AU2022100062A4 - Crystal form for treating liver disease and use thereof - Google Patents

Abstract

The invention provides crystal forms of Tenofovir phosphate (9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]a denine fumarate), Pradefovir mesylate ((+)-cis-9-{2-[4-[(S)-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphacyclo-2-methylene] -1-ethyl}adenine mesylate) and Cytarabine prodrug (4-Amino-I-[5-0-(2R,4S)-2-oxy-4-(4-pyridine)-1,3,2-dioxaphosphine-heterocyclohexane-2]-B D-arabinofuranosyl-2(lH)-pyrimidinone; MB07133) for treating a liver disease. The invention also provides a pharmaceutical composition comprising the crystal form and a use thereof in the preparation of medicines.

Description

Crystal form for treating liver disease and use thereof

Prior application

The application claims the priority of Chinese patent applications 202010376833.3

(application date: May 7, 2020), 202010376938.9 (application date: May 7, 2020) and

202010391424.0 (application date: May 11, 2020).

Technical Field The invention is associated with the field of medicinal chemistry; in particular, the invention

is related to Tenofovir phosphate

(9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy] propyl]

adenine fumarate), Pradefovir mesylate

((+)-cis-9-{2-[4-[(S)-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphacyclo-2-methylene]

-1-ethyl}adenine methanesulfonate) and Cytarabine prodrug

(4-Amino-i-[5-0-(2R,4S)-2-oxy-4-(4-pyridine)-1,3,2-dioxaphosphine-heterocyclohexane-2]-B

D-arabinofuranosyl-2(1H)-pyrimidinone) and other forms of compound crystals used in the

treating the liver diseases and their uses thereof, etc.

Background Techniques

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]ad

enine fumarate (also referred to as Tenofovir phosphate in the invention) has a structure as shown

in formula (I):

0 H2N HOA O N4 N 0 KNN

05

(1)

The compound is a prodrug compound (HTS) of Tenofovir among the prodrug compounds

disclosed in Chinese Patent No. 200580018611.8, which can be used to treat or prevent liver diseases or metabolic diseases, including hepatitis B and others. Chinese Patent No.

201310283713.9 discloses a crystal of Tenofovir prodrug (HTS), and specifically describes a

crystal-1 type crystal preparation method for the succinate of Tenofovir prodrug

(9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine succinate).

The chemical name of Pradefovir mesylate is

(+)-cis-9-{2-[4-[(S)-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphacyclo-2-methylene]

-1-ethyl}adenine mesylate; its structure is shown in formula (I):

H 3CO 3SHH 2N

N N (I )

The compound is a PMEA liver-targeted prodrug disclosed in Chinese patent CN1964967A.

It is converted into a pharmacologically active PMEA under the catalysis of the liver CYP3A4

enzyme. It fits for treating adult chronic hepatitis B patients with hepatitis B virus active

replication, and/or with compensation of liver function with persistent elevation of serum amino

acid transferase or active lesions of liver histology. It can significantly inhibit the replication of the

hepatitis B virus; the curative effect is remarkable. It also has obvious inhibitory effect on

lamivudine-resistant mutants, which solves the problem of resistance to anti-HBV drugs;

compared with Adefovir dipivoxil, its liver targeting avoids nephrotoxicity caused by drug

metabolism. Chinese patent CN102827206A reports two crystal forms i.e. crystal forms I and II;

these can be obtained by crystallization in aqueous acetonitrile and in aqueous methanol,

respectively. Since the description of these two, no new crystal forms of Pradefovir mesylate have

been reported.

MB07133, i.e.

4-Amino-I-[5-0-(2R,4S)-2-oxy-4-(4-pyridine)-1,3,2-dioxaphosphine-heterocyclohexane-2]-B-

D-arabinofuranosyl-2(1H)-pyrimidinone, also known as Cytarabine prodrug, its structure is shown

in formula (I):

NH2

H6 OH

(1)

The compound is a hepdirect prodrug compound of Cytarabine among the prodrug

compounds disclosed in Chinese patent CN1711278A, which can be used for the treating of or

prevention of liver disease or metabolic disease, especially for the treating of advanced

hepatocarcinoma. However, the patent does not disclose or suggest that crystallographic studies of

the compound should be carried out.

The inventors efficiently prepared

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine fumarate; in cases where it is not yet possible to predict whether the fumarate salt of

Tenofovir prodrugs can form a new (mono) crystalline form, likewise, in the case that whether

there is a new (mono) crystal form of Pradefovir sulfonate is still unpredictable, and whether there

is a new (mono) crystal form of MB07133 is also unpredictable; the existing solvents that can be

used for crystallization and the number of mixed solvents is astronomical. However, the inventors

did not flinch, and carried out long-term and painstaking research, and found that

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine fumarate, Pradefovir mesylate and MB07133 were crystallized respectively, and the

crystals had a complex distribution; polycrystals were often crystallized, and it was not easy to

obtain mono-crystals, but the inventors finally obtained a series of crystals by unexpectedly

effective crystallization, and selected 5 or 5 and 6 kinds of different mono crystals, respectively.

These crystals offer advantages in terms of stability, thereby facilitating production, storage and/or

improving the safety of liver-targeted therapy.

Description of the Invention

The technical problem to be solved by the invention is to provide new crystal forms of

compounds for treating liver diseases, including

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine fumarate, Pradefovir mesylate or new crystal forms of MB07133. In addition, the

invention also provides a preparation method of the crystal form, a medicine comprising the

crystal form, and a therapeutic use thereof and detection method.

Specifically, in a first aspect, the invention provides a crystal of a compound for treating

liver disease, which is a crystal of

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine fumarate, a crystal of Pradefovir mesylate, or a crystal of MB07133.

In the crystal of the first aspect of the invention, the crystal of

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]ad

enine fumarate is selected from one of the crystal forms a, b, c, d and e:

Crystal form a has an X-ray powder diffraction pattern substantially as shown in Figure 1-1.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

a has diffraction peaks at 20(, ±0.2): 5.9, 12.1°, 24.8°, and 31.2°; in addition, the differential

thermal analysis curve of the type crystal has a sharp endothermic peak at 151.7°C;

Crystal form b has an X-ray powder diffraction pattern substantially as shown in Figure 1-2.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

b has diffraction peaks at 20(, ±0.2): 9.8°, 10.4°, 11.6°, 12.5°, 13.3°, 15.2°, 15.5°, 16.9°, 17.3°,

19.5°, 22.1°, 23.3°, 24.9°, and 31.2°; in addition, the differential thermal analysis curve of the type

crystal has a sharp endothermic peak at 152.3°C;

Crystal form c has an X-ray powder diffraction pattern substantially as shown in Figure 1-3.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

c has diffraction peaks at 20(, ±0.2): 9.70, 10.4°, 11.6°, 12.5°, 13.3°, 15.2°,16.8°, 17.2°,

19.5°,21.5°, 22.1°, 23.2°, 24.8°, 26. 0 °and 31.1; in addition, the differential thermal analysis curve

of the type crystal has a sharp endothermic peak at 152.6°C;

Crystal form d has an X-ray powder diffraction pattern substantially as shown in Figure 1-4.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

d has diffraction peaks at 20(, ±0.2): 9.9°, 10.4°, 12.6°, 13.4°, 13.8°, 17.2°,18.3°, 19.5°,

20.2°,20.9°, 22.0°, 23.3°, and 25.1°; in addition, the differential thermal analysis curve of the type

crystal has a sharp endothermic peak at 149.2°C;

Crystal form e has an X-ray powder diffraction pattern substantially as shown in Figure 1-5.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

e has diffraction peaks at 20(, ±0.2): 9.8°, 10.5°, 11.6°, 12.6°, 13.4°, 13.9°, 15.3°, 17.4°, 19.6°,

21.0°, 22.1°, 23.4°, 25.0°, and 26.2°; in addition, the differential thermal analysis curve of the type

crystal has a sharp endothermic peak at 151.4°C;

In the crystal of the first aspect of the invention, the crystal of Pradefovir mesylate is selected

from one of crystal forms A, B, C, D and E:

Crystal form A has an X-ray powder diffraction pattern substantially as shown in Figure 2-1.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

A has diffraction peaks at 20(, ±0.2): 11.0°, 12.0°, 13.0°, 16.5°, 17.6°, 19.7°, 20.5°, 22.0°, 24.2°,

26.4°, 27.5°, 27.9°, 28.4°, 33.6°, 41.5°, and 45.6°; in addition, the differential thermal analysis

curve of the type crystal has a sharp endothermic peak at 196.9°C;

Crystal form B has an X-ray powder diffraction pattern substantially as shown in Figure 2-3.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

B has diffraction peaks at 20(, ±0.2): 11.8°, 12.8°, 16.3°, 17.4°, 19.7°, 20.2°, 21.9°, 24.0°, 25.3°,

26.1°, 26.9°, 27.2°, 27.7°, and 28.1° ; in addition, the differential thermal analysis curve of the

type crystal has a sharp endothermic peak at 191.8°C;

Crystal form C has an X-ray powder diffraction pattern substantially as shown in Figure 2-5.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

C has diffraction peaks at 20(, ±0.2): 13.0°, 16.6°, 17.6°, 19.7°, 22.2°, 24.2°,26.4°, and 28.3°; in

addition, the differential thermal analysis curve of the type crystal has a sharp endothermic peak at

193.4°C;

Crystal form D has an X-ray powder diffraction pattern substantially as shown in Figure 2-7.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

D has diffraction peaks at 20(, ±0.2): 15.5°, 17.2°, 17.6°, 21.9°, and 26.1°; in addition, the

differential thermal analysis curve of the type crystal has a sharp endothermic peak at 192.6°C;

Crystal form E has an X-ray powder diffraction pattern substantially as shown in Figure 2-9.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

D has diffraction peaks at 20(, ±0.2): 12.9°, 16.4°, 17.4°, 19.5°, 22.0°, 26.2°, and 28.2°; in

addition, the differential thermal analysis curve of the type crystal has a sharp endothermic peak at

193.2°C;

In the crystal of the first aspect of the invention, the crystal of MB07133 is selected from one

of crystal form A, B, C, D, E or F:

Crystal form A has an X-ray powder diffraction pattern substantially as shown in Figure 3-1.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

A has diffraction peaks at 20(, ±0.2): 10.6°, 12.5°, 13.7°, 16.0, 16.3°, 17.2°, 17.4°, 18.6°, 20.3°,

21.8°, 22.2°, 23.1°, 23.4°, 24.9°, 25.6°, 26.0°, 28.7°, 29.4°, 30.1°, 31.0°, 32.9°, and 37.8°; in

addition, the differential thermal analysis curve of the type crystal has a sharp endothermic peak at

239.81°C;

Crystal form B has an X-ray powder diffraction pattern substantially as shown in Figure 3-2.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

B has diffraction peaks at 20(, ±0.2): 10.4°, 12.°, 15.8°, 16.°, 17.3°, 18.6°, 20.2°, 21.6°, 22.°,

22.9°, 23.2°, 24.8°, 25.4°, 25.8°, and 30.8°; in addition, the differential thermal analysis curve of

the type crystal has a sharp endothermic peak at 253.21°C;

Crystal form C has an X-ray powder diffraction pattern substantially as shown in Figure 3-3.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

C has diffraction peaks at 20(, ±0.2): 10.5°, 12.4°, 13.5°, 15.8°, 16.3°, 17.4°, 18.6°, 20.2°, 21.6°,

22.1°, 23.0°, 23.3°, 24.8°, 25.5°, 28.6°, 29.3°, and 31.0°; in addition, the differential thermal

analysis curve of the type crystal has a sharp endothermic peak at 247.95°C;

Crystal form D has an X-ray powder diffraction pattern substantially as shown in Figure 3-4.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

D has diffraction peaks at 20(, ±0.2): 12.5°, 15.50, 15.9°, 16.3°, 18.2°, 18.6°, 19.6°, 20.3°, 21.2°,

23.1°, 23.4°, 24.2°, 24.4°, 24.8°, and 27.4°; in addition, the differential thermal analysis curve of

the type crystal has a sharp endothermic peak at 251.10°C;

Crystal form E has an X-ray powder diffraction pattern substantially as shown in Figure 3-5.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

E has diffraction peaks at 20(,±0.2): 10.6°, 12.5°, 13.7°, 15.5°, 16.0°, 16.5°, 17.2°, 17.5°, 18.2°,

18.7°, 20.4°, 21.7°, 22.2°, 23.1°, 23.4°, 24.3°, 24.9°, 25.6°, 26.0°, 27.2°, 29.4°, 31.0°, and 41.5°;

in addition, the differential thermal analysis curve of the type crystal has a sharp endothermic peak

at 246.59°C;

Crystal form F has an X-ray powder diffraction pattern substantially as shown in Figure 3-6.

