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BENZIMIDAZOLE DERIVATIVES, PREPARATION METHOD THEREFOR AND MEDICAL USE THEREOF

20230250095 · 2023-08-10

    Inventors

    Cpc classification

    International classification

    Abstract

    The present disclosure relates to benzimidazole derivatives, a preparation method therefor and a medical use thereof. Specifically, the present disclosure relates to a benzimidazole derivative represented by the general formula (I), a preparation method therefor, a pharmaceutical composition containing the derivative, and its use as a therapeutic agent, in particular its use for the treatment of diseases related to P2X3 activity.

    ##STR00001##

    Claims

    1. A compound of formula (I) or a pharmaceutically acceptable salt or isomer thereof, ##STR00092## wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, halogen, C.sub.1-C.sub.6 hydroxyalkyl, C.sub.1-C.sub.6 alkyl optionally substituted with halogen or deuterium, and C.sub.1-C.sub.6 alkoxy optionally substituted with halogen or deuterium; R.sub.2 are each independently selected from the group consisting of hydrogen, deuterium, halogen, C.sub.1-C.sub.6 hydroxyalkyl, C.sub.1-C.sub.6 alkyl optionally substituted with halogen or deuterium, and C.sub.1-C.sub.6 alkoxy optionally substituted with halogen or deuterium; R.sub.3 and R.sub.4 are each independently selected from the group consisting of hydrogen, halogen and C.sub.1-C.sub.4 alkyl optionally substituted with halogen, or R.sub.3 and R.sub.4 form, together with the carbon atom to which they are attached, C.sub.3-C.sub.6 cyclohydrocarbylene optionally substituted with halogen, or R.sub.3 and R.sub.4 on adjacent carbon atoms together form C.sub.3-C.sub.8 cyclohydrocarbyl optionally substituted with halogen; R.sub.5 is selected from the group consisting of C.sub.1-C.sub.6 alkyl optionally substituted with halogen or cyano, C.sub.3-C.sub.6 cyclohydrocarbyl optionally substituted with halogen or cyano, heterocyclyl optionally substituted with halogen or cyano, C.sub.1-C.sub.6 alkoxy optionally substituted with halogen or cyano, and amino optionally substituted with alkyl; R.sub.6 are each independently selected from the group consisting of hydrogen, deuterium, halogen, cyano, cyclopropyl, and C.sub.1-C.sub.6 alkyl optionally substituted with halogen or deuterium; R.sub.7 and R.sub.8 are each independently selected from the group consisting of: a) hydrogen, deuterium, halogen, cyano, amino, hydroxy, C.sub.1-C.sub.6 alkyl optionally substituted with halogen, sulfone, sulfoxide, sulfonamide, sulfenamide, C.sub.1-3 carboxyl, and C.sub.1-C.sub.6 alkoxy optionally substituted with halogen; ##STR00093## wherein p is selected from the group consisting of 0, 1 and 2; R.sub.9 and R.sub.10 are each independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, and C.sub.3-C.sub.8 cyclohydrocarbyl, or R.sub.9 and R.sub.10 form, together with the nitrogen atom to which they are attached, 4- to 6-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, C.sub.1-C.sub.6 haloalkyl, and C.sub.1-C.sub.6 alkyl; R′ is selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.5 cycloalkyl, aryl and heteroaryl; and in ##STR00094## when p is 0 and R.sub.9 is hydrogen, R.sub.10 is not methyl; and when p is 0 and R.sub.9 is methyl, R.sub.10 is not hydrogen; ##STR00095## d) heterocyclyl and heteroaryl, wherein the heterocyclyl and heteroaryl are each optionally substituted with one or more substituents selected from the group consisting of oxo, halogen, hydroxy, carbonyl, C.sub.1-C.sub.6 alkyl and cyano, wherein the C.sub.1-C.sub.6 alkyl is optionally substituted with one or more halogens; and ##STR00096## wherein R.sub.11 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, aryl, heteroaryl, C.sub.3-C.sub.8 cyclohydrocarbyl, heterocyclyl, C.sub.1-C.sub.6 cyanoalkyl, C.sub.3-C.sub.8 cyclohydrocarbyloxy, and amino optionally substituted with C.sub.1-C.sub.6 alkyl; or R.sub.7 and R.sub.8 form, together with the atom to which they are attached, an optionally substituted aromatic or non-aromatic heterocyclic ring; X is selected from the group consisting of an oxygen atom, —NH— and methylene, wherein the methylene is optionally substituted with one or more substituents selected from the group consisting of halogen, C.sub.3-C.sub.8 cyclohydrocarbyl, C.sub.3-C.sub.6 cyclohydrocarbylene, and C.sub.1-C.sub.6 alkyl; m is an integer of 1-3; and n is an integer of 1-4.

    2. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.7 and R.sub.8 are each independently selected from the group consisting of: a) hydrogen, deuterium, halogen, cyano, amino, sulfone, sulfonamide, sulfenamide, and C.sub.1-C.sub.3 alkyl substituted with one or more halogen; ##STR00097## wherein p is selected from the group consisting of 0, 1 and 2; R.sub.9 and R.sub.10 are each independently selected from the group consisting of hydrogen, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkoxy, and C.sub.3-C.sub.6 cyclohydrocarbyl, or R.sub.9 and R.sub.10 form, together with the nitrogen atom to which they are attached, 4- to 6-membered heterocyclyl, wherein the heterocyclyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy and C.sub.1-C.sub.3 alkyl; R′ is selected from the group consisting of hydrogen, C.sub.1-C.sub.3 alkyl, C.sub.3-C.sub.6 cycloalkyl, aryl and heteroaryl; and in ##STR00098## when p is 0, ##STR00099## d) 4- to 6-membered heterocyclyl and heteroaryl, wherein the heterocyclyl and heteroaryl are each optionally substituted with one or more substituents selected from the group consisting of oxo, halogen, hydroxy, carbonyl, C.sub.1-C.sub.3 alkyl and cyano, wherein the C.sub.1-C.sub.3 alkyl is optionally substituted with one or more halogens; and ##STR00100## wherein R.sub.11 is selected from the group consisting of C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkoxy, 5- to 6-membered aryl or heteroaryl, 3- to 8-membered cyclohydrocarbyl, 3- to 8-membered heterocyclyl, C.sub.1-C.sub.3 cyanoalkyl, C.sub.3-C.sub.6 cyclohydrocarbyloxy, and amino optionally substituted with C.sub.1-C.sub.3 alkyl.

    3. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.7 is 4- to 6-membered heterocyclyl or heteroaryl, wherein the heterocyclyl or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of oxo, halogen, hydroxy, carbonyl, C.sub.1-C.sub.3 alkyl and cyano, wherein the C.sub.1-C.sub.3 alkyl is optionally substituted with one or more halogens; and R.sub.8 is selected from the group consisting of hydrogen, deuterium, halogen, cyano, and C.sub.1-C.sub.3 alkyl substituted with one or more halogens.

    4. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.7 is 4- to 6-membered heterocyclyl, wherein the heterocyclyl comprises —NH—C(═O)— or —NH—S(═O).sub.2—, and the heterocyclyl is optionally substituted with one or more substituents selected from the group consisting of oxo, halogen, hydroxy, carbonyl, C.sub.1-C.sub.3 alkyl and cyano, wherein the C.sub.1-C.sub.3 alkyl is optionally substituted with one or more halogens.

    5. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.7 and R.sub.8 form, together with the atom to which they are attached, a 3- to 12-membered aromatic or non-aromatic heterocyclic ring, wherein the heterocyclic ring is monocyclic or bicyclic, and the heterocyclic ring is optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.6 alkylamide, halogen, oxo, C.sub.1-C.sub.6 alkyl optionally substituted with halogen, and C.sub.1-C.sub.6 alkoxy.

    6. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.5 is C.sub.1-C.sub.6 alkyl optionally substituted with halogen or cyano or C.sub.1-C.sub.6 alkoxy optionally substituted with halogen or cyano; R.sub.6 are each independently selected from the group consisting of hydrogen, deuterium, halogen and cyano; and n is an integer of 1-4.

    7. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.2 are each independently selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.3 and R.sub.4 are each independently hydrogen or halogen, or R.sub.3 and R.sub.4 form, together with the carbon atom to which they are attached, C.sub.3-C.sub.6 cyclohydrocarbylene optionally substituted with halogen; R.sub.5 is C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy; R.sub.6 are each independently selected from the group consisting of hydrogen, deuterium, halogen and cyano; m is an integer of 1-3; and n is an integer of 1-4.

    8. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.7 is the following group optionally substituted with one or more substituents selected from the group consisting of methyl, a fluorine atom, a chlorine atom, halomethyl and cyano: ##STR00101## ##STR00102## and R.sub.8 is selected from the group consisting of hydrogen, deuterium, halogen, cyano, and C.sub.1-C.sub.3 alkyl substituted with one or more halogens.

    9. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.7 and R.sub.8 form, together with the atom to which they are attached, a heterocyclic ring A ##STR00103## wherein the heterocyclic ring A is selected from the group consisting of the following structures: ##STR00104## ##STR00105## ##STR00106## R.sub.6 are each independently selected from the group consisting of hydrogen, deuterium, halogen and cyano; R.sub.12 is independently selected from the group consisting of halogen, C.sub.1-C.sub.3 alkyl, and C.sub.3-C.sub.6 cyclohydrocarbylene, or adjacent R.sub.12 together form a ring, wherein the ring is optionally substituted with one or more halogens or C.sub.1-C.sub.3 alkyl; n is an integer selected from the group consisting of 1-3; and q is an integer of 0-6.

    10. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.2 are each independently selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.3 and R.sub.4 are each independently hydrogen or halogen, or R.sub.3 and R.sub.4 form, together with the carbon atom to which they are attached, C.sub.3-C.sub.6 cyclohydrocarbylene optionally substituted with halogen; R.sub.5 is C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy; R.sub.7 and R.sub.8 form, together with the atom to which they are attached, a heterocyclic ring A ##STR00107## wherein the heterocyclic ring A is selected from the group consisting of the following structures: ##STR00108## ##STR00109## ##STR00110## R.sub.6 are each independently selected from the group consisting of hydrogen, deuterium, halogen and cyano; m is an integer of 1-3; and n is an integer of 1-3.

    11. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, being ##STR00111## wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.2 are each independently selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.5 is C.sub.1-C.sub.3 alkyl or C.sub.1-C.sub.3 alkoxy; R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, deuterium, a chlorine atom, a fluorine atom and cyano; R.sub.7 is a 4- to 6-membered heterocyclyl, wherein the heterocyclyl comprises —NH—C(═O)— or —NH—S(═O).sub.2—, and the heterocyclyl is optionally substituted with one or more substituents selected from the group consisting of oxo, halogen, hydroxy, carbonyl, C.sub.1-C.sub.3 alkyl and cyano, wherein the C.sub.1-C.sub.3 alkyl is optionally substituted with one or more halogens; R.sub.8 is selected from the group consisting of hydrogen, deuterium, halogen, cyano and C.sub.1-C.sub.3 alkyl substituted with one or more halogens; and m is an integer of 1-3.

    12. The compound of formula (I-1) or the pharmaceutically acceptable salt or isomer thereof according to claim 11, wherein R.sub.7 is the following group optionally substituted with one or more substituents selected from the group consisting of methyl, a fluorine atom, a chlorine atom, halomethyl and cyano: ##STR00112## ##STR00113## and R.sub.8 is selected from the group consisting of hydrogen, deuterium, halogen and cyano.

    13. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, being ##STR00114## wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.2 are each independently selected from the group consisting of hydrogen, deuterium, C.sub.1-C.sub.3 alkyl optionally substituted with halogen or deuterium, and halogen; R.sub.5 is C.sub.1-C.sub.3 alkyl or C.sub.1-C.sub.3 alkoxy; R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, deuterium, a chlorine atom, a fluorine atom and cyano; R.sub.7 and R.sub.8 form, together with the atom to which they are attached, a 4- to 8-membered non-aromatic heterocyclic ring, wherein the heterocyclic ring is monocyclic or bicyclic, the heterocyclic ring comprises —NH—C(═O)— or —NH—S(═O).sub.2—, and the heterocyclic ring is optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.3 alkylamide, halogen, oxo, C.sub.1-C.sub.3 alkyl optionally substituted with halogen, and C.sub.1-C.sub.3 alkoxy; and m is an integer of 1-3.

