WO2016086798A1 - Nep inhibitor crystalline free acid, calcium salt polymorph of same, preparation method for same, and applications thereof - Google Patents

Abstract

Disclosed are a crystalline free acid of a NEP inhibitor, a calcium salt polymorph of same, a preparation method for same, and applications thereof. The present invention relates to a crystalline free acid of the NEP inhibitor compound (also known as HAS-000129) as represented by formula 1, a calcium salt polymorph of same, a preparation method for same, and applications thereof. The powder x-ray diffraction pattern of the HAS-000129 crystalline free acid comprises peaks located at diffraction angles (2θ) 6.4±0.2°, 15.9±0.2°, 20.8±0.2°, and 19.0±0.2°. The present invention also relates to a pharmaceutical composition containing an efficacious dose of the HAS-000129 crystalline free acid and the calcium salt polymorph and uses of the composition in preparing medicaments for treatment or prevention of neutral endopeptidase-related diseases, cardiovascular diseases, hypertension, acute and chronic heart failure, left ventricular dysfunction, hypertrophic cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter or detrimental vascular remodeling. The HAS-000129 process of the present invention has great operability, physical and chemical stability, and facilitated preservation and pharmaceutical application.

Description

Crystallized free acid and calcium salt polymorph of NEP inhibitor, preparation method and application thereof Technical field

The invention belongs to the technical field of medicines, and particularly relates to a NEP inhibitor HAS-000129 crystalline free acid, calcium salt polymorph and a preparation method and application thereof.

Background technique

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 family of vasodilatation, diuretic and antihypertensive peptides, one form - ANF 99-126 is a circulating 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 circulation: receptor-mediated clearance and enzyme inactivation at NEP. NEP inhibitors enhance hypotension, diuresis, natriuresis, and plasma ANF response in experimental animals following pharmacological ANF injection. 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.

Shanghai Hansen Company Patent CN201410001940.2 discloses a class of biaryl substituted 4-aminobutyric acid derivatives, which are found to have significant NEP inhibitory activity, and one of the most representative compounds is Formula 1 Show (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3,5 , 7-trioxa-12-azahexadecane-16-acid (also known as HAS-000129).

Equation 1 (HAS-000129)

In the prior art, HAS-000129 is an oily substance, which is difficult to purify, solvent residue is difficult to reach the standard, and there are great problems in material stability, storage, and weighing, and it is difficult to prepare an oral solid preparation, which is disadvantageous for pharmaceutical development and industrial production.

Summary of the invention

In order to solve the problems existing in the prior art, the physical and chemical properties of HAS-000129 are improved. There is an urgent need to transform HAS-000129 oil into a solid state, particularly in a crystalline state, to meet the needs of pharmaceutical development and industrial production. The inventors have intensively studied the different aggregation states of HAS-000129, and developed HAS-000129 crystalline free acid and calcium salt crystalline solids based on the prior art, which makes the crystallinity, solubility, hygroscopicity, etc. of HAS-000129. The physical and chemical properties have been completely changed, and the chemical stability of the materials is conducive to the development of storage and oral solid preparations. By converting HAS-000129 from an oil to a crystalline solid, significant progress can be made in separating and purifying it, removing residual organic solvents, improving chemical stability, and facilitating the preparation of solid preparations.

In one aspect, the invention provides a HAS-000129 crystalline free acid (designated as Form I) having a powder X-ray diffraction pattern comprising at 6.4 ± 0.2°, 15.9 ± 0.2°, 20.8 ± 0.2°, and 19.0 ± 0.2°. The peak at the diffraction angle (2θ).

As a further preferred embodiment, the powder X-ray diffraction pattern further includes peaks at diffraction angles (2θ) at 20.4 ± 0.2 °, 19.2 ± 0.2 °, 26.0 ± 0.2 °, 18.0 ± 0.2 °, and 7.9 ± 0.2 °.

As a still further preferred embodiment, the powder X-ray diffraction pattern further includes diffraction angles (2θ at 25.70±0.2°, 7.5±0.2°, 24.7±0.2°, 20.2±0.2°, 10.2±0.2°, and 16.5±0.2°). ) The peak at the place.

