HDAC6 selective inhibitors, preparation method therefor, and application thereof

Abstract

Compounds serving as histone deacetylase 6 (HDAC6) selective inhibitors, and applications thereof in the preparation of drugs for treating HDAC6-related diseases. Specifically disclosed are a compound as represented by formula (I) and a pharmaceutically acceptable salt thereof. ##STR00001##

Claims

1. A compound represented by formula (I), a pharmaceutically acceptable salt or a isomer thereof, ##STR00098## ##STR00099## is a single bond or a double bond; n is 0 or 1; each of T.sub.1, T.sub.2 is independently selected from the group consisting of CH, CH.sub.2, C(O) and N; T.sub.3 is C or N; each of Z.sub.1, Z.sub.2, Z.sub.3 is CH; L.sub.1 is selected from the group consisting of a single bond, NH and C(O)NH; R.sub.1 is selected from the group consisting of C.sub.1-3 alkyl, phenyl or 6-membered heteroaryl, each of which is optionally substituted by 1, 2 or 3 R; R.sub.2 is H, F, Cl, Br or I; Ring A is 4 to 7-membered heterocycloalkyl; R is F, Cl, Br or I; the hetero in 6-membered heteroaryl or 4 to 7-membered heterocycloalkyl is independently NH, N or O; in any of the cases above, the number of heteroatom or heteroatom group is independently 1, 2 or 3, respectively.

2. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 1, wherein R.sub.1 is selected from the group consisting of methyl, ethyl, isopropyl, phenyl and pyridyl, each of which is optionally substituted by 1, 2 or 3 R; or, ring A is selected from the group consisting of oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, 1,3-dioxolanyl, 1,4-dioxepinyl, 1,4-dioxanyl, 1,4-oxazacycloheptyl and morpholinyl; or, the structural unit ##STR00100##

3. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 2, wherein R.sub.1 is selected from the group consisting of CH.sub.3, ##STR00101## each of which is optionally substituted by 1, 2 or 3 R; or, ring A is selected from the group consisting of ##STR00102## or, the structural unit ##STR00103##

4. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 3, wherein R.sub.1 is selected from the group consisting of CH.sub.3, ##STR00104##

5. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 3, wherein the structural unit ##STR00105##

6. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 1, wherein the structural unit ##STR00106## or, the structural unit ##STR00107## is selected from the group consisting of CH.sub.2, NH, C(O)NH, ##STR00108##

7. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 6, wherein the structural unit ##STR00109##

8. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 7, wherein the structural unit ##STR00110## is selected from the group consisting of ##STR00111## ##STR00112##

9. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 8, wherein the structural unit ##STR00113## is selected from the group consisting of ##STR00114## ##STR00115##

10. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 1, wherein the structural unit ##STR00116## is selected from the group consisting of ##STR00117## ##STR00118##

11. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 10, wherein the structural unit ##STR00119## is selected from the group consisting of ##STR00120## ##STR00121##

12. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 1, wherein the structural unit ##STR00122##

13. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 1, which is selected from the group consisting of ##STR00123## ##STR00124## wherein, ring A, R, R.sub.2, L.sub.1 and n is as defined in claim 1; R.sub.1 is as defined in claim 1.

14. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 13, which is ##STR00125## wherein, each of E.sub.1, E.sub.2 is independently O, CH.sub.2 or CH.sub.2CH.sub.2; R, R.sub.2, L.sub.1 and n is as defined in claim 1.

15. The compounds or the pharmaceutically acceptable salt or the isomer thereof as defined in claim 1, which is selected from the group consisting of ##STR00126## ##STR00127## ##STR00128## ##STR00129##

16. The compound, the pharmaceutically acceptable salt or the isomer thereof as defined in claim 15 is selected from the group consisting of ##STR00130##

17. A pharmaceutical composition comprising a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof as defined in claim 1 as an active ingredient, as well as a pharmaceutically acceptable carrier.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(1) The following examples further illustrate the present invention, but the present invention is not limited thereto. The present invention has been described in detail herein, wherein the specific embodiments thereof has been disclosed, for the man skilled in the art, it's obvious that various modifications and improvements could be made to the embodiments of the present invention without departing from the spirit and scope of the present invention.

Embodiment 1

(2) ##STR00049## ##STR00050##

(3) step 1: Compound 1-1 (11.30 g, 80.14 mmol) was added into anhydrous THF (120.00 mL), then iPrMgCl (2M, 40.07 mL) was added dropwise at 78 C., and stirred at 15 C. for 16 hours under nitrogen atmosphere. When the reaction was completed, saturated NH.sub.4Cl (50 mL) was added, then after extraction and concentration, the residue was purified by silica gel chromatography to give the compound 1-2. .sup.1HNMR (400 MHz, CDCl.sub.3) 3.57 (t, J=6.0 Hz, 2H), 2.592.66 (m, 3H), 2.022.07 (m, 2H), 1.11 (d, J=6.8 Hz, 6H).

(4) step 2: Compound 1-2 (3.70 g, 24.89 mmol) and 2-chloro-4-bromopyridine (4.79 g, 24.89 mmol) were added into anhydrous THF (50.00 mL), then n-BuLi (2.5 M, 9.96 mL) was added dropwise at 78 C., and stirred at 20 C. for 18 hours under nitrogen protection. After the completion of the reaction, saturated NH.sub.4Cl was added, followed by extraction and concentration, the residue was purified by silica gel chromatography to give the compound 1-3. .sup.1HNMR (400 MHz, CDCl.sub.3) 8.31 (d, J=6.8 Hz, 1H), 7.33 (d, J=1.6 Hz, 1H), 7.157.19 (m, 1H), 3.923.98 (m, 1H), 3.753.81 (m, 1H), 2.08-2.17 (m, 2H), 1.892.01 (m, 2H), 1.601.72 (m, 2H), 0.78 (d, J=6.8 Hz, 3H), 0.72 (d, J=6.8 Hz, 3H).

(5) Step 3: Compound 1-3 (1.60 g, 7.09 mmol), KOH (795.64 mg, 14.18 mmol) was added into the mixed solvent of dioxane (15.00 mL)/H.sub.2O (5.00 mL), then Pd.sub.2(dba).sub.3 (324.62 mg, 354.50 mol) and tBuXPhOS (301.07 mg, 709.00 mol) were added and stirred at 115 C. for 18 hours under nitrogen protection. After the completion of the reaction, the mixture was diluted by water and extracted by EtOAc, the extract was dried by brine and anhydrous and then concentrated to give the compound 1-4. MS ESI calculated value C.sub.12H.sub.16ClNO [M+H].sup.+ 208, measured value 208.

(6) Step 4: Compound 1-4 (200.00 mg, 964.92 mol) was added into anhydrous DMF (50.00 mL), then NaH (60%, 57.90 mg, 1.45 mmol) was added at 0 C., and stirred at 25 C. for 0.5 hours, then methyl 4-bromomethylbenzoate (221.04 mg, 964.92 mol) was added at 0 C. and stirred at 25 C. for 0.5 hours. After the completion of the reaction, 20 mL water was added and the residue was filtered off and dried then purified by column chromatography to give the compound 1-5. MS ESI calculated value C.sub.21H.sub.25NO.sub.4 [M+H].sup.+ 356, measured value 356.

(7) Step 5: Compound 1-5 (100 mg, 281.35 mol) was added into the mixed solvent DCM/H.sub.2O (15.00 mL), then 2M NaOH (2 mL) was added at 0 C., followed by addition of 50% NH.sub.2OH (2 mL), and then stirred at 25 C. for 0.5 hours. Preparative HPLC was used for separation to give the target compound 1-6. .sup.1H NMR (400 MHz, MeOD) 7.74 (d, J=8.0 Hz, 2H), 7.68 (d, J=6.8 Hz, 1H), 7.40 (d, J=8.0 Hz, 2H), 6.61 (d, J=0.8 Hz, 1H), 6.46 (dd, J=6.8 Hz & 0.8 Hz, 1H), 5.25 (s, 2H), 3.913.97 (m, 1H), 3.793.84 (m, 1H), 2.102.19 (m, 3H), 2.012.05 (m, 1H), 1.791.94 (m, 1H), 0.95 (d, J=7.2 Hz, 3H), 0.83 (d, J=7.2 Hz, 3H). MS ESI calculated value C.sub.20H.sub.24N.sub.2O.sub.4 [M+H].sup.+ 357, measured value 357.

Embodiment 2

(8) ##STR00051## ##STR00052##

(9) Step 1: At 78 C. and under nitrogen atmosphere, N-butyllithium (2.5 M, 24.94 mL, 1.20 eq) was added dropwise into the compound 2-1 (10.00 g, 51.96 mmol, 1.00 eq) and 5-chloropentan-2-one (6.27 g, 51.96 mmol, 1.00 eq) in tetrahydrofuran (100.00 mL), and then stirred at 78-15 C. for 5 hours under nitrogen atmosphere. The reaction solution was then concentrated and dispersed in water (100 mL) and EtOAc (100 mL), after extraction, the organic phases were combined and washed by saturated brine (100 mL*3), dried by Na.sub.2SO.sub.4, concentrated and then purified by silica get column to give the compound 2-2. MS ESI calculated value C.sub.10H.sub.12ClNO [M+H].sup.+ 198, measured value 198.

(10) Step 2: Under nitrogen atmosphere, Pd.sub.2(dba) 3 (1.39 g, 1.52 mmol, 0.10 eq) and t-BuXphos (1.29 g, 3.04 mmol, 0.20 eq) were added into the compound 2-2 (3.00 g, 15.18 mmol, 1.00 eq) and potassium hydroxide (1.28 g, 22.77 mmol, 1.50 eq) in dioxane (15.00 mL) and water (3.00 mL) solution. Ater 3 times of nitrogen displacement, the mixture was stirred at 110 C. for 12 hours under nitrogen atmosphere. The reaction mixture was suction filtered and was added into water, then extracted by dichloromethane:methanol (10:1, 20 mL*3), washed by saturated brine and dried over sodium sulfate, then concentrated and purified by silica gel column to give the compound 2-3. MS ESI calculated value C.sub.10H.sub.13NO [M+H].sup.+ 180, measured value 180.

(11) Step 3: Compound 2-3 (230.00 mg, 1.28 mmol, 1.00 eq), methyl 4-(bromomethyl) benzoate (293.97 mg, 1.28 mmol, 1.00 eq), barium carbonate (627.21 mg, 1.93 mmol, 1.50 eq) and potassium iodide (10.65 mg, 64.17 mol, 0.05 eq) in tetrahydrofuran (20.00 mL) was displaced by nitrogen for 3 times, and then stirred at 66 C. for 12 hours under nitrogen atmosphere. The reaction mixture was concentrated, dissolved in water then extracted by EtOAc, after washed by saturated brine, it was dried over sodium sulfate and concentrated to give the compound 2-4. MS ESI calculated value C.sub.19H.sub.21NO.sub.4 [M+H].sup.+ 328, measured value 328.

(12) Step 4: At 0 C. under nitrogen atmosphere, sodium hydroxide solution (2 M, 2.29 mL, 5.00 eq) was added dropwise into the compound 2-4 (300.00 mg, 916.39 mol, 1.00 eq) in methanol (15.00 mL). The reaction mixture was stirred at 15 C. for 2.5 hours. Then the reaction mixture was concentrated and dissolved into water (15 mL), extracted by dichloromethane:methanol (10:1, 15 mL*3), and washed by saturated brine, then dried over sodium sulfate, followed by concentration, finally purified by preparative HPLC to give the compound 2-5. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.40 (m, 2H), 7.19 (d, J=7.2, 1H), 6.87 (m, 2H), 6.72 (s, 1H), 6.27 (d, J=5.6 Hz, 2H), 4.01 (m, 2H), 3.86 (d, J=6.0, 2H), 2.46 (m, 2H), 2.16 (s, 2H), 1.96 (m, 2H), 1.65 (m, 4H), 1.32 (s, 4H). MS ESI calculated value C.sub.18H.sub.20N.sub.2O.sub.4 [M+H].sup.+ 329, measured value 329.

Embodiment 3

(13) ##STR00053##

(14) Step 1: At 65 C. to 75 C., N-butyllithium in cyclohexane solution (2.5 M, 49.89 mL, 1.20 eq) was added dropwise into 4-bromo-2-chloro-pyridine (20.00 g, 103.93 mmol, 1.00 eq) in toluene (30.00 mL), and then stirred at 65 C. to 75 C. for 1 hours, then the compound 3-1 (20.85 g, 103.93 mmol, 17.09 mL, 1.00 eq) in toluene (100.00 mL) solution was added into the reaction system, the temperature was raised to 25 C. and stirred for 2 hours. Saturated ammonium chloride solution was added into the reaction mixture, then extracted by EtOAc, then dried over anhydrous sodium sulfate, followed by filtration and concentration. After concentrated, the solution was purified by silica gel column to give the compound 3-2. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.29 (d, J=5.3 Hz, 1H), 7.43-7.37 (m, 3H), 7.24 (dd, J=1.6, 5.2 Hz, 1H), 7.06-7.00 (m, 2H), 4.13-4.00 (m, 2H), 2.64-2.56 (m, 1H), 2.46 (m, 1H), 2.04-1.91 (m, 2H). MS ESI calculated value C.sub.15H.sub.13ClFNO [M+H]+ 278, measured value 278.

