USE OF JAK INHIBITORS IN PREPARATION OF DRUGS FOR TREATING JAK KINASE-RELATED DISEASES

Abstract

Disclosed is the use of a JAK inhibitor [1,2,4]-triazolo-[1,5-a]0pyridine compound in the preparation of drugs for treating autoimmune, inflammatory or allergic diseases, or diseases such as transplant rejection. The JAK inhibitor [1,2,4]-triazolo-[1,5-a]-pyridine compound comprises a compound as shown in formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof. The JAK inhibitor has a good efficacy in animal model tests of diseases such as autoimmune, inflammatory or allergic diseases.

Claims

1. A method for the treatment of autoimmune diseases, inflammatory diseases, allergic diseases, or graft-versus-host diseases, comprising administering to a patient in need thereof a JAK inhibitors [1,2,4]triazolo [1,5-a]pyridine compound of formula (I), isomers, or pharmaceutically acceptable salts thereof: ##STR00062## wherein, E.sub.1 and E.sub.2 are independently selected from single bond, —CH.sub.2— or —(CH.sub.2).sub.2—, respectively; L.sub.1 is selected from single bond, —(CH.sub.2).sub.g—, —C(═O)— or —C(═O)—(CH.sub.2).sub.h—; m is 1 or 2; n is 1 or 2; g is 1, 2 or 3; h is 1, 2 or 3; R.sub.1 is selected from H, CN, C.sub.1-6 alkyl or 3- to 6-membered cycloalkyl, wherein the C.sub.1-6 alkyl and 3- to 6-membered cycloalkyl are optionally substituted by 1, 2 or 3 R.sub.a; R.sub.2 is selected from H, F, Cl, Br, I or C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 R.sub.b; R.sub.3, R.sub.4 and R.sub.5 are independently selected from H, F, Cl, Br, I or C.sub.1-3 alkyl, respectively, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 Re; R.sub.6, R.sub.7 and R.sub.8 are independently selected from H, F, Cl, Br, I or C.sub.1-3 alkyl, respectively, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 R.sub.d; Each R.sub.a is independently selected from H, F, Cl, Br, I, CN or C.sub.1-3 alkyl, respectively, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 R; Each R.sub.b is independently selected from F, Cl, Br or I, respectively; Each R.sub.c is independently selected from F, Cl, Br or I, respectively; Each R.sub.d is independently selected from F, Cl, Br or I, respectively; and Each R is independently selected from F, Cl, Br or I, respectively.

2. The method according to claim 1, wherein the autoimmune diseases comprise systemic lupus erythematosus, psoriasis, psoriatic arthritis or lupus nephritis; the inflammatory diseases include inflammatory bowel disease, ankylosing spondylitis or primary cholangitis; and the allergic diseases include allergic dermatitis, contact dermatitis, allergic purpura or bronchial asthma.

3. The method according to claim 2, wherein the autoimmune disease is systemic lupus erythematosus.

4. The method according to claim 2, wherein the autoimmune disease is psoriasis.

5. The method according to claim 2, wherein the allergic disease is allergic dermatitis.

6. The method according to claim 1, wherein the graft-versus-host disease comprises anti acute rejection, anti-chronic rejection or induction of immune tolerance.

7. The method according to claim 2, wherein the inflammatory disease is inflammatory bowel disease.

8. The method according to claim 1, wherein each R.sub.a in the compounds of formula (I) is independently selected from H, F, Cl, Br, I or CN, respectively.

9. The method according to claim 1, wherein R.sub.1 in the compounds of formula (I) is selected from H, CN, C.sub.1-3 alkyl or 3- to 5-membered cycloalkyl, wherein the C.sub.1-3 alkyl and 3- to 5-membered cycloalkyl are optionally replaced by 1, 2 or 3 R.sub.a.

10. (canceled)

11. The method according to claim 24, wherein R.sub.1 in the compounds of formula (I) is selected from H, CN, CF.sub.3, CHF.sub.2, ##STR00063##

12. The method according to claim 1, wherein R.sub.2 in the compounds of formula (I) is selected from H, F, Cl, Br or I.

13. The method according to claim 1, wherein R.sub.3, R.sub.4 and R.sub.5 in the compounds of formula (I) are independently selected from H, F, Cl, Br or I, respectively.

14. The method according to claim 1, wherein R.sub.6, R.sub.7 and R.sub.8 in the compounds of formula (I) are independently selected from H, F, Cl, Br or I, respectively.

15. The method according to claim 1, wherein L.sub.1 in the compounds of formula (I) is selected from single bond, —CH.sub.2—, —(CH.sub.2).sub.2—, —C(═O)— or —C(═O)—(CH.sub.2)—.

16. The method according to claim 1, wherein the structural unit ##STR00064## in the compounds of formula (I) is selected from ##STR00065##

17. The according to claim 1, wherein the structural unit ##STR00066## in the compounds of formula (I) is selected from ##STR00067##

18. The method according to claim 1, wherein the structural unit ##STR00068## in the compounds of formula (I) is selected from ##STR00069## ##STR00070##

19. The method according to claim 1, wherein the compounds of formula (I), their isomers or their pharmaceutically acceptable salts are selected from ##STR00071##

20. The method according to claim 19, wherein the compounds of formula (I), their isomers or their pharmaceutically acceptable salts are selected from ##STR00072##

21. The method according to claim 1, wherein the compounds of formula (I), their isomers or their pharmaceutically acceptable salts are selected from: ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##

22. The method according to claim 21, wherein the compounds of formula (I), their isomers or their pharmaceutically acceptable salts are selected from: ##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##

23. A pharmaceutical composition, comprising a therapeutically effective amount of compounds of formula (I) according to claim 1 as an active ingredient, an isomer or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

24. The method according to claim 9, wherein R.sub.1 in the compounds of formula (I) is selected from H, CN, CH.sub.3, ##STR00085## wherein the CH.sub.3, ##STR00086## are optionally replaced by 1, 2 or 3 R.sub.a.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0112] FIG. 1: curve diagram of DAI scoring results in experimental example 3.

[0113] FIG. 2: curve diagram of weight change results of mice in experimental example 3.

[0114] FIG. 3: histogram of colon length in experimental example 3.

[0115] FIG. 4: histogram of spleen index in experimental example 3.

[0116] FIG. 5: curve diagram of skin score results in experimental example 4.

[0117] FIG. 6: right ear thickness histogram in experimental example 4.

[0118] FIG. 7: histogram of spleen index in experimental example 4.

[0119] FIG. 8: histogram of serum lgE concentration in experimental example 4.

[0120] FIG. 9: curve diagram of weight change of mice in experimental example 4.

DETAILED DESCRIPTION

[0121] The present invention will be described in detail below by way of examples, but does not imply any adverse limitation to the present invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. It will be apparent to those skilled in the art to make various changes and improvements to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

Example 1

[0122] ##STR00035## ##STR00036##

[0123] Step 1: LiHMDS (1.0 M, 51.2 mL) was added dropwise into THF (150 mL) solution containing Compound 1-1 (10.2 g, 42.6 mmol) at −78° C., then the mixture was stirred at −78° C. for 1 hour, THF (150 mL) solution of 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl) methanesulfonamide (16.7 g, 46.9 mmol) was added to the reaction solution, and then stirred at 15° C. for 12 hours. TLC (PE:EA=10:1) showed that raw materials were completely consumed and new points were generated. The reaction solution was quenched with 250 mL of saturated ammonium chloride, diluted with 200 mL of water, and extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed with saturated salt water, dried with sodium sulfate, then filtered and concentrated to obtain Compound 1-2. The crude product was directly used for the next reaction without purification.

[0124] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.63 (br s, 1H), 3.50-3.65 (m, 4H), 2.34 (br s, 4H), 1.88 (br t, J=5.90 Hz, 2H), 1.37 (s, 9H).

[0125] Step 2: a DMF (100 mL) solution containing Compound 1-2 (16.0 g, 43.1 mmol) and pinacol borate (12.0 g, 47.4 mmol) was added with potassium acetate (12.7 g, 129.3 mmol) and Pd (dppf)Cl.sub.2CH.sub.2Cl.sub.2 (3.5 g, 4.3 mmol), replaced with nitrogen for 3 times and stirred at 70° C. in nitrogen atmosphere for 3 hours. TLC showed that raw materials were consumed completely and new points were generated. The reaction solution was dispersed in a mixture of 300 mL of water and 400 mL of ethyl acetate. The organic phase was separated and washed with saturated salt water, dried with sodium sulfate, then filtered and concentrated to obtain a crude product. The crude product was purified by silica gel chromatography to obtain Compound 1-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ6.46 (br s, 1H), 3.71-3.53 (m, 4H), 2.31 (br d, J=3.0 Hz, 2H), 2.24-2.16 (m, 2H), 1.74 (t, J=6.3 Hz, 2H), 1.44 (s, 9H), 1.26 (s, 12H).

[0126] Step 3: potassium carbonate (3.8 g, 27.3 mmol) and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (744 mg, 911.0 μmol) were added to a dioxane (60 mL) and water (15 mL) solution containing Compound 1-3 (3.5 g, 10.0 mmol) and N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl) cyclopropane formamide (2.6 g, 9.1 mmol) in nitrogen atmosphere. The reaction solution was stirred at 90° C. for 3 hours. LCMS showed that the raw materials were completely consumed and the target molecular ion peak was monitored. The reaction solution was concentrated, and the obtained crude product was separated and purified by column chromatography to obtain Compound 1-4. LCMS (ESI) m/z: 424.3[M+H].sup.+.

[0127] Step 4: hydrochloric acid/ethyl acetate (4.0 M, 30 mL) was added to a dichloromethane (10 mL) solution containing Compound 1-4 (3.5 g, 8.2 mmol), and the mixture was stirred at 25° C. for 0.5 hours. LCMS showed that the raw material was consumed and the target molecular ion peak was monitored. The solid was precipitated, then filtered and dried to obtain Compound 1-5 (3.3 g hydrochloride, crude product), which was not purified and used for the next reaction directly. LCMS (ESI) m/z: 324.1[M+H].sup.+.

[0128] Step 5: Pd/C (1.0 g, 10%) was added to a methanol (100 mL) solution containing Compound 1-5 (3.0 g, 8.34 mmol, hydrochloride) in nitrogen atmosphere. The suspension was replaced with hydrogen for 3 times and then stirred for 12 hours at 30° C. in hydrogen atmosphere (30 psi). LCMS showed that the raw material was consumed and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain Compound 1-6 (3.0 g hydrochloride, crude product), which was not purified and used for the next reaction directly. LCMS (ESI) m/z: 326.2 [M+H].sup.+.

[0129] Step 6: Compound 1-6 (0.87 g, 2.40 mmol, hydrochloride) was dissolved in N, N-dimethylformamide (10 mL), HOBt (487 mg, 3.6 mmol,) and EDCI (691 mg, 3.6 mmol) were added, and then (1S)-2,2-difluorocyclopropyl formic acid (323 mg, 2.6 mmol) and diisopropyl ethylamine (621 mg, 4.8 mmol) were added into the reaction solution. The reaction solution was reacted at 15° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was concentrated under reduced pressure, and a residue was subjected to preparative HPLC (neutral system) to obtain Compound 1-13: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ7.32-7.73 (m, 2H), 6.95 (br s, 1H), 3.62-4.22 (m, 4H), 3.45 (br s, 1H), 3.18-3.37 (m, 1H), 2.61 (br s, 1H), 1.45-2.27 (m, 10H), 0.78-1.17 (m, 4H). LCMS (ESI) m/z: 430.0[M+H].sup.+.

[0130] Compound 1-6 was used as the common intermediate, the following compounds were obtained by the same synthesis and separation method as compound 1-13 (i.e. replacing the carboxylic acid of compound 1-13 with the corresponding carboxylic acid in the following target molecules in the acid amine condensation reaction step). The characterization data are as follows:

##STR00037## ##STR00038##

[0131] Compound 1-7: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ11.08 (br s, 1H), 7.48-7.78 (m, 2H), 7.03 (d, J=7.0 Hz, 1H), 3.86-4.25 (m, 2H), 3.61-3.80 (m, 2H), 3.29-3.38 (m, 1H), 2.69-2.88 (m, 1H), 1.85-2.19 (m, 7H), 1.51-1.79 (m, 4H), 0.83-0.96 (m, 4H). LCMS (ESI) m/z: 430.0[M+H].sup.+.

