Crystalline form of HDAC6 inhibitor and preparation method therefor

Abstract

A crystalline form of a compound represented by formula (I) and a preparation method therefor. The crystal form can be used for preventing and / or treating HDAC6-mediated or -dependent diseases or conditions.

Description

A crystalline form of an HDAC6 inhibitor and a preparation method thereof

[0001] Related applications

[0002] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on September 28, 2023, entitled “A crystalline form of an HDAC6 inhibitor and a method for preparing the same” and application number 202311277270.2. The entire contents of the above application are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to a crystalline form of an HDAC6 inhibitor and a preparation method thereof. Specifically, a crystalline form of a compound represented by formula (I) and a preparation method thereof are provided. Background Art

[0004] Histone deacetylases (HDACs) catalyze the deacetylation of histones and other proteins, playing a vital role in various biological processes, primarily through transcriptional repression. HDAC6 primarily catalyzes the deacetylation of non-histone substrates, such as α-tubulin and Hsp90. HDAC6 is involved in the pathogenesis of numerous diseases, including cancer, neurological disorders, infections, cardiovascular diseases, and immune and inflammatory disorders.

[0005] The crystalline form of a known compound in the pharmaceutical field affects the compound's solubility, stability, compressibility, and other factors, thereby affecting the drug's safety and efficacy. Therefore, discovering new crystalline forms of drugs can provide multiple advantages.

[0006] Summary of the Invention

[0007] The present disclosure provides a crystalline form, preparation method, and application of a compound represented by formula (I). The crystalline form disclosed herein has excellent stability and high crystallinity, and is of great value to the optimization and development of drugs.

[0008] Specifically, the present disclosure provides a crystalline form I of a compound represented by formula (I),

[0009] The X-ray powder diffraction pattern expressed as a 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°, 22.0595±0.2° and 26.3492±0.2°; characteristic peaks at 0.2°, 22.0595±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°, Characteristic peaks are found at 21.4220±0.2°, 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°, 17.09 There are characteristic peaks at 31±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°;

[0010] Furthermore, 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°, 24.245 0±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°;

[0011] Furthermore, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is shown in FIG1 ;

[0012] Furthermore, the TGA spectrum of Form I is shown in Figure 2;

[0013] Furthermore, the DSC spectrum of Form I is shown in FIG3 .

[0014] Effects of the Invention

[0015] The crystalline form I of the compound represented by formula (I) provided in the present disclosure has advantages in terms of physical and chemical properties, formulation processing performance and bioavailability, for example, it has advantages in at least one of the aspects of melting point, solubility, stability, and bioavailability. The crystalline form I of the compound represented by formula (I) provided in the present disclosure has good crystallinity and process operability, and has excellent stability, providing a better option for the development of HDAC6 inhibitor drugs, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1: XRPD pattern of Form I of the compound represented by formula (I).

[0017] Figure 2: TGA data of Form I of the compound represented by formula (I).

[0018] Figure 3: DSC data of Form I of the compound represented by formula (I).

[0019] Figure 4: XRPD patterns of Form I of the compound represented by formula (I) before and after storage at 40°C / 75% RH / open for 1 month. DETAILED DESCRIPTION

[0020] To make the technical solutions and beneficial effects of the present disclosure more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which the present disclosure belongs.

[0021] The present disclosure provides a crystalline form I of a compound represented by formula (I),

[0022] The X-ray powder diffraction pattern expressed in terms of 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 present 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°, 21.4220±0.2°, Characteristic peaks are found 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°, 17.0931±0.2 There are characteristic peaks at 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°;

[0023] In certain embodiments, 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°. 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°, 24.2 450±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 There are characteristic peaks at 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°;

[0024] In certain embodiments, the X-ray powder diffraction pattern expressed in diffraction angle 2θ is shown in FIG1 ;

[0025] In certain embodiments, the TGA spectrum of Form I is shown in Figure 2;

[0026] In certain embodiments, the DSC spectrum of Form I is shown in FIG3 .

[0027] The present disclosure further provides a method for preparing the crystalline form I of the compound represented by formula (I), comprising the steps of: mixing the compound represented by formula (I) with solvent I, stirring, centrifuging, and drying.

