Crystal forms, salts, production methods and uses of phenothiazine compounds

Abstract

The present invention provides a crystalline form, salt, and preparation method and use thereof of a phenothiazine compound, the hydrochloride salt of which and its crystals can be used as a ferroptosis inhibitor, and has good solubility, stability, oral bioavailability and drug formability.

Description

[Technical field]

[0001] This application claims priority to Chinese patent applications with application numbers 202210108728.0 and 202210108746.9 filed with the State Intellectual Property Office of China on January 28, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of medicinal chemistry, and more particularly to a crystalline form, salt, preparation method and use of a medicinal phenothiazine compound. [Background technology]

[0003] Ferroptosis is an iron-dependent and lipid peroxidation-driven programmed cell death. The morphological characteristics of ferroptosis are mainly manifested by a reduction in mitochondrial volume, an increase in mitochondrial membrane density, a reduction or disappearance of mitochondrial cristae, and a rupture of the outer mitochondrial membrane but a normal size of the cell nucleus. This is the main morphological feature that distinguishes ferroptosis from apoptosis, necrosis, and autophagy. The biochemical characteristics of ferroptosis are mainly manifested by the accumulation of intracellular iron and ROS, activation of the mitogen-activated protein kinase (MAPK) signaling pathway, inhibition of the cystine / glutamate transporter pathway, and increased NADPH oxidation.

[0004] More and more research is confirming that cell ferroptosis is closely related to many diseases, disorders and disorders. This cell death mode was first discovered in 2012 by Dixon et al. to be related to small molecule-induced RAS tumor cell death (Scott J Dixon et al., Cell. 2012 May 25; 149(5): 1060-72.). The role of ferroptosis in cancer, organ damage, and degenerative diseases has been further demonstrated in recent studies (Xuejun Jiang et al., Nat Rev Mol Cell Biol. 2021 Apr; 22(4): 266-282). Specifically, this includes the occurrence and development of various cancers, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, immune-related diseases, hepatic and renal failure, inflammation, metabolic diseases, etc. In particular, it plays an important role in diseases such as Alzheimer's disease, Parkinson's disease, tumors, stroke, ischemia-reperfusion injury, atherosclerosis, hepatic and renal failure, inflammation, and diabetic complications. Since stimulating or inhibiting the occurrence of ferroptosis allows for the occurrence and development of associated diseases, ferroptosis inhibitors are considered as potential pharmaceuticals for treating these diseases.

[0005] International application WO2019205854A1 discloses a compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine having excellent ferroptosis inhibitory activity and a method for producing the same. However, no research has been conducted on its crystal form in the prior art.

[0006] Drug crystal polymorphism is a common phenomenon in pharmaceutical research and development, and is an important factor affecting the quality of pharmaceuticals. Different crystal forms often have different solubility, stability, hygroscopicity, and bioavailability, which directly affect the quality of pharmaceutical preparations of pharmaceuticals, the absorption behavior in the human body, and ultimately affect the therapeutic effect and side effect benefit ratio of this preparation in the human body. Therefore, in order to maximize the effectiveness of pharmaceuticals, it is necessary to conduct research on the crystal form of this compound and find a crystal form with high purity, good solubility, good stability, and high bioavailability, which is very important for pharmaceutical development. Summary of the Invention

[0007] One object of the present invention is to provide crystalline forms of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, particularly crystalline forms of its hydrochloride salt, which have improved druggability, such as stability, solubility, hygroscopicity, and bioavailability.

[0008] Another object of the present invention is to provide pharmaceutical compositions, processes for preparation and uses of said crystalline forms.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions.

[0010] In one or more embodiments, there is provided a crystalline form of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride having the following crystalline form A: The X-ray powder diffraction pattern of said form A has characteristic peaks at 10.68±0.2°, 14.36±0.2°, 18.57±0.2°, 21.08±0.2°, 22.14±0.2°, 23.40±0.2°, and 29.03±0.2° when expressed in 2θ angles using Cu-Ka radiation.

[0011] In one or more embodiments, the X-ray powder diffraction pattern of crystalline form A has characteristic peaks at 10.68±0.2°, 14.36±0.2°, 17.84±0.2°, 18.57±0.2°, 21.08±0.2°, 22.14±0.2°, 23.40±0.2°, 27.50±0.2°, 29.03±0.2° when expressed in 2θ angles using Cu-Ka radiation.

[0012] In one or more embodiments, the X-ray powder diffraction pattern of crystalline form A, expressed in 2θ angles using Cu-Ka radiation, has characteristic peaks at 10.68±0.2°, 14.36±0.2°, 16.54±0.2°, 17.84±0.2°, 18.57±0.2°, 20.89±0.2°, 21.08±0.2°, 22.14±0.2°, 22.92±0.2°, 23.40±0.2°, 25.88±0.2°, 27.50±0.2°, 29.03±0.2.

[0013] In one or more embodiments, the X-ray powder diffraction pattern of crystalline form A, expressed in 2θ angles using Cu-Ka radiation, has characteristic peaks at 10.68±0.2°, 12.63±0.2°, 14.36±0.2°, 16.06±0.2°, 16.54±0.2°, 17.84±0.2°, 18.57±0.2°, 20.89±0.2°, 21.08±0.2°, 22.14±0.2°, 22.92±0.2°, 23.40±0.2°, 25.11±0.2°, 25.51±0.2°, 25.88±0.2°, 27.50±0.2°, 28.54±0.2°, 29.03±0.2°, 33.55±0.2°.

[0014] In one or more embodiments, the X-ray powder diffraction pattern of the crystalline form A, expressed in 2θ angles using Cu-Ka radiation, is shown in FIG.

[0015] In one or more embodiments, there is provided a crystalline form of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride having the following crystalline form B. The X-ray powder diffraction pattern of said crystalline form B has characteristic peaks at 16.46±0.2°, 22.01±0.2° when expressed in 2θ angles using Cu-Ka radiation.

[0016] In one or more embodiments, the X-ray powder diffraction pattern of crystalline form B has characteristic peaks at 16.46±0.2°, 22.01±0.2°, 27.62±0.2°, 28.91±0.2°, and 33.41±0.2°, expressed in 2θ angles using Cu-Ka radiation.

[0017] In one or more embodiments, the X-ray powder diffraction pattern of crystalline form B has characteristic peaks at 5.41±0.2°, 10.92±0.2°, 16.46±0.2°, 22.01±0.2°, 27.62±0.2°, 28.91±0.2°, and 33.41±0.2°, expressed in 2θ angles using Cu-Ka radiation.

[0018] In one or more embodiments, the X-ray powder diffraction pattern of crystalline form B, expressed in 2θ angles using Cu-Ka radiation, is shown in FIG.

[0019] In one or more embodiments, there is provided a pharmaceutical composition comprising crystalline form A of the hydrochloride salt, or crystalline form B of the hydrochloride salt, as described above, and a pharma- ceutically acceptable carrier.

[0020] In one or more embodiments, there is provided the use of crystalline form A of the hydrochloride salt, or crystalline form B of the hydrochloride salt, or a pharmaceutical composition thereof, as described above, in the manufacture of a medicament for the prevention and / or treatment of cancer, organ damage and degenerative diseases.

[0021] In one or more embodiments, there is provided a use of crystalline form A of the hydrochloride salt, or crystalline form B of the hydrochloride salt, or a pharmaceutical composition thereof, in the manufacture of a medicament for the prevention and / or treatment of cancer, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, immune-related diseases, hepatic and renal failure, inflammation, and metabolic diseases. In one or more embodiments, the disease may be selected from, but is not limited to, cancer, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, atherosclerosis, immune-related diseases, hepatic and renal failure, inflammation, diabetes, and diabetic complications. In one or more embodiments, the stroke is a hemorrhagic stroke and / or an ischemic stroke, which is also called cerebral infarction or cerebral infarction.

[0022] In one or more embodiments, there is provided the use of crystalline form A of the hydrochloride salt, or crystalline form B of the hydrochloride salt, or a pharmaceutical composition thereof, in the manufacture of a ferroptosis inhibitor.

