Sulfonyl-substituted isoquinolines as inhibitors of RHO kinase and hERG potassium channel activity

Abstract

Disclosed are a class of isoquinoline compounds acting as RHO protein kinase inhibitors, and the use thereof in the preparation of a drug for treating diseases associated with RHO protein kinase. Particularly disclosed are compounds as shown in formula (I) and pharmaceutically acceptable salts thereof. ##STR00001##

Claims

1. A compound represented by formula (I): ##STR00042## or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: R.sub.1 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.2 is H, F, Cl, Br, I, NH.sub.2, or OH; R.sub.3 and R.sub.4, together with the carbon atom to which they are attached, form a 3-membered ring, wherein the 3-membered ring is optionally substituted by 1, 2, or 3 independently selected R substituents; each R is independently F, Cl, Br, I, NH.sub.2, or OH; T.sub.1 is —CH.sub.2— or —NH—; and T.sub.2 is —CH.sub.2— or —NH—.

2. The compound of claim 1, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: ##STR00043##

3. A compound selected from the group consisting of: ##STR00044## ##STR00045## ##STR00046## or a pharmaceutically acceptable salt or tautomer thereof.

4. A method for inhibiting rho-associated protein kinase activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound represented by formula (I): ##STR00047## or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: R.sub.1 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.2 is H, F, Cl, Br, I, NH.sub.2, or OH; R.sub.3 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.4 is C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; or R.sub.3 and R.sub.4, together with the carbon atom to which they are attached, form a 3- to 6-membered ring, wherein the 3- to 6-membered ring is optionally substituted by 1, 2, or 3 independently selected R substituents; each R is independently F, Cl, Br, I, NH.sub.2, or OH; T.sub.1 is —CH.sub.2— or —NH—; and T.sub.2 is —CH.sub.2— or —NH—.

5. The method of claim 4, wherein the subject has a disease or disorder caused by vasoconstriction selected from the group consisting of angina, cerebral angiospasm caused by subarachnoid hemorrhage, cerebral embolism, cerebral injury, cerebral ischemia, fibrosis, glaucoma, hypertension, and vertebrobasilar insufficiency.

6. A method for inhibiting rho-associated protein kinase activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient a compound represented by formula (I): ##STR00048## or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: R.sub.1 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.2 is H, F, Cl, Br, I, NH.sub.2, or OH; R.sub.3 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.4 is C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; or R.sub.3 and R.sub.4, together with the carbon atom to which they are attached, form a 3- to 6-membered ring, wherein the 3- to 6-membered ring is optionally substituted by 1, 2, or 3 independently selected R substituents; each R is independently F, Cl, Br, I, NH.sub.2, or OH; T.sub.1 is —CH.sub.2— or —NH—; and T.sub.2 is —CH.sub.2— or —NH—.

7. The method of claim 6, wherein the subject has a disease or disorder caused by vasoconstriction selected from the group consisting of angina, cerebral angiospasm caused by subarachnoid hemorrhage, cerebral embolism, cerebral injury, cerebral ischemia, fibrosis, glaucoma, hypertension, and vertebrobasilar insufficiency.

8. A method for inhibiting human Ether-à-go-go-Related Gene potassium channel activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound represented by formula (I): ##STR00049## or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: R.sub.1 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.2 is H, F, Cl, Br, I, NH.sub.2, or OH; R.sub.3 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.4 is C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; or R.sub.3 and R.sub.4, together with the carbon atom to which they are attached, form a 3- to 6-membered ring, wherein the 3- to 6-membered ring is optionally substituted by 1, 2, or 3 independently selected R substituents; each R is independently F, Cl, Br, I, NH.sub.2, or OH; T.sub.1 is —CH.sub.2— or —NH—; and T.sub.2 is —CH.sub.2— or —NH—.

9. The method of claim 8, wherein the subject has a disease or disorder caused by vasoconstriction selected from the group consisting of angina, cerebral angiospasm caused by subarachnoid hemorrhage, cerebral embolism, cerebral injury, cerebral ischemia, fibrosis, glaucoma, hypertension, and vertebrobasilar insufficiency.

10. A method for inhibiting human Ether-à-go-go-Related Gene potassium channel activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient a compound represented by formula (I): ##STR00050## or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: R.sub.1 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.2 is H, F, Cl, Br, I, NH.sub.2, or OH; R.sub.3 is H, F, Cl, Br, I, C.sub.1-3 alkyl, NH.sub.2, or OH, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; R.sub.4 is C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 independently selected R substituents; or R.sub.3 and R.sub.4, together with the carbon atom to which they are attached, form a 3- to 6-membered ring, wherein the 3- to 6-membered ring is optionally substituted by 1, 2, or 3 independently selected R substituents; each R is independently F, Cl, Br, I, NH.sub.2, or OH; T.sub.1 is —CH.sub.2— or —NH—; and T.sub.2 is —CH.sub.2— or —NH—.

11. The method of claim 10, wherein the subject has a disease or disorder caused by vasoconstriction selected from the group consisting of angina, cerebral angiospasm caused by subarachnoid hemorrhage, cerebral embolism, cerebral injury, cerebral ischemia, fibrosis, glaucoma, hypertension, and vertebrobasilar insufficiency.

Description

DRAWINGS

(1) FIG. 1: Pulmonary fibrosis score

(2) One-way ANOVA: .sup.###p<0.001 vs. sham-operated group; *p<0.05 vs. model group; ** p<0.01 vs. model group; ***p<0.001 vs. model group; T-test: .sup.$p<0.05 vs. model group.

(3) FIG. 2: Percentage of pulmonary fibrosis score

(4) Two-way ANOVA: .sup.###p<0.001 vs. sham operation group; *p<0.05 vs. model group; **p<0.01 vs. model group; ***p<0.001 vs. model group.

DETAILED DESCRIPTION

(5) The following examples further illustrate the present invention, but the present disclosure is not limited thereto. The present disclosure has been described in detail in the text, and its specific embodiments have also been disclosed, for one skilled person in the art, it is obvious to modify and improve the embodiments of the present disclosure within the spirit and scope of the present invention.

Embodiment 1

(6) ##STR00024## ##STR00025##

The First Step

(7) A solution of 1a (30.00 g, 173.24 mmol) and triethylamine (43.83 g, 433.00 mmol) in N, N dimethylformamide (500 mL) was cooled down to 0° C., and then O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphine salt (79.05 g, 208.00 mmol) was added. The reaction mixture obtained was stirred for 10 min at 0° C. Then N-methoxymethylamine hydrochloride (18.59 g, 191.00 mmol) was added, and stirred at 20° C. for 16 hrs. The reaction mixture was poured into water (1.00 L), then extracted with EtOAc (2.00 L×2), the organic phases were combined and dried over anhydrous sodium sulfate, then filtered and evaporated, the crude product was purified by silica gel column (PE/EtOAc=100-0%) to give 1b.

(8) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 4.13-4.07 (m, 2H), 3.74 (s, 3H), 3.37 (s, 1H), 3.21 (s, 3H), 1.37-1.35 (m, 2H), 1.26-1.22 (m, 3H), 1.03-1.00 (m, 2H).

The Second Step

(9) Sodium hydrogen (7.55 g, 189.00 mmol, 60%) was added into a solution of 1b (34.00 g, 157.24 mmol) in N, N-dimethylformamide (300 mL) at 0° C. The reaction mixture obtained was stirred at 0° C. for 10 mins, then allyl bromide (28.53 g, 235.86 mmol) was added dropwise. The reaction mixture was stirred at 20° C. for 5 hrs. The reaction mixture was poured into water (1.00 L) and extracted with EtOAc (300 mL×3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated, then the crude product was purified by silica gel column (PE/EtOAc=100-0%) to give 1c.

