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SPIRO COMPOUND AND MEDICAL USES THEREOF

20220041616 · 2022-02-10

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to a spiro compound and medical uses thereof. Specifically, the present invention relates to a spiro compound represented by the general formula (I), a preparation method thereof, a pharmaceutical composition containing the same, and is used as an acetyl-Coenzyme A carboxylase (ACC) inhibitor and is used for treating diseases associated with ACC activity. Each substituent in the general formula (I) is as defined in description.

    ##STR00001##

    Claims

    1. A compound of formula (I): ##STR00097## or a mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein, X is selected from —O—, —S— and —NR—; Y is selected from N and CR.sup.1; Z is selected from N and CR.sup.2; ring A is cycloalkyl or heterocyclyl; L.sup.1 is selected from a single bond and a C.sub.1-C.sub.3 alkylene, wherein said alkylene is optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.4 is selected from the group consisting of hydrogen, halogen, cyano, —R.sup.a, —OR.sup.a, —S(O).sub.nR.sup.a, —NR.sup.aR.sup.b, —N(R.sup.a)C(O)R.sup.b, —C(O)NR.sup.aR.sup.b, —C(O)N(R.sup.a)S(O).sub.nR.sup.b, —N(R.sup.a)C(O)NR.sup.aR.sup.b, —N(R.sup.a)C(O)OR.sup.b, —OC(O)NR.sup.aR.sup.b, —N(R.sup.a)S(O).sub.nR.sup.b, —S(O).sub.nNR.sup.aR.sup.b, —C(O)R.sup.a, —C(O)OR.sup.a, —OC(O)R.sup.a, —P(O)(OR.sup.a)(OR.sup.b) and —B(OH).sub.2; R.sup.5 is each independently selected from the group consisting of hydrogen, halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R is selected from the group consisting of hydrogen, halogen, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally further substituted by one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.1 is selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl and heterocyclyl, wherein said alkyl, cycloalkyl, and heterocyclyl are optionally further substituted by one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.2 is selected from the group consisting of hydrogen, halogen, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally further substituted by one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.3 is selected from alkyl, said alkyl is further substituted with one or more Q groups; each Q is independently selected from the group consisting of halogen, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.a and SR.sup.a; wherein said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally further substituted by one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxy, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.a and R.sup.b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, nitro, cyano, oxo, carboxyl, ester, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally further substituted by one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R.sup.a and R.sup.b together with the nitrogen atom attached to them form a N-containing heterocyclic group, said N-containing heterocyclic group is optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, oxo, hydroxyl, thiol, carboxyl, ester, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; m is an integer from 0 to 5; n is an integer from 0 to 2.

    2. The compound of formula (I) according to claim 1, wherein X is —S—.

    3. The compound of formula (I) according to claim 1, which is a compound of formula (II), ##STR00098## wherein, R.sup.1, R.sup.2, R.sup.3, ring A, L.sup.1, R.sup.4, R.sup.5 and m are as defined in claim 1.

    4. The compound of formula (I) according to claim 1, wherein, R.sup.3 is selected from alkyl, preferably C.sub.1-C.sub.6 alkyl, said alkyl is further substituted by one or more Q groups; each Q is independently selected from the group consisting of aryl, heteroaryl, OR.sup.a and SR.sup.a; wherein said aryl and heteroaryl are optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.a is selected from the group consisting of alkyl, cycloalkyl and heterocyclyl, wherein said alkyl, cycloalkyl and heterocyclic are optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl.

    5. The compound of formula (I) according to claim 1, which is a compound of formula (III), ##STR00099## wherein, Q.sub.1 is selected from aryl and heteroaryl, preferably C.sub.5-C.sub.10 aryl or 5- to 10-membered heteroaryl, said aryl and heteroaryl are optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; Q.sub.2 is selected from OR.sup.a and SR.sup.a, preferably OR.sup.a; R.sup.a is selected from cycloalkyl and heterocyclyl, preferably C.sub.3-C.sub.7 cycloalkyl or 5- to 7-membered heterocyclyl; wherein said cycloalkyl and heterocyclyl are optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; wherein, R.sup.1, R.sup.2, ring A, L.sup.1, R.sup.4, R.sup.5 and m are as defined in claim 1.

    6. The compound of formula (I) according to claim 1, which is a compound of formula (IV), ##STR00100## wherein, R.sup.6 is selected from the group consisting of hydrogen, halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.a is selected from cycloalkyl and heterocyclyl, preferably C.sub.3-C.sub.7 cycloalkyl or 5- to 7-membered heterocyclyl; wherein said cycloalkyl and heterocyclyl are optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; p is an integer from 1 to 4; wherein, R.sup.1, R.sup.2, ring A, L.sup.1, R.sup.4, R.sup.5 and m are as defined in claim 1.

    7. The compound of formula (I) according to claim 1, wherein, ring A is a C.sub.3-C.sub.7 cycloalkyl group or a 5- to 7-membered heterocyclic group; L.sup.1 is selected from a single bond and a C.sub.1-C.sub.3 alkylene, preferably a single bond; R.sup.4 is selected from the group consisting of hydrogen, halogen, cyano, —R.sup.a, —OR.sup.a, —S(O).sub.nR.sup.a, —NR.sup.aR.sup.b, —N(R.sup.a)C(O)R.sup.b, —C(O)NR.sup.aR.sup.b, —C(O)N(R.sup.a)S(O).sub.nR.sup.b, —N(R.sup.a)C(O)NR.sup.aR.sup.b, —N(R.sup.a)C(O)OR.sup.b, —OC(O)NR.sup.aR.sup.b, —N(R.sup.a)S(O).sub.nR.sup.b, —S(O).sub.nNR.sup.aR.sup.b, —C(O)R.sup.a, —C(O)OR.sup.a, —OC(O)R.sup.a, —P(O)(OR.sup.a)(OR.sup.b) and —B(OH).sub.2; R.sup.5 is each independently selected from the group consisting of hydrogen, halogen, amino, hydroxyl, thiol, carboxyl, ester, oxo and alkyl, wherein said alkyl is optionally further substituted with halogen; R.sup.a and R.sup.b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, nitro, cyano, oxo, carboxyl, ester, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally further substituted by one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R.sup.a and R.sup.b together with the nitrogen atom attached to them form a N-containing heterocyclic group, wherein said N-containing heterocyclic group is optionally further substituted with one or more groups selected from the group consisting of halogen, amino, nitro, cyano, oxo, hydroxyl, thiol, carboxyl, ester, alkyl, alkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; m is 0, 1 or 2; n is an integer from 0 to 2; preferably 1 or 2.

    8. The compound of formula (I) according to claim 1, wherein, ring A is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, piperidinyl and piperazinyl; L.sup.1 is selected from a single bond; R.sup.4 is selected from the group consisting of hydrogen, —Ra, —C(O)R.sup.a, —C(O)OR.sup.a, —C(O)NR.sup.aR.sup.b, —S(O).sub.nR.sup.a and —P(O)(OR.sup.a)(OR.sup.b); R.sup.5 is each independently selected from the group consisting of hydrogen, halogen, amino, hydroxyl, thiol, carboxyl, ester, oxo and alkyl, wherein said alkyl is optionally further substituted with halogen; R.sup.a and R.sup.b are each independently selected from the group consisting of hydrogen, hydroxyl, carboxyl, ester and alkyl, wherein said alkyl is optionally further substituted with one or more groups selected from the group consisting of hydroxyl, thiol, carboxyl and ester; m is 0, 1 or 2; n is 1 or 2.

    9. The compound of formula (I) according to claim 1, wherein, R.sup.1 is selected from the group consisting of hydrogen, halogen and alkyl, wherein said alkyl is optionally further substituted with halogen.

    10. The compound of formula (I) according to claim 1, wherein, R.sup.2 is selected from aryl and heteroaryl, preferably C.sub.5-C.sub.10 aryl or 5- to 10-membered heteroaryl, more preferably oxazolyl, imidazolyl, pyrazolyl, thiazolyl, said aryl and heteroaryl are optionally further substituted by one or more groups selected from the group consisting of halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, alkoxyl, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl and heteroaryl.

    11. The compound of formula (I) according to claim 1 or a mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of: ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112##

    12. A method for preparing the compound of formula (I) according to claim 1, which comprises the following steps: when Z is CR.sup.2, ##STR00113## the compound IF is reacted with R.sup.2Sn(C.sub.4H.sub.9).sub.3 in the presence of a catalyst to obtain a compound of formula (I), wherein said catalyst is preferably bis(triphenylphosphorus)palladium dichloride; X, Y, ring A, L.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and m are as defined in claim 1.

    13. A pharmaceutical composition comprising the compound of formula (I) according to claim 1 and a pharmaceutically acceptable carrier or excipient.

    14. Use of the compound of formula (I) according to claim 1 or a pharmaceutical composition in the preparation of acetyl-Coenzyme A carboxylase inhibitor.

    15. Use of the compound of formula (I) according to claim 1 or a pharmaceutical composition in the preparation of medicaments for the prevention or treatment of diseases associated with acetyl-Coenzyme A carboxylase activity; said disease is preferably metabolic disease, cardiovascular disease or cancer; said metabolic disease is for example dyslipidemia, obesity, diabetes, insulin resistance, metabolic syndrome, fatty liver disease or steatohepatitis, preferably fatty liver disease or steatohepatitis; said cardiovascular disease is for example atherosclerosis, angina pectoris, acute coronary syndrome or heart failure; said cancer is for example breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanoma, solid tumor, glioma, glioblastoma, hepatocellular carcinoma, mastoid nephroma, head and neck tumors, leukemia, lymphoma, myeloma or non-small cell lung cancer, preferably liver cancer.

    Description

    DESCRIPTION OF THE DRAWINGS

    [0136] FIG. 1 is a single crystal X-ray diffraction pattern of the compound 8c prepared in Example 8, which is an overlay spectrum of the simulated pattern and the test pattern of the single crystal, wherein the upper line is the test pattern, and the lower line is the simulated pattern.

    [0137] FIG. 2 is the crystal structure of compound 8c, which shows atomic shift parameters with a probability of 50%.

    [0138] FIG. 3 is the crystal structure of compound 8c in one unit crystal cell, with hydrogen omitted for clarity.

    DETAILED DESCRIPTION OF THE INVENTION

    [0139] The compounds of the present invention and their preparation are further illustrated with reference to the following examples. These examples illustrate some methods of preparing or using said compounds. However, it should be understood that these examples are not intended to limit the scope of the invention. Variations of the invention now known or to be further developed are considered to fall within the scope of the invention described and claimed herein.

    [0140] The compound of the present invention is prepared by using convenient starting materials and general preparation procedures. The present invention provides typical or preferential reaction conditions, such as reaction temperature, time, solvent, pressure, and molar ratio of reactants. However, unless otherwise specified, other reaction conditions can also be adopted. Optimal conditions may vary with the use of specific reactants or solvents, but under normal circumstances, reaction optimization steps and conditions can be determined.

    [0141] In addition, some protecting groups may be used in the present invention to protect certain functional groups from unnecessary reactions. The protecting groups suitable for various functional groups and their protection or deprotection conditions are well known to those skilled in the art. For example, “Protective Groups in Organic Synthesis” by T. W. Greene and G. M. Wuts (3rd edition, Wiley, New York, 1999 and citations in the book) describes in detail the protection or deprotection of a large number of protective groups.

    [0142] The separation and purification of compounds and intermediates adopt appropriate methods and steps according to specific needs, such as filtration, extraction, distillation, crystallization, column chromatography, preparative thin-layer chromatography, preparative high performance liquid chromatography or a combination of the above methods. The examples described in the present invention can be referred for the specific method of use. Of course, other similar separation and purification methods can also be used. They can be characterized using conventional methods (including physical constants and spectral data).

    [0143] The structure of the compounds are determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). The NMR shift is given in units of 10.sup.−6 (ppm). The NMR is determined by using a Brukerdps 300 nuclear magnetic instrument. The solvent is deuterated dimethyl sulfoxide (DMSO-d.sub.6), deuterated chloroform (CDCl.sub.3), deuterated methanol (CD.sub.3OD), and the internal standard is tetramethylsilane (TMS).

    [0144] MS is determined using a LC/MS-2020 mass spectrometer (manufacturer: Shimadzu) (QDa Detector).

    [0145] The preparative liquid chromatography applies Waters high performance liquid chromatograph (Waters 2545 binary gradient pump, 2767 sample manager, 2489 UV/visual detector, single C18, 5 μm, 19 mm×250 mm) (manufacturer: Waters).

    [0146] The thin layer chromatography (TLC) applies Qingdao Ocean Chemical GF254 silica gel plate, and the specification is 0.15 mm to 0.2 mm for analysis, and 0.4 mm to 0.5 mm for separation and purification.

    [0147] Column chromatography generally applies Qingdao Ocean Silicone 100 to 200 mesh and 200 to 300 mesh silica gel as carrier.

    [0148] The known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from WHmall, Beijing Ouhe, Sigma, J&K Chemicals, Yi Shiming, Shanghai Shuya, Shanghai Innochem, Energy Chemical, Shanghai Bide Pharmatech and other companies.

    [0149] Unless otherwise specified in the examples, the reactions can all be carried out under nitrogen atmosphere.

    [0150] Argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon having a volume of about 1 L.

    [0151] The reaction solvent, organic solvent or inert solvent are each expressed as the solvent used that does not participate in the reaction under the described reaction conditions, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, dichloromethane, ether, methanol, N-methylpyrrolidone (NMP), pyridine, and the like. Unless otherwise specified in the examples, a solution means an aqueous solution.

    [0152] The chemical reaction described in the present invention is generally carried out under normal pressure. The reaction temperature is between −78° C. and 200° C. The reaction time and conditions are, for example, between −78° C. and 200° C. under one atmospheric pressure, and completed within about 1 to 24 hours. If the reaction is overnight, then the reaction time is generally 16 hours. Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20° C. to 30° C.

    [0153] The progress of the reaction in the examples is monitored by thin layer chromatography (TLC). The developing system includes: A: dichloromethane and methanol system, B: petroleum ether and ethyl acetate system, C: acetone. The volume ratio of the solvents is adjusted depending on the polarity of the compound.

    [0154] The eluent system of column chromatography and the developing system of TLC for the purification of the compound include: A: dichloromethane and methanol system, B: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted depending on the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and trifluoroacetic acid may be added for adjustment.

    [0155] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the content described herein can be applied to the method of the present invention.

    Abbreviations

    [0156] μL=microliter

    [0157] μM=micromole

    [0158] NMR=nuclear magnetic resonance;

    [0159] Boc=tert-butoxycarbonyl

    [0160] br=broad peak

    [0161] d=doublet

    [0162] δ=chemical shift

    [0163] ° C.=degrees celsius

    [0164] dd=double doublet

    [0165] DIPEA=diisopropylethylamine

    [0166] DMF=N,N-dimethylformamide

    [0167] DMSO=dimethyl sulfoxide

    [0168] DCM=dichloromethane

    [0169] EA=ethyl acetate

    [0170] EDCI=1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride

    [0171] HATU=2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate

    [0172] HOBt=1-hydroxybenzotriazole

    [0173] HPLC=high performance liquid chromatography

    [0174] Hz=hertz

    [0175] IC.sub.50=the concentration that inhibits 50% of the activity

    [0176] J=coupling constant (Hz)

    [0177] LC-MS=liquid chromatography-mass spectrometry

    [0178] LDA=lithium diisopropylamide

    [0179] m=multiplet

    [0180] M+H.sup.+=mass of parent compound+one proton

    [0181] mg=milligram

    [0182] mL=milliliter

    [0183] mmol=millimole

    [0184] MS=mass spectrum

    [0185] m/z=mass-to-charge ratio

    [0186] nM=nanomole

    [0187] NBS=N-bromosuccinimide

    [0188] PE=petroleum ether

    [0189] ppm=parts per million

    [0190] PyBrOP=tripyrrolidinyl phosphonium bromide hexafluorophosphate

    [0191] s=singlet

    [0192] t=triplet

    [0193] TEA=triethylamine

    [0194] TBDPS=tert-butyl diphenyl silicon

    [0195] TFA=trifluoroacetic acid

    [0196] THF=tetrahydrofuran

    [0197] LDA=lithium diisopropylamide

    [0198] HMPA=hexamethyl phosphoric acid triamide.

