CN112028889B

CN112028889B - Crystal form of deoxyguanosine analogue

Info

Publication number: CN112028889B
Application number: CN201910477671.XA
Authority: CN
Inventors: 翟立海; 路来菊; 马超; 杨宇
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2019-06-03
Filing date: 2019-06-03
Publication date: 2023-03-17
Anticipated expiration: 2039-06-03
Also published as: CN112028889A

Abstract

The invention belongs to the technical field of crystal form drug molecules, and particularly provides a crystal form of a deoxyguanosine analogue, which uses Cu-Kalpha radiation, and has characteristic peaks at 14.84 +/-0.2 degrees, 16.98 +/-0.2 degrees, 20.19 +/-0.2 degrees, 20.95 +/-0.2 degrees, 25.94 +/-0.2 degrees, expressed by 2 theta, of an X-ray powder diffraction spectrum. The crystal form provided by the invention has the characteristics of better chemical stability, solubility and the like; provides better basis for the application of the compound in the aspect of drug treatment, thereby exerting greater medicinal value.

Description

Crystal form of deoxyguanosine analogue

Technical Field

The invention belongs to the technical field of crystal form drug molecules, and particularly relates to a crystal form of a deoxyguanosine analogue.

Background

Entecavir is a 2, -cyclopenta deoxyguanosine analog with the chemical name [1S- (1 α,3 α,4 β) ] -2-amino-1, 9-dihydro-9- [ 4-hydroxy-3- (hydroxymethyl) -2-methylenecyclopentyl ] -6H-purin-6-one, sparingly soluble in water with a solubility of 2.4mg/mL. The chemical structural formula is as follows:

entecavir is mainly used for treating adult chronic hepatitis B infection accompanied by active virus replication and continuous increase of serum transaminase, or active pathological changes of liver histology. Entecavir can be phosphorylated to active entecavir triphosphate. Entecavir triphosphate competes with deoxyguanosine triphosphate, a natural substrate of HBV polymerase, thereby inhibiting the activity of viral polymerase (reverse transcriptase). Entecavir is a guanine nucleoside analogue oral drug developed by Baishimeibao corporation. Entecavir is approved by the U.S. food and drug administration in 3 months in 2005 and approved by the food and drug administration in China at the end of 2005, and is marketed in China by Shi Guibao pharmaceutical Co., ltd, shanghai, china at the beginning of 2006.

The different crystal forms of the medicine can affect the physicochemical properties of the medicine, directly affect the dissolution and absorption efficiency of the medicine under the physiological pH7.4 condition, and further affect the bioavailability, clinical curative effect and the like of the medicine. By analyzing the crystallization mode of the drug, on one hand, the crystallographic parameters of the drug molecules in the crystal form can be determined, and on the other hand, whether the crystal form contains a solvent can be determined, which has very important effects on understanding and mastering the spatial arrangement and the physicochemical properties of the drug molecules.

CN200410077396.6 discloses entecavir in an amorphous state and a preparation method thereof. CN200910236408.8 discloses a crystal form of entecavir, which is good in stability but low in solubility. Cn201080045378.X discloses a DMF solvate of entecavir, which is generated during the entecavir refining process, and the crystal form stability is poor. CN201310370917.6 discloses an entecavir monohydrate crystal form which has good stability and poor solubility. CN201310286506.9 discloses a new crystalline entecavir, which is a DMF solvate of entecavir, wherein 1 molecule of entecavir is combined with 3 molecules of DMF, and the crystalline form has relatively good solubility relative to entecavir monohydrate, but the stability problem still cannot be solved. CN201510372885.2 discloses an entecavir crystalline compound and a capsule preparation thereof, wherein 15min dissolution rate of the capsule prepared by the crystalline form reaches more than 90%, which is higher than that of an entecavir tablet of a sample on the market, but the crystalline form can generate enantiomer impurities after long-term placement, so that potential threat exists in clinical medication, and the stability of the capsule needs to be further improved. CN201510864425.1 discloses an entecavir dispersible tablet which is prepared by using a crystal form of entecavir, wherein the dissolution rate of the prepared dispersible tablet in 5min reaches more than 98%, but the stability problem of the crystal form is still not solved. Therefore, a crystal form of entecavir with high solubility and good stability is needed.