    14. The compound of formula (I-1) or the pharmaceutically acceptable salt or isomer thereof according to claim 13, wherein R.sub.7 and R.sub.8 form, together with the atom to which they are attached, a heterocyclic ring A ##STR00115## wherein the heterocyclic ring A is selected from the group consisting of the following structures: ##STR00116## ##STR00117## ##STR00118## R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, deuterium, a chlorine atom and a fluorine atom; R.sub.12 are each independently selected from the group consisting of halogen, C.sub.1-C.sub.3 alkyl, and C.sub.3-C.sub.6 cyclohydrocarbylene, or adjacent R.sub.12 together form a ring, wherein the ring is optionally substituted with one or more halogens or C.sub.1-C.sub.3 alkyl; and q is an integer of 0-6.

    15. The compound of formula (I-1) or the pharmaceutically acceptable salt or isomer thereof according to claim 14, wherein R.sub.7 and R.sub.8 form, together with the atom to which they are attached, a heterocyclic ring A ##STR00119## wherein the heterocyclic ring A is selected from the group consisting of the following structures: ##STR00120## ##STR00121## ##STR00122## R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, deuterium, a chlorine atom and a fluorine atom.

    16. The compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, being ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130##

    17. (canceled)

    18. A method for preparing the compound of formula (I) or the pharmaceutically acceptable salt or isomer thereof according to claim 1, comprising the following steps: ##STR00131## subjecting a compound of formula (I-a) to a reaction with a compound of formula (I-b) under an alkaline condition to give a compound of formula (I-c); subjecting the compound of formula (I-c) to a reduction reaction to give a compound of formula (I-d); subjecting the compound of formula (I-d) to a ring closure reaction with a compound of formula (I-e) under an acidic condition to give a compound of formula (I-g); and subjecting the compound of formula (I-g) under the action of a catalyst to give the compound of formula (I); wherein the catalyst is selected from the group consisting of palladium/carbon, Raney Ni, tetrakis(triphenylphosphine)palladium(0), palladium dichloride, palladium acetate, [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride, 1,1′-bis(dibenzylphosphino)ferrocene-palladium(II)dichloride, tris(dibenzylideneacetone)dipalladium(0), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl, [1,1′-bis(di-tert-butylphosphino)ferrocene]palladium (II) dichlorine, cuprous iodide, cuprous bromide, cuprous chloride and copper(II) trifluoromethanesulphonate; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, X, m, and n are as defined in claim 1; and Y and Z are each independently selected from the group consisting of halogen, sulfonyl and sulfinyl.

    19. A pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt or isomer thereof according to claim 1, and at least one pharmaceutically acceptable carrier, diluent or excipient.

    20. A method for treating a disease related to P2X3 activity in a subject in need thereof, the method comprising: administering to the subject an effective amount of the compound or the pharmaceutically acceptable salt or isomer thereof according to claim 1.

    21. The method of claim 20, wherein the disease is selected from the group consisting of pain, urinary tract diseases and cough.

    Description

    DETAILED DESCRIPTION

    [0244] The structure of the compound is determined by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS). NMR shift (δ) is given in a unit of 10.sup.−6 (ppm). NMR spectra are determined using a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d.sub.6), deuterated chloroform (CDCl.sub.3) and deuterated methanol (CD.sub.3OD) as determination solvents and tetramethylsilane (TMS) as an internal standard.

    [0245] HPLC analysis is performed using a Waters ACQUITY ultra high performance LC, Shimadzu LC-20A systems, Shimadzu LC-2010HT series, or Agilent 1200 LC high performance liquid chromatograph (ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm column, Ultimate XB-C18 3.0×150 mm column, or Xtimate C18 2.1×30 mm column).

    [0246] MS analysis is performed using Waters SQD2 mass spectrometer in positive/negative ion mode with a mass scan range of 100-1200.

    [0247] Chiral HPLC analysis is performed using Chiralpak IC-3 100×4.6 mm I.D., 3 μm, Chiralpak AD-3 150×4.6 mm I.D., 3 μm, Chiralpak AD-3 50×4.6 mm I.D., 3 μm, Chiralpak AS-3 150×4.6 mm I.D., 3 μm, Chiralpak AS-3 100×4.6 mm I.D., 3 μm, ChiralCel OD-3 150×4.6 mm I.D., 3 μm, ChiralCel OD-3 100×4.6 mm I.D., 3 μm, ChiralCel OJ-H 150×4.6 mm I.D., 5 μm, ChiralCel OJ-3 150×4.6 mm I.D., 3 μm chromatographic columns.

    [0248] Huanghai HSGF254 or Qingdao GF254 silica gel plates of specifications 0.15 mm to 0.2 mm are adopted for thin layer chromatography (TLC) analysis and 0.4 mm to 0.5 mm for TLC separation and purification.

    [0249] Yantai Huanghai silica gel of 100-200 mesh, 200-300 mesh or 300-400 mesh is generally used as a carrier in column chromatography.

    [0250] Chiral HPLC preparation is performed using a DAICEL CHIRALPAK IC (250×30 mm, 10 μm) or Phenomenex-Amylose-1 (250×30 mm, 5 μm) column.

    [0251] Known starting materials described herein may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Chembee Chemicals, and other companies.

    [0252] In the examples, all reactions can be performed under argon atmosphere or nitrogen atmosphere unless otherwise specified.

    [0253] The argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of argon or nitrogen.

    [0254] The hydrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of hydrogen.

    [0255] Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator is used in the pressurized hydrogenation reactions.

    [0256] The hydrogenation reactions usually involve 3 cycles of vacuumization and hydrogen purge.

    [0257] In the examples, a solution refers to an aqueous solution unless otherwise specified.

    [0258] In the examples, the reaction temperature is room temperature, i.e., 20° C. to 30° C., unless otherwise specified.

    [0259] The monitoring of the reaction progress in the examples is conducted by thin layer chromatography (TLC). The developing solvent for reactions, the eluent system for column chromatography for purification of compounds, the developing solvent system for thin layer chromatography system and the volume ratio of the solvents are adjusted according to the polarity of the compound, or by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

    [0260] The positive compound MK-7264 is prepared by referring to the experimental procedures in the patent WO2005095359.

    Example 1

    1-[4-(1-{[(2R)-4-acetylmorpholin-2-yl]methyl}-5-methyl-1H-1,3-benzooxadiazol-2-yl)-3,5-difluorophenyllpyrrolidin-2-one (1)

    [0261] ##STR00069## ##STR00070##

    Step 1: tert-butyl (2S)-2-[(1,3-dicarbonyl-2,3-dihydro-1H-isoindol-2-yl)methyl]morpholine-4-carboxylate (1a)

    [0262] Tert-butyl (2R)-2-(hydroxymethyl)morpholine-4-carboxylate (3.00 g, 13.81 mmol), 2,3-dihydro-1H-isoindole-1,3-dione (2.23 g, 15.19 mmol) and triphenylphosphine (5.43 g, 20.71 mmol) were dissolved in THF (50 mL), diisopropyl azodicarboxylate (4.1 mL, 20.71 mmol) was added dropwise at 0° C. under nitrogen atmosphere, and the mixture was stirred at room temperature for 12 h. After LCMS showed that the starting material was reacted completely, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-30%, 60 mL/min) to give the title compound 1a (6.00 g, yield: 87.8%).

    [0263] MS (ESI) m/z=291.1 [M+H].sup.+.

    [0264] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.83-7.76 (m, 2H), 7.69-7.63 (m, 2H), 4.91 (spt, J=6.2 Hz, 3H), 3.96-3.84 (m, 1H), 3.83-3.77 (m, 2H), 3.75-3.64 (m, 2H), 3.60 (dd, J=4.6, 13.6 Hz, 1H), 3.37 (dt, J=2.9, 11.4 Hz, 1H), 2.92 (br s, 1H), 2.69 (br s, 1H), 1.39 (s, 9H), 1.21 (s, 11H).

    Step 2: tert-butyl (2S)-2-(aminomethyl)morpholine-4-carboxylate (1b)

    [0265] To a solution of compound 1a (6.00 g, 12.12 mmol) in ethanol (150 mL) was added hydrazine hydrate (1.07 g, 18.19 mmol) at room temperature, and the mixture was stirred at 80° C. for 1 h. After LCMS showed that the reaction was completed, the reaction mixture was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (80 mL) and filtered, and the filtrate was concentrated under reduced pressure to give the title compound 1b (3.00 g, yield: 91.5%).

    [0266] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.27 (br s, 2H), 3.82 (br d, J=10.9 Hz, 3H), 3.51-3.41 (m, 1H), 3.34-3.23 (m, 1H), 3.29 (br dd, J=3.7, 6.7 Hz, 1H), 2.85 (br s, 1H), 2.73-2.63 (m, 2H), 2.57 (br s, 1H), 1.40 (s, 9H).

    Step 3: tert-butyl (2S)-2-{[(4-methyl-2-nitrophenyl)amino]methyl}morpholine-4-carboxylate (1c)

    [0267] To a solution of compound 1b (3.00 g, 11.10 mmol) in 1,4-dioxane (60 mL) were added 1-fluoro-4-methyl-2-nitrobenzene (1.72 g, 11.10 mmol) and ethyl diisopropylamine (3.7 mL, 22.19 mmol) at room temperature, and the mixture was stirred at 100° C. for 12 h. After LCMS showed that the starting material was consumed completely, water (50 mL) was added, and the mixture was extracted with ethyl acetate (80 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product, which was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-17%, 60 mL/min) to give the title compound 1c (2.35 g, yield: 60%).

    [0268] MS (ESI) m/z=374.2 [M+Na]*.

    [0269] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.11 (br s, 1H), 7.99 (d, J=0.8 Hz, 1H), 7.30-7.27 (m, 1H), 6.77 (d, J=8.8 Hz, 1H), 4.06-3.81 (m, 3H), 3.75-3.65 (m, 1H), 3.64-3.53 (m, 1H), 3.47-3.29 (m, 2H), 3.09-2.91 (m, 1H), 2.80 (br s, 1H), 2.28 (s, 3H), 1.47 (s, 9H).

    Step 4: tert-butyl (2S)-2-{[(2-amino-4-methylphenyl)amino]methyl}morpholine-4-carboxylate (1d)

    [0270] To a solution of compound 1c (2.00 g, 5.69 mmol) in methanol (20 mL) was added 10% Pd/C (100 mg) at room temperature, and the resulting mixture was purged 3 times with hydrogen and stirred at room temperature for 12 h. After LCMS showed that the starting material was consumed completely, the reaction mixture was filtered, and the filtrate was concentrated in vacuum to give the title compound 1d (1.73 g, yield: 94.4%).

    [0271] MS (ESI) m/z=322.2 [M+H].sup.+.

    [0272] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.65-6.50 (m, 3H), 3.92 (br d, J=10.0 Hz, 3H), 3.70-3.62 (m, 1H), 3.60-3.51 (m, 1H), 3.44 (br s, 2H), 3.32-3.00 (m, 3H), 2.97 (br s, 1H), 2.79 (br s, 1H), 2.23 (s, 3H), 1.48 (s, 9H).

    Step 5: tert-butyl (2S)-2-{[2-(4-bromo-2,6-difluorophenyl)-5-methyl-1H-1,3-benzooxadiazol-1-yl]methyl}morpholine-4-carboxylate (1e)

    [0273] To a solution of compound 1d (810.0 mg, 2.52 mmol) in n-butanol (14 mL) were added 4-bromo-2,6-difluorobenzaldehyde (612.6 mg, 2.77 mmol) and acetic acid (144 μL, 2.52 mmol) at room temperature, and the resulting mixture was purged 3 times with nitrogen and stirred at 90° C. for 14 h. After LCMS showed that the starting material was reacted completely, the reaction solution was concentrated in vacuum to give a crude product, which was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-17%) to give the title compound 1e (189.8 mg, yield: 14.4%).

    [0274] MS (ESI) m/z=524.2 [M+H].sup.+.

    [0275] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.63 (br s, 1H), 7.37 (br d, J=7.6 Hz, 1H), 7.26 (br d, J=6.0 Hz, 2H), 7.19 (br d, J=7.2 Hz, 1H), 4.10 (br s, 2H), 3.88-3.53 (m, 4H), 3.30 (br s, 1H), 2.77 (br s, 1H), 2.51 (br s, 3H), 2.48-2.38 (m, 1H), 1.42 (br s, 9H).

