NEUROKININ-1 ANTAGONIST

Abstract

A compound represented by formula II or a pharmaceutically acceptable salt thereof, and a preparation method therefor. The compound represented by formula II is an antagonist of a neurokinin-1 receptor, can be used for treating diseases related to the neurokinin-1 receptor, and can avoid hemolytic effects of drugs and reduce the side effects of drug administration.

##STR00001##

Claims

1. A compound of formula II, ##STR00077## or a pharmaceutically acceptable salt thereof, or a stereoisomer, rotamer or tautomer thereof, or a deuteride thereof, wherein, X is selected from the group consisting of hydrogen, heterocyclyl, aryl, heteroaryl, —C(O)OA.sub.mR.sup.3, —C(O)NR.sup.4A.sub.mR.sup.3, -A.sub.m[C(R.sup.1)(R.sup.2)]C(O)OA.sub.nR.sup.3, -A.sub.mOC(O)[C(R.sup.1)(R.sup.2)]A.sub.nR.sup.3, A.sub.mC(O)NR.sup.4A.sub.nR.sup.3, -A.sub.mNR.sup.4C(O)A.sub.nR.sup.3 and -A.sub.mR.sup.5, said heterocyclyl, aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of alkyl, cycloalkyl, alkoxyl, hydroxyalkyl, alkenyl, alkynyl, aryl, heteroaryl, nitro, cyano, hydroxyl, halogen, SR′, NR(R″), COOR′ and CONR′(R″); Y is selected from the group consisting of hydrogen, —C(O)OA.sub.mR.sup.3, —C(O)NR.sup.4A.sub.mR.sup.3, -A.sub.m[C(R.sup.1)(R.sup.2)]C(O)OA.sub.nR.sup.3, -A.sub.mOC(O)[C(R.sup.1)(R.sup.2)]A.sub.nR.sup.3, -A.sub.mC(O)NR.sup.4A.sub.nR.sup.3, -A.sub.mNR.sup.4C(O)A.sub.nR.sup.3 and -A.sub.mR.sup.5; A is independently selected from the group consisting of —C(R.sup.1)(R.sup.2)(B).sub.p— and —(B).sub.qC(R.sup.1)(R.sup.2)—; R.sup.1, R.sup.2 and R.sup.4 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of alkyl, cycloalkyl, alkoxyl, hydroxyalkyl, alkenyl, alkynyl, aryl, heteroaryl, nitro, cyano, hydroxyl, halogen, SR′, NR′(R″), COOR′ and CONR′(R″); R.sup.3 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, poly(ethyleneoxy) ##STR00078##  poly(oxy ethyleneoxy) ##STR00079##  OPO(R.sup.6).sub.2, PO(R.sup.6).sub.2, OSO.sub.2(R.sup.6).sub.2, SO.sub.2(R.sup.6).sub.2, OC(O)R.sup.6 and C(O)OR.sup.6, said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of alkyl, cycloalkyl, alkoxyl, hydroxyalkyl, alkenyl, alkynyl, aryl, heteroaryl, nitro, cyano, hydroxyl, halogen, SR′, NR′(R″), COOR′ and CONR′(R″); R.sup.5 is selected from the group consisting of heterocyclyl, heteroaryl, OSO.sub.2R.sup.7, OC(O)R.sup.7, SR′, ##STR00080##  SO.sub.2R′ and NR′(R″); each of R.sup.6 is independently selected from the group consisting of hydrogen, hydroxyl, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxyl, hydroxyalkyl and NR′(R″), said alkyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of alkyl, cycloalkyl, alkoxyl, hydroxyalkyl, alkenyl, alkynyl, aryl, heteroaryl, nitro, cyano, hydroxyl, halogen, SR′, NR′(R″), COOR′ and CONR′(R″); each of R.sup.7 is independently selected from the group consisting of alkyl, hydroxyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl and NR′(R″), said alkyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of alkyl, cycloalkyl, alkoxyl, hydroxyalkyl, alkenyl, alkynyl, aryl, heteroaryl, nitro, cyano, hydroxyl, halogen, SR′, NR′(R″), COOR′ and CONR′(R″); R′ and R″ are each independently selected from the group consisting of hydrogen, hydroxyl, alkyl, alkoxyl, alkenyl and acyl; each of B is independently selected from the group consisting of O, N and SC(O); m, n and o are each independently selected from the group consisting of 1˜10; p and q are each independently selected from the group consisting of 0 and 1; and X and Y are not hydrogen at the same time.

2. The compound according to claim 1, wherein X is selected from the group consisting of hydrogen, heterocyclyl, aryl, heteroaryl, —C(O)O[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mR.sup.3, —C(O)NR.sup.4[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mR.sup.3, —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mC(O)[C(R.sup.1)(R.sup.2)(O).sub.p].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2) (O).sub.p].sub.m[C(R.sup.1)(R.sup.2)]C(O)[(O).sub.qC(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mC(O)NR.sup.4[C(R.sup.1)(R.sup.2)(O).sub.p].sub.nR.sup.3 and —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mR.sup.5, said cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of alkyl, cycloalkyl, alkoxyl, hydroxyalkyl, alkenyl, alkynyl, aryl, heteroaryl, nitro, cyano, hydroxyl, halogen, SR′, NR′ (R″), COOR′ and CONR′(R″); Y is selected from the group consisting of hydrogen, —C(O)O[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mR.sup.3, —C(O)NR.sup.4[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mR.sup.3, —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mC(O)[C(R.sup.1)(R.sup.2)(O).sub.p].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mC(O)[(O).sub.qC(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mC(O)NR.sup.4[C(R.sup.1)(R.sup.2)(O).sub.p].sub.nR.sup.3 and —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mR.sup.5; and X and Y are not hydrogen at the same time.

3. The compound according to claim 1, wherein Y is selected from the group consisting of —C(O)O[C(R.sup.1)(R.sup.2)].sub.mR.sup.3, —C(O)NR.sup.4[C(R.sup.1)(R.sup.2)].sub.mR.sup.3, —[C(R.sup.1)(R.sup.2)O].sub.mC(O)[C(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)].sub.mC(O)[OC(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)N].sub.mC(O)[C(R.sup.1)(R.sup.2)].sub.nR.sup.3, [C(R.sup.1)(R.sup.2)N].sub.mC(O)[OC(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)N].sub.mC(O)[NC(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.mC(O)NR.sup.4[C(R.sup.1)(R.sup.2)(O).sub.p].sub.nR.sup.3 and —[C(R.sup.1)(R.sup.2)(O).sub.p].sub.nR.sup.5; X is hydrogen or 3 to 6 membered heterocyclyl.

