SULFUR-CONTAINING ISOINDOLINE DERIVATIVE, AND PREPARATION METHOD THEREFOR AND MEDICAL USE THEREOF

Abstract

The present application relates to a sulfur-containing isoindoline derivative, and a preparation method therefor and medical use thereof. In particular, the present disclosure relates to a sulfur-containing isoindoline derivative as represented by general formula (I), a preparation method therefor, a pharmaceutical composition containing the derivative, and use thereof as a therapeutic agent, particularly use thereof as a Cereblon modulator in the field of treatment of multiple myeloma.

##STR00001##

Claims

1. A compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer or diastereomer thereof or a mixture thereof, or a pharmaceutically acceptable salt thereof: ##STR00188## wherein: ring A is aryl or heteroaryl; ring B is cycloalkyl or heterocyclyl; Y is CH.sub.2 or C(O); R.sup.1 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro and hydroxy; R.sup.2 is selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.3 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro and hydroxy; R.sup.4 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.5 and R.sup.6 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; n is 0, 1, 2 or 3; p is 0, 1, 2, 3 or 4; q is 0, 1 or 2; and t is 0, 1, 2 or 3.

2. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1 wherein the compound comprises general formula (II) or a tautomer, mesomer, racemate, enantiomer or diastereomer thereof or a mixture thereof, or a pharmaceutically acceptable salt thereof: ##STR00189## wherein: ring A, ring B, Y, R.sup.1 to R.sup.4, n, p and t are as defined in claim 1.

3. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound comprises general formula (II-1) or general formula (II-2) or a tautomer, mesomer, racemate, enantiomer or diastereomer thereof or a mixture thereof, or a pharmaceutically acceptable salt thereof: ##STR00190## wherein: ring A, ring B, Y, R.sup.1 to R.sup.4, n, p and t are as defined in claim 1.

4. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is phenyl or 5- to 6-membered heteroaryl.

5. (canceled)

6. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein ring B is 3- to 8-membered heterocyclyl.

7. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein ##STR00191## is selected from the group consisting of ##STR00192## and R.sup.3 and p are as defined in claim 1; ##STR00193## R.sup.4a and R.sup.4b are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; and r is 0, 1 or 2.

8-9. (canceled)

10. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.4 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, C.sub.1-6 alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl, wherein the 3- to 8-membered cycloalkyl, 3- to 8-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl are each independently and optionally substituted with one or more substituents selected from the group consisting of halogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, cyano, amino, nitro, hydroxy and C.sub.1-6 hydroxyalkyl.

11. (canceled)

12. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen and C.sub.1-6 alkyl; R.sup.2 is selected from the group consisting of a hydrogen atom, C.sub.1-6 alkyl, and 3- to 8-membered cycloalkyl; R.sup.3 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen and C.sub.1-6 alkyl.

13-14. (canceled)

15. The compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of the following compounds: ##STR00194## ##STR00195## ##STR00196## ##STR00197## ##STR00198## ##STR00199## ##STR00200## ##STR00201## ##STR00202## ##STR00203## ##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213## ##STR00214## ##STR00215## ##STR00216## ##STR00217##

16. A compound of general formula (IA) or a tautomer, mesomer, racemate, enantiomer or diastereomer thereof or a mixture thereof, or a salt thereof, ##STR00218## wherein: ring A is selected from the group consisting of phenyl, pyridinyl and pyrimidinyl; ring B is heterocyclyl; and R.sup.2 to R.sup.6, p, q and t are as defined in claim 1.

17. The compound of general formula (IA) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the salt thereof according to claim 16, selected from the group consisting of the following compounds: ##STR00219## ##STR00220## ##STR00221##

18. A compound of general formula (IC) or a tautomer, mesomer, racemate, enantiomer or diastereomer thereof or a mixture thereof, or a salt thereof, ##STR00222## wherein: R.sup.m is C.sub.1-6 alkyl; and ring A, ring B, Y, R.sup.1 to R.sup.6, n, p, q and t are as defined in claim 1.

19. The compound or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the salt thereof according to claim 18, selected from the group consisting of the following compounds: ##STR00223## ##STR00224## ##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229## ##STR00230## ##STR00231##

20. A method for preparing the compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, comprising: ##STR00232## subjecting a compound of general formula (IA) and a compound of general formula (IB) to a reaction to obtain the compound of general formula (I), wherein: ring A, ring B, Y, R.sup.1 to R.sup.6, n, p, q and t are as defined in claim 1.

21. A method for preparing the compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, comprising: ##STR00233## subjecting a compound of general formula (IC) to an intramolecular ring closure reaction to obtain the compound of general formula (I), wherein: R.sup.m is C.sub.1-6 alkyl; and ring A, ring B, Y, R.sup.1 to R.sup.6, n, p, q and t are as defined in claim 1.

22. A pharmaceutical composition, comprising the compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1, and one or more pharmaceutically acceptable carriers, diluents or excipients.

23. (canceled)

24. A method for treating and/or preventing a cancer, an angiogenesis-related disorder, pain, macular degeneration, a skin disease, a pulmonary disease, an asbestos-related disease, a parasitic disease, an immunodeficiency disease, a CNS disease, a CNS injury, atherosclerosis, sleep disorder, an infectious disease, hemoglobinopathy, or a TNFα-related disorder in a subject in need thereof, the method comprising: administering to the subject an effective amount of the compound of general formula (I) or the tautomer, mesomer, racemate, enantiomer or diastereomer thereof or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 1.

25. The method according to claim 24, wherein the cancer is selected from the group consisting of leukemia, myeloma, lymphoma, melanoma, skin cancer, liver cancer, kidney cancer, lung cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, gallbladder cancer, bile duct cancer, chorionic epithelioma, pancreatic cancer, polycythemia vera, pediatric tumor, cervical cancer, ovarian cancer, breast cancer, bladder cancer, urothelial cancer, ureteral tumor, prostate cancer, seminoma, testicular tumor, head and neck tumor, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, sarcoma, osteoma, neuroblastoma, neuroendocrine cancer, brain tumor, CNS cancer, astrocytoma, and glioma.

26. The method according to claim 25, wherein the myeloma is multiple myeloma (MM) and myelodysplastic syndrome (MDS).

27. (canceled)

28. The method according to claim 26, wherein the multiple myeloma is relapsed, refractory or resistant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0178] FIG. 1: data on the efficacy of the compound of Example 6 and the control example CC-92480 on NCI-H929 xenograft tumor in CB-17 SCID mice.

[0179] FIG. 2: effect of the compound of Example 6 and the control example CC-92480 on the body weight of CB-17 SCID mice.

DETAILED DESCRIPTION

[0180] The present disclosure is further described below with reference to examples below, which are not intended to limit the scope of the present disclosure.

EXAMPLES

[0181] The structure of the compound is determined by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS). NMR shift (δ) is given in a unit of 10.sup.−6 (ppm). NMR spectra are determined using a Bruker AVANCE-400 nuclear magnetic resonance instrument or Bruker AVANCE NEO 500M, with deuterated dimethyl sulfoxide (DMSO-d.sub.6), deuterated chloroform (CDCl.sub.3) and deuterated methanol (CD.sub.3OD) as determination solvents and tetramethylsilane (TMS) as an internal standard.

[0182] Mass spectra are determined using Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent; MS model: 6110/6120 Quadrupole MS), Waters ACQuity UPLC-QD/SQD (manufacturer: Waters, MS model: Waters ACQuity Qda Detector/Waters SQ Detector) and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0183] High performance liquid chromatography (HPLC) analysis is performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high performance liquid chromatographs.

