CRYSTAL FORM OF NUCLEOPROTEIN INHIBITOR AND USE THEREOF
20230026869 · 2023-01-26
Assignee
Inventors
Cpc classification
International classification
Abstract
A crystal form of a compound of formula (I), a hydrate thereof, a solvate thereof, or a co-complex of water and a solvent, and the use thereof in the preparation of a drug for treating a disease associated with HBV.
##STR00001##
Claims
1. A crystal form of a compound of formula (I), a hydrate thereof, a solvate thereof, or a combination of the hydrate and the solvate ##STR00012##
2. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.20±0.20°, 8.90±0.20°, 16.30±0.20° and 24.78±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.20±0.20°, 8.90±0.20°, 14.22±0.20°, 16.30±0.20°, 22.32±0.20° and 24.78±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.20±0.20°, 8.90±0.20°, 11.26±0.20°, 14.22±0.20°, 16.30±0.20°, 17.89±0.20°, 22.32±0.20° and 24.78±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.20±0.20°, 8.90±0.20°, 10.09±0.20°, 11.26±0.20°, 14.22±0.20°, 16.30±0.20°, 17.89±0.20°, 20.35±0.20°, 22.32±0.20°, 24.78±0.20° and 27.78±0.20°.
3. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.13±0.20°, 10.53±0.20°, 21.17±0.20° and 22.64±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.13±0.20°, 10.53±0.20°, 11.67±0.20°, 20.09±0.20°, 21.17±0.20° and 22.64±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.13±0.20°, 10.53±0.20°, 11.67±0.20°, 13.52±0.20°, 20.09±0.20°, 21.17±0.20° and 22.64±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.72±0.20°, 8.53±0.20°, 17.76±0.20° and 20.38±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.72±0.20°, 8.53±0.20°, 10.50±0.20°, 13.53±0.20°, 17.76±0.20°, 18.83±0.20° and 20.38±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.72±0.20°, 8.53±0.20°, 10.50±0.20°, 13.53±0.20°, 17.76±0.20°, 18.83±0.20°, 20.38±0.20°, 21.06±0.20° and 24.00±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.72±0.20°, 8.53±0.20°, 10.50±0.20°, 11.41±0.20°, 13.53±0.20°, 17.76±0.20°, 18.83±0.20°, 19.99±0.20°, 20.38±0.20°, 21.06±0.20°, 22.23±0.20°, 24.00±0.20°, 24.42±0.20° and 25.90±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.05±0.20°, 9.10±0.20°, 10.78±0.20° and 22.90±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.05±0.20°, 9.10±0.20°, 10.78±0.20°, 21.24±0.20°, 21.74±0.20° and 22.90±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.05±0.20°, 9.10±0.20°, 10.78±0.20°, 13.07±0.20°, 19.57±0.20°, 21.24±0.20°, 21.74±0.20°, 22.24±0.20° and 22.90±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.05±0.20°, 9.10±0.20°, 10.78±0.20°, 11.34±0.20°, 13.07±0.20°, 14.07±0.20°, 14.99±0.20°, 15.86±0.20°, 16.17±0.20°, 18.60±0.20°, 19.57±0.20°, 21.24±0.20°, 21.46±0.20°, 21.74±0.20°, 22.24±0.20°, 22.72±0.20° and 22.90±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.53±0.20°, 11.09±0.20°, 22.34±0.20° and 23.12±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.53±0.20°, 11.09±0.20°, 15.00±0.20°, 20.76±0.20°, 22.34±0.20° and 23.12±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.53±0.20°, 11.09±0.20°, 14.14±0.20°, 15.00±0.20°, 20.76±0.20°, 22.34±0.20°, 23.12±0.20° and 26.85±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.53±0.20°, 10.50±0.20°, 11.09±0.20°, 14.14±0.20°, 15.00±0.20°, 20.76±0.20°, 22.34±0.20°, 23.12±0.20° and 26.85±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.32±0.20°, 10.43±0.20°, 12.46±0.20° and 19.62±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.32±0.20°, 10.43±0.20°, 10.81±0.20°, 12.46±0.20°, 19.62±0.20° and 21.03±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.32±0.20°, 10.43±0.20°, 10.81±0.20°, 12.46±0.20°, 17.55±0.20°, 17.99±0.20°, 19.62±0.20°, 21.03±0.20° and 22.90±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.32±0.20°, 10.43±0.20°, 10.81±0.20°, 12.46±0.20°, 13.00±0.20°, 15.06±0.20°, 17.55±0.20°, 17.99±0.20°, 19.62±0.20°, 21.03±0.20° and 22.90±0.20°.
4. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.94±0.20°, 9.83±0.20° and 10.99±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.94±0.20°, 9.83±0.20°, 10.99±0.20°, 18.62±0.20° and 19.82±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.94±0.20°, 9.83±0.20°, 10.99±0.20°, 13.36±0.20°, 17.21±0.20°, 18.62±0.20°, 19.82±0.20° and 21.56±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.94±0.20°, 9.39±0.20°, 9.83±0.20°, 10.48±0.20°±0.20°, 10.99±0.20°, 13.36±0.20°, 14.29±0.20°, 17.21±0.20°, 18.14±0.20°, 18.62±0.20°, 19.82±0.20° and 21.56±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.52±0.20°, 11.21±0.20°, 12.40±0.20° and 14.41±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.52±0.20°, 8.70±0.20°, 11.21±0.20°, 12.40±0.20°, 14.41±0.20°, 17.49±0.20° and 22.92±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.52±0.20°, 8.70±0.20°, 11.21±0.20°, 12.40±0.20°, 14.41±0.20°, 16.06±0.20°, 17.49±0.20°, 20.98±0.20°, 21.98±0.20° and 22.92±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.17±0.20°, 7.52±0.20°, 7.99±0.20°, 8.70±0.20°, 9.99±0.20°, 10.74±0.20°, 11.21±0.20°, 12.40±0.20°, 14.41±0.20°, 14.88±0.20°, 16.06±0.20°, 17.05±0.20°, 17.49±0.20°, 20.98±0.20°, 21.98±0.20°, 22.48±0.20°, 22.92±0.20°, 23.50±0.20°, 26.47±0.20°, 27.05±0.20° and 28.04±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 4.18±0.20°, 8.35±0.20°, 10.58±0.20° and 16.86±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 4.18±0.20°, 8.35±0.20°, 10.58±0.20°, 11.87±0.20°, 16.86±0.20° and 21.16±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 4.18±0.20°, 8.35±0.20°, 10.58±0.20°, 11.87±0.20°, 12.32±0.20°, 16.86±0.20°, 21.16±0.20°, 25.47±0.20° and 29.17±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 5.56±0.20°, 11.25±0.20° and 14.09±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 5.56±0.20°, 7.54±0.20°, 11.25±0.20, 14.09±0.20° and 19.64±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 5.56±0.20°, 7.54±0.20°, 11.25±0.20°, 14.09±0.20°, 18.07±0.20°, 19.64±0.20° and 20.33±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 5.56±0.20°, 7.54±0.20°, 11.25±0.20°, 14.09±0.20°, 18.07±0.20°, 19.64±0.20°, 20.33±0.20°, 21.65±0.20° and 22.31±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.50±0.20°, 10.65±0.20° and 11.10±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.18±0.20°, 7.50±0.20°, 10.65±0.20°, 11.10±0.20°, 14.04±0.20° and 21.48±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.18±0.20°, 7.50±0.20°, 10.65±0.20°, 11.10±0.20°, 14.04±0.20°, 21.48±0.20°, 22.79±0.20° and 27.02±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.18±0.20°, 7.50±0.20°, 10.65±0.20°, 11.10±0.20°, 14.04±0.20°, 15.67±0.20°, 19.16±0.20°, 21.48±0.20°, 22.79±0.20°, 23.39±0.20°, 26.28±0.20° and 27.02±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.22±0.20° and 14.10±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.22±0.20°, 10.34±0.20°, 14.10±0.20°, 14.66±0.20° and 21.61±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.22±0.20°, 8.53±0.20°, 10.34±0.20°, 14.10±0.20°, 14.66±0.20°, 16.47±0.20°, 17.07±0.20° and 21.61±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.22±0.20°, 8.53±0.20°, 8.95±0.20°, 10.34±0.20°, 14.10±0.20°, 14.66±0.20°, 15.90±0.20°, 16.47±0.20°, 17.07±0.20°, 19.45±0.20°, 21.61±0.20°, 22.89±0.20° and 23.36±0.20°.
