Crystal forms of diaminocyclopentyl-substituted heteroaryl derivative and preparation method therefor

Abstract

The present disclosure relates to crystal forms of a diaminocyclopentyl-substituted heteroaryl derivative and a preparation method therefor. Specifically, the present disclosure provides crystal form A, crystal form B, crystal form C, and crystal form D of 1-(6-{[(1S,3S)-3-(6,7-dihydro[1,4]dioxino[3,2-d]pyrimidin-2-ylamino)cyclopentyl]amino}pyridin-3-yl)-1,2-dihydropyridin-2-one, which have good stability and can be better used for clinical treatment.

Description

Crystalline form and preparation method of a diaminocyclopentyl-substituted heteroaryl derivative Technical Field

[0001] This disclosure belongs to the field of pharmaceutical technology and relates to the crystalline form of a diaminocyclopentyl-substituted heteroaryl derivative and its preparation method. Background Technology

[0002] PCSK9, also known as "kexin9," is a member of the secreted proteotransferase family and plays a crucial role in cholesterol metabolism. PCSK9 increases circulating LDL cholesterol levels by enhancing LDL receptor degradation, independent of its catalytic activity. Secreted PCSK9 binds to the epidermal growth factor domain A (EGFA) of the LDL receptor (LDLR) on the cell surface, and the PCSK9 / LDL receptor complex is internalized into an endosome / lysosomal compartment. The enhanced affinity of PCSK9 for the LDL receptor at the acidic pH of late endosome / lysosomes reduces LDL receptor recycling, while targeting the LDL receptor for lysosomal degradation. Genetic association studies have demonstrated that loss-of-function mutations in PCSK9 are associated with lower plasma LDL-C levels and a reduced incidence of adverse cardiovascular events.

[0003] PCT / CN2024 / 107781 provides a PCSK9 inhibitor with the chemical name 1-(6-{[(1S,3S)-3-(6,7-dihydro[1,4]dioxane[3,2-d]pyrimidin-2-ylamino)cyclopentyl]amino}pyridin-3-yl)-1,2-dihydropyridin-2-one, having the structure of Formula 1.

[0004] The crystal form of a pharmaceutical active ingredient often affects its chemical stability. Different crystallization and storage conditions can lead to changes in the crystal structure of the compound, sometimes even resulting in other crystal forms. Polymorphism of drugs places different requirements on product storage, production, and scale-up. Therefore, in-depth research into the crystal forms of the aforementioned compounds is essential to improve their various properties. Summary of the Invention

[0005] This disclosure provides a novel crystal form of a compound of Formula 1, which exhibits good stability and can be better applied in clinical practice.

[0006] The crystal form A of the compound of Formula 1 disclosed herein has characteristic peaks at 5.008, 14.411, 17.012, 18.441, and 20.941 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0007] In some embodiments, the X-ray powder diffraction pattern of crystal form A of compound of formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.008, 14.411, 14.973, 17.012, 18.441, 19.593, 20.941, 21.419, 24.736, and 25.279.

[0008] In some embodiments, the X-ray powder diffraction pattern of crystal form A of compound of formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.008, 13.700, 14.411, 14.973, 15.210, 17.012, 18.441, 18.885, 19.593, 20.941, 21.419, 22.594, 23.531, 24.736, and 25.279.

[0009] In some embodiments, the X-ray powder diffraction pattern of crystal form A of compound of formula 1, expressed in terms of diffraction angle 2θ, is shown in Figure 1.

[0010] This disclosure also provides a method for preparing crystal form A of compound of formula 1, the method comprising any of the following methods:

[0011] Method 1: Mix the compound of Formula 1 with solvent I and stir. Solvent I is selected from one or more of ethyl acetate, isopropyl acetate, ethanol, methyl tert-butyl ether, n-heptane, cyclohexane, and isopropyl ether.

[0012] Method 2: Mix the compound of Formula 1 with solvent II, then add solvent III and stir. Solvent II is selected from acetone and tetrahydrofuran, and solvent III is selected from n-heptane and methyl tert-butyl ether.

[0013] In an optional embodiment, the method for preparing compound A of formula 1 further includes a centrifugation step.

[0014] The crystal form B of the compound of Formula 1 disclosed herein has characteristic peaks at 5.834, 9.606, 10.122, 15.516, and 17.142 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0015] In some embodiments, the crystal form B of the compound of Formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 5.834, 9.606, 10.122, 11.061, 15.516, 17.142, and 24.992.

[0016] In some embodiments, the X-ray powder diffraction pattern of crystal form B of compound 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.834, 9.606, 10.122, 11.061, 11.656, 15.516, 17.142, 19.229, 20.319, 24.992, and 25.616.

[0017] In some embodiments, the X-ray powder diffraction pattern of crystal form B of compound of formula 1, expressed in terms of diffraction angle 2θ, is shown in Figure 2.

[0018] This disclosure also provides a method for preparing crystal form B of compound of formula 1, the method comprising any of the following methods:

[0019] Method 1: Mix the compound of Formula 1 with solvent IV and stir. Solvent IV is selected from one or more of water, methanol, ethanol, isopropanol, 2-butanone, ethyl acetate, acetone, dichloromethane, and 2-methyltetrahydrofuran.

[0020] Method 2: Mix the compound of Formula 1 with solvent V, and then add solvent VI, wherein solvent V is selected from one or more of ethanol, acetone, acetonitrile, tetrahydrofuran, dichloromethane, and methanol, and solvent VI is selected from n-heptane, methyl tert-butyl ether, and isopropyl ether;

[0021] Method 3: Mix the compound of Formula 1 with solvent VII and evaporate the solvent, wherein solvent VII is selected from one or more of water, methanol, ethanol, n-propanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol methyl ether, N,N-dimethylformamide, and dimethyl sulfoxide;

[0022] Method 4: Mix the compound of Formula 1 with solvent VIII, heat to dissolve, and cool to crystallize. The solvent VIII is selected from one or more of water, methanol, ethanol, acetone, 2-methyltetrahydrofuran, and ethyl acetate.