(10) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.85-5.77 (m, 1H), 5.06-5.03 (m, 2H), 4.17-4.08 (m, 2H), 3.98-3.94 (m, 2H), 3.63 (s, 3H), 3.15 (s, 3H), 1.53 (brs, 2H), 1.26-1.21 (m, 5H).

The Third Step

(11) A solution of 1c (22.30 g, 87.01 mmol) in THF (220 mL) was added into a solution of lithium tetrahydroaluminum (3.63 g, 95.71 mmol) in THF (440 mL) at 0° C., then stirred at 0° C. for 1 hr. The reaction was quenched by saturated ammonium chloride solution (300 mL) slowly, then extracted with EtOAc (300 mL×3), the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by silica gel column (PE/EtOAc=100-0%) to give 1d.

(12) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.15 (brs, 1H), 5.89-5.81 (m, 1H), 5.14-5.11 (m, 2H), 4.19-4.11 (m, 2H), 3.81 (brs, 2H), 1.53 (brs, 2H), 1.42 (brs, 2H), 1.24 (brs, 3H).

The Fourth Step

(13) 1e (4.50 g, 25.10 mmol) was added into a solution of 1d (4.50 g, 22.82 mmol) in toluene (45 mL), then stirred for 72 hrs at 130° C. The reaction was quenched by 1N diluted hydrochloric acid (150 mL), and washed with EtOAc (50 mL×2). The pH of the remaining aqueous phase was adjusted to 12 with sodium hydroxide, and then extracted with the mixture of dichloromethane/methane=10:1 (100 mL×3), the organic phases were combined, and dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by silica gel column (PE/EtOAc=100-0%) to give 1f.

The Fifth Step

(14) Wet palladium on carbon (1.20 g, 10%) was added into a solution of if (4.30 g, 13.68 mmol) and BOC.sub.2O (4.48 g, 20.52 mmol) in methanol (100 mL). The reaction mixture obtained was stirred for 20 hrs at 50° C. under 50 psi hydrogen atmosphere, then the reaction mixture was filtered and concentrated, the crude product was purified by silica gel column (PE/EtOAc=100-0%) to give 1g.

The Sixth Step

(15) Potassium hydroxide (22.24 g, 396.30 mmol) was added into a solution of 1g (4.10 g, 13.21 mmol) in ethanol (120 mL) and water (30 mL), the reaction mixture was stirred at 95° C. for 40 hrs. The reaction mixture was concentrated to eliminate ethanol, then extracted with dichloromethane (150 mL×5), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by silica gel column (PE/EtOAc=100-0%) to give 1h.

(16) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.13-4.08 (m, 1H), 3.75-3.58 (m, 1H), 3.34-3.29 (m, 1H), 3.11-3.06 (m, 2H), 2.73-2.69 (m, 1H), 2.00-1.95 (m, 2H), 1.70-1.67 (m, 1H), 1.49-1.44 (m, 10H), 0.66-0.59 (m, 2H).

The Seventh Step

(17) Triethylamine (170 mg, 1.68 mmol) was added dropwise into a solution of the compound 1h (200 mg, 0.84 mmol) and the compound 1i (287 mg, 1.26 mmol) in dichloromethane (10 mL). The reaction mixture obtained was reacted at 15° C. for 5 hrs. After completion of the reaction, the dichloromethane was eliminated directly, the crude product obtained was purified by preparative thin layer plate (EtOAc) to give the compound 1j.

(18) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.36-9.34 (m, 1H), 8.71-8.69 (m, 1H), 8.48-8.47 (m, 2H), 8.25-8.20 (m, 1H), 7.74-7.70 (m, 1H), 4.15-3.96 (m, 2H), 3.73-3.25 (m, 4H), 1.84-1.62 (m, 1H), 1.60 (brs, 3H), 1.38 (s, 9H), 1.26-0.68 (m, 2H).

The Eighth Step

(19) At 20° C., HCl/EtOAc (4 mL, 4 M) was added into a solution of the compound 1j (130 mg, 0.30 mmol) in EtOAc (1 mL). The reaction mixture obtained was stirred at the same temperature for 2 hrs. After completion of the reaction, the mixture was filtered and dried to give the compound 1.

(20) MS-ESI calculated value [M+H].sup.+ 330, measured value 330.

(21) .sup.1H NMR (400 MHz, D.sub.2O) δ 9.75 (s, 1H), 8.81-8.65 (m, 4H), 8.08 (t, J=8.0 Hz, 1H), 4.10-4.07 (m, 1H), 3.93-3.91 (m, 1H), 3.79-3.74 (m, 1H), 3.51-3.48 (m, 1H), 3.42-3.25 (m, 2H), 2.41-2.39 (m, 1H), 2.03-2.02 (m, 1H), 1.39-1.36 (m, 1H), 1.14-1.12 (m, 1H), 0.81-0.78 (m, 1H), 0.54-0.53 (m, 1H).

Embodiment 2

(22) ##STR00026##

The First Step

(23) The compound 2 was obtained from the compound 1h and the compound 2a using the synthesis method of the compound 1.

(24) MS-ESI calculated value [M+H].sup.+ 364, measured value 364.

(25) .sup.1H NMR (400 MHz, D.sub.2O) δ 9.30 (s, 1H), 8.67 (s, 1H), 8.62 (d, J=7.6 Hz, 1H), 8.46 (d, J=8.4 Hz, 1H), 7.86 (t, J=8.0 Hz, 1H), 4.05-3.94 (m, 2H), 3.82-3.77 (m, 1H), 3.57-3.55 (m, 1H), 3.38-3.22 (m, 2H), 2.42-2.39 (m, 1H), 2.08-2.04 (m, 1H), 1.06-0.79 (m, 4H).

Embodiment 3

(26) ##STR00027##

The First Step

(27) The compound 3 was obtained from compound 1h and the compound 3a using the synthesis method of the compound 1.

(28) MS-ESI calculated value [M+H].sup.+ 348, measured value 348.

(29) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 9.25 (s, 1H), 8.67 (d, J=8.0 Hz 1H), 8.53 (d, J=8.0 Hz, 1H), 8.45 (d, J=8.0 Hz, 1H), 7.86 (t, J=8.0 Hz, 1H), 3.85-3.73 (m, 2H), 3.43 (s, 1H), 3.06-2.98 (m, 1H), 2.72-2.65 (m, 1H), 2.15-2.05 (m, 1H), 1.73-1.71 (m, 1H), 0.97-0.87 (m, 1H), 0.82-0.79 (m, 2H), 0.69-0.60 (m, 2H).

(30) ##STR00028## ##STR00029##

Embodiment 4

The First Step

(31) Benzyl glycine 4a (7.06 g, 42.74 mmol), acetone (6.21 g, 106.85 mmol), 1-benzyl-2,5-dihydropyrrole-2,5-dione 4b (4.00 g, 21.37 mmol) and toluene (40 mL) were added into a 200 mL pot. The reaction mixture obtained was stirred at 140° C. for 48 hrs. After completion of the reaction, the reaction mixture was concentrated, the crude product was purified by silica gel chromatography (PE/EtOAc=100-0%) to give the compound 4c.

(32) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.40-7.32 (m, 10H), 4.77-4.72 (t, J=5.2, 2H), 3.96-3.74 (m, 3H), 2.90-2.66 (m, 3H), 2.31 (s, 3H), 1.60 (s, 3H).

The Second Step

(33) The compound 4c (5.28 g, 15.15 mmol) and 350 mL methanol was added into a 1000 mL hydrogenated bottle, under the protection of nitrogen, wet palladium on carbon (2.00 g, purity 10%) and Boc.sub.2O (6.61 g, 30.30 mmol) were added, then the suspension was replaced with hydrogen for 3 times. The mixture obtained was stirred under hydrogen atmosphere (50 psi) at 50° C. for 16 hrs. After completion of the reaction, the reaction mixture was filtered, concentrated, the crude product was purified by silica gel chromatography (PE/EtOAc=100-0%) to give the compound 4d.

