TRI-HETEROCYCLIC COMPOUND AS JAK INHIBITOR, AND USE THEREOF

Abstract

Disclosed are a tri-heterocyclic compound as a JAK inhibitor, and the use thereof in the preparation of a drug for treating JAK1- or/and JAK2-related diseases. Specifically, the present invention relates to a compound as shown in formula (I′) or a pharmaceutically acceptable salt thereof.

##STR00001##

Claims

1. A compound of formula (I′) or a pharmaceutically acceptable salt thereof, ##STR00037## wherein, m is 1, 2, 3, 4 or 5; n is 1, 2, 3 or 4; each R.sub.1 is independently H, F, Cl, Br, I, CN, C.sub.1-8 alkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkyl S—, NH.sub.2, C.sub.1-8 alkyl NH—, (C.sub.1-8 alkyl).sub.2N—, —COOH, or —C(O)OC.sub.1-8 alkyl, wherein the C.sub.1-8 alkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkyl S—, C.sub.1-8 alkyl NH—, (C.sub.1-8 alkyl).sub.2N— or —C(O)OC.sub.1-8 alkyl is each independently optionally substituted with 1, 2, 3 or 4 R.sub.a; each R.sub.2 is independently H, F, Cl, Br, I, CN, NH.sub.2, C.sub.1-8 alkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkyl S—, C.sub.1-8 alkyl NH—, (C.sub.1-8 alkyl).sub.2N—, or C.sub.3-12 cycloalkyl; each R.sub.a is independently H, F, Cl, Br, I, OH, CN or NH.sub.2.

2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein m is 1, 2 or 3; alternatively, m is 1 or 2; alternatively, m is 1.

3. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein n is 1, 2 or 3; alternatively, n is 1 or 2; alternatively, n is 1; alternatively, m and n are both 1.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural fragment ##STR00038## alternatively, the structural fragment ##STR00039##

5. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural fragment ##STR00040## alternatively, the structural fragment ##STR00041##

6. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl S—, NH.sub.2, C.sub.1-6 alkyl NH—, (C.sub.1-6 alkyl).sub.2N—, —COOH, or —C(O)OC.sub.1-6 alkyl, wherein the C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl S—, C.sub.1-6 alkyl NH—, (C.sub.1-6 alkyl).sub.2N— or —C(O)OC.sub.1-6 alkyl is each independently optionally substituted with 1, 2, 3 or 4 R.sub.a; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl S—, NH.sub.2, C.sub.1-6 alkyl NH—, or (C.sub.1-6 alkyl).sub.2N—, wherein the C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl S—, C.sub.1-6 alkyl NH— or (C.sub.1-6 alkyl).sub.2N— is each independently optionally substituted with 1, 2, 3 or 4 R.sub.a; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S—, NH.sub.2, C.sub.1-3 alkyl NH—, (C.sub.1-3 alkyl).sub.2N—, —COOH, or —C(O)OC.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S—, C.sub.1-3 alkyl NH—, (C.sub.1-3 alkyl).sub.2N— or —C(O)OC.sub.1-3 alkyl is each independently optionally substituted with 1, 2, 3 or 4 R.sub.a; alternatively, wherein each R.sub.1 above is independently H, F, Cl, Br, I, CN, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S—, NH.sub.2, C.sub.1-3 alkyl NH—, or (C.sub.1-3 alkyl).sub.2N—, wherein the C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S—, C.sub.1-3 alkyl NH— or (C.sub.1-3 alkyl)2N— is each independently optionally substituted with 1, 2, 3 or 4 R.sub.a; alternatively, wherein each R.sub.1 above is independently H, F, Cl, Br, I, CN, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S—, or C.sub.1-3 alkyl NH—, wherein the C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S— or C.sub.1-3 alkyl NH— is each independently optionally substituted with 1, 2, 3 or 4 R.sub.a; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, C.sub.1-3 alkyl, or C.sub.1-3 alkoxy, wherein the C.sub.1-3 alkyl or C.sub.1-3 alkoxy is each independently optionally substituted with 1, 2, 3 or 4 R.sub.a.

7. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein each R.sub.2 is independently H, F, Cl, Br, I, CN, NH.sub.2, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl S—, C.sub.1-6 alkyl NH—, (C.sub.1-6 alkyl).sub.2N—, or C.sub.3-10 cycloalkyl; alternatively, wherein each R.sub.2 is independently H, F, Cl, Br, I, CN, NH.sub.2, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S—, C.sub.1-3 alkyl NH—, (C.sub.1-3 alkyl).sub.2N—, or C.sub.3-6 cycloalkyl; alternatively, wherein each R.sub.2 is independently H, F, Cl, Br, I, CN, NH.sub.2, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkyl S—, or C.sub.1-3 alkyl NH—; alternatively, wherein each R.sub.2 is independently H, F, Cl, Br, I, CN or NH.sub.2; alternatively, wherein each R.sub.2 is independently H, F, Cl, Br, I or CN.

8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein each R.sub.a is independently F, Cl, Br, I or OH.

9. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I′) is represented by formula (I), ##STR00042## wherein, R.sub.1 is H, F, Cl, Br, I, CN, C.sub.1-3 alkyl or C.sub.1-3 alkoxy, wherein the C.sub.1-3 alkyl and C.sub.1-3 alkoxy are optionally substituted with 1, 2, 3 or 4 R.sub.a; R.sub.2 is H, F, Cl, Br, I or CN; each R.sub.a is independently H, F, Cl, Br, I or OH.

10. The compound or the pharmaceutically acceptable salt thereof according to claim 9, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, —CH.sub.3, —CH.sub.2CH.sub.3, or —OCH.sub.3, wherein the —CH.sub.3, —CH.sub.2CH.sub.3 and —OCH.sub.3 are optionally substituted with 1, 2, 3 or 4 Ra; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, —CH.sub.3, —CH.sub.2CH.sub.3 or —OCH.sub.3, wherein the —CH.sub.3, —CH.sub.2CH.sub.3 and —OCH.sub.3 are each independently optionally substituted with 1, 2 or 3 R.sub.a; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, —CH.sub.3, —CH.sub.2CH.sub.3, or —OCH.sub.3, wherein the —CH.sub.3 or —CH.sub.2CH.sub.3 is each independently optionally substituted with 1, 2 or 3 R.sub.a; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, —CH.sub.3, —CH.sub.2CH.sub.3, or —OCH.sub.3, wherein the —CH.sub.3 or —CH.sub.2CH.sub.3 is each independently optionally substituted with 1, 2 or 3 F or OH; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, —CH.sub.3, —CF.sub.3, —CH.sub.2CH.sub.3, —CH(OH)CH.sub.3, or —OCH.sub.3; alternatively, wherein each R.sub.1 is independently H, F, Cl, Br, I, CN, —CH.sub.3, —CF.sub.3, —CH.sub.2CH.sub.3, or —OCH.sub.3; alternatively, wherein each R.sub.1 is independently H, CN, —CH.sub.3, —CF.sub.3, —CH(OH)CH.sub.3, or —OCH.sub.3; alternatively, wherein each R.sub.1 is independently H, CN, —CH.sub.3, —CF.sub.3, —CH.sub.2CH.sub.3, or —OCH.sub.3.

11. The compound or the pharmaceutically acceptable salt thereof according to claim 9, wherein each R.sub.a is independently F, Cl, Br, I or OH; alternatively, wherein each R.sub.a is independently F or OH.

12. The compound or the pharmaceutically acceptable salt thereof according to claim 9, wherein each R.sub.2 is independently F, Cl, Br, I or CN; alternatively, wherein each R.sub.2 is independently CN.

13. The compound or the pharmaceutically acceptable salt thereof according to claim 1, which is selected from the group consisting of compounds of formula (I′-A) and formula (I′-B) or pharmaceutically acceptable salts thereof: ##STR00043##

14. The compound or the pharmaceutically acceptable salt thereof according to claim 1, which is selected from the group consisting of compounds of formula (I-A) and formula (I-B) or pharmaceutically acceptable salts thereof: ##STR00044##

15. The compound or the pharmaceutically acceptable salt thereof according to claim 1, which has a structure of formula (I-1): ##STR00045## or which is selected from the group consisting of formula (I-1-A) and formula (I-1-B) or pharmaceutically acceptable salts thereof: ##STR00046##

16. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is ##STR00047##

17. The compound or the pharmaceutically acceptable salt thereof according to claim 16, wherein the compound is ##STR00048## ##STR00049## ##STR00050##

18. A pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt thereof according to claim 1.

19. (canceled)

20. A method of treating a JAK1- and/or JAK2-associated disease, comprising: administering a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition thereof.

