TW201625520A - Polymorphic free acid, hemi-calcium salt and [alpha] -phenethylamine salt of AHU-377 and preparation method and use thereof - Google Patents

Abstract

The present invention discloses new polymophys of a NEP inhibitor AHU-377, including polymorphic free acid, hemi-calcium salt and [alpha] -phenethylamine salt, as well as preparation method and use thereof. These polymophys improve the physical and chemical properties of AHU-377. The preparation of the invention has the advantage of maturity and strong operability. The product of this method is high quality, uniform and stable, it improves chemical stability, which is conducive to storage and has broad application prospect.

Description

AHU-377 crystalline free acid, hemi-calcium salt, α-phenethylamine salt, and preparation method and application thereof

The invention belongs to the technical field of medicines, and particularly relates to a NEP inhibitor AHU-377 crystalline free acid, a semi-calcium salt, an α-phenethylamine salt, a preparation method and application thereof.

Neutral endopeptidase (EC 3.4.24.11; enkephalinase; peptidase; NEP) is a zinc-containing metalloproteinase that cleaves various peptide substrates at the amino terminus of an aromatic amino acid. Substrates of such enzymes include, but are not limited to, atrial natriuretic peptide (ANF, also known as ANP), brain natriuretic peptide (BNP), met and leu enkephalin, bradykinin, neurokinin A, and substance P.

ANF is a vasodilator, diuretic, and antihypertensive peptide. One form, ANF 99-126, is a loop peptide hormone released by the heart during heart expansion. The function of ANF is to maintain the homeostasis of salt and water and regulate blood pressure. ANF is rapidly inactivated by at least two processes in the loop: receptor-mediated clearance and enzyme inactivation at NEP. NEP inhibitors enhance the hypotension and benefit of experimental animals after pharmacological ANF injection Urine, natriuretic and plasma ANF responses. The enhancement of ANF by two specific NEP inhibitors is generally disclosed in U.S. Patent No. 4,749, 688, which is incorporated herein by reference. In the same year, U.S. Patent 4,740,499 discloses that thiorphan and ceratophene can also be used to enhance the application of atrial peptides. In addition, NEP inhibitors can lower blood pressure and exert ANF-like effects such as diuresis in some forms of experimental hypertension and increase in the excretion of cyclic guanosine 3', 5'-monophosphate (cGMP). Since the antibodies to ANF will counteract the decrease in blood pressure, the antihypertensive effect of NEP inhibitors is mediated through ANF. Long-term and uncontrolled hypertensive vascular disease will eventually lead to various pathological changes in target organs such as heart and kidney. Sustained high blood pressure can also increase the incidence of stroke. Therefore, there is a strong need to evaluate the efficacy of antihypertensive treatment by examining other cardiovascular endpoints other than blood pressure reduction to further discover the benefits of combination therapy.

In 1993, Gary Ksander et al. No. 5,217,996 discloses a class of biaryl substituted 4-aminobutyric acid derivatives which have been found to have significant NEP inhibitory activity, the most representative of which is N-(3-carboxy-1-oxo). Propyl)-(4S)-p-phenylphenylmethyl)-4-amino-2R-methylbutyrate (also known as AHU-377).

AHU-377 is usually oiled during actual production preparation The appearance of the substance is not conducive to preservation and subsequent drug development. AHU-377 crude product The AHU-377 sodium salt obtained after the third butanol esterification and sodium hydroxide hydrazine hydrolysis was recrystallized from dichloromethane/n-hexane, and the aggregation state was unknown. The inventors further studied and found that AHU-377 sodium salt has comparable hygroscopicity. Large, materially unstable and unfavorable for storage. Therefore, AHU-377 is a medicinal active ingredient, and its product quality, material stability, storage or weighing have great problems, and its sodium salt does not solve the above problems, although the United States Both US Pat. No. 5,217,996 and WO2007056546 disclose that AHU-377 can also be prepared into various salt-type compounds, such as alkali metal salts, alkaline earth metal salts, amine salts, alkylamine salts, hydroxyalkylamine salts, etc., but mainly from AHU- The chemical structure of 377 containing a carboxyl group inferred that various salt-type compounds other than sodium salts were not actually obtained, and the problems of poor quality, unstable materials, difficulty in storage, and weighing of AHU-377 products could not be solved.

Therefore, in view of the problems existing in the prior art, it is necessary A stable free acid aggregation state was developed for AHU-377 to meet the needs of drug development.

In addition, the application of the drug is hypertension and heart failure. In patients with cardiovascular disease, sodium intake also increases the cardiovascular burden, so it is necessary to replace the sodium salt with a salt that does not contain sodium ions, or other salts that are harmless or beneficial to the cardiovascular. Therefore, in view of the problems in the prior art, it is necessary to develop a more stable free acid aggregation state for AHU-377 or its sodium salt in order to meet the needs of drug development.

The beneficial effects of calcium on cardiovascular are widely recognized. The effect of calcium ion lowering blood pressure may be caused by the following mechanisms: 1. Calcium membrane stabilization, calcium binding on the cell membrane, can reduce cell membrane permeability, increase the threshold of excitability, and relax vascular smooth muscle; 2. Calcium itself can resist Broken calcium channel, so that extracellular calcium ions can not enter the cell; 3, high blood calcium can increase the potassium excretion caused by high sodium, and potassium ions play an important role in stabilizing cell membrane. People maintain adequate high calcium intake, which can counter the harmful effects of high sodium; 4, some scholars believe that 40% of blood pressure is related to the parathyroid gland. The parathyroid glands produce a high-heat-resistant peptide substance, which is the chief culprit in causing high blood pressure, known as the "hypertensive factor." The "hypertensive factor" is stimulated by a low-calcium diet, which inhibits its "hypertensive factor." Modern research has shown that middle-aged and elderly people who eat calcium-rich foods can prevent arteriosclerosis. Hypertensive patients who take antihypertensive drugs with calcium supplementation can better reduce excessive blood pressure to normal. Therefore, for people who are deficient in calcium, calcium supplementation is maintained for a long time, blood calcium is maintained at a normal level, the balance of calcium metabolism inside and outside the smooth muscle cells is maintained, and sputum of vascular smooth muscle is avoided, thereby achieving the purpose of effectively preventing arteriosclerosis and lowering blood pressure. Therefore, replacing the sodium salt of AHU-377 with a calcium salt not only reduces the side effects caused by the sodium salt, but also brings additional beneficial effects on cardiovascular diseases.

Although WO2008031567 also discloses a synthetic route for the preparation of calcium salts of AHU-377 free acid, as shown in the following figure:

However, those skilled in the art will appreciate that the above-mentioned common salt-forming reaction route is based on the general technical route of formulating a salt-containing compound with a calcium salt, but the technical route is not suitable for all carboxyl compounds, and it is not known whether this can be passed. Reflected to prepare the calcium salt of AHU-377. This technical route is supported by no examples, so it is even more unknown whether the technical route can produce a crystalline calcium salt. In addition, the technical route is only established under special conditions, especially the second step in the synthetic route, which is the ion exchange reaction between AHU-377 sodium salt and CaCl ₂ , if it is impossible to create AHU-377 calcium salt or sodium chloride from the system. For the special conditions of precipitation, the above synthetic route is not established. Secondly, from this general technical route, it is impossible to speculate whether a solid form of AHU-377 calcium salt can be prepared, and whether the obtained calcium salt is crystalline or not. Therefore, this general technical route cannot obtain AHU-377 calcium salt or crystalline form of AHU-337 calcium salt crystal.

In order to solve the problems existing in the prior art, the physical properties of AHU-377 are improved, such as improved crystallinity, increased solubility, reduced hygroscopicity, enhanced technical operability, improved chemical stability, and the like. Through in-depth research, the inventors developed a kind of AHU-377 crystalline free acid based on the prior art, which greatly improved the physicochemical properties of AHU-377 free acid, such as product quality, solubility, hygroscopicity and chemical stability. Wait. At the same time, the inventors prepared AHU-377 semi-calcium salt crystal form and AHU-377 α-phenethylamine salt crystal form (α-phenethylamine, the molecular formula is C ₈ H ₁₁ N, α-). Phenylethylamine has two stereoisomers: (R)-(+)-α-phenethylamine and (S)-(-)-α-phenethylamine. The physicochemical properties of AHU-377 free acid or its sodium salt, such as crystallinity, solubility, hygroscopicity and the like, are altered by preparing AHU-377 as a calcium salt or an alpha-phenethylamine salt. And to solve the problem of cardiovascular side effects caused by sodium salt, especially in the treatment of heart failure with complications of depression, has a very broad prospects.

A first aspect of the invention provides an AHU-377 crystalline free acid (designated as Form I) having an X-ray powder diffraction pattern comprising 23.70 ± 0.2°, 15.84 ± 0.2°, 20.84 ± 0.2°, and 8.56 ± 0.2°, The peak at the diffraction angle (2θ).

As a further preferred embodiment, preferably, the X-ray powder diffraction pattern further includes diffraction angles (2θ) at 17.06±0.2°, 25.98±0.2°, 18.01±0.2°, 9.42±0.2°, and 26.91±0.2°. Peak.

As a still further preferred solution, it is preferred that the X-ray powder diffraction pattern further comprises 34.70±0.2°, 21.83±0.2°, 25.69±0.2°, 25.33±0.2°, 11.67±0.2° and 26.24±0.2°. The peak at the diffraction angle (2θ).

As a still further preferred embodiment, the optimum X-ray powder diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in Fig. 1, and the X-ray powder diffraction data is as follows:

As used herein, the position of the X-ray diffraction peak The phrase "substantially identical" means to consider typical peak position and intensity variability. For example, those skilled in the art will appreciate that the peak position (2 theta) will vary depending on the XRPD instrument, sometimes varying up to as much as 0.2 degrees. In addition, those skilled in the art will appreciate that XRPD sample preparation methods, XRPD instruments, sample crystallinity, sample amount, and crystal orientation will lead to relative peak intensities in the XRPD diffraction pattern of the sample. Change.