    Example 1: Preparation of 3 ‘-methyl-2’-(oxazol-2-yl)-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (1)

    [0199] ##STR00055##

    Step 1: Preparation of 2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethan-1-ol (Compound 1-2)

    [0200] Ferric chloride (12.0 g, 0.750 mol) was added to 4-tetrahydropyranol (102 g, 1.00 mol) at room temperature. The mixed solution was cooled to 0° C., and phenyl oxirane (1-1) (60.0 g, 0.500 mol) was slowly added dropwise. After the addition, the reaction mixture was warmed to room temperature and stirred for 4 hours. The reaction was quenched by adding water (2000 mL) and was extracted with ethyl acetate (3×1000 mL). The organic phases were combined and then washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 50%) to obtain the title compound (18.0 g, 16.2%, a mixture of two optical isomers) as a colorless oil.

    [0201] .sup.1H-NMR (CDCl.sub.3) δ: 7.41-7.29 (m, 5H), 4.85 (m, 1H), 4.62 (dd, J=4.2, 8.1 Hz, 1H), 4.03-3.89 (m, 2H), 3.73-3.50 (m, 3H), 3.46-3.34 (m, 2H), 2.03-1.96 (m, 1H), 1.79-1.61 (m, 3H).

    [0202] LC-MS: m/z 245.15 [M Na].sup.+.

    Step 2: Preparation of 5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (Compound 1-4)

    [0203] The compound ethyl 2-amino-4-methylthiophen-3-carboxylate (1-3) (20.0 g, 0.108 mol) was dissolved in water (400 mL) at room temperature, and potassium hydroxide solid (12.1 g, 0.216 mol) was added. The reaction solution was heated to 100° C. and refluxed for 6 hours. The resulting solution was cooled to 0° C., and slowly added with diphosgene (21.2 g, 0.108 mol) dropwise. After addition, the reaction mixture was warmed to room temperature and stirred for 3 hours. A solid was gradually precipitated out during the reaction. After the reaction, the obtained solid was filtered, washed with water to neutrality, then washed with petroleum ether (200 mL), and dried under reduced pressure to obtain the title compound (7.00 g, 35.3%) as a brown solid. LC-MS: m/z 184.25 [M+H].sup.+.

    Step 3: Preparation of 5-methyl-1-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (Compound 1-5)

    [0204] 5-Methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (1-4) (7.00 g, 38.2 mmol) was dissolved in dry tetrahydrofuran (150 mL) under nitrogen atmosphere at room temperature, and 2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethan-1-ol (1-2) (16.9 g, 76.5 mmol) and triphenylphosphorus (20.0 g, 76.5 mmol) was added successively. The reaction solution was cooled to 0° C., and slowly added with diisopropyl azodicarboxylate (15.5 g, 76.5 mmol) dropwise. After addition, the reaction mixture was warmed to room temperature and reacted for 16 hours. After the reaction, the reaction solution was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 50%) to obtain the title compound (4.50 g, 30.4%) as a white solid.

    [0205] LC-MS: m/z 388.15 [M+H].sup.+.

    Step 4: Preparation of 6-bromo-5-methyl-1-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (Compound 1-6)

    [0206] 5-Methyl-1-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (1-5) (4.50 g, 11.6 mmol) was dissolved in chloroform (100 mL) at room temperature, and then added with N-bromosuccimide (2.07 g, 11.6 mmol). The reaction mixture was stirred at room temperature for 3 hours, and then quenched by adding 5% aqueous solution of sodium thiosulfate (10 mL). The mixture was diluted with dichloromethane (200 mL) and water (100 mL). After separation of the organic phase, the aqueous phase was extracted with dichloromethane (3×100 mL), and the combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 40%) to obtain the title compound (3.1 g, 57.3%, a mixture of two optical isomers) as a pale yellow solid.

    [0207] .sup.1H-NMR (CDCl.sub.3) δ: 7.47-7.37 (m, 5H), 5.00 (dd, J=2.4, 7.5 Hz, 1H), 4.20 (dd, J=1.8, 10.8 Hz, 1H), 3.80-3.69 (m, 2H), 3.64 (dd, J=7.5, 10.8 Hz, 1H), 3.51-3.45 (m, 1H), 3.38-3.31 (m, 2H), 1.80-1.71 (m, 2H), 1.57-1.50 (m, 2H), 1.42-1.34 (m, 1H).

    [0208] LC-MS: m/z 466.10 [M+H].sup.+.

    Step 5: Preparation of 2′-bromo-3′-methyl-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (Compound 1-7)

    [0209] Methyl cyclobutanecarboxylate (98.6 mg, 0.860 mmol) was dissolved in dry THF (5 mL) under nitrogen atmosphere, and then added with LDA (0.860 mmol, 2 mol/L) at −78° C. The mixture was stirred at this temperature for 1 hour, and then added with 6-bromo-5-methyl-1-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (1-6) (200 mg, 0.430 mmol). The reaction solution was warmed to room temperature and stirred for 1 hour. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 40%) to obtain the title compound (110 mg, 50.8%) as a white solid.

    [0210] LC-MS: m/z 504.05 [M+H].sup.+.

    Step 6: Preparation of 3′-methyl-2′-(oxazol-2-yl)-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (Compound 1)

    [0211] 2-(Tributylstannyl)oxazole (53 mg, 0.15 mmol) and bis(triphenylphosphorus) palladium dichloride (0.01 mmol, 7.1 mg) were added to 2′-bromo-3′-methyl-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (1-7) (50 mg, 0.099 mmol) in a dry toluene solution (3 mL) under nitrogen atmosphere at room temperature. The reaction solution was heated and refluxed overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×10 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered The filtrate was concentrated under reduced pressure. The residues were purified by preparative column chromatography (column type: XSelect CSH Prep C18 OBD column 5 μm, 19×150 mm; mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL/min; gradient: 50% to 53% acetonitrile; duration: 8 minutes; 254 nm) to obtain the title compound (10 mg, 20.4%, a mixture of two optical isomers) as a white solid.

    [0212] .sup.1H-NMR (CDCl.sub.3) δ: 8.00 (s, 1H), 7.53-7.36 (m, 5H), 7.30 (s, 1H), 5.02-4.97 (m, 1H), 4.31-4.25 (m, 1H), 3.95-3.87 (m, 1H), 3.77-3.61 (m, 2H), 3.55-3.47 (m, 1H), 3.42-3.33 (m, 2H), 2.84 (s, 3H), 2.70-2.47 (m, 4H), 2.24-2.13 (m, 2H), 1.83-1.64 (m, 2H), 1.55-1.38 (m, 2H).

    [0213] LC-MS: m/z 493.15 [M+H].sup.+.

    Example 2: Preparation of 3′-methyl-2′-(oxazol-2-yl)-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (2)

    [0214] ##STR00056##

    [0215] The preparation method was the same as in Example 1, except that methyl cyclopentanecarboxylate was used instead of methyl cyclobutanecarboxylate, to obtain the title compound 2.

    [0216] .sup.1H-NMR (CDCl.sub.3) δ: 7.73 (d, J=0.6 Hz, 1H), 7.44-7.50 (m, 2H), 7.36-7.42 (m, 3H), 7.24 (d, J=0.6 Hz, 1H), 4.96 (dd, J=3.9, 9.6 Hz, 1H), 4.26 (dd, J=3.6, 14.4 Hz, 1H), 3.70-3.88 (m, 3H), 3.41-3.50 (m, 1H), 3.30-3.36 (m, 2H), 2.84 (s, 3H), 2.17-2.27 (m, 3H), 2.03-2.07 (m, 1H), 1.88-1.93 (m, 4H), 1.67-1.77 (m, 2H), 1.49-1.61 (m, 1H), 1.37-1.47 (m, 1H).

    [0217] LC-MS: m/z 507.35 [M+H].sup.+.

    Example 3: Preparation of

    3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (3)

    [0218] ##STR00057##

    Step 1: Preparation of methyl 2′-bromo-3′-methyl-2′-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (3-1)

    [0219] Methyl cyclopentane-1,3-dicarboxylate (120 mg, 0.643 mmol) was dissolved in dry THF (5 mL) under nitrogen atmosphere, and then added with LDA (0.643 mmol, 2 mol/L) at −78° C. The mixture was stirred at this temperature for 1 hour and then added with 6-bromo-5-methyl-1-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-2H-thieno[2,3-d][1,3]oxazine-2,4 (1H)-dione (1-6) (250 mg, 0.536 mmol). The reaction solution was warmed to room temperature and stirred overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by column chromatography silica gel (mobile phase: ethyl acetate/petroleum ether=30% to 70%) to obtain the title compound (136 mg, 44.0%) as a white solid.

    [0220] LC-MS: m/z 598.15 [M+Na].sup.+.

    Step 2: Preparation of methyl 3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (3-2)

    [0221] 2-(Tributylstannyl)oxazole (348 mg, 0.971 mmol) and bis(triphenylphosphorus) palladium dichloride (170 mg, 0.243 mmol) were added to methyl-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-4′H-spiro[cyclopentane-1, 5′-thieno[2,3-b]pyridine]-3-carboxylate (3-1) (280 mg, 0.486 mmol) in a dry toluene solution (10 mL) under nitrogen atmosphere at room temperature. The reaction solution was heated and refluxed overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by column chromatography silica gel (mobile phase: ethyl acetate/petroleum ether=20% to 40%) to obtain the title compound (136 mg, 50.0%) as a pale yellow oil.

    [0222] LC-MS: m/z 579.35 [M+H].sup.+.

    Step 3: Preparation of 3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (3)

    [0223] Lithium hydroxide (3.4 mg, 0.14 mmol) was added to methyl 3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (3-2) (40 mg, 0.071 mmol) in a methanol/water mixed solution (2 mL, methanol/water=1/1) at room temperature. The reaction solution was stirred overnight at room temperature. After completion of the reaction, the organic solvent was removed under reduced pressure, then the reaction solution was adjusted to pH 2 with dilute hydrochloric acid (pH=1). The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 60%) to obtain the title compound (14 mg, 36.0%, a mixture of eight optical isomers) as a white solid.

    [0224] .sup.1H-NMR (CDCl.sub.3) δ: 7.75 (d, J=0.6 Hz, 1H), 7.35-7.49 (m, 5H), 7.28 (d, J=0.6 Hz, 1H), 4.94-5.01 (m, 1H), 4.16-4.40 (m, 1H), 3.71-3.83 (m, 3H), 3.42-3.49 (m, 1H), 3.26-3.38 (m, 2H), 2.84 (m, 3H), 2.48-2.70 (m, 2H), 2.18-2.30 (m, 4H), 1.70-1.83 (m, 2H), 1.56-1.58 (m, 1H), 1.41-1.45 (m, 1H).

    [0225] LC-MS m/z 551.35+Hr.

    Example 4: Preparation of

    3,3′-dimethyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (4)

    [0226] ##STR00058##

    Step 1: Preparation of methyl 1-methyl-cyclopentane-1,3-dicarboxylate (4-1)

    [0227] Methyl cyclopentane-1,3-dicarboxylate (0.552 g, 5.52 mmol) and hexamethyl phosphate triamide (3.10 g, 17.2 mmol) were dissolved in dry THF (10 mL) under nitrogen atmosphere, and then added with LDA (16.5 mmol, 2 mol/L) at −78° C. The mixture was stirred at this temperature for 1 hour and then added with methyl iodide (1.22 g, 8.59 mmol). The reaction solution was warmed to room temperature and stirred overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×40 mL), and the combined organic phase was washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 40%) to obtain the title compound (630 mg, 73.0%) as a pale yellow oil.

    [0228] .sup.1H-NMR (CDCl.sub.3) δ: 3.69 (s, 6H), 2.87-3.00 (m, 1H), 2.37-2.52 (m, 1H), 2.12-2.31 (m, 1H), 1.83-2.02 (m, 2H), 1.49-1.77 (m, 2H), 1.30 (s, 3H).

    Step 2: Preparation of methyl 2′-bromo-3,3′-dimethyl-2′-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (4-2)

    [0229] Methyl 1-methyl-cyclopentane-1,3-dicarboxylate (4-1) (322 mg, 1.61 mmol) was dissolved in dry THF (20 mL) under nitrogen atmosphere, and then added with LDA (2.04 mmol, 2 mol/L) at −78° C. The mixture was stirred at this temperature for 1 hour and then added with 6-bromo-5-methyl-1-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (1-6) (250 mg, 0.536 mmol). The reaction solution was warmed to room temperature and stirred overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×40 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 40%) to obtain the title compound (260 mg, 82.0%) as a pale yellow oil.

    [0230] LC-MS: m/z 590.25 [M+H].sup.+.

    Step 3: Preparation of methyl 3,3′-dimethyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (4-3)

    [0231] 2-(Tributylstannyl)oxazole (303 mg, 0.847 mmol) and bis(triphenylphosphorus) palladium dichloride (149 mg, 0.212 mmol) were added to methyl 2′-bromo-3,3′-dimethyl-2′-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-4′H-spiro[cyclopentane-2,5′-thieno[2,3-b]pyridine]-3-carboxylate (4-2) (0.423 mmol, 250 mg) in a dry toluene solution (10 mL) under nitrogen atmosphere at room temperature. The reaction solution was heated and refluxed overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 40%) to obtain the title compound (170 mg, 69.0%) as a pale yellow oil.

    [0232] .sup.1H-NMR (MeOD) δ: 7.74 (m, 1H), 7.37-7.50 (m, 5H), 7.28 (m, 1H), 4.89-5.02 (m, 1H), 4.29-4.37 (m, 1H), 4.12-4.19 (m, 1H), 3.71-3.87 (m, 5H), 3.42-3.49 (m, 1H), 3.28-3.37 (m, 2H), 2.88-3.01 (m, 1H), 2.82 (s, 3H), 2.43-2.52 (m, 1H), 2.18-2.35 (m, 2H), 2.03-2.14 (m, 1H), 1.68-1.84 (m, 4H), 1.54-1.61 (m, 1H), 1.40 (s, 3H).

    [0233] LC-MS: m/z 579.25 [M+H].sup.+.

    Step 4: Preparation of 3,3′-dimethyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (4)

    [0234] Lithium hydroxide (13 mg, 0.56 mmol) was added to 3,3′-dimethyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (4-3) (0.16 g, 0.28 mmol) in a methanol/water mixed solution (6 mL, methanol/water=1/1) at room temperature. The reaction solution was stirred overnight at room temperature. After completion of the reaction, the organic solvent was removed under reduced pressure, then the reaction solution was adjusted to pH 2 with dilute hydrochloric acid (pH=1). The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 60%) to obtain the title compound (60 mg, 38%, a mixture of eight optical isomers) as a white solid.

    [0235] .sup.1H-NMR (CDCl.sub.3) δ: 7.74 (m, 1H), 7.37-7.50 (m, 5H), 7.28 (m, 1H), 4.92-4.97 (m, 1H), 4.35-4.41 (m, 1H), 3.67-3.83 (m, 3H), 3.43-3.48 (m, 1H), 3.29-3.38 (m, 2H), 2.90-2.97 (m, 1H), 2.79 (s, 3H), 2.39-2.47 (m, 2H), 2.23-2.34 (m, 2H), 2.09-2.18 (m, 1H), 1.78-1.99 (m, 2H), 1.43-1.62 (m, 2H), 1.54 (s, 3H).

    [0236] LC-MS: m/z 565.35 [M+H].sup.+.

