PREPARATION AND APPLICATION OF TRICYCLIC PYRIMIDINONE COMPOUND AND ITS COMPOSITION

Abstract

Disclosed is a tricyclic pyrimidinone compound of Formula (I) or a pharmaceutically acceptable salt thereof, which is an entirely new Lp-PLA2 inhibitor useful in treating neurodegeneration-related diseases such as Alzheimer's disease (AD), glaucoma and age-related macular degeneration (AMD), or cardiovascular diseases including atherosclerosis.

Claims

1. A compound of Formula (I) or a pharmaceutically acceptable salt thereof, ##STR00037## wherein n.sub.1, n.sub.2, and n.sub.3 are each independently 0, 1, or 2; R.sub.1 and R.sub.2 are each independently selected from —H, hydroxyl, cyano, halogen, alkyl, deuterated alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, alkoxy, haloalkoxy, deuterated alkoxy; X.sub.1 and X.sub.2 are each independently selected from alkylene, —O—, —S—, or —NR′—, R′ is selected from —H, alkyl, deuterated alkyl, or cycloalkyl; Ar is an arylene group or a heteroarylene group, wherein hydrogen atoms in the arylene or heteroarylene are optionally substituted by 0, 1 or more substituents, and the substituents are each independently selected from halogen, alkyl, deuteroalkyl, haloalkyl, alkoxy, deuteroalkoxy, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, monoalkyl- or dialkyl-substituted amino, nitro, carboxyl, aldehyde, cycloalkyl, heterocyclyl, aryl, or heteroaryl; Y is —H, halogen, alkyl, haloalkyl, haloalkoxy, cycloalkyl, alkoxy, deuterated alkyl, deuterated alkoxy, —OAr′, —SAr′, —NH—Ar′, —NMe—Ar′, —NR″, or —R′″—Ar′; Ar′ is selected from aryl or heteroaryl, wherein hydrogen atoms in the aryl or heteroaryl are optionally substituted with one or more substituents, the substituents are each independently selected from halogen, alkyl, deuterated alkyl, haloalkyl, alkoxy, deuterated alkoxy, hydroxy, hydroxyalkyl, haloalkoxy, cyano, amino, nitro, carboxyl, aldehyde, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R″ is alkyl; R″′ is alkylene; Z is O or S.

2. The compound or a salt thereof according to claim 1, wherein Optionally, halogens in the “halogen” “haloalkyl” and “haloalkoxy” are each independently selected from F, Cl, Br, or I; optionally, alkyls in the “alkyl” “deuterated alkyl” “hydroxyalkyl” “haloalkyl” “haloalkoxy”, “alkoxy” and “mono- or di-alkyl substituted amino” are each independently C.sub.1-C.sub.10 linear or branched alkyl; optionally each independently C.sub.1-C.sub.7 linear or branched alkyl; optionally each independently C.sub.1-C.sub.4 linear or branched alkyl; and optionally selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3-ethylpentyl, or 2,2,3-trimethylbutyl; optionally, “alkylenes” are each independently C.sub.1-C.sub.10 linear or branched alkylene; optionally each C.sub.1-C.sub.7 linear or branched alkylene; optionally each C.sub.1-C.sub.5 linear or branched alkylene; and optionally each selected from methylene, ethylene, n-propylene, iso-propylene, n-butylene, iso-butylene, tert-butylene, sec-butylene, n-pentylene, 1-methylbutylene, 2-methylbutylene, 3-methylbutylene, isopentylene, 1-ethylpropylene, neopentylene, n-hexylene, 1-methylpentylene, 2-methylpentylene, 3-methylpentylene, isohexylene, 1,1-dimethylbutylene, 2,2-dimethylbutylene, 3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene, 2,3-dimethylbutylene, 2-ethylbutylene, n-heptylene, 2-methylhexylene, 3-methylhexylene, 2,2-dimethylpentylene, 3,3-dimethylpentylene, 2,3-dimethylpentylene, 2,4-dimethylpentylene, 3-ethylpentylene, or 2,2,3-trimethylbutylene; optionally, “cycloalkyl” is C.sub.3-C.sub.10 monocyclic or bicyclic cycloalkyl, optionally C.sub.3-C.sub.7 monocyclic cycloalkyl, and optionally cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl; optionally, “heterocyclyl” is 3- to 10-membered non-aromatic heterocycle ring containing 1, 2, or 3 heteroatoms selected from N, O, and S; optionally 3- to 10-membered non-aromatic ring containing 1 or 2 heteroatoms selected from N and O; optionally 3- to 6-membered non-aromatic ring containing 1 or 2 heteroatoms selected from N and O; optionally 3- to 10-membered non-aromatic ring containing 1 or 2 heteroatoms selected from N and S; and optionally 3- to 6-membered non-aromatic ring containing 1 or 2 heteroatoms selected from N and S; optionally, “aryl” is 6- to 10-membered aryl; optionally phenyl or naphthyl, and optionally phenyl, 1-naphthyl, or 2-naphthyl; optionally, “arylene” is 6- to 10-membered arylene; and optionally phenylene or naphthylene; optionally, “heteroaryl” is 5- to 10-membered heteroaryl ring containing 1-3 heteroatoms selected from N, O, and S; optionally 5- to 10-membered heteroaryl ring containing 1-2 heteroatoms selected from N, O, and S; optionally the heteroaryl ring is selected from pyridine ring, pyrrole ring, pyrazole ring, pyrimidine ring, pyrazine ring, pyridazine ring, thiophene ring, and furan ring; optionally selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyridazin-3-yl, pyridazin-4-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, pyrazin-2-yl, pyrazin-3-yl, indolyl, isoindolyl, indazolyl, indolizinyl, purinyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, quinazolinyl, quinoxalinyl, thieno[2,3-b] furanyl, furo[3,2-b]-pyranyl, pyrido[2,3-d]oxazinyl, pyrazolo[4,3-d]oxazolyl, imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl, imidazo[1,2-b][1,2,4]triazinyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzoxepinyl, benzoxazinyl, benzofuranyl, benzotriazolyl, pyrrolo[2,3-b]pyridyl, pyrrolo[3,2-c]pyridinyl, pyrrolo[3,2-b]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrazolo[4,3-d]pyridyl, pyrazolo[4,3-c]pyridyl, pyrazolo[3,4-c]pyridinyl, pyrazolo[3,4-d]pyridyl, pyrazolo[3,4-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrrolo[1,2-b]pyridazinyl, imidazo[1,2-c]pyrimidinyl, pyrido[3,2-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl, pyrido[2,3-b]pyrazinyl, pyrido[3,4-b]pyrazinyl, pyrimido[5,4-d]pyrimidinyl, pyrazolo[2,3-b]pyrazinyl, or pyrimido[4,5-d]pyrimidinyl; and is optionally selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-2-yl, pyrimidin-4-yl, or pyrimidin-5-yl; optionally, “heteroarylene” is 5- to 10-membered heteroarylene ring containing 1-3 heteroatoms selected from N, O, and S; optionally 5- to 10-membered heteroaromatic ring containing 1-2 heteroatoms selected from N, O, and S; and optionally the heteroarylene ring is selected from pyridine ring, pyrrole ring, pyrazole ring, pyrimidine ring, pyrazine ring, pyridazine ring, thiophene ring, furan ring.