    Step 6: 1-[4-(1-{[(2R)-4-acetylmorpholin-2-yl]methyl}-5-methyl-1H-1,3-benzooxadiazol-2-yl)-3,5-difluorophenyl]pyrrolidin-2-one (1)

    [0276] To a solution of compound 1e (90.0 mg, 0.172 mmol) and pyrrolidin-2-one (29.3 mg, 0.345 mmol) in 1,4-dioxane (3 mL) were added Cs.sub.2CO.sub.3 (112.3 mg, 0.345 mmol), Pd(OAc).sub.2 (5.8 mg, 0.026 mmol) and Xantphos (20.0 mg, 0.034 mmol) at room temperature, and the resulting mixture was purged with nitrogen for 5 min and stirred under microwave irradiation at 100° C. for 35 min. After LCMS showed that the reaction was completed, water (5 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (10 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give a crude product, which was dissolved in dichloromethane (2 mL). Trifluoroacetic acid (400 μL) was added in an ice-water bath, and the mixture was stirred at room temperature for 1 h. After LCMS showed that the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in methanol (2 mL). Triethylamine (238 μL, 1.71 mmol) was added in an ice-water bath to adjust pH to 8, followed by the addition of acetic anhydride (80 μL, 0.854 mmol), and the mixture was reacted at room temperature for 1 h. After LCMS showed that the reaction was completed, the reaction mixture was concentrated. The residue was purified by C18 reverse phase chromatography (acetonitrile/water (containing 0.05% ammonia water)=10%-75%) and lyophilized to give the title compound 1 (52.0 mg, yield: 64%).

    [0277] MS (ESI) m/z=469.2 [M+H].sup.+.

    [0278] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.43 (br d, J=7.0 Hz, 1H), 7.63 (dd, J=3.3, 10.3 Hz, 2H), 7.37 (s, 1H), 6.93-6.73 (m, 1H), 4.09 (br dd, J=9.2, 18.9 Hz, 2H), 3.99-3.85 (m, 3H), 3.77-3.52 (m, 2H), 3.51-3.41 (m, 2H), 3.09-2.99 (m, 2H), 2.90-2.82 (m, 1H), 2.73-2.65 (m, 1H), 2.63-2.58 (m, 2H), 2.41 (s, 3H), 2.18-2.03 (m, 2H).

    Example 2

    Methyl 2-((2-(2,6-difluoro-4-(2-carbonylpyrrolidin-1-yl)phenyl)-5-methyl-1H-benzo[d]imidazol-1-yl)methyl)morpholine-4-carboxylate (2)

    [0279] ##STR00071##

    [0280] Example 2 was synthesized by referring to the synthetic procedures for Example 1, with tert-butyl (2R)-2-(hydroxymethyl)morpholine-4-carboxylate replaced by tert-butyl 2-(hydroxymethyl)morpholine-4-carboxylate in step 1, and acetic anhydride replaced by methyl chloroformate in step 6.

    [0281] MS (ESI) m/z=486.4 [M+H].sup.+.

    Example 3

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chlorophenyl)pyrrolidin-2-one (3)

    [0282] ##STR00072##

    [0283] Example 3 was synthesized by referring to the synthetic procedures for Example 1, with 4-bromo-2,6-difluorobenzaldehyde replaced by 4-bromo-2-chlorobenzaldehyde in step 5.

    [0284] MS (ESI) m/z=467.2 [M+H].sup.+.

    Example 4

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one (4)

    [0285] ##STR00073##

    [0286] Example 4 was synthesized by referring to the synthetic procedures for Example 1, with 4-bromo-2,6-difluorobenzaldehyde replaced by 4-bromo-2-chloro-6-fluorobenzaldehyde in step 5.

    [0287] MS (ESI) m/z=485.5 [M+H].sup.+.

    [0288] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.88 (br s, 1H), 7.79 (br d, J=11.6 Hz, 1H), 7.70-7.60 (m, 1H), 7.49 (br s, 1H), 7.20-7.12 (m, 1H), 4.33-4.16 (m, 2H), 4.03 (br d, J=15.0 Hz, 1H), 3.97-3.87 (m, 3H), 3.78 (br d, J=17.6 Hz, 1H), 3.68-3.55 (m, 1H), 3.12 (br d, J=11.0 Hz, 1H), 2.99 (br d, J=11.6 Hz, 1H), 2.81-2.70 (m, 2H), 2.58 (br d, J=7.9 Hz, 2H), 2.44 (br s, 3H), 2.14-2.06 (m, 2H), 1.93 (s, 2H).

    Example 5

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-(difluoromethyl)-1H-benzo[d]imidazol-2-yl)-3,5-difluorophenyl)pyrrolidin-2-one (5)

    [0289] ##STR00074##

    [0290] Example 5 was synthesized by referring to the synthetic procedures for Example 1, with 1-fluoro-4-methyl-2-nitrobenzene replaced by 4-(difluoromethyl)-1-fluoro-2-nitrobenzene in step 3.

    [0291] MS (ESI) m/z=505.5 [M+H].sup.+.

    Example 6

    Methyl (S)-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-6-methyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)morpholine-4-carboxylate (6)

    [0292] ##STR00075## ##STR00076##

    Step 1: methyl (R)-2-(hydroxymethyl)morpholine-4-carboxylate (6a)

    [0293] To a solution of tert-butyl (2R)-2-(hydroxymethyl)morpholine-4-carboxylate (10.00 g, 46.03 mmol) in methanol (50 mL) was added dropwise a solution of 4 N HCl in methanol (57.5 mL, 230.13 mmol) at room temperature, and the mixture was stirred at room temperature for 4 h. After TLC (petroleum ether/ethyl acetate=3/1, sample was treated with a saturated NaHCO.sub.3 solution) showed that the starting material was reacted completely, the reaction mixture was concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane (50 mL), and ethyl diisopropylamine (38.0 mL, 230.13 mmol) and methyl chloroformate (4.3 mL, 55.23 mmol) were added dropwise slowly and successively in an ice-water bath. The mixture was stirred at room temperature for 2 h. After TLC (petroleum ether/ethyl acetate=1/2) showed that the starting material was reacted completely, water (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×5). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 6a (9.45 g, yield: 94%).

    [0294] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.02-3.85 (m, 3H), 3.73 (s, 3H), 3.69-3.66 (m, 1H), 3.63-3.53 (m, 3H), 3.05-2.92 (m, 1H), 2.90-2.72 (m, 1H).

    Step 2: methyl (S)-2-((1,3-dicarbonylisoindolin-2-yl)methyl)morpholine-4-carboxylate (6b)

    [0295] Compound 6b was synthesized by referring to step 1 in Example 1, with tert-butyl (2R)-2-(hydroxymethyl)morpholine-4-carboxylater replaced by compound 6a.

    [0296] MS (ESI) m/z=305.0 [M+H].sup.+.

    [0297] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91-7.84 (m, 2H), 7.77-7.71 (m, 2H), 3.99 (br s, 1H), 3.93-3.87 (m, 2H), 3.82-3.80 (m, 1H), 3.77 (br s, 1H), 3.71 (s, 3H), 3.70-3.64 (m, 1H), 3.47 (dt, J=2.4, 11.2 Hz, 1H), 3.04 (br s, 1H), 2.83 (br t, J=11.6 Hz, 1H).

    Step 3: methyl (S)-2-(aminomethyl)morpholine-4-carboxylate (6c)

    [0298] Compound 6c was synthesized by referring to step 2 in Example 1, with compound 1a replaced by compound 6b.

    [0299] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.91 (br d, J=9.2 Hz, 3H), 3.72 (s, 3H), 3.61-3.48 (m, 1H), 3.37 (br d, J=2.4 Hz, 1H), 2.99 (br s, 1H), 2.76 (br d, J=6.4 Hz, 1H), 2.74-2.63 (m, 1H).

    Step 4: methyl (S)-2-(((5-methyl-3-nitropyridin-2-yl)amino)methyl)morpholine-4-carboxylate (6d)

    [0300] To a solution of 2-chloro-5-methyl-3-nitropyridine (200.0 mg, 1.16 mmol) and compound 6c (356.2 mg, 1.39 mmol) in dimethyl sulfoxide (2 mL) was added ethyl diisopropylamine (0.57 mL, 3.48 mmol) at room temperature, and the mixture was stirred at 60° C. for 12 h. After LCMS showed that the starting material was consumed completely, the reaction mixture was diluted with ethyl acetate (30 mL) and filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-50%) to give the title compound 6d (237.2 mg, yield: 66%).

    [0301] MS (ESI) m/z=310.9 [M+H].sup.+.

    [0302] .sup.1H NMR (400 MHz, CDCl.sub.3) (8.42-8.19 (m, 3H), 4.09-3.83 (m, 3H), 3.73 (s, 3H), 3.70-3.51 (m, 3H), 3.11-2.93 (m, 1H), 2.89-2.69 (m, 1H), 2.28 (s, 3H).

    Step 5: methyl (S)-2-(((3-amino-5-methylpyridin-2-yl)amino)methyl)morpholine-4-carboxylate (6e)

    [0303] Compound 6e was synthesized by referring to step 4 in Example 1, with compound 1c replaced by compound 6d.

    [0304] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.55 (s, 1H), 6.71 (s, 1H), 4.31 (br s, 1H), 4.07-3.82 (m, J=12.8 Hz, 3H), 3.72 (s, 3H), 3.71-3.63 (m, 2H), 3.61-3.51 (m, 1H), 3.44-3.31 (m, 1H), 3.21 (br s, 2H), 3.06-2.96 (m, 1H), 2.86-2.75 (m, 1H), 2.16 (s, 3H).

    Step 6: methyl (S)-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-6-methyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)morpholine-4-carboxylate (6)

    [0305] Compound 6 synthesized by referring to step 5 in Example 1, with compound 1d replaced by compound 6e, and 4-bromo-2,6-difluorobenzaldehyde replaced by 3,5-difluoro-4-formyl-N-methyl benzamide.

    [0306] MS (ESI) m/z=460.1 [M+H].sup.+.

    [0307] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.32 (s, 1H), 7.92 (s, 1H), 7.53 (br s, 1H), 7.45 (br d, J=8.8 Hz, 2H), 4.36 (dd, J=3.2, 14.4 Hz, 1H), 4.16 (br dd, J=7.6, 14.4 Hz, 1H), 3.98 (br s, 1H), 3.83-3.69 (m, 1H), 3.66 (s, 3H), 3.65-3.61 (m, 1H), 3.57 (br d, J=10.8 Hz, 1H), 3.23 (br t, J=10.8 Hz, 1H), 3.03 (d, J=4.4 Hz, 3H), 2.76 (br s, 1H), 2.53 (s, 3H), 2.47 (br s, 1H).

    Example 7

    Methyl (S)-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-5-methyl-1H-imidazo[4,5-b]pyridin-1-yl)methyl)morpholine-4-carboxylate (7)

    [0308] ##STR00077##

    Step 1: 6-methyl-2-nitropyridin-3-yl trifluoromethanesulfonate (7a)

    [0309] To a solution of 6-methyl-2-nitropyridin-3-ol (1.00 g, 6.50 mmol) in dichloromethane (10 mL) were added triethylamine (1.4 mL, 9.70 mmol) and trifluoromethanesulfonic anhydride (1.3 mL, 7.80 mmol) at 0° C. under nitrogen atmosphere, and the resulting solution was stirred at 0° C. for 2 h. Water (80 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×3). The combined organic phase was washed with brine (80 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 7a (1.70 g, yield: 92%).

    [0310] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.82 (d, J=8.4 Hz, 1H), 7.60 (d, J=8.4 Hz, 1H), 2.70 (s, 3H).

    Step 2: methyl (S)-2-(((6-methyl-2-nitropyridin-3-yl)amino)methyl)morpholine-4-carboxylate (7b)

    [0311] To a solution of compound 7a (1.20 g, 4.2 mmol) and compound 6c (870 mg, 3.5 mmol) in acetonitrile (15 mL) was added dropwise triethylamine (1.10 g, 10.5 mmol) at room temperature, and the resulting solution was stirred at 80° C. for 12 h. After LCMS showed that the starting material was consumed completely, the reaction solution was concentrated under reduced pressure, and the resulting crude product was purified by C18 reverse phase column chromatography (methanol/water solution (0.10% TFA)=50%) and lyophilized to give the title compound 7b (275.0 mg, yield: 25%).

