Preparation method for salt of cyclic carbonate nucleoside compound and crystal form thereof

Abstract

The present invention relates to the technical field of medicine and relates to a salt of a cyclic carbonate nucleoside compound and a crystal thereof, and a preparation method therefor and a use thereof. The salt of the cyclic carbonate nucleoside compound has a structure represented by formula I. When Y is hydrobromic acid and n=1, the salt of the nucleoside compound exists in crystal form A or crystal form B. Crystal form A has the advantages of good physical and chemical stability, good solid-state properties, good water solubility, low hygroscopicity, high oral bioavailability, etc., and can be used in the preparation of a drug for treating and / or alleviating related diseases caused by viruses (especially novel coronavirus, feline infectious peritonitis virus, respiratory syncytial virus, porcine epidemic diarrhea virus, feline calicivirus, etc.).

Description

Preparation method of salt of cyclic carbonate nucleoside compound and crystal form thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024110689279 entitled "Preparation method of salt of cyclic carbonate nucleoside compound and crystal form thereof" filed on August 6, 2024, the entire disclosure of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the technical field of medicine, in particular to a salt of cyclic carbonate nucleoside compound, a crystal thereof and a preparation method and application thereof. BACKGROUND

[0004] The novel coronavirus (SARS-CoV-2), a new coronavirus with high similarity to the SARS-CoV genetic sequence in 2003, has rapidly spread globally since its discovery. This type of virus not only has high transmissibility and variability, but also is difficult to prevent through vaccination due to its high proliferation capacity. Respiratory syncytial virus (RSV) currently has no effective chemical drugs for treatment, and is the primary factor for viral respiratory tract infection in infants and young children to be hospitalized, seriously endangering children's health. In this context, antiviral drugs, especially nucleoside analogs targeting viral genetic material (DNA or RNA), have become a key response strategy.

[0005] Some coronaviruses can also infect various mammals, including cats, pigs, dogs, cows, horses, and camels. For example, it is estimated that 40-80% of cats worldwide carry feline coronavirus (FCoV), which is divided into two subtypes, and in particular, the feline infectious peritonitis virus (FIPV) type caused by feline coronavirus mutation has a very high mortality rate. Similarly, porcine epidemic diarrhea virus (PEDV) also belongs to the coronavirus family, and causes a high incidence and mortality rate in sows. In addition, feline calicivirus (FCV) is a highly variable, single-stranded RNA virus that mainly causes acute upper respiratory tract infection in cats, has a high incidence rate, and is currently believed to be a major cause of cat stomatitis.

[0006] Therefore, there is an urgent need to develop nucleoside analogs with stable physical properties and excellent pharmacokinetic characteristics to treat various infectious diseases caused by these viruses.

[0007] SUMMARY

[0008] In view of the deficiencies in the prior art, the present application provides a salt of cyclic carbonate nucleoside compound, a crystal thereof and a preparation method and application thereof. The salt of cyclic carbonate nucleoside compound and the crystal thereof provided by the present application have the characteristics of simple preparation, high reproducibility, good physical and chemical stability, etc.

[0009] In a first aspect, the present application provides a salt of a cyclic carbonate nucleoside compound having the structure of Formula I,

[0010] wherein: Y is hydrochloric acid, sulfuric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, maleic acid, succinic acid, tartaric acid, hydroiodic acid, fumaric acid, benzoic acid, phthalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, camphoric acid, camphorsulfonic acid, salicylic acid, trifluoroacetic acid, acetic acid, perchloric acid, malic acid, formic acid, propionic acid, malonic acid, oxalic acid, naphthalenesulfonic acid, acetylsalicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, ascorbic acid, gallic acid, mandelic acid, sorbic acid, taurine, homotaurine, cinnamic acid, mucic acid, or a combination thereof, preferably hydrobromic acid or hydrochloric acid; and n is 0.5 to 2, preferably 1.

[0011] Preferably, when the positively charged hydrogen ion in Y forms a salt with the nitrogen atom at position 3 in the free base heterocyclic 4-amino pyrrolo[2,1-f][1,2,4]triazine structure of the compound of Formula I, the compound of Formula I is a compound of Formula I-1, wherein Y-H represents Y minus one H atom.

[0012] wherein: n is 0.5 to 2, preferably 1.

[0013] In particular, when n is 1 and Y is hydrobromic acid in the compound of Formula I-1, the compound is a compound of Formula I-1’.

[0014] In a second aspect, the present application provides a crystal of the compound of Formula I-1’. The crystal has a crystal form A or a crystal form B.

[0015] The X-ray powder diffraction pattern of the crystal form A has characteristic peaks at at least 3, preferably at least 5, more preferably at least 7 of the following 2-theta values: 5.42±0.2°, 10.87±0.2°, 16.56±0.2°, 18.88±0.2°, 21.83±0.2°, 23.81±0.2°, and 27.39±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystal form A further has characteristic peaks at at least 3, preferably at least 5, more preferably at least 8 of the following 2-theta values: 25.09±0.2°, 27.02±0.2°, 28.26±0.2°, 29.05±0.2°, 29.84±0.2°, 31.71±0.2°, 33.52±0.2°, and 37.17±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystal form A is shown in FIG. 1.

[0016] The DSC pattern of the crystal form A has an absorption peak at 217±5°C; in some embodiments, the DSC pattern of the crystal form A is shown in FIG. 2.

[0017] The thermal gravimetric analysis (TGA) spectrum of the crystal form A only has a weight loss of 0.04% before 105°C, and slowly loses weight to about 180°C, and then rapidly loses weight due to sample decomposition, indicating that there is no free water and crystallization solvent in the sample, as shown in Figure 3.

[0018] The polarized light microscope graph of the crystal form A is shown in Figure 4, and the sample is a needle-shaped crystal, and the particle size range is: D10=5 μm; D50=13 μm; D90=27 μm.

[0019] The crystal form A of the compound represented by Formula I-1' has small hygroscopicity. After being placed at 25°C, RH 80% for 24 hours, the moisture increase is less than 1%, preferably less than 0.2%, and more preferably less than 0.1%.

[0020] The X-ray powder diffraction spectrum of the crystal form B has characteristic peaks at at least 3, preferably at least 5, and more preferably at least 8 of the following 2θ values: 5.33±0.2°, 11.07±0.2°, 19.49±0.2°, 20.19±0.2°, 21.04±0.2°, 25.50±0.2°, 30.31±0.2° and 31.47±0.2°. In some embodiments, the X-ray powder diffraction spectrum of the crystal form B also has characteristic peaks at at least 3, preferably at least 5, and more preferably at least 8 of the following 2θ values: 14.44±0.2°, 16.41±0.2°, 17.20±0.2°, 19.72±0.2°, 21.46±0.2°, 22.88±0.2°, 23.58±0.2° and 24.26±0.2°. In some embodiments, the X-ray powder diffraction spectrum of the crystal form B is substantially as shown in Figure 5.

[0021] In a third aspect, the present application provides a preparation method of a compound represented by Formula I or Formula I-1', which comprises the following steps:

[0022] The compound of Formula II is dissolved in solvent A to obtain solution A, and acid or solution B obtained by dissolving acid in solvent is mixed with solution A to obtain a mixed solution, and the mixed solution is continuously stirred to wait for solid precipitation, filtered, eluted with an appropriate amount of solvent A, and dried to obtain the target product.

[0023] The compound of formula II is described in CN202210372580.1 Chinese patent application, such compounds have significant anti-SARS-COV-2 virus and feline infectious peritonitis virus activity, and also have good inhibitory effect on other various RNA viruses. However, at room temperature, the compound is often a viscous gel, insoluble in water, easy to degrade, poor chemical stability, poor drug property, especially in the application as a pharmaceutical preparation, which causes certain difficulties to the preparation process. Therefore, it is particularly important to find a solid form of the compound with good water solubility and chemical stability.

