Salts of ponabulin and uses thereof

Abstract

The invention discloses a salt of ponabulin and application thereof, relates to the field of medicinal chemistry, and solves the problem that the curative effect is influenced by poor water solubility of ponabulin in the prior art. The invention provides a plurality of ponabulin salts, the salts are formed by ponabulin and inorganic acid or organic acid, and the structure of the ponabulin is shown as a formula I. The invention further provides a preparation method of the ponabulin salt. In addition, the invention finds that the solubility of p-toluenesulfonate and 1, 5-naphthalene disulfonate of ponabulin in water is remarkably improved by more than 2000 times compared with free alkali, so that the requirement that a solubilizer is not added or a few solubilizers are added in the injection preparation process can be met, and potential harm to human health in the long-term use process of the solubilizers can be prevented. Pharmacodynamic tests show that the salt of the ponabulin has good anti-tumor activity and an effect of relieving neutropenia, and is suitable for preparing anti-tumor drugs. # imgabs0 #

Description

[0001] Related applications: This application claims priority to the invention patent with the invention name “Salts of Plinabulin and Uses Thereof” and patent number CN202210448071.2 filed on April 27, 2022, and is hereby expressly incorporated herein by reference. Technical Field

[0002] The invention relates to the field of pharmaceutical chemistry, and in particular to a salt of Plinabulin and a crystal form and use thereof. Background Art

[0003] Plinabulin, chemical name is (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione), and its structure is shown in Formula I:

[0004] Plinabulin is a tubulin binder, a synthetic derivative of the low molecular weight cyclic dipeptide phenylahistin or halimide from marine Aspergillus. It can bind to the vicinity of the colchicine binding site of tubulin and has good anti-tumor activity, especially in non-small cell lung cancer, but has poor solubility and low oral bioavailability. Currently, Plinabulin has entered Phase III clinical studies, and the preparation used is a concentrated solution for injection: 40% Solutol HS-15 / 60% propylene glycol as a solution, an amber vial containing 80 mg of the drug, and a capacity of 20 mL. In clinical practice, a large amount of solubilizer Solutol HS-15 is added to increase the solubility of the compound. However, combined injection with a solubilizer and long-term use may cause unexpected safety issues or affect the patient's quality of life.

[0005] In order to improve the water solubility of Plinabulin, Hayashi et al. designed and synthesized a series of water-soluble prodrugs of Plinabulin, which can be hydrolyzed to release Plinabulin under the action of esterase. However, the activation of prodrugs requires the participation of enzymes. The enzymes that activate prodrugs may have genetic polymorphisms, resulting in differences between different subjects; in addition, drug-drug interactions may also occur. Usually, prodrug strategies are only considered when the existing pharmacophores cannot achieve the required pharmaceutical and pharmacokinetic properties. In addition, since the structure of Plinabulin is too rigid, even through means such as prodrugs or their pharmaceutically acceptable salts, it is difficult to predict the possibility of success in improving solubility and the possibility of preparation, which undoubtedly brings greater challenges to the screening of target molecules. Therefore, how to improve the water solubility of Plinabulin without affecting or even optimizing its efficacy has become a problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] One of the purposes of the present invention is to provide a stable, highly soluble and highly pure (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazine dione salt, so as to solve the problem of poor water solubility of Plinabulin in the prior art.

[0007] The second object of the present invention is to provide the application of the salt.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The present invention provides a salt of Plinabulin, the structure of which is shown in Formula I, and the salt is formed by Plinabulin and an inorganic acid or an organic acid; .

[0009] In some embodiments, the inorganic acid is selected from hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, carbonic acid, phosphoric acid or phosphorous acid; the organic acid is selected from methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, naphthalenesulfonic acid, trifluoroacetic acid, fumaric acid, tartaric acid, oxalic acid, malic acid, citric acid, citric acid, glucuronic acid, succinic acid, maleic acid, ascorbic acid, benzoic acid, salicylic acid, caffeic acid, phthalic acid, terephthalic acid, acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, suberic acid, mandelic acid, galactonic acid or amino acid; Preferably, the organic acid is selected from methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid or naphthalenesulfonic acid; more preferably p-toluenesulfonic acid or 1,5-naphthalene disulfonic acid.

[0010] In some embodiments, the salt is selected from the following compounds: , , , , , or .

[0011] In some embodiments, the salt is a p-toluenesulfonate salt represented by Formula V: .