[0028] In certain embodiments, the solvent I is selected from ethanol.

[0029] The present disclosure further provides a pharmaceutical composition comprising the crystalline form I of the compound represented by formula (I) and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0030] The present disclosure further provides a method for preparing a pharmaceutical composition, comprising the step of mixing Form I of the compound represented by formula (I) with a pharmaceutically acceptable carrier, diluent or excipient.

[0031] The present disclosure further provides a use of the aforementioned crystalline form I of the compound represented by formula (I) or the aforementioned composition, or the composition prepared by the aforementioned method, in the preparation of a medicament for preventing and / or treating HDAC6-mediated or dependent diseases or conditions.

[0032] In certain embodiments, the HDAC6-mediated or -dependent disease or condition is selected from cancer, nervous system disease, cardiovascular disease, immune and inflammation-related diseases.

[0033] The "2θ or 2θ angle" mentioned in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2θ is ±0.2 (including the case where the number exceeding 1 decimal place is rounded off), which can 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.

[0034] The precipitation methods disclosed herein include, but are not limited to, stirring, cooling, volatilization, beating, and precipitation.

[0035] The "differential scanning calorimetry or DSC" described in this disclosure refers to measuring the temperature difference and heat flow difference between a sample and a reference object during the process of heating or maintaining the sample at a constant temperature to characterize all physical and chemical changes related to thermal effects and obtain phase change information of the sample.

[0036] The drying temperature in the present disclosure is generally 25° C. to 100° C., preferably 40° C. to 70° C., and the drying can be performed under normal pressure or reduced pressure.

[0037] A "pharmaceutical composition" refers to a composition comprising one or more crystalline forms of a compound of formula (I) described herein, and optionally a pharmaceutically acceptable carrier, diluent, or excipient. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0038] The method of the present invention is described below by means of specific examples. It should be understood that these examples are used 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 following examples. The implementation conditions adopted in the examples can be further adjusted according to specific requirements. The implementation conditions not specified are generally the conditions in routine experiments.

[0039] The abbreviations used in this disclosure are explained as follows:

[0040] XRPD: X-ray powder diffraction

[0041] DSC: Differential Scanning Calorimetry

[0042] TGA: Thermogravimetric analysis

[0043] Instruments and methods used to collect data:

[0044] The X-ray powder diffraction patterns described in the examples of this disclosure were collected on an Empyrean X-ray powder diffractometer from Panalytical. The X-ray powder diffraction method parameters described in this disclosure are as follows:

[0045] X-ray: Cu, Kα; 1.54060; 1.54443

[0046] X-ray tube setting: 45kV, 40mA

[0047] Scanning range (°2TH): 3° to 40°

[0048] Scan step (°2TH): 0.0263

[0049] Scan time per step (s): 46.665

[0050] The differential scanning calorimetry (DSC) patterns described in the present disclosure were collected on a METTLERDSC3 differential scanner. The method parameters for the differential scanning calorimetry (DSC) described in the present disclosure are as follows:

[0051] Method: Linear temperature ramp

[0052] Sample pan: aluminum pan, pressure cover

[0053] Temperature range: 35-325℃

[0054] Heating rate: 10℃ / min

[0055] Protective gas: nitrogen

[0056] The thermogravimetric analysis (TGA) graphs described in the present disclosure were collected on a TA Discovery TGA5500 thermogravimetric analyzer. The method parameters for the thermogravimetric analysis (TGA) described in the present disclosure are as follows:

[0057] Method: Linear temperature ramp

[0058] Sample pan: Aluminum pan, open

[0059] Temperature range: room temperature-350℃

[0060] Heating rate: 10℃ / min

[0061] Protective gas: nitrogen

[0062] The high performance liquid chromatography (HPLC) data described in the present disclosure were collected from a Waters 2489 UV / Vis Detector, and the setting parameters are shown in Table 1.

[0063] Table 1: HPLC parameters

[0064] Stability testing methods

[0065] Appropriate amounts of crystalline samples of the compound represented by Formula (I) were subjected to stability experiments at 40°C / 75% RH / unprotected for one month. Solid samples isolated under different conditions were subjected to XRPD analysis to assess physical stability and HPLC analysis to assess chemical stability.