5.-7. (canceled)
8. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.31±0.20°, 19.18±0.20° and 25.99±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.31±0.20°, 15.86±0.20°, 19.18±0.20°, 21.05±0.20° and 25.99±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.31±0.20°, 11.62±0.20°, 12.91±0.20°, 15.86±0.20°, 19.18±0.20°, 21.05±0.20°, 24.67±0.20° and 25.99±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.03±0.20°, 8.31±0.20°, 11.62±0.20°, 12.91±0.20°, 15.86±0.20°, 17.17±0.20°, 18.20±0.20°, 19.18±0.20°, 19.74±0.20°, 21.05±0.20°, 21.30±0.20°, 24.67±0.20° and 25.99±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.28±0.20°, 10.34±0.20°, 22.66±0.20° and 26.12±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.28±0.20°, 10.34±0.20°, 19.45±0.20°, 20.93±0.20°, 22.66±0.20° and 26.12±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 9.28±0.20°, 10.34±0.20°, 12.35±0.20°, 14.95±0.20°, 17.88±0.20°, 19.45±0.20°, 20.93±0.20°, 22.66±0.20° and 26.12±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 3.47±0.20°, 9.28±0.20°, 10.34±0.20°, 10.92±0.20°, 12.35±0.20°, 14.95±0.20°, 17.62±0.20°, 17.88±0.20°, 19.09±0.20°, 19.45±0.20°, 20.08±0.20°, 20.93±0.20°, 22.66±0.20°, 23.98±0.20°, 26.12±0.20° and 28.63±0.20°.
9.-11. (canceled)
12. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.49±0.20°, 8.46±0.20°, 15.99±0.20° and 17.02±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.49±0.20°, 8.46±0.20°, 13.18±0.20°, 14.43±0.20°, 15.99±0.20° and 17.02±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.49±0.20°, 8.46±0.20°, 9.91±0.20°, 13.18±0.20°, 14.43±0.20°, 15.99±0.20°, 17.02±0.20°, 20.97±0.20° and 24.20±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.49±0.20°, 8.46±0.20°, 9.12±0.20°, 9.91±0.20°, 10.67±0.20°, 13.18±0.20°, 14.43±0.20°, 15.99±0.20°, 17.02±0.20°, 18.34±0.20°, 19.95±0.20°, 20.29±0.20°, 20.97±0.20°, 21.66±0.20°, 23.03±0.20°, 24.20±0.20°, 24.94±0.20° and 25.69±0.20°.
13.-14. (canceled)
15. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.47°±0.20°, 12.62±0.20°, 15.70±0.20° and 18.41±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 8.47±0.20°, 11.23±0.20°, 12.62±0.20°, 15.70±0.20°, 18.41±0.20° and 21.49±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.04±0.20°, 8.47±0.20°, 10.01±0.20°, 11.23±0.20°, 12.62±0.20°, 15.70±0.20°, 18.41±0.20°, 21.49±0.20° and 22.53±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.04±0.20°, 8.47±0.20°, 10.01±0.20°, 11.23±0.20°, 12.62±0.20°, 15.70±0.20°, 17.32±0.20°, 18.41±0.20°, 20.31±0.20°, 21.49±0.20°, 22.53±0.20° and 26.34±0.20°.
16. (canceled)
17. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20° and 21.46±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 21.28±0.20° and 21.46±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 20.38±0.20°, 21.28±0.20°, 21.46±0.20°, 26.72±0.20° and 27.48±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 20.38±0.20°, 21.46±0.20°, 26.72±0.20° and 27.48±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 12.96±0.20°, 18.66±0.20°, 20.38±0.20°, 21.46±0.20°, 22.74±0.20°, 26.72±0.20°, 27.48±0.20° and 27.82±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 12.96±0.20°, 18.66±0.20°, 20.38±0.20°, 21.28±0.20°, 21.46±0.20°, 22.74±0.20°, 24.84±0.20°, 26.72±0.20°, 27.48±0.20° and 27.82±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 15.40±0.20°, 21.28±0.20°, 21.46±0.20° and 22.74±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 12.96±0.20°, 15.40±0.20°, 21.28±0.20°, 21.46±0.20°, 22.74±0.20° and 27.48±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 12.96±0.20°, 15.40±0.20°, 20.38±0.20°, 21.28±0.20°, 21.46±0.20°, 22.74±0.20°, 26.72±0.20° and 27.48±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 12.96±0.20°, 15.40±0.20°, 18.66±0.20°, 20.38±0.20°, 21.28±0.20°, 21.46±0.20°, 22.74±0.20°, 26.72±0.20°, 27.48±0.20° and 27.82±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 8.74±0.20°, 12.96±0.20°, 15.40±0.20°, 15.92±0.20°, 18.66±0.20°, 20.38±0.20°, 21.28±0.20°, 21.46±0.20°, 22.74±0.20°, 26.72±0.20° and 27.48±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 8.74±0.20°, 11.66±0.20°, 12.96±0.20°, 15.40±0.20°, 15.92±0.20°, 16.22±0.20°, 18.66±0.20°, 20.38±0.20°, 21.28±0.20°, 21.46±0.20°, 22.74±0.20°, 26.72±0.20°, 27.48±0.20° and 27.82±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.12±0.20°, 8.74±0.20°, 9.29±0.20°, 11.66±0.20°, 12.96±0.20°, 15.40±0.20°, 15.92±0.20°, 16.22±0.20°, 17.54±0.20°, 18.66±0.20°, 20.38±0.20°, 21.28±0.20°, 21.46±0.20°, 22.74±0.20°, 26.72±0.20°, 27.48±0.20° and 27.82±0.20°.
18. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form belongs to a triclinic crystal system, with a space group being P-1, unit cell parameters being a=9.407(2)Å, b=11.531(3)Å, c=13.574(4)Å, α=66.982(8)°, β=75.337(8)° and γ=68.492(8)°, a volume of a unit cell being V=1250.4(6)Å{circumflex over ( )}3, and a number of asymmetric units in a unit cell being Z=2.
19. (canceled)
20. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.60±0.20°, 8.55±0.20° and 15.21±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.60±0.20°, 8.55±0.20°, 11.67±0.20°, 15.21±0.20°, 17.60±0.20° and 24.56±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.60±0.20°, 8.55±0.20°, 11.67±0.20°, 15.21±0.20°, 17.12±0.20°, 17.60±0.20°, 23.25±0.20°, 24.56±0.20° and 27.31±0.20°.
21. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.15±0.20°, 8.43±0.20°, 21.57±0.20° and 23.90±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.15±0.20°, 8.43±0.20°, 14.94±0.20°, 16.29±0.20°, 16.84±0.20°, 21.57±0.20° and 23.90±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.15±0.20°, 6.57±0.20°, 8.43±0.20°, 11.27±0.20°, 14.94±0.20°, 16.29±0.20°, 16.84±0.20°, 17.71±0.20°, 21.57±0.20° and 23.90±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.15±0.20°, 6.57±0.20°, 8.43±0.20°, 8.83±0.20°, 11.27±0.20°, 13.97±0.20°, 14.23±0.20°, 14.94±0.20°, 16.29±0.20°, 16.84±0.20°, 17.20±0.20°, 17.71±0.20°, 18.48±0.20°, 19.19±0.20°, 20.36±0.20°, 20.74±0.20°, 21.57±0.20°, 22.61±0.20°, 23.02±0.20°, 23.90±0.20°, 26.16±0.20°, 26.67±0.20° and 27.74±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.15±0.20°, 6.57±0.20°, 8.43±0.20°, 8.83±0.20°, 9.90±0.20°, 11.27±0.20°, 11.54±0.20°, 13.13±0.20°, 13.97±0.20°, 14.23±0.20°, 14.94±0.20°, 16.29±0.20°, 16.84±0.20°, 17.20±0.20°, 17.71±0.20°, 18.48±0.20°, 19.19±0.20°, 20.36±0.20°, 20.74±0.20°, 21.57±0.20°, 22.20±0.20°, 22.61±0.20°, 23.02±0.20°, 23.90±0.20°, 24.92±0.20°, 25.58±0.20°, 26.16±0.20°, 26.67±0.20°, 27.74±0.20°, 28.25±0.20° and 31.28±0.20°.
22. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.23±0.20°, 8.45±0.20°, 16.18±0.20° and 26.35±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.23±0.20°, 8.45±0.20°, 12.85±0.20°, 16.18±0.20°, 19.41±0.20° and 26.35±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.23±0.20°, 8.45±0.20°, 12.85±0.20°, 16.18±0.20°, 18.46±0.20°, 19.41±0.20°, 19.97±0.20°, 21.46±0.20° and 26.35±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.23±0.20°, 8.45±0.20°, 9.72±0.20°, 12.85±0.20°, 14.41±0.20°, 16.18±0.20°, 18.46±0.20°, 19.41±0.20°, 19.97±0.20°, 21.46±0.20°, 24.95±0.20° and 26.35±0.20°.
23. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 1, wherein the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 3.50±0.20°, 6.97±0.20°, 9.51±0.20° and 19.13±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 3.50±0.20°, 6.97±0.20°, 9.51±0.20°, 11.55±0.20°, 17.53±0.20°, 19.13±0.20° and 19.62±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 3.50±0.20°, 6.97±0.20°, 9.51±0.20°, 10.29±0.20°, 11.55±0.20°, 14.00±0.20°, 17.53±0.20°, 19.13±0.20°, 19.62±0.20° and 21.09±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 3.50±0.20°, 6.97±0.20°, 9.51±0.20°, 9.98±0.20°, 10.29±0.20°, 11.55±0.20°, 14.00±0.20°, 17.53±0.20°, 19.13±0.20°, 19.62±0.20°, 20.71±0.20°, 21.09±0.20°, 21.41±0.20°, 22.32±0.20°, 24.35±0.20°, 26.98±0.20° and 35.53±0.20°; or, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.17±0.20°, 9.44±0.20°, 19.06±0.20° and 19.56±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 7.17±0.20°, 9.44±0.20°, 10.22±0.20°, 11.48±0.20°, 19.06±0.20°, 19.56±0.20° and 21.35±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.90±0.20°, 7.17±0.20°, 8.99±0.20°, 9.44±0.20°, 9.91±0.20°, 10.22±0.20°, 11.48±0.20°, 19.06±0.20°, 19.56±0.20° and 21.35±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.90±0.20°, 7.17±0.20°, 8.99±0.20°, 9.44±0.20°, 9.91±0.20°, 10.22±0.20°, 11.48±0.20°, 14.50±0.20°, 17.46±0.20°, 19.06±0.20°, 19.56±0.20°, 21.01±0.20°, 21.35±0.20°, 21.85±0.20°, 22.25±0.20° and 24.27±0.20°; typically, the crystal form has characteristic diffraction peaks in an X-ray powder diffraction pattern thereof at the following 2θ: 6.90±0.20°, 7.17±0.20°, 8.99±0.20°, 9.44±0.20°, 9.91±0.20°, 10.22±0.20°, 10.41±0.20°, 10.57±0.20°, 11.48±0.20°, 13.93±0.20°, 14.50±0.20°, 17.46±0.20°, 19.06±0.20°, 19.56±0.20°, 20.64±0.20°, 21.01±0.20°, 21.35±0.20°, 21.85±0.20°, 22.25±0.20°, 24.27±0.20°, 26.88±0.20° and 29.30±0.20°.
24. (canceled)
25. A crystal form composition comprising the crystal form according to claim 1, wherein the crystal form accounts for 50% or more, preferably 80% or more, more preferably 90% or more and most preferably 95% or more of the weight of the crystal form composition.
26. A pharmaceutical composition, comprising a therapeutically effective amount of the crystal form according to claim 1 or the crystal form composition thereof.
27. A method for inhibiting nucleoprotein, comprising administering an effective amount of the crystal according to claim 1 to a subject in need thereof.
28. A method for treating or preventing HBV infection related diseases, comprising administering an effective amount of the crystal according to claim 1 to a subject in need thereof.
29. A method for treating or preventing HBV infection related diseases, comprising administering an effective amount of the pharmaceutical composition according to claim 26 to a subject in need thereof.
30. The crystal form of the compound of formula (I), the hydrate thereof, the solvate thereof, or the combination of the hydrate and the solvate according to claim 17, wherein the crystal form belongs to a triclinic crystal system, with a space group being P-1, unit cell parameters being a=9.407(2)Å, b=11.531(3)Å, c=13.574(4)Å, α=66.982(8)°, β=75.337(8)° and γ=68.492(8)°, a volume of a unit cell being V=1250.4(6)Å{circumflex over ( )}3, and a number of asymmetric units in a unit cell being Z=2.
31. A preparation method for the crystal form according to claim 17, wherein the preparation method comprises: 1) adding a compound of formula (I) to a mixed solvent of MTBE and MeOH; and 2) precipitating a solid and separating to give the crystal form; or wherein the preparation method comprises: 1) dissolving a compound of formula (I) in methanol or acetone; and 2) precipitating a solid and separating to give the crystal form P.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0324] In order to better understand the content of the present application, further description is given with reference to specific examples, but the specific embodiments are not intended to limit the content of the present application.
Example 1: Compound of Formula (I)
[0325] ##STR00003##
[0326] Synthetic Route:
##STR00004## ##STR00005##
##STR00006##
Step 1
[0327] ##STR00007##
[0328] Compound 13-1 (1 g, 4.40 mmol, 1 eq.) was dissolved in tetrahydrofuran (20 mL) in a single-neck flask, and sodium hydride (615.93 mg, purity: 60%, 3.5 eq.) was added at 0° C. under nitrogen atmosphere. After stirring for 0.5 h, compound 13-2 (2.33 g, 9.24 mmol, 2.1 eq.) was added, and the reaction system was stirred for 30 min at 30° C. After the reaction was completed as indicated by TLC, the reaction system was poured into 0.5 M diluted hydrochloric acid (100 mL), and the crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate=50:1 to 20:1) to give compound 13-3.
[0329] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=5.55 (s, 1H), 4.43 (s, 1H), 3.32-3.26 (m, 1H), 2.41-2.38 (m, 1H), 2.30-2.28 (m, 1H), 2.15-2.14 (m, 1H), 2.13-2.04 (m, 1H), 2.03-2.01 (m, 1H), 1.54-1.49 (m, 2H), 1.46 (s, 9H), 1.43 (s, 9H), 1.33 (s, 3H).
Step 2
[0330] ##STR00008##
[0331] Wet palladium on carbon (200 mg, content: 10%) was dissolved in tetrahydrofuran (40 mL) in a hydrogenation flask, and compound 13-3 (1.35 g, 4.15 mmol, 1 eq.) was added. The reaction system was stirred at 25° C. for 2 h under H.sub.2 atmosphere (15 psi). After the reaction was completed as indicated by TLC, the reaction solution was filtered and concentrated to give compound 13-4.
Step 3
[0332] ##STR00009##
[0333] Compound 13-4 (1.3 g, 3.97 mmol, 1 eq.) was dissolved in ethyl acetate (30 mL) in a dry single-neck flask, and hydrochloric acid/ethyl acetate (4 M, 10 mL, 10.08 eq.) was added. The reaction system was stirred at 30° C. for 16 h. The reaction solution was directly concentrated to give compound 13-5.
[0334] .sup.1H NMR (400 MHz, MeOH-d.sub.4) δ=1.85-1.83 (m, 4H), 1.77-1.76 (m, 2H), 1.63-1.60 (m, 3H), 1.45 (s, 9H), 1.35-1.31 (m, 5H).
Step 4
[0335] ##STR00010##
[0336] Compound 13-5 (250 mg, 861.77 μmol, 1 eq.) was dissolved in dichloromethane (10 mL) in a dry single-neck flask, and triethylamine (261.61 mg, 2.59 mmol, 359.85 μL, 3 eq.) was added. The resulting mixture was added with a solution of compound 11-7 (293.88 mg, 1.29 mmol, 1.5 eq.) in dichloromethane (10 mL) dropwise under nitrogen atmosphere at 0° C. The reaction system was stirred at 30° C. for 1 h. After the reaction was completed as indicated by LCMS, the reaction solution was poured into water (50 mL). Then the aqueous phase was extracted with dichloromethane (20 mL×3), and the organic phase was washed with 0.5 M diluted hydrochloric acid (20 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give compound 13-6.
[0337] MS(ESI) m/s:425.2 [M+H-(t-Bu)].sup.+.
[0338] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=5.99 (s, 1H), 4.32 (t, J=7.6 Hz, 2H), 3.84 (s, 2H), 3.14 (t, J=7.6 Hz, 2H), 2.52-2.48 (m, 2H), 2.31-2.28 (m, 2H), 2.14-2.12 (m, 2H), 1.68-1.65 (m, 2H), 1.45-1.42 (m, 2H), 1.39-1.36 (m, 12H), 1.24-1.22 (m, 2H), 1.18-1.14 (m, 2H).
[0339] Compound 13-6 was separated by SFC (column: DAICELCHIRALCELOJ (250 mm×30 mm, 10 μm); mobile phase: Neu-ETOH; B %: 30%-30%, 9 min) to give compound 1-1 (SFC retention time: 1.7 min), SFC analysis conditions (column: Daicel OJ-3 chiral column, with a specification of 0.46 cm id×5 cm; mobile phase: A: carbon dioxide, B: ethanol for chromatography (containing 0.05% isopropylamine); B %: 5%-40%; flow rate: 4 mL/min; 4 min; system back pressure: 100 bar)).
