Crystalline forms, salts, manufacturing 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 the Chinese patent applications No. 202210108728.0 and No. 202210108746.9 filed with the China National Intellectual Property Administration on 28 January 2022, and the entire contents of those applications are incorporated herein by reference.

[0002] This invention belongs to the field of medicinal chemistry and, more specifically, relates to the crystalline form, salts, methods for producing the same, and uses of pharmacopoeial phenothiazine compounds. [Background technology]

[0003] Ferroptosis is a programmed cell death mechanism driven by iron-dependent and lipid peroxidation. Morphological features of ferroptosis primarily manifest as a decrease in mitochondrial volume, an increase in mitochondrial membrane density, a decrease or absence of mitochondrial cristae, and rupture of the mitochondrial outer membrane while the cell nucleus remains normal in size. These are the main morphological features that distinguish ferroptosis from apoptosis, necrosis, and autophagy. Biochemical features of ferroptosis primarily manifest as intracellular iron and ROS accumulation, activation of the mitogen-activated protein kinase (MAPK) signaling pathway, inhibition of the cystine / glutamate transporter system, and increased NADPH oxidation.

[0004] Research is increasingly confirming the close link between cellular ferroptosis and many diseases, disorders, and illnesses. This mode of cell death was first discovered by Dixon et al. in 2012 to be associated with 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 injury, 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 development and progression of various cancers, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, immune-related diseases, hepatic and renal failure, inflammation, and metabolic diseases. In particular, it plays a crucial role in diseases such as Alzheimer's disease, Parkinson's disease, tumors, stroke, ischemia-reperfusion injury, atherosclerosis, hepatic and renal failure, inflammation, and complications of diabetes. Because stimulating or inhibiting the development of ferroptosis makes the development and progression of related diseases tolerable, ferroptosis inhibitors are considered drugs that have the potential to treat these diseases.

[0005] International patent application WO2019205854A1 discloses the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, which has excellent ferroptosis inhibitory activity, and a method for producing the same. However, prior art has not studied its crystalline form.

[0006] Drug crystalline polymorphism is a common phenomenon in pharmaceutical research and development and is an important factor influencing the quality of pharmaceuticals. Different crystalline forms often result in differences in solubility, stability, hygroscopicity, and bioavailability, directly affecting the quality of the pharmaceutical formulation, its absorption behavior in the human body, and ultimately the benefit-to-benefit ratio of the therapeutic effect and side effects of the formulation in the human body. Therefore, in order to maximize the effectiveness of a drug, it is necessary to study the crystalline form of the compound and find a crystalline form that is highly pure, has good solubility, good stability, and high bioavailability, which is extremely important for drug development. [Overview of the project]

[0007] One of the objectives of the present invention is to provide multiple crystalline forms of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, particularly multiple crystalline forms of its hydrochloride salt, in which the drug-forming properties of the pharmaceutical, such as stability, solubility, hygroscopicity, and bioavailability, are improved.

[0008] Another object of the present invention is to provide the above-mentioned crystalline pharmaceutical composition, manufacturing method, and uses.

[0009] To achieve the above objective, the present invention employs the following technical solutions.

[0010] In one or more embodiments, crystals of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride having the following crystal form A are provided. When expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal 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°.

[0011] In one or more embodiments, when expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal 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°, and 29.03±0.2°.

[0012] In one or more embodiments, when expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal form A 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°, and 29.03±0.2°.

[0013] In one or more embodiments, when expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal form A 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°, and 33.55±0.2°.

[0014] In one or more embodiments, when expressed in terms of a 2θ angle using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal form A is shown in Figure 1.

[0015] In one or more embodiments, crystals of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride having the following crystal form B are provided. When expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal form B has characteristic peaks at 16.46±0.2° and 22.01±0.2°.

[0016] In one or more embodiments, when expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal 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°.

[0017] In one or more embodiments, when expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal 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°.

[0018] In one or more embodiments, when expressed in terms of a 2θ angle using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal form B is shown in Figure 3.

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

[0020] In one or more embodiments, 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 drug for the prevention and / or treatment of cancer, organ damage, and degenerative diseases is provided.

[0021] In one or more embodiments, the use of crystalline form A of the hydrochloride, crystalline form B of the hydrochloride, or a pharmaceutical composition thereof is provided in the manufacture of agents 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 disorders, hepatic and renal failure, inflammation, diabetes, and complications of diabetes. In one or more embodiments, the stroke is a hemorrhagic stroke and / or an ischemic stroke. The ischemic stroke is also called a cerebral infarction.

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

[0023] In one or more embodiments, a method for producing crystalline form A of the hydrochloride of the present application is provided, which includes dispersing 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine in methanol at 10 to 40 °C, then 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 under vacuum to obtain a crystalline substance. Preferably, the weight ratio of the solvent methanol to 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine is 5:1 to 15:1, such as 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 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is 1:1 to 8:1, such as 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, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours.