The second aspect of the present invention provides a method for preparing AHU-377 crystalline free acid, comprising the steps of: 1) dissolving AHU-377 free acid in a suitable organic solvent; 2) lowering the temperature of the solution system, and/or adding crystal And/or adding an appropriate amount of anti-solvent until the solution appears turbid and crystallization; 3) solid-liquid separation to obtain AHU-377 crystalline free acid.

As a further preferred embodiment, the method comprises the steps of: 1) dissolving AHU-377 free acid in a suitable organic solvent at normal temperature or under heating; 2) reducing the temperature of the solution system in step 1), and/or adding crystals. Species, crystallization; 3) solid-liquid separation to obtain AHU-377 crystalline free acid.

As a further preferred embodiment, the following steps are included, 1) dissolving AHU-377 free acid in a suitable organic solvent; 2) mixing the solution in step 1) with an anti-solvent; 3) solid-liquid separation to obtain AHU- 377 crystalline free acid.

Preferably, the AHU-377 crystalline free acid seed crystal is previously added to the anti-solvent before mixing with the anti-solvent in step 2).

By "phase mixing" is meant that the solution in step 1) is added dropwise to the anti-solvent or the anti-solvent is added dropwise to the solution in step 1).

As a still further preferred embodiment, the above preparation method The suitable organic solvent in the step 1) is selected from the group consisting of ethers, cyclic ethers, esters, halogenated alkanes, benzene organic solvents or mixtures thereof, including but not limited to the following solvents: from ethyl acetate, isopropyl acetate Ester, dichloromethane, trichloroethane, carbon tetrachloride, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene or a combination thereof, preferably from isopropyl acetate, dichloroacetic acid Methane, methyl tert-butyl ether, isopropyl ether or a combination thereof.

As a still further preferred embodiment, the above preparation method The anti-solvent in step 2) refers to a less polar organic solvent, including but not limited to the following solvents: n-heptane, n-hexane, isooctane, pentane, cyclohexane, cyclopentane, diethyl ether or A composition; preferably from n-heptane, n-hexane, cyclohexane, diethyl ether or a combination thereof.

As a further preferred embodiment, in the above preparation method "Dissolved" means the general operation of one of ordinary skill in the art, usually The AHU-377 free acid raw material is dissolved or dissolved by appropriate heating, or the amount of the solvent is increased at a normal temperature (20 to 25 ° C) to dissolve or dissolve the AHU-377 free acid raw material.

As a still further preferred solution, the appropriate addition The heat is preferably such that the solvent is heated to 40 to 80 ° C to dissolve or dissolve the AHU-377 free acid material.

As a still further preferred embodiment, the amount of the increasing solvent is preferably a crystalline solvent.

The amount of AHU-377 free acid is added or dissolved in a 5-20 volume ratio of AHU-377 free acid.

As a further preferred embodiment, in the above preparation method Step 2) adding the seed crystal means adding the homologous crystal in the AHU-377 free acid solution, and adding the seed crystal amount is preferably 0.1-20.0% of the AHU-377 free acid quality ratio, and the quality ratio is 0.5-5.0%. For the best. It can be understood that those skilled in the art can further increase the amount of seed crystals added, and the same crystal can be obtained by sub-batch addition, and the same crystal can be obtained. Therefore, modifications or equivalent substitutions of the technical solutions for the addition of seed crystals are intended to be encompassed within the spirit of the invention.

As a further preferred solution, the step 3) of the present invention The powder X-ray diffraction pattern of the obtained AHU-377 crystalline free acid includes peaks at diffraction angles (2θ) at 23.70 ± 0.2 °, 15.84 ± 0.2 °, 20.84 ± 0.2 °, and 8.56 ± 0.2 °.

A third aspect of the invention provides an AHU-377 hemi-calcium salt a crystalline form having an X-ray powder diffraction pattern comprising peaks at diffraction angles (2θ) of 12.70 ± 0.2°, 7.32 ± 0.2°, 15.90 ± 0.2°, and 18.56 ± 0.2°, or an X-ray powder diffraction pattern thereof, including 4.02 Peaks at diffraction angles (2θ) of ±0.2°, 3.62±0.2°, and 17.82±0.2°.

A fourth aspect of the invention provides an AHU-377 hemi-calcium salt The trihydrate crystalline form (designated as Form I) has an X-ray powder diffraction pattern comprising peaks at diffraction angles (2 theta) at 12.70 ± 0.2 °, 7.32 ± 0.2 °, 15.90 ± 0.2 °, and 18.56 ± 0.2 °.

As a further preferred solution, it is preferably X-ray The powder diffraction pattern also includes peaks at diffraction angles (2θ) at 14.74 ± 0.2 °, 15.42 ± 0.2 °, 7.82 ± 0.2 °, 19.42 ± 0.2 °, 16.64 ± 0.2 °, and 17.54 ± 0.2 °.

As a still further preferred embodiment, the optimum X-ray powder diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in FIG. 4, and the X-ray powder diffraction data is as shown in Table 2:

According to a fifth aspect of the present invention, there is provided an AHU-377 semi-calcium salt anhydrous form (designated as Form II), the X-ray powder diffraction pattern comprising diffraction angles at 4.02 ± 0.2 °, 3.62 ± 0.2 ° and 17.82 ± 0.2 ° ( The peak at 2θ).

As a further preferred embodiment, it is preferred that the X-ray powder diffraction pattern further includes peaks at diffraction angles (2θ) of 20.48 ± 0.2 °, 14.50 ± 0.2 ° and 19.66 ± 0.2 °.

As a still further preferred embodiment, the optimum X-ray powder diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in FIG. 6, and the X-ray powder diffraction data is as shown in Table 3:

A sixth aspect of the present invention provides a method for preparing a crystalline form of AHU-377 hemi-calcium salt trihydrate, comprising: 1) dissolving or suspending AHU-377 free acid in a suitable solvent, and being soluble in an equal amount or excess The caustic or weakly basic sodium or potassium salt of the system is mixed to prepare a corresponding salt of AHU-377, preferably a weakly basic sodium or potassium salt is mixed; 2) the above AHU-377 salt system is The water-soluble calcium salt is mixed in water or an organic solvent-containing water system to produce AHU-377 calcium salt precipitate; 3) collecting the product in the above salt-forming reaction to obtain AHU-377 hemi-calcium salt trihydrate crystal form.

As a further preferred embodiment, the suitable solvent of step 1) is a crystallization solvent, including water, a water-soluble solvent or a mixture thereof.

As a still further preferred embodiment, the water-soluble solvent is selected from the group consisting of alcohols, ketones, cyclic ethers, guanamines, sulfonium-based organic solvents or mixtures thereof, preferably from methanol, ethanol, n-propanol, Isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl hydrazine or a mixture thereof; as a further preferred embodiment, step 1 Caustic The base is sodium hydroxide or potassium hydroxide.

As a further preferred embodiment, the weakly basic sodium salt of the step 1) is selected from the group consisting of sodium carbonate, sodium hydrogencarbonate, sodium acetate, sodium formate, sodium propionate, sodium acrylate, sodium benzoate or a mixture thereof; preferably sodium hydrogencarbonate And sodium acetate or a mixture thereof; the weakly basic potassium salt of the step 1) is selected from the group consisting of potassium carbonate, potassium hydrogencarbonate, potassium acetate, potassium formate, potassium propionate, potassium acrylate, potassium benzoate or a mixture thereof; Potassium hydrogen, potassium acetate or a mixture thereof; as a further preferred embodiment, the water-soluble calcium salt of the step 2) is selected from the group consisting of calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium chlorate, calcium hypochlorite. Calcium perchlorate, calcium lactate, calcium gluconate or a mixture thereof; as a still further preferred embodiment, the water-soluble calcium salt of step 2) is selected from the group consisting of calcium chloride, calcium chlorate or a mixture thereof.

As a further preferred embodiment, the dissolution refers to the general operation of a person of ordinary skill in the art, and the AHU-377 free acid raw material can usually be dissolved or dissolved by appropriate heating, or the amount of the solvent can be increased to make the AHU-377. The free acid material is dissolved or dissolved.

As a still further preferred embodiment, the appropriate heating is preferably carried out by heating the crystalline positive solvent to 30 to 80 ° C to dissolve or dissolve the AHU-377 free acid material.

As a still further preferred embodiment, the amount of the increasing solvent is preferably such that the amount of the crystallization solvent added is 2-20 times by volume of the AHU-377 free acid, so that the AHU-377 free acid raw material is dissolved or dissolved.

As a further preferred embodiment, the step of collecting the product in the salt formation reaction process described in the step 3) may specifically be directly collected. The AHU-377 calcium salt polymorph is obtained by describing the solid product precipitated during the calcium salt reaction, or by volatilizing the solvent, or adding an anti-solvent, or directly cooling the crystal, or adding a seed crystal.

As a still further preferred embodiment, the anti-solvent is Refers to less polar organic solvents including, but not limited to, the following solvents: n-heptane, n-hexane, isooctane, pentane, cyclohexane, cyclopentane, diethyl ether or combinations thereof. The amount of the anti-solvent added is 1-10 times by volume of the crystallization solvent.

As a further preferred solution, the step 3) of the present invention The powder X-ray diffraction pattern of the obtained AHU-377 calcium salt crystal form I includes peaks at diffraction angles (2θ) of 12.70 ± 0.2 °, 7.32 ± 0.2 °, 15.90 ± 0.2 °, and 18.56 ± 0.2 °.

A seventh aspect of the invention provides an AHU-377 hemi-calcium salt In the non-crystal type preparation method, AHU-377 hemi-calcium salt trihydrate is heated to 50 ° C or higher, and dehydrated to be crystal-free to obtain AHU-377 hemi-calcium salt crystal-free type.