[0340] Compound 1-1: .sup.1H NMR (400 MHz, CDCl.sub.3) δ=6.08 (s, 1H), 4.31 (t, J=7.4 Hz, 2H), 3.83 (s, 3H), 3.12 (t, J=7.6 Hz, 2H), 2.51-2.47 (m, 2H), 2.17-2.15 (m, 2H), 2.14-2.00 (m, 2H), 1.83-1.70 (m, 6H), 1.48 (s, 3H), 1.47-1.45 (m, 11H), 1.45-1.26 (m, 3H).
Step 5
[0341] Compound 1-1 (550.00 mg, 1.14 mmol, 1 eq.) was dissolved in tetrahydrofuran (10 mL) in a single-neck flask, and water (10 mL) and methanol (10 mL) and lithium hydroxide monohydrate (239.91 mg, 5.72 mmol, 5 eq.) were added. The reaction system was stirred at 30° C. for 16 h. After the reaction was completed as indicated by LCMS, the reaction solution was adjusted to pH=1 with 0.5 M diluted hydrochloric acid, the aqueous phase was extracted with ethyl acetate (20 mL×3), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give compound 1-2.
Step 6
[0342] Compound 1-2 (500 mg, 1.07 mmol, 1 eq.) was dissolved in dichloromethane (20 mL), and oxalyl chloride (271.83 mg, 2.14 mmol, 187.47 μL, 2 eq.) and N,N-dimethylformamide (7.83 mg, 107.08 μmol, 8.24 μL, 0.1 eq.) were added dropwise at 0° C. under nitrogen atmosphere. The reaction system was stirred at 25° C. for 1 h. Two drops of the reaction solution were taken and added with methanol to quench the reaction, and after the reaction was completed as indicated by LCMS, the reaction solution was concentrated under reduced pressure to give compound 1-3.
Step 7
[0343] Compound 1-3 was dissolved in dichloromethane (15 mL), TEA (312.70 mg, 3.09 mmol, 430.12 μL, 3 eq.) was added, and then compound 1-4 (500.00 mg, 1.03 mmol, 1 eq.) was added dropwise into the resulting dichloromethane (5 mL) solution at 0° C. under nitrogen atmosphere. The reaction system was stirred at 25° C. for 1 h. After the reaction was completed as indicated by LCMS, the reaction solution was poured into 0.5 mol/L diluted hydrochloric acid (50 mL), the aqueous phase was extracted with dichloromethane (20 mL×3), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (100-200 mesh silica gel, petroleum ether:ethyl acetate=10:1 to 4:1, V/V) to give compound 1-5. MS(ESI) m/s:540.1 [M+H-(t-Bu)].sup.+.
[0344] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=8.47 (s, 1H), 7.38-7.34 (m, 2H), 6.23 (s, 1H), 4.35-4.31 (m, 2H), 3.28-3.23 (m, 2H), 2.52-2.49 (m, 2H), 2.18-2.14 (m, 2H), 2.05-2.00 (m, 2H), 1.93-1.81 (m, 3H), 1.80-1.71 (m, 2H), 1.55 (s, 3H), 1.50-1.45 (m, 9H), 1.32-1.26 (m, 2H).
Step 8
[0345] Compound 1-5 (100.00 mg, 167.78 μmol, 1 eq.) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (382.59 mg, 3.36 mmol, 248.44 μL, 20 eq.) was added. The reaction system was stirred at 25° C. for 1 h. After the reaction was completed as indicated by LCMS and HPLC, the reaction solution was concentrated under reduced pressure. The crude product was separated and purified by prep-HPLC (neutral system, column: Xtimate C18 150 mm×25 mm×5 Wm; mobile phase: water (containing 10 mM ammonium bicarbonate)-acetonitrile; acetonitrile %: 10%-50%, 20 min) to give the compound of formula (I). MS(ESI) m/s:540.2 [M+H].sup.+.
[0346] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=9.99 (s, 1H), 8.29 (s, 1H), 7.65-7.57 (m, 2H), 4.30-4.26 (m, 2H), 3.09-3.05 (m, 2H), 2.47-2.43 (m, 2H), 2.16-2.12 (m, 2H), 1.85-1.80 (m, 2H), 1.75-1.61 (m, 5H), 1.35 (s, 3H), 1.21-1.16 (m, 2H).
Example 2: Preparation Method for Crystal Form
[0347] The preparation method for the crystal form A was as follows: 7.5 g of compound 1-5 was dissolved in dichloromethane (80 mL), and then trifluoroacetic acid (40 mL) was added. The reaction system was stirred at 15° C. for 1 h. After the reaction was completed as indicated by HPLC, the reaction solution was directly concentrated under reduced pressure. The crude product was purified by slurrying with methyl tert-butyl ether (100 mL), filtered and concentrated, and lyophilized to give the product in the form of a white solid which was separated and subjected to XRPD analysis to give the crystal form A.
[0348] The preparation method for the crystal form B was as follows: about 15 mg of the crystal form A sample was dissolved in 0.04 mL of DMSO, and water was added dropwise until a solid was precipitated. The solid was suspended and stirred overnight, and then separated and subjected to XRPD analysis to give the crystal form B.
[0349] The preparation method for the crystal form C was as follows: about 15 mg of the crystal form A sample was dissolved in 0.18 mL of THF, and MTBE was added dropwise until a solid was precipitated. The solid was suspended and stirred overnight, and then separated and subjected to XRPD analysis to give the crystal form C.
[0350] The preparation method for the crystal form D was as follows: about 15 mg of the crystal form A sample was dissolved in 0.13 mL of THF, and DCM was added dropwise until a solid is precipitated. The solid was suspended and stirred overnight, and then separated and subjected to XRPD analysis to give the crystal form D.
[0351] The preparation method for the crystal form E was as follows: about 15 mg of the crystal form A sample was dissolved in 0.6 mL of 1,4-dioxane, the above solution was placed in a 3 mL glass bottle, the 3 mL glass bottle containing the above solution was placed in a 20 mL glass bottle containing 3 mL of ACN (acetonitrile) to enable ACN (acetonitrile) to be slowly evaporated into the 1,4-dioxane solution to give a solid in the 1,4-dioxane solution, and the solid was then separated and subjected to XRPD analysis to give the crystal form E.
[0352] The preparation method for the crystal form F was as follows: about 15 mg of the crystal form A sample was dissolved in 0.3 mL of DMF to give a clear solution which was then added dropwise into 3 mL of H.sub.2O. The reaction system was suspended and stirred overnight at room temperature, and the solid was separated and subjected to XRPD analysis to give the crystal form F.
[0353] The preparation method for the crystal form G was as follows: about 15 mg of the crystal form A sample was dispersed in 0.1 mL of CHCl.sub.3/THF (9:1, v/v), suspended and stirred at room temperature for about 2 weeks, and then separated and subjected to XRPD analysis to give the crystal form G.
[0354] The preparation method for the crystal form H was as follows: about 15 mg of the crystal form A sample was dispersed in 0.1 mL of EtOH/DMF (19:1, v/v), suspended and stirred at room temperature for about 2 weeks, and then separated and subjected to XRPD analysis to give the crystal form H.
[0355] The preparation method for the crystal form I was as follows: about 15 mg of the crystal form A sample was dispersed in 0.1 mL of water, suspended and stirred at 50° C. for about 2 weeks, and then separated and subjected to XRPD analysis to give the crystal form I.
[0356] The preparation method for the crystal form J was as follows: about 15 mg of the crystal form A sample was dispersed in 0.1 mL of 2-MeTHF, suspended and stirred at 50° C. for about 2 weeks, and then separated and subjected to XRPD analysis to give the crystal form J.
[0357] The preparation method for the crystal form K was as follows: about 15 mg of the crystal form A sample was dissolved in 0.2 mL of DCM/MeOH (4:1, v/v) at 50° C., directly cooled to 5° C., and then separated and subjected to XRPD analysis to give the crystal form K.
[0358] The preparation method for the crystal form L was as follows: about 15 mg of the crystal form A sample was dissolved in 0.1 mL of NMP (N-methylpyrrolidone), the above solution was placed in a 3 mL glass bottle, the 3 mL glass bottle containing the above solution was placed in a 20 mL glass bottle containing 3 mL of EtOAc (ethyl acetate) to enable EtOAc (ethyl acetate) to be slowly evaporated into the NMP (N-methylpyrrolidone) solution to give a solid in the NMP (N-methylpyrrolidone) solution, and the solid was separated and subjected to XRPD analysis to give the crystal form L.
[0359] The preparation method for the crystal form M was as follows: about 15 mg of the crystal form A sample was dissolved in 0.4 mL of THF and slowly evaporated at room temperature to give a solid, and the solid was separated and subjected to XRPD analysis to give the crystal form M.