3. The compound or a salt thereof according to claim 1, wherein Optionally, n.sub.1, n.sub.2, and n.sub.3 are each independently 0, 1, or 2; optionally, n.sub.1 is 0 or 1; optionally, n.sub.2 is 1; optionally, n.sub.3 is 1. Optionally, R.sub.1 and R.sub.2 are each independently selected from —H, fluorine, chlorine, bromine, hydroxyl, cyano, C.sub.1-C.sub.7 alkyl (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3-ethylpentyl, or 2,2,3-trimethylbutyl), C.sub.1-C.sub.3 deuteroalkyl (such as —CD.sub.3, —C.sub.2D.sub.5, or —C.sub.3D.sub.7), C.sub.1-C.sub.3 deuteroalkoxyl (such as —OCD.sub.3, —OC.sub.2D.sub.5, or —OC.sub.3D.sub.7), haloalkyl, haloalkoxyl, cyclopropanyl, cyclobutanyl, cyclopentanyl; optionally, R.sub.1 is —H or —CH.sub.3; and optionally, R.sub.1 is —H, R.sub.2 is —H; Optionally, X.sub.1 and X.sub.2 are each independently selected from C.sub.1-C.sub.7 alkylene (optionally, —CH.sub.2—, ethylene, n-propylene, isopropylene, n-butylene, or isobutylene), —O—, —S—, or —NR′—; optionally, X.sub.1 is —CH.sub.2—, or —O—; optionally, X.sub.1 is —O—; optionally, X.sub.2 is —O—; optionally, R′ is selected from —H, C.sub.1-C.sub.7 alkyl (optionally, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3-ethylpentyl, or 2,2,3-trimethylbutyl), deuterated alkyl (optionally, —CD.sub.3, —C.sub.2D.sub.5, or —C.sub.3D.sub.7), or C.sub.3-C.sub.6 cycloalkyl (optionally, cyclopropanyl, cyclobutanyl, cyclopentanyl, or cyclohexanyl); Optionally, Ar is phenylene or pyridyl, wherein hydrogen atoms in the phenylene or pyridyl are optionally substituted with 0, 1, 2, or 3 substituents, the substituents are each independently selected from F, Cl, Br, I, —CN, -Me, —C.sub.2H.sub.5, cyclopropyl, —CD.sub.3, —OMe, —OCD.sub.3, —CF.sub.3, or —OCF.sub.3; optionally, Ar is phenylene, wherein the hydrogen atom in the phenylene is optionally substituted by 2 substituents, and the substituent is F; Optionally, Y is —H, —F, —Cl, —Br, methyl, ethyl, n-propyl, isopropyl, —CD.sub.3, —CF.sub.3, —CH.sub.2CF.sub.3, —OCF.sub.3, —OCHF.sub.2, —OCH.sub.2F, cyclopropyl, cyclobutyl, cyclopentyl, —OCH.sub.3, —OCD.sub.3, —OC.sub.2H.sub.5, —OC.sub.3H.sub.7 or —OAr′; Optionally, Y is H, halogen, or —OAr′; and optionally, Y is H, —F, or —OAr′; Ar′ is selected from phenyl, pyridyl, pyrimidyl, pyrrolyl, pyrazolyl, thienyl or quinolinyl, and the phenyl, pyridyl, pyrimidyl, pyrrolyl, pyrazolyl, thienyl or quinoline Optionally, Ar′ is selected from phenyl, pyridyl, pyrimidyl, pyrrolyl, pyrazolyl, thienyl or quinolinyl, wherein hydrogen atoms in the phenyl, pyridyl, pyrimidyl, pyrrolyl, pyrazolyl, thienyl or quinolinyl ring are each independently optionally substituted with 1, 2, or 3 substituents, the substituents are each independently selected from F, Cl, Br, —CN, C.sub.1-C.sub.7 alkyl (optionally, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 172-dimethylbutyl, 173-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3-ethylpentyl, or 2,2,3-trimethylbutyl), —CD.sub.3, C.sub.1-C.sub.6 haloalkyl, —OCH.sub.3, —OCD.sub.3, —OC.sub.2H.sub.7—OC.sub.3H.sub.7, C.sub.1-C.sub.6 haloalkoxyl, or C.sub.3-C.sub.6 cycloalkyl (optionally, cyclopropanyl, cyclobutanyl, cyclopentanyl, or cyclohexanyl); Optionally, Ar′ is selected from phenyl, pyridin-3-yl, pyridin-4-yl, or pyrimidin-5-yl, and is optionally substituted with 1 or 2 substituents, the substituents are selected from F, Cl, —CH.sub.3, —CF.sub.3, or —OCF.sub.3; Optionally, Z is O.

4. The compound of Formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of Formula (I) is selected from the following compounds: ##STR00038## ##STR00039## ##STR00040## ##STR00041##

5. The compound of Formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt includes an anionic salt or cationic salt of the compound of Formula (I); optionally, the pharmaceutically acceptable salt includes alkali metal salt, alkaline earth metal salt, or ammonium salt of the compound of Formula (I); optionally, the alkali metal includes sodium, potassium, lithium, or cesium, and the alkaline earth metal includes magnesium, calcium, or strontium; optionally, the pharmaceutically acceptable salt includes salt formed by the compound of Formula (I) and an organic base; optionally, the organic base includes trialkylamine, pyridine, quinoline, piperidine, imidazole, picoline, dimethylaminopyridine, dimethylaniline, N-alkylmorpholine, 1,5-diazabicyclo[4.3.0]nonene-5, 1,8-diazabicyclo[5.4.0]undecene-7, 1,4-diazabicyclo[2.2.2] octane; optionally, the trialkylamine includes trimethylamine, triethylamine, or N-ethyldiisopropylamine; and optionally, the N-alkyl morpholine includes N-methylmorpholine; optionally, the pharmaceutically acceptable salt includes salt formed by the compound of Formula (I) and an acid; optionally, the acid includes inorganic acid, or organic acid; optionally, the inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, or carbonic acid; optionally, the organic acid includes formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, citric acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, glutamic acid, or pamoic acid.