[0184] Chiral HPLC analysis is performed using an Agilent 1260 DAD high performance liquid chromatograph.

[0185] HPLC preparation is performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

[0186] Chiral preparative HPLC is performed using a Shimadzu LC-20AP preparative chromatograph.

[0187] A CombiFlash rapid preparation instrument used is Combiflash Rf200 (TELEDYNE ISCO).

[0188] Huanghai HSGF254 or Qingdao GF254 silica gel plates of specifications 0.15 mm to 0.2 mm are adopted for thin layer chromatography (TLC) analysis and 0.4 mm to 0.5 mm for TLC separation and purification.

[0189] Yantai Huanghai silica gel of 200-300 mesh is generally used as a carrier in silica gel column chromatography.

[0190] The mean inhibition of kinase and the IC.sub.50 value are determined using a NovoStar microplate reader (BMG, Germany).

[0191] Known starting materials described herein may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Shanghai Bide Pharmatech, Chembee Chemicals, and other companies.

[0192] In the examples, the reactions can be performed in an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

[0193] The argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of argon or nitrogen.

[0194] The hydrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of hydrogen.

[0195] Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator is used in the pressurized hydrogenation reactions.

[0196] The hydrogenation reactions usually involve 3 cycles of vacuumization and hydrogen purge.

[0197] A CEM Discover-S 908860 microwave reactor is used in the microwave reactions.

[0198] In the examples, a solution refers to an aqueous solution unless otherwise specified.

[0199] In the examples, the reaction temperature is room temperature, i.e., 20° C. to 30° C., unless otherwise specified.

[0200] The monitoring of the reaction progress in the examples is conducted by thin layer chromatography (TLC). The developing solvent for reactions, the eluent system for column chromatography purification and the developing solvent system for thin layer chromatography include: A: dichloromethane/methanol system, and B: n-hexane/ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound, or by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

Example 1

4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 1

[0201] ##STR00158## ##STR00159## ##STR00160##

Step 1

Tert-butyl 4-((4-formylphenyl)thio)piperidine-1-carboxylate 1b

[0202] Tert-butyl 4-mercaptopiperidine-1-carboxylate 1a (2.05 g, 9.44 mmol, Bide Pharmatech Ltd.), 4-fluorobenzaldehyde (1.17 g, 9.44 mmol) and potassium carbonate (2.86 g, 20.71 mmol) were added to N,N-dimethylformamide (40 mL). The mixture was heated to 120° C. and reacted overnight. After the reaction was completed, the reaction solution was diluted with water (200 mL) and extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 1b (2.66 g, yield: 88%).

[0203] MS m/z (ESI): 266.1 [M−55].

Step 2

4-(piperidin-4-ylthio)benzaldehyde Hydrogen Chloride 1c

[0204] Compound 1b (2.66 g, 8.28 mmol) was dissolved in solution of 4 M hydrogen chloride in 1,4-dioxane (20 mL), and the solution was stirred for about 3 h. The reaction solution was filtered, and the filter cake was rinsed with ethyl acetate (3 mL×3) and dried naturally to give the title compound 1c (crude product), which was directly used in the next step.

[0205] MS m/z (ESI): 221.8 [M+1].

Step 3

3-fluoro-4-(4-((4-formylphenyl)thio)piperidin-1-yl)benzonitrile 1d

[0206] Compound 1c (1.89 g, 7.33 mmol), 3,4-difluorobenzonitrile (1.07 g, 7.70 mmol, Bide Pharmatech Ltd.) and potassium carbonate (4.05 g, 29.33 mmol) were added to N,N-dimethylacetamide (15 mL), and the mixture was heated to 120° C. and reacted overnight. The reaction was added with water (50 mL) and extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with saturated sodium chloride solution (50 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography with an eluent system B to give the title compound 1d (2.2 g, yield: 93%).

[0207] MS m/z (ESI): 341.0 [M+1].

Step 4

3-fluoro-4-(4-((4-(hydroxymethyl)phenyl)thio)piperidin-1-yl)benzonitrile 1e

[0208] Compound 1d (150 mg, 0.44 mmol) was added to methanol (8 mL) under an ice bath, followed by the addition of potassium borohydride (29 mg, 0.53 mmol), and the mixture was reacted for 2 h under an ice bath. The reaction solution was diluted with water (20 mL) and extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated aqueous sodium chloride solution (30 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with an eluent system B to give the title compound 1e (150 mg, yield: 99%).

[0209] MS m/z (ESI): 343.1 [M+1].

Step 5

4-(4-((4-(bromomethyl)phenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 1f

[0210] Compound 1e (150 mg, 0.43 mmol) was added to dichloromethane (8 mL) under an ice bath, followed by the sequential addition of triphenylphosphine (150 mg, 0.57 mmol) and carbon tetrabromide (189 mg, 0.57 mmol), and the mixture was reacted for 20 min under an ice bath, and heated to room temperature and reacted for 2 h. The reaction solution was concentrated. The residue was purified by column chromatography with an eluent system B to give the title compound 1f (120 mg, yield: 68%).

[0211] MS m/z (ESI): 405.0 [M+1]; 407.0 [M+3].

Step 6

4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 1

[0212] 3-(4-hydroxy-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1g (25 mg, 0.096 mmol, Jiangsu Aikon Biopharmaceutical R&D Co., Ltd.) and anhydrous potassium carbonate (27 mg, 0.096 mmol) were added to N,N-dimethylacetamide (3 mL), and compound 1f (38 mg, 0.093 mmol) was added and reacted for 16 h. The reaction solution was filtered and purified by high performance liquid chromatography (Waters 2767-SQ Detecor2, elution system: 10 mmol/L aqueous ammonium bicarbonate solution and acetonitrile, gradient of acetonitrile: 55%-70%, flow rate: 30 mL/min) to give the title compound 1 (16 mg, yield: 28%).

[0213] MS m/z (ESI): 585.1 [M+1].

[0214] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.99 (s, 1H), 7.69 (dd, 1H), 7.55 (dd, 1H), 7.52-7.43 (m, 5H), 7.37-7.29 (m, 2H), 7.13 (t, 1H), 5.25 (s, 2H), 5.12 (dd, 1H), 4.43 (d, 1H), 4.27 (d, 1H), 3.58-3.42 (m, 3H), 3.05-2.83 (m, 3H), 2.63-2.52 (m, 1H), 2.48-2.38 (m, 1H), 2.06-1.93 (m, 3H), 1.68-1.55 (m, 2H).

Example 2

4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)methyl)phenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 2

[0215] ##STR00161##

[0216] 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione 2a (30 mg, 0.113 mmol, Bide Pharmatech Ltd.) and anhydrous potassium carbonate (21 mg, 0.152 mmol) were added to N,N-dimethylacetamide (3 mL), followed by the addition of compound if (30 mg, 0.074 mmol), and the mixture was reacted for 16 h. The reaction solution was filtered and purified by high performance liquid chromatography (Waters 2767-SQ Detecor2, eluent system: 10 mmol/L aqueous ammonium bicarbonate solution and acetonitrile, gradient of acetonitrile: 55%-75%, flow rate: 30 mL/min) to give the title compound 2 (30 mg, yield: 68%).

[0217] MS m/z (ESI): 599.1 [M+1].