[0066] Solubility test method

[0067] Weigh an appropriate amount of a crystalline form sample of the compound represented by formula (I) into a vial, add an appropriate amount of different media, and stir in a 37°C water bath for observation; if the sample is completely dissolved, continue to add sample until insoluble matter remains after rotation and mixing; seal the sample vial, stir in a 37°C water bath for 24 hours, centrifuge, filter the supernatant, and measure the HPLC solubility.

[0068] Example 1: Preparation of the compound represented by formula (I)

[0069] Step 1.

[0070] A 50L jacketed reactor equipped with a reflux condenser, mechanical stirring, and a thermocouple thermometer was charged with: 16L of dichloromethane, 2kg of CPD7, 1kg of triethylamine, and 127g of water. Stirring was started at 0°C under nitrogen protection. 1.6kg of difluoroacetic anhydride in dichloromethane was added dropwise to the reaction solution. Stirring was continued after the addition was complete. After the reaction was completed, the solvent was evaporated under reduced pressure, 5L of methanol was added, and stirring was continued until the solution was clear. 20L of water was added dropwise. Solids precipitated and were filtered, the filter cake was washed with water, and dried to obtain CPD9. Material information is shown in Table 2.

[0071] ESIm / z[MH] - =501.2.

[0072] Table 2 Material information

[0073] Step 2.

[0074] In a 50L jacketed reactor equipped with a reflux condenser, mechanical stirring, and a thermocouple thermometer, 16L acetonitrile, 2.25kg cpd9, 1.35kg triethylamine were added, and stirring was started at room temperature under nitrogen protection. 1.02kg of acetonitrile solution of p-toluenesulfonyl chloride was added dropwise to the reaction solution. After the dropwise addition, stirring was continued, 22L ethyl acetate and 22L water were added, stirred, and allowed to stand for stratification. The aqueous phase was extracted with ethyl acetate, the organic phases were combined, the solvent was evaporated under reduced pressure, 7L isopropanol was added, the mixture was heated to reflux and slurried, the temperature was lowered and filtered, washed with an isopropanol-water mixed solvent, and dried to obtain cpd10.

[0075] ESIm / z[MH] - =483.2.

[0076] Step 3.

[0077] In a 50L jacketed reactor equipped with a reflux condenser, mechanical stirring, and a thermocouple thermometer, 16L acetonitrile and 1.6kg cpd10 were added. Stirring was started at room temperature, and 1.15kg of acetonitrile solution of p-toluenesulfonic acid monohydrate was added dropwise. Stirring was continued after the addition was completed. After the reaction was completed, the mixture was filtered, the filter cake was washed with acetonitrile, and dried to obtain cpd11.

[0078] ESIm / z[M+H] + =385.2.

[0079] Step 4.

[0080] In a 50L jacketed reactor equipped with a reflux condenser, mechanical stirring, a thermocouple thermometer, and nitrogen protection, 18L of dichloromethane, 1.8kg of cpd11, and 2.1kg of DIPEA were added, and stirring was started at -15°C under nitrogen protection. 1.13kg of a dichloromethane solution of difluoroacetic anhydride was added dropwise, and stirring was continued after the dropwise addition was completed. The temperature was raised to 25°C, 18L of water was added dropwise, and after stirring, the mixture was allowed to stand for stratification. The organic phase was washed with saturated ammonium chloride and water, and the organic phase was collected. Part of the solvent was evaporated under reduced pressure, ethyl acetate was added to replace dichloromethane, isopropanol was added, part of the solvent was evaporated under reduced pressure, the temperature was raised to reflux and slurry was beaten, the temperature was lowered, n-heptane was added dropwise, and stirring was continued after the dropwise addition was completed. The mixture was filtered, and the filter cake was washed with a mixed solvent of n-heptane-isopropanol and dried to obtain a compound of formula (I).