6. A preparation method of the compound of Formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, comprising the step of reacting a compound of Formula (II) with a compound of Formula (III) to produce the compound of Formula (I): ##STR00042##

7. A pharmaceutical composition, comprising a therapeutically effective amount of one or more of the compound of Formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, and optionally, pharmaceutically acceptable excipient(s).

8. The pharmaceutical composition according to claim 7, wherein the dosage form of the pharmaceutical composition includes oral, rectal, or parenteral formulation; optionally, the oral formulation includes solid or liquid formulation; optionally, the solid formulation includes tablet, powder, granule, or capsule; optionally, the liquid formulation includes aqueous or oily suspension, or syrup; optionally, the parenteral formulation includes solution for injection, or aqueous or oily suspension.

9. Use of the compound of Formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an Lp-PLA2 inhibitor.

10. Use of the compound of Formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treatment of neurodegeneration-related diseases; optionally, the neurodegeneration-related diseases include Alzheimer's disease (AD), glaucoma, and age-related macular degeneration (AMD).

11. Use of the compound of Formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for the treatment of cardiovascular diseases, diabetic macular edema (DME), or prostate diseases; optionally, the cardiovascular diseases include atherosclerosis.

Description

DETAILED DESCRIPTION

[0073] The present invention is further illustrated by the following examples. It should be understood that the embodiments herein are only used to illustrate the present invention, and do not limit the scope of the present invention in any way.

[0074] The starting materials of the present invention can be synthesized by a method known in the art, or be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., and Darry Chemicals, among other companies.

[0075] Unless otherwise specified, the solution in the examples refers to an aqueous solution.

[0076] Unless otherwise specified, the temperature in the examples at which the reaction is carried out is room temperature, e.g., 20° C. to 30° C.

Example 1 Preparation of Compound 1

[0077] ##STR00007##

[0078] Step I: Preparation of Compound 1c

##STR00008##

[0079] At room temperature, 2,4,6-trichloro-5-methoxypyrimidine 1b (1.5 g, 7.03 mmol), diethanolamine 1a (1.1 g, 10.5 mmol) and diisopropylethylamine (1.36 g, 10.54 mmol) were dissolved in acetonitrile (50 mL), stirred and reacted for 3 h, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=20/1) afforded the title compound 1c (1.6 g, Yield: 81.0%) as a white solid.

[0080] 1H NMR (400 MHz, CDCl.sub.3) δ3.94-3.89 (m, 4H), 3.88-3.83 (m, 4H), 3.77 (s, 3H), 3.17 (s, 2H).

[0081] Step II: Preparation of Compound 1d

##STR00009##

[0082] At room temperature, chloro-5-methoxypyrimidine-4-yl)azanediyl)diethanol 1c (1.0 g, 3.54 mmol), and lithium chloride (0.75 g, 17.73 mmol) were dissolved in N,N-dimethylformamide (5 mL), heated in microwave at 130° C. for 1 h, concentrated under reduced pressure, extracted with ethyl acetate (60 mL×3). The combined organic phases were washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered to remove desiccant, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=20/1) afforded the title compound 1d (0.39 g, Yield: 44.0%) as a white solid.

[0083] .sup.1H NMR (400 MHz, CDCl.sub.3) δ4.32-4.27 (m, 2H), 3.92 (m, 2H), 3.83-3.76 (m, 2H), 3.76-3.69 (m, 2H).

Steps III and IV: Preparation of Compound 1f

[0084] ##STR00010##

[0085] At room temperature, 2-(2,4-dichloro-6hydro-pyrimido[5,4-b][1,4]oxazin-8(7H)-yl)ethanol 1d (0.77 g, 3.1 mmol) and Triethylamine (0.94 g, 9.3 mmol) were dissolved in dichloromethane (30 mL), then methanesulfonyl chloride (0.39 g, 3.4 mmol) was added dropwise at 0° C. The reaction was stirred and reacted at 0° C. for 1 h, concentrated and proceeded directly to the next reaction. The crude product was dissolved in a mixed solvent of 1/1 dioxane/water (60 mL), followed by addition of potassium carbonate (1.3 g, 9.3 mmol) at room temperature. The reaction mixture was stirred at 90° C. overnight, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 1f (0.38 g, Yield: 57.4%) as a white solid.

[0086] .sup.1H NMR (400 MHz, MeOD) δ4.32-4.25 (m, 2H), 4.25-4.17 (m, 2H), 3.99 (m, 2H), 3.60-3.52 (m, 2H).

Step V: Preparation of Compound 1

[0087] ##STR00011##

[0088] To a solution of (2,4,5-trifluorophenoxy)methanol 1g (39 mg, 0.24 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 16 mg, 0.4 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (43 mg, 0.20 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 1 (13.6 mg, 20%) as a white solid.

[0089] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.30 (m, 1H), 6.96 (m, 1H), 5.43 (s, 2H), 4.32-4.22 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 3.48-3.39 (m, 2H). MS (ESI): m/z 340.1 [M+H].sup.+.

Example 2 Preparation of Compound 2

[0090] ##STR00012##

[0091] To a solution of (2,3-Difluorophenyl)methanol (35 mg, 0.24 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 16 mg, 0.4 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (43 mg, 0.20 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 1 (15 mg, 23.3%) as a white solid.

[0092] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.45 (m, 1H), 6.96-6.82 (m, 2H), 5.43 (s, 2H), 4.33-4.22 (m, 2H), 4.19 (m, 2H), 3.77 (m, 2H), 3.46-3.39 (m, 2H). MS (ESI): m/z 322.1 [M+H].sup.+.

Example 3 Preparation of Compound 3

[0093] ##STR00013##

[0094] To a solution of (3,4,5-Trifluorophenyl)methanol (39 mg, 0.24 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 16 mg, 0.4 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (43 mg, 0.20 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 3 (11.5 mg, 17%) as a white solid.

