Indoleamine 2,3-dioxygenase inhibitor, method for preparation and use thereof

Abstract

A novel indoleamine 2,3-dioxygenase inhibitor and/or its pharmaceutically acceptable salts can used, in the preparation of a drug for indoleamine 2,3-dioxygenase (IDO) related diseases, such as cancer, Alzheimer's, depression, cataract, etc.

Claims

1. A compound having the structure as shown in formula I or formula II, and a salt or stereoisomer thereof: ##STR00090## wherein, in formula I and formula II, R.sub.1 is independently selected from hydrogen atom, halogen, and trifluoromethyl; n represents 0 or 1; Y is independently selected from oxygen atom, sulfur atom, nitrogen atom, and ##STR00091## Z is independently selected from oxygen atom, sulfur atom, nitrogen atom, and ##STR00092## R.sub.3 is independently selected from substituted C.sub.1-10 alkyl and C.sub.3-10cycloalkyl, and unsubstituted C.sub.1-10 alkyl and C.sub.3-10 cycloalkyl, and the substituent is selected from one or more of amino, oxy, C.sub.3-6 cycloalkyl, C.sub.2-6 ester group, C.sub.1-6 alkyl hydroxyl group, —CONH.sub.2; R.sub.4 is independently selected from one of hydrogen atom, ##STR00093## substituted C.sub.1-10 alkyl, and unsubstituted C.sub.1-10 alkyl; wherein the substituent of C.sub.1-10 alkyl is independently selected from one or more of ##STR00094## unsubstituted phenyl, and phenyl substituted with C.sub.1-4 alkyl; R.sub.2 is independently selected from hydrogen atom, C.sub.1-6 alkyl, C.sub.2-6 ester group, ##STR00095## sulfonyl substituted with C.sub.1-4 alkyl, sulfonyl substituted with amino, sulfonamido substituted with amino, ##STR00096## substituted and unsubstituted C.sub.4-8 aryl, substituted and unsubstituted heteroaryl, ##STR00097## said R.sub.5 is independently selected from C.sub.1-6 alkyl, C.sub.1-6 alkoxy, substituted and unsubstituted anilino, substituted and unsubstituted C.sub.5-12 aryl, and substituted and unsubstituted C.sub.4-10 heteroaryl, and wherein each of the substituted anilino, the substituted C.sub.5-12 aryl, and the substituted C.sub.4-10 is independently substituted by halogen, amino, or both.

2. The compound, the salt or stereoisomer thereof according to claim 1, wherein Z and R.sub.3 together form a 5-8 membered heterocycloalkyl.

3. The compound, the salt or stereoisomer thereof according to claim 1, wherein Y, Z, and R.sub.3 together form a 5-8 membered heterocycloalkyl.

4. The compound, the salt or stereoisomer thereof according to claim 1, wherein R.sub.2 is ##STR00098##

5. The compound, the salt or stereoisomer thereof according to claim 1, wherein n is 1, the group R.sub.2 is ##STR00099##

6. The compound, the salt or stereoisomer thereof according to claim 1, wherein R.sub.4 is selected from a plurality of C.sub.1-10 alkyls, each terminated ##STR00100## unsubstituted phenyl, and phenyl substituted with C.sub.1-4 alkyl.

7. A compound, a salt or stereoisomer thereof, wherein the compound is selected from ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109##

8. A pharmaceutical composition, comprising a therapeutically effective amount of the compound of formula I or formula II, or the pharmaceutically acceptable salt or stereoisomer thereof as an active ingredient, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

9. A method of treating a disease associated with indoleamine 2,3-dioxygenase, comprising administering a therapeutically effective amount of the compound according to claim 1 or a salt or stereoisomer thereof to a subject in need thereof, wherein the disease is a cervical cancer, Alzheimer's disease, depression, or cataract.

10. The method according to claim 9, wherein the compound is selected from ##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115## ##STR00116## ##STR00117## ##STR00118##

11. A method of treating a disease associated with indoleamine 2,3-dioxygenase, comprising administering a therapeutically effective amount of the compound according to the pharmaceutical composition according to claim 8 to a subject in need thereof, wherein the disease is cervical cancer, Alzheimer's disease, depression, or cataract.

Description

DETAILED DESCRIPTION

(1) The following examples further describe the invention. However, these examples are only for illustrating the invention without limiting the scope of the invention.

Example 1

(2) ##STR00023## ##STR00024##
Step 1: 632 mg of compound I-24-1 was dissolved in 20 ml tetrahydrofuran, and added with 486 mg of carbonyldiimidazole (CDI), and then the mixture was placed at 50° C. for 0.5 h. After the reaction was completed, the solvent was removed by rotary evaporating under reduced pressure. The residual was added with 30 ml water, adjusted to pH 4-5 with diluted hydrochloric acid and then extracted twice with ethyl acetate. The organic phases were combined, washed twice with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to give compound I-24-2 (650 mg, off-white solid);
Step 2: 650 mg of compound I-24-2 was added to a mixed solvent of 20 ml trifluoroacetic acid and 12 ml hydrogen peroxide. The mixture was placed for reaction at 70° C. overnight. After the reaction was completed, the reaction mixture was added with 20 ml water to precipitate a large amount of solid, which was filtered off with suction and dried to obtain compound I-24-3 (315 mg, light yellow solid);
Step 3: 315 mg of compound I-24-3 was dissolved in 20 ml tetrahydrofuran, and added dropwise with 224 mg/10 ml compound 1 in tetrahydrofuran. The mixture was reacted at room temperature for 12.0 h. After the reaction was completed, the solvent was removed by rotary evaporating under reduced pressure, and ethyl acetate and water were added to the residual to give separated phases. The organic phase was washed twice with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to give compound I-24-4 (320 mg, off-white solid);
Step 4: 320 mg of compound I-24-4 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and the residual was added with 20 ml water, adjusted to pH 8 with saturated sodium bicarbonate solution and the extracted twice with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution twice, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-24-5 (205 mg, off-white solid);
Step 5: 15 ml dichloromethane was cooled to below 0° C., added with 108 mg of chlorosulfonyl isocyanate, and then added with 56 mg of tert-butanol dropwise under the temperature controlled below 0° C. After the addition was completed, the mixture was reacted at the same temperature for 15 min and at room temperature for 2.0 h to obtain the reaction solution A. 205 mg of compound I-24-5 was dissolved in 20 ml dichloromethane, at the temperature lowered below 0° C., added dropwise with reaction solution A, then added dropwise with 155 mg of triethylamine, and reacted for 2.0 h. After the reaction was completed, the mixture was added with saturated sodium bicarbonate to quench the reaction and for liquid separation. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-24-6 (225 mg, off-white solid);
Step 6: 225 mg of compound I-24-6 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure and the residual was added with 20 ml water, adjusted to pH 8 with saturated sodium bicarbonate, and extracted twice with ethyl acetate. Organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-24-7 (160 mg, off-white solid);
Step 7: 160 mg of compound I-24-7 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-24 (18 mg, off-white solid, purity: 98.2%).

