Crystalline form of HDAC6 inhibitor and method for preparing it

Abstract

The present invention relates to the crystalline form of a compound represented by the following formula (I) and a method for preparing the same, wherein the crystal can be used to prevent and / or treat HDAC6-mediated or dependent diseases or conditions. [Formula 1] TIFF2026533887000022.tif45166

Description

Technical Field

[0001] The present application relates to a crystalline form of an HDAC6 inhibitor and a preparation method thereof, and specifically relates to a crystalline form of the compound represented by Formula (I) and a preparation method thereof.

[0002] <Cross-Reference to Related Applications> This application claims the priority of the Chinese patent application filed with the China National Intellectual Property Administration on September 28, 2023, with the title of the invention being "Crystalline Form of HDAC6 Inhibitor and Preparation Method Thereof" and the application number being 202311277270.2, the entire text of which is incorporated herein by reference in its entirety.

Background Art

[0003] Histone deacetylase (HDAC) catalyzes the deacetylation of histones and other proteins, and plays an important role in various biological processes mainly through transcriptional repression. HDAC6 mainly catalyzes the deacetylation of non-histone substrates such as α-tubulin and Hsp90. HDAC6 is involved in the pathology of various diseases including cancers, neurological diseases, infectious diseases, cardiovascular diseases, immune diseases, inflammation-related diseases and the like. In the pharmaceutical field, the crystalline form of a known compound affects the solubility, stability, compressibility and the like of the compound, and further affects the safety and effectiveness of pharmaceutical products. Therefore, discovering new crystalline forms of pharmaceutical agents brings various advantages.

Summary of the Invention

[0004] The present application provides a crystalline form of the compound represented by Formula (I), a preparation method and use thereof. The crystalline form of the present application has excellent stability and high crystallinity, and is of great significance for the optimization and development of pharmaceutical products.

[0005] Specifically, the present application provides crystalline form I of the compound represented by Formula (I),

[0006]

Chemical Formula

[0007] The X-ray powder diffraction pattern, represented by the diffraction angle 2θ, has characteristic peaks at 17.0931±0.2°, 18.3337±0.2°, 21.4220±0.2°, 22.0595±0.2°, and 26.3492±0.2°, preferably at 12.7282±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 21.4220±0.2°, 2 Characteristic peaks are found at 2.0595±0.2°, 24.2450±0.2°, 25.7696±0.2°, and 26.3492±0.2°, preferably at 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 19.8849±0.2°, and 21.4220±0.2°. Characteristic peaks are located at 22.0595±0.2°, 23.0060±0.2°, 24.2450±0.2°, 25.7696±0.2°, 26.3492±0.2° and 29.5926±0.2°, preferably at 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 14.8709±0.2°, 16.1708±0.2°, 16.8430±0.2° and 17.0931±0. Characteristic peaks are found at 0.2°, 18.3337±0.2°, 19.6726±0.2°, 19.8849±0.2°, 21.4220±0.2°, 22.0595±0.2°, 23.0060±0.2°, 24.2450±0.2°, 25.4835±0.2°, 25.7696±0.2°, 26.3492±0.2°, 27.5202±0.2°, 29.5926±0.2°, and 30.0442±0.2°. Furthermore, 8.5090±0.2°, 11.4131±0.2°, 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 14.8709±0.2°, 15.8155±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 18.9 954±0.2°, 19.6726±0.2°, 19.8849±0.2°, 20.6845±0.2°, 20.8631±0.2°, 21.4220±0.2°, 22.0595±0.2°, 22.2748±0.2°, 23.0060±0.2°, 23.6149±0.2°, 23.8802±0.2°, 24.2450±0.2° , 25.4835±0.2°, 25.7696±0.2°, 26.3492±0.2°, 27.2456±0.2°, 27.5202±0.2°, 27.8593±0.2°, 28.0475±0.2°, 29.5926±0.2°, 30.0442±0.2°, 30.7152±0.2°, 30.9672±0.2°, 31.6849 Characteristic peaks are found at ±0.2°, 31.9795±0.2°, 32.4073±0.2°, 33.1395±0.2°, 34.0516±0.2°, 34.8350±0.2°, 35.4536±0.2°, 35.9251±0.2°, 36.5791±0.2°, 37.2047±0.2°, and 38.9204±0.2°. Furthermore, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is shown in Figure 1. Furthermore, the TGA pattern of crystal form I is shown in Figure 2. Furthermore, the DSC pattern of crystal form I is shown in Figure 3. [Effects of the Invention]