[0360] The preparation method for the crystal form N was as follows: about 15 mg of the crystal form A sample was dissolved in 1.5 mL of EtOH at 50° C., directly cooled to 5° C., and then separated and subjected to XRPD analysis to give the crystal form N.
[0361] The preparation method for the crystal form O was as follows: the crystal form D was dried for about 1 h at room temperature under vacuum, and then subjected to XRPD analysis to give the crystal form O.
[0362] Preparation method 1 for the crystal form P was as follows: about 15 mg of the crystal form A sample was dissolved in 1.0 mL of MTBE/MeOH (3:2, v/v) and slowly evaporated at room temperature to give a solid, and the solid was separated and subjected to XRPD analysis to give the crystal form P. The XRPD measurement results of the resulting crystal form P are shown in
[0363] The preparation method for the crystal form Q was as follows: about 15 mg of the crystal form A sample was dissolved in 1.5 mL of MeOH, about 3 mL of ACN was added dropwise to give a clear solution, and the clear solution was suspended and stirred at −20° C. The solid was separated and subjected to XRPD analysis to give the crystal form Q.
[0364] The preparation method for the crystal form R was as follows: the crystal form O was heated to 150° C., then cooled to room temperature, and then subjected to XRPD analysis to give the crystal form R.
[0365] The preparation method for the crystal form S was as follows: the crystal form E was heated to 210° C., and then subjected to in-situ XRPD analysis to give the crystal form S.
[0366] The preparation method for the crystal form T was as follows: the crystal form G was heated to 150° C., and then cooled to room temperature to give the crystal form T.
[0367] The preparation method for the crystal form U was as follows: about 15 mg of the crystal form A sample was dissolved in 0.7 mL of DMAc, toluene was slowly evaporated into the DMAc solution at room temperature to give a clear solution, and the clear solution was evaporated at room temperature, and then separated and subjected to XRPD analysis to give the crystal form U.
[0368] The preparation method for the crystal form V was as follows: the crystal form U was dried for 2-3 h at 50° C. under vacuum, and then subjected to XRPD analysis to give the crystal form V.
Example 3: Preparation Method 2 for Crystal Form P
[0369] Acetone (10.06 L) was added into a reaction kettle, the temperature was controlled at 10-25° C., and then the crystal form A sample (1342.02 g, 2.49 mol) was added. The reaction system was then stirred at 40° C. for 16-24 h. The reaction was stopped, the reaction solution was directly filtered, and the filter cake was concentrated under reduced pressure at 45° C. to give the compound of formula (I) (1142.51 g, crude). The above procedure was repeated to homogenize the sample twice, the reaction solution was directly filtered, and the filter cake was concentrated under reduced pressure at 45° C. to give the compound of formula (I) (989.01 g, 73.44% yield). The sample was taken and subjected to XRPD analysis, the results are shown in
Example 4: Single Crystal Preparation Method for Crystal Form P
[0370] The compound of formula (I) was dissolved in methanol and then incubated at room temperature for 10 days by solvent evaporation.
[0371] The unit cell parameters, crystallographic data and atomic coordinates and the like of a single crystal of the crystal form P of the compound of formula (I) are shown in Tables 23 and 24 below.
TABLE-US-00026 TABLE 2 Crystallographic data and structure refinement Experimental molecular formula C.sub.25H.sub.25ClF.sub.3N.sub.3O.sub.5 Molecular weight 539.93 Temperature 295(2) K Wavelength 1.54178 A Crystal system Triclinic crystal system Space group P-1 Unit cell parameters a = 9.407(2) Å b = 11.531(3) Å c = 13.574(4) Å a = 66.982(8)° β = 75.337(8)° γ = 68.492(8)° Volume of unit cell 1250.4(6) Å.sup.3 Z 2 Calculating density 1.434 Mg/m.sup.3 Absorption correction parameter 1.925 mm.sup.−1 F(000) 560 Size of crystal 0.220 × 0.180 × 0.150 mm Angle range for data collection 4.368 degrees to 66.594 degrees Collection range for hkl −11 <= h <= 11, −13 <= k <= 13, −16 <= l <= 16 Reflection data collection/ 18006/4315 [R(int) = 0.0827] independence Data integrity for theta = 66.66 97.4% Absorption correction From equivalent semi- experience Maximum and minimum transmission 0.7531 and 0.6141 Refinement method F2 full matrix least square method Number of data/number of 4315/0/334 usage restrictions/number of parameters Degree of fitting of F.sub.2 1.951 Final R index [I > 2sigma(I)] R1 = 0.0687, wR2 = 0.1999 R index (all data) R1 = 0.2322, wR2 = 0.2821 Extinction coefficient n/a Maximum difference (peak top 1.119 and −1.571 e.A{circumflex over ( )}−3 and valley)
TABLE-US-00027 TABLE 24 Atomic coordinate (×10.sup.4) and equivalent isotropic displacement parameters (Å2 × 10.sup.3) x y z U(eq) Cl(1) 3668(1) 4061(1) 5464(1) 54(1) F(1) 5177(4) −811(4) 3807(4) 111(1) F(2) 3136(4) −790(4) 2711(3) 103(1) F(3) 885(4) 1471(4) 1938(3) 98(1) O(1) 8263(5) −175(5) 10621(4) 101(2) O(2) 8996(5) 1614(5) 10024(4) 106(2) O(3) 4357(3) 6198(4) 6898(2) 69(1) O(4) 2029(5) 8251(4) 5707(3) 89(1) O(5) 261(3) 5018(3) 3128(2) 64(1) N(1) 2472(3) 5198(3) 7459(2) 50(1) N(2) 786(3) 7559(3) 4396(2) 48(1) N(3) 2618(3) 3658(4) 3581(3) 55(1) C(1) 3034(4) 4130(5) 8462(3) 56(1) C(2) 4581(6) 3212(6) 8146(3) 82(2) C(3) 5392(7) 2176(6) 9124(4) 87(2) C(4) 5584(4) 2857(5) 9832(3) 60(1) C(5) 4032(4) 3694(5) 10193(3) 62(1) C(6) 3228(4) 4756(5) 9223(3) 53(1) C(7) 1787(7) 3447(7) 8968(4) 95(2) C(8) 6404(5) 1802(6) 10816(3) 78(2) C(9) 7968(6) 1008(6) 10475(3) 73(2) C(10) 3168(4) 6069(5) 6781(3) 51(1) C(11) 2340(4) 7095(5) 5805(3) 56(1) C(12) 1896(3) 6658(4) 5080(2) 47(1) C(13) 2329(3) 5496(4) 4843(2) 42(1) C(14) 1498(3) 5674(4) 4032(2) 47(1) C(15) 549(4) 6971(4) 3800(3) 48(1) C(16) −686(5) 7898(5) 3106(3) 64(1) C(17) −1022(7) 9185(6) 3308(5) 96(2) C(18) −205(4) 8923(5) 4253(3) 58(1) C(19) 1405(3) 4766(4) 3538(2) 47(1) C(20) 2720(4) 2578(5) 3305(3) 54(1) C(21) 1689(4) 2592(5) 2718(3) 56(1) C(22) 1870(5) 1469(6) 2524(3) 67(1) C(23) 3013(6) 313(7) 2887(4) 76(2) C(24) 4044(5) 323(7) 3451(4) 79(2) C(25) 3928(5) 1423(6) 3654(4) 67(1) H(2A) 8688 2331 10118 159 H(1A) 1611 5252 7309 59 H(3A) 3419 3615 3806 66 H(2B) 5250 3738 7684 98 H(2C) 4417 2759 7734 98 H(3B) 4788 1578 9547 105 H(3C) 6395 1663 8875 105 H(4A) 6227 3433 9402 72 H(5A) 4164 4117 10642 74 H(5B) 3388 3132 10625 74 H(6A) 3831 5361 8825 63 H(6B) 2223 5258 9482 63 H(7A) 2082 2743 9622 142 H(7B) 834 4074 9127 142 H(7C) 1658 3095 8473 142 H(8A) 6490 2240 11270 94 H(8B) 5784 1217 11239 94 H(16A) −1593 7600 3323 77 H(16B) −324 7992 2352 77 H(17A) −2124 9556 3475 115 H(17B) −662 9816 2665 115 H(18A) 396 9523 4069 70 H(18B) −931 8989 4894 70 H(21A) 892 3354 2464 67 H(25A) 4648 1406 4023 81
The XRPD pattern obtained by calculation through software simulation based on single crystal data is shown in
Experimental Example 1: Study on Solid Stability of Crystal Form P
[0372] In order to evaluate the solid stability of the crystal form P, the crystal form P was investigated for influence factors (high temperature, high humidity and light), stability under accelerated conditions and stability under intermediate conditions. The crystal form P was placed at high temperature (60° C., closed), high humidity (room temperature, 92.5% RH, sealing film wrapping and pricking 5-10 small holes) for 5 days and 10 days, then placed under visible light and ultraviolet light (a light-shielding control group was wrapped by tinfoil paper) according to ICH conditions (visible light illumination reached 1.2E+06 Lux hrs, ultraviolet light illumination reached 200 W.Math.hrs/m) in a closed manner, and meanwhile, placed with stability under accelerated conditions (60° C./75% RH, sealing film wrapping and pricking 5-10 small holes) for 10 days and 1-2 months. XRPD and HPLC characterizations were carried out on the placed sample so as to detect the change of crystal form and purity; the results of Table 25 show that the crystal form of the crystal form P was unchanged under all stability conditions.
