CN115572298B - Crystal form, preparation method and application of a nucleoside analog and its salt - Google Patents

Abstract

The present invention relates to a crystal form, preparation method and application of a nucleoside analog and a salt thereof. The nucleoside analog is a compound of formula A, the compound of formula A is in a crystalline form or an amorphous form, and the crystalline form is crystal form I. The crystal form of the nucleoside analog and a salt thereof of the present invention has stable physical properties, good drugability, and a simple and repeatable preparation method.

Description

Crystal form, preparation method and application of a nucleoside analog and its salt

Technical Field

The present invention belongs to the field of medical technology, and specifically relates to a crystal form, a preparation method and application of a nucleoside analog and a salt thereof.

Background Art

The history of human social development is a history of fighting against viral infectious diseases. In recent years, due to the complex interactions between humans and animals and changes in the earth's ecosystem, the prevalence of new and re-emerging infectious diseases has intensified, posing a serious threat to human health. These viral infectious diseases have great social harm and have always been a huge challenge that the public health system must face.

The genetic material of the virus is DNA or RNA, and a large number of nucleotides are required during its replication process. Nucleoside (acid) analogs can mimic endogenous nucleosides and, during the process of viral synthesis of DNA or RNA, compete with the corresponding nucleosides to bind to the viral reverse transcriptase or RNA polymerase, terminating the extension of the viral DNA or RNA chain, thereby inhibiting viral replication.

The invention patent application with application publication number CN 113735862A discloses a nucleoside compound for treating viral infection and its use, the nucleoside compound and its prodrug have the compound described in formula I, its prodrug and/or its pharmaceutically acceptable salt. The compound and its composition have the use of preventing, alleviating or treating coronavirus infection, or the replication or reproduction of its homologous variant virus and the cytopathic effect produced by it.

Different crystal forms of the same compound as a drug often have different stabilities. However, so far, there are very few studies on the crystal forms of nucleoside compounds. Therefore, there is an urgent need in the art to develop crystal forms of nucleoside analogs with stable physical properties and good drugability for treating related diseases caused by viral infections.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a crystal form, preparation method and application of a nucleoside analog and its salt. The crystal form of the nucleoside analog and its salt has stable physical properties and good drugability, and the preparation method is simple and easy to repeat.

In order to solve the above technical problems, the technical solution adopted by the present invention is:

A compound of formula A,

The compound of formula A is in a crystalline form, the crystalline form is Form I, and the X-ray powder diffraction pattern of Form I has characteristic peaks at at least 3, preferably at least 5, and more preferably at least 7 of the following 2θ values: 9.85°±0.2°, 17.42°±0.2°, 17.75°±0.2°, 18.72°±0.2°, 19.39°±0.2°, 23.90°±0.2° and 24.43°±0.2°.

Preferably, the X-ray powder diffraction pattern of Form I also has characteristic peaks at at least 3, preferably at least 5, and more preferably at least 9 of the following 2θ values: 12.94°±0.2°, 13.18°±0.2°, 15.62°±0.2°, 19.64°±0.2°, 21.32°±0.2°, 23.57°±0.2°, 28.20°±0.2°, 30.57°±0.2° and 31.18°±0.2°.

Preferably, the X-ray powder diffraction pattern of Form I is shown in Figure 1.

A compound of formula A,

The compound of formula A is in an amorphous state.

Preferably, the X-ray powder diffraction pattern of the amorphous state is as shown in FIG3 .

In order to solve the above technical problems, another technical solution adopted by the present invention is:

A method for preparing a crystalline form of the compound of formula A as described above comprises the following steps:

1) dispersing the compound of formula A in solvent S1, heating and stirring to dissolve, to obtain solution S1';

2) Slowly cooling the solution S1', stirring for crystallization, and filtering to obtain the crystalline form I of the compound of formula A.

Preferably, in step 1), the usage ratio of the compound of formula A to the solvent S1 is 1 g: 20 to 100 mL, preferably 1 g: 50 to 80 mL.

The stirring time is 0.5 to 5 hours, preferably 0.5 to 1 hour.

The heating temperature is 30 to 120°C, preferably 50 to 90°C;

In step 2), the crystallization temperature is -20 to 30°C, preferably -10 to 10°C.

The stirring time is 0.5 to 5 hours, preferably 0.5 to 1 hour.

