# Patent

## Title
Crystal forms of azetidinone compounds and preparing methods thereof

## Abstract

The present invention provides crystal forms of the compound of (3R,4S)-4-(4-hydroxyphenyl)-3-[3-(4-fluorophenyl)-4-hydroxybut-2(Z)-enyl]-1-(4-fluorophenyl)-2-azetidinone (formula A). The crystal forms can be characterized by X-ray powder diffraction (XRPD) spectra, differential scanning calorimetry (DSC) spectra, infrared absorption spectra and so on. Meanwhile, the present invention also provides methods for preparing the crystal forms of the compound of formula A, pharmaceutical compositions and uses thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a divisional of U.S. patent application Ser. No. 14/426,085, filed on Mar. 4, 2015, which application is a national phase entry under 35 U.S.C. §371 of International Application No. PCT/CN2013/082998, filed on Sep. 5, 2013, published in Chinese, which claims priority from Chinese Patent Application No. 201210323683.5, filed Sep. 5, 2012, all of which are hereby incorporated herein by reference.


FIELD OF THE INVENTION
The present invention belongs to the field of chemistry, and more specifically, the present invention relates to new crystal forms of (3R,4S)-4-(4-hydroxyphenyl)-3-[3-(4-fluorophenyl)-4-hydroxybut-2(Z)-enyl]-1-(4-fluorophenyl)-2-azetidinone and preparation methods thereof.
BACKGROUND OF THE INVENTION
A compound of azetidinone has a structure of formula A as follows and a chemical name of (3R,4S)-4-(4-hydroxyphenyl)-3-[3-(4-fluorophenyl)-4-hydroxybut-2(Z)-enyl]-1-(4-fluorophenyl)-2-azetidinone. The compound of formula A is a novel plasma cholesterol reducing agent, which is used for reducing plasma cholesterol contents, thus being used for treating diseases highly correlated to the plasma cholesterol contents. WO2011017907 discloses a compound of formula A and preparation methods and applications thereof.




No report about the crystal forms of the compound of formula A is found at present. The inventor obtains the crystal forms of the compound of formula A through conducting a large number of experiments.
BRIEF SUMMARY OF THE INVENTION
Therefore, the object of the present invention is to provide crystal forms of the compound of (3R,4S)-4-(4-hydroxyphenyl)-3-[3-(4-fluorophenyl)-4-hydroxybut-2(Z)-enyl]-1-(4-fluorophenyl)-2-azetidinone of formula A structured as follows and the preparation methods thereof.




According to the object of the present invention, the present invention provides a crystal form I of the compound of formula A (hereinafter referred to as “crystal form I”).
An X-ray powder diffraction (XRPD) spectrum of the crystal form I radiated by Cu-Kα and characterized in degrees 2θ has following characteristic peaks at 8.17±0.20°, 13.53±0.20°, 16.67±0.20°, 18.13±0.20°, 19.14±0.20°, 19.57±0.20°, 20.26±0.20°, 22.32±0.20°, 23.05±0.20°, 23.77±0.20°, 25.03±0.20°, 27.69±0.20° and 30.99±0.20°.
Preferably, the X-ray powder diffraction spectrum of the crystal form I characterized in degrees 2θ further has following characteristic peaks at 15.76±0.20°, 16.25±0.20°, 17.14±0.20°, 21.12±0.20°, 25.50+0.20°, 26.02±0.20°, 28.95±0.20°, 29.84±0.20°, 33.34±0.20°, 36.32±0.20° and 37.57±0.20°.
More preferably, the X-ray powder diffraction spectrum of the crystal form I characterized in degrees 2θ has following characteristic peaks and relative intensity at:














Diffraction angle 2θ
Relative intensity %



















 8.17 ± 0.20°
10



13.53 ± 0.20°
13



15.76 ± 0.20°
3



16.25 ± 0.20°
6



16.67 ± 0.20°
11



17.14 ± 0.20°
3



18.13 ± 0.20°
38



19.14 ± 0.20°
47



19.57 ± 0.20°
14



20.26 ± 0.20°
71



21.12 ± 0.20°
3



22.32 ± 0.20°
33



23.05 ± 0.20°
100



23.77 ± 0.20°
21



25.03 ± 0.20°
13



25.50 ± 0.20°
7



26.02 ± 0.20°
4



27.69 ± 0.20°
21



28.95 ± 0.20°
4



29.84 ± 0.20°
6



30.99 ± 0.20°
9



33.34 ± 0.20°
5



36.32 ± 0.20°
3



37.57 ± 0.20°
4










More preferably, the XRPD spectrum of the crystal form I is shown in FIG. 1.
Further, the differential scanning calorimetry (DSC) spectrum of the crystal form I is shown in FIG. 2.
Further, the infrared (IR) spectrum of the crystal form I is shown in FIG. 3.
The infrared absorption peak and relative absorption intensity data of the crystal form I are shown as follows.