[0023] In one or more embodiments, there is provided a method for producing Crystalline Form A of the hydrochloride salt of the present application, comprising dispersing the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in methanol at 10 to 40° C., adding a mixed solution of concentrated hydrochloric acid and methanol dropwise thereto, stirring and crystallizing the mixture, filtering, washing the filter cake with methanol, and then vacuum drying the filter cake to obtain a crystalline substance. Preferably, the weight ratio of the solvent methanol to the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine is 5:1 to 15:1, for example 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.46:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, or 15:1. Preferably, the weight ratio of the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is 1:1 to 8:1, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.15:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1. Preferably, the stirring and crystallization is carried out for 1 to 8 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours.

[0024] In one or more embodiments, there is provided a method for preparing Crystalline Form A of the hydrochloride salt of the present application, comprising dispersing compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in acetone and water at 10 to 40° C., adding dropwise a mixed solution of concentrated hydrochloric acid, acetone and water, stirring and crystallizing, filtering, washing the filter cake with acetone, and then vacuum drying the filter cake to obtain a crystalline substance. Preferably, the weight ratio of the solvent acetone, water and the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine is (1-8):(0.5-3):(0.5-3), preferably (4-6):(0.5-1):(0.5-1), for example, 4:1:1, 4.74:1:1, 5:1:1. Preferably, the weight ratio of the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is 1:1 to 8:1, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.66:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1. The stirring and crystallization is preferably carried out for 1 to 8 hours, and more preferably for 2 to 5 hours. The mass fraction of concentrated hydrochloric acid in the above production method is 20% or more, for example, 20%, 25%, 30%, 35%, 36%, 37%, 38%, 39%, or 40%.

[0025] The organ injury of the present invention refers to any damage, injury, reduction or loss of one or more functions of an organ or tissues associated with said organ. The injury includes, but is not limited to, changes in organ tissue or structure, such as the development of tissue necrotic areas, or the destruction or loss of cellular or tissue structural integrity, or damage or changes to the normal state, such as abnormal accumulation of cellular material or debris, for example, from cellular inflammatory processes or apoptosis. It is understood that the pathology of injury may differ depending on the type of organ or tissue. Organ injury may also result in the mechanism of onset of illness or disease associated with said injury (i.e., illness associated with organ injury).

[0026] In the hydrochloride crystals of the present invention, the molar ratio of the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to hydrochloric acid is about 1:(1±0.5), for example, 1:(1±0.4), 1:(1±0.2), 1:(1±0.1), or 1:1.

[0027] The crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride in the present invention is also called crystalline form A. The crystalline form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride in the present invention is also called crystalline form B.

[0028] Compared with the prior art, the present invention provides the following beneficial effects: 1. In the present invention, through extensive experimental research, two novel crystalline forms of compounds have been obtained whose physical and chemical properties are significantly improved compared to the free compounds. 1) The crystalline form A of the hydrochloride salt has a very high purity, for example, in one embodiment of the present invention, the purity reaches 99.76%. The solubility in water is 10,000 times higher than that of the free compound, and while the solubility is greatly improved, the crystalline form A also maintains good stability. For example, in one embodiment of the present invention, the crystalline form A still maintains its crystalline form under polishing conditions and has good mechanical stability. In another embodiment of the present invention, the melting point of the crystalline form A is about 265.31°C, which indicates that the crystalline form has good thermal stability. In yet another embodiment of the present invention, the crystalline form A still maintains its crystalline form under accelerated and stable conditions, and the purity is basically unchanged. In yet another embodiment of the present invention, the crystalline form A was dissolved in different solvents, but no crystalline form transition occurred even after magnetic stirring. In addition, the bioavailability of the crystalline form A was also greatly improved compared to the free compound.

[0029] 2) Crystalline form B of the hydrochloride salt also achieved unexpected beneficial effects in terms of solubility compared to the free compound. For example, in one embodiment of the present invention, crystalline form B was 1000 times more soluble in water than the free compound.

[0030] 2. The crystalline form A of the hydrochloride of the present invention has superior physical and chemical properties to the crystalline form B of the hydrochloride. Regarding hygroscopicity, the water adsorption rate of the hydrochloride crystalline form A at 25°C / 80%RH is only 0.36%, which indicates that the crystalline form A has a slight hygroscopicity and is superior to the crystalline form B. Regarding solubility, the crystalline form A of the hydrochloride has a superior solubility in both water and hydrochloric acid to the crystalline form B, for example, the solubility of the hydrochloride crystalline form A in water and in a hydrochloric acid solution with a pH value of 1.0 is 10 times or more than that of the hydrochloride crystalline form B. However, regarding stability, the hydrochloride crystalline form A, which has better solubility, has better stability. For example, in one embodiment of the present invention, the crystalline form A maintained its crystalline form under accelerated and stable conditions, while the crystalline form B began to transform to the crystalline form A within 2 days at room temperature. Regarding stability, the crystalline form A of the hydrochloride has almost no change in purity after being left under long-term 25°C / 60%RH and accelerated 40°C / 75%RH conditions for 1 week, and has good chemical stability, and no change in the crystalline form of the sample was observed. After the hydrochloride salt was left for one week under RT-75%RH and RT-97%RH conditions, no change in the crystal shape of the sample was observed. Also, the hydrochloride salt form A maintained its crystal shape after being polished. Also, under accelerated conditions (40°C±2°C, 75%RH±10%RH, 6 months), form A had a significantly lower content of total impurities than form B, and the stability of form A was superior to that of form B.

[0031] It is another object of the present invention to provide multiple pharma- ceutically acceptable salts of the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine which have significantly improved both solubility and stability over the compound. Another object of the present invention is to provide a method for preparing and using the salt of the above compound. In response to this, the specific technical solution of the present invention is as follows:

[0032] In one or more embodiments of the present invention, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine methanesulfonate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine ethanesulfonate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine-p-toluenesulfonate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine tartrate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)- 10H-Phenothiazine hydrobromide, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine oxalate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine phosphate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine sulfate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine acetate, 2-(1-(4-(4-methylpiperazin-1 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine propionate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine perchlorate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine malate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine salicylate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine mandelate,2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine lactate and 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine succinate are provided.

[0033] In one or more embodiments, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride is provided.

[0034] In one or more embodiments, the molar ratio of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to hydrochloric acid in the hydrochloride salt is about 1:(0.5 to 2), for example, 1:1 or 1:2.

[0035] In the salt of the present invention, the molar ratio of the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to the acid may vary appropriately within an acceptable range, as exemplified below. In one or more embodiments, the molar ratio of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to acid may be 1:(2±0.4), such as 1:(2±0.3), 1:(2±0.2), 1:(2±0.1), or 1:2. The acid is optionally selected from hydrochloric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid, fumaric acid, maleic acid, tartaric acid, hydrobromic acid, oxalic acid, phosphoric acid, sulfuric acid, acetic acid, propionic acid, perchloric acid, malic acid, salicylic acid, mandelic acid, lactic acid, and succinic acid.

[0036] In one or more embodiments, the molar ratio of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to acid may be 1:(1±0.2), such as 1:(1±0.15), 1:(1±0.1), 1:(1±0.05), or 1:1. The acid is optionally selected from hydrochloric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid, fumaric acid, maleic acid, tartaric acid, hydrobromic acid, oxalic acid, phosphoric acid, sulfuric acid, acetic acid, propionic acid, perchloric acid, malic acid, salicylic acid, mandelic acid, lactic acid, and succinic acid.

[0037] In one or more embodiments, there is provided a method for preparing a salt of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, comprising reacting 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine with an acid to obtain the salt. Preferably, the reaction is carried out in water and / or an organic solvent. The solvent is one or a mixture of one or more selected from ketones having 2 to 6 carbon atoms, ethyl acetate, lower aliphatic alcohols, and tetrahydrofuran. The lower aliphatic alcohols refer to alcohols containing 1 to 8 carbon atoms. Furthermore, the ketone having 2 to 6 carbon atoms is preferably acetone. Further still, the lower fatty alcohol is optionally selected from methanol, ethanol, propanol, and isopropanol. Preferably, the reaction is carried out in one or a mixture of one or more of the following solvents: acetone, methanol, and ethanol. Preferably, the acid is one selected from hydrochloric acid, HCl, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid, fumaric acid, maleic acid, and tartaric acid, and more preferably, hydrochloric acid. Note that the hydrochloric acid refers to an aqueous solution of HCl, and HCl refers to HCl gas.

