PYRIDONE DERIVATIVE CRYSTAL FORM AND PREPARATION METHOD AND APPLICATION THEREFOR
20230144122 · 2023-05-11
Inventors
Cpc classification
A61K31/5383
HUMAN NECESSITIES
International classification
Abstract
Provided are crystals of (((R)-12′-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl)-6′,8′-dioxo-6′,8′,12′,12a′-tetrahydro-1′H,4′H-spiro[cyclopropane-1,3′-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin]-7′-yl)oxy)methyl methyl carbonate, i.e. compound of Formula (1) or a solvate thereof and a preparation method therefor, the crystals comprising crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, and single crystal. The crystal of the compound of Formula (1) or a solvate thereof can be individually used for clinical treatment or in combination with other anti-influenza drugs such as neuraminidase inhibitors, nucleoside drugs, or PB2 inhibitors, being capable of clinically curing influenza patients quickly, and having very good activity and good bioavailability compared to existing pyridone derivatives. In addition, the stability, hygroscopicity, and storability of the crystals meet the requirements for pharmaceutical use.
##STR00001##
Claims
1. A crystal of a compound of Formula (1) or a solvate thereof: ##STR00006##
2. The crystal as claimed in claim 1, is characterized in that, the crystal is in crystal form A of the compound of formula (1), which has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 3.10°±0.2°, 8.74°±0.2°, 15.44°±0.2° and 21.91°±0.2°.
3. The crystal as claimed in claim 2, is characterized in that, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (1) further has one or more characteristic peaks at 2θ of 13.08°±0.2°, 26.35°±0.2°, and 30.83°±0.2°.
4. The crystal as claimed in claim 2 or 3, is characterized in that, a spectrum of the crystal form A of the compound of formula (1) determined by thermogravimetric analysis shows that it loses 1.8±0.2% in weight when heated to 150±2° C.; and/or, a spectrum of the crystal form A of the compound of formula (1) determined by differential scanning calorimetry shows one endothermic peak, indicating that a melting point onset temperature of the crystal form A is 230.5±2° C.
5. The crystal as claimed in claim 2 or 3, is characterized in that, the crystal form A of the compound of formula (1) is an anhydrous crystal form.
6. The crystal as claimed in claim 1, is characterized in that, the crystal is in crystal form B of the compound of formula (1), which has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 8.42°±0.2°, 14.27°±0.2°, 16.04°±0.2° and 25.41°±0.2°.
7. The crystal as claimed in claim 6, is characterized in that, the X-ray powder diffraction pattern of the crystal form B of the compound of formula (1) further has one or two characteristic peaks at 2θ of 10.75°±0.2° and 16.87°±0.2°.
8. The crystal as claimed in claim 6 or 7, is characterized in that, the X-ray powder diffraction pattern of the crystal form B of the compound of formula (1) further has one or more characteristic peaks at 2θ of 18.21°±0.2°, 18.78°±0.2°, 19.26°±0.2°, 19.60°±0.2°, 20.40°±0.2°, 21.39°±0.2°, 21.66°±0.2°, 23.38°±0.2°, 27.32°±0.2°, 29.17°±0.2% and 34.08°±0.2°.
9. The crystal as claimed in claim 6 or 7, is characterized in that, a spectrum of the crystal form B of the compound of formula (1) determined by thermogravimetric analysis shows that it loses 2.2±0.2% in weight when heated to 150±2° C.; and/or, a spectrum of the crystal form B of the compound of formula (1) determined by differential scanning calorimetry shows two endothermic peaks, and onset temperatures of the two endothermic peaks are 208.5±2° C. and 233.8±2° C., respectively.
10. The crystal as claimed in claim 6 or 7, is characterized in that, the crystal form B of the compound of formula (1) is an anhydrous crystal form.
11. The crystal as claimed in claim 1, is characterized in that, the crystal is in crystal form C of the compound of formula (1), which has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 7.73°±0.2°, 17.13°±0.2°, 20.08°±0.2° and 21.74°±0.2°.
12. The crystal as claimed in claim 11, is characterized in that, the X-ray powder diffraction pattern of the crystal form C of the compound of formula (1) further has one or more characteristic peaks at 2θ of 13.13°±0.2°, 13.65°±0.2°, 20.98°±0.2°, and 23.22°±0.2°.
13. The crystal as claimed in claim 11 or 12, is characterized in that, the X-ray powder diffraction pattern of the crystal form C of the compound of formula (1) further has one or more characteristic peaks at 2θ of 12.51°±0.2°, 14.76°±0.2°, 15.21°±0.2°, 18.39°±0.2°, and 24.13°±0.2°.
14. The crystal as claimed in claim 11 or 12, is characterized in that, a spectrum of the crystal form C of the compound of formula (1) determined by thermogravimetric analysis shows that it loses 12.05±0.2% in weight when heated to 150±2° C.; and/or, a spectrum of the crystal form C of the compound of formula (1) determined by differential scanning calorimetry shows two endothermic peaks, and onset temperatures of the two endothermic peaks are 86.8±2° C. and 233.9±2° C., respectively.
15. The crystal as claimed in claim 11 or 12, is characterized in that, the crystal form C of the compound of formula (1) is a tetrahydrofuran solvate of the compound of formula (1).
16. The crystal as claimed in claim 1, is characterized in that, the crystal is in crystal form D of the compound of formula (1), which has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 7.94°±0.2°, 22.16°±0.2°, and 28.03°±0.2°.
17. The crystal as claimed in claim 16, is characterized in that, the X-ray powder diffraction pattern of the crystal form D of the compound of formula (1) further has one or more characteristic peaks at 2θ of 15.09°±0.2°, 15.50°±0.2°, 19.63°±0.2°, 23.56°±0.2% and 25.86°±0.2°.
18. The crystal as claimed in claim 16 or 17, is characterized in that, a spectrum of the crystal form D of the compound of formula (1) determined by thermogravimetric analysis shows that it loses 7.5±0.2% in weight when heated to 150±2° C.; and/or, a spectrum of the crystal form D of the compound of formula (1) determined by differential scanning calorimetry shows two endothermic peaks, and onset temperatures of the two endothermic peaks are 113.2±2° C. and 230.6±2° C., respectively.
