Ammonium carboxylate compound, crystalline form, amorphous form and preparation method thereof

Abstract

The present disclosure belongs to the field of chemical synthesis, and in particular relates to an ammonium carboxylate compound, a crystalline form and an amorphous form, and a preparation method thereof. The present disclosure prepares the compound and the crystalline form I and its single crystal, amorphous form and crystalline form II thereof. The compound, the crystalline forms, the single crystal and the amorphous form can stably exist and exhibit good solid forms, suitable for medicine-making. Furthermore, these products possess high purity and less single impurity. Moreover, the preparation methods of the present disclosure are easy to implement due to the simple processes with mild reaction conditions, and could produce products of high yield and high purity without complex purification steps. Furthermore, the preparation methods may facilitate safety, environmental protection, and industrial production.

Claims

1. A crystalline form I of the compound of formula (A): ##STR00016## having an X-ray powder diffraction pattern with characteristic peaks at 2θ values of 10.04°±0.20°, 16.66°±0.20°, and 21.89°±0.20°.

2. A preparation method MA of the crystalline form I of the compound of formula (A) according to claim 1, comprising the following steps: ##STR00017## and 4) dissolving the compound of formula (A) in an organic solvent, cooling the solvent to form the crystalline form I of the compound of formula (A), wherein, in step 1), 5-(biphenyl-4-yl)-4-[(tert-butoxycarbonyl)amino]-2-methylpentanoic acid is reacted with ethanol and thionyl chloride to produce ethyl 5-([1, 1-biphenyl)-4-amino-2-methylpentanoate hydrochloride; in step 2), ethyl 5-([1,1-biphenyl)-4-amino-2-methylpentanoate hydrochloride is reacted with succinic anhydride in the presence of a base to produce ethyl 5-(biphenyl-4-yl)-4-[(3-carboxypropionyl) amino]-2-methylpentanoate; in step 3), ethyl 5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate is reacted with ammonia water or ammonia gas to produce the compound of formula (A); and in step 4), the organic solvent is selected from alcohol solvents, ketone solvents, ether solvents, nitrile solvents, ester solvents, hydrocarbon solvents, halogenated hydrocarbon solvents, and mixtures thereof.

3. The crystalline form I of the compound of formula (A) according to claim 1 having an X-ray powder diffraction pattern with characteristic peaks at 2θ values of 10.04°±0.20°, 14.51°±0.20°, 16.66°±0.20°, 17.60°±0.20°, 20.47°±0.20°, 21.89°±0.20°, 24.70°±0.20°, 26.31°±0.20°, and 29.35°±0.20°.

4. A preparation method of the crystalline form I according to claim 1, comprising one or more of the following methods M1, M2 and M3: wherein the preparation method M1 comprises the following steps: mixing the compound of formula (A) with an organic solvent to form a suspension, filtering and drying to obtain the crystalline form I according to claim 1, wherein the preparation method M2 comprises the following steps: dissolving the compound of formula (A) in a good solvent, then adding a poor solvent, filtering, and drying to give obtain the crystalline form I according to claim 1, and wherein the preparation method M3 comprises the following steps: mixing the compound of formula (A) with an organic solvent, heating to dissolve, cooling to crystallize, filtering, and drying to obtain the crystalline form I according to claim 1.

5. A single crystal of the crystalline form I of the compound of formula (A) according to claim 1, wherein the crystal structure of which is monoclinic, with the space group of P2.sub.1, the unit cell parameters of a=12.382 (8) Å, b=6.126 (4) Å, c=15.883 (10) Å, a=γ=90°, and β=102.35 (4)°.

6. A preparation method of the single crystal according to claim 5, comprising the following steps: D1) dissolving the compound of formula (A) in a good solvent to obtain a solution of the compound of formula (A); and D2) placing the solution obtained in the step D1) in a vapor of a poor solvent to obtain a single crystal.

7. An amorphous form of the compound of formula (A) ##STR00018## having an X-ray powder diffraction pattern substantially as shown in FIG. 10.

8. A preparation method MW of the amorphous form according to claim 7, comprising the following steps: volatilizing or evaporating the solution of the compound of formula (A) to dryness to remove the solvent, optionally subjected or not subjected to milling, so as to obtain the amorphous form.

9. A crystalline form II of the compound of formula (A), ##STR00019## having an X-ray powder diffraction pattern with characteristic peaks at 2θ values: 16.68°±0.20°, 19.59°±0.20°, 21.91°±0.20°.

10. A preparation method M4 of the crystalline form II according to claim 9, comprising the following steps: mixing the compound of formula (A) with an organic solvent to form a suspension, filtering and drying to obtain the crystalline form II of the compound of formula (A); wherein the organic solvent is n-hexane, petroleum ether, or mixtures thereof.

11. A pharmaceutical composition, comprising a therapeutically effective amount of the crystalline form I of the compound of formula (A) according to claim 1 as an active ingredient and a pharmaceutically acceptable excipient.