[0218] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 11.13 (s, 1H), 7.84 (dd, 1H), 7.68 (dd, 1H), 7.60 (d, 1H), 7.55 (dd, 1H), 7.53-7.45 (m, 5H), 7.13 (t, 1H), 5.37 (s, 2H), 5.10 (dd, 1H), 3.60-3.44 (m, 3H), 3.06-2.82 (m, 3H), 2.65-2.55 (m, 1H), 2.54-2.45 (m, 1H), 2.10-1.95 (m, 3H), 1.68-1.54 (m, 2H).

Example 3

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 3

[0219] ##STR00162## ##STR00163##

Step 1

Tert-butyl (S)-5-amino-4-(4-((4-((1-(4-cyano-2-fluorophenyl)piperidin-4-yl)thio)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate 3b

[0220] Tert-butyl (S)-5-amino-4-(4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate 3a (318 mg, 0.951 mmol, prepared by a known method described in “Journal of Medicinal Chemistry, 2020, 63 (13), 6648-6676”) and anhydrous potassium carbonate (239 mg, 1.729 mmol) were added to N,N-dimethylacetamide (8 mL), followed by the addition of compound 1f (350 mg, 0.863 mmol), and the mixture was reacted for 16 h. The reaction solution was poured into ice water (20 mL), and the mixture was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 3b (500 mg, yield: 88%).

[0221] MS m/z (ESI): 659.2 [M+1].

Step 2

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 3

[0222] Compound 3b (500 mg, 0.758 mmol) was added to acetonitrile (30 mL), followed by the addition of p-toluenesulfonic acid monohydrate (187 mg, 0.983 mmol), and the mixture was stirred at 85° C. for 10 h. The reaction solution was concentrated under reduced pressure to remove the solvent, and water (20 mL) was added. The resulting mixture was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed sequentially with a saturated sodium bicarbonate solution (20 mL) and a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography (Waters 2767-SQ Detecor2, elution system: 10 mmol/L aqueous ammonium bicarbonate solution and acetonitrile, gradient of acetonitrile: 45%-60%, flow rate: 30 mL/min) to give the title compound 3 (30 mg, yield: 7%, ee: 99.6%). Chiral HPLC analysis: retention time 31.81 min, chiral purity: 99.6% (column: Chiralpak IE 150×4.6 mm, 5 μm; column temperature: 35° C., flow rate: 1.0 mL/min, mobile phase: n-hexane/ethanol/diethylamine=20/80/0.08 (v/v/v)).

[0223] MS m/z (ESI): 585.2 [M+1].

[0224] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.98 (s, 1H), 7.68 (dd, 1H), 7.55 (dd, 1H), 7.52-7.43 (m, 5H), 7.37-7.28 (m, 2H), 7.13 (t, 1H), 5.25 (s, 2H), 5.12 (dd, 1H), 4.43 (d, 1H), 4.27 (d, 1H), 3.58-3.42 (m, 3H), 3.05-2.84 (m, 3H), 2.63-2.54 (m, 1H), 2.48-2.37 (m, 1H), 2.06-1.93 (m, 3H), 1.68-1.55 (m, 2H).

Example 4

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-2-fluorophen yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 4

[0225] ##STR00164## ##STR00165## ##STR00166##

Step 1

Tert-butyl 4-((2-fluoro-4-formylphenyl)thio)piperidine-1-carboxylate 4b

[0226] Compound 1a (600 mg, 2.76 mmol), 3,4-difluorobenzaldehyde 4a (470 mg, 3.3 mmol) and potassium carbonate (954 mg, 6.90 mmol) were added to N,N-dimethylformamide (12 mL). The mixture was heated to 80° C. and reacted for 2 h. The reaction solution was diluted with water (200 mL) and extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 4b (920 mg, yield: 98%).

[0227] MS m/z (ESI): 283.9 [M−55].

Step 2

3-fluoro-4-(piperidin-4-ylthio)benzaldehyde Trifluoroacetate 4c

[0228] Compound 4b (920 mg, 2.71 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was slowly added under an ice bath. The mixture was reacted for 1 h. The reaction solution was concentrated and dried to give the title compound 4c (crude product), which was directly used in the next step without purification.

[0229] MS m/z (ESI): 239.9 [M+1].

Step 3

3-fluoro-4-(4-((2-fluoro-4-formylphenyl)thio)piperidin-1-yl)benzonitrile 4d

[0230] Compound 4c (960 mg, 2.72 mmol), 3,4-difluorobenzonitrile (756 mg, 5.43 mmol; Bide Pharmatech Ltd.) and potassium carbonate (1.50 g, 10.86 mmol) were added to N,N-dimethylformamide (12 mL), and the mixture was heated to 80° C. and reacted overnight. The reaction solution was added with water (50 mL) and extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (50 mL×3), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated. The resulting residue was purified by column chromatography with an eluent system B to give the title compound 4d (890 mg, yield: 91%).

[0231] MS m/z (ESI): 358.9 [M+1].

Step 4

3-fluoro-4-(4-((2-fluoro-4-(hydroxymethyl)phenyl)thio)piperidin-1-yl)benzonitrile 4e

[0232] Compound 4d (260 mg, 0.725 mmol) was added to methanol (8 mL) under an ice bath, followed by the addition of sodium borohydride (55 mg, 1.45 mmol), and the mixture was stirred for 1 h under an ice bath. The reaction was quenched with a saturated ammonium chloride solution (10 mL), and the reaction solution was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 4e (250 mg, yield: 96%).

[0233] MS m/z (ESI): 360.9 [M+1].

Step 5

4-(4-((4-(bromomethyl)-2-fluorophenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 4f

[0234] Compound 4e (250 mg, 0.694 mmol) was added to dichloromethane (5 mL), followed by the sequential addition of triphenylphosphine (237 mg, 0.901 mmol) and carbon tetrabromide (299 mg, 0.901 mmol), and the mixture was reacted for 1 h. The reaction solution was concentrated. The residue was purified by column chromatography with an eluent system B to give the title compound 4f (130 mg, yield: 44%).

[0235] MS m/z (ESI): 423.0 [M+1]; 425.0 [M+3].

Step 6

Tert-butyl (S)-5-amino-4-(4-((4-(((1-(4-cyano-2-fluorophenyl)piperidin-4-yl)thio)-3-fluorobenzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate 4g

[0236] Compound 3a (56 mg, 0.169 mmol) and anhydrous potassium carbonate (42 mg, 0.307 mmol) were added to N,N-dimethylformamide (3 mL), followed by the addition of compound 4f (65 mg, 0.153 mmol), and the mixture was reacted for 1 h. The reaction solution was poured into ice water (20 mL), and the resulting mixture was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 4g (95 mg, yield: 91%).

[0237] MS m/z (ESI): 677.0 [M+1].

Step 7

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-2-fluorophen yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 4

[0238] Compound 4g (50 mg, 0.074 mmol) was added to acetonitrile (6 mL), followed by the addition of benzenesulfonic acid (17 mg, 0.096 mmol) at room temperature, and the mixture was reacted at 80° C. for 12 h. The reaction solution was concentrated under reduced pressure to remove the solvent. The resulting residue was purified by high performance liquid chromatography (Waters 2767-SQ Detecor2, elution system: one-thousandth volume of aqueous trifluoroacetic acid solution and acetonitrile, gradient of acetonitrile: 50%-67%, flow rate: 30 mL/min) to give the title compound 4 (13 mg, yield: 29%).

[0239] MS m/z (ESI): 603.2 [M+1].

