tumor graft became palpable, measurements were taken. The average tumor volume reached approximately 85 mm². 3 Animals were randomly assigned to different treatment groups to administer the drug, while maintaining tumor volume and animal numbers so that the average tumor volume remained the same across all groups.

[0431]

[0432] Research Design

[0433] The compound was formulated with 0.5% CMC + 0.1% Tween 80 and administered twice daily (BID) for 12 days at 8-hour intervals. The results of tumor growth inhibition are shown in Table 11 below.

[0434] Table 11

[0435]

[0436] Antitumor efficacy of compound 131 in a xenograft model derived from the MV.4.11 cell (B-granulocyte monocytic leukemia) line in nude mice

[0437] 5×10⁶ cells were subcutaneously implanted in the right flank region of nude mice. 6 MV.4.11 cells. Mice were randomly assigned to three groups (n=8 per group) on day 12 post-cell engraftment. The mediator control was administered the test compound formulation along with the control group, and the treatment groups received compound 131 orally at doses of 75 and 150 mg / kg in suspension in 0.1% Tween-80 + 0.5% CMC (carboxymethyl cellulose), twice daily (bid) for 24 days. Tumor measurements and body weight were recorded three times weekly throughout the study until the study ended (day 24). Tumor growth inhibition was 73.6% and 86.3% in the dose groups (75 mg / kg and 150 mg / kg), respectively. See [link to relevant documentation]. Figure 4 .

[0438] All references cited in this application (including papers, published patents, disclosed patent applications, and co-pending patent applications) are expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have meanings commonly known to one of ordinary skill in the art.

Claims

1. Compounds having formula I or formula III: (I) or (III) Or its pharmaceutically acceptable salt, wherein Z 1 and Z 2 Each can be either N or CH independently; X is either N or CH; Ring A is a phenyl or a 5- to 9-membered heteroaryl group, each optionally surrounded by 1 to 3 groups selected from R. 4 Substitution of groups; Y represents CH2, -CHR a or -CR a R b ; R a and R b Each is independently a halogen, (C1-C6) alkyl, or halo(C1-C6) alkyl; or R a and R b Together with the carbon atoms they are bonded to, they form 3 to 6-membered cycloalkyl groups, which are optionally substituted by 1 to 3 groups selected from halogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, (C1-C6)alkylOH, (C1-C6)alkylO, (C1-C6)alkyl and OH groups; R 1 It is a halogenated (C1-C6)alkoxy or -NR c R d ; R c and R d Each of these can be independently hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkylO(C1-C6)alkyl, halo(C1-C6)alkylO(C1-C6)alkyl, (C1-C6)alkyl-O-halo(C1-C6)alkyl, halo(C1-C6)alkyl-O-halo(C1-C6)alkyl, or (C1-C6)alkylOH; or R c and R d Together with the nitrogen atoms they are bonded to, they form 4 to 7-membered heterocyclic groups, which are optionally substituted by 1 to 3 groups selected from halogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy and oxo groups; R 2 CN, halogen, OH, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, or halo(C1-C6)alkoxy; or R 1 and R 2 When on adjacent carbon atoms, it forms a 5- or 6-membered oxygen-containing heterocyclic group together with the carbon atoms to which they are attached, which is optionally substituted by 1 to 3 groups selected from halogens, (C1-C6)alkyls and halo(C1-C6)alkyls; R 3 It is hydrogen, (C1-C6)alkyl, or halo(C1-C6)alkyl; R 4 CN, halogen, OH, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, -NH(C1-C6)alkyl or -N[(C1-C6)alkyl]2; and p is 0 or 1.

2. The compound according to claim 1, wherein the compound has the following formula: (II); Or its pharmaceutically acceptable salt.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 3 It is hydrogen.

4. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Y is CH2.

5. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Z 1 For N and Z 2 CH; Z 1 For CH and Z 2 For N; or Z 1 and Z 2 Each is CH.

6. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Z 1 and Z 2 Each is CH.

7. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl or a 5- to 6-membered heteroaryl group, each optionally surrounded by 1 to 3 groups selected from R 4 Substitution of groups.

8. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl, pyridyl, furanyl, or pyrazolyl, each optionally surrounded by 1 to 3 derivatives selected from R 4 Substitution of groups.

9. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl or furanyl, each optionally surrounded by 1 to 3 derivatives selected from R 4 Substitution of groups.

10. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein ring A is optionally surrounded by 1 to 3 molecules selected from R 4 phenyl groups substituted with .

11. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 A five-membered oxygen-containing heterocyclic group is formed on adjacent carbon atoms and together with the carbon atoms to which they are attached, which is optionally substituted with one or two halogens.

12. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 A dioxolane group is formed on adjacent carbon atoms and together with the carbon atoms to which they are attached, which is optionally substituted with one or two halogens.

13. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 1 It is a halogenated (C1-C4)alkoxy or -NR c R d And R c It is hydrogen and R d It is a haloalkyl (C1-C4) group; or R c and R d Together they form a 4- to 7-membered heterocyclic group, which may be optionally substituted by 1 to 3 groups selected from halogens, (C1-C4) alkyl groups and oxo groups.

14. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 1 It is -OCF3, -OCHF2, -OCH2CF3, piperidinyl, pyrrolidinyl, azircyclic heptyl, morpholinyl, thiomorpholinyl, piperazineyl, or azircyclic butyl, and each of said heterocycles is optionally substituted by 1 to 3 groups selected from halogens, (C1-C4) alkyl and oxo groups.

15. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 2 It can be CN, halogen, (C1-C4)alkyl, halo(C1-C4)alkyl, or (C1-C4)alkoxy.

16. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 2 It is either CN or halogen.

17. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 2 It is fluorine.

18. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein p is 0.

19. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 4 It is a halogen, (C1-C4)alkyl, halo(C1-C4)alkyl, (C1-C4)alkoxy or -N[(C1-C4)alkyl]2.

20. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 4 It is F, Br, Cl, -OCH3, -OCH2CH3, OH, -O(CH2)2CH3, -NMe2, -CH(CH3)2, -C(CH3)3, -OCH(CH3)2, -CH3, or -CF3.

21. A compound, wherein the compound is selected from... Or a pharmaceutically acceptable salt of any of the aforementioned.

22. The compound according to claim 1, wherein the compound is a compound of the following formula: or its pharmaceutically acceptable salt.

23. The compound according to claim 1, wherein the compound is a compound of the following formula: or its pharmaceutically acceptable salt.

24. A pharmaceutical composition comprising the compound of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

25. Use of any compound of claims 1 to 23 or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating a subject’s cancer by modulating or inhibiting UBE2K activity, wherein the cancer is selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pancreatic cancer, ovarian cancer, breast cancer, and colon cancer.

26. Use of the compound of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating a subject’s cancer by modulating or inhibiting UBE2K activity, wherein said cancer is gastrointestinal cancer.

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