Process for preparing crystalline form I of CHKI-03 compound

Abstract

The application discloses a preparation method of a crystal form I of CHKI-03 compound and belongs to the technical field of drug crystal forms. The technical problem to be solved is that the biological availability of the amorphous form of CHKI-03 compound is poor, and a new crystal form is needed to improve the biological availability of the CHKI-03 compound. The prior art does not disclose how to prepare the new crystal form of the CHKI-03 compound. The technical solution is that the preparation method of the crystal form I of the CHKI-03 compound comprises the following steps: adopting a volatile crystallization, a cooling crystallization or a reverse solvent precipitation method to crystallize to obtain the crystal form I. The X-ray powder diffraction pattern of the crystal form I comprises at least one characteristic peak in the 2theta value of 6.524+ / -0.2°, 20.142+ / -0.2° and 20.326+ / -0.2°.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical crystal form technology, specifically to a method for preparing crystal form I of the CHKI-03 compound. Background Technology

[0002] Choline kinase (ChoK) is a cytoplasmic enzyme and the first-step reactive enzyme in the choline metabolic pathway. It phosphorylates choline to produce phosphocholine, which then enters the Kennedy pathway for the synthesis of phosphatidylcholine (PtdCho), a highly abundant phospholipid in mammalian cell membranes. In choline metabolism, choline is first converted to phosphocholine (PCho), which then reacts with CTP to form 3-phosphocholine. Increased ChoK activity leads to increased PCho levels, and PCho acts as a second messenger in proliferation. PCho can be transferred to diacylglycerol to generate PtdCho, and this pathway is the main source of PtdCho. In mammals, the choline kinase family consists of two subtypes: choline kinase α (ChoKα) and choline kinase β (ChoKβ). ChoKα has been identified as a proto-oncogene that mediates human cell transformation and induces tumorigenesis in vivo.

[0003] Compound CHKI-03 is an N-methylaniline quinoline compound with Mw = 981.65, and its structural formula is shown in Formula I: .

[0004] Relevant patent documents retrieved: Chinese Patent Publication No. CN116178258A discloses the CHKI-03 compound and its synthesis method, and experimentally confirms that the CHKI-03 compound can act as a choline kinase (ChoK) inhibitor to inhibit ChoK activity. Furthermore, experiments have also demonstrated that the CHKI-03 compound can serve as an active ingredient in anticancer drugs, such as for inhibiting pancreatic tumor metastasis.

[0005] The prior art represented by the aforementioned literature has at least the following unresolved technical problems or defects: The CHKI-03 compound prepared by current synthetic methods is in an amorphous form, and its bioavailability needs to be further improved.

[0006] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: Obtaining high-purity CHKI-03 during its preparation is challenging and requires column chromatography. After column chromatography purification, the eluent is concentrated to obtain an amorphous product. Since the sample is in quaternary ammonium salt form, a highly polar solvent is needed for dissolution. During product development, we found it difficult to obtain a satisfactory solid form; some samples were oily, while others were semi-solid. Therefore, obtaining the crystalline form of CHKI-03 from the samples is quite difficult.

[0007] Therefore, it is essential to develop a method for preparing crystal form I of the CHKI-03 compound that can solve the above-mentioned technical problems. Summary of the Invention

[0008] The purpose of this invention is to provide: A method for preparing crystal form I of CHKI-03 compound, and related technologies thereof, to solve technical problems such as improving the bioavailability of CHKI-03 compound, or a combination thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] The definition of standard chemical terms can be found in the reference “Sun Jihui. Pharmaceutical Chemistry [M]. Beijing: People’s Medical Publishing House, 1986.11.”

[0012] Unless otherwise specified, conventional methods within the scope of the art, such as XRPD (X-ray powder diffraction pattern), TGA (thermogravimetric analysis), and DSC (differential scanning calorimetry), shall be used. 1 Methods such as H-NMR (hydrogen nuclear magnetic resonance spectroscopy).

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The CHKI-03 compound of the present invention is also known as the YAN-001 compound.

[0015] The terms "optional / arbitrary" or "optionally / arbitrarily" refer to events or situations described subsequently that may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below, "optionally 2θ value" means "at least one of the various 2θ values".

[0016] The term "at least one" includes various cases such as "at least one", "at least two", "at least three", or "at least four", etc.

