Crystal form of inhibitor compound and preparation method therefor

Abstract

The present invention belongs to the technical field of chemical pharmaceuticals. Provided are a free base crystal form of a compound as represented by general formula (C) and a preparation method therefor, a pharmaceutical composition containing the crystal form, and the pharmaceutical use of the crystal form and pharmaceutical composition.

Description

Crystal form of an inhibitor compound and its preparation method Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, and provides a free base crystal form of a compound as shown in general formula (C), its preparation method and application. Background Technology

[0002] Proprotein convertase subtilisin / kexin type 9 (PCSK9), also known as neuronal apoptosis-regulating convertase 1 (NARC-1), is a pro-proprotein convertase belonging to the subtilisin (S8) family of serine proteases. It is expressed in cells capable of proliferation and differentiation, including hepatocytes, renal interstitial cells, ileal and colonic epithelial cells, and embryonic telencephalon neurons. Studies have found that PCSK9 plays a role in the differentiation of hepatocytes and nerve cells. It not only specifically acts on cholesterol biosynthesis or uptake, but circulating PCSK9 can also directly bind to the low-density lipoprotein receptor (LDLR) on the surface of hepatocytes, being phagocytosed by hepatocytes along with LDLR. This promotes the degradation of LDLR in hepatocytes, hinders its recycling, and thus increases the level of LDL cholesterol (LDL-C) in plasma. Elevated LDL-C expression is closely related to dyslipidemia and cardiovascular diseases in humans.

[0003] Current research focuses on inhibiting PCSK9 function or production. For example, attempts have been reported to inhibit PCSK9 function using monoclonal antibodies targeting PCSK9, and to inhibit PCSK9 production through RNA interference. However, for patients with cardiovascular disease, effective small-molecule inhibitors are needed to suppress PCSK9 function. Summary of the Invention

[0004] In one aspect, this invention provides a free base crystal form of a compound as shown in general formula (C), characterized in that:

[0005] Ring A is selected from Wherein, X and Y form a 5-7 member saturated or unsaturated ring, wherein the 5-7 member saturated or unsaturated ring contains 0, 1, and 2 heteroatoms, wherein the heteroatoms are selected from O, N, and S; B is selected from

[0006] Q is selected from N or CR1, and R1 is selected from H or halogen;

[0007] T1 is selected from N or CH;

[0008] R2 is selected from H, alkyl, or halogen, and there are one or more R2s.

[0009] R3 is selected from hydrogen or indicates that the hydrogen on ring A is further replaced by an oxo, alkyl, halogen, alkoxy, alkylthio, haloalkyl, haloalkoxy, cycloalkyl, cycloalkylalkyl, or alkynyl group. R3 can be one or more, or adjacent R3 groups can form an alkoxy group.

[0010] R4 is selected from hydrogen, halogen, hydroxyl, alkoxy, haloalkoxy, substituted or unsubstituted alkyl, cyano, -C(O)-O-alkyl, phenylalkoxy, carboxyl, hydroxymethyl, or cycloalkyl, and the substituent is selected from hydroxyl, amide, halogen, or... The components that are replaced are: U1, U2, and U4, which are independently selected from CH or N; U3, which is selected from CH2 or NH; and R4, which is one or more.

[0011] And when ring A is selected At that time, ring B is not

[0012] As one embodiment of the present invention:

[0013] The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 1-ethylpropyl, 2-methylbutyl, tert-pentyl, 1,2-dimethylpropyl, isopentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, tert-hexyl, neohexyl, 2-methylpentyl, 1,2-dimethylbutyl, and 1-ethylbutyl.

[0014] The alkoxy group is selected from methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0015] The cycloalkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl;

[0016] The halogen is selected from fluorine, chlorine, bromine, and iodine.

[0017] In one embodiment of the present invention, Q is selected from N; and / or Q is selected from CH, R2 is selected from F; and / or T1 is selected from N;

[0018] And / or R3 is selected from hydrogen, methyl, methoxy, cyclopropyl, cyclopropylmethyl, fluorine, chlorine, oxo, CHF2-O-; R4 is selected from hydrogen, methyl, hydroxy, hydroxymethyl, cyano, F, Cl, Br, -O-CH2-benzene, -COOH, -COOCH2CH3, Amides, formamide, CH3-C(O)-, ethynyl, trifluoromethyl, difluoromethoxy, methoxy.

[0019] As one embodiment of the present invention, ring A is selected from...

[0020] Furthermore, the A ring replaced by R3 is selected from:

[0021] In one embodiment of the present invention, the B ring replaced by R4 is selected from:

[0022] As one embodiment of the present invention, the compound is selected from:

[0023] Further, the compound is selected from compound A, and a crystal form A of compound A is provided. Compound A is 6'-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one. The crystal form A of compound A has characteristic peaks at 17.99°, 18.80°, and 19.69° in the X-ray diffraction pattern, represented by a 2θ angle, with an error of ±0.2°.

[0024] As a preferred embodiment of the present invention, the crystal form A has characteristic peaks at 12.21°, 13.42°, 17.99°, 18.80°, 19.69°, 25.58°, and 25.85° in the X-ray diffraction pattern, with an error of ±0.2°.

[0025] As a preferred embodiment of the present invention, crystal form A is represented by a 2θ angle in the X-ray diffraction pattern at 4.79°, 9.45°, 12.21°, ...

[0026] Characteristic peaks are found at 13.42°, 16.23°, 17.99°, 18.80°, 19.69°, 20.64°, 24.43°, 25.85°, 27.78°, and 28.76°, with an error of ±0.2°.

[0027] As a preferred embodiment of the present invention, the X-ray diffraction pattern of crystal form A of compound A is shown in Figure 1 or Figure 3.

[0028] As a preferred embodiment of the present invention, the crystal form A of compound A has an endothermic peak at 154.3℃ in the DSC spectrum, with an error of ±5℃.

[0029] As a preferred embodiment of the present invention, the DSC spectrum of crystal form A of compound A is shown in Figure 2 or Figure 4.

[0030] The present invention further provides a pharmaceutical composition comprising a free base crystal form of a compound of general formula (C) in a therapeutically effective amount, specifically including crystal form A of compound A, and one or more pharmaceutically acceptable carriers.

[0031] The present invention further provides a free base crystal form of a compound as shown in general formula (C), and the use of crystal form A of compound A and a pharmaceutical composition containing crystal form A of compound A in the preparation of a medicament for PCSK9 inhibitor-related diseases.

[0032] As a preferred embodiment of the present invention, the PCSK9 inhibitor-related diseases are specifically selected from conditions such as hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, dyslipoproteinemia, atherosclerosis, hepatic steatosis, metabolic syndrome, and coronary artery disease.

[0033] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium capable of delivering an effective amount of the active substance of this invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, ointment bases, lotion bases, and ointment bases. These bases include suspending agents, thickeners, and transdermal penetration enhancers. Their formulations are well known to those skilled in the art of cosmetics or topical pharmaceuticals. For further information on carriers, see Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0034] PCTCN2024120513 introduces this invention, including the preparation, identification and evaluation of compounds. Attached Figure Description

[0035] Figure 1 is the X-ray diffraction pattern of crystal form A of compound A obtained in Example 5 of the present invention.

[0036] Figure 2 is the DSC spectrum of crystal form A of compound A obtained in Example 5 of the present invention.

[0037] Figure 3 is the X-ray diffraction pattern of crystal form A of compound A obtained in Example 6 of the present invention.

[0038] Figure 4 is the DSC spectrum of crystal form A of compound A obtained in Example 6 of the present invention.

[0039] Figure 5 is a schematic diagram of the dynamic moisture test of the crystal form and amorphous form of compound A in the embodiment of the present invention. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the invention is not limited thereto.

[0041] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ increments. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-III NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3), with tetramethylsilane (TMS) as the internal standard.

[0042] MS measurements were performed using an ISQ EC mass spectrometer (manufacturer: Thermo, model: ISQ EC).

[0043] High-performance liquid chromatography (HPLC) analysis was performed using a Thermo U3000 HPLC DAD high-performance liquid chromatograph.

[0044] The CombiFlash rapid preparation system uses CombiFlash Rf+LUMEN (TELEDYNE ISCO).

[0045] Thin-layer chromatography silica gel plates used are from Yantai Yinlong HSGF. 254 or GF 254 For thin-layer chromatography (TLC), the silica gel plates used are 0.17 mm to 0.23 mm in size, while those used for TLC separation and purification are 0.4 mm to 0.5 mm in size.

[0046] Silica gel column chromatography generally uses 100-200 mesh silica gel from Rushan Shangbang as the carrier.

[0047] This invention relates to the following reagents: DMF (N,N-dimethylformamide), KI (potassium iodide), Cs2CO3 (cesium carbonate), DCM (dichloromethane), n-hexane, EA (ethyl acetate), Py (pyridine), CF3COOH (trifluoroacetic acid), IPA (isopropanol), isopropyl ether, and acetone.

[0048] Unless otherwise stated, the polymorphs of this invention were detected using the following equipment and conditions: X-ray powder diffraction (XRPD). The XRPD patterns were acquired using an X-ray powder diffractometer manufactured by PANalytacal, and the scanning parameters are shown in the table below:

[0049] Differential scanning calorimetry (DSC) was performed using a Netzsch DSC 200F3 differential scanning calorimeter. The test parameters are shown in the table below:

[0050] Dynamic moisture adsorption (DVS) curves were acquired using the DVS Intrinsic on the SMS (Surface Measurement Systems) device. The DVS test parameters are listed below:

[0051] Example 1: Preparation of Compound A

[0052] Synthesis of 6'-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one (compound A, C17)

[0053] Step 1: Synthesis of tert-butyl N-[(1S,3S)-3-[(5-bromo-3-fluoro-2-pyridyl)amino]cyclopentyl]carbamate

[0054] N-[(1S,3S)-3-aminocyclopentyl]carbamate tert-butyl ester (3.00 g, 15.0 mmol, 1 eq), 5-bromo-2,3-difluoropyridine (2.91 g, 15.0 mmol, 1.0 eq), and N,N-diisopropylethylamine (4.84 g, 37.5 mmol, 2.5 eq) were dissolved in dimethyl sulfoxide (40 mL) and stirred at 100 °C for 12 h. The MS value of the product was monitored by LCMS (RT = 0.568 min). The reaction solution was cooled to room temperature, then diluted with water (200 mL), and extracted with ethyl acetate (100 mL * 2). The concentrated organic phase was washed with saturated brine (500 mL * 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain tert-butyl N-[(1S,3S)-3-[(5-bromo-3-fluoro-2-pyridinyl)amino]cyclopentyl]carbamate (4.00 g). LCMS: RT = 0.568 min, MS (ESI) m / z = 374.0 [M+1] +

[0055] Step 2: Synthesis of tert-butyl N-[(1S,3S)-3-[[3-fluoro-5-(2-oxo-1-pyridyl)-2-pyridyl]amino]cyclopentyl]carbamate

[0056] tert-butyl N-[(1S,3S)-3-[(5-bromo-3-fluoro-2-pyridinyl)amino]cyclopentyl]carbamate (1.5 g, 4.01 mmol, 1.0 eq), 1H-pyridin-2-one (457 mg, 4.81 mmol, 1.2 eq), potassium phosphate (2.55 g, 12.0 mmol, 3.0 eq), cuprous iodide (153 mg, 802 μmol, 0.20 eq), and (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (114 mg, 802 μmol, 0.2 eq) were dissolved in dioxane (20 mL) and stirred at 100 °C for 12 hours. TLC (petroleum ether / ethyl acetate = 3 / 1) showed that the reaction was complete. The reaction solution was cooled to room temperature, then diluted with water (100 mL) and extracted with ethyl acetate (30 mL * 2). The concentrated organic phase was washed with saturated brine (100 mL * 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain tert-butyl N-[(1S,3S)-3-[[3-fluoro-5-(2-oxo-1-pyridyl)-2-pyridyl]amino]cyclopentyl]carbamate (1.00 g).

[0057] Step 3: Synthesis of 6'-((((1S,3S)-3-aminocyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one

[0058] 1.00 g, 2.57 mmol, 1.0 eq of tert-butyl N-[(1S,3S)-3-[[3-fluoro-5-(2-oxo-1-pyridyl)-2-pyridyl]amino]cyclopentyl]carbamate was dissolved in 10 mL of dichloromethane, and then ethyl acetate hydrochloride (HCl / EtOAc) (2 M, 20 mL, 15.5 eq) was added. The mixture was stirred at 20 °C for 2 hours. The MS value of the product was monitored by LCMS (RT = 0.320 min). The reaction solution was directly concentrated and dried to obtain 6'-((((1S,3S)-3-aminocyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one (600 mg, 2.08 mmol, 80.8% yield). No purification was performed, and it was used directly in the next step. LCMS: RT = 0.320 min, MS (ESI) m / z = 577.3 [2M+1] +

[0059] Step 4: Synthesis of 6'-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one

[0060] 6'-((((1S,3S)-3-aminocyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one (120 mg, 416 μmol, 1.0 eq), 2-bromo-[1,2,4]triazolo[1,5-a]pyridine (90.7 mg, 458 μmol, 1.1 eq), sodium tert-butoxide (120 mg, 1.25 mmol, 3.0 eq), and tBuXPhos Pd G3 (33.1 mg, 41.6 μmol, 0.1 eq) were dissolved in dioxane (4 mL), and then stirred at 140 °C for 1 h in a microwave reactor. The MS value of the product was monitored by LCMS (RT = 0.399 min). The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by reverse-phase preparation (column: Phenomenex luna C18). 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 15%-45% B over 15min) 6'-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one (36.6 mg) was obtained twice. LCMS: RT = 0.399 min, MS (ESI) m / z = 406.1 [M+1] + The resulting compound was amorphous.