As a most preferred embodiment, the powder X-ray 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 shown in Table 1:

Table 1

2θ(°) strength% 2θ(°) strength% 6.4 100 20.2 10 7.5 12.1 20.4 27.5 7.9 16.6 20.8 42.9 10.2 9.4 21.8 8.4 12.0 4.4 22.1 5.8 15.0 6.3 22.9 5.2 15.9 62.8 24.6 7.9

16.5 8.5 24.7 10.6 18.0 18.9 25.7 13.5 19.0 27.5 26.0 20.2 19.2 20.9 27.7 7.3 19.6 6

The melting point of the HAS-000129 crystalline free acid (Form I) was about 131.5 ° C (onset point) as measured by a differential calorimeter.

Another aspect of the invention also provides a HAS-000129 calcium salt polymorph (designated as Form I) having a powder X-ray diffraction pattern comprising at 20.8 ± 0.2°, 11.3 ± 0.2°, 3.9 ± 0.2° and 19.2 ± A peak at a diffraction angle (2θ) of 0.2°.

As a further preferred embodiment, the powder X-ray diffraction pattern further includes 11.7±0.2°, 14.8±0.2°, 20.3±0.2°, 5.6±0.2°, and 19.9±0.2°.

As a still further preferred embodiment, the powder X-ray diffraction pattern further includes diffraction angles at 13.5±0.2°, 5.9±0.2°, 12.7±0.2°, 24.8±0.2°, 14.6±0.2°, and 31.6±0.2° ( The peak at 2θ).

As a most preferred embodiment, the powder X-ray 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:

Table 2

2θ(°) strength% 2θ(°) strength% 3.9 55.2 14.8 40.9 5.6 34.5 19.2 45 5.9 31.7 19.9 33.2 8.7 26.3 20.3 39.6 11.3 70.1 20.8 100 11.7 41.7 23.8 27.1 12.7 30.9 24.8 30.9 13.5 32.2 31.6 27.4 14.6 28.1

The term "substantially the same" as used herein with respect to the position of the X-ray diffraction peak 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 usage, and crystal orientation will result in changes in relative peak intensities in the XRPD diffraction pattern of the sample.

Another aspect of the present invention provides a method for preparing a HAS-000129 crystalline free acid, comprising

1) dissolving HAS-000129 free acid in a suitable organic solvent;

2) adding an appropriate amount of anti-solvent under stirring until the solution becomes cloudy or a small amount of seed crystal is added, and stirring and crystallization are continued;

3) Solid-liquid separation gave HAS-000129 crystalline free acid.

As a further preferred embodiment, the organic solvent in the step 1) is selected from the group consisting of alcohols, chlorinated alkanes, ketones, ethers, cyclic ethers, esters, alkanes, cycloalkanes, benzenes, amides, sulfoxides. 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-dimethyl Carboxamide, dimethyl sulfoxide, ethyl acetate, isopropyl acetate, dichloromethane, trichloroethane, carbon tetrachloride, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene Or a composition thereof; preferably ethyl acetate, isopropyl acetate, dichloromethane, methyl tert-butyl ether, isopropyl ether or a combination thereof.

The anti-solvent includes, but is not limited to, the following solvents: n-heptane, n-hexane, isooctane, pentane, cyclohexane, cyclopentane, diethyl ether, water or a combination thereof; preferably n-heptane, n-hexane, Ether or a combination thereof.

The dissolution refers to the general operation of a person of ordinary skill in the art, and the raw material can be usually dissolved or dissolved by appropriate heating, or the amount of the solvent is increased to dissolve or dissolve the raw material, or the technical scheme is modified or equivalent. Instead, it should be covered by the inventive content of the present invention.

As a further preferred embodiment, the specific operation is as follows: 1) Dissolving in a suitable organic solvent in HAS-000129 free acid to form a solution having a concentration of 1-200 mg/mL, which is selected from ethyl acetate and acetic acid. Isopropyl ester, dichloromethane, methyl tert-butyl ether, diisopropyl ether or a combination thereof; 2) adding an appropriate amount of anti-solvent n-heptane, n-hexane, diethyl ether or a combination thereof to the solution to cause turbidity, anti-solvent The amount is 1-10 times by volume of the crystallization solvent, and the agitation is continued; 3) solid-liquid separation yields a free acid of HAS-000129 crystal form.

As a further preferred embodiment, the specific operation is as follows: 1) adding a suitable organic solvent to the HAS-000129 free acid, heating to 30-80 ° C to dissolve it to form a solution having a concentration of 1-200 mg/mL, The organic solvent is selected from the group consisting of ethyl acetate, isopropyl acetate, dichloromethane, methyl tert-butyl ether, isopropyl ether or a combination thereof; 2) slowly cooling to 20 to 30 ° C, and adding a mass ratio of 0.1 to 10.0 under stirring % of the homologous crystals act as seed crystals, and continue to stir and crystallize; 3) solid-liquid separation to obtain HAS-000129 crystalline free acid.

