Crystalline form, manufacturing method, and use of tetramethylpyrazinenitrone

Abstract

The present disclosure relates to a crystalline form of tetramethylpyrazine nitrone, a preparation method thereof, and applications thereof, and provides crystalline form A of tetramethylpyrazine nitrone, which exhibits characteristic diffraction peaks in its XRPD spectrum at 2θ angle positions of 10.60±0.2, 11.03±0.2, 15.31±0.2, 15.55±0.2, 17.14±0.2, 17.93±0.2, and 23.81±0.2. Crystalline form A has a low risk of crystal rearrangement and is highly stable, which is advantageous for stability during preparation manufacturing and storage, effectively ensuring the consistency of the crystalline form content in preparations, and is safe, effective, and allows for quality control. It also has high biological activity and good drug discovery potential, with high bioavailability, rapid onset of effect, and high biological activity.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and specifically to the crystalline forms of tetramethylpyrazine nitrone, its production method, and its use.

Background Art

[0002] Tetramethylpyrazine nitrone (abbreviated as TBN) is a nitrone derivative of tetramethylpyrazine (TMP), and is a new compound chemically synthesized by adding a nitrone pharmacophore to the structure of tetramethylpyrazine. Its chemical name is (cis)-2-methyl-N-[(3,5,6-trimethylpyrazin-2-)methine]2-propylamine oxide, the molecular formula is C 12 H 19 N3O, the molecular weight is 221.30, and it has a chemical structure shown in the following formula.

[0003]

Chemical Formula

[0004] Tetramethylpyrazine nitrone plays a role in protecting nerve cells by suppressing oxidative damage of nerve cells caused by ischemia, and can reduce neurological symptoms and functional disorders associated with cerebral embolism. Clinically, it can be used for the treatment of nervous system diseases, cardiovascular and cerebrovascular diseases, and degenerative aging diseases, etc.

Summary of the Invention

[0005] An object of the present invention is to provide a crystalline form of tetramethylpyrazine nitrone, its production method, and its use.

[0006] One object of the present invention is to provide crystalline form A of tetramethylpyrazine nitrone, the XRPD spectrum of which shows characteristic diffraction peaks at 2θ angular positions of 10.60±0.2, 11.03±0.2, 15.31±0.2, 15.55±0.2, 17.14±0.2, 17.93±0.2, 23.81±0.2.

[0007] In one specific embodiment, crystalline form A of tetramethylpyrazinenitrone exhibits characteristic diffraction peaks in its XRPD spectrum at 2θ angular positions of 10.60±0.2, 11.03±0.2, 13.51±0.2, 15.31±0.2, 15.55±0.2, 17.14±0.2, 17.93±0.2, 21.22±0.2, 23.81±0.2, 25.23±0.2, and 27.08±0.2.

[0008] In one specific embodiment, crystalline form A of tetramethylpyrazinenitrone has essentially the same XRPD spectrum as that shown in Figure 1 of the specification.

[0009] Furthermore, the crystalline form A of tetramethylpyrazinenitrone described in the present invention has three crystalline states: needle-like, massive, and rod-like, and micrographs of these are shown in Figures 5 and 6.

[0010] Furthermore, the crystalline form A of tetramethylpyrazinenitrone described in the present invention has a melting point of 76°C to 78°C.

[0011] Furthermore, crystalline form A of tetramethylpyrazinenitrone described in the present invention has essentially the same DSC spectrum as shown in Figure 3.

[0012] Furthermore, Figure 7 shows the TGA spectrum of crystalline form A of tetramethylpyrazinenitrone as described in the present invention.

[0013] Furthermore, the present invention provides the infrared spectrum of crystalline form A of tetramethylpyrazinenitrone, as shown in Figure 9.