[0023] In an optional embodiment, the method for preparing compound B of formula 1 further includes a filtration or centrifugation step.

[0024] The crystal form C of the compound of Formula 1 provided in this disclosure has characteristic peaks at 8.096, 18.515, 21.509, 23.739, and 24.274 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0025] In some embodiments, the X-ray powder diffraction pattern of crystal form C of compound 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.096, 9.166, 15.945, 18.515, 19.708, 21.509, 22.088, 23.739, 24.274, and 26.668.

[0026] In some embodiments, the X-ray powder diffraction pattern of crystal form C of compound 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.096, 9.166, 10.826, 15.568, 15.945, 16.360, 18.515, 19.708, 21.509, 22.088, 23.739, 24.274, and 26.668.

[0027] In some embodiments, the X-ray powder diffraction pattern of the crystal form C of compound 1, expressed as a diffraction angle 2θ, is shown in Figure 3.

[0028] This disclosure also provides a method for preparing crystal form C of compound of formula 1, the method comprising the steps of mixing compound of formula 1 with water and stirring.

[0029] In an optional embodiment, the method for preparing compound crystal form C of formula 1 further includes a centrifugation step.

[0030] The crystal form D of the compound of Formula 1 disclosed herein has characteristic peaks at 13.532, 13.889, 19.679, 22.352, and 24.910 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0031] In some embodiments, the crystal form D of the compound of Formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 12.410, 13.532, 13.889, 15.952, 19.679, 22.352, 24.910, and 25.346.

[0032] In some embodiments, the X-ray powder diffraction pattern of the crystal form D of the compound of Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 12.410, 13.532, 13.889, 15.952, 16.532, 19.679, 22.352, 23.104, 24.910, and 25.346.

[0033] In some embodiments, the X-ray powder diffraction pattern of the crystal form D of the compound of Formula 1, expressed in terms of the diffraction angle 2θ, is shown in Figure 4.

[0034] This disclosure also provides a method for preparing crystal form D of compound of formula 1, the method comprising the steps of mixing compound of formula 1 with 2-butanone and stirring.

[0035] In an optional embodiment, the method for preparing compound D of formula 1 further includes a centrifugation step.

[0036] In some embodiments, the preparation method described in this disclosure further includes any one of the steps of crystallization, centrifugation (filtration), washing, or drying.

[0037] The crystallization methods disclosed herein include, but are not limited to, stirred crystallization, static crystallization, or evaporative crystallization. In some embodiments, the crystallization is stirred crystallization. In some embodiments, the crystallization is static crystallization.

[0038] This disclosure also provides a pharmaceutical composition comprising the aforementioned crystal form A, crystal form B, crystal form C, and crystal form D, and a pharmaceutical excipient optionally selected from pharmaceutically acceptable excipients.

[0039] This disclosure also provides a pharmaceutical composition prepared from the aforementioned crystal forms A, B, C, and D, and optionally a pharmaceutically acceptable excipient.

[0040] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the aforementioned crystal forms A, B, C, and D with a pharmaceutically acceptable excipient.

[0041] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients based on the total weight of the composition; in some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients; in some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients; in some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients; and in some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0042] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, transporter, diluent, and / or delivery polymer.

[0043] This disclosure also provides the use of the aforementioned crystal form A, crystal form B, crystal form C, crystal form D, or the aforementioned composition in the preparation of a medicament for the prevention and / or treatment of dyslipidemia, dyslipoproteinemia, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, hyperlipoproteinemia, xanthomas, hypoalpha-lipoproteinemia, sitosterolemia, atherosclerosis, arteriosclerosis, metabolic syndrome, coronary heart disease, peripheral vascular disease, congestive heart failure, stroke, vascular dementia, coronary artery disease, chronic kidney disease, retinopathy, inflammation, diabetic complications, or thrombosis.

[0044] On the other hand, this disclosure provides the use of the aforementioned crystal form A, crystal form B, crystal form C, crystal form D, or the aforementioned composition in the preparation of PCSK9 inhibitors.

[0045] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.20 (including the case where the number has more than one decimal place after rounding), specifically -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.

[0046] The numerical values ​​in this disclosure, such as those relating to the content of certain substances, are calculated data and inevitably contain a certain degree of error. Generally, ±10% is within the reasonable error range. The error may vary to some extent depending on the context in which it is used, but this variation shall not exceed ±10%, and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0047] The starting material used in the crystal form preparation method disclosed herein can be any form of compound, including but not limited to: amorphous, arbitrary crystal form, hydrate, solvate, etc.

[0048] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 40℃-70℃, and can be dried under normal pressure or reduced pressure.

[0049] The crystallization methods described in this disclosure include room temperature crystallization, cooling crystallization, solvent evaporation crystallization, and seed crystallization induction. The cooling temperature is selected from below 65°C, preferably from -10°C to 60°C. Stirring can also be performed during the crystallization process.

[0050] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0051] According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "Guiding Principles on Hygroscopicity of Drugs" in Part IV of the 2020 edition of the Chinese Pharmacopoeia,

[0052] Deliquescence: Absorbs sufficient moisture to form a liquid;

[0053] Extremely hygroscopic: the weight gain due to hygroscopic absorption is not less than 15%;

[0054] It has hygroscopic properties: the weight gain due to hygroscopic absorption is less than 15% but not less than 2%;

[0055] Slightly hygroscopic: the weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0056] It has little or no hygroscopicity: the weight gain due to moisture absorption is less than 0.2%.