[0024] In one or more embodiments, a method for producing crystalline form A of the hydrochloride salt of this application is provided, comprising dispersing compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine in acetone and water at 10-40°C, then adding concentrated hydrochloric acid and a mixed solution of acetone and water dropwise, stirring and crystallizing, filtering, washing the filtered cake with acetone, and then vacuum drying the filtered cake to obtain a crystalline substance. Preferably, the weight ratio of the solvent acetone, water, and compound 2-(1-(4-(4-methylpiperazine-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, or 5:1:1. Preferably, the weight ratio of the compound 2-(1-(4-(4-methylpiperazine-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, 3.6:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, and 8:1. Preferably, the stirring and crystallization is carried out for 1 to 8 hours, and more preferably for 2 to 5 hours. In the above manufacturing method, the mass fraction of concentrated hydrochloric acid is 20% or more, for example, 20%, 25%, 30%, 35%, 36%, 37%, 38%, 39%, and 40%.

[0025] In this invention, the organ injury refers to any damage, injury, reduction, or loss of function of one or more organs or tissues associated with such organs. The injury is, but is not limited to, a change in organ tissue or structure, which may take the form of, for example, the development of tissue necrosis, or the destruction or loss of cellular or tissue structural integrity, or damage or alteration to a normal state, such as the abnormal accumulation of cellular material or debris from inflammatory processes or apoptosis. It is understood that the pathogenesis of the injury may differ depending on the type of organ or tissue. Furthermore, organ injury may also lead to the development of diseases or disorders associated with the injury (i.e., diseases associated with organ injury).

[0026] In the hydrochloride crystal of the present invention, the molar ratio of compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to hydrochloric acid is approximately 1:(1±0.5), and examples include 1:(1±0.4), 1:(1±0.2), 1:(1±0.1), and 1:1.

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

[0028] Compared to the prior art, the present invention provides the following beneficial effects. 1. In this invention, through extensive experimental research, two novel crystalline forms of compounds were obtained in which their physical and chemical properties were significantly improved compared to the free compounds. 1) Crystalline form A of the hydrochloride salt has extremely high purity, for example, in one embodiment of the present invention, this purity reaches 99.76%. Its solubility in water is 10,000 times higher than that of the free compound, and while the solubility is significantly improved, good stability is also maintained. For example, in one embodiment of the present invention, 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 crystalline form A is approximately 265.31°C, which indicates that the crystalline form has good thermal stability. In yet another embodiment of the present invention, crystalline form A still maintains its crystalline form under accelerated and stabilized conditions, and its purity remains essentially unchanged. In yet another embodiment of the present invention, crystalline form A was dissolved in different solvents, but no crystalline transformation occurred even after magnetic stirring. Furthermore, crystalline form A also showed significantly improved bioavailability compared to the free compound.

[0029] 2) Crystalline form B of the hydrochloride salt also achieved unexpectedly beneficial effects in terms of solubility compared to the free compound. For example, in one embodiment of the present invention, crystalline form B showed a 1000-fold improvement in solubility in water compared to the free compound.

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

[0031] Another objective of the present invention is to provide several pharmaceutically acceptable salts of compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, which are significantly improved in both solubility and stability compared to the compound. Another object of the present invention is to provide a method for producing salts of the above compounds and their uses. In contrast, the specific technical proposal of the present invention is as follows:

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

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

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

[0035] In the salt of the present invention, the molar ratio of compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to the acid is appropriately varied within an acceptable range, as exemplified below. In one or more embodiments, the molar ratio of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to the acid may be 1:(2±0.4), for example, 1:(2±0.3), 1:(2±0.2), 1:(2±0.1), or 1:2. The acid is arbitrarily 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-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to the acid may be 1:(1±0.2), for example, 1:(1±0.15), 1:(1±0.1), 1:(1±0.05), or 1:1. The acid is arbitrarily 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, a method for producing a salt of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine is provided, which includes reacting 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine with an acid to obtain a salt. Preferably, the reaction is carried out in water and / or an organic solvent. The solvent is one or more of the following, optionally selected from ketones having 2 to 6 carbon atoms, ethyl acetate, lower fatty alcohols, and tetrahydrofuran. The lower fatty alcohols refer to alcohols containing 1 to 8 carbon atoms. Furthermore, the ketones having 2 to 6 carbon atoms are preferably acetone. Furthermore, the lower fatty alcohol may be optionally selected from methanol, ethanol, propanol, and isopropanol. Preferably, the reaction is carried out in one solvent or a mixture of one or more solvents selected from acetone, methanol, and ethanol. Preferably, the acid is one of any type 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 hydrochloric acid refers to an aqueous solution of HCl, and HCl refers to HCl gas.

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

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

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

[0041] In one or more embodiments, the use of a salt of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, or a pharmaceutical composition comprising a salt of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, in the manufacture of agents 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 is provided.

[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 disorders, hepatic and renal failure, inflammation, atherosclerosis, diabetes, or complications of diabetes.