[0024] The acid is hydrochloric acid, sulfuric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, maleic acid, succinic acid, tartaric acid, hydroiodic acid, fumaric acid, benzoic acid, phthalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, camphoric acid, camphorsulfonic acid, salicylic acid, trifluoroacetic acid, acetic acid, perchloric acid, malic acid, formic acid, propionic acid, malonic acid, oxalate, naphthalenesulfonic acid, acetylsalicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, ascorbic acid, gallic acid, apricot acid, sorbic acid, taurine, homotaurine, cinnamic acid, mucic acid or a combination thereof, preferably hydrobromic acid, hydrochloric acid.

[0025] Preferably, in the preparation method, the amount of solvent A used is 2-20 mL per 1 g of the compound of formula II, preferably 1 g: 10-14 mL; the temperature for preparing the mixed solution and waiting for the solid to precipitate is -15-50°C, preferably -5-15°C; the molar ratio of the compound of formula II to the acid is 1:0.5-2, preferably 1:1; the amount of solvent B used for diluting the acid is 1-4 times, preferably 2-3 times, the volume of the acid; the stirring time is 0.5-12 hours, preferably 0.5-4 hours; the amount of solvent A used for washing the filter cake is 4-20 mL, preferably 8-12 mL. The solvent A and solvent B are each independently selected from ethyl acetate, acetonitrile, acetone, water and homogeneous mixtures thereof, and both solvent A and solvent B are preferably ethyl acetate.

[0026] The prepared hydrobromide salt X-ray powder diffraction pattern at a salt formation temperature of 38-42°C is as shown in Figure 6, and the 2θ value is basically consistent with that of the crystal form A of Figure 1.

[0027] The X-ray powder diffraction pattern of the crystal form A sample baked at 80°C for 24 hours is as shown in Figure 7, and the 2θ value is basically consistent with that of the crystal form A of Figure 1.

[0028] The single crystal diffraction of the crystal form A is as shown in Figure 8, which is consistent with the chemical structure of ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-cyano-2-oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)isobutyric acid methyl ester hydrobromide.

[0029] It should be noted that when the acid is hydrobromic acid, the above preparation method produces Form A. As for Form B of the hydrobromide salt of compound II, the preparation method comprises:

[0030] The compound of formula II is dissolved in solvent A to obtain solution A, and hydrobromic acid or a solution of hydrobromic acid in solvent B is mixed with the solution A to obtain a mixed solution, and the mixed solution is continuously stirred, no solid is precipitated, the mixed solution is concentrated under reduced pressure to obtain a gum. A large amount of solid is precipitated by stirring the gum with solvent C, and the filter cake is washed with an appropriate amount of solvent C, and then dried to obtain the target product.

[0031] Preferably, for every 1 g of the compound of formula II, the amount of solvent A is 1-5 mL, preferably 2-3 mL; the amount of solvent B used for diluting the acid is 1-4 times, preferably 2-3 times, the volume of the acid; the stirring time is 10-60 minutes, preferably 30 minutes; the amount of solvent C added to the gum is 4-20 mL, preferably 8-12 mL; the amount of solvent C used for washing the filter cake is 4-20 mL, preferably 8-12 mL. The solvent A, the solvent B and the solvent C are each independently selected from ethyl acetate, acetonitrile, acetone, water and a homogeneous mixture thereof, and the solvent A is preferably acetonitrile and the solvent C is preferably ethyl acetate.

[0032] In a fourth aspect, the present application provides a pharmaceutical composition comprising the salt of the above cyclocarbonate nucleoside compound or the crystal of the hydrobromide salt of the cyclocarbonate nucleoside compound.

[0033] In a fifth aspect, the present application provides the use of the salt of the above cyclocarbonate nucleoside compound, the crystal of the hydrobromide salt of the cyclocarbonate nucleoside compound or the pharmaceutical composition in the treatment of diseases caused by infectious viruses.

[0034] Preferably, the infectious viruses include one or more of the novel coronavirus, feline infectious peritonitis virus, respiratory syncytial virus, porcine epidemic diarrhea virus and feline calicivirus.

[0035] In a sixth aspect, the present application provides the use of the salt of the above cyclocarbonate nucleoside compound, the crystal of the hydrobromide salt of the cyclocarbonate nucleoside compound or the pharmaceutical composition in the preparation of a drug for treating diseases caused by infectious viruses.

[0036] Preferably, the infectious viruses include one or more of the novel coronavirus, feline infectious peritonitis virus, respiratory syncytial virus, porcine epidemic diarrhea virus and feline calicivirus.

[0037] The application provides a salt of a cyclic carbonate nucleoside compound, a crystal thereof and a preparation method and application of the salt, the salt of the cyclic carbonate nucleoside compound has a structure shown in formula I. When the salt is a hydrobromide, the salt includes a crystal form A or a crystal form B, wherein the crystal form A has the advantages of good physical and chemical stability, good solid properties, good water solubility, small hygroscopicity and high oral bioavailability, and can be used for preparing a medicine for treating and / or relieving diseases caused by viruses (in particular, a novel coronavirus, a feline infectious peritonitis virus, a respiratory syncytial virus, a porcine epidemic diarrhea virus and a feline calicivirus). BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 shows an X-ray powder diffraction spectrum of a hydrobromide (crystal form A) salt of compound II.

[0039] FIG. 2 shows a DSC spectrum of the hydrobromide (crystal form A) salt of compound II.

[0040] FIG. 3 shows a TGA spectrum of the hydrobromide (crystal form A) salt of compound II.

[0041] FIG. 4 shows a polarizing microscope image of the hydrobromide (crystal form A) salt of compound II.

[0042] FIG. 5 shows an X-ray powder diffraction spectrum of a hydrobromide (crystal form B) salt of compound II.

[0043] FIG. 6 shows an X-ray powder diffraction spectrum of a hydrobromide salt of compound II when the salting temperature is 38-42 DEG C.

[0044] FIG. 7 shows an X-ray powder diffraction spectrum of the hydrobromide (crystal form A) salt of compound II after being baked at 80 DEG C for 24 hours.

[0045] FIG. 8 shows a single crystal diffraction image of the hydrobromide (crystal form A) salt of compound II. DETAILED DESCRIPTION

[0046] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0047] Compound II

[0048] Reagent and consumable description

[0049] In the embodiments of the present application, the solvents such as ethyl acetate involved in the preparation method are all of analytical purity, and the reagents used are not specially treated unless otherwise specified.

[0050] In embodiments of the application, the room temperature refers to 15-25 °C.

[0051] General test methods

[0052] 1. X-ray powder diffraction (XRPD) test method

[0053] Instrument: X-ray polycrystalline diffractometer; Target: Cu-Kβ (40 kV, 40 mA); Sample-to-detector distance: 30 cm; Scan step width: 0.02°; Scan range: 3°-60°; Scan step length: 0.1 s.

[0054] Generally, the X-ray powder diffraction angle (2θ value) can have an error within the range of ±0.2°, therefore the values related to the diffraction angle in the present application should be understood as also including values within the range of about 0.2°. Therefore, the present application not only covers the crystal form that exactly matches the characteristic signal peaks in the specific X-ray powder diffraction pattern, but also covers the crystal form that has an error of about ±0.2° with the characteristic signal peaks in the specific X-ray powder diffraction pattern.

[0055] 2. DSC test method:

[0056] Instrument: Thermo plus EVO2 differential scanning calorimeter; Temperature range: 30-300 °C; Scanning rate: 10 °C / min.

