IDO1 INHIBITOR AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

A compound as an indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor, and an application thereof in the field of IDO1-related diseases, and in particular a compound as shown in formula (I) and a pharmaceutically acceptable salts thereof.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, ##STR00344## wherein D is O, S or S(O); L is selected from a single bond, or selected from the group consisting of C.sub.1-10 alkyl- group, C.sub.3-6 cycloalkyl- group, C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl- group, -phenyl-group, 3- to 6-membered heterocycloalkyl- group, and 3- to 6-membered heterocycloalkyl-C.sub.1-3 alkyl- group, one or more of which are optionally substituted by 1, 2, or 3 R groups; R.sub.1 is selected from the group consisting of H, F, Cl, Br, I, OH, and NH.sub.2, or selected from the group consisting of C.sub.1-6 alkyl group, C.sub.3-6 cycloalkyl group, C.sub.1-6 heteroalkyl group, N,N-bis(C.sub.1-6 alkyl)amino group, 3- to 6-membered heterocycloalkyl group, C.sub.2-6 alkenyl group, phenyl group, 5- to 9-membered heteroaryl group, ##STR00345## one or more of which are optionally substituted by 1, 2, or 3 R groups; R.sub.2 is OH or CN; R.sub.3, R.sub.4, and R.sub.5 are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, and NH.sub.2, or selected from the group consisting of C.sub.1-6 alkyl group, C.sub.3-6 cycloalkyl group, C.sub.1-6 heteroalkyl group, N,N-bis(C.sub.1-6 alkyl)amino group, and 3- to 6-membered heterocycloalkyl group, one or more of which are optionally substituted by 1, 2, or 3 R groups; R is selected from the group consisting of H, F, Cl, Br, I, OH, CN, and NH.sub.2, or selected from the group consisting of C.sub.1-6 alkyl group, C.sub.1-6 heteroalkyl group, N,N-bis(C.sub.1-6 alkyl)amino group, C.sub.3-6 cycloalkyl group, C.sub.2-6 alkenyl group, phenyl group, and thienyl group, one or more of which are optionally substituted by 1, 2, or 3 R groups; R is selected from the group consisting of F, Cl, Br, I, OH, CN, and NH.sub.2; hetero moieties in the -3- to 6-membered heterocycloalkyl, the -3- to 6-membered heterocycloalkyl-C.sub.1-3 alkyl-, the C.sub.1-6 heteroalkyl, the 3- to 6-membered heterocycloalkyl, or the 5- to 9-membered heteroaryl groups are each independently selected from the group consisting of C(O)NH, NH, S(O).sub.2NH, S(O)NH, N, O, S, O, S, C(O)O, C(O), C(S), S(O), S(O).sub.2, NHC(O)NH, NHC(S)NH, and H.sub.2P(O)NH groups; and in any of above cases, number of heteroatom or heteroatom group is each independently selected from 1, 2 or 3.

2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R is selected from the group consisting of H, F, Br, I, OH, CN, and NH.sub.2, or selected from the group consisting of C.sub.1-6 alkyl group, C.sub.1-6 alkoxy group, C.sub.1-6 alkylamino group, N,N-bis(C.sub.1-6 alkyl)amino group, C.sub.2-6 alkenyl group, C.sub.3-6 cycloalkyl group, phenyl group, and thienyl group, one or more of which are optionally substituted by 1, 2, or 3 R groups.

3. The compound or the pharmaceutically acceptable salt thereof according to claim 2, wherein R is selected from the group consisting of H, F, Cl, Br, I, OH, CN, and NH.sub.2, or selected from the group consisting of Me, Et, ##STR00346## one or more of which are optionally substituted by 1, 2, or 3 R groups.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 3, wherein R is selected form the group consisting of H, F, Cl, Br, I, OH, CN, NH2, Me, Et, CF3, CHF2, CH2F, ##STR00347##

5. The compound or the pharmaceutically acceptable salt thereof according to any claim 1, wherein L is selected from a single bond, or selected from the group consisting of C.sub.1-5 alkyl- group, C.sub.3-6 cycloalkyl- group, C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl- group, and -3- to 6-membered azacycloalkyl-C.sub.1-3 alkyl- group, one or more of which are optionally substituted by 1, 2 or 3 R groups.

6. The compound or the pharmaceutically acceptable salt thereof according to claim 5, wherein L is selected from a single bond, or selected from the group consisting of CH.sub.2 group, ##STR00348## one or more of which are optionally substituted by 1, 2, or 3 R groups.

7. The compound or the pharmaceutically acceptable salt thereof according to claim 6, wherein L is selected from the group consisting of a single bond, CH.sub.2 group, ##STR00349##

8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 is selected from the group consisting of H, F, Cl, Br, I, OH, and NH.sub.2, or selected from the group consisting of C.sub.1-6 alkyl group, C.sub.1-6 alkoxy group, C.sub.1-6 alkylthio group, C.sub.1-6 alkylamino group, N,N-bis(C.sub.1-3 alkyl)amino group, C.sub.3-6 cycloalkyl group, tetrahydrofuryl group, oxetanyl group, C.sub.2-6 alkenyl group, phenyl group, thienyl group, pyridyl group, imidazolyl group, thiazolyl group, 2-oxo-imidazolidinyl group, NH.sub.2C(S)NH group, C.sub.1-6 alkyl-S(O) group, C.sub.1-6 alkyl-S(O).sub.2 group, C.sub.1-6 alkoxy-C(O)NH group, NH.sub.2S(O)NH group, NH.sub.2C(O) group, NH.sub.2C(O)NH group, HC(O)NH group, HS(O).sub.2NH group, ##STR00350## one or more of which are optionally substituted by 1, 2 or 3 R groups.

9. The compound or the pharmaceutically acceptable salt thereof according to claim 8, wherein R.sub.1 is selected from the group consisting of H, F, Cl, Br, I, OH, and NH.sub.2, or selected from the group consisting of Me, Et, BOCNH, CH.sub.2CH, ##STR00351## one or more of which are optionally substituted by 1, 2, or 3 R groups.

10. The compound or the pharmaceutically acceptable salt thereof according to claim 9, wherein R.sub.1 is selected from the group consisting of H, F, Cl, Br, I, OH, NH.sub.2, ##STR00352## Me, CF.sub.3, BOCNH, CH.sub.2CH, ##STR00353## ##STR00354##

11. The compound or the pharmaceutically acceptable salt thereof according to claim 7, wherein a structural unit R.sub.1-L- is selected from the group consisting of H, NH.sub.2, ##STR00355## Me, BOCNH, ##STR00356## ##STR00357## ##STR00358##

12. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.3, R.sub.4, and R.sub.5 are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, and NH.sub.2, or selected from the group consisting of Me, Et, C.sub.3-6 cycloalkyl, and C.sub.1-3 alkoxy, one or more of which are optionally substituted by 1, 2, or 3 R groups.

13. The compound or the pharmaceutically acceptable salt thereof according to claim 12, wherein R.sub.3, R.sub.4, and R.sub.5 are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, and NH.sub.2, or selected from the group consisting of Me, Et, ##STR00359## one or more of which are optionally substituted by 1, 2, or 3 R groups.

14. The compound or the pharmaceutically acceptable salt thereof according to claim 13, wherein R.sub.3, R.sub.4, and R.sub.5 are each independently selected from the group consisting of H, F, Cl, Br, I, CN, CH.sub.2F, CHF.sub.2, CF.sub.3, ##STR00360##

15. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein a structural unit ##STR00361## is selected from ##STR00362##

16. The compound or the pharmaceutically acceptable salt thereof according to claim 14, wherein the structural unit ##STR00363## is selected from the group consisting of ##STR00364##

17. The compound or the pharmaceutically acceptable salt thereof according to claim 1, represented by: ##STR00365## wherein R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are as defined claim 1; R.sub.6 is selected from H, or selected from the group consisting of C.sub.1-3 alkyl group and C.sub.3-6 cycloalkyl group, one or more of which are optionally substituted by 1, 2, or 3 R groups; and provided that R.sub.6 is not Me.

18. The compound or the pharmaceutically acceptable salt thereof according to claim 17, wherein R.sub.6 is selected from the group consisting of H, CF.sub.3, Et, ##STR00366##

19. The compound or the pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of ##STR00367## ##STR00368## ##STR00369## ##STR00370## ##STR00371## ##STR00372## ##STR00373## ##STR00374## ##STR00375## ##STR00376## ##STR00377##

20. A pharmaceutical composition, comprising a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according claim 1 as an active ingredient and a pharmaceutical acceptable carrier.

21. A method for treatment of IDO1-related diseases in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0075] FIG. 1 shows the effect of a tested drug on the body weight of tumor-bearing mice;

[0076] FIG. 2 shows the effect of the tested drug on the volume of a transplanted tumor; and

[0077] FIG. 3 shows the effect of the tested drug on the weight of the transplanted tumor.

DETAILED DESCRIPTION OF EMBODIMENTS

Reference Example 1

Segment BB-1

[0078] ##STR00035##

Synthetic Route:

[0079] ##STR00036##

[0080] Step 1: Synthesis of Compound BB-1-2

[0081] BB-1-1 (20.00 g, 302.76 mmol, 19.05 mL, 1.00 eq) was dissolved in water (436.00 mL), and stirred for 5 minutes. The reaction solution was cooled in an ice bath to 0 C. Sodium nitrite (22.98 g, 333.04 mmol, 18.09 mL, 1.10 eq) was added, and then hydrochloric acid (6 M, 3.53 mL, 0.07 eq) was added. After 15 minutes, the ice bath was removed. After the reaction solution was stirred at 25 C. for 1.5 hours, a 50% hydroxylamine aqueous solution (60.00 g, 908.28 mmol, 3.00 eq) was added all at once. After the reaction solution was stirred continuously at 25 C. for 1 hour, the reaction solution was slowly heated to reflux. The reaction was carried out at reflux for 2 hours before being slowly cooled to 25 C., and was allowed to further react for 16 hours. At 0 C., the reaction solution was adjusted to have pH=7.0 with 6 N hydrochloric acid (70 mL, slowly added dropwise for about 30 minutes), and continued to be stirred at 0 C. for 1 hour. A solid precipitate appeared and was filtered and washed with water. A light yellow solid was collected and dried in the air, without the need of further purification. The product BB-1-2 (38.08 g, yield: 87.89%, purity: 100%) was finally obtained as a light yellow solid. MS (ESI) m/z:144 [M+H].sup.+.

[0082] Step 2: Synthesis of Compound BB-1-3

[0083] The compound BB-1-2 (38.08 g, 266.11 mmol, 1.00 eq) was dissolved in a mixed solution of water (532.00 mL), acetic acid (270.00 mL) and hydrochloric acid (6 M, 133.06 mL, 3.00 eq). The resultant mixture was heated to 45 C. and stirred until the solution was completely clear (about 0.5 hours). Sodium chloride (46.65 g, 798.33 mmol, 3.00 eq) was added. The mixed reaction solution was cooled to 0 C. Sodium nitrite (17.99 g, 260.79 mmol, 14.17 mL, 0.98 eq) (dissolved in 63 mL of water) was slowly added dropwise to the reaction solution (more than 0.5 hours), while maintaining the temperature at 0 C. during addition. After the addition, the reaction solution continued to stir at 0 C. for 2 hours. With LCMS monitoring showing completion of reaction of raw materials, a precipitated solid was filtered and washed with water (6*60 mL). The solid obtained from the suction filtration was dissolved in ethyl acetate (400 mL), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation, without the need of purification. A light yellow solid product BB-1-3 (18.83 g, yield: 40.63%, purity: 93.33%) was finally obtained. MS (ESI) m/z: 163 [M+H].sup.+

[0084] Step 3: Synthesis of Compound BB-1-5

[0085] The compound BB-1-3 (2.00 g, 12.30 mmol, 1.00 eq) was dissolved in ethanol (25.00 mL), and compound BB-1-4 (4.67 g, 24.60 mmol, 2.00 eq) was added. After the mixed reaction solution was allowed to react at 85 C. for 16 hours. While being heated the reaction solution gradually turned brown. Complete reaction of raw materials was observed by LCMS monitoring and a desired compound was generated. The reaction solution was dried by rotary evaporation under reduced-pressure distillation. A crude product was separated and purified by flash silica gel column chromatography (petroleum ether:ethyl acetate=2:1), to yield a light gray solid product BB-1-5 (3.60 g, yield: 88.39%, purity: 95.46%). MS (ESI) m/z:316, 318 [M+H].sup.+.

[0086] Step 4: Synthesis of Compound BB-1-6

[0087] The compound BB-1-5 (3.60 g, 11.39 mmol, 1.00 eq) was dissolved in tetrahydrofuran (30.00 mL), and carbonyl diimidazole (2.03 g, 12.53 mmol, 1.10 eq) was added. The mixed reaction solution was allowed to react at 65 C. for 1 hour. With LCMS monitoring showing completion of reaction of raw materials, 20 mL of water was added, followed by extraction with ethyl acetate (25 mL*3). Organic phases were combined, washed with 1 M hydrochloric acid (20 mL*2), washed with brine, dried over anhydrous sodium sulfate, filtered, and dried by rotary evaporation under reduced-pressure distillation, without further purification. A dust-gray solid product BB-1-6 (3.55 g, yield: 91.11%, purity: 100%) was obtained. MS (ESI) m/z:342, 344 [M+H].sup.+.

[0088] Step 5: Synthesis of Compound BB-1

[0089] At 0 C., sulfuric acid (35.00 mL) was slowly added to hydrogen peroxide (41.30 g, 364.30 mmol, 35.00 mL, 30% purity, 41.97 eq), then sodium tungstate (2.55 g, 8.68 mmol, 1.00 eq) was added followed by the compound BB-1-6 (2.97 g, 8.68 mmol, 1.00 eq). The mixture was heated to 25 C. and stirred for 16 hours. With LCMS monitoring showing about half of raw materials remaining, the mixture was diluted by addition of 250 mL of water, and subjected to suction filtration. A resultant white solid was rinsed with water (25 mL*3). The solid was dissolved in ethyl acetate (200 mL), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation. Purification was performed by flash silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain a light yellow solid product BB-1 (1.29 g, yield: 39.20%, purity: 98.14%). MS (ESI) m/z: 372, 374 [M+H].sup.+.

Reference Example 2

Segment BB-2

[0090] ##STR00037##

Synthetic Route:

[0091] ##STR00038##

[0092] Step 1: Synthesis of Compound BB-2

[0093] The compound BB-2-1 (25.00 mg, 328.73 umol, 20.00 uL, 1.00 eq) and methyl amine (44.39 mg, 657.46 umol, 2.00 eq, hydrochloride) were dissolved in N,N-dimethyl formamide (1.00 mL). Diisopropylethylamine (254.91 mg, 1.97 mmol, 344.47 uL, 6.00 eq) and HATU (187.49 mg, 493.10 umol, 1.50 eq) were added. The reaction solution turned yellow from colorless, and the reaction solution was allowed to react at 4 C. for 16 hours. Complete reaction of raw materials was observed by TLC monitoring (petroleum ether:ethyl acetate=1:1). Five mL of water was added to the reaction solution, followed by extraction with ethyl acetate (5 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation to obtain a crude product. The reaction succeeded, and a light yellow liquid product BB-2 (30.00 mg, crude product) was obtained.