Infrared absorption wave number (cm−1)
Relative absorption



















430.1189
0.86744



514.9855
0.46846



636.4989
0.86912



721.3654
0.85935



783.0865
0.49801



827.4485
0.12002



898.8136
0.91525



941.2468
0.81022



1010.683
0.29323



1105.194
0.51903



1157.271
0.4074



1222.849
0.11491



1355.935
0.36791



1392.582
0.2545



1440.802
0.64795



1508.309
0.01676



1602.82
0.50812



1724.333
0.02433



1888.28
0.86998



2821.812
0.94387



2883.533
0.86289



2945.254
0.90718



3032.049
0.8926



3381.159
0.4183










According to the object of the present invention, the present invention provides the preparation methods of the crystal form I. The preparation method for obtaining the crystal form I can be selected from any one of the following methods.
Method (1), comprising the following steps of:
1) dissolving the compound of formula A into an organic solvent to prepare a saturated solution, wherein the organic solvent is selected from C1-C4 linear alcohol, acetonitrile, furans, carboxylic acid, ester, amide or ketone;
2) adding water or C2-C8 ether anti-solvent more than one time of the volume of the organic solvent in step 1) into the foregoing saturated solution all in once to crystallize out the solids of the crystal form I; and
3) collecting the obtained solids, thus obtaining the crystals in form I.
In step 1) of the method (1), the carboxylic acid is preferably C1-6 carboxylic acid, the ester is preferably C1-6 alkyl C1-6 carboxylate, the amide is preferably N—C1-6 alkylamide or N,N-bi-C1-6 alkylamide, and the ketone is preferably C3-6 ketone.
In a further embodiment, the organic solvent in step 1) of the method (1) is selected from methanol, ethanol, n-propanol, iso-propanol, n-butylalcohol, tetrahydrofuran, acetic acid, ethyl acetate, acetonitrile, N,N-dimethylformamide or acetone; and is more preferably methanol or ethanol.
The volume of the water or C2-C8 ether anti-solvent added in step 2) of the method (1) is 1-10 times of the volume of the organic solvent, and is preferably 1.5 times. Further preferably, the anti-solvent is water.
In a further embodiment, the preparation method (1) of the crystal form I comprises the following steps of: dissolving one portion of the compound of formula A in g into one portion of methanol in mL to prepare a saturated solution thereof under the normal temperature, then adding 10 portions of anti-solvent water in mL into the foregoing saturated solution all at once, immediately filtrating solids that are crystallized out, placing the solids in a vacuum drying oven for drying, wherein the dried solids are namely the crystal form I.
Method (2), comprising the following steps of:
1) dissolving the compound of formula A into C1-6 alkyl C1-6 carboxylate to obtain a solution, wherein the ratio of the mass of the compound of formula A in g to the volume of the C1-6 alkyl C1-6 carboxylate in mL is 1:1.5-5;
2) adding C6-C7 alkane anti-solvent dropwise 1-3 times of the volume of the C1-6 alkyl C1-6 carboxylate in step 1) into the foregoing solution to crystallized out the solids in the crystal form I; and
3) collecting the obtained solids, thus obtaining the crystals in form I.
In step 1) of the method (2), the C1-6 alkyl C1-6 carboxylate is preferably ethyl acetate.
In step 2) of the method (2), the C6-C7 alkane is preferably n-hexane or n-heptane.
In a further embodiment, the preparation method (2) of the crystal form I comprises the following steps of: adding one portion of the compound of formula A in g into a crystallizer (for example, 10 times of the volume), adding two portions of ethyl acetate in mL thereinto, stirring at the room temperature till complete dissolving to obtain a solution, adding two portions of n-heptane in mL dropwise into the foregoing solution to crystallize a large amount of solids, then performing vacuum drying on the solids after the solids are filtrated out, wherein the dried solids are namely the crystal form I.
Method (3), comprising the following steps of:
adding one portion of the compound of formula A in g into five portions of methylene chloride in mL, completely dissolving under heating and refluxing conditions, then slowly cooling to the normal temperature, separating out solids, filtrating, and drying at the normal temperature and the normal pressure, wherein the dried solids are namely the crystal form I.
Method (4), comprising the following steps of:
adding one portion of the compound of formula A in g into 10 portions of toluene in mL, completely dissolving under heating and refluxing conditions, then slowly cooling to the normal temperature, crystallizing out solids, filtrating, and drying at the normal temperature and the normal pressure, wherein the dried solids are namely in the crystal form I.
According to the object of the present invention, the present invention provides a crystal form II of the compound of formula A (hereinafter referred to as “crystal form II”).
An X-ray powder diffraction spectrum of the crystal form II radiated by Cu-Kα and characterized in degrees 2θ has following characteristic peaks at 5.20±0.20°, 7.46±0.20°, 8.70±0.20°, 10.60±0.20°, 11.84±0.20°, 17.14±0.20°, 20.36±0.20° and 26.48±0.20°.
Preferably, the X-ray powder diffraction spectrum of the crystal form II characterized in degrees 2θ further has following characteristic peaks at 6.29±0.20°, 6.96±0.20°, 12.82±0.20°, 13.28±0.20°, 14.21±0.20°, 15.02±0.20°, 15.40±0.20°, 16.40±0.20°, 17.61±0.20°, 18.34±0.20°, 18.62±0.20°, 19.06±0.20°, 19.58±0.20°, 20.02±0.20°, 21.50±0.20°, 22.18±0.20°, 22.74±0.20°, 23.41±0.20, 24.06±0.20°, 24.34±0.20°, 24.70±0.20°, 25.16±0.20°, 27.34±0.20°, 28.00±0.20°, 28.88±0.20°, 29.36±0.20°, 31.39±0.20°, 32.02±0.20°, 32.60±0.20° and 34.42±0.20°.
More preferably, the X-ray powder diffraction spectrum of the crystal form II characterized in degrees 2θ has following characteristic peaks and relative intensity at:














Diffraction angle 2θ
Relative intensity %



















 5.20 ± 0.20°
39



 6.29 ± 0.20°
4



 6.96 ± 0.20°
29



 7.46 ± 0.20°
84



 8.70 ± 0.20°
28



10.60 ± 0.20°
18



11.84 + 0.20°
27



12.82 ± 0.20°
3



13.28 ± 0.20°
3



14.21 ± 0.20°
4



15.02 ± 0.20°
13



15.40 ± 0.20°
7



16.40 ± 0.20°
33



17.14 ± 0.20°
25



17.61 ± 0.20°
13



18.34 ± 0.20°
28



18.62 ± 0.20°
33



19.06 ± 0.20°
11



19.58 ± 0.20°
17



20.02 ± 0.20°
23



20.36 ± 0.20°
28



21.50 ± 0.20°
100



22.18 ± 0.20°
17



22.74 ± 0.20°
20



23.41 ± 0.20°
6



24.06 ± 0.20°
13



24.34 ± 0.20°
11



24.70 ± 0.20°
12



25.16 ± 0.20°
23



26.48 ± 0.20°
16



27.34 ± 0.20°
6



28.00 ± 0.20°
11



28.88 ± 0.20°
11



29.36 ± 0.20°
4



31.39 ± 0.20°
5



32.02 ± 0.20°
4



32.60 ± 0.20°
3



34.42 ± 0.20°
3










More preferably, the XRPD spectrum of the crystal form II is shown in FIG. 4.
Further, the differential scanning calorimetry (DSC) spectrum of the crystal form II is shown in FIG. 5.
Further, the infrared (IR) spectrum of the crystal form II is shown in FIG. 6.
The infrared absorption peak and relative absorption intensity data of the crystal form II are shown as follows.