[0057] The “excipients” described in this disclosure include, but are not limited to, any adjuvants, carriers, flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0058] In this disclosure, "mixing" means that the order in which the components are added is not limited. For example, mixing A and B can mean either A is added to B or B is added to A. Attached Figure Description

[0059] Figure 1 shows the XRPD spectrum of crystal form A of compound of formula 1.

[0060] Figure 2 shows the XRPD spectrum of crystal form B of compound 1.

[0061] Figure 3 shows the XRPD spectrum of crystal form C of compound 1.

[0062] Figure 4 shows the XRPD spectrum of crystal form D of compound of formula 1.

[0063] Figure 5 shows the XRPD spectrum of the amorphous compound of Formula 1. Detailed Implementation

[0064] The present disclosure will be explained in more detail below with reference to embodiments or experimental examples. The embodiments or experimental examples in the present disclosure are only used to illustrate the technical solutions in the present disclosure and are not intended to limit the substance and scope of the present disclosure.

[0065] Test conditions of the instruments used in the experiment:

[0066] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE NEO 500M NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard. The spatial configurations of the optical isomers (isomers) of the compounds were further confirmed by measuring single-crystal parameters.

[0067] HPLC determinations were performed using a Waters ACQUITY ultra high performance LC, Shimadzu LC-20A systems, Shimadzu LC-2010HT series, or Agilent 1200LC high performance liquid chromatograph (ACQUITY UPLC BEH C18 1.7UM 2.1×50MM column, Ultimate XB-C18 3.0×150mm column, or Ultimate C18 2.1×30mm column).

[0068] MS measurements were performed using a Waters SQD2 mass spectrometer in positive / negative ion mode, with a mass scan range of 100–1200.

[0069] Chiral HPLC analysis was performed using Chiralpak IC-3 100×4.6mm ID, 3µm, Chiralpak AD-3 150×4.6mm ID, 3µm, Chiralpak AD-3 50×4.6mm ID, 3µm, Chiralpak AS-3 150×4.6mm ID, 3µm, Chiralpak AS-3 100×4.6mm ID, 3µm, ChiralCel OD-3 150×4.6mm ID, 3µm, Chiralcel OD-3 100×4.6mm ID, 3µm, ChiralCel OJ-H 150×4.6mm ID, 5µm, and Chiralcel OJ-3 150×4.6mm ID, 3µm columns.

[0070] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0071] Rapid column purification systems use either the Combiflash Rf150 (TELEDYNE ISCO) or Isolara One (Biotage).

[0072] Normal column chromatography generally uses Yantai Huanghai silica gel of 100-200 mesh, 200-300 mesh or 300-400 mesh as the carrier, or Changzhou Santai pre-filled ultrapure normal phase silica gel column (40-63μm, 60, 12g, 25g, 40g, 80g or other specifications).

[0073] Reversed-phase column chromatography typically uses Changzhou Sante pre-packed ultrapure C18 silica gel columns (20-45μm). 40g, 80g, 120g, 220g or other sizes).

[0074] The high-pressure column purification system uses Waters AutoP, in conjunction with the Waters XBridge BEH C18 OBD Prep Column. 5μm, 19mm×150mm or Atlantis T3 OBD Prep Column, 5μm, 19mm×150mm.

[0075] Chiral preparation columns used were DAICL CHIRALPAK IC (250 mm × 30 mm, 10 μm) or Phenomenex-Amylose-1 (250 mm × 30 mm, 5 μm).

[0076] XRPD (X-ray Powder Diffraction) was used for analysis: measurements were performed using a BRUKER D8 X-ray diffractometer. Specific data collected included: Cu anode (40 kV, 40 mA), Cu-Kα1 rays. Kα2 rays Kβ rays Scanning mode: θ / 2θ, scanning range (2θ range): 3°~45°.

[0077] DSC stands for Differential Scanning Calorimetry: Measurements were performed using a METTLER TOLEDO DSC 3+ differential scanning calorimeter with a heating rate of 10℃ / min. The specific temperature range was referenced from the corresponding spectra (mostly 25-250℃), and the nitrogen purging rate was 50mL / min.

[0078] TGA is thermogravimetric analysis: the test was performed using a METTLER TOLEDO TGA2 thermogravimetric analyzer, with a heating rate of 10℃ / min, and the specific temperature range was referenced from the corresponding spectrum (mostly 30-400℃). The nitrogen purging rate was 50mL / min.

[0079] DVS stands for Dynamic Moisture Adsorption: The detection method is SMSDVS Advantage, with humidity changing from 50% to 95% to 0% to 95% to 50% at 25℃, in 10% increments (the final step is 5%) (the specific humidity range is subject to the corresponding spectrum; the methods listed here are the most commonly used). The judgment criterion is Tmax 360min, and dm / dt not greater than 0.002%.

[0080] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0081] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0082] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0083] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: n-hexane / ethyl acetate system, B: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0084] Example 1

[0085] 1-(6-{[(1S,3S)-3-(6,7-dihydro[1,4]dioxane[3,2-d]pyrimidin-2-ylamino)cyclopentyl]amino}pyridin-3-yl)-1,2-dihydropyridin-2-one (Compound 1)

[0086] Step 1:

[0087] Pyridine-2(1H)-one 1b (1.51 g, 15.9 mmol), 2-chloro-5-pyridineboronic acid 1a (5 g, 31.8 mmol), copper acetate (5.77 g, 31.8 mmol), pyridine (2.51 g, 331.8 mol), and molecular sieve (6 g) were added to a mixed solution (DMF / DCM = 1:6) (210 mL). The mixture was aerated for one minute and reacted at room temperature for 16 hours. The reaction mixture was filtered through diatomaceous earth. 100 mL of water and 50 mL of ethyl acetate were added to the filtrate. The mixture was separated, and the aqueous phase was extracted again with ethyl acetate (50 mL * 2). The organic phases were combined, concentrated under reduced pressure, and the residue was separated by normal-phase column chromatography to obtain compound 1c (1.03 g, yield: 31.4%).