Example 2

(3) ##STR00025##
Step 1: 320 mg of compound I-24-3 was dissolved in 20 ml tetrahydrofuran, added dropwise with 262 mg/10 ml compound 2 in tetrahydrofuran, and reacted at room temperature for 6.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure; ethyl acetate and water were added to separate liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-20-1 (364 mg, off-white solid);
Step 2: 364 mg of compound I-20-1 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was complete, the solvent was rotary evaporated under reduced pressure, and the residual was added with 20 ml water, adjusted to pH 8 with saturated sodium bicarbonate, and extracted with ethyl acetate twice. The organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-20-2 (235 mg, off-white solid);
Step 3: 15 ml dichloromethane was cooled to below 0° C., added with 118 mg of chlorosulfonyl isocyanate, and then added dropwise with 60 mg of tert-butanol under the temperature controlled below 0° C. After the addition was completed, the mixture was reacted at the same temperature for 15 min and at room temperature for 2.0 h to obtain the reaction solution A.
235 mg of compound I-20-2 was dissolved in 20 ml dichloromethane, the temperature was reduced below 0° C., added dropwise with reaction solution A, then added dropwise with 166 mg of triethylamine, and reacted for 2.0 h. After the reaction was completed, the mixture was added with saturated sodium bicarbonate to quench the reaction and for liquid separation. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-20-3 (260 mg, off-white solid);
Step 4: 260 mg of compound I-20-3 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure and the residual was added with 20 ml water, adjusted to pH 8 with saturated sodium bicarbonate, and extracted twice with ethyl acetate. Organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-20-4 (184 mg, off-white solid);
Step 5: 184 mg of compound I-20-4 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-20 (25 mg, off-white solid, purity: 97.8%).

Example 3

(4) ##STR00026##
Step 1: 200 mg of compound I-24-3 was dissolved in 20 ml tetrahydrofuran, and added dropwise with 97 mg/10 ml compound 3 in tetrahydrofuran. The mixture was reacted at room temperature for 6.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and ethyl acetate and water were added to the residual to give separated phases. The organic phase was washed twice with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to give compound I-1-1 (180 mg, off-white solid);
Step 2: 180 mg of compound I-1-1 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-1 (15 mg, off-white solid, purity: 96.4%).

Example 4

(5) ##STR00027##
Step 1: 200 mg of compound I-24-3 was dissolved in 20 ml tetrahydrofuran, added dropwise with 128 mg/10 ml compound 4 in tetrahydrofuran, and reacted at room temperature for 6.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure; ethyl acetate and water were added to separate liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-15-1 (196 mg, off-white solid);
Step 2: 196 mg of compound I-15-1 was dissolved in 20 ml dichloromethane, cooled to −70° C., slowly added dropwise with 220 mg of boron tribromide, and slowly warmed to −10° C. for 0.5 h after the addition. After the reaction was completed, the mixture was kept at room temperature, added with saturated sodium bicarbonate to quench the reaction, and extracted with dichloromethane. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry, and purified by column chromatography to obtain compound I-15-2 (120 mg, oil);
Step 3: 120 mg of compound I-15-2 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, added with saturated ammonium chloride solution and ethyl acetate to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-15 (11 mg, off-white solid, purity: 96.5%);

Example 5

(6) ##STR00028##
Step 1: 200 mg of compound I-24-5 was dissolved in 20 ml dichloromethane, and added with 100 mg of triethylamine. The mixture was placed at 0° C., added with 115 mg of methanesulfonyl chloride dropwise and kept reacting for 2.0 h after the addition was completed. After the reaction was completed, the reaction mixture was added with water, and adjusted to pH 4-5 with dilute hydrochloric acid. The separated organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain crude compound I-13-1 (180 mg, oil), which was used directly in the next step;
Step 2: 180 mg of compound I-13-1 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, the mixture was added with saturated ammonium chloride solution and ethyl acetate to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-13 (19 mg, off-white solid, purity: 96.8%);

Example 6

(7) ##STR00029##
Step 1: 370 mg of compound I-24-3 was dissolved in 20 ml tetrahydrofuran, added dropwise with 496 mg/10 ml compound 6 in tetrahydrofuran, and reacted at room temperature for 6.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure; ethyl acetate and water were added to separate liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-16-1 (430 mg, off-white solid);
Step 2: 430 mg of compound I-16-1 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was complete, the solvent was rotary evaporated under reduced pressure, and the residual was added with 20 ml water, adjusted to about pH 8 with saturated sodium bicarbonate, and extracted with ethyl acetate twice. The organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-16-2 (284 mg, off-white solid);
Step 3: 284 mg of compound I-16-2 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-16 (20 mg, off-white solid, purity: 98.5%).

Example 7

(8) ##STR00030## ##STR00031##
Step 1: 542 mg of compound I-5-1 was dissolved in 20 ml tetrahydrofuran, and added with 486 mg of carbonyldiimidazole (CDI), and then the mixture was placed at 50° C. for 0.5 h. After the reaction was completed, the solvent was removed by rotary evaporating under vacuum. The residual was added with 30 ml water, adjusted to pH 4-5 with diluted hydrochloric acid and then extracted twice with ethyl acetate. The organic phases were combined, washed twice with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate and rotary evaporated to dry under reduced pressure to give compound I-5-2 (564 mg, off-white solid);
Step 2: 564 mg of compound I-5-2 was added to a mixed solvent of 20 ml trifluoroacetic acid and 12 ml hydrogen peroxide. The mixture was placed for reaction at 70° C. overnight. After the reaction was completed, the reaction mixture was added with 20 ml water to precipitate a large amount of solid, which was filtered off with suction and dried to obtain compound I-5-3 (402 mg, light yellow solid);
Step 3: 402 mg of compound I-5-3 was dissolved in 20 ml tetrahydrofuran, and added dropwise with 324 mg/10 ml compound 1 in tetrahydrofuran. The mixture was reacted at room temperature for 12.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and ethyl acetate and water were added to the residual to give separated phases. The organic phase was washed twice with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to give compound I-5-4 (465 mg, off-white solid);
Step 4: 465 mg of compound I-5-4 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and the residual was added with 20 ml water, adjusted to pH 8 with saturated sodium bicarbonate and the extracted twice with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution twice, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-5-5 (308 mg, off-white solid);
Step 5: 308 mg of compound I-5-4 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-7-5 (256 mg, off-white solid), which was used directly in the next step.
Step 6: 256 mg of compound I-5-5 was dissolved in 20 ml ethanol, added with 115 mg of maleic anhydride, and reacted at reflux for 6.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure and the residual was added with water and ethyl acetate to separate liquid phases. The organic phase was washed twice with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-5 (13 mg, off-white solid, purity: 97.2%).