Reference Example 3

Segment BB-3

[0094] ##STR00039##

Specific Route

[0095] ##STR00040##

[0096] Step 1: Synthesis of Compound BB-3

[0097] The compound BB-3-1 (571.78 mg, 4.04 mmol, 350.79 uL, 1.00 eq) was added to dichloromethane (5 mL), to which a dichloromethane (8 mL) solution containing tertiary butanol (314.42 mg, 4.24 mmol, 403.10 uL, 1.05 eq) was added dropwise at 0 C. The reaction solution was stirred at 0 C. for 1 hour. A dichloromethane solution (13 mL) of a target product BB-3 (871.00 mg, crude product) was obtained and directly used for reaction in the next step.

Reference Example 4

Segment BB-4

[0098] ##STR00041##

Synthetic Route:

[0099] ##STR00042##

[0100] Step 1: Synthesis of Compound BB-4-2

[0101] The compound BB-1 (1.00 g, 2.69 mmol, 1.00 eq) was dissolved in tetrahydrofuran (15.00 mL) and water (500.00 uL), to which the compound BB-4-1 (650.44 mg, 4.04 mmol, 625.42 uL, 1.50 eq) and sodium hydroxide (118.36 mg, 2.96 mmol, 1.10 eq) were added. The reaction solution was stirred at 25 C. for 16 hours. Complete reaction of raw materials was observed by LCMS monitoring and a desired compound was generated. Five mL of water was added, followed by extraction with ethyl acetate (5 mL*3), drying over anhydrous sodium sulfate, and filtration. A filtrate was dried by rotary evaporation under reduced-pressure distillation, without further purification, to obtain a liquid product BB-4-2 as yellow oil (1.73 g, crude product).

[0102] Step 2: Synthesis of Compound BB-4

[0103] The compound BB-4-2 (1.73 g, 3.56 mmol, 1.00 eq) was dissolved in dichloromethane (10.00 mL). Hydrochloric acid/dioxane (4 M, 889.46 uL, 1.00 eq) was added. The reaction solution turned turbid and white from yellow, and reacted at 25 C. for 1 hour. A white solid precipitated. Complete reaction of raw materials was observed by LCMS monitoring, and a main product peak was generated. The reaction solution was dried by rotary evaporation to obtain a crude product, without purification. The reaction succeeded. A white solid product BB-4 (1.48 g, crude product, hydrochloride) was obtained. MS (ESI) m/z:386, 388 [M+H].sup.+.

Reference Example 5

Segment BB-5

[0104] ##STR00043##

Synthetic Route:

[0105] ##STR00044##

[0106] Step 1: Synthesis of Compound BB-5-2

[0107] The compound BB-1 (2.50 g, 6.72 mmol, 1.00 eq) was dissolved in tetrahydrofuran (20.00 mL) and water (1.00 mL). Sodium bicarbonate (846.74 mg, 10.08 mmol, 392.01 uL, 1.50 eq) was added. The mixed solution was allowed to react at 14 C. for 16 hours. Complete reaction of raw materials was observed by LCMS monitoring and a main new product peak was generated. Twenty mL of water was added to the reaction, followed by extraction with ethyl acetate (30 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation. Purification was performed by flash silica gel column chromatography (petroleum ether:ethyl acetate=4:1). The reaction succeeded, and a white solid product BB-5-2 (3.29 g, yield: 97.47%) was obtained. MS (ESI) m/z: 502, 504 [M+H].sup.+.

[0108] Step 2: Synthesis of Compound BB-5

[0109] The compound BB-5-2 (4.09 g, 8.14 mmol, 1.00 eq) was dissolved in dichloromethane (30.00 mL). Hydrochloric acid/dioxane (4 M, 30.00 mL, 14.74 eq) was added. The reaction solution was allowed to react at 14 C. for 1 hour. It is observed by LCMS monitoring that 9.4% of raw materials remained, and a target compound was generated. The reaction solution was directly dried by rotary evaporation under reduced-pressure distillation to obtain a crude product. The reaction succeeded, and a white solid product BB-5 (3.57 g, crude product, hydrochloride) was obtained. MS (ESI) m/z: 402, 404 [M+H].sup.+.

Reference Example 6

Segment BB-6

[0110] ##STR00045##

Synthetic Route:

[0111] ##STR00046##

[0112] Step 1: Synthesis of Compound BB-6-2

[0113] The compound BB-1 (200.00 mg, 537.55 umol, 1.00 eq) was dissolved in tetrahydrofuran (4.00 mL) and water (800.00 uL), and then sodium bicarbonate (112.90 mg, 1.34 mmol, 52.27 uL, 2.50 eq) and the compound BB-6-1 (68.47 mg, 645.06 umol, 58.52 uL, 1.20 eq) were added. The reaction solution was allowed to react at 15 C. for 14 hours. The reaction solution was combined with another batch of reaction solution in an amount of 30 mg and the combined reaction solution was concentrated to remove the tetrahydrofuran solvent, then diluted by addition of 5 mL of water, extracted with ethyl acetate (10 mL*3), and combined. Organic phases were dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was dissolved by 10 mL of ethyl acetate, mixing with silica gel, and separated by an automatic column chromatography device (petroleum ether:ethyl acetate=1:03:1) to obtain a white solid product BB-6-2 (200.00 mg, yield: 71.90%). MS (ESI) m/z:431, 433 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.66 (dd, J=5.77, 2.51 Hz, 1H), 7.29-7.39 (m, 2H), 4.09 (s, 2H), 3.83 (s, 3H).

[0114] Step 2: Synthesis of Compound BB-6

[0115] The compound BB-6-2 (100.00 mg, 231.92 umol, 1.00 eq) was dissolved in methanol (2.00 mL) and water (1.00 mL), to which sodium hydroxide (37.11 mg, 927.68 umol, 4.00 eq) was added. The reaction solution was allowed to react at 15 C. for 1.5 hours. The reaction solution was concentrated to remove the solvent, diluted by addition of 5 mL of water, extracted with ethyl acetate (5 mL*3), and combined and concentrated to obtain a liquid crude product as light yellow oil. Five mL of dichloromethane was added to the crude product, and the mixture was concentrated to remove the solvent and to obtain a white solid product BB-6 (90.00 mg, crude product) which was directly used for reaction in a next step. MS (ESI) m/z: 413, 415 [M+Na].sup.+.

Reference Example 7

Segment BB-7

[0116] ##STR00047##

Synthetic Route:

[0117] ##STR00048##

[0118] Taking compounds BB-1 and BB-7-1 as raw materials, BB-7 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 412, 414 [M+H].sup.+.

Reference Example 8

Segment BB-8

[0119] ##STR00049##

Synthetic Route:

[0120] ##STR00050##

[0121] Taking the compounds BB-1 and BB-8-1 as raw materials, BB-8 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 400, 402 [M+H].sup.+.

Reference Example 9

Segment BB-9

[0122] ##STR00051##

Synthetic Route:

[0123] ##STR00052##

[0124] Taking compounds BB-1 and BB-9-1 as raw materials, BB-9 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 462, 464 [M+H].sup.+.

Reference Example 10

Segment BB-10

[0125] ##STR00053##

Synthetic Route:

[0126] ##STR00054##

[0127] Taking compounds BB-1 and BB-10-1 as raw materials, BB-10 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 400, 402 [M+H].sup.+.

Reference Example 11

Segment BB-11

[0128] ##STR00055##

Synthetic Route:

[0129] ##STR00056##

[0130] Taking compounds BB-1 and BB-11-1 as raw materials, BB-11 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 400, 402 [M+H].sup.+.

Reference Example 12

Segment BB-12

[0131] ##STR00057##

Synthetic Route:

[0132] ##STR00058##

[0133] Step 1: Synthesis of Compound BB-12-2

[0134] Taking compounds BB-1 and BB-11-1 as raw materials, a segment BB-12-2 was synthesized according to the synthesis step 1 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 534, 536 [M+Na].sup.+.

[0135] Step 2: Synthesis of Compound BB-12

[0136] The compound BB-12-2 (110.00 mg, 214.72 umol, 1.00 eq) was dissolved in dichloromethane (10.00 mL), to which trifluoroacetic acid (1.54 g, 13.51 mmol, 1.00 mL, 62.90 eq) was added, followed by stirring at 25 C. for 1 hour. No remaining raw materials are observed by LCMS monitoring and a desired product was generated. The reaction solution was adjusted to basic with pH of about 89, and subsequently 100 milliliters of dichloromethane wad added. The resultant mixture was washed with water (30 mL*3). Organic phases were dried over anhydrous sodium sulfate, and concentrated under a reduced pressure created by a water pump to obtain a liquid product BB-12 as gray oil (85.00 mg, crude product) which was ready for next step without further purification. MS (ESI) m/z: 412, 414 [M+H].sup.+.

Reference Example 13

Segment BB-13

[0137] ##STR00059##

Synthetic Route:

[0138] ##STR00060##

[0139] Taking compounds BB-1 and BB-13-1 as raw materials, BB-13 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 440, 442 [M+H].sup.+.

Reference Example 14

Segment BB-14

[0140] ##STR00061##

Synthetic Route:

[0141] ##STR00062##

[0142] Taking compounds BB-1 and BB-14-1 as raw materials, BB-14 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-4 in Reference Example 4. MS (ESI) m/z: 412, 414 [M+H].sup.+.

Reference Example 15

Segment BB-15

[0143] ##STR00063##

Synthetic Route:

[0144] ##STR00064##

[0145] Step 1: Synthesis of Compound BB-15-2

[0146] Taking compounds BB-1 and BB-15-1 as raw materials, a segment BB-15-2 was synthesized according to the synthesis step 1 for the segment BB-6 in Reference Example 6. MS (ESI) m/z: 459, 461 [M+H].sup.+.

[0147] Step 2: Synthesis of Compound BB-15

[0148] The compound BB-15-2 (400.00 mg, 870.99 umol, 1.00 eq) was dissolved in water (600.00 uL) and tetrahydrofuran (1.80 mL). Lithium hydroxide hydrate (73.09 mg, 1.74 mmol, 2.00 eq) was then added. The resultant mixture was stirred and reacted at 20 C. for 3 hours, whereby a product was generated albeit in a small amount. The reaction continued to stir and react for another 20 hours. The reaction solution was adjusted to pH of 67 by addition of hydrochloric acid (6 M), and dried by rotary evaporation. Methanol (5 mL) was added, followed by filtration, isolation by high performance liquid chromatography (Phenomenex Synergi C18 150*30 mm*4 um, water (0.05% HCl)-ACN), and lyophilization. The target compound BB-15 was obtained as a yellow solid (60.00 mg, yield: 17.00%, purity:100%). MS (ESI) m/z: 405, 407 [M+H].sup.+.

Reference Example 16

Segment BB-16

[0149] ##STR00065##

Synthetic Route:

[0150] ##STR00066##

[0151] Taking compounds BB-1-3 and BB-16-1 as raw materials, a segment BB-16 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 319 [M+H].sup.+.

Reference Example 17

Segment BB-17

[0152] ##STR00067##

Synthetic Route:

[0153] ##STR00068##

[0154] Taking compounds BB-1-3 and BB-17-1 as raw materials, a segment BB-17 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1, and the synthesis steps 1-2 for the segment BB-4. MS (ESI) m/z: 376 [M+H].sup.+.

Reference Example 18

Segment BB-18

[0155] ##STR00069##

Synthetic Route:

[0156] ##STR00070##

[0157] Step 1: Synthesis of Compound BB-18

[0158] The compounds BB-1-4 (1.00 g, 5.26 mmol, 1.00 eq), BB-18-1 (587.38 mg, 6.84 mmol, 1.30 eq), potassium phosphate (3.91 g, 18.41 mmol, 3.50 eq), triphenylphosphine (137.96 mg, 526.00 umol, 0.10 eq), and palladium acetate (59.05 mg, 263.00 umol, 0.05 eq) were dissolved in toluene (24.00 mL) and water (2.00 mL), heated to 100 C. in nitrogen ambient and reacted for 16 hours. After being heated, the reaction solution gradually turned dark brown from brown. Complete reaction of raw materials was observed by LCMS monitoring and a target compound was generated. The reaction solution was cooled to 23 C., followed by addition of 20 mL of water, extraction with ethyl acetate (20 mL*3), drying over anhydrous sodium sulfate, and filtration. A filtrate was dried by rotary evaporation under reduced-pressure distillation, and purified by flash silica gel column chromatography (petroleum ether:ethyl acetate=10:1). The reaction succeeded, and a yellow liquid product BB-18 (790.00 mg, yield: 99.35%) was obtained. MS (ESI) m/z: 152 [M+H].sup.+.

Reference Example 19

Segment BB-19

[0159] ##STR00071##

Synthetic Route:

[0160] ##STR00072##

[0161] Taking the compounds BB-1-3 and BB-18 as raw materials, a segment BB-19 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 334 [M+H].sup.+.

Reference Example 20

Segment BB-20

[0162] ##STR00073##

Synthetic Route:

[0163] ##STR00074##

[0164] Step 1: Synthesis of Compound BB-20-2

[0165] At 0 C., to a dichloromethane solution (20 mL) of the compound BB-3 (5.39 g, 24.99 mmol, 1.00 eq), triethylamine (7.59 g, 75.03 mmol, 10.40 mL, 3.00 eq) was added. After the mixture was stirred at 0 C. for 30 minutes, the compound BB-20-1 (1.60 g, 26.13 mmol, 1.58 mL, 1.05 eq) was added. The mixed solution was heated to 23 C. and reacted for 16 hours. Complete reaction of raw materials was observed by TLC (petroleum ether:ethyl acetate=1:1) monitoring. The reaction solution was then subjected to rotary evaporation under reduced-pressure distillation to remove dichloromethane, adjusted to pH=5 with 1 M hydrochloric acid, and extracted with ethyl acetate (20 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation to obtain a white solid product BB-20-2 (5.44 g, crude product). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.83 (s, 1H) 5.88 (t, 1H) 3.73-3.80 (t, 2H) 3.26 (q, 2H) 1.48-1.50 (m, 9H).

[0166] Step 2: Synthesis of Compound BB-20

[0167] The compound BB-20-2 (2.00 g, 8.32 mmol, 1.00 eq) was dissolved in dichloromethane (10.00 mL), and hydrochloric acid/dioxane (4 M, 10.00 mL, 4.81 eq) was added. The mixed solution was allowed to react at 24 C. for 2 hours. Complete reaction of raw materials was observed by TLC (petroleum ether:ethyl acetate=1:1) monitoring. The reaction solution was then directly dried by rotary evaporation under reduced-pressure distillation to obtain a brown liquid product BB-20 (1.10 g, crude product). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 6.49 (s, 2H) 3.81-4.19 (s, 1H) 3.43-3.52 (t, 2H) 2.91-2.99 (t, 2H).

