Pharmaceutically acceptable salt of diaminocyclopentyl-substituted heteroaryl derivative, crystalline form thereof, and use thereof

Abstract

The present disclosure relates to a pharmaceutically acceptable salt of a diaminocyclopentyl-substituted heteroaryl derivative, a crystalline form thereof, and use thereof. Specifically, the present disclosure provides a pharmaceutically acceptable salt 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, a crystal form thereof, and a preparation method therefor. The corresponding salt has good stability and can be better used for clinical treatment.

Description

A pharmaceutically acceptable salt of a diaminocyclopentyl-substituted heteroaryl derivative, its crystalline form and uses Technical Field

[0001] This disclosure pertains to the field of pharmaceutical technology and relates to a pharmaceutically acceptable salt of a diaminocyclopentyl-substituted heteroaryl derivative, its crystalline form, and its uses. 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 pharmaceutically acceptable salt of compound 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, wherein the pharmaceutically acceptable salt is selected from fumarate, succinate, hydrochloride, phosphate, tartrate, citrate, acetate, malate, methanesulfonate, benzoate, p-toluenesulfonate, maleate, and sulfate.

[0006] In some embodiments, the chemical ratio of the compound of Formula 1 to the acid is 3:1 to 1:3, including but not limited to 3:1, 2:1, 1:1, 1:2, and 1:3.

[0007] In an optional embodiment, the chemical ratio of the compound of Formula 1 to the acid is 2:1 to 1:2.

[0008] In an optional embodiment, the chemical ratio of the compound of Formula 1 to the acid is 1:1 or 1:2.

[0009] In an optional embodiment, the chemical ratio of the compound of Formula 1 to hydrochloric acid is 2:1 to 1:2.

[0010] In an optional embodiment, the chemical ratio of the compound of Formula 1 to fumaric acid is 2:1 to 1:2.

[0011] In an optional embodiment, the chemical ratio of the compound of Formula 1 to fumaric acid is 1:1.

[0012] In an optional embodiment, the chemical ratio of the compound of Formula 1 to succinic acid is 2:1 to 1:2.

[0013] In an optional embodiment, the chemical ratio of the compound of Formula 1 to succinic acid is 1:1.

[0014] In an optional embodiment, the chemical ratio of the compound of Formula 1 to phosphoric acid is 2:1 to 1:2.

[0015] In an optional embodiment, the chemical ratio of the compound of Formula 1 to tartaric acid is 2:1 to 1:2.

[0016] In an optional embodiment, the chemical ratio of the compound of Formula 1 to citric acid is 2:1 to 1:2.

[0017] This disclosure also provides a method for preparing a pharmaceutically acceptable salt of a compound of formula 1, comprising the step of reacting the compound of formula 1 with an acid selected from fumaric acid, succinic acid, hydrochloric acid, phosphoric acid, tartaric acid, citric acid, acetic acid, malic acid, methanesulfonic acid, benzoic acid, p-toluenesulfonic acid, maleic acid, and sulfuric acid.

[0018] In some embodiments, the method for preparing a pharmaceutical salt of compound 1 is described, wherein the chemical ratio of compound 1 to acid is 3:1 to 1:3, including but not limited to 3:1, 2:1, 1:1, 1:2, and 1:3.

[0019] In an optional embodiment, the chemical ratio of the compound of Formula 1 to the acid is 2:1 to 1:2.

[0020] In an optional embodiment, the chemical ratio of the compound of Formula 1 to the acid is 1:1 or 1:2.

[0021] The method for preparing pharmaceutically acceptable salts of the compound of Formula 1 involves reacting the compound in a conventional solvent selected from, but not limited to, acetone, ethyl acetate, acetonitrile, ethanol, or 2-methyltetrahydrofuran.

[0022] Furthermore, in an optional embodiment, the method for preparing the aforementioned pharmaceutically acceptable salt also includes steps such as crystallization, filtration, washing, or drying.

[0023] The fumarate crystal form I of the compound of Formula 1 provided in this disclosure has characteristic peaks at 6.009, 10.840, 15.694, and 20.881 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0024] In some embodiments, the fumarate crystal form I of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 6.009, 10.238, 10.840, 11.972, 13.639, 15.694, 17.962, and 20.881.

[0025] In some embodiments, the fumarate crystal form I of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 6.009, 10.238, 10.840, 11.972, 13.639, 15.694, 17.609, 17.962, 20.881, and 21.693.