[0012] In some embodiments, the present invention also provides a crystalline form A of the p-toluenesulfonate salt represented by the above formula V, which uses Cu-Ka radiation and has characteristic peaks in X-ray powder diffraction expressed in 2θ angles at 6.22±0.2°, 6.91±0.2°, 17.93±0.2°, 18.43±0.2°, 22.56±0.2°, and 22.90±0.2°.

[0013] Preferably, the above-mentioned form A uses Cu-Ka radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 6.22±0.2°, 6.91±0.2°, 7.31±0.2°, 7.47±0.2°, 15.04±0.2°, 17.93±0.2°, 18.43±0.2°, 22.56±0.2°, 22.90±0.2°, 23.26±0.2°, and 23.63±0.2°.

[0014] Further preferably, the above-mentioned form A uses Cu-Ka radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 6.22±0.2°, 6.91±0.2°, 7.31±0.2°, 7.47±0.2°, 11.74±0.2°, 11.98±0.2°, 12.34±0.2°, 15.04±0.2°, 17.93±0.2°, 18.43±0.2°, 22.56±0.2°, 22.90±0.2°, 23.26±0.2°, 23.63±0.2°, 24.20±0.2°, and 24.56±0.2°.

[0015] Furthermore, the above-mentioned crystal form A, using Cu-Ka radiation, X-ray powder diffraction includes the following positions and relative peak intensities represented by 2θ angles: 2θ <![CDATA[Relative peak intensity (I / I0)]]> 6.22±0.2° 69.9±5% 6.91±0.2° 100.0±5% 17.93±0.2° 25.9±5% 18.43±0.2° 35.9±5% 22.56±0.2° 44.7±5% 22.90±0.2° 37.6±5% .

[0016] Furthermore, the above-mentioned crystal form A, using Cu-Ka radiation, X-ray powder diffraction includes the following positions and relative peak intensities represented by 2θ angles: 2θ <![CDATA[Relative peak intensity (I / I0)]]> 6.22±0.2° 69.9±5% 6.91±0.2° 100.0±5% 7.31±0.2° 63.3±5% 7.47±0.2° 49.9±5% 15.04±0.2° 28.4±5% 17.93±0.2° 25.9±5% 18.43±0.2° 35.9±5% 22.56±0.2° 44.7±5% 22.90±0.2° 37.6±5% 23.26±0.2° 47.6 ±5% 23.63±0.2° 42.4±5% .

[0017] Preferably, the X-ray powder diffraction pattern of the above-mentioned crystal form A is as follows Figure 1 shown.

[0018] In some embodiments, the melting point of the above-mentioned crystal form A is 305°C~306°C.

[0019] In some embodiments, the salt is a 1,5-naphthalene disulfonate salt represented by formula IV: .

[0020] In some embodiments, the present invention also provides a crystalline form I of the 1,5-naphthalene disulfonate salt shown in the above formula IV, which uses Cu-Ka radiation and has characteristic peaks in X-ray powder diffraction expressed in 2θ angles at 12.52±0.2°, 15.31±0.2°, 17.14±0.2°, 18.62±0.2°, 20.75±0.2°, 22.83±0.2°, and 25.84±0.2°.

[0021] Preferably, the above-mentioned form I uses Cu-Ka radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 12.52±0.2°, 15.31±0.2°, 17.14±0.2°, 17.60±0.2°, 18.62±0.2°, 20.75±0.2°, 21.31±0.2°, 22.35±0.2°, 22.83±0.2°, 25.06±0.2°, and 25.84±0.2°.

[0022] Further preferably, the above-mentioned Form I uses Cu-Ka radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 6.20±0.2°, 7.96±0.2°, 8.63±0.2°, 12.52±0.2°, 15.31±0.2°, 16.31±0.2°, 17.14±0.2°, 17.60±0.2°, 18.62±0.2°, 20.75±0.2°, 21.31±0.2°, 22.35±0.2°, 22.83±0.2°, 23.48±0.2°, 25.06±0.2°, and 25.84±0.2°.

[0023] Furthermore, the above-mentioned Form I, using Cu-Ka radiation, X-ray powder diffraction includes the following positions and relative peak intensities represented by 2θ angles: 2θ <![CDATA[Relative peak intensity (I / I0)]]> 12.52±0.2° 76.3±5% 15.31±0.2° 55.5±5% 17.14±0.2° 61.2±5% 18.62±0.2° 60.7±5% 20.75±0.2° 51.6±5% 22.83±0.2° 100.0±5% 25.84±0.2° 61.7±5% .

