QUINOLINE DERIVATIVE HAVING INDOLEAMINE-2,3-DIOXYGENASE INHIBITORY ACTIVITY

Abstract

Provided is a quinoline derivative having indoleamine-2,3-dioxygenase inhibitory activity, specifically, provided is a compound of general Formula (I) or pharmaceutically acceptable salt thereof, its pharmaceutical composition, preparation method and use in the manufacture of a medicament for immunomodulating and preventing and/or treating of a disease associated with IDO expression abnormality and/or tryptophan metabolism abnormality. Also provided is use of a combination medication of the quinoline derivative and HDAC inhibitor and its use in the manufacture of an anti-tumor drug.

A-X—B—Y-M   Formula (I)

Claims

1. A compound of general Formula (I) or pharmaceutically acceptable salt thereof,
A-X—B—Y-M   (I) wherein, A represents a 9- to 15-membered fused aromatic heterocycle containing 1 to 5 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; optionally, the fused aromatic heterocycle is substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halogenated C.sub.1-6 alkyl and hydroxyl-substituted C.sub.1-6 alkyl; X represents a single covalent bond, —C(R.sup.1R.sup.2)—, ##STR00146## —O—, —O—C.sub.1-6 alkylene-, —NR.sup.1—, —S—, —S(O)— or ##STR00147## wherein R.sup.1 and R.sup.2 are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halogenated C.sub.1-6 alkyl and hydroxyl-substituted C.sub.1-6 alkyl; B represents a 6- to 12-membered polycyclic aliphatic heterocycle containing 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; Y represents ##STR00148## wherein, D represents a single covalent bond or C.sub.1-6 alkylene, optionally, the C.sub.1-6 alkylene is substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halogenated C.sub.1-6 alkyl and hydroxyl-substituted C.sub.1-6 alkyl; E represents a single covalent bond, C.sub.1-6 alkylene, C.sub.2-6 alkenylene, —NR.sup.3— or —C.sub.1-6 alkylene-NR.sup.3—, wherein, R.sup.3 is selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl and hydroxyl-substituted C.sub.1-6 alkyl; M represents a 6- to 10-membered aromatic ring; 3- to 7-membered aliphatic carbocycle; 5- to 10-membered monocyclic or polycyclic aromatic heterocycle, each of which contains 1 to 4 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; 3 to 7-membered saturated or partially unsaturated monocyclic aliphatic heterocycle or 6- to 12-membered polycyclic aliphatic heterocycle, each of which contains 1 to 5 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; optionally, the aromatic ring, carbocycle, aromatic heterocycle and aliphatic heterocycle are substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.1-6 alkoxy, C.sub.1-6 alkylamino, ##STR00149## halogenated C.sub.1-6 alkoxy, hydroxyl-substituted C.sub.1-6 alkoxy and C.sub.1-6 alkoxy-substituted C.sub.2-6 alkenyl.

2. The compound of general Formula (I) or pharmaceutically acceptable salt thereof according to claim 1; wherein, A represents a 9- to 10-membered fused aromatic heterocycle containing 1 to 2 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; optionally, the fused aromatic heterocycle is substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, halogenated C.sub.1-4 alkyl and hydroxyl-substituted C.sub.1-4 alkyl; preferably, A represents a 9- to 10-membered fused aromatic heterocycle containing 1 to 2 nitrogen atoms; optionally, the fused aromatic heterocycle is substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-2 alkyl, C.sub.1-2 alkoxy, halogenated C.sub.1-2 alkyl and hydroxyl-substituted C.sub.1-2 alkyl; preferably, A is selected from the following groups optionally substituted with one or more halogens: benzimidazolyl, imidazopyridyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl and naphthyridinyl; preferably, A is 6-fluoroquinolinyl.

3. The compound of general Formula (I) or pharmaceutically acceptable salt thereof according to claim 1; wherein, X represents a single covalent bond, —C(R.sup.1R.sup.2)—, ##STR00150## —O—, —O—C.sub.1-4 alkylene-, —NR.sup.1—, —S—, —S(O)— or ##STR00151## wherein R.sup.1 and R.sup.2 are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, halogenated C.sub.1-4 alkyl and hydroxyl-substituted C.sub.1-4 alkyl; preferably, X represents a single covalent bond, —O—, —O—C.sub.1-2 alkylene- or —NR.sup.1—; wherein R.sup.1 and R.sup.2 are each independently selected from the group consisting of hydrogen and C.sub.1-2 alkyl; preferably, X represents a single covalent bond, —O—, —OCH.sub.2—, —NH— or —N(CH.sub.3)—; preferably, X represents —O—, —NH— or —N(CH.sub.3)—; preferably, X represents —O— or —NH—; preferably, X represents —O—.

4. The compound of general Formula (I) or pharmaceutically acceptable salt thereof according to claim 1; wherein, B represents a 6- to 9-membered polycyclic aliphatic heterocycle containing 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; preferably, at least one of the heteroatoms is nitrogen; preferably, B represents a 6- to 9-membered bicyclic aliphatic heterocycle containing 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; preferably, at least one of the heteroatoms is nitrogen; preferably, B represents a 6- to 9-membered spiro heterocycle or 6- to 9-membered fused heterocycle, each of which contains 1 to 3 nitrogen atoms; preferably, B is connected to Y through a nitrogen atom on its ring; preferably, B is selected from ##STR00152##

5. The compound of general Formula (I) or pharmaceutically acceptable salt thereof according to claim 1; wherein, Y represents ##STR00153## wherein D represents a single covalent bond or C.sub.1-4 alkylene, optionally, the C.sub.1-4 alkylene is substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, halogenated C.sub.1-4 alkyl and hydroxyl-substituted C.sub.1-4 alkyl; E represents a single covalent bond, C.sub.1-4 alkylene, C.sub.2-4 alkenylene, —NR.sup.3— or —C.sub.1-4 alkylene-NR.sup.3—, wherein R.sup.3 is selected from the group consisting of hydrogen, halogen, hydroxyl, amino, C.sub.1-4 alkyl, halogenated C.sub.1-4 alkyl and hydroxyl-substituted C.sub.1-4 alkyl; preferably, Y represents ##STR00154## wherein D represents a single covalent bond or C.sub.1-2 alkylene, optionally, the C.sub.1-2 alkylene is substituted with one or more hydroxyl groups; E represents a single covalent bond, vinylidene, —NH— or —C.sub.1-2 alkylene-NH—; preferably, Y represents —C(O)—, ##STR00155##

6. The compound of general Formula (I) or pharmaceutically acceptable salt thereof according to claim 1; wherein, M represents a 6- to 10-membered aromatic ring; 3- to 7-membered aliphatic carbocycle; 5- to 6-membered monocyclic aromatic heterocycle or 8- to 10-membered bicyclic aromatic heterocycle, each of which contains 1 to 4 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; 3- to 6-membered monocyclic aliphatic heterocycle or 7- to 10-membered bicyclic aliphatic heterocycle, each of which contains 1 to 5 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; optionally, the aromatic ring, carbocycle, aromatic heterocycle, or aliphatic heterocycle is substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.1-4 alkoxy, C.sub.1-4 alkylamino, ##STR00156## halogenated C.sub.1-4 alkoxy, hydroxyl-substituted C.sub.1-4 alkoxy and C.sub.1-4 alkoxyl-substituted C.sub.2-4 alkenyl; preferably, M represents a benzene ring; 3- to 7-membered aliphatic carbocycle; 5- to 6-membered monocyclic aromatic heterocycle or 8- to 10-membered bicyclic aromatic heterocycle, each of which contains 1 to 2 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; 3- to 6-membered monocyclic aliphatic heterocycle containing 1 to 2 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; optionally, the benzene ring, carbocycle, 5- to 6-membered monocyclic aromatic heterocycle, 8- to 10-membered bicyclic aromatic heterocycle, or 3- to 6-membered monocyclic aliphatic heterocycle is substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C.sub.1-2 alkyl, vinyl, C.sub.1-2 alkoxy, C.sub.1-2 alkylamino, ##STR00157## halogenated C.sub.1-2 alkoxy, hydroxyl-substituted C.sub.1-2 alkoxy and C.sub.1-2 alkoxy-substituted C.sub.2-4 alkenyl; preferably, M represents a benzene ring; 3- to 7-membered aliphatic carbocycle; 5- to 6-membered monocyclic aromatic heterocycle or 8- to 10-membered bicyclic aromatic heterocycle, each of which contains 1 to 2 heteroatoms selected from the group consisting of nitrogen and oxygen; optionally, the benzene ring, carbocycle, 5- to 6-membered monocyclic aromatic heterocycle or 8- to 10-membered bicyclic aromatic heterocycle is substituted with one or more substituents selected from the group consisting of: halogen, C.sub.1-2 alkoxy, ##STR00158## and C.sub.1-2 alkoxy-substituted C.sub.2-4 alkenyl; preferably, M represents a benzene ring, cyclohexane ring, pyridine ring, 2,3-dihydrobenzofuran ring, benzo[1,3]dioxole ring or 2,3-dihydrobenzo[1,4]dioxane ring; optionally, the benzene ring is substituted with one or more substituents selected from the group consisting of fluorine, chlorine, methoxy, dimethylamino and methoxy-substituted propenyl.

7. The compound of general Formula (I) or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of: ##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166##

8. A pharmaceutical composition, comprising the compound or pharmaceutically acceptable salt thereof according to claim 1, and at least one pharmaceutically acceptable carriers or excipients.

9. The pharmaceutical composition according to claim 8, wherein the compound is selected from the group consisting of N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide, N-(benzo[d][1,3]dioxol-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide, 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide, and 2-(benzo[d][1,3]dioxol-5-yl-amino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one.

10. The pharmaceutical composition according to claim 8, further comprising one or more other Indoleamine 2,3-dioxygenase (IDO) inhibitors, and/or one or more epigenetic modulators; preferably, the IDO inhibitor is selected from the group consisting of a tryptophan analog (e.g., Indoximod) and an IDO1 inhibitor (e.g., Epacadostat, NLG919); preferably, the epigenetic modulator is selected from the group consisting of a DNA methyltransferase inhibitor (e.g., Azacitidine (5-Aza), Decitabine), a histone methyltransferase inhibitor (e.g., EZH2 inhibitor), a histone demethylase inhibitor (e.g., LSD inhibitor), and a histone deacetylase (HDAC) inhibitor; preferably, the HDAC inhibitor is selected from the group consisting of a benzamide HDAC inhibitor (e.g., Chidamide), a hydroxamic acid HDAC inhibitor (e.g., Vorinostat), a cyclic peptide HDAC inhibitor (e.g., Romidepsin) and a short-chain fatty acid HDAC inhibitor (e.g., valproic acid); preferably, the pharmaceutical composition further comprises Chidamide.

11. A combination medication, comprising a first active ingredient and a second active ingredient, and optionally a pharmaceutically acceptable carrier or excipient; wherein, the first active ingredient is selected from the group consisting of the compound or pharmaceutically acceptable salt thereof according claim 1, and other IDO inhibitors; the second active ingredient is selected from epigenetic modulators; preferably, the IDO inhibitor is selected from the group consisting of a tryptophan analog (e.g., Indoximod) and an IDO1 inhibitor (e.g., Epacadostat, NLG919); preferably, the epigenetic modulator is selected from the group consisting of a DNA methyltransferase inhibitor, such as Azacitidine (5-Aza) or Decitabine; a histone methyltransferase inhibitor, such as EZH2 inhibitor; a histone demethylase inhibitor, such as LSD inhibitor; and a HDAC inhibitor; preferably, the HDAC inhibitor is selected from the group consisting of a benzamide HDAC inhibitor (e.g., Chidamide), a hydroxamic acid HDAC inhibitor (e.g., Vorinostat), a cyclic peptide HDAC inhibitor (e.g., Romidepsin) and a short-chain fatty acid HDAC inhibitor (e.g., valproic acid); preferably, the first active ingredient is selected from the group consisting of N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide, N-(benzo[d][1,3]dioxol-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide, 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl) propionamide, 2-(benzo[d][1,3]dioxol-5-yl-amino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a] isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one, and a pharmaceutically acceptable salt thereof; the second active ingredient is Chidamide; preferably, the first active ingredient and the second active ingredient are in the same preparation unit; preferably, the first active ingredient and the second active ingredient are in different preparation units; preferably, the first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially.

12. (canceled)

13. A method for regulating immunity, comprising administering to a subject in need thereof an effective amount of the compound or pharmaceutically acceptable salt thereof according to claim 1.

14. A method for preventing and/or treating a disease associated with IDO expression abnormality and/or tryptophan metabolism abnormality, comprising administering to a subject in need thereof an effective amount of the compound or pharmaceutically acceptable salt thereof according claim 1; preferably, the disease is selected from the group consisting of tumor, autoimmune disease, cataract, Alzheimer's disease, depression disorder and anxiety disorder.

15. (canceled)

16. A method for preparing the compound of general Formula (I) according claim 1, which is selected from the group consisting of Methods 1, 2 and 3, wherein: in Method 1: when Y is ##STR00167## D is a single covalent bond, and E is —NR.sup.3—, the compound of general Formula (I) is prepared by the following method: in an organic solvent, in the presence of a base, reacting a compound of Formula I-1, a compound of Formula I-2 and an ureation reagent to obtain a compound of general Formula (I), ##STR00168## in Method 2: when Y is ##STR00169## D is a single covalent bond, E is a single covalent bond, C.sub.1-6 alkylene, C.sub.2-6 alkenylene or —C.sub.1-6 alkylene-NR.sup.3—, the compound of general Formula (I) is prepared by the following method: subjecting a compound of Formula I-1 and a compound of Formula I-3 to a coupling reaction to obtain a compound of Formula (I); ##STR00170## in Method 3: when Y is ##STR00171## D is a substituted or unsubstituted C.sub.1-6 alkylene, and E is —NR.sup.3—, the compound of general Formula (I) is prepared by the following method: subjecting a compound of Formula I-4 and a compound of Formula I-2 to a coupling reaction to obtain a compound of general Formula (I), ##STR00172## wherein, A, X, B, R.sup.3 and M are as defined in claim 1.