[0095] 1H NMR (400 MHz, CDCl.sub.3) δ7.31 (m, 2H), 5.42 (s, 2H), 4.31-4.22 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 3.46-3.38 (m, 2H). MS (ESI): m/z 340.1 [M+H].sup.+.

Example 4 Preparation of Compound 4

[0096] ##STR00014##

[0097] Step I: Preparation of Compound 4c

[0098] 2-(Trifluoromethyl)pyridin-4-ol 4b (0.85 g, 5.2 mmol), 3,4,5-trifluorobenzaldehyde 4a (1 g, 6.2 mmol) and potassium carbonate (0.93 g, 6.76 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 1 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=5/1) afforded the title compound 4c (1.47 g, yield: 93.2%) as a yellow solid.

[0099] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.97 (s, 1H), 8.65 (m, 1H), 7.63 (m, 2H), 7.27 (m, 1H), 7.01 (m, 1H).

Step II: Preparation of Compound 4d

[0100] At room temperature, 3,5-difluoro-4-((2-(trifluoromethyl)pyridin-4-yl)oxy)benzaldehyde 4c (1.47 g, 4.85 mmol) was dissolved in ethanol (50 mL), then NaBH.sub.4 (184 mg, 4.84 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous Sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=2/1) afforded the title compound 4d (1.04 g, yield: 70.3%) as a white solid.

[0101] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.59 (m, 1H), 7.24 (m, 1H), 7.11 (m, 2H), 6.99 (m, 1H), 4.75 (m, 2H), 2.19 (m, 1H).

Step III: Preparation of Compound 4

[0102] To a solution of (3,5-Difluoro-4-((2-(trifluoromethyl)pyridin-4-yl)oxy)phenyl)methanol 4d (68 mg, 0.22 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 16 mg, 0.4 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (43 mg, 0.20 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 4 (31 mg, 32.1%) as a white solid.

[0103] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.58 (m, 1H), 7.24 (m, 1H), 7.16 (m, 2H), 6.96 (m, 1H), 5.45 (s, 2H), 4.32-4.23 (m, 2H), 4.19 (m, 2H), 3.78 (m, 2H), 3.46-3.37 (m, 2H). MS (ESI): m/z 482.9 [M+H].sup.+.

Example 5 Preparation of Compound 5

[0104] ##STR00015##

[0105] Step I: Preparation of Compound 5b

[0106] 6-(trifluoromethyl)pyridin-3-ol 5a (0.85 g, 5.2 mmol), 3,4,5-trifluorobenzaldehyde 4a (1 g, 6.2 mmol) and potassium carbonate (0.93 g, 6.76 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 1 h, cooled to room temperature, followed by addition of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=5/1) afforded the title compound 5b (1.34 g, yield: 85.0%) as a yellow solid.

Step II: Preparation of Compound 5c

[0107] At room temperature, 3,5-Difluoro-4-((6-(trifluoromethyl)pyridin-3-yl)oxy)benzaldehyde 5b (1.34 g, 4.4 mmol) was dissolved in ethanol (50 mL), then NaBH.sub.4 (167 mg, 4.4 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=2/1) afforded the title compound 5c (0.77 g, yield: 57.3%) as a colorless oil.

[0108] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.46 (m, 1H), 7.63 (m, m 1H), 7.30 (m, 1H), 7.09 (m, 2H), 4.73 (m, 2H), 2.40 (m, 1H).

Step III: Preparation of Compound 5

[0109] To a solution of (3,5-Difluoro-4-((6-(trifluoromethyl)pyridin-3-yl)oxy)phenyl)methanol 5c (43 mg, 0.14 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (30 mg, 0.14 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 5 (11 mg, 16.3%) as a white solid.

[0110] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.50 (m, 1H), 7.63 (m, 1H), 7.28 (m, m 1H), 7.15 (m, 2H), 5.45 (s, 2H), 4.33-4.27 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 3.46-3.39 (m, 2H). MS (ESI): m/z 482.9 [M+H].sup.+.

Example 6 Preparation of Compound 6

[0111] ##STR00016##

[0112] Step I: Preparation of Compound 6b

[0113] 6-Methylpyridin-4-ol 6a (0.5 g, 4.6 mmol), 3,4,5-trifluorobenzaldehyde 4a (0.88 g, 5.5 mmol) and potassium carbonate (0.82 g, 5.95 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 2 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 6b (0.4 g, yield: 34.8%) as a yellow solid.

[0114] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.94 (m, 1H), 8.39 (m, 1H), 7.63-7.55 (m, 2H), 6.72-6.64 (m, 2H), 2.50 (s, 3H).

[0115] Step II: Preparation of Compound 6c

[0116] At room temperature, 3,5-Difluoro-4-((2-methylpyridin-4-yl)oxy)benzaldehyde 6b (0.4 g, 1.6 mmol) was dissolved in ethanol (50 mL), then NaBH.sub.4 (71 mg, 1.87 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 6c (0.34 g, yield: 85.7%) as a colorless oil.

[0117] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.29 (m, 1H), 7.07 (m, 2H), 6.70 (m, 1H), 6.65 (m, 1H), 4.73 (s, 2H), 3.20 (m, 1H), 2.50 (s, 3H).

[0118] Step III: Preparation of Compound 6

[0119] To a solution of (3,5-Difluoro-4-((2-methylpyridin-4-yl)oxy)phenyl)methanol 6c (35 mg, 0.14 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (30 mg, 0.14 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 6 (6 mg, 10%) as a white solid.

[0120] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.35 (m, 1H), 7.12 (m, 2H), 6.67 (m, 2H), 5.44 (s, 2H), 4.33-4.27 (m, 2H), 4.19 (m, 2H), 3.78 (m, 2H), 3.45-3.38 (m, 2H), 2.50 (s, 3H). MS (ESI): m/z 429.0 [M+H].sup.+.

Example 7 Preparation of Compound 7

[0121] ##STR00017##

[0122] Step I: Preparation of Compound 7b

[0123] 6-Methylpyridin-3-ol 7a (0.57 g, 5.2 mmol), 3,4,5-trifluorobenzaldehyde 4a (1 g, 6.2 mmol) and potassium carbonate (0.93 g, 6.76 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 1 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 7b (0.91 g, yield: 70.2%) as a yellow solid.

[0124] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.92 (s, 1H), 8.28 (s, 1H), 7.62-7.49 (m, 2H), 7.18-7.10 (m, 2H), 2.54 (s, 3H).