[0038] In one or more embodiments, there is provided a method for preparing 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, comprising reacting 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine with hydrochloric acid in acetone, or ethanol, or isopropanol, or tetrahydrofuran, stirring, and filtering to obtain the hydrochloride salt.

[0039] In one or more embodiments, there is provided a pharmaceutical composition comprising a salt of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine and one or more pharma- ceutically acceptable carriers and / or diluents. Preferably, the salt is the hydrochloride salt.

[0040] In one or more embodiments there is provided the use of a salt of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine or a pharmaceutical composition comprising a salt of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in the manufacture of a medicament for the prevention and / or treatment of cancer, organ damage and degenerative diseases.

[0041] In one or more embodiments, there is provided use of a salt of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine or a pharmaceutical composition comprising a salt of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in the manufacture of a medicament for the prevention and / or treatment of cancer, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, immune-related diseases, hepatic and renal failure, inflammation, and metabolic diseases.

[0042] In one or more embodiments, the disease is cancer, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, immune-related disease, hepatic and renal failure, inflammation, atherosclerosis, diabetes, or a diabetic complication.

[0043] In one or more embodiments, the stroke is a hemorrhagic stroke and / or an ischemic stroke, which is also called cerebral infarction or cerebral infarction.

[0044] Compared with the prior art, the present invention provides the following beneficial effects: 1. After 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was converted into the hydrochloride salt, the solubility, stability and hygroscopicity were all improved. The hydrochloride has a significantly improved solubility compared to the free compound. For example, in one embodiment of the present invention, the hydrochloride has a water solubility of about 7700 times higher than that of the free compound, and other salts such as citrate also have a significantly improved solubility compared to the free compound. The hydrochloride has a significantly improved stability compared to the free compound. For example, in one embodiment of the present invention, the hydrochloride has a better stability under high temperature, high humidity, and light irradiation conditions than the free compound, and has a significantly lower content of total impurities. In addition, the hydrochloride has a lower hygroscopicity than the free compound.

[0045] 2. 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride shows significant improvement in performance over other salts. In terms of solubility, hydrochloride has better solubility than other salts. For example, in one embodiment of the present invention, hydrochloride has 7 times the solubility of citrate, which has good solubility, and 35 times the solubility of maleate and fumarate. In terms of stability, hydrochloride has better effect than other salts. For example, in one embodiment of the present invention, hydrochloride has higher stability under high temperature, high humidity, and light irradiation conditions than other salts, and the total impurity content is obviously lower than other salts. In another embodiment, hydrochloride did not change the color of the solution in a hydrochloric acid solution of pH 1.0, whereas other salts such as citrate, fumarate, and maleate changed obviously, indicating that hydrochloride has better stability. In terms of hygroscopicity, hydrochloride has lower hygroscopicity than other salts. In addition, hydrochloride can be well solidified during the manufacturing process, while other salts such as methanesulfonate and p-toluenesulfonate become sticky paste-like. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 is an XRPD pattern of crystalline form A obtained in Example 1. [Diagram 2] FIG. 2 is an XRPD pattern of crystalline form A obtained in Example 2. [Diagram 3] FIG. 3 is an XRPD pattern of crystalline form B. [Figure 4] FIG. 4 is a purity detection pattern of crystalline form A. [Diagram 5] FIG. 5 is the 1H NMR spectrum of crystalline form A. [Figure 6] FIG. 6 is a TGA curve of crystalline form A. [Figure 7] FIG. 7 is a DSC curve of crystalline form A. [Figure 8] FIG. 8 is an XRPD pattern of a stability study of crystalline form A. [Figure 9] FIG. 9 is an XRPD pattern of the stability study of crystalline form B. [Figure 10]FIG. 10 is a 1H NMR spectrum of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. [Figure 11] FIG. 11 is a C NMR spectrum of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. [Figure 12] FIG. 12 is a 1H NMR spectrum of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate. [Figure 13] FIG. 13 is a 1H NMR spectrum of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate. [Figure 14] FIG. 14 is a 1H NMR spectrum of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] For a specific synthesis method of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free state), please refer to the production method of International Application WO2019205854A1.

[0048] Example 1: Method 1 for preparing crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride [ka]

[0049] Nitrogen was introduced into a 50 L reaction vessel to replace the gas inside the reaction vessel, and then under a nitrogen atmosphere, 1.70 kg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free state) and 17.78 kg of methanol were added to the reaction vessel, stirring was started, and a mixed solution of 0.41 kg of concentrated hydrochloric acid (mass fraction 36%) and 0.82 kg of methanol was added dropwise to the reaction vessel under conditions of 25±5°C, and the addition was completed in about 1 hour. After the dropwise addition was completed, the reaction mixture was crystallized while being kept at 25±5°C for 2-4 hours with stirring, then the reaction mixture was filtered, the filter cake was washed with 1.50 kg of methanol, and the filter cake was thermally dried at 40°C-50°C under vacuum (pressure -0.08MPa--0.10MPa) for 12-16 hours, and a total of 1.64 kg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystal form A was collected. The crystal yield was 88.3%. XRPD detection revealed that it was crystal form A (XRPD pattern is shown in Figure 1).

[0050] The prepared crystalline form A had a purity of 99.76%, the maximum single impurity content was 0.068%, and the total impurities and single impurities were less than the control limit ≦0.15% (the maximum daily dosage of this product ≦2g / day) specified by the "Technical Guidelines for the Study of Impurities in Chemical Drugs" issued by the National Medical Products Administration and ICH Q3A. (In the purity detection pattern shown in Figure 4, the main peak named DA414 is crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride.)

[0051] Nuclear magnetic data: 1H NMR (400 MHz, DMSO) δ=11.27 (s, 1H), 8.64 (s, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.92 (m, 4H), 6.80 (d, J = 7.9 Hz, 1H), 6.71 (t, J = 8.6 Hz, 2H), 6.64 (d, J = 7.9 Hz, 1H), 6.56 (d, J = 1.2 Hz, 1H), 3.90 (m, 1H), 3.60 (d, J = 73.7 Hz, 2H), 3.39 (s, 2H), 3.16 (d, J = 9.1 Hz, 4H), 2.77 (s, 3H), 1.45 (d, J = 7.2 Hz, 3H),( 1 1 H NMR pattern is shown in Figure 5).

[0052] Example 2: Method 2 for preparing crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride [ka]

[0053] Nitrogen was introduced into a 50L reaction kettle to replace the gas inside the reaction kettle, and then under a nitrogen atmosphere, 1.50 kg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free state), 7.11 kg of acetone and 1.50 kg of water were added to the reaction kettle, stirring was started, and a mixed solution of 0.41 kg of concentrated hydrochloric acid (mass fraction 36%), 0.68 kg of acetone and 0.15 kg of water was added dropwise to the reaction kettle under conditions of 25±5°C, and the addition was completed in about 1 hour. After the dropwise addition was completed, the reaction mixture was stirred at 25±5°C for 2-4 hours to crystallize, then the reaction mixture was filtered, the filter cake was washed with 1.50 kg of acetone, and the filter cake was thermally dried at 40°C-50°C under vacuum (pressure -0.08MPa--0.10MPa) for 12-16 hours to collect a total of 1.30 kg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. The crystal yield was 79.5%. XRPD detection revealed that it was crystalline form A (XRPD pattern is shown in Figure 2).

[0054] Example 3: Method for preparing crystalline form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride Approximately 5 mg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed out, 0.2 mL of N,N-dimethylformamide was added thereto, and the mixture was dissolved by ultrasonication. The solution was filtered into a 5 mL vial, and a stirrer was added. Under stirring conditions at room temperature, 4 mL of ethyl acetate was added dropwise, and a large amount of precipitate was precipitated. The precipitate was filtered, and the filter cake was vacuum dried at room temperature overnight. XRPD detection revealed that the precipitate was crystalline form B (the XRPD pattern of crystalline form B is shown in FIG. 3).

