N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal and a preparation method and use thereof

Abstract

The application discloses a N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl) acethydrazine tartrate crystal, characterized in that a structural formula is as shown in formula 1: The application further provides a preparation method and application of the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl) acethydrazine tartrate crystal, and the crystal has good solubility and higher bioactivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystal and a preparation method and application thereof. BACKGROUND

[0002] Tumor is a malignant disease with extremely high morbidity and mortality and relatively poor treatment effect in the world. In the global range, anti-tumor drugs have become the first treatment field in the drug market. N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine, chemical formula C 23 H 28 N4O2, is the first small molecule compound capable of directly activating procaspase-3, and is mainly used for treating malignant tumors such as lung cancer, breast cancer, gastrointestinal cancer and the like. However, due to the low solubility and biological activity of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine, the bioavailability thereof is not ideal, and a high dose often needs to be given to exert good biological activity, but with the increase of the dose, toxic side effects will appear, thereby limiting the better clinical effect. SUMMARY

[0003] In view of the above defects of the prior art, the purpose of the present application is to provide a N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystal and a preparation method and application thereof, and the crystal has good solubility and higher biological activity.

[0004] The present application provides the following technical solutions.

[0005] The present application provides a N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystal, characterized in that the structural formula is as shown in formula 1:

[0006]

[0007] Further, the X powder diffraction spectrum thereof has characteristic peaks with diffraction angles 2θ of 19.90±0.2°, 20.89±0.2°, 21.69±0.2°, 22.98±0.2°, 23.27±0.2°, 25.40±0.2°, 25.62±0.2°.

[0008] Further, the X powder diffraction spectrum thereof is as shown in Figure 1 .

[0009] Further, the crystal has a platelet structure.

[0010] The present application also provides a method for preparing N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal, comprising the following steps:

[0011] mixing N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)hydrazide and tartaric acid to react to form the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal, which has a structure of formula 1

[0012]

[0013] Further, the molar ratio of the tartaric acid and the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)hydrazide is 1:(1-5), preferably 1:(1-2).

[0014] Further, the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)hydrazide and the tartaric acid are mixed, stirred, separated and dried in a reaction medium to obtain the crystal.

[0015] Further, the reaction medium is selected from one of anhydrous ethanol, methanol, acetonitrile, n-butanol, tetrahydrofuran and acetone.

[0016] Further, the reaction temperature is -20-80℃, preferably 10-30℃;

[0017] Preferably, the stirring time is 1-36 hours, more preferably 1-12 hours,

[0018] Preferably, the drying temperature is 10-50℃, more preferably 10-30℃,

[0019] Preferably, the drying time is 5-48 hours, more preferably 5-24 hours,

[0020] Preferably, the concentration of the crystal in the reaction medium is 0.5-1.2 times, more preferably 0.8-1 times, of the solubility of the crystal in the reaction medium at the reaction temperature.

[0021] Further, the purity of the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal is ≥95%.

[0022] Further, the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal prepared by the method has the crystal form as defined in any one of claims 1-4.

[0023] The application also provides a pharmaceutical composition comprising the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal as defined above.

[0024] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, adjuvant or carrier.

[0025] Further, the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal has a mass fraction of 10%-50%.

[0026] Further, the pharmaceutical composition is in the form of a tablet, capsule or pill.

[0027] The application also provides use of the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal in the preparation of a medicament for treating tumors.

[0028] Further, the tumor is lung cancer, liver cancer, colon cancer or melanoma and complications thereof.

[0029] The N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal provided by the application has better solubility than N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide, and thus has better bioavailability. It also has better anti-tumor effect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The X-ray diffraction pattern of the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal.

[0031] Figure 2 The DSC pattern of the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal.

[0032] Figure 3 The HPLC pattern of the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)hydrazide tartrate crystal.

[0033] Figure 4 UV spectrum of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal.

[0034] Figure 5 H-NMR spectrum of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal.