In a specific implementation of the invention, the X-ray powder diffraction pattern of crystal form

E has diffraction peaks at 20(, ±0.2): 10.6°, 12.3°, 12.5°, 13.7°, 16.0°, 16.5°, 17.2°, 17.5°, 17.7°,

18.7°, 20.4°, 21.8°, 22.2°, 23.1°, 23.4°, 24.9°, 25.6°, 28.7°, 29.4°, 30.1°, and 31.0°; in addition, the

differential thermal analysis curve of the type crystal has a sharp endothermic peak at 249.26°C;

In the document, unless indicated to the contrary, the terms "crystal" and "crystal form" are

used interchangeably and refer to a solid whose internal particles are periodically repeated in

three-dimensional space; the terms (of a compound) "(X) crystal form", "crystal form (X)",

"crystal form (X) crystal" and "(X) form crystal" can be used interchangeably, referring to the

(X)-kind of crystal. Preferably, the crystal of the first aspect of the invention is a mono crystal.

There are many kinds of solvents that can be used for crystallization in the existing art. In

addition, mixed solvents composed of different kinds and proportions of solvents are even more

uncountable, the crystallization practice is basically still left to experience, and generally it is

impossible to predict the crystallized crystal types of the crystallization conditions. While the

inventors engaged in long and painstakingly research studies on

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine fumarate, Pradefovir mesylate, and MB07133, and with some luck, finally found solvents

that can be used to crystallize

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]

adenine fumarate, Pradefovir mesylate and MB07133 and discovered appropriate preparation

methods. Thus in a second aspect, the invention provides a method for the preparation of the

crystals of the first aspect of the invention.

For Tenofovir phosphate crystal form a, its preparation method includes:

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]ad

enine fumarate was mixed with methanol and heated to 60-70 0 C, dissolved and cooled to 20

25 0 C, and the crystals were collected and dried. Wherein,

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]ad

enine fumarate can be amorphous, or it can be in crystal form b, c, d or e.

For Tenofovir phosphate crystal form b, its preparation method includes:

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]ad

enine fumarate was mixed with water and acetonitrile and heated to 65°C, dissolved and cooled to

20-25°C, and the crystals were collected and dried. Wherein,

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]ad

enine fumarate can be amorphous, or it can be in crystal form a or c.

For Tenofovir phosphate crystal form c, its preparation method includes:

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]ad

enine fumarate was mixed with tetrahydrofuran and heated to 65°C, dissolved and cooled to 20

25°C, and the crystals were collected and dried. Wherein,

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]ad

enine fumarate can be amorphous, or it can be in crystal form a or e.

For Tenofovir phosphate crystal form d, its preparation method includes:

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]ad

enine fumarate was mixed with water and heated to 80°C, dissolved and cooled to 20-25°C, and

the crystals were collected and dried. Wherein,

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]ad

enine fumarate can be amorphous, or it can be in crystal form a.

For Tenofovir phosphate crystal form e, its preparation method includes:

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine fumarate was mixed with isopropanol and heated to 70°C, and the crystals were collected

and dried. Wherein,

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxophosphahexacyclo-2-methoxy]propyl]ad

enine fumarate can be amorphous, or it can be in crystal form a or c.

For Pradefovir mesylate crystal form A, its preparation method includes: Dissolve Pradefovir

mesylate in water or methanol, add acetone, methyl tert-butyl ether or ethyl acetate dropwise with

stirring, then stir at 10-30°C, cool down to 0-10°C and stir, and the crystals were collected and

dried. Wherein, Pradefovir mesylate can be amorphous, or it can be crystal form I or II reported in

CN102827206A, and it can also be crystal form B, C, D or E.

For Pradefovir mesylate crystal form B, its preparation method includes: Pradefovir mesylate

was dissolved in 1,4-dioxane, stirred at 10-30°C, and the crystals were collected and dried.

Wherein, Pradefovir mesylate can be amorphous, or can be crystal form I or II reported in

CN102827206A, and it can also be crystal formA, C, D or E.

For Pradefovir mesylate crystal form C, its preparation method includes: Pradefovir mesylate

was dissolved in DMF, MTBE was added dropwise with stirring, and then the mixture was stirred

at 10-30°C, and the crystals were collected and dried. Wherein, Pradefovir mesylate can be

amorphous, or can be crystal form I or II reported in CN102827206A, and it can also be crystal

form A, B, D, or E.

For Pradefovir mesylate crystal form D, its preparation method includes: Heat and dissolve

Pradefovir mesylate in a THF solution containing water, cool down to 10-30°C and stir, and the

crystals were collected and dried. Wherein, Pradefovir mesylate can be amorphous, or can be

crystal form I or crystal form II reported in CN102827206A, and it can also be crystal form A, B,

D, or E.

For Pradefovir mesylate crystal form E, its preparation method includes: Mix Pradefovir

mesylate with acetonitrile, heat under reflux to dissolve, cool to 10-30°C and stir, and the crystals

were collected and dried. Wherein, Pradefovir mesylate can be amorphous, or can be crystal form

I or II reported in CN102827206A, and it can also be crystal form A, B, D or E.

For MB07133 crystal form A, the preparation method includes: Dissolve MB07133 in

sulfuric acid solution, stir at 15-25°C, add sodium dihydrogen phosphate, then slowly add sodium

hydroxide solution, adjust pH value to 5.0-8.0, stir at 10-20°C, and the crystals were collected

and dried. Whereas the MB07133 can be amorphous, or can be crystal form B, C, D, E or F.

For MB07133 crystal form B, the preparation method includes: MB07133 was heated and

stirred in dimethyl sulfoxide until dissolved, toluene was added dropwise, cooled to -10°C,

stirred, and the crystals were collected and dried. Whereas the MB07133 can be amorphous, or can

be crystal form A, C, D, E or F.

For MB07133 crystal form C, the preparation method includes: MB07133 was heated and

stirred in dimethyl sulfoxide until dissolved, acetone was added dropwise, cooled to -10°C,

stirred, and the crystals were collected and dried. Whereas the MB07133 can be amorphous, or can be crystal form A, B, D, E or F.

For MB07133 crystal form D, the preparation method includes: MB07133 was heated and

stirred in N,N-dimethylformamide until dissolved, ethyl acetate was added dropwise, cooled to 0

10°C, stirred, and the crystals were collected and dried. Whereas the MB07133 can be amorphous,

or can be crystal form A, B, C, E, or F.

For MB07133 crystal form E, the preparation method includes: MB07133 was heated and

stirred in N-methylpyrrolidone and water until dissolved, acetone was added dropwise, cooled to

0-10°C, stirred, and the crystals were collected and dried. Whereas the MB07133 can be

amorphous, or can be crystal form A, B, C, D or F.

For MB07133 crystal form F, the preparation method includes: MB07133 was heated and

stirred in dimethyl sulfoxide and water until dissolved, isopropanol was added dropwise, cooled to

0-10°C, stirred, and the crystals were collected and dried. Whereas the MB07133 can be

amorphous, or can be crystal form A, B, C, D or E.

In a third aspect, the invention provides a pharmaceutical composition for treating or

preventing liver disease or metabolic disease, which comprises the crystal of the first aspect of the

invention and a pharmaceutically acceptable excipient, preferably composed of the composition of

crystals and pharmaceutically acceptable excipients as stated in the first aspect of the invention. In

the paper, pharmaceutically acceptable excipients refer to nontoxic fillers, stabilizers, diluents,

adjuvants or other formulation excipients. For example, diluents and excipients, such as water,

normal saline, microcrystalline cellulose, etc.; fillers, such as starch, sucrose, etc.; adhesives, such

as starch, cellulose derivatives, alginates, gelatin, and/or polyamides vinylpyrrolidone; wetting

agents, such as glycerin; disintegrating agents, such as agar, calcium carbonate and/or sodium

bicarbonate; absorption enhancers, such as quaternary ammonium compounds; surfactants, such as

cetyl alcohol; adsorption carriers, such as kaolin and/or bentonite; lubricants, such as talc,

calcium/magnesium stearate, and polyethylene glycol, etc. In addition, the pharmaceutical

composition of the invention may further contain other adjuvants, such as flavoring agents,

sweeteners, etc. The pharmaceutical composition of the third aspect of the invention may further

comprise other active ingredients for the treating or prevention of liver disease or metabolic

disease.

Preferably, in the pharmaceutical composition of the third aspect of the invention, the pharmaceutically acceptable excipients include mannitol, pregelatinized starch, magnesium stearate and/or silicon dioxide.

The pharmaceutical composition of the third aspect of the invention is preferably used for the

treating or prevention of hepatitis B, and also preferably for reducing the level of hepatitis B virus

in a patient. When the pharmaceutical composition of the third aspect of the invention comprises

MB07133 crystal, it is preferably used for the treating advanced hepatocarcinoma, more

preferably used for the targeted treating advanced hepatocarcinoma.

Based on the known technology in the field, the pharmaceutical composition can be made

into various dosage forms based on the needs of the therapeutic purpose and the route of

administration, preferably the composition is in the form of a unit dosage form, such as

lyophilisate, tablet, capsule, powder, emulsion, water injection or spray, more preferably the

pharmaceutical composition is an injection dosage form (e.g., the lyophilized powder for injection)

or an oral dosage form, more preferably an oral dosage form (e.g., the tablet or capsule). The drug

can be administered by conventional routes, especially enterally, e.g. orally, e.g. in the form of

tablets or capsules; or parenterally, e.g. in the form of injectable solutions or suspensions; or nasal

administration. In particular among the implementations of the invention, an exemplary

pharmaceutical composition comprising crystals of Tenofovir phosphate is an oral dosage form,

such as a tablet; another exemplary pharmaceutical composition comprising crystals of Tenofovir

phosphate is injections, such as freeze-dried powder injections.

In a fourth aspect, the invention provides the use of the crystal of the first aspect of the

invention in the preparation of a medicament for the treating or prevention of liver disease or

metabolic disease. Accordingly, in a fifth aspect, the invention provides a method of treating or

preventing liver disease or metabolic disease comprising administering to a patient in need thereof

an effective dose of the crystal of the first aspect of the invention.

The medicine of the invention is administered in an effective dose, wherein the effective

dose is usually based on the amount of crystals of the first aspect of the invention. An effective

dose may be the content of a drug in a unit administration dosage form (i.e., a tablet, a shot, a pill,

or a dose), or a unit dose (e.g., a unit body weight dose) for a patient in need of treating/prevention.

The drug manufacturer can easily convert the unit body weight dose for the desired

treating/prevention of the patient to the content in the unit administered dosage form of the drug by the average body weight of the patient population desiring the treating or prevention, for example, an average adult patient may have the body weight at 60 kg, so by multiplying the average body weight by the unit body weight dose for adults, the content of the drug in unit dosage form for adults can be obtained. In the paper, the patient may be a mammal, such as a human, rabbit, dog or mouse, preferably a human. Based on the equivalent dose conversion relationship between experimental animals and humans well known in the art (usually refer to the guidance of FDA, SFDA and other drug regulatory agencies, and also refer to "Huang Jihan et al.

The equivalent dose conversion between animals and between animals and humans in the

pharmacological tests". China Clinical Pharmacology and Therapeutics, 2004, 9 (9): 1069-1072")

can be derived from the dose for unit body weight of experimental animals. For example, for the

commonly used experimental animal mouse, based on the above literature, its conversion

relationship with adult human is about 12:1; for the commonly used experimental animal rats,

based on the above literature, its conversion relationship with adult human is about 6:1. For

example, the dosage of MB07133 crystals is 300-1200 mg/m 2/d, intravenous infusion for seven

consecutive days, and repeated administration after three weeks of discontinuation. Through

calculation, the average daily dose of the patients is about 500-2000mg. Dose escalation was

performed at the following dose levels: 300, 600, 1200, 1800, 2400 and 3000 mg/m 2 /d until a clear

dose-limiting toxicity (DLT) occurred and the maximum tolerated dose (MTD) was determined.

Preferably, the use of the fourth aspect of the invention is in the preparation of a medicament

for treating or preventing hepatitis B, and accordingly, the method of the fifth aspect of the

invention is preferably a method for treating or preventing hepatitis B. It is also preferred that the

use of the fourth aspect of the invention is in the preparation of a medicament for reducing the

level of hepatitis B virus in a patient, and accordingly, the method of the fifth aspect of the

invention is preferably a method for reducing the level of hepatitis B virus in a patient. In addition,

for the MB07133 crystal, it is also preferred that the use of the fourth aspect of the invention is in

the preparation of a drug for treating the advanced hepatocarcinoma. Correspondingly, the method

of the fifth aspect of the invention is also preferably a method for treating the advanced

hepatocarcinoma.