[0125] Step II: Preparation of Compound 7c

[0126] At room temperature, 3,5-Difluoro-4-((6-methylpyridin-3-yl)oxy)benzaldehyde 7b (0.91 g, 3.6 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (161 mg, 4.2 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 7c (0.89 g, yield: 98.4%) as a colorless oil.

[0127] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.20 (m, 1H), 7.16-6.98 (m, 4H), 4.69 (m, 2H), 2.88 (m, 1H), 2.50 (s, 3H).

Step III: Preparation of Compound 7

[0128] To a solution of (3,5-Difluoro-4-((6-methylpyridin-3-yl)oxy)phenyl)methanol 7c (35 mg, 0.14 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (30 mg, 0.14 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 7 (12 mg, 20%) as a white solid.

[0129] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.27 (m, 1H), 7.14-7.02 (m, 4H), 5.42 (s, 2H), 4.32-4.25 (m, 2H), 4.19 (m, 2H), 3.77 (m, 2H), 3.45-3.36 (m, 2H), 2.51 (s, 3H). MS (ESI): m/z 429.0 [M+H].sup.+.

Example 8 Preparation of Compound 8

[0130] ##STR00018##

[0131] Step I: Preparation of Compound 8b

[0132] 2-methylpyrimidin-5-ol 8a (0.25 g, 2.3 mmol), 3,4,5-trifluorobenzaldehyde 4a (0.44 g, 2.7 mmol) and potassium carbonate (0.41 g, 2.9 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 2 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 8b (0.24 g, yield: 41.7%) as a yellow solid.

[0133] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.93 (s, 1H), 8.39 (s, 2H), 7.64-7.54 (m, 2H), 2.72 (s, 3H).

Step II: Preparation of Compound 8c

[0134] At room temperature, 3,5-Difluoro-4-((2-methylpyrimidin-5-yl)oxy)benzaldehyde 8b (0.24 g, 0.96 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (30 mg, 0.79 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 8c (0.17 g, yield: 70.2%) as a colorless oil.

[0135] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.33 (s, 2H), 7.04 (m, 2H), 4.71 (m, 2H), 2.70 (s, 3H).

Step III: Preparation of Compound 8

[0136] To a solution of (3,5-Difluoro-4-((2-methylpyrimidin-5-yl)oxy)phenyl)methanol 8c (35 mg, 0.14 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (30 mg, 0.14 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 8 (8 mg, 13.3%) as a white solid.

[0137] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.34 (s, 2H), 7.13 (m, 2H), 5.43 (s, 2H), 4.32-4.24 (m, 2H), 4.19 (m, 2H), 3.77 (m, 2H), 3.42 (m, 2H), 2.69 (s, 3H). MS (ESI): m/z 429.9 [M+H].sup.+.

Example 9 Preparation of Compound 9

[0138] ##STR00019##

[0139] Step I: Preparation of Compound 9b

[0140] 4-(Trifluoromethyl)phenol 9a (0.84 g, 5.2 mmol), 3,4,5-trifluorobenzaldehyde 4a (1 g, 6.2 mmol) and potassium carbonate (0.93 g, 6.76 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 1 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=5/1) afforded the title compound 9b (1.33 g, yield: 84.6%) as a yellow solid.

[0141] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.94 (m, 1H), 7.59 (m, 4H), 7.04 (m, 2H).

Step II: Preparation of Compound 9c

[0142] At room temperature, 3,5-Difluoro-4-(4-(trifluoromethyl)phenoxy)benzaldehyde 9b (1.33 g, 4.4 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (166 mg, 4.4 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=2/1) afforded the title compound 9c (0.85 g, yield: 63.4%) as a colorless oil.

[0143] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.57 (m, 2H), 7.09-7.00 (m, 4H), 4.72 (m, 2H), 2.03 (m, 1H).

Step III: Preparation of Compound 9

[0144] To a solution of 3,5-Difluoro-4-(4-(trifluoromethyl)phenoxy)phenyl)methanol 9c (43 mg, 0.14 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (30 mg, 0.14 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 9 (9 mg, 13.4%) as a white solid.

[0145] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.56 (m, 2H), 7.16-7.09 (m, 2H), 7.00 (m, 2H), 5.44 (s, 2H), 4.30 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 3.46-3.35 (m, 2H). MS (ESI): m/z 481.9 [M+H].sup.+.

Example 10 Preparation of Compound 10

[0146] ##STR00020##

[0147] Step I: Preparation of Compound 10b

[0148] 3-(Trifluoromethoxy)phenol 10a (0.50 g, 2.8 mmol), 3,4,5-trifluorobenzaldehyde 4a (0.5 g, 3.1 mmol) and potassium carbonate (0.5 g, 3.64 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 2 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 10b (0.73 g, yield: 81.9%) as a yellow solid.

[0149] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.94 (s, 1H), 7.64-7.54 (m, 2H), 7.34 (m, 1H), 7.00 (m, 1H), 6.87 (m, 2H).

Step II: Preparation of Compound 10c

[0150] At room temperature, 4-(3-(Trifluoromethoxy)phenoxy)-3,5-difluorobenzaldehyde 10b (0.73 g, 2.3 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (86 mg, 2.26 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 10c (0.57 g, yield: 77.4%) as a colorless oil.

[0151] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.30 (m, 1H), 7.06 (m, 2H), 6.94 (m, 1H), 6.85 (m, 1H), 6.81 (s, 1H), 4.72 (m, 2H), 1.94 (m, 1H).

Step III: Preparation of Compound 10

[0152] To a solution of 4-(3-(Trifluoromethoxy)phenoxy)-3,5-difluorophenyl)methanol 10c (48 mg, 0.15 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (30 mg, 0.14 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 10 (18 mg, 25.8%) as a white solid.

[0153] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.30 (m, 1H), 7.11 (m, 2H), 6.93 (m, 1H), 6.83 (m, 2H), 5.44 (s, 2H), 4.34-4.27 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 3.46-3.38 (m, 2H). MS (ESI): m/z 497.9 [M+H].sup.+.

Example 11 Preparation of Compound 11

[0154] ##STR00021##

[0155] Step I: Preparation of Compound 11b

[0156] 3-Chloro-4-(trifluoromethyl)phenol 11a (0.25 g, 1.27 mmol), 3,4,5-trifluorobenzaldehyde 4a (0.22 g, 1.4 mmol) and potassium carbonate (0.23 g, 1.65 mmol) were dissolved in N,N-dimethylformamide (DMF) (20 mL) at room temperature. The reaction mixture was stirred at 90° C. for 2 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 11b (0.32 g, yield: 74.8%) as a yellow solid.