[0035] Figure 6 SEM image of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal. DETAILED DESCRIPTION

[0036] The exemplary embodiments of the present application will be described herein below with reference to the accompanying drawings, in which various details of the present application are set forth to facilitate an understanding; however, it should be appreciated that the detailed disclosure herein is provided for the purposes of illustration only, and not for the purpose of limiting the present application. Thus, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the spirit and scope of the present application. Also, for the purpose of clarity and a concise description, descriptions of well-known functions and constructions are omitted herein.

[0037] The present application provides a N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal, the structural formula of which is shown as Formula 1:

[0038]

[0039] In the present application, the X powder diffraction spectrum of the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal has characteristic peaks at diffraction angles 2Θ of 19.90±0.2°, 20.89±0.2°, 21.69±0.2°, 22.98±0.2°, 23.27±0.2°, 25.40±0.2°, and 25.62±0.2°.

[0040] In the present application, the X powder diffraction spectrum of the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal is shown as Figure 1

[0041] ​The crystals described in the present application are characterized by X-ray powder diffraction patterns (XRD) that demonstrate a unique order of molecular arrangement or configuration within the crystal lattice. Those skilled in the art are aware that experimental error is dependent on the instrument conditions, sample preparation, and sample purity. The 2 theta angle of the peaks in the XRD pattern will vary slightly depending on the instrument and sample. The difference in peak angle can vary by 1°, 0.8°, 0.5°, 0.3°, 0.1°, etc. depending on the instrument, sample, etc. and is generally allowed to vary by ± 0.2°, so the difference in peak angle cannot be used as the sole criterion. The relative intensities of the peaks can vary depending on the sample, sample preparation, and other experimental conditions, so the order of peak intensities cannot be used as the sole or determinative factor. The influence of sample height and other experimental factors can cause a shift in the overall peak angle, and some shift is generally allowed. Thus, those skilled in the art will understand that any crystal having peaks with the same or similar characteristics as the X-ray powder diffraction patterns described in the present application are within the scope of the present application.

[0042] The present application provides a method for preparing N'-[(3-allyl-2-hydroxyphenyl)methyl]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal, comprising the following steps:

[0043] Mixing N'-[(3-allyl-2-hydroxyphenyl)methyl]-2-(4-benzyl-1-piperazinyl)acetohydrazide and tartaric acid to react to form the N'-[(3-allyl-2-hydroxyphenyl)methyl]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal, which has the structure of Formula 1

[0044]

[0045] Specifically, the molar ratio of the N'-[(3-allyl-2-hydroxyphenyl)methyl]-2-(4-benzyl-1-piperazinyl)acetohydrazide and tartaric acid is 1:(1-5).

[0046] In some embodiments, the molar ratio of the N'-[(3-allyl-2-hydroxyphenyl)methyl]-2-(4-benzyl-1-piperazinyl)acetohydrazide and tartaric acid can be 1:(1-2).

[0047] In some embodiments, the molar ratio of the N'-[(3-allyl-2-hydroxyphenyl)methyl]-2-(4-benzyl-1-piperazinyl)acetohydrazide and tartaric acid can be 1:(1-2.5).

[0048] In some embodiments, the molar ratio of the N'-[(3-allyl-2-hydroxyphenyl)methyl]-2-(4-benzyl-1-piperazinyl)acetohydrazide and tartaric acid can be 1:(1-3).

[0049] In some embodiments, the molar ratio of N'-[(3-allyl-2-hydroxyphenyl)methyliden]-2-(4-benzyl-l-piperazinyl)hydrazide and tartaric acid can be 1 : (1-3.5).

[0050] In some embodiments, the molar ratio of N'-[(3-allyl-2-hydroxyphenyl)methyliden]-2-(4-benzyl-l-piperazinyl)hydrazide and tartaric acid can be 1 : (1-4).

[0051] In particular, the molar ratio of N'-[(3-allyl-2-hydroxyphenyl)methyliden]-2-(4-benzyl-l-piperazinyl)hydrazide and tartaric acid can be 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9, 1 : 2, 1 : 2.1, 1 : 2.2, 1 : 2.3, 1 : 2.4, 1 : 2.5, 1 : 2.6, 1 : 2.7, 1 : 2.8, 1 : 2.9, 1 : 3, 1 : 3.1, 1 : 3.2, 1 : 3.3, 1 : 3.4, 1 : 3.5, 1 : 3.6, 1 : 3.7, 1 : 3.8, 1 : 3.9, 1 : 4, 1 : 4.1, 1 : 4.2, 1 : 4.3, 1 : 4.4, 1 : 4.5, 1 : 4.6, 1 : 4.7, 1 : 4.8, 1 : 4.9, or 1 : 5.