TABLE-US-00028 TABLE 25 Evaluation results of solid stability of the crystal form P Crystal Relative Point taking form purity* Conditions conditions change (%) Starting crystal form P / Unchanged — 60° C. Day 5 Unchanged 100.0 Day 10 Unchanged 100.0 92.5% RH Day 5 Unchanged 100.0 Day 10 Unchanged 100.0 Visible light # The illumination Unchanged 100.0 reaches 1.2E+06 Lux .Math. hrs Light-shielding Taking points Unchanged 99.9 control group simultaneously with the visible light group Visible + The illumination Unchanged 100.0 ultraviolet # reaches 200 W .Math. hrs/m.sup.2 Light-shielding Taking points Unchanged 100.0 control group simultaneously with the visible light + ultraviolet groups 60° C./75% RH Day 10 Unchanged 100.0 1 month Unchanged 100.0 Month 2 Unchanged 100.0 Note: # indicates ICH conditions. *Relative purity = ratio of stability sample purity to starting sample purity. The aforementioned results indicate that the crystal form P has good stability.
Experimental Example 2: qPCR Assay for In Vitro Testing of HBV
[0373] 1. Objective
[0374] HBV DNA content in HepG2.2.15 cells was detected by a real time-qPCR assay, and the inhibitory action of the compound on HBV was evaluated by taking the EC.sub.50 value of the compound as an index.
[0375] 2. Experimental materials
[0376] 2.1. cell lines: HepG2.2.15 cells
[0377] HepG2.2.15 cell culture medium (DMEM/F12, Invitrogen-11330057; 10% serum, Invitrogen-10099141; 100 units/mL penicillin and 10 μg/mL streptomycin, Invitrogen-15140122; 1% non-essential amino acids, Invitrogen-11140076; 2 mM L-Glutamine, Invitrogen-25030081; 300 μg/mL geneticin, Invitrogen-10131027)
[0378] 2.2. reagents
[0379] Pancreatin (Invitrogen-25300062)
[0380] DPBS (Hyclone-SH30028.01B)
[0381] DMSO (Sigma-D2650-100ML)
[0382] High-throughput DNA purification Kit (QIAamp 96 DNA Blood Kit, Qiagen-51162) Quantitative faststart universal probe reagent (FastStart Universal Probe Master, Roche-04914058001)
[0383] 2.3. consumables and instrument
[0384] 96-well cell culture plate (Corning-3599)
[0385] CO.sub.2 incubator (HERA-CELL-240)
[0386] Optical sealing film (ABI-4311971)
[0387] Quantitative PCR 96-well plate (Applied Biosystems-4306737)
[0388] Fluorescent quantitative PCR instrument (Applied Biosystems-7500 real time PCR system)
[0389] 3. Experimental procedures and method
[0390] 3.1. HepG2.2.15 cells (4×10.sup.4 cells/well) were plated in a 96-well plate and cultured overnight at 37° C. and 5% CO.sub.2.
[0391] 3.2. On the next day, the compound was diluted for a total of 8 concentrations, with 3-fold gradient dilutions. The compound at different concentrations was added into the culture wells in duplicate. The final concentration of DMSO in the culture medium was 1%. 1 μM GLS4 served as 100% inhibition control (WO2008154817A1 discloses the structure of GLS4 as follows:
##STR00011##
DMSO at 1% served as 0% inhibition control.
[0392] 3.3. On the fifth day, the original culture medium was replaced with a fresh culture medium containing the compound.
[0393] 3.4. On the eighth day, the culture medium in the culture wells was collected, and the high-throughput DNA purification kit (Qiagen-51162) was used to extract DNA. For specific steps, refer to the product manual.
[0394] 3.5 The preparation of the PCR reaction solution is shown in Table 26.
TABLE-US-00029 TABLE 26 Preparation of PCR reaction solution Volume Volume required for required for Items 1 well (μL) 80 wells (μL) Quantitative faststart 12.5 1000 universal probe reagent Upstream primer (10 mol) 1 80 Downstream primer (10 mol) 1 80 Probe (10 mol) 0.5 40
TABLE-US-00030 The sequence of the upstream primer is as follows: GTGTCTGCGGCGTTTTATCA. The sequence of the downstream primer is as follows: GACAAACGGGCAACATACCTT. The sequence of the probe is as follows: 5′ + FAM + CCTCTKCATCCTGCTGCTATGCCTCATC + TAMRA-3′
[0395] 3.6. 15 μL of the reaction mixture was added into each well of the 96-well PCR plate, and then 10 μL of sample DNA or HBV DNA standard was added into each well.
[0396] 3.7. Reaction conditions for PCR were as follows: heating at 95° C. for 10 min; then denaturating at 95° C. for 15 s and extending at 60° C. for 1 min for 40 cycles.
[0397] 3.8. Data analysis
[0398] 3.8.1. Calculation of inhibition percentage: % Inh.=[1−(DNA copy number in the sample−DNA copy number in 1 μM GLS4)/(DNA copy number in the DMSO control−DNA copy number in 1 μM GLS4)]×100.
[0399] 3.8.2. Calculation of EC.sub.50: the concentration for 50% of maximal effect (EC.sub.50) values of the compound against HBV were calculated using GraphPad Prism software.
[0400] 3.8.3. The experimental results are shown in Table 27.
TABLE-US-00031 TABLE 27 EC.sub.50 results of the qPCR assay Sample Concentration for 50% of maximal (title compound) effect (EC.sub.50) of HBV Compound of formula (I) 4 nM
Experimental Example 3: Inhibition Test of the hERG Potassium Channel
[0401] 1. Objective
[0402] The effect of the compound disclosed herein on hERG potassium channel was detected by using automatic patch-clamp method.
[0403] 2. Experimental method
[0404] 2.1. Cell culture
[0405] The cells stably expressing the hERG potassium channel used in the experiment were CHO-hERE from Aviva Biosciences. CHO-hERG was cultured at 37° C. and 5% CO.sub.2. CHO hERG culture medium is shown in Table 28.
TABLE-US-00032 TABLE 28 CHO hERG culture medium Reagent Supplier Volume (mL) F12 culture medium Invitrogen 500 Fetal bovine serum Invitrogen 50 G418/Geneticin Invitrogen 1 Hygromycin B Invitrogen 1
[0406] 2.2. Preliminary preparation for cells
[0407] The CHO-hERG cells used in the experiment were cultured for at least two days, and the cell density reached 75% or more. Before the experiment, the cells were digested with TrypLE, and then the collected cells were resuspended in extracellular fluid.
[0408] 2.3. Preparation of the intracellular and extracellular fluids
[0409] The extracellular fluid needed to be prepared once a month. The intracellular fluid must be frozen in aliquots at −20° C. Compositions of the intracellular and extracellular fluids are shown in Table 29.
TABLE-US-00033 TABLE 29 Compositions of the intracellular and extracellular fluids Extracellular Intracellular Composition fluid (mM) fluid (mM) NaCl 145 — KCl 4 120 KOH — 31.25 CaCl.sub.2 2 5.374 MgCl.sub.2 1 1.75 Glucose 10 — Na.sub.2ATP — 4 HEPES 10 10 EGTA — 10 pH pH was adjusted to 7.4 pH was adjusted to 7.4 with sodium hydroxide with potassium hydroxide Osmotic pressure 295 mOsm 285 mOsm
[0410] 2.4. Preparation of Compound
[0411] The test compound and its positive control amitriptyline were dissolved in DMSO to obtain a stock solution at a certain concentration. Then the stock solution was diluted for different gradients, and finally added to an extracellular fluid at a certain ratio to be diluted to a concentration for test. Precipitation was checked with the naked eye before the experiment. Finally, the concentration of DMSO in the solution to be tested and the positive control amitriptyline should not exceed 0.3%.
[0412] 2.5. Voltage stimulation scheme
[0413] With a holding potential of −80 mv, a voltage stimulation of −50 my was applied for 80 ms to record the cell leakage current value; then hERG channel was opened by a depolarization to +20 my for 4800 ms and hERG tail current was elicited by a repolarization to −50 my for 5000 ms and recorded; and finally, the voltage was restored to the holding potential of −80 my and maintained for 3100 ms. The above voltage stimulation was repeated every 15000 ms.