[0014] The present invention is obtained through systematic crystal form screening experiments. These screening methods include evaporation crystallization (single-solvent and mixed-solvent methods), heating / dissolution / cooling / precipitation, and suspension / slurrying. Screening solvents include tetrahydrofuran, ethyl acetate, toluene, acetone, dioxane, isopropanol, petroleum ether, n-hexane, isopropyl acetate, isooctane, and isobutyl acetate. All screening results are of crystal form A.

[0015] Furthermore, the present invention also provides a method for producing crystalline form A of tetramethylpyrazinenitrone, comprising the following steps. Step (1): Mix the crude tetramethylpyrazine nitrone with an organic solvent, heat in a water bath to 60-80°C, stir, filter, and cool the filtrate to crystallize and obtain crystalline solids. Step (2): The crystalline solid obtained in step (1) is mixed with n-heptane, heated to dissolve, and cooled to crystallize, thereby obtaining crystalline form A of tetramethylpyrazinenitrone.

[0016] Furthermore, in the manufacturing method described in the present invention, the organic solvent in step (1) is one or more selected from ethyl acetate, n-hexane, n-heptane, and cyclohexane, and more preferably the organic solvent is n-hexane or n-heptane, or a mixed solvent of n-hexane and ethyl acetate is selected as the organic solvent. The applicant has found that by selecting a preferred organic solvent, the content of impurities can be significantly reduced.

[0017] Furthermore, in the manufacturing method described in the present invention, the weight-to-volume ratio of crude tetramethylpyrazine nitrone to organic solvent in step (1) is 1:5 to 20, preferably 1:8 to 12.

[0018] Furthermore, in the manufacturing method described in the present invention, the temperature at which the cooling and crystallization takes place in step (1) is selected from 2 to 12°C, more preferably from 3 to 10°C, and most preferably from 3 to 5°C. The applicant has found that when the temperature at which the cooling and crystallization takes place is within the most preferred range, the amount of impurities can be significantly reduced.

[0019] Furthermore, in the manufacturing method described in the present invention, the weight-to-volume ratio of crude tetramethylpyrazine nitrone to n-heptane in step (2) is preferably 1:1 to 5, and most preferably 1:1 to 3.

[0020] Furthermore, in the manufacturing method described in the present invention, the temperature at which the crystalline solids are mixed with n-heptane and heated in step (2) is 60 to 80°C, preferably 65 to 75°C.

[0021] Furthermore, in the manufacturing method described in the present invention, the temperature at which the cooling and crystallization are performed in step (2) is selected from 2 to 12°C, and more preferably from 4 to 10°C.

[0022] Another object of the present invention is to provide a tetramethylpyrazinenitrone dihydrate whose XRPD spectrum exhibits characteristic diffraction peaks at 2θ angular positions of 8.91±0.2, 11.46±0.2, 14.29±0.2, 17.60±0.2, 21.19±0.2, 22.02±0.2, 23.19±0.2, 24.30±0.2, 24.92±0.2, 29.20±0.2, and 31.41±0.2.

[0023] In one specific embodiment, the dihydrate of tetramethylpyrazinenitrone exhibits characteristic diffraction peaks in its XRPD spectrum at 2θ angular positions of 8.91±0.2, 11.46±0.2, 12.00±0.2, 14.29±0.2, 17.60±0.2, 19.50±0.2, 21.19±0.2, 22.02±0.2, 23.19±0.2, 24.30±0.2, 24.92±0.2, 26.70±0.2, 29.20±0.2, 31.41±0.2, and 36.20±0.2.

[0024] In one specific embodiment, the dihydrate of tetramethylpyrazine nitrone has an XRPD spectrum that is basically the same as that shown in Figure 2 of the specification.

[0025] Furthermore, the dihydrate of tetramethylpyrazine nitrone described in the present invention has a melting point of 37 - 40 °C.

[0026] Furthermore, the DSC spectrum of the dihydrate of tetramethylpyrazine nitrone described in the present invention is basically the same as that shown in Figure 4.

[0027] Furthermore, the TGA spectrum of the dihydrate of tetramethylpyrazine nitrone described in the present invention is shown in Figure 8.