19. The crystal as claimed in claim 16 or 17, is characterized in that, the crystal form D of the compound of formula (1) is a N-methyl-2-pyrrolidone solvate of the compound of formula (1).
20. The crystal as claimed in claim 1, is characterized in that, the crystal is in crystal form E of the compound of formula (1), which has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 8.01°±0.2°, 8.78°±0.2°, and 26.33°±02°.
21. The crystal as claimed in claim 20, is characterized in that, the X-ray powder diffraction pattern of the crystal form E of the compound of formula (1) further has one or more characteristic peaks at 2θ of 4.44°±0.2°, 17.56°±0.2°, 21.95°±0.2°, and 22.25°±0.2°.
22. The crystal as claimed in claim 20 or 21, is characterized in that, the X-ray powder diffraction pattern of the crystal form E of the compound of formula (1) further has one or more characteristic peaks at 2θ of 5.87°±0.2°, 19.64°±0.2°, 28.15°±0.2°, 29.07°±0.2% and 30.86°±0.2°.
23. The crystal as claimed in claim 1, is characterized in that, the crystal is in crystal form F of the compound of formula (1), which has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 5.77°±0.2% 8.69°±0.2°, 17.48°±0.2° and 22.84°±0.2°.
24. The crystal as claimed in claim 23, is characterized in that, the X-ray powder diffraction pattern of the crystal form F of the compound of formula (1) further has one or two characteristic peaks at 2θ of 11.61°±0.2° and 19.47°±0.2°.
25. The crystal as claimed in claim 23 or 24, is characterized in that, the X-ray powder diffraction pattern of the crystal form F of the compound of formula (1) further has one or more characteristic peaks at 2θ of 11.98°±0.2% 13.84°±0.2% 14.30°±0.2% 18.33°±0.2% and 18.95°±0.2°.
26. The crystal as claimed in claim 23 or 24, is characterized in that, a spectrum of the crystal form F of the compound of formula (1) determined by thermogravimetric analysis shows that it loses 1.16±0.2% in weight when heated to 150±2° C.; and/or, a spectrum of the crystal form F of the compound of formula (1) determined by differential scanning calorimetry shows an endothermic peak, and an onset temperature of the endothermic peak is 231.1±2° C.
27. The crystal as claimed in claim 23 or 24, is characterized in that, the crystal form F of the compound of formula (1) is an anhydrous crystal form.
28. The crystal as claimed in claim 1 or 23, is characterized in that, the crystal is in a single crystal form, which belongs to orthorhombic crystal system, with space group P2.sub.12.sub.12.sub.1, and unit-cell parameters are: a=11.7±0.2 Å, b=19.6±0.2 Å, c=23.4±0.2 Å, α=90°±0.2°, β=90°±0.2°, γ=90°±0.2°.
29. The crystal as claimed in claim 28, is characterized in that, the unit-cell parameters are: a=11.65-11.75 Å, b=19.50-19.60 Å, c=23.33-23.43 Å, α=89.9-90.1°, β=89.9-90.1°, γ=89.9-90.1°.
30. The crystal as claimed in claim 28 or 29, is characterized in that, a method for preparing the single crystal comprises: dissolving the compound of formula (1) in an alcohol solvent, filtering, volatilizing the solvent in a filtrate at room temperature to precipitate crystals, collecting the resulting crystals by filtration, and drying at room temperature to obtain.
31. A preparation method for the crystal as claimed in any one of claims 1-5, is characterized in that, the preparation method comprises: 1) adding the compound of formula (1) to one of or a mixture of more of an ester solvent, an alcohol solvent, and a ketone solvent to dissolve, then mixing with a hydrocarbon solvent, crystallizing, filtering, and drying to give a crystal form A of the compound of formula (1); or 2) adding the compound of formula (1) to a ketone solvent, volatilizing the solvent at room temperature to obtain a solid, and drying the resulting solid to give a crystal form A of the compound of formula (1); or 3) adding the compound of formula (1) to an ether solvent, stirring at room temperature, filtering to obtain a solid, and drying the resulting solid to give a crystal form A of the compound of formula (1); or 4) adding the compound of formula (1) to water, stirring at 45-55° C., filtering to obtain a solid, and drying the resulting solid to give a crystal form A of the compound of formula (1); or 5) adding the compound of formula (1) to a mixture solvent of a hydrocarbon solvent and an ether solvent, stirring at a set temperature for 1 to 3 h, then heating up or cooling at a rate of 0.1±0.05° C./min to circulate the temperature of the system between the set temperature and 5° C. for several times, and finally stirring at 3-7° C., filtering to obtain a solid, and drying the resulting solid to give a crystal form A of the compound of formula (1), the set temperature is 45 to 55° C.
32. A preparation method for the crystal as claimed in any one of claims 1 and 6-10, is characterized in that, the preparation method comprises: 1) adding the compound of formula (1) to an alcohol solvent or a ketone solvent or a mixture thereof to dissolve, mixing with water, crystallizing, filtering to obtain a solid, and drying the resulting solid to give a crystal form B of the compound of formula (1); or 2) adding the compound of formula (1) to a halohydrocarbon solvent, volatilizing the solvent at room temperature to obtain a solid, and drying the resulting solid to give a crystal form B of the compound of formula (1); or 3) adding the compound of formula (1) to a hydrocarbon solvent, stirring at room temperature, filtering to obtain a solid, and drying the resulting solid to give a crystal form B of the compound of formula (1); or 4) adding the compound of formula (1) to 2-methyltetrahydrofuran, stirring at a first set temperature, filtering and collecting a supernatant, and cooling the supernatant from a first set temperature to a second set temperature at a rate of 0.1±0.05° C./min and maintaining at the second set temperature, collecting a precipitated solid, and drying the resulting solid to give a crystal form B of the compound of formula (1), the first set temperature is 45 to 55° C., and the second set temperature is 0 to 10° C.
33. A preparation method for the crystal as claimed in any one of claims 1 and 11-15, is characterized in that, the method comprises: adding the compound of formula (1) to a mixture system of tetrahydrofuran and a hydrocarbon solvent to form a turbid solution, stirring at a first set temperature, filtering and collecting a supernatant, and cooling the supernatant from a first set temperature to a second set temperature at a rate of 0.1±0.05° C./min and maintaining at the second set temperature, collecting a precipitated solid, and drying the resulting solid to give a crystal form C of the compound of formula (1), the first set temperature is 45 to 55° C., and the second set temperature is 0 to 10° C.