[0017] This invention is achieved through the following technical solutions: In a first aspect, the present invention provides a method for preparing crystal form I of CHKI-03 compound, wherein the structure of CHKI-03 compound is shown in Formula I, and the preparation method includes the following steps: dissolving CHKI-03 compound in solvent A to form a clear liquid, and allowing it to evaporate and crystallize at room temperature, wherein the evaporation rate is 0.01-0.1 mL / h, and the solid obtained is crystal form I; .

[0018] In a preferred embodiment of the present invention, the evaporation rate is any value among 0.01 mL / h, 0.02 mL / h, 0.03 mL / h, 0.04 mL / h, 0.05 mL / h, 0.06 mL / h, 0.07 mL / h, 0.08 mL / h, 0.09 mL / h, and 0.1 mL / h, or a range between any two values, and is more preferably 0.042 mL / h.

[0019] In one embodiment of the present invention, solvent A includes at least one of methanol and ethanol.

[0020] In one embodiment of the present invention, the temperature of solvent A is 20-50°C.

[0021] In one embodiment of the present invention, the mass-to-volume ratio of the CHKI-03 compound to solvent A is 0.05-1 g: 1 mL.

[0022] Secondly, the present invention provides a method for preparing crystal form I of CHKI-03 compound, wherein the structure of CHKI-03 compound is shown in Formula I, and the preparation method includes the following steps: dissolving CHKI-03 compound in solvent B to form a clear liquid, cooling, and the precipitated solid is crystal form I.

[0023] In one embodiment of the present invention, solvent B is ethanol.

[0024] In one embodiment of the present invention, the temperature of solvent B is 45-50°C.

[0025] In one embodiment of the present invention, the temperature after cooling is 0-25°C, preferably 0-5°C.

[0026] In one embodiment of the present invention, the cooling time is 0.5-1.5 hours, preferably 1 hour.

[0027] Thirdly, the present invention provides a method for preparing crystal form I of CHKI-03 compound, wherein the structure of CHKI-03 compound is shown in Formula I, and the preparation method includes the following steps: dissolving CHKI-03 compound in solvent C to form a clear liquid, adding antisolvent D to the clear liquid, and the precipitated solid is crystal form I.

[0028] In one embodiment of the present invention, the solvent C includes at least one of DMF, ethanol and DMSO.

[0029] In one embodiment of the present invention, the antisolvent D includes at least one of water and n-heptane.

[0030] In one embodiment of the present invention, the volume ratio of solvent C to antisolvent D is 1:4-20, preferably 1:4-10.

[0031] In one embodiment of the present invention, the addition parameters of antisolvent D are as follows: The temperature of the antisolvent D is 10-30℃ and / or the addition rate is 5 mL / h; after adding the antisolvent D, stirring is continued at 10-30℃ for 2-48 h, preferably for 2-22 h.

[0032] Fourthly, the present invention provides a crystal form I of the CHKI-03 compound prepared by the above preparation method, wherein the X-ray powder diffraction pattern of the crystal form I includes at least one characteristic peak at 2θ values ​​of 6.524±0.2°, 20.142±0.2° and 20.326±0.2°.

[0033] As one embodiment of the present invention, the X-ray powder diffraction pattern of crystal form I includes at least one characteristic peak at 2θ values ​​of 6.524±0.2°, 18.096±0.2°, 20.142±0.2°, 20.326±0.2°, 22.105±0.2°, and 23.507±0.2°.

[0034] In one embodiment of the present invention, the X-ray powder diffraction pattern of crystal form I includes at least one characteristic peak at 2θ values ​​of 6.524±0.2°, 17.913±0.2°, 18.096±0.2°, 20.142±0.2°, 20.326±0.2°, 22.105±0.2°, 22.720±0.2°, 23.507±0.2°, and 25.466±0.2°.

[0035] In one embodiment of the present invention, the X-ray powder diffraction pattern of crystal form I includes at least one characteristic peak among 2θ values ​​of 6.524±0.2°, 14.402±0.2°, 17.913±0.2°, 18.096±0.2°, 20.142±0.2°, 20.326±0.2°, 22.105±0.2°, 22.720±0.2°, 23.507±0.2°, 24.351±0.2°, 25.265±0.2°, and 25.466±0.2°.

[0036] As one embodiment of the present invention, the X-ray powder diffraction pattern of crystal form I is basically as follows: Figure 2 As shown.

[0037] As one embodiment of the present invention, the differential scanning calorimetry of the crystal form I includes a single endothermic event with an initial temperature range of 145°C to 159°C.