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

[0044] Compared to the prior art, the present invention provides the following beneficial effects. 1,2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine showed improved solubility, stability, and hygroscopicity after being converted to its hydrochloride salt. Hydrochloride salts exhibit significantly improved solubility compared to the free compound. For example, in one embodiment of the present invention, the hydrochloride salt exhibits approximately 7700 times greater solubility in water than the free compound, and other salts such as citrate also exhibit significantly improved solubility compared to the free compound. Hydrochloride salts exhibit significantly improved stability compared to the free compound. For example, in one embodiment of the present invention, the hydrochloride salt exhibits superior stability under high temperature, high humidity, and light irradiation conditions compared to the free compound, and has a clearly lower total impurity content. Furthermore, hydrochloride salts are also less hygroscopic than the free compound.

[0045] 2.2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride exhibits significantly improved performance compared to other salts. Regarding solubility, hydrochloride salts have superior solubility compared to other salts. For example, in one embodiment of the present invention, hydrochloride salts exhibit seven times greater solubility than citrate salts, which have good solubility, and more than 35 times greater solubility than maleate and fumarate salts. Regarding stability, hydrochloride salts have better performance than other salts. For example, in one embodiment of the present invention, hydrochloride salts have higher stability under high temperature, high humidity, and light irradiation conditions than other salts, and their total impurity content is significantly lower than that of other salts. In another embodiment, the hydrochloride salt did not change the color of the solution in a pH 1.0 hydrochloric acid solution, while other salts such as citrate, fumarate, and maleate salts clearly changed color, demonstrating the superior stability of hydrochloride salts. Regarding hygroscopicity, hydrochloride salts are less hygroscopic than other salts. Furthermore, hydrochloride salts can solidify well during the manufacturing process, while other salts such as methanesulfonate and p-toluenesulfonate become sticky paste-like. [Brief explanation of the drawing]

[0046] [Figure 1] Figure 1 shows the XRPD pattern of crystal form A obtained in Example 1. [Figure 2] Figure 2 shows the XRPD pattern of crystal form A obtained in Example 2. [Figure 3] Figure 3 shows the XRPD pattern of crystal form B. [Figure 4] Figure 4 shows the purity detection pattern for crystal form A. [Figure 5] Figure 5 shows the 1H NMR spectrum of crystal form A. [Figure 6] Figure 6 shows the TGA curve for crystal form A. [Figure 7] Figure 7 shows the DSC curve for crystal form A. [Figure 8] Figure 8 shows the XRPD pattern for the stability study of crystal form A. [Figure 9] Figure 9 shows the XRPD pattern of crystal form B in stability studies. [Figure 10]Figure 10 shows the 1H NMR spectrum of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. [Figure 11] Figure 11 shows the 13C NMR spectrum of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride. [Figure 12] Figure 12 shows the 1H NMR spectrum of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine citrate. [Figure 13] Figure 13 shows the 1H NMR spectrum of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate. [Figure 14] Figure 14 shows the 1H NMR spectrum of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine maleate. [Modes for carrying out the invention]

[0047] For a specific synthesis method of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine (in its free state), refer to the manufacturing method in international application WO2019205854A1.

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

[0049] Nitrogen was introduced into a 50L reaction vessel to replace the gas inside the vessel. Then, under a nitrogen atmosphere, 1.70 kg of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine (in its free state) and 17.78 kg of methanol were added to the reaction vessel, and stirring was started. Under conditions of 25±5°C, 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, and the dropwise addition was completed in about 1 hour. After the dropwise addition was complete, the reaction mixture was crystallized under constant temperature stirring at 25±5°C for 2-4 hours. The reaction mixture was then filtered, the filtered cake was washed with 1.50 kg of methanol, and the filtered cake was heat-dried under vacuum conditions (pressure -0.08 MPa to -0.10 MPa) at 40°C-50°C for 12-16 hours. A total of 1.64 kg of crystalline form A of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was collected. The crystallization yield was 88.3%. XRPD detection confirmed crystalline form A (XRPD pattern shown in Figure 1).

[0050] The manufactured crystal form A had a purity of 99.76%, with a maximum single impurity content of 0.068%. The total impurities and single impurities were below the control limits ≤0.15% (maximum daily dose of this product ≤2 grams / day) stipulated by the "Technical Guidelines on the Study of Impurities in Chemical Substances" issued by the National Medical Products Administration and ICHQ3A (in the purity detection pattern shown in Figure 4, the main peak named DA414 is crystal form A of 2-(1-(4-(4-methylpiperazine-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 The 1H NMR pattern is shown in Figure 5.

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

[0053] Nitrogen was introduced into a 50L reaction vessel to replace the gas inside the vessel. Then, under a nitrogen atmosphere, 1.50 kg of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine (in its free state), 7.11 kg of acetone, and 1.50 kg of water were added to the reaction vessel, and stirring was started. Under 25±5°C conditions, 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 vessel, and the dropwise addition was completed in about 1 hour. After the dropwise addition was complete, the reaction mixture was crystallized under constant heating and stirring conditions at 25±5°C for 2-4 hours. The reaction mixture was then filtered, the filtered cake was washed with 1.50 kg of acetone, and the filtered cake was heat-dried under vacuum conditions at 40°C-50°C (pressure -0.08 MPa to -0.10 MPa) for 12-16 hours. A total of 1.30 kg of crystalline form A of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was collected. The crystallization yield was 79.5%. XRPD detection confirmed that it was crystalline form A (XRPD pattern shown in Figure 2).