34. A preparation method for the crystal as claimed in any one of claims 1 and 16-19, is characterized in that, the method comprises: adding the compound of formula (1) to a mixture of N-methyl-2-pyrrolidone and water to form a turbid solution, stirring at 45-55° C., filtering to obtain a solid, and drying the resulting solid to give a crystal form D of the compound of formula (1).
35. A preparation method for the crystal as claimed in any one of claims 1 and 20-22, is characterized in that, the method comprises: dissolving the compound of formula (1) in N,N-dimethylacetamide, obtaining a solid by gas-liquid diffusion in an atmosphere of water or an alcohol solvent, and filtering to give a crystal form E of the compound of formula (1).
36. A preparation method for the crystal as claimed in any one of claims 1 and 23-27, is characterized in that, the method comprises: adding the compound of formula (1) to an ester solvent or an alcohol solvent or a mixture thereof to form a turbid solution, stirring at 0-10° C., filtering to obtain a solid, and drying the resulting solid to give a crystal form F of the compound of formula (1).
37. The preparation method as claimed in claim 36, is characterized in that, the ester solvent is ethyl acetate; and/or, the alcohol solvent is methanol and/or ethanol; and/or, stirring time is 2-4 h.
38. The preparation method as claimed in claim 36 or 37, is characterized in that, before stirring the turbid solution at 0-10° C., heating the turbid solution to 30-60° C., and stirring at this temperature for more than 5 min.
39. A pharmaceutical composition containing the crystal as claimed in any one of claims 1-30 and a pharmaceutically acceptable carrier.
40. Use of the crystal as claimed in any one of claims 1-30 in the preparation of anti-influenza virus drugs.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0090] The “room temperature” mentioned in the present application refers to the temperature of the natural environment that can be reached without additional heating or cooling, and the corresponding specific temperature may be between 10-30° C.
[0091] In the present application, the test instruments and conditions used in the experiment are as follows:
1. Single crystal X-ray diffraction
Instrument model: Bruker D8 Venture single-crystal diffractometer
Light source: Mo target
X-ray: Mo-K (=0.71073 Å)
[0092] Detector: CMOS based detector
Resolution: 0.84 Å
[0093] Current and voltage: 50 kV, 1.4 A
2. X-ray powder diffraction (XRPD)
Instrument model: PANalytical X'Pert3 Power
X-ray: Cu, Kα, Kα1(Å): 1.540598; Kα2(Å): 1.544426
[0094] X-ray tube setting: 45 kV, 40 mA
Divergence slit: ⅛°
Scan pattern: continuous
Scanning range (2θ): 3°-40°
Scan step size (2θ): 0.0263°
3. Differential Scanning calorimetry (DSC)
Instrument model: TA Q2000/2500 differential scanning calorimeter
Heating rate: 10° C./min
Temperature range: 25° C. to setting end-point temperature
Shielding gas: nitrogen
4. Thermogravimetric analysis (TGA)
Instrument model: TA Q5000/5500 thermogravimetric analyzer
Heating rate: 10° C./min
Temperature range: room temperature to setting end-point temperature
Shielding gas: nitrogen
5. Dynamic vapour sorption (DVS)
Instrument model: SMS DVS Intrinsic vapour sorption analyzer
Temperature: 25° C.
[0095] RH range: 0% RH-95% RH
Shielding gas: nitrogen
[0096] The application will be further explained below in conjunction with specific embodiments.
Embodiment 1: Preparation of the Compound of Formula (1)
[0097] The Route is as Follows:
##STR00003## ##STR00004## ##STR00005##
[0098] Preparation of Compound of formula (10): Compound of formula (11) (388 mg, 1 mmol) was dissolved in 3 mL dichloromethane and added with 1 mL trifluoroacetic acid, and the mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete, and the mixture was added with 3N sodium hydroxide solution to adjust pH=9-10. The mixture was extracted with dichloromethane, and organic phases were merged, washed with saturated salt solution, dried, and concentrated to give 270 mg solid, which was directly used in the next step.
[0099] Preparation of Compound of formula (8): Compound of formula (9) (1.0 g, 7.8 mmol) was dissolved in 10 mL anhydrous tetrahydrofuran, and dropwise added with 2.5M n-butyllithium solution (3.1 mL, 7.8 mmol) slowly at −78° C. and under nitrogen protection. After addition, the mixture was stirred and reacted at the temperature for 2 hours. Then allyl formate (0.94 g, 7.8 mmol) was dropwise added. After addition, the mixture was stirred and reacted for 2 hours, and TLC showed the raw materials were substantially completely reacted, and then the reaction mixture was poured into saturated ammonium chloride solution to quench, and then extracted with ethyl acetate (15 mL×3). The organic phases were merged, dried over anhydrous sodium sulfate, and concentrated to dry to give 1.65 g oily product.
[0100] Preparation of Compound of formula (7): Compound of formula (8) (1.65 g, 7.8 mmol) was dissolved in 15 mL anhydrous tetrahydrofuran, and dropwise added with 1M diisobutylaluminum hydride solution (11.7 mL, 11.7 mmol) slowly at −78° C. and under nitrogen protection. After addition, the mixture was stirred and reacted at the temperature for 2 hours. TLC showed the raw materials were substantially completely reacted, and then the reaction mixture was poured into saturated potassium sodium tartrate solution to quench, and then extracted with ethyl acetate (20 mL×3). The organic phases were merged, dried over anhydrous sodium sulfate, and concentrated to dry to give 1.57 g oily product.
[0101] Preparation of Compound of formula (6): Compound of formula (7) (1.57 g, 7.4 mmol) was dissolved in 15 mL methanol, and p-toluenesulfonic acid monohydrate (140 mg, 0.74 mmol) was added, and the mixture was stirred at room temperature overnight. TLC showed the raw materials were substantially completely reacted, and then the mixture was added with saturated sodium bicarbonate solution to adjust to be neutral, and concentrated. The residue was separated by column chromatography to give 0.86 g yellow oily product.