Infrared absorption wave number (cm−1)
Relative absorption



















430.1189
0.9543



520.7718
0.69298



628.7837
0.91287



734.8669
0.82243



827.4485
0.46604



879.5257
0.95653



948.962
0.91914



1012.612
0.67808



1101.336
0.74233



1155.342
0.66551



1224.778
0.38758



1354.007
0.69178



1390.654
0.5504



1444.66
0.76162



1510.238
0.12598



1604.749
0.72333



1726.262
0.23374



1888.28
0.9551



2684.868
0.967



2819.883
0.94866



2902.821
0.89406



2966.471
0.8802



3068.696
0.89403



3240.358
0.81058



3379.23
0.75873



3489.171
0.73966










According to the object of the present invention, the present invention provides a preparation method of the crystal form II. The preparation method comprises the following steps of:
1) adding the compound of formula A into C1-C3 alcohol, optionally heating to dissolve the compound and obtain a solution, wherein the ratio of the mass of the compound of formula A in g to the volume of the C1-C3 alcohol in mL is 1:2-10;
2) adding water into the foregoing solution and ensuring that no crystal is crystallized out; and
3) naturally volatilizing the solvent at the normal temperature and the normal pressure, and collecting solids that are crystallized out, thus obtaining the crystals in form II.
Preferably, the C1-C3 alcohol in step 1) is ethanol or iso-propanol.
Preferably, the ratio of the volume of the water added in step 2) to the volume of the C1-C3 alcohol is 10:1-6, and is more preferably 10:2.
Preferably, the ratio of the mass of the compound of formula A in g to the volume of the C1-C3 alcohol in mL in step 1) is 1:2-5.
In a further embodiment, the preparation method of the crystal form II comprises the following steps of: adding 0.7 portion of the compound of formula A in g into a crystallizer (e.g., 18 times of volume), then adding 1.7 portions of ethanol in mL thereinto, heating to 50° C. for complete dissolving, then adding one portion of water (e.g., purified water) in mL thereinto, stopping heating, cooling naturally, volatilizing the solvent at the normal temperature and pressure, crystallizing out crystals (acicular), filtrating, and drying (for example, 24 h) the crystals at the normal temperature to obtain the crystals in form II.
According to the object of the present invention, the present invention provides a crystal form III of the compound of formula A (hereinafter referred to as “crystal form III”).
An X-ray powder diffraction spectrum of the crystal form III radiated by Cu-Kα and characterized in degrees 2θ has following characteristic peaks at: 8.16±0.20°, 12.02±0.20°, 14.38±0.20°, 17.60±0.20°, 18.36±0.20° and 20.98±0.20°.
Preferably, the X-ray powder diffraction spectrum of the crystal form III characterized in degrees 20 further has following characteristic peaks at 6.06±0.20°, 8.74±0.20°, 10.32±0.20°, 13.22±0.20°, 14.78±0.20°, 15.36±0.20°, 16.12±0.20°, 16.52±0.20°, 17.08±0.20°, 18.70±0.20°, 19.04±0.20°, 20.06±0.20°, 21.52±0.20°, 22.36±0.20°, 22.84±0.20°, 23.50±0.20°, 24.20±0.20°, 24.84±0.20°, 25.10±0.20°, 26.18±0.20°, 26.66±0.20°, 27.12±0.20°, 27.44±0.20°, 28.44±0.20°, 29.02±0.20°, 29.62±0.20°, 30.62±0.20°, 31.16±0.20°, 31.58±0.20°, 33.47±0.20° and 33.73±0.20°.
More preferably, the X-ray powder diffraction spectrum of the crystal form III characterized in the degrees 2θ has following characteristic peaks and relative intensity at:














Diffraction angle 2θ
Relative intensity %



















 6.06 ± 0.20°
4



 8.16 ± 0.20°
53



 8.74 ± 0.20°
9



10.32 ± 0.20°
8



12.02 ± 0.20°
37



13.22 ± 0.20°
13



14.38 ± 0.20°
34



14.78 ± 0.20°
19



15.36 ± 0.20°
34



16.12 ± 0.20°
11



16.52 ± 0.20°
40



17.08 ± 0.20°
38



17.60 ± 0.20°
94



18.36 ± 0.20°
65



18.70 ± 0.20°
100



19.04 ± 0.20°
37



20.06 ± 0.20°
91



20.98 ± 0.20°
42



21.52 ± 0.20°
34



22.36 ± 0.20°
28



22.84 ± 0.20°
31



23.50 ± 0.20°
13



24.20 ± 0.20°
24



24.84 ± 0.20°
19



25.10 ± 0.20°
41



26.18 ± 0.20°
8



26.64 ± 0.20°
12



27.12 ± 0.20°
11



27.44 ± 0.20°
13



28.44 ± 0.20°
8



29.02 ± 0.20°
9



29.62 ± 0.20°
6



30.62 ± 0.20°
5



31.16 ± 0.20°
6



31.58 ± 0.20°
6



33.47 ± 0.20°
4



33.73 ± 0.20°
4










More preferably, the XRPD spectrum of the crystal form III is shown in FIG. 7.
Further, the differential scanning calorimetry (DSC) spectrum of the crystal form III is shown in FIG. 8.
Further, the infrared (IR) spectrum of the crystal form III is shown in FIG. 9.
The infrared absorption peak and relative absorption intensity data of the crystal form III are shown as follows.