[0052] In particular, under certain conditions, N'-[(3-allyl-2-hydroxyphenyl)methyliden]-2-(4-benzyl-l-piperazinyl)hydrazide and tartaric acid are mixed in a reaction medium, stirred, separated, dried, and finally the crystal is obtained.

[0053] In particular, the reaction medium is selected from one of anhydrous ethanol, methanol, acetonitrile, n-butanol, tetrahydrofuran, and acetone.

[0054] In particular, the concentration of the crystal in the reaction medium is 0.5-1.2 times, preferably 0.8-1 times, of the solubility of the crystal in the reaction medium at the reaction temperature. For example, it can be 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1.0 times, 1.1 times, or 1.2 times.

[0055] In particular, the reaction temperature of N'-[(3-allyl-2-hydroxyphenyl)methyliden]-2-(4-benzyl-l-piperazinyl)hydrazide and tartaric acid is -20-80°C, more preferably room temperature. For example, the reaction temperature can be -20°C, -10°C, 0°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C.

[0056] Specifically, the stirring time is 1-36 hours, more preferably 1-12 hours. For example, the stirring time can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h or 12 h.

[0057] Specifically, the stirring can be carried out by using conventional methods in the art, such as mechanical stirring, magnetic stirring, etc., and the stirring speed is preferably 200-800 rpm. For example, it can be 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm, 750 rpm or 800 rpm.

[0058] Specifically, the separation can be carried out by using conventional methods in the art, such as filtration, centrifugation or concentration under reduced pressure, preferably filtration under reduced pressure, and the filtration pressure under reduced pressure is less than 0.09 MPa, for example, it can be 0.08 MPa or 0.07 MPa, etc.

[0059] Specifically, the drying can be carried out by using conventional methods in the art, such as air drying, freeze drying or drying under reduced pressure, preferably drying under reduced pressure, and the drying pressure under reduced pressure is less than 0.09 MPa, for example, it can be 0.08 MPa or 0.07 MPa, etc.

[0060] Specifically, the drying temperature is 10-50°C, more preferably 10-30°C. For example, the drying temperature can be 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C.

[0061] Specifically, the drying time is 5-48 hours, more preferably 5-24 hours. For example, the drying time can be 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h or 48 h.

[0062] Specifically, the reaction process of tartaric acid and N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetohydrazide is as follows:

[0063]

[0064] Specifically, the purity of the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal is ≥95%. For example, the purity can be 95%, 96%, 97%, 98%, etc.

[0065] The N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal prepared by the method described in the present application is the aforementioned N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal, and specific reference can be made to the foregoing description.

[0066] In the present application, the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)acetohydrazide is abbreviated as PAC-1, and the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal is abbreviated as PAC-1 tartrate crystal.

[0067] The present application provides a pharmaceutical composition containing the aforementioned N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal.

[0068] In the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, adjuvant or carrier.

[0069] Specifically, the mass fraction of the N'-[(3-allyl-2-hydroxyphenyl)methylidene]-2-(4-benzyl-1-piperazinyl)acetohydrazide tartrate crystal is 10%-50%. For example, it can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% or 50%.

[0070] In the present application, the pharmaceutical composition is a tablet, a capsule or a pill.

[0071] The pharmaceutical compositions can be in a form suitable for oral use, for example, as tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, etc. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets can contain effective ingredients in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, binding agents and lubricating agents. Tablets can be uncoated or they can be coated by known techniques so as to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

[0072] The present application provides a use of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal in the manufacture of a medicament for treating a tumor.

[0073] In particular, the tumor is lung cancer, liver cancer, colon cancer or melanoma and complications thereof. The patient includes, but is not limited to, a mammal.