17. The method according to claim 16, wherein, in the compound of general Formula (I), B represents a 6- to 12-membered polycyclic heterocyclic alkylene containing 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one of the heteroatoms is nitrogen, and B is connected to Y through a nitrogen atom on its ring; preferably, in Method 1, the organic solvent may be dichloromethane, THF, toluene, ethyl acetate or benzene; preferably, in Method 1, the base is triethylamine, pyridine, DIPEA or NaOH; preferably, in Method 1, the ureation reagent is selected from isocyanate, triphosgene, phosgene, diphosgene, chloroformamide, carbonyldiimidazole (CDI) and potassium isocyanate; preferably, in Method 2 and/or Method 3, using a peptide condensing agent as a catalyst for the coupling reaction, such as 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-dicyclohexylcarbodiimide (DCC), N,N′-carbonyldiimidazole (CDI), 2-(7-aza-benzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU); preferably, in Method 2 and/or Method 3, conducting the coupling reaction at 0° C. to 60° C.; preferably, in Method 2 and/or Method 3, conducting the coupling reaction for 2 to 72 hours; preferably, in Method 2 and/or Method 3, the solvent used in the coupling reaction is selected from the group consisting of benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform and N,N′-dimethylformamide; preferably, in Method 2 and/or Method 3, if necessary, using a base, such as sodium hydroxide, triethylamine, DMAP or pyridine for the coupling reaction preferably, the compound of Formula I-1 is prepared through the following method: (a) under the action of a base, subjecting Compound 1 and Compound 2 to a nucleophilic substitution reaction to obtain Compound 3: ##STR00173## (b) under the action of an acid, deprotecting Compound 3 to obtain Formula I-1: ##STR00174## wherein, A, X and B are as defined in claim 1; preferably, the compound of Formula I-4 is prepared through the following method: (c) subjecting a compound of Formula I-1 and a halogenated carboxylic acid ester 4 (e.g., chloroformate, 2-bromoacetate, 2-bromopropionate, 1-hydroxyl-2-bromopropionate) to a nucleophilic substitution reaction under the action of a base to obtain Compound 5; ##STR00175## (d) subjecting Compound 5 to a hydrolysis reaction under alkaline condition to obtain a compound of Formula I-4: ##STR00176## wherein, R.sup.4 represents C.sub.1-6 alkyl, A, X, B and D are as defined in claim 1; preferably, the compound of Compound F is prepared through the following method: (e) under the action of a base, subjecting a compound of Formula I-1 and 2-halopropionate (e.g., ethyl 2-bromopropionate) to a nucleophilic substitution reaction to obtain Compound 6; ##STR00177## (f) subjecting Compound 4 to a hydrolysis reaction under alkaline condition to obtain a compound of Formula F; ##STR00178## wherein, A, X and B are as defined in claim 1; preferably, KHMDS, NaH, NaOH, Na.sub.2CO.sub.3 or K.sub.2CO.sub.3 is used as a deacidification agent in the nucleophilic substitution reaction of step (a); preferably, conducting the reaction of step (a) at 25° C. to 140° C.; preferably, conducting the reaction of step (a) for 2 to 72 hours; preferably, the solvent used in the nucleophilic substitution reaction of step (a) is selected from the group consisting of water, methanol, ethanol, acetonitrile, benzene, xylene, acetone, N,N′-dimethylformamide and DMSO; preferably, the acid of step (b) is selected from the group consisting of trifluoroacetic acid and hydrochloride solution; preferably, the base of step (b) is NaOH; preferably, conducting the reaction of step (b) at 25° C. to 140° C.; preferably, conducting the reaction of step (b) for 2 to 72 hours; preferably, the solvent used in step (b) is selected from the group consisting of water, methanol, ethanol, acetonitrile, benzene, xylene, acetone, N,N′-dimethylformamide and DMSO; preferably, NaOH, Na.sub.2CO.sub.3, or K.sub.2CO.sub.3 is used as a deacidification agent for the nucleophilic substitution reaction of step (c) and/or step (e); preferably, conducting the reaction of step (c) and/or step (e) at 25° C. to 140° C.; preferably, conducting the reaction of step (c) and/or step (e) for 2 to 72 hours; preferably, the solvent used in the reaction in step (c) and/or step (e) is selected from the group consisting of water, methanol, ethanol, acetonitrile, benzene, xylene, acetone, N,N′-dimethylformamide and DMSO; preferably, NaOH or LiOH is used as the base for the hydrolysis reaction of step (d) and/or step (f); preferably, conducting the reaction of step (d) and/or step (f) at 0° C. to 60° C.; preferably, conducting the reaction of step (d) and/or step (f) for 0.5 to 2 hours; preferably, the solvent used in step (d) and/or step (f) is selected from the group consisting of water, methanol, ethanol, tetrahydrofuran and N,N′-dimethylformamide.

18.-22. (canceled)

23. A method for regulating immunity, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to claim 8.

24. A method for preventing and/or treating a disease associated with IDO expression abnormality and/or tryptophan metabolism abnormality, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to claim 8; preferably, the disease is selected from the group consisting of tumor, autoimmune disease, cataract, Alzheimer's disease, depression disorder and anxiety disorder.

25. A method for regulating immunity, comprising administering to a subject in need thereof an effective amount of the combination medication according to claim 11.

26. A method for preventing and/or treating a disease associated with IDO expression abnormality and/or tryptophan metabolism abnormality, comprising administering to a subject in need thereof an effective amount of the combination medication according to claim 11; preferably, the disease is selected from the group consisting of tumor, autoimmune disease, cataract, Alzheimer's disease, depression disorder and anxiety disorder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0124] FIG. 1 shows the inhibition of IDO enzyme activity of HeLa cells by Chidamide in combination with Example 3.

[0125] FIG. 2 shows the inhibition of IDO enzyme activity of HeLa cells by Chidamide in combination with Example 20.

[0126] FIG. 3 shows the inhibition of IDO enzyme activity of HeLa cells by Chidamide in combination with Example 72.

[0127] FIG. 4 shows the inhibition of IDO enzyme activity of HeLa cells by Chidamide in combination with Example 87.

[0128] FIG. 5 shows the growth inhibition curve of Chidamide in combination with Example 3 on CT-26 tumor in mice.

[0129] FIG. 6 shows the weight inhibition of Chidamide in combination with Example 3 on CT-26 tumor in mice.

[0130] FIG. 7 shows the growth inhibition curve of Chidamide in combination with Example 20 on CT-26 tumor in mice.

[0131] FIG. 8 shows the growth inhibition curve of Chidamide in combination with Example 72 on CT-26 tumor in mice.

[0132] FIG. 9 shows the growth inhibition curve of Chidamide in combination with Example 87 on CT-26 tumor in mice.

DETAILED DESCRIPTION

[0133] The embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art shall understand that the following examples are only used for illustration, and should not be regarded as limiting the scope of the present invention. In the following, if not particularly specified, the materials and operation methods in the present application are well-known in the art. Those without specific conditions in the examples are carried out in accordance with the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used without indicating manufacturers are all conventional products obtained from the market.

[0134] The complete names represented by the abbreviations of reagents used in the examples are as follows:

[0135] KHMDS potassium hexamethyldisilazide

[0136] HATU 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

[0137] DIPEA diisopropylethylamine

[0138] HOBt 1-hydroxybenzotriazole

[0139] EDCI.HCl 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0140] DMF N,N-dimethylformamide

[0141] THF tetrahydrofuran

[0142] EA ethyl acetate

[0143] DCM dichloromethane

Example 1: Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3] heptan-2-carboxylate

[0144] ##STR00059##

[0145] Tert-butyl 6-Hydroxy-2-aza-spiro[3.3]heptan-2-carboxylate (2.04 g, 9.6 mmol) was dissolved in dioxane (16 mL) and stirred at room temperature, added with KHMDS (9.6 mL, 9.6 mmol), stirred at room temperature for 5 minutes, and then added with a solution of 4-chloro-6-fluoroquinoline (1.46 g, 8.0 mmol) in dioxane (5 mL). The reaction solution was stirred at 60° C. for 30 minutes, cooled to room temperature by stopping heating and stirred for 15 hours, then poured into water, adjusted with aqueous solution of NaHCO.sub.3 to reach a pH value of 8-10, extracted with ethyl acetate, the organic phases were combined and dried, filtered, and distilled under reduced pressure to obtain the residue, which was then separated by column chromatography [ethyl acetate/petroleum ether=3/2] to obtain a light yellow viscous oily substance tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxylate (1.3 g, yield: 45%), LC-MS (m/z) 359 (M+1).

Example 2: Preparation of 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline Hydrochloride

[0146] ##STR00060##

[0147] Tert-butyl ((6-Fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxylate (1.3 g, 3.6 mmol) was dissolved in 20 mL of dichloromethane, then added with concentrated hydrochloric acid (1 mL). The reaction solution was stirred at room temperature for 16 hours and then concentrated directly to obtain an off-white solid 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (1.0 g, yield: 94%), LC-MS (m/z) 259 (M+1).

Example 3: Preparation of N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0148] ##STR00061##

[0149] p-Chloroaniline (50 mg, 0.39 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL), added with triphosgene (116 mg, 0.39 mmol) under ice bath, and then slowly added dropwise with triethylamine (0.2 mL, 1.6 mmol). The reaction solution was stirred for 25 minutes under ice bath, then concentrated under reduced pressure, added with ultra-dry N,N-dimethylformamide (5 mL), and under ice bath added with a solution of 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (115 mg, 0.43 mmol) in N,N-dimethylformamide (1 mL), and then slowly added dropwise with triethylamine (0.3 mL, 2.3 mmol). After the reaction solution was stirred under ice bath for 10 minutes, the ice bath was removed, the reaction solution was stirred at room temperature for 40 minutes, and then poured into water. The solid was precipitated, filtered, washed with water, and dried, the obtained solid was subjected to column chromatography separation [methanol/dichloromethane=1/20] to obtain a white solid N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (120 mg, yield: 70%).

[0150] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J=5.1 Hz, 1H), 8.55 (s, 1H), 8.02 (dd, J=9.2, 5.4 Hz, 1H), 7.79 (dd, J=9.7, 3.0 Hz, 1H), 7.69-7.62 (m, 1H), 7.56-7.50 (m, 2H), 7.30-7.24 (m, 2H), 6.92 (d, J=5.2 Hz, 1H), 4.97 (p, J=6.6 Hz, 1H), 4.06 (s, 2H), 4.01 (s, 2H), 2.95-2.84 (m, 2H), 2.47-2.39 (m, 2H).

[0151] LC-MS (m/z) 412 (M+1).

Example 4: Preparation of N-(3-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0152] ##STR00062##

[0153] According to the steps similar to those in Example 3, a white solid N-(3-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (32 mg, 57% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (40 mg, 0.14 mmol) and 3-methoxyaniline (18 mg, 0.14 mmol).

[0154] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J=5.1 Hz, 1H), 8.41 (s, 1H), 8.02 (dd, J=9.2, 5.4 Hz, 1H), 7.79 (dd, J=9.7, 2.9 Hz, 1H), 7.66 (td, J=8.8, 3.0 Hz, 1H), 7.19 (t, J=2.2 Hz, 1H), 7.14-7.03 (m, 2H), 6.92 (d, J=5.3 Hz, 1H), 6.49 (ddd, J=8.0, 2.7, 1.2 Hz, 1H), 4.97 (p, J=6.7 Hz, 1H), 4.05 (s, 2H), 3.99 (s, 2H), 3.69 (s, 3H), 2.92-2.81 (m, 2H), 2.49-2.36 (m, 2H).

[0155] LC-MS (m/z) 408 (M+1).

Example 5: Preparation of N-(4-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0156] ##STR00063##

[0157] According to the steps similar to those in Example 3, a white solid N-(4-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (26 mg, 47% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (40 mg, 0.14 mmol) and 4-methoxyaniline (18 mg, 0.14 mmol).

[0158] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J=5.1 Hz, 1H), 8.25 (s, 1H), 8.02 (dd, J=9.4, 5.3 Hz, 1H), 7.83-7.75 (m, 1H), 7.66 (t, J=8.8 Hz, 1H), 7.37 (d, J=8.7 Hz, 2H), 6.92 (d, J=5.2 Hz, 1H), 6.81 (d, J=8.6 Hz, 2H), 5.02-4.92 (m, 1H), 4.03 (s, 2H), 3.97 (s, 2H), 3.69 (s, 3H), 3.32 (s, 5H), 2.87 (s, 2H), 2.44 (d, J=6.7 Hz, 2H).

[0159] LC-MS (m/z) 408 (M+1).

Example 6: Preparation of (4-chlorophenyl)(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl) methanone

[0160] ##STR00064##

[0161] 4-((2-Aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (60 mg, 0.2 mmol) was dissolved in 10 ml of DMF, then added with p-chlorobenzoic acid (38.2 mg, 0.24 mmol), EDCI (77.8 mg, 0.4 mmol), HOBt (55 mg, 0.4 mmol), DIPEA 1 ml, stirred at room temperature for 16 hours, the reaction solution was poured into 40 ml of ice water, extracted with ethyl acetate (20 ml) twice, the organic phases were combined, washed with saturated brine (40 ml), dried over anhydrous sodium sulfate, subjected to rotary evaporation to remove the organic phase, and separated by column chromatography (dichloromethane/methanol=20/1) to obtain a white solid (20 mg, 25% yield).

[0162] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.68 (d, J=5.1 Hz, 1H), 8.01 (dd, J=9.2, 5.4 Hz, 1H), 7.76 (t, J=7.4 Hz, 1H), 7.70-7.59 (m, 3H), 7.51 (t, J=9.2 Hz, 2H), 6.87 (dd, J=17.2, 5.2 Hz, 1H), 4.93 (p, J=6.6 Hz, 1H), 3.54-3.40 (m, 4H), 2.88 (t, J=9.9 Hz, 2H), 2.43 (d, J=8.5 Hz, 2H).

[0163] LC-MS (m/z) 397 (M+1).

Example 7: Preparation of (E)-3-(4-chlorophenyl)-1-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)prop-2-en-1-one

[0164] ##STR00065##

[0165] According to the steps similar to those in Example 6, a white solid (E)-3-(4-chlorophenyl)-1-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)prop-2-en-1-one (18 mg, 21% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (40 mg, 0.14 mmol) and 4-chlorocinnamic acid (18 mg, 0.14 mmol).

[0166] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.71 (d, J=5.1 Hz, 1H), 8.03 (dd, J=9.2, 5.4 Hz, 1H), 7.92 (td, J=6.4, 3.7 Hz, 1H), 7.80 (dd, J=9.7, 2.9 Hz, 1H), 7.75 (dd, J=15.6, 1.7 Hz, 1H), 7.66 (td, J=8.8, 3.0 Hz, 1H), 7.57-7.49 (m, 1H), 7.47-7.35 (m, 2H), 6.92 (t, J=4.9 Hz, 1H), 6.77 (dd, J=15.6, 8.5 Hz, 1H), 4.99 (p, J=6.6 Hz, 1H), 4.40 (d, J=27.1 Hz, 2H), 4.07 (d, J=27.4 Hz, 2H), 2.92 (ddd, J=13.1, 6.2, 2.6 Hz, 2H), 2.46 (ddt, J=9.7, 6.2, 3.2 Hz, 2H).

[0167] LC-MS (m/z) 423 (M+1).

Example 8: Preparation of p-nitrocinnamyl Methyl Ether

[0168] ##STR00066##

[0169] p-Nitrocinnamyl alcohol (200 mg, 1.1 mmol) was dissolved in 20 ml of dry tetrahydrofuran, stirred under nitrogen protection until fully dissolved, added with sodium hydride (178 mg, 4.4 mmol), reacted under stirring for 20 minutes, then added with methyl iodide (174 mg, 1.2 mmol), reacted at room temperature for 16 hours, the reaction solution was poured into 20 ml of ice water, extracted twice with ethyl acetate (20 ml), dried over anhydrous sodium sulfate, subjected to rotary evaporation to remove the organic phase, and separated by solid column chromatography [ethyl acetate/petroleum ether=1/2] to obtain a light yellow solid (60 mg, 28% yield).