12. A pharmaceutical composition, comprising the crystalline form I of the compound of formula (A) according to claim 1 and the compound of formula (B): ##STR00020##

13. The crystalline form I of the compound of formula (A) according to claim 1 having an X-ray powder diffraction pattern with characteristic peaks at 2θ values of 5.58°±0.20°, 7.21°±0.20°, 10.04°±0.20°, 12.06°±0.20°, 14.51°±0.20°, 15.44°±0.20°, 16.12°±0.20°, 16.66°±0.20°, 16.98°±0.20°, 17.60°±0.20°, 18.34°±0.20°, 18.84°±0.20°, 19.95°±0.20°, 20.27°±0.20°, 20.47°±0.20°, 21.89°±0.20°, 22.39°±0.20°, 22.77°±0.20°, 23.79°±0.20°, 24.70°±0.20°, 24.98°±0.20°, 25.61°±0.20°, 26.31°±0.20°, 26.80°±0.20°, 27.97°±0.20°, 28.69°±0.20°, 29.35°±0.20°, 30.12°±0.20°, 30.66°±0.20°, 32.31°±0.20°, 35.12°±0.20°, 36.82°±0.20°, and 39.17°±0.20°.

14. The crystalline form I of the compound of formula (A) according to claim 1 having an X-ray powder diffraction pattern as shown in FIG. 1.

15. The preparation method according to claim 4, wherein the preparation method M1 comprises stirring the compound of formula (A) and the organic solvent at 15° C. to 60° C. for equilibrium for 24 to 48 h filtering, and drying the solid filtride to obtain the crystalline form I according to claim 1, wherein the organic solvent is selected from alcohol solvents, ketone solvents, ether solvents, nitrile solvents, ester solvents, hydrocarbon solvents, halogenated hydrocarbon solvents, and mixtures thereof, and wherein a ratio of the mass (g) of the compound of formula (A) to a total volume (L) of the organic solvent ranges from 1:1 to 50:1.

16. The preparation method according to claim 4, wherein the preparation method M2 comprises dissolving the compound of formula (A) in a good solvent at 5-30° C., adding a poor solvent, allowing the mixture to stand at 5° C. to 30° C. for 1 to 10 days, filtering, and drying the solid filtride to obtain the crystalline form I according to claim 1, wherein the good solvent is selected from methanol, ethanol, isopropanol, dichloromethane, chloroform, acetone, methyl ethyl ketone and mixtures thereof, wherein the poor solvent is selected from n-hexane, diethyl ether, petroleum ether and mixtures thereof, and wherein a ratio of the mass (g) of the compound of formula (A) to a total volume (L) of the good solvent ranges from 5:1 to 50:1.

17. The preparation method according to claim 4, wherein, in the preparation method M3 the organic solvent is selected from alcohol solvents, ketone solvents, ether solvents, nitrile solvents, ester solvents, hydrocarbon solvents, halogenated hydrocarbon solvents, and mixtures thereof, and wherein a ratio of the mass (g) of the compound of formula (A) to a total volume (L) of the organic solvent ranges from 1:1 to 50:1.

18. The preparation method of the single crystal according to claim 6, wherein the good solvent is methanol or ethanol, the poor solvent is hexane or diethyl ether, and the solution obtained in step (D1) and the vapor of a poor solvent are placed in a container sealed from air.

19. The preparation method of the single crystal according to claim 6, wherein a mass ratio (g) of the compound of formula (A) to a total volume (L) of the poor solvent ranges from 200:1 to 10:1.

20. The crystalline form II of the compound of formula (A) according to claim 9 having an X-ray powder diffraction pattern with characteristic peaks at 2θ values of 6.07°±0.20°, 14.52°±0.20°, 16.68°±0.20°, 19.59°±0.20°, 21.91°±0.20°, and 29.37°±0.20°.

21. The crystalline form II of the compound of formula (A) according to claim 9 having an X-ray powder diffraction pattern with characteristic peaks at 20 values: 6.07°±0.20°, 7.07°±0.20°, 10.05°±0.20°, 14.52°±0.20°, 16.68°.±0.20°, 19.59°±0.20°, 21.91°±0.20°, and 29.37°±0.20°.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an X-ray powder diffraction (XRPD) pattern of the crystalline form I of the present disclosure.

(2) FIG. 2 is a differential scanning calorimetry (DSC) chart of the crystalline form I of the present disclosure.

(3) FIG. 3 is a graph showing the thermogravimetric analysis (TG) of the crystalline form I of the present disclosure.

(4) FIG. 4 is an infrared spectrum (IR) diagram of the crystalline form I of the present disclosure.

(5) FIG. 5 is a Raman spectrum of the crystalline form I of the present disclosure.

(6) FIG. 6 is a graph showing the hygroscopicity analysis (DVS) of the crystalline form I of the present disclosure.

(7) FIG. 7 is a polarized photograph of the crystalline form I of the present disclosure.

(8) FIG. 8 is a view showing the crystal structure of the single crystal of crystalline form I of the present disclosure.

(9) FIG. 9 is an interaction diagram of the single crystal of the crystalline form I of the present disclosure.