Preferably, the solvent S1 is selected from water, hydrocarbons, alcohols, ethers, ketones, esters, nitriles and homogeneous mixtures thereof;

The hydrocarbon is selected from the group consisting of n-pentane, n-hexane, n-heptane, petroleum ether, methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, toluene, xylene, chlorobenzene and dichlorobenzene;

The alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, ethylene glycol and propylene glycol;

The ether is selected from the group consisting of ethyl ether, n-propyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol dimethyl ether, tetrahydrofuran, dioxane, dimethoxyethane and diethylene glycol dimethyl ether;

The ketone is selected from the group consisting of acetone, butanone and diethyl ketone;

The ester is selected from the group consisting of methyl formate, ethyl formate, propyl formate, butyl formate, methyl acetate, ethyl acetate, propyl acetate and butyl acetate;

The nitrile is selected from acetonitrile and propionitrile.

More preferably, the solvent S1 is selected from methanol.

In order to solve the above technical problems, another technical solution adopted by the present invention is:

A salt of a compound of formula A, which is a compound of formula B,

wherein X is maleic acid, succinic acid, citric acid, tartaric acid, fumaric acid, formic acid, acetic acid, propionic acid, malonic acid, oxalic acid, benzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, camphoric acid, camphorsulfonic acid, salicylic acid, acetylsalicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, ascorbic acid, gallic acid, mandelic acid, malic acid, sorbic acid, trifluoroacetic acid, taurine, homotaurine, 2-hydroxyethanesulfonic acid, cinnamic acid, mucic acid, hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, or a combination thereof;

X is preferably maleic acid, hydrogen bromide, nitric acid, methanesulfonic acid, sulfuric acid or a combination thereof;

n is 0.5～2.

Preferably, the salt is a compound of formula B-1,

Among them, n is 0.5~2.

Preferably, the salt is a compound of formula B-1',

Preferably, the salt is in a crystalline form, the crystalline form is Form II, and the X-ray powder diffraction pattern of Form II has characteristic peaks at at least 3, preferably at least 5, and more preferably at least 7 of the following 2θ values: 3.38°±0.2°, 3.75°±0.2°, 13.17°±0.2°, 16.77°±0.2°, 25.50°±0.2°, 26.39°±0.2° and 26.76°±0.2°.

Preferably, the X-ray powder diffraction pattern of Form II also has characteristic peaks at at least 3, preferably at least 5, and more preferably at least 9 of the following 2θ values: 13.67°±0.2°, 15.22°±0.2°, 17.49°±0.2°, 18.28°±0.2°, 20.17°±0.2°, 23.76°±0.2°, 29.51°±0.2°, 32.59°±0.2° and 33.16°±0.2°.

Preferably, the X-ray powder diffraction pattern of Form II is shown in FIG5 .

Preferably, the salt is in an amorphous state.

Preferably, the X-ray powder diffraction pattern of the amorphous state is as shown in FIG7 .

In order to solve the above technical problems, another technical solution adopted by the present invention is:

A method for preparing a crystalline form of a salt of a compound of formula A as described above comprises the following steps:

1) mixing an acid or solution S2' with solution S1' to obtain a mixed solution, wherein solution S2' is obtained by dissolving an acid in solvent S2, and solution S1' is obtained by dispersing a compound of formula A in solvent S1, heating, stirring and dissolving;

2) stirring the mixed solution under reflux conditions and concentrating;

3) The product in step 2) is mixed with solvent S3, stirred at room temperature and/or under heating conditions, and cooled to precipitate a solid to obtain the target product.

Preferably, in step 1), the acid content in solution S2' is 40 wt% to 50 wt%,

The molar ratio of the compound of formula A to the acid is 1:0.9-1,

The usage ratio of the compound of formula A and the solvent S1 is 1 g: 20 to 100 mL, preferably 1 g: 50 to 80 mL.

The stirring time is 0.5 to 5 hours, preferably 0.5 to 1 hour.

The heating temperature is 30 to 120°C, preferably 50 to 90°C;

In step 2), the stirring time is 0.5 to 5 hours, preferably 0.5 to 1 hour;

In step 3), the ratio of the compound of formula A to the solvent S3 is 1 g: 10-50 mL, preferably 1 g: 20-30 mL.

The heating condition is 35 to 100°C, preferably 50 to 60°C.

The stirring time is 0.5 to 5 hours, preferably 0.5 to 2 hours.

The crystallization temperature is -20 to 30°C, preferably -10 to 10°C.