[0132] Compound 1-8: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.14 (br s, 1H), 7.52-7.66 (m, 2H), 7.00 (d, J=7.03 Hz, 1H), 3.54-3.83 (m, 6H), 3.29 (br t, J=11.54 Hz, 1H), 1.94-2.09 (m, 5H), 1.41-1.70 (m, 4H), 0.77-0.90 (m, 4H). LCMS (ESI) m/z: 393.1[M+H].sup.+.

[0133] Compound 1-9: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.99 (br s, 1H), 7.49-7.65 (m, 2H), 6.99 (br d, J=7.03 Hz, 1H), 4.02-4.20 (m, 2H), 3.61-3.78 (m, 2H), 1.94-2.13 (m, 5H), 1.48-1.72 (m, 4H), 1.14-1.32 (m, 4H), 0.77-0.87 (m, 4H). LCMS (ESI) m/z: 412.1[M+H].sup.+.

[0134] Compound 1-10: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.77-7.87 (m, 1H), 7.62 (d, J=8.78 Hz, 1H), 7.20 (dd, J=7.28, 11.80 Hz, 1H), 4.08 (s, 1H), 3.96 (s, 1H), 3.83 (s, 1H), 3.72 (s, 1H), 3.43-3.56 (m, 1H), 3.22 (dq, J=6.90, 10.75 Hz, 2H), 2.06-2.23 (m, 4H), 1.94 (br s, 1H), 1.56-1.84 (m, 3H), 1.56-2.00 (m, 1H), 0.94-1.14 (m, 4H). LCMS (ESI) m/z: 436.1[M+H].sup.+.

[0135] Compound 1-11: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.56-7.67 (m, 1H), 7.50 (d, J=8.78 Hz, 1H), 7.00 (t, J=7.15 Hz, 1H), 4.26-4.48 (m, 2H), 3.70-3.90 (m, 2H), 3.42-3.59 (m, 1H), 2.08-2.24 (m, 4H), 1.48-1.99 (m, 9H), 0.87-1.10 (m, 4H). LCMS (ESI) m/z: 419.1[M+H].sup.+.

[0136] Compound 1-12: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.56-7.64 (m, 1H), 7.48 (d, J=9.03 Hz, 1H), 6.97 (d, J=7.28 Hz, 1H), 3.91-4.17 (m, 2H), 3.78-3.86 (m, 1H), 3.67-3.75 (m, 1H), 3.40-3.54 (m, 1H), 2.53-2.69 (m, 1H), 1.92-2.21 (m, 6H), 1.72-1.85 (m, 3H), 1.50-1.69 (m, 2H), 0.86-1.08 (m, 4H). LCMS (ESI) m/z: 430.1[M+H].sup.+.

[0137] Compound 1-14: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.57-7.66 (m, 1H), 7.50 (d, J=8.78 Hz, 1H), 6.95-7.03 (m, 1H), 6.01-6.38 (m, 1H), 4.62 (s, 1H), 3.65-4.10 (m, 4H), 3.43-3.58 (m, 1H), 2.76-2.93 (m, 2H), 2.04-2.21 (m, 4H), 1.51-1.87 (m, 4H), 1.01-1.07 (m, 2H), 0.93 (qd, J=3.74, 7.34 Hz, 2H). LCMS (ESI) m/z: 418.1[M+H].sup.+.

[0138] Compound 1-15: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.01 (br s, 1H), 7.48-7.66 (m, 2H), 7.00 (dd, J=7.53, 9.79 Hz, 1H), 4.11-4.33 (m, 2H), 3.60-3.81 (m, 2H), 3.25-3.32 (m, 1H), 2.03 (br t, J=9.03 Hz, 5H), 1.53-1.73 (m, 4H), 1.49 (d, J=4.77 Hz, 6H), 0.76-0.88 (m, 4H). LCMS (ESI) m/z: 421.1[M+H].sup.+.

##STR00039##

[0139] Synthesis of compound 1-16: Compound 1-6 (100 mg, 227.6 μmol, TFA) was dissolved in N,N-dimethylformamide (5 mL), potassium carbonate (94 mg, 682.7 μmol) and 2-bromoacetonitrile (30 mg, 250.3 μmol) were added, and it was stirred at 10° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was diluted with water (5 mL), and extracted with dichloromethane/methanol (10/1, 10 mL). The organic phase was washed with saturated salt water (10 mL), dried with anhydrous sodium sulfate, then filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (neutral system) to obtain Compound 1-16. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.83 (t, J=8.03 Hz, 1H), 7.64 (br d, J=8.78 Hz, 1H), 7.21 (d, J=7.53 Hz, 1H), 4.51 (s, 2H), 4.23 (s, 2H), 4.08 (s, 2H), 3.49 (br t, J=11.92 Hz, 1H), 2.14-2.30 (m, 4H), 1.79-1.97 (m, 3H), 1.59-1.74 (m, 2H), 0.95-1.12 (m, 4H). LCMS (ESI) m/z: 365.0[M+H].sup.+.

[0140] Compounds 1-6 was used as a common intermediate, the following compounds were obtained by the same synthesis and separation methods as for Compound 1-16 (replacing bromoacetonitrile with corresponding bromopropiononitrile in the target molecule).

[0141] Compound 1-17: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ11.00 (br s, 1H), 7.50-7.63 (m, 2H), 6.96 (d, J=6.27 Hz, 1H), 3.33-3.34 (m, 2H), 3.23-3.30 (m, 1H), 2.95 (s, 2H), 3.05 (s, 2H), 2.58-2.69 (m, 2H), 1.99 (br d, J=10.29 Hz, 5H), 1.40-1.65 (m, 4H), 0.74-0.88 (m, 4H). LCMS (ESI) m/z: 379.0[M+H].sup.+.

Example 2

[0142] ##STR00040## ##STR00041##

[0143] Step 1: tert butyl 9-oxygen-3-azaspiro[5.5] undecane-3-carboxylic acid (3-1) (5 g, 18.7 mmol) was dissolved in anhydrous tetrahydrofuran (150 mL) at −78° C. under the protection of nitrogen, dropped with bis(trimethylsilyl) lithium amino (1 M, 22.4 mL) slowly, and reaction solution was stirred at −78° C. for 1 hour. Then, the reaction solution was added with anhydrous tetrahydrofuran (50 mL) solution of 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl) sulfonyl) methanesulfonamide (7.35 g, 20.6 mmol), and the reaction solution was stirred at 15° C. for 12 hours. TLC showed the reaction was complete. The reaction solution was quenched with saturated ammonium chloride (50 mL) and extracted with ethyl acetate (200 mL*2). The combined organic phases were washed with saturated salt water (50 mL), dried with anhydrous sodium sulfate, then filtered and concentrated under reduced pressure to obtain Compound 3-2, which was directly used in the next step without purification.

[0144] Step 2: Compound 3-2 (8 g, 20.0 mmol) and pinacol diboroate (5.59 g, 22.0 mmol) were dissolved in N, N-dimethylformamide (100 mL), potassium acetate (5.90 g, 60.1 mmol) and [1,1-bis (diphenylphosphine)ferrocene]palladium dichloride dichloromethane (1.64 g, 2.0 mmol) were added, and stirred at 70° C. for 3 hours. TLC showed the reaction was complete. The reaction solution was diluted with water (300 mL) and extracted with ethyl acetate (200 mL*2). The combined organic phases were washed with saturated salt water (150 mL), dried with anhydrous sodium sulfate, then filtered and concentrated under reduced pressure. The residue was separated by rapid silica gel column (0˜10% ethyl acetate/petroleum ether) to obtain compound 3-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ6.41 (br s, 1H), 6.34-6.47 (m, 1H), 3.32-3.44 (m, 2H), 3.14-3.29 (m, 2H), 2.00-2.10 (m, 2H), 1.90 (br d, J=3.01 Hz, 2H), 1.38 (s, 9H), 1.28 (br t, J=5.52 Hz, 4H), 1.19 (s, 12H).

[0145] Step 3: under the protection of nitrogen, N-(5-bromo-[1,2,4]triazole[1,5-a] pyridin-2-yl) cyclopropylformamide (2 g, 7.1 mmol) was dissolved in mixed solution of dioxane (40 mL) and water (10 mL), Compound 3-3 (3.49 g, 9.3 mmol), potassium carbonate (2.95 g, 21.3 mmol), and [1,1-bis (diphenylphosphine) ferrocene] palladium dichloride dichloromethane (581 mg, 711.5 μmol) were added into the solution. It was replaced with nitrogen for 3 times, and heated to 90° C. to react for 3 hours. LC-MS showed the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was separated by rapid silica gel column (0˜4% methanol/dichloromethane) to obtain Compound 3-4. LCMS (ESI) m/z: 452.4[M+H].sup.+.

[0146] Step 4: Compound 3-4 (3.5 g, 7.8 mmol) was dissolved in dichloromethane (15 mL), hydrochloric acid/ethyl acetate (4 M, 30 mL) was added, and the reaction solution was stirred at 20° C. for 30 minutes. LC-MS showed the reaction was complete. The solid was precipitated, filtered and dried to obtain Compounds 3-5. LCMS (ESI) m/z: 352.2[M+H].sup.+.

[0147] Step 5: under the protection of N2, Compound 3-5 (2.9 g, 7.4 mmol, hydrochloride) was dissolved in methanol (100 mL) solution, catalyst dry palladium/carbon (1 g, 10%) was added, and the reaction solution was replaced with hydrogen for 3 times. The reaction solution was stirred for 12 hours under hydrogen pressure (30 psi) and reaction temperature of 25° C. LC-MS showed the reaction was complete. The solid was filtered using diatomite and the filtrate was concentrated under reduced pressure to obtain Compound 3-6 (2.6 g hydrochloride). LCMS (ESI) m/z: 354.7[M+H].sup.+.

[0148] Step 6: Compound 3-6 (1 g, 2.6 mmol, hydrochloride) was dissolved in N, N-dimethylformamide (20 mL), HOBt (573 mg, 4.2 mmol) and EDCI (813 mg, 4.2 mmol) were added, and then (ES)-2,2-difluorocyclopropyl carboxylic acid (380 mg, 3.1 mmol) and diisopropyl ethylamine (731 Mg, 5.7 mmol) were added. The reaction solution was reacted at 15° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was diluted with water (100 mL), and extracted with dichloromethane/methanol (10/1, 150 mL*2). The combined organic phase was washed with saturated salt water (100 mL), dried with anhydrous sodium sulfate, then filtered and concentrated under reduced pressure. The residue was subjected to a preparative HPLC (neutral system) to obtain Compound 3-7.

[0149] .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.57-7.64 (m, 1H), 7.48 (d, J=8.78 Hz, 1H), 7.02 (d, J=7.28 Hz, 1H), 3.63-3.77 (m, 3H), 3.41-3.61 (m, 2H), 2.93 (dt, J=8.28, 11.80 Hz, 1H), 1.36-2.06 (m, 15H), 1.04 (quin, J=3.76 Hz, 2H), 0.93 (qd, J=3.66, 7.34 Hz, 2H). LCMS (ESI) m/z: 458.1[M+H].sup.+.

[0150] Compound 3-6 was used as a common intermediate, the following compounds were obtained by the same synthesis and separation method as Compound 3-7 (i.e. replacing the carboxylic acid of compound 3-7 with the corresponding carboxylic acid in the following target molecules in the acid amine condensation reaction step),

##STR00042##

[0151] The characterization data are as follows:

[0152] Compound 3-8: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ7.57-7.67 (m, 1H), 7.49 (d, J=8.53 Hz, 1H), 7.03 (dd, J=3.76, 6.78 Hz, 1H), 4.61 (s, 1H), 3.83-3.91 (m, 1H), 3.62 (td, J=3.76, 7.53 Hz, 2H), 3.42-3.54 (m, 3H), 1.68-2.08 (m, 9H), 1.40-1.56 (m, 4H), 1.05 (quin, J=3.76 Hz, 2H), 0.88-0.97 (m, 2H). LCMS (ESI) m/z: 421.1[M+H].sup.+.