(10) FIG. 10 is an X-ray powder diffraction (XRPD) pattern of the amorphous form of the present disclosure.

(11) FIG. 11 is a differential scanning calorimetry (DSC) chart of the amorphous form of the present disclosure.

(12) FIG. 12 is a graph showing the thermogravimetric analysis (TG) of the amorphous form of the present disclosure.

(13) FIG. 13 is an infrared spectrum (IR) image of the amorphous form of the present disclosure.

(14) FIG. 14 is a Raman spectrum of the amorphous form of the present disclosure.

(15) FIG. 15 is a graph showing the hygroscopicity analysis (DVS) of the amorphous form of the present disclosure.

(16) FIG. 16 is a polarized photograph of an amorphous form of the present disclosure.

(17) FIG. 17 is an X-ray powder diffraction (XRPD) pattern of the crystalline form II of the present disclosure.

(18) FIG. 18 is a differential scanning calorimetry (DSC) pattern of the crystalline form II of the present disclosure.

(19) FIG. 19 is a graph showing the thermogravimetric analysis (TG) of the crystalline form II of the present disclosure.

(20) FIG. 20 is an infrared spectrum (IR) of the crystalline form II of the present disclosure.

(21) FIG. 21 is a Raman spectrum of the crystalline form II of the present disclosure.

(22) FIG. 22 is a graph showing the hygroscopicity analysis (DVS) of the crystalline form II of the present disclosure.

(23) FIG. 23 is a polarized photograph of the crystalline form II of the present disclosure.

(24) FIG. 24 is a carbon spectrum of the compound (A) prepared in example 1 of the present disclosure.

(25) FIG. 25 is a hydrogen spectrum of the compound (A) prepared in example 1 of the present disclosure.

(26) FIG. 26 is a graph showing the hygroscopicity analysis of the compound (A) prepared in example 1 of the present disclosure.

DETAILED DESCRIPTION

(27) The compounds of the general formula of the present disclosure, as well as the preparation methods and applications thereof, will be further described in detail below in conjunction with specific examples. The following examples are merely illustrative of the disclosure and are not to be construed as limiting the scope of the present disclosure. The technology implemented based on the above-described contents of the present disclosure is encompassed within the scope of the present disclosure.

(28) The starting materials and reagents used in the following examples are commercially available or can be prepared by known methods unless otherwise stated.

(29) Testing Instruments and Testing Methods

(30) TGA method: instrument model: Netzsch TG 209F3, temperature range: 30-400° C., scanning rate: 10 K/min, purge gas: 25 mL/min; protective gas: 15 mL/min.

(31) DSC method: instrument model: Perkin Elmer DSC 1200, temperature range: −40-400° C., scanning rate: 10° C./min, nitrogen flow rate: 50 ml/min.

(32) XRPD method: instrument model: Bruker D8 advance, target: Cu Kα (40 kV, 40 mA), distance of samples to the detector: 30 cm; scanning range: 3°-40° (2θ value), scanning step: 0.05 s.

(33) IR method: resolution 4.0, KBr pellets.

(34) DVS detection of the ammonium salt: instrument model: TA Q5000SA, balance at 25° C., humidity start from 0.00%, balance for 180 min, balance for 15 min when the weight gain is less than 0.01%, 10% gradient humidity increase from 0.00% to 90.00%, each gradient maintained for 120 min; a 10% gradient humidity reduction procedure performed when the humidity is from 90.00% to 0.00%, each gradient maintained for 120 min.

(35) DVS detection of other products: instrument model: SMS DVS Intrinsic, 0-95% RH, temperature: 25° C.

Example 1 Synthesis of the Compound of Formula (A)

1.1 Synthesis of ethyl (2R,4S)-5-([1,1-biphenyl)-4-amino-2-methylpentanoate hydrochloride

(36) ##STR00006##

(37) (2R,4S)-5-(biphenyl-4-yl)-4-[(tert-butoxycarbonyl)amino]-2-methylpentanoic acid (50 g, 0.13 mol) and absolute ethanol (500 ml) were added to a 1 L three-necked flask equipped with a magnetic stirrer and a condenser tube, dissolved by stirring at room temperature, added dropwise with thionyl chloride (23.3 g, 0.195 mol). After the addition was completed, the reaction was carried out for 3-3.5 h when the temperature was raised to 50-60° C. The reaction mixture was cooled and concentrated to dryness under reduced pressure. The residue was washed with ethyl acetate, with not more than 15% residual ethanol detected by GC, then added with ethyl acetate (500 ml), stirred at room temperature to a slurry for 3 h, filtered and dried to give ethyl (2R, 4S)-5-([1,1-biphenyl)-4-amino-2-methylpentanoate hydrochloride, with a yield of 88%-96% and a purity higher than 98.5%.

