TW202308646A - Crystal form of a compound for treating influenza and use thereof - Google Patents

Abstract

The present invention relates to the field of biomedicine, in particular to a crystal form of a compound and use thereof. The compound is the compound represented by Formula A, and the crystal form has XRPD characteristic peaks of 10.7 ± 0.2, 13.1 ± 0.2, 15.6 ± 0.2, 16.0 ± 0.2, 17.5 ± 0.2, 20.1 ± 0.2, 21.9 ± 0.2, 24.1 ± 0.2, 26.5 ± 0.2 and 29.7 ± 0.2 degree 2-theta. The provided crystal form of the compound is useful treating influenza.

Description

Crystal form and application of compound for treating influenza

The invention relates to the field of anti-infective drugs, in particular to a crystal form and application of a compound for treating influenza.

Influenza outbreaks occur every autumn and winter. Influenza usually causes respiratory illness with fever lasting 3 to 7 days. Although influenza vaccines are available, seasonal influenza epidemics can cause 5% to 10% of adults and 20% to 30% of children to suffer from influenza each year, causing 3 to 5 million severe cases and 290,000 to 650,000 deaths. The mortality rate is 4.0-8.8/100,000, causing a huge humanitarian disaster and economic burden (WHO: Global Influenza Strategy 2019-2030 and Iuliano AD et al., Lancet. 2018, 391, 1285-300).

Influenza viruses belong to the Orthomyxoviridae family, which are enveloped viruses containing a single-stranded negative-sense RNA genome. In the past few decades, two classes of therapies have generally been used to treat influenza viruses: M2 ion channel inhibitors and neuraminidase inhibitors. However, influenza viruses not only show widespread drug resistance to M2 ion channel inhibitors, but also gradually develop antiviral resistance to neuraminidase inhibitors.

Cap (cap)-dependent endonuclease inhibitor controls the key link of influenza virus replication and inhibits the virus from obtaining the Cap structure at the 5' end of host mRNA from the host cell, thereby inhibiting the transcription of influenza virus's own mRNA and achieving the effect of treating influenza . And since there is no protease with a similar mechanism in the host cell, the Cap-dependent endonuclease inhibitor will not affect the host cell. In 2018, the first cap-dependent endonuclease (CEN) inhibitor, baloxavir marboxil (trade name: Xofluza), was approved in the United States and Japan for the treatment of influenza A and B. The prodrug baloxavir dipivoxil is converted to its active ingredient baloxavir by hydrolysis. Baroxavir inhibits influenza virus polymerase acid (PA) protein endonuclease, inhibits the synthesis of viral RNA, and thus can effectively inhibit the replication of influenza virus. However, the oral availability of baloxavir dipivoxil and its active component baloxavir is low.

The patent application with publication number WO2021/007506A1 provides a Cap-dependent endonuclease inhibitor, which can be used to treat influenza virus, exhibits excellent biological activity and pharmacokinetic properties including good oral bioavailability, and Unaffected by eating. However, further improvements are needed to obtain stable and manufacturable therapeutics.

The present invention aims to solve one of the above-mentioned technical problems at least to a certain extent. The invention provides a crystal form and application of a compound for treating influenza. The provided compound crystal form is more stable, has low hygroscopicity, and is more suitable for GMP scale-up production. The crystal form of the compound provided by the invention can be used as an influenza virus endonuclease inhibitor to inhibit the replication of influenza virus. The crystalline form of the compound is especially useful for treating or preventing human influenza virus infection.

式A 所述晶型具有至少兩個選自2θ為10.7±0.2、13.1 ±0.2、15.6±0.2，16.0±0.2、17.5±0.2、20.1±0.2、21.9±0.2、24.1±0.2、26.5±0.2和29.7±0.2的XRPD特徵峰。 The first aspect of the present invention provides a crystal form of a compound, the compound is a compound of formula A,

The crystal form of Formula A has at least two 2θ selected from 10.7±0.2, 13.1±0.2, 15.6±0.2, 16.0±0.2, 17.5±0.2, 20.1±0.2, 21.9±0.2, 24.1±0.2, 26.5±0.2 and Characteristic XRPD peak of 29.7 ± 0.2.

式A 所述晶型具有如下晶胞參數： a為約7.1埃、b為約20.0埃、c為約9.4埃、α為約90.0∘、β為約108.8∘、γ為約90.0 ∘。 The second aspect of the present invention provides a crystal form of a compound, the crystal form is a monoclinic crystal, and the compound is a compound represented by formula A,

The crystal form of Formula A has the following unit cell parameters: a is about 7.1 angstroms, b is about 20.0 angstroms, c is about 9.4 angstroms, α is about 90.0∘, β is about 108.8∘, and γ is about 90.0 ∘.

The third aspect of the present invention provides a pharmaceutical composition, which includes the above crystal form and a pharmaceutically acceptable carrier.

The fourth aspect of the present invention provides a method for inhibiting the activity of influenza virus in vitro, comprising: contacting cells with an effective amount of the above crystal form or the pharmaceutical composition in vitro.

The fifth aspect of the present invention provides a method for preventing or treating influenza, comprising administering an effective amount of the above-mentioned crystal form or the above-mentioned pharmaceutical composition to a subject in need.

The present invention also provides a method for preparing the above crystal form. In some embodiments, the provided crystal form of Compound A can be prepared by the following method: Compound A is dissolved in a single solvent or a mixed solvent; Crystallization is evaporated to remove the solvent in the solution of Compound A to obtain the crystalline form of Compound A.

In some embodiments, the provided crystalline form of Compound A can be prepared by the following method: Compound A is dissolved in a single solvent or a mixed solvent; Part of the solvent in the solution of compound A is concentrated by distillation, so as to obtain a concentrated solution of compound A; The temperature is lowered to crystallize so as to obtain the crystalline form of Compound A.

In at least some specific embodiments, the single solvent is an organic solvent, and the mixed solvent is a mixed solution of an organic solvent and water. Organic solvents mentioned include, but are not limited to, acetonitrile, acetone, isopropanol, n-heptane, methyl tertiary butyl ether, isopropyl acetate, ethanol, toluene, methanol, ethyl acetate, and the like.