(34) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.23-7.20 (m, 5H), 4.57 (s, 2H), 2.93-2.83 (m, 4H), 1.41-1.36 (m, 15H).

The Third Step

(35) The compound 4d (300 mg, 0.84 mmol) and 7 mL THF was added into a 50 mL three-neck round bottom flask, under the protection of nitrogen, borane-THF (1 M, 3.4 mL) was then added dropwise at 0° C., the reaction mixture obtained was stirred at 50° C. for 2.5 hrs. After completion of the reaction, the temperature was cooled to 0° C., methanol (10 mL) was added slowly to quench the reaction, then the reaction mixture was concentrated. Preparative thin layer chromatography was used for purification (PE/EtOAc=2:1) to give the compound 4e.

(36) MS-ESI calculated value [M+H].sup.+ 331, measured value 331.

(37) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.32-7.26 (m, 5H), 3.62 (s, 2H), 3.25 (s, 1H), 2.98-2.95 (m, 1H), 2.72-2.66 (m, 2H), 2.51-2.44 (m, 2H), 2.32-2.29 (m, 1H), 2.11 (m, 1H), 1.46-1.34 (m, 15H).

The Fourth Step

(38) The compound 4e (100 mg, 0.30 mmol) and 5 mL methanol were added into a 50 mL hydrogenated bottle, under the protection of nitrogen, wet palladium on carbon (100 mg, purity 10%) was added, the suspension was replaced with nitrogen and hydrogen for 3 times sequentially. Then the mixed solution was stirred under hydrogen atmosphere (50 psi) at 50° C. for 12 hrs. After completion of the reaction, the reaction mixture was filtered and concentrated to give the compound 4f, which was used directly in the next step.

(39) MS-ESI calculated value [M+H].sup.+ 241, measured value 241.

The Fifth Step

(40) The compound 4f (72 mg, the crude product obtained in previous step), N,N-diisopropylethylamine (77 mg, 0.60 mmol) and 1 mL dichloromethane were added into a 50 mL round bottom flask, benzyl chloroformate (77 mg, 0.45 mmol) was added dropwise slowly at 0° C. and under the protection of nitrogen. The reaction mixture was stirred at 25° C. for 3 hrs. After completion of the reaction, the reaction mixture was washed with N, N, N-trimethylethylenediamine (2 mL, 10%), then extracted with dichloromethane (5 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by preparative thin layer chromatography (PE/EtOAc=2:1) to give the compound 4g.

(41) MS-ESI calculated value [M−56+H].sup.+ 319, measured value 319.

(42) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.37-7.36 (m, 5H), 5.18-5.09 (m, 2H), 3.68-3.59 (m, 2H), 3.48-3.39 (m, 2H), 3.67-3.35 (m, 1H), 2.84-2.80 (m, 1H), 1.99-1.95 (m, 2H), 1.47-1.29 (m, 15H).

The Sixth Step

(43) The compound 4g (62 mg, 0.17 mmol) and 1 mL dichloromethane were added into a 25 mL round bottom flask, then under the protection of nitrogen, trifluoroacetate (190 mg, 1.67 mmol) was added dropwise, then stirred at 25° C. for 1 hr. After completion of the reaction, the reaction mixture was concentrated directly to give the compound 4h (46 mg, a crude product).

(44) MS-ESI calculated value [M+H].sup.+ 275, measured value 275.

The Seventh Step

(45) The compound 4h (46 mg, 0.17 mmol), N, N-diisopropylethylamine (65 mg, 0.5 mmol) and 1.5 mL dichloromethane were added into a 25 mL round bottom flask, and under the protection of nitrogen, isoquinoline sulfonyl chloride 1i (49 mg, 0.22 mmol) was added dropwise slowly at 0° C., then stirred at 25° C. for 2 hrs. After completion of the reaction, the reaction mixture was concentrated directly, the crude product was purified by preparative thin layer chromatography (PE/EtOAc=1:1) to give the compound 4i.

(46) MS-ESI calculated value [M+H].sup.+ 466, measured value 466.

(47) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.34-9.33 (m, 1H), 8.68-8.66 (m, 1H), 8.49-8.39 (m, 2H), 8.21-8.19 (m, 1H), 7.71-7.66 (m, 1H), 7.34-7.28 (m, 5H), 5.09-5.05 (m, 2H), 3.64-3.49 (m, 2H), 3.33-3.28 (m, 1H), 3.08-2.97 (m, 3H), 2.81-2.80 (m, 1H), 2.54-2.52 (m, 1H), 1.32-1.27 (m, 6H).

The Eighth Step

(48) The compound 4i (32 mg, 0.07 mmol) and 1 mL trifluoroacetate were added into a 5 mL microwave tube, sealed, then stirred at 100° C. in a microwave reactor for 1 hr. After completion of the reaction, the reaction mixture was concentrated directly, the crude product was purified by preparative liquid chromatography HPLC to give the compound 4.

(49) MS-ESI calculated value [M+H].sup.+ 332, measured value 332.

(50) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.42 (s, 1H), 8.69-8.68 (m, 1H), 8.60-8.59 (m, 1H), 8.56-8.54 (m, 1H), 8.45 (d, J=8.0 Hz, 1H), 7.86 (t, J=8.0 Hz, 1H), 3.82-3.77 (m, 1H), 3.26-3.20 (m, 2H), 2.98 (m, 1H), 2.97 (m, 1H), 2.54-2.44 (m, 3H), 1.41 (s, 6H).

Embodiment 5

(51) ##STR00030## ##STR00031##

The First Step

(52) 5a (4.00 g, 18.33 mmol) was dissolved in 20 mL N, N-dimethylformamide, sodium hydrogen (0.88 g, 21.99 mmol, 60%) was added dropwise slowly under the protection of nitrogen, the temperature was maintained at 0° C. The mixture was stirred at 25° C. for 10 mins, bromopropene (4.43 g, 36.66 mmol) was added into the reaction mixture. The mixture was continued to be stirred at 25° C. for 3 hrs. After completion of the reaction, the reaction mixture was quenched by saturated ammonium chloride solution 20 mL at 0° C., water (40 mL), EtOAc (40 mL×3) were added, and the organic phases were combined and washed with saturated brine (50 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by column chromatography (PE/EtOAc=100-0%) to give 5b.

The Second Step

(53) 5b (2.50 g, 9.68 mmol) was dissolved in THF (40 mL), under the protection of nitrogen, diisobutylaluminum hydride (1 M, 17.4 mL) was added dropwise at −78° C. The reaction mixture obtained was stirred at −78° C. for 6 hrs. After completion of the reaction, the reaction mixture was quenched by saturated ammonium chloride solution (20 mL) and HCl (1 N, 10 mL) at 25° C., water (20 mL) and EtOAc (40 mL×3) were added, the organic phases were combined and washed with saturated brine (40 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by column chromatography (PE/EtOAc=100-0%) to give 5c.

(54) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.32 (s, 1H), 5.90-5.78 (m, 1H), 5.23-5.11 (m, 2H), 4.14 (q, J=8.0 Hz, 2H), 3.95 (d, J=4.0 Hz, 2H), 1.27 (s, 6H), 1.23 (t, J=8.0 Hz, 3H).

The Third Step

(55) 5c (1.50 g, 7.53 mmol) and glycine 4a (2.49 g, 15.06 mmol) were dissolved in 20 mL toluene. The reaction mixture obtained was stirred at 130° C. for 16 hrs. After completion of the reaction, water (10 mL) was added into the reaction mixture, then the mixture was extracted with EtOAc (15 mL×3), the organic phases were combined and washed with saturated brine (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by column chromatography (PE/EtOAc=100-0%) to give 5d.