[0054] Example 3: Method for producing crystalline form B of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride Approximately 5 mg of crystalline form A of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed, 0.2 mL of N,N-dimethylformamide was added, and the solution was sonicated. 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 formed. The precipitate was filtered, and the filtered cake was vacuum-dried overnight at room temperature. XRPD detection revealed that it was crystalline form B (the XRPD pattern of crystalline form B is shown in Figure 3).

[0055] Example 4: XRPD measurement of crystalline form A of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride Measurements were performed according to the "Chinese Pharmacopoeia" (2020 edition) -- Part 4 -- General Provisions 0451 -- X-ray Diffraction -- Method 2 -- Powder X-ray Diffraction.

[0056] [Table 1]

[0057] The XRPD pattern of the crystalline form of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride is shown in Figure 1, and the XRPD data is shown in Table 2. This crystalline form was named Crystal 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 Measurements were performed according to the "Chinese Pharmacopoeia" (2020 edition) -- Part 4 -- General Provisions 0451 -- X-ray Diffraction -- Method 2 -- Powder X-ray Diffraction.

[0060] [Table 3]

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

[0062] [Table 4]

[0063] Example 6: TGA measurement of crystal form A [Table 5]

[0064] The TGA detection pattern for crystal form A is shown in Figure 6. The TGA measurement results showed that when crystal form A was heated to 120°C, the sample weight decreased by approximately 0.1586%, indicating that crystal form A was anhydrous.

[0065] Example 7: DSC measurement of crystal form A [Table 6]

[0066] The DSC detection pattern for crystal form A is shown in Figure 7. The DSC pattern revealed that crystal form A has a single endothermic peak and a melting point of approximately 265.31°C. This result indicates that crystal form A has good thermal stability.

[0067] Example 8: Ion chromatography (IC) of crystalline form A [Table 7]

[0068] Ion chromatography (IC) measurements showed a salt formation ratio of 1:1, indicating that 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine and HCl are bound in a 1:1 ratio.

[0069] Example 9: Another method for producing crystal form A (1) Volatile crystallization method Experimental procedure: Approximately 10 mg of crystalline form A of the starting material 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into 5 mL vials and dissolved in a fixed volume of the mixed solvent shown in Table 8. The mixture was filtered into another 5 mL vial using a 0.45 μm PTFE filter and left open to allow volatilization at various temperatures. The resulting solids were collected and subjected to XRPD measurement. Based on the measurement results shown in Table 8, the hydrochloride crystalline form A product was obtained.

[0070] [Table 8]

[0071] (2) Poor solvent addition method Approximately 5 mg of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed and added to 5 mL vials. Each vial was completely dissolved in the corresponding first solvent shown in Table 9, and the mixture was filtered into another 5 mL vial using a 0.45 μm PTFE filter. The second solvent shown in Table 9 was added dropwise to the clarified solution while stirring until the solid precipitated. If the solid did not precipitate even after adding a total volume of 5 mL of solvent, the mixture was transferred to a magnetic stirrer at 5°C and stirred overnight. If the sample was still clear, it was transferred to room temperature for evaporation or rotational drying. The solid was collected by centrifugation and subjected to XRPD measurement. Based on the measurement results shown in Table 9, the hydrochloride crystalline form A product was obtained.

[0072] [Table 9]

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

[0074] [Table 10]

[0075] (4) Cooling crystallization method Crystalline form A of the starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed out in approximately 10 mg portions and added to 5 mL vials. A fixed volume of the corresponding single or mixed solvent from Table 11 was added to each vial, and the mixture was dissolved under ultrasonic conditions at 50°C. The mixture was then filtered into another 5 mL vial using a 0.45 μm PTFE filter. The clarified filtrate was placed in a biochemical incubator and kept at a constant temperature of 50°C for 120 min, then cooled to 5°C at a rate of 0.1°C / min, with stirring continued during the temperature change. If a solid precipitated, it was collected by centrifugation and subjected to XRPD measurement. The measurement results are shown in Table 11.

[0076] [Table 11]

[0077] (5) Gas-liquid diffusion method Crystalline form A of the starting material 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into approximately 10 mg portions and placed in 5 mL vials. Each portion was dissolved in a fixed volume of the first solvent (single or mixed solvent) from Table 12. The solution was filtered into another 5 mL vial using a 0.45 μm PTFE filter. A 20 mL vial was then taken, and approximately 3 mL of the second solvent was added. The 5 mL vial containing the clarified solution was placed on top of the 20 mL vial with the lid open, then sealed and left to stand at room temperature for approximately 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 crystalline form A of the starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed and placed in 5 mL vials. Each vial was dissolved in a fixed volume of mixed solvent (filtered using a 0.45 μm PTFE filter), 1-2 mg of polymer was added, the vials were sealed with a perforated sealing film, and allowed to evaporate slowly at room temperature. Based on the measurement results shown in Table 13, the hydrochloride crystalline form A product was obtained. [Table 13]

[0080] The XRPD results showed that all the samples prepared in Example 9 were of crystal form A (XRPD patterns are not shown).