[0153] Compound 3-9: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.61 (dd, J=7.28, 8.78 Hz, 1H), 7.49 (d, J=8.78 Hz, 1H), 7.02 (dd, J=4.52, 6.78 Hz, 1H), 3.63 (td, J=3.83, 7.40 Hz, 2H), 3.43-3.58 (m, 5H), 1.67-2.07 (m, 9H), 1.39-1.54 (m, 4H), 1.01-1.08 (m, 2H), 0.93 (qd, J=3.68, 7.28 Hz, 2H). LCMS (ESI) m/z: 464.1[M+H].sup.+.

Example 3

[0154] ##STR00043## ##STR00044##

[0155] Step 1: 5-bromo-[1,2,4]triazole[1,5-a]pyridin-2-amino(4-1)(5 g, 23.5 mmol) was dissolved in acetonitrile (50 mL) at 0° C., triethylamine (11.87 g, 117.4 mmol) and cyclopropyl formyl chloride (6.13 g, 58.7 mmol) were added, and reaction solution reacted at 25° C. for 12 hours. TLC showed the reaction was complete. The acetonitrile was removed by vacuum concentration, and the residue was separated by rapid silica gel column (0-5% methanol/dichloromethane) to obtain Compound 4-2. LCMS (ESI) m/z: 350.8[M+H].sup.+.

[0156] Step 2: under the protection of N2, Compound 4-2 (1.99 g, 5.7 mmol) and Compound 1-1 (1.5 g, 6.3 mmol) were dissolved in anhydrous tetrahydrofuran (30 mL), n-butyl lithium (2.5 m, 5.7 mL) solution was added at −70° C. slowly, and reaction solution was stirred at 10° C. for 30 minutes. LC-MS showed the reaction was complete. The reaction solution was quenched with saturated ammonium chloride (50 mL) at 0° C., and extracted with ethyl acetate (150 mL*2). The combined organic phases were washed with saturated salt water (10 mL), dried with anhydrous sodium sulfate, then filtered and concentrated under reduced pressure. The residue was separated by rapid silica gel column (0-3% methanol/dichloromethane) to obtain Compound 4-3. LCMS (ESI) m/z: 442.3[M+H].sup.+.

[0157] Step 3: Compound 4-3 (0.8 g, 1.81 mmol) was dissolved in anhydrous dichloromethane (10 mL) at 0° C., diethylamino sulfur trifluoride (DAST) (351 mg, 2.17 mmol) was added and reacted at 0° C. for 15 minutes, and then heated to 25° C. to react for 1 hour. LC-MS showed the reaction was complete. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution (5 mL) at 0° C., diluted with water (10 mL), and extracted with dichloromethane (50 mL*3). The combined organic phases were washed with saturated salt water (20 mL), dried with anhydrous sodium sulfate, then filtered and concentrated under reduced pressure. The residue was separated by rapid silica gel column (0-100% ethyl acetate/petroleum ether) to obtain Compound 4-4. LCMS (ESI) m/z: 444.3[M+H].sup.+.

[0158] Step 4: Compound 4-4 (410 mg, 924.4 μmol) was dissolved in dichloromethane (5 mL), hydrochloric acid/ethyl acetate (4 M, 10 mL) was added, and reaction solution reacted at 20° C. for 30 minutes. LC-MS showed the reaction was complete. The solid was precipitated, filtered and dried to obtain Compound 4-5 (390 mg hydrochloride). LCMS (ESI) m/z: 344.2[M+H].sup.+.

[0159] Step 5: Compound 4-5 (130 mg, 342.2 μmol, hydrochloride) was dissolved in N,N-dimethylformamide (10 mL), HOBt (77 mg, 567.9 μmol) and EDCI (109 mg, 567.9 μmol) were added, then 2-cyanoacetic acid (35 mg, 416.4 μmol) and diisopropyl ethylamine (98 mg, 757.1 μmol) were added, and reaction solution reacted at 15° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was concentrated under reduced pressure and the residue was subjected to preparative HPLC (neutral condition) to obtain Compounds 4-6. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.66-7.72 (m, 1H), 7.56-7.62 (m, 1H), 7.25 (t, J=7.28 Hz, 1H), 4.29 (s, 1H), 4.03 (s, 1H), 3.97 (s, 1H), 3.76 (s, 1H), 3.26-3.30 (m, 2H), 2.97-3.28 (m, 2H), 1.74-2.08 (m, 7H), 0.89-1.13 (m, 4H). LCMS (ESI) m/z: 411.1[M+H].sup.+.

[0160] Compound 4-5 was used as the common intermediate, Compounds 4-7 and 4-8 were prepared by the same synthesis and separation method of acid amine condensation as Compound 4-6 (added with carboxylic acid compounds with different substitutions from Compound 4-6). The characterization data of compounds 4-7 and 4-8 are as follows:

##STR00045##

[0161] Compound 4-7: .sup.1H NR (400 MHz, METHANOL-d.sub.4) δ=7.65-7.73 (m, 1H), 7.59 (dt, J=1.13, 9.72 Hz, 1H), 7.20-7.29 (m, 1H), 4.33 (s, 1H), 4.01 (d, J=11.29 Hz, 2H), 3.76 (s, 1H), 2.99-3.30 (m, 4H), 1.74-2.10 (m, 7H), 0.88-1.12 (m, 4H). LCMS (ESI) m/z: 454.1[M+H].sup.+.

[0162] Compound 4-8: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ=7.64-7.73 (m, 1H), 7.59 (dt, J=1.25, 9.16 Hz, 1H), 7.20-7.28 (m, 1H), 6.01-6.44 (m, 1H), 4.30 (s, 1H), 3.99 (d, J=11.80 Hz, 2H), 3.73 (s, 1H), 2.99-3.27 (m, 2H), 2.76-2.99 (m, 2H), 1.75-2.10 (m, 7H), 0.89-1.10 (m, 4H). LCMS (ESI) m/z: 436.1[M+H].sup.+.

Example 4

[0163] ##STR00046## ##STR00047##

[0164] Step 1: THF (8 mL) solution of Compound 5-1 (0.15 g, 592.1 μmol) was dropped with LiHMDS (1 M, 770 μL) at −78° C. The mixture was stirred at −78° C. for 1 hour. The reaction solution was added with tetrahydrofuran (4 mL) solution of 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl) methanesulfonamide (233 mg, 651 μmol) dropwise at −78° C. μmol) and stirred at 15° C. for 12 hours. TLC (PE:EA=5:1) showed that the reaction of raw materials was complete and new points were formed. The reaction was quenched with 10 mL of saturated ammonium chloride solution, then 20 mL of water was added, and extract with ethyl acetate (30 mL*3). The organic phases were combined and washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, then filtered and concentrated to obtain Compound 5-2, which was directly used in the next reaction without purification.

[0165] Step 2: DMF (10 mL) solution of Compound 5-2 (0.25 g, 648.7 μmol) and pinacol borate (165 mg, 648.7 μmol) was added with KOAc (191 mg, 2.0 mmol) and Pd (dppf)Cl.sub.2 (48 mg, 64.9 μmol). The reaction solution was stirred at 70° C. for 12 hours. TLC (PE:EA=5:1) showed that the reaction of raw materials was complete, and new points were detected. The reaction solution was added with 20 mL of water and extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, and then it was separated and purified by column chromatography (SiO.sub.2, PE:EA=50:0-20:1) to obtain colorless oily Compound 5-3. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ6.50 (br s, 1H), 3.35-3.49 (m, 2H), 3.07-3.15 (m, 2H), 2.02-2.22 (m, 4H), 1.54-1.81 (m, 4H), 1.47 (s, 9H), 1.27 (s, 12H).

[0166] Step 3: Compound 5-3 (0.13 g, 357.8 μmol) was dissolved in dioxane (4 mL) and water (1 mL) solution, N-(5-bromo-[1,2,4]triazolo[1,5-a] pyridin-2-yl) cyclopropane formamide (101 mg, 357.8 μmol), K.sub.2CO.sub.3 (149 mg, 1.1 mmol), Pd(dppf)Cl.sub.2 (26 mg, 35.8 μmol) were added, it was replaced with nitrogen for 3 times. The mixture was stirred at 90° C. for 12 hours in a nitrogen atmosphere. LCMS showed that the raw materials were consumed and a target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent, then dispersed in 10 mL of water and extracted with DCM/MeOH (10:1, 30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, and filtered, with the filtrate being distilled under reduced pressure. Compound 5-4 was purified by silica gel chromatography (SiO.sub.2, DCM:MeOH=1:0 to 20:1). LCMS (ESI) m/z: 438.3[M+H].sup.+.

[0167] Step 4: methanol (10 mL) solution of Compound 5-4 (0.2 g, 457.1 μmol) was added with Pd/C (10%, 50 mg) in argon atmosphere. It was replaced with hydrogen 3 times and then stirred at 25° C. in hydrogen atmosphere (15 psi) for 2 hours. LCMS showed that the raw material was consumed and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain Compound 5-5, which was directly used in the next step without purification. LCMS (ESI) m/z: 440.4[M+H].sup.+.

[0168] Step 5: Compound 5-5 (150 mg, 341.3 μmol) and trifluoroacetic acid (4 mL) were dissolved in dichloromethane (10 mL), it was replaced with nitrogen for 3 times, and then the reaction solution was stirred at 25° C. for 30 minutes. LCMS showed that the raw material was consumed and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent to obtain Compound 5-6 (0.15 g, trifluoroacetate), which was directly used in the next step without purification. LCMS (ESI) m/z: 340.2[M+H].sup.+.

[0169] Step 6: DMF (4 mL) solution of (1S)-2,2-difluorocyclopropyl formic acid (44 mg, 360.7 μmol) was added with EDCI (104 mg, 541.1 μmol), HOBt (73 mg, 541.1 μmol) and DIEA (140 mg, 1.1 mmol, 189 μL). It was stirred at 25° C. for 5 minutes, then Compound 5-6 (122 mg, 270 μmol, trifluoroacetate) was added, and it was stirred at 25° C. for 16 hours. LCMS showed that the raw materials were consumed and the target molecular ion peak was monitored. Crude product was obtained by preparative HPLC (neutral separation condition, chromatographic column: Waters XBridge 150 mm*25 mm 5 μm; Mobile phase: [H.sub.2O (10 mM NH.sub.4HCO.sub.3)-ACN]; B (CH.sub.3CN) %: 25%-55%, 7 min) and SFC chiral separation (chromatographic column: DAICEL CHIRALCEL OD-H (250 mm*30 mm, 5 μm); Mobile phase: [0.1% NH.sub.3H.sub.2O EtOH]; B (C.sub.02)%: 40%) to obtain Compound 5-7, SFC retention time: 3.685 min. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.48-7.55 (m, 1H), 7.39 (d, J=8.78 Hz, 1H), 6.92 (dd, J=6.90, 3.39 Hz, 1H), 3.35-3.76 (m, 5H), 2.66-2.94 (m, 1H), 1.51-2.10 (m, 13H), 0.94 (br s, 2H), 0.78-0.88 (m, 2H). LCMS (ESI) m/z: 444.1[M+H].sup.+. Compound 5-8, SFC retention time: 4.283 min. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.48-7.59 (m, 1H), 7.40 (br d, J=8.53 Hz, 1H), 6.94 (br d, J=6.78 Hz, 1H), 3.23-3.78 (m, 5H), 2.65-2.81 (m, 1H), 1.54-2.06 (m, 13H), 0.95 (br s, 2H), 0.77-0.87 (m, 2H). LCMS (ESI) m/z: 444.2[M+H].sup.+.

[0170] Compound 5-6 was used as a common intermediate, the following compounds were prepared by the same synthesis and separation method of acid amine condensation as Compound 5-7 (added with carboxylic acids different from Compound 5-7). The characterization data are as follows:

##STR00048##

[0171] Compound 5-9: It was separated by preparative HPLC (neutral separation condition, chromatographic column: Waters XBridge 150 mm*25 mm 5 μm; Mobile phase: [H.sub.2O (10 mM NH.sub.4HCO.sub.3)-ACN]; B (CH.sub.3CN) %: 18%-32%, 9 min), retention time 2.117 min. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.60-7.68 (m, 1H), 7.52 (d, J=8.6 Hz, 1H), 7.05 (dd, J=3.4, 6.8 Hz, 1H), 3.44-3.73 (m, 4H), 3.31 (br s, 2H), 2.11 (td, J=7.6, 14.9 Hz, 3H), 2.01 (br t, J=7.3 Hz, 1H), 1.96 (br s, 1H), 1.66-1.88 (m, 6H), 1.31 (br s, 1H), 1.02-1.10 (m, 2H), 0.91-0.99 (m, 2H). LCMS (ESI) m/z: 407.2[M+H].sup.+.