4. The compound according to claim 1, wherein X is selected from the group consisting of —C(O)O[C(R.sup.1)(R.sup.2)].sub.mR.sup.3, —C(O)NR.sup.4[C(R.sup.1)(R.sup.2)].sub.mR.sup.3, —[C(R.sup.1)(R.sup.2)O].sub.mC(O)[C(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)].sub.mC(O)[OC(R.sup.1)(R.sup.2)].sub.nR.sup.3, —[C(R.sup.1)(R.sup.2)].sub.mC(O)NR.sup.4[C(R.sup.1)(R.sup.2)].sub.nR.sup.3 and —[C(R.sup.1)(R.sup.2)].sub.mR.sup.5, Y is hydrogen.

5. The compound according to claim 1, wherein R.sup.3 is selected from the group consisting of hydrogen, poly(oxyethyleneoxy) ##STR00081## poly(ethyleneoxy) ##STR00082## OPO(R.sup.6).sub.2, PO(R.sup.6).sub.2, OSO.sub.2(R.sup.6).sub.2, SO.sub.2(R.sup.6).sub.2, OC(O)R.sup.6 and C(O)OR.sup.6, R.sup.6 is as defined in claim 1.

6. The compound according to claim 1, wherein R.sup.3 is selected from OPO(R.sup.6).sub.2, R.sup.6 is selected from the group consisting of hydroxyl, C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, C.sub.1-6 alkoxyl and 3 to 7 membered heterocyclyl.

7. The compound according to claim 1, wherein m=1, 2, 3 or 4.

8. The compound according to claim 1, wherein R.sup.1 and R.sup.2 are each independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl and C.sub.3-7 cycloalkyl.

9. The compound according to claim 1, wherein m=1, 2, 3 or 4, n=1, 2, 3 or 4, o=1˜8.

10. The compound according to claim 1, wherein the compound is formula (III): ##STR00083## or a pharmaceutically acceptable salt thereof, or a stereoisomer, rotamer or tautomer thereof, wherein, m=1, 2, 3 or 4; R.sup.1 and R.sup.2 are as defined in claim 1.

11. The compound according to claim 1, wherein the compound of formula (II) is selected from the group consisting of: ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## or a pharmaceutically acceptable salt thereof, a stereoisomer, rotamer or tautomer thereof, or a deuteride thereof.

12. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent or excipient.

13. A method of treating a physiological disorder, condition, or disease in a subject in need thereof, the method comprising administering to subject, the compound according to claim 1.

14. The method of claim 13, wherein the physiological disorder, condition or disease is selected from the group consisting of asthma, vomiting, nausea, depression, anxiety, cough and migraine.

15. The method of claim 13, wherein the physiological disorder, condition or disease is a respiratory disease, cough, inflammatory disease, skin disorder, ophthalmological disorder, depression, anxiety, phobias, bipolar disorder, alcohol dependence, substance abuse with significant effect on nerves, epilepsy, nociception, psychosis, schizophrenia, Alzheimer's disease, AIDs-related dementia, Towne's disease, stress-related disorder, obsessive/compulsive disorder, bulemia, anorexia nervosa, binge eating, mania, premenstrual syndrome, gastrointestinal dysfunction, atherosclerosis, fibrotic disorder, obesity, type II diabetes, headache, neuropathic pain, post-action pain, chronic pain syndrome, bladder disorder, genitourinary disorder or vomiting or nausea.

Description

DESCRIPTION OF THE DRAWINGS

[0098] FIG. 1: Graph of the transformation trend of the compound of Example 5 in human plasma.

DETAILED DESCRIPTION

[0099] The present disclosure will be further described with reference to the following examples, but the examples should not be considered as limiting the scope of the present disclosure.

[0100] The experimental methods with unspecified conditions in the examples of the present disclosure generally follow conventional conditions, or according to the conditions recommended by the manufacturer of the raw material or product. The reagents with unspecified sources are conventional reagents purchased from the market.

Example 1

[0101] ##STR00038##

Step 1:

[0102] ##STR00039##

[0103] Compound 1 (2.43 g, 4.86 mmol, 1 eq) was weighed and dissolved in dichloromethane (36 mL) in a 100 mL three-necked flask under N.sub.2 atmosphere. Diisopropylethylamine (5 g, 38.76 mmol, 8 eq) was added and the mixture was cooled to −30° C. Trimethylchlorosilane (1.36 g, 12.52 mmol, 2.6 eq) was added and the mixture was stirred at room temperature for 2 h. The reaction mixture was cooled to −25° C. A solution of chloromethyl chloroformate (0.77 g, 6 mmol, 1.23 eq) in dichloromethane was added dropwise and the mixture was stirred under controlled temperature at −20° C.˜−5° C. until completion of the reaction. The reaction solution was poured into ice water, put to separation, and extracted with dichloromethane. Water and 1 N hydrochloric acid solution were added and put to separation. The organic layer was then successively washed with brine, saturated aqueous solution of sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and concentrated to give 3.0 g yellow jelly with a yield of 104%.

Step 2:

[0104] ##STR00040##

[0105] Compound 2 (2.8 g, 4.53 mmol, 1 eq), tetrabutylammonium iodide (1.68 g, 4.55 mmol, 1 eq), di-tert-butylphosphate potassium salt (5.63 g, 22.67 mmol, 5 eq) and dioxane (84 mL) were added into a 500 mL three-necked flask under N.sub.2 atmosphere. The reaction mixture was heated to 55° C. and stirred for 4 h. The reaction solution was cooled, poured into ethyl acetate and water, put to separation, and extracted with ethyl acetate. The organic layer was washed with aqueous solution of sodium sulfite, and then successively washed with water and brine, dried over anhydrous sodium sulfate, filtered and concentrated to give 3.73 g yellow foam with a yield of 107%.