    [0312] MS (ESI) m/z=310.9 [M+H].sup.+.

    [0313] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.92-7.80 (m, 1H), 7.35-7.29 (m, 2H), 3.74 (s, 3H), 3.63-3.52 (m, 3H), 3.51-3.40 (m, 2H), 3.39-3.32 (m, 1H), 3.25 (dd, J=7.6, 13.2 Hz, 1H), 3.11-2.93 (m, 2H), 2.52 (s, 3H).

    Step 3: methyl (S)-2-(((2-amino-6-methylpyridin-3-yl)amino)methyl)morpholine-4-carboxylate (7c)

    [0314] Compound 7c was synthesized by referring to step 4 in Example 1, with compound 1c replaced by compound 7b.

    [0315] MS (ESI) m/z=280.9 [M+H].sup.+.

    [0316] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.75 (d, J=8.0 Hz, 1H), 6.53 (d, J=7.6 Hz, 1H), 3.74 (s, 3H), 3.61-3.53 (m, 3H), 3.46 (dd, J=3.6, 13.6 Hz, 1H), 3.25 (dd, J=7.6, 13.6 Hz, 1H), 3.11-3.02 (m, 2H), 2.91-2.69 (m, 2H), 2.34 (s, 3H).

    Step 4: methyl (S)-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-5-methyl-1H-imidazo[4,5-b]pyridin-1-yl)methyl)morpholine-4-carboxylate (7)

    [0317] Compound 7 was synthesized by referring to step 5 in Example 1, with compound 1d replaced by compound 7c, and 4-bromo-2,6-difluorobenzaldehyde replaced by 3,5-difluoro-4-formyl-N-methyl benzamide.

    [0318] MS (ESI) m/z=460.1 [M+H].sup.+.

    [0319] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.91 (br s, 1H), 7.78 (d, J=6.4 Hz, 1H), 7.52 (d, J=8.4 Hz, 2H), 7.22 (d, J=8.4 Hz, 1H), 4.17-3.98 (m, 2H), 3.95-3.59 (m, 6H), 3.59-3.46 (m, 1H), 3.36-3.18 (m, 1H), 3.05 (d, J=4.8 Hz, 3H), 2.86-2.77 (m, 1H), 2.75 (s, 3H), 2.56-2.42 (m, 1H).

    Example 8

    Methyl (S)-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-6-methyl-3H-imidazo[4,5-c]pyridin-3-yl)methyl)morpholine-4-carboxylate (8)

    [0320] ##STR00078##

    Step 1: (S)-5-(((4-(methoxycarbonyl)morpholin-2-yl)methyl)amino)-2-methyl-4-nitropyridine 1-oxide (8a)

    [0321] 5-bromo-2-methyl-4-nitropyridine 1-oxide (200.0 mg, 0.86 mmol) and compound 6c (329.0 mg, 1.89 mmol) were dissolved in tetrahydrofuran (5 mL) at room temperature, and the resulting solution was stirred at 80° C. for 12 h. After LCMS showed that the starting material was consumed completely, the reaction solution was concentrated under reduced pressure, and methanol (2 mL) was added to the resulting crude product. The mixture was filtered, and the solid was dried to give the title compound 8a (50.0 mg, yield: 18%).

    [0322] MS (ESI) m/z=327.0 [M+H].sup.+.

    [0323] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.06 (s, 1H), 8.01 (s, 1H), 7.90-7.80 (m, 1H), 4.23-3.81 (m, 4H), 3.74 (s, 3H), 3.61-3.55 (m, 1H), 3.42-3.35 (m, 1H), 3.32-3.24 (m, 1H), 3.14-3.00 (m, 2H), 2.41 (s, 3H).

    Step 2: methyl (S)-2-(((4-amino-6-methylpyridin-3-yl)amino)methyl)morpholine-4-carboxylate (8b)

    [0324] Compound 8b was synthesized by referring to step 4 in Example 1, with compound 1c replaced by compound 8a.

    [0325] MS (ESI) m/z=281.2 [M+H].sup.+.

    [0326] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.76 (s, 1H), 6.46 (s, 1H), 4.03-3.81 (m, 4H), 3.73 (s, 3H), 3.69-3.53 (m, 3H), 3.16-3.07 (m, 2H), 2.40 (s, 3H).

    Step 3: methyl (S)-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-6-methyl-3H-imidazo[4,5-c]pyridin-3-yl)methyl)morpholine-4-carboxylate (8)

    [0327] Compound 8 was synthesized by referring to step 5 in Example 1, with compound 1d replaced by compound 8b, and 4-bromo-2,6-difluorobenzaldehyde replaced by 3,5-difluoro-4-formyl-N-methyl benzamide.

    [0328] MS (ESI) m/z=460.2 [M+H].sup.+.

    [0329] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.87 (s, 1H), 7.58 (s, 1H), 7.49 (d, J=8.4 Hz, 2H), 6.92-6.75 (m, 1H), 4.25-4.09 (m, 2H), 3.98-3.71 (m, 3H), 3.69 (s, 3H), 3.65-3.55 (m, 1H), 3.36-3.25 (m, 1H), 3.06 (d, J=4.8 Hz, 3H), 2.90-2.77 (m, 1H), 2.72 (s, 3H), 2.60-2.46 (m, 1H).

    Example 9

    Methyl (S)-2-((2-(2,6-difluoro-4-(2-carbonylpyrrolidin-1-yl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate (9)

    [0330] ##STR00079##

    Step 1: tert-butyl (2R)-2-formylmorpholine-4-carboxylate (9a)

    [0331] To a solution of oxalyl chloride (9.9 mL, 115.07 mmol) in anhydrous dichloromethane (200 mL) was added dropwise dimethyl sulfoxide (9.8 mL, 138.08 mmol) at −70° C. under nitrogen atmosphere. The mixture was stirred at −70° C. for 30 min. A solution of tert-butyl (2R)-2-(hydroxymethyl)morpholine-4-carboxylate (10.00 g, 46.03 mmol) in dichloromethane (30 mL) was added dropwise, and the mixture was stirred at −70° C. for 2 h. Triethylamine (32.0 mL, 230.13 mmol) was added dropwise at −70° C., and the mixture was stirred at −70° C. for 30 min. After TLC (petroleum ether/ethyl acetate=1/1) showed that the reaction was completed, the mixture was warmed to room temperature and washed with a saturated NaHCO.sub.3 solution (80 mL×2) and brine (80 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 9a (9.90 g, yield: 99.9%).

    [0332] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.67 (s, 1H), 4.15-4.04 (m, 1H), 3.95-3.82 (m, 2H), 3.70-3.53 (m, 2H), 3.13-2.97 (m, 2H), 1.50 (s, 9H).

    Step 2: tert-butyl (2R)-2-formylmorpholine-4-carboxylate (9b)

    [0333] To a solution of compound 9a (9.90 g, 45.99 mmol) in methanol (200 mL) were added potassium carbonate (25.43 g, 183.97 mmol) and dimethyl (1-diazo-2-oxopropyl) phosphonate (17.67 g, 91.99 mmol) successively at 0° C. under nitrogen atmosphere. The mixture was stirred at room temperature for 12 h. After TLC (petroleum ether/ethyl acetate=3/1) showed that the reaction was completed, water (80 mL) was added to the reaction mixture, and the resulting mixture was concentrated under reduced pressure to remove methanol. The residue was extracted with ethyl acetate (100 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-25%) to give compound 9b (6.4 g, yield: 65.9%).

    [0334] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.29-4.25 (m, 1H), 3.99-3.96 (m, 1H), 3.93-3.63 (m, 1H), 3.64-3.55 (m, 2H), 3.33-3.27 (m, 2H), 2.50 (d, J=2.4 Hz, 1H), 1.49 (s, 9H).

    Step 3: methyl (S)-2-ethynylmorpholine-4-carboxylate (9c)

    [0335] To a solution of compound 9b (6.4 g, 30.29 mmol) in dichloromethane (60 mL) was added dropwise a solution of 4 N HCl in methanol (60 mL, 240.00 mmol) at room temperature, and the mixture was stirred at room temperature for 12 h. After TLC (petroleum ether/ethyl acetate=3/1) showed that the starting material was reacted completely, the reaction mixture was concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane (80 mL), and ethyl diisopropylamine (14.8 mL, 89.43 mmol) and methyl chloroformate (2.8 mL, 35.98 mmol) were added dropwise slowly and successively in an ice-water bath. The mixture was stirred at room temperature for 12 h. After TLC (petroleum ether/ethyl acetate=1/2) showed that the starting material was reacted completely, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-25%) to give the title compound 9c (5.00 g, yield: 97.6%).

    [0336] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.31-4.28 (m, 1H), 4.01-3.97 (m, 1H), 3.90-3.82 (m, 1H), 3.75 (s, 3H), 3.68-3.56 (m, 2H), 3.74-3.34 (m, 2H), 2.52 (d, J=2.0 Hz, 1H).

    Step 4: methyl (S)-2-((2-(4-bromo-2,6-difluorophenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate (9d)

    [0337] Compound 9c (7.5 g, 44.33 mmol), 4-bromo-2,6-difluorobenzaldehyde (9.80 g, 44.33 mmol) and 4-methylpyridin-2-amine (4.79 g, 44.33 mmol) were dissolved in toluene (75 mL), and cuprous chloride (1.32 g, 13.30 mmol) and copper(II) trifluoromethanesulphonate (4.81 g, 13.30 mmol) were added under nitrogen atmosphere. The mixture was stirred at 85° C. for 5 min, and N,N-dimethylacetamide (1.2 mL, 13.30 mmol) was added. The mixture was stirred at 85° C. for 12 h. After TLC showed that the starting material was consumed completely, water (50 mL) and ammonia water (30 mL) were added. The reaction mixture was extracted with dichloromethane (100 mL×3). The combined organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0-85%) to give compound 9d (6.00 g, yield: 26.8%).

    [0338] MS (ESI) m/z=480.1 [M+H].sup.+.

    [0339] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.17 (d, J=6.8 Hz, 1H), 7.38 (s, 1H), 7.25-7.18 (m, 2H), 6.67 (br d, J=6.8 Hz, 1H), 4.01-3.74 (m, 3H), 3.68 (s, 3H), 3.56 (br s, 1H), 3.44-3.34 (m, 1H), 3.06-2.85 (m, 3H), 2.60 (dd, J=11.2, 12.8 Hz, 1H), 2.42 (s, 3H).

    Step 5: methyl (S)-2-((2-(2,6-difluoro-4-(2-carbonylpyrrolidin-1-yl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate (9)

    [0340] To a solution of compound 9d (48.0 mg, 0.10 mmol) in 1,4-dioxane (2 mL) were added 2-pyrrolidone (34.0 mg, 0.40 mmol), N,N′-dimethylethylenediamine (6 μL, 0.05 mmol), cesium carbonate (195.5 mg, 0.60 mmol), and cuprous iodide (9.5 mg, 0.05 mmol) successively in a microwave tube. The mixture was heated to 150° C. under microwave irradiation for 1 h. After LCMS showed that the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in acetonitrile and filtered, and the filtrate was directly purified by C18 reverse phase chromatography (acetonitrile/water (containing 0.05% NH.sub.3.Math.H.sub.2O)=5%-95%, flow rate: 60 mL/min) and lyophilized to give the title compound 9 (26.0 mg, yield: 53.7%).

    [0341] MS (ESI) m/z=485.3 [M+H].sup.+.

    [0342] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J=7.3 Hz, 1H), 7.60 (d, J=10.0 Hz, 2H), 7.34 (s, 1H), 6.82 (dd, J=1.4, 7.2 Hz, 1H), 3.88 (t, J=7.2 Hz, 2H), 3.75-3.60 (m, 3H), 3.55 (s, 3H), 3.46 (br d, J=7.3 Hz, 1H), 3.28-3.19 (m, 1H), 3.01 (br d, J=6.0 Hz, 2H), 2.80 (br s, 1H), 2.57 (t, J=8.0 Hz, 3H), 2.37 (s, 3H), 2.08 (quin, J=7.6 Hz, 2H).

