PHENYLPROPIONAMIDE COMPOUND AND USE THEREOF
20230174487 · 2023-06-08
Inventors
- Yang Zhang (Shanghai, CN)
- Wentao Wu (Shanghai, CN)
- Zhixiang Li (Shanghai, CN)
- Wenyuan Zhu (Shanghai, CN)
- Jian Qin (Shanghai, CN)
- Shuhui Chen (Shanghai, CN)
Cpc classification
C07K5/1008
CHEMISTRY; METALLURGY
International classification
Abstract
The use of a phenylpropionamide compound in the preparation of a drug for treating diseases associated with pain and pruritus. Specifically, the present invention relates to a compound as represented by formula (I) or a pharmaceutically acceptable salt thereof.
##STR00001##
Claims
1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, ##STR00062## wherein, R.sub.1 is selected from C.sub.3-5 cycloalkyl and —C.sub.1-3 alkyl-C.sub.3-5 cycloalkyl, and the C.sub.3-5 cycloalkyl and —C.sub.1-3 alkyl-C.sub.3-5 cycloalkyl are optionally substituted by 1, 2 or 3 R.sub.a; R.sub.2 is selected from ##STR00063## and the ##STR00064## are optionally substituted by 1, 2 or 3 R.sub.b; L.sub.1 is selected from —N(R.sub.e)—C(R.sub.c)(R.sub.d)— with the nitrogen atom connected to R.sub.3 and the carbon atom connected to —C(═O); R.sub.3 is selected from —C.sub.1-3 alkyl-phenyl, and the —C.sub.1-3 alkyl-phenyl is optionally substituted by 1, 2 or 3 R.sub.f; R.sub.a and R.sub.f are selected from F, Cl, Br and CH.sub.3; R.sub.b is selected from F, Cl, Br, NH.sub.2, COOH, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, —C(═O)—C.sub.1-3 alkyl and —NH—C(═O)NH—C.sub.1-3 alkyl, and the C.sub.1-3 alkyl, C.sub.1-3 alkoxy, —C(═O)—C.sub.1-3 alkyl and —NH—C(═O)NH—C.sub.1-3 alkyl are optionally substituted by 1, 2 or 3R; R.sub.c and R.sub.d are each independently selected from H, NH.sub.2 and CH.sub.3; R.sub.e is selected from H and CH.sub.3; R is selected from F, Cl and Br.
2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.1 is selected from cyclopropyl, cyclopentyl and —CH.sub.2-cyclopropyl, and the cyclopropyl, cyclopentyl and —CH.sub.2-cyclopropyl are optionally substituted by 1, 2 or 3 R.sub.a.
3. The compound or the pharmaceutically acceptable salt thereof according to claim 2, wherein, R.sub.1 is selected from ##STR00065##
4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.b is selected from F, Cl, Br, NH.sub.2, COOH, CH.sub.3, CF.sub.3, OCH.sub.3, —C(═O)—CH.sub.3 and —NH—C(═O)NH—CH.sub.3.
5. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.2 is selected from ##STR00066## and the ##STR00067## are optionally substituted by 1, 2 or 3 R.sub.b.
6. The compound or the pharmaceutically acceptable salt thereof according to claim 5, wherein, R.sub.2 is selected from ##STR00068##
7. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.3 is selected from ##STR00069## and the ##STR00070## are optionally substituted by 1, 2 or 3 R.sub.f.
8. The compound or the pharmaceutically acceptable salt thereof according to claim 7, wherein, R.sub.3 is selected from ##STR00071##
9. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, L.sub.1 is selected from —NH—CH.sub.2—.
10. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.3-L.sub.1- is selected from ##STR00072##
11. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, the compound is selected from ##STR00073## wherein, R.sub.1, R.sub.2 and R.sub.3 are as defined above; the carbon atoms with “*” are chiral carbon atoms and exist in the form of (R) or (S) single enantiomer or enriched in one enantiomer.
12. A compound represented by the following formula or a pharmaceutically acceptable salt thereof, wherein, the compound is selected from: ##STR00074## ##STR00075##
13. The compound or the pharmaceutically acceptable salt thereof according to claim 12, wherein, the compound is selected from: ##STR00076## ##STR00077##
14. A method for activating kappa receptor in a subject in need thereof, comprising: administering an effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.
15. A method for treating pain and pruritus in a subject in need thereof, comprising: administering an effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.
Description
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0092] The present disclosure is described in detail by the embodiments below, but it does not mean that there are any adverse restrictions on the present disclosure. The compounds of the present disclosure can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific implementations listed below, the implementations formed by their combination with other chemical synthesis methods, and equivalent alternatives known to those skilled in the art, and preferred implementations include but are not limited to the embodiments of the present disclosure. It will be apparent to those skilled in the art that various variations and improvements can be made to specific implementations of the present disclosure without departing from the spirit and scope of the present disclosure.
Reference Embodiment 1: Synthesis of Intermediate M1
[0093] ##STR00039##
Step 1: Preparation of Intermediate M1-2
[0094] Compound M1-1 (1.1 g, 8.52 mmol) was dissolved in 1,4-dioxane (10 mL), and sodium hydroxide (408.80 mg, 10.22 mmol) aqueous solution (10 mL) was added thereto, then di-tert-butyl dicarbonate (2.23 g, 10.22 mmol) was added thereto, and the reaction solution was continued to stir at 20° C. for 15 hours. 30 mL of water was added to the reaction solution, and the pH was adjusted to about 3 with saturated potassium hydrogen sulfate solution; the mixture was extracted with ethyl acetate (50 mL*2), and the organic phases obtained by extraction were combined, washed with 60 mL of saturated brine, and dried over anhydrous sodium sulfate, and then the mixture was filtrated, and the organic solvent was removed under reduced pressure to obtain compound M1-2, and the compound was used directly in the next reaction without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 5.09-5.07 (d, J=8.0 Hz, 1H), 4.28-4.26 (m, 1H), 1.63-1.53 (m, 2H), 1.32 (s, 9H), 0.70-0.58 (m, 1H), 0.42-0.32 (m, 2H), 0.05-−0.05 (m, 2H).