Step 3:

[0106] ##STR00041##

[0107] Compound 3 (1.95 g, 2.543 mmol, 1 eq) was added into a 100 mL single-necked flask and dissolved in dichloromethane (40 mL) under N.sub.2 atmosphere. Trifluoroacetic acid (1.45 mL, 19.52 mmol, 8 eq) was added slowly under ice water cooling. The reaction mixture was stirred until completion of the reaction, and then concentrated to give 2.29 g oil which was then purified purified to give 1.39 g white foamy solid with a yield of 83.5%.

[0108] .sup.1H-NMR (400 MHz, CD.sub.3OD): δ(ppm) 7.89 (s, 2H), 7.86 (s, 1H), 7.41-7.27 (m, 5H), 5.66 (d, J=12 Hz, 1H), 5.50-5.47 (m, 1H), 4.60 (d, J=8 Hz, 1H), 4.20-3.88 (m, 3H), 2.51-2.10 (m, 5H), 1.86-1.66 (m, 3H), 1.44-1.31 (m, 4H).

Step 4:

[0109] ##STR00042##

[0110] Compound 4 (111 mg, 0.17 mmol) and meglumine (59.6 mg, 0.305 mmol) were added to a 50 mL single-neck flask and dissolved in methanol (5 mL). The reaction mixture was stirred at room temperature for 1.5 h and then concentrated to give 174 mg white solid salt.

Example 2

[0111] ##STR00043##

Step 1:

[0112] ##STR00044##

[0113] Compound 1 (5 g, 10 mmol, 1 eq) was placed in a 250 mL three-neck flask and 50 mL of dichloromethane was added. The reaction mixture was replaced with N.sub.2. Diisopropylethylamine (5.1 g, 40 mmol, 4 eq) was added and the mixture was cooled to 0° C. 3-Chloropropyl chloroformate (4.71 g, 30 mmol, 3 eq) was added slowly dropwise and the reaction mixture was stirred until completion of the reaction. The reaction solution was washed with 20 mL×2 water, dried over anhydrous sodium sulfate and concentrated. The crude product was pulped with 20 mL of tert-butyl methyl ether, filtered and dried to give 5.3 g product as a white solid, with a yield of 85.5% and a HPLC purity of 95.2%.

Step 2:

[0114] ##STR00045##

[0115] Compound 2 (500 mg, 0.833 mmol, 1 eq) was placed in a 25 mL round bottom flask. 5 mL of dimethylformamide, 5 mg potassium iodide and di-tert-butyl phosphate tetrabutyl quaternary ammonium (564 mg, 1.25 mmol, 1.5 eq) were added. The temperature was raised to 100° C. until completion of the reaction. The reaction mixture was concentrated and the residues were purified by HPLC to give 370 mg product with a yield of 57.8% and a HPLC purity of 97%.

Step 3:

[0116] ##STR00046##

[0117] Compound 3 (2 g, 2.52 mmol) was dissolved in a solution of hydrochloric acid in dioxane (25 mL, 4 M), and stirred at room temperature for 30 minutes. The reaction mixture was evaporated to dry under reduced pressure to give compound 4 (1.4 g, 2.05 mmol) with a yield of 81%.

Step 4:

[0118] ##STR00047##

[0119] Compound 4 (700 mg, 1.025 mmol) and meglumine (310 mg, 2 mmol) were dissolved in methanol (10 mL) at 25° C. and stirred for 1 h. The reaction mixture was concentrated under reduced pressure to give a crude product of compound 5 (1.1 g), which was then pulped with methyl tert-butyl ether and filtered and then dried to give the pure product of compound 5 (1 g, 0.932 mmol) with a yield of 91%.

[0120] .sup.1H-NMR (400 MHz, CD.sub.3OD): δ7.90-7.84 (m, 3H), 7.32-7.25 (m, 5H), 4.14-3.61 (m, 25H), 2.81 (m, 5H), 2.47-2.29 (m, 12H), 1.79-1.63 (m, 5H), 1.46-1.29 (m, 3H), 1.21-1.12 (m, 6H).

Example 3

[0121] ##STR00048##

[0122] 2 ml of acetone and 100 mg compound 1 were added into a 25 ml flask and stirred. Potassium carbonate (42 mg, 0.3 mmol, 1.5 eq) was added in batches and the mixture was stirred at room temperature for half an hour. 36 mg compound 2 was added to the reaction flask. The reaction mixture was stirred at room temperature for about 18 hours until completion of the reaction, and purified by column chromatography to give 50 mg 3 (yield 40.8%).

[0123] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.79 (s, 1H), 7.72 (s, 2H), 7.42-7.40 (d, J=8 Hz, 2H), 7.31-7.27 (m, 2H), 7.27-7.21 (m, 1H), 5.58 (s, 1H), 4.55-4.53 (m, 1H), 4.06-3.99 (m, 2H), 3.69-3.67 (d, J=8 Hz, 1H), 3.53-3.49 (d, J=16 Hz, 1H), 3.25-3.21 (d, J=16 Hz, 1H), 2.77-2.74 (d, J=12 Hz, 1H), 2.59-2.57 (d, J=8 Hz, 1H), 2.34-2.31 (m, 3H), 1.97-1.71 (m, 7H), 1.46-1.45 (d, J=4 Hz, 3H).

##STR00049##

Example 4

Step 1:

[0124] ##STR00050##

[0125] 750 mg compound 1 and 2.1 ml of diisopropylethylamine were added into a three-necked flask under nitrogen atmosphere. Then 12 ml of anhydrous dichloromethane was added. The mixture was cooled to −40° C. and 0.5 ml of trimethylchlorosilane was added dropwise. The mixture was stirred at room temperature for two hours. Then the reaction mixture was cooled to −30° C. to −20° C. and 0.024 ml of chloroethyl chloroformate dissolving in 3 ml of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred at −20° C. to 5° C. until completion of the reaction. The reaction was quenched by adding water. The liquid was put to separation and washed successively with 1 N diluted hydrochloric acid, saturated brine, saturated aqueous solution of sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and dried under vacuum to give 1 g 2 as white solid.

Step 2:

[0126] ##STR00051##

[0127] 1 g compound 2 and 0.99 g sodium iodide were dissolved in 10 ml of dimethylformamide. 1.15 ml of diisopropylethylamine and 0.75 ml of methylpiperazine were added. The reaction mixture was heated to 90° C. and stirred until completion of the reaction. The reaction solution was directly concentrated and the residues were purified by HPLC to give 430 mg compound 3.