[0026] In some embodiments, the X-ray powder diffraction pattern of the fumarate crystal form I of compound 1, expressed as a diffraction angle 2θ, is shown in Figure 2.

[0027] This disclosure also provides a method for preparing fumarate crystal form I of compound formula 1, wherein the method is selected from any of the following methods:

[0028] Method 1: Mix the compound of Formula 1 with ethanol, then add a methanol solution of fumaric acid and stir.

[0029] Method 2: Mix the compound of Formula 1 with solvent I, then add fumaric acid and stir. Solvent I is selected from ethanol, acetone and ethyl acetate.

[0030] In an optional embodiment, the method for preparing compound formula 1 fumarate crystal form I further includes a filtration or centrifugation step.

[0031] The fumarate crystal form II of the compound of formula 1 provided in this disclosure has characteristic peaks at 6.666, 15.702, 20.023, 22.931, and 24.668 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0032] In some embodiments, the fumarate crystal form II of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 6.666, 12.336, 15.702, 18.200, 20.023, 21.641, 22.931, and 24.668.

[0033] In some embodiments, the fumarate crystal form II of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 6.666, 12.336, 13.907, 15.702, 18.200, 20.023, 21.641, 22.340, 22.931, and 24.668.

[0034] In some embodiments, the X-ray powder diffraction pattern of the fumarate crystal form II of compound 1, expressed in terms of diffraction angle 2θ, is shown in Figure 3.

[0035] This disclosure also provides a method for preparing fumarate crystal form II of compound formula 1, the method comprising the steps of mixing compound formula 1 with ethanol, and then adding fumaric acid and stirring.

[0036] The succinate crystal form I of the compound of formula 1 provided in this disclosure has characteristic peaks at 6.256, 9.500, 10.016, 14.960, and 15.880 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0037] In some embodiments, the succinate crystal form I of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 5.972, 6.256, 9.500, 10.016, 10.803, 14.960, 15.880, and 20.215.

[0038] In some embodiments, the succinate crystal form I of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 5.972, 6.256, 9.500, 10.016, 10.803, 11.796, 14.960, 15.880, 19.172, and 20.215.

[0039] In some embodiments, the X-ray powder diffraction pattern of the succinate crystal form I of compound 1, expressed in terms of diffraction angle 2θ, is shown in Figure 4.

[0040] This disclosure also provides a method for preparing succinate crystal form I of compound formula 1, the method comprising the steps of mixing compound formula 1 with solvent II, and then adding succinic acid and stirring, wherein solvent II is selected from acetone and ethanol.

[0041] In an optional embodiment, the method for preparing succinate crystal form I of compound formula 1 further includes a centrifugation step.

[0042] This disclosure provides the succinate crystal form II of the compound of formula 1, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, with characteristic peaks at 6.248, 9.742, 15.752, 20.152, and 23.119.

[0043] In some embodiments, the succinate form II of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 6.248, 9.742, 10.575, 11.724, 15.752, 20.152, 23.119, and 31.652.

[0044] In some embodiments, the succinate form II of the compound of formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 6.248, 9.742, 10.575, 11.724, 15.752, 17.507, 20.152, 23.119, 27.110, and 31.652.

[0045] In some embodiments, the X-ray powder diffraction pattern of the succinate form II of compound 1, expressed in terms of diffraction angle 2θ, is shown in Figure 5.

[0046] This disclosure also provides a method for preparing succinate form II of the compound of formula 1, the method comprising the steps of mixing the compound of formula 1 with ethyl acetate, and then adding succinic acid and stirring.

[0047] In an optional embodiment, the method for preparing succinate form II of the compound of formula 1 further includes a centrifugation step.

[0048] This disclosure provides the hydrochloride crystal form I of the compound of formula 1, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, which has characteristic peaks at 8.304, 16.789, 19.065, 22.082, and 22.753.

[0049] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride crystal form I of the compound of formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.304, 16.789, 19.065, 20.461, 22.082, 22.753, 24.296, and 27.115.

[0050] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride crystal form I of compound 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.304, 16.789, 19.065, 20.461, 22.082, 22.753, 24.296, 26.282, 27.115, 29.038, and 30.211.

[0051] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride crystal form I of compound of formula 1, expressed in terms of diffraction angle 2θ, is shown in Figure 1.