[0038] In a preferred embodiment of the present invention, the differential scanning calorimetry (DSC) of crystal form I includes a single endothermic event with an initial temperature range of 145.1°C to 158.6°C.

[0039] As a further preferred embodiment of the present invention, the differential scanning calorimetry of crystal form I has a peak value of an endothermic peak at 150.8±3℃, such as 150.8℃, 150.8±0.5℃, 150.8±1℃, 150.8±1.5℃, 150.8±2℃, 150.8±2.5℃, and 150.8±3℃.

[0040] As one embodiment of the present invention, the differential scanning calorimetry of crystal form I further includes a single exothermic event having an initial temperature range of 222°C to 257°C.

[0041] In a preferred embodiment of the present invention, the differential scanning calorimetry of crystal form I further includes a single exothermic event with an initial temperature range of 222.5°C to 256.9°C.

[0042] As a further preferred embodiment of the present invention, the differential scanning calorimetry of crystal form I has a peak value of an exothermic peak at 241.8±3℃, such as 241.8℃, 241.8±0.5℃, 241.8±1℃, 241.8±1.5℃, 241.8±2℃, 241.8±2.5℃, and 241.8±3℃.

[0043] As one embodiment of the present invention, the differential scanning calorimetry (DSC) image of crystal form I is basically as follows: Figure 3 As shown.

[0044] As one embodiment of the present invention, the thermogravimetric analysis curve of crystal form I includes a weight loss of 1.858% at 105±3℃, for example, at 105℃, 105±0.5℃, 105±1℃, 105±1.5℃, 105±2℃, 105±2.5℃, and 105±3℃.

[0045] In a preferred embodiment of the present invention, the thermogravimetric analysis curve of crystal form I also includes a weight loss of 4.391% at 160±3℃, for example, at 160℃, 160±0.5℃, 160±1℃, 160±1.5℃, 160±2℃, 160±2.5℃, and 160±3℃.

[0046] As one embodiment of the present invention, the thermogravimetric analysis curve of crystal form I is basically as follows: Figure 4 As shown.

[0047] The beneficial effects of this invention are: Compared with the prior art, the present invention provides a crystal form of CHKI-03 compound and has better technical effects in terms of bioavailability.

[0048] According to experimental tests, after oral administration of 50 mg / kg of different forms of CHKI-03 to rats, only trace amounts of CHKI-03 were detectable in the plasma of amorphous rats at 0.25-0.5 h (close to the lower limit of quantitation), while at other time points, the levels were below the limit of detection. Compared to the amorphous group, the plasma concentration and in vivo exposure of CHKI-03 were significantly increased in the crystalline group I animals. The C60 of CHKI-03... max The value was 39.26 ± 5.09 ng / mL, T max The exposure time is 0.25-0.5 h, and the AUC in vivo is... 0-t The mean retention time (MRT) was 113.32 ± 31.11 ng / mL·h.(0-t) It is 2.99±0.56h. Attached Figure Description

[0049] Figure 1 The image shows the XRPD diagrams of the solids obtained by different methods of volatilization and crystallization in Example 1.

[0050] Figure 2 This is the XRPD diagram of crystal form I of the CHKI-03 compound.

[0051] Figure 3 This is the DSC diagram of crystal form I of the CHKI-03 compound.

[0052] Figure 4 This is a TGA image of crystal form I of the CHKI-03 compound.

[0053] Figure 5 For CHKI-03 compound crystal form I 1 H-NMR spectrum. Detailed Implementation

[0054] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0055] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0056] The CHKI-03 amorphous compound used in the embodiments of the present invention was prepared according to the steps disclosed in Example 1 of Chinese Patent CN116178258B to obtain crude CHKI-03, which was then purified by column chromatography to finally obtain CHKI-03 amorphous compound with a purity of 99.3% and batch number XZH-26-27-2.

[0057] The column chromatography method is as follows: Dissolve crude CHKI-03 in a 10:2 volume ratio of dichloromethane / methanol (denoted as 12 volumes, or 12V). Add silica gel with a weight twice that of the crude CHKI-03 and mix to obtain the sample to be chromatographically analyzed. Add silica gel with a weight twice that of the crude CHKI-03 to the chromatography column. Add the sample to be chromatographically analyzed to the column and elute with a 40:1 volume ratio of dichloromethane / methanol at 30V. Elute again with a 30:1 volume ratio of dichloromethane / methanol at 30V. Elute again with a 20:1 volume ratio of dichloromethane / methanol at 30V, and collect the eluent based on TLC determination. Finally, elute with a 10:1 volume ratio of dichloromethane / methanol at 60V, collect the eluent, combine the eluents containing the product, and concentrate to dryness below 45°C to obtain the amorphous CHKI-03 compound.