[0057] 3. Hygroscopicity test method:

[0058] First, place the stoppered glass weighing bottle in an artificial climate chamber (temperature of 25 °C±1 °C, relative humidity of 80%±2%) for 24 h, and accurately weigh the weight of the stoppered glass weighing bottle (m1). Take an appropriate amount of the test sample, and spread it in the stoppered glass weighing bottle that has been placed in the artificial climate chamber (temperature of 25 °C±1 °C, relative humidity of 80%±2%) for 24 h, and accurately weigh the weight of the stoppered glass weighing bottle at this time (m2). Place the weighing bottle open and together with the bottle cap in the artificial climate chamber (temperature of 25 °C±1 °C, relative humidity of 80%±2%) for 24 h, and then take it out, and accurately weigh the weight of the stoppered glass weighing bottle at this time (m3). Calculate the weight gain percentage of the sample.

[0059] 4. Stability test method:

[0060] Place the test sample in a suitable clean container, and place it in high temperature (80 °C), accelerated (40 °C, 75% RH), and long-term (25 °C / 60% RH) conditions, respectively, for 7 days, take the sample on the 7th day, and detect the appearance of the sample by visual method according to the stability test item, and detect the content (purity) by HPLC.

[0061] 5. Solubility test method:

[0062] Take about 10 mg of the test sample, place it in a 10 mL volumetric flask, add deionized water to constant volume, shake vigorously for 30 seconds every 5 minutes at room temperature, after 30 minutes, filter with a 0.45 μm microporous filter, take 2 mL of the filtrate to a 10 mL volumetric flask, add acetonitrile to constant volume, as the test sample solution, perform HPLC analysis, and calculate the solubility of the test sample in pure water by the external standard method.

[0063] Example 1: Preparation of the hydrochloride salt of compound II

[0064] Methyl ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-cyano-2- oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)isobutyrate (1.0 g, 2.58 mmol) was dissolved in ethyl acetate (12 mL), and 36% hydrochloric acid (0.26 g, 2.58 mmol) was added dropwise at room temperature. After the addition was completed, stirring was continued at room temperature for 30 minutes. A solid was precipitated, which was filtered, the filter cake was rinsed with 8 mL of ethyl acetate, and the filter cake was dried to obtain the title compound as a white solid (708 mg, yield 64.7%, HPLC purity 99.76%).

[0065] 1 H-NMR (400 MHz, DMSO-d6): δ 9.87 (s, 1H), 9.20 (s, 1H), 8.27 (s, 1H), 7.43 (d, J = 4.7 Hz, 1H), 7.03 (d, J = 4.7 Hz, 1H), 5.94 (d, J = 7.7 Hz, 1H), 5.51 (dd, J = 7.7, 3.7 Hz, 1H), 4.87 (q, J = 4.0 Hz, 1H), 4.35 (dd, J = 12.3, 4.0 Hz, 1H), 4.26 (dd, J = 12.3, 5.2 Hz, 1H), 2.45 (h, J = 7.0 Hz, 1H), 1.02 (dd, J = 12.6, 7.0 Hz, 6H).

[0066] Example 2: Preparation of the hemisulfate salt of compound II

[0067] Methyl ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6- cyano-2-oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)isobutyrate (1.0 g, 2.58 mmol) was dissolved in ethyl acetate (12 mL) and 98% sulfuric acid (0.13 g, 1.29 mmol) was added dropwise to 300 μL of water diluted at room temperature. After addition, stirring was continued at room temperature for 4 h and the reaction liquid became gummy. Filtration was performed and the filter cake was washed with 8 mL of ethyl acetate. The filter cake was dried to obtain the title compound as a white solid (870 mg, yield 77.2%, HPLC purity 99.26%).

[0068] 1 H-NMR (400 MHz, DMSO-d6): δ 9.11 (s, 1H), 8.73 (s, 1H), 8.18 (s, 1H), 7.21 (d, J = 4.7 Hz, 1H), 7.00 (d, J = 4.7 Hz, 1H), 5.95 (d, J = 7.7 Hz, 1H), 5.50 (dd, J = 7.7, 3.7 Hz, 1H), 4.86 (q, J = 3.9 Hz, 1H), 4.35 (dd, J = 12.3, 3.9 Hz, 1H), 4.25 (dd, J = 12.3, 5.2 Hz, 1H), 2.44 (h, J = 7.0 Hz, 1H), 1.01 (dd, J = 13.0, 7.0 Hz, 6H).

[0069] Example 3: Preparation of phosphate salt of compound II

[0070] Methyl ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6- cyano-2-oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)isobutyrate (1.0 g, 2.58 mmol) was dissolved in ethyl acetate (12 mL) and 85% phosphoric acid (0.15 g, 2.58 mmol) was added dropwise at room temperature. After addition, stirring was continued at room temperature for 30 min and the reaction liquid became gummy. Filtration was performed and the filter cake was washed with 8 mL of ethyl acetate. The filter cake was dried to obtain the title compound as a white solid (934 mg, yield 74.5%, HPLC purity 99.42%).

[0071] 1H-NMR (400 MHz, DMSO-d6): δ 8.06 (d, 2H), 7.98 (s, 1H), 6.96 (d, J = 4.6 Hz, 1H), 6.91 (d, J = 4.6 Hz, 1H), 6.00 (d, J = 7.7 Hz, 1H), 5.49 (dd, J = 7.7, 3.7 Hz, 1H), 4.82 (q, J = 4.0 Hz, 1H), 4.34 (dd, J = 12.2, 4.0 Hz, 1H), 4.23 (dd, J = 12.2, 5.2 Hz, 1H), 2.44 (p, J = 7.0 Hz, 1H), 1.00 (dd, J = 13.6, 7.0 Hz, 6H).

[0072] Example 4: Preparation of nitrate salt of compound II

[0073] ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-cyano-2- oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)isobutyrate methyl ester (1.0 g, 2.58 mmol) was dissolved in ethyl acetate (12 mL) and 65% nitric acid (0.25 g, 2.58 mmol) was added dropwise at room temperature. After the addition was completed, stirring was continued at room temperature for 30 minutes. A solid was precipitated, which was filtered, the filter cake was washed with 8 mL of ethyl acetate, and the filter cake was dried to obtain the title compound as a white solid (878 mg, yield 75.5%, HPLC purity 99.85%).

[0074] 1 H-NMR (400 MHz, DMSO-d6): δ 9.49 (s, 1H), 8.98 (s, 1H), 8.25 (s, 1H), 7.29 (d, J = 4.7 Hz, 1H), 7.03 (d, J = 4.7 Hz, 1H), 5.93 (d, J = 7.7 Hz, 1H), 5.51 (dd, J = 7.7, 3.7 Hz, 1H), 4.88 (q, J = 4.0 Hz, 1H), 4.35 (dd, J = 12.3, 4.0 Hz, 1H), 4.26 (dd, J = 12.3, 5.1 Hz, 1H), 2.45 (h, J = 7.0 Hz, 1H), 1.02 (dd, J = 12.7, 7.0 Hz, 6H).

[0075] Example 5: Preparation of maleate salt of compound II

[0076] Methyl ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6- cyano-2-oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)isobutyrate (1.0 g, 2.58 mmol) was dissolved in ethyl acetate (12 mL) and maleic acid (0.30 g, 2.58 mmol) was added dropwise at room temperature. After the addition was complete, stirring was continued at room temperature for 3 h. A solid precipitated, which was filtered, the filter cake was rinsed with 8 mL of ethyl acetate, and the filter cake was dried to give the title compound as a white solid (878 mg, 75.5% yield, 99.60% HPLC purity).

