A crystal form of a fumarate salt of a diaminocyclopentyl-substituted heteroaryl derivative and uses thereof

Abstract

The present disclosure relates to a crystalline form of a fumarate salt of a diaminocyclopentyl-substituted heteroaryl derivative and uses thereof. In particular, the present disclosure provides a crystalline form A of a fumarate 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 and a preparation method thereof, which has good stability and can be better used for clinical treatment.

Description

Technical Field

[0001] This disclosure belongs to the field of pharmaceutical technology and relates to the fumarate crystal form and uses of a diaminocyclopentyl-substituted heteroaryl derivative. 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] .

[0005] 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

[0006] This disclosure provides a crystal form A of the fumarate 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,

[0007] .

[0008] The fumarate crystal form A of the compound of Formula 1 disclosed herein has characteristic peaks at 3.768, 15.774, 18.610, 21.441, and 24.025 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0009] In some embodiments, the fumarate crystal form A of the compound of Formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 3.768, 15.308, 15.774, 16.755, 17.635, 18.610, 19.191, 21.441, 22.805, and 24.025.

[0010] In some embodiments, the fumarate crystal form A of the compound of Formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 3.768, 10.076, 11.277, 15.308, 15.774, 16.755, 17.635, 18.610, 19.191, 21.037, 21.441, 22.805, 24.025, and 24.722.

[0011] In some embodiments, the X-ray powder diffraction pattern of fumarate crystal form A of compound of formula 1, expressed as a diffraction angle 2θ, is as follows: Figure 1 As shown.

[0012] This disclosure also provides a method for preparing fumarate crystal form A of compound of formula 1, the method comprising the steps of mixing compound of formula 1 with ethanol, and then adding fumaric acid and stirring.

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

[0014] This disclosure also provides a pharmaceutical composition comprising the fumarate crystal form A of the aforementioned compound of formula 1, and optionally a pharmaceutically acceptable excipient.

[0015] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the fumarate crystal form A of the aforementioned compound of Formula 1 with a pharmaceutically acceptable excipient.

[0016] In some embodiments, the pharmaceutical composition contains 0.01%-99.99% pharmaceutically acceptable excipients based on the total weight of the composition; in some embodiments, the pharmaceutical composition contains 0.1%-99.9% pharmaceutically acceptable excipients; in some embodiments, the pharmaceutical composition contains 0.5%-99.5% pharmaceutically acceptable excipients; in some embodiments, the pharmaceutical composition contains 1%-99% pharmaceutically acceptable excipients; and in some embodiments, the pharmaceutical composition contains 2%-98% pharmaceutically acceptable excipients.

[0017] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, transporter, diluent, and / or delivery polymer.

[0018] This disclosure also provides the use of the fumarate crystal form A of the aforementioned compound of formula 1, or the aforementioned pharmaceutical 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.

[0019] On the other hand, this disclosure provides a fumarate crystal form A of the aforementioned compound of formula 1, or the use of the aforementioned pharmaceutical composition in the preparation of PCSK9 inhibitors.

[0020] 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.

[0021] 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 a certain extent depending on the context in which it is used, but this error variation shall not exceed ±10%, and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0022] 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.

[0023] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 40℃-70℃, and can be dried under normal pressure or reduced pressure.

[0024] 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.

[0025] 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.

[0026] 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,

[0027] Deliquescence: Absorbs sufficient moisture to form a liquid;

[0028] Extremely hygroscopic: hygroscopic weight gain is no less than 15%;

[0029] It has hygroscopic properties: the weight gain due to hygroscopic absorption is less than 15% but not less than 2%;

[0030] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0031] It has little or no hygroscopicity: the weight gain due to moisture absorption is less than 0.2%.

[0032] 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.

[0033] 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

[0034] Figure 1 The image shows the XRPD spectrum of fumarate crystal form A of compound 1. Detailed Implementation

[0035] 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.

[0036] Test conditions of the instruments used in the experiment:

[0037] 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.

[0038] HPLC determinations were performed using a Waters ACQUITY ultra high performance LC, Shimadzu LC-20A systems, Shimadzu LC-2010HT series, or Agilent 1200 LC high performance liquid chromatograph (ACQUITY UPLC BEH C18 1.7UM 2.1×50 MM column, Ultimate XB-C18 3.0×150 mm column, or Ultimate C18 2.1×30 mm column).

[0039] MS measurements were performed using a Waters SQD2 mass spectrometer in positive / negative ion mode, with a mass scan range of 100–1200.