[0240] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.99 (s, 1H), 7.69 (dd, 1H), 7.62-7.53 (m, 2H), 7.50 (t, 1H), 7.43 (dd, 1H), 7.39-7.27 (m, 3H), 7.13 (t, 1H), 5.28 (s, 2H), 5.13 (dd, 1H), 4.46 (d, 1H), 4.30 (d, 1H), 3.53-3.48 (m, 3H), 3.01-2.88 (m, 3H), 2.65-2.56 (m, 1H), 2.46-2.41 (m, 1H), 2.03-1.96 (m, 3H), 1.67-1.57 (m, 2H).

Example 5

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-3-fluorophen yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 5

[0241] ##STR00167##

[0242] The title compound 5 (90 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 2,4-difluorobenzaldehyde.

[0243] MS m/z (ESI): 603.1 [M+1].

[0244] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.98 (s, 1H), 7.70 (dd, 1H), 7.60-7.48 (m, 3H), 7.42-7.31 (m, 3H), 7.27 (dd, 1H), 7.14 (t, 1H), 5.27 (s, 2H), 5.11 (dd, 1H), 4.39 (d, 1H), 4.23 (d, 1H), 3.68-3.62 (m, 1H), 3.56-3.49 (m, 2H), 3.01 (t, 2H), 2.95-2.87 (m, 1H), 2.61-2.54 (m, 1H), 2.46-2.41 (m, 1H), 2.02-1.94 (m, 3H), 1.70-1.59 (m, 2H).

Example 6

(S)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)pyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 6

[0245] ##STR00168## ##STR00169## ##STR00170##

Step 1

Tert-butyl 4-((5-formylpyridin-2-yl)thio)piperidine-1-carboxylate 6b

[0246] Compound 1a (700 mg, 3.22 mmol), 6-fluoropyridine-3-carbaldehyde 6a (443 mg, 3.54 mmol) and potassium carbonate (1.11 g, 8.05 mmol) were added to N,N-dimethylformamide (10 mL). The mixture was heated to 80° C. and reacted for 1 h. The reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (40 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 6b (1.0 g, yield: 96%).

[0247] MS m/z (ESI): 267.1 [M−55].

Step 2

6-(piperidin-4-ylthio)nicotinaldehyde Trifluoroacetate 6c

[0248] Compound 6b (950 mg, 2.95 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was slowly added under an ice bath. The mixture was reacted for 1 h. The reaction solution was concentrated and dried to give the title compound 6c (crude product), which was directly used in the next step without purification.

[0249] MS m/z (ESI): 223.1 [M+1].

Step 3

3-fluoro-4-(4-((5-formylpyridin-2-yl)thio)piperidin-1-yl)benzonitrile 6d

[0250] Compound 6c (760 mg, 2.94 mmol), 3,4-difluorobenzonitrile (817 mg, 5.87 mmol) and potassium carbonate (1.22 g, 8.81 mmol) were added to N,N-dimethylformamide (15 mL), and the mixture was heated to 80° C. and reacted overnight. The reaction solution was added with water (50 mL) and extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (50 mL×3), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography with an eluent system B to give the title compound 6d (850 mg, yield: 84%).

[0251] MS m/z (ESI): 342.1 [M+1].

Step 4

3-fluoro-4-(4-((5-(hydroxymethyl)pyridin-2-yl)thio)piperidin-1-yl)benzonitrile 6e

[0252] Compound 6d (600 mg, 1.76 mmol) was added to methanol (10 mL) under an ice bath, followed by the slow addition of sodium borohydride (133 mg, 3.51 mmol), and the mixture was reacted for 1 h. The reaction was quenched with water (10 mL), and the reaction solution was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 6e (580 mg, yield: 96%).

[0253] MS m/z (ESI): 344.1 [M+1].

Step 5

4-(4-((5-(bromomethyl)pyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 6f

[0254] Compound 6e (200 mg, 0.582 mmol) was added to dichloromethane (6 mL), followed by the sequential addition of triphenylphosphine (199 mg, 0.757 mmol) and carbon tetrabromide (251 mg, 0.757 mmol), and the mixture was reacted for 2 h. The reaction solution was concentrated. The residue was purified by column chromatography with an eluent system B to give the title compound 6f (190 mg, yield: 80%).

[0255] MS m/z (ESI): 406.0 [M+1]; 408.0 [M+3].

Step 6

Tert-butyl (S)-5-amino-4-(4-((6-((1-(4-cyano-2-fluorophenyl)piperidin-4-yl)thio)pyridin-3-yl)methoxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate 6g

[0256] Compound 3a (82 mg, 0.246 mmol) and anhydrous potassium carbonate (65 mg, 0.468 mmol) were added to N,N-dimethylformamide (3 mL), followed by the addition of compound 6f (95 mg, 0.244 mmol), and the mixture was reacted for 2 h. The reaction solution was poured into ice water (10 mL), and the resulting mixture was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 6g (145 mg, yield: 94%).

[0257] MS m/z (ESI): 660.2 [M+1].

Step 7

(S)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)pyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 6

[0258] Compound 6g (60 mg, 0.091 mmol) was added to acetonitrile (5 mL), followed by the addition of benzenesulfonic acid (16 mg, 0.091 mmol) at room temperature, and the mixture was reacted at 80° C. for 8 h. The reaction solution was concentrated under reduced pressure to remove the solvent. The resulting residue was subjected to high performance liquid chromatography (Gilson GX-281, elution system: 10 mmol/L aqueous ammonium bicarbonate solution and acetonitrile, gradient of acetonitrile: 60%-80%, flow rate: 30 mL/min) to give the title compound 6 (42 mg, yield: 78%).

[0259] MS m/z (ESI): 586.4 [M+1].

[0260] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.98 (s, 1H), 8.60 (s, 1H), 7.78 (dd, 1H), 7.69 (dd, 1H), 7.59-7.48 (m, 2H), 7.38-7.33 (m, 3H), 7.16 (t, 1H), 5.24 (s, 2H), 5.12 (dd, 1H), 4.42 (d, 1H), 4.26 (d, 1H), 4.07-3.99 (m, 1H), 3.56-3.48 (m, 2H), 3.13-3.03 (m, 2H), 2.96-2.87 (m, 1H), 2.63-2.55 (m, 1H), 2.46-2.38 (m, 1H), 2.21-2.12 (m, 2H), 2.02-1.94 (m, 1H), 1.82-1.70 (m, 2H).

Example 6′

(R)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)pyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 6′

[0261] ##STR00171## ##STR00172##

[0262] Compound 6 (500 mg, 0.853 mmol) was dissolved in tetrahydrofuran (30 mL), followed by the addition of a saturated aqueous sodium carbonate solution (30 mL). After the addition, the mixture was vigorously stirred for 1.5 h. The reaction solution was subjected to liquid separation. The organic phase was diluted with dichloromethane (120 mL), washed sequentially with a saturated ammonium chloride solution (20 mL) and a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give the title compound 6′a (crude product, chiral HPLC showed a content of compound 6′ of 42.8%).

[0263] The resulting crude 6′a was subjected to preparative chiral separation (separation conditions: chiral preparation column CHIRALPAK IE 20×250 mm (Daicel); mobile phase: n-hexane and ethanol, gradient of ethanol: 80%; flow rate: 20 mL/min), and the resulting preparation solution was concentrated under reduced pressure under a water bath at less than 20° C. to give the title compound 6′ (25 mg, yield: 5.0%).

[0264] MS m/z (ESI): 586.1 [M+1].