[0088] MS m / z(ESI): 207.1 [M+H] + .

[0089] Step 2:

[0090] Compound 1c (1.03 g, 4.99 mmol), (1S,3S)-3-aminocyclopentylcarbamate tert-butyl ester 1d (1.2 g, 5.98 mmol), cesium carbonate (4.87 g, 14.96 mmol), and methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II) (0.42 g, 0.5 mmol) were added sequentially to 1,4-dioxane (10 mL), and the mixture was microwaved at 130 °C for 4 hours. The reaction solution was preparatively separated by normal-phase column chromatography to give compound 1e (440 mg, yield: 23.8%).

[0091] MS m / z (ESI): 371.3 [M+H] + .

[0092] Step 3:

[0093] Compound 1e (440 mg, 1.2 mmol) was added to dichloromethane (5 mL), followed by trifluoroacetic acid (2 mL). The mixture was reacted at room temperature for 1 hour and then concentrated under reduced pressure to give compound 1f (800 mg, yield: 135.1%).

[0094] MS m / z(ESI): 271.2 [M+H] + .

[0095] Step 4:

[0096] 2,4-Dichloro-5-hydroxypyrimidine 3a (520 mg, 3.2 mmol), 2-bromoethanol (1.18 g, 9.5 mmol), and potassium carbonate (2.18 g, 15.8 mmol) were added sequentially to DMF (5 mL). The reaction mixture was microwaved at 100 °C for 1 hour. The reaction solution was poured into water (15 mL), extracted with ethyl acetate (20 mL x 2), the organic phases were combined, and concentrated under reduced pressure to obtain a crude product. The crude product was preparatively separated by normal-phase column chromatography to give compound 3b (120 mg, yield: 22.1%).

[0097] MS m / z (ESI): 173.1 [M+H] + .

[0098] Step 5:

[0099] Compound 3b (100 mg, 0.37 mmol), compound 1f (53 mg, 0.24 mmol), cesium carbonate (361.6 mg, 1.11 mmol), and dichloro[1,3-bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) (29.3 mg, 0.04 mmol) were added sequentially to 1,4-dioxane (1 mL). The reaction was microwaved at 100 °C for 2 hours. After filtration, the crude product was concentrated and then separated by reversed-phase column chromatography to obtain compound 1 (5 mg, yield: 3.33%).

[0100] MS m / z (ESI): 407.3 [M+H] + .

[0101] 1 H NMR(400MHz,MeOD)δ7.93(d,J=2.7Hz,1H),7.78(s,1H),7.60(ddt,J=11.1,6.7 ,2.0Hz,2H),7.44(dd,J=9.0,2.7Hz,1H),6.61(ddd,J=9.0,2.9,1.7Hz,2H),6.4 6(td,J=6.8,1.3Hz,1H),4.49–4.43(m,2H),4.30(dp,J=13.4,6.5Hz,2H),4.21– 4.15(m,2H),2.31–2.15(m,2H),1.95(qd,J=6.5,2.6Hz,2H),1.63–1.51(m,2H).

[0102] X-ray powder diffraction analysis showed that the product was amorphous, and the XRPD spectrum, as shown in Figure 5, had no obvious characteristic peaks.

[0103] Test Example 1. Test on the binding force of the disclosed compound to PCSK9.

[0104] Table 1. Main experimental materials and instruments

[0105] 1. Experimental Procedure

[0106] 1.1 Reagent Preparation

[0107] 1) Protein: PCSK9 (Biotin-labeled) (Acro).

[0108] Dissolve the protein in water to a concentration of 200 μg / mL and store at -80°C.

[0109] 2) Chip: SA chip.

[0110] 3) Run buffer: 1x HBSP + 4% DMSO + 0.1mM CaCl2.

[0111] 1.2 Protein fixation

[0112] 1) Preheat the chip to room temperature;

[0113] 2) Prepare the required buffer solution and filter it using a 0.22 μm filter membrane;

[0114] 3) Load the chip and write the chip parameters;

[0115] 4) Dilute PCSK9 protein to 50 μg / mL with PBS;

[0116] 5) Set up a fixed program in the BIAcore S200 instrument;

[0117] 6) Run the fixed program;

[0118] 7) The final fixed response value is 8000RU, and the maximum response value is 49.4RU.

[0119] 1.3 Compound Testing

[0120] 1) Dilute the compounds 3-fold with running buffer. Start with 0.4 μM of the reference compound and dilute 3-fold, and start with 10 μM of the test compound and dilute 3-fold.

[0121] 2) Refer to the table below to prepare the dissolution and correction buffer solution:

[0122] Table 2. Dissolution Correction Buffer

[0123] 3) Set the instrument's "LMW multi kinetics" program as the running program for compound testing.

[0124] a) Set the binding time to 100s and the dissociation time to 300s.

[0125] 4) Run the compound testing program.

[0126] 1.4 Data Analysis

[0127] 1) Rmax = (MW analyte / MW ligand) * RL * Sm

[0128] When using affinity fitting to fit a curve, the response value at the highest concentration should be close to the optimal Rmax value of the fit.