As a further preferred embodiment, the heating is generally used by one of ordinary skill in the art.

The operation can usually be carried out under nitrogen or under vacuum.

As a further preferred embodiment, the heating temperature ranges from 50 to 100 °C.

As a further preferred embodiment, the AHU-377 calcium salt amorphous form (Form II) obtained by the present invention has an X-ray powder diffraction pattern including diffraction angles at 4.02±0.2°, 3.62±0.2°, and 17.82±0.2°. (2θ) Peak.

As a further preferred solution, it is preferably X-ray The powder diffraction pattern also includes peaks at diffraction angles (2 theta) at 20.48 ± 0.2 °, 14.50 ± 0.2 °, and 19.66 ± 0.2 °.

An eighth aspect of the present invention provides an AHU-377 α-phenylethyl An amine salt having an X-ray powder diffraction pattern comprising peaks at diffraction angles (2θ) of 20.58 ± 0.2 °, 24.28 ± 0.2 °, 8.38 ± 0.2 °, and 23.20 ± 0.2 °, or including at 23.28 ± 0.2 °, 18.9 ± Peaks at diffraction angles (2θ) of 0.2°, 13.7±0.2° and 14.72±0.2°.

A ninth aspect of the present invention provides a AHU-377(R)-(+)-α-phenethylamine salt crystal form (designated as Form I), the X-ray powder diffraction pattern of which includes 20.58±0.2°, 24.28±0.2°, 8.38±0.2° and A peak at a diffraction angle (2θ) of 23.20 ± 0.2°.

As a further preferred embodiment, the X-ray powder diffraction pattern further includes peaks at diffraction angles (2θ) at 19.36 ± 0.2 °, 15.16 ± 0.2 °, 16.78 ± 0.2 °, 18.84 ± 0.2 °, and 22.06 ± 0.2 °.

As a still further preferred embodiment, the X-ray powder diffraction pattern further includes diffraction angles at 17.34 ± 0.2 °, 7.52 ± 0.2 °, 28.10 ± 0.2 °, 29.66 ± 0.2 °, 28.94 ± 0.2 °, and 10.96 ± 0.2 ° ( The peak at 2θ).

As a still further preferred embodiment, the optimum X-ray powder diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in Fig. 7, and the X-ray powder diffraction data are as shown in Table 4:

A tenth aspect of the present invention provides a AHU-377(S)-(-)-α-phenethylamine salt crystal form (designated as Form II), its X-ray powder diffraction pattern is included at 23.28±0.2°, 18.9±0.2°, 13.7±0.2° and A peak at a diffraction angle (2θ) of 14.72 ± 0.2°.

As a further preferred solution, it is preferably X-ray The powder diffraction pattern also includes peaks at diffraction angles (2 theta) at 3.13 ± 0.2 °, 15.98 ± 0.2 °, 18.36 ± 0.2 °, 9.12 ± 0.2 °, and 32.38 ± 0.2 °.

As a still further preferred solution, it is preferably X-ray The line powder diffraction pattern also includes peaks at diffraction angles (2 theta) at 10.88 ± 0.2 °, 22.18 ± 0.2 °, 21.92 ± 0.2 °, 27.66 ± 0.2 °, 20.34 ± 0.2 °, and 27.24 ± 0.2 °.

As a still further preferred embodiment, the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in Fig. 9, and the X-ray powder diffraction data is as shown in Table 5:

The eleventh aspect of the present invention provides a method for preparing a crystalline form of AHU-377 α-phenethylamine salt, comprising: 1) dissolving or suspending AHU-377 free acid in a suitable solvent with an equal amount or excess of α- AHU-377 α-phenethylamine salt is prepared by mixing phenethylamine; 2) AHU-377 α-benzene is obtained by collecting the product in the above salt formation reaction Ethylamine salt crystal form.

The α-phenethylamines include (R)-(+)-α-phenethylamine and (S)-(-)-α-phenethylamine.

As a further preferred embodiment, the suitable solvent in the step 1) is a crystallization solvent, including water, a water-soluble solvent or a water-insoluble solvent.

In a still further preferred embodiment, the water-soluble solvent is selected from the group consisting of alcohols, ketones, cyclic ethers, guanamines, sulfonium-based organic solvents or mixtures thereof, including but not limited to the following solvents: methanol, ethanol, N-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl hydrazine or mixtures thereof; The solvent is selected from the group consisting of chlorinated alkanes, ethers, esters, alkanes, cycloalkanes, benzene organic solvents or mixtures thereof, including but not limited to the following solvents: ethyl acetate, isopropyl acetate, dichloromethane, three Ethyl chloride, carbon tetrachloride, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene or a combination thereof.

As a further preferred embodiment, the dissolution refers to the general operation of a person of ordinary skill in the art, and the AHU-377 free acid raw material can usually be dissolved or dissolved by appropriate heating, or the amount of the solvent can be increased to make the AHU-377. The free acid material is dissolved or dissolved.

As a still further preferred embodiment, the appropriate heating is preferably carried out by heating the crystallization solvent to 30 to 80 ° C to dissolve or dissolve the AHU-377 free acid material.

As a still further preferred embodiment, the amount of the increasing solvent is preferably such that the amount of the crystallization solvent added is AHU-377 free acid. The AHU-377 free acid material is dissolved or dissolved by a 1-200 volumetric mass ratio.

As a further preferred solution, the step 2) The step of collecting the product in the salt formation reaction may specifically be directly collecting the solid product precipitated during the reaction of forming the α-phenethylamine salt, or by volatilizing the solvent, or adding the anti-solvent, or directly cooling the crystal, or adding the crystal. The AHU-377 α-phenethylamine salt crystal form is obtained; preferably, the solid product precipitated during the reaction of the above α-phenethylamine salt is directly collected to obtain a crystal form of AHU-377 α-phenethylamine salt.

As a still further preferred embodiment, the anti-solvent is Refers to less polar organic solvents including, but not limited to, the following solvents: n-heptane, n-hexane, isooctane, pentane, cyclohexane, cyclopentane, diethyl ether or combinations thereof.

As a further preferred solution, the step 2) of the present invention The X-ray powder diffraction pattern of the obtained AHU-377(R)-(+)-α-phenethylamine salt crystal form includes diffraction at 20.58±0.2°, 24.28±0.2°, 8.38±0.2° and 23.20±0.2°. The peak at the angle (2θ), or the obtained AHU-377(S)-(-)-α-phenethylamine salt, has an X-ray powder diffraction pattern including 23.28±0.2°, 18.9±0.2°, and 13.7±0.2. ° and a peak at a diffraction angle (2θ) of 14.72 ± 0.2°.

A twelfth aspect of the present invention provides a pharmaceutical composition, The pharmaceutical compositions comprise a therapeutically effective amount of the aforementioned AHU-377 crystalline free acid, AHU-377 hemi-calcium salt crystalline form, AHU-377 hemi-calcium salt trihydrate crystalline form, AHU-377 semi-calcium salt amorphous form, AHU-377 α-phenethylamine salt crystal form, AHU-377(R)-(+)-α-phenethylamine salt crystal form, AHU-377(S)-(-)-α-phenethylamine salt crystal And a pharmaceutically acceptable carrier or excipient.

As a further preferred embodiment, the pharmaceutically acceptable carriers Or the excipient is selected from the group consisting of a diluent or filler, a disintegrant, a binder, a glidant, a lubricant, a colorant, or a combination thereof.

As a still further preferred embodiment, the diluents or The filler is selected from the group consisting of powdered sugar, compressible sugar, glucose, sucrose, lactose, dextrin, mannitol, microcrystalline cellulose, sorbitol, starch or a combination thereof, and the diluent or filler is used in an amount of 4.0% by weight of the composition. ~60.0%, preferably 10.0%~40.0%; the disintegrants are selected from the group consisting of starch, clay, cellulose, alginate, gum, crosslinked polymer, soybean polysaccharide, guar gum or combinations thereof. The crosslinked polymer is preferably self-crosslinking polyvinylpyrrolidone, croscarmellose sodium, croscarmellose calcium or a combination thereof, and the disintegrant is used in an amount of from 0% to 65.0% by weight of the composition. Preferably, the binder is selected from the group consisting of starch, cellulose and derivatives thereof, sucrose, glucose, corn syrup, gelatin, povidone or a combination thereof, and the cellulose and its derivatives are more preferred. Preferred from microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose or a combination thereof; more preferred is hydroxypropyl cellulose; the best low-substituted hydroxypropyl cellulose The binder is used in an amount of 1.0% to 60.0% by weight of the composition, preferably 5.0% to 40.0%, more preferably 10.0% to 30.0%; The flow aid or lubricant is selected from the group consisting of colloidal cerium oxide, magnesium triacetate, starch, talc, calcium orthophosphate, magnesium stearate, aluminum stearate, calcium stearate, calcium carbonate, magnesium oxide, Polyethylene glycol, powdered cellulose, glyceryl behenate, stearic acid, hydrogenated castor oil, glyceryl monostearate, sodium stearyl fumarate or combinations thereof, glidant or lubricant The amount is from 0% to 10.0% by weight of the composition, preferably from 0.5% to 5.0%.

As a still further preferred solution, the drug combinations The diuretic comprises a diuretic; the diuretic is selected from the group consisting of furosemide, ethenic acid, bumetanide, torsemide, hydrochlorothiazide, chlorthalidone, benzfluorothiazide, cyclopentazine, boratio The azine, metoprazine, indapamide or a combination thereof; the diuretic is used in an amount of from 0.1% to 10.0% by weight of the composition, preferably from 0.5% to 5.0%.

As a still further preferred embodiment, the pharmaceutical compositions can be prepared into tablets, capsules, granules.