[0077] 1 H-NMR (400 MHz, DMSO-d6): δ 8.13 (d, 2H), 8.00 (s, 1H), 6.97 (d, J = 4.6 Hz, 1H), 6.92 (d, J = 4.6 Hz, 1H), 6.27 (s, 2H), 5.99 (d, J = 7.7 Hz, 1H), 5.49 (dd, J = 7.7, 3.7 Hz, 1H), 4.82 (q, J = 4.0 Hz, 1H), 4.34 (dd, J = 12.3, 4.0 Hz, 1H), 4.23 (dd, J = 12.3, 5.2 Hz, 1H), 2.43 (h, J = 7.0 Hz, 1H), 1.01 (dd, J = 13.5, 7.0 Hz, 6H).

[0078] Example 6: Preparation of hydrobromide (crystal form A) salt of compound II

[0079] Methyl ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6- cyano-2-oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)isobutyrate (1.0 g, 2.58 mmol) was dissolved in ethyl acetate (12 mL) and 48% hydrobromic acid (0.43 g, 2.58 mmol) was added dropwise at room temperature. After the addition was complete, stirring was continued at room temperature for 30 min. A solid precipitated, which was filtered, the filter cake was rinsed with 8 mL of ethyl acetate, and the filter cake was dried to give the title compound as a white crystalline solid ((1115 mg, 92.1% yield, 99.78% HPLC purity).

[0080] 1H-NMR (400 MHz, DMSO-d6): δ 11.34 (s, 1H), 10.31 (s, 1H), 9.54 (s, 1H), 8.44 (s, 1H), 7.64 (d, J = 4.8 Hz, 1H), 7.12 (d, J = 4.8 Hz, 1H), 6.00 (d, J = 7.7 Hz, 1H), 5.59 (dd, J = 7.7, 3.6 Hz, 1H), 4.92 (q, J = 3.9 Hz, 1H), 4.48 - 4.21 (m, 2H), 2.47 (h, J = 7.0 Hz, 1H), 1.03 (dd, J = 12.8, 7.0 Hz, 6H).

[0081] 13 C-NMR (100 MHz, DMSO-d6): δ 176.10, 153.33, 149.84, 139.06, 125.53, 115.62, 114.08, 112.86, 109.46, 83.66, 82.63, 80.75, 79.61, 62.64, 33.46, 19.17, 19.07.

[0082] MS: m / s 388.1255 [M+1] + .

[0083] X-ray powder diffraction (XRPD) results showed that the obtained solid was in crystalline form, corresponding to Form A, as shown in Figure 1.

[0084] Differential scanning calorimetry (DSC) results of Form A showed that the obtained solid started to have an endothermic peak at 214.4 °C and reached a peak at 217.6 °C, as shown in Figure 2.

[0085] Thermogravimetric analysis (TGA) and polarized light microscope images of Form A are shown in Figures 3 and 4, respectively.

[0086] Single crystal diffraction results of Form A showed that the positively charged hydrogen ion in hydrobromic acid formed a salt with the nitrogen atom at position 3 in the heterocyclic pyrrolo[2,1-f][1,2,4]triazine structure of Compound II, as shown in Figure 8.

[0087] Example 7: Preparation of hydrobromic acid (Form B) salt of Compound II

[0088] Methyl ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6- cyano-2-oxotetrahydrofuran-4-yl)isobutyrate (1.0 g, 2.58 mmol) was dissolved in acetonitrile (2.5 mL), 48% hydrobromic acid (0.43 g, 2.58 mmol) was added dropwise at room temperature, and the reaction was stirred at room temperature for 30 minutes. The reaction was concentrated under reduced pressure to an oily residue, 12 mL of ethyl acetate was added, and the mixture was stirred to precipitate a large amount of solid. The solid was filtered, the filter cake was washed with 8 mL of ethyl acetate, and the filter cake was dried to obtain a white crystalline solid (1028 mg, 85.0% yield, HPLC purity 99.30%).

[0089] X-ray powder diffraction (XRPD) results showed that the obtained solid was in a crystalline form, corresponding to Form B, as shown in Figure 5.

[0090] Example 8: Preparation of a hydrobromide salt of Compound II with a salt formation temperature of 38-42°C

[0091] Methyl ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6- cyano-2-oxotetrahydrofuran-4-yl)isobutyrate (0.5 g, 1.29 mmol) was dissolved in ethyl acetate (6 mL), and 48% hydrobromic acid (0.22 g, 1.29 mmol) was added dropwise at 38-42°C. The reaction was stirred at 38-42°C for 30 minutes, and a solid was precipitated. The solid was filtered, the filter cake was washed with 4 mL of ethyl acetate, and the filter cake was dried to obtain a white solid (337 mg, 55.8%).

[0092] The obtained solid was in a crystalline form, and the X-ray powder diffraction (XRPD) results are shown in Figure 6, which are substantially consistent with those of Form A in Figure 1.

[0093] Example 9: Comparison of the physicochemical properties and preparation of Compound II and its salts

[0094] (1) Comparison of the properties and preparation of Compound II and its salts

[0095] Compound II and its different salts (prepared by Examples 1-7) were selected for comparison of properties and ease of preparation, and the results are shown in Table 1.

[0096] Table 1: Properties and preparation of Compound II and its salts

[0097] As shown in Table 1 above, except for the acetate salt, other acid addition salt forms of Compound II are all in good condition. In addition, the hydrobromide salt, the hydrochloride salt and the nitrate salt of Compound II are easier to prepare, especially the hydrobromide salt presents obvious white needle-like crystalline solid.

[0098] (2) Comparison of hygroscopicity and stability of the salts of Compound II

[0099] Different salts of Compound II (prepared by Examples 1-7) are selected to compare the hygroscopicity and stability. The high temperature condition is (80°C), the accelerated condition is (40°C / 75% RH) and the long-term condition is (25°C / 60% RH). The stability results are shown in Table 2, and the hygroscopicity and stability evaluation results are shown in Table 3.

[0100] Table 2 Stability data of Compound II and the salts of Compound II

[0101] As shown in Table 2 above, the hydrobromide salt (crystal form A and crystal form B) of Compound II is very stable under the conditions of high temperature, long-term and acceleration, the stability of the hydrochloride salt of Compound II is general; and the stability of the hemisulfate salt, the phosphate salt, the nitrate salt and the maleate salt of Compound II is relatively poor.

[0102] Table 3 Hygroscopicity and stability of the salts of Compound II

[0103] As shown in Table 3 above, the hydrobromide salt (crystal form A and crystal form B) of Compound II has no hygroscopicity under the condition of 25°C and RH 80%; the hydrochloride salt, the hemisulfate salt, the phosphate salt, the nitrate salt and the maleate salt of Compound II have obvious hygroscopicity.

[0104] (3) Comparison of solubility of Compound II and its salts

[0105] Compound II and its different salts (prepared by Examples 1-7) are selected to compare the solubility. The results are shown in Table 4.

[0106] Table 4 Solubility of the salts of Compound II in deionized water

[0107] As shown in the above table, the salt formation obviously increases the water solubility of Compound II, and the water solubility and chemical stability of the compound have important influence on the preparation of pharmaceutical preparations and oral bioavailability. Therefore, the salt formation of Compound II can significantly improve the water solubility, which is more conducive to the development of pharmaceutical preparations.

[0108] Example 10: Determination of the composition ratio of the hydrobromide salt of Compound II

[0109] The ion chromatography method is used to determine the composition ratio of the hydrobromide salt of Compound II, and the results are shown in Table 5.