(15) Step 2: The temperature of the reaction mixture formed by compound 3-2 (8.50 g, 30.61 mmol, 1.00 eq), potassium hydroxide (3.44 g, 61.22 mmol, 2.00 eq), tris(dibenzylideneacetone) dipalladium (5.61 g, 6.12 mmol, 0.20 eq) and 2-di-tert-butylphosphine-2, 4, 6-triisopropylbiphenyl (2.60 g, 6.12 mmol, 0.20 eq) in dioxane (2.00 mL) and water (1.00 mL) was raised to 100 C. and the mixture was stirred for 2 hours. 1M dilute hydrochloric acid was added into the reaction mixture to adjust the PH to 7, then water was added, EtOAc was used for extraction and then the mixture was dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The mixture was then slurry purify () to give the compound 3-3. .sup.1H NMR (400 MHz, CDCl.sub.3) 12.84 (brs, 1H), 7.40 (dd, J=5.3, 8.7 Hz, 2H), 7.25 (d, J=6.8 Hz, 1H), 7.01 (t, J=8.8 Hz, 2H), 6.69 (s, 1H), 6.29 (dd, J=1.4, 6.8 Hz, 1H), 4.12-3.98 (m, 2H), 2.48 (t, J=7.2 Hz, 2H), 1.98 (m, 2H), 1.25 (dd, J=6.9, 9.5 Hz, 2H). MS ESI calculated value C.sub.15H.sub.14FNO.sub.2 [M+H].sup.+ 260, measured value 260.

(16) Step 3: At 0-5 C., sodium hydride (1.08 g, 27.00 mmol, purity 60%, 1.00 eq) was added into the compound 3-3 (7.00 g, 27.00 mmol, 1.00 eq) in N, N-dimethylformamide (70.00 mL) solution, and stirred at 0-5 C. for 10 mins, Then methyl 4-bromomethylbenzoate (6.18 g, 27.00 mmol, 1.00 eq) in N, N-dimethylformamide (20.00 mL) was added into the reaction system, and stirred at 10-25 C. for 2 mins. Saturated ammonium chloride solution was added into the reaction mixture, extracted with EtOAc and dried over anhydrous sodium sulfate then concentrated. After concentration the compound 3-4 was obtained and used directly in the next step.

(17) Step 4: Sodium hydroxide solution (2 M, 27.00 mL, 2.00 eq) and oxyammonia solution (1.00 mL, 50% purity) were added into the mixture of the compound 3-4 (11.00 g, 27.00 mmol, 1.00 eq), oxyammonia solution (100.00 mL, 50% purity), methanol (20.00 mL) and dichloromethane (10.00 mL), the temperature was raised to 40-50 C., and stirred for 1 hours. After the reaction mixture was concentrated, 1M hydrochloric acid was added to adjust the PH to 7, and then extracted by dichloromethane, After concentration, reverse phase preparative HPLC was used for purification to give the compound 3-5. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 11.17 (s, 1H), 9.04 (br s, 1H), 7.74-7.64 (m, 3H), 7.55-7.45 (m, 2H), 7.32 (d, J=8.3 Hz, 2H), 7.14 (t, J=8.9 Hz, 2H), 6.50 (d, J=1.9 Hz, 1H), 6.25 (dd, J=2.0, 7.2 Hz, 1H), 6.29-6.22 (m, 1H), 5.05 (s, 2H), 3.98-3.83 (m, 2H), 2.48-2.41 (m, 2H), 1.85 (m, 2H). MS ESI calculated value C.sub.23H.sub.21FN.sub.2O.sub.4 [M+H].sup.+ 409, measured value 409.

Embodiment 4

(18) ##STR00054## ##STR00055## ##STR00056##

(19) Step 1: Sodium hydride (347.13 mg, 8.69 mmol, 60% purity) was added into the compound 4-1 (1.50 g, 5.79 mmol) in dimethylformamide (20.00 mL) solution, and stirred at 0 C. for 30 mins, then methyl 4-bromomethylbenzoate (1.99 g, 8.69 mmol) was added, raise to room temperature, and stirred for 2 hours. At 0 C. The mixture was quenched with saturated ammonium chloride solution (15 mL) and diluted with water (20 mL) then extracted with EtOAc (20 mL*4). The organic phases were combined and washed with water (20 mL*2) and saturated brine (20 mL*1), then dried and concentrated, purified with silica get column chromatography (PE/EtOAc=3/1, 1:1 to 0/1) to give the crude product. The mixture of crude product was chiral resoluted (chiral column type: OJ (250 mm*30 mm, 10 m); mobile phase: A: CO2 B: 0.05% diethylamine/EtOH, EtOH (0.05% diethylamine) with supercritical fluid CO2 from 5% to 40%, flow velocity 60 mL/min), after resolution, the compound 4-2 and the compound 5-1 was obtained, remaining times were 2.226 min and 2.835 min respectively. The compound 4-2 has 1H NMR (400 MHz, D MSO-d6) =7.95-7.88 (m, 2H), 7.71 (d, J=7.2 Hz, 1H), 7.54-7.46 (m, 2H), 7.38 (d, J=8.4 Hz, 2H), 7.19-7.09 (m, 2H), 6.51 (d, J=2.0 Hz, 1H), 6.27 (dd, J=2.0, 7.2 Hz, 1H), 5.09 (s, 2H), 3.98-3.85 (m, 2H), 3.83 (s, 3H), 2.49-2.41 (m, 2H), 1.91-1.75 (m, 2H). MS ESI calculated value C24H22FNO4 [M+H]+ 407.43, measured value 408.1.

(20) Step 2: Sodium hydroxide (103.20 mg, 2.58 mmol) and hydroxylamine (5.00 mL, 50% solution) were added into the compound 4-2 (700.00 mg, 1.72 mmol) in methanol (5.00 mL) solution, then stirred at rt for 30 mins. After concentration, diluted hydrochloric acid (1M) was added to adjust pH to 7, then purified by reverse phase preparative HPLC (0.225% FA) to give the compound 4-3. .sup.1H NMR (400 MHz, D MSO-d6) =11.49-10.50 (br s, 1H), 9.04 (br s, 1H), 7.69 (m, 3H), 7.49 (m, 2H), 7.33 (m, 2H), 7.14 (m, 2H), 6.50 (m, 1H), 6.25 (m, 1H), 5.05 (s, 2H), 3.90 (m, 2H), 2.45 (m, 2H), 1.85 (m, 2H). MS ESI calculated value C.sub.23H.sub.21N.sub.2O.sub.4F [M+H].sup.+ 408.42, measured value 408.9.

Embodiment 5

(21) ##STR00057## ##STR00058##

(22) Step 1: Sodium hydroxide (132.60 mg, 3.32 mmol) and hydroxylamine (5.00 mL, 50% solution) were added into the compound 5-1 (900.00 mg, 2.21 mmol) in methanol (5.00 mL) solution, then stirred at rt for 30 mins. The mixture was concentrated and was added diluted hydrochloric acid 1M) to adjust the pH to 7, then extracted with EtOAc (50 mL*4), the organic phases were combined and concentrated, then purified by reverse phase preparative HPLC ((0.225% FA) to give the compound 5-2. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 12.07-10.09 (br s, 1H), 9.08 (br s, 1H), 7.68 (m, 3H), 7.56-7.44 (m, 2H), 7.32 (m, 2H), 7.14 (m, 2H), 6.50 (m, 1H), 6.25 (m, 1H), 5.01 (s, 2H), 4.09-3.74 (m, 2H), 2.45 (m, 2H), 1.94-1.76 (m, 2H). MS ESI calculated value C.sub.23H.sub.21N.sub.2O.sub.4F [M+H].sup.+ 408.42, measured value 408.8.

Embodiment 6

(23) ##STR00059## ##STR00060##

(24) Step 1: At 5 C., 4-chlorobutyryl chloride (500.00 mg, 3.55 mmol, 396.83 L, 0.50 eq) was added into the mixture of the compound 6-1 (809.15 mg, 7.09 mmol, 697.54 L, 1.00 eq) and alcohol (633.60 mg, 4.75 mmol, 259.67 L, 0.67 eq), then stirred fat 25 C. for 3 hours, the reaction mixture was added into ice water, then stirred, extracted with EtOAc, and washed by saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the compound 6-2. The liquid was used directly in the next reaction.

(25) Step 2: At 68 C., N-butyllithium in cyclohexane solution (2.5 M, 1.21 mL, 1.10 eq) was added into 4-bromo-2-chloro-pyridine (527.29 mg, 2.74 mmol, 1.00 eq) in toluene (5.00 mL) solution, and stirred at 68 C. for 10 mins, the compound 6-2 (600.00 mg, 2.74 mmol, 1.00 eq) in toluene (5.00 mL) solution was added into the reaction system, and stirred at 68 C. for 1 hours then at 25 C. for 14 hours. Saturated ammonium chloride solution and water were added into the reaction mixture sequentially, extracted by EtOAc, then washed by saturated brine, dried over anhydrous sodium sulfate then concentrated. After concentration, TLC plate was used for purification and give the compound the compound 6-3. MS ESI calculated value C.sub.15H.sub.12F.sub.2ONCl [M+H]+ 296, measured value 296.

(26) Step 3: Potassium hydroxide (33.02 mg, 588.42 mol, 2.00 eq), tris(dibenzylideneacetone) dipalladium (26.94 mg, 29.42 mol, 0.10 eq) and 2-di-tertbutylphosphine-2, 4, 6-triisopropylbiphenyl (12.49 mg, 29.42 mol, 0.10 eq) were added into the compound 6-3 (87.00 mg, 294.21 mol, 1.00 eq) in dioxane (2.00 mL) and water (1.00 mL) solution, then stirred at 80 C. for 3 hours. Water was added into the reaction mixture, then the mixture was extracted by EtOAc, washed by saturated brine, dried over anhydrous sodium sulfate and filtrated and concentrated. After concentration, TLC plate was used for purification to give the compound 6-4. MS ESI calculated value C.sub.15H.sub.13F.sub.2O.sub.2N [M+H].sup.+ 278, measured value 278.

(27) Step 4: At 0 C., sodium hydride (17.31 mg, 432.80 mol, purity 60%, 2.00 eq) was added into the compound 6-4 (60.00 mg, 216.40 mol, 1.00 eq) in N, N-dimethylformamide (1.00 mL) solution, then methyl 4-bromomethylbenzoate (49.57 mg, 216.40 mol, 1.00 eq) in N, N-dimethylformamide (1.00 mL) solution was added into the reaction system, then stirred at 25 C. for 2 hours. Water was added into the reaction mixture, then the mixture was extracted by EtOAc, washed by saturated brine, dried over anhydrous sodium sulfate and filtrated and concentrated. After concentration, TLC plate was used for purification to give the compound 6-5. MS ESI calculated value C.sub.24H.sub.21NO.sub.4F.sub.2 [M+H].sup.+ 426, measured value 426.

(28) Step 5: Sodium hydroxide solution (6 M, 50.00 L, 3.99 eq) and hydroxylamine solution (1.00 mL, 50% solution) were added into the compound 6-5 (32.00 mg, 75.22 mol, 1.00 eq) in methanol (2.00 mL) solution, then stirred at 25 C. for 17 hours. After the concentration of the reaction mixture, the mixture was purified by reverse phase column to give the compound 6-6. .sup.1H NMR (400 MHz, MeOD) ppm 1.89-2.08 (m, 2H) 2.48-2.74 (m, 2H) 3.93-4.11 (m, 2H) 5.20 (s, 2H) 6.46 (dd, J=7.09, 1.82 Hz, 1H) 6.66 (s, 1H) 6.88-7.03 (m, 2H) 7.37 (d, J=8.16 Hz, 2H) 7.63 (d, J=7.15 Hz, 1H) 7.67-7.72 (m, 2H) 7.72-7.75 (m, 1H). MS ESI calculated value C.sub.23H.sub.20N.sub.2O.sub.4F.sub.2 [M+H].sup.+ 427, measured value 427.

Embodiment 7

(29) ##STR00061##

(30) Step 1: Sodium hydride (21.60 mg, 539.98 mol, 60% purity) was added into the compound 7-1 (70.00 mg, 269.99 mol) in dimethylformamide (5.00 mL) solution, then stirred at 0 C. for 30 mins, and 6-bromomethyl-3-methyl formate pyridine (124.23 mg, 539.98 mol) was added into the mixture, then stirred at 0 C. for 30 mins. The temperature was raised to rt and keep stirring for 30 mins. The mixture was added saturated ammonium chloride solution (1 mL) and water (15 mL), then extracted by EtOAc (10 mL*4). The organic phases were combined and washed by water (10 mL*3) and saturated brine (10 mL), then dried, filtered and concentrated. Separation with plate (PE/EA=1/1) gave the compound 7-2. MS ESI calculated value C.sub.23H.sub.21FN.sub.2O.sub.4 [M+H].sup.+ 409, measured value 409.

(31) Step 2: Hydroxylamine (1.00 mL, 50% solution) and sodium hydroxide NaOH (6.44 mg, 161.11 mol) were added into the compound 7-2 (70.00 mg, 161.11 mol) in methanol (5.00 mL) solution, then stirred at rt for 30 mins. The mixture was added into diluted hydrochloric acid (1M) to adjust pH to 7, followed by purification to give the compound 7-3. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 8.77 (d, J=1.8 Hz, 1H), 8.05 (dd, J=2.4, 8.2 Hz, 1H), 7.69 (d, J=7.2 Hz, 1H), 7.57-7.48 (m, 2H), 7.30 (d, J=8.2 Hz, 1H), 7.21-7.11 (m, 2H), 6.48 (d, J=1.8 Hz, 1H), 6.28 (dd, J=2.0, 7.2 Hz, 1H), 5.14 (s, 2H), 4.01-3.83 (m, 2H), 2.49-2.44 (m, 2H), 1.92-1.79 (m, 2H). MS ESI calculated value C.sub.22H.sub.20FN.sub.3O.sub.4 [M+H].sup.+ 410, measured value 410.