Example 8

(9) ##STR00032## ##STR00033##
Step 1: 740 mg of compound I-24-3 was dissolved in 20 ml tetrahydrofuran, added dropwise with 738 mg/10 ml compound 7 in tetrahydrofuran, and reacted at room temperature for 6.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure; ethyl acetate and water were added to separate liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-23-1 (855 mg, off-white solid);
Step 2: 855 mg of compound I-23-1 was dissolved in 20 ml methanol, added with 115 mg of p-toluenesulfonic acid hydrate, and reacted at room temperature for 12.0 h. After the reaction was completed, the mixture was added with saturated sodium bicarbonate solution to neutralizing acids, concentrated under reduced pressure, and extracted with ethyl acetate. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-23-2 (395 mg, oil);
Step 3: 395 mg of compound I-23-2 was dissolved in 20 ml dichloromethane, added with 148 mg of triethylamine, cooled to below 0° C., then added dropwise with 116 mg of acetyl chloride, and reacted for 1.0 h. After the reaction was completed, the mixture was added with saturated sodium bicarbonate for liquid separation. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-23-3 (400 mg, oil), which was directly used in the next step;
Step 4: 400 mg of compound I-23-3 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and the residual was added with 20 ml water, adjusted to about pH 8 with saturated sodium bicarbonate, and extracted with ethyl acetate twice. The organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-23-4 (284 mg, off-white solid);
Step 5: 15 ml dichloromethane was cooled to below 0° C., added with 127 mg of chlorosulfonyl isocyanate, and then added dropwise with 65 mg of tert-butanol under the temperature controlled below 0° C. After the addition was completed, the mixture was reacted at the same temperature for 15 min and at room temperature for 2.0 h to obtain the reaction solution A.
284 mg of compound I-23-4 was dissolved in 20 ml dichloromethane, at the temperature lowered below 0° C., added dropwise with reaction solution A, then added dropwise with 240 mg of triethylamine, and reacted for 2.0 h. After the reaction was completed, the mixture was added with saturated sodium bicarbonate to quench the reaction and for liquid separation. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-23-5 (293 mg, off-white solid);
Step 6: 293 mg of compound I-23-5 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure and the residual was added with 20 ml water, adjusted to pH 8 with saturated sodium bicarbonate, and extracted twice with ethyl acetate. Organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-23-6 (198 mg, off-white solid);
Step 7: 198 mg of compound I-23-6 was dissolved in 20 ml methanol, added with 248 mg of potassium carbonate, and reacted at 50° C. for 2.0 h. After the reaction was completed, the mixture was added with saturated ammonium chloride solution and ethyl acetate to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-23 (18 mg, off-white solid, purity: 97.6%).

Example 9

(10) ##STR00034##
Step 1: 710 mg of compound I-7-1 was dissolved in 20 ml tetrahydrofuran, and added with 486 mg of CDI, and then the mixture was placed at 50° C. for 0.5 h. After the reaction was completed, the solvent was removed by rotary evaporating under vacuum. The residual was added with 30 ml water, adjusted to pH4-5 with diluted hydrochloric acid and then extracted twice with ethyl acetate. The organic phases were combined, washed twice with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate and rotary evaporated to dry under reduced pressure to give compound I-7-2 (610 mg, off-white solid);
Step 2: 610 mg of compound I-7-2 was added to a mixed solvent of 20 ml trifluoroacetic acid and 12 ml hydrogen peroxide. The mixture was placed for reaction at 70° C. overnight. After the reaction was completed, the reaction mixture was added with 20 ml water to precipitate a large amount of solid, which was filtered and dried to obtain compound I-7-3 (365 mg, light yellow solid);
Step 3: 365 mg of compound I-7-3 was dissolved in 20 ml tetrahydrofuran, and added dropwise with 215 mg/10 ml compound 5 in tetrahydrofuran. The mixture was reacted at room temperature for 12.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and ethyl acetate and water were added to the residual to give separated phases. The organic phase was washed twice with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to give compound I-7-4 (375 mg, off-white solid);
Step 4: 375 mg of compound I-7-4 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-7 (26 mg, off-white solid, purity: 97.6%).

Example 10

(11) ##STR00035## ##STR00036##
Step 1: 494 mg of compound I-27-1 was dissolved in 20 ml tetrahydrofuran, and added with 486 mg of CDI, and then the mixture was placed at 50° C. for 0.5 h. After the reaction was completed, the solvent was removed by rotary evaporating under vacuum. The residual was added with 30 ml water, adjusted to pH4-5 with diluted hydrochloric acid and then extracted twice with ethyl acetate. The organic phases were combined, washed twice with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate and rotary evaporated to dry under reduced pressure to give compound I-27-2 (518 mg, off-white solid);
Step 2: 518 mg of compound I-27-2 was added to a mixed solvent of 20 ml trifluoroacetic acid and 12 ml hydrogen peroxide. The mixture was placed for reaction at 70° C. overnight. After the reaction was completed, the reaction mixture was added with 20 ml water to precipitate a large amount of solid, which was filtered and dried to obtain compound I-27-3 (345 mg, light yellow solid);
Step 3: 345 mg of compound I-27-3 was dissolved in 20 ml tetrahydrofuran, and added dropwise with 300 mg/10 ml compound 1 in tetrahydrofuran. The mixture was reacted at room temperature for 12.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and ethyl acetate and water were added to the residual to give separated phases. The organic phase was washed twice with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to give compound I-27-4 (334 mg, off-white solid);
Step 4: 334 mg of compound I-27-4 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and the residual was added with 20 ml water, adjusted to about pH8 with saturated sodium bicarbonate, and extracted with ethyl acetate twice. The organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-27-5 (225 mg, off-white solid);
Step 5: 15 ml dichloromethane was cooled to below 0° C., added with 150 mg of chlorosulfonyl isocyanate, and then added dropwise with 73 mg of tert-butanol under the temperature controlled below 0° C. After the addition was completed, the mixture was reacted at the same temperature for 15 min and at room temperature for 2.0 h to obtain the reaction solution A. 225 mg of compound I-27-5 was dissolved in 20 ml dichloromethane, at the temperature lowered below 0° C., added dropwise with reaction solution A, then added dropwise with 205 mg of triethylamine, and reacted for 2.0 h. After the reaction was completed, the mixture was added with saturated sodium bicarbonate to quench the reaction and for liquid separation. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-27-6 (263 mg, off-white solid);
Step 6: 263 mg of compound I-27-6 was dissolved in 20 ml dichloromethane, added with 4 ml trifluoroacetic acid, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure and the residual was added with 20 ml water, adjusted to pH 8 with saturated sodium bicarbonate, and extracted twice with ethyl acetate. Organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to obtain compound I-27-7 (180 mg, off-white solid);
Step 4: 180 mg of compound I-27-7 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-27 (22 mg, off-white solid, purity: 96.9%).