    Example 10

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-chloro-1H-benzo[d]imidazol-2-yl)-3,5-difluorophenyl)pyrrolidin-2-one (10)

    [0343] ##STR00080## ##STR00081##

    Step 1: (R)-2-((4-acetylmorpholin-2-yl)methyl)isoindoline-1,3-dione (10a)

    [0344] Compound 1a (5.00 g, 14.44 mmol) was dissolved in a solution of 4 M HCl in 1,4-dioxane (20 mL), and the mixture was stirred at 25° C. for 2 h. After TLC (petroleum ether/ethyl acetate=1/1) showed that the reaction was completed, the reaction mixture was concentrated, and the residue was dissolved in tetrahydrofuran (30 mL). Triethylamine (9.8 mL, 70.74 mmol) and acetic anhydride (4.0 mL, 42.44 mmol) were added in an ice-water bath, and the mixture was reacted at room temperature for 1.5 h. After TLC (petroleum ether/ethyl acetate=1/1) showed that the reaction was completed, water (50 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-100%) to give compound 10a (2.50 g, yield: 54.0%).

    [0345] MS (ESI) m/z=314.1 [M+H].sup.+.

    [0346] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.88 (br s, 2H), 7.75 (br d, J=5.4 Hz, 2H), 4.55-4.28 (m, 1H), 3.94 (br d, J=7.2 Hz, 2H), 3.83-3.64 (m, 3H), 3.60-3.42 (m, 1H), 3.38-3.02 (m, 1H), 2.93-2.61 (m, 1H), 2.10 (br d, J=6.0 Hz, 3H).

    Step 2: (S)-1-(2-(aminomethyl)morpholino)ethan-1-one (10b)

    [0347] To a solution of compound 10a (1.00 g, 3.47 mmol) in ethanol (6 mL) was added hydrazine hydrate (206 μL, 4.16 mmol) at room temperature, and the mixture was stirred at 80° C. for 1 h. After TLC (petroleum ether/ethyl acetate=1/1) showed that the reaction was completed, the reaction mixture was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (20 mL) and filtered, and the filtrate was concentrated under reduced pressure to give the title compound 10b (600.0 mg, yield: 98.4%).

    [0348] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.42-4.32 (m, 1H), 3.95-3.87 (m, 1H), 3.68-3.43 (m, 3H), 3.42-3.18 (m, 2H), 3.03-2.91 (m, 1H), 2.88-2.57 (m, 3H), 2.46 (dd, J=10.8, 13.2 Hz, 1H), 2.07 (s, 3H).

    Step 3: (S)-1-(2-(((4-chloro-2-nitrophenyl)amino)methyl)morpholino)ethane-1-one (10c)

    [0349] To a solution of compound 10b (300.0 mg, 1.90 mmol) in tetrahydrofuran (5 mL) were added 1-fluoro-4-chloro-2-nitrobenzene (399.5 mg, 2.28 mmol) and potassium carbonate (523.0 mg, 3.79 mmol) at room temperature, and the mixture was stirred at 25° C. for 2 h. After LCMS showed that the starting material was consumed completely, water (10 mL) was added, and the mixture was extracted with ethyl acetate (15 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product, which was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-50%, 35 mL/min) to give the title compound 10c (300.0 mg, yield: 30.2%).

    [0350] MS (ESI) m/z=314.1 [M+H].sup.+.

    [0351] .sup.1H NMR (400 MHz, CDCl.sub.3) (8.27-8.15 (m, 2H), 7.46-7.37 (m, 1H), 6.89-6.79 (m, 1H), 4.54 (br d, J=13.2 Hz, 1H), 4.02 (dd, J=2.8, 10.8 Hz, 1H), 3.77-3.53 (m, 3H), 3.51-3.28 (m, 3H), 2.67 (dd, J=10.8, 13.2 Hz, 1H), 2.15-2.10 (m, 3H).

    Step 4: (S)-1-(2-(((2-amino-4-chlorophenyl)amino)methyl)morpholino)ethane-1-one (10d)

    [0352] To a mixed solution of compound 10c (400.0 mg, 1.28 mmol) in tetrahydrofuran (5 mL), ethanol (5 mL) and water (5 mL) were added ammonium chloride (675.7 mg, 12.75 mmol) and iron powder (356.0 mg, 6.38 mmol) at room temperature, and the mixture was stirred at 60° C. for 2 h. After LCMS showed that the starting material was consumed completely, the mixture was filtered, and the filtrate was extracted with ethyl acetate (30 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 10d (380.0 mg, yield: 94.5%).

    [0353] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.78-6.67 (m, 2H), 6.57-6.51 (m, 1H), 4.56-4.38 (m, 1H), 4.03-3.93 (m, 1H), 3.71-3.50 (m, 5H), 3.36-3.25 (m, 1H), 3.23-3.07 (m, 3H), 2.87-2.60 (m, 1H), 2.11 (d, J=3.2 Hz, 3H).

    Step 5: (S)-1-(2-((2-(4-bromo-2,6-difluorophenyl)-5-chloro-1H-benzo[d]imidazol-1-yl)methyl)morpholino)ethan-1-one (10e)

    [0354] To a solution of compound 10d (190.0 mg, 0.67 mmol) in n-butanol (5 mL) were added 4-bromo-2,6-difluorobenzaldehyde (163.0 mg, 0.74 mmol) and acetic acid (238 μL, 1.34 mmol) at room temperature, and the resulting mixture was stirred at 90° C. for 12 h. After LCMS showed that the starting material was reacted completely, the reaction solution was concentrated in vacuum to give a crude product, which was purified by flash silica gel chromatography (petroleum ether/ethyl acetate=0-80%) to give the title compound 10e (300.0 mg, yield: 83.1%).

    [0355] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.86-7.79 (m, 1H), 7.46-7.40 (m, 1H), 7.38-7.27 (m, 3H), 4.50-4.31 (m, 1H), 4.16-4.10 (m, 3H), 3.85-3.70 (m, 1H), 3.55-3.47 (m, 1H), 3.35-3.25 (m, 1H), 3.18-3.06 (m, 1H), 2.37-2.26 (m, 1H), 2.05-2.05 (m, 3H).

    Step 6: (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-chloro-1H-benzo[d]imidazol-2-yl)-3,5-difluorophenyl)pyrrolidin-2-one (10)

    [0356] To a solution of compound 10e (50.0 mg, 0.172 mmol) and pyrrolidin-2-one (8.7 mg, 0.10 mmol) in 1,4-dioxane (3 mL) were added Cs.sub.2CO.sub.3 (67.2 mg, 0.21 mmol), Pd(dba).sub.2 (5.9 mg, 0.01 mmol) and Xantphos (11.9 mg, 0.02 mmol) at room temperature, and the resulting mixture was purged with nitrogen for 5 min and stirred at 90° C. for 12 h. After LCMS showed that the reaction was completed, the reaction mixture was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: YMC-Actus Triart C18 150×30 mm×7 μm, acetonitrile/water (containing 0.05% ammonia water)=47%-70%, 9 min) and lyophilized to give the title compound 10 (13.5 mg, yield: 26.8%).

    [0357] MS (ESI) m/z=489.1 [M+H].sup.+.

    [0358] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.82-7.73 (m, 2H), 7.66 (d, J=10.8 Hz, 2H), 7.36 (dd, J=2.0, 8.8 Hz, 1H), 4.33 (br dd, J=3.2, 15.2 Hz, 1H), 4.17 (br dd, J=7.6, 15.2 Hz, 1H), 3.92 (t, J=7.2 Hz, 2H), 3.69-3.48 (m, 3H), 3.21 (br d, J=10.0 Hz, 2H), 3.10 (br s, 1H), 3.00 (br s, 1H), 2.60 (t, J=8.0 Hz, 2H), 2.17-2.11 (m, 2H), 1.93 (s, 3H).

    Example 11

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-6-fluoro-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one (11)

    [0359] ##STR00082##

    Step 1: (S)-1-(2-(((5-fluoro-4-methyl-2-nitrophenyl)amino)methyl)morpholino)ethane-1-one (11a)

    [0360] Compound 11a was synthesized by referring to step 3 in Example 10, with 1-fluoro-4-chloro-2-nitrobenzene replaced by 1,5-difluoro-2-methyl-4-nitrobenzene.

    [0361] MS (ESI) m/z=312.1 [M+H].sup.+.

    [0362] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.24 (br s, 1H), 8.11-8.04 (m, 1H), 6.53-6.45 (m, 1H), 4.54 (br d, J=13.3 Hz, 1H), 4.07-4.01 (m, 1H), 3.74-3.58 (m, 3H), 3.46-3.26 (m, 3H), 2.66 (dd, J=10.8, 13.1 Hz, 1H), 2.20-2.12 (m, 6H).

    Step 2: (S)-1-(2-((2-(4-bromo-2-chloro-6-fluorophenyl)-6-fluoro-5-methyl-1H-benzo[d]imidazol-1-yl)methyl)morpholino)ethan-1-one (11b)

    [0363] To a mixed solution of compound 11a (100.0 mg, 0.32 mmol) in ethanol (2 mL) and DMSO (2 mL) were added 4-bromo-2-chloro-6-fluorobenzaldehyde (83.9 mg, 0.35 mmol) and Na.sub.2S.sub.2O.sub.4 (447.4 mg, 2.57 mmol) at room temperature, and the resulting mixture was stirred at 90° C. for 12 h. After LCMS showed that the starting material was reacted completely, water (15 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by C18 reverse phase chromatography (acetonitrile/water (containing 0.05% NH.sub.3.Math.H.sub.2O)=10%-75%, flow rate: 40 mL/min) and lyophilized to give the title compound 11b (100.0 mg, yield: 62.5%).

    [0364] MS (ESI) m/z=498.1 [M+H].sup.+.

    Step 3: (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-6-fluoro-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one (11)

    [0365] Compound 11 was synthesized by referring to step 6 in Example 10, with compound 10e replaced by compound 11b.

    [0366] MS (ESI) m/z=503.4 [M+H].sup.+.

    Example 12

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-chloro-6-fluoro-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one (12)

    [0367] ##STR00083## ##STR00084##

    Step 1: tert-butyl (S)-2-(((4-chloro-5-fluoro-2-nitrophenyl)amino)methyl)morpholine-4-carboxylate (12a)

    [0368] Compound 12a was synthesized by referring to step 3 in Example 10, with 1-fluoro-4-chloro-2-nitrobenzene replaced by 1-chloro-2,4-difluoro-5-nitrobenzene.

    [0369] .sup.1H NMR (400 MHz, CDCl.sub.3) (8.36-8.25 (m, 2H), 6.65 (d, J=11.6 Hz, 1H), 4.09-3.80 (m, 3H), 3.75-3.67 (m, 1H), 3.63-3.53 (m, 1H), 3.43-3.35 (m, 1H), 3.34-3.25 (m, 1H), 2.99 (br s, 1H), 2.79 (br s, 1H), 1.48 (s, 9H).

    Step 2: tert-butyl (S)-2-(((2-amino-4-chloro-5-fluorophenyl)amino)methyl)morpholine-4-carboxylate (12b)

    [0370] Compound 12b was synthesized by referring to step 4 in Example 10, with compound 10c replaced by compound 12a.

    [0371] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.81-7.65 (m, 1H), 6.63-6.37 (m, 1H), 4.68 (br s, 1H), 4.40-4.18 (m, 1H), 3.84 (br d, J=12.0 Hz, 1H), 3.76-3.61 (m, 2H), 3.12-3.00 (m, 1H), 2.85 (br s, 1H), 2.53 (br s, 2H), 1.40 (d, J=1.6 Hz, 9H), 1.27-1.20 (m, 3H).

    Step 3: tert-butyl (S)-2-((2-(4-bromo-2-chloro-6-fluorophenyl)-5-chloro-6-fluoro-1H-benzo[d]imidazol-1-yl)methyl)morpholine-4-carboxylate (12c)

    [0372] Compound 12c was synthesized by referring to step 5 in Example 10, with compound 10d replaced by compound 12b, and 4-bromo-2,6-difluorobenzaldehyde replaced by 4-bromo-2-chloro-6-fluorobenzaldehyde.

    [0373] MS (ESI) m/z=578.1 [M+H].sup.+.

    [0374] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.87 (d, J=6.8 Hz, 1H), 7.59-7.55 (m, 1H), 7.40-7.33 (m, 2H), 4.08-3.97 (m, 2H), 3.75 (br s, 3H), 3.62-3.50 (m, 1H), 3.33 (br s, 1H), 2.81 (br s, 1H), 2.47 (br t, J=11.6 Hz, 1H), 0.96 (s, 9H).