[0128] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ7.77 (s, 1H), 7.73 (s, 2H), 7.37-7.26 (m, 5H), 6.56 (s, 1H), 4.44-4.40 (m, 1H), 4.29-4.24 (m, 2H), 4.10-4.07 (m, 1H), 3.90-3.87 (d, J=12 Hz, 1H), 3.79-3.76 (d, J=12 Hz, 1H), 3.01-2.97 (d, J=16 Hz, 1H), 2.52-2.32 (m, 15H), 1.93-1.65 (m, 6H), 1.29-1.28 (d, J=4 Hz, 3H).

Example 5

[0129] ##STR00052##

Step 1:

[0130] ##STR00053##

[0131] 750 mg compound 1 and 2.1 ml of diisopropylethylamine were dissolved in 13 ml of anhydrous dichloromethane under nitrogen atmosphere. The reaction mixture was cooled to −10° C. and 0.5 ml of trimethylchlorosilane was added dropwise. The mixture was then warmed to room temperature and stirred for three hours. The mixture was cooled to −10° C. again And 3 ml of solution of chloromethyl chloroformate (0.288 g) in dichloromethane was added. The reaction mixture was then reacted at −10° C. until completion of the reaction. The reaction was quenched by water. The liquid was put to separation and washed successively with diluted hydrochloric acid, saturated brine, saturated aqueous solution of sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residues were purified to give 400 mg compound 2 as a white solid.

Step 2:

[0132] ##STR00054##

[0133] 147 mg compound 3 and 203 mg sodium iodide were added to 2 ml of dimethylformamide, followed by addition of 136 mg potassium bicarbonate. The reaction mixture was stirred at room temperature for half an hour, and then 400 mg compound 2 dissolving in 10 ml of dimethylformamide was added dropwise. The mixture was reacted overnight. The reaction was quenched by adding water. The reaction mixture was extracted twice with ethyl acetate. The organic phases were collected, dried over anhydrous sodium sulfate, filtered and concentrated. The concentrate was purified by silica gel column to give 450 mg compound 4 as an oil.

Step 3:

[0134] ##STR00055##

[0135] 405 mg compound 4 was dissolved in 18 ml of dichloromethane. 4.5 ml of trifluoroacetic acid was added dropwise under cooling in an ice bath. After addition, the reaction was warmed to room temperature and stirred for two hours. The reaction mixture was concentrated and the residues were purified by column chromatography to give 330 mg of product, compound 5, with a yield of 80%.

[0136] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 8.34 (s, 1H), 7.72 (s, 1H), 7.63 (s, 2H), 7.40-7.28 (m, 5H), 6.19 (s, 1H), 5.68-5.67 (d, J=4 Hz, 1H), 4.30-4.29 (d, J=4 Hz, 1H), 4.20-4.17 (d, J=12 Hz, 1H), 3.99-3.91 (m, 2H), 3.79 (s, 1H), 2.70-2.67 (d, J=12 Hz, 1H), 2.49-2.21 (m, 8H), 1.83-1.70 (m, 4H), 1.29-1.28 (m, 3H), 1.09-1.07 (m, 6H).

Example 6

[0137] ##STR00056##

Step 1:

[0138] ##STR00057##

[0139] 1.294 g triphosgene was dissolved in 7.5 ml of anhydrous tetrahydrofuran. The solution was cooled in an ice bath and replaced with nitrogen for three times. Then 0.33 ml of pyridine was added dropwise. After addition, a solution of 500 mg compound 1 dissolving in 7.5 ml of anhydrous tetrahydrofuran was added dropwise. After addition, the reaction was stirred at 5° C. for 3 hours. 30 ml of dichloromethane was added for dilution. The solution was washed successively with diluted hydrochloric acid, water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give 700 mg crude product.

Step 2:

[0140] ##STR00058##

[0141] 128 mg compound 3 was added to 2 ml of anhydrous tetrahydrofuran. The reaction mixture was replaced with nitrogen for three times. The reaction was cooled to about −65° C., and 0.28 ml of lithium hexamethyldisilazide (1 mol/L, dissolving in n-hexane) was added dropwise. The mixture was stirred for half an hour. Meanwhile, 60 mg crude product of compound 2 from the previous step was dissolved in 1 ml of anhydrous tetrahydrofuran. The mixture was cooled to −65° C. after replacing with nitrogen for three times. The lithium salt of compound 3 prepared above was transferred into the reaction flask containing acyl chloride compound 2. After addition, the reaction was completed at −65° C. and quenched by adding saturated aqueous solution of ammonium chloride. The reaction mixture was extracted with ethyl acetate. The organic phase was concentrated and directly purified by column chromatography to give 62 mg compound 4.

Step 3:

[0142] ##STR00059##

[0143] 62 mg compound 4 and 31 mg of 20% wet palladium hydroxide were added to 1.5 ml of ethyl acetate and stirring was started. The reaction mixture was replaced with hydrogen for three times, stirred at room temperature for 5 hours and filtered. The filter cake was washed with ethyl acetate. The filtrate was concentrated and the residues were purified to give 41 mg of compound 5 as a white solid.

[0144] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ=7.72 (s, 1H), 7.57 (s, 2H), 7.50-7.41 (m, 5H), 4.80-4.59 (m, 3H), 4.17-4.13 (d, J=16 Hz, 1H), 3.83-3.80 (d, J=12 Hz, 1H), 3.62-3.59 (d, J=12 Hz, 1H), 3.26-3.22 (d, J=16 Hz, 1H), 2.60-2.47 (m, 4H), 2.26-2.18 (m, 2H), 1.85-1.80 (m, 2H), 1.45-1.43 (d, J=8 Hz, 3H), 0.89-0.83 (m, 1H).

Example 7

[0145] ##STR00060##

[0146] Potassium carbonate (11.7 g, 84.66 mmol, 8.47 eq) was dissolved in water (40 mL) until use. Compound 1 (5.55 g, 10 mmol, 1 eq) was suspended in ethyl acetate (80 mL) under N.sub.2 atmosphere, to which the aforementioned aqueous solution of potassium carbonate was added under cooling by an ice-bath. The reaction solution gradually became clear under stirring, and then Cbz-Cl (1.7 mL, 12 mmol, 1.2 eq) was added dropwise. After addition, the reaction mixture was stirred for 10 min, and stirred at room temperature overnight. The reaction solution was put to separation and extracted with ethyl acetate. The organic phases were collected, dried over anhydrous sodium sulfate, filtered and concentrated. The residues were purified by column chromatography to give 4.3 g white solid with a yield of 56%.

