Crystal form of pyridone multi-ring derivative and preparation method thereof

Abstract

The invention discloses a crystal form of a pyridone multi-ring derivative and a preparation method of the crystal form, and particularly discloses a crystal form of a compound shown as a formula (I) and a preparation method of the crystal form. # imgabs0 #

Description

Crystal form of pyridone polycyclic derivatives and preparation method thereof

[0001] The present invention claims the following priority:

[0002] CN202210812490.X, application date: July 5, 2022. Technical Field

[0003] The present invention relates to a crystal form of a pyridone polycyclic derivative and a preparation method thereof. Background Art

[0004] Influenza virus (IFV) is a segmented, single-stranded, negative-sense RNA virus that causes the common cold in humans and animals. Influenza viruses can cause significant morbidity and mortality, and influenza A viruses, in particular, can cause global pandemics.

[0005] Influenza A virus is a single negative-strand RNA virus with a genome divided into 8 segments that encode 8 proteins. The 5' and 3' ends of the influenza virus genome segments are highly conserved, and the sequences of the two ends are complementary to form a handle-loop structure, which plays an important role in initiating viral RNA replication. The proteins encoded by each viral gene segment are of different sizes and play different roles in the life cycle of the influenza virus. The basic functions of several major proteins are first introduced below. The HA of the influenza virus is the ligand for the influenza virus to recognize the host receptor. It binds to the virus-specific receptor on the cell surface, mediates the fusion of the viral outer membrane with the intracellular small body membrane, and releases the viral nucleocapsid into the cytoplasm. The receptors of influenza viruses are specific, and the receptors of influenza A viruses are sialic acid glycoproteins. The NA protein of the influenza virus can remove sialic acid from the surface of the virus particles during the replication process, so that the virus particles cannot continue to aggregate on the surface of the host cell, thereby facilitating the release of virions and further infection of more host cells.

[0006] The role of the influenza virus's M2 protein: The influenza virus's HA protein binds to sialic acid, allowing the virus to be internalized by host cells. The pH within the phagocytic vesicle plays a crucial role in viral uncoating. The ion channel of the M2 protein on the viral membrane gradually lowers the pH of the phagocytic vesicle. When the pH drops to 5.0-6.0, the HA2 protein undergoes conformational changes, shifting the fusion peptide at its amino terminus. This activates the fusion process, leading to fusion of the viral bilayer with the cell membrane and the release of RNPs within the viral particle into the host cell cytoplasm. The M2 protein is a transmembrane ion channel found only in influenza A viruses, with a portion extending to the surface of the viral outer membrane.

[0007] Influenza virus protein synthesis also utilizes the host cell translation machinery; the virus can even pause host protein translation to accelerate its own protein synthesis. Polyadenylation of host cell mRNA is accomplished by specific adenylases. In contrast, the adenylate tail of viral mRNA is formed by transcribed 5-7 consecutive uracils from the negative-strand vRNA. Capping of each viral messenger RNA (mRNA) is accomplished in a similar manner: the PA and PB2 proteins grab the 5' capping primer of the host pre-mRNA transcript and initiate viral mRNA synthesis. This process, known as "cap snatching," is primarily accomplished by the viral RNA-dependent RNA polymerase (RdRp). Its PA subunit possesses RNA endonuclease activity, responsible for cleaving host mRNA. After completing the polyadenylation and capping processes, the viral mRNA exits the nucleus, enters the cytoplasm, and is translated like the host cell's mRNA. The nuclear export of viral vRNA fragments is mediated by the viral M1 protein and NS2 protein. When the M1 protein interacts with vRNA and NP protein, it also interacts with the nuclear export protein NS2. Thus, the nuclear export protein NS2 mediates the M1-RNP to exit the nucleus in the form of a nucleoprotein and enter the cytoplasm of the host cell.

[0008] Influenza incurs direct costs due to lost productivity and associated medical resources, as well as indirect costs associated with preventive measures. In the United States, influenza cumulatively causes approximately $10 billion in losses annually, and it is estimated that future influenza pandemics could incur hundreds of billions of dollars in direct and indirect costs. Prevention costs are also substantial. Governments worldwide have spent billions of dollars preparing and planning for a potential H5N1 avian influenza pandemic, with costs associated with purchasing drugs and vaccines, developing disaster drills, and improving border control strategies.

[0009] Current treatment options for influenza include vaccination and chemotherapy and chemoprevention with antiviral drugs. Flu vaccines against influenza are often recommended for high-risk groups, such as children and the elderly, or those with asthma, diabetes, or heart disease, but even vaccination does not completely protect against influenza. Vaccines are prepared for specific influenza strains each season, but it is impossible to cover the various strains actively infecting people worldwide during that season. In addition, because influenza viruses undergo a certain degree of antigenic drift, if more than one virus infects a single cell, the eight separate vRNA segments in the genome mix or reassort, and the resulting rapid changes in viral genetics can produce antigenic shifts and enable the virus to infect new host species and quickly overcome protective immunity.

[0010] Antiviral drugs can also be used to treat influenza. Neuraminidase inhibitors, such as oseltamivir (Tamiflu), are highly effective against influenza A viruses. However, clinical observations have revealed the emergence of virus strains resistant to these neuraminidase inhibitors. In the field of influenza virus treatment, there is an urgent need for antiviral drugs with novel mechanisms of action that can be used alone to treat influenza A, or in combination with other marketed antiviral drugs with other mechanisms of action for the prevention and treatment of influenza A.

[0011] Summary of the Invention

[0012] The present invention provides a crystal form A of a compound of formula (I),

[0013] The X-ray powder diffraction pattern of the A crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 10.68±0.20°, and 16.90±0.20°.

[0014] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 10.68±0.20°, 16.90±0.20°, 19.42±0.20°, and 20.72±0.20°.

[0015] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 16.90±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, and 29.12±0.20°.

[0016] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20°, 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, and 29.12±0.20°.

[0017] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20°, 14.52±0.20°, 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, 26.26±0.20°, 28.24±0.20°, 29.12±0.20°, and 29.98±0.20°.

[0018] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, and / or 11.80±0.20°, and / or 14.52±0.20°, and / or 16.56±0.20°, and / or 16.90±0.20°, and / or 17.84±0.20°, and / or 19. 42±0.20°, and / or 19.94±0.20°, and / or 20.30±0.20°, and / or 20.72±0.20°, and / or 22.66±0.20°, and / or 24.30±0.20°, and / or 26.26±0.20°, and / or 28.24±0.20°, and / or 29.12±0.20°, and / or 29.98±0.20°.

[0019] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56°, 9.08°, 10.68°, 11.80°, 13.94°, 14.52°, 14.92°, 15.28°, 15.74°, 16.56°, 16.90°, 17.84°, 18.42°, 19.42°, 19.94°, 20.3 0°, 20.72°, 21.12°, 22.66°, 23.80°, 24.30°, 24.68°, 25.46°, 25.80°, 26.26°, 26.86°, 28.24°, 29.12°, 29.98°, 30.88°, 31.94°, 33.60°, 35.00°, 37.88°, 38.48°.

[0020] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form A is shown in Figure 1.

[0021] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form A is basically as shown in Figure 1.