[0008] Crystalline form I of the compound represented by formula (I) provided in this application offers advantages in terms of physicochemical properties, formulation processability, and bioavailability, and also offers advantages in at least one aspect of melting point, solubility, stability, and bioavailability. Crystalline form I of the compound represented by formula (I) provided in this application has good crystallinity, good processability, and excellent stability, providing a superior option in the drug development of HDAC6 inhibitors and is of great importance. [Brief explanation of the drawing]

[0009] [Figure 1] XRPD pattern of crystalline form I of the compound represented by formula (I). [Figure 2] The TGA data pattern of crystalline form I of the compound represented by formula (I). [Figure 3] DSC data pattern of crystalline form I of the compound represented by formula (I). [Figure 4] XRPD patterns of crystalline form I of the compound represented by formula (I) before and after 1 month of storage at 40°C / 75%RH / open conditions. [Modes for carrying out the invention]

[0010] To make the technical solutions and beneficial effects of this application clearer and easier to understand, specific embodiments are described in detail below. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show their details. Unless otherwise defined, technical and scientific terms used herein have the same meanings as those in the art to which this application relates.

[0011] This application provides crystalline form I of a compound represented by formula (I),

[0012] [ka]

[0013] The X-ray powder diffraction pattern, represented by the diffraction angle 2θ, has characteristic peaks at 17.0931±0.2°, 18.3337±0.2°, 21.4220±0.2°, 22.0595±0.2°, and 26.3492±0.2°, preferably at 12.7282±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 21.4220±0.2°, 2 Characteristic peaks are found at 2.0595±0.2°, 24.2450±0.2°, 25.7696±0.2°, and 26.3492±0.2°, preferably at 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 19.8849±0.2°, and 21.4220±0.2°. Characteristic peaks are located at 22.0595±0.2°, 23.0060±0.2°, 24.2450±0.2°, 25.7696±0.2°, 26.3492±0.2° and 29.5926±0.2°, preferably at 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 14.8709±0.2°, 16.1708±0.2°, 16.8430±0.2° and 17.0931±0. Characteristic peaks are found at 0.2°, 18.3337±0.2°, 19.6726±0.2°, 19.8849±0.2°, 21.4220±0.2°, 22.0595±0.2°, 23.0060±0.2°, 24.2450±0.2°, 25.4835±0.2°, 25.7696±0.2°, 26.3492±0.2°, 27.5202±0.2°, 29.5926±0.2°, and 30.0442±0.2°. In some embodiments, there are characteristic peaks at 8.5090±0.2°, 11.4131±0.2°, 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 14.8709±0.2°, 15.8155±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 18.9954±0.2°, 19.6726±0.2°, 19.8849±0.2°, 20.6845±0.2°, 20.8631±0.2°, 21.4220±0.2°, 22.0595±0.2°, 22.2748±0.2°, 23.0060±0.2°, 23.6149±0.2°, 23.8802±0.2°, 24.2450±0.2°, 25.4835±0.2°, 25.7696±0.2°, 26.3492±0.2°, 27.2456±0.2°, 27.5202±0.2°, 27.8593±0.2°, 28.0475±0.2°, 29.5926±0.2°, 30.0442±0.2°, 30.7152±0.2°, 30.9672±0.2°, 31.6849±0.2°, 31.9795±0.2°, 32.4073±0.2°, 33.1395±0.2°, 34.0516±0.2°, 34.8350±0.2°, 35.4536±0.2°, 35.9251±0.2°, 36.5791±0.2°, 37.2047±0.2° and 38.9204±0.2°, In some embodiments, the X-ray powder diffraction pattern expressed as diffraction angle 2θ is shown in Figure 1, In some embodiments, the TGA pattern of Crystal Form I is shown in Figure 2, In some embodiments, the DSC pattern of Crystal Form I is shown in Figure 3.

[0014] The present application also provides a process for preparing Crystal Form I of the compound represented by Formula (I), comprising the steps of mixing, stirring, centrifuging and drying the compound represented by Formula (I) with Solvent I.

[0015] In some embodiments, the aforementioned Solvent I is selected from ethanol.

[0016] The present application also provides a pharmaceutical composition comprising crystalline Form I of the compound represented by Formula (I), and an optionally selected pharmaceutically acceptable carrier, diluent or excipient.

[0017] The present application also provides a method for preparing a pharmaceutical composition, comprising the step of mixing crystalline Form I of the compound represented by Formula (I) with a pharmaceutically acceptable carrier, diluent or excipient. The present application also provides the use of crystalline Form I of the compound represented by the aforementioned Formula (I), or the aforementioned composition, or the composition obtained by preparation via the aforementioned method, in the preparation of a medicament for preventing and / or treating HDAC6-mediated or dependent diseases or conditions.