[0129] 2) Check solvent correction and compound concentration gradient.

[0130] 3) Check the baseline, binding, and positive compound results in the test report.

[0131] 4) Select the appropriate fitting method to fit the curve based on the actual situation.

[0132] 5) Check whether the fitting results meet the instrument data quality inspection standards.

[0133] Table 3. Binding affinity of the compounds disclosed herein to PCSK9 (K) D )

[0134] The results show that the binding force of compound 1 is more than 10 times stronger than that of reference compound 2.

[0135] Reference point 2 Example 487 is from patent applications WO2020150473 and WO2020150474.

[0136] Test Example 2. Pharmacokinetic Experiment in Mice

[0137] Using C57BL6J mice as test animals, the plasma drug concentrations at different time points after oral administration of reference compound 1 (WO2020150473 and WO2020150474, Example 458B) and the examples were determined by LC / MS / MS. The pharmacokinetic behavior of reference compound 1 and the compounds of the examples in mice was studied to evaluate their pharmacokinetic characteristics.

[0138] Experimental animals: Two healthy male mice (20-30g) aged 6-8 weeks per group.

[0139] Drug preparation: Weigh a certain amount of drug and prepare a colorless and clear solution of 1 mg / mL (solvent: 5% DMSO / 40% PEG400 / 55% physiological saline).

[0140] Administration: Mice were fasted overnight and then administered the drug by gavage. The dosage of both reference compound 1 and the compound in the example was 10 mg / kg.

[0141] Procedure: Reference 1 and the compound of the example were administered to mice by gavage. At 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration, approximately 0.03 mL of blood was collected by peripheral vein puncture and placed in a test tube containing EDTA-K2. The plasma was separated by centrifugation at 4000g per minute for 5 minutes at approximately 4°C and stored at -75°C.

[0142] To determine the content of the target compound in mouse plasma after oral administration of different concentrations of the drug: 10 μL of mouse plasma was collected at each time point after administration, 5 μL of blank solution and 200 μL of acetonitrile solution containing internal standard dexamethasone were added, vortexed for 30 s, centrifuged for 15 minutes (3900 rpm), and 12 μL of the supernatant of the plasma sample was analyzed by LC / MS / MS.

[0143] Table 4. Pharmacokinetic parameters of reference 1 and compound 1

[0144] Note: All values ​​listed are averages of two animals. Reference 1 is Example 458B in patent applications WO2020150473 and WO2020150474.

[0145] The results show that compound 1 has better oral pharmacokinetic properties in mice, with blood drug exposure nearly three times that of reference compound 1 and a longer half-life, indicating that the pharmacokinetic properties of the compound of the present invention are superior to those of reference compound 1.

[0146] Test Example 3. Effect of the reference compound and the compound of the examples on LDLR levels in HepG2 cells.

[0147] Table 5. Main experimental materials and instruments

[0148] Experimental steps

[0149] HepG2 cells were seeded at 30,000 cells / well in 96-well plates. The next day, 4 nM PCSK9 D374Y was added to each well to stimulate the cells, and the cells were simultaneously treated with the same concentration of the test compound. Each sample was in triplicate. Cells were cultured at 37°C and 5% CO2 for 48 hours. Cells were washed twice with PBS, and the supernatant was discarded. Cells were then placed on ice and treated with 50 μL / well of RIPA lysis buffer containing protease and phosphatase inhibitors. The cell culture plates were frozen at -80°C overnight. After thawing, the cells were lysed on ice for 30 minutes, followed by centrifugation at low temperature for 15 minutes. The cell lysis buffer was diluted 20-fold for ELISA detection. 50 μL / well of sample or standard was added to the coated plates and incubated at room temperature for 2 hours. The cells were washed four times with washing buffer, and 200 μL / well of human LDLR-conjugated antibody was added. The cells were incubated at room temperature for 2 hours. The washing steps were repeated, and 200 μL / well of substrate solution was added each time. After 30 minutes, 50 μL of stop solution was added per well, and the OD value was measured at 450 nm. The LDLR concentration in the sample wells was calculated based on the standard curve, and the LDLR increase percentage was calculated using the formula (sample value - control well mean) / control well mean. Data were processed using GraphPad Prism 8.

[0150] Table 6. Effects of Reference 1 and Example Compounds on LDLR Protein Levels in HepG2 Cells at 75 μM Concentration

[0151] Note: a The average value of the three-hole composite is represented by Example 458B from patent applications WO2020150473 and WO2020150474.

[0152] The results showed that both reference 1 and compound 1 upregulated LDLR levels in HepG2 cells, with compound 1 showing a greater upregulation of LDLR.

[0153] Test Example 4. Metabolite testing of reference 1 and compound 1 in mouse hepatocytes

[0154] 1. Experimental Objective

[0155] The aim of this study was to identify the possible metabolites of the test substance in hepatocytes of different species and to infer its possible metabolic pathways. The test concentration of the test substance was 10 μM.

[0156] 2. Materials and Reagents

[0157] Mouse hepatocytes were preserved in liquid nitrogen. See the table below for details.

[0158] 3. Experimental Design

[0159] 3.1 Preparation of the compound working solution

[0160] The test substance and the control drug verapamil powder were prepared into a high-concentration stock solution with DMSO. Before use, the stock solution was diluted with DMSO to a working solution of 2 mM. The final concentration of the test substance and verapamil was 10 μM.

[0161] 3.2 Preparation of hepatocytes

[0162] 1) Preheat the hepatocyte resuscitation solution and incubation solution in a 37°C water bath for at least 15 minutes before use.