[0265] Chiral HPLC analysis: retention time: 24.426 min, chiral purity: 100% (column: CHIRALPAK IE 150×4.6 mm, 5 μm; mobile phase: n-hexane/ethanol=20/80 (v/v))

[0266] .sup.1H NMR (500 MHz, DMSO-d.sub.6): 10.97 (s, 1H), 8.59 (s, 1H), 7.78 (dd, 1H), 7.68 (d, 1H), 7.56 (d, 1H), 7.50 (t, 1H), 7.35 (t, 3H), 7.15 (t, 1H), 5.24 (s, 2H), 5.11 (dd, 1H), 4.41 (d, 1H), 4.25 (d, 1H), 4.02 (t, 1H), 3.51 (d, 2H), 3.07 (t, 2H), 2.91 (ddd, 1H), 2.60-2.55 (m, 1H), 2.43 (dd, 1H), 2.20-2.12 (m, 2H), 1.99 (s, 1H), 1.76 (tt, 2H).

Example 7

(S)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)pyrimidin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 7

[0267] ##STR00173##

[0268] The title compound 7 (38 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 2-chloropyrimidine-5-carbaldehyde.

[0269] MS m/z (ESI): 587.2 [M+1].

[0270] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.97 (s, 1H), 8.82 (s, 2H), 7.70 (dd, 1H), 7.57 (dd, 1H), 7.53 (t, 1H), 7.38 (t, 2H), 7.17 (t, 1H), 5.26 (s, 2H), 5.12 (dd, 1H), 4.44 (d, 1H), 4.27 (d, 1H), 3.99-3.92 (m, 1H), 3.58-3.47 (m, 2H), 3.14-3.04 (m, 2H), 2.96-2.87 (m, 1H), 2.64-2.56 (m, 1H), 2.44-2.37 (m, 1H), 2.26-2.15 (m, 2H), 2.03-1.96 (m, 1H), 1.85-1.75 (m, 2H).

Example 8

(S)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxoisoindolin-4-yl)oxy)methyl)pyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 8

[0271] ##STR00174##

[0272] The title compound 8 (33 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 2-fluoropyridine-5-carbaldehyde and compound 3a in step 6 replaced by tert-butyl (S)-5-amino-4-(6-fluoro-4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate (prepared by the method disclosed in “Example 7 on page 99 of the description of the patent application WO2019040274A1”).

[0273] MS m/z (ESI): 604.1 [M+1].

[0274] .sup.1H NMR (500 MHz, DMSO-d6): δ 10.99 (s, 1H), 8.60 (d, 1H), 7.78 (dd, 1H), 7.70 (dd, 1H), 7.57 (dd, 1H), 7.39-7.31 (m, 2H), 7.20-7.11 (m, 2H), 5.25 (s, 2H), 5.11 (dd, 1H), 4.40 (d, 1H), 4.23 (d, 1H), 4.09-3.98 (m, 1H), 3.58-3.46 (m, 2H), 3.13-3.02 (m, 2H), 2.96-2.83 (m, 1H), 2.63-2.55 (m, 1H), 2.46-2.34 (m, 1H), 2.21-2.12 (m, 2H), 2.04-1.93 (m, 1H), 1.82-1.68 (m, 2H).

Example 9

(S)-4-(4-((6-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)pyridin-3-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 9

[0275] ##STR00175##

[0276] The title compound 9 (45 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 5-fluoropyridine-2-carbaldehyde.

[0277] MS m/z (ESI): 586.3 [M+1].

[0278] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.99 (s, 1H), 8.63 (d, 1H), 7.95 (dd, 1H), 7.68 (dd, 1H), 7.61-7.51 (m, 2H), 7.49 (t, 1H), 7.35 (d, 1H), 7.32 (d, 1H), 7.13 (t, 1H), 5.33 (s, 2H), 5.13 (dd, 1H), 4.47 (d, 1H), 4.32 (d, 1H), 3.65-3.45 (m, 3H), 3.05-2.85 (m, 3H), 2.68-2.55 (m, 1H), 2.44-2.35 (m, 1H), 2.07-1.93 (m, 3H), 1.71-1.55 (m, 2H).

Example 10

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-2,3-difluorophenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 10

[0279] ##STR00176##

[0280] The title compound 10 (63 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced with the starting compound 2,3,4-trifluorobenzaldehyde.

[0281] MS m/z (ESI): 621.1 [M+1].

[0282] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.98 (s, 1H), 7.69 (dd, 1H), 7.58-7.48 (m, 2H), 7.47-7.33 (m, 4H), 7.13 (t, 1H), 5.35 (s, 2H), 5.12 (dd, 1H), 4.40 (d, 1H), 4.25 (d, 1H), 3.67-3.46 (m, 3H), 3.06-2.82 (m, 3H), 2.64-2.56 (m, 1H), 2.46-2.37 (m, 1H), 2.07-1.90 (m, 3H), 1.73-1.56 (m, 2H).

Example 11

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-2,5-difluorophenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 11

[0283] ##STR00177##

[0284] The title compound 11 (70 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 2,4,5-trifluorobenzaldehyde.

[0285] MS m/z (ESI): 621.1 [M+1].

[0286] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.98 (s, 1H), 7.89 (dd, 1H), 7.63-7.46 (m, 4H), 7.42-7.32 (m, 2H), 7.14 (t, 1H), 5.27 (s, 2H), 5.12 (dd, 1H), 4.42 (d, 1H), 4.27 (d, 1H), 3.72-3.61 (m, 1H), 3.58-3.46 (m, 2H), 3.07-2.96 (m, 2H), 2.95-2.83 (m, 1H), 2.65-2.56 (m, 1H), 2.44-2.38 (m, 1H), 2.09-1.90 (m, 3H), 1.74-1.58 (m, 2H).

Example 12

(S)-4-(4-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-2,6-difluorophenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 12

[0287] ##STR00178##

[0288] The title compound 12 (20 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 3,4,5-trifluorobenzaldehyde.

[0289] MS m/z (ESI): 621.1 [M+1].

[0290] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.99 (s, 1H), 7.67 (dd, 1H), 7.54 (dd, 1H), 7.50 (t, 1H), 7.43-7.33 (m, 3H), 7.29 (d, 1H), 7.11 (t, 1H), 5.31 (s, 2H), 5.13 (dd, 1H), 4.50 (d, 1H), 4.34 (d, 1H), 3.58-3.42 (m, 2H), 3.40-3.34 (m, 1H), 3.01-2.84 (m, 3H), 2.68-2.55 (m, 1H), 2.46-2.35 (m, 1H), 2.05-1.85 (m, 3H), 1.65-1.52 (m, 2H).

Example 13

(S)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-6-methylpyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 13

[0291] ##STR00179##

[0292] The title compound 13 (60 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 6-fluoro-2-methyl-3-pyridinecarboxaldehyde.

[0293] MS m/z (ESI): 600.2 [M+1].

[0294] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.97 (s, 1H), 7.78-7.63 (m, 2H), 7.61-7.47 (m, 2H), 7.44-7.30 (m, 2H), 7.24-7.07 (m, 2H), 5.24 (s, 2H), 5.11 (dd, 1H), 4.41 (d, 1H), 4.25 (d, 1H), 4.06-3.92 (m, 1H), 3.60-3.43 (m, 2H), 3.14-3.02 (m, 2H), 2.97-2.83 (m, 1H), 2.68-2.56 (m, 1H), 2.52 (s, 3H), 2.48-2.37 (m, 1H), 2.22-2.11 (m, 2H), 2.04-1.93 (m, 1H), 1.83-1.68 (m, 2H).

Example 14

(S)-4-(4-((2-chloro-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phen yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 14

[0295] ##STR00180##

[0296] The title compound 14 (22 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 3,4-difluorobenzaldehyde replaced by the starting 3-chloro-4-fluorobenzaldehyde.