    Example 5: Preparation of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (5)

    [0237] ##STR00059##

    Step 1: Preparation of 2-methoxyphenyl oxirane (5-2)

    [0238] Sodium hydride (35.3 g, 0.882 mol) was dissolved in dry dimethyl sulfoxide (800 mL) at room temperature. The mixed solution was cooled to 0° C., and then added with trimethylsulfoxide iodide (194 g, 0.882 mol). After stirring for 2 hours at 0° C., o-methoxybenzaldehyde (5-1) (100 g, 0.735 mol) was slowly added. The mixture was stirred at room temperature for 3 hours, and quenched by adding water (3000 mL). The reaction was extracted with ethyl acetate (3×3000 mL). The organic phases were combined and then washed with saturated brine (2000 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The title compound (105 g) was obtained as a pale yellow oil. The crude product was directly used in the next reaction without purification.

    Step 2: Preparation of 2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethane-1-ol (5-3)

    [0239] Ferric chloride (16.9 g, 0.105 mol) was added to 4-tetrahydropyranol (204 g, 1.40 mol) at room temperature. The mixed solution was cooled to 0° C., and then slowly added with crude 2-methoxyphenyl oxirane (5-2) (105 g, 0.700 mol) dropwise. The reaction mixture was warmed to room temperature and stirred for 4 hours. The reaction was quenched by adding water (2000 mL). The reaction system was extracted with ethyl acetate (3×1000 mL). The organic phases were combined and then washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were separated and purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 50%). The title compound (30.0 g, 17.0%, a mixture of two optical isomers) was obtained as a colorless oil.

    [0240] .sup.1H-NMR (CD.sub.3OD) δ: 7.43 (dd, J=1.8, 7.5 Hz, 1H), 7.32-7.27 (m, 1H), 7.00 (t, J=7.5 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 5.09 (dd, J=3.6, 8.4 Hz, 1H), 4.03-3.90 (m, 2H), 3.85 (s, 3H), 3.70 (dd, J=3.6, 11.4 Hz, 1H), 3.59-3.51 (m, 2H), 3.46-3.37 (m, 3H), 2.07-1.98 (m, 1H), 1.84-1.79 (m, 1H), 1.72-1.61 (m, 2H).

    [0241] LC-MS: m/z 275.15 [M+Na].sup.+.

    Step 3: Preparation of 1-(2-(2-methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2H-thieno[2, 3-d][1,3]oxazine-2,4(1H)-dione (5-4)

    [0242] 5-Methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (1-4) (5.00 g, 27.3 mmol) was dissolved in dry tetrahydrofuran (150 mL) under nitrogen atmosphere at room temperature, and added with 2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethane-1-ol (5-3) (13.77 g, 54.6 mmol) and triphenylphosphorus (14.3 g, 54.6 mmol) successively. The reaction solution was cooled to 0° C., and then slowly added with diisopropyl azodicarboxylate (11.0 g, 54.6 mmol) dropwise. After addition, the reaction solution was warmed to room temperature and reacted for 16 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 50%) to obtain the title compound (3.50 g, 30.6%) as a white solid.

    [0243] LC-MS: m/z 418.15 [M+H].sup.+.

    Step 4: Preparation of 6-bromo-1-(2-(2-methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (5-5)

    [0244] 1-(2-(2-Methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (5-4) (3.50 g, 8.37 mmol) was dissolved in chloroform (100 mL) at room temperature, and then added with N-bromosuccimide (1.49 g, 8.37 mmol). The mixture was reacted at room temperature for 3 hours. The reaction was quenched by adding 5% aqueous solution of sodium thiosulfate (10 mL), and diluted with dichloromethane (200 mL) and water (100 mL). After separation of the organic phase, the aqueous phase was extracted with dichloromethane (3×100 mL), and the combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 40%) to obtain the title compound (2.50 g, 60.2%, a mixture of two optical isomers) as a pale yellow solid.

    [0245] .sup.1H-NMR (d.sup.6-DMSO) 7.46 (dd, J=1.8, 7.5 Hz, 1H), 7.32 (dt, J=1.85, 8.1 Hz, 1H), 7.06-6.99 (m, 2H), 5.24 (dd, J=4.8, 8.4 Hz, 1H), 4.01-3.97 (m, 2H), 3.79 (s, 3H), 3.67-3.56 (m, 2H), 3.46-3.48 (m, 1H), 3.29-3.21 (m, 2H), 2.52-2.50 (m, 1H), 1.71-1.63 (m, 2H), 1.40-1.18 (m, 2H).

    [0246] LC-MS: m/z 518.10 [M+Na].sup.+.

    Step 5: Preparation of methyl 2′-bromo-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-4 ‘,6’-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylat e (5-6)

    [0247] Methyl cyclopentane-1,3-dicarboxylate (563 mg, 3.02 mmol) was dissolved in dry THF (20 mL) under nitrogen atmosphere, and then added with LDA (3.02 mmol, 2 mmol/L) at −78° C. The mixture was stirred at this temperature for 1 hour and then added with 6-bromo-1-(2-(2-methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (5-5) (500 mg, 1.01 mmol). The reaction solution was warmed to room temperature and stirred overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×40 mL), and the combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 40%) to obtain the title compound (392 mg, 64.0%) as a white solid.

    [0248] LC-MS: m/z 628.15 [M+Na].sup.+.

    Step 6: Preparation of methyl 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (5-7)

    [0249] 2-(Tributylstannyl)oxazole (463 mg, 1.29 mmol) and bis(triphenylphosphorus) palladium dichloride (227 mg, 0.323 mmol) were added to methyl 2′-bromo-7′-(2-(2-methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (5-6) (392 mg, 0.646 mmol) in a dry toluene solution (10 mL) under nitrogen atmosphere at room temperature. The reaction solution was heated and refluxed overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were separated and purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 40%) to obtain the title compound (162 mg, 42.0%) as a pale yellow oil.

    [0250] LC-MS: m/z 617.35 [M+Na].sup.+.

    Step 7: Preparation of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (5)

    [0251] Methyl 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (5-7) (160 mg, 0.277 mmol) was dissolved in 6N hydrochloric acid (5 mL) at room temperature. The reaction solution was stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain a crude product as colorless oil which was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 60%) to obtain the title compound (60.1 mg, 37.1%, a mixture of eight optical isomers) as a white solid.

    [0252] .sup.1H-NMR (CD.sub.3OD) δ: 7.73 (d, J=0.9 Hz, 1H), 7.57 (t, J=4.5 Hz, 1H), 7.29-7.34 (m, 2H), 7.04 (t, J=6 Hz, 1H), 6.89 (t, J=6.3 Hz, 1H), 5.38-5.45 (m, 1H), 4.17-4.27 (m, 1H), 3.98-4.10 (m, 1H), 3.81 (s, 3H), 3.77-3.84 (m, 2H), 3.43-3.47 (m, 1H), 3.23-3.36 (m, 3H), 2.84 (s, 3H), 2.53-2.72 (m, 2H), 2.13-2.48 (m, 5H), 1.73-1.94 (m, 2H), 1.53-1.59 (m, 1H), 1.44-1.48 (m, 1H).

    [0253] LC-MS: m/z 581.15 [M+H].sup.+.

    Step 8: Separation of optical isomers of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (5)

    [0254] ##STR00060##

    [0255] 7′-(2-(2-Methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclopentane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic acid (compound 5) (465 mg) was separated by preparative chiral column chromatography (column type: XSelect CSH Prep C18 OBD column, 5 μm, 19×150 mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL/min; gradient: 68% to 73% acetonitrile; duration: 15 minutes; detection wavelength: 254/220 nm), to obtain compound 5x (119 mg, a mixture of 6 optical isomers) as a white solid and compound 5y (100 mg, a mixture of 2 optical isomers) as a white solid.

    [0256] Compound 5x: .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.56-7.53 (m, 1H), 7.32-7.28 (m, 2H), 7.05-6.96 (m, 2H), 5.49-5.42 (m, 2H), 4.16-4.13 (m, 2H), 3.87 (s, 3H) 3.86-3.75 (m, 2H), 3.50-3.47 (m, 1H), 3.39-3.36 (m, 2H), 3.15-3.09 (m, 1H), 2.78 (s, 3H), 2.56-2.53 (m, 1H), 2.16-2.09 (m, 3H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 1H), 1.56-1.44 (m, 2H).

    [0257] LC-MS: m/z 581.15 [M+H].sup.+.

    [0258] Compound 5y: .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.55 (dd, J=1.2, 5.4 Hz, 1H), 7.33-7.29 (m, 2H), 7.03 (t, J=5.7 Hz, 1H), 6.98 (d, J=6.3 Hz, 1H), 5.46-5.43 (m, 1H), 4.18-4.09 (m, 1H), 3.89 (s, 3H), 3.80-3.73 (m, 2H), 3.50-3.46 (m, 1H), 3.38-3.36 (m, 2H), 3.11-3.09 (m, 1H), 2.79 (s, 3H), 2.44-2.42 (m, 2H), 2.30-2.23 (m, 2H), 2.16-2.12 (m, 2H), 1.83-1.81 (m, 1H), 1.76-1.72 (m, 1H), 1.54-1.44 (m, 2H).

    [0259] LC-MS: m/z 581.15 [M+H].sup.+.

    Step 9: Separation of Optical Isomers of Compound 5y

    [0260] ##STR00061##

    [0261] Compound 5y (100 mg) was separated by preparative chiral column chromatograph (column type: CHIRALPAK IF-3, 2.0×25 cm L (5 μm); mobile phase A: tert-butyl methyl ether (0.1% formic acid), mobile phase B: ethanol; flow rate: 20 mL/min; gradient: 10% isogradient; duration: 40 minutes; detection wavelength: 220/320 nm), to obtain compound 5a (22.1 mg, single configuration) as a white solid and compound 5b (18.3 mg, single configuration) as a white solid, respectively.

    [0262] The .sup.1H NMR and LC/MS data of compounds 5a and 5b were completely consistent with those of compound 5y.

    [0263] Chiral analysis: Column type: CHIRALPAK IF-3, 10×0.46 cm L (3 μm); mobile phase A: tert-butyl methyl ether (0.1% formic acid), mobile phase B: ethanol; flow rate: 1 mL/min; gradient: 10% isogradient; detection wavelength 220/320 nm, retention time: 5a: 1.35 min; 5b: 1.55 min.

    Step 10: Separation of Optical Isomers of Compound 5x

    [0264] ##STR00062##

    [0265] Compound 5x (119 mg) was separated by preparative chiral column chromatography (column type: CHIRALPAK ID-3, 2.0×25 cm L (5 μm); mobile phase A: n-hexane (0.1% formic acid), mobile phase B: ethanol; flow rate: 20 mL/min; gradient: 15% isogradient; duration: 40 min; detection wavelength: 220/320 nm), to obtain compound 5c (9.1 mg, single configuration) as a white solid, compound 5d (4.6 mg, single configuration) as a white solid, and compound 5e (20 mg, a mixture of two optical isomers) as a white solid, respectively.

    [0266] Compound 5c: .sup.1H-NMR (CD.sub.3OD) δ: 7.97 (s, 1H), 7.54 (dd, J=1.5, 9 Hz, 1H), 7.32-7.27 (m, 2H), 7.05-6.91 (m, 2H), 5.44-5.40 (m, 1H), 4.12-4.09 (m, 2H), 3.86 (s, 3H), 3.80-3.72 (m, 2H), 3.50-3.44 (m, 1H), 3.39-3.36 (m, 1H), 3.10-3.07 (m, 1H), 2.76 (s, 3H), 2.53-2.50 (m, 2H), 2.20-2.10 (m, 4H), 1.85-1.70 (m, 2H), 1.56-1.42 (m, 2H), 1.35-1.30 (m, 2H).

    [0267] LC-MS: m/z 581.15 [M+H].sup.+.

    [0268] Compound 5d: .sup.1H-NMR (CD.sub.3OD) δ: 7.98 (s, 1H), 7.54 (dd, J=1.5, 7.5 Hz, 1H), 7.33-7.27 (m, 2H), 7.05-6.96 (m, 2H), 5.45-5.41 (m, 1H), 4.15-4.12 (m, 2H), 3.86 (s, 3H), 3.81-3.73 (m, 2H), 3.51-3.45 (m, 1H), 3.40-3.37 (m, 1H), 3.13-3.08 (m, 1H), 2.77 (s, 3H), 2.60-2.53 (m, 1H), 2.45-2.38 (m, 1H), 2.25-2.04 (m, 4H), 1.86-1.72 (m, 2H), 1.57-1.45 (m, 2H), 1.35-1.33 (m, 1H).

    [0269] LC-MS: m/z 581.15 [M+H].sup.+.

    [0270] Compound 5e: .sup.1H-NMR (CD.sub.3OD) δ: 7.97 (s, 1H), 7.54-7.52 (m, 1H), 7.32-7.27 (m, 2H), 7.05-6.95 (m, 2H), 5.44-5.40 (m, 1H), 4.13-4.10 (m, 1H), 3.87 (s, 3H), 3.80-3.72 (m, 2H), 3.48-3.45 (m, 1H), 3.10-3.02 (m, 1H), 2.76 (s, 3H), 2.55-2.50 (m, 2H), 2.23-2.03 (m, 4H), 1.81-1.71 (m, 2H), 1.56-1.40 (m, 2H), 1.32-1.30 (m, 1H).

    [0271] LC-MS: m/z 581.15 [M+H].sup.+.

    [0272] Chiral analysis: Column type: CHIRALPAK ID-3, 10×0.46 cm L (3 μm); mobile phase A: n-hexane (0.1% formic acid), mobile phase B: ethanol; flow rate: 1.5 mL/min; gradient: 15% isogradient; detection wavelength: 220/320 nm, retention time: 5c: 9.1 min; 5d: 10.2 min; 5e: 12.5 min.

    Example 6: Preparation of 3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-7′-(2-phenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-4-carboxylic acid (6)

    [0273] ##STR00063##

    [0274] The preparation method was the same as in Example 3, except that methyl cyclohexane-1,4-dicarboxylate was used instead of methyl cyclopentane-1,3-dicarboxylate, to obtain the title compound 6 (a mixture of four optical isomers).

    [0275] .sup.1H-NMR (CDCl.sub.3) δ: 7.73 (1H, d, J=0.9 Hz), 7.49 (1H, dd, J=1.5, 7.5 Hz), 7.37-7.46 (3H, m), 7.25 (1H, d, J=0.9 Hz), 4.93 (1H, dd, J=6, 9.6 Hz), 4.28 (1H, dd, J=3.6, 14.4 Hz), 3.73-3.84 (3H, m), 3.42-3.48 (1H, m), 3.28-3.70 (2H, m), 2.82 (3H, s), 2.45-2.49 (1H, m), 2.06-2.25 (5H, m), 2.01 (3H, m), 1.70-1.82 (2H, m), 1.50-1.62 (1H, m), 1.36-1.47 (1H, m).

    [0276] LC-MS: m/z 565.40 [M+H].sup.+.

    Example 7: Preparation of

    7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (7)

    [0277] ##STR00064##

    Step 1: Preparation of Compound 7-1

    [0278] The preparation method was the same as for compound 5-7 in Example 5, except that methyl cyclohexane-1,3-dicarboxylate was used instead of methyl cyclopentane-1,3-dicarboxylate to obtain the title compound 7-1.

    Step 2: Preparation of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (7)

    [0279] Methyl 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxylate (50 mg, 0.086 mmol) was dissolved in 6N hydrochloric acid (3 mL). The reaction solution was stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain the title compound as a colorless oil.

    Step 3: Separation of optical isomers of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (7)

    [0280] 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic acid was separated by preparative column chromatography (column type: XSelect CSH Prep C18 OBD column, 5 μm, 19×150 mm; mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL/min; gradient: 25% to 75% acetonitrile; duration: 8 min; detection wavelength: 254 nm), to obtain compound 7a (5.6 mg, 11.2%, a mixture of optical isomers) as a white solid and compound 7b (2.5 mg, 5.0%, a mixture of optical isomers) as a white solid, respectively.