21. The method according to claim 20, wherein the disease is inflammatory conditions.

Description

DETAILED DESCRIPTION

[0099] The present application is described in detail by way of examples. However, this is by no means disadvantageously limiting the scope of the present application. Although the present application has been described in detail herein and specific embodiments have also been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments without departing from the spirit and scope of the present application.

EXAMPLE 1

Synthesis of Compounds 1A and 1B

[0100] ##STR00021## ##STR00022## ##STR00023## ##STR00024##

[0101] Step 1: (4-methoxyphenyl)methanol (50 g, 361.89 mmol, 45.05 mL, 1 eq) was added into a suspension of sodium hydride (15.92 g, 398.08 mmol, 60% purity, 1.1 eq) in DMF (200 mL) at 0° C.; after 0.5 h of stirring, 3-bromoprop-1-yne (59.19 g, 398.08 mmol, 42.89 mL, 1.1 eq) was added slowly into the reaction system; the resulting solution was stirred at 0° C. for 0.5 h and stirred at 25° C. for 16 h; the reaction was completed and a new major product was produced, as indicated by TLC (PE:EA=10:1); saturated aqueous ammonium chloride solution (50 mL) was added into the reaction solution, and the aqueous phase was extracted with ethyl acetate (500 mL×3); the organic phases were pooled, washed successively with water (200 mL×2) and brine (200 mL×1), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO.sub.2, PE:EA=1:0 to 5:1) to give compound 1-2. MS (ESI) calcd. for C.sub.11H.sub.12O.sub.2: 176; found: 177 [M+H].sup.+.

[0102] Step 2: compound 1-2 (34.14 g, 193.74 mmol, 25.00 mL, 1.1 eq), copper(I) iodide (3.35 g, 17.59 mmol, 0.1 eq), piperidine (44.99 g, 528.36 mmol, 52.18 mL, 3 eq) and dichlorobis(triphenylphosphine)palladium(II) (6.18 g, 8.80 mmol, 0.05 eq) were added into a solution of 5-bromo-2-iodo-pyridine (50 g, 176.12 mmol, 1 eq) in THF (500 mL) at 25° C., and the reaction system was subjected to nitrogen purging three times; the resulting solution was stirred at 25° C. for 16 h; the reaction was completed and a new major product was produced, as indicated by TLC (PE:EA=10:1); the reaction solution was filtered through diatomite, and the resulting filtrate was concentrated under reduced pressure; the residue was dissolved in 800 mL of ethyl acetate, and then washed successively with 300 mL of water and 300 mL of brine; the organic phase was dried over anhydrous sodium sulfate and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was subjected to column chromatography (SiO.sub.2, PE:EA=1:0 to 30:1) to give compound 1-3. NMR (400 MHz, CCl.sub.3-d) δ=8.61 (d, J=1.8 Hz, 1H), 7.75 (dd, J=2.4, 8.4 Hz, 1H), 7.32-7.28 (m, 1H), 7.32-7.26 (m, 1H), 7.27 (s, 1H), 6.89-6.84 (m, 2H), 4.58 (s, 2H), 4.35 (s, 2H), 3.79-3.76 (m, 1H), 3.77 (s, 2H). MS (ESI) calcd. for C.sub.16H.sub.14BrNO.sub.2: 332; found: 334. MS (ESI) calcd. for C.sub.16H.sub.14BrNO.sub.2: 331, 333; found: 331 [M].sup.+, 334 [M+H].sup.+.

[0103] Step 3: ammonium 2,4,6-trimethylbenzenesulfonate (972 mg, 4.52 mmol, 1.5 eq) was dissolved in dry DCM (15 mL) containing anhydrous Na.sub.2SO.sub.4, and then compound 1-3 (1 g, 3.01 mmol, 1 eq) was added into the resulting mixture at 0° C., which was then stirred at 25° C. for 16 h. A small amount of the reaction materials remained and a new major product was produced, as indicated by TLC (PE:EA=2:1). 32 mL of tert-butyl methyl ether was slowly added into the system with stirring at 0° C., and a large amount of off-white solid slowly precipitated; the solid was filtrated and dried to give compound 1-4. MS (ESI) calcd. for C.sub.16H.sub.16BrN.sub.2O.sub.2: 348; found: 348 [M].sup.+.

[0104] Step 4: silver carbonate (10.07 g, 36.52 mmol, 1.66 mL, 2 eq) was added into a solution of compound 1-4 (10 g, 18.27 mmol, 1 eq) in DMF (160 mL) at 25° C., and the resulting solution was stirred at 40° C. for 16 h. The starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was filtered through diatomite, and the resulting filtrate was concentrated under reduced pressure; the residue was dissolved in 200 mL of ethyl acetate, and the resulting solution was washed successively with 100 mL of water and 100 mL of brine; the organic phase was then dried over anhydrous sodium sulfate and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was subjected to column chromatography (SiO.sub.2, PE:EA=1:0 to 5:1) to give compound 1-5. .sup.1H NMR (400 MHz, CCl.sub.3-d) δ=8.50-8.46 (m, 1H), 7.34-7.27 (m, 1H), 7.26-7.21 (m, 2H), 7.10-7.06 (m, 1H), 6.84-6.78 (m, 2H), 6.50-6.46 (m, 1H), 4.67-4.60 (m, 2H), 4.51-4.46 (m, 2H), 3.75-3.71 (m, 3H). MS (ESI) calcd. for C.sub.16H.sub.15BrN.sub.2O.sub.2: 347; found: 348.8 [M+H].sup.+.

[0105] Step 5: potassium hydroxide (1.63 g, 29.05 mmol, 2.15 eq), 5-(di-tert-butylphosphino)-1,3,5-triphenyl-1H-[1,4]bipyrazole (783.26 mg, 1.55 mmol, 0.1 eq) and tris(dibenzylideneacetone)dipalladium(0) (619.79 mg, 676.83 μmol, 0.05 eq) were sequentially added into a mixed solution of compound 1-5 (4.7 g, 13.54 mmol, 1 eq) and tert-butyl carbamate (1.9 g, 16.22 mmol, 1.2 eq) in isoamyl alcohol (15 mL) and water (15 mL) at 25° C., and the reaction system was subjected to nitrogen purging three times; the resulting solution was stirred at 100° C. for 2 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was filtered through diatomite to obtain the filtrate, and the filter cake was washed with 100 mL of ethyl acetate; the organic phases were pooled, washed successively with 100 mL of water and 100 mL of brine, dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO.sub.2, PE:EA=1:0 to 20:1) to give compound 1-6. MS (ESI) calcd. for C.sub.21H.sub.25N.sub.3O.sub.4: 383; found: 384 [M+H].sup.+.

[0106] Step 6: platinum dioxide (933.33 mg, 4.11 mmol, 0.1 eq) was added to a solution of compound 1-6 (4 g, 40.43 mmol, 1 eq) in EtOH (60 mL) under nitrogen atmosphere at 25° C., and the reaction system was subjected to H2 purging three times; the resulting solution was stirred at 70° C. under H2 (3 MPa) for 72 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was filtered through diatomite, and the resulting filtrate was concentrated under reduced pressure to give compound 1-7. MS (ESI) calcd. for C.sub.21H.sub.29N.sub.3O.sub.4: 387; found: 388 [M+H].sup.+. Step 7: trifluoroacetic acid (12.33 g, 108.14 mmol, 8.00 mL, 10.85 eq) was added into a solution of compound 1-7 (4 g, 10.32 mmol, 1 eq) in DCM (40 mL) at 0° C., and the resulting solution was stirred at 25° C. for 2 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction mixture was concentrated under reduced pressure to give compound 1-8. MS (ESI) calcd. for C.sub.8H.sub.13N.sub.3O: 167; found: 168 [M+H].sup.+.

[0107] Step 8: p-toluenesulfonyl chloride (27.49 g, 144.19 mmol, 1.1 eq), DMAP (1.6 g, 13.11 mmol, 0.1 eq) and triethylamine (19.9 g, 196,66 mmol, 27.37 mL) were added into a solution of compound 1-9 (20 g, 131.08 mmol, 1 eq) in DCM (50 mL) at 25° C., respectively, and the resulting solution was stirred at 25° C. for 16 h; the starting materials were completely reacted as indicated by LCMS; the solvent was removed under reduced pressure, and a saturated solution of NaHCO.sub.3 (50 mL) was added thereto; the resulting mixture was filtered, and the filter cake was washed with water and dried to give compound 1-10. MS (ESI) calcd. for C.sub.14H.sub.11N.sub.2ClSO.sub.2: 306; found: 307 [M+H].sup.+.