Preferably, solvent S1, solvent S2 and solvent S3 are each independently selected from water, hydrocarbons, alcohols, ethers, ketones, esters, nitriles and homogeneous mixtures thereof, and solvents S1, S2 and S3 are miscible with each other;

The hydrocarbon is selected from the group consisting of n-pentane, n-hexane, n-heptane, petroleum ether, methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, toluene, xylene, chlorobenzene and dichlorobenzene;

The alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, ethylene glycol and propylene glycol;

The ether is selected from the group consisting of ethyl ether, n-propyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol dimethyl ether, tetrahydrofuran, dioxane, dimethoxyethane and diethylene glycol dimethyl ether;

The ketone is selected from the group consisting of acetone, butanone and diethyl ketone;

The ester is selected from the group consisting of methyl formate, ethyl formate, propyl formate, butyl formate, methyl acetate, ethyl acetate, propyl acetate and butyl acetate;

The nitrile is selected from acetonitrile and propionitrile.

Preferably, the solvent S1 is selected from methanol, the solvent S2 is selected from water, and the solvent S3 is selected from methyl tert-butyl ether.

In order to solve the above technical problems, another technical solution adopted by the present invention is:

Use of the crystalline form of the compound of formula A as described above or the crystalline form of the salt of the compound of formula A as described above in the preparation of a medicament for treating and/or alleviating a disease caused by a virus,

The virus is preferably a coronavirus,

The coronavirus is preferably SARS-CoV-2.

In order to solve the above technical problems, another technical solution adopted by the present invention is:

A pharmaceutical composition comprising:

1) a crystalline form of a compound of formula A as described above or a crystalline form of a salt of a compound of formula A as described above;

2) Pharmaceutically acceptable excipients.

Preferably, the pharmaceutical composition is an oral preparation or a non-oral preparation;

Preferably, the oral preparation is selected from tablets, capsules, granules, powders and syrups;

Preferably, the non-oral preparation is selected from injections, powder injections, sprays and suppositories.

Preferably, the excipients include fillers, disintegrants and lubricants.

Preferably, the filler is selected from one or more of lactose, starch, modified starch, mannitol, sorbitol, dextrin derivatives, cellulose derivatives, calcium carbonate, calcium hydrogen phosphate and calcium sulfate; wherein the dextrin derivative is selected from dextrin and/or maltodextrin; and the cellulose derivative is selected from microcrystalline cellulose and/or cellulose.

Preferably, the disintegrant is selected from one or more of cross-linked carboxymethyl cellulose sodium, cross-linked polyvinylpyrrolidone, starch, pregelatinized starch, sodium carboxymethyl starch, hydroxypropyl starch, microcrystalline cellulose and low-substituted hydroxypropyl cellulose.

Preferably, the lubricant is selected from one or more of stearic acid, calcium stearate, magnesium stearate, hydrogenated vegetable oil, carnauba wax, talc, polyethylene glycol and sodium stearyl fumarate.

Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

1. The nucleoside analogs of the present invention have a crystal form of Form I, which has the advantages of good solid properties and high stability;

2. The acid addition salts (e.g., hydrogen bromide salts) of the nucleoside analogs of the present invention and their corresponding crystalline forms (e.g., crystalline form II) have good solubility;

3. The crystal forms of the nucleoside analogs and their salts of the present invention have stable physical properties and are easy to store, and can be used to prepare drugs for treating and/or alleviating diseases related to viruses (especially SARS-CoV-2);

4. The preparation method of the present invention also has the advantages of stable product crystal form, high purity, easy storage, simple method and easy repeatability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an XRPD spectrum of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate Form I;

Figure 2 is an XRPD spectrum of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexanecarboxylate Form I after high temperature at 80°C for 24 hours;

FIG3 is an XRPD pattern of the amorphous form of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate;

FIG4 is a DSC spectrum of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate Form I;

FIG5 is an XRPD spectrum of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate hydrobromide Form II;

Figure 6 is an XRPD spectrum of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrobromide salt Form II after high temperature at 80°C for 24 hours;

FIG7 is an XRPD pattern of the amorphous form of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate hydrobromide;

Figure 8 is the DSC spectrum of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate hydrobromide Form II.

DETAILED DESCRIPTION

In order to make the technical scheme and beneficial effects of the present invention more obvious and easy to understand, the following is described in detail by combining the accompanying drawings and listing specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually based on conventional experimental conditions.