[0081] ESIm / z[M+H] +=463.2.1HNMR(400MHz,Chloroform-d)δ8.53(d,J=1.5Hz,1H), 8.41(dd,J=8.1,1.6Hz,1H),7.67(d,J=8.1Hz,1H),6.99(t,J=51.6Hz,1H),6.8 3(d,J=9.2Hz,1H),5.74(t,J=54.2Hz,1H),4.64(dd,J=72.0,16.0Hz2H),4.07( qd,J=10.8,3.9Hz,1H),3.75(td,J=11.4,3.8Hz,1H),2.20(ddd,J=37.9,14.1, 4.0Hz, 2H), 2.03–1.86 (m, 2H), 1.78 (qd, J=12.3, 3.4Hz, 1H), 1.57–1.34 (m, 3H).

[0082] Experimental Example 1: Evaluation of the inhibitory activity of the compound represented by formula (I) on HDAC6 using enzyme activity assay

[0083] HDAC6 (Abcam), the compound represented by formula (I), and 20 μM substrate solution (Ac-GAK(Ac)-AMC) were added to a 384-well plate, incubated at 37°C for 30 min, 1 μM Trypsin and 10 μM TSA were added, and incubated at room temperature for 15 min. The fluorescence emission intensity was detected at 455 nm with excitation at 360 nm, and the inhibition rate at each concentration was calculated, IC 50 The experimental results were obtained by fitting with GraphPadPrism7.0 software and are shown in Table 3.

[0084] Table 3: Compounds represented by formula (I) IC 50 value

[0085] Experimental Example 2: Evaluation of the inhibitory activity of the compound represented by formula (I) against HDAC1-5 and 7-11 using enzyme activity assay

[0086] Different HDAC subtype proteins (purchased from BPS), the compound represented by formula (I), and 2.5-40 μM substrate solution (purchased from BPS) were added to a 384-well plate, incubated at 37°C for 30 min, 1 μM Trypsin and 10 μM TSA were added, and incubated at room temperature for 15 min. The fluorescence emission intensity was detected at 455 nm with excitation at 360 nm, and the inhibition rate at each concentration, IC 50 The experimental results were obtained by fitting with GraphPadPrism7.0 software and are shown in Tables 4 and 5.

[0087] Table 4: HDAC1 inhibition rate of compounds represented by formula (I) / %

[0088] Table 5: Compounds represented by formula (I) IC 50 value

[0089] Experimental Example 3: Testing of α-Tublin Acetylation Levels

[0090] Take A375 / HCT116 / HCC827 cells in the logarithmic growth phase, digest them with trypsin cell digestion solution, centrifuge, count, and plate them into 96-well plates (30,000 cells / well) at an appropriate cell density, 100 μL per well, and seal them with an appropriate amount of PBS. The next day, treat the cells with different concentrations of compounds (starting concentration is 100 μM, 5-fold dilution, and 9 concentration gradients are set). After 6 hours, aspirate the culture medium and wash them twice with 100 μL of PBST. Then fix the cells with 4% paraformaldehyde and incubate at room temperature for 20 minutes. Discard the fixative and wash the cells with PBST. Add 150 μL of special blocking solution (containing 1% Triton-100) to each well, block for 2 hours at room temperature, and wash away the excess blocking solution with PBS. α-tubulin (DM1A) mouse mAb and acetyl-α-tubulin (Lys40) (D20G3) XP rabbit mAb were diluted at a ratio of 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 set up. The next day, the plates were washed twice with PBST and the secondary antibody anti-mouse IgG (H+L) (DyLight TM 680 conjugate) and anti-rabbit IgG (H+L) (DyLight TM 800 conjugate) and added to a 96-well plate and incubated at room temperature for 2 h. The wells were washed twice with PBST and once with PBS. The fluorescence signals at 700 nm and 800 nm were detected using a Li-COROdyssey dual-color near-infrared laser imager. The experimental results are shown in Table 6.

[0091] Table 6: Test results of α-Tublin acetylation level

[0092] Experimental Example 4: Stability Test

[0093] The test compounds were assessed for their one-phase metabolic stability in liver microsomes of CD-1 mice (MLM), Sprague-Dawley rats (RLM), beagle dogs (DLM), cynomolgus monkeys (CLM), and humans (HLM).

[0094] Experimental system:

[0095] The animal and human liver microsomes used in this test system were purchased from Xenotech, Corning or other qualified suppliers and stored in a freezer below -60°C before use.