Reference Example 21

Segment BB-21

[0168] ##STR00075##

Synthetic Route:

[0169] ##STR00076##

[0170] Step 1: Synthesis of Compound BB-21

[0171] A compound BB-21-1 (2.00 g, 10.47 mmol, 1.00 eq) was dissolved in methanol (20.00 mL), and then 10% carbon-supported palladium (200.00 mg) was added. The reaction solution was allowed to react at 25 C. in 15 Psi hydrogen gas for 16 hours. LCMS showed that reactant 1 was completely consumed and a product peak appeared. The reaction solution was filtered. A filtrate was dried by rotary evaporation to obtain a product BB-21 as yellow oil (1.60 g, crude product) which was directly used for reaction in a next step. MS (ESI) m/z: 162 [M+H].sup.+.

Reference Example 22

Segment BB-22

[0172] ##STR00077##

Synthetic Route:

[0173] ##STR00078##

[0174] Taking the compounds BB-1-3 and BB-21 as raw materials, a segment BB-22 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 344 [M+H].sup.+.

Reference Example 23

Segment BB-23

[0175] ##STR00079##

Synthetic Route:

[0176] ##STR00080##

[0177] Taking compounds BB-1-3 and BB-23-1 as raw materials, a segment BB-23 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 346 [M+H].sup.+.

Reference Example 24

Segment BB-24

[0178] ##STR00081##

Synthetic Route:

[0179] ##STR00082##

[0180] Taking compounds BB-1-3 and BB-24-1 as raw materials, a segment BB-24 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 342 [M+H].sup.+.

Reference Example 25

Segment BB-25

[0181] ##STR00083##

Synthetic Route:

[0182] ##STR00084##

[0183] Taking compounds BB-1-3 and BB-25-1 as raw materials, a segment BB-25 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 344 [M+H].sup.+.

Reference Example 26

Segment BB-26

[0184] ##STR00085##

Synthetic Route:

[0185] ##STR00086##

[0186] Taking compounds BB-1-3 and BB-26-1 as raw materials, a segment BB-26 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 312 [M+H].sup.+.

Reference Example 27

Segment BB-27

[0187] ##STR00087##

Synthetic Route:

[0188] ##STR00088##

[0189] Taking compounds BB-1-3 and BB-27-1 as raw materials, a segment BB-27 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 378 [M+H].sup.+.

Reference Example 28

Segment BB-28

[0190] ##STR00089##

Synthetic Route:

[0191] ##STR00090##

[0192] Taking compounds BB-1-3 and BB-28-1 as raw materials, a segment BB-28 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 390, 392 [M+H].sup.+.

Reference Example 29

Segment BB-29

[0193] ##STR00091##

Synthetic Route:

[0194] ##STR00092##

[0195] Taking compounds BB-1-3 and BB-29-1 as raw materials, a segment BB-29 was synthesized according to the synthesis steps 3-5 for the segment BB-1 in Reference Example 1. MS (ESI) m/z: 328 [M+H].sup.+.

Reference Example 30

Segment BB-30

[0196] ##STR00093##

Synthetic Route:

[0197] ##STR00094##

[0198] Taking the compounds BB-17-4 and BB-5-1 as raw materials, BB-30 in the reference example was synthesized according to the synthesis steps 1-2 for the segment BB-5 in Reference Example 5. MS (ESI) m/z: 392 [M+H].sup.+.

Reference Example 31

Segment BB-31

[0199] ##STR00095##

Synthetic Route:

[0200] ##STR00096## ##STR00097##

[0201] Step 1: Synthesis of Compound BB-31-2

[0202] At 0 C., sulfuric acid (30.00 mL) was slowly added to hydrogen peroxide (35.40 g, 312.26 mmol, 30.00 mL, 30% purity, 27.93 eq), then sodium tungstate (3.28 g, 11.18 mmol, 1.00 eq) was added, to which the compound BB-31-1 (1.60 g, 11.18 mmol, 1.00 eq) was added. The resultant mixture was heated to 15 C. and reacted for 3 hours. With LCMS monitoring showing 1.88% of raw materials remaining, 30 mL of water was added to the reaction solution, followed by extraction with ethyl acetate (50 mL*3). Organic phases were combined, washed with water (50 mL*3), washed with a saturated sodium chloride solution (50 mL*3), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation to obtain a yellow liquid product BB-31-2 (1.40 g, yield: 72.35%, crude product). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 4.00-4.02 (s, 3H).

[0203] Step 2: Synthesis of Compound BB-31-3

[0204] The compound BB-31-2 (1.40 g, 8.09 mmol, 1.00 eq) was dissolved in tetrahydrofuran (10.00 mL) and water (1.00 mL), the compound BB-5-1 (1.58 g, 8.90 mmol, 1.10 eq) was added, and then sodium bicarbonate (1.36 g, 16.18 mmol, 629.63 uL, 2.00 eq) was added. The mixed solution was allowed to react at 15 C. for 2 hours. With LCMS monitoring showing completion of reaction of raw materials, 10 mL of water was added, followed by extraction with ethyl acetate (15 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation. Purification was performed by flash silica gel column chromatography (petroleum ether:ethyl acetate=4:1). The reaction succeeded, and a light pink solid product BB-31-3 (2.57 g, yield: 96.28%, purity: 91.93%) was obtained. MS (ESI) m/z: 304 [M+H].sup.+.

[0205] Step 3: Synthesis of Compound BB-31-4

[0206] The compound BB-31-3 (2.57 g, 8.47 mmol, 1.00 eq) was dissolved in tetrahydrofuran (10.00 mL) and water (5.00 mL), and lithium hydroxide hydrate (888.50 mg, 21.18 mmol, 2.50 eq) was added. The reaction solution was allowed to react at 15 C. for 1 hour. With LCMS monitoring showing completion of reaction of raw materials, 5 mL of water was added. The resultant mixture was adjusted to have pH=6 with concentrated hydrochloric acid, and extracted with ethyl acetate (15 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation. The reaction succeeded, and a light yellow solid product BB-31-4 (2.70 g, crude product) was obtained. MS (ESI) m/z: 290 [M+H].sup.+.

[0207] Step 4: Synthesis of Compound BB-31-5

[0208] The compound BB-31-4 (590.00 mg, 2.04 mmol, 1.00 eq) was added to N,N-dimethylformamide (10.00 mL), then the compound BB-33 (531.68 mg, 2.24 mmol, 1.10 eq), HATU (930.50 mg, 2.45 mmol, 1.20 eq), and diisopropylethylamine (527.13 mg, 4.08 mmol, 712.33 uL, 2.00 eq) were added. The reaction solution was allowed to react at 15 C. for 2 hours. With LCMS showing completion of the reaction, the reaction solution was adjusted to 5 in pH value by addition of 1 N hydrochloric acid, extracted with ethyl acetate (30 mL2), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation to obtain a product BB-31-5 as yellow oil (850.00 mg, yield: 81.97%). MS (ESI) m/z: 531 [M+Na].sup.+.

[0209] Step 5: Synthesis of Compound BB-31-6

[0210] The compound BB-31-5 (100.00 mg, 196.73 umol, 1.00 eq) was added to toluene (3.00 mL), and then pyridine (147.00 mg, 1.86 mmol, 150.00 uL, 9.45 eq), and phosphorus pentachloride (81.93 mg, 393.46 umol, 2.00 eq) were added. The reaction solution was allowed to react at 80 C. for 2 hours. With TLC (petroleum ether:ethyl acetate=4:1) showing completion of consumption of a reactant 1 and generation of a main product peak, the reaction solution was dried by rotary evaporation to obtain a yellow solid product BB-31-6 (105.00 mg, crude product).

[0211] Step 6: Synthesis of Compound BB-31-7

[0212] The compound BB-31-6 (100.00 mg, 189.84 umol, 1.00 eq) was added to ethanol (3.00 mL), and cooled to 0 C., and then 50% hydroxylamine aqueous solution (251.03 mg, 3.80 mmol, 20.02 eq) was added. The reaction solution was allowed to react at 0 C. for 2 hours. With LCMS showing completion of the reaction, the reaction solution was extracted with ethyl acetate (30 mL2), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation to obtain a crude product as yellow oil. The crude product was separated (ethyl acetate) through a thick preparative plate and purified to obtain a white solid product BB-31-7 (70.00 mg, yield: 64.82%, purity: 92%). MS (ESI) m/z: 546 [M+Na].sup.+.

[0213] Step 7: Synthesis of Compound BB-31-8

[0214] The compound BB-31-7 (80.00 mg, 152.87 umol, 1.00 eq) was added to tetrahydrofuran (3.00 mL), and then carbonyl diimidazole (27.27 mg, 168.16 mmol, 1.10 eq) was added. The reaction solution was allowed to react at 60 C. for 1 hour. With LCMS showing completion of reaction, the reaction solution was diluted by addition of ethyl acetate (50 mL), washed with a saturated saline (10 mL), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation to obtain a yellow solid product BB-31-8 (80.00 mg, crude product). MS (ESI) m/z: 550 [M+H].sup.+.

[0215] Step 8: Synthesis of Compound BB-31

[0216] The compound BB-31-8 (80.00 mg, 145.64 umol, 1.00 eq) was added to dichloromethane (2.00 mL), and then hydrochloric acid/dioxane (4 M, 1.90 mL, 52.31 eq) was added. The reaction solution was allowed to react at 15 C. for 2 hours. With LCMS showing completion of reaction, the reaction solution was dried by rotary evaporation to obtain a product BB-31 as yellow oil (71.00 mg, crude product, hydrochloride). MS (ESI) m/z: 450 [M+H].sup.+.

Reference Example 32

Segment BB-32

[0217] ##STR00098##

Synthetic Route:

[0218] ##STR00099##

[0219] Taking the compounds BB-31-4 and BB-32-1 as raw materials, BB-32 in the reference example was synthesized according to the synthesis steps 4-8 for the segment BB-31 in Reference Example 31. MS (ESI) m/z: 432 [M+H].sup.+.

Reference Example 33

Segment BB-33

[0220] ##STR00100##

Synthetic Route:

[0221] ##STR00101##

[0222] Step 1: Synthesis of Compound BB-33-2

[0223] The compound BB-33-1 (1.20 g, 7.69 mmol, 1.00 eq) was dissolved in hydrochloric acid (4.00 mL), and cooled to 0 C. Sodium nitrite (583.67 mg, 8.46 mmol, 459.58 uL, 1.10 eq) was dissolved in 2.6 mL of water, and added dropwise to the reaction solution. After being stirred for 15 minutes, the mixed solution was slowly added to an aqueous solution (16 mL) of potassium iodide (4.47 g, 26.92 mmol, 3.50 eq), and heated to 10 C. and stirred for 16 hours. With TLC (petroleum ether:ethyl acetate=10:1) monitoring showing completion of reaction of raw materials, a target compound was generated. The resulting compound was diluted by addition of ethyl acetate (30 mL), followed by washing with 10% sodium hydroxide (25 mL*2), washing with 5% sodium sulfite (25 mL*2), drying over anhydrous sodium sulfate, and filtration. A filtrate was dried by rotary evaporation under reduced-pressure distillation. Purification was performed by flash silica gel column chromatography (petroleum ether:ethyl acetate=10:1). The reaction succeeded, and a yellow solid product BB-33-2 (550.00 mg, yield: 21.22%, purity: 79.22%) was obtained. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.60 (dd, J=5.3, 2.8 Hz, 1H), 8.19 (ddd, J=9.0, 4.3, 2.8 Hz, 1H), 7.09-7.17 (m, 1H).

[0224] Step 2: Synthesis of Compound BB-33

[0225] The compound BB-33-2 (1.45 g, 5.43 mmol, 1.00 eq) was dissolved in acetic acid (10.00 mL) and ethanol (10.00 mL), and an iron powder (1.52 g, 27.15 mmol, 5.00 eq) was added. The reaction solution was allowed to react at 60 C. for 20 minutes. Complete reaction of raw materials was observed by LCMS monitoring, and a target compound was generated. The reaction solution was filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation, dissolved in 40 mL of ethyl acetate, washed with saturated sodium bicarbonate (30 mL*3), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation. Purification was performed by flash silica gel column chromatography (petroleum ether:ethyl acetate=4:1). The reaction succeeded, and a brown liquid product BB-33 (900.00 mg, yield: 53.92%, purity: 77.1%) was obtained. MS (ESI) m/z: 238 [M+H].sup.+.

Reference Example 34

Segment BB-34

[0226] ##STR00102##

Synthetic Route:

[0227] ##STR00103##

[0228] Step 1: Synthesis of Compound BB-34

[0229] The compound BB-34-1 (293.00 mg, 2.00 mmol, 1.00 eq, HCl) was dissolved in DMF (500.00 uL), and DIEA (258.49 mg, 2.00 mmol, 349.31 uL, 1.00 eq) was added. The reaction solution was allowed to react at 25 C. for 64 hr, and the reaction solution gradually turned into a white turbid solution to obtain a DMF solution (0.5 ml) of segment BB-34 (200.00 mg, crude, HCl).

Example 1

Compound 0052

[0230] ##STR00104##

Synthetic Route:

[0231] ##STR00105##

[0232] Step 1: Synthesis of Compound 0052

[0233] A compound BB-1-7 (300.00 mg, 806.32 umol, 1.00 eq) was dissolved in methanol (4.00 mL). Sodium hydroxide (129.01 mg, 3.23 mmol, 4.00 eq) was dissolved in water (1.00 mL) and added to the reaction solution. After the addition of sodium hydroxide, the reaction solution turned yellow from colorless, and a solid precipitated. Upon continuous stirring, the solid gradually disappeared. The reaction was carried out at a room temperature for 16 hours. Complete reaction of raw materials was observed by LCMS monitoring, and a desired compound was generated. The resulting compound mixture was adjusted to pH=5 with 1 M hydrochloric acid, followed by rotary evaporation under reduced-pressure distillation to remove methanol, extraction with ethyl acetate (10 mL*3), drying over anhydrous sodium sulfate, and filtration. A filtrate was dried by rotary evaporation under reduced-pressure distillation, and dissolved in methanol, and filtered. A filtrate was separated by high performance liquid chromatography (Phenomenex Synergi C18 150*30 mm*4 um water (0.05% HCl)-ACN), to obtain a product 0052 (167.72 mg, yield: 62.82%, purity: 100%). MS (ESI) m/z: 331, 333 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) 7.10-7.15 (m, 1H), 7.06 (t, 1H), 6.77-6.81 (m, 1H), 3.95 (s, 3H).

Example 2

Compound 0103

[0234] ##STR00106##

Synthetic Route:

[0235] ##STR00107##

[0236] Taking a compound BB-16 as raw material, a compound 0103 was synthesized according to the synthesis step 1 for the compound 0052 in Example 1. MS (ESI) m/z: 278 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.69 (s, 1H), 9.14 (s, 1H), 7.34 (t, 1H), 7.23 (dd, 1H), 7.07-7.17 (m, 1H), 4.00 (s, 2H).