[0024] Furthermore, the above-mentioned Form I, using Cu-Ka radiation, X-ray powder diffraction includes the following positions and relative peak intensities represented by 2θ angles: 2θ <![CDATA[Relative peak intensity (I / I0)]]> Optionally 6.20±0.2° 16.7±5% Optional 7.96±0.2° 17.9±5% Optionally 8.63±0.2° 27.3±5% 12.52±0.2° 76.3±5% 15.31±0.2° 55.5±5% 17.14±0.2° 61.2±5% Optionally 17.60±0.2° 39.7±5% 18.62±0.2° 60.7±5% 20.75±0.2° 51.6±5% Optionally 21.31±0.2° 13.9±5% Optionally 22.35±0.2° 47.6±5% 22.83±0.2° 100.0±5% 25.84±0.2° 61.7±5% Optionally 25.06±0.2° 28.3±5% .

[0025] Preferably, the X-ray powder diffraction pattern of the above-mentioned crystal form I is as follows: Figure 2 shown.

[0026] In some embodiments, the melting point of the above-mentioned Form I is greater than 330°C.

[0027] The invention provides a method for preparing the salt, which comprises the steps of stirring Plinabulin and an inorganic acid or an organic acid in an organic solvent for reaction, filtering, washing and drying to obtain the salt.

[0028] In some embodiments, the inorganic acid in the preparation method of the above-mentioned salt is selected from hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, carbonic acid, phosphoric acid or phosphorous acid; the above-mentioned organic acid is selected from methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, naphthalenesulfonic acid, trifluoroacetic acid, fumaric acid, tartaric acid, oxalic acid, malic acid, citric acid, citric acid, glucuronic acid, succinic acid, maleic acid, ascorbic acid, benzoic acid, salicylic acid, caffeic acid, phthalic acid, terephthalic acid, acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, suberic acid, mandelic acid, galactonic acid or amino acid; preferably, the above-mentioned organic acid is selected from methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid or naphthalenesulfonic acid; more preferably, p-toluenesulfonic acid or 1,5-naphthalene disulfonic acid.

[0029] In some embodiments, the organic solvent in the above-mentioned method for preparing the salt includes, but is not limited to, tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, ethyl acetate or dichloromethane.

[0030] The present invention also provides a method for preparing the crystalline form A of the p-toluenesulfonate salt represented by the above formula V, comprising the following steps: stirring Plinabulin and p-toluenesulfonic acid in an organic solvent for reaction, filtering, washing, and drying to obtain the crystalline form A.

[0031] In some embodiments, the preparation method of the above-mentioned crystal form A includes: taking Plinabulin, adding an organic solvent to dissolve it, dropping p-toluenesulfonic acid monohydrate dissolved in the organic solvent, stirring at room temperature for 10 to 40 minutes, filtering, washing with an organic solvent, and drying to obtain.

[0032] In some embodiments, in the preparation method of the above-mentioned crystalline form A, the molar mass ratio of Plinabulin to p-toluenesulfonic acid is 1:(0.5-3); preferably 1:(0.5-1.5); more preferably 1:1.

[0033] In some embodiments, the organic solvent in the preparation method of the above-mentioned crystal form A includes but is not limited to tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, ethyl acetate or dichloromethane.

[0034] The present invention also provides a method for preparing the crystalline form I of the 1,5-naphthalene disulfonate salt shown in the above formula IV, comprising the following steps: stirring Plunabulin and 1,5-naphthalene disulfonic acid in an organic solvent for reaction, filtering, washing, and drying to obtain the crystalline form I.

[0035] In some embodiments, the preparation method of the above-mentioned crystal form I includes: adding plinabulin and 1,5-naphthalene disulfonic acid to tetrahydrofuran, stirring at room temperature for 0.5 to 2 hours, filtering, washing with an organic solvent, and drying to obtain.

[0036] In some embodiments, the molar mass ratio of Plinabulin to 1,5-naphthalene disulfonic acid in the preparation method of the above-mentioned crystal form I is 1: (0.1~2); preferably 1: (0.2~1.0); more preferably 1: (0.5~0.8).

[0037] In some embodiments, the organic solvent in the preparation method of the above-mentioned Form I includes but is not limited to tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, ethyl acetate or dichloromethane.

[0038] The present invention also provides use of any of the above salts or crystal forms in the preparation of anti-tumor drugs, preferably in the preparation of drugs for non-small cell lung cancer, pancreatic cancer, skin cancer or leukemia.