Another aspect of the present invention provides a method for preparing a calcium salt polymorph of HAS-000129, comprising

1) Dissolving or dispersing HAS-000129 free acid in a water-soluble organic solvent or an aqueous water-soluble organic solvent, and mixing with an appropriate amount or an excess of a suitable base to prepare a corresponding salt of HAS-000129;

2) mixing the above HAS-000129 salt system with the calcium salt, and continuing to stir and precipitate the HAS-000129 calcium salt crystal;

3) Solid-liquid separation gives the HAS-000129 calcium salt polymorph.

Another aspect of the present invention provides a method for preparing a calcium salt polymorph of HAS-000129, comprising

1) Dissolving HAS-000129 free acid in a water-soluble organic solvent, mixing with an equal amount or excess of a suitable base to prepare a corresponding salt of HAS-000129;

2) mixing the above HAS-000129 salt system with the calcium salt, stirring the reaction, depositing the precipitate, and removing the precipitate by filtration;

3) The above mother liquor is collected, and the HAS-000129 calcium salt polymorph is obtained by a volatile solvent or concentration.

As a further preferred embodiment, the water-soluble organic solvent in the step 1) is selected from the group consisting of alcohols, ketones, amides, sulfoxide 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 sulfoxide or a combination thereof.

As a further preferred embodiment, the suitable base is selected from the group consisting of sodium hydroxide, sodium carbonate, sodium hydrogencarbonate, sodium acetate, sodium formate, sodium propionate, sodium acrylate, sodium benzoate, potassium hydroxide, potassium carbonate, potassium hydrogencarbonate, Potassium acetate, potassium formate, potassium propionate, potassium acrylate, potassium benzoate or a mixture thereof; preferably sodium hydrogencarbonate or potassium hydrogencarbonate; the calcium salt 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 mixtures thereof; preferably from calcium chloride.

The above two methods for preparing the calcium salt polymorph of HAS-000129, step 2) continue to stir until the precipitate is precipitated, which may be HAS-000129 calcium salt, or may be added to the corresponding salt of step 1) adding a base. The difference is mainly whether the entire crystallization system contains water, and the crystallization system contains water to precipitate HAS-000129 calcium salt. The added calcium salt may be added as a solid, or the calcium salt may be added after being dissolved in water or the calcium salt may be dissolved in a suitable organic solvent.

Another aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned HAS-000129 crystalline free acid, a calcium salt polymorph thereof, and a pharmaceutically acceptable carrier or excipient.

Another aspect of the present invention provides the aforementioned HAS-000129 crystalline free acid, a calcium salt polymorph thereof or a pharmaceutical composition thereof for the preparation of a disease for treating or preventing neutral endopeptidase, cardiovascular, antihypertensive Use in medicine.

More effective antihypertensive treatments through improved efficacy and higher response rates, whether for malignant hypertension, essential hypertension, renovascular hypertension, diabetic hypertension, and isolated systolic hypertension It is still true for other secondary hypertension.

Another aspect of the present invention provides the aforementioned HAS-000129 crystalline free acid, a calcium salt polymorph thereof or a pharmaceutical composition thereof for treating or preventing acute or chronic heart failure such as congestive heart failure, left ventricular dysfunction, hypertrophy Use in drugs for cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter or harmful vascular remodeling.

"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 carrier or excipient. 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.

DRAWINGS

1 is a powder X-ray diffraction pattern of the HAS-000129 free acid crystal form in Example 1, the abscissa is an angle 2θ (°), and the ordinate is an intensity;

Figure 2 is a DSC chart of the free acid crystal form of HAS-000129 in Example 1, with the abscissa being the temperature (°C), ordinate hot flow (W/g).

3 is a TGA chart of the HAS-000129 free acid crystal form in Example 1, the abscissa is temperature (° C.), and the ordinate is weight loss ratio (%).

Figure 4 is a powder X-ray diffraction pattern of the calcium salt crystal form of HAS-000129 in Example 5, the abscissa is an angle 2θ (°), and the ordinate is an intensity;

Fig. 5 is a TGA diagram of the calcium salt crystal form of HAS-000129 in Example 5, wherein the abscissa is temperature (°C) and the ordinate is weight loss ratio (%).

Fig. 6 is a dissolution curve of the HAS-000129 calcium salt capsule of Example 8, the abscissa is time (minutes), and the ordinate is release degree (%).