[0297] MS m/z (ESI): 618.9 [M+1].

[0298] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.98 (s, 1H), 7.74-7.62 (m, 2H), 7.60 (d, 1H), 7.56 (dd, 1H), 7.53-7.44 (m, 2H), 7.35 (d, 1H), 7.32 (d, 1H), 7.14 (t, 1H), 5.25 (s, 2H), 5.12 (dd, 1H), 4.43 (d, 1H), 4.28 (d, 1H), 3.71-3.61 (m, 1H), 3.58-3.47 (m, 2H), 3.08-2.98 (m, 2H), 2.96-2.85 (m, 1H), 2.66-2.54 (m, 1H), 2.46-2.37 (m, 1H), 2.12-1.93 (m, 3H), 1.75-1.60 (m, 2H).

Example 15

4-(3-((5-(((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)pyridin-2-yl)thio)pyrrolidin-1-yl)-3-fluorobenzonitrile 15

[0299] ##STR00181##

[0300] The title compound 15 (39 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 1a (tert-butyl 4-mercaptopiperidine-1-carboxylate) replaced by tert-butyl 3-mercaptopyrrolidine-1-carboxylate, and the starting compound 3,4-difluorobenzaldehyde replaced by the starting compound 2-fluoropyridine-5-carbaldehyde.

[0301] MS m/z (ESI): 572.3 [M+1].

[0302] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.97 (s, 1H), 8.81 (d, 1H), 7.80 (dd, 1H), 7.58 (dd, 1H), 7.52 (t, 1H), 7.44 (dd, 1H), 7.42-7.32 (m, 3H), 6.79 (t, 1H), 5.25 (s, 2H), 5.11 (dd, 1H), 4.49-4.36 (m, 2H), 4.26 (d, 1H), 4.12-4.04 (m, 1H), 3.68-3.54 (m, 2H), 3.52-3.44 (m, 1H), 3.00-2.84 (m, 1H), 2.64-2.56 (m, 1H), 2.44-2.35 (m, 2H), 2.10-1.94 (m, 2H).

Example 16

(S)-4-(3-((4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)thio)azetidin-1-yl)-3-fluorobenzonitrile 16

[0303] ##STR00182##

[0304] The title compound 16 (15 mg) was prepared by following the synthetic route for compound 4 in Example 4, with the starting compound 1a (tert-butyl 4-mercaptopiperidine-1-carboxylate) replaced by tert-butyl 3-mercaptoazetidine-1-carboxylate and the starting compound 3,4-difluorobenzaldehyde replaced by 4-fluorobenzaldehyde.

[0305] MS m/z (ESI): 557.0 [M+1].

[0306] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.97 (s, 1H), 7.60 (dd, 1H), 7.54-7.42 (m, 4H), 7.37-7.27 (m, 4H), 6.62 (t, 1H), 5.24 (s, 2H), 5.12 (dd, 1H), 4.64-4.54 (m, 2H), 4.47-4.35 (m, 2H), 4.26 (d, 1H), 4.02-3.90 (m, 2H), 3.00-2.84 (m, 1H), 2.66-2.56 (m, 1H), 2.44-2.35 (m, 1H), 2.08-1.90 (m, 1H).

Example 17

(S)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-6-fluoropyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 17

[0307] ##STR00183## ##STR00184## ##STR00185##

Step 1

Tert-butyl 4-((6-fluoro-5-formylpyridin-2-yl)thio)piperidine-1-carboxylate 17b

[0308] Compound 1a (1.5 g, 6.90 mmol), 2,6-difluoropyridine-3-carbaldehyde 17a (1.04 g, 7.27 mmol) and potassium carbonate (2.4 g, 17.36 mmol) were added to N,N-dimethylformamide (15 mL), and the mixture was reacted for 2 h. The reaction solution was diluted with water (200 mL) and extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 17b (1.0 g, yield: 42%).

[0309] MS m/z (ESI): 285.0 [M−55].

Step 2

Tert-butyl 4-((6-fluoro-5-(hydroxymethyl)pyridin-2-yl)thio)piperidine-1-carboxylate 17c

[0310] Compound 17b (1.0 g, 2.94 mmol) was dissolved in a mixed solvent of tetrahydrofuran (20 mL) and methanol (2 mL), followed by the slow addition of sodium borohydride (223 mg, 5.90 mmol) under an ice bath, and the mixture was reacted for 2 h. The reaction was quenched with water (50 mL), and the reaction solution was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 17c (910 mg, yield: 90%).

[0311] MS m/z (ESI): 287.0 [M−55].

Step 3

(2-fluoro-6-(piperidin-4-ylsulfanyl)pyridin-3-yl)methanol Hydrochloride 17d

[0312] Compound 17c (910 mg, 2.66 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of a solution of 4 M hydrogen chloride in 1,4-dioxane (3 mL, 12 mmol), and the mixture was reacted for 1 h. The reaction solution was concentrated and dried to give the title compound 17d (625 mg), which was directly used in the next step.

[0313] MS m/z (ESI): 243.1 [M+1].

Step 4

3-fluoro-4-(4-((6-fluoro-5-(hydroxymethyl)pyridin-2-yl)thio)piperidin-1-yl)benzonitrile 17e

[0314] Compound 17d (625 mg, 2.24 mmol) was added to N,N-dimethylformamide (20 mL), followed by the addition of 3,4-difluorobenzonitrile (312 mg, 2.24 mmol) and potassium carbonate (930 mg, 6.73 mmol), and the mixture was heated to 80° C. and reacted for 2 h. The reaction solution was diluted with water (100 mL) and extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate and filtered and concentrated. The resulting residue was purified by column chromatography with an eluent system B to give the title compound 17e (400 mg, yield: 50%).

[0315] MS m/z (ESI): 362.0 [M+1].

Step 5

4-(4-((5-(bromomethyl)-6-fluoropyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 17f

[0316] Compound 17e (310 mg, 0.86 mmol) was added to dichloromethane (10 mL), followed by the addition of triphenylphosphine (360 mg, 1.37 mmol) and carbon tetrabromide (455 mg, 1.37 mmol), and the mixture was reacted for 12 h. The reaction solution was diluted with water (50 mL) and extracted with dichloromethane (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography with an eluent system B to give the title compound 17f (340 mg, yield: 93%).

[0317] MS m/z (ESI): 423.9 [M+1].

Step 6

Tert-butyl (S)-5-amino-4-(4-((6-((1-(4-cyano-2-fluorophenyl)piperidin-4-yl)thio)-2-fluoropyridin-3-yl) methoxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate 17g

[0318] Compound 3a (268 mg, 0.80 mmol) and anhydrous potassium carbonate (223 mg, 1.61 mmol) were added to N,N-dimethylformamide (10 mL), followed by the addition of compound 17f (340 mg, 0.80 mmol), and the mixture was reacted for 1 h. The reaction solution was poured into ice water (20 mL), and the resulting mixture was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with a saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with an eluent system B to give the title compound 17g (540 mg, yield: 99%).

[0319] MS m/z (ESI): 678.2 [M+1].

Step 7

(S)-4-(4-((5-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-6-fluoropyridin-2-yl)thio)piperidin-1-yl)-3-fluorobenzonitrile 17

[0320] Compound 17g (540 mg, 0.80 mmol) was added to acetonitrile (20 mL), followed by the addition of benzenesulfonic acid (252 mg, 1.59 mmol) at room temperature, and the mixture was reacted at 80° C. for 12 h. The reaction solution was concentrated under reduced pressure to remove the solvent. The resulting residue was subjected to high performance liquid chromatography (Waters 2545-SQ Detecor2, elution system: 10 mmol/L aqueous ammonium bicarbonate solution and acetonitrile, gradient of acetonitrile: 53%-95%, flow rate: 30 mL/min) to give the title compound 4 (270 mg, yield: 56%).