Example 3

Compound 0124

[0237] ##STR00108##

Synthetic Route:

[0238] ##STR00109##

[0239] Step 1: Synthesis of Compound 0124

[0240] The compound BB-1-7 (50.00 mg, 134.39 umol, 1.00 eq) was added to water (100.00 uL) and tetrahydrofuran (4.00 mL), and then the compound 0124-1 (23.68 mg, 268.78 umol, 21.73 uL, 2.00 eq) and sodium hydroxide (21.50 mg, 537.56 umol, 4.00 eq) were added. The reaction solution was stirred at 25 C. for 16 hours. With LCMS showing completion of consumption of a reactant 1 and generation of a main product peak, the reaction solution was adjusted to pH1=5 with 6 M hydrochloric acid, and filtered. A filtrate was purified by preparative high performance liquid chromatography (column: Boston Green ODS 150*30 5 u; mobile phase: [water (0.05% HCl)-ACN]; B %:40%-70%, 10 min) to obtain a product 0124 (35.00 mg, yield: 61.48%, purity: 100%, hydrochloride). MS (ESI) m/z: 387, 389 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD):=7.19-7.11 (m, 1H), 7.10-7.01 (m, 1H), 6.82-6.72 (m, 1H), 5.19-5.09 (m, 1H), 3.88-3.63 (m, 4H), 2.25-2.13 (m, 1H), 1.92-1.81 (m, 1H).

[0241] Various examples in the following table were synthesized according to the synthetic method of step 1 in Example 3 (compound 0124):

TABLE-US-00001 Example Structure Segment 1 Segment 2 MS m/z Compound 4 [00110] [00111] [00112] 432 434 [M + H].sup.+ 0078 5 [00113] [00114] [00115] 403 405 [M + H].sup.+ 0128 6 [00116] [00117] [00118] 387 389 [M + H].sup.+ 0129 7 [00119] [00120] [00121] 422 424 [M + H].sup.+ 0131 8 [00122] [00123] [00124] 464 466 [M + H].sup.+ 0133 9 [00125] [00126] [00127] 400 402 [M + H].sup.+ 0135 10 [00128] [00129] [00130] 389 491 [M + H].sup.+ 0139 11 [00131] [00132] [00133] 389 491 [M + H].sup.+ 0140 12 [00134] [00135] [00136] 397 399 [M + H].sup.+ 0143 13 [00137] [00138] [00139] 414 416 [M + H].sup.+ 0144 14 [00140] [00141] [00142] 427 429 [M + H].sup.+ 0150 15 [00143] [00144] [00145] 361 363 [M + H].sup.+ 0177 16 [00146] [00147] [00148] 459 461 [M + H].sup.+ 0178 17 [00149] [00150] [00151] 429 431 [M + H].sup.+ 0179 18 [00152] [00153] [00154] 437 439 [M + H].sup.+ 0180 19 [00155] [00156] [00157] 445 447 [M + H].sup.+ 0182 20 [00158] [00159] [00160] 430 432 [M + H].sup.+ 0183 21 [00161] [00162] [00163] 457 459 [M + H].sup.+ 0134 22 [00164] [00165] [00166] 415 417 [M + H].sup.+ 0137 23 [00167] [00168] [00169] 373 375 [M + H].sup.+ 0138 24 [00170] [00171] [00172] 386 [M + H].sup.+ 0120 25 [00173] [00174] [00175] 401 [M + H].sup.+ 0122 26 [00176] [00177] [00178] 411 [M + H].sup.+ 0151 27 [00179] [00180] [00181] 413 [M + H].sup.+ 0156 28 [00182] [00183] [00184] 409 [M + H].sup.+ 0158 29 [00185] [00186] [00187] 411 [M + H].sup.+ 0160 30 [00188] [00189] [00190] 379 [M + H].sup.+ 0162 31 [00191] [00192] [00193] 445 [M + H].sup.+ 0184 32 [00194] [00195] [00196] 457 459 [M + H].sup.+ 0189 33 [00197] [00198] [00199] 395 [M + H].sup.+ 0190

Example 34

0026

[0242] ##STR00200##

Synthetic Route:

[0243] ##STR00201##

[0244] Step 1: Synthesis of Compound 0026

[0245] The compound BB-1 (100.00 mg, 268.77 umol, 1.00 eq) was dissolved in tetrahydrofuran (2.00 mL) and water (1.00 mL), then sodium hydroxide (43.00 mg, 1.08 mmol, 4.00 eq) was added. The resultant mixture was stirred at 25 C. for 2 hours. The reaction solution was dried by rotary evaporation to obtain a crude product as yellow oil. The crude product was purified by preparative high performance liquid chromatography (column: Boston Green ODS 150*30 5 u, condition: water (0.05% HCl)-ACN) to obtain a product 0026 (15.00 mg, yield: 17.60%, purity: 100%). MS (ESI) m/z: 317, 319 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.25-7.13 (m, 1H), 7.12-6.99 (m, 1H), 6.94-6.77 (m, 1H).

Example 35

0077

[0246] ##STR00202##

Synthetic Route:

[0247] ##STR00203##

[0248] Step 1: Synthesis of Compound 0077

[0249] The compound 0078 (20.00 mg, 46.27 umol, 1.00 eq) was dissolved in dichloromethane (500.00 uL), then trifluoroacetic acid (256.69 mg, 2.25 mmol, 166.68 uL, 48.65 eq) was added, and then the resultant mixture was stirred at 25 C. for 1 hour. With LCMS showing completion of most of reaction, the reaction solution was adjusted to pH=7 by addition of a saturated sodium bicarbonate solution, and filtered. A filtrate was dried by rotary evaporation to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (water (0.05% ammonia hydroxide v/v)-ACN, column: DuraShell 150*25 mm*5 um) to obtain a product 0077 (8.00 mg, yield: 51.16%, purity: 98.27%). MS (ESI) m/z: 332, 334 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3): 7.21-7.15 (m, 1H), 7.08-6.97 (m, 2H), 6.85-6.79 (m, 1H), 6.76-6.67 (m, 2H).

Example 36

0147

[0250] ##STR00204##

Synthetic Route:

[0251] ##STR00205##

[0252] Step 1: Synthesis of Compound 0147

[0253] The compound BB-4 (560.00 mg, 1.33 mmol, 1.00 eq, hydrochloric acid) was added to methanol (6.00 mL) and water (2.00 mL), and then sodium hydroxide (4 M, 1.33 mL, 4.00 eq) was added. The reaction solution was stirred at 25 C. for 16 hours. With LCMS showing completion of reaction, the reaction solution was diluted by addition of 60 mL of ethyl acetate, washed with saline (20 mL2), dried over anhydrous sodium sulfate, filtered, and dried by rotary evaporation to obtain a crude product. 80.00 mg of the crude product was subjected to preparative high performance liquid chromatography (column: Boston Green ODS 150*30 5 u; mobile phase:[water (0.05% HCl)-ACN]; B %: 13%-43%, 10 min) to obtain the product 0147 (60.00 mg, yield: 67.76%, purity: 99.5%, hydrochloride). MS (ESI) m/z: 360, 362 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.23-7.14 (m, 1H), 7.11-7.03 (m, 1H), 6.88-6.78 (m, 1H), 4.60-4.49 (m, 2H), 3.42-3.36 (m, 2H).

Example 37

0108

[0254] ##STR00206##

Synthetic Route:

[0255] ##STR00207##

[0256] Step 1: Synthesis of Compound 0108

[0257] The compound 0147 (80.00 mg, 222.14 umol, 1.00 eq) was added to dichloromethane (2.00 mL), and then diisopropylethylamine (86.13 mg, 666.42 umol, 116.39 uL, 3.00 eq) and benzoyl chloride (37.47 mg, 266.57 umol, 30.97 uL, 1.20 eq) were added. The reaction solution was stirred at 25 C. for 16 hours. Though LCMS showed completion of reactants, there were some dibenzoyl byproducts. The reaction solution was dried by rotary evaporation to obtain a crude product as yellow oil (120.00 mg, crude product). The crude product (110.00 mg, 193.54 umol, 1.00 eq) was added to methanol (2.00 mL) and water (1.00 mL), and then sodium hydroxide (23.23 mg, 580.63 umol, 3.00 eq) was added. The reaction solution was stirred at 25 C. for 1 hour. With LCMS showing completion of reaction, the reaction solution was filtered. A filtrate was subjected to preparative high performance liquid chromatography (Boston Green ODS 150*30 5 u, water (0.1% TFA)-ACN) for separation to obtain a product 0108 (20.00 mg, yield: 22.26%). MS (ESI) m/z: 464, 466 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.89-7.80 (m, 2H), 7.61-7.53 (m, 1H), 7.52-7.45 (m, 2H), 7.14-7.09 (m, 1H), 6.94-6.88 (m, 1H), 6.83-6.74 (m, 1H), 4.48-4.33 (m, 2H), 3.79-3.65 (m, 2H).

Example 38

0015

[0258] ##STR00208##

Synthetic Route:

[0259] ##STR00209##

[0260] Step 1: Synthesis of Compound 0015-1

[0261] The compound BB-4 (1.48 g, 3.50 mmol, 1.00 eq, hydrochloric acid) was added to dichloromethane (15 mL), and diisopropylethylamine (452.63 mg, 3.50 mmol, 611.66 uL, 1.00 eq) was added. The reaction solution turned into brown clear liquid from a turbid state. Then a dichloromethane (13 mL) solution of the compound BB-3 (830.25 mg, 3.85 mmol, 1.10 eq) was added dropwise, and the reaction solution gradually turned yellow. After being stirred at 0 C. for 2 hours, the reaction solution turned into a white turbid state. With LCMS monitoring showing about 40% of raw materials remaining, diisopropylethylamine (1.36 g, 10.50 mmol, 1.83 mL, 3.00 eq) was further added, the reaction solution turned into a brown clear state, and then the compound BB-3 (830.25 mg, 3.85 mmol, 1.10 eq) was added dropwise. The reaction was allowed to continue at 25 C. for 16 hours. Complete reaction of raw materials was observed by TLC (petroleum ether:ethyl acetate=1:1) monitoring. The reaction solution was then dried by rotary evaporation to obtain a liquid product 0015-1 as yellow oil (2.14 g, crude product). MS (ESI) m/z: 565, 567 [M+H].sup.+.

[0262] Step 2: Synthesis of Compound 0015-2

[0263] The compound 0015-1 (2.14 g, 3.79 mmol, 1.00 eq) was dissolved in dichloromethane (10.00 mL), and hydrochloric acid/dioxane (4 M, 10.00 mL, 10.55 eq) was added, followed by reacting at 25 C. for 1 hour. With LCMS monitoring showing completion of reaction of raw materials, the reaction solution was dried by rotary evaporation under reduced-pressure distillation to obtain a yellow liquid product 0015-2 (1.78 g, crude product). MS (ESI) m/z: 465, 467 [M+H].sup.+.

[0264] Step 3: Synthesis of Compound 0015

[0265] The compound 0015-2 (1.78 g, 3.83 mmol, 1.00 eq) was dissolved in methanol (8.00 mL) and water (4.00 mL), and sodium hydroxide (612.20 mg, 15.30 mmol, 4.00 eq) was added. The mixed solution was allowed to react at 25 C. for 20 hours. With LCMS showing completion of reaction, the mixed solution was adjusted to have pH=5 with 1 M hydrochloric acid, and extracted with ethyl acetate (15 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation, dissolved in methanol, and filtered. A filtrate was subjected to high performance liquid chromatography (Phenomenex Synergi C18 150*30 mm*4 um water (0.05% HCl)-ACN) for separation to obtain a product 0015 (532.65 mg, yield: 31.66%, purity: 100%). MS (ESI) m/z: 439, 441 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) 7.12-7.18 (m, 1H), 7.05-7.11 (m, 1H), 6.85 (ddd, 1H), 4.36 (t, 2H), 3.38 (t, 2H).

[0266] Various examples in the following table were synthesized according to the synthetic method of steps 1-3 in Example 38 (compound 0015):

TABLE-US-00002 Example Structure Segment 1 39 [00210] [00211] 40 [00212] [00213] 41 [00214] [00215] 42 [00216] [00217] 43 [00218] [00219] 44 [00220] [00221] 45 [00222] [00223] 46 [00224] [00225] 47 [00226] [00227] 48 [00228] [00229] 49 [00230] [00231] 50 [00232] [00233] Example Segment 2 MS m/z Compound 39 [00234] 465 467 [M + H].sup.+ 0070 40 [00235] 453 455 [M + H].sup.+ 0071 41 [00236] 514 517 [M + H].sup.+ 0089 42 [00237] 453 455 [M + H].sup.+ 0118 43 [00238] 453 455 [M + H].sup.+ 0119 44 [00239] 429 [M + H].sup.+ 0121 45 [00240] 455 457 [M + H].sup.+ 0117 46 [00241] 445 [M + H].sup.+ 0157 47 [00242] 465 467 [M + H].sup.+ 0222 48 [00243] 465 467 [M + H].sup.+ 0225 49 [00244] 503 [M + H].sup.+ 0273 50 [00245] 485 [M + H].sup.+ 0295

Example 51

0068

[0267] ##STR00246##

Synthetic Route:

[0268] ##STR00247##

[0269] Step 1: Synthesis of Compound 0068-1

[0270] A compound BB-1 (50.00 mg, 134.39 umol, 1.00 eq) was dissolved in water (100.00 uL) and tetrahydrofuran (5.00 mL), then sodium thiomethoxide (18.84 mg, 268.78 umol, 17.13 uL, 2.00 eq) was added, and then the resultant mixture was stirred at 25 C. for 16 hours. With LCMS showing completion of reaction, the reaction solution was dried by rotary evaporation to obtain a product 0068-1 as yellow oil (51.00 mg, crude product) which was directly used for reaction in a next step. MS (ESI) m/z: 373, 375 [M+H].sup.+.

[0271] Step 2: Synthesis of Compound 0068

[0272] The compound 0068-1 (50.00 mg, 133.99 umol, 1.00 eq) was dissolved in water (300.00 uL) and tetrahydrofuran (2.00 mL), then sodium hydroxide (18.76 mg, 468.97 umol, 17.13 uL, 3.50 eq) was added, and then the resultant mixture was stirred at 25 C. for 3 hours. With LCMS showing completion of most of reaction, the reaction solution was adjusted to have a pH value of 7 by addition of 1 M hydrochloric acid, and filtered. A filtrate was subjected to high performance liquid chromatography (water (0.05% HCl)-ACN, column: Boston Green ODS 150*30 5 u) to obtain a product 0068 (15.00 mg, yield: 32.25%, purity: 100%). MS (ESI) m/z: 347, 349 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.08-7.04 (m, 2H), 6.78-6.77 (m, 1H), 2.62 (s, 3H).

