Pharmaceutically acceptable salts of polyamine derivatives, their crystalline forms, and production methods

Abstract

The present invention discloses bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, its crystal form, and preparation method. Compared with the free alkoxy and other salts of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine, it has excellent stability and high dynamic solubility, and is more advantageous in terms of safety as a drug and industrial production.

Description

[Technical Field]

[0001] The present invention relates to the technical field of chemical medicine, specifically to bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, its crystalline form, and preparation method. [Background technology]

[0002] Systemic inflammatory response syndrome (SIRS) and autoimmune disorder-related diseases, such as sepsis and autoimmune diseases, are two types of diseases caused by the body's own excessive immune response. There are currently no effective therapeutic drugs, and targeted prevention and treatment have become a topic of clinical interest.

[0003] Sepsis, a systemic inflammatory response syndrome (SIRS) mediated by infectious agents, affects 19 million patients worldwide each year. Despite significant advances in antibiotics and emergency medical technology, sepsis remains a leading cause of death in infected patients, and an ideal treatment has yet to be developed. Research has shown that the pathogenesis of sepsis is as follows: pathogen-associated molecular patterns (PAMPs) released by pathogens such as bacteria, viruses, and fungi are recognized by pattern recognition receptors (PRRs) in the host's innate immune system, mediating the activation of inflammatory cells, resulting in an exaggerated systemic inflammatory response. Epidemiological studies have shown that the PAMP molecules that cause sepsis primarily include bacterial lipopolysaccharide (LPS), bacterial genomic DNA (CpG DNA), peptidoglycan (PGN), lipoteichoic acid (LTA), viral RNA, and zymosan.

[0004] Patent document CN105348137B discloses a pharmaceutically acceptable salt of a polyamine derivative, its preparation method, and its use in the treatment of sepsis. The pharmaceutically acceptable salt of the polyamine derivative is the hydrochloride salt of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine with an acid / alkali ratio of 3.

[0005] However, in actual production, the hydrochloride salt of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine with an acid / alkali ratio of 3 disclosed in the prior art has the drawback of serious adhesion to the vessel wall during production and manufacturing, making it difficult to discharge the product and reducing operability, making it useless for actual production.

[0006] Therefore, it is important to develop a stable pharmaceutically acceptable salt of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine that is suitable for industrial production. Summary of the Invention [Problem to be solved by the invention]

[0007] To solve the shortcomings of the prior art, the present invention provides bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, its crystalline form, and a preparation method thereof. [Means for solving the problem]

[0008] A first aspect of the present invention provides bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate.

[0009] Furthermore, the bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate has the following structure: [ka]

[0010] x represents the number of moles of phosphoric acid bound to one mole of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine, and further, the x is 1 to 3 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0), preferably 1.5 to 2.5, more preferably 1.9 to 2.1, and most preferably 2.0.

[0011] Preferably, the phosphate salt is: [ka]

[0012] Furthermore, the phosphate salt may be in crystalline, amorphous form, or a mixed crystalline and amorphous form.

[0013] A second aspect of the present invention provides a method for producing bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to the first aspect, comprising the step of mixing bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine with phosphoric acid.

[0014] Furthermore, the manufacturing method includes: bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine is mixed with phosphoric acid and reacted; Furthermore, the reaction solvent is one or more selected from ethyl acetate, methylene chloride, methanol, water, chloroform, ethanol, acetone, acetonitrile, butanol, dimethylformamide, dimethyl sulfoxide, methyl tert-butyl ether, isopropyl amine, and isopropanol, preferably, the reaction solvent is one or more selected from methanol, ethanol, ethyl acetate, acetone, and methylene chloride, more preferably, the reaction solvent is methanol or ethanol.

[0015] Furthermore, the reaction temperature is 0 to 30°C (for example, 0°C, 5°C, 15°C, 20°C, 25°C, 30°C), preferably 0 to 15°C.

[0016] Furthermore, the reaction time is 2 to 6 hours (for example, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours), preferably 3 to 5 hours, and more preferably 4 hours.

[0017] Furthermore, seed crystals are added during the reaction.

[0018] Furthermore, the equivalent ratio of the bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine to phosphoric acid is 1:1 to 2.5 (e.g., 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5), preferably 1:1 to 2, more preferably 1:1.8 to 2, and the equivalent ratio is a molar ratio.

[0019] Furthermore, the method for producing bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate further comprises a step of separating the product.

[0020] Furthermore, the separation may include a filtration or centrifugation step.

[0021] Furthermore, the filtration operation means filtering to react, obtaining a reaction system, obtaining a filter cake, and optionally washing the filter cake.

[0022] Furthermore, the solvent used for washing is one selected from methanol, ethyl acetate, ethanol, and isopropanol, and preferably, the washing solvent is methanol or ethyl acetate.

[0023] Furthermore, the method for producing bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate further comprises a drying step.

[0024] Furthermore, the drying step means that the substance obtained by separation is dried at normal pressure or under reduced pressure.

[0025] Furthermore, the drying temperature is 0 to 35°C (for example, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C), preferably 25 to 35°C.

[0026] wherein the bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine may be prepared on a gram or kilogram scale by any of several different methods, such as the preparation method described in patent document CN201510729318.8, which is incorporated herein by reference.