[0321] MS m/z (ESI): 604.0 [M+1].

[0322] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.98 (s, 1H), 8.01 (t, 1H), 7.70 (dd, 1H), 7.57 (dd, 1H), 7.52 (t, 1H), 7.43-7.30 (m, 3H), 7.17 (t, 1H), 5.25 (s, 2H), 5.12 (dd, 1H), 4.39 (d, 1H), 4.24 (d, 1H), 4.00-3.88 (m, 1H), 3.58-3.45 (m, 2H), 3.16-3.04 (dd, 2H), 2.98-2.84 (m, 1H), 2.66-2.55 (m, 1H), 2.48-2.35 (m, 1H), 2.23-2.12 (m, 2H), 2.08-1.92 (m, 1H), 1.82-1.70 (m, 2H).

Example 18

(S)-4-(44(4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)-3,5-difluorophenyl)thio)piperidin-1-yl)-3-fluorobenzonitrile 18

[0323] ##STR00186##

[0324] The title compound 18 (27 mg) was prepared by following the synthetic route for compound 17 in Example 17, with the starting compound 2,6-difluoropyridine-3-carbaldehyde replaced by the starting compound 2,4,6-trifluorobenzaldehyde.

[0325] MS m/z (ESI): 621.0 [M+1].

[0326] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 10.95 (s, 1H), 7.70 (dd, 1H), 7.59-7.51 (m, 2H), 7.45 (d, 1H), 7.38 (d, 1H), 7.27 (d, 2H), 7.16 (t, 1H), 5.24 (dd, 2H), 5.09 (dd, 1H), 4.32 (d, 1H), 4.16 (d, 1H), 3.86-3.73 (m, 1H), 3.60-3.47 (m, 2H), 3.13-3.00 (m, 2H), 2.96-2.82 (m, 1H), 2.62-2.53 (m, 1H), 2.44-2.34 (m, 1H), 2.17-2.04 (m, 2H), 2.01-1.90 (m, 1H), 1.75-1.60 (m, 2H).

Biological Evaluation

Test Example 1. Biological Evaluation of NCI-H929 Proliferation Experiment

[0327] The following method was used to determine the inhibitory activity of the compounds of the present disclosure on the proliferation of NCI-H929 cells. The experimental method was briefly described as follows.

[0328] NCI-H929 cells (ATCC, CRL-9068) were cultured in a complete medium (RPMI 1640 medium (Hyclone, SH30809.01) containing 10% fetal bovine serum (Corning, 35-076-CV) and 0.05 mM 2-mercaptoethanol (Sigma, M3148)). On the first day of the experiment, NCI-H929 cells were seeded in a 96-well plate at a density of 6000 cells/well with a complete medium to form 100 μL of cell suspension per well, and 10 μL of test compound prepared in a complete medium and diluted in a gradient was added to each well. The compound was firstly dissolved in DMSO, with an initial concentration of 10 mM, and then subjected to serial dilution at a 5-fold concentration gradient for a total of 9 concentration points, with a blank control of 100% DMSO. Another 5 μL of the compound dissolved in DMSO was added to 95 μL of complete medium, i.e., the compound was diluted 20-fold with the complete medium. Finally the compound diluted in the complete medium at 10 μL/well was added to the cell suspension, wherein the final concentrations of the compound were 9 concentration points obtained by 5-fold gradient dilution starting from 50 μM. A blank control containing 0.5% DMSO was set. The plate was incubated in a cell incubator at 37° C. with 5% CO.sub.2 for 5 days. On the sixth day, the 96-well cell culture plate was taken out, added with a CellTiter-Glo® luminescent cell activity detect reagent (Promega, G7573) at 50 μL/well, left to stand at room temperature for 10 min, and read for the luminescence signal values using a multi-mode microplate reader (PerkinElmer, EnVision 2015). IC.sub.50 values for the inhibitory activity of the compounds were calculated using Graphpad Prism software, with the results shown in Table 1.

TABLE-US-00004 TABLE 1 IC.sub.50 values for the inhibition of proliferation of NCI- H929 cells by the compounds of the present disclosure Compound IC.sub.50 (nM) 1 0.34 2 4.36 3 0.10 4 0.17 5 0.08 6 0.02 7 0.13 8 0.28 9 0.90 10 0.18 11 0.22 12 0.61 13 1.78 14 0.27 15 0.75 16 1.13 17 0.21 18 0.34

[0329] Conclusion: the compounds of the present disclosure have good inhibitory activity for the proliferation of NCI-H929 cells.

Test Example 2. Pharmacodynamic Test

1. Objective

[0330] This experiment was performed to evaluate the inhibition effect of the compound of Example 6 and the control example CC-92480 on the growth of human multiple myeloma cell NCI-H929 xenograft tumor in CB-17 SCID mice.

2. Test Compounds

[0331] Compound of Example 6; control example CC-92480 (see compound 2 of WO2019014100A1, synthesized according to the method disclosed therein)

##STR00187##

Solutions of the compound of Example 6 and the control example CC-92480 were prepared with 5% DMSO+20% PEG400+70% (10% TPGS)+5% (1% HPMC K100LV).

3. Experimental Procedures and Materials

3.1 Experimental Animals and Housing Conditions

[0332] Thirty CB-17 SCID female mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (certificate No. 20170011006049, SCXK (Shanghai) 2017-0011), weighing about 19 g at the time of purchase, were bred at 5 mice/cage (in 12/12 hour light/dark cycle, at a constant temperature of 23±1° C. and humidity of 50%-60%) and had free access to food and water.

3.2 Group of Animals:

[0333] After adaptive feeding, the CB-17 SCID mice were grouped as follows:

TABLE-US-00005 Group n Route of administration Vehicle control 7 5% DMSO + 20% PEG400 + 70%(10% TPGS) + 5%(1% HPMC K100LV) (i.g/qd) Example 6 7 1 mg/kg (i.g/qd) CC-92480 7 1 mg/kg (i.g/qd) Note: qd denotes once a day; i.g. denotes intragastric administration.

3.3 Procedures:

[0334] NCI-H929 cells in a logarithmic growth phase were inoculated subcutaneously into the right flank of 30 female CB-17 SCID mice at 5×10.sup.6 cells/mouse/100 μL (containing 50 μL of Matrigel). After 10 days, when the tumor volume of the tumor-bearing mice reached about 200 mm.sup.3, the mice were randomly divided into 3 groups according to the tumor volume and body weight: vehicle control group, CC-92480-1 mpk, compound of Example 6-1 mpk, with 7 mice in each group. The day of grouping was set as Day 0 (D0), and intragastric administration was performed once a day for 11 days (Table 2). For the tumor-bearing mice, the tumor volume was measured with a vernier caliper and the body weight was measured with a balance twice a week, and the data were recorded. The tumor-bearing animals were euthanized as the experimental endpoint when the tumor volume reached 2000 mm.sup.3 or when most tumors showed rupture or when the tumor-bearing animals showed 20% of body weight loss.

3.4 Data Statistics

[0335] All data were plotted and statistically analyzed using Excel and GraphPad Prism 5 software.

[0336] The tumor volume (V) was calculated as follows: V=½×a×b.sup.2, where a and b represent length and width, respectively.