[0096] Experiment Introduction:

[0097] Test and control compounds were incubated with animal and human liver microsomes at 37 ± 1°C for a specified period of time, with a maximum incubation time of 60 minutes. Samples were removed at designated time points and the reaction was terminated 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).

[0098] Experimental methods:

[0099] 1. Preparation of buffer

[0100] Dissolve 73.21 g of dipotassium phosphate trihydrate and 10.78 g of potassium dihydrogen phosphate in 4000 mL of ultrapure water. Adjust the pH of the solution to 7.40 ± 0.10 using 10% phosphoric acid or 1 M potassium hydroxide, with a final concentration of 100 mM.

[0101] 2. Preparation of working solution

[0102] The test sample powder is prepared into a stock solution of a certain concentration using DMSO or other organic solvents, and then further diluted with a suitable organic solvent.

[0103] The control compounds testosterone, diclofenac, and propafenone were prepared as 10 mM stock solutions in DMSO and then further diluted in appropriate organic solvents.

[0104] 3. Preparation of Liver Microsome Solution

[0105] Dilute each microsome to a 2x working solution using 100 mM potassium phosphate buffer. The final concentration of microsomes in the reaction system is 0.5 mg / mL.

[0106] 4. Preparation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system

[0107] Weigh an appropriate amount of nicotinamide adenine dinucleotide phosphate (NADP) and isocitrate (ISO) powder, dissolve in magnesium chloride solution, and vortex to mix thoroughly. Add an appropriate amount of isocitrate dehydrogenase (IDH) and gently invert the solution to mix thoroughly. The final concentrations in the reaction system are: 1 mM NADP, 1 mM magnesium chloride, 6 mM ISO, and 1 unit / mL IDH.

[0108] 5. Preparation of Stop Solution

[0109] The stop solution is prepared with acetonitrile or other organic solvent containing an internal standard (tolbutamide or other suitable compound) and stored in a refrigerator at 2-8°C.

[0110] 6. Incubation Process

[0111] Incubations will be performed in 96-well plates. Prepare eight incubation plates, designated T0, T5, T15, T30, T45, T60, Blank60, and NCF60. The first six plates correspond to reaction time points of 0, 5, 15, 30, 45, and 60 minutes, respectively. In the Blank60 plate, no test or control compound is added, and a sample is taken after 60 minutes of incubation. In the NCF60 plate, potassium phosphate buffer is used instead of the NADPH regeneration system solution and incubated for 60 minutes. All conditions are replicated in triplicate.

[0112] Mix the microsomes with the test article or control compound, then preincubate the Blank60, T5, T15, T30, T45, and T60 plates (excluding T0 and NCF60) in a 37°C waterbath for approximately 10 minutes. Add the stop solution to the T0 plate, followed by the NADPH regeneration system working solution. In the NCF60 plate, 98 μL of potassium phosphate buffer was added to each well to initiate the reaction. After the preincubation period, 98 μL of the NADPH regeneration system working solution was added to each well of the Blank60, T5, T15, T30, T45, and T60 plates to initiate the reaction. The reaction temperature was 37 ± 1°C, and the final reaction volume was 200 μL. The reaction system contained 0.5 mg / mL microsomes, 1.0 μM substrate, 1 mM NADP, 6 mM ISO, and 1 unit / mL IDH.

[0113] The reaction was terminated by adding cold stop solution containing internal standard to the reaction plate at 5, 15, 30, 45 and 60 minutes, respectively.

[0114] All reaction plates were shaken and centrifuged at 3220 × g for 20 minutes at 4°C. The supernatant was diluted to a certain ratio and then analyzed by LC-MS / MS.

[0115] Sample analysis

[0116] Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) without a standard curve or quality control samples. Semiquantitative determination was performed using the ratio of the analyte peak area to the internal standard peak area. Analyst software (Sciex, Framingham, Massachusetts, USA) was used for analyte and internal standard retention times, chromatogram acquisition, and chromatogram integration.

[0117] The CV of the internal standard peak area in each matrix should be within 20% for each analytical run.