[0052] This disclosure also provides a method for preparing the hydrochloride crystal form I of compound of formula 1, the method comprising the steps of mixing compound of formula 1 with solvent III, and then adding hydrochloric acid ethanol solution and stirring, wherein solvent III is selected from acetone and ethyl acetate.

[0053] In an optional embodiment, the method for preparing the hydrochloride crystal form I of compound formula 1 further includes a centrifugation step.

[0054] In some embodiments, the phosphate of the compound of formula 1 provided in this disclosure is amorphous, and its X-ray powder diffraction pattern has no obvious characteristic peaks in the diffraction angle 2θ range of 3-45°.

[0055] This disclosure also provides a method for preparing an amorphous phosphate of compound of formula 1, comprising the steps of mixing compound of formula 1 with a solvent selected from acetone, ethanol, and ethyl acetate, and then adding a phosphate ethanol solution and stirring.

[0056] In an optional embodiment, the method for preparing the amorphous phosphate of compound 1 further includes a centrifugation step.

[0057] In some embodiments, the tartrate salt of the compound of formula 1 provided in this disclosure is amorphous, and its X-ray powder diffraction pattern has no obvious characteristic peaks in the diffraction angle 2θ range of 3-45°.

[0058] This disclosure also provides a method for preparing the amorphous tartrate salt of compound 1, comprising the steps of mixing the compound of formula 1 with a solvent selected from acetone, ethanol, and ethyl acetate, and then adding a tartaric acid ethanol solution and stirring.

[0059] In an optional embodiment, the method for preparing the amorphous tartrate salt of compound formula 1 further includes a centrifugation step.

[0060] In some embodiments, the citrate of the compound of formula 1 provided in this disclosure is amorphous, and its X-ray powder diffraction pattern has no obvious characteristic peaks in the diffraction angle 2θ range of 3-45°.

[0061] This disclosure also provides a method for preparing the amorphous citrate of compound of formula 1, comprising the steps of mixing compound of formula 1 with ethyl acetate and then adding a citric acid ethanol solution and stirring.

[0062] In an optional embodiment, the method for preparing the amorphous citrate of compound 1 further includes a centrifugation step.

[0063] This disclosure also provides a pharmaceutical composition comprising a fumarate, succinate, hydrochloride, phosphate, tartrate, citrate, acetate, malate, methanesulfonate, benzoate, p-toluenesulfonate, maleate, sulfate, or a crystal form thereof of the aforementioned compound of formula 1, and optionally a pharmaceutically acceptable excipient.

[0064] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the fumarate, succinate, hydrochloride, phosphate, tartrate, citrate, acetate, malate, methanesulfonate, benzoate, p-toluenesulfonate, maleate, sulfate, or a crystal form thereof of the aforementioned compound of formula 1 with a pharmaceutically acceptable excipient.

[0065] 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.

[0066] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, transporter, diluent, and / or delivery polymer.

[0067] This disclosure also provides the use of the fumarate, succinate, hydrochloride, phosphate, tartrate, citrate, acetate, malate, methanesulfonate, benzoate, p-toluenesulfonate, maleate, sulfate, or their crystal forms thereof, or the aforementioned pharmaceutical compositions, in the preparation of medicaments 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.

[0068] On the other hand, this disclosure provides a fumarate, succinate, hydrochloride, phosphate, tartrate, citrate, acetate, malate, methanesulfonate, benzoate, p-toluenesulfonate, maleate, sulfate or a crystal form thereof of the aforementioned compound of formula 1, or the use of the aforementioned pharmaceutical composition in the preparation of a PCSK9 inhibitor.

[0069] 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.

[0070] 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%.

[0071] 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.

[0072] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 40℃-70℃, and can be dried under normal pressure or reduced pressure.

[0073] 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.

[0074] 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.

[0075] 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,

[0076] Deliquescence: Absorbs sufficient moisture to form a liquid;

[0077] Extremely hygroscopic: the weight gain due to hygroscopic absorption is not less than 15%;

[0078] It has hygroscopic properties: the weight gain due to hygroscopic absorption is less than 15% but not less than 2%;

[0079] Slightly hygroscopic: the weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0080] It has little or no hygroscopicity: the weight gain due to moisture absorption is less than 0.2%.

[0081] 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.