    [0281] Compound 7a: .sup.1H-NMR (CDCl.sub.3) δ: 7.73 (1H, s), 7.48-7.50 (2H, m), 7.37-7.44 (3H, m), 7.25 (1H, s), 3.42-3.47 (1H, m), 3.31-3.39 (3H, m), 2.81 (3H, s), 2.31 (1H, t, 9.6 Hz), 2.10-2.17 (2H, m), 1.91-1.95 (1H, m), 1.70-1.82 (5H, m), 1.53-1.61 (1H, m), 1.42-1.45 (2H, m).

    [0282] LC-MS: m/z 565.25 [M+H].sup.+.

    [0283] Compound 7b: .sup.1H-NMR (CDCl.sub.3) δ: 7.73 (1H, s), 7.48-7.50 (2H, m), 7.37-7.44 (3H, m), 7.24 (1H, s), 4.95-4.97 (1H, m), 4.12-4.16 (1H, s), 3.90-3.96 (1H, m), 3.73-3.80 (2H, m), 3.41-3.46 (1H, m), 3.27-3.32 (2H, m), 3.13-3.19 (1H, m), 2.82 (3H, s), 2.10-2.23 (4H, m), 1.91-1.97 (4H, m), 1.76-1.84 (3H, m), 1.67-1.73 (2H, m), 1.38-1.57 (4H, m), 1.28-1.30 (1H, m), 0.88-0.91 (1H, m).

    [0284] LC-MS: m/z 565.25 [M+H].sup.+.

    Example 8: Preparation of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (8)

    [0285] ##STR00065## ##STR00066##

    Step 1: Preparation of methyl 3-hydroxymethylcyclobutane-carboxylate (8-2)

    [0286] Methyl 3-methylenecyclobutane-1-carboxylate (8-1) (10.0 g, 78.1 mmol) was dissolved in dry tetrahydrofuran (100 mL) under a nitrogen atmosphere, and borane dimethyl sulfide (32.0 mL, 64.0 mmol) was slowly added dropwise at −10° C. The reaction solution was warmed to room temperature and stirred for 3 hours to obtain a colorless transparent liquid. Anhydrous methanol (5 mL) was added to the reaction solution at −10° C., and the reaction solution was warmed to room temperature and stirred for 30 minutes. Sodium hydroxide solution (15.0 mL, 2 mol/L) and hydrogen peroxide (8.96 g, 78.1 mol, 30%) were slowly added dropwise at −10° C., stirred at room temperature for 2 hours, and the reaction solution was evaporated under reduced pressure to dryness to obtain the title compound (5.60 g, 49.0%) as a colorless oil.

    [0287] LC-MS: m/z 145.15 [M+H].sup.+.

    Step 2: Preparation of methyl 3-tert-butyldimethylsiloxymethylcyclobutane-carboxylate (8-3)

    [0288] Methyl 3-hydroxymethylcyclobutane-carboxylate (8-2) (5.60 g, 38.7 mmol) was dissolved in dichloromethane (50 mL) at room temperature. The reaction solution was cooled to −10° C. and then slowly added with tert-butyl dimethylchlorosilane (7.00 g, 46.7 mmol), triethylamine (5.89 g, 58.3 mmol) and 4-dimethylaminopyridine (0.470 g, 3.87 mmol). The reaction solution was warmed to room temperature and stirred for 12 hours, and then quenched by adding water (100 mL). The reaction system was extracted with ethyl acetate (3×150 mL), and the combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound (5.00 g, 50.0%) as a colorless oil. The obtained product was directly used in the next step without purification.

    [0289] LC-MS: m/z 259.20 [M+H].sup.+.

    Step 3: Preparation of 2′-bromo-3-(((tert-butyldimethylsilyl)methyl)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (8-4)

    [0290] Methyl 3-tert-butyldimethylsiloxymethylcyclobutane-carboxylate (2.30 g, 9.00 mmol) was dissolved in dry THF (5 mL) under nitrogen atmosphere, and then added with LDA (9.00 mmol, 2 mol/L) at −78° C. The mixture was stirred at this temperature for 1 hour and then added with 6-bromo-1-(2-(2-methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (5-5) (3.00 g, 6.00 mmol). The reaction solution was warmed to room temperature and stirred for 2 hours. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×50 mL), and the combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=30% to 70%) to obtain the title compound (1.40 g, 34.0%) as a yellow oil.

    [0291] LC-MS: m/z 702.00 [M+H].sup.+.

    Step 4: Preparation of 3-(((tert-butyldimethylsilyl)methyl)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (8-5)

    [0292] 2-(Tributylstannyl)oxazole (0.810 g, 2.30 mmol) and bis(triphenylphosphorus) palladium dichloride (0.730 g, 1.00 mmol) were added to 2′-bromo-3-(((tert-butyldimethylsilyl)methyl)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (8-4) (1.40 g, 2.10 mmol) in a dry toluene solution (10 mL) under nitrogen atmosphere at room temperature. The reaction solution was heated and refluxed overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×30 mL), and the combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=40% to 70%) to obtain the title compound (0.800 g, 58.0%) as a pale yellow oil.

    [0293] LC-MS: m/z 689.30 [M+H].sup.+.

    Step 5: Preparation of 3-(hydroxymethyl)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (8-6)

    [0294] 3-(((Tert-butyldimethylsilyl)methyl)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′methyl-2′-(oxazol-2-yl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (8-5) (0.800 g, 1.20 mmol) was dissolved in dry tetrahydrofuran (5 mL) at room temperature, and then added with tetrabutylamine fluoride (0.370 g, 1.40 mmol). The mixture was stirred at room temperature for 12 hours, and then quenched by adding water (50 mL). The reaction mixture was extracted with ethyl acetate (3×50 mL). The organic phases were combined and then washed with saturated brine (40 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=50% to 90%) to obtain the title compound (0.500 g, 75.0%) as a colorless oil.

    [0295] LC-MS: m/z 553.20 [M+H].sup.+.

    Step 6: Preparation of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (8)

    [0296] Jones reagent (0.43 mL, 2 mol/L) was slowly added dropwise to a solution of 3-(hydroxymethyl)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-4′,6′(7′H)-dione (8-6) (220 mg, 0.400 mmol) in acetone (5 mL) at 0° C., and stirred at this temperature for 1.5 hours. The reaction was quenched with isopropanol (5 mL). The reaction solution was concentrated under reduced pressure, and added with water (20 mL). The mixture was extracted with ethyl acetate (3×20 mL). The organic phases were combined and then washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by high performance liquid chromatography (column type: XBridge Shiels RP18 OBD column, 5 μm, 19×150 nm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL/min; gradient: 45% to 65% in 8 min; detection wavelength: 254/220 nm) to obtain the title compound (82 mg, 36.3%, a mixture of four optical isomers) as a white powder.

    [0297] LC-MS: m/z 567.15 [M+H].sup.+.

    Step 7: Separation of Optical Isomers of 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic Acid (8)

    [0298] ##STR00067##

    [0299] 7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carboxylic acid (compound 8) (82 mg) was separated by preparative column chromatography (column type: CHIRALPAK IC, 2×25 cm, 5 μm; mobile phase A: n-hexane (0.1% formic acid), mobile phase B: ethanol; flow rate: 18 mL/min; gradient: 18%; duration: 40 min; detection wavelength: 210 nm), to obtain compound 8a (13.8 mg, single configuration) and 8b (5.8 mg, single configuration) and a mixture of 8c and 8d (30 mg, a mixture of two optical isomers) as white solid, respectively.

    [0300] Compound 8a: .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.56-7.53 (m, 1H), 7.33-7.28 (s, 2H), 7.06-6.97 (m, 2H), 5.43 (t, J=6.9 Hz, 1H), 4.13 (d, J=6.9 Hz, 2H), 3.87 (s, 3H), 3.81-3.69 (m, 2H), 3.50-3.44 (m, 1H), 3.40-3.34 (m, 3H), 2.95-2.88 (m, 1H), 2.84 (s, 3H), 2.81-2.62 (m, 3H), 1.84-1.70 (m, 2H), 1.57-1.40 (m, 2H).

    [0301] LC-MS: m/z 567.15 [M+H].sup.+.

    [0302] Compound 8b: .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.56-7.53 (m, 1H), 7.34-7.28 (s, 2H), 7.07-6.97 (m, 2H), 5.47 (t, J=6.9 Hz, 1H), 4.22-4.12 (m, 2H), 3.87 (s, 3H), 3.81-3.69 (m, 2H), 3.54-3.44 (m, 1H), 3.40-3.34 (m, 3H), 2.99-2.83 (m, 2H), 2.80 (s, 3H), 2.81-2.62 (m, 2H), 1.86-1.72 (m, 2H), 1.57-1.42 (m, 2H).

    [0303] LC-MS: m/z 567.15 [M+H].sup.+.

    [0304] The mixture of compound 8c and 8d (30 mg) was separated by preparative column chromatography (column type: CHIRALPAK IE, 2×25 cm, 5 μm; mobile phase A: n-hexane (0.1% formic acid), mobile phase B: ethanol; flow rate: 20 mL/min; gradient: 20%; duration: 15 min; detection wavelength: 210 nm), to obtain compound 8c (10.2 mg, retention time: 8.55 min, single configuration) and compound 8d (8.3 mg, retention time: 10.10 min, single configuration) as a white solid, respectively.

    [0305] The NMR and LC/MS data of compound 8c were completely consistent with those of compound 8a.

    [0306] The NMR and LC/MS data of compound 8d were completely consistent with those of compound 8b.

    [0307] Chiral analysis: Column type: CHIRALPAK IC, 2×25 cm, 5 μm; mobile phase A: n-hexane (0.1% formic acid), mobile phase B: ethanol; flow rate: 1 mL/min; gradient: 30%; detection wavelength: 210 nm, retention time: 8a: 5.83 min; 8b: 7.38 min; 8c: 10.09 min; 8d: 10.09 min.

    Step 8: Confirmation of the Configuration of Compound 8c

    [0308] About 10 mg of compound 8c isolated in Step 7 was dissolved in 1 mL of MeOH solvent, and the solvent was naturally evaporated at room temperature to obtain a rod-shaped single crystal. The single crystal was analyzed by X-ray diffraction. The X-ray diffraction pattern (XRD) is shown in FIG. 1. The simulated and test XRD patterns of the single crystal were overlaped and compared in FIG. 1. Between the simulated and test XRD patterns, the main peaks are well matched.

    [0309] X-ray diffraction analysis parameters are as follows:

    [0310] Instrument model: Rigaku XtaLAB PRO 007HF (Mo)

    [0311] Ray: Mo Kα radiation (λ=0.71073 Å)

    [0312] Temperature: 180 K

    [0313] Scanning method: 2θ/θ

    [0314] Scanning range: 3.000°˜40.006°

    [0315] The crystal structure data is summarized in Table 1 below.

    TABLE-US-00002 TABLE 1 Crystal data and structure data of compound 8c Empirical formula C.sub.29H.sub.30N.sub.2O.sub.8S Molecular weight 566.61 Temperature/K 180.00 (10) Crystal system Orthogonal Space group P2.sub.12.sub.12.sub.1 a/Å 5.83670 (10) b/Å 17.8465 (3) c/Å 25.7535 (4) α/° 90 β/° 90 γ/° 90 volume/Å.sup.3 2682.60 (8) Z 4 ρ.sub.calc g/cm.sup.3 1.403 μ/mm.sup.−1 0.176 F(000) 1192.0 Crystal size/mm.sup.3 0.51 × 0.07 × 0.03 Radiation Mo Kα (λ = 0.71073 Å) Data collection range of 2θ/° 4.564 to 54.97 Index range −7 ≤ h ≤ 7, −23 ≤ k ≤ 23, −33 ≤ l ≤ 33 Diffraction points collected 62490 Independent diffraction spot 6172 [R.sub.int = 0.0397, R.sub.sigma = 0.0181] Data/limits/parameters 6172/1/366 Goodness of fit on F.sup.2 1.061 Final R index [I ≥ 2 σ (I)] R.sub.1 = 0.0323, wR.sub.2 = 0.0853 Final R index [all data] R.sub.1 = 0.0338, wR.sub.2 = 0.0862 Maximum difference peak/ 0.29/−0.15 hole/e Å.sup.−3 Flack parameter 0.014 (14)

    [0316] The CrysAlisPro program was used for data reduction and empirical absorption correction. The SHELXT program was used to analyze the structure through a dual-space algorithm. All non-hydrogen atoms can be located directly from the differential Fourier diagram. The skeleton hydrogen atoms were geometrically placed and a riding model was used to confine them to the parent atoms. The SHELXL program was used to complete the refinement of the final structure by using the full matrix technique to minimize the sum of the square deviations of F.sup.2.

    [0317] The fine single crystal structure is shown in FIG. 2 and FIG. 3. The chemical formula unit of the single crystal is C.sub.32H.sub.42N.sub.2O.sub.4S, which crystallizes in an orthogonal form in the P2.sub.12.sub.12.sub.1 space group. Each asymmetric unit has one molecule, which shows a cis conformation, and the chiral carbon is shown as “S”. Each unit crystal cell has four molecules. The hydrogen bond of O5-H5 . . . O4 was found through symmetrical operation.

    [0318] From the above analysis, it can be determined that the configurational formula of compound 8c is as follows:

    ##STR00068##

    Example 9: Preparation of (1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-N,3′-dimethyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxyamide (9)

    [0319] ##STR00069##

    [0320] Compound 8c (30 mg, 0.035 mmol) was dissolved in DMF (2 mL) at room temperature. Methylamine hydrochloride (7.2 mg, 0.107 mmol), HATU (20.0 mg, 0.053 mmol) and DIPEA (23.1 mg, 0.178 mmol) were added to the reaction solution. The mixture was stirred at room temperature overnight, and quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (7 mg, 32.7%) as a white solid.

    [0321] .sup.1H-NMR (CD.sub.3OD) δ: 7.98 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.8 Hz, 1H); 7.34-7.28 (m, 2H), 7.06-8.97 (m, 2H), 5.44-5.39 (m, 1H), 4.12-4.09 (m, 2H), 3.87 (s, 3H), 3.80-3.67 (m, 2H), 3.51-3.42 (m, 1H), 3.40-3.37 (m, 3H), 2.96-2.90 (m, 1H), 2.82 (s, 3H), 2.77 (s, 3H), 2.71-2.54 (m, 2H), 1.82-1.71 (m, 2H), 1.57-1.37 (m, 2H).

    [0322] LC-MS: m/z 580.20 [M+H].sup.+.

    Example 10: Preparation of ((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-meth yl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)glycine (10)

    [0323] ##STR00070##

    Step 1: Preparation of methyl ((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)glycinate (10-1)

    [0324] Compound 8c (30 mg, 0.053 mmol) was dissolved in DMF (2 mL) at room temperature. Methyl glycinate hydrochloride (9.4 mg, 0.106 mmol), HATU (30.2 mg, 0.079 mmol) and triethylamine (21.4 mg, 0.212 mmol) were added to the reaction solution. The mixture was stirred at room temperature overnight, and quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (5 mg, 15%) as a white solid.

    [0325] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.5 Hz, 1H), 7.34-7.28 (m, 2H), 7.06-6.97 (m, 2H), 5.44-5.40 (m, 1H), 4.13-4.10 (m, 2H), 4.00-3.98 (m, 2H), 3.88 (s, 1H), 3.80-3.67 (m, 5H), 3.51-3.42 (m, 2H), 3.40-3.35 (m, 2H), 3.01-2.94 (m, 1H), 2.89-2.83 (m, 4H), 2.74-2.58 (m, 2H), 1.81-1.72 (m, 2H), 1.57-1.41 (m, 1H).