[0039] Furthermore, the present invention provides the use of any of the above salts or crystal forms in the preparation of a drug for alleviating chemotherapy bone marrow suppression and / or reducing neutropenia.

[0040] The present invention also provides a pharmaceutical composition comprising any of the above salts or crystal forms, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0041] In some embodiments, the above-mentioned pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable additives.

[0042] In some embodiments, the above-mentioned additives include but are not limited to fillers, disintegrants, lubricants, solubilizers, adhesives, diluents, glidants, etc. The dosage form of the above-mentioned pharmaceutical composition includes but is not limited to injections, capsules, tablets, granules, nanoformulations, etc. The preparation of the above-mentioned pharmaceutical composition can be obtained by using the common knowledge of those skilled in the art.

[0043] In some embodiments, the pharmaceutical composition contains an effective amount of any salt or crystalline form, and calculated as the free base of Formula I, the effective amount includes but is not limited to 1-120 mg, 1-110 mg, 1-100 mg, 1-90 mg, 1-80 mg, 1-70 mg, 1-60 mg, 10-50 mg, 10-60 mg, 10-70 mg, 10-80 mg, 10-90 mg, 10-10 0mg, 20~40mg, 20~50mg, 20~60mg, 20~70mg, 20~80mg, 20~90mg, 20~100mg, 30~40mg, 30~50m g, 30~60mg, 30~70mg, 30~80mg, 30~90mg, 30~100mg, 40~50mg, 40~60mg, 40~70mg, 40~80mg, 40~90mg, 40~100mg, 50~60mg, 50~70mg, 50~80mg, 50~90mg, 50~100mg, 60~70mg, 60~80mg, 6 0~90mg, 60~100mg, 70~80mg, 70~90mg, 70~100mg, 80~90mg, 80~100mg, 90~100mg, 1~10mg, 1 ~30mg, 1~40mg, 1~50mg, 25~35mg, 1mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg , 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 120mg.

[0044] Compared with the prior art, the present invention has the following beneficial effects: After a large number of experimental studies, the inventor unexpectedly found that the p-toluenesulfonate and 1,5-naphthalene disulfonate of the present invention have a significant improvement in solubility in water relative to the free base, which is greater than 2000 times, and can meet the requirements of adding no or less solubilizer in the preparation of injections, reducing the potential harm to human health during the long-term use of the solubilizer; and pharmacodynamic tests show that the salt of Plinabulin also has good anti-tumor activity and the effect of alleviating neutropenia. In addition, the p-toluenesulfonate and 1,5-naphthalene disulfonate of the present invention have good hygroscopicity, and show better stability than the free base in stability tests under high temperature, high humidity and strong light, especially solving the problem of photosensitivity stability of the free base, providing a more advantageous choice for clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The X-ray powder diffraction pattern of Plunabulin p-toluenesulfonate.

[0046] Figure 2 This is the X-ray powder diffraction pattern of Plunabulin 1,5-naphthalene disulfonate. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with embodiments and test examples. The embodiments and test examples of the present invention are only used to illustrate the technical scheme of the present invention, and are not intended to limit the present invention. Any equivalent substitutions in the art made in accordance with the contents disclosed in the present invention shall fall within the protection scope of the present invention.

[0048] The compounds and salts of the present invention can be prepared by selecting the synthetic routes of the embodiments, and the conventional conditions of the reaction raw materials and the reaction solvent can be adjusted according to the needs of salt formation, which can be realized by those skilled in the art based on the disclosure of the present invention. In addition, the column chromatography of the present invention refers to silica gel column chromatography unless otherwise specified, and the elution solvent can be determined as a single or mixed elution solvent in combination with the reaction solvent and the common knowledge or common means of those skilled in the art unless otherwise specified.

[0049] The structures of the compounds were determined by nuclear magnetic resonance ( 1 H NMR) or liquid chromatography-mass spectrometry (LC-MS).

[0050] The liquid chromatography-mass spectrometer (LC-MS) was Agilent G6120B (used with Agilent 1260 liquid phase); the nuclear magnetic resonance instrument ( 1 HNMR) is Bruker AVANCE~400 or Bruker AVANCE~800, NMR ( 1 H NMR) shift ( δ ) is given in parts per million (ppm), the solvent used for determination is DMSO or CDCl3, the internal standard is tetramethylsilane (TMS), and the chemical shift is 10 ~6 (ppm) is given as the unit.

[0051] The term "room temperature" in the present invention means a temperature between 10 and 30°C.