[0172] Compound 5-10: SFC chiral separation conditions, chromatographic column: DAICEL CHIRALCEL OD-H (250 mm*30 mm, 5 μm); Mobile phase: [0.1% NH.sub.3H.sub.2O EtOH]; B(CO.sub.2)%: 40%-40%, retention time 4.114 min. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ=7.52 (br d, J=8.0 Hz, 1H), 7.40 (br d, J=8.8 Hz, 1H), 6.89-6.98 (m, 1H), 3.57 (t, J=7.0 Hz, 1H), 3.25-3.51 (m, 6H), 1.78-2.09 (m, 5H), 1.51-1.76 (m, 6H), 0.94 (br d, J=3.8 Hz, 2H), 0.79-0.88 (m, 2H). LCMS (ESI) m/z: 450.2[M+H].sup.+.

Example 5

[0173] ##STR00049## ##STR00050##

[0174] Step 1: THF (8 mL) solution of Compound 6-1 (250 mg, 986.8 μmol) was added with LiHMDS (1 M, 1.3 mL) dropwise. The mixture was stirred at −78° C. for 1 hour. The reaction solution was added with tetrahydrofuran (4 mL) solution of 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl) methanesulfonamide (388 mg, 1.1 mmol) dropwise at −78° C., and then stirred at 25° C. for 12 hours. TLC (PE:EA=5:1) showed that the reaction of raw materials were complete and new points were formed. The reaction was quenched with 10 mL of saturated ammonium chloride solution, then 20 mL of water was added and extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, then filtered and concentrated to obtain the crude product, and it was separated and purified by column chromatography (SiO.sub.2, PE:EA=20:1-10:1) to obtain Compound 6-2.

[0175] Step 2: DMF (5 mL) solution of Compound 6-2 (386 mg, 1.0 mmol) and pinacol borate (254 mg, 1.0 mmol) was added with KOAc (295 mg, 3.0 mmol) and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (82 mg, 100 μmol). The reaction solution was stirred at 70° C. for 12 hours. TLC (PE:EA=5:1) showed that the reaction of raw material was complete, and new points were detected. The solution was added with 20 mL of water and extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, then filtered and concentrated to obtain the crude product, and then separated and purified by column chromatography (SiO.sub.2, PE:EA=50:0-20:1) to obtain Compound 6-3.

[0176] Step 3: Compound 6-3 (186 mg, 512 μmol) was dissolved in dioxane (4 mL) and water (1 mL) solution, N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl) cyclopropane formamide (144 mg, 512 μmol), K.sub.2CO.sub.3 (212 mg, 1.5 mmol), and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (42 mg, 51.2 μmol) were added, it was replaced with nitrogen for 3 times. The mixture was stirred at 90° C. for 12 hours in nitrogen atmosphere. LCMS showed that the raw materials were consumed and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent, then dispersed in 10 mL of water and extracted with DCM:MeOH (10:1, 30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, and filtered. The filtrate was distilled under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (SiO.sub.2, DCM:MeOH=1:0-20:1) to obtain Compound 6-4. LCMS (ESI) m/z: 438.7[M+H].sup.+.

[0177] Step 4: Methanol (10 mL) solution of Compound 6-4 (196 mg, 448 μmol) was added with Pd/C (10%, 50 mg). It was replaced with hydrogen for three times and stirred for 16 hours at 25° C. in a hydrogen atmosphere (15 psi). LCMS showed that the raw materials were consumed and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain Compound 6-5, which was directly used in the next step without purification. LCMS (ESI) m/z: 440.3[M+H].sup.+.

[0178] Step 5: Compound 6-5 (130 mg, 296 μmol) and trifluoroacetic acid (4 mL) were dissolved in dichloromethane (10 mL) solution, it was replaced with nitrogen for three times, and then the reaction solution was stirred at 25° C. for 30 minutes. LCMS showed that the raw materials were consumed and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent to obtain Compound 6-6 (134 mg, trifluoroacetate), which was directly used in the next step reaction without purification. LCMS (ESI) m/z: 340.2[M+H].sup.+.

[0179] Step 6: DMF (4 mL) solution of (1S)-2,2-difluorocyclopropyl formic acid (36 mg, 295.5 μmol) was added with EDCI (85 mg, 443.3 μmol), HOBt (60 mg, 443.3 μmol), and DIEA (115 mg, 886.5 μmol, 154.4 μL), it was stirred at 25° C. for 5 minutes, then Compound 6-6 (134 mg, 295.5 μmol, trifluoroacetate) was added, and the mixture was stirred at 25° C. for 16 hours. LCMS showed that the raw materials were consumed and the target molecular ion peak was monitored. Crude product was subjected to preparative separation (neutral condition. Waters Xbridge 150*25 5 μm; Mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN];B %: 30%-50%, 7 min) and SFC chiral separation (chromatographic column: YMC CHIRAL Amylose-C (250 mm*30 mm, 10 μm; Mobile phase: [0.1% NH.sub.3H.sub.2O EtOH];B %: 50%). Compound 6-7 was obtained. SFC retention time: 2.339 min. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.01 (br s, 1H), 7.51-7.63 (m, 2H), 7.08 (br d, J=6.78 Hz, 1H), 3.83 (br s, 1H), 3.41-3.66 (m, 4H), 3.15 (br d, J=5.02 Hz, 1H), 2.14-2.35 (m, 2H), 2.06 (br s, 1H), 1.75-1.95 (m, 4H), 1.40-1.73 (m, 6H), 0.84 (br s, 4H). LCMS (ESI) m/z: 444.1[M+H].sup.+. Compound 6-8: SFC retention time: 4.142 min. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.01 (br s, 1H), 7.54-7.65 (m, 2H), 7.08 (br s, 1H), 3.80-3.90 (m, 1H), 3.45-3.66 (m, 4H), 3.09-3.22 (m, 1H), 2.18-2.36 (m, 2H), 2.06 (br s, 1H), 1.75-1.95 (m, 4H), 1.68 (br d, J=7.28 Hz, 3H), 1.50 (br d, J=4.77 Hz, 3H), 0.77-0.88 (m, 4H). LCMS (ESI) m/z: 444.1[M+H].sup.+.

Example 6

[0180] ##STR00051## ##STR00052##

[0181] Step 1: THF (8 mL) solution of Compound 7-1 (0.3 g, 1.33 mmol) was added with LiHMDS (1 M, 1.7 mL) dropwise at −78° C. The mixture was stirred at −78° C. for 1 hour, tetrahydrofuran (4 mL) solution of 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl) methanesulfonamide (523 mg, 1.46 mmol) was added dropwise, and then the mixture was stirred at 25° C. for 12 hours. TLC (PE:EA=5:1) showed that the reaction of raw materials was complete and new points were formed. The reaction solution was quenched with saturated ammonium chloride (10 mL) solution, then 20 mL of water was added and it was extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, and then separated and purified by column chromatography (SiO.sub.2, PE:EA=20:1-10:1) to obtain Compound 7-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.72 (s, 1H), 3.86-3.92 (m, 2H), 3.76-3.82 (m, 2H), 2.53-2.61 (m, 2H), 2.18-2.25 (m, 2H), 1.37 (s, 9H).

[0182] Step 2: DMF (5 mL) solution of Compound 7-2 (0.46 g, 1.3 mmol) and pinacol borate (327 mg, 1.3 mmol) was added with KOAc (379 mg, 3.9 mmol) and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (105 mg, 128.7 μmol). The reaction solution was stirred at 70° C. for 12 hours. TLC (PE:EA=5:1) showed that the reaction of raw materials was complete, and new points were detected. The reaction solution was quenched with 20 mL of water and extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, and then it was separated and purified by column chromatography (SiO.sub.2, PE:EA=50:0-20:1) to obtain Compound 7-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.45 (t, J=1.88 Hz, 1H), 3.83-3.88 (m, 2H), 3.73-3.78 (m, 2H), 2.36-2.42 (m, 2H), 2.04 (t, J=7.03 Hz, 2H), 1.37 (s, 9H), 1.21 (s, 12H).

[0183] Step 3: Compound 7-3 (0.15 g, 447.43 μmol) was dissolved in dioxane (4 mL) and water (1 mL) solution, N-(5-bromo-[1,2,4]triazolo[1,5-a] pyridin-2-yl) cyclopropane formamide (126 mg, 447.43 μmol), K.sub.2CO.sub.3 (186 mg, 1.34 mmol), and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (37 mg, 44.7 μmol) were added, it was replaced with nitrogen for 3 times. The mixture was stirred at 90° C. for 12 hours in a nitrogen atmosphere. LCMS showed that the raw materials were completely consumed and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent, then dispersed in 10 mL of water and extracted with DCM:MeOH (10:1, 30 mL*3). The organic phases were combined, washed with saturated salt water (40 mL), dried with anhydrous sodium sulfate, and filtered. The filtrate was distilled under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography (SiO.sub.2, DCM:MeOH=1:0-20:1) to obtain Compound 7-4. LCMS (ESI) m/z: 410.2[M+H].sup.+.

[0184] Step 4: Methanol (10 mL) solution of Compound 7-4 (0.15 g, 366.3 μmol) was added with Pd/C (10%, 0.05 g). The mixture was replaced with hydrogen for three times and stirred in a hydrogen atmosphere (15 psi) at 25° C. for 16 hours. LCMS showed that the raw materials were completely consumed and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain Compound 7-5, which was directly used in the next step without purification. LCMS (ESI) m/z: 412.2[M+H].sup.+.

[0185] Step 5: Compound 7-5 (0.13 g, 315.9 μmol) and trifluoroacetic acid (4 mL) were dissolved in dichloromethane (10 mL) solution, it was replaced with nitrogen for three times, and then the reaction solution was stirred at 25° C. for 30 minutes. LCMS showed that the raw materials were completely consumed and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent to obtain Compound 7-6 (130 mg, trifluoroacetate), which was directly used in the next step without purification. LCMS (ESI) m/z: 312.1[M+H].sup.+.

[0186] Step 6: DMF (4 mL) solution of (1S)-2,2-difluorocyclopropyl formic acid (37 mg, 305.6 μmol) was added with EDCI (88 mg, 458.4 μmol), HOBt (62 mg, 458.4 μmol), DIEA (119 mg, 916.8 μmol, 160 μL). The mixture was stirred at 25° C. for 5 minutes, then Compound 7-6 (0.13 g, 305.6 μmol, trifluoroacetate) was added, and it was stirred at 25° C. for 16 hours. LCMS showed that the raw materials were completely consumed and the target molecular ion peak was monitored. The crude product was subjected to preparative HPLC separation (chromatographic column: Waters Xbridge 150*25 5 μm; Mobile phase: [H.sub.2O (10 mM NH.sub.4HCO.sub.3)-ACN]; B (CH.sub.3CN) %: 20%-50%, 7 min) and chiral separation (chromatographic column: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 μm; Mobile phase, A %: (0.1% NH.sub.3H.sub.2O EtOH); B(CO.sub.2)%: (40%-40%) to obtain Compound 7-7, SFC retention time: 3.714 min. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=9.59 (br d, J=12.05 Hz, 1H), 7.34-7.57 (m, 2H), 6.70-6.84 (m, 1H), 4.06-4.21 (m, 2H), 3.92-3.99 (m, 1H), 3.74-3.92 (m, 2H), 1.81-2.38 (m, 8H), 1.59 (dtd, J=11.36, 7.62, 7.62, 3.76 Hz, 1H), 1.08-1.24 (m, 2H), 0.79-0.96 (m, 2H). LCMS (ESI) m/z: 416.0[M+H].sup.+.

[0187] Compounds 7-8 was isolated and the SFC retention time was 4.468 min. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=9.48 (br s, 1H), 7.34-7.54 (m, 2H), 6.76 (br d, J=7.03 Hz, 1H), 4.03-4.25 (m, 2H), 3.73-3.99 (m, 3H), 2.50 (ddd, J=16.81, 13.18, 8.16 Hz, 1H), 1.80-2.40 (m, 8H), 1.53-1.66 (m, 1H), 1.05-1.23 (m, 2H), 0.80-0.95 (m, 2H). LCMS (ESI) m/z: 416.0[M+H].sup.+.