    [0326] LC-MS: m/z 638.30 [M+H].sup.+.

    Step 2: Preparation of ((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)glycine (10)

    [0327] Compound 10-1 (30 mg, 0.047 mmol) was dissolved in methanol/water mixed solution (1/1, 2 mL) at room temperature. Lithium hydroxide (4.0 mg, 0.094 mmol) was added to the reaction solution. The reaction mixture was reacted at room temperature for 2 hours and then added with diluted hydrochloric acid until the pH of the reaction solution reached 2. The organic solvent was removed under reduced pressure, and the system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=60% to 70%) to obtain the title compound (11 mg, 61.2%) as a white solid.

    [0328] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.6 Hz, 1H), 7.54 (dd, J=1.2, 5.7 Hz, 1H), 7.33-7.29 (m, 2H), 7.03 (td, J=0.6, 5.4 Hz, 1H), 6.98 (dd, J=0.6, 6.3 Hz, 1H), 5.44-5.40 (m, 1H), 4.17-4.06 (m, 2H), 3.96 (s, 3H), 3.88 (s, 3H), 3.79-3.67 (m, 2H), 3.50-3.43 (m, 2H), 3.39-3.35 (m, 2H), 3.00-2.96 (m, 1H), 2.88-2.79 (m, 4H), 2.73-2.67 (m, 2H), 2.65-2.59 (m, 2H).

    [0329] LC-MS: m/z 624.20 [M+H].sup.+.

    Example 11: Preparation of 2-((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carboxyamido)-2-methylpropionic Acid (11)

    [0330] ##STR00071##

    [0331] Compound 8c (20 mg, 0.035 mmol) was dissolved in DMF (2 mL) at room temperature. HATU (20.1 mg, 0.053 mmol) and triethylamine (10.7 mg, 0.106 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 1 hour. Methyl 2-aminoisobutyrate hydrochloride (7.3 mg, 0.071 mmol) was added and then stirred at room temperature overnight. After completion of the reaction, the reaction was quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL), and the organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (3.6 mg, 15.3%) as a white solid.

    [0332] .sup.1H-NMR (CD.sub.3OD) δ: 7.97 (d, J=0.9 Hz, 1H), 7.52 (dd, J=1.5, 7.5 Hz, 1H), 7.33-7.25 (m, 2H), 7.05-6.96 (m, 2H), 5.41-5.37 (m, 1H), 4.14-4.00 (m, 1H), 3.86 (s, 3H), 3.79-3.67 (m, 2H), 3.48-3.43 (m, 1H), 3.37-3.32 (m, 3H), 3.02-2.80 (m, 4H), 2.68-2.54 (m, 2H), 1.80-1.70 (m, 2H), 1.53-1.31 (m, 8H).

    [0333] LC-MS: m/z 652.40 [M+H].sup.+.

    Example 12: Preparation of (1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-N,N,3′-trimethyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carboxamide (12)

    [0334] ##STR00072##

    [0335] Compound 8c (30 mg, 0.053 mmol) was dissolved in DMF (2 mL) at room temperature. Dimethylamine hydrochloride (14.3 mg, 0.176 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (8.0 mg, 0.042 mmol), 1-hydroxybenzotriazole (HOBt) (6.0 mg, 0.044 mmol) and DIPEA (36.1 mg, 0.279 mmol) were added to the reaction solution. The reaction mixture was stirred at room temperature overnight, and quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL), and the organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (7.6 mg, 35.1%) as a white solid.

    [0336] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.5 Hz, 1H), 7.30-7.28 (m, 2H), 7.06-6.97 (m, 2H), 5.44-5.40 (m, 1H), 4.16-4.09 (m, 2H), 3.86 (s, 3H), 3.80-3.59 (m, 3H), 3.51-3.44 (m, 1H), 3.40-3.37 (m, 3H), 3.23 (dd, J=1.2, 14.7 Hz, 1H), 3.03 (s, 3H), 2.98 (s, 3H), 2.92-2.88 (m, 1H), 2.84 (s, 3H), 2.06-2.03 (m, 1H), 1.83-1.71 (m, 2H), 1.57-1.43 (m, 2H).

    [0337] LC-MS: m/z 594.30 [M+H].sup.+.

    Example 13: Preparation of ((1R,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclohexane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)-L-alanine (13)

    [0338] ##STR00073##

    [0339] Compound 8c (20 mg, 0.035 mmol) was dissolved in DMF (2 mL) at room temperature. HATU (20.1 mg, 0.053 mmol) and DIPEA (10.7 mg, 0.106 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 1 hour, and then added with L-alanine (7.3 mg, 0.071 mmol) and stirred overnight at room temperature. After completion of the reaction, the reaction was quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (3.6 mg, 15.3%) as a white solid.

    [0340] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.54 (dd, J=1.8, 7.5 Hz, 1H), 7.33-7.28 (m, 2H), 7.06-6.97 (m, 2H), 5.44-5.40 (m, 1H), 4.43 (q, J=7.5 Hz, 1H), 4.13-4.11 (m, 2H), 3.87 (s, 3H), 3.80-3.68 (m, 2H), 3.50-3.36 (m, 3H), 3.00-2.94 (m, 1H), 2.88-2.79 (m, 4H), 2.73-2.57 (m, 2H), 1.84-1.72 (m 2H), 1.57-1.38 (m, 5H).

    [0341] LC-MS: m/z 638.35 [M+H].sup.+.

    Example 14: Preparation of ((1S,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)-D-alanine (14)

    [0342] ##STR00074##

    [0343] Compound 8c (20 mg, 0.035 mmol) was dissolved in DMF (2 mL) at room temperature. HATU (26.8 mg, 0.071 mmol) and DIPEA (18.3 mg, 0.14 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 1 hour, and then added with D-alanine (6.3 mg, 0.071 mmol) and stirred overnight at room temperature. After completion of the reaction, the reaction was quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (5 mg, 21.7%) as a white solid.

    [0344] .sup.1H-NMR (CD.sub.3OD) δ: 7.98 (s, 1H), 7.53 (dd, J=1.8, 7.5 Hz, 1H), 7.33-7.28 (m, 1H), 7.05-6.96 (m, 2H), 5.41 (dd, J=4.8, 7.2 Hz, 1H), 4.44 (q, J=7.2 Hz, 1H), 4.17-4.02 (m, 2H), 3.87-3.67 (m, 2H), 3.49-3.36 (m, 3H), 2.96 (t, J=9 Hz, 1H), 2.88-2.78 (m, 4H), 2.73-2.56 (m, 2H), 1.82-1.71 (m, 2H), 1.55-1.40 (m, 5H).

    [0345] LC-MS: m/z 638.35 [M+H].sup.+.

    Example 14-1: Preparation of Methyl ((1S,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-meth yl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)-D-alaninate

    [0346] ##STR00075##

    [0347] Compound 8c (20 mg, 0.035 mmol) was dissolved in DMF (2 mL) at room temperature. HATU (26.8 mg, 0.071 mmol) and DIPEA (18.3 mg, 0.14 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 1 hour, and then added with methyl D-alaninate (9.9 mg, 0.071 mmol) and stirred overnight at room temperature. After completion of the reaction, the reaction was quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=30% to 50%) to obtain the title compound (6.7 mg, 29.1%) as a white solid.

    [0348] .sup.1H-NMR (CD.sub.3OD) 7.99 (s, 1H), 7.55 (dd, J=1.8, 7.5 Hz, 1H), 7.34-7.28 (m, 2H), 7.06-6.97 (m, 2H), 5.44-5.39 (m, 1H), 4.45 (q, J=7.2 Hz, 1H), 4.12-4.03 (m, 2H), 3.88 (s, 3H), 3.75 (s, 3H), 3.80-3.67 (m, 2H), 3.48-3.36 (m, 4H), 3.03-2.93 (m, 1H), 2.87-2.81 (m, 1H), 2.83 (s, 3H), 2.73-2.56 (m, 2H), 1.82-1.72 (m, 2H), 1.57-1.45 (m, 2H), 1.42 (d, J=7.5 Hz, 3H).

    [0349] LC-MS: m/z 652.25 [M+H].sup.+.

    Example 15: Preparation of N-((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)-N-methylglycine (15)

    [0350] ##STR00076##

    Step 1: Preparation of N-((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)-N-methylglycine (15-1)

    [0351] Compound 8c (30 mg, 0.053 mmol) was dissolved in DMF (2 mL) at room temperature. Methyl sarcosinate hydrochloride (10.9 mg, 0.106 mmol), HATU (30.2 mg, 0.079 mmol) and triethylamine (21.4 mg, 0.212 mmol) were added to the reaction solution. The reaction mixture was stirred at room temperature overnight, and then quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 50%) to obtain the title compound (8.0 mg, 23.1%) as a white solid.

    [0352] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.55 (dd, J=1.5, 7.5 Hz, 1H), 7.34-7.29 (m, 2H), 7.06-8.97 (m, 2H), 5.45-5.40 (m, 1H), 4.21-4.11 (m, 4H), 3.89 (s, 3H), 3.80-3.66 (m, 6H), 3.56-3.46 (m, 2H), 3.10 (s, 3H), 3.02-2.96 (m, 2H), 2.92-2.83 (m, 4H), 2.79-2.63 (m, 2H), 1.84-1.72 (m, 2H), 1.55-1.40 (m, 2H).

    [0353] LC-MS: m/z 652.35 [M+H].sup.+.

    Step 2: Preparation of N-((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)-N-methylglycine (15)

    [0354] Compound 15-1 (8.0 mg, 0.014 mmol) was dissolved in methanol/water mixed solution (1/1, 1 mL) at room temperature. Lithium hydroxide (0.7 mg, 0.028 mmol) was added to the reaction solution. The reaction mixture was stirred at room temperature for 2 hours, and then added with diluted hydrochloric acid until the pH of the reaction solution reached 2. The organic solvent was removed under reduced pressure, and the system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (3.4 mg, 42.1%) as a white solid.

    [0355] .sup.1H-NMR (CD.sub.3OD) δ: 7.99-7.98 (m, 1H), 7.55 (dd, J=1.2, 7.8 Hz, 1H), 7.34-7.28 (m, 2H), 7.06-6.97 (m, 2H), 5.44-5.40 (m, 1H), 4.13-4.04 (m, 4H), 3.87 (s, 3H), 3.80-3.70 (m, 3H), 3.57-3.45 (m, 2H), 3.40-3.37 (m, 1H), 3.09-3.08 (m, 2H), 3.04-2.97 (m, 2H), 2.93-2.89 (m, 1H), 2.87-2.83 (m, 3H), 2.78-2.63 (m, 2H), 1.83-1.72 (m, 2H), 1.58-1.40 (m, 2H).

    [0356] LC-MS: m/z 638.40 [M+H].sup.+.

    Example 16: Preparation of 2-(((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carbonyl)oxy)acetic Acid (16)

    [0357] ##STR00077##

    [0358] Compound 8c (20 mg, 0.035 mmol) was dissolved in DMF (2 mL) at room temperature. HATU (26.8 mg, 0.071 mmol) and DIPEA (18.3 mg, 0.14 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 1 hour, and then added with glycolic acid (5.4 mg, 0.071 mmol) and stirred at room temperature overnight. After completion of the reaction, the reaction was quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=60% to 70%) to obtain the title compound (5.0 mg, 22.7%) as a white solid.

    [0359] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.56-7.54 (m, 1H), 7.34-7.29 (m, 2H), 7.06-6.97 (m, 2H), 5.45-5.41 (m, 1H), 4.65 (m, 2H), 4.14-4.12 (m, 2H), 3.88 (s, 3H), 3.88-3.70 (m, 3H), 3.62-3.34 (m, 5H), 3.27-3.20 (m, 1H), 3.11-3.09 (m, 1H), 3.02-2.96 (m, 1H), 2.90-2.71 (m, 7H), 1.84-1.71 (m, 2H), 1.46-1.31 (m, 2H).

    [0360] LC-MS: m/z 625.25 [M+H].sup.+.

    Example 17: Preparation of ((1s,3S)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carboxamide (17)

    [0361] ##STR00078##

    [0362] Compound 8c (20 mg, 0.035 mmol) was dissolved in THF (2 mL) at room temperature. Triethylamine (10.7 mg, 0.106 mmol) and methyl chloroformate (6.7 mg, 0.07 mmol) were added to the reaction solution at 0° C. successively. The mixture was stirred at room temperature for 1 hour, and then added with ammonia (7.42 mg, 0.212 mmol) and stirred at room temperature overnight. After completion of the reaction, the reaction was quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=60% to 70%) to obtain the title compound (6 mg, 30.1%) as a white solid.

    [0363] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.56-7.53 (m, 1H), 7.33-7.29 (m, 2H), 7.06-6.97 (m, 2H), 5.44-5.40 (m, 1H), 4.14-4.11 (m, 2H), 3.88 (s, 3H), 3.79-3.69 (m, 2H), 3.51-3.33 (m, 4H), 2.97-2.90 (m, 1H), 2.83-2.74 (m, 4H), 2.70-2.58 (m, 2H), 1.83-1.72 (m, 2H), 1.56-1.42 (m, 2H).

    [0364] LC-MS: m/z 566.15 [M+H].sup.+.

    Example 18: Preparation of (1s,3S)—N-(2-hydroxyethyl)-7′-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl) oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[cyclobutane-1,5′-thieno[2,3-b]pyridine]-3-carboxamide (18)

    [0365] ##STR00079##

    [0366] Compound 8c (30 mg, 0.053 mmol) was dissolved in DMF (2 mL) at room temperature. Ethanolamine (4.3 mg, 0.071 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (11.5 mg, 0.06 mmol), HOBt (8.1 mg, 0.06 mmol) and DIPEA (20.6 mg, 0.159 mmol) were added to the reaction solution. The reaction mixture was stirred at room temperature overnight, and quenched by adding water (5 mL). The reaction system was extracted with ethyl acetate (3×10 mL). The organic phases were combined and then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate=40% to 70%) to obtain the title compound (5.9 mg, 26.3%) as a white solid.

    [0367] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (s, 1H), 7.56-7.54 (m, 1H), 7.34-7.29 (m, 2H), 7.06-6.97 (m, 2H), 5.45-5.40 (m, 1H), 4.18-4.10 (m, 2H), 3.88 (s, 3H), 3.80-3.56 (m, 4H), 3.50-3.32 (m, 4H), 2.99-2.92 (m, 1H), 2.87-2.80 (m, 4H), 2.72-2.56 (m, 2H), 1.82-1.71 (m, 2H), 1.57-1.41 (m, 2H).

    [0368] LC-MS: m/z 610.55 [M+H].sup.+.

    Example 19: Preparation of tert-butyl (R)-2′-bromo-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-meth yl-4′,6′-dione-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxylate (19)

    [0369] ##STR00080## ##STR00081##

    Step 1: Preparation of 2-methoxyphenyl oxirane (19-1)

    [0370] Sodium hydride (35.3 g, 0.882 mol) was dissolved in dry dimethyl sulfoxide (800 mL) at room temperature. The mixed solution was cooled to 0° C., and then added with trimethylsulfoxide iodide (194 g, 0.882 mol). After stirring for 2 hours at 0° C., o-methoxybenzaldehyde (100 g, 0.735 mol) was slowly added. The mixture was stirred at room temperature for 3 hours, and then quenched by adding water (3000 mL). The reaction mixture was extracted with ethyl acetate (3×3000 mL). The organic phases were combined and then washed with saturated brine (2000 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure to obtain the crude title compound as a pale yellow oil (105 g). The crude product was used in the next step directly without purification.