As a most preferred solution, the capsule groups are assigned as follows:

As a best solution, the capsule groups are assigned as follows:

A thirteenth aspect of the present invention provides the aforementioned AHU-377 Crystalline free acid, AHU-377 hemi-calcium salt crystal form, AHU-377 hemi-calcium salt trihydrate crystal form, AHU-377 hemi-calcium salt crystal form, AHU-377 α-phenethylamine salt crystal form, AHU- 377(R)-(+)-α-phenethylamine salt crystal form, AHU-377(S)-(-)-α-phenethylamine salt crystal form or the aforementioned pharmaceutical composition for preparing treatment or prevention and neutrality Use in endopeptidase-related diseases, cardiovascular, antihypertensive drugs.

As a further preferred solution, the antihypertensive The drug refers to a drug that produces more effective antihypertensive treatment by improving efficacy and having a higher corresponding rate, including but not limited to anti-malignant hypertension, essential hypertension, renal vascular hypertension, and diabetes. Hypertension, isolated systolic hypertension, or other secondary hypertension medications.

A fourteenth aspect of the present invention provides the aforementioned AHU-377 Crystalline free acid, AHU-377 hemi-calcium salt crystal form, AHU-377 hemi-calcium salt trihydrate crystal form, AHU-377 hemi-calcium salt crystal form, AHU-377 α-phenethylamine salt crystal form, AHU- 377(R)-(+)-α-phenethylamine salt crystal form, AHU-377(S)-(-)-α-phenethylamine salt crystal form or the aforementioned pharmaceutical composition for treating or preventing acute and chronic heart Use in debilitating, congestive heart failure, left ventricular dysfunction, hypertrophic cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmia, atrial fibrillation, atrial flutter, or harmful vascular remodeling drugs.

A fifteenth aspect of the present invention provides a pharmaceutical composition, The pharmaceutical compositions comprise a therapeutically effective amount of the aforementioned AHU-377 crystalline free acid, AHU-377 hemi-calcium salt crystalline form, AHU-377 hemi-calcium salt trihydrate. Crystal form, AHU-377 semi-calcium salt crystal-free type, AHU-377 α-phenethylamine salt crystal form, AHU-377(R)-(+)-α-phenethylamine salt crystal form, AHU-377(S a -(-)-α-phenethylamine salt crystal form and an angiotensin II (AT1) receptor antagonist, and a pharmaceutically acceptable carrier or excipient.

As a further preferred embodiment, the angiotensin II antagonists are selected from the group consisting of losartan, irbesartan, olmesartan, telmisartan, valsartan, azilsartan, candesartan, yi Prosartan, losartan, saprisartan, ilartartan, tamsaltan, elsartan or a pharmaceutically acceptable salt thereof.

As a still further preferred embodiment, the angiotensin II antagonist is selected from the group consisting of valsartan, azilsartan or a pharmaceutically acceptable salt thereof.

A sixteenth aspect of the present invention provides the pharmaceutical composition of the present invention for the preparation or treatment of myocardial infarction and its sequelae, atherosclerosis, angina pectoris, diabetic or non-diabetic renal insufficiency, secondary aldosteronism, primary Or secondary pulmonary hypertension, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary nephropathy, renal vascular hypertension, diabetic retinopathy, migraine, peripheral vascular disease Use in Raynaud's disease, hyperplasia of the cavity, cognitive dysfunction, glaucoma or stroke medication.

"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof with other chemical components, or other components such as physiological/pharmaceutical Accepted carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

A pharmaceutical composition containing an active ingredient may be suitable for use in Oral forms such as tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions can be prepared according to any method known in the art for preparing pharmaceutical compositions, such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring, coloring, and preservatives to provide a pleasing And delicious pharmaceutical preparations. Tablets contain the active ingredient and non-toxic pharmaceutically acceptable excipients suitable for the preparation of a tablet for admixture. These excipients may be inert excipients such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrating agents such as microcrystalline cellulose, croscarmellose sodium, corn Starch or alginic acid; a binder such as starch, gelatin, polyvinylpyrrolidone or gum arabic and a lubricant such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or may be coated by masking the taste of the drug or delaying disintegration and absorption in the gastrointestinal tract, thus providing a sustained release effect over a longer period of time. For example, water-soluble taste masking materials such as hydroxypropylmethylcellulose or hydroxypropylcellulose, or extended-time materials such as ethylcellulose, cellulose acetate butyrate may be used.

Examples of active ingredients and inert solid diluents may also be employed. A hard gelatin capsule such as calcium carbonate, calcium phosphate or kaolin mixed, or a soft gelatin capsule in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oil vehicle such as peanut oil, liquid paraffin or olive oil provides an oral preparation.

Aqueous suspensions contain active substances and are suitable for mixing An excipient for the preparation of an aqueous suspension. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone and acacia; dispersing or wetting agents can be natural a phospholipid produced, such as lecithin, or a condensation product of an alkylene oxide with a fatty acid, such as polyoxyethylene stearate, or a condensation product of ethylene oxide with a long chain fatty alcohol, such as heptadecyl ethyleneoxy cetyl wax An alcohol (heptadecaethyleneoxy cetanol), or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol, such as polyethylene oxide sorbitol monooleate, or ethylene oxide with a fatty acid and a hexitol anhydride A condensation product of a derivatized partial ester, such as polyethylene oxide sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives such as ethylparaben or n-propylparaben, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents such as sucrose, saccharin or arsenic. Spartan.

An oil suspension can be obtained by suspending the active ingredient in vegetable oil Formulated in peanut oil, olive oil, sesame oil or coconut oil, or mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. The above sweeteners and flavoring agents may be added to provide a palatable preparation. These compositions can be preserved by the addition of an anti-oxidant such as butylated hydroxyanisole or alpha-tocopherol.

It can be applied to the preparation of water suspension by adding water. Dispersing powders and granules provide the active ingredient and dispersing or wetting agents, suspending agents or one or more preservatives. Suitable dispersing or wetting agents and suspending agents can be used to illustrate the above examples. Other excipients such as sweetening, flavoring, and coloring agents may also be added. These compositions are preserved by the addition of an anti-oxidant such as ascorbic acid.

The pharmaceutical composition of the present invention may also be an oil-in-water emulsion form. The oil phase can be a vegetable oil such as olive oil or peanut oil, or a mineral Oils such as liquid paraffin or mixtures thereof. Suitable emulsifiers may be naturally occurring phospholipids, such as soy lecithin and esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of the partial esters and ethylene oxide, For example, polyethylene oxide sorbitol monooleate. The emulsions may also contain sweeteners, flavoring agents, preservatives, and antioxidants. Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a colorant, and an antioxidant.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin to form a microemulsion. The injection or microemulsion can be injected into the bloodstream of the patient by a local injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant loop concentration of the compound of the invention. To maintain this constant concentration, a continuous intravenous delivery device can be used.

The pharmaceutical composition can be for intramuscular and subcutaneous administration In the form of sterile injectable water or oil suspension. The suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension, such as a solution prepared in 1,3-butanediol, in a non-toxic parenterally acceptable diluent or solvent. In addition, sterile fixed oils may conveniently be employed as a solvent or suspension medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid can also be prepared as an injection.

The invention can be administered in the form of a suppository for rectal administration Compound. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and thus dissolves in the rectum to release the drug. Such materials include mixtures of cocoa butter, glycerin gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.

Drug-administering agents well known to those skilled in the art The amount depends on a variety of factors, including but not limited to the following factors: the activity of the particular compound used, the age of the patient, the weight of the patient, the health of the patient, the living conditions of the patient, the diet of the patient, the time of administration, the mode of administration, and the excretion. The rate, combination of drugs, and the like; in addition, the optimal mode of treatment, such as the mode of treatment, the daily amount of AHU-377 or its salt, or the type of pharmaceutically acceptable salt, can be verified according to conventional treatment regimens.

Compared with the prior art, the present invention has the following advantages:

1) The present invention provides a novel crystalline form of AHU-377 free acid. AHU-377 free acid is an oily compound, and it is technically difficult to obtain a pure compound because the solvent residue is difficult to remove, and AHU-377 can be crystallized to obtain high purity AHU-377, and the preparation technique is simple. Oily compounds are highly detrimental to the use and production of oral solid preparations, while solids make it very easy to use oral solid preparations. The crystal is superior to the oil in stability.

2) The present invention changes the physicochemical properties of AHU-377 free acid, such as crystallinity, solubility, hygroscopicity, etc., by preparing AHU-377 as α-phenethylamine salt, and the technology of the present invention is mature and operable. The resulting product is of high quality and Uniform stability, chemical stability, conducive to storage and separation and purification of AHU-377 and improved chiral purity.

3) The present invention provides a novel AHU-377 calcium salt trihydrate, which has high purity, stable and reliable quality, improved hygroscopicity of AHU-377 or its sodium salt, improved chemical stability, and is convenient for storage and industrialization. Production needs.

4) The AHU-377 calcium salt crystal form I obtained by the invention is suitable for clinical drug application, and the prepared capsule can meet medical or pharmaceutical requirements and is suitable for industrialization.

5) The crystallization method of the invention has strong operability, mature technology and easy control.

6) Compared with the sodium salt of AHU-377 obtained in the present invention, in the treatment of hypertension and heart failure, sodium intake can be reduced, and the cardiovascular burden of the patient can be alleviated. And calcium has a soothing effect on blood vessels, which is conducive to the treatment of cardiovascular diseases.

Therefore, the drug safety and effectiveness of AHU-377 as a calcium salt in the treatment of hypertension and heart failure diseases has obvious advantages over sodium salt.

Figure 1 is an X-ray powder diffraction pattern of AHU-377 free acid crystal form I, with the abscissa being the angle 2θ (°) and the ordinate being the intensity.