[0108] Step 9: a solution of tetrabutylammonium nitrate (29.78 g, 97.81 mmol, 3 eq) in dichloromethane (50 mL) was added dropwise into a solution of compound 1-10 (10 g, 32.60 mmol, 1 eq) in DCM (50 mL) at −5° C., and trifluoroacetic anhydride (20.54 g, 97.79 mmol, 13.60 mL, 3 eq) was slowly added dropwise; the resulting solution was stirred at −5° C. for 30 min, and then stirred at 25° C. for 16 h; the starting materials were completely reacted as indicated by LCMS; ethyl acetate (500 mL×3) was used for extraction, and the organic phases were pooled, washed successively with water (200 mL×2) and brine (200 mL×1), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the residue was recrystallized from dichloromethane to give product 1-11. MS (ESI) calcd. for C.sub.14H.sub.10N.sub.3ClSO.sub.4: 351; found: 352 [M+H].sup.+.

[0109] Step 10: N,N-diisopropylethylamine (15.43 g, 119.40 mmol, 20.8 mL, 10 eq) was added into a solution of compound 1-11 (4.2 g, 11.94 mmol, 1 eq) and compound 1-8 (3.69 g, 13.12 mmol, 1.1 eq) in isopropanol (50 mL) at 25° C., and the resulting solution was stirred at 90° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction mixture was concentrated by rotary evaporation, and water (500 mL) was added; the aqueous phase was extracted with ethyl acetate (500 mL×3), and the organic phases were pooled, washed successively with water (200 mL×2) and brine (200 mL×1), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO.sub.2, DCM:MeOH=100:1 to 50:1) to give compound 1-12. MS (ESI) calcd. for C.sub.22H.sub.22N.sub.6O.sub.5S: 482; found: 483 [M+H].sup.+.

[0110] Step 11: palladium on carbon (0.1 g, 10%) was added into a solution of compound 1-12 in methanol (20 mL) at 25° C. under nitrogen, and the reaction system was subjected to hydrogen purging three times; the resulting solution was stirred at 25° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was filtered through diatomite, and the resulting filtrate was concentrated under reduced pressure to give compound 1-13. MS (ESI) calcd. for C.sub.22H.sub.24N.sub.6O.sub.3S: 452; found: 453 [M+H].sup.+.

[0111] Step 12: compound 1-13 (2 g, 4.42 mmol, 1 eq) was dissolved in formic acid (10 mL), and the resulting solution was stirred at 110° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was concentrated by rotary evaporation to give compound 1-14. MS (ESI) calcd. for C.sub.24H.sub.22N.sub.6O.sub.4S: 490; found: 491 [M+H].sup.+.

[0112] Step 13: 10% aqueous sodium hydroxide solution (10 mL) was added dropwise into a solution of compound 1-14 (1.50 g, 3.06 mmol, 1 eq) in methanol (10 mL) at 25° C., and the resulting solution was stirred at 25° C. for 4 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was neutralized with 10% diluted hydrochloric acid, and the aqueous phase was extracted with dichloromethane (100 mL×3); the organic phases were pooled, washed with saturated brine (100 mL×1), and concentrated by rotary evaporation to give compound 1-15. MS (ESI) calcd. for C.sub.16H.sub.16N.sub.6O: 308; found: 309 [M+H].sup.+.

[0113] Step 14: manganese dioxide (2.83 g, 32.6 mmol, 10 eq) was added into a solution of compound 1-15 (1 g, 3.24 mmol, 1 eq) in a mixed solvent of DCM (20 mL) and MeOH (2 mL) at 25° C., and the resulting solution was stirred at 65° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was filtered through diatomite, and the resulting filtrate was concentrated under reduced pressure to give compound 1-16. MS (ESI) calcd. for C.sub.16H.sub.14N.sub.6O: 306; found: 307 [M+H].sup.+.

[0114] Step 15: hydroxylamine hydrochloride (106.99 mg, 1.66 mmol, 1.2 eq) and sodium acetate (126.29 mg, 1.54 mmol, 1.2 eq) were added into a solution of compound 1-16 (0.39 g, 1.27 mmol, 1 eq) in methanol (15 mL) at 25° C., and the resulting solution was stirred at 25° C. for 0.5 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction mixture was concentrated under reduced pressure, and the resulting residue was diluted with 25 mL of saturated brine, and extracted with THF (25 mL×3); the organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give compound 1-17. MS (ESI) calcd. for C.sub.16H.sub.15N.sub.7O: 321; found: 322 [M+H].sup.+.

[0115] Step 16: thiocarbonyldiimidazole (388.2 mg, 2.18 mmol, 2 eq) was added into a solution of compound 1-17 (0.35 g, 1.09 mmol, 1 eq) in THF (15 mL) at 25° C., and the resulting solution was stirred at 25° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; water (50 mL) was added to the reaction solution, and the aqueous phase was extracted with dichloromethane (100 mL×3); the organic phases were pooled, dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO.sub.2, DCM:MeOH=50:0 to 5:1) to give compound 1-18. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=11.91 (s, 1H), 8.62 (s, 1H), 8.22 (s, 1H), 7.50-7.48 (s, 1H), 6.92-6.88 (m, 1H), 5.53-5.20 (s, 1H), 4.84-4.80 (m, 1H), 4.73-4.67 (m, 1H), 3.34-3.32 (m, 2H), 3.02-2.49 (m, 2H). MS (ESI) calcd. for C.sub.16H.sub.13N.sub.7: 303; found: 304 [M+H].sup.+.

[0116] Step 17: compound 1-18 was subjected to chiral resolution (chiral column: Chiralcel OJ-3 50×4.6 mm I.D., 3 μm; mobile phases: supercritical CO.sub.2 as phase A, and MeOH (0.05% DEA) as phase B; gradient: B content in A from 5% to 40%; flow rate: 3 mL/min; wavelength: 220 nm; column temperature: 35° C.; back pressure: 100 Bar) to give compound 1A (retention time: 0.917 min) and compound 1B (retention time: 2.790 min). Compound 1A: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=11.91 (s, 1H), 8.62 (s, 1H), 8.22 (s, 1H), 7.50-7.48 (s, 1H), 6.92-6.88 (m, 1H), 5.53-5.20 (s, 1H), 4.84-4.80 (m, 1H), 4.73-4.67 (m, 1H), 3.34-3.32 (m, 2H), 3.02-2.49 (m, 2H). C.sub.16H.sub.13N.sub.7: 303; found: 304 [M+H].sup.+.

EXAMPLE 2

Synthesis of Compounds 2A and 2B

[0117] ##STR00025## ##STR00026##

[0118] Step 1: compound 1-13 (2.0 g, 4.40 mmol, 1 eq) was dissolved in acetic acid (20 mL), and the resulting solution was stirred at 120° C. for 15 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction mixture was concentrated by rotary evaporation to give compound 2-1. MS (ESI) calcd. for C.sub.26H.sub.26N.sub.6O.sub.4S: 518; found: 519 [M+H].sup.+.

[0119] Step 2: 10% aqueous sodium hydroxide solution (10 mL) was added dropwise into a solution of compound 2-1 (1.50 g, 2.89 mmol, 1 eq) in methanol (10 mL) at 25° C., and the resulting solution was stirred at 25° C. for 4 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was neutralized with 10% diluted hydrochloric acid, and the aqueous phase was extracted with dichloromethane (100 mL×3); the organic phases were pooled, washed with saturated brine (100 mL×1), and concentrated by rotary evaporation to give compound 2-2. MS (ESI) calcd. for C.sub.17H.sub.18N.sub.6O: 322; found: 323 [M+H].sup.+.

[0120] Step 3: manganese dioxide (2.7 g, 31.0 mmol, 10 eq) was added into a solution of compound 2-2 (1 g, 3.1 mmol, 1 eq) in a mixed solvent of DCM (20 mL) and MeOH (2 mL) at 25° C., and the resulting solution was stirred at 65° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was filtered through diatomite, and the resulting filtrate was concentrated under reduced pressure to give compound 2-3. MS (ESI) calcd. for C.sub.17H.sub.6N.sub.6O: 320; found: 321 [M+H].sup.+.

[0121] Step 4: hydroxylamine hydrochloride (119.3 mg, 1.54 mmol, 1.1 eq) and sodium acetate (192.1 mg, 2.34 mmol, 1.5 eq) were added into a solution of compound 2-3 (0.5 g, 1.56 mmol, 1 eq) in methanol (50 mL) at 25° C., and the resulting solution was stirred at 25° C. for 0.5 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was concentrated under reduced pressure, and the resulting residue was diluted with 25 mL of saturated brine, and extracted with THF (25 mL×3); the organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give compound 2-4. MS (ESI) calcd. for C.sub.17H.sub.17N.sub.7O: 335; found: 336 [M+H].sup.+.