[0022] In some embodiments of the present invention, the peak positions and relative intensities of the diffraction peaks in the Cu Kα radiation XRPD pattern of the above-mentioned crystal form A are shown in Table 1:

[0023] Table 1 XRPD diffraction data of the crystal form A of compound of formula (I)

[0024] In some embodiments of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form A shows an onset point of an endothermic peak at 194.2°C±5°C and an onset point of an exothermic peak at 201.4°C±5°C.

[0025] In some embodiments of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form A shows an endothermic peak at 198.4°C±3°C.

[0026] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is shown in Figure 2.

[0027] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is basically as shown in Figure 2.

[0028] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystal form A shows a weight loss of 2.87% at 200.0°C±3°C.

[0029] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form A is shown in Figure 3.

[0030] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form A is basically as shown in Figure 3.

[0031] The present invention also provides a crystal form A of the compound of formula (I),

[0032] The preparation method comprises the following steps:

[0033] (a) adding compound Z to a mixed solvent of solvent X and solvent Y;

[0034] (b) stirring at 10-35° C. for 5-24 hours after the addition is complete;

[0035] (c) filtering, and vacuum drying the filter cake at 25-60° C. for 1-24 hours;

[0036] in,

[0037] The compound Z is selected from the group consisting of the compound of formula (I), the crystal form of compound B of formula (I), the crystal form of compound C of formula (I), the crystal form of compound D of formula (I) and the crystal form of compound E of formula (I); preferably, the compound Z is the crystal form of compound B of formula (I);

[0038] The solvent X is ethyl acetate;

[0039] The solvent Y is selected from n-heptane, n-hexane and cyclohexane; preferably, the solvent Y is n-heptane;

[0040] The volume ratio of the solvent X to the solvent Y is 1:1 to 1:6; preferably, the volume ratio of the solvent X to the solvent Y is 1:3.

[0041] The present invention also provides a method for preparing crystal form A of the compound of formula (I), comprising the following steps:

[0042] (a) adding compound Z to a mixed solvent of solvent X and solvent Y;

[0043] (b) stirring at 10-35° C. for 5-24 hours after the addition is complete;

[0044] (c) filtering, and vacuum drying the filter cake at 25-60° C. for 1-24 hours;

[0045] in,

[0046] Compound Z is a crystalline form of compound B of formula (I);

[0047] Solvent X is ethyl acetate;

[0048] Solvent Y is selected from n-heptane, n-hexane and cyclohexane;

[0049] The volume ratio of solvent X to solvent Y is 1:1 to 1:6.

[0050] In some embodiments of the present invention, the compound Z is a compound of formula (I).

[0051] In some embodiments of the present invention, the compound Z is the crystal form B of the compound of formula (I) or the crystal form C of the compound of formula (I).

[0052] In some embodiments of the present invention, the compound Z is the crystalline form B of the compound of formula (I).

[0053] In some embodiments of the present invention, the above step (b) is to continue stirring at 10-25° C. for 5-12 hours after the addition is completed.

[0054] In some embodiments of the present invention, the solvent Y is n-heptane.

[0055] In some embodiments of the present invention, the volume ratio of the solvent X to the solvent Y is 1:3.

[0056] In some embodiments of the present invention, the mass ratio of the total volume of the solvent X and the solvent Y to the compound Z is 1:8 to 1:15.

[0057] In some embodiments of the present invention, the mass ratio of the total volume of the solvent X and the solvent Y to the compound Z is 1:10.

[0058] The present invention also provides a crystal form B of the compound of formula (I),

[0059] The X-ray powder diffraction pattern of the B crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, and 18.08±0.20°.

[0060] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, and 21.58±0.20°.

[0061] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, 17.02±0.20°, 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, 21.58±0.20°, 22.23±0.20°, 24.87±0.20°, and 26.93±0.20°.

[0062] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.19±0.20°, 14.91±0.20°, 17.02±0.20°, 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, 21.58±0.20°, 22.23±0.20°, 22.66±0.20°, 23.81±0.20°, 24.87±0.20°, 26.93±0.20°, and 28.74±0.20°.

[0063] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, and / or 8.98±0.20°, and / or 17.02±0.20°, and / or 18.08±0.20°, and / or 18.54±0.20°, and / or 19.07±0.20°, and / or 21.58±0.20°, and / or 22. 23±0.20°, and / or 22.66±0.20°, and / or 23.81±0.20°, and / or 24.87±0.20°, and / or 26.93±0.20°, and / or 28.74±0.20°, and / or 10.48±0.20°, and / or 25.38±0.20°, and / or 23.38±0.20°, and / or 20.12±0.20°, and / or 32.87±0.20°.

[0064] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 7.09°, 7.92°, 8.98°, 9.70°, 10.48°, 12.16°, 13.57°, 13.84°, 14.19°, 14.91°, 15.90°, 17.02°, 18.08°, 18.54°, 19.07°, 20.12°, 2 1.18°, 21.58°, 21.74°, 22.23°, 22.66°, 23.38°, 23.81°, 24.47°, 24.87°, 25.38°, 25.87°, 26.93°, 28.74°, 29.27°, 31.17°, 32.04°, 32.87°, 34.35°, 36.05°, 39.04°.

[0065] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form B is shown in Figure 4.

[0066] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form B is basically as shown in Figure 4.

[0067] In some embodiments of the present invention, the peak positions and relative intensities of the diffraction peaks in the Cu Kα radiation XRPD pattern of the above-mentioned B crystal form are shown in Table 2:

[0068] Table 2 XRPD diffraction data of the crystal form B of compound of formula (I)

[0069] In some embodiments of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned Form B shows an endothermic peak at 154.2°C±3°C.

[0070] In some embodiments of the present invention, the DSC spectrum of the above-mentioned Form B is shown in Figure 5.

[0071] In some embodiments of the present invention, the DSC spectrum of the above-mentioned Form B is basically as shown in Figure 5.

[0072] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned Form B shows a weight loss of 4.82% at 150.0°C±3°C.

[0073] In some embodiments of the present invention, the TGA spectrum of the above-mentioned B crystal form is shown in Figure 6.

[0074] In some embodiments of the present invention, the TGA spectrum of the above-mentioned Form B is basically as shown in Figure 6.

[0075] The present invention also provides a crystal form B of the compound of formula (I),

[0076] The preparation method comprises the following steps:

[0077] (a) adding compound Z to solvent M;

[0078] (b) stirring at 40-60° C. for 1 to 12 hours;

[0079] (c) cooling to 10-30°C, adding solvent N, and continuing stirring for 1-12 hours;

[0080] (d) filtering, and drying the filter cake under vacuum at 25-60° C. for 1-24 hours;

[0081] in,

[0082] Compound Z is selected from compounds of formula (I);

[0083] Solvent M is toluene;

[0084] Solvent N is absent, or solvent N is selected from n-heptane, n-hexane and cyclohexane; preferably, solvent N is n-heptane;

[0085] When the solvent N is selected from n-heptane, n-hexane and cyclohexane, the volume ratio of the solvent M to the solvent N is 1:0.5 to 1:3.