[0028] When the temperature of the dihydrate of tetramethylpyrazine nitrone described in the present invention reaches the melting point (about 37 - 40 °C), its weight begins to decrease, and the weight loss rate is 13.67%.

[0029] The dihydrate of tetramethylpyrazine nitrone described in the present invention is obtained by cooling and precipitating tetramethylpyrazine nitrone in a binary system of a saturated ethanol aqueous solution. The volume percentage of ethanol in the ethanol aqueous solution is preferably 5 - 50%, more preferably 5 - 20%.

[0030] In another aspect, the present invention provides a pharmaceutical composition comprising one or more kinds of crystalline form A or dihydrate of the present invention. Optionally, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier, excipient, filler, binder, disintegrant, glidant and / or medium, etc.

[0031] In another aspect, the crystalline form A and / or dihydrate described in the present invention, or the pharmaceutical composition described in the present invention, can be administered by an administration route such as oral administration or injection.

[0032] In another embodiment, the present invention further provides dosage forms comprising crystalline form A and / or dihydrate of the present invention. The dosage forms include, but are not limited to, tablets, capsules, powder injections, dispersants, etc., and are preferably tablets and powder injections.

[0033] In another aspect, the present invention further provides the use of the crystalline form A and / or dihydrate or drug composition described in the present invention in the manufacture of drugs for the treatment of neurological disorders, cardiovascular and cerebrovascular diseases and degenerative aging diseases.

[0034] The beneficial effects of this invention are as follows: (1) The crystalline form A of tetramethylpyrazinenitrone and the dihydrate of tetramethylpyrazinenitrone described in the present invention are easy to manufacture and readily available for large-scale industrial production.

[0035] (2) The crystalline form A of tetramethylpyrazinenitrone described in the present invention is stable as crystalline form A in hot treatment, mechanical treatment and accelerated testing (50°C, 75%RH), has a low risk of crystallization dislocation, is highly stable, is advantageous for stability during the manufacture and storage of the formulation, effectively ensures consistency in the content of the crystalline form in the formulation, is safe and effective, and allows for quality control.

[0036] (3) Crystal form A of tetramethylpyrazinenitrone described in the present invention is readily soluble in most solvents (e.g., acetonitrile, methanol, ethanol, acetone, and ethyl acetate), very soluble in water, has high biological activity and good drug discovery potential, has high bioavailability, a rapid onset of action and high biological activity. [Brief explanation of the drawing]

[0037] [Figure 1] This is the XRD spectrum of crystalline form A of tetramethylpyrazinenitrone. [Figure 2] This is a comparative XRD spectrum of tetramethylpyrazinenitrone in crystalline form A and dihydrate. [Figure 3]This is the DSC spectrum of tetramethylpyrazinenitrone crystal form A. [Figure 4] These are the DSC spectra of tetramethylpyrazinenitrone in crystalline form A and its dihydrate. [Figure 5] This is a biological microscope image of crystalline form A of tetramethylpyrazinenitrone. [Figure 6] This is an observation diagram of the crystalline state of tetramethylpyrazinenitrone crystal form A. [Figure 7] This is the TGA spectrum of crystalline form A of tetramethylpyrazinenitrone. [Figure 8] This is a comparative TGA spectrum of tetramethylpyrazinenitrone in crystalline form A and dihydrate. [Figure 9] This is the infrared spectrum of crystalline form A of tetramethylpyrazinenitrone. [Figure 10] This is the XRD spectrum of tetramethylpyrazinenitrone crystal form A after being suspended for one week and then formed into a slurry. [Figure 11] This is the XRD spectrum of tetramethylpyrazinenitrone crystal form A after polishing for 10 minutes. [Figure 12] This is the XRD spectrum of tetramethylpyrazinenitrone dihydrate after polishing for 10 minutes. [Figure 13] This is the XRPD analysis spectrum after tableting treatment of crystalline form A. [Figure 14] This is the XRPD analysis spectrum of the hydrate after tablet compression testing. [Figure 15] This is the DSC spectrum of crystal form A after cold working. [Figure 16] This is the XRPD spectrum of crystal form A after cold working. [Figure 17] This is the DSC spectrum of crystal form A after hot treatment. [Figure 18] This is the XRPD spectrum of crystal form A after hot working. [Figure 19] This is the DSC spectrum of tetramethylpyrazine nitrone hydrate after hot treatment. [Figure 20]This is the XRPD spectrum of tetramethylpyrazine nitrone hydrate after hot treatment. [Figure 21] This is the XRPD spectrum of crystal form A after a 7-day accelerated test. [Figure 22] This shows the tissue distribution results after intravenous administration of tetramethylpyrazine nitrone to SD rats (30 mg / kg, n=6, half male and half female). [Modes for carrying out the invention]