Infrared absorption wave number (cm−1)
Relative absorption



















426.2614
0.88521



524.6294
0.69233



609.4959
0.89427



736.7957
0.87285



825.5198
0.24393



920.0302
0.63294



1012.612
0.38132



1095.55
0.70228



1163.057
0.45052



1220.921
0.2604



1379.081
0.32645



1460.09
0.63607



1512.167
0.02957



1608.606
0.63564



1728.191
0.26212



1888.28
0.91463



2609.645
0.92228



2696.441
0.91122



2817.954
0.88812



2922.108
0.70643



2968.399
0.43922



3066.767
0.7935



3332.94
0.47441



3404.305
0.4607










According to the object of the present invention, the present invention provides a preparation method of the crystal form III. The preparation method comprises the following steps of:
1) adding the compound of formula A into a mixed solvent of C4-C10 alcohol and ethanol, optionally heating to dissolve the compound and obtain a solution, wherein the ratio of the volume of ethanol to the volume of C4-C10 alcohol is less than or equal to 0.2;
2) adding water more than one time of the volume of the foregoing mixed solvent into the foregoing solution; and
3) Crystallizing out the crystals, filtrating, and optionally drying to obtain the crystals in form III.
Preferably, the C4-C10 alcohol in step 1) is tert-butyl alcohol.
Preferably, the volume of the water added in step 2) is 2-10 times of the volume of the mixed solvent, and is preferably 2-3 times.
Preferably, the ratio of the mass of the compound of formula A in g to the volume of the C4-C10 alcohol in mL in step 1) is 1:8-15.
In a further embodiment, the preparation method of the crystal form III comprises the following steps of: adding 0.4 portion of the compound of formula A in g into a mixed solvent of four portions of tert-butyl alcohol and 0.3 portion of ethanol in mL, completely dissolving at the normal temperature to obtain a solution, adding 10 portions of water (for example, distilled water) in mL into the foregoing solution, crystallizing out the crystals, filtrating, and drying at the normal temperature and pressure to obtain the crystals in form III.
The present invention further provides another preparation method of the crystal form III, comprising the following steps of: adding one portion of the compound of formula A in g into a crystallizer (for example, 25 times of the volume), then adding 15 portions of tert-butyl alcohol in mL thereinto, dissolving sufficiently at 50° C., then naturally cooling to the room temperature to crystallizing out solids, filtrating the solid, and drying at the normal temperature and pressure, thus obtaining the crystals in form III.
In addition, the present invention further provides an amorphous form of the compound of formula A (hereinafter referred to as “amorphous form”).
The amorphous form radiated by Cu-Kα has an X-ray powder diffraction spectrum as shown in FIG. 10.
Further, the DSC spectrum of the amorphous form is shown in FIG. 11.
Further, the IR spectrum of the amorphous form is shown in FIG. 12.
The infrared absorption peak and relative absorption intensity data of the amorphous form are shown as follows.














Infrared absorption wave number (cm−1)
Relative absorption



















426.2614
94%



513.0567
68%



543.9172
83%



592.1368
92%



619.1398
87%



638.4277
89%



704.0063
89%



727.1517
87%



781.1577
80%



817.8046
47%



833.2349
30%



935.4605
89%



991.3952
73%



1012.612
59%



1070.475
88%



1103.265
69%



1157.271
57%



1224.778
32%



1271.069
63%



1355.935
66%



1392.582
48%



1429.229
74%



1450.446
67%



1510.238
11%



1598.962
59%



1614.393
61%



1722.405
23%



1886.351
93%



2858.459
91%



2925.966
85%



2956.827
86%



3024.334
92%



3078.34
91%



3350.299
70%



3367.658
70%










According to the object of the present invention, the present invention provides a preparation method of the amorphous form. The preparation method comprises the following steps of:
1) dissolving the compound of formula A into an alcohol organic solvent to prepare a saturated solution;
2) adding alkane anti-solvent more than one time of the volume of the saturated solution into the foregoing saturated solution to form a slurry; and
3) lyophilizing the obtained slurry to obtain the solids in amorphous form.
Preferably, the alcohol organic solvent in step 1) is C1-C10 alcohol, and is more preferably C1-C4 alcohol, and is further preferably methanol or ethanol.
Preferably, the volume of the anti-solvent added in step 2) is 2-10 times of the volume of the saturated solution, and is more preferably 5 times.
Preferably, the alkane anti-solvent in step 2) is n-hexane or n-pentane.
In a further embodiment, the preparation method of the amorphous form comprises the following steps of: adding one portion of the compound of formula A in g into a crystallizer (for example, 18 times of the volume), then adding one portion of ethanol in mL thereinto, dissolving sufficiently at 50° C., then cooling to the room temperature, quickly adding five portions of n-hexane in mL thereinto under a quick stirring state to obtain a slurry (vaporous), lyophilizing (for example, one hour) the slurry to obtain the solids in amorphous form (white solid).
In this application, the steps of dissolving and crystal separating involved in all the above-mentioned methods generally require stirring unless otherwise noted. A known manner may be employed for stirring, for example, stirring via a magnetic force stirrer, mechanical stirring, and the like may be employed.
In this application, unless otherwise noted, the step of drying involved in all the above-mentioned methods may be carried out by employing a known manner, for example, vacuum drying, and the like. The drying temperature is generally 35-40° C. unless otherwise noted.
In this application, the normal temperature or the room temperature refers to 10-30° C., and is preferably 25° C.
In addition, the present invention provides a pharmaceutical composition/pharmaceutical preparation that comprises a therapeutically effective amount of the crystal form I or the crystal form II or the crystal form III. The pharmaceutical composition/pharmaceutical preparation includes but is not limited to general dosage form administration, for example, oral preparation form and injection preparation form, including capsule, tablet, powder, cachet, suspension and solution, wherein oral preparation form administration is preferable, and the tablet and capsule administration in the oral preparation form is more preferable.
In the pharmaceutical composition/pharmaceutical preparation, a pharmaceutically acceptable carrier or vehicle is further included. The pharmaceutically acceptable carrier or vehicle/additive includes but is not limited to avirulent compatible filler, adhesive, disintegrating agent, buffer, preservative, antioxidant, lubricant, corrigent, thickener, stain, emulsifying agent, and the like.
In addition, the present invention further provides an application of the crystal form I or the crystal form II or the crystal form III of the compound of formula A in preparing drugs for reducing plasma cholesterol contents.
The crystal form I or the crystal form II or the crystal form III of the compound of formula A according to the present invention may be used as a drug for reducing serum cholesterol. Therefore, the crystal form I or the crystal form II or the crystal form III of the compound of formula A according to the present invention can be used for treating or preventing such diseases as atherosclerosis and hypercholesterolemia, and the like.
In addition, the present invention further provides a method for reducing plasma cholesterol contents, which comprises the steps of giving the therapeutically effective amount of the crystal form I or the crystal form II or the crystal form III to a sufferer in need.
The compound of formula A according to the present invention can be prepared according to the process described in WO2011017907A1.
Advantages of the present invention: the present invention changes the solid-state physical properties of the drug compound of formula A through the foregoing manners. The solid-state physical properties comprise solid lapping liquidity, solid density, apparent crystal habit, solid melting point, and the like. These properties will affect the difficulty or facility of processing raw materials into drugs, the stability of the drugs during storage process, and the like. The drugs are easy to be processed into stable preparations to ensure the stability of the drugs only when the solid-state physical properties are well.
Another important solid-state property of drug compound is the dissolution rate of the drug compound in an aqueous fluid. The dissolution rate of active ingredients in the gastric juice of the sufferer has the result of therapeutics because the dissolution rate is the upper limit of the rate of the active ingredients of oral administration arriving at the blood. When preparing syrup, elixir and other liquid drugs, the dissolution rate is also a factor to be considered.
The molecular conformation and orientation of a unit cell may affect the foregoing actual physical properties. A specific heteromorphic form of a substance can be determined using the unit cell. The heteromorphic form enables the thermal behavior of the crystal to differ from amorphous substances or other heteromorphic forms.
The finding of the officinal new crystal forms of the compound provides a new opportunity for improving the behavior characteristics of drugs. It expands the range of materials that can be selected for designing by pharmaceutists, for example, a pharmaceutical preparation has a targeted release performance or other performance in need.
Compared with the amorphous form, the crystal form I or the crystal form II or the crystal form III of the compound of formula A according to the present invention have the advantages of high crystal form stability, high purity, and convenience for the storage and transportation. With regard to the handling process, the process of handling solids in the crystal forms can be safer as the hazard of dust explosion can be reduced; and the crystal form has better performances on pharmaceutics than that of the amorphous form.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the XRPD spectrum of a crystal form I prepared in embodiment 2.
FIG. 2 is the DSC spectrum of the crystal form I prepared in embodiment 2.
FIG. 3 is the IR spectrum of the crystal form I prepared in embodiment 2.
FIG. 4 is the XRPD spectrum of a crystal form II prepared in embodiment 9.
FIG. 5 is the DSC spectrum of the crystal form II prepared in embodiment 9.
FIG. 6 is the IR spectrum of the crystal form II prepared in embodiment 9.
FIG. 7 is the XRPD spectrum of a crystal form III prepared in embodiment 13.
FIG. 8 is the DSC spectrum of the crystal form III prepared in embodiment 13.
FIG. 9 is the IR spectrum of the crystal form III prepared in embodiment 13.
FIG. 10 is the XRPD spectrum of an amorphous form prepared in embodiment 16.
FIG. 11 is the DSC spectrum of the amorphous form prepared in embodiment 16.
FIG. 12 is the IR spectrum of the amorphous form prepared in embodiment 16.
FIG. 13 is the XRPD spectrum of a crystal form I prepared in embodiment 7.
FIG. 14 is the XRPD spectrum of a crystal form I prepared in embodiment 8.
FIG. 15 is the XRPD spectrum of a compound of formula A prepared in embodiment 1.