Embodiment 8

(32) ##STR00062## ##STR00063##

(33) Step 1: Sodium hydride (21.60 mg, 539.98 mol, purity 60%) was added into the compound 8-1 (70.00 mg, 269.99 mol) in dimethylformamide (5.00 mL) solution, then stirred at 0 C. for 30 mins, Then 2-bromomethyl-5-methyl formate pyrimidine (132.33 mg, 539.98 mol) was added and keep stirring at 0 C. for 30 mins, raised the temperature to rt and keep stirring for 30 mins. The mixture was quenched by saturated ammonium chloride (1 mL), water was added (15 mL) then extracted by EtOAc (10 mL*4). The combined organic phases were washed by water (5 mL*3) and saturated brine (10 mL), and was dried, filtered and concentrated, then purified by preparative thin layer chromatography (PE/EA=1/1) to give the compound 8-2. MS ESI calculated value C22H20FN3O4 [M+H].sup.+ 424, measured value 424.

(34) Step 2: Hydroxylamine (1.00 mL, 50% solution) and sodium hydroxide NaOH (6.43 mg, 160.72 mol) were added into the compound 8-2 (70.00 mg, 160.72 mol,) in methanol (5.00 mL) solution, then stirred at 0 C. for 30 mins. The mixture was added hydrochloric acid to adjust pH to 7, then purified to give the compound 8-3. .sup.1H NMR (400 MHz, D MSO-d6) 8.97 (s, 2H), 7.69 (d, J=7.2 Hz, 1H), 7.59-7.51 (m, 2H), 7.20-7.13 (m, 2H), 6.47 (d, J=2.0 Hz, 1H), 6.31 (dd, J=2.0, 7.2 Hz, 1H), 5.26 (s, 2H), 4.00-3.87 (m, 2H), 2.53 (m, 2H), 1.93-1.82 (m, 2H). MS ESI calculated value C.sub.21H.sub.19FN.sub.4O.sub.4 [M+H].sup.+ 411, measured value 411.

Embodiment 9

(35) ##STR00064## ##STR00065##

(36) Step 1: Sodium hydride (21.60 mg, 539.98 mol, purity 60%) was added into the compound 9-1 (70.00 mg, 269.99 mol) in dimethylformamide (5.00 mL) solution, then stirred at 0 C. for 30 mins, then 2-methylformate-5-bromomethylpyridine (124.23 mg, 539.98 mol) was added and was continued stirring at 0 C. for 30 mins, the temperature was raised to rt and stirred for 30 mins. The mixture was quenched by saturated ammonium chloride (1 mL*1), water (15 mL) was added then extracted with EtOAc (10 mL*3). The combined organic phases were washed by water (10 mL*2) and saturated brine (10 mL), then was dried, filtered and concentrated, followed by purification with preparative TLC (PE/EA=1/1) to give the compound 9-2. MS ESI calculated value C.sub.23H.sub.21FN.sub.2O.sub.4 [M+H].sup.+ 409, measured value 409.

(37) Step 2: Hydroxylamine (1.00 mL, 50% solution) and sodium hydroxide NaOH (3.06 mg, 76.39 mol) were added into the compound 9-2 (50.00 mg, crude product) in methanol (5.00 mL) solution, then stirred at 0 C. for 30 mins. The mixture was added hydrochloric acid to adjust pH to 7, then purified to give the compound 9-3. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 8.53 (s, 1H), 7.97-7.87 (m, 1H), 7.86-7.72 (m, 2H), 7.50 (m, 2H), 7.14 (m, 2H), 6.51 (m, 1H), 6.28 (m, 1H), 5.10 (s, 2H), 4.01-3.81 (m, 2H), 2.48-2.42 (m, 2H), 1.92-1.76 (m, 2H). MS ESI calculated value C.sub.22H.sub.20FN.sub.3O.sub.4 [M+H].sup.+410, measured value 410.

Embodiment 10

(38) ##STR00066## ##STR00067##

(39) Step 1: 4-chlorobutyryl chloride (2.50 g, 17.73 mmol, 1.98 mL, 1.00 eq) was added dropwise into the mixture of the compound 10-1 (4.68 g, 35.46 mmol, 3.66 mL, 2.00 eq) and alcohol (3.14 g, 23.58 mmol, 1.29 mL, 1.33 eq), then stirred at 30 C. for 6 hours, the reaction mixture was added into ice water, then was stirred and extracted by EtOAc, washed by saturated brine, dried over anhydrous sodium sulfate, and was filtered and concentrated, then purified by silica gel column to give the compound 10-2. This liquid was used directly in the next reaction.

(40) Step 2: At 68 C., N-butyllithium in cyclohexane solution (2.5 M, 1.12 mL, 1.10 eq) was added into 4-bromo-2-chloro-pyridine (488.80 mg, 2.54 mmol, 1.00 eq) in toluene (3.00 mL) solution, then the compound 10-2 (600.00 mg, 2.54 mmol, 1.00 eq) in toluene (1.00 mL) solution was added into the reaction system, temperature was raised to 25 C. and stirred for 6 hours. Saturated ammonium chloride solution and water were added into the reaction mixture, then the mixture was extracted by EtOAc, and washed by saturated brine, dried over anhydrous sodium sulfate, and was concentrated. After concentration, the mixture was purified with silica gel column to give the compound 10-3. MS ESI calculated value C.sub.15H.sub.11F.sub.3ONCl [M+H].sup.+ 314, measured value 314.

(41) Step 3: Potassium hydroxide (146.67 mg, 2.61 mmol, 2.00 eq), tris(dibenzylideneacetone) dipalladium (119.96 mg, 131.00 mol, 0.10 eq) and 2-di-tert-butylphosphine-2, 4, 6-triisopropylbiphenyl (55.50 mg, 130.70 mol, 0.10 eq) were added into the compound 10-3 (410.00 mg, 1.31 mmol, 1.00 eq) in dioxane (5.00 mL) and water (1.00 mL) solution, then stirred at 80 C. for 4 hours. Saturated ammonium chloride solution and water were added into the reaction mixture, then the mixture was extracted by EtOAc, and washed by saturated brine, dried over anhydrous sodium sulfate, then filtered and concentrated. After concentration, the mixture was purified by silica gel column to give the compound 10-4.

(42) Step 4: The temperature of the compound 10-4 (100.00 mg, 338.68 mol, 1.00 eq), methyl 4-bromomethylbenzoate (155.16 mg, 677.36 mol, 2.00 eq) and barium carbonate (220.70 mg, 677.37 mol, 2.00 eq) in acetonitrile (2.00 mL) solution was raised to 80 C., the mixture was stirred for 2 hours. The reaction mixture was then added water, extracted by EtOAc, washed by saturated brine and dried over anhydrous sodium sulfate, followed by concentration. After concentration, the mixture was purified by preparative thin layer chromatography to give the compound 10-5.

(43) Step 5: Sodium hydroxide (3.61 mg, 90.21 mol, 1.00 eq) and hydroxylamine solution (1.00 mL, 50% purity) were added into the compound 10-5 (40.00 mg, 90.21 mol, 1.00 eq) methanol (2.00 mL) solution, then stirred at 25 C. for 17 hours. After the reaction mixture was concentrated, it was purified by reverse phase preparative column to give the compound 10-6. .sup.1H NMR (400 MHz, MEOD) ppm 1.75-2.11 (m, 2H) 2.53-2.84 (m, 2H) 3.90-4.20 (m, 2H) 5.41 (s, 2H) 6.79-6.88 (m, 2H) 6.92 (s, 1H) 6.97 (dd, J=5.40, 1.25 Hz, 1H) 7.54 (d, J=8.28 Hz, 2H) 7.76 (d, J=8.28 Hz, 2H) 8.05 (d, J=5.40 Hz, 1H), MS ESI calculated value C23H19N2O4F3 [M+H].sup.+ 445, measured value 445.

Embodiment 11

(44) ##STR00068##

(45) Step 1: Under nitrogen protection, at 70 C., n-BuLi (2.5 M, 9.12 mL, 1.20 eq) was added dropwise into 4-bromo-2-chloro-3-fluoropyridine (4.00 g, 19.01 mmol, 1.00 eq) in toluene (20.00 mL) solution, after the completion of the adding, the mixture was stirred at the temperature for 30 mins, then the compound 11-1 (3.81 g, 19.01 mmol, 819.67 L, 1.00 eq) was added into the suspension above. The temperature of the reaction system was raised slowly to 40 C. and stirred for 12 hours. The reaction mixture was poured into water (80 mL), then extracted by EtOAc (40 mL3), the combined extract was washed by brine (50 mL), and dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The solid obtained was purified by silica gel column chromatography (PE/EtOAc=10/1) to give the compound 11-2. MS ESI calculated value Cs.sub.15H.sub.12ClF.sub.2NO [M+H].sup.+ 296, measured value 296.

(46) Step 2: Under nitrogen protection, the mixture of compound 11-2 (3.86 g, 13.05 mmol, 1.00 eq), t-BuXPhOS (554.16 mg, 1.31 mmol, 0.10 eq), Pd.sub.2(dba).sub.3 (1.20 g, 1.31 mmol, 0.10 eq) and KOH (1.46 g, 26.10 mmol, 2.00 eq) in dioxane (100 mL) and H.sub.2O (20 mL) was heated to 100 C. and stirred for 2 hours. The reaction mixture was then poured into H.sub.2O (80 mL), then extracted by EtOAc/MeOH (10/1, 30 mL3), the combined extract was washed by brine (40 mL), then dried over anhydrous Na.sub.2SO.sub.4, and filtrated and concentrated under reduced pressure. The solid obtained was slurried by PE (20 mL) and filtered, dried over to give the compound 11-3. MS ESI calculated value C.sub.15H.sub.13F2NO.sub.2 [M+H].sup.+ 278, measured value 278.

(47) Step 3: Under nitrogen protection, at 0 C., NaH (553.97 mg, 13.85 mmol, purity 60%, 1.20 eq) was added into the compound 11-3 (3.20 g, 11.54 mmol, 1.00 eq) in DMF (60 mL) solution, then stirred at the temperature for 20 mins, then methyl 4-bromomethylbenzoate (2.64 g, 11.54 mmol, 1.10 eq) was added into the suspension above and the temperature was raised to 20 C., continued to stir for 1 hours. The reaction mixture was poured into H.sub.2O (250 mL), and extracted by EtOAc/MeOH (10/1, 50 mL3), the combined extract was washed by brine (100 mL), then dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The solid obtained was pass through silica gel column chromatography (PE/EtOAc=3/1) to give the compound 11-4. MS ESI calculated value C.sub.24H.sub.21F.sub.2NO.sub.4 [M+H].sup.+ 426, measured value 426.

(48) Step 4: At 0 C., NH.sub.2OH.H.sub.2O (20.0 mL, 50% solution) and NaOH (2 M, 20.0 mL) solution were added dropwise in sequence into the compound 11-4 (2.00 g, 4.70 mmol, 1.00 eq) in MeOH (20.0 mL) and DCM (10.0 mL) solution, then continued to stir the reaction at 0 C. for 1 hours. Most of the solvent was eliminated by concentration under reduced pressure, the rest of the solution was cooled down to 0 C. and adjusted the pH=7-8 with concentrated hydrochloric acid, after treatment the compound 11-5 was obtained. 1H NMR (400 MHz, D MSO-d.sub.6) 11.18 (s, 1H), 9.90-9.90 (m, 1H), 9.04 (brs, 1H), 7.98 (d, J=6.78 Hz, 1H), 7.70 (d, J=8.03 Hz, 2H), 7.44 (dd, J=8.41, 5.65 Hz, 2H), 7.37 (d, J=8.03 Hz, 2H), 7.15 (t, J=8.91 Hz, 2H), 6.60 (d, J=7.53 Hz, 1H), 4.90-5.15 (m, 2H), 3.80-4.05 (m, 2H), 2.60-2.75 (m, 1H), 2.38-2.44 (m, 1H), 1.93 (q, J=7.09 Hz, 2H). MS ESI calculated value C.sub.23H.sub.20F.sub.2N.sub.2O.sub.4 [M+H]+ 427, measured value 427.

Embodiment 12

(49) ##STR00069## ##STR00070##

(50) Step 1: Under N.sub.2 protection, at 70 C., n-BuLi (2.5 M, 1.29 mL, 1.20 eq) was added dropwise into 4-bromo-2-chloropyridine (515.62 mg, 2.68 mmol, 1.00 eq) in toluene (3.00 mL) solution, after completion of adding, the mixture was stirred at the temperature for 20 mins, then the compound 12-1 (500.00 mg, 2.68 mmol, 1.00 eq) was added into the suspension above. The temperature of the reaction system was raised slowly to 25 C. and stirred for 6 hours. Toluene was eliminated by concentration under reduced pressure, the remaining solid was dissolved in DMF (3.00 mL), at 25 C., NaH (160.76 mg, 4.02 mmol, purity 60%, 1.50 eq) was added, then the system was heated to 60 C. and stirred for 2 hours. Then the reaction mixture was poured into H.sub.2O (80 mL), then EtOAc (30 mL3) was used for extraction, the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was passed through silica gel column chromatography (PE/EtOAc=10/1) to give the compound 12-2. MS ESI calculated value C.sub.14H.sub.11ClFNO [M+H].sup.+ 264, measured value 264.