[0082] In this disclosure, "mixing" means that the order of addition of the components is not limited. Mixing A and B can mean either adding A to B or adding B to A. Attached Figure Description

[0083] Figure 1 shows the XRPD spectrum of the hydrochloride crystal form I of compound formula 1.

[0084] Figure 2 shows the XRPD spectrum of fumarate crystal form I of compound formula 1.

[0085] Figure 3 shows the XRPD spectrum of fumarate form II of compound 1.

[0086] Figure 4 shows the XRPD spectrum of succinate crystal form I of compound 1.

[0087] Figure 5 shows the XRPD spectrum of succinate form II of compound 1.

[0088] Figure 6 shows the XRPD spectrum of the amorphous phosphate of compound 1.

[0089] Figure 7 shows the XRPD spectrum of the amorphous form of compound tartrate of Formula 1.

[0090] Figure 8 shows the XRPD spectrum of the amorphous citrate of compound 1. Detailed Implementation

[0091] 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.

[0092] Test conditions of the instruments used in the experiment:

[0093] 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 ohms. -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.

[0094] 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).

[0095] MS measurements were performed using a Waters SQD2 mass spectrometer in positive / negative ion mode, with a mass scan range of 100–1200.

[0096] 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.

[0097] 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.

[0098] Rapid column purification systems use either the Combiflash Rf150 (TELEDYNE ISCO) or Isolara One (Biotage).

[0099] 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).

[0100] Reversed-phase column chromatography typically uses Changzhou Sante pre-packed ultrapure C18 silica gel columns (20-45μm). 40g, 80g, 120g, 220g or other sizes).

[0101] 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 T3OBD Prep Column, 5μm, 19mm×150mm.

[0102] Chiral preparation columns used were DAICL CHIRALPAK IC (250 mm × 30 mm, 10 μm) or Phenomenex-Amylose-1 (250 mm × 30 mm, 5 μm).

[0103] 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°.

[0104] 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 25-350℃), and the nitrogen purging rate was 50mL / min.

[0105] TGA is thermogravimetric analysis: the test was performed using a METTLER TOLEDO TGA 2 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.

[0106] DVS stands for Dynamic Moisture Adsorption: The detection method uses SMSDVS Advantage, with humidity changes of 50%-95%-0%-95%-50% at 25℃, in 10% increments (5% in the final step). The judgment criterion is Tmax 360min, and dm / dt not greater than 0.002%.

[0107] 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.

[0108] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0109] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0110] 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.

[0111] Example 1

[0112] 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)

[0113] Step 1:

[0114] 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%).

[0115] MS m / z(ESI): 207.1 [M+H] + .

[0116] Step 2:

[0117] 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%).

[0118] MS m / z (ESI): 371.3 [M+H] + .

[0119] Step 3:

[0120] 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%).

[0121] MS m / z(ESI): 271.2 [M+H] + .

[0122] Step 4:

[0123] 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%).

[0124] MS m / z (ESI): 173.1 [M+H] + .

[0125] Step 5:

[0126] 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%).

[0127] MS m / z (ESI): 407.3 [M+H] + .

[0128] 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).

[0129] X-ray powder diffraction analysis revealed that the product was amorphous, and the XRPD spectrum showed no obvious characteristic peaks.

[0130] Test Example 1. Test on the binding force of the disclosed compound to PCSK9.

[0131] Table 1. Main experimental materials and instruments

[0132] 1. Experimental Procedure

[0133] 1.1 Reagent Preparation

[0134] 1) Protein: PCSK9 (Biotin-labeled) (Acro).

[0135] Dissolve the protein in water to a concentration of 200 μg / mL and store at -80°C.

[0136] 2) Chip: SA chip.

[0137] 3) Run buffer: 1x HBSP + 4% DMSO + 0.1mM CaCl2.

[0138] 1.2 Protein fixation

[0139] 1) Preheat the chip to room temperature;

[0140] 2) Prepare the required buffer solution and filter it using a 0.22 μm filter membrane;

[0141] 3) Load the chip and write the chip parameters;

[0142] 4) Dilute PCSK9 protein to 50 μg / mL with PBS;

[0143] 5) Set up a fixed program in the BIAcore S200 instrument;

[0144] 6) Run the fixed program;

[0145] 7) The final fixed response value is 8000RU, and the maximum response value is 49.4RU.