[0163] 2) Take a tube of cryopreserved hepatocytes, ensuring that the hepatocytes remain frozen before thawing. Quickly place the hepatocytes in a 37°C water bath and gently shake until all ice crystals are dispersed. Spray with 70% ethanol and transfer to a biosafety cabinet.

[0164] 3) Pour the contents of the hepatocyte tubules into a centrifuge tube containing 50 mL of resuscitation medium and centrifuge at 100 g for 10 minutes. After centrifugation, aspirate the resuscitation medium and add sufficient incubation medium to obtain a cell density of approximately 1.5 × 10⁻⁶ cells / mL. 6 Cell suspension of cells per mL.

[0165] 4) Use Cellometer Vision to count hepatocytes and determine viable cell density. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 1 × 10⁻⁶. 6 Cells / mL.

[0166] 3.3 Test Methods

[0167] 1) Pipette 1 μL of the 2 mM analyte solution into a 24-well incubation plate, then add 199 μL of hepatocyte suspension to initiate the reaction. Set the time points to 0, 2, and 4 hours. Different time points correspond to different wells. Place the incubation plate back onto the vortex mixer in the incubator (37℃, 5% CO2, 90-95% relative humidity) and incubate at 500 rpm.

[0168] 2) After incubation for the appropriate time, immediately add 400 μL of cold acetonitrile containing 0.1% formic acid to terminate the reaction. Mix well and transfer to the appropriate EP.

[0169] 3) Incubate and process the control drug verapamil according to the above method, and prepare samples only for 0 and 4 hours. The remaining percentage of verapamil at 4 hours will be used to detect enzyme activity.

[0170] 4) All samples were vortexed for 100 seconds, followed by centrifugation at 16,000g for 15 minutes to precipitate proteins. 60 μL of the supernatant was mixed with 60 μL of pure water and analyzed by UHPLC-MS / MS.

[0171] 4. Instrument Configuration

[0172] The Vanquish ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA) is combined with a Thermo Scientific Q Ex-active mass spectrometer (Thermo Fisher Scientific, USA) and equipped with a HESI ion source.

[0173] 5. Mass spectrometry detection

[0174] UHPLC-MS / MS data acquisition and processing were performed using a Q Exactive high-resolution mass spectrometer. A full MS scan was used to trigger data-dependent acquisition (DDA) MS / MS daughter ion scans.

[0175] The relevant mass spectrometry parameters of the test analyte are optimized based on its own properties.

[0176] 6. Data Analysis

[0177] Data processing and analysis were performed using Xcalibur (v3.0 / 4.1, Thermo Fisher Scientific), Compound Discov-erer 3.0 (Thermo Fisher Scientific), and Microsoft Excel 2016.

[0178] Table 7. Residual percentage of parent drug for reference 1 and compound 1 (%)

[0179] Note: Reference 1 is Example 458B from patent applications WO2020150473 and WO2020150474.

[0180] The experimental results show that compound 1 has better stability in mouse hepatocytes than reference compound 1.

[0181] Test Example 5. Stability test of the test compound in hepatocytes

[0182] 1. Experimental Procedure

[0183] 1) Prepare a high-concentration stock solution of the test substance and the control drug verapamil powder with DMSO. Before use, dilute with DMSO to a working solution of 100 μM. The final concentration of the test substance and verapamil is 1 μM.

[0184] 2) Take a tube of cryopreserved hepatocytes, ensuring that the hepatocytes remain frozen before thawing. Quickly place the hepatocytes in a 37°C water bath and gently shake until all ice crystals are dispersed. Spray with 70% ethanol and transfer to a biosafety cabinet.

[0185] 3) Pour the contents of hepatocyte tubules from different species into centrifuge tubes containing 50 mL of resuscitation medium and centrifuge at 100 g for 10 minutes. After centrifugation, aspirate the resuscitation medium and add sufficient incubation medium to obtain a cell density of approximately 1.0 × 10⁻⁶ cells / year. 6 Cell suspension of cells per mL.

[0186] 4) Use Cellometer Vision to count hepatocytes and determine viable cell density. The hepatocyte viability must be greater than 75%. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 0.5 × 10⁻⁶ cells / day. 6 Cells / mL.

[0187] 5) Transfer 198 μL of live cell suspension to a 96-well deep-well plate, place the plate on a vortex and preheat in an incubator for 10 minutes. Perform double parallel incubation.

[0188] 6) Add 2 μL of 100 μM test substance or verapamil to each well to initiate the reaction, and then place the deep well plate back onto the incubator vortex.

[0189] 7) Incubate the sample. At 0, 15, 30, 60, 90, and 120 minutes, respectively, take 25 μL of the suspension and add 150 μL of acetonitrile containing the internal standard to terminate the reaction. Vortex for 10 minutes, then centrifuge at 3220 g and 4 °C for 45 minutes. Transfer 100 μL of the supernatant to the sample plate, add 100 μL of pure water and mix well for UPLC-MS / MS analysis.

[0190] 2. Data Analysis

[0191] All calculations were performed using Microsoft Excel. Peak areas were detected by extracting ion spectra. The in vitro half-life (T0) of the parent drug was determined by linearly fitting the natural logarithm of the elimination percentage of the parent drug to time. 1 / 2 ).

[0192] In vitro half-life (T 1 / 2 ) Calculated by slope: In vitro T 1 / 2 =0.693 / k

[0193] In vitro clearance rate (unit: μL / min / 10) 6 (per cell) is calculated using the following formula: In vitro CL int =kV / NV = Incubation volume per well (0.2 mL); N = Number of cells per well (0.1 × 10⁻⁶) 6 (cells)

[0194] Table 8. Metabolic clearance rate of the tested compounds in mouse hepatocytes

[0195] The results showed that compound 1 had better stability in mouse hepatocytes than reference 1 and reference 2.