[0086] The present invention also provides a method for preparing the crystal form B of the compound of formula (I), comprising the following steps:

[0087] (a) adding compound Z to solvent M;

[0088] (b) stirring at 40-60° C. for 1 to 12 hours;

[0089] (c) cooling to 10-30°C, adding solvent N, and continuing stirring for 1-12 hours;

[0090] (d) filtering, and drying the filter cake under vacuum at 25-60° C. for 1-24 hours;

[0091] in,

[0092] Compound Z is selected from compounds of formula (I);

[0093] Solvent M is toluene;

[0094] Solvent N is absent, or, alternatively, solvent N is selected from n-heptane, n-hexane and cyclohexane.

[0095] In some embodiments of the present invention, the step (b) is performed by stirring at 50-60° C. for 1 to 5 hours.

[0096] In some embodiments of the present invention, the solvent N is selected from n-heptane, n-hexane and cyclohexane, and the volume ratio of solvent M to solvent N is 1:0.5 to 1:3.

[0097] In some embodiments of the present invention, the solvent N is n-heptane.

[0098] In some embodiments of the present invention, the volume ratio of the solvent M to the solvent N is 1:1.

[0099] In some embodiments of the present invention, the mass ratio of the total volume of the solvent M and the solvent N to the compound Z is 1:15 to 1:30.

[0100] In some embodiments of the present invention, the mass ratio of the total volume of the solvent M and the solvent N to the compound Z is 1:20.

[0101] The present invention also provides a hydrate of the compound of formula (I) in crystal form C,

[0102] The X-ray powder diffraction pattern of the C crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 5.85±0.20°, 11.70±0.20°, and 17.59±0.20°.

[0103] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned C crystal form using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 5.85°, 11.70°, and 17.59°.

[0104] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C is shown in Figure 7.

[0105] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C is basically as shown in Figure 7.

[0106] In some embodiments of the present invention, the peak positions and relative intensities of the diffraction peaks in the Cu Kα radiation XRPD pattern of the above-mentioned C crystal form are shown in Table 3:

[0107] Table 3 XRPD diffraction data of hydrate C form of compound of formula (I)

[0108] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystal form C shows a weight loss of 12.61% at 150.0°C±3°C.

[0109] In some embodiments of the present invention, the TGA spectrum of the above-mentioned C crystal form is shown in Figure 8.

[0110] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form C is basically as shown in Figure 8.

[0111] The crystal form A, crystal form B and crystal form C of the compound of formula (I) described in the present invention may be in the form of a non-solvate or a solvate, such as a hydrate.

[0112] The present invention also provides use of the crystal form A, crystal form B, crystal form C of the above-mentioned compound or the crystal form obtained by the preparation method thereof in the preparation of a drug for treating influenza.

[0113] The present invention also provides the use of the crystal form A of the above compound or the crystal form obtained by the preparation method thereof in the preparation of a drug for treating influenza.

[0114] As an RNA polymerase inhibitor, the compound of this invention has demonstrated positive effects in cellular inhibition of influenza virus replication tests. In animal models of efficacy, it has shown excellent weight loss and early weight loss recovery. Plasma protein binding tests have shown moderate plasma protein binding, and PK results have demonstrated favorable pharmacokinetic properties and good drugability. The compound of this invention has a simple and stable crystalline form, facilitating formulation, and holds great promise for the prevention and / or treatment of influenza.

[0115] Definition and Description

[0116] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0117] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0118] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0119] Unless otherwise indicated, in the differential scanning calorimetry curves of the compounds of the present invention, upward direction indicates exothermic heat (Exo Up).

[0120] Unless otherwise noted, the positions or relative intensities of peaks in powder X-ray diffraction (XRPD) spectra may vary due to factors such as the instrument, method, and conditions. For any particular crystalline form, peak positions may vary, and the 2θ values ​​may have an error of ±0.2°. Therefore, this error should be taken into account when determining each crystalline form, and determinations within this error are within the scope of this application.

[0121] In the present invention, the expression "substantially as shown in..." for a specific XRPD pattern means that the positions of the diffraction peaks in the XRPD pattern are substantially the same within the range of visual inspection or with the aid of a selected diffraction peak list (±0.20°, 2θ). It is understood by ordinary technicians that the intensity may vary from sample to sample.

[0122] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0123] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data from a cultured single crystal using a Bruker D8venture diffractometer, using CuKα radiation and a φ / ω scanning mode. After collecting relevant data, the crystal structure can be further analyzed using a direct method (Shelxs97) to confirm the absolute configuration.

[0124] The following abbreviations are used in this disclosure: ACN stands for acetonitrile; DMSO stands for dimethyl sulfoxide; N2: nitrogen; RH: relative humidity; mL: milliliter; L: liter; min: minute; °C: degrees Celsius; μm: micrometer; mm: millimeter; μL: microliter; moL / L: mole per liter; mg: milligram; s: second; nm: nanometer; MPa: megapascal; lux: lux; μw / cm 2 : microwatt per square centimeter; h: hour; Kg: kilogram; nM: nanomole, rpm: rotational speed; XRPD stands for X-ray powder diffraction; DSC stands for differential scanning calorimetry; TGA stands for thermogravimetric analysis; 1 H NMR stands for proton nuclear magnetic resonance.

[0125] The compounds of the present invention are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names, and all solvents used in the present invention are commercially available.

[0126] Instrument and analysis method of the present invention

[0127] 1.1 X-ray powder diffractometer (XRPD) method

[0128] About 10 mg of sample was used for XRPD analysis. The detailed instrument information and parameters are shown in Table 4:

[0129] Table 4 XRPD instrument information and parameters

[0130] 1.2 Differential Scanning Calorimeter (DSC) and Thermogravimetric Analysis (TGA)

[0131] The detailed parameters are shown in Table 5 below:

[0132] Table 5 DSC and TGA instrument parameters

[0133] 1.3 Dynamic Vapor Sorption (DVS) method

[0134] Detailed instrument information and parameters are shown in Table 6 below:

[0135] Table 6 DVS instrument parameters

[0136] The moisture absorption evaluation is classified as follows:

[0137] Absorbs sufficient water to form a liquid: deliquescent; ΔW% ≥ 15%: extremely hygroscopic; 15% > ΔW% ≥ 2%: somewhat hygroscopic; 2% > ΔW% ≥ 0.2%: slightly hygroscopic; ΔW% < 0.2%: no or almost no hygroscopicity. ΔW% represents the weight gain of the test article due to moisture absorption at 25 ± 1°C and 80 ± 2% relative humidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] FIG1 is an XRPD spectrum of the crystalline form A of compound of formula (I).

[0139] FIG2 is a DSC spectrum of the crystalline form A of compound of formula (I).

[0140] FIG3 is a TGA spectrum of the crystalline form A of compound of formula (I).

[0141] FIG4 is an XRPD spectrum of the crystalline form B of compound of formula (I).

[0142] FIG5 is a DSC spectrum of the crystalline form B of compound of formula (I).

[0143] FIG6 is a TGA spectrum of the crystalline form B of compound of formula (I).

[0144] FIG7 is an XRPD spectrum of the hydrate C crystal form of the compound of formula (I).

[0145] FIG8 is a TGA spectrum of the hydrate C crystal form of the compound of formula (I).

[0146] FIG9 is a DVS spectrum of the crystalline form A of compound of formula (I).