[0018] In some embodiments, the aforementioned HDAC6-mediated or dependent diseases or conditions are selected from cancer, neurological diseases, neovascular diseases, immune and inflammation-related diseases.

[0019] As described in the present application, "2θ or 2θ angle" refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degrees. The 2θ error range of each characteristic peak is ±0.2 (values exceeding one digit after the decimal point include the situation after rounding), and may be -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0020] The precipitation methods described in the present application include, but are not limited to, stirring, cooling, volatilization, slurrying, and precipitation.

[0021] The "differential scanning calorimetry or DSC" described in this application refers to measuring the temperature difference and heat flow difference between a sample and a reference object during a heating or constant-temperature process of the sample, characterizing all physical and chemical changes related to thermal effects, and obtaining phase transition information of the sample.

[0022] The drying temperature described in this application is generally 25°C to 100°C, preferably 40°C to 70°C, and can be dried under normal pressure or reduced pressure.

[0023] A "pharmaceutical composition" refers to a composition comprising the crystalline form of a compound represented by formula (I) described herein, and one or more of optionally selected pharmaceutically acceptable carriers, diluents, or excipients. The purpose of a pharmaceutical composition is to exert biological activity by facilitating administration to a living organism and contributing to the absorption of the active ingredient.

[0024] The method of the present invention will be described through the following specific examples. These examples are intended to illustrate the basic principles, main features, and advantages of the present invention, and the present invention is not limited to the scope of the examples below. The conditions used in the examples can be further adjusted according to specific requirements, and conditions not specified are generally typical test conditions.

[0025] The abbreviations used in this application are interpreted as follows: XRPD: X-ray Powder Diffraction DSC: Differential Scanning Calorimetry TGA: Thermogravimetric analysis

[0026] Equipment and methods used for data collection: The X-ray powder diffraction patterns described in the embodiments of this application were collected using an Empyrean-type X-ray powder diffractometer manufactured by Panalytical. The X-ray powder diffraction method described in this application...

[0027] The parameters are as follows: TIFF2026533887000004.tif12166 X-ray tube settings: 45kV, 40mA Scanning range (°2TH): 3°~40° Scanning step width (°2TH): 0.0263 Scan time per step (s): 46.665

[0028] The differential scanning calorimetry (DSC) patterns described in this application were acquired using a METTLER DSC 3 differential scanning calorimetry meter. The differential scanning calorimetry (DSC) method and parameters described in this application are as follows: Method: Linear temperature rise Sample tray: Aluminum tray with lid Temperature range: 35~325℃. Heating rate: 10℃ / min Protective gas: Nitrogen gas

[0029] The thermogravimetric analysis (TGA) patterns described in this application were obtained using a TA Discovery TGA 5500 thermogravimetric analyzer. The thermogravimetric analysis (TGA) method and parameters described in this application are as follows: Method: Linear temperature rise Sample tray: Aluminum tray, open type Temperature range: Room temperature to 350℃ Heating rate: 10℃ / min Protective gas: Nitrogen gas

[0030] The high-performance liquid chromatography (HPLC) data described in this application were obtained using a Waters 2489UV / Vis Detector, and the settings are shown in Table 1.

[0031] [Table 1]

[0032] Stability measurement method Appropriate amounts of crystalline samples of the compound represented by formula (I) were weighed and stability tests were conducted under the conditions of 40°C / 75%RH / open / 1 month. For solid samples separated under different conditions, the physical stability was evaluated by XRPD testing of the crystalline form, and the chemical stability was evaluated by HPLC testing of purity.

[0033] Solubility measurement method A suitable amount of crystalline sample of the compound represented by formula (1) was weighed into a vial, and appropriate amounts of various solvents were added. The mixture was then stirred in a 37°C water bath and observed. If the sample was completely dissolved, the sample was continued to be added after rotary stirring until insoluble matter remained. The sample vial was sealed and stirred in a 37°C water bath for 24 hours. After centrifugation, the supernatant was filtered, and the solubility was measured by HPLC.

[0034] Example 1: Preparation of the compound represented by formula (I)

[0035] [ka]

[0036] Step 1. In a 50L jacketed reaction vessel equipped with a reflux condenser, mechanical stirrer, and thermocouple thermometer, 16L of dichloromethane, 2kg of cpd7, 1kg of triethylamine, and 127g of water were added. Stirring was started at 0°C under a nitrogen atmosphere, and 1.6kg of a dichloromethane solution of difluoroacetic anhydride was added dropwise to the reaction mixture. After the addition was complete, stirring was continued. After the reaction was complete, the solvent was removed under reduced pressure, 5L of methanol was added, and the mixture was stirred until clarification was achieved. When 20L of water was added dropwise, a solid precipitate formed, which was filtered by suction. The filter cake was washed with water and dried to obtain cpd9. Material information is shown in Table 2. ESI m / z [MH] - = 501.2.