[0074] Examples

[0075] The materials used in the examples and test methods used in the present application are described generally and / or specifically below. In the following examples, % means wt%, i.e. weight percent, unless otherwise specified. The reagents or instruments used are not specified by the manufacturer, and are all conventional reagent products available on the market.

[0076] Example 1

[0077] N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide (prepared by referring to the method of Quinn P. Peterson et al., J. Med. Chem. 2009, 52, 5721-5731) (2.00 g, 1 eq) was weighed into a reaction bottle, 30 ml of anhydrous ethanol was added and stirred at 50°C, and tartaric acid (1.53 g, 2 eq) was dissolved in 30 ml of ethanol; the solution became turbid and a transparent white solid adhered to the wall of the container during the dropwise addition process, and stirring was continued for 1.5 h; the temperature was lowered to 4°C, and a white solid was washed out, filtered, and the filter cake was dried under reduced pressure for 3 h to obtain a white flaky solid 3.36 g. The parameters thereof are shown in Table 1, and the characterization results of the white flaky solid are shown in Table 2. Figures 1-6 It can be known from Figures 1-6 that the white flaky solid is N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal.

[0078] The N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal prepared in Example 2 is different from Example 1 in that the molar ratio of tartaric acid to PAC-1 is different, and the parameters of this example are shown in Table 1.

[0079] The N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal prepared in Example 3-Example 4 is different from Example 2 in that the reaction medium is different, and the parameters of this example are shown in Table 1.

[0080] The N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal prepared in Example 5-Example 7 is different from Example 2 in that the reaction temperature is different, and the parameters of this example are shown in Table 1.

[0081] The N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal prepared in Example 8-Example 9 is different from Example 2 in that the drying temperature is different, and the parameters of this example are shown in Table 1.

[0082] Table 1 is the parameters of each example and comparative example

[0083]

[0084] Summary: Examples 1-7 of the present application have all prepared N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-l-piperazinyl)hydrazide tartrate crystal (PAC-1 tartrate crystal), and the yield of PAC-1 tartrate crystal obtained by the preparation method of the present application is high.

[0085] Test Example

[0086] Test Example 1 Solubility Determination

[0087] Take 10 mL of water in a beaker, and weigh an appropriate amount of solid to be tested (PAC-1 tartrate prepared in Example 1, PAC-1 prepared by the method of reference Quinn P. Peterson et al., J. Med. Chem. 2009, 52, 5721-5731). Gradually add the solid sample, constantly stirring, fully dissolving, until just saturated, and calculate the amount.

[0088] Weigh the total mass of the undissolved solid, and calculate the mass of the solid that has been dissolved in 10 mL (10 g) of water, which is the solubility of the solid, and the detailed data are shown in Table 2.

[0089] Table 2 Solubility of PAC-1 and PAC-1 tartrate

[0090]

[0091] Summary: From Table 2, it can be seen that the solubility of PAC-1 tartrate described in the present application is greater than that of PAC-1.

[0092] Test Example 2 Anti-tumor activity study

[0093] The PAC-1 tartrate described in the present application has inhibitory activity on human non-small cell lung cancer cells (A549), human hepatoma cells (HepG2), human melanoma cells (A375), and human colon cancer cells (HCT-116).

[0094] 1. Test method: MTT method was used to study the inhibitory effect of the compound on the proliferation of cancer cells.

[0095] 2. Instruments and reagents are shown in Table 3 and Table 4.

[0096] Table 3 List of reagents

[0097]

[0098] Table 4 List of instruments

[0099]

[0100] 3. Compound preparation

[0101] PAC-1 tartrate prepared in Example 1 of the present application, PAC-1 hydrochloride (laboratory-made salt crystal, the preparation method of which is the same as that of Example 1, except that hydrochloric acid is used instead of tartaric acid, and the molar ratio of PAC-1 to hydrochloric acid is 1:2), PAC-1 acetate (laboratory-made salt crystal, the preparation method of which is the same as that of Example 1, except that acetic acid is used instead of tartaric acid, and the molar ratio of PAC-1 to acetic acid is 1:2), PAC-1 sulfate (laboratory-made salt crystal, the preparation method of which is the same as that of Example 1, except that sulfuric acid is used instead of tartaric acid, and the molar ratio of PAC-1 to sulfuric acid is 1:2), and PAC-1 prepared by the method of Quinn P. Peterson et al., J. Med. Chem. 2009, 52, 5721-5731 were dissolved in DMSO, respectively, and stored at room temperature under a nitrogen environment for standby, and the samples in DMSO were sequentially diluted with culture solution to the required concentration, thereby obtaining the test compound group.