[0157] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.95 (s, 1H), 7.69-7.56 (m, 3H), 7.10 (m, 1H), 6.92 (m, 1H).

Step II: Preparation of Compound 11c

[0158] At room temperature, 4-(3-Chloro-4-(trifluoromethyl)phenoxy)-3,5-difluorobenzaldehyde 11b (0.32 g, 0.95 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (36 mg, 0.95 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 11c (0.15 g, yield: 46.6%) as a white solid.

[0159] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.62 (m, 1H), 7.13-7.00 (m, 3H), 6.90 (m, 1H), 4.74 (m, 2H), 1.88 (m, 1H).

Step III: Preparation of Compound 11

[0160] To a solution of (4-(3-Chloro-4-(trifluoromethyl)phenoxy)-3,5-difluorophenyl)methanol 11c (67 mg, 0.20 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 15 mg, 0.38 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (40 mg, 0.19 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 11 (21 mg, 21.4%) as a white solid.

[0161] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.62 (m, 1H), 7.14 (m, 2H), 7.06 (m, 1H), 6.89 (m, 1H), 5.45 (s, 2H), 4.34-4.26 (m, 2H), 4.21 (m, 2H), 3.78 (m, 2H), 3.52-3.37 (m, 2H). MS (ESI): m/z 515.9 [M+H].sup.+.

Example 12 Preparation of Compound 12

[0162] ##STR00022##

[0163] Step I: Preparation of Compound 12b

[0164] 3-Chloro-4-(trifluoromethoxy)phenol 12a (0.5 g, 2.4 mmol), 3,4,5-trifluorobenzaldehyde 4a (0.41 g, 2.6 mmol) and potassium carbonate (0.42 g, 3.04 mmol) were dissolved in N,N-dimethylformamide (DMF) (20 mL) at room temperature. The reaction mixture was stirred at 90° C. for 2 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 12b (0.62 g, yield: 73.2%) as a yellow solid.

[0165] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.94 (s, 1H), 7.63-7.54 (m, 2H), 7.29 (m, 1H), 7.07 (m, 1H), 6.90 (m, 1H).

Step II: Preparation of Compound 12c

[0166] At room temperature, 4-(3-Chloro-4-(trifluoromethoxy)phenoxy)-3,5-difluorobenzaldehyde 12b (0.62 g, 1.8 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (62 mg, 1.63 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 12c (0.53 g, yield: 83.0%) as a colorless oil.

[0167] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.25 (m, 1H), 7.06 (m, 2H), 7.01 (m, 1H), 6.87 (m, 1H), 4.72 (s, 2H), 2.04 (m, 1H).

Step III: Preparation of Compound 12

[0168] To a solution of (4-(3-Chloro-4-(trifluoromethoxy)phenoxy)-3,5-difluorophenyl)methanol 12c (67 mg, 0.19 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 15 mg, 0.38 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (40 mg, 0.19 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 12 (19 mg, 18.8%) as a white solid.

[0169] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.23 (m, 1H), 7.16-7.08 (m, 2H), 7.03 (m, 1H), 6.86 (m, 1H), 5.44 (s, 2H), 4.30 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 3.46-3.39 (m, 2H). MS (ESI): m/z 531.8 [M+H].sup.+.

Example 13 Preparation of Compound 13

[0170] ##STR00023##

[0171] Step I: Preparation of Compound 13b

[0172] 4-Chloro-3-(trifluoromethyl)phenol 13a (0.5 g, 2.5 mmol), 3,4,5-trifluorobenzaldehyde 4a (0.41 g, 2.8 mmol) and potassium carbonate (0.46 g, 3.3 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 2 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 13b (0.6 g, yield: 71.3%) as a yellow solid.

[0173] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.94 (s, 1H), 7.64-7.55 (m, 2H), 7.45 (m, 1H), 7.31 (m, 1H), 7.05 (m, 1H).

Step II: Preparation of Compound 13c

[0174] At room temperature, 4-(4-Chloro-3-(trifluoromethyl)phenoxy)-3,5-difluorobenzaldehyde 13b (0.6 g, 1.78 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (67 mg, 1.76 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 13c (0.28 g, yield: 46.4%) as a white solid.

[0175] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.41 (m, 1H), 7.28 (m, 1H), 7.08-7.00 (m, 3H), 4.73 (m, 2H), 1.94 (m, 1H).

Step III: Preparation of Compound 13

[0176] To a solution of (4-(4-Chloro-3-(trifluoromethyl)phenoxy)-3,5-difluorophenyl)methanol 13c (64 mg, 0.19 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 15 mg, 0.38 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (40 mg, 0.19 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 13 (29 mg, 29.6%) as a white solid.

[0177] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.41 (m, 1H), 7.30 (m, 1H), 7.13 (m, 2H), 6.99 (m, 1H), 5.44 (s, 2H), 4.30 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 3.57-3.35 (m, 2H). MS (ESI): m/z 515.9 [M+H].sup.+.

Example 14 Preparation of Compound 14

[0178] ##STR00024##

[0179] Step I: Preparation of Compound 14b

[0180] 4-chloro-3-methylphenol 14a (1 g, 7.0 mmol), 3,4,5-trifluorobenzaldehyde 4a (1.2 g, 7.5 mmol) and potassium carbonate (1.3 g, 9.1 mmol) were dissolved in N,N-dimethylformamide (DMF) (30 mL) at room temperature. The reaction mixture was stirred at 90° C. for 2 h, cooled to room temperature, followed by addtion of ice water (100 mL), extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=10/1) afforded the title compound 14b (1.2 g, yield: 60.6%) as a yellow solid.

[0181] .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.92 (s, 1H), 7.61-7.51 (m, 2H), 7.30-7.23 (m, 1H), 6.85 (m, 1H), 6.73 (m, 1H), 2.34 (s, 3H).

Step II: Preparation of Compound 14c

[0182] At room temperature, 4-(4-Chloro-3-methylphenoxy)-3,5-difluorobenzaldehyde 14b (1.2 g, 4.24 mmol) was dissolved in methanol (50 mL), then NaBH.sub.4 (161 mg, 4.2 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 0.5 h, concentrated under reduced pressure, followed by addition of water, extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove desiccant. The filtrate was concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (petroleum ether/ethyl acetate=4/1) afforded the title compound 14c (0.89 g, yield: 73.7%) as a colorless oil.