Example 8

[0147] ##STR00061##

[0148] Compound 1 (317 mg, 0.5 mmol) and THF (7.2 mL) were added into a 50 ml of single-necked flask under nitrogen atmosphere and then stirred to dissolve and cooled to −20° C. NaHMDS (2 M, 0.5 mL, 1 mmol) was added dropwise and the reaction mixture was stirred until completion of the reaction. The reaction was quenched by saturated ammonium chloride. The reaction mixture was extracted with methyl tert-butyl ether. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residues were purified by column chromatography to give compound 2 (250 mg) with a yield of 68%.

##STR00062##

[0149] Compound 2 (250 mg, 0.34 mmol), methanol (10 mL) and palladium on carbon (10%, 250 mg) were added into a 50 ml of single-neck flask. The reaction mixture was stirred at room temperature under hydrogen atmosphere until completion of the reaction. The reaction mixture was filtered and the residues were concentrated to give compound 3 (150 mg) with 74% yield.

[0150] LCMS: 601 [M+1].

Example 9

[0151] ##STR00063##

[0152] Compound 1 (215 mg, 0.339 mmol, 1 eq), anhydrous potassium carbonate (55 mg, 0.396 mmol, 1.1 eq), paraformaldehyde (37 mg, 1.23 mmol, 3.3 eq) and THF (5 ml) were added into a 50 mL reaction flask under N.sub.2 atmosphere. The reaction mixture was heated and stirred until completion of the reaction. The reaction mixture was filtered and concentrated to give a crude product, which was purified by column chromatography to give 216 mg of oil 2 with a yield of 95%.

##STR00064##

[0153] Compound 2 (66 mg, 0.1 mmol, 1 eq) and THF (5 ml) were added to a reaction flask under N.sub.2 atmosphere. LiHMDS (1 M in THF, 0.2 ml, 0.2 mmol, 2 eq) and then compound 3 (40 mg, 0.37 mmol, 3.7 eq) were added dropwise and the reaction mixture was stirred until completion of the reaction. The reaction mixture was extracted with ethyl acetate and concentrated to give a crude product, which was purified by column chromatography to give 27 mg of crude product 4 as an oil with a yield of 36%.

##STR00065##

[0154] Compound 4 (27 mg, 0.367 mmol, 1 eq), Pd/C (33 mg) and methanol (5 ml) were added to a reaction flask at room temperature, and stirred under hydrogen atmosphere until completion of the reaction. The reaction mixture was filtered and concentrated to give a crude product, which was subjected to column chromatography to give 13 mg of oil 5 with a yield of 58.8%.

[0155] LCMS: 602 [M+1].

Example 10

[0156] ##STR00066##

[0157] The target compound was synthesized according to the method of Example 5 with replacing chloromethyl chloroformate by chloropropyl chloroformate. LCMS: 702 [M+1].

Example 11

[0158] ##STR00067##

[0159] The target compound was synthesized according to the method of Example 5 with replacing chloromethyl chloroformate by chloroethyl chloroformate. LCMS: 688 [M+1].

Example 12

[0160] ##STR00068##

[0161] The target compound was synthesized according to the method of Example 5 with replacing Boc-L-valine by N-Boc-glycine. LCMS: 632 [M+1].

Example 13

[0162] ##STR00069##

[0163] The target compound was synthesized according to the method of Example 5 with replacing Boc-L-valine by Boc-L-alanine. LCMS: 646 [M+1].

Example 14

[0164] ##STR00070##

[0165] The target compound (isomers approximately 1/1) was synthesized according to the method of Example 5 with replacing chloromethyl chloroformate by 1-chloroethyl chloroformate. LCMS: 688 [M+1].

Example 15

[0166] ##STR00071##

[0167] The target compound was synthesized according to the method of Example 5 with replacing Boc-L-valine by Boc-L-methionine. LCMS: 706 [M+1].

Example 16

[0168] ##STR00072##

[0169] The target compound was synthesized according to the method of Example 5 with replacing Boc-L-valine by Boc-L-proline. LCMS: 672 [M+1].

Example 17

[0170] ##STR00073##

[0171] The target compound was synthesized according to the method of Example 5 with replacing Boc-L-valine by (S)-2,6-di-tert-butylcarbonylaminocaproic acid. LCMS: 703 [M+1].

Example 18

[0172] ##STR00074##

[0173] The target compound was synthesized according to the method of Example 5 with replacing Boc-L-valine by Boc-D-valine. LCMS: 674 [M+1].

Test Example 1: Water Solubility Data and Chemical Stability

[0174] 1.1. Preparation of Reagents

[0175] Reagent: NaH.sub.2PO.sub.4.2H.sub.2O

[0176] 1.2. Preparation Method

[0177] The 100 mL strength reagents were prepared as follows:

[0178] pH=3.0: phosphate buffer solution: 100 ml of 20 mmol/L NaH.sub.2PO.sub.4, 0.1M H.sub.3PO.sub.4, adjusting pH to 3.0.

[0179] pH=4.0: phosphate buffer solution: 100 ml of 20 mmol/L NaH.sub.2PO.sub.4, 0.1M H.sub.3PO.sub.4, adjusting pH to 4.0.

[0180] pH=7.0: ultrapure water

[0181] pH=9.0: phosphate buffer solution: 100 ml of 20 mmol/L Na.sub.2HPO.sub.4, 0.1M NaOH solution, adjusting pH to 9.0.

[0182] 1.3. Test Method

[0183] An appropriate amount of test compound was accurately weighed. The solution was added at a small amount each time for several times and stirred until the compound was dissolved, and the content of the compound in the solution was determined. The data are shown in Table 1.

[0184] 2.1 Test of Stability of the Compounds

[0185] About 1 mg of sample was weighed into a vial, then the vial was placed in a vacuum bag and the bag was vacuumized. Then the bag was put into a container containing color-changing silica gel and sealed. Two parallel samples were prepared. Enough samples were prepared according to the sampling time point and placed at 4° C. and room temperature respectively. The data are shown in Table 1.

