Crystal form of small molecule immune compound, preparation method thereof and pharmaceutical composition containing the same

The invention belongs to the technical field of medicine, and specifically relates to a new crystal form of a novel small molecule immune compound;

The invention also relates to the preparation method of the above new crystal form and the pharmaceutical composition containing the new crystal form of the compound.

As the main means of tumor treatment, anti-tumor drugs have made considerable contributions to prolonging the survival time of patients and improving their quality of life. Among them, the compound of the present invention, as a novel small molecule compound, can attach to the colchicine binding site of tubulin, thereby inhibiting the binding of tubulin. Studies have shown that it can be used in combination with docetaxel to increase efficacy and reduce toxicity, and can exert multiple effects in the tumor microenvironment, including: 1. directly inducing apoptosis of tumor cells by activating Caspase-3; 2. promoting dendritic cell maturation, thereby further promoting T cell-mediated tumor killing; 3. promoting the release of a series of cytokines that protect neutrophils from apoptosis.

Chinese patent application CN106565686 discloses (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl)methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene) piperazine-2,5-dione, the compound of formula (1). The compound is used to reduce vascular proliferation and vascular density, acts on tumor blood vessels, and has an anticancer effect.

An object of the present invention is to provide a new crystal form of the above compound, namely crystal form B of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl)methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene)piperazine-2,5-dione (as shown in formula (1));

The crystal form B is characterized by melting point, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TG), infrared spectroscopy (IR), and elemental analysis. The crystal form has the properties required for the preparation of pharmaceutical preparations.

Another object of the present invention is to provide a method of preparing the new crystal form of the compound.

Yet another object of the present invention is to provide a pharmaceutical composition comprising the crystal form of the compound.

According to one aspect of the present invention, crude product of the compound (as shown in formula (1)) is first prepared, and then the crude product is crystallized by recrystallization to obtain the crystal form of the compound.

By melting point measurement, X-ray powder diffraction, DSC, TG, IR and elemental analysis of this crystal, it is confirmed that the obtained crystal is a new type of crystal, called crystal form B.

Specifically, when X-ray powder diffraction is performed with a Cu radiation source, the crystal form B indicates characteristic diffraction peaks at 2θ of 5.5±0.2°, 8.7±0.2°, 11.0±0.2°, 13.5±0.2°, 16.0±0.2°, 16.4±0.2°, and the relative intensities (I/I 0 ) of these peaks are greater than or equal to 30%. Furthermore, the X-ray powder diffraction pattern of the crystal may also comprise characteristic diffraction peaks at 2θ° of 18.5±0.2°, 21.9±0.2°, 22.1±0.2°, and the relative intensities of these peaks are greater than or equal to 15% (see FIG. 1 ).

Wherein “±0.2°” is the allowable measurement error range.

The crystal form B of the present invention can be characterized by X-ray powder diffraction pattern. Itis characterized in that its X-ray powder diffraction pattern has the above-mentioned characteristic diffraction peaks represented by 2θ°, as shown in Table 1, and their relative intensities are close to the following values.

The term “close” herein refers to the uncertainty of measurement value of the relative intensity. Those skilled in the art understand that the uncertainty of the relative intensity is very dependent on the measurement conditions. The relative intensity value may change, for example, within a range of ±25% or preferably within the range of ±10%.

The above-mentioned crystal form B has an X-ray powder diffraction pattern shown in FIG. 1 .

The present invention uses differential scanning calorimetry (DSC) technology to characterize the crystal form B (see FIG. 2 ), wherein the differential scanning calorimetry analysis results show that the test sample has no discernible endothermic or exothermic process in the detection temperature range (20-310° C.).

The present invention uses thermogravimetric analysis technology to characterize the crystal form B (see FIG. 3 ), wherein the thermogravimetric spectrum (TG) shows a weight loss of 0.87% from room temperature to 326° C., a weight loss of 39.8% from 326° C. to 436° C., a weight loss of 54.48% from 436° C. to 700° C., and a remaining amount of 0.85% at 700° C. This indicates that as the temperature increases, the compound degrades.

The infrared spectrum of the crystal form B of the compound of the present invention is shown in FIG. 4 , wherein it comprises relatively strong absorption peaks at 3175, 3060, 3007, 2969, 2905, 1691-1634, 1600, 1579, 1555, 1503, 1491, 1472, 1376, 1277, 956, 810, 775, and 681 cm −1 .

Element analysis data of crystal form B of the present invention are in agreement with the theoretical values (with difference within ±0.3%), which further confirms that the compound does not co-crystallize with other solvents (see Table 2).

According to another aspect of the present invention, the method of preparing the crystalline form B comprises: adding the crude product of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl)methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene) piperazine-2,5-dione into a mixed solvent of C1-C4 alkyl alcohol and C3-C4 alkyl ketone, heating under reflux to achieve dissolution; after the solution is clarified, cooling the solution to precipitate, filtrating, collecting the precipitate, and drying the collected precipitate with air blasts to obtain the crystal form B. The alkyl alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol and butanol, preferably ethanol; the ketone is selected from the group consisting of acetone, methyl ethyl ketone and n-butanone, etc., preferably acetone; the volume ratio (V/V) of alcohol to ketone is 5:1˜10:1, preferably 8:1; the ratio of the crude product to the solvent is, based on g/ml, 1:5˜20 by weight-volume ratio, preferably 1:10. The solution is preferably heated to 40˜80° C., more preferably, the mixed solvent of C1-C4 alkyl alcohol and C3-C4 alkyl ketone to 50° C.; according to this embodiment, the precipitation is carried out for 2˜8 hours, more preferably 4 hours. The precipitation temperature is 0˜40° C., preferably 10˜20° C. After the precipitation followed by filtration, the precipitate is dried at temperature 30˜60° C., preferably 50° C.