[0147] FIG10 is an ellipsoid diagram of a single crystal of the compound of formula (I). DETAILED DESCRIPTION

[0148] The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any way. While the present invention has been described in detail herein, and specific embodiments thereof have been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0149] Example 1: Preparation of compound of formula (I)

[0150] Step 1: Synthesis of compound 1-2

[0151] Compound 1-1 (66 g, 383.43 mmol) was dissolved in dichloromethane (460 mL), and N,N-dimethylformamide (280.27 mg, 3.83 mmol, 295.02 μL) was added. Oxalyl chloride (73.00 g, 575.15 mmol, 50.35 mL) was added dropwise to the reaction solution. After completion of the addition, the reaction solution was stirred at 20°C for 30 minutes and then concentrated under reduced pressure. The crude product was added to dichloromethane (460 mL), and triethylamine (77.60 g, 766.87 mmol, 106.74 mL) and N,O-dimethylhydroxylamine hydrochloride (37.40 g, 383.43 mmol) were added with stirring. The reaction solution was stirred at 20°C for 1 hour. Water (100 mL) was added, the layers were separated, and the aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, washed with dilute hydrochloric acid (0.2 M, 50 mL), saturated aqueous sodium bicarbonate solution (50 mL) and saturated brine (50 mL), respectively, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1-2. 1 H NMR (400 MHz, deuterated chloroform) δ 7.02-7.05 (m, 2H), 3.81 (brs, 3H), 3.49 (s, 3H), 2.28 (s, 3H).

[0152] Step 2: Synthesis of Compounds 1-3

[0153] Compound 1-2 (20 g, 92.94 mmol) was dissolved in tetrahydrofuran (200 mL), and methylmagnesium bromide (3 M, 37.18 mL) was added dropwise at 0°C. After completion of the addition, the reaction mixture was warmed to 20°C and stirred for 2 hours. The reaction mixture was quenched with 1 M hydrochloric acid, the pH was adjusted to 7, and extracted with ethyl acetate (100 mL × 2). The organic phases were combined and washed with dilute hydrochloric acid (0.2 M, 50 mL), saturated sodium bicarbonate solution (50 mL), and saturated brine (50 mL), respectively. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1-3. 1 H NMR (400 MHz, deuterated chloroform) δ 7.47-7.50 (m, 1H), 7.03-7.07 (m, 1H), 2.60 (s, 3H), 2.47 (s, 3H).

[0154] Step 3: Synthesis of Compounds 1-4

[0155] Compound 1-3 (15 g, 88.15 mmol) was dissolved in pyridine (90 mL), and selenium dioxide (19.56 g, 176.31 mmol) was added. The reaction mixture was stirred at 110°C for 12 hours. The reaction mixture was cooled to room temperature, filtered, and concentrated under reduced pressure. Water (50 mL) was added to the crude product, and the pH was adjusted to 4 with 1M hydrochloric acid. The product was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1-4. 1 H NMR (400 MHz, deuterated methanol) δ 7.64-7.68 (m, 1H), 7.30-7.32 (m, 1H), 2.52 (s, 3H).

[0156] Step 4: Synthesis of Compounds 1-5

[0157] Compound 1-4 (15 g, 74.95 mmol) was dissolved in dichloromethane (60 mL) and methanol (60 mL). The temperature was maintained at 0-20°C, and trimethylsilyldiazomethane (2 M, 44.97 mL) was added dropwise. The reaction mixture was stirred at 20°C for 2 hours, followed by the addition of acetic acid (3 mL) and stirring for 5 minutes. The reaction mixture was concentrated under reduced pressure, water (50 mL) was added, and the mixture was extracted with dichloromethane (50 mL x 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-20%) to obtain compound 1-5. 1 H NMR (400 MHz, deuterated chloroform) δ 7.51-7.55 (m, 1H), 7.12-7.16 (m, 1H), 3.98 (s, 3H), 2.54 (s, 3H).

[0158] Step 5: Synthesis of Compounds 1-6

[0159] Compound 1-5 (5 g, 23.35 mmol) was dissolved in 1,2-dichloroethane (50 mL). N-bromosuccinimide (8.31 g, 46.69 mmol) and azobisisobutyronitrile (383.37 mg, 2.33 mmol) were added. The reaction mixture was stirred at 80°C for 12 hours. The reaction mixture was cooled to room temperature and washed with saturated sodium sulfite solution (20 mL), water (20 mL), and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-5%) to obtain compound 1-6. 1 H NMR (400 MHz, deuterated chloroform) δ 7.61-7.64 (m, 1H), 7.26-7.31 (m, 1H), 4.94 (s, 2H), 3.99 (s, 3H).

[0160] Step 6: Synthesis of Compounds 1-7

[0161] Sodium dihydrogen phosphate (11.52 g, 96.05 mmol) was dissolved in water (60 mL), followed by acetonitrile (30 mL). 3-(3-pyridyldiselenyl)pyridine (3.62 g, 11.53 mmol) was added, and zinc powder (1.88 g, 28.82 mmol) was added portionwise. The reaction mixture was stirred at 20°C for 30 minutes. Compound 1-6 (5.63 g, 19.21 mmol) was added, and the reaction mixture was stirred at 20°C for 3 hours. The reaction mixture was filtered, and the filtrate was extracted with ethyl acetate (30 mL x 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-60%) to obtain compound 1-7. MS (ESI) m / z: 373.8 [M+H] + .

[0162] Step 7: Synthesis of Compounds 1-8

[0163] Compound 1-7 (4.2 g, 11.28 mmol) was dissolved in dichloromethane (80 mL), and Dess-Martin periodinane (7.18 g, 16.93 mmol) was added. The reaction solution was stirred at 20°C for 12 hours. Saturated sodium sulfite solution (30 mL) was added to the reaction solution and stirred for 5 minutes. The mixture was extracted with dichloromethane (30 mL × 2), and the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 1-8. MS (ESI) m / z: 371.9 [M+H] + .

[0164] Step 8: Synthesis of Compounds 1-9

[0165] Compound 1-8 (2.9 g, 7.83 mmol) was dissolved in tetrahydrofuran (16 mL), and aqueous sodium hydroxide solution (626.67 mg, 15.67 mmol, 4 mL) was added. The reaction mixture was stirred at 20°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove most of the tetrahydrofuran. The aqueous phase was adjusted to pH 6 with 1N hydrochloric acid. The solid was filtered, and the filter cake was dried under reduced pressure to obtain compound 1-9. MS (ESI) m / z: 357.9 [M+H] + .

[0166] Step 9: Synthesis of Compounds 1-10

[0167] Compound 1-9 (2.3 g, 6.46 mmol) was dissolved in dimethyl sulfoxide (23 mL). Ammonium persulfate (2.95 g, 12.91 mmol), silver nitrate (109.69 mg, 645.74 μmol), and concentrated sulfuric acid (633.34 mg, 6.46 mmol) were added, respectively. The reaction mixture was stirred at 50°C for 3 hours. Saturated aqueous sodium bicarbonate (20 mL), water (10 mL), and dichloromethane (20 mL) were added, respectively, and the mixture was stirred for 5 minutes. The mixture was filtered, and the filtrate was separated. The aqueous phase was extracted with dichloromethane (10 mL). The organic phases were combined, washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 1-10. MS (ESI) m / z: 311.8 [M+H] + .