[0061] 1 H NMR (400MHz, DMSO-d6) δ=8.58(d,J=6.9Hz,1H),7.84(d,J=2.0Hz,1H),7.64(dd,J=1.7,6.7Hz,1 H),7.54(dd,J=2.1,11.9Hz,1H),7.49(ddd,J=2.1,6.8,9.0Hz,1H),7.44-7.34(m,2H),6.94(br d,J=6.9Hz,1H),6.85(dt,J=1.4,6.7Hz,1H),6.64(d,J=7.3Hz,1H),6.46(d,J=9.0Hz,1H),6.32-6.25( m,1H),4.57-4.47(m,1H),4.25-4.14(m,1H),2.19-2.10(m,2H),2.01-1.94(m,2H),1.65-1.51(m,2H).

[0062] Example 2: Pharmacokinetic Experiment

[0063] 1. Reagents and Instruments

[0064] Polyethylene glycol 400 (batch number R22040588, Shanghai Shaoyuan Reagent Co., Ltd.), DMSO (batch number 20200319, Guangdong Guanghua Technology Co., Ltd.), physiological saline (batch number 2011110727, Chenxin Pharmaceutical Co., Ltd.). LC-MS instruments (Thermo Fisher Ultimate 3000UPLC, TSQ QUANTUM ULTRA triple quadrupole mass spectrometer, AB SCIEX 5500+QTARP).

[0065] 2. Laboratory animals

[0066] SD rats: male, 180-250g, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.

[0067] 3. Formulation preparation

[0068] Accurately weigh the sample powder, dissolve it completely in DMSO, add PEG-400, vortex and sonicate to mix, then add physiological saline and vortex and sonicate to make a concentration of 0.5 mg / mL (DMSO:PEG-400:NS=5:60:35, V / V / V). Administer 10 mL / kg by gavage or 2 mL / kg by intravenous administration.

[0069] 4. Blood sample collection

[0070] After intravenous or gavage administration to rats, 200 μL of venous blood was collected at 5 min (no blood was collected after gavage), 15 min, 30 min, 1 h, 2 h, 5 h, 7 h, and 24 h and placed in EDTA-K2-anticoagulant EP tubes. The tubes were centrifuged at 10,000 rpm for 2 min, and the plasma was frozen at -80℃ for later testing.

[0071] 5. Biological Analysis

[0072] Accurately weigh a certain amount of the test sample and dissolve it in DMSO to a concentration of 2 mg / mL to prepare a stock solution. Accurately pipette an appropriate amount of the stock solution and dilute it with acetonitrile to prepare a series of standard solutions. Accurately pipette 4 μL of each of the above standard solutions and add 36 μL of blank plasma. Vortex to mix, preparing plasma samples equivalent to concentrations of 1, 3, 5, 10, 30, 100, 300, 1000, and 3000 ng / mL. Perform dual-sample analysis for each concentration to establish a standard curve. Take 30 μL of plasma and add 200 μL of acetonitrile solution containing the internal standard propranolol (5 ng / mL). Vortex to mix and centrifuge at 4000 rpm for 10 min. Analyze the supernatant by LC-MS. The LC-MS detection conditions are as follows:

[0073] Chromatographic column: YMC-Triart C18, 50×2.1mm, S-3μm 12nm.

[0074] Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile, flow rate: 0.5 mL / min, gradient elution as shown in Table 1 below:

[0075] Table 1

[0076] 6. Data Processing

[0077] After detecting blood drug concentrations by LC-MS, the pharmacokinetic parameters of rats after drug administration were calculated using WinNonlin 6.1 software and a non-compartmental model. The results are shown in Table 2 below.

[0078] Table 2: Pharmacokinetic parameters of the compounds of this invention in rats (PO administration)

[0079] The oral exposure of the compounds of this invention is improved compared with the control compounds, and is superior to compounds 458B and 464 of CN113574055A, as well as compound B, as shown in Table 3.

[0080] Table 3

[0081] Example 3: Effect of hERG currents on hERG-HEK293 cells

[0082] Experimental Methods: Human embryonic kidney cells (hERG-HEK293 cells) stably expressing the hERG channel were used in the experiment. hERG-HEK293 cells were clamped in a whole-cell voltage-clamp configuration using an automated patch-clamp system, and hERG currents were induced by appropriate voltages. Cells were administered extracellular fluid containing 0.3% DMSO (negative control) and 30 μM of the compound, or 1, 10, 100, and 1000 nM of cisapride (positive control). The tail current of the hERG channel was recorded, and the peak value of the tail current at each concentration was obtained. Using the peak value of the tail current recorded under the negative control (0.3% DMSO) as 100%, the inhibition rate of hERG current by the 30 μM compound and different concentrations of cisapride was calculated. Cisapride concentration-response curve fitting and IC50 analysis were performed. 50 The calculations were performed using GraphPad Prism software, and the results are shown in Table 4.

[0083] Table 4. Effects of hERG currents on hERG-HEK293 cells.

[0084] The risk of the Herg compound of this invention is improved compared to the control compound, and is superior to the control compound 458B in CN113574055A.

[0085] Example 4: Affinity test of the compound with PCSK9 protein

[0086] The binding affinity of the compounds of the present invention to PCSK9 protein was determined by fluorescence polarization method.

[0087] All compounds were dissolved in DMSO to prepare 10 mM stock solutions. Positive and test compounds were serially diluted 5-fold with DMSO, starting at 10 mM, for a total of 8 concentration gradients. First, a certain volume of fluorescent probe solution was prepared to a concentration of 5 nM using test buffer (20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, and 0.01% Tween-20). Then, the serial DMSO solutions of the test compounds were diluted 50-fold with the fluorescent probe solution. Finally, a solution of recombinant human PCSK9 protein (ACRO, Cat#PC9-H5223) at a concentration of 4.5 μg / mL was prepared using test buffer. After the test solution was prepared, 5 μL of PCSK9 protein was added to a black 384-well plate (PerkinElmer, Cat#6008260), along with 5 μL of different concentrations of the compound (DMSO final concentration 1%). A positive control group (test buffer + equal volume of target protein + equal proportion of fluorescent probe molecules) and a negative control group (test buffer + equal proportion of fluorescent probe molecules) were also set up. The final concentration of the probe molecules in the system was 2.5 nM, and the median concentration of PCSK9 protein was 2.25 μg / mL. After incubation at room temperature with shaking for 15 minutes, the fluorescence polarization values ​​were read using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The inhibition rate of the drug = [1-(mP...] (药物筛选组) -mP (阴性对照 组) ]÷[mP (阳性对照组) -mP (阴性对照组) []×100. Using the logarithm of compound concentration as the x-axis and the inhibition rate as the y-axis, a 4-parameter nonlinear regression curve was fitted to calculate the IC. 50 Value(Y=Bottom+(Top-Bottom) / (1+10^((LogIC A50 -X)*HillSlope)), where: Hillslope represents the slope of this curve, IC 50 This indicates the half-maximal inhibitory concentration (MCI).

[0088] Table 5. FP-IC 50 data

[0089] B < 300nm. *Incubate for 18 hours.

[0090] Example 5 Preparation of crystal form A

[0091] 500 mg of the product from step 4 of Example 1 was dissolved in 5 ml of ethanol, heated to 50°C, stirred for 2 h, and then 20 ml of isopropyl ether was slowly added dropwise. The mixture was kept warm and stirred for 3 h, then cooled to room temperature and stirred for another 4 h. The mixture was then filtered, and the solid was dried under vacuum at 45°C to obtain solid of crystal form A.

[0092] The XRD pattern of crystal form A, expressed at 2θ angles, shows the strongest characteristic absorption peaks at 17.99°, 18.80°, and 19.69°, with an error of ±0.2° and a relative absorption intensity greater than 50%. Further, the crystal form exhibits characteristic peaks at 12.21°, 13.42°, 17.99°, 18.80°, 19.69°, and 25.85°, with an error of ±0.2° and a relative absorption intensity greater than 30%. Additionally, the crystal form shows characteristic peaks at 4.79°, 9.45°, 12.21°, 13.42°, 16.23°, 17.99°, 18.80°, 19.69°, 20.64°, 24.43°, 25.85°, 27.78°, and 28.76°, with an error of ±0.2° and a relative absorption intensity greater than 5%, exhibiting good peak shapes.

[0093] The crystal form A of compound A has an endothermic peak at 154.3℃ in the DSC spectrum, with an error of ±5℃.

[0094] Specifically, the XRD pattern is shown in Figure 1, and the DSC pattern is shown in Figure 2.

[0095] Example 6 Preparation of crystal form A

[0096] 500 mg of the product from step 4 of Example 1 was dissolved in 10 ml of acetone and stirred at room temperature for 24 h. A solid precipitated out, was filtered, and the solid was dried under vacuum at 45 °C to obtain solid of crystal form A.

[0097] The XRD pattern of crystal form A, expressed at 2θ angles, shows the strongest characteristic absorption peaks at 18.14°, 18.89°, and 19.84°, with an error of ±0.2° and a relative absorption intensity greater than 50%. Further, the crystal form exhibits characteristic peaks at 12.31°, 13.39°, 18.14°, 18.89°, 19.84°, and 25.96°, with an error of ±0.2° and a relative absorption intensity greater than 30%. Additionally, the crystal form shows characteristic peaks at 4.91°, 9.55°, 12.31°, 13.39°, 16.36°, 18.14°, 18.89°, 19.84°, 20.76°, 24.51°, 25.96°, 27.90°, and 28.84°, with an error of ±0.2° and a relative absorption intensity greater than 5%, exhibiting good peak shapes.

[0098] The crystal form A of compound A has an endothermic peak at 154.3℃ in the DSC spectrum, with an error of ±5℃.

[0099] The XRD pattern is shown in Figure 3, and the DSC pattern is shown in Figure 4.

[0100] The comparison of the main absorption peaks in Figures 1 and 3 is shown in Table 6 below, with an error of ±0.2°.

[0101] Table 6

[0102] Where No. = serial number, Rel.Int. = Relative Intensity, Pos.[°2Th.] = Position[°2Theta], with an error of ±0.2°. Rel.Int. = Relative Intensity only indicates the approximate intensity of the characteristic peak and should not be used as a limitation on a specific crystal form.

[0103] In summary, based on the XRD spectra and characteristic peak data in Figures 1 and 3, the strongest characteristic absorption peaks are found at 17.99°, 18.80°, and 19.69°, represented by a 2θ angle, with an error of ±0.2° and a relative absorption intensity greater than 50%.

[0104] Furthermore, the crystal form has characteristic peaks at 12.21°, 13.42°, 17.99°, 18.80°, 19.69°, and 25.85° with an error of ±0.2° and a relative absorption intensity greater than 30%.

[0105] Furthermore, the described crystal form exhibits characteristic peaks at 4.79°, 9.45°, 12.21°, 13.42°, 16.23°, 17.99°, 18.80°, 19.69°, 20.64°, 24.43°, 25.85°, 27.78°, and 28.76°, with an error of ±0.2°. The relative absorption intensity is greater than 5%, and the peak shape is good, allowing for more detailed differentiation of other substances representing this crystal form. Other weak absorption peaks may vary significantly due to experimental errors; for those skilled in the art, these other absorption peaks are unnecessary for characterizing this crystal form.

[0106] Example 7 Crystal Transformation Observation Experiment

[0107] Add 20 mg of amorphous compound A sample to 0.5 ml of solvent and observe the dissolution.

[0108] Note: * After stirring, a solid precipitates out, changing from amorphous to crystalline form A.

[0109] Example 8 Stability Test

[0110] Compound A sample was left exposed at 50°C to examine product stability.

[0111] At 50°C, crystal form A exhibits better stability than amorphous form.

[0112] Example 9: Water Absorption Study

[0113] The crystalline and amorphous forms of compound A were determined by dynamic moisture testing. At 95% humidity, crystalline form A absorbed 1.93% of the water without any change in crystal form. At 95% humidity, the amorphous form absorbed 19.84% of the water and deliquesced after the test. The water absorption is shown in Figure 5.

[0114] Example 10 Pharmacokinetic Experiment

[0115] 1. Reagents and Instruments

[0116] Polyethylene glycol 400 (PEG-400), TPGS, CMC-Na. LC-MS / MS instruments (Thermo Fisher Ultimate 3000UPLC, TSQ QUANTUM ULTRA triple quadrupole mass spectrometer, AB SCIEX 5500+QTARP).

[0117] 2. Laboratory animals

[0118] SD rats: male, 180-250g.

[0119] 3. Formulation preparation

[0120] Accurately weigh a certain amount of crystalline or amorphous compound A test sample powder, add PEG-400 and vortex mix, then add the pre-prepared TPGS solvent and continue vortexing, finally add 0.5% CMC-Na and vortex mix to make a 10 mg / mL crystalline and amorphous suspension (PEG-400:TPGS:CMC-Na=20:30:50, V / V / V), and administer by gavage at 10 mL / kg.

[0121] 4. Blood sample collection

[0122] After oral administration to rats, 200 μL of venous blood was collected at 15 min, 30 min, 1 h, 2 h, 5 h, 7 h, and 24 h and placed in EDTA-K2-anticoagulant EP tubes. The tubes were centrifuged at 10,000 rpm for 2 min, and the plasma was frozen at -80℃ for later analysis.