[0122] Step 5: thiocarbonyldiimidazole (371.9 mg, 2.09 mmol, 2 eq) was added into a solution of compound 2-4 (0.35 g, 1.04 mmol, 1 eq) in THF (15 mL) at 25° C., and the resulting solution was stirred at 25° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; water (50 mL) was added into the reaction solution, and the aqueous phase was extracted with dichloromethane (100 mL×3); the organic phases were pooled, dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was subjected to prep-HPLC (column: Phenomenex Gemini-NX 80×40 mm×3 μm; mobile phase: [Water (0.05% NH.sub.3H.sub.2+10 mM NH.sub.4HCO.sub.3)-ACN]; ACN: 17%-37%, 8 min) to give compound 2-5. NMR (400 MHz, DMSO-d.sub.6) δ=11.88 (s, 1H), 8.52 (s, 1H), 7.43 (s, 1H), 6.90 (s, 1H), 6.35-6.34 (m, 1H), 5.36-5.34 (m, 1H), 4.82-4.68 (m, 2H), 3.52-3.37 (m, 2H), 2.69-2.58 (m, 4H), 2.50-2.29 (m, 1H); MS (ESI) calcd. for C.sub.14H.sub.15N.sub.7: 317; found: 318 [M+H].sup.+.

[0123] Step 6: compound 2-5 was subjected to chiral resolution (chiral column: Chiralcel OJ-3 50×4.6 mm I.D., 3 μm; mobile phases: supercritical CO.sub.2 as phase A, and MEOH (0.05% DEA) as phase B; gradient: B content in A from 5% to 40%; flow rate: 3 mL/min; wavelength: 220 nm; column temperature: 35° C.; back pressure: 100 Bar) to give compound 2A (retention time: 3.198 min) and compound 2B (retention time: 3.947 min).

EXAMPLE 3

Synthesis of Compounds 3A and 3B

[0124] ##STR00027## ##STR00028##

[0125] Step 1: compound 1-13 (2 g, 4.40 mmol, 1 eq) was dissolved in trifluoroacetic acid (10 mL), and the resulting solution was stirred at 110° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction mixture was concentrated by rotary evaporation to give compound 3-1. MS (ESI) calcd. for C.sub.26H.sub.20F.sub.6N.sub.6O.sub.4S: 626; found: 627 [M+H].sup.+.

[0126] Step 2: 10% aqueous sodium hydroxide solution (10 mL) was added dropwise into a solution of compound 3-1 (1.50 g, 2.39 mmol, 1 eq) in methanol (10 mL) at 25° C., and the resulting solution was stirred at 25° C. for 4 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was neutralized with 10% diluted hydrochloric acid, and the aqueous phase was extracted with dichloromethane (100 mL×3); the organic phases were pooled, washed with saturated brine (100 mL×1), and concentrated by rotary evaporation to give compound 3-2. MS (ESI) calcd. for C.sub.17H.sub.15F.sub.3N.sub.6O: 376; found: 377 [M+H].sup.+.

[0127] Step 3: manganese dioxide (2.3 g, 27.0 mmol, 10 eq) was added into a solution of compound 3-2 (1 g, 2.7 mmol, 1 eq) in a mixed solvent of DCM (20 mL) and MeOH (2 mL) at 25° C., and the resulting solution was stirred at 65° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was filtered through diatomite, and the resulting filtrate was concentrated under reduced pressure to give compound 3-3. MS (ESI) calcd. for C.sub.17H.sub.13F.sub.3N.sub.6O: 374; found: 375 [M+H].sup.+.

[0128] Step 4: hydroxylamine hydrochloride (111.4 mg, 1.60 mmol, 1.1 eq) and sodium acetate (131.5 mg, 1.60 mmol, 1.5 eq) were added into a solution of compound 3-3 (0.5 g, 1.34 mmol, 1 eq) in methanol (50 mL) at 25° C., and the resulting solution was stirred at 25° C. for 0.5 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; the reaction solution was concentrated under reduced pressure, and the resulting residue was diluted with 25 mL of saturated brine, and extracted with THF (25 mL×3); the organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give compound 3-4. MS (ESI) calcd. for C.sub.17H.sub.14F.sub.3N.sub.7O: 389; found: 390 [M+H].sup.+.

[0129] Step 5: thiocarbonyldiimidazole (320 mg, 1.80 mmol, 2 eq) was added into a solution of compound 3-4 (0.35 g, 0.89 mmol, 1 eq) in THF (15 mL) at 25° C., and the resulting solution was stirred at 25° C. for 16 h; the starting materials were completely reacted and a product was produced, as indicated by LCMS; water (50 mL) was added into the reaction solution, and the aqueous phase was extracted with dichloromethane (100 mL×3); the organic phases were pooled, dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO.sub.2, DCM:MeOH=50:0 to 5:1) to give compound 3-5. MS (ESI) calcd. for C.sub.17H.sub.12F.sub.3N.sub.7O: 371; found: 372 [M+H].sup.+. NMR (400 MHz, DMSO-d.sub.6) δ=12.34 (s, 1H), 8.83 (s, 1H), 7.59-7.58 (m, 1H), 6.96-6.93 (s, 1H), 6.32 (s, 1H), 5.52 (s, 1H), 4.87-4.79 (m, 2H), 3.14-3.02 (m, 2H), 2.73-2.68 (m, 1H), 2.34-2.33 (m, 1H); MS (ESI) calcd. for C.sub.17H.sub.12F.sub.3N.sub.7: 371; found: 372 [M+H].sup.+. Step 6: compound 3-5 was subjected to chiral resolution (chiral column: Chiralcel OJ-3 50×4.6 mm I.D., 3 μm; mobile phases: supercritical CO.sub.2 as phase A, and MEOH (0.05% DEA) as phase B; gradient: B content in A from 5% to 40%; flow rate: 3 mL/min; wavelength: 220 nm; column temperature: 35° C.; back pressure: 100 Bar) to give compound 3A (retention time: 1.166 min) and compound 3B (retention time: 1.339 min).

EXAMPLE 4

Synthesis of Compound 4

[0130] ##STR00029## ##STR00030## ##STR00031##

[0131] Step 1: a solution of n-butyllithium in n-hexane (2.5 M, 427.54 mL) was added dropwise into a solution of tert-butyldimethyl(2-propynyloxy)silane (200 g, 1174.24 mmol) in tetrahydrofuran (2 L) at -78° C. under nitrogen atmosphere, and the reaction solution was stirred at -78° C. for 30 min; a solution of compound 4-1 (250 g, 971.7 mmol) in tetrahydrofuran (2 L) was then added dropwise to the reaction solution at -78° C.; the reaction solution was allowed to react at −78° C. for 3 h; the reaction was completed as indicated by TLC (PE:EA=3:1); the reaction was quenched with saturated aqueous ammonium chloride solution (2 L) and water (1 L), and extracted with EA (2 L×3); the reaction solutions were pooled, washed with saturated brine (2 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure to give compound 4-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=5.11 (br d, J=7.3 Hz, 1H), 4.48 (s, 2H), 4.35-4.27 (m, 1H), 4.21 (q, J=7.2 Hz, 2H), 2.80-2.59 (m, 2H), 2.30-2.13 (m, 1H), 1.98 (br dd, J=6.4, 14.2 Hz, 1H), 1.55-1.42 (s, 9H), 1.36-1.27 (m, 3H), 0.93 (s, 9H), 0.19-0.07 (s, 6H).

[0132] Step 2: hydrazine hydrate (34.71 g, 1.03 mol, 98%) was added into a solution of compound 4-2 (400 g, 935.44 mmol) in DMF (3 L) under ice bath; the reaction was conducted at 25° C. for 2 h; the reaction was completed as indicated by LC-MS; the reaction solution was diluted with water (10 L), and extracted with EA (2 L×2); the reaction solutions were pooled, washed with saturated brine (2 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure to give compound 4-3. MS (ESI) calcd. for C.sub.21H.sub.39N.sub.3O.sub.5Si: 441; found: 442 [M+H].sup.+.

[0133] Step 3: NaBH.sub.4 (77.71 g, 2.05 mol) was added in portions to a solution of compound 4-3 (432 g, 978.18 mmol) in THF (3 L) under ice bath; methanol (0.6 L) was then slowly added dropwise; the reaction mixture was stirred at 25° C. for 12 h, and the reaction was completed as indicated by LC-MS; the reaction was quenched with saturated aqueous ammonium chloride solution (300 mL) under ice bath, and then diluted with water (2 L); the dilution was extracted with EA (2 L×2), and the reaction solutions were pooled, washed with saturated brine (2 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography (SiO.sub.2, DCM:MeOH=50:1 to 20:1) to give compound 4-4. MS (ESI) calcd. for C.sub.19H.sub.37N.sub.3O.sub.4Si: 399; found: 400 [M+H].sup.+.