In a sixth aspect, the invention provides a method for detecting the crystal of the first aspect

of the invention, characterized in that, X-ray powder diffraction detection is performed on the suspected crystal, and the obtained X-ray powder diffraction pattern is the same as that shown in

Figs. 1-1 or 1-2 or 1-3 or 1-4 or 1-5 or 2-1 or 2-3 or 2-5 or 2-7 or 2-9 or 3-1 or 3-2 or 3-3 or 3-4 or

3-5 or 3-6 as comparison. Based on the spectral line position of the spectrum (usually expressed in

degrees of Bragg's 20 angle), spectral line height, relative abundance and/or the distance d

between crystal planes (usually expressed as A), those skilled in the art can compare and conclude

if the suspected crystal is the crystal of the first aspect of the invention.

The beneficial effect of the invention is that the crystals with excellent properties of

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propyl]

adenine fumarate are obtained, which has good temperature and humidity stability, high purity, no

solvent and moisture, and is more convenient for the preparation process adaptivity, and is

convenient for long-term storage; the crystals with excellent properties of Pradefovir mesylate are

obtained, which have good temperature and humidity stability and high purity, which is more

convenient for the adaptability of the preparation process and also facilitates long-term storage;

the crystals with excellent properties of MB07133 are obtained, which have good stability, high

purity, and no solvent and moisture, which facilitates the adaptability of the preparation process

and helps the storage.

For ease of understanding, the disclosure cites published documents for the purpose of more

clearly describing the invention, the entire contents of which are incorporated herein by reference

as if they were fully recited herein.

The invention will be described in detail below through specific embodiments/examples and

accompanying drawings. It should be particularly pointed out that these descriptions are only

exemplary and do not limit the scope of the invention. Numerous variations and modifications of

the invention will be apparent to those skilled in the art from the discussion in the specification.

Description of the Drawings

Figure 1-1: X-ray powder diffraction pattern of Tenofovir phosphate crystal form a.

Figure 1-2: X-ray powder diffraction pattern of Tenofovir phosphate crystal form b.

Figure 1-3: X-ray powder diffraction pattern of Tenofovir phosphate crystal form c.

Figure 1-4: X-ray powder diffraction pattern of Tenofovir phosphate crystal form d.

Figure 1-5: X-ray powder diffraction pattern of Tenofovir phosphate crystal form e.

Figure 1-6: DSC patterns of Tenofovir phosphate crystal form a.

Figure 1-7: DSC patterns of Tenofovir phosphate crystal form b.

Figure 1-8: DSC patterns of Tenofovir phosphate crystal form c.

Figure 1-9: DSC patterns of Tenofovir phosphate crystal form d.

Figure 1-10: DSC patterns of Tenofovir phosphate crystal form e.

Figure 2-1: X-ray powder diffraction pattern of Pradefovir mesylate in crystal form A.

Figure 2-2: The DSC spectrum of Pradefovir mesylate in crystal form A.

Figure 2-3: X-ray powder diffraction pattern of Pradefovir mesylate in crystal form B.

Figure 2-4: The DSC spectrum of Pradefovir mesylate in crystal form B.

Figure 2-5: X-ray powder diffraction pattern of Pradefovir mesylate in crystal form C.

Figure 2-6: The DSC spectrum of Pradefovir mesylate in crystal form C.

Figure 2-7: X-ray powder diffraction pattern of Pradefovir mesylate in crystal form D.

Figure 2-8: The DSC spectrum of Pradefovir mesylate in crystal form D.

Figure 2-9: X-ray powder diffraction pattern of Pradefovir mesylate in crystal form E.

Figure 2-10: The DSC spectrum of Pradefovir mesylate in crystal form E.

Figure 3-1: X-ray powder diffraction pattern of MB07133 Form A.

Figure 3-2: X-ray powder diffraction pattern of MB07133 Form B.

Figure 3-3: X-ray powder diffraction pattern of MB07133 Form C.

Figure 3-4: X-ray powder diffraction pattern of MB07133 Form D.

Figure 3-5: X-ray powder diffraction pattern of MB07133 Form E.

Figure 3-6: X-ray powder diffraction pattern of MB07133 Form F.

Figure 3-7: Differential thermal analysis curve of MB07133 Form A.

Figure 3-8: Differential thermal analysis curve of MB07133 Form B.

Figure 3-9: Differential thermal analysis curve of MB07133 Form C.

Figure 3-10: Differential thermal analysis curve of MB07133 Form D.

Figure 3-11: Differential thermal analysis curve of MB07133 Form E.

Figure 3-12: Differential thermal analysis curve of MB07133 Form F.

Examples The invention will be explained in detail below with references to examples. The

embodiments/examples of the invention are only used to illustrate the technical solutions of the

invention, and the protection scope of the invention is not limited thereto.

Example 1 Crystals of Tenofovir Phosphate

Instruments used in the experiment

1. X-ray powder diffraction spectrum

Instrument: PHI-5400 X-ray photoelectron analyzer (supplied by PE Company)

The test parameters are: Voltage: 46 kv, Current: 40 mA, Copper Ka radiation, k: 1.5405A.

2. Differential scanning calorimetry (DSC) spectrum

Instrument: SII Nano, EXSTAR, DSC6220

Heating rate: 10°C/min

Temperature range: 50-250°C

Carrier gas: High purity nitrogen

Example 1-1

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxylpropy

]Preparation of Adenine Fumarate

Specific steps are as follows:

OH 0

Ni, OH HON HO

N N N> 0 N OH

1H NO

Regent MW. Feeding Mol Mol Ratio Remark Tenofovir 287 28.7 g 0.1 1 Diol 186 1 8.6 0.1 1_ Oxalyl chloride 127 50.8 g 0.4 4

DEF 101 10.9 g 0.11 1.08 Titanium tetrachloride 189.7 19.0 g 0.1 1 Triethylamine 100 42.0 g 0.42 4.2 Dichloromethane 1 / 900 mL /

/ Methanol / 1000 mL /

/ Water / 370 mL /

/ Acetone 200 mL

/ Methyl tert-butyl ether (MTBE) / 510 mL /

/ Succinic acid 118.1 11.8 R 0.1 1 Fumaric acid 116.1 11.6 g 0.1 1 Add 18.6 g of diol and 90 mL of dichloromethane to a dry and clean 250 mL flask, stir and

cool down to 0-5°C; start adding 19.0 g of titanium tetrachloride dropwise into the reaction

system, and then add triethylamine of 42.0 g dropwise to the reaction solution. The mixture is

identified as reaction solution A.

Add dichloromethane 370 mL to the 1.0 L flask at ambient temperature, turn on stirring and

add Tenofovir 28.7 g, DEF50.8 g, then at 10-25°C add dropwise oxalyl chloride 10.9 g, complete

the dropwise addition, start heating to reflux; reflux reaction for 2-3 h, stop heating, cool down to

below 10°C and add dropwise reaction solution A to the 1.0 L reaction system; after the dropwise

addition, stir and react for 1 h.

Methanol and water are added to the reaction system, stir for 5 min to separate the layers,

the aqueous phase is extracted with dichloromethane four times, the organic phases are combined,

washed with saturated brine, separated, and the organic phase is dried with anhydrous magnesium

sulfate; filter, and the filter cake is washed with dichloromethane; the filtrate is concentrated under

reduced pressure to no fractions; the concentrate is dissolved in ethanol, transferred to a 500 mL

flask, and 32 mL of acetic acid is added and heated to reflux for 5-6 h. Concentrate under reduced

pressure at 60-70°C until there is basically no fraction, add methanol and succinic acid, stir and

react for 1 h; stir to cool down for crystallization, filter, and blast the filter cake at 55-65°C for 5h

to obtain 30 g of succinate.

Add 300 mL of water to the reaction flask, add 30 g of HTS succinate, and 200 mL of

acetone, heat to 30-40°C to dissolve, and extract with methyl tert-butyl ether (MTBE). The

organic phase is discarded, and the aqueous phase is adjusted to pH 8-9 with saturated sodium

bicarbonate solution; the aqueous phase is extracted with dichloromethane. The organic phases are combined and washed once with saturated sodium chloride. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filter cake is washed with dichloromethane. The filtrate is concentrated under reduced pressure at 30-40°C until there is no fraction. About 18 g of an oily substance is obtained, which is added to the reaction flask after being dissolved in methanol, and about 11.6 g of fumaric acid is added while stirring; the mixture is allowed to react while stirred for 30 minutes below 30°C. Stir to cool down and crystallize, filter, and blast the filter cake at 55-65°C for more than 10 h. About 15 g of white powder are obtained.

The HNMR data of the compound are as follows:

'HNMR(600MHz,DMSO):613.1-13.2(2H,s)

68.145(1H,s),68.082(1H,s),7.45(2H,m),7.395-7.403(2H,m),7.259(2H,s)7.216-7.233(1H,m),6.647(

2H,s),5.633-5.648(lH,d),4.488-4.513(lH,m),4.29-4.297(lH,m),4.213-4.247(lH,m),4.042-4.062(1

H,t),3.942-3.999(2H,m),2.051-2.055(2H,d),1.117-1.127(3H,d).

Example 1-2 Preparation and identification of Tenofovir phosphate a-type crystal

Take 5 g of the white powder obtained in Example 1-1 and add it to 25 ml of methanol, heat

it to 60-70°C while stirring until it is completely dissolved, then cool to 20-25°C; when crystals

have precipitated, filter with suction, keep the crystals, and then the crystals are directly dried in

an oven at 55°C. X-ray powder diffraction and DSC detection reveal a-type monocrystal, as

shown in Figures 1 and 6.

Example 1-3 Preparation and identification of Tenofovir phosphate a-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate b-type crystals of 3 g is added into 18 ml of methanol, the mixture is heated

to 60-70°C while stirring until completely dissolved, and then it is cooled to 20-25°C, and when

the crystals have precipitated, suction filter, and keep the crystals which are then directly dried in

an oven at 55°C. X-ray powder diffraction and DSC detection reveal a-type monocrystal.

Example 1-4 Preparation and identification of Tenofovir phosphate a-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate c-type crystals of 2 g is added into 11 ml of methanol, the mixture is heated

to 60-70°C while stirring until completely dissolved, and then it is cooled to 20-25°C, and when

crystals have precipitated, suction filter, and keep the crystals which are then directly dried in an

oven at 55°C. X-ray powder diffraction and DSC detection reveal a-type monocrystal.

Example 1-5 Preparation and identification of Tenofovir phosphate a-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propy

1] adenine fumarate d-type crystals of 2 g is added into 11 ml of methanol, the mixture is heated to

60-70°C while stirring until completely dissolved, and then it is cooled to 20-25°C, and when

crystals have precipitated, suction filter, and keep the crystals which are then directly dried in an

oven at 55°C. X-ray powder diffraction and DSC detection reveal a-type monocrystal.

Example 1-6 Preparation and identification of Tenofovir phosphate a-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]propy

1] adenine fumarate e-type crystals of 2 g is added into 10 ml of methanol, the mixture is heated to

60-70°C while stirring until completely dissolved, and then it is cooled to 20-25°C, and when

crystals have precipitated, suction filter, and keep the crystals which are then directly dried in an

oven at 55°C. X-ray powder diffraction and DSC detection reveal a-type monocrystal.

Example 1-7 Preparation and identification of Tenofovir phosphate b-type crystal

Take 6g of the white powder obtained in Example 1-1, add 5 ml of water, 60ml of acetonitrile,

the mixture is heated to 65°C to dissolve, cool to 20-25°C when crystals have precipitated, suction

filter, retain the crystals which are then directly dried in an oven at 55°C. X-ray powder diffraction

and DSC detection reveal b-type monocrystal, as shown in Figures 2 and 7.

Example 1-8 Preparation and identification of Tenofovir phosphate b-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate a-type crystal of 3 g is added to 2 ml of water and 30 ml of acetonitrile, the

mixture is heated to 65°C to dissolve, cooled to 20-25°C and when crystals have precipitated,

suction filter, the crystals are retained and directly dried in an oven at 55°C. X-ray powder

diffraction and DSC detection reveal b-type monocrystal.

Example 1-9 Preparation and identification of Tenofovir phosphate b-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate c-type crystal of 3 g is added to 2 ml of water and 30 ml of acetonitrile, the

mixture is heated to 65°C to dissolve, cooled to 20-25°C and when crystals have precipitated,

suction filter, the crystals are retained and directly dried in an oven at 55°C. X-ray powder

diffraction and DSC detection reveal b-type monocrystal.

Example 1-10 Preparation and identification of Tenofovir phosphate c-type crystal

Take 3 g of the white powder obtained in Example 1-1, add 35 ml of tetrahydrofuran, the

mixture is heated to 65°C to dissolve, cool the solution to 20-25°C and when crystals have

precipitated, suction filter, retain the crystal which are then directly dried in a 55°C oven. X-ray

powder diffraction and DSC detection reveal c-type monocrystal, as shown in Figures 3 and 8.

Example 1-11 Preparation and identification of Tenofovir phosphate c-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate a-type crystals of 3 g is added into 30 ml of tetrahydrofuran, the mixture is

heated to 65°C to dissolve, cooled to 20-25°C when crystals have precipitated, filter with

suction, keep the crystals which are directly dried in a 55°C oven. X-ray powder diffraction and

DSC detection reveal c-type monocrystal.