[0123] 5. Biological Analysis

[0124] Accurately weigh a certain amount of the test sample and dissolve it in DMSO to a concentration of 2 mg / mL to prepare a stock solution. Accurately pipette an appropriate amount of the stock solution and dilute it with acetonitrile to prepare a series of standard solutions. Accurately pipette 4 μL of each of the above standard solutions and add 36 μL of blank plasma. Vortex to mix, preparing plasma samples equivalent to concentrations of 10, 30, 100, 300, 1000, 3000, 5000, 10000, 15000, and 30000 ng / mL. Perform dual-sample analysis for each concentration to establish a standard curve. Take 30 μL of plasma and add 200 μL of propranolol in acetonitrile solution. Vortex to mix and centrifuge at 4000 rpm for 10 min. Analyze the supernatant by LC-MS / MS.

[0125] 6. Data Processing

[0126] After detecting blood drug concentrations by LC-MS / MS, the pharmacokinetic parameters of mice after drug administration were calculated using WinNonlin software and a non-compartmental model.

[0127] 7. Experimental Results

[0128] Comparison of PK data after drug administration between crystalline and amorphous samples

[0129] Data showed that the exposure levels after administration of the crystalline sample were higher than those in the amorphous sample group.

[0130] Example 11: Preparation and Pharmacological Study of Compounds

[0131] Example C1

[0132] Synthesis of 6'-(((1S,3S)-3-((5-(difluoromethoxy)thiazo[5,4-b]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0133] Step 1: Synthesis of 5-bromo-2-(difluoromethoxy)pyridine

[0134] At room temperature, 5-bromopyridin-2-ol (10.0 g, 57.5 mmol, 1.00 eq) was dissolved in DMF (200 mL), and cesium carbonate (22.5 g, 69.0 mmol, 1.20 eq) was added. The mixture was stirred at 25 °C for 1.5 h under nitrogen protection, and then sodium 2-chloro-2,2-difluoroacetate (26.3 g, 172 mmol, 3.00 eq) was added. The mixture was then heated to 100 °C and stirred for 1.5 h.

[0135] LCMS showed the target product's MS (RT = 0.537 min, m / z = 223.9 [M+H]). +The mixture was cooled to room temperature, and 600 ml of water was added. It was extracted with 600 ml of ethyl acetate (600 ml x 5). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The sample was mixed with silica gel and purified by column chromatography. The product was extracted with pure petroleum ether. A colorless oily liquid, 5-bromo-2-(difluoromethoxy)pyridine (3.01 g, 13.4 mmol, 23.38% yield), was obtained. LCMS (ESI) m / z = 223.9 [M+H]. + .

[0136] Step 2: Synthesis of 6-(difluoromethoxy)pyridine-3-amine

[0137] At room temperature, 5-bromo-2-(difluoromethoxy)pyridine (3.00 g, 13.4 mmol, 1.00 eq), ammonia (2.35 g, 20.1 mmol, 2.58 mL, 30% purity, 1.50 eq), cuprous iodide (510 mg, 2.68 mmol, 0.2 eq), potassium carbonate (2.78 g, 20.09 mmol, 1.50 eq), and L-proline (616.76 mg, 5.36 mmol, 0.40 eq) were added to N-methylpyrrolidone (30 mL), and the mixture was heated to 140 °C in a sealed container and stirred for 12 hours.

[0138] TLC (petroleum ether / ethyl acetate = 5 / 1) showed that the reactants had reacted completely. 20 mL of aqueous solution was added, and the mixture was extracted with ethyl acetate (30 mL * 4). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was stirred with silica gel and separated by column chromatography (petroleum ether / ethyl acetate = 5 / 1) to give a yellow oily product, 6-(difluoromethoxy)pyridine-3-amine (1.3 g, 8.12 mmol, 60.62% yield).

[0139] Step 3: Synthesis of 5-(difluoromethoxy)thiazo[5,4-b]pyridine-2-amine

[0140] Under 0°C ice bath conditions, 6-(difluoromethoxy)pyridine-3-amine (1.00 g, 6.25 mmol, 1.00 eq) and potassium thiocyanate (4.86 g, 50.0 mmol, 4.84 mL, 8.00 eq) were dissolved in glacial acetic acid (3.00 mL). A solution of bromine (2.99 g, 18.8 mmol, 965 μL, 3.00 eq) in glacial acetic acid (1.00 mL) was slowly added dropwise, keeping the reaction solution temperature below 0°C. After the addition was complete, the temperature was naturally raised to 20°C, and the reaction was stirred at 20°C for 8 hours.

[0141] TLC (petroleum ether / ethyl acetate = 2 / 1) showed that the starting material reacted completely. 5 ml of water was added, the temperature was raised to 85°C, and the mixture was filtered while hot. 1.5 ml of acetic acid was added to the filter cake, the temperature was raised to 85°C, and the mixture was filtered while hot. The two filtrates were combined, and the pH was adjusted to 8 with ammonia in an ice-water bath. A large amount of yellow solid precipitated out. The mixture was filtered under reduced pressure, and the filter cake was dried to give the yellow target product 5-(difluoromethoxy)thiazo[5,4-b]pyridine-2-amine (0.9 g, 4.14 mmol, 66.35% yield).

[0142] Step 4: Synthesis of 2-chloro-5-(difluoromethoxy)thiazo[5,4-b]pyridine

[0143] At room temperature, 5-(difluoromethoxy)thiazo[5,4-b]pyridine-2-amine (0.90 g, 4.14 mmol, 1.00 eq) was dissolved in acetonitrile (15 mL) under nitrogen protection. Isoamyl nitrite (728 mg, 6.22 mmol, 837 μL, 1.50 eq) and copper chloride (669 mg, 4.97 mmol, 161 μL, 1.20 eq) were added, and the mixture was stirred at 25 °C for 3 hours.

[0144] TLC (petroleum ether / ethyl acetate = 2 / 1) showed that the reactants reacted completely. 8 ml of saturated ammonium chloride solution was added and stirred for 10 minutes. 25 ml of water was added for dilution. The mixture was extracted with ethyl acetate in 80 ml solutions (4 times). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. 。 The sample was mixed with silica gel and purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 - 0 / 1). The target product, 2-chloro-5-(difluoromethoxy)thiazo[5,4-b]pyridine, was obtained as a white solid (0.7 g, 2.96 mmol, 71.39% yield). LCMS (ESI) m / z = 236.9 [M+H] + .

[0145] Step 5: Synthesis of 6'-(((1S,3S)-3-((5-(difluoromethoxy)thiazo[5,4-b]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0146] At room temperature, 2-chloro-5-(difluoromethoxy)thiazo[5,4-b]pyridine (200 mg, 845 μmol, 1 eq), 6'-(((1S,3S)-3-aminocyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one hydrochloride (259 mg, 845 μmol, 1.00 eq, HCl) and triethylamine (257 mg, 2.54 mmol, 352 μL, 3.00 eq) were added to DMSO (10.0 mL), and the mixture was heated to 60 °C for 12 hours under nitrogen protection.

[0147] LCMS showed the target product's MS time (RT = 0.423 min, m / z = 471.1 [M+H]). + The reaction mixture was quenched with 30 ml of aqueous solution, extracted with 60 ml of ethyl acetate (4 times), and the organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was then stirred with silica gel and purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0–1 / 1). A yellow solid product, 6'-(((1S,3S)-3-((5-(difluoromethoxy)thiazo[5,4-b]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one, was obtained (124 mg, 264 μmol, 31.18% yield).

[0148] LCMS(ESI)m / z = 471.1[M+H] +

[0149] 1 H NMR (400MHz, CDCl3): δ8.41(d,J=6.6Hz,1H),7.93(d,J=2.6Hz,1H),7.84-7.77(m,1H ),7.64-7.56(m,1H),7.47(ddd,J=2.1,6.8,9.1Hz,1H),7.44-7.37(m,1H),7.02-6.8 8(m,2H),6.53(d,J=9.0Hz,1H),6.44(d,J=9.1Hz,1H),6.27(dt,J=1.2,6.7Hz,1H),4 .47-4.26(m,2H),2.26-2.11(m,2H),1.96(tq,J=6.6,13.7Hz,2H),1.65-1.49(m,2H).

[0150] Example C2

[0151] Synthesis of 6'-(((1S,3S)-3-((7-(difluoromethoxy)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0152] Step 1: Synthesis of 2,7-dibromo-[1,2,4]triazolo[1,5-a]pyridine

[0153] (7-bromo-[1,2,4]triazolo[1,5-a]pyridine-2-amine (2.00 g, 9.39 mmol, 1 equivalent), copper bromide (3.15 g, 14.1 mmol, 1.5 equivalent), and tert-butyl nitrite (1.45 g, 14.1 mmol, 1.5 equivalent) were dissolved in acetonitrile (120 mL) and stirred at 80 °C for 3 hours. The MS value of the product was monitored by LCMS (RT = 0.452 min). The reaction solution was then cooled to room temperature. The reaction mixture was then diluted with water (200 mL) and extracted with ethyl acetate (80 mL x 2). The concentrated organic phase was washed with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give 2,7-dibromo-[1,2,4]triazolo[1,5-a]pyridine (2.00 g, 7.22 mmol, 76.9% yield). No purification was required; it was used directly for the next step. LCMS (ESI) m / z = 277.9 [M+1] +

[0154] Step 2: Synthesis of 2-bromo-[1,2,4]triazolo[1,5-a]pyridine-7-ol

[0155] 2,7-Dibromo-[1,2,4]triazolo[1,5-a]pyridine (2.00 g, 7.22 mmol, 1 equivalent), potassium hydroxide (1.2 g, 21.7 mmol, 3 equivalent), t-Bu Xphos (675 mg, 1.59 mmol, 0.22 equivalent), and Pd2(dba)3 (728 mg, 794 μmol, 0.11 equivalent) were dissolved in 1-4,dioxane (20 mL) and water (5 mL) and stirred at 100 °C for 4 hours. The MS value of the product was monitored by LCMS (RT = 0.358 min). The reaction solution was cooled to room temperature, then diluted with water (60 mL) and ethyl acetate (40 mL). The organic phase was separated and discarded. The pH of the aqueous phase was adjusted to pH = 5 with dilute hydrochloric acid, and then the aqueous phase was extracted with ethyl acetate (60 mL * 2). The concentrated organic phase was washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give 2-bromo-[1,2,4]triazolo[1,5-a]pyridine-7-ol (860 mg, 3.97 mmol, 54.9% yield, 98.7% purity). It was used directly as the next step without purification. LCMS (ESI) m / z = 215.9 [M+1] +

[0156] Step 3: Synthesis of 2-bromo-7-(difluoromethoxy)-[1,2,4]triazolo[1,5-a]pyridine

[0157] 2-Bromo-[1,2,4]triazolo[1,5-a]pyridin-7-ol (400 mg, 1.84 mmol, 1 equivalent) and cesium carbonate (721 mg, 2.21 mmol, 1.2 equivalent) were dissolved in N,N-dimethylformamide (10 mL), and stirred at 20 °C for 1 hour. Sodium difluorochloroacetate (844 mg, 5.53 mmol, 3 equivalent) was then added to the reaction mixture, and the mixture was stirred at 100 °C for 3 hours. TLC (petroleum ether / ethyl acetate = 3 / 1) showed that the reaction was complete. The reaction mixture was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (20 mL x 2). The concentrated organic phase was washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by reverse-phase preparative chromatography (column: C18 150 × 30 mm; mobile phase: [water(FA)-ACN]; gradient: 30%-60% B over 7 min) to give 2-bromo-7-(difluoromethoxy)-[1,2,4]triazolo[1,5-a]pyridine (230 mg, 871 μmol, 47.2% yield, 100% purity). LCMS (ESI) m / z = 263.9 [M+1] +

[0158] Step 4: Synthesis of 6'-(((1S,3S)-3-((7-(difluoromethoxy)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0159] 2-Bromo-7-(difluoromethoxy)-[1,2,4]triazolo[1,5-a]pyridine (100 mg, 379 μmol, 1 equivalent), 6'-(((1S,3S)-3-aminocyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (123 mg, 455 μmol, 1.2 equivalent), Xantphos (17.5 mg, 30.3 μmol, 0.08 equivalent), sodium phenolate (66.0 mg, 568 μmol, 1.5 equivalent), and Pd2(dba)3 (13.9 mg, 15.2 μmol, 0.04 equivalent) were dissolved in dioxane (4 mL), and then stirred in a microwave at 135 °C for 40 min. The MS value of the product was monitored by LCMS (RT = 0.391 min). The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by reverse-phase preparation (column: C18 150×30mm; mobile phase: [water(FA)-ACN]; gradient: 12%-42% B over 7min) to obtain 6'-(((1S,3S)-3-((7-(difluoromethoxy)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (49.0 mg, 103 μmol, 27.1% yield, 95.1% purity).

[0160] LCMS(ESI)m / z = 454.2[M+1] +

[0161] 1 H NMR: (400MHz, DMSO-d6): δ8.63(d,J=7.3Hz,1H),7.92(d,J=2.5Hz,1H),7.63-7.22( m,4H),7.14(d,J=2.6Hz,1H),6.91(d,J=6.9Hz,1H),6.78-6.70(m,2H),6.52(d,J=9 .0Hz,1H),6.44(d,J=8.9Hz,1H),6.26(dt,J=1.3,6.7Hz,1H),4.38-4.26(m,1H),4. 15(qd,J=6.8,13.4Hz,1H),2.22-2.09(m,2H),2.02-1.82(m,2H),1.64-1.43(m,2H).

