Crystalline form of tetramethylpyrazine nitrone, method of manufacture and use thereof

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a crystal form of tetramethylpyrazine nitrone, and a preparation method and use thereof.BACKGROUND

[0002] Tetramethylpyrazine nitrone, abbreviated as TBN, is a nitrone derivative of tetramethylpyrazine (TMP), and a new compound chemically synthesized by adding a nitrone pharmacophore to the structure of tetramethylpyrazine. It has a chemical name of (Z)-2-methyl-N-((3,5,6-trimethylpyrazin-2-yl)methylene)propan-2-amine oxide, a molecular formula of C 12 H 19 N 3 O, a molecular weight of 221.30, and a chemical structure shown below: Structural formula of tetramethylpyrazine nitrone (TBN)

[0003] TBN can inhibit the oxidative damage of nerve cells caused by ischemia, thereby protecting the nerve cells and alleviating the neurological symptoms and dysfunctions associated with cerebral embolism. It can be used clinically in the treatment of neurological diseases, cardiovascular and cerebrovascular diseases, and aging-associated degenerative diseases.

[0004] WO 2009 / 129726 A1 describes nitrone compounds, a process of preparation thereof, and use thereof in medicament manufacture.

[0005] SUN et al. Antioxidative and thrombolytic TMP nitrone for treatment of ischemic stroke. Bioorg. Med. Chem. August 2008, Vol. 16, No. 19, Pages 8868-8874, ISSN: 0968-0896, DOI: 10.1016 / J.BMC.2008.08.075 describes antioxidative and thrombolytic TMP nitrone for treatment of ischemic stroke.SUMMARY

[0006] The present invention aims to provide a crystal form of TBN, and a preparation method and use thereof.

[0007] The present invention provides a crystal form A of tetramethylpyrazine nitrone (TBN), having characteristic diffraction peaks in the XRPD pattern of the crystal form at 2θ°: 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 melting point of the crystal form A of TBN is 76-78°C; and preferably having characteristic diffraction peaks in the XRPD pattern of the crystal form at 2θ°: 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 a specific embodiment, the crystal form A of TBN has the same XRPD pattern as shown in Fig. 1.

[0009] Further, the crystal form A of TBN according to the present invention has needle-, cube-, or rod-shaped crystalline structure, as shown in the micrographs of Figs. 5 and 6.

[0010] Further, the melting point of the crystal form A of TBN is 76-78°C, respectively.

[0011] Further, the DSC profile of the crystal form A of TBN is shown in Figure 3.

[0012] Further, the TGA pattern of the crystal form A of TBN is shown in Fig. 7.

[0013] Further, the present invention provides an infrared spectrum of the crystal form A of TBN, as shown in Fig. 9.

[0014] In the present invention, a systematic crystal form screening experiment is carried out by using a screening method including evaporative crystallization (single solvent method and mixed solvent method), hot dissolution-cold precipitation, and suspension beating. The screening involves solvents such as tetrahydrofuran, ethyl acetate, toluene, acetone, dioxane, isopropanol, petroleum ether, n-hexane, isopropyl acetate, isooctane and isobutyl acetate, and the screening results all reveal crystal form A.

[0015] Further, the present invention also provides a method for preparing the crystal form A of TBN, which includes the following steps: (1) mixing the API (active pharmaceutical ingredient) TBN with an organic solvent, heating in a water bath to 60-80°C, stirring and filtering, and cooling the filtrate for crystallization to obtain a crystalline solid; and (2) mixing the crystalline solid obtained in Step (1) and n-heptane, heating to dissolve it, and cooling for crystallization to obtain the crystal form A of TBN.

[0016] Further, in the preparation method of the present invention, the organic solvent in Step (1) is one or more selected from ethyl acetate, n-hexane, n-heptane and cyclohexane, and further preferably, n-hexane or n-heptane, or a mixed solvent of n-hexane and ethyl acetate. The applicant finds that the use of a preferred organic solvent can significantly reduce the impurity content.

[0017] Further, in the preparation method of the present invention, the weight-to-volume ratio of the API TBN to the organic solvent in Step (1) is 1:5-20, and preferably 1:8-12.

[0018] Further, in the preparation method of the present invention, the temperature of cooling for crystallization in Step (1) is 2-12°C, more preferably 3-10°C, and most preferably 3-5°C. The applicant finds that in the most preferred range, the impurity content can be significantly reduced.