Example 52

0148

[0273] ##STR00248##

Synthetic Route:

[0274] ##STR00249##

[0275] Step 1: Synthesis of Compound 0148-2

[0276] The compound BB-4 (50.00 mg, 118.32 umol, 1.00 eq, hydrochloric acid) was dissolved in dichloromethane (1.00 mL), and diisopropylethylamine (45.88 mg, 354.96 umol, 61.99 uL, 3.00 eq) and a compound 0148-1 (15.17 mg, 130.15 umol, 8.67 uL, 1.10 eq) were added. The mixed solution was allowed to react at 23 C. for 2 hours. With LCMS monitoring showing completion of reaction of raw materials, the reaction solution was directly dried by rotary evaporation under reduced-pressure distillation to obtain a colorless liquid product 0148-2 (56.00 mg, crude product).

[0277] Step 2: Synthesis of Compound 0148

[0278] The compound 0148-2 (56.00 mg, 120.12 umol, 1.00 eq) was dissolved in methanol (1.00 mL) and water (120.00 uL), and sodium hydroxide (28.83 mg, 720.72 umol, 6.00 eq) was added. The reaction solution gradually turned into a brown solution. The mixed solution was allowed to react at 21 C. for 16 hours. With LCMS monitoring showing completion of reaction of raw materials, the reaction solution was subjected to rotary evaporation under reduced-pressure distillation to remove methanol, followed by addition of 3 mL of water, and adjusted to pH=2 with 1 M hydrochloric acid, and extracted with ethyl acetate (5 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation, dissolved in 3 mL of methanol, and filtered. The filtrate was separated by high performance liquid chromatography (Phenomenex Synergi C18 150*30 mm*4 um water (0.05% HCl)-ACN) to obtain a product 0148 (15.73 mg, yield: 27.47%, purity: 100%, hydrochloride). MS (ESI) m/z: 438, 440 [MH].sup.. .sup.1H NMR (400 MHz, CD.sub.3OD) 7.18 (dd, 1H), 7.08 (t, 1H), 6.81-6.89 (m, 1H), 4.56-4.63 (m, 2H), 3.55-3.66 (m, 2H).

[0279] Various examples in the following table were synthesized according to the synthetic method of steps 1-2 in Example 52 (compound 0148):

TABLE-US-00003 Example Structure Segment 1 53 [00250] [00251] 54 [00252] [00253] 55 [00254] [00255] 56 [00256] [00257] 57 [00258] [00259] 58 [00260] [00261] 59 [00262] [00263] 60 [00264] [00265] 61 [00266] [00267] 62 [00268] [00269] 63 [00270] [00271] 64 [00272] [00273] 65 [00274] [00275] 66 [00276] [00277] Example Segment 2 MS m/z Compound 53 [00278] 438 440 [M + H].sup.+ 0106 54 [00279] 500 502 [M + H].sup.+ 0107 55 [00280] 464 466 [M + H].sup.+ 0141 56 [00281] 492 494 [M + H].sup.+ 0142 57 [00282] 436 438 [M + H].sup.+ 0149 58 [00283] 418 420 [M + H].sup.+ 0227 59 [00284] 468 470 [M + H].sup.+ 0228 60 [00285] 454 456 [M + H].sup.+ 0229 61 [00286] 480 482 [M + H].sup.+ 0230 62 [00287] 483 484 [M + H].sup.+ 0232 63 [00288] 433 435 [M + H].sup.+ 0233 64 [00289] 459 461 0234 [M + H].sup.+ 65 [00290] 469 471 [M + H].sup.+ 0240 66 [00291] 434 436 [M + H].sup.+ 0250

Example 67

0153

[0280] ##STR00292##

Synthetic Route:

[0281] ##STR00293##

[0282] Step 1: Synthesis of Compound 0153-1

[0283] The compound BB-4 (50.00 mg, 129.49 umol, 1.00 eq) and formic acid (8.94 mg, 194.24 umol, 7.33 uL, 1.50 eq) were dissolved in N,N-dimethylformamide (1.00 mL), and diisopropylethylamine (66.94 mg, 517.96 umol, 90.46 uL, 4.00 eq) and HATU (59.08 mg, 155.39 umol, 1.20 eq) were added. The reaction mixture was allowed to react at 20 C. for 16 hours. Complete reaction of raw materials was observed by LCMS monitoring, and a target compound was generated. Five mL of water was added, followed by extraction with ethyl acetate (5 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation to obtain a light yellow liquid product 0153-1 (55.00 mg, crude product). MS (ESI) m/z: 414, 416 [M+H].sup.+.

[0284] Step 2: Synthesis of Compound 0153

[0285] The compound 0153-1 (55.00 mg, 132.81 umol, 1.00 eq) was dissolved in methanol (1.00 mL) and water (200.00 uL), and sodium hydroxide (21.25 mg, 531.22 umol, 4.00 eq) was added. The mixed solution was allowed to react at 21 C. for 16 hours. Complete reaction of raw materials was observed by LCMS monitoring, and a target compound was generated. The resulting compound is subjected to rotary evaporation under reduced-pressure distillation to remove methanol, followed by adjustment to pH1=2 with 1 M hydrochloric acid, and extraction with ethyl acetate (5 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation, dissolved in 3 mL of methanol, and filtered. A filtrate was separated by high performance liquid chromatography (Phenomenex Synergi C18 150*30 mm*4 um water (0.05% HCl)-ACN) to obtain a product 0153 (40.09 mg, yield: 71.09%, purity: 100%, hydrochloride). MS (ESI) m/z: 388, 390 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) 8.12 (s, 1H), 7.34 (dd, 1H), 7.09-7.19 (m, 1H), 6.86-7.02 (m, 1H), 4.27 (t, 2H), 3.54 (t, 2H).

[0286] Various examples in the following table were synthesized according to the synthetic method of steps 1-2 in Example 67 (compound 0153):

TABLE-US-00004 Example Structure Segment 1 68 [00294] [00295] 69 [00296] [00297] 70 [00298] [00299] 71 [00300] [00301] 72 [00302] [00303] Example Segment 2 MS m/z Compound 68 [00304] 444 446 [M + H].sup.+. 0236 69 HCOOH 404 0245 406 [M + H].sup.+. 70 [00305] 443 445 [M + H].sup.+. 0249 71 [00306] 454 456 [M + H].sup.+. 0287 72 [00307] 434 436 [M + H].sup.+. 0293

[0287] Various examples in the following table were synthesized according to the synthetic method of step 1 in Example 67 (compound 0153):

TABLE-US-00005 Example Structure Segment 1 Segment 2 73 [00308] [00309] CH.sub.3NH.sub.2 74 [00310] [00311] CH.sub.3NH.sub.2 75 [00312] [00313] [00314] 76 [00315] [00316] NH.sub.2OH 77 [00317] [00318] [00319] 78 [00320] [00321] [00322] 79 [00323] [00324] [00325] Example MS m/z Compound 73 404 0237 406 [M + H].sup.+ 74 418 0238 420 [M + H].sup.+. 75 430 0247 432 [M + H].sup.+ 76 406 0260 408 [M + H].sup.+. 77 418 0300 420 [M + H].sup.+ 78 472 0301 474 [M + H].sup.+ 79 454 0302 456 [M + H].sup.+

##STR00326##

Synthetic Route:

[0288] ##STR00327##

[0289] Step 1: Synthesis of Compound 0251

[0290] A methanol/ammonia solution (5.00 mL, 4 M) was added to a round-bottom flask containing the compound BB-6-2 (50.00 mg, 115.96 umol, 1.00 eq), followed by stirring and reaction at 25 C. for 3 hours. Methanol (2 mL) was added thereto, followed by filtration. Upon high performance liquid chromatography (Kromasil 150*25 mm*10 um, water (0.05% ammonia hydroxide v/v)-ACN) separation, a product 0251 (24.50 mg, yield: 54.15%, purity: 100%) was obtained. MS (ESI) m/z: 390, 392 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.13 (dd, 5.9 Hz, 1H), 7.05 (t, J=8.7 Hz, 1H), 6.81 (ddd, 4.0, 8.9 Hz, 1H), 3.96 (s, 2H).

##STR00328##

Synthetic Route:

[0291] ##STR00329##

[0292] Step 1: Synthesis of Compound 0262

[0293] To a round-bottom flask containing a compound BB-15-2 (180.00 mg, 392.79 umol, 1.00 eq), a methanol/ammonia solution (13.00 mL) (4 M) was added. Upon stirring and reaction at 15 C. for 6 hours, no product was generated. Upon continuous stirring and reaction for 20 hours, a product was generated, and the reaction was continued for 20 hours. After half of the solvent was removed upon rotary evaporation at 20 C., the remaining was filtered, and subjected to high performance liquid chromatography (DuraShell 150*25 mm*5 um, water (0.05% HCl)-ACN) for separation, to obtain a product 0262 (15.85 mg, yield: 9.98%, purity: 100%). MS (ESI) m/z: 404, 406 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.10 (dd, 5.9 Hz, 1H), 7.05 (t, J=8.7 Hz, 1H), 6.77 (ddd, 4.0, 8.8 Hz, 1H), 3.40 (t, J=6.9 Hz, 2H), 2.74 (t, J=6.9 Hz, 2H).

##STR00330##

Synthetic Route:

[0294] ##STR00331##

[0295] Step 1: Synthesis of Compound 0231-1

[0296] A compound BB-5 (50.00 mg, 113.98 umol, 1.00 eq, hydrochloride) and potassium cyanate (9.25 mg, 113.98 umol, 1.00 eq) were dissolved in water (2.00 mL), and heated to 100 C. and reacted for 2 hours. Water (5 mL) was added to the reaction solution, and the resultant mixture was extracted with ethyl acetate (5 mL*3). Organic phases were combined and then dried over anhydrous sodium sulfate, and dried by rotary evaporation to obtain a product 0231-1 as light yellow oil (50.00 mg, crude product). MS (ESI) m/z: 445, 447 [M+H].sup.+.

[0297] Step 2: Synthesis of Compound 0231

[0298] The compound 0231-1 (50.00 mg, 112.30 umol, 1.00 eq) was dissolved in methanol (1.50 mL) and water (1.00 mL), and sodium hydroxide (17.97 mg, 449.20 umol, 4.00 eq) was added, followed by stirring and reaction at 20 C. for 1.5 hours. The reaction solution was adjusted to have pH of 67 by addition of hydrochloric acid (6 M), followed by addition of methanol (2 mL) and filtration. A filtrate was subjected to high performance liquid chromatography (Phenomenex Synergi C18 150*30 mm*4 um, water (0.05% HCl)-ACN) for separation, to obtain a product 0231 (23.00 mg, yield: 48.51%, purity: 99.3%). MS (ESI) m/z: 419, 421 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.11 (dd, J=2.8, 6.0 Hz, 1H), 7.06 (t, J=8.7 Hz, 1H), 6.79 (ddd, J=2.8, 4.0, 8.8 Hz, 1H), 3.59 (t, J=6.4 Hz, 3H), 3.36 (br.s., 1H).

Example 83

0309

[0299] ##STR00332##

Synthetic Route:

[0300] ##STR00333##

[0301] Taking a compound BB-32 as raw material, a compound 0309 was synthesized according to the synthesis steps 1-2 for the compound 0231 in Example 82. MS (ESI) m/z: 471 [M+Na].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) 7.33 (d, 1H), 7.18 (t, 1H), 6.95 (t, 1H), 6.75 (dd, 1H), 3.51 (t, 2H), 3.26-3.31 (t, 2H).

Example 84

0239

[0302] ##STR00334##

Synthetic Route:

[0303] ##STR00335##

[0304] Step 1: Synthesis of Compound 0239

[0305] A compound 0117 (40.00 mg, 87.86 umol, 1.00 eq) was dissolved in dichloromethane (1.00 mL), and m-chloroperbenzoic acid (27.57 mg, 87.86 umol, purity: 55%, 1.00 eq) was added. The reaction solution was allowed to react at 10 C. for 1 hour. Complete reaction of raw materials was observed by LCMS monitoring, and a target compound was generated. Three mL of methanol was added to the reaction solution, followed by filtration. A filtrate was subjected to high performance liquid chromatography (Phenomenex Synergi C18 150*30 mm*4 um water (0.05% HCl)-ACN) for separation, to obtain a product 0239 (11.57 mg, yield: 25.94%, purity: 100%, hydrochloride). MS (ESI) m/z: 471, 473 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) 7.27 (d, 1H), 7.09 (t, 1H), 6.96 (d, 1H), 3.73-3.85 (m, 1H), 3.53-3.71 (m, 3H).

Example 85

0069

[0306] ##STR00336##

Synthetic Route:

[0307] ##STR00337##

[0308] Step 1: Synthesis of Compound 0069-2

[0309] The compound 0069-1 (2.50 g, 19.37 mmol, 1.00 eq) was dissolved in N,N-dimethylformamide (60.00 mL), then a compound BB-1-4 (3.68 g, 19.37 mmol, 1.00 eq), HATU (8.84 g, 23.24 mmol, 1.20 eq), and diisopropylethylamine (5.01 g, 38.74 mmol, 6.77 mL, 2.00 eq) were sequentially added. The reaction solution was allowed to react at 25 C. for 1 hour. The reaction solution turned yellow. With LCMS showing completion of reaction, 240 mL of water was added to the resulting mixture, followed by filtration. A filter cake was washed with water (20 mL*3), and naturally dried to obtain a gray solid product 0069-2 (5.50 g, yield: 92.43%, purity: 98%) which was directly used for reaction in a next step. MS (ESI) m/z: 301, 303 [M+H].sup.+.

[0310] Step 2: Synthesis of Compound 0069-3

[0311] At 0 C., sulfuric acid (9.00 mL) was slowly added to a hydrogen peroxide (10.62 g, 93.64 mmol, 9.00 mL, purity: 30%, 56.41 eq), then sodium tungstate (487.96 mg, 1.66 mmol, 1.00 eq) was added, following by the compound 0069-2 (500.00 mg, 1.66 mmol, 1.00 eq). The resultant mixture was heated and stirred at 25 C. for 16 hours. With LCMS showing completion of reaction, the reaction solution was diluted by addition of 20 mL of water, and filtered. A filter cake was dried by rotary evaporation to obtain a white solid product 0069-3 (520.00 mg, yield: 89.89%, purity: 95%) which was directly used for reaction in a next step. MS (ESI) m/z: 331, 333 [M+H].sup.+.

[0312] Step 3: Synthesis of Compound 0069-4

[0313] The compound 0069-3 (500.00 mg, 1.51 mmol, 1.00 eq) was dissolved in tetrahydrofuran (5.00 mL), water (100.00 uL) and methanol (1.00 mL), then sodium hydroxide (84.58 mg, 2.11 mmol, 1.40 eq) was added, and then the resultant mixture was stirred at 25 C. for 16 hours. With LCMS showing completion of most of the reaction, the reaction solution was adjusted to 7 in pH value by addition of 1 N hydrochloric acid. The reaction solution was diluted by addition of 50 mL of ethyl acetate, washed with a saturated saline (10 mL3), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation to obtain a yellow solid product 0069-4 (430.00 mg, crude product) which was directly used for reaction in a next step. MS (ESI) m/z: 316, 318 [M+H].sup.+.