[0168] Step 10: Synthesis of Compounds 1-11

[0169] Compound 1-10 (390 mg, 1.26 mmol) was dissolved in isopropanol (8 mL), and sodium borohydride (95.14 mg, 2.51 mmol) was added. The reaction mixture was stirred at 20°C for 1 hour. 1N hydrochloric acid was added to adjust the pH to 7, and the mixture was extracted with ethyl acetate (15 mL x 2). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 1-11. MS (ESI) m / z: 313.8 [M+H] + .

[0170] Step 11: Synthesis of Compounds 1-12

[0171] Compound 1-11 (330 mg, 1.06 mmol) was dissolved in dichloromethane (6 mL), and thionyl chloride (251.53 mg, 2.11 mmol, 153.37 μL) was added. The reaction mixture was stirred at 20°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain compound 1-12, which was used directly in the next reaction.

[0172] Step 12: Synthesis of Compounds 1-14

[0173] Compound 1-13 (340 mg, 1.04 mmol) was dissolved in acetonitrile (6 mL), and compound 1-12 (343.41 mg, 1.04 mmol) and cesium carbonate (676.86 mg, 2.08 mmol) were added. The reaction solution was stirred at 60°C for 12 hours. The reaction solution was cooled to room temperature, water (5 mL) was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column (methanol / dichloromethane, methanol ratio: 0-5%), and the obtained compound was subjected to chiral separation (column model: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 μm); mobile phase: [A: carbon dioxide, B: 0.1% ammonia / ethanol]; gradient: mobile phase B was maintained at 40%) to give compound 1-14 (analytical method: column model: Chiralpak AD-3 (50 mm*4.6 mm, 3 μm); mobile phase: [A: carbon dioxide, B: 0.05% diethylamine / ethanol]; gradient: mobile phase B concentration increased from 5% to 40% within 2 minutes, 40% was maintained for 1.2 minutes, and then 5% was maintained for 0.8 minutes, retention time of compound 1-14 was 1.831 min, ee = 96.1%).

[0174] Step 13: Synthesis of Compound 1-15

[0175] Compound 1-14 (6 mg, 9.65 μmol) was added to N,N-dimethylacetamide (1 mL), followed by lithium chloride (2.05 mg, 48.27 μmol). The reaction mixture was stirred at 80°C for 12 hours. The reaction mixture was cooled to room temperature and diluted with acetonitrile (1 mL). The crude reaction mixture was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75*30mm*3μm; mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; gradient: acetonitrile %: 30%-53%) to obtain compound 1-15. MS (ESI) m / z: 533.1 [M+H] + ; 1H NMR (400 MHz, deuterated methanol) δ 7.98-8.09 (m, 1H), 7.70 (dd, J = 1.51, 8.03 Hz, 1H), 7.41 (d, J = 7.53 Hz, 1H), 7.18-7.31 (m, 2H), 7.13 (dd, J = 4.52, 8.03 Hz, 1H), 5.75-5.88 (m, 2H), 5.40-5.53 (m, 1H), 4.71 (dd, J = 3.01, 10.04 Hz, 1H), 4.63 (br s,1H),4.17(d,J=12.55Hz,1H),4.07(dd,J=3.01,11.04Hz,1H),3.77(dd,J=3.01,11. 54Hz, 1H), 3.66 (t, J = 10.54Hz, 1H), 3.48 (dt, J = 2.51, 11.80Hz, 1H), 3.04-3.18 (m, 1H).

[0176] Step 14: Synthesis of compound of formula (I)

[0177] Compound 1-15 (130.00 mg, 244.65 μmol) was added to N,N-dimethylacetamide (2 mL), followed by the addition of chloromethyl methyl carbonate (45.70 mg, 366.98 μmol), potassium carbonate (67.63 mg, 489.30 μmol), and potassium iodide (40.61 mg, 244.65 μmol). The reaction solution was stirred at 70°C for 3 hours. The reaction solution was cooled to room temperature, water (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL × 2). The organic phase was washed with saturated brine (10 mL × 4), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column (dichloromethane:methanol = 1:0 to 20:1) to obtain the compound of formula (I). MS (ESI) m / z: 621.0 [M+H] + ; 1H NMR (400 MHz, deuterated chloroform) δ 8.03 (dd, J = 1.00, 4.52 Hz, 1H), 7.44 (dd, J = 1.51, 8.03 Hz, 1H), 6.99-7.16 (m, 3H), 6.95 (dd, J = 4.52, 8.03 Hz, 1H), 5.90 (d, J = 6.53 Hz, 1H), 5.74-5.85 (m, 1H), 5.22-5.35 (m, 3H), 4.60 (dd, J = 2.01, 13.55 Hz, 1H),4.50(dd,J=3.01,10.04Hz,1H),4.03(d,J=12.55Hz,1H),3.95(dd,J=3.01,11.04Hz,1H),3.77-3.83(m,3H ), 3.73 (dd, J = 3.01, 12.05Hz, 1H), 3.54 (t, J = 10.54Hz, 1H), 3.41 (dt, J = 2.51, 11.80Hz, 1H), 2.85-2.97 (m, 1H).

[0178] Example 2: Preparation of Crystalline Form B of Compound (I)

[0179] Toluene (590 mL) was added to the compound of formula (I) (59 g) at 20°C and stirred at 50°C for 1 hour. The mixture was then cooled to 20°C, and n-heptane (590 mL) was added dropwise. Stirring was continued for 1 hour. The mixture was filtered, and the filter cake was vacuum-dried at 45°C for 2 hours to obtain crystalline Form B of the compound of formula (I). The XRPD spectrum is shown in Figure 4, the DSC spectrum is shown in Figure 5, and the TGA spectrum is shown in Figure 6.

[0180] Example 3: Preparation of Crystalline Form A of Compound (I)

[0181] Crystalline Compound B of Formula (I) (approximately 55 g) was added to a mixed solvent of ethyl acetate (125 mL) and n-heptane (375 mL) and stirred at 20°C for 12 hours. The mixture was filtered, and the filter cake was vacuum-dried at 45°C for 2 hours to obtain Crystalline Compound A of Formula (I). The XRPD spectrum is shown in Figure 1 , the DSC spectrum is shown in Figure 2 , and the TGA spectrum is shown in Figure 3 .

[0182] Example 4: Preparation of Crystal Form C of the Hydrate of the Compound of Formula (I)

[0183] Approximately 15 mg of the compound of formula (I) was weighed and added to 0.5 mL of methanol / methyl tert-butyl ether (1:9 volume ratio). The suspension was stirred at room temperature for 8 days. Centrifugation was performed, and the solid was collected to obtain Hydrate Form C of the compound of formula (I). The XRPD spectrum is shown in Figure 7, and the TGA spectrum is shown in Figure 8.