Example 11

(12) ##STR00037##
Step 1: 981 mg of compound I-5 was dissolved in 20 ml tetrahydrofuran, added dropwise with 10 ml solution of 20% methylhydrazine in tetrahydrofuran, and reacted at room temperature for 2.0 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure; ethyl acetate and water were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry and purified by column chromatography under reduced pressure to obtain compound I-36-1 (780 mg, off-white solid);
Step 2: 780 mg of compound I-36-1 was dissolved in 20 ml tetrahydrofuran, added dropwise with a solution of 405 mg of phenylacetyl chloride in 5 ml tetrahydrofuran, then added 263 mg of triethylamine, and reacted at room temperature for 0.5 h. After the reaction was completed, the solvent was rotary evaporated under reduced pressure, and the residual was added with 30 ml water, and extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to dry under reduced pressure to give compound I-36-2 (818 mg, off-white solid);
Step 3: 818 mg of compound I-36-2 was dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-36 (38 mg, off-white solid, purity: 98.9%).

Example 12

(13) ##STR00038##
Step 1: 401 mg of compound I-24-5, 198 mg of 3,5-difluorobenzene isocyanate and 200 mg of triethylamine were dissolved in 10 ml DMF, and reacted overnight at room temperature. After the reaction was completed, the mixture was added with 50 ml water and then extracted twice with addition of ethyl acetate. The organic phases were combined, washed twice with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure, and purified by column chromatography to obtain compound I-40-1 (283 mg, off-white solid);
Step 2: 283 mg of compound I-40-1 was completely dissolved in 20 ml tetrahydrofuran, added with 1 ml 2.5M sodium hydroxide solution, and reacted at room temperature for 2.0 h. After the reaction was completed, saturated ammonium chloride solution and ethyl acetate were added to separate the liquid phases. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, rotary evaporated to dry under reduced pressure and purified by column chromatography to obtain compound I-40 (12 mg, off-white solid, purity: 95.8%).

(14) With reference to the above examples of compound preparation methods, the following compounds were prepared through a series of reactions under suitable solvents and reaction temperatures. NMR and mass spectrometry were tested for compounds including but not limited to those shown in the table below.