In some preferred embodiments, by the method of recrystallization, the easily soluble solvent acetone is used as the recrystallization solvent, under reflux conditions, the crude product of compound A is dissolved, then the acetone is concentrated, and the poor solvent methyl tertiary butyl ether ( MTBE) was crystallized, and it was found that the purity of the crystal form of compound A could be significantly improved, and the solvent residue was significantly reduced.

In some preferred embodiments, the crude product of Compound A is dissolved by refluxing acetone, the acetone solvent is concentrated to 0.2-0.3 times the original volume, the temperature is lowered to 10-30 degrees Celsius, and then the volume of the original acetone solvent is added dropwise to 0.4-0.6 Double the amount of methyl tertiary butyl ether, continue to crystallize, and filter to obtain the crystalline product.

In a preferred embodiment, the crude product of compound A is dissolved by reflux with acetone (7V), concentrated to a volume of about 1.5V, cooled to 20 degrees Celsius, and then added dropwise with 3V methyl tertiary butyl ether to continue crystallization, The crystalline product was obtained by filtration; wherein "V" represents the volume-to-mass ratio of the solvent to compound A, and the unit is mL/g.

The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention. At the same time, some terms appearing in the text are explained and illustrated, and these explanations and illustrations are only used to facilitate the understanding of those skilled in the art, and should not be regarded as limiting the protection scope of the present invention.

For the description of the crystal form of the compound herein, unless otherwise specified, the numerical values in the specification and scope of claims are all approximate values. Taking into account factors such as instrument calibration and instrument error, the values of different determinations may vary slightly, as long as those skilled in the art can judge that the crystal form of the compound is the same. Herein, the term "about" or "approximately" means that the parameter recognized by those skilled in the art may be similar to the crystal form of the provided compound, as long as it can be confirmed as the same crystal form. In some embodiments, the term "about" or "approximately" is 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, of a recited value or a recited range of values. 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%.

Among the present invention, X-ray powder diffraction measurement method is a commonly used measurement method for those skilled in the art, for example as follows: Utilize PANalytical EMPYREAN equipped with PIXceI ^1D detector to analyze the crystal form of the solid obtained in the experiment, and the instrument parameters are as follows : Scanning range: 3 ^o (2θ) to 40 ^o (2θ); step size: 0.013 ^o (2θ); light tube voltage and current are 45 KV and 40 mA, respectively.

Crystal form of compound

式A 所述晶型具有至少一個選自2θ為約10.7、約13.1、約15.6、約16.0、約17.5、約20.1、約21.9、約24.1、約26.5和約29.7的XRPD特徵峰。 The present invention provides a crystal form of a compound, the compound is a compound represented by formula A,

The crystalline form of Formula A has at least one XRPD characteristic peak selected from the group consisting of about 10.7, about 13.1, about 15.6, about 16.0, about 17.5, about 20.1, about 21.9, about 24.1, about 26.5 and about 29.7 in 2θ.

X-ray powder diffraction (XPRD) is a method to determine the crystallinity of drugs. It irradiates the sample with X-rays, and the X-rays irradiated on the crystal are reflected by the lattice planes and interfere with each other, showing different laws. The intensity and distribution of diffraction lines of different crystals have their own special regulations, and information such as changes in crystal forms and crystallinity can also be determined.

The crystal forms of the compounds provided by the present invention have distinguishable X-ray powder diffraction curves. For example, whether it is the crystal form of the compound shown in Formula A can be distinguished by the characteristics of the diffraction peaks. The crystalline form has at least one XRPD characteristic peak selected from the group consisting of about 10.7, about 13.1, about 15.6, about 16.0, about 17.5, about 20.1, about 21.9, about 24.1, about 26.5, and about 29.7 in 2Θ. Preferably having at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine selected from the group consisting of about 10.7, about 13.1, about 15.6, about 16.0 in 2θ , about 17.5, about 20.1, about 21.9, about 24.1, about 26.5 and about 29.7 XRPD characteristic peaks. In some embodiments, the crystalline form has XRPD characteristic peaks of about 10.7, about 13.1, about 15.6, about 16.0, about 17.5, about 20.1, about 21.9, about 24.1, about 26.5, and about 29.7 2Θ.

In some embodiments, the crystal form has at least one selected from 2θ of 10.7±0.2, 13.1±0.2, 15.6±0.2, 16.0±0.2, 17.5±0.2, 20.1±0.2, 21.9±0.2, 24.1±0.2, 26.5 XRPD characteristic peaks of ±0.2 and 29.7±0.2. Preferably there are at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine selected from 2θ of 10.7±0.2, 13.1±0.2, 15.6±0.2 , 16.0±0.2, 17.5±0.2, 20.1±0.2, 21.9±0.2, 24.1±0.2, 26.5±0.2 and 29.7±0.2 XRPD characteristic peaks.

In some embodiments, the crystalline form has a 2θ of 10.7±0.2, 13.1±0.2, 15.6±0.2, 16.0±0.2, 17.5±0.2, 20.1±0.2, 21.9±0.2, 24.1±0.2, 26.5±0.2, and 29.7 XRPD characteristic peaks of ±0.2.

In some embodiments, the crystal form has at least one selected from the group consisting of 2θ of 10.7±0.1, 13.1±0.1, 15.6±0.1, 16.0±0.1, 17.5±0.1, 20.1±0.1, 21.9±0.1, 24.1±0.1, 26.5 XRPD characteristic peaks of ±0.1 and 29.7±0.1. Preferably there are at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine selected from the group consisting of 2θ of 10.7±0.1, 13.1±0.1, 15.6±0.1 , 16.0±0.1, 17.5±0.1, 20.1±0.1, 21.9±0.1, 24.1±0.1, 26.5±0.1 and 29.7±0.1 XRPD characteristic peaks.

In some embodiments, the crystalline form has a 2θ of 10.7±0.1, 13.1±0.1, 15.6±0.1, 16.0±0.1, 17.5±0.1, 20.1±0.1, 21.9±0.1, 24.1±0.1, 26.5±0.1, and 29.7 XRPD characteristic peaks of ±0.1.