Disclosure of Invention

Based on the problems of low solubility and poor stability of entecavir in the prior art, the invention provides an entecavir DMSO solvate and simultaneously provides a method for preparing the high-purity entecavir DMSO solvate, which is simple and easy to operate. The entecavir DMSO solvate has the characteristics of good chemical stability, good solubility and the like, and provides a better basis for the application of entecavir in the aspect of drug therapy, so that the medicinal value of entecavir is exerted more efficiently.

The crystal form of the deoxyguanosine analogue in the invention is entecavir DMSO solvate, and the deoxyguanosine analogue is entecavir.

In a first aspect, the present invention provides a crystalline form of an entecavir solvate. The crystal form uses Cu-Kalpha radiation, and an X-ray powder diffraction spectrum of the crystal form has characteristic peaks at 14.84 +/-0.2 degrees, 16.98 +/-0.2 degrees, 20.19 +/-0.2 degrees, 20.95 +/-0.2 degrees and 25.94 +/-0.2 degrees expressed by 2 theta.

Preferably, the crystal form of the entecavir solvate has characteristic peaks expressed in terms of 2 theta at 10.44 +/-0.2 degrees, 14.53 +/-0.2 degrees, 14.84 +/-0.2 degrees, 16.98 +/-0.2 degrees, 17.62 +/-0.2 degrees, 20.19 +/-0.2 degrees, 20.95 +/-0.2 degrees, 24.48 +/-0.2 degrees, 25.06 +/-0.2 degrees, 25.94 +/-0.2 degrees, 26.32 +/-0.2 degrees, 29.94 +/-0.2 degrees and 35.30 +/-0.2 degrees when Cu-Ka radiation is used.

More preferably, the crystal form of the entecavir DMSO solvate has an X-ray powder diffraction spectrum expressed by 2 theta at 10.44 +/-0.2 degrees, 14.05 +/-0.2 degrees, 14.53 +/-0.2 degrees, 14.84 +/-0.2 degrees, 16.04 +/-0.2 degrees, 16.98 +/-0.2 degrees, 17.62 +/-0.2 degrees, 18.36 +/-0.2 degrees, 20.19 +/-0.2 degrees, 20.95 +/-0.2 degrees, 21.20 +/-0.2 degrees, 23.56 +/-0.2 degrees, 24.48 +/-0.2 degrees, 25.06 +/-0.2 degrees, 25.94 +/-0.2 degrees, 26.32 +/-0.2 degrees, 29.94 +/-0.2 degrees, 31.91 +/-0.2 degrees, 34.32 +/-0.2 degrees, 35.30 +/-0.2 degrees, 35.70 +/-0.2 degrees, 37.06 +/-0.2 degrees, 38.09 +/-0.2 degrees, 31.41 +/-0.2 degrees and 2 degrees.

Further preferred is a crystalline form of entecavir DMSO solvate having an X-ray powder diffraction spectrum as shown in figure 1 using Cu-ka radiation.

The second aspect of the present invention provides a preparation method of the entecavir DMSO solvate crystal form, which specifically includes the following steps: adding entecavir into a mixed solvent of an organic solvent A containing DMSO, heating and stirring until the solid is completely dissolved, adding an organic solvent B, naturally cooling to room temperature, standing for crystallization, filtering, and drying to obtain the crystal form of the entecavir DMSO solvate.

Preferably, the organic solvent A is selected from one or more of methanol, ethanol, formic acid, acetic acid and water.

Preferably, the volume content of DMSO in the mixed solvent containing DMSO and the organic solvent A is 5-100%.

When the volume content of DMSO is 100%, the entecavir is only dissolved in DMSO.

Preferably, the organic solvent B is one or more selected from n-hexane, n-heptane, isopropanol, acetone and ethyl acetate.

Preferably, the heating temperature is 10-85 ℃.

Preferably, the mass volume ratio of the entecavir to the mixed solvent is 6-36, preferably 6-20; wherein the mass is in mg and the volume is in ml.

Preferably, the volume ratio of the organic solvent A to the organic solvent B is 1.

Preferably, the crystallization time is 3 to 10 days.