[0038] The present invention will be further described below with reference to examples. The specific examples described herein should be understood to be for interpretive purposes only and not to limit the present invention. Any simple modifications made to the manufacturing method of the present invention within the scope of the concept of the present invention are all covered by the present invention. Experimental methods in the following examples where specific conditions are not given will be in accordance with methods commonly known in the art. Unless otherwise specified, the test materials used in the following examples are all obtained from a standard biochemical supply store.

[0039] In the following examples and drawings, unless otherwise specified, the abbreviation TBN refers to the tetramethylpyrazine nitrone described in the present invention.

[0040] In the following examples, characteristic diffraction peaks in the XRD spectra of crystalline form A of tetramethylpyrazinenitrone and tetramethylpyrazinenitrone dihydrate were measured under the following experimental conditions.

[0041] A powder X-ray diffraction analyzer (Bruker D2PHASER) was used. The voltage and tube current were 30kV and 10mA, respectively, the 2θ scan angle of the sample was 3° to 40°, and the scan step was 0.02°. [Examples]

[0042] In a 250 ml round-bottom flask, 10 g of crude tetramethylpyrazine nitrone, 100 ml of cyclohexane, and 2 ml of ethyl acetate were added. The mixture was heated to 70°C in a water bath at a rotation speed of 140 ± 5 rpm, and continuously stirred. The solution was then filtered while still hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cooled to ambient temperature (10°C), then placed in a refrigerator at 4°C to stand, filtered, and the filter cake was washed with n-hexane. The solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0043] In a 250 ml round-bottom flask, the solid portion of the crude tetramethylpyrazine nitrone and twice its volume of n-heptane were added. The rotation speed was adjusted to 140 ± 5 rpm, and the mixture was heated to 70°C in a water bath with continuous stirring to obtain an orange-yellow clarified solution. The water bath was removed, stirring continued, and the mixture was cooled to 10°C to crystallize. The solution was filtered by suction, the solid was washed with n-heptane, and the mixture was vacuum-dried at 38°C for 24 hours so that the dry weight loss rate was less than 1.0% to obtain tetramethylpyrazine nitrone crystal form A. [Examples]

[0044] In a 250 ml round-bottom flask, 10 g of crude tetramethylpyrazine nitrone, 100 ml of n-hexane, and 2 ml of ethyl acetate were added. The mixture was heated to 70°C in a water bath at a rotation speed of 140 ± 5 rpm, and continuously stirred. The solution was then filtered while still hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cooled to ambient temperature (10°C), then placed in a refrigerator at 4°C to stand, filtered, and the filter cake was washed with n-hexane. The solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0045] In a 250 ml round-bottom flask, the solid portion of the crude tetramethylpyrazine nitrone and twice its volume of n-heptane were added. The rotation speed was adjusted to 140 ± 5 rpm, and the mixture was heated to 70°C in a water bath with continuous stirring to obtain an orange-yellow clarified solution. The water bath was removed, stirring continued, and the mixture was cooled to 10°C to crystallize. The solution was filtered by suction, the solid was washed with n-heptane, and the mixture was vacuum-dried at 38°C for 24 hours so that the dry weight loss rate was less than 1.0% to obtain tetramethylpyrazine nitrone crystal form A. [Examples]