[0118] Data Analysis

[0119] The in vitro elimination rate constant ke of the compound was obtained by converting the ratio of the compound to the internal standard peak area into a residual rate using the following formula:

[0120] CLint(mic) = 0.693 / T1 / 2 / microsomal protein content (microsomal concentration during incubation mg / mL)

[0121] CLint(liver) = CLint(mic) × amount of microsomal protein in the liver (mg / g) × liver weight to body weight ratio

[0122] According to the wellstir model, the hepatic intrinsic clearance and hepatic clearance can be converted using the following formula.

[0123] CL(liver)=(CLint(liver)*Qh) / (CLint(liver)+Qh)

[0124] The parameters in the formula are shown in Table 7 below.

[0125] Table 7: Formula parameters

[0126] The experimental results are shown in Table 8.

[0127] Table 8: Stability test results

[0128] Experimental Example 5: Pharmacokinetic Test in Mice

[0129] In this experiment, the pharmacokinetic behavior of the test compound was investigated in BALB / c mice after intravenous (IV) and oral (PO) administration.

[0130] On the day of administration, the actual body weight of the mice was weighed and the administration volume was calculated. There were 3 mice in each group, and the compound was tested in two groups, one group was administered with a single intravenous injection, and the other group of mice was administered with a single oral gavage. Whole blood samples were collected at the specified time (0.25, 0.5, 1, 2, 4, 8, 24h after administration) by orbital bleeding. After blood sample collection, it was immediately transferred to a labeled commercial sample tube containing K2-EDTA (0.85-1.15mg), followed by centrifugation (3200xg, 4°C, 10 minutes) and plasma was collected. The plasma was transferred to a pre-cooled centrifuge tube, quick-frozen in dry ice, and then stored in an ultra-low temperature freezer at -60°C or lower until LC-MS / MS analysis.

[0131] Plasma concentrations were determined using LC-MS / MS. Plasma drug concentration data for the compound of Formula (I) were analyzed using WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software using a non-compartmental model. The relevant pharmacokinetic parameters were calculated using the linear-log trapezoidal method. The results are shown in Table 9.

[0132] Table 9: Experimental data of mouse pharmacokinetic test

[0133] Experimental Example 6: Ratio of drug concentration in brain and blood of mice

[0134] On the day of administration, the actual body weight of the mice was weighed and the administration volume was calculated. The compound was administered by a single intravenous injection (IV) or oral gavage (PO) to each group of 9 mice. Whole blood samples were collected at the specified time by orbital bleeding. After blood sample collection, it was immediately transferred to a labeled commercial sample tube containing K2-EDTA (0.85-1.15 mg), followed by centrifugation (3200xg, 4°C, 10 minutes) and plasma was collected. The plasma was transferred to a pre-cooled centrifuge tube, quick-frozen in dry ice, and then stored in an ultra-low temperature freezer at -60°C or lower until LC-MS / MS analysis. 20 μL of plasma sample or brain tissue homogenate was added to 80 μL of internal standard solution (50 ng / mL propafenone in acetonitrile), the plate was sealed with a film sealer at 165°C, oscillated with a micro-oscillator (maximum vibration speed) for 10 minutes, and centrifuged at 4000 rpm in a low-speed desktop centrifuge for 20 minutes. 10 μL of the supernatant was removed and added to 90 μL of acetonitrile. After vortex mixing, the plate was sealed with a film sealer at 165°C. After vortex mixing, 1 μL of the supernatant was taken for LC-MS / MS analysis. The plasma drug concentration data of the compound were processed using WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software using a non-compartmental model. The relevant pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. The experimental results are shown in Table 10.

[0135] Data processing:

[0136] At the same time point, the ratio of drug concentration in brain tissue to drug concentration in plasma

[0137] Table 10: Experimental data of drug concentration ratio between brain and blood in mice

[0138] Example 2: Preparation of Form I of the Compound of Formula (I)

[0139] About 20 mg of sample was weighed into a 5 mL sample bottle, 500 μL of ethanol solvent was added, and the mixture was placed on a magnetic stirrer (200 rpm). After stirring for 24 hours, the mixture was centrifuged at 10,000 r / min for 10 min. The supernatant was poured out and the precipitate was dried in a 40° oven. The obtained solid was collected and subjected to XRD analysis. The test results are shown in Table 11.