Step III: Preparation of Compound 14

[0183] To a solution of (3,5-Difluoro-4-((2-(trifluoromethyl)pyridin-4-yl)oxy)phenyl)methanol 14c (40 mg, 0.14 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 1f (30 mg, 0.14 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 14 (16 mg, 24.7%) as a white solid.

[0184] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.23 (m, m 1H), 7.13-7.05 (m, 2H), 6.80 (m, 1H), 6.70 (m, 1H), 5.43 (s, 2H), 4.33-4.25 (m, 2H), 4.20 (m, 2H), 3.77 (m, 2H), 3.46-3.36 (m, 2H), 2.32 (s, 3H). MS (ESI): m/z 461.9 [M+H].sup.+.

Example 15 Preparation of Compound 15

[0185] ##STR00025## ##STR00026##

[0186] Step I: Preparation of Compound 15b

##STR00027##

[0187] At room temperature, 2,4,6-trichloro-5-methoxypyrimidine 1b (1.5 g, 7.03 mmol), ethanolamine 15a (0.64 g, 10.48 mmol) and diisopropylethylamine (1.36 g, 10.54 mmol) were dissolved in acetonitrile (70 mL), stirred and reacted for 3 h, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=20/1) afforded the title compound 15b (1.35 g, Yield: 80.6%) as an oil.

[0188] .sup.1H NMR (400 MHz, DMSO) δ7.99 (m, 1H), 4.77 (m, 1H), 3.73 (s, 3H), 3.52 (m, 2H), 3.39 (m, 2H).

[0189] Step II: Preparation of Compound 15c

##STR00028##

[0190] Under the protection of argon, 15b (1.35 g, 5.7 mmol) was dissolved in anhydrous dichloromethane (50 mL), then boron tribromide (28.5 mL, 1M) was slowly added dropwise, and the reaction was stirred at 0° C. for 4 h. The reaction was quenched by adding methanol, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 15c (1.09 g, Yield: 85.4%) as a white solid.

[0191] Step III: Preparation of Compound 15d

##STR00029##

[0192] Under the protection of nitrogen, triphenylphosphine (3.8 g, 14.5 mmol) was dissolved in anhydrous THF (50 mL), then DIAD (2.87 mL, 14.6 mmol) was slowly added dropwise. The reaction mixture was stirred at 0° C. for 15 min, and 15c was added dropwise (1.09 g, 4.87 mmol) in THF/DMF solution (30 mL/5 mL), then stirred at 0° C. for 4 h, quenched by addition of water dropwise, extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=40/1) afforded the title compound 15d (1.0 g, Yield: 99.6%) as a white solid.

[0193] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.14 (s, 1H), 4.30 (m, 2H), 3.75-3.66 (m, 2H).

[0194] Step IV: Preparation of Compound 15f

##STR00030##

[0195] At room temperature, 15d (1.0 g, 4.85 mmol) was dissolved in DMF (40 mL), followed by addition of potassium carbonate (0.92 g, 6.64 mmol) and 3-iodopropanol (1.23 g, 6.61 mmol). The reaction mixture was stirred at 80° C. for 2 h, quenched by addition of water the reaction, extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=20/1) afforded the title compound 15f (0.58 g, Yield: 45.3%) as a white solid.

[0196] .sup.1H NMR (400 MHz, CDCl.sub.3) δ4.26 (m, 2H), 3.77-3.70 (m, 2H), 3.61-3.54 (m, 3H), 3.30 (m, 1H), 1.84 (m, 2H).

Steps V and VI: Preparation of Compound 15h

[0197] ##STR00031##

[0198] At room temperature, 15f (0.58 g, 2.2 mmol) and triethylamine (0.67 g, 6.6 mmol) were dissolved in dry dichloromethane (30 mL), then methanesulfonyl chloride (0.30 g, 2.6 mmol) was added dropwise at 0° C. The reaction mixture was stirred at 0° C. for 1 h, concentrated under reduced pressure. The crude product from the previous step was dissolved in mixed solvent of dioxane/water (60 mL, 1/1), followed by addition of potassium carbonate (0.9 g, 6.6 mmol) at room temperature. The reaction mixture was stirred at 90° C. overnight, concentrated under reduced pressure. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 15h (85 mg, Yield: 16.8%) as a white solid.

[0199] .sup.1H NMR (400 MHz, CDCl.sub.3) δ4.19-4.14 (m, 2H), 3.97-3.90 (m, 2H), 3.56 (m, 2H), 3.44 (m, 2H), 2.14 (m, 2H).

[0200] Step VII: Preparation of Compound 15

##STR00032##

[0201] To a solution of (3,5-Difluoro-44(2-(trifluoromethyl)pyridin-4-yl)oxy)phenyl)methanol 4d (79 mg, 0.26 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 15h (40 mg, 0.18 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 15 (12 mg, 13.4%) as a white solid.

[0202] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.61 (m, 1H), 7.20 (m, 3H), 6.99 (m, 1H), 5.47 (m, 2H), 4.19 (m, 2H), 4.05-3.97 (m, 2H), 3.58-3.50 (m, 2H), 3.40 (m, 2H), 2.17 (m, 2H). MS (ESI): m/z 497.1 [M+H].sup.+.

Example 16 Preparation of Compound 16

[0203] ##STR00033##

[0204] To a solution of (3,5-Difluoro-44(6-(trifluoromethyl)pyridin-3-yl)oxy)phenyl)methanol 5c (61 mg, 0.2 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 8 mg, 0.2 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 15h (30 mg, 0.13 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 16 (11 mg, 17.0%) as a white solid.

[0205] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.49 (m, 1H), 7.62 (m, 1H), 7.28 (m, m 1H), 7.17 (m, 2H), 5.43 (s, 2H), 4.17 (m, 2H), 4.03-3.95 (m, 2H), 3.55-3.49 (m, 2H), 3.37 (m, 2H), 2.15 (m, 2H). MS (ESI): m/z 497.1 [M+H].sup.+.

Example 17 Preparation of Compound 17

[0206] ##STR00034##

[0207] To a solution of (3,5-Difluoro-4-((6-methylpyridin-3-yl)oxy)phenyl)methanol 7c (50 mg, 0.2 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 8 mg, 0.2 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 15h (30 mg, 0.13 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 17 (18 mg, 31.3%) as a white solid.