[0110] Table 5 Composition ratio of hydrobromide salt of compound II

[0111] As can be seen from the above Table 5, compound II is salted with hydrogen bromide in a molar ratio of 1:1.

[0112] Test Example 1: Toxicity of compound II, hydrobromide (crystal form A) salt of compound II, hydrochloride salt of compound II, GS-441524 and Ribavirin on HEp-2 cells.

[0113] HEp-2 cells were inoculated in a 96-well plate at 6,000 cells per well and 100 μL per well, and cultured overnight in a 37°C, 5% CO2 incubator. The next day, 50 μL of gradient-diluted compound II, hydrobromide (crystal form A) salt of compound II, hydrochloride salt of compound II and GS-441524 at concentrations of 50 μM, 16.667 μM, 5.556 μM, 1.852 μM, 0.617 μM, 0.206 μM, 0.069 μM, 0.023 μM, and positive control Ribavirin at corresponding concentrations of 300 μM, 100 μM, 33.333 μM, 11.111 μM, 3.704 μM, 1.235 μM, 0.412 μM, 0.137 μM (8 concentration points, 3-fold gradient dilution, triplicate wells) were added to each well, and a blank cell control group (normal cells without compound treatment) was set. The final concentration of DMSO in the cell culture solution was 0.5%. The cells were cultured in a 37°C, 5% CO2 incubator for 5 days. Cell viability was detected using CCK-8 reagent, and the survival rate of the cells was calculated. The results showed that compound II, hydrobromide (crystal form A) salt of compound II, hydrochloride salt of compound II and GS-441524 had no cytotoxicity to HEp-2 cells at 50 μM, and had good safety.

[0114] Test Example 2: In vitro anti-respiratory syncytial virus (RSV) A2 strain activity of compound II, hydrobromide (crystal form A) salt of compound II, hydrochloride salt of compound II, GS-441524 and Ribavirin.

[0115] HEp-2 cells were inoculated in a 96-well plate at 6,000 cells per well and 100 μL per well, and cultured overnight in a 37°C, 5% CO2 incubator. The next day, 50 μL of gradient-diluted compound (8 concentration points, same as in Test 1, 3-fold gradient dilution, triplicate wells) and 50 μL of virus (200 TCID 50). Cell control (normal cells, no virus infection or compound treatment) and virus control (cells infected with virus, no compound treatment) were set up. The final concentration of DMSO in cell culture medium was 0.5%. Cells were incubated in a 37℃, 5% CO2 incubator for 5 days.

[0116] The expression of virus F protein in each well was detected by ELISA. After 5 days of infection, the supernatant was discarded, and 75 μL of 80% acetone was added to each well to fix the cells. After removing the acetone, 75 μL of rabbit anti-RSV F protein antibody was added to each well, and incubated at 37℃ for 1 h, and the plate was washed with TBST for three times. 75 μL of HRP-labeled goat anti-rabbit IgG antibody was added to each well, and incubated at 37℃ for 1 h, and the plate was washed with TBST for three times. 100 μL of TMB color developing solution was added to each well, and reacted at room temperature for 20-30 min, and then 100 μL of 1% HCl was added to each well to stop the color developing reaction. After shaking evenly, the absorbance value (OD450-650) of each well was read using a microplate reader. 450 The antiviral activity of the compounds was calculated by the obtained raw data. The selectivity index (SI) was calculated using the formula "SI = CC 50 / EC 50 ", and the results of the antiviral activity test are shown in Table 6.

[0117] Table 6 Experimental results of cytotoxicity and anti-RSV A2 activity of all compounds

[0118] As can be seen from Table 6, the compound II, the hydrochloride salt of the compound II, the hydrobromide (crystal form A) salt of the compound II and GS-441524 do not show cytotoxicity in the test range, and have good safety; the compound II, the hydrochloride salt of the compound II, the hydrobromide (crystal form A) salt of the compound II and GS-441524 all have antiviral activity against the virus strain RSV A2, and the inhibitory effect is significantly better than the positive control drug Ribavirin, among which the hydrobromide (crystal form A) salt has the best activity and the highest selectivity index.

[0119] Test Example 3: Toxicity of the compound II, the hydrobromide (crystal form A) salt of the compound II, the hydrochloride salt of the compound II and GS-441524 to Vero-E6 cells

[0120] Vero-E6 cells in good growth state were digested with trypsin-EDTA, counted, and the cell concentration was adjusted to 2 x 105 / mL with DMEM culture solution containing 10% fetal bovine serum. The above cell suspension was inoculated in a 96-well plate for culture, 100 μL per well, and a monolayer was formed after 24 h of culture at 37°C in a 5% CO2 incubator. After 24 h, the culture solution was discarded, washed with PBS, and dried before being replaced with new DMEM culture solution. Compound II, the hydrobromic acid (crystal form A) salt of compound II, the hydrochloric acid salt of compound II, and GS-441524 were added, respectively, at a final concentration of 50 μM, 10 μM, 5 μM, 1 μM, and 0.1 μM. A blank cell control group was also set up. After 48 h of culture, cell viability was detected with a reagent, and the survival rate of the cells was calculated. The results showed that all the above compounds had no cytotoxicity to Vero-E6 cells and good safety at a final concentration of 50 μM, 10 μM, 5 μM, 1 μM, and 0.1 μM. The results showed that all the above compounds had no cytotoxicity to Vero-E6 cells and good safety at a final concentration of 50 μM, 10 μM, 5 μM, 1 μM, and 0.1 μM.

[0121] Test Example 4: In vitro anti-SARS-CoV-2 virus activity of compound II, the hydrobromic acid (crystal form A) salt of compound II, the hydrochloric acid salt of compound II, and GS-441524.

[0122] Cell preparation: Vero-E6 cells were counted at 2 x 10 5 / ml of cell suspension was plated in a 96-well plate, and the plate was incubated overnight in a 37°C, 5% CO2 incubator. When the cells grew into a monolayer, they were ready for use.

[0123] Virus dilution: 0.9 mL of DMEM containing 5% fetal bovine serum was added to the first tube, and the rest was added to the second tube. Then, 0.1 mL of virus preservation solution was added to the first tube, and 0.2 mL was taken from the first tube with a new gun head and added to the second tube. This was repeated in the same way to dilute to the highest dilution.

[0124] Virus inoculation: the cells were washed once with serum-free DMEM; 100 μL of different virus dilutions was added to each well, with 8 parallel wells for each dilution. The highest dilution was added first, and a negative control was set up by adding 100 μL of DMEM containing 5% fetal bovine serum to the negative control well. The survival of the cells was detected, and the plate was incubated at 37°C in a 5% CO2 incubator for 5 days. After 5 days, the plate was observed under a microscope, and the number of wells with CPE in each column was counted. The virus TCID 50 / mL was calculated by the KARBER method.

[0125] Virus viability detection: VeroE6 cells were counted at 2 x 10 4Compound II, the hydrobromic acid (crystal form A) salt of Compound II, the hydrochloric acid salt of Compound II and GS-441524 were diluted to an initial concentration of 6.0 μM (0.5% DMSO in DMEM medium solution) and then diluted by 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 after inactivation, with 3 parallel holes for each dilution. Then the above-mentioned each dilution of the test drug was mixed with 100 TCID 50 The virus solution was mixed in equal amounts and incubated at 37°C for 1 h, then inoculated into Vero E6 cells and cultured at 37°C, 5% CO2 for 5 days. The highest virus dilution that caused 50% of the cells to show cytopathic effect was observed. At the same time, control groups were set up, including cell control (cells only without virus serum in the culture medium), negative control (culture medium containing 100 TCID 50 virus, cells and blank serum) and blank control (culture medium containing 100 TCID 50 virus and cells without serum). According to the degree of cytopathic effect, the 50% drug concentration was calculated as the endpoint using the Reed-Muench method, i.e. the drug dilution at which 50% of the cells did not show cytopathic effect (CPE). The test results are shown in Table 7.