Fig. 7 is a dissolution curve of the crystalline HAS-000129 free acid capsule of Example 9, the abscissa is time (minutes), and the ordinate is release degree (%).

detailed description

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

Methods and materials

以反射方式操作的GADDS(一般面积衍射检测器系统)CS的Bruker D8Discover X射线粉末衍射仪上采集所述盐的X射线粉末衍射图。管电压和电流量分别设置为40kV和40mA采集扫描。在3.0°至40°的2θ范围内扫描样品60秒的时期。针对2θ表示的峰位置，使用刚玉标准品校准衍射仪。在通常是20℃-30℃的室温下实施所有分析。使用用于4.1.14T版WNT软件的GADDS，采集和积分数据。使用2003年发行的具有9.0.0.2版Eva的DiffracPlus软件，分析衍射图。XRPD样品的制备，通过是将样品至于单晶硅片上，用玻璃片或等效物压样品粉末以确保样品的表面平坦并有适当的高度。然后将样品支架放入Bruker XRPD仪器，并使用上文描述的仪器参数采集粉末X射线衍射图。由包括以下的多种因素产生与这类X射线粉末衍射分析结果相关的测量差异：(a)样品制备物(例如样品高度)中的误差，(b)仪器误差，(c)校准差异，(d)操作人员误差(包括在测定峰位置时出现的那些误差)，和(e)物质的性质(例如优选的定向误差)。校准误差和样品高度误差经常导致所有峰在相同方向中的位移。一般地说，这个校准因子将使测量的峰位置与预期的峰位置一致并且可以在预期的2θ值±0.2°的范围中。The HAS-000129 crystalline free acid and its calcium salt polymorph are characterized by their X-ray powder diffraction pattern. Therefore, in the presence of Cu Kα radiation

The X-ray powder diffraction pattern of the salt was taken on a Bruker D8 Discover X-ray powder diffractometer of GADDS (General Area Diffraction Detector System) CS operating in a reflective manner. 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. Data was acquired and integrated using GADDS for the 4.1.14T version of WNT software. The diffraction pattern was analyzed using the DiffracPlus software released in 2003 with version 9.0.0.2 Eva. The XRPD sample is prepared by passing the sample onto a single crystal silicon wafer and 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 a powder X-ray 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 variety of factors including: (a) errors in sample preparation (eg, sample height), (b) instrument error, (c) calibration differences, ( d) operator error (including those that occur 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 align the measured peak position to the expected peak position and may be in the range of the expected 2θ value ± 0.2°.

The angle 2θ (°) value and the intensity value (as the % of the highest peak) of each polymorph obtained in the examples of the present invention are listed in Tables 1 to 2.

The raw material HAS-000129 free acid oil used in the examples of the present invention was prepared according to Example 11 of the Chinese patent CN201410001940.2, and other raw materials or municipal grades were commercially available products.

Example 1

Weigh 20mg HAS-000129 free acid (oily) in a 4.0mL glass bottle, then add 0.5mL of normal solvent methyl tert-butyl ether, stir and dissolve, slowly add 2.0mL anti-solvent n-heptane, room temperature (20 -25 ° C) stirring for 48 hours, solid-liquid separation to obtain HAS-000129 crystalline free acid, the powder X-ray diffraction pattern shown in Figure 1.

The melting point of the free acid crystal form was measured using a differential calorimeter (DSC, model Perkins Elmer 8500). The measurement conditions were from room temperature to 160 ° 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 free acid crystal form is shown in Figure 2. The melting point (onset point) of the free acid crystal form is: 131.5 degrees Celsius.

The thermal weight loss of the free acid crystal form was measured using a thermogravimetric analyzer (TGA, model Netzsch F3). The measurement conditions were from room temperature to 350 ° 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 free acid crystal form is shown in Figure 3. There is almost no weight loss within 100 degrees Celsius, so it can be judged that the free acid crystal form of the compound is an amorphous type.

Example 2

Weigh 20mg HAS-000129 free acid (oily) in a 4.0mL glass bottle, then add 0.3mL of normal solvent isopropyl acetate, heat to 50 ° C, stir to dissolve, slowly add 2.0mL anti-solvent n-hexane, cool to room temperature (20-25 ° C), stirring was continued for 48 hours, and solid-liquid separation gave HAS-000129 crystalline free acid, and the powder X-ray diffraction pattern thereof was substantially identical to that of FIG.