[0196] Example 2 Preparation of free crystal form A

[0197] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of ethyl acetate, stir to induce crystallization, centrifuge, collect the solid and dry it under vacuum to obtain the product.

[0198] X-ray powder diffraction analysis identified the product as crystal form A. The XRPD spectrum is shown in Figure 1, and the positions of its characteristic peaks are listed in Table 9. The DSC spectrum shows an endothermic peak at 153.72℃. The TGA spectrum shows no significant weight loss.

[0199] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.28%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.40%; and under extreme conditions (90% RH), the moisture absorption weight gain was approximately 0.89%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0200] Table 9

[0201] Example 3 Preparation of free crystal form A

[0202] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of Table 10 solvent, stir to induce crystallization, centrifuge, collect the solid and dry it under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product is crystal form A.

[0203] Table 10

[0204] Example 4: Preparation of free crystal form A

[0205] 5 mg of the compound shown in Formula 1 was added to 0.1 mL of solvent 1 (Table 11) to dissolve it. Then, 0.3 mL of solvent 2 (Table 11) was added, and the mixture was stirred to induce crystallization. The crystals were then centrifuged, and the solid was collected and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was crystal form A.

[0206] Table 11

[0207] Example 5: Preparation of free crystal form B

[0208] 100 mg of the compound shown in Formula 1 was dissolved in 2 mL of acetone, 2 mL of MTBE was added, the mixture was stirred to induce crystallization, filtered under reduced pressure, and the solid was collected and dried under vacuum at 40 °C to obtain the product.

[0209] X-ray powder diffraction analysis identified the product as crystal form B. The XRPD spectrum is shown in Figure 2, and the positions of its characteristic peaks are listed in Table 12. The DSC spectrum shows an endothermic peak at 134.97℃. The TGA spectrum shows a weight loss of 1.04% between 30℃ and 100℃.

[0210] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.46%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.89%; and under extreme conditions (90% RH), the moisture absorption weight gain was approximately 2.29%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0211] Table 12

[0212] Example 6 Preparation of free crystal form B

[0213] Add 5 mg of compound 1 to 0.5 mL of solvent from Table 13, stir to induce crystallization, centrifuge, collect the solid and dry under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product is crystal form B.

[0214] Table 13

[0215] Example 7 Preparation of free crystal form B

[0216] 5 mg of the compound shown in Formula 1 was dissolved in 0.1 mL of Table 14 solvent 3, followed by the addition of 0.3 mL of Table 14 solvent 4. The mixture was centrifuged, and the solid was collected and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was crystal form B.

[0217] Table 14

[0218] Example 8: Preparation of Free Crystal Form B

[0219] 5 mg of the compound shown in Formula 1 was dissolved in 0.5 mL of solvent from Table 15 and allowed to evaporate slowly at room temperature to obtain the product. X-ray powder diffraction analysis showed that the product was crystal form B.

[0220] Table 15

[0221] Example 9: Preparation of Free Crystal Form B

[0222] 10 mg of the compound shown in Formula 1 was dissolved in 0.1 mL of solvent from Table 16 at room temperature, stirred to induce crystallization, filtered, and the solid was dried under vacuum at 40 °C to obtain a solid product. X-ray powder diffraction analysis showed that the product was crystal form B.

[0223] Table 16

[0224] Example 10 Preparation of free crystal form B

[0225] 10 mg of the compound shown in Formula 1 was dissolved in 0.1 mL of solvent from Table 17 at 85 °C. The solution was then cooled to 25 °C with stirring to induce crystallization. The solution was filtered, and the solid was dried under vacuum at 40 °C to obtain the solid product. X-ray powder diffraction analysis showed that the product was crystal form B.

[0226] Table 17

[0227] Example 11 Preparation of free crystalline form C

[0228] 120 mg of the compound shown in Formula 1 was added to 1.2 mL of water, stirred to induce crystallization, centrifuged, and the solid was collected and dried at 40 °C to obtain the product.

[0229] X-ray powder diffraction analysis identified the product as crystal form C. The XRD pattern is shown in Figure 3, and the positions of its characteristic peaks are listed in Table 18. The DSC spectrum shows endothermic peaks at 80.36℃ and 152.66℃. The TGA spectrum shows a weight loss of 13.28% between 30℃ and 120℃.

[0230] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 5.07%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 7.79%; and under extreme conditions (90% RH), the moisture absorption weight gain was approximately 16.89%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0231] Table 18

[0232] Example 12 Preparation of free crystal form D

[0233] Add 5 mg of the compound shown in Formula 1 to 0.1 mL of 2-butanone, stir to induce crystallization, centrifuge, and obtain the product as a wet sample.

[0234] X-ray powder diffraction analysis determined the product to be crystal form D. The XRPD spectrum is shown in Figure 4, and the positions of its characteristic peaks are shown in Table 19.

[0235] Table 19

[0236] Example 13: Study on Crystal Form Stability

[0237] The aforementioned free A and B crystal forms were laid flat in the open, and the stability of the samples was investigated under light (4500 Lux), high temperature (40℃, 60℃), and high humidity (RH 75%, RH 92.5%) conditions. The sampling and investigation period was 1 month.

[0238] Table 20 Factors Affecting the Stability of Crystal Form A

[0239] Conclusion: Crystal form A exhibits good chemical stability after 30 days of exposure to light, high temperature, and high humidity.

[0240] Table 21 Factors Affecting Stability of B Crystal Form

[0241] Conclusion: Crystal form B exhibits good physicochemical stability after being placed under light, high temperature, and high humidity conditions for 30 days.