Example 1-12 Preparation and identification of Tenofovir phosphate c-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate b-type crystals of 2 g is added into 22 ml of tetrahydrofuran, the mixture is

heated to 65°C to dissolve, cooled to 20-25°C when crystals have precipitated, filter with suction,

keep the crystals which are directly dried in a 55°C oven. X-ray powder diffraction and DSC

detection reveal c-type monocrystal.

Example 1-13 Preparation and identification of Tenofovir phosphate c-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate d-type crystals of 2 g is added into 18 ml of tetrahydrofuran, the mixture is

heated to 65°C to dissolve, cooled to 20-25°C when crystals have precipitated, filter with suction,

keep the crystals which are directly dried in a 55°C oven. X-ray powder diffraction and DSC

detection reveal c-type monocrystal.

Example 1-14 Preparation and identification of Tenofovir phosphate d-type crystal

Take 3 g of the white powder obtained in Example 1-1, add 30 ml of water, heat to 80°C to

dissolve, cool to 20-25°C when crystals have precipitated, suction filter, retain the crystals which

are directly dried in a 55°C oven. X-ray powder diffraction and DSC detection reveal d-type

monocrystal, as shown in Figures 4 and 9.

Example 1-15 Preparation and identification of Tenofovir phosphate d-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate a-type crystals of 5 g is added into 40 ml of water, heat to 80°C to dissolve, cool down to 20-25°C when crystals have precipitated, filter with suction, keep the crystals which are then directly dried in an oven at 55C. X-ray powder diffraction and DSC detection reveal d-type monocrystal.

Example 1-16 Preparation and identification of Tenofovir phosphate d-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate b-type crystals of 2g is added into 22 ml of water, heat to 80C to dissolve,

cool down to 20-250 C when crystals have precipitated, filter with suction, keep the crystals which

are then directly dried in an oven at 55C. X-ray powder diffraction and DSC detection reveal

d-type monocrystal.

Example 1-17 Preparation and identification of Tenofovir phosphate e-type crystal

Take 3 g of the white powder obtained in Example 1-1, add 30 ml of isopropanol, heat to

70 0C to dissolve, then cool to 20-250 C and when crystals have precipitated, suction filter, retain

the crystals which are then directly dried in a 550 C oven. X-ray powder diffraction and DSC

detection reveal e-type monocrystal, as shown in Figures 5 and 10.

Example 1-18 Preparation and identification of Tenofovir phosphate e-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate a-type crystal of 5 g is added with 45 ml of isopropanol, heated to 700 C to

dissolve into solution which is cooled to 20-250 C and when crystals have precipitated, suction

filtered, retained the crystals which are then directly dried in an oven at 55C. X-ray powder

diffraction and DSC detection reveal e-type monocrystal.

Example 1-19 Preparation and identification of Tenofovir phosphate e-type crystal

9-[(2R)-2-[(2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphahexacyclo-2-methoxy]prop

yl] adenine fumarate c-type crystal of 3 g is added with 35 ml of isopropanol, heated to 700 C to

dissolve into solution which is cooled to 20-250 C and when crystals have precipitated, suction

filtered, retained the crystals which are then directly dried in an oven at 55C. X-ray powder

diffraction and DSC detection reveal e-type monocrystal.

Example 1-20 Stability of Tenofovir phosphate a-type crystal

The example describes the stability experiments of a-type crystal.

The stability test of the a-type crystal is carried out under three conditions i.e. high

temperature, high humidity and light. The results are shown in Table 1-1, which shows that the

crystal is stable under the conditions of high temperature, high humidity and light.

Table 1-1 Stability test results of a-type crystal (high temperature, high humidity, light)

Condition Time Total impurities Impurity A Impurity B Impurity C Impurity D

/ 0 day 1.82 0.71 0.45 0.29 0.11

5 days 1.89 0.72 0.42 0.27 0.13 High temperature 10 days 1.91 0.74 0.43 0.27 0.13 (60°C + 2°C) 30 days 1.96 0.70 0.40 0.28 0.10

5 days 1.84 0.73 0.43 0.27 0.13 High humidity 10 days 1.87 0.72 0.42 0.27 0.13 (90% + 5% RH) 30 days 1.88 0.71 0.42 0.28 0.11

5 days 1.84 0.72 0.42 0.27 0.12 Light 10 days 1.88 0.73 0.43 0.27 0.13 (4500 lx ±500 lx) 30 days 1.81 0.70 0.41 0.27 0.11

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 1-2,

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 1-2 Stability test for 6 months at 40°C

Sample 1 Total impurities Impurity A Impurity B Impurity C Impurity D

0 month 1.82 0.71 0.45 0.29 0.11

3 months 1.89 0.70 0.44 0.28 0.13

6 months 1.98 0.74 0.42 0.27 0.10

Example 1-21 Stability of Tenofovir phosphate b-type crystal

The example describes the stability experiments of b-type crystal.

The stability test of the b-type crystal is carried out under three conditions i.e. high

temperature, high humidity and light. The results are shown in Table 1-3, which shows that the

crystal is stable under the conditions of high temperature, high humidity and light.

Table 1-3 Stability test results of b-type crystal (high temperature, high humidity, light)

Condition Time Total impurities Impurity A Impurity B Impurity C Impurity D

/ 0 day 1.82 0.71 0.45 0.29 0.11

High temperature 5 days 1.88 0.72 0.45 0.30 0.13

(60°C + 2°C) 10 days 1.93 0.74 0.46 0.31 0.13

30 days 1.99 0.73 0.40 0.27 0.11

5 days 1.89 0.73 0.43 0.32 0.13 High humidity 10 days 1.92 0.72 0.42 0.32 0.13 (90% + 5% RH) 30 days 1.90 0.71 0.42 0.30 0.11

5 days 1.88 0.73 0.43 0.29 0.12 Light 10 days 1.90 0.73 0.43 0.29 0.13 (4500 lx + 500 lx) 30 days 1.86 0.70 0.42 0.28 0.12

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 1-4,

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 1-4 Stability test for 6 months at 40°C

Sample 1 Total impurities Impurity A Impurity B Impurity C Impurity D

0 month 1.82 0.71 0.45 0.29 0.11

3 months 1.91 0.72 0.44 0.29 0.13

6 months 1.99 0.74 0.43 0.27 0.11

Example 1-22 Stability of Tenofovir phosphate c-type crystal

The example describes the stability experiments ofc-type crystal.

The stability test of the c-type crystal is carried out under three conditions i.e. high

temperature, high humidity and light. The results are shown in Table 1-5, which shows that the

crystal is stable under the conditions of high temperature, high humidity and light.

Table 1-5 Stability test results ofc-type crystal (high temperature, high humidity, light)

Condition Time Total impurities Impurity A Impurity B Impurity C Impurity D

/ 0 day 1.82 0.71 0.45 0.29 0.11

5 days 1.89 0.73 0.44 0.30 0.12 High temperature 10 days 1.94 0.74 0.48 0.32 0.11 (60°C + 2°C) 30 days 1.98 0.72 0.43 0.30 0.13

5 days 1.89 0.73 0.45 0.30 0.12 High humidity 10 days 1.91 0.73 0.44 0.31 0.13 (90% + 5% RH) 30 days 1.92 0.71 0.42 0.32 0.11

5 days 1.89 0.72 0.43 0.31 0.12 Light 10days 1.90 0.73 0.41 0.29 0.13 (4500 lx + 500 lx) 30days 1.88 0.72 0.42 0.28 0.12

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 1-6,

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 1-6 Stability test for 6 months at 40°C

Sample 1 Total impurities Impurity A Impurity B Impurity C Impurity D

0 month 1.82 0.71 0.45 0.29 0.11

3 months 1.91 0.73 0.44 0.29 0.12

6 months 1.99 0.74 0.45 0.30 0.11

Example 1-23 Stability of Tenofovir phosphate d-type crystal

The example describes the stability experiments of d-type crystal.

The stability test of the d-type crystal is carried out under three conditions i.e. high

temperature, high humidity and light. The results are shown in Table 1-7, which shows that the

crystal is stable under the conditions of high temperature, high humidity and light.

Table 1-7 Stability test results of d-type crystal (high temperature, high humidity, light)

Condition Time Total impurities Impurity A Impurity B Impurity C Impurity D

/ 0 day 1.82 0.71 0.45 0.29 0.11

5 days 1.87 0.72 0.44 0.27 0.13 High temperature 10 days 1.92 0.74 0.43 0.28 0.13 (60°C + 2°C) 30 days 1.97 0.72 0.41 0.27 0.10

5 days 1.85 0.73 0.42 0.28 0.12 High humidity 10 days 1.88 0.72 0.44 0.28 0.13 (90% + 5% RH) 30 days 1.88 0.71 0.42 0.28 0.11

5 days 1.83 0.73 0.42 0.27 0.12 Light 10 days 1.86 0.73 0.43 0.27 0.11 (4500 lx ±500 lx) 30 days 1.81 0.70 0.41 0.27 0.10

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 1-8, which

indicates that the crystal has good stability and is suitable for long-term storage.

Table 1-8 Stability test for 6 months at 40°C

Sample 1 Total impurities Impurity A Impurity B Impurity C Impurity D

Month 1.82 0.71 0.45 0.29 0.11

3 months 1.89 0.72 0.44 0.29 0.12

6 months 1.98 0.73 0.43 0.28 0.10

Example 1-24 Stability of Tenofovir phosphate e-type crystal

The example describes the stability experiments of e-type crystal.

The stability test of the e-type crystal is carried out under three conditions i.e. high temperature, high

humidity and light. The results are shown in Table 1-9, which shows that the crystal is stable under the

conditions of high temperature, high humidity and light.

Table 1-9 Stability test results of e-type crystal (high temperature, high humidity, light)

Condition Time Total impurities Impurity A Impurity B Impurity C Impurity D

/ 0 day 1.82 0.71 0.45 0.29 0.11

5 days 1.87 0.72 0.47 0.30 0.13 High temperature 10 days 1.93 0.73 0.46 0.33 0.12 (60°C + 2°C) 30 days 1.98 0.72 0.48 0.36 0.12

5 days 1.90 0.73 0.47 0.34 0.13 High humidity 10 days 1.96 0.73 0.48 0.33 0.12 (90% + 5% RH) 30 days 2.01 0.72 0.48 0.36 0.12

5 days 1.85 0.72 0.44 0.30 0.11 Light 10 days 1.92 0.73 0.45 0.32 0.11 (4500 lx + 500 lx) 30 days 1.97 0.74 0.46 0.33 0.10

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 1-10, which

indicates that the crystal has good stability and is suitable for long-term storage.

Table 1-10 Stability test for 6 months at 40°C

Sample 1 Total impurities Impurity A Impurity B Impurity C Impurity D

0 month 1.82 0.71 0.45 0.29 0.11

3 months 1.90 0.72 0.47 0.32 0.11

6 months 1.99 0.72 0.46 0.34 0.10

Examples 1-25 Pharmaceutical compositions comprising Tenofovir phosphate a-type crystal

Based on the formula in Table 1-11, take Tenofovir phosphate a-type crystals and silicon dioxide,

mix and sieve, then add mannitol and pregelatinized starch and mix well, then add magnesium stearate,

press the mixture into tablets, then coat them. The tablets are thus prepared.

Table 1-11 Formulation of a-type crystal tablet

Content per tablet (mg)* Composition Effect 75 mg 50 mg 30 mg 12.5 mg

Crystal form a API 75 50 30 12.5

Mannitol Diluent 213 160 105 90

Pregelatinized starch Adhesive 15 80 50 45

Magnesium stearate Lubricant 6 9 5 4.5

Silicon dioxide Filler 4 3 1 2

Examples 1-26 Pharmaceutical compositions comprising Tenofovir phosphate b-type crystal

Based on the formula in Table 1-12, take Tenofovir phosphate b-type crystals and silicon dioxide,

mix and sieve, then add mannitol and pregelatinized starch and mix well, then add magnesium stearate,

press the mixture into tablets, then coat them. The tablets are thus prepared.

Table 1-12 Formulation of b-type crystal tablet

Content per tablet (mg)* Composition Effect 75 mg 50 mg 30 mg 12.5 mg

Crystal form b API 75 50 30 12.5

Mannitol Diluent 210 182 105 90

Pregelatinized starch Adhesive 15 60 50 45

Magnesium stearate Lubricant 6 6 4 4.5

Silicon dioxide Filler 4 2 1 3

Examples 1-27 Pharmaceutical compositions comprising Tenofovir phosphate c-type crystal

Based on the formula in Table 1-13, take Tenofovir phosphate c-type crystals and silicon dioxide,

mix and sieve, then add mannitol and pregelatinized starch and mix well, then add magnesium stearate,

press the mixture into tablets, then coat them. The tablets are thus prepared.