[Definition of terms]

Nucleoside analogs

Nucleoside analogs are the most important class of antiviral drugs. They have played an important role in the clinical treatment of viral diseases for a long time. Nucleoside drugs can be converted into the corresponding triphosphate form in the body, especially in the viral replication stage, where nucleoside triphosphate can be "disguised" as a substrate and incorporated into the viral DNA or RNA chain, thereby inhibiting the replication of genetic material and exerting an antiviral effect.

Reagents and consumables description

The methanol, methyl tert-butyl ether and other reagents involved in the embodiments and preparation methods of the present invention are all analytically pure and provided by Sinopharm Chemical Reagent Co., Ltd. Unless otherwise specified, the reagents used are not specially treated. The triethylamine and phosphoric acid involved in the high performance liquid chromatography experiment are chromatographically pure and provided by Sinopharm Chemical Reagent Co., Ltd. The nuclear magnetic resonance spectra were measured on Brucker 500 Hz and Brucker 600 Hz nuclear magnetic resonance instruments.

General test methods

1. X-ray powder diffraction (XRPD) test method:

Instrument: Bruker D8 advance X-ray polycrystal diffractometer; target: Cu-Kα (40 kV, 40 mA); sample to detector distance: 30 cm; scanning type: two-axis linkage; scanning step width: 0.02°; scanning range: 3°～40°; scanning step: 0.1 s.

Generally, the diffraction angle (2θ value) in XRPD may have an error within the range of ±0.2°, and therefore, the numerical values related to the diffraction angle in the present invention should be understood as being also included in the range of about ±0.2°. Therefore, the present invention not only covers the crystal form that completely matches the characteristic signal peak in the specific XRPD spectrum, but also covers the crystal form that has an error of about ±0.2° with the characteristic signal peak in the specific XRPD spectrum.

2.DSC test method:

Instrument: TA Discovery DSC 2500 differential scanning calorimeter; temperature range: 25-280°C; scanning rate: 10°C/min; protective gas: nitrogen.

3. Stability test method:

Place the test sample in a suitable clean container and place it under high temperature (80°C), high humidity (25°C, relative humidity 92.5%), and light (light intensity 4500±500lx and 90μw/ ^cm2 ) conditions for 7 days. Take samples on the 7th day and test them according to the stability inspection items.

4. Solubility test method:

Take about 10 mg of the test sample, place it in a 1.5 ml sample tube, add 1 ml of deionized water, place it in an ultrasonic instrument, and ultrasonicate at 37°C for 30 minutes; after the ultrasonication, take 500 μl of the supernatant, dilute it with acetonitrile to an appropriate concentration as the test sample solution, perform HPLC analysis, and calculate the solubility of the test sample in water (37°C) by the external standard method.

Example 1

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate Form I

Disperse ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate (400 mg, 1 mmol) in methanol (32 ml, 80 V), heat to reflux, stir for 30 min until the product is completely dissolved, stop heating, cool naturally to room temperature, continue stirring at 0-10 ° C for 30 min, filter, and pull the filter cake dry with an oil pump to obtain a white powdery solid, which is Form I (345 mg, yield 86%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ7.92(m,3H),6.92(d,J＝4.5Hz,1H),6.81(d,J＝4.5Hz,1H),6.33(d,J＝5.7Hz,1H ),5.37(d,J＝5.9Hz,1H),4.70(t,J＝5.3Hz,1H),4.30(dd,J＝12.1,2.8Hz,1H),4.2 2(ddd,J＝7.3,4.8,2.8Hz,1H),4.14(dd,J＝12.1,4.9Hz,1H),3.96(q,J＝5.8Hz,1H ),2.25(dt,J＝10.7,5.9Hz,1H),1.64(dd,J＝39.6,31.8Hz,5H),1.36–1.07(m,5H).

¹³ C NMR (101MHz, DMSO-d ₆ )δ175.15,156.05,148.40,124.02,117.41,117.04,110.67,101.21,81.6 5,79.41,74.53,70.58,62.99,42.60,28.98,28.89,25.73,25.24,25.22.

MS: m/z 402.21[M+1] ⁺ .

The differential scanning calorimetry (DSC) results showed that the obtained solid began to have an endothermic peak at 227.1°C and reached a peak at 227.4°C, as shown in FIG4 .

X-ray powder diffraction (XRPD) results showed that the obtained solid was in crystalline form, corresponding to Form I, as shown in Table 1 and Figure 1.

Table 1. XRPD data of Form I of Formula A compound

Example 2

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate amorphous form

((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate (100 mg) was added to tetrahydrofuran (THF) (8 mL), refluxed and stirred to dissolve, and then concentrated to remove the solvent, and the oil pump was pumped dry to obtain a foamy solid (100 mg). X-ray powder diffraction (XRPD) results showed that the obtained solid was amorphous, as shown in Figure 3.