1.2 Synthesis of ethyl (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate

(38) ##STR00007##

(39) Ethyl (2R,4S)-5-([1,1-biphenyl)-4-amino-2-methylpentanoate hydrochloride (43.6 g, 0.125 mol) and pyridine (87.2 ml) were added to a 250 ml three-necked flask equipped with a magnetic stirrer and a condenser tube, dissolved by stirring at room temperature; added with succinic anhydride (18.7 g, 0.186 mol) at room temperature, and after the addition reacted for 1-1.5 h when the temperature was raised to 60-70° C., cooled, and concentrated under reduced pressure until pyridine-free, dissolved with ethyl acetate (515 ml), adjusted to pH 1-2 with 2N hydrochloric acid, stirred for 20-30 min, allowed to stand for 10-15 min, and separated to different liquid phases; The organic phase was acidified with 0.1N hydrochloric acid (515 ml), washed successively with saturated aqueous sodium chloride (515 ml), water (515 ml) and separated to different liquid phases, dried over anhydrous magnesium sulfate, filtered, and concentrated to dryness under reduced pressure, added with absolute ethanol (103 ml), concentrated to dryness, then added with acetone (256 ml), concentrated to dryness at 40-45° C. to give a crude product of 52.4 g ethyl (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate, with a yield of 95%-105% and purity higher than 97.0%.

1.3 Synthesis of ammonium 4(((2S, 4R)-1-([1, l′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl) amino)-4-oxobutanoate

(40) ##STR00008##

(41) The crude product of ethyl (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate (52.4 g, 0.125 mol) and acetone (629 ml) were added to a 1 L three-necked flask equipped with a mechanical stirrer, dissolved by stirring at room temperature; cooled to 0-10° C. for 5-10 min, added dropwise with concentrated ammonia water (21.3 g, 0.313 mol), after the addition, kept stirring for 4 h and filtered. The filter cake was washed with acetone (63 ml), vacuum dried at 40-50° C., −0.09-0.1 MPa for 6-8 h to give 37.7 g solid of ammonium 4-((2S,4R)-1-([1, l′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoate, the compound of formula (A), with a yield of 70%-75% and a purity of more than 99.5%. MS: in/z=412.3 (M+H).sup.+. The nuclear magnetic carbon spectrum and hydrogen spectrum of the product are shown in FIG. 24 and FIG. 25. The compound of formula (A) was used as a raw material for the following experiments unless otherwise stated.

Example 2 Synthesis of ammonium 4(((2S,4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-ethyl)amino)-4-oxobutanoate

(42) ##STR00009##

(43) The crude product of ethyl (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate (60.0 g, 0.145 mol) obtained by the method of step 1.2 of example 1 and acetone (720 ml) were added to a 1 L three-necked flask equipped with a mechanical stirrer, dissolved by stirring at room temperature; cooled to 0-10° C. for 5-10 min, added dropwise with concentrated ammonia water (24.4 g, 0.358 mol), after the addition, kept stirring for 4 h and filtered. The filter cake was washed with acetone (72 ml), vacuum dried at 10-30° C., −0.09 to −0.1 MPa for 4-6 h to give 46.86 g solid of ammonium 4-((2S, 4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoate, the compound of formula (A), with a yield of 70%-75% and a purity higher than 99.5%. The process repeatability of the procedure is higher than that of step 1.3 and the energy consumption can be reduced under room temperature heating.

Example 3 Preparation of Form I of Compound of Formula (A)

(44) 3.1 20 mg compound of formula (A) was weighted each time, placed in a glass bottle, then respectively added with acetonitrile (1 ml), tetrahydrofuran (1 ml), nitromethane (1 ml), ethyl acetate (1 ml), methyl t-butyl ether (1 ml), methyl isobutyl ketone (1 ml), n-heptane (1 ml), diethyl ether (1 ml), dichloromethane (1 ml), chloroform (1 ml), isopropyl acetate (1 ml), chloroform/methyl tert-butyl ether (500 μl/500 μl), isopropyl acetate/methyl tert-butyl ether (500 μl/500 μl), dichloromethane/toluene (500 μl/500 μl), acetonitrile/n-hexane (500 μl/500 μl), nitromethane/n-hexane (500 μl/500 μl), ethyl acetate/n-heptane (500 μl/500 μl), methyl isobutyl ketone/n-heptane (500 μl/500 μl), ethyl acetate/diethyl ether (500 μl/500 μl), ethyl acetate/petroleum ether (500 μl/500 μl), dichloromethane/petroleum ether (500 μl/500 μl), stirred for equilibrium at 25° C. for at least 24 h, and filtered. The obtained solid was dried in air for 10 min. 3.2 20 mg compound of formula (A) was weighted each time, placed in a glass bottle, then respectively added with acetonitrile (1 ml), tetrahydrofuran (1 ml), nitromethane (1 ml), ethyl acetate (1 ml), methyl t-butyl ether (1 ml), methyl isobutyl ketone (1 ml), n-heptane (1 ml), diethyl ether (1 ml), dichloromethane (1 ml), chloroform (1 ml), isopropyl acetate (1 ml), chloroform/methyl tert-butyl ether (500 μl/500 μl), isopropyl acetate/methyl tert-butyl ether (500 μl/500 μl), dichloromethane/toluene (500 μl/500 acetonitrile/n-hexane (500 μl/500 μl), nitromethane/n-hexane (500 μl/500 μl), ethyl acetate/n-heptane (500 μl/500 μl), methyl isobutyl ketone/n-heptane (500 μl/500 μl), ethyl acetate/diethyl ether (500 μl/500 μl), ethyl acetate/petroleum ether (500 μl/500 μl), dichloromethane/petroleum ether (500 μl/500 μl), stirred for equilibrium at 50° C. for at least 24 h, and filtered. The obtained solid was dried in air for 10 min.