Table 1-13 Formulation of c-type crystal tablet

Content per tablet (mg)* Composition Effect 75 mg 50 mg 30 mg 12.5 mg

Crystal form c API 75 50 30 12.5

Mannitol Diluent 226 142 100 30

Pregelatinized starch Adhesive 15 60 45 32

Magnesium stearate Lubricant 6 5 4 3.5

Silicon dioxide Filler 3 3 1 2

Examples 1-28 Pharmaceutical compositions comprising Tenofovir phosphate d-type crystal

Based on the formula in Table 1-14, take Tenofovir phosphate d-type crystals and silicon dioxide,

mix and sieve, then add mannitol and pregelatinized starch and mix well, then add magnesium stearate,

press the mixture into tablets, then coat them. The tablets are thus prepared.

Table 1-14 Formulation of d-type crystal tablet

Content per tablet (mg)* Composition Effect 75 mg 50 mg 30 mg 12.5 mg

Crystal form d API 75 50 30 12.5

Mannitol Diluent 213 122 85 35

Pregelatinized starch Adhesive 12 121 15 22

Magnesium stearate Lubricant 6 6 4 3.5

Silicon dioxide Filler 4 1 1 2

Examples 1-29 Pharmaceutical compositions comprising Tenofovir phosphate e-type crystal

Based on the formula in Table 1-15, take Tenofovir phosphate e-type crystals and silicon dioxide,

mix and sieve, then add mannitol and pregelatinized starch and mix well, then add magnesium stearate,

press the mixture into tablets, then coat them. The tablets are thus prepared.

Table 1-15 Formulation of e-type crystal tablet

Content per tablet (mg)* Composition Effect 75 mg 50 mg 30 mg 12.5 mg

Crystal form e API 75 50 30 12.5

Mannitol Diluent 220 100 90 40

Pregelatinized starch Adhesive 9 64 35 22

Magnesium stearate Lubricant 5 5 4 3.5

Silicon dioxide Filler 1 1 1 2

Example 2 Crystals of Pradefovir mesylate

Instruments used in the experiment

1. X-ray powder diffraction spectrum

Instrument: PHI-5400 X-ray photoelectron analyzer (supplied by PE Company)

The test parameters are: Voltage: 46 kv, Current: 40 mA, Copper k radiation, k: 1.5405A.

2. Differential scanning calorimetry (DSC) spectrum

Instrument: SII Nano, EXSTAR, DSC6220

Heating rate: 10°C /min

Temperature range: 50-250°C

Carrier gas: High purity nitrogen

3. Material source: Pradefovir mesylate crystal form I, which is of the Chinese patent

CN102827206A Crystal form I prepared in Example 1.

Example 2-1 Pradefovir mesylate A-type crystal

10 g of Pradefovir mesylate crystal form I is added to 8 ml of purified water, stirred to dissolve, and

92 ml of acetone is added dropwise with stirring. After the dropwise addition, the mixture is stirred at

10-30°C for 2 h. Cool to 0-10°C and stir for 2 h. Filter, wash the filter cake with 13 ml of acetone, and

dry the cake at 55-65°C for more than 18 hours to obtain 7.9 g of a white solid. It was detected to belong

to the crystal form A (as shown in Figures 1 and 2).

Example 2-2 Pradefovir mesylate A-type crystal

10 g of Pradefovir mesylate crystal form B is added to 20 ml of purified water, stirred to dissolve,

and 300 ml of acetone is added dropwise with stirring. After the dropwise addition, the mixture is stirred

at 10-30°C for 2 h. Cool to 0-10°C and stir for 2h. Filter, wash the filter cake with 20 ml of acetone, and

dry the cake at 55-65°C for more than 18 hours to obtain 7.2 g of a white solid. It was detected to belong

to the crystal form A.

Example 2-3 Pradefovir mesylate A-type crystal

10 g of Pradefovir mesylate crystal form C is added to 30 ml of purified water, stirred to dissolve,

and 400 ml of acetone is added dropwise with stirring. After the dropwise addition, the mixture is stirred

at 10-30°C for 2 h. Cool to 0-10°C and stir for 2 h. Filter, wash the filter cake with 13 ml of acetone, and

dry the cake at 55-65°C for more than 18 hours to obtain 7.0 g of a white solid. It was detected to belong

to the crystal form A.

Example 2-4 Pradefovir mesylate A-type crystal

Take 10 g of Pradefovir mesylate crystal form D, add 170 ml of methanol, stir to dissolve, and add

250 ml of methyl tert-butyl ether dropwise with stirring. After the dropwise addition, the mixture is stirred

at 10-30°C for 2 h. Cool to 0-10°C and stir for 2h. Filter, wash the filter cake with 20 ml of methyl

tertiary butyl ether, and dry the cake at 55-65°C for more than 18 hours to obtain 6.5 g of a white solid. It

was detected to belong to the crystal form A.

Example 2-5 Pradefovir mesylate A-type crystal

Take 10 g of Pradefovir mesylate crystal form E, add 200 ml of methanol, stir to dissolve, and add

300 ml of ethyl acetate dropwise with stirring. After the dropwise addition, the mixture is stirred at 10

30°C for 2 h. Cool to 0-10°C and stir for 2h. Filter, wash the filter cake with 20 ml of ethyl acetate, and

dry the cake at 55-65°C for more than 18 hours to obtain 7.1 g of a white solid. It was detected to belong

to the crystal form A.

Example 2-6 Pradefovir mesylate B-type crystal

Take 5g of Pradefovir mesylate crystal form I, add 350 ml of 1,4-dioxane, stir to dissolve, and stir at

10-30°C for 2 h. Filter, wash, and dry at 55-65°C for more than 18 hours to obtain 2.5 g of a white solid.

It was detected to belong to the crystal form B (as shown in Figures 3 and 4).

Example 2-7 Pradefovir mesylate B-type crystal

Take 5 g of Pradefovir mesylate crystal form A, add 500 ml of 1,4-dioxane, stir to dissolve, and stir

at 10-30°C for 2 h. Filter, wash, and dry at 55-65°C for more than 18 hours to obtain 2.3 g of a white

solid. It was detected to belong to the crystal form B.

Example 2-8 Pradefovir mesylate C-type crystal

10 g of Pradefovir mesylate crystal form I is added to 50 ml of DMF, the mixture is stirred to

dissolve, and 50 ml of MTBE is added dropwise with stirring. After the dropwise addition, the mixture is

stirred at 10-30°C for 2 h. Filter, wash the filter cake with 10 ml of MTBE, and dry the cake at 55-65°C

for more than 18 hours to obtain 8.4 g of a white solid. It was detected to belong to the crystal form C (as

shown in Figures 5 and 6).

Example 2-9 Pradefovir mesylate C-type crystal

Take 10 g of Pradefovir mesylate crystal form A, add DMF 60 ml, stir to dissolve, and add MTBE

100 ml dropwise with stirring. After the dropwise addition, the mixture is stirred at 10-30°C for 2 h.

Filter, wash the filter cake with 10 ml of MTBE, and dry the cake at 55-65°C for more than 18 hours to

obtain 8.6 g of a white solid. It was detected to belong to the crystal form C.

Table 2-10 Pradefovir mesylate D-type crystal

10 g of Pradefovir mesylate crystal form I is added to 40 ml of a THF solution containing 2.5%

water, the mixture is heated to 55°C and dissolved with stirring. Stir at 10-30°C for 2h. Filter,

wash the filter cake with 5 ml of THF, and dry the cake at 55-65°C for more than 18 hours to

obtain 7.1 g of a white solid. It was detected to belong to the crystal form D (as shown in Figures 7

and 8).

Table 2-11 Pradefovir mesylate D-type crystal

Take 10 g of Pradefovir mesylate crystal form A, add 60 ml of THF solution containing 2% water,

heat to 55°C and stir to dissolve. Stir at 10-30°C for 2h. Filter, wash the filter cake with 5 ml of THF, and

dry the cake at 55-65°C for more than 18 hours to obtain 6.8 g of a white solid. It was detected to belong

to the crystal form D.

Table 2-12 Pradefovir mesylate E-type crystal

Take 2g of Pradefovir mesylate crystal form I, add 200 ml of acetonitrile, heat under reflux and stir

to dissolve, and stir at 10-30°C for 2h. Filter, wash the filter cake with 10 ml of acetonitrile, and dry the

cake at 55-65 0C for more than 18 hours to obtain 1.5 g of a white solid. It was detected to belong to the

crystal form E (as shown in Figures 9 and 10).

Table 2-13 Pradefovir mesylate E-type crystal

Take 5 g of Pradefovir mesylate crystal form A, add 600 ml of acetonitrile, heat under reflux and stir

to dissolve, and stir at 10-30°C for 2h. Filter, wash the filter cake with 10 ml of acetonitrile, and dry the

cake at 55-65°C for more than 18 hours to obtain 4.3 g of a white solid. It was detected to belong to the

crystal form E.

Example 2-14 Stability study experiment of Pradefovir mesylate crystal form A

Stability test data of Pradefovir mesylate crystal form A.

The stability test is carried out under three conditions i.e. high temperature, high humidity and light.

The results are shown in Table 2-1, which shows that the crystal has long-term high temperature, high

humidity and light stability.

Table 2-1 Stability test under high temperature, high humidity and light

Relative substance(%) Condition Impurity A Impurity B Impurity C Impurity D Impurity F

0 day 0.10 0.02 0.01 ND ND

High temperature 5 days 0.10 0.04 0.02 ND ND

(60 0 C) 10 days 0.10 0.03 0.02 ND ND

High humidity 5 days 0.10 0.05 0.04 ND ND

(92.5%) 10 days 0.13 0.12 0.18 ND ND

Light test 5 days 0.10 0.04 0.03 ND ND

(4500 lx) 10 days 0.10 0.03 0.02 ND ND

The results of a long-term high-humidity stability test are shown in Table 2-2; the amount of

impurities do not increase significantly over time, which indicates that the crystal has long-term

high-humidity stability.

Table 2-2 Long-term stability test of Pradefovir mesylate A-type crystal

Item 0 month 6 months 9 months 12 months

0.23 0.22 0.18 0.21 Relative Impurity A 0.02 0.01 0.03 substance Impurity B ND Impurity C ND 0.02 0.03 0.04 (%) Impurity D ND ND ND ND

Impurity F 0.19 0.21 0.19 0.21

Example 2-15 Stability study experiment of Pradefovir mesylate crystal form B

Stability test data of Pradefovir mesylate crystal form B.

A stability test was carried out under three conditions i.e. high temperature, high humidity and light.

The results are shown in Table 2-3, which shows that the crystal has long-term high temperature, high

humidity and light stability.

Table 2-3 Stability test under high temperature, high humidity and light

Relative substance(%) Condition Impurity A Impurity B Impurity C Impurity D Impurity F

0 day 0.10 0.01 0.01 ND ND

High temperature 5 days 0.10 0.03 0.02 ND ND

(60°C) 10 days 0.10 0.03 0.02 ND ND

High humidity 5 days 0.10 0.05 0.03 ND ND

(92.5%) 10 days 0.12 0.12 0.15 ND ND

Light test 5 days 0.10 0.04 0.03 ND ND

(4500 lx) 10 days 0.10 0.03 0.03 ND ND

The results of a long-term high-humidity stability test are shown in Table 2-4; the impurities do not

increase significantly over time, which indicates that the crystal has long-term high-humidity stability.

Table 2-4 Long-term stability test of Pradefovir mesylate B-type crystal

Item 0 month 6 months 9 months 12 months

Impurity A 0.22 0.22 0.19 0.21

Impurity B ND 0.02 0.02 0.03 Relative Impurity C ND 0.02 0.02 0.04 substance Impurity D ND ND ND ND (%) Impurity F 0.19 0.21 0.19 0.20

Example 2-16 Stability study experiment of Pradefovir mesylate crystal form C

Stability test data of Pradefovir mesylate crystal form C.

A stability test was carried out under three conditions i.e. high temperature, high humidity and light.

The results are shown in Table 2-5, which shows that the crystal has long-term high temperature, high

humidity and light stability.

Table 2-5 Stability test under high temperature, high humidity and light

Relative substance (%) Condition Impurity A Impurity B Impurity C Impurity D Impurity F

0 day 0.09 0.01 0.01 ND ND

High temperature 5 days 0.10 0.03 0.02 ND ND

(60°C) 10 days 0.10 0.03 0.02 ND ND

High humidity 5 days 0.10 0.05 0.04 ND ND

(92.5%) 10 days 0.13 0.10 0.18 ND ND

Light test 5 days 0.10 0.04 0.03 ND ND

(4500 lx) 10 days 0.10 0.04 0.02 ND ND

The results of a long-term high-humidity stability test are shown in Table 2-6; the impurities did not

increase significantly over time, which indicates that the crystal has long-term high-humidity stability.