Example 7

[0188] ##STR00053## ##STR00054##

[0189] Step 1: under the protection of nitrogen Compound 8- (1.11 g 5.6 mmol) and Compound 1-3 were dissolved in dioxane (40 mL) and water (10 mL) solution, potassium carbonate (2.16 g, 15.6 mmol) and [1,1-bis (diphenylphosphine) ferrocene]palladium dichloride dichloromethane (425 mg, 520.6 mol) were added, it was replaced with nitrogen for 3 times, and reaction solution was heated to 90° C. for 3 hours. LC-MS showed the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was subjected to rapid silica gel column separation (0˜4% methanol/dichloromethane) to obtain Compound 8-2. LCMS (ESI) m/z: 356.3[M+H].sup.+.

[0190] Step 2: under the protection of nitrogen, Compound 8-2 (2 g, 5.6 mmol) was dissolved in methanol (100 mL) solution, catalyst dry palladium/carbon (0.5 g, 10%) was added, and it was replaced with hydrogen for 3 times. Under hydrogen pressure (30 psi) and reaction temperature of 30° C., the reaction solution was stirred for 12 hours. LC-MS showed that 50% of raw materials were remained. The catalyst was filtered out and replaced with new catalyst dry palladium/carbon (1 g). The reaction continued for 3 hours, and LCMS showed that the reaction was complete. The solid was filtered by diatomite and the filtrate was concentrated under reduced pressure to obtain Compound 8-3. LCMS (ESI) m/z: 358.2[M+H].sup.+.

[0191] Step 3: (1R)-2,2-difluorocyclopropyl carboxylic acid (282 mg, 2.3 mmol) was dissolved in pyridine (10 mL), EDCI (4.0 g, 21.0 mmol) and Compound 8-3 (0.75 g, 2.1 mmol) were added, and reaction solution was stirred at 10° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was diluted with water (30 mL), extracted with dichloromethane/methanol (10/1, 50 mL*3), washed with saturated salt water (30 mL), dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to rapid silica gel column separation (0-3% methanol/dichloromethane), and then purified by beating with ethyl acetate to obtain Compound 8-4. LCMS (ESI) m/z: 462.3[M+H].sup.+.

[0192] Step 4: Compound 8-4 (300 mg, 650.1 μmol) was dissolved in dichloromethane (5 mL), hydrochloric acid/ethyl acetate (4 M, 10 mL) was added, and reacted at 15° C. for half an hour. LC-MS showed the reaction was complete. The reaction solution was concentrated to obtain Compound 8-5 (hydrochloride). LCMS (ESI) m/z: 362.2[M+H].sup.+.

[0193] Step 5: Compound 8-5 (100 mg, 251.4 μmol, HCl) was dissolved in N,N-dimethylformamide (5 mL), HOBt (51 mg, 377.0), EDCI (72.28 mg, 377.0 μmol), (1S)-2,2-difluorocyclopropyl carboxylic acid (34 mg, 276.5 μmol) and diisopropyl ethylamine (65 mg, 502.7 μmol) were added, and reaction solution reacted at 15° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was concentrated under reduced pressure and the residue was subjected to preparative HPLC (neutral condition) to obtain Compound 8-6. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ7.59-7.67 (m, 1H), 7.51 (d, J=8.78 Hz, 1H), 7.01 (br d, J=7.53 Hz, 1H), 3.92-4.20 (m, 2H), 3.79-3.88 (m, 1H), 3.67-3.77 (m, 1H), 3.43-3.57 (m, 1H), 2.81 (br s, 1H), 2.62 (dq, J=7.78, 11.96 Hz, 1H), 2.07-2.24 (m, 5H), 1.52-2.05 (m, 7H). LCMS (ESI) m/z: 466.2[M+H].sup.+.

[0194] Compound 8-5 was used as a common intermediate, Compounds 8-7 and 8-8 were prepared by the same synthesis and separation method as Compound 8-6 by acid amine condensation (added with carboxylic acids substituted differently from Compound 8-6). The characterization data of Compounds 8-7 and 8-8 are as follows:

##STR00055##

[0195] Compounds 8-7, crude product was purified by preparative HPLC (neutral condition). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.59-7.66 (m, 1H), 7.51 (d, J=8.78 Hz, 1H), 6.97-7.04 (m, 1H), 4.30 (d, J=4.27 Hz, 1H), 4.18 (d, J=4.27 Hz, 1H), 3.87 (s, 1H), 3.76 (s, 1H), 3.49 (br t, J=11.80 Hz, 1H), 2.81 (br s, 1H), 2.05-2.28 (m, 5H), 1.74-1.96 (m, 3H), 1.53-1.70 (m, 2H), 1.23-1.33 (m, 4H). LCMS (ESI) m/z: 448.2[M+H].sup.+.

[0196] Compounds 8-8, crude product was purified by preparative HPLC (neutral condition). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ7.59-7.67 (m, 1H), 7.51 (d, J=8.78 Hz, 1H), 7.00 (t, J=7.40 Hz, 1H), 4.61 (s, 2H), 3.69-4.11 (m, 4H), 3.41-3.54 (m, 1H), 2.82 (br s, 1H), 2.04-2.26 (m, 5H), 1.72-1.93 (m, 3H), 1.61 (q, J=11.80 Hz, 2H). LCMS (ESI) m/z: 429.0[M+H].sup.+.

##STR00056##

[0197] Synthesis of compound 8-9: intermediate 8-5 (100 mg, 227.6 μmol, TFA) was dissolved in N,N-dimethylformamide (5 mL), potassium carbonate (94 mg, 682.7 μmol) and 2-bromoacetonitrile (30 mg, 250.3 μmol) were added, and it was stirred at 10° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was diluted with water (5 mL), and extracted with dichloromethane/methanol (10/1, 10 mL). The organic phase was washed with saturated salt water (10 mL), dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (neutral condition) to obtain Compound 8-9. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.59-7.66 (m, 1H), 7.50 (d, J=8.78 Hz, 1H), 6.99 (d, J=7.53 Hz, 1H), 3.62 (s, 2H), 3.46 (br t, J=12.05 Hz, 1H), 3.33 (s, 2H), 3.21 (s, 2H), 2.80 (br s, 1H), 2.14 (br d, J=9.79 Hz, 5H), 1.82-1.95 (m, 1H), 1.52-1.77 (m, 4H). LCMS (ESI) m/z: 401.0[M+H].sup.+.

Example 8

[0198] ##STR00057##

[0199] Step 1: (S)-2,2-difluorocyclopropyl carboxylic acid (1.13 g, 9.2 mmol) was dissolved in pyridine (150 mL), EDCI (16.1 g, 84 mmol) and Compound 8-3 (3 G, 8.4 mmol) were added, and reaction solution was stirred at 10° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was diluted with water (100 mL), extracted with dichloromethane/methanol (10/1, 100 mL*3), washed with saturated salt water (30 mL), dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to rapid silica gel column separation (0-3% methanol/dichloromethane), and then purified by beating with ethyl acetate to obtain Compound 9-1. LCMS (ESI) m/z: 462.3[M+H].sup.+.

[0200] Step 2: Compound 9-1 (2.3 g, 4.9 mmol) was dissolved in dichloromethane (5 mL), hydrochloric acid/ethyl acetate (4 M, 20 mL) was added, and it was stirred at 15° C. for half an hour. LC-MS showed that the reaction was complete and the target molecular ion peak was detected. The precipitated solid was filtered and dried to obtain Compound 9-2 (hydrochloride). LCMS (ESI) m/z: 362.2[M+H].sup.+.

[0201] Step 3: Compound 9-2 (1.23 g, 3.1 mmol, HCl) was dissolved in N,N-dimethylformamide (20 mL), HOBt (626 mg, 4.6 mmol) and EDCI (889 mg, 4.6 mmol) were added, then (1S)-2,2-difluorocyclopropyl carboxylic acid (414.92 mg, 3.40 mmol) and diisopropyl ethylamine (798.70 mg, 6.18 mmol) were added. The reaction solution was stirred at 15° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was diluted with water (10 mL), and extracted with dichloromethane/methanol (10/1, 50 mL). The organic phase was washed with saturated salt water (10 mL), dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to preparative HPLC (neutral condition) to obtain Compound 9-3. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.63 (dd, J=7.53, 8.78 Hz, 1H), 7.51 (d, J=8.78 Hz, 1H), 7.01 (br d, J=7.28 Hz, 1H), 3.92-4.19 (m, 2H), 3.79-3.87 (m, 1H), 3.67-3.76 (m, 1H), 3.44-3.55 (m, 1H), 2.52-2.92 (m, 2H), 1.53-2.25 (m, 12H). LCMS (ESI) m/z: 466.1[M+H].sup.+.

[0202] Compound 9-2 was used as a common intermediate, Compounds 9-4 and 9-5 were prepared by the same synthesis and separation method as Compound 9-3 by acid amine condensation (added with carboxylic acids substituted differently from Compound 9-3). The characterization data are as follows:

##STR00058##

[0203] Compound 9-4: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.58-7.68 (m, 1H), 7.51 (d, J=8.78 Hz, 1H), 6.97-7.05 (m, 1H), 4.30 (d, J=4.02 Hz, 1H), 4.18 (d, J=4.27 Hz, 1H), 3.87 (s, 1H), 3.76 (s, 1H), 3.49 (br t, J=11.80 Hz, 1H), 2.82 (br s, 1H), 2.07-2.25 (m, 5H), 1.73-1.95 (m, 3H), 1.51-1.70 (m, 2H), 1.24-1.35 (m, 4H). LCMS (ESI) m/z: 448.2[M+H].sup.+.

[0204] Compound 9-5: .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.58-7.68 (m, 1H), 7.51 (d, J=9.03 Hz, 1H), 7.00 (t, J=7.53 Hz, 1H), 4.61 (s, 2H), 3.69-4.10 (m, 4H), 3.43-3.55 (m, 1H), 2.82 (br s, 1H), 2.05-2.25 (m, 5H), 1.72-1.97 (m, 3H), 1.51-1.69 (m, 2H). LCMS (ESI) m/z: 429.0[M+H].sup.+.

##STR00059##

[0205] Synthesis of compound 9-6: compound 9-2 (190 mg, 525.8 μmol) was dissolved in N,N-dimethylformamide (5 mL), potassium carbonate (218 mg, 1.6 mmol) and 2-bromoacetonitrile (70 mg, 578.3 μmol) were added, and reaction solution was stirred at 10° C. for 12 hours. LC-MS showed the reaction was complete. The reaction solution was diluted with water (5 mL), and extracted with dichloromethane/methanol (10/1, 10 mL). The organic phase was washed with saturated salt water (10 mL), dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (neutral condition) to obtain compounds 9-6. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ7.58-7.66 (m, 1H), 7.50 (d, J=8.53 Hz, 1H), 6.99 (d, J=7.28 Hz, 1H), 3.62 (s, 2H), 3.46 (br t, J=11.42 Hz, 1H), 3.33 (s, 2H), 3.21 (s, 2H), 2.81 (br s, 1H), 2.14 (br d, J=10.29 Hz, 5H), 1.81-1.95 (m, 1H), 1.51-1.78 (m, 4H). LCMS (ESI) m/z: 401.2[M+H].sup.+.

Example 9

[0206] ##STR00060## ##STR00061##

[0207] Step 1: Compound 10-1 (100 mg, 334.3 μmol), Compound 3-3 (126 mg, 334.3 μmol), Pd(dppf)Cl.sub.2 (25 mg, 33.4 μmol) and potassium carbonate (139 mg, 1.00 mmol) were added into the mixed solution of dioxane (12 mL) and H.sub.2O (3 mL). It was replaced with nitrogen for 3 times. The reaction solution was stirred in nitrogen atmosphere at 90° C. for 2 hours. LCMS shows that the raw materials were consumed, and the main peak was the target molecular ion peak. The reaction solution was filtered and concentrated to remove the solvent, and then subjected to preparation plate separation and purification to obtain Compound 10-2. LCMS (ESI) m/z: 470.4[M+H].sup.+.