[0046] In a 250 ml round-bottom flask, 10 g of crude tetramethylpyrazine nitrone and 100 ml of cyclohexane were added. The mixture was heated to 70°C in a water bath at a rotation speed of 140 ± 5 rpm, and continuously stirred. The solution was then filtered while still hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cooled to ambient temperature (10°C), then placed in a refrigerated room at 4°C to stand, filtered, and the filter cake was washed with n-hexane. The solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0047] In a 250 ml round-bottom flask, the solid portion of the crude tetramethylpyrazine nitrone and twice its volume of n-heptane were added. The rotation speed was adjusted to 140 ± 5 rpm, and the mixture was heated to 70°C in a water bath with continuous stirring to obtain an orange-yellow clarified solution. The water bath was removed, stirring continued, and the mixture was cooled to 10°C to crystallize. The solution was filtered by suction, the solid was washed with n-heptane, and the mixture was vacuum-dried at 38°C for 24 hours so that the dry weight loss rate was less than 1.0% to obtain tetramethylpyrazine nitrone crystal form A. [Examples]

[0048] In a 250 ml round-bottom flask, 10 g of crude tetramethylpyrazine nitrone and 100 ml of n-hexane were added. The mixture was heated to 70°C in a water bath at a rotation speed of 140 ± 5 rpm, and continuously stirred. The solution was then filtered while still hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cooled to ambient temperature (10°C), then placed in a refrigerator at 4°C to stand, filtered, and the filter cake was washed with n-hexane. The solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0049] In a 250 ml round-bottom flask, the solid portion of the crude tetramethylpyrazine nitrone and twice its volume of n-heptane were added. The rotation speed was adjusted to 140 ± 5 rpm, and the mixture was heated to 70°C in a water bath with continuous stirring to obtain an orange-yellow clarified solution. The water bath was removed, stirring continued, and the mixture was cooled to 10°C to crystallize. The solution was filtered by suction, the solid was washed with n-heptane, and the mixture was vacuum-dried at 38°C for 24 hours so that the dry weight loss rate was less than 1.0% to obtain tetramethylpyrazine nitrone crystal form A. [Examples]

[0050] In a 250 ml round-bottom flask, 10 g of crude tetramethylpyrazine nitrone and 100 ml of n-heptane were added. The mixture was heated to 70°C in a water bath at a rotation speed of 140 ± 5 rpm, and continuously stirred. The solution was then filtered while still hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cooled to ambient temperature (10°C), then placed in a refrigerator at 4°C to stand, filtered, and the filter cake was washed with n-hexane. The solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0051] In a 250 ml round-bottom flask, the solid portion of the crude tetramethylpyrazine nitrone and twice its volume of n-heptane were added. The rotation speed was adjusted to 140 ± 5 rpm, and the mixture was heated to 70°C in a water bath with continuous stirring to obtain an orange-yellow clarified solution. The water bath was removed, stirring continued, and the mixture was cooled to 10°C to crystallize. The solution was filtered by suction, the solid was washed with n-heptane, and the mixture was vacuum-dried at 38°C for 24 hours so that the dry weight loss rate was less than 1.0% to obtain tetramethylpyrazine nitrone crystal form A. [Examples]

[0052] In a 250 ml round-bottom flask, 10 g of crude tetramethylpyrazine nitrone and 100 ml of n-hexane were added. The mixture was heated to 70°C in a water bath at a rotation speed of 140 ± 5 rpm, and continuously stirred. The solution was then filtered while still hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cooled to ambient temperature (10°C), then placed in a refrigerated room at 10°C to stand, filtered, the filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0053] In a 250 ml round-bottom flask, the solid portion of the crude tetramethylpyrazine nitrone and twice its volume of n-heptane were added. The rotation speed was adjusted to 140 ± 5 rpm, and the mixture was heated to 70°C in a water bath with continuous stirring to obtain an orange-yellow clarified solution. The water bath was removed, stirring continued, and the mixture was cooled to 10°C to crystallize. The solution was filtered by suction, the solid was washed with n-heptane, and the mixture was vacuum-dried at 38°C for 24 hours so that the dry weight loss rate was less than 1.0% to obtain tetramethylpyrazine nitrone crystal form A. [Examples]