In some embodiments, the crystalline form further has at least one XRPD characteristic peak selected from the group consisting of about 14.1, about 18.8, about 20.6, and about 21.6 in 2Θ. For example, having one, two, three or four XRPD characteristic peaks selected from the group consisting of about 14.1, about 18.8, about 20.6, and about 21.6 in 2Θ.

In some embodiments, the crystalline form further has at least one XRPD characteristic peak selected from 2θ of 14.1±0.2, 18.8±0.2, 20.6±0.2 and 21.6±0.2. For example, having one, two, three or four XRPD characteristic peaks selected from 2Θ of 14.1±0.2, 18.8±0.2, 20.6±0.2 and 21.6±0.2.

In some embodiments, the crystalline form further has at least one XRPD characteristic peak selected from 2θ of 14.1±0.1, 18.8±0.1, 20.6±0.1 and 21.6±0.1. For example, having one, two, three or four XRPD characteristic peaks selected from 2Θ of 14.1±0.1, 18.8±0.1, 20.6±0.1 and 21.6±0.1.

In some embodiments, the crystalline form further has at least one characteristic XRPD peak selected from the group consisting of about 9.7, about 16.4, about 19.1, about 22.2, about 23.7, about 25.6, about 28.0, about 28.3, and about 31.3 2Θ. For example, having one, two, three, four, five, six, seven, eight or nine selected from the group consisting of about 9.7, about 16.4, about 19.1, about 22.2, about 23.7, about 25.6, Characteristic XRPD peaks at about 28.0, about 28.3 and about 31.3.

In some embodiments, the crystal form further has at least one selected from the group consisting of 2θ of 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and XRPD characteristic peak of 31.3 ± 0.2. For example, having one, two, three, four, five, six, seven, eight or nine selected from 2θ of 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2 XRPD characteristic peaks at 0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3±0.2.

In some embodiments, the crystal form further has at least one selected from the group consisting of 2θ of 9.7±0.1, 16.4±0.1, 19.1±0.1, 22.2±0.1, 23.7±0.1, 25.6±0.1, 28.0±0.1, 28.3±0.1 and XRPD characteristic peak of 31.3 ± 0.1. For example, having one, two, three, four, five, six, seven, eight or nine selected from 2θ of 9.7±0.1, 16.4±0.1, 19.1±0.1, 22.2±0.1, 23.7±0.1 XRPD characteristic peaks at 0.1, 25.6±0.1, 28.0±0.1, 28.3±0.1 and 31.3±0.1.

In some embodiments, the crystalline form has an X-ray powder diffraction pattern substantially as shown in FIG. 1 .

In some embodiments, the crystalline form has a melting peak at 221-224 degrees Celsius.

In some embodiments, the crystalline form has a DSC and TGA profile substantially as shown in FIG. 2 .

式A 所述晶型具有如下晶胞參數： a為約7.1埃、b為約20.0埃、c為約9.4埃、α為約90.0∘、β為約108.8∘、γ為約90.0 ∘。 The present invention also provides a crystal form of a compound, the crystal form is a monoclinic crystal, and the compound is a compound shown in formula A,

The crystal form of Formula A has the following unit cell parameters: a is about 7.1 angstroms, b is about 20.0 angstroms, c is about 9.4 angstroms, α is about 90.0∘, β is about 108.8∘, and γ is about 90.0 ∘.

In a specific measurement, the crystal form has the following unit cell parameters: a is 7.1465 angstroms, b is 19.9876 angstroms, c=9.4303 angstroms, α is 90.0∘, β is 108.762∘, and γ is 90.0 ∘.

Compound A is provided in a substantially pure crystalline form. Reference to substantially pure crystalline form means that the formed crystals contain no more than 10% impurities, for example less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1% impurities. Preparation of the crystal form of the compound The substantially pure crystal form of the compound can be obtained by common methods in the art. In some embodiments, substantially pure crystalline forms of Compound A are provided at levels of purity of 97.0%, 97.5%, 98.0%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1% , 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or higher.

In some embodiments, the provided crystalline form of Compound A can be prepared by the following method: Compound A is dissolved in a single solvent or a mixed solvent; Crystallization is evaporated to remove the solvent in the solution of Compound A to obtain the crystalline form of Compound A.

In some embodiments, the provided crystalline form of Compound A can be prepared by the following method: Compound A is dissolved in a single solvent or a mixed solvent; Part of the solvent in the solution of compound A is concentrated by distillation, so as to obtain a concentrated solution of compound A; The temperature is lowered to crystallize so as to obtain the crystalline form of Compound A.

In at least some specific embodiments, the single solvent is an organic solvent, and the mixed solvent is a mixed solution of an organic solvent and water. Organic solvents mentioned include, but are not limited to, acetonitrile, acetone, isopropanol, n-heptane, methyl tertiary butyl ether, isopropyl acetate, ethanol, toluene, methanol, ethyl acetate, and the like.

pharmaceutical composition

The present invention also provides a pharmaceutical composition, which comprises any one of the crystal forms described above and a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier refers to an ingredient other than an active ingredient that is non-toxic to a subject, and these pharmaceutically acceptable carriers may be carriers commonly used in the art. Pharmaceutically acceptable carriers may include buffers, excipients, stabilizers or preservatives, etc. according to their functions. For example, it can be adjuvants commonly used in the art as binders, disintegrants, lubricants, suspending agents and the like.

Treatment and Use

The present invention also provides the use of the above-mentioned crystal form or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing or treating influenza.

The present invention also provides a method for preventing or treating influenza, comprising administering an effective amount of the above-mentioned crystal form or the above-mentioned pharmaceutical composition to a subject in need.

The present invention also provides a method for preventing or treating a subject infected with type A, B or C influenza virus, comprising administering an effective amount of the above crystal form or the above pharmaceutical composition to the subject in need.

The present invention also provides the above-mentioned crystal form or the above-mentioned pharmaceutical composition, which is used for preventing or treating influenza, especially type A influenza virus, type B influenza virus or type C influenza virus.