[0208] Step 2: Dichloromethane (1 mL) solution of Compound 10-2 (130 mg, 276.9 μmol) and HCL/EtOAc (4 M, 2 mL) was stirred at 25° C. for 5 minutes. LCMS showed that the raw materials were consumed, and the main peak was the target molecular ion peak. The reaction solution was concentrated under reduced pressure to obtain yellow solid Compound 10-3 (120 mg, hydrochloride), which was directly used in the next step without purification. LCMS (ESI) m/z: 370.6[M+H].sup.+.

[0209] Step 3: under nitrogen atmosphere, MeOH (25 mL) solution of Compound 10-3 (120 mg, 295.6 μmol, hydrochloride) was added with Pd/C (20 mg, 10%). The suspension was replaced with hydrogen for 3 times, and then stirred in hydrogen atmosphere (15 Psi) at 25° C. for 12 hours. LCMS showed that the raw materials were consumed, and the main peak was the target molecular ion peak. The reaction solution was filtered and concentrated under reduced pressure to remove the solvent to obtain Compound 10-4 (130 mg, hydrochloride), which was directly used in the next step of the reaction without further purification. LCMS (ESI) m/z: 372.3[M+H].sup.+.

[0210] Step 4: Compound 10-4 (130 mg, 318.7 μmol, hydrochloride) was dissolved in DMF (5 mL) solution, 2-cyanoacetic acid (33 mg, 382.4 μmol), EDCI (92 mg, 478 μmol), HOBt (65 mg, 478 μmol) and DIEA (206 mg, 1.6 mmol, 277.6 μL) were added, the mixture was stirred at 25° C. for 12 hours. LCMS showed that the raw materials were consumed and the target molecular ion peak was monitored. The reaction solution was concentrated under reduced pressure to remove the solvent, and then subjected to preparative separation to obtain (neutral system) Compound 10-5. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ=7.57-7.65 (m, 1H), 7.49-7.55 (m, 1H), 3.59-3.81 (m, 3H), 3.44-3.54 (m, 2H), 3.34-3.38 (m, 2H), 2.48 (br s, 2H), 1.81-2.04 (m, 5H), 1.64-1.77 (m, 2H), 1.36-1.58 (m, 4H), 0.86-1.12 (m, 4H). LCMS (ESI) m/z: 439.1 [M+H].sup.+.

Biological Activity Test

Experimental Example 1: In Vitro Activity Test of JAK1, JAK2, JAK3 and TYK2 Kinases

Experimental Materials

[0211] Recombinant human JAK1, JAK2, JAK3, TyK2 protease, main instruments and reagents were provided by Eurofins in the UK

Experimental Method

[0212] Diluting JAK2, JAK3 and TYK2: 20 mM 3-(N-morpholine) propanesulfonic acid (MOPS), 1 mM EDTA, 0.01% Brij-35.5% glycerol, 0.1% β-mercaptoethanol, 1 mg/mL BSA; JAK1 dilution: 20 mM Tris, 0.2 mM EDTA, 0.1% β-mercaptoethanol, and 0.01% Brij-35.5% glycerol. All compounds were prepared into a 100% DMSO solution and reached 50 times the final determined concentration. The test compound was diluted by 3 times the concentration gradient, and the final concentration was 10 μM to 0.001 μM, 9 concentrations in total, and the content of DMSO in the detection reaction was 2%. The working stock solution of the compound was added to a determination hole as the first component of the reaction, and then the remaining components were added according to the detailed scheme determination below.

[0213] JAK11 (h) Enzyme Reaction

[0214] JAK1 (h) was incubated with 20 mm Tris/HCl pH7.5, 0.2 mM EDTA, 500 μM MGEEPLYWSFPAKKK, 10 mm magnesium acetate and [γ-.sup.33P]-ATP (activity and concentration as required) together. Mg/ATP mixture was added to start the reaction. After incubation at room temperature for 40 minutes, 0.5% phosphoric acid was added to stop the reaction. Then 10 μL of the reactant was placed on a P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried and counted by scintillation.

[0215] JAK2 (h) Enzyme Reaction

[0216] JAK2 (h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 100 μM KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC, 10 mM magnesium acetate and [γ-.sup.33P]-ATP (activity and concentration as required) together. Mg/ATP mixture was added to start the reaction. After incubation at room temperature for 40 minutes, 0.5% phosphoric acid was added to stop the reaction. Then add 10 μL of the reactant point was placed on the P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried and counted by scintillation.

[0217] JAK3 (h) Enzyme Reaction

[0218] JAK3 (h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 500 μM GGEEEEYFELVKKKK, 10 mM magnesium acetate and [γ-.sup.33P]-ATP (activity and concentration as required) together. Mg/ATP mixture was added to start the reaction. After incubation at room temperature for 40 minutes, 0.5% phosphoric acid was added to stop the reaction. Then 10 μL of the reactant point was placed on the P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried and counted by scintillation.

[0219] TYK2 (h) Enzyme Reaction

[0220] TYK2 (h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM GGMEDIYFEFMGGKKK, 10 mM magnesium acetate and [γ-.sup.33P]-ATP (activity and concentration as required) together. Mg/ATP mixture was added to start the reaction. After incubation at room temperature for 40 minutes, 0.5% phosphoric acid was added to stop the reaction. Then 10 μL of the reactant point was placed on the P30 filter pad, washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried and counted by scintillation.

[0221] Data Analysis

[0222] IC.sub.50 results were obtained by analysis using XLFIT5 (Formula 205) of IDBS company. See Table 1 for details.

TABLE-US-00001 TABLE 1 in vitro screening test results of compounds of the present invention TYK2 JAK1 JAK2 JAK3 Compounds (IC.sub.50, nM) (IC.sub.50, nM) (IC.sub.50, nM) (IC.sub.50, nM) 1-7 38 6 60 3834 1-8 27 14 78 2939 1-9 366 34 315 >10000 1-10 311 32 426 >10000 1-11 18 15 198 >10000 1-12 360 45 527 >10000 1-13 36 3 37 1517 1-14 134 12 144 5035 1-15 106 20 208 9669 1-16 581 114 1020 >10000 1-17 371 65 791 >10000 3-7 26 3 40 1002 3-8 26 20 80 2323 3-9 155 54 294 >10000 4-6 307 170 2008 8448 4-7 194 436 7685 >10000 4-8 >10000 244 3711 364 5-7 67 31 324 5759 5-8 692 75 789 7349 5-9 278 68 520 >10000 5-10 1526 131 2730 >10000 6-7 829 63 998 9391 6-8 570 381 1924 >10000 7-7 830 63 1632 >10000 7-8 404 176 2313 >10000 8-6 127 14 110 7637 8-7 1032 63 5463 >10000 8-8 109 60 1376 >10000 8-9 1329 52 3672 >10000 9-3 105 7 292 >10000 9-4 1186 253 982 >10000 9-5 175 224 624 >10000 9-6 1388 432 1174 >10000 10-5 430 336 812 5410

[0223] Conclusion: the compounds of the present invention shows good selective inhibition to Jak1 and/or TYK2 in in vitro activity test of four kinase subtypes JAK1, JAK2, JAK3 and TYK2.

Experimental Example 2: Pharmacokinetic (PK) Test

[0224] A clear solution obtained by dissolving test compounds was administered into male mice (C.sub.57BL/6) or rats (SD) via tail vein and oral administration (overnight fasting, 7-8 weeks old). After administration of the test compounds, blood was collected from mandibular vein and centrifuged to obtain plasma, at 0.117, 0.333, 1, 2, 4, 7 and 24 hours for the tail vein injection group (2 mg/kg) and 0.25, 0.5, 1, 2, 4, 8 and 24 hours for the oral administration group (15 mg/kg). The blood drug concentration was determined by LC-MS/MS, and WinNonlin™ Version 6.3 pharmacokinetic software was used to calculate relevant pharmacokinetic parameters by non atrioventricular model linear logarithmic ladder method. The test results are as follows:

TABLE-US-00002 TABLE 2-1 PK test results of Compounds 1-11 in mice PK parameters Results T.sub.1/2 (hr) 2.99 C.sub.max (nM) 5745 AUC.sub.0-inf (nM .Math. hr) 9918 Bioavailability (%).sup.a 42.1%

TABLE-US-00003 TABLE 2-2 PK test results of Compounds 1-13 in mice PK parameters Results T.sub.1/2 (hr) 1.61 C.sub.max (nM) 5105 AUC.sub.0-inf (nM .Math. hr) 9917 Bioavailability (%).sup.a 38.1%

TABLE-US-00004 TABLE 2-3 PK test results of Compounds 3-7 in mice PK parameters Results T.sub.1/2 (h) 4.74 C.sub.max (nM) 7380 AUC.sub.0-inf (nM .Math. h) 17969 Bioavailability (%).sup.a 50.1%

[0225] Note: T.sub.1/2: half life: C.sub.max: peak concentration;

[0226] AUC.sub.o-inf: area under the plasma concentration time curve extrapolated from time 0 to infinity;

[0227] Bioavailability: bioavailability.

[0228] Conclusion: the compounds of the present invention have good oral bioavailability and high exposure in mice, which is conducive to producing good in vivo drug efficacy.

Experimental Example 3: In Vivo Pharmacodynamic Study of the Compounds on Inflammatory Bowel Disease (IBD)

Experiment Purpose:

[0229] Inflammatory bowl disease (IBD) is a kind of recurrent disease. It is difficult to cure, has a high recurrence rate, and has a poor prognosis, it has a certain correlation with the incidence of colon cancer. The development of drugs for the treatment of IBD will help alleviate the symptoms of ulcerative colitis and Crohn's disease and improve the quality of life of patients. The mouse inflammation model induced by dextran sulfate sodium (DSS) is similar to the human disease ulcerative colitis and Crohn's disease. It is a common classical model for preclinical evaluation of drug efficacy. The purpose of this experiment is to investigate the therapeutic effects of compounds 1-8, 1-11, 9-3 and 1-13 on DSS induced ulcerative colitis and Crohn's disease in mice, and to provide preclinical pharmacodynamic information for clinical research.

Test method:

[0230] 1. Preparation of Modeling Solution and Drugs for Intragastric Administration

[0231] Preparation method of DSS: 20 g DSS was weighed, 1000 mL drinking water was added to form 2% DSS solution, and stored in a 4° C. refrigerator. It is effective within one month.

[0232] Preparation method of a solvent: Vehicle solution is an aqueous solution containing 0.5% methylcellulose and 0.5% Tween 80. Firstly, 5 g of methylcellulose was dissolved in 990 mL of pure water, then 5 g of Tween 80 was added in small amount for many times, mixed evenly and stored at room temperature.

[0233] Preparation method of compounds: the compound was weighed, an aqueous solution with a mass ratio of 0.5% methylcellulose+0.5% Tween 80 was added, and it was processed by ultrasonic until it was dissolved. After the solution is fully mixed, it was loaded into glass bottles and stored in a 4° C. refrigerator.

[0234] 2. Induction of IBD and Administration

[0235] Seventy male C57BL/6 mice were weighed and randomly divided into 7 groups with mice in each group. The grouping and dose design are shown in Table 3-1.

[0236] The adaptation period of the animals was 3 days. After adaptation, the animals were weighed every day. The blank group was given drinking water, and the other groups were given drinking water containing 2% DSS. The water volume was 5 mL/animal/day. The animals in the test drug group were administrated by gavage once a day. The blank group and DNCB group were given the same volume of vehicle, with the administration volume of 10 mL/kg. Every other day, the fecal viscosity was evaluated, feces were collected for fecal occult blood detection, and the disease activity index (DAI) score was calculated. DSS consumption was detected every day. Fresh DSS solution was replaced every two days. The dosing cycle was continuous intragastric administration for 7 days, followed by 4 days of drug withdrawal, followed by 7 days of continuous intragastric administration. Normal drinking water was changed when intragastric administration was stopped.