Example 9: Preparation of p-aminocinnamyl Methyl Ether

[0170] ##STR00067##

[0171] p-Nitrocinnamyl methyl ether (60 mg, 0.31 mmol) was dissolved in 10 ml of ethanol, added with iron powder (52 mg, 0.93 mmol), stirred for 10 minutes, heated to reflux for 2 hours, poured into ice water (20 ml), adjusted with sodium bicarbonate to pH=8, extracted twice with ethyl acetate (20 ml), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the residue, which was then separated by column chromatography [ethyl acetate/petroleum ether=3/2] to obtain a brown solid (20 mg, 39% yield), LC-MS (m/z) 164 (M+1).

Example 10: Preparation of (E)-(6-((6-fluoroquinolin-4-yl)oxy)-N-(4-(3-methoxyprop-1-en-1-yl)phenyl)-2-aza-spiro[3.3]heptan-2-carboxamide

[0172] ##STR00068##

[0173] According to the steps similar to those in Example 3, a white solid (E)-(6-((6-fluoroquinolin-4-yl)oxy)-N-(4-(3-methoxyprop-1-en-1-yl)phenyl)-2-aza-spiro[3.3]heptan-2-carboxamide (6 mg, 11% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (36 mg, 0.12 mmol) and p-aminocinnamyl methyl ether (20 mg, 0.12 mmol).

[0174] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J=5.1 Hz, 1H), 8.48 (s, 1H), 8.03 (dd, J=9.3, 5.4 Hz, 1H), 7.80 (dd, J=9.7, 3.0 Hz, 1H), 7.66 (ddd, J=9.3, 8.4, 3.0 Hz, 1H), 7.48 (d, J=8.7 Hz, 2H), 7.32 (d, J=8.6 Hz, 2H), 6.93 (d, J=5.2 Hz, 1H), 6.51 (d, J=16.2 Hz, 1H), 6.20 (dt, J=16.0, 6.0 Hz, 1H), 5.76 (s, 2H), 5.07-4.89 (m, 1H), 4.07 (s, 2H), 4.04-3.94 (m, 2H), 3.27 (s, 3H), 2.95-2.76 (m, 2H), 2.48-2.34 (m, 2H).

[0175] LC-MS (m/z) 448 (M+1).

Example 11: Preparation of 1-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-cyclo[3.3]heptan-2-yl)-2-(anilino)ethyl-1-one

[0176] ##STR00069##

[0177] According to the steps similar to those in Example 6, a white solid 1-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-cyclo[3.3]heptan-2-yl)-2-(anilino)ethyl-1-one (35 mg, 52% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (50 mg, 0.17 mmol) and 2-(anilino)acetic acid (30.8 mg, 0.2 mmol).

[0178] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.69 (d, J=5.1 Hz, 1H), 8.02 (dd, J=9.4, 5.4 Hz, 1H), 7.77 (t, J=6.9 Hz, 1H), 7.64 (t, J=8.6 Hz, 1H), 7.08 (q, J=7.5 Hz, 2H), 6.92-6.86 (m, 1H), 6.62-6.52 (m, 2H), 5.75 (s, 2H), 5.00-4.89 (m, 1H), 4.02 (s, 2H), 3.94 (s, 2H), 3.67 (d, J=10.4 Hz, 2H), 2.94-2.80 (m, 2H), 2.45-2.40 (m, 2H).

[0179] LC-MS (m/z) 492 (M+1).

Example 12: Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-3-aza-cyclo[3.1.0]hexan-3-carboxylate

[0180] ##STR00070##

[0181] Tert-butyl 6-hydroxy-3-aza-bicyclo[3.1.0]hexan-3-carboxylate (603 mg, 3.03 mmol) was dissolved in 30 ml of DMF, added with sodium hydride (550 mg, 13.7 mmol) under ice bath, stirred for 30 minutes, then added with 6-fluoro-4-chloroquinoline (500 mg, 2.75 mmol), heated to 75° C. and reacted for 2 hours, poured into ice water (20 ml), and extracted twice with ethyl acetate (20 ml), dried over anhydrous sodium sulfate, subjected to rotary evaporation to remove the solvent, separated by column chromatography (ethyl acetate:petroleum ether=1:1) to obtain a white solid (230 mg, 24% yield), LC-MS (m/z) 345 (M+1).

Example 13: Preparation of 4-((3-aza-cyclo[3.1.0]hex-4-yl)oxy)-6-fluoroquinoline Hydrochloride

[0182] ##STR00071##

[0183] Tert-butyl ((6-fluoroquinolin-4-yl)oxy)-3-aza-cyclo[3.1.0]hexan-3-carboxylate (50 mg, 0.145 mmol) was dissolved in 20 mL of dichloromethane, then added with concentrated hydrochloric acid (0.5 mL), stirred at room temperature for 3 hours, and then concentrated directly to obtain a white solid (38.8 mg, 95% yield), LC-MS (m/z) 245 (M+1).

Example 14: Preparation of 1-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-cyclo[3.1.0]hept-3-yl)-2-(anilino)ethyl-1-one

[0184] ##STR00072##

[0185] According to the steps similar to those in Example 6, a white solid 1-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-cyclo[3.1.0]hept-3-yl)-2-(anilino)ethyl-1-one (60 mg, 89% yield) was prepared from 4-((3-aza-cyclo[3.1.0]hex-4-yl)oxy)-6-fluoroquinoline hydrochloride (50 mg, 0.18 mmol) and 2-(anilino)acetic acid (33.7 mg, 0.21 mmol).

[0186] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.47 (d, J=5.2 Hz, 1H), 8.01 (dd, J=11.0, 2.8 Hz, 1H), 7.86 (dd, J=9.2, 5.8 Hz, 1H), 7.52 (ddd, J=9.3, 8.1, 2.7 Hz, 1H), 7.43 (s, 1H), 7.09 (dd, J=8.4, 7.1 Hz, 2H), 6.74 (d, J=5.2 Hz, 1H), 6.68-6.61 (m, 2H), 6.56 (tt, J=7.2, 1.1 Hz, 1H), 5.53 (t, J=5.1 Hz, 1H), 3.99 (d, J=10.4 Hz, 1H), 3.91-3.86 (m, 2H), 3.78-3.69 (m, 2H), 3.53-3.47 (m, 2H), 2.32 (d, J=2.4 Hz, 2H).

[0187] LC-MS (m/z) 377 (M+1).

Example 15: Preparation of N-(4-chlorobenzyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0188] ##STR00073##

[0189] According to the steps similar to those in Example 3, a white solid N-(4-chlorobenzyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (20 mg, 14% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (100 mg, 0.36 mmol) and 4-chlorobenzylamine (40 mg, 0.36 mmol).

[0190] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.72 (d, J=5.1 Hz, 1H), 8.04 (dd, J=9.2, 5.4 Hz, 1H), 7.80 (dd, J=9.7, 3.0 Hz, 1H), 7.67 (td, J=8.8, 3.0 Hz, 1H), 7.42-7.35 (m, 2H), 7.29 (d, J=8.4 Hz, 2H), 6.94 (dd, J=7.7, 5.5 Hz, 2H), 4.97 (p, J=6.7 Hz, 1H), 4.20 (d, J=6.1 Hz, 2H), 3.96 (s, 2H), 3.89 (s, 2H), 2.91-2.80 (m, 2H), 2.46-2.34 (m, 2H).

[0191] LC-MS (m/z) 426 (M+1).

Example 16: Preparation of N-(4-fluorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0192] ##STR00074##

[0193] According to the steps similar to those in Example 3, a white solid N-(4-fluorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (20 mg, 14% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (100 mg, 0.36 mmol) and 4-fluoroaniline (40 mg, 0.36 mmol).

[0194] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J=5.1 Hz, 1H), 8.47 (s, 1H), 8.03 (dd, J=9.2, 5.4 Hz, 1H), 7.79 (dd, J=9.7, 3.0 Hz, 1H), 7.66 (m, 1H), 7.49 (m, 2H), 7.05 (m, 2H), 6.92 (d, J=5.1 Hz, 1H), 4.97 (t, J=6.7 Hz, 1H), 4.05 (s, 2H), 4.00 (s, 2H), 2.91-2.86 (m, 2H), 2.46-2.41 (m, 2H).

[0195] LC-MS (m/z) 396 (M+1).

Example 17: Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.4]octan-2-carboxylate

[0196] ##STR00075##

[0197] 4-Chloro-6-fluoroquinoline (181 mg, 1.0 mmol), tert-butyl 6-amino-2-azo-azole[3.4]octan-2-carboxylate (226 mg, 1.0 mmol) and cesium carbonate (980 mg, 3.0 mmol) were dissolved in dioxane (5 mL), then added with 2-dicyclohexylphospho-2,4,6-triisopropylbiphenyl (48 mg, 0.1 mmol) and tri(dibenzylideneacetone) dipalladium (46 mg, 0.05 mmol), protected by nitrogen atmosphere. The reaction solution was heated to 123° C. and stirred for 7 hours, then poured into water, extracted with ethyl acetate, washed with water, and the organic phase was concentrated under reduced pressure to obtain a residue, which was separated by column chromatography to obtain tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.4]octan-2-carboxylate (100 mg, 27% yield), LC-MS (m/z) 372 (M+1).

Example 18: Preparation of 4-(2-aza-spiro[3.4]oct-6-yl)amino-6-fluoroquinoline Hydrochloride

[0198] ##STR00076##

[0199] Tert-butyl ((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.4]octan-2-carboxylate (100 mg, 0.27 mmol) was dissolved in 5 mL of dichloromethane, then added with concentrated hydrochloric acid (1 mL). The reaction solution was stirred at room temperature for 16 hours and then concentrated directly to obtain an off-white solid 4-(2-aza-spiro[3.4]oct-6-yl)amino-6-fluoroquinoline hydrochloride (80 mg, 94% yield), LC-MS (m/z) 272 (M+1).

Example 19: Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.4]octan-2-carboxamide

[0200] ##STR00077##

[0201] p-Chloroaniline (30 mg, 0.24 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL), added with triphosgene (70 mg, 0.24 mmol) under ice bath, then slowly added dropwise with triethylamine (0.2 mL, 1.6 mmol). The reaction solution was stirred under ice bath for 25 minutes, then concentrated under reduced pressure, added with ultra-dry N,N-dimethylformamide (5 mL), added under ice bath with a solution of 4-(2-aza-spiro[3.4]oct-6-yl)amino-6-fluoroquinoline hydrochloride (65 mg, 0.21 mmol) in N,N-dimethylformamide (1 mL), then slowly added dropwise with triethylamine (0.3 mL, 2.3 mmol). After the reaction solution was stirred for 10 minutes under ice bath, the ice bath was removed. The reaction solution was stirred at room temperature for 40 minutes, then poured into water to precipitate a solid, which was filtered, washed with water, and dried and separated by solid column chromatography [methanol/dichloromethane=1/20] to obtain a white solid N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.4]octan-2-carboxamide (68 mg, 70% yield).

[0202] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.53 (s, 1H), 8.41 (d, J=5.3 Hz, 1H), 8.18 (dd, J=11.2, 2.9 Hz, 1H), 7.85 (dd, J=9.2, 5.8 Hz, 1H), 7.54 (d, J=9.0 Hz, 2H), 7.28 (d, J=8.9 Hz, 2H), 6.81 (d, J=6.0 Hz, 1H), 6.51 (d, J=5.4 Hz, 1H), 3.98-3.81 (m, 3H), 2.43-2.33 (m, 1H), 2.21-2.11 (m, 2H), 2.10-1.96 (m, 2H), 1.96-1.84 (m, 2H), 1.83-1.73 (m, 2H).

[0203] LC-MS (m/z) 425 (M+1).

Example 20: Preparation of N-(benzo[d][1,3]dioxol-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0204] ##STR00078##

[0205] According to the steps similar to those in Example 3, a white solid N-(benzo[d] [1,3]dioxol-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (45 mg, 45% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (65 mg, 0.22 mmol) and 3,4-methylenedioxyaniline (33 mg, 0.242 mmol).

[0206] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J=5.1 Hz, 1H), 8.31 (s, 1H), 8.02 (dd, J=9.2, 5.4 Hz, 1H), 7.79 (dd, J=9.7, 3.0 Hz, 1H), 7.66 (td, J=8.8, 3.0 Hz, 1H), 7.18 (d, J=2.0 Hz, 1H), 6.92 (d, J=5.2 Hz, 1H), 6.85 (dd, J=8.4, 2.1 Hz, 1H), 6.77 (d, J=8.4 Hz, 1H), 5.93 (s, 2H), 4.97 (p, J=6.6 Hz, 1H), 4.03 (s, 2H), 3.97 (s, 2H), 2.94-2.79 (m, 2H), 2.47-2.38 (m, 2H).

[0207] LC-MS (m/z) 422 (M+1).

Example 21: Preparation of tert-butyl 6-((quinolin-4-yl)oxy)-2-aza-spiro[3.4]heptan-2-carboxylate

[0208] ##STR00079##

[0209] Tert-butyl 6-hydroxy-2-aza-spiro[3.3]heptan-2-carboxylate (0.717 g, 3.3 mmol) was dissolved in dioxane (20 mL) and stirred at room temperature, added with KHMDS (3.6 mL, 3.6 mmol), stirred at room temperature for 30 minutes, then added with a solution of 4-chloroquinoline (0.5 g, 3.0 mmol) in dioxane (5 mL). The reaction solution was stirred at 75° C. for 60 minutes, cooled to room temperature by stopping heating and stirred for 15 hours, then poured into 20 ml of water, extracted twice with ethyl acetate (20 ml), the organic phases were combined, dried, filtered, and distilled under reduced pressure to obtain a residue, which was then separated by column chromatography (ethyl acetate/petroleum ether=3/2) to obtain a light yellow oil (0.35 g, 33% yield), LC-MS (m/z) 341.16 (M+1).

Example 22: Preparation of 4-(2-aza-spiro[3.3]heptan-6-yl)oxyquinoline Hydrochloride

[0210] ##STR00080##

[0211] Tert-butyl 6-((quinolin-4-yl)oxy)-2-aza-spiro[3.4]heptan-2-carboxylate (0.35 g, 1.028 mmol) was dissolved in 20 mL of dichloromethane, then added with concentrated hydrochloric acid (0.5 mL), stirred at room temperature for 3 hours, and then directly concentrated to obtain an off-white solid (0.235 g, 95% yield), LC-MS (m/z) 241.2 (M+1).

Example 23: Preparation of N-(4-chlorophenyl)-6-(quinolin-4-yloxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0212] ##STR00081##

[0213] p-Chloroaniline (25.3 mg, 0.198 mmol) was dissolved in ultra-dry dichloromethane (10 mL), protected by nitrogen, added with triphosgene (21.4 mg, 0.072 mmol) under ice bath, then added with triethylamine (0.5 mL, 3.6 mmol), stirred under ice bath for 25 minutes, then added under ice bath with a solution of 4-(2-aza-spiro[3.3]heptan-6-yl)oxyquinoline hydrochloride (50 mg, 0.18 mmol) in dichloromethane (5 mL) and triethylamine (0.5 ml), the reaction solution was stirred under ice bath for 10 minutes, then the ice bath was removed, the reaction solution was stirred at room temperature for 120 minutes, added with 5 ml of methanol and stirred for 5 minutes, subjected to rotary evaporation to remove the solvent, and separated by solid column chromatography [methanol/dichloromethane=1/20] to obtain a white solid (38 mg, 53.6% yield).