[0058] Chemical reagent abbreviations: DMSO: dimethyl sulfoxide; DMF: N,N-dimethylformamide; IPA: isopropanol; DCM: dichloromethane.

[0059] Example 1: Investigation of Volatile Crystallization (1) At room temperature, 50 mg of the starting material CHKI-03 amorphous compound was dissolved in 1 mL of DCM (with 1 drop of methanol) to form a clear solution. (2) At room temperature, 1 g of the starting material CHKI-03 amorphous compound was dissolved in 1 mL of methanol to form a clear solution. (3) At 50 °C, 0.2 g of the starting material CHKI-03 amorphous compound was dissolved in 1 mL of ethanol to form a clear solution. These three portions were dried with nitrogen at room temperature for rapid evaporation, and evaporation was completed after 1 h (evaporation rate 1 mL / h). Three other clear solutions were prepared in the same way and placed under a perforated film at room temperature for slow evaporation, and evaporation was completed after 1 day (evaporation rate 0.042 mL / h). After the evaporation and crystallization were completed, the solids were collected for XRPD characterization, and the results are shown in Table 1 and Figure 1 As shown.

[0060] Table 1 Results of the investigation on volatile crystallization

[0061] As can be seen from the above, the solid obtained by rapid evaporation is amorphous, and only by using a specific solvent with slow evaporation can crystal form I be obtained.

[0062] Example 2: Investigation of Cooling Crystallization 200 mg of the amorphous compound CHKI-03 was weighed into a sample vial and added to different solvents (1 mL or 2 mL, see Table 2). Methanol dissolved the compound completely at room temperature; after the solution clarified, it was cooled to 0°C over 1 hour. Ethanol dissolved the compound completely by stirring at 50°C for 10 minutes; after the solution clarified, it was cooled uniformly from 50°C to room temperature over 1 hour, and crystals precipitated. Isopropanol IPA remained insoluble even after heating to 50°C, and after adding 1 mL, it still did not dissolve, becoming gel-like; this was not further investigated. The precipitated solid was characterized by XRPD. The results are shown in Table 2.

[0063] Table 2 Results of Cooling Crystallization Investigation

[0064] As can be seen from the above, only by using ethanol for cooling crystallization can crystal form I be obtained.

[0065] Example 3: Investigation of Antisolvent Precipitation At room temperature, 500 mg of the amorphous compound CHKI-03 was dissolved in DMF (2.5 mL), DMSO (2.5 mL), methanol (1 mL), and ethanol (2.5 mL), respectively, and filtered to obtain clear solutions. The antisolvents, water (10 mL), water (10 mL), n-heptane (10 mL), and n-heptane (10 mL), were added dropwise to the obtained clear solutions, respectively. The precipitated solids were characterized by XRPD. The results are shown in Table 3.

[0066] Table 3 Results of antisolvent precipitation investigation

[0067] Note: "Wet sample - 2h" refers to the result of solid detection of the wet sample obtained after stirring for 2 hours after solid precipitation; "Wet sample-22h" indicates the result of solid analysis of the wet sample obtained after stirring for 22 hours after solid precipitation; “N / A” indicates that it was not examined.

[0068] As can be seen from the above, only by using a specific combination of solvent and antisolvent system and specific processing methods can crystal form I be obtained.

[0069] The purity of crystal form I prepared in Examples 1-3 is all above 99%.

[0070] Test Example 1: Hygroscopicity Test The test was conducted according to Method 1 of the Guidelines for Hygroscopicity Testing of Drugs in Section IV, General Chapter 9103 of the 2025 Edition of the Chinese Pharmacopoeia.

[0071] Comparison of hygroscopicity (RH 80%±2%) between CHKI-03 amorphous compound and crystal form I (batch number: LCB-80-17-1): The hygroscopicity of the amorphous compound is 1.85%, and that of crystal form I is 1.75%.

[0072] The above test results show that there is no significant difference in hygroscopicity among different crystal forms of active pharmaceutical ingredients.

[0073] Test Example 2: Bioavailability Test I. Experimental Objective The pharmacokinetic characteristics of different forms of the compound CHKI-03 were studied in rats after oral administration, and the relative bioavailability was calculated.