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

[0082] [Table 14]

[0083] (2) Polymer induction method Crystalline form A of the starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed out in approximately 10 mg portions and placed in 5 mL vials. Each vial was dissolved in a fixed volume of mixed solvent (filtered using a 0.45 μm PTFE filter), 1-2 mg of polymer was added, the vials were sealed with a sealing film with a hole in it, and allowed to evaporate slowly at room temperature. Crystalline form B of the hydrochloride was obtained based on the measurement results shown in Table 15.

[0084] [Table 15]

[0085] The XRPD results showed that all the sample crystals produced in Example 10 were crystal form B (XRPD patterns are not shown).

[0086] Example 11: Study on hygroscopic properties The results of evaluating the hygroscopicity using dynamic water vapor sorption (DVS) showed that crystal form A of the hydrochloride salt had a water adsorption rate of approximately 0.36% at 25°C / 80%RH. This indicates that crystal form A has slight hygroscopic properties, 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 had lower hygroscopicity and better drug-forming properties than crystalline form B of the hydrochloride salt.

[0088] Example 12: Solubility Comparison 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 (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, added to a fixed volume of solvent under conditions of 25°C ± 2°C, shaken vigorously for 30 seconds every 5 minutes, and the solubility was measured by observing dissolution within 30 minutes.

[0089] [Table 16]

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

[0091] As a result, in aqueous solutions, the solubility of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was clearly 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. In a pH 1.0 hydrochloric acid solution (approximately the pH of human stomach acid), the solubility of crystalline form A of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was clearly 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 superior physical and chemical properties and better drug-forming potential. Crystalline form B of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride exhibited 1000 times greater solubility in water than its free state, yielding an unexpected effect.

[0092] Furthermore, the inventors discovered during their research that the crystalline forms obtained by crystallizing other salt forms of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, such as citrate, fumarate, and maleate, had almost the same solubility in water and hydrochloric acid solution at pH 1.0 as those salt forms, and did not show significant improvement.

[0093] Example 13: Crystalline Stability Test (1) Stability experiment of crystal form A Experiment 1: Stability experiment against temperature and humidity

[0094] Test Procedure 1: Sample A of the hydrochloride salt was left for one week under long-term conditions of 25°C / 60%RH and accelerated conditions of 40°C / 75%RH, respectively. The change in crystal form was then tested. The results showed that after one week under both test conditions, the purity of sample A of the hydrochloride salt remained essentially unchanged, demonstrating good chemical stability, and no change in the crystal form of the sample was observed. Figure 8 shows a comparison of XRPD values ​​of the sample stability before and after the period of storage.

[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. 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 about one week, the solid was collected and subjected to XRPD measurement. 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: Experiment on Mechanical Stability Approximately 10 mg of crystalline form A of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into a mortar, and 0.05 mL of wetting agent was added in three separate additions. The mixture was polished for 5 minutes each time, and the solid was collected and subjected to test XRPD. The sample crystalline 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 the single solvent or mixed solvent listed in Table 19 was added to each vial. The resulting suspension was magnetically stirred at 5°C for approximately 3 days, then centrifuged to collect the solid, which was subjected to XRPD measurement. No crystal form transition occurred in the sample. The measurement results are shown in Table 19.

[0100] [Table 19]

[0101] Test Procedure 2: Approximately 10 mg of crystalline form A of the starting material 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was weighed into 5 mL vials, and 0.5 mL of a single solvent or mixed solvent listed in Table 20 was added to each to obtain a suspension. The suspension was magnetically stirred at room temperature for approximately 3 days, then centrifuged to collect the solid, which was subjected to XRPD measurement. No crystal form transition occurred in the sample. 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 5 mL vials, and 0.5 mL of a single solvent or mixed solvent listed in Table 21 was added to each vial. The resulting suspensions were magnetically stirred at 50°C for approximately 3 days, then centrifuged to collect the solid, which was subjected to XRPD measurement. No crystal form transition occurred in the sample. The measurement results are shown in Table 21.

[0104] [Table 21]

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

[0106] Furthermore, crystal form A exhibits excellent properties such as compressibility and fluidity, and its specific surface area, bulk density, porosity, and particle size are highly suitable for the development and manufacture of pharmaceutical formulations. On the other hand, crystal form B lacks these characteristics.

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

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

[0109] (3) Comparative test of the stability of crystal forms A and B Based on the Chinese Pharmacopoeia 2020 edition and the ICH Q1A Stability Study Guidelines, crystal forms A and B were stored for 6 months under accelerated conditions (40°C ± 2°C, 75% RH ± 10% RH), and the change in total impurities in crystal forms A and B was detected. The results are shown in Table 22. Table 22 shows the stability experiments for various crystal forms. As a result, after 6 months under accelerated conditions, the total impurities of crystal form A increased from 0.25% to 0.41%, while the total impurities of crystal form B increased from 0.28% to 0.88%. This result indicates that the stability of crystal form B is very poor, and the stability of crystal form A is superior to that of crystal form B.