[0019] Further, in the preparation method of the present invention, the weight-to-volume ratio of API TBN to n-heptane in Step (2) is preferably 1:1-5, and most preferably 1:1-3.

[0020] Further, in the preparation method of the present invention, the mixing and heating temperature of the crystalline solid and n-heptane in Step (2) is 60-80°C, and preferably 65-75°C.

[0021] Further, in the preparation method of the present invention, the temperature of cooling for crystallization in Step (2) is 2-12°C, and more preferably 4-10°C.

[0022] Also described herein, but not claimed is a TBN dihydrate, having characteristic diffraction peaks in the XRPD pattern at 2θ°: 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] The TBN dihydrate can have characteristic diffraction peaks in the XRPD pattern at 2θ°: 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] The TBN dihydrate can have basically the same XRPD pattern as shown in Fig. 2.

[0025] Further, the melting point of the TBN dihydrate is 37-40°C.

[0026] Further, the DSC profile of the TBN dihydrate is basically as shown in Fig. 4.

[0027] Further, the TGA pattern of the TBN dihydrate is shown in Fig. 8.

[0028] When the described, but not claimed TBN dihydrate reaches the melting point (about 37-40 °C), the weight begins to decrease, and the weight loss is 13.67%.

[0029] The described, but not claimed TBN dihydrate is obtained by cold precipitation of TBN in a binary system of a saturated ethanol-water solution, where preferably the content of ethanol in percentages by weight in the ethanol-water solution is 5-50%, and further preferably 5-20%.

[0030] In another aspect, the present invention provides a pharmaceutical composition comprising one or more of the crystal form A. The pharmaceutical composition may also optionally comprise a pharmaceutically acceptable carrier, excipient, filler, binder, disintegrant, glidant and / or medium, etc.

[0031] On the other hand, the crystal form A or the pharmaceutical composition of the present invention can be given by oral administration or injection or other routes of administration.

[0032] In another aspect, the present invention also provides a dosage form comprising the crystal form A, including but not limited to tablets, capsules, powder injections, and dispersions, etc., preferably tablets and powder injections.

[0033] In another aspect described herein, but not claimed, the crystal form A and / or dihydrate or pharmaceutical composition can be used in the preparation of drugs for the treatment of neurological diseases, cardiovascular and cerebrovascular diseases and aging-related degenerative diseases.

[0034] The present invention has the following beneficial effects. (1) The preparation method of the crystal form A of TBN described in the present invention is simple and easy for industrial production. (2) The crystal form A of TBN according to the present invention is stable in the heat treatment, mechanical treatment and acceleration (50°C, 75% RH), is at low risk of polymorphism transformation, and has good stability, which contribute to the stability of the preparations during the production and storage processes, and effectively guarantee the consistency of the content of the crystal form in the preparations. Therefore, the preparations are safe, effective, and controllable in quality. (3) The crystal form A of TBN according to the present invention is readily soluble in most solvents (such as acetonitrile, methanol, ethanol, acetone and ethyl acetate, etc.) and very soluble in water, and has high biological activity, good druggability, high bioavailability, fast onset of action, and high biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Fig. 1 shows an XRD pattern of the crystal form A of TBN; Fig. 2 compares XRD patterns of the crystal form A and the reference compound dihydrate of TBN; Fig. 3 shows an DSC pattern of the crystal form A of TBN; Fig. 4 shows a DSC profile of the crystal form A and the reference compound dihydrate of TBN; Fig. 5 shows a biological microscope image of the crystal form A of TBN; Fig. 6 shows the structure of the crystal form A of TBN; Fig. 7 shows an TGA pattern of the crystal form A of TBN; Fig. 8 compares TGA patterns of the crystal form A and the reference compound dihydrate of TBN; Fig. 9 shows an IR spectrum of the crystal form A of TBN; Fig. 10 shows an XRD pattern of the crystal form A of TBN one week after suspension beating; Fig. 11 shows an XRD pattern of the crystal form A of TBN after being ground for 10 min; Fig. 12 shows an XRD pattern of the reference compound TBN dihydrate after being ground for 10 min; Fig. 13 shows an XRD pattern of the crystal form A after being tableted; Fig. 14 shows an XRD pattern of the reference compound dihydrate after being tableted; Fig. 15 shows a DSC profile of the crystal form A after a cooling treatment; Fig. 16 shows an XRPD profile of the crystal form A after a cooling treatment; Fig. 17 shows a DSC profile of the crystal form A after thermal treatment 1; Fig. 18 shows an XRPD profile of the crystal form A after thermal treatment 1; Fig. 19 shows a DSC profile of the reference compound TBN dihydrate after a thermal treatment; Fig. 20 shows an XRPD profile of the reference compound TBN dihydrate after a thermal treatment; Fig. 21 shows an XRPD profile of the crystal form A after 7 days of acceleration test; Fig. 22 shows the distribution in tissues after intravenous administration of TBN (30 mg / kg, n=6, male:female 1:1) to SD rats. DETAILED DESCRIPTION