[0102] Preparation of Compound of formula (5): Compound of formula (10) (270 mg, 0.94 mmol) and Compound of formula (6) (255 mg, 1.13 mmol) were dissolved in 5 mL acetonitrile. 1M solution of tin tetrachloride in dichloromethane (1.4 mL, 1.41 mmol) was added dropwise under nitrogen protection and at −20° C. After addition, the mixture was stirred and reacted at the temperature for 3 hours. The mixture was added with saturated sodium bicarbonate solution, stirred for 30 min, and stood to layer, and the aqueous phase was extracted with dichloromethane. The organic phases were merged, washed with saturated salt solution, dried, and concentrated to give 428 mg crude product.
[0103] Preparation of Compound of formula (4): Compound of formula (5) (428 mg, 0.89 mmol) was dissolved in 5 mL tetrahydrofuran, and tetrakis(triphenylphosphine)palladium (104 mg, 0.09 mmol) and morpholine (774 mg, 8.9 mmol) were added and reacted at room temperature for 2 hours. The reaction was shown complete through TLC spot plate analysis. The mixture was concentrated, and the residue was separated by column chromatography to give 216 mg product.
[0104] Preparation of Compound of formula (3): Compound of formula (4) (216 mg, 0.61 mmol) and Compound of formula (12) (242 mg, 0.92 mmol) were reacted at 100° C. for 3 hours in 3 mL solution of T.sub.3P in ethyl acetate under airtight condition. The mixture was cooled, diluted with saturated NaHCO.sub.3, and then extracted with ethyl acetate. The organic phases were merged, dried and concentrated, and separated by column chromatography to give 200 mg crude product, which was separated by chiral column to give 40 mg product.
[0105] Preparation of Compound of formula (2): Compound of formula (3) (40 mg, 0.067 mmol) and lithium chloride (20 mg, 0.48 mmol) were reacted at 100° C. for 3 hours in 1 mL DMA. After the reaction finished, the mixture was diluted with 10 mL water, and added with 2N hydrochloric acid to adjust pH to 3-4. The mixture was filtered, and the solid was pumped to dry to give 25 mg product. .sup.1HNMR (400 MHz, CDCl.sub.3) δ: 7.05-7.15 (m, 5H), 6.85 (m, 1H), 6.70 (d, 1H, J=7.6 Hz), 5.78 (d, 1H, J=7.6 Hz), 5.3 (m, 2H), 4.69 (d, 1H, J=6.8 Hz), 4.17 (d, 1H, J=14 Hz), 4.09 (d, 1H, J=14 Hz), 3.90 (m, 1H), 3.69 (m, 1H), 3.44 (d, 1H, J=15.2 Hz), 0.95 (m, 1H), 0.74 (m, 3H); ESI-MS m/z (M+H).sup.+ 510.1.
[0106] Preparation of Compound of formula (1): Compound of formula (2) (40 mg, 0.08 mmol), chloromethyl methyl carbonate (25 mg, 0.2 mmol), potassium carbonate (28 mg, 0.2 mmol) and potassium iodide (3 mg, 0.02 mmol) were reacted at 60° C. for 5 hours in 1 mL N,N-dimethylacetamide. The reaction was shown complete through TLC spot plate analysis, the mixture was added with water to quench, and added with 1N diluted hydrochloric acid to adjust pH to 3-4. The solid was filtered, dried, and separated by column chromatography to give 35 mg product, which was the compound of formula (1), and the solid was analyzed to be amorphous and used as the starting material for preparing various crystal forms in subsequent embodiments. .sup.1HNMR (400 MHz, DMSO-d6) δ: 7.40-7.42 (m, 2H), 7.25 (d, 1H, J=7.6 Hz), 7.15 (m, 1H), 7.10 (m, 1H), 7.00 (d, 1H, J=7.2 Hz), 6.84 (t, 1H, J=7.6 Hz), 5.75 (m, 4H), 5.43 (d, 1H, J=16.4 Hz), 4.57 (dd, 1H, J=3.2, 9.6 Hz), 3.96-4.03 (m, 3H), 3.73 (s, 3H), 3.51 (t, 1H, J=10.0 Hz), 3.41 (s, 1H), 0.75 (t, 2H, J=8.4 Hz), 0.50 (m, 2H); ESI-MS m/z (M+H).sup.+ 598.1.
Embodiment 2: Pharmacodynamic Experiment of the Compound of Formula (1)
[0107] 2.1 In Vitro Bioactivity and Cytotoxicity Study
[0108] In actual drug applications, the compound of formula (1) is a prodrug of the compound of formula (2), and is converted into an active drug (the compound of formula (2)) in the body to exert its efficacy. For the activity data and toxicity data of the compound of formula (2), see Table a.
[0109] Test method for In vitro bioactivity study: MDCK cells were seeded into 384-well cell culture plate at a density of 2,000 cells/well, and then incubated at 37° C. overnight in a 5% CO.sub.2 incubator. On the following day, the compounds were diluted and added into the wells (3-fold multiple proportion dilution, 8 test concentration points), and the influenza virus A/PR/8/34 (H1N1) strain was then added to the cell culture wells at 2*TCID90 per well, and the final concentration of DMSO in the medium was 0.5%. The cell plate was incubated at 37° C. for 5 days in the 5% CO.sub.2 incubator. After 5 days of culture, the cell viability was measured using the cell viability detection kit CCK8. The raw data were subjected to nonlinear fitting analysis of the inhibition rate and cytotoxicity of the compounds using GraphPad Prism software to obtain EC.sub.50 values (see Table a for the results).
[0110] Method for cytotoxicity study: the cytotoxicity assay and antiviral activity assay of the compounds were performed in parallel, except for the absence of virus, other experimental conditions were consistent with the antiviral activity assay. After 5 days of culture, the cell viability was measured using the cell viability detection kit CCK8. Raw data were used for calculating compound cytotoxicity (CC.sub.50) (see Table a for results).
TABLE-US-00001 TABLE a Inhibitory activity and toxicity of the compounds against influenza virus A/PR/8/34 (H1N1) Results (nM) EC.sub.50 CC.sub.50 Compound of formula (2) 0.26 >1000 Comparative compound 1.4 >1000 Baloxavir acid
[0111] The results showed that when compared with the comparative compound, the compound of the present application has more excellent activity against H1N1 and has low cytotoxicity.