    Step 4: (5)-1-(2-((2-(4-bromo-2-chloro-6-fluorophenyl)-5-chloro-6-fluoro-1H-benzo[d]imidazol-1-yl)methyl)morpholino)ethan-1-one (12d)

    [0375] Compound 12c was synthesized by referring to step 1 in Example 10, with compound 1a replaced by compound 12c.

    [0376] MS (ESI) m/z=518.0 [M+H].sup.+.

    [0377] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.90-7.85 (m, 1H), 7.61-7.56 (m, 1H), 7.41-7.32 (m, 2H), 4.50-4.38 (m, 1H), 4.08-4.01 (m, 2H), 3.86-3.79 (m, 1H), 3.55 (br d, J=11.2 Hz, 2H), 3.41-3.32 (m, 1H), 3.24-3.11 (m, 1H), 2.42-2.27 (m, 1H), 2.09 (s, 3H).

    Step 5: (5)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-chloro-6-fluoro-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one (12)

    [0378] Compound 12 was synthesized by referring to step 6 in Example 10, with compound 10e replaced by compound 12d.

    [0379] MS (ESI) m/z=523.3 [M+H].sup.+.

    [0380] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.93-7.87 (m, 3H), 7.78 (dd, J=2.0, 12.0 Hz, 1H), 4.38-3.99 (m, 3H), 3.93 (t, J=7.2 Hz, 2H), 3.72-3.44 (m, 3H), 3.33-3.13 (m, 2H), 3.06-2.85 (m, 1H), 2.60 (t, J=8.4 Hz, 2H), 2.13 (quin, J=7.6 Hz, 2H), 1.95 (s, 3H).

    Example 13

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-chloro-7-fluoro-1H-benzo[d]imidazol-2-yl)-3,5-difluorophenyl)pyrrolidin-2-one (13)

    [0381] ##STR00085## ##STR00086##

    Step 1: (S)-1-(2-(((4-chloro-2-fluoro-6-nitrophenyl)amino)methyl)morpholino)ethane-1-one (13a)

    [0382] Compound 13a was synthesized by referring to step 3 in Example 10, with 1-fluoro-4-chloro-2-nitrobenzene replaced by 5-chloro-1,2-difluoro-3-nitrobenzene.

    [0383] MS (ESI) m/z=332.2 [M+H].sup.+.

    [0384] .sup.1H NMR (400 MHz, CDCl.sub.3) (8.05-7.92 (m, 2H), 7.27-7.20 (m, 1H), 4.54-4.40 (m, 1H), 4.05-3.97 (m, 1H), 3.83-3.73 (m, 1H), 3.71-3.60 (m, 2H), 3.60-3.51 (m, 2H), 3.36-3.06 (m, 1H), 2.87-2.55 (m, 1H), 2.12 (s, 3H).

    Step 2: (S)-1-(2-(((2-amino-4-chloro-6-fluorophenyl)amino)methyl)morpholino)ethan-1-one (13b)

    [0385] Compound 13b was synthesized by referring to step 4 in Example 10, with compound 10c replaced by compound 13a.

    [0386] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.53-6.45 (m, 2H), 4.42 (br d, J=13.2 Hz, 1H), 4.13 (q, J=7.2 Hz, 3H), 4.00 (br d, J=11.6 Hz, 1H), 3.63-3.44 (m, 3H), 3.36-3.25 (m, 1H), 3.09-2.92 (m, 2H), 2.85-2.53 (m, 1H), 2.09 (d, J=9.2 Hz, 3H).

    Step 3: (S)-1-(2-((2-(4-bromo-2,6-difluorophenyl)-5-chloro-7-fluoro-1H-benzo[d]imidazol-1-yl)methyl)morpholino)ethan-1-one (13c)

    [0387] Compound 13c was synthesized by referring to step 5 in Example 10, with compound 10d replaced by compound 13b.

    [0388] MS (ESI) m/z=503.8 [M+H].sup.+.

    [0389] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.67-7.60 (m, 1H), 7.33-7.27 (m, 2H), 7.14-7.05 (m, 1H), 4.51 (br d, J=13.2 Hz, 1H), 4.41-4.27 (m, 2H), 3.73-3.58 (m, 2H), 3.28 (br t, J=11.6 Hz, 1H), 3.15-3.03 (m, 1H), 2.81 (br t, J=12.2 Hz, 1H), 2.64-2.53 (m, 1H), 2.34-2.25 (m, 1H), 2.05 (s, 3H).

    Step 4: (5)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-chloro-7-fluoro-1H-benzo[d]imidazol-2-yl)-3,5-difluorophenyl)pyrrolidin-2-one (13)

    [0390] Compound 13 synthesized by referring to step 6 in Example 10, with compound 10e replaced by compound 13c.

    [0391] MS (ESI) m/z=507.2 [M+H].sup.+.

    [0392] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.79-7.65 (m, 3H), 7.41 (d, J=11.2 Hz, 1H), 4.51-4.37 (m, 1H), 4.23 (br d, J=13.2 Hz, 1H), 4.11 (br dd, J=8.4, 15.2 Hz, 1H), 4.02 (br d, J=13.2 Hz, 1H), 3.90 (t, J=7.2 Hz, 2H), 3.73 (br d, J=12.8 Hz, 1H), 3.63-3.50 (m, 2H), 3.28-3.07 (m, 1H), 3.02-2.72 (m, 1H), 2.59 (t, J=8.0 Hz, 2H), 2.30-2.20 (m, 1H), 2.57-2.07 (m, 2H), 1.93 (s, 3H).

    Example 14

    (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-(methyl-d3)-1H-benzo[d]imidazol-2-yl)-3,5-difluorophenyl)pyrrolidin-2-one (14)

    [0393] ##STR00087##

    Step 1: tert-butyl (S)-2-(((4-bromo-2-nitrophenyl)amino)methyl)morpholine-4-carboxylate (14a)

    [0394] To a solution of compound 1b (2.00 g, 7.40 mmol) in 1,4-dioxane (10 mL) were added 4-bromo-1-fluoro-2-nitrobenzene (1.63 g, 7.40 mmol) and triethylamine (2.0 mL, 14.80 mmol) at room temperature, and the mixture was stirred at 80° C. for 12 h. After LCMS showed that the starting material was consumed completely, the reaction mixture was directly concentrated under reduced pressure to give a crude product, which was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0%-15%, 60 mL/min) to give the title compound 14a (2.00 g, yield: 58.4%).

    [0395] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.25 (d, J=2.4 Hz, 1H), 8.15 (br s, 1H), 7.44 (dd, J=2.4, 9.2 Hz, 1H), 6.71 (d, J=9.2 Hz, 1H), 3.96-3.72 (m, 3H), 3.63 (tdd, J=3.2, 7.2, 10.4 Hz, 1H), 3.55-3.46 (m, 1H), 3.39-3.23 (m, 2H), 2.92 (br s, 1H), 2.71 (br s, 1H), 1.40 (s, 9H).

    Step 2: tert-butyl (S)-2-(((4-(methyl-d3)-2-nitrophenyl)amino)methyl)morpholine-4-carboxylate (14b)

    [0396] To a mixed solution of compound 14a (1.10 g, 2.64 mmol) and 4,4,5,5-tetramethyl-2-(methyl-d3)-1,3,2-dioxaborolan (1.15 g, 7.93 mmol) in 1,4-dioxane (15 mL) and water (5 mL) were added Cs.sub.2CO.sub.3 (2.58 g, 7.93 mmol) and Pd(dppf)Cl.sub.2CH.sub.2Cl.sub.2 (58.8 mg, 0.072 mmol) at room temperature, and the resulting mixture was purged with nitrogen for 5 min and stirred at 100° C. for 12 h under nitrogen atmosphere. After LCMS showed that the reaction was completed, the reaction mixture was cooled to room temperature. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (50 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product, which was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-20%, 35 mL/min) to give the title compound 14b (470.0 mg, yield: 45.2%).

    [0397] MS (ESI) m/z=299.2 [M+H-56].sup.+.

    [0398] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.09 (br t, J=5.6 Hz, 1H), 7.87 (d, J=2.4 Hz, 1H), 7.39 (dd, J=2.0, 8.8 Hz, 1H), 7.04 (d, J=8.8 Hz, 1H), 3.87 (br d, J=11.2 Hz, 2H), 3.70 (br d, J=13.2 Hz, 1H), 3.63-3.50 (m, 2H), 3.47-3.36 (m, 2H), 3.27-3.24 (m, 1H), 2.88 (br s, 1H), 1.39 (s, 9H).

    Step 3: tert-butyl (S)-2-((2-(4-bromo-2,6-difluorophenyl)-5-(methyl-d3)-1H-benzo[d]imidazol-1-yl)methyl)morpholine-4-carboxylate (14c)

    [0399] To a mixed solution of compound 14b (200.0 mg, 0.56 mmol) in ethanol (6 mL) and water (2 mL) were added 4-bromo-2,6-difluorobenzaldehyde (124.7 mg, 0.56 mmol) and Na.sub.2S204 (294.8 mg, 1.69 mmol) at room temperature, and the resulting mixture was stirred at 80° C. for 4 h under nitrogen atmosphere. After LCMS showed that the starting material was reacted completely, the reaction mixture was filtered, and water (3 mL) was added to the filtrate. The resulting mixture was extracted with ethyl acetate (10 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 14c (340.0 mg, yield: 114.7%).

    [0400] MS (ESI) m/z=525.1 [M+H].sup.+.

    Step 4: (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-(methyl-d3)-1H-benzo[d]imidazol-2-yl)-3,5-difluorophenyl)pyrrolidin-2-one (14)

    [0401] To a solution of compound 14c (300.0 mg, 0.57 mmol) and pyrrolidin-2-one (48.4 mg, 0.57 mmol) in 1,4-dioxane (8 mL) were added Cs.sub.2CO.sub.3 (372.1 mg, 1.14 mmol), Pd(dba).sub.2 (32.8 mg, 0.057 mmol) and Xantphos (66.1 mg, 0.11 mmol) at room temperature, and the resulting mixture was purged with nitrogen for 5 min and stirred at 100° C. for 2 h. After LCMS showed that the reaction was completed, the reaction mixture was cooled to room temperature, followed by the addition of p-toluenesulfonic acid (981.5 mg, 5.70 mmol). The resulting mixture was stirred at 100° C. for 1 h. After LCMS showed that the reaction was completed, the reaction mixture was cooled to room temperature, methanol (5 mL) was added, and triethylamine (1.2 mL, 8.55 mmol) was added in an ice-water bath to adjust the pH to 8, followed by the addition of acetic anhydride (267 μL, 2.85 mmol). The resulting mixture was reacted at room temperature for 1 h. After LCMS showed that the reaction was completed, water (10 mL) was added, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: YMC Triart C18 250×50 mm×7 μm, acetonitrile/water (containing 0.05% ammonia water)=12%-52%, 9 min) and lyophilized to give the title compound 14 (90.2 mg, yield: 33.3%).

    [0402] MS (ESI) m/z=472.2 [M+H].sup.+.

    [0403] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.69-7.58 (m, 3H), 7.49 (d, J=1.2 Hz, 1H), 7.16 (dd, J=1.6, 8.4 Hz, 1H), 4.27 (br dd, J=3.6, 15.2 Hz, 1H), 4.12 (br dd, J=7.2, 15.2 Hz, 1H), 3.92 (t, J=7.2 Hz, 2H), 3.64 (br dd, J=2.0, 11.6 Hz, 1H), 3.54 (br s, 2H), 3.23 (br s, 2H), 3.14-3.09 (m, 2H), 2.60 (t, J=8.0 Hz, 2H), 2.18-2.08 (m, 2H), 1.93 (s, 3H).

    Example 15

    (S)-1-(3-chloro-4-(1-((4-(cyclopropylcarbonyl)morpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-5-fluorophenyl)pyrrolidin-2-one (15)

    [0404] ##STR00088##

    Step 1: tert-butyl (S)-2-((2-(4-bromo-2-chloro-6-fluorophenyl)-5-methyl-1H-benzo[d]imidazol-1-yl)methyl)morpholine-4-carboxylate (15a)

    [0405] Compound 15a was synthesized by referring to step 5 in Example 1, with 4-bromo-2,6-difluorobenzaldehyde replaced by 4-bromo-2-chloro-6-fluorobenzaldehyde. MS (ESI) m/z=540.1 [M+H+2].sup.+.