Table 2-6 Long-term stability test of Pradefovir mesylate C-type crystal

Item 0 month 6 months 9 months 12 months

Impurity A 0.22 0.23 0.19 0.22

Impurity B ND 0.02 0.02 0.03 Relative Impurity C ND 0.02 0.03 0.03 substance Impurity D ND ND ND ND (%) Impurity F 0.19 0.20 0.19 0.20

Example 2-17 Stability study experiment of Pradefovir mesylate crystal form D

Stability test data of Pradefovir mesylate crystal form D.

The results of stability test under three conditions, i.e. high temperature, high humidity and light, are

shown in Table 2-7, which shows that the crystal has long-term high temperature, high humidity and light

stability.

Table 2-7 Stability test under high temperature, high humidity and light

Condition Relative substance (%)

Impurity A Impurity B Impurity C Impurity D Impurity F

0 day 0.10 0.01 0.01 ND ND

High temperature 5 days 0.10 0.03 0.03 ND ND

(60°C) 10 days 0.10 0.04 0.03 ND ND

High humidity 5 days 0.10 0.04 0.04 ND ND

(92.5%) 10 days 0.12 0.11 0.15 ND ND

Light test 5 days 0.10 0.04 0.04 ND ND

(4500 x) 10 days 0.10 0.03 0.04 ND ND

The results of a long-term high-humidity stability test are shown in Table 2-8; the impurities did not

increase significantly over time, which indicates that the crystal has long-term high-humidity stability.

Table 2-8 Long-term stability test of Pradefovir mesylate D-type crystal

Item 0 month 6 months 9 months 12 months

Impurity A 0.21 0.23 0.19 0.20 Relative Impurity B ND 0.02 0.01 0.03

substance Impurity C ND 0.02 0.03 0.04

(%) Impurity D ND ND ND ND

Impurity F 0.19 0.21 0.19 0.21

Example 2-18 Stability study experiment of Pradefovir mesylate crystal form E

Stability test data of Pradefovir mesylate crystal form E.

The results of a stability test carried out under three conditions i.e. high temperature, high humidity

and light, are shown in Table 2-9, which shows that the crystal has long-term high temperature, high

humidity and light stability.

Table 2-9 Stability test under high temperature, high humidity and light

Relative substance(%) Condition Impurity A Impurity B Impurity C Impurity D Impurity F

0 day 0.10 0.02 0.01 ND ND

High temperature 5 days 0.10 0.04 0.02 ND ND

(60°C) 10 days 0.10 0.03 0.02 ND ND

High humidity 5 days 0.10 0.05 0.04 ND ND

(92.5%) 10 days 0.13 0.12 0.18 ND ND

Light test 5 days 0.10 0.04 0.03 ND ND

(4500 lx) 10 days 0.10 0.03 0.02 ND ND

The results of a long-term high-humidity stability test are shown in Table 2-10; the impurities do not

increase significantly over time, which indicates that the crystal has long-term high-humidity stability.

Table 2-10 Long-term stability test of Pradefovir mesylate E-type crystal

Item 0 month 6 months 9 months 12 months

Impurity A 0.22 0.22 0.18 0.20

Relative Impurity B ND 0.02 0.01 0.03

substance Impurity C ND 0.02 0.03 0.04

Impurity D ND ND ND ND (%)

Impurity F 0.19 0.20 0.19 0.20

Example 3 MB07133 crystal

Instruments used in the experiment

1. X-ray powder diffraction spectrum

Instrument: PHI-5400 X-ray photoelectron analyzer (supplied by PE Company)

The test parameters are: Voltage: 46 kv, Current: 40 mA, Copperka radiation, k: 1.5405A.

2. Differential scanning calorimetry (DSC) spectrum

Instrument: SII Nano, EXSTAR, DSC6220

Heating rate: 10°C /min

Temperature range: 50-250°C

Carrier gas: High purity nitrogen

3. Material source:

The compound MB07133 is synthesized with reference to the preparation method of Chinese patent

CN1711278A. Details as follows:

Under nitrogen protection, 2,3-diO-TBS-cytarabine-N,N-dimethylformamidine (250g, 0.47moL) and

tetrahydrofuran (2.5L) are added to the reaction kettle, cooled to 4°C. The temperature is controlled not to

exceed 8°C, and a tert-butylmagnesium chloride solution (617 mL, 0.62 moL) is added dropwise; the

reaction takes place for 1.25 h. Phosphate ester reagent (262 g, 0.78 moL) is added at one time, and the

reaction mixture is stirred at room temperature for 16 h. Ammonium chloride solution (20%, 2.5 L) is

added dropwise, and ethyl acetate (2.5 L) is added with stir and the layers are separated; then the aqueous

phase is extracted with ethyl acetate (1.1 L); the organic phases are combined, and a sodium chloride

solution (15%, 1.6 L) is used to backwash and the mixture is separated, dried over magnesium sulfate

(260g), filtered and concentrated to dryness to obtain 526 g of dark orange slurry.

The dark orange slurry is dissolved in methanol (2.5 L) and HCl-dioxane solution (790 mL, 3.16moL)

at 50-55°C, reacted at 50-55°C for 16 h, and concentrated under reduced pressure to obtain a thick

orange tar. The above tar is partitioned with water (800 mL) and ethyl acetate (800 mL); sodium

bicarbonate solid is slowly added until the pH value of the water is 7; the liquids are separated, extracted

with ethyl acetate (800 mL) again, the aqueous phase is filtered and concentrated under reduced pressure,

and the ethanol is used to remove the water to dryness. The reaction yields 445 g of oil. Add ethanol

(700mL), stir at room temperature for 2 h, filter and dry to obtain 199 g of solid, and then make a slurry

with water (650 mL), add hydrochloric acid (20 mL); stir the mixture, and then add sodium bicarbonate

solid (36 g) to adjust the pH value to 6-7, filter; it is then dried to obtain MB07133 solid of 160 g. 1 H NMR (600 MHz, DMSO) of compound MB07133: 6:8.61(2H, dd), 7.54(1H, d), 7.43(2H, dd),

7.07-7.14(2H, s), 6.12(1H, d), 5.75-5.78(1H, m), 5.63-5.64(2H, d), 5.60(1H, d), 4.41-4.59(2H, m),

4.25-4.33(2H, m), 4.00(1H, m), 3.95-3.97(1H, m), 3.92(1H, s), 2.13-2.28(2H, m).

Example 3-1 Preparation and identification of crystal form A of MB07133

To a 100 mL three-necked flask, add 30 mL of purified water, add 10 g of the white powder of

MB07133 obtained above, and add 3 mol/L aqueous sulfuric acid solution dropwise with stirring to adjust

the pH to 2.0-5.0. The temperature is controlled at 15-25°C and the mixture is stirred for 1 h, 0.14 g of

sodium dihydrogen phosphate is added, and 14% aqueous sodium hydroxide solution is slowly added

dropwise to the reaction system to adjust the pH to 5.0-8.0. After adjusting the pH, control the

temperature to 10-20°C, stir for 3 hours, centrifuge, collect the filter cake, keep the crystals which are

then directly dried at 50°C. X-ray powder diffraction and DSC detection show that it is crystal form A as

shown in Figures 1 and 7, indicating that stable crystal form A could be obtained by the method.

Example 3-2 Preparation and identification of crystal form A of MB07133

To a 100 mL three-necked flask, add 24 mL of purified water and 8 g of MB07133 crystal form B,

and add 3 mol/L aqueous sulfuric acid solution dropwise with stirring to adjust the pH to 2.0-5.0. The

temperature is controlled at 15-25°C and stirred for 1 h, 1.2 g of sodium dihydrogen phosphate is added,

and a 14% aqueous sodium hydroxide solution is slowly added dropwise to the reaction system to adjust

the pH to 5.0-8.0. After adjusting the pH, control the temperature to 10-20°C, stir for 3 hours, centrifuge,

collect the filter cake, keep the crystals which are then directly dried at 50°C. X-ray powder diffraction

and DSC detection reveal crystal form A.

Example 3-3 Preparation and identification of crystal form A of MB07133

To a 100 mL three-necked flask, add 24 mL of purified water and 8 g of MB07133 crystal form C,

and add 3 mol/L aqueous sulfuric acid solution dropwise with stirring to adjust the pH to 2.0-5.0. The

temperature is controlled at 15-25°C and stirred for 1 h, 1.2 g of sodium dihydrogen phosphate is added,

and a 14% aqueous sodium hydroxide solution is slowly added dropwise to the reaction system to adjust

the pH to 5.0-8.0. After adjusting the pH, control the temperature to 10-20°C, stir for 3 hours, centrifuge,

collect the filter cake, keep the crystals which are then directly dried at 50°C. X-ray powder diffraction

and DSC detection reveal crystal form A.

Example 3-4 Preparation and identification of crystal form A of MB07133

To a 100 mL three-necked flask, add 30 mL of purified water and 10 g of MB07133 crystal form D,

and add 3 mol/L aqueous sulfuric acid solution dropwise with stirring to adjust the pH to 2.0-5.0. The

temperature is controlled at 15-25°C and stirred for 1 h, 1.2 g of sodium dihydrogen phosphate is added,

and a 14% aqueous sodium hydroxide solution is slowly added dropwise to the reaction system to adjust

the pH to 5.0-8.0. After adjusting the pH, control the temperature to 10-20°C, stir for 3 hours, centrifuge, collect the filter cake, keep the crystals which are then directly dried at 50°C. X-ray powder diffraction and DSC detection reveal crystal form A.

Example 3-5 Preparation and identification of crystal form A of MB07133

To a 100 mL three-necked flask, add 24 mL of purified water and 8 g of MB07133 crystal form E,

and add 3 mol/L aqueous sulfuric acid solution dropwise with stirring to adjust the pH to 2.0-5.0. The

temperature is controlled at 15-25°C and stirred for 1 h, 1.2 g of sodium dihydrogen phosphate is added,

and a 14% aqueous sodium hydroxide solution is slowly added dropwise to the reaction system to adjust

the pH to 5.0-8.0. After adjusting the pH, control the temperature to 10-20°C, stir for 3 hours, centrifuge,

collect the filter cake, keep the crystals which are then directly dried at 50°C. X-ray powder diffraction

and DSC detection reveal crystal form A.

Example 3-6 Preparation and identification of crystal form A of MB07133

To a 100 mL three-necked flask, add 30 mL of purified water and 10 g of MB07133 crystal form F,

and add 3 mol/L aqueous sulfuric acid solution dropwise with stirring to adjust the pH to 2.0-5.0. The

temperature is controlled at 15-25°C and stirred for 1 h, 1.2 g of sodium dihydrogen phosphate is added,

and a 14% aqueous sodium hydroxide solution is slowly added dropwise to the reaction system to adjust

the pH to 5.0-8.0. After adjusting the pH, control the temperature to 10-20°C, stir for 3 hours, centrifuge,

collect the filter cake, keep the crystals which are then directly dried at 50°C. X-ray powder diffraction

and DSC detection reveal crystal form A.

Example 3-7 Preparation and identification of crystal form B of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g of the previously

obtained MB07133 white powder, stir and heat to 130°C to completely dissolve the solid, then dropwise

add 100 mL of toluene, after the addition, cool the system to 0-10°C, which is kept at 0-10°C and stirred

for 3 h, filtered with suction to keep the crystals which are then directly dried at 50°C by blasting. X-ray

powder diffraction and DSC detection show that it is crystal form B as shown in Figures 2 and 8,

indicating that stable crystal form B could be obtained by the method.

Example 3-8 Preparation and identification of crystal form B of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g MB07133 crystal form

A, stir and heat to 125°C to completely dissolve the solid, then dropwise add 100 mL toluene, after the

addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h, filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form B.

Example 3-9 Preparation and identification of crystal form B of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g MB07133 crystal form

C, stir and heat to 130°C to completely dissolve the solid, then dropwise add 100 mL toluene, after the

addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h, filter with suction to keep

the crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and DSC

detection reveal crystal form B.

Example 3-10 Preparation and identification of crystal form B of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g MB07133 crystal form

D, stir and heat to 128°C to completely dissolve the solid, then dropwise add 100 mL toluene, after the

addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h, filter with suction to keep

the crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and DSC

detection reveal crystal form B.

Example 3-11 Preparation and identification of crystal form B of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g MB07133 crystal

form E, stir and heat to 125°C to completely dissolve the solid, then dropwise add 100 mL toluene,

after the addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h, filter with

suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray powder

diffraction and DSC detection reveal crystal form B.

Example 3-12 Preparation and identification of crystal form B of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g MB07133

crystal form F, stir and heat to 128°C to completely dissolve the solid, then dropwise add 100 mL

toluene, after the addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h,

filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray

powder diffraction and DSC detection reveal crystal form B.

Example 3-13 Preparation and identification of crystal form C of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g of MB07133

white powder obtained above, stir and heat to 115°C to dissolve the solid completely, then

dropwise add 150 mL acetone, after the addition is completed, cool the system to 0-10°C, and incubate it at 0-10°C and stir for 3 h, filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection show that it is crystal form B as shown in Figures 3 and 9, indicating that stable crystal form B could be obtained by the method.