[0112] 2.2 Rat PK Study
[0113] Intravenous injection: about 2 mg of samples, namely the compound of formula (2) was accurately weighed, appropriate amount of DMA was added, and the system was vortex oscillated to completely dissolve the solid matter; an appropriate volume of 30% Solutol HS-15 aqueous solution was added, and the system was vortex oscillated and then saline was added such that DMA: 30% Solutol HS-15: saline=20:20:60 (v/v/v), the liquid was vortex oscillated to mix evenly, and filtered to give a pharmaceutical preparation of a concentration of 0.05 mgmL.sup.−1. SD rats were given a single intravenous injection of 0.25 mgmL.sup.−1 of the compound of formula (2) intravenously. 0.20 mL of blood was collected from the jugular vein before administration and 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after administration, and placed in an EDTA-K2 anticoagulation tube. 150 μL of whole blood was accurately aspirated immediately, and added into a test tube to which 450 μL of acetonitrile has been added to precipitate proteins, and the tube was vortex oscillated, and placed on wet ice. It was stored in a −90˜−60° C. refrigerator for biological sample analysis. The concentration of the corresponding compound in the plasma of S-D rats was determined by LC-MS/MS analysis. The corresponding pharmacokinetic parameters were calculated using a non-compartmental model in Pharsight Phoenix 7.0. See Table b for the results.
[0114] Gavage administration: about 4 mg of sample, the compound of formula (1) was accurately weighed, appropriate amount of PEG400 was added, and the system was vortex oscillated to completely dissolve the solid matter; an appropriate volume of 30% Solutol HS-15 aqueous solution was added, the system was vortex oscillated and then saline was added such that PEG400: 30% Solutol HS-15: saline=2:2:6 (v/v/v), the liquid was vortex oscillated to mix evenly, to give a pharmaceutical preparation of a concentration of 0.3 mgmL.sup.−1. SD rats were given a single oral administration of 3.0 mgmL.sup.−1 of the compound of formula (1) by gavage, and the concentration of the compound of formula (2) in the plasma of S-D rats was measured before administration and 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after administration. See Table c for the results.
TABLE-US-00002 TABLE b PK Parameters (Intravenous Injection) of the Compound of formula (2) PK (i.v.) Compound of formula (2) T.sub.1/2 (h) 2.97 AUC.sub.0-t (ng .Math. h .Math. mL.sup.−1) 276 CL (mL .Math. kg.sup.−1 .Math. min.sup.−1) 13.6 Vd.sub.ss (L .Math. kg.sup.−1) 3.12
TABLE-US-00003 TABLE c PK Parameters (Gavage) of the Tested Compound of formula (1) PK (i.g.) Compound of formula (1) T.sub.1/2 (h) 3.32 T.sub.max (h) 1.67 C.sub.max (ng .Math. mL.sup.−1) 253 AUC.sub.0-t (ng .Math. h .Math. mL.sup.−1) 1377 F (%) 47.2
[0115] The above results indicate that the compound of formula (1) of the present application have a low in vivo clearance rate, has a long half-life, high bioavailability, and has high absorption in the body.
[0116] 2.3 Efficacy on Mice
[0117] Female BALB/c mice were inoculated with influenza A virus (H1N1, A/WSN/33) by intranasal administration to establish an IAV mouse infection model. Menstruum, the compound of formula (1) (15 mpk) or oseltamivir phosphate (15 mpk) were orally administered twice a day. Animal weight and survival status were monitored daily during the test, and on the 5th day, some animals were sacrificed to take lung tissue for virus titer detection, and the remaining mice were used for survival rate monitoring. The in vivo anti-influenza virus efficacy of the test compound was determined by virus titer in lung tissue, mouse body weight change and survival rate. Virus titer in lung tissue: on the 5th day after virus infection, the average virus titer in the lung tissue of mice in the menstruum group reached 7.20 Log 10 (number of plaques per gram of lung tissue), the average virus titer in the lung tissue of mice in the oseltamivir phosphate group was 3.74 Log 10 (number of plaques per gram of lung tissue). Compared with the menstruum group, oseltamivir phosphate significantly inhibited the replication of the virus in mice, and the average virus titer decreased by 3.46 Log 10 (number of plaques per gram of lung tissue), and the difference was very statistically significant (p<0.01) between the results, showing the expected efficacy; the average virus titer in the lung tissue of mice on the 5th day after treatment with test compound of formula (1) was 3.28 Log 10 (number of plaques per gram of lung tissue)), and compared with the menstruum group, the test compound significantly inhibited the replication of the virus in mice, and the average virus titer decreased by 3.92 Log 10 (number of plaques per gram of lung tissue), and the difference was extremely statistically significant (p<0.001) between the results, which is superior to the control compound oseltamivir phosphate (Table d).
TABLE-US-00004 TABLE d Virus Titer in Lung Tissue Statistical analysis Influenza virus titer (Compared with the Log10 (plaques menstruum group) number/gram Mean Statistic Group of lung) difference difference Menstruum 7.20 ± 0.1024 NA NA Oseltamivir 3.74 ± 0.5205 3.46 **(p < 0.01) phosphate Compound of 3.28 ± 0.2813 3.92 ***(p < 0.001) formula (1) **P < 0.01 means very significant difference, ***P < 0.001 means extremely significant difference
[0118] Body weight change and result analysis: the mice in the menstruum group showed significant weight loss on the 3rd day after infection, and then continued to decline or even die; the weight of the mice in the oseltamivir phosphate group and the compound of formula (1) group remained stable during the experiment, had no significant decline, and the mice were in good health.
[0119] Survival rate and result analysis: the mice in the menstruum group were found dead on the 7th day after infection, and on the 10th day, all mice died or were euthanized due to weight loss to the humane end point, and the survival rate was 0%; the mice in the oseltamivir phosphate group and in the compound of formula (1) group maintained healthy during the experiment, and all animals survived to the predetermined experimental end point with a survival rate of 100%, indicating excellent anti-influenza efficacy in vivo.