In the preparation method provided by the invention, the deoxynucleotide analogue is entecavir, and the raw material of the entecavir can be selected from entecavir monohydrate sold in the market or entecavir amorphous prepared by the method according to the prior art or other crystal forms of the entecavir; the crystal form of the deoxynucleotide analogue prepared by the invention is an entecavir crystal form, and further is a DMSO solvate crystal form of the entecavir.

The third aspect of the invention provides an application of the entecavir crystal form as an active ingredient in treating adult chronic hepatitis B infection accompanied by active virus replication, continuously increased serum transaminase or active lesion in liver histology.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising a crystalline form of the entecavir DMSO solvate of the present invention in admixture with other ingredients.

Preferably, the pharmaceutical composition of the present invention is prepared as follows: the compounds of the present invention are combined with pharmaceutically acceptable solid or liquid carriers and optionally with pharmaceutically acceptable adjuvants or excipients using techniques conventional in the art to prepare useful dosage forms.

Preferably, the other components include other active ingredients, excipients, fillers, diluents, binders, disintegrants, lubricants, glidants, and the like that may be used in combination.

Preferably, the pharmaceutical composition is a spray, a tablet, a capsule, a powder injection, a liquid injection, a pill and the like.

The specific structure of the entecavir crystal form prepared by the invention is confirmed as follows:

the entecavir crystal form provided by the invention is further subjected to X-ray single crystal diffraction test analysis. The test conditions were: a pharmacological XtaLAB Synergy X-ray single crystal diffractometer; light source copper target, temperature 293K, voltage 50kv, current 1mA, data were collected in omega scan mode and Lp correction was performed. Analyzing the structure by a direct method, finding out all non-hydrogen atoms by a difference Fourier method, obtaining all hydrogen atoms on carbon and nitrogen by theoretical hydrogenation, and refining the structure by a least square method. Further, the structure was analyzed to obtain an X-ray single crystal diffraction pattern (SXRD) as shown in FIG. 2.

The crystallographic parameters are as follows: monoclinic crystal system, space group is P2 ₁ (ii) a The unit cell parameters are as follows: a =10.6143 (2), b =14.5566

(3) C =38.400 (3), α =90 °, β =101.052 (2) °, γ =90 °, z =2, z' =1, unit cell volume

3. The ORTEP chart of the entecavir crystal shows that one molecule of entecavir shares one DMSO molecule, and the crystal form can be confirmed to be an entecavir DMSO solvate. The main crystallographic parameters are shown in table 1.

TABLE 1 main crystallographic data for entecavir DMSO solvate

The X-ray powder diffraction test instrument and the test conditions of the invention are as follows: x-ray powder diffractometer PANalytical Emprev; cu-K alpha; a sample stage: a flat plate; the incident light path is BBHD; diffraction light path: PLXCEL; voltage 45kv and current 40mA; 1/4 of divergent slit; 1, an anti-scattering slit; 0.04rad of rope pulling slit; step length: 0.5s; scanning range: 3 to 50 degrees.

According to the crystallographic data, the characteristic peak in the corresponding X-ray powder diffraction pattern (Cu-K alpha) is shown in the attached figure 1 and the table 2.

TABLE 2 major XRD peaks for entecavir DMSO solvate crystals

Tests prove that the entecavir crystal form provided by the invention has better solubility, 10ml of water is measured and put into a penicillin bottle, excessive medicine is added, the penicillin bottle is sealed and placed into a thermostatic water bath at 25 ℃ for stirring for 1 hour, the mixture is filtered through a 0.45-micron filter membrane, filtrate is taken to measure absorbance at the wavelength of 254nm, and the solubility of the entecavir DMSO solvate reaches 15.6mg/ml which is 6-7 times of the solubility of the commercially available entecavir through the calculation of the absorbance of a standard reference substance. Provides a raw material with high solubility for preparing an entecavir preparation.

The method for preparing the entecavir DMSO solvate crystal is simple and convenient to operate, the prepared crystal is high in purity, the entecavir DMSO solvate crystal provided by the invention has good chemical stability and good solubility, and the prepared tablet can be quickly dissolved out.

Drawings

FIG. 1 is an X-ray powder diffraction pattern of entecavir DMSO solvate crystals;

FIG. 2 is a diagram of ORTEP crystals of entecavir DMSO solvate;

figure 3 is a crystal hydrogen bond diagram of entecavir DMSO solvate.