[0027] Furthermore, the method for producing bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate further includes a step of purifying (purifying) bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate.

[0028] Furthermore, the purification step may include recrystallization and / or washing.

[0029] Furthermore, the solvent for the recrystallization is one or a combination of two selected from methanol, ethanol, and water, particularly methanol-water, ethanol-water, water-methanol, or water-ethanol.

[0030] Furthermore, the washing solvent is one selected from methanol, ethanol, ethyl acetate, and isopropanol, and preferably the solvent for washing the crystals is methanol or ethyl acetate.

[0031] Furthermore, the purification step includes adding seed crystals.

[0032] A third aspect of the present invention provides a method for producing the amorphous form of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to the first aspect.

[0033] Furthermore, the production method is one or a combination of methods selected from a gas-liquid diffusion method, a polymer induction method, a slow evaporation method, and a slow cooling method, preferably one or a combination of methods selected from a gas-liquid diffusion method, a polymer induction method, and a slow evaporation method.

[0034] Furthermore, the gas-liquid diffusion method involves placing the target product in an open container, dissolving it in a good solvent, then placing the open container in a sealed container containing a poor solvent, leaving it to stand (room temperature), and finally collecting it to obtain a solid.

[0035] Furthermore, the polymer derivatization method involves dissolving the desired product in a solvent, filtering through a pin film, adding a polymer to the filtrate, and evaporating it at room temperature to obtain a solid.

[0036] Furthermore, the slow evaporation method involves dissolving the desired product in a solvent and then drying to obtain a solid.

[0037] Furthermore, the slow cooling method includes dissolving the target product in a solvent, heating at 40°C to 60°C, stirring for 1 to 4 hours to allow equilibration, filtering the solution when it becomes clear, slowly cooling the filtrate from 40°C to 60°C to 0°C to 10°C, and leaving it at -10 to 30°C (e.g., 10°C, 15°C, 20°C, 25°C, 30°C) for 3 to 8 days while keeping it clear, and finally evaporating it at 40 to 60°C (e.g., 40°C, 45°C, 50°C, 55°C, 60°C).

[0038] In one embodiment of the present invention, the amorphous form of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate is prepared by a slow evaporation method. Specifically, 10-20 mg of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate is weighed into a 4 mL glass bottle and dissolved in a 2:1 volumetric mixture of DMSO and water. The solution is filtered through a pin film (pore size 0.22 μm), and the filtrate is sealed with Parafilm™, with five pinholes drilled through it, and allowed to slowly evaporate at a predetermined temperature. The final solid is then characterized by XRPD. Experimental results show that amorphous bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate can be obtained by the slow evaporation method at room temperature.

[0039] A fourth aspect of the present invention provides crystalline form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to the first aspect.

[0040] Furthermore, in the XRPD pattern of crystalline form A, characteristic peaks (major characteristic diffraction peaks) are present at at least three (or all) of the following positions: 2θ values ​​of 6.4°±0.2°, 9.7°±0.2°, 16.2°±0.2°, 19.5°±0.2°, 22.9°±0.2°, and 26.1°±0.2°.

[0041] Furthermore, in the XRPD pattern of crystalline form A, characteristic peaks (major characteristic diffraction peaks) are present at at least three (or all) of the following positions: 2θ values: 6.4°±0.2°, 9.7°±0.2°, 11.80°±0.2°, 13.0°±0.2°, 14.8°±0.2°, 16.2°±0.2°, 19.5°±0.2°, 22.9°±0.2°, and 26.1°±0.2°.

[0042] Furthermore, in the XRPD pattern of crystalline form A, characteristic peaks (minor characteristic diffraction peaks) are present at at least three (at least four or all) of the following positions: 11.80°±0.2°, 13.0°±0.2°, 14.8°±0.2°, 15.3°±0.2°, 18.7°±0.2°, 20.8°±0.2°, 22.6°±0.2°, and 23.7°±0.2° 2θ values.

[0043] Furthermore, said crystalline form A has an XRPD pattern substantially as shown in FIG.

[0044] Furthermore, in the DSC pattern of the crystalline form A, there are endothermic peaks at 175 to 195°C (for example, about 175°C, 180°C, 185°C, 190°C, and 195°C).

[0045] Furthermore, the crystalline form A has a DSC pattern substantially as shown in FIG.

[0046] Additionally, the crystalline form A exhibits a weight loss of about 3% (eg, 1.5%, 2.0%, 2.5%, 3.0%) when heated from 22°C to 150°C.

[0047] Furthermore, the crystalline form A has a TGA pattern substantially as shown in FIG.

[0048] A fifth aspect of the present invention provides a method for preparing crystalline form A according to the fourth aspect.

[0049] Furthermore, the production methods are anti-solvent crystallization and anti-anti-solvent crystallization.

[0050] Furthermore, the anti-solvent crystallization method involves dissolving the target product in a good solvent and then adding an anti-solvent to the system. The anti-solvent does not dissolve or only slightly dissolves the substance to be crystallized, thereby reducing the solubility of the crystallization substance and causing it to precipitate from the mixed solution.

[0051] Furthermore, the poor-poor solvent crystallization method is a method comprising adding a solution of a target product dissolved in a good solvent to one or more poor solvents, so that the product is slightly dissolved in the solution, thereby making the solution supersaturated and precipitating crystals.