Embodiment 3: Preparation of the Crystal Form a of the Compound of Formula (1)
[0120] Method 1: 15 mg of the compound of formula (1) was weighted, and completely dissolved in 1 mL of isopropyl acetate. 4 mL of n-heptane was slowly added to the clear solution while stirring, and a solid was precipitated. The system was stirred for 1 h and filtered, and the solid was forced-air dried at 50° C. for 4 h, to give 13.6 mg product, with a yield of 90.7% and an HPLC purity of 99.3%.
[0121] Method 2: 15 mg of the compound of formula (1) was weighted, and completely dissolved in 1 mL of ethyl acetate. To a 20 mL bottle, 4 mL of n-heptane was added, and the solution of the compound in ethyl acetate was slowly added into the n-heptane while stirring, and a solid was precipitated. The system was stirred for 1 h and filtered, and the solid was forced-air dried at 50° C. for 4 h, to give 14.0 mg product, with a yield of 93.3% and an HPLC purity of 99.1%.
[0122] Method 3: 15 mg of the compound of formula (1) was weighted, and completely dissolved in 1 mL of acetone, then the solvent was volatilized at room temperature, and the solid was collected and forced-air dried at 50° C. for 4 h.
[0123] Method 4: 15 mg of the compound of formula (1) was weighted, and 0.5 mL of methyl tert-butyl ether was added, then the resulting turbid solution was magnetically stirred at room temperature for 3 days, and centrifuged to collect the solid, and the solid was forced-air dried at 50° C. for 4 h.
[0124] Method 5: 15 mg of the compound of formula (1) was weighted, and 0.5 mL of water was added, then the resulting turbid solution was magnetically stirred at 50° C. for 3 days, and centrifuged to collect the solid, and the solid was forced-air dried at 50° C. for 4 h.
[0125] Method 6: 15 mg of the compound of formula (1) was weighted, and 0.1 mL of methylbenzene and 0.4 mL of methyl tert-butyl ether were added, then the resulting turbid solution was magnetically stirred at 50° C. for 2 h, and the sample was place in a biochemical incubator, the temperature was increased and decreased at a rate of 0.1° C./min, and 50-5° C. temperature rise-fall cycle test was performed for three times. The sample was finally stirred at 5° C., and centrifuged to collect the solid, and the solid was forced-air dried at 50° C. for 4 h. XRPD test was performed on the solid obtained in Method 1, and the pattern is shown in
[0126] The results of TGA (
TABLE-US-00005 TABLE 1 XRPD pattern details of the crystal form A of the compound of formula (1) Position [2θ (°)] D spacing [Å] Relative intensity [%] 3.10 28.50 8.72 8.74 10.12 100.00 13.08 6.77 7.10 14.20 6.24 3.17 15.44 5.74 9.68 16.03 5.53 4.68 17.50 5.07 5.34 19.20 4.62 3.75 19.58 4.53 4.61 21.91 4.06 28.01 22.89 3.89 2.91 26.35 3.38 7.05 30.83 2.90 6.21
[0127] XRPD tests were performed on the solids obtained by other methods, and the test patterns were substantially the same as depicted in
Embodiment 4: Preparation of the Crystal Form B of the Compound of Formula (1)
[0128] Method 1: 15 mg of the compound of formula (1) was weighted, and completely dissolved in 1 mL of methanol. 4 mL of water was slowly added to the clear solution while stirring, and a solid was precipitated. The system was stirred for 1 h and filtered, and the solid was forced-air dried at 50° C. for 4 h, to give 14.1 mg product, with a yield of 94% and an HPLC purity of 99.2%. Method 2: 15 mg of the compound of formula (1) was weighted, and completely dissolved in 1 mL of acetone. To a 20 mL bottle, 4 mL of water was added, and the solution of the compound in acetone was slowly added into the water while stirring, and a solid was precipitated. The system was stirred for 1 h and filtered, and the solid was forced-air dried at 50° C. for 4 h, to give 13.8 mg product, with a yield of 92% and an HPLC purity of 99.2%.
[0129] Method 3: 15 mg of the compound of formula (1) was weighted, and completely dissolved in 1 mL of chloroform, then the solvent was volatilized at room temperature, and the solid was collected and forced-air dried at 50° C. for 4 h.
[0130] Method 4: 15 mg of the compound of formula (1) was weighted, and 0.5 mL of methylbenzene was added, then the resulting turbid solution was magnetically stirred at room temperature for 3 days, and centrifuged to collect the solid, and the solid was forced-air dried at 50° C. for 4 h. Method 5: 15 mg of the compound of formula (1) was weighted, and 0.5 mL of 2-methyltetrahydrofuran was added, then the resulting turbid solution was stirred at 50° C. for 2 h, and filtered to collect the supernatant. The supernatant was cooled from 50° C. to 5° C. at a rate of 0.1° C./min, and maintained at 5° C. The precipitated solid was collected, and forced-air dried at 50° C. for 4 h.
[0131] XRPD test was performed on the solid obtained in Method 1, and the pattern is shown in
[0132] The results of TGA (
TABLE-US-00006 TABLE 2 XRPD pattern details of the crystal form B of the compound of formula (1) Position [2θ (°)] D spacing [Å] Relative intensity [%] 8.42 10.50 100.00 10.75 8.23 27.14 14.27 6.21 36.91 16.04 5.53 44.93 16.87 5.25 26.64 18.21 4.87 18.81 18.78 4.72 13.79 19.26 4.61 11.00 19.60 4.53 22.26 19.92 4.46 5.65 20.40 4.35 15.37 21.39 4.15 25.90 21.66 4.10 19.03 23.38 3.81 26.50 23.67 3.76 7.85 24.91 3.58 9.49 25.41 3.50 43.22 27.32 3.26 13.04 28.01 3.19 4.24 29.17 3.06 11.55 31.35 2.85 4.60 34.08 2.63 11.15
[0133] XRPD tests were performed on the solids obtained by other methods, and the test patterns were substantially the same as depicted in
Embodiment 5: Preparation of the Crystal Form C of the Compound of Formula (1)
[0134] Method: 15 mg of the compound of formula (1) was weighted, and 0.25 mL of tetrahydrofuran and 0.25 mL of n-heptane were added, then the resulting turbid solution was stirred at 50° C. for 2 h, and filtered to collect the supernatant. The supernatant was cooled from 50° C. to 5° C. at a rate of 0.1° C./min, and maintained at 5° C. The precipitated solid was collected, and forced-air dried at 50° C. for 4 h.