[0037] [Table 2]

[0038] Step 2. In a 50L jacketed reaction vessel equipped with a reflux condenser, mechanical stirrer, and thermocouple thermometer, 16L of acetonitrile, 2.25kg of cpd9, and 1.35kg of triethylamine were added. Stirring was started under a nitrogen atmosphere at room temperature, and 1.02kg of acetonitrile solution of p-toluenesulfonyl chloride was added dropwise to the reaction mixture. After the addition was complete, stirring was continued, and 22L of ethyl acetate and 22L of water were added and stirred. The mixture was allowed to stand and separated into layers. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined. The solvent was removed under reduced pressure, and 7L of isopropanol was added. The mixture was heated, refluxed, and slurryed. It was cooled and filtered, washed with an isopropanol-water mixed solvent, and dried to obtain cpd10. ESI m / z [MH] - = 483.2.

[0039] Step 3. In a 50L jacketed reaction vessel equipped with a reflux condenser, mechanical stirrer, and thermocouple thermometer, 16L of acetonitrile and 1.6kg of cPD10 were added, and stirring was started at room temperature. 1.15kg of acetonitrile solution of p-toluenesulfonic acid monohydrate was added dropwise. After the addition was complete, stirring was continued. After the reaction was complete, the mixture was filtered, the filtered cake was washed with acetonitrile, and dried to obtain cPD11. ESI m / z [M+H] + = 385.2.

[0040] Step 4. In a 50 L jacketed reaction vessel with a reflux condenser, mechanical stirrer, and thermocouple thermometer, under a nitrogen atmosphere, 18 L of dichloromethane, 1.8 kg of cpd11, and 2.1 kg of DIPEA were added. Stirring was started under a nitrogen atmosphere at -15°C, and 1.13 kg of a dichloromethane solution of difluoroacetic anhydride was added dropwise. After the addition was complete, stirring was continued and the temperature was raised to 25°C. 18 L of water was added, stirred, and then allowed to stand to separate the layers. The organic phase was washed with saturated ammonium chloride and water, and the organic phase was recovered. Some of the solvent was removed under reduced pressure, ethyl acetate was added to replace the dichloromethane, isopropanol was added, some of the solvent was removed under reduced pressure, and the temperature was raised, refluxed, and slurry was formed. The temperature was lowered, n-heptane was added dropwise, and stirring was continued after the addition was complete. The mixture was filtered by suction, and the filter cake was washed with a mixed solvent of n-heptane and isopropanol and dried to obtain the compound represented by formula (I).

[0041] ESI m / z [M+H] + = 463.2.1H NMR (400 MHz, Chloroform-d) δ 8.53 (d, J = 1.5 Hz, 1H), 8.41 (dd, J = 8.1, 1.6 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 6.99 (t, J = 51.6 Hz, 1H), 6.83 (d, J = 9.2 Hz, 1H), 5.74 (t, J = 54.2 Hz, 1H), 4.64 (dd, J = 72.0, 16.0 Hz 2H), 4.07 (qd, J = 10.8, 3.9 Hz, 1H), 3.75 (td, J = 11.4, 3.8Hz, 1H), 2.20 (ddd, J = 37.9, 14.1, 4.0 Hz, 2H), 2.03 - 1.86 (m, 2H), 1.78 (qd, J = 12.3, 3.4 Hz, 1H), 1.57 - 1.34 (m, 3H).

[0042] experiment HDAC6 (Abcam), the compound represented by formula (I), and a 20 μM substrate solution (Ac-GAK(Ac)-AMC) were each added to a 384-well plate and incubated at 37°C for 30 minutes. 1 μM Trypsin and 10 μM TSA were then added, and the mixture was incubated at room temperature for 15 minutes. Excitation was performed at 360 nm, and the fluorescence emission intensity at 455 nm was detected. Inhibition rates at each concentration were calculated, and fitting was performed using GraphPadPrism 7.0 software. IC50 was then calculated. 50 We sought [the value]. The experimental results are shown in Table 3.