(51) Step 2: Under N.sub.2 protection, the mixture of compound 12-2 (320.00 mg, 1.21 mmol, 1.00 eq), t-BuXPhOS (102.76 mg, 242.00 mol, 0.20 eq), Pd.sub.2(dba).sub.3 (221.60 mg, 242.00 mol, 0.20 eq) and KOH (135.79 mg, 2.42 mmol, 2.00 eq) in dioxane (10.00 mL) and H.sub.2O (3.00 mL) was heated to 100 C. and stirred for 2 hours. The reaction mixture was poured into H.sub.2O (100 mL), Then EtOAc (50 mL3) was used for extraction, the combine extracts was washed by brine (60 mL), and dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was passed through preparative TLC (PE/EtOAc=3/1) to give the compound 12-3. MS ESI calculated value C.sub.14H.sub.12FNO.sub.2 [M+H].sup.+ 246, measured value 246.

(52) Step 3: Under N.sub.2 protection, at 0 C., NaH (56.80 mg, 1.42 mmol, purity 60%, 1.20 eq) was added into the compound 12-3 (290.00 mg, 1.18 mmol, 1.00 eq) in DMF (5.00 mL) solution, and stirred at the temperature for 20 mins, then methyl 4-bromomethylbenzoate (297.33 mg, 1.30 mmol, 1.10 eq) was added into the suspension above, the temperature of the reaction system was raised to 20 C. and continued to stir for 2 hours. The reaction mixture was poured into water (80 mL), then extracted by EtOAc/MeOH (10/1, 30 mL3), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=1/1) to give the compound 12-4. MS ESI calculated value C.sub.23H.sub.20FNO.sub.4 [M+H].sup.+ 394, measured value 394.

(53) Step 4: At 0 C., NH.sub.2OH.H.sub.2O (150.00 L, 50% solution) and NaOH (2 M, 150.00 L) solution was added dropwise in sequence into the compound 12-4 (150.00 mg, 381.28 mol, 1.00 eq) in DCM (2.00 mL) and MeOH (2.00 mL) solution, then the reaction was stirred at 0 C. for 2 hours. Most of the solvent was eliminated by concentration under reduced pressure, the rest of the solution was purified by preparative HPLC (0.1% NH.sub.4OH) to give the compound 12-5. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 10.99 (brs, 1H), 8.67-9.79 (brs, 1H), 7.77 (d, J=7.28 Hz, 1H), 7.69 (d, J=8.03 Hz, 2H), 7.46 (dd, J=8.66, 5.40 Hz, 2H), 7.34 (d, J=8.03 Hz, 2H), 7.20 (t, J=8.91 Hz, 2H), 6.52 (d, J=1.51 Hz, 1H), 6.18 (dd, J=7.03, 1.76 Hz, 1H), 5.08 (s, 2H), 4.39-4.58 (m, 2H), 3.01-3.19 (m, 2H). MS ESI calculated value C.sub.22H.sub.19FN.sub.2O.sub.4 [M+H]+ 395, measured value 395.

Embodiment 13

(54) ##STR00071## ##STR00072##

(55) Step 1: Under N.sub.2 protection, at 70 C., n-BuLi (2.5 M, 1.03 mL, 1.20 eq) was added dropwise into 4-bromo-2-chloro-3-fluoropyridine (450.32 mg, 2.14 mmol, 1.00 eq) in toluene (8.00 mL) solution, after completion of adding, the mixture was stirred at the temperature for 20 mins, then compound 13-1 (400.00 mg, 2.14 mmol, 1.00 eq) was added into the suspension above. The temperature of the system was raised slowly to 25 C. and stirred for 6 hours. Toluene was eliminated by concentration under reduced temperature, the remaining solid was dissolved in DMF (8.00 mL), at 25 C., NaH (128.40 mg, 3.21 mmol, purity 60%, 1.50 eq) was added and then the system was heated to 60 C. and stirred for 2 hours. The reaction mixture was poured into water (80 mL), then extracted by EtOAc (30 mL3), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was passed through column chromatography (PE/EtOAc=10/1) to give the compound 13-2. MS ESI calculated value C.sub.14H.sub.10ClF.sub.2NO [M+H].sup.+ 282, measured value 282.

(56) Step 2: Under N.sub.2 protection, the mixture of compound 13-2 (440.00 mg, 1.56 mmol, 1.00 eq), t-BuXPhOS (132.49 mg, 312.00 mol, 0.20 eq), Pd.sub.2(dba).sub.3 (285.70 mg, 312.00 mol, 0.20 eq) and KOH (175.06 mg, 3.12 mmol, 2.00 eq) in dioxane (10.00 mL) and H.sub.2O (3.00 mL) was heated to 100 C. and stirred for 2 hours. The reaction mixture was poured into water (100 mL), and extracted by EtOAc (50 mL3), the combined extracts was washed by brine (60 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=3/1) to give the compound 13-3. MS ESI calculated value C.sub.14H.sub.11F2NO.sub.2 [M+H].sup.+ 264, measured value 264.

(57) Step 3: Under N.sub.2 protection, at 0 C., NaH (69.29 mg, 1.73 mmol, purity 60%, 1.20 eq) was added into the compound 13-3 (380.00 mg, 1.44 mmol, 1.00 eq) in DMF (5.00 mL) solution, then the mixture was stirred at the temperature for 20 mins, then methyl 4-bromomethylbenzoate (363.74 mg, 1.59 mmol, 1.10 eq) was added into the suspension above and the temperature of the system was raised to 20 C., then continued stirring for 2 hours. The reaction mixture was poured into water (80 mL), and extracted by EtOAc/MeOH (10/1, 30 mL3), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=1/1) to give the compound 13-4. MS ESI calculated value C.sub.23H.sub.19F.sub.2NO.sub.4 [M+H].sup.+ 412, measured value 412.

(58) Step 4: At 0 C., NH.sub.2OH.H.sub.2O (3.00 mL, 50% solution) and NaOH (2 M, 3.00 mL) solution was added dropwise in sequence into the compound 13-4 (350.00 mg, 850.75 mol, 1.00 eq) in DCM (3.00 mL) and MeOH (3.00 mL) solution, then the reaction was stirred at 0 C. for 1 hours. Most of the solvent was eliminated by concentration under reduced pressure, the rest of the solution was purified by preparative HPLC (0.1% TFA) to give the compound 13-5. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.19 (brs, 1H), 8.06 (d, J=6.52 Hz, 1H), 7.71 (d, J=8.28 Hz, 2H), 7.38-7.50 (m, 5H), 7.22 (t, J=8.91 Hz, 2H), 6.65 (d, J=7.28 Hz, 1H), 4.98-5.08 (m, 2H), 4.49-4.59 (m, 2H), 3.25-3.34 (m, 1H), 3.05-3.15 (m, 1H). MS ESI calculated value C.sub.22H.sub.18F.sub.2N.sub.2O.sub.4 [M+H]+ 413, measured value 413.

Embodiment 14

(59) ##STR00073##

(60) Step 1: Under N.sub.2 protection, at 70 C., n-BuLi (2.5 M, 6.84 mL, 1.20 eq) was added dropwise into 4-bromo-2-chloro-5-fluoropyridine (3.00 g, 14.26 mmol, 1.00 eq) in toluene (80.00 mL) solution, after completion of adding, the mixture was stirred at the temperature for 30 mins, then the compound 14-1 (2.86 g, 14.26 mmol, 2.34 mL, 1.00 eq) was added into the suspension above. the temperature of the system was raised slowly to 40 C. and stirred for 12 hours. The reaction mixture was poured into water (300 mL), and extracted by EtOAc (100 mL3), the combined extracts was washed by brine (100 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was passed through column chromatography (PE/EtOAc=10/1) to give the compound 14-2. MS ESI calculated value C.sub.15H.sub.12ClF.sub.2NO [M+H].sup.+ 296, measured value 296.

(61) Step 2: Under N.sub.2 protection, the mixture of compound 14-2 (300.00 mg, 1.01 mmol, 1.00 eq), t-BuXPhOS (85.78 mg, 202.00 mol, 0.20 eq), Pd.sub.2(dba).sub.3 (184.98 mg, 202.00 mol, 0.20 eq) and KOH (113.34 mg, 2.02 mmol, 2.00 eq) in dioxane (10.00 mL) and H.sub.2O (3.00 mL) was heated to 100 C. and stirred for 2 hours. The reaction mixture was poured into water (100 mL), and extracted by EtOAc (50 mL3), the combined extracts was washed by brine (60 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=3/1) to give the compound 14-3. MS ESI calculated value C.sub.15H.sub.13F.sub.2NO.sub.2 [M+H].sup.+ 278, measured value 278.

(62) Step 3: Under N.sub.2 protection, at 0 C., NaH (34.62 mg, 865.58 mol, purity 60%, 1.20 eq) was added into the compound 14-3 (200.00 mg, 721.32 mol, 1.00 eq) in DMF (5.00 mL) solution, and the mixture was stirred at the temperature for 20 mins, then methyl 4-bromomethylbenzoate (165.23 mg, 721.32 mol, 1.00 eq) was added into the suspension above and the temperature of the system was raised to 20 C., then continued stirring for 1 hours. The reaction mixture was poured into water (50 mL), and extracted by EtOAc/MeOH (10/1, 20 mL3), the combined extracts was washed by brine (30 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=1/1) to give the compound 14-4. MS ESI calculated value C.sub.24H.sub.21F.sub.2NO.sub.4 [M+H].sup.+ 426, measured value 426.

(63) Step 4: At 0 C., NH.sub.2OH.H.sub.2O (2.00 mL, 50% solution) and NaOH (2.00 mL, 2 M) solution were added dropwise in sequence into the compound 14-4 (100.00 mg, 235.06 mol, 1.00 eq) in DCM (2.00 mL) and MeOH (4.00 mL) solution, then the reaction was stirred at 0 C. for 1 hours. Most of the solvent was eliminated by concentration under reduced pressure, the rest of the solution was cooled down to 0 C., concentrated hydrochloric acid was added to adjust the pH=6-7, aftertreatment to give the compound 14-5. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 11.17 (brs, 1H), 9.05 (brs, 1H), 8.61-9.43 (m, 1H), 7.64-7.73 (m, 3H), 7.46 (dd, J=8.5, 5.5 Hz, 2H), 7.34 (d, J=8.0 Hz, 2H), 7.16 (t, J=8.9 Hz, 2H), 6.48 (t, J=6.9 Hz, 1H), 5.07-5.18 (m, 2H), 3.90 (t, J=7.2 Hz, 2H), 2.64-2.70 (m, 1H), 2.41-2.47 (m, 1H), 1.82-1.97 (m, 2H). MS ESI calculated value C.sub.23H.sub.20F.sub.2N.sub.2O.sub.4 [M+H]+ 427, measured value 427.

Embodiment 15

(64) ##STR00074## ##STR00075##

(65) Step 1: At 0 C., compound 15-1 (6.20 g, 64.50 mmol, 6.08 mL, 2.00 eq) and alcohol (4.73 g, 35.48 mmol, 1.10 eq) was added into a 100 mL 3-neck flask, then added also 5-chlorovaleryl chloride (5.00 g, 32.25 mmol, 4.17 mL, 1.00 eq), the mixture was heated to rt and stirred for 2 hours. The mixture was quenched by ice water (20 mL), water (100 mL) was added and extracted by EtOAc (100 mL*2), the organic phases were combined, then washed by saturated brine, dried over sodium sulfate, followed by filtration and concentration, to give the compound 15-2. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.08-7.91 (m, 2H), 7.13 (m, 2H), 3.63-3.55 (m, 2H), 2.99 (m, 2H), 1.98-1.82 (m, 4H).

(66) Step 2: At 78 C. nitrogen atmosphere, tert-butyllithium (2.5 M, 13.32 mL, 1.10 eq) was slowly added dropwise into 4-bromo-2-chloropyridine (5.83 g, 30.28 mmol, 1.00 eq) in toluene (100.00 mL) solution, then stirred for 10 mins and added the compound 15-2 (6.50 g, 30.28 mmol, 1.00 eq), then continued stirring for 30 mins. Saturated ammonium chloride (20 mL) was used to quench the reaction, water (100 mL) was then added, and EtOAc (100 mL*3) was used for extraction, the combined organic phases were washed by saturated brine (100 mL), dried over sodium sulfate, filtered and concentrated, separated with column (silica gel column, PE/EtOAc=10/1) to give the compound 15-3. MS ESI calculated value C.sub.16H.sub.16Cl.sub.2FNO [M+H].sup.+ 328.2, measured value 328.2.

(67) Step 3: At 0 C. nitrogen atmosphere, NaH (877.49 mg, 21.94 mmol, 60% purity, 1.20 eq) was added into the compound 15-3 (6.00 g, 18.28 mmol, 1.00 eq) in N, N-dimethylformamide (30.00 mL) solution, then stirred at 0-5 C. for 30 mins. The reaction was quenched by saturated ammonium chloride (30 mL), then water (100 mL) was added, and extracted by EtOAc (80 mL*3), the combined organic phases were washed by water (100 mL*3) and saturated brine (50 mL*1), dried over sodium sulfate, filtered and concentrated to give the compound 15-4. MS ESI calculated value C.sub.16H.sub.15ClFNO [M+H].sup.+ 291.75, measured value 292.0.

(68) Step 4: Under nitrogen protection, the compound 15-4 (1.00 g, 3.43 mmol, 1.00 eq), potassium hydroxide (384.64 mg, 6.86 mmol, 2.00 eq), tris(dibenzylideneacetone) dipalladium (627.74 mg, 686.00 mol, 0.20 eq) and 2-di-tertbutylphosphine-2,4,6-triisopropylbiphenyl (291.10 mg, 686.00 mol, 0.20 eq) were added into the mixed solution of water (5.00 mL) and 1,4-dioxane (10.00 mL), the mixture was then heated to 90 C., then stirred for 3 hours. The mixture was concentrated and water (50 mL) was added, then was extracted by EtOAc (20 mL*3), the combined organic phases were washed by saturated brine (50 mL), dried over sodium sulfate, filtered and concentrated, then purified by column (silica gel column, PE/EtOAc=10/1 to 2/1, dichloromethane/methanol=20/1) to give the compound 15-5. MS ESI calculated value C.sub.16H.sub.16FNO.sub.2 [M+H].sup.+ 273.30, measured value 274.1.