The crystalline form of the compound disclosed herein exhibits the ability to inhibit or prevent influenza virus replication and pharmaceutical properties, as shown by the following test data, can be indicated for the inhibition of influenza virus (especially type A influenza virus, type B influenza virus or influenza C virus) replicates. Accordingly, the compounds of the present invention are useful in the treatment of infections caused by influenza viruses (particularly influenza A, B or C), especially in human subjects having or at risk of contracting an influenza virus infection. As a further aspect, the invention provides the use of a crystalline form of a compound as described herein as a therapeutic agent. The crystalline form of the compound is suitable for the treatment of subjects who have or are at particularly high risk of influenza virus infection, especially influenza A, influenza B or influenza C.

An "effective amount" or "effective dose" referred to herein is an amount sufficient to achieve a beneficial or desired result. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of disease, including biochemical, histological, and/or behavioral symptoms of disease, Its complications and intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired results include amelioration, alleviation, alleviation, delay or reduction of the symptoms produced by one or more diseases, improving the quality of life of those suffering from the diseases, reducing the dosage of other agents needed to treat the diseases, such as Via targeting to enhance the effect of another agent, delay disease progression and/or prolong survival. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay relapse. An effective dose can be administered in one or more administrations. For the purposes of this disclosure, an effective dosage of a crystalline form of a compound or a pharmaceutical composition containing the crystalline form is an amount sufficient to achieve, directly or indirectly, a prophylactic or therapeutic purpose. It should be understood that an effective dosage of a crystalline form of a compound or a pharmaceutical composition containing the crystalline form may be achieved alone or in combination with another drug, compound or pharmaceutical composition. Thus, an "effective dose" can be understood in the context of the administration of one or more therapeutic agents, and a single agent can be considered to be administered in an effective amount if the desired result is achieved or achieved in combination with one or more other therapeutic agents. .

Herein, the term "subject" is a mammal, including a human. Subjects include, but are not limited to, humans, cows, horses, cats, dogs, rodents or primates. In some embodiments, the subject is a human.

Products and Kits

The present disclosure also provides articles of manufacture comprising, in suitable packaging, the crystalline forms, pharmaceutical compositions and unit dosages of the compounds described herein. In certain embodiments, the articles of manufacture are used in any of the methods described herein. Suitable packaging is known in the art and includes, for example, vials, vessels, ampoules, bottles, jars, flexible packaging, and the like. The articles can be further sterilized and/or sealed.

The disclosure also provides kits for practicing the methods of the disclosure comprising one or more crystalline forms of the compounds described herein or a pharmaceutical composition comprising a crystalline form of the compounds described herein. The kits may employ crystalline forms of the compounds disclosed herein. The kits may be used for any one or more of the uses described herein, and thus the kits may contain instructions for the treatment of the diseases described herein.

Kits are usually included in suitable packaging. A kit may comprise one or more containers comprising a crystalline form of a compound described herein. If more than one component is present, the different components may be packaged in separate containers, or some components may be combined in one container.

Kits can be in unit dosage form, bulk packs or subunit doses. For example, a kit may be provided containing sufficient doses of a crystalline form of a compound as disclosed herein and/or a second pharmaceutically active compound useful for a disease as detailed herein. The kit may also include multiple unit doses of the compound and instructions for use, and be packaged in an amount sufficient for storage and use in a pharmacy.

The kit may also optionally include a set of instructions, usually written instructions, or electronic storage media, such as magnetic or optical disks, related to the use of one or more components of the method of the present invention. Instructions included in the kit generally include information about the components and their administration to an individual.

Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

The synthesis of the compound of the present invention can be carried out with reference to methods known in the art. As the starting compound, commercially available compounds, compounds described in the specification, compounds described in references cited in the specification, and other known compounds can be utilized.

synthetic example

Reagents such as solvents used in the examples are available from commercial sources. ¹ H NMR spectra were recorded on a Varian III plus 300 MHz and TMS was used as internal standard. List valid peaks in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet), in hertz (Hz) Calculated coupling constant and proton number. Mass spectral results are reported as mass-to-charge ratios, followed by the relative abundance of each ion in brackets. Electron spectroscopy was performed on an Agilent LC/MSD 1200 series quadrupole mass spectrometer (column: Welchrom XB-C18 (50 × 4.6 mm, 5 μm); T=30°C; flow rate=1.5 ml/min; detection wavelength: 214 nm). Spray ionization (ESI) mass spectrometry.

The term "solvent" refers to a solvent that is inert under the described reaction conditions, ie, does not take part in the reaction process. Solvents involved in the following examples include, for example, acetonitrile, acetone, isopropanol, n-heptane, methyl tertiary butyl ether, isopropyl acetate, ethanol, toluene, methanol, ethyl acetate, water, dichloromethane , dimethylsulfoxide, butanone, tetrahydrofuran, etc. Unless stated to the contrary, the solvents used in the reactions of the present invention are inert organic solvents.

During the research process, the salt of compound A and its original drug and its formed crystal form were studied, and it was found that the formed salt was unstable, which did not help to further improve and enhance the stability of compound A and scale up production. . During the research process, it was creatively found that by preparing the crystal form of Compound A, a crystal form product with high stability and extremely low hygroscopicity was obtained, which is suitable for scale-up production.

Example 1

式A Example 1 The crystalline form of Compound A was prepared according to the following method. Firstly, the following compound A (that is, the compound C-1 mentioned in the WO2021007506A1 publication) was prepared by referring to the method described in the publication number WO2021007506A1.

Formula A

The acetonitrile/water solution containing the product was purified by preparative HPLC (Pre-HPLC), and then the acetonitrile in the solution was removed by rotary evaporation to obtain the crystalline product, and the corresponding crystal was obtained by filtration, that is, sample B was obtained.

The sample B prepared above was characterized by XRPD, DSC and TGA.

The X-ray powder diffraction determination method is as follows: The crystal form of the solid obtained in the experiment was analyzed by using PANalytical EMPYREAN equipped with a PIXceI ^1D detector. The instrument parameters are as follows: scanning range: 3 ^o (2θ) to 40 ^o (2θ); step size: 0.013 ^o (2θ); light tube voltage and current are 45 KV and 40 mA, respectively.