Example 3

Weigh 20mg HAS-000129 free acid (oily) in a 4.0mL glass bottle, then add 0.5mL ethyl acetate / n-hexane (20:80, v / v), heat to 60 ° C, stir and dissolve, cool to room temperature (20-25 ° C), 0.2 mg of the crystal of Example 1 was slowly added, stirring was continued for 72 hours, and solid-liquid separation was carried out to obtain a free acid of HAS-000129 crystal form, and the powder X-ray diffraction pattern thereof was substantially identical to that of FIG.

Example 4

Weigh 20mg HAS-000129 free acid (oily) in a 4.0mL glass bottle, then add 0.2mL dichloromethane, heat to 40 ° C, stir to dissolve, cool to room temperature (20-25 ° C), slowly add 0.2mg The crystal of Example 1 was stirred for 72 hours, and solid-liquid separation gave HAS-000129 crystalline free acid, and the powder X-ray diffraction pattern thereof was substantially identical to that of FIG.

Example 5

To 30 g (58.45 mmol) of HAS-000129 free acid (oily), 300 mL of acetone was added and stirred to dissolve. 4.91 g (58.45 mmol) of NaHCO _{3 was} weighed, dissolved in 550 mL of water, slowly added dropwise to the above acetone solution, and the addition was completed. Stirring reaction was continued for 1 h, and the solution was rotary evaporated to no fraction at 40 ° C (acetone was removed, and the fraction was collected to be about 250 mL). ). 3.24 g of anhydrous CaCl ₂ (29.3 mmol) was weighed and dissolved in 450 mL of water, and slowly added dropwise to the above fraction. After the dropwise addition was completed, the reaction was further stirred for 24 hours, filtered, and dried under vacuum at 40 ° C for 20 hours to obtain a white solid 29.72 g. The ray diffraction pattern is shown in Figure 4.

The thermal weight loss of the HAS-000129 calcium salt crystal form was measured using a thermogravimetric analyzer (TGA, model TA Q500). The measurement conditions were from room temperature to 350 ° 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 HAS-000129 calcium salt crystal form is shown in the figure. The weight loss of about 3.0% within 100 degrees Celsius is due to dehydration, so it can be judged that the crystal form is HAS-000129 calcium salt monohydrate (the monohydrate theoretical dehydration weight loss is 3.2%).

Example 6

To 20 mg (0.039 mmol) of HAS-000129 free acid (oily), 0.4 mL of methanol was added and stirred to dissolve. 2.4 mg (0.043 mmol) of KOH was weighed, dissolved in 1.0 mL of methanol, and slowly added dropwise to the above-mentioned HAS-000129 methanol solution. After the dropwise addition was completed, the reaction was further stirred for 2 hours. 2.6 mg (0.023 mmol) of anhydrous CaCl _{2 was} added and dissolved in 1.0 mL of methanol, and the mixture was slowly added dropwise to the above reaction system. After the dropwise addition was completed, the reaction was further stirred for 24 hours, and the solid was collected by filtration, and the filtrate was concentrated to dryness. Then, it was dried under vacuum at 40 ° C for 20 hours to obtain 20.2 mg of a white solid. The powder X-ray diffraction pattern was consistent with that of FIG.

Example 7

In order to compare the chemical stability of the oil, crystal form and calcium salt of HAS-000129 free acid, HAS-000129 free acid oil, crystalline HAS-000129 free acid (prepared in this patent example 1) and HAS-000129 The calcium salt (prepared in the fifth embodiment of the patent) was placed in an incubator at 80 degrees Celsius, and after one week of standing, the purity was measured by a high performance liquid phase method, and the stability test results were as follows:

The test proves that the chemical stability of the crystalline form and calcium salt of HAS-000129 free acid after thermal acceleration test is much better than that of HAS-000129 free acid oil.

Example 8

(HAS-000129 calcium salt capsule)

Component Weight (mg) Weight percentage HAS-000129 calcium salt monohydrate 25 62.2% Microcrystalline cellulose 7 17.4% Low substituted hydroxypropyl cellulose (L-HPC) 5 12.4% Cross-linked povidone (PVP) 2 5.0% Talc powder (Talc) 0.4 1.0% Colloidal silica 0.2 0.5% Magnesium stearate 0.6 1.5% total 40.2 100%

1. Capsule preparation method

Magnesium stearate, colloidal silica and microcrystalline cellulose were first sieved through a 30 mesh screen. The above mixture, active ingredient HAS-000129 calcium salt, low substituted hydroxypropyl cellulose (L-HPC) and povidone, talc, colloidal silica 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.