[0102] 4. Test method

[0103] Specific steps are human non-small cell lung cancer cells (A549), human hepatoma cells (HepG2), human melanoma cells (A375), human colon cancer cells (HCT-116) in DMEM medium containing 10% FBS (both 100 kU / L of penicillin and streptomycin), in a 5% CO2 37°C constant temperature incubator, and when the cells grow to 80-90% of the plate, they are passaged every 24 hours, and the fresh culture medium is replaced.

[0104] A549, HepG2, HCT-116, A375 cells were cultured in a 37°C, 5% CO2 incubator to the logarithmic growth phase, and inoculated in a 96-well plate at a cell density of 4000 cells / well. After 24 hours, they were divided into blank control group (NC), test compound group (2 μM), and cultured for 48 hours. Then 20 μL of MTT solution (5 mg·mL -1 ) was added to each well, and incubated for 4 hours. The culture medium was carefully aspirated, and 150 μL of DMSO solution was added to each well, and shaken for 10 minutes to completely dissolve it. The absorbance value (OD) of each well was measured by a microplate reader at 490 nm, and the cell inhibition rate was calculated, as shown in Table 5 below.

[0105] Table 5 is the effect parameter

[0106] Compound / 10 μM A549 inhibition rate % HepG2 inhibition rate % HCT116 inhibition rate % A375 inhibition rate % PAC-1 tartrate 59.37％ 55.13％ 65.73％ 89.09％ PAC-1 hydrochloride 52.49％ 65.66％ 55.36％ 78.87％ PAC-1 acetate 42.33％ 39.33％ 49.81％ 70.06％ PAC-1 sulfate 45.60％ 33.82％ 43.65％ 63.45％ PAC-1 43.23％ 30.39％ 50.69％ 58,62％

[0107] Summary: The inhibitory effects of PAC-1 different salts and PAC-1 on non-small cell lung cancer cells (A549), human hepatoma cells (HepG2), human melanoma cells (A375), and human colon cancer cells (HCT-116) were compared. The results showed that the tartrate salt showed better tumor cell inhibition effect than other salts and PAC-1, suggesting that it has better anti-tumor activity.

[0108] Test Example 3 Toxicity Test

[0109] The PAC-1 tartrate salt described in the present application was studied for its effect on the behavior of C57 / BL6 mice (neurotoxicity).

[0110] 1. Toxicity verification and neurotoxicity scoring

[0111] (1) Neurological function score of epilepsy

[0112] Grade 0: no response; Grade I: rhythmic mouth or facial twitching; Grade II: nodding or tail flicking; Grade III: single limb twitching; Grade IV: multiple limb twitching or rigidity; and Grade V: generalized tonic-clonic seizures.

[0113] (2) C57 / BL6 mice were injected intraperitoneally with low, medium and high doses of PAC-1, PAC-1 hydrochloride, PAC-1 acetate, PAC-1 sulfate and PAC-1 tartrate. The mice were observed for 24 hours for toxicity, and a table was prepared according to the neurological score of epilepsy.

[0114] 2. Observation of the behavior of C57 / BL6 mice

[0115] To study the effect of the test compound on the neurotoxicity of mice, C57 / BL6 mice were injected intraperitoneally with low, medium and high doses of the compound (ZYS-1 (amorphous PAC-1 prepared by WO2017012113A1), PAC-1, PAC-1 hydrochloride, PAC-1 acetate, PAC-1 sulfate and PAC-1 tartrate), and the mice were observed for 24 hours. The results showed that PAC-1 caused symptoms of epilepsy in mice at medium and high doses; PAC-1 sulfate and PAC-1 hydrochloride caused symptoms of epilepsy in mice at high doses; ZYS-1, PAC-1 acetate and PAC-1 tartrate did not exhibit similar conditions or had milder conditions, and the specific results are shown in Table 6.