[0052] In one embodiment of the present invention, the anti-solvent crystallization method for bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate crystalline form A is specifically Step (1) of mixing the reaction product obtained in the second aspect of the present invention with a good solvent; Step (2) of adding a poor solvent dropwise to the mixed solution obtained in step (1); Step (3) of cooling and separating the solution obtained in step (2) to obtain a wet product; and step (4) of drying the wet product obtained in step (3) to obtain dry phosphate crystals A.

[0053] Furthermore, the good solvent described in step (1) is selected from water, a mixture of methanol and water, or a mixture of ethanol and water, and preferably, the good solvent described in step (1) is a mixture of methanol and water.

[0054] Furthermore, the anti-solvent described in step (1) is one or more selected from methanol, ethanol, tetrahydrofuran, ethyl acetate, or toluene.

[0055] Furthermore, the temperature-lowering treatment described in step (3) means lowering the temperature to 0 to 15°C (e.g., 0°C, 5°C, 10°C, 15°C), and preferably the temperature-lowering treatment described in step (3) means lowering the temperature to 5°C.

[0056] Furthermore, the time for the temperature-lowering treatment described in step (3) is 3 to 16 hours (e.g., 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours), preferably 8 to 14 hours (e.g., 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours), and more preferably 12 hours.

[0057] Furthermore, the separation method described in step (3) includes centrifugation, filtration, and / or suction filtration. Preferably, the centrifugation means centrifuging the mixed solution after the reaction to obtain a solid.

[0058] Furthermore, in the drying treatment described in step (4), the drying temperature is 25 to 35°C (for example, 25°C, 30°C, 35°C), preferably 30°C.

[0059] A sixth aspect of the present invention provides use of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate and its crystalline form A in the manufacture of an anti-PAMP drug.

[0060] Furthermore, the PAMP is one or more selected from bacterial lipopolysaccharide (LPS), bacterial genomic DNA (CpG DNA), peptidoglycan (PGN), lipoteichoic acid (LTA), viral RNA, and zymosan.

[0061] A seventh aspect of the present invention provides use of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate and its crystalline form A in the manufacture of a medicament for preventing and / or treating systemic inflammatory response syndrome (SIRS).

[0062] Furthermore, the systemic inflammatory response syndrome is sepsis.

[0063] An eighth aspect of the present invention provides use of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate and its crystalline form A in the manufacture of a medicament for preventing and / or treating an autoimmune disease.

[0064] Furthermore, the autoimmune disease is one or more selected from organ-specific autoimmune diseases, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis.

[0065] The bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate described in the present application has excellent stability and high dynamic solubility compared to the free alkali and other salts of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine, making it advantageous for industrial production. Here, bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine diphosphate has higher stability and is more advantageous in terms of safety as a drug compared to bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine triphosphate. In the production of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, when the equivalent ratio of phosphoric acid described in the present application is used, the salt is easily precipitated from the reaction system and can be separated from the system by a simple filtration operation, which is advantageous for industrial mass production. [Brief explanation of the drawings]

[0066] [Figure 1] 1 shows the XRPD pattern of phosphate salt crystalline form A. [Figure 2] 1 shows the TGA / DSC pattern of crystalline form A of the phosphate salt. DETAILED DESCRIPTION OF THE INVENTION

[0067] Some of the abbreviations used in this invention are explained below.

[0068] XRPD: X-ray powder diffraction

[0069] DSC: Differential scanning calorimetry

[0070] TGA: Thermogravimetric analysis

[0071] In the present invention, the term "crystalline form" is confirmed by characterization of the powder X-ray diffraction pattern. Those skilled in the art will understand that the physical and chemical properties discussed herein can be characterized, and that the experimental error depends on instrument conditions, sample preparation, sample purity, and the like. In particular, it is well known to those skilled in the art that X-ray diffraction patterns often vary depending on instrument conditions. In particular, the relative intensities of powder X-ray diffraction patterns can also vary depending on experimental conditions, so the order of peak intensities cannot be used as the sole or decisive factor. In fact, the relative intensities of diffraction peaks in an XRPD pattern are related to the preferred orientation of the crystal. The peak intensities shown herein are illustrative and should not be used for absolute comparison. Furthermore, experimental errors in peak angles are typically less than 5%, and errors in these angles must also be taken into account; typically, an error of ±0.2° is acceptable. Experimental factors such as sample thickness can cause overall deviations in peak angles, but a certain degree of deviation is usually acceptable. Therefore, those skilled in the art will understand that the powder X-ray diffraction pattern of the crystalline form of the present invention does not necessarily have to be completely identical to the powder X-ray diffraction pattern in the examples referred to herein. The phrase "the XRPD pattern is the same" used herein does not mean that the patterns are identical, but rather that the peak positions are the same with an error of ±0.2° and that the peak intensities may vary to a certain extent. Any crystalline form having the same or similar characteristic peaks in these patterns is within the scope of the present invention. Those skilled in the art can compare the patterns listed in the present invention with those of an unknown crystalline form to determine whether the two patterns reflect the same or different crystalline forms. In some embodiments, crystalline form A of the present invention is pure and single, and is substantially free from other crystalline forms mixed with it. In the present invention, "substantially free" refers to a new crystalline form in which the crystalline form contains less than 20% (by weight), particularly less than 10% (by weight), even less than 5% (by weight), and even less than 1% (by weight) of other crystalline forms.