[0146] 1.3 Compound Testing

[0147] 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.

[0148] 2) Refer to the table below to prepare the dissolution and correction buffer solution:

[0149] Table 2. Dissolution Correction Buffer

[0150] 3) Set the instrument's "LMW multi kinetics" program as the running program for compound testing.

[0151] a) Set the binding time to 100s and the dissociation time to 300s.

[0152] 4) Run the compound testing program.

[0153] 1.4 Data Analysis

[0154] 1) Rmax = (MW analyte / MW ligand) * RL * Sm

[0155] 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.

[0156] 2) Check solvent correction and compound concentration gradient.

[0157] 3) Check the baseline, binding, and positive compound results in the test report.

[0158] 4) Select the appropriate fitting method to fit the curve based on the actual situation.

[0159] 5) Check whether the fitting results meet the instrument data quality inspection standards.

[0160] Table 3. Binding affinity of the compounds disclosed herein to PCSK9 (K) D )

[0161] The results show that the binding force of compound 1 is more than 10 times stronger than that of reference compound 2.

[0162] Reference point 2 Example 487 is from patent applications WO2020150473 and WO2020150474.

[0163] Test Example 2. Pharmacokinetic Experiment in Mice

[0164] 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.

[0165] Experimental animals: Two healthy male mice (20-30g) aged 6-8 weeks per group.

[0166] 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).

[0167] 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.

[0168] 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.

[0169] 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.

[0170] Table 4. Pharmacokinetic parameters of reference 1 and compound 1 Note: All values ​​listed are averages of two animals. Reference 1 is Example 458B in patent applications WO2020150473 and WO2020150474.

[0171] 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.

[0172] Test Example 3. Effect of the reference compound and the compound of the examples on LDLR levels in HepG2 cells.

[0173] Table 5. Main experimental materials and instruments

[0174] Experimental steps

[0175] 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.

[0176] Table 6. Effects of Reference 1 and Example Compounds on LDLR Protein Levels in HepG2 Cells at 75 μM Concentration Note: a The average value of the three-hole composite is represented by Example 458B from patent applications WO2020150473 and WO2020150474.

[0177] 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.

[0178] Test Example 4. Metabolite testing of reference 1 and compound 1 in mouse hepatocytes

[0179] 1. Experimental Objective

[0180] 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.

[0181] 2. Materials and Reagents

[0182] Mouse hepatocytes were preserved in liquid nitrogen. See the table below for details.

[0183] 3. Experimental Design

[0184] 3.1 Preparation of the compound working solution

[0185] 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.

[0186] 3.2 Preparation of hepatocytes

[0187] 1) Preheat the hepatocyte resuscitation solution and incubation solution in a 37°C water bath for at least 15 minutes before use.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 3.3 Test Methods

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 4. Instrument Configuration

[0197] The Vanquish ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA) is combined with a Thermo Scientific Q Exact-tive mass spectrometer (Thermo Fisher Scientific, USA) and equipped with a HESI ion source.

[0198] 5. Mass spectrometry detection

[0199] 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.

[0200] The relevant mass spectrometry parameters of the test analyte are optimized based on its own properties.

[0201] 6. Data Analysis

[0202] 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.

[0203] Table 7. Residual percentage of parent drug for reference 1 and compound 1 (%) Note: Reference 1 is Example 458B from patent applications WO2020150473 and WO2020150474.

[0204] The experimental results show that compound 1 has better stability in mouse hepatocytes than reference compound 1.

[0205] Test Example 5. Stability test of the test compound in hepatocytes

[0206] 1. Experimental Procedure

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 2. Data Analysis

[0215] 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 ).

[0216] In vitro half-life (T 1 / 2 ) Calculated by slope: In vitro T 1 / 2 =0.693 / k

[0217] 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)

[0218] Table 8. Metabolic clearance rate of the tested compounds in mouse hepatocytes

[0219] The results showed that compound 1 had better stability in mouse hepatocytes than reference 1 and reference 2.

[0220] Example 2 Preparation of Hydrochloride Crystal Form I

[0221] Add 8 mg of the compound shown in Formula 1 to 0.2 mL of the solvent in Table 9, add 21.0 μl of 2M hydrochloric acid-ethanol solution, stir to induce crystallization, centrifuge, collect the solid and dry it under vacuum to obtain the product.