[0040] Chiral HPLC analysis was performed using the following columns: Chiralpak IC-3 100×4.6 mm ID, 3 μm; Chiralpak AD-3 150×4.6 mm ID, 3 μm; Chiralpak AD-3 50×4.6 mm ID, 3 μm; Chiralpak AS-3 150×4.6 mm ID, 3 μm; Chiralpak AS-3 100×4.6 mm ID, 3 μm; ChiralCel OD-3 150×4.6 mm ID, 3 μm; Chiralcel OD-3 100×4.6 mm ID, 3 μm; ChiralCel OJ-H 150×4.6 mm ID, 5 μm; and Chiralcel OJ-3 150×4.6 mm ID, 3 μm.

[0041] 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 size of 0.15 mm to 0.2 mm, and the size used for thin-layer chromatography separation and purification of products is 0.4 mm to 0.5 mm.

[0042] Rapid column purification systems use either the Combiflash Rf150 (TELEDYNE ISCO) or Isolara one (Biotage).

[0043] 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 (40-63 μm, 60, 12 g, 25 g, 40 g, 80 g or other specifications).

[0044] Reversed-phase column chromatography generally uses Changzhou Santai pre-packed ultrapure C18 silica gel columns (20-45 μm, 100 Å, 40 g, 80 g, 120 g, 220 g or other specifications).

[0045] The high-pressure column purification system used was Waters AutoP, with Waters XBridge BEH C18 OBDPrep Column, 130 Å, 5 µm, 19 mm × 150 mm, or Atlantis T3 OBD Prep Column, 100 Å, 5 µm, 19 mm × 150 mm.

[0046] Chiral preparation columns used were DAICL CHIRALPAK IC (250 mm × 30 mm, 10 μm) or Phenomenex-Amylose-1 (250 mm × 30 mm, 5 μm).

[0047] XRPD (X-ray Powder Diffraction) was used for measurement. Measurements were performed using a BRUKER D8 X-ray diffractometer. Specific data collected included: Cu anode (40 kV, 40 mA), Cu-Kα1 rays (λ = 1.54060 Å), Kα2 rays (λ = 1.54439 Å), and Kβ rays (λ = 1.39222 Å). The scanning mode was θ / 2θ, and the scanning range (2θ range) was 3°–45°.

[0048] 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 50 mL / min.

[0049] 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 50 mL / min.

[0050] The known starting materials disclosed herein can be synthesized using or according to 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.

[0051] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0052] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0053] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used 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.

[0054] Example 1

[0055] 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)

[0056]

[0057]

[0058]

[0059] Step 1:

[0060] 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%).

[0061] MS m / z (ESI): 207.1 [M+H] + .

[0062] Step 2:

[0063] 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%).

[0064] MS m / z (ESI): 371.3 [M+H] + .

[0065] Step 3:

[0066] 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%).

[0067] MS m / z (ESI): 271.2 [M+H] + .

[0068] Step 4:

[0069] 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%).

[0070] MS m / z (ESI): 173.1 [M+H] + .

[0071] Step 5:

[0072] 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 carried out in a microwave environment 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%).

[0073] MS m / z (ESI): 407.3 [M+H] + .

[0074] 1H NMR (400 MHz, MeOD) δ 7.93 (d, J = 2.7 Hz, 1H), 7.78 (s, 1H), 7.60 (ddt, J = 11.1, 6.7, 2.0 Hz, 2H), 7.44 (dd, J = 9.0, 2.7 Hz, 1H), 6.61 (ddd,J = 9.0, 2.9, 1.7 Hz, 2H), 6.46 (td, J = 6.8, 1.3 Hz, 1H), 4.49 – 4.43 (m,2H), 4.30 (dp, J = 13.4, 6.5 Hz, 2H), 4.21 – 4.15 (m, 2H), 2.31 – 2.15 (m,2H), 1.95 (qd, J = 6.5, 2.6 Hz, 2H), 1.63 – 1.51 (m, 2H).

[0075] X-ray powder diffraction analysis revealed that the product was amorphous, and the XRPD spectrum showed no obvious characteristic peaks.

[0076] Test Example 1. Test on the binding affinity of the disclosed compound to PCSK9.

[0077] Table 1. Main experimental materials and instruments

[0078]

[0079] 1. Experimental Procedure

[0080] 1.1 Reagent Preparation

[0081] (1) Protein: PCSK9 (Biotin-labeled) (Acro).

[0082] Dissolve the protein in water to a concentration of 200 µg / mL and store at -80°C.

[0083] (2) Chip: SA chip.

[0084] (3) Run buffer: 1x HBSP + 4% DMSO + 0.1 mM CaCl2.