[0043] [Table 3]

[0044] Experimental Example 2: Enzyme activity test on the inhibitory activity of compounds represented by formula (I) against HDAC1-5 and 7-11 Different HDAC isoform proteins (purchased from BPS), the compound represented by formula (I), and substrate solutions at concentrations of 2.5–40 μM (purchased from BPS) were added to 384-well plates and incubated at 37°C for 30 minutes. 1 μM Trypsin and 10 μM TSA were added, and the mixture was incubated at room temperature for 15 minutes. Excitation was performed at 360 nm, and fluorescence emission intensity at 455 nm was detected. Inhibition rates at each concentration were calculated and fitted using GraphPadPrism 7.0 software. IC50 was calculated. 50 The results were obtained. The experimental results are shown in Tables 4 and 5.

[0045] [Table 4]

[0046] [Table 5]

[0047] Experimental Example 3: Measurement of α-tubular phosphoacetylation levels Logarithmically growing A375 / HCT116 / HCC827 cells were digested with trypsin-based cell digestant, centrifuged, and the cell count was measured. The cells were then seeded in 100 μL portions into 96-well plates at an appropriate cell density (30,000 cells / well). The plates were sealed with an appropriate amount of PBS. The following day, the cells were treated with compounds of different concentrations (initial concentration 100 μM, with a 9-step concentration gradient using 5-fold dilutions). After 6 hours, the medium was aspirated and removed, and the cells were washed twice with 100 μL of PBST. The cells were then fixed with 4% formaldehyde and incubated at room temperature for 20 minutes. The fixative was discarded, and the cells were washed with PBST. 150 μL of a dedicated blocking solution (containing 1% Triton-100) was added to each well, and the cells were blocked at room temperature for 2 hours. Excess blocking solution was then washed off with PBS. α-tubulin (DM1A) mouse mAb and acetyl-α-tubulin (Lys40) (D20G3) XP rabbit mAb were diluted 1:2000 with 0.033% Triton-100 blocking solution, added to 96-well plates, and incubated overnight at 4°C. A blank control group was also prepared simultaneously. The following day, the plates were washed twice with PBST and the secondary antibody, anti-mouse IgG (H+L) (DyLight) was used. TM 680 conjugate) and anti-rabbit IgG(H+L)(DyLight) TM The 800 conjugate was diluted 1:2000 with a 0.033% Triton-100 blocking solution, added to a 96-well plate, and incubated at room temperature for 2 hours. The plates were washed twice with PBST and once with PBS, and fluorescence signals at 700 nm and 800 nm were detected, respectively, using a Li-COR Odyssey dual-color near-infrared laser imaging system. The experimental results are shown in Table 6.

[0048] [Table 6]

[0049] Experimental Example 4: Stability Test The metabolic stability of the test compound in hepatic microsomes in CD-1 mice (MLM), Sprague-Dawley rats (RLM), beagle dogs (DLM), cynomolgus monkeys (CLM), and humans (HLM) was evaluated in a Phase 1 trial.

[0050] Experimental system: The animal and human liver microsomes used in this study were purchased from Xenotech, Corning, or other authorized suppliers and stored in a refrigerator at -60°C or below before use.

[0051] Experiment Overview: The test compound and control compound were incubated with animal and human liver microsomes at 37±1°C for a specified time. The maximum incubation time was 60 minutes. At the specified time, the samples were removed and the reaction was stopped with acetonitrile or other organic solvent containing an internal standard. After centrifugation, the resulting supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0052] Experimental method: 1. Preparation of buffer solution 73.21 g of dipotassium hydrogen phosphate trihydrate and 10.78 g of potassium dihydrogen phosphate were dissolved in 4000 mL of ultrapure water. The pH of the solution was adjusted to 7.40 ± 0.10 using 10% phosphoric acid or 1 M potassium hydroxide, and the final concentration was 100 mM. 2. Preparation of standard solutions The test compound powder was prepared into a stock solution of a certain concentration using DMSO or other organic solvents, and then further diluted with a suitable organic solvent. Using DMSO, 10 mM stock solutions of the control compounds testosterone, diclofenac, and propafenone were prepared and further diluted with a suitable organic solvent. 3. Preparation of liver microsome solution Microsomes from each animal species were diluted 2× with 100 mM potassium phosphate buffer to prepare standard solutions. The final concentration of microsomes in the reaction system was set to 0.5 mg / mL. 4. Preparation of a reduced nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system Appropriate amounts of nicotinamide adenine dinucleotide phosphate (NADP) and isocitrate (ISO) powder were weighed, dissolved in magnesium chloride solution, and mixed by shaking. Appropriate amounts of isocitrate dehydrogenase (IDH) were added, and the mixture was thoroughly mixed by gently inverting the mixture. The final concentrations in the reaction system were 1 mM NADP, 1 mM magnesium chloride, 6 mM ISO, and 1 unit / mL IDH, respectively. 5. Preparation of stop solution The stop solution was prepared using acetonitrile or other organic solvent containing an internal standard (tolbutamide or other suitable compound). The prepared stop solution was stored in a refrigerator at 2–8°C. 6. Incubation process Incubation was performed in 96-well plates. Eight culture plates were prepared and named T0, T5, T15, T30, T45, T60, Blank60, and NCF60. The first six plates corresponded to reaction times of 0, 5, 15, 30, 45, and 60 minutes, respectively. The Blank60 plate had neither the test compound nor the control compound added and was sampled after 60 minutes of incubation. The NCF60 plate was incubated for 60 minutes using potassium phosphate buffer instead of NADPH regeneration solution. Three parallel tests were performed for samples under all conditions.