TABLE-US-00001 TABLE 1 No. Water solubility Chemical stability Example 1 >10 mg/ml (PH = 5) Good Example 2 9.05 mg/ml (PH = 5) Good Example 3 <0.1 mg/ml (PH = 5) Good Example 4 2.08 mg/mL (PH = 3) Good Example 5 >10 mg/ml (PH = 4) Good Example 6 1.12 mg/ml (PH = 3) Good Example 7 NA NA Example 8 1.37 mg/ml (PH = 5) Moderate Example 9 <0.1 mg/ml (PH = 3) Good Example 10 1.86 mg/mL(PH = 3) Example 11 2.81 mg/mL(PH = 3) Good Example 12 NA Poor Example 13 NA Moderate Example 14 4.5 mg/ml (PH = 4) Good Example 15 NA Poor Example 16 NA Poor Example 17 NA Moderate Example 18 2.62 mg/mL (PH = 4) Good Note: Good: purity reduced by <0.5% after placing for 7 days; Moderate: purity reduced by 0.5%~2.0% after placing for 7 days; Poor: purity reduced by >2.0% after placing for 7 days.

Test Example 2: Plasma Stability Test

[0186] Test Protocol

[0187] 1.1 Test Drug

[0188] The compound of Example 5 and the compound of formula I.

[0189] 1.2 Test Plasma

[0190] Human fresh plasma was donated by volunteers with informed consent.

[0191] 1.3 Preparation of Solution of the Compound

[0192] A certain amount of the compound of Example 5 was weighed and DMSO was added to prepare a 30 mM stock solution. A certain volume of the stock solution was diluted with DMSO to prepare solution I at a concentration of 1600 μM. Then a certain volume of the 1600 μM solution I was diluted with 45% methanol to prepare working solution II at a concentration of 16 μM. The 30 mM stock solution and the 1600 μM solution II for the compound of formula I were also prepared by the above method.

[0193] 1.4 Sample Incubation

[0194] 5 μL of the 16 μM working solution of the compound of Example 5 was added to 75 μL plasma at a final concentration of 1 μM. The samples were incubated in a 37° C. water bath for 0, 15, 30, 60, 90, 120 and 180 min. After completion of the incubation, 240 μL of internal standard containing acetonitrile was added into the samples which was then shaken on a shaker at 800 rpm for 10 min and centrifuged at 3700 rpm at 4° C. for 20 min. The supernatant was analyzed by LC-MS, and the injection volume was 2 μL.

[0195] 1.5 Preparation of Standard Curve

[0196] The previously 1600 μM solution I was diluted with acetonitrile to prepare the standard curve working solution with concentrations of 160, 400, 1600, 4000, 8000, 16000 and 32000 ng/mL. The concentration of QC working solution was 480, 1920 and 25600 ng/mL. 5 μL of the standard curve working solution and QC working solution were added to 75 μL plasma to obtain the standard curve samples with final concentrations of 10, 25, 100, 250, 500, 1000 and 2000 ng/mL and QC samples with final concentrations of 30, 120 and 1600 ng/mL. 240 μL of acetocyano containing the internal standard was then added into the samples quickly, which was then shaken in a shaker at 800 rpm for 10 min and centrifuged at 3700 rpm at 4° C. for 20 min. The supernatant was collected and analyzed by LC-MS, and the injection volume was 2 μL.

[0197] The standard curve of the compound of formula I and QC sample were prepared by the above method.

[0198] 2. Results

[0199] The conversion of the compound of Example 5 of the present invention in fresh human plasma is as follows, and the data are shown in Table 2:

TABLE-US-00002 TABLE 2 Time point Compound of Compound of (min) Example 5 (μM) formula 1 (μM) 0 1 0.00 15 0.53 0.78 30 0.12 1.18 60 0.01 1.22 90 0.00 1.31 120 0.00 1.28 180 0.00 1.02

[0200] Conclusion: the compound was completely converted into the compound of formula I in human fresh plasma in about 30 min.

Test Example 3: Plasma Stability Test

[0201] 1.1 Test Drug

[0202] The compounds of Example 4, Example 6, Example 10 and Example 11.

[0203] 1.2 Test Plasma

[0204] Human fresh plasma was donated by volunteers with informed consent.

[0205] 1.3 Experimental Steps

[0206] 1) The test compounds in Table 3 were respectively prepared into 30 mM stock solutions with DMSO for later use.

[0207] 2) The 30 mM stock solution was diluted with DMSO solution to solution I at a concentration of 1600 μM. Then the 1600 μM solution I was diluted with acetonitrile (ACN) to a working solution II at a concentration of 16 μM.

[0208] 3) 7 time points of 0, 15, 30, 60, 90, 120 and 180 min were set in the experiment with two parallel samples for each time point. Two sample groups were set for each compound. 75 μL plasma and 5 μL of the above prepared working solution II with a concentration of 16 μM were added to each group. The reaction system was incubated at 37° C. until the preset time when the reaction was stopped by 300 μL of the ACN solution containing the internal standard. The reaction mixture was centrifuged at 3700 rpm for 10 min, and the supernatant was collected for analysis.

[0209] 4) Preparation of the standard curve: the previously diluted 1600 μM solution I was diluted with acetonitrile to 1.5 μM/mL solution III as the standard curve for later use. The standard curve concentration was set to 0.32, 0.8, 1.6, 4.0, 8, 12, 16 and 42 uM. After dilution of each concentration of the standard curve, 75 μL plasma was added to 5 μL of each concentration point at a final concentration of 0.02, 0.05, 0.1, 0.25, 0.75, 1.0 and 1.5 uM, respectively. 300 μL stop solution was then added to the samples quickly which was then centrifuged at 3700 rpm for 10 min, and the supernatant was collected for LC-MSMS analysis. The data are shown in Table 3.