[0162] Example C3

[0163] Synthesis of 6'-(((1S,3S)-3-((6-(difluoromethoxy)thiazo[5,4-b]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0164] Step 1: Synthesis of 3-bromo-5-(difluoromethoxy)pyridine

[0165] At room temperature, 3-bromo-5-hydroxypyridine (40.0 g, 230 mmol, 1.00 eq) was dissolved in DMF (800 mL).

[0166] Add Cs2CO3 (89.9 g, 276 mmol, 1.20 eq) to the mixture, and stir at 25 °C for 1.5 hours under nitrogen protection. Then add sodium 2-chloro-2,2-difluoroacetate (105 g, 690 mmol, 3.00 eq), and heat to 100 °C and stir for 1.5 hours.

[0167] TLC (petroleum ether / ethyl acetate = 5 / 1) showed complete reaction of the starting material and formation of the target product. 60 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The product was purified by column chromatography (pure petroleum ether yield) using silica gel. A yellow oily compound, 3-bromo-5-(difluoromethoxy)pyridine (6.3 g, 28.1 mmol, 12.23% yield), was obtained.

[0168] Step 2: Synthesis of 5-(difluoromethoxy)pyridine-3-amine

[0169] At room temperature, 3-bromo-5-(difluoromethoxy)pyridine (5.00 g, 22.3 mmol, 1.00 eq), ammonia (3.91 g, 33.5 mmol, 4.30 mL, 30% purity, 1.50 eq), CuI (850 mg, 4.46 mmol, 0.20 eq), K₂CO₃ (4.63 g, 33.48 mmol, 1.5 eq), and N,N'-bis(2-furanmethyl)oxalamide (2.22 g, 8.93 mmol, 0.4 eq) were added to NMP (10 mL), and the mixture was allowed to react in a sealed container. The temperature was raised to 145 °C, and the mixture was stirred for 36 hours.

[0170] TLC (petroleum ether / ethyl acetate = 1 / 1) showed complete reaction of the starting material and formation of a new principal spot. The reaction mixture was quenched with 100 mL of water, extracted with ethyl acetate (100 mL * 4), and the organic phases were combined. The mixture was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Column chromatography (petroleum ether / ethyl acetate = 1 / 1) was used for purification to obtain the target product 5-(difluoromethoxy)pyridine-3-amine (3 g, 18.7 mmol, 83.94% yield).

[0171] Step 3: Synthesis of 6-(difluoromethoxy)thiazo[5,4-b]pyridine-2-amine

[0172] Under an ice bath at 0°C, 5-(difluoromethoxy)pyridine-3-amine (2.00 g, 12.5 mmol, 1.00 eq) and KSCN (9.71 g, 100 mmol, 9.68 mL, 8.00 eq) were dissolved in AcOH (6 mL), and Br2 (5.99 g, 37.5 mmol, 1.93 mL, 3.00 eq) in AcOH (2 mL) was slowly added dropwise, keeping the temperature of the reaction solution below 0°C. After the addition was complete, the temperature was naturally raised to 20°C and the reaction was stirred for 8 hours.

[0173] TLC (petroleum ether / ethyl acetate = 2 / 1) showed incomplete reaction of the starting material, with a new spot of the target product forming. 5 ml of water was added, the temperature was raised to 85°C, and the mixture was filtered while hot. 1.5 ml of acetic acid was added to the filter cake, the temperature was raised to 85°C, and the mixture was filtered while hot. The two filtrates were combined, and the pH was adjusted to 8 with ammonia in an ice-water bath. A large amount of yellow solid precipitated. The mixture was filtered under reduced pressure, and the filter cake was dried. The sample was mixed with silica gel, and purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1). The target product, 6-(difluoromethoxy)thiazo[5,4-b]pyridine-2-amine (460 mg, 2.12 mmol, 16.96% yield), was obtained as a yellow solid. LCMS (ESI) m / z = 218.0 [M+H] +

[0174] Step 4: Synthesis of 2-chloro-6-(difluoromethoxy)thiazo[5,4-b]pyridine

[0175] At room temperature, 6-(difluoromethoxy)thiazo[5,4-b]pyridine-2-amine (450 mg, 2.07 mmol, 1.00 eq) was dissolved in acetonitrile (10 mL) under nitrogen protection. Isoamyl nitrite (364 mg, 3.11 mmol, 418 μL, 1.50 eq) and CuCl2 (334 mg, 2.49 mmol, 1.20 eq) were added, and the mixture was stirred at 25 °C for 12 hours.

[0176] LCMS showed the target product's MS time (RT = 0.524 min, m / z = 236.9 [M+H]). + Add 20 ml of saturated ammonium chloride solution and stir for 10 minutes. Dilute with 100 ml of water, extract with 150 ml of ethyl acetate (4 times), combine the organic phases, wash with saturated sodium chloride solution, dry to anhydrous sodium sulfate, and concentrate under reduced pressure. Purify by column chromatography (petroleum ether / ethyl acetate = 5 / 1). The target product, 2-chloro-6-(difluoromethoxy)thiazo[5,4-b]pyridine, is given as a white solid (275 mg, 1.16 mmol, 56.09% yield). LCMS (ESI) m / z = 236.9 [M+H]. +

[0177] Step 5: Synthesis of 6'-(((1S,3S)-3-((6-(difluoromethoxy)thiazo[5,4-b]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0178] At room temperature, 2-chloro-6-(difluoromethoxy)thiazo[5,4-b]pyridine (100 mg, 423 μmol, 1.00 eq), 1-[6-[[[(1S,3S)-3-aminocyclopentyl]amino]-3-pyridyl]pyridin-2-one (114 mg, 423 μmol, 1 eq), and TEA (128 mg, 1.27 mmol, 176 μL, 3.00 eq) were added to DMSO (2.00 mL), and the mixture was stirred at 70 °C for 6 hours.

[0179] LCMS showed the target product's MS (RT = 0.417 min, m / z = 471.2 [M+H]). + The solution was cooled to room temperature, diluted with 10 ml of water, and extracted with 60 ml of ethyl acetate (4 times). The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by column chromatography (petroleum ether / ethyl acetate = 0 / 1). The target product, 6'-(((1S,3S)-3-((6-(difluoromethoxy)thiazo[5,4-b]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (198 mg, 421 μmol, 99.58% yield), was obtained as a pale yellow solid.

[0180] LCMS(ESI)m / z = 471.2[M+H] +

[0181] 1 H NMR (400MHz, DMSO-d6): δ8.67(d,J=6.8Hz,1H), 8.01(d,J=2.4Hz,1H), 7.93(d,J=2. 6Hz,1H),7.60(dd,J=1.6,6.8Hz,1H),7.56(d,J=2.5Hz,1H),7.51-7.07(m,3H),6.97 (d,J=6.9Hz,1H),6.53(d,J=8.9Hz,1H),6.44(d,J=8.9Hz,1H),6.27(dt,J=1.2,6.7 Hz,1H),4.46-4.28(m,2H),2.28-2.11(m,2H),2.06-1.87(m,2H),1.69-1.45(m,2H).

[0182] Example C4

[0183] Synthesis of 6'-(((1S,3S)-3-((5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)chloro)cyclopentyl)chloro)-2H-[1,3'-bipyridin]-2-one

[0184] Step 1: 5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-a]pyridine-2-amine

[0185] At 25°C, [1,2,4]triazolo[1,5-a]pyridine-2-amine (2.50 g, 18.6 mmol, 1.00 eq), wet palladium on carbon (0.40 g, 10% purity) and HCl (3.65 mL, 36.5% purity, 2.00 eq) were dissolved in ethanol (30 mL), purged three times with hydrogen, and then reacted under a hydrogen (50 psi) atmosphere for 72 hours.

[0186] LC-MS showed the MS value of the product. After the reaction was complete, the product was concentrated by filtration to give 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridine-2-amine (2.50 g, 97.1% yield). MS (ESI) m / z = 139.1 [M+1] +

[0187] Step 2: 2-Iodo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridine

[0188] 5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-a]pyridine-2-amine (0.20 g, 1.45 mmol, 1.00 eq), potassium iodide (601 mg, 3.62 mmol, 2.50 eq), p-toluenesulfonic acid (997 mg, 5.79 mmol, 4.00 eq), and sodium nitrite (200 mg, 2.89 mmol, 2.00 eq) were dissolved in acetonitrile (2 mL) and water (0.40 mL), and then the mixture was heated to 50 °C and reacted for 2 hours.

[0189] TLC (dichloromethane:methanol = 20:1) shows the feedstock (R) f =0.24) completely consumed.

[0190] The reaction solution was concentrated, then diluted with ethyl acetate (50 mL) and water (70 mL), and the aqueous phase was extracted with ethyl acetate (20 mL * 3). The concentrated organic phase was washed with saturated brine (30 mL * 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 50:1 to 1:1) to obtain the product 2-iodo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridine (250 mg, 69.4% yield).

[0191] Step 3: 6'-(((1S,3S)-3-((5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0192] 2-Iodo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridine (200 mg, 1.00 eq), 1-[6-[[[(1S,3S)-3-aminocyclopentyl]amino]-3-pyridyl]pyridin-2-one (217 mg, 1.00 eq), sodium tert-butoxide (232 mg, 2.41 mmol, 3.00 eq) and t-BuXPhos Pd G3 (63.8 mg, 0.10 eq) were dissolved in dioxane (15 mL), purged three times with nitrogen, and then heated to 90 °C and reacted for 12 hours.

[0193] LCMS displays the MS value of the detected product.

[0194] The reaction solution was concentrated to obtain a crude product, which was then prepared by reverse phase reaction (column: C18 150×30mm; mobile phase: [water(FA)-ACN]; gradient: 2%-32% B over 7min) to obtain the product 6'-(((1S,3S)-3-((5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (45mg, 13.80% yield, 96.4% purity).

[0195] LCMS(ESI)m / z = 392.2[M+1] +

[0196] 1H NMR (400MHz, CD3OD): δ7.98-7.89(m,1H),7.66-7.54(m,2H),7.49-7.36(m ,1H),6.61(dd,J=5.2,8.8Hz,2H),6.47(dt,J=1.2,6.8Hz,1H),4.40-4.27( m,1H),4.06(quin,J=6.4Hz,1H),3.94(t,J=6.0Hz,2H),2.74(t,J=6.4Hz, 2H),2.66(s,1H),2.35-2.16(m,2H),2.09-1.87(m,5H),1.66-1.51(m,2H).

[0197] Example C5

[0198] Synthesis of 6'-(((1S,3S)-3-((7-cyclopropyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0199] Step 1: 2-Bromo-[1,2,4]triazolo[1,5-a]pyrazine

[0200] [1,2,4]triazolo[1,5-a]pyrazin-2-amine (7.00 g, 51.8 mmol, 1.00 eq) and hydrobromic acid (29.3 mL, 48% purity, 5.00 eq) were dissolved in acetic acid (40 mL) at 0°C. Then, a solution of sodium nitrite (7.15 g, 2.00 eq) dissolved in water (40 mL) was added dropwise to the reaction solution. After the addition was complete, the reaction was kept at the temperature for 2 hours.

[0201] LCMS displays the MS value of the detected product.

[0202] The reaction solution was concentrated, then diluted with water (100 mL) and ethyl acetate (60 mL), and the aqueous phase was extracted with ethyl acetate (20 mL x 3). The concentrated organic phase was washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 20:1 to 1:1) to obtain the product 2-bromo-[1,2,4]triazolo[1,5-a]pyrazine (3.50 g, 33.9% yield). LCMS (ESI) m / z = 200.9 [M+1] +

[0203] Step 2: 2-Bromo-5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine

[0204] At 20°C, 2-bromo-[1,2,4]triazolo[1,5-a]pyrazine (3.00 g, 1.00 eq) was dissolved in ethanol (20 mL), and then lithium borohydride (2.00 M, 30.2 mL, 4.00 eq) was added to the solution. After the addition was complete, the reaction solution was heated to 50°C and reacted for 5 hours.

[0205] LCMS displays the MS value of the detected product.

[0206] After the reaction was complete, the reaction solution was quenched with 1M hydrochloric acid (800 mL), followed by washing with ethyl acetate (30 mL * 2), and the organic phase was discarded directly. The aqueous phase was adjusted to pH = 9 with saturated sodium carbonate aqueous solution, and then extracted with dichloromethane (40 mL * 5). The organic phase from the second extraction was washed with saturated brine (30 mL * 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was prepared by reverse-phase chromatography (column: Waters Xbridge 150 * 25 mm 5 μm; mobile phase: [water(NH4HCO3)-ACN]; gradient: 1%-30% B over 9 min) to obtain the product 2-bromo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (1.60 g, 52.3% yield). MS (ESI) m / z = 203.0 [M+1] +

[0207] Step 3: 2-Bromo-7-cyclopropyl-6,8-dihydro-5H-[1,2,4]triazolo[1,5-a]pyrazine

[0208] 2-Bromo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (0.70 g, 3.45 mmol, 1.00 eq), cyclopropylboronic acid (4441 mg, 5.17 mmol, 1.50 eq), copper acetate (1.25 g, 6.90 mmol, 2.00 eq), and N,N-diisopropylethylamine (891 mg, 1.20 mL, 2.00 eq) were dissolved in dichloromethane (2 mL), and then reacted at 20°C for 12 hours.