[0052] Comparative Example: Plunabulin: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione

[0053] Step 1: Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione Take 69.2g (349mmol) N,N-diacetylpiperazine-2,5-dione and 26.6g (175mmol) 5-(tert-butyl)-1H-imidazole-4-carboxaldehyde, add them to 500ml DMF, replace with argon, add 85.4g (262mmol) cesium carbonate, replace with argon, and react at room temperature in the dark overnight. The next day, pour the reaction solution into 3L water, stir for 15 minutes, filter, wash the filter cake with 200ml water, 200ml ethyl acetate: petroleum ether = 1:5, and vacuum dry to obtain 24.3g yellow solid, with a yield of 47.8%.

[0054] Step 2: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione Take 23.5g (81mmol) of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazole-4-yl)methylene)piperazine-2,5-dione and 17.2g (162mmol) of benzaldehyde, add them to 250ml of DMF, replace with argon, add 39.6g (121.5mmol) of cesium carbonate, replace with argon, protect from light, slowly heat to 80℃ and stir to react for 5-6 hours, TLC (DCM:MeOH=10:1) tracks the completion of the reaction, cool to room temperature, pour the reaction solution into 1.5L of water, stir for 15 minutes, filter, and wash the filter cake with 200ml of water, 200ml Ethyl acetate: petroleum ether = 1:5, washed, and vacuum dried to obtain 27 g of crude product, which was dissolved in 2 L of ethyl acetate and ethanol, filtered to remove insoluble matter, and the filtrate was evaporated to dryness, 150 ml of ethyl acetate and 150 ml of petroleum ether were added, slurried at room temperature for 2 hours, filtered, and the filter cake was dried to obtain 23.4 g of yellow solid, with a yield of 86.0% and a purity of 97.82%.

[0055] ESI-MS: m / z = 337.2(M+H) + .

[0056] 1 H NMR (400 MHz, CDCl3) δ1.45 (s, 9H),7.00 (s, 1H), 7.02 (s, 1H),7.30-7.50 (m, 5H), 7.59 (s, 1H), 8.11 (s, 1H), 9.60 (s, 1H), 12.40 (s, 1H).

[0057] Example 1: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione p-toluenesulfonate (compound of formula V)

[0058] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione, add 20ml of tetrahydrofuran to dissolve, add dropwise 566mg (2.98mmol) of p-toluenesulfonic acid monohydrate dissolved in 5ml of tetrahydrofuran, stir at room temperature for 30 minutes, filter, wash with ethyl acetate, and dry to obtain 1.5g of light yellow solid (compound of formula V), with a yield of 95.5% and a purity of 99.7%.

[0059] Melting point: 305℃~306℃.

[0060] ESI-MS: m / z = 337.2(M+H) + .

[0061] 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.14 (s, 1H),11.50 (s, 1H), 10.27 (s, 1H),8.47 (s, 1H), 7.59 – 7.50 (d, 2H), 7.50 – 7.45 (d, 2H), 7.45 – 7.40 (t, 2H),7.38 – 7.29 (t, 1H), 7.15 – 7.07 (d, 2H), 6.79 (s, 1H), 6.71 (s, 1H), 2.29 (s, 3H), 1.36 (s, 9H).

[0062] X-ray powder diffraction using Cu-Ka radiation is as Figure 1 shown.

[0063] Example 2: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione-1,5-naphthalene disulfonate (Formula IV compound)

[0064] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione and 429mg (1.49mmol) of 1,5-naphthalene disulfonic acid, add them into 25ml of tetrahydrofuran, stir at room temperature for 1 hour, filter, wash with ethyl acetate, and dry to obtain 1.3g of off-white solid (compound of formula IV), with a yield of 91.5% and a purity of 99.6%.

[0065] Melting point: greater than 330°C.

[0066] ESI-MS: m / z = 337.2(M+H) + .

[0067] 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.21 (s, 1H), 11.42(s, 1H), 10.29 (s,1H), 9.07 – 8.57 (d, 1H), 8.55 (s, 1H), 8.09 – 7.77 (d, 1H), 7.62 – 7.51 (d,2H), 7.48 – 7.36 (m, 3H), 7.37 – 7.29 (t, 1H), 6.80 (s, 1H), 6.68 (s, 1H), 1.39 – 1.33 (s, 9H).

[0068] X-ray powder diffraction using Cu-Ka radiation is as Figure 2 shown.