[0222] X-ray powder diffraction analysis determined the product to be hydrochloride crystal form I. The XRPD spectrum is shown in Figure 1, and the positions of its characteristic peaks are shown in Table 10.

[0223] Ion chromatography showed a chloride ion content of 13.16%. DSC chromatograms showed endothermic peaks at 163.51℃, 184.18℃, 215.35℃, and 284.68℃. TGA chromatograms showed a weight loss of 4.74% between 30℃ and 120℃, and a weight loss of 6.05% between 120℃ and 220℃.

[0224] Table 9

[0225] Table 10

[0226] Example 3 Preparation of fumarate crystal form I

[0227] 100 mg of the compound shown in Formula 1 was dissolved in 2 mL of ethanol, and then 0.5 mL of 0.5 M fumaric acid methanol solution was added. The mixture was stirred to induce crystallization, filtered under reduced pressure, and the solid was collected and dried under vacuum to obtain the product.

[0228] X-ray powder diffraction analysis identified the product as fumarate crystal form I. The XRPD spectrum is shown in Figure 2, and the characteristic peak positions are listed in Table 11. Ion chromatography determined the fumaric acid content to be 22.16%. The DSC spectrum showed an endothermic peak at 221.24℃. The TGA spectrum showed no significant weight loss between 30℃ and 101℃, and a weight loss of 15.29% between 100℃ and 261℃.

[0229] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.30%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.37%; and under extreme conditions (90% RH), the moisture absorption weight gain was approximately 0.69%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0230] Table 11

[0231] Example 4 Preparation of fumarate crystal form I

[0232] Add 8 mg of the compound shown in formula (I) to 0.2 mL of Table 12 solvent, add 2.2 mg of fumaric acid solid, stir to induce crystallization, centrifuge, collect the solid and dry it under vacuum to obtain the product.

[0233] Table 12

[0234] Example 5: Preparation of fumarate crystal form IⅠ

[0235] 100 mg of the compound shown in Formula 1 was added to 2 mL of ethanol, followed by 29 mg of solid fumaric acid. The mixture was stirred for 2 h to obtain a wet sample. X-ray powder diffraction analysis identified the product as fumarate crystal form II. The XPRD spectrum is shown in Figure 3, and the positions of its characteristic peaks are shown in Table 13.

[0236] Table 13

[0237] Example 6 Preparation of Succinate Crystal Form I

[0238] 100 mg of the compound shown in Formula 1 was added to 2 mL of solvent from Table 14, followed by 29.5 mg of succinic acid solid. The mixture was stirred to induce crystallization, centrifuged, and the solid was collected and dried under vacuum to obtain the product.

[0239] X-ray powder diffraction analysis identified the product as succinate crystal form I. The XRPD spectrum is shown in Figure 4, and the characteristic peak positions are listed in Table 15. Ion chromatography determined the succinic acid content to be 24.27%. The DSC spectrum showed an endothermic peak at 176.53℃. The TGA spectrum showed no significant weight loss between 31℃ and 100℃, and a weight loss of 19.22% between 100℃ and 260℃.

[0240] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.57%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.96%; and under extreme conditions (90% RH), the moisture absorption weight gain was approximately 2.14%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0241] Table 14

[0242] Table 15

[0243] Example 7 Preparation of Succinate Crystal Form II

[0244] 100 mg of the compound shown in Formula 1 was added to 2 mL of ethyl acetate, followed by 29.5 mg of succinic acid solid. The mixture was stirred to induce crystallization, centrifuged, and the solid was collected and dried under vacuum to obtain the product.

[0245] X-ray powder diffraction analysis identified the product as succinate crystal form II. The XRPD spectrum is shown in Figure 5, and the characteristic peak positions are listed in Table 16. Ion chromatography determined the succinic acid content to be 23.56%. The DSC spectrum showed an endothermic peak at 174.67℃. The TGA spectrum showed no significant weight loss between 30℃ and 100℃, and a weight loss of 17.46% between 100℃ and 260℃.

[0246] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.41%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.68%; and under extreme conditions (90% RH), the moisture absorption weight gain was approximately 1.63%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0247] Table 16

[0248] Example 8: Preparation of amorphous phosphate

[0249] Add 8 mg of the compound shown in Formula 1 to 0.2 mL of the solvent in Table 17, add 21.0 μl of 2M phosphate ethanol solution, stir to induce crystallization, centrifuge, collect the solid and dry it under vacuum to obtain the product.