TABLE-US-00003 TABLE 3 Time point Example 4 Example 6 Example 10 Example 11 (min) μM.sup.a μM.sup.b μM.sup.a μM.sup.b μM.sup.a μM.sup.b μM.sup.a μM.sup.b 0 1.16 0 0.98 0.01 1.04 0.00 1.01 0.01 15 0.98 0 0.85 0.01 0.92 0.00 0.99 0.00 30 0.99 0 0.91 0.01 0.97 0.00 0.91 0.00 60 1.00 0 0.85 0.01 0.96 0.00 0.92 0.01 90 0.97 0 0.77 0.01 0.93 0.00 1.03 0.00 120 0.81 0 0.70 0.01 0.95 0.00 0.97 0.00 180 0.78 0 0.53 0.01 0.88 0.00 0.95 0.00 Note: .sup.athe plasma concentration of the compound of the examples, .sup.bthe plasma concentration of rolapitant after metabolism of the compound of the examples.

[0210] Conclusion: The compounds in Example 4, Example 10 and Example 11 are relatively stable in plasma with relatively longer half-life in plasma, but only a small portion of these three compounds are metabolized to rolapitant in plasma. The compound in Example 6 can be metabolized to rolapitant in plasma, but it can be seen from the above data that the overall amount of metabolism in plasma is relatively small.

Test Example 4

[0211] The metabolism of the compounds of Example 1, Example 2 and Example 8 in the plasma of mouse, rat and human was determined by referring to the test method in Test Example 2. The data are shown in Table 4.

##STR00075##

TABLE-US-00004 TABLE 4 Example 1 Example 2 Example 8 μM.sup.a μM.sup.b μM.sup.a μM.sup.b μM.sup.a μM.sup.b Mouse 72.84 27.16 91.17 8.83 61.67 38.33 Rat 62.04 37.96 97.70 2.30 59.89 40.11 Human plasma 93.47 6.53 99.00 1.00 54.54 45.46 Note: .sup.athe plasma concentration of the compounds of the examples, .sup.bthe plasma concentration of rolapitant after metabolism of the compounds of the examples.

[0212] Conclusions: The compound of Example 8 can be converted into rolapitant in the plasma of mouse, rat and human, notably, the conversion rate in human plasma is nearly 46%. Meanwhile, the compounds of Example 1 and Example 2 had basically no convertion into rolapitant in human plasma, or only a slight conversion.

Test Example 5: In Vivo Pharmacokinetic Test in Rats

[0213] Rats were used as the test animals. The plasma drug concentration at different time points after administration of the compounds of Example 1 and Example 2 by injection was determined using LC/MS/MS method. The in vivo pharmacokinetic of the compounds in rats was studied, and the pharmacokinetic characteristics were evaluated.

[0214] Preparation of Drug

[0215] A certain amount of the compounds of Example 1 and Example 2 were weighed and prepared into a pH=4.0 solution by using 20 mmol/L sodium dihydrogen phosphate for later use.

[0216] 1.1 Drug Administration

[0217] The drug was administered by intravenous bolus injection with an injection time of about 5 min, administration dose of 2 mg/kg, administration concentration of 0.4 mg/ml and administration volume of 5 ml/kg.

[0218] 1.2 Operation

[0219] Blood was collected from the orbital vein before administration and 5 min, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h and 48 h after administration. About 0.6 mL was collected for each sample which was subject to anticoagulation using heparin sodium and placed on ice immediately after collection. The blood samples were placed in labeled centrifuge tubes after collection, and plasma was separated by centrifugation (centrifugation conditions: centrifugal force 2200 g, centrifugation at 2-8° C. for 10 min).

[0220] 1.3 Results of Pharmacokinetic Parameters

TABLE-US-00005 TABLE 5 Compound of Example 1 Compound of Example 2 (ng/ml).sup.a (ng/ml).sup.b (ng/ml).sup.a (ng/ml).sup.b AUC.sub.0-t (ng/mL *h) 1076 3638 41.6 1834 AUC.sub.0-∞ (ng/mL * h) 1081 27738 42.2 796 T.sub.1/2(h) 0.297 81.4 0.087 4.80 MRT 0-∞ (h) 0.092 118 0.073 7.68 Note: .sup.athe in vivo pharmacokinetic parameters of the compounds of the examples in rat, .sup.bthe in vivo pharmacokinetic parameters of rolapitant after metabolism of the compounds of examples in rat.

[0221] Conclusions: Although the compound of Example 1 is basically not metabolized in vitro, especially in human plasma, to the active substance rolapitant, it shows excellent pharmacokinetic data of rolapitant in rats, which indicates that the compound of Example 1 has been metabolized to rolapitant in vivo. Moreover, from the data of AUC.sub.0-∞, AUC.sub.0-t and T.sub.1/2, the in vivo metabolic cycle of compound 1 after administration is longer, and the absorption and exposure level of compound 1 are comparable to that of rolapitant.

Test Example 6: Pharmacokinetic Test of the Compounds in Cynomolgus Monkeys

[0222] Cynomolgus monkeys were used as the test animals. The plasma drug concentration at different time points after iv administration of the compound of Example 5 by injection was determined using LC/MS/MS method. The in vivo pharmacokinetic of the compound of the present invention in cynomolgus monkeys was studied, and the pharmacokinetic characteristics were evaluated.

[0223] Preparation of Drug

[0224] A certain amount of the compound of Example 5 was weighed and prepared into a pH=4.0 solution by using 20 mmol/L sodium dihydrogen phosphate for later use.

[0225] 1.1 Drug Administration

[0226] The drug was administered by intravenous with injection time of about 30 min, administration dose of 2 mg/kg, administration concentration of 0.4 mg/ml and administration volume of 5 ml/kg.

[0227] 1.2 Operation

[0228] Blood was collected from the femoral vein before administration and 5 min, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h and 48 h after administration. About 0.6 ml of was collected for each sample, which was subject to anticoagulation using heparin sodium and placed on ice immediately after collection. The blood samples were placed in labeled centrifuge tubes after collection, and plasma was separated by centrifugation (centrifugation conditions: centrifugal force 2200 g, centrifugation at 2-8° C. for 10 min).

[0229] The content of the compound of Example 5 and rolapitant in plasma samples was determined by LC/MS/MS.

[0230] 1.3 Results of Pharmacokinetic Parameters

TABLE-US-00006 TABLE 6 Compound of Rolapitant (compound of Compound Example 5 formula I) Tmax (h) 0.11 ± 0.12 0.14 ± 0.10 Cmax (ng/mL) 2.28 ± 0.39 315.25 ± 97.08  AUC.sub.0-t (ng/mL * h) 0.77 ± 0.29 4326.87 ± 1820.65

[0231] Conclusions: In the pharmacokinetic study of the compound of Example 5 in cynomolgus monkeys, most of the compound were rapidly transformed into the active metabolite rolapitant in cynomolgus monkeys, which has good pharmacokinetic properties.