[0414] 2.6. QPatch.sup.HTX whole-cell patch clamp recording
[0415] The hERG QPatch.sup.HTX experiment was performed at room temperature. Whole-cell scheme, voltage stimulation scheme and compound detection scheme were established on QPatch Assay Software 5.2 (Sophion Bioscience). First, 30 repetitive set voltage stimulations were performed, of which the section on a current spectrum was the baseline section for subsequent analysis. Then 5 μL of extracellular fluid was added, and the voltage stimulation was repeated three times. Each compound at the action concentration was added in sequence with a volume of 5 μL, and the voltage stimulation was repeated three times. The cells were incubated for at least 5 min with the compound at each tested concentration. During the entire recording process, all indicators needed to meet the data analysis acceptance standard. If the standard was not met, the cell would not be included in the analysis range and the compound would be tested again. The above recording process was automatically operated by the Qpatch analysis software. The tested concentrations of the compounds were 0.24 μM, 1.20 μM, 6.00 μM and 30.00 μM, and each concentration was repeated for at least two cells.
[0416] 2.7. Data Analysis
[0417] In each complete current recording, the inhibition percentage of each compound at the action concentration could be calculated based on the percentage of peak current in the negative control. The dose-effect relationship curve was obtained by fitting with the standard Hill Equation, and the specific equation is as follows:
I.sub.(C)=I.sub.b+(I.sub.fr−I.sub.b)*c.sup.n/(IC.sub.50.sup.n+c.sup.n)
[0418] C is the tested concentration of the compound, n is the slope, and I is the current.
[0419] The curve fitting and inhibition rate calculation were all completed by Qpatch analysis software. If the inhibition rate exceeded 50% at the lowest concentration or the inhibition rate did not reach 50% at the highest concentration, the corresponding IC.sub.50 of the compound was lower than the lowest concentration or the IC.sub.50 value was greater than the highest concentration.
[0420] 2.8. Test results
[0421] The hERG IC.sub.50 values of the compounds in the examples are shown in Table 30.
TABLE-US-00034 TABLE 30 hERG IC.sub.50 values of the compounds in the examples Sample hERG IC50 (μM) Compound of formula (I) >40
Experimental Example 4: Study on Inhibition Against Cytochrome P450 Isoenzyme
[0422] Objective: The inhibitory effect of the test compound on the activity of human liver microsomal cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was determined.
[0423] Experimental procedures: firstly, the test compound (10 mM) was diluted in gradient to prepare working solutions (100×final concentration) at concentrations of: 5 mM, 1.5 mM, 0.5 mM, 0.15 mM, 0.05 mM, 0.015 mM and 0.005 mM, and working solutions of positive inhibitors for P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and the specific substrate mixtures thereof were prepared simultaneously; human liver microsomes frozen in a −80° C. refrigerator were thawed on ice, and after all thawed, the human liver microsomes were diluted with PB (phosphate buffered saline) to prepare a working solution at a specific concentration (0.253 mg/mL); 20 μL of the substrate mixture (20 μL of PB was added into the blank well) and 158 μL of the working solution of human liver microsomes were added into the reaction plate which was then placed on ice for use; then 2 μL of the test compound at each concentration (N=1) and a specific inhibitor (N=2) were added into the corresponding well, and the group without the inhibitor (test compound or the positive inhibitor) was added with a corresponding organic solvent as a control sample (the test compound control sample was 1:1 DMSO:MeOH; the positive control sample was 1:9 DMSO:MeOH); after pre-incubation under a 37° C. water bath for 10 min, 20 μL of a coenzyme factor (NADPH) solution was added into the reaction plate and incubated under a 37° C. water bath for 10 min, 400 μL of a cold acetonitrile solution (the internal standard was 200 ng/mL Tolbutamide and Labetalol) was added to terminate the reaction, and the reaction plate was placed on a shaker and shaken for 10 min; after centrifugation at 4,000 rpm for 20 min, 200 μL of the supernatant was collected and added to 100 μL of water to dilute the sample, and, finally, the plate was sealed, oscillated, shaken evenly, and subjected to LC/MS/MS detection. The experimental results are shown in Table 31.
TABLE-US-00035 TABLE 31 Results of inhibitory effect of the test compound on the activity of human liver microsomal cytochrome P450 isoenzymes IC.sub.50 (μM) CYP3A4- Compound CYP1A2 CYP2C9 CYP2C19 CYP2D6 M Compound >50 >50 >50 >50 >50 of formula (I)
Experimental Example 5: Experiment on Cytotoxicity
[0424] 1. The compound was diluted with DMSO (dimethyl sulfoxide) in a 3-fold gradient for 9 concentrations, and added into a 96-well plate in duplicate. The compound concentration was 200-fold of the final test concentration. [0425] 2. The cells were rinsed with PBS (phosphate buffered saline) once, added with 0.25% trypsin and digested for about 2-5 min in a 37° C., 5% CO.sub.2 incubator. Then the digestion was terminated with a cell culture medium and the cells were dispersed into single cells by pipetting with a pipettor. [0426] 3. The cell density was counted with a cell counter, and adjusted to the required density with the medium. [0427] 4. The cells were added into a 96-well plate that had been added with the compound. The final concentration of DMSO in each well was 0.5%. Wells containing 0.5% DMSO were used as non-toxic negative controls, and wells containing the cell culture medium were used as 100% cytotoxicity controls. Then the cell plate was incubated in a 37° C., 5% CO.sub.2 cell incubator for 3 days. [0428] 5. The chemiluminescence signal (RLU, relative chemiluminescence unit) of each well in the cell plate was detected with the cell viability detection kit CellTiter-Glo using the multi-functional microplate reader Envision, with instructions on the kit followed. [0429] 6. The raw data (RLU) were substituted into the following formula to calculate the cell viability of each well (cell viability %):
Cell viability %=(RLU.sub.sample−AverageRLU.sub.Mediumcont rol)/(AverageRLU.sub.Cellcont rol−AverageRLU.sub.Mediumcont rol)×100% [0430] RLU.sub.sample: signal value of the sample well; Average RLU.sub.Cell control: average signal value of the cell control well; Average RLU.sub.Mediumcontrol: average signal value of the medium control well. [0431] 7. Using GraphPad Prism software, the cell viability data were nonlinearly fitted to draw a dose-response curve and the 50% cytotoxic concentration (CC.sub.50) value of the compound was obtained. The results are shown in Table 32.
TABLE-US-00036 TABLE 32 Test results of 50% cytotoxic concentration (CC.sub.50) Compound CC.sub.50(μM) Compound of formula (I) >50
Experimental Example 6: Study on In Vitro Microsomal Stability
[0432] Metabolic Stability of the Compound in CD-1 Mice and Human Liver Microsomes
[0433] Objective: The metabolic stability of the test compound in CD-1 mice and human liver microsomes was determined.
[0434] Experimental procedures: firstly, the test compound (10 mM) was subjected to a two-step dilution, where the compound was diluted to an intermediate concentration of 100 μM with 100% methanol, and the working solution was diluted to 10 μM with a potassium phosphate buffer; eight 96-well incubation plates were prepared, and named T0, T5, T10, T20, T30, T60, Blank and NCF60, respectively; the reaction time points corresponding to the first 6 incubation plates were 0 min, 5 min, 10 min, 20 min, 30 min and 60 min, respectively; for the Blank plate, neither the test compound nor a control compound was added, and for the NCF60 plate, potassium phosphate buffer was used in an incubation of 60 min in place of a NADPH regeneration system solution; 10 μL of the test compound working solution and 80 μL of the microsome working solution (liver microsome protein concentration was 0.625 mg/mL) were added to the T0, T5, T10, T20, T30, T60 and NCF60 plates, while only the microsome working solution was added to the Blank plate, and all the incubation plates were then placed in a 37° C. water bath for pre-incubation for about 10 min; after the pre-incubation, 10 μL of a NADPH regeneration system working solution was added into each sample well of all the plates except the NCF60 plate and T0 plate to start the reaction, and 10 μL of potassium phosphate buffer was added to each well of the NCF60 plate; therefore, in the test compound or control compound samples, the final reaction concentrations of the compound, testosterone, diclofenac and propafenone were 1 μM, the concentration of the liver microsomes was 0.5 mg/mL, and the final concentrations of DMSO and acetonitrile in the reaction system were 0.01% (v/v) and 0.99% (v/v), respectively; after incubation for an appropriate time (such as 5 min, 10 min, 20 min, 30 min, and 60 min), 300 μL of a stop solution (acetonitrile solution containing 100 ng/mL tolbutamide and 100 ng/mL labetalol) was added to each sample well to stop the reaction; 300 μL of the stop solution and then 10 μL of the NADPH working solution were added to the T0 plate, all the sample plates were shaken and centrifuged in a centrifuge (3220×g) for 20 min, and then 100 μL of supernatant was taken from each well and diluted with 300 μL of pure water for liquid chromatography-tandem mass spectrometry analysis.