[0069] Example 3: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione methanesulfonate (compound of formula II)

[0070] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione, add 20ml of tetrahydrofuran to dissolve, add dropwise 286mg (2.98mmol) of methanesulfonic acid dissolved in 5ml of tetrahydrofuran, stir at room temperature for 30 minutes, filter, wash with ethyl acetate, and dry to obtain 1.2g of light yellow solid (compound of formula II), with a yield of 93.0% and a purity of 99.8%.

[0071] ESI-MS: m / z = 337.2(M+H) + .

[0072] 1 H NMR (400 MHz, DMSO- d 6) δ 13.21 (s, 1H),11.60 (s, 1H), 10.37 (s, 1H),8.51 (s, 1H), 7.55 -7.31 (m, 5H), 6.81 (s, 1H), 6.71 (s, 1H), 3.11 (s,3H),1.38 (s,9H).

[0073] Example 4: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedionebenzenesulfonate (Formula III compound)

[0074] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione, add 20ml of tetrahydrofuran to dissolve, drop 471mg (2.98mmol) of benzenesulfonic acid dissolved in 5ml of tetrahydrofuran, stir at room temperature for 30 minutes, filter, wash with ethyl acetate, and dry to obtain 1.3g of light yellow solid (compound of formula III), with a yield of 88.4% and a purity of 99.2%.

[0075] ESI-MS: m / z = 337.2(M+H) + .

[0076] 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.84 (s, 1H),11.20 (s, 1H), 10.17 (s, 1H),8.41 (s, 1H), 7.60 -7.45 (m, 5H), 7.45 – 7.05 (m, 5H), 6.77 (s, 1H), 6.68 (s,1H), 1.41 (s,9H).

[0077] Example 5: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione trifluoroacetate (compound of formula VI)

[0078] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione, add 20ml of tetrahydrofuran to dissolve, drop 339mg (2.97mmol) of trifluoroacetic acid dissolved in 5ml of tetrahydrofuran, stir at room temperature for 30 minutes, filter, wash with ethyl acetate, and dry to obtain 1.2g of light yellow solid (compound of formula VI), yield, purity 99.9%.

[0079] ESI-MS: m / z = 337.2(M+H) + .

[0080] 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.35 (s, 1H), 11.68 (s, 1H), 10.47 (s, 1H), 8.21 (s, 1H), 7.60 -7.41 (m, 5H), 6.89 (s, 1H), 6.75 (s, 1H), 1.39 (s, 9H).

[0081] Example 6: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione hydrochloride (Formula VII compound)

[0082] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione, add 20ml of tetrahydrofuran to dissolve, add dropwise 1ml of 4M hydrogen chloride dioxane solution, stir at room temperature for 30 minutes, filter, wash with ethyl acetate, and dry to obtain 1.0g of yellow solid (compound of formula VII), with a yield of 90.1% and a purity of 99.5%.

[0083] ESI-MS: m / z = 337.2(M+H) + .

[0084] 1 H NMR (400 MHz, DMSO- d 6) δ 12.85 (s, 1H), 11.35 (s, 1H), 10.56 (s, 1H), 8.28 (s, 1H), 7.59 -7.35 (m, 5H), 6.85 (s, 1H), 6.71 (s, 1H), 1.39 (s, 9H).

[0085] Example 7: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione hydrobromide (Formula VIII compound)

[0086] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione, add 20ml of tetrahydrofuran to dissolve, drop 502mg (2.98mmol) of 48% hydrobromic acid aqueous solution dissolved in 5ml of tetrahydrofuran, stir at room temperature for 30 minutes, filter, wash with ethyl acetate, and dry to obtain 1.0g of yellow solid (compound of formula VIII), with a yield of 80.6% and a purity of 99.3%.

[0087] ESI-MS: m / z = 337.2(M+H) + .

[0088] 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.88 (s, 1H),10.88 (s, 1H), 10.17 (s, 1H),8.41 (s, 1H), 7.55 -7.32(m, 5H), 6.81 (s, 1H), 6.69 (s, 1H),1.36 (s,9H).

[0089] Example 8: Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione sulfate (Formula IX compound)

[0090] Take 1g (2.97mmol) of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione, add 20ml of tetrahydrofuran to dissolve, drop 146mg (1.47mmol) of concentrated sulfuric acid dissolved in 5ml of tetrahydrofuran, stir at room temperature for 30 minutes, filter, wash with ethyl acetate, and dry to obtain 1.0g of yellow solid (compound of formula IX), with a yield of 87.0% and a purity of 99.3%.

[0091] ESI-MS: m / z = 337.2(M+H) + .