According to yet another aspect of the present invention, a pharmaceutical composition is provided, which comprises the novel crystal form of the compound and optionally a pharmaceutically acceptable carrier and/or excipient.

The pharmaceutical composition can be further formulated into forms for administration according to conventional formulation methods, including oral or parenteral administration forms. In a form available for administration, a therapeutically effective amount of the crystal form B should be included. The so-called “therapeutically effective amount” means that at this dose, the compound of the present invention can improve or alleviate the symptoms of a disease, or can inhibit or block the development of a disease.

Based on experience and considering standard methods and references in the art, those skilled in the art can easily select various carriers and/or excipients and determine their dosage.

According to yet another aspect of the present invention, the crystal form of the present invention may be used alone in the preparation of drugs for the treatment of hyperproliferative diseases, or it may be prepared and acts synergistically in combination with other therapeutic drugs.

The crystal form B of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl) methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene)piperazine-2,5-dione of the present invention may be used for treatment of hyperproliferative diseases, preferably cancer, including but not limited to non-small cell lung cancer, colorectal cancer, refractory non-small cell lung cancer, pancreatic cancer, ovarian cancer, breast cancer, neuroglioma, brain cancer, or neck cancer.

The compound is a cell cycle inhibitor, may be used as a tumor growth inhibitor or fungal inhibitor, and may be used to treat or prevent related diseases such as cancer, especially non-small cell lung cancer, colorectal cancer, refractory non-small cell lung cancer, ovarian cancer, pancreatic cancer, breast cancer, neuroglioma, brain tumor or neck cancer.

Beneficial Effect:

The new crystal form of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl) methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene)piperazine-2,5-dione obtained by the present invention has a stable morphology, good chemical stability, and high temperature resistance. This new crystal form of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl)methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene)piperazine-2,5-dione possesses the properties necessary for preparation of the formulation, and is easy to store. Its production operation is simpler, and the quality is easier to control.

FIG. 1 is an X-ray diffraction pattern of the new crystal form B obtained in Example 1 of the present invention;

FIG. 2 is a DSC pattern of the new crystal form B obtained in Example 1 of the present invention;

FIG. 3 is a TG pattern of the new crystal form B obtained in Example 1 of the present invention;

FIG. 4 is an IR spectrum of the new crystal form B obtained in Example 1 of the present invention;

FIG. 5 is a HPLC chromatogram of the new crystal form B obtained in Example 1 of the present invention.

All raw materials and reagents are purchased commercially.

Crude Product Preparation:

The crude product of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl) meth ylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene)piperazine-2,5-dione was prepared using 1,4-diacetylpiperazine-2,5-dione, 5-tert-butyl-1H-imidazole-4-carbaldehyde as starting materials, by referring to the method of the patent publication (CN 106565686).

200 g of crude product of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl) methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene)piperazine-2,5-dione was added to a reaction flask, added with 2000 ml of mixed solvent of ethanol and acetone (V/V=8:1), heated to reflux at 50° C. under stirring. After the crude product was dissolved, the solution was stirred for 10 min, cooled to 20˜25° C. to precipitate, and then stirred for 4 hours for crystallization, and filtrated. The filter cake was leached with acetone, and then dried with air blasts at 50° C. with assistance of phosphorus pentoxide. 182 g off-white solid was obtained with a yield of 91.0%, the water content of which was found to be 0.1% by Karl Fischer titrator. The obtained compound was crystal form B of (3Z,6Z)-3-[((E)-3-(5-tert-butyl)-1H-imidazolyl-4-yl)methylene]-6-((E)-3-(3-fluorophenyl)-2-propenylene)piperazine-2,5-dione.

The characteristics of the compound were identified as shown in FIGS. 1 to 5 .

Test conditions for the samples of the example:

(1) XRD:

Detection instrument: Empyrean X-ray diffractometer

Detection conditions: Cu target Kα ray, Voltage 40 kV, Current 40 mA, Divergence slit 1/32°, Anti-scatter slit 1/16°, Anti-scatter slit 7.5 mm, 2θ range: 3°-50°, Step size 0.02°, counting time: 40 s/step

Detection basis: 0451 X-ray powder diffraction method, Pharmacopoeia of the People's Republic of China (2015 Edition, Part IV)

Detection results: see FIG. 1 .

(2) DSC:

Detection instrument: DSC 204F1 differential scanning calorimeter, NETZSCH Company, Germany

Detection conditions: Atmosphere: N 2 , 40 ml/min

Scanning procedures: The temperature curve was recorded by heating from room temperature to 250° C. at a rate of 10° C./min

Detected sample quality: Sample 1: 2.48 mg (Aluminum sample tray)

Detection basis: General rules for thermal analysis JY/T 014-1996

Detection results: see FIG. 2 .

(3) TG:

Detection instrument: TG209 thermal gravimetric analyzer, NETZSCH company, Germany

Detection condition: Atmosphere: air, 20 ml/min; scanning procedures: room temperature˜800° C., heating rate: 10° C./min

Detection basis: General rules for thermal analysis JY/T 014-1996

Detection results: see FIG. 3 .

Infrared Spectrum:

Detection instrument: FT-IR NICOLET6700 (U.S.)

Detection condition: potassium bromide disc

Detection basis: General rules for infrared analysis GB/T 6040˜2002

Detection results: see FIG. 4 .

Detection instrument: Agilent 1260 series (U.S.)

Detection Conditions:

Chromatographic column: Waters Sunfire C18;

Mobile phase A: water-mobile phase acetonitrile B (80:20);

Column temperature: 40° C.; Detection wavelength: 390 nm.

Detection basis: High performance liquid chromatography method, Pharmacopoeia of the People's Republic of China, Part II Appendix VD

Detection results: see FIG. 5 .