[0054] In a 250 ml round-bottom flask, 10 g of crude tetramethylpyrazine nitrone and 100 ml of n-hexane were added. The mixture was heated to 70°C in a water bath at a rotation speed of 140 ± 5 rpm, and continuously stirred. The solution was then filtered while still hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cooled to ambient temperature (10°C), then placed in a refrigerator at 4°C to stand, filtered, and the filter cake was washed with n-hexane. The solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0055] In a 250 ml round-bottom flask, the solid portion of the crude tetramethylpyrazine nitrone and twice its volume of n-heptane were added. The rotation speed was adjusted to 140 ± 5 rpm, and the mixture was heated to 70°C in a water bath with continuous stirring to obtain an orange-yellow clarified solution. The water bath was removed, stirring continued, and the mixture was cooled to 10°C to crystallize. The solution was filtered by suction, the solid was washed with n-heptane, and the mixture was vacuum-dried at 38°C for 24 hours so that the dry weight loss rate was less than 1.0% to obtain tetramethylpyrazine nitrone crystal form A.

[0056] Figure 1 and Table 1 show the XRD spectra of crystalline form A of tetramethylpyrazine nitrone produced in Examples 1 to 7 of the present invention. The XRD spectrum of crystalline form A shows characteristic diffraction peaks at 2θ angular positions of 10.60±0.2, 11.03±0.2, 15.31±0.2, 15.55±0.2, 17.14±0.2, 17.93±0.2, and 23.81±0.2. The DSC spectrum, as shown in Figure 3, has one endothermic peak, which is the melting peak, with a melting point of 76°C to 78°C. As can be seen from the micrographs in Figures 5 and 6, tetramethylpyrazine nitrone has three crystalline states: needle-like, massive, and rod-like. The TGA spectrum is shown in Figure 7. The infrared spectrum is shown in Figure 9. [Examples]

[0057] Tetramethylpyrazine nitrone was cooled and precipitated in a two-component system of saturated ethanol aqueous solution (ethanol volume 9%) to obtain tetramethylpyrazine nitrone dihydrate. The XRD spectrum of the prepared dihydrate shows characteristic diffraction peaks at 2θ angular positions of 8.91±0.2, 11.46±0.2, 14.29±0.2, 17.60±0.2, 21.19±0.2, 22.02±0.2, 23.19±0.2, 24.30±0.2, 24.92±0.2, 29.20±0.2, and 31.41±0.2, as shown in Figure 2 and Table 1. The DSC spectrum is shown in Figure 4, where the melting point of tetramethylpyrazine nitrone dihydrate is 37-40°C. The TGA spectrum is shown in Figure 8.

[0058] [Table 1]

[0059] Investigation of manufacturing methods: 1. The results of the investigation into the effect of the solvent on the yield and quality of the tetramethylpyrazine nitrone final product are shown in Table 2.

[0060] [Table 2]

[0061] 2. The results of our investigation into the effect of crystallization temperature on the yield and quality of the final tetramethylpyrazine nitrone product are shown in Table 3.

[0062] [Table 3]

[0063] Investigation of effects: 1. Stability investigation 1.1 Crystallographic rearrangement test using suspension (1) Crystal form A of tetramethylpyrazinenitrone was suspended in petroleum ether (1.0 mL) at room temperature for 7 days to form a slurry. The suspension was then centrifuged to collect a solid sample, which was then subjected to XRPD characterization.