[0053] Microsomes were mixed with the test compound or control compound, and incubation plates Blank60, T5, T15, T30, T45, and T60 (excluding T0 and NCF60) were pre-incubated in a 37°C water bath for approximately 10 minutes. After adding stop solution to the T0 incubation plate, NADPH regeneration system standard solution was added. 98 μL of potassium phosphate buffer was added to each well of the NCF60 incubation plate to initiate the reaction. After pre-incubation of incubation plates Blank60, T5, T15, T30, T45, and T60, 98 μL of NADPH regeneration system standard solution was added to each sample well to initiate the reaction. The reaction temperature was 37±1°C, the final reaction volume was 200 μL, and the reaction system contained 0.5 mg / mL of microsomes, 1.0 μM of substrate, 1 mM of NADP, 6 mM of ISO, and 1 unit / mL of IDH.

[0054] The reaction was stopped by adding a cold stop solution containing an internal standard to the reaction plate at 5, 15, 30, 45, and 60 minutes, respectively. After stopping, all reaction plates were thoroughly shaken and centrifuged at 3220 × g for 20 minutes at 4°C. The supernatant was diluted to a specific ratio and analyzed by LC-MS / MS.

[0055] Sample analysis Sample analysis, with the exception of standard curves and quality control samples, was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Semi-quantification was performed using the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard. Retention times of the analyte and internal standard, chromatographic data acquisition, and chromatographic integration were processed using Analyst software (Sciex, Framingham, Massachusetts, USA). The coefficient of variation (CV) of the peak area of ​​the internal standard in each matrix within each analysis batch was kept within 20%.

[0056] Data Analysis The in vitro elimination rate constant ke of the compound was calculated by converting the ratio of the peak area of ​​the compound to the peak area of ​​the internal standard substance into a residue rate using the following formula.

[0057]

number

[0058] CLint (mic) = 0.693 / T1 / 2 / microsomal protein content (microsomal concentration mg / mL during incubation) CLint (liver) = CLint (mic) × Microsomal protein content in liver (mg / g) × Liver weight / body weight ratio

[0059] Based on the well-stir model, intrinsic liver clearance and hepatic clearance can be converted using the following formula. CL(liver) = (CLint(liver) * Qh) / (CLint(liver) + Qh)

[0060] The parameters in the formula are shown in Table 7 below.

[0061] [Table 7]

[0062] The experimental results are shown in Table 8.

[0063] [Table 8]

[0064] Experimental Example 5: Mouse Pharmacokinetic Study In this experiment, the pharmacokinetic behavior of the test compound was examined after intravenous (IV) and oral (PO) administration to BALB / c mice. The body weight of the mice was measured on the day of administration to calculate the dosage. Three mice were used in each group, and the compound was tested in two groups: one group received a single intravenous injection, and the other group received a single oral injection. Whole blood samples were collected by orbital collection at specified time points (0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration). Immediately after collection, the samples were transferred to commercially available labeled sample tubes containing K2-EDTA (0.85-1.15 mg), and plasma was collected by centrifugation (3200 xg, 4°C, 10 minutes). The plasma was transferred to a pre-cooled centrifuge tube, rapidly frozen with dry ice, and stored in an ultra-low temperature refrigerator at -60°C or below until LC-MS / MS analysis. Plasma concentrations were measured using LC-MS / MS. Plasma drug concentration data for the compound represented by equation (I) were processed using WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software with a non-compartmental model. Relevant pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. The experimental results are shown in Table 9.