Example 3

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate hydrobromide salt form II

Disperse ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methylhexylcarboxylate (300 mg, 0.75 mmol) in methanol (24 ml, 80 V), heat to reflux, stir for 30 min until the product is completely dissolved, add 48% hydrobromic acid solution (60 mg, 0.75 mmol), continue stirring for 30 min, wait for it to cool to room temperature, concentrate to a small volume (about 5 ml), slowly add methyl tert-butyl ether (6 ml, 20 V) under stirring, continue stirring at 50 ° C for 30 min, cool to 0-10 ° C and continue stirring for 30 min, filter, and pull the filter cake dry with an oil pump to obtain a white powdery solid (256 mg, yield 71%).

¹ H NMR (400MHz, DMSO-d ₆ )δ9.49(s,1H),8.91(s,1H),8.20(s,1H),7.33(d,J＝4.6Hz,1H),6.97(d,J＝4.7Hz,1H),4.64( d,J＝4.8Hz,1H),4.35–4 .21(m,2H),4.20–4.06(m,1H),3.94(dd,J＝7.0,4.6Hz,1H),2.35–2.17(m,1H),1.87–1.50(m,5H),1.35 –1.12(m,5H).

¹³ C NMR (101MHz, DMSO-d ₆ )δ173.01,140.76,127.95,127.61,116.93,115.34,112.15,106.94,82.6 4,79.54,75.21,69.88,62.94,44.59,29.00,28.90,25.73,25.25,25.22.

MS: m/z 402.23[M+1] ⁺ .

The differential scanning calorimetry (DSC) results showed that the obtained solid began to have an endothermic peak at 170.5°C and reached a peak at 178.4°C, as shown in FIG8 .

X-ray powder diffraction (XRPD) results showed that the obtained solid was in crystalline form, corresponding to Form II, as shown in Table 2 and Figure 5.

Table 2. XRPD data of the compound of formula B-1', crystal form II

Example 4

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate hydrobromide amorphous form

((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methylcyclohexylcarboxylate hydrobromide (50 mg) was added to THF (4 mL), refluxed and stirred to dissolve, and then concentrated to remove the solvent, and the oil pump was pumped dry to obtain a foamy solid (50 mg). X-ray powder diffraction (XRPD) results showed that the obtained solid was amorphous, as shown in Figure 7.

Example 5

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate maleate

Disperse ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate (300 mg, 0.75 mmol) in methanol (24 ml, 80 V), heat to reflux, stir for half an hour until the product is completely dissolved, add maleic acid (87 mg, 0.75 mmol), continue stirring for 30 min, cool to room temperature, concentrate to a small volume (about 5 ml), slowly add methyl tert-butyl ether (6 ml, 20 V) under stirring, continue stirring at 50 ° C for 30 min, cool to 0-10 ° C and continue stirring for 30 min, filter, and pull the filter cake dry with an oil pump to obtain a white powdery solid (256 mg, yield 71%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ7.94(s,3H),6.93(d,J＝4.5Hz,1H),6.82(d,J＝4.5Hz,1H),6.34(s,1H),6.2 4(s,1H),5.38(s,1H),4.70(d,J＝4.8Hz,1H),4.30(dd,J＝12.1,2.8Hz,1H),4.2 3(dq,J＝7.2,2.9Hz,1H), 4.15(dd,J＝12.1,4.9Hz,1H), 3.96(dd,J＝6.7,4.8Hz, 1H),2.24(ddt,J＝10.8,7.2,3.6Hz,1H),1.85–1.48(m,5H),1.41–1.01(m,5H).

MS: m/z 402.23[M+1] ⁺ .

Example 6

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methylcyclohexylcarboxylate methanesulfonate

Disperse ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate (300 mg, 0.75 mmol) in methanol (24 ml, 80 V), heat to reflux, stir for half an hour until the product is completely dissolved, add methanesulfonic acid (72 mg, 0.75 mmol), continue stirring for 30 min, cool to room temperature, concentrate to a small volume (about 5 ml), slowly add methyl tert-butyl ether (6 ml, 20 V) under stirring, continue stirring at 50 ° C for 30 min, cool to 0-10 ° C and continue stirring for 30 min, filter, and pull the filter cake dry with an oil pump to obtain a white viscous solid (288 mg, yield 77%).