Detailed Description

The invention is further illustrated by the following examples, which should be properly understood: the examples of the present invention are intended to be illustrative only and not limiting, and the present invention may be modified in a simple manner within the scope of the invention as claimed.

The raw material entecavir 1 to 8 used in the following embodiments is commercially available as entecavir monohydrate, and other materials are commercially available without specific reference.

Example 1

Adding 100mg of entecavir into 10ml of DMSO, heating to 60 ℃, stirring until the solid is completely dissolved, adding 40ml of n-hexane, filtering, naturally cooling to room temperature, standing for crystallization for 7 days, filtering, collecting the solid, and drying in an oven at 50 ℃ for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 92.3%, and the HPLC purity is 99.98%.

Example 2

Adding 120mg of entecavir into 20ml of mixed solvent of DMSO and water (the volume content of the DMSO is 5%), heating to 85 ℃, stirring until the solid is completely dissolved, adding 160ml of ethyl acetate, filtering, naturally cooling to room temperature, standing for 3 days, filtering, collecting the solid, and drying in an oven at 50-60 ℃ for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 91.7%, and the HPLC purity is 99.97%.

Example 3

Adding 400mg of entecavir into 20ml of mixed solvent of DMSO and formic acid (the volume content of the DMSO is 20%), stirring at 10 ℃ until the solid is completely dissolved, adding 200ml of n-heptane, filtering, standing for crystallization for 7 days, filtering, collecting the solid, and drying in an oven at 50-60 ℃ for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 93.1%, and the HPLC purity is 99.99%.

Example 4

Adding 300mg of entecavir into 20ml of a mixed solvent of DMSO and methanol (the volume content of DMSO is 30%), heating to 40 ℃, stirring until the solid is completely dissolved, adding 100ml of acetone, filtering, naturally cooling to room temperature, standing for 10 days, filtering, collecting the solid, and drying in a 50-60 ℃ oven for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 90.8%, and the HPLC purity is 99.95%.

Example 5

Adding 360mg of entecavir into 10ml of a mixed solvent of DMSO and ethanol (the volume content of DMSO is 60%), heating to 50 ℃, stirring until the solid is completely dissolved, adding 160ml of isopropanol, filtering, naturally cooling to room temperature, standing for 7 days, filtering, collecting the solid, and drying in an oven at 50-60 ℃ for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 90.5%, and the HPLC purity is 99.92%.

Example 6

Adding 100mg of entecavir into 10ml of a mixed solvent of DMSO and acetic acid (the volume content of DMSO is 10%), stirring at room temperature until the solid is completely dissolved, adding 40ml of n-hexane, filtering, standing for crystallization for 7 days, filtering to collect the solid, and drying in an oven at 50-60 ℃ for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 92.6%, and the HPLC purity is 99.96%.

Example 7

Adding 100mg of entecavir into 2ml of DMSO, heating to 60 ℃, stirring until the solid is completely dissolved, adding 40ml of n-hexane, filtering, naturally cooling to room temperature, standing for crystallization for 7 days, filtering to collect the solid, and drying in an oven at 50-60 ℃ for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 85.3%, and the HPLC purity is 99.90%.

Example 8

Adding 100mg of entecavir into 20ml of mixed solvent of DMSO and acetic acid (the volume content of the DMSO is 50%), stirring at room temperature until the solid is completely dissolved, adding 40ml of n-hexane, filtering, standing for crystallization for 7 days, filtering, collecting the solid, and drying in an oven at 50-60 ℃ for 24 hours to obtain the entecavir DMSO solvate crystal form, wherein the yield is 80.6%, and the HPLC purity is 99.92%.

Comparative example 1

Adding 200mg of entecavir into 5ml of DMF, heating until the mixture is dissolved, stirring and naturally cooling for crystallization, cooling to room temperature, continuously stirring for crystallization for 4 hours, filtering, leaching with a small amount of purified water, draining, and vacuum drying at 50 ℃ to obtain the entecavir combined 3-molecule DMF crystal form. The yield was 65.3% and the HPLC purity was 99.76%.