(69) Step 5: At 0 C. under nitrogen protection, sodium hydride (17.56 mg, 439.08 mol, 60% purity, 1.00 eq) was added into the compound 15-5 (150.00 mg, 439.08 mol, 1.00 eq) in N, N-dimethylformamide (5.00 mL) solution, the mixture was stirred for 10 mins then methyl 4-bromomethylbenzoate (125.72 mg, 548.85 mol, 1.00 eq) was added, then the mixture was heated to rt, and continued stirring for 30 mins. Saturated ammonium chloride (2 mL) and water (15 mL) was added into the system, then extracted by EtOAc (10 mL*3), the combined organic phases were washed by saturated brine (15 mL), dried over sodium sulfate, then filtered, concentrated and purified to give the compound 15-6. MS ESI calculated value C.sub.25H.sub.24FNO.sub.4 [M+H].sup.+ 421.46, measured value 422.2.

(70) Step 6: Hydroxylamine solution (2.00 mL, 50% solution) and sodium hydroxide (35.09 mg, 877.32 mol, 3.00 eq) were added into the compound 15-6 (145.00 mg, 292.44 mol, 1.00 eq) in methanol (10.00 mL) solution, the mixture was stirred at rt for 1 hours, then concentrated and purified by preparative HPLC (0.225% FA) to give the compound 15-7. .sup.1H NMR (400 MHz, D MSO-d.sub.6) =7.73-7.62 (m, 3H), 7.46 (m, 2H), 7.32 (d, J=8.2 Hz, 2H), 7.16 (t, J=8.8 Hz, 2H), 6.47 (d, J=1.6 Hz, 1H), 6.20 (dd, J=1.6, 7.2 Hz, 1H), 5.04 (s, 2H), 3.713.53 (m, 2H), 2.342.07 (m, 2H), 1.65-1.46 (m, 4H). MS ESI calculated value C.sub.24H.sub.23FN.sub.2O.sub.4 [M+H].sup.+ 422.45, measured value 423.4.

Embodiment 16

(71) ##STR00076## ##STR00077##

(72) Step 1: At 78 C. under nitrogen protection, N-butyllithium (2.5 M, 88.63 mL, 1.10 eq) was slowly added dropwise into the compound 16-1 (38.76 g, 201.43 mmol, 1.00 eq) in toluene (250.00 mL) solution, then stirred for 30 mins, then p-fluorobenzaldehyde was slowly added dropwise into the reaction system (25.00 g, 201.43 mmol, 21.19 mL, 1.00 eq), the system temperature was raised to rt in 30 mins, saturated ammonium chloride (100 mL) solution was added into the system, followed by water (200 mL), then extracted by EtOAc (150 mL*3), the organic phases were combined and washed by saturated brine (150 mL), dried over sodium sulfate, filtered and concentrated, then purified by column (silica gel column, PE:EtOAc=5:1) to give the compound 16-2. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.30 (d, J=5.2 Hz, 1H), 7.43-7.37 (m, 1H), 7.35-7.29 (m, 2H), 7.20 (dd, J=0.8, 5.2 Hz, 1H), 7.11-7.03 (m, 2H), 5.78 (d, J=2.6 Hz, 1H), 2.67 (d, J=3.4 Hz, 1H). MS ESI calculated value C.sub.12H9ClFNO [M+H].sup.+ 237.66, measured value 238.2.

(73) Step 2: At 0 C., Dess-Martin Periodinane (45.15 g, 106.46 mmol, 32.96 mL, 1.10 eq) was added into the compound 16-2 (23.00 g, 96.78 mmol, 1.00 eq) in dichloromethane (230.00 mL), the system temperature was raised to 25 C., then stirred for 2 hours. The system was then filtered, the filtrate was concentrated, chromatographic purified by silica gel column (PE:EtOAc=5:1) to give the compound 16-3. MS ESI calculated value C.sub.12H7ClFNO [M+H].sup.+ 235.64, measured value 236.2.

(74) Step 3: P-toluenesulfonic acid (7.27 g, 38.19 mmol, 3.00 eq) and ethanediol (1.58 g, 25.46 mmol, 1.42 mL, 2.00 eq) were added into the compound 16-3 (3.00 g, 12.73 mmol, 1.00 eq) in toluene (100.00 mL) solution, the system was heated to 110 C., then stirred for 5 hours. The mixture was concentrated then purified by column (PE:EtOAc=10:1 5:1) to give the compound 16-4. MS ESI calculated value C.sub.14H.sub.11ClFNO.sub.2 [M+H].sup.+ 279.69, measured value 280.0.

(75) Step 4: Tris(dibenzylideneacetone) dipalladium (61.55 mg, 67.22 mol, 0.20 eq), potassium hydroxide (40.12 mg, 715.08 mol, 2.00 eq) and 2-di-tertbutylphosphine-2, 4, 6-triisopropylbiphenyl (28.54 mg, 67.22 mol, 0.20 eq) were added into the mixture of the compound 16-4 (100.00 mg, 336.09 mol, 1.00 eq) in 1, 4-dioxane (5.00 mL), the system was heated to 90 C. and stirred for 3 hours. The mixture was concentrated and water (10 mL) was added, then extracted by EtOAc (10 mL*3), the organic phases were combined then washed with saturated brine (10 mL), dried over sodium sulfate, filtered and concentrated, then purified to give the compound 16-5. MS ESI calculated value C.sub.14H.sub.12FNO.sub.3 [M+H].sup.+ 261.25, measured value 262.2.

(76) Step 5: At 0 C. under nitrogen protection, sodium hydride (22.54 mg, 563.44 mol, 60% purity, 2.00 eq) was added into the mixture of the compound 16-5 (80.00 mg, 281.72 mol, 1.00 eq) in N, N-dimethylformamide (10.00 mL), the system was stirred for 10 mins, then methyl 4-bromomethylbenzoate (129.07 mg, 563.44 mol, 2.00 eq) was added, and continued stirring for 30 mins. The mixture was quenched by water (5 mL), and extracted by EtOAc (5 mL*3), the combined organic phases was then washed by water (10 mL) and saturated brine (10 mL), dried over sodium sulfate, filtered and concentrated then purified to give the compound 16-6. MS ESI calculated value C.sub.23H.sub.20FNO.sub.5 [M+H].sup.+ 409.41, measured value 410.3.

(77) Step 6: Hydroxylamine solution (1.00 mL, 50% purity) and sodium hydroxide (17.03 mg, 425.73 mol, 3.00 eq) were added into the compound 16-6 (70.00 mg, 141.91 mol, 1.00 eq) in methanol (5.00 mL) solution, the mixture was stirred at rt for 1 hours. the mixture was concentrated then purified by preparative HPLC (0.225% FA) to give the compound 16-7. .sup.1H NMR (400 MHz, D MSO-d.sub.6) =7.77 (d, J=7.2 Hz, 1H), 7.68 (d, J=8.2 Hz, 2H), 7.53-7.45 (m, 2H), 7.33 (d, J=8.2 Hz, 2H), 7.19 (t, J=8.8 Hz, 2H), 6.48 (d, J=1.8 Hz, 1H), 6.20 (dd, J=1.8, 7.2 Hz, 1H), 5.08 (s, 2H), 3.99 (m, 4H). MS ESI calculated value C.sub.22H.sub.19FN.sub.2O.sub.5 [M+H].sup.+ 410.40, measured value 411.1.

Embodiment 17

(78) ##STR00078## ##STR00079##

(79) Step 1: Under N.sub.2 protection, methyltriphenylphosphonium iodide (2.83 g, 7.01 mmol, 1.10 eq) and t-BuOK (1 M, 7.01 m, 1.10 eq) THF (20.00 mL) solution were heated and refluxed for 1 hours. Then the suspension was cooled down to 0 C. and added the compound 17-1 (1.50 g, 6.37 mmol, 1.00 eq), and the reaction mixture obtained was stirred at 20 C. for 12 hours. The reaction mixture was poured into water (100 mL), and extracted by EtOAc (50 mL3), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was passed through column chromatography (PE/EtOAc=20/1) to give the compound 17-2. MS ESI calculated value C.sub.13H.sub.19ClFN [M+H]+ 234, measured value 234.

(80) Step 2: Compound 17-2 (800.00 mg, 3.42 mmol, 1.00 eq) and OsO.sub.4 (869.47 mg, 3.42 mmol, 177.44 L, 1.00 eq) in THF (10.00 mL) and H.sub.2O (2.00 mL) solution was stirred at 20 C. for 4 hours. The reaction mixture was washed by 2M Na.sub.2SO.sub.3 solution (80 mL), and extracted by EtOAc (30 mL3), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced temperature to give the compound 17-3. MS ESI calculated value C.sub.13H.sub.11ClFNO.sub.2 [M+H]+ 268, measured value 268.

(81) Step 3: Compound 17-3 (800.00 mg, 2.99 mmol, 1.00 eq), 1,2-dibromoethane (561.45 mg, 2.99 mmol, 225.48 L, 1.00 eq) and t-BuOK (503.03 mg, 4.49 mmol, 1.50 eq) in D MSO (10.00 mL) was heated to 70 C. and stirred for 6 hours. The reaction mixture was poured into water (100 mL), and extracted by EtOAc/MeOH (10/1, 50 mL3), the combined extracts was washed by brine (80 mL), then dried over anhydrous Na2SO4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=2/1) to give the compound 17-4. MS ESI calculated value C.sub.15H.sub.13ClFNO.sub.2 [M+H]+ 294, measured value 294.

(82) Step 4: Under N.sub.2 protection, compound 17-4 (180.00 mg, 612.83 mol, 1.00 eq), t-BuXPhOS (26.02 mg, 61.28 mol, 0.10 eq), Pd.sub.2(dba).sub.3 (56.12 mg, 61.28 mol, 0.10 eq) and KOH (68.77 mg, 1.23 mmol, 2.00 eq) in dioxane (10.00 mL) and H.sub.2O (2.00 mL) solution was heated to 100 C. and stirred for 1 hours. The reaction mixture was poured into water (80 mL), and 2M HCl was used to adjust pH=6, then extracted by EtOAc (30 mL3), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na2SO4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=3/1) to give the compound 17-5. MS ESI calculated value C.sub.15H.sub.14FNO.sub.3 [M+H]+ 276, measured value 276.

(83) Step 5: Under N.sub.2 protection, at 0 C., NaH (15.69 mg, 392.34 mol, purity 60%, 1.20 eq) was added into the compound 17-5 (90.00 mg, 326.95 mol, 1.00 eq) in DMF (5.00 mL) solution, then continued stirring at this temperature for 20 mins. Then methyl 4-(bromomethyl) benzoate (74.89 mg, 326.95 mol, 1.00 eq) was added into the suspension above, the reaction mixture was heated to 15 C. and stirred for 1 hours. The reaction mixture was poured into water (50 mL), and extracted by EtOAc (20 mL3), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The solid obtained was purified by preparative TLC (PE/EtOAc=1/2) to give the compound 17-6. MS ESI calculated value C.sub.24H.sub.22FNO.sub.5 [M+H]+ 424, measured value 424.

(84) Step 6: At 0 C., NH.sub.2OH.H.sub.2O (1.00 mL, 50% solution) and NaOH solution (2 M, 1.00 mL) were added dropwise in sequence into the compound 17-6 (100.00 mg, 236.17 mol, 1.00 eq) in DCM (1.00 mL) and MeOH (2.00 mL) solution. When the addition was completed, the reaction mixture was stirred at 0 C. for 6 hours. Most of the solvent was eliminated by concentration under reduced pressure. The rest of the solution was cooled down to 0 C. and 2 M HCl was used to adjust pH=8, then filtered and rinsed repeatedly with H.sub.2O (5 mL3), finally dried in vacuum to give the compound 17-7. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 11.17 (brs, 1H), 9.04 (brs, 1H), 7.74 (d, J=7.28 Hz, 1H), 7.69 (d, J=8.28 Hz, 2H), 7.46 (dd, J=9.03, 5.52 Hz, 2H), 7.33 (d, J=8.28 Hz, 2H), 7.19 (t, J=8.91 Hz, 2H), 6.57 (d, J=1.76 Hz, 1H), 6.13 (dd, J=7.15, 1.88 Hz, 1H), 5.07 (s, 2H), 4.11-4.24 (m, 1H), 3.99-4.08 (m, 1H), 3.54-3.71 (m, 4H). MS ESI calculated value C.sub.23H.sub.21FN.sub.2O.sub.5 [M+H]+ 425, measured value 425.

Embodiment 18

(85) ##STR00080## ##STR00081##

(86) Step 1: At 0 C., sodium hydride (1.75 g, 43.75 mmol, purity 60%, 1.20 eq) was added into 1, 4-butyrolactone (3.45 g, 40.11 mmol, 3.05 mL, 1.10 eq) in toluene (30.00 mL) solution, then the compound 18-1 (5.00 g, 36.46 mmol, 4.39 mL, 1.00 eq) in toluene (10.00 mL) solution was added at 0 C., the mixture then was stirred to react at 20 C. for 12 hours. Saturated ammonium chloride solution and water were added into the reaction mixture, then extracted by EtOAc. The organic phase was washed by saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give the compound the compound 18-2, which was used directly in the next reaction.