[0135] XRPD test was performed on the solid obtained in the above-mentioned method, and the pattern is shown in
[0136] The results of TGA (
TABLE-US-00007 TABLE 3 XRPD pattern details of the crystal form C of the compound of formula (1) Position [2θ (°)] D spacing [Å] Relative intensity [%] 7.73 11.43 100.00 10.61 8.34 7.14 12.51 7.08 24.74 13.13 6.74 40.04 13.65 6.49 47.88 14.76 6.00 29.00 15.21 5.83 28.24 17.13 5.18 47.51 18.39 4.82 21.94 20.08 4.42 52.03 20.98 4.23 46.41 21.74 4.09 70.10 22.72 3.91 16.41 23.22 3.83 30.03 24.13 3.69 24.24 25.27 3.52 8.23 25.57 3.48 12.66 26.55 3.36 12.88 29.08 3.07 5.05 31.19 2.87 11.46
Embodiment 6: Preparation of the Crystal Form D of the Compound of Formula (1)
[0137] Method: 15 mg of the compound of formula (1) was weighted, and 0.05 mL of N-methyl-2-pyrrolidone and 0.45 mL of water were added, then the resulting turbid solution was stirred at 50° C. for 3 days, and centrifuged to collect the solid, and the solid was forced-air dried at 50° C. for 4 h.
[0138] XRPD test was performed on the solid obtained in the above-mentioned method, and the pattern is shown in
[0139] The results of TGA (
TABLE-US-00008 TABLE 4 XRPD pattern details of the crystal form D of the compound of formula (1) Position [2θ (°)] D spacing [Å] Relative intensity [%] 7.94 11.13 100.00 10.37 8.53 3.59 12.44 7.11 5.58 13.06 6.78 9.84 13.63 6.50 2.97 15.09 5.87 29.92 15.50 5.72 17.90 17.45 5.08 7.18 19.23 4.62 9.53 19.63 4.52 10.37 20.88 4.25 7.33 22.16 4.01 46.64 23.56 3.78 19.29 23.96 3.71 9.93 25.86 3.45 13.02 28.03 3.18 21.48 30.75 2.91 2.46
Embodiment 7: Preparation of the Crystal Form E of the Compound of Formula (1)
[0140] Method: 15 mg of the compound of formula (1) was weighted, and 0.5 mL of N,N-dimethylacetamide was added to dissolve it, the bottle containing the resulting solution was placed in a 20 mL bottle with 4 mL of water, then the 20 mL bottle was sealed and stood at room temperature for 7 days, to give 14.3 mg while solid, with a yield of 95.2% and an HPLC purity of 99.2%.
[0141] XRPD test was performed on the solid obtained in the above-mentioned method, and the pattern is shown in
TABLE-US-00009 TABLE 5 XRPD pattern details of the crystal form E of the compound of formula (1) Position [2θ (°)] D spacing [Å] Relative intensity [%] 4.44 19.90 9.67 5.87 15.06 7.32 8.01 11.03 40.52 8.78 10.08 100.00 17.56 5.05 10.44 19.64 4.52 10.13 21.95 4.05 30.07 22.25 4.00 10.80 26.33 3.39 20.19 28.15 3.17 8.35 29.07 3.07 7.12 30.86 2.90 9.45
Embodiment 8: Preparation of the Crystal Form F of the Compound of Formula (1)
[0142] Method 1: 33 g of the compound of formula (1) was weighted, and 40 mL of methanol was added, then the solution was stirred at 0-10° C. for 3 h, and filtered. The solid was forced-air dried at 50° C. for 8 h, to give 31.88 g white solid, with a yield of 96.6% and an HPLC purity of 99.4%.
[0143] Method 2: 33 g of the compound of formula (1) was weighted, and 40 mL of ethanol was added, then the solution was stirred at 0-10° C. for 3 h, and filtered. The solid was forced-air dried at 50° C. for 8 h, to give 30.76 g white solid, with a yield of 93.2% and an HPLC purity of 99.5%. Method 3: 33 g of the compound of formula (1) was weighted, and 40 mL of ethyl acetate was added, then the solution was stirred at 0-10° C. for 3 h, and filtered. The solid was forced-air dried at 50° C. for 8 h, to give 31.24 g white solid, with a yield of 94.7% and an HPLC purity of 99.5%.
[0144] XRPD test was performed on the solid obtained in Method 1, and the pattern is shown in
[0145] The results of TGA (
TABLE-US-00010 TABLE 6 XRPD pattern details of the crystal form F of the compound of formula (1) Position [2θ (°)] D spacing [Å] Relative intensity [%] 5.77 15.32 41.05 8.69 10.18 26.61 11.61 7.62 33.54 11.98 7.39 8.38 13.84 6.40 8.10 14.30 6.19 8.45 15.37 5.76 3.55 16.14 5.49 4.14 16.64 5.33 4.41 17.48 5.07 35.95 18.33 4.84 7.10 18.95 4.68 8.63 19.47 4.56 17.70 20.22 4.39 2.89 20.69 4.29 5.94 21.16 4.20 3.49 21.47 4.14 6.97 21.64 4.11 5.41 22.84 3.89 100.00 24.06 3.70 4.29 25.31 3.52 4.35
[0146] XRPD tests were performed on the solids obtained by other methods, and the test patterns were substantially the same as depicted in
[0147] The single crystal of the crystal form F of the compound of formula (1) may be prepared by the following method: dissolving the compound of formula (1) in methanol, filtering, volatilizing methanol in the filtrate at room temperature to precipitate crystals, collecting the resulting crystals by filtration, and airing at room temperature to obtain it.