[0184] Example 5: Single crystal X-ray diffraction analysis of the compound of formula (I)

[0185] Single crystals were grown using the n-hexane vapor diffusion method. About 5 mg of the compound of formula (I) was weighed, dissolved in isopropanol (0.3 mL), filtered with a filter membrane (0.45 μM), and the filtrate was placed in a 2 mL sample bottle. The sample bottle was sealed with aluminum foil, and then 3-5 small holes were pierced with a syringe and placed in a sealed brown bottle. 20 mL of n-hexane was added to the brown bottle, about 0.3 cm high, and the bottle was sealed and allowed to stand at 20-25 ° C for 2 days to precipitate square columnar crystals. The crystals were collected and the diffraction intensity data were collected using a single crystal X-ray diffractometer (SC-XRD) (D8-VENTURE). The single crystal data showed that the absolute configuration of the compound of formula (I) could be determined, and the molecular formula of the compound was C 26 H 22 The ellipsoidal diagram of the three-dimensional structure of the compound of formula (I) is shown in Figure 10. The crystal structure data of the single crystal of the compound of formula (I) are shown in Table 7.

[0186] Table 7 Crystal data of single crystal of compound of formula (I)

[0187] Example 6: Solid Stability Test of Crystalline Form A of Compound (I)

[0188] According to the "Guidelines for Stability Testing of APIs and Preparations" (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 9001), in order to evaluate the solid stability of the crystal form A of compound of formula (I), the stability of the crystal form A was investigated under the influencing factors (high temperature, high humidity and light), 40°C / 75%RH and 25°C / 60%RH conditions. The crystal form A was placed under high temperature (60°C, open) and high humidity (25°C / 92.5%RH, open) conditions for 30 days each, and the total illumination of visible light reached 1200000Lux·hrs and the total illumination of ultraviolet light reached 200W·hrs / m 2 ) were exposed to visible light and UV light (a light-shielded control sample was also placed and wrapped in tin foil) at 40°C / 75% RH (exposed) for 1, 2, and 3 months, and at 25°C / 60% RH (exposed) for 3 months. XRPD analysis was performed on all stability samples to detect changes in crystal form, and the results are shown in Table 8.

[0189] Accurately weigh about 10 mg of the crystal form and place it in a dry and clean glass bottle, spread it into a thin layer, and place it open under the influencing factor test conditions and accelerated conditions. 2) conditions were placed in transparent glass bottles, completely exposed, and samples for XRPD detection were placed separately.

[0190] After the sample was removed at the time point, the lid was secured, sealed with parafilm, and stored in a -20°C refrigerator. For sample preparation, the sample was removed from the refrigerator, returned to room temperature, and 25 mL of diluent (acetonitrile / water, 1:1, v / v) was added to dissolve the sample to a concentration of approximately 0.5 mg / mL. Liquid chromatography was used for sample injection analysis, and the test results were compared with the initial test results on day 0. The test results are shown in Table 8 below.

[0191] Preparation of Day 0 standard solution: Weigh approximately 25 mg of the crystalline form into a 50 mL volumetric flask, dissolve it in 50% acetonitrile and dilute to the mark.

[0192] At the same time, HPLC tests were performed on all stability samples. The specific results are summarized in Table 8. The HPLC test instrument and analysis conditions are shown in Tables 9 and 10.

[0193] Table 8 Solid stability test results of the crystal form of compound A of formula (I)

[0194] Note*: The compound of formula (I) is a prodrug, which can be converted into compound 1-15 in vivo and partially degraded into compound 1-15 during storage. The purity data here includes the compound of formula (I) and compound 1-15.

[0195] Table 9 HPLC instrument information and analysis method

[0196] Table 10 HPLC instrument information and analysis method for compounds 1-15

[0197] Conclusion: The crystal form A of compound of formula (I) is stable under all stability conditions (high temperature, high humidity, and light) and has good chemical stability.

[0198] Example 7: Hygroscopicity study of the crystalline form of compound A of formula (I)

[0199] Experimental materials: SMS DVS intrinsic plus dynamic moisture adsorption instrument

[0200] Experimental method: Take the crystal form A of compound of formula (I) (10-20 mg) and place it in a DVS sample tray for testing.

[0201] Experimental Results: The moisture absorption weight gain of Form A of Compound (I) at 25°C / 80% RH was ΔW% = 1.37%, and the DVS spectrum is shown in Figure 9. Experimental Conclusion: The moisture absorption weight gain of Form A of Compound (I) at 25°C / 80% RH was 2% > ΔW% ≥ 0.2%, indicating that it is slightly hygroscopic.

[0202] Biological test data:

[0203] Experimental Example 1: Influenza virus cytopathic effect (CPE) experiment

[0204] The half effective concentration (EC50) of the compound was determined 50 ) value to evaluate the antiviral activity of compounds against influenza virus (IFV). Cytopathic effect assay is widely used to measure the protective effect of compounds on virus-infected cells to reflect the antiviral activity of compounds.

[0205] Influenza virus CPE test

[0206] MDCK cells were seeded at a density of 2,000 cells per well in black 384-well cell culture plates and then cultured overnight in a 37°C, 5% CO2 incubator. Compounds were diluted using an Echo555 non-contact nanoliter acoustic pipetting system and added to the wells (4-fold serial dilutions, 8 test concentrations). Influenza virus A / PR / 8 / 34 (H1N1) strain was then added to the wells at 1-2 90% tissue culture infectious doses (TCID90) per well, with a final DMSO concentration of 0.5% in the culture medium. Virus control wells (DMSO and virus added, no compound), cell control wells (DMSO added, no compound or virus added), and culture medium control wells (culture medium only, no cells) were set up. Cytotoxicity assays and antiviral activity assays of the compounds were performed in parallel, using the same experimental conditions as the antiviral activity assay, except that virus was omitted. The plates were cultured in a 37°C, 5% CO2 incubator for 5 days. After 5 days of culture, cell viability was assessed using the CCK8 cell viability assay kit. The raw data were used to calculate the antiviral activity and cytotoxicity of the compounds.

[0207] The antiviral activity and cytotoxicity of the compound are expressed by the inhibition rate (%) of the compound on the cytotoxic effect caused by the virus. The calculation formula is as follows:

[0208] GraphPad Prism software was used to perform nonlinear fitting analysis on the inhibition rate and cytotoxicity of the compound to obtain the EC 50 The experimental results are shown in Table 11.

[0209] Table 11 Inhibitory activity of compounds against influenza virus A / PR / 8 / 34 (H1N1)

[0210] Conclusion: The compounds of the present invention showed positive effects in the test of inhibiting influenza virus replication at the cellular level.

[0211] Experimental Example 2: In vivo efficacy study

[0212] Objective: To evaluate the efficacy of the compound in a mouse model of influenza A virus H1N1 infection

[0213] Experimental Protocol: Mice were infected intranasally with influenza A virus A / PR / 8 / 34 (H1N1). Compounds were then orally administered twice daily for 7 consecutive days, beginning 48 hours after infection. The anti-influenza A virus (H1N1) activity of the compounds in this model was evaluated by observing changes in mouse body weight and survival rates.

[0214] The experiment used SPF-level BALB / c mice, 6-7 weeks old, female. The experiment began after the mice arrived at the BSL-2 animal room and adapted for at least 3 days. The day of infection was set as day 0 of the experiment. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg / kg, 10 mL / kg). After the animals entered a deep anesthesia state, they were infected with A / PR / 8 / 34 (H1N1) virus through intranasal drops with an infection volume of 50 μL. From the second day to the eighth day, 5 mg / kg (dosage volume 10 mL / kg) of the test compound was orally administered twice a day. The first administration time was 48 hours after infection. The status of the mice was observed every day, and the weight and survival rate of the mice were recorded. On the 14th day, all surviving animals were euthanized.