[0072] It should be noted that the numerical values ​​and numerical ranges referred to in the present invention should not be understood in a narrow sense as numerical values ​​or numerical ranges themselves. Those skilled in the art will understand that specific values ​​may vary depending on the specific technical environment without departing from the spirit and principles of the present invention. In the present invention, such a variation range that can be predicted by a person skilled in the art is often expressed using the term "about." In the present invention, when the term "about" is used before a numerical value to refer to the numerical value, it means any value within a range of ±10%, preferably within a range of ±5%, more preferably within a range of ±2%, and more preferably within a range of ±1% of the value. For example, "about 10" should be interpreted as meaning 9 to 11, preferably 9.5 to 10.5, more preferably 9.8 to 10.2, and more preferably 9.9 to 10.1.

[0073] In powder X-ray diffraction patterns of samples, diffraction patterns obtained from crystalline compounds are often characteristic of specific crystal forms, and the relative intensities of bands (especially at low angles) may vary due to predominant orientation effects resulting from differences in crystallization conditions, particle size, relative content of the mixture, and other test conditions. Therefore, the relative intensities of diffraction peaks are not characteristic of the target crystal, and when determining whether a given crystal form is the same as a known crystal form, attention should be paid to the peak positions rather than the relative intensities.

[0074] For purposes of the present invention, the term "substantially as shown in the figure" means that at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the peaks in an XRPD pattern, DSC pattern, or TGA pattern are represented in that pattern.

[0075] In the present invention, the term "room temperature" means that the temperature of the item is close to or the same as the temperature of the space (e.g., the location of the ventilation hood where the item is placed). Typically, room temperature is about 20°C to about 30°C, about 22°C to 27°C, or about 25°C.

[0076] Anti-solvent crystallization (also known as anti-solvent addition, precipitation crystallization, salting out, or forced crystallization) is a method in which one or more anti-solvents are added to a solution in which the target product is dissolved in a good solvent, causing the solution to become supersaturated with the product, resulting in the precipitation of crystals. Anti-anti-solvent crystallization is a method in which one or more anti-solvents are added to a solution in which the target product is dissolved in a good solvent, causing the solution to become supersaturated with the product, resulting in the precipitation of crystals.

[0077] The dissolving ability of the poor solvent for the target product is inferior to that of the good solvent, for example, by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. Therefore, the poor solvent in a system is relative. The good solvent and the poor solvent may be polar or nonpolar solvents, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water, alcoholic solvents, etheric solvents, ketone solvents, ester solvents, alkane solvents, aromatic hydrocarbon solvents, and nitrile solvents. Among them, alcoholic solvents include, but are not limited to, methanol, ethanol, propanol, isopropanol, 1,3-propanediol, 1,2-propanediol, chlorobutanol, or combinations thereof. Etheric solvents include, but are not limited to, tetrahydrofuran, methyl tert-butyl ether, 1,4-dioxane, or combinations thereof. Ketone solvents include, but are not limited to, acetone, methyl ethyl ketone, or 4-methyl-2-pentanone, or a combination thereof. Ester solvents include, but are not limited to, ethyl acetate, isopropyl acetate, n-butyl acetate, or tert-butyl acetate, or a combination thereof. Alkane solvents include, but are not limited to, methylene chloride, chloroform, n-hexane, cyclohexane, pentane, n-heptane, or a combination thereof. Aromatic hydrocarbon solvents include, but are not limited to, benzene, toluene, or a combination thereof. Nitrile solvents include, but are not limited to, acetonitrile and malononitrile.

[0078] Anti-solvent crystallization, or poor-anti-solvent crystallization, may be a batch, semi-batch, or continuous crystallization operation. The anti-solvent may be added to the solution (anti-solvent crystallization) or the product solution to the anti-solvent (poor-poor anti-solvent crystallization) at a constant rate, or slowly at first and then gradually increased in speed.

[0079] The disclosures of various publications, patents and published patent specifications cited herein are hereby incorporated by reference in their entireties.

[0080] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, but obviously, the described embodiments are only a part, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0081] The information on the equipment and methods used in the experiments in the examples are as follows:

[0082] 1. X-ray powder diffraction (XRPD) TIFF0007784008000003.tif43170

[0083] 2. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) Thermogravimetric data for phosphate salts was collected using a TA Discovery Series Thermogravimetric Apparatus (TGA). A few milligrams of sample was placed in a Tzero aluminum pan and heated from room temperature to the target temperature under N2 protection, with an N2 flow rate of 25 mL / min and a heating rate of 10 °C / min. Thermal data for the samples was collected using a TA Discovery Series Differential Scanning Calorimeter (DSC). For crystalline samples, a few milligrams of sample was weighed into a Tzero aluminum pan, sealed with a Tzero sealing lid, and heated under N2 protection, with an N2 flow rate of 50 mL / min and a heating rate of 10 °C / min. For amorphous samples, the modulated mode was set for testing. Approximately 10 mg of the sample was weighed into a Tzero aluminum pan, sealed with a Tzero sealing lid, and heated under N2 protection with an N2 flow rate of 50 mL / min, a temperature amplitude of ±1 °C, a modulation period of 40 seconds, a heating rate of 1 °C / min, and a test temperature range of 25 °C to 200 °C.