Comparative example 2

Adding 1.0g of entecavir, 18ml of anhydrous methanol and 3ml of DMF into a 50ml volumetric flask, heating to 70 ℃, stirring for 30min, cooling to 0 ℃, standing for 8h, performing suction filtration to obtain white crystalline powder, washing for 2 times by using a small amount of methanol, and performing vacuum drying to obtain the entecavir crystal form. Yield 66.4% and HPLC purity 99.68%.

Comparative example 3

Adding 1g of entecavir into 5ml of DMF, stirring for 2h at room temperature, stirring for 1h at 0 ℃, filtering the solution to obtain a white solid, and drying in vacuum to obtain the entecavir combined 1 molecular DMF crystal form. Yield 85.7%, HPLC purity: 98.36 percent.

Comparative example 4

Dissolving 2g of entecavir in 40ml of ethanol solvent which is heated to boiling, filtering the prepared hot solution into 30ml of isopropanol solvent which is boiling, cooling the solution to room temperature, keeping the obtained suspension solution at 20 ℃ for 3h, collecting the precipitate by a centrifugal filtration method, washing the precipitate with ethanol at 4 ℃, heating the precipitate to 40 ℃ in vacuum, and drying the precipitate for 15h to obtain the entecavir solid. The yield was 75.3% and the HPLC purity was 97.29%.

Comparative example 5

Adding 15g of entecavir solid into 100ml of a mixed solvent of dimethyl acetamide and methanol, wherein the volume ratio of dimethyl acetamide to methanol is 1; controlling the temperature to be 35-40 ℃, and adding an isopropanol water solution into the obtained solution, wherein the isopropanol water solution contains 30% of isopropanol ethanol by mass; adding an isopropanol water solution, cooling to-10 ℃ at the speed of 2.5 ℃/10min, standing at-10 ℃ for 18 hours, separating out crystals, filtering, washing a filter cake with diethyl ether, and drying in vacuum to obtain the entecavir crystals. The yield was 80.2% and the HPLC purity was 99.85%.

Comparative example 6

Preparing 100ml of mixed solution A by using dimethylformamide and acetone according to the volume ratio of 1; taking 10g of crude entecavir, adding the mixed solution A, heating to 40 ℃, stirring to completely dissolve, adding 0.1g of activated carbon into the obtained solution for decolorization, and filtering to obtain a clear solution; (3) Preparing 600ml of mixed solution B from isopropyl ether and isopropanol in a volume ratio of 2.5; after the ultrasonic treatment is finished, the temperature is reduced to-5 ℃, the mixture is kept stand for 3 hours, crystals are separated out, and the entecavir crystals are obtained after drying. The yield was 66.7% and the purity 98.63%.

Verification examples

(1) Stability test

The stability tests of the crystal forms of Entecavir monohydrate sold in examples 1-8 and comparative examples 1-6 and the crystal forms of Entecavir monohydrate sold in market are carried out under the conditions of illumination, high temperature and high humidity, the specific stability test method refers to the guidance method related to stability investigation in the fourth part of the Chinese pharmacopoeia 2015 edition, the purity detection is carried out by an HPLC method, and the specific test results are shown in Table 3.

TABLE 3 stability test results of entecavir crystalline forms under light, high temperature and high humidity conditions

As can be seen from the results in Table 3, the entecavir DMSO solvate crystal form has good stability, and the purity of the entecavir DMSO solvate crystal form does not change significantly after being placed for 5 days or 10 days under the conditions of illumination, high temperature and high humidity.

(2) Dissolution test

Entecavir tablets were prepared according to the following formulation using the crystalline forms of the present invention (example 1) and the crystalline form samples of entecavir prepared in comparative examples 1-6, respectively.

Prescription:

the preparation method comprises the following steps: weighing lactose in a prescription amount, sieving for later use, weighing PVPK30 in the prescription amount to prepare a binder, adding entecavir in the prescription amount to dissolve, granulating the lactose by using the solution, drying, adding sodium carboxymethyl starch and magnesium stearate in the prescription amount, and tabletting to obtain the tablet.