[0134] Step 4: tributyl phosphine (340.24 g, 1.68 mol) was added into a solution of compound 4-4 (336 g, 840.84 mmol) in tetrahydrofuran (4 L) under ice bath; the reaction solution was stirred under ice bath for 30 min, and ADDP (424.31 g, 1.68 mol) was added to the reaction solution. The reaction solution was stirred at 20° C. for 12 h. The reaction was completed as indicated by LC-MS. The reaction solution was diluted with water (2 L), and extracted with EA (2 L×2); the reaction solutions were pooled, washed with saturated brine (1.5 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography (SiO.sub.2, PE:EA=20:1 to 2:1) to give compound 4-5. MS (ESI) calcd. for C.sub.19H.sub.35N.sub.3O.sub.3Si: 381; found: 382 [M+H].sup.+.

[0135] Step 5: TBAF (1 M, 1.02 L, 1.02 mol) was added into a solution of compound 4-5 (390 g, 1.02 mol) in tetrahydrofuran (1 L) at room temperature, and the reaction was conducted at 20° C. for 1.5 h; the reaction was completed as indicated by LC-MS; the reaction solution was diluted with water (1 L), and the dilution was adjusted to pH=8 with saturated aqueous NaHCO.sub.3 solution and extracted with EA (1 L×3); the reaction solutions were pooled, washed with saturated brine (1 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the concentrate was then dissolved in ethyl acetate (1 L); HC.sub.1/EtOAc (4 M, 200 mL) was slowly added dropwise to the resulting solution, which was then stirred for 1 h, with a white solid forming; filtration was performed to give compound 4-6. MS (ESI) calcd. for C.sub.1-3H.sub.21N.sub.3O.sub.3: 267; found: 268 [M+H].sup.+.

[0136] Step 6: manganese dioxide (37.40 g, 430.19 mmol) was added into a solution of compound 4-6 (11.5 g, 43.02 mmol) in DCM (150 mL) and MeOH (15 mL), which was subjected to nitrogen purging three times and then stirred at 65° C. for 12 h; the reaction was completed as indicated by LC-MS; the reaction solution was filtered, and the filtrate was concentrated to give compound 4-7. MS (ESI) calcd. for C.sub.1-3H.sub.19N.sub.3O.sub.3: 265; found: 266 [M+H].sup.+.

[0137] Step 7: NH.sub.3.H.sub.2O (51.89 g, 414.61 mmol, 57.03 mL, 28% purity) and 1.sub.2 (31.57 g, 124.38 mmol) were added into a solution of compound 4-7 (11 g, 41.46 mmol) in THF (150 mL), and the reaction solution was subjected to nitrogen purging three times and then stirred at 25° C. for 12 h; the reaction was completed as indicated by LC-MS; the reaction was quenched by adding saturated aqueous sodium sulfite solution into the reaction solution, into which 20 mL of water was added for dilution, and the dilution was extracted with ethyl acetate (50 mL×2); the organic phases were pooled, washed with saturated brine (30 mL), dried over sodium sulfate, and filtered; the filtrate was concentrated to give a crude product; the crude product was purified by column chromatography (SiO.sub.2, PE:EA=3:1) to give compound 4-8; MS (ESI) calcd. for C.sub.13H.sub.18N.sub.4O.sub.2: 262; found: 263 [M+H].sup.+.

[0138] Step 8: TMSI (10.91 g, 54.52 mmol, 7.42 mL) was added to a solution of compound 4-8 (11 g, 41.94 mmol) in DCM (150 mL) at 0° C.; the reaction solution was stirred at 0° C. for 1 h; the starting materials disappeared and a new spot occurred as indicated by TLC; the reaction solution was concentrated under reduced pressure to give the hydroiodide salt of compound 4-9. MS (ESI) calcd. for C.sub.8H.sub.10N.sub.4: 162; found: 163 [M+H].sup.+.

[0139] Step 9: DIEA (26.73 g, 206.82 mmol, 36.0 mL) was added to a solution of compound 4-9 (12 g, 41.36 mmol, hydroiodide salt) and compound 1-11 (11.64 g, 33.09 mmol) in isopropanol (200 mL); the reaction solution was subjected to nitrogen purging three times, and then stirred at 90° C. for 12 h; The reaction was completed as indicated by LC-MS. The reaction solution was cooled, and H.sub.2O (200 mL) was added; filtration and drying were performed to give compound 4-10. MS (ESI) calcd. for C.sub.22H.sub.19N.sub.7SO.sub.4: 477; found: 478 [M+H].sup.+.

[0140] Step 10: Fe (9.36 g, 167.54 mmol) and NH.sub.4C.sub.1 (12.55 g, 234.56 mmol) were added into a solution of compound 4-10 (16 g, 33.51 mmol) in THF (200 mL) and H.sub.2O (50 mL), which was subjected to nitrogen purging three times and then stirred at 100° C. for 1 h. The reaction was completed as indicated by LCMS. The reaction solution was filtered, and the filtrate was diluted with H.sub.2O (100 mL); the dilution was then extracted with ethyl acetate (150 mL×2). The filter cake was washed with DCM:MeOH (20:1, 100 mL×3). The extracts and filtrate were pooled, dried over sodium sulfate, and filtered; the filtrate was concentrated to give compound 4-11. MS (ESI) calcd. for C.sub.22H.sub.21N.sub.7SO.sub.2: 447; found: 448 [M+H].sup.+.

[0141] Step 11: tetramethyl orthocarbonate (456.4 mg, 3.35 mmol) was added into a solution of compound 4-11 (150 mg, 335.19 μmop and TsOH (5.8 mg, 33.52 μmop in AcOH (5 mL); the reaction solution was subjected to nitrogen purging three times, and then stirred at 50° C. for 2 h; the reaction was completed as indicated by LCMS; the reaction solution was concentrated to remove the solvent, and the residue was diluted with H.sub.2O (5 mL); the dilution was extracted with dichloromethane (5 mL×3); the organic phases were pooled, washed with saturated brine, dried over sodium sulfate, and filtered; the filtrate was concentrated to give 4-12. MS (ESI) calcd. for C.sub.24H.sub.21N.sub.7O.sub.3S: 487; found: 488 [M+H].sup.+. Step 12: compound 4-12 (180 mg, 369.21 μmol) was dissolved in THF (10 mL), and TBAF (1 M, 738.4 μL) was then added. The reaction solution was subjected to nitrogen purging three times, and stirred at 70° C. for 12 h. The reaction was completed as indicated by LC-MS; the reaction solution was concentrated, and the residue was then diluted with aqueous NaHCO.sub.3 solution (15 mL); the dilution was extracted with DCM (15 mL×3), and the organic phases were pooled, dried over sodium sulfate, and filtered; the filtrate was concentrated to give a crude product. The crude product was subjected to prep-HPLC (Phenomenex Gemini NX 80×30 mm×μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B(ACN) %: 20%-50%, 9 min) to isolate compound 4. MS (ESI) calcd. for C.sub.17H.sub.15N.sub.7O: 333; found: 334 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=11.72 (br s, 1H), 8.38 (br s, 1H), 7.42 (br s, 1H), 6.85 (br s, 1H), 6.71 (br s, 1H), 5.41 (br s, 1H), 4.69 (br s, 2H), 4.09 (br s, 3H), 3.05 (br s, 2H), 2.68 - 2.55 (m, 1H), 2.28 (br s, 1H).

EXAMPLE 5

Synthesis of Compound 5

[0142] ##STR00032## ##STR00033##

[0143] Step 1: compound 4-11 (250 mg, 558.64 μmol, 1 eq), (2R)-2-hydroxypropionic acid (221.41 mg, 1.68 mmol), EDCI (321.28 mg, 1.68 mmol) and HOBt (226.46 mg, 1.68 mmol) were dissolved in THF (10 mL), and DIEA (361.00 mg, 2.79 mmol, 486.53 μL) was then added; the reaction solution was subjected to nitrogen purging three times, and then stirred at 20° C. for 12 h; the reaction was completed as indicated by LC-MS; the reaction solution was diluted with H.sub.2O (15 mL), and the dilution was extracted with EA (15 mL×3); the organic phases were pooled, washed with saturated brine, dried over sodium sulfate, and filtered; the filtrate was concentrated to give compound 5-1. MS (ESI) calcd. for C.sub.27H.sub.27N.sub.7O.sub.5S: 561; found: 562 [M+H].sup.+.