[0214] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.71 (d, J=5.1 Hz, 1H), 8.57 (s, 1H), 8.17-8.10 (m, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.74 (ddd, J=8.6, 6.8, 1.5 Hz, 1H), 7.62-7.48 (m, 3H), 7.30-7.24 (m, 2H), 6.88 (d, J=5.2 Hz, 1H), 4.97 (t, J=6.6 Hz, 1H), 4.07 (s, 2H), 4.01 (s, 2H), 2.96-2.84 (m, 2H), 2.48-2.37 (m, 2H).

[0215] LC-MS (m/z) 394 (M+1).

Example 24: Preparation of tert-butyl 6-((quinolin-3-yl)oxy)-2-aza-spiro[3.4]heptan-2-carboxylate

[0216] ##STR00082##

[0217] Tert-butyl 6-hydroxy-2-aza-spiro[3.3]heptan-2-carboxylate (98.7 mg, 0.458 mmol) was dissolved in 10 ml of DMF, added with sodium hydride (61.1 mg, 1.5 mmol) and stirred for 30 minutes, then added with 3-chloroquinoline (50 mg, 0.3 mmol), heated to 75° C. and reacted for 1 hour, poured into ice water (20 ml), extracted twice with ethyl acetate (20 ml), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain a residue, which was then separated by column chromatography [ethyl acetate/petroleum ether=3/2] to obtain a white solid (30 mg, 49% yield), LC-MS (m/z) 341 (M+1).

Example 25: Preparation of 3-(2-aza-spiro[3.3]heptan-6-yl)oxyquinoline Hydrochloride

[0218] ##STR00083##

[0219] Tert-butyl 6-((quinolin-3-yl)oxy)-2-aza-spiro[3.4]heptan-2-carboxylate (30 mg, 0.088 mmol) was dissolved in 20 mL of dichloromethane, added with concentrated hydrochloric acid (0.5 mL) stirred at room temperature for 3 hours and then concentrated directly to obtain a white solid (23.1 mg, 95% yield), LC-MS (m/z) 241 (M+1).

Example 26: Preparation of N-(4-chlorophenyl)-6-(quinolin-3-yloxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0220] ##STR00084##

[0221] p-Chloroaniline (13.5 mg, 0.1 mmol) was dissolved in ultra-dry dichloromethane (10 mL), protected by nitrogen, added with triphosgene (13 mg, 0.043 mmol) under ice bath, then added with triethylamine (0.5 mL, 3.6 mmol), stirred under ice bath for 25 minutes, then added under ice bath with a solution of 3-(2-aza-spiro[3.3]heptan-6-yl)oxyquinoline hydrochloride (23.1 mg, 0.08 mmol) in dichloromethane (5 mL) and triethylamine (0.5 ml), the reaction solution was stirred under ice bath for 10 minutes, then the ice bath was removed, the reaction solution was stirred at room temperature for 120 minutes, added with 5 ml of methanol and stirred for 5 minutes, subjected to rotary evaporation to remove the solvent, and separated by solid column chromatography [methanol/dichloromethane=1/20] to obtain a white solid (25 mg, 79% yield).

[0222] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.60 (d, J=2.8 Hz, 2H), 7.95-7.88 (m, 2H), 7.63-7.52 (m, 5H), 7.27 (d, J=8.9 Hz, 2H), 4.86-4.82 (m, 1H), 4.06 (s, 2H), 3.99 (s, 2H), 2.89-2.84 (m, 2H), 2.37-2.32 (m, 2H).

[0223] LC-MS (m/z) 394.2 (M+1).

Example 27: Preparation of tert-butyl 6-(quinazolin-4-yloxy)-2-aza-spiro[3.3]heptan-2-carboxylate

[0224] ##STR00085##

[0225] Tert-butyl 6-hydroxy-2-aza-spiro[3.3]heptan-2-carboxylate (155.5 mg, 0.729 mmol) was dissolved in 20 ml of DMF, added with sodium hydride (120 mg, 3 mmol) under ice bath and stirred for 30 minutes, then added with 3-chloroquinoline (100 mg, 0.6 mmol), reacted at room temperature for 0.5 hours, poured into ice water (30 ml), extracted twice with ethyl acetate (20 ml), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and subjected to rotary evaporation under reduced pressure to obtain a brown liquid (120 mg, 58% yield). LC-MS (m/z) 342 (M+1).

Example 28: Preparation of 4-(2-aza-spiro[3.3]heptan-6-yl)oxyquinazoline Trifluoroacetate

[0226] ##STR00086##

[0227] Tert-butyl 6-(quinazolin-4-yloxy)-2-aza-spiro[3.3]heptan-2-carboxylate (70 mg, 0.2 mmol) was dissolved in 20 mL of dichloromethane, then added with trifluoroacetic acid (0.5 mL), stirred at room temperature for 3 hours and concentrated directly to obtain a gray liquid (58.7 mg, 95% yield). LC-MS (m/z) 241 (M+1).

Example 29: Preparation of N-(4-chlorophenyl)-6-(quinazolin-4-yloxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0228] ##STR00087##

[0229] p-Chloroaniline (31.3 mg, 0.24 mmol) was dissolved in ultra-dry dichloromethane (10 mL), protected by nitrogen, added with triphosgene (24 mg, 0.08 mmol) under ice bath, then added with triethylamine (0.5 mL, 3.6 mmol), stirred under ice bath for 25 minutes, added under ice bath with a solution of 4-(2-aza-spiro[3.3]heptan-6-yl)oxyquinazoline trifluoroacetate (58.7 mg, 0.195 mmol) in dichloromethane (5 mL) and triethylamine (0.5 ml), the reaction solution was stirred under ice bath for 10 minutes, then the ice bath was removed, the reaction solution was stirred at room temperature for 120 minutes, added with 5 ml of methanol, stirred for 5 minutes, subjected to rotary evaporation to remove the solvent, and separated by solid column chromatography [methanol/dichloromethane=1/20] to obtain a white solid (30 mg, 39% yield).

[0230] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.78 (s, 1H), 8.56 (s, 1H), 8.21-8.13 (m, 1H), 8.01-7.86 (m, 2H), 7.70 (ddd, J=8.2, 6.6, 1.5 Hz, 1H), 7.57-7.49 (m, 2H), 7.31-7.20 (m, 2H), 5.40 (p, J=6.9 Hz, 1H), 5.00 (d, J=6.2 Hz, 4H), 2.90-2.80 (m, 2H), 2.49-2.42 (m, 2H).

[0231] LC-MS (m/z) 395 (M+1).

Example 30: Preparation of N-(4-fluorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-3-aza-cyclo[3.1.0]hexan-3-carboxamide

[0232] ##STR00088##

[0233] p-Fluoroaniline (19.3 mg, 0.174 mmol) was dissolved in ultra-dry dichloromethane (10 mL), protected by nitrogen, added with triphosgene (17.2 mg, 0.058 mmol) under ice bath, then added with triethylamine (0.5 mL, 3.6 mmol), stirred under ice bath for 25 minutes, added under ice bath with a solution of 4-((3-aza-cyclo[3.1.0]hex-4-yl)oxy)-6-fluoroquinoline hydrochloride (38.8 mg, 0.138 mmol) in dichloromethane (5 mL) and triethylamine (0.5 ml), the reaction solution was stirred under ice bath for 10 minutes, then the ice bath was removed, the reaction solution was stirred at room temperature for 120 minutes, added with 5 ml of methanol, stirred for 5 minutes, subjected to rotary evaporation to remove the solvent, and separated by solid column chromatography [methanol/dichloromethane=1/20] to obtain a white solid (18 mg, 34% yield).

[0234] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.77 (d, J=5.1 Hz, 1H), 8.23 (s, 1H), 8.04 (dd, J=9.2, 5.4 Hz, 1H), 7.73 (dd, J=9.7, 2.9 Hz, 1H), 7.70-7.62 (m, 1H), 7.56-7.47 (m, 2H), 7.30 (d, J=5.1 Hz, 1H), 7.11-7.02 (m, 2H), 3.97 (d, J=1.5 Hz, 1H), 3.92 (d, J=10.5 Hz, 2H), 3.56 (dt, J=10.3, 2.3 Hz, 2H), 2.17 (dt, J=3.0, 1.5 Hz, 2H).

[0235] LC-MS (m/z) 382 (M+1).

Example 31: Preparation of N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-3-aza-cyclo[3.1.0]hexan-3-carboxamide

[0236] ##STR00089##

[0237] p-Chloroaniline (22.3 mg, 0.174 mmol) was dissolved in ultra-dry dichloromethane (10 mL), protected by nitrogen, added with triphosgene (17.2 mg, 0.058 mmol) under ice bath, then added with triethylamine (0.5 mL, 3.6 mmol), stirred under ice bath for 25 minutes, added under ice bath with a solution of 4-((3-aza-cyclo[3.1.0]hex-4-yl)oxy)-6-fluoroquinoline hydrochloride (38.8 mg, 0.138 mmol) in dichloromethane (5 mL) and triethylamine (0.5 ml), the reaction solution was stirred under ice bath for 10 minutes, then the ice bath was removed, and the reaction solution was stirred at room temperature for 120 minutes, added with 5 ml of methanol, stirred for 5 minutes, subjected to rotary evaporation to remove the solvent, and separated by solid column chromatography [methanol/dichloromethane=1/20] to obtain a white solid N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-3-aza-cyclo[3.1.0]hexan-3-carboxamide (25 mg, 44.9% yield).

[0238] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.77 (d, J=5.1 Hz, 1H), 8.32 (s, 1H), 8.04 (dd, J=9.2, 5.4 Hz, 1H), 7.72 (dd, J=9.7, 2.9 Hz, 1H), 7.70-7.62 (m, 1H), 7.61-7.54 (m, 2H), 7.32-7.25 (m, 3H), 3.97 (t, J=1.5 Hz, 1H), 3.93 (d, J=10.5 Hz, 2H), 3.57 (dt, J=10.5, 2.2 Hz, 2H), 2.16 (dt, J=3.0, 1.5 Hz, 2H).

[0239] LC-MS (m/z) 398 (M+1).

Example 32: Preparation of N-(2,3-dihydrobenzofuran-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0240] ##STR00090##

[0241] According to the steps similar to those in Example 3, a white solid N-(2,3-dihydrobenzofuran-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (55 mg, 85% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (45 mg, 0.15 mmol) and 2,3-dihydrobenzofuran-5-amine (24.9 mg, 0.18 mmol).

[0242] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J=5.1 Hz, 1H), 8.18 (s, 1H), 8.02 (dd, J=9.2, 5.4 Hz, 1H), 7.79 (dd, J=9.7, 3.0 Hz, 1H), 7.69-7.60 (m, 1H), 7.35 (d, J=2.2 Hz, 1H), 7.09 (dd, J=8.6, 2.3 Hz, 1H), 6.92 (d, J=5.2 Hz, 1H), 6.62 (d, J=8.5 Hz, 1H), 4.97 (p, J=6.6 Hz, 1H), 4.46 (t, J=8.7 Hz, 2H), 4.02 (s, 2H), 3.96 (s, 2H), 3.12 (t, J=8.6 Hz, 2H), 2.92-2.83 (m, 2H), 2.47-2.37 (m, 2H).

[0243] LC-MS (m/z) 420 (M+1).

Example 33: Preparation of N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0244] ##STR00091##

[0245] According to the steps similar to those in Example 3, a white solid N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-carboxamide (35 mg, 52% yield) was prepared from 4-((2-aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquine hydrochloride (45 mg, 0.15 mmol) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (27.8 mg, 0.18 mmol).

[0246] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J=5.1 Hz, 1H), 8.21 (s, 1H), 8.02 (dd, J=9.2, 5.4 Hz, 1H), 7.79 (dd, J=9.7, 2.9 Hz, 1H), 7.65 (td, J=8.8, 2.9 Hz, 1H), 7.09 (d, J=2.5 Hz, 1H), 6.97-6.85 (m, 2H), 6.70 (d, J=8.7 Hz, 1H), 4.97 (p, J=6.6 Hz, 1H), 4.25-4.13 (m, 4H), 4.02 (s, 2H), 3.96 (s, 2H), 2.96-2.80 (m, 2H), 2.48-2.37 (m, 2H).

[0247] LC-MS (m/z) 436 (M+1).

Example 34: Preparation of tert-butyl 6-(isoquinolin-1-yloxy)-2-aza-spiro[3.3]heptan-2-carboxylate

[0248] ##STR00092##

[0249] Tert-butyl 6-hydroxy-2-aza-spiro[3.3]heptan-2-carboxylate (106 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (10 ml), and added with sodium hydride (92 mg, 60% in mineral, 2.5 mmol), the reaction solution was stirred at room temperature for 5 minutes, and then added with 1-chloroisoquinoline (82 mg, 0.5 mmol). The reaction solution was stirred at room temperature for 15 hours, and quenched by adding water. The solid was precipitated by adding water, filtered and washed with water to obtain a pale yellow solid tert-butyl 6-(isoquinolin-1-yloxy)-2-aza-spiro[3.3]heptan-2-carboxylate (80 mg, 47% yield), LC-MS (m/z) 341 (M+1).

Example 35: Preparation of 1-(2-aza-spiro[3.3]heptan-6-yl)oxyisoquinoline Hydrochloride

[0250] ##STR00093##

[0251] Tert-butyl 6-(isoquinolin-1-yloxy)-2-aza-spiro[3.3]heptan-2-carboxylate (80 mg, 0.23 mmol) was dissolved in 5 mL of dichloromethane, then added with concentrated hydrochloric acid (1 mL). The reaction solution was stirred at room temperature for 16 hours and then concentrated directly to obtain an off-white solid 1-(2-aza-spiro[3.3]heptan-6-yl)oxyisoquinoline hydrochloride (100 mg, 95% yield), LC-MS (m/z) 241 (M+1).

Example 36: Preparation of N-(4-chlorophenyl)-6-(isoquinolin-1-yloxy)-2-aza-spiro[3.3]heptan-2-carboxamide

[0252] ##STR00094##

[0253] p-Chloroaniline (30 mg, 0.24 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL), added with triphosgene (70 mg, 0.24 mmol) under ice bath, then slowly added dropwise with triethylamine (0.2 mL, 1.6 mmol). The reaction solution was stirred under ice bath for 25 minutes, then concentrated under reduced pressure, added with ultra-dry N,N-dimethylformamide (5 mL), added under ice bath with a solution of 1-(2-aza-spiro[3.3]heptan-6-yl) oxyisoquinoline hydrochloride (60 mg, 0.21 mmol) in N,N-dimethylformamide (1 mL), and then slowly added dropwise with triethylamine (0.3 mL, 2.3 mmol). After the reaction solution was stirred under ice bath for 10 minutes, the ice bath was removed. The reaction solution was stirred at room temperature for 40 minutes, and then poured into water. The solid was precipitated, filtered, washed with water, dried, and separated by solid column chromatography [methanol/dichloromethane=1/25] to obtain a white solid N-(4-chlorophenyl)-6-(isoquinolin-1-yloxy)-2-aza-spiro[3.3]heptan-2-carboxamide (68 mg, 70% yield).