The results show that the prepared sample B is crystal form 1, and its XRPD pattern is basically shown in FIG. 1 . The XRPD results are shown in Table 1 below:

Table 1 XRPD characterization results 2θ value strength(%) 2θ value strength(%) 8.69 100 22.17 7.6 9.74 6.5 22.84 1.1 10.68 25.6 23.75 7.3 12.91 6.6 24.12 20.8 13.10 14.7 25.04 3.1 13.63 0.9 25.61 6.4 14.11 10.2 25.87 1 15.61 14.9 26.46 40.5 16.04 18.1 26.98 1.8 16.38 5.9 27.37 0.9 17.52 35.8 27.54 1.1 18.47 3.1 28.04 4 18.83 9.7 28.28 4.4 19.15 7 28.59 1.8 19.63 3.7 29.31 1.5 20.12 41.4 29.68 19.7 20.62 9.4 30.04 1.9 21.55 9.7 31.28 4.7 21.86 16.2 - -

Differential Scanning Calorimetry (DSC) is one of the main measurement methods used in thermal analysis and can be used to measure the thermal properties of substances as aggregates of atoms/molecules. The differential scanning calorimetry curve can be obtained by measuring the heat capacity change of a substance with temperature or time by DSC, and plotting the obtained data against temperature or time. Based on this curve, the initial concentration, endothermic maximum, and melting peak of the substance, etc. can be obtained. The analysis method is as follows: Thermal analysis of the samples was carried out using Discovery DSC 250 (TA Instruments, US). The sample was weighed and placed in the DSC sample pan and punched. The sample was equilibrated at 25 ^° C and heated to the final temperature at a rate of 10 ^° C/min.

The thermogravimetric analysis (TGA) method is as follows: The samples were subjected to thermogravimetric analysis using TGA 55 (TA Instruments, US). Place the sample in a peeled open aluminum sample pan, and after the quality of the sample is automatically weighed in the TGA heating furnace, the sample is heated to the final temperature at a rate of 10°C/min.

The experimental results show that sample B has an endothermic peak at about 225 degrees Celsius, which is caused by the endothermic melting of the crystalline form, and the melting point is about 221 degrees Celsius; TGA results show that the sample has a slight weight loss (about 1.8%) before melting, as shown in Figure 2.

In addition, in Example 1, a single crystal of Compound A was prepared by slow cooling and crystallization. About 50 mg of the compound A sample prepared in Example 1 was dissolved in 0.5 mL of methanol under heating. The solution was filtered and the filtrate was left at room temperature overnight. Cooling and crystallization in methanol gave rod-like crystals.

Single crystal X-ray diffraction data of compound A were collected by Bruker D8 X-ray diffractometer with Ga Ka rays (λ=1.34139Å) at 169.99 K. Using Olex2, the SHELXT structure analysis program was used to analyze the structure using the intrinsic phase method, and the SHELXL optimization package was used to optimize the structure using the least square method.

The single crystal sample belongs to the monoclinic system, the space group P 1 21 1, and the molecular formula is C27H23F2N3O7Se. Each asymmetric unit contains one compound A molecule, and each unit cell contains 2 molecules. The molecular structure contains two chiral carbon atoms in "S" (C7) and "R" (C15) configurations. The single crystal structure is consistent with the molecular structure. Its single crystal structure parameters are shown in Table 2 below.

Table 2 Single crystal structure parameters chemical formula C ₂₇ H ₂₃ F ₂ N ₃ O ₇ Se Molecular weight (g/mol) 617.43 temperature (K) 169.99K Wavelength (Å) 1.34139 Å crystal system monoclinic crystal space group P1 21 1 a (Å) a=7.1465(3) Å b (Å) b=19.9876(8) Å c (Å) c=9.4303(4) Å α=90∘ β=108.762(2)∘ γ=90 ∘ unit cell volume 1275.46(9) Å ³ Z 2 ρcalc (g/cm3) 1.608Mg/ ^m3 Absorption Coefficient(mm-1) 1.753 mm F(000) 626 crystal size 0.08x0.08x0.05mm ³ Data acquisition in theta range 4.308<θ<54.929 ∘ Index range -8<=h<=8, 0<=k<=24, 0<=l<=11 The number of diffractions collected 2484 independent diffraction number 2484 to θ equals 53.594 data integrity 99.7% Absorption correction Semi-empirical from equivalents Maximum and minimum transmittance 0.7508 and 0.5274 Refining method Full Matrix Least Squares Method for F2 Data, Convergence and Parameters 2484/1/363 Goodness of fit for F2 1.084 final R index R1=0.0638, wR2=0.1740 R index R1=0.0651, wR2=0.1785

The XRPD fitted by Mercury through the single crystal is compared with the XRPD measured by the above experiment, and it is found that the two are highly matched.

Example 2

Example 2 uses the crystal form 1 obtained in Example 1 as the starting material, and explores the influence of different experimental methods on the crystal form 1, as follows:

1. Suspension stirring

Put the starting material into a single solvent (isopropanol, n-heptane, water, methyl tertiary butyl ether, isopropyl acetate, ethanol, toluene, methanol or ethyl acetate) to form a suspension, and in room Suspended and stirred under warm or high temperature conditions (50°C) for 3 days, and filtered the obtained solid for XRPD characterization. By comparing the XPRD results of crystal form 1 and crystal form 1 as the starting material after suspension and stirring, it was shown that all solvents obtained A stable crystal form 1 with good crystallinity. The specific results are shown in Table 3 and Table 4.

Table 3 Stirring results of single suspension at room temperature serial number solvent XRPD results evaluate S1 Isopropanol Form 1 Good crystallinity S2 n-heptane Form 1 Good crystallinity S3 water Form 1 Good crystallinity S4 Methyl tertiary butyl ether Form 1 Good crystallinity S5 Isopropyl acetate Form 1 Good crystallinity S6 ethanol Form 1 Good crystallinity S7 toluene Form 1 Good crystallinity S8 Methanol Form 1 Good crystallinity S9 ethyl acetate Form 1 Good crystallinity

Table 4 High temperature single suspension stirring results serial number solvent XRPD results evaluate S10 Isopropanol Form 1 Good crystallinity S11 n-heptane Form 1 Good crystallinity S12 water Form 1 Good crystallinity S13 Methyl tertiary butyl ether Form 1 Good crystallinity S14 Isopropyl acetate Form 1 Good crystallinity S15 ethanol Form 1 Good crystallinity S16 Toluene Form 1 Good crystallinity S17 Methanol Form 1 Good crystallinity S18 ethyl acetate Form 1 Good crystallinity

The above results show that the prepared crystal form 1 is not easily affected by the solvent, has good stability and good crystallinity.