[0110] [Table 22]

[0111] Example 14: Comparative study of pharmacokinetics Experimental method: Three healthy male SD rats were selected and fasted overnight. Then, 10 mg / kg of either 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 administered intragastricly. The dosage volume was 10 ml / kg. Approximately 200 μL 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. This blood was placed in a centrifuge tube containing EDTA-K2 anticoagulant, and then centrifuged for 10 minutes under 3000-4000 rpm conditions. All of the upper plasma was transferred to another clean centrifuge tube, and the blood concentration of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine in the plasma sample was detected by LC-MS / MS. Pharmacokinetic parameters were calculated using a non-compartment 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 state). This indicates that crystalline form A of the hydrochloride has significantly improved drug-forming properties compared to the free state.

[0114] Example 15: Study on the inhibition rate of the compound of the present invention on ferroptosis. Experimental Method: In this example, a ferroptosis screening model was constructed as follows to study ferroptosis inhibitors. The ferroptosis screening model primarily employed the CCK8 cell viability detection method. First, mouse hippocampal neurons HT22 and human neuroblastoma SH-SY5Y cells were cultured in dishes. A specific number of cells in the logarithmic growth phase (5000 cells / well) were seeded into a 96-well plate at 80 μL per well. Subsequently, the cells were cultured in an incubator at 37°C and 5% CO2 to ensure cell adhesion to the wall. After 24 hours, 10 μL of the compound of the present invention at various concentrations prepared in a predetermined medium and 10 μL of the ferroptosis inducer elastin at a final concentration of 10 μM were added. To ensure the accuracy of the results, three duplicate wells were provided for each compound. Then, a positive control group (a fixed concentration of the compound ferrostein-1 and 10 μL of the ferroptosis inducer elastin, prepared in 10 μL of the same medium as above, were added), a blank control group (equal volumes of the medium and DMSO as specified above were added, but no cells were included), and a solvent control group (equal volumes of the medium and DMSO as specified above were added, but no cells were included). To ensure the accuracy of the results, three duplicate wells were also provided in each group. After adding the drugs, the wells were incubated for 24 hours. 10 μL of CCK8 solution was added to each well, and the wells were incubated for 2-4 hours. After that, 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)] × 100% Using GraphPadPrism6 software, the inhibition rate change curve was fitted, and IC 50 The result was calculated.

[0116] Experimental results: [Table 24]

[0117] As shown in the measurement results in Table 24, crystalline form A of the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride of the present invention was found to have an inhibitory effect on ferroptosis in multiple cell lines, and its inhibitory activity was significantly superior to that of the positive control group ferrostein-1.

[0118] Example 16: Preparation of 2-(1-(4-(4-methylpiperazine-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, refer to the manufacturing method in international application WO2019205854A1. The product was analyzed for impurities, and the total impurity content was found to be 1.36%.

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

[0121] Method 1: Under conditions of 20-25°C, 5.0 g of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine (12.45 mmol, 1.0 eq) was dissolved in 50 mL of acetone. 1.14 mL of 36% concentrated hydrochloric acid (13.70 mmol, 1.1 eq) was diluted to 4.5 mL with acetone, and then slowly added dropwise to the reaction mixture. The mixture was stirred for 1 hour, after which it was filtered, and the filtered cake was dried. 4.4 g of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obtained in 80.7% yield.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 The 1H 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 13C NMR pattern is shown in Figure 11. Structural identification revealed that 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine and hydrochloric acid are bound in a 1:1 ratio in the obtained hydrochloride salt.

[0122] Method 2: Except for substituting the solvent acetone with ethanol, the product 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obtained by referring to the procedure of Method 1. Structural identification confirmed that this product was identical to the product obtained by Method 1.

[0123] Method 3: Except for substituting the solvent acetone with isopropanol, the product 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obtained by referring to the procedure of Method 1. Structural identification confirmed that this product was identical to the product obtained by Method 1.

[0124] Method 4: Except for substituting the solvent acetone with tetrahydrofuran, the product 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride was obtained by referring to the procedure of Method 1. Structural identification confirmed that this product was identical to the product obtained by Method 1.

[0125] When the impurities in the above hydrochloride product were measured, the total impurity content was found to be between 0.10% and 0.25%.

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

[0127] Under conditions of 20-25°C, 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2 mL of ethyl acetate and stirred until clear. Next, a mixed solution of 2 mL of ethyl acetate and methanesulfonic acid (36.0 mg, 0.374 mmol, 1.0 eq) was added dropwise to the reaction mixture. A large amount of cotton-like solid precipitated during the addition. The mixture was stirred for 1 hour under conditions of 20-25°C, then filtered, and the filtration cake (which was sticky) was collected and dried. 90.15 mg of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine methanesulfonate was obtained in a yield of 48.4%.

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

[0129] Under conditions of 20-25°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 stirred until clear. Next, 65 mg of p-toluenesulfonic acid (0.374 mmol, 1.0 eq) was added to the reaction mixture, and a small amount of solid precipitated. The mixture was stirred for 1 hour under conditions of 20-25°C, then filtered, and the filtration cake (which was sticky) was collected and dried. 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-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine citrate [ka]

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

[0132] Method 2: Under the condition of 20 - 25°C, 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 4 mL of acetone and dissolved until clear while stirring. 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 continuing stirring for 2 hours under the condition of 20 - 25°C, filtration was carried out, 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). After structure identification, it was found that 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine and citric acid in the obtained citrate were combined in a ratio of 1:2.