[0095] The following intermediate was synthesized using the similar method to the intermediate M1-2:
TABLE-US-00001 Intermediate number Structural formula Spectrum Intermediate M2-2
Step 2: Preparation of Intermediate M1-3
[0096] Intermediate M1-2 (1.95 g, 8.51 mmol) was dissolved in N,N-dimethylformamide (15 mL), and potassium carbonate (1.41 g, 10.21 mmol) and benzyl bromide (1.75 g, 10.21 mmol) were added thereto, and the reaction solution was continued to stir at 20° C. for 15 hours. The reaction solution was diluted with 80 mL of ethyl acetate, washed with water (50 mL*2) and saturated brine (50 mL) in turn, and the mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain a crude product, then the obtained crude product was purified by silica gel column chromatography (eluent: 0 to 10% of ethyl acetate/petroleum ether) to obtain compound M1-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.38-7.25 (m, 5H), 5.12 (d, J=2.4 Hz, 2H), 4.40-4.36 (m, 1H), 1.64-1.60 (m, 2H), 1.39 (s, 9H), 0.68-0.56 (m, 1H), 0.46-0.29 (m, 2H), 0.07-−0.08 (m, 2H).
[0097] The following intermediate was synthesized using the similar method to the intermediate M1-3:
TABLE-US-00002 Inter- mediate number Structural formula Spectrum Inter- mediate M2-3
Step 3: Preparation of Intermediate M1-4
[0098] Compound M1-3 (2.4 g, 7.51 mmol) was dissolved in ethyl acetate (10 mL), then 4 M of hydrochloric acid-ethyl acetate solution (10 mL) was added thereto, and the reaction solution was continued to react at 20° C. for 0.5 hours. The organic solvent was removed under reduced pressure to obtain the hydrochloride of a crude product of compound M1-4, and the compound was used directly in the next reaction without further purification. .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.32-7.19 (m, 5H), 5.19-5.08 (m, 2H), 4.02 (t, J=6.4 Hz, 1H), 1.82-1.54 (m, 2H), 0.70-0.55 (m, 1H), 0.47-0.32 (m, 2H), 0.08-−0.09 (m, 2H).
[0099] The following intermediate was synthesized using the similar method to the intermediate M1-4:
TABLE-US-00003 Inter- mediate number Structural formula Spectrum Inter- mediate M2-4
Step 4: Preparation of Intermediate M1-5
[0100] The hydrochloride of compound M1-4 (1.9 g, 7.43 mmol) and Boc-D-phenylalanine (1.97 g, 7.43 mmol) were dissolved in N,N-dimethylformamide (20 mL), and then diisopropylethylamine (2.88 g, 22.29 mmol, 3.88 mL) and HATU (4.24 g, 11.14 mmol) were added thereto, and the reaction solution was stirred at 20° C. for 16 hours. The reaction solution was diluted with 80 mL of ethyl acetate, and washed with water (40 mL*2) and saturated brine (50 mL) in turn. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure, then the obtained crude product was purified by silica gel column chromatography (eluent: 10 to 25% of ethyl acetate/petroleum ether) to obtain compound M1-5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.44-7.21 (m, 10H), 6.53 (d, J=7.2 Hz, 1H), 5.17 (q, J=12.0 Hz, 2H), 5.10-5.00 (m, 1H), 4.72-4.61 (m, 1H), 4.42-4.38 (m, 1H), 3.13-3.02 (m, 2H), 1.79-1.70 (m, 1H), 1.68-1.56 (m, 1H), 1.44 (s, 9H), 0.63-0.49 (m, 1H), 0.46-0.28 (m, 2H), 0.07-−0.09 (m, 2H).
[0101] The following intermediate was synthesized using the similar method to the intermediate M1-5:
TABLE-US-00004 Intermediate number Structural formula Spectrum Intermediate M2-5
Step 5: Preparation of Intermediate M1
[0102] Compound M1-5 (3.4 g, 7.29 mmol) was dissolved in ethyl acetate (30 mL), then 4 M of hydrochloric acid-ethyl acetate solution (30 mL) was added thereto, and the reaction solution was continued to react at 20° C. for 1 hour. The organic solvent was removed under reduced pressure to obtain the hydrochloride of compound M1, and the compound was used directly in the next reaction without further purification. .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.35-7.11 (m, 10H), 5.08 (s, 2H), 4.49-4.46 (m, 1H), 4.09-4.00 (m, 1H), 3.16-3.12 (m, 1H), 2.87-2.84 (m, 1H), 1.92-1.89 (d, J=8.8 Hz, 1H), 1.73-1.50 (m, 2H), 0.79-0.60 (m, 1H), 0.43-0.22 (m, 2H), 0.09-−0.12 (m, 2H).
[0103] The following intermediate was synthesized using the similar method to the intermediate M1:
TABLE-US-00005 Reference Intermediate Embodiment number Structural formula Spectrum Reference Embodiment 2 Intermediate M2
Reference Embodiment 3: Synthesis of Intermediate A1
[0104] ##STR00045##
Step 1: Preparation of Intermediate A1-1
[0105] The hydrochloride of compound M1 (1.2 g, 3.27 mmol) was suspended in dichloromethane (15 mL), and triethylamine (994.08 mg, 9.82 mmol) was added thereto, then chloroacetyl chloride (554.77 mg, 4.91 mmol, 390.68 μL) was slowly added dropwise, and the reaction solution was continued to stir at 20° C. for 18 hours. The reaction solution was diluted with 50 mL of dichloromethane, and washed with 30 mL of saturated ammonium chloride solution and 30 mL of saturated sodium chloride solution in turn. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain a crude product of compound A1-1, and the compound was used directly in the next reaction without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.51-7.38 (m, 5H), 7.35-7.24 (m, 5H), 6.30 (d, J=7.6 Hz, 1H), 5.38-5.14 (m, 2H), 4.81-4.59 (m, 2H), 3.29-3.02 (m, 3H), 1.83-1.67 (m, 2H), 1.48 (t, J=7.2 Hz, 1H), 0.66-0.50 (m, 1H), 0.45-0.39 (m, 2H), 0.08-−0.09 (m, 2H); MS m/z=443.2 [M+H].sup.+.