Example 19

[0232] Compound 3 was prepared by referring to steps 1-2 in Example 1. Then compound 3 (6.65 g, 8.67 mmol, 1 eq) was dissolved in dichloromethane (200 mL) in a 500 mL single-necked flask under N.sub.2 atmosphere, and trifluoroacetic acid (9.89 g, 86.7 mmol, 10.0 eq) was added slowly under cooling in ice water. The reaction mixture was stirred until completion of the reaction. The reaction mixture was concentrated to give 2.29 g oil, which was purified by reversed-phase silica gel column (C18) (A solution: 20 mmol aqueous solution of NH.sub.4HCO.sub.3, B solution: acetonitrile) and then adjusted to pH=1˜2 with 1 M phosphoric acid, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give 2.7 g of the target product.

##STR00076##

Test Example 7: Solubility

[0233] Solubility of the compound of Example 19 at different pH values were measured by referring to the solubility test method of Test Example 1. The data are as follows:

TABLE-US-00007 TABLE 7 pH Solubility Saturation solubility 7.4 26 mg/ml 19.8 mg/ml 9.0 28 mg/ml 21.4 mg/ml

Test Example 8: Hemolytic Effect

[0234] Red blood cells (RBC) were collected from the jugular vein or central ear artery of rabbits (10 ml of EDTA whole blood). The blood was put in a conical flask with glass beads and shaken for 10 minutes to remove fibrinogen, resulting in defibrinated blood. About 10 times the amount of sodium chloride solution was added to the defibrinated blood, which was then shaken well and centrifuged at 1500 rpm for 10 minutes. The supernatant was removed and the precipitated red blood cells were washed with sodium chloride injection for 3 times according to the above method, until the supernatant was observed colorless. The obtained red blood cells were prepared into a 2% (v/v) suspension with sodium chloride injection for later use.

[0235] The test samples (the compound of Example 5 and the compound of Example 19) were respectively dissolved in PBS (pH 7.4 or pH 5) and filtered to prepare 0.4 mg/ml, 0.8 mg/ml, 1.2 mg/ml, 1.6 mg/ml and 2 mg/ml solutions for later use.

[0236] A certain amount of test sample solution was added to the above hemoglobin for testing in the supernatant.

[0237] If the solution in the test tube is clear and red, and there are no remaining cells or a small amount of remaining red blood cells at the bottom of the tube, it indicates that hemolysis has occurred. If all the red blood cells sink and the supernatant liquid is colorless and clear, it indicates that no hemolysis has occurred. If there are brown-red or red-brown flocculent precipitates in the solution, and still do not disperse after gently inverting for 3-5 times, it indicates that coagulation of red blood cell may occur. The sample should be further observed under a microscope, and if red blood cells can be seen as aggregated, then coagulation has occurred. The hemolytic effect of the compounds of the present disclosure was determined by using this method.

[0238] Conclusion: The compound of Example 19 has no hemolytic effect at a concentration up to 2 mg/ml, and the compound of Example 5 has hemolytic effect at a concentration of 0.04 mg/ml and higher.

Test Example 9: Hemolytic Effect of Rolapitant Emulsion

[0239] Rolapitant emulsion was prepared by referring to the method in CN102573475 (formula: 4.4% polyethylene glycol-15 hydroxystearate, 1.1% medium chain triglyceride and 0.66% soybean oil), and prepared into 0.18 mg/ml, 0.09 mg/ml, 0.045 mg/ml, 0.023 mg/ml, 0.011 mg/ml, 0.056 mg/ml and 0.028 mg/ml with PBS for later use.

[0240] Hemolytic effect was determined by referring to the method in Test Example 8.

[0241] Conclusion: rolapitant emulsions at all concentrations showed hemolytic effect.

Test Example 10: Pharmacokinetic Test in Cynomolgus Monkeys

[0242] Cynomolgus monkeys were used as the test animals. The plasma drug concentration at different times after administration of the compound prepared by referring to Example 19 by injection was determined by using the LC/MS/MS method. The in vivo pharmacokinetics of the compounds of the present invention in cynomolgus monkeys was studied, and the pharmacokinetic characteristics were evaluated.

[0243] Preparation of Drug

[0244] A certain amount of the test compound was weighed and prepared into a pH=4.0 solution by using 20 mmol/L sodium dihydrogen phosphate for later use.

[0245] 1.1 Drug Administration

[0246] The drug was administered by intravenous drip with injection time of about 30 min, administration dose of 3.54 mg/kg, administration concentration of 2 mg/ml and administration volume of 5 ml/kg.

[0247] 1.2 Operation

[0248] Blood was collected from the femoral vein before administration and 5 min, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h and 24 h after administration. About 0.6 mL was collected for each sample, which was subject to anticoagulation using heparin sodium and placed on ice immediately after collection. The blood samples were placed in labeled centrifuge tubes after collection, and plasma was separated by centrifugation (centrifugation conditions: centrifugal force 2200 g, centrifugation at 2-8° C. for 10 min).

[0249] The content of the compound of Example 24 and rolapitant in plasma samples was determined by LC/MS/MS.

[0250] 1.3 Results of Pharmacokinetic Parameters

TABLE-US-00008 TABLE 8 Compounds (ng/ml).sup.a (ng/ml).sup.b AUC.sub.0-24 h 1434.78 8410.94 (ng/mL * h) T.sub.1/2(h) 0.47 13.16 MRT 0-∞ (h) 0.26 8.17 Note: .sup.athe in vivo pharmacokinetic parameters of the compounds of the examples in cynomolgus monkey, .sup.bthe in vivo pharmacokinetic parameters of rolapitant after metabolism of the compounds of the examples in cynomolgus monkey.

[0251] Conclusion: in the in vivo pharmacokinetic study of the compound in cynomolgus monkeys, most of the compound is rapidly converted to the active metabolite rolapitant in cynomolgus monkeys, and the compound has good pharmacokinetic properties. In addition, compared with the compound of Example 5, the compound has a higher in vivo bioavailability in cynomolgus monkeys.