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

[0134] When the impurities in the above citrate product were measured, the total impurity content was found to be between 0.64% and 0.80%.

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

[0136] Method 1: Under conditions of 20-25°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 stirred until clear. Next, 43.41 mg of fumaric acid (trans-2-butenioic acid; 0.374 mmol, 1.0 eq) was added to the reaction mixture, and a solid gradually precipitated while stirring. After continuing stirring for 4-5 hours under conditions of 20-25°C, a large amount of solid 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 1H NMR pattern is shown in Figure 13. Structural identification revealed that in the obtained fumarate, 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine and fumaric acid are bonded in a 1:1 ratio.

[0137] Method 2: Under conditions of 20-25°C, 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 2 mL of acetone and stirred until clear. Next, 43.41 mg of fumaric acid (trans-2-butenioic acid; 0.374 mmol, 1.0 eq) was added to the reaction mixture. Initially, a small amount of solid precipitated at the bottom of the reaction bottle, and then it gradually turned into an oily substance. After stirring continued for 4-5 hours under conditions of 20-25°C, 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] When the impurities in the above fumarate product were measured, the total impurity content was found to be between 0.76% and 0.84%.

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

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

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

[0142] Under conditions of 20-25°C, 150 mg (0.374 mmol) of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine was dissolved in 3 mL of ethyl acetate and stirred until clear. Next, 43.41 mg of maleic acid (cis-2-butenioic acid; 0.374 mmol, 1.0 eq) was added to the reaction mixture and stirred. During the process, a small amount of solid precipitated at the bottom of the reaction bottle, and then gradually turned into an oily substance. After stirring continued at 20-25°C for 4-5 hours, a large amount of solid precipitated. The mixture was filtered, and the filter cake was dried. Then, 132.8 mg of 2-(1-(4-(4-methylpiperazin-1-yl)phenyl)ethyl)-10H-phenothiazine maleate was obtained in a yield of 68.6%, with a total impurity content of 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 1H NMR pattern is shown in Figure 14. Structural identification revealed that in the obtained maleate, 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine and maleic acid are bonded in a 1:1 ratio.

[0143] Example 24: Preparation of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrobromide Referring to the manufacturing method of Example 17, hydrochloric acid was substituted with hydrobromic acid to produce 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrobromide.

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

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

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

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

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

[0149] Example 30: Preparation of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine propionate Referring to the manufacturing method of Example 21, fumaric acid was substituted with propionic acid to produce 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine propionate.

[0150] Example 31: Comparative study of solubility 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 eutectic salt, 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, added to a fixed volume of solvent under conditions of 25°C ± 2°C, and vigorously shaken for 30 seconds every 5 minutes. Solubility was measured by observing dissolution within 30 minutes.

[0151] [Table 25]

[0152] Solubility experiments showed that 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride exhibited superior solubility in water and pH 1.0 hydrochloric acid solution, particularly in water, compared to 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine. 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine citrate showed 1000 times higher solubility in water than 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine.

[0153] Furthermore, the solubility of the hydrochloride salt was significantly better than that of other salts of this compound, being seven times more solubility than the citrate salt, which has better solubility, and more than 35 times more solubility than the maleate and fumarate salts.

[0154] Example 32: Stability Test 1 Test (1): Test against high temperatures In accordance with the method of "Guidelines for Stability Testing of Raw Materials and Formulations" in the Chinese Pharmacopoeia (2020 edition) 9001, the test samples were placed in a suitable constant temperature device with the lid open and left at a temperature of 60°C for 30 days, and samples were taken on the 5th, 10th, and 30th days.

[0155] High-temperature tests revealed that 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, after being converted to a salt, exhibited higher stability under high-temperature conditions than the free state of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine. Among these, the hydrochloride salt showed the best stability under high-temperature conditions, exhibited no change in properties, and produced a low amount of total impurities.

[0156] [Table 26]

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

[0158] High humidity tests revealed that 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, after being converted to a salt, exhibited higher stability under high humidity conditions than the free state of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine. Among these, the hydrochloride salt showed superior stability under high humidity conditions compared to other salt forms, exhibiting no change in properties and resulting in a lower total impurity generation.

[0159] [Table 27]

[0160] Test (3): Light Irradiation Test In accordance with the method of "Guidelines for Stability Testing of Raw Materials and Formulations" 9001 of the Chinese Pharmacopoeia (2020 edition), the test specimens were placed in a stability test box with an illuminance of 45001x ± 5001x and a total illuminance of 1.2x10⁻¹⁰. 6 lux·hr or higher, near-ultraviolet lamp energy 200W·hr / m 2 The samples were left in place for 30 days under the above conditions, and samples were taken on the 5th, 10th, and 30th days.

[0161] The light irradiation test revealed that 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine, after being converted to a salt, exhibited higher stability under light irradiation conditions than the free state of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine. Among these, the citrate salt changed in appearance from pale yellow to gray on day 5 and to dark gray on day 10 under light irradiation conditions. The hydrochloride salt showed superior stability under light irradiation conditions compared to the other salt forms, exhibiting no change in properties and resulting in a low amount of total impurity formation.