TABLE-US-00005 TABLE 3-1 Grouping and dose design Concen- Name of drugs to be Route of tration Dosage Frequency of Groups tested Number administration mg/mL mg/kg administration Blank Blank (solvent 10 p.o. N/A N/A qd, 14 days control group) DSS Blank (solvent 10 p.o. N/A N/A qd, 14 days control group) DSS+001 Compounds 1-8 10 p.o. 1.5 15 qd, 14 days DSS+002 Compounds 1-11 10 p.o. 1.5 15 qd, 14 days DSS+003 Compounds 9-3 10 p.o. 1.5 15 qd, 14 days DSS+004 Compounds 1-13 10 p.o. 1.5 15 qd, 14 days DSS+005 Filgotinib 10 p.o. 3.0 30 qd, 14 days Note: P.O.: oral; dq: once a day.

[0237] 3. IBD Disease Severity Test

[0238] DAI is the sum of the scores of body weight, fecal viscosity and fecal occult blood. The scoring criteria are shown in Table 3-2. Among them, the semi quantitative detection method of piramine cave was adopted for detecting fecal occult blood, including adding piramine cave to the sample first, then adding hydrogen peroxide and ethanol solution, and reading and scoring the color within 2 min. Immediately generating purple blue is marked as 4+, generating the purple blue within 10s is marked as 3+, generating purple red within 1 min is marked as 2+, and gradually generating the purple red within 1-2 min is marked as 1+, and generating no color reaction of purple orchid or purple red within the reading time is recorded as negative (−). After the experiment, the colon and spleen were taken, and the length of colon and the weight of spleen were measured. The spleen index was the ratio of spleen weight to body weight.

TABLE-US-00006 TABLE 3-2 DAI scoring standard Scores Weight loss Fecal viscosity Occult blood 0  <1% Normal — 1  1-5% Soft but shaped stool 1+ 2  6-10% Soft and unshaped stool 2+ 3 11-18% Wet stool 3+ 4 >18% Watery stool 4+

[0239] 4. Statistical Processing

[0240] The experimental data were expressed by means±standard error (Mean±SEM), and all data were statistically analyzed by one-way ANOVA, with P<0.05 as the statistical difference.

Experimental Results:

[0241] 1. DAI Score

[0242] From the DAI score results, as shown in Table 3-3 and FIG. 1, the DAI score of DSS group increased rapidly after 4 days of administration. Compared with the blank control group, the DAI score of DSS group increased significantly on the 6th day and continued until the end of the experiment. No significant difference was found between each administration group and DSS group within 1 week of administration. Compared with before and after drug withdrawal (i.e. day 8 and day 12), the DAI of all DSS drinking water groups decreased significantly. After administration on the 12th day, the scores of Compounds 1-8, 1-13 and Filgotinib DAI began to decrease, and there were significant differences between the DSS group and the DSS group on day 16 and day 18. At the same time, on day 16 and day 18, the DAI score of Compounds 1-13 was significantly lower than that of the positive drug Filgotinib, indicating that Compounds 1-13 has a better effect on reducing DAI score than the positive drug Filgotinib, suggesting that Compounds 1-13 may have a better therapeutic effect on IBD.

TABLE-US-00007 TABLE 3-3 DAI scores of the present application Days Blank DSS DSS+001 DSS+002 DSS+003 DSS+004 DSS+005 2 0.89 ± 0.49 0.60 ± 0.27 0.90 ± 0.28 0.30 ± 0.15 0.40 ± 0.16 0.60 ± 0.38 1.20 ± 0.42 4 0.78 ± 0.34 1.10 ± 0.23 1.70 ± 0.42 1.50 ± 0.34 1.70 ± 0.54 1.50 ± 0.31 2.20 ± 0.61 6 0.89 ± 0.33 3.10 ± 0.60 # 4.40 ± 0.33 3.50 ± 0.27 2.40 ± 0.37 2.50 ± 0.73 2.90 ± 0.43 8 0.56 ± 0.23 5.60 ± 0.58 ## 4.44 ± 0.57 4.20 ± 0.79 4.70 ± 0.94 5.00 ± 0.69 6.40 ± 0.69 12 0.00 ± 0.00 2.78 ± 0.49 ## 2.75 ± 0.50 3.11 ± 0.49 4.11 ± 0.53 2.43 ± 0.31 3.56 ± 0.82 14 0.22 ± 0.14 3.44 ± 0.51 ## 2.63 ± 0.83 3.22 ± 0.79 4.88 ± 0.44 2.07 ± 0.31 3.00 ± 0.57 16 0.22 ± 0.14 5.78 ± 0.60 ## 2.25 ± 0.58 ** 4.44 ± 0.67 3.67 ± 0.86 * 1.58 ± 0.44 **α 3.00 ± 0.96 ** 18 0.11 ± 0.10 6.75 ± 0.51 ## 2.57 ± 0.51 ** 5.89 ± 0.51 4.83 ± 0.68 1.05 ± 0.56 **αα 3.52 ± 0.73 ** Note: compared with the blank group, # P < 0.05, ## P < 0.01; compared with DSS group, * p < 0.05, ** p < 0.01; and compared with DSS+005 group α p < 0.05 αα p < 0.01

[0243] 2. Weight

[0244] The experimental results are shown in Table 3-4 and FIG. 2. From day 5, the weight of mice in the DSS group began to decrease. Compared with the blank control group, the weight of mice in the DSS group began to decrease on day 5, and there was a significant difference on day 7 and lasted until the end of the experiment. Compared with DSS group, Compounds 1-8 and 1-13 can reduce the weight loss caused by DSS. Compounds 1-13 showed significant difference on day 11, day 15, day 16 and day 18, while Compounds 1-8 had no significant difference. Compared with the positive drug Filgotinib, Compound 1-13 can reduce the weight loss of mice caused by DSS and significantly increase on day 9, day 10, day 14 and day 18, indicating that Compounds 1-13 had a better effect on reducing the weight loss of mice caused by DSS, suggesting that Compounds 1-13 may have a better therapeutic effect on IBD.

TABLE-US-00008 TABLE 3-4 Weight of mice in the invention Days Blank DSS DSS+001 DSS+002 DSS+003 DSS+004 DSS+005 1 21.87 ± 0.55 22.43 ± 0.43    22.30 ± 0.30 22.63 ± 0.29 22.26 ± 0.29 22.46 ± 0.40  22.10 ± 0.42 2 22.00 ± 0.55 22.40 ± 0.39    22.43 ± 0.25 22.64 ± 0.28 22.48 ± 0.29 22.60 ± 0.54  22.35 ± 0.41 3 22.03 ± 0.58 22.54 ± 0.41    22.43 ± 0.31 22.20 ± 0.28 22.39 ± 0.27 22.48 ± 0.51  22.11 ± 0.31 4 22.12 ± 0.58 22.48 ± 0.38    22.56 ± 0.37 21.75 ± 0.25 22.26 ± 0.31 22.63 ± 0.41  22.07 ± 0.38 5 22.23 ± 0.50 22.15 ± 0.35    21.99 ± 0.49 20.90 ± 0.30 22.18 ± 0.40 22.38 ± 0.38  21.61 ± 0.47 6 22.69 ± 0.46 21.47 ± 0.45    21.29 ± 0.60 20.33 ± 0.28 22.00 ± 0.43 21.54 ± 0.51  21.21 ± 0.55 7 22.71 ± 0.45 20.50 ± 0.52 ## 20.48 ± 0.47 20.64 ± 0.38 21.21 ± 0.53 21.18 ± 0.53  20.28 ± 0.47 8 22.77 ± 0.49 19.97 ± 0.63 ## 19.96 ± 0.51 20.44 ± 0.58 20.72 ± 0.67 20.56 ± 0.49  19.65 ± 0.47 9 22.68 ± 0.50 19.24 ± 0.72 ## 19.85 ± 0.47 19.49 ± 0.77 19.89 ± 0.37 20.78 ± 0.39α 18.57 ± 0.49 10 22.72 ± 0.46 18.66 ± 0.80 ## 19.84 ± 0.50 19.00 ± 0.87 18.92 ± 0.47 20.24 ± 0.45α 17.98 ± 0.62 11 22.87 ± 0.50 19.28 ± 0.66 ## 20.23 ± 0.67 19.73 ± 0.77 18.60 ± 0.62 21.86 ± 0.50 * 17.85 ± 0.80 12 23.06 ± 0.47 19.70 ± 0.71 ## 20.69 ± 0.77 20.09 ± 0.90 18.50 ± 0.74 21.34 ± 0.60   18.27 ± 0.91 13 23.00 ± 0.49 20.59 ± 0.71 #  21.51 ± 0.67 20.88 ± 0.86 19.42 ± 0.78 22.38 ± 0.69   19.21 ± 0.94 14 23.19 ± 0.47 20.54 ± 0.88 #  21.77 ± 0.72 20.67 ± 0.84 19.26 ± 0.93 22.29 ± 0.70α  20.16 ± 0.61 15 23.11 ± 0.44 20.34 ± 0.91 #  21.67 ± 0.84 20.82 ± 0.91 19.96 ± 0.84 22.26 ± 0.69 * 21.11 ± 0.58 16 23.21 ± 0.45 20.32 ± 0.85 #  21.39 ± 0.94 20.41 ± 1.05 20.84 ± 0.46 22.39 ± 0.74 * 21.09 ± 0.53 17 23.39 ± 0.43 19.60 ± 0.89 ## 20.87 ± 0.92 19.85 ± 1.05 20.42 ± 0.50 21.02 ± 0.76   20.58 ± 0.38 18 23.71 ± 0.46 19.10 ± 0.88 ## 20.99 ± 0.28 18.84 ± 1.06 19.78 ± 0.43   21.82 ± 0.89**α 19.59 ± 0.34 Note: compared with the blank group, # P < 0.05, ## P < 0.01; compared with DSS group, * p < 0.05, **p < 0.01; and compared with DSS+005 group αp < 0.05 αα p < 0.01

[0245] 3. Colon Length

[0246] The experimental results are shown in Table 3-5 and FIG. 3. Compared with the blank control group, the colon length of DSS group mice was significantly reduced. After drug intervention, compared with DSS group, Compounds 1-8, 1-13, 9-3, 1-11 and Filgotinib can reduce colon shortening in mice caused by DSS. Compounds 1-8 and 1-13 have significant effects, and the effect of Compounds 1-13 is significantly better than that of Filgotinib.

TABLE-US-00009 TABLE 3-5 Colon length of the present application Blank DSS DSS+001 DSS+002 DSS+003 DSS+004 DSS+005 5.42 ± 0.15 2.58 ± 0.21## 3.57 ± 0.15* 2.84 ± 0.21 2.87 ± 0.11 4.39 ± 0.24*α 3.13 ± 0.10 Note: compared with the blank group, ##P < 0.01; compared with DSS group *p < 0.05; and compared with DSS+005 group αp < 0.05

[0247] 4. Spleen Index

[0248] The experimental results are shown in Table 3-6 and FIG. 4. Compared with the blank control group, DSS can significantly increase the spleen index of mice (DSS group). After drug intervention, Compounds 1-8, 1-13, 9-3, 1-11 and Filgotinib could reduce the increase of spleen index in mice induced by DSS, and Compounds 1-8, 1-13 and Filgotinib had significant inhibitory effect.

TABLE-US-00010 TABLE 3-6 Spleen index of the present invention Blank DSS DSS+001 DSS+002 DSS+003 DSS+004 DSS+005 3.06 ± 0.19 5.34 ± 0.34## 4.17 ± 0.09* 4.72 ± 0.28 4.98 ± 0.40 3.72 ± 0.22* 4.04 ± 0.18* Note: compared with the blank group, ##P < 0.01; and compared with DSS group *p < 0.05

[0249] Conclusion: in the model of inflammatory bowel disease (IBD) in mice induced by DSS, the compounds of the present invention show good disease treatment effect, and have the same or even better drug effect in mice at half the dose of Filgotinib.

Experimental Example 4: In Vivo Pharmacodynamic Study of Compounds on Allergic Dermatitis (AD)

Experiment Purpose:

[0250] Atopic dermatitis (AD) is a chronic inflammatory skin disease, which is prone to recurrent attacks, its clinical manifestations include severe pruritus, pleomorphic lesions and dry skin disease like symptoms. Its pathogenesis is related to many factors such as heredity, immunity and infection. In recent years, the incidence rate of AD has been increasing year by year. The development of drugs for the treatment of AD will help to alleviate the skin symptoms and improve the quality of life of patients. The animal allergic dermatitis model induced by 1-chloro-2,4-dinitrobenzene (DNCB) is basically consistent with the clinical manifestations of AD patients. It is a classic model for preclinical evaluation of drug efficacy. The purpose of this experiment is to investigate the therapeutic effect of Compounds 1-8, 1-11, 9-3 and 1-13 on DNCB induced allergic dermatitis in mice, and to provide preclinical pharmacodynamic information for clinical research.