(56) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.38-7.34 (m, 2H), 7.30 (t, J=8.0 Hz, 2H), 7.26-7.20 (m, 1H), 4.16 (d, J=8.0 Hz, 2H), 3.97 (d, J=4.0 Hz, 2H), 3.64-3.55 (m, 1H), 3.48-3.40 (m, 1H), 3.29 (d, J=4.0 Hz, 1H), 3.11-2.98 (m, 2H), 2.84-2.73 (m, 1H), 2.45-2.36 (m, 1H), 1.87-1.93 (m, 1H), 1.30 (s, 6H), 1.25 (s, 3H).

The Fourth Step

(57) 5d (400 mg, 1.32 mmol) was dissolved in 20 mL acetonitrile, then trimethyl iodosilane (2.65 g, 13.23 mmol) was added dropwise. The reaction mixture obtained was stirred at 25° C. for 6 hrs. After completion of the reaction, the reaction mixture was quenched by adding 20 mL water, EtOAc (20 mL×3), the organic phases were combined and washed with saturated brine (30 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product of 5e, which was used directly in the next step.

The Fifth Step

(58) 5e (250 mg, 1.09 mmol) was dissolved in 10 mL dichloromethane, then di-tert-butyl dicarbonate (474 mg, 2.17 mmol) and diisopropylethylamine (281 mg, 2.17 mmol) were added dropwise sequentially. The mixture obtained was stirred at 25° C. for 16 hrs. After completion of the reaction, water (10 mL) was added to quench the reaction, dichloromethane (20 mL×2), the organic phases were combined and washed with saturated brine (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product obtained was purified by preparative thin layer chromatography (EtOAc/PE=5/1) to give 5f.

The Sixth Step

(59) 5f (300 mg, 0.91 mmol) and acetic anhydride (185 mg, 1.82 mmol) were dissolved in EtOAc (30 mL), under the protection of nitrogen protection, palladium on carbon (60 mg, 10%) was added. The reaction mixture obtained was replaced with hydrogen and stirred under hydrogen atmosphere (50 PSI) at 50° C. for 3 hrs. After completion of the reaction, the reaction mixture was filtered and concentrated to give a crude product of 5g.

(60) MS-ESI calculated value [M+H].sup.+ 283, measured value 283.

The Seventh Step

(61) Ethyl acetate-hydrochloride (20 mL, 4 M) was added dropwise into a solution of 5g (250 mg, 0.89 mmol) in 5 mL EtOAc at 25° C. The reaction mixture obtained was continued to be stirred for 0.5 hrs. After completion of the reaction, the solvent was removed directly to give a crude product of 5h, which was used directly in the next step.

The Eighth Step

(62) The compound 1i (150 mg, 0.66 mmol) and 5h (200 mg, hydrochloride) were dissolved in 5 mL dichloromethane, then diisopropylethylamine (142 mg, 1.10 mmol) was added dropwise. The reaction mixture obtained was stirred for 16 hrs at 25° C. After completion of the reaction, the solvent was removed by concentration, water (5 mL) was added, then extracted by EtOAc (10 mL×2), the organic phases were combined and washed by saturated brine (15 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product obtained was purified by preparative thin layer chromatography (EtOAc) to give 5i.

(63) MS-ESI calculated value [M+H].sup.+ 374, measured value 374.

The Ninth Step

(64) 5i (100 mg, 0.66 mmol) was dissolved in the mixture of ethanol (0.5 mL) and water (1 mL), sodium hydrochloride (321 mg, 8.03 mmol) was added. The reaction mixture obtained was stirred at 100° C. for 16 hrs. After completion of the reaction, the pH of the mixture was adjusted to neutral by diluted hydrochloric acid (1 N), then purified by high performance liquid chromatography to give the compound 5.

(65) MS-ESI calculated value [M+H].sup.+ 332, measured value 332.

(66) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 9.38 (s, 1H), 8.66-8.54 (m, 3H), 8.43-8.37 (m, 1H), 7.82 (t, J=8.0 Hz, 1H), 3.75-3.80 (m, 1H), 3.29-3.20 (m, 2H), 3.13-3.07 (m, 1H), 2.89-2.68 (m, 3H), 1.97-1.88 (m, 1H), 1.49 (s, 3H), 1.36 (s, 3H).

(67) ##STR00032## ##STR00033##

Embodiment 6

The First Step

(68) At 0° C., N-methoxymethylamine hydrochloride (8.83 g, 90.57 mmol) was added into a solution of the compound 6a (15.58 g, 82.34 mmol), HATU (32.87 g, 86.46 mmol) and diisopropylethylamine (22.35 g, 172.91 mmol) in 200 mL dichloromethane. The reaction mixture obtained was stirred at 25° C. for 16 hrs. After completion of the reaction, 200 mL water was added into the reaction mixture, and the pH of the mixture was adjusted to 14 with 1 N aqueous sodium hydrochloride solution, then extracted with dichloromethane (200 mL×2), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by silica gel column (PE/EtOAc=100-0%) to give the compound 6b.

(69) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.25 (s, 1H), 4.69 (s, 1H), 3.78 (s, 3H), 3.22 (s, 3H), 1.45 (s, 9H), 1.32 (d, J=8.0 Hz, 3H).

The Second Step

(70) At 0° C. and under the protection of nitrogen, sodium hydrogen (4.30 g, 107.51 mmol, 60%) was added in batch into a solution of the compound 6b (14.69 g, 63.24 mmol) in 200 mL N, N-dimethylformamide, when the addition was finished, the mixture was continued stirring for another 10 mins, then 3-bromopropene (19.13 g, 158.10 mmol) was added dropwise at 0° C. The reaction mixture obtained was reacted at 15° C. for 22 hrs. After completion of the reaction, 200 mL saturated ammonium chloride aqueous solution was added into the mixture, 200 mL water was added, the mixture was extracted by EtOAc (200 mL×2). The organic phase was washed with saturated brine (300 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product obtained was purified by silica gel column (PE/EtOAc=100-0%) to give the compound 6c.

(71) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.86-5.81 (m, 1H), 5.30-5.25 (m, 1H), 5.14-5.04 (m, 2H), 3.95-3.83 (m, 2H), 3.74 (s, 3H), 3.16 (s, 3H), 1.44 (s, 9H) 1.31 (d, J=8.0 Hz, 3H).

The Third Step

(72) At −78° C. and under the protection of nitrogen, DIBAL-H (97.90 mmol, 97.9 mL, 1 M) was added into a solution of the compound 6c (13.33 g, 48.95 mmol) in 200 mL THF. The reaction mixture obtained was stirred at 20° C. for 2 hrs. After completion of the reaction, 400 mL saturated potassium tartrate, 200 mL water and 300 mL EtOAc were added into the reaction mixture, then extracted with EtOAc (300 mL×2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give directly a crude product of the compound 6d.

(73) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.56 (s, 1H), 5.87-5.81 (m, 1H), 5.30-5.10 (m, 3H), 3.85-3.76 (m, 1H), 3.56-3.49 (m, 1H), 1.46 (s, 9H) 1.34 (d, J=8.0 Hz, 3H).

The Fourth Step

(74) A solution of the compound 6d (12.96 g, 60.77 mmol) and the compound 4a (25.10 g, 151.93 mmol) in 307 mL toluene was heated to 135° C. then reacted for 24 hrs. After completion of the reaction, 300 mL water was added into the reaction system, then extracted with EtOAc (300 mL×4), the organic phase was washed with saturated brine (300 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product obtained was purified by silica gel column (PE/EtOAc=100-0%) to give the compound 6e.

(75) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.26-7.16 (m, 5H), 3.96-3.61 (m, 2H), 3.45-3.20 (m, 3H), 2.98-2.84 (m, 1H), 2.73-2.59 (m, 2H), 2.20-2.13 (m, 1H) 1.97-1.84 (m, 1H), 1.58-1.46 (m, 1H), 1.38 (s, 9H), 1.01-0.85 (m, 3H).