[0162] [Table 28]

[0163] Based on the stability experiments, 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride exhibited higher stability under high temperature, high humidity, and light irradiation conditions than the free compound and other salts, and its total impurity content was significantly lower than that of the other salts and free compound.

[0164] Example 33: Stability Test 2 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine citrate, 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate, and 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine maleate were weighed by the same mass and added to a constant volume of pH 1.0 hydrochloric acid solution (similar to the acidity of human stomach acid) under conditions of 25℃±2℃. The solution was shaken vigorously for 30 seconds every 5 minutes, and the color of the solution was observed after 0.5 hours and within 24 hours.

[0165] [Table 29]

[0166] The experimental results showed that the 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine citrate, 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine fumarate, and 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine maleate samples showed a clear change in solution color in a pH 1.0 hydrochloric acid solution, indicating that their stability was worse than that of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride.

[0167] Example 34: Comparative experiment on hygroscopicity In accordance with the "Guidelines for the Hygroscopicity of Pharmaceuticals" in Part 4 of the "Chinese Pharmacopoeia" (2020 edition), hygroscopicity tests were performed 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 weight increase due to hygroscopicity are as follows: Deliquescence: The process of absorbing sufficient water to form a liquid. It is extremely hygroscopic: the weight increase due to moisture absorption is 15% or more. It is hygroscopic: the weight increase due to moisture is less than 15%, but more than 2%. It is slightly hygroscopic: the weight increase due to moisture is less than 2%, but can be 0.2% or more. No or minimal hygroscopicity: Weight increase due to moisture absorption should be less than 0.2%.

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

[0170] The embodiments described above are used solely to illustrate the technical spirit and features of the present invention and are intended to enable those familiar with the art to understand and implement the invention accordingly, and are not intended to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be included within the scope of protection of the present invention.

Claims

1. A crystal having crystalline form A of 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine hydrochloride, When expressed in terms of 2θ angle using Cu-Ka radiation, the X-ray powder diffraction pattern of the crystal 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°, as described above.

2. The crystal according to claim 1, wherein, when expressed in terms of 2θ angle using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal 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°, and 29.03±0.2°.

3. The crystal according to claim 1, wherein, when expressed in terms of 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal form A 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°, and 29.03±0.2°.

4. The crystal according to claim 1, wherein, when expressed in 2θ angles using Cu-Ka radiation, the X-ray powder diffraction pattern of crystal form A 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°, and 33.55±0.2°.

5. A pharmaceutical composition comprising a crystal having crystal form A as described in any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, used for the prevention and / or treatment of a disease selected from the group consisting of cancer, organ injury, degenerative diseases, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, immune-related diseases, hepatic and renal failure, ischemia-reperfusion injury, inflammation, and metabolic diseases.

7. The neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis. The aforementioned immune-related disease is multiple sclerosis. The aforementioned cardiovascular disease or cerebrovascular disease is atherosclerosis or stroke. The pharmaceutical composition according to claim 6, wherein the metabolic disease is diabetes or a complication of diabetes.

8. The pharmaceutical composition according to claim 7, wherein the stroke is a hemorrhagic stroke and / or an ischemic stroke.

9. The pharmaceutical composition according to claim 5, which is used as a ferroptosis inhibitor.

10. A method for producing crystals having the crystalline form A described in any one of claims 1 to 4, comprising dispersing compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine in methanol at 10 to 40°C, adding a mixed solution of concentrated hydrochloric acid and methanol dropwise, stirring and crystallizing, filtering, washing the filtered cake with methanol, and then vacuum drying the filtered cake to obtain a crystalline substance, The weight ratio of methanol as the solvent to the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine is 5:1 to 15:

1. The weight ratio of the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is 1:1 to 8:

1. The stirring and crystallization described above is carried out for 1 to 8 hours.

11. The weight ratio of methanol as the solvent to compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine is 8:1 to 12:

1. The weight ratio of the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is 3:1 to 5:

1. The method according to claim 10, wherein the stirring and crystallization are carried out for 2 to 5 hours.

12. A method for producing crystals having the crystalline form A described in any one of claims 1 to 4, comprising dispersing compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine in acetone and water at 10 to 40°C, then dropping a mixed solution of concentrated hydrochloric acid, acetone and water, stirring and crystallizing, filtering, washing the filtered cake with acetone, and then vacuum drying the filtered cake to obtain a crystalline substance, The weight ratio of the solvents acetone and water to the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine is (1-8):(0.5-3):(0.5-3). The weight ratio of the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is 1:1 to 8:

1. The stirring and crystallization described above is carried out for 1 to 8 hours.

13. The weight ratio of the solvent acetone and water to the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine is (4-6):(0.5-1):(0.5-1). The weight ratio of the compound 2-(1-(4-(4-methylpiperazine-1-yl)phenyl)ethyl)-10H-phenothiazine to concentrated hydrochloric acid is 3:1 to 5:

1. The method according to claim 12, wherein the stirring and crystallization are carried out for 2 to 5 hours.

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