[0337] The relative tumor proliferation rate T/C (%)=(T−T.sub.0)/(C−C.sub.0)×100(%), where T and C are the tumor volume of animals at the end of the experiment in the treatment group and control group, respectively; T.sub.0 and C.sub.0 are the tumor volume of animals at the beginning of the experiment in the treatment group and control group, respectively.

[0338] Tumor growth inhibition TGI (%)=1−T/C (%), and when TGI (%) exceeds 100%, no specific value will be shown, and it is expressed only by >100%.

Tumor regression (%)=[(T.sub.0−T)/T.sub.0]×100(%).

4. Results

[0339] The data on the efficacy of the compound of Example 6 and the control example CC-92480 on NCI-H929 xenograft tumor in CB-17 SCID mice are shown in Table 2 below and FIG. 1.

[0340] The effect of the compound of Example 6 and the control example CC-92480 on the body weight of CB-17 SCID mice is shown in FIG. 2.

TABLE-US-00006 TABLE 2 Efficacy of the compound of the present disclosure on NCI-H929 xenograft tumor in CB-17 SCID mice % tumor p Number of of Mean tumor volume (mm.sup.3) regression (vs. vehicle remaining Group Route administration D0 SEM D11 SEM D11 control) animals/group Vehicle qd/11d po 156.3 15.9 1711.7 190.1 / / 7/7 control Example 6 qd/11d po 155.4 16.5 18.6 1.8 88 <0.001 7/7 CC-92480 qd/11d po 157.2 13.6 103.6 27.1 34 <0.001 7/7 Note: qd denotes once a day; po denotes oral administration.

5. Conclusion

[0341] The compound of Example 6 was administered once a day starting from 10 days after tumor cell transplantation, and significant tumor volume regressions occurred 11 days after administration. Upon calculation, the tumor inhibition rate is >100%, and the tumor regression rate is 88%. The statistical difference (p<0.05) is obtained when compared with an equal dose of CC-92480 at the end point of the experiment, and the administration has no influence on the body weight of mice. Under the same conditions, the tumor regression rate of the control example CC-92480 is 34%.

Test Example 3. Pharmacokinetic Evaluation

1. Overview

[0342] The drug concentration in the plasma of the test animals (mice) at different time points after intragastric administration of the compound of Example 6 and the control example CC-92480 was determined by an LC/MS/MS method. The pharmacokinetic behavior of the compound of the present disclosure in mice was studied and its pharmacokinetic profile was evaluated.

2. Test Protocol

2.1 Test Compounds

[0343] Compound of Example 6 and control example CC-92480.

2.2 Test Animals

[0344] Eighteen mice, female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with animal production license number of SCXK (Shanghai) 2017-0005.

2.3 Preparation of Compound Solutions

[0345] A certain amount of compound of Example 6 was weighed, dissolved by adding 5% by volume of DMSO and 5% Tween 80 (Shanghai Titan Scientific Co., Ltd.), and then prepared into a 0.1 mg/mL clear solution by adding 90% normal saline.

[0346] A certain amount of the control example CC-92480 was weighed, dissolved by adding 5% by volume of DMSO and 5% Tween 80 (Shanghai Titan Scientific Co., Ltd.), and then prepared into a 0.1 mg/mL clear solution by adding 90% normal saline.

2.4 Administration

[0347] Nine mice were intragastrically administered with the compound of Example 6 at a dose of 2 mg/kg and at a volume of 0.2 mL/10 g.

[0348] Nine mice were intragastrically administered with the control example CC-92480 at a dose of 2 mg/kg and at a volume of 0.2 mL/10 g.

3. Procedures

[0349] Mice were intragastrically administered with the compound of Example 6 and the control example CC-92480, and 0.2 mL of blood was collected before the administration and 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h, 11.0 h and 24.0 h after the administration (3 animals at each time point), placed in EDTA-K2 anticoagulation tubes, and centrifuged at 10,000 rpm for 1 min(4° C.). Plasma was separated within 1 h, and stored at −20° C. for testing. The process from blood collection to centrifugation was performed under an ice bath.

[0350] The content of the test compounds at different concentrations in mouse plasma after the administration was determined: 25 μL of mouse plasma at each time point after the administration was mixed with 50 μL (100 ng/mL) of internal standard solution camptothecin (National Institutes for Drug Control) and 175 μL of acetonitrile. The mixture was vortexed for 5 min and centrifuged for 10 min (3700 rpm/min), and 1 μL of the supernatant of the plasma sample was taken for LC/MS/MS assay (API4000 triple quadrupole tandem mass spectrometer, Applied Biosystems, USA; Shimadzu, LC-30AD ultra high performance liquid chromatography system, Shimadzu, Japan).

4. Pharmacokinetic Parameters

[0351] Pharmacokinetic parameters for the compound of the present disclosure are shown in Table 3 below.

TABLE-US-00007 TABLE 3 Pharmacokinetic parameters of the compound of the present disclosure Pharmacokinetic experiment (2 mg/kg) Plasma Area Resi- Apparent concen- under Half- dence Clearance volume of tration curve life time CL/F distribution Cmax AUC T½ MRT (mL/min/ Vz/F No. (ng/mL) (ng/mL*h) (h) (h) kg) (mL/kg) Example 6 799 4686 2.9 4.6 7.1 1785 CC-92480 692 1937 1.3 2.3 17.2 1961

[0352] Conclusion: The compound of the present disclosure demonstrates good absorption profile and has significant pharmacokinetic advantages.

Test Example 4. Evaluation of Plasma Stability of the Compound of the Present Disclosure

1. Abstract

[0353] The stability of the compound of Example 6 and the control example CC-92480 after incubation in cryopreserved monkey plasma at 37° C. for 0 min, 15 min, 30 min, 60 min, 120 min, 180 min and 240 min was determined quantitatively by LC-MS/MS.

2. Test Protocol

2.1 Test Compounds

[0354] Compound of Example 6 and control example CC-92480.

2.2 Test Plasma

[0355] Monkey plasma was purchased from Shanghai Medicilon Inc.

2.3 Preparation of Compound Solutions

[0356] A certain amount of the compound of Example 6 was weighed and prepared with DMSO to obtain a 30 mM stock solution. A certain volume of the stock solution was diluted into a solution I at a concentration of 1600 μM with DMSO; and a certain volume of the solution I at 1600 μM was diluted with 50% methanol to obtain a working solution II at a concentration of 16 μM. A 30 mM stock solution, a 1600 μM solution I′, and a 16 μM working solution II′ of CC-92480 were prepared as described above.

2.4 Sample Incubation

[0357] 5 μL of the working solutions at 16 μM of the compound of Example 6 and the control example CC-92480 were each added to 75 μL of plasma to make the final concentration of the compounds of 1 μM. The samples were incubated under a 37° C. water bath for 0 min, 15 min, 30 min, 60 min, 90 min, 120 min, and 180 min. After the incubation, 240 μL of acetonitrile containing the internal standard was added, and the plate was shaken on a shaker at 800 rpm for 10 min and centrifuged on a centrifugation at 4° C. at 3700 rpm for 20 min. The supernatant was analyzed by LC-MS with a sample injection volume of 2 μL.

3. Results

[0358] The conversion of the compound of the present disclosure in monkey plasma is shown in Table 4 below.

TABLE-US-00008 TABLE 4 Stability data of the compound of the present disclosure in monkey plasma No. T.sub.1/2 (min)/monkey Example 6 806 CC-92480 199

[0359] Conclusion: the compound of the present disclosure has stability advantages in monkey plasma.