[0209] LCMS showed that the product was detected.

[0210] The reaction solution was diluted with dichloromethane (50 mL) and water (30 mL), and the aqueous phase was subsequently extracted with dichloromethane (10 mL * 4). The concentrated organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 20:1 to 1:1) to obtain the product 2-bromo-7-cyclopropyl-6,8-dihydro-5H-[1,2,4]triazolo[1,5-a]pyrazine (160 mg, 19.09% yield). MS (ESI) m / z = 243.0 [M+1] +

[0211] Step 4: 6'-(((1S,3S)-3-((7-cyclopropyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0212] 2-Bromo-7-cyclopropyl-6,8-dihydro-5H-[1,2,4]triazolo[1,5-a]pyrazine (140 mg, 1.00 eq), 1-[6-[[[(1S,3S)-3-aminocyclopentyl]amino]-3-pyridyl]pyridin-2-one (156 mg, 1.00 eq), sodium tert-butoxide (166 mg, 3.00 eq), and t-BuXPhos Pd G3 (45.7 mg, 0.10 eq) were dissolved in dioxane (12 mL), purged three times with nitrogen, and then heated to 90 °C and reacted for 12 hours.

[0213] LCMS displays the MS value of the detected product.

[0214] The reaction solution was concentrated to obtain a crude product, which was then prepared by reverse-phase chromatography (column: C18 150×30mm; mobile phase: [water(FA)-ACN]; gradient: 5%-35% B over 7min and column: C18 150×30mm; mobile phase: [water(FA)-ACN]; gradient: 5%-35% B over 7min) to obtain the product 6'-(((1S,3S)-3-((7-cyclopropyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (20 mg, 8.03% yield).

[0215] LCMS(ESI)m / z = 433.2[M+1] +

[0216] 1H NMR (400MHz, CDCl3): δ8.03(d,J=2.4Hz,1H),7.52(dd,J=2.4,8.8Hz,1H),7.39(ddd,J=2.4,6.8,9.2Hz,1H),7 .30(dd,J=2.0,7.2Hz,1H),6.65(d,J=9.2Hz,1H),6.44(d,J=8.8Hz,1H),6.23(dt,J=1.2,6.8Hz,1H),4.77(br dd,J=1.2,5.6Hz,1H),4.28-4.12(m,2H),4.07-3.95(m,3H),3.79(s,2H),3.12(t,J=5.6Hz,2H) ,2.39-2.23(m,2H),2.11-1.97(m,2H),1.95-1.86(m,1H),0.62-0.55(m,2H),0.54-0.48(m,2H).

[0217] Example C6

[0218] 1-(5-(((1S,3S)-3-((5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)pyrazin-2-yl)pyridin-2(1H)-one

[0219] Step 1: 1-[6-[[[(1S,3S)-3-[(7-cyclopropyl-6,8-dihydro-5H-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)amino]cyclopentyl]amino]-3-pyridyl]pyridin-2-one

[0220] 2-Iodo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridine (300 mg, 1.00 eq), 1-[5-[[[(1S,3S)-3-aminocyclopentyl]amino]pyrazin-2-yl]pyridin-2-one (327 mg, 1.00 eq), sodium tert-butoxide (347 mg, 3.61 mmol, 3.00 eq) and t-BuXPhos Pd G3 (95.7 mg, 0.10 eq) were dissolved in dioxane (10 mL), purged three times with nitrogen, and then heated to 90 °C and reacted for 12 hours.

[0221] LCMS displays the MS value of the detected product.

[0222] The reaction solution was concentrated to obtain a crude product, which was then prepared by reverse phase chromatography (column: Waters Xbridge 150*25mm*5um; mobile phase: [water(ammonia hydroxide v / v)-ACN]; gradient: 0%-25% B over 10min) to obtain the product 1-(5-(((1S,3S)-3-((5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)pyryl-2-yl)pyridin-2(1H)-one (30 mg, 6.35% yield).

[0223] LCMS(ESI)m / z=391.1[M-1] +

[0224] 1 H NMR: (400MHz, CDCl3): δ7.76 (d, J=1.2Hz, 1H), 7.63 (dd, J=2.0, 6.8Hz, 1H), 7.38 (ddd,J=2.0,6.8,9.2Hz,1H),6.64(d,J=9.2Hz,1H),6.32-6.22(m,1H),4.93(br d,J=6.8Hz,1H),4.41-4.29(m,1H),4.24-4.12(m,1H),4.04(br d,J=6.8Hz,1H),3.97(t,J=6.0Hz,2H),2.78(t,J=6.4Hz,2H),2.42-2.25(m,2H),2.12-1.99(m,4H),1.96-1.87(m,2H),1.60-1.51(m,2H).

[0225] Example C7

[0226] Synthesis of 6'-(((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0227] Step 1: Synthesis of 2-bromo-[1,2,4]triazolo[1,5-a]pyridine

[0228] [1,2,4]triazolo[1,5-a]pyridine-2-amine (1.90 g, 14.2 mmol, 1.0 eq), copper bromide (4.75 g, 21.3 mmol, 1.5 eq), and tert-butyl nitrite (2.19 g, 21.3 mmol, 1.50 eq) were dissolved in acetonitrile (30 mL) and stirred at 70 °C for 4 h. TLC (petroleum ether / ethyl acetate = 1 / 1) showed complete reaction. The reaction mixture was cooled to room temperature and then diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 2). The concentrated organic phase was washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give 2-bromo-[1,2,4]triazolo[1,5-a]pyridine (2.17 g, 11.0 mmol, 77.4% yield). No purification was required; it was used directly for the next step. LCMS MS(ESI)m / z=200.0[M+1] + .

[0229] Step 2: Synthesis of 6'-(((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0230] 2-Bromo-[1,2,4]triazolo[1,5-a]pyridine (100 mg, 505 μmol, 1.0 eq), 6'-(((1S,3S)-3-aminocyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (150 mg, 556 μmol, 1.1 eq), Xantphos (23.4 mg, 40.4 μmol, 0.08 eq), sodium phenolate (87.9 mg, 758 μmol, 1.5 eq), and Pd2(dba)3 (18.5 mg, 20.2 μmol, 0.04 eq) were dissolved in dioxane (4 mL), and then stirred in a microwave oven at 140 °C for 1 h. The MS value of the product was monitored by LCMS (RT = 0.347 min). The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was first purified by reverse-phase preparation (column: Waters Xbridge 150*25mm*5um; mobile phase: [water(NH4HCO3)-ACN]; gradient: 12%-42% B over 9min) to obtain 6'-(((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (microwave parallel injection was performed in 4 batches, 83.1mg, 211μmol, 10.5% yield, 98.4% purity).

[0231] LCMS MS(ESI)m / z = 388.2[M+1] +.

[0232] 1 H NMR, (400MHz, DMSO-d6): δ8.61-8.55(m,1H),7.92(d,J=2.8Hz,1H),7.60(dd,J=1.6,6.8Hz,1H),7. 47(ddd,J=2.1,6.7,9.1Hz,1H),7.44-7.34(m,2H),6.92(d,J=6.9Hz,1H),6.85(dt,J=1.6,6.8Hz,1H ),6.64(d,J=7.3Hz,1H),6.53(d,J=8.8Hz,1H),6.44(d,J=8.8Hz,1H),6.26(dt,J=1.3,6.7Hz,1H),4 .40-4.26(m,1H),4.18(sxt,J=6.7Hz,1H),2.21-2.09(m,2H),2.00-1.84(m,2H),1.63-1.43(m,2H).

[0233] Among them, the deuterated compound C7A of compound 7 was prepared by combining this preparation method with a deuteration preparation method, and its structure is as follows:

[0234] The identification data is as follows: 1 H NMR(400MHz, DMSO-d6)δ8.59(s,1H),7.92(d,J=2.7Hz,1H),7.61(dd,J=6.8,2.1Hz,1H),7.53–7.4 6(m,1H),7.46–7.33(m,3H),6.94(d,J=6.9Hz,1H),6.66(d,J=7.3Hz,1H),6.53(d,J=8.9Hz,1H),6 .49–6.40(m,1H),6.27(td,J=6.7,1.4Hz,1H),4.33(q,J=6.6Hz,1H),4.19(q,J=6.7Hz,1H),2.15( ddt,J=12.1,7.0,4.5Hz,2H),1.97(dt,J=13.4,6.8Hz,1H),1.93–1.83(m,1H),1.65–1.40(m,2H).

[0235] Example C8

[0236] Synthesis of 6'-(((1S,3S)-3-((7-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0237] Step 1: Synthesis of 2,7-dibromo-[1,2,4]triazolo[1,5-a]pyridine

[0238] 7-Bromo-[1,2,4]triazolo[1,5-a]pyridine-2-amine (2.00 g, 9.39 mmol, 1.0 eq), copper bromide (3.15 g, 14.1 mmol, 1.5 eq), and tert-butyl nitrite (1.45 g, 14.1 mmol, 1.5 eq) were dissolved in acetonitrile (120 mL) and stirred at 80 °C for 3 h. The MS value of the product was monitored by LCMS (RT = 0.452 min). The reaction mixture was cooled to room temperature, diluted with water (200 mL), and extracted with ethyl acetate (80 mL x 2). The concentrated organic phase was washed with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give 2,7-dibromo-[1,2,4]triazolo[1,5-a]pyridine (2.00 g, 7.22 mmol, 76.9% yield). No purification was performed; it was used directly for the next step. LCMS MS(ESI)m / z=277.9[M+1] + .

[0239] Step 2: Synthesis of 2-bromo-[1,2,4]triazolo[1,5-a]pyridine-7-ol

[0240] 2,7-Dibromo-[1,2,4]triazolo[1,5-a]pyridine (2.00 g, 7.22 mmol, 1.0 eq), potassium hydroxide (1.20 g, 21.7 mmol, 3.0 eq), t-Bu Xphos (675 mg, 1.59 mmol, 0.22 eq), and Pd2(dba)3 (728 mg, 794 μmol, 0.11 eq) were dissolved in 1-4, dioxane (20 mL) and water (5 mL), and stirred at 100 °C for 4 hours. The MS value of the product was monitored by LCMS (RT = 0.358 min). The reaction solution was cooled to room temperature, then diluted with water (60 mL) and ethyl acetate (40 mL). The organic phase was separated and discarded. The pH of the aqueous phase was adjusted to pH = 5 with dilute hydrochloric acid, and then the aqueous phase was extracted with ethyl acetate (60 mL * 2). The concentrated organic phase was washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give 2-bromo-[1,2,4]triazolo[1,5-a]pyridine-7-ol (860 mg, 3.97 mmol, 54.9% yield, 98.7% purity). It was used directly as the next step without purification. LC-MS MS (ESI) m / z = 215.9 [M+1] + .

[0241] Step 3: Synthesis of 2-bromo-7-methoxy-[1,2,4]triazolo[1,5-a]pyridine

[0242] 2-Bromo-[1,2,4]triazolo[1,5-a]pyridin-7-ol (1.30 g, 6.07 mmol, 1.0 eq) and cesium carbonate (4.95 g, 15.2 mmol, 2.5 eq) were dissolved in N,N-dimethylformamide (20 mL), and then methyl iodoformane (1.72 g, 12.2 mmol, 2.0 eq) was added to the reaction solution. After the addition was complete, the mixture was stirred at 40 °C for 2 hours. TLC (petroleum ether / ethyl acetate = 3 / 1) showed that the reaction was complete. The reaction solution was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (30 mL * 2). The concentrated organic phase was washed with saturated brine (50 mL * 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain 2-bromo-7-methoxy-[1,2,4]triazolo[1,5-a]pyridine (1.00 g, 4.39 mmol, 72.2% yield).

[0243] Step 4: Synthesis of 6'-(((1S,3S)-3-((7-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0244] 2-Bromo-7-methoxy-[1,2,4]triazolo[1,5-a]pyridine (100 mg, 439 μmol, 1 eq), 6'-(((1S,3S)-3-aminocyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (130 mg, 482 μmol, 1.1 eq), Xantphos (20.3 mg, 35.1 μmol, 0.08 eq), sodium phenolate (76.4 mg, 658 μmol, 1.5 eq), and Pd2(dba)3 (16.1 mg, 17.5 μmol, 0.04 eq) were dissolved in dioxane (5 mL), and then stirred in a microwave oven at 140 °C for 1 h. The MS value of the product was monitored by LCMS (RT = 0.366 min). The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was first purified by reverse-phase preparative purification (acidic conditions) (column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 20%-50% B over 15min), and then purified again by reverse-phase preparative purification (alkaline conditions) (column: Waters Xbridge 150*25mm*5um; mobile phase: [water(ammonia hydroxide v / v)-ACN]; gradient: 0%-30% B over (10 min) to obtain 6'-(((1S,3S)-3-((7-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one (8 batches were microwaved in parallel, 69.9 mg, 166 μmol, 4.72% yield, 98.9% purity).

[0245] LCMS MS(ESI)m / z=418.2[M+1] + . 1H NMR, (400MHz, DMSO-d6): δ8.39(d,J=7.4Hz,1H),7.91(d,J=2.6Hz,1H),7.59(dd,J=1.7,6.8Hz ,1H),7.47(ddd,J=2.0,6.7,9.1Hz,1H),7.39(dd,J=2.6,8.9Hz,1H),6.89(d,J=6.9Hz,1H),6. 81(d,J=2.6Hz,1H),6.58-6.38(m,4H),6.26(dt,J=1.3,6.7Hz,1H),4.31(sxt,J=6.4Hz,1H),4 .13(sxt,J=6.7Hz,1H),3.82(s,3H),2.19-2.08(m,2H),1.99-1.82(m,2H),1.62-1.44(m,2H).