[0208] .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.26 (s, 1H), 7.09 (m, 4H), 5.42 (s, 2H), 4.16 (m, 2H), 3.99 (m, 2H), 3.51 (m, 2H), 3.36 (m, 2H), 2.51 (s, 3H), 2.14 (m, 2H). MS (ESI): m/z 443.1 [M+H].sup.+.

Example 18 Preparation of Compound 18

[0209] ##STR00035##

[0210] To a solution of (2,3-Difluorophenyl)methanol (38 mg, 0.26 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 15h (40 mg, 0.18 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 18 (16 mg, 26.5%) as a white solid.

[0211] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.45 (m, 1H), 6.95-6.83 (m, 2H), 5.42 (s, 2H), 4.20 (m, 2H), 4.04-3.95 (m, 2H), 3.56-3.49 (m, 2H), 3.40 (m, 2H), 2.16 (m, 2H). MS (ESI): m/z 336.1 [M+H].sup.+.

Example 19 Preparation of Compound 19

[0212] ##STR00036##

[0213] To a solution of (3,4,5-Trifluorophenyl)methanol (42 mg, 0.26 mmol) in dry N,N-dimethylformamide (5 mL) was added sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol) at 0° C., and stirred at room temperature for 5 min. Then compound 15h (40 mg, 0.18 mmol) was added, and stirred for 1 h, quenched with a small amount of water. Purification via silica gel column chromatography with an eluent system (dichloromethane/methanol=10/1) afforded the title compound 19 (15 mg, 23.6%) as a white solid.

[0214] .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.33 (m, 2H), 5.43 (s, 2H), 4.20 (m, 2H), 4.06-3.96 (m, 2H), 3.57-3.49 (m, 2H), 3.41 (m, 2H), 2.18 (m, 2H). MS (ESI): m/z 354.1 [M+H].sup.+.

[0215] Biological Evaluation

[0216] The biological activity of the compounds can be determined by using any suitable assay and tissue and in vivo models for determining the activity of the compounds as LpPLA2 inhibitors.

[0217] (1) Recombinant Human Lp-PLA2 (rhLp-PLA2) Assay (Also Known as PED6 Assay)

[0218] PED6 is a fluorescently labeled phospholipid that can be purchased directly from Invitogene or Molecular Probes. There is a fluorescence-quenching p-nitrophenyl group on the Sn3 position, and a Bodipy fluorescein (FL) group on the sn2 position. Once it is cleaved by the Lp-PLA2 enzyme, the FL group is released, resulting in enhanced fluorescence. However, Lp-PLA2 inhibitors can prevent this cleavage, so that no fluorescence enhancement is observed

[0219] Assay method: The compound to be tested (as shown in Table 1) was mixed with DMSO solution at a volume ratio of 1:3, diluted to prepare a source plate of a 384-well microplate. Then 0.01 μl of the compound was transferred via an ECHO liquid dispenser from the source plate to a 384-well Greiner 784076 plate, and 5 microliters of a buffer composed of 50 mM HEPES, pH7.4, 150 mM NaCl, 1 mM CHAPS (the buffer solution contains Recombinant human Lp-PLA2 enzyme at a concentration of 4 nM or 110 pM) was added to each well on the plate. The plate was centrifuged at 500 rpm for 10 seconds. After 30 minutes of pre-incubation, 5 microliters of the above buffer solution was added to a 384-well Greiner784076 plate, and the plate was centrifuged at 500 rpm for 10 seconds. After the plate was incubated at room temperature for 20 min in the dark, the fluorescence intensity was read at ex 480/em 540 with a ViewLux microplate imager, and the Excel's XL fitting model was used to performe curve and QC analysis to calculate pIC50. The results are listed in Table 1.

TABLE-US-00001 TABLE 1 Compound No. rhLp-PLA2 (pIC.sub.50) 1 7.8 2 8.0 3 8.5 4 9.9 5 9.7 6 9.8 7 9.6 8 9.0 9 9.8 10 9.7 11 10.0 12 10.1 13 9.8 14 9.7 15 10.2 16 10.1 17 10.3 18 8.0 19 8.4 Positive Compound 8.9 Rilapladib

[0220] (2) Human Plasma Lp-PLA2 Assay (Also Known as Thio-PAF Assay)

[0221] The human plasma assay was conducted using the sulphatide analog of PAF (phosphatidylcholine), which is hydrolyzed to produce phospholipids containing free sulfhydryl groups, subjected to Michael addition with CPM to generate fluorescence-enhancing maleimide.

Continuous quantitative analysis of thiol could be conducted by detecting the fluorescence intensity.
This assay can be used to detect the inhibitory activity of the Lp-PLA2 inhibitor on the Lp-PLA2 enzyme in human plasma.

[0222] Assay Method: The compound to be tested (as shown in Table 2) was mixed with a DMSO solution in a volume ratio of (1:3), and diluted to prepare a source plate of a 384-well microplate. Then 0.01 μl of the compound was transferred via an ECHO liquid dispenser from the source plate to a 384-well Greiner 784076 low-volume plate, and 8 μl of pre-aliquoted and frozen mixed human plasma was added. The plate was centrifuged at 500 rpm for 10 seconds. After a 30 min pre-incubation, 2 μl of a substrate solution, and containing 2.5 mM 2-thio-PAF (a solution in ethanol), 32 μM CPM (a solution in DMSO) and a buffer of 3.2 mM N-ethylmaleimide (NEM) (a buffer solution consisting of 50 mM HEPES, pH7.4, 150 mM NaCl, 1 mM CHAPS) was added by a BRAVO liquid handling station to a 384-well Greiner 784076 low-volume plate. After 2 min, the reaction was quenched with 5 μl of 5% trifluoroacetic acid. After the plate was incubated at room temperature for 40 min in a dark place, the fluorescence intensity was read at ex 380/em 485 with an Envision microplate reader, and the XL fitting model in Excel was used to perform the curve analysis and QC analysis to calculate pIC50. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Compound No. Thio-PAF (pIC.sub.50) 1 6.5 2 6.8 3 7.3 4 7.9 5 7.7 6 7.8 7 7.7 8 7.4 9 8.0 10 7.8 11 8.0 12 7.8 13 7.9 14 7.8 15 8.1 16 8.0 17 8.1 18 6.9 19 7.2 Positive compound 7.8 Rilapladib