2, dissolution determination method

The dissolution test of the obtained capsules was carried out on the ERWEKA DT827LH dissolution apparatus. The specific dissolution methods and conditions were as follows: Take this product, according to the Chinese Pharmacopoeia (2010 edition, part 2) dissolution measurement method (Appendix X C second method), pH 6.8 phosphate buffer 900ml is the dissolution medium, the rotation speed is 100 rpm, and it is operated according to law. 10 ml was sampled at 10, 20, 40, and 60 minutes, respectively, and the drug concentration was determined by HPLC to calculate the release percentage.

3. Test process and results

25 g of HAS-000129 calcium salt monohydrate was weighed, and the auxiliary materials were weighed according to the above ratio, and the weighed drugs and auxiliary materials were mixed by a V-type powder mixer for 24 hours, and the capsules were filled with capsule plates. The capsule is a gelatin capsule with a model number of zero. The loading of each capsule was 40.2 mg ± 5%. The obtained capsules were taken for dissolution test. The test conditions were as follows: the dissolution medium was pH 6.8 phosphate buffer, the temperature was 37 ° C, the basket method, and the rotation speed was 100 rpm, and samples were taken at 10, 20, 40, and 60 minutes, respectively. The capsule dissolution results of HAS-000129 calcium salt are shown in Fig. 6.

Example 9

(crystalline HAS-000129 free acid capsule)

Component Weight (mg) Weight percentage Crystalline HAS-000129 free acid 25 62.2% Microcrystalline cellulose 7 17.4% Low substituted hydroxypropyl cellulose (L-HPC) 5 12.4% Cross-linked povidone (PVP) 2 5.0% Talc powder (Talc) 0.4 1.0% Colloidal silica 0.2 0.5%

Magnesium stearate 0.6 1.5% total 40.2 100%

1. Capsule preparation method

Magnesium stearate, colloidal silica and microcrystalline cellulose were first sieved through a 30 mesh screen. The above mixture, active ingredient crystalline HAS-000129 free acid, low substituted hydroxypropyl cellulose (L-HPC) and povidone, talc, colloidal silica 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.

2, dissolution determination method

The dissolution test of the obtained capsules was carried out on the ERWEKA DT827LH dissolution apparatus. The specific dissolution methods and conditions were as follows: Take this product, according to the Chinese Pharmacopoeia (2010 edition, part 2) dissolution measurement method (Appendix X C second method), pH 6.8 phosphate buffer 900ml is the dissolution medium, the rotation speed is 100 rpm, and it is operated according to law. 10 ml was sampled at 10, 20, 40, and 60 minutes, respectively, and the drug concentration was determined by HPLC to calculate the release percentage.

3. Test process and results

The crystalline HAS-000129 free acid 25 g was weighed, and the auxiliary materials were weighed according to the above ratio, and the weighed drugs and auxiliary materials were mixed with a V-type powder mixer for 24 hours, and the capsules were filled with capsule plates. The capsule is a gelatin capsule with a model number of zero. The loading of each capsule was 40.2 mg ± 5%. The obtained capsules were taken for dissolution test. The test conditions were as follows: the dissolution medium was pH 6.8 phosphate buffer, the temperature was 37 ° C, the basket method, and the rotation speed was 100 rpm, and samples were taken at 10, 20, 40, and 60 minutes, respectively. The dissolution results of the crystalline HAS-000129 free acid capsule are shown in Fig. 7.

The inventors also attempted to prepare the oily HAS-000129 free acid into a capsule. However, since the oily substance was mixed with the above-mentioned auxiliary material to form a briquettes, it was not uniformly dispersed into a fluid powder, and the filling of the capsule could not be performed. Therefore, the oily HAS-000129 free acid cannot be made into a capsule.

Tests have shown that the HAS-000129 crystalline free acid and its calcium salt prepared by the present invention can be conveniently prepared into an oral solid preparation, and the oily substance cannot be developed into an oral solid preparation.

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 invention, and are intended to be included within the scope of the appended claims.