[0144] Step 2: compound 5-1 was dissolved in AcOH (10 mL), and stirred at 120° C. for 0.5 h; the reaction was completed as indicated by LCMS; the reaction solution was diluted with water (15 mL), and extracted with ethyl acetate (15 mL×3); the organic phases were pooled, washed with saturated brine, dried over sodium sulfate, and filtered; the filtrate was concentrated to give compound 5-2. MS (ESI) calcd. for C.sub.27H.sub.25N.sub.7O.sub.4S: 543; found: 544 [M+H].sup.+.

[0145] Step 3: compound 5-2 (0.3 g, 551.88 μmol) was dissolved in MeOH (12 mL) and THF (3 mL), and K.sub.2CO.sub.3 (152.55 mg, 1.10 mmol) was then added. The reaction solution was subjected to nitrogen purging three times, and then stirred at 25° C. for 10 min. The reaction was completed as indicated by LC-MS. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was diluted with H.sub.2O (10 mL); the dilution was then extracted with DCM:MeOH (10:1, 10 mL×3), and the organic phases were pooled, washed with saturated brine, dried over sodium sulfate, and filtered; the filtrate was concentrated to give compound 5-3. MS (ESI) calcd. for C.sub.25H.sub.23N.sub.7O.sub.3S: 501; found: 502 [M+H].sup.+.

[0146] Step 4: compound 5-3 (150 mg, 299.07 μmol) was dissolved in THF (5 mL), and TBAF (1 M, 598.1 μL) was then added; the reaction solution was subjected to nitrogen purging three times, and stirred at 70° C. for 12 h; the reaction was completed as indicated by LC-MS; the reaction solution was concentrated, and then diluted with aqueous NaHCO.sub.3 solution (15 mL); the dilution was extracted with DCM (15 mL×3), and the organic phases were pooled, dried over sodium sulfate, and filtered; the filtrate was concentrated to give a crude product; the crude product was subjected to prep-HPLC (Phenomenex Gemini NX 80×30 mm×3 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; ACN %: 15%-45%, 9 min) to isolate and obtain compound 5. MS (ESI) calcd. for C.sub.18H.sub.17N.sub.7O: 347; found: 348 [M+H].sup.+. NMR (400 MHz, CD.sub.3OD) δ=8.64 (s, 1H), 7.44 (br d, J=2.8 Hz, 1H), 6.74 (s, 1H), 6.35 (br s, 1H), 5.79 (br d, J=10.4 Hz, 1H), 5.41-5.29 (m, 1H), 4.99-4.90 (m, 2H), 4.84-4.76 (m, 1H), 3.31-3.22 (m, 1H), 3.18-3.07 (m, 1H), 3.01-2.87 (m, 1H), 2.37 (br d, J=12.4 Hz, 1H), 1.82 (br d, J=6.3 Hz, 3H).

EXAMPLE 6

Synthesis of Compound 6

[0147] ##STR00034## ##STR00035##

[0148] Step 1: a solution of n-butyllithium in n-hexane (2.5 M, 85.5 mL) was added dropwise into a solution of tert-butyldimethyl(2-propynyloxy)silane (38.07 g, 223.49 mmol, 45.3 mL) in tetrahydrofuran (500 mL) at −78° C. under nitrogen atmosphere, and the reaction solution was stirred at −78° C. for 30 min; a solution of compound 6-1 (50 g, 194.34 mmol) in tetrahydrofuran (500 mL) was then added dropwise to the reaction solution at −78° C.; the reaction solution was allowed to react at −78° C. for 3 h; the reaction was completed as indicated by TLC (PE:EA=3:1); the reaction was quenched with saturated aqueous ammonium chloride solution (500 mL) and water (0.5 L), and extracted with EA (0.5 L×3); the reaction solutions were pooled, washed with saturated brine (1 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure to give compound 6-2.

[0149] Step 2: hydrazine hydrate (7.81 g, 231.52 mmol, 8.81 mL, 95% purity) was added into a solution of compound 6-2 (90 g, 210.47 mmol) dissolved in DMF (700 mL) at 0° C.; the reaction was conducted at 25° C. for 2 h. The reaction was completed as indicated by LC-MS; the reaction solution was diluted with water (1 L), and extracted with EA (1 L×2); the reaction solutions were pooled, washed with H.sub.2O (1 L×3) and saturated brine (1 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure to give compound 6-3. MS (ESI) calcd. for C.sub.21H.sub.39N.sub.3O.sub.5Si: 441; found: 442 [M+H].sup.+.

[0150] Step 3: NaBH.sub.4 (16.01 g, 423.20 mmol) was added in portions to a solution of compound 6-3 (432 g, 978.18 mmol) in THF (800 mL) at 0° C.; methanol (200 mL) was then slowly added dropwise; the reaction solution was stirred at 25° C. for 12 h, and the reaction was completed as indicated by LC-MS; the reaction was quenched with saturated aqueous ammonium chloride solution (300 mL) under ice bath, and then diluted with water (1 L); the dilution was extracted with EA (1 L×2), and the reaction solutions were pooled, washed with saturated brine (1 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography (SiO.sub.2, DCM:MeOH=50:1 to 20:1) to give compound 6-4. MS (ESI) calcd. for C.sub.19H37N.sub.3O.sub.4Si: 399; found: 400 [M+H].sup.+.

[0151] Step 4: tributyl phosphine (68.86 g, 340.34 mmol, 84.0 mL) was added into a solution of compound 6-4 (68 g, 170.17 mmol) in tetrahydrofuran (1.4 L) at 0° C.; the reaction solution was stirred at 0° C. for 30 min, and ADDP (85.87 g, 340.34 mmol) was added to the reaction solution; the reaction solution was stirred at 20° C. for 12 h; the reaction was completed as indicated by LC-MS; the reaction solution was filtered, and the filtrate was diluted with water (1 L); the dilution was extracted with EA (1 L×2), and the reaction solutions were pooled, washed with saturated brine (1.5 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography (SiO.sub.2, PE:EA=20:1 to 2:1) to give compound 6-5. MS (ESI) calcd. for C.sub.19H.sub.35N.sub.3O.sub.3Si: 381; found: 382 [M+H].sup.+.

[0152] Step 5: TBAF (1 M, 230.62 mL) was added into a solution of compound 6-5 (80 g, 209.65 mmol) dissolved in tetrahydrofuran (800 mL) at room temperature, and the reaction was conducted at 20° C. for 0.5 h; the reaction was completed as indicated by LC-MS; the reaction solution was diluted with water (1 L), and the dilution was extracted with EA (1 L×2); the reaction solutions were pooled, washed with saturated brine (1 L), dried over anhydrous sodium sulfate, and filtered; the filtrate was concentrated under reduced pressure to give a crude product; the resulting crude product was dissolved in 800 mL of EA, and 200 mL of HCl/EA was added; the resulting mixture was stirred for 1 h to precipitate 28 g of a crude product; the crude product was dissolved in 250 mL of water, and the resulting solution was adjusted to pH 8 with sodium bicarbonate and then extracted with EA (300 mL×2); the organic phases were pooled and concentrated to give compound 6-6. MS (ESI) calcd. for C.sub.13H.sub.21N.sub.3O.sub.3: 267; found: 268 [M+H].sup.+.

[0153] Step 6: manganese dioxide (71.55 g, 822.97 mmol) was added into a solution of compound 6-6 (22 g, 82.30 mmol, 1 eq) in DCM (300 mL) and MeOH (30 mL), which was subjected to nitrogen purging three times and then stirred at 60° C. for 12 h; the reaction was completed as indicated by LCMS; the reaction solution was filtered, and the filtrate was concentrated to give compound 6-7. MS (ESI) calcd. for C.sub.13H.sub.19N.sub.3O.sub.3: 265; found: 266 [M+H].sup.+.

[0154] Step 7: NH.sub.3H.sub.2O (28.31 g, 226.15 mmol, 31.11 mL, 28% purity) and 1.sub.2 (17.22 g, 67.85 mmol, 13.7 mL) were added into a solution of compound 6-7 (6 g, 22.62 mmol) dissolved in THF (90 mL), and the reaction solution was subjected to nitrogen purging three times and then stirred at 20° C. for 12 h. The reaction was completed as indicated by TLC; the reaction was quenched by adding saturated aqueous sodium sulfite solution into the reaction solution and diluted with 10 mL of water, and the dilution was extracted with ethyl acetate (15 mL×2). The organic phases were pooled, washed with saturated brine (15 mL), dried over sodium sulfate, and filtered; the filtrate was concentrated to give a crude product; the crude product was purified by column chromatography (SiO.sub.2, PE:EA=3:1) to isolate compound 6-8. MS (ESI) calcd. for C.sub.13H.sub.18N.sub.4O.sub.2: 262; found: 263 [M+H].sup.+.