[0092] 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.12 (s, 1H), 11.15 (s, 1H), 10.35 (s, 1H), 8.22 (s, 1H), 7.52 -7.28 (m, 5H), 6.83 (s, 1H), 6.68 (s, 1H), 1.42 (s, 9H).

[0093] Test Example 1: Determination of equilibrium solubility Weigh appropriate amounts of compounds and dissolve them in DMSO to prepare a 1 mg / mL stock solution. Dilute the stock solution with methanol to different concentrations to prepare a standard curve for the samples.

[0094] Take a 1.5mL brown EP tube and add 1mL of ultrapure water. Then take an appropriate amount of the compound, add it to the brown EP tube, vortex and oscillate, and sonicate until the drug no longer dissolves (the solution is turbid or has suspended particles). Put it in an incubation oscillator, maintain the temperature at (37±1)℃, 100r / min, and shake for 24h to achieve full dissolution equilibrium. After 24h, take out the saturated solution, take the supernatant and quickly filter it with a 0.45um microporous filter membrane, discard the initial filtrate, and take the subsequent filtrate and dilute it by half with methanol. Prepare 3 samples in parallel for each group. Inject according to the chromatographic conditions, measure the peak area and calculate the equilibrium solubility of each compound in pure water. The test results are shown in Table 1.

[0095]

[0096] The results show that not all salts can greatly improve the solubility of Plinabulin. The water solubility of Plinabulin p-toluenesulfonate (compound of formula V) and Plinabulin naphthalene disulfonate (compound of formula IV) is more than 2000 times higher than that of the free base Plinabulin.

[0097] Experimental Example 2: Study on Adriamycin-induced Neutropenia Animals and groups: Male Sprague-Dawley rats were divided into 7 groups, namely: blank control group, model group, comparative example group, Example 1 group, Example 2 group, Example 6 group, and Example 8 group; each group included 10 rats.

[0098] Modeling: Rats in the model group, comparative example group, example 1 group, example 2 group, example 6 group, and example 8 group were intravenously administered with doxorubicin (3.0 mg / kg); the blank control group was intravenously administered with the same volume of vehicle, which contained 7.1% Tween 80 (v / v), 25.5% propylene glycol (v / v) and 67.4% D5W (v / v).

[0099] One hour after modeling, the rats in each group were intraperitoneally injected with the same mole of the corresponding drugs, and the control group 1 (Plinabulin 7.5 mg / kg) and the other groups were calculated with the same mole of each drug. The model group and the blank control group were intraperitoneally injected with the same volume of normal saline.

[0100] Blood samples were collected from rats in each group on the 1st day before administration, the 2nd day, the 3rd day, the 5th day and the 8th day to measure the number of neutrophils (ANC) in peripheral blood. The results are shown in Table 2: .

[0101] Note: *Statistically significant compared with the model group alone (P < 0.05); # There was statistical significance compared with the blank control group (P<0.05).

[0102] As shown in the table above, ANC decreased after a duration of at least 8 days of doxorubicin administration, and an increase in ANC was noted in all groups 24 hours after doxorubicin administration. The change was rapidly normalized in the group treated with the blank control group. Compared with the blank control group, the model group had P<0.05, indicating that the doxorubicin-induced neutropenia model was successfully established. Compared with the model group, the comparative group, the example 1 group, the example 2 group, and the example 6 group had P<0.05, indicating that the formula V compound of Example 1, the formula IV compound of Example 2, and the formula VII compound of Example 6, like the comparative example compound, all had the effect of significantly alleviating neutropenia, and the effect of the example compound of the present invention on increasing neutrophils was also slightly better than that of the comparative example compound, proving that the compound of the present invention has the potential to treat neutropenia-related diseases. The formula IX compound group of Example 8 had a smaller increase in neutrophils than the model group, and the neutrophils at each time point were also far below normal levels, proving that the effect of the formula IX compound on alleviating neutropenia was very limited, far inferior to the comparative example compound.

[0103] Test Example 3: Moisture absorption study 1. Test samples: Comparative Example, Example 1, Example 2.

[0104] 2. The specific test methods are as follows: (1) Take a dry stoppered glass weighing bottle (outer diameter 50 mm, height 15 mm) and place it in a suitable 25℃±1℃ constant temperature desiccator (with ammonium chloride or ammonium sulfate saturated solution placed at the bottom) or an artificial climate box (set temperature 25℃±1℃, relative humidity 80%±2%) one day before the test, and accurately weigh the weight (m1); (2) Take an appropriate amount of the test sample and spread it evenly in the above-mentioned weighing bottle. The thickness of the test sample is generally about 1 mm. Accurately weigh the weight (m2); (3) Open the weighing bottle and place it with the bottle cap under the above constant temperature and humidity conditions for 24 hours; (4) Cover the weighing bottle and accurately weigh the weight (m3); (5) Weight gain percentage = (m3-m2) / (m2-m1)×100%.