¹ H NMR (400MHz, DMSO-d ₆ )δ9.31(s,1H),8.83(s,1H),8.17(d,J＝6.0Hz,1H),7.32–7.18(m,1H),6.95(d,J＝4.6Hz,1H), 4.60(dd,J＝30.3,4.9Hz,1H),4.3 3–4.21(m,2H),4.18–4.10(m,1H),3.94(t,J＝5.8Hz,1H),2.34(s,3H),2.26–2.23(m,1H),1.87–1.51( m,5H),1.35–1.19(m,5H).

MS: m/z 402.24 [M+1] ⁺ .

Example 7

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate sulfate

Disperse ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate (100 mg, 0.25 mmol) in methanol (8 ml, 80 V), heat to reflux, stir for half an hour until the product is completely dissolved, add sulfuric acid (25 mg, 0.25 mmol), continue stirring for 30 min, cool to room temperature, concentrate to a small volume (about 1 ml), slowly add methyl tert-butyl ether (2 ml, 20 V) under stirring, continue stirring at 50 ° C for 30 min, cool to 0-10 ° C and continue stirring for 30 min, filter, and pull the filter cake dry with an oil pump to obtain a white foamy solid (98 mg, yield 79%).

¹ H NMR (400MHz, DMSO-d ₆ )δ9.39(d,J＝30.9Hz,1H),8.87(s,1H),8.18(d,J＝6.0Hz,1H),7.32–7.23(m,1H),7.00(dd,J＝33.4 ,4.6Hz,1H),4.57(d,J=4. 9Hz,1H),4.31–4.22(m,2H),3.66–3.60(m,1H),3.56–3.49(m,1H),2.30–2.11(m,1H),1.95–1.43(m,5H), 1.44–0.98(m,5H).

MS: m/z 402.21[M+1] ⁺ .

Example 8

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate hemisulfate

Disperse ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate (100 mg, 0.25 mmol) in methanol (8 ml, 80 V), heat to reflux, stir for half an hour until the product is completely dissolved, add sulfuric acid (13 mg, 0.13 mmol), continue stirring for 30 min, cool to room temperature, concentrate to a small volume (about 1 ml), slowly add methyl tert-butyl ether (2 ml, 20 V) under stirring, continue stirring at 50 ° C for 30 min, cool to 0-10 ° C and continue stirring for 30 min, filter, and pull the filter cake dry with an oil pump to obtain a white foamy solid (98 mg, yield 88%).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.80(s,1H),8.45(s,1H),8.08(d,J＝5.7Hz,1H),7.13(d,J＝4.5Hz,1H),6.90(d,J＝4.6Hz, 1H),4.66(d,J＝4.8Hz,1H),4.33–4 .20(m,2H),4.15(dd,J＝12.0,4.7Hz,1H),3.95(dd,J＝6.6,4.8Hz,1H),2.30–2.20(m,1H),1.83–1.51(m ,5H),1.35–1.14(m,5H).

MS: m/z 402.23[M+1] ⁺ .

Example 9

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate phosphate

Disperse ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexylcarboxylate (100 mg, 0.25 mmol) in methanol (8 ml, 80 V), heat to reflux, stir for half an hour until the product is completely dissolved, add phosphoric acid (25 mg, 0.25 mmol), continue stirring for 30 min, cool to room temperature, concentrate to a small volume (about 1 ml), slowly add methyl tert-butyl ether (2 ml, 20 V) under stirring, continue stirring at 50 ° C for 30 min, cool to 0-10 ° C and continue stirring for 30 min, filter, and pull the filter cake dry with an oil pump to obtain a white foamy solid (85 mg, yield 69%).

¹ H NMR (400MHz, DMSO-d ₆ )δ7.92(s,3H),6.91(dd,J＝9.6,5.4Hz,1H),6.81(d,J＝4.5Hz,1H),4.70(d,J＝4.8Hz,1H),4.30( dd,J＝12.1,2.8Hz ,1H),4.22(dt,J＝7.2,3.8Hz,2H),4.16(d,J＝4.9Hz,1H),2.27–2.18(m,1H),1.82–1.49(m,5H),1.35– 1.11(m,5H).

MS: m/z 402.34[M+1] ⁺ .

Example 10

Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methylcyclohexylcarboxylate hydrochloride

Referring to the method of Example 2-7, the compound ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexanecarboxylate (300 mg, 0.75 mmol) was salified with hydrochloric acid (76 mg, 0.75 mmol). The precipitated solid adhered to the wall and could not be filtered.