[0314] Step 4: Synthesis of Compound 0069-5

[0315] The compound 0069-4 (200.00 mg, 632.75 umol, 1.00 eq) was dissolved in toluene (2.00 mL), then Lawesson's reagent (511.86 mg, 1.27 mmol, 2.00 eq) was added, and then the resultant mixture was stirred at 90 C. for 16 hours. With LCMS showing completion of most of reaction, the reaction solution was dried by rotary evaporation to obtain a crude product. The crude product was purified by a flash column chromatography (0-40% ethyl acetate in petroleum ether) to obtain a red solid product 0069-5 (120.00 mg, yield: 51.96%, purity: 91%). MS (ESI) m/z: 332, 334 [M+H].sup.+.

[0316] Step 5: Synthesis of Compound 0069-6

[0317] The compound 0069-5 (150.00 mg, 451.60 umol, 1.00 eq) was dissolved in dichloromethane (3.00 mL), then diisopropylethylamine (175.09 mg, 1.35 mmol, 236.61 uL, 3.00 eq) was added, then methyl trifluoromethansulfonate (111.16 mg, 677.40 umol, 74.11 uL, 1.50 eq) was added dropwise, and then the resultant mixture was stirred at 25 C. for 3 hours. With TLC (petroleum ether:ethyl acetate=3:1) showing completion of reaction, the reaction solution was dried by rotary evaporation to obtain a crude product. The crude product was purified by flash column chromatography (0-40% ethyl acetate in petroleum ether) to obtain a product 0069-6 as yellow oil (130.00 mg, yield: 82.82%, purity: 99.6%). MS (ESI) m/z: 346, 348 [M+H].sup.+.

[0318] Step 6: Synthesis of Compound 0069

[0319] The compound 0069-6 (100.00 mg, 288.87 umol, 1.00 eq) was dissolved in ethanol (3.00 mL), then diisopropylethylamine (112.00 mg, 866.60 mmol, 151.35 uL, 3.00 eq) was added, then cyanamide (36.43 mg, 866.60 umol, 36.43 uL, 3.00 eq) was added, and the resultant mixture was transferred into a microwave tube, to be subjected to microwave reaction at 100 C. for 1 hour. With LCMS monitoring showing completion of reaction, a main product peak was generated. Filtration was performed, and a filtrate was dried by rotary evaporation to obtain a crude product. The crude product was purified by high performance liquid chromatography (water (0.05% HCl)-ACN, Boston Green ODS 150*30 5 u) to obtain a product 0069 (18.00 mg, yield: 18.32%, purity: 100%). MS (ESI) m/z: 340, 342 [M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD): =8.22-7.98 (m, 1H), 7.82-7.62 (m, 1H), 7.43-7.26 (m, 1H), 4.25 (br.s., 3H).

Example 85

Compound 0310

[0320] ##STR00338##

Synthetic Route:

[0321] ##STR00339##

[0322] Step 1: Synthesis of Compound 0310-1

[0323] The segment BB-5 (100.00 mg, 227.97 umol, 1.00 eq, HCl) and sodium dicyanamide (60.89 mg, 683.91 umol, 3.00 eq) were dissolved in DMF (2.00 mL), and HCl (2 M, 115.12 uL, 1.01 eq) was added. The reaction solution was heated to 110 C. and reacted for 2 hr. Eight mL of water was added to the reaction solution, followed by extraction with ethyl acetate (8 mL*3). Organic phases were combined, washed with a saline (20 ml*3), dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation to obtain a compound 0310-1. MS (ESI) m/z: 469.0, 471.0 [M+H].sup.+.

[0324] Step 2: Synthesis of Compound 0310

[0325] The compound 0310-1 (106.00 mg, 225.89 umol, 1.00 eq) was dissolved in MeOH (1.00 mL) and H.sub.2O (500.00 uL), and NaOH (36.14 mg, 903.56 umol, 4.00 eq) was added. The reaction solution was allowed to react at 22 C. for 2 hr. The reaction solution was filtered. A filtrate was purified by Pre-HPLC (Kromasil 150*25 mm*10 um water (0.05% ammonia hydroxide v/v)-ACN) for separation to obtain a compound 0310. MS (ESI) m/z: 464.9, 466.9 [M+Na].sup.+. .sup.1H NMR (400 MHz, MeOD): =7.01-7.14 (m, 2H), 6.77 (ddd, 1H), 3.51-3.66 (m, 2H), 3.28-3.32 (m, 2H).

Example 86

Compound 0383

[0326] ##STR00340##

Synthetic Route:

[0327] ##STR00341##

[0328] Step 1: Synthesis of Compound 0383-2

[0329] The segment BB-5 (50.00 mg, 113.98 umol, 1.00 eq, HCl) was dissolved in DMF (1.00 mL), DIEA (103.12 mg, 797.86 umol, 139.35 uL, 7.00 eq) was added, and then the compound 0383-1 (33.41 mg, 227.96 umol, 2.00 eq) was added. The reaction solution was allowed to react at 23 C. for 2 hr. Five mL of water was added, followed by extraction with ethyl acetate (5 mL*3). Organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A filtrate was dried by rotary evaporation under reduced-pressure distillation to obtain a compound 0383-2. MS (ESI) m/z: 444.0, 446.0 [M+H].sup.+.

[0330] Step 2: Synthesis of Compound 0383

[0331] The compound 0383-2 (100.00 mg, 225.10 umol, 1.00 eq) was dissolved in THF (1.00 mL) and H.sub.2O (500.00 uL), and NaOH (18.01 mg, 450.20 umol, 2.00 eq) was added. The reaction solution was allowed to react at 25 C. for 1 hr. The reaction solution was adjusted to pH=5 with concentrated hydrochloric acid, followed by addition of 3 ml of methanol and filtration. A filtrate was separated and purified by pre-HPLC (YMC-Actus Triart C18 150*30 5 u water (0.05% HCl)-ACN) to obtain a compound 0383. MS (ESI) m/z: 417.9, 419.9 [M+H].sup.+ .sup.1H NMR (400 MHz, MeOD): =7.13-7.03 (m, 2H), 6.85-6.73 (m, 1H), 3.70-3.54 (m, 2H), 3.45-3.35 (m, 2H).

Example 87

Compound 0384

[0332] ##STR00342##

Synthetic Route:

[0333] ##STR00343##

[0334] Step 1: Synthesis of Compound 0384-1

[0335] The segment BB-5 (50.00 mg, 113.98 umol, 1.00 eq, HCl) was dissolved in CH.sub.3CN (1.00 mL), DIEA (58.92 mg, 455.92 umol, 79.63 uL, 4.00 eq) was added, and then a DMF solution (0.25 ml) of the segment BB-34 (107.49 mg, 569.90 umol, 5.00 eq, HCl) was added. The reaction solution was allowed to react at 20 C. for 64 hr. The reaction solution was directly dried by rotary evaporation under reduced-pressure distillation to obtain a compound 0384-1. MS (ESI) m/z: 485.9, 487.9 [M+H].sup.+.

[0336] Step 2: Synthesis of Compound 0384

[0337] The compound 0384-1 (55.00 mg, 113.10 umol, 1.00 eq) was dissolved in THF (1.00 mL) and H.sub.2O (500.00 uL), and NaOH (36.19 mg, 904.80 umol, 8.00 eq) was added. The reaction solution was allowed to react at 20 C. for 1 hr. The reaction solution was adjusted to pH=6 with concentrated hydrochloric acid, followed by addition of 3 ml of methanol and filtration. A filtrate was separated and purified by pre-HPLC (YMC-Actus Triart C18 150*30 5 u water (0.05% HCl)-ACN) to obtain a compound 0384. MS (ESI) m/z: 460.0, 462.0 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD): =7.13-7.04 (m, 2H), 6.85-6.77 (m, 1H), 3.80 (t, J=6.8 Hz, 2H), 3.49 (br t, J=6.5 Hz, 2H).

Test Example 1

Bioactivity Test

[0338] I. Test of hIDO1 for In Vitro Activity

[0339] 1. Test of hIDO1 for Enzymatic Activity In Vitro

[0340] 1.1 Purpose of Experiment:

[0341] Changes in production of NFK, an IDO1 enzymatic metabolite were detected with NFK Green fluorescent molecules. With an IC50 value of a compound as an index, an inhibitory effect of the compound on recombinant human IDO1 enzyme was evaluated.

[0342] 1.2 Experimental Materials:

[0343] NFK Green reagent, Netherlands Translational research center

[0344] IDO1 enzymatic activity detection reagent kit, NTRC#NTRC-hIDO-10K

[0345] 384-well enzymatic reaction plate, PerkinElmer#6007279

[0346] 384-well compound plate, Greiner#781280

[0347] plate sealing film, PerkinElmer#6050185

[0348] Envision multi-functional plate reader, PerkinElmer

[0349] Bravo automatic liquid processing platform, Agilent

[0350] 1.3 Experimental Steps and Method:

[0351] 1.3.1 Addition of Compound Sample:

[0352] The compound was diluted with dimethyl sulfoxide (DMSO) to 1 mM, with 10 gradients, each 3-fold dilution, in duplicate. 48 L of 50 mM phosphate buffer, having pH of 6.5, was transferred by the Bravo automatic liquid processing platform to the compound plate. Then 2 L of an already diluted compound/DMSO solution was added. After even mixing, 10 L of the resultant mixture was transferred to the enzymatic reaction plate.

[0353] 1.3.2 Experiment for Detecting IDO1 Enzymatic Activity:

[0354] IDO1 enzyme was diluted to 20 nM in a reaction buffer (50 mM phosphate buffer with pH of 6.5, 0.1% of Tween-20, 2% of glycerol, 20 mM ascorbic acid, 20 g/ml catalase, and 20 M methylene blue), 20 L of the diluted IDO1 enzyme was transferred to the enzymatic reaction plate, and incubated at 23 C. for 30 minutes. 10 L of 400 M L-type tryptophan substrate was added to start reaction. The incubation lasted at 23 C. for 90 minutes. 10 L of NFK Green fluorescent dye was added. The resultant mixture was sealed with the plate sealing film. After incubation at 37 C. for 4 hours, an Envision multi-functional plate reader was used for reading (Ex 400 nm/Em 510 nm).

[0355] 1.3.3 Data Analysis:

[0356] A reference well to which the IDO1 enzyme was added but no compound was added was set to have an inhibition rate of 0%, and a reference well to which no IDO1 enzyme was added was set to have an inhibition rate of 100%. Data was analyzed with XLFit 5 to calculate an IC50 value of the compound. Test results thereof are as shown in Table 1.

[0357] 2. Test of hIDO1 for Cytological Activity

[0358] 2.1 Purpose of Experiment:

[0359] Changes in Hela cell kynurenine were detected by an LCMS method. With an IC50 value of a compound as an index, an inhibitory effect of the compound on IDO1 enzyme was evaluated.

[0360] 2.2 Experimental Materials:

[0361] Cell lines: Hela cells

[0362] Culture medium: RPMI 1640 phenol red free, Invitrogen#11835030

[0363] 10% fetal bovine serum, Gibco#10099141

[0364] 1XPenicillin-Streptomycin, Gibco#15140-122

[0365] Precipitation agent: 4 M L-kynurenine-d4 dissolved in 100% acetonitrile, CacheSyn#CSTK008002

[0366] Trypsin, Invitrogen#25200-072

[0367] DPBS, Hyclone#SH30028.01B

[0368] Recombinant human interferon-, Invitrogen#PHC4033

[0369] 5% (w/v) trichloroacetic acid, Alfa Aesar#A11156

[0370] 96-well cell plate, Corning#3357

[0371] 96-well compound plate, Greiner#781280

[0372] 96-well V-bottom plate, Axygen#WIPP02280

[0373] CO.sub.2 incubator, Thermo#371

[0374] Centrifuge, Eppendorf#5810R

[0375] Vi-cell cell counter, Beckman Coulter

[0376] 2.3 Experimental Steps and Method:

[0377] 2.3.1 Hela Cell Inoculation:

[0378] The culture medium, trypsin, and DPBS were preheated in water bath at 37 C. The culture medium was removed by sucking from cell culture, followed by cleaning the cell culture with 10 mL of DPBS; the preheated trypsin was added to the culture flask, which was revolved to make the culture flask covered by trypsin uniformly, and placed into an incubator at 37 C. with 5% CO.sub.2 for digestion for 1-2 minutes; 10-15 mL of the culture medium was used to suspend cells each T150, followed by centrifugation at 800 rpm for 5 minutes. 10 mL of the culture medium was used to resuspend the cells. 1 mL of the cell resuspension was pipetted, and counted with Vi-cell; the Hela cells were diluted to 510.sup.5/mL with the culture medium, 80 L of the diluted Hela cells were added to the 96-well cell plate, and incubated in the an incubator at 37 C. with 5% CO.sub.2 for 5-6 hours.

[0379] 2.3.2 Addition of Compound Sample:

[0380] The compound was diluted with DMSO to 1 mM, with 9 gradients, each 3-fold dilution, in duplicate. 5 L of the already diluted compound DMSO solution was added to the compound plate containing 95 L of the culture medium. After even mixing, 10 L of the mixture was transferred to the cell plate.

[0381] 1) Test for Cytological Activity:

[0382] 10 L of recombinant human interferon- was added to result in a final concentration of 100 ng/ml, to induce expression of IDO1. Incubation was carried out in the 5% CO.sub.2 incubator at 37 C. for 20 hours. 4 L of 5% (w/v) trichloroacetic acid was added, followed by even mixing and incubation at 50 C. for 30 minutes. After centrifugation at 2400 rpm for 10 minutes, 40 L of a supernatant was added to a 96-well V-bottom plate, followed by addition of a precipitant. Centrifugation was carried out at 4000 rpm for 10 minutes after even mixing. 100 L of the supernatant was transferred to a new 96-well V-bottom plate. A content of kynurenine was detected through LCMS.

[0383] 2) Data Analysis:

[0384] A reference well to which the interferon- was added but no compound was added was set to have an inhibition rate of 0%, and a reference well to which no Hela cell was added was set to have an inhibition rate of 100%. Data was analyzed with XLFit 5 to calculate an IC50 value of the compound. Test results thereof are as shown in Table 1.