[0036] The present invention will be further described by way of examples. It should be understood that the specific examples described herein are merely provided for illustrating, instead of limiting the present invention. Any simple modifications can be made to the preparation method of the present invention without departing from the concept of the present invention, which all fall within the protection scope of the present invention. In the examples where no specific conditions are indicated for the experimental procedures, generally known means are employed. Test materials used in the following examples are commercially available, unless otherwise specified.

[0037] In the following examples and accompanying drawings, unless otherwise specified, TBN represents TBN according to the present invention.

[0038] The characteristic diffraction peaks in the XRD patterns of the crystal form A of TBN and the TBN dihydrate, which is not claimed by but described as it contributes to a better understanding of the present invention, in the following examples are measured under the following experimental conditions: The instrument is X-ray powder diffraction analyzer (Bruker D2PHASER), the voltage and tube current are 30 KV and 10 mA, respectively, the 2θ scanning angle of the sample is from 3°-40°, and the scanning step is 0.02°.Example 1

[0039] To a 250 ml round-bottom flask, API TBN (10 g), cyclohexane (100 ml) and ethyl acetate (2 ml) were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (10°C), and then stood in a freezer at 4°C and filtered. The filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0040] To a 250 ml round-bottom flask, the above crude solid TBN and 2 volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to 10°C for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for 24 hrs at 38°C under vacuum to a drying loss of <1.0%, to obtain the crystal form A of TBN.Example 2

[0041] To a 250 ml round-bottom flask, API TBN (10 g), n-hexane (100 ml) and ethyl acetate (2 ml) were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (10°C), and then stand in a freezer at 4°C and filtered. The filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0042] To a 250 ml round-bottom flask, the above crude solid TBN and 2 volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to 10°C for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for 24 hrs at 38°C under vacuum to a drying loss of <1.0%, to obtain the crystal form A of TBN.Example 3

[0043] To a 250 ml round-bottom flask, API TBN (10 g) and cyclohexane (100 ml) were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (10°C), and then stood in a freezer at 4°C and filtered. The filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0044] To a 250 ml round-bottom flask, the above crude solid TBN and 2 volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to 10°C for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for 24 hrs at 38°C under vacuum to a drying loss of <1.0%, to obtain the crystal form A of TBN.Example 4

[0045] To a 250 ml round-bottom flask, API TBN (10 g) and n-hexane (100 ml) were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (10°C), and then stood in a freezer at 4°C and filtered. The filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0046] To a 250 ml round-bottom flask, the above crude solid TBN and 2 volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to 10°C for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for 24 hrs at 38°C under vacuum to a drying loss of <1.0%, to obtain the crystal form A of TBN.Example 5

[0047] To a 250 ml round-bottom flask, API TBN (10 g) and n-heptane (100 ml) were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (10°C), and then stood in a freezer at 4°C and filtered. The filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0048] To a 250 ml round-bottom flask, the above crude solid TBN and 2 volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to 10°C for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for 24 hrs at 38°C under vacuum to a drying loss of <1.0%, to obtain the crystal form A of TBN.Example 6

[0049] To a 250 ml round-bottom flask, API TBN (10 g) and n-hexane (100 ml) were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (10°C), and then stood in a freezer at 10°C and filtered. The filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0050] To a 250 ml round-bottom flask, the above crude solid TBN and 2 volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to 10°C for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for 24 hrs at 38°C under vacuum to a drying loss of <1.0%, to obtain the crystal form A of TBN.Example 7

[0051] To a 250 ml round-bottom flask, API TBN (10 g) and n-hexane (100 ml) were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (10°C), and then stood in a freezer at 4°C and filtered. The filter cake was washed with n-hexane, and the solvent was removed under reduced pressure to obtain a pale yellow crystalline solid.