Figure 2 is a differential calorimetry (DSC) diagram of AHU-377 free acid crystal form I, with the abscissa being temperature (°C) and the ordinate bit heat flow (W/g).

Figure 3 is a thermogravimetric analysis (TGA) of AHU-377 free acid crystal form I, with the abscissa being temperature (°C) and the ordinate being weight loss ratio (%).

Figure 4 is an X-ray powder diffraction pattern (XRPD pattern) of the AHU-377 calcium salt trihydrate crystal form.

Figure 5 is a thermogravimetric analysis (TGA chart) of the AHU-377 calcium salt trihydrate crystal form.

Figure 6 is an X-ray powder diffraction pattern (XRPD pattern) of the AHU-377 calcium salt crystal-free type.

Fig. 7 is an XRPD pattern of the crystal form of AHU-377(R)-(+)-α-phenethylamine salt, the abscissa is the angle 2θ (°), and the ordinate is the intensity.

Figure 8 is a DSC chart of the AHU-377(R)-(+)-α-phenethyl salt crystal form, with the abscissa being the temperature (°C) and the ordinate being the heat flow (w/g).

Figure 9 is an XRPD pattern of the crystalline form of AHU-377(S)-(-)-α-phenethylamine salt. The abscissa is the angle 2θ (°) and the ordinate is the intensity.

Figure 10 is a DSC chart of the AHU-377(S)-(-)-α-phenethylamine salt type, with the abscissa being the temperature (°C) and the ordinate being the heat flow (w/g).

Figure 11 is a chiral chromatogram of the enantiomeric mixture of AHU-377 and 2-methyl S. The abscissa is time (minutes) and the ordinate is absorbance (mAU).

Figure 12 is a chiral chromatogram of AHU-377(S)-(-)-α-phenethylamine salt prepared from the enantiomeric mixture of AHU-377 and 2-methyl S. The abscissa is Time (minutes), ordinate is absorbance (mAU).

Specific examples and preparation methods provided below will be Further specific aspects of embodiments of the invention are further illustrated. The scope of the following examples is not intended to limit the scope of the invention in any way.

Methods and materials

AHU-377 crystalline free acid, AHU-377 calcium salt polymorph, and AHU-377 alpha-phenethylamine crystal form are characterized by their X-ray powder diffraction pattern. Thus, an X-ray powder diffraction pattern of the compound was taken on a Bruker D8 Discover X-ray powder diffractometer with GADDS (General Area Diffraction Detector System) CS operating in a reflective manner using Cu K alpha radiation (1.54 Å). The tube voltage and current quantities were set to 40kV and 40mA acquisition scans, respectively. The sample was scanned for a period of 60 seconds in the range of 2θ from 3.0° to 40°. The diffractometer was calibrated using a corundum standard for the peak position indicated by 2θ. All analyses were performed at room temperature, typically between 20 ° C and 30 ° C. Collect and integrate data using GADDS for the 4.1.14T version of WNT software. The diffraction pattern was analyzed using the 2003 DiffracPlus software version 9.0.0.2 released by Eva. The XRPD sample is prepared by applying the sample to a single crystal wafer, pressing the sample powder with a glass slide or equivalent to ensure that the surface of the sample is flat and of a suitable height. The sample holder was then placed in a Bruker XRPD instrument and an X-ray powder diffraction pattern was acquired using the instrument parameters described above. Measurement differences associated with such X-ray powder diffraction analysis results are produced by a number of factors including: (a) errors in sample preparations (eg, sample height), (b) instrument errors, (c) calibration differences, (d) Operator error (including those occurring when determining peak position), and (e) properties of the substance (eg, preferred orientation error). Calibration errors and sample height errors often result in displacement of all peaks in the same direction. In general, this calibration factor will cause the measured peak position to coincide with the expected peak position and may be in the range of the expected 2[Theta] value ± 0.2[deg.].

The raw material of the invention AHU-377 free acid is based on the patent US 5,217,996 A reports the preparation process and the product is an oil. The angle 2θ (°) value and the intensity value (as % of the highest peak) of the crystal form obtained in the examples of the present invention are listed in Tables 1-5.

Differential sweep on the TA Instruments DSC Q2000 Tracing calorimetry (DSC). Calibrate the instrument for cell constants and heat capacity using indium and sapphire, respectively. Samples were prepared by weighing 1-3 mg of the sample into an aluminum pan and then capping the aluminum pan with an aluminum cap. Analyze data using Universal Analysis 2000. The test was started at room temperature, and the sample was heated to 300 ° C at 10 ° C / min under a nitrogen purge (flow rate of 50 ml / min).

Analysis of diastereomers by HPLC, chiral Chromatographic conditions were: column: Daicel 璐 Chiralpak AD-H (4.6*250mm, 5um); mobile phase A: cyclohexane: isopropanol: glacial acetic acid = 1000:10:1; mobile phase B: cyclohexane : isopropanol: glacial acetic acid = 800:200:1; mobile phase A: mobile phase B = 50:50; running time per needle: 22 min; column temperature: 30 ° C; wavelength: 254 nm; flow rate: 1.0 ml/min.

Example 1

Weigh 200mg of AHU-377 free acid (oily) in a 20.0mL glass bottle, then add 4mL of methyl tert-butyl ether, stir and dissolve, slowly add n-heptane to turbidity (about 5mL), room temperature The mixture was further stirred at (20-25 ° C) for 48 hours, and solid-liquid separation was carried out to obtain AHU-377 crystalline free acid, and the X-ray powder diffraction pattern thereof is shown in Fig. 1.

Differential calorimeter scanner (DSC, model TA Q2000) The melting point of the free acid of AHU-377 crystalline form was measured. The measurement conditions were from room temperature to 250 ° C, a heating rate of 10 ° C per minute, and heating under a nitrogen atmosphere at a nitrogen flow rate of 20 mL per minute. The DSC chart of the AHU-377 crystalline free acid is shown in Figure 2. The melting point (onset point) of the AHU-377 crystalline free acid is about 94.3 °C.

Measured with a thermogravimetric analyzer (TGA, model TA Q500) Thermal weight loss of AHU-377 crystalline free acid. The measurement conditions were from room temperature to 300 ° C, a heating rate of 10 ° C per minute, and heating under a nitrogen atmosphere at a nitrogen flow rate of 50 mL per minute. The TGA pattern of the AHU-377 crystalline free acid is shown in Figure 3. There is almost no weight loss within 100 ° C, so it can be judged that the AHU-377 crystalline free acid is an amorphous type.

Example 2

Weigh 50mg of AHU-377 free acid (oily) in a 2.0mL glass bottle, then add 0.5mL of isopropyl acetate, heat to 50 ° C, stir to dissolve, slowly add 5.0mL of n-hexane, then cool to room temperature (20-25 ° C), stirring was continued for 48 hours, and solid-liquid separation obtained AHU-377 crystalline free acid, and the X-ray powder diffraction pattern thereof was consistent with FIG.

Example 3

Weigh 50mg of AHU-377 free acid (oily) in a 2.0mL glass bottle, then add 0.25mL of toluene, heat to 80 ° C, stir to dissolve, then cool to room temperature (20-25 ° C), slowly add 2.0 mL anhydrous ether, stirred for 48 hours, solid-liquid separation to obtain AHU-377 crystalline free acid, The X-ray powder diffraction pattern is identical to that of Figure 1.

Example 4

Weigh 50 mg of AHU-377 free acid (oily) in a 2.0 mL glass vial, then add 0.5 mL of methyl tert-butyl ether, heat to 55-60 ° C, stir to dissolve, and then cool to room temperature (20 -25 ° C), stirring was continued for 48 hours, and solid-liquid separation obtained AHU-377 crystalline free acid, and the X-ray powder diffraction pattern thereof was consistent with FIG.

Example 5

Weigh 20 mg of AHU-377 free acid (oily) in a 2.0 mL glass vial, then add 0.5 mL of ethyl acetate, stir to dissolve, slowly add 0.2 mg of the crystal of Example 1, at room temperature (20-25 ° C) Stirring was continued for 24 hours, and solid-liquid separation gave AHU-377 crystalline free acid, and its X-ray powder diffraction pattern was consistent with FIG.

Example 6

Weigh 50mg of AHU-377 free acid (oily) in a 2.0mL glass bottle, then add 0.5mL of isopropyl acetate, heat to 55-60 ° C, stir to dissolve, and then cool to room temperature (20-25 ° C 0.5 mg of the crystal of Example 1 was slowly added, and stirring was continued for 24 hours at room temperature (20-25 ° C), and solid-liquid separation was carried out to obtain AHU-377 crystalline free acid, and the X-ray powder diffraction pattern thereof was consistent with FIG.

Example 7

20 mg of AHU-377 free acid (oily) was weighed into a 20.0 mL glass vial, and then 2 mL of methyl tert-butyl ether was added to dissolve to obtain a clear solution, which was used. 2 mg of the crystal of Example 1 was suspended in 10 mL of n-heptane, and the AHU-377 methyl tert-butyl ether solution was slowly added dropwise to the n-heptane solution, and added dropwise for about 1 hour while stirring. The AHU-377 crystalline free acid was obtained by liquid separation, and its X-ray powder diffraction pattern was consistent with FIG.

Example 8

Weigh 500 mg (1.215 mmol) of AHU-377 free acid (oily) in a 50.0 mL round bottom flask, add 5.0 mL of acetone, stir to dissolve, add 12.2 mL of a 0.10 mmol/mL aqueous solution of sodium bicarbonate, and stir. After reacting for 12 hours, 0.65 mL of a calcium chloride aqueous solution having a concentration of 1.0 mmol/mL was added, and then 15 mL of water was further added thereto, followed by stirring for 24 hours to sufficiently react. Solid-liquid separation gave AHU-377 calcium salt crystal form I, and its X-ray powder diffraction pattern (XRPD pattern) is shown in Fig. 4.