[0106] The following intermediate was synthesized using the similar method to the intermediate A1-1:
TABLE-US-00006 Intermediate number Structural formula Spectrum Intermediate A2-1
Step 2: Preparation of Intermediate A1-2
[0107] Compound A1-1 (1.42 g, 3.21 mmol) was dissolved in N,N-dimethylformamide (15 mL), and then compound (R)-(+)-β-methylphenethylamine (660.41 mg, 3.85 mmol), potassium iodide (1.06 g, 6.41 mmol) and potassium carbonate (1.33 g, 9.62 mmol) were added thereto, and the reaction solution was continued to stir at 60° C. for 16 hours. The mixture was cooled, and the organic solvent was removed under reduced pressure, and the mixture was added to 30 mL of water, extracted with dichloromethane (30 mL*2). The organic phases obtained by extraction were combined, and washed with saturated brine (30 mL), then the mixture was dried over anhydrous sodium sulfate, filtrated, and the organic solvent was removed under reduced pressure to obtain a crude product of compound A1-2, and the compound was used directly in the next reaction without further purification. MS m/z=542.3 [M+H].sup.+.
[0108] The following intermediate was synthesized using the similar method to the intermediate A1-2:
TABLE-US-00007 Intermediate number Structural formula Spectrum Intermediate A2-2
Step 3: Preparation of Intermediate A1-3
[0109] Compound A1-2 (1.7 g, 3.14 mmol) was dissolved in dichloromethane (15 mL), and then diisopropylethylamine (1.22 g, 9.42 mmol, 1.64 mL) and Boc anhydride (1.37 g, 6.28 mmol) were added, and the reaction solution was continued to stir at 20° C. for 16 hours. The organic solvent was removed under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (eluent: 10 to 30% of ethyl acetate/petroleum ether) to obtain compound A1-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.46-7.28 (m, 10H), 7.25-7.18 (m, 5H), 6.77-6.32 (m, 2H), 5.25-5.05 (m, 2H), 4.77-4.54 (m, 2H), 3.90-3.06 (m, 7H), 1.75-1.61 (m, 2H), 1.44 (s, 9H), 1.26 (d, J=6.8 Hz, 3H), 0.66-0.48 (m, 1H), 0.41-0.33 (m, 2H), 0.09-−0.11 (m, 2H).
[0110] The following intermediate was synthesized using the similar method to the intermediate A1-3:
TABLE-US-00008 Intermediate number Structural formula Spectrum Intermediate A2-3
Step 4: Preparation of Intermediate A1
[0111] Compound A1-3 (1.05 g, 1.64 mmol) was dissolved in ethanol (30 mL), then 10% palladium/carbon (150 mg, 155.33 μmol) was added thereto under the protection of nitrogen, and the reaction solution was continued to stir at 20° C. for 15 hours under 15 psi hydrogen pressure. The reaction solution was filtered through diatomite, and the organic solvent was removed under reduced pressure to obtain compound A1, and the compound was used directly in the next reaction without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.30-7.09 (m, 10H), 7.00-6.64 (m, 2H), 4.80-4.66 (m, 1H), 4.52-4.44 (m, 1H), 3.83-3.59 (m, 2H), 3.55-2.83 (m, 5H), 1.78-1.52 (m, 2H), 1.36 (s, 9H), 1.18 (d, J=6.8 Hz, 3H), 0.60-0.52 (m, 1H), 0.40-0.30 (m, 2H), 0.05-−0.05 (m, 2H).
[0112] The following intermediate was synthesized using the similar method to the intermediate A1:
TABLE-US-00009 Reference Intermediate Embodiment number Structural formula Spectrum Reference Embodi- ment 4 Interme- diate A2
Reference Embodiment 5: Synthesis of Intermediate B1
[0113] ##STR00050##
Step 1: Preparation of Intermediate B1-1
[0114] Compound A1 (1.9 g, 3.44 mmol) and compound A1-A (1.16 g, 3.44 mmol) were dissolved in DMF (20 mL), and then diisopropylethylamine (1.34 g, 10.33 mmol, 1.80 mL) and HATU (1.96 g, 5.17 mmol) were added thereto, and the reaction solution was continued to stir at 20° C. for 15 hours. The reaction solution was diluted with 150 mL of ethyl acetate, and the organic phase was washed with water (60 mL*2) and saturated brine (80 mL) in turn. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent: 10 to 40% of ethyl acetate/petroleum ether) to obtain compound B1-1. MS m/z=870.9 [M+H].sup.+.
Step 2: Preparation of Intermediate B1
[0115] Compound B1-1 (1.4 g, 1.61 mmol) was dissolved in ethanol (20 mL), and then palladium/carbon (0.2 g, 10% of purity) was added thereto, and the reaction solution was continued to stir at 20° C. for 15 hours under 15 psi hydrogen pressure. The reaction solution was filtered through diatomite, and the organic solvent was removed under reduced pressure to obtain compound B1, and the compound was used directly in the next reaction without further purification. MS m/z=802.6 [M+Na].sup.+.