[0435] The experimental results are shown in Table 33.
TABLE-US-00037 TABLE 33 Metabolic stability results of the test compound in CD-1 mice and human liver microsomes T.sub.1/2 CL.sub.int(mic) CL.sub.int(liver) Compound Species (min) (μL/min/mg) (mL/min/kg) Compound of CD-1 mice >145 <9.6 <38.0 formula (I) Human >145 <9.6 <8.6
[0436] Metabolic Stability of the Compound in Liver Microsomes of SD Rats, Beagle Dogs and Cynomolgus Monkeys
[0437] Objective: The metabolic stability of the tested compound of formula (I) in liver microsomes of rats, beagle dogs and cynomolgus monkeys was determined.
[0438] Experimental procedures: firstly, the test compound (10 mM) was subjected to a two-step dilution, where the compound was diluted to an intermediate concentration of 100 μM with 100% methanol, and the working solution was diluted to 10 μM with a potassium phosphate buffer; eight 96-well incubation plates were prepared, and named T0, T5, T10, T20, T30, T60, Blank and NCF60, respectively; the reaction time points corresponding to the first 6 incubation plates were 0 min, 5 min, 10 min, 20 min, 30 min and 60 min, respectively; for the Blank plate, neither the test compound nor a control compound was added, and for the NCF60 plate, potassium phosphate buffer was used in an incubation of 60 min in place of a NADPH regeneration system solution; 10 μL of the test compound working solution and 80 μL of the microsome working solution (liver microsome protein concentration was 0.625 mg/mL) were added to the T0, T5, T10, T20, T30, T60 and NCF60 plates, while only the microsome working solution was added to the Blank plate, and all the incubation plates were then placed in a 37° C. water bath for pre-incubation for about 10 min; after the pre-incubation, 10 μL of a NADPH regeneration system working solution was added into each sample well of all the plates except the NCF60 plate and T0 plate to start the reaction, and 10 μL of potassium phosphate buffer was added to each well of the NCF60 plate; therefore, in the test compound or control compound samples, the final reaction concentrations of the compound, testosterone, diclofenac and propafenone were 1 μM, the concentration of the liver microsomes was 0.5 mg/mL, and the final concentrations of DMSO and acetonitrile in the reaction system were 0.01% (v/v) and 0.99% (v/v), respectively; after incubation for an appropriate time (such as 5 min, 10 min, 20 min, 30 min, and 60 min), 300 μL of a stop solution (acetonitrile solution containing 100 ng/mL tolbutamide and 100 ng/mL labetalol) was added to each sample well to stop the reaction; 300 μL of the stop solution and then 10 μL of the NADPH working solution were added to the T0 plate, all the sample plates were shaken and centrifuged in a centrifuge (3220×g) for 20 min, and then 100 μL of supernatant was taken from each well and diluted with 300 μL of pure water for liquid chromatography-tandem mass spectrometry analysis.
[0439] The experimental results are shown in Table 34.
TABLE-US-00038 TABLE 34 Metabolic stability results of the test compound in liver microsomes of SD rats, beagle dogs and cynomolgus monkeys T.sub.1/2 CL.sub.int(mic) CL.sub.int(liver) Compound Species (min) (μL/min/mg protein) (mL/min/kg) Compound of SD rats >145 <9.6 <17.3 formula (I) Beagle dogs >145 <9.6 <13.8 Cynomolgus >145 <9.6 <13.0 monkeys
Experimental Example 7: Pharmacokinetic Study
[0440] Pharmacokinetic Study of the Test Compound in Balb/c Mice by Oral Administration and Intravenous Injection:
[0441] The test compound was mixed with a solution containing dimethyl sulfoxide (101), polyethylene glycol 400 (600%) and water (300), and the mixture was vortexed and sonicated to prepare a 0.2 mg/mL clear solution, which was filtered through a millipore filter for later use. Balb/c female mice aged 7 to 10 weeks were intravenously injected with the candidate compound solution at a dose of 1 mg/kg.
[0442] The test compound was mixed with an aqueous solution containing 10% polyoxyethylene stearate, and the mixture was vortexed and sonicated to prepare a 0.3 mg/mL clear solution for later use. Balb/c female mice aged 7 to 10 weeks were orally administered with the candidate compound solution at a dose of 3 mg/kg.
[0443] Whole-blood was collected and plasma was prepared. Drug concentration was analyzed by LC-MS/MS and pharmacokinetic parameters were calculated with Phoenix WinNonlin software. The results are shown in Table 35.
TABLE-US-00039 TABLE 35 Pharmacokinetic results of the test compound Compound of Dosage Pharmacokinetic parameters formula (I) IV(1 mg/kg) Half-life T.sub.1/2 (h) 1.95 Clearance (CL, mL/min/kg) 20 Apparent volume of distribution 3.1 (Vd.sub.ss, L/kg) Area under the plasma concentration- 1488 time curve AUC.sub.0-24 h (nM .Math. h) PO(3 mpk) Peak time T.sub.max (h) 0.5 Peak concentration C.sub.max (nM) 1480 Area under the plasma concentration- 2274 time curve AUC (nM .Math. h) Bioavailability (F %) 51%
Experimental Example 8: Study on Liver-to-Blood Ratio in Mice
[0444] Experiment on the Liver-to-Blood Ratio in Balb/c Mice Orally Administered with the Test Compound
[0445] The compound of formula (I) was mixed with an aqueous solution containing 100% polyethylene glycol-15 hydroxystearate, and the mixture was vortexed and sonicated to prepare a 0.3 mg/mL clear solution for later use. Balb/c female mice aged 7 to 10 weeks were orally administered with the candidate compound solution at a dose of 3 mg/kg.
[0446] Whole blood at a certain time point was collected and plasma was prepared. Liver tissues at the corresponding time point were collected to prepare a tissue homogenate. Drug concentration was analyzed by LC-MS/MS and pharmacokinetic parameters were calculated with Phoenix WinNonlin software. The results are shown in Table 36.
TABLE-US-00040 TABLE 36 Liver-to-blood ratio of the test compound Compound Compound of formula (I) Matrix Plasma Liver AUC.sub.0-last (nM .Math. h) or (nmol/kg .Math. h) 3476 86036 AUC ratio (L/P) Liver/plasma — 24.7 exposure ratio
Experimental Example 9: In Vivo Efficacy Study
[0447] HDJ/HBV Model
[0448] Objective: The efficacy of the compound against hepatitis B virus in mice was determined through the HDJ/HBV mouse model.
[0449] Preparation of compound: the solvent was 10% polyethylene glycol-15 hydroxystearate; a certain amount of the tested compound of formula (I) was dissolved in an aqueous solution containing 10% polyethylene glycol-15 hydroxystearate, and the mixture was vortexed and sonicated to prepare a homogeneous suspension, and the suspension was stored at 4° C. for later use.
[0450] High pressure injection of the HBV plasmid DNA solution via the tail vein of mice: the day of plasmid injection was day 0, the day after injection was day 1, and so on. On day 0, all animals were injected by tail vein with a saline solution containing 10 μg of plasmid DNA at a volume of 80% of body weight, and the injection was completed within 5 seconds.
[0451] Administration: all mice were administered intragastrically twice a day (8/16 h interval) on day 1-6 and once on day 7. All mice were euthanized in the afternoon on day 7. The body weight of the mice, which was monitored every day, remained stable throughout the experiment.
[0452] Sample collection: blood was collected from the submandibular vein 4 h after the first administration in the morning on days 1, 3, and 5. All blood samples were collected in K.sub.2-EDTA anticoagulation tubes, and centrifuged for 10 min at 4° C., 7000 g to prepare about 40 μL of plasma. All mice were euthanized by CO.sub.2 four hours after administration in the morning on day 7. Blood was collected from the heart, and the plasma preparation method was the same as above. Two liver tissues were collected with 70-100 mg each, and quick-frozen by liquid nitrogen. After all samples were collected, they were stored in a refrigerator (−80° C.) for HBV DNA content detection.
[0453] Sample analysis: all plasma samples and liver samples were detected for HBV DNA by qPCR.
[0454] The experimental results are shown in Table 37.
TABLE-US-00041 TABLE 37 HBV-DNA reduction Plasma (day 5) Liver (day 7) Δ Log10 Δ Log10 Test compound Dosage copies/μL copies/100 ng Compound of 30 mg/kg 3.57 2.36 formula (I) 10 mg/kg 2.0 0.94