(87) Step 2: The mixture of the compound 18-2 (3.22 g, 16.84 mmol, 1.00 eq) and 36% hydrochloric acid (10.20 g, 100.71 mmol, 10.00 mL, 5.98 eq) was heated to 80 C., and stirred to react for 3 hours. The reaction mixture was poured into 0 C. saturated potassium carbonate solution, water was added then, and extracted by EtOAc, the organic phase was washed by brine, dried over anhydrous sodium sulfate, filtered and concentrated, then purified by silica gel column to give the compound 18-3, which was used directly in the next reaction.

(88) Step 3: At 68 C., N-butyllithium hexanesolution (2.5 M, 1.34 mL, 1.10 eq) was added dropwise into 2-fluoro-4-iodo-pyridine (730.20 mg, 3.05 mmol, 1.00 eq) toluene (5.00 mL) solution, then the compound 18-3 (560.00 mg, 3.05 mmol, 1.00 eq) in toluene (1.00 mL) solution was added, the reaction gradually heated to 20 C., then stirred to react for 2 hours. Saturated ammonium chloride solution and water were added into the reaction mixture, then extracted by EtOAc, the organic phase was washed by saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, then purified by silica gel column to give the compound 18-4. MS ESI calculated value C.sub.14H.sub.13ClN.sub.2O [M+H]+ 261.0, measured value 261.0.

(89) Step 4: Potassium hydroxide (322.82 mg, 5.75 mmol, 2.00 eq), tris(dibenzylideneacetone) dipalladium (263.42 mg, 287.66 mol, 0.10 eq) and 2-di-tertbutylphosphine-2, 4, 6-triisopropylbiphenyl (122.15 mg, 287.66 mol, 0.10 eq) were added into the compound 18-4 (750.00 mg, 2.88 mmol, 1.00 eq) in dioxane (5.00 mL) and water (1.00 mL) solution, then stirred at 100 C. for 4 hours. Saturated ammonium chloride and water were added into the reaction mixture, then extracted by EtOAc, washed by brine, dried over anhydrous sodium sulfate, filtered and concentrated. After concentration, it was purified by silica gel column to give the compound 18-5. MS ESI calculated value C.sub.14H.sub.14N.sub.2O.sub.2 [M+H]+ 243, measured value 243.

(90) Step 5: At 0 C., sodium hydride (32.29 mg, 807.36 mol, 60% purity, 1.20 eq) was added into the compound 18-5 (163.00 mg, 672.80 mol, 1.00 eq) in N, N-dimethylformamide (2.00 mL) solution, methyl 4-bromomethylbenzoate (154.12 mg, 672.80 mol, 1.00 eq) in N, N-dimethylformamide (1.00 mL) solution was added into the system, then stirred at 20 C. for 2 hours. Saturated ammonium chloride solution and water was added into the reaction mixture, extracted by EtOAc, washed by brine, dried over anhydrous sodium sulfate, followed by concentrated to give the compound 18-6. MS ESI calculated value C.sub.23H.sub.22N.sub.2O.sub.4 [M+H]+ 391.0, measured value 391.0.

(91) Step 6: Sodium hydroxide (22.54 mg, 563.48 mol, 1.00 eq) and hydroxylamine solution (1.00 mL, 50% solution) were added into the compound 18-6 (220.00 mg, 563.48 mol, 1.00 eq) in methanol (2.00 mL) solution, then stirred at 20 C. for 6 hours. After concentration of the reaction mixture, the mixture was purified by reverse phase preparative HPLC to give the compound 18-7. .sup.1H NMR (400 MHz, METHANOL-d4) =8.53 (dd, J=0.8, 4.8 Hz, 1H), 7.82-7.74 (m, 1H), 7.70 (d, J=8.2 Hz, 2H), 7.67 (d, J=7.9 Hz, 1H), 7.60 (d, J=7.1 Hz, 1H), 7.36 (d, J=8.3 Hz, 2H), 7.27 (ddd, J=1.1, 4.9, 7.5 Hz, 1H), 6.79 (d, J=1.8 Hz, 1H), 6.60 (dd, J=2.0, 7.1 Hz, 1H), 5.18 (s, 2H), 4.17-3.96 (m, 2H), 3.04-2.91 (m, 1H), 2.38 (td, J=7.6, 12.6 Hz, 1H), 2.06-1.80 (m, 2H). MS ESI calculated value C.sub.22H.sub.21N.sub.3O.sub.4 [M+H]+ 392, measured value 392.

Embodiment 19

(92) ##STR00082##

(93) Step 1: At 0 C., sodium hydride (1.06 g, 26.38 mmol, purity 60%, 2.27 eq) was added into 1, 4-butyrolactone (1.00 g, 11.62 mmol, 884.96 L, 1.00 eq) in tetrahydrofuran (5.00 mL) solution, then stirred at 0 C. for 30 mins, the compound 19-1 (2.39 g, 17.43 mmol, 2.06 mL, 1.50 eq) in tetrahydrofuran (2.00 mL) solution was added at 0 C., then stirred at 20 C. for 2 hours. Saturated ammonium chloride solution water and EtOAc were added into the reaction mixture. The aqueous phase was concentrated and added acetone, then stirred and filtered, the mother solution was concentrated to give the compound 19-2, which was used directly in the next step.

(94) Step 2: The mixture of the compound 19-2 (1.50 g, 7.85 mmol, 1.00 eq) and 36% hydrochloric acid (15.30 g, 151.11 mmol, 15.00 mL, 19.26 eq) was heated to 80 C., the reaction was stirred for 2 hours. Then the reaction mixture was poured into 0 C. ice water, then extracted by EtOAc, the organic phase was washed by brine, dried over anhydrous sodium sulfate, filtered and concentrated, then purified by silica gel column to give the compound 19-3, which was used directly in the next step.

(95) Step 3: At 68 C., N-butyllithium hexanesolution (2.5 M, 1.44 mL, 1.10 eq) was added dropwise into 2-fluoro-4-iodo-pyridine (782.97 mg, 3.27 mmol, 1.00 eq) toluene (5.00 mL) solution, followed by addition of the compound 19-3 (600.00 mg, 3.27 mmol, 1.00 eq) in toluene (1.00 mL) solution, the reaction gradually heated to 20 C., the reaction was stirred for 12 hours. Saturated ammonium chloride solution and water were added into the reaction mixture, then extracted by EtOAc, the organic phase was washed by saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, then purified by silica gel column to give the compound 19-4. MS ESI calculated value C.sub.14H.sub.13ClN.sub.2O [M+H]+ 261.0, measured value 261.0.

(96) Step 4: Potassium hydroxide (221.07 mg, 3.94 mmol, 2.00 eq), tris(dibenzylideneacetone) dipalladium (180.40 mg, 197.00 mol, 0.10 eq) and 2-di-tertbutylphosphine-2, 4, 6-triisopropylbiphenyl (83.65 mg, 197.00 mol, 0.10 eq) were added into the compound 19-4 (513.00 mg, 1.97 mmol, 1.00 eq) in dioxane (5.00 mL) and water (1.00 mL) solution, then stirred at 100 C. for 2 hours. Water was added into the reaction mixture, then extracted by EtOAc, washed by brine, dried over anhydrous sodium sulfate, filtered and concentrated. After concentration, the mixture was then purified by silica gel column to give the compound 19-5. MS ESI calculated value C.sub.14H.sub.14N.sub.2O.sub.2 [M+H]+ 243, measured value 243.

(97) Step 5: At 0 C., sodium hydride (47.55 mg, 1.19 mmol, purity 60%, 1.20 eq) was added into 19-5 (240.00 mg, 990.63 mol, 1.00 eq) in N, N-dimethylformamide (5.00 mL) solution, methyl 4-bromomethylbenzoate (226.92 mg, 990.63 mol, 1.00 eq) in N, N-dimethylformamide (1.00 mL) solution was added into the reaction system, and stirred at 20 C. for 2 hours. Saturated ammonium chloride solution and water was added into the reaction mixture, then extracted by EtOAc, washed by brine, dried over anhydrous sodium sulfate, followed by concentration and then purified by chromatography to give the compound 19-6. MS ESI calculated value C.sub.23H.sub.22N.sub.2O.sub.4 [M+H]+ 391.1, measured value 391.1

(98) Step 6: Sodium hydroxide (13.52 mg, 338.08 mol, 2.00 eq) and hydroxylamine solution (1.00 mL, 50% purity) were added into the compound 19-6 (66.00 mg, 169.04 mol, 1.00 eq) in methanol (5.00 mL) solution, and stirred at 20 C. for 12 hours. After the reaction mixture was concentrated, it was purified by reverse phase preparative column to give the compound 19-7. .sup.1H NMR (400 MHz, MeOD) 8.51 (d, J=6.1 Hz, 2H), 8.45 (brs, 1H), 8.47-8.41 (m, 1H), 7.71 (d, J=8.3 Hz, 2H), 7.65 (d, J=7.2 Hz, 1H), 7.62-7.56 (m, 2H), 7.37 (d, J=8.3 Hz, 2H), 6.76 (d, J=1.9 Hz, 1H), 6.49 (dd, J=1.9, 7.2 Hz, 1H), 5.20 (s, 2H), 4.13-3.99 (m, 2H), 2.58 (t, J=7.2 Hz, 2H), 2.09-1.88 (m, 2H). MS ESI calculated value C.sub.22H.sub.21N.sub.3O.sub.4 [M+H]+ 392, measured value 392.

Embodiment 20

(99) ##STR00083## ##STR00084##

(100) Step 1: Under N.sub.2 protection, at 70 C., n-BuLi (2.5 M, 23.33 mL, 1.10 eq) was added dropwise into 1-bromo-4-iodobenzene (15.00 g, 53.02 mmol, 1.00 eq) in THF (150.00 mL) solution, after the addition was completed, the reaction was continued stirring at the temperature for 30 mins. Compound 20-1 (10.64 g, 53.02 mmol, 8.72 mL, 1.00 eq) was then added dropwise into the yellow suspension above, when the addition is complete, the system temperature was raised to 20 C. and stirred for 12 hours. When the reaction was completed, it was quenched by water (300 mL) and extracted by EtOAc (100 mL3), the combined extracts was washed by brine (100 mL), then dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced temperature to give the compound 20-2. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.25-7.50 (m, 6H), 6.90-7.00 (m, 2H), 4.03 (t, J=6.8 Hz, 2H), 2.40-2.55 (m, 2H), 1.85-2.00 (m, 2H).

(101) Step 2: Under N.sub.2 protection, at 70 C., n-BuLi (2.5 M, 1.24 mL, 1.00 eq) was added dropwise into the compound 20-2 (1.00 g, 3.11 mmol, 1.00 eq) in THF (15.00 mL) solution, when the addition was completed, the mixture was continued stirring at the temperature for 30 mins. Then drikold (about 5 g) was added into the system above and gradually raise the temperature to 20 C. and stirred for 2 hours. When the reaction was completed, the mixture was poured into water (30 mL), 2 M KOH was used to adjust pH=9, then extracted by EtOAc (10 mL2). Then the aqueous phase was adjusted to pH=5 using 2M HCl, then extracted by EtOAc (20 mL3. The extracts was washed by brine (30 mL), then dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced temperature to give the compound 20-3. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 7.87 (d, J=8.0 Hz, 2H), 7.56 (d, J=7.6 Hz, 2H), 7.40-7.50 (m, 2H), 7.12 (t, J=8.8 Hz, 2H), 3.90-4.00 (m, 2H), 2.51-2.60 (m, 2H), 1.75-1.90 (m, 2H).

(102) Step 3: Compound 20-3 (100.00 mg, 349.28 mol, 1.00 eq), HATU (159.37 mg, 419.14 mol, 1.20 eq) and DIEA (67.71 mg, 523.92 mol, 91.50 L, 1.50 eq) in MeCN (10.00 mL) solution were stirred at 20 C. for 30 mins, then 4-(aminomethyl) methyl benzoate (70.43 mg, 349.28 mol, 1.00 eq, hydrochloride) was added into the above solution then continued stirring at 20 C. for 12 hours. When the reaction was completed, the reaction mixture was concentrated, the residue was slurried by H.sub.2O (3 mL), and filtered and dried in vacuum to give the compound 20-4, which was used directly in the next step. MS ESI calculated value C.sub.26H.sub.24FNO.sub.4 [M+H].sup.+ 434, measured value 434.

(103) Step 4: At 20 C., NH.sub.2OH.H.sub.2O (3.00 mL, 50% solution) and NaOH (2 M, 1.50 mL) solution was added dropwise in sequence into the compound 20-4 (150.00 mg, 346.04 mol, 1.00 eq) in DCM (4.00 mL) and MeOH (2.00 mL) solution. When the addition was completed, the reaction mixture was heated and refluxed for 6 hours. Then the mixture was directly purified by preparative HPLC (0.1% TFA) to give the compound 20-5. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 11.15 (brs, 1H), 8.95-9.05 (m, 2H), 7.80 (d, J=8.0 Hz, 2H), 7.67 (d, J=8.0 Hz, 2H), 7.52 (d, J=8.4 Hz, 1H), 7.40-7.50 (m, 2H), 7.33 (d, J=8.4 Hz, 2H), 7.02-7.10 (m, 2H), 4.67 (d, J=5.6 Hz, 2H), 3.92 (t, J=6.8 Hz, 2H), 2.51-2.60 (m, 2H), 1.85-1.95 (m, 2H). MS ESI calculated value C.sub.25H.sub.23FN.sub.2O.sub.4 [M+H]+ 435, measured value 435.