Reference Embodiment 6: Synthesis of Intermediate D1
[0116] ##STR00051##
Step 1: Preparation of Intermediate D1-2
[0117] Compound D1-1 (3 g, 7.93 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then potassium carbonate (1.31 g, 9.51 mmol) and benzyl bromide (1.63 g, 9.51 mmol, 1.13 mL) were added thereto, and the reaction solution was continued to stir at 20° C. for 15 hours. The reaction solution was added with 50 mL of water, and extracted with ethyl acetate (60 mL*2). The organic phases obtained by extraction were combined, washed with saturated brine (60 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain a crude product, then the obtained crude product was purified by silica gel column chromatography (eluent: 5 to 25% of ethyl acetate/petroleum ether) to obtain compound D1-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.33-7.19 (m, 10H), 5.06 (s, 2H), 4.98 (s, 2H), 4.95 (s, 1H), 3.75-3.71 (m, 2H), 3.06-3.00 (m, 2H), 2.02-1.92 (m, 4H), 1.37 (s, 9H).
Step 2: Preparation of Intermediate D1-3
[0118] Compound D1-2 (3.7 g, 7.90 mmol) was dissolved in ethyl acetate (10 mL), then 4 M of hydrochloric acid/ethyl acetate (10 mL) was added thereto, and the reaction solution was continued to stir at 20° C. for 1 hour. The organic solvent was removed under reduced pressure to obtain the hydrochloride of compound D1-3, and the compound was used directly in the next reaction without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.37-7.31 (m, 10H), 5.16 (s, 2H), 5.06 (s, 2H), 3.37-3.30 (m, 4H), 3.27-3.11 (m, 2H), 2.40-2.21 (m, 4H).
Step 3: Preparation of Intermediate D1-5
[0119] The hydrochloride of compound D1-3 (3 g, 7.41 mmol) and compound D1-4 (3.47 g, 7.41 mmol) were dissolved in N,N-dimethylformamide (30 mL), and then diisopropylethylamine (2.87 g, 22.23 mmol, 3.87 mL) and HATU (4.23 g, 11.11 mmol) were added thereto, and the reaction solution was continued to stir at 20° C. for 14 hours. The reaction solution was diluted with 100 mL of ethyl acetate, then washed with water (50 mL*2) and saturated brine (60 mL) in turn. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain a crude product, then the obtained crude was purified by silica gel column chromatography (eluent: 15 to 30% of ethyl acetate/petroleum ether) to obtain compound D1-5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.78 (d, J=7.4 Hz, 2H), 7.67-7.57 (m, 2H), 7.47-7.28 (m, 14H), 5.84-5.68 (m, 1H), 5.26-5.01 (m, 5H), 4.74-4.53 (m, 2H), 4.39 (d, J=7.0 Hz, 2H), 4.27-4.19 (m, 1H), 4.03-3.57 (m, 2H), 3.51-3.25 (m, 1H), 3.20-3.01 (m, 2H), 2.30-2.14 (m, 2H), 2.02-1.96 (m, 2H), 1.79-1.65 (m, 2H), 1.60-1.30 (m, 14H).
[0120] The following intermediate was synthesized using a similar method to the intermediate D1-5:
TABLE-US-00010 Intermediate number Structural formula Spectrum Intermediate D2-5
Step 4: Preparation of Intermediate D1
[0121] Compound D1-5 (3 g, 3.66 mmol) was dissolved in dichloromethane (15 mL), and then piperidine (3.12 g, 36.63 mmol, 3.62 mL) was added thereto, and the reaction solution was continued to stir at 20° C. for 16 hours. The organic solvent was removed under reduced pressure, and 30 mL of acetonitrile was added to slurry, then the mixture was filtered, and the filtrate was evaporated to dryness by rotary evaporation, then the obtained crude product was purified by silica gel column chromatography (eluent: 35% of ethyl acetate/petroleum ether to 10% of methanol/dichloromethane) to obtain the hydrochloride of compound D1. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.41-7.29 (m, 10H), 5.16 (s, 2H), 5.08 (s, 2H), 4.62-4.58 (m, 1H), 3.75-3.60 (m, 2H), 3.43-3.08 (m, 4H), 2.29-2.13 (m, 2H), 2.05-1.87 (m, 2H), 1.68-1.54 (m, 4H), 1.54-1.39 (m, 15H); MS m/z=597.3 [M+H].sup.+.
[0122] The following intermediate was synthesized using the similar method to the intermediate D1:
TABLE-US-00011 Reference Intermediate Embodiment number Structural formula Spectrum Reference Embodiment 7 Intermediate D2
Reference Embodiment 8: Synthesis of Intermediate E1
[0123] ##STR00054##
Step 1: Preparation of Intermediate E1-2
[0124] Compound E1-1 (2 g, 9.99 mmol) and pyridine (880.00 mg, 11.13 mmol, 897.96 μL) were dissolved in tetrahydrofuran (45 mL), then phenyl chloroformate (1.56 g, 9.99 g, 1.25 mL) was added dropwise at 0° C. under the protection of nitrogen, and the reaction solution was continued to react at 20° C. for 2 hours after the addition was completed. After the reaction was completed, the organic solvent was removed under reduced pressure, and the crude product was diluted with ethyl acetate, and then washed with water and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure, and the obtained crude product was purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound E1-2. MS m/z=301.9 [M-56+Na].sup.+.
Step 2: Preparation of Intermediate E1-3
[0125] Compound E1-2 (0.6 g, 1.87 mmol) was dissolved in methanol (15 mL), and then methylamine solution (2 M, 842.74 μL) was added thereto, and the temperature was raised to 50° C. and the reaction was continued to react for 15 hours after the addition was completed. The mixture was cooled, and the organic solvent was removed under reduced pressure, and then the obtained crude product was purified by column chromatography (petroleum ether:ethyl acetate=1:1) to obtain compound E1-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 4.26-4.10 (m, 2H), 4.03-3.80 (m, 2H), 3.75-3.61 (m, 1H), 2.79 (br t, J=12.4 Hz, 2H), 2.70 (d, J=4.8 Hz, 3H), 1.91-1.80 (m, 2H), 1.38 (s, 9H).