[0084] 3. High-Performance Liquid Chromatography (HPLC) The stability of the samples was confirmed using HPLC. Specifically, an Agilent 1260 model high performance liquid chromatograph (equipped with a DAD detector) was used to collect data on the purity and solubility of the samples. The test method is shown in Table 1.

[0085] [Table 1]

[0086] 4. Phosphate ion content For bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, the phosphate ion content data of the sample was collected using a DIONEX ICS-6000+DP ion chromatography system, and the test method is shown in Table 2.

[0087] [Table 2]

[0088] 5. Biosolvent Preparation The specific procedure for producing the biosolvent is shown in Table 3.

[0089] [Table 3]

[0090] 6. Hygroscopicity Measurement

[0091] The measurement of hygroscopicity is based on the Guiding Principles for Drug Hygroscopicity Testing in the 2015 edition of the Chinese Pharmacopoeia, and the specific test methods are as follows:

[0092] 1. Prepare a dry stoppered glass weighing bottle (outer diameter 50 mm, height 15 mm) and, on the day before the test, place it in an appropriate constant temperature dryer at 25°C ± 1°C (put a saturated solution of ammonium chloride or ammonium sulfate in the bottom) or an artificial climate box (set to a temperature of 25°C ± 1°C and a relative humidity of 80% ± 2%), and accurately weigh out its weight (m1).

[0093] 2. Take an appropriate amount of the test product and place it flat in the weighing bottle. The thickness of the test product is usually about 1 mm, and the weight (m2) is accurately measured.

[0094] 3. Open the weighing bottle and leave it with the lid on under the above constant temperature and humidity conditions for 24 hours.

[0095] 4. Close the lid of the weighing bottle and accurately measure the weight (m3). Weight increase rate = (m3-m2) / (m2-m1)×100%

[0096] 5. Explain the characteristics of hygroscopicity and define the weight gain due to hygroscopicity Deliquescence: Absorbing enough water to form a liquid. Very hygroscopic: Weight gain due to moisture absorption is 15% or more. Hygroscopic: Weight increase due to moisture absorption is less than 15% and more than 2%. Slightly hygroscopic: Weight gain due to moisture absorption is less than 2% and more than 0.2%. No or little moisture absorption: Weight increase due to moisture absorption is less than 0.2%.

[0097] Example 1 Salt type selection An ethanol solution of the free alkoxylate of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine (hereafter referred to as "free alkoxy") was prepared at room temperature. The acid solution, with an acid / alkoxy molar ratio of 1:1, was placed in an HPLC vial, and the ethanol solution of the free alkoxylate was added. The mixture was then magnetically stirred at room temperature. After 2–4 days of magnetic stirring at room temperature, a small amount of the mixture turned cloudy, and some of the mixture showed adhesion to the vessel wall or colloidal formation, but the remaining mixture remained clear. In subsequent processing steps, 1) the cloudy mixture was centrifuged to separate the solids. 2) For mixtures that showed adhesion to the vessel wall or colloidal formation, a circulating temperature-ramp (25–50°C) was performed to improve the appearance and obtain solids (see Table 5 for the temperature-ramp procedure). 3) For clear or slightly cloudy mixtures, magnetic stirring was performed at 5°C for approximately 5 hours, followed by overnight storage at -20°C in an attempt to collect a sufficient amount of solid for further characterization. If the amount of solid was small, it was slowly evaporated at room temperature. If the system remained clear, anti-solvent (n-heptane) was added or slowly evaporated at room temperature. The solid obtained above was separated by centrifugation. The test results are shown in Table 4.

[0098] [Table 4] α The acid / alkali molar input ratio is 1:1. β The acid / alkali molar input ratio is 3:1. *: Because the amount of solids is small or the system is clear, process at a low temperature of 5°C / -20°C, then evaporate slowly at room temperature (18-22°C). # : If the amount of solid is small or the system is clear, treat at low temperature 5°C / -20°C and then add anti-solvent. †The system was colloidal. Improvement in the state was also observed during the circulating temperature increase / decrease treatment at 25-50°C. & : A small amount of solid was obtained by the reaction, but it became a colloid when dried in vacuum at 30°C for 17 hours. ※ : The solid obtained by the reaction becomes a colloid when left sealed at room temperature for one week.

[0099] [Table 5]

[0100] Stability Testing To evaluate the solid-state stability of various salts, approximately 15 mg of each sample was weighed into an HPLC vial and left open at 25°C / 60% RH. Chemical purity (HPLC) and hygroscopicity tests were performed on the initial sample and on samples stored for 5 days and 2 weeks. The stability data are shown in Table 6. The results clearly show that the phosphate salts prepared with an acid / alkali ratio of 1 showed no significant changes in their properties or purity after 5 days and 2 weeks of storage under the specified conditions. Meanwhile, the hydrochloride, sulfate, and oxalate salts prepared with an acid / alkali ratio of 1 and the phosphate salt prepared with an acid / alkali ratio of 3 transformed from powdery solids to liquids by the fourth day, and their purity also decreased to some extent. The phosphate salt prepared with an acid / alkali ratio of 1 was slightly hygroscopic, with a hygroscopicity of 1.29%. The other samples were all very hygroscopic, with hygroscopicities exceeding 15%. From the above experimental data, it was found that, among the five salt forms, only the phosphate produced at an acid / alkali ratio of 1 was relatively stable. Samples were taken from the phosphate solids produced at an acid / alkali ratio of 1 or 3 to measure the acid / alkali ratio, and the test method for phosphate ion content is shown in Table 2. The test results clearly show that the phosphate produced at an acid / alkali ratio of 1 has an acid / alkali ratio of 1.9, and the phosphate produced at an acid / alkali ratio of 3 has an acid / alkali ratio of 3.0.