[0055] Example 4: XRPD Measurement of Crystalline Form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine Hydrochloride The measurements were performed according to "Chinese Pharmacopoeia" (2020 edition)--Part 4--General Provisions 0451--X-ray Diffraction Method--Method 2--Powder X-ray Diffraction Method.

[0056] [Table 1]

[0057] The XRPD pattern of the 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystal form is shown in Figure 1 and the XRPD data is shown in Table 2. The crystal form was designated as Form A.

[0058] [Table 2]

[0059] Example 5: XRPD Measurement of Crystalline Form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine Hydrochloride The measurements were performed according to "Chinese Pharmacopoeia" (2020 edition)--Part 4--General Provisions 0451--X-ray Diffraction Method--Method 2--Powder X-ray Diffraction Method.

[0060] [Table 3]

[0061] The XRPD pattern of the 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystalline form is shown in Figure 3, and the XRPD data is shown in Table 4. The crystalline form was designated as Form B.

[0062] [Table 4]

[0063] Example 6: TGA measurements of crystalline form A [Table 5]

[0064] The TGA detection pattern of crystalline form A is shown in FIG. The TGA measurement showed that when the crystalline form A was heated to 120° C., the weight of the sample decreased by about 0.1586%, indicating that the crystalline form A was anhydrous.

[0065] Example 7: DSC Measurement of Crystal Form A [Table 6]

[0066] The DSC pattern of crystalline form A is shown in FIG. The DSC pattern showed that crystalline form A had a single endothermic peak and a melting point of about 265.31° C. The results indicated that crystalline form A had good thermal stability.

[0067] Example 8: Ion Chromatography (IC) of Crystalline Form A [Table 7]

[0068] The results of ion chromatography (IC) measurement showed that the salt formation ratio was 1:1, indicating that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine and HCl were combined in a ratio of 1:1.

[0069] Example 9: Another method for preparing crystalline form A (1) Evaporative crystallization method Experimental procedure: Starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystalline form A was weighed out in portions of about 10 mg into 5 mL vials, and dissolved in a volume of the mixed solvent in Table 8, respectively, and filtered into another 5 mL vial using a 0.45 μm PTFE filter, and left open to volatilize at various temperatures. The resulting solid was collected and subjected to XRPD measurement. According to the measurement results shown in Table 8, the product was hydrochloride crystalline form A.

[0070] [Table 8]

[0071] (2) Antisolvent addition method Approximately 5 mg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into a 5 mL vial, and the solid was completely dissolved in the corresponding first solvent in Table 9, and filtered into another 5 mL vial using a 0.45 μm PTFE filter. The second solvent in Table 9 was added dropwise to the clear solution while stirring until a solid precipitated. If no solid precipitated even after adding the total volume of the solvent to 5 mL, the sample was transferred to magnetic stirring at 5° C. and stirred overnight, and if the sample was still clear, it was transferred to room temperature and evaporated or spun dry. The solid was collected by centrifugation and subjected to XRPD measurement. The product was hydrochloride crystalline form A, with the measurement results shown in Table 9.

[0072] [Table 9]

[0073] (3) Anti-poor solvent addition method Approximately 5 mg of starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystalline form A was weighed into a 5 mL vial, and the solid was completely dissolved in the corresponding first solvent in Table 10, respectively, and filtered into another 5 mL vial using a 0.45 μm PTFE filter. Under stirring conditions, the clear solution was added dropwise to the second solvent in Table 10. The volume of the second solvent was 3 mL. If no solid precipitated, it was transferred to magnetic stirring at 5° C. and stirred overnight, after which the still clear sample was transferred to room temperature for evaporation or spin drying. The solid was collected by centrifugation and subjected to XRPD measurement. The measurement results shown in Table 10 showed that only the hydrochloride crystalline form A was obtained.

[0074] [Table 10]

[0075] (4) Cooling crystallization method The starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystalline form A was weighed out in an amount of about 10 mg and added to a 5 mL vial, and a certain volume of the corresponding single solvent or mixed solvent in Table 11 was added, and dissolved under ultrasonic conditions at 50°C, and then filtered into another 5 mL vial using a 0.45 μm PTFE filter. The clear filtrate was placed in a biochemical incubator and kept at 50°C for 120 min, and then cooled to 5°C at a rate of 0.1°C / min, and stirring was continued during the temperature change. When solids precipitated, they were centrifuged to collect the solids and subjected to XRPD measurement. The measurement results are shown in Table 11.

[0076] [Table 11]

[0077] (5) Gas-liquid diffusion method The starting material, crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, was weighed out in an amount of about 10 mg each and placed in a 5 mL vial, and dissolved by adding a certain volume of the first solvent (single solvent or mixed solvent) in Table 12, and filtered into another 5 mL vial using a 0.45 μm PTFE filter. Another 20 mL vial was taken and about 3 mL of the second solvent was added thereto, and the 5 mL vial containing the clear solution was placed in the 20 mL vial with the lid open, and then sealed and allowed to stand at room temperature for about one week. The resulting solid was collected and subjected to XRPD measurement. The measurement results are shown in Table 12.

[0078] [Table 12]

[0079] (6) Polymer induction method Approximately 10 mg of starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystalline form A was weighed into 5 mL vials, and dissolved in a certain volume of mixed solvent (filtered using a 0.45 μm PTFE filter), 1-2 mg of polymer was added, and the vial was sealed with a sealing film with holes on it, and left at room temperature to slowly evaporate. The product of hydrochloride crystalline form A was obtained, with the measurement results shown in Table 13. [Table 13]

[0080] XRPD results showed that the sample crystal forms prepared in Example 9 were all Form A (XRPD patterns not shown).

[0081] Example 10: Another method for preparing crystalline form B (1) Antisolvent addition method Approximately 5 mg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into a 5 mL vial, and the solid was completely dissolved with the corresponding first solvent in Table 14, and filtered into another 5 mL vial using a 0.45 μm PTFE filter. The second solvent in Table 14 was added dropwise to the clear solution while stirring until the solid precipitated. The solid was collected by centrifugation and subjected to XRPD measurement. According to the measurement results shown in Table 14, the hydrochloride crystalline form B product was obtained.

[0082] [Table 14]

[0083] (2) Polymer induction method Approximately 10 mg of the starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystalline form A was weighed into 5 mL vials, and dissolved in a certain volume of mixed solvent (filtered using a 0.45 μm PTFE filter), 1-2 mg of polymer was added, and the vial was sealed with a sealing film with holes on it, and left at room temperature to slowly evaporate. The hydrochloride crystalline form B product was obtained according to the measurement results shown in Table 15.

[0084] [Table 15]

[0085] XRPD results showed that the sample crystal forms prepared in Example 10 were all crystal form B (XRPD patterns not shown).

[0086] Example 11: Hygroscopicity Studies The hygroscopicity of crystalline form A of the hydrochloride salt was evaluated by dynamic vapor sorption (DVS), and the moisture adsorption rate at 25oC / 80%RH was approximately 0.36%, indicating that crystalline form A has slight hygroscopicity and that the crystal form did not change before and after the hygroscopicity experiment.

[0087] The results of the hygroscopicity study showed that crystalline form A of the hydrochloride salt was less hygroscopic than crystalline form B of the hydrochloride salt and had better drug formability.

[0088] Example 12: Comparative solubility study According to the general solubility test method of "Chinese Pharmacopoeia" (2020 edition), 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free state), crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, and crystalline form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride were weighed and added to a certain volume of solvent under conditions of 25°C ± 2°C, vigorously shaken for 30 seconds every 5 minutes, and observed for dissolution within 30 minutes to measure the solubility.

[0089] [Table 16]

[0090] Here, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obtained by Method 1 in Example 17.

[0091] As a result, in an aqueous system, the solubility of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obviously superior to the solubilities of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free state), 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, and crystalline form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. In a hydrochloric acid solution system of pH 1.0 (approximately the pH of human gastric juice), the solubility of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obviously superior to that of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free state), 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, and crystalline form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. These results indicate that crystalline form A has better physical and chemical properties and better drug formability. Crystalline form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride is 1000 times more soluble in water than the free form, which is an unexpected effect.

[0092] In addition, the inventors have found during their research that the crystalline forms obtained by crystallizing other salt forms of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, such as the citrate, fumarate and maleate salts, have almost the same solubility in water and in a hydrochloric acid solution of pH 1.0 as compared with the salt forms, and the solubility is not significantly improved.