[0105] 3. Test results:

[0106] The results of hygroscopicity are shown in the following table: The results show that Examples 1 and 2 of the present invention have slight hygroscopicity, while the comparative example has no or almost no hygroscopicity.

[0107]

[0108] Test Example 4: Stability Study 1. Test samples: Comparative Example, Example 1, Example 2.

[0109] 2. The specific test methods are as follows: Take an appropriate amount of each sample and investigate the stability of the influencing factors according to the guiding principles of high temperature (60°C), high humidity (RH90%±5%), and strong light (45001x±5001x) in the 2020 edition of the Chinese Pharmacopoeia.

[0110] 3. Test results:

[0111] After investigating the influence of high temperature, high humidity and strong light, the content of each sample, calculated as free alkali, is statistically analyzed as follows: .

[0112] It can be seen that the comparative example compound has a certain stability under high temperature and high humidity conditions, but the content is greatly reduced under strong light conditions, the stability is poor, and it has high photosensitivity; while the compound of formula V in Example 1 of the present invention and the compound of formula IV in Example 2 have less content changes under high temperature, high humidity and strong light conditions, and have higher stability.

[0113] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A salt of Plenabulin, characterized in that The structure of Plunabulin is shown in Formula I: , the salt is formed by Plinabulin and an inorganic acid or an organic acid.

2. The salt according to claim 1, characterized in that The inorganic acid is selected from hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, carbonic acid, phosphoric acid or phosphorous acid; The organic acid is selected from methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, naphthalenesulfonic acid, trifluoroacetic acid, fumaric acid, tartaric acid, oxalic acid, malic acid, citric acid, citric acid, glucuronic acid, succinic acid, maleic acid, ascorbic acid, benzoic acid, salicylic acid, caffeic acid, phthalic acid, terephthalic acid, acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, suberic acid, mandelic acid, galactonic acid or amino acid; Preferably, the organic acid is selected from methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid or naphthalenesulfonic acid; more preferably p-toluenesulfonic acid or 1,5-naphthalene disulfonic acid.

3. The salt according to claim 2, characterized in that The salt is selected from the following compounds: , , , , , or .

4. A crystalline form A of a p-toluenesulfonate salt of formula V, ， It is characterized in that The crystal form A has characteristic peaks at 6.22±0.2°, 6.91±0.2°, 17.93±0.2°, 18.43±0.2°, 22.56±0.2°, and 22.90±0.2° in X-ray powder diffraction expressed as 2θ angle using Cu-Ka radiation; Preferably, the crystal form A, using Cu-Ka radiation, X-ray powder diffraction comprises the following positions and relative peak intensities represented by 2θ angles: 。 5. The crystalline form A according to claim 4, characterized in that: The melting point of the crystal form A is 305°C to 306°C.

6. A crystalline form I of a 1,5-naphthalene disulfonate salt of formula IV, ， It is characterized in that The crystal form I has characteristic peaks at 12.52±0.2°, 15.31±0.2°, 17.14±0.2°, 18.62±0.2°, 20.75±0.2°, 22.83±0.2°, and 25.84±0.2° in X-ray powder diffraction expressed as 2θ angle using Cu-Ka radiation; Preferably, the crystal form I, using Cu-Ka radiation, has an X-ray powder diffraction pattern comprising the following positions and relative peak intensities represented by 2θ angles: 。 7. The crystalline form I according to claim 6, characterized in that The melting point of the crystalline form I is greater than 330°C.

8. Use of the salt according to any one of claims 1 to 3 or the crystal form according to any one of claims 4 to 7 in the preparation of anti-tumor drugs; preferably in the preparation of drugs for non-small cell lung cancer, pancreatic cancer, skin cancer or leukemia.

9. Use of the salt according to any one of claims 1 to 3 or the crystal form according to any one of claims 4 to 7 in the preparation of a drug for alleviating chemotherapy bone marrow suppression or reducing neutropenia.

10. A pharmaceutical composition comprising the salt according to any one of claims 1 to 3 or the crystalline form according to any one of claims 4 to 7, characterized in that: The composition further contains pharmaceutically acceptable excipients.

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