Claims (12)

(9R,11S)-11-([1,1'-Biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-three represented by Formula 1 a crystalline free acid of oxo-3,5,7-trioxa-12-azahexadecane-16-acid,

The powder X-ray diffraction pattern includes peaks at diffraction angles (2θ) of 6.4 ± 0.2°, 15.9 ± 0.2°, 20.8 ± 0.2°, and 19.0 ± 0.2°; preferably, the powder X-ray diffraction pattern thereof also includes 20.4 ± 0.2 °, 19.2 ± 0.2 °, 26.0 ± 0.2 °, 18.0 ± 0.2 ° and 7.9 ± 0.2 ° the peak at the diffraction angle 2θ; more preferably the powder X-ray diffraction pattern further includes 25.70 ± 0.2 °, 7.5 ± 0.2 ° , a peak at a diffraction angle 2θ of 24.7±0.2°, 20.2±0.2°, 10.2±0.2°, and 16.5±0.2°; most preferably a powder X-ray diffraction pattern and a peak at a diffraction angle (2θ) shown in FIG. Basically the same. (9R,11S)-11-([1,1'-Biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-three represented by Formula 1 a calcium salt polymorph of oxo-3,5,7-trioxa-12-azahexadecane-16-acid,

The powder X-ray diffraction pattern includes peaks at diffraction angles 2θ of 20.8 ± 0.2°, 11.3 ± 0.2°, 3.9 ± 0.2°, and 19.2 ± 0.2°; preferably, the powder X-ray diffraction pattern further includes at 11.7 ± 0.2°. 14.8±0.2°, 20.3±0.2°, 5.6±0.2°, 19.9±0.2°; more preferably, the powder X-ray diffraction pattern further includes 13.5±0.2°, 5.9±0.2°, 12.7±0.2°, 24.8±0.2. °, 14.6 ± 0.2°, and a peak at a diffraction angle 2θ of 31.6 ± 0.2°; most preferably, the powder X-ray diffraction pattern thereof is substantially the same as the peak at the diffraction angle 2θ shown in FIG. (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4 represented by Formula 1 according to Claim 1. A method for preparing a crystalline free acid of 8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid, comprising: 1) (9R,11S)-11-([1,1'-Biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3 , 5,7-trioxa-12-azahexadecane-16-acid free acid is dissolved in a suitable organic solvent; 2) adding an appropriate amount of anti-solvent to the solution after turbidity, or adding seed crystals, and stirring and crystallization; 3) Solid-liquid separation gives (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxane Generation of -3,5,7-trioxa-12-azahexadecane-16-acid crystalline free acid; Step 1) The organic solvent is selected from the group consisting of alcohols, chlorinated alkanes, ketones, ethers, cyclic ethers, esters, alkanes, cycloalkanes, benzenes, amides, sulfoxides, organic solvents, or combinations thereof. Preferred from methanol, ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl sulfoxide, acetic acid Ethyl ester, isopropyl acetate, dichloromethane, trichloroethane, carbon tetrachloride, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene or a combination thereof; more preferably from ethyl acetate , isopropyl acetate, dichloromethane, methyl tert-butyl ether, isopropyl ether or a combination thereof; Step 2) The anti-solvent is selected from the group consisting of n-heptane, n-hexane, isooctane, pentane, cyclohexane, cyclopentane, diethyl ether, water or a combination thereof; preferably from n-heptane, n-hexane, diethyl ether or Its composition. The preparation method according to claim 3, comprising 1) In (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3 , 5,7-trioxa-12-azahexadecane-16-acid free acid is dissolved in a suitable organic solvent to form a solution having a concentration of 1-200 mg/mL, the organic solvent being selected from the group consisting of acetic acid Ethyl ester, isopropyl acetate, dichloromethane, methyl tert-butyl ether, isopropyl ether or a combination thereof; 2) adding an appropriate amount of anti-solvent to the solution to be turbid, the anti-solvent is selected from the group consisting of n-heptane, n-hexane, diethyl ether or a combination thereof, and the volume of the anti-solvent is 1-10 times that of the organic solvent in the step 1). Continue to stir and crystallize; 3) Solid-liquid separation gives (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxane Generation-3,5,7-trioxa-12-azahexadecane-16-acid crystalline free acid. The preparation method according to claim 3, comprising 1) In (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3 , 5,7-trioxa-12-azahexadecane-16-acid free acid is added to a suitable organic solvent, heated to 30-80 ° C to dissolve to form a solution having a concentration of 1-200 mg / mL, The organic solvent is selected from the group consisting of ethyl acetate, isopropyl acetate, dichloromethane, methyl tert-butyl ether, isopropyl ether or a combination thereof; 2) Slowly cool down to 20 ~ 30 ° C, and add (9R, 11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl- The mass ratio of 4,8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid free acid is 0.1-10.0% of the homologous crystal as a seed crystal, and stirring is continued. Crystallization 3) Solid-liquid separation gives (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxane Generation-3,5,7-trioxa-12-azahexadecane-16-acid crystalline free acid. (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4 represented by Formula 1 according to Claim 2. a method for preparing a calcium salt