TABLE-US-00006 TABLE 1 Test Results of in vitro Screening of Compounds of the Present Disclosure Enzyme Hela Cell Compound IC50(nM) IC50(nM) 0015 115.37 14.01 0026 >10000 / 0052 109.41 24.87 0068 112.62 28.65 0069 >10000 / 0070 1376.52 / 0071 798.52 / 0077 >10000 / 0078 >10000 / 0089 981.00 / 0103 537.32 / 0106 105.56 36.95 0107 1298.73 / 0108 387.42 / 0117 26.19 5.85 0118 55.26 64.51 0119 2067.84 / 0120 615.82 / 0121 187.84 / 0122 564.64 / 0124 1026.54 / 0128 601.05 / 0129 426.13 / 0131 701.55 / 0133 1028.69 / 0134 7150.29 / 0135 3291.28 / 0137 5714.89 / 0138 364.38 / 0139 265.41 100.1 140 197.23 / 0141 452.46 / 0142 1310.71 / 0143 >10000 / 0144 >10000 / 0147 71.51 123 0148 147.21 158.7 0149 290.21 / 0150 >10000 / 0151 149.09 25.22 0153 153.46 82.37 0156 5702.94 / 0157 159.86 13.39 0383 100 104.7 0158 4358.44 / 0160 3830.14 / 0162 928.12 / 0177 278.81 54.25 0178 3077.61 / 0179 593.82 / 0180 >10000 / 0182 672.53 / 0183 1124.05 / 0184 4833.48 / 0189 590.30 / 0190 944.74 / 0222 466.47 / 0225 273.95 18.71 0227 59.27 3.10 0228 101.37 3.97 0229 80.11 11.21 0230 203.23 / 0231 42.20 4.35 0232 372.66 / 0233 108.56 19.19 0234 76.95 9.58 0236 128.70 55.42 0237 42.27 8.51 0238 85.38 13.88 0239 845.76 / 0240 128.22 26.24 0245 74.40 14.84 0247 95.72 17.07 0249 164.2 30.34 0250 137.59 27.02 0251 44.33 7.15 0260 74.64 31.54 0262 89.56 70.80 0273 144.02 34.40 0287 69.36 12.89 0293 60.13 29.45 0295 122.26 47.22 0300 72.57 / 0301 125.28 / 0302 88.03 / 0309 54.48 23.48 0310 97.3 12.8 0384 173 152

[0385] It is concluded that the compounds of the present disclosure have good in vitro activity.

II. Measurement of Thermodynamic Solubility

[0386] 1. Solutions for Thermodynamic Solubility

[0387] Buffer A (pH 2.0): 50 mM phosphate buffer, with a pH value of 2.0.

[0388] Buffer B (pH 7.4): 50 mM phosphate buffer, with a pH value of 7.4.

[0389] 2. Preparation of Standard Solution

[0390] 50% acetonitrile solution and 50% buffer (A, B) were mixed together to obtain a diluent. 10 mM (20 L/compound) stock solution was added to acetonitrile (480 L/compound), and mixed with the buffer (A, B) (500 L/compound) to obtain 200 M UV detection standard solution. The 200 M UV detection standard solution was diluted with the diluent 10 times or 200 times the amount of the standard solution, so as to acquire 20 M or 1 M UV standard solution. 1, 20, and 200 M UV standard solutions were taken as standard samples for thermodynamic solubility test.

[0391] 3. Method

[0392] 3.1 Sample Preparation, Shaking, and Filtering

[0393] No less than 2 mg of a sample powder was weighed and placed in a Whatman miniuniprep vial. If it was required to test the thermodynamic solubility of the sample in multiple buffers (A, B), an independent vial was demanded for each test. 450 L of the buffer (A, B) was respectively added to each Whatman miniuniprep vial. After the buffer was added, a piston cover of the Whatman miniuniprep functioning to filter was put on and pressed above a liquid surface, such that a filter screen was in contact with the buffer (A, B) during the shaking. The solubility sample was shaken in a vortex manner for 1 minute, and phenomenon of the solution was recorded. The sample was shaken at 600 rpm at a room temperature (approximately 2225 C.) for 24 hours. The filtering cover of Whatman Miniunipreps was pressed to a bottom, to obtain a filtrate of the sample solubility solution. All sample vials should be recorded for insoluble substances before and after the filtration and leakage phenomenon thereof. The buffer (A, B) was 50-fold diluted to obtain a sample diluent.

[0394] 3.2 Analysis and Detection

[0395] 3 UV standard solutions, from low concentration to high concentration, were injected into HPLC, and then the diluent and the supernatant of the compound to be tested were injected. The sample to be tested was in duplicate. Integration was carried out for UV chromatographic peaks. A standard curve was established by simulation and the thermodynamic solubility of the sample was calculated. See Table 2 for results. HPLC conditions were as follows.

[0396] Test method: HPLC-UV detection

[0397] Instrument: Agilent 1200

[0398] Mobile phase: A: water+0.69% TFA; B: acetonitrile+0.62% TFA

[0399] Chromatographic column: Agilent TC C18 (2.150 mm, 4.6 m)

[0400] Proportions:

TABLE-US-00007 Time (min) B % Flow Velocity (mL/min) 0.00 5 1 2.00 90 1 2.50 90 1 2.60 5 1 4.00 5 1

TABLE-US-00008 TABLE 2 Solubility of Compounds of the Present Disclosure thermodynamic solubility thermodynamic solubility Compound (pH: 2.0) (pH: 7.4) 360 65 160 0015 138 298 0117 2616 2607 0231 155 125

[0401] It is concluded that the compounds of the present disclosure have relatively good water solubility.

III. Permeability Test

[0402] Test for Permeability Across MDR1

[0403] 1.1 Preparation of Stock Solution

[0404] A sample for test was dissolved in dimethyl sulfoxide (DMSO) or other suitable solvents, to be prepared into 10 mM stock solution. A suitable internal standard (IS) was dissolved in acetonitrile (ACN) or other organic solvents as a stop solution. Specific information would be described in a research report.

[0405] Fenoterol, propranolol, and digoxin acted as low-permeability control, high-permeability control, and P-gp substrate in the present research. The stock solutions of these compounds were prepared with DMSO, stocked at 2-8 C., and valid for use within 3 months.

[0406] 1.2 Preparation of Donor Solution and Receiver Solution

[0407] In the present project, a Hank's balanced salt buffer containing 10 mM HEPES was used as a transport buffer. Preparation methods for the donor solution and the receiver solution are as shown in Table 3.

TABLE-US-00009 TABLE 3 Preparation Methods for Donor Solution and Receiver Solution Final DMSO Concentration Solution Name Components pH (v/v) Apical and Prepare a control or sample ND 0.4% basolateral for test with a concentration donor solutions of 2 M from the transport buffer Apical and Transport buffer ND .sup.0% basolateral receiver solutions Notes: ND represents undetected.

[0408] 1.3 Cell Culturing

[0409] MDR1-MDCK II cells were cultivated using -MEM culture media (-Minimum Essential Media), with a culture condition of 371 C., 5% CO.sub.2, and saturated relative humidity. Afterwards, the cells were inoculated into a BD Transwell-96-well plate (BD Gentest), with an inoculation density of 2.310.sup.5 cells/cm.sup.2, then the cells were placed in a carbon dioxide incubator to be incubated for 4-7 days, and then used for a transport experiment.

[0410] 1.4 Transport Experiment

[0411] The sample for test and digoxin had a donor concentration of 2 M, and were administered in two directions (direction A-B and direction B-A), each in duplicate. Fenoterol and propranolol each had a test concentration of 2 M, and were administered in a single direction (direction A-B), each in duplicate.

[0412] A solution to be used was placed in a 371 C. water bath kettle to be pre-incubated for 30 minutes. The donor solution and the receiver solution were respectively added to corresponding sides of wells of cell plate (to an apical side and a basolateral side, 75 L and 250 L of samples were added respectively), and experiment of bidirectional transport was started. After the addition of the samples, the cell plate was placed in a 371 C., 5% CO.sub.2 incubator with saturated relative humidity to be incubated for 150 minutes. Information of sample collection is as shown in Table 4.

TABLE-US-00010 TABLE 4 Information of Sample Collection Volume of Volume of Sample Received Volume of Transport in Each Well Stop Solution Buffer Sample Type (L) (L) (L) A-B Donor side 50 250 100 A-B Receiver side 150 250 0 B-A Donor side 50 250 100 B-A Receiver side 50 250 100 T0 50 250 100

[0413] The samples were all centrifuged at 3220 g for 10 minutes after being shaken in a vortex manner. A suitable volume of a supernatant was transferred to a sample analyzing plate. If analysis was not carried out immediately after the plate was sealed, the plate was stored at 2-8 C. The analysis was carried out through a method of LC/MS/MS.

[0414] 1.5 Test for Cell Membrane Integrity

[0415] After the transport experiment was ended, integrity of MDR1-MDCK II cells was tested through Lucifer Yellow Rejection Assay. After a Lucifer Yellow solution was incubated for 30 minutes, a Lucifer Yellow sample was collected, and relative fluorescence intensity (the relative fluorescence unit, RFU) of the Lucifer Yellow in the sample was detected at 425/528 nm (excitation/emission) with a 2.sup.e plate reader.

[0416] 1.6 Sample Analysis

[0417] The sample for test, fenoterol control, propranolol control, and digoxin were analyzed in a semiquantitative manner, and specific values of analytes to a peak area of the internal standard were taken as concentration of the controls.

TABLE-US-00011 TABLE 5 Permeability of Compounds of the Present Disclosure across MDR1: Compounds A to B (10.sup.6 cm/s) B to A (10.sup.6 cm/s) 360 1.61 13.32 0015 1.76 13.59 0117 3.2 11.3 0227 12.53 18.03 0228 6.78 10.46 0231 3.79 17.85

[0418] 2. Test for Permeability Across Caco2

[0419] 2.1 Preparation of Stock Solution

[0420] A sample for test was dissolved in dimethyl sulfoxide (DMSO) or other suitable solvents, to be prepared into 10 mM stock solution. A suitable internal standard

[0421] (IS) was dissolved in acetonitrile (ACN) or other organic solvents as a stop solution. Specific information would be described in a research report.

[0422] Fenoterol, propranolol, and digoxin acted as low-permeability control, high-permeability control, and P-gp (P-glycoprotein) substrate, respectively, in the present research. The stock solutions of these compounds were prepared with DMSO, stocked at 2-8 C., and valid for use within 3 months.

[0423] 2.2 Preparation of Donor Solution and Receiver Solution

[0424] In the present project, a Hank's balanced salt buffer containing 10 mM HEPES was used as a transport buffer. Preparation methods for the donor solution and the receiver solution are as shown in Table 6.

[0425] HEPES: 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, supplier: gibco, article number: 15630-080

[0426] Hank's balanced salt buffer: Hank's balanced salt solution, referred to as HBSS for short, purchased from gibco, with an article number 14025-076

TABLE-US-00012 TABLE 6 Preparation Methods for Donor Solution and Receiver Solution Final DMSO Concentration Solution Name Components pH (v/v) Apical and Prepare a control or a sample ND 0.5% basolateral for test with a concentration donor solutions of 2 M from the transport buffer Apical and Transport buffer ND .sup.0% basolateral receiver solutions Notes: ND represents undetected.

[0427] 2.3 Cell Culturing

[0428] Caco-2 cells were cultivated using MEM culture media (Minimum Essential Media), with a culture condition of 371 C., 5% CO.sub.2, and saturated relative humidity. Afterwards, the cells were inoculated into a BD Transwell-96-well plate, with an inoculation density of 110.sup.5 cells/cm.sup.2, then the cells were placed in a carbon dioxide incubator to be incubated for 21-28 days, and then used for a transport experiment.

[0429] 2.4 Transport Experiment

[0430] The sample for test and digoxin with donation concentration of 2 M were administered in two directions (direction A-B and direction B-A), each in duplicate. Fenoterol and propranolol each with test concentration of 2 M were administered in a single direction (direction A-B), each in duplicate.

[0431] A solution to be used was placed in a 371 C. water bath kettle to be pre-incubated for 30 minutes. The donor solution and the receiver solution were respectively added to corresponding sides of well of cell plate (to each apical sides and each basolateral sides, 75 L and 250 L of samples were added respectively), and experiment of bidirectional transport was started. After addition of the samples, the cell plate was placed in a 371 C., 5% CO.sub.2 incubator with saturated relative humidity to be incubated for 120 minutes. Information of sample collection is as shown in Table 7.

TABLE-US-00013 TABLE 7 Information of Sample Collection Volume of Volume of Sample Received Volume of Transport in Each Well Stop Solution Buffer Sample Type (L) (L) (L) A-B Donor side 50 250 100 A-B Receiver side 150 250 0 B-A Donor side 50 250 100 B-A Receiver side 50 250 100 T0 50 250 100

[0432] The samples were all centrifuged at 3220 g for 10 minutes after being shaken in a vortex manner. A suitable volume of a supernatant was transferred to a sample analyzing plate. If analysis was not carried out immediately after the plate was sealed, the plate was stored at 2-8 C. The analysis was carried out through a method of LC/MS/MS.

[0433] 2.5 Test for Cell Membrane Integrity

[0434] After the transport experiment was ended, integrity of Caco-2 cells was tested through Lucifer Yellow Rejection Assay. After a Lucifer Yellow solution was incubated for 30 minutes, a Lucifer Yellow sample was collected, and relative fluorescence intensity (the relative fluorescence unit, RFU) of the Lucifer Yellow in the sample was detected at 425/528 nm (excitation/emission) with a 2.sup.e plate reader.

[0435] 2.6 Sample Analysis

[0436] The sample for test, the fenoterol control, propranolol control, and digoxin were analyzed in a semiquantitative manner, and specific values of analytes to a peak area of the internal standard were taken as concentration of the controls.

TABLE-US-00014 TABLE 8 Permeability of Compounds of the Present Disclosure across Caco2: Compounds A to B (10.sup.6 cm/s) B to A (10.sup.6 cm/s) 360 0.39 19.63 0117 2.97 12.19 0231 2.58 20.13

[0437] It is concluded that the compounds of the present disclosure have good permeability.

Test Example 2

[0438] Research on Efficacy of Anti-Mice Colon Cancer Ct26

[0439] 1. Purpose of Research

[0440] In the present research, colon cancer CT26 models were employed to compare differences between compound 231 and compounds 360 and 117 in antitumor effects.

[0441] 2. Experimental Materials

[0442] 2.1 Cells

TABLE-US-00015 TABLE 9 Basic Information of Cell Line Cell Line Name CT26.WT (ATCC CRL-2638) Tumor Type Mouse colorectal cancer cell Conditions of Culture RPMI 1640 culture medium, 10% fetal bovine serum, 37 C., 5% CO.sub.2 Culturing Process Replacing the medium every other day, passage every 2-3 at a ratio of 1:2-1:5 Cell Source Type Culture Collection, Chinese Academy of Sciences

[0443] 2.2 Experimental Animals

TABLE-US-00016 TABLE 10 Basic Information of Experimental Animals Animal Breed/Species BALB/c mice Level SPF Level Sex Male Number of Animals 200 Weeks of Age 4-5 w Weight 16-20 g Quarantine/adaptation At least 1 week of pre-adaptation before start of experiment Feeding Condition Feed: common feed for growth and reproduction of mice (Beijing Keao Xieli), water: deionized purifed water temperature: 21 2 C., humidity: 30-70%, lighting: in the alternation of day and night of 12 hr Animal Source Beijing Vital River Laboratory Animal Technol- ogy Co., Ltd; animal certificate number: SCXK (JING) 2012-0001

[0444] 2.3 Samples for Test

TABLE-US-00017 TABLE 11 Basic Information of Different IDO Inhibitors Molecular Compounds Weight Supplier Batch Number 360 438.2 Shanghai Send SEND20160920-37-3 Pharmaceutical Technology Co., Ltd 117 455.3 231 419.2

[0445] 3. Dose Design and Administration Route and Frequency

[0446] Preliminary experimental research showed that via gavage administration by 100 mg/kg bid, the compound 360 had a tumor inhibitory rate of 30-50% for CT26, substantially reaching a maximum efficacy plateau. Therefore, the dose of the two compounds, compound 360 and compound 117, in the present experiment was set as 100 mg/kg. In order to observe dose-efficacy relevance of 231, a dose of the compound 231 was set as 25, 50, 100, 200 mg/kg in the present experiment. All drugs were administered by gavage, twice a day.