Step 2: Preparation of Intermediate E1
[0126] Compound E1-3 (249.03 mg, 967.73 μmol) was dissolved in ethyl acetate (3 mL), and then hydrochloric acid-ethyl acetate solution (4 M, 2.42 mL) was added thereto, and the reaction solution was continued to react at 20° C. for 1 hour after the addition was completed. The organic solvent was removed under reduced pressure, and the obtained crude product was purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain the hydrochloride of compound E1. .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 3.84-3.70 (m, 1H), 3.41 (br d, J=13.2 Hz, 2H), 3.17-3.05 (m, 2H), 2.72 (s, 3H), 2.12 (br dd, J=3.2, 14.0 Hz, 2H), 1.75-1.62 (m, 2H); MS m/z=158.1[M+H].sup.+.
Embodiment 1: Preparation of Compound 1
[0127] ##STR00055##
Step 1: Preparation of Intermediate 1-1
[0128] Compound A1 (220 mg, 398.79 μmol) and compound D1 (237.96 mg, 398.79 μmol) were dissolved in N,N-dimethylformamide (5 mL), and then diisopropylethylamine (154.62 mg, 1.20 mmol, 208.38 μL) and HATU (227.45 mg, 598.18 μmol) were added thereto, and the reaction solution was continued to stir at 20° C. for 16 hours. The reaction solution was diluted with 60 mL of ethyl acetate, washed with water (30 mL*2) and saturated brine (40 mL) in turn. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure, then the obtained crude was purified by silica gel column chromatography (eluent: 50% of ethyl acetate/petroleum ether to 5% of methanol/dichloromethane) to obtain compound 1-1. MS m/z=1130.6 [M+H].
[0129] The following intermediate was synthesized using the similar method to the intermediate 1-1:
TABLE-US-00012 Intermediate number Structural formula Spectrum Intermediate 2-1
Step 2: Preparation of Intermediate 1-2
[0130] Compound 1-1 (200 mg, 176.93 μmol) was dissolved in ethanol (10 mL), and then 10% palladium/carbon (20 mg, 176.93 μmol) was added thereto, and the reaction solution was continued to stir at 20° C. for 15 hours under 15 psi hydrogen pressure. The reaction solution was filtered through diatomite, and the organic solvent was removed under reduced pressure to obtain compound 1-2, and the compound was used directly in the next reaction without further purification. MS m/z=906.6 [M+H].sup.+.
[0131] The following intermediate was synthesized using the similar method to the intermediate 1-2:
TABLE-US-00013 Intermediate number Structural formula Spectrum Intermediate 2-2
Step 3: Preparation of Compound 1
[0132] Compound 1-2 (155 mg, 171.06 μmol) was dissolved in ethyl acetate (5 mL), then 4 M of hydrochloric acid/ethyl acetate (5 mL) was added thereto, and the reaction solution was continued to stir at 20° C. for 0.5 hours. Most of the solvent was removed under reduced pressure, and 20 mL of ethyl acetate was added to slurry, then the mixture was filtered, and the crude product of the filter cake was purified by high performance liquid chromatography (column: Venusil ASB Phenyl 150*30 mm*5 μm; mobile phase: [water (0.05% hydrochloric acid)-acetonitrile]; B (acetonitrile) %: 8% to 38%, 9 minutes) to obtain the hydrochloride of compound 1. .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.26-7.08 (m, 9H), 7.07-6.97 (m, 1H), 4.62-4.50 (m, 1H), 4.31-4.18 (m, 1H), 4.08-3.83 (m, 1H), 3.80-3.49 (m, 4H), 3.25-3.24 (m, 1H), 3.12-2.88 (m, 4H), 2.86-2.68 (m, 3H), 2.25-2.03 (m, 2H), 1.93-1.50 (m, 7H), 1.49-1.25 (m, 4H), 1.24-1.15 (m, 3H), 0.77-0.56 (m, 1H), 0.49-0.23 (m, 2H), 0.12-−0.10 (m, 2H); MS m/z=706.4 [M+H].sup.+.
[0133] The following compound was synthesized using the similar method to the compound 1:
TABLE-US-00014 Embodi- Intermediate ment number Structural formula Spectrum 2 Compound 2
Embodiment 3: Preparation of Compound 3
[0134] ##STR00059##
Step 1: Preparation of Intermediate 3-1
[0135] Compound B1 (0.2 g, 256.42 μmol) and the hydrochloride of compound E1 (70.81 mg, 307.71 μmol) were dissolved in DMF (5 mL), and then HATU (146.25 mg, 384.64 μmol) and diisopropylethylamine (99.42 mg, 769.27 μmol, 133.99 μL) were added thereto, and the reaction solution was continued to stir at 20° C. for 15 hours. The reaction solution was diluted with 50 mL of ethyl acetate, and the organic phase was washed with water (30 mL*2) and saturated brine (30 mL) in turn. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure, then the obtained crude product was purified by silica gel column chromatography (eluent: 0 to 5% of methanol/dichloromethane) to obtain compound 3-1. MS m/z=919.6 [M+H].sup.+.