[0074] II. Laboratory Animals Male SD rats, weighing 180-200g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0075] III. Experimental Materials CHKI-03 amorphous compound and CHKI-03 crystalline form I compound (batch number: LCB-80-17-1, purity: 99%). The internal standard carboxyamine triazole (CAI, batch number: 69921050801, purity: 100.4%) was obtained from Guangdong Yinzhu Pharmaceutical Co., Ltd. The solvents used in LC-MS analysis were chromatographic grade methanol / acetonitrile (Fisher, USA), all other reagents were analytical grade, and the water used for analysis was purified water prepared using a Milli-Q water purifier (Millipore, USA).

[0076] IV. Experimental Methods 1. Preparation of standard curve and quality control samples for rat plasma Take 50 μL of heparin-anticoagulated blank SD rat plasma sample, add 50 μL of mixed working solution of different concentration standard curve or quality control sample (containing drug powder and solvent acetonitrile), 50 μL of internal standard working solution (solvent acetonitrile, concentration of 1.5 μg / mL), and 150 μL of acetonitrile to prepare CHKI-03 rat plasma standard curve or quality control sample. After shaking and mixing for 30 s, centrifuge twice at high speed (14,000 rpm × 5 min). Take 2 μL of supernatant for LC-MS / MS analysis.

[0077] The final concentrations of the CHKI-03 rat plasma standard curve were 2, 5, 20, 50, 100, 200, 500 and 1000 ng / mL, respectively; the final concentrations of the CHKI-03 rat plasma quality control samples were 5, 400 and 800 ng / mL, respectively.

[0078] 2. Plasma pharmacokinetic studies of rats after oral administration of different forms of CHKI-03 Six male rats were divided into two groups. Before the experiment, the rats were fasted for 12 hours but had free access to water. The two groups were orally administered CHKI-03 (a suspension prepared with 0.5 wt% CMC, where CMC represents carboxymethyl cellulose) in either amorphous form (amorphous group) or crystalline form I (crystalline form I group), at a dose of 50 mg / kg. Continuous blood collection was used. 200 μL of blood was collected from the orbital venous plexus at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after oral administration. The blood samples were placed on ice, centrifuged, and 50 μL of plasma was separated. The concentration of CHKI-03 in the plasma was determined according to the established analytical method. The experimental data were analyzed using a non-compartmental model with WinNonLin software (Pharsight, version 8.3) to calculate plasma pharmacokinetic parameters.

[0079] 3. LC-MS determination Chromatographic and mass spectrometry conditions: Chromatographic conditions: Instrument: Shimadzu LC-30a, equipped with a binary high-pressure pump, autosampler and column oven; Column: Zorbax SB C 18 (2.1 × 100 mm, 3.5 μm, Angilent), with a 0.5 μm in-line filter (Upchurch Scientific Ltd.); Column temperature: 35℃; Sample injection chamber temperature: 15℃; Mobile phase: Phase A: Water (0.2% formic acid); Phase B: Acetonitrile (0.2% formic acid); Flow rate: 0.25 mL / min; Injection volume: 2 μL; The gradient elution procedure is shown in Table 4.

[0080] Table 4 Gradient elution program

[0081] Note: % indicates volume percentage.

[0082] Mass spectrometry conditions: API4000 mass spectrometer, specific parameters are shown in Table 5.

[0083] Table 5 Mass Spectrometry Conditions

[0084] V. Experimental Results 1. Sample determination standard curve and sample determination Based on the LC-MS chromatograms of each sample, the determination of the analyte CHKI-03 showed no interference. Correlation analysis was performed with the ratio of the analyte peak area to the internal standard peak area as the ordinate and the compound concentration as the abscissa. Within the range of 2-1000 ng / mL, the concentration of CHKI-03 in plasma samples showed a good correlation with the peak area ratio, with a correlation coefficient >0.99.

[0085] 2. Plasma pharmacokinetics of rats after oral administration of different crystalline forms of CHKI-03 The blood concentration data of different crystal forms of CHKI-03 in rats after oral administration are shown in Tables 6 and 7, and the relevant pharmacokinetic parameters are shown in Table 8.