    Step 2: Preparation of 2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethan-1-ol (19-2)

    [0371] Ferric chloride (16.9 g, 0.105 mol) was added to 4-tetrahydropyranol (204 g, 1.40 mol) at room temperature. The mixed solution was cooled to 0° C., and then slowly added with crude 2-methoxyphenyl oxirane (19-1) (105 g, 0.700 mol) dropwise. The reaction mixture was warmed to room temperature and stirred for 4 hours. The reaction was quenched by adding water (2000 mL). The reaction system was extracted with ethyl acetate (3×1000 mL). The organic phases were combined and then washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 50%). The title compound (30.0 mg, 17.0%, a mixture of two optical isomers) was obtained as a colorless oil.

    [0372] .sup.1H-NMR (CD.sub.3OD) δ: 7.43 (dd, J=1.8, 7.5 Hz, 1H), 7.32-7.27 (m, 1H), 7.00 (t, J=7.5 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 5.09 (dd, J=3.6, 8.4 Hz, 1H), 4.03-3.90 (m, 2H), 3.85 (s, 3H), 3.70 (dd, J=3.6, 11.4 Hz, 1H), 3.59-3.51 (m, 2H), 3.46-3.37 (m, 3H), 2.07-1.98 (m, 1H), 1.84-1.79 (m, 1H), 1.72-1.61 (m, 2H).

    [0373] LC-MS: m/z 275.15 [M+Na].sup.+.

    Step 3: Preparation of (R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl butyrate (19-3)

    [0374] Compound 19-2 (30.0 g, 0.12 mol), Candida antarctica enzyme (Beijing Cliscent Technology Co., Ltd.) (3.00 g) and vinyl n-butyrate (7.50 g, 0.07 mol) were dissolved in dry acetonitrile (150 mL) at room temperature. The reaction solution was stirred at room temperature for 2 hours and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=20% to 40%) to obtain the title compound (15.0 g, 39.1%) as a colorless oil.

    [0375] .sup.1H-NMR (CDCl.sub.3) δ: 7.50 (d, J=7.2 Hz, 1H), 7.33-7.27 (m, 1H), 7.10-6.98 (m, 1H), 6.92-6.87 (m, 1H), 5.23-5.04 (m, 1H), 4.21-4.13 (m, 2H), 4.02-3.85 (m, 5H), 3.51-3.40 (m, 3H), 2.38-2.30 (m, 2H), 1.98-1.66 (m, 6H), 1.02-0.95 (m, 3H).

    [0376] LC-MS: m/z 345.18 [M+Na].sup.+.

    Step 4: Preparation of (R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethan-1-ol (19-4)

    [0377] Compound 19-3 (15.0 g, 0.05 mol) and sodium hydroxide (3.10 g, 0.08 mol) were dissolved in a mixed solution of methanol (30 mL) and water (30 mL) at room temperature. The reaction solution was stirred at room temperature for 2 hours. The organic phase was concentrated under reduced pressure and then filtered to obtain the title compound (10.0 g, 85.2%) as a white solid.

    [0378] .sup.1H-NMR (CD.sub.3OD) δ: 7.43 (dd, J=1.8, 7.5 Hz, 1H), 7.32-7.27 (m, 1H), 7.00 (t, J=7.5 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 5.09 (dd, J=3.6, 8.4 Hz, 1H), 4.03-3.90 (m, 2H), 3.85 (s, 3H), 3.70 (dd, J=3.6, 11.4 Hz, 1H), 3.59-3.51 (m, 2H), 3.46-3.37 (m, 3H), 2.07-1.98 (m, 1H), 1.84-1.79 (m, 1H), 1.72-1.61 (m, 2H).

    [0379] LC-MS: m/z 275.15 [M+Na].sup.+.

    Step 5: Preparation of 5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (19-5)

    [0380] Ethyl 2-amino-methylthiophene-3-carboxylate (20.0 g, 0.108 mol) was dissolved in water (400 mL) at room temperature, and then added with potassium hydroxide solid (12.1 g, 0.216 mol). The reaction solution was heated to 100° C. and refluxed for 6 hours. The resulting solution was cooled to 0° C., and then slowly added with diphosgene (21.2 g, 0.108 mol) dropwise. After the addition, the reaction mixture was stirred for 3 hours at room temperature. A solid was gradually precipitated out during the reaction. After completion of the reaction, the resulted solid was filtered, washed with water to neutrality, and then washed with petroleum ether (200 mL). The solid was dried under reduced pressure to obtain the title compound (7.00 g, 35.3%) as a brown solid.

    [0381] LC-MS: m/z 184.25 [M+H].sup.+.

    Step 6: Preparation of (R)-1-(2-(2-methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (19-6)

    [0382] Compound 19-5 (5.00 g, 27.3 mmol) was dissolved in anhydrous tetrahydrofuran (150 mL) under nitrogen protection at room temperature, and then added with compound 19-4 (13.77 g, 54.6 mmol) and triphenylphosphine (14.3 g, 54.6 mmol) successively. The reaction solution was cooled to 0° C., and then slowly added with diisopropyl azodicarboxylate (11.0 g, 54.6 mmol) dropwise. After the addition, the reaction mixture was warmed to room temperature and stirred for 16 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 50%) to obtain the title compound (3.50 g, 30.6%) as a white solid.

    [0383] LC-MS: m/z 418.15 [M+H].sup.+.

    Step 7: Preparation of (R)-6-bromo-1-(2-(2-methoxyphenyl-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2H-thieno[2,3-d][1,3]oxazine-2,4(1H)-dione (19-7)

    [0384] Compound 19-6 (3.50 g, 8.37 mmol) was dissolved in chloroform (100 mL) at room temperature. N-bromosuccinimide (1.49 g, 8.37 mmol) was added to the solution. The reaction solution was stirred at room temperature for 3 hours, and then quenched by adding 5% aqueous solution of sodium thiosulfate (10 mL). The solution was diluted with dichloromethane (200 mL) and water (100 mL). After separation of the organic phase, the aqueous phase was extracted with dichloromethane (3×100 mL), and the combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 40%) to obtain the title compound (2.50 g, 60.2%) as a pale yellow solid.

    [0385] .sup.1H-NMR (d6-DMSO) δ: 7.46 (dd, J=1.8, 7.5 Hz, 1H), 7.32 (dt, J=1.85, 8.1 Hz, 1H), 7.06-6.99 (m, 2H), 5.24 (dd, J=4.8, 8.4 Hz, 1H), 4.01-3.97 (m, 2H), 3.79 (s, 3H), 3.67-3.56 (m, 2H), 3.46-3.48 (m, 1H), 3.29-3.21 (m, 2H), 2.52-2.50 (m, 1H), 1.71-1.63 (m, 2H), 1.40-1.18 (m, 2H).

    [0386] LC-MS: m/z 518.10 [M+Na].sup.+.

    Step 8: Preparation of tert-butyl (R)-2′-bromo-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-4′,6′-dione-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxylate (19)

    [0387] Methyl N-Boc-4-piperidine-carboxylate (243.3 mg, 1.21 mmol) was dissolved in dry THF (5 mL) under nitrogen atmosphere, and then added with LDA (1.29 mmol, 2 mol/L) at −78° C. The mixture was stirred at this temperature for 1 hour, and then added with compound 19-7 (200 mg, 0.40 mmol). The reaction solution was warmed to room temperature and stirred for 1 hour. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 40%) to obtain the title compound (243.0 mg, 90.5%) as a white solid.

    [0388] .sup.1H-NMR (CD.sub.3OD) 7.49 (dd, J=3, 7.5 Hz, 1H), 7.32 (td, J=1.8, 7.5 Hz, 1H), 7.05-6.97 (m, 2H), 5.38 (t, J=6.6 Hz, 1H), 4.09-4.06 (m, 2H), 3.86 (s, 3H), 3.86-3.73 (m, 2H), 3.72-3.60 (m, 2H), 3.50-3.35 (m, 5H), 2.33 (s, 3H), 2.03-1.70 (m, 6H), 1.56-1.39 (m, 11H).

    [0389] LC-MS: m/z 686.95 [M+H].sup.+.

    Example 20: Preparation of tert-butyl (R)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dione-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxylate (20)

    [0390] ##STR00082##

    [0391] 2-(Tributylstannyl)oxazole (43.2 mg, 0.12 mmol) and bis(triphenylphosphorus)palladium dichloride (0.03 mmol, 21.2 mg) were added to a solution (3 mL) of compound 19 in dry toluene (40.0 mg, 0.06 mmol) under nitrogen atmosphere at room temperature. The reaction solution was heated and refluxed overnight. After completion of the reaction, the reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=10% to 40%) to obtain the title compound (38.1 mg, 95.6%) as a white solid.

    [0392] .sup.1H-NMR (CD.sub.3OD) 7.99 (s, 1H), 7.53 (dd, J=1.5, 7.5 Hz, 1H), 7.35-7.29 (m, 2H), 7.06-6.98 (m, 2H), 5.45 (t, J=6.3 Hz, 1H), 4.15 (m, 2H), 3.88 (s, 3H), 3.82-3.76 (m, 2H), 3.72-3.61 (m, 2H), 3.60-3.53 (m, 1H), 3.51-3.43 (m, 2H), 3.40-3.36 (m, 2H), 2.00-1.72 (m, 6H), 1.56-1.40 (m, 11H).

    [0393] LC-MS: m/z 652.25 [M+H].sup.+.

    Example 21: Preparation of (R)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-4′,6′(7′H)-dione (21)

    [0394] ##STR00083##

    [0395] Compound 20 (35.0 mg, 0.054 mmol) was added to a solution of hydrochloric acid in 1,4-dioxane (4 mol/L, 2 mL) at room temperature. After stirring at room temperature for 1 hour, the reaction solution was concentrated under reduced pressure to remove the hydrochloric acid and organic solvent. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=50% to 60%) to obtain compound 21 (28.1 mg, 90.2%) as a white solid.

    [0396] .sup.1H-NMR (CD.sub.3OD) 8.00 (s, 1H), 7.53 (dd, J=1.5, 7.5 Hz, 1H), 7.35-7.29 (m, 2H), 7.06-6.98 (m, 2H), 5.46 (t, J=6.3 Hz, 1H), 4.12-4.11 (m, 2H), 3.88 (s, 3H), 3.83-3.76 (m, 2H), 3.68-3.35 (m, 3H), 3.22-3.08 (m, 4H), 2.77 (m, 3H), 2.21-2.06 (m, 3H), 2.03-1.93 (m, 1H), 1.89-1.83 (m, 1H), 1.78-1.73 (m, 1H), 1.56-1.39 (m, 2H).

    [0397] LC-MS: m/z 552.30 [M+H].sup.+.

    Example 22: Preparation of methyl (R)-2-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-yl)carboxylate (22)

    [0398] ##STR00084##

    [0399] Methyl bromoacetate (6.1 mg, 0.036 mmol) and potassium carbonate (10.0 mg 0.073 mmol) were added to a solution (3 mL) of compound 21 in dry acetonitrile (20.0 mg, 0.036 mmol) under nitrogen atmosphere at room temperature. The reaction solution was stirred for 2 hours at room temperature, and then quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=30% to 40%) to obtain compound 22 (15.3 mg, 62.9%) as a white solid.

    [0400] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.53 (dd, J=1.8, 7.5 Hz, 1H), 7.34-7.28 (m, 2H), 7.06-6.97 (m, 2H), 5.46-5.42 (m, 2H), 7.25 (q, J=4.8 Hz, 2H), 4.17-4.12 (m, 2H), 3.86 (s, 3H), 3.81-3.77 (m, 2H), 3.51-3.42 (m, 1H), 3.39-3.35 (m, 4H), 2.93-2.88 (m, 2H), 2.85-2.81 (m, 2H), 2.76 (s, 3H), 2.27-2.12 (m, 3H), 2.09-1.98 (m, 1H), 1.87-1.73 (m, 2H), 1.58-1.40 (m, 2H), 1.31 (t, J=7.2 Hz, 3H).

    [0401] LC-MS: m/z 638.20 [M+H].sup.+.

    Example 23: Preparation of (R)-2-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridin]-1-yl)acetic Acid (23)

    [0402] ##STR00085##

    [0403] Compound 22 (20.0 mg, 0.03 mmol) and lithium hydroxide (1.4 mg, 0.06 mmol) were dissolved in a methanol/water mixed solution (6 mL, methanol/water=1/1) under nitrogen atmosphere at room temperature. The reaction solution was stirred overnight at room temperature. After completion of the reaction, the organic solvent was removed under reduced pressure. The reaction solution was adjusted to pH 2 with dilute hydrochloric acid (pH=1). The system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=30% to 40%) to obtain compound 23 (8.5 mg, 43.3%) as a white solid.

    [0404] .sup.1H-NMR (CD.sub.3OD) 8.01 (d, J=0.6 Hz, 1H), 7.52 (dd, J=1.5, 7.5 Hz, 1H), 7.35-7.30 (m, 2H), 7.06-7.00 (m, 2H), 5.49-5.45 (m, 1H), 4.34-4.25 (m, 1H), 4.19-4.11 (m, 3H), 3.89 (s, 3H), 3.94-3.77 (m, 2H), 3.62-3.44 (m, 5H), 3.40-3.35 (m, 2H), 2.79 (s, 3H), 2.46-2.33 (m, 4H), 1.90-1.86 (m, 1H), 1.79-1.74 (m, 1H), 1.57-1.39 (m, 2H).

    [0405] LC-MS: m/z 610.20 [M+H].sup.+.

    Example 24: Preparation of (2-methoxy-2-oxoethyl) (R)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxylate (24)

    [0406] ##STR00086##

    [0407] P-nitrophenyl chloroformate (21.9 mg, 0.109 mmol) was added to a solution (3 mL) of 2-hydroxyethyl acetate (11.3 mg, 0.109 mmol) and pyridine (0.01 mL, 0.126 mmol) in dry dichloromethane at 0° C. under nitrogen atmosphere. The reaction solution was stirred at 0° C. for 2 hours, and then concentrated under reduced pressure to obtain a crude product. The crude product was added to a solution (3 mL) of compound 21 (30.0 mg, 0.054 mmol) and diisopropylethylamine (0.02 mL, 0.155 mmol) in dry dichloromethane. The reaction solution was stirred for 2 hours at room temperature, and then quenched by adding water. The system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=50% to 70%) to obtain compound 24 (7.8 mg, 20.8%) as a white solid.

    [0408] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.5, 7.5 Hz, 1H), 7.32-7.29 (m, 2H), 7.06-6.98 (m, 2H), 5.48-5.44 (m, 2H), 4.86 (s, 3H), 4.27 (q, J=7.2 Hz, 4.20-4.16 (m, 2H), 3.88 (s, 3H), 3.84-3.75 (m, 3H), 3.71-3.57 (m, 2H), 3.50-3.43 (m, 1H), 3.39-3.33 (m, 2H), 2.77 (s, 3H), 2.11-2.01 (m, 3H), 1.94-1.84 (m, 2H), 1.80-1.73 (m, 1H), 1.57-1.40 (m, 2H), 1.32 (t, J=6.9 Hz, 3H).

    [0409] LC-MS: m/z 682.25 [M+H].sup.+.

    Example 25: Preparation of (R)-2-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridin]-1-yl)-2-methylpropionic Acid (25)

    [0410] ##STR00087##

    Step 1: Preparation of methyl (R)-2-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridin]-1-yl)-2-methylpropanoate (25-1)

    [0411] Methyl 2-bromoisobutyrate (26.5 mg, 0.136 mmol) and potassium carbonate (15.0 mg 0.109 mmol) were added to a solution (3 mL) of compound 21 in dry acetonitrile (15.0 mg, 0.027 mmol) under nitrogen atmosphere at room temperature. The reaction solution was stirred at 80° C. for 16 hours, and then quenched by adding water. The reaction system was extracted with ethyl acetate (3×20 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=30% to 40%) to obtain the title compound (8.5 mg, 46.0%) as a white solid.