(45) 3.3 3 mg compound of formula (A) was weighted each time, placed in a glass bottle, then respectively added with ethanol (200 μl), isopropanol (200 μl), dichloromethane (200 μl), chloroform (200 μl), dissolved by stirring at room temperature, when completely dissolved, added with 6 ml n-hexane separately, allowed to stand at room temperature for one week, and filtered. The obtained solid was dried in air for 10 min.

(46) 3.4 3 mg compound of formula (A) was weighted each time, placed in a glass bottle, then respectively added with ethanol (200 μl), isopropanol (200 μl), dichloromethane (200 μl), chloroform (200 μl), dissolved by stirring at room temperature, when completely dissolved, added with 6 ml petroleum ether separately, allowed to stand at room temperature for one week, and filtered. The obtained solid was dried in air for 10 min.

(47) 3.5 3 mg compound of formula (A) was weighted each time, placed in a glass bottle, then respectively added with methanol (200 μl), ethanol (200 μl), isopropanol (200 μl), acetone (200 μl), methyl ethyl ketone (200 μl), dichloromethane (200 μl), chloroform (200 μl), dissolved by stirring at room temperature, when completely dissolved, added with 6 ml ethyl ether separately, allowed to stand at room temperature for one week, and filtered. The obtained solid was dried in air for 10 min.

(48) 3.6 3 mg compound of formula (A) was weighted each time, placed in a glass bottle, then respectively added with acetonitrile (10 ml), ethyl acetate (10 ml), tetrahydrofuran (10 ml), acetone (10 ml), methyl tert-butyl ether (20 ml), methyl isobutyl ketone (10 ml), dichloromethane (10 ml), chloroform (5 ml), nitromethane (10 ml), heated to reflux and dissolved under stirring, cooled to room temperature, and filtered. The obtained solid was dried in air for 10 min.

(49) These experiments testified that the products prepared by the above methods were all crystalline form I.

Example 4 Preparation of the Single Crystal of the Crystalline Form I of the Compound of Formula (A)

(50) About 50 mg compound of formula (A) was dissolved in 0.5 mL ethanol, then added to a large glass bottle containing 4 mL ether, sealed with a parafilm, and placed the bottle in a larger bottle to allow the ether continuously evaporate into the ethanol solution. The mixture was allowed to stand at room temperature. Two days later, a colorless columnar single crystal was obtained, the crystal structure and interaction of which are shown in FIG. 8 and FIG. 9.

Example 5 X-Ray Powder Diffraction Test of the Crystalline Form I of the Compound of Formula (A)

(51) The crystalline form I of the compound of formula (A) prepared in example 3 was ground into powder, and the powder diffraction test was carried out by an X-ray diffractometer. The X-ray powder diffraction pattern of the crystalline form I of the compound of formula (A) is shown in FIG. 1.

(52) The specific data of crystal parameters of the crystalline form I of formula (A) such as diffraction angle (2θ) and relative intensity (%), is shown in Table 1 below:

(53) TABLE-US-00001 TABLE 1 X-ray powder diffraction (XRPD) data of the crystalline form I 2θ Relative intensity (%) 5.58 5 7.21 4 10.04 51.5 12.06 10.7 14.51 17.6 15.44 4.6 16.12 9.6 16.66 69.3 16.98 14.1 17.60 16.4 18.34 4.2 18.84 4.6 19.95 4.7 20.27 16.6 20.47 27 21.89 100 22.39 18.4 22.77 8.9 23.79 6 24.70 28.3 24.98 5.5 25.61 3.8 26.31 20.9 26.80 12 27.97 5.7 28.69 5 29.35 18.1 30.12 3.3 30.66 5.2 32.31 7.3 35.12 4.7 36.82 4.7 39.17 4.3