[0086] Following oral administration of 50 mg / kg of different forms of CHKI-03 to rats, only trace amounts of CHKI-03 were detectable in the plasma of amorphous rats at 0.25–0.5 h (close to the lower limit of quantitation), with levels below the detection limit at other time points. Compared to the amorphous group, the plasma concentration and in vivo exposure of CHKI-03 were significantly increased in the crystalline group. The C60 of CHKI-03 was also significantly higher. max The value was 39.26 ± 5.09 ng / mL, T max The exposure time is 0.25-0.5 h, and the AUC in vivo is... 0-t The mean retention time (MRT) was 113.32 ± 31.11 ng / mL·h. (0-t) It is 2.99±0.56h.

[0087] 3. Summary After oral administration to rats, the peak concentration and exposure of CHKI-03 crystalline form I in rats were significantly higher than those of the amorphous form of CHKI-03, suggesting that the bioavailability of CHKI-03 crystalline form I in vivo is significantly higher than that of the amorphous form.

[0088] Table 6. Changes in plasma concentrations of amorphous CHKI-03 (50 mg / kg) in rats over time after oral administration.

[0089] Note: BLQ indicates not detected.

[0090] Table 7. Changes in plasma concentrations of CHKI-03 (50 mg / kg) of crystal form I in rats over time.

[0091] Note: BLQ indicates not detected.

[0092] Table 8 Plasma pharmacokinetic parameters of rats after oral administration of different forms of CHKI-03

[0093] Note: "—" indicates that there is insufficient blood drug concentration data, and pharmacokinetic parameters cannot be calculated.

[0094] Characterization of compound CHKI-03 (Crystal Form I) in Test Example 3 The following characterization was performed on the solid sample of compound CHKI-03, crystal form I, prepared by batch number LCB-80-17-1.

[0095] 3.1 XRPD The XRPD detection methods used in the various embodiments of this invention are as follows: Solid samples are detected using an X-ray powder diffractometer. The sample is laid flat on a zero-background single-crystal silicon sample disk, and after being gently pressed flat, analysis is performed according to the parameters in Table 9. The XRPD results of compound CHKI-03 crystal form I prepared by LCB-80-17-1 are as follows: Figure 2 As shown in Table 10.

[0096] Table 9 Test parameters for XRPD

[0097] Table 10 XRPD data for CHKI-03 (lot number: LCB-80-17-1)

[0098] 3.2 TGA TGA data for the samples were collected using a TA instrument. 3 mg of sample was placed in a peeled, open aluminum sample dish, and the sample was heated according to the instrument parameters in Table 11. The data were then analyzed using TRIOS software. The TGA chromatogram of compound CHKI-03 (crystal form I) prepared by LCB-80-17-1 is shown below. Figure 4 As shown.

[0099] Table 11 TGA Test Parameters

[0100] 3.3 DSC DSC data of the samples were collected using a differential calorimeter. Two mg of sample was placed in a perforated DSC sample dish, and heating measurements were performed according to the parameters in Table 12. The data were analyzed using NETZSCH Proteus software. The DSC chromatogram of compound CHKI-03 crystal form I prepared by LCB-80-17-1 is shown below. Figure 3 As shown.

[0101] Table 12 Test parameters for DSC

[0102] 3.41 H-NMR 1 H-NMR data were acquired using a Bruker 400 MHz instrument. Samples were analyzed using DMSO- d6 As a solvent, the test parameters are shown in Table 13, and the data were analyzed and processed using MestReNova software. The compound CHKI-03 crystal form I prepared by LCB-80-17-1... 1 H-NMR spectrum as follows Figure 5 As shown.

[0103] Table 13 1 H-NMR test parameters

[0104] Characterization results show that the sample exhibits distinct crystal form diffraction characteristic peaks on XRPD. Figure 2 ), named crystal form I. 1 H-NMR showed no obvious organic solvent residue (approximately 0.28% acetone residue). Figure 5 TGA showed a weight loss of 1.858% in the range of room temperature to 105°C. Figure 4 The weight loss was attributed to the removal of free water and a small amount of residual solvent. A total weight loss of 4.391% was observed from room temperature to 150℃, therefore the weight loss of 2.533% from 105℃ to 150℃ is likely due to the thermal decomposition of the compound at high temperatures to formaldehyde. DSC analysis showed an endothermic peak at 150.8℃ (145.1℃ / 158.6℃) and an exothermic peak at 241.8℃ (222.5℃ / 256.9℃), indicating a possible phase transition during heating. Based on the values ​​of the endothermic peaks, this crystal form is relatively stable. Figure 3 Therefore, crystal form I is inferred to be an anhydrous and solvent-free substance.