[0250] X-ray powder diffraction analysis revealed that the product was an amorphous phosphate, as shown in Figure 6 (XRPD pattern). Ion chromatography analysis showed that its phosphate content was 29.34%.

[0251] Table 17

[0252] Example 9: Preparation of amorphous tartrate salts

[0253] Add 8 mg of the compound shown in formula (I) to 0.2 mL of Table 18 solvent, add 10.5 μl of 2M tartaric acid ethanol solution, stir to induce crystallization, centrifuge, collect the solid and dry it under vacuum to obtain the product.

[0254] X-ray powder diffraction analysis revealed that the product was an amorphous tartrate salt, as shown in Figure 7 (XRPD pattern). Ion chromatography analysis showed that its tartrate content was 28.97%.

[0255] Table 18

[0256] Example 10 Preparation of amorphous citrate

[0257] Add 8 mg of the compound shown in Formula 1 to 0.2 mL of ethyl acetate, add 21.0 μl of 2 M citric acid ethanol solution, stir to induce crystallization, centrifuge, collect the solid and dry under vacuum to obtain the product.

[0258] X-ray powder diffraction analysis revealed that the product was an amorphous citrate, as shown in Figure 8 (XRPD pattern). Ion chromatography analysis showed a citrate content of 32.01%.

[0259] Example 11: Study on Crystal Form Stability

[0260] Fumarate crystal form I, succinate crystal form I, and succinate crystal form II were laid out in an open manner, 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 period was one month.

[0261] Table 19 Factors Affecting the Stability of Fumarate Crystal Form I

[0262] Conclusion: Fumarate crystal form I exhibits good physicochemical stability after being placed under high temperature, high humidity, and light conditions for 30 days.

[0263] Table 20 Factors Affecting the Stability of Succinate Crystal Form I

[0264] Conclusion: Succinate crystal form I exhibits good physicochemical stability after being placed under high temperature, high humidity, and light conditions for 30 days.

[0265] Table 21 Factors affecting the stability of succinate crystal form II

[0266] Conclusion: Succinate crystal form II exhibits good physicochemical stability after 30 days of exposure to high temperature, high humidity, and light.

[0267] Example 12 Long-term / Accelerated Stability

[0268] The stability of fumarate crystal form I and succinate crystal forms I / II was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH, respectively.

[0269] Table 22 Long-term / Accelerated Stability of Fumarate Crystal Form I

[0270] Conclusion: Fumarate crystal form I exhibits good physical and chemical stability under accelerated and long-term conditions for 6 months.

[0271] Table 23 Long-term / Accelerated Stability of Succinate Crystal Form I

[0272] Conclusion: Succinate crystal form I exhibits good physical and chemical stability under accelerated and long-term conditions for 6 months.

[0273] Table 24 Long-term / Accelerated Stability of Succinate Crystal Form II

[0274] Conclusion: Succinate crystal form II exhibits good physical and chemical stability under accelerated and long-term conditions for 6 months.

Claims

1. A pharmaceutically acceptable salt of 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 as shown in Formula 1, wherein the pharmaceutically acceptable salt is selected from fumarate, succinate, hydrochloride, phosphate, tartrate, citrate, acetate, malate, methanesulfonate, benzoate, p-toluenesulfonate, maleate, and sulfate.

2. The medicinal salt according to claim 1, characterized in that, The chemical ratio of the compound shown in Formula 1 to the acid is 3:1-1:3, preferably 2:1-1:2, and more preferably 1:1 or 1:

2.

3. The method for preparing the pharmaceutically acceptable salt according to claim 1 or 2, comprising the step of reacting the compound of formula 1 with an acid, wherein the acid is selected from fumaric acid, succinic acid, hydrochloric acid, phosphoric acid, tartaric acid, citric acid, acetic acid, malic acid, methanesulfonic acid, benzoic acid, p-toluenesulfonic acid, maleic acid, and sulfuric acid.

4. The crystal form I of the fumarate of formula 1 according to claim 1 or 2, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 6.009, 10.840, 15.694, and 20.881, preferably at 6.009, 10.238, 10.840, 11.972, 13.639, 15.694, 17.962, and 20.881, and more preferably at 6.009, 10.238, 10.840, 11.972, 13.639, 15.694, 17.609, 17.962, 20.881, and 21.