Embodiment 11

Comparison of properties and stability of compound A and its acid addition salt.

(1) Comparison of properties and preparation of compound A and its acid salt

Compound A and its different salts (prepared by Examples 1-8) were selected to compare their properties and preparation difficulty. The results are shown in Table 3.

Table 3. Properties and preparation of compound A and its salts

Compound Characteristics Preparation Compound A Crystalline Form I White powdery solid Simple operation, natural sedimentation of solids, easy to filter Hydrobromide (B-1') White powdery solid Simple operation, natural sedimentation of solids, easy to filter Maleate (B-2') White powdery solid Simple operation, natural sedimentation of solids, easy to filter Methanesulfonate (B-3') White sticky solid Solids stick to the wall, easily absorb moisture, and are difficult to filter Sulfate (B-4') Foamy solid Solids cannot settle naturally, easy to filter, slightly hygroscopic Hemisulfate (B-5') Foamy solid Solids cannot settle naturally, easy to filter, slightly hygroscopic Phosphate (B-6') Poor solid properties Solids stick to the wall, difficult to filter, easy to absorb moisture Hydrochloride (B-7') No solid was obtained /

As can be seen from the above table, the hydrobromic acid and maleic acid addition salt forms of compound A have good properties and are easier to prepare.

(2) Comparison of the stability of the addition salt of compound A

Different salts of compound A (prepared by Examples 2-7) were selected to compare their stabilities. The results are shown in Table 4.

Table 4. Stability of Compound A Addition Salt

As can be seen from the above table, the hydrobromide and maleate of compound A are very stable under high temperature and light conditions, and slightly absorb moisture under high humidity conditions, while the stability of the methanesulfonate, sulfate, hemisulfate and phosphate of compound A is relatively poor.

(3) Comparison of solubility of compound A and its salt

Compound A and its different salts (prepared in Examples 1, 2 and 3) were selected and their solubilities were compared.

Weigh an appropriate amount of sample, place it in a glass test tube, gradually add the selected solvent, and observe the clarification. The solubility of compound A and its different salts in deionized water was measured, and the results are shown in Table 5.

Table 5. Solubility of Compound A and its salts in deionized water

As can be seen from the above table, the water solubility of compound A is significantly lower than that of the salt of compound A, among which the hydrobromide salt has the best water solubility, and the water solubility has a significant impact on pharmaceutical preparations, oral biological use, etc. Therefore, converting compound A into a hydrobromide salt is more conducive to preparing pharmaceutical preparations and broadening its pharmaceutical uses.

Example 12

Stability study of Form I of Compound A

As shown in Figures 1 and 2, after 24 hours at a high temperature of 80°C, Form I did not change and had good stability.

Embodiment 13

Stability study of Form II of Compound A hydrobromide

As shown in Figures 5 and 6, after 24 hours at a high temperature of 80°C, Form II remained essentially unchanged.

The present invention synthesizes and separates the crystal form of a nucleoside analogue having antiviral activity (especially anti-SARS-CoV-2 activity), its acid addition salt, and salt-type crystals obtained based on the acid addition salt, as well as studies on related physical and chemical properties.

It has been found that the nucleoside analog has a crystalline form of Form I, which has the advantages of good solid properties and high stability. In addition, the acid addition salt (e.g., hydrogen bromide salt) of the nucleoside analog and its corresponding crystalline form (e.g., Form II) are found to have good solubility. The crystalline form of nucleoside analogs and their salts can be used to prepare drugs for treating and/or alleviating related diseases caused by viruses (especially SARS-CoV-2). In addition, the preparation method in the present invention also has the advantages of stable product crystal form, high purity, easy storage, simple method and easy repetition.

It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims (18)