    Step 2: (R)-1-(3-chloro-5-fluoro-4-(5-methyl-1-(morpholin-2-ylmethyl)-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidin-2-one (15b)

    [0406] To a solution of compound 15a (870.0 mg, 1.62 mmol) and pyrrolidin-2-one (549.7 mg, 6.46 mmol) in 1,4-dioxane (10 mL) were added Cs.sub.2CO.sub.3 (1.05 g, 3.23 mmol), Pd(dba).sub.2 (92.9 mg, 0.16 mmol) and Xantphos (186.8 mg, 0.32 mmol) at room temperature, and the resulting mixture was purged with nitrogen for 5 min and stirred at 100° C. for 4 h. After LCMS showed that the reaction was completed, the reaction mixture was cooled to room temperature, followed by the addition of p-toluenesulfonic acid monohydrate (3.07 g, 16.14 mmol). The resulting mixture was stirred at 100° C. for 1 h. After LCMS showed that the reaction was completed, water (15 mL) was added, and the mixture was extracted with ethyl acetate (20 mL×3). The aqueous phase was adjusted to pH 8 with 1 N aqueous NaOH solution and extracted with ethyl acetate (20 mL×3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound 15b (680.0 mg, yield: 95.1%).

    [0407] MS (ESI) m/z=443.5 [M+H].sup.+.

    Step 3: (S)-1-(3-chloro-4-(1-((4-(cyclopropylcarbonyl)morpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-5-fluorophenyl)pyrrolidin-2-one (15)

    [0408] To a solution of compound 15b (50.0 mg, 0.11 mmol) in DMF (1 mL) were added cyclopropanecarboxylic acid (19.4 mg, 0.23 mmol), HATU (85.8 mg, 0.0.23 mmol) and N,N-diisopropylethylamine (75 μL, 0.45 mmol) successively, and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly purified by C18 reverse phase chromatography (acetonitrile/water (containing 0.05% ammonia water)=10%-75%) and lyophilized to give the title compound 15 (32.0 mg, yield: 55.5%).

    [0409] MS (ESI) m/z=469.4 [M+H].sup.+.

    [0410] .sup.1H NMR (400 MHz, DMSO-d6, t=75° C.) δ 7.87 (s, 1H), 7.77 (dd, J=12.0, 2.1 Hz, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.49 (s, 1H), 7.16 (dd, J=8.4, 1.6 Hz, 1H), 4.27-4.11 (m, 2H), 4.08 (t, J=5.1 Hz, 2H), 3.99 (d, J=13.5 Hz, 1H), 3.93 (t, J=7.1 Hz, 2H), 3.68 (br s, 1H), 3.56 (br s, 1H), 3.47 (dd, J=7.0, 5.1 Hz, 2H), 3.25 (t, J=11.7 Hz, 1H), 2.59 (dd, J=8.5, 7.6 Hz, 2H), 2.46 (s, 3H), 2.13 (p, J=7.5 Hz, 2H), 1.08 (t, J=7.0 Hz, 4H).

    Example 16

    (S)-1-(3-chloro-5-fluoro-4-(5-methyl-1-((4-(3,3,3-trifluoropropionyl)morpholin-2-yl)methyl)-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidin-2-one (16)

    [0411] ##STR00089##

    [0412] Compound 16 was synthesized by referring to step 3 in Example 15, with cyclopropanecarboxylic acid replaced by 3,3,3-trifluoropropionic acid.

    [0413] .sup.1H NMR (400 MHz, DMSO-d6, t=75° C.) δ 7.86 (s, 1H), 7.76 (dd, J=12.0, 2.1 Hz, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.49 (s, 1H), 7.16 (dd, J=8.5, 1.6 Hz, 1H), 4.41-4.13 (m, 2H), 4.08 (t, J=5.1 Hz, 2H), 3.93 (t, J=7.1 Hz, 2H), 3.78-3.64 (m, 1H), 3.59-3.44 (m, 5H), 3.35-3.16 (m, 1H), 2.59 (t, J=8.1 Hz, 2H), 2.46 (s, 3H), 2.13 (p, J=7.6 Hz, 2H).

    Example 17

    (S)-6-chloro-8-fluoro-7-(5-methyl-1-((4-propionylmorpholin-2-yl)methyl)-1H-benzo[d]imidazol-2-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (17)

    [0414] ##STR00090##

    Step 1: 4-chloro-2-fluoro-6-nitrophenol (17a)

    [0415] Fuming nitric acid (4.51 g, 71.65 mmol) was slowly added dropwise to a solution of 4-chloro-2-fluorophenol (10.00 g, 68.24 mmol) in acetic acid (100 mL) in an ice-water bath, and the mixture was stirred in the ice-water bath for 1 h. After LCMS showed that the starting material was consumed completely, the reaction mixture was slowly poured into water (1.5 L) while stirring. The resulting mixture was stirred for 1 h and filtered in vacuum to give a solid, which was dried to obtain the title compound 17a (11.00 g, yield: 84%).

    [0416] .sup.1H NMR (400 MHz, CDCl.sub.3) (10.38 (s, 1H), 7.94 (t, J 2.4 Hz, 1H), 7.45 (dd, J 2.4, 9.6 Hz, 1H).

    Step 2: 2-amino-4-chloro-6-fluorophenol (17b)

    [0417] To a mixed solution of compound 17a (5.00 g, 26.10 mmol) in tetrahydrofuran (60 mL), ethanol (60 mL) and water (30 mL) were added ammonium chloride (13.96 g, 261.04 mmol) and iron powder (11.69 g, 208.83 mmol) at room temperature, and the mixture was stirred at 60° C. for 2 h. After LCMS showed that the starting material was consumed completely, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was dispersed in ethyl acetate (500 mL) and filtered, and the filtrate was concentrated under reduced pressure to give the title compound 17b (4.20 g, yield: 99.6%).

    [0418] MS (ESI) m/z=162.0 [M+H].sup.+.

    [0419] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.56-6.49 (m, 2H), 5.03 (br s, 1H), 3.93 (br s, 2H).

    Step 3: 6-chloro-8-fluoro-2H-benzo[b][1,4]oxazin-3(4H)-one (17c)

    [0420] To a solution of compound 17b (4.20 g, 26.00 mmol) in tetrahydrofuran (50 mL) were added potassium carbonate (10.78 g, 77.99 mmol) and chloroacetyl chloride (3.1 mL, 38.99 mmol) in an ice-water bath, and the mixture was stirred at 40° C. for 15 h. After LCMS showed that the starting material was consumed completely, water (100 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (100 mL×3). The organic phase was dried and concentrated, and the crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=30%, flow rate: 30 mL/min) to give the title compound 17c (4.30 g, 82.0%).

    [0421] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.00 (br s, 1H), 7.10 (dd, J 2.4, 10.4 Hz, 1H), 6.74 (t, J 2.0 Hz, 1H), 4.68 (s, 2H).

    Step 4: 6-chloro-8-fluoro-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-carbaldehyde (17d)

    [0422] To a solution of 2,2,6,6-tetramethylpiperidine (3.8 mL, 22.32 mmol) in anhydrous tetrahydrofuran (4 mL) was added a solution of 2.5 M n-butyllithium in n-hexane (8.9 mL, 22.32 mmol) dropwise at −78° C. After the addition, the mixture was stirred for 20 min with the temperature maintained at −78° C., and then a solution of compound 17c (1.50 g, 7.44 mmol) in anhydrous tetrahydrofuran (15 mL) was slowly added dropwise. The mixture was stirred at −78° C. for 3 h, and then N,N-dimethylformamide (1.09 g, 14.88 mmol) was slowly added dropwise. The resulting mixture was slowly warmed to room temperature and reacted for 30 min. After TLC (petroleum ether/ethyl acetate=1/1) showed that the reaction was completed, a saturated aqueous ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the title compound 17d (600.0 mg, 35.0%).

    [0423] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.38 (br s, 1H), 10.16 (d, J 0.8 Hz, 1H), 6.85 (d, J 1.6 Hz, 1H), 4.76 (s, 2H).

    Step 5: tert-butyl (S)-2-((2-(6-chloro-8-fluoro-3-carbonyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-5-methyl-TH-benzo[d]imidazol-1-yl)methyl)morpholine-4-carboxylate (17e)

    [0424] Compound 17e was synthesized by referring to step 3 in Example 14, with compound 14b replaced by compound 1c, and 4-bromo-2,6-difluorobenzaldehyde replaced by compound 17d.

    [0425] MS (ESI) m/z=531.2 [M+H].sup.+.

    [0426] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.81 (br s, 1H), 7.69 (s, 1H), 7.41 (dd, J 2.4, 8.4 Hz, 1H), 7.21 (d, J 8.4 Hz, 1H), 6.79 (dd, J 1.2, 5.6 Hz, 1H), 4.75-4.71 (m, 2H), 4.12-4.02 (m, 2H), 3.99-3.69 (m, 3H), 3.66-3.56 (m, 1H), 3.41-3.26 (m, 1H), 2.90-2.76 (m, 1H), 2.52 (s, 3H), 2.52-2.44 (m, 1H), 1.43 (s, 9H).

    Step 6: (S)-6-chloro-8-fluoro-7-(5-methyl-1-((4-propionylmorpholin-2-yl)methyl)-1H-benzo[d]imidazol-2-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (17)

    [0427] To a solution of compound 17e (182.0 mg, 0.34 mmol) in dichloromethane (5 mL) was slowly added dropwise a solution of 4 M HCl in 1,4-dioxane (0.9 mL) in an ice-water bath, and the mixture was stirred at room temperature for 4 h. After TLC (petroleum ether/ethyl acetate=1/1) showed that the reaction was completed, the reaction mixture was concentrated, and the residue was dissolved in dichloromethane (3 mL). Triethylamine (240 μL, 1.72 mmol) and propionyl chloride (33 μL, 0.38 mmol) were added in an ice-water bath, and the mixture was reacted at room temperature for 2 h. After TLC (petroleum ether/ethyl acetate=1/1) showed that the reaction was completed, water (20 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (20 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ethyl acetate/petroleum ether=0-100%) to give compound 17 (74.0 mg, yield: 44.7%).

    [0428] MS (ESI) m/z=487.1 [M+H].sup.+.

    Biological Assay

    Test Example 1. Evaluation of In Vitro Biological Activity

    [0429] The compounds were screened for antagonistic activity against hP2X3 and hP2X2/3 receptors (changes in calcium flux signals represent the effect of the compounds on ion channels) by an FLIPR assay.

    1. Experimental Instruments and Materials

    [0430]

    TABLE-US-00001 Instrument Equipment manufacturer Model FLIPR Molecular Devices TETRA CO.sub.2 incubator Thermo Fisher 3111 Name of reagents and Source Cat. No. consumables FLIPR® calcium 4 assay kit Molecular Devices R8141 FLIPR pipettor tip Molecular Devices 9000-0764 Matrix D.A.R.T.s Tips-30 ul Thermo 5416 FBS Gibco 10099-141 DMEM Gibco 11965 Hygromycin B Invitrogen 10687010 G418 disulfate salt SIGMA G5013 384-well assay plate Corning CC3712 384-well compound plate Corning CC3657 Echo qualified 384-well LABCYTE P-05525 polypropylene microplate Probenecid Sigma P8761-25G 1x HBSS Invitrogen 14025 ATP hydrolase Sigma A7646 HEPES Invitrogen 15630-080 Versene Gibco 15040066 aß-meATP Sigma M6517 Stable cell lines Chempartner 1321N1/hP2X3, 1321N1/hP2X2/3

    2. Experimental Procedures

    [0431] 1321N1 cells (adherent cells) stably transfected with hP2X3 and hP2X2/3 receptors were digested, centrifuged, resuspended in a plating medium (DMEM+10% DFBS) and counted, adjusted to 2×10.sup.5 cells/mL. The cells were seeded in a 384-well assay plate at 50 μL/well, and incubated in an incubator at 37° C. with 5% CO.sub.2 for 16-24 h. Test compounds (20 mM DMSO stock solution) that were 180 times the desired concentration were prepared with DMSO, and added to a 384-well compound plate at 500 nL/well. Then, each well was supplemented with 30 μL of FLIPR buffer (lx HBSS containing 1.26 mM Ca.sup.2++2 mM CaCl.sub.2)+20 mM HEPES), and the mixture was shaken for 20-40 min to be mixed well. The agonist α,β-meATP (final concentration of 500 nM for hP2X3 cells and final concentration of 1000 nM for hP2X2/3 cells) that was 3 times the desired concentration was prepared with an FLIPR buffer, and added to another 384-well compound plate at 35 μL/well. The cell plate in which cells had been plated and cultured for 16-24 h was taken out, and the cell supernatant was removed by pipetting. 30 μL of dye (FLIPR® calcium 4 assay kit, diluted with an FLIPR buffer) was added to each well, and the plate was incubated for 1 h. 15 μL of the compound was added to each well containing cells (dosing using an FLIPR instrument), and after 15 min, 22.5 μL of agonist was added to each well and the fluorescent signal was detected (excitation wavelength: 470-495 nm, emission wavelength: 515-575 nm). With the difference between the peak and the trough of the signal taken as the basic data, the data of the highest concentration of the positive drug taken as the 100% inhibition rate, and the data of DMSO taken as the 0% inhibition rate, the inhibition effect curves of the compounds were fitted on the software Graphpad Prism 6 and IC.sub.50 values were calculated.