[0105] Test Example 4 Stability Test The content of compound CHKI-03 was determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2025 Edition), as detailed below.

[0106] Solvent: Acetonitrile-water (V / V=50 / 50); Test solution: Take an appropriate amount of the test sample, dissolve and dilute it with solvent to prepare a solution containing 0.3 mg per 1 mL.

[0107] Sensitivity solution: Take an appropriate amount of CHKI-03 reference standard, dissolve and dilute it with solvent to prepare a solution containing 0.09 μg per 1 mL.

[0108] System suitability solution: Take an appropriate amount of CHK03 system suitability reference standard, dissolve and dilute it with solvent to prepare a solution containing 0.3 mg of CHKI-03 per 1 mL.

[0109] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Waters Xbridge C18 250×4.6mm, 5μm, or equivalent column); mobile phase A was 0.01 mol / L potassium dihydrogen phosphate aqueous solution (containing 0.5wt% triethylamine), pH=2.5, and mobile phase B was acetonitrile-methanol (V / V=50 / 50), with gradient elution according to Table 14; the flow rate was 1.0 mL / min; the detection wavelength was 230 nm; the sample chamber temperature was 5℃, the column temperature was 40℃, and the injection volume was 5 μL.

[0110] Table 14 Gradient elution program

[0111] System suitability requirements: In the sensitivity solution chromatogram, the signal-to-noise ratio of the CHKI-03 peak should be no less than 10; in the system suitability solution chromatogram, the resolution between the main peak and impurities, and between impurities, should be no less than 2.0.

[0112] Assay: Accurately measure the test solution, sensitivity solution, and system suitability solution, and inject them into the liquid chromatograph separately, and record the chromatograms.

[0113] Limits: If impurity peaks are present in the test solution, calculated using the area normalization method, the impurity concentrations shall not exceed 0.3% for RRT0.96, 0.3% for RRT0.84, 0.5% for RRT0.90, 0.3% for RRT1.06, and 0.5% for other individual impurities. The total impurity concentration shall not exceed 1.5%, and the purity shall not be less than 98.5%.

[0114] The accelerated stability and long-term stability results are shown in Tables 15 and 16, respectively. RRT represents the relative retention time, where RRT = retention time of the analyte / retention time of the reference compound, and the reference compound is CHKI-03.

[0115] Table 15 Results of accelerated stability studies (40±2℃, 75±5%RH) of different crystalline forms of CHKI-03

[0116] As can be seen from the above, after being placed under accelerated testing conditions for 6 months, the appearance and related substances of the two types of active pharmaceutical ingredients showed no significant changes, indicating that there was no significant difference in chemical stability between the different types of active pharmaceutical ingredients.

[0117] Table 16 Statistical Table of Long-Term Stability Test Results for Crystal Form I (Batch No.: LCB-80-17-1) for 6 Months (25±2℃, 60±5%RH)

[0118] Note: " / " indicates that it was not detected.

[0119] Test Example 5: Solubility Test The results of the saturated solubility tests of different forms of CHKI-03 compounds in water are shown in Table 17.

[0120] Table 17 Results of saturation solubility tests of different forms of CHKI-03 compounds in water

[0121] To further investigate the influence of the active pharmaceutical ingredient's crystal form on formulation production and performance, CHKI-03 injection was prepared using the two different forms of active pharmaceutical ingredient described above, following the same formulation process. The preparation information of the samples is shown in Table 18.

[0122] Table 18. Preparation formula and process of CHKI-03 injection

[0123] The properties, pH value and related substances of CHKI-03 injection prepared using different forms of raw materials in Table 18 were tested, and the test results of the samples are shown in Table 19.

[0124] Table 19 Detection Results of CHKI-03 Injection Samples

[0125] Experimental conclusion: (1) CHKI-03 has poor solubility in water. The saturated solubility of amorphous active pharmaceutical ingredient in water is higher than that of crystal form I active pharmaceutical ingredient, but it has no effect on the dissolution time during the preparation of the injection. According to the preparation process of CHKI-03 injection, the dissolution time of the two batches of samples is basically the same. After the active pharmaceutical ingredient is added, it is necessary to stir at room temperature for about 90 minutes to ensure that CHKI-03 is completely dissolved. This indicates that the crystal form of the active pharmaceutical ingredient has no significant effect on the production of the formulation.