[0215] Experimental results:

[0216] Animal survival rate and weight loss rate were measured: weight loss rate = (weight on day 0 - weight on day N) / weight on day 0 * 100%. The results are shown in Table 12: Compound (I) protected the animals, achieving a maximum weight loss rate of 7.03% on day 3, after which recovery began. By the end of the experiment, the mouse survival rate was 100%.

[0217] Table 12 Animal survival rate and weight loss rate results

[0218] Conclusion: The compound of the present invention showed excellent weight protection in the animal in vivo efficacy model and the recovery time was early.

[0219] Experimental Example 3: Cytopathic Effect (CPE) Experiment on Baloxavir-Resistant A / PR / 8 / 34 (H1N1) I38T Influenza Virus Strain

[0220] The purpose of the experiment was to determine the effective concentration (EC50) of the compound. 50) values ​​were used to evaluate the antiviral activity of the compounds against the baloxavir-resistant A / PR / 8 / 34(H1N1)I38T influenza virus strain.

[0221] Experimental protocol: MDCK cells were seeded at a density of 15,000 cells per well in a 96-well cell culture plate and cultured overnight at 37°C in a 5% CO2 incubator. The following day, compound solution (3-fold serial dilutions, 8 concentration points, in triplicate) and baloxavir-resistant influenza A / PR / 8 / 34 (H1N1) virus were added to the cell culture medium at a final DMSO concentration of 0.5%. Cells were cultured in a 5% CO2, 37°C incubator for 5 days until the cytopathic effect (CPE) in the virus-infected control wells without compound reached 80–95%. Cell viability in each well was then measured using CCK8. If cell viability in the compound-containing wells was higher than that in the virus-infected control wells, indicating a decrease in CPE, the compound demonstrated inhibitory activity against the tested virus.

[0222] Experimental results:

[0223] The antiviral activity of a compound is expressed as the inhibitory activity (%) of the compound on the cytoviral effect caused by the virus. The calculation formula is as follows:

[0224] EC 50 GraphPad Prism (version 5) software was used to perform nonlinear fitting analysis of the inhibitory activity and cell viability of the compounds using the "log (inhibitor) vs. response - variable slope" fitting method. The experimental results are shown in Table 13.

[0225] Table 13 Inhibitory activity of the compounds of the present invention against Baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T influenza virus strain

[0226] Conclusion: The compounds of the present invention showed positive effects in inhibiting the replication of Baloxavir-resistant A / PR / 8 / 34 (H1N1) influenza virus strain at the cellular level.

[0227] Experimental Example 4: In vivo efficacy study

[0228] Objective: To evaluate the efficacy of the compound in a mouse infection model with a drug-resistant strain of influenza A virus H1N1

[0229] Experimental Protocol: Mice were intranasally infected with the baloxavir-resistant influenza A virus strain A / PR / 8 / 34(H1N1)I38T. Compounds were then orally administered twice daily for 7 consecutive days, starting 2 hours before infection. The anti-influenza A virus (H1N1) activity of the compounds in this model was evaluated by observing changes in mouse body weight and survival rate.

[0230] The experiment used SPF-level BALB / c mice, 6-7 weeks old, female. The experiment began after the mice arrived at the BSL-2 animal room and adapted for at least 3 days. The day of infection was set as day 0 of the experiment. After the mice were deeply anesthetized by intraperitoneal injection of Shutai 50 / Xylazine Hydrochloride, they were infected with Baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T virus strain by intranasal drops, and the infection volume was 50 μL. From the 2nd day to the 8th day, 15 mg / kg or 50 mg / kg (dosing volume 10 mL / kg) of the test compound was orally administered twice a day. The first administration time was 2 hours before infection. The status of the mice was observed every day, and the weight and survival rate of the mice were recorded. On the 14th day, all surviving animals were euthanized.

[0231] Experimental results:

[0232] Animal survival rate and weight loss rate were measured: weight loss rate = (weight on day 0 - weight on day N) / weight on day 0 * 100%. The experimental results are shown in Table 14 below. The mice showed little weight loss at a dose of 50 mg / kg of the compound of formula (I) and the crystalline form A of the compound of formula (I). By the end of the experiment, the survival rate was 100%.

[0233] Table 14 Animal survival rate and weight loss rate results

[0234] Conclusion: The compound of the present invention showed excellent weight protection in the animal in vivo efficacy model and the recovery time was early.

[0235] Experimental Example 5 Plasma protein binding test of the compound

[0236] Experimental purpose: Equilibrium dialysis was used to evaluate the protein binding rate of the compounds of the present invention in CD-1 mice, SD rats and human plasma.

[0237] Experimental Protocol: Test compounds were diluted in dialysis buffer into plasma from each of the five species to a final concentration of 2 μM. The samples were then placed in a 96-well equilibrium dialysis apparatus and dialyzed against phosphate buffer at 37°C for 4 hours. Warfarin was used as a control compound. Concentrations of test compounds and warfarin in plasma and buffer were determined by LC-MS / MS.

[0238] Experimental results: The results are shown in Table 15.

[0239] Table 15 Plasma protein binding rate results of the compounds of the present invention

[0240] Note: H stands for human, R stands for rat, M stands for mouse, D stands for dog, and C stands for cynomolgus monkey

[0241] Conclusion: The compounds of the present invention have moderate plasma protein binding rates in the plasma of the five species, indicating that the free drug concentration ratio of the test compounds in the plasma of the above five species is moderate and has good drug-forming properties.

[0242] Experimental Example 6: Pharmacokinetic Study in Rats

[0243] Experimental purpose: To investigate the plasma pharmacokinetics of the compound of the present invention in male SD rats after single intravenous injection and oral administration.

[0244] Experimental animals: Male SD rats, 6-8 weeks old, weighing 200-300 g;

[0245] Experimental procedures: Injection (iv) at a dose of 1 mpk, a concentration of 0.50 mg / mL, and a solvent consisting of 40% DMAC + 40% PG + 20% (20% HP-β-CD + water); oral administration (po) at a dose of 10 mpk, a concentration of 1 mg / mL, and a solvent consisting of 3% DMSO + 10% solutol HS + 87% water).

[0246] Sample Collection: 0.03 mL of blood was collected from the experimental animals via saphenous vein puncture at each time point, and the actual time of blood collection was recorded. All blood samples were placed in 1.5 mL commercial EDTA-K2 anticoagulant tubes. After blood sample collection, DDV was added to the plasma matrix as a stabilizer, with a plasma:DDV solution ratio of 40:1 (40 mM DDV in acetonitrile / water (1:1)). Within half an hour, the samples were centrifuged at 3000 g for 10 minutes at 4°C. The supernatant plasma was aspirated, immediately placed on dry ice, and stored at -80°C for LC-MS / MS analysis.

[0247] Data analysis: The plasma concentration was processed using a non-compartmental model using Phoenix WinNonlin 6.3 pharmacokinetic software, and the pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method: Cl (apparent clearance), T 1 / 2 (time required to clear half of the compound), C max (peak concentration), AUC 0-last (0-integrated area of ​​concentration during the last sampling time), the results are shown in Table 16.