(15) TABLE-US-00001 Compound structures and numbers MS .sup.1H NMR [M + H].sup.+ = 389.0 1H NMR (400 MHz, d6-DMSO): 11.25 (s, 1H), 9.08 (s, 1H), 7.53- 7.51 (m, 2H), 7.10-7.06 (m, 1H), 6.82 (s, 1H), 2.02 (s, 2H), 1.52- 1.50 (m, 4H), 1.23-1.20 (dd, 3H) ppm. 0 [M + H].sup.+ = 446.2 1H NMR (400 MHz, d6-DMSO): 10.93 (s, 1H), 9.02 (s, 1H), 7.15- 7.09 (m, 1H), 6.99-6.95 (m, 2H), 6.45 (s, 1H), 1.82-1.79 (m, 2H), 1.75-1.73 (m, 2H), 1.62-1.60 (m, 4H), 1.55-1.53 (m, 4H) ppm. [M + H].sup.+ = 430.6 1H NMR (400 MHz, d6-DMSO): 11.09 (s, 1H), 9.15 (s, 1H), 7.18- 7.12 (m, 1H), 6.95-6.93 (m, 2H), 6.43 (s, 1H), 1.80-1.78 (m, 2H), 1.65-1.63 (m, 2H), 1.48-1.45 (m, 4H), 1.25-1.23 (m, 4H) ppm. [M + H].sup.+ = 458.3 1H NMR (400 MHz, d6-DMSO): 11.21 (s, 1H), 9.80 (s, 1H), 7.66-7.65 (m, 1H), 6.78-6.73 (m, 2H), 6.27 (s, 1H), 3.35-3.30 (m, 2H), 3.13-3.10 (m, 2H), 2.98-2.95 (m, 4H) ppm. [M + H].sup.+ = 412.5 1H NMR (400 MHz, d6-DMSO): 11.35 (s, 1H), 9.92 (s, 1H), 7.65- 7.62 (m, 1H), 6.93 (s, 2H), 6.86- 6.83 (m, 2H), 6.75 (s, 1H), 3.45- 3.43 (m, 2H), 3.18-3.16 (m, 2H) ppm. [M + H].sup.+ = 418.2 1H NMR (400 MHz, d6-DMSO): 11.26 (s, 1H), 9.82 (s, 1H), 7.65- 7.63 (m, 1H), 6.79-6.76 (m, 2H), 6.37 (s, 1H), 4.35 (s, 1H), 3.42- 3.38 (m, 2H), 3.10-3.08 (m, 2H), 2.85 (s, 3H) ppm. [M + H].sup.+ = 432.1 1H NMR (400 MHz, d6-DMSO): 11.20 (s, 1H), 9.15 (s, 1H), 7.50- 7.48 (m, 2H), 7.11-7.08 (m, 2H), 6.95-6.93 (m, 2H), 6.80 (s, 1H), 5.05-5.02 (m, 2H), 2.02 (dd, 2H) ppm. [M + H].sup.+ = 450.1 1H NMR (400 MHz, d6-DMSO): 11.12 (s, 1H), 9.32 (s, 1H), 7.60- 7.55 (m, 2H), 7.18-7.12 (m, 2H), 6.96-6.94 (m, 2H), 6.76-6.73 (m, 2H), 4.62 (m, 2H), 2.21 (dd, 2H), 1.93 (s, 3H) ppm. [M + H].sup.+ = 486.9 1H NMR (400 MHz, d6-DMSO): 11.35 (s, 1H), 9.95 (s, 1H), 8.23 (s, 1H), 7.75-7.70 (m, 3H), 6.82- 6.79 (m, 4H), 6.35 (s, 1H), 3.48- 3.42 (m, 2H), 3.15-3.13 (m, 2H) ppm. [M + H].sup.+ = 457.8 1H NMR (400 MHz, d6-DMSO): 11.53 (s, 1H), 9.86 (s, 1H), 8.36 (s, 1H), 8.16 (s, 1H), 7.85-7.80 (m, 1H), 6.86-6.82 (m, 2H), 6.33 (s, 1H), 5.85 (s, 2H), 3.48-3.42 (m, 2H), 3.15-3.13 (m, 2H) ppm. [M + H].sup.+ = 453.3 1H NMR (400 MHz, d6-DMSO): 11.17 (s, 1H), 9.32 (s, 1H), 7.50- 7.52 (m, 1H), 6.88-6.83 (m, 2H), 6.28 (s, 1H), 2.10-2.08 (m, 2H), 1.98-1.93 (m, 2H) ppm. 0 [M + H].sup.+ = 376.3 1H NMR (400 MHz, d6-DMSO): 11.30 (s, 1H), 10.02 (s, 1H), 7.68- 7.66 (m, 1H), 6.87-6.83 (m, 2H), 6.87 (s, 1H), 3.53 (t, 2H), 3.40- 3.36 (m, 2H), 3.14-3.11 (m, 2H) ppm. [M − H].sup.− = 451.3 1H NMR (400 MHz, d6-DMSO): 11.05 (s, 1H), 9.86 (s, 1H), 7.65- 7.61 (m, 1H), 6.78-6.74 (m, 2H), 6.48 (s, 1H), 4.32 (s, 1H), 3.48- 3.46 (m, 2H), 3.14-3.12 (m, 2H), 2.85 (s, 3H) ppm. [M − H]− 375.2 1H-NMR (400 MHz, d6-DMSO): δ = 11.47 (s, 1H), 8.88 (s, 1H), 7.25-7.24 (m, 1H), 7.17-7.09 (m, 1H), 6.88-6.84 (m, 1H), 5.88 (s, 1H), 3.28-3.26 (s, 1H), 1.88-1.84 (m, 2H), 1.65-1.60 (m, 2H) ppm. [M − H]− 344.6 1H-NMR (400 MHz, d6-DMSO): δ = 11.48 (s, 1H), 8.93 (s, 1H), 7.26-7.23 (m, 1H), 7.18-7.12 (m, 1H), 6.88-6.82 (m, 1H), 5.85 (s, 1H), 3.38-3.36 (s, 1H), 1.87-1.86 (m, 2H), 1.64-1.62 (m, 2H), 1.23- 1.20 (m, 2H) ppm. [M − H]− 432.3 1H-NMR (400 MHz, d6-DMSO): δ = 11.52 (s, 1H), 11.08 (s, 1H), 7.21-7.17 (m, 1H), 7.14-7.11 (m, 1H), 6.36-6.31 (m, 1H), 5.13-5.11 (m, 2H), 4.63-4.59 (m, 2H), 3.59- 3.53 (m, 2H), 3.49-3.47 (m, 1H), 1.95-1.92 (m, 2H) ppm [M + H]+ 433.1 1H-NMR (400 MHz, d6-DMSO): δ = 11.58 (s, 1H), 8.05 (s, 2H), 7.28-7.26 (m, 1H), 7.17-7.14 (m, 1H), 6.42-6.39 (m, 1H), 5.05-5.02 (m, 2H), 4.68-4.65 (m, 2H), 3.63- 3.58 (m, 2H), 3.50-3.47 (m, 1H), 1.88-1.82 (m, 2H) ppm [M − H]− 417.2 1H-NMR (400 MHz, d6-DMSO): δ = 11.43 (s, 1H), 11.25 (s, 1H), 7.35-7.31 (m, 1H), 7.27-7.25 (m, 1H), 6.32-6.29 (m, 1H, 4.05-4.02 (m, 2H), 3.53-3.51 (m, 2H), 2.30- 2.26 (, 2H), 1.83-1.81 (m, 2H) ppm [M + H]+ 448.3 1H-NMR (400 MHz, d6-DMSO): δ = 11.48 (s, 1H), 7.25-7.21 (m, 1H), 7.18-7.15 (m, 1H), 6.38-6.36 (m, 1H), 5.23-5.19 (m, 2H), 4.66- 4.60 (m, 2H), 3.70 (s, 3H), 3.63- 3.60 (m, 2H), 3.44-3.41 (m, 1H), 1.85-1.83 (m, 2H) ppm [M − H]− 480.1 1H-NMR (400 MHz, d6-DMSO): δ = 11.51 (s, 1H), 9.62 (s, 1H), 8.95 (s, 1H), 7.19-7.07 (m, 3H), 6.76-6.64 (m, 3H), 3.95 (s, 2H), 1.24-1.23 (m, 2H), 0.96-0.89 (m, 2H) ppm. [M + H]+ 495.3 1H-NMR (400 MHz, d.sub.6-DMSO): 11.53 (s, 1H), 8.96 (s, 1H), 7.20-7.18 (m, 1H), 7.15-7.13 (m, 1H), 6.78-6.75 (m, 1H), 6.70 (t, 1H), 6.45 (s, 2H), 6.16 (t, 1H), 3.25 (d, 2H), 2.83 (d, 2H), 1.03 (s, 4H) ppm. 0 [M − H]+ 466.4 1H-NMR (400 MHz, d.sub.6-DMSO): δ = 11.56 (s, 1H), 9.67 (s, 1H), 8.86 (s, 1H), 7.28-7.26 (m, 1H), 7.15-7.12 (m, 1H), 6.99-6.95 (m, 1H), 6.62-6.60 (m, 2H), 6.58- 6.55 (m, 1H), 3.96-3.93 (m, 2H), 3.73 (s, 3H), 3.65-3.60 (m, 1H), 3.11-3.08 (m, 2H) ppm [M + H].sup.+ 406.1 1H-NMR (400 MHz, d6-DMSO): δ = 11.32 (s, 1H), 8.95 (s, 1H), 7.19-7.15 (m, 2H), 6.84-6.82 (m, 1H), 6.66-6.65 (m, 1H), 6.45- 6.40 (m, 1H), 6.31 (m, 1H), 6.26- 6.23 (m, 1H), 4.62 (t, 1H), 4.01- 3.98 (m, 1H), 3.44-3.41 (m, 3H), 3.13-3.08 (m, 1H) ppm [M − H]− 453.7 1H-NMR (400 MHz, d6-DMSO): δ = 11.54 (s, 1H), 9.68 (s, 1H), 8.95 (s, 1H), 7.22-7.18 (t, 1H), 7.12-7.10 (m, 1H), 6.79-6.75 (m, 1H), 6.62 (s, 2H), 6.56-6.53 (t, 1H), 3.96-3.93 (m, 2H), 3.17- 3.15 (m, 2H) ppm. [M − H]− 370.2 1H-NMR (400 MHz, d6-DMSO): δ = 11.51 (s, 1H), 9.64 (s, 1H), 8.93 (s, 1H), 7.26-7.19 (m, 2H), 7.15-7.06 (m, 3H), 6.82-6.78 (m, 1H), 6.54-6.50 (m, 2H), 3.86- 3.83 (m, 2H), 3.62-3.59 (m, 2H), 3.22-3.18 (m, 2H) ppm [M − H]− 481.6 1H-NMR (400 MHz, d6-DMSO): δ = 11.55 (s, 1H), 9.66 (s, 1H), 8.89 (s, 1H), 7.27-7.25 (m, 1H), 7.16-7.13 (m, 1H), 6.88-6.83 (m, 1H), 6.65 (s, 2H), 6.59-6.57 (m, 1H), 3.92-3.89 (m, 2H), 3.87-3.85 (m, 2H), 3.33-3.28 (m, 2H), 3.20- 3.14 (m, 2H) ppm [M − H]− 384.4 1H-NMR (400 MHz, d6-DMSO): δ = 11.50 (s, 1H), 9.67 (s, 1H), 8.98 (s, 1H), 7.26-7.22 (m, 2H), 7.19-7.08 (m, 3H), 6.86-6.84 (m, 1H), 6.55-6.51 (m, 2H), 3.90-3.87 (m, 2H), 3.58-3.53 (m, 1H), 3.25- 3.20 (m, 2H), 1.28-1.22 (m, 3H) ppm. [M + H].sup.