[0093] Example 13: Stability study of crystalline forms (1) Stability experiment of crystalline form A Experiment 1: Temperature and humidity stability experiment

[0094] Test procedure 1: The hydrochloride crystalline form A sample was left for one week under long-term 25℃ / 60%RH and accelerated 40℃ / 75%RH conditions, respectively, to test the change in crystalline form. The results showed that the purity of the hydrochloride crystalline form A sample was basically unchanged after one week under the two test conditions, the chemical stability was good, and no change in the crystalline form of the sample was observed. The XRPD comparison diagram of the sample stability before and after leaving is shown in Figure 8.

[0095] Test procedure 2: Approximately 10 mg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into a 5 mL vial, and the 5 mL vial was placed in a humidity chamber with the lid open, and the humidity chamber was then sealed. After standing at room temperature for approximately one week, the solid was collected and subjected to XRPD measurement, and no change in the crystalline form of the sample was observed. The measurement results are shown in Table 17.

[0096] [Table 17]

[0097] Experiment 2: Mechanical stability experiment About 10mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystal form A was weighed into a mortar, and 0.05mL of wetting agent was added in three portions and ground, each time for 5min, and the solid was collected and subjected to test XRPD, the sample crystal form did not change. The measurement results are shown in Table 18.

[0098] [Table 18]

[0099] Experiment 3: Other stability experiments Test procedure 1: Approximately 10 mg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into a 5 mL vial, and 1 mL of each of the single solvents or mixed solvents listed in Table 19 was added. The resulting suspension was magnetically stirred at 5° C. for approximately 3 days, and then centrifuged to collect the solid. The sample was subjected to XRPD measurement, and no crystalline form transition occurred. The measurement results are shown in Table 19.

[0100] [Table 19]

[0101] Test procedure 2: Approximately 10 mg of starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride crystalline form A was weighed into each 5 mL vial, and 0.5 mL of each of the single solvents or mixed solvents listed in Table 20 was added to obtain a suspension. The suspension was magnetically stirred at room temperature for about 3 days, and then centrifuged to collect the solid. The XRPD measurement showed that the sample did not undergo crystalline form transition. The measurement results are shown in Table 20.

[0102] [Table 20]

[0103] Test procedure 3: Approximately 10 mg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into each 5 mL vial, and 0.5 mL of the single solvent or mixed solvent listed in Table 21 was added to each vial. The resulting suspension was magnetically stirred at 50° C. for approximately 3 days, and then centrifuged to collect the solid. The sample was subjected to XRPD measurement, and no crystalline form transition occurred. The measurement results are shown in Table 21.

[0104] [Table 21]

[0105] (2) Stability test of crystalline form B Test procedure: A certain amount of the hydrochloride crystalline form B sample was weighed into a sample bottle, and then the sample bottle was left sealed at room temperature for 2 days and 5 days, respectively, and then a certain amount of the sample in the sample bottle was taken for XRPD measurement. The measurement results showed that the hydrochloride crystalline form B sample started to transform into the hydrochloride crystalline form A after being left sealed at room temperature for 2 days, and after being left for 5 days, the crystalline form B partially transformed into the hydrochloride crystalline form A, indicating that the hydrochloride crystalline form B is an unstable crystalline form. The XRPD pattern obtained from the stability study of crystalline form B is shown in Figure 9. It was found that crystalline form A has better crystalline form stability than crystalline form B.

[0106] In addition, crystalline form A has good properties such as compressibility and flowability, and its properties such as specific surface area, bulk density, porosity and particle size are very suitable for the development and manufacture of pharmaceutical preparations, whereas crystalline form B does not have these characteristics.

[0107] In addition, a study on the influencing factors of crystalline form A of the hydrochloride salt and crystalline form B of the hydrochloride salt was conducted, and it was found that crystalline form A was more stable and produced less impurities than crystalline form B when left for 30 days under conditions of high temperature (60°C), high humidity (25°C / 90%±5%RH) and light exposure (45001x±5001x).

[0108] At the same time, compared to the free compound, crystalline form A of the hydrochloride salt exhibited superior stability and a lower content of total impurities was measured.

[0109] (3) Comparative study of the stability of crystalline forms A and B Based on the Chinese Pharmacopoeia 2020 edition and ICH Q1A stability study guideline, crystalline forms A and B were stored under accelerated conditions (40℃±2℃, 75%RH±10%RH) for 6 months to detect the changes in total impurities in crystalline forms A and B. The results are shown in Table 22. Table 22 shows the stability experiments of various crystalline forms. As a result, after 6 months under accelerated conditions, crystalline form A had an increase in total impurities from 0.25% to 0.41%, while crystalline form B had an increase in total impurities from 0.28% to 0.88% after 6 months under accelerated conditions. The results showed that the stability of crystalline form B was very poor, and the stability of crystalline form A was better than that of crystalline form B.

[0110] [Table 22]

[0111] Example 14: Comparative Pharmacokinetic Study Experimental method: Three healthy male SD rats were selected, and after fasting overnight, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free state) or a suspension of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride (solvent: 0.5% CMC-Na) was intragastrically administered at 10 mg / kg. The administration volume was 10 ml / kg. Approximately 200μL of whole blood was collected from the rat orbital venous plexus before administration and 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration, and placed in a centrifuge tube containing EDTA-K2 anticoagulant. The blood was then centrifuged at 3000-4000 rpm for 10 min, and the upper plasma was transferred to another clean centrifuge tube. The blood concentration of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in the plasma sample was detected by LC-MS / MS, and the pharmacokinetic parameters were calculated using a non-compartmental model.

[0112] [Table 23]

[0113] As a result, crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was well absorbed after oral administration to rats, and its blood exposure and bioavailability were superior to those of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (free form), indicating that crystalline form A of the hydrochloride has significantly improved drug formability compared to the free form.

[0114] Example 15: Study on the inhibitory rate of ferroptosis of the compounds of the present invention Experimental method: In this embodiment, a screening model of ferroptosis was constructed to study inhibitors of ferroptosis as follows. The ferroptosis screening model mainly adopted the CCK8 cell viability detection method. First, mouse hippocampal neuronal cells HT22 and human neuroblastoma SH-SY5Y cells were cultured in a dish, and a certain number of cells in the logarithmic growth phase (5000 cells / well) were seeded in a 96-well plate at 80 μL per well, and then cultured in an incubator at 37 °C and 5% CO2 so that the cells were attached to the wall. After 24 hours, 10 μL of the compounds of the present invention at various concentrations prepared in a specified medium and 10 μL of the ferroptosis inducer elastin at a final concentration of 10 μM were added. In addition, three duplicate wells were set up for each compound to ensure the accuracy of the results. Then, a positive control group (10 μL of the compound Ferrostein-1 at a fixed concentration prepared in the same medium and 10 μL of the ferroptosis inducer Elastin were added as above), a blank control group (an equal volume of medium and medium and an equal volume of DMSO were added as above, but no cells were included), and a solvent control group (an equal volume of medium and medium and an equal volume of DMSO were added as above, but no cells were included) were set up. In addition, three duplicate wells were set up for each group to ensure the accuracy of the results. After adding the drug, the wells were placed in an incubator and cultured for 24 hours. After adding 10 μL of CCK8 solution to each well and continuing to culture in an incubator for 2 to 4 hours, the absorbance value at 450 nm was detected using a microplate reader, and the inhibition rate of the drug to ferroptosis was calculated. The inhibition rate was calculated using the following formula.

[0115] Inhibition rate (IR) = [1-(A experimental group-A blank) / (A solvent-A blank)] x 100% The inhibition curves were fitted using GraphPad Prism 6 software, and IC 50 was calculated.

[0116] Test Results: [Table 24]

[0117] The measurement results shown in Table 24 indicate that the crystalline form A of the compound of the present invention, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, has an inhibitory effect on ferroptosis in multiple cell systems, and its inhibitory activity is significantly superior to that of the positive control group, Ferrostein-1.

[0118] Example 16: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine [ka]

[0119] For a specific synthesis method of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, please refer to the preparation method in International Application WO2019205854A1. When the impurities of the product were measured, the total impurity content was 1.36%.