The Fifth Step

(76) Under the protection of nitrogen, dry palladium on carbon (1.00 g, 10%) was added into a solution of the compound 6e (7.00 g, 22.12 mmol) and Ac.sub.2O (4.52 g, 4.1 mL, 44.24 mmol) in 100 mL EtOAc. The reaction mixture was replaced with hydrogen for 3 times. The reaction mixture obtained was stirred to react under hydrogen atmosphere (50 PSI) at 50° C. for 10 hrs. After completion of the reaction, the reaction mixture was filtered and concentrated, the crude product was purified by column chromatography (PE/EtOAc=100-0%) to give the compound 6f. MS-ESI calculated value [M+H−100].sup.+ 269, measured value 269.

(77) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.03-4.01 (m, 2H), 3.59-3.50 (m, 2H), 3.48-3.35 (m, 2H), 2.94-2.93 (m, 1H), 2.09-2.02 (m, 4H), 1.80-1.70 (m, 1H), 1.45 (s, 9H), 1.28-1.12 (m, 3H).

The Sixth Step

(78) At 0° C. and under the protection of nitrogen, trifluoroacetate (36.87 g, 323.40 mmol) was added dropwise into a solution of the compound 6f (4.34 g, 16.17 mmol) in 20 mL dichloromethane. The reaction mixture obtained was stirred at 25° C. for 12 hrs. After completion of the reaction, the mixture was directly concentrated, 20 mL saturated aqueous sodium carbonate solution was added slowly into the crude product obtained at 0° C., extracted with EtOAc (50 mL×3), then washed with saturated brine (20 mL×3), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product of the compound 6g.

(79) MS-ESI calculated value [M+H].sup.+ 169, measured value 169.

(80) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 4.00-3.91 (m, 2H), 3.69-3.52 (m, 2H), 3.41-3.04 (m, 3H), 2.71-2.61 (m, 3H), 2.09-2.02 (m, 4H), 1.83-1.76 (m, 1H).

The Seventh Step

(81) The compound 6h was obtained from 6g (2.08 g, 12.37 mmol) and the compound 3a (3.04 g, 12.37 mmol) using the synthesis method in the seventh step of the embodiment 1.

(82) MS-ESI calculated value [M+H].sup.+ 378, measured value 378.

(83) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.17 (s, 1H), 8.62-8.55 (m, 2H), 8.23 (d, J=8.0 Hz, 1H), 7.73 (t, J=8.0 Hz, 1H), 4.33-4.22 (m, 1H), 4.12-4.02 (m, 1H) 3.81-3.71 (m, 1H), 3.70-3.60 (m, 1H), 3.58-3.42 (m, 1H), 3.07-2.96 (m, 1H), 2.05 (s, 3H), 1.95-1.83 (m, 1H), 1.55-1.45 (m, 1H), 1.44-1.35 (m, 1H), 1.07 (d, J=8.0 Hz, 3H).

The Eighth Step

(84) Concentrated hydrochloric acid 25 mL (12 M) was added into a solution of the compound 6h (2.26 g, 5.99 mmol) in the mixed solvent of 12.5 mL ethanol and 25 mL water. The reaction mixture obtained was reacted at 100° C. for 24 hrs. After completion of the reaction, the reaction mixture was concentrated to remove ethanol, then the pH of the mixture was adjusted to 7 with saturated sodium bicarbonate aqueous solution, and a solid precipitated, filtered to give a crude product, then purified by high performance liquid chromatography to give the compound 6.

(85) MS-ESI calculated value [M+H]+ 336, measured value 336.

(86) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.15 (s, 1H), 8.82 (d, J=8.0 Hz, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.22 (d, J=8.0 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 4.04-3.92 (m, 1H), 3.81-3.76 (m, 1H), 3.47 (d, J=8.0 Hz, 1H), 3.34-3.30 (m, 1H), 3.15-3.05 (m, 1H), 2.95-2.85 (m, 2H), 2.03-1.96 (m, 1H), 1.60-1.54 (m, 1H), 1.20 (d, J=8.0 Hz, 3H).

6-1 and 6-2

(87) ##STR00034##

(88) SFC Analysis Conditions:

(89) column: Chiralpak AD-3 100×4.6 mm I.D., 3 μm

(90) mobile phase: A: CO.sub.2 B: methanol (0.05% DEA)

(91) gradient: B went from 5% to 40% in 4.5 mins, then was maintained at 40% for 2.5 mins, then B was maintained at 5% for 1 min, flow rate: 2.8 mL/min

(92) column temperature: 40° C.

(93) 6-1 retention time t=3.818 mins

(94) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.13 (s, 1H), 8.81-8.80 (m, 1H), 8.56 (brs, 1H), 8.21-8.19 (m, 1H), 7.70 (brs, 1H), 4.01-4.00 (m, 1H), 3.77-3.75 (m, 1H), 3.46-3.45 (m, 1H), 3.31 (brs, 1H), 3.06 (brs, 1H), 2.86 (brs, 2H), 2.07 (brs, 1H), 1.57 (brs, 1H), 1.20-1.19 (m, 3H). MS-ESI calculated value [M+H].sup.+ 336, measured value 336.

(95) 6-2 retention time t=4.111 mins

(96) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.13 (s, 1H), 8.81-8.80 (m, 1H), 8.55 (brs, 1H), 8.20-8.19 (m, 1H), 7.70 (brs, 1H), 4.01 (brs, 1H), 3.77 (brs, 1H), 3.46 (brs, 1H), 3.31 (brs, 1H), 3.06 (brs, 1H), 2.87 (brs, 2H), 2.07 (brs, 1H), 1.56 (brs, 1H), 1.19 (brs, 3H).

(97) MS-ESI calculated value [M+H].sup.+ 336, measured value 336.

Embodiment 7

(98) ##STR00035##

The First Step

(99) The compound 7 was obtained from the compound 6g (1.00 g, 5.94 mmol) and the compound 1i (1.73 g, 6.53 mmol) by two-step of reaction using the synthesis in embodiment 6. MS-ESI calculated value [M+H].sup.+ 360, measured value 360.

(100) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.35 (s, 1H), 8.71-8.69 (m, 1H), 8.56-8.50 (m, 2H), 8.20 (d, J=8.4 Hz, 1H), 7.70 (d, J=8.0 Hz, 1H), 3.88-3.87 (m, 1H), 3.68-3.64 (m, 1H), 3.42-3.40 (m, 1H), 3.18-3.17 (m, 1H), 2.82-2.73 (m, 3H), 1.90-1.86 (m, 1H), 1.43-1.41 (m, 1H), 1.22-1.21 (m, 3H).

(101) 7-1 and 7-2

(102) ##STR00036##

(103) SFC Analysis Conditions:

(104) column: Chiralpak AD-3 100×4.6 mm I.D., 3 um;

(105) mobile phase: A: CO.sub.2 B: methanol (0.05% DEA);

(106) gradient: B went from 5% to 40% in 5 mins, then was maintained at 40% for 2.5 mins, then B was maintained at 5% for 1 min;

(107) flow rate: 2.5 mL/min;

(108) column temperature: 35° C.

(109) 7-1 retention time t=4.062 mins

(110) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.27 (s, 1H), 8.63-8.61 (m, 1H), 8.49-8.42 (m, 2H), 8.13 (d, J=8.0 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 3.78-3.77 (m, 1H), 3.61-3.56 (m, 1H), 3.32-3.31 (m, 1H), 3.10-3.06 (m, 1H), 2.67-2.64 (m, 3H), 1.81-1.76 (m, 1H), 1.33-1.32 (m, 1H), 1.15-1.14 (m, 3H).

(111) MS-ESI calculated value [M+H].sup.+ 318, measured value 318.