[0065] [Table 9]

[0066] Experimental Example 6: Drug concentration ratio in mouse brain / plasma The body weight of the mice was measured on the day of administration to calculate the dosage. Nine mice were used in each group, and the compound was administered as a single intravenous injection (IV) or orally (PO). Whole blood samples were collected by orbital collection at the designated time. Immediately after collection, the samples were transferred to commercially available labeled sample tubes containing K2-EDTA (0.85-1.15 mg), and plasma was collected by centrifugation (3200 xg, 4°C, 10 minutes). The plasma was transferred to a pre-cooled centrifuge tube, rapidly frozen with dry ice, and stored in an ultra-low temperature refrigerator below -60°C until LC-MS / MS analysis. 20 μL of plasma sample or brain tissue homogenate was collected and added to 80 μL of internal standard solution (50 ng / mL propafenone acetonitrile solution). The plate was sealed at 165°C using a sealing device, shaken for 10 minutes with a microshaker (maximum shaking speed), and then centrifuged at 4000 rpm for 20 minutes using a low-speed benchtop centrifuge. Ten μL of supernatant was collected, ninety μL of acetonitrile was added, and the mixture was mixed using a vortex mixer. The mixture was sealed in a sealing device at 165°C, mixed again using a vortex mixer, and then one μL of supernatant was collected and analyzed by LC-MS / MS. Plasma drug concentration data of the compounds were processed using WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software with a non-compartment model. Relevant pharmacokinetic parameters were calculated using the linear logarithmic trapezoid method. The experimental results are shown in Table 10.

[0067] Data processing: The ratio of drug concentration in mouse brain / plasma at the same time point = drug concentration in brain tissue / drug concentration in plasma

[0068] [Table 10]

[0069] Example 2: Preparation of crystalline form I of the compound represented by formula (I) Approximately 20 mg of the sample was weighed into a 5 mL sample vial, and 500 μL of ethanol solvent was added. The vial was placed in a magnetic stirrer (200 rpm) and stirred for 24 hours. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was dried in an oven at 40°C. XRD analysis was performed on the recovered solid, and the results are shown in Table 11. The XRPD pattern is shown in Figure 1, and the location of its characteristic peaks is shown in Table 11. The TGA pattern is shown in Figure 2, and its weight loss from 26.8 to 190°C was 1.35%. The DSC pattern is shown in Figure 3, and it showed endothermic peaks at 111.59°C and 194.07°C.

[0070] [Table 11]

[0071] Example 3: Solubility in water of crystalline form I of the compound represented by formula (I) Approximately 1-2 mg of a crystalline sample of the compound represented by formula (I) was weighed into a 5 mL vial, 1 mL of water was added, and the mixture was stirred in a 37°C water bath and observed. If the sample was completely dissolved, the sample was continued to be added after rotary stirring until insoluble matter remained. The sample vial was sealed and stirred in a 37°C water bath for 24 hours, then centrifuged, the supernatant was filtered, and the solubility was measured by HPLC. The results are shown in Table 12.

[0072] [Table 12]

[0073] Example 4: Dynamic solubility of crystalline form I of the compound represented by formula (I) in a biological solvent FaSSGF (fasting-simulated gastric juice), FeSSGF (supply-simulated gastric juice), FaSSIF (fasting-simulated intestinal fluid), and FeSSIF (supply-simulated intestinal fluid) are biocompatible media. These media better reflect the influence of the gastrointestinal physiological environment on drug release, and the solubility tested in these media is closer to the solubility in the human environment. Approximately 1-2 mg of a crystalline sample of the compound represented by formula (I) was weighed into a 5 mL vial, 1 mL of a different medium was added, and the mixture was stirred in a 37°C water bath and observed. If the sample was completely dissolved, the sample was continued to be added after rotary stirring until insoluble matter remained. The sample vial was sealed and stirred in a 37°C water bath for 24 hours, then centrifuged, the supernatant was filtered, and the solubility was measured by HPLC. The results are shown in Table 13.

[0074] [Table 13]

[0075] Example 5: Stability of crystalline form I of the compound represented by formula (I) A suitable amount of crystalline form I sample was taken, and stability testing was performed under accelerated conditions of 40°C / 75%RH / open / 1 month. Physical stability was evaluated by XRPD testing of the crystalline form. The XRPD patterns before and after installation are shown in Figure 4, and the chemical stability is shown in Table 14.

[0076] [Table 14]

[0077] The XRPD pattern results showed that crystal form I exhibited good stability, with no change in characteristic peaks under accelerated conditions of 40°C / 75%RH / open / 1 month.

[0078] The above embodiments are illustrative and do not encompass all possible embodiments included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this application. Similarly, various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the invention not expressly described. Therefore, the above embodiments are merely illustrative of some embodiments of the invention and do not limit the scope of protection of this patent.