DETAILED DESCRIPTION
The present invention will be further described hereinafter with references to the embodiments. It should be understood that the preparation methods according to the embodiments of the present invention are for explanation merely, but not to limit the present invention. Any simple improvement figured out on the preparation methods of the present invention under the concept of the present invention shall all fall within the protection scopes claimed by the present invention.
Instruments used for determining the XRPD, DSC and IR spectra of various new crystal forms and amorphous form of the present invention and the determining conditions are as follows.
The instruments used for determining the XRPD spectra and the determining conditions are seen in Table 1 and Table 2. FIG. 4, FIG. 7 and FIG. 10 are obtained by using the instruments and determining conditions as shown in Table 1. FIG. 1, FIG. 13, FIG. 14 and FIG. 15 are obtained by using the instruments and determining conditions as shown in Table 2.







TABLE 1





Instruments used for determining XRPD spectra and


determining conditions


















Model of
Ri gaku D/max-2500
Scanning range
5°-50° (2θ)


instrument


Emitting target
Cu-Kα (1.5405 Å)
Scanning rate
1 step/s


Power supply
40 kV, 100 mA
Scanning step
0.02°


settings


Receiving slit
0.15 mm
Measuring
(25 ± 1)° C.




temperature
















TABLE 2





Instruments used for determining XRPD spectra


and determining conditions


















Model of instrument
Ri gaku D/max-2200
Scanning range
3°-60°(2θ)


Emitting target
Cu-Kα (1.54056 Å)
Scanning rate
3°/min


Power supply
40 kV, 40 mA
Scanning step
0.01°


settings


Measuring
(25 ± 1)° C.


temperature









Infrared (IR) spectral analysis: Bruker TENSOR 27 Fourier infrared spectrometer and DTGS detector are employed. The scanning range is 4000-400 cm−1, and KBr tabletting method is adopted. 1.5 mg of samples is evenly mixed with 100 mg of KBr and pressed into sheets having a diameter of 13 mm, wherein the experimental data is collected and stored through an OPUS program.
DSC method: it is measured by employing the DSC1 of METTLER TOLEDO at a heating rate of 10° C./min. Specifically, DSC Mettler 1/700 differential scanning calorimeter is employed to analyze the samples. The weight of the samples ranges from 4 mg to 6 mg, the heating rate is 10° C./min, the protective gas is N2, and the flow rate of the protective gas is 120-150 mL/min.
Embodiment 1: preparation of (3R,4S)-4-(4-hydroxyphenyl)-3-[3-(4-fluorophenyl)-4-hydroxybut-2(Z)-enyl]-1-(4-fluorophenyl)-2-azetidinone (the Compound of Formula A)
Refer to the method of WO2011017907A1 for the preparation of the compound of formula A, wherein the details are as follows:




Step 1: add 32.5 mmol of compound II (formula Z), 250 mL of methanol, and 4.89 g (35.8 mmol) of potassium carbonate into a 500 mL reaction flask, and stir for 30 min at the room temperature. After the reaction is completed, extract for three times (300 mL×3) with ethyl acetate, combine organic phases, wash with a saturated saline solution, dry with anhydrous sodium sulfate, concentrate till it is dry, and keep stand-by.
Step 2: dissolve the product of step 1 with 200 mL of tetrahydrofuran, use 6 mol/L hydrochloric acid to adjust the pH at around 1, stir for 30 mins at the room temperature, then extract for three times (250 mL×3) with ethyl acetate, combine the organic phases, wash with a saturated with saline solution, dry with anhydrous sodium sulfate, concentrate till it is dry, use a solvent system of n-heptane and ethyl acetate (the volume ratio is 3:1) to carry out column chromatography separation, collect a target project, decompress, concentrate, and dry to obtain 8.14 g of the compound of formula A. The XRPD spectrum is shown in FIG. 15, indicating that it is an amorphous form.
1H NMR (400 MHz, DMSO-d6): δ2.72-2.84 (m, 2H, —CH2-), 3.20-3.25 (m, 1H, —CH—), 4.39 (d, 2H, J=5.2 Hz, —CH2-), 4.85 (t, 1H, J=5.2 Hz, —OH), 4.93 (d, 1H, J=2.3 Hz, —CH—), 5.80 (t, 1H, J=7.6 Hz, —CH—), 6.73-6.76 (m, 2H, Cpr-H), 7.10-7.20 (m, 4H, Cpr-H), 7.21-7.39 (m, 4H, Cpr-H), 8.40-7.42 (m, 2H, Cpr-H), 9.48 (s, 1H, —OH); MS (m/z): 422 [M+H].
Embodiment 2: Preparation of Crystal Form I
At the normal temperature, dissolve 1 g of the compound of formula A into 1 mL of methanol to prepare a saturated solution thereof, then add 10 mL of water into the saturated solution all at once, immediately filter out an obtained solid, place the solid into a vacuum drying oven for drying (the drying temperature is 35-40° C.), wherein the dried solids are namely the crystal form I. The output is 0.73 g, and the yield is 73%.
The XRPD spectrum thereof is shown in FIG. 1.
The DSC spectrum is shown in FIG. 2. Display: the melting point is 154.8° C.
The infrared spectrum is shown in FIG. 3.
Embodiment 3: Preparation of Crystal Form I
The operating process is identical to that of the embodiment 2, excepting respectively replacing the methanol with tetrahydrofuran, iso-propanol, acetone, n-propanol, acetic acid, n-butylalcohol, ethyl acetate, acetonitrile and N,N-dimethylformamide to respectively obtain the crystal form I. The XRPD spectrum of the obtained crystal form I is identical to the XRPD spectrum of the crystal form I prepared in embodiment 2.
Embodiment 4: Preparation of Crystal Form I
Add 1 g of the compound of formula A into 1 mL of ethanol, completely dissolve at the normal temperature, then add 10 mL of distilled water thereinto all at once, separate out a solid, filter the solid out, and dry at the normal temperature and pressure, wherein the obtained solid is the crystal form I. The output is 0.88 g, and the yield is 88%. The XRPD spectrum of the obtained crystal form I is identical to the XRPD spectrum of the crystal form I prepared in embodiment 2.
Embodiment 5: Preparation of Crystal Form I
Add 1 g of the compound of formula A into 1 mL of ethanol, warmly dissolve, then add 1.5 mL of distilled water thereinto at the normal temperature, crystallize solids out, filtrating the solids out, and drying the solids (the drying temperature is 35-40° C.), wherein the obtained solids are the crystals in form I. The output is 0.91 g, and the yield is 91%. The XRPD spectrum of the obtained crystal form I is identical to the XRPD spectrum of the crystal form I prepared in embodiment 2.
Embodiment 6: Preparation of Crystal Form I
Add 1 g of the compound of formula A into a 10 mL crystallizer, add 2 mL of ethyl acetate thereinto, stir at the room temperature till complete dissolving, dropwise add 2 mL of n-heptane thereinto to crystallize out a large amount of solids, filtrate the solids out, then perform vacuum drying (the drying temperature is 35-40° C.), wherein the dried solids are namely the crystal form I. The output is 0.75 g, and the yield is 75%. The XRPD spectrum of the obtained crystal form I is identical to the XRPD spectrum of the crystal form I prepared in embodiment 2.
Embodiment 7: Preparation of Crystal Form I
Add 1 g of the compound of formula A into 5 mL of methylene chloride, completely dissolve under heating and refluxing conditions, then slowly cool the solution to the normal temperature, crystallize out solids, filtrate the solids out, and dry them at the normal temperature and pressure, wherein the dried solids are namely the crystal form I. The output is 0.68 g, and the yield is 68%. The XRPD spectrum thereof is shown in FIG. 13.
Embodiment 8: Preparation of Crystal Form I
Add 1 g of the compound of formula A into 10 mL of toluene, completely dissolve under heating and refluxing conditions, then slowly cool to the normal temperature, crystallize out solids, filtrate the solids out, and dry them at the normal temperature and the normal pressure, wherein the dried solids are namely the crystal form I. The output is 0.77 g, and the yield is 77%. The XRPD spectrum thereof is shown in FIG. 14.
Embodiment 9: Preparation of Crystal Form II
Add 0.7 g of the compound of formula A into a 18 mL crystallizer, then add 1.7 mL of ethanol thereinto, completely dissolve at 50° C., then add 1 mL of purified water thereinto, stop heating, cool naturally, volatilize the solvent, at the normal temperature and the normal pressure, and filtrate after a large amount of acicular crystals are crystallized out naturally. Place the solids at the normal temperature for drying around 24 h, thus obtaining the crystal form II, wherein the output is 0.54 g, and the yield is 77%.
The XRPD spectrum thereof is shown in FIG. 4.
The DSC spectrum is shown in FIG. 5. Display: the melting point is 152.14° C.
The infrared spectrum is shown in FIG. 6.
Embodiment 10: Preparation of Crystal Form II
Add 0.7 g of the compound of formula A into a 18 mL crystallizer, then add 1.7 mL of iso-propanol thereinto, completely dissolve at 50° C., then add 1 mL of purified water thereinto, stop heating, cool naturally, volatilize the solvent, at the normal temperature and the normal pressure, and filtrate after a large amount of acicular crystals are crystallized out naturally. Place the solids at the normal temperature for drying around 24 h, thus obtaining the crystal form II, wherein the output is 0.60 g, and the yield is 85%. The XRPD spectrum of the obtained crystal form II is identical to the XRPD spectrum of the crystal form II prepared in embodiment 9.