1. A crystalline form II of a hydrobromide salt of a compound of formula A, characterized in that the hydrobromide salt of the compound of formula A is a compound of formula B-1', and the X-ray powder diffraction pattern of the crystalline form II has characteristic peaks at the following 2θ values: 3.38°±0.2°, 3.75°±0.2°, 13.17°±0.2°, 16.77°±0.2°, 25.50°±0.2°, 26.39°±0.2° and 26.76°±0.2°, 2. The crystalline form II of the hydrobromide salt of the compound of formula A according to claim 1, characterized in that the X-ray powder diffraction pattern of the crystalline form II also has characteristic peaks at at least 3 of the following 2θ values: 13.67°±0.2°, 15.22°±0.2°, 17.49°±0.2°, 18.28°±0.2°, 20.17°±0.2°, 23.76°±0.2°, 29.51°±0.2°, 32.59°±0.2° and 33.16°±0.2°. 3. The crystalline form II of the hydrobromide salt of the compound of formula A according to claim 1, characterized in that the X-ray powder diffraction pattern of the crystalline form II also has characteristic peaks at at least 5 of the following 2θ values: 13.67°±0.2°, 15.22°±0.2°, 17.49°±0.2°, 18.28°±0.2°, 20.17°±0.2°, 23.76°±0.2°, 29.51°±0.2°, 32.59°±0.2° and 33.16°±0.2°. 4. The crystalline form II of the hydrobromide salt of the compound of formula A according to claim 1, characterized in that the X-ray powder diffraction pattern of the crystalline form II also has characteristic peaks at at least 9 of the following 2θ values: 13.67°±0.2°, 15.22°±0.2°, 17.49°±0.2°, 18.28°±0.2°, 20.17°±0.2°, 23.76°±0.2°, 29.51°±0.2°, 32.59°±0.2° and 33.16°±0.2°. 5. The crystalline form II of the hydrobromide salt of the compound of formula A according to claim 1, characterized in that the X-ray powder diffraction pattern of the crystalline form II is shown in Figure 5. 6. A method for preparing the crystalline form II of the hydrobromide salt of the compound of formula A as claimed in any one of claims 1 to 5, characterized in that it comprises the following steps: 1) mixing an acid or solution S2' with a solution S1' to obtain a mixed solution, wherein the solution S2' is obtained by dissolving an acid in a solvent S2, and the solution S1' is obtained by dispersing a compound of formula A in a solvent S1, heating, stirring and dissolving; 2) stirring and concentrating the mixed solution under reflux conditions; 3) mixing the product in step 2) with solvent S3, stirring at room temperature and/or under heating conditions, cooling to precipitate a solid, and obtaining the target product; Step 1) the heating temperature is 50-90°C; The temperature of the heating condition in step 3) is 50-60°C; Step 3) the temperature of the cooling to precipitate the solid is -10°C to 10°C; The solvent S1 is selected from one or more of methanol, ethanol, n-propanol, and isopropanol; Solvent S2 is selected from water; The solvent S3 is selected from one or more of n-propyl ether, isopropyl ether and methyl tert-butyl ether. 7. The preparation method according to claim 6, characterized in that, in the step 1), the content of acid in the solution S2' is 40wt% to 50wt%, The molar ratio of the compound of formula A to the acid is 1:0.9-1, The usage ratio of the compound of formula A and the solvent S1 is 1g:20-100mL, The stirring time is 0.5 to 5 hours; In the step 2), the stirring time is 0.5 to 5 hours; In the step 3), the ratio of the compound of formula A to the solvent S3 is 1 g: 10-50 mL. The stirring time is 0.5 to 5 hours. 8. The preparation method according to claim 7, characterized in that, in the step 1), the amount ratio of the compound of formula A to the solvent S1 is 1g:50-80mL. 9. The preparation method according to claim 7, characterized in that in the step 1), the stirring time is 0.5 to 1 hour. 10. The preparation method according to claim 7, characterized in that in the step 2), the stirring time is 0.5 to 1 hour. 11. The preparation method according to claim 7, characterized in that, in the step 3), the usage ratio of the compound of formula A to the solvent S3 is 1 g: 20-30 mL. 12. The preparation method according to claim 7, characterized in that in the step 3), the stirring time is 0.5 to 2 hours. 13. Use of the crystalline form II of the hydrobromide salt of the compound of formula A as claimed in any one of claims 1 to 5 in the preparation of a medicament for treating and/or alleviating a disease caused by a virus. 14. The use according to claim 13, characterized in that the virus is a coronavirus. 15. The use according to claim 14, characterized in that the coronavirus is SARS-CoV-2. 16. A pharmaceutical composition, characterized in that it comprises: 1) Form II of the hydrobromide salt of the compound of formula A according to any one of claims 1 to 5; 2) Pharmaceutically acceptable excipients. 17. The pharmaceutical composition according to claim 16, characterized in that the pharmaceutical composition is an oral preparation or a non-oral preparation. 18. The pharmaceutical composition according to claim 17, characterized in that the oral preparation is selected from tablets, capsules, granules, powders and syrups, and the non-oral preparation is selected from injections, powder injections, sprays and suppositories.