+ 430.6 1H-NMR (400 MHz, d6-DMSO): δ = 11.51 (s, 1H), 9.64 (s, 1H), 8.93 (s, 1H), 7.19-7.16 (m, 1H), 6.94-6.92 (m, 1H), 6.01 (m, 1H), 3.26-3.23 (m, 2H), 3.05-3.02 (d, 2H), 2.13-2.08 (m, 4H), 1.56-1.52 (m, 4H) ppm. [M + H].sup.+ 416.3 1H-NMR (400 MHz, d6-DMSO): δ = 11.45 (s, 1H), 8.90 (s, 1H), 7.22-7.18 (t, 1H), 7.12-7.09 (m, 1H), 6.78-6.75 (m, 1H), 6.18-6.15 (m, 1H), 3.90-3.89 (m, 2H), 3.57- 3.55 (m, 1H), 3.33-3.28 (m, 2H), 2.20-2.14 (m, 1H), 1.93-1.92 (m, 2H), 1.45-1.43 (m, 6H) ppm. [M + H]+ 495.0 1H-NMR (400 MHz, d6-DMSO): δ = 11.50 (s, 1H), 8.93 (s, 1H), 7.18-7.16 (m, 1H), 6.90-6.79 (m, 1H), 6.37-6.35 (m, 1H), 6.13 (s, 1H), 3.42-3.40 (m, 1H), 3.21 (s, 2H), 3.08-3.05 (m, 1H), 2.11-2.08 (m, 4H), 1.86-1.82 (m, 4H) ppm. [M + H]+ 448.2 1H-NMR (400 MHz, d6-DMSO): δ = 11.51 (s, 1H), 9.64 (s, 1H), 8.93 (s, 1H), 7.19-7.16 (m, 1H), 6.94-6.92 (m, 1H), 6.67-6.65 (m, 3H), 6.01 (m, 1H), 3.26-3.23 (m, 1H), 3.05-3.02 (m, 1H), 2.13- 2.08 (m, 4H), 1.56-1.52 (m, 4H) ppm. 0 [M + H]+ = 333.5 1H NMR (400 MHz, d6-DMSO): 11.30 (s, 1H), 8.92 (s, 1H), 7.18- 7.15 (m, 2H), 7.06-7.05 (d, 1H), 6.90-6.88 (m, 1H), 4.50 (d, 1H) ppm. [M + H]+ = 272.2 1H NMR (400 MHz, d6-DMSO): 11.42 (s, 1H), 8.98 (s, 1H), 7.16- 7.14 (m, 2H), 7.05-7.02 (d, 1H), 6.90-6.86 (m, 1H), 4.55 (d, 1H) ppm. [M + H]+ = 304.8 1H NMR (400 MHz, d6-DMSO): 11.22 (s, 1H), 8.96 (s, 1H), 7.15- 7.13 (m, 2H), 7.08-7.06 (d, 1H), 6.91-6.88 (m, 1H), 3.58 (d, 1H) ppm. [M − H]− 370.2 1H-NMR (400 MHz, d6-DMSO): δ = 11.52 (s, 1H), 8.93 (s, 1H), 7.26-7.22 (m, 1H), 7.19-7.08 (m, 1H), 6.86-6.84 (m, 1H), 5.89- 5.85 (m, 2H), 4.59-4.56 (m, 1H), 3.46-3.45 (m, 1H), 3.28-3.26 (m, 1H), 1.85-1.83 (m, 2H), 1.25- 1.20 (m, 4H) ppm. [M + H]+ 462.9 1H-NMR (400 MHz, d6-DMSO): δ = 11.08 (s, 1H), 10.37 (s, 1H), 8.99 (s, 1H), 7.27-7.11 (m, 8H), 6.72-6.70 (m, 1H), 3.40 (s, 2H), 3.07 (s, 3H) ppm. [M + H]+ = 318.7 1H NMR (400 MHz, d6-DMSO): 11.62 (s, 1H), 9.21 (s, 1H), 7.55- 7.53 (m, 2H), 7.18-7.16 (d, 1H), 6.81-6.80 (m, 1H), 2.12 (s, 3H) ppm. [M + H]+ = 326.1 1H NMR (400 MHz, d6-DMSO): 11.03 (s, 1H), 9.12 (s, 1H), 7.18- 7.12 (m, 1H), 6.76-6.73 (m, 2H), 2.23-2.21 (m, 4H), 1.90-1.88 (m, 4H) ppm. [M + H]+ = 356.0 1H NMR (400 MHz, d6-DMSO): 11.16 (s, 1H), 9.11 (s, 1H), 7.16- 7.12 (m, 1H), 6.98-6.96 (m, 2H), 6.73 (s, 1H), 2.25-2.23 (t, 1H), 1.90-1.88 (m, 4H), 1.20-1.16 (m, 4H) ppm. [M + H]+ = 531.3 1H NMR (400 MHz, d6-DMSO): 11.35 (s, 1H), 9.76 (s, 1H), 8.56 (s, 1H), 8.26-8.23 (m, 1H), 7.82- 7.81 (m, 2H), 6.96-6.94 (m, 4H), 6.43 (s, 1H), 3.47-3.42 (m, 2H), 3.16-3.13 (m, 2H) ppm. [M + H]+ = 487.3 1H NMR (400 MHz, d6-DMSO): 11.13 (s, 1H), 9.66 (s, 1H), 7.64- 7.60 (m, 1H), 6.76-6.74 (m, 2H), 6.43 (s, 1H), 4.30-4.28 (m, 2H), 3.49-3.45 (m, 2H), 3.15-3.13 (m, 2H), 2.82-2.80 (m, 1H), 1.78-1.75 (m, 4H), 1.34-1.30 (m, 4H) ppm. 0 [M + H]+ = 462.5 1H NMR (400 MHz, d6-DMSO): 11.09 (s, 1H), 9.76 (s, 1H), 7.65- 7.63 (m, 1H), 6.82-6.80 (m, 2H), 6.45 (s, 1H), 4.33-4.29 (m, 2H), 3.48-3.46 (m, 2H), 3.23-3.20 (m, 1H), 2.77-2.75 (m, 1H), 1.43-1.41 (m, 6H) ppm. [M − H]− 689.2 1H-NMR (400 MHz, d6-DMSO): δ = 11.49 (s, 2H), 8.90 (s, 2H), 7.29-7.11 (m, 4H), 6.77-6.73 (m, 2H), 6.36-6.32 (m, 2H), 3.47-3.46 (m, 4H) ppm. [M − H]− 485.5 1H-NMR (400 MHz, d6-DMSO): δ = 11.56 (s, 1H), 9.69 (s, 1H), 8.85 (s, 1H), 7.20-7.18 (t, 1H), 7.12-7.10 (m, 1H), 6.79-6.76 (m, 1H), 6.68 (s, 2H), 6.55-6.54 (t, 1H), 3.96-3.95 (m, 2H), 3.16-3.15 (m, 2H) ppm. [M + H].sup.+ = 374.9 1H NMR (400 MHz, d6-DMSO): 11.05 (s, 1H), 9.65 (s, 1H), 7.53- 7.50 (m, 1H), 6.86-6.83 (m, 2H), 6.18 (s, 1H), 2.18 (s, 3H) ppm. [M + H]+ = 437.0 1H NMR (400 MHz, d6-DMSO): 11.26 (s, 1H), 9.78 (s, 1H), 7.50- 7.45 (m, 4H), 6.85-6.82 (m, 4H), 6.78 (s, 1H) ppm. [M + H]+ = 452.0 1H NMR (400 MHz, d6-DMSO): 11.06 (s, 1H), 9.98 (s, 1H), 7.51- 7.48 (m, 4H), 7.12-7.08 (s, 1H), 6.65-6.62 (m, 4H), 6.78 (s, 1H) ppm. [M + H]+ 575.6 1H-NMR (400 MHz, d6-DMSO): δ = 11.47 (s, 1H), 8.93 (s, 1H), 7.19-7.16 (m, 3H), 6.94-6.92 (m, 1H), 6.67-6.65 (m, 3H), 6.01 (m, 1H), 3.26-3.23 (m, 1H), 2.32-2.30 (m, 2H), 2.13-2.08 (m, 4H), 1.86- 1.82 (m, 2H) ppm. [M + H]+ 438.4 1H-NMR (400 MHz, d6-DMSO): δ = 11.53 (s, 1H), 8.82 (s, 1H), 7.21-7.19 (m, 1H), 6.93-6.89 (m, 1H), 6.47-6.45 (m, 1H), 6.23 (s, 1H), 3.62-3.61 (s, 2H), 3.45-3.43 (m, 1H), 1.96-1.93 (m, 4H), 1.84- 1.82 (m, 4H) ppm. [M + H]+ 519.5 1H-NMR (400 MHz, d6-DMSO): δ = 11.43 (s, 1H), 9.44 (s, 1H), 8.93 (s, 1H), 7.19-7.16 (m, 1H), 6.94-6.92 (m, 3H), 6.65-6.61 (m, 3H), 6.12 (s, 1H), 3.26-3.23 (m, 2H), 3.15-3.12 (m, 2H), 1.85- 1.83 (m, 1H), 1.54-1.50 (m, 8H) ppm. [M + H].sup.+ = 342.6 1H NMR (400 MHz, d6-DMSO): 10.87 (s, 1H), 9.31 (s, 1H), 7.54- 7.51 (m, 1H), 6.88-6.85 (m, 2H), 6.38 (s, 1H), 3.43 (s, 3H), 3.10 (s, 2H) ppm.
Biological Evaluation