[0126] Table 7 Dilution of drug concentration at which 50% of the cells showed cytopathic effect (CPE)

[0127] The microplate method was used to evaluate the inhibitory effect of the drugs on the novel coronavirus (the greater the dilution factor, the higher the inhibitory activity of the virus). As can be seen from Table 7, Compound II, the hydrobromic acid (crystal form A) salt of Compound II, the hydrochloric acid salt of Compound II and GS-441524 all had significant inhibitory effects on the three variants of the novel coronavirus (BA.5, BF.7 and XBB). Among them, Compound II, the hydrobromic acid (crystal form A) salt of Compound II and the hydrochloric acid salt of Compound II had significantly better inhibitory effects than GS-441524.

[0128] Test Example 5: Toxicity of Compound II, the hydrobromic acid (crystal form A) salt of Compound II, the hydrochloric acid salt of Compound II and GS-441524 on CRFK cells

[0129] Gradient dilution of drugs: Compound II, Compound II hydrobromide (crystal form A) salt, Compound II hydrochloride and GS-441524 were weighed and dissolved in DMSO, respectively, and then GS-441524 was diluted to 25 mM, 12.5 mM, 6.25 mM, 3.125 mM, 1.5625 mM, 0.390 mM, 0.098 mM solutions with maintenance medium. The above test drugs were diluted to 16 mM, 8 mM, 4 mM, 2 mM, 1 mM, 0.25 mM, 0.0625 mM solutions with maintenance medium, and the DMSO concentration was 1.0%. The solutions were filtered with a 0.22 μm filter to remove bacteria and stored at 4°C for use.

[0130] Drug cytotoxicity assay: CRFK cells were pre-cultured in a 96-well plate with complete medium in a 37°C, 5% CO2 incubator. After the cells grew into a monolayer, the culture medium in the wells was discarded. 0.1 mL of Compound II, Compound II hydrobromide (crystal form A) salt, Compound II hydrochloride and GS-441524 at different dilutions were added to the cell culture wells, respectively, with 6 wells in parallel for each concentration, and cell control wells and blank control wells were set up at the same time. After 48 h of culture, 10 μL of CCK8 reagent was added to each well, and after 0.5 h of further incubation, the absorbance value (OD 450 ) at 450 nm wavelength was determined by a microplate reader. The cell survival percentage was calculated according to the control group, and the cell survival percentage (%) = (average OD of the test group - average OD of the blank control group) / (average OD of the cell control group - average OD of the blank control group) x 100%.

[0131] Drug cytotoxicity assay results: Statistical analysis showed that when the concentration of Compound II was 0.87≤1 mM, the concentration of Compound II hydrobromide (crystal form A) salt was ≤1 mM, the concentration of Compound II hydrochloride was ≤0.51 mM, and the concentration of GS-441524 was ≤0.39 mM, the OD 450 values of the drug groups at each concentration had no difference from the cell control group (P>0.05), and had no obvious inhibitory effect on the growth of CRFK cells, i.e., the maximum safe concentrations of Compound II, Compound II hydrobromide (crystal form A) salt, Compound II hydrochloride and GS-441524 were 0.87 mM, 1.0 mM, 0.51 mM and 0.39 mM, respectively.

[0132] Test Example 6: In vitro anti-feline infectious peritonitis virus (FIPV) activity of Compound II, Compound II hydrobromide (crystal form A) salt, Compound II hydrochloride and GS-441524.

[0133] Virus dilution: The virus solution was diluted by 10 times. First, 0.9 mL of blank DMEM medium was dispensed into 10 test tubes, and 0.1 mL of virus suspension was added to the first test tube. After mixing, the first test tube contained 10 -1, in 10 -1 0.1 mL of the virus liquid was taken into a second tube and shaken to make 10 -2 , and diluted to 10 -7 .

[0134] Virus inoculation: The culture solution of the 96-well plate with the CRFK cell monolayer was poured out, and the diluted virus liquid was inoculated into the 96-well plate, 0.1 mL per well, 6 replicates for each dilution, and normal cell controls were set. After inoculation, they were placed in a 37°C, 5% CO2 incubator for 3 hours of adsorption. After adsorption, the virus liquid was poured out, 0.1 mL of maintenance medium (containing 2% FBS) was added to each well of cells, and it was placed in a 37°C, 5% CO2 incubator for culture. The results were observed daily, the number of lesions was recorded, and the TCID 50 / 100 μL was calculated by the Reed-Muench method.

[0135] Virus activity detection: 100 TCID 50 0.1 mL of FIPV virus was added to each well, adsorbed for 3 hours, and the virus liquid was poured out. Compound II, Compound II hydrobromide (crystal form A) salt, Compound II hydrochloride salt, and GS-441524 were diluted to different compound concentrations of 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.5 μM, 0.3125 μM, and 0.25 μM with maintenance medium containing 0.1% DMSO, 100 μL was added to each well, and 5 replicates were set for each concentration. At the same time, virus controls (added virus, no drug liquid) and cell controls (no virus, no drug liquid) were set. They were placed in a 37°C, 5% CO2 incubator for culture, and cell lesions (CPE) were observed daily under a microscope. When the virus control well cell lesions reached 100%, the CCK-8 reagent was added to detect the OD 450 value of each well, the virus reproduction inhibition rate of different concentrations of compounds was calculated, a curve was drawn, and the half effective concentration was calculated. The test results are shown in Table 8.

[0136] Table 8 Inhibition results of all compounds on feline infectious peritonitis virus

[0137] As can be seen from Table 8, the compound II, the compound II hydrobromide (crystal form A) salt, the compound II hydrochloride salt, and the GS-441524 all have significant inhibition effect on feline infectious peritonitis virus, and the inhibition effect of the compound II hydrobromide (crystal form A) salt is the best.

[0138] Test Example 7: In vitro anti-feline calicivirus (FCV) activity of compound II, compound II hydrobromide (crystal form A) salt, compound II hydrochloride salt, and GS-441524.

[0139] Cell preparation: Cat kidney cell (CRFK) cells were counted at 1x105 / ml, 100ul cell suspension was plated per well in a 96-well plate, and incubated overnight at 37℃ in a 5% CO2 incubator. The cells were divided into test groups (different drug concentrations), normal cell groups (without virus and drugs), and virus groups (without drugs), with 3 replicate wells in each group.

[0140] Virus activity detection: 100TCID 50 of feline calicivirus (FCV) was added to each well, and after overnight adsorption, the supernatant was discarded. The cells were washed with PBS, and 400uM of the drug was prepared, filtered to remove bacteria, and diluted with maintenance medium to prepare different drug concentrations of 100uM, 50uM, 25uM, 12.5uM, 6.25uM, 3.125uM, 1.5625uM, and 0.78125uM of Compound II, Compound II hydrobromide (crystal form A) salt, Compound II hydrochloride salt, and GS-441524. 100ul was added to each well, and the CPE of the virus group was observed when the CPE reached 75%-100%. The CPE was observed, with 0%-25% of the lesions counted as 1, 25%-50% as 2, 50%-75% as 3, and 75%-100% as 4. The results are shown in Table 9.

[0141] Table 9 Percentage of cell cytopathic effect (CPE) of all compounds at different drug concentrations

[0142] The inhibitory effect of the drugs on feline calicivirus was evaluated by cell cytopathic effect (CPE). As can be seen from Table 9, all the compounds had a significant inhibitory effect on feline calicivirus at a drug concentration of 12.5uM, with a cell cytopathic rate of less than 50%.