Measured with a thermogravimetric analyzer (TGA, model TA Q500) AHU-377 calcium salt crystal form I thermal weight loss. The measurement conditions were from room temperature to 400 ° C, a heating rate of 10 ° C per minute, and heating under a nitrogen atmosphere at a nitrogen flow rate of 50 mL per minute. The TGA pattern of AHU-377 calcium salt crystal form I is shown in Fig. 5. The weight loss within 100 ° C is 10%, which is similar to the theoretical weight loss of AHU377 hemi-calcium salt trihydrate (theoretical weight loss is 11%). Therefore, it can be judged that AHU-377 calcium salt crystal form I is AHU377 hemi-calcium tricrystal.

Example 9

Weigh 50 mg (0.12 mmol) of AHU-377 free acid (oily) in a 10.0 mL round bottom flask, add 1.0 mL of isopropanol, stir to dissolve, and add 0.25 mL of a 0.50 mmol/mL sodium acetate aqueous solution. The reaction was stirred for 12 hours, 0.15 mL of a 0.5 mmol/mL calcium bromide aqueous solution was added, and then 5 mL of water was added thereto, and the mixture was stirred for 24 hours to sufficiently react. AHU-377 calcium salt crystal form I was obtained by solid-liquid separation, and its X-ray powder diffraction pattern (XRPD pattern) was substantially identical to that shown in Fig. 4.

Example 10

Weigh 50 mg (0.12 mmol) of AHU-377 free acid (oily) in a 10.0 mL round bottom flask, add 0.5 mL of tetrahydrofuran, stir to dissolve, add 1.0 mL of a 0.10 mmol/mL potassium bicarbonate solution, and stir. After reacting for 12 hours, 0.25 mL of a 0.5 mmol/mL aqueous solution of calcium chloride solution was added, and then 5 mL of water was added thereto, and the mixture was stirred for 24 hours to sufficiently react. AHU-377 calcium salt crystal form I was obtained by solid-liquid separation, and its X-ray powder diffraction pattern (XRPD pattern) was substantially identical to that shown in Fig. 4.

Example 11

Weigh 50 mg (0.12 mmol) of AHU-377 free acid (oily) in a 10.0 mL round bottom flask, add 0.3 mL of N,N-dimethylformamide, stir to dissolve, and add 0.65 mL to a concentration of 0.20 mmol. /mL potassium potassium propionate solution, stirring reaction for 12 hours, adding 0.30mL of calcium chloride aqueous solution with a concentration of 0.5mmol / mL, then adding 5mL of water, stirring for 24 hours to charge Sub-reaction. AHU-377 calcium salt crystal form I was obtained by solid-liquid separation, and its X-ray powder diffraction pattern (XRPD pattern) was substantially identical to that shown in Fig. 4.

Example 12

Weigh 50 mg (0.12 mmol) of AHU-377 free acid (oily) in a 10.0 mL round bottom flask, add 1.0 mL of ethanol, heat to 60 ° C, stir to dissolve, and add 1.4 mL of hydrogen carbonate at a concentration of 0.10 mmol/mL. The potassium aqueous solution was stirred for 12 hours, and 0.15 mL of a calcium lactate aqueous solution having a concentration of 0.5 mmol/mL was added thereto, and then 5 mL of water was further added thereto, followed by stirring for 24 hours to sufficiently react. AHU-377 calcium salt crystal form I was obtained by solid-liquid separation, and its X-ray powder diffraction pattern (XRPD pattern) was substantially identical to that shown in Fig. 4.

Example 13

Weigh 50 mg (0.12 mmol) of AHU-377 free acid (oily) in a 10.0 mL round bottom flask, add 1.0 mL of acetonitrile, heat to 40 ° C, stir to dissolve, add 1.3 mL of sodium benzoate at a concentration of 0.10 mmol/mL. The aqueous solution was stirred for 12 hours, and 0.2 mL of a calcium lactate aqueous solution having a concentration of 0.5 mmol/mL was added thereto, and then 5 mL of water was further added thereto, followed by stirring for 24 hours to sufficiently react. AHU-377 calcium salt crystal form I was obtained by solid-liquid separation, and its X-ray powder diffraction pattern (XRPD pattern) was substantially identical to that shown in Fig. 4.

Example 14

Weigh 20 mg of AHU-377 calcium salt crystal form I, heat it to 90 ° C in a thermogravimetric analyzer, and then slowly remove it after natural cooling. The crystal form is detected by XRPD. Its X-ray powder diffraction pattern (XRPD pattern) is basically identical to that of Figure 6.

Example 15

Weigh 20 mg (0.049 mmol) of AHU-377 free acid (oily) in a 5.0 mL glass vial, add 1.0 mL of isopropyl acetate, stir to dissolve, and add 5.9 mg (0.049 mmol) of (R)-(+) -α-phenethylamine, stirring reaction for 48 hours, collecting the reaction product to obtain a powder X-ray diffraction pattern of AHU-377(R)-(+)-α-phenethylamine salt crystal form as shown in Fig. 7. Melting point: 126.6 ° C (onset point) as shown in Fig. 8.

Example 16

Weigh 20 mg (0.049 mmol) of AHU-377 free acid (oily) in a 5.0 mL glass vial, add 0.4 mL of isopropanol, stir to dissolve, and add 6.5 mg (0.053 mmol) of (R)-(+)- The α-phenylethylamine was further stirred for 48 hours, and the AHU-377(R)-(+)-α-phenethylamine crystal form obtained by the reaction product was collected, and the powder X-ray diffraction pattern thereof was substantially the same as in Fig. 7.

Example 17

Weigh 20 mg (0.049 mmol) of AHU-377 free acid (oily) in a 5.0 mL glass vial, add 0.2 mL of methyl tert-butyl ether, heat to 50 ° C, stir to dissolve, add 5.9 mg (0.049 mmol) (R)-(+)-α-phenethylamine, cooling to room temperature (20~25 ° C), stirring the reaction for 48 hours, collecting the reaction product to obtain AHU-377(R)-(+)-α-phenylethyl The amine salt crystal form has a powder X-ray diffraction pattern substantially identical to that of Figure 7.

Example 18

Weigh 20 mg (0.049 mmol) of AHU-377 free acid (oily) in a 5.0 mL glass vial, add 0.2 mL of dichloromethane, heat to 40 ° C, stir to dissolve, add 6.2 mg (0.051 mmol) (R)- (+)-α-phenethylamine, cooling to room temperature (20~25 ° C), stirring the reaction for 48 hours, collecting the reaction product to obtain AHU-377(R)-(+)-α-phenethylamine salt crystal form The powder X-ray diffraction pattern is substantially identical to that shown in Fig. 7.

Example 19

Weigh 20 mg (0.049 mmol) of AHU-377 free acid (oily) in a 5.0 mL glass vial, add 0.2 mL of 95% ethanol, heat to 60 ° C, stir to dissolve, and add 6.0 mg (0.049 mmol) of (R)-( +)-α-phenethylamine, cooling to room temperature (20~25 ° C), stirring reaction for 48 hours, collecting the AHU-377(R)-(+)-α-phenethylamine salt crystal form obtained by the reaction product. Its powder X-ray diffraction pattern is substantially identical to that shown in Fig. 7.

Example 20

Weigh 20 mg (0.049 mmol) of AHU-377 free acid (oily) in a 5.0 mL glass vial, add 1.0 mL of isopropyl acetate, stir to dissolve, and add 5.9 mg (0.049 mmol) of (S)-(-) -α-phenethylamine, stirring reaction for 48 hours, collecting the reaction product to obtain a powder X-ray diffraction pattern of AHU-377(S)-(-)-α-phenethylamine salt crystal form as shown in Fig. 9. Melting point: 140.16 ° C (onset point) as shown in Fig. 10.

Example 21

Weigh 40 mg (0.096 mmol) of AHU-377 free acid (the ratio of AHU-377 to 2S-methyl diastereomer is about 50:50, as shown in Figure 11, AHU-377 retention time is 10 minutes) Left and right, 2S-methyl diastereomer retention time of about 9 minutes) was placed in a 5.0 mL glass bottle, 2.0 mL of isopropyl acetate was added, stirred and dissolved, and 1 mL of 5.9 mg/mL (0.049 mmol) was added ( The solution of S)-(-)-α-phenethylamine acetate isopropyl ester was further stirred for 24 hours, and the AHU-377(S)-(-)-α-phenethylamine salt crystal form obtained by the reaction product was collected. 1 mg of AHU-377(S)-(-)-α-phenethylamine salt was added to 1 mL of 1% aqueous acetic acid solution, and then 1 mL of ethyl acetate was added thereto. After shaking vigorously, the layer was allowed to stand and the ethyl acetate layer was taken. The purity of the diastereomer of AHU-377 was analyzed by chiral chromatography. The HPLC results showed that the chiral purity of AHU-377 was greatly improved. The ratio of AHU-377 to 2S-methyl diastereomer was about 99.5. :0.5 as shown in Figure 12.

Example 22

First, magnesium stearate, colloidal cerium oxide and microcrystalline fiber The vitamins were sieved through a 30 mesh sieve. The above mixture, the active ingredient AHU-377 calcium salt Form I, crosslinked polyvinylpyrrolidone and povidone were then mixed in a hopper mixer for about 120 revolutions. The mixture was pressed with a roller press using a pressure of 30 kN. After pressing, the mixture was milled using a grinder and sieved through a 18 mesh screen to give the final internal phase or granules. The granules are filled in capsules to form capsules.

Example 23

First screening the active ingredient through a 40 mesh sieve AHU-377 calcium salt crystal form I. Microcrystalline cellulose and crosslinked polyvinylpyrrolidone were added to the active ingredient, and the mixture was sieved through a 20 mesh sieve. The mixture was then mixed and rotated about 100 revolutions in a hopper mixer. The low-substituted hydroxypropylcellulose and colloidal ceria were then added to the hopper mixer and rotated 100 revolutions. Finally add magnesium stearate. The powdered mixture is then compressed into tablets.