Embodiment 21

(104) ##STR00085## ##STR00086## ##STR00087##

(105) Step 1: Compound 21-1 (1 g, 2.31 mmol) purified by preparative SFC (chiral separation column type: OJ (250 mm*30 mm, 5 um), eluent: A: supercutical fluid CO2 B: 0.1% NH.sub.3H.sub.2O MeOH, phase B was increased from 5% to 40% in 5.5 mins, then eluted by keeping 40% phase B for 3 mins, finally 5% phase B was kept for 1.5 mins, flow velocity 60 mL), the two isomers were obtained, 21-2(P1) and 22-1(P2), remaining times were 6.474 min and 7.256 min, respectively.

(106) Step 2: At 0 C., NH.sub.2OH.H.sub.2O (4.00 mL, 50% solution) and NaOH (2 M, 4.00 mL) were added dropwise in sequence into the compound 21-2(P1) (450.00 mg, 1.04 mmol, 1.00 eq) in DCM (4.00 mL) and MeOH (4.00 mL) solution, when the addition was completed, the mixture was continued stirring at that temperature for 2 hours. Most of the solvent was eliminated by concentration under reduced pressure, the solvent remained was cooled down to 0 C. and by using 8 M HCl, the pH=7-8 were adjusted, the precipitates were filtered, the residue was washed by H.sub.2O (5 mL2), then dried under reduced pressure to give the compound 21-3. .sup.1H NMR (400 MHz, MeOD) 7.79 (d, J=8.4 Hz, 2H), 7.71 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.40-7.05 (m, 4H), 6.95-7.05 (m, 2H), 4.60 (d, J=4.0 Hz, 2H), 4.02 (t, J=7.2 Hz, 2H), 2.50-2.60 (m, 2H), 1.85-2.00 (m, 2H). MS ESI calculated value C.sub.25H.sub.23FN.sub.2O.sub.4 [M+H].sup.+ 435, measured value 435.

Embodiment 22

(107) ##STR00088## ##STR00089##

(108) Step 1: At 0 C., NH.sub.2OH.H.sub.2O (4.00 mL, 50% solution) and NaOH (2 M, 4.00 mL) were added dropwise in sequence into the compound 22-1(P2) (450.00 mg, 1.04 mmol, 1.00 eq) in DCM (4.00 mL) and MeOH (4.00 mL) solution, when the addition was complete, the mixture was continued stirring at that temperature for 2 hours. Most of the solvent was eliminated by concentration under reduced pressure, the solvent remained was cooled down to 0 C. then adjusted pH=7-8 using 8 M HCl, aftertreatment to give the compound 22-2(P2). .sup.1H NMR (400 MHz, MeOD) 7.79 (d, J=8.4 Hz, 2H), 7.71 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.40-7.05 (m, 4H), 6.95-7.05 (m, 2H), 4.60 (s, 2H), 4.02 (t, J=7.2 Hz, 2H), 2.50-2.60 (m, 2H), 1.85-2.00 (m, 2H). MS ESI calculated value C.sub.25H.sub.23FN.sub.2O.sub.4 [M+H].sup.+ 435, measured value 435.

Embodiment 23

(109) ##STR00090## ##STR00091##

(110) Step 1: Under N.sub.2 protection, compound 23-1 (1.00 g, 3.11 mmol, 1.00 eq), tert-butoxycarbonyl ammonia (546.50 mg, 4.67 mmol, 1.50 eq), Xantphos (179.95 mg, 311.00 mol, 0.10 eq), Pd.sub.2(dba).sub.3 (142.39 mg, 155.50 mol, 0.05 eq) and Cs.sub.2CO.sub.3 (1.52 g, 4.67 mmol, 1.50 eq) in DMF (15.00 mL) solution were heated to 100 C. and stirred for 12 hours. The reaction mixture was poured into water (100 mL), and extracted by EtOAc (50 mL3), the combined extracts was washed by brine (80 mL), then dried over anhydrous Na2SO4, followed by filtration and concentration under reduced pressure. The product was passed through silica gel column chromatography (PE/EtOAc=9/1) to give the compound 23-2. MS ESI calculated value C21H24FNO3 [M+H]+ 358, measured value 358.

(111) Step 2: At 25 C., TFA (2.00 mL) was added into the compound 23-2 (250.00 mg, 699.46 mol, 1.00 eq) in DCM (5.00 mL) solution and stirred for 30 mins, then concentrated under reduced temperature to give the compound 23-3. MS ESI calculated value C.sub.16H.sub.16FNO [M+H].sup.+ 258, measured value 258.

(112) Step 3: Compound 23-3 (250.00 mg, 673.26 mol, 1.00 eq, TFA salt), methyl 4-(bromomethyl) benzoate (154.22 mg, 673.26 mol, 1.00 eq) and K.sub.2CO.sub.3 (139.58 mg, 1.01 mmol, 1.50 eq) in DMF (5.00 mL) solution were stirred at 25 C. for 6 hours. The reaction mixture was then poured into water (50 mL), and extracted by EtOAc (30 mL2, the combined extracts was washed by brine (30 mL), then dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced temperature to give the compound 23-4, which was used directly in the next step. MS ESI calculated value C.sub.25H.sub.24FNO.sub.3 [M+H].sup.+406, measured value 406.

(113) Step 4: At 25 C., NH.sub.2OH.H.sub.2O (3.00 mL, 50% solution) and NaOH (2 M, 1.50 mL) solution were added dropwise in sequence into the compound 23-4 (250.00 mg, 616.58 mmol, 1.00 eq) in DCM (4.00 mL) and MeOH (2.00 mL) solution, then the reaction system temperature was raised to 50 C. and stirred for 2 hours. Most of the solvent was eliminated by concentration under reduced pressure, the rest of the solution was purified by preparative HPLC (0.1% NH.sub.4OH) to give the compound 23-5. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 11.04 (brs, 1H), 9.03 (brs, 1H), 7.67 (d, J=8.0 Hz, 2H), 7.35-7.45 (m, 4H), 6.95-7.05 (m, 4H), 6.45 (d, J=8.4 Hz, 2H), 6.28 (t, J=5.6 Hz, 1H), 4.25 (d, J=6.0 Hz, 2H), 3.70-3.90 (m, 2H), 2.20-2.45 (m, 4H), 1.65-1.90 (m, 2H). MS ESI calculated value C.sub.25H.sub.24FNO.sub.3 [M+H]+ 406, measured value 406.

Embodiment 24

(114) ##STR00092## ##STR00093##

(115) Step 1: Under N.sub.2 protection, compound 24-1 (300.00 mg, 934.06 mol, 1.00 eq), methyl 4-aminobenzoate (141.19 mg, 934.06 mol, 1.00 eq), Xantphos (108.09 mg, 186.81 mol, 0.20 eq), Cs.sub.2CO.sub.3 (456.50 mg, 1.40 mmol, 1.50 eq) and Pd.sub.2(dba) 3 (85.53 mg, 93.41 mol, 0.10 eq) in DMF (15.00 mL) solution were heated to 100 C. and stirred for 8 hours. The reaction mixture was poured into water (150 mL), and extracted by EtOAc (80 mL2), the combined extracts was washed by brine (50 mL), then dried over anhydrous Na.sub.2SO.sub.4, followed by filtration and concentration under reduced pressure. The product was purified by silica gel column chromatography (PE/EtOAc=5/1) to give the compound 24-2. MS ESI calculated value C.sub.24H.sub.22FNO.sub.3 [M+H]+ 392, measured value 392.

(116) Step 2: At 25 C., NH2OH.H2O (2.00 mL, 50% solution) and NaOH (2 M, 2.00 mL) solution were added dropwise in sequence into the compound 24-2 (250.00 mg, 638.67 mol, 1.00 eq) in DCM (2.00 mL) and MeOH (4.00 mL) solution. When the addition was complete, the reaction was continued stirring at 20 C. for 12 hours. Most of the solvent was eliminated by concentration under reduced pressure, the residue was dissolved by DMSO (5 mL), the solution obtained was purified by preparative HPLC (0.1% NH.sub.4OH) to give the compound 24-3.

(117) .sup.1H NMR (400 MHz, D MSO-d6) 10.88 (brs, 1H), 8.85 (brs, 1H), 8.50 (brs, 1H), 6.90-7.80 (m, 12H), 3.80-4.00 (m, 2H), 2.30-2.45 (m, 2H), 1.80-1.90 (m, 2H). MS ESI calculated value C.sub.23H.sub.21FN.sub.2O.sub.3 [M+H]+ 393, measured value 393.

Embodiment 25

(118) ##STR00094## ##STR00095##

(119) Step 1: Compound 25-1 (800.00 mg, 2.79 mmol, 1.00 eq), HATU (1.59 g, 4.19 mmol, 1.50 eq) and DIEA (722.26 mg, 5.59 mmol, 976.03 L, 2.00 eq) in MeCN (5.00 mL) solution were stirred at 20 C. for 20 mins, then methyl 4-aminobenzoate (422.38 mg, 2.79 mmol, 1.00 eq) was added into above solution, then continued stirring at that temperature for 12 hours. The solvent was eliminated by concentration under reduced temperature, the solid remained was dissolved by EtOAc (80 mL), and then washed, in turn, by 2M NaOH solution (30 mL2) and brine (50 mL), then dried over anhydrous Na2SO4, and filtered and concentrated under reduced temperature. The product was purified by silica gel column chromatography (PE/EtOAc=10/1) to give the compound 25-2. MS ESI calculated value C.sub.25H.sub.22FNO.sub.4 [M+H]+ 420, measured value 420.

(120) Step 2: At 20 C., NH.sub.2OH.H.sub.2O (11.00 mL, 50% solution) and NaOH (2 M, 10.00 mL) solution were added dropwise in sequence into the compound 25-2 (1.10 g, 2.62 mmol, 1.00 eq) in DCM (10.00 mL) and MeOH (20.00 mL) solution, when the addition was completed, the mixture was continued stirring at that temperature for 12 hours. Most of the solvent was eliminated by concentration under reduced pressure, the residue was dissolved to clarification () by DMSO (5 mL), solution obtained was purified by preparative HPLC (0.1% HCl) to give the compound 25-3.

(121) .sup.1H NMR (400 MHz, D MSO-d.sub.6) 11.14 (brs, 1H), 10.39 (brs, 1H), 7.86 (d, J=8.4 Hz, 2H), 7.82 (d, J=8.4 Hz, 2H), 7.40 (d, J=8.8 Hz, 2H), 7.61 (d, J=8.8 Hz, 2H), 7.45-7.55 (m, 2H), 7.13 (t, J=8.8 Hz, 2H), 3.96 (t, J=7.6 Hz, 2H), 2.58 (t, J=6.8 Hz, 2H), 1.80-1.95 (m, 2H). MS ESI calculated value C.sub.24H.sub.21FN.sub.2O.sub.4 [M+H]+ 421, measured value 421.

Embodiment 26

(122) ##STR00096## ##STR00097##

(123) Step 1: Under N.sub.2 protection, at 70 C., n-BuLi (2.5 M, 12.47 mL, 1.20 eq) was added dropwise into the compound 26-1 (5.00 g, 25.98 mmol, 1.00 eq) in toluene (50.00 mL) solution, when the addition was completed, the mixture was continued stirring at that temperature for 30 mins, then 4-chloro-1-(4-fluorophenyl)butyl-1-one (5.21 g, 25.98 mmol, 4.27 mL, 1.00 eq) was added into the yellow suspension above. And the reaction system temperature was raised to 15 C., then continued stirring for 12 hours. The reaction mixture was poured into water (100 mL), and extracted by EtOAc (50 mL3), the combined extracts were washed by brine (80 mL), then dried over anhydrous Na2SO4, followed by filtration and concentration under reduced pressure. The product was purified by silica gel column chromatography (PE/EtOAc=10/1) to give the compound 26-2. MS ESI calculated value C.sub.15H.sub.13ClFNO [M+H]+ 278, measured value 278.

(124) Step 2: The mixture of compound 26-2 (1.00 g, 3.60 mmol, 1.00 eq), DPPP (148.51 mg, 360.00 mol, 0.10 eq), Pd.sub.2(dba).sub.3 (164.86 mg, 180.00 mol, 0.05 eq) and TEA (546.54 mg, 5.40 mmol, 748.68 L, 1.50 eq) in D MSO (10.00 mL) and MeOH (3.00 mL) solution was heated to 80 C., then stirred under CO (50 psi) atmosphere for 12 hours. The reaction mixture was poured into water (100 mL), and extracted by EtOAc (50 mL3), the combined extracts were washed by brine (80 mL), then dried over anhydrous Na.sub.2SO.sub.4, then filtered and concentrated under reduced temperature to give the compound 26-3. MS ESI calculated value C.sub.17H.sub.16FNO.sub.3 [M+H]+ 302, measured value 302.

(125) Step 3: The mixed solution of compound 26-3 (1.00 g, 3.32 mmol, 1.00 eq) and NaOH (265.60 mg, 6.64 mmol, 2.00 eq) MeOH (10.00 mL) and H.sub.2O (10.00 mL) was heated, refluxed and stirred for 2 hours. Then MeOH was eliminated by concentration under reduced temperature, the aqueous phase was extracted by EtOAc (10 mL2), then the aqueous phase was adjusted the pH=6 by using hydrochloric acid. Finally the mixture was concentrated under reduced temperature to give the compound 26-4. MS ESI calculated value C.sub.16H.sub.14FNO.sub.3 [M+H]+ 288, measured value 288.