Example 3-14 Preparation and identification of crystal form C of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g of MB07133

crystal form A, stir and heat to 120°C to completely dissolve the solid, then dropwise add 150 mL

of acetone, after the addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h,

filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray

powder diffraction and DSC detection reveal crystal form C.

Example 3-15 Preparation and identification of crystal form C of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g of MB07133

crystal form B, stir and heat to 118°C to completely dissolve the solid, then dropwise add 150 mL

of acetone, after the addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h,

filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray

powder diffraction and DSC detection reveal crystal form C.

Example 3-16 Preparation and identification of crystal form C of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g of MB07133

crystal form D, stir and heat to 118°C to completely dissolve the solid, then dropwise add 150 mL

of acetone, after the addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h,

filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray

powder diffraction and DSC detection reveal crystal form C.

Example 3-17 Preparation and identification of crystal form C of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g of MB07133

crystal form E, stir and heat to 115°C to completely dissolve the solid, then dropwise add 150 mL

of acetone, after the addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h,

filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray

powder diffraction and DSC detection reveal crystal form C.

Example 3-18 Preparation and identification of crystal form C of MB07133

Into a 500 mL three-necked flask, add 100 mL dimethyl sulfoxide, add 10 g of MB07133

crystal form F, stir and heat to 120°C to completely dissolve the solid, then dropwise add 150 mL

of acetone, after the addition, cool the system to 0-10°C, and incubate at 0-10°C and stir for 3 h,

filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray

powder diffraction and DSC detection reveal crystal form C.

Example 3-19 Preparation and identification of crystal form D of MB07133

In a 500 mL three-necked flask, add 100 mL N,N-dimethylformamide, add 10 g of MB07133

white powder obtained above, stir and heat to 120°C to completely dissolve the solid, then drop

the temperature and add 200 mL of ethyl acetate dropwise. After the addition is completed, the

system is cooled to 0-10°C, keep at 0-10°C and stir for 3 hours, filter with suction to keep the

crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and DSC

detection show that it is crystal form D as shown in Figures 4 and 10, indicating that stable crystal

form D could be obtained by the method.

Example 3-20 Preparation and identification of crystal form D of MB07133

Into a 500 mL three-necked flask, add 100 mL N,N-dimethylformamide, add 10 g of

MB07133 crystal form A, stir and heat to 125°C to completely dissolve the solid, then drop the

temperature and add 200 mL of ethyl acetate dropwise. Then cool the system to 0-10°C, keep at

0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then directly dried at

50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form D.

Example 3-21 Preparation and identification of crystal form D of MB07133

Into a 500 mL three-necked flask, add 100 mL N,N-dimethylformamide, add 10 g of

MB07133 crystal form B, stir and heat to 120°C to completely dissolve the solid, then drop the

temperature and add 200 mL of ethyl acetate dropwise. Then cool the system to 0-10°C, keep at

0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then directly dried at

50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form D.

Example 3-22 Preparation and identification of crystal form D of MB07133

Into a 500 mL three-necked flask, add 100 mL N,N-dimethylformamide, add 10 g of

MB07133 crystal form C, stir and heat to 118°C to completely dissolve the solid, then drop the

temperature and add 200 mL of ethyl acetate dropwise. Then cool the system to 0-10°C, keep at

0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then directly dried at

50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form D.

Example 3-23 Preparation and identification of crystal form D of MB07133

Into a 500 mL three-necked flask, add 100 mL N,N-dimethylformamide, add 10 g of

MB07133 crystal form E, stir and heat to 116°C to completely dissolve the solid, then drop the

temperature and add 200 mL of ethyl acetate dropwise. Then cool the system to 0-10°C, keep at

0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then directly dried at

50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form D.

Example 3-24 Preparation and identification of crystal form D of MB07133

Into a 500 mL three-necked flask, add 100 mL N,N-dimethylformamide, add 10 g of

MB07133 crystal form F, stir and heat to 118°C to completely dissolve the solid, then drop the

temperature and add 200 mL of ethyl acetate dropwise. Then cool the system to 0-10°C, keep at

0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then directly dried at

50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form D.

Example 3-25 Preparation and identification of crystal form E of MB07133

In a 500 mL three-necked flask, add 20mL of N-methylpyrrolidone, add 20mL of water, add

10 g of MB07133 white powder obtained above, stir and heat to 100°C to completely dissolve the

solid, then drop the temperature and add 100 mL of acetone dropwise, and after the addition, cool

the system to 0-10°C, keep the temperature at 0-10°C and stir for 3 hours, filter with suction to

keep the crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and

DSC detection show that it is crystal form D as shown in Figures 5 and 11, indicating that stable

crystal form E could be obtained by the method.

Example 3-26 Preparation and identification of crystal form E of MB07133

To a 500 mL three-necked flask, add 20mL of N-methylpyrrolidone, add 20mL of water, then

add 10 g of MB07133 crystal form A, stir and heat to 105°C to completely dissolve the solid, then

dropwise add 100 mL of acetone, and after the addition, cool the system to 0-lOC, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

E.

Example 3-27 Preparation and identification of crystal form E of MB07133

To a 500 mL three-necked flask, add 20mL of N-methylpyrrolidone, add 20mL of water, then

add 10 g of MB07133 crystal form B, stir and heat to110°C to completely dissolve the solid, then

dropwise add 100 mL of acetone, and after the addition, cool the system to -10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

E.

Example 3-28 Preparation and identification of crystal form E of MB07133

To a 500 mL three-necked flask, add 20mL of N-methylpyrrolidone, add 20mL of water, then

add 10 g of MB07133 crystal form C, stir and heat to110°C to completely dissolve the solid, then

dropwise add 100 mL of acetone, and after the addition, cool the system to 0-10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

E.

Example 3-29 Preparation and identification of crystal form E of MB07133

To a 500 mL three-necked flask, add 20mL of N-methylpyrrolidone, add 20mL of water, then

add 10 g of MB07133 crystal form D, stir and heat to 107°C to completely dissolve the solid, then

dropwise add 100 mL of acetone, and after the addition, cool the system to 0-10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

E.

Example 3-30 Preparation and identification of crystal form E of MB07133

To a 500 mL three-necked flask, add 20mL of N-methylpyrrolidone, add 20mL of water, then

add 10 g of MB07133 crystal form F, stir and heat to 120°C to completely dissolve the solid, then

dropwise add 100 mL of acetone, and after the addition, cool the system to 0-10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

E.

Example 3-31 Preparation and identification of crystal form F of MB07133

To a 250mL three-necked flask, add 10mL purified water, add 10mL DMSO, add 5g of the

previously obtained MB07133 white powder, stir and heat to 100°C until the solid is completely dissolved, then dropwise add 150 mL isopropanol, and after the addition, cool the system to 0

10°C, and keep the temperature at 0-10°C and stir for 3 hours, filter with suction to keep the

crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and DSC

detection show that it is crystal form F as shown in Figures 6 and 12, indicating that stable crystal

form F could be obtained by the method.

Example 3-32 Preparation and identification of crystal form F of MB07133

To a 500 mL three-necked flask, add 20mL purified water, 20mL DMSO, and then add 10 g

of MB07133 crystal form A, stir and heat to 110°C until the solid is completely dissolved, then

dropwise add 300 mL isopropanol, and after the addition, cool the system to 0-10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

F.

Example 3-33 Preparation and identification of crystal form F of MB07133

To a 250mL three-necked flask, add 20mL purified water, 20mL DMSO, and then add 10 g

of MB07133 crystal form B, stir and heat to 120°C until the solid is completely dissolved, then

dropwise add 300 mL isopropanol, and after the addition, cool the system to 0-10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

F.

Example 3-34 Preparation and identification of crystal form F of MB07133

To a 250mL three-necked flask, add 20mL purified water, 20mL DMSO, and then add 10 g

of MB07133 crystal form C, stir and heat to 110°C until the solid is completely dissolved, then

dropwise add 300 mL isopropanol, and after the addition, cool the system to 0-10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

F.

Example 3-35 Preparation and identification of crystal form F of MB07133

To a 250mL three-necked flask, add 20mL purified water, 20mL DMSO, and then add 10 g

of MB07133 crystal form D, stir and heat to 115°C until the solid is completely dissolved, then

dropwise add 300 mL isopropanol, and after the addition, cool the system to 0-10°C, and keep the temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

F.

Example 3-36 Preparation and identification of crystal form F of MB07133

To a 250mL three-necked flask, add 20mL purified water, 20mL DMSO, and then add 10 g

of MB07133 crystal form E, stir and heat to 112°C until the solid is completely dissolved, then

dropwise add 300 mL isopropanol, and after the addition, cool the system to 0-10°C, and keep the

temperature at 0-10°C and stir for 3 hours, filter with suction to keep the crystals, which are then

directly dried at 50°C by blasting. X-ray powder diffraction and DSC detection reveal crystal form

F.

Example 3-37 Stability of MB07133 crystal form A compound

The example describes the stability experiments for crystal form A compound.

The stability test of the crystal compound of crystal form A under three conditions i.e. high

temperature, high humidity and light; the results are shown in Table 3-1. Under the condition of

high temperature (60°C), high humidity (90%±5%) and light (4500 lx), the related substances of

the sample basically did not change, indicating that the crystal form A is stable to high

temperature 60°C, high humidity and light.

Table 3-1 Table of crystal form A stability results

High temperature High humidity Light

Item Standard 0 day 60 0C RH92.5% 4500 lx

5 days 10 days 5 days 10 days 5 days 10 days

Not more than Isomer 0.10 0.10 0.10 0.11 0.10 0.11 0.10 0.2%

Relative Not more than Maximum impurity 0.24 0.25 0.24 0.24 0.24 0.25 0.23 substance 0.5%

Not more than Total impurities 0.70 0.68 0.64 0.76 0.71 0.74 0.66 1.5%

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 3-2),

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 3-2 Long-term test results of crystal form A

Time Relative substance (%) Isomer Maximum impurity Total impurities 5 Limit specification Not more than 0.2% Not more than 0.5% Not more than 1.

% 0 0.10 0.24 0.70 3 0.11 0.23 0.72 6 0.12 0.28 0.73

Example 3-38 Stability of MB07133 crystal form B compound

The example describes the stability experiments for crystal form B compound.

The stability test of the crystal compound of crystal form B under three conditions, i.e. high

temperature, high humidity and light; the results are shown in Table 3-3. Under the condition of

high temperature (60°C), high humidity (90%±5%) and light (4500 lx), the related substances of

the sample basically did not change, indicating that the crystal form B is stable to high temperature

60°C, high humidity and light.

Table 3-3 Table of crystal form B stability results

High temperature High humidity Light

Item Standard 0 day 60 0 C RH92.5% 4500 lx

5 days 10 days 5 days 10 days 5 days 10 days

Isomer Not more than 0.2% 0.10 0.10 0.10 0.11 0.11 0.10 0.10

Maximum Relative Not more than 0.5% 0.24 0.24 0.24 0.25 0.23 0.25 0.24 impurity substance Total Not more than 1.5% 0.70 0.69 0.66 0.72 0.71 0.73 0.68 impurities

The stability test is carried out at 40 0 C for 6 months, and the results are shown in Table 3-4,

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 3-4 Long-term test results of crystal form B

Time Relative substance (%) Isomer Maximum impurity Total impurities 2 Limit specification Not more than 0. % Not more than 0.5% Not more than 1.5% 0 0.10 0.24 0.70 3 0.11 0.23 0.72

6 0.11 0.25 0.71 Example 3-39 Stability of MB07133 crystal form C compound

The example describes the stability experiments for crystal form C compound.

The stability test of the crystal compound of crystal form C under three conditions, i.e. high

temperature, high humidity and light; the results are shown in Table 3-5. Under the condition of

high temperature (60°C), high humidity (90%±5%) and light (4500 lx), the related substances of

the sample basically did not change, indicating that the crystal form B is stable to high temperature

60°C, high humidity and light.

Table 3-5 Table of crystal form C stability results

High temperature High humidity Light

Item Standard 0 day 60 0 C RH92.5% 45001x

5 days 10 days 5 days 10 days 5 days 10 days

Isomer Not more than 0.10 0.10 0.10 0.11 0.10 0.11 0.10

Maximum Relative Not more than 0.24 0.24 0.24 0.25 0.24 0.25 0.24 impurity substance Total Not more than 0.70 0.69 0.70 0.72 0.71 0.73 0.68 impurities

The stability test is carried out at 40 0 C for 6 months, and the results are shown in Table 3-6,

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 3-6 Long-term test results of crystal form C

Time Relative substance (%) Isomer Maximum impurity Total impurities Limit specification Not more than 0.2% Not more than 0.5% Not more than 1.5% 0 0.10 0.24 0.70 3 0.12 0.24 0.71 6 0.11 0.26 0.70 Example 3-40 Stability of MB07133 crystal form D compound

The example describes the stability experiments for crystal form D compound.