[0064] As a result, crystalline form A of tetramethylpyrazinenitrone remained crystalline form A even after being suspended in petroleum ether for one week and formed into a slurry. Its XRD spectrum is shown in Figure 10.

[0065] 1.2 Stability testing by mechanical treatment (2) Crystal form A of tetramethylpyrazinenitrone and the dihydrate of tetramethylpyrazinenitrone were polished for 10 minutes, and the treated samples were analyzed by XRPD.

[0066] As a result, the characteristic peak in the XRPD spectrum of crystal form A described in the present invention did not change after polishing for 10 minutes. The crystal form of tetramethylpyrazinenitrone dihydrate also did not change after polishing for 10 minutes. These spectra are shown in Figures 11 and 12.

[0067] 1.3 Tablet Compression Test (3) Crystalline form A and tetramethylpyrazine nitrone dihydrate were compressed into tablets at a pressure of 2 tons for 2 minutes, and then analyzed by XRPD after being pressurized into tablets.

[0068] As a result, as shown in Figures 13 and 14, the crystal forms of both crystalline form A and tetramethylpyrazine nitrone dihydrate remained unchanged after being pressurized at a pressure of 2 tons for 2 minutes (the degree of crystallinity decreased in both cases).

[0069] 1.4 Stability experiments using cold and hot working treatments (4) Crystal form A was cooled to -55°C and then heated to 25°C. As a result, as shown in Figures 15 and 16, crystal form A did not change.

[0070] (5) Crystal form A was heated to 110°C (below the decomposition point), then cooled to 25°C to obtain a yellow oily liquid. After stirring, a white solid precipitated. XRPD characterization results showed that it was crystal form A, as shown in Figures 17 and 18.

[0071] (6) Tetramethylpyrazine nitrone hydrate was heated to 110°C (below the decomposition point), then cooled to 25°C to obtain a yellow oily liquid. After stirring, a white solid precipitated. XRPD characterization showed that it was crystal form A, as shown in Figures 19 and 20.

[0072] 1.5 Experiments on the effects of acceleration Crystal form A was accelerated for 7 days in an environment of 40°C and RH=75%. XRPD characterization results showed that it was crystal form A, as shown in Figure 21.

[0073] 2. Investigation of biological activity We conducted pharmacodynamic studies on a stroke animal model in cynomolgus monkeys, and simultaneously studied the pharmacokinetics of crystalline form A of tetramethylpyrazinenitron in the cerebrospinal fluid of cynomolgus monkeys. After the second administration (30 mg / kg administered intravenously 6 hours after the first administration), 0.5 ml of cerebrospinal fluid was collected at 10, 30, 60, and 120 minutes, and pharmacokinetic studies were performed.

[0074] [Table 4]

[0075] According to the experimental results in Table 4, the concentration of tetramethylpyrazine nitrone in the cerebrospinal fluid 10 minutes after the second administration was 176 μM, which is almost equal to the drug concentration in plasma (195 μM) and exceeds the effective protective concentration of tetramethylpyrazine nitrone of 30 μM in in vitro cell experiments. As is clear from these results, crystalline form A of tetramethylpyrazine nitrone can cross the blood-brain barrier and reach the effective protective concentration.

[0076] 3. Clearance of metabolic data SD rats were administered tetramethylpyrazinenitrone crystalline form A at a dose of 30 mg / kg by intravenous injection, and the distribution of tetramethylpyrazinenitrone crystalline form A in various tissues was investigated.

[0077] The experimental results are shown in Figure 22. The results indicate that after intravenous administration of tetramethylpyrazine nitrone at 30 mg / kg to male and female rats, it rapidly and widely distributed to all tissues almost simultaneously with the plasma, was excreted in vitro almost simultaneously with the plasma after 24 hours, and there was no tendency for tetramethylpyrazine nitrone to accumulate in the test tissues. The tetramethylpyrazine nitrone concentration in brain tissue 0.25 hours after administration was 0.6 to 0.7 times the plasma concentration, 0.8 to 1.1 times the plasma concentration after 4 hours, and tetramethylpyrazine nitrone was completely removed after 24 hours, similar to the plasma concentration.

