Patent Description:
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)-<NUM>-methyl-N-((<NUM>,<NUM>,<NUM>-trimethylpyrazin-<NUM>-yl)methylene)propan-<NUM>-amine oxide, a molecular formula of C<NUM>H<NUM>N<NUM>O, a molecular weight of <NUM>, and a chemical structure shown below:
<CHM>.

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.

<CIT> describes nitrone compounds, a process of preparation thereof, and use thereof in medicament manufacture.

<NPL> describes antioxidative and thrombolytic TMP nitrone for treatment of ischemic stroke.

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

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°: <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, and <NUM>±<NUM>, the melting point of the crystal form A of TBN is <NUM>-<NUM>; and preferably having characteristic diffraction peaks in the XRPD pattern of the crystal form at 2θ°: <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, and <NUM>±<NUM>.

In a specific embodiment, the crystal form A of TBN has the same XRPD pattern as shown in <FIG>.

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 <FIG> and <FIG>.

Further, the melting point of the crystal form A of TBN is <NUM>-<NUM>, respectively.

Further, the DSC profile of the crystal form A of TBN is shown in <FIG>.

Further, the TGA pattern of the crystal form A of TBN is shown in <FIG>.

Further, the present invention provides an infrared spectrum of the crystal form A of TBN, as shown in <FIG>.

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.

Further, the present invention also provides a method for preparing the crystal form A of TBN, which includes the following steps:.

Further, in the preparation method of the present invention, the organic solvent in Step (<NUM>) 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.

Further, in the preparation method of the present invention, the weight-to-volume ratio of the API TBN to the organic solvent in Step (<NUM>) is <NUM>:<NUM>-<NUM>, and preferably <NUM>:<NUM>-<NUM>.

Further, in the preparation method of the present invention, the temperature of cooling for crystallization in Step (<NUM>) is <NUM>-<NUM>, more preferably <NUM>-<NUM>, and most preferably <NUM>-<NUM>. The applicant finds that in the most preferred range, the impurity content can be significantly reduced.

Further, in the preparation method of the present invention, the weight-to-volume ratio of API TBN to n-heptane in Step (<NUM>) is preferably <NUM>:<NUM>-<NUM>, and most preferably <NUM>:<NUM>-<NUM>.

Further, in the preparation method of the present invention, the mixing and heating temperature of the crystalline solid and n-heptane in Step (<NUM>) is <NUM>-<NUM>, and preferably <NUM>-<NUM>.

Further, in the preparation method of the present invention, the temperature of cooling for crystallization in Step (<NUM>) is <NUM>-<NUM>, and more preferably <NUM>-<NUM>.

Also described herein, but not claimed is a TBN dihydrate, having characteristic diffraction peaks in the XRPD pattern at 2θ°: <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, and <NUM>±<NUM>.

The TBN dihydrate can have characteristic diffraction peaks in the XRPD pattern at 2θ°: <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, and <NUM>±<NUM>.

The TBN dihydrate can have basically the same XRPD pattern as shown in <FIG>.

Further, the melting point of the TBN dihydrate is <NUM>-<NUM>.

Further, the DSC profile of the TBN dihydrate is basically as shown in <FIG>.

Further, the TGA pattern of the TBN dihydrate is shown in <FIG>.

When the described, but not claimed TBN dihydrate reaches the melting point (about <NUM>-<NUM>), the weight begins to decrease, and the weight loss is <NUM>%.

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 <NUM>-<NUM>%, and further preferably <NUM>-<NUM>%.

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..

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.

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.

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.

The present invention has the following beneficial effects.

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.

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

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 <NUM> KV and <NUM> mA, respectively, the 2θ scanning angle of the sample is from <NUM>°-<NUM>°, and the scanning step is <NUM>°.

To a <NUM> round-bottom flask, API TBN (<NUM>), cyclohexane (<NUM>) and ethyl acetate (<NUM>) were added, stirred continuously at a rotation speed adjusted to <NUM> ± <NUM> rpm, and heated in a water bath at <NUM>. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (<NUM>), and then stood in a freezer at <NUM> 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.

To a <NUM> round-bottom flask, the above crude solid TBN and <NUM> volumes of n-heptane were added, stirred continuously at a rotation speed adjusted to <NUM> ± <NUM> rpm, and heated in a water bath at <NUM>, to obtain an orange-yellow clear solution. The water bath was removed, and the solution was continuously stirred, cooled to <NUM> for crystallization, and filtered with suction. The solid was washed with n-heptane, and dried for <NUM> hrs at <NUM> under vacuum to a drying loss of <<NUM>%, to obtain the crystal form A of TBN.