2. Anti-solvent precipitation

Take a certain amount of starting material and dissolve it in 0.1-1.2 mL of good solvent, and add the filtered clear liquid into a poor solvent for precipitation experiment. For example, anti-solvent precipitation was carried out in a system in which dichloromethane, acetonitrile, dimethylsulfoxide, butanone, acetone, and tetrahydrofuran were used as good solvents, and isopropanol, n-heptane, water, etc. were used as anti-solvents, and crystals were obtained. Type 1. The solid obtained by filtration was characterized, and the results are shown in Table 5.

Table 5 Anti-solvent precipitation results serial number solvent temperature Solvent volume (mL) Anti-solvent Anti-solvent volume (mL) result W1 Dichloromethane RT 0.25 Isopropanol 1 Form 1 W2 Dichloromethane RT 0.25 n-heptane 0.7 Form 1 W3 Dichloromethane RT 0.25 Methyl tertiary butyl ether 1 Form 1 W4 Acetonitrile RT 0.5 water 1.5 Form 1 W5 DMSO RT 0.9 water 0.9 Form 1 W6 butanone RT 1 n-heptane 3 Form 1 W7 acetone RT 1.2 n-heptane 5 Form 1 W8 acetone RT 1.2 water 3 Form 1 W9 Tetrahydrofuran 50℃ 1 n-heptane 1 Form 1

3. Cooling and crystallization

Rapid cooling: Weigh about 50 mg of sample B and dissolve it in 0.4 mL of methanol, dissolve at 50°C, filter the solution while it is hot, and store the filtrate directly in a refrigerator at 5°C for about 20 h. XRPD test was carried out on the precipitated crystals.

Slow cooling: Weigh about 50 mg of sample B and dissolve it in 0.4 mL of methanol, dissolve it at 50°C, filter the solution while it is hot, and place the filtrate at room temperature to cool down slowly for about 20 h. XRPD test was carried out on the precipitated crystals.

The experimental results show that crystal form 1 was obtained by cooling and crystallizing at two speeds.

4. Evaporation crystallization

Evaporation crystallization experiments were carried out using mixed solvents to find out whether other crystals could be obtained. For example, weigh about 20 mg of sample B into a sample bottle, add 0.1-1 mL of a selected solvent (mixed solvent), filter the solution, place the filtrate in a sample bottle, and dry at room temperature at different rates . Prepare slow evaporating samples by covering vials with Parafilm with small holes. The obtained solid was characterized by PLM or XRPD.

The results show that in mixed solvent evaporation crystallization experiments, mixed systems such as dichloromethane/butanone and dichloromethane/acetone binary solvent systems can still obtain crystal form 1 with better crystallinity, and in some binary solvent systems such as The solids obtained in acetonitrile/butanone, butanone/tetrahydrofuran were amorphous samples. No new crystal form was found in the experiment.

Example 3

Example 3 evaluates the effect of different mechanical treatments on the crystal form. In the experiment, the effect on the crystal form 1 was investigated by grinding experiment and pressure experiment respectively. As follows:

1. Grinding experiment

An appropriate amount of sample B was placed in a mortar and ground for 2-5 minutes at room temperature, and the solids were collected at two and five minutes for XRPD detection. Experimental results show that the crystal form remains unchanged after grinding.

2. Pressurization experiment

Put an appropriate amount of starting material under a pressure of 40 Mpa, pressurize for about 1 minute, and collect the solid for XRPD detection. The experimental results show that the crystal form remains unchanged after pressurization, all of which are crystal form 1.

3. Solid state stability test

Sample B was subjected to 7-day closed solid stability and chemical stability experiments at 40°C/75%RH and 60°C, respectively, and analyzed by XRPD and HPLC.

The measuring instruments used in high performance liquid chromatography analysis (HPLC) are Agilent HPLC 1260 series instruments. The HPLC measurement method is shown in Table 6.

Table 6 HPLC measurement method parameters Column YMC-Triart C18 EXRS, 4.6*150 mm, 3 μm mobile phase A: 0.02% trifluoroacetic acid in water B: 0.02% trifluoroacetic acid in acetonitrile Time/Component (T/B %) 0/5%, 8/50%, 15/50%, 20/95%, 22/95%, 22.1/5% Column temperature 30℃ Detector DAD; 220 nm flow rate 1.0 mL/min Injection volume 5 μL keep time 14.3 minutes cleaning time 4 minutes Thinner Acetonitrile

The XRPD results showed that the crystal form of Form 1 remained unchanged after 7 days under closed conditions of 40°C/75%RH and 60°C (as shown in Figure 3). The HPLC results showed that the chemical purity of the crystal form 1 would not decrease when placed under the condition of 40°C/75%RH for 7 days, and the stability was good. When placed under closed conditions at 60°C for 7 days, its chemical purity only decreased by 0.14%. The results are shown in Table 7.

Table 7 HPLC results of stability samples of crystal form 1 condition XRPD HPLC detection of purity 0 days Form 1 100% 40℃/75% for one week Form 1 100% 60°C/closed mouth for one week Form 1 99.86%

Example 4

First, Sample C was prepared as follows:

Obtain the crude product of compound A, use acetone (7V) to reflux to dissolve, distill and crystallize, concentrate to a volume of about 1.5V, cool down to 20 degrees Celsius, then add 3V methyl tertiary butyl ether dropwise to continue crystallization, and filter to obtain the product. Named Sample C. The prepared sample C is crystal form 1.

The moisture absorption weight gain of sample C at 25°C/80%RH was measured by dynamic water vapor adsorption. The test results showed that the moisture absorption weight gain of sample C was only 0.03% at 25°C/80%RH Hygroscopicity.