[0126] (2) There were no significant differences between the related substances and the active pharmaceutical ingredient (API) of the two batches of injection solution before sterilization, indicating that there were no significant changes in the related substances during the preparation of the drug solution. At the same time, there were no significant differences in the properties, pH value and osmolar concentration of the two batches of injection solution before sterilization, indicating that the crystal form of the API had no significant effect on the properties, pH value and osmolar concentration of the injection solution. In addition, there were no significant changes in the properties and osmolar concentration of the two batches of injection solution after sterilization. Although the pH value of both batches increased after sterilization, the increasing trend was basically the same. There was no significant difference in the pH value after sterilization. Although the related substances of both batches increased after sterilization, the increasing trend was still basically the same. This indicates that the crystal form of the API also had no significant effect on the performance of the formulation.

[0127] In summary, there are no significant differences in solubility and chemical stability among CHKI-03 active pharmaceutical ingredients (APIs) with different crystal forms, and the crystal form of the API has no significant impact on formulation production and formulation performance.

[0128] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing crystal form I of a CHKI-03 compound, wherein the structure of the CHKI-03 compound is shown in Formula I, characterized in that, The preparation method includes the following steps: dissolving CHKI-03 compound in solvent A to form a clear liquid, and allowing it to evaporate and crystallize at room temperature, wherein the evaporation rate is 0.01-0.1 mL / h, and the obtained solid is crystal form I; the preferred evaporation rate is 0.042 mL / h. 。 2. The preparation method according to claim 1, characterized in that, Solvent A includes at least one of methanol and ethanol.

3. The preparation method according to claim 1, characterized in that, The temperature of solvent A is 20-50℃, and / or the mass-volume ratio of the CHKI-03 compound to solvent A is 0.05-1g:1mL.

4. A method for preparing crystal form I of a CHKI-03 compound, wherein the structure of the CHKI-03 compound is shown in Formula I, characterized in that, The preparation method includes the following steps: dissolving CHKI-03 compound in solvent B to form a clear liquid, cooling the temperature, and the precipitated solid is crystal form I; 。 5. The preparation method according to claim 4, characterized in that, Solvent B is ethanol.

6. The preparation method according to claim 4, characterized in that, The temperature of solvent B is 45-50℃, and / or the temperature after cooling is 0-25℃, preferably 0-5℃; and / or the cooling time is 0.5-1.5h, preferably 1h.

7. A method for preparing crystal form I of a CHKI-03 compound, wherein the structure of the CHKI-03 compound is shown in Formula I, characterized in that, The preparation method includes the following steps: dissolving CHKI-03 compound in solvent C to form a clear liquid, adding antisolvent D to the clear liquid, and the precipitated solid is crystal form I; 。 8. The preparation method according to claim 7, characterized in that, The solvent C includes at least one of DMF, ethanol and DMSO; and / or the antisolvent D includes at least one of water and n-heptane; and / or the volume ratio of the solvent C to the antisolvent D is 1:4-20, preferably 1:4-10.

9. The preparation method according to claim 7, characterized in that, The parameters for adding antisolvent D are: the temperature of antisolvent D is 10-30℃ and / or the addition rate is 5 mL / h; after adding antisolvent D, stirring is continued at 10-30℃ for 2-48 h, preferably for 2-22 h.

10. The preparation method according to any one of claims 1-9, characterized in that, The X-ray powder diffraction pattern of crystal form I includes at least one characteristic peak at 2θ values ​​of 6.524±0.2°, 20.142±0.2°, and 20.326±0.2°. Preferably, the characteristic peak is present at least at one of the following 2θ values: 6.524±0.2°, 18.096±0.2°, 20.142±0.2°, 20.326±0.2°, 22.105±0.2°, and 23.507±0.2°. More preferably, it has a characteristic peak at least at one of the following 2θ values: 6.524±0.2°, 17.913±0.2°, 18.096±0.2°, 20.142±0.2°, 20.326±0.2°, 22.105±0.2°, 22.720±0.2°, 23.507±0.2°, and 25.466±0.2°. More preferably, it has a characteristic peak at least at one of the following 2θ values: 6.524±0.2°, 14.402±0.2°, 17.913±0.2°, 18.096±0.2°, 20.142±0.2°, 20.326±0.2°, 22.105±0.2°, 22.720±0.2°, 23.507±0.2°, 24.351±0.2°, 25.265±0.2°, and 25.466±0.2°. The most preferred X-ray powder diffraction pattern of crystal form I is basically as shown in Figure 2.

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