[0254] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.56 (s, 1H), 8.18 (d, J=8.3 Hz, 1H), 7.98 (d, J=5.8 Hz, 1H), 7.89 (d, J=8.2 Hz, 1H), 7.80-7.72 (m, 1H), 7.63 (t, J=7.6 Hz, 1H), 7.56-7.51 (m, 2H), 7.38 (d, J=5.9 Hz, 1H), 7.30-7.24 (m, 2H), 5.32 (p, J=6.9 Hz, 1H), 4.07 (s, 2H), 4.00 (s, 2H), 2.88-2.76 (m, 2H), 2.45-2.36 (m, 2H).

[0255] LC-MS (m/z) 394 (M+1).

Example 37: Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)propionate

[0256] ##STR00095##

[0257] 4-((2-Aza-spiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (600 mg, 2.0 mmol), ethyl 2-bromopropionate (841 mg, 4.0 mmol) and potassium carbonate (962 mg, 6.0 mmol) were dissolved in acetonitrile (30 mL). The reaction solution was stirred at room temperature for 16 hours, then poured into water, extracted with ethyl acetate, the organic phases were combined, dried, filtered, and distilled under reduced pressure to obtain a residue, which was then separated by column chromatography [methanol/dichloromethane=1/25] to obtain a colorless oily substance ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)propionate (450 mg, 61% yield), LC-MS (m/z) 259 (M+1).

Example 38: Preparation of 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)propionic Acid

[0258] ##STR00096##

[0259] Ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)propionate (380 mg, 1.0 mmol) was dissolved in ethanol (8 mL) and water (0.5 mL), then added with sodium hydroxide (85 mg, 2.0 mmol). The reaction solution was stirred at room temperature for 5 hours, then added with concentrated hydrochloric acid to adjust pH to 3-4, and directly concentrated to obtain a crude off-white solid 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)propionic acid (460 mg, crude product), which was used directly in the next step. LC-MS (m/z) 331 (M+1).

Example 39: Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)oxo)-2-aza-spiro[3.3]heptan-2-yl)propionamide

[0260] ##STR00097##

[0261] 2-(6-((6-Fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)propionic acid (350 mg, 1.1 mmol), p-chloroaniline (270 mg, 2.2 mmol) and N,N-diisopropylethylamine (0.66 mL, 3.6 mmol) were dissolved in N,N-dimethylformamide (15 mL), and then added with 1-hydroxybenzotriazole (223 mg, 1.6 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (420 mg, 2.2 mmol). The reaction solution was stirred at room temperature for 15 hours, then poured into water and adjusted with sodium hydrogen carbonate aqueous solution to pH 8-10, a solid was precipitated, filtered, washed with water, and dried to obtain a yellowish solid, which was separated by column chromatography [methanol/dichloromethane=1/25] to obtain a white solid N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl)propionamide (130 mg, 28% yield), LC-MS (m/z) 440 (M+1).

[0262] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.72 (s, 1H), 8.68 (d, J=5.2 Hz, 1H), 8.01 (dd, J=9.6, 5.2 Hz, 1H), 7.77 (dd, J=9.6, 2.8 Hz, 1H), 7.72-7.62 (m, 3H), 7.38-7.34 (m, 2H), 6.90 (d, J=5.2 Hz, 1H), 4.96-4.90 (m, 1H), 3.37-3.25 (m, 4H), 2.97 (dd, J=13.2, 6.8 Hz, 1H), 2.84-2.77 (m, 2H), 2.34 (dd, J=13.2, 6.8 Hz, 2H), 1.11 (d, J=6.8 Hz, 3H).

Example 40: Preparation of N-cyclohexyl-2-(6-((6-fluoroquinolin-4-yl)oxo)-2-aza-spiro[3.3]heptan-2-yl)propionamide

[0263] ##STR00098##

[0264] According to the steps similar to those in Example 39, a white solid N-cyclohexyl-2-(6-((6-fluoroquinolin-4-yl)oxo)-2-aza-spiro[3.3]heptan-2-yl)propionamide (56 mg, 37% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl) propionic acid (120 mg, 0.36 mmol) and cyclohexylamine (30 mg, 0.36 mmol). LC-MS (m/z) 412 (M+1).

[0265] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.71 (d, J=5.2 Hz, 1H), 8.03 (dd, J=9.6, 5.2 Hz, 1H), 7.78 (dd, J=9.6, 2.8 Hz, 1H), 7.69-7.64 (m, 1H), 7.28 (d, J=8.4 Hz, 1H), 6.92 (d, J=5.2 Hz, 1H), 4.98-4.92 (m, 1H), 3.56-3.53 (m, 1H), 3.32-3.23 (m, 2H), 3.21-3.19 (m, 2H), 2.84-2.70 (m, 3H), 2.33 (dd, J=12.0, 6.8 Hz, 2H), 1.71-1.67 (m, 4H), 1.57 (d, J=12.0 Hz, 1H), 1.30-1.12 (m, 6H), 0.99 (d, J=6.8 Hz, 3H).

Example 41: Preparation of 2-(6-((6-fluoroquinolin-4-yl)oxo)-2-aza-spiro[3.3]heptan-2-yl)-N-(pyridin-3-yl)propionamide

[0266] ##STR00099##

[0267] According to the steps similar to those in Example 39, a white solid N-cyclohexyl-2-(6-((6-fluoroquinolin-4-yl)oxo)-2-aza-spiro[3.3]heptan-2-yl)propionamide (15 mg, 10% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptan-2-yl) propionic acid (120 mg, 0.36 mmol) and 3-aminopyridine (30 mg, 0.31 mmol). LC-MS (m/z) 407 (M+1).

[0268] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.81 (s, 1H), 8.82 (d, J=2.4 Hz, 1H), 8.68 (d, J=5.2 Hz, 1H), 8.26 (dd, J=4.8, 1.6 Hz, 1H), 8.10-8.07 (m, 1H), 8.01 (dd, J=9.6, 5.4 Hz, 1H), 7.77 (dd, J=9.6, 2.8 Hz, 1H), 7.67-7.62 (m, 1H), 7.33 (dd, J=8.4, 4.8 Hz, 1H), 6.90 (d, J=5.2 Hz, 1H), 4.97-4.905 (m, 1H), 3.380-3.27 (m, 4H), 3.00 (q, J=6.8 Hz, 1H), 2.87-2.76 (m, 2H), 2.34 (dd, J=12.0, 6.8 Hz, 2H), 1.13 (d, J=6.8 Hz, 3H).

Example 42: Preparation of tert-butyl 6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-carboxylate

[0269] ##STR00100##

[0270] According to the steps similar to those in Example 1, a white solid tert-butyl 6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexa-3-carboxylate (50 mg, 9% yield) was prepared from tert-butyl 6-(hydroxymethyl)-3-aza-bicyclo[3.1.0]hexa-3-carboxylate (388 mg, 1.82 mmol) and 4-chloro-6-fluoroquinoline (270 mg, 1.5 mmol). LC-MS (m/z) 359 (M+1).

Example 43: Preparation of 4-((3-aza-bicyclo[3.1.0]hexan-6-yl)methoxy)-6-fluoroquinoline Hydrochloride

[0271] ##STR00101##

[0272] According to the steps similar to those in Example 2, a white solid 4-((3-aza-bicyclo[3.1.0]hexan-6-yl)methoxy)-6-fluoroquinoline hydrochloride (50 mg, 90% yield) was prepared from tert-butyl 6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-carboxylate (50 mg, 0.14 mmol). LC-MS (m/z) 259 (M+1).

Example 44: Preparation of ethyl 2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate

[0273] ##STR00102##

[0274] According to the steps similar to those in Example 37, a white solid ethyl 2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate (30 mg, 60% yield) was prepared from 4-((3-aza-bicyclo[3.1.0]hexan-6-yl)methoxy)-6-fluoroquinoline hydrochloride (50 mg, 0.19 mmol) and ethyl 2-bromopropionate (63 mg, 0.38 mmol). LC-MS (m/z) 359 (M+1).

Example 45: Preparation of 2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic Acid

[0275] ##STR00103##

[0276] According to the steps similar to those in Example 38, a white solid 2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (25 mg, 92% yield) was prepared from ethyl 2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate (30 mg, 0.08 mmol). LC-MS (m/z) 331 (M+1).

Example 46: Preparation of N-(4-chlorophenyl)-2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionamide (12659)

[0277] ##STR00104##

[0278] According to the steps similar to those in Example 39, a light yellow solid N-(4-chlorophenyl)-2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0]hexan-3-yl) propionamide (8 mg, 24% yield) was prepared from 2-(6-(((6-fluoroquinolin-4-yl)oxo)methyl)-3-aza-bicyclo[3.1.0)hexan-3-yl)propionic acid (25 mg, 0.08 mmol) and 4-chloropropionamide (20 mg, 0.16 mmol). LC-MS (m/z) 440 (M+1).

[0279] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.76 (s, 1H), 8.70 (d, J=5.2 Hz, 1H), 8.02 (dd, J=9.2, 5.2 Hz, 1H), 7.78 (dd, J=9.2, 3.0 Hz, 1H), 7.70-7.61 (m, 3H), 7.37-7.31 (m, 2H), 7.04 (d, J=5.2 Hz, 1H), 4.25-4.07 (m, 2H), 3.19 (q, J=6.8 Hz, 1H), 3.01 (dd, J=14.4, 8.4 Hz, 2H), 2.65 (td, J=9.6, 8.4, 2.8 Hz, 1H), 2.58-2.53 (m, 1H), 1.85 (dt, J=7.2, 4.4 Hz, 1H), 1.59 (tq, J=7.2, 4.4, 3.6 Hz, 2H), 1.22 (d, J=6.8 Hz, 3H).

Example 47: Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate

[0280] ##STR00105##

[0281] According to the steps similar to those in Example 37, a white solid ethyl 2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate (90 mg, 90% yield) was prepared from 4-((3-aza-bicyclo[3.1.0]hexan-6-yl)oxo)-6-quinoline hydrochloride (80 mg, 0.29 mmol) and ethyl 2-bromopropionate (103 mg, 0.58 mmol). LC-MS (m/z) 345 (M+1).

Example 48: Preparation of 2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic Acid

[0282] ##STR00106##

[0283] According to the steps similar to those in Example 38, a white solid 2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (100 mg, 73% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate (90 mg, 0.26 mmol). LC-MS (m/z) 317 (M+1).

Example 49: Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionamide

[0284] ##STR00107##

[0285] According to the steps similar to those in Example 39, a white solid 2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (45 mg, 36% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)oxo)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (100 mg, 0.32 mmol) and 4-chloroaniline (37 mg, 0.29 mmol). LC-MS (m/z) 426 (M+1).

[0286] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.77 (s, 1H), 8.78 (d, J=5.2 Hz, 1H), 8.03 (dd, J=9.2, 5.2 Hz, 1H), 7.74-7.61 (m, 4H), 7.41-7.31 (m, 2H), 7.25 (d, J=5.2 Hz, 1H), 4.39 (s, 1H), 3.24-3.21 (m, 3H), 2.78-2.68 (m, 2H), 1.93 (s, 1H), 1.26 (d, J=6.8 Hz, 3H).

Example 50: Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-carboxylate

[0287] ##STR00108##

[0288] 4-Chloro-6-fluoroquinoline (271 mg, 1.5 mmol), tert-butyl 6-amino-2-aza-spiro[3.3]heptan-2-carboxylate (318 mg, 1.5 mmol) and cesium carbonate (1.5 g, 4.5 mmol) were dissolved in dioxane (5 mL), then added with 2-dicyclohexylphospho-2,4,6-triisopropylbiphenyl (71 mg, 0.15 mmol) and tris(dibenzylideneacetone) dipalladium (70 mg, 0.075 mmol), and protected by nitrogen atmosphere. The reaction solution was heated to 123° C. and stirred for 7 hours, then poured into water, extracted with ethyl acetate, washed with water, and the organic phase was concentrated under reduced pressure to obtain a residue, which was separated by column chromatography [EA] to obtain a white solid tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-carboxylate (280 mg, 52% yield). LC-MS (m/z) 358 (M+1).

Example 51: Preparation of 6-fluoro-N-(2-aza-spiro[3.3]heptan-6-yl)quinolin-4-amine Hydrochloride

[0289] ##STR00109##

[0290] According to the steps similar to those in Example 2, a white solid 6-fluoro-N-(2-aza-spiro[3.3]heptan-6-yl)quinolin-4-amine hydrochloride (128 mg, 94% yield) was prepared from tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-carboxylate (180 mg, 0.51 mmol). LC-MS (m/z) 258 (M+1). LC-MS (m/z) 258 (M+1).

Example 52: Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionate

[0291] ##STR00110##

[0292] According to the steps similar to those in Example 37, a white solid ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionate (150 mg, 83% yield) was prepared from 6-fluoro-N-(2-aza-spiro[3.3]heptan-6-yl)quinolin-4-amine hydrochloride (128 mg, 0.5 mmol) and ethyl 2-bromopropionate (100 mg, 0.55 mmol). LC-MS (m/z) 358 (M+1).

Example 53: Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.1.0]hexan-2-yl)propionic Acid

[0293] ##STR00111##

[0294] According to the steps similar to those in Example 38, a white solid 2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionic acid (100 mg, 72% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro(3.3)heptan-2-yl)propionate (150 mg, 0.42 mmol). LC-MS (m/z) 330 (M+1).

Example 54: Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionamide

[0295] ##STR00112##

[0296] According to the steps similar to those in Example 39, a white solid N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionamide (5 mg, 4% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionic acid (100 mg, 0.3 mmol) and 4-chloroaniline. LC-MS (m/z) 439 (M+1).

[0297] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.15 (s, 1H), 8.44 (d, J=5.6 Hz, 1H), 8.23 (dd, J=10.8, 2.8 Hz, 1H), 7.89 (dd, J=10.2, 5.6 Hz, 1H), 7.72-7.60 (m, 3H), 7.38 (d, J=8.4 Hz, 2H), 6.46 (d, J=5.6 Hz, 1H), 4.05 (dd, J=14.0, 6.8 Hz, 1H), 3.72-3.51 (m, 5H), 2.74 (d, J=10.0 Hz, 2H), 2.33-2.28 (m, 2H), 2.06-1.92 (m, 1H), 1.24 (d, J=4.8 Hz, 3H).

Example 55: Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-carboxylate

[0298] ##STR00113##

[0299] Tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-aza-spiro[3.3]heptan-2-carboxylate (150 mg, 0.42 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL), added with sodium hydride (84 mg, 2.1 mmol), protected by nitrogen atmosphere. The reaction solution was stirred at room temperature for 5 minutes, then added with iodomethane (89 mg, 0.63 mmol), protected by nitrogen atmosphere. The reaction solution was heated to 60° C. and stirred for 7 hours, then quenched by adding water, distilled under reduced pressure to remove tetrahydrofuran, extracted with ethyl acetate, washed with water, dried, concentrated under reduced pressure to obtain a residue, which was separated by column chromatography [EA] to obtain a colorless oil tert-butyl 6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-carboxylate (50 mg, 32% yield). LC-MS (m/z) 372 (M+1).