[0447] 4. Selection and Preparation of Model

[0448] According to document reports, mice colon cancer CT26 is a commonly used tumor model for evaluating immunological drugs. The IDO inhibitor compound 360 can effectively inhibit growth of tumors in the above model. Therefore, CT26-bearng BALB/c models were chosen in the present experiment for research on efficacy and tissue distribution.

[0449] Tumor cells in logarithmic phase were collected, and re-suspended in a serum-free medium, and adjusted in cell concentration to be 510.sup.5/mL, followed by addition of Matrigel of an equal volume to the cell suspension, such that the cells had a final concentration of 510.sup.5/mL. In a super-clean bench, each mouse was subcutaneously inoculated with 0.2 mL of the tumor cell suspension at shoulder, with an inoculation amount of 110.sup.5 per mouse. When a tumor block grew to be 500-1000 mm.sup.3, the tumor block was taken out, cut into pieces with scissors, and subcutaneously inoculated at shoulder at the mouse's back using a subcutaneous embedding implantation puncture needle (with a diameter of 1.2 mm).

[0450] 5. Grouping and Administration

[0451] The day of tumor inoculation was defined as Day 0, and the inoculated animals were grouped randomly on the day of inoculation. One day after the inoculation (Day 1), the administration was started. The experiment had 7 groups in total, with 12 animals in each group. See details in Table 12 for information on animal grouping and administration.

TABLE-US-00018 TABLE 12 Grouping of Animals and Administration (CT26 Models) for Efficacy analysis Dose of Drug Administration Administration Quantity Administration Concentration Route and Volume of Groups Compounds (mg/kg) (mg/mL) Frequency (mL/10 g) Animals Control 20% solutol gavage, BID 0.1 12 360 100 mg/kg 360 100 10 gavage, BID 0.1 12 117 100 mg/kg 117 100 10 gavage, BID 0.1 12 231 25 mg/kg 231 40 4 gavage, BID 0.1 12 231 50 mg/kg 231 100 10 gavage, BID 0.1 12 231 100 mg/kg 231 100 10 gavage, BID 0.1 12 231 200 mg/kg 231 250 95 gavage, BID 0.1 12

[0452] 6. Observation of Indexes

[0453] It was an experimental cycle when the tumor in the control group grew to have a volume of 3000 mm.sup.3. The following indexes were observed during the experiment.

[0454] (1) As for tumor growth curve, when the tumor was measurable, a maximum diameter (a) and a minimum diameter (b) of the tumor were measured once every other day, to calculate the tumor volume (a calculation formula is V=ab.sup.2), and anti-tumor effect of the tested drug was observed dynamically.

[0455] (2) As for outcome rate, when the experiment was ended, the outcome rate of each group of animals was observed.

[0456] (3) As for animal weight, body weight of the mouse was weighed each time when the tumor diameter was measured or before the administration, and death of the animals was observed once a day.

[0457] (4) As for tumor inhibitory rate, when the experiment was ended, the mice were killed by cervical dislocation, the tumor blocks were taken out and weighed, to calculate the tumor inhibitory rate, where tumor inhibitory rate=(mean tumor weight of the control group-mean tumor weight of the treatment group)/mean tumor weight of the control group100%), and the tumor blocks were photographed with a digital camera for recording.

[0458] 7. Experimental Results

[0459] 7.1 Death and Body Weight of Tumor-Bearing Mice

[0460] During the whole test, no animal died, surviving animals in each group had an increased body weight compared with those before administration. When the test was ended, the animals in the control group were increased by 16.0% in body weight, while the magnitude of increase in body weight of the animals in each administration group was reduced (4.4%-12.0%). Results were shown in FIG. 1 and Table 12. When the experiment was ended, the animals in group 117 and group 231 50 mg/kg, group 231 100 mg/kg, and group 231 200 mg/kg had remarkably decreased weight compared to those in the control group (P<0.05), and the body weight of the animals in group 360 and group 231 25 mg/kg had no significant difference than the control group (P>0.05). Therefore, with a relatively high dose of the compound 231, the increase magnitude of the animal weight could be modestly reduced, but the animal weight was still increased.

TABLE-US-00019 TABLE 12 Effect of Tested Drug on Body Weight of Tumor-bearing Mice (unit: g, x s) Rate of Weight Change of D17 vs Day1 Day3 Day6 Day8 Day10 D12 D14 D17 D1 (%) Control 22.5 1.3 23.3 1.3 24.3 1.1 24.4 1.1 24.3 1.1 24.5 1.3 25.0 1.2 26.1 1.6 16.0 360 23.2 0.8 23.5 0.8 23.7 1.0 23.5 1.2 23.9 1.4 24.5 1.2 25.0 1.5 25.3 1.3 9.4 117 22.3 1.2 22.7 1.4 22.7 1.5 21.8 1.7 22.9 2.6 23.1 1.4 23.3 1.4 24.2 1.1* 8.5 231 22.5 1.1 21.5 1.0 23.3 1.4 23.0 1.3 23.1 1.0 23.9 1.1 24.4 1.3 25.2 1.5 12.0 25 mg/kg 231 22.8 1.5 23.0 1.5 22.7 1.2 22.6 1.3 23.2 1.4 23.4 1.3 24.2 1.6 24.5 1.6* 7.5 50 mg/kg 231 22.5 1.3 22.7 1.1 22.8 1.5 23.2 1.4 23.4 1.4 23.5 1.1 24.3 0.9 24.4 0.9* 8.4 100 mg/kg 231 22.7 0.9 23.2 0.8 20.8 1.8 21.9 1.9 22.0 1.9 22.3 1.4 22.9 1.5 23.7 1.3* 4.4 200 mg/kg Notes: *P < 0.05, compared with the body weight in the control group.

[0461] 7.2 Tumor Volume

[0462] During the test, mean tumor volumes of group 360 and group 117 were always less than that of the control group, but without statistical difference (P>0.05). The tumor volume of the group 231 25 mg/kg (low dose), was remarkably less than that of the control group at Day 10 and Day 12 (P<0.05); the tumor volumes of 231 groups with a dose equal to or greater than 50 mg/kg were always remarkably less than that of the control group (P<0.05) from Day 10 to the end of the test. See details in FIG. 2 and Table 13 for results and statistical analysis.

TABLE-US-00020 TABLE 13 Mean Tumor Volumes (unit: mm.sup.3, s) of Various Groups of Tumor-bearing Models at Various Time Points Day 8 Day 10 D 12 D 14 D 17 Control 107.5 55.9 362.9 160.6 755.7 338.2 1116.8 455.9 1973.9 851.1 360 95.2 36.6 303.5 233.3 512.0 240.2 949.2 773.3 1531.0 606.0 117 130.1 31.1 262.3 131.2 517.3 261.9 760.9 335.7 1410.5 734.0 231 25 mg/kg 69.5 21.9 229.1 76.7* 411.7 275.4* 721.9 575.3 1471.2 1269.4 231 50 mg/kg 94.5 52.5 234.6 117.6* 436.8 204.9* 661.5 255.6* 1242.2 500.9* 231 100 mg/kg 97.9 86.9 168.2 145.5* 372.4 308.9* 598.6 442.3* 1195.4 779.8* 231 200 mg/kg 74.2 32.2 160.5 114.9* 300.4 231.8* 512.2 351.8* 909.7 544.6* Notes: *P < 0.05, compared with the control group.

[0463] 7.3 Tumor Weight

[0464] When the experiment was ended, mean tumor weight of group 360 and group 117 was less than that of the control group, but without statistical difference. The tumor inhibitory effect of the compound 231 was shown to be dose-dependent, and with 25, 50, 100 mg/kg, the mean tumor weight was less than that of the control group, but without statistical difference; and with 200 mg/kg of 231, the mean tumor weight was remarkably less than that of the control group (P<0.05), with a tumor inhibitory rate of 56.8%. With the same dose of 100 mg/kg, 231 and 360 had equivalent tumor inhibitory effect, superior to 117; for group 117, there was one animal on which no tumor block grew when the test was ended. See details in FIG. 3, accompanying drawings, and Table 14 for results and statistical analysis.

TABLE-US-00021 TABLE 14 Effect of Tested Drug on Transplanted Tumor Weight ( s) Inhibitory Rate for Group Tumor Weight (g) Tumor Weight (%) Control 2.01 0.84 360 1.41 0.81 29.5 117 1.71 0.76 14.4 231 25 mg/kg 1.49 1.16 25.9 231 50 mg/kg 1.42 0.56 29.2 231 100 mg/kg 1.32 1.09 34.0 231 200 mg/kg .sup.0.87 0.74 * 56.8 Notes: * P < 0.05, compared with the control group.

[0465] 8. Conclusion

[0466] The purpose of research in the present experiment is assessing phase I metabolic stability of the sample for test in CD-1 mice, SD rats, and human liver microsomes. Under the conditions of the present experiment, the compound 231 could inhibit growth of CT26 transplanted tumor in a dose-dependent manner, with effect superior to the compound 117 and the compound 360, but could modestly decrease the increase in body weight of the tumor-bearing mice.

Test Example 3

Stability Experiment for Human Liver Microsomes

[0467] The animals and human liver microsomes used in this test system were purchased from BD Gentest, Xenotech, Corning or BioreclamationIVT, and stored in a refrigerator at 80 C. before use.

[0468] The sample for test and control were incubated together with the microsomes under a condition of 37 C. for 60 minutes, and a cold acetonitrile solution (or other organic solvents) containing an internal standard substance was added at a designated time point to terminate the reaction. After centrifugation, a resultant supernatant was assayed in a semiquantitative manner through liquid chromatography tandem mass spectrometry (LC/MS/MS). Software Analyst (AB Sciex, Framingham, Mass., USA) was used for processing retention time of analyte and internal standard, and achieving chromatogram collection, and chromatogram integration

[0469] An in vitro elimination rate constant ke of the sample was obtained by converting a ratio of the sample to a peak area of the internal standard to remaining percentage, to calculate an in vitro elimination rate and half-life of the sample for test. Results are as shown in Table 15:

TABLE-US-00022 TABLE 15 Test Compounds 231 227 Remaining % (60 min), H/R/M 81.6/64.8/35.8 56.9/21.6/11.0 Intrinsic clearance (mL/min/kg) <8.6, 23.7, 134.3 15.3, 91.9, 274.8

[0470] Discussion: the compound 231 is metabolized in a relatively steady manner in human and rat liver microsomes, but metabolized quickly in mice. The compound 227 is metabolized in a moderate manner in human liver microsomes, and metabolized quickly in both rats and mice, inferior to the compound 231 in metabolic stability.

Test Example 4

Experiment for Inhibition on Human Liver Microsome CYP

[0471] The research project aimed at evaluating inhibitory ability of a sample for test on human liver microsome cytochrome P450 isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) with use of a 5-in-1 probe substrate for CYP isozymes.

[0472] Mixed human liver microsomes (HLM) were purchased from Corning Inc. (Steuben, N.Y., USA) or XenoTech, LLC. (Lenexa, Kans., USA) or other suppliers, and stored in a condition below 70 C. before use.

[0473] Diluted working solutions of the sample for test in serial concentrations were added to an incubation system containing the human liver microsomes, probe substrate, and accessory factors of a cycling system, and a control containing no sample for test but a solvent was taken as a control for enzymatic activity (100%). Concentration of metabolites generated by the probe substrate in the sample was measured with a method of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nonlinear regression analysis of mean percentage activity vs concentration of the sample for test was carried out using SigmaPlot (V.11). An IC.sup.50 value was calculated through a three-parameter or four-parameter inflection logarithmic equation. Test results are in Table 16:

TABLE-US-00023 TABLE 16 Compounds Tested 231 227 CYP1A2, 2C9, >50, >50, 36.4, >50, >50 13.0, 15.9, 3.61, 26.1, 39.2 2C19, 2D6, 3A4 (IC50, M)

[0474] Discussion: the compound 231 had relatively weak effect in inhibiting all of the five CYP isozymes. The inhibiting effects of the compound 227 on the five CYP isozymes were all superior to the compound 231, and the inhibiting effect on CYP2C19 was in an intermediate degree.

Test Example 5

Pharmacokinetics Experiment of Mice In Vivo

[0475] The present experiment aimed at researching status of pharmacokinetics of a sample for test in plasma of male CD-1 mice after single intravenous injection.

[0476] 1. Test Method

[0477] Three animals in an intravenous group were intravenously injected with the sample for test by 1 mg/kg, and a formulation was 0.2 mg/mL settled solution of 5% DMSO/95% (10% HP--CD). 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after the administration, blood was collected and prepared into a plasma specimen, with an anticoagulant of EDTA-K.sub.2. Plasma concentration data was obtained by analyzing the specimen through LC-MS/MS.

[0478] 2. Data Analysis

[0479] The plasma drug concentration data was processed with a non-compartment model using WinNonlin Version 6.3 (Pharsight, Mountain View, Calif.) pharmacokinetics software. Following pharmacokinetics parameters were calculated using a log-linear trapezoidal method: elimination phase half-life (T.sub.1/2), apparent volume of distribution (V.sub.dss), and clearance rate (CL), mean retention time of drug in body from point 0 to end time point (MRT.sub.0-last), mean retention time of drug in body from point 0 to infinite time (MRT.sub.0-inf), area under a time-plasma concentration curve from point 0 to end time point (AUC.sub.0-last), area under a time-plasma concentration curve from point 0 to infinite time (AUC.sub.0-inf), and initial concentration (C.sub.0). Results are shown in Table 17:

TABLE-US-00024 TABLE 17 Pharmacokinetics Parameters 231 227 Mouse PK Cl (mL/min/kg) 55.1 64.2 (IV 1 mg/kg) Vd (L/kg) 4.85 2.66 AUC.sub.0-last/inf (nM .Math. h) 852/881 624/628 Conc. (8 h/24 h, nM) 8.16/ND ND/ND T.sub.1/2 (h): 2.44 0.867

[0480] Discussion: the two compounds both had a medium clearing rate in bodies of mice, the compound 231 had higher AUC than the compound 227, and also had apparent volume of distribution and half-life much greater than those of the compound 227.