    TABLE-US-00002 TABLE 1 The half maximal inhibitory concentrations (IC.sub.50) of the compounds of the present disclosure against hP2X3 and hP2X2/3 receptors Compound No. hP2X3 (IC.sub.50, nM) hP2X2/3 (IC.sub.50, nM) MK-7264 35.4 116.2 1 36.8 5566 2 105.0 18990 3 41.2 1805 4 31.6 1927 5 100.4 NT 6 >10000 NT 7 5843 NT 8 >10000 NT 9 75.8 30270 10 54.1 7407 11 79.4 NT 12 87.7 NT 13 765.9 NT 14 33.0 NT 15 85.1 NT 16 70.5 NT 17 45.3 3501 NT: not tested.

    Test Example 2. CYP Inhibition Assay

    [0432] The metabolic reactions of representative substrates of 5 major human CYP subtypes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5) were evaluated using 150-donor pooled human liver microsomes (purchased from Corning, Cat. No. 452117). The effects of different concentrations of the test compounds on the metabolic reactions of phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-mephenytoin (CYP2C19), bufuralol hydrochloride (CYP2D6) and midazolam (CYP3A4/5) were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS).

    [0433] 30 μM phenacetin, 10 μM diclofenac sodium, 35 μM S-mephenytoin, 5 μM bufuralol hydrochloride, 3 μM midazolam, 1 mM NADPH, and test compounds (at concentrations of 0.1 μmol/L, 0.3 μmol/L, 1 μmol/L, 3 μmol/L, 10 μmol/L, and 30 μmol/L, respectively) or positive compound or blank were incubated with 200 μL of a reaction system of pooled human liver microsomes (0.2 mg/mL) (100 mmol/L phosphate buffer, pH 7.4, containing 0.3% by volume of DMSO, 0.6% by volume of acetonitrile, and 0.1% by volume of methanol) at 37° C. for 5 min. Then, 200 μL of acetonitrile containing 3% formic acid and 40 nM internal standard verapamil was added, and the mixture was centrifuged at 4000 rpm for 50 min. The mixture was cooled on ice for 20 min and centrifuged at 4000 rpm for 20 min to precipitate the protein. 200 μL of the supernatant was analyzed by LC/MS/MS.

    [0434] The peak area was calculated from the chromatogram. The residual activity rate (%) was calculated by the following formula:


    peak area rate=peak area of metabolite/peak area of internal standard


    residual activity rate (%)=peak area proportion of the test compound group/peak area proportion of the blank group

    [0435] The half maximal inhibitory concentrations (IC.sub.50) against CYP were calculated by Excel XLfit 5.3.1.3.

    [0436] The determined half maximal inhibitory concentrations (IC.sub.50) values against CYP were shown in the table below.

    TABLE-US-00003 TABLE 2 Half maximal inhibitory concentrations (IC.sub.50) of part of the compounds of the present disclosure against CYP Examples CYP 1A2 CYP 2C9 CYP 2C19 CYP 2D6 CYP 3A4/5 No. (μM) (μM) (μM) (μM) (μM) 1 >30 21.7 13.0 >30 >30 2 >30    3.06 14.6 >30 >30 3 >30 >30   >30   >30 >30 4 >30 >30   >30   >30 >30 9 >30    1.48 12.9 >30    12.9 10 >30 >30  >30   >30 >30 14 >30 21.9 >30   >30 >30

    Test Example 3. Assay on In Vitro Metabolic Stability in Human Hepatocytes

    [0437] The concentrations of the test compounds in the reaction system were determined by LC/MS/MS, so that the intrinsic clearance of the test compounds was calculated, and the in vitro metabolic stability in human hepatocytes was evaluated.

    [0438] 247.5 μL of a mixed solution of human hepatocytes at 1×10.sup.6 cells/mL (purchased from BioreclamationIVT, Cat. No. S01205) and 2.5 μL of 100 μM test compound or positive control was added to a plate to initiate the reaction. The plate was incubated at 37° C. and 600 rpm. 20 μL of incubation system was transferred to a stop plate at 0.5 min, 5 min, 15 min, 30 min, 45 min, 60 min, 80 min, 100 min and 120 min. Then, the mixture was vortexed for 2 min to be mixed well. The stop plate was centrifuged at 4000 rpm for 20 min. 40 μL of supernatant of each compound was transferred to a 96-well sample template, after which 160 μL of pure water was added to dilute the samples.

    [0439] The resulting samples were quantified from ion chromatogram. The residual rate was calculated from the peak area of the test compound or the positive control. Slope k was determined by linear regression of the natural pair values of residual rates versus incubation time using Microsoft Excel.

    [0440] Intrinsic clearance (in vitro CL.sub.int, μL/min/10.sup.6 cells) was calculated from the slope value according to the following equation:


    in vitro CL.sub.int=kV/N


    V=incubation volume (0.25 mL);


    N=number of cells per well (0.25×10.sup.6 cells)

    [0441] The determined values of intrinsic clearance in human hepatocytes are shown in Table 3.

    TABLE-US-00004 TABLE 3 Intrinsic clearance of part of the compounds of the present disclosure in human hepatocytes Intrinsic clearance Compound No. (uL/min/10.sup.6 cells) 1 <1     2 2.98 3 4.08 4 <1     9 3.86 10 <1     14 <1     17 4.1 

    Test Example 4. Caco-2 Permeability Assay

    [0442] The apparent permeability coefficient (P.sub.app) of the analytical drug was determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS) through a Caco-2 cell model.

    [0443] The transfer rate of drug from the apical end to the basal end was determined. 108 μL of HBSS (25 mM HEPES, pH 7.4) containing 10 μM test compound was added to the apical end of the upper chamber of Transwell (purchased from Corning) containing Caco-2 cells (purchased from ATCC) at a density of 6.86×10.sup.5 cells/cm.sup.2, and meanwhile, 8 μL of the sample was added, as an initial dosing-end sample (A-B), to a new 96-well plate to which 72 μL of HBSS (25 mM HEPES, pH 7.4) and 240 μL of acetonitrile (containing 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide) had been added, and the plate was vortexed at 1000 rpm for 10 min. 300 μL of HBSS (25 mM HEPES, pH 7.4) was added to the basal end.

    [0444] The transfer rate of drug from the basal end to the apical end was determined. 308 μL of HBSS (25 mM HEPES, pH 7.4) containing 10 μM test compound was added to wells of the basal end of the plate, and meanwhile, 8 μL of the sample was added, as an initial dosing-end sample (B-A), to a new 96-well plate to which 72 μL of HBSS (25 mM HEPES, pH 7.4) and 240 μL of acetonitrile (containing 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide) had been added. The plate was vortexed at 1000 rpm for 10 min, and 100 μL of HBSS (25 mM HEPES, pH 7.4) was added to the apical end.

    [0445] The transfer rate from the apical end to the basal end and the transfer rate from the basal end to the apical end need to be determined simultaneously.

    [0446] At the end of the transfer cycle, 8 μL of the sample was taken from the dosing end (apical end of A-B flow and basal end of B-A flow) and added to 72 μL of HBSS (25 mM HEPES, pH 7.4) and 240 μL of acetonitrile (containing 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide), and the mixture was added to a new 96-well plate. 80 μL of the liquid was taken directly from each of the basal end of the A-B flow and the apical end of the B-A flow, and added together with 240 μL of acetonitrile (containing 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide) to a new 96-well plate. The plates were vortexed at 1000 rpm for 10 min. The samples were centrifuged at 4000 rpm for 30 min. 100 μL of the supernatant was transferred to a new 96-well plate. All samples were analyzed by LC-MS/MS with 100 μL of pure water.

    [0447] Data were calculated using Microsoft Excel and peak areas were calculated from the chromatograms. The apparent permeability coefficient (Papp) is in cm/s and is calculated using the following formula:

    [00001] P app = dQ / Dt A × D 0

    [0448] Papp is apparent permeability (cm/s×10.sup.−6);

    [0449] dQ/dt is the drug delivery rate (pmol/s);

    [0450] A is the surface area of the film (cm.sup.2);

    [0451] D.sub.0 is the initial supply-end drug concentration (nM; pmol/cm.sup.3).

    [0452] The outflow ratio can be determined by the following equation:

    [00002] Outflow ⁢ ratio = P app ⁡ ( B - A ) P app ⁡ ( A - B )

    [0453] Papp (B-A) is the apparent permeability coefficient in the direction from the basal end to the apical end;

    [0454] Papp (A-B) is the apparent permeability coefficient in the direction from the apical end to the basal end.

    [0455] The apparent permeability coefficient values of the Caco-2 cells are shown in Table 4.

    TABLE-US-00005 TABLE 4 P.sub.app (A-B) Compound No. (10.sup.−6, cm/s) Outflow ratio 4 8.1 3.8 BLU-5937 2.4 1.2 Note: [00091]embedded image

    Test Example 5. In Vivo Pharmacokinetic Experiment in Rats

    [0456] The drug concentrations in the plasma of the test animals (rats) at different time points after intragastric administration of the compound of the present disclosure were determined by an LC/MS/MS method. The pharmacokinetic behavior in rats of the compound of the present disclosure was studied and its pharmacokinetic profile was evaluated.

    1. Test Protocol

    1.1 Test Compounds

    [0457] Compound 4 and BLU-5937.

    1.2 Test Animals

    [0458] Male, healthy SD rats of SPF grade, 6-8 weeks old, 3 rats in each group.

    1.3 Drug Preparation

    [0459] Intragastric administration: a certain amount of compound was weighed and prepared into a 1 mg/mL white suspension by adding 0.5% by volume of hydroxypropyl methylcellulose, 0.1% by volume of tween 80 and 99.4% by volume of water.

    1.4 Administration

    [0460] SD rats were administered intragastrically with compound 4 or BLU-5937 at a dose of 5 mg/kg after fasting overnight.

    2. Operation

    [0461] Rats were administered intragastrically with the compound of the present disclosure. 0.2 mL of blood was collected from the jugular vein at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after the administration, placed in a tube containing EDTA-K2, separated by centrifugation at 4° C. at 4000 rpm for 5 min to obtain plasma, which was stored at −75° C.

    [0462] Determination of the content of the test compounds at different concentrations in the plasma of rats after intragastric administration: 50 μL of rat plasma at each time point after the administration was taken, 200 μL of a solution of internal standard dexamethasone (50 ng/mL) in acetonitrile was added thereto, and the mixture was vortexed for 30 s to be mixed well, centrifuged at 4° C. at 4700 rpm for 15 min. The supernatant of the plasma sample was subjected to 3-fold dilution with water, and 2.0 μL of the dilution was taken for LC/MS/MS analysis.

    3. Results of Pharmacokinetic Parameters

    [0463] The pharmacokinetic parameters for part of the compounds of the disclosure in rats are as follows:

    TABLE-US-00006 TABLE 5 Compound 4 BLU-5937 V (L/kg) 2.05 1.09 AUC.sub.0-t 408 361 (h*ng/ml) Cmax (ng/ml) 356 151 T.sub.1/2 (h) 0.96 2.90 T.sub.max (h) 0.42 0.25