(112) 7-2 retention time t=4.303 mins

(113) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.27 (s, 1H), 8.63-8.61 (m, 1H), 8.49-8.42 (m, 2H), 8.12 (d, J=8.4 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 3.78-3.76 (m, 1H), 3.61-3.56 (m, 1H), 3.32-3.30 (m, 1H), 3.09-3.05 (m, 1H), 2.67-2.64 (m, 3H), 1.81-1.75 (m, 1H), 1.33-1.32 (m, 1H), 1.15-1.14 (m, 3H).

(114) MS-ESI calculated value [M+H].sup.+ 318, measured value 318.

Embodiment 8

(115) ##STR00037## ##STR00038##

The First Step

(116) At 25° C., ethyl chloroformate (84.9 g, 782.34 mmol) was added dropwise into a solution of 8a (50.00 g, 561.23 mmol) and sodium bicarbonate (141.45 g, 1.68 mol) in the mixed solvent of 250 mL THF and 250 mL water. The reaction mixture obtained was stirred to react at 25° C. for 48 hrs. After completion of the reaction, the mixture was filtered and concentrated to remove THF, after the concentration, water (50 mL) was added, then extracted with methyl ter-butyl ether (200 mL×1). The pH of the aqueous phase was adjusted to 1 then extracted with EtOAc (200 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product of the compound 8b.

(117) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.19 (s, 1H), 4.43-4.38 (m, 1H), 4.16-4.12 (m, 2H), 1.47 (d, J=7.6 Hz, 3H), 1.27 (t, J=7.2 Hz, 3H).

The Second Step

(118) At 0° C. and under the protection of nitrogen, propanephosphonic anhydride solution (473.83 g, 744.6 mmol, 50%) and diisopropylethylamine (128.31 g, 992.80 mmol) were added sequentially into a solution of the compound 8b (40.00 g, 248.20 mmol) in 500 mL EtOAc. The reaction mixture obtained was stirred at 25° C. for 10 mins, then N-methoxymethylamine hydrochloride (26.63 g, 273.02 mmol) was added. The reaction mixture was stirred to react at 25° C. for 16 hrs. After completion of the reaction, water (300 mL) was added into the reaction mixture, then the mixture was extracted with EtOAc (200 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by column chromatography (PE/EtOAc=100-0%) to give the compound 8c.

(119) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.43-5.42 (m, 1H), 4.73-4.69 (m, 1H), 4.08 (t, J=6.8 Hz, 2H), 3.76 (s, 3H), 3.20 (s, 3H), 1.32 (d, J=6.8 Hz, 3H), 1.22 (d, J=7.2 Hz, 3H).

The Third Step

(120) At 0° C. and under the protection of nitrogen, sodium hydrogen (8.46 g, 211.54 mmol, 60%) was added in batch into a solution of 3-bromopropene (31.99 g, 264.42 mmol) in N, N-dimethylformamide (400 mL). The reaction mixture was stirred for 10 mins then the compound 8c (36.00 g, 176.28 mmol) was added. The reaction mixture obtained was continued stirring and reacting at 20° C. for 5 hrs. After the completion of the reaction, saturated ammonium chloride solution (300 mL) and water (200 mL) were added into the mixture, then extracted with EtOAc (400 mL×3), the organic phases were combined and washed with saturated brine (400 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by column chromatography (PE/EtOAc=100-0%) to give the compound 8d.

(121) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.83-5.77 (m, 1H), 5.26-5.01 (m, 3H), 4.11-4.07 (m, 2H), 3.97-3.90 (m, 2H), 3.73-3.66 (m, 3H), 3.17-2.95 (m, 3H), 1.36-1.29 (m, 3H), 1.24-1.19 (m, 3H).

The Fourth Step

(122) At −78° C. and under the protection of nitrogen, diisobutylaluminum hydride (81.9 mL, 1 M) was added dropwise into a solution of the compound 8d (10.00 g, 40.93 mmol) in 150 mL THF. After completion of the addition, the mixture was stirred to react at 20° C. for 3 hrs. After completion of the reaction, saturated potassium sodium tartrate solution (500 mL) and water (200 mL) were added slowly into the reaction mixture, then extracted with EtOAc (300 mL×3), the organic phases were combined and washed by saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by column chromatography (PE/EtOAc=100-0%) to give the compound 8e.

(123) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.51 (s, 1H), 5.81-5.74 (m, 1H), 5.18-5.09 (m, 2H), 4.12-4.04 (m, 3H), 3.95-3.74 (m, 2H), 1.34-1.28 (m, 3H), 1.22-1.13 (m, 3H).

The Fifth Step

(124) The compound 8f was obtained from the compound 8e (4.65 g, 25.11 mmol) and the compound 4a (8.29 g, 50.21 mmol) using the synthesis method in the third step of the embodiment 5.

(125) MS-ESI calculated value [M+H].sup.+ 289, measured value 289.

(126) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.21-7.13 (m, 5H), 4.06-4.01 (m, 2H), 3.81 (brs, 2H), 3.49-3.46 (m, 1H), 3.44-3.38 (m, 2H), 2.88 (s, 1H), 2.69 (brs, 2H), 2.17-2.11 (m, 1H), 1.95-1.85 (m, 1H), 1.55-1.43 (m, 1H), 1.19-1.15 (m, 3H), 1.02-0.95 (m, 3H).

The Sixth Step

(127) Under the protection of nitrogen, wet palladium on carbon (200 mg, 10%) was added into a solution of the compound 8f (2.00 g, 6.94 mmol) and di-tert-butyl dicarbonate (3.03 g, 13.88 mmol) in 150 mL methanol. The reaction mixture obtained was replaced with hydrogen then under hydrogen atmosphere (50 PSI) and stirred to react at 50° C. for 24 hrs. After completion of the reaction, filtered and concentrated, the crude product was purified by silica get column (PE/EtOAc=100-0%) to give the compound 8g.

(128) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.15-4.09 (m, 3H), 3.86-5.75 (m, 1H), 3.60-3.55 (m, 2H), 3.36-3.34 (m, 2H), 3.02-2.91 (m, 1H), 1.98-1.93 (m, 1H), 1.73 (s, 1H), 1.48 (s, 9H), 1.28-1.23 (m, 3H).

The Seventh Step

(129) The compound 8g (250 mg, 0.84 mmol) was dissolved in the mixed solvent of ethanol (4 mL) and water (3 mL), potassium hydroxide (1.50 g, 26.81 mmol) was then added. The reaction mixture obtained was stirred at 120° C. for 40 hrs. After completion of the reaction, the reaction mixture was concentrated to remove ethanol, then water (5 mL) was added, extracted with dichloromethane (5 mL×2), the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by column chromatography (dichloromethane/methanol=100-0%) to give the compound 8h.

(130) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.86-3.57 (m, 2H), 3.28-3.23 (m, 1H), 3.07-3.10 (m, 2H), 2.77 (s, 1H), 2.60-2.55 (m, 1H), 1.83-2.75 (m, 2H), 1.65-1.53 (m, 1H), 1.41 (s, 9H), 1.20-1.13 (m, 3H).

The Eighth Step

(131) The compound 8j was obtained from the compound 8h (81 mg, 0.36 mmol) and the compound 8i (200 mg, 0.72 mmol) using the synthesis method in the seventh step of embodiment 1.

(132) MS-ESI calculated value [M+H].sup.+ 432, measured value 432.

The Ninth Step

(133) At 20° C., trifluoroacetate (2 mL) was added dropwise into a solution of the compound 8j (109 mg, 0.25 mmol) in 6 mL dichloromethane, the reaction mixture was continued stirring for 2 hrs. After completion of the reaction, the reaction mixture was directly concentrated, the crude product was purified by high performance liquid chromatography to give the compound 8.

(134) MS-ESI calculated value [M+H].sup.+ 332, measured value 332.