[0052] To a 250 ml round-bottom flask, the above crude solid TBN and 2 volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to 140 ± 5 rpm, and heated in a water bath at 70°C, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to 10°C for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for 24 hrs at 38°C under vacuum to a drying loss of <1.0%, to obtain the crystal form A of TBN.

[0053] The XRD pattern of the crystal form A of TBN prepared in Examples 1-7 is shown in Fig. 1 and Table 1. The crystal form A has characteristic diffraction peaks in the XRD pattern at 20°: 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 profile is shown in Fig. 3, in which there is an endothermic peak, which is a melting peak, and the melting point is between 76-78°C. It can be seen from the micrographs of Fig. 5 and Fig. 6 that TBN has three crystalline structures, namely needle-, cube-, or rod-shaped crystalline structure. The TGA pattern is shown in Fig. 7. The IR spectrum is shown in Fig. 9.Reference Example 8

[0054] TBN is cold precipitated in a binary system of a saturated ethanol / water solution (9% ethanol by volume) to obtain a TBN dihydrate. The XRD pattern of the prepared dihydrate is shown in Fig. 2 and Table 1. The dihydrate has characteristic diffraction peaks in the XRD pattern at 2θ°: 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. The DSC profile is shown in Fig. 4, and the melting point of the TBN dihydrate is between 37-40°C. The TGA pattern is shown in Fig. 8. Table 1. Comparison of characteristic diffraction peaks of crystal form A and dihydrateCrystal form ADihydrate*Angle of diffraction 20Interplanar spacing (Å)Relative diffraction intensity (I / I 0 %)Angle of diffraction 20Interplanar spacing (Å)Relative diffraction intensity (I / I 0 %)10.5988.34060100%8.9059.9220817.1%11.0258.0190227.5%11.4597.71598100%15.3085.7835218.2%14.2896.1936242.9%15.5515.693475.4%17.6025.0346466.0%17.1355.1707513.4%21.1914.1893532.2%17.9264.9441411.5%22.0194.0336038.7%23.8133.733555.1%23.1923.8321340.0%24.2963.6603933.1%24.9183.5705357.4%29.1973.0562342.0%31.4052.8461531.7%*Reference compound Investigation of preparation method

[0055] 1. The influence of solvent on the yield and quality of final product TBN. The results are shown in Table 2: 2. The influence of crystallization temperature on the yield and quality of final product TBN. The results are shown in Table 3: Investigation of performances: 1. Stability investigation1.1. Polymorph transformation test of suspension

[0056] (1) The crystal form A of TBN was beaten in petroleum ether (1.0 mL) at room temperature for 7 days, and then the suspension was centrifuged, and the solid sample was collected for characterization by XRPD.

[0057] The results show that after the crystal form A of TBN is beaten in suspension in petroleum ether for a week, it is still crystal form A. The XRD pattern is shown in Fig. 10.1.2. Test of stability against mechanical treatment

[0058] (2) The crystal form A of TBN and the reference compound TBN dihydrate were ground for 10 min respectively, and the treated samples were analyzed by XRPD.

[0059] The results show that the XRPD characteristic peaks of the crystal form A of the present invention have no change after grinding for 10 min. The crystal form of the reference compound TBN dihydrate has no change after grinding for 10 min. The patterns are shown in Fig. 11 and Fig. 12.1.3. Tableting test

[0060] (3) The crystal form A and the reference compound TBN dihydrate were tableted under a pressure of 2 tons for 2 min. The tablets thus obtained were analyzed by XRPD.

[0061] The results show that the crystal form A and the reference compound TBN dihydrate do not undergo polymorph transformation after 2 min of pressurization under 2 tons (where the crystallinity became smaller), as shown in Fig. 13 and 14.1.4. Test of stability against cold / heat treatment

[0062] (4) The crystal form A was cooled to -55°C, and then heated to 25°C. The results show that the crystal form A does not undergo polymorph transformation, as shown in Fig. 15 and 16.

[0063] (5) The crystal form A was heated to 110°C (before the decomposition point), and then cooled to 25°C to obtain a yellow oily liquid. After stirring, a white solid was precipitated out, which is characterized by XRPD as crystal form A, as shown in Fig. 17 and 18.