Claims

1. A condensed ring compound in crystalline form I, wherein the structural formula of the condensed ring compound is as follows: 【Chemistry 1】 The X-ray powder diffraction pattern, represented by the diffraction angle 2θ, has characteristic peaks at 17.0931±0.2°, 18.3337±0.2°, 21.4220±0.2°, 22.0595±0.2°, and 26.3492±0.2°, preferably at 12.7282±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 21.4220±0.2°, 2 Characteristic peaks are located at 2.0595±0.2°, 24.2450±0.2°, 25.7696±0.2°, and 26.3492±0.2°, preferably at 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, 18.3337±0.2°, 19.8849±0.2°, and 21.4220±0.2°. Characteristic peaks are located at 22.0595±0.2°, 23.0060±0.2°, 24.2450±0.2°, 25.7696±0.2°, 26.3492±0.2° and 29.5926±0.2°, preferably at 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 14.8709±0.2°, 16.1708±0.2°, 16.8430±0.2° and 17.0931± Characteristic peaks are located at 0.2°, 18.3337±0.2°, 19.6726±0.2°, 19.8849±0.2°, 21.4220±0.2°, 22.0595±0.2°, 23.0060±0.2°, 24.2450±0.2°, 25.4835±0.2°, 25.7696±0.2°, 26.3492±0.2°, 27.5202±0.2°, 29.5926±0.2°, and 30.0442±0.2°. The crystal form I characterized by the above.

2. The aforementioned X-ray powder diffraction patterns were 8.5090±0.2°, 11.4131±0.2°, 12.7282±0.2°, 13.0778±0.2°, 13.8888±0.2°, 14.8709±0.2°, 15.8155±0.2°, 16.1708±0.2°, 16.8430±0.2°, 17.0931±0.2°, and 18.3337±0. 2°, 18.9954±0.2°, 19.6726±0.2°, 19.8849±0.2°, 20.6845±0.2°, 20.8631±0.2°, 21.4220±0.2°, 22.0595±0.2°, 22.2748±0.2°, 23.0060±0.2°, 23.6149±0.2°, 23.8802±0.2°, 24.2450 ±0.2°, 25.4835±0.2°, 25.7696±0.2°, 26.3492±0.2°, 27.2456±0.2°, 27.5202±0.2°, 27.8593±0.2°, 28.0475±0.2°, 29.5926±0.2°, 30.0442±0.2°, 30.7152±0.2°, 30.9672±0.2°, 31.6 Characteristic peaks are located at 849±0.2°, 31.9795±0.2°, 32.4073±0.2°, 33.1395±0.2°, 34.0516±0.2°, 34.8350±0.2°, 35.4536±0.2°, 35.9251±0.2°, 36.5791±0.2°, 37.2047±0.2°, and 38.9204±0.2°. Crystal form I as described in claim 1.

3. The aforementioned X-ray powder diffraction pattern is shown in Figure 1. Crystal form I as described in claim 2.

4. The TGA pattern of crystal form I is shown in Figure 2. Preferably, the DSC spectrum of crystal form I is shown in Figure 3. Crystal form I as described in claim 3.

5. The procedure includes mixing the compound represented by formula (I) with solvent I, stirring, centrifuging, and drying, A method for preparing crystalline form I of a compound represented by formula (I) as described in any one of claims 1 to 4.

6. The solvent I is selected from ethanol. A method for preparing crystal form I according to claim 5.

7. The crystalline form I of the compound represented by formula (I) according to any one of claims 1 to 4 was prepared. A pharmaceutical composition characterized by the following features.

8. A crystalline form I of a compound represented by formula (I) as described in any one of claims 1 to 4, and an optionally selected pharmaceutically acceptable carrier, diluent, or excipient. A pharmaceutical composition characterized by the following features.

9. The procedure includes mixing crystalline form I of a compound represented by formula (I) as described in any one of claims 1 to 4 with a pharmaceutically acceptable carrier, diluent, or excipient. A method for preparing a pharmaceutical composition characterized by the following.

10. The use of crystalline form I of a compound represented by formula (I) according to any one of claims 1 to 4, or a composition according to claim 7 or 8, or a composition prepared by the method according to claim 9, in the preparation of a pharmacopoeia for the prevention and / or treatment of HDAC6-mediated or dependent diseases or conditions, Preferably, the HDAC6-mediated or dependent disease or condition is selected from cancer, neurological disorders, cardiovascular diseases, immune disorders, and inflammation-related diseases. A characteristic use.

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