[0136] The following intermediate was synthesized using the similar method to the intermediate 3-1:
TABLE-US-00015 Intermediate number Structural formula Spectrum Intermediate 4-1
Step 2: Preparation of Compound 3
[0137] Compound 3-1 (0.19 g, 206.71 μmol) was dissolved in ethyl acetate (4 mL), and hydrogen chloride-ethyl acetate solution (4 M, 2 mL) was added thereto, and the reaction solution was continued to stir at 20° C. for 2 hours. The organic solvent was removed under reduced pressure, and the crude product was purified by high performance liquid chromatography (column: Venusil ASB Phenyl 150*30 mm*5 μm; mobile phase: [water (0.05% HCl)-ACN]; B (acetonitrile) %: 15%-45%, 9 min) to obtain compound 3. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ: 7.41-7.37 (m, 2H), 7.33-7.26 (m, 7H), 7.20-7.11 (m, 1H), 4.77-4.69 (m, 1H), 4.45-4.27 (m, 2H), 4.08-3.94 (m, 1H), 3.91-3.82 (m, 1H), 3.81-3.74 (m, 1H), 3.68 (br d, J=15.6 Hz, 1H), 3.24 (br dd, J=4.0, 14.0 Hz, 2H), 3.18-3.05 (m, 3H), 3.01-2.86 (m, 4H), 2.73 (s, 3H), 2.03-1.88 (m, 2H), 1.83-1.67 (m, 6H), 1.58 (td, J=6.8, 14.0 Hz, 1H), 1.47 (br s, 3H), 1.38-1.32 (m, 4H), 0.79 (br d, J=7.2 Hz, 1H), 0.56-0.41 (m, 2H), 0.23-0.10 (m, 2H); MS m/z=719.2[M+H].sup.+.
[0138] The following compound was synthesized using the similar method to the compound 3:
TABLE-US-00016 Embodi- Intermediate ment number Structural formula Spectrum 4 Compound 4
Experimental Embodiment 1: cAMP Test of Kappa Receptor
[0139] Solution and Buffer:
[0140] Experimental buffer: 1*HBSS (+/+) (SIGMA #H1387)+20 mM HEPES (LONZA #17-737E)
[0141] Stimulation buffer (STB): 200 μM IBMX (SIGMA #I7018) and 3 μM NKH477 (SIGMA #N3290) experimental buffer
Experimental Methods and Steps
Preparation of Compound
[0142] The compound powder was dissolved with DMSO (Amresco #0231), and the concentration of the compound solution was 10 mM. 1 μL of the test compound was taken, and 49 μL of DMSO was added thereto, and the mixture was diluted to 200 μM of solution.
[0143] A LDV 384-well plate was taken, and the compound solution diluted in the previous step was respectively added to wells A1 to H1; a pre-prepared 200 μM of U-50488 (Tocris #0495/25) was taken and added to well P1; 10.8 μL of DMSO was added to each well of A2 to A11, B2 to H22, and P2 to P11, respectively, and the mixture was centrifuged at 1000 rpm for 30 seconds.
[0144] Continuous gradient dilutions of compounds were performed with bravo, and the first column of LDV plate prepared in the previous step was taken as the initial concentration column, and 5 μL was sucked into the next column each time and blown and mixed evenly.
[0145] After the dilution was completed, 10 μL 200 μM of U-50488 was added to well A24 as HPE (100% inhibition rate activity), and 10 μL of DMSO was added to well P24 as ZPE (0% inhibition rate activity), and the mixture was centrifuged at 1000 rpm for 30 seconds.
[0146] According to the experimental layout, the compound solution diluted in the previous step was transferred to the experimental plate Corning 3824 with Echo 550, 50 nL per well.
Experimental Steps
[0147] A set of frozen KOR cAMP cells was taken from the −80° C. refrigerator and thawed in a 37° C.-water bath.
[0148] After thawing, the cells in the tube were aspirated into a 15 mL centrifuge tube, and 4 mL of experimental buffer was added thereto.
[0149] The mixture was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded, and then another 5 mL of experimental buffer was added thereto, and the mixture was blown and mixed well gently with pipette.
[0150] Cell density was counted and adjusted on a cell counter to 1.5*10.sup.6 cells/mL.
[0151] STB was added to the experimental plates containing the compound with Multidrop Combi, 5 μL per well.
[0152] The prepared cell suspension was added to the experimental plates containing the compound with Multidrop Combi, 5 μL per well.
[0153] The mixture was centrifuged at 300 rpm for 30 seconds and incubated in a 37° C. incubator for 40 minutes.
[0154] A standard curve of cAMP was prepared, and a pre-prepared and sub-packed frozen 2848 nM cAMP solution was thawed, and 2848 nM was used as the highest concentration point for 4 times continuous gradient dilution, with a total of 16 concentration points.
[0155] The diluted cAMP solution was then transferred to a new Corning 3824 experiment plate with a multichannel pipettor at 10 μL per well in triplicate.
[0156] After 40 min of incubation, D2 (Cisbio #62AM4PEJ) was added to the experimental plate (including the standard curve experiment plate) with Multidrop Combi at5 μL per well.
[0157] Ac (Cisbio #62AM4PEJ) was added to the experimental plate (including the standard curve experimental plates) with Multidrop Combi, 5 μL per well, and incubated for 60 min at room temperature.
[0158] Experimental plates were read with Envision.
Experimental Results
[0159] The results are shown in Table 1.
TABLE-US-00017 TABLE 1 EC.sub.50 value of the compound to Kappa receptor Compound number EC.sub.50 (nM) Compound 1 0.085 Compound 2 0.311 Compound 3 0.040 Compound 4 0.034
[0160] The results show that the compound of the present disclosure has an obvious Kappa receptor agonistic effect.
Experimental Embodiment 2: Plasma Protein Binding Rate (PPB) Test
1. Experimental Objectives
[0161] To test the protein binding rate of the test compound in human and SD rat plasma.
2. Experimental Method
2.1 Preparation of Plasma
[0162] The frozen plasma was thawed in flowing cold tap water, and then the plasma was centrifuged at 3220×g for 5 minutes to remove the suspension and precipitate. The pH of plasma was determined, and only the plasma with a pH between 7.0 to 8.0 could be used for experiments.