(135) 1H NMR (400 MHz, CD3OD) δ 9.20 (s, 1H), 8.80 (d, J=7.2 Hz, 1H), 8.48 (s, 1H), 8.37 (d, J=8.4 Hz, 1H), 7.77 (t, J=8.0 Hz, 1H), 4.12-4.10 (m, 1H), 3.82-3.78 (m, 1H), 3.62-3.60 (m, 1H), 3.41-3.37 (m, 1H), 3.62-3.60 (m, 1H), 3.18 (brs, 1H), 3.08-3.05 (m, 5H), 2.09-2.04 (m, 1H), 1.80-1.79 (m, 1H), 1.34-1.32 (m, 3H).

Embodiment 9

(136) ##STR00039##

The First Step

(137) The compound 7a (80 mg, 0.22 mmol) was dissolved in 1 mL dichloromethane, then m-chloroperbenzoic acid (68 mg, 0.33 mmol, 85%) was added into the reaction mixture at 0° C. and under the protection of nitrogen. The reaction mixture obtained was stirred at 25° C. for 4 hrs. After completion of the reaction, the reaction was quenched by adding saturated sodium carbonate aqueous solution (20 mL) and saturated sodium thiosulfate aqueous solution (20 mL) at 0° C., extracted with EtOAc (50 mL×3), the organic phases were combined and then washed by saturated brine (20 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated then purified by thin layer chromatography (methanol/dichloromethane=1:10) to give the compound 9a. MS-ESI calculated value [M+H].sup.+ 376, measured value 376.

(138) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.82 (s, 1H), 8.61 (d, J=7.6 Hz, 1H), 8.25 (d, J=7.6 Hz, 2H), 7.91 (d, J=8.4 Hz, 1H), 7.71 (t, J=8.0 Hz, 1H), 4.31-4.29 (m, 1H), 3.97 (d, J=7.2 Hz, 1H), 3.71-3.66 (m, 1H), 3.35-3.33 (m, 1H), 3.30-3.26 (m, 1H), 3.04-3.03 (m, 2H), 2.04-1.93 (m, 1H), 1.86 (s, 3H), 1.76-1.70 (m, 1H), 1.21 (d, J=6.8 Hz, 3H).

The Second Step

(139) The compound 9a (66 mg, 0.18 mmol) was dissolved in 1 mL acetic anhydride, under the protection of nitrogen, the mixture was stirred at 120° C. for 4 hrs. After completion of the reaction, the reaction mixture was concentrated, then quenched by saturated sodium carbonate aqueous solution (20 mL) at 0° C., then extracted with EtOAc (50 mL×3), the organic phases were combined and was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by thin layer chromatography (methanol/dichloromethane=1:10) to give the compound 9b.

(140) MS-ESI calculated value [M+H].sup.+ 376, measured value 376.

(141) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.68 (d, J=7.2 Hz, 1H), 8.37 (d, J=7.6 Hz, 1H), 7.61-7.57 (m, 1H), 7.53-7.48 (m, 1H), 7.25-7.23 (m, 1H), 4.32-4.31 (m, 1H), 3.97-3.95 (m, 1H), 3.71-3.67 (m, 1H), 3.36-3.28 (m, 1H), 3.20-3.16 (m, 1H), 3.02-2.98 (m, 2H), 1.93-1.92 (m, 1H), 1.84 (s, 3H), 1.74-1.70 (m, 1H), 1.26 (d, J=6.8 Hz, 3H).

The Third Step

(142) The compound 9 was obtained from the compound 9b (53 mg, 0.14 mmol) using the synthesis method in the eighth step of embodiment 6.

(143) MS-ESI calculated value [M+H].sup.+ 334, measured value 334.

(144) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.61 (d, J=8.0 Hz, 1H), 8.41 (d, J=6.4 Hz, 1H), 7.66 (t, J=8.0 Hz, 1H), 7.45 (d, J=7.6 Hz, 1H), 7.35 (d, J=7.2 Hz, 1H), 3.81-3.79 (m, 1H), 3.64-3.59 (m, 1H), 3.50-3.39 (m, 1H), 3.11-3.07 (m, 1H), 2.88-2.86 (m, 1H), 2.81-2.68 (m, 2H), 1.90-1.83 (m, 1H), 1.45 (brs, 1H), 1.22 (d, J=6.4 Hz, 3H).

Embodiment 10

(145) ##STR00040##

The First Step

(146) The compound 10 was obtained by a three-step of reaction from the compound 6h using the synthesis method in embodiment 9.

(147) MS-ESI calculated value [M+H].sup.+ 352, measured value 352.

(148) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.66-8.63 (m, 2H), 7.74 (t, J=8.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H), 4.08-4.06 (m, 1H), 3.72-3.64 (m, 2H), 3.37-3.34 (m, 1H), 3.18-3.10 (m, 1H), 3.04-3.02 (m, 2H), 2.09-2.04 (m, 1H), 1.75-1.72 (m, 1H), 1.21 (d, J=6.4 Hz, 3H).

Embodiment 11

(149) ##STR00041##

The First Step

(150) The compound 11a was obtained from the compound 1j using the synthesis method in the first step of embodiment 9.

The Second Step

(151) Benzoyl chloride (25 mg, 0.18 mmol) was added into a solution of the compound 11a (40 mg, 0.09 mmol), tetrabutylammonium bromide (6 mg, 0.02 mmol) and sodium acetate (22 mg, 0.27 mmol) in a mixed solvent of 3 mL water and 3 mL dichloromethane. The reaction mixture obtained was reacted at 20° C. for 1 hr. After completion of the reaction, the liquid was separated, while the aqueous phase was extracted with dichloromethane (5 mL×2), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, then the crude product was purified by thin layer chromatography (EtOAc) to give the compound 11b.

The Third Step

(152) The compound 11 was obtained from the compound 11b (15 mg, 0.03 mmol) using the synthesis method in the eight step of embodiment 1.

(153) MS-ESI calculated value [M+H].sup.+ 346, measured value 346.

(154) .sup.1H NMR (400 MHz, D.sub.2O) δ 8.54 (brd, J=8.0 Hz, 1H), 8.38 (brd, J=7.6 Hz, 1H), 7.65 (t, J=8.0 Hz, 1H), 7.39 (d, J=7.6 Hz, 1H), 7.14 (d, J=7.6 Hz, 1H), 4.00-3.83 (m, 2H), 3.75-3.65 (m, 1H), 3.54-3.39 (m, 1H), 3.35-3.14 (m, 2H), 2.41-2.28 (m, 1H), 2.08-1.93 (m, 1H), 1.36-1.24 (m, 1H), 1.13-0.99 (m, 1H), 0.84-0.72 (m, 1H), 0.65-0.53 (m, 1H).

(155) Evaluation of ROCK Protein Kinase Inhibitory Activity In Vitro

(156) Experimental object: detecting the inhibitory IC.sub.50 value of ROCK protein kinase of the compounds.

(157) Experimental Materials:

(158) Assay buffer solution: 20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT, 1% DMSO

(159) Experimental Operation:

(160) ROCK protein kinase substrate Long S6 Kinase substrate peptide was added to the freshly prepared buffer solution at a concentration of 20 μM. Then 1 nM ROCK protein kinase was added and stirred evenly. A series of DMSO dilutions containing the test compound or positive reference (starting at 1004, 3-fold serial dilution) was added using a Echo550. After pre-incubating at room temperature for 20 minutes, .sup.33P-ATP (radiation intensity 10 μCi/μL) was added to initiate the reaction and the reaction was carried out at room temperature for two hours. It was then filtered using P81 ion exchange paper (Whatman #3698-915) and washed with 0.75% phosphoric acid. The Filter-Binding method was used to detect radiation intensity.