In addition, during the research process, it was found that the crystal form 1 of compound A is more stable during the scale-up production process, and is more suitable for production scale-up. For example, through the method of recrystallization, the easily soluble solvent acetone is used as the recrystallization solvent, and the crude product is dissolved at 55°C, then the acetone is concentrated, and the poor solvent methyl tertiary butyl ether (MTBE) is added for crystallization. It is found that it can The purity of the product containing compound A crystal form 1 is significantly improved, and the solvent residue is significantly reduced.

Example 5

Example 5 The in vivo antiviral efficacy of sample C prepared in Example 4 was evaluated using the influenza A virus ferret infection model.

In the experiment, female M.putorius ferrets (purchased from Wuxi Coral Reef Biotechnology Co., Ltd.) were infected with influenza A virus (influenza virus A/PR/8/34, purchased from ATCC, article number: VR-1469 ), the infection amount was 5.4*10 ⁶ PFU (plaque forming unit, plaque forming unit) per animal, and the inoculation volume was 0.2 mL (0.1 mL/nostril); The nasal lavage fluid of experimental ferrets was collected every hour, and the virus titer in it was detected to evaluate the inhibitory effect of the samples on virus replication.

Experimental animals were divided into 4 groups. The treatment groups are as follows: Sample C administration group: Weigh an appropriate amount of sample C and dissolve it in 5 vol % DMSO + 40 vol % PEG400 + 55 vol % DI water, and the prepared concentrations are 0.3 mg/mL and 2.0 mg/mL respectively. Positive control group: weigh baloxavir dipivoxil (product number: 1627-125-22) powder, dissolve it in an appropriate amount of 20% by volume PEG400 + 20% by volume (30% solutol HS 15 aqueous solution) + 60% by volume normal saline (0.9 %), the preparation concentration is 2.0mg/mL. Vehicle group: comprising 5 vol% DMSO + 40 vol% PEG400 + 55 vol% DI water.

The delivery method is as follows: Continuous administration at an administration volume of 5 mL/kg was administered continuously for 3 days in groups 1-4, and the first administration time was 2 hours before virus inoculation. Administer twice a day with an interval of 8/16 hours by gavage.

(1) Determination of body weight changes of ferrets in different treatment groups, the results are shown in Figure 4. Taking day 0 body weight as the baseline body weight, the percentage of daily body weight change of ferrets was calculated. Error bars are standard errors, and dashed lines are humane endpoints below which any ferret weight loss (20%) will be euthanized.

(2) Score the clinical symptoms of ferrets in different treatment groups. The scoring criteria are as follows: Activity level: 0 points for normal activity level, 1 point for decreased activity level, 2 points for inactivity and lethargy; Secretion: 0 points for no nasal secretions, 1 point for nasal secretions; Sneezing: 0 points for no sneezing, 1 point for sneezing.

The clinical symptoms were observed 4 times a day (9:00--21:00), once every 4 hours, and 20 minutes each time.

After virus inoculation, the clinical symptom score results of the ferrets in each group are shown in Figure 5. The ferrets in the vehicle group were affected by the virus infection, and the frequency of lethargy, nasal secretions, and sneezing was significantly increased clinically. The clinical symptoms of ferrets in the control compound baloxavir group (10mg/kg) were relieved, and the frequency of lethargy, nasal secretions, and sneezing symptoms decreased. While administering sample C, under the set dose (1.5 mg/kg, 10 mg/kg), the clinical symptoms of the ferrets were better than those of the vehicle group, and the symptoms of lethargy, nasal secretions, and sneezing were significantly improved, and the symptoms increased with the dose , the milder the clinical symptoms.

Wherein in Fig. 5, compared with vehicle group: * (the 2nd group), p＜0.05; # (the 3rd group), p＜0.05; ^ (the 4th group), p＜0.05, ^^, p＜ 0.01, ^^^, p<0.001, Two Way ANOVA.

(3) The samples of nasal lavage fluid were collected 72 hours after virus inoculation. The titers of influenza A virus in the nasal cavity lavage fluid of ferrets in each group are summarized in Figure 6, and were determined by plaque method. The first group is the vehicle group , the second group is baloxavir dipivoxil 10 mg/kg treatment group, the third group is sample C 1.5 mg/kg treatment group, the fourth group is sample C 10 mg/kg treatment group, LLOQ in Figure 6 is the lower limit of quantitation. Among them, the average virus titer in the ferret nasal lavage fluid of the vehicle group was 5.056 Log 10 (plaque number/mL nasal cavity lavage fluid, the same below), and the reference compound baloxavir dipivoxil (10 mg/kg) The average virus titer in the samples of the group was 1.402 Log 10; compared with the vehicle group, the differences were statistically significant (p<0.001), showing the expected antiviral efficacy.

And the administration of sample C (1.5 mg/kg, 10mg/kg) group: the first administration 2 hours before infection, the virus titer in ferret nasal lavage fluid at the set dose (1.5 mg/kg, 10mg/kg) 1.280 Log 10 and lower detection limit 1.00 Log 10 , respectively. Compared with the vehicle group, the average virus titers decreased by 3.776 Log 10 and 4.056 Log 10, respectively, and all the differences were statistically significant (p<0.001). Compared with the baloxavir dipivoxil group, the mean virus titers decreased by 0.122 Log 10 and 0.402 Log 10 respectively, and all the differences were statistically significant (p<0.001).

The error bars in Figure 6 show the standard error. One Way ANOVA, Tukey’s multiple comparisons test was used for statistical analysis, compared with the vehicle group: ***, p<0.001.

The above results show that compared with the control group and 10mg/kg baloxavir group, in the 1.5mg/kg and 10mg/kg sample C groups, the animal body weight increased slightly, showing that sample C protected the infected animals from weight loss, while sample C It can improve the clinical symptoms of animals and significantly reduce the viral load in nasal lavage fluid, reflecting excellent antiviral efficacy in vivo.