2.2 Preparation of Solution
[0163]
TABLE-US-00018 Storage Solution name Composition pH condition Working solution 2 μM of DMSO ND 2-8° C. solution of test compound and control compound Blank plasma Sprague-Dawley Between 7.0 Below −30° C. rat, human to 8.0 Dialysis buffer 100 mM sodium 7.4 ± 0.1 2-8° C. phosphate and 150 mM sodium chloride buffer Plasma 2 μM of plasma ND Room containing sample of test temperature test compound compound or or control control compound compound
2.3 Experimental Operation
[0164] The experiment was used a 96-well balanced dialysis plate (HTDialysis device) to determine the plasma protein binding rate of the test compound and control compound. Before the experiment was started, the dialysis membrane was pretreated according to the description, and then the dialysis device was assembled as required. The blank plasma of human and SD rats (plasma purchased from BioreclamationIVT) was taken, and the test compound working solution or the warfarin working solution was added to make the final concentration of the test compound and warfarin in the plasma samples both 2 μM. The sample was mixed well. 50 μL of the test compound and warfarin plasma sample were removed into the sample receiving plate, and the corresponding volume blank plasma or buffer was immediately added, so that the final volume of each sample well was 100 μL, and the volume ratio of the plasma:the dialysis buffer was 1:1, and the termination solution was added to these T.sub.0 samples of the test compound and the control compound. The plate was sealed and shaken at 800 rpm for 10 minutes. Then, these T.sub.0 samples were stored at 2 to 8° C. together with other dialyzed samples, waiting for after-treatment together with other dialyzed samples; 150 μL of test compound and warfarin plasma sample were added to the administration end of each dialysis well, and 150 μL of blank dialysis buffer was added to the corresponding receiving end of each dialysis well. Then, the dialysis plate was sealed with a gas permeable membrane, and then placed in a moist 5% CO.sub.2 incubator, and incubated with shaking at 37° C. and 100 rpm for 4 hours. At the end of dialysis, 50 μL of sample aliquots from the buffer side and plasma (matrix) side of the dialysis device were put into a new 96-well plate (sample collection plate). An equal volume of relative blank matrix (buffer or plasma) was added to each sample to reach a final volume of 100 μL, and the volume ratio of the plasma (matrix):the dialysis buffer in each well was 1:1 (v:v). All samples were further treated by protein precipitation for LC/MS/MS analysis. The unbound rate (Unbound) %, bound rate (Bound) % and recovery rate (Recovery) % of the compound were calculated by the following formula:
% Unbound=100*F.sub.C/T.sub.C,
% Bound=100−% Unbound,
% Recovery=100*(F.sub.C+T.sub.C)/T.sub.0.
[0165] Where F.sub.C is the concentration of the compound at the buffer end of the dialysis plate; T.sub.C is the concentration of the compound at the plasma end of the dialysis plate; T.sub.0 is the concentration of the compound in the plasma sample at time zero.
3. Experimental Results
[0166] The results are shown in Table 2.
TABLE-US-00019 TABLE 2 Unbound rates of the compound in human and rat plasma Compound Unbound rate in plasma number Human plasma SD rat plasma Compound 1 55.2% 44.7%
[0167] Conclusion: The compound of the present disclosure shows very high plasma protein unbounding rate in human and SD rat plasma.
Experimental Embodiment 3: Cytochrome P450 Isoenzyme Inhibition Activity Test
1. Experimental Objectives
[0168] To test the inhibitory activities of test compound against different subtypes of human cytochrome P450 isoenzymes
2. Experimental Method
[0169] The test compound, standard inhibitor (100× final concentration) and mixed substrate working solution were prepared; and the microsomes (purchased from Corning Inc) frozen in the −80° C. refrigerator were taken out and thawed. 20 μL of the test compound and standard inhibitor solution were added to the corresponding well position, and at the same time, 20 μL of the corresponding solvent was added to the control well position (NIC) without inhibitor and the blank control well position (Blank) without inhibitor; secondly, 20 μL of mixed substrate solution was added to the corresponding well position except the Blank well position (20 μL of PB was added to the Blank well position); human liver microsome solution was prepared (the solution was put back in the refrigerator immediately after marking the date after use), and then 158 μL of human liver microsome solution was added to all well positions; the sample plate was put in a 37° C.-water bath for pre-incubation, and then a coenzyme factor (NADPH) solution was prepared; after 10 minutes, 20 μL of NADPH solution was added to all wells, and then the sample plate was shaken well, and incubated in a 37° C.-water bath for 10 minutes; at the corresponding time point, 400 μL of cold acetonitrile solution (internal standard was 200 ng/mL tolbutamide and labetalol) was added to stop the reaction; after the sample plates were mixed well, the mixture was centrifuged at 4000 rpm for 20 minutes to precipitate protein; 200 μL of supernatant was added into 100 μL of water, and the mixture was shaken well and sent to LC/MS/MS for detection.
3. Experimental Results
[0170] The results are shown in Table 3.
TABLE-US-00020 TABLE 3 IC.sub.50 value of compound for P450 isoenzyme inhibition Compound Cytochrome P450 isoenzyme IC.sub.50 (nM) number CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4-M Compound 1 >50 >50 >50 >50 >50
[0171] Experimental conclusion: The inhibitory IC.sub.50 of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4-M are greater than 50 μM, and there is no risk of related drug interaction.
Experimental Embodiment 4: Metabolic Stability (HMS) Study in Hepatocytes
1. Experimental Objectives
[0172] To test the metabolic stability of the test compound in human and rat hepatocytes.
2. Experimental Materials
[0173] 2.1 Test Compound (10 mM), Reference Substance: 7-Ethoxycoumarin, 30 mM, 7-Hydroxycoumarin, Reference Substance, 30 mM
[0174] 2.2 Cells
TABLE-US-00021 Hepatocyte Cell viability Supplier Cat No. Rat hepatocyte 85% BioreclamationIVTM00005 Human hepatocyte 84% BioreclamationIVTX008001
[0175] 2.3 Buffer System:
[0176] Culture medium for thawing: Williams' E medium containing 5% fetal bovine serum and 30% Percoll solution and other auxiliary materials.
[0177] Culture medium for incubation: Williams' E medium (without phenol red), wherein containing 2 mM L-glutamine and 25 mM of hydroxyethyl piperazine ethanesulfonic acid.