[0101] [Table 6]

[0102] The stability of the samples was measured by HPLC, specifically, the purity and solubility data of the samples were collected by an Agilent 1260 model high performance liquid chromatograph (equipped with a DAD detector), and the test method is shown in Table 1. The hygroscopicity of the samples was measured according to the method described in the Chinese Pharmacopoeia, and the specific measurement method is described in "6. Measurement of Hygroscopicity" in the specification.

[0103] dynamic solubility The dynamic solubility of phosphate salts in water and three biosolvents (simulated gastric fluid SGF, fasted simulated intestinal fluid FaSSIF, and fed simulated intestinal fluid FeSSIF) was tested at 37°C. The specific solubility testing method was as follows: Four 30 mg portions of sample were weighed, and 3 mL of water and one of the three biosolvents was added to each portion. The mixture was incubated at 37°C and shaken (100 rpm). At 1, 4, and 24 hours, 1.0 mL of suspension (or clear solution) was taken. The suspension was centrifuged for 3 minutes, and the supernatant / clear solution was filtered before being used for solubility testing (HPLC) and pH measurement (pH meter). The specific procedure for preparing the biosolvents is shown in Table 3.

[0104] The solubility of phosphate in water / SGF / FaSSIF / FeSSIF is greater than 9 mg / mL. The sample solubility was measured by HPLC, specifically, an Agilent 1260 model high performance liquid chromatograph (equipped with a DAD detector) was used to collect sample purity and solubility data, and the test method is shown in Table 1 above. The pH value remained unchanged within 24 hours. Phosphate is highly soluble in both water and the simulated biological environment.

[0105] Conclusion: Compared to bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, the free alkali of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine is less stable, with its purity decreasing from 97.56% to 73.73% after 7 days at room temperature. Therefore, it must be stored at -20°C. Bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine hydrochloride is extremely hygroscopic and becomes colloidal at room temperature. Non-vacuum drying yielded a sample with good solid properties. The sulfate salt prepared with an acid / alkali ratio of 1 becomes solid at room temperature, but the salt formed is very hygroscopic and unstable at room temperature. The L-tartrate salt can become a solid at room temperature, but the resulting solid becomes a colloid when left sealed at room temperature for a week. The oxalate salt and the phosphate salt produced with an acid / alkali ratio of 3 are highly hygroscopic and have poor stability. Because they are highly hygroscopic, adhesion to the container wall occurs during production, although to varying degrees, which is disadvantageous for industrial production.

[0106] The phosphate produced at an acid / alkali input ratio of 1 is well suspended during formation and easily precipitates and separates from the system. Moreover, the produced phosphate is highly stable, with no obvious changes in its properties or purity observed even after being left for two weeks. It also has low hygroscopicity, making it particularly advantageous for industrial mass production.

[0107] Salt production Example 2 A 100 ml three-neck flask was charged with 2 g of the free alkane of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine at room temperature. Next, methylene chloride (20 g) was added, stirred, dissolved, and clarified to obtain a methylene chloride solution of the free alkane. The solution was then cooled to 0-5°C.

[0108] Phosphoric acid (695 mg, 85%) was added to the centrifuge tube, and then 10 g of ethyl acetate was added to obtain a solution of phosphoric acid in ethyl acetate, which was then shaken uniformly and prepared for use.

[0109] A solution of phosphoric acid in ethyl acetate was slowly added dropwise to a solution of free ally in methylene chloride. The mixture was stirred at 0-5°C for 4 hours, during which time a white solid gradually precipitated, the system became cloudy, and began to form spheres. A white suspension formed during stirring. The mixture was filtered, and the filter cake was washed with ethyl acetate to obtain a wet product. The solid was placed in an oven at 30°C and baked for 16 hours, yielding 2.1 g of a white solid. A sample of the solid was taken to measure its purity, content, and acid / alkali ratio; the test results are shown in Table 7.

[0110] Example 3 A 100 ml three-neck flask was charged with 1 g of the free alkane of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine at room temperature. Methanol (10 g) was then added, stirred, dissolved, and clarified to obtain a methanol solution of the free alkane. The solution was then cooled to 0-5°C.

[0111] Phosphoric acid (347.8 mg, 85%) was added to the centrifuge tube, and then 5 g of methanol was added to obtain a solution of phosphoric acid in methanol, which was then shaken uniformly and prepared for use.

[0112] A solution of phosphoric acid in methanol was slowly added dropwise to a solution of free alkali in methanol. The mixture was stirred at 0-5°C for 4 hours. The reaction mixture was initially clear, but after 0.5 hours it gradually became cloudy, followed by the precipitation of a large amount of solid. The mixture was filtered and the filter cake was washed with ethyl acetate to obtain a wet product. The solid was placed in an oven at 30°C and baked for 16 hours, yielding 1.1 g of a white solid. A solid sample was taken and its purity, content, and acid / alkali ratio were measured. The test results are shown in Table 7.