[0155] Step 8: TMSI (9.92 g, 49.56 mmol, 6.8 mL) was added to a solution of compound 6-8 (10 g, 38.12 mmol) in DCM (100 mL) at 0° C.; the reaction solution was stirred at 0° C. for 1.5 h. The reaction was completed as indicated by LCMS. 100 mL EA was added into the reaction solution, which was then filtered to give the hydriodide salt of compound 6-9. MS (ESI) calcd. for C.sub.8H.sub.10N.sub.4: 162; found: 163 [M+H].sup.+.

[0156] Step 9: DIEA (9.36 g, 72.39 mmol, 12.6 mL) was added to a solution of compound 6-9 (7 g, 24.13 mmol, hydroiodide salt), 4-chloro-5-nitro-1-(p-toluenesulfonyl)pyrrolo[2,3 -b]pyridine (8.49 g, 24.13 mmol) dissolved in isopropanol (140 mL). The reaction solution was subjected to nitrogen purging three times, and then stirred at 90° C. for 12 h. The reaction was completed as indicated by LCMS. The reaction solution was cooled, and H.sub.2O (100 mL) was added to precipitate a large amount of yellow solid; filtration and drying were performed to give compound 6-10. MS (ESI) calcd. for C.sub.22H.sub.19N.sub.7SO.sub.4: 477; found: 478 [M+H].sup.+.

[0157] Step 10: Fe powder (8.19 g, 146.60 mmol) and NH.sub.4Cl (10.98 g, 205.24 mmol) were added into a solution of compound 6-10 (14 g, 29.32 mmol) in THF (168 mL) and H.sub.2O (42 mL), which was stirred at 90° C. for 1 h. The reaction was completed as indicated by LC-MS. The reaction solution was filtered, and the filtrate was diluted with H.sub.2O (100 mL); the dilution was then extracted with ethyl acetate (150 mL×2); the filter cake was washed with DCM:MeOH (20:1, 100 mL×3); the extracts and filtrate were pooled, dried over sodium sulfate, and filtered. The filtrate was concentrated to give compound 6-11. MS (ESI) calcd. for C.sub.22H.sub.21N.sub.7SO.sub.2: 447; found: 448 [M+H].sup.+.

[0158] Step 11: tetramethyl orthocarbonate (304.23 mg, 2.23 mmol) was added into a solution of compound 6-11 (100 mg, 223.46 μmop and TsOH (3.85 mg, 22.35 μmop dissolved in AcOH (5 mL). The reaction solution was subjected to nitrogen purging three times, and stirred at 50° C. for 12 h. The reaction was completed as indicated by LC-MS. The reaction solution was concentrated to remove the solvent, and the residue was diluted with H.sub.2O (5 mL); the dilution was extracted with DCM (5 mL×3). The organic phases were pooled, washed with saturated brine (5 mL), dried over sodium sulfate, and filtered; the filtrate was concentrated to give compound 6-12. MS (ESI) calcd. for C.sub.24H.sub.21N.sub.7O.sub.3S: 487; found: 488 [M+H].sup.+.

[0159] Step 12: compound 6-12 (100 mg, 205.11 μmol, 1 eq) was dissolved in THF (5 mL), and TBAF (1 M, 410.2 μL) was then added; the reaction solution was subjected to nitrogen purging three times, and stirred at 70° C. for 12 h. The reaction was completed as indicated by LC-MS; the reaction solution was concentrated, and the residue was then diluted with aqueous NaHCO.sub.3 solution (5 mL); the dilution was extracted with DCM (5 mL×3), and the organic phases were pooled, dried over sodium sulfate, and filtered; the filtrate was concentrated to give a crude product. The crude product was subjected to prep-HPLC (Phenomenex Gemini NX 80×30 mm×3 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B(ACN) %: 20%-50%, 9 min) to isolate compound 6. MS (ESI) calcd. for C.sub.17H.sub.15N.sub.7O: 333; found: 334 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=11.73 (br s, 1H), 8.38 (s, 1H), 7.43 (br s, 1H), 6.85 (s, 1H), 6.71 (br s, 1H), 5.41 (br s, 1H), 4.81-4.61 (m, 2H), 4.09 (s, 3H), 3.05 (br s, 2H), 2.64-2.54 (m, 2H).

EXAMPLE 7

Synthesis of Compound 7

[0160] ##STR00036##

[0161] Step 1: compound 6-11 (150 mg, 335.19 μmop and (2R)-2-hydroxypropionic acid (75.48 mg, 837.97 μmop were dissolved in THF (5 mL), and EDCI (160.64 mg, 837.97 μmol), HOBt (113.23 mg, 837.97 μmop and DIEA (129.96 mg, 1.01 mmol, 175.2 μL) were added; the mixed solution was stirred at 20° C. for 12 h. The reaction was completed as indicated by LC-MS. H.sub.2O (15 mL) was added into the reaction solution, which was then extracted with ethyl acetate (15 mL×3); the organic phases were pooled, washed with saturated brine (15 mL), dried over sodium sulfate, and filtered; the filtrate was concentrated to give compound 7-1. MS (ESI) calcd. for C.sub.25H.sub.25N.sub.7O.sub.4S: 519; found: 520 [M+H].sup.+.

[0162] Step 2: compound 7-1 (200 mg, 384.93 μmop was dissolved in AcOH (4.20 g, 69.94 mmol, 4 mL), and stirred under nitrogen atmosphere at 120° C. for 3 h. The reaction was completed as indicated by LC-MS. The reaction solution was concentrated, and the residue was diluted with aqueous NaHCO.sub.3 solution (5 mL); the dilution was extracted with DCM (5 mL×3), and the organic phases were pooled, washed with saturated brine (15 mL), dried over sodium sulfate, and filtered; the filtrate was concentrated to give compound 7-2. MS (ESI) calcd. for C.sub.25H.sub.23N.sub.7O.sub.3S: 501; found: 502 [M+H].sup.+.

[0163] Step 3: compound 7-2 (200 mg, 398.76 μmop was dissolved in THF (5 mL), and TBAF (1 M, 797.51 μL) was then added; the reaction solution was subjected to nitrogen purging three times, and stirred at 70° C. for 12 h. The reaction was completed as indicated by LC-MS. The reaction solution was concentrated, and the residue was then diluted with aqueous NaHCO.sub.3 solution (15 mL); the dilution was extracted with DCM (15 mL×3), and the organic phases were pooled, dried over sodium sulfate, and filtered; the filtrate was concentrated to give a crude product. The crude product was subjected to prep-HPLC (Phenomenex Gemini NX 80×30 mm×3 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B(ACN) %: 17%-47%, 9 min) to isolate compound 7. MS (ESI) calcd. for C.sub.18H.sub.17N.sub.7O: 347; found: 348 [M+H].sup.+. NMR (400 MHz, DMSO-d.sub.6) δ=11.97 (br s, 1H), 8.62 (s, 1H), 7.60-7.34 (m, 1H), 6.92 (s, 1H), 6.31 (br s, 1H), 5.80 (br d, J=7.0 Hz, 1H), 5.63 (br s, 1H), 5.23 (br s, 1H), 4.88-4.64 (m, 2H), 3.24 (br d, J=12.3 Hz, 1H), 3.09-2.88 (m, 1H), 2.76-2.62 (m, 1H), 2.28 (br s, 1H), 1.67 (br d, J=6.4 Hz, 3H).

Bioactivity Assay

Experimental Example 1

In Vitro Activity Assays of JAM, JAK2, JAK3 and Tyk2 Kinases

Experimental Materials

[0164] Recombinant human JAK1, JAK2, JAK3 and Tyk2 proteases, and main instruments and reagents were all provided by Eurofins, UK.

Experimental Method

[0165] Dilution of JAK2, JAK3 and TYK2: 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 1 mM EDTA, 0.01% Brij-35.5% glycerol, 0.1% β-mercaptoethanol, and 1 mg/mL BSA; dilution of JAK1: 20 mM TRIS, 0.2 mM EDTA, 0.1% β-mercaptoethanol, and 0.01% Brij-35.5% glycerol. All compounds were prepared as 100% DMSO solutions, and the concentration was brought to 50 times the final assay concentration. A test compound was diluted with a 3-fold gradient, with a total of 9 final concentrations ranging from 10 μM to 0.001 μM. The DMSO content in the assay reaction was 2%. The working stock solution of the compound was added into the assay wells as a first component of the reaction, and the remaining components were then added according to the protocol detailed for the assay below.