[0178] Termination solution: acetonitrile containing 200 ng/mL of tolbutamide and labetalol as internal standard.
[0179] Dilution solution: ultrapure water.
3. Experimental Method
[0180] 1) An accurate amount of positive control compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 30 mM solution.
[0181] 2) 10 mM of test compound and 30 mM of positive control compound were diluted to 1 mM and 3 mM with DMSO on a 96-well plate.
[0182] 3) 1 mM of test compound and 3 mM of positive control compound were diluted to 100 μM and 300 μM quantitative solution with acetonitrile.
[0183] 4) The frozen cells were melted, separated and suspended in the culture medium, and then diluted to 0.5×10.sup.6 cells/mL with preheated culture solution.
[0184] 5) 198 μL of preheated cell suspension was added to a 96-well plate.
[0185] 6) 100 μL of termination solution was transferred to a set of pre-labeled 96-well plates (acetonitrile containing 200 ng/mL tolbutamide and 200 ng/mL labetalol as internal standards).
[0186] 7) 2 μL 100 μM of test compound or 300 μM positive control quantitative solution was added in duplicate to each well of a 96-well plate.
[0187] 8) For T.sub.0 samples, the samples were mixed to achieve uniform suspension for about 1 minute, then immediately transferred 20 μL of each sample to a well containing 100 μL of ice-cold termination solution, and then mixed well.
[0188] 9) All plates were incubated at 37° C. in 5% CO.sub.2 in a 95% humidified incubator, and the reaction was started with a constant shaking of about 600 rpm.
[0189] 10) At 15, 30, 60 and 90 min, the samples were mixed, then 20 μL of each sample was transferred to wells containing 100 μL of ice-cold termination solution at each time point and then mixed.
[0190] 11) Culture medium control (MC) sample plates (labeled as T.sub.0-MC and T.sub.90-MC) were prepared at T.sub.0 and T.sub.90 by adding the same ingredients except for the cell suspension in each well. The final concentration table was generated.
[0191] 12) At each corresponding time point, the reaction was stopped by removing the plate from the incubator and mixed with 100 μL of ice-cold termination solution.
[0192] 13) The plate was immediately shaken at a 500 rpm vortex on a plate oscillator for 10 minutes. Then, all sample plates were centrifuged at 3220×g at 4° C. for 20 minutes.
[0193] 14) After centrifugation, the supernatant from the 35 μL/well sample plate was transferred to another set of pre-labeled 96-well plates containing 70 μL of ultrapure water according to the plate chart.
[0194] 15) The analysis plate was sealed and stored at 4° C. until LC-MS-MS analysis.
[0195] The residual rate of the test compound and control compound was obtained by the following formula:
[0196] The elimination rate constant k of the test compound and the control compound in hepatocyte was calculated by plotting the logarithm of the time to the residual rate, and the half-life (T.sub.1/2) and the intrinsic clearance rate in vitro (CL.sub.int) were obtained by the elimination rate constant k with the following formula:
T.sub.1/2=0.693/k
CL.sub.int(hep)=k/number of cells per mL (million cells/mL)
CL.sub.int(liver)=CL.sub.int(hep)×ratio of liver weight to body weight×number of hepatocytes per gram of liver
[0197] The parameters for the various species in the formula are listed below:
TABLE-US-00022 Ratio of liver weight Hepatic blood Number of to body weight flow (Q.sub.h) hepatocytes Specie (g/kg Body Weight) (mL/min/kg) (of cells/g liver) Mice 88 90.0 135 × 10.sup.6 Rat 40 55.2 117 × 10.sup.6 Dog 32 30.9 215 × 10.sup.6 Monkey 30 43.6 120 × 10.sup.6 Human 20 20.7 139 × 10.sup.6
4. Experimental Results
[0198] The results are shown in Table 4.
TABLE-US-00023 TABLE 4 Intrinsic clearance rate of compound in human and rat liver Compound Intrinsic clearance rate in liver (mL/min/Kg) number Human Rat Compound 1 <17.8 <29.9
[0199] Experimental conclusion: The compound of the present disclosure has low clearance in both human and rat.
Experimental Embodiment 5: In Vivo Pharmacokinetic Study in SD Rats
1. Experimental Objectives
[0200] To test the in vivo pharmacokinetics of test compound in SD rats
2. Experimental Materials
[0201] Sprague Dawley rats (male, 6 to 10 weeks old, purchased from Beijing Vital River Laboratory Animal Co., LTD)
3. Experimental Method
[0202] Compound 1 was mixed with normal saline to prepare a clear solution of 1 mg/mL for injection group administration, filtered by microporous membranes and ready for use. The test compound was administered intravenously at 3 mg/kg, and the solvent for intravenous administration was normal saline. Plasma samples were collected at 0, 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 24 hours after administration. The plasma concentration was determined by LC-MS/MS method, and the relevant pharmacokinetic parameters were calculated by non-compartmental model linear logarithmic trapezoidal method using WinNonlin™ Version 6.3 (Pharsight, Mountain View, Calif.) pharmacokinetic software.
4. Experimental Results
[0203] The experimental results are shown in Table 5 below:
TABLE-US-00024 TABLE 5 Pharmacokinetic data of compound 1 in rats C.sub.0 T.sub.1/2 Vd.sub.ss Cl AUC.sub.0-last (μM) (hr) (L/kg) (mL/min/kg) (μM .Math. hr) Compound 1 17.6 1.33 0.29 9.90 7.2 Note: C.sub.0 is the initial concentration, T.sub.1/2 is the elimination half-life, Vd.sub.ss is the steady-state apparent volume of distribution, Cl is the total clearance rate, and AUC.sub.0-last is the area under the plasma concentration-time curve from 0 time to the last quantifiable time point.
[0204] Experimental conclusion: The compound of the present disclosure has good pharmacokinetic properties.