[0120] Example 17: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride [ka]

[0121] Method 1: 5.0g of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (12.45mmol, 1.0eq) was dissolved in 50mL of acetone under the condition of 20-25°C. 1.14mL of concentrated hydrochloric acid (13.70mmol, 1.1eq) with a mass fraction of 36% was diluted to 4.5mL with acetone, and then slowly added dropwise to the reaction solution to react, and after stirring for 1 hour, it was filtered and the filter cake was dried. Then, 4.4g of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obtained with a yield of 80.7%.1 H NMR (400 MHz, DMSO-d6) δ=11.27 (s, 1H), 8.65 (s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 6.93 (m, 4H), 6.81 (d, J = 7.9 Hz, 1H), 6.72 (t, J = 8.6 Hz, 2H), 6.65 (d, J = 7.9 Hz, 1H), 6.56 (d, J = 1.2 Hz, 1H), 3.91 (m, 1H), 3.70 (s, 2H), 3.35 (s, 4H), 3.17 (d, J = 9.1 Hz, 2H), 2.77 (s, 3H), 1.46 (d, J = 7.2 Hz, 3H),( 1 1 H NMR pattern is shown in Figure 10); 13 C NMR (101 MHz, DMSO-d6) δ=148.29, 146.87, 142.56, 138.00, 128.37, 127.87, 126.54, 122.09, 121.26, 116.89, 116.57, 114.91, 113.98, 52.47, 46.11, 43.12, 42.35, 21.91,( 13 The C NMR pattern is shown in Figure 11. The structure was identified and it was found that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine and hydrochloric acid in the obtained hydrochloride were bound in a 1:1 ratio.

[0122] Method 2: Except for replacing the solvent acetone with ethanol, the procedure of Method 1 was followed to obtain the product 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. The product was identified as identical to the product obtained by Method 1.

[0123] Method 3: The product 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obtained by referring to the procedure of Method 1, except that the solvent acetone was replaced with isopropanol. The product was identified by its structure and was found to be identical to the product obtained by Method 1.

[0124] Method 4: Except for replacing the solvent acetone with tetrahydrofuran, the procedure of Method 1 was followed to obtain the product 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. The product was identified as identical to the product obtained by Method 1.

[0125] The impurities of the above hydrochloride products were measured and the total impurity content was found to be 0.10%-0.25%.

[0126] Example 18: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine methanesulfonate [ka]

[0127] 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2 mL of ethyl acetate under 20-25°C conditions, and the solution was dissolved to become clear while stirring. Next, a mixed solution of 2 mL of ethyl acetate and methanesulfonic acid (36.0 mg, 0.374 mmol, 1.0 eq) was dropped into the reaction solution, and a large amount of flocculent solid precipitated during the dropwise addition. After stirring for 1 hour under 20-25°C conditions, the solution was filtered, and the filter cake (the filter cake was sticky) was collected and dried. Then, 90.15 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine methanesulfonate was obtained in a yield of 48.4%.

[0128] Example 19: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine-p-toluenesulfonate [ka]

[0129] 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2 mL of ethyl acetate under 20-25°C conditions, and the solution was dissolved to become clear while stirring. Then, 65 mg of p-toluenesulfonic acid (0.374 mmol, 1.0 eq) was added to the reaction solution, a small amount of solid precipitated, and the mixture was stirred for 1 hour under 20-25°C conditions, filtered, and the filter cake (the filter cake was sticky) was collected and dried. Then, 100.20 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine-p-toluenesulfonate was obtained in a yield of 46.5%.

[0130] Example 20: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate [ka]

[0131] Method 1: 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 4 mL of methanol under 20-25°C conditions, and the solution was dissolved to a clear solution while stirring (a solid was slowly precipitated after dissolution). Next, 72 mg of citric acid (0.374 mmol, 1.0 eq) was added to the reaction solution, and after dissolving the turbidity, a solid was gradually precipitated. After stirring for 4 hours under 20-25°C conditions, the solution was filtered, and the filter cake was dried. Then, 183.10 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate was obtained.

[0132] Method 2: 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 4 mL of acetone under conditions of 20 to 25°C, and the solution was dissolved while stirring until it became clear. Next, 72 mg of citric acid (0.374 mmol, 1.0 eq) was added to the reaction solution, and a solid was gradually precipitated during stirring. After stirring for 2 hours under conditions of 20 to 25°C, the solution was filtered, and the filter cake was dried. Then, 179.63 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate was obtained. 1 H NMR (400 MHz, DMSO-d6) δ=10.88 (s, 2H), 8.51 (s, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.99 - 6.93 (m, 1H), 6.89 (d, J = 8.4 Hz, 3H), 6.82 (d, J = 7.6 Hz, 1H), 6.73 (m, 1H), 6.66 (m, 2H), 6.52 (d, J = 1.6 Hz, 1H), 3.91 (m, 1H), 3.22 (s, 4H), 2.92 (d, J = 4.4 Hz, 4H), 2.65 (d, J = 15.2 Hz, 2H), 2.57 (m, 4H), 2.53 (s, 2H), 2.09 (s, 3H), 1.47 (d, J = 7.2 Hz, 3H),( 1 The H NMR pattern is shown in Figure 12. The structure was identified and it was found that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine and citric acid were bound in a 1:2 ratio in the obtained citrate.

[0133] Method 3: Under conditions of 20 to 25°C, 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2 mL of ethyl acetate, and the solution was stirred until it became clear. Next, 72 mg of citric acid (0.374 mmol, 1.0 eq) was added to the reaction solution, and a solid was gradually precipitated during stirring. After stirring for 4 hours under conditions of 20 to 25°C, the solution was filtered, and the filter cake was dried. Then, 189.4 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate was obtained.

[0134] The impurities of the above citrate product were measured, and the total impurity content was found to be 0.64%-0.80%.

[0135] Example 21: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate [ka]

[0136] Method 1: 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2 mL of ethyl acetate under 20-25°C conditions, and the solution was stirred until it became clear. Next, 43.41 mg of fumaric acid (trans-2-butenedioic acid; 0.374 mmol, 1.0 eq) was added to the reaction solution, and a solid was gradually precipitated under stirring. After stirring for 4 to 5 hours under 20-25°C conditions, a large amount of solid was precipitated. The mixture was filtered and the filter cake was dried. Then, 146.3 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate was obtained in a yield of 75.8%. 1H NMR (400 MHz, DMSO-d6) δ=8.51 (s, 1H), 7.06 (d, J = 8.6 Hz, 2H), 6.99 - 6.93 (m, 1H), 6.91 - 6.84 (m, 3H), 6.81 (d, J = 8.0 Hz, 1H), 6.73 (m, 1H), 6.65 (m, 2H), 6.59 (s, 2H), 6.51 (d, J = 1.6 Hz, 1H), 3.90 (m, 1H), 3.22 - 3.08 (m, 4H), 2.70 - 2.58 (m, 4H), 2.35 (s, 3H), 1.46 (d, J = 7.2 Hz, 3H),( 1 The H NMR pattern is shown in Figure 13. The structure was identified and it was found that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine and fumaric acid were bound in a 1:1 ratio in the obtained fumarate salt.

[0137] Method 2: 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2 mL of acetone under 20-25°C conditions, and the solution was stirred until it became clear. Then, 43.41 mg of fumaric acid (trans-2-butenedioic acid; 0.374 mmol, 1.0 eq) was added to the reaction solution, and initially, a small amount of solid precipitated on the bottom of the reaction bottle, and then it gradually became an oily substance. After stirring for 4 to 5 hours under 20-25°C conditions, a large amount of solid precipitated. The mixture was filtered and the filter cake was dried. Then, 121 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate was obtained in a yield of 62.5%.

[0138] The impurities of the above fumarate product were measured, and the total impurity content was 0.76%-0.84%.

[0139] Example 22: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine tartrate Method 1: Under the condition of 20-25°C, 150mg (0.374mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2mL of acetone, and the solution was stirred to become clear. Then, 56mg (DL type; 0.374mmol, 1.0eq) of tartaric acid was added to the reaction solution, and a small amount of solid precipitated at the bottom of the reaction bottle during stirring. Stirring was continued at 20-25°C for 2 hours, and a solid precipitated. The solution was filtered, and the filter cake was dried. Then, 155.3mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine tartrate was obtained.