Preparation stability and dissolution test

The capsule of Example 22 and the tablet of Example 23 were each placed under different conditions, and the stability of the preparation was examined. The results are shown in the table:

The experiment proves that the AHU-377 calcium salt crystal provided by the invention Type I (AHU-377 calcium salt trihydrate) has high purity, stable and reliable quality, greatly improved the hygroscopicity of AHU-377 or its sodium salt, improved chemical stability, etc., which is conducive to product storage and meets industrial production needs. . The obtained AHU-377 calcium salt crystal form I is suitable for clinical drug application, and the prepared capsule can meet medical or pharmaceutical needs. In the treatment of hypertension and heart failure, it can reduce the intake of sodium, reduce the cardiovascular burden of patients, and is conducive to the treatment of cardiovascular diseases. Therefore, the safety and effectiveness of AHU-377 as a calcium salt in the treatment of hypertension and heart failure diseases has obvious advantages over sodium salt.

It should be noted that the above embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art The technical solutions are modified or equivalent, without departing from the spirit and scope of the present invention, and are intended to be included within the scope of the claims.

The picture in this case is experimental data, not the representative figure of this case. Therefore, there is no designated representative map in this case.

Claims (35)

An AHU-377 crystalline free acid having an X-ray powder diffraction pattern comprising peaks at diffraction angles (2θ) at 23.70 ± 0.2 °, 15.84 ± 0.2 °, 20.84 ± 0.2 °, and 8.56 ± 0.2 °; The X-ray powder diffraction pattern further includes peaks at diffraction angles (2θ) of 17.06±0.2°, 25.98±0.2°, 18.01±0.2°, 9.42±0.2°, and 26.91±0.2°; more preferably, its X-ray powder The diffraction pattern further includes peaks at diffraction angles (2θ) of 34.70±0.2°, 21.83±0.2°, 25.69±0.2°, 25.33±0.2°, 11.67±0.2°, and 26.24±0.2°; The ray powder diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in Fig. 1. A method for preparing a crystalline free acid of AHU-377 according to claim 1, wherein the method comprises the steps of: 1) dissolving AHU-377 free acid in a suitable organic solvent; 2) lowering the temperature of the solution system, and / or adding seed crystals, and / or adding an appropriate amount of anti-solvent until the solution appears turbid, crystallization; 3) solid-liquid separation to obtain AHU-377 crystalline free acid. The preparation method according to claim 2, which comprises the steps of: 1) dissolving AHU-377 free acid in a suitable organic solvent at normal temperature or heating; 2) reducing the solution system in step 1) Temperature, and / or seed crystals, crystallization; 3) solid-liquid separation to obtain AHU-377 crystalline free acid. The preparation method according to claim 2, comprising the steps of: 1) dissolving AHU-377 free acid in a suitable organic solvent; 2) mixing the solution in step 1) with an anti-solvent 3) solid-liquid separation to obtain AHU-377 crystalline free acid; preferably, AHU-377 crystalline free acid seed crystal is pre-added to the anti-solvent before mixing with the anti-solvent in step 2). The preparation method according to any one of claims 2-4, wherein the suitable organic solvent in the step 1) is selected from the group consisting of a halogen ether, a cyclic ether, an ester, a halogenated alkane, and a benzene organic. a solvent or a mixture thereof; preferably from ethyl acetate, isopropyl acetate, dichloromethane, trichloroethane, carbon tetrachloride, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene Or a combination thereof, more preferably from isopropyl acetate, dichloromethane, methyl tert-butyl ether, isopropyl ether or a combination thereof. The preparation method according to the second aspect of the invention, wherein the anti-solvent of the step 2) is selected from the group consisting of n-heptane, n-hexane, isooctane, pentane, cyclohexane, Cyclopentane, diethyl ether or a combination thereof; preferably from n-heptane, n-hexane, cyclohexane, diethyl ether or a combination thereof. The preparation method according to any one of claims 2 to 4, wherein the seeding in the step 2) means adding a homologous crystal to the AHU-377 free acid solution, and the seed crystal amount is AHU. The -377 free acid quality ratio is from 0.1 to 20.0%, preferably from 0.5 to 5.0% by mass ratio. The preparation method according to any one of claims 2-4, which The X-ray powder diffraction pattern of the AHU-377 crystalline free acid obtained in the step 3) includes diffraction angles (2θ) at 23.70±0.2°, 15.84±0.2°, 20.84±0.2° and 8.56±0.2°. Peak. An AHU-377 semi-calcium salt crystal form having an X-ray powder diffraction pattern comprising peaks at diffraction angles (2θ) of 12.70 ± 0.2 °, 7.32 ± 0.2 °, 15.90 ± 0.2 ° and 18.56 ± 0.2 °, or X thereof The ray powder diffraction pattern includes peaks at diffraction angles (2 theta) at 4.02 ± 0.2 °, 3.62 ± 0.2 °, and 17.82 ± 0.2 °. An AHU-377 hemi-calcium salt trihydrate crystal form having an X-ray powder diffraction pattern comprising peaks at diffraction angles (2θ) of 12.70 ± 0.2 °, 7.32 ± 0.2 °, 15.90 ± 0.2 °, and 18.56 ± 0.2 °; Preferably, the X-ray powder diffraction pattern further comprises peaks at diffraction angles (2θ) of 14.74 ± 0.2 °, 15.42 ± 0.2 °, 7.82 ± 0.2 °, 19.42 ± 0.2 °, 16.64 ± 0.2 ° and 17.54 ± 0.2 °. More preferably, the X-ray powder diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in FIG. An AHU-377 semi-calcium salt crystalless type, the X-ray powder diffraction pattern of which includes peaks at diffraction angles (2θ) of 4.02±0.2°, 3.62±0.2° and 17.82±0.2°; preferably X-ray powder thereof The diffraction pattern also includes peaks at diffraction angles (2θ) of 20.48±0.2°, 14.50±0.2°, and 19.66±0.2°; more preferably, the X-ray powder diffraction pattern and the diffraction angle (2θ) shown in FIG. The peaks at the place are basically the same. A method for preparing a crystalline form of AHU-377 hemi-calcium salt trihydrate, which comprises the following steps: 1) dissolving or suspending AHU-377 free acid in a suitable solvent Preparing a corresponding salt of AHU-377 with an equal or excess amount of caustic or weakly basic sodium or potassium salt soluble in the system, preferably with an equal or excess amount of weakly basic sodium salt Or the potassium salt phase is mixed; 2) mixing the above AHU-377 salt system with the water-soluble calcium salt in water or an organic solvent-containing water system to produce AHU-377 calcium salt precipitation; 3) collecting the above salt formation reaction process The product gives the AHU-377 hemi-calcium salt trihydrate crystalline form; the suitable solvent for step 1) is a crystallization solvent, including water, a water soluble solvent or a mixture thereof. The preparation method according to claim 12, wherein the water-soluble solvent is selected from the group consisting of alcohols, ketones, cyclic ethers, guanamines, sulfonium-based organic solvents or mixtures thereof, preferably from methanol. Ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl hydrazine or a mixture thereof. The preparation method according to claim 12, wherein the caustic alkali of the step 1) is sodium hydroxide or potassium hydroxide, and the weakly basic sodium salt is selected from the group consisting of sodium carbonate, sodium hydrogencarbonate, sodium acetate, Sodium formate, sodium propionate, sodium acrylate, sodium benzoate or a mixture thereof, preferably sodium hydrogencarbonate, sodium acetate or a mixture thereof, the weakly basic potassium salt is selected from the group consisting of potassium carbonate, potassium hydrogencarbonate, potassium acetate, potassium formate, Potassium propionate, potassium acrylate, potassium benzoate or a mixture thereof, preferably potassium hydrogencarbonate, potassium acetate or a mixture thereof; step 2) the water-soluble calcium salt is selected from the group consisting of calcium chloride, calcium bromide, calcium iodide, Calcium nitrate, calcium chlorate, calcium hypochlorite, calcium perchlorate, lactic acid Calcium, calcium gluconate or a mixture thereof is preferably selected from the group consisting of calcium chloride, calcium chlorate or mixtures thereof. The preparation method according to claim 12, wherein the powder X-ray diffraction pattern of the AHU-377 calcium salt crystal form I obtained in the step 3) is located at 12.70±0.2°, 7.32±0.2°, 15.90±0.2. The peak at the diffraction angle (2θ) of ° and 18.56±0.2°, preferably the X-ray powder diffraction pattern is also included at 14.74±0.2°, 15.42±0.2°, 7.82±0.2°, 19.42±0.2°, 16.64± Peak at diffraction angle (2θ) of 0.2° and 17.54±0.2°. The invention discloses a preparation method of AHU-377 semi-calcium salt crystal-free type, wherein AHU-377 hemi-calcium salt trihydrate is heated to above 50 ° C to be dehydrated and converted into an anhydrous crystal type; preferably, the heating temperature range is 50- 100 ° C. The preparation method according to claim 16, wherein the obtained AHU-377 hemi-calcium salt crystal-free type has an X-ray powder diffraction pattern of 4.02±0.2°, 3.62±0.2° and 17.82±0.2°. The peak at the diffraction angle (2θ); preferably, the X-ray powder diffraction pattern further includes peaks at diffraction angles (2θ) of 20.48 ± 0.2 °, 14.50 ± 0.2 °, and 19.66 ± 0.2 °. An AHU-377 alpha-phenethylamine salt having an X-ray powder diffraction pattern comprising peaks at diffraction angles (2 theta) at 20.58 ± 0.2 °, 24.28 ± 0.2 °, 8.38 ± 0.2 ° and 23.20 ± 0.2 °, or The peak at the diffraction angle (2θ) at 23.28 ± 0.2 °, 18.9 ± 0.2 °, 13.7 ± 0.2 °, and 14.72 ± 0.2 °. A crystalline