(126) Step 4: 26-4 (900.00 mg, 3.13 mmol, 1.00 eq), HATU (1.79 g, 4.70 mmol, 1.50 eq) and DIEA (1.21 g, 9.40 mmol, 3.00 eq) in MeCN (15.00 mL) solution were stirred at 20 C. for 30 mins. Then 4-(aminomethyl) methyl benzoate (631.71 mg, 3.13 mmol, 1.00 eq, hydrochloride) was added into the above solution and stirred at 20 C. for 3 hours. The mixture was concentrated under reduced temperature, then diluted by H.sub.2O (80 mL), then extracted by EtOAc (30 mL3), the combined extracts were washed by brine (30 mL), then dried over anhydrous Na2SO4, followed by filtration and concentration under reduced pressure. The product was finally passed through column chromatography (PE/EtOAc=5/1 to 3/1) to give 26-5. MS ESI calculated value C.sub.25H.sub.23FN.sub.2O.sub.4 [M+H]+ 435, measured value 435.

(127) Step 5: At 0 C., NH.sub.2OH.H.sub.2O (47.04 mg, 460.34 mol, 2.00 mL, 50% solution) and NaOH (2 M, 230.17 L) solution were added dropwise in sequence into 26-5 (200.00 mg, 460.34 mol, 1.00 eq) in DCM (2.00 mL) and MeOH (4.00 mL) solution, after the completion of the addition, the mixture was continued stirring at that temperature for 3 hours. Most of the solvent was eliminated by concentration under reduced pressure, then dissolve to clarification by DMSO (5 mL), the solution obtained was purified by preparative HPLC (0.05% HCl) to give 26-6. .sup.1H NMR (400 MHz, D MSO-d.sub.6) 11.15 (brs, 1H), 10.22 (brs, 1H), 9.35 (t, J=6.4 Hz, 1H), 8.72 (d, J=1.5 Hz, 1H), 8.01-8.07 (m, 1H), 7.94-8.00 (m, 1H), 7.68 (d, J=8.3 Hz, 2H), 7.49-7.56 (m, 2H), 7.34 (d, J=8.3 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 4.51 (d, J=6.3 Hz, 2H), 3.98 (t, J=7.2 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.89 (qd, J=7.2, 5.1 Hz, 2H). MS ESI calculated value C.sub.24H.sub.22FN.sub.3O.sub.4 [M+H]+ 436, measured value 436.

Experimental Example 1: In Vitro Evaluation

(128) The degree of deacetylation of the acetylated substrate was determined by detecting the changes in the intensity of the fluorescent signal in the 384-well microplate using EnVision microplate reader, the half inhibitory comcentration IC50 value of the compound inhibitor was used as a reference to evaluate the inhibitory effect of the compound on the histone deacetylase (HDAC).

(129) 1. Steps and Methods of the Assay:

(130) 1.1 Compound Dilution and Sample Loading:

(131) The compound was diluted by DMSO to 2 mM, and then added to 384 compound plate, then the solution was diluted 3 times, 10 gradients and double duplicate wells using a Bravo automated liquid workstation; then 0.15 L of the compound was transferred from 384 compound plate to 384 assay plate by Echo liquid handler.

(132) 1.2 configuration of the assay buffer: the configuration contains 20 mM Hepes, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl.sub.2, 0.05% BSA, 0.5 mM TCEP of 1 assay buffer, each of which is represented by final concentration thereof.

(133) 1.3 Configuration of the 1.5 Enzyme Mixture:

(134) The rapidly dissolving histone deacetylase was added to the 1 assay buffer and mixed well. The 1.5 enzyme mixture was added to a 20 L to 384-well assay plate, mixed by centrifugation, placed in a 23 C. incubator, and the enzyme was incubated with the compound for 20 minutes.

(135) Configuration of 3 Substrate Mixture:

(136) The 3 substrate solution was configured by 1 assay buffer, and then mixed well. 10 L was added to a 384-well assay plate, mixed by centrifugation, and placed in a 23 C. incubator for 90 minutes for reaction.

(137) 1.5 Preparation of Terminating the Mixture:

(138) The terminating mixture of trypsin and the positive compound SAHA (Vorinostat, vorinostat) was configured with 1 assay buffer and mixed well. The reaction was terminated by adding 30 L to a 384-well assay plate, then centrifuged, and placed in a 23 C. incubator for 60 minutes.

(139) 1.6 Readings:

(140) After termination of the reaction for 60 minutes, the 384-well assay plate was placed on an Envision micro-plate reader and the fluorescence value was read.

(141) 1.7 Data analysation: the data was analyzed by using XLfit5 software to calculate the IC50 value of the compound.

(142) TABLE-US-00001 TABLE 1 In vitro screening test results of the compounds of the invention The compounds formed by the HDAC 6 HDAC 1 embodiment IC50(nM) IC50(nM) Embodiment1 A D Embodiment2 A D Embodiment3 A C Embodiment4 A C Embodiment5 A C Embodiment6 A C Embodiment7 A D Embodiment8 A D Embodiment9 C D Embodiment10 B D Embodiment11 A C Embodiment12 A C Embodiment13 A C Embodiment14 A C Embodiment15 A C Embodiment16 A C Embodiment17 A C Embodiment18 A C Embodiment19 A C Embodiment20 A C Embodiment21 A C Embodiment22 A C Embodiment23 B D Embodiment24 B D Embodiment25 B D Embodiment26 A C Remarks: A 10 nM; 10 nM < B 50 nM; 50 nM < C 200 nM; D > 200 nM; Conclusion: the inhibitory effect of the compound of the present invenion on HDAC6 is signicicant, while the inhibitory effect on HDAC1 is weak, which shows the property of high selectivity.

Experimental Example 2: Pharmacokinetic Evaluation of the Compounds

(143) Purpose of the experiment: test the compound's pharmacokinetics in male beagle dogs

(144) Experimental materials: Beagle (male, 6-15 kg, 6 months-4 age, Marshall Bioresources, Beijing, China)

(145) Experimental Operation:

(146) The Beagle's pharmacokinetic characteristics after oral administration of the compound were tested by a standard scheme. In the experiment, the candidate compound was formulated into a uniform suspension and administered to a beagle dog for single oral administration. Oral menstruum was formed by a certain proportion of polyethylene glycol-polyoxyethylene castor oil aqueous solution. Blood samples within 24 hours was collected, then centrifuged at 3000 g for 10 minutes, the supernatant was separated to obtain plasma samples, 30 L of plasma samples was added into 300 L of internal standard acetonitrile solution to precipitate protein, 30 L of supernatant was obtained after vortex and centrifugation, and then equal volume of water was added, followed by vortex and centrifugation for another time to collect the supernatant, and the plasma concentration was quantitatively analyzed by LC-MS/MS analysis, and the pharmacokinetic parameters such as peak concentration, peak-reached time, half-life, and area under the administrating curve were calculated.

(147) The experimental results are shown in the Table 2

(148) TABLE-US-00002 TABLE 2 pharmacokinetic test results Half- Candidate life Integrated bio- (componds of the Clearance rate T.sub.1/2 concentration availability embodiment) (mL/min/kg) (h) AUC (nM .Math. hr) F (%) Embodiment 26 16.0 2.14 13340 53.1 Conclusion: the compond of the present invention shows good single or partial canine pharmacokinetic index.

Experimental Example 3: In Vivo Pharmacodynamic Study of Administration Combining with Ixazomib on CB-17 SCID Model of Human Myeloma MM.1S Cells Subcutaneous Xenograft Tumor

(149) 1. Purpose of the Experiment:

(150) HDAC inhibitors are widely used in a variety of cancers and can be combined with a variety of drugs to enhance therapeutic effect of the drug, which is a well-recognized anti-tumor target. HDAC inhibitors are widely used in a variety of cancers, and can be combined with a variety of drugs to enhance the therapeutic effect of the drug, such as the combination of panobinostat and the proteasome inhibitor bortezomib can enhance the therapeutic effect against the multiple myeloma and significantly reduce the toxicity.

(151) The purpose of this experiment was to investigate the in vivo therapeutic effect of the compounds of the present invention in combination with proteasome inhibitor Ixazomib on human myeloma MM.1S cell subcutaneous xenografts in the CB-17 SCID model.

(152) 2. Methods and Steps of the Experiments

(153) 2.1 Cell Culture

(154) Human myeloma MM. S cells were cultured in vitro in a single layer, culture environment was RPMI-1640 medium (supplier: Gibco; Cat. No. 22400-089) supplemented with 10% fetal bovine serum, 37 C. 5% CO2 incubator. Then passage was done by routinely digested with trypsin-EDTA twice a week. When the cell saturation rate reached 80%-90%, the cells were collected, counted, and inoculated.

(155) 2.2 Tumor Cell Inoculation

(156) 0.2 mL of 510.sup.6 MM.1S cells were subcutaneously inoculated into the right back of each mouse (Beijing Weitong Lihua Biotechnology Co., Ltd., female, 6-8 weeks old) (PBS: Matrigel=1:1)). Administration by groups was initiated when the average tumor volume reached 100-150 mm.sup.3.

(157) 2.3 Configuration of the Candidate Compounds

(158) TABLE-US-00003 TABLE 3 Methods of configuration of the candidate compounds concentration Storage Compound packing Methods of configuration (mg/mL) condition menstruum 2.5 mL of dimethyl sulfoxide was 4 C. placed in a 50.0 mL volumetric flask, and 47.5 mL of 10% hydroxypropyl--cyclodextrin was added and vortexed to a clear solution. Embodiment 3501.04 53.38 mg of the compound of 3.0 4 C. 26, mg/vial embodiment 26 was weighted in a 30 mg/kg brown dispensing bottle, 875 L of dimethyl sulfoxide was added, vortexed to mix well, then 16.625 mL of 10% hydroxypropyl-- cyclodextrin was added followed by vortexing and ultrasound to give a clear and transparent solution. Ixazomib, 100.80 2.83 mg of Ixazomib was weighed 0.4 4 C. 4 mg/kg mg/vial into a brown dispensing vial, and 7.001 mL of 5% hydroxypropyl-- cyclodextrin was added followed by vortexing and ultrasound to give a clear and transparent solution. Remarks: the drugs need to be gently mixed well before being administrated to the animal.

(159) 2.4 Daily Observation of the Experimental Animals

(160) Animals were monitored daily for health and mortality, routine tests includes observations of tumor growth and drug treatment effects on animal performance, such as behavioral activities, food intake (visual inspection only), weight change (measured every other day), appearance signs or other abnormal conditions. The number of animal deaths and side effects in the group were recorded based on the number of animals in each group.

(161) 2.5 Tumor Measurement and Experimental Index

(162) The experimental index is to investigate whether tumor growth is inhibited, delayed or cured. Tumor diameters were measured with vernier calipers three times a week. The tumor volume is calculated by: V=0.5ab.sup.2, where a and b represent the long and short diameters of the tumor, respectively.

(163) The antitumor effect of the compound was evaluated by TGI (%) or relative tumor growth rate T/C (%). TGI (%), reflecting the tumor growth inhibition rate. TGI (%) is calculated by: TGI (%)=((1(the average tumor volume at the end of a treatment groupthe average tumor volume at the beginning of the treatment group))/(the average tumor at the end of the treatment of the solvent control group)(the average tumor at the beginning of the treatment of the solvent control group))100%.

(164) Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C %=T.sub.RTV/C.sub.RTV100% (T.sub.RTV: RTV of the treatment group; C.sub.RTV: RTV of the negative control group). The relative tumor volume (RTV) was calculated according to the results of tumor measurement, the calculation formula was RTV=V.sub.t/V.sub.0, where V.sub.0 is the average tumor volume measured at the time of group administration (ie, d.sub.0), and V.sub.t is the average tumor volume measured for a certain measurement, T.sub.RTV and C.sub.RTV took data from the same day.

(165) Tumor were weighted after the end of the experiment and the percentage of T/C.sub.weight was calculated. T.sub.weight and C.sub.weight represent the tumor weights of the drug-administered group and the menstruum control group, respectively.

(166) 3. Statistic Analysis

(167) Statistical analysis includes the mean and standard error (SEM) of tumor volume at each time point for each group. The treatment group showed the best therapeutic effect on the 21st day after the administration at the end of the trial, therefore statistical analysis was performed based on this data to evaluate the difference between the groups. T-test was used for comparison between the two groups, and one-way ANOVA was used for comparison between three or more groups. If there was a significant difference in F values, the test was performed by Games-Howell method. If there is no significant difference in F values, the Dunnet (2-sided) method was used for analysis. All data analysis was performed with SPSS 17.0. A significant difference was considered if p<0.05.

(168) 4. Experimental Results and Discussion

(169) In the experiments, we have evaluated the in vivo therapeutic effect of the compound from embodiment 26 administrated in combination with Ixazomib on human myeloma MM.1s cell xenograft model. The tumor volume datas of this group at different time points indicated. On the 21st day after the administration started, the tumor volume of the tumor-bearing mice in the solvent control group reached 2611 mm.sup.3, and the candidate compound Ixazomib (4 mg/kg) administered alone had a significant antitumor effect compared with the solvent control group (T/C).=34.97%, TGI=68.65%, p=0.030), with a tumor volume of 916 mm.sup.3. The compound of the embodiment 26 (30 mg/kg) and Ixazomib (4 mg/kg) combined administrated group had a significant antitumor effect compared with the solvent control group (T/C=8.34%, TGI=96.88%, p=0.001). The tumor volume was 218 mm.sup.3.

(170) In summary, in the human myeloma MM. S cell xenograft model, comparing with the solvent control group, the compound of embodiment 26 (30 mg/kg) and Ixazomib (4 mg/kg) combined administrated group have the best tumor inhibition effect. The mice showed good tolerance and no significant weight loss.