Example 56: Preparation of 6-fluoro-N-methyl-N-(2-aza-spiro[3.3]heptan-6-yl)quinolin-4-amine Hydrochloride

[0300] ##STR00114##

[0301] According to the steps similar to those in Example 2, a white solid 6-fluoro-N-methyl-N-(2-aza-spiro[3.3]heptan-6-yl)quinolin-4-amine hydrochloride (50 mg, 57% yield) was prepared from tert-butyl 6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptane-2-carboxylate (50 mg, 0.13 mmol). LC-MS (m/z) 272 (M+1).

Example 57: Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionate

[0302] ##STR00115##

[0303] According to the steps similar to those in Example 37, a white solid ethyl 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionate (50 mg, 83% yield) was prepared from 6-fluoro-N-methyl-N-(2-aza-spiro[3.3]heptan-6-yl)quinolin-4-amine hydrochloride (50 mg, 0.16 mmol) and ethyl 2-bromopropionate (60 mg, 0.32 mmol). LC-MS (m/z) 372 (M+1).

Example 58: Preparation of 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionic Acid

[0304] ##STR00116##

[0305] According to the steps similar to those in Example 38, a white solid 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionic acid (40 mg, 82% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionate (50 mg, 0.13 mmol). LC-MS (m/z) 344 (M+1).

Example 59: Preparation of N-(4-chloro)-2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionamide

[0306] ##STR00117##

[0307] According to the steps similar to those in Example 39, a white solid N-(4-chloro)-2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionamide (6 mg, 11% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-aza-spiro[3.3]heptan-2-yl)propionic acid (40 mg, 0.12 mmol) and 4-chloroaniline (20 mg, 0.16 mmol). LC-MS (m/z) 453 (M+1).

[0308] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.74 (s, 1H), 8.62 (d, J=5.2 Hz, 1H), 8.01 (dd, J=9.2, 5.2 Hz, 1H), 7.75-7.65 (m, 3H), 7.63-7.58 (m, 1H), 7.40-7.32 (m, 2H), 6.86 (d, J=5.2 Hz, 1H), 3.90-3.83 (m, 1H), 3.23 (s, 2H), 3.01 (s, 1H), 2.84 (s, 3H), 2.16-2.06 (m, 2H), 1.31-1.25 (m, 4H), 1.12 (d, J=6.8 Hz, 3H).

Example 60: Preparation of tert-butyl 2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-carboxylate

[0309] ##STR00118##

[0310] According to the steps similar to those in Example 50, a white solid tert-butyl 2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-carboxylate (400 mg, 60% yield) was prepared from tert-butyl octahydro-5H-pyrrolo[3,4-c]pyridin-5-carboxylate (457 mg, 1.7 mmol) and 4-chloro-6-fluoroquinoline (350 mg, 1.9 mmol). LC-MS (m/z) 372 (M+1).

Example 61: Preparation of 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline Hydrochloride

[0311] ##STR00119##

[0312] According to the steps similar to those in Example 2, a white solid 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline hydrochloride (380 mg, 93% yield) was prepared from tert-butyl 2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-carboxylate (400 mg, 1.0 mmol). LC-MS (m/z) 272 (M+1).

Example 62: Preparation of ethyl 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionate

[0313] ##STR00120##

[0314] According to the steps similar to those in Example 37, a pale yellow oily substance ethyl 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionate (380 mg, 73% yield) was prepared from 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline hydrochloride (380 mg, 1.2 mmol) and ethyl 2-bromopropionate (362 mg, 2.4 mmol). LC-MS (m/z) 372 (M+1).

Example 63: Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionic Acid

[0315] ##STR00121##

[0316] According to the steps similar to those in Example 38, a white solid 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionic acid (300 mg, 85% yield) was prepared from ethyl 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionate (380 mg, 1.0 mmol). LC-MS (m/z) 344 (M+1).

Example 64: Preparation of N-(4-chlorophenyl)-2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionamide

[0317] ##STR00122##

[0318] According to the steps similar to those in Example 39, a white solid N-(4-chlorophenyl)-2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionamide (20 mg, 15% yield) was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo(3,4-c)pyridin-5-yl)propionic acid (100 mg, 0.29 mmol) and 4-chloroaniline (74 mg, 0.58 mmol). LC-MS (m/z) 453 (M+1).

[0319] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.98 (s, 0.5H), 9.91 (s, 0.5H), 8.38 (dd, J=5.6, 1.6 Hz, 1H), 8.11-8.06 (m, 1H), 7.87 (dd, J=9.2, 6.0 Hz, 1H), 7.80-7.73 (m, 2H), 7.58-7.53 (m, 1H), 7.38-7.35 (m, 2H), 6.54 (dd, J=5.6, 3.2 Hz, 1H), 4.11-4.05 (m, 1H), 3.77-3.72 (m, 1H), 3.68-3.60 (m, 1H), 3.50-3.40 (m, 2H), 3.26-3.21 (m, 1H), 2.80-2.67 (m, 1H), 2.62-2.51 (m, 2H), 2.32-2.28 (m, 2H), 1.76-1.61 (m, 2H), 1.23-1.16 (m, 3H).

Example 65: Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(3-methoxyphenyl)propionamide

[0320] ##STR00123##

[0321] According to the steps similar to those in Example 39, a white solid 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(3-methoxyphenyl)propionamide (15 mg, 12% yield) was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionic acid (100 mg, 0.29 mmol) and 3-methoxyaniline (80 mg, 0.60 mmol). LC-MS (m/z) 449 (M+1).

[0322] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.81 (s, 0.5H), 9.76 (s, 0.5H), 8.37 (d, J=5.6 Hz, 1H), 8.10-8.00 (m, 1H), 7.87 (dd, J=9.2, 6.0 Hz, 1H), 7.56-7.38 (m, 2H), 7.23-7.20 (m, 2H), 6.66-6.62 (m, 1H), 6.55-6.53 (m, 1H), 4.03-3.96 (m, 1H), 3.78-3.73 (m, 3H), 3.66-3.60 (m, 1H), 3.52-3.48 (m, 1H), 3.28-3.23 (m, 1H), 2.78-2.54 (m, 3H), 2.33-2.28 (m, 2H), 2.03-1.97 (m, 1H), 1.74-1.41 (m, 3H), 1.23-1.16 (m, 3H).

Example 66: Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(4-methoxyphenyl)propionamide

[0323] ##STR00124##

[0324] According to the steps similar to those in Example 39, a white solid 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(3-methoxyphenyl)propionamide (12 mg, 11% yield) was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionic acid (85 mg, 0.24 mmol) and 4-methoxyaniline (65 mg, 0.48 mmol). LC-MS (m/z) 449 (M+1).

[0325] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.70 (s, 0.5H), δ 9.64 (s, 0.5H), 8.38 (d, J=5.6 Hz, 1H), 8.11-8.06 (m, 1H), 7.89-7.85 (m, 1H), 7.63-7.53 (m, 3H), 6.92-6.87 (m, 2H), 6.55-6.53 (m, 1H), 4.08-4.04 (m, 1H), 3.78-3.72 (m, 4H), 3.68-3.62 (m, 1H), 3.50-3.46 (m, 1H), 3.23-3.18 (m, 1H), 2.79-2.67 (m, 1H), 2.615-2.55 (m, 2H), 2.33-2.296 (m, 2H), 1.78-1.61 (m, 3H), 1.23-1.16 (m, 3H).

Example 67: Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-phenyl-propionamide

[0326] ##STR00125##

[0327] According to the steps similar to those in Example 39, a white solid 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-phenyl-propionamide (10 mg, 10% yield) was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propionic acid (80 mg, 0.23 mmol) and aniline (45 mg, 0.46 mmol). LC-MS (m/z) 419 (M+1).

[0328] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.87 (s, 0.5H), δ 9.79 (s, 0.5H), 8.42 (d, J=6.0 Hz, 1H), 8.20-8.15 (m, 1H), 7.93-7.90 (m, 1H), 7.74-7.63 (m, 3H), 7.35-7.29 (m, 2H), 7.09-7.04 (m, 1H), 6.62-6.60 (m, 1H), 4.16-4.12 (m, 1H), 3.87-3.72 (m, 2H), 3.61-3.57 (m, 1H), 3.43-3.268 (m, 3H), 2.82-2.58 (m, 3H), 2.34-2.29 (m, 2H), 1.76-1.61 (m, 2H), 1.26-1.15 (m, 3H).

Example 68: Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-carboxylate

[0329] ##STR00126##

[0330] According to the steps similar to those in Example 50, a white solid tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-carboxylate (390 mg, 57% yield) was prepared from tert-butyl 6-amino-3-aza-bicyclo[3.1.0]hexan-3-carboxylate (440 mg, 2.2 mmol) and 4-chloro-6-fluoroquinoline (365 mg, 2.0 mmol). LC-MS (m/z) 344 (M+1).

Example 69: Preparation of N-(3-aza-bicyclo[3.1.0]hexan-6-yl)-6-fluoroquinolin-4-amine hydrochloride

[0331] ##STR00127##

[0332] According to the steps similar to those in Example 2, a white solid N-(3-aza-bicyclo[3.1.0]hexan-6-yl)-6-fluoroquinolin-4-amine hydrochloride (300 mg, 95% yield) was prepared from tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-carboxylate (390 mg, 1.1 mmol). LC-MS (m/z) 244 (M+1).

Example 70: Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate

[0333] ##STR00128##

[0334] According to the steps similar to those in Example 37, a white solid ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate (350 mg, 90% yield) was prepared from 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline hydrochloride (300 mg, 1.1 mmol) and ethyl 2-bromopropionate (402 mg, 2.2 mmol). LC-MS (m/z) 344 (M+1).

Example 71: Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic Acid

[0335] ##STR00129##

[0336] According to the steps similar to those in Example 38, a white solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (320 mg, 95% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionate (350 mg, 1.0 mmol). LC-MS (m/z) 316 (M+1).

Example 72: Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide

[0337] ##STR00130##

[0338] According to the steps similar to those in Example 39, a white solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide (7 mg, 7% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (80 mg, 0.25 mmol) and 4-chloroaniline (64 mg, 0.5 mmol). LC-MS (m/z) 421 (M+1).

[0339] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.77 (s, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.99 (dd, J=11.2, 2.8 Hz, 1H), 7.83 (dd, J=9.2, 5.2 Hz, 1H), 7.68-7.65 (m, 2H), 7.53-7.48 (m, 1H), 7.39 (s, 1H), 7.36-7.32 (m, 2H), 6.69 (d, J=5.2 Hz, 1H), 3.24-3.12 (m, 3H), 2.91 (s, 1H), 2.73 (d, J=8.4 Hz, 1H), 2.65 (d, J=8.4 Hz, 1H), 1.68 (s, 2H), 1.24 (d, J=6.8 Hz, 3H).

Example 73: Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide

[0340] ##STR00131##

[0341] According to the steps similar to those in Example 39, a white solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(3-methoxyphenyl)propionamide (12 mg, 11% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (80 mg, 0.25 mmol) and 4-methoxyaniline (65 mg, 0.5 mmol). LC-MS (m/z) 421 (M+1).

[0342] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.49 (s, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.99 (dd, J=11.2, 2.8 Hz, 1H), 7.83 (dd, J=9.2, 5.6 Hz, 1H), 7.54-7.46 (m, 3H), 7.38 (s, 1H), 6.90-6.82 (m, 2H), 6.69 (d, J=5.2 Hz, 1H), 3.70 (s, 3H), 3.21-3.10 (m, 3H), 2.91 (s, 1H), 2.73 (d, J=8.4 Hz, 1H), 2.65 (d, J=8.4 Hz, 1H), 1.68 (s, 2H), 1.23 (d, J=6.8 Hz, 3H).

Example 74: Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-phenyl-propionamide

[0343] ##STR00132##

[0344] According to the steps similar to those in Example 39, a white solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-phenyl-propionamide (10 mg, 9% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (80 mg, 0.25 mmol) and aniline (60 mg, 0.5 mmol). LC-MS (m/z) 391 (M+1).

[0345] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.63 (s, 1H), 8.45 (d, J=5.2 Hz, 1H), 8.00 (dd, J=11.2, 2.8 Hz, 1H), 7.83 (dd, J=9.2, 5.6 Hz, 1H), 7.62-7.62 (m, 2H), 7.54-7.49 (m, 1H), 7.42 (s, 1H), 7.28 (t, J=7.2 Hz, 2H), 7.04 (t, J=7.2 Hz, 1H), 6.70 (d, J=5.2 Hz, 1H), 3.23-3.140 (m, 3H), 2.91 (s, 1H), 2.74 (d, J=8.4 Hz, 1H), 2.66 (d, J=8.4 Hz, 1H), 1.69 (s, 2H), 1.25 (d, J=6.8 Hz, 3H).

Example 75: Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide

[0346] ##STR00133##

[0347] According to the steps similar to those in Example 39, a white solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide (20 mg, 19% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)propionic acid (80 mg, 0.25 mmol) and 3-methoxyaniline (65 mg, 0.5 mmol). LC-MS (m/z) 421 (M+1).

[0348] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.65 (s, 1H), 8.46 (d, J=5.2 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H), 7.86 (dd, J=9.2, 5.6 Hz, 1H), 7.54 (t, 1H), 7.45 (s, 1H), 7.37 (s, 1H), 7.21-7.19 (m, 2H), 6.72-6.53 (m, 2H), 3.72 (s, 3H), 3.22-3.19 (m, 3H), 2.91 (s, 1H), 2.77 (d, J=8.4 Hz, 1H), 2.67 (d, J=8.4 Hz, 1H), 1.70 (s, 2H), 1.25 (d, J=6.8 Hz, 3H).

Example 76: Preparation of 2-bromo-3-hydroxypropionic Acid

[0349] ##STR00134##

[0350] L-serine (500 mg, 4.8 mmol) and sodium bromide were dissolved in 2.5M sulfuric acid aqueous solution (10 mL), and sodium nitrite (460 mg, 6.7 mmol) was slowly added under ice-salt bath. The reaction solution was slowly warmed to room temperature and reacted for 15 hours, then added with water and ethyl acetate for extraction, washed with brine and water respectively, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain a pale yellow oily substance 2-bromo-3-hydroxypropionic acid (400 mg, 30% yield). LC-MS (m/z) 169 (M+1).

Example 77: Preparation of 2-bromo-N-(4-chlorophenyl)-3-hydroxypropionamide

[0351] ##STR00135##

[0352] 2-Bromo-3-hydroxypropionic acid (200 mg, 1.2 mmol), p-chloroaniline (100 mg, 0.78 mmol) and N,N-diisopropylethylamine (0.6 mL, 3.1 mmol) were dissolved in tetrahydrofuran (5 mL), and then added with 2-(7-oxobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (600 mg, 1.6 mmol). The reaction solution was stirred at room temperature for 5 hours, then poured into water, adjusted with sodium bicarbonate aqueous solution to pH 8-10, extracted with ethyl acetate, washed with water, dried, and concentrated under reduced pressure to obtain a residue, which was separated by column chromatography [ethyl acetate/petroleum ether=1/2] to obtain a light brown solid 2-bromo-N-(4-chlorophenyl)-3-hydroxypropionamide (100 mg, 46% yield). LC-MS (m/z) 279 (M+1).