[0064] (6) The reference compound TBN dihydrate was heated to 110°C (before the decomposition point), and then cooled to 25°C to obtain a yellow oily liquid. After stirring, a white solid was precipitated out, which is characterized by XRPD as crystal form A, as shown in Fig. 19 and 20.1.5. Test of influence of acceleration

[0065] When the crystal form A is accelerated for 7 days at 40°C and RH=75 %, the XRPD characterization result shows crystal form A, as shown in Fig. 21.2. Investigation of biological activity

[0066] The pharmacodynamic study in cynomolgus monkey animal model of cerebral stroke and also the pharmacokinetic study of the crystal form A of TBN in the cerebrospinal fluid of cynomolgus monkey were carried out. 10 min, 30 min, 60 min, and 120 min after the second administration (30 mg / kg, 6 hrs after the first intravenous administration of 30 mg / kg), 0.5 ml of cerebrospinal fluid was collected for pharmacokinetic test. Table 4. Drug concentration-time data in cerebrospinal fluid of cynomolgus monkeys given TBN at 30 mg / kg intravenouslyRoute of administrationAnimal No.TBN at various time (min)Drug concentration in cerebrospinal fluid (ng / ml)103060120Intravenous injectionMonkey-0801353430025100200007800Monkey-07036148800411002750016500Monkey-07151533700322002530014300

[0067] The test results in Table 4 show that 10 min after the second administration, the concentration of TBN in the cerebrospinal fluid is 176 µM, which is almost equal to the concentration of the drug in plasma (195 µM), and higher than the effective protection concentration of 30 µM of TBN in in vitro cell experiments. This result shows that the crystal form A of TBN penetrate through the blood-brain barrier and reach an effective protection concentration.3. Metabolic data: clearance rate

[0068] SD rats were given the crystal form A of TBN at a dose of 30 mg / kg by intravenous injection and the distribution of the crystal form A of TBN in various tissues was investigated.

[0069] The experimental results are shown in Fig. 22. The results show that after intravenous administration of TBN to female and male rats at 30 mg / kg, rapid and widespread distribution is reached in all tissues and plasma almost at the same time, and the drug is cleared away from the tissues and plasma almost at the same time in 24 hrs. TBN does not tend to accumulate in the detected tissues. 0.25 hrs after administration, the concentration of TBN in the brain tissue is 0.6-0.7 time the plasma concentration, and 0.8-1.1 times the plasma concentration after 4 hrs, and TBN is completely cleared as is in the plasma at 24 hrs.

Claims

1. A crystal form A of tetramethylpyrazine nitrone (TBN), having characteristic diffraction peaks in the XRPD pattern of the crystal form at 2θ°: 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 melting point of the crystal form A of TBN is 76-78°C; and preferably having characteristic diffraction peaks in the XRPD pattern of the crystal form at 2θ°: 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.

2. The crystal form A of TBN according to claim 1, having the same XRPD pattern as shown in Fig. 1.

3. The crystal form A of TBN according to claim 1, having the same DSC profile as shown in Fig. 3, the same TGA pattern as shown in Fig. 7, and the same IR spectrum as shown in Fig. 9.

4. A method for preparing the crystal form A of TBN, comprising the following steps: (1) mixing the API TBN with an organic solvent, heating in a water bath to 60-80°C, stirring and filtering, and cooling the filtrate for crystallization to obtain a crystalline solid, the organic solvent is one or more selected from n-hexane, cyclohexane, and n-heptane, or a mixed solvent of n-hexane and ethyl acetate; and (2) mixing the crystalline solid obtained in Step (1) and n-heptane, heating to dissolve it, and cooling for crystallization to obtain the crystal form A of TBN, the crystal form A of TBN having characteristic diffraction peaks in the XRPD pattern of the crystal form at 2θ°: 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 the melting point of the crystal form A of TBN being 76-78°C;.

5. The preparation method according to claim 4, wherein the weight-to-volume ratio of the API TBN to the organic solvent in Step (1) is 1:5-20, and preferably 1:8-12; the temperature of cooling for crystallization in Step (1) is 2-12°C, more preferably 3-10°C, and most preferably 3-5°C; the weight-to-volume ratio of the API TBN to n-heptane in Step (2) is preferably 1:1-5, and most preferably 1:1-3; the mixing and heating temperature of the crystalline solid and n-heptane in Step (2) is 60-80°C, and preferably 65-75°C; and the temperature of cooling for crystallization in Step (2) is 2-12°C, and more preferably 4-10°C.

6. A pharmaceutical composition, comprising the crystal form A according to any one of claims 1 to 3.