[0248] Table 16 PK results of the compounds of the present invention in rats

[0249] Experimental conclusion: The compound of the present invention has a high oral plasma exposure and good pharmacokinetic properties.

[0250] Experimental Example 7: Pharmacokinetic Study in Beagle Dogs

[0251] Experimental purpose: To investigate the plasma pharmacokinetics of the compound of the present invention in male beagle dogs after single intravenous injection and oral administration.

[0252] Experimental animals: Male beagle dogs, ≥6 months of age, weighing 6–12 kg;

[0253] Experimental procedure: Injection (iv) at a dose of 1 mpk at a concentration of 1 mg / mL, with a vehicle of 10% DMAC + 90% (20% HP-β-CD + water); oral administration (po) of the compound of formula (I) at a dose of 10 mpk at a concentration of 2 mg / mL, with a vehicle of 3% DMSO + 10% solutol HS + 87% water); oral administration (po) of the crystalline form A of the compound of formula (I) at a dose of 5 mpk at a concentration of 1 mg / mL, with a vehicle of 3% DMSO + 10% solutol HS + 87% water.

[0254] Sample Collection: 0.8 mL of blood was collected from the experimental animals via saphenous vein puncture at each time point, and the actual time of blood collection was recorded. All blood samples were placed in 1.5 mL commercial EDTA-K2 anticoagulant tubes. After blood sample collection, DDV was added to the plasma matrix as a stabilizer, with a plasma:DDV solution ratio of 40:1 (40 mM DDV in acetonitrile / water (1:1)). Within half an hour, the samples were centrifuged at 3000 g for 10 minutes at 4°C. The supernatant plasma was aspirated, immediately placed on dry ice, and stored at -80°C for LC-MS / MS analysis.

[0255] Data analysis: The plasma concentrations were analyzed using a non-compartmental model using Phoenix WinNonlin 6.3 pharmacokinetic software, and the pharmacokinetic parameters Cl and T were calculated using the linear-log trapezoidal method. 1 / 2 ,C max ,AUC 0-last , the results are shown in Table 17.

[0256] Table 17 PK results of the compounds of the present invention in beagle dogs

[0257] Experimental conclusion: The compound of the present invention has a low clearance rate, a long half-life, a high oral plasma exposure, and good pharmacokinetic properties.

Claims

1. Crystal Form A of the compound of formula (I), It is characterized by: Its Cu Kα radiation X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 10.68±0.20°, and 16.90±0.20°.

2. The crystal form A according to claim 1, wherein its X-ray powder diffraction pattern using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 16.90±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, and 29.12±0.20°.

3. The crystal form A according to claim 2, wherein its X-ray powder diffraction pattern using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20°, 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, and 29.12±0.20°.

4. The crystal form A according to claim 3, wherein the X-ray powder diffraction pattern of the crystal form A using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20°, 14.52±0.20°, 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, 26.26±0.20°, 28.24±0.20°, 29.12±0.20°, and 29.98±0.20°.

5. The crystal form A according to claim 4, wherein the X-ray powder diffraction pattern of the crystal form A using Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56°, 9.08°, 10.68°, 11.80°, 13.94°, 14.52°, 14.92°, 15.28°, 15.74°, 16.56°, 16.90°, 17.84°, 18.42°, 19.42°, 19.94°, 20.3 0°, 20.72°, 21.12°, 22.66°, 23.80°, 24.30°, 24.68°, 25.46°, 25.80°, 26.26°, 26.86°, 28.24°, 29.12°, 29.98°, 30.88°, 31.94°, 33.60°, 35.00°, 37.88°, 38.48°.

6. The crystal form A according to any one of claims 1 to 5, characterized in that Have any of the following characteristics: (1) Its XRPD pattern is basically as shown in Figure 1; (2) its differential scanning calorimetry curve has an endothermic peak at 198.4°C ± 3°C; (3) Its DSC spectrum is basically as shown in Figure 2; (4) Its thermogravimetric analysis curve shows a weight loss of 2.87% at 200.0℃±3℃; (5) Its TGA spectrum is basically as shown in Figure 3.

7. A method for preparing the crystal form A of the compound of formula (I) according to any one of claims 1 to 5, The steps include: (a) adding compound Z to a mixed solvent of solvent X and solvent Y; (b) stirring at 10-35° C. for 5-24 hours after the addition is complete; (c) filtering, and vacuum drying the filter cake at 25-60° C. for 1-24 hours; in, Compound Z is a crystalline form of compound B of formula (I); Solvent X is ethyl acetate; Solvent Y is selected from n-heptane, n-hexane and cyclohexane; The volume ratio of solvent X to solvent Y is 1:1 to 1:

6.

8. The preparation method according to claim 7, wherein Compound Z is a crystalline form of compound B of formula (I), solvent Y is n-heptane, and the volume ratio of solvent X to solvent Y is 1:

3.

9. Crystal Form B of the compound of formula (I), It is characterized by: Its Cu Kα radiation X-ray powder diffraction pattern has characteristic diffraction peaks at any of the following 2θ angles: (1)9.70±0.20°，12.16±0.20°，14.91±0.20°，18.08±0.20°； (2)8.98±0.20°，9.70±0.20°，12.16±0.20°，14.91±0.20°，18.08±0.20°，18.54±0.20°，19.07±0.20°，21.58±0.20°； (3)8.98±0.20°，9.70±0.20°，12.16±0.20°，14.91±0.20°，17.02±0.20°，18.08±0.20°，18.54±0.20°，19.07±0.20°，21.58±0.20°，22.23±0.20°，24.87±0.20°，26.93±0.20°。 10. The crystal form B according to claim 9, characterized in that Have any of the following characteristics: (1) Its XRPD pattern is basically as shown in Figure 4; (2) its differential scanning calorimetry curve has an endothermic peak at 154.2°C ± 3°C; (3) Its DSC spectrum is basically as shown in Figure 5; (4) Its thermogravimetric analysis curve shows a weight loss of 4.82% at 150.0°C ± 3°C; (5) Its TGA spectrum is basically as shown in Figure 6.

11. A method for preparing the crystal form B of the compound of formula (I) according to claim 9 or 10, The steps include: (a) adding compound Z to solvent M; (b) stirring at 40-60° C. for 1 to 12 hours; (c) cooling to 10-30° C., adding solvent N, and stirring for 1 to 12 hours; (d) filtering, and drying the filter cake under vacuum at 25-60° C. for 1-24 hours; in, Compound Z is selected from compounds of formula (I); Solvent M is toluene; Solvent N is absent, or, alternatively, solvent N is selected from n-heptane, n-hexane and cyclohexane.

12. The preparation method according to claim 11, wherein Solvent N is n-heptane.

13. Crystalline Form C of a hydrate of a compound of formula (I), It is characterized by: Its Cu Kα radiation X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.85±0.20°, 11.70±0.20°, and 17.59±0.20°.

14. The crystal form C according to claim 13, whose XRPD pattern is substantially as shown in Figure 7.

15. Use of the crystal form A according to any one of claims 1 to 6, the crystal form B according to claim 9 or 10, the crystal form C according to claim 13 or 14, or the crystal form prepared according to the method according to any one of claims 7 to 8, 11 to 12 in the preparation of a medicament for treating influenza.

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