[0140] Method 2: Under the condition of 20-25°C, 150mg (0.374mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2mL of ethyl acetate, and the solution was stirred to become clear. Next, 56mg (DL type; 0.374mmol, 1.0eq) of tartaric acid was added to the reaction solution, and a small amount of solid precipitated at the bottom of the reaction bottle during stirring. Stirring was then continued at 20-25°C for 2 hours, and a large amount of solid precipitated. The solution was filtered, and the filter cake was dried. Then, 166.5mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine tartrate was obtained.

[0141] Example 23: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate [ka]

[0142] Under the condition of 20-25°C, 150mg (0.374mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 3mL of ethyl acetate, and the solution was stirred to become clear. Then, 43.41mg of maleic acid (cis-2-butenedioic acid; 0.374mmol, 1.0eq) was added to the reaction solution, and a small amount of solid precipitated at the bottom of the reaction bottle while stirring, and then gradually became oily. After stirring was continued for 4-5 hours at 20-25°C, a large amount of solid precipitated. The mixture was filtered and the filter cake was dried. Then, 132.8mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate was obtained in a yield of 68.6%, and the total impurity content was 0.42%. 1 H NMR (400 MHz, DMSO-d6) δ=8.51 (s, 1H), 7.11 (d, J = 8.8 Hz, 2H), 7.00 - 6.87 (m, 4H), 6.82 (d, J = 8.0 Hz, 1H), 6.73 (m, 1H), 6.66 (dd, J = 8.0, 2.0 Hz, 2H), 6.54 (d, J = 1.6 Hz, 1H), 6.05 (s, 2H), 3.92 (m, 1H), 3.28 (s, 8H), 2.83 (s, 3H), 1.47 (d, J = 7.2 Hz, 3H),( 1 The H NMR pattern is shown in Figure 14. The structure was identified and it was found that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine and maleic acid were bonded in a 1:1 ratio in the obtained maleate.

[0143] Example 24: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrobromide By referring to the preparation method of Example 17, hydrochloric acid was replaced with hydrobromic acid to prepare 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrobromide.

[0144] Example 25: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine phosphate Referring to the preparation method of Example 17, hydrochloric acid was replaced with phosphoric acid to prepare 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine phosphate.

[0145] Example 26: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine oxalate Referring to the preparation method of Example 17, hydrochloric acid was replaced with oxalic acid to prepare 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine oxalate.

[0146] Example 27: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine ethanesulfonate Referring to the preparation method of Example 21, fumaric acid was replaced with ethanesulfonic acid to prepare 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine ethanesulfonate.

[0147] Example 28: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine malate Referring to the preparation method of Example 21, fumaric acid was replaced with malic acid to prepare 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine malate.

[0148] Example 29: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine succinate Referring to the preparation method of Example 21, fumaric acid was replaced with succinic acid to prepare 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine succinate.

[0149] Example 30: Preparation of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazinepropionate Referring to the preparation method of Example 21, fumaric acid was replaced with propionic acid to prepare 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine propionate.

[0150] Example 31: Comparative solubility study According to the general solubility test method of the "Chinese Pharmacopoeia" (2020 edition), 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine The enoate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate, and 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate were weighed out, added to a certain volume of a solvent at 25°C ± 2°C, and vigorously shaken for 30 seconds every 5 minutes. Dissolution within 30 minutes was observed to measure the solubility.

[0151] [Table 25]

[0152] As a result of solubility experiments, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was more soluble in water and in a hydrochloric acid solution of pH 1.0, especially in water, than 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine. 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate was 1000 times more soluble in water than 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine.

[0153] Furthermore, the solubility of the hydrochloride salt is clearly superior to that of other salts of this compound, including a 7-fold improvement over the more soluble citrate salt and a 35-fold improvement over the maleate and fumarate salts.

[0154] Example 32: Stability Study 1 Test (1): High temperature test In accordance with the method of "Chinese Pharmacopoeia" (2020 edition) 9001 "Guideline for Stability Testing of Raw Drugs and Preparations," the test samples were placed in an appropriate thermostatic device with the lid open and left at 60°C for 30 days, and samples were taken on the 5th, 10th, and 30th days.

[0155] As a result of the high temperature test, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, after being converted into a salt, was more stable under high temperature conditions than 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in its free state. Among them, the hydrochloride salt was found to have the best stability under high temperature conditions, no change in properties, and a low amount of total impurities.

[0156] [Table 26]

[0157] Test (2): High humidity test In accordance with the method of "Guideline for Stability Testing of Raw Drugs and Preparations" in "Chinese Pharmacopoeia" (2020 edition) 9001, the test samples were placed in a humidity-controlled sealed container with the lid open and left at 25°C for 30 days under conditions of relative humidity of 90% ± 5%, and samples were taken on the 5th, 10th, and 30th days.

[0158] As a result of the high humidity test, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, after becoming a salt, was more stable under high humidity conditions than 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in the free state. Among them, it was found that the hydrochloride salt was more stable under high humidity conditions than other salt forms, did not change in properties, and produced a low amount of total impurities.

[0159] [Table 27]

[0160] Test (3): Light irradiation test According to the method of "Chinese Pharmacopoeia" (2020 edition) 9001 "Guideline for Stability Testing of Raw Materials and Preparations", the test sample is placed in a stability test box, and the illuminance is 45001x ± 5001x, the total illuminance of the light source is 1.2x10 6 lux·hr or more, near-ultraviolet lamp energy 200W·hr / m 2 The samples were left under the above conditions for 30 days, and samples were taken on the 5th, 10th, and 30th days.

[0161] As a result of the light irradiation test, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, after becoming a salt, was more stable under light irradiation conditions than 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in the free state. Among them, the appearance of the citrate salt changed from light yellow to gray on the 5th day and to dark gray on the 10th day under light irradiation conditions, while the hydrochloride salt was more stable under light irradiation conditions than the other salt forms, did not change in properties, and produced a low amount of total impurities.

[0162] [Table 28]

[0163] In conclusion, the results of the stability experiment showed that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was more stable under high temperature, high humidity and light exposure conditions than the free compound and other salts, and the total impurity content was obviously lower than the other salts and the free compound.

[0164] Example 33: Stability Study 2 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate, and 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate were weighed out in equal amounts and added to a constant volume of a hydrochloric acid solution of pH 1.0 (similar to the acidity of human gastric juice) at 25°C ± 2°C. The solution was vigorously shaken for 30 seconds every 5 minutes, and the color state of the solution was observed within 0.5 hours and 24 hours.

[0165] [Table 29]

[0166] The experimental results showed that the 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate and 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate samples showed obvious change in color in a hydrochloric acid solution of pH 1.0, and were less stable than 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride.

[0167] Example 34: Comparative experiment on hygroscopicity In accordance with the "Guideline for Drug Hygroscopicity" in Part 4 of the "Chinese Pharmacopoeia" (2020 edition), hygroscopicity tests were conducted on 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate, etc. obtained in the above examples.

[0168] The description of hygroscopic characteristics and the definition of hygroscopic weight gain are as follows: deliquescence absorb enough water to form a liquid Extremely hygroscopic: Weight increase due to moisture absorption is 15% or more. Hygroscopic: Weight increase due to moisture absorption is less than 15% but not less than 2%. Slightly hygroscopic: Weight increase due to moisture absorption is less than 2% but not less than 0.2%. No or almost no moisture absorption: Weight increase due to moisture absorption is less than 0.2%.

[0169] The experimental results showed that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride had low hygroscopicity, excellent physical and chemical properties, and the best drug formability.