Claims

1. A crystal of tetramethylpyrazinenitrone in crystalline form A, characterized in that its XRPD spectrum shows characteristic diffraction peaks at 2θ angular positions of 10.60±0.2, 11.03±0.2, 15.31±0.2, 15.55±0.2, 17.14±0.2, 17.93±0.2, and 23.81±0.2, and has a melting point of 76°C to 78°C.

2. The crystal of crystal form A according to claim 1, characterized in that the XRPD spectrum shows characteristic diffraction peaks at 2θ angular positions of 10.60±0.2, 11.03±0.2, 13.51±0.2, 15.31±0.2, 15.55±0.2, 17.14±0.2, 17.93±0.2, 21.22±0.2, 23.81±0.2, 25.23±0.2, and 27.08±0.

2.

3. A crystal of crystal form A according to claim 1 or 2, characterized in that it has the same XRPD spectrum as the XRPD spectrum shown below. 【Chemistry 1】

4. The DSC spectrum of the crystal of crystal form A is as follows: 【Chemistry 2】 It is the same as shown in, The TGA spectrum is as follows: 【Transformation 3】 It is the same as shown in, The infrared spectrum is as follows: 【Chemistry 4】 A crystal of crystal form A according to claim 1, characterized in that it is the same as that shown.

5. A method for producing crystals of crystal form A as described in any one of claims 1 to 4, characterized by comprising the following steps. Step (1): The crude tetramethylpyrazine nitrone is mixed with an organic solvent, heated in a water bath to 60-80°C, stirred, filtered, and the filtrate is cooled to crystallize and obtain crystalline solids. The organic solvent is one or more selected from ethyl acetate, n-hexane, n-heptane, and cyclohexane. Step (2): The crystalline solid obtained in step (1) is mixed with n-heptane, heated to dissolve, and cooled to crystallize, thereby obtaining crystals of tetramethylpyrazinenitrone in crystalline form A.

6. The manufacturing method according to claim 5, characterized in that the organic solvent in step (1) is n-hexane or n-heptane, the weight-to-volume ratio of crude tetramethylpyrazinenitrone to the organic solvent in step (1) is 1:5 to 20, and the temperature at which the cooling and crystallization in step (1) is selected from 2 to 12°C.

7. The manufacturing method according to claim 6, characterized in that the weight-to-volume ratio of crude tetramethylpyrazine nitrone to organic solvent in step (1) is 1:8 to 12.

8. The manufacturing method according to claim 6, characterized in that the temperature at which the cooling and crystallization occurs in step (1) is 3 to 10°C.

9. The manufacturing method according to claim 6, characterized in that the temperature at which the cooling and crystallization occurs in step (1) is 3 to 5°C.

10. The manufacturing method according to claim 5, characterized in that in step (2) above, the weight-to-volume ratio of crude tetramethylpyrazine nitrone to n-heptane is 1:1 to 5.

11. The manufacturing method according to claim 5, characterized in that in step (2) above, the weight-to-volume ratio of crude tetramethylpyrazine nitrone to n-heptane is 1:1 to 3.

12. The manufacturing method according to claim 5, characterized in that the temperature at which the crystalline solids are mixed with n-heptane and heated in step (2) is 60 to 80°C.

13. The manufacturing method according to claim 5, characterized in that the temperature at which the crystalline solids are mixed with n-heptane and heated in step (2) is 65 to 75°C.

14. The manufacturing method according to claim 5, characterized in that the temperature at which the cooling and crystallization occurs in step (2) is 2 to 12°C.

15. The manufacturing method according to claim 5, characterized in that the temperature at which the cooling and crystallization occurs in step (2) is 4 to 10°C.

16. A drug composition comprising crystals of crystalline form A as described in any one of claims 1 to 4.

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