Reaction of JAK1(h) Enzyme

[0166] JAK1(h) was incubated with 20 mM Tris/HC pH 7.5, 0.2 mM EDTA, 500 μM MGEEPLYWSFPAKKK (SEQ ID NO: 1), 10 mM magnesium acetate and [γ-.sup.33P]-ATP (the activity and concentration were tailored as required). The reaction was started by adding a Mg/ATP mixture, and terminated by adding 0.5% phosphoric acid after 40 min of incubation at room temperature. 10 μL of the reactant was then dotted on a P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, and dried, and scintillation counting was performed.

Reaction of JAK2(h) Enzyme

[0167] JAK2(h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 100 μM KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC (SEQ ID NO: 2), 10 mM magnesium acetate and [γ-.sup.33P]-ATP (the activity and concentration were tailored as required). The reaction was started by adding a Mg/ATP mixture, and terminated by adding 0.5% phosphoric acid after 40 min of incubation at room temperature. 10 μL of the reactant was then dotted on a P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, and dried, and scintillation counting was performed.

Reaction of JAK3(h) Enzyme

[0168] JAK3(h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 500 μM GGEEEEYFELVKKKK (SEQ ID NO: 3), 10 mM magnesium acetate and [γ-.sup.33P]-ATP (the activity and concentration were tailored as required). The reaction was started by adding a Mg/ATP mixture, and terminated by adding 0.5% phosphoric acid after 40 min of incubation at room temperature. 10 μL of the reactant was then dotted on a P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, and dried, and scintillation counting was performed.

Reaction of TYK2(h) Enzyme

[0169] TYK2(h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM GGMEDIYFEFMGGKKK (SEQ ID NO:4), 10 mM magnesium acetate and [γ-.sup.33P]-ATP (the activity and concentration were tailored as required). The reaction was started by adding a Mg/ATP mixture, and terminated by adding 0.5% phosphoric acid after 40 min of incubation at room temperature. 10 μL of the reactant was then dotted on a P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, and dried, and scintillation counting was performed.

Data Analysis

[0170] The IC50 results were obtained by analysis using XLFITS (formula 205) of IDBS company and detailed in Table 1.

TABLE-US-00001 TABLE 1 Results of in vitro screening assays of the compounds of the present application JAK1 JAK2 JAK3 TYK2 Compound (IC.sub.50, nM) (IC.sub.50, nM) (IC.sub.50, nM) (IC.sub.50, nM) 1A 1.3 27.0 30.8 35.8 2A <1 2 25 2 3A 3 62 217 57 4 0.9 7.0 27.3 50.3 5 9 250 576 231 6 2.3 70.2 73.8 130 7 0.3 53.9 38.4 12.4

Experimental Example 2

Pharmacokinetic (PK) Experiment

[0171] A test compound was dissolved [5% DMSO, 95% (12% SBE-β-CD)] to give a clear solution, which was administered into male rats (SD) (overnight fasting, 7-8 weeks old) by tail vein injection and by gavage, respectively. After administration of the test compound, blood was collected from the mandibular vein and centrifuged to obtain plasma at 0.117, 0.333, 1, 2, 4, 7 and 24 h in the tail vein injection group (1 mg/kg), and at 0.25, 0.5, 1, 2, 4, 8 and 24 h in the gavage group (5 mg/kg). The plasma concentration was measured using LC-MS/MS method, and the relevant pharmacokinetic parameters were calculated using WinNonlin™ Version 6.3 pharmacokinetic software with non-compartmental model linear logarithmic trapezoidal method. The test results were as follows:

TABLE-US-00002 TABLE 2-1 PK test results of compounds 1A and 4 in rats PK parameters Compound 1A Compound 4 T.sub.1/2 (hr) 1.35 3.65 C.sub.max (nM) 2944 10152 AUC.sub.0 − .sub.inf (nM .Math. hr) 8509 67786 Bioavailability (%) 41.8% 119% Note: T.sub.1/2: half-life; C.sub.max: peak concentration; AUC.sub.0 − .sub.inf: area under plasma concentration-time curve from time 0 extrapolated to infinite time.

Experimental Example 3

In Vivo Efficacy Study of Adjuvant-Induced Arthritis (AIA) in Rats

[0172] Experimental procedures: the compounds of the present application were tested for their therapeutic effect on arthritis using a rat adjuvant arthritis model. Female Lewis rats weighing 160-180 g were anesthetized with isoflurane and injected with 0.1 mL of Mycobacterium tuberculosis suspension subcutaneously in the left hind paw. 13 days after the modeling, the rats were divided into groups and given corresponding test compounds. For example, the female Lewis rats were orally administered different doses (see Table 3-2 for specific doses) of a solution of test compound 1A in a mixed solvent [5% DMSO, 95% (12% SBE-β-CD)] twice a day (10 test animals in each dose group). During two consecutive weeks of administration, the states of the rats were observed, and the foot volume swellings were recorded and scored. The scoring criteria were shown in Table 3-1.

TABLE-US-00003 TABLE 3-1 Clinical scoring criteria for arthritis Score Clinical symptoms 0 No erythema and swelling with redness 1 Erythema or mild swelling with redness near the tarsal bones or at the ankle or metatarsus, or erythema and swelling with redness at one toe 2 Mild erythema and swelling at the ankle and metatarsus, swelling with redness and erythema at two or more toes 3 Moderate erythema and swelling at the ankle, wrist, and metatarsus 4 Severe swelling with redness at all of the ankle, wrist, metatarsus and toes

[0173] Experimental results: the two doses treatment groups of compound 1A show significant relieving effect on the downward trend of the weight in animals caused by the disease attack, and the low and medium dose groups (3, 10 mg/kg) show significant difference from the solvent control group from day 20, showing good weight recovery effect. Compound 1A has inhibited the increase in arthritis clinical score and foot volume, and the inhibition is dose-dependent. Compound 1A shows the most significant effect at 10 mg/kg (significant difference from the solvent control group from day 15). The mean arthritis clinical score for this group decreases from a peak of 5.8 at day 13 to 1.3 at day 27 at the experimental endpoint.

TABLE-US-00004 TABLE 3-2 Inhibition rate of area under clinical score curve* Compound Dose (mg/kg) AUC (%) Solvent control group 0 0 Compound 1A 3 49.8 10 60.3 *Note: P < 0.05 for each group compared to the solvent control group (one-way analysis of variance).

Experimental Example 4

In Vivo Efficacy Study of Collagen-Induced Arthritis (CIA) in Rats

Experimental Procedures:

[0174] The compounds of the present application were tested for their therapeutic effect on arthritis using a rat collagen-induced arthritis model. Lewis rats were immunized. The day of the first immunization was recorded as day 0, and the subsequent days were recorded in order. The Lewis rats were anesthetized with isoflurane and injected with 50 μL of prepared collagen emulsion (containing 200 μg of CII) subcutaneously in the tail (2-3 cm away from the base of the tail). On day 21, the same volume of the collagen emulsion was injected in the tail in the same manner. Rats in the normal group were not immunized. On day 27 of the modeling, the rats were divided into groups and administered corresponding test compounds. For example, the female Lewis rats were orally administered different doses (see Table 4-1 for specific doses) of a solution of test compound 4 in a mixed solvent [0.5% HPMC E5/0.5% PVP K30/0.2% SLS in water] twice a day (8 test animals in each dose group). During 14 consecutive days of administration, the states of the rats were observed, and the foot volume swellings were recorded and scored. The scoring criteria were shown in Table 3-1.

Experimental Results:

[0175] Compound 4 reduces the clinical scores of the arthritic rats in a dose-dependent manner when being administered BID at the doses of 1 and 3 mg/kg, and shows significant difference from the solvent control group. By the end of the administration at day 14, the 3 mg/kg BID group reduces the clinical scores of the rats to zero (Table 4-1). Meanwhile, the degree of the foot volume swelling is also reduced in a dose-dependent manner, and by the end of the administration at day 14, the 3 mg/kg BID group shows significant difference from the solvent control group in that it reduces the foot volume swelling of the rat to 1.28 μL. The body weight in the treatment group also recovers in a dose-dependent manner, and by the end of the administration at day 14, the body weight in the 3 mg/kg BID dose group recovers to the level in the normal group.

TABLE-US-00005 TABLE 4-1 Main parameters for in vivo efficacy study of CIA in rats* Clinical score Foot volume (μL, (on day 14 on day 14 of Dose of administration) administration) Compound (mg/kg) Mean ± SEM Mean ± SEM Normal 0 0 1.298 ± 0.022 group Solvent 0 13.20 ± 0.82  2.096 ± 0.064 control group Dexa- 0.1 0.00 ± 0.00 1.224 ± 0.042 methasone group Compound 1 0.38 ± 0.16 1.324 ± 0.040 4 3 0.00 ± 0.00 1.280 ± 0.012 *Note: P < 0.001 for each group compared to the solvent control group (two-way analysis of variance).