polymorph of 8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid, comprising 1) (9R,11S)-11-([1,1'-Biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3 , 5,7-trioxa-12-azahexadecane-16-acid free acid is dissolved or dispersed in a water-soluble organic solvent or an aqueous water-soluble organic solvent, and mixed with an equivalent amount or an excess of a suitable base phase Generation of (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3,5 a corresponding salt of 7-trioxa-12-azahexadecane-16-acid; wherein, when the water-soluble organic solvent does not contain water, the solution of the base contains water; 2) The above (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo- The 3,5,7-trioxa-12-azahexadecane-16-acid salt system is mixed with the calcium salt, and the reaction is further stirred to precipitate (9R, 11S)-11-([1,1'-biphenyl) ]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid Calcium salt crystals; 3) Solid-liquid separation gives (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxane Generation of a polymorphic form of the -3,5,7-trioxa-12-azahexadecane-16-acid calcium salt. (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4 represented by Formula 1 according to Claim 2. a method for preparing a calcium salt polymorph of 8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid, comprising 1) (9R,11S)-11-([1,1'-Biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3 , 5,7-trioxa-12-azahexadecane-16-acid free acid is dissolved in a water-soluble organic solvent, and mixed with an equivalent amount or an excess of a suitable base to prepare (9R, 11S)-11- ([1,1'-Biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3,5,7-trioxa-12- a corresponding salt of azahexadecane-16-acid; 2) The above (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo- The 3,5,7-trioxa-12-azahexadecane-16-acid salt system is mixed with the calcium salt, the stirring reaction is continued, the precipitate is precipitated, and the precipitate is removed by filtration; 3) Collecting the above mother liquor, and obtaining (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4 by a volatile solvent or concentration. 8,13-Trioxo-3,5,7-trioxa-12-azahexadecane-16-acid calcium salt polymorph. The preparation method according to claim 6 or 7, wherein the water-soluble organic solvent in the step 1) is selected from the group consisting of alcohols, ketones, amides, sulfoxide-based organic solvents or combinations thereof; preferably from methanol, Ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl sulfoxide or combinations thereof. The preparation method according to any one of claims 6 to 8, wherein the suitable base is Selected from sodium hydroxide, sodium carbonate, sodium hydrogencarbonate, sodium acetate, sodium formate, sodium propionate, sodium acrylate, sodium benzoate, potassium hydroxide, potassium carbonate, potassium hydrogencarbonate, potassium acetate, potassium formate, potassium propionate, acrylic acid Potassium, potassium benzoate or a mixture thereof; preferably from sodium hydrogencarbonate or potassium hydrogencarbonate; the calcium salt is selected from the group consisting of calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium chlorate, calcium hypochlorite, high Calcium chlorate, calcium lactate, calcium gluconate or mixtures thereof; preferably from calcium chloride. A pharmaceutical composition comprising a therapeutically effective amount of the aforementioned (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6 , 9-trimethyl-4,8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid crystalline free acid, its calcium salt polymorph and A pharmaceutically acceptable carrier or excipient. (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4 represented by Formula 1 according to Claim 1. a crystalline free acid of 8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid, (9R, 11S represented by Formula 1 described in Claim 2) )-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3,5,7-trioxane A calcium salt polymorph of hetero-12-azahexadecane-16-acid, the pharmaceutical composition according to claim 10 for the preparation or treatment of a disease associated with a neutral endopeptidase, cardiovascular, antihypertensive Use in the drug; the hypertension is preferably selected from malignant hypertension, essential hypertension, renal vascular hypertension, diabetic hypertension, isolated systolic hypertension or other secondary hypertension. (9R,11S)-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4 represented by Formula 1 according to Claim 1. a crystalline free acid of 8,13-trioxo-3,5,7-trioxa-12-azahexadecane-16-acid, (9R, 11S represented by Formula 1 described in Claim 2) )-11-([1,1'-biphenyl]-4-ylmethyl)-2,6,9-trimethyl-4,8,13-trioxo-3,5,7-trioxane A calcium salt polymorph of hetero-12-azahexadecane-16-acid, the pharmaceutical composition of claim 10 for the treatment or prevention of acute or chronic heart failure, congestive heart failure, left ventricular dysfunction, hypertrophy Use in drugs for cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter or harmful vascular remodeling.

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