[0251] 1. Preparation of Modeling Solution and Drugs for Intragastric Administration

[0252] Preparation method of solvent: vehicle solution is 30% PEG-400+70% (5% HP-β-CD), pH 4-5. Firstly, 35 g HP-β-CD was dissolved in 700 mL MilliQ pure water, 300 mL PEG-400 was added and mixed well, the pH was adjusted to 4-5, and stored at room temperature.

[0253] Preparation method of compounds: the compounds were weighed and added to 30% PEG-400+ 70% (5% HP-β-CD) solution (by volume), and ultrasonically processed. After the solution was fully mixed, it is load into glass bottles and stored in a 4° C. refrigerator.

[0254] 2. Induction of Allergic Dermatitis and Administration

[0255] Seventy male Balb/c mice were weighed and randomly divided into 7 groups with 10 mice in each group. See Table 4-1 for detailed grouping and dose design information.

[0256] The animal adaptation period was 3 days. It was adapted to shaving on the back. After 2 days, it was sensitized with the modeling solution, coated with 20 μL 0.5% DNCB solution on the right ear of the mouse, applied with 200 μL 0.5% DNCB solution on the back, once a day for 3 days. The mice were stimulated from day 4, and applied with 10 μL 1% DNCB solution on the right ear of mice, 100 μL 1% DNCB solution on the back of mice, respectively, once every 3 days. At the same time, from day 4, the mice were given Compounds 1-8, 1-11, 9-3 and 1-13 by gavage, respectively, and the positive drug alonson was applied to the ear and back skin.

TABLE-US-00011 TABLE 4-1 Grouping and dose design Names of drug Route of Frequency of Group to be tested Number administration Concentration Dosage administration Blank Blank (solvent 10 p.o. N/A N/A qd, 28 days control group) DNCB Blank (solvent 10 p.o. N/A N/A qd, 28 days control group) DNCB+ Eloson 10 Right ear/skin N/A 0.01 g/ Once 3 days, Eloson (external use) 0.1 g 28 days DNCB+A Compounds 1-8 10 p.o. 3.0 30 mg/kg qd, 28 days mg/mL DNCB+B Compounds 1-11 10 p.o. 3.0 30 mg/kg qd, 28 days mg/mL DNCB+C Compounds 9-3 10 p.o. 3.0 30 mg/kg qd, 28 days mg/mL DNCB+D Compounds 1-13 10 p.o. 3.0 30 mg/kg qd, 28 days mg/mL Note: P.O.: oral administration; QD: once a day.

[0257] 3. Disease Severity Test

[0258] Indicators of successful DNCB atopic dermatitis modeling: DNCB successfully induced BALB/c mice to produce typical atopic dermatitis like lesions such as erythema, erosion, bleeding, edema, epidermal exfoliation, epidermal thickening, etc. On days 3, 9, 15, 21, and 27, the severity of specific dermatitis was evaluated by evaluating four clinical inflammatory indicators of atopic dermatitis: Erythema/bleeding, scar/dryness, edema, and infiltration/erosion. Each evaluation index has four grades: 0, none; 1. slight; 2. moderate; 3. obvious; 4. very obvious. Sum the scores represents score of the skin. After the experiment, the thickness of the right ear of mice was measured, blood was taken from the eyes, and the serum was separated by centrifugation (3000 rpm, 15 min) to detect the concentration of LgE. The spleen was taken and the spleen index was calculated: spleen index=spleen weight (mg)/body weight (g).

[0259] 4. Statistical Processing

[0260] The experimental data were expressed by means±standard error (Mean±SEM), and all data were statistically analyzed by one-way ANOVA, with P<0.05 as the statistical difference.

Experimental Results:

[0261] 1. Skin Score

[0262] The skin score results showed that, as shown in Table 4-2 and FIG. 5, after DNCB induction, the skin score of DNCB group increased continuously. From day 9, the skin score was significantly higher than that of the blank control group, and continued until the end of the experiment. Compared with DNCB group, the skin score decreased gradually after drug intervention, and the Compounds 1-13 group significantly decreased at day 21 of administration, showing a better effect than the positive drug Alonson. At day 27 of administration, all treatment groups, including Compounds 1-8, 1-11, 9-3 and 1-13, and the alonson group, showed a significant reduction in skin scores.

TABLE-US-00012 TABLE 4-2 skin score of the present application DNCB+ Days Blank DNCB Eloson DNCB+A DNCB+B DNCB+C DNCB+D 3 0.0 ± 0.0 0.8 ± 0.4    0.5 ± 0.5 0.6 ± 0.5 0.9 ± 0.3 0.7 ± 0.5    0.5 ± 0.5 9 0.0 ± 0.0 3.5 ± 1.1 ## 3.5 ± 0.5 3.1 ± 0.8 3.6 ± 1.1 3.4 ± 0.7    3.9 ± 1.6 15 0.0 ± 0.0 6.8 ± 0.8 ## 4.4 ± 1.1 5.3 ± 1.2 4.4 ± 0.8 4.2 ± 0.6    3.7 ± 0.6 21 0.0 ± 0.0 9.5 ± 1.0 ## 6.2 ± 0.9 6.7 ± 1.4 5.4 ± 0.7 5.4 ± 0.9       5.0 ± 1.0 * 27 0.0 ± 0.0 10.5 ± 1.1 ##     3.3 ± 1.3 **    3.2 ± 1.5 **    2.9 ± 1.2 ** 2.2 ± 0.9 **     1.5 ± 0.5 **  Note: compared with the blank group, ## P < 0.01; and compared with DNCB group, * p < 0.05, ** p < 0.01

[0263] 2. Thickness of Right Ear

[0264] The experimental results showed that (as shown in Table 4-3 and FIG. 6), the thickness of the right ear of mice after DNCB induction (DNCB group) significantly increased, and the thickness of the right ear of mice after drug intervention (including Compounds 1-8, 1-11, 9-3 and 1-13 and Eloson group) significantly decreased, indicating that Compounds 1-8, 1-11, 9-3 and 1-13 have a significant therapeutic effect on allergic dermatitis in mice induced by DNCB, which is equivalent to the effect of Eloson. Four compounds (compounds 1-8, 1-11, 9-3 and 1-13) had the strongest effect on the reduction of right ear thickness, but there was no significant difference between the groups.

TABLE-US-00013 TABLE 4-3 thickness of the right ear of the present application DNCB+ Blank DNCB Eloson DNCB+A DNCB+B DNCB+C DNCB+D 0.169 ± 0.005 0.349 ± 0.006 ## 0.231 ± 0.008 ** 0.254 ± 0.007 ** 0.283 ± 0.006 ** 0.252 ± 0.006 ** 0.232 ± 0.004 ** Note: compared with the blank group, ## P < 0.01; and compared with DNCB group ** p < 0.01

[0265] 3. Spleen Index

[0266] The experimental results showed that (as shown in Table 4-4 and FIG. 7), the spleen of mice after DNCB induction (DNCB group) was enlarged, which showed that the spleen index was significantly increased, and the spleen index of mice was significantly decreased through drug intervention (including Compounds 1-8, 1-11, 9-3 and 1-13 and elosone group), which showed that Compounds 1-8, 1-11, 9-3 and 1-13 have a significant therapeutic effect on allergic dermatitis in mice induced by DNCB, which is equivalent to the effect of elosone. The four compounds (Compounds 1-8, 1-11, 9-3 and 1-13) had the strongest effect on the reduction of spleen index, but there was no significant difference between the groups.

TABLE-US-00014 TABLE 4-4 spleen index of the present application DNCB+ Blank DNCB Eloson DNCB+A DNCB+B DNCB+C DNCB+D 2.98 ± 0.23 7.16 ± 1.37 ## 3.37 ± 1.34 ** 4.16 ± 0.48 ** 3.45 ± 0.48 ** 4.17 ± 0.65 ** 2.76 ± 0.33 ** Note: compared with the blank group, ## P < 0.01; and compared with DNCB group ** p < 0.01

[0267] 4. IgE Concentration in Serum

[0268] The experimental results showed that (as shown in Table 4-5 and FIG. 8), after DNCB induction (DNCB group), the concentration of lgE in serum of mice significantly increased, and the concentration of lgE in serum of mice significantly decreased through drug intervention (including Compounds 1-8, 1-11, 9-3 and 1-13 and Eloson group), indicating that Compounds 1-8, 1-11, 9-3 and 1-13 have significant therapeutic effect on allergic dermatitis in mice induced by DNCB.

TABLE-US-00015 TABLE 4-5 lgE concentration of the present application in serum DNCB+ Blank DNCB Eloson DNCB+A DNCB+B DNCB+C DNCB+D 4.39 ± 0.66 36.44 ± 8.53 ## 5.26 ± 1.07 ** 5.25 ± 0.99 ** 7.46 ± 1.34 ** 6.39 ± 1.04 ** 6.79 ± 1.46 ** Note: compared with the blank group, ## P < 0.01; and compared with DNCB group ** p < 0.01

[0269] 5. Weight

[0270] The experimental results showed that (as shown in Table 4-6 and FIG. 9), the weight of mice in the blank group and DNCB group increased slowly, and the weight of mice in DNCB group was slightly lower than that in the blank group, and there was no statistical difference between the groups. The weight of the four compounds (Compounds 1-8, 1-11, 9-3 and 1-13) group was the same as that of the DNCB group and higher than that of the alonson group, but there was no statistical difference between the groups, indicating that Compounds 1-8, 1-11, 9-3 and 1-13 had no significant effect on the weight of mice.

TABLE-US-00016 TABLE 4-6 weight of mice in the invention DNCB+ Days Blank DNCB Eloson DNCB+A DNCB+B DNCB+C DNCB+D 1 18.08 ± 0.70 19.69 ± 0.44 19.42 ± 0.45 18.55 ± 0.83 19.45 ± 0.59 19.32 ± 0.46 19.11 ± 0.62 4 19.62 ± 0.76 19.36 ± 0.42 19.68 ± 0.45 19.84 ± 0.70 20.15 ± 0.46 20.02 ± 0.41 19.50 ± 0.50 7 21.68 ± 0.54 19.60 ± 0.49 18.29 ± 0.48 19.29 ± 0.84 19.84 ± 0.55 19.24 ± 0.47 20.52 ± 0.58 10 22.53 ± 0.55 20.91 ± 0.43 18.02 ± 0.42 19.89 ± 0.51 20.21 ± 0.62 19.41 ± 0.58 20.49 ± 0.48 13 23.48 ± 0.55 21.27 ± 0.41 18.24 ± 0.39 19.95 ± 0.45 20.24 ± 0.41 20.83 ± 0.42 21.72 ± 0.48 16 24.66 ± 0.58 21.41 ± 0.52 17.67 ± 0.41 21.05 ± 0.36 20.63 ± 0.43 21.49 ± 0.45 22.14 ± 0.53 19 24.95 ± 0.51 22.30 ± 0.43 17.85 ± 0.42 21.40 ± 0.44 22.50 ± 0.40 23.36 ± 0.38 23.24 ± 0.44 22 25.85 ± 0.61 22.76 ± 0.42 20.03 ± 0.47 23.60 ± 0.47 23.97 ± 0.42 23.67 ± 0.37 24.38 ± 0.42 25 26.24 ± 0.60 22.68 ± 0.33 18.92 ± 0.52 22.96 ± 0.46 22.45 ± 0.36 23.14 ± 0.42 23.35 ± 0.36 28 27.08 ± 0.62 22.97 ± 0.57 18.79 ± 0.55 24.28 ± 0.47 22.66 ± 0.34 24.15 ± 0.50 23.70 ± 0.33

[0271] Conclusion: in the mouse allergic dermatitis (AD) model induced by DNCB, Compounds 1-8, 1-11, 9-3 and 1-13 of the present invention can significantly reduce the skin score, right ear thickness, spleen index and serum lgE concentration of mice, and have no significant effect on body weight, showing a good disease treatment effect, which is equivalent to the effect of glucocorticoid alonson.