[0085] 1.2 Protein fixation

[0086] (1) Preheat the chip to room temperature;

[0087] (2) Prepare the required buffer solution and filter it using a 0.22 µm filter membrane;

[0088] (3) Load the chip and write the chip parameters;

[0089] (4) Dilute PCSK9 protein to 50 µg / mL with PBS;

[0090] (5) Set a fixed program in the BIAcore S200 instrument;

[0091] (6) Run the fixed program;

[0092] (7) The final fixed response value is 8000 RU, and the maximum response value is 49.4 RU.

[0093] 1.3 Compound Testing

[0094] (1) Dilute the compounds 3-fold with running buffer. Start with 0.4 μM of the reference compound and dilute 3-fold. Start with 10 μM of the test compound and dilute 3-fold.

[0095] (2) Refer to the table below to prepare the dissolution and correction buffer solution:

[0096] Table 2. Dissolution Correction Buffer

[0097]

[0098] (3) Set the instrument’s “LMW multi kinetics” program as the running program for compound testing.

[0099] a) Set the binding time to 100s and the dissociation time to 300s.

[0100] (4) Run the compound test program.

[0101] 1.4 Data Analysis

[0102] (1) Rmax = (MW analyte / MW ligand) * RL * Sm

[0103] 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.

[0104] (2) Check solvent correction and compound concentration gradient.

[0105] (3) Check the baseline, binding and positive compound results in the test report.

[0106] (4) Select a suitable fitting method to fit the curve based on the actual situation.

[0107] (5) Check whether the fitting results meet the instrument data quality test standards.

[0108] Table 3. Binding affinity of the compounds disclosed herein to PCSK9 (K) D )

[0109]

[0110] The results show that the binding force of compound 1 is more than 10 times stronger than that of reference compound 2.

[0111] Reference point 2 Example 487 is from patent applications WO2020150473 and WO2020150474.

[0112] Test Example 2. Pharmacokinetic Experiment in Mice

[0113] Using C57BL6J mice as test animals, the plasma drug concentrations at different time points after gavage 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.

[0114] Experimental animals: 2 healthy male mice (20-30g) aged 6-8 weeks per group

[0115] 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).

[0116] Administration: Mice were fasted overnight and then administered the drug by gavage. The dosage for both reference compound 1 and the compound in the examples was 10 mg / kg.

[0117] 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.

[0118] 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.

[0119] Table 4. Pharmacokinetic parameters of reference material 1 and compound 1

[0120]

[0121] Note: All values ​​listed are averages of two animals. Reference 1 is Example 458B in patent applications WO2020150473 and WO2020150474.

[0122] 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.

[0123] Test Example 3. Effect of the reference compound and the compound of the examples on LDLR levels in HepG2 cells.

[0124] Table 5. Main experimental materials and instruments

[0125]

[0126] Experimental steps

[0127] HepG2 cells were seeded at 30,000 cells / well in 96-well plates. The next day, 4 nM PCSK9D374Y 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 overnight at -80°C. 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.

[0128] Table 6. Effects of Reference 1 and Example Compounds at 75 μM Concentration on LDLR Protein Levels in HepG2 Cells

[0129]

[0130] Note: aThe average value of the three-hole composite is represented by Example 458B from patent applications WO2020150473 and WO2020150474.

[0131] 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.

[0132] Test Example 4. Metabolite testing of reference substance 1 and compound 1 in mouse hepatocytes

[0133] 1. Experimental Objective

[0134] 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.

[0135] 2. Materials and Reagents

[0136] Mouse hepatocytes were preserved in liquid nitrogen. See the table below for details.

[0137] 3. Experimental Design

[0138]

[0139] 3.1 Preparation of the compound working solution

[0140] 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.

[0141] 3.2 Preparation of hepatocytes

[0142] 1) Preheat the hepatocyte resuscitation solution and incubation solution in a 37 °C water bath for at least 15 minutes before use.

[0143] 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.

[0144] 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.

[0145] 4) Count hepatocytes and determine viable cell density using Cellometer Vision. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 1 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0146] 3.3 Test Methods

[0147] 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 an incubator (37°C, 5% CO2, 90-95% relative humidity) and incubate at 500 rpm.

[0148] 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.

[0149] 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.

[0150] 4) All samples were vortexed for 100 seconds, followed by centrifugation at 16,000 g 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.

[0151] 4. Instrument Configuration

[0152] The Vanquish ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA) is combined with a ThermoScientific Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) and equipped with a HESI ion source.

[0153] 5. Mass spectrometry detection

[0154] 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.

[0155] The relevant mass spectrometry parameters of the test analyte are optimized based on its own properties.

[0156] 6. Data Analysis

[0157] Data processing and analysis were performed using Xcalibur (v3.0 / 4.1, Thermo Fisher Scientific) software, Compound Discoverer 3.0 (Thermo Fisher Scientific) software, and Microsoft Excel 2016.