Test Example 1. Determination of the IDO1 Inhibitory Activity of Compounds

(16) The present disclosure is further explained below in combination with test examples, but these test examples are not meant to limit the present disclosure. The following disclosures show the inhibitory activity of some compounds of the invention on IDO1 enzyme. The structure formula of the compounds is shown in the examples above.

(17) 1. Materials, Kits and Equipments

(18) Sodium L-ascorbate (Cat: A4034-100G, SIGMA)

(19) 4-(dimethylamino)benzaldehyde (Cat: 156477-25g, SIGMA)

(20) Trichloroacetic acid (Cat: T0699-100ML, SIGMA)

(21) L-Tryptophan (Cat: T8941-25G, SIGMA)

(22) Methylene blue (Cat: M9140-25G, SIGMA)

(23) Potassium dihydrogen phosphate (Cat: 10017618, Sinopharm Chemical Reagent)

(24) Disodium hydrogen phosphate (Cat: 20040618, Sinopharm Chemical Reagent)

(25) Constant temperature water tank (Cat: DK-8D, Shanghai Jinghong Experimental Equipment)

(26) Multifunctional microplate reader (Cat: M5, Molecular Devices)

(27) 96-well reaction plate (Cat: 3590, costar)

(28) IDO1 protease (commercially available)

(29) Desktop Microplate Reader: SpectraMax M5 Microplate Reader (Molecular Devices)

(30) Test compounds: self-made

(31) Positive control agent: INCB024360 (commercially available)

(32) 2. Reagent Preparation

(33) 100 mM PBS:

(34) 100 mM disodium hydrogen phosphate and 100 mM potassium dihydrogen phosphate mixed in a ratio of 3:5, pH 6.5

(35) IDO1 assay buffer:

(36) 100 mM PBS containing 400 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue and 1000 U/ml catalase, pH 6.5

(37) 30% trichloroacetic acid

(38) ddH.sub.2 O solution of 30% trichloroacetic acid

(39) Ehrlich reagent

(40) 1% (w/v) diluted solution of 4-(dimethylamino) benzaldehyde compound

(41) All compounds were dissolved with DMSO. During the assay, each compound was diluted to a concentration as needed. The compound of each concentration was added to multi-wells, and the final concentration of DMSO was controlled at 1%.

(42) 3. Test Method

(43) a.) the reaction mixture was prepared by adding 50 nM IDO1 and the desired concentration of the test compound to 100 μL of IDO1 assay buffer. IDO1 and assay buffer need to be preheated to 37° C.

(44) b.) The mixture was reacted in a constant temperature water tank at 37° C. for 30 minutes.

(45) c.) 50 μL of 30% trichloroacetic acid was added.

(46) d.) The above mixture was reacted in a constant temperature water tank at 52° C. for 30 minutes.

(47) e.) The reaction mixture was centrifuged at 12000 g for 10 minutes at room temperature.

(48) f.) 100 μL of the obtained supernatant and 100 μL of Ehrlich reagent were mixed.

(49) g.) the absorbance at 480 nm was measured using an M5 microplate reader.

(50) 4. Data Analysis
Inhibition rate=(OD.sub.postive−OD.sub.sample)/(OD.sub.positive−OD.sub.negative)*100%
5. Results and Discussion

(51) In this experiment, the inhibitory activity of the test compounds on the IDO1 enzyme was tested. Compounds of each diluted concentration were tested in multi-wells. The final concentration of the reaction system in DMSO was controlled to 1%. The inhibition rate was tested twice, and the average value was taken. The experimental results are in the table below, which show that the compounds of the present disclosure exhibit good inhibitory activity against IDO1 protease.