[0246] Example C9

[0247] Synthesis of 1-(5-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)pyrazin-2-yl)pyridin-2(1H)-one

[0248] Step 1: Synthesis of 2-bromo-[1,2,4]triazolo[1,5-a]pyridine

[0249] [1,2,4]triazolo[1,5-a]pyridine-2-amine (1.90 g, 14.2 mmol, 1.0 eq), copper bromide (4.75 g, 21.3 mmol, 1.5 eq), and tert-butyl nitrite (2.19 g, 21.3 mmol, 1.50 eq) were dissolved in acetonitrile (30 mL) and stirred at 70 °C for 4 h. TLC (petroleum ether / ethyl acetate = 1 / 1) showed complete reaction. The reaction mixture was cooled to room temperature and then diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 2). The concentrated organic phase was washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give 2-bromo-[1,2,4]triazolo[1,5-a]pyridine (2.17 g, 11.0 mmol, 77.4% yield). No purification was required; it was used directly for the next step. LCMS MS(ESI)m / z=200.0[M+1] + .

[0250] Step 2: Synthesis of 1-(5-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)pyrazin-2-yl)pyridin-2(1H)-one

[0251] 2-Bromo-[1,2,4]triazolo[1,5-a]pyridine (300 mg, 1.51 mmol, 1.0 eq), 1-(5-((((1S,3S)-3-aminocyclopentyl)amino)pyrazin-2-yl)pyridin-2(1H)-one (411 mg, 1.51 mmol, 1.0 eq), sodium tert-butoxide (437 mg, 4.54 mmol, 3.0 eq), and tBuXPhos Pd G3 (120 mg, 152 μmol, 0.10 eq) were dissolved in dioxane (10 mL), and the mixture was stirred at 100 °C for 12 hours. The MS value of the product was monitored by LCMS (RT = 0.394 min). The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by reverse-phase preparative chromatography (column: Phenomenex luna C18). 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 10%-40% B over 15min) to obtain 1-(5-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)pyrazin-2-yl)pyridin-2(1H)-one (79.6mg, 201μmol, 13.2% yield, 97.9% purity).

[0252] LCMS MS(ESI)m / z=389.2[M+1] + . 1 H NMR, (400MHz, DMSO-d6): δ8.62-8.54(m,1H),8.25(d,J=1.4Hz,1H),7.87(d,J= 1.3Hz,1H),7.72(dd,J=1.5,6.9Hz,1H),7.56-7.32(m,4H),6.87(dt,J=1.5,6.8 Hz,1H),6.66(d,J=7.3Hz,1H),6.47(d,J=9.0Hz,1H),6.32(dt,J=1.3,6.7Hz,1 H),4.41-4.15(m,2H),2.25-2.11(m,2H),2.06-1.86(m,2H),1.67-1.47(m,2H).

[0253] Example C10

[0254] Synthesis of 1-(5-(((1S,3S)-3-((6,7-dihydro-5H-[1,2,4]triazolo[1,3]thiazin-2-yl)amino)cyclopentyl)amino)pyrazin-2-yl)pyridin-2(1H)-one

[0255] Step 1: 2-Bromo-6,7-dihydro-5H-[1,2,4]triazolo[5,1-b][1,3]thiazine

[0256] Compound 10-1 (3.00 g, 13.2 mmol, 1.00 eq), 3-chloropropane-1-thiol (1.76 g, 15.8 mmol, 1.55 mL, 1.20 eq), and K2CO3 (3.60 g, 26.5 mmol, 2.00 eq) were dissolved in DMF (20 mL), and the reaction mixture was stirred at 50 °C for 5 hours.

[0257] The formation of the target product was detected by LCMS. The reaction solution was quenched with 200 mL of water, then extracted with ethyl acetate (100 mL x 3 mL). The organic phases were combined, washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, and concentrated by filtration. The solid organic phase reaction solution was purified by normal silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to give compound 10⁻² (800 mg, 3.63 mmol, yield: 27.5%) as a white solid.

[0258] Step 2: Synthesis of 1-(5-(((1S,3S)-3-((6,7-dihydro-5H-[1,2,4]triazolo[1,3]thiazin-2-yl)amino)cyclopentyl)amino)pyrazin-2-yl)pyridin-2(1H)-one

[0259] Under nitrogen protection, compound 10-3 (300 mg, 1.11 mmol, 1.00 eq), compound 10-2 (4988 mg, 2.27 mmol, 2.05 eq), t-BuONa (318 mg, 3.32 mmol, 3.00 eq), [2-(2-aminophenyl)phenyl]-methylsulfonyloxy-palladium; di-tert-butyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphine (175 mg, 221 μmol, 0.20 eq) were dissolved in 1,4-dioxane (20 mL), and the reaction mixture was stirred at 90 °C for 16 hours.

[0260] The formation of the target product was detected by LC-MS and HPLC. The reaction solution was quenched with 200 mL of water, followed by extraction with ethyl acetate (100 mL x 3 mL). The organic phases were combined, washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, and concentrated by filtration. The organic phase was purified using reverse preparative chromatography (column: Phenomenex Luna C18 150 x 25 mm x 10 μm; mobile phase: [water(TFA)-ACN]; gradient: 12%-42% B over 11 min) to obtain the target compound (60.0 mg, 146 μmol, yield: 13.2%) as a pale yellow gelatinous compound. LC-MS: [M+H]+ = 411.

[0261] 1H NMR(400MHz,Chloroform-d)δ8.52(s,1H),7.73(d,J=1.6Hz,1H),7.62(dd,J=7.2,2.1Hz,1H),7.41–7.33(m,1H),6.62(d,J=9.3Hz,1H),6.33–6.21( m,1H),5.01(d,J=6.8Hz,1H),4.31(q,J=6.6Hz,1H),4.22–4.04(m,4H),3. 23–3.06(m,2H),2.43–2.22(m,4H),2.07–1.99(m,2H),1.63–1.49(m,3H).

[0262] Example C11

[0263] Synthesis of 1-(5-(((1S,3S)-3-((4-oxy-6,7-dihydro-5H-[1,2,4]triazolo[5,1-b][1,3]thioazin-2-yl)amino)cyclopentyl)amino)pyrazin-2-yl)piperidin-2(1H)-one

[0264] Compound 10 (20.0 mg, 48.7 μmol, 1.00 eq) was dissolved in THF (8.00 mL), and then NaIO4 (10.4 mg, 48.7 μmol, 2.70 μL, 1.00 eq) dissolved in H2O (3 mL) was added. The reaction mixture was stirred at 70 °C for 12 hours.

[0265] The formation of the target product was detected by LC-MS and HPLC. The reaction solution was directly concentrated under reduced pressure. The compound was purified by reverse preparation (column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [water(TFA)-ACN]; gradient: 10%-40% B over 11min) to obtain the target compound (18.0 mg, 42.2 μmol, yield: 86.6%) as a white solid. LC-MS: [MH]-=427.1.

[0266] 1 H NMR, (CHLOROFORM-d, 400MHz): 8.54 (dd, 1H, J = 1.0, 4.3Hz), 8.04 (d, 1H, J = 5.6H z),7.7-7.8(m,1H),7.52(ddd,1H,J=1.9,6.9,9.0Hz),6.81(d,1H,J=9.1Hz),6 .46(dt,1H,J=0.9,6.8Hz),4.3-4.4(m,2H),4.1-4.2(m,2H),3.42(dd,1H,J=5. 9,12.6Hz),2.9-3.2(m,2H),2.3-2.5(m,3H),2.0-2.2(m,2H),1.6-1.8(m,2H).

[0267] Example C12

[0268] Following the preparation methods of Examples C1 and C3 described above, compounds C12, C12A, and C12B were prepared via the following synthetic route:

[0269] Compound 12 was chirally resolved (column type: CHIRALCEL OD-H 5µm 10mm*250mm; mobile phase: n-hexane / ethanol = 70:30; flow rate: 5ml / min; column temperature: 30℃) to give white solids C12A and C12B:

[0270] Compound C12A, 1H NMR: (400MHz, DMSO-d6): δ8.59-8.33(m,1H),8.08(dd,J=1.6,4.8Hz,1H),7.92(d,J=2.4Hz,1H),7.66(dd,J=1.4,8.0Hz,1H),7.55-7.48(m ,2H),7.40(dd,J=2.8,8.9Hz,1H),7.24(dd,J=4.8,8.0Hz,1H),6.96(d,J=6.9Hz,1H),6.53(d,J=8.9Hz,1H),6.31(t,J=6.8Hz,1H),5.06(br s,1H),4.79-4.62(m,1H),4.37(dt,J=6.4,13.8Hz,2H),2.27-2.12(m,2H),2.04-1.90(m,2H),1.65-1.48(m,2H),1.26(d,J=6.4Hz,3H);

[0271] Compound C12B, 1 H NMR: (400MHz, DMSO-d6): δ8.54-8.37(m,1H),8.08(dd,J=1.4,4.8Hz,1H),7.92(d,J=2.6Hz,1H),7.66( dd,J=1.4,8.1Hz,1H),7.55-7.48(m,2H),7.40(dd,J=2.6,8.9Hz,1H),7.24(dd,J=4.8,8.0Hz,1H),6.9 6(d,J=6.9Hz,1H),6.53(d,J=8.9Hz,1H),6.31(t,J=6.8Hz,1H),5.06(d,J=4.6Hz,1H),4.75-4.67(m,1 H),4.44-4.29(m,2H),2.27-2.13(m,2H),2.03-1.91(m,2H),1.66-1.49(m,2H),1.25(d,J=6.4Hz,3H).

[0272] Example C13

[0273] Compounds C13, C13A, and C13B were prepared by referring to the preparation methods of Examples C1, C3, and C12 described above:

[0274] Compound C13A, 1H NMR: (400MHz, DMSO-d6): δ8.49(br d,J=6.4Hz,1H),8.16-8.08(m,1H),7.89(d,J=2.0Hz,1H),7.70(dd,J=1.2,8.0Hz,1H),7.62-7.55(m,3H),7.28(dd,J=4.8,8.0Hz,1H),7.03(br d,J=7.2Hz,1H),6.37(t,J=6.8Hz,1H),5.12(d,J=4.4Hz,1H),4.76(quin,J=5.6Hz,1H),4.66-4.53(m, 1H),4.51-4.39(m,1H),2.31-2.16(m,2H),2.11-2.02(m,2H),1.74-1.60(m,2H),1.30(d,J=6.4Hz,3H);

[0275] Compound C13B, 1 H NMR: (400MHz, DMSO-d6): δ8.45(br d,J=4.4Hz,1H),8.08(dd,J=1.2,4.8Hz,1H),7.84(d,J=2.0Hz,1H),7.66(dd ,J=1.2,8.0Hz,1H),7.60-7.51(m,3H),7.24(dd,J=4.8,8.0Hz,1H),6.98(br d,J=6.8Hz,1H),6.33(t,J=6.8Hz,1H),5.08(d,J=4.4Hz,1H),4.80-4.65(m,1H),4.55(sxt,J=7.2Hz,1H ),4.46-4.35(m,1H),2.27-2.15(m,2H),2.02(t,J=7.2Hz,2H),1.69-1.56(m,2H),1.26(d,J=6.4Hz,3H).

[0276] Example C14

[0277] Compound C14 was prepared by referring to the preparation method of Example C2 described above:

[0278] Example C45

[0279] Synthesis of 6'-(((1S,3S)-3-((7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0280] Step 1: Synthesis of 2-((6-bromo-4-iodopyridin-3-yl)oxy)ethane-1-ol

[0281] Add t-BuOK (5.95 g, 53.0 mmol, 2.00 eq) to a solution of C45-1 (8.00 g, 26.5 mmol, 1.00 eq) and ethylene glycol (55.6 g, 896 mmol, 50 mL, 33.8 eq) in 50 mL of NMP. Stir the mixture at 60 °C for 3 hours.

[0282] The formation of the target product was detected by LCMS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by reversed-phase column HPLC (0.1% FA condition) to give compound C45-2 (3.50 g, 10.1 mmol, 38.4% yield) as a white solid.

[0283] Step 2: Synthesis of 7-bromo-2,3-dihydro-[1,4]dioxo[2,3-c]pyridine

[0284] CuI (116 mg, 610 μmol, 0.06 eq), t-BuOK (1.60 g, 14.2 mmol, 1.40 eq), and 3,4,7,8-tetramethyl-1,10-phenanthroline (192 mg, 814 μmol, 0.08 eq) were added to a 35 mL solution of C45-2 (3.50 g, 10.1 mmol, 1.00 eq) in isopropanol (35 mL). The mixture was stirred at 80 °C for 1 hour under nitrogen protection.

[0285] The formation of the target product was detected by LCMS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to give compound C45-3 (2.20 g, 9.12 mmol, 89.6% yield) as a brown solid.

[0286] Step 3: Synthesis of 2,3-dihydro-[1,4]dioxo[2,3-c]pyridine-7-amine

[0287] To a 20 mL solution of C45-3 (2.20 g, 10.1 mmol, 1.00 eq) in ethylene glycol, add Cu2O (14.5 mg, 101 μmol, 10.4 μL, 0.01 eq), NH3H2O ​​(50.9 g, 407 mmol, 56.0 mL, 40.0 eq), K2CO3 (281 mg, 2.04 mmol, 0.200 eq), and N',N'-dimethylethane-1,2-diamine (89.7 mg, 1.02 mmol, 111 μL, 0.100 eq). Stir the mixture at 80 °C for 4 hours.