Embodiment 11: Preparation of Crystal Form II
Add 1 g of the compound of formula A into 10 mL of ethanol, completely dissolve at the normal temperature, then add 1 mL of distilled water thereinto, and volatilize the solvent at the normal temperature and the normal pressure. After the solvent is volatilized completely, the solids obtained are the crystals in form II. The XRPD spectrum of the obtained crystal form II is identical to the XRPD spectrum of the crystal form II prepared in embodiment 9.
Embodiment 12: Preparation of Crystal Form II
Weigh 1 g of the compound of formula A and add into a crystallizer, add 5 mL of anhydrous ethanol to stir, vibrate, and dissolve clearly, then add 1 mL of distilled water, wherein white casse is generated at the bottom of the flask. Shake the crystallizer until the casse disappears. Open the cover of the crystallizer to let the solvent volatilize naturally, thus obtaining the crystal form II. The XRPD spectrum of the obtained crystal form II is identical to the XRPD spectrum of the crystal form II prepared in embodiment 9.
Embodiment 13: Preparation of Crystal Form III
Add 0.4 g of the compound of formula A into the mixed solvent of 4 mL of tert-butyl alcohol and 0.3 mL of ethanol, completely dissolve at the normal temperature, then add 10 mL of distilled water thereinto, crystallize out solids, filtrate the solid out, and dry at the normal temperature and the normal pressure, wherein the obtained solids are the crystals in form III. The output is 0.3 g, and the yield is 75%.
The XRPD spectrum thereof is shown in FIG. 7.
The DSC spectrum is shown in FIG. 8. Display: the temperatures corresponding to three heat absorption peaks are respectively 105.94° C., 112.53° C., and 157.11° C.
The infrared spectrum is shown in FIG. 9.
Embodiment 14: Preparation of Crystal Form III
Add 0.2 g of the compound of formula A and 2 mL of t tert-butyl alcohol into a 20 mL crystallizer, stir, heat to 50° C., add 0.4 mL of ethanol, dissolve clearly, add 5 mL of distilled water all at once under the condition of 50° C., wherein a large amount of white solids crystallized out immediately. Pour the solid suspension from the crystallizer into a culture dish, and volatilize naturally, wherein the volatilizing time is no less than 24 h, thus obtaining the crystal form III. The XRPD spectrum of the obtained crystal form III is identical to the XRPD spectrum of the crystal form III prepared in embodiment 13.
Embodiment 15: Preparation of Crystal Form III
Add 1 g of the compound of formula A into a 25 mL crystallizer, then add 15 mL of tert-butyl alcohol thereinto, dissolve sufficiently at 50° C., then naturally cool to the room temperature, wherein solids are crystallized out. Filtrate the solid and dry at the normal temperature and the normal pressure, wherein the obtained solids are the crystals in form III. The output is 0.89 g, and the yield is 89%. The XRPD spectrum of the obtained crystal form III is identical to the XRPD spectrum of the crystal form III prepared in embodiment 13.
Embodiment 16: Preparation of the Amorphous Form
Add 1.0 g of the compound of formula A into a 18 mL crystallizer, then add 1 mL of ethanol thereinto, and dissolve sufficiently at 50° C. After the dissolving is completed, cool it to the room temperature, quickly add 5 mL of n-hexane thereinto under a quick stirring state to obtain vaporous slurry, take the suspended solids out, and lyophilize the solids for 1 hour, wherein an obtained white solids are in the amorphous form. The output is 0.92 g, and the yield is 92%.
The XRPD spectrum thereof is shown in FIG. 10.
The DSC spectrum is shown in FIG. 11.
The infrared spectrum is shown in FIG. 12.
Embodiment 17: Preparation of the Amorphous Form
Add 1.0 g of the compound of formula A into a 18 mL crystallizer, then add 1 mL of propanol thereinto, and dissolve sufficiently at 50° C. After the dissolving is completed, cool the solution to the room temperature, quickly add 5 mL of n-pentane thereinto under a quick stirring state to obtain a vaporous suspension, take the slurry out, and lyophilize the slurry for 1 hour, wherein the obtained white solids are in the amorphous form. The output is 0.96 g, and the yield is 96%. The XRPD spectrum of the obtained amorphous form is identical to the XRPD spectrum of the amorphous form prepared in embodiment 16.
Embodiment 18: Preparation of the Amorphous Form
Completely dissolve 1 g of the compound of formula A into 1 mL of ethanol at the normal temperature, then add 5 mL of n-hexane thereinto, mix sufficiently, and then lyophilize the slurry (placing a cold source at the temperature of −51° C. with the degree of vacuum of 208 Pa) for 40 min, wherein an obtained solids are namely in the amorphous form. The XRPD spectrum of the obtained amorphous form is identical to the XRPD spectrum of the amorphous form prepared in embodiment 16.
Embodiment 19: Preparation of the Amorphous Form
Weigh 1 g of the compound of formula A and put into a crystallizer, add 1 mL of anhydrous ethanol to stir, heat up to 50° C., dissolve clearly in 1 min, dropwise add two drops of DMF, then pour the suspension into a culture dish, add 5 mL of n-hexane, and shake evenly, then white casse is produced. Put the culture dish into a lyophilizer for lyophilizing, take the culture dish out, thus obtaining the amorphous form. The XRPD spectrum of the obtained amorphous form is identical to the XRPD spectrum of the amorphous form prepared in embodiment 16.
Stability Test Data
1. Long Term Stability Test
In accordance with the stability test guiding principles (Chinese Pharmacopoeia 2010 Edition 2—Annex XIX C), prepare the samples of the crystal form I of the compound of formula A described in embodiment 6 in a commercial package, place the samples in a thermotank at the temperature of 25° C.±2° C. with the relative humidity of 60%±10%, take each sample from the samples once at the ends of the third month, the sixth month and the ninth month respectively to detect the key stability inspection items of each sample and compare with the result of the sample at the zero day, wherein see Table 3 for the comparisons.