Inhibitory Activity of Test Compounds on IDO1 Enzyme

(52) TABLE-US-00002 Compound No. IC.sub.50(nm) I-1 80 I-2 43 I-3 108 I-4 96 I-5 32 I-6 162 I-7 113 I-8 94 I-9 85 I-10 90 I-11 120 I-12 35 I-13 124 I-14 81 I-15 93 I-16 42 I-17 85 I-18 183 I-19 140 I-20 18 I-21 82 I-22 64 I-23 36 I-24 8 I-25 68 I-26 92 I-27 42 I-28 184 I-29 80 I-30 65 I-31 42 I-32 125 I-33 99 I-34 104 I-35 94 I-36 82 I-37 53 I-38 78 I-39 142 I-40 12 I-41 69 I-42 36 I-43 35 I-44 78 I-45 92 I-46 65 I-47 48 I-48 72 I-49 66 I-50 28 I-51 83 INCB024360 32
Conclusion: the assay results show that the compound of the invention has a significant inhibitory effect on IDO, and the effect is comparable or even better than INCB024360.

Test Example 2. Determination of the Inhibitory Activity of Compounds on IDO Protease in Highly Expressed HeLa Cells

(53) This method was used to determine the inhibitory effect of the compounds of the invention on the inhibitory activity of IDO protease in highly expressed HeLa cells.

(54) 1. Reagents and Materials:

(55) Desktop Microplate Reader SpectraMax M5 Microplate Reader (Molecular Devices)

(56) Multifunctional microplate reader (Cat: M5, Molecular Devices)

(57) L-Tryptophan (Cat: T8941-25G, SIGMA)

(58) 4-(dimethylamino)benzaldehyde (Cat: 156477-25g, SIGMA)

(59) Trichloroacetic acid (Cat: T0699-100ML, SIGMA)

(60) Highly expressed HeLa cell line

(61) 2. Test Method

(62) a.) 50 μL of 30% trichloroacetic acid was added.

(63) b.) The mixture was reacted in a constant temperature water tank at 52° C. for 30 minutes.

(64) c.) The above mixture was centrifuge at 12,000 g for 10 minutes at room temperature.

(65) d.) 100 μL of the supernatant was mixed with 100 μL of Ehrlich reagent.

(66) e.) the absorbance was measured at 480 nm using an M5 microplate reader.

(67) 3. Data Processing

(68) The inhibition rate of the tumor cell growth was calculated by the following formula: tumor cell growth inhibition rate %=[(Ac−As)/(Ac−Ab)]×100%

(69) The software Graphpad Prism 5 was used with the calculation formula of log(inhibitor) vs. normalized response to perform IC.sub.50 curve fitting and the IC.sub.50 value was calculated. The results are shown in the following table:

(70) TABLE-US-00003 Compound No. IC.sub.50(nm) I-2 38 I-5 58 I-12 43 I-16 35 I-20 8 I-23 38 I-24 5 I-27 26 I-40 12 I-42 28 I-43 38 I-47 47 I-50 21 INCB024360 33
Conclusion: the test results show that the compound of the invention has a significant inhibitory effect on HeLa intracellular IDO protease.

Test Example 3. Pharmacokinetic Evaluation

(71) Pharmacokinetic tests were conducted on compounds I-20, I-24, I-40 of the invention and the compound INCB024360 to study their pharmacokinetic behavior in rats and evaluate their pharmacokinetic characteristics.

(72) 1. Experimental animals: 100 (half male and half female) SPF grade SD rats were purchased from Shanghai Cypre-Bikai Experimental Animal Co., Ltd. Among them, 72 medically qualified and healthy SD rats without abnormality (half male and half female) were used for the study.
2. Animal Administration

(73) 72 SD rats (half male and half female) were tested according to the following table. The drug samples were stirred at least 10 minutes before administration.

(74) TABLE-US-00004 Adminis- Adminis- tration tration Mode of Group Test Dosage.sup.a concentration volume adminis- No. substance mg/kg mg/mL mL/kg tration 1 I-20 75 7.5 10 PO.sup.b 2 I-24 75 7.5 10 PO.sup.b 3 I-40 75 7.5 10 PO.sup.b 4 INCB024360 75 7.5 10 PO.sup.b Note: *Before oral administration, all animals were fasted overnight (10-14 hours) and fed 4 hours after administration.
3. Sample Collection and Processing

(75) Blood samples were obtained through the jugular vein puncture, and each sample was collected for about 0.25 mL and added with heparin sodium for anticoagulation. The blood sampling was performed at time points as follows:

(76) for oral administration group, before administration, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h after administration.

(77) After collection, the blood samples were placed on ice and the plasma was separated by centrifugation (centrifugation conditions: 8000 rpm, 6 minutes, 2-8° C.). The collected plasma was stored at −80° C. before analysis. Plasma samples were analyzed by the analysis department of the experimental institution using LC-MS/MS for the content of the test compound in the rat plasma. The LLOQ of the detection substance was 1 ng/mL.

(78) 4. Pharmacokinetic Analysis

(79) According to the blood plasma concentration data of the drugs, the pharmacokinetic calculation software WinNonlin5.2 non-compartmental model was used to calculate the pharmacokinetic parameters AUC.sub.0-t, Cmax, Tmax, T.sub.1/2 d and other parameters of the test samples and their average value and standard deviation.

(80) When pharmacokinetic parameters were calculated for samples with lower concentration than the lower limit of quantification, samples taken before the curve reached Cmax should be calculated with zero value, and samples taken after the curve reached Cmax should be calculated with non-quantitative (BLQ).

(81) 5. Results and Discussion

(82) Main Pharmacokinetic Parameters

(83) According to the blood plasma concentration data of the drugs, the pharmacokinetic parameters of I-20, I-24, I-40, INCB024360 were respectively calculated using the pharmacokinetic calculation software WinNonlin5.2 non-compartment model, and provided in the table below.

(84) The main pharmacokinetic parameters of compounds in SD rats plasma after oral administration by single gavage

(85) TABLE-US-00005 Gender t½ T.sub.max C.sub.max AUC.sub.(0-∞) Group No. F/M h h ng/mL ng/mL*h I-24 M 2.12 1.0 8761.8 20653.4 F 2.34 1.0 7243.3 18743.9 I-20 M 1.98 1.0 4238.3 2385.3 F 1.42 0.5 3325.8 1985.5 I-40 M 1.67 1.0 4761.8 8253.4 F 1.45 1.0 4243.3 7543.9 INCB024360 M 2.24 0.5 3937.4 12164.3 F 2.51 1.0 3412.8 11296.9
Conclusion: The compound of the invention has good pharmacokinetic absorption and obvious pharmacokinetic absorption effect. Compared with INCB024360, the compound of the invention has better pharmacokinetic properties and broad market prospects.