(161) The protein kinase inhibitory activity of the compound was expressed as the residual protein kinase activity of a relatively blank substrate (DMSO alone). IC.sub.50 values and curves were calculated using Prism software package (GraphPad Software, san Diego Calif., USA). The results are shown in Table 1.

(162) In this experiment, Fasudil was used as a positive reference.

(163) Experimental Results:

(164) TABLE-US-00001 TABLE 1 Test results of protein kinase inhibitory activity Samples (compounds obtained Protein kinase inhibitory in the embodiments) activity (nM) embodiment 1 20 embodiment 2 32 embodiment 3 93 embodiment 6 12 embodiment 6-1/6-2 65/8  embodiment 7 28 embodiment 7-1/7-2 63/15 embodiment 8 18 embodiment 10 783  Fasudil 116 

(165) The results show that the compounds of the present disclosure have significant and unexpected protein kineses inhibitory activity.

(166) Evaluation of Pharmacokinetics in Rats

(167) Experimental Object

(168) Male SD rats were used as test animals, after a single administration, the blood concentrations of the compounds were measured and the pharmacokinetic behavior was evaluated.

(169) Experimental Operation

(170) Six healthy adult male SD rats (7-10 weeks of age, purchased from Shanghai Slack Experimental Animal Co., Ltd.) were randomly divided into two groups of three animals each, and one group was administered the test compound intravenously at 2 mg/kg, the other group was administered orally by gavage the test compound at 10 mg/kg. The vehicle in the intravenous administration group and the gastric administration group was both 10% DMSO+18% HP-β-CD+72% physiological saline. Blood samples were collected from the animals in the intravenous group at 0.0833, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after the administration, and blood samples were collected from the animals in the gavage group at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after the administration. LCMS-MS method was used to determine the plasma drug concentration. WinNonlin™ version 6.3 (Pharsight Mountain View, Calif.) pharmacokinetic software was used to calculate the relevant pharmacokinetic parameters by non-compartment model logarithmic trapezoid method.

(171) Experimental Results

(172) The test results are shown in Table 2.

(173) TABLE-US-00002 TABLE Evaluation of pharmacokinetics in rats Embodiment 38 Embodiment 6 (WO2015/165341) Clearance rate (Cl, mL/min/kg) 43.8 153 Half-life period (T.sub.1/2, hr) 1.12 0.48

(174) The results show that the half-life of the compound of the present disclosure was increased by about 3 times, and the clearance rate was significantly reduced, which proves that the present disclosure has more superior properties than the prior art.

(175) Pharmacodynamics in Rats

(176) Experimental Object

(177) To investigate the effect of the test compound (Embodiment 6) on unilateral pulmonary fibers in the left lung of SD rats, with reference to fasudil and the clinical treatment drugs pirfenidone and nintedanib, which empoly similar mechanism of action.

(178) Experimental Operation

(179) Male SD rats were randomly divided into eleven groups based on body weight, namely the sham-operated group, the model group, the nintedanib 100 and 30 mg/kg/d-qd group, the pirfenidone 50 and 15 mg/kg/d-bid group, fasudil 25 mg/kg/d-qd group, test compound (Embodiment 6) 1, 3, 10 mg/kg/d-bid group and test compound (Embodiment 6) 3 mg/kg/d-qd group. Animals in each group started to be administered orally by gavage on the 8.sup.th day of modeling for a total of 14 days. All animals were euthanized the next day after the last administration, and the left lung was taken, and the same amount of formalin solution was infused into the lungs, the lung fibrosis score was analyzed by weighing and lung pathology.

(180) Experimental Results

(181) Masson Trichrome staining was used for pathological evaluation of pulmonary fibrosis lesion area, pulmonary fibrosis pathology score, and fibrosis grade parameters for left lung pulmonary fibrosis lesions. Pulmonary fibrosis ashcraft score results showed that the positive drugs nintedaniband pirfenidone significantly improved the degree of pulmonary fibrosis compared with the model group (p<0.05) (FIG. 1), the test compound (embodiment 6) was orally administered twice a day at three different doses for 14 consecutive days, showing significant inhibition of pulmonary fibrosis, which was significantly different from the model group (p<0.001) (FIG. 1), but no clear dose-dependent efficacy response was observed. The test compound (embodiment 6) was administrated orally once a day at 3 mg/kg also showed a significant effect on inhibiting pulmonary fibrosis, which was consistent with the effect of twice daily oral administration of the same dose, and no significant difference was observed (FIG. 1). The test compound fasudil was administered orally once a day at 25 mg/kg for 14 consecutive days and achieved the same effect as the positive drug in inhibiting pulmonary fibrosis (p<0.001) (FIG. 1). The percentage of pulmonary fibrosis was calculated based on ashcraft score, with a boundary line of the score of 3, for the score below 3 (including 3), or above 4 (including 4), the result showed that 65% or more of the lesion area had a score of 4 or more than 4, after drug therapy, more than 70% of the lesion area of the animals in each drug therapy group scored below 3. The statistical results showed that the positive drugs nintedanib and pirfenidone had significant differences compared with the model group (p<0.001); the test compound (Embodiment 6) had a significant statistical difference compared with the model group and different doses treatment groups, but no significant dose-dependent effect was observed. (FIG. 2).

(182) Experimental conclusion: In the bleomycin-induced rat pulmonary fibrosis model, the test compound (Embodiment 6) was administered continuously for two weeks and showed a dose-dependent effect on inhibiting pulmonary fibrosis, and it was effective at as low as 1 mg/kg BID. The test compound (Embodiment 6) could achieve a pulmonary fibrosis-improving effect comparable to that of nintedanib, pirfenidone, and fasudil at lower doses.

(183) hERG Experiment

(184) The cells stably expressing hERG potassium channel used in the experiment were derived from CHO-hERE of Aviva Biosciences, CHO-hERG was cultured under 5% CO.sub.2 at 37° C. hERGQPatch.sup.HTX experiments were performed at room temperature. QPatch AssaySoftware 5.2 (Sophion Bioscience) software was used to establish whole-cell protocols, voltage stimulation protocols and compound detection protocols. First, the voltage stimulation was repeated for 30 times, this section was used as the baseline area for subsequent analysis, then 5 μL extracellular fluid was added and repeated three times. The effect concentrations of each compound were added one after the other, repeating three times by the addition of 5 μL volume. The cells were incubated at each test concentration for at least 5 mins. During the entire recording process, each indicator must meet the data analysis acceptance criteria, if the criterion is not met, the cell will not be counted in the analysis range, and the compound will be tested again, the above recording process is automatically operated by Qpatch analysis software. Each compound was tested at a concentration of 0.24 μM, 1.2004, 6.00 μM, and 30.00 μM, each concentration was repeated for at least two cells. In each complete current record, based on the percentage of peak current in the negative control group, the percentage inhibition of the effect concentration of each compound can be calculated. The standard Greek equation is used to fit the dose-response curve, and the specific equation is as follows:
I.sub.(C)=I.sub.b+(I.sub.fr−I.sub.b)*c.sup.n/(IC.sub.50.sup.n+c.sup.n)

(185) C is the test concentration of the compound, n is the slope.

(186) The curve fitting and inhibition rate calculations were completed by Qpatch analysis software, if the inhibition rate exceeds the half inhibition at the lowest concentration or the inhibition rate does not reach the half inhibition at the highest concentration, then the corresponding IC.sub.50 of the compound is lower than the lowest concentration or the IC.sub.50 value is greater than the highest concentration.

(187) Experimental Results

(188) The results of hERG inhibitory activity of compounds of the Embodiments are shown in Table 3.

(189) TABLE-US-00003 TABLE 3 Evaluation of hERG inhibitory activity Embodiment 38 Embodiment 6 (WO2015/165341) hERG (μM) >30 4.6

(190) The results show that the compounds of the present disclosure have a lower potential risk for hERG than the prior art.