[0113] Example 4 5 g of the free alkoxylate bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine was added to a 100 ml three-neck flask at room temperature. Next, 50 g of methanol was added, stirred, dissolved, and clarified to obtain a methanol solution of the free alkoxylate, which was then cooled to 0-5°C.

[0114] Phosphoric acid (1.65 g, 85%) was added to a 50 ml beaker, and then 25 g of methanol was added to obtain a solution of phosphoric acid in methanol, which was then shaken uniformly and prepared for use.

[0115] A solution of phosphoric acid in methanol was slowly added dropwise to a solution of free alkali in methanol. The mixture was stirred at 0-5°C for 4 hours. The reaction mixture was initially clear, but after 0.5 hours it gradually became cloudy, followed by the precipitation of a large amount of solid. The mixture was filtered and the filter cake was washed with methanol to obtain a white wet product. The solid was placed in an oven at 30°C and baked for 16 hours, yielding 5.5 g of a white solid. A solid sample was taken and its purity, content, and acid / alkali ratio were measured. The test results are shown in Table 7.

[0116] Example 5 A 100 ml three-neck flask was charged with 3.4 g of the free alkane of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine at room temperature. Methanol (34 g) was then added, stirred, dissolved, and clarified to obtain a methanol solution of the free alkane. The solution was then cooled to 0-5°C.

[0117] Phosphoric acid (1.12 g, 85%) was added to a 50 ml beaker, and then 17 g of methanol was added to obtain a solution of phosphoric acid in methanol, which was then shaken uniformly and prepared for use.

[0118] A solution of phosphoric acid in methanol was slowly added dropwise to a solution of free alkali in methanol. The mixture was stirred at 0-5°C for 4 hours. The reaction mixture was initially clear, but after 0.5 hours it gradually became cloudy, followed by the precipitation of a large amount of solid. The mixture was filtered and the filter cake was washed with methanol to obtain a white wet product. The solid was placed in a 30°C oven and baked for 16 hours, yielding 5.5 g of a white solid. A solid sample was taken and its purity, content, and acid / alkali ratio were measured. The test results are shown in Table 7.

[0119] Example 6 A 100 ml three-neck flask was charged with 1.0 g of the free alkane of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine at room temperature. Methanol (10 g) was then added, stirred, dissolved, and clarified to obtain a methanol solution of the free alkane. The solution was then cooled to 0-5°C.

[0120] Phosphoric acid (183 mg, 85%) was added to the centrifuge tube, and then 5 g of methanol was added to obtain a methanol solution of phosphoric acid, which was then shaken uniformly and prepared for use.

[0121] A solution of phosphoric acid in methanol was slowly added dropwise to a solution of free alkali in methanol. The mixture was stirred at 0-5°C for 4 hours, then filtered. The filter cake was washed with methanol to obtain a white wet product. The solid was placed in a 30°C oven and baked for 16 hours, yielding 0.8 g of a white solid. A solid sample was taken and its purity, content, and acid / alkali ratio were measured. The test results are shown in Table 7.

[0122] The researchers found that when the produced phosphate salts with an acid / alkali ratio of approximately 2 were recrystallized in a methanol-water or water-methanol recrystallization system, the acid / alkali ratio remained almost unchanged, making them stable during the recrystallization process.

[0123] Comparative Example 1 A 100 ml three-neck flask was charged with 1.0 g of the free alkane of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine at room temperature. Methanol (10 g) was then added, stirred, dissolved, and clarified to obtain a methanol solution of the free alkane. The solution was then cooled to 0-5°C.

[0124] Phosphoric acid (558.3 ​​mg, 85%) was added to the centrifuge tube, and then 5 g of methanol was added to obtain a methanol solution of phosphoric acid, which was then shaken uniformly and prepared for use.

[0125] A methanol solution of phosphoric acid was slowly added dropwise to a methanol solution of free alkali. The mixture was stirred at 0-5°C for 4 hours, after which significant adhesion to the vessel wall occurred. The methanol was concentrated to dryness, and the solids adhering to the vessel wall were scraped off with a spatula. The mixture was then slurried with ethyl acetate and filtered. The solid rapidly absorbed moisture and turned into an oil. The oil was placed in an oven at 30°C and baked for 16 hours to obtain a solid. A sample was taken and analyzed for purity, content, and acid / alkali ratio. The acid / alkali ratio is the molar amount of phosphoric acid divided by the molar amount of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine, i.e., the acid / alkali molar ratio. The test method for phosphate ion content is shown in Table 2, and the test results are shown in Table 7.

[0126] The researchers found that when the produced phosphate with an acid / alkali ratio of 3 was recrystallized in a methanol-water or water-methanol recrystallization system, the acid / alkali ratio decreased by 10%, which means that the phosphate produced with a phosphoric acid equivalent of 3.05 eq and an acid / alkali ratio of 3 was not stable.

[0127] [Table 7]

[0128] From the above table, it is clear that the process for forming free alkali and phosphate produced a product with high purity, which was not less than that of the raw material but was improved by 1%. In other words, the process for forming free alkali and phosphate had a significant impurity removal effect.