To a <NUM> round-bottom flask, API TBN (<NUM>), n-hexane (<NUM>) and ethyl acetate (<NUM>) were added, stirred continuously at a rotation speed adjusted to <NUM> ± <NUM> rpm, and heated in a water bath at <NUM>. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (<NUM>), and then stand in a freezer at <NUM> 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.

To a <NUM> round-bottom flask, API TBN (<NUM>) and cyclohexane (<NUM>) were added, stirred continuously at a rotation speed adjusted to <NUM> ± <NUM> rpm, and heated in a water bath at <NUM>. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (<NUM>), and then stood in a freezer at <NUM> 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.

To a <NUM> round-bottom flask, API TBN (<NUM>) and n-hexane (<NUM>) were added, stirred continuously at a rotation speed adjusted to <NUM> ± <NUM> rpm, and heated in a water bath at <NUM>. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (<NUM>), and then stood in a freezer at <NUM> 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.

To a <NUM> round-bottom flask, API TBN (<NUM>) and n-heptane (<NUM>) were added, stirred continuously at a rotation speed adjusted to <NUM> ± <NUM> rpm, and heated in a water bath at <NUM>. The solution was filtered while hot to obtain an orange-yellow solution. The filtrate was allowed to stand and cool to ambient temperature (<NUM>), and then stood in a freezer at <NUM> 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.

The XRD pattern of the crystal form A of TBN prepared in Examples <NUM>-<NUM> is shown in <FIG> and Table <NUM>. The crystal form A has characteristic diffraction peaks in the XRD pattern at <NUM>°: <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, and <NUM>±<NUM>. The DSC profile is shown in <FIG>, in which there is an endothermic peak, which is a melting peak, and the melting point is between <NUM>-<NUM>. It can be seen from the micrographs of <FIG> and <FIG> that TBN has three crystalline structures, namely needle-, cube-, or rod-shaped crystalline structure. The TGA pattern is shown in <FIG>. The IR spectrum is shown in <FIG>.

TBN is cold precipitated in a binary system of a saturated ethanol/water solution (<NUM>% ethanol by volume) to obtain a TBN dihydrate. The XRD pattern of the prepared dihydrate is shown in <FIG> and Table <NUM>. The dihydrate has characteristic diffraction peaks in the XRD pattern at 2θ°: <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, <NUM>±<NUM>, and <NUM>±<NUM>. The DSC profile is shown in <FIG>, and the melting point of the TBN dihydrate is between <NUM>-<NUM>. The TGA pattern is shown in <FIG>.

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>.

(<NUM>) The crystal form A of TBN and the reference compound TBN dihydrate were ground for <NUM> respectively, and the treated samples were analyzed by XRPD.

The results show that the XRPD characteristic peaks of the crystal form A of the present invention have no change after grinding for <NUM>. The crystal form of the reference compound TBN dihydrate has no change after grinding for <NUM>. The patterns are shown in <FIG> and <FIG>.

(<NUM>) The crystal form A and the reference compound TBN dihydrate were tableted under a pressure of <NUM> tons for <NUM>. The tablets thus obtained were analyzed by XRPD.

The results show that the crystal form A and the reference compound TBN dihydrate do not undergo polymorph transformation after <NUM> of pressurization under <NUM> tons (where the crystallinity became smaller), as shown in <FIG> and <FIG>.

(<NUM>) The crystal form A was cooled to -<NUM>, and then heated to <NUM>. The results show that the crystal form A does not undergo polymorph transformation, as shown in <FIG> and <FIG>.

(<NUM>) The crystal form A was heated to <NUM> (before the decomposition point), and then cooled to <NUM> 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> and <FIG>.

(<NUM>) The reference compound TBN dihydrate was heated to <NUM> (before the decomposition point), and then cooled to <NUM> 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> and <FIG>.

When the crystal form A is accelerated for <NUM> days at <NUM> and RH=<NUM> %, the XRPD characterization result shows crystal form A, as shown in <FIG>.

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. <NUM>, <NUM>, <NUM>, and <NUM> after the second administration (<NUM>/kg, <NUM> hrs after the first intravenous administration of <NUM>/kg), <NUM> of cerebrospinal fluid was collected for pharmacokinetic test.

The test results in Table <NUM> show that <NUM> after the second administration, the concentration of TBN in the cerebrospinal fluid is <NUM>, which is almost equal to the concentration of the drug in plasma (<NUM>), and higher than the effective protection concentration of <NUM> 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.

SD rats were given the crystal form A of TBN at a dose of <NUM>/kg by intravenous injection and the distribution of the crystal form A of TBN in various tissues was investigated.

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