Example 6 Preparation of the Amorphous Form

(54) 1) 24 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol (400 μl), ethanol (400 μl), isopropanol (400 μl), acetone (400 μl), acetonitrile (400 μl), tetrahydrofuran (400 μl), nitromethane (400 μl), ethyl acetate (400 μl), methyl isobutyl ketone (400 μl), dichloromethane (400 μl), chloroform (400 μl), isopropyl acetate (400 μl), mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(55) 2) 24 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol/water (400 μl/200 μl), ethanol/water (400 μl/200 μl), isopropanol/water (400 μl/200 μl), acetone/water (400 μl/200 μl), acetonitrile/water (400 μl/200 μl), tetrahydrofuran/water (400 μl/200 μl), nitromethane/water/ethanol (400 μl/200 μl/200 μl), ethyl acetate/water/ethanol (400 μl/200 μl/200 μl), methyl isobutyl ketone/water/ethanol (400 μl/200 μl/200 μl), dichloromethane/water/ethanol (400 μl/200 μl/200 μl), chloroform/water/ethanol (400 μl/200 μl/200 μl), isopropyl acetate/water/ethanol (400 μl/200 μl/200 μl), mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(56) 3) 20 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol/methyl tert-butyl ether (400 μl/400 μl), ethanol/methyl tert-butyl ether (400 μl/400 μl), isopropanol/methyl tert-butyl ether (400 μl/400 μl), acetone/methyl tert-butyl ether (400 μl/400 μl), acetonitrile/methyl tert-butyl ether (400 μl/400 μl), tetrahydrofuran/methyl tert-butyl ether (400 μl/400 μl), nitromethane/methyl tert-butyl ether (400 μl/400 μl), ethyl acetate/methyl tert-butyl ether (400 μl/400 μl), methyl isobutyl ketone/methyl tert-butyl ether (400 μl/400 μl), dichloromethane/methyl tert-butyl ether (400 μl/400 μl), mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(57) 4) 22 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol/toluene (400 μl/400 μl), ethanol/toluene (400 μl/400 μl), isopropanol/toluene (400 μl/400 μl), acetone/toluene (400 μl/400 μl), acetonitrile/toluene (400 μl/400 μl), tetrahydrofuran/toluene (400 μl/400 μl), nitromethane/toluene (400 μl/400 μl), ethyl acetate/toluene (400 μl/400 μl), methyl isobutyl ketone/toluene (400 μl/400 μl), chloroform/toluene (400 μl/400 μl), isopropyl acetate/toluene (400 μl/400 μl), mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(58) 5) 18 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol/n-hexane (400 μl/400 μl), ethanol/n-hexane (400 μl/400 μl), isopropanal/n-hexane (400 μl/400 μl), acetone/n-hexane (400 μl/400 μl), tetrahydrofuran/n-hexane (400 μl/400 μl)), nitromethane/n-hexane (400 μl/400 μl), ethyl acetate/n-hexane (400 μl/400 μl), methyl isobutyl ketone/n-hexane (400 μl/400 μl), isopropyl acetate/n-hexane (400 μl)/400 μl, mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(59) 6) 20 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol/n-heptane (400 μl/400 μl), ethanol/n-heptane (400 μl/400 μl), isopropanol/n-heptane (400 μl/400 μl), acetone/n-heptane (400 μl/400 μl), acetonitrile/n-heptane (400 μl/400 μl), tetrahydrofuran/n-heptane (400 μl/400 μl), nitromethane/n-heptane (400 μl/400 μl), dichloromethane/n-heptane (400 μl/400 μl), chloroform/n-heptane (4000/4000), isopropyl acetate/n-heptane (400 μl/400 μl), mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(60) 7) 18 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol/diethyl ether (400 μl/400 μl), ethanol/diethyl ether (400 μl/400 μl), isopropanol/diethyl ether (400 μl/400 μl), acetone/diethyl ether (400 μl/400 μl), acetonitrile/diethyl ether (400 μl/400 μl), tetrahydrofuran/diethyl ether (400 μl/400 μl), nitromethane/diethyl ether (400 μl/400 μl), methyl isobutyl ketone/diethyl ether (400 μl/400 μl), isopropyl acetate/diethyl ether (400 μl/400 μl), mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(61) 8) 20 portions of the compound of formula (A) (3 mg each portion) were weighted, added to glass bottles respectively and divided into two groups. Each portion in each group was respectively added with methanol/petroleum ether (400 μl/400 μl), ethanol/petroleum ether (400 μl/400 μl), isopropanol/petroleum ether (400 μl/400 μl), acetone/petroleum ether (400 μl/400 μl), acetonitrile/petroleum ether (400 μl/400 μl), tetrahydrofuran/petroleum ether (400 μl/400 μl), nitromethane/petroleum ether (400 μl/400 μl), methyl isobutyl ketone/petroleum ether (400 μl/400 μl), chloroform/petroleum ether (400 μl/400 μl), isopropyl acetate/petroleum ether (400 μl/400 μl), mixed and dissolved. The two groups were respectively placed at 25° C. and 50° C. to slowly evaporate to dryness.

(62) These experiments testified that the products prepared by the above methods were all amorphous form.

Example 7 Preparation of the Crystalline Form II of the Compound

(63) 1) 20 mg compound of formula (A) was weighted each time, added to a glass bottle, and added with 1 ml n-hexane and 1 ml petroleum ether respectively to form a suspension, stirred for equilibrium at 25° C. for at least 24 h, and filtered. The obtained solid was dried in air for 10 min.

(64) 2) 20 mg compound of formula (A) was weighted each time, added to a glass bottle, and added with 1 ml n-hexane and 1 ml petroleum ether respectively to form a suspension, stirred for equilibrium at 50° C. for at least 24 h, and filtered. The obtained solid was dried in air for 10 min.

(65) The resulting products were all crystalline form II.