It should be noted that although the examples of the present invention have been shown and described above, it can be understood that the above-mentioned examples are exemplary and cannot be construed as limitations of the present invention. Variations, modifications, substitutions and variations can be made to the examples described above.

none

Fig. 1 is the XRPD spectrum result of the crystal form 1 of compound A provided according to Example 1 of the present invention. Fig. 2 is the DSC and TGA spectrum results of the crystal form 1 of Compound A provided in Example 1 of the present invention. Fig. 3 is the XRPD pattern results of sample B (ie, the crystal form 1 of compound A) provided according to Example 1 of the present invention after being placed under different conditions. Fig. 4 is a graph showing the results of body weight changes of ferrets in different treatment groups according to Example 5 of the present invention. Fig. 5 is a graph showing the score results of clinical symptoms of ferrets in different treatment groups according to Example 5 of the present invention. Fig. 6 is a graph showing the determination results of influenza A virus titers in different treatment groups according to Example 5 of the present invention.

none.

Claims (16)

A crystal form of a compound, characterized in that the compound is a compound shown in formula A,

The crystal form of Formula A has at least two 2θ selected from 10.7±0.2, 13.1±0.2, 15.6±0.2, 16.0±0.2, 17.5±0.2, 20.1±0.2, 21.9±0.2, 24.1±0.2, 26.5±0.2 and Characteristic XRPD peak of 29.7 ± 0.2. The crystal form according to claim 1, wherein the crystal form has at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine selected from 2θ being XRPD characteristic peaks of 10.7±0.2, 13.1±0.2, 15.6±0.2, 16.0±0.2, 17.5±0.2, 20.1±0.2, 21.9±0.2, 24.1±0.2, 26.5±0.2 and 29.7±0.2; Preferably, the crystal form has 2θ of 10.7±0.2, 13.1±0.2, 15.6±0.2, 16.0±0.2, 17.5±0.2, 20.1±0.2, 21.9±0.2, 24.1±0.2, 26.5±0.2 and 29.7±0.2 XRPD characteristic peaks. The crystal form according to claim 1, wherein the crystal form further has at least one XRPD characteristic peak selected from 2θ of 14.1±0.2, 18.8±0.2, 20.6±0.2 and 21.6±0.2; Preferably, the crystal form further has at least two XRPD characteristic peaks selected from 2θ of 14.1±0.2, 18.8±0.2, 20.6±0.2 and 21.6±0.2; Preferably, the crystal form further has at least three XRPD characteristic peaks selected from 2θ of 14.1±0.2, 18.8±0.2, 20.6±0.2 and 21.6±0.2; Preferably, the crystal form further has XRPD characteristic peaks with 2θ of 14.1±0.2, 18.8±0.2, 20.6±0.2 and 21.6±0.2. The crystal form according to Claim 1, wherein the crystal form further has at least one selected from the group consisting of 2θ of 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, XRPD characteristic peaks of 28.0±0.2, 28.3±0.2 and 31.3±0.2; Preferably, the crystal form further has at least two 2θ selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3 XRPD characteristic peaks of ±0.2; Preferably, the crystal form further has at least three 2θ selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3 XRPD characteristic peaks of ±0.2; Preferably, the crystal form further has at least four 2θ selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3 XRPD characteristic peaks of ±0.2; Preferably, the crystal form further has at least five 2θ selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3 XRPD characteristic peaks of ±0.2; Preferably, the crystal form further has at least six 2θ selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3 XRPD characteristic peaks of ±0.2; Preferably, the crystal form further has at least seven 2θ selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3 XRPD characteristic peaks of ±0.2; Preferably, the crystal form further has at least eight 2θ selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3 XRPD characteristic peaks of ±0.2; Preferably, the crystal form further has a 2θ value selected from 9.7±0.2, 16.4±0.2, 19.1±0.2, 22.2±0.2, 23.7±0.2, 25.6±0.2, 28.0±0.2, 28.3±0.2 and 31.3±0.2 XRPD characteristic peaks. The crystal form according to claim 1, wherein the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG. 1 . The crystal form according to Claim 1, wherein the crystal form has a melting peak at 221-224 degrees Celsius. The crystal form as claimed in claim 1, characterized in that the crystal form has the results of DSC and TGA spectra basically shown in FIG. 2 . A crystal form of a compound, characterized in that the crystal form is a monoclinic crystal, and the compound is a compound shown in Formula A,

The crystal form of Formula A has the following unit cell parameters: a is about 7.1 angstroms, b is about 20.0 angstroms, c is about 9.4 angstroms, α is about 90.0∘, β is about 108.8∘, and γ is about 90.0 ∘. A preparation method of the crystal form described in any one of claim items 1 to 8, characterized in that compound A is dissolved in a single solvent or a mixed solvent, and part of the solvent in the solution of compound A is distilled and concentrated, so as to obtain compound A Concentrated solution of the crystallization at lower temperature in order to obtain the crystal form of compound A. The preparation method as described in claim item 9, is characterized in that, the single solvent is an organic solvent, and the mixed solvent is a mixed solution of an organic solvent and water, wherein the organic solvent is selected from acetonitrile, acetone, isopropanol, n-heptyl alkanes, methyl tertiary butyl ether, isopropyl acetate, ethanol, toluene, methanol and ethyl acetate. The preparation method as described in claim item 9, is characterized in that, the method further includes, using the easily soluble solvent acetone as the recrystallization solvent, under reflux conditions, dissolving the crude product of compound A, then concentrating the acetone, adding the poor solvent methyl Tertiary butyl ether (MTBE) crystallization. The preparation method as described in claim item 9, is characterized in that, the method further includes, using acetone to reflux the crude product of compound A to dissolve, concentrating the acetone solvent to 0.2 to 0.3 times the original volume, cooling to 10 to 30 Celsius, then dropwise add methyl tertiary butyl ether in an amount 0.4 to 0.6 times the volume of the original acetone solvent, continue to crystallize, and filter to obtain a crystalline product. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the crystal form described in any one of Claims 1 to 8, and a pharmaceutically acceptable carrier. A method for inhibiting the activity of influenza virus in vitro, characterized by comprising: in vitro, contacting cells with an effective amount of the crystal form described in any one of claims 1 to 8 or the pharmaceutical composition described in claim 13 . A method for preventing or treating influenza, characterized by comprising administering an effective amount of the crystal form described in any one of Claims 1 to 8 or the pharmaceutical composition described in Claim 13 to a subject in need. A use of the crystal form described in any one of claim items 1 to 8 or the pharmaceutical composition described in claim item 13 in the preparation of a medicament for preventing or treating influenza.

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