[0288] The formation of the target product was detected by LCMS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to obtain compound C45-4 (1.50 g, crude) as a yellow oil.

[0289] Step 4: Synthesis of N-(2,3-dihydro-[1,4]dioxo[2,3-c]pyridin-7-ylaminothio)carbamate

[0290] Add N-(thiomethylene)carbamate (1.29 g, 9.86 mmol, 1.00 eq) to a solution of C45-4 (1.50 g, 9.86 mmol, 1.00 eq) in dichloromethane (15 mL). Stir the mixture at 25 °C for 1 hour.

[0291] The formation of the target product was detected by LCMS. The reaction solution was poured into water and extracted with dichloromethane. The organic phase was washed with a saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to give compound C45-5 (2.20 g, 7.77 mmol, 78.7% yield) as a yellow solid.

[0292] Step 5: Synthesis of 7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridine-2-amine

[0293] Add NH₂OH·HCl (2.43 g, 34.9 mmol, 4.50 eq) and DIEA (3.01 g, 23.30 mmol, 4.06 mL, 3 eq) to a methanol solution (15 mL) of C45-5 (2.20 g, 7.77 mmol, 1.00 eq). Stir the mixture at 70 °C for 2 hours.

[0294] The formation of the target product was detected by LCMS. The reaction mixture was cooled to 25°C and filtered. The residue was compound C45-6 (1.30 g, 6.76 mmol, 87.1% yield) as a white solid.

[0295] Step 6: Synthesis of 2-bromo-7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridine

[0296] At 0 °C, NaNO2 (359 mg, 5.20 mmol, 2.00 eq) and HBr (1.21 g, 5.98 mmol, 812 μL, 2.30 eq) were added to a 5 mL solution of acetonitrile containing C45-6 (500 mg, 2.60 mmol, 1.00 eq). The mixture was stirred at 25 °C for 2 hours.

[0297] The formation of the target product was detected by LCMS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to give compound C45-7 (300 mg, 1.17 mmol, 45.0% yield) as a yellow solid.

[0298] Step 7: Synthesis of 6'-(((1S,3S)-3-((7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0299] Add t-BuONa (225 mg, 2.34 mmol, 2.00 eq), Pd2(dba)3 (107 mg, 117 μmol, 0.100 eq) and Xantphos (135 mg, 234 μmol, 0.200 eq) to a dioxane (5 mL) solution of C45-7 (300 mg, 1.17 mmol, 1.00 eq) and C45-8 (316 mg, 1.17 mmol, 1.00 eq), and stir the mixture at 100 °C for 12 hours under nitrogen protection.

[0300] The formation of the target product was detected by LCMS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by reverse preparation (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water(NH4HCO3)-ACN]; gradient: 10%-40% B over 52 min) and (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 1%-25% B over 10 min) to obtain the target compound (36.6 mg).

[0301] LC-MS:[M+H]-=446.1. 1 H NMR: (DMSO-d6, 400MHz): δ (ppm) = 8.38 (s, 1H), 7.91 (d, J = 2.4Hz, 1H), 7.60 (dd, J = 2.0, 6.8Hz, 1H),7.49-7.45(m,1H),7.39(dd,J=2.8,8.8Hz,1H),6.90(d,J=6.8Hz,1H),6.78(s,1H),6.52 (d,J=9.2Hz,1H),6.44(d,J=9.2Hz,1H),6.36(d,J=7.2Hz,1H),6.26(dt,J=1.2,6.8Hz,1H),4 .37-4.24(m,5H),4.18-4.05(m,1H),2.20-2.06(m,2H),1.99-1.78(m,2H),1.64-1.41(m,2H).

[0302] Example C46

[0303] Synthesis of 6'-((((1S,3S)-3-((7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one

[0304] Step 1: Synthesis of 2-iodo-7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridine

[0305] At 0 °C, NaNO2 (215 mg, 3.12 mmol, 2.00 eq) and HI (1.02 g, 3.59 mmol, 600 μL, 2.30 eq) were added to a 5 mL solution of C46-1 (300 mg, 1.56 mmol, 1.00 eq). The mixture was stirred at 25 °C for 2 hours.

[0306] The formation of the target product was detected by LCMS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed with an aqueous sodium bicarbonate solution, dried with anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to give compound C46-2 (480 mg, 1.11 mmol, 71.0% yield) as a brown solid.

[0307] Step 2: Synthesis of 6'-((((1S,3S)-3-((7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one

[0308] Add t-BuONa (190.26 mg, 1.98 mmol, 2 eq) and tBuBrettphos Pd G3 (84.5 mg, 98.9 μmol, 0.100 eq) to a dioxane (5 mL) solution of C46-2 (300 mg, 989 μmol, 1.00 eq) and C46-3 (285 mg, 989 μmol, 1.00 eq). Stir the mixture at 100 °C for 4 hours under nitrogen protection.

[0309] The formation of the target product was detected by LCMS. The reaction solution was filtered, and the filtrate was concentrated and evaporated to dryness. The crude product was purified by reverse preparation (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water(NH4HCO3)-ACN]; gradient: 16%-46% B over 15min) to obtain the target compound (20.0 mg).

[0310] LC-MS:[M+H] - =464.1. 1H NMR: (DMSO-d6, 400MHz): δ (ppm) = 8.38 (s, 1H), 7.84 (d, J = 2.0Hz, 1H), 7.64 (dd, J = 1.6, 6.8Hz, 1H), 7.57-7.45 (m, 2H), 6.90 (br d,J=7.2Hz,1H),6.78(s,1H),6.46(d,J=8.8Hz,1H),6.35(d,J=7.2Hz,1H),6.28(t,J=6.4Hz,1H),4.57-4.47(m,1H),4.35(br dd,J=2.0,5.2Hz,2H),4.31-4.24(m,2H),4.17-4.07(m,1H),2.18-2.06(m,2H),1.99-1.90(m,2H),1.63-1.50(m,2H).

[0311] Example C47

[0312] Synthesis of 2-(6-(((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5-fluoropyridin-3-yl)pyridazine-3(2H)-one

[0313] Step A: tert-butyl((1S,3S)-3-((5-bromo-3-fluoropyridin-2-yl)amino)cyclopentyl)carbamate

[0314] At room temperature, 100.0 g (0.52 mol) of 5-bromo-2,3-difluoropyridine and tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate (114.58 g, 0.57 mol) were added to DMF (800 mL), followed by the addition of triethylamine (156.99 g, 1.55 mol), and the mixture was heated to 100 °C and reacted for 8 hours.

[0315] After the reaction was complete, the mixture was cooled to room temperature, and water (500 mL) was added to the system. Extraction was performed with ethyl acetate (400 mL × 3). The organic phases were combined, washed twice with saturated brine (300 mL), dried, filtered, and concentrated under reduced pressure. The residue was slurried with isopropyl ether (300 mL) to give 130 g of a white solid product, tert-butyl((1S,3S)-3-((5-bromo-3-fluoropyridin-2-yl)amino)cyclopentyl)carbamate (yield 67%). LC-MS: [M+H] + =374.

[0316] Step B: tert-butyl((1S,3S)-3-((3-fluoro-5-(6-oxopyridazin-1(6H)-yl)pyridin-2-yl)amino)cyclopentyl)amino)carbamate

[0317] At room temperature, tert-butyl((1S,3S)-3-((5-bromo-3-fluoropyridin-2-yl)amino)cyclopentyl)carbamate (120 g, 0.32 mol), pyridazine-3(2H)-one (40 g, 0.41 mol), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (9.1 g, 0.064 mol), and potassium carbonate (133.19 g, 0.96 mol) were added to NMP (960 mL), followed by the addition of cuprous iodide (12.3 g, 0.064 mol). After the addition was complete, the nitrogen atmosphere was replaced and the temperature was raised to 120°C for 12 hours.

[0318] After the reaction was complete, the mixture was cooled to room temperature, and water (1000 mL) was added to the system. The mixture was extracted with ethyl acetate (400 mL × 3). The organic phases were combined, washed three times with saturated brine (400 mL), dried, filtered, and concentrated under reduced pressure. The residue was purified by normal-phase column chromatography (dichloromethane / methanol = 50:1) to give 70 g of a black oily product, tert-butyl((1S,3S)-3-((3-fluoro-5-(6-oxopyridazin-1(6H)-yl)pyridin-2-yl)amino)cyclopentyl)amino)carbamate (yield 56%). LC-MS: [M+H] + =390.

[0319] Step C: 2-(6-(((1S,3S)-3-aminocyclopentyl)amino)-5-fluoropyridin-3-yl)pyrazin-3(2H)-one

[0320] At room temperature, tert-butyl((1S,3S)-3-((3-fluoro-5-(6-oxopyridazin-1(6H)-yl)pyridin-2-yl)amino)cyclopentyl)amino)carbamate (70 g, 0.17 mol) was dissolved in hydrochloric acid / 1,4-dioxane (2 M, 300 mL) and reacted at room temperature for 4 hours.

[0321] After the reaction was completed, the solution was concentrated to dryness to obtain 2-(6-(((1S,3S)-3-aminocyclopentyl)amino)-5-fluoropyridin-3-yl)pyrazin-3(2H)-one hydrochloride. This hydrochloride was then dissolved in methanol (200 mL), and the pH of the system was adjusted to approximately 9 using a basic ion exchange resin. The solution was filtered, and the filter cake was washed three times with methanol (100 mL). The filtrate was collected and then evaporated to dryness to obtain 55 g of a brown solid product, 2-(6-(((1S,3S)-3-aminocyclopentyl)amino)-5-fluoropyridin-3-yl)pyrazin-3(2H)-one. This product was used directly in the next reaction without further processing. LC-MS: [M+H] + =290.

[0322] Step D: 2-(6-(((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5-fluoropyridin-3-yl)pyridazine-3(2H)-one

[0323] At room temperature, 55 g (0.19 mol) of 2-(6-(((1S,3S)-3-aminocyclopentyl)amino)-5-fluoropyridin-3-yl)pyrazin-3(2H)-one and 2-bromo-[1,2,4]triazolo[1,5-a]pyridine (37.4 g, 0.19 mol) were dissolved in 1,4-dioxane (550 mL), followed by the addition of sodium tert-butoxide (36.5 g, 0.38 mol) and methanesulfonic acid (2-di-tert-butylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (30 g, 0.037 mol). After the addition was complete, nitrogen was purged, and the system was slowly heated to 100°C and reacted for 1 hour.

[0324] After the reaction was complete, the mixture was cooled to room temperature, and water (400 mL) was added to the system. Extraction was performed with ethyl acetate (300 mL × 3). The organic phases were combined, washed with saturated brine (200 mL), dried, filtered, and concentrated under reduced pressure. The residue was purified by normal-phase column chromatography (dichloromethane / methanol = 10:1) to give 20.5 g of 2-(6-(((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5-fluoropyridin-3-yl)pyridazin-3(2H)-one. LC-MS: [M+H] + =407.

[0325] 1 H NMR(400MHz, DMSO-d6)δ8.61–8.58(dd,J=6.7,1.1Hz,1H),8.06–8.04(q,J=1.7Hz,2H),7.67–7.61(dd,J=12.1 ,2.2Hz,1H),7.52–7.47(dd,J=9.5,3.9Hz,1H),7.46–7.41(m,1H),7.40–7.37(dt,J=8.8,1.3Hz,1H),7.09–7.0 5(dd,J=9.5,1.6Hz,1H),6.99–6.94(m,1H),6.90–6.84(td,J=6.7,1.6Hz,1H),6.68–6.63(d,J=7.2Hz,1H),4.6 1–4.51(p,J=7.0Hz,1H),4.26–4.17(p,J=6.6Hz,1H),2.22–2.12(m,2H),2.02–1.97(m,2H),1.67–1.54(m,2H).

[0326] Example C54

[0327] Synthesis of 2-(6-((((1S,3S)-3-((7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)pyridazin-3(2H)-one

[0328] Step 1: Synthesis of 2-(6-((((1S,3S)-3-((7,8-dihydro-[1,4]dioxo[2,3-d][1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)pyridazin-3(2H)-one

[0329] Add t-BuONa (190 mg, 1.98 mmol, 5.00 eq) and tBuBrettphos PD G3 (33.8 mg, 39.60 μmol, 0.100 eq) to a dioxane solution (4 mL) of C54-1 (120 mg, 395 μmol, 1.00 eq) and C54-2 (107 mg, 395 μmol, 1.00 eq). Stir the mixture at 100 °C for 12 hours under nitrogen protection.

[0330] The formation of the target product was detected by LCMS. The reaction solution was filtered, and the filtrate was concentrated and evaporated to dryness. The crude product was purified by reverse-phase preparation (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 1%-30% B over 10min) to obtain the target compound (18.6 mg).

[0331] LC-MS:[M+H] - =447.1. 1H NMR: (DMSO-d6, 400MHz): δ (ppm) = 8.37 (s, 1H), 8.09 (d, J = 2.4Hz, 1H), 8.01 (dd, J = 1.6, 4.0 Hz,1H),7.51(dd,J=2.4,8.8Hz,1H),7.46(dd,J=4.0,9.6Hz,1H),7.02(dd,J=1.6,9.6Hz,1 H),6.91(d,J=7.2Hz,1H),6.77(s,1H),6.52(d,J=8.8Hz,1H),6.35(d,J=7.6Hz,1H),4.37 -4.25(m,5H),4.16-4.07(m,1H),2.18-2.08(m,2H),1.99-1.83(m,2H),1.56-1.44(m,2H).