And (3) dissolution rate determination: referring to the dissolution rate determination method of the year edition of the Chinese pharmacopoeia 2015, 900mL of 0.1mol/L hydrochloric acid solution is used as a dissolution medium, the rotating speed is 75 revolutions per minute, the contents of entecavir are determined and analyzed by HPLC (high performance liquid chromatography) by taking 10mL of the solution (10 mL of the dissolution medium with the same temperature is supplemented after each sampling) at 5min, 15min and 30min respectively, and the dissolution rates of the tablets prepared by using the crystal form of the example 1 and the comparative examples 1-6 and the commercially available entecavir tablets are calculated. The dissolution rate results of tablets prepared from different entecavir crystal forms in 0.1mol/L hydrochloric acid solution are shown in Table 4.

TABLE 4 dissolution rate determination test results for tablets prepared from different entecavir samples

As can be seen from the results in Table 4, the tablet prepared from the entecavir DMSO solvate crystal form can be quickly dissolved out, and is far larger than the commercially available entecavir tablets; the dissolution rate of the tablets prepared by the crystal forms of the comparative examples 1 to 6 is lower than that of the entecavir DMSO solvate.

Claims

1. A crystal form of entecavir DMSO solvate is characterized in that Cu-Ka radiation is used, and an X-ray powder diffraction spectrum of the crystal form has characteristic peaks expressed by 2 theta at 14.84 +/-0.2 degrees, 16.98 +/-0.2 degrees, 20.19 +/-0.2 degrees, 20.95 +/-0.2 degrees and 25.94 +/-0.2 degrees.

2. A crystalline form according to claim 1, characterized in that, using Cu-ka radiation, its X-ray powder diffraction spectrum has characteristic peaks, expressed in 2 Θ, at 10.44 ± 0.2 °,14.53 ± 0.2 °,14.84 ± 0.2 °,16.98 ± 0.2 °,17.62 ± 0.2 °,20.19 ± 0.2 °,20.95 ± 0.2 °,24.48 ± 0.2 °,25.06 ± 0.2 °,25.94 ± 0.2 °,26.32 ± 0.2 °,29.94 ± 0.2 °,35.30 ± 0.2 °.

3. A crystalline form according to claim 1, characterized in that, using Cu-ka radiation, its X-ray powder diffraction spectrum has peaks, expressed in 2 Θ, at 10.44 ± 0.2 °,14.05 ± 0.2 °,14.53 ± 0.2 °,14.84 ± 0.2 °,16.04 ± 0.2 °,16.98 ± 0.2 °,17.62 ± 0.2 °,18.36 ± 0.2 °,20.19 ± 0.2 °,20.95 ± 0.2 °,21.20 ± 0.2 °,23.56 ± 0.2 °,24.48 ± 0.2 °,25.06 ± 0.2 °,25.94 ± 0.2 °,26.32 ± 0.2 °,29.94 ± 0.2 °,31.91 ± 0.2 °,34.32 ± 0.2 °,35.30 ± 0.2 °,35.70 ± 0.2 °,37.06 ± 0.2 °,38.09 ± 0.2 °, 31.04 ± 0.2 °.

4. The crystalline form of claim 1, having an X-ray powder diffraction spectrum as shown in figure 1 using Cu-ka radiation.

5. A method for preparing the crystal form of any one of claims 1 to 4, which is characterized by comprising the following steps: adding entecavir into a mixed solvent containing DMSO and an organic solvent A, heating and stirring until the solid is completely dissolved, adding an organic solvent B, naturally cooling to room temperature, standing for crystallization, filtering, and drying to obtain a crystal form of an entecavir DMSO solvate;

the organic solvent A is selected from one or more of methanol, ethanol, formic acid, acetic acid and water; the volume content of DMSO in the mixed solvent is 5-100%;

the organic solvent B is selected from one or more of n-hexane, n-heptane, isopropanol, acetone and ethyl acetate;

the mass volume ratio of the entecavir to the mixed solvent of the DMSO and the organic solvent A is 6 to 36; wherein the mass is in mg and the volume is in ml; the volume ratio of the organic solvent A to the organic solvent B is 1.

6. The preparation method according to claim 5, wherein the mass volume ratio of the entecavir to the mixed solvent of the DMSO and the organic solvent A is 6 to 20:1; wherein the mass is in mg and the volume is in ml.

7. Use of the crystalline form of any one of claims 1-4 for the manufacture of a medicament for the treatment of chronic hepatitis B infection in adults with active viral replication, persistent elevation of serum transaminases, or active lesions of hepatic histology.