Example 78: Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-3-aza-bicyclo[3.1.0]hexan-3-yl)-3-hydroxypropionamide

[0353] ##STR00136##

[0354] 2-Bromo-N-(4-chlorophenyl)-3-hydroxypropionamide (65 mg, 0.23 mmol), N-(3-aza-bicyclo[3.1.0]hexan-6-yl)-6-fluoroquinolin-4-amine hydrochloride (40 mg, 0.16 mmol) and potassium carbonate (68 mg, 48 mmol) were dissolved in acetonitrile (5 mL). The reaction solution was stirred at 80° C. for 8 hours, then poured into water, and a solid was precipitated out, filtered, and the residue was separated by column chromatography [methanol/dichloromethane=1/25] to obtain a white solid ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3)heptan-2-yl)propionate (13 mg, 18% yield). LC-MS (m/z) 441 (M+1).

Example 79: Preparation of tert-butyl 5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-carboxylate

[0355] ##STR00137##

[0356] According to the steps similar to those in Example 50, a white solid tert-butyl 5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-carboxylate (200 mg, 65% yield) was prepared from tert-butyl 5-amino-hexahydro-cyclopenta[c]pyrrol-2(1H)-carboxylate (210 mg, 0.9 mmol) and 4-chloro-6-fluoroquinoline (150 mg, 0.82 mmol). LC-MS (m/z) 372 (M+1).

Example 80: Preparation of 6-fluoro-N-(octahydro-cyclopenta[c]pyrrol-5-yl) quinolin-4-amine Hydrochloride

[0357] ##STR00138##

[0358] According to the steps similar to those in Example 2, a white solid 6-fluoro-N-(octahydro-cyclopenta[c]pyrrol-5-yl)quinolin-4-amine hydrochloride (150 mg, 91% yield) was prepared from tert-butyl 5-((6-fluoroquinolin-4-yl)amino)-hexahydro-cyclopenta[c]pyrrol-2(1H)-carboxylate (200 mg, 0.54 mmol). LC-MS (m/z) 272 (M+1).

Example 81: Preparation of ethyl 2-(5-((6-fluoroquinolin-4-yl)amino)-hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)propionate

[0359] ##STR00139##

[0360] According to the steps similar to those in Example 37, a white solid ethyl 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)propionate (100 mg, 55% yield) was prepared from 6-fluoro-N-(octahydro-cyclopenta[c]pyrrol-5-yl)quinolin-4-amine hydrochloride (150 mg, 0.49 mmol) and ethyl 2-bromopropionate (177 mg, 0.98 mmol). LC-MS (m/z) 372 (M+1).

Example 82: Preparation of 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)propionic Acid

[0361] ##STR00140##

[0362] According to the steps similar to those in Example 38, a white solid 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)propionic acid (80 mg, 87% yield) was prepared from ethyl 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)propionate (100 mg, 0.27 mmol). LC-MS (m/z) 344 (M+1).

Example 83: Preparation of N-(4-chlorophenyl)-2-(5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)propionamide

[0363] ##STR00141##

[0364] According to the steps similar to those in Example 39, a light yellow solid N-(4-chlorophenyl)-2-(5-((6-fluoroquinolin-4-yl)amino)hexahydro-cyclopenta[c]pyrrol-2(1H)-yl) propionamide (15 mg, 14% yield) was prepared from 2-(5-((6-fluoroquinolin-4-yl)amino) hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)propionic acid (80 mg, 0.23 mmol) and 4-chloroaniline (60 mg, 0.46 mmol). LC-MS (m/z) 453 (M+1).

[0365] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.84 (s, 1H), 8.37 (d, J=5.6 Hz, 1H), 8.20 (dd, J=11.2, 2.8 Hz, 1H), 7.84 (dd, J=9.2, 5.6 Hz, 1H), 7.70 (d, J=8.8 Hz, 2H), 7.57-7.52 (m, 1H), 7.37 (d, J=8.8 Hz, 2H), 6.93 (d, J=6.4 Hz, 1H), 6.56 (d, J=5.6 Hz, 1H), 4.24-4.17 (m, 1H), 3.10-3.05 (m, 1H), 2.69-2.66 (m, 4H), 2.44-2.37 (m, 1H), 2.03-1.83 (m, 5H), 1.29 (d, J=6.8 Hz, 3H).

Example 84: Preparation of methyl benzo[d][1,3]dioxol-5-yl-glycinate

[0366] ##STR00142##

[0367] 3,4-Methylenedioxyaniline (160 mg, 1.16 mmol) was dissolved in 20 ml of DMF, added with potassium carbonate (480 mg, 3.48 mmol) and ethyl bromoacetate (213 mg, 1.4 mmol), and reacted at room temperature for 16 hours. The reaction solution was poured into 40 ml of water, extracted twice with 20 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, subjected to rotary evaporation to remove the solvent and separated by column chromatography [ethyl acetate/petroleum ether=1/6] to obtain a brown oily substance methyl benzo[d][1,3]dioxol-5-yl-glycinate (175 mg, 72% yield). LC-MS (m/z) 210 (M+H).

Example 85: Preparation of benzo[d][1,3]dioxol-5-yl-glycine

[0368] ##STR00143##

[0369] Methyl benzo[d][1,3]dioxol-5-yl-glycinate (175 mg, 3.42 mmol) was dissolved in 20 ml of methanol, added with sodium hydroxide (164 mg, 4.1 mmol), reacted at room temperature for 16 hours, poured into 20 ml of water, adjusted with hydrochloric acid to pH=4, then extracted twice with 20 ml of ethyl acetate, the organic phases were combined, dried with anhydrous sodium sulfate, filtered, the organic phase was subjected to rotary evaporation to obtain a brown solid benzo[d][1,3]dioxol-5-yl-glycine (160 mg, 98% yield). LC-MS (m/z) 196 (M+H).

Example 86: Preparation of 2-(benzo[d][1,3]dioxol-5-yl-amino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl)piperidin-1-yl)ethyl-1-one

[0370] ##STR00144##

[0371] 1-Boc-4-[2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl]piperidine (100 mg, 0.25 mmol) was dissolved in 20 ml of dichloromethane, then added with 0.5 ml of hydrochloric acid, reacted at room temperature for 2 hours, subjected to rotary evaporation to remove the solvent, added with 10 ml of DMF and stirred until the dissolution was completed, then added with benzo[d][1,3]dioxol-5-yl-glycine (58.5 mg, 0.3 mmol) and 1-hydroxybenzotriazole (67.7 mg, 0.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (95.6 mg, 0.5 mmol), 1 ml of N,N-diisopropylethylamine, reacted at room temperature for 16 hours, poured into 40 ml of water, extracted twice with 30 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the organic phase was subjected to rotary evaporation to remove the solvent and separated by column chromatography [dichloromethane/methanol=100/3] to obtain a white solid 2-(benzo[d][1,3]dioxol-5-yl-amino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl)piperidin-1-yl)ethyl-1-one (50 mg, 42% yield). LC-MS (m/z) 477 (M+H).

Example 87: Preparation of 2-(benzo[d][1,3]dioxol-5-yl-amino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a)isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one

[0372] ##STR00145##

[0373] 2-(Benzo[d][1,3]dioxol-5-yl-amino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl)piperidin-1-yl)ethyl-1-one (50 mg, 0.1 mmol) was dissolved in 10 ml of methanol, cooled under nitrogen protection to 0° C. with ice bath, then added with sodium borohydride (11.3 mg, 0.1 mmol), reacted at room temperature for 2 hours, added with 1 ml of saturated ammonium chloride aqueous solution, stirred for 5 minutes, poured into 20 ml of water, extracted twice with 20 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, the organic phase was subjected to rotary evaporation to remove the solvent and separated by column chromatography (dichloromethane/methanol=20/1) to obtain a light brown solid 2-(benzo[d][1,3]dioxol-5-yl-amino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one (15 mg, 30% yield).

[0374] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 0.7H), 7.97 (s, 0.3H), 7.47-7.42 (m, 2H), 7.22 (s, 0.3H), 7.20 (s, 0.7H), 7.14-7.07 (m, 1H), 6.65 (d, J=8.4 Hz, 1H), 6.42 (d, J=2.0 Hz, 1H), 6.07 (dd, J=8.4, 2.0 Hz, 1H), 5.83 (s, 2H), 5.72-5.67 (m, 0.3H), 5.64-5.57 (m, 0.7H), 5.19-5.11 (m, 0.3H), 4.73 (t, J=4.8 Hz, 0.8H), 4.42 (t, J=12.5 Hz, 1H), 3.94 (s, br, 1H), 3.80 (s, 2H), 3.43 (s, br, 1H), 3.00-2.82 (m, 1H), 2.41-2.23 (m, 1H), 2.04-1.86 (m, 0.7H), 1.87-1.76 (m, 0.3H), 1.74-1.42 (m, 3H), 1.37-0.97 (m, 4H).

[0375] LC-MS (m/z) 479 (M+H).

[0376] In Vitro Biological Evaluation

[0377] The subject of this assay was to comprehensively evaluate different compounds on their enzymatic inhibitory activity in vitro and on cell models against human indoleamine 2,3-dioxygenase 1 (hIDO1).

Example 88: Inhibitory Activity Test at Cellular Level

[0378] In addition to the constitutive expression in immune cells such as myeloid-derived suppressor cells (MDSC), IDO1 is also up-regulated in many tumor cells, or is induced by cytokine such as IFN-γ. In the present application, the inventors used Hela cells expressing IDO1 induced by IFN-γ as a model, to test the IDO1 inhibitory activity of the compounds at the cellular level.

[0379] Main Experimental Principle

[0380] HeLa cells are human cervical cancer cell lines, which can up-regulate the expression of endogenous IDO1 under the induction of human IFN-γ. By adding the substrate L-tryptophan to cell culture medium, the enzymatic catalysis product kynurenine in cell supernatant was detected. In this experiment, the cultured Hela cells were used and co-incubated with different concentrations of test compounds under stimulation of human IFN-γ for a specified period of time, and the color reaction method of enzyme product and p-dimethylaminobenzaldehyde was used to detect the effect on IDO enzyme activity of cells treated by the test compounds.

[0381] Experimental Materials and Reagents

[0382] Recombinant human IFN-γ cytokine was purchased from Sangon Biotechnology Co., Ltd., and phenol red-free DMEM for cell culture was purchased from Gibco. Detection reagents such as L-tryptophan (Sangon, A601911-0050), Kynurenine (Sigma, K8625-100MG), trichloroacetic acid (Sangon, A600968-0250), p-dimethylaminobenzaldehyde (Tianjin Damao Chemical Reagent Factory), 96-well flat bottom plate for cell culture (CORNING, costar 3599).

[0383] Experimental Method

[0384] According to the conventional cell culture experiment procedure, the experiment was carried out in a 96-well flat bottom plate.

[0385] (1) Hela cells were inoculated into 96-well culture plates at an appropriate concentration (about 20,000 cells/well). After adherent culture overnight, the culture medium was replaced with phenol red-free DMEM medium containing 200 μM L-tryptophan, 50 ng/ml cytokine human IFN-γ and test compounds with different concentration gradients (maximum final concentration was 25 μM) and NLG919 were added simultaneously, the vehicle control (DMSO) and negative control wells without cytokine and L-tryptophan were set at the same time, and triplicate wells were set. The cells were continuously cultured for 48 hours before detection.

[0386] (2) 200 μL of the supernatant in the culture well was pipetted out, added with 40 μL of the pre-prepared 30% (w/v) trichloroacetic acid solution, and reacted at 65° C. for 20 minutes, and then centrifuged at 12000 rpm for 15 minutes.

[0387] (3) 100 μL of the supernatant after centrifugation was pipetted out, added into a 96-well flat bottom plate, then added with an equal volume of 2% p-dimethylaminobenzaldehyde in glacial acetic acid and mixed well, and stood at room temperature for 10 minutes.

[0388] (4) microplate reader (ELX800NB) was used to detect the absorbance value of each well, and the detection wavelength was 492 nm.

[0389] (5) the calculation formula of enzymatic inhibition rate of test compound at cellular level was:

Enzyme activity inhibition rate (%)=(OD.sub.solvent control−OD.sub.compound detection well)/(OD.sub.solvent control−OD.sub.negative control)*100%.

[0390] In addition, after the cell enzyme activity inhibition rates of test compounds with different concentration gradients were separately calculated, their cell enzyme activity half inhibition concentrations (EC.sub.50) were calculated by using EC.sub.50 calculator.

[0391] According to the above-mentioned experimental method, the compounds of the present application were subjected to IDO1 enzymatic evaluation at cytological level (the concentration of each test compound was all 100 nM). The data summary was shown in Table 1.

TABLE-US-00001 TABLE 1 Data of hIDO1-cell base inhibition rates of the representative compounds of the present application hIDO1-cell base Example inhibition rate (%) 3 90 4 49 5 63 6 87 7 31 10 90 11 25 14 13 15 18 16 85 19 61 20 64 23 74 26 15 29 10 30 11 31 46 32 44 33 10 36 26 39 91 40 19 41 23 46 49 49 99 54 69 59 85 64 96 65 59 66 90 67 94 72 88 73 83 74 85 75 47 78 42 83 45 87 23 NLG919 10

[0392] According to the above-mentioned experimental method, the compound of the present application was subjected to hIDO1-cell base EC.sub.50 detection. The data summary was shown in Table 2.

TABLE-US-00002 TABLE 2 Data of hIDO1-cell base EC.sub.50 (nM) of the representative compounds of the present application Example EC.sub.50 (nM) 3 14 6 36 16 33 20 46 23 26 39 20 49 5 59 25 72 8

Example 89: Experiment of Inhibiting Enzyme Activity of IDO in HeLa Cell Using Chidamide in Combination with the Compound of Example 3

[0393] Experimental Materials

[0394] Human cervical cancer cell line HeLa was purchased from the Cell Resource Center, Shanghai Institute of Biological Science, Chinese Academy of Sciences, and was routinely cultured at 37° C. and 5% CO.sub.2; the culture medium was DMEM (Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% Penicillin-Streptomycin (HyClone). Trypsin was purchased from Gibco. Recombinant human IFN-γ cytokine was purchased from Sangon Biotech. Phenol red-free DMEM for cell culture was purchased from Gibco. Detection reagents included L-tryptophan (Sangon, A601911-0050), kynurenine (Sigma, K8625-100MG), trichloroacetic acid (Sangon, A600968-0250), p-dimethylaminobenzaldehyde (Tianjin Damao Chemical Reagent Factory). 96-Well flat bottom plates (CORNING, costar 3599) for cell culture.