[0116] Table 6 Neurotoxicity behavior classification of C57 / BL6 mice

[0117] Dose (mg / kg) PAC-1 ZYS-1 PAC-1 hydrochloride PAC-1 acetate PAC-1 hydrochloride PAC-1 tartrate 0 0 0 0 0 0 0 5 0 0 0 0 0 0 25 Ⅰ 0 0 0 0 0 50 Ⅲ Ⅰ / 0 Ⅱ Ⅰ / 0 Ⅱ Ⅰ / 0

[0118] Although the embodiments of the present application are described above in conjunction with the drawings, the present application is not limited to the specific embodiments and application fields described above, and the specific embodiments described above are merely illustrative and instructive, but not restrictive. Those of ordinary skill in the art can make many forms under the guidance of the present specification and without departing from the scope of the claims of the present application, and all of these are within the scope of protection of the present application.

Claims

1. A crystal of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate, characterized in that, Its structural formula is as shown in Formula 1: Formula 1; Its X-ray powder diffraction pattern has characteristic peaks with diffraction angles 2θ of 19.90±0.2°, 20.89±0.2°, 21.69±0.2°, 22.98±0.2°, 23.27±0.2°, 25.40±0.2°, and 25.62±0.2°.

2. The crystal according to claim 1, characterized in that, Its X-ray powder diffraction pattern is shown in Figure 1.

3. The crystal according to claim 1, characterized in that, The crystal has a plate-like structure.

4. A method for preparing N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystals, characterized in that, Includes the following steps: N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine and tartaric acid were mixed, stirred, separated, and dried in a reaction medium to produce the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystals, the structure of which is shown in Formula 1. Formula 1; The molar ratio of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine and tartaric acid is 1:(1-5); The reaction medium is selected from one of anhydrous ethanol, methanol, and acetonitrile; The drying temperature is 10-30℃; The X-ray powder diffraction pattern of the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystal has characteristic peaks with diffraction angles 2θ of 19.90±0.2°, 20.89±0.2°, 21.69±0.2°, 22.98±0.2°, 23.27±0.2°, 25.40±0.2°, and 25.62±0.2°.

5. The preparation method according to claim 4, characterized in that, The molar ratio of N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine and tartaric acid is 1:(1-2).

6. The preparation method according to claim 4, characterized in that, The reaction temperature is -20 to 80°C.

7. The preparation method according to claim 4, characterized in that, The reaction temperature is 10-30℃.

8. The preparation method according to claim 4, characterized in that, The stirring time is 1-36 hours.

9. The preparation method according to claim 4, characterized in that, The stirring time is 1-12 hours.

10. The preparation method according to claim 4, characterized in that, The drying time is 5-48 hours.

11. The preparation method according to claim 4, characterized in that, The drying time is 5-24 hours.

12. The preparation method according to claim 4, characterized in that, The concentration of the crystal in the reaction medium is 0.5-1.2 times its solubility in the reaction medium at the reaction temperature.

13. The preparation method according to claim 4, characterized in that, The concentration of the crystal in the reaction medium is 0.8-1 times its solubility in the reaction medium at the reaction temperature.

14. The preparation method according to claim 4, characterized in that, The purity of the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystals is ≥95%.

15. The preparation method according to claim 4, characterized in that, The N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystals prepared therefrom are the crystals described in any one of claims 1-3.

16. A pharmaceutical composition, characterized in that, The crystals contain N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate as described in any one of claims 1-3.

17. The pharmaceutical composition according to claim 16, wherein, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

18. The pharmaceutical composition according to claim 16, wherein, The mass fraction of the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystals is 10%-50%.

19. The pharmaceutical composition according to claim 16, wherein, The pharmaceutical composition is in the form of tablets, capsules, or pills.

20. Use of the N'-[(3-allyl-2-hydroxyphenyl)methylene]-2-(4-benzyl-1-piperazinyl)acetylhydrazine tartrate crystal according to any one of claims 1-3 in the preparation of a medicament for treating tumors; The tumor is lung cancer, liver cancer, colon cancer, or melanoma.

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