Example 8 X-Ray Powder Diffraction Test

(66) The obtained crystalline form II was ground into powder, and the powder diffraction test was carried out by an X-ray diffractometer. The X-ray powder diffraction pattern of the crystalline form II of the compound of formula (A) is shown in FIG. 16.

(67) The specific data of crystal parameters of the crystalline form II such as diffraction angle (2θ) and relative intensity (%), is shown in Table 2 below:

(68) TABLE-US-00002 TABLE 2 X-ray powder diffraction (XRPD) data for the crystalline form II 2θ Relative intensity (%) 5.79 4 6.07 14.8 7.07 7.8 10.05 7.6 11.46 3.8 11.74 3.9 11.96 5.6 12.28 3.7 13.45 5.2 14.52 16.7 15.09 9.1 16.16 8.2 16.68 41.5 17.85 1.9 19.59 33.6 20.29 2.9 21.42 2.6 21.91 100 22.76 2.5 23.79 5.4 24.72 2 24.91 2.4 25.87 2.4 26.80 1.9 29.37 16.7

Comparative example 1 Synthesis of potassium 4(((2S,4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoate

(69) ##STR00010##

(70) A crude product of ethyl (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate (52.4 g, 0.125 mol) and acetone (629 ml) were added to a 1 L three-necked flask equipped with a mechanical stirrer, stirred and dissolved at room temperature; cooled to 0-10° C. for 5-10 min, added dropwise with potassium hydroxide solution (25 ml, 5 mol/L), after the addition was completed, kept stirring for 1 h, concentrated to dryness, added with methanol (125 ml) or ethanol (125 ml), concentrated to dryness to give potassium 4((2S,4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoate (AHU-377 potassium salt). The product failed to exhibit a good solid form and is highly hygroscopic.

Comparative example 2 Synthesis of sodium 4-(((2S,4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutyrate

(71) ##STR00011##

(72) A crude product of ethyl (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate (52.4 g, 0.125 mol) and acetone (629 ml) were added to a 1 L three-necked flask equipped with a mechanical stirrer, stirred and dissolved at room temperature; cooled to 0-10° C. for 5-10 min and added dropwise with sodium hydroxide solution (25 ml, 5 mol/L), after the addition was completed, kept stirring for 1 h, concentrated to dryness, added with methanol (125 ml) or ethanol (125 ml), concentrated to dryness to give sodium 4-(((2S,4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutyrate (AHU-377 sodium salt). The product also does not exhibit a good solid form, and is highly hygroscopic despite less hygroscopic than potassium salt.

Comparative Example 3 Synthesis of Organic Ammonium Salt of 4-(((2S,4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoic acid

(73) ##STR00012##

(74) A crude product of ethyl (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoate (52.4 g, 0.125 mol) and acetone (629 ml) were added to a 1 L three-necked flask equipped with a mechanical stirrer, stirred and dissolved at room temperature: cooled to 0-10° C. for 5-10 min and added dropwise with a basic ammonium salt such as choline (15 g, 0.125 mol), after the addition was completed, kept stirring for 1 h, concentrated to dryness, added with methanol (125 ml) or ethanol (125 ml), and concentrated to dryness. Since high hygroscopicity, the organic ammonium salt of 4 ((2S,4R)-1-([1,1′-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoic acid cannot be obtained in a solid form.

(75) The product results obtained above turn out that the compound of formula (A) of the present disclosure is more suitable for process synthesis, compared to AHU-377 potassium salt, AHU-377 sodium salt and the organic ammonium salt of 4-(((2S,4R)-1-([1,1′-biphenyl) 4-amino)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoic acid.

Example 9 Stability Comparison Test

(76) The AHU-377 sodium salt prepared in comparative example 2, the AHU-377 potassium salt prepared in comparative example 1, and the compound of formula (A) in example 1 were respectively divided into 5 portions. Each portion was about 100 mg, placed in a zip-lock bag and added with a desiccant, then three-layer packaged by a two-layer aluminum plastic material with each layer vacuumed, and stored at 25° C. (relative humidity 60%) and 40° C. (relative humidity 75%). Relevant substances of the samples were respectively determined at 0 day, 30 days, and 60 days later.

(77) TABLE-US-00003 TABLE 3 stability result of main peak purity of the samples at 25° C. Test time (day) Sample 0 30 60 Compound of formula (A) 99.82% 99.80% 99.82% AHU-377 sodium salt 94.12% 94.16% 93.75% AHU-377 potassium salt 93.63% 93.31% 93.28%

(78) TABLE-US-00004 TABLE 4 stability result of main peak purity of the samples at 40° C. Test time(day) Sample 0 30 60 Compound of formula (A) 99.82% 99.68% 99.58% AHU-377 sodium salt 94.12% 94.06% 93.86% AHU-377 potassium salt 94.11% 94.05% 93.37%

(79) The above results show:

(80) 1. The purity of the compound of formula (A) was almost unchanged at 25° C. 60 days later, while the purity of AHU-377 sodium salt was decreased by 0.37%, and the purity of AHU-377 potassium salt was decreased by 0.35%.