    [0412] .sup.1H-NMR (CD.sub.3OD) δ: 7.98 (d, J=0.9 Hz, 1H), 7.53 (dd, J=1.8, 7.5 Hz, 1H), 7.34-7.28 (m, 2H), 7.05-8.97 (m, 2H), 5.46-5.42 (t, J=6.6 Hz, 1H), 4.25 (q, J=6.9 Hz, 2H), 4.18-4.10 (m, 2H), 3.87 (s, 3H), 3.83-3.76 (m, 2H), 3.49-3.38 (m, 3H), 2.93-2.83 (m, 4H), 2.76 (s, 3H), 2.24-1.96 (m, 4H), 1.87-1.73 (m, 2H), 1.57-1.42 (m, 2H), 1.38-1.33 (m, 9H).

    [0413] LC-MS: m/z 666.50 [M+H].sup.+.

    Step 2: Preparation of (R)-2-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridin]-1-yl)-2-methylpropionic Acid (Compound 25)

    [0414] Compound 25-1 (13.0 mg, 0.019 mmol) was added to a solution of hydrochloric acid in 1,4-dioxane (3 mL) under nitrogen atmosphere at room temperature. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to remove the hydrochloric acid and organic solvent. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=50% to 70%) to obtain compound 25 (3.0 mg, 25.1%) as a white solid.

    [0415] .sup.1H-NMR (CD.sub.3OD) δ: 8.01 (s, 1H), 7.52 (d, J=7.5 Hz, 1H), 7.36-7.31 (m, 2H), 7.05-6.98 (m, 2H), 5.48-5.44 (m, 1H), 4.34-4.18 (m, 2H), 3.87-3.80 (m, 5H), 3.52-3.45 (m, 1H), 3.42-37 (m, 5H), 3.27-3.10 (m, 1H), 2.79 (s, 3H), 2.48-2.03 (m, 4H), 1.93-1.89 (m, 1H), 1.78-1.73 (m, 1H), 1.58-1.49 (m, 8H).

    [0416] LC-MS: m/z 638.20 [M+H].sup.+.

    Example 26: Preparation of methyl (R)-2-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridin]-1-yl)-2-oxoacetate (26)

    [0417] ##STR00088##

    [0418] Methyl 2-chloro-2-oxoacetate (5.0 mg, 0.041 mmol) was added to a solution (3 mL) of compound 21 (15.0 mg, 0.027 mmol) and triethylamine (5.5 mg, 0.054 mmol) in dry dichloromethane at 0° C. under nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, and then quenched by adding water. The reaction system was extracted with ethyl acetate (3×10 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=30% to 40%) to obtain compound 26 (8.5 mg, 46.0%) as a white solid.

    [0419] .sup.1H-NMR (CD.sub.3OD) δ: 8.00 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.5 Hz, 1H), 7.35-7.29 (m, 2H), 7.06-6.99 (m, 2H), 5.48-5.43 (m, 1H), 4.29-4.08 (m, 2H), 3.92-3.87 (m, 7H), 3.84-3.77 (m, 3H), 3.71-3.61 (m, 3H), 3.51-3.45 (m, 3H), 2.78 (s, 3H), 2.22-2.15 (m, 1H), 2.10-2.04 (m, 2H), 1.94-1.74 (m, 3H), 1.47-1.45 (m, 2H), 1.36-1.25 (m, 2H).

    [0420] LC-MS: m/z 638.20 [M+H].sup.+.

    Example 27: Preparation of (R)-2-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridin]-1-yl)-2-oxoacetic Acid (27)

    [0421] ##STR00089##

    [0422] Oxalyl chloride (5.2 mg, 0.041 mmol) was added to a solution (3 mL) of compound 21 (28.0 mg, 0.051 mmol) and triethylamine (10.3 mg, 0.102 mmol) in dry dichloromethane at −10° C. under nitrogen atmosphere. The reaction solution was stirred at −10° C. for 20 minutes, and then warmed to room temperature and further stirred for 1 hour. The reaction was quenched by adding water. The reaction system was extracted with ethyl acetate (3×10 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=70% to 80%) to obtain compound 27 (15.0 mg, 46.4%) as a white solid.

    [0423] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.5 Hz, 1H), 7.34-7.28 (m, 2H), 7.05-6.97 (m, 2H), 5.45 (t, J=6.6 Hz, 1H), 4.19-4.17 (m, 1H), 3.87 (s, 3H), 3.86-3.78 (m, 4H), 3.66-3.45 (m, 3H), 3.36-3.33 (m, 2H), 2.76 (s, 3H), 3.19-2.07 (m, 3H), 1.99-1.85 (m, 3H), 1.77-1.72 (m, 1H), 1.56-1.31 (m, 2H).

    [0424] LC-MS: m/z 624.40 [M+H].sup.+.

    Example 28: Preparation of methyl (R)-7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxylate (28)

    [0425] ##STR00090##

    [0426] The preparation method was the same as in Example 26, except that methyl chloroformate was used instead of methyl 2-chloro-2-oxoacetate to obtain compound 28.

    [0427] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.8 Hz, 1H), 7.35-7.27 (m, 2H), 7.06-6.98 (m, 2H), 5.45 (t, J=6.6 Hz, 1H), 4.49-4.16 (m, 1H), 3.88 (m, 3H), 3.83-3.76 (m, 3H), 3.73-3.70 (m, 4H), 3.57-3.36 (m, 5H), 2.77 (s, 3H), 2.09-1.99 (m, 3H), 1.88-1.83 (m, 2H), 1.77-1.73 (m, 1H), 1.56-1.40 (m, 2H).

    [0428] LC-MS: m/z 610.25 [M+H].sup.+.

    Example 29: Preparation of (R)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carbonyl)glycine (29)

    [0429] ##STR00091##

    Step 1: Preparation of tert-butyl (R)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carbonyl)glycinate (29-1)

    [0430] Triphosgene (8.0 mg, 0.027 mmol) was added to a solution (3 mL) of tert-butyl glycinate (14.2 mg, 0.108 mmol) and triethylamine (27.3 mg, 0.27 mmol) in dry dichloromethane at 0° C. under nitrogen atmosphere. The reaction solution was stirred at 0° C. for 3 hours, and then added with compound 21 (30.0 mg, 0.054 mmol). The reaction solution was warmed to room temperature and stirred overnight. The reaction was quenched by adding water and the reaction system was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=30% to 40%) to obtain compound 29-1 (24.1 mg, 65.2%) as a white solid.

    [0431] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.5, 7.5 Hz, 1H), 7.35-7.29 (m, 2H), 7.08-6.98 (m, 2H), 5.45 (t, J=6.6 Hz, 1H), 4.17-4.15 (m, 2H), 3.88 (s, 3H), 3.84-3.71 (m, 5H), 3.68-3.44 (m, 4H), 2.77 (s, 3H), 2.12-2.04 (m, 3H), 1.94-1.84 (m, 2H), 1.78-1.73 (m, 1H), 1.54-1.39 (m, 11H).

    [0432] LC-MS: m/z 708.60 [M+H].sup.+.

    Step 2: Preparation of (R)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carbonyl)glycine (29)

    [0433] Compound 29-1 (20.0 mg, 0.028 mmol) was dissolved in a solution of hydrochloric acid in 1,4-dioxane (3 mL) under nitrogen atmosphere at room temperature. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to remove the hydrochloric acid and organic solvent. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=50% to 70%) to obtain compound 29 (13.0 mg, 64.4%) as a white solid.

    [0434] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.5, 7.5 Hz, 1H), 7.35-7.29 (m, 2H), 7.08-6.98 (m, 2H), 5.47-5.43 (m, 1H), 4.18-4.15 (m, 2H), 3.89-3.88 (m, 4H), 3.84-3.71 (m, 4H), 3.68-3.44 (m, 4H), 2.77 (s, 3H), 2.12-2.04 (m, 3H), 1.94-1.84 (m, 2H), 1.78-1.73 (m, 1H), 1.54-1.39 (m, 2H).

    [0435] LC-MS: m/z 653.20 [M+H].sup.+.

    Example 30: Preparation of (R)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-sulfonic Acid (30)

    [0436] ##STR00092##

    [0437] Chlorosulfonic acid (8.5 mg, 0.073 mmol) was added to a solution (3 mL) of compound 21 (20.0 mg, 0.036 mmol) and diisopropyl ethyl amine (14.1 mg, 0.109 mmol) in dry dichloromethane at 0° C. under nitrogen atmosphere. The reaction solution was stirred at room temperature overnight, and then quenched by adding water. The reaction system was extracted with ethyl acetate (3×10 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=60% to 80%) to obtain compound 30 (5.1 mg, 21.8%) as a white solid.

    [0438] .sup.1H-NMR (CD.sub.3OD) δ: 8.00 (s, 1H), 7.52-7.51 (d, J=7.5 Hz, 1H), 7.35-7.30 (m, 2H), 7.05-6.98 (m, 2H), 5.48-5.43 (m, 1H), 4.30-4.12 (m, 2H), 3.88 (s, 3H), 3.84-3.79 (m, 3H), 3.62-3.46 (m, 5H), 3.41-3.34 (m, 3H), 2.77 (s, 3H), 2.37-2.26 (m, 2H), 2.15-2.06 (m, 2H), 1.90-1.86 (m, 1H), 1.78-1.74 (m, 1H), 1.57-1.42 (m, 2H).

    [0439] LC-MS: m/z 632.45 [M+H].sup.+.

    Example 31: Preparation of (R)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxamide (31)

    [0440] ##STR00093##

    [0441] Trimethylsilyl isocyanate (52.2 mg, 0.453 mmol) was added to a solution (3 mL) of compound 21 (25.0 mg, 0.045 mmol) and triethylamine (101.2 mg, 0.227 mmol) in dry dichloromethane at 0° C. under nitrogen atmosphere. The reaction solution was stirred at room temperature overnight, and then quenched by adding water. The reaction system was extracted with ethyl acetate (3×10 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=60% to 80%) to obtain compound 31 (9.1 mg, 32.5%) as a white solid.

    [0442] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.2, 5.7 Hz, 1H), 7.34-7.29 (m, 2H), 7.06-6.98 (m, 2H), 5.45 (t, J=7.8 Hz, 1H), 4.18-4.14 (m, 2H), 3.88 (s, 3H), 3.84-3.76 (m, 3H), 3.72-3.63 (m, 2H), 3.60-3.56 (m, 1H), 3.52-3.44 (m, 2H), 3.39-3.34 (m, 2H), 2.77 (s, 3H), 2.12-2.00 (m, 3H), 1.93-1.84 (m, 2H), 1.77-1.74 (m, 1H), 1.5-1.40 (m, 2H).

    [0443] LC-MS: m/z 595.20 [M+H].sup.+.

    Example 32: Preparation of (R)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-N,3′-dimethyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxamide (32)

    [0444] ##STR00094##

    [0445] The preparation method was the same as in Example 31, except that methylcarbamic chloride was used instead of trimethylsilyl isocyanate to obtain compound 32.

    [0446] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.6 Hz, 1H), 7.54 (dd, J=1.5, 7.5 Hz, 1H), 7.34-7.29 (m, 2H), 7.06-6.96 (m, 2H), 5.46-5.43 (m, 1H), 4.19-4.14 (m, 2H), 3.88 (s, 3H), 3.82-3.77 (m, 2H), 3.73-3.61 (m, 2H) 3.58-3.44 (m, 3H), 2.77 (s, 3H), 2.76 (s, 3H), 2.10-1.99 (m, 3H), 1.91-1.84 (m, 2H), 1.77-1.73 (m, 1H), 1.55-1.40 (m, 2H).

    [0447] LC-MS: m/z 609.20 [M+H].sup.+.

    Example 33: Preparation of (R)—N-(2-hydroxyethyl)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-carboxamide (33)

    [0448] ##STR00095##

    [0449] Triphosgene (8.0 mg, 0.027 mmol) was added to a solution (3 mL) of aminoethanol (6.6 mg, 0.108 mmol) and triethylamine (27.3 mg, 0.27 mmol) in dry dichloromethane at 0° C. under nitrogen atmosphere. The reaction solution was stirred at 0° C. for 3 hours, and then added with compound 21 (30.0 mg, 0.054 mmol). The reaction solution was warmed to room temperature and stirred overnight. The reaction was quenched by adding water and the system was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=60% to 70%) to obtain compound 33 (7.2 mg, 22.5%) as a white solid.

    [0450] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.5 Hz, 1H), 7.35-7.29 (m, 2H), 7.06-6.98 (m, 2H), 5.45 (t, J=7.8 Hz, 1H), 4.18-4.16 (m, 2H), 3.88 (s, 3H), 3.83-3.76 (m, 2H), 3.72-3.61 (m, 4H), 3.57-3.44 (m, 3H), 3.39-3.34 (m, 3H), 2.77 (s, 3H), 2.14-2.00 (m, 3H), 1.94-1.84 (m, 2H), 1.78-1.73 (m, 1H), 1.57-1.40 (m, 2H).

    [0451] LC-MS: m/z 639.20 [M+H].sup.+.

    Example 34: Preparation of diethyl (R)-(7′-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-3′-methyl-2′-(oxazol-2-yl)-4′,6′-dioxo-6′,7′-dihydro-4′H-spiro[piperidine-4,5′-thieno[2,3-d]pyridine]-1-yl)phosphate (34)

    [0452] ##STR00096##

    [0453] Diethyl chlorophosphate (4.7 mg, 0.027 mmol) was added to a solution (3 mL) of compound 21 (10.0 mg, 0.018 mmol) and triethylamine (3.7 mg, 0.036 mmol) in dry chloroform at 0° C. under nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, and was quenched by adding water. The reaction system was extracted with ethyl acetate (3×10 mL), and the combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated under reduced pressure. The residues were purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether=40% to 80%) to obtain compound 34 (4.5 mg, 30.8%) as a white solid.

    [0454] .sup.1H-NMR (CD.sub.3OD) δ: 7.99 (d, J=0.9 Hz, 1H), 7.54 (dd, J=1.8, 7.5 Hz, 1H), 7.34-7.29 (m, 2H), 7.06-6.98 (m, 2H), 5.45 (t, J=6.6 Hz, 1H), 4.17-4.14 (m, 1H), 4.12-4.02 (m, 4H), 3.89 (s, 3H), 3.83-3.78 (m, 3H), 3.50-3.34 (m, 4H), 2.77 (s, 3H), 2.06-1.97 (m, 3H), 1.88-1.84 (m, 2H), 1.78-1.74 (m, 1H), 1.56-1.42 (m, 2H), 1.39-1.31 (m, 6H).

    [0455] LC-MS: m/z 688.25 [M+H].sup.+.

    Biological Tests

    Test Example 1: Analysis of the In Vitro Inhibition of the Present Compound on Acetyl-CoA Carboxylase (ACC) Activity

    [0456] ADP-Glo™ Kinase Analysis Kit (Promega) is used for the in vitro analysis of the inhibitory effect of the present compound on ACC1 or ACC2. ADP-Glo™ Kinase Analysis is a kinase detection analysis of luminescence method. ADP is produced in the process of catalyzing the substrate by ACC. ADP is converted into ATP which is then converted into light by Ultra-Glo™ luciferase. ADP is quantified by detecting the light signal, thus the enzyme activity can be measured by quantification of the ADP amount produced during the enzyme reaction. The analysis is carried out in two steps: in the first step, after the kinase reaction, a portion of ADP-Glo™ reagent equal in volume to the kinase reaction system is added to terminate the reaction and consume the remaining ATP; in the second step, a kinase detection reagent is added, which not only converts ADP into ATP, but also uses a coupled luciferase/luciferin reaction to detect newly synthesized ATP.

    [0457] Test Methods:

    [0458] a. 10 mM compound stock solution (the present compound was dissolved in 100% DMSO to prepare the 10 mM stock solution) was diluted 50-fold to 200 μM with 100% DMSO, and then diluted in equal proportions (1:3) in a 96-well dilution plate (249944, Nunc). The gradient concentrations of the compound were 200 μM, 66.67 μM, 22.22 μM, 7.41 μM, 2.47 μM, 0.823 μM, 0.274 μM, 0.0914 μM, 0.0305 μM, 0.0102 μM, 0.00339 μM and 0.00113 μM.