The stability test of the crystal compound of crystal form D under three conditions, i.e. high

temperature, high humidity and light; the results are shown in Table 3-7. Under the condition of

high temperature (60 0C), high humidity (90%±5%) and light (4500 lx), the related substances of the sample basically did not change, indicating that the crystal form D is stable to high temperature 60°C, high humidity and light.

Table 3-7 Table of crystal form D stability results

High temperature High humidity Light

Item Standard 0 day 60 0C RH92.5% 45001x

5 days 10 days 5 days 10 days 5 days 10 days

Isomer Not more than 0.10 0.11 0.10 0.10 0.10 0.11 0.10

Maximum Not more than Relative 0.24 0.24 0.26 0.24 0.25 0.24 0.23 impurity substance Total Not more than 0.70 0.69 0.68 0.72 0.71 0.73 0.69 impurities

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 3-8,

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 3-8 Long-term test results of crystal form D

Time Relative substance (%) Isomer Maximum impurity Total impurities Limit specification Not more than 0.2% Not more than 0.5% Not more than 1.5% 0 0.10 0.24 0.70 3 0.11 0.22 0.71 6 0.12 0.24 0.70 Example 3-41 Stability of MB07133 crystal form E compound

The example describes the stability experiments for crystal form E compound.

The stability test of the crystal compound of crystal form E under three conditions, i.e. high

temperature, high humidity and light; the results are shown in Table 3-9. Under the condition of

high temperature (60°C), high humidity (90%±5%) and light (4500 lx), the related substances of

the sample basically did not change, indicating that the crystal form E is stable to high temperature

60°C, high humidity and light.

Table 3-9 Table of crystal form E stability results

High temperature High humidity Light

Item Standard 0 day 60 0 C RH92.5% 45001x

5 days 10 days 5 days 10 days 5 days 10 days

Isomer Not more than 0.10 0.10 0.10 0.11 0.11 0.10 0.10

Maximum Relative Not more than 0.24 0.23 0.24 0.25 0.23 0.24 0.24 impurity substance Total Not more than 0.70 0.68 0.67 0.72 0.71 0.72 0.68 impurities

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 3-10,

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 3-10 Long-term test results of crystal form E

Time Relative substance (%) Isomer Maximum impurity Total impurities Limit specification Not more than 0.2% Not more than 0.5% Not more than 1.5% 0 0.10 0.24 0.70 3 0.11 0.22 0.70 6 0.11 0.24 0.70 Example 3-42 Stability of MB07133 crystal form F compound

The example describes the stability experiments for crystal form F compound.

The stability test of the crystal compound of crystal form F under three conditions i.e. high

temperature, high humidity and light; the results are shown in Table 3-11. Under the condition of

high temperature (60°C), high humidity (90%±5%) and light (4500 lx), the related substances of

the sample basically did not change, indicating that the crystal form F is stable to high temperature

60°C, high humidity and light.

Table 3-11 Table of crystal form F stability results

High temperature High humidity Light

Item Standard 0 day 60 0 C RH92.5% 45001x

5 days 10 days 5 days 10 days 5 days 10 days

Isomer Not more than 0.10 0.09 0.10 0.10 0.11 0.11 0.10

Maximum Relative Not more than 0.24 0.23 0.22 0.24 0.24 0.22 0.24 impurity substance Total Not more than 0.70 0.69 0.69 0.70 0.68 0.70 0.68 impurities

The stability test is carried out at 40°C for 6 months, and the results are shown in Table 3-12),

which indicates that the crystal has good stability and is suitable for long-term storage.

Table 3-12 Long-term test results of crystal form F

Time Relative substance (%) Isomer Maximum impurity Total impurities 2 5 5 Limit specification Not more than 0. % Not more than 0. % Not more than 1. %

0 0.10 0.24 0.70 3 0.11 0.23 0.68 6 0.11 0.23 0.70 Example 3-43 Pharmaceutical composition comprising crystal form A of MB07133

Weigh or measure the materials of Table 3-13, add the crystal form A of MB07133 to the

water for injection at a temperature of (2-8°C), stir and add phosphoric acid to adjust the pH to

3.8-4.0, continue stirring to make the solution clear. Put it in a low temperature water bath to keep

the temperature at (2-8°C), add 0.1% (w/v) activated carbon for needles, stir for 30 min, remove

the carbon, filter through a 0.45 pm screen, and then filter through a 0.22 pm screen. After the initial and fine filtration, it is filled and half plugged, and then is placed in a freeze dryer, freeze-dried, fully plugged, and capped. Obtain MB07133 freeze-dried powder injection.

Table 3-13 Pharmaceutical formulations of crystal form A

Composition Dosage Overdose Effect Mg

% MB07133 crystal form A 500 100 No Active ingredient Activated carbon 0.5 / No Depyrogenation by adsorption Phosphoric acid Moderate / No pH adjuster Water for injection 10 mL / No Solvent Example 3-44 Pharmaceutical composition comprising crystal form B of MB07133

Weigh or measure the materials of Table 3-14, add the crystal form B of MB07133 to the

water for injection at a temperature of (2-8°C), stir and add phosphoric acid to adjust the pH to

3.8-4.0, continue stirring to make the solution clear. Put it in a low temperature water bath to keep

the temperature at (2-8°C), add 0.1% (w/v) activated carbon for needles, stir for 30 min, remove

the carbon, filter through a 0.45 tm screen, and then filter through a 0.22 tm screen. After the

initial and fine filtration, it is filled and half plugged, and then is placed in a freeze dryer,

freeze-dried, fully plugged, and capped. Obtain MB07133 freeze-dried powder injection.

Table 3-14 Pharmaceutical formulations of crystal form B

Composition Dosage Overdose Effect mg %

MB07133 crystal form B 500 100 No Active ingredient Activated carbon 0.5 / No Depyrogenation by adsorption Phosphoric acid Moderate / No pH adjuster Water for injection 10 mL / No Solvent

Example 3-45 Pharmaceutical composition comprising crystal form C of MB07133

Weigh or measure the materials of Table 3-15, add the crystal form C of MB07133 to the

water for injection at a temperature of (2-8°C), stir and add phosphoric acid to adjust the pH to

3.8-4.0, continue stirring to make the solution clear. Put it in a low temperature water bath to keep

the temperature at (2-8°C), add 0.1% (w/v) activated carbon for needles, stir for 30 min, remove

the carbon, filter through a 0.45 pm screen, and then filter through a 0.22 pm screen. After the

initial and fine filtration, it is filled and half plugged, and then is placed in a freeze dryer,

freeze-dried, fully plugged, and capped. Obtain MB07133 freeze-dried powder injection.

Table 3-15 Pharmaceutical formulations of crystal form C

Composition Dosage Overdose Effect mg

% MB07133 crystal form C 500 100 No Active ingredient Activated carbon 0.5 / No Depyrogenation by adsorption Phosphoric acid Moderate / No pH adjuster Water for injection 10 mL / No Solvent

Example 3-46 Pharmaceutical composition comprising crystal form D of MB07133

Weigh or measure the materials of Table 3-16, add the crystal form D of MB07133 to the

water for injection at a temperature of (2-8°C), stir and add phosphoric acid to adjust the pH to

3.8-4.0, continue stirring to make the solution clear. Put it in a low temperature water bath to keep

the temperature at (2-8°C), add 0.1% (w/v) activated carbon for needles, stir for 30 min, remove

the carbon, filter through a 0.45 pm screen, and then filter through a 0.22 pm screen. After the

initial and fine filtration, it is filled and half plugged, and then is placed in a freeze dryer,

freeze-dried, fully plugged, and capped. Obtain MB07133 freeze-dried powder injection.

Table 3-16 Pharmaceutical formulations of crystal form D

Composition Dosage Overdose Effect mg %

MB07133 crystal form D 500 100 No Active ingredient Activated carbon 0.5 / No Depyrogenation by adsorption Phosphoric acid Moderate / No pH adjuster Water for injection 10 mL / No Solvent Example 3-47 Pharmaceutical composition comprising crystal form E of MB07133

Weigh or measure the materials of Table 3-17, add the crystal form E of MB07133 to the

water for injection at a temperature of (2-8°C), stir and add phosphoric acid to adjust the pH to

3.8-4.0, continue stirring to make the solution clear. Put it in a low temperature water bath to keep

the temperature at (2-8°C), add 0.1% (w/v) activated carbon for needles, stir for 30 min, remove

the carbon, filter through a 0.45 tm screen, and then filter through a 0.22 tm screen. After the

initial and fine filtration, it is filled and half plugged, and then is placed in a freeze dryer,

freeze-dried, fully plugged, and capped. Obtain MB07133 freeze-dried powder injection.

Table 3-17 Pharmaceutical formulations of crystal form E

Composition Dosage Overdose Effect mg

% MB07133 crystal form E 500 100 No Active ingredient Activated carbon 0.5 / No Depyrogenation by adsorption Phosphoric acid Moderate / No pH adjuster Water for injection 10 mL / No Solvent Example 3-48 Pharmaceutical composition comprising crystal form F of MB07133

Weigh or measure the materials of Table 3-18, add the crystal form F of MB07133 to the

water for injection at a temperature of 2-8°C), stir and add phosphoric acid to adjust the pH to

3.8-4.0, continue stirring to make the solution clear. Put it in a low temperature water bath to keep

the temperature at (2-8°C), add 0.1% (w/v) activated carbon for needles, stir for 30 min, remove

the carbon, filter through a 0.45 tm screen, and then filter through a 0.22 tm screen. After the

initial and fine filtration, it is filled and half plugged, and then is placed in a freeze dryer,

freeze-dried, fully plugged, and capped. Obtain MB07133 freeze-dried powder injection.

Table 3-18 Pharmaceutical formulations of crystal form F

Composition Dosage Overdose Effect mg %

MB07133 crystal form D 500 100 No Active ingredient Activated carbon 0.5 / No Depyrogenation by adsorption Phosphoric acid Moderate / No pH adjuster Water for injection 10 mL / No Solvent

Claims (12)

The claims defining the invention are as follows:

1. A crystal for treating a liver disease, which is a crystal of Tenofovir phosphate, a crystal

of Pradefovir mesylate, or a crystal of MB07133, wherein the crystal of Tenofovir phosphate has

an X-ray powder diffraction pattern substantially as shown in Figures 1-1, 1-2, 1-3, 1-4 or 1-5, the

crystal of Pradefovir mesylate has an X-ray powder diffraction pattern substantially as shown in

Figures 2-1, 2-3, 2-5, 2-7 or 2-9, the crystal of MB07133 has an X-ray powder diffraction pattern

substantially as shown in Figures 3-1, 3-2, 3-3, 3-4, 3-5 or 3-6.

2. The crystal of claim 1, characterized in that a differential thermal analysis curve of the

crystal of Tenofovir phosphate has sharp endothermic peaks at 151.7°C, 152.3°C, 152.6°C,

149.2°C or 151.4°C, respectively, a differential thermal analysis curve of the crystal of Pradefovir

mesylate has sharp endothermic peaks at 196.9°C, 191.8°C, 193.4°C, 192.6°C or 193.2°C,

respectively, or, a differential thermal analysis curve of the crystal of MB07133 has sharp

endothermic peaks at 239.81°C, 253.21°C, 247.95°C, 251.10°C, 246.57°C or 249.26°C.

3. The crystal of claim 1 or 2, which is a mono-crystal.

4. A pharmaceutical composition for treating or preventing a liver disease or metabolic

disease, comprising the crystal of any one of claims 1-3 and a pharmaceutically acceptable

excipient.

5. The pharmaceutical composition of claim 4, which is in an oral dosage form, preferably

a tablet.

6. The pharmaceutical composition of claim 4, which is an injection.

7. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable

excipient comprises mannitol, pregelatinized starch, magnesium stearate and/or silicon dioxide.

8. A use of the crystal of any one of claims 1-3 in the preparation of a medicine for treating

or preventing a liver disease or metabolic disease.

9. The use of claim 8, which is a use in the preparation of a medicine for treating or

preventing hepatitis B, or a use in the preparation of a medicine for reducing a level of hepatitis B

virus in a patient.

10. The use of claim 8, which is a use in the preparation of a medicine for the treating

advanced hepatocarcinoma.

11. The use of claim 10, which is a use of the crystal of MB07133.

12. A method for detecting the crystal of any one of claims 1-3, wherein a suspected crystal

is subjected to X-ray powder diffraction detection, and the obtained X-ray powder diffraction

pattern is compared with those in one of Figures 1-1, 1-2, 1-3, 1-4, 1-5, 2-1, 2-3, 2-5, 2-7, 2-9, 3-1,

3-2, 3-3, 3-4, 3-5 and 3-6.