[0143] Test Example 8: Pharmacokinetic evaluation in rats

[0144] Pharmacokinetic process: 21 SD male rats (body weight 180-220g) were taken, 3 rats per group, and fasted for 12h before the experiment, with free access to water. Compound II, Compound II hydrobromide (crystal form A) salt, and GS-441524 were dissolved in appropriate solvents, and the solvent for administration was DMSO / propylene glycol / 0.9% NaCl solution. The administration dose was 0.103umol / kg, and the rats were administered by gavage. After administration, the rats were taken from each group, and 0.3-0.5ml of blood was taken from the orbit at 0.25, 0.5, 1, 2, 3, 4, 8, and 12h, and placed in 1.5ml EP tubes (heparin pretreated), centrifuged at 4℃, and the upper drug-containing plasma was separated and frozen at -80℃ for testing. The concentration of GS-441524 in the plasma was determined by LC-MS-MS method, and the pharmacokinetic parameters were calculated.

[0145] Standard curve: Take about 5 ml of rat blank blood, centrifuge, separate the plasma, take 1.5 ml EP tube 6, respectively, add 0.1, 0.5, 1, 5, 10, 20, 50 μl of stock solution, working solvent to 450 μl, mix well, respectively, add 50 μl of blank plasma, vortex for 5 min, centrifuge for 5 min, take the supernatant, centrifuge again for 5 min, take the supernatant in 1.5 ml EP tube, get 10, 50, 100, 500, 1000, 2000, 5000 ng / ml GS-441524 standard curve samples (containing internal standard 6,7-Dimethyl-2,3-Bis(2-pyridyl)quinoxaline, QX 100 ng / ml).

[0146] Detection sample processing: Take 50 μl of drug-containing plasma obtained from the pharmacokinetic process into a 1.5 ml EP tube containing 450 μl of working solvent, vortex for 5 min, take the supernatant, centrifuge again for 5 min, take the supernatant in a 1.5 ml EP tube, get the test compound pharmacokinetic detection sample.

[0147] LC-MS detection: chromatographic column, Kinetex C18 core-shell universal chromatographic column (Phenomenex, USA), column temperature 30℃, injection volume 10 μL, mobile phase acetonitrile-0.6% ammonium formate, flow rate 1.0 ml / min, isocratic elution; ion source, electrospray ion source ESI; detection mode, multiple reaction monitoring (MRM); scanning mode, positive ion mode; ESI spray voltage, ion source temperature 400℃, atomization gas flow rate 150 μL / min, backflushing drying gas flow rate 100 μL / min.

[0148] The pharmacokinetic results are shown in Table 10.

[0149] Table 10 Pharmacokinetic parameter results of all compounds

[0150] Test Example 9: Toxicity test of compound II hydrobromide (crystal form A) salt single gavage administration to SD rats

[0151] Method: A total of 50 SD rats (25 / sex) were used, and were randomly divided into 5 groups according to body weight, 10 rats per group (5 / sex / group). Respectively, single gavage of solvent (0.5% carboxymethylcellulose sodium aqueous solution) and compound II hydrobromide (crystal form A) salt dissolved in the solvent (1-5 groups were 5000, 3344, 2236, 1495 and 1000 mg / kg, respectively), the volume of administration was 10 mL / kg. After administration, continuous observation for 14 days, all animals were dissected on D15. During the test period, the animals were clinically observed, body weight, food intake, gross dissection and histopathological examination of abnormal organs were observed.

[0152] Results: During the experiment, no death or near-death was observed in male animals of each administration group and in female animals of 1000 mg / kg dose group. Abnormal appearance (urine color was red) was observed in female animals of ≥1000 mg / kg dose group; 7 female animals died in total in ≥1495 mg / kg dose group, and excessive salivation, eye discharge, emaciation, listlessness, hunched posture, prone position, reduced spontaneous activity, nasal discharge, general body hair fluffiness and / or toe walking, reduced food intake were observed in female animals, and excessive salivation and eye discharge were observed in male animals. Except for the death animals, no abnormal changes related to the test product were observed in the body weight and body weight gain of animals in each administration group.

[0153] In summary, the responses of male and female animals to the test product were significantly different, the acute oral median lethal dose (LD50) of the test product to female rats was about 3481 mg / kg, the 95% confidence interval of LD10was 2539-4772 mg / kg, and it was low toxicity; no animal death was observed in male rats, which was practically non-toxic. 50 ) of the test product to female rats was about 3481 mg / kg, the 95% confidence interval of LD10was 2539-4772 mg / kg, and it was low toxicity; no animal death was observed in male rats, which was practically non-toxic. 50

[0154] Test Example 10: Dose exploration test of Compound II hydrobromide (crystal form A) salt by repeated gavage administration to SD rats for 30 days

[0155] Methods: A total of 64 SD rats (32 / sex) were used in the test, and were randomly divided into 7 groups according to body weight, the first 4 groups were used for toxicological study (5 / sex / group), and the 5th to 7th groups (4 / sex / group) were used for toxicokinetic study. The animals in the first group were given 0.5% carboxymethylcellulose sodium aqueous solution as a solvent control group, and the second and fifth groups, the third and sixth groups, and the fourth and seventh groups were given 0.5% carboxymethylcellulose sodium aqueous solution and Compound II hydrobromide (crystal form A) salt dissolved in the solvent (30, 100 and 300 mg / kg, respectively) as a solvent. The animals were administered by gavage, the administration volume was 10 mL / kg, and the administration was once a day for 30 consecutive days. The animals in groups 1-4 were dissected the day after the last drug (D31). The animals in the toxicokinetic study group were euthanized the day after the last drug (D31) without dissection.

[0156] During the test, the animals were subjected to clinical observation, body weight, food intake, body temperature, ophthalmic examination, clinical pathology, gross anatomy, organ weight and histopathology. At the same time, the content of Compound II hydrobromide (crystal form A) salt and its metabolite GS441524 in the samples of the toxicokinetic study animals on D1 and D30 were determined and toxicokinetic analysis was performed.

[0157] ​Results: During the test period, no death or moribundity was observed in all animals. No abnormal changes related to the test product were observed in the clinical observation of 30 mg / kg animals. No abnormal changes related to the test product were observed in the ophthalmic examination, urine analysis and gross autopsy of 100 mg / kg and 300 mg / kg animals. The main changes included: excessive salivation was observed in animals of 100 mg / kg dose group; excessive salivation, reduced body weight and food intake, and slightly elevated hemoglobin (HGB) were observed in animals of 300 mg / kg dose group.

[0158] In summary, under the conditions of this test, the hydrobromic acid (crystal form A) salt of compound II was repeatedly administered by gavage to SD rats at doses of 30, 100 and 300 mg / kg, once a day for 30 consecutive days. The maximum tolerated dose (MTD) was 300 mg / kg. At this dose, the average peak concentration (C max ) and area under the curve AUC last of GS441524 in D30 male animals were 7398.30 ng / mL and 41061.39 h·ng / mL, respectively, and the average peak concentration (C max ) and area under the curve AUC last of GS441524 in female animals were 9549.49 ng / mL and 36641.16 h·ng / mL, respectively.

[0159] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial applicability

[0160] The present application provides a salt of a cyclic carbonate nucleoside compound, a crystal thereof, and a preparation method and application thereof. The salt of the cyclic carbonate nucleoside compound has a structure shown in formula I. When Y is hydrobromic acid and n = 1, the salt of the nucleoside compound exists in crystal form A or crystal form B, wherein the crystal form A has the advantages of good physical and chemical stability, good solid properties, good water solubility, small hygroscopicity, and high oral bioavailability, and can be used for preparing a medicine for treating and / or alleviating diseases caused by viruses (especially novel coronavirus, feline infectious peritonitis virus, respiratory syncytial virus, porcine epidemic diarrhea virus, and feline calicivirus, etc.), and has good economic value and application prospect.