form of AHU-377(R)-(+)-α-phenethylamine salt having an X-ray powder diffraction pattern comprising diffraction at 20.58±0.2°, 24.28±0.2°, 8.38±0.2° and 23.20±0.2° a peak at an angle (2θ); preferably an X-ray powder The final diffraction pattern also includes peaks at diffraction angles (2θ) at 19.36 ± 0.2 °, 15.16 ± 0.2 °, 16.78 ± 0.2 °, 18.84 ± 0.2 °, and 22.06 ± 0.2 °; more preferably, the X-ray powder diffraction pattern is complex. Also included are peaks at diffraction angles (2θ) at 17.34±0.2°, 7.52±0.2°, 28.10±0.2°, 29.66±0.2°, 28.94±0.2°, and 10.96±0.2°; optimal X-ray powder diffraction The graph is substantially the same as the peak at the diffraction angle (2θ) shown in Fig. 7. An AHU-377(S)-(-)-α-phenethylamine salt crystal form having an X-ray powder diffraction pattern comprising diffraction at 23.28±0.2°, 18.9±0.2°, 13.7±0.2° and 14.72±0.2° a peak at an angle (2θ); preferably a peak at a diffraction angle (2θ) of 3.13 ± 0.2°, 15.98 ± 0.2°, 18.36 ± 0.2°, 9.12 ± 0.2°, and 32.38 ± 0.2°; more preferably The X-ray powder diffraction pattern further includes peaks at diffraction angles (2θ) at 10.88±0.2°, 22.18±0.2°, 21.92±0.2°, 27.66±0.2°, 20.34±0.2°, and 27.24±0.2°; The optimum is that the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in Fig. 9. A method for preparing AHU-377 α-phenethylamine salt crystal form, comprising: 1) dissolving or suspending AHU-377 free acid in a suitable solvent, and mixing with an equal amount or excess of α-phenethylamine to prepare AHU-377 α-phenethylamine salt; 2) collecting the product in the above salt formation reaction to obtain AHU-377 α-phenethylamine salt crystal form; the α-phenethylamine includes (R)-(+)-α - phenethylamine, (S)-(-)-α-phenethylamine; a suitable solvent for the step 1) is a crystallization solvent, including water, A water soluble solvent or a water insoluble solvent. The preparation method according to claim 21, wherein the water-soluble solvent is selected from the group consisting of alcohols, ketones, cyclic ethers, guanamines, hydrazine-based organic solvents or mixtures thereof, preferably from methanol. , ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl hydrazine or mixtures thereof; The water-insoluble solvent is selected from the group consisting of chloroalkanes, ethers, esters, alkanes, cycloalkanes, benzene organic solvents or mixtures thereof, preferably from ethyl acetate, isopropyl acetate, dichloromethane, trichloroethylene. Ethane, carbon tetrachloride, methyl tertiary butyl ether, diisopropyl ether, benzene, toluene, xylene or a combination thereof. The preparation method according to claim 21, wherein the AHU-377(R)-(+)-α-phenethylamine salt obtained in the step 2) has an X-ray powder diffraction pattern of 20.58±0.2°. , a peak at a diffraction angle (2θ) of 24.28±0.2°, 8.38±0.2° and 23.20±0.2°, or an X-ray powder of the obtained AHU-377(S)-(-)-α-phenethylamine salt The diffraction pattern includes peaks at diffraction angles (2θ) at 23.28 ± 0.2 °, 18.9 ± 0.2 °, 13.7 ± 0.2 °, and 14.72 ± 0.2 °. A pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective dose of AHU-377 crystalline free acid as described in claim 1 of the patent application, AHU-377 hemi-calcium salt as described in claim 9 The AHU-377 hemi-calcium salt trihydrate crystal form as described in claim 10 of the patent application, and the AHU-377 hemi-calcium salt crystal form as described in claim 11 of the patent application, as claimed in the patent application 18 items The AHU-377 α-phenethylamine salt crystal form, the AHU-377(R)-(+)-α-phenethylamine salt crystal form described in claim 19 or as claimed in claim 20 The AHU-377(S)-(-)-α-phenethylamine salt crystal form and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to claim 24, wherein the pharmaceutically acceptable carrier or excipient is selected from the group consisting of a diluent or a filler, a disintegrant, a binder, a glidant, a lubricant, and a colorant. Or a combination thereof. The pharmaceutical composition according to claim 24, wherein the diluent or filler is selected from the group consisting of powdered sugar, compressible sugar, glucose, sucrose, lactose, dextrin, mannitol, microcrystalline cellulose, and sorbus The alcohol, the starch or a combination thereof, the diluent or the filler is used in an amount of 4.0% to 60.0% by weight of the composition, preferably 10.0% to 40.0%; and the disintegrants are selected from the group consisting of starch, clay, cellulose, and alginic acid. Salt, gum, crosslinked polymer, soybean polysaccharide, guar gum or a combination thereof, preferably crosslinked polyvinylpyrrolidone, croscarmellose sodium, crosslinked carboxymethyl fiber Calcium or a combination thereof, the disintegrant is used in an amount of 0% to 65.0% by weight of the composition, preferably 1.0% to 40.0%; and the binder is selected from the group consisting of starch, cellulose and derivatives thereof, sucrose, glucose, Corn syrup, gelatin, povidone or a combination thereof, preferably from cellulose microcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose or Combination; better hydroxypropyl cellulose; best low-substituted hydroxypropyl cellulose, adhesive 1.0% to 60.0% of the composition by weight, preferably 5.0 to 40.0% based, more preferably 10.0% ~ 30.0% based; such glidant or lubricant selected from colloidal silica Barium, magnesium triacetate, starch, talc, calcium orthophosphate, magnesium stearate, aluminum stearate, calcium stearate, calcium carbonate, magnesium oxide, polyethylene glycol, powdered cellulose, behenic acid Glyceride, stearic acid, hydrogenated castor oil, glyceryl monostearate, sodium stearyl fumarate or a combination thereof, the amount of glidant or lubricant is from 0% to 10.0% by weight of the composition, Good system 0.5%~5.0%. The pharmaceutical composition according to claim 24, wherein the pharmaceutical composition comprises a diuretic; the diuretic is selected from the group consisting of furosemide, etalic acid, bumetanide, torasemide , hydrochlorothiazide, chlorthalidone, benzyl fluorothiazide, cyclopentazine, policothiazine, metoprazine, indapamide or a combination thereof; the diuretic is used in an amount of 0.1% to 10.0% by weight of the composition, Preferably, it is 0.5% to 5.0%. The pharmaceutical composition according to claim 24, wherein the pharmaceutical composition can be prepared into tablets, capsules, granules. The pharmaceutical composition according to claim 24, wherein the capsule group is distributed as follows: The pharmaceutical composition according to claim 24, wherein the capsule group is distributed as follows: The AHU-377 crystalline free acid as described in claim 1 of the patent application, the AHU-377 hemi-calcium salt crystal form according to claim 9 of the patent application, and the AHU-377 as described in claim 10 The semi-calcium salt trihydrate crystal form, the AHU-377 hemi-calcium salt crystal-free type as described in claim 11, the AHU-377 α-phenethylamine salt crystal form as described in claim 18 AHU-377(R)-(+)-α-phenethylamine salt crystal form as described in claim 19, AHU-377(S)-(- as described in claim 20 The ?-phenethylamine salt crystal form or the pharmaceutical composition according to any one of claims 24 to 30 for the treatment or prevention of diseases associated with neutral endopeptidase, cardiovascular, high resistance The use of blood pressure drugs; such antihypertensive drugs are selected from the group consisting of anti-malignant hypertension, essential hypertension, renal vascular hypertension, diabetic hypertension, solitary systolic hypertension or other secondary high Blood pressure drugs. The AHU-377 crystalline free acid as described in claim 1 of the patent application, the AHU-377 hemi-calcium salt crystal form according to claim 9 of the patent application, and the AHU-377 as described in claim 10 Semi-calcium salt three Hydrate crystal form or AHU-377 hemi-calcium salt crystal form as described in claim 11 of the patent application, AHU-377 α-phenethylamine salt crystal form as described in claim 18, if applied AHU-377(R)-(+)-α-phenethylamine salt crystal form described in claim 19, AHU-377(S)-(-)-α as described in claim 20 Use of a phenethylamine salt crystal form or a pharmaceutical composition according to any one of claims 24 to 30 for the preparation of a medicament for treating or preventing acute or chronic heart failure or harmful vascular remodeling, preferably These acute and chronic heart failures are selected from the group consisting of congestive heart failure, left ventricular dysfunction, hypertrophic cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmia, atrial fibrillation, and atrial flutter. The pharmaceutical composition according to claim 24, wherein the pharmaceutical composition further comprises an angiotensin II receptor antagonist. The pharmaceutical composition according to claim 33, wherein the angiotensin II antagonist is selected from the group consisting of losartan, irbesartan, olmesartan, telmisartan, valsartan, and azide. Sartan, candesartan, eprosartan, losartan, saprisartan, ilartartan, tamsaltan, elsartan or a pharmaceutically acceptable salt thereof; preferably from valsartan, It is valsartan or a pharmaceutically acceptable salt thereof. A pharmaceutical composition according to any one of claims 33 to 34 for the treatment or prevention of myocardial infarction and its sequelae, atherosclerosis, angina pectoris, diabetic or non-diabetic renal insufficiency, secondary Hyperaldosteronism, primary or secondary pulmonary hypertension, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, Use of proteinuria, renal vascular hypertension, diabetic retinopathy, migraine, peripheral vascular disease, Raynaud's disease, hyperplasia of the cavity, cognitive dysfunction, glaucoma or stroke medication of primary nephropathy.