    [0459] b. 4.5 μL ACC1 working solution (BPS bioscience) (2.22 nM) was added to each well of a 384-well test plate (6007290, Perkin Elmer).

    [0460] c. 0.5 μL of the serially diluted compound (prepared in step a) was added to each well of the 384-well test plate, which was centrifuged at 1000 rpm for 1 min and then incubated at room temperature for 15 min. The reaction system with compound GS-0976 at a final concentration of 0.1 μM was used as positive control; the reaction system with no compound but DMSO at a final concentration of 5% was used as negative control.

    [0461] d. In the 384-well test plate (prepared in step c), 5 μL of the substrate working solution was added to each well to start the enzymatic reaction. The plate was centrifuged at 1000 rpm for 1 min and then incubated at room temperature for 30 min. The final concentration of each component in the ACC1 reaction system: 1 nM ACC1, 10 μM acetyl-CoA, 30 mM NaHCO.sub.3, and 20 μM ATP. The final concentration of each component in the ACC2 reaction system: 1.1 nM ACC2, 20 μM acetyl-CoA, 20 mM NaHCO.sub.3, and 15 μM ATP. Final concentrations of test compounds: 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.123 μM, 0.0411 μM, 0.0137 μM, 0.00457 μM, 0.00152 μM, 0.00051 μM, 0.00017 μM and 0.000056 μM. The final concentration of DMSO was 5% (v/v).

    [0462] e. 10 μL of ADP-Glo reagent was added to each well of the 384-well test plate (prepared in step d), which was centrifuged at 1000 rpm for 1 min and then incubated at room temperature for 40 min.

    [0463] f. 20 μL of kinase detection reagent was added to each well of the 384-well test plate (prepared in step e), which was centrifuged at 1000 rpm for 1 min and then incubated at room temperature for 40 min.

    [0464] g. The chemiluminescence signal was detected with Perkin Elmer EnVision 2104.

    [0465] ACC1 working solution and substrate working solution were both prepared with 1× kinase reaction buffer. The buffer was consisting of 50 mM HEPES buffer, 2 mM magnesium chloride, 0.01% BRIJ-35, 2 mM potassium citrate, 50 μg/mL BSA and 2 mM DTT.

    [0466] The average value of the positive and negative control well and the standard deviation were calculated. The inhibition percentage was calculated according to the following formula: 100×[1−(compound−average value of positive control)/(average value of negative control-average value of positive control)].

    [0467] IC.sub.50 of the compound was obtained using GraphPad Prism 6 software according to the following nonlinear fitting formula:


    Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC.sub.50−X)×Hillslope)),

    [0468] wherein X is the logarithm of the compound concentration, Y is the inhibition rate; Top and Bottom are the Y values at the highest and lowest plateau of the curve; Hillslope is the Hill constant.

    [0469] The inhibitory activities of the present compound on ACC1 and ACC2 are shown in Table 2 below.

    [0470] In Table 2, A means the inhibitory activity of the compound with IC.sub.50<10 nM; B means 10 nM<IC.sub.50<100 nM; C means 100 nM<IC.sub.50<500 nM; D means IC.sub.50>500 nM.

    TABLE-US-00003 TABLE 2 The inhibitory IC.sub.50 values of the present compound on ACC1 and ACC2 Compound IC.sub.50 (nM) No. ACC1 ACC2  1 D  2 D  3 C  4 C  5 A  5x A  5y A  5a B  5b A A  5c A  5d A  5e B  6 C  7a D  7b C  8 A  8a B  8b C  8c A A  8d A A  9 A 10 A 10-1 A 11 A 12 A 13 A 14 A 14-1 A 15 A 15-1 A 16 A 17 A 18 A 20 B 21 A 22 B 23 A 24 B 25-1 B 25 B 26 A 27 A 28 A 29 A 30 A 31 A 32 A 33 A

    [0471] The present compound can inhibit the enzyme activity of ACC1 and ACC2.

    Test Example 2: In Vitro Inhibitory Effect of the Present Compound on Fatty Acid Synthesis in HepG2 Cells

    [0472] Human liver tumor cells (HepG2 cells) with strong fatty acid synthesis ability were selected. The sample to be tested was added and incubated for 1 hour. Isotope-labeled [.sup.14C]-sodium acetate was added to the cell culture medium and incubated for 5 hours. Then the cells were lysed, and the fatty acid components in the cell were extracted and separated. The relative content of the isotope integrated into the fatty acid was analyzed, so as to detect the inhibitory effect of the compound on fatty acid synthesis in HepG2 cells at different concentrations, and the IC.sub.50 value was calculated.

    [0473] Test Methods:

    [0474] a. HepG2 cells were purchased from the American type culture collection (ATCC) resource bank. Cells were cultured in DMEM (GIBCO) containing 10% fetal bovine serum (GIBCO), penicillin (100 units/mL) and streptomycin (100 μg/mL) (GIBCO), and then incubated in a 37° C. incubator containing 5% carbon dioxide, and passaged once every 2 to 3 days.

    [0475] b. On the first day, HepG2 cells were inoculated in a 24-well cell culture plate at 2×10.sup.5 per well, and were incubated in a 37° C. incubator containing 5% carbon dioxide.

    [0476] c. On the fourth day, the medium was changed to that containing the compound. The initial concentration of the compound was 3 μM or 0.3 μM, 3-fold dilution, 8 concentration gradients, and the final concentration of DMSO was 0.5% (v/v). The plate was incubated in a 37° C. incubator containing 5% carbon dioxide for 1 hour. GS-0976 was positive compound control; the reaction system with no compound but DMSO at a final concentration of 0.5% was negative control.

    [0477] d. 1 μCi [2-.sup.14C]-acetic acid was added to each well, and incubated for 5 hours in a 37° C. incubator containing 5% carbon dioxide.

    [0478] e. The medium was first transferred to 15 mL centrifuge tubes. 0.5 mL of 0.1 M NaOH was added to each well of cells, and the lysed cell suspension was added to the corresponding 15 mL centrifuge tubes. 1 mL of ethanol and 0.17 mL of 50% KOH were added to each tube, and incubated in a water bath at 90° C. for 1 hour.

    [0479] f. The sample were taken out and cooled to room temperature, then 5 mL petroleum ether was added to each tube, inverted several times, and centrifuged at 1000 rpm for 5 min. The upper organic phase was discarded and the aqueous phase was kept for fatty acid extraction. 1 mL of concentrated hydrochloric acid (its pH was ensured to be below 1) was added to each tube.

    [0480] g. 5 mL petroleum ether was added to each tube, invert several times and centrifuged at 1000 rpm for 5 min. 4 mL petroleum ether layer was transferred to a new glass tube (18×180 mm). This step was repeated once.

    [0481] h. The extracts were collected and placed in a water bath at 64° C. to evaporate for 30 min. Fatty acids were dissolved with 400 μL chloroform/n-hexane (1:1).

    [0482] i. 50 μL sample were placed in iso-plate (Perkinelmer), 200 μL ULTIMA GOLD (Perkinelmer) was added to each well and incubated for 10 min at room temperature.

    [0483] j. MicroBeta (Perkinelmer) was used to record the CPM scintillation signal.

    [0484] The average value of the positive and negative control well and the standard deviation were calculated. IC.sub.50 was fitted by using XLFit 5.3.1.3 (2006-2011 ID Business Solutions Limited) with 4-Parameter Logistic Model or Sigmoidal Dose-Response Model:


    Y=(Bottom+((Top−Bottom)/(1+((Log IC.sub.50/X){circumflex over ( )}Hillslope)))),

    [0485] wherein X is the logarithm of the compound concentration, Y is CPM (count per minute); Top and Bottom are the Y values at the highest and lowest plateau of the curve; Hillslope is the Hill constant.

    [0486] The inhibitory activities of the present compound on fatty acid synthesis in HepG2 cells are shown in Table 3 below.

    [0487] In Table 3, A means the inhibitory activity of the compound with IC.sub.50<50 nM; B means 50 nM<IC.sub.50<500 nM; C means IC.sub.50>500 nM.

    TABLE-US-00004 TABLE 3 The inhibitory IC.sub.50 values of the present compound on fatty acid synthesis in HepG2 cells Example IC.sub.50 (nM) 5x B 5y A 5b A 8 A 8c A 8d A

    [0488] The compound of the invention can inhibit fatty acid synthesis in HepG2 cells.

    Test Example 3: Analysis of the In Vitro Inhibition Effect of the Present Compound on Pyruvate Carboxylase (PC) Activity

    [0489] The in vitro analysis of the inhibitory effect of the present compound on PC activity was carried out by a coupling method. The PC reaction product oxaloacetate and β-NADH undergo a redox reaction under the catalysis of malate dehydrogenase, generating malic acid and NAD.sup.+. Accordingly, the inhibition ability of the compound on PC can be reflected by detecting the change of absorbance value at 340 nm.

    [0490] Test Methods:

    [0491] a. 10 mM compound stock solution (the present compound was dissolved in 100% DMSO to prepare the 10 mM stock solution) was geometrically diluted with 100% DMSO in 1:3 in a 96-well dilution plate (249944, Nunc). The gradient concentrations of the compound were: 10 mM, 3.333 mM, 1.111 mM, 0.370 mM, 0.123 mM, 0.0412 mM, 0.0137 mM, 0.00457 mM, 0.00152 mM, 0.000508 mM, 0.000169 mM, and 0.0000565 mM.

    [0492] b. 65.7 μL PC enzyme reaction buffer (201 mM triethanolamine, 7.5 mM magnesium sulfate, 0.18% bovine serum albumin) was added to each well of a 96-well dilution plate (249944, Nunc), and then 1 μL of the serially diluted compound to be tested in step a was added to each well.

    [0493] c. 10 μL of compound (prepared in step b) was added to each well of a 384-well test plate (3702, Corning). The reaction system with ZnCl.sub.2 at a final concentration of 500 μM was used as positive control; the reaction system with no compound but DMSO at a final concentration of 0.5% was used as negative control.

    [0494] d. 10 μL of PC enzyme (Sigma, 0.15 U/ml stock solution of PC enzyme dissolved in 0.15 mM Tris-HCl pH 7.4, 0.006 mM Mg(AcO).sub.2, 0.15% glycerol, and 0.003 mM EDTA) was added to each well of the 384-well test plate (prepared in step c), centrifuged at 1000 rpm for 1 min, and incubated at room temperature for 15 min.

    [0495] e. 10 μL of substrate mixture (1.5 mM sodium pyruvate, 1.5 mM ATP, 0.15 mM acetyl-CoA, 45 mM sodium bicarbonate, 0.69 mM NADH, 3 U/ml malate dehydrogenase, 201 mM triethanolamine, 7.5 mM magnesium sulfate, and 0.18% bovine serum albumin) was added to each well of the 384-well test plate (prepared in step d), and centrifuged at 1000 rpm for 1 min. The final concentrations of each component in the PC enzymatic reaction system: 0.05 U/ml pyruvate carboxylase, 0.5 mM sodium pyruvate, 0.5 mM ATP, 15 mM sodium bicarbonate, 0.23 mM β-NADH, 1 U/ml malic acid dehydrogenase, and 0.05 mM acetyl-CoA. Test compound concentrations: 50 μM, 16.667 μM, 5.556 μM, 1.852 μM, 0.617 μM, 0.206 μM, 0.0686 μM, 0.0229 μM, 0.00763 μM, 0.00254 μM, 0.000847 μM and 0.000282 μM. The final concentration of DMSO was 0.5% (v/v).

    [0496] f. The absorbance at 340 nm was read by using EnVision 2104 plate reader (Perkin Elmer) with reading every 1 min for 40 min.

    [0497] GraphPad Prism 6 software was used to perform a linear fit, and the concentration of each compound and the slope of the positive and negative controls were calculated. The inhibition percentage was calculated according to the following formula: 100×[1−(compound−average value of positive control)/(average value of negative control−average value of positive control)].

    [0498] IC.sub.50 of the compound was obtained using GraphPad Prism 6 software according to the following nonlinear fitting formula:


    Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC50−X)×HillSlope)),

    [0499] wherein X is the logarithm of the compound concentration, Y is the inhibition rate; Top and Bottom are the Y values at the highest and lowest plateau of the curve; Hillslope is the Hill constant.

    [0500] The inhibitory activities of the present compound on PC are shown in Table 4 below.

    TABLE-US-00005 TABLE 4 The inhibitory IC.sub.50 values of the present compound on PC Example IC.sub.50 (μM) 5b >50 8 >50 8c >50 8d >50

    [0501] The compound of the invention has no significant inhibitory effect on PC, suggesting a relatively high selectivity.

    Test Example 4: In Vitro Inhibition Effect of the Present Compound on the Proliferation of HepG2 Cells

    [0502] CellTiter-Glo Analysis Kit (Promega) was used for the detection of the inhibitory effect of the present compound on the proliferation of HepG2 cells. The CellTiter-Glo detection method uses luciferase, which requires ATP for its reaction and luminescence. The intensity of the luminescent signal depends on the amount of ATP, and the amount of ATP depends on the number of living cells. Said analysis only requires that ATP in living cells is converted to luminescence by UltraGlo™ luciferase after treatment with the tested drug, so that the inhibitory effect of the compound is detected based on the luminescence intensity, and its IC.sub.50 value is calculated.

    [0503] Test Methods:

    [0504] a. HepG2 cells were purchased from the American type culture collection (ATCC) resource bank. Cells were cultured in MEM (GIBCO) containing 10% fetal bovine serum, penicillin (100 units/mL) and streptomycin (100 μg/mL), and then incubated in a 37° C. incubator containing 5% carbon dioxide, and passaged once every 2 to 3 days.

    [0505] b. On the first day, 800 HepG2 cells were inoculated in each well of a 384-well cell culture plate, and then incubated in a 37° C. incubator containing 5% carbon dioxide.

    [0506] c. On the second day, the medium was changed to that containing the compound. The concentrations of the compound were 30 μM, 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.123 μM, 0.0411 μM, 0.0137 μM, 0.00457 μM and 0.00152 μM. The final concentration of DMSO was 0.3% (v/v). The plate was incubated for 3 days in a incubator containing 5% carbon dioxide at 37° C. The reaction system with paclitaxel at a final concentration of 1 μM was used as positive control; the reaction system with no compound but DMSO at a final concentration of 0.3% was used as negative control.

    [0507] d. The cell plate to be tested was placed at room temperature to equilibrate for 30 min.

    [0508] e. 30 μL of CTG reagent (CelTiter Glo reagent) was added to each well, placed on a fast shaker for 2 min, and placed in the dark at room temperature for 10 min.

    [0509] f. The chemiluminescence signal value was read with Envision 2104 instrument.

    [0510] The average value of the positive and negative control well and the standard deviation were calculated. The percentage inhibition rate was calculated according to the following formula: 100×[1−(compound−average value of positive control)/(average value of negative control−average value of positive control)].

    [0511] IC.sub.50 of the compound was obtained using GraphPad Prism 6 software according to the following nonlinear fitting formula:


    Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC.sub.50−X)×HillSlope)),

    [0512] wherein X is the logarithm of the compound concentration, Y is the inhibition rate; Top and Bottom are the Y values at the highest and lowest plateau of the curve; Hillslope is the Hill constant.

    [0513] The inhibitory activities of the present compound on proliferation of HepG2 cells are shown in Table 5 below.

    TABLE-US-00006 TABLE 5 The inhibitory IC.sub.50 values of the present compound on proliferation of HepG2 cells Example IC.sub.50 (μM) 5x >30 5y >30 8 >30

    [0514] The compound of the invention has no significant inhibitory effect on the proliferation of HepG2 cells, suggesting a relatively high liver safety.