[0170] The above embodiments are merely used to describe the technical intent and features of the present invention, and are intended to enable those skilled in the art to understand and implement the contents of the present invention accordingly, and are not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the spirit and essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A crystal of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride having the following crystalline form A. The X-ray powder diffraction pattern of said crystalline form A, expressed in 2θ angles using Cu—Ka radiation, has characteristic peaks at 10.68±0.2°, 14.36±0.2°, 18.57±0.2°, 21.08±0.2°, 22.14±0.2°, 23.40±0.2°, and 29.03±0.2°; Preferably, the X-ray powder diffraction pattern of said crystalline form A, expressed in 2θ angles using Cu—Ka radiation, has characteristic peaks at 10.68±0.2°, 14.36±0.2°, 17.84±0.2°, 18.57±0.2°, 21.08±0.2°, 22.14±0.2°, 23.40±0.2°, 27.50±0.2°, 29.03±0.2°; Preferably, the X-ray powder diffraction pattern of said crystalline form A, expressed in 2θ angles using Cu—Ka radiation, has characteristic peaks at 10.68±0.2°, 14.36±0.2°, 16.54±0.2°, 17.84±0.2°, 18.57±0.2°, 20.89±0.2°, 21.08±0.2°, 22.14±0.2°, 22.92±0.2°, 23.40±0.2°, 25.88±0.2°, 27.50±0.2°, 29.03±0.2°; Preferably, the X-ray powder diffraction pattern of said crystalline form A, expressed in 2θ angles using Cu—Ka radiation, has characteristic peaks at 10.68±0.2°, 12.63±0.2°, 14.36±0.2°, 16.06±0.2°, 16.54±0.2°, 17.84±0.2°, 18.57±0.2°, 20.89±0.2°, 21.08±0.2°, 22.14±0.2°, 22.92±0.2°, 23.40±0.2°, 25.11±0.2°, 25.51±0.2°, 25.88±0.2°, 27.50±0.2°, 28.54±0.2°, 29.03±0.2°, 33.55±0.2°; More preferably, the X-ray powder diffraction pattern of said crystalline form A, when expressed in 2θ angles using Cu—Ka radiation, is shown in FIG.

2. A crystal of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride having the following crystalline form B. The X-ray powder diffraction pattern of said crystalline form B has characteristic peaks at 16.46±0.2° and 22.01±0.2° when expressed in 2θ angles using Cu—Ka radiation; Preferably, the X-ray powder diffraction pattern of said crystalline form B, expressed in 2θ angles using Cu—Ka radiation, has characteristic peaks at 16.46±0.2°, 22.01±0.2°, 27.62±0.2°, 28.91±0.2°, 33.41±0.2°; Preferably, the X-ray powder diffraction pattern of said crystalline form B, expressed in 2θ angles using Cu—Ka radiation, has characteristic peaks at 5.41±0.2°, 10.92±0.2°, 16.46±0.2°, 22.01±0.2°, 27.62±0.2°, 28.91±0.2°, 33.41±0.2°; More preferably, the X-ray powder diffraction pattern of said crystalline form B, when expressed in 2θ angles using Cu—Ka radiation, is shown in FIG.

3. 10. A pharmaceutical composition comprising crystalline form A of the hydrochloride salt of claim 1 or crystalline form B of the hydrochloride salt of claim 2 and a pharma- ceutically acceptable carrier.

4. Use of crystalline form A of the hydrochloride salt according to claim 1, or crystalline form B of the hydrochloride salt according to claim 2, or a pharmaceutical composition according to claim 3, in the manufacture of a medicament for the prevention and / or treatment of cancer, organ damage and degenerative diseases.

5. Use of crystalline form A of the hydrochloride salt according to claim 1, or crystalline form B of the hydrochloride salt according to claim 2, or the pharmaceutical composition according to claim 3, in the manufacture of a medicament for the prevention and / or treatment of cancer, neurodegenerative diseases, cardiovascular diseases, cerebrovascular diseases, immune-related diseases, hepatic and renal failure, inflammation, and metabolic diseases, comprising Preferably, the disease is cancer, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, atherosclerosis, immune-related disease, hepatic and renal failure, inflammation, diabetes, or diabetic complications; Preferably, said stroke is a hemorrhagic stroke and / or an ischemic stroke.

6. Use of the crystalline form A of the hydrochloride salt of claim 1, or the crystalline form B of the hydrochloride salt of claim 2, or the pharmaceutical composition of claim 3 in the manufacture of a ferroptosis inhibitor.

7. A method for preparing the crystalline form A of the hydrochloride salt according to claim 1, comprising dispersing the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in methanol at 10-40° C., dropping a mixed solution of concentrated hydrochloric acid and methanol, stirring and crystallizing, filtering, washing the filter cake with methanol, and then drying the filter cake in a vacuum to obtain a crystalline material, Preferably, the weight ratio of the solvent methanol to the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine is from 5:1 to 15:1, preferably from 8:1 to 12:1; Preferably, the weight ratio of said compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is from 1:1 to 8:1, preferably from 3:1 to 5:1; Preferably, the stirring and crystallization is carried out for 1 to 8 hours, preferably for 2 to 5 hours.

8. A method for preparing the crystalline form A of the hydrochloride salt according to claim 1, comprising dispersing the compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in acetone and water at 10-40° C., dropping a mixed solution of concentrated hydrochloric acid, acetone and water, stirring and crystallizing, filtering, washing the filter cake with acetone, and then vacuum drying the filter cake to obtain a crystalline material, Preferably, the weight ratio of the solvent acetone, water and compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine is (1-8):(0.5-3):(0.5-3), preferably (4-6):(0.5-1):(0.5-1); Preferably, the weight ratio of said compound 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is from 1:1 to 8:1, preferably from 3:1 to 5:1; Preferably, the stirring and crystallization is carried out for 1 to 8 hours, preferably for 2 to 5 hours.

9. 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine salt, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine methanesulfonate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine ethanesulfonate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine-p-toluenesulfonate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine citrate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine tartrate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrobromide, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine oxalate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine phosphate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine sulfate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine acetate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazinepropionate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine perchlorate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine malate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine salicylate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine mandelate, 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine lactate, and A salt arbitrarily selected from 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine succinate.

10. 10. The salt according to claim 9, characterized in that the salt is 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride.

11. The salt according to claim 9, characterized in that the molar ratio of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to hydrochloric acid in the hydrochloride is 1:(0.5-2), preferably said molar ratio is 1:1 or 1:

2.

12. A method for producing the salt according to claim 9, characterized in that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine is reacted with an acid to produce the salt, Preferably, the reaction is carried out in water and / or an organic solvent, and the organic solvent is one or a mixture of one or more selected from ketones having 2 to 6 carbon atoms, ethyl acetate, lower aliphatic alcohols, and tetrahydrofuran. Furthermore, it is preferable that the ketones having 2 to 6 carbon atoms are acetone, and more preferably, the lower aliphatic alcohols are selected from methanol, ethanol, propanol, and isopropanol. Preferably, the reaction is carried out in one or a mixture of one or more solvents selected from acetone, methanol and ethanol; Preferably, the acid is any one selected from hydrochloric acid, HCl, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid, fumaric acid, maleic acid, tartaric acid, hydrobromic acid, oxalic acid, phosphoric acid, sulfuric acid, acetic acid, propionic acid, perchloric acid, malic acid, salicylic acid, mandelic acid, lactic acid, and succinic acid, more preferably hydrochloric acid.

13. The method according to claim 12, characterized in that the salt is obtained by reacting 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine with hydrochloric acid in acetone, ethanol, isopropanol, or tetrahydrofuran, stirring, and filtering.

14. A pharmaceutical composition comprising a salt according to any one of claims 9 to 12, preferably said salt being a hydrochloride salt, and one or more pharma- ceutically acceptable carriers and / or diluents.

15. Use of a salt according to any one of claims 9 to 12, or a pharmaceutical composition according to claim 14, in the manufacture of a medicament for the prevention and / or treatment of cancer, organ damage and degenerative diseases.

16. Use of the salt according to claims 9 to 12 or the pharmaceutical composition according to claim 14 in the manufacture of a medicament for the prevention and / or treatment of cancer, neurodegenerative diseases, cardiovascular diseases, cerebrovascular diseases, immune-related diseases, hepatic and renal failure, inflammation, and metabolic diseases.

17. 16. The use according to claim 15, characterized in that the disease is cancer, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, immune-related disease, hepatic and renal failure, inflammation, atherosclerosis, diabetes, or diabetic complications.

18. 18. The use according to claim 17, characterized in that the stroke is a hemorrhagic stroke and / or an ischemic stroke.

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