[0158] Table 7. Remaining percentage of parent drug for reference 1 and compound 1 (%)

[0159]

[0160] Note: Reference 1 is Example 458B from patent applications WO2020150473 and WO2020150474.

[0161] The experimental results show that compound 1 has better stability in mouse hepatocytes than reference compound 1.

[0162] Test Example 5. Stability test of the test compound in hepatocytes

[0163] 1. Experimental Procedure

[0164] (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.

[0165] (2) Take a tube of cryopreserved hepatocytes and ensure that the hepatocytes are still frozen before revival. 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.

[0166] (3) The contents of hepatocyte tubules from different species were poured into centrifuge tubes containing 50 mL of resuscitation medium and centrifuged at 100 g for 10 minutes. After centrifugation, the resuscitation medium was removed and sufficient incubation medium was added to obtain a cell density of approximately 1.0 × 10⁻⁶ cells / year. 6 Cell suspension of cells per mL.

[0167] (4) Use Cellometer Vision to count hepatocytes and determine the 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.

[0168] (5) Transfer 198 μL of live cell suspension to a 96-well deep plate and preheat the plate in an incubator for 10 minutes on a vortex. Perform double parallel incubation.

[0169] (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.

[0170] (7) Incubate the sample. Take 25 μL of the suspension at 0, 15, 30, 60, 90 and 120 minutes respectively, and add 150 μL of acetonitrile containing internal standard to terminate the reaction. Vortex for 10 minutes, and centrifuge at 3220 g, 4°C for 45 minutes. Transfer 100 μL of supernatant to the sample plate, add 100 μL of pure water and mix well for UPLC-MS / MS analysis.

[0171] 2. Data Analysis

[0172] 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 ).

[0173] In vitro half-life (T 1 / 2 ) Calculated by slope:

[0174] In vitro T 1 / 2 = 0.693 / k

[0175] In vitro clearance rate (unit µL / min / 10) 6 (Number of cells) is calculated using the following formula:

[0176] In vitro CL int = kV / N

[0177] V = Incubation volume per well (0.2 mL);

[0178] N = Number of cells per well (0.1 × 10⁻⁶) 6 (cells)

[0179] Table 8. Metabolic clearance rate of the tested compounds in mouse hepatocytes

[0180]

[0181] The results showed that compound 1 had better stability in mouse hepatocytes than reference 1 and reference 2.

[0182] Example 2: Preparation of fumarate crystal form A

[0183] 200 mg of the compound shown in Formula 1 was added to 4 mL of ethanol and stirred at room temperature to dissolve. After the solution was cleared, 58 mg of fumaric acid was added, stirred to induce crystallization, centrifuged, and the solid was collected and dried under vacuum to obtain the product.

[0184] X-ray powder diffraction analysis identified the product as fumarate crystal form A, and the XRPD spectrum is shown below. Figure 1 The positions of its characteristic peaks are shown in Table 1. The DSC spectrum shows that the endothermic peak has a peak value of 219.83℃. The TGA spectrum shows that the weight loss is 0.35% from 30℃ to 120℃ and 1.26% from 120℃ to 190℃.

[0185] Table 1

[0186]

Claims

1. A fumarate crystal form A 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, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 3.768, 15.774, 18.610, 21.441, and 24.025 Å, preferably at 3.768, 15.308, 15.774, 16.755, 17.635, 18.610, 19.191, 21.441, 22.805, and 24.025 Å, and more preferably at 3.768, 10.076, 11.277, 15.308, 15.774, 16.755, 17.635, 18.610, 19.191, 21.037, 21.441, 22.805, 24.025, and 24.722 Å. 。 2. The fumarate 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 fumarate crystal form A as described in claim 1 or 2, the method comprising the steps of mixing the compound of formula 1 with ethanol, and then adding fumaric acid and stirring.

4. The fumarate crystal form A according to claim 1 or 2, wherein the 2θ angle error range is ±0.20°.

5. A pharmaceutical composition comprising fumarate crystal form A as described in any one of claims 1-2, 4 and optionally a pharmaceutically acceptable excipient.

6. A method for preparing a pharmaceutical composition, comprising the step of mixing fumarate crystal form A as described in any one of claims 1-2, 4 with a pharmaceutically acceptable excipient.

7. Use of fumarate crystal form A according to any one of claims 1-2, 4, or the pharmaceutical composition according to claim 5 in the preparation of PCSK9 inhibitors.

8. The use of fumarate crystal form A according to any one of claims 1-2, 4, or the pharmaceutical composition according to claim 5 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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