TABLE 3







Long term stability test results










Limitation
Time (month)












Inspection item
requirements
0
3
6
9





Character
White or off-white
Off-white
Off-white
Off-white
Off-white



crystalline powder
crystalline
crystalline
crystalline
crystalline




powder
powder
powder
powder


X-diffraction
Crystalline
Having
Consistent
Consistent with
Consistent



powder, consistent
crystalline
with that
that of zero
with that of



with that of zero

of zero month
month
zero month



month


Melting point
149-153° C.
149.8-152.1° C.
149.5-151.6° C.
149.3-151.2° C.
149.7-152.0° C.


Relevant
Diastereomer shall
Not detected
Not detected
Not detected
Not detected


substances
not exceed 1.0%



Single maximum
0.097
0.1013
0.0956
0.0940



unknown foreign



matter shall not



exceed 0.3%



Total foreign
0.342
0.3488
0.3551
0.3581



matter shall not



exceed 1.5%


Isomer
E-isomer shall not
/
/
/
0.0161



exceed 0.5%



Enantiomer shall
/
/
/
0.0587



not exceed 0.5%


Moisture
Moisture shall not
0.23
0.41
0.56
0.32



exceed 2.0%


Content
Based on
98.74
98.99
98.83
98.91



anhydride, the



content of



C25H21F2NO3 shall



be no less than



97.0%









Conclusion: after the stability test of nine month long term, various inspection indexes of the samples do not change apparently and all meet the specifications.
2. Accelerated Stability Test
In accordance with the stability test guiding principles (Chinese Pharmacopoeia 2010 Edition 2—Annex XIX C), prepare the sample of the crystal form I of the compound of formula A described in embodiment 6 in a commercial package, place the samples in a thermotank at the temperature of 40° C.±2° C. with the relative humidity of 75%±5%, with the duration of 6 months, take each sample once at the ends of the first month, the second month, the third month and the sixth month respectively to detect the key stability inspection items and compare with the results of the sample at the zero day, wherein see Table 4 for the comparisons.







TABLE 4







Accelerated stability test results









Inspection
Limitation
Time (month)













item
requirements
0
1
2
3
6





Character
White or off-
Off-
Off-
Off-white
Off-
Off-white



white crystalline
white
white
crystalline
white
crystalline



powder
crystalline
crystalline
powder
crystalline
powder




powder
powder

powder


X-diffraction
Crystalline
Having
/
/
Consistent
Consistent



powder,
crystalline


with that
with that



consistent with



of zero
of zero



that of zero



month
month



month


Melting point
149-153° C.
149.8-152.1° C.
149.3-151.5° C.
149.7-152.1° C.
149.7-152.1° C.
149.5-151.9° C.


Relevant
Diastereomer
Not
0.0128
Not detected
Not detected
Not detected


substances
shall not
detected



exceed 1.0%



Single
0.097
0.1004
0.0856
0.0983
0.2017



maximum



unknown



foreign matter



shall not exceed



0.3%



Total foreign
0.342
0.3757
0.3650
0.3717
1.0828



matter shall not



exceed 1.5%


Moisture
Moisture shall
0.20
0.30
0.28
0.37
0.44



not exceed 2.0%


Content
Based on
98.71
99.11
98.82
98.78
97.97



anhydride, the



content of



C25H21F2NO3



shall be no less



than 97.0%









Conclusion: the accelerated stability test results indicate that the relevant substances are grown significantly in the sixth month and the content is reduced at a certain level; however, both of the two are within the acceptable range, and other indexes are substantially not changed apparently.
Those skilled in the art can understand that some modifications or alternations may be made to the present invention under the teaching of this specification. These modifications and alternations shall also fall within the scope defined in the claims of the present invention.

## Claims

1. The crystal form II of the compound of formula A structured as follows,



wherein an X-ray powder diffraction spectrum radiated by Cu-Kα and characterized in degrees 2θ has following characteristic peaks at 5.20±0.20°, 7.46±0.20°, 8.70±0.20°, 10.60±0.20°, 11.84±0.20°, 17.14±0.20°, 20.36±0.20° and 26.48±0.20°.

2. The crystal form II according to claim 1, wherein the X-ray powder diffraction spectrum characterized in degrees 2θ further has following characteristic peaks at: 6.29±0.20°, 6.96±0.20°, 12.82±0.20°, 13.28±0.20°, 14.21±0.20°, 15.02±0.20°, 15.40±0.20°, 16.40±0.20°, 17.61±0.20°, 18.34±0.20°, 18.62±0.20°, 19.06±0.20°, 19.58±0.20°, 20.02±0.20°, 21.50±0.20°, 22.18±0.20°, 22.74±0.20°, 23.41±0.20, 24.06±0.20°, 24.34±0.20°, 24.70±0.20°, 25.16±0.20°, 27.34±0.20°, 28.00±0.20°, 28.88±0.20°, 29.36±0.20°, 31.39±0.20°, 32.02±0.20°, 32.60±0.20° and 34.42±0.20°.

3. A preparation method of the crystal form II of the compound of formula A according to claim 1, comprising the following steps of:
1) adding the compound of formula A into C1-C3 alcohol, optionally heating to dissolve the compound and obtain a solution, wherein the ratio of the mass of the compound of formula A in g to the volume of the C1-C3 alcohol in mL is 1:2-10;
2) adding water into the foregoing solution and ensuring that no crystal is crystallized out; and
3) naturally volatilizing the solvent at the normal temperature and the normal pressure, and collecting solids that are crystallized out, thus obtaining the crystals in form II.

4. The preparation method according to claim 3, wherein the C1-C3 alcohol is ethanol or iso-propanol.

5. The preparation method according to claim 3, wherein the ratio of the volume of the water added in step 2) to the volume of the C1-C3 alcohol is 10:1-6.

6. The preparation method according to claim 3, wherein the ratio of the mass of the compound of formula A in g to the volume of the C1-C3 alcohol in mL in step 1) is 1:2-5.

7. The preparation method according to claim 6, comprising the following steps of: adding the compound of formula A in g into a crystallizer, then adding ethanol in mL thereinto, heating to 50° C. for complete dissolving, then adding water in mL thereinto, stopping heating, cooling naturally, volatilizing the solvent at the normal temperature and pressure, crystallizing out crystals, filtrating, and drying the crystals at the normal temperature to obtain the crystals in form II, wherein the ratio of the mass of the compound of formula A in g to the volume of the ethanol in mL to the volume of the water in mL is 0.7:1.7:1.

8. A pharmaceutical composition, comprising a therapeutically effective amount of the crystal form II according to claim 1.

9. A method for reducing plasma cholesterol contents, comprising the steps of administering to a sufferer in need the therapeutically effective amount of the crystal form II according to claim 1.

10. A method for reducing plasma cholesterol contents, comprising the steps of administering to a sufferer in need the therapeutically effective amount of the pharmaceutical composition according to claim 8.

11. The preparation method according to claim 3, wherein the ratio of the volume of the water added in step 2) to the volume of the C1-C3 alcohol is 10:2.