[0148] The obtained single crystal was subjected to single crystal X-ray diffraction data collection and the single crystal structure was analyzed. See Table 7 for the single crystal structure data of the compound of formula (1). The single crystal structure analysis determined the absolute configuration of the chiral center of the compound of formula (1). As shown in
TABLE-US-00011 TABLE 7 single crystal structure data of the crystal form F of the compound of formula (1) Empirical formula C.sub.29H.sub.25F.sub.2N.sub.3O.sub.7S Formula weight 597.58 Temperature 100K Wavelength Mo K□ radiation, □ = 0.71073 Å Crystal system, space group orthorhombic crystal system, P2.sub.12.sub.12.sub.1 Unit cell dimensions a = 11.7243 (3) Å, b = 19.5693 (6) Å, c = 23.3801 (7) Å, α = β = γ = 90° Volume 5364.2 (3) Å.sup.3 Z, Calculated density 8, 1.480 g/cm.sup.3 Absorption coefficient 0.189 mm.sup.−1 F(000) 2480 Crystal size 0.19 × 0.12 × 0.08 mm
Embodiment 9: Evaluation of the Solid State Stability of the Crystal Form
[0149] The crystal form F samples of the compound of formula (1) were placed at 60° C. for 30 days, at 25° C./92.5% RH for 30 days, and exposed to light for 15 days, respectively. The physical and chemical stability of the samples were tested by XRPD and HPLC. The results show that under the various stability test conditions, the crystal form F samples of the compound of formula (1) have not undergone crystal form transformation and HPLC purity reduction, indicating that it has good physical and chemical stability under the test conditions (Table 8).
TABLE-US-00012 TABLE 8 Solid state stability of the crystal form F of the compound of formula (1) Purity Purity/ Crystal form Appearance Conditions (area %) initial (%) change and character Initial 98.6 — No White powder 60° C./ 98.7 100.1 No White 30 days powder 25° C./ 99.0 100.4 No White 92.5% RH/ powder 30 days Light*/ 98.5 99.9 No White 15 days powder *White light: 4500 Lux, ultraviolet light: 90 μw/cm.sup.2
Embodiment 10: Evaluation of Hygroscopicity of the Crystal Forms
[0150] The hygroscopicity of the crystal form F sample of the compound of formula (1) was tested by dynamic vapour sorption (DVS) test at 25° C., and the result showed that the moisture-absorption weight gain of the sample under the condition of 25° C./80% RH was 0.10% (
[0151] The stability and hygroscopicity of other crystal forms have also been studied, and the results show that they all meet the requirements for medicinal use, wherein, the crystal form F of the compound of formula (1) is the most stable and has the best hygroscopicity.
Embodiment 11: Large-Scale Production of the Crystal Form F of the Compound of Formula (1)
[0152] 243.31 g of the compound of formula (2), 89.90 g of chloromethyl dimethyl carbonate, 133.20 g of potassium carbonate and 79.19 g of potassium iodide were added to 1215 mL of N,N-dimethylacetamide in a 2 L three-neck flask, and the system was heated to 45 to 55° C. to react for 5 h. HPLC monitored the reaction being complete. The system was filtered while hot, the solid was rinsed with 1215 mL of N,N-dimethylacetamide, the filtrate was merged, cooled and maintained at 0 to 10° C. Then 240 mL of 1N hydrochloric acid solution was added to adjust the pH to 1 to 2, then 4860 mL of water was added dropwise, and the system was stirred for 0.5 h and then filtered, the filter cake was rinsed with 1 L of water and suction filtered to dryness to give a crude product of the compound of formula (1). The crude product was dissolved in 3600 mL of dichloromethane, the aqueous layer was separated, the aqueous layer was extracted with 480 mL of dichloromethane, the organic layers were merged, washed with 1200 mL of water, vacuum concentrated to dryness at 40 to 50° C., and 1215 mL of acetone and 500 mL of methanol were added, then the system was heated to 50 to 60° C. to reflux, 480 mL of acetone was added to dissolve it, the system was vacuum concentrated to dryness at 40 to 50° C., and 730 mL of methanol was added, the system was heated to 50 to 60° C. and stirred for 1 h, then cooled to 0 to 10° C. to crystallize for 1 h, filtered, and the filter cake was rinses with 310 mL of cold methanol, and forced-air dried at 50° C. for 8 h to give 237.18 g of the compound of formula (1). It is the crystal form F determined by XRPD, and the pattern is shown in
TABLE-US-00013 TABLE 9 XRPD pattern details of the crystal form F obtain in the large-scale production Position [2θ (°)] D spacing [Å] Relative intensity [%] 5.83 15.16 60.14 8.75 10.11 100.00 11.70 7.57 68.81 12.03 7.36 27.35 13.88 6.38 29.27 14.31 6.19 22.08 15.45 5.74 11.60 16.20 5.47 34.26 16.71 5.31 19.89 17.54 5.06 44.66 18.00 4.93 10.00 18.43 4.81 81.67 19.03 4.66 50.91 19.56 4.54 18.75 20.74 4.28 34.86 21.70 4.10 29.17 22.89 3.88 59.88 23.18 3.84 11.84 24.14 3.69 33.36 25.38 3.51 12.78 27.69 3.22 25.96 28.81 3.10 14.44
[0153] In addition, 224.90 g of the above compound of the formula (1) was jet milled (feed pressure 0.4 to 0.5 MPa, milling pressure 0.18 to 0.22 MPa) to give 212.00 g sample with a particle size of D90≤10 um. The milled sample was tested by XRPD, as shown in
TABLE-US-00014 TABLE 10 XRPD pattern details after milling Position [2θ (°)] D spacing [Å] Relative intensity [%] 5.84 15.14 25.37 8.75 10.11 100.00 11.70 7.56 30.63 12.03 7.36 14.15 13.90 6.37 30.36 14.28 6.20 13.17 15.47 5.73 9.20 15.92 5.57 16.53 16.19 5.48 27.80 16.73 5.30 16.21 17.52 5.06 14.00 17.94 4.94 9.48 18.43 4.81 27.35 19.04 4.66 40.97 19.55 4.54 12.76 20.67 4.30 19.44 21.70 4.09 26.34 22.89 3.89 31.87 23.19 3.84 12.56 24.13 3.69 20.40 25.39 3.51 8.15 25.73 3.46 9.24 27.70 3.22 15.63 28.80 3.10 11.19
[0154] The explanation on the above embodiments is only to help understanding of the method and its core concept of the present application. It should be noted that, for those ordinary skilled in the art, various improvements and modifications can be made without depart from the technical principle of the present application, and these improvements and modifications should be covered by the protective scope of the present application.