[0395] Experimental Method

[0396] HeLa cells were digested by trypsin, collected and counted, then inoculated in a 96-well cell culture plate at 2×10.sup.4 cells per well and cultured at 5% CO.sub.2 and 37° C. After the cells were inoculated overnight, the medium was replaced with 200 μM L-tryptophan phenol red-free DMEM medium, and 50 ng/ml cytokine human IFN-γ and drugs with different concentrations (administered according to the group and final concentration shown in FIG. 1) were added simultaneously, the vehicle control (DMSO) and cytokine-free and L-tryptophan negative control wells were set at the same time, and triplicate wells were set. After 48 hours of drug treatment, 200 μL of the supernatant from culture well was pipetted out, added with 40 μL of pre-prepared 30% (w/v) trichloroacetic acid solution, reacted at 65° C. for 20 minutes, and then centrifuged at 12000 rpm for 15 minutes. 100 μL of the supernatant was pipetted out and added into a 96-well flat bottom plate, then equal volume of 2% p-dimethylaminobenzaldehyde in glacial acetic acid was added and mixed well, and stood at room temperature for 10 minutes. The absorbance value of each well at wavelength of 492 nm was read with microplate reader (ELX800NB). After subtracting the cell-free medium OD492-BLK as a blank control from the reading of each well, the OD492-T of each dosing well with subtraction of the detection background and the OD490-T0 of drug-free positive control well were obtained.

[0397] The relative enzyme activity inhibition rate against IDO of each dosing well cell was calculated according to the following formula:

enzyme activity inhibition rate against IDO=(OD.sub.492-T0−OD.sub.492-T)/OD.sub.492-T0×100%

[0398] Experimental Results

[0399] As shown in FIG. 1 and Table 3, as compared with the vehicle control, both chidamide and the compound of Example 3 showed a certain inhibitory effect on enzyme activity of IDO; while the combination of the two drugs showed a significantly synergistic or superimposed inhibitory effect. Considering that HDAC inhibitor did not directly bind to and inhibited the catalytic activity of IDO protease, but indirectly inhibited IDO activity by down-regulating IDO mRNA to reduce protein expression thereof (He Y W, et al. Life Sci, 2013, 93(15):509-515), therefore, the combination of epigenetic inhibitor and IDO inhibitor actually exert a synergistic inhibitory effect on IDO enzyme activity via different mode of action.

TABLE-US-00003 TABLE 3 enzyme inhibition rate against IDO of chidamide and the compound of Example 3 alone and in combination vehicle Combination control Chidamide Example 3 of two drugs Average 0.5 24 31 49 inhibition rate (%)

Example 90: Experiment of Inhibiting Enzyme Activity of IDO in HeLa Cell Using Chidamide in Combination with the Compound of Example 20

[0400] The method similar to Example 89 was applied. The experimental results obtained were shown in FIG. 2 and Table 4.

TABLE-US-00004 TABLE 4 enzyme inhibition rate against IDO of chidamide and the compound of Example 20 alone and in combination vehicle Combination control Chidamide Example 20 of two drugs Average 0.5 22 16 35 inhibition rate (%)

Example 91: Experiment of Inhibiting Enzyme Activity of IDO in HeLa Cell Using Chidamide in Combination with the Compound of Example 72

[0401] The method similar to Example 89 was applied. The experimental results obtained were shown in FIG. 3 and Table 5.

TABLE-US-00005 TABLE 5 enzyme inhibition rate against IDO of chidamide and the compound of Example 72 alone and in combination vehicle Combination control Chidamide Example 72 of two drugs Average 0.5 23 36 58 inhibition rate (%)

Example 92: Experiment of Inhibiting Enzyme Activity of IDO in HeLa Cell Using Chidamide in Combination with the Compound of Example 87

[0402] The method similar to Example 89 was applied. The experimental results obtained were shown in FIG. 4 and Table 6.

TABLE-US-00006 TABLE 6 enzyme inhibition rate against IDO of chidamide and the compound of Example 87 alone and in combination vehicle Combination control Chidamide Example 87 of two drugs Average 0.5 23 20 47 inhibition rate (%)

Example 93: Efficacy Experiment of Chidamide in Combination with Example 3 on Balb/c Mouse CT-26 Tumor Model

[0403] Experimental Materials

[0404] The mouse colon cancer cell line CT-26 was purchased from the Cell Resource Center, Shanghai Institute of Biological Science, Chinese Academy of Sciences, and was routinely cultured at 37° C., 5% CO.sub.2, and the culture medium was DMEM (Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% Penicillin-Streptomycin (HyClone); trypsin was purchased from Gibco. Normal Balb/c mice were purchased from Guangdong Medical Experimental Animal Center.

[0405] Experimental Method

[0406] CT-26 cells were subjected to multiplication culture and kept in a logarithmic growth state. After the number of cells met the requirement, the cells were digested with trypsin and collected, washed sufficiently with a large amount of PBS twice to remove trypsin and serum components, centrifuged at room temperature and 800 rpm for 10 minutes, and the supernatant was discarded. The cells were resuspended in FBS-free DMEM medium and adjusted to reach a cell concentration of 3×10.sup.7/mL.

[0407] Under aseptic condition, the cell suspension was injected into subcutaneous of back of nude mouse at a dose of 100 μL/needle, one injection per Balb/c mouse. During the injection, 1 mL disposable medical syringe was used to ensure that the position and direction for inserting needle to each mouse was basically the same.

[0408] Eight days after cell inoculation, the tumor grew to an average volume of about 100 mm.sup.3. The tumor-bearing mice were randomly divided into four groups (9 mice in each group), namely the vehicle control group, the chidamide group (25 mg/kg, intragastric administration, once per day), Example 3 group (20 mg/kg, intragastric administration, once per day), and the combination of Chidamide and Example 3 group, and then the mice were raised in cages after labeling, the mice was administered according to groups and observed for tumor formation every day. The body weight of each mouse was weighted before administration, and the dose was calculated according to per kilogram of body weight, that was, 10 μL of CMC-Na solution per gram of body weight for the vehicle control group, 10 μL of 2.5 mg/mL Chidamide-CMC-Na suspension per gram of body weight for the chidamide group (25 mg/kg), 10 μL of 2 mg/mL the compound of Example 3-CMC-Na suspension per gram of body weight for the Example 3 group (20 mg/kg), and 10 μL of CMC-Na suspension containing 2.5 mg/ml chidamide and 2 mg/ml the compound of Example 3 per gram of body weight for the combination group were administered intragastrically. The longest diameter of tumor (length) and the widest diameter (width) perpendicular thereto were measured by vernier caliper every 2 days, and the tumor volume was calculated by the formula TS=length×(width).sup.2/2 and recorded. Each mouse was intragastrically administered once per day at regular time; after the last administration on the 17.sup.th day, the mice were sacrificed and the tumors of tumor-bearing mice were stripped and weighed with a meter.

[0409] The relative tumor inhibition rate of each administration group was calculated according to the following formula:

Relative tumor inhibition rate=(average tumor volume of the vehicle control group−average tumor volume of the administration group)/average tumor volume of the vehicle control group×100%

[0410] Experimental Results

[0411] As shown in FIG. 5 and Table 7, compared with the vehicle control group, both of the Chidamide group (25 mg/kg) and the Example 3 group (20 mg/kg) as two single-drug groups showed certain inhibition on tumor volume of mice, and the tumor inhibition rates were 33% and 20%, respectively; while the final relative tumor inhibition rate of the combination group (80%) was significantly higher than the sum of the inhibition rates of the two single-drug groups. As shown in FIG. 6, compared with the average tumor weight of 2.7 g of the vehicle group, the average tumor weights of the Chidamide group and the Example 3 group as single-drug groups were 1.6 g and 2.3 g, respectively, which showed certain inhibition; while the average tumor weight of the combination group was only 0.35 g, which was significantly smaller than the two single-drug groups. The above results showed that Chidamide and IDO inhibitor have good synergistic anti-tumor activity in tumor-bearing mice.

TABLE-US-00007 TABLE 7 Relative tumor inhibition rate of chidamide and the compound of Example 3 alone and in combination Combination Chidamide Example 3 of two drugs Relative tumor 33.6 20.1 80.8 inhibition rate (%)

Example 94: Efficacy Experiment of Chidamide in Combination with Example 20 on Balb/c Mouse CT-26 Tumor Model

[0412] The method similar to that of Example 93 was applied. The experimental results obtained were shown in FIG. 7 and Table 8.

TABLE-US-00008 TABLE 8 Relative tumor inhibition rate of chidamide and the compound of Example 20 alone and in combination Combination Chidamide Example 20 of two drugs Relative tumor 30 8.3 87.7 inhibition rate (%)

Example 95: Efficacy Experiment of Chidamide in Combination with Example 72 on Balb/c Mouse CT-26 Tumor Model

[0413] The method similar to that of Example 93 was applied. The experimental results obtained were shown in FIG. 8 and Table 9.

TABLE-US-00009 TABLE 9 Relative tumor inhibition rate of chidamide and the compound of Example 72 alone and in combination Combination Chidamide Example 72 of two drugs Relative tumor 25.4 7.6 86.9 inhibition rate (%)

Example 96: Efficacy Experiment of Chidamide in Combination with Example 87 on Balb/c Mouse CT-26 Tumor Model

[0414] The method similar to that of Example 93 was applied. The experimental results obtained were shown in FIG. 9 and Table 10.

TABLE-US-00010 TABLE 10 Relative tumor inhibition rate of chidamide and the compound of Example 87 alone and in combination Combination Chidamide Example 87 of two drugs Relative tumor 55 45 89.8 inhibition rate (%)

Example 97: hERG Inhibitory Activity Assay (Patch Clamp Method)

[0415] Non-cardiac drugs may cause the prolongation of myocardial action potential duration by inhibiting the hERG (IKr) channel, increasing the possibility of life-threatening torsade de pointes (TdP) ventricular arrhythmia. In this experiment, HEK293 cell line without endogenous IKr current was used as host cell (this cell line was widely used in the detection of hERG) to evaluate the cardiotoxicity of Example 85 in patent CN108203438A and Example 87 of the present application.

[0416] 1. Cell Culture

[0417] (1) HEK293 cell line stably expressing hERG potassium channel was cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg/mL G418 under the condition of 37° C. and 5% CO.sub.2.

[0418] (2) Cell passage: the old medium was removed, the cell was washed with PBS for one time, then 1 mL of TrypLE™ Express solution was added, and incubated at 37° C. for 0.5 minutes. When the cells were detached from the bottom of the dish, 5 mL of complete medium preheated at 37° C. was added. The cell suspension was gently blown and beaten by pipette for dissociation of the aggregated cells. The cell suspension was transferred into a sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes to collect the cells. After amplifying or maintaining the culture, the cells were inoculated in 6 cm cell-culture dishes at an incubation amount of 2.5*10.sup.5 cells (final volume: 5 mL) for each cell culture dish.

[0419] (3) In order to maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0420] (4) Patch clamp detection: before the experiment, the cells were separated by TrypLE™ Express, 3*10.sup.3 cells were spread on the cover glass, cultured in a 24-well plate (final volume: 500 μL); 18 hours later, the detection was performed.

[0421] 2. Liquid Formulation

[0422] The components of the extracellular fluid used in the whole cell patch clamp experiment (mM) were: 140 NaCl; 1 MgCl.sub.2; 3.5 KCl; 10 Glucose; 10 HEPES; 1.25 NaH.sub.2PO.sub.4 and 2 CaCl.sub.2, NaOH was used to adjust pH value to 7.4, and sucrose was used to adjust osmotic pressure value to 300 mOsm.

[0423] The components of the intracellular fluid (mM) were: 20 KCl; 115 K-Aspartic; 1 MgCl.sub.2; 5 EGTA; 10 HEPES; and 2 Na.sub.2ATP, KOH was used to adjust pH value to 7.2, and sucrose was used to adjust osmotic pressure value to 290 mOsm.

[0424] 3. Patch Clamp Detection

[0425] The voltage stimulation scheme for recording whole-cell hERG potassium current with whole-cell patch clamp was as follows: when a whole-cell seal was formed, the cell membrane voltage was clamped at −80 mV, then the clamp voltage was depolarized from −80 mV to −50 mV and maintained for 0.5 s (used for detection of leakage current), and then stepped to 30 mV and maintained for 2.5 s, and then quickly restored to −50 mV and maintained for 4 s to stimulate the tail current of hERG channel. Data were repeatedly collected every 10 s to observe the effect of drug on the hERG tail current. The stimulation of −50 mV for 0.5 s was used for detection of leakage current. The experimental data were collected by EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0426] A capillary glass tube was drawn into a recording electrode with a microelectrode puller. A microelectrode manipulator was operated under an inverted microscope, the recording electrode was put to contact the cells, and negative pressure suction was conducted to form a GΩ seal. After the GΩ seal was formed, fast capacitance compensation was performed, and then negative pressure was continuously conducted to suck and break cell membrane to form a whole-cell recording mode. Then, slow capacitance compensation was performed, and the membrane capacitance and series resistance were recorded. No leakage compensation was given.

[0427] When the hERG current of the whole-cell recording was stabilized, the drug was administered. After each drug concentration was applied for 5 minutes (or until the current was stable), the next concentration was tested, and a plurality of concentrations for each test sample were tested. The cover glass with spread cells was placed in a recording chamber of the inverted microscope, a working fluid of the test sample and the extracellular fluid without the compound were flowed through the recording chamber from low concentration to high concentration by gravity perfusion to act on the cell, and liquid exchange was performed by a vacuum pump during recording. The current detected for each cell in the compound-free extracellular fluid was used as its own control group. A plurality of cells were tested independently. All electrophysiological experiments were performed at room temperature.

[0428] The drug concentration was selected as 0.3, 1, 3, 10 and 30 μM.

[0429] 4. Data Analysis

[0430] Firstly, the current after action of each concentration and the current of blank control were normalized

[00001]$\left(\frac{\mathrm{Peak}\mathrm{tail}\mathrm{current}\mathrm{compound}}{\mathrm{Peak}\mathrm{tail}\mathrm{current}\mathrm{vehicle}}\right),$

and then the inhibition rate corresponding to each concentration was calculated

[00002]$\left(1-\frac{\mathrm{Peak}\mathrm{tail}\mathrm{current}\mathrm{compound}}{\mathrm{Peak}\mathrm{tail}\mathrm{current}\mathrm{vehicle}}\right).$

For each concentration, mean number and standard error were calculated, and the following equation was used to calculate the half inhibitory concentration of each compound:

[00003]$\mathrm{inhibition}=\frac{1}{1+{\left(\frac{\mathrm{IC}50}{C}\right)}^h}$

[0431] The above equation was used to perform a non-linear fitting of the dose-dependent effect, wherein C represented the concentration of the test sample, IC50 was the half-inhibitory concentration, and h represented the Hill coefficient. The curve fitting and the calculation of IC50 were performed using IGOR software.

[0432] According to the above-mentioned experimental method, the hERG inhibition EC50 value of Example 85 in patent CN108203438A was determined, and the EC50 value was 1.9 uM. The hERG inhibition EC50 value of Example 87 of the present application was 4.6 uM.

[0433] Although the specific embodiments of the present invention have been described in detail, according to all the teachings that have been disclosed, those skilled in the art can make various modifications and substitutions to the details of the technical solutions of the present invention, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.