[0140] The XRPD spectrum is shown in Figure 1, and the positions of its characteristic peaks are shown in Table 11. The TGA spectrum is shown in Figure 2, showing a weight loss of 1.35% from 26.8 to 190°C. The DSC spectrum is shown in Figure 3, showing endothermic peaks at 111.59°C and 194.07°C.

[0141] Table 11: XRPD diffraction peak data of Form I

[0142] Example 3: Solubility of Form I of the compound represented by formula (I) in water

[0143] Approximately 1-2 mg of a crystalline form of the compound of 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 for observation. If the sample was completely dissolved, further sample was added until insoluble matter remained after rotational mixing. The sample vial was sealed, stirred in a 37°C water bath for 24 h, centrifuged, and the supernatant was filtered and the HPLC solubility was measured. The results are shown in Table 12.

[0144] Table 12: Solubility of the crystalline form of the compound represented by formula (I) in water

[0145] Example 4: Dynamic Solubility of Form I of the Compound of Formula (I) in Biological Solvents

[0146] FaSSGF (simulated fasting gastric juice), FeSSGF (simulated fed gastric juice), FaSSIF (simulated fasting intestinal juice), and FeSSIF (simulated fed intestinal juice) are bio-relevant media that can better reflect the impact of the gastrointestinal physiological environment on drug release. The solubility tested in this type of media is closer to the solubility in the human body environment.

[0147] Approximately 1-2 mg of a crystalline form sample of the compound represented by formula (I) was weighed into a 5 mL vial, 1 mL of different media was added, and the mixture was stirred in a 37°C water bath and observed. If the sample was completely dissolved, further sample was added until insoluble matter remained after rotational mixing. The sample vial was sealed, stirred in a 37°C water bath for 24 h, centrifuged, and the supernatant was filtered and the HPLC solubility was measured. The results are shown in Table 13.

[0148] Table 13: Dynamic solubility of the crystalline form in biological media

[0149] Example 5: Stability of Form I of the Compound of Formula (I)

[0150] Appropriate amounts of Form I samples were taken and subjected to stability experiments under accelerated conditions of 40°C / 75% RH / open air / 1 month. The physical stability of the Form I was evaluated by XRPD testing. The XRPD patterns before and after storage are shown in Figure 4, and the chemical stability is shown in Table 14.

[0151] Table 14: Stability of Form I

[0152] The XRPD spectrum results show that the characteristic peaks of Form I remain unchanged under the accelerated conditions of 40°C / 75% RH / open / 1 month, and the stability is good.

[0153] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A crystalline form I of a condensed ring compound, wherein the structural formula of the condensed ring compound is: It is characterized in that The X-ray powder diffraction pattern expressed as a 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 present 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°, 21.4220±0.2°, Characteristic peaks are present 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°, 17.0931±0.2 There are characteristic peaks at 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°.

2. The crystalline form I according to claim 1, characterized in that The X-ray powder diffraction pattern is 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. There are characteristic peaks at 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°.

3. The crystalline form I according to claim 2, characterized in that The X-ray powder diffraction pattern is shown in FIG1 .

4. The crystalline form I according to claim 3, characterized in that The TGA spectrum of Form I is shown in Figure 2; Preferably, the DSC spectrum of Form I is shown in Figure 3.

5. A method for preparing the crystalline form I of the compound represented by formula (I) according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing the compound represented by formula (I) with solvent I, stirring, centrifuging and drying.

6. The method for preparing the crystal form I according to claim 5, characterized in that: The solvent I is selected from ethanol.

7. A pharmaceutical composition prepared from the crystalline form I of the compound represented by formula (I) as described in any one of claims 1 to 4.

8. A pharmaceutical composition comprising the crystalline form I of the compound of formula (I) as claimed in any one of claims 1 to 4 and a pharmaceutically acceptable carrier, diluent or excipient.

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

10. Use of the crystalline form I of the compound represented by formula (I) as described in any one of claims 1 to 4, or the composition as described in claim 7 or 8, or the composition prepared by the method as described in claim 9 in the preparation of a medicament for preventing and / or treating HDAC6-mediated or dependent diseases or conditions; Preferably, the HDAC6-mediated or -dependent disease or disorder is selected from cancer, nervous system disease, cardiovascular disease, immune and inflammation-related diseases.

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