[0332] Examples C15-C95

[0333] Following the aforementioned preparation method, compounds C15-C95 were prepared:

[0334] Example C96 related activity test

[0335] Test method:

[0336] At 25°C in Biacore TMSurface plasmon resonance data were collected on an 8K system (GE Healthcare). Streptavidin was immobilized on an SA (Cytiva) sensor chip using standard amine coupling chemistry at 25 °C with NBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the run buffer. Briefly, the carboxymethyldextrose glycoside surface was activated by injecting 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 μl / min for 12 min. To capture streptavidin, the protein was diluted to 0.5 mg / mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 100 μL onto the active chip surface. Excess residual activating groups were blocked by injecting 1 M ethanolamine (pH 8.5) for 7 min. Protein was diluted to 5 μL / mL in NBS-N, 0.05% Tween-20, and 0.1 mM CaCl2 at a flow rate of 5 μL / min for 60 s. Aci-tagged PCSK9 protein was captured on streptavidin surfaces. Typical surface densities obtained were 2900–3200 RU. SPR binding data were obtained using appropriate dilution series for each compound at a flow rate of 30 μL / min, with a capture time of 60 s / concentration point and a dissociation time of 3600 s. The run buffer used for compound binding studies was 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P2O, and 2% DMSO. Data for DMSO correction do not include volume effects. Standard processing procedures were used, all data were double-referenced for blank injection and reference surfaces, and data processing and kinetic fitting were performed using Scrubber software version 2.0c (Biological Software). A simple 1:1 binding model was used to fit the data to determine KB. D value.

[0337] Experimental results:

[0338] K D As shown in Table 1 below:

[0339] Table 1. PCSK9 Kinetics Data

[0340] A < 1 * E - 8.

[0341] Example C97: Pharmacokinetic Experiment

[0342] 1. Reagents and Instruments

[0343] Polyethylene glycol 400 (batch number R22040588, Shanghai Shaoyuan Reagent Co., Ltd.), DMSO (batch number 20200319, Guangdong Guanghua Technology Co., Ltd.), physiological saline (batch number 2011110727, Chenxin Pharmaceutical Co., Ltd.). LC-MS instruments (Thermo Fisher Ultimate 3000UPLC, TSQ QUANTUM ULTRA triple quadrupole mass spectrometer, AB SCIEX 5500+QTARP).

[0344] 2. Laboratory animals

[0345] SD rats: male, 180-250g, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.

[0346] 3. Formulation preparation

[0347] Accurately weigh the sample powder, dissolve it completely in DMSO, add PEG-400, vortex and sonicate to mix, then add physiological saline and vortex and sonicate to make a concentration of 0.5 mg / mL (DMSO:PEG-400:NS=5:60:35, V / V / V). Administer 10 mL / kg by gavage or 2 mL / kg by intravenous administration.

[0348] 4. Blood sample collection

[0349] After intravenous or gavage administration to rats, 200 μL of venous blood was collected at 5 min (no blood was collected after gavage), 15 min, 30 min, 1 h, 2 h, 5 h, 7 h, and 24 h and placed in EDTA-K2-anticoagulant EP tubes. The tubes were centrifuged at 10,000 rpm for 2 min, and the plasma was frozen at -80℃ for later testing.

[0350] 5. Biological Analysis

[0351] Accurately weigh a certain amount of the test sample and dissolve it in DMSO to a concentration of 2 mg / mL to prepare a stock solution. Accurately pipette an appropriate amount of the stock solution and dilute it with acetonitrile to prepare a series of standard solutions. Accurately pipette 4 μL of each of the above standard solutions and add 36 μL of blank plasma. Vortex to mix, preparing plasma samples equivalent to concentrations of 1, 3, 5, 10, 30, 100, 300, 1000, and 3000 ng / mL. Perform dual-sample analysis for each concentration to establish a standard curve. Take 30 μL of plasma and add 200 μL of acetonitrile solution containing the internal standard propranolol (5 ng / mL). Vortex to mix and centrifuge at 4000 rpm for 10 min. Analyze the supernatant by LC-MS. The LC-MS detection conditions are as follows:

[0352] Chromatographic column: YMC-Triart C18, 50×2.1mm, S-3μm 12nm.

[0353] Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile, flow rate: 0.5 mL / min, gradient elution as shown in Table 2 below:

[0354] Table 2

[0355] 6. Data Processing

[0356] After detecting blood drug concentrations by LC-MS, the pharmacokinetic parameters of mice after drug administration were calculated using WinNonlin 6.1 software and a non-compartmental model. The results are shown in Table 3 below.

[0357] Table 3: Pharmacokinetic parameters of the compounds of this invention in rats (IV and PO administration)

[0358] The compounds of this invention have improved half-life and in vivo exposure compared to control compounds, and are superior to compounds 458B and 464 of CN113574055A, as well as compound C14.

[0359] Effect of Example C98 on hERG currents in hERG-HEK293 cells

[0360] Experimental Methods: Human embryonic kidney cells (hERG-HEK293 cells) stably expressing the hERG channel were used in the experiment. hERG-HEK293 cells were clamped in a whole-cell voltage-clamp configuration using an automated patch-clamp system, and hERG currents were induced by appropriate voltages. Cells were administered extracellular fluid containing 0.3% DMSO (negative control) and 30 μM of the compound, or 1, 10, 100, and 1000 nM of cisapride (positive control). The tail current of the hERG channel was recorded, and the peak value of the tail current at each concentration was obtained. Using the peak value of the tail current recorded under the negative control (0.3% DMSO) as 100%, the inhibition rate of hERG current by the 30 μM compound and different concentrations of cisapride was calculated. Cisapride concentration-response curve fitting and IC50 analysis were performed. 50 The calculations were performed using GraphPad Prism software, and the results are shown in Table 4.

[0361] Table 4. Effects of hERG currents on hERG-HEK293 cells.

[0362] The risk of the compound Herg of this invention is improved compared with the control compound, and is superior to CN113574055A control compound 458B, superior to WO2024078620A1 compound 27, and superior to WO2025007915A1 compound 591.

[0363] Example: Binding affinity test of C99 substitute to PCSK9 protein

[0364] The binding affinity of the compounds of the present invention to PCSK9 protein was determined by fluorescence polarization method.

[0365] All compounds were dissolved in DMSO to prepare 10 mM stock solutions. Positive and test compounds were serially diluted 5-fold with DMSO, starting at 10 mM, for a total of 8 concentration gradients. First, a certain volume of fluorescent probe solution was prepared to a concentration of 5 nM using test buffer (20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, and 0.01% Tween-20). Then, the serial DMSO solutions of the test compounds were diluted 50-fold with the fluorescent probe solution. Finally, a solution of recombinant human PCSK9 protein (ACRO, Cat#PC9-H5223) at a concentration of 4.5 μg / mL was prepared using test buffer. After the test solution was prepared, 5 μL of PCSK9 protein was added to a black 384-well plate (PerkinElmer, Cat#6008260), along with 5 μL of different concentrations of the compound (DMSO final concentration 1%). A positive control group (test buffer + equal volume of target protein + equal proportion of fluorescent probe molecules) and a negative control group (test buffer + equal proportion of fluorescent probe molecules) were also set up. The final concentration of the probe molecules in the system was 2.5 nM, and the median concentration of PCSK9 protein was 2.25 μg / mL. After incubation at room temperature with shaking for 15 minutes, the fluorescence polarization values ​​were read using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The inhibition rate of the drug = [1-(mP...] (药物筛选组) -mP (阴性对照 组) ]÷[mP (阳性对照组) -mP (阴性对照组) []×100. Using the logarithm of compound concentration as the x-axis and the inhibition rate as the y-axis, a 4-parameter nonlinear regression curve was fitted to calculate the IC. 50 Value(Y=Bottom+(Top-Bottom) / (1+10^((LogIC A50 -X)*HillSlope)), where: Hillslope represents the slope of this curve, IC 50 This indicates the half-maximal inhibitory concentration (MCI).

[0366] Table 5. FP-IC 50 data

[0367] B < 300nm. *Incubate for 18 hours.

[0368] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A free base crystalline form of a compound of Formula (C): ###0001### (C) characterized in that: A ring is selected from wherein X and Y form a 5-7 membered saturated or unsaturated ring containing 0, 1 and 2 heteroatoms selected from O, N and S; B is selected from Q is selected from N or CR1, and R1 is selected from H or halogen; T1 is selected from N or CH; R2 is selected from H, alkyl, or halogen, and there are one or more R2s. R3is selected from hydrogen or represents a hydrogen on the A ring which is further substituted by oxo, alkyl, halo, alkoxy, alkylthio, haloalkyl, haloalkoxy, cycloalkyl, cycloalkylalkyl, alkynyl, R3is one or more, or adjacent R3form an alkoxy group R4is selected from hydrogen, halogen, hydroxyl, alkoxy, haloalkoxy, substituted or unsubstituted alkyl, cyano, -C(O)-O-alkyl, phenylalkoxy, carboxyl, hydroxymethyl, or cycloalkyl, the substituents being selected from hydroxyl, amide, halogen, or The components that are replaced are: U1, U2, and U4, which are independently selected from CH or N; U3, which is selected from CH2 or NH; and R4, which is one or more. and when the A ring is selected from at the time, B ring is not 2. The compound according to claim 1, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that, The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 1-ethylpropyl, 2-methylbutyl, tert-pentyl, 1,2-dimethylpropyl, isopentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, tert-hexyl, neohexyl, 2-methylpentyl, 1,2-dimethylbutyl, and 1-ethylbutyl. The alkoxy group is selected from methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. The cycloalkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; The halogen is selected from fluorine, chlorine, bromine, and iodine.

3. The free base crystalline form of a compound as represented by Formula (C) according to claim 1, characterized in that, Q is selected from N; and / or Q is selected from CH, R2 is selected from F; and / or T1 is selected from N; and / or R3is selected from hydrogen, methyl, methoxy, cyclopropyl, cyclopropylmethyl, fluoro, chloro, oxo, CHF2-O-; R4is selected from hydrogen, methyl, hydroxyl, hydroxymethyl, cyano, F, Cl, Br, -O-CH2-phenyl, -COOH, -COOCH2CH3, Amides, formamide, CH3-C(O)-, ethynyl, trifluoromethyl, difluoromethoxy, methoxy.

4. The free base crystalline form of a compound as represented by Formula (C) according to claim 1, characterized in that, A ring is selected from Further, the R3substituted A ring is selected from:

5. The free base crystalline form of a compound as represented by Formula (C) according to claim 1, characterized in that, R4-substituted B ring is selected from:

6. A crystalline form of a free base of a compound of Formula (C) according to any one of claims 1-5, wherein, The compounds are selected from C1-C95.

7. A crystalline form of a free base of a compound of Formula (C) according to any one of claims 1-5, wherein, The compound is selected from compound A, and the free base crystal form is selected from crystal form A of compound A. Compound A is 6'-((((1S,3S)-3-([1,2,4]triazolo[1,5-a]pyridin-2-ylamino)cyclopentyl)amino)-5'-fluoro-2H-[1,3'-bipyridine]-2-one. Crystal form A of compound A has characteristic peaks at 17.99°, 18.80°, and 19.69° in the X-ray diffraction pattern, represented by a 2θ angle, with an error of ±0.2°.

8. The free base crystalline form of a compound as represented by Formula (C) according to claim 7, characterized in that, The crystal form A has characteristic peaks at 12.21°, 13.42°, 17.99°, 18.80°, 19.69°, and 25.85° in the X-ray diffraction pattern, represented by a 2θ angle, with an error of ±0.2°.

9. The free base crystalline form of a compound as represented by Formula (C) according to claim 7, characterized in that, The crystal form A, represented by a 2θ angle in the X-ray diffraction pattern, has characteristic peaks at 4.79°, 9.45°, 12.21°, 13.42°, 16.23°, 17.99°, 18.80°, 19.69°, 20.64°, 24.43°, 25.85°, 27.78°, and 28.76°, with an error of ±0.2°.

10. The free base crystalline form of a compound as represented by Formula (C) according to claim 7, characterized in that, The X-ray diffraction pattern of crystal form A of compound A is shown in Figure 1 or Figure 3.

11. A crystalline form of a free base of a compound of Formula (C) according to any one of claims 7-10, wherein, The crystal form A of compound A has an endothermic peak at 154.3℃ in the DSC spectrum, with an error of ±5℃.

12. A crystalline form of a free base of a compound of Formula (C) according to any one of claims 7-10, wherein, The DSC spectrum of crystal form A of compound A is shown in Figure 2 or Figure 4.

13. A pharmaceutical composition, characterized by, The pharmaceutical composition contains a free base crystal form of a compound of general formula (C) according to any one of claims 1-12 in a therapeutically effective amount, and one or more pharmaceutically acceptable carriers.

14. Use of the free base crystalline form of a compound as represented by Formula (C) according to any one of claims 1-12, or the pharmaceutical composition of claim 13, in the manufacture of a medicament for the treatment and / or prevention of a disease associated with PCSK9 inhibition.

15. Use according to claim 14, characterized in that, The disease includes a hypercholesterolemia disease.

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