[0242] Example 14 Long-term / Accelerated Stability

[0243] The stability of crystal forms A and B was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH, respectively.

[0244] Table 22 Long-term / accelerated stability of crystal form A

[0245] Conclusion: Crystal form A exhibits good physical and chemical stability under long-term / accelerated conditions for 6 months.

[0246] Table 23 Long-term / accelerated stability of B crystal form

[0247] Conclusion: Crystal form B exhibits good physical and chemical stability under long-term / accelerated conditions for 6 months.

Claims

1. A crystal form A of a compound of formula 1, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 5.008, 14.411, 17.012, 18.441, and 20.941, preferably at 5.008, 14.411, 14.973, 17.012, 18.441, 19.593, 20.941, 21.419, 24.736, and 25.279, and more preferably at 5.008, 13.700, 14.411, 14.973, 15.210, 17.012, 18.441, 18.885, 19.593, 20.941, 21.419, 22.594, 23.531, 24.736, and 25.

279.

2. The crystal form A according to claim 1, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 1.

3. A method for preparing crystal form A as described in claim 1 or 2, the method comprising any of the following methods: Method 1: Mix the compound of Formula 1 with solvent I and stir. Solvent I is selected from one or more of ethyl acetate, isopropyl acetate, ethanol, methyl tert-butyl ether, n-heptane, cyclohexane, and isopropyl ether. Method 2: Mix the compound of Formula 1 with solvent II, then add solvent III and stir. Solvent II is selected from acetone and tetrahydrofuran, and solvent III is selected from n-heptane and methyl tert-butyl ether.

4. A crystal form B of a compound of formula 1, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 5.834, 9.606, 10.122, 15.516, and 17.142, preferably at 5.834, 9.606, 10.122, 11.061, 15.516, 17.142, and 24.992, and more preferably at 5.834, 9.606, 10.122, 11.061, 11.656, 15.516, 17.142, 19.229, 20.319, 24.992, and 25.

616.

5. The crystal form B according to claim 4, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 2.

6. A method for preparing crystal form B as described in claim 4 or 5, the method comprising any of the following methods: Method 1: The compound of Formula 1 is mixed with solvent IV and stirred. Solvent IV is selected from one or more of water, methanol, ethanol, isopropanol, 2-butanone, ethyl acetate, acetone, dichloromethane, and 2-methyltetrahydrofuran. Method 2: Mix the compound of Formula 1 with solvent V, and then add solvent VI, wherein solvent V is selected from one or more of ethanol, acetone, acetonitrile, tetrahydrofuran, dichloromethane, and methanol, and solvent VI is selected from n-heptane, methyl tert-butyl ether, and isopropyl ether; Method 3: The compound of Formula 1 is mixed with solvent VII, and the solvent is evaporated. Solvent VII is selected from one or more of water, methanol, ethanol, n-propanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, propylene glycol methyl ether, N,N-dimethylformamide, and dimethyl sulfoxide. Method 4: The compound of Formula 1 is mixed with solvent VIII, heated to dissolve, and cooled to crystallize. Solvent VIII is selected from one or more of water, methanol, ethanol, acetone, 2-methyltetrahydrofuran, and ethyl acetate.

7. A crystal form C of a compound of formula 1, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 8.096, 18.515, 21.509, 23.739, and 24.274, preferably at 8.096, 9.166, 15.945, 18.515, 19.708, 21.509, 22.088, 23.739, 24.274, and 26.668, and more preferably at 8.096, 9.166, 10.826, 15.568, 15.945, 16.360, 18.515, 19.708, 21.509, 22.088, 23.739, 24.274, and 26.

668.

8. The crystal form C according to claim 7, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 3.

9. A method for preparing crystal form C as described in claim 7 or 8, the method comprising the steps of mixing the compound of formula 1 with water and stirring.

10. A crystal form D of a compound of formula 1, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 13.532, 13.889, 19.679, 22.352, and 24.910, preferably at 12.410, 13.532, 13.889, 15.952, 19.679, 22.352, 24.910, and 25.346, and more preferably at 12.410, 13.532, 13.889, 15.952, 16.532, 19.679, 22.352, 23.104, 24.910, and 25.

346.

11. The crystal form D according to claim 10, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 4.

12. A method for preparing crystal form D as described in claim 10 or 11, the method comprising the steps of mixing the compound of formula 1 with 2-butanone and stirring.

13. The crystal form according to any one of claims 1-2, 4-5, 7-8, 10-11, wherein the 2θ angle error range is ±0.

20.

14. A pharmaceutical composition comprising the crystal form as described in any one of claims 1-2, 4-5, 7-8, 10-11, and 13, and optionally a pharmaceutically acceptable excipient.

15. A method for preparing a pharmaceutical composition, comprising the step of mixing the crystal form according to any one of claims 1-2, 4-5, 7-8, 10-11, and 13 with a pharmaceutically acceptable excipient.

16. Use of a crystal form according to any one of claims 1-2, 4-5, 7-8, 10-11, 13, or the pharmaceutical composition according to claim 14 in the preparation of a PCSK9 inhibitor.

17. The use of the crystal form according to any one of claims 1-2, 4-5, 7-8, 10-11, and 13, or the pharmaceutical composition according to claim 14, in the preparation of a medicament for the treatment and / or prevention of dyslipidemia, dyslipoproteinemia, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, hyperlipoproteinemia, xanthoma, hypoalpha-lipoproteinemia, sitosterolemia, atherosclerosis, arteriosclerosis, metabolic syndrome, coronary heart disease, peripheral vascular disease, congestive heart failure, stroke, vascular dementia, coronary artery disease, chronic kidney disease, retinopathy, inflammation, diabetic complications, or thrombosis.

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