[0129] When the equivalent weight of phosphoric acid was 1.0 eq, 1.8 eq, or 1.9 eq, the acid / alkali ratio of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate produced by the reaction was 1.9 to 2.1. Furthermore, since the salt easily precipitates from the reaction system during the production process, it can be separated from the system by simple filtration, making it advantageous for industrial mass production. On the other hand, when the equivalent weight of phosphoric acid was 3.05 eq, the acid / alkali ratio of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate produced by the reaction was 3.0, and serious adhesion to the vessel wall occurred during the reaction process. However, when the equivalent weight of phosphoric acid was 1.9 eq, the reaction system was favorable and a uniform solid was produced. When the equivalent weight of phosphoric acid is 3.05 eq, the mixture in the reaction system is obviously not uniform, the bottom of the mixture is white, and in the center there is a slight buttery substance attached to the inner wall of the flask. In the whole reaction system, the phosphate cannot be separated by direct filtration, and it is necessary to make a slurry with a solvent and then filter it. In addition, the solid obtained by filtration is very hygroscopic at room temperature and quickly becomes oily, which is unfavorable for industrial mass production of the product.

[0130] Preparation of crystalline forms Example 7 Using the method of Example 4, bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate was prepared. 3.27 g of the prepared bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate was added to 11.4 g of purified water, and 13.3 g of methanol was added to the reaction vessel. The temperature inside the reaction vessel was adjusted to 0-10°C, and then 193.0 g of methanol was slowly added to the reaction vessel. After the addition was completed, the temperature inside the reaction vessel was controlled to 0-10°C and the mixture was stirred for 3-16 hours to obtain a white solid. This was separated by centrifugation to obtain a wet product, which was then vacuum dried at 25°C and characterized by XRPD. The XRPD pattern of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, crystalline form A of the phosphate salt, is shown in FIG. 1, and Table 8 lists the corresponding powder X-ray diffraction pattern data, including the diffraction angle 2θ and the relative intensity (expressed as a percentage of the most intense peak).

[0131] [Table 8]

[0132] The crystalline form A of the phosphate salt was characterized by TGA and DSC, and the results are shown in Figure 2. The TGA results indicate that when the sample was heated from 22°C to 150°C, a weight loss of 2.714% was observed. This weight loss was due to the desorption of adsorbed water. The DSC results indicate that the sample exhibited an endothermic peak at 71.28°C, which was due to the desorption of adsorbed water. The sample also exhibited an endothermic peak at 185.64°C, which was due to the crystal form transformation of the sample. The DSC heating onset signal is presumed to be an instrumental signal.

[0133] As a result of the detection, the produced crystalline form A of the phosphate salt had an acid / alkali ratio of 2.0, was slightly hygroscopic, and had good stability. After being left for 2 weeks under conditions of 25°C / 60% RH, light exposure, 60°C, and 80°C, the crystalline form did not change.

Claims

1. 1. Bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, characterized by having the following structure: 【Chemistry 1】 (where x is 1.9 to 2.1.)

2. Bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate, wherein x is 2.

0.

3. A method for producing bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 1, comprising the steps of: A production method characterized by mixing and reacting bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine with phosphoric acid to obtain a solid.

4. The manufacturing method described in Claim 3, characterized in that the solvent for the reaction is one or more selected from ethyl acetate, methylene chloride, methanol, water, chloroform, ethanol, acetone, acetonitrile, butanol, dimethylformamide, dimethyl sulfoxide, methyl tert-butyl ether, isopropyl amine, and isopropanol.

5. The manufacturing method described in Claim 3, characterized in that the reaction temperature is 0 to 35°C.

6. 2. Crystalline Form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 1, characterized in that the XRPD pattern has characteristic peaks at at least three of the following positions with 2θ values: 6.4°±0.2°, 9.7°±0.2°, 16.2°±0.2°, 19.5°±0.2°, 22.9°±0.2°, and 26.1°±0.2°.

7. 7. Crystalline Form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 6, characterized in that the XRPD pattern has characteristic peaks at at least three of the following 2θ values: 11.80°±0.2°, 13.0°±0.2°, 14.8°±0.2°, 15.3°±0.2°, 18.7°±0.2°, 20.8°±0.2°, 22.6°±0.2°, and 23.7°±0.2°.

8. The crystalline form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 6, characterized in that it has an XRPD pattern as shown in FIG.

1.

9. Crystalline form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 6, characterized in that it has a DSC pattern as shown in Figure 2.

10. The crystalline form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 6, characterized in that it has a TGA pattern as shown in FIG.

2.

11. The method for producing crystalline form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 6, characterized in that bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate is mixed with a good solvent, and then mixed with a poor solvent, followed by crystallization to obtain phosphate crystal A.

12. 12. The method for preparing crystalline form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 11, wherein the reaction temperature is 0 to 30°C.

13. 12. The method for producing crystalline form A of bis((N-3-aminopropyl)-(N-(3,4-dimethoxyphenylpropionyl)))aminopropylamine phosphate according to claim 11, wherein the good solvent is water, a mixture of methanol and water, or a mixture of ethanol and water, and the poor solvent is one or more solvents selected from the group consisting of methanol, ethanol, tetrahydrofuran, ethyl acetate, and toluene.

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