Claims

1. A salt of a cyclic carbonate nucleoside compound having the structure of Formula I, ###00001### Formula I wherein Y is hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, maleic acid, succinic acid, tartaric acid, hydroiodic acid, fumaric acid, benzoic acid, phthalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, camphoric acid, camphorsulfonic acid, salicylic acid, trifluoroacetic acid, acetic acid, perchloric acid, malic acid, formic acid, propionic acid, malonic acid, oxalic acid, naphthalenesulfonic acid, acetyl salicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, ascorbic acid, gallic acid, mandelic acid, sorbic acid, taurine, homotaurine, cinnamic acid, mucic acid, or a combination thereof, preferably hydrobromic acid or hydrochloric acid; n is 0.5-2, preferably 1.

2. A salt of a cyclic carbonate nucleoside compound, characterized by, The structural formula is shown in formula I-1: Y is hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, maleic acid, succinic acid, tartaric acid, hydroiodic acid, fumaric acid, benzoic acid, phthalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, camphoric acid, camphorsulfonic acid, salicylic acid, trifluoroacetic acid, acetic acid, perchloric acid, malic acid, formic acid, propionic acid, malonic acid, oxalic acid, naphthalenesulfonic acid, acetyl salicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, ascorbic acid, gallic acid, mandelic acid, sorbic acid, taurine, homotaurine, cinnamic acid, mucic acid, or a combination thereof, preferably hydrobromic acid or hydrochloric acid; n is 0.5-2, preferably 1.

3. A crystal of a cyclic carbonate nucleoside compound hydrobromide salt, characterized in that, The structural formula is shown in formula I-1': The crystal has a crystal form A or a crystal form B; wherein the crystal form A has at least one of the following characteristics: 1) its X-ray powder diffraction pattern has characteristic peaks at at least 3, preferably at least 5, more preferably at least 7 of the following 2Θ values: 5.42±0.2°, 10.87±0.2°, 16.56±0.2°, 18.88±0.2°, 21.83±0.2°, 23.81±0.2° and 27.39±0.2°; Further preferably, the X-ray powder diffraction pattern of the crystal form A further has characteristic peaks at at least 3, preferably at least 5, more preferably at least 8 of the following 2Θ values: 25.09±0.2°, 27.02±0.2°, 28.26±0.2°, 29.05±0.2°, 29.84±0.2°, 31.71±0.2°, 33.52±0.2° and 37.17±0.2°; 2) its DSC pattern has an absorption peak at 217±5℃; The X-ray powder diffraction pattern of the crystal form B has characteristic peaks at at least 3, preferably at least 5, more preferably at least 8 of the following 2Θ values: 5.33±0.2°, 11.07±0.2°, 19.49±0.2°, 20.19±0.2°, 21.04±0.2°, 25.50±0.2°, 30.31±0.2° and 31.47±0.2°; Further preferably, the X-ray powder diffraction pattern of the crystal form B further has characteristic peaks at at least 3, preferably at least 5, more preferably at least 8 of the following 2Θ values: 14.44±0.2°, 16.41±0.2°, 17.20±0.2°, 19.72±0.2°, 21.46±0.2°, 22.88±0.2°, 23.58±0.2° and 24.26±0.2°.

4. The cyclic carbonate nucleoside compound hydrobromide salt crystal according to claim 3, characterized by, The X-ray powder diffraction pattern of the crystal form A is substantially as shown in Figure 1. The X-ray powder diffraction pattern of the crystal form B is substantially as shown in Figure 5.

5. The process for preparing the crystal of the hydrobromide salt of the cyclic carbonate nucleoside compound according to claim 3 or 4, characterized by, The crystal has a crystal form A, and the preparation method comprises the following steps: The compound of formula II is dissolved in solvent A to obtain solution A. Hydrobromic acid or a solution of hydrobromic acid in solvent B is mixed with the solution A to obtain a mixed solution. The mixed solution is continuously stirred and solid is precipitated. The solid is filtered, washed with an appropriate amount of solvent A, and dried to obtain the target product. Further, The use amount ratio of the compound of formula II to the solvent A is 1 g: 2-20 mL, preferably 10-14 mL; The temperature for preparing the mixed solution and waiting for the solid to precipitate is -15-50°C, preferably -5-15°C; The molar ratio of the compound of formula II to the hydrobromic acid is 1: 0.5-2, preferably 1: 1; The stirring time is 0.5-12 hours, preferably 0.5-4 hours; The use amount ratio of the compound of formula II to the elution filter cake solvent A is 1 g: 4-20 mL, preferably 1 g: 8-12 mL; The solvent A and the solvent B are each independently selected from ethyl acetate, acetonitrile, acetone, water and a homogeneous mixture thereof, and the solvent A and the solvent B are both preferably ethyl acetate.

6. A process for preparing the crystalline hydrobromide salt of the cyclic carbonate nucleoside compound of claim 3 or 4, characterized in that, The crystal has a crystal form B, and the preparation method comprises the following steps: The compound of formula II is dissolved in the solvent A to obtain a solution A, and the hydrobromic acid or the solution of hydrobromic acid in the solvent B is mixed with the solution A to obtain a mixed solution, and the mixed solution is continuously stirred, no solid is precipitated, the mixed solution is concentrated under reduced pressure to obtain a gum, and the solvent C is added to the gum, and stirred until a large amount of solid is precipitated, and then filtered, and the filter cake is eluted with an appropriate amount of the solvent C, and then dried to obtain the target product.

7. The preparation method according to claim 6, wherein, The use amount ratio of the compound of formula II to the solvent A is 1 g: 1-5 mL, preferably 1 g: 2-3 mL; The use amount of the solvent B for diluting the acid is 1-4 times, preferably 2-3 times, of the volume of the acid; The stirring time is 10-60 minutes, preferably 30 minutes; The use amount ratio of the compound of formula II to the elution solvent C is 1 g: 4-20 mL, preferably 1 g: 8-12 mL; The solvent A, the solvent B and the solvent C are each independently selected from ethyl acetate, acetonitrile, acetone, water and a homogeneous mixture thereof, and the solvent A and the solvent B are both preferably acetonitrile, and the solvent C is preferably ethyl acetate.

8. A pharmaceutical composition comprising the salt of the cyclic carbonate nucleoside compound according to claim 1 or 2, or the crystal of the hydrobromide salt of the cyclic carbonate nucleoside compound according to claim 3 or 4.

9. Use of the salt of the cyclic carbonate nucleoside compound according to claim 1 or 2, or the crystal of the hydrobromide salt of the cyclic carbonate nucleoside compound according to claim 3 or 4, or the pharmaceutical composition according to claim 8 in the treatment of diseases caused by infectious viruses. Preferably, the infectious viruses include one or more of the novel coronavirus, feline infectious peritonitis virus, respiratory syncytial virus, porcine epidemic diarrhea virus and feline calicivirus.

10. Use of the salt of the cyclic carbonate nucleoside compound according to claim 1 or 2, or the crystal of the hydrobromide salt of the cyclic carbonate nucleoside compound according to claim 3 or 4, or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating diseases caused by infectious viruses. Preferably, the infectious virus comprises one or more of a novel coronavirus, feline infectious peritonitis virus, respiratory syncytial virus, porcine epidemic diarrhea virus, and feline calicivirus.

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