693.

5. The fumarate crystal form I 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 fumarate crystal form I as described in claim 4 or 5, wherein the method is selected from any of the following methods: Method 1: Mix the compound of Formula 1 with ethanol, then add a methanol solution of fumaric acid and stir. Method 2: Mix the compound of Formula 1 with solvent I, then add fumaric acid and stir. Solvent I is selected from ethanol, acetone and ethyl acetate.

7. Crystal form II of the fumarate of formula 1 according to claim 1 or 2, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 6.666, 15.702, 20.023, 22.931, and 24.668, preferably at 6.666, 12.336, 15.702, 18.200, 20.023, 21.641, 22.931, and 24.668, and more preferably at 6.666, 12.336, 13.907, 15.702, 18.200, 20.023, 21.641, 22.340, 22.931, and 24.

668.

8. The fumarate crystal form II 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 fumarate crystal form II as described in claim 7 or 8, the method comprising the steps of mixing the compound of formula 1 with ethanol, and then adding fumaric acid and stirring.

10. Crystal form I of the succinate of formula 1 according to claim 1 or 2, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 6.256, 9.500, 10.016, 14.960, and 15.880, preferably at 5.972, 6.256, 9.500, 10.016, 10.803, 14.960, 15.880, and 20.215, and more preferably at 5.972, 6.256, 9.500, 10.016, 10.803, 11.796, 14.960, 15.880, 19.172, and 20.

215.

11. The succinate crystal form I 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 succinate crystal form I as described in claim 10 or 11, the method comprising the steps of mixing the compound of formula I with solvent II, and then adding succinic acid and stirring, wherein solvent II is selected from acetone and ethanol.

13. Crystal form II of the succinate of formula 1 according to claim 1 or 2, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 6.248, 9.742, 15.752, 20.152, and 23.119, preferably at 6.248, 9.742, 10.575, 11.724, 15.752, 20.152, 23.119, and 31.652, and more preferably at 6.248, 9.742, 10.575, 11.724, 15.752, 17.507, 20.152, 23.119, 27.110, and 31.

652.

14. The succinate crystal form II according to claim 13, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 5.

15. A method for preparing succinate crystal form II as described in claim 13 or 14, the method comprising the steps of mixing the compound of formula 1 with ethyl acetate, and then adding succinic acid and stirring.

16. The crystal form I of the hydrochloride salt of formula 1 according to claim 1 or 2, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 8.304, 16.789, 19.065, 22.082, and 22.753, preferably at 8.304, 16.789, 19.065, 20.461, 22.082, 22.753, 24.296, and 27.115, and more preferably at 8.304, 16.789, 19.065, 20.461, 22.082, 22.753, 24.296, 26.282, 27.115, 29.038, and 30.

211.

17. The hydrochloride crystal form I according to claim 16, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 1.

18. A method for preparing hydrochloride crystal form I as described in claim 16 or 17, the method comprising the steps of mixing the compound of formula I with solvent III, and then adding hydrochloric acid ethanol solution and stirring, wherein solvent III is selected from acetone and ethyl acetate.

19. The crystal form according to any one of claims 4-5, 7-8, 10-11, 13-14, 16-17, wherein the 2θ angle error range is ±0.

20.

20. A pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula 1 as claimed in claim 1 or 2, or the crystal form as claimed in any one of claims 4-5, 7-8, 10-11, 13-14, 16-17, 19, and optionally a pharmaceutically acceptable excipient.

21. A method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt of a compound of formula 1 as claimed in claim 1 or 2, or a crystal form as claimed in any one of claims 4-5, 7-8, 10-11, 13-14, 16-17, 19, with a pharmaceutically acceptable excipient.

22. The use of a pharmaceutically acceptable salt of the compound of formula 1 as claimed in claim 1 or 2, or the crystal form as claimed in any one of claims 4-5, 7-8, 10-11, 13-14, 16-17, or 19, or the use of the pharmaceutical composition as claimed in claim 20 in the preparation of a PCSK9 inhibitor.

23. The use of a pharmaceutically acceptable salt of the compound of formula 1 as claimed in claim 1 or 2, or the crystal form as claimed in any one of claims 4-5, 7-8, 10-11, 13-14, 16-17, 19, or the use of the pharmaceutical composition of claim 20 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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