(81) 2. The purity of the compound of formula (A) was decreased by 0.24% at 60° C. 60 days later, while the purity of AHU-377 sodium salt was decreased by 0.26%, and the purity of AHU-377 potassium salt was decreased by 0.74%.

(82) Therefore, the stability of the compound of formula (A) of the present invention is far superior to that of the potassium salt and the sodium salt of AHU-377. Moreover, the stability of the compound of formula (A) of the present disclosure is also superior to the organic ammonium salt of AHU-377 by comparison in the same experiment.

Example 10 Drug Interaction Test

(83) The Chinese Patent Application with the publication number CN103709154A firstly disclosed the following compound of formula (I):

(84) ##STR00013##

(85) The above compound is a sartan drug which is coupled with a ligustrazine or NO donor, as a prodrug of angiotensin II receptor antagonist azisartan (TAK-536). The compound can release hydroxyligustrazine or NO in vivo, which thereby causing an effective synergistic action with azilsartan, so as to enhance its antihypertensive effect, lower heart rate, reduce adverse effect, along with an ideal protective effect on the heart and kidney of patients. A potassium salt of compound (I), represented by the compound of formula (II) as below, has been discovered by the present applicant in further studies to show better solubility, higher bioavailability, more potent and longer-lasting antihypertensive effect, more significant and sustainable effect of lowering heart rate, higher safety, as well as desired protective effect on the heart and kidney function of patients, and can be used for preventing and/or treating hypertension, chronic heart failure, diabetic nephropathy, and the like.

(86) ##STR00014##

(87) The applicant has found through repeated studies in the research of drug compatibility that the compound of formula (A), the crystalline form I, single crystal, amorphous form, crystalline form II thereof have good compatibility with the compound of formula (I) described in CN103709154A. A specific compound of formula (B) was selected to be mixed with the crystalline form I of the compound of formula (A) prepared in example 3 of the present disclosure for further stability studies.

(88) ##STR00015##

(89) TABLE-US-00005 TABLE 5 Results of the stability of the mixture of the crystalline form I of the compound of formula (A) + the compound of formula (B) Test time (day) Sample 0 30 60 90 Maximum unknown 0.07% 0.06% 0.07% 0.04% single impurity of the mixture of Form I + compound of formula(B) Total impurities of the 0.74% 1.20% 1.44% 1.42% mixture of Form I + compound of formula(B) Maximum unknown single 0.05% 0.05% 0.06% 0.04% impurity of Formula (B) as a single component Total impurities of 0.72% 1.29% 1.67% 1.99% the mixture of Formula (B) as the single component

(90) The above results indicate that after the crystalline form I of the compound of formula (A) was mixed with the compound of formula (B), the stability of the potassium salt of formula (B) in the mixture was superior to that of the potassium salt of formula (B) as a single component.

(91) Moreover, because of the moisture sensitivity of the compound of formula (B), the crystalline form I of the compound of formula (A) was significantly more hygroscopic than the above-mentioned AHU-377 sodium salt and AHU-377 potassium salt. Therefore, during the process of compounding, warehousing and transportation, the combination drug formed by the compound of formula (B) and the compound of formula (A) had a stability superior to that of the AHU-377 sodium salt or the AHU-377 potassium salt.

Example 11 Water Adsorption and Desorption Test

(92) The water adsorption and desorption tests of the compound (A) of example 1 at 25° C., 0 to 95% relative humidity were carried out by a dynamic water adsorber (DVS) to determine the hygroscopicity of the compound of formula (A). The result is as shown in FIG. 26, which shows the hygroscopicity of the compound of formula (A) is less than 2%.

(93) The water adsorption and desorption experiments of the crystalline form I of the compound of formula (A) prepared by methods of example 3 at 25° C., 0 to 95% relative humidity were carried out by a dynamic water adsorber (DVS) to determine the hygroscopicity of the crystalline form I of the compound of formula (A). The result is as shown in FIG. 6, which shows under the condition of 80% humidity, the hygroscopicity do not change much.

(94) The water adsorption and desorption experiments of the sample of example 6 at 25° C., 0 to 95% relative humidity were carried out by a dynamic water adsorber (DVS) to determine the hygroscopicity of the amorphous form of the compound of formula (A). The result is as shown in FIG. 15, which shows the crystalline form of the amorphous form do not change in the range of 0 to 95% relative humidity.

(95) The hygroscopicity of the crystalline form II of the present invention was examined with the result shown in FIG. 22, which shows that the hygroscopicity changes very little under 80% humidity.

(96) In the description of the present specification, the description of the terms “embodiment”, “example” and the like means that a specific feature, structure, material or characteristic described in connection with the embodiment or example is comprised in at least one embodiment or example of the present invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or features described may be combined in a suitable manner in any one or more embodiments or examples. Although, the embodiments of the present invention have been shown and described in the foregoing, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the present invention, and those skilled in the art can make changes, modifications